open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1559sociobiology 64(2): 225-227 (june, 2017) first observations of a social wasp preying on termite workers termites are an abundant potential food source throughout the tropics and subtropics (e.g. lubin et al., 1977). during their nuptial flights, the vulnerable reproductive termites are taken by a wide variety of predators, including several social wasps of the neotropical genus polybia. in some cases, polybia are known to hunt reproductives in such quantity that they store them in their nests (noll et al., 1997; richards & richards, 1951:45; wasmann, 1897; pers. comms. from r.l. jeanne, s. o’donnell, and j.w. wenzel). in contrast, worker termites appear to be relatively safe from all except the several specialist mammalian and ant predators (hölldobler & wilson, 1990: table 15-1; nowak, 1999). under ordinary circumstance, those of most species are consistently enclosed in the nest and foraging galleries and usually accompanied by the soldier caste. it is mainly when this protection is breached that they are preyed upon by a variety of generalist predators. nasutitermes corniger (motschulsky) is found throughout most of the new world tropics, where its distinctive arboreal nests are often a conspicuous part of the abstract the social wasp polybia quadricincta is observed preying opportunistically on workers of the termite nasutitermes corniger in trinidad, west indies. several polybia spp. and other social wasps are known to prey on winged reproductive termites, but this appears to be the first report of any preying on workers. sociobiology an international journal on social insects ck starr, k heera article history edited by reginaldo constantino, unb, brazil received 03 march 2017 initial acceptance 01 may 2017 final acceptance 04 may 2017 publication date 21 september 2017 keywords nasutitermes corniger, polybia quadricincta, predation. corresponding author christopher k. starr dep’t of life sciences, univ. of the west indies st augustine, trinidad & tobago e-mail: ckstarr@gmail.com environment (constantino, 2009). both the nest surface and gallery walls are thin and brittle, so that it seems certain that any bird or lizard and many insects could breach them without difficulty. nonetheless, none is known to do so. however, reduviid bugs (hemiptera), web-building spiders (araneae: theridiidae) and anolis lizards are known opportunistically to prey on n. corniger and the very similar n. ephratae (holmgren) (marshall et al., 2015, mcmahan, 1982; pers. obs.) when such breaches occur. we report here on polybia quadricincta saussure preying on workers of n. corniger under similar conditions. as far as we know, this is the first description of any social wasp preying on worker termites in a systematic fashion. all observations are from trinidad, west indies, where p. quadricincta is uncommon but n. corniger is probably the most abundant termite species. as seen in fig 1, the wasp is roughly 100 times the size of the termite workers. initial observations by one of us (cks) were made at an iron gate with a long-standing n. corniger gallery running along it. because the gallery crosses the latch, it is broken university of the west indies, st augustine, trinidad & tobago short note ck starr, k heera – polybia preying on nasutitermes 226 whenever the gate is opened, requiring some minutes for the termites to re-seal it when the gate is closed again. for a period of several weeks in late 2013 and early 2014 a p. quadricincta worker appeared at the site on many mornings, lingering close to the breach in the gallery. there was never more than one wasp at a time, presumably the same individual each day. no colony of this species appeared to be within 50 m of the gate. the wasp moved actively about the breach, frequently lunging at termites involved in repair in a way that suggested that she was hunting workers while avoiding the chemically-defended (and defending) soldiers. this had the appearance of a practiced activity, although during the moments before the observer hurried off to work the wasp was not seen to make a capture. direct observation of captures came during a class exercise in june 2014 in which a live n. corniger nest was opened during the middle part of the day in an open-air classroom. exposing termites in this way commonly brings many ants and lizards to the windfall. on this occasion, it also attracted p. quadricincta foragers to an uncovered column of workers and soldiers along a railing and outer wall of the building. foragers oriented close and actively to the column (fig 1), making open-mandible lunges at workers, while shying away from any soldiers. we repeatedly saw wasps grab and fly off with workers, always just one worker at a time. at least three wasps hunted at the column, each apparently making several trips. although several other social wasp species are more abundant than p. quadricincta at the locality, none came to prey on the termites. the wasps took both live workers and some that had been crushed. several neotropical social wasps are known to take carrion (o’donnell, 1995), and it seems likely that p. quadricincta does so at least occasionally. our observations are consistent with the hypothesis that this particular wasp is alert to opportunities to prey on termite workers exposed by significant damage to the nest or galleries. the strong, distinctive odour of an opened nasutitermes nest is readily perceived by humans and is almost certainly enough to alert scouting wasps. why does p. quadricincta apparently not create its own hunting opportunities by biting open n. corniger’s gallery walls? even if the wasp cannot detect intact galleries by odour, these are so abundant in its environment that it seems very likely that it could find them by visual and tactile search. however, colonies of the nasutitermitinae characteristically have an extraordinarily high proportion of soldiers, often accounting for more than 15% of adults (haverty, 1977; merritt & starr, 2010). these come quickly to any breach, so that it is unlikely that the wasp could open a wide enough span of gallery quickly enough to afford access to workers without contacting distasteful soldiers. acknowledgments in addition to those whose personal communications are mentioned above, we thank m.j. west-eberhard for comments and joshua spiers for graphic assistance. references constantino, r. 2009. catalog of the living termites of the new world. http://www.termitologia.unb.br (accessed 1 may 2016) haverty, m.i. 1977. the proportion of soldiers in termite colonies: a list and a bibliography (isoptera). sociobiology, 2: 199-216. https://www.treesearch.fs.fed.us/pubs/9893 hölldobler, b. & wilson, e.o., 1990. the ants. cambridge: harvard univ. press, 732 p. lubin, y.d., montgomery g.g. & young o.p. 1977. food resources of anteaters (edentata: myrmecophagidae). i. a year’s census of arboreal nests of ants and termites on barro colorado island, panama canal zone. biotropica, 9: 26-34. marshall, s.a., borkent, a., agnarsson, i., otis, g.w., fraser, l. & ‘entremont, d. d’ 2015. new observations on a neotropical termite-hunting theridiid spider: opportunistic nest raiding, prey storage, and ceratopogonid kleptoparasites. journal of fig 1. polybia quadricincta interacting with exposed individuals of nasutitermes corniger. a) feeding on a worker. b) backing away from soldiers. the wasp’s body length is about 8 mm. sociobiology 64(2): 225-227 (june, 2017) 227 arachnology, 43: 419-421. http://www.americanarachnology. org/joa_free/joa_v43_n3/arac-43-03-419.pdf mcmahan, e.a. (1982). bait-and-capture strategy of a termite-eating assassin bug. insectes sociaux, 29: 346-351. merritt, n.r.c. & starr, c.k. (2010). comparative nesting habits and colony composition of three arboreal termites (isoptera: termitidae) in trinidad & tobago, west indies. sociobiology, 56: 611-622. http://ckstarr.net/cks/2010termites.pdf noll, f.b., zucchi, r. & mateus, s. (1997). morphological caste differences in the neotropical swarm-founding and polygynous polistine wasp polybia scutellaris. studies on neotropical fauna and environment, 32: 76-80. nowak, r.m. (1999). walker’s mammals of the world. 6th ed. vol. 1-2. baltimore: johns hopkins univ. press, 1936 p. o’donnell, s. (1995). necrophagy by neotropical swarmfounding social wasps (hymenoptera: vespidae, epiponini). biotropica, 27: 133-136. richards, o.w. & richards, m.j. (1951). observations on the social wasps of south america (hymenoptera vespidae). transactions of the royal entomological society of london, 102: 1-169. wasmann, e. (1897). beutetiere von polybia scutellaris white. zoologisher anzeiger 20: 276-279. (cited from richards & richards 1951:53) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2073sociobiology 65(2): 333-336 (june, 2018) neotropical swarm-founding wasps (vespidae: polistinae: epiponini) accept expelled queens in case of queen loss epiponini, a tribe of neotropical social wasps, presents complex caste differences, which vary from no differences between queens and workers to conspicuous differences (noll et al., 2004). in addition, colonies have multiple queens which decrease in number through the colony cycle from several (polygyny) to a few (olygogyny), or only one (monogyny) (west-eberhard, 1978), leading to an increase in kinship among colony mates, as predicted by kin-selection theory (hamilton, 1964 a, b) and reported for several epiponines (see hastings et al., 1998 for a review). in the first stages of the colony cycle, there are several queens, since more are necessary to produce enough workers to maintain the colony (west-eberhard, 1978). nevertheless, as the colony grows queen number diminishes (west-eberhard, 1978; noll & zucchi, 2000; 2002). new queens will be produced during the reproductive phase or when colony requires, for example, in abstract in the epiponini, queen number declines through colony cycle, because some queens are expelled from colonies. here we demonstrate that epiponini wasps may accept expelled queens in situations of queenlessness. one colony of protopolybia exigua was observed at the university of são paulo in brazil; and another of metapolybia docilis was observed at la selva biological station in costa rica. queen removal tests were performed to study workers’ acceptance of expelled queens and queens from other colonies. in p. exigua, the experimental queen elimination caused a change in the workers’ behavior, ranging from aggressive expulsion of non-selected queens to re-acceptance. in m. docilis workers were willing to accept queens from other colonies after queen elimination. our results indicate that because of a decrease in workers aggressiveness during the colony cycle, workers may accept expelled queens (even foreign ones, in experimental situations) in order to ensure colony survival. sociobiology an international journal on social insects l chavarria-pizarro1,2, m silva3, fb noll3 article history edited by gilberto m. m. santos, uefs, brazil received 11 september 2017 initial acceptance 03 october 2017 final acceptance 08 january 2018 publication date 09 july 2018 keywords queen selection, behavior, flexibility, queen elimination. corresponding author laura chavarría pizarro escuela de biología instituto tecnológico de costa rica 159-7050, cartago, costa rica. e-mail: laurachp@gmail.com case of queen loss (noll & zucchi, 2000; 2002). according to west-eberhard (1978) rejected queens are expelled from colonies by workers, who can distinguish reproductive from non-reproductive females (naumann, 1970; west-eberhard, 1977; nascimento et al., 2004; chavarria-pizarro, 2013). epiponini wasps are also characterized by their behavioral flexibility, which is evident in certain stages of colony cycle (west-eberhard, 1978). during periods of a large number of queens, the level of aggressiveness of workers towards them is extremely high and workers perform aggressive displays toward some of them, probably those with less fitness. this was first observed by westeberhard (1978), who showed that queens who behaved submissively in response to this display are usually removed from the nest. in some cases, the targeted queens remain in the nest and take up worker activities (west-eberhard, 1978). 1 escuela de biología, instituto tecnológico de costa rica, cartago, costa rica 2 departamento de biologia, ffclrp-usp, ribeirão preto, são paulo, brazil 3 departamento de zoologia e botânica, ibilce-unesp, são josé do rio preto, são paulo, brazil short note l chavarria-pizarro, m silva,fb noll – neotropical wasps re-accept expelled queens334 on the other hand, some workers may lay eggs in case of queen loss (chavarria-pizarro, 2013) and also replace the queens in some situations (mateus et al., 1997). it is interesting that the level of aggressiveness is extremely high during the periods of high queen density only and workers are constantly removing non-qualified reproductives. in contrast, during periods of colony orphanage (queenlessness), the need for new reproductives forces workers to be less exigent in terms of reproduction (noll & wenzel, 2008; noll, 2013), even for a short period of time before new gynes will be produced (noll, 2013). consequently, behavioral flexibility is important for colony survival over critical phases. here we provide observations from two species of epiponini, protopolybia exigua and metapolybia docilis, which indicates that in cases of orphanage these wasps accept expelled queens. protopolybia exigua one colony of protopolybia exigua with approximately 50 individuals, was observed from 17/12/2011 to 01/02/2012, twice a week, four hours a day, at the campus of university of são paulo in ribeirão preto, brazil (21°10’s; 47°48’w). at the beginning of the observations, the colony was in a period of pre-emergence of adults, phase in which it remained since no adult offspring was produced. in this nest, all queens and a sample of nine workers were individually marked with quick-drying paint. the queens were identified based on the observations of oviposition and bending display behaviors (west-eberhard, 1978). queen and worker behavior through colony establishment and pre-emergence phase, was observed during the first month, to determine queen selection dynamics. after four weeks of observation, selected queens (i.e. egg layers which were not expelled from colony by workers) were removed in order to study workers behavior and their flexibility to accept eliminated queens. during the first week of observations, nine females were observed to behave as queens. after one month, the workers selected three of the nine putative queens, and expelled the remaining (n=6) using aggressive behaviors (biting) (west-eberhard, 1978). when the expelled queens tried to access the nest, workers aggressively bit them, forcing these individuals to fly away. after the experimental queen elimination, workers’ behavior changed. the unselected queens were not bitten and expelled by workers, which also reaccepted them into the colony. metapolybia docilis one colony (n-2) of metapolybia docilis (50 individuals) was observed for three days (23/03/2012 to 25/03/2012), six hours a day, at la selva biological station in sarapiquí, heredia, costa rica (10°26’n, 83°59’w). additionally, other three colonies (n-1, n-3, n-4), with around 100 wasps, were used to obtain individuals to perform treatments. all m. docilis colonies were in the workerproduction stage. a section of nest envelope was cut, and folded back (as a door) every day to perform observations. to identify queens, some eggs were removed from the nest to stimulate oviposition. in colony n-2, five females were observed behaving as queens and all of them were marked. in order to observe the behavior of wasps, four treatments (t1 to t4) were performed. in t1, two marked workers from nest n-4 were introduced in n-2 to observe workers’ and queens’ behavior towards foreign individuals. n-2 workers behaved aggressively by biting these foreign individuals. nevertheless, after few minutes of aggression, n-2 workers carefully groomed the foreign workers for several minutes. after grooming, one n-4 worker flew away, and one was accepted by colony n-2 for one day. in t2, four marked queens from n-1 were introduced in n-2 to observe workers’ behavior towards “foreign queens” when the original queens were present. two of these flew away before workers recognized them and two were expelled by workers through aggressive behaviors. in t3, four queens of n-3 were introduced to n-2 after the queens’ removal (same procedure performed for protopolybia) to observe n-2 workers’ behavior under queen’s absence. unlike in the protopolybia procedure, the original queens were kept individually in a 50ml plastic vial where they remained for approximately 24 hours. as in t2, workers at first aggressively bit the foreign queens, but after a few minutes they groomed and accepted three of the four queens for at least one day. in t4, after 24 hours, the original n-2 queens were re-introduced into their colony to observe workers’ behavior towards their own queens. when the original queens (n-2) were reintroduced in t4, they were all accepted (workers did not behave aggressively) into the colony. our observations indicate that in both species workers were not willing to accept expelled queens when selected queens were present in the colony. unlike other insect societies, queen selection is performed by workers in epiponini (noll & wenzel, 2008), who usually test queens by ritualized behaviors, using their response as an indicator of reproductive capacity (west-eberhard, 1978; nascimento et al., 2004; chavarriapizarro, 2013). selected queens are probably recognized by workers as having a higher reproductive capacity. nevertheless, after selected queens were experimentally removed, workers re-accepted expelled (p. exigua) and foreign queens (m. docilis). social wasps can recognize queen absence (nascimento et al., 2004; chavarria-pizarro, 2013) and, for this reason, expelled queens were accepted only when queen removal test were performed. if unselected queens have a good reproductive output, or belong to the original swarm, workers could be willing to re-accept them to replace queen(s) loss, as we observed in p. exigua. similarly, foreign queens of m. docilis n-3 may have high reproductive capacity and because n-2 workers could perceive it, they were accepted sociobiology 65(2): 333-336 (june, 2018) 335 even belonging to another colony. these findings do not oppose kin-selection theory, because orphanage is very likely to lead to colony death. in these scenarios, the possibility of colony survival, even with a cost of lowing the relationship among colony members, is probably adaptive. as mentioned above (see introduction), low aggressiveness during periods of low queen number may be an important adaptive strategy to allow colony survival, because during orphanage workers with some reproductive potential (intermediates) can assume colony reproduction until new queens can be produced (noll, 2013). in this scenario, it may be that queens that were previously rejected but remained on the nest acting as workers might find a window to reassume the queen role again. the acceptance of foreign queens by queenless workers shows that workers can recognize they are without a queen and modify their behavior to assure colony survival. it is not known how often queen loss in these species occurs under natural conditions. although in m. docilis we could not be sure if foreign queens remain permanently in the colony, or, if they were accepted temporarily under queen loss, the workers’ behavioral flexibility was evident. these observations indicate that under certain situations it is convenient to accept expelled queens (even foreign queens under experimental conditions), possibly when queen-worker conflicts and the levels of aggressiveness are low. even when selected queen(s) would be favored over expelled and foreign queens to increase workers’ fitness, behavioral flexibility seems to be an important feature because it allows colonies to distribute individuals to perform tasks according to colony needs, and this feature is very important to ensure colony survival (karsai & wenzel, 2000; nowak et al., 2010). acknowledgments we especially thank fundação de amparo à pesquisa do estado de são paulo (fapesp) (2009/07526-2; 2011/060585) for financial support; to organization of tropical studies (ots) for scholarship during short course on neotropical social insect biology; the entomology graduate program of ffclrp-universidade de são paulo, brazil; to sergio jansen, for field assistance, their comments and suggestions essential for manuscript improvement. we are grateful to the two anonymous reviewers for their suggestions, editing and comments. references chavarria-pizarro, l. (2013). sobre a produção e seleção de rainhas em diferentes fases do ciclo colonial em epiponini (vespidae: polistinae). dissertation, university of são paulo, brazil. hamilton, w.d. (1964a). the genetical evolution of social behaviour i. journal of theoretical biology. 7:1-16. doi: 10.1016/0022-5193(64)90038-4 hamilton, w.d. (1964b). the genetical evolution of social behaviour i. journal of theoretical biology, 7:17-52. doi: 10.1016/0022-5193(64)90039-6 hastings, m.d., queller, d.c., eischen, f.& strassmann, j.e. (1998). kin selection, relatedness and worker control of reproduction in a large-colony epiponine wasp, brachygastra mellifica. behavioral ecology, 9: 573-581. doi: 10.1093/ beheco/9.6.573 karsai, i. & wenzel, j. (2000). organization and regulation of nest construction behavior in metapolybia wasps. journal of insect behavior, 13: 111-40. doi: 10.1023/ a:1007771727503 mateus, s., noll, f.b. & zucchi, r. (1997). morphological caste differences in the neotropical swarm-founding polistine wasps: parachartergus smithii (hymenoptera: vespidae). journal of the new york entomological society, 105: 129-139. nascimento, f.s., tannure-nascimento, i.c. & zucchi, r. (2004). behavioral mediators of cyclical oligogyny in the amazonian swarm-founding wasp asteloeca ujhelyii (vespidae, polistinae, epiponini). insectes sociaux, 51: 17-23. doi: 10.1007/ s00040-003-0696-y naumann, m.g. (1970). the nesting behaviour of protopolybia pumilia in panama (hymenoptera: vespidae). dissertation. university of kansas, usa. noll, f.b. (2013). “marimbondos”: a review on the neotropical swarm-founding polistines. sociobiology, 60: 347-352. doi: 10.13102/sociobiology.v60i4.347-354 noll, f. & zucchi, r. (2000). increasing caste differences related to life cycle progression in some neotropical swarmfounding polygynic polistinae wasps (hymenoptera vespidae epiponini). ethology ecology & evolution, 12: 43-65. doi: 10.1080/03949370.2000.9728322 noll, f.b. & zucchi, r. (2002). castes and the influence of the colony cycle in swarm-founding polistine wasps (hymenoptera, vespidae, epiponini). insectes sociaux, 49: 62–74. doi: 10.1007/s00040-002-8281-3. noll, f.b., wenzel, j. & zucchi, r. (2004). evolution of caste in neotropical swarm-founding wasps (hymenoptera: vespidae; epiponini). american museum novitates, 25(3467): 1-24. doi: 10.1206/0003-0082(2004)467<0001:eocinw>2.0.co;2. noll, f.b. & wenzel, j.w. (2008). caste in the swarming wasps: “queenless” societies in highly social insects. biological journal of the linnean society, 93: 509-522. doi: 10.1111/j.1095-8312.2007.00899.x. noll, f.b. (2013). “marimbondos”: a review on the neotropical swarm-founding polistines. sociobiology, 60: 347-354. doi: 10.13102/sociobiology.v60i4.347-354 nowak, m., tarnita, c.e. & wilson, e.o. (2010). the evolution of eusociality. nature. nature publishing group,466 (7310): 1057-62. doi: 10.1038/nature09205 l chavarria-pizarro, m silva,fb noll – neotropical wasps re-accept expelled queens336 queller, d.c., negrón-stomayor, h.c.r. & strassmann, j.e. (1993). queen number and genetic relatedness in a neotropical wasp, polybia occidentalis. behavioral ecology, 4:7-13. doi: 10.1093/beheco/4.1.7 west-eberhard, m.j. (1977). the establishment of dominance in social wasps. proceedings of the international union for study of social insects. wageningen; 223–227. west-eberhard, m.j. (1978). temporary queens in metapolybia wasps: nonreproductive helpers without altruism? science, 200(4340): 441-443. doi: 10.1126/science.200.4340.441. doi: 10.13102/sociobiology.v65i1.1845sociobiology 65(1): 10-14 (march, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 estimating global termite species richness using extrapolation introduction wilson (1971) suggested that termite taxonomy was nearly complete almost half a century ago, and that relatively few genera and species remained to be described. nevertheless, new genera and species continued to be discovered and described in considerable numbers. a partial analysis of termite description rates was presented by eggleton (1999), covering the second half of the 20th century. complete description curves have never been published. it is possible to estimate total species richness using extrapolation based on species accumulation curves (colwell & coddington, 1994). for instance, paxton (1998) estimated the total number of large marine animals using this approach. the major difficulty with this method is to assemble adequate datasets. it requires a nearly complete and accurate taxonomic catalog of the taxon being studied, which takes considerable effort, even for relatively small groups such as termites. in this paper, complete and accurate cumulative description curves of termite species and genera are presented. total richness was estimated by maximum likelihood using the michaelis-menten model. abstract cumulative species description curves since 1758 are given for all termites of the world and for each biogeographical region (australian, ethiopian, nearctic, neotropical, oriental, palearctic, and papuan). a cumulative description curve is also given for world genera. estimation by maximum likelihood using the michaelis-menten model suggests a maximum of 5366 ± 175 species (p < 2.2e-16) and 704 ± 77 genera (p < 4.387e-13). model fitting was poor for most individual biogeographical regions, with the exception of the ethiopian region (estimate = 1295 ± 57 species, p < 2.2e-16). world war i and world war ii had marked negative impacts on termite description rates. data from china was treated separately due to the atypical rate of description of new termites in that country during the last two decades of the 20th century. sociobiology an international journal on social insects r constantino article history edited by paulo f. cristaldo, ufs, brazil received 07 july 2017 initial acceptance 31 july 2017 final acceptance 29 august 2017 publication date 30 march 2018 keywords isoptera, diversity, michaelis-menten, curve-fitting, maximum likelihood. corresponding author reginaldo constantino ib, depto de zoologia, unb cep 70910-900, brasília-df, brasil. e-mail: constant@unb.br methods all taxonomic data were extracted from the termite database (constantino, 2016), which is available online since 2002, and has been continuously updated. the current version incorporates taxonomic changes from krishna et al. (2013) and includes living and fossil termites from all regions of the world, and nomenclatural data from linnaeus (1758) to the present. the data itself is maintained in a custom relational database using mysql as the database engine. data were extrated from the database using sql queries and perl scripts, converted to csv tables, and imported into r software (r core team, 2017). fossil taxa and nomina dubia were excluded from the dataset. curve fitting was conducted using maximum likelihood with r package drc (ritz et al., 2015) using the michaelismenten model mm.2(), and data from 1900 to the present. description rates from 1758 to 1899 were too low due to the very small number of active taxonomists and were excluded from model fitting (but are presented in the graphs). all graphs were plotted using standard r graphics functions (plot(), lines(), points()). universidade de brasília (unb), brasília-df, brazil research article termites sociobiology 65(1): 10-14 (march, 2018) special issue 11 the michaelis-menten model is usually represented by the formula s(n) = smax·n/(b + n), where, in this case, s(n) = expected number of species in year n; smax = total number of species (asymptote); b = second parameter of the model (colwell & coddington, 1994). the rationale for the use of this model is that there is a fixed species pool in the planet from which new species can be discovered. under a relatively constant sampling effort, the rate of new discoveries should decrease as the proportion of known species increases. results the total current numbers are the following (excluding fossils): world total 2951 species (2516 excluding china) and 297 genera (290 excluding china); australian region 272 spp.; ethiopian 754 spp.; nearctic 51 spp.; neotropical 597 spp.; oriental 1148 spp. (741 excluding china); palearctic 169 spp. (126 excluding china); and papuan 127 spp. a few species occur in more than one region. the current total number of junior subjective synonyms in the species category is 661 (18%), excluding fossils. these are names that were regarded as valid species for some time, have their own types, and can be revalidated. the number of objetive synonyms in the same category is 49; these are nomenclatural synonyms that were never treated as separate species. another 15 names are classified as nomina dubia, with uncertain status; most of these are probably synonyms. initial global analyses resulted in poor model fitting. after some testing it became clear that the problematic data were from china (see discussion below). excluding these data considerably improved model fitting (fig 1) and resulted in estimates of 5366 ± 175 species (p < 2.2e-16, fig. 1) and 704 ± 77 genera (p < 4.387e-13, fig 2). these results suggest that 2675–3025 species and 337–491 genera remain undescribed. curve fitting for separate biogeographical regions was poor in most cases (fig 3), with the only exception of the ethiopian region. the smax estimate for this region was 1295 ± 57 species (p < 2.2e-16). world war i (1914–1918) and world war ii (1939– 1945) had marked negative impacts on termite description rates (figs 1 and 2). discussion the number of undescribed termite species estimated in this paper is considerably higher than suggested by wilson (1971). the shape of the species description curve suggests that termite taxonomy is far from complete and that more than half of the species remains undescribed and nameless. this is not surprising considering that the estimated total number of insect species on the planet is nearly 5 million (mora et al., 2011; stork et al., 2015), which indicates that approximately 80% of them remain undescribed. termite description rates in china, especially from 1980–2000, were very unusual and artificial. many new “species” were described during a period when the chinese scientific community was isolated from the rest of the world, with limited access to the literature and collections. fig 1. cumulative number of termite species since linnaeus (1758). a) number of species, excluding china; b) number of species, including china; c) total number of specific names, including those currently regarded as synonyms; d) michaelis-menten model fitted on curve a. wwi: world war i (1914-1918); wwii: world war ii (1939-1945). r constantino – global termite species richness12 this resulted in artificial taxonomic inflation (crosland, 1995; eggleton, 1999), visible as a strong deviation in the species accumulation curve (see fig 3, oriental region). they also described several endemic genera such as sinonasutitermes and sinocapritermes which do not seem to be valid. this taxonomic inflation is best illustrated by the number of reticulitermes species in china (117) compared to the entire nearctic region (6) and to india (5). the most likely explanation for this discrepancy is taxonomic error. this abnormal activity seems to have ceased, but they still need to revise the status of all these names. most of them will eventually be synonymized, but currently they might be regarded as nomina dubia. the separate cumulative species description curves for the biogeographical regions showed more variation than the global one, and most of them did not fit the michaelismenten model. this variation reflects the number of active taxonomists studying the fauna of each region and also the fact that smaller samples are expected to show more variation than larger ones. for instance, in the neotropical region the description rate has accelerated in recent years because the number of active taxonomists increased. on the other hand, taxonomic activity has been very low in australian and papuan regions in recent decades. the cumulative description curve of genera also showed more variation than the species one. the delimitation of genera is based on phylogeny, but ranking is arbitrary and subjective, and for this reason we should not expect a perfect correlation between the accumulation curves of genera and species. the average number of species per genus has changed considerably along history. in the classification scheme presented by desneux (1904), there was an average of about 35 species per genus, while in snyder’s (1949) catalog the average was 14 species per genus, and the current average is about 10 species per genus. estimates based on the method used here may be influenced by several factors (may, 1994; mora et al., 2011) including operational criteria used for species delimitation and changes in taxonomic effort. incorrect species delimitation may both result in overestimated species richness due to synonyms, or underestimate it due to cryptic (=sibling) species. molecular data will probably reveal cryptic species (e.g. hausberger et al., 2011) and increase total richness, but this may be counterbalanced by the discovery of new synonymies, which will reduce the number of valid species in some genera (e.g. coptotermes, see chouvenc et al., 2016). the number of active taxonomists in each period also affects the rate of new discoveries. we currently do not have a good assessment of the relative importance of these factors, but their net effect is probably small. several taxonomic revisions also suggest a large number of undescribed taxa in some groups. the revision of the soldierless termites of africa (sands, 1972) resulted in the description of 16 new genera and 51 new species. before that there were only 9 species, all classified in the genus anoplotermes. similarly, the revision of the australian termites of the termes-capritermes group (miller, 1991) resulted in the recognition of 13 genera (8 new) and 54 species (27 new). in the neotropical region, a revision of the soldierless genus ruptitermes resulted the recognition of 13 species, 9 of them new (acioli & constantino, 2015). there also many undescribed kalotermitidae. for instance, a revision of cryptotermes from the west indies (scheffrahn & krecek, 1999) listed 17 species, 12 of them new. fig 2. cumulative number of termite genera since linnaeus (1758). a) number of genera, excluding china; b) number of genera, including china; c) michaelis-menten model fitted on curve a. wwi: world war i (1914-1918); wwii: world war ii (1939-1945). sociobiology 65(1): 10-14 (march, 2018) special issue 13 the data presented here suggests that there is still a large number of undescribed termite species. however, this should not be seen as encouragement for the publication of a large number of small and purely descriptive taxonomic papers. isolate descriptions of new taxa, especially when based on small and incomplete series, are particularly undesirable because they cause fragmentation and are more prone to error. more investment is needed in integrative revisionary taxonomic work based both on traditional and modern methods. acknowledgments this work was supported by the brazilian national research council (cnpq, grants 481360/2013-1 and 311016/ 2016-2). references acioli, a.n.s. & constantino, r. (2015). a taxonomic revision of the neotropical termite genus ruptitermes (isoptera, termitidae, apicotermitinae). zootaxa, 4032: 451492. doi: 10.11646/zootaxa.4032.5.1. chouvenc, t., li, h.-f., austin, j., bordereau, c., bourguignon, t., cameron, s.l., cancello, e.m., constantino, r., costaleonardo, a.m., eggleton, p., evans, t.a., forschler, b., grace, j.k., husseneder, c., krecek, j., lee, c.-y., lee, t., lo, n., messenger, m., mullins, a., robert, a., roisin, y., scheffrahn, r.h., sillam-dussès, d., sobotník, j., szalanski, a., takematsu, y., vargo, e.l., yamada, a., yoshimura, t. & su, n.-y. (2016). revisiting coptotermes (isoptera: rhinotermitidae): a global taxonomic road map for species validity and distribution of an economically important subterranean termite genus. systematic entomology, 41: 299-306. doi: 10.1111/syen.12157. colwell, r.k. & coddington, j.a. (1994). estimating terrestrial biodiversity through extrapolation. philosophical transactions of the royal society of london b, 345: 101118. doi: 10.1098/rstb.1994.0091. constantino, r. (2016). termite database. http://termitologia. unb.br (accessed date: 30 june, 2017). crosland, m. (1995). taxonomic splitters in china. isoptera newsletter, 5: 1. desneux, j. (1904). isoptera fam. termitidae. in p. wytsman (ed.), genera insectorum (pp. 1–52). vol. 25. brussels: v. verteneuil & l. desmet,. fig 3. cumulative number of termite species since linnaeus (1758) for each biogeographic region: australian, ethiopian, nearctic, neotropical, oriental, palearctic, and papuan. data from china is shown as a separate line for the oriental and palearctic regions. the michaelis-menten model is shown only for the ethiopian region. r constantino – global termite species richness14 eggleton, p. (1999). termite species description rates and the state of termite taxonomy. insectes sociaux, 46: 1-5. doi: 10.1007/s000400050105. hausberger, b., kimpel, d., van neer, a. & korb, j. (2011). uncovering cryptic species diversity of a termite community in a west african savanna. molecular phylogenetics and evolution, 61: 964–969. doi: 10.1016/j.ympev.2011.08.015. krishna, k., grimaldi, d.a., krishna, v. & engel, m.s. (2013). treatise on the isoptera of the world. bulletin of the american museum of natural history, 377: 1-2704. doi: 10.1206/377.1. linnaeus, c. (1758). systema naturae. 10th ed. stockholm: laurentii salvii, 823 p. may, r.m. (1994). conceptual aspects of the quantification of the extent of biological diversity. philosophical transactions of the royal society of london. series b: biological sciences, 345: 13-20. doi: 10.1098/rstb.1994.0082. miller, l.r. (1991). a revision of the termes capritermes branch of the termitinae in australia (isoptera: termitidae). invertebrate taxonomy, 4: 1147-1282. doi: 10.1071/it9901147. mora, c., tittensor, d.p., adl, s., simpson, a.g.b. & worm, b. (2011). how many species are there on earth and in the ocean? plos biology, 9: 1-8. doi: 10.1371/journal.pbio.1001127. paxton, c. (1998). a cumulative species description curve for large open water marine animals. journal of the marine biological association of the united kingdom, 78: 13891391. doi: 10.1017/s0025315400044611. r core team. (2017). r: a language and environment for statistical computing. vienna, austria: r foundation for statistical computing,. http://www.r-project.org ritz, c., baty, f., streibig, j.c. & gerhard, d. (2015). doseresponse analysis using r. plos one, 10: 1-13. doi: 10.1371/journal.pone.0146021. sands, w.a. (1972). the soldierless termites of africa (isoptera: termitidae). bulletin of the british museum of natural history, entomological supplement, 18: 1-244. scheffrahn, r.h. & krecek, j. (1999). termites of the genus cryptotermes banks (isoptera: kalotermitidae) from the west indies. insecta mundi, 13: 111-171. snyder, t.e. (1949). catalog of the termites (isoptera) of the world. smithsonian miscellaneous collections, 112: 1–490. stork, n.e., mcbroom, j., gely, c. & hamilton, a.j. (2015). new approaches narrow global species estimates for beetles, insects, and terrestrial arthropods. pnas, 112: 7519-7523. doi: 10.1073/pnas.1502408112. wilson, e.o. (1971). the insect societies. cambridge, ma: harvard university press, 548 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1153sociobiology 64(4): 437-441 (december, 2017) phylogenetic position of the western bangladesh populations of weaver ant, oecophylla smaragdina (fabricius) (hymenoptera: formicidae) introduction the weaver ant genus oecophylla (hymenoptera, formicidae) has two broadly distributed species: o. smaragdina and o. longinoda. both species are distributed in tropical and subtropical asia and africa, respectively. their colonies are arboreal, large and polydomous in nature. workers show polymorphic characters with diversified organizing behavior in the colony. they are aggressive and well known for their predatory behavior (hölldobler & wilson, 1977). weaver ants form their nest in the tree canopy by their unique nest building behavior. workers construct pendulous bag-like nests from cluster of green leaves which are bound together with silk produced by their mature larvae (chapuisat & keller, 2002). oecophylla ants are hosts to a variety of inquilines, such as spiders, which mimic the colony odor to escape detection (schlüns et al., 2009). oecophylla smaragdina and o. longinoda are very similar in morphology and behavior abstract the weaver ant species, oecophylla smaragdina is distributed from india through southeast asia to northern australia including many tropical western pacific islands. a recent phylogenetic study of o. smaragdina revealed the central bangladesh population as belonging to the southeast asian mainland clade despite of its geographical proximity to india. however, the bangladeshi analyzed sample was limited to a single site and the geographical border between indian and southeast asian groups has not been presented. in this study, 19 samples collected from western parts of bangladesh have been used to infer the phylogenetic position. a total of 20 o. smaragdina colonies were sampled from bangladesh during 2013 to 2014. their haplotype and phylogenetic relationships were determined by analyzing 2 mitochondrial loci: cytochrome b (cytb) consisting of 606 bp and cytochrome c oxidase subunit i (coi) consisting of 775 bp. bayesian analysis inferred that the western parts of bangladesh were occupied by mitochondrial haplotype usually found in india, which is recorded first time in the country. the present study revealed that, both the indian and southeast asian mitochondrial haplotypes were occurred on either side of ganges river. sociobiology an international journal on social insects mm rahman, s hosoishi, k ogata article history edited by rodrigo feitosa, ufpr, brazil received 08 july 2016 initial acceptance 04 february 2017 final acceptance 15 november 2017 publication date 27 december 2017 keywords mitochondrial dna, cytb, geographical distribution, coi, ganges river. corresponding author md mamunur rahman institute of tropical agriculture kyushu university 6-10-1 hakozaki, higashi-ku, fukuokashi, fukuoka, japan 812-8581 e-mail: mamunur111@gmail.com (bolton, 1995). according to the fossil records, oecophylla might have originated in the early paleogene (ca. 60 ma) in the palaearctic region, and dispersed during the climatic changes of the eocene–oligocene transition at ca. 43 ma (dlussky et al., 2008). recently, blamier et al (2015) estimated the divergence time of the genus oecophylla based on the fossil records and ultra conserved elements (uces). they estimated that oecophylla crown group evolved during oligocene at ca. < 30 ma and stem-group evolved during early eocene at ca.50 ma. azuma et al. (2002) first analyzed populations of o. smaragdina using molecular data and samples of o. smaragdina from bangladesh. including additional populations of o. smaragdina from india, southeast asia and australia, azuma et al. (2006) proposed an outline of the phylogeography of o. smaragdina and categorized the sampled populations into 7 major clades: group 1 from india; group 2 from southeast asian mainland including the indochinese and malayan peninsulas, as well as the greater institute of tropical agriculture, kyushu university, fukuoka, japan research article ants mm rahman, s hosoishi, k ogata – phylogenetic position of western bangladesh weaver ant438 sunda islands; group 3 from the philippines; group 4 from flores; group 5 from sulawesi; group 6 from halmahera; group 7 from australia and new guinea. hereafter we call their group 1 the indian clade and group 2 as southeast asian clade. asaka (2010) extended the survey of o. smaragdina to south asia, and collected several samples from india and sri lanka. her phylogenetic analysis showed that all analyzed samples belong to indian clade with low levels of sequence divergence. azuma et al. (2006) characterized the mitochondrial sequence identity of the bangladesh populations as belonging to the southeast asian clade in spite of geographical proximity of bangladesh to india. based on those data, bangladesh is considered a major transition zone between indian and southeast asian populations. this is the unique case of population boundaries without any distinguished geographical borders (e.g., deep sea or high mountains) although the seven groups of o. smaragdina based on haplotype grouping by azuma et al. (2006) are geographically bordered by the sea. as bangladesh is a riverine country with three main rivers ganges, jamuna and meghna crisscrossed throughout the mostly flat territories of the country, the river might have some influence of separating the indian clade and the southeast asian clade in bangladesh. the goal of the present study is to test whether the western bangladesh populations of o. smaragdina all belong to the se asian clade. the previous sampling by azuma et al. (2002) was limited to a single site, nurbag, gazipur, which is located in the central part of bangladesh. surveying the western part of bangladesh provides additional information on the phylogeography of o. smaragdina. the analysis of mitochondrial haplotype identity of these populations will shed light on the geographic distribution of the southeast asian clade in bangladesh. materials and methods sampling and preparation of specimens in 2013 to 2014 we collected adult oecophylla smaragdina workers from 20 colonies at 19 localities in 12 districts belonging to 4 divisions of bangladesh (fig 1 and table 1). the specimens were preserved in 99% ethanol prior to dna extraction. molecular studies genomic dna was extracted from the fore, middle and hind legs of specimens that were preserved in alcohol by using qiagen dneasy blood and tissue kit (qiagen, meryland, usa). amplification of mitochondrial dna was done by polymerase chain reaction (pcr). the thermal cycling parameters for cytb and coi basically followed the protocols established by crozier and crozier (1993) and sameshima et al. (1999), including 95 °c for 5 min for initial denaturation, 35 cycles of dissociation (92 °c, 1 min), locality code locality name no. of colonies upazila district division collection date accession number coi cytb l01 ishwardi 1 ishwardi pabna rajshahi 18 mar. 2014 kx385842 kx430217 l02 bonpara 1 baraigram natore rajshahi 19 mar. 2014 kx385843 kx430218 l03 tarash 1 tarash sirajganj rajshahi 18 mar. 2014 kx385841 kx430216 l04 chauhali 1 belkuchi sirajganj rajshahi 19 mar. 2014 kx389168 kx398946 l05 w side of jamuna bridge 1 sirajganj sadar sirajganj rajshahi 18 mar. 2014 kx385840 kx430215 l06 panjia 1 keshabpur jessore khulna 04 mar. 2014 kx371575 kx398943 l07 manirampur 1 manirampur jessore khulna 14 sep. 2013 kx355139 kx430212 l08 khulna univ. campus 1 batiaghata khulna khulna 03 mar. 2014 kx379493 kx398942 l08 khulna univ. campus 1 batiaghata khulna khulna 03 mar. 2014 kx379494 kx430213 l09 chuknagar 1 dumuria khulna khulna 04 mar. 2014 kx385837 kx398944 l10 batiaghata 1 batiaghata khulna khulna 15 sep. 2013 kx389167 l11 atulia 1 shyamnagar satkhira khulna 24 mar. 2014 kx385844 kx398947 l12 modonpur 1 tala satkhira khulna 25 mar. 2014 kx385845 kx430219 l13 mollarhat bazar 1 mollarhat bagerhat khulna 29 oct. 2014 kx430220 l14 bhanga 1 bhanga faridpur dhaka 09 nov. 2014 kx389172 l15 elenga 1 kalihati tangail dhaka 18 mar. 2014 kx385839 kx398945 l16 kumrail 1 dharmrai dhaka dhaka 19 oct. 2014 kx389169 l17 thanamore 1 dohar dhaka dhaka 21 oct. 2014 kx389170 l18 bhawal national park 1 joydebpur gazipur dhaka 17 mar. 2014 kx385838 kx430214 l19 nurbag 1 kaliakoir gazipur dhaka 22 oct. 2014 kx389171 kx430221 table 1. specimen data and genbank accession numbers.the list of figures. sociobiology 64(4): 437-441 (december, 2017) 439 annealing (50 °c for cytb and 54 °c for coi, 1 min), and extension (70 °c, 2 min). the primers used for amplification are identical to primers reported by crozier et al. (1994), lunt et al. (1996), azuma et al. (2002), and azuma et al. (2006). primers for the cytb gene fragment were cb1 (5‘tatgtactaccatgaggacaaatatc’3) and trs (5’tatttctttattatgttttcaaaac’3). for the coi gene fragment, coi 1-3 (5’ataattttttttatagttatacc’3) and coi 2-4 (5’tcctaaaaaatgttgaggaaa’3) were used as forward and reverse primers, respectively (crozier & crozier, 1993). illustra and exoprostar were followed according to the instruction of the manufacturer ge healtcare. for cycle sequencing, abi prism big dye terminator v3.1 cycle sequencing kits from applied biosystems were used in an automated sequencer. primers for the sequencing reaction were identical to those used in the amplification step. sequencing reaction was performed by using abi 3100 avant dna sequencer (applied biosystems). for the phylogenetic analysis of western bangladeshi o. smaragdina populations, 16 samples for cytb and 19 samples for coi genes have been used with 606 bp and 775 bp, respectively. in addition, in this analysis, sequence data of both coi and cytb were used from azuma et al. (2002), azuma et al. (2006) and asaka (2010) retrieved from ddbj genbank. sequence data of both coi and cytb of oeocophylla longinoda from cameroon were used as outgroup fig 1. the sampling sites of oecophylla smaragdina in bangladesh. locality codes correspond to those in table 1. mm rahman, s hosoishi, k ogata – phylogenetic position of western bangladesh weaver ant440 in this analysis. the sequencing analysis was done by using vector nti advance ver. 11.5 software. haplotypes of cytb and coi were aligned by using mega 6.0 software (tamura et al., 2013). phylogenetic trees inferred from concatenated matrix conducted by bayesian methods based on mrbayes 3.1.2. for the selection of bestfit model mrmodeltest 2.3 was performed with paup*4.0 beta version10. for both mitochondrial coi and cytb genes, substitution model gtr + i + g with 1,000,000 generations were used. the nucleotide sequences for both cytb and coi have been deposited in the genbank with accession number mentioned in table 1. results and discussion we recognized a total of 211 variable characters including 140 in coi and 71 in cytb from o. smaragdina samples, of which 80 and 40 characters were parsimony informative in coi and cytb, respectively. the phylogenetic tree obtained from the bayesian analysis of the mitochondrial concatenated matrix dataset showed that the samples collected from the western part of bangladesh were nested within the indian clade (posterior probability 100%) (fig 2). this is the first record of indian mitochondrial haplotypes in bangladesh. we recognized that indian mitochondrial haplotypes occurred on both sides of ganges river. oecophylla species may disperse via nuptial flight of queens and/or rafting method which is very effective between island to island dispersal (peng et al., 1998). since o. smaragdina is an arboreal species, the inseminated queen dispersed more likely by wind than ground-dwelling ants (azuma et al., 2006). thornton (1996) also reported that the relatively frequent colonization by rafting between two neighboring islands is very common. it is interesting that our bangladesh samples of bhawal national park (l18) and nurbag (l19) were geographically close to the former sampling site of azuma et al. (2002). all of these are located in the gazipur district, but our samples were inferred to fall into the indian clade. in contrast, o. smaragdina previously sampled from gazipur was inferred to belong to the southeast asian clade (azuma et al., 2002). provided that our results are inconsistent with the result previously reported by azuma et al. (2002) it is possible that: (1) there might be a misidentification of former bangladesh samples caused by contamination, (2) the former record of southeast asian type in bangladesh might be an exceptional case. in our opinion, both the first and second cases are less plausible because additional samples from bangladesh were inferred to belong to the southeast asian clade (asaka, personal communication). the third possible case is that the materials of azuma et al. (2002) would express the western geographic distribution limit of southeast asian mitochondrial haplotypes. we here confirm that o. smaragdina populations with indian mitochondrial haplotypes exist in the western part of bangladesh. the result of present study suggested the importance of comprehensive surveys, also taking into account the central and eastern populations of bangladeshi o. smaragdina. acknowledgements we are thankful to mr. helal uddin, bangabandhu shiekh mujibur rahman agricultural university (bsmrau); mr. ataur rahman, bangladesh sugarcane resource and training institute (bsrti), for helping us to collect samples. we express our sincere gratitude to dr. masaru matsumoto, fig 2. bayesian phylogenetic tree of bangladeshi o. smaragdina populations as inferred from the mitochondrial gene fragments of the coi and the cytb genes. numbers adjacent to internal nodes represent bootstrap values (%). black circles indicate the samples from bangladesh in the present study. additional dna sequence data were downloaded from ddbj genbank. the underlined locality shows the bangladeshi sample in the previous study of azuma et al. (2006). the number ahead of each locality indicates the locality number. sociobiology 64(4): 437-441 (december, 2017) 441 institute of tropical agriculture of kyushu university, japan for his technical support and useful suggestions and ms. yukiko asaka, sapporo, japan for her valuable information. we are also thankful to dr. akinori ozaki, institute of tropical agriculture for providing facilities during sampling in bangladesh. this work was supported in part by jsps kakenhi (grant-in-aid for scientific research (b)) grant number 26304014, mext, japan. references asaka y. (2010). phylogeography of the weaver ant oecophylla smaragdina supporting southern indian refugia hypothesis. (thesis). hokkaido university, hokkaido, japan. azuma, n., kikuchi, t., ogata, k., higashi, s. (2002). molecular phylogeny among local populations of weaver ant oecophylla smaragdina. zoological science, 19: 1321-1328. doi: 10.2108/zsj.19.1321 azuma, n., ogata, k., kikuchi, t., higashi, s. 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(1998). how many queens are there in mature colonies of the green ant, oecophylla smaragdina (fabricius)? australian journal of entomology, 37: 249-253. doi: 10.1111/j.1440-6055.1998.tb01579.x. sameshima, s., hasegawa, e., kitade, o., minaka, n., matsumoto, t. (1999). phylogenetic comparison of endosymbionts with their host ants based on molecular evidence. zoological science, 16: 993-1000. doi: 10.2108/zsj.16.993. schlüns, e.a., wegener, b.j., schlüns, h., azuma, n., robson, s.k.a., crozier, r.h. (2009). breeding system, colony and population structure in the weaver ant oecophylla smaragdina. molecular ecology, 18: 156-167. doi: 10.1111/ j.1365-294x. 2008.04020.x tamura, k., stecher, g., peterson, d., filipski, a., kumar, s. (2013). mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution, 18: 156-167. doi: 10.1111/j.1365-294x.2008.04020.x. thornton, i. (1996). krakatau: the destruction and reassembly of an island ecosystem, in: tropical ecology, pp. 147-148. doi: 10.13102/sociobiology.v64i2.1223sociobiology 64(2): 212-216 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 performance of soil termiticides in open field and under roof overhang introduction the distribution and sale of soil termiticides in the philippines for structural protection against subterranean termites requires registration with the food and drug administration (fda) of the philippines. registration of termiticides with the fda involves field efficacy trial against endemic species of subterranean termites in at least three sites. the trial is conducted by government agencies or an accredited researcher. after successful performance in field trial for 2 and 5 years, conditional and full registration can be granted, respectively. the protocol currently used is the concrete slab test as described by beal (1986) and kard et al. (1989). the test involves application of the termiticide at the recommended rate to a plot of soil under concrete slabs with a capped pvc pipe placed at the center. a block of nondurable wood is placed on the treated soil at the bottom of the pipe and the degree of termite damage is evaluated on a yearly basis. for registration purposes, a candidate termiticide should prevent termites from penetrating treated soil in all test plots for at least five years. the concrete slab test is normally installed in open field on tests conducted by the us department abstract the study investigated the performance of cypermethrin and chlorpyrifos based soil termiticides using concrete slab test installed in open field and under roof overhang in the philippines over a 5 year study period. plots treated with cypermethrin and chlorpyrifos in open field showed higher proportion of termite attacks compared to that installed under roof overhang. the high proportion of plots attacked by termites in open field could be attributed to environmental factors such as presence of a wider diversity of termite species, moisture, temperature, soil properties, microbial communities common in tropical climates, etc. the results of these two methods of installation could affect protocol used for field trial and the granting of registration or performance warranty to candidate termiticides in philippines and other tropical countries. sociobiology an international journal on social insects mn acda article history edited by og de souza, ufv, brazil received 01 november 2016 initial acceptance 07 march 2017 final acceptance 01 may 2017 publication date 21 september 2017 keywords termiticide, concrete slab test, roof overhang. corresponding author menandro n. acda department of forest products and paper science, university of the philippines los banos college, laguna 4031, philippines e-mail: mnacda@yahoo.com of agriculture forest service (usda/fs) in the unites states (wagner, 2003; shelton et al., 2016). however, there are debates in the philippines between registrants, regulators and the pest control industry on whether concrete slabs test for candidate termiticides should be installed in open field or in soil under cover from the weather. the debate stemmed from the premise that structures in the philippines and other tropical countries are built with wide roof overhang or eaves (fig 1). overhang on all four sides of the structure are common to protect exterior doors, windows, and siding from the weather. consequently, the soil under roof overhang is relatively dry and free of vegetation. considering that soil termiticides used for chemical barrier treatments are generally applied along the perimeter of structures under wide roof overhang, many would argue that concrete slab test should be installed in soil under cover from weather to simulate actual service condition. however, no data is available in the literature to make comparison between termiticide efficacy in open and covered concrete slab test. efficacy of termiticides is reported to be affected by various environmental factors such as moisture, temperature, soil properties (e.g. type, organic matter and ph), microbial communities, etc. (gold et al., department of forest products and paper science, university of the philippines los banos, laguna, philippines research article termites sociobiology 64(2): 212-216 (june, 2017) 213 1996; baskaran et al., 1999; saran & kamble, 2008; spomer et al., 2009; wiltz, 2010). considering that environmental factors are more severe in tropical countries, many candidate termiticides experience high percentage of failure and consequently fail the 5 year efficacy requirement. the present paper reports on the performance of soil termiticide chlorfyripos and α-cypermethrin in concrete slab tests placed in open field and under roof overhang to compare efficacy and how they affected results of field trial. materials and methods termiticides commercial soil termiticides with active ingredient consisting of chorpyrifos (dursban® tc, dowagrosciences) and α-cypermethrin (probuild® tc, syngenta philippines inc.) were used in this study. solution strength of 2.0% for dursban® tc and 0.5% for probuild® tc were prepared in water following label instructions. field sites the experiment was conducted in six selected buildings with wide roof overhang (1.2-1.4 m) and adjacent to an open field within university of the philippines los banos (uplb) campus (14.1655° n latitude, 121.2396° e longitude). the trial sites were atleast 1 km away from each other and have not been treated with soil termiticides. four of the buildings have been treated along the perimeter with pyrethroid based termiticides (rodding 1 m apart) in 2002 and two buildings received no prior treatment. it is assumed that the pyrethroid used to treat said buildings have already been depleted before the start of this study. the experimental sites have loamy soil with ph of about 7.2-7.6. an average temperature of 23-31°c with annual rainfall of about 1,942 mm prevailed in uplb during the study period (2011-2015). in general, the soil is dry to moist during the summer months and very wet during the rainy season under roof overhang and open field sites, respectively. each trial site was treated with only one type of termiticide. heavy and sometimes multiple infestations of coptotermes gestroi wasmann, macrotermes gilvus hagen, microcerotermes spp., and nasutitermes spp. are present in all sites. concrete slab test two groups of concrete slab tests were conducted simultaneously in two conditions viz., under roof overhang of a built structure and in open field. one set of concrete slabs were installed along the side of buildings (30 cm from the wall, 2 m apart) under roof overhang and an identical number in open field opposite (3 m apart) to that placed under cover (fig 2). the concrete slab test was conducted similar to that described by beal (1986) and kard et al. (1989) with some modifications. briefly, termiticide solutions were applied at a preconstruction rate of 4.07 liters m-2 to a plot of soil (100 x 432 x 432 mm) cleared of debris and vegetation. after the chemical has soaked into the soil, the plot was covered by a polyethylene sheet vapor barrier. a precast concrete slab (100 x 432 x 432 mm) with a 100 mm pvc cleanout adapter and plug at the center was placed on top of the treated plot to serve as inspection port. the vapor barrier was cut from inside and a block of wood (paraserianthes falcataria l, 50 x 64 x 64 mm) was placed on the soil inside the pipe. ten (10) replicates for each termiticide were installed in concrete slabs under roof overhang and 10 replicates in the adjacent open field as described above for each of the field sites. water only control slabs (10 replicates) were randomly installed between termiticide in both open field and under overhang. boards were inspected annually and replaced if attacked by termites. termite attack was defined as presence of termites or damage to wood at the end of each year of inspection. termite damage was rated according to the u.s. forest service “gulfport” scale, where 0 is no damage, 1 is nibbles to surface etching, 2 is light damage with penetration, 3 is moderate damage, 4 is heavy damage, and 5 is destroyed. data from the three field sites were pooled and the ratio of the number plots attacked by termites (i.e. wood blocks receiving ratings of 2 to 5) to the total number of slabs installed was used to determine the proportion of plots attacked for each year and termiticide used. goodness of fit using chi-squared (χ2) test was performed to compare differences in plots attacked by termites in open field and under roof overhang. an analysis of means (anom) plot was also used to determine which time period are significantly different from the grand mean for each termiticide used (statgraphics, 2010). results and discussion the percentage of plots attacked by termites in soil treated with cypermethrin and chlorpyrifos located in open field and under roof overhang over the 5-year study period is shown in figs 3 and 4. the number of plots penetrated by termites in soil treated with cypermethrin under roof overhang showed significant difference compared to that installed in open field (χ2 = 17.70, p-value <0.001). plots in open field experienced higher termite penetration (10-50%) compared to that under roof overhang (fig 3). similar results were observed with plots treated with chlorpyrifos (χ2 = 16.04, fig 1. schematic of roof overhang commonly used for built structures in the philippines. mn acda – termiticide performance in concrete slab test214 p-value <0.003) with 13-40% higher penetration in slabs installed in open field. in comparison, control plots of both termiticides were readily penetrated and wood blocks heavily attacked by termites (97-100%) indicating the high termite pressure in the selected test sites. apparently, the results confirmed the claim that performance of repellent or contact poison termiticides vary under cover by roof overhang and in open field. studies have shown that efficacy of termiticides is affected by various environmental factors such as moisture, temperature, soil properties (e.g. type, organic matter and ph), microbial communities, etc. (gold et al., 1996; baskaran et al., 1999, saran & kamble, 2008; spomer et al., 2009; wiltz, 2010). in open field condition, these factors are present in elevated levels contributing to the early degradation of termiticides and consequently breach of chemical barrier. termite species in the tropics are also more diverse (e.g., termitidae) and active throughout the year (tho, 1992; acda, 2004a). furthermore, diffuse root system of trees and woody shrubs often penetrate treated soil and serve as conduit for the entry of termites into concrete slabs in open field. apparently, these factors may have contributed to the relatively higher percentage of plots penetrated by termites in open field compared to those under roof overhang. the results further showed that termite penetrated 1 to 4 treated plots during the first year of trial in concrete slabs treated with cypermethrin and chlorpyrifos in both open field and under overhang. the percentage of termite attacks increased rapidly for both termiticides every year thereafter (figs 3 and 4). analysis of means (anom) showed significant increase in the mean proportion of plots penetrated by termites during each year compared to that of the grand mean for both cypermethrin (χ2 = 14.54, p-value <0.001) and chlorpyrifos (χ2 = 16.03, p-value <0.004). m. gilvus was the predominant termite species observed attacking wood blocks inside concrete slabs. m. gilvus is a very large, aggressive species that dominate the soil and open field in the philippines (acda, 2004a; rojo & acda, 2016). termite species observed attacking wood blocks under roof overhang were mostly nasutitermes and microcerotermes spp., although m. gilvus and c. gestroi were also common. this is consistent with foraging behavior observed with destructive philippine subterranean termites (acda, 2004b; 2007). in comparison, concrete slab tests of cypermethrin and chlorpyrifos conducted in the u.s. showed lower percentage of termite penetration over longer period of time (mulrooney et al., 2007). for registration purposes in the philippines, the current practice of using the 100% passing rate of concrete slabs in open field would present a stringent criterion for new termiticide registration if this requirement would be the only fig 2. schematic position of concrete slabs in three field sites used for cypermethrin in open field and under roof overhang. fig 3. percentage proportion of concrete slab plots treated with cypermethrin attacked by termites over 5 years. sociobiology 64(2): 212-216 (june, 2017) 215 basis of evaluation. however, other factors such as product chemistry, toxicology, environmental data, toxicity to nontarget organisms, etc. are commonly used for evaluation purposes. more often, regulating agencies do not prescribe assessment criteria and efficacy data is reduced to a lower status in the evaluation process or used as basis in providing period of service warranty. similar problems and issues were reported in australia, brazil, european union and other countries for evaluation and registration of new termiticides (krygsman, 2005). conclusions generally, the study showed that there was a significant difference in the performance of termiticide using concrete slab test placed under roof overhang and those installed in open field. plots treated with cypermethrin and chlorpyrifos in open field showed higher proportion of termite penetration and attack compared to those under roof overhang during each year of evaluation. in both conditions termites were able to penetrate treated plots during the first year of trial albeit at lower percentage then increased rapidly for both termiticides every year thereafter. the high percentage of plots attacked by termites in open field compared to that under overhang could be attributed to severe environmental factors such as moisture, temperature, soil properties, microbial communities, etc. common in tropical climates. the presence of wide diversity of termite species in the philippines could also be a factor. the results in both sites could affect trial protocol to determine performance of termiticide or performance warranty to candidate termiticides in the philippines. acknowlegements the author wishes to thank syngenta philippines for providing termiticides for this study and mr. nu valguna and mr. ba villanueva of the department of forest products and paper science, cfnr-uplb for their assistance in concrete slab installation. references acda, m, n. (2004a). economically important termites (isoptera) of the philippines and their control. sociobiology, 43: 159-169. acda, m. n. (2004b). foraging population and territories of the tropical subterranean termite macrotermes gilvus (isoptera: macrotermitinae). sociobiology, 43: 169-177. acda, m. n. (2007). foraging populations and territories of two species of subterranean termite (isoptera: termitidae) in the philippines. asia life sciences, 16: 71-80. baskaran, s., kookana, r.s. & naidu, r. (1999). degradation of bifenthrin, chlopyrifos, and imidacloprid in soil and bedding materials at termiticidal application rates. pesticide science, 55: 1222-1228. beal, r.h. (1986). field testing of soil insecticides as termiticides. international research group on wood preservation document irg/wp/1294. gold, r.e., howell, h.n., pawson, b.m., wright, m.s. & lutz, j.c. (1996). persistence and bioavailability of termiticides to subterranean termites (isoptera: rhinotermitidae) from five soil types in texas. sociobiology, 28: 337-363. kard, b.m., mauldin, j.k. & jones, s.c. (1989). evaluation of soil termiticides for control of subterranean termites (isoptera: rhinotermitidae). sociobiology, 15: 285-297. krygsman, a. international regulation of termiticides: an industry perspective. in proceedings of the fifth international conference on urban pests. lee cy & robinson wh (editors). july 10-13, 2005, singapore. mulrooney, j.e., wagner, t.l., shelton, t.g., peterson, c.j. & gerard, p.d. (2007). historical review of termite activity at forest service termiticide test sites from 1971 to 2004. journal of economic entomology, 100: 488-494. doi 10.1603/0022-0493. rojo, m.j.a. & acda, m.n. (2016). interspecific agonistic behavior of macrotermes gilvus (isoptera: termitidae): implication on termite baiting in the philippines. journal of insect behavior, 29: 273-282. doi 10.1007/s10905-016-9564-2 saran, r.k. & kamble, s.t. (2008). concentration-dependent degradation of three termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (isoptera: rhinotermitidae). journal of economic entomology, 101: 1373-1383. doi: 10.1093/jee/101.4.1373 shelton, t., fye, d., mankowski, m. & tang, j. termiticide report. pest management professional, april 11, 2016. pp 52-70. spomer, n.a, kamble, s.t. & siegfried, b.d. (2009). bioavailability of chlorantraniliprole and indoxacarb to eastern subterranean termites (isoptera: rhinotermitidae) in various soils. journal of economic entomology, 102: 19221927. doi: 10.1603/029.102.0524. fig 4. percentage proportion of concrete slab plots treated with chorpyrifos attacked by termites over 5 years. mn acda – termiticide performance in concrete slab test216 statgraphics centurion xvi: user’s manual. (2010). manugistics inc., rockville, md. usa. wagner, t.l. ( 2003). u.s. forest service termiticide tests. sociobiology, 41: 131-141. wiltz, b.a. (2010). laboratory evaluation of effects of soil properties on termiticide performance against formosan subterranean termites (isoptera: rhinotermitidae). sociobiology, 56: 755-773. tho, y.p. (1992). termites of peninsular malaysia. forest research institute malaysia, kepong. doi: 10.13102/sociobiology.v63i4.1074sociobiology 63(4): 1058-1062 (december, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the reallocation of the ant species dinoponera lucida emery (formicidae: ponerinae) population increasing its local genetic diversity introduction the atlantic forest is one of the richest biomes in the world and one of the 25 biodiversity hotspots (myers et al., 1999), but most of its remaining biome is distributed in small forest fragments (ribeiro et al., 2009). this habitat fragmentation process may represent a constant threat to endemic species (brooks et al., 2002), such as the giant ant dinoponera lucida emery 1901, whose geographic distribution is limited to bahia and espírito santo states, in northeastern and southeastern brazil, respectively (campiolo & delabie, 2008). this region is also known as the central corridor of the atlantic forest. female ants belonging to this species have no wings, whereas the male ants are winged (paiva & brandão, 1995), although they do not perform an efficient flight (teixeira, m.c. personal communication, february, 2009). new colonies are abstract the aim of the current study is to describe the genetic consequences of the reallocation process in order to preserve an entire dinoponera lucida emery (1901) population living in a forest fragment supposed to be deforested to give area for a seaport construction site. the sample collection and the mitochondrial data analysis were conducted before and after the reallocation of all found nests to two conservation units in espírito santo state, southeastern brazil. it was hypothesized that the reallocation of an exogenous population of d. lucida would increase the genetic variability which is naturally very low of populations living in isolated forest fragments. the data analysis showed that the genetic variability within the receiving populations has considerably increased, above the natural levels, due to the introduction of a new population. the biological viability of this effect was observed during the five years of monitoring program. the importance of the present study relies on information and reports from an unprecedented study on insect populations, and it will provide essential knowledge to future studies. sociobiology an international journal on social insects ss simon1, tm fernandes-salomão2, mc teixeira3 article history edited by evandro nascimento silva, uefs, brazil received 21 may 2016 initial acceptance 07 august 2016 final acceptance 15 december 2016 publication date 13 january 2017 keywords conservation strategies, endemic genetic variability, metapopulation, rescue effect, atlantic forest. corresponding author sabrina soares simon faculdade pitágoras de linhares avenida são mateus, 1458 araçá, cep 29.901-398, linhares-es, brasil e-mail: simonssabrina@gmail.com created through the fission of bigger colonies. thus, nests are established in an aggregated spatial distribution pattern inside the forest fragment (mariano et al., 2008; peixoto et al., 2010), and are probably genetically related to each other (araújo, 1994). such distribution pattern increasingly limits the species’ dispersal ability. habitat fragmentation causes the isolation of populations and increases endogamy, which results in low genetic variability inside inbreeding groups and high genetic divergence between isolated groups (packer & owen, 2001). in addition, populations with dispersion limitations, such as d. lucida, are under high extinction risks due to stochastic events (burkey, 1989). although the process that causes stochastic events may naturally occur, the anthropic exploration accelerates its natural course and leads to the extinction of populations haplotypes. because of the high forest fragmentation, not just entire forest fragments 1 faculdade pitágoras de linhares, linhares-es, brazil 2 universidade federal de viçosa, viçosa-mg, brazil 3 universidade federal do espírito santo, são mateus-es, brazil short note sociobiology 63(4): 1058-1062 (december, 2016) 1059 disappear, but a habitat might become no longer suitable to host highly adapted populations (fahrig, 1997; brooks et al., 2002). endemic species are even more threatened under these circumstances, and this is why the species d. lucida requires ultimate population genetic studies. the ecological theory features fragmented populations as a set of subpopulations form a metapopulation. these metapopulations experience extinction and recolonization events within subpopulations, and they persist in landscapes depending on the dynamic flow of the individuals. from a genetic point of view, small subpopulations are prone to extinction because of endogamy. however, individuals from other large subpopulations can migrate to a small subpopulation, and it could increase the genetic diversity in and avoid the extinction of this small subpopulation. such effect is known as rescue effect (begon, 2006); the large populations are called “donor” or “source”, and the small ones, “sink” or “receiver”. as it was previously mentioned, d. lucida populations have special dispersion limitations. in the framework of fragmentation and metapopulation theory, it is important understanding how the migration of individuals from a donor (or source) population affects the genetics of a receiver (or sink) population. therefore, a rescue and reallocation process was designed using an entire d. lucida population. the experiment took place in 2009, when a seaport construction site was set in an atlantic forest fragment. we tested the hypothesis that the reallocation of an exogenous d. lucida population would increase the genetic variability of populations located in isolated forest fragments. material and methods the forest fragments that have donated and received the reallocated population are located in aracruz county, espírito santo state, southeastern brazil. the research team visited conservation units in aracruz county aiming to assess the viability of reallocating the rescued nests to these units, before moving the population in. the criteria for selection were fragments under environmental protection policies, the shortest distance from the original area as possible, and the occurrence of the species. the barra do riacho terminal waterway (brtw, -19o50.548’, -40o03.827’) was the donor forest fragment supposed to be deforested, and it comprised 11 hectares. the conservation units selected to host the nests were: david victor farina municipal natural park (dvfmnp, -19o55.850’, -40o07.774’, 44 hectares) and aricanga waldemar devens municipal natural park (awdmnp, -19o48.827’, -40o19.959’, 515 hectares). these fragments belong to a highly fragmented landscape, and the two cu fragments are located approximately 25 kilometers from each other (in a straight line) (fig 1). all the 19 nest found in the brtw forest fragment were open, and every individual (eggs, young and adults forms) were removed and reallocated to artificial nests constructed in two conservation units (cu), according to the protocol designed by ferreira et al. (in prep). the reallocated population was monitored throughout the following five years in order to observe its behavioral and ecological reactions. fig 1. map of espírito santo state indicating the study area. (1) brtw: barra do riacho terminal waterway; (2) awdmnp: aricanga waldemar devens municipal natural park; (3) dvfmnp: david victor farina municipal natural park. ss simon, tm fernandes-salomão, mc teixeira – increasing genetic diversity on ant population1060 back to 2009, at the time the procedure was put in place, one sample was collected from each nest for genetic analysis, as well as samples from each of the 18 awdmnp and 17 dvfmnp nests of the native populations in the cu, in order to gather genetic data of the d. lucida populations before the reallocation. the research team collected new samples from all monitored nests in the receiving fragments in 2011. the dna extraction was conducted according to the protocol by waldschmidt et al. (1997) in the insect molecular biology laboratory of federal university of viçosa. sequences of the cox1-cox2 mitochondrial gene and intergenic trnaleu regions were obtained using primers developed by resende et al. (2010), which were later aligned in the mega 5.0 software (tamura et al., 2011). the haplotype network analysis was conducted in the network 4.6 software, through the median-joining method (bandelt et al., 1999). the analysis of molecular variance (amova) was performed using the arlequin 3.5 (excoffier & lischer, 2010) applied to the data collected before and after the reallocation process. the “among populations” hierarchical level corresponded to the brtw, awdmnp and dvfmnp populations in the amova conducted before the reallocation. the same hierarchical level consisted of awdmnp and dvfmnp populations in the amova conducted after the reallocation process, since the brtw population was reallocated to those both receivers. the amova was applied to three hierarchical levels in order to take the potential subpopulations into account, because previous reviews refer to aggregate distribution patterns within a single forest fragment. results four haplotypes were identified in the data set: one in each park (named h1 in awdmnp; h4 in dvfmnp) and two in the reallocating population (h2 and h3, both from brtw) (fig 2). as it was expected, the amova conducted before the reallocation process has shown high genetic variance between populations from different forest fragments (83%) when the three populations, namely: brtw, awdmnp and dvfmnp, where studied as a single one (table 1). the second amova has shown that the genetic variance between awdmnp and dvfmnp has decreased to 55% after the reallocation, whereas the diversity within populations and subpopulations has increased. it is worth mentioning that after the five-year monitoring, 82% of the reallocated nests has survived in the receiver fragments (ferreira et al., in prep). discussion the main aim of the present study was to describe the genetic consequences of the reallocation process involving an entire d. lucida population. instead of collecting genetic samples from all populations and simulate the genetic diversity in order to predict the reallocation impacts, it was made the choice for effectively performing the reallocation procedure and monitoring its effects for the following five years. it was demanding to put this intervention in place in order to save brtw haplotypes from extinction. the haplotype network analysis showed that the haplotypes h2 and h3, both from the brtw fragment, were more similar to each other than the h1 and h4, from awdmnp and dvfmnp, respectively. such finding may be justified by the geographic distance, because h2 and h3 come from the same area, and it suggests the existence of recent divergence. d. lucida populations living in a certain forest fragment often have one or few haplotypes (resende et al., 2010). there is high genetic diversity from population to population, as well as low variability inside a single population. since it is common finding one or few haplotypes in a single population, it is possible inferring that the smaller table 1. analysis of molecular variance (amova) applied to three hierarchical levels on data set before reallocation. vs: variation source; df: degrees of freedom; ss: sum of squares; e (ms): estimate medium squares variance components. vs df ss e (ms) % variation among populations 2 17.500 0.42226 83.00 within population 5 1.724 0.04212 8.28 within the potential sub-population 53 2.350 0.04434 8.72 total 60 21.574 0.50873 100.00 10.000 permutations, p<0.000001 fst = 0.91284 fig 2. the haplotype network of the cox1-trnaleu-cox2 region from 54 dinoponera lucida individuals. the numbers represent mutational steps. h1, h2, h3 and h4 represent the haplotypes. mv1 and mv2 (median vector) represent the extant or the non-sampled haplotypes. sociobiology 63(4): 1058-1062 (december, 2016) 1061 vs df ss e(ms) %variation among populations 2 13.162 0.25457 55.10 within population 5 6.062 0.16306 35.30 within the potential sub-population 53 2.350 0.04434 9.60 total 60 21.574 0.46196 100.00 10.000 permutations, p<0.000001 fst = 0.90402 table 2. analysis of molecular variance (amova) applied to three hierarchical levels on data set after reallocation. vs: variation source; df: degrees of freedom; ss: sum of squares; e(ms): estimate medium squares variance components. the area, the lower the genetic diversity of d. lucida. the genetic diversity within the populations and subpopulations of both receivers in the cu has increased after the donor population was reallocated, whereas the variation between awdmnp and dvfmnp populations decreased, due to the homogenization of nests from the brtw population (table 2). thus, the haplotypic diversity in the receiver populations in the present study is higher than natural. it means that the herein studied hypothesis was accepted, because of the high survival rate presented by the reallocated population. the reallocation effectively increased the genetic diversity of subsequent generations. it is a satisfactory result, since the literature shows that the rate of successful reallocation, repatriation and translocation processes is low (dodd & seigel, 1991). variation may enable enhancing the population’s survival, thus simulating a rescue effect. this result may be representative for the entire d. lucida species. in addition, the previously conducted search for suitable and similar environments may have prevented or minimized adaptation issues. genetic studies about reallocation and translocation process are rare, mainly studies of this nature involving groups of invertebrates (sherley et al., 2010). yet, these studies are essential to assess the persistence of the introduced population (armstrong & seddon, 2008). the reallocation program appears to have succeeded after five years monitoring the studied populations. yet, the herein adopted reallocation procedures shall not be replicated if the basic information provided in the current study are not followed. this is the first time a reallocation program involving an invertebrate population is conducted based on a genetic data analysis associated with a middle time monitoring. it is strongly recommended that future studies do not ignore the importance of the herein presented information. acknowledgments the authors thank filipe pola vargas, who have been supportive in many steps of this work. references araújo, c.z.d., jaisson, p. (1994). modes de fondation des colonies chez la fourmi sans reine dinoponera quadriceps santschi (hymenoptera, formicidae, ponerinae). actes des colloques insectes sociaux, 9:79-88. armstrong, d.p., seddon, p.j. (2007). directions in reintroduction biology. trends in ecology and evolution 23:20-25. doi: 10.1016/j.tree.2007.10.003 bandelt, h.j., forster, p., röhl, a. (1999). median-joining networks for inferring intraspecific phylogenies. molecular biology and evolution 16:37-48. distributed by network © copyright fluxus technology ltd. retrived from: http://mbe. oxfordjournals.org/content/16/1/37.abstract begon, m., townsend, c.r., harper, j. l. (2006). ecology: from individuals to ecosystems. oxford: blackwell publishing, 738p. brooks, t. m., mittermeier, r. a., mittermeier, c. g., da fonseca, g. a. b., rylands, a. b., konstant, w. r., flick, p., pilgrim, j., oldfield, s., magin, g. and hiltontaylor, c. (2002). habitat loss and extinction in the hotspots of biodiversity. conservation biology, 16: 909–923. doi: 10.1046/j.1523-1739.2002.00530.x. burkey, t. v. (1989). extinction in nature reserves: the effect of fragmentation and the importance of migration between reserve fragments. oikos 55:75-81. retrived from: http:// www.jstor.org/stable/3565875?seq=1#page_scan_tab_contents campiolo, s. & delabie, j.h.c. (2008). dinoponera lucida it is worth noticing that, although the aggregate distribution within a single forest fragment has been observed in the field (peixoto et al., 2010), the genetic relationship predicted by araújo et al. (1994) has not been confirmed by the mitochondrial sequence analysis. the variation within a potential subpopulation and between subpopulation found in the whole fragment is not clearly consistent with this idea (table 1). it means that nests from the same subpopulations are related to each other, as well as to other nests in the population, alike. it suggests that the aggregation pattern may due to suitable environmental conditions inside the forest, and that such pattern may be dynamic if these conditions change. however, the predicted genetic relationship between close nests may be assessed through nuclear dna analysis, which takes into account the disperser role played by the male individuals. if the low diversity between close nests is confirmed, it may be explained by their limited dispersal ability. reintroduction programs are a frequent alternative to the recolonization of a sink area when the metapopulation dynamics fails. these interventions often fail due to the founder effect, if the introduced group is highly inbreed (armstrong & seddon, 2008). such problem was avoided in the current study, because the introduction was performed in an area hosting its own population and haplotypes, and it increased the total genetic variation. this increased genetic ss simon, tm fernandes-salomão, mc teixeira – increasing genetic diversity on ant population1062 emery, 1901. in a.b.m. machado, g. m. drummond, a.p. paglia (eds.), livro vermelho da fauna brasileira ameaçada de extinção (pp. 388-389). ministério do meio ambiente: fundação biodiversitas. dodd jr, c.k., seigel, r.a. (1991). relocation, repatriation, and translocation of amphibians and reptiles: are they conservation strategies that work? herpetologica, 47:336-350. excoffier, l. & lischer, h.e. l. (2010). arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under linux and windows. molecular ecology resources. 10:564-567. doi: 10.1111/j.17550998.2010.02847.x. fahrig, l. (1997). relative effects of habitat loss and fragmentation on population extinction. the journal of wildlife management. 61. retrived from: http://www.jstor. org/stable/3802168. mariano, c.s.f., pompolo, s.g., barros, l.a.c., marianoneto, e., campiolo, s., delabie, j.h.c. (2008). a biogeographical study of the threatened ant dinoponera lucida emery (hymenoptera: formicidae: ponerinae) using a cytogenetic approach. insect conservation and diversity, 1:161-168. doi: 10.1111/j.1752-4598.2008.00022.x. myers, n., mittermeier, r. a., mittermeier, c. g., fonseca, g. a. b., kent, j. (1999). biodiversity hotspots for conservation priorities. nature 403:853-858. doi: 10.1038/35002501 packer, l. & owen, r. (2001). population genetic aspects of pollinator decline. conservation ecology, 5(4). retrived from: http:// www.ecologyandsociety.org/vol5/iss1/art/4. paiva, r.v.s., brandão, c.r.f. (1995). nests, worker population, and reproductive status of workers, in the giant queenless ponerine ant dinoponera roger (hymenoptera formicidae). ethology, ecology and evolution, 7: 297-312. doi: 10.1080/08927014.1995.9522938. peixoto, a. v., campiolo, s., delabie, j. h. c. (2010). basic ecological information about the threatened ant, dinoponera lucida emery (hymenoptera: formicidae: ponerinae), aiming effective long-term conservation. species diversity and extinction. g. h. tepper, nova science publishers, inc: 183-213. retrived from: https://www.researchgate.net/ publication/287738303_basic_ecological_information_ about_the_threatened_ant_dinoponera_lucida_emery_ hymenoptera_formicidae_ponerinae_aiming_its_effective_ long-term_conservation resende, h.c., yotoko, k.s.c., costa, m.a., delabie, j.h.c., tavares, m.g., campos, l.a.o., fernandes-salomão, t.m. (2010). pliocene and pleistocene events shaping the genetic diversity within the central corridor of the brazilian atlantic forest. biological journal of the linnean society. vol 101: 949-960. doi: 10.1111/j.1095-8312.2010.01534.x ribeiro, m.c., metzger, j.p., martensen, a.c., ponzoni, f.j., hirota, m.m. (2009). the brazilian atlantic forest: how much is left, and how is the remaining forest distributed? implications for conservation. biological conservation 142:1141-1153. doi: 10.1016/j.biocon.2008.10.012 sherley, g.h., stringer, i.a.n., parrish, g.r. (2010). summary of native bat, reptile, amphibian and terrestrial invertebrate translocations in new zealand. science for conservation. wellington: department of conservation, 39 p. tamura, k., peterson d., peterson n., stecher g., nei m., kumar s. (2011). mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution, 28:2731-2739. doi: 10.1093/molbev/msr121 waldschmidt, a. m., salomão, t. m. f., barros, e. g., campos, l. a. o. (1997). extraction of genomic dna from melipona quadrifasciata (hymenoptera: apidae, meliponinae). brazilian journal of genetics 20:421-423. retrived from: http://www.scielo.br/scielo.php?script=sci_ arttext&pid=s0100-84551997000300011 co-authors contribution tânia maria fernandes-salomão and marcos da cunha teixeira have substantially contributed to every step of this study, since its conception, the acquisition, analysis, and interpretation of data. in addition, they have contributed to the text drafting, revising and its final approval, as well as they have agreed to be accountable for all aspects of the work, ensuring its integrity. a1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1195sociobiology 63(4): 1063-1068 (december, 2016) ants visible from space influence soil properties and vegetation in steppe rangelands of iran introduction information concerning iranian ant’s effects on environment has tended to be sparse or is often published in iran.this situation is likely to change due to the opening up of the country and removal of sanctions, although a further stimulus is the recent translation into persian (farsi) and online availability of the book ants: standard methods for measuring and monitoring biodiversity (agosti et al., 2000) by mahsa ghobadi and mohammad mahdavi (agosti et al., 2015, down-loadable at https://zenodo.org/record/16183#. vu-hzlkfsjg). a recent series of researches published in iran by ghobadi (2013) and ghobadi et al.(2015; 2016) have quantified the influence of one of the most prominent messor spp. on soil properties and vegetation in steppe rangelands of iran. in view of the importance of this work, we here summarize and synthesise the findings of these two papers in order to bring them to the attention of the wider readership. abstract messor nests in iranian steppe rangelands can be so large that they are visible from space. when compared with reference soils, nest soil is higher in nutrients and lower in ph. ant nests also homogenise the nutrients throughout the upper soil profile, although this effect diminished when nests are abandoned. the denuded circles around nests are surrounded by rings of vegetation that differ in species composition from that of the surrounding vegetation, while abandoned nests are colonized by a different range of plant species. data on the density and abundance of messor cf. intermedius nests indicate that the soil in less than 1% of the area is impacted, although the cumulative effect of so many nests influences the plant species and vegetation structure of the region.the data indicate the importance of these ants in altering soil chemical composition and plant diversity, which could have flow-on effects to the diversity of animals. sociobiology an international journal on social insects m ghobadi1, d agosti2, m mahdavi1, mh jouri1, j majer3 article history edited by evandro nascimento silva, uefs, brazil received 22 september 2016 initial acceptance 08 october 2016 final acceptance 09 october 2016 publication date 13 january 2017 keywords formicidae, ant nest, soil chemistry, plant composition, nest abandonment. corresponding author jonathan majer school of plant biology, the university of western australia perth, australia e-mail: jonathan.majer@uwa.edu.au the study harvester ants from the genus messor count among that selected group of invertebrates whose manifestations are clearly visible from space by satellite imagery (fig 1). the study was performed in a 30 ha site near roodshoor, saveh, iran, located at a point 60 km along the tehran-saveh highway (35.43802° n; 50.89633°e) in an area from which sheep and goats had been excluded for 40 years. the vegetation is a chenopod steppe shrubland whose dominant plant cover includes artemisia sieberi and stipa hohenackeriana, along with salsola tomentosa, brassica deflexa, and poasinaica as co-dominant species (mahdavi et al., 2009). prominent ant nests were visually located, mapped and measured along ten 200 m long transects. identification of ants was mainly to species group, since the taxonomy of iranian ants has not been fully defined. voucher specimens of these species are deposited in the naturhistorisches museum, bern, (code nmbe http://grbio.org/cool/5hwi-0wgz). 1 islamic azad university, noor branch, iran 2 american museum of natural history, new york, united states of america 3 the university of western australia, perth, australia short note m ghobadi et al. – iranian ants visible from space1064 the species recorded represented three genera (messor (3 species), cataglyphis (3 species) and formica (1 species) (table 1). other ant species with more cryptic nests may also be present, but messor cf. intermedius had by far the largest (mean diameter 2.3 m, modal diameter 3.5 m) and most abundant nests (table 1), which were flat and circular in shape, as evidenced by the light circles in figure 1. some of the nests of this species were abandoned, but still visible (fig 2). the study measured soil physical and chemical properties on 18 live and seven abandoned ant nests and also in un-nested control areas 3 m away from the live nests of messor cf. intermedius. chemical properties were measured at four depths and averaged across nests. fig 1. location of the study area at roodshoor, saveh, iran, showing location of messor cf. intermedius ant colonies (light circles like the one in the red square). (photo courtesy of google earth). species functional group typical habitat color size mound shape density (no./h) mound diameter mean (m) ±se material composition messor cf. intermedius. harvester dry areas black small & medium flat 8.3 2.3± 0.03 seed, plant, soil messor cf. subgracilinodis harvester dry areas black & red medium dome 0.9 1.46± 0.10 seed, plant, soil messor cf. structor harvester dry areas black small pore 0.1 soil cataglyphis bellicosus scavenger steppe & desert black large pore 1.7 soil cataglyphis cf. nodus. scavenger steppe & desert black & red medium flat 0.5 0.95± 0.05 soil cataglyphis cf. lividus. scavenger steppe & desert orange small pore 0.2 soil formica cf. epinotalis general forager unknown brown small pore 0.1 soil table 1. properties of ant species found in the study site. active mound soil has a significantly lower ph than controls (table 2) and significantly higher levels of nitrogen, phosphorus, potassium, magnesium, calcium, organic matter and carbon and electrical conductivity (ec) (table 2). percentage sand was higher on active ant nests than on controls but percentage clay was lower. soil temperatures were significantly higher on ant nests than control areas in both dry and humid seasons, as was soil moisture. water infiltration, as measured by timing water in cylinders to percolate into soil, was significantly higher on ant nests in both dry and humid seasons (table 2). none of the chemical or physical soil properties differed significantly between abandoned nests and control areas (table 2), indicating that it had been the activity of ants that was responsible for the differences observed on active nests. sociobiology 63(4): 1063-1068 (december, 2016) 1065 this was further illustrated when the organic matter and nutrient (n, p and k) levels throughout the soil profile were considered. duncan’s multiple range test indicated that levels of all four measures were considerably higher in active nest soil compared to control soil at all four measured depths (0-10, 10.1-20, 20-1-30 and 30.1-40 cm), while levels soil of abandoned nests were virtually the same as in control soil (fig 3). furthermore, active ant nests had the effect of homogenizing the nutrient distribution throughout the profile, fig 2. mounds of (a) active and (b) abandoned messor cf. intermedius nests at roodshoor, saveh, iran. even though concentrations declined with depth in control area soils. soil nutrient levels and profiles had regressed to control area levels in abandoned nests (fig 3). unlike the control areas, which were dominated by plants stipa hohenackeriana, brassica deflexa, and artemisia sieberi, the active nest areas were dominated by campanula stricta, lepidium vesicarium, achillea tenuifolia, brassica deflexa, papaver tenuifolium and scabiosa oliveri (table 3), the major part of which were concentrated in 1m wide rings fig 3. nutrient concentrations of soil at four depths in active (n = 18) and abandoned (n = 7) messor cf. intermedius mounds and control areas (n = 18). m ghobadi et al. – iranian ants visible from space1066 properties mound control site abandoned mound ph 7.47 ± 0.01b* 8.30 ± 0.03 a 8.40 ± 0.04 a ec 3.05 ± 0.03 a 1.43 ± 0.06 b 1.30 ± 0.06 b % oc 0.59 ± 0.04 a 0.18 ± 0.03 b 0.19 ± 0.02 b % om 1.13 ± 0.08 a 0.33 ± 0.05 b 0.35 ± 0.06 b %n 0.05 ± 0.005 a 0.01 ± 0.03 b 0.01 ± 0.003 b p (ppm) 15.70 ± 0.7 a 4.01 ± 0.03 b 4.08 ± 0.1 b k (ppm) 551.90 ± 3.8 a 320.10 ± 1.02 b 322.50 ± 7.81 b mg (mg g–1) 110.50 ± 0.2 a 90.20 ± 0.25 b 91.30 ± 0.01 b ca (mg g–1) 78.20 ± 0.1 a 60.40 ± 0.01 b 61.20 ± 0.03 b % sand 80.53 ± 0.19 a 77.93 ± 0.18 b 78.00 ± 0.15 b % silt 8.4 0± 0.16 a 8.90 ± 0.2 a 8.93 ± 0.2 a % clay 11.06 ± 0.2 b 13.13 ± 0.25 a 13.00 ± 0.21 a temperate dry season 32.00± 0.1 a 28.10 ± 0.01 b 27.50 ± 0.1 b temperate humid season 25.20 ± 0.01a 22.00 ± 0.2 b 23.40 ± 0.003 b moisture dry season 6.50 ± 0.6 a 4.20 ± 0.01 b 4.90 ± 0.02 b moisture humid season 9.30 ± 0.3 a 7.40 ± 0.05 b 7.80 ± 0.001 b infiltration rate dry season (mm ) 34.74 ± 1.30 a 21.66 ± 0.56 b ** infiltration rate humid season (mm ) 40.32 ± 1.88 a 22.94 ± 0.65 b ** table 2. line 1-16: physical and chemical properties of soils (mean from 0 to 40 cm) from messor cf. intermedius mounds, dead mounds and control sites in the roodshoor and significance of the comparison between sites by duncan’s multiple range test (mean±s.e; control and live nest each n=18, abandoned nest n= 7). line 17-18: results of soil infiltration measuring under conditions of high and low soil moisture contents by double rings method (t-test, mean±s.e; control and colony each n=5). * the means of the rows with same letters were shown not significantly different by duncan’s multiple range test ** not measured around the periphery of the nest, which itself was largely devoid of plant cover (fig 2). when these surrounding rings were included for comparison with control areas, the active nests had significantly higher vegetation cover, plant species richness and diversity, lower grass cover, higher forb cover and an absence of shrubs. the situation changed markedly on abandoned nests, which were totally dominated by the forb campanula stricta, leading to significantly higher vegetation cover but lower plant species richness, diversity and evenness than on active nests (table 3). furthermore, the vegetation on abandoned nests was more evenly distributed, with no vegetated ring or bare centre being evident (fig 2). discussion these observations on the influence of messor ants add to those from southern europe (e.g., cammeraat et al., 2002), africa (e.g., dean & yeaton, 1993) and parts of western asia (e.g. ginzberg et al., 2008; brown et al., 2012), the other regions of the world where this genus of seed harvester ants is found. as in these other studies, the iranian messor nests have a profound influence on soil chemistry, although this effect diminishes once the nests are abandoned and the ants cease their harvesting and soil-moving activities. the time taken for this effect to diminish was not measured. considering the nest diameter and density of messor cf. intermedius nests, the area family species name ivi control active nest abandoned nest campanulaceae* campanula stricta 1.03 18.78 45.03 silene chaetodonta 1.11 1.1 chenopodiaceae salsola laricina 2.07 salsola tomentosa 2.54 compositae achillea tenuifolia 1.47 7.11 1.13 anthemis gilanica 2.35 3.56 1.07 artemisia sieberi 5.54 centaurea behen 3.84 3.01 centaurea bruguierana 1.04 1.21 cousinia belangeri 1.07 1.25 echinops pungens 1.18 1.05 table 3. above: mean importance value index (ivi) for plants species and mean (± se) percentage cover of different functional groups of plants. below: plant species richness (s), diversity (h'), and evenness (e) for active and abandoned messor cf. intermedius nests and control sites (mean ± se). sociobiology 63(4): 1063-1068 (december, 2016) 1067 under the influence of active nests represents less than 1% of the total area, even if the additional but unmeasured influence of messor cf. subgracilinodis is included. however, the implications to biodiversity are profound. the active nests, and to a lesser extent, the abandoned nests, produce changes in the dominance of the vegetation and, as a result, increase the heterogeneity or patchiness of the environment. this is likely to enhance the diversity of invertebrate (siemann, 1998), and possibly vertebrate animals (murkin & batt, 1987) that directly or indirectly depend on the plant species and vegetation structure of the region or the invertebrates on which they may feed. references agosti, d., majer, j., alonso, e. & schultz t, (eds.) (2000). ants: standard methods for measuring and monitoring biodiversity. biological diversity handbook series. smithsonian institution press, washington d.c., 20+280 pp. doi: 10.5281/zenodo.11736 agosti, d., majer, j., alonso, e. & schultz t, (eds.) (2015). ants: standard methods for measuring and monitoring biodiversity. nasr, tehran, 533 pp. (farsi version translated by m. ghobadi and m. mahdavi.). doi: 10.5281/zenodo.16183 brown, g., scherber, c., ramos, jr. p. & ebrahim, e. k. (2012). the effects of harvester ant (messor ebenius forel) nests on vegetation and soil properties in a desert dwarf shrub community in northeastern arabia. flora, 207: 503-511. doi: 10.1016/j.flora.2012.06.009 cammeraat, l. h., willmott, s. j., compton, s. g. & incoll l. d.(2002). the effect of ants’ nests on the physical, chemical and hydrological properties of a rangeland soil in semi-arid spain. geoderma, 105: 1-20. doi:10.1016/s0016-7061(01)00085-4 dean, w. r. j.&yeaton, r. i.(1993). the effects of harvester ant messor capensis nest-mounds on the physical and chemical properties of soils in the southern karoo, south africa. journal of arid environments, 25: 249–260. doi: 10.1006/ jare.1993.1059 ghobadi, m.(2013). the effects of harvester ant nest activities (messor spp.) on some properties of soil structure and function of plants in steppe rangeland of roodshoor. msc thesis, islamic azad university of noor. 156 pp. (in farsi.) ghobadi, m., agosti, d., mahdavi, m. & jouri, m. h. (2015). effects of harvester ant nest activity (messor spp.) on structure and function of plant community in a steppe rangeland (case study: roodshoor, saveh, iran). journal of rangeland science, 5: 269-283. doi: 10.5281/zenodo.231998 ghobadi, m., agosti, d. & mahdavi, m. (2016).change in soil properties by harvester ant’s activity (messor spp.) in roodshoor steppe rangeland of saveh, iran). journal of rangeland science, 6: 273-285. doi: 10.5281/zenodo.232012 family species name ivi control active nest abandoned nest compositae senecio vernalis 1.06 1.12 taraxacum vulgare complex 1.32 1.61 cruciferae alyssum marginatum 2.11 3.17 brassica deflexa 7.07 7 1.03 lepidium vesicarium 1.21 15.07 sisymbrium officinale 1.05 1.06 gramineae stipa hohenackeriana 9.14 1.12 aegilops columnaris 1.07 1.17 anisantha tectorum 1.21 2.09 hordeum murinum 2.11 1.2 poa sinaica 2.15 1.07 ephedraceae ephedra strobilacea 2.15 euphorbiaceae euphorbia sororia 1.14 2.02 dipsacaceae scabiosa oliveri 1.1 5.89 1.03 geraniaceae erodium oxyrrhynchum 2.2 3.4 labiatae ziziphora tenuior 1.62 2.73 papaveraceae papaver tenuifolium 1.09 6.07 1.05 fabaceae astragalus chaborasicus 2.08 1.1 apiaceae ferula hirtella 2.12 1.11 valerianaceae valerianella oxyrrhyncha 1.02 1.04 percentage plant cover ** grass 14.83 ± 2.30 a 4.44 ± 0.01 b forb 21.83 ± 0.42 c 62.94 ± 0.94 b 82.57 ± 1.30 a shrub 9.22 ± 0.01 indices ** vegetation cover (%) 45.88 c 67.88 b 82.57a richness (s) 6.44 b 12.33 a 2.85 c diversity (h’) 1.73 b 2.26 a 0.39 c evenness (e) 0.40 a 0.45 a 0.13 b * the underlined data has higher values of ivi for treatments ** the means of the rows with same letters were shown not significantly different by duncan’s multiple range test. table 3. above: mean importance value index (ivi) for plants species and mean (± se) percentage cover of different functional groups of plants. below: plant species richness (s), diversity (h'), and evenness (e) for active and abandoned messor cf. intermedius nests and control sites (mean ± se). (continuation) m ghobadi et al. – iranian ants visible from space1068 ginzburg, o., whitford, w. g. & steinberger, y. (2008). effects of harvester ant (messor spp) activity on soil properties and microbial communities in a negev desert ecosystem). biology and fertility of soils, 45: 165-173. doi: 10.1007/ s00374-008-0309-z mahdavi, m., arzani, h. & jouri, m. h. (2009). analysis of rangeland condition changes using a qualitative measure of rangeland health. rangeland journal, 3: 397-411. (in persian). murkin, r. h. & batt, b. d. j. (1987). the interactions of vertebrates and invertebrates in peatlands and marshes. memoirs of the entomological society of canada 119: supplement s140: 15-30. doi: 10.4039/entm119140015-1 siemann, e. (1998). experimental tests of effects of plant productivity and diversity on grassland arthropod diversity. ecology, 79: 2057-2070. doi: 10.2307/176709 authors contribution mahsa ghobardi conducted this work for a higher degree thesis under the supervision of donat agosti, mohammad mahdaviand mohammad hassan jouri. jonathan majer assisted with the preparation of this review of the work. all data have been cleared for copyright with the journal in which the primary papers were published. doi: 10.13102/sociobiology.v67i4.5833sociobiology 67(4): 604-609 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the brazilian atlantic rainforest is a biodiversity hotspot with a notable number of endemic species; however, anthropogenic actions have led to the substantial reduction and fragmentation of most of this biome, and also the loss of this biodiversity (morellato & haddad, 2000; paviolo et al., 2016). many unknown species were, or may be extinct even before their description (morellato & haddad, 2000). in this scenario, cytogenetics is a key tool in discovering biodiversity of different organisms (di-nizo et al., 2017; cioffi et al., 2018) and generates useful information that can be used in conservation strategies for endangered species (mariano et al., 2008; potter & deakin, 2018). considering ant communities, even with area regeneration strategies, only pristine native areas can maintain a substantial proportion of biodiversity (silva et al., 2007). ants show a significant karyotype diversity and the variation in the chromosome number observed for the abstract the brazilian atlantic rainforest is an endangered biome and a biodiversity hotspot. ant cytogenetic studies from this biome showed remarkable chromosomal diversity among species, and provided useful insights on phylogeny, chromosomal evolution, and taxonomy. in this study, we karyotyped five ant taxa from the atlantic rainforest. the observed chromosome numbers were pheidole germaini 2n=22, pheidole sp. flavens group 2n=20, brachymyrmex admotus 2n=18, camponotus atriceps 2n=40, and odontomachus haematodus 2n=44. the data obtained for pheidole spp. represent the first chromosomal records for the genus in brazil and provide information on the chromosomal evolution of pheidole. karyotypic information from b. admotus brings the genus back to the cytogenetic scenario after decades of neglect. similar karyotype was observed among the c. atriceps from different populations already studied, corroborating its status as a good species. chromosomal variations were observed between populations of o. haematodus, which need to be investigated in further researches. this study increased the cytogenetic knowledge of ants from the brazilian atlantic rainforest. sociobiology an international journal on social insects ga teixeira1,2, lac barros3, dm lopes2, hjac aguiar3 article history edited by jacques delabie, uesc, brazil cléa dos santos f. mariano, uesc, brazil received 04 september 2020 initial acceptance 15 october 2020 final acceptance 27 october 2020 publication date 28 december 2020 keywords karyotype, classical cytogenetics, formicidae, biodiversity, brachymyrmex, pheidole. corresponding author luísa antônia campos barros https://orcid.org/0000-0002-1501-4734 universidade federal do amapá campus binacional, oiapoque, br 156, n˚ 3051 bairro universitário, 68980-000, amapá, brasil. e-mail: luufv@yahoo.com.br neotropical region is 2n=4 in strumigenys louisianae roger, 1863 to 2n=120 in dinoponera lucida emery, 1901 (reviewed by mariano et al., 2019). both species are from the brazilian atlantic rainforest, with d. lucida endemic and endangered of this biome. the minimum interaction theory (mit), the most widely used in ants, proposes that centric fissions are the main rearrangements that explain this significant chromosomal variation among species (imai et al., 1994). however, inversions, translocations, and robertsonian fusions have also been important in the origin of numerical and structural chromosomal polymorphisms and chromosome evolution in ant species (reviewed by lorite & palomeque, 2010; mariano et al., 2019; teixeira et al., 2020; micolino et al., 2020). we described the karyotypes of five ant taxa, from four genus and three subfamilies, which live in the brazilian atlantic rainforest, suggesting insights on chromosomal evolution for some of these species. additionally, a comparative 1 programa de pós-graduação em biologia celular e estrutural, universidade federal de viçosa, viçosa, minas gerais, brazil 2 laboratório de citogenética de insetos, departamento de biologia geral, universidade federal de viçosa, viçosa, minas gerais, brazil 3 universidade federal do amapá, campus binacional, oiapoque, amapá, brazil karyotypic data of five ant taxa from the brazilian atlantic rainforest short note sociobiology 67(4): 604-609 (december, 2020) 605 approach with other available populations was performed for camponotus atriceps (smith, 1858) and odontomachus haematodus (linnaeus, 1758). colonies were collected from the southeastern brazilian atlantic forest in the cities of viçosa, in the horto botânico of universidade federal de viçosa, and in a rural area at ubá (table 1, fig 1) (icmbio/sisbio accession number 32459). adult vouchers were identified by dr. jacques delabie and deposited in the ant collection at the laboratório de mirmecologia do centro de pesquisas do cacau (cpdc/brazil), in bahia, brazil, under records #5815 and #5817. mitotic chromosomes were obtained from the cerebral ganglia of larvae after meconium elimination, using hypotonic colchicine solution (0,005%) and fixatives according to imai et al. (1988) and submitted to 4% giemsa staining. chromosomes were measured, arranged in order of decreasing size and classified as m=metacentric (r=1–1.7), sm=submetacentric (r=1.7–3), st=subtelocentric (r=3–7) and a=acrocentric (r>7), according to levan et al. (1964). chromosomes were organized using adobe photoshop® cs6 and measured using image pro plus®. ant species locality coordinates col./ind. 2n karyotypic formula subfamily myrmicinae pheidole germaini emery, 1896* viçosa, minas gerais, brazil -20.757041, -42.873516 1/8 22 18m+2sm+2st pheidole sp. (flavens group) viçosa, minas gerais, brazil -20.757041, -42.873516 1/4 20 18m+2sm subfamily formicinae brachymyrmex admotus mayr, 1887* viçosa, minas gerais, brazil -20.757041, -42.873516 1/6 18 16m+2sm camponotus atriceps (smith, 1858) viçosa, minas gerais, brazil -20.757041, -42.873516 1/5 40 4sm+34st+2a subfamily ponerinae odontomachus haematodus (linnaeus, 1758) ubá, minas gerais, brazil -21.128880, -42.937646 1/5 44 8sm+18st+18a * first cytogenetic report table 1. ant species from the brazilian atlantic rainforest karyotyped in this study. collection site, sample size (number of colonies/ individuals), diploid chromosome number, and karyotypic formula. pheidole germaini emery, 1896 (tristis group) showed 2n=22; 2k=18m+2sm+2st (fig 2a). this is the first record of occurrence of p. germaini for the state of minas gerais since published records are restricted to the states of mato grosso and mato grosso do sul (guénard et al., 2017). pheidole is a monophyletic hyperdiverse genus with over 1,100 species described on all continents (except antarctica) and possibly originated in new world (moreau, 2008; bolton, 2020). karyotype data are available for 75 taxa and most of them showed 2n=20 with representatives in the old and new world (reviewed by lorite & palomeque, 2010). pheidole spininodis mayr, 1887 and pheidole subarmata mayr, 1884 (as p. cornutula), also included in the tristis group, presented 2n=20 metacentric chromosomes. the former has two larger metacentric pairs compared to the other chromosomes and the latter has three pairs (goñi et al., 1983). pheidole germaini showed only a single larger metacentric pair, in addition to one submetacentric and another subtelocentric pair, both of medium size. considering the karyotype configuration observed in p. germaini, it is more likely that chromosomal fission involving the large metacentric pair resulted in two acrocentrics pairs, according to mit, increasing the chromosomal number from 2n=20 to 22. subsequent differential heterochromatin growth in the short arms of the acrocentric chromosomes seems to have originated a subtelocentric and a submetacentric pair observed in p. germaini. pheidole sp. (flavens group) revealed 2n=20; 2k=18m +2sm (fig 2b). the same karyotype was observed in pheidole dentigula smith, 1927 from florida, usa, also included in the flavens group (crozier, 1970). further studies in brazil will investigate karyotypic variations in this group and the understanding of chromosome evolution. brachymyrmex admotus mayr, 1887 showed 2n=18; 2k=16m+2sm (fig 2c). brachymyrmex is a monophyletic genus with 40 described species and taxonomically neglected due to small body size (3 mm), soft mesosoma, and superficially similar external morphology among species (ortiz-sepulveda et al., 2019). to date, only a single unidentified taxon of this genus from são paulo, brazil, was karyotyped (crozier, 1970), showing this genus was also overlooked cytogenetically. the karyotype of b. admotus is similar to brachymyrmex sp., despite that the latter species showed only metacentric chromosomes based in visual comparison (crozier, 1970). in this study, mensuration of chromosome arms was performed making chromosomal classification more accurate. considering the difficulties in species identification in brachymyrmex due to their complex morphology and their extremely small size, molecular cytogenetics can bring important insights in taxonomy as observed in other ant species (aguiar et al., 2017; micolino et al., 2019). camponotus (myrmothrix) atriceps, exhibited 2n=40; 2k=4sm+34st+2a (fig 2d). similar karyotypes were observed for individuals collected in the atlantic rainforest at ilhéus, ga teixeira, lac barros, dm lopes, hjac aguiar – cytogenetics of five ant taxa from the atlantic rainforest606 bahia (mariano et al., 2001) and in the brazilian cerrado (aguiar et al., 2017). although mariano et al. (2001) used a chromosomal classification different to the present study, it is possible to recognize the same chromosomal pairs in the two localities from the atlantic rainforest. it suggested karyotype stability of c. atriceps since several chromosomal polymorphisms were observed for different populations of camponotus (myrmothrix) spp. (aguiar et al., 2017). a secondary constriction was observed in the short arm of the second submetacentric pair of c. atriceps (fig 2d, arrow), which possibly corresponds to the nucleolus organizing region. this region coincides with the location of gc-rich bands observed for the same species from cerrado, and with ribosomal genes mapped for other camponotus spp. (aguiar et al., 2017). odontomachus haematodus presented 2n=44; 2k=8sm+ 18st+18t (fig 2e). the monophyletic genus odontomachus includes 74 species described (larabee et al., 2016; bolton, 2020), with 13 taxa studied cytogenetically (reviewed by santos et al., 2010; mariano et al., 2019). the karyotype formula varies among species, specifically, the number of acrocentric chromosomes, due to differential growth of heterochromatin chromosomal evolution of the genus (aguiar et al., 2020). although the karyotype formula is the same, variations were observed between the karyotype of o. haematodus from the atlantic rainforest (present study) and the amazon rainforest (aguiar et al., 2020). the former has the first submetacentric pair with almost twice the size of the other submetacentric chromosomes, while the latter karyotype has all submetacentric pairs with a similar size. duplications/deletions of chromosomal segments and other complex rearrangements may be involved in the origin of this difference. fig 1. map of the sampling localities of the ant taxa cytogenetically studied in this report from the atlantic rainforest. modified from dinerstein et al. (2017). sociobiology 67(4): 604-609 (december, 2020) 607 and by the programa de auxílio ao pesquisador – papesq/ unifap/2019. authors' contribution ga teixeira conceptualization, methodology, resources, investigation, data curation, visualization and writing lac barros conceptualization, methodology, resources, investigation, data curation, visualization, writing, supervision, project administration and funding acquisition dm lopes methodology, writing, visualization and funding acquisition hjac aguiar conceptualization, methodology, resources, investigation, visualization, writing, supervision, project administration and funding acquisition references aguiar, h.j.a.c., barros, l.a.c., alves, d.r., mariano, c.s.f., delabie, j.h.c. & pompolo, s.g. 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(2010). a cytogenetic approach to the study of neotropical odontomachus and anochetus ants (hymenoptera: formicidae). annales de la société entomologique de france, 103: 424-429. doi: 10.1603/an09101 silva, r.r., feitosa, r.s.m. & eberhardt, f. (2007). reduced ant diversity along a habitat regeneration gradient in the southern brazilian atlantic forest. forest ecology and management, 240: 61-69. doi: 10.1016/j.foreco teixeira, g.a., barros, l.a.c., lopes, d.m. & aguiar, h.j.a.c. (2020). cytogenetic variability in four species of gnamptogenys roger, 1863 (formicidae: ectatomminae) showing chromosomal polymorphisms, species complex, and cryptic species. protoplasma, 257: 549-560. doi: 10.1007/s00709-019-01451-6 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.1433sociobiology 65(2): 325-329 (june, 2018) a founder-controlled, social wasp assemblage, and a recent severe fall in numbers introduction the study began as a simple survey of the species of social wasps encountered at a convenient place. when it transpired that there were large differences in the species composition between the initial surveys, the investigation was continued. the high species richness of social wasps in the neotropics is well-known (richards, 1978). in the federal district of brasília 56 species have been recorded (raw, 2016). although they are predators, neotropical species often occur at relatively high densities (raw, 1998a, 1998b; silveira, 2002; elpino-campos et al., 2007; souza et al., 2014a; souza et al., 2014b). neotropical social wasps have preferences for particular habitats (raw, 1992, 1998b) and the study site was selected because it includes the three common natural vegetation forms of the region, and a garden. the wasps possess characteristics that make them suitable subjects for surveys of this nature. they fly slowly and almost all are diurnal so they are easily seen and collected. they abstract the 35 species of social wasps surveyed in an 8 ha plot in the cabeça de veado valley near brasilia (1979 and 1997) averaged 17.9 species per survey. from 1/3 to 1/2 of the species disappeared between consecutive surveys and only two were present in every survey. on average, 43% of the 40 species known to inhabit the valley were recorded on each survey. this high rate of turn-over demonstrates that the wasps comprised a “founder-controlled” assemblage. on two more surveys, nine species were recorded in 2010 and ten in 2015; numbers which reflect recent reports on the global trend of losses of social wasps. sociobiology an international journal on social insects a raw article history edited by gilberto m. m. santos, uefs, brazil received 14 february 2017 initial acceptance 16 february 2017 final acceptance 16 march 2017 publication date 09 july 2018 keywords species richness, neotropics, central brazil, hymenoptera, paper wasps, species losses. corresponding author anthony raw departamento de ciências biológicas universidade estadual de santa cruz cep 45650-662 ilhéus-ba, brazil. e-mail: anthonyraw2@gmail.com are social and philopatric, and an encounter with an individual is proof of the presence of a colony nearby. they occupy a nest continuously often for lengthy periods. their generation turnover is fast enough for sufficient individuals to be available to recolonise sites. each colony can be regarded as a subpopulation of a metapopulation and the death of a colony as the extinction of that subpopulation but, when there are colonies of several species close by, a vacated site can be recolonised. social wasps initiate new colonies by two processes. the members of polistes and mischocyttarus are independent founders in which a lone female begins a nest and her daughters remain to enlarge the colony. the colony lasts a few months and the largest ones comprise a few dozen adult females (pers. obs). in the second group (tribe epiponini) a swarm of dozens to hundreds of wasps leaves the natal nest and may fly a kilometre to find a new nest site (pers. obs). a mature colony is active for a few months to several years and comprises hundreds to thousands of wasps (pers. obs). evidence is emerging, some of it anecdotal, of dramatic losses of social wasps in many parts of the world, including departamento de zoologia, universidade de brasília, brasília-df, brazil research article wasps a raw – assemblage of neotropical paper wasps326 the neotropics; in french guiana (dejean et al., 2010; dejean et al., 2011), trinidad (c. k. starr pers. com.), são paulo state, brazil (e. giannotti pers. com.) and paraguay (b. r. garcete-barrett pers. com). while these losses might be reported as good news in the popular press, the consequences will be detrimental for farming and forestry. social wasps are important predators of innumerable insect pests (rabb & lawson, 1957; nakasuji et al., 1976; dowell & johnson, 1986; raw, 1988). the recent reports of losses prompted me to publish these results. study area and methods the study area comprises 8 hectares in the cabeça de veado valley (15º 52’ s; 47º52’ w), 12 km south of the centre of brasília. the area is described in detail in a previous study (raw, 1998a). three hectares of evergreen forest line a permanent stream, four are cerrado (see eiten, 1972), one hectare is forest margin and a ½ hectare garden lies next to a house. the area was subject to very little disturbance, except maintenance of the garden, and sometimes parts of the cerrado were burnt. the study area outside the garden is part of the brasília botanical garden, an area of 4,518 hectares, almost all under natural vegetation. the area was searched visually for wasps and their nests during ten surveys. baits and traps were not used. eight surveys were from 1979 to 1997 and two were in 2010 and 2015. in the first two (1979 and 1981) the area was searched for 60 and 40 hours. however, all the species recorded had been encountered during 16 and 18 hours so, thereafter, the area was searched for 20 hours each survey. these were conducted between 20 jan and 16 march which is towards the end of summer when social wasp populations are at their highest (pers. obs). it is often difficult to locate the wasps’ nests in dense vegetation and in tree tops, but many wasps hunt at ground level where they are easily encountered so the presence or absence of the adults was recorded and searches were not made specifically for the nests. however, colonies attached to the house were recorded during each survey. the results of each survey were compared with the next one using jaccard’s similarity indices calculated from the total number of species per survey (after real, 1999). results species composition a total of 37 species of social wasps were collected during the ten surveys. several of them were encountered elsewhere in the valley when they were not found in the study area. i found three additional species, but not in the study area during the survey period. polistes occipitalis ducke was collected in the study area in 1977, before the investigation began. polybia dimidiata (olivier) and mischocyttarus latior (fox) were collected in forest 250 m and 450 m away, but were never seen in the study area. thus, a total of 40 species are known to have inhabited the valley. by 2010, the numbers had dropped severely, with only ten species in 2010 and nine in 2015 (table 1). of the 14 recorded in these two surveys, m. cassununga, m. cerberus and polybia ignobilis had been found in all or almost all the previous surveys, but two other frequent species, p. occidentalis and p. sericea, were not seen in 2015. agelaia pallipes and polybia ruficeps had not been recorded previously in the study area, but both were present in 2010 and a. pallipes also in 2015. nests and habitats the wasps occupied three types of natural habitat. twothirds of the species (24 of 37) were restricted to one habitat in their nesting and foraging. these were 2 to forest, 16 to the forest margin and 6 to the open cerrado. none nested or hunted in all three habitats. the wasps nested in several places. most suspended their nests from branches and twigs (29 of 37). synoeca surinama and parachartergus fraternus attach their nests to tree trunks and thick boughs. agelaia pallipes, polybia flavifrons and p. ignobilis nest in cavities. seven species attached their nests to the undersides of leaves, but none was restricted to that surface. active colonies of 27 species were discovered. five (p. ignobilis, p. liliacea, p. rejecta, e. tatua and s. surinama) nested outside the study area and hunted within it. the longevity of individual colonies encountered during this investigation was not recorded, but it varies greatly among species, from three to six months in polistes, mischocyttarus and polybia occidentalis to more than ten years in epipona tatua (pers. obs). some of the species encountered on every survey had short-lived colonies that were continually replaced. the numbers of colonies of the three species which nested on the house increased and decreased together between 1979 and 1997 (figure 1). however, in 2010, the numbers had plummeted, though m. cerberus returned strongly in 2015. polistes satan was present in all the surveys except that of 2015. fig 1. numbers of colonies of mischocyttarus cerberus (b), m. cassununga (n) and polistes satan (s) attached to the house in the study area. sociobiology 65(2): 325-329 (june, 2018) 327 year recorded 1979 1981 1984 1987 1989 1991 1994 1997 2010 2015 number of occurrences loners mischocyttarus cassununga (von ihering) c c c c c c c c c c 10 mischocyttarus cerberus ducke c c c c c c c c c c 10 mischocyttarus drewseni de saussure w c c w 4 mischocyttarus goyanus zikán c c c 3 mischocyttarus lecointei (ducke) c 1 mischocyttarus marginatus (fox) c c c c w 5 mischocyttarus mattogrossoensis zikán w c c w 4 mischocyttarus rotundicollis (cameron) w w w 3 polistes billardieri f w w w w w w w 7 polistes cinerascens de saussure w w w w w 5 polistes davillae richards w w 2 polistes geminatus fox w w w w w 5 polistes satan bequaert c c c c c c c c c 9 polistes subsericeus de saussure w w w w 4 swarmers agelaia pallipes (olivier) w w 2 apoica pallens (f) w c w w w c 6 apoica thoracica du buysson w 1 brachygastra augusti (de saussure) c 1 brachygastra lecheguana (latreille) w c w 3 chartergellus communis richards c c w 3 epipona tatua (cuvier) w w 2 parachartergus fraternus (gribodo) w c c w w c 6 polybia chrysothorax (lichtenstein) c c 2 polybia emaciata lucas c c c 3 polybia fastidiosuscula de saussure w c c w c 5 polybia flavifrons smith w c 2 polybia ignobilis (haliday) w w w w w w w w 8 polybia liliacea (f) w w w 3 polybia occidentalis (olivier) c w c c w c c c w 9 polybia paulista von ihering w c c c c w 6 polybia rejecta (f) w 1 polybia ruficeps schrottky c 1 polybia sericea (olivier) w w w c w w w w w 9 protonectarina sylveirae (de saussure) c c w 3 protopolybia exigua (de saussure) c w c c c c w 7 pseudopolybia vespiceps (de saussure) c w 2 synoeca surinama (l) w w w w 4 total species per survey 19 17 16 17 20 17 17 19 10 9 jaccard’s similarity index 0.69 0.58 0.63 0.60 0.69 0.59 0.66 0.63 0.61 species present on previous survey 13 8 9 8 13 8 11 7 5 % similarity between consecutive surveys 0,68 0,47 0,56 0,47 0,65 0,47 0,65 0,37 0,50 table 1. presence of workers (w) and colonies (c) of social wasps in eight surveys in 8 hectares near brasilia. a raw – assemblage of neotropical paper wasps328 turnover the numbers of species per survey of the first eight surveys (1979-1997) were similar (16 to 20; mean 17.9 (sd 1.4)) (table 1) so they were analysed without the surveys of 2010 and 2015. on average, 43% of the 40 species known to inhabit the valley were recorded on each survey. the proportion of the species recorded in a survey which had been present in the previous one varied from 37% to 65%, while the values of jaccard’s similarity indices calculated for all the species in the surveys ranged from 0.58 to 0.69 (table 1). of the 30 species which were not present on all eight surveys, some missed an occasional survey, but others were absent for longer periods. protonectarina sylveirae and polybia emaciata were not recorded for 12 years. similarly, brachygastra lecheguana and synoeca surinama were not recorded for ten years. agelaia pallipes had been recorded 1½ km outside the study area in 1987 and 1989, but was not seen again in the valley until 2010. only two species were recorded on all ten surveys (table 1). the 14 independent-founder species were recorded more regularly than the 23 swarming species. indices of jaccard’s similarity were calculated for the two groups. those for the independent founders ranged from 0.58 to 0.69 (mean 0.50), while those for the swarmers ranged from 0.19 to 0.43 (mean 0.32). discussion species composition the total number of species recorded was substantial (71% of the total recorded for the federal district (raw, 2016)) so it is considered unlikely that species had been overlooked. furthermore, independent founders with their small, scattered colonies were recorded more regularly than the swarmers. variable factors no direct competition between any pair of species was detected, that is to say, the presence of one species did not coincide with the absence of another. indeed, there are examples to the contrary. two species of the nocturnal genus apoica were present in the same survey, and absent from most. nor was any successional tendency detected in the disappearance and appearance of species. nest sites might be a limiting factor for the three cavity nesters, but it is doubtfully so for the other species. a species might have disappeared and reappeared between consecutive surveys. conversely, a species might have been present only between surveys so would not be recorded. however, these possibilities would increase the rate of turn-over which is already considered to be high. the index of similarity of species between consecutive surveys ranged from ½ to 2/3. all the values of the similarity indices of jaccard were low (< 0.7) indicating a strong turnover. founder-controlled assemblage less than half of the species known to inhabit the valley were recorded on any survey. all the species had patchy dispersions and the locations of these patches continually change. this disappearance and reappearance of colonies is a major influence on the species composition at this site. swarms rarely travel more than a kilometre (pers. obs.) so all these species were likely present somewhere in the cabeça de veado valley during the periods when the wasps were not recorded. these findings demonstrate that it is an open system with attributes typical of a founder-controlled assemblage (after yodzis, 1978). the species occupy gaps as they become available and apparently almost any one may occupy any gap regardless of which species had vacated it and no species is dominant. the high diversity of species is maintained in a “competitive lottery” (sale, 1977) and the assemblage is maintained through the continuing births and deaths of colonies. hence, the number of species per survey was relatively constant, though the species composition changed markedly. recent losses of species presumably, many of these species continue to exist in the valley, but their numbers have dropped. ten species were recorded only in the earlier surveys, but it is not known if they had become locally extinct. all are widely dispersed in the region’s forests and open cerrado. conversely, chartergellus communis and polybia liliacea were present in three of the last four surveys and were not seen before that. both have established themselves in several parts of the federal district in the last 20 years (pers. obs). the numbers of the three species nesting on the house increased and decreased together between 1979 and 1997 so it is thought that they did not compete and that the environmental factors regulating their numbers affected all three similarly. absences are considered to be real disappearances as the nests are easily seen under the eaves. conversely, it is not known why the number of colonies of mischocyttarus cerberus recuperated so well. it is difficult to suggest an explanation for these losses. the whole of the surrounding land is a protected area and not subject to the use of agricultural chemicals. all the species involved range into regions with cooler or warmer climates than brasília so it is doubtful that the losses are related directly to a change of climate. increased predation was not seen. it might be the availability of prey. it could not have been a dearth of nest sites. sociobiology 65(2): 325-329 (june, 2018) 329 practical implications the results of this study have two practical implications. first, when conducting surveys to investigate the real losses of species it is important to distinguish between temporary and prolonged absences and that requires long-term studies (though maybe not for twenty or thirty years). secondly, the planning and management of areas for conservation should take into account that the survival of an assemblage depends on the protection of neighbouring habitats harbouring sub-populations. a founder-controlled assemblage can exist only when the reservoir of species in the surrounding area is high enough to facilitate recolonization (sale & douglas, 1984; lima et al., 1996). even with insects, these patchy dispersions can be large. the cabeça de veado valley is a triangle 6 km long and 5 km wide. it is likely that all the 40 species are involved in the lottery of a foundercontrolled assemblage throughout the valley. acknowledgements i thank mr. john n. landers whose garden comprised part of the study area and who often provided accommodation. dr alain dejean, université de toulouse; dr christopher k. starr, university of the west indies, trinidad and tobago; dr edilberto giannotti, universidade estadual paulista, rio claro, são paulo state and dr bolívar rafael garcete-barrett, universidad nacional de asunción, san lorenzo, paraguay kindly provided personal information on the losses of social wasps. the following colleagues provided invaluable comments on the manuscript: dr dejean; dr starr; dr martin cody, university of california in los angeles; dr roberto cavalcanti and dr john d. hay, universidade de brasília. the late professor o. w. richards very kindly encouraged my studies on neotropical social wasps and his support was unswerving. references dejean, a., j. m. carpenter, m. gibernau, m. leponce and b. corbara. 2010. nest relocation and high mortality rate in a neotropical social wasp: impact of an exceptionally rainy la niña year. comptes rendus de l’academie des science, biologie, 333: 35-40. dejean a., r. céréghino, j. m. carpenter, b. corbara, b. hérault, v. rossi, m. leponce, j. orivel and d. bonal. 2011. climate change impact on neotropical social wasps. plos one, 6 (11): 1-8. doi: 10. 1371/journal.pone.0027004 dowell, r. v. and m. johnson. 1986. polistes major (hymenoptera: vespidae) predation of the treehopper, umbonia crassicornis (homoptera: membracidae). pan pacific entomologist, 62: 150-152. eiten, g. 1972. the cerrado vegetation of brazil. botanical review, 38: 201-341. elpino-campos, a., k. del-claro and f. prezoto. 2007. diversity of social wasps (hymenoptera: vespidae) in cerrado fragments of uberlândia, minas gerais state, brazil. neotropical entomology, 36: 685-692. lima, m., p. a. marquet and f. m. jaksic. 1996. extinction and colonisation processes in subpopulations of five neotropical small mammal species. oecologia, 107: 197-203. nakasuji, f., h. yamanaka and k. kiritani. 1976. predation of larva of the tobaccocutworm spodoptera litura (lepidoptera:noctuidae) by polistes wasps. biological control, 44: 205-213. rabb, r. l. and f. r. lawson. 1957. some factors influencing the predation of polistes wasps on tobacco hornworm. journal of economic entomology, 50: 778-84. raw, a. 1988. social wasps (hymenoptera: vespidae) and insect pests of crops of the suruí and cinta larga indians in rondônia, brazil. the entomologist, 107: 104-109. raw, a. 1992. the forest: savanna margin and habitat selection by brazilian social wasps (hymenoptera: vespidae). in: the nature and dynamics of the forest-savanna boundary (eds. p. a. furley, j. a. ratter and j. proctor). chapman & hall, london, pp. 499-511. raw, a. 1998a. population densities and biomass of neotropical social wasps (hymenoptera, vespidae) related to colony size, hunting range and wasp size. revista brasileira de zoologia, 15: 815-822. raw, a. 1998b. social wasps (hymenoptera, vespidae) of the ilha de maracá. in maracá: the biodiversity and environment of an amazonian rainforest, [eds. j.a. ratter and w. milliken], john wiley & sons, chichester, england, pp. 307-321. raw, a. 2016. new records of social wasps around brasília (hymenoptera; vespidae; polistinae). sociobiology, 63: 1073-1075. real, r. 1999. tables of significant values of jaccard’s index of similarity. miscellania zoologica, 22: 29-40. richards, o. w. 1978. the social wasps of the americas. british museum (natural history), london, 580 pp. sale, p. f. 1977. maintenance of high of high diversity in coral reef fish communities. american naturalist, 111: 337-359. sale, p. f. and w. a. douglas. 1984. temporal variability in the community structure of fish on coral reef patches and the relation of community structure to coral reef structure. ecology, 65: 409-422. silveira, o. t. 2002. surveying neotropical social wasps. an evaluation of methods in the “ferreira penna” research station (ecfpn), in caxiuanã, pa, brazil (hym., vespidae, polistinae). papéis avulsos de zoologia, museu de zoologia da universidade de são paulo, 42: 299-323. souza, m. m., e. p. pires, a. elpino-campos and j. n. c. louda. 2014a. nesting of social wasps (hymenoptera: vespidae) in a riparian forest of rio das mortes in southeastern brazil. acta scientiarum. biological sciences, maringá, brazil, 36: 189-196. souza, m. m., e. p. pires and f. prezoto. 2014b. seasonal richness and composition of social wasps (hymenoptera, vespidae) in areas of cerrado biome in barroso, minas gerais, brazil. bioscience journal, 30: 539-545. yodzis, p. 1978. founder-controlled communities (pp. 28-38). in competition for space and the structure of ecological communities. springer-verlag, berlin and new york. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.283-288sociobiology 60(3): 283-288 (2013) food niche overlap among neotropical carpenter bees (hymenoptera: apidae: xylocopini) in an agricultural system dm carvalho, cml aguiar, gmm santos introduction most studies on bee niches have focused on the diversity of floral resources used and the partitioning of these resources (e.g., wilms et al., 1996; goulson & darvill, 2004; aguiar, 2003; aguiar et al., 2013; andena et al., 2012; figueiredo et al., 2013). however, guilds of flower visitors can also be studied from other perspectives, such as activity time (carvalho et al., 2010; santos et al., 2013). the partitioning of temporal niches may facilitate the coexistence of ecologically similar species (kronfeld-schor & dayan, 2003). the level of complementarity or redundancy in the niches of flower-visiting insects is an important mechanism underlying the relationship between biodiversity and ecosystem functioning (blüthgen et al., 2006; hoehn et al., 2008; santos et al., 2013). however, little empirical evidence exists about the level of complementarity among pollinators (hoehn et al., 2008). different species may have complementary niches and perform different functional roles, (santos et al., 2010), thereby presenting different realized niches (begon abstract in the present study, we used niche overlap analysis and a network approach to investigate the use of floral resources by carpenter bees (xylocopa spp.). we assessed the frequency of visit to different plant species and the activity time of carpenter bees in an agricultural system. six species of carpenter bees were collected visiting flowers. among the 48 interactions that were theoretically possible in the interaction network, only 19 were recorded (connectance = 39.58%). the temporal overlap between pairs of species measured by the shannon index (0 to 0.648) was lower than dietary overlap (0 to 0.967). the network analysis also showed that bees separated their niches more strongly in the temporal dimension (e = 0.72, p < 0.001) than in the dietary dimension (e = 0.55, p < 0.001). the levels of dietary and temporal overlap were strongly correlated with each other, as well as the time of highest frequency of visit coincided with the time of availability of resources by the most important plants (moringa oleifera, passiflora edulis, and solanum palinacanthum). the correlation between dietary and temporal overlap is biologically explained by the presence of plants that structure the system by exerting a strong influence not only on the plant choice by foraging bees, but also on the time of resource collection. sociobiology an international journal on social insects universidade estadual de feira de santana feira de santana, bahia, brazil article history edited by kleber del-claro, ufu, brazil received 19 june 2013 initial acceptance 30 july 2013 final acceptance 07 august 2013 key words carpenter bees, bee-plant network, trophic niches, temporal activity corresponding author gilberto m. m. santos departamento de ciências biológicas univ. estadual de feira de santana feira de santana, bahia, brazil 44036-900 e-mail gmms.uefs@gmail.com et al., 2006). niche complementarity requires some degree of trophic or temporal specialization, whereas niche redundancy is associated with high niche overlap and functional redundancy (blüthgen & klein, 2011). however, two species may present redundancy on a dimension (e.g., diet) and complementarity in another dimension (e.g., temporal patterns of resource use) of their trophic niches (santos et al., 2013). in the present study, we used niche overlap analysis and a network approach to investigate the use of floral resources by carpenter bees (xylocopa spp.). we assessed the frequency of visit to different plant species and the activity time of bees in an agricultural system. these bees are attractive candidates for crop pollination, because they have broad diet, long activity season, and are active under low illumination levels (keasar, 2010). indeed, several crops, such as passion fruits, tomato, and melons, can be favored by the pollination service rendered by carpenter bees (hogendoorn et al., 2000; sadeh et al., 2007; benevides et al., 2009; yamamoto et al., 2010; keasar, 2010; benevides et al., in press). research article bees dm carvalho, cml aguiar, gmm santos food niche overlap among neotropical carpenter bees 284 plant species carpenter bee species x. frontalis (x01) x. grisescens (x02) x. nigrocincta (x03) x. ordinaria (x04) xylocopa sp.1 (x05) x. suspecta (x06) anacardium occidentale l. (p01) 1 bixa orellana l. (p02) 5 1 cucurbita pepo l. (p03) 1 moringa oleifera lam. (p04) 23 3 4 1 7 passiflora edulis sims (p05) 51 2 1 4 portulaca oleracea l. (p06) 1 solanum palinacanthum dunal (p07) 3 1 1 2 solanum stipulaceum willd. ex roem.& schult. (p08) 1 material and methods study area we collected data on bee–plant interactions in the municipality of feira de santana, bahia, northeastern brazil (12º15’25”s, 38º57’54”w). the climate of the region is semiarid, with an average annual temperature of 23.5 °c and an average annual rainfall of 867 mm (mma 2009). the study was carried out at the bocaiúvas farm, that covers an area of 23.4 ha, where crops of fruits and vegetables dominate the landscape, including citrus sinensis l (orange), citrus limonum l. (lime), mangifera indica l. (mango), psidium guajava l. (guava), passiflora edulis sims (passion fruit), capsicum annuum l. (papikra), cucurbita sp. (pumpkin), and cucumis anguria l. (bur cucumber). all plants are cultivated with no chemical additives. sampling we used as a sampling unit each record of bee-plant interaction. we recorded flower-visiting bees, plants visited, visit time, temperature, relative air humidity, and luminosity each hour during each sampling. in order to characterize the guild of carpenter bees that forages on flowers in an agricultural system, we carried out monthly sampling from august 2011 to july 2012. before the first monthly sampling, we data analysis we used the importance index (ij) to evaluate the importance of each plant species for each bee species, as well as the importance of each bee species to each plant species. this index varies from 0 to 1, and it tends to 1 when a plant species (or a bee species) has many interactions in the community or a large number of exclusive interactions (murray, 2000). we measured the overlap in both trophic niche dimensions (dietary and temporal) for each pair of bee species with the schoener index (1986). this index (noih) varies from 0 to 1, and we considered the level of overlap low when the value found was lower than 0.3, moderate when the value was higher than 0.3 and lower than 0.5, and high when the value was higher than 0.5. following santos et al. (2013), we used a network approach to determine the degree of specialization in the diet and temporal activity of flower-visiting bees with the e index (araújo et al. 2008). we organized the data on bee species and activity time as adjacency matrices and represented them as bipartite graphs using the package bipartite 2.00 for r (dormann et al., 2009). we used linear regressions and spearman correlations to test for relationships between temperature, air humidity, bee richness, bee abundance, and degree of niche overlap in pairs of bee species. table 1. carpenter bee species and plants visited for floral resources in an agricultural system. selected a pathway crossing places rich in flowering plants. in each sampling session, two collectors captured carpenter bees visiting flowers, using entomological nets, along the same transect 1 km long. the sampling effort per specimen of plant in blossom was 5 min. we carried out sampling from 0600 to 1800, summing up 12 samples of 12 h, or 244 h of sampling per collector. all flowering plant species were sampled each hour, as collectors walked the entire transect once every hour. results we collected six species of carpenter bees (113 individuals) visiting flowers of eight plant species (table 1). in the interaction network we recorded 19 out of 48 theoretically possible bee-plant interactions (connectance = 39.58%). the low connectance of the bee-plant network is related to singletons, as four plant species were visited only by a single bee species, and one bee species visited only one plant species (fig. 1). moringa oleifera (p04), passiflora edulis (p05), and sociobiology 60(3): 283-288 (2013) 285 solanum palinacanthum (p07) stood out by their number of interactions with bees. these species received 91% of the bee visits. xylocopa frontalis (x01) was the most abundant bee (n = 83, 73%) and visited the highest richness of plant species (tables 1 and 2) carpenter bees visited flowers throughout the day, but with higher frequency of visits at three times (fig. 2). the first peak of individuals visiting flowers occurred between 0600 and 0800 (32% of the individuals), the second between 1100 and 1300 (41%), and the third at 1500 (17%). we compared niche overlap (dietary and temporal) for six xylocopa species (table 2). the values calculated for species with few individuals should be interpreted with caution, since they may not reflect the real local overlap in the use of the resources. the dietary overlap varied from 0 to 0.967. out of 15 pairs of species analyzed, three pairs showed low overlap table 2. richness of visited plants (spla), abundance of individuals in each bee species (nbees), and trophic niche overlap (noih, schoener index) between pairs of species of carpenter bees flower-visiting in an agricultural system. temporal niche overlap dimension (upper triangle) and dietary niche overlap dimension (lower triangle). spla nbees x. frontalis x. grisescens x. nigrocincta x. ordinaria xylocopa sp1 x. suspecta xylocopa frontalis (olivier, 1789) 5 83 0.108 0.611 0.325 0.145 0.568 xylocopa grisescens lepeletier, 1841 4 5 0.337 0.143 0.000 0.000 0.385 xylocopa nigrocincta smith, 1854 3 7 0.599 0.500 0.286 0.143 0.648 xylocopa ordinaria smith, 1874 3 3 0.369 0.000 0.429 0.333 0.308 xylocopa sp.1 1 1 0.277 0.500 0.571 0.000 0.154 xylocopa suspecta moure & camargo, 1988 3 13 0.621 0.500 0.967 0.462 0.538 0,00 0,25 0,50 p06 (01) p01 (01) p03 (01) p02 (06) p08 (01) p07 (07) p05 (58) p04 (38) 0,00 0,25 0,50 0,75 x05 (01) x03 (07) x06 (13) x04 (03) x02 (06) x01 (83) fig. 1 importance index (ij; murray) of the plant species that provide floral resources for carpenter bees (ij = 0.002 to 0.361) and of the flower-visiting bees (ij = 0.004 to 0.624). bee abundance is provided in parenthesis. plants and bees codes on table 1. dm carvalho, cml aguiar, gmm santos food niche overlap among neotropical carpenter bees 286 (noih < 30%), seven pairs showed intermediate overlap (30% < noih < 50%), and five pairs showed high overlap (noih > 50%). temporal overlap was lower than dietary overlap, varying from 0 to 0.648. out of 15 pairs of species analyzed in terms of temporal overlap, eight showed low overlap (noih < 30%), four pairs showed intermediate overlap (30% < noih < 50%), and only three pairs showed high overlap (noih > 50%; table 2). the highest value of overlap in the dietary niche (0.967) was found between x. nigrocincta and x. suspecta, which resulted primarily from the concentration of visits both in the flowers of m. oleifera and p. edulis. strongly in the temporal dimension (e = 0.72, p < 0.001) than in the dietary dimension (e = 0.55, p < 0.001; fig. 4). discussion among xylocopa bees, x. frontalis was the species with the highest number of interactions and with the broadest trophic niche. this species is known for its generalistic foraging behavior and for exploring a relatively broad diversity of floral resources (silva, 2009; yamamoto et al., 2012). however, an analysis of the pollen collected by female x. frontalis, x. grisescens, and other carpenter bee species revealed that, in spite of being generalistic, carpenter bees tend to explore more intensively a subset of the floral resources available, which contributes to the narrowing of their dietary niche and increases dietary overlap between species (silva, 2009). the three peaks of carpenter bee foraging on flowers observed in the present study and the high overlap in bee fig. 2 – number of individuals of carpenter bees (xylocopa spp) captured visiting flowers in an agricultural system at each sampling time. 0 5 10 15 20 25 30 06:00 07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 n um be r of in di vi du al s time other x. suspecta x. frontalis fig. 3 – correlation between dietary and temporal overlap among carpenter bees (xylocopa spp) (y = 0.063 + 0.4815*x; r = 0.5557; p = 0.0315) in an agricultural system. there was no significant relationship between bee abundance, bee species richness, plant species richness, temperature, and relative air humidity. however, we found a significant positive relationship between the level of temporal overlap and the level of dietary overlap (y = 0.063 + 0.4815*x; r = 0.56; p = 0.03) (fig. 3). the network analysis showed similar results; bees separated their niches more fig. 4 – graphs showing a larger weighted clustering coefficient in the bee-plant network than in the bee-time network. dark gray pentagons represent carpenter bees (xylocopa spp), light gray circles represent in graph a the plants visited, and in graph b the time of visit. lines represent links and their thickness is proportional to the number of observations. plants and bee codes on table 1. a b sociobiology 60(3): 283-288 (2013) 287 activity at these times seem to be related to the temporal availability of the floral resources of three important plants. in the morning, the foraging activity of bees was more intensive at early hours, a period that coincides with the opening of m. oleifera flowers, which occurs from 0300 on (jyothi et al., 1990) and of s. palinacanthum flowers, whose anthesis begins around 0600 (carvalho et al., 2001). the second peak was recorded around midday, which coincides with the anthesis and availability of the resources of p. edulis flowers at 1200 (benevides et al., 2009; siqueira et al., 2009). in the mid afternoon, there was an increase in the frequency of carpenter bees on flowers, which is consistent with the observations made by carvalho et al. (2001), who studied the visitors of s. palinacanthum. the availability of floral resources by p. edulis from midday on strongly influenced the overlap of carpenter bee niches, since this plant received the highest number of visitors. as in our study, benevides et al. (2009) recorded the highest frequency of bee visits to p. edulis flowers between 12:30 and 13:30 in passion fruit crops in southeastern brazil, in which x. frontalis and x. ordinaria were the most frequent carpenter bees. on the other hand, varassin et al. (2010) found a constant visit rate of x. frontalis to p. edulis flowers between 14:00 and 18:00, and concluded that the visit rate of this carpenter bee was not influenced by anthesis timing or nectar volume or concentration. at times of low visit frequency of xylocopa to the monitored plants, we presume that the females changed their foraging routes to explore other plants in the surroundings. likewise, jyothi et al. (1990) observed that the temporal activity of xylocopa on m. oleifera depended not only on the availability of nectar provided by this plant, but also on the availability of resources produced by other plants in the surroundings, which could be more attractive to foraging females at some times of the day. as pointed out by silva (2009) and yamamoto et al. (2012), in agricultural systems, xylocopa species interact with both cultivated and wild plants. in the present study, abiotic factors (temperature and relative humidity) did not affect the visit frequency of carpenter bees or the overlap in dietary and temporal niches. although there are several examples of a relationship between the foraging activity of bees and abiotic factors, there are also examples of the daily activity of bees explained by the temporal availability of floral resources. according to stone et al. (1999), the time of resource production by plants represents a window of opportunity for foraging bees and may also be a driver of bee activity. in the studied system, the levels of dietary and temporal overlap were strongly correlated with each other, as well as the time of highest visit frequency of bees coincided with the time of availability of resources by most important plants. the correlation between dietary and temporal overlap is biologically explained by the presence of plants that structure the system by exerting a strong influence not only on the plant choice by foraging bees (bee diets), but also on the time of resource collection. acknowledgments several colleagues contributed to the present study in different ways: j.j. resende helped us in fieldwork, maise silva identified bee species, efigenia melo identified plant species and marco a.r. mello gave invaluable suggestions to an early version of the manuscript. the brazilian council for scientific and technological development (cnpq) and the bahia research foundation (fapesb) funded the present study. cnpq granted g.m.m. santos and c.m.l. aguiar research productivity fellowships. references aguiar, c.m.l. 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(1999). windows of opportunity and temporal structuring of foraging activity in a desert solitary bee. ecol. entomol., 24: 208-221. varassin, i.g., ximenes, b.m.s., moreira, p.a., zanon, m.m.f., elbl, p., löwenberg-neto, p., & melo, g.a.r. (2012). produção de néctar e visitas por abelhas em duas es-produção de néctar e visitas por abelhas em duas espécies cultivadas de passiflora l. (passifloraceae). acta bot. brasilica, 26: 251-255. yamamoto, m., silva, c.i., augusto, s.c., barbosa, a.a.a. & oliveira, p.e. (2012). the role of bee diversity in pollina-the role of bee diversity in pollination and fruit set of yellow passion fruit (passiflora edulis forma flavicarpa, passifloraceae) crop in central brazil. api-apidologie, 43: 515-526, doi: 10.1007/s13592-012-0120-6. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1936sociobiology 64(4): 477-483 (december, 2017) polistes canadensis (linnaeus, 1758) (vespidae: polistinae) in the western amazon: a potential biological control agent introduction wasps are hymenoptera that present different behaviour, ranging from solitary species to social groups with female caste variations (prezoto et al., 2007). the wasps reveal an opportunistic characteristic: they return to places with a great supply of resources or food, in search of the optimization of the foraging and reduction of the search effort (raveret-richter, 2000). in general, wasps perform several ecological functions; a relevant point among these ecological functions is their role as natural enemies, since predatory wasps contribute to the regulation of insect populations and consequently to the reduction of insecticide use, with natural biological control being a significant contribution in abstract wasps of the genus polistes (vespidae: polistinae) are eusocial, considered valuable biological control agents. the objective of this work was to determine the resources collected by polistes canadensis wasps, evaluate their performance and importance as a natural enemy and possible agent of biological control in the brazilian amazon. between october 8th and november 20th, 2014, 20 evaluations were performed, totalizing 101 hours of observations of the foraging activity of an aggregation out in stage of development post-emergence with approximately 50 adult individuals distributed in 15 colonies. additionally, observations of the predatory activity of polistes canadensis on plutella xylostella on a small organic plantation of kale (brassica oleracea l. var. acephala dc), were also made. during the evaluations 1742 returns were recorded, 11.72% of them with prey, 3.10% with plant fiber, 16.76% with nectar, 45.17% with water and 23.25% without any visible load. all the preys identified were classified as lepidoptera, belonging to ten morphospecies. only one morphospecies was identified as spodoptera frugiperda, which was the most commonly resource used by the wasps in 37 % in immature feeding. only returns with nectar had statistically significant difference between the evaluated schedules. polistes canadensis wasps did not prey plutella xylostella caterpillars. the wasp aggregation studied was able to prey an average of 10.2 caterpillars per day, which demonstrates the potential of this species for the biological control of pests in the amazon region. sociobiology an international journal on social insects m montefusco1, fb gomes2, a somavilla1, c krug2 article history edited by gilberto m. m. santos, uefs, brazil received 10 august 2017 initial acceptance 03 october 2017 final acceptance 13 october 2017 publication date 27 december 2017 keywords foraging activity, resources, caterpillars, agricultural pests. corresponding author matheus montefusco instituto nacional de pesquisas da amazônia (inpa), programa de pósgraduação em entomologia av. andré araújo nº 2936, aleixo cep 69080-971 caixa-postal 2223 manaus-am, brasil. e-mail: matheus.montefusco10@gmail.com this process (gallo et al., 2002). they use a large source of resources present in the environment: water (used to cool the colony); plant fibers (construction and maintenance of the colony); nectar and pollen (a rich source of energy) and prey (used to feed the immature). polistes (vespidae: polistinae) is one of the most common genus among social wasps (carpenter, 1996). it is a cosmopolitan genus, with 222 valid species distributed in all biogeographical zones, except in antarctica, most of which occur in the tropics (carpenter, 1996; pickett et al., 2006). some authors pointed out the importance of polistes in the regulation of insect-plague populations of crops of economic importance and for the facility of manipulation and translocation of their colonies to artificial shelters (figueiredo 1 instituto nacional de pesquisas da amazônia (inpa), manaus-am, brazil 2 empresa brasileira de pesquisa agropecuária (embrapa), manaus-am, brazil research article wasps mailto:matheus.montefusco10@gmail.com m montefusco et al. – polistes canadensis: a potential biological control agent478 et al., 2006; elisei et al., 2010; prezoto, 1999; prezoto et al., 1994; giannotti et al., 1995; prezoto & machado, 1999a, 1999b; andrade & prezoto, 2001). the species polistes canadensis (linnaeus, 1758), is a social wasp found in the new world, with a wide distribution, occurring from the southern united states to southern brazil and argentina (carpenter, 1996). the nests are established by independent foundation, where only one or a few queens begin to construct the nest, oviposit and feed the immature forms (wenzel, 1998). they are able to obtain resources at 650 meters from the colony, but with radius of action of 125 meters, foraging in an area of approximately 49,000 m² (santos et al., 1994). this species is relatively non-aggressive, which allows translocate their nests easily (marques et al., 1992; marques, 1996). the pattern of foundations and dropouts of colonies of p. canadensis canadensis occurring all year round, presents an asynchronous pattern between nesting and environmental variables (masques et al., 1992; torres et al., 2009b). the social wasp p. canadensis presents several characteristics which qualifies it as important natural enemies of pest insects. studying aspects related to their foraging activity and the resources collected by them, may contribute to ecological and agricultural benefits. therefore, the objective with this work was to determine the resources collected by p. canadensis and to evaluate their performance and importance as a natural enemy and possible agent of biological control. material and methods the experiment was carried out at the experimental field of embrapa western amazon called caldeirão (fig 1), located at coordinates 03º 14’22 “and 03º15’47” south latitude and at 60º 13’02 “and 60º 13’50” west longitude, in the municipality of iranduba, amazonas, during the period from october 8 to november 20, 2014. were evaluated an aggregation of p. canadensis colonies with approximately 50 adult individuals distributed in 15 colonies (fig 2 a). besides, observations on an organic small plantation of kale (brassica oleracea l. var. acephala dc), were also realized. resources collected by polistes canadensis in total, 101 hours of observations of foraging activity of the aggregation were carried out in stage of development the colony post-emergence (jeanne, 1972). the method used in the evaluations was adapted from prezoto et al. (1994), where the identification of the material collected by the wasps was carried out by observing the behaviour presented by them when they returned to the nest: nectar collection, when there were trophallaxis, adult-adult or adult-larvae (fig 2b); water collection (fig 2c), when the liquid was deposited directly on the cell walls; the returns with wood pulp used in cells construction (fig 2d) and returns with prey used in feeding of the immatures (fig 2e, f and g) were identified by the observation of the resources in the mouthparts besides the slow flight. the evaluation of foraging activities occurred in 16 non-consecutive days. on each day, the foraging activity was observed and registered every 30 minutes, with intervals of the same time, during the hours of highest foraging activity of the colonies, between 10 am and 15:30 pm. the wasps that returned to the colony with some material between the jaws were intercepted with the use of entomological fig 1. location of the experimental field of caldeirão of embrapa western amazon, iranduba, amazonas, brazil. sociobiology 64(4): 477-483 (december, 2017) 479 net and the material that they carried was collected. the preys carried by the wasps were collected and fixed in 70% alcohol, inside microtubes. in the entomology laboratory of embrapa western amazonia the samples were screened using a stereomicroscope equipped with a camera and categorized in morphospecies, based on corporeal structures such as cephalic capsule, pseudopodia and seta, according to elisei et al. (2010). some data on atmosphere conditions such as mean temperature, relative air humidity, precipitation and wind speed were obtained at the weather station of the same experimental field. based in an empirical knowledge that p. canadensis could be a regulated agent of plutella xylostella (linnaeus, 1758), during the intervals between evaluations of foraging activity (every 30 minutes), some observations of this expected predatory activity were performed. two small kale plantation measuring 6m long and 1m wide were observed, each plantation had 20 plants in 2.8 liter pots, spaced 50 x 50cm, distant approximately 12m from the wasps colonies evaluated. the plantations were covered with voile cloth to ensure the fixation and spread of p. xylostella insects that were later released. the observations were made very close to the plants allowing the record of the predatory action of the wasps and their foraging behaviour. data analysis variance analysis (anova) and a tukey’s posttest (5% significance) were done in order to compare the data collected by the wasps in every 30 minutes per hour observed. the analyses were performed with r core team software (2017). results and discussion polistes canadensis foraging wasps presented homogenously return activity with resources practically at all times evaluated, with no peaks of returns (table 1). in total, 1742 returns of foraging wasps to the colonies were recorded during this study. of these returns, 45.17 % were returns with water, 11.72 % with prey, 3.10 % with vegetal fiber, 16.76 % with nectar and 23.25 %, returns were considered frustrated based on behavior during the arrival at the colony. following, these returns will be better evaluated individually. resources collected by polistes canadensis water: most of the recorded returns during the evaluations were identified as water 45.17 % (n = 787). assessments water prey fiber nectar frustaded 10:00-10:30 h 7.37±5.61 a 1.62±1.58 a 0.5±0.83 a 3.81±2.26 a 3.93±2.95 a 11:00-11:30 h 7.06±4.40 a 2.25±1.61 a 0.37±0.5 a 2.62±1.96 a 4.12±2.44 a 12:00-12:30 h 8.12±6.11 a 1.75±1.73 a 0.62±0.80 a 3.12±1.66 ab 3.62±2.5 a 13:00-13:30 h 5.87±5.09 a 1.81±1.47 a 0.25±0.44 a 1.81±2.00 ac 3.18±3.35 a 14:00-14:30 h 7.5±6.11 a 1.68±1.13 a 0.37±0.61 a 2.25±1.65 a 4.31±2.62 a 15:00-15:30 h 6.43±5.98 a 2.18±1.60 a 0.5±0.81 a 1.18±1.37 cd 2.93±2.37 a table 1. average and standard error of returns with water, prey, plant fiber, nectar and frustrated returns of the foragers of polistes canadensis. values followed by the same letter in the column do not differ from each other, by the tukey test at 5% significance. this resource was provided to the immature or divided between the wasps and placed inside the cells, mainly with immature ones, being also deposited in the back part of the colonies. the water is used to cool the colony and is collected by the foragers in large quantity, mainly in hours with higher temperature (akre, 1982; greene, 1991). the effort employed by p. canadensis in the collection was higher than compared to other species of polistes: p. simillimus, p. versicolor, p. lanio, in studies conducted on days with temperature between 22 and 35°c (eg: prezoto & machado 1999a; prezoto et al., 1994; elisei et al., 2010; giannotti et al., 1995). the large amount of water collected shows that the availability of this resource nearby the colonies is fundamental for the maintenance of the nest microclimate and colony equilibrium. preys: 11.72 % (n = 204) of worker wasps returns were recorded as returns with preys. the behavior presented by the wasps as soon as they arrived at the colony was to remain still while chewing the prey. after a few moments, the macerate was divided with other wasps, which later fed the larvae. this same behavior was recorded and described by torres et al. (2009a) for this same species of wasp. an average of 10.2 returns with prey per day was recorded, a similar value to this one was found by prezoto et al. (2006) for p. versicolor and prezoto et al. (1994) for p. simillimus, with averages of 11 and 16.9 returns with preys per day. these results demonstrate the potential of p. canadensis as a biological control agent, when compared to the previously mentioned species, as a controlling agent of herbivores of agricultural importance (elisei et al., 2010; prezoto & machado, 1999b). preys captured by p. canadensis: was possible to characterize 49 % (n = 100) of the prey captured by the size, color and body structures, for 51 % (n = 104) of the preys specimens, the identification was not possible due to the high level of maceration of the samples. all samples with possible identification were classified as immature of lepidoptera order, being categorized as belonging to 10 different morphospecies m montefusco et al. – polistes canadensis: a potential biological control agent480 fig 2. a – evaluated colonies of polistes canadensis.; bnectar collection, when there were trophallaxis adultlarvae.; cwater collection.; dgathering of vegetable fiber in the vicinity of the colonies.; e and f returns with prey used in feeding of the immatures.; gprey being shared. sociobiology 64(4): 477-483 (december, 2017) 481 (fig 3). only one morphospecies was identified, as spodoptera frugiperda (smith, 1797) (morphospecies 1), which was the most commonly resource used by the wasps in 37 % in immature feeding. caterpillars (lepidoptera) are the major food source to the immature of p. canadensis and other species of this genus (prezoto et al., 1994; giannotti et al., 1995; andrade & prezoto, 2001; torres et al., 2009a). in other studies showing the efficiency of polistes spp. as a predator, wasps were also related to the predation of large caterpillars, such as s. frugiperda predated by p. simillimus (prezoto & machado, 1999a) and chlosyne lacinia saundersii doubleday & hewitson, 1849 predated by p. versicolor (prezoto et al., 2006). considering the agricultural importance of the fall armyworm s. frugiperda and the resistance presented by the insect to both insecticides (omoto & diez-rodriguez, 2001) and transgenic crops (farias et al., 2014), a biological control agent stands out as a great option in the management this pest. from the results obtained with this study, it can be estimated that an aggregation of a colony containing 50 individuals is able to prey on an average of 10.2 caterpillars per day, which demonstrates the potential of p. canadensis as a biological control agent, including the possibility of agent control of the armyworm s. frugiperda. plant fiber: 3.10 % (n = 54) of the foraging wasps returns were characterized as returns with fibers of vegetal material. approximately 60 % (n = 34) of the foragers returning to the colony with fibers, divided this resource with other wasps, which later deposited it in the cells, mainly with immature ones. the objective of this activity and behavior is probably due to the need to increase the height of the cells, as a function of immature development, as proposed for this species by torres et al. (2009a) and for other species of the genus, such as p. lanio (giannotti et al., 1995) and p. versicolor (zara & balestieri, 2000). trophallaxis: 16.76 % (n = 292) of the returns were considered as returns with nectar to the colony followed by trophallaxis. the foraging wasps performed the behavior considered as trophallaxis when they returned to the colony, apparently with no visible recourse between mouthparts, but always followed, soon after, by trophallaxis with the immature or with the adult present at the colony. elisei et al. (2010) quantified this behavior associated with the nectar collection for p. versicolor in 51.6 % (934) and andrade & prezoto (2001) in 54.2 % for p. ferreri in the post-emergence period. yet prezoto et al. (1994) quantified a lower percentage to p. simillimus (28.5 %). and torres et al. (2009a), studying the division of labor of 15 colonies of p. canadensis under natural conditions in mundo novo, mato grosso do sul, showed that 35% of returns of wasps were with nectar. the percentage of returns with nectar followed by trophallaxis in this study was lower than the others mentioned previously, however, the differences of species, biome and experimental conditions prevent further generalizations or conclusions in this respect. frustrated returns: practically ¼ of the returns (23.25 %, n = 405) were considered frustrated, in other words, without any behavior or apparent material or prey at the return, performing an efficient foraging index of 76.75 %. similar value was found by prezoto et al. (1994) and elisei et al. (2010) for p. simillimus and p. versicolor, with 72.93 % and 80.13 % returns with resources. foraging activity the average of returns with prey at the different times did not present significant differences (table 1). foraging wasps collected prey at all times evaluated. despite the postemergence development stage of the 15 colonies assessed, fig 3. caterpillar morphospecies collected through flight interception of p. canadensis returning to the colony: aspodoptera frugiperda (n = 37) (morphospecies 1); bmorphospicies 2 (n = 7); cmorphospecies 3 (n = 3); dmorphospecies 4 (n = 6); emorphospecies 5 (n = 3); fmorphospecies 6 (n = 15); gmorphospecies 7 (n = 2); hmorphospecies 8 (n = 18); imorphospecies 9 (n = 4); j morphospecies 10 (n = 5). m montefusco et al. – polistes canadensis: a potential biological control agent482 plant fiber was the least used resource, approximately 3.1 %, with no difference between the 30-minute evaluations (table 1). return with nectar was the only resource with statistically significant difference between the evaluated schedules (p = 0.001). although there was a tendency to decrease the amount of nectar returns, a significant difference was observed only in the half hour evaluations performed from 12:00 to 12:30, 13:00 to 13:30 and 15:00 to 15:30 (table 1). the production and availability of floral resources, such as nectar, may be directly related to the number of floral visitors/pollinators visited during the day (waddington, 1983). therefore, the decrease in the amount of nectar returns may be related to the availability of this resource in the environment. water was the most used resource, also with no significant difference. according to torres et al. (2009a), p. canadensis collects more water at temperatures above 22°c. during our evaluations of the foraging activity, the temperature was higher than that, ranging from 23.85 to 35.29ºc, with an average of 26.59ºc and a standard deviation of 2.90ºc, therefore, requiring a large amount of water returns to maintain the thermal comfort of the colonies. there was also no significant difference in frustrated returns, showing that the resources used by p. canadensis are available throughout all the period studied. predatory activity of p. canadensis on p. xylostella during the evaluations of the foraging activity of p. canadensis in the small plantation of kale, the predation of p. xylostella caterpillar wasn’t observed. however, other social wasps of the genus polybia, which were collected and identified as p. bistriata, p. rejecta and p. sericea, preyed p. xylostella in small amounts. p. canadensis wasps were frequently observed above the kale plants seeking drops of water and predating another larger caterpillar species identified as pieridae family, which used kale as host. most of the p. canadensis wasps observed in kale presented the behavior of removing the cephalic capsule from the caterpillar. in addition, cephalic capsules of caterpillars were also found on kale leaves, possibly predated by this species or other social wasps. conclusion in several regions of the amazon, the advance of agriculture is undeniable and crescent, thought this activity presents great gaps in the knowledge of sustainable agricultural practices adapted to the local reality, especially in the phytosanitary area. for this reason, the biological control with predatory wasps is an excellent strategy for the amazon, in order to regulated the pest insect population, reducing the use of insecticides and consequently reducing the environmental impacts. the species p. canadensis is widely distributed in the new world, showed intense foraging activity and generalist habit of predation for the selection of caterpillars in the amazon region studied, predating caterpillars of several species and preferring larger caterpillars. due to the high index of captured prey and the intense foraging activity, this species should be considered as a potential species for the biological control of agricultural pests in crops in the amazon region. acknowledgements to fapeam and embrapa (03.13.00.012.00.00) for the financing of the project and to marinice cardoso for leading the bigger project. cnpq (150029/2017-9) for the posdoctoral scholarship (pdj) to as. references akre, d. 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(2000). behavioural catalogue of polistes versicolor olivier (vespidae: polistinae) postemergent colonies. naturalia, 25: 301-319. doi: 10.13102/sociobiology.v65i1.1784sociobiology 65(1): 24-30 (march, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 termites of iranian date palm orchards and their spatial and temporal distribution introduction several species of termites cause damage in date palm plantations in different regions of the world. twenty-five species of termites (isoptera: termitidae) cause damage to wood tissue of date palm trees (al-jboory, 2007). studies show that both temperature and rainfall affect the termite’s geographical distribution. that’s why termites are generally spread between 45 and 50 degrees north and south latitude. therefore, most species of termites are found in tropical and subtropical regions (sands, 1992). iran has variety of climates. southern and southeastern of iran have desert climate based on emberger classification and their ecological conditions are similar to the arabian peninsula and southwestern pakistan (yousof, 2010). khuzestan province in the southwest is one of iran’s warmest provinces. this province has suitable climate for the growth and development of termites. termite abstract this study was conducted during 2013-2014 to determine the dominant species and behavior of termites foraging in date palm orchards in khuzestan province, iran. starting in early march, the ‘cluster random sampling’ method was used within sixtyfive sampling plots. termite species were sampled by breaking open mud tubing and collecting different castes, then bringing them to the laboratory for identification using the scientific keys. glass microscope slides of different body parts, especially the labium, maxillae, palps, mandibles, labrum, and clypeus. the simpson diversity index was used to determine the level of dominance. results showed three species of termites, microcerotermes diversus silvestri, microcerotermes buettikeri chhotani and bose and amitermes vilis hagen, all family termitidae, are active in khuzestan date palm plantations. microcerotermes diversus was dominant with a correlation coefficient 0.997 and 8.87, respectively. seasonal population fluctuations of workers, nymphs, soldiers and winged castes had several peaks over the years. according to kriging map spatial distribution, four geographic severity groupings for m. diversus can be considered. these groups include low, medium, and high risk, and hotspot infestation geographic regions, with severity indices of 2.3 to 3.96, 3.97 to 4.82, 4.83 to 5.68, and 5.96 to 6.53, respectively. sociobiology an international journal on social insects m latifian1, b rad1, b habibpour2 article history edited by paulo f. cristaldo, ufs, brazil received 06 june 2017 initial acceptance 28 july 2017 final acceptance 13 august 2017 publication date 30 march 2018 keywords date palm, iranian termites, temporal distribution, spatial distribution. corresponding author masoud latifian horticulture research institute date palm and tropical fruit research center, agricultural research education and extension organization (areeo) ahvaz, iran. e-mail: masoud_latifian@yahoo.com diversity of this province is affected by termite fauna in nearby geographic areas such as persian gulf and oman sea countries including iraq, saudi arabia, kuwait, oman, bahrain, qatar, and the united arab emirates (pearce, 1997). termite distribution has been studied in iran, iraq, and the arabian peninsula. there are similar species in these countries as are found in khuzestan provence (badawiet al., 1987; el-shafie, 2012). microcerotermes diversus silvestri and psammotermes rajasthanicus roonwal and bose have been reported from all areas with date palm plantations of iran and chabahar, respectively (ghauryfaret al. 2005).termites from the genera coptotermes, psammotermes, bellicostitermes, and anacanthotermes ochraceus burmeister have been reported from mauritania and libya, respectively (pearce, 1997). odontotermes nilensis emerson, odontotermes smeathmani fuller, and odontotermes obesus rambur have been reported in date palm orchards in northern sudan. amitermes desertorum 1 agricultural research, education and extension organization (areeo),horticulture research institute, date palm and tropical fruit research center, ahvaz, iran 2 department of plant protection, college of agriculture, shahid chamran university of ahvaz, ahvaz, iran research article termites sociobiology 65(1): 24-30 (march, 2018) special issue 25 desneux and psammotermes hybostoma desneux from egypt, o. obesus from india, and m. diversus from saudi arabia and iraq have been reported in date palm orchards (engel & krishna, 2004). according to other studies, caste densities of different species of termites fluctuate during the year depending on needs of for different castes, and also due to environmental conditions (ghauryfar, 2005). spatial and temporal knowledge of the pest’s population dynamics are essential to development of ipm programs in each region over time. so that, the pest management professionals can be applied ipm offers as quickly as possible and with low cost (rad & latifian, 2005). the objective of this research was to determine the dominant date palm termite species and their spatial and temporal distribution of in date palm orchards of khuzestan province, iran, based on viewpoints of integrated pest management. materials and methods location sampling time. sampling of date palm termites was done from march when air temperature increased in date palm orchards. the ‘regional’ or ‘cluster’ random sampling method was applied (latifian & solymannejadian, 2002). this sampling method was selected because the overall date palm sampling ‘society’ was too large. the geographical distribution of society members was such that sampling was not possible from all date palms due to resource constraints. therefore, sampling societies were plotted, and a 1.0-ha date palm orchard was randomly selected within each society plot. according to previous studies, the number of necessary 1.0-hasampling plots was 65. a date palm garden was randomly selected and marked in each plot, then sampling was done at specific time intervals throughout the season (latifian and solymannejadian, 2002; latifian & zare, 2004). fifteen date palm trees were selected randomly in each 1.0-ha orchard and severity of termite infestations were estimated (table 1).the infestation index in each date palm orchard was estimated by using equation 1: in equation1, a, b, c, and d were the number of trees with infestation severity 1, 2, 3 and 4, respectively. the frond mid rib or petiole is triangular in cross section with two lateral angles and one dorsal (fig 1). dead old fronds are not shed naturally, but remain attached to the mother palm and are usually removed by farmers during pruning (nixon & wedding, 1956). the symptoms of the injuries were recorded as a biological index for pest activity level. all eight locations annotated on the date palm tree in figure 1 were evaluated for termite injury, with damage severity ratings based on codes provided in table 1. the different termite species castes were sampled by destruction of muddy channels on date palm trees. the samplings were done on base of petioles on the trunks of date palm because the maximum of channels termites were observed there. then collected termites were transmitted into a container containing 80% ethanol by using a finer brush and requirements specification such as the name and date of collections were recorded on them (longan & el bakri, 1990). in addition, seasonal population fluctuations of different castes and their frequency relative to the total population were evaluated. also, the dominant termite species were studied by similar sampling methods in date palm orchards located in areas within longitudes 48°34’ to 48°39’ east, and latitudes 31°14’ to 31°19’ north, every 15 days during the year. identification method some glass microscope slides were prepared for different parts of the termite body including mandibles, antennae, and labium. termites were dissected using a subtle needle and then boiled in 10% koh solution in water bath for 5 minutes. then fig 1. diagram showing the morphology of date palm: (1) aerial roots, (2) trunk or stem, (3) fruit bunch, (4) leaf or frond, (5) leaflet, (6) crown, (7) scars at frond base or petiole, and (8) basal offshoot (source: oihabi, 1991). infestation severity selective code infestation % none 0 0 low 1 1-25 medium 2 26-50 high 3 51-75 hot spot 4 76-100 table 1. estimation of termite infestation severity. infestation index = a + 2b + 3c + 4d 15 m latifian, b rad, b habibpour – termites of iranian date palm orchards26 slides were autoclaved for one week at 50°c. termite parts were measured by using a microscope with an ocular micrometer, and compared using identification keys (longan & el bakri, 1990; strewart & zalucki, 2006; habibpouret al., 2010). dominant species determination. the correlations between total termite populations and the relative density of each species were calculated. for this purpose, the total and individual termite species population density were estimated in each area by sampling of 15 date palm trees as utilizing previous methods (varely & gradwell, 1963). equation 2: x = x1 + x2 + …. + xn in equation 2, x= total termite species population density, xn= individual termite species population density. the species with greater correlation coefficient was designated as the dominant species in the region. simpson diversity index was used for determining dominance. d is calculated by equation 3 in an unlimited society (zhang, 2016). equation 3: 2pid σ= distribution models geostatistical methods were used for simulating distribution models. geostatistical method is based on the spatial variables theory (story & congalton 1994; wright et al., 2002). spatial correlation between samples can be described as a mathematical model known as the variogram (ellbsuryet al., 1998). if it is assumed that the total number of each pair of samples n (h) located at the distance h, then: equation 4: is called ‘semi-variance’ in the above equation. the values of semi-variance are plotted on the vertical axis for different distances to select the best models. the obtained curves drawn by this method known as variograms (journel et al., 1978). some variogram models (eg., gaussian and spherical) had limited threshold levels, and others such as linear models did not. this means that variogram values were increased by increasing distance between samples (h) but not to a fixed extent (katheriene, 2001). the distance after which semi-variance is constant is known as range of effects and is shown by r. the effect range is interval more than it; samples are independent and not affected to each other. this parameter determines the characteristic correlation and allowable interval sampling distance, and therefore the lag distance at which the semi-variogram (or semivariogram component) reaches the ‘sill’ value. presumably, autocorrelation is essentially zero beyond the range. most of variograms don’t show rapid changes at very short distances, so much semi-variance is not zero at the base of avariogram curve. so, in theory the semi-variogram value at the origin should be zero. if it is significantly different from zero for distance very close to zero, then this semi-variogram value is referred to as the ‘nugget’. the nugget represents variability at distances smaller than the typical sample spacing, including measurement error (goovaets, 1997). the variogram model parameters can be used to estimate the distribution of insects. all interpolation algorithms estimate the value at a given location as a weighted sum of data values at surrounding locations. almost all assign weights according to functions that give a decreasing weight with increasing separation distance. kriging is a commonly used method of interpolation (prediction) for spatial data. the data are a set of observations of some variable(s) of interest, with some spatial correlation present (katheriene, 2001). results and discussion termite species dominance. three species of termites were collected from date palm orchards of khuzestan province in iran that all of them were from termitinae (termitidae). these species were microcerotermes diversus silvestri, microcerotermes buettikeri chhotani and bose, and amitermes vilis hagen (blumberg 2008; carpenter & elmer, 1978; yousof, 2010). correlation coefficients between the relative abundance of each species with a total population of termite’s species and their dominance index were calculated based on equations 2 and 3 in each studied area that the results showed in figure 2. according to figure 2, m. diversus with correlation ∑ = +−= )( 1 2)]()([ )(2 1 )(ˆ hn i hxizxiz hn hγ )(ˆ hγ fig 2. correlation between the relative densities of each species with the total density. coefficients and dominant index species 0.997 and 8.87 was introduced as the dominant species. other species m. buettikeri and a. vilis were the second and third priority respectively. twenty-two species of termites were reported in date palmrich countries in persian gulf littoral (el-shafie, 2012). one or more species have been dominant despite the several reported species that active in each region. heterotermes aethiopicus sjostedt is the have maximum frequency species in united arab emirates (kaakeh, 2006). twelve species, 6 genera and 3 families were reported from date palms orchards of saudi arabia but termites of the genus microcerotemes were have maximum frequency (faragalla & al qhtani, 2013). more than 45 percent of infections were related to m. diversus in date palm orchards of iraq (ali, 2007). sociobiology 65(1): 24-30 (march, 2018) special issue 27 seasonal population fluctuation of dominant species. fluctuations in populations of workers had two periods abundance during the year. the first period started from late march and continued to late may, and its peak was in late april. the second period started from late june and continued to early april of next year, and its peak was in late december. nymph population also has four peak periods over the years. the first period lasted from late may to early august and had its peak in late july. the second period lasted from early august to early october and the peak was in late september. the third period started from early october to late november and its peak was in early november. the fourth period started from late october to late november and its peak was at early december (fig 3). soldiers had the lowest population density in their colonies. soldier number peaks corresponded with the peak of reproductive caste. swarmer population fluctuations had four peak periods over the years. the first peak period started from early june to late september. the second period continued from late october to early december and peaked in late november. the third period started from early december, peaked in late december, and continued to early january. the fourth period started from early january, peaked in late january, and continued through late february (fig 3). the dominant species castes including alates, nymphes, soldier and worker had different fluctuations over the years in the two studied areas. the worker caste was more frequent in most cases. but the highest percentage of the population fig 3. seasonal variation of population density of various castes of dominant species termite in khuzestan date palm orchards. m latifian, b rad, b habibpour – termites of iranian date palm orchards28 is over 90% of total population in the spring. abundance of some castes including alates, nymphes and soldier decreased to near 0% during the year. the frequency percent of nymphs and alates decreased to less than 5% at the beginning of spring and during the summer, respectively (fig 4). geographical distribution. geographical distributions of three dominant species in khuzestan date palm orchards are shown in figure 5. results showed that m. diversus had the greatest maximum range fig 4. percent of the population abundance of castes dominant termitespecies in khuzestan date palm orchards. of distribution in the most date palm gardens in khuzestan province. microcerotermes buettikeri is distributed in the south and southwest of khuzestan province, including abadan, khorramshahr, shadegan, sosangerd, and azadegan counties. amitermes vitis had limited distribution in abadan and arvandkenar counties. the variogram of distribution m. diversus severity as dominant species were calculated based on spherical, exponential, linear, and gaussian to sill models. based on results (table 2), spherical model of severity distribution was the more suitable fit than other models. the nugget effect of the sampling was 0.82 for geographic distances, which was a less effect than sampling intervals. variogram effective range was 53.5 kilometers. this distance means that there is little correlation between the termite damage severity data when sampling intervals were more than this threshold level. r2nuggetsill (c0+c)e. rangemodel 0.850.221.1953.5spherical 0.450.00061.1724.7exponential 0.410.691.2278.9linear 0.290.291.1942.7linear to sill 0.280.341.1872.57gaussian table 2. distribution variogram models of date palm termite m. diversus severity. fig 5. geographical distribution of termite species indate palm orchards of khuzestan province, iran. green =m. diversus, blue=m. buettikeri, orange=a. vitis this parameter showed the relationship between the density of date palm trees and injury severity by termites dates indicated in each area. sill was 1.19, which indicates the aerial coverage ratio of date palm orchard areas by m. diversus. variograms showed that there was a suitable trend for termite injury severity of this termite at the regional and local levels. the nugget effect was 0.2, which indicates that termite damage estimation error was extremely low by this model. based on the model, four categories of termite injury severity were considered for regional control management planning in date palms orchards in khuzestan province. sociobiology 65(1): 24-30 (march, 2018) special issue 29 the first pathosystem included low-risk areas, where injury indices varied between 2.3 to 3.96. termite injury to date palms was tolerable in this region. the second pathosystem included medium-risk areas where injury indices varied from 3.97 to 4.82. the termite injury can be reduced to a tolerable level by good horticultural management in this region. the third pathosystem included high-risk areas where injury indices varied from 4.83 to 5.68. chemical control measures are necessary in these regions. the fourth pathosystem included the main foci injury areas 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(1963). the interpretation of insect population change. proc-ceylon. the american association for the advancement of science, 18: 471-477. wright. r. j., t. a. devries, l. j. young, k. j. jarvi, & r. c. seymout. (2002). geostatistical analysis of smallscale distribution of european corn borer (coleoptera: chrisomelidae) larvae and damage in whorl stage corn. environmental entomology, 31: 160-167. yousof, d. e. (2010). insect and mite pest species and some insect natural enemies on date palm in the northern state, sudan. msc. thesis, sudan academy of sciences, khartoum, sudan. zhang, z. (2016). estimation of simpson’s indices, in statistical implications of turing’s formula. john wiley and sons, inc., hoboken, nj, usa. doi: 10.1002/9781119237150.ch2 doi: 10.13102/sociobiology.v61i1.82-87sociobiology 61(1): 82-87 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 comparative evaluation of three chitin synthesis inhibitor termite baits using multiple bioassay designs bk gautam, g henderson introduction present termite control methods mainly include application of liquid termiticides and baiting systems. subterranean termite baiting systems exploit the termites’ foraging behavior and their food transfer system (trophallaxis) to reduce or eliminate the colony population from an area (french, 1991; su et al., 1995). for a typical bait to succeed, foraging termites must find the bait station, recruit more termites, consume sufficient amount of bait matrix containing a toxicant, carry the toxicant back to the colony and transfer a sufficient dose to the rest of the colony members by trophallaxis (su et al., 1995; grace et al., 1996; su & scheffrahn, 1996; grace & su, 2001). it is suggested that bait treatments have advantages over alternative termite control strategies both by affecting a greater foraging area and by reducing the amount of toxicants placed in the environment (su & lees, 2009). the interconnected nests and galleries of a subterranean termite colony often spread over dozens of meters in the field. an excavation conducted by king and spink (1969) revealed that underabstract use of chitin synthesis inhibitors has revolutionized the potential impact of termite baiting systems. several chitin synthesis inhibitors have been used or tested against subterranean termites. we evaluated the effect of lufenuron on bait matrix consumption and mortality of coptotermes formosanus and compared it with 2 other chitin synthesis inhibitors presently used for termite control: diflubenzuron and noviflumuron. laboratory no-choice and multi-chamber bioassay designs were employed. at the end of 6 weeks, in both the no-choice and multi-chamber tests, mortality was significantly higher in all the chitin synthesis inhibitor treatments as compared to the controls; however, lufenuron treatment had significantly higher mortality than the other chitin synthesis inhibitors. multi-chamber tests suggested no sign of feeding deterrence with any of the chitin synthesis inhibitors at the concentrations tested. consumption of lufenuron cardboard or noviflumuron bait matrix was similar to that of control cardboard in the no-choice tests. we conclude that, based on the overall bait consumption and mortality data, lufenuron was at least as effective as noviflumuron and diflubenzuron. sociobiology an international journal on social insects department of entomology, louisiana state university, baton rouge, la, usa. article history edited by evandro n silva, uefs, brazil received 22 july 2013 initial acceptance 26 august 2013 final acceptance 08 octuber 2013 keywords benzoylphenylurea, termite baits, coptotermes formosanus, termite control corresponding author gregg henderson 404 life sciences building, department of entomology, louisiana state university baton rouge, la, 70803, usa e-mail: grhenderson@agcenter.lsu.edu ground tunneling system of coptotermes formosanus shiraki extended over 60 m and covered an area of over 0.58 ha. su and scheffrahn (1988) estimated 1.4 to 6.8 million foraging individuals in one colony of c. formosanus. due to the huge population size and extensive foraging range of subterranean termite colony, a non-repellent and slow acting control agent has the best potential to impact the majority of individuals in a colony (su, 1994; su, 2005). chitin synthesis inhibitors (csi) fall within the benzoylphenylurea group and are slow acting insect growth regulators (igr) that induce malformation of cuticle and significant reduction of chitin synthesis (van eck, 1979; merzendorfer et al., 2012). inhibition of chitin synthesis disrupts the molting process in insects leading to their death. csi also prevents normal formation of peritrophic membrane (zimmermann & peters, 1987); as a result, the insects become more susceptible to infection by microorganisms such as nuclear polyhedrosis virus (arakawa et al., 2002). several chitin synthesis inhibitors such as diflubenzuron, hexaflumuron, noviflumuron, lufenuron, and novaluron have been used or tested with various degree of success in termite baiting systems (su, 1994; research article termites sociobiology 61(1): 82-87 (march, 2014) 83 cabrera and thoms, 2006; vahabzadeh et al., 2007; lewis & forschler, 2010; osbrink et al., 2011). there are quite a few studies, both laboratory and field, about the noviflumuron based termite bait (though not the durable bait we tested), however, very limited published studies have evaluated the lufenuron and diflubenzuron based termite bait. lufenuron based termite bait was developed by syngenta about a decade ago but is still not commercially available for termite control. this could possibly be because of the conflicting reports of the effect of lufenuron on subterranean termites (see for example, su & scheffrahn, 1996; lovelady et al., 2008). the objective of this study was to evaluate the effect of lufenuron on bait matrix consumption and mortality of c. formosanus and compare it with 2 other csis: diflubenzuron and noviflumuron in the laboratory using two different bioassay designs. we believe that a comparative study in the lab using different bioassay designs would help to determine the efficacy of these chemicals against subterranean termites. materials and methods test materials test materials included three chitin synthesis inhibitor bait materials and untreated cardboard as controls. lufenuron treated cardboard (company code: nb 3401-91; 0.15% lufenuron) and control cardboard were received from fmc corporation (philadelphia, pa). they also provided samples of diflubenzuron bait (advance® compressed termite bait ii, basf corporation, st. louis, mo; 0.25% diflubenzuron) and noviflumuron bait (recruit® hd termite bait, dow agrosciences llc, indianapolis, in; 0.5% noviflumuron). termites formosan subterranean termites were collected in june 2012 from brechtel park, new orleans, louisiana using milkcrate trap methods as described in gautam and henderson (2011). in brief, the trap consists of a milkcrate filled with pine 44 wood (pinus sp.) sticks arranged in a lattice structure. the crate is buried in the ground and checked after 1-2 months. when infested with ~10,000 termites or more, the crate is retrieved, brought back to the laboratory and maintained in a trash can by adding water whenever necessary. the termite groups used in the experiment were collected from such laboratory stocks. termites from two colonies (separated by >500m) were used. no-choice tests no-choice tests were conducted in individual petri dishes (100 × 15 mm) each containing 50 g of autoclaved sand added with 6 ml of deionized water (12% moisture wt/wt). a pre-weighed cardboard piece or bait material (the lufenuron treated cardboard and untreated control cardboard were cut into pieces with separate scissors, whereas the diflubenzuron and noviflumuron baits were cut with a power operated band saw) was placed on moist sand surface in the dish. the cardboard or bait materials were pressed gently to the moist sand. one hundred termites (90 undifferentiated workers of at least the third instar and 10 soldiers) were released in each dish and the dishes were sealed with parafilm® to retain moisture. there were 6 replications for each treatment (3/termite colony group) and control. the dishes were then placed in an incubator maintained at 27°c. moisture content of the dishes was monitored regularly. termite survival count was made at 6 weeks when the experiment was ended. the cardboard and bait materials were cleaned of debris, dried and weighed to determine the consumption. consumption of diflubenzuron bait was not recorded as it was difficult to quantify due to its being spread throughout the dish by the termites and mixed with sand. multi-chamber tests to test how additional food sources may affect bait toxicity, an especially designed 16-chambered test arena with a slight modification from gautam et al. (2013) was used. the arena consisted of one center chamber (8.5 cm diam × 3.4 cm, pioneer plastics©, north dixon, ky) connected to 5 first peripheral chambers (5.08 cm diam × 3.63 cm, pioneer plastics©, north dixon, ky) with tygon tubing (5 cm long, 0.64 cm inside diameter, watts co., north andover, ma). the first peripheral containers again connected to 10 second peripheral containers with the same length tubing making two peripheries of the chambers around the center chamber. the side chambers in the first periphery were connected to each other with 10 cm tubing and similarly the side chambers in the second periphery were also connected (fig. 1). this test arena represented multiple feeding sites as might occur in a typical subterranean termite infested area. the center chamber contained 60 g of autoclaved sand and the peripheral chambers contained 30 g each. deionized water was added to each chamber to make the sand moisture content ~15% (wt/wt). the center chamber was the termite release chamber and contained nothing except the moist sand whereas the peripheral chambers contained 3 bait pieces of either lufenuron, diflubenzuron or noviflumuron in 3 randomly selected chambers and the remaining 12 chambers contained pinewood pieces pre-soaked in water for 1 h. after 1 d of arena set up, 300 termites (90% undifferentiated workers of at least the 3rd instar and 10% soldiers) were introduced in the center chamber. the lids were put back and the arenas were placed on a laboratory bench at 27 ± 1.5ºc. there were 4 replications for each treatment and control. control arenas contained no cardboard or bait, instead wood pieces were placed in all 15 peripheral chambers. bk gautam, g henderson chitin synthesis inhibitors against termites84 the test arenas were monitored regularly for moisture and water was added when necessary. live termites were counted at 6 weeks when the experiment ended, and wood and bait material consumption was recorded as in the no-choice tests. in each test arena, consumption of all 12 wood blocks or all 3 bait materials was added to calculate the total consumption. in controls, consumption of all 15 wood blocks was determined. unlike no-choice tests where consumption of diflubenzuron bait was not quantified, a best effort attempt of consumption was quantified in the multi-chamber tests. unconsumed diflubenzuron bait was carefully separated from sand and scrapped off the container, then dried and weighed to determine consumption. statistical analysis data analysis was done using proc mixed model in sas 9.3. data were transformed using either arcsine of the square root method (mortality data) or log transformation method (consumption data) when necessary, especially to improve the normality. post anova comparisons were made using tukey’s hsd and significance level was determined when α < 0.05. results and discussion no-choice tests there was no significant difference in consumption among lufenuron, noviflumuron and control baits (f = 1.18; df = 2, 15; p = 0.334) (fig. 2). csis had a significant impact on termite mortality (f = 17.53; df = 3, 20; p < 0.0001). all three csis caused significantly higher mortality as compared to the control. when compared among the three csis, lufenuron caused significantly higher mortality (87%) than either diflubenzuron (55%) or noviflumuron (65%) at 6 weeks (fig. 3). multi-chamber tests among the 3 csis, bait matrix consumption was significantly different (f = 44.54; df = 2, 9; p < 0.0001). consumption of diflubenzuron bait was significantly higher than noviflumuron bait or lufenuron bait and the consumption of noviflumuron bait was significantly higher than the lufenuron bait (fig. 4). similarly, wood consumption was significantly impacted by the csi baits (f = 247.19; df = 3, 10; p < 0.0001). wood consumption was significantly lower in all the csi tests at 6 weeks (range: 120-333 mg) compared to the controls (4190 mg). consumption of wood was not significantly different among the csi baits (fig. 5). as in no-choice tests, termite mortality was significantly impacted by the csi baits in multi-chamber tests (f = 21.25; df = 3, 10; p < 0.0001) and the mortality varied depending on the type of csi. approximately 100% mortality was observed in lufenuron tests at 6 weeks, which was significantly higher than the mortality in diflubenzuron or noviflumuron tests. there was no significant difference in mortality between diflubenzuron and noviflumuron (fig. 6). the results showed that the 3 csis tested induced significant mortality of c. formosanus foragers with lufenuron causing the highest percentage mortality in 6-week test. it is interesting to note that the relatively low concentration of fig. 1. image of a laboratory test arena consisting of multiple feeding chambers connected with tubings. fig. 2. consumption (mean ± sem) of 2 chitin synthesis inhibitor baits and control cardboard in no-choice tests at 6 weeks after exposure. same letters above the bar indicate not significantly different from each other. lufe = lufenuron, novi = noviflumuron. fig. 3. percentage mortality (mean ± sem) of c. formosanus exposed to 3 different baits and control in no-choice tests at 6 weeks after exposure. different letters above the bars indicate significantly different from each other. lufe = lufenuron, diflu = diflubenzuron, novi = noviflumuron. sociobiology 61(1): 82-87 (march, 2014) 85 4000 ppm or 8000 ppm lufenuron in 6 weeks after exposure. contrary to the reports that lufenuron treatment elicited feeding deterrence at >1000 ppm for c. formosanus and >50 ppm for r. flavipes (su & scheffrahn, 1996), the present multi-chamber tests indicate that lufenuron does not cause noticeable feeding deterrence to c. formosanus at 1500 ppm. feeding deterrence was not reported by vahabzadeh et al. (2007) at any concentration (0.1-1000 ppm) tested. in fact, the authors mentioned that lufenuron was highly acceptable to r. flavipes and was the most palatable of the 4 chemicals evaluated: lufenuron, diflubenzuron, hexaflumuron and triflumuron. similarly, lovelady et al. (2008) and haverty et al. (2010) stated that r. flavipes and r. hesperus readily consumed the cardboard bait matrix loaded at a rate of 1500 ppm lufenuron. noviflumuron, a csi which replaced hexaflumuron used in recruit iii termite bait (sentricon system, dow agrosciences), is a more extensively studied chemical. dow agroscience scientists reported that this chemical is non-deterrent at up to 10,000 ppm on filter paper to r. flavipes (karr et al., 2004) and successful results in suppressing the populations of subterranean termites by others has also been reported (su 2005; cabrera & thoms, 2006; thoms et al., 2009). fig. 5. consumption (mean ± sem) of wood in treated and control arenas in multi-chamber tests at 6 weeks after exposure. different letters above the bars indicate significantly different from each other. lufe = lufenuron, diflu = diflubenzuron, novi = noviflumuron. lufenuron (1500 ppm) inflicted a significantly higher mortality than the higher concentrations of either diflubenzuron (2500 ppm) or noviflumuron (5000 ppm) in 6 weeks. these results are consistent with the findings by vahabzadeh et al. (2007) who reported that lufenuron caused significantly higher mortality than diflubenzuron in 6 weeks for reticulitermes flavipes (kollar). rojas and ramos (2004) found significantly lower queen fecundity in the lufenuron treatments but not in the diflubenzuron or hexaflumuron treatments when compared with the control. in 6 weeks, only lufenuron treatment caused ~100% mortality of c. formosanus individuals in multi-chamber tests suggesting a faster knock-down of lufenuron. the multi-chamber test, where only 3 of 16 chambers were provisioned with bait matrix, was particularly designed to dilute the baiting effect which may occur in the field. we observed that there was no noticeable impact on termite mortality due to the presence of other food sources. there are successful field results of lufenuron termite bait developed by syngenta (greensboro, nc), using a cardboard matrix containing 1500 ppm lufenuron (lovelady et al. 2008, haverty et al. 2010). lovelady et al. (2008), in their review paper, reported that all of the 4 baited r. flavipes colony sites in columbus, oh were eliminated of termites within 3.5-10.5 months of lufenuron bait placement. the authors further reported that the lufenuron bait successfully eliminated subterranean termites from 78 out of ~100 infested structures in a large multi-site study covering all of the major termite regions across the us (22 states). likewise, haverty et al. (2010) reported the cessation of termite activity within 70 days of the lufenuron bait placement in all the 6 baited colony sites containing 21 colonies of r. hesperus banks in placerville, ca. successful elimination of an aerial colony of r. flavipes from a six-story apartment building has also been reported using 1500 ppm lufenuron bait (bowen & kard 2012). wang et al. (2013) reported that a combination of a lower concentration (1000 ppm) of lufenuron and opportunistic pathogens such as pseudomonas aeruginosa (schroeter) migula caused higher percentage mortality of c. formosanus in a relatively short period. su and scheffrahn (1996), however, reported only ~60% mortality of c. formosanus individuals in the laboratory with fig. 6. percentage mortality (mean ± sem) of c. formosanus exposed to 3 different baits and control in multi-chamber tests at 6 weeks after exposure. different letters above the bars indicate significantly different from each other. lufe = lufenuron, diflu = diflubenzuron, novi = noviflumuron. fig. 4. consumption (mean ± sem) of 3 chitin synthesis inhibitor baits in multi-chamber tests at 6 weeks after exposure. different letters above bars indicate significantly different from each other. lufe = lufenuron, diflu = diflubenzuron, novi = noviflumuron. bk gautam, g henderson chitin synthesis inhibitors against termites86 relatively low mortality caused by diflubenzuron in both the no-choice and multi-chamber tests in 6 weeks indicated a slow action of this chemical similar to that of noviflumuron. osbrink et al. (2011) reported that field tests of bait matrix containing 1000 ppm diflubenzuron had no noticeable impact on c. formosanus or r. flavipes populations when checked regularly for 3 years after bait placement. we are not sure, however, if the lower concentration of diflubenzuron used in their study was the reason for not achieving a significant mortality. however, other studies have also reported not so promising results with diflubenzuron (green et al., 2008; su & scheffrahn; 1993). although rojas and ramos (2001) reported that diflubenzuron caused 100% mortality of c. formosanus in 9 weeks after exposure in the laboratory tests, their field testing was less impressive (rojas et al., 2008). nevertheless, the present results demonstrated that lufenuron is an effective csi against c. formosanus and is relatively fast acting. acknowledgements we thank louisiana department of agriculture and forestry and fmc corporation for partial funding and material support in this study. we thank two other anonymous referees for their comments and criticisms on the manuscript. this manuscript is approved for publication by the director of the louisiana agricultural experiment station as manuscript number 2013-234-9630. references arakawa, t., furuta, y., miyazawa, m. & kato, m. 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(1987). fine structure and permeability of peritrophic membranes of calliphora erythrocephala (meigen) (insecta: diptera) after inhibition of chitin and protein synthesis. comparative biochemistry and physiology b, 86: 353–360. 435 how do scale insects settle into the nests of plant-ants on macaranga myrmecophytes? dispersal by wind and selection by plant-ants by chihiro handa1,2, shouhei ueda3, hirotaka tanaka4, takao itino5 & takao itioka1 abstract this report elucidates the process of settlement by coccus scale insects into crematogaster plant-ant nests formed inside the hollow stems of a myrmecophytic species, macaranga bancana, in a tropical rain forest. we collected wafting scale insect nymphs from the canopy using sticky traps and characterized the dna sequence of the trapped nymphs. in addition, we experimentally introduced first-instar nymphs of both symbiotic and nonsymbiotic scale insects to m. bancana seedlings with newly formed plant-ant colonies. nymphs of symbiotic species were generally carried by ants into their nests within a few minutes of introduction. most nymphs of nonsymbiotic species were thrown to the ground by ants. our results suggest that in crematogaster–macaranga myrmecophytism, symbiotic coccids disperse by wind onto host plant seedlings at the nymphal stage, and plant-ants actively carry the nymphs landing on seedlings into their nests in discrimination from nonsymbiotic scale insects. key words: ant-plant, ant–hemipteran interaction, mutualism, southeast asian tropical rain forest, borneo introduction mutualisms between ants and honeydew-producing hemipterans are sometimes so developed and intensive that both partners have evolved extensive 1graduate school of human and environmental studies, kyoto university, sakyo, kyoto 606-8501, japan 2corresponding author: e-mail: c.h.chihiro@gmail.com 3institute of mountain science, shinshu university, asahi 3-1-1, matsumoto, nagano 390-8621, japan 4entomological laboratory, faculty of agriculture, university of the ryukyus, okinawa 903-0213, japan 5department of biology, faculty of science, shinshu university, asahi 3-1-1, matsumoto, nagano 3908621, japan 436 sociobiolog y vol. 59, no. 2, 2012 coadaptations (way 1963; buckley 1987). myrmecophilous hemipterans appear to adapt to associations with ants by modifying their behaviors, body structures, and life cycles (way 1963; buckley 1987; gullan 1997; gullan & kosztarab 1997). hemipterans that are frequently associated with ants tend to have poorly developed structures for movement and defense against natural enemies (way 1963; buckley 1987). meanwhile, partner ants sometimes have behaviors adapted to the hemipterans. for example, some ants have been reported to not only tend, but also transport, their hemipteran symbionts (hölldobler & wilson 1990). in some species, workers carry symbiotic hemipterans to the appropriate part of the host plants (way 1963; maschwitz & hänel 1985; hölldobler & wilson 1990; gullan 1997). in some other species, foundress queens carry symbiotic hemipterans during the nuptial flight and colony foundation (hölldobler & wilson 1990; klein et al. 1992; gullan 1997; gaume et al. 2000; johnson et al. 2001). these transport behaviors are thought to play an important role in ensuring the foundress queens maintain symbioses with hemipterans that can provide the ants with a nutritious food resource in the form of honeydew. ant–hemipteran mutualisms are observed in ant nests in some myrmecophytes, plants that provide their partner ants (plant-ants) with nesting spaces (domatia) (ward 1991; gullan 1997). in a few cases of mutualism between hemipterans and “plant-ants,” i.e., in ant species symbiotic with a myrmecophyte, the foundress queens transport the hemipteran symbionts during the nuptial flight or colony foundation (klein et al. 1992; gaume et al. 2000). however, how most hemipteran species colonize the nests of their partner plant-ants remains unknown. macaranga myrmecophytes (euphorbiaceae) harbor scale insects of the genus coccus (coccidae) inside hollow stems that are occupied by partner plant-ants (heckroth et al. 1998; ueda et al. 2008, 2010). some macaranga species depend on plant-ants for anti-herbivore defense, and they are thought to be unable to survive without symbiotic plant-ants (fiala et al. 1989, 1994; itioka et al. 2000; heil et al. 2001). in addition, symbiotic coccids are considered to benefit the performance of plant-ants (heckroth et al. 1999; handa & itioka 2011). however, like many symbiotic hemipterans, how coccids disperse and settle into newly founded colonies of their partner plant-ants is not fully known. coccids have never been previously observed being carried by 437 handa, c. et al. —settlement of plant-ant nests by scale insects alate foundress queens (fiala & maschwitz 1990; maschwitz et al. 1996, and handa & itioka personal observation of >30 alate plant-ant queens founding new colonies). in addition, coccids have rarely been observed in early-stage nests of plant-ant colonies, even in several macaranga myrmecophyte species that almost always harbor symbiotic coccids (fiala & maschwitz 1990; heckroth et al. 1998). in this study, we aimed to elucidate the process of settlement by coccids into plant-ant nests on macaranga myrmecophytic species. many species of scale insects in widespread taxa are known to be wind-dispersed as first-instar nymphs (washburn & washburn 1984; gullan & kosztarab 1997). therefore, we hypothesized that coccids inhabiting plant-ant nests on macaranga myrmecophytes might disperse by wind. to test this hypothesis, we used sticky traps in the forest canopy to catch small airborne insects such as dispersing symbiotic coccids of macaranga myrmecophytes. next, we experimentally introduced first-instar nymphs of both symbiotic and nonsymbiotic scale insect species onto the surface of macaranga seedlings and observed the behavior of plant-ant workers in response to each. materials and methods study area and macaranga myrmecophytes the study was carried out in lambir hills national park, sarawak, malaysia (4°2’ n, 113°50’ e; 50–200 m above sea level), which is mainly primary lowland mixed dipterocarp forest. the uppermost canopy layer ranges from 30 to 40 m, with emergent trees penetrating the layer to heights of >70 m (kato et al. 1995). rainfall and temperature do not follow clear seasonal patterns (ashton & hall 1992; kato et al. 1995; roubik et al. 2005). more than 16 species of macaranga plants, including at least 10 species of myrmecophytes that can reach 20 m in height (davies et al. 1998; davies 2001), are found around the study site (itioka 2005). the stems of most macaranga myrmecophytes species become hollow when seedlings reach approximately 10–20 cm in height, allowing an alate foundress queen of crematogaster ant species to settle into the stem and produce workers (fiala & maschwitz 1990). workers then make exit holes on the exterior surface of stems. coccids have rarely been observed inside the nests of plant-ant colonies in the early stage of foundation just after the workers begin to emerge on the 438 sociobiolog y vol. 59, no. 2, 2012 surface of seedlings (handa & itioka, personal observations on >200 seedlings at this stage). first-instar “crawling” nymphs of the superfamily coccoidea disperse over relatively long distances by wind or walking. older nymphs and adults, except for alate males, tend to stay at a site or move only short distances (miller & kosztarab 1979; gullan & kosztarab 1997). partnerships between macaranga myrmecophytes and plant-ants are remarkably species-specific (fiala et al. 1999; itino et al. 2001). however, partnerships between plant-ants and coccids are not highly species-specific, although several coccus spp. exclusively inhabit ant nests in macaranga myrmecophytes (heckroth et al. 1998; ueda et al. 2008, 2010). capture of wafting coccids in january 2008, we selected nine trees of four macaranga myrmecophytic species (one m. lamellata, one m. trachyphylla, two m. winkleri, and five m. beccariana), which ranged from 3 to 10 m in height, at the crane site (4 ha, 200 × 200 m) where a crane provided access to the canopy (ozanne et al. 2003; roubik et al. 2005). to capture small wafting insects, we strung three 6 × 25 cm2 sheets coated on one side with adhesive (shimada co., higashiomi, japan) from three lateral branches of each tree. we collected all 27 adhesive sheets after 7 days. we collected 27 suspected coccid insects from eight sheets that were hung from one m. trachyphylla, one m. winkleri, and three m. beccariana trees. dna sequencing and phylogenetic analyses we extracted dna from a whole body of each of the 27 wafting insects collected using the dneasy blood & tissue kit (qiagen, hilden, germany). a partial cytochrome oxidase i (coi) gene (521 bp) of mitochondrial dna genome was amplified by polymerase chain reaction (pcr) and sequenced. methods of pcr, dna purification, and sequencing followed ueda et al. (2008). we obtained 16 sequences among 27. in the other 11 samples dna fragments could not be amplified using pcr. we inferred a molecular phylogeny by adding the sequences to the dna alignment of coi (dataset 2) in ueda et al. (2010), which is thought to cover almost all species of symbiotic coccids associated with macaranga myrmecophytes. maximum likelihood (ml) analysis was performed with phyml version 2.4.4 (guindon & gascuel 2003). the best-fitting substitution model 439 handa, c. et al. —settlement of plant-ant nests by scale insects was selected for each data set based on hierarchical likelihood ratio tests (hlrt, huelsenbeck & rannala 1997) using modeltest version 3.7 (posada & crandall 1998). the gtr+i+g substitution model was used as selected by hlrt in both data sets. clade support was assessed with 1000 bootstrap pseudoreplications. in addition, bayesian posterior probabilities and maximum parsimony (mp) bootstrap support were obtained using mrbayes version 3.1.2 (huelsenbeck & ronquist 2001) and paup* 4.0b10 (swofford 2002), respectively. for more details on phylogenetic methods, see ueda et al. (2008). introduction of symbiotic and nonsymbiotic scale insects to plant-ants we haphazardly selected one m. bancana tree that was approximately 10 m high and growing in a forest gap. we cut off and dissected a few lateral branches of the tree and collected crawling coccid nymphs from under sessile mature adult coccids in hollow stems. we also collected crawling nymphs of nonsymbiotic scale insect species from five m. bancana trees that had no plant-ant workers on the surface. plant-ant workers on these saplings were thought to have disappeared or become inactive due to accidental causes such as damage by falling trees, large litterfalls, and floods. we found three species of nonsymbiotic scale insects (coccus sp. nr. celatus, pseudococcus sp. 1, and iceryini sp. 1) on leaves and twigs of m. bancana trees. we collected an additional scale insect species (iceryini sp. 2) from the leaves of one mature non-myrmecophyte m. gigantea tree. we observed these four species of scale insects being tended by opportunistic (non-plant-ant) ant species, but they were never found in plant-ant nests. we collected m. bancana seedlings of 10 to 30 cm height from the forest and planted them in pots in a meshed nursery. we selected 45 vigorous seedlings that had active ant colonies. plant-ants were seen walking on the plant surface and several exit holes occurred near the ant nest. the plant-ant activities and the density of exit holes were clearly higher around the apical part, compared to the other parts. we introduced one crawling nymph of either a symbiotic or nonsymbiotic scale insect onto one of the 45 seedlings. for each trial, a crawling nymph was placed on the apical part of the stem surface within 3 cm of an exit hole. we 440 sociobiolog y vol. 59, no. 2, 2012 observed the behavior of the nymph and the behavioral responses by plantants. each seedling was used for one to five introduction trials. within 3 min after introduction, most nymphs were contacted by ant workers, and the others entered the nests without any contact with ants. the ants’ behaviors after contacting introduced nymphs were categorized into two types: “carrying into nests,” in which plant-ants carried nymphs into nests with their mandibles, and “removal,” in which they dropped nymphs off the plant. we observed all exit holes of a target seedling for 3 min after a nymph entered or was carried into a nest. in addition, for 10 individuals of symbiotic species and two individuals of nonsymbiotic species, we checked the ground for nymphs for 15 min after the nymphs entered a nest. overall, we obtained observational data for 32 symbiotic coccid nymphs and 73 nonsymbiotic nymphs. results dna characterization of wafting scale insects all of the sequences obtained from the 16 individuals constituted one haplotype. the monophyly of the clade including these obtained sequences and those belonging to l6 from ueda et al. (2010), which consists of macaranga-inhabiting coccus scale insects, was strongly supported by all of the three methods: ml bootstrapping (100%), bayesian posterior probability (100%), and mp bootstrapping (100%). plant-ants’ responses to experimentally introduced scale insect nymphs of the 32 introduced symbiotic coccid nymphs, 30 were directly contacted by plant-ants. within 30 s after the first contact of nymphs, plant-ants always grasped nymphs in their mandibles and carried them into hollow stems, except for one case in which a plant-ant dropped the nymph down (table 1). the other two nymphs walked into nests were not recognized by the plant-ants and entered nests on their own after searching around exit holes. one of these was observed being grasped by an ant just after it entered an exit hole. the proportion of symbiotic nymphs taken into nests was significantly greater than nymphs removed from plants (table 1; binominal test, p < 0.001). regardless 441 handa, c. et al. —settlement of plant-ant nests by scale insects of the mode of entry, all symbiotic nymphs stayed inside for at least 3 min and none of the 10 target nymphs were found on the ground 15 min later. of the 73 nonsymbiotic scale insects introduced, 71 were removed and the other two were carried into nests. for each of the four nonsymbiotic species, the proportion of removed nymphs was significantly higher than that of nymphs carried into nests (table 1; binominal test, coccus sp. nr. celatus: p < 0.001; pseudococcus sp. 1: p < 0.001; iceryini sp. 1: p = 0.003; iceryini sp. 2: p < 0.001). seventy percent of the removed nymphs were dropped off the plant within 10 s after their first contact with plant-ants. the others were carried by plant-ants from 10 s to 7 min before eventually being dropped from the plant. one nymph of pseudococcus sp. 1 and one nymph of iceryini sp. 1 were carried into nests and stayed inside for more than 3 min, but were then found on the ground within 15 min. discussion because no case of foundress queens of plant-ants on macaranga myrmecophytes carrying their symbiont coccids to new nests had been reported, the symbiont coccids were assumed to disperse independently of their partner plant-ants (fiala & maschwitz 1990; maschwitz et al. 1996). however, the dispersal of the symbiont coccids has not been empirically studied. this study presents some evidence on details of the dispersal of the symbiont coccids. we collected symbiotic coccid nymphs in the canopy dispersing by wind to host plant seedlings, where plant-ants develop new colonies. first-instar table 1. behavioral responses of the plant-ants to experimentally introduced first-instar nymphs of symbiotic and nonsymbiotic scale insects on seedlings of macaranga bancana and the significance probability (p) of binomial tests on the null hypothesis that the two types of behaviors might be randomly adopted. responses of plant-ants carrying into nests removal p symbiotic coccids 29 1 <0.001 nonsymbiotic scale insects coccus sp. nr. celatus 0 22 <0.001 pseudococcus sp. 1 1 22 <0.001 iceryini sp. 1 1 12 0.003 iceryini sp. 2 0 15 <0.001 442 sociobiolog y vol. 59, no. 2, 2012 coccid nymphs are difficult to identify to species morphologically. dna sequencing compared with a molecular phylogeny (ueda et al. 2010) suggested that many of the trapped coccids were in one of the clades known to be symbionts of plant-ants on macaranga myrmecophytes. crawlers of these symbiotic coccids have no physical adaptations for flight, so they are believed to disperse passively by wind. our experimental introductions of crawling nymphs suggest that in crematogaster–macaranga myrmecophytism, plant-ants actively carry winddispersed nymphs of symbiotic coccids into their nests and clearly discriminate against morphologically similar nonsymbiotic nymphs. crematogaster plant-ants are known to exclude almost all extraneous materials, including those that would be food for the majority of other ants, by carrying them in their mandibles and throwing them off their host plants (fiala & maschwitz 1990; hashimoto et al. 1997). this habit of plant-ants was confirmed in our experiments; most of the introduced nymphs of non-symbiont scale insects were removed by plant-ants soon after first contact. this indicates that symbiont coccids are among the few organisms not excluded by plant-ants. symbiont coccid nymphs also seem to be able to enter plant-ant nests by walking into them after alighting on a seedling. in our observations, none of the symbiont coccid nymphs that entered ant nests by either method were excluded by plant-ants, whereas all of the non-symbiont scale insect nymphs were excluded even if they were carried into nests. thus, plant-ant workers in the macaranga myrmecophytism appear to actively accept nymphs of only symbiotic coccid species. some symbiotic scale insects have been suggested to enter the domatia of myrmecophytes without any help from plant-ants (wheeler 1921; bequaert 1922; moog et al. 2005). to the best of our knowledge, no previous report has described plant-ants actively carrying symbiotic hemipterans into their nests. the active transportation of symbiotic coccids by the plant-ants inhabiting macaranga myrmecophytes suggests they may have a stronger dependence on their symbiotic coccids than in many other ant–hemipteran interactions, except for ones in which foundress queens transport symbiotic scale insects (klein et al. 1992; gullan 1997; gaume et al. 2000). positive effects of symbiotic coccids on the growth of plant-ant colonies in macaranga 443 handa, c. et al. —settlement of plant-ant nests by scale insects myrmecophytism (handa & itioka 2011) may be associated with the plantants’ heavy dependence on coccids. the limited range of symbiotic hemipteran species of macaranga plant-ants (heckroth et al. 1998; ueda et al. 2008, 2010) can no doubt be maintained by such rejecting behaviors as observed in our system. selection of scale insects by plant-ants may be adjusted in the evolutionary process on the basis not only of food intake from the honeydew or meat of scale insects but also of the total effects, including herbivory damage caused by the scale insects on the host plants. acknowledgments our study was conducted in accordance with the memorandum of understanding signed between the sarawak forestry corporation and the japan research consortium for tropical forests in sarawak in november 2005. we thank j. j. kendawang, l. chong, and other staff members of the forest research centre, sarawak, and forest department of sarawak for providing access to the study site as well as kind assistance. we are also grateful to t. nakashizuka, m. ichikawa, n. yamamura, and s. sakai for supporting our research activities and a. yoneyama for assistance in sampling. this work was supported by grants-in-aid from the japan society for the promotion of science ( jsps) to t. i. 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oxford university press, oxford, 335–352. washburn, j.o. & l. washburn 1984. active aerial dispersal of minute wingless arthropods: exploitation of boundary-layer velocity gradients. science 223: 1088–1089. way, m.j. 1963. mutualism between ants and honeydew-producing homoptera. annual review of entomolog y 8: 307–344. wheeler, w.m. 1921. a study of some social beetles in british guiana and of their relations to the ant-plant tachigalia. zoologica 3: 35–126. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.355-361sociobiology 60(4): 355-361 (2013) molecular cloning, characterization and expression analysis of calreticulin gene in the ant polyrhachis vicina roger (hymenoptera: formicidae) lp liu, l dang, gs xi, f wang introduction calreticulin (crt) is located both inside and outside of the endoplasmic reticulum, and also distributed on the cell surface (ostwald et al., 1974). crt belongs to a multifunctional calcium-binding protein, which can regulate calcium balance, assist the protein folding, processing and presenting the antigen. usually, the gene of crt composition and the amino acid sequence are highly conservative. many studies show that the crt protein may play a critical role in the maintenance of cell functions (lee et al., 2001; lynch et al., 2006). crt was initially purified from hare sarcoplasmic reticulum of muscle cells by ostwald and maclennan in 1974. however, crt gene’s cdna sequence was cloned from the liver of mice by smith and koch in 1989. currently, crt gene’s cdna sequence was cloned from vertebrates, invertebrates and higher plants. at the same time, the molecular structure and related functions of crt were studied. in insects, crt was detected as an encapsulative protein. crt may be relevant to the identification of foreign objects in abstract calreticulin (crt) as a ubiquitous and highly conserved calcium-binding protein that exists in endoplasmic reticulum (er), which possesses a variety of biological functions in the regulation of cell calcium homeostasis, molecular chaperoning and innate immunity. in our research, the calreticulin homologous gene (refered as pvcrt) was cloned from the ant polyrhachis vicina roger (hymenoptera: formicidae), the full-length cdna of pvcrt is 1584bp base pairs (bp), contains a 5’-untranslated region of 87bp and a 3’untranslated region of 246bp. the open reading frame (orf) of 1251bp encodes 416 amino acids. using real-time quantitative rt-pcr to study pvcrt mrna expression levels indicates that this gene was expressed in different developmental stages of castes of p. vicina. the mrna expression level in both embryos and adults shows that pvcrt gene may play some essential roles in the ant’s development. sociobiology an international journal on social insects college of life sciences, shaanxi normal university, xi’an, china research article ants article history edited by: kleber del-claro ufu, brazil received 07 january 2013 initial acceptance 04 april 2013 final acceptance 26 july 2013 keywords calreticulin homologous gene, molecular cloning, polyrhachis vicina real-time quantitative rt-pcr corresponding author gengsi xi college of life sciences shaanxi normal university, xi’an 710062, china e-mail: xigengsi@snnu.edu.cn the cell defense response, since higher amounts of crt can be found around the foreign substance. in drosophila melanogaster meigen crt named dmcrt acts as a marker for phagocytosis of apoptotic cells by d. melanogaster phagocytes (kuraishi et al. 2007). in .addition, dmcrt plays an important role in odor-guided behavior (stoltzfus et al., 2003) and also is involved in mediating anesthetic sensitivity (gamo et al., 2003). in anopheles gambiae giles agcrt is known to interact with malaria parasite surface protein (rodriguezmdel et al., 2007). here, the full-length cdna sequences of the calreticulin gene were cloned from the ant polyrhachis vicina roger and the homologs were named pvcrt. a phylogenetic tree was constructed using mega 5.0. the results of the phylogenetic analysis indicated that pvcrt shares high identity with pteromalus puparum l and nasonia vitripennis walker calreticulin. the expression pattern of pvcrt mrnas was studied by using real-time rt-pcr. our data indicate that this gene is expressed differentially in distinct development stages and different castes. lp liu, l dang, gs xi, f wang calreticulin gene investigation in polyrhachis vicina roger356 material and methods experimental insects polyrhachis vicina, which belongs to genus polyrhachis (hymenoptera:formicidae), is a species of typical eusocial insect with the characteristic of castes differentiated in females, males and workers. it is a good material for studying the mechanisms of insect development and behavior. p. vicina as a complete metamorphosis insect, is mainly distributed in east and south of china, burma, cambodia, japan, australia and papua new guinea. the colonies of p. vicina were bought from hongfa edible ant research center in nanning city, guangxi province, in people’s republic of china. then, the ants were cultured in boxes at the laboratory, under a controled temperature of 28ºc, relative humidity of 40% and natural light-dark periods. the ants were usually reared on fruits, meat and honeydew. egg, larvae, pupae and adults (workers, winged males and winged females ) were collected from the two colonies, 6 individuals were used respectively for each caste, immersed immediately in liquid nitrogen, stored at -80°c for rna extraction (lv et al., 2008; guo et al., 2010). rna preparation and cdna synthesis total rna was extracted from pooled samples of 12 frozen larvae selected randomly with rnaiso plus (takara bio inc., shiga, japan), and then immediately reverse-transcribed in order to generate cdna using the first-strand cdna sythesis kit with oligo (dt) primer (fermentas life sciences, burlington, ontario; http://www.fermentas.com/). all steps were followed through the manufacturer’s instructions. molecular cloning and sequencing of pvcrt the full-length cdna of crt from p. vicina named pvcrt was cloned based on the scheme shown in table 1. first, a partial cdna fragment of pvcrt was gained using degenerated primers (s1 and a4), according to the above fragment, primers p1 and l4 were designed and used these pair primers, and then seq2 fragment was obtained. the four primers were designed on the basis of the conserved motifs of published pvcrt of other insect species (pteromalus puparum l., bombyx mori l, culex quinquefasciatus (say), nasonia vitripennis walker, galleria mellonella l. pcr products were purified from agarose gel using gel extraction kit (qiagen, germany http://www.qiagen.com/) and purified products were cloned into pmd19-t vector (takara bio inc.), then pcr positive clones were obtained for sequencing. after that, using 3’ and 5’ rapid amplification of cdna ends (race) to get the full length of pvcrt, the specific primers of race are shown in table 1. all the primers were designed using primerpremier 5.0 and oligo 6.0. structural and phylogenetic analysis of pvcrt the orf of pvcrt was obtained using the national center for biotechnology information orf finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). signal peptide prediction was completed using the singalp program (http://www. cbs.dtu.dk/services/signalp/ bendtsen j.d. et al. 2004; center for biological sequence analysis;). the secondary structure of the pvcrt protein was predicted usingsopm (http://npsapbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/npsa/npsa_hnn. html). the tertiary structure of p. vicina crt was also pretable1. oligonucleotide primers used for cdna cloning and real-time quantitative rt-pcr. note: y: c or t; v: a or g or c; m: a or c; w: a or t; d: a or g or t; r: a or g; h: a or c or t; tm: annealing temperature. target name primer sequence 5′-3′ expected size(bp) tm(°c) crt fragment s1 a4 p1 l4 gaytcvtgggaamacmattgg cttgccayaaatcvagwccdac tgaggataaaaaaccagaggac catcrtgctcttcaathtcagg 873 606 52 52 3’race 3outer f1 3inner q4 3’oligo(dt) gctgtcaacgatacgctacgtaacg catttgtggcccaggaattagg cgctacgtaacggcatgacagtg tacccgatagcgacgatgat gctgtcaacgatacgctacgtaacggcatgacagtg(t)18 310 60 5’race 5outer h9 5inner m1 catggctacatgctgacagccta tcgccatccatttcgtcatcc cgcggatccacagcctactgatgatcagtcgatg atcattttctgggtcattcc 300 56 real time e13 g13 cgacaacgaggaagtagagt attcgccatccatttcgtcatc 224 60 β-actin b1 b2 tgaacaggagatggctacag ccgatggtgatgacttgac 80 60 sociobiology 60(4): 355-361 (2013) 357 dicted using phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/ html/page.cgi?id=index). potential functional motifs were analyzed using prosite database (expert protein analysis system, swiss institute of bioinformatics, basel; http:// myhits.isb-sib.ch/cgi-bin/motif_scan). sequence alignments based on the amino acid sequences of published crt genes were performed with clustal x 2 and dnaman. from alignments, a phylogenetic tree of pvcrt was generated by mega 5.0, based on the neighbor-joining method with a bootstrap test calculated with 500 replicates and a poisson correction model. real-time quantitative rt-pcr real-time quantitative rt-pcr was used to quantify the mrna expression levels of pvcrt from two colonies at different development stages and different castes including egg, wholly body of one to four instars larvae’s, pupae and wholly body of caste adult ants (ergate, winged male ant and winged female ant) of p. vicina were used in this experiment. for each of these developmental stages, samples were taken from 6 individuals. reactions volume, reactions temperature are 25.5ul and 60ºc, respectively. such reactions were performed in abi stepone with a sybr premix ex taq kit (takara bio inc). β-actin was used as an endogenous control to research the gene expression. primers (b1, b2) of β-actin and primers (e13, g13) of pvcrt were used in real-time rt-pcr (table 1). the amplified fragments of two pairs of primers are 80bp and 224bp, respectively. the detailed protocol was as follows: 95ºc for 10min, 40 cycles of 95ºc for 15s and 60ºc for 1min, followed by a melting-curve program from 59 to 95ºc with a heating rate of 0.3ºc every step and continuous-fluorescence acquisition. all rt-pcr reactions were completed in triplicate. the relative expression quantification of pvcrt was determined by the formula f=10∆ct,t/at-∆ct,r/ar (zhang et al., 2005). one of cdna samples was chose to construct standard curve of pvcrt gene. in this study, we chose the third instar as calibrator to analyze pvcrt expression at different development stages and different castes. the relative expression levels of pvcrt were analyzed using one way analysis of variance (anova) with dunnett’s multiple comparision test. results cloning and characterization of pvcrt cdna the full-length cdna of pvcrt was obtained by rtpcr and race. the full-length cdna of pvcrt is 1584bp. the pvcrt cdna includes a 5’-untranslated region of 87bp, an orf of 1251bp and a 3’-untranslated region of 246bp (fig.1). a putative polyadenylation signal (aataaa) was found upstream from the 18-nucleotide poly (a) tail, which fig. 1. the nucleotide and deduced amino acid from the pvcrt. the full sequence of pvcrt is 1584bp, which encodes 416 amino acid. the start codon atg and the stop codon taa are underlined and shaded, and the polyadenylation signal (aataaa) is underlined. a lot of important functional motifs of pvcrt protein are marked. six casein kinase ii phosphorylation sites at 67-70 (tsqd), 84-87 (snkd), 223-226 (sidd), 247250 (tepe), 324-327 (tifd), and 401-404 (sddd); one n-myristoylation site at 64-69 (giqtsq); four protein kinase c phosphorylation sites at 77-79 (skk), 84-86 (snk), 95-97 (tvk), and 180-182 (tyk); two tyrosine kinase phosphorylation sites at 161167 (rckddiy), and 277-284 (kqidnpny); calreticulin family signature 1 at 97-112 (kheqnidcgggylkvf); calreticulin family signature 2 at 129-137 (imfgpdicg); calreticulin family repeated motif signature at 224-236 (iddpedkkpedwd); endoplasmic reticulum targeting sequence at 413-416 (hdel). lp liu, l dang, gs xi, f wang calreticulin gene investigation in polyrhachis vicina roger358 coincides with the fact that the polyadenylation signal is most often present 11-30 nucleotide upstream from the poly (a) tail (fitzgerald et al., 1981). the deduced proteins of pvcrt have a calculated molecular mass of 48.5kda and an isoelectric point (pi) of 4.37. nucleotide sequence of pvcrt has been deposited into the genbank database and obtained the accession number of jq783054. protein structure prediction the orf of pvcrt encodes 416 amino acid, and the predicted protein shares 99% similarity with n. vitripennis and 88% with plutella xylostella l. analysis with the signalp program showed that there was n-terminal signal sequence, and the cleavage site between 18 and 19 amino acid, so the protein was determined secreted protein. a series of predicted functional motifs were found by prosite program, including six casein kinase ii phosphorylation sites at 67-70 (tsqd), 84-87 (snkd), 223-226 (sidd), 247-250 (tepe), 324-327 (tifd), and 401-404 (sddd); one n-myristoylation site at 64-69 (giqtsq); four protein kinase c phosphorylation sites at 77-79 (skk), 84-86 (snk), 95-97 (tvk), and 180-182 (tyk); two tyrosine kinase phosphorylation sites at 161-167 (rckddiy), and 277-284 (kqidnpny); calreticulin family signature 1 at 97-112 (kheqnidcgggylkvf); calreticulin family signature 2 at 129-137 (imfgpdicg); calreticulin family repeated motif signature at 224-236 (iddpedkkpedwd); endoplasmic reticulum targeting sequence at 413-416 (hdel). the predicted secondary structure of the pvcrt protein contains 13.70% alpha helix, 14.66% extended strand and 71.63% random coil (fig. 2). the prediction of tertiary structure is shown in fig.4. alignment analysis and phylogenetic-tree construction in insects a multiple alignment of the deduced amino acid sequence of pvcrt with other known calreticulin homologues was performed by clustal x 2 and dnaman (fig.3). a phylogenetic tree was constructed using mega 5.0 based on the neighbor-joining method (tumura et al., 2007) in fig. 5. the results of the phylogenetic analysis indicated that pvcrt shares high identity with p. puparum and n. vitripennis calreticulin (fig.5). expression analysis of pvcrt mrna the expression level of pvcrt mrna at different developmental stages and in different castes was analyzed by means of real-time quantative rt-pcr. previous studies revealed that the analyzed β-actin mrna levels remain constant in tissues of insects regardless of their developmental and physiological condition (simonet et al., 2004). the analyzed results indicated that pvcrt was expressed in all templates (fig.6). during the developmental stages, the highest expression level was found in the third instars and the lowest expression level was found in embryos. among the three adult’s castes, the pvcrt gene expression was highest in workers and lowest in males. discussion in this study, the full-length cdna of calreticulin gene was cloned and characterized from p. vicina. the cdna sequence of pvcrt is highly conserved with calreticulin homologs found from other insects, which indicated that this sequence is indeed calreticulin of p. vicina. the results of predicted functional motifs found that in pvcrt protein exists an er targeting sequence hdel, which is closely related to the cellular localization of crt in er. this result may shows that the pvcrt possesses function of calregulin in er mainly involved in directing proper conformation of the proteins, controlling calcium level, and participating in immune responses (wang et al., 2012; byung-jae park et al, 2001;) including: regulation of intracellular ca2+ homeostasis chaperone activity, steroid-mediated gene regulation, and cell adhesion (mesaeli et al., 1999). the phylogenetic tree (fig.5) was analyzed and found that pvcrt is most closely related to that of p. puparum and n. vitripennis. usually, p. civica is similar to p. puparum and n. vitripennis in classification status. all of them belong to hymenoptera insects, which may indicate that the calregulin possesses conservative properties. khanna et al. (1987) demonstrated that“the physicochemical and structural properties of calregulin from a single tissue have been highly conserved during vertebrate evolution after comparison of calregulins from bovine, rabbit and chicken livers”luana et al. (2007) cloned the full length cdna of calreticulin from fenneropenaeus chinensis osbeck (fccrt). disply that calreticulin belong to a highly conserved calcium-binding protein. the deduced amino acid sequence of fccrt showed high identity with those of b. mori (88%), drosophila melanogaster meigen (83%), mus musculus l. (82%) and homo sapiens l.(82%). in this paper the analysis of real-time rt-pcr indicated that pvcrt gene is expressed in each template, but the expresfig. 2. predicted secondary structure of the pvcrt protein, where c is random coil; h is alpha helix; e is extended strand; t is beta turn. sociobiology 60(4): 355-361 (2013) 359 fig.3. amino acid sequence alignments of pvcrt with the homologues sequences of other insects and one crustacean. species and genbank accession numbers for alignmental and phylogenetic analysis are as follows: n. vitripennis (nvi) nm_001161679; p. puparum (ppu) fj882064; p. vicina (pvi) jq783054; b. mori (bmo) fj360528; pieris rapae l. (pra) eu826537; plutela xylostella l. (pxy) hm240516; cotesia rubecula (marshall) (cru) ay150370; d. melanogaster (dme) nm_079569; anopheles stephensi liston (ast) jn559458; culex quinquefasciatus (say) (cqu) xm_001848772; litopenaeus vannamei boone (lva) (crustacea) jq682618. similarity of 100%, (>=75%), (>=50%) in all crts are marked in black, dark green and gray, respectively. lp liu, l dang, gs xi, f wang calreticulin gene investigation in polyrhachis vicina roger360 sion levels are different. during the developmental stages, the highest expression level was found in the third instars and the lowest expression level was found in embryos. according to those results, we guess that third instars’s nervous system and sense organs may reach an development peak due to calregulin can promote the development of nerve and sense organs (silerová et al., 2007; gamo et al.,1998). in the three adult’s castes, the pvcrt gene expression was highest in workers and lowest in males. this result coincide with stoltzfus and horton’s conclusion (2003) “the calregulin plays a key role in olfactory system function, possibly by establishing its overall sensitivity to odorants”. among p. civica adult’s castes, the worker olfactory system is the most developed especially in antenna and chest foot aspects because of the main responsibilities related to building and defending nests, feeding the young ants and queens. the main task of male ants is only to mate with the queen, so its olfactory system function may be the weakest among the castes. from the above results we can deduce that pvcrt gene may play critical roles for the ant growth and development. more study of pvcrt functions will be further continueding. in conclusion, the full-length pvcrt cdna for the first time was cloned from p. vicina, and the characterization of pvcrt protein was analyzed. the differential pvcrt mrna expression levels of pvcrt in different developmental stages and different castes, may indicate the physiological importance of pvcrt for this ant species. acknowledgment this work was supported by the national natural science foundation of china, nsfc (31171195). we thank two anonymous reviewers for contributions that helped to improve this article. references bendtsen, j.d., h. nielsen, g., von heijine & s. brunak (2004). improved prediction of signal peptides: signalp 3.0. j. biochem. mol. biol., 40:783-795. doi: 10.1016/j. jmb.2004.05.028 fitzgerald m. & t. shenk (1981). the sequence 5’-aauaaa3’forms parts of the recognition site for polyadenylation of late sv40 mrnas. cell, 24(1): 251-260. gamo, s., dodo, k., matakatsu, h. & tanaka, y. 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(2005). a novel and convenient relative quantitative method of fluorescence real time rt-pcr assay based on slope of standard curve. prog. biochem. biophysiol., 32: 883-888. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1179sociobiology 64(1): 33-41 (march, 2017) morphometric diversity and phylogenetic relationships among iranian honey bee (apis mellifera meda skorikow, 1829) populations using morphological characters introduction honey bee (apis mellifera l.) as a natural component of the local biota and beneficial insects make an important contribution to honey, wax, propolis, royal jelly, bee poison production and they are most important pollinators, too by helping plants reproduce and increases in agricultural productivity and environmental conservation (maganus & tripodi, 2011; rahimi et al., 2014 b). honey bees expanded to northward to the south of scandinavia, southward to the cape of good hope, westward to the dakar in senegal, eastward to mashad in iran and oman in the gulf of oman with human-assisted migration. in sixteenth century by the human migrations to oceanic islands and to north and south american continents, abstract in this study, the morphometric diversity and phylogenetic relationships of iranian honey bee populations, were investigated using 14 morphometric characteristics. a total of 2250 young adult worker bees from 20 different populations in 20 different provinces of iran were collected during june to october 2014. the results of nested analysis of variance showed that there were significant differences (p<0.01) between the provinces for all analyzed morphometric traits indicating the existence of a diversity among them. correlation coefficient analysis showed a high degree of association among the most of the traits. this correlation coefficient should be a putative mean to improve of certain characters in breeding of honey bee. principal component analysis revealed three principal components explained 81.5% of the total variation. cluster analysis using ward method classified honey bee populations into two main groups. the first group includes the honey bees collected from north, northwest and west portions of iran. the second group was represented by the honey bees from eastern north, central and southern regions of iran. the phylogenetic tree based on upgma method divided 29 subspecies of honey bee to 5 distinct clusters. the iranian subspecies honey bee composed of a shared clade with subspecies of eastern mediterranean, near east and eastern parts of middle east (o branch). sociobiology an international journal on social insects a rahimi1,2, a mirmoayedi1, d kahrizi3, l zarei3, s jamali1 article history edited by yi juan-xu, south china agricultural university, china received 07 august 2016 initial acceptance 23 october 2016 final acceptance 10 march 2017 publication date 29 may 2017 keywords dendrogram, honey bee, morphometric, morphometric diversity, phylogenetic relationships. corresponding author ataollah rahimi department of plant protection, college of agriculture, campus of agriculture and natural resources, razi university, kermanshah, iran e-mail: rahimi.ata.1@gmail.com the honey bees were transported into the new continents. in such a distribution over vast area, many factors such as geographical isolation and ecological adaptations caused the appearance of different populations and emergence of new subspecies (ruttner et al., 1978; ruttner, 1988; tahmasebi et al., 1998; rahimi & asadi, 2010). twenty nine subspecies and many ecotypes of honey bees recorded from different locations in the world. these subspecies were characterized based on a set of characteristic (morphological, behavioral and molecular) and divided into five evolutionary groups; subspecies from west mediterranean and north-west european group (m), subspecies from south and central africa group (a), subspecies from central mediterranean and south eastern european group (c), subspecies from east mediterranean, near east and eastern 1department of plant protection, college of agriculture, campus of agriculture and natural resources, razi university, kermanshah, iran 2department of animal science, college of agriculture kifri, garmian university, kalar, as sulaymaniyah, krg of iraq 3-department of plant breeding and biotechnology, college of agriculture, campus of agriculture and natural resources, razi university, kermanshah, iran research article bees a rahimi et al. – morphometric diversity and phylogenetic relationships in iranian honey bee34 parts of middle eastern group (o), subspecies from north-east africa and eastern part of ethiopia group (y) (ruttner, 1988, 1992; garnery et al., 1993; sheppard et al., 1997; engel, 1999; sheppard & meixner, 2003; arias & sheppard, 2005; meixner et al., 2011). in the middle east there are eight subspecies, and between them, apis mellifera meda exist in iran (ruttner et al., 1985; kandemir et al., 2004; rahimi et al., 2014 a). the iranian subspecies honey bee (apis mellifera meda) are spread over northern part of iraq, south eastern part of turkey (ruttner, 1988), north part of syria (fetayeh et al., 1994) and in all parts of iran except eastern desert regions (tahmasebi et al., 1996). the iranian subspecies honey bee has similar morphological characters with italian subspecies honey bee (apis mellifera ligustica spinola, 1806). in other cases, however, biological, molecular and behavioral traits seem to be different between them for example: a high rate of reproduction, a very good tolerance for hibernation and high aggressiveness (ruttner, 1988). morphological characters in zoology is routinely used to differentiate different subspecies from each other, still is the common way to characterize honey bee populations in many countries of the world (ruttner et al., 1978). ruttner et al. (1978) collected subspecies of honey bees from different countries and showed that the european subspecies (apis mellifera carnica pallmann, 1879 and apis mellifera caucasica pallmann, 1889) with a bigger body size located the right part of the axis using principal component analysis (pca), the african subspecies (apis mellifera jemenitica ruttner, 1976 and apis mellifera lamarckii cockerell, 1906) formed the left side of axis and the iranian subspecies honey bee occupied the central part of the axis. kence et al. (2009) grouped seven populations of iranian and caucasian subspecies of honey bee existing in turkey based on morphological characters. the iranian subspecies honey bee formed a distinct group whereas caucasian and other populations formed two other groups. in the meral (2010) study of genetic diversity among turkish honey bee populations, different turkish populations formed three distinct groups. tahmasebi et al. (1998) studied morphological characters of different populations of iranian honey bees and showed that the most of honey bee populations in iran belonged to iranian subspecies honey bee which had significant differences from introduced european subspecies. in another study, iranian subspecies honey bee have some overlapping characters with turkish honey bee, but the samples collected from different regions of iran (urmia, tabriz and tehran regions) formed a distinct group (farshineh-adl et al., 2007). rahimi and mirmoayedi, (2013) reported that the populations of honey bee from mazendaran province (north of iran) formed three groups, and they concluded that the climatic factors should be blamed for the population separation. iran is a vast country comprising a land area about 1648195 km2. from the geographical point of view, iran has a very diverse climatic condition with diverse patterns of plant communities forming four climatic zones simultaneously; therefore it has a high potential for beekeeping and honey production. therefore, genetic structure maintenance and preservation of iranian honey bees is the first step to explore of this potential. the objective of this study was to determine the morphometric variation and phylogenetic relationships among honey bee samples collected from different locations of from iran using morphometric characters analysis. fig 1. the geographical locations of honey bee populations collected in this study. sociobiology 64(1): 33-41 (march, 2017) 35 materials and methods sampling a total of 2250 young adult worker bees from 150 colonies of apis mellifera meda were sampled from 100 different localities distributed in 20 iranian provinces during june to october 2014 (fig 1, table 1). samples were taken from honey bee colonies in most active apiaries from five cities in each province, one apiary in each city and one to three hives per apiary were selected for sampling. young adult worker bees directly were collected from the brood areas on the combs, using an open mouth jar containing the cotton soaked to the ether and poured into pumpel solution (30 parts distilled water, 6 parts formaldehyde, 15 parts ethanol 65% and 2 parts acetic acid). selected morphometric characters and morphometric measurements from every mccarthy glass tube, which contained 20 bees, fifteen bees were randomly selected to measure the morphometric traits. we have chosen fourteen morphometric characters of honey bees to distinguish the local subspecies from widely used forty characters. the measured characters were as follows; fwl forewing length, fww forewing width, hwl hind wing length, hww hind wing width, a4, d7 and g18: angle in the forewing, pi proboscis length, ci cubital index, si sternite index, sc scutellum color, tftl third & forth tergite length, hll hind leg length, ttc third tergite color. all measurements were done based on ruttner et al. (1985, 1988). to measure morphometric table 1. sampling localities, geographical positions and number of honey bees used for morphometric analysis. province the number of apiary the number of colonies the number of bees latitude longitude altitude kermanshah 5 7 140 34° 29′ 15.62 n 047° 05′ 57.65 e 1664 hamadan 5 8 160 34° 43′ 24.64 n 048° 31′ 01.27 e 2464 ilam 5 5 100 33° 34′ 31.97 n 046° 27′ 26.44 e 1548 lorestan 5 6 120 33° 32′ 01.81 n 048° 14′ 12.03 e 1802 esfahan 5 10 200 32° 36′ 56.09 n 051° 34′ 04.20 e 1618 chaharmahal and bakhtiari 5 7 140 32° 28′ 19.40 n 050° 06′ 30.53 e 2582 fars 5 10 200 28° 59′ 19.33 n 053° 39′ 56.47 e 1563 sistan and baluchestan 5 5 100 29° 30′ 53.89 n 060° 50′ 17.17 e 1407 kerman 5 6 120 29° 18′ 19.25 n 057° 08′ 29.72 e 2838 kurdistan 5 7 140 34° 42′ 48.06 n 046° 51′ 34.16 e 1775 west azerbaijan 5 10 200 37° 34′ 04.14 n 044° 44′ 17.19 e 2138 east azerbaijan 5 10 200 38° 06′ 13.12 n 046° 11′ 10.99 e 1345 ardabil 5 9 180 38° 13′ 58.28 n 048° 10′ 05.22 e 1455 zanjan 5 6 120 36° 42′ 37.00 n 048° 22′ 25.99 e 1546 tehran 5 6 120 35° 43′ 44.11 n 052° 07′ 00.70 e 2233 razavi khorasan 5 7 140 36° 25′ 56.06 n 059° 31′ 26.22 e 922 north khorasan 5 6 120 37° 29′ 16.88 n 057° 09′ 56.89 e 1454 golestan 5 5 100 36° 50′ 47.46 n 054° 40′ 12.88 e 181 mazandaran 5 10 200 36° 25′ 22.19 n 052° 14′ 39.71 e 135 gilan 5 10 200 36° 50′ 23.28 n 049° 25′ 57.83 e 329 characters of the wing, the right side wings were separated and put into a mixture of alcohol and honey, then transferred to two layers glass slides and arranged in a numbered regular manner. the wings were attached firmly to glass slides following the evaporation of alcohol. slide projector was used to measure the wings dimensions in each sample analyzed. stereomicroscope with calibrated eyepiece was used to measure other body characters. color characters were measured according ruttner’s international index. statistical analysis a nested analytical design was used. the analysis of variance and means comparisons were done with sas statistical package v.14 (harvey, 1990). multivariate analytical techniques including correlation analysis, principal component analysis, factor analysis and cluster analysis were performed using spss v. 13.0 (2004). to draw a phylogenetic tree between apis mellifera meda and other subspecies, a rahimi et al. – morphometric diversity and phylogenetic relationships in iranian honey bee36 morphological information of other honey bee subspecies obtained from the world bank database. results in order to study morphometric diversity and investigate phylogenetic relationships, 2250 worker honey bees from different iranian honey bee populations were used to analyze fourteen characters. the results were checked with keys provided by ruttner et al. (1985). the results showed that there were five subspecies of honey bee in our collected samples from different regions of iran as follows; apis mellifera meda, apis mellifera ligustica, apis mellifera carnica, subspecies hybrid midnite and subspecies hybrid star-line. analysis of variance the analysis of variance showed a significant difference among all different iranian provinces for all characters (p<0.01). there were no differences between cities in the same province for all character, except for the sternite index (p<0.05). besides, analysis of variance revealed that there were significant differences among hives in each cities for the following characters: forewing length, forewing width, hind wing length, hind wing width, a4 angle, d7 angle, g18 angle, proboscis length, cubital index and third & forth tergite length. no significant differences were observed among hives within cities for sternite index and scutellum color (p>0.05) (table 2). g18d7a4hwwhwlfwwfwldfs.o.v 188.970 **197.502 **10.185 **0.245 **0.228 **0.043 **1.817 **19province (a) 30.321 n.s32.846 n.s1.977 n.s0.056 n.s0.061 n.s0.016 n.s0.072 n.s80city (province) (b) 61.888 **61.936 **2.601 **0.200 **0.124 **0.022 **0.091 **50error fwl: forewing length, fww: forewing width, hwl: hind wing length, hww: hind wing width, a4, d7 and g18: angle in the forewing *: significant at p<0.05, **: significant at p<0.01, n.s: non – significant, df: degree of freedom, s.o.v: source of variation fwl, fww, hwl and hww(mm), a4, d7 and g18 (degree). table 2. continue ttchlltftlscsicipldfs.o.v 20.436 **1.089 **0.176 **3.887 **0.007 **0.839 **0.728 **19province (a) 1.989 n.s0.179 n.s0.035 n.s1.266 n.s0.002 *0.256 n.s0.150 n.s80city (province) (b) 3.616 **0.385 **0.047 **0.880 n.s0.001 n.s0.225 **0.214 **50error pi: proboscis length, ci: cubital index, si: sternite index, sc: scutellum color, tftl: third & forth tergite length, hll: hind leg length, ttc: third tergite color *: significant at p<0.05, **: significant at p<0.01, n.s: non – significant, df: degree of freedom, s.o.v: source of variation pl, tftl and hll (mm). table 2. analysis of variance of 14 morphometric traits in apis mellifera meda. correlation coefficient pearson correlation coefficients between fourteen characters were calculated (table 3). length of fore wing showed a positive correlation with width of fore wing, length of hind wing, angle a4, angle d7, angle g18, length of proboscis, sternite index, hind leg length and third tergite color, but the forewing length had a significant negative correlation with cubital index. cluster analysis dendrogram constructed using ward method divided the provinces into two groups. twelve provinces including kermanshah, hamadan, mazandaran, ardabil, golestan, kurdistan, ilam, east azerbaijan, west azerbaijan, gilan, zanjan and tehran clustered were grouped together, and other provinces including fars, sistan and beluchistan, esfahan, chaharmahal and bakhtiari, razavi khorasan, lorestan, kerman and north khorasan provinces were placed together in the second group (fig 2). the results of cluster analysis confirmed by discriminant analysis. fig 2. dendrogram following ward method for clustering populations of honey bees from iranian provinces. sociobiology 64(1): 33-41 (march, 2017) 37 principal component analysis (pca) pca is a variable reduction technique which maximizes the amount of variance accounted for in the observed variables by a smaller group of variables called components. the first three axes of the principal component analysis explained 81.5 % of the total variation (table 4). the first two components suggested that diversity in honey bee populations were influenced by forewing length, d7 angle, g18 angle, cubital index, hind leg length, third tergite color, forewing width, a4 angle, proboscis length, scutellar color and third & forth tergite length (fig 3). factor analysis principal factors were determined as the variables with the highest projection scores on the principal components (table 5). results showed that six main factors accounted for 93.4 % of the total variability. the first factor which explained 23.1 % of total variation emphasized the forewing length, d7 angle and g18 angle, and can be named as the flight factor (table 5). the results of correlation analysis also confirmed the existence of negative correlation between the length of fore wing, the index of cubital and the length of hind leg which are in accordance with the results obtained by the factor analysis. the 2nd factor covered 18.9 % of the data variation and had negative loadings for length of proboscis, scutellar color, length of hind wing and the third tergite color. the 3td factor justified 16.2 % of the data variability and had the maximum of negative loading for the characters the width of fore wing, length of hind wing and a4 angle. the 4th factor covered 15.2 % of the data variance and had positive loading. the 5th factor with 10.7 % of the data variation showed negative loading for the width of hind wing and a7 angle. finally, the 6th factor covered 9.2 % of the variation and had a positive loading for sternite index (table 5). ttchlltftlscsicipig18d7a4hwwhwlfwwfwl 1fwl 10.225 *fww 10.228*0.355 **hwl 10.236*0.0040.358 **hww 10.616**0.626**0.241*0.452 **a4 10.575**0.298*0.532**0.1960.575 **d7 10.636**0.665**0.259*0.579**0.1320.549 **g18 10.261*0.206*-0.1470.1210.1460.1240.335 **pi 10.148-0.114-0.252*-0.144-0.226*-0.1200.1440.327 **ci 10.141-0.142-0.106-0.1690.1390.1270.116-0.1840.301**si 10.115-0.583**-0.389**0.0890.1120.026-0.136-0.1770.238*-0.010sc 1-0.122-0.278*-0.297*0.257*-0.1280.0510.1110.1020.1190.249*0.054tftl 10.250*0.600**-0.260*0.609**0.467**-0.224*0.544**-0.180-0.138-0.1570.1960.384**hll 10.651**0.519**0.660**0.245*0.258*-0.427**-0.1840.1240.309**-0.157-0.1560.1810.264 *tcc table 3. correlation coefficient between fourteen morphometric traits in honey bee apis mellifera meda. fig 3. two dimensional biplot composed of the 1st and the 2nd principal components of morphometric characters of iranian a.mellifera meda honey bee populations. a rahimi et al. – morphometric diversity and phylogenetic relationships in iranian honey bee38 phylogenetic tree the phylogenetic trees are generally plotted to find the genetic distances between the collected samples of honey bees. phylogenetic tree based on morphometric character was drawn using upgma method to compare phylogenetic relationships iranian subspecies honey bee with other subspecies. phylogenetic tree showed 29 honey bee subspecies could be divided into 5 distinct clusters (fig 4). discussion there is one native subspecies of honey bee in iran, apis mellifera meda, which during the past thousands years of iranian civilization, are adapted to the different climate conditions and plant flora varieties in this area. also, certain resistance against diseases and pests which attack to honey bees in iran have been acquired by this subspecies (ruttner, 1978; tahmasebi et al., 1998). we have compared morphometric traits the first component the second component the third component fwl -0.309 0.158 -0.258 fww 0.004 0.385 0.437 hwl -0.268 0.249 0.210 hww -0.270 0.176 -0.360 a4 -0.250 0.354 -0.015 d7 -0.357 0.152 0.132 g18 -0.319 0.239 0.179 pi 0.053 0.375 -0.396 ci 0.324 0.148 0.275 si -0.260 -0.174 -0.322 sc 0.264 0.301 0.067 tftl 0.136 0.391 -0.039 hll 0.337 0.190 -0.248 ttc 0.302 0.237 -0.339 eigenvalue 5.89 4.16 1.33 proportion 42.1 29.8 9.6 cumulative variance (%) 42.1 71.8 81.4 table 4. principal component analysis of fourteen morphometric traits in populations of apis mellifera meda of different parts of iran. traits factor 1 factor 2 factor 3 factor 4 factor 5 factor 6 fwl 0.821 -0.142 -0.117 -0.199 -0.268 0.240 fww 0.249 -0.209 -0.698 0.432 0.213 -0.301 hwl 0.312 0.066 -0.881 -0.078 -0.230 0.107 hww 0.396 -0.031 -0.205 -0.098 -0.850 0.130 a4 0.474 -0.141 -0.682 0.101 -0.477 0.055 d7 0.869 0.190 -0.359 -0.129 -0.106 0.109 g18 0.857 0.147 -0.410 0.058 -0.185 -0.016 pi 0.174 -0.912 -0.278 0.085 -0.105 0.000 ci -0.439 -0.205 -0.048 0.697 0.331 -0.223 si 0.223 0.176 0.002 -0.328 -0.107 0.879 sc -0.009 -0.537 0.032 0.640 0.224 -0.360 tftl -0.006 -0.401 -0.205 0.730 -0.353 -0.238 hll -0.458 -0.723 0.096 0.432 0.125 -0.063 ttc -0.251 -0.787 0.223 0.363 -0.054 -0.288 proportion 23.2 18.9 16.2 15.2 10.7 9.2 cumulative variance (%) 23.2 42.1 58.3 73.5 84.2 93.4 table 5. matrix of factors rotation (varimex rotation) for fourteen traits of apis mellifera meda in various populations of different locations in iran. fig 4. phylogenetic tree based on upgma method to compare subspecies of apis mellifera meda with different subspecies of a.mellifera. sociobiology 64(1): 33-41 (march, 2017) 39 characters of iranian honey bee samples with keys provided for study of subspecies of honey bees by ruttner et al. (1978), five subspecies of honey bee have been identified in different regions of iran including apis mellifera meda, apis mellifera ligustica, apis mellifera carnica, subspecies hybrid midnite and subspecies hybrid star-line. the only native honey bee subspecies in iran is apis mellifera meda (ruttner et al., 1985; kandemir et al., 2004; rahimi et al., 2014 a) and other identified subspecies in this study are not native to iran. probably, these subspecies had entered iran as a result of trafficking queen. the existence of other subspecies in iran can be a warning to beekeeping community because it eliminates racial purity iranian honey bee. moreover the results of other studies (tahmasebi et al., 1998; rahimi & mirmoayedi, 2013; rahimi et al., 2014 b) support the particular identity of iranian subspecies honey bee. conservation of genetic purity in iranian subspecies honey bee as genetic resources and most expensive natural heritage should be considered in order to preserve them against admixture with other subspecies. probably, the admixture of iranian subspecies honey bee with other regional subspecies is occurring spontaneously in border regions; however we should be worried about the illegal trafficking of queens from other honey bee subspecies into iran. the results of analysis of variance showed that there was a significant difference between the provinces for all the honey bee characters (p<0.01) which could support the existence of genetic diversity among bees from iranian different provinces. moreover, most of the fourteen morphometric characters had significant positive correlations to each other. shykon et al. (1991) showed significant positive correlations between length of fore wing and characters such as length of proboscis, length of hind leg and third and forth tergite length. tahmasebi et al. (1996) observed significant positive correlations between length of fore wings with characters such as length of proboscis, length of hind leg and third and forth tergite length. rahimi et al. (2015) have found positive correlations between the length of fore wings and the honey production of the iranian honey bee. cluster analysis classified honey bee populations into two groups; a group consisted of populations from north, northwest and west provinces of iran while the other group comprised of populations from eastern north, central and the southern iranian provinces. in the study of kence et al. (2009) on five honey bee populations from north, northwest and west of iran, all the populations composed single group in agreement with our results. tahmasebi et al. (1998) have found different three groups by cluster analysis of iranian honey bee populations; the first group consisted of the populations from north and northeast, the second included the populations from northwest and west and the third consisted of the populations from central parts of iran. the different results in our findings is likely due to the event of dryness which iran encountered during last twenty years (1995-2014), this is because of scarcity of raining and the obligation of beekeepers to displace their hives to new sites. these hive migrations influenced the trade of queens among beekeepers and induced genetic introgression of the bee populations and disrupting the previously groups detected by tahmasebi et al. (1998). principal component analysis (pca) revealed three principal components accounted for 81.5 % of the total variance. most of the variation could be explained by the two first components (71.9 %) supporting the existence of two distinct groups. the first group composed the provinces tehran, mazandaran, gilan, east and west azerbaijan and kermanshah had the bees with higher cubital index, the longer hind leg and the lighter color of scutellum. the second group comprised populations belonged to the provinces kurdistan, ilam, zanjan, kerman and sistan and beluchistan with the wide forewing, longer hindwing, wide a4 and g18 angles, longer proboscis, longer third & forth tergite length and the biggest abdomen. as an exploratory tool to reduce many dependent variables to a few principal ones, we used factor analysis for fourteen morphometric characters. the results revealed six hidden factors that explained 93.4 % of variation between studied populations. phylogenetic tree drawn based on upgma method divided 29 honey bee subspecies into 5 distinct clusters (fig 4). dupraw (1965) has grouped the honey bee subspecies based on geographical distribution into five groups. iranian honey bee subspecies grouped with east mediterranean, near east and eastern middle east subspecies (o branch). ruttner (1988), garnery et al. (1993), engel (1997), sheppard et al. (1997), meixner (2003), arias and sheppard, (2005) and meixner et al. (2011) have used cluster analysis to grouping 29 honey bee subspecies based on morphological, behavioral and molecular traits; they detect five evolutionary groups. the iranian subspecies honey bee formed a group with east mediterranean, near and middle eastern subspecies (o branch). in the current study, the phylogenic tree drawn based on measured morphological characters along with other subspecies data, grouped 29 honey bees subspecies into five groups. iranian honey bee subspecies with subspecies apis mellifera ligustica, apis mellifera anatolica, apis mellifera syriaca, apis mellifera cyprica, apis mellifera adami, apis mellifera armeniaca, apis mellifera caucasica and apis mellifera pomonella were placed in the same group. these subspecies except for apis mellifera ligustica are the same subspecies of evolutionary group (o branch). the results of this study were in accordance with previous studies. many authors have speculated about a close relationship for the morphological characters between iranian honey bee subspecies and italian subspecies (apis mellifera ligustica). whereas these subspecies were clustered in the same group, but the italian honey bee subspecies has biological, behaviorial and molecular characters different from iranian honey bee subspecies, so honey bee researchers have grouped apis mellifera ligustica with central mediterranean and south east european subspecies into one group (ruttner, a rahimi et al. – morphometric diversity and phylogenetic relationships in iranian honey bee40 1988, 1992; garnery et al., 1993; sheppard et al., 1997; engel, 1999; sheppard & meixner, 2003; arias & sheppard, 2005; meixner et al., 2011). acknowledgements the authors appreciate the kindness of those beekeepers who allowed us to collect honey bee samples from their apiaries and equally thank the authorities of plant protection and biotechnology departments of razi university who let us a free access to laboratory facilities which led to fulfill of the present study. references arias, m. & sheppard, w. 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(1993). a simple test usingrestricted pcr amplified mitochondrial dna to study the genetic structure of apismellifera l. experienta, 49: 1016-1021. harvey, w.r. (1990). users guide for lsmlmw and mixmdl, po-2 version. the ohio state university, u.s. kandemir, i., ozkan, a., & mohammad, g.m. (2004). a scientific note on allozyme in persian honey bee (apis mellifera meda) from the elburz mountains in iran. apidologie, 35: 521-522. kence, m., jabbari farhoud, h. & tunca, r.i. (2009). morphometric and genetic variability of honey bee (apis mellifera l.) populations from northern iran. journal of apicultural research, 48: 247 – 255. maganus, r. & tripodi, a. (2011). mitochondrial dna diversity of honey bees, apis mellifera l. (hymenoptera: apidae) from queen breeders in the united states. journal of apicultural science, 55: 5-17. meixner, m.d., leta, m.a., koeniger, n. & fuchs, s. (2011). the honey bees of ethiopia represent a new subspecies of apis mellifera apis mellifera simensis ssp. apidologie, 42: 425-437. meral, k. (2010). honey bee (apis mellifera l.) in turkey: biodiversity using geometric morphometrics analysis. 4th european conference of apidologie, september 2010, metu ankara, turkey. page: 134. rahimi, a. & asadi, m. (2010). morphological characteristics of apis mellifera meda (hymenoptera: apidae) in saghez (west of iran). nature montenegro, 10: 101-107. rahimi, a. & mirmoayedi, a. (2013). evaluation of morphlogical characteristics of honey bee apis mellifera meda (hymenoptera: apidae) in mazandaran (north of iran). technical journal of engineering and applied sciences, 3: 1280-1284. rahimi, a., asadi, m. & abdolshahi, r. (2014) a. genetic diversity of honey bee (apis mellifera meda) populations using microsatellite markers in jiroft. journal of science and tactic of honey bee, 6: 26-33. rahimi, a., miromayedi, a., kahrizi, d., abdolshahi, r., kazemi, e. & yari kh. (2014) b. microsatellite genetic diversity of apis mellifera meda skorikov. molecular biology reports, 41: 7755–7761. rahimi, a., hasheminasab, h. & azati, n. (2015). predicting honey production based on morphological characteristics of honey bee (apis mellifera l.) using multiple regression model. ecology-environment-conservation, 21: 29-33. ruttner, f., tassencourt, l. & louvaux, j. (1978). biometrical – statistical analysis of the geographic variability of apis mellifera l. apidologie, 9: 363 – 381. ruttner, f., pourasghar, d. & kauhausen, d. (1985). honey bees of iran .2. apis mellifera meda skorikoow. the persian bee. apidologie, 9: 363-381. ruttner, f. (1988). biogeography and taxonomy ofhoneybees. springer-verlag. berlin., germany, 285 pp. ruttner, f. (1992). naturgeschichte der honigbienen. ehrenwirth verlag. münich. germany. 357 pp. sheppard, w.s., arias, m.c., greech, a. & meixner, m.d. (1997). apis mellifera ruttneri, a new honey bee subspecies from malta. apidologie, 28: 287-293. sheppard, w.s. & meixner, m.d. (2003). apis mellifera pomonella, a new honey bee subspecies from central asia. apidologie, 34: 367–375. shykon, j.a., poschmid, h. & stort, a. (1991). genetic relatedness and eusociality. parastie mediated selection on the genetic composition of groups. behavioral ecology and sociobiology, 28: 371-376. spss. (2004). spss for windows, release 13.0 standard version, spss inc., 19892004. sociobiology 64(1): 33-41 (march, 2017) 41 tahmasebi, gh. (1996). morpological and biochemical survey of honey bee (apis mellifera l) populations in iran. ph.d. thesis, tarbit modares university, tahran, iran. tahmasebi, gh., abadi, r., esmaili, m. & kambozia, j. (1998). morphological study of honey bee (aips mellifare l) in iran. journal of agricultural sciences and natural resources, 2: 89-100. doi: 10.13102/sociobiology.v64i2.1277sociobiology 64(2): 166-173 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 bee life in the city: an analysis of the pollen provisions of centris (centris) flavifrons (centridini) in an urban area introduction palynological analysis is an important tool in the study of bee food resources, and in the determination of the floral resources used by bees (dórea et al., 2009; 2010a; 2010b; 2013; santos et al., 2013; cruz et al., 2015). the pollen stored in nests provides many data on bee foraging and help to fill the knowledge gap concerning the floral resources needed to maintain bee populations (dórea et al., 2013). this is particularly important for bee populations that inhabit urban areas where food resources are generally sparse and its diversity can be quite limited. although most of the studies indicate a decrease in species richness and abundance of pollinators (e.g. zanette et al., 2005) even in the cities a substantial number of bee species can be found (cane et al., 2006; banaszak-cibicka & zmihorski, 2011). with the continuous and rapid growth of cities, ecosystems depend increasingly on urban development patterns (alberti, 2005). abstract due to deforestation and fragmentation of ecosystems, the management of bee populations targeting pollination services is increasingly urgent. because urban environments are stressful, the dietary knowledge in such areas can help to cope with this issue in the near future. using palynological analysis the floral resources used by centris flavifrons, an important pollinator of crops and native plants was studied in an urban area. byrsonima sericea type, solanum paniculatum type, cestrum type, and myrcia type 1 together accounted for more than 93% of pollen grains foraged by females. it is noteworthy that this bee population depends on few plant species both for pollen and for oil. furthermore, females showed flexibility to replace the primary pollen source in different breeding seasons, as well as one female could provision different cells in the same nest with different predominant pollen types. the importance of wastelands and natural areasis highlighted for keeping bee populations in urban areas. sociobiology an international journal on social insects mc dórea1, far santos1, cm laguiar1, cf martins2 article history edited by denise alves, esalq-usp, brazil received 15 december 2016 initial acceptance 22 february 2017 final acceptance 17 march 2017 publication date 21 september 2017 keywords oil-collecting bees, trophic niche, diet, pollinators, oil-flowers. corresponding author marcos da c. dórea departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina s/nº, 44036-900 feira de santana-ba, brasil e-mail: mcdorea@gmail.com amid discussions about loss of biodiversity, urban areas are seen with a potential to promote diversity as a result of designing and planning the cities to foster interaction between people and nature (miller, 2005). centris (centris) flavifrons (fabricius) has mainly a neotropical distribution, occurring also in the nearctic region in sinaloa, mexico (moure et al., 2012) and is an important pollinator of malpighia emarginata l., called west indian cherry, and others agricultural crops, for example passiflora alata curtis (gaglianone et al., 2010; vilhena et al., 2012). it has also an important role in native plant species pollination in the neotropical region, especially of malpighiaceae, e.g. byrsonima sericea dc. (teixeira & machado, 2000), b. gardneriana a. juss. (bezerra et al., 2009) and b. cydoniifolia mart. (sazan et al., 2014). the nesting biology and ecology of this ground-nesting solitary bee species were initially studied by vinson and frankie (1988), rêgo et al. (2006), gonzalez et al. (2007) and, 1 universidade estadual de feira de santana, bahia, brazil 2 universidade federal da paraíba, joão pessoa-pb, brazil research article bees sociobiology 64(2): 166-173 (june, 2017) 167 recently in a more detailed study by martins et al. (2014). at open areas of coastal savanna in the atlantic forest dominium, including urban areas in the northeastern coast of brazil, females nest mainly during the dry season, associated to flowering that occurs mainly during the dry period and at the beginning of the rainy season (lima et al., 2008), and exhibit notable flexibility for time each nest is active and the number of brood cells produced (martins et al., 2014). females were observed building until three nests, however the period of female activity in each nest, ranged from 2 to19 days (mean=6.9±4.2, n=141) and, consequently, the number of cells constructed per nest by each female ranged from 2 to 12 (martins et al., 2014). furthermore, a previous study suggested that c. flavifrons presents two to three generations per year (martins et al., 2014). considering the lack of knowledge about the diet of this pollinator, and the existence of an aggregation of c. flavifrons resident in an urban area (martins et al., 2014),where fragmented vegetation areas were available, the trophic resources exploited by this population were investigated. material and methods study area the study area was located in an urban area of joão pessoa city (7°9′11″ s; 34°50′28″ w), paraiba state, northeastern brazil. the climate is hot and humid, tropical (as’, accordingly to köppen classification), with annual average temperatures of 26 °c. the rainy season lasts from march to august, and the average annual rainfall is 1,700 mm (lima & heckendorff, 1985). the nest aggregation of c. flavifrons was established in a home garden (642.46 m2 total area) planted with fruit trees (malpighia emarginata dc., mangifera indica l., anacardium occidentale l., spondias purpurea l., eugenia uniflora l., syzygium malaccense (l.) merr. & l.m. perry, psidiumguajava l., citrus limon (l.) osbeck, citrus sinensis (l.) osbeck, cocos nucifera l., carica papaya l., and morus sp.) and grass. the garden had some small areas with bare soil: 22 bare ground circles, approximately 1 m in diameter, surrounding each of the orchard trees, and a narrow strip of land along the walls of the garden (20 cm×167.6 m) used for nesting by the female bees (see more details in martins et al. (2014). surrounding the garden area with the aggregation of nests there are some vacant lots, and at about 1,200 m distant there is a fragment of a well conserved atlantic rainforest with 471 ha, and smaller forest fragments. pollen analysis twelve broodcells from six nests of c. flavifrons from three different generations (nov 2011, feb 2012, and nov/dec 2012) were randomly sampled in this aggregation and selected to the pollen analysis of the provisions. nest content of each sample (a cell) was treated with the acetolysis method (erdtman, 1960). after the chemical treatment, the samples from each cell were mounted in glycerine jelly on five slides. the pollen types were identified using the palynological catalogs (roubik & moreno, 1991;carreira et al., 1996; carreira& barth, 2003; melhem et al., 2003; silva et al., 2010) and the reference slide collection of the palynotheca of plant micromorphology laboratory from universidade estadual de feira de santana (lamiv/uefs). in addition, lists of floristic surveys surrounding the study area were used in order to have knowledge of the local flora (barbosa, 2008; amazonas & barbosa, 2011; gadelha-neto & barbosa, 2012). at least 1,000 pollen grains were counted per sample (and at least 200 pollen grains per slide) for the quantitative analysis, following vergeron (1964). we calculated the occurrence frequency of each pollen type in each sampled brood cell, as well as the averages of the frequencies of each pollen type among all the samples (according with villanuevagutiérrez & roubik, 2004). results from the pollen contents of twelve brood cells from c. flavifrons nests it was elaborated a pollen spectrum (fig 1) containing 16 pollen types. fifteen pollen types were related to eight plant families and only one could not be identified (table 1). byrsonima sericea type, solanum paniculatum type, cestrum type, and myrcia type 1 together accounted for more than 93% of pollen grains foraged by the females of c. flavifrons.thus, the plant species related to these pollen types were the main protein sources in the diet of this bee population. myrtaceae (4 pollen types) and solanaceae (3) were the families with the highest number of different pollen types. other families ranged from one to two different types of pollen grains. however, malpighiaceae was the only family represented in all samples. moreover, it was the family with the second most foraged pollen type (byrsonima sericea) amounting 30.44% of the total number of pollen grains counted. the type b. sericea was identified in eleven of the twelve samples analyzed, ranging from very low percentages (0.10%) to nearly the entire sample (97.07%). although they have been identified in nearly half of the samples, cestrum (23.11%) and solanum paniculatum (30.75%) types had high percentages in the total pollen grains counted. in only one sample, no pollen grains of solanaceae were present. myrtaceae was the most represented family in number of distinct pollen types, however they were present in only six samples. in addition, in one sample a high percentage (97.30%) of myrcia type 1 was found. among the total of 16 pollen types, ten pollen types showed percentages below 10% in the samples analyzed. six types of pollen grains were exclusively found in only one sample and at least five have been identified in six or more samples. mc dórea, far santos, cmlaguiar, cfmartins – pollen sources for centris flavifrons in the city168 fig 1. pollen types identified in nests of centris (centris) flavifrons in an urban area. fabaceae: a. dioclea. lythraceae: b. cuphea. myrtaceae: c-d. myrcia type 1. e. psidium. malpighiaceae: f-g. byrsonima sericea. h. stigmaphyllum blanchetii. solancaceae: i-j. cestrum. k-l. solanum paniculatum. it is noteworthy that brood cells provisioned in the same period by different females could contain a high percentage of the same pollen type (cells from nests 1 to 4 with more than 90% of solanum paniculatum type, fig 2), and cells provisioned by the one female in the same nest showed, or not, a high percentage of the same pollen type (cells 30 and 32 from nest 5 contained 82 to 90% of byrsonima sericea type; while in cells 14 and 31, from the same nest, predominated 97.3% of myrcia type 1 and 82.6% of cestrum type, respectively; a similar pattern occurred in cells from nest 6, table 1, fig 2). 0 20 40 60 80 100 n1 n2 n3 n4 n5 n5 n5 n5 n6 n6 n6 n6 solanum paniculatum type myrcia type 1 byrsonima sericea type cestrum type psidium others fig 2. proportion (%) of different pollen types in brood cells from six nests (n1 to n6) of centris (centris) flavifrons. each column represents one cell, and the sequence of cells is the same presented in table1. sociobiology 64(2): 166-173 (june, 2017) 169 discussion the main feature of the c. flavifrons diet in this urban landscape is the high dependence on a few (four) species of plants as protein sources for rearing offspring, suggesting that this bee population has a narrow realized trophic niche. based on literature data on floral resources available from plant species, chamaecrista type, solanum type, myrcia types 2 and 3, psidium type, brosimum type and laporteaaestuans type are probably related to other pollen sources (machado & lopes, 2006; gressler et al., 2006; pederneiras et al., 2011; gaglioti et al., 2016), but they were little exploited by this population of c. flavifrons in the temporal window analyzed. although stigmaphyllon species are exploited for pollen by centridini bees (sigrist & sazima, 2004), the frequency of occurrence s. blanchetii type in our samples suggested that the related plant was exploited only by oil. cuphea type is related to plant species that supply nectar (cavalcanti & graham, 2002), as also cestrum type (silva et al., 2003). similarly, in an agricultural area surrounded by savanna vegetation, c. flavifrons females collected pollen from different plant species, but they showed stronger interactions with only three species of malpighiaceae and anacardium sp. (vilhena et al., 2012), which were more quantitatively represented in pollen loads of foraging females. although the number of studies on the diet of this species is very low, there are clues that the pollen diet of the larvae tends to be based on a few plant species in different types of landscapes, such as natural vegetation (gaglianone, 2003; rego et al., 2006), crops surrounded by native vegetation (vilhena et al., 2012) and urban areas (this study). a similar trend has been reported for other species of this genus, as centris flavofasciata friese and centris nitida smith also showed a high concentration of foraging for pollen in a few plants pecies (3 to 2 species) in tropical deciduous forest vegetation in mexico (quiroz-garcia et al., 2001; quiroz-garcia & arreguinsánches, 2006). although many pollen types have been collected by the females in these studies, two malpighiaceae species only accounted for most of foraged pollen. nov 2011 nov 2011 nov 2011 nov 2011 feb 2012 feb 2012 feb 2012 feb 2012 novdec 2012 novdec 2012 novdec 2012 novdec 2012 cellcode/nest code 41 62 153 114 145 305 315 325 176 246 256 266 pollen type/samples i ii iii iv v vi vii viii ix x xi xii leg. caesalpinioideae chamaecrista type 0.10 leg. papilionoideae clitoria racemosa type 0.09 0.20 diocleatype 0.20 1.80 1.00 0.20 1.05 0.38 lytraceae cupheatype 2.15 malpighiaceae byrsonima sericea type 1.70 0.50 0.10 0.30 82.45 0.43 90.34 5.77 97.07 37.36 47.71 stigmaphyllon blanchetii type 0.10 0.50 1.10 0.20 1.69 0.19 0.29 1.14 2.39 moraceae brosimumtype 0.17 myrtaceae myrciatype 1 0.10 0.30 97.30 11.44 myrciatype 2 2.10 myrciatype 3 4.89 psidium 8.12 22.15 solanaceae cestrumtype 15.85 82.63 77.79 2.44 38.21 49.52 solanum paniculatum type 94.50 96.50 91.90 91.30 3.10 2.64 0.10 solanumtype 2 3.80 3.10 5.20 6.20 urticaceae laportea type 0.10 0.39 0.10 0.10 und. type 9.47 total 100 100 100 100 100 100 100 100 100 100 100 100 table 1. frequency of pollen types (%), recorded in brood cells/nests of centris (centris) flavifrons within an urban area in the municipality of joão pessoa, paraíba state, brazil. und= undetermined. mc dórea, far santos, cmlaguiar, cfmartins – pollen sources for centris flavifrons in the city170 our findings also indicate that the breeding females of c. flavifrons have the flexibility to replace the primary pollen source in different breeding seasons, as indicated by the replacement of s. paniculatum in nov/2011 season by b. sericea or by cestrum type in the other two nesting seasons. moreover, females of the same generation may focus the foraging for pollen in the same plant species (e.g. nov/2011) or may use two or three different plant species (e.g. feb/2012, nov-dec/2012), which suggests an adjustment of nesting females to the local availability of some preferred sources of pollen. similarly, rêgo et al. (2006) analyzed the pollen content of four cells from different nests of c. flavifrons and found that in each cell the most important pollen type was different. furthermore, this flexibility is reinforced by the provisioning of different cells with different predominant pollen types by the same female in the same nest, observed in this study. our study also showed a low diversity of floral oil sources in the diet of this c. flavifrons population, an essential resource for centridini nesting females, since it is used as material for lining the brood cells, nests waterproofing, and as larval food for many centris species (vinson & frankie, 1988; jesus & garófalo, 2000; aguiar & gaglianone, 2003). another population of c. flavifrons in a savanna vegetation also showed dependence on a single source of oil and pollen (byrsonima intermedia), although several other malpighiaceae species were available during the nesting season (gaglianone, 2003). despite the dependence on a limited number of plant species, the reactivation of the aggregation for several consecutive years, and the production of several hundred nests by this species of bee in the same nest site (martins et al., 2014) indicate that this urban landscape meets an adequate abundance of resources to support this population of c. flavifrons. to the maintenance of this bee population it is noteworthy the existence of solanaceae species, used as a pollen source by this and other species of bees, that grow spontaneously in wastelands (vacant lots or unbuilt areas inside the city), as well as the proximity to areas where there are pioneer species, such as byrsonima sericea, which can reach high densities and provide pollen and oil for the centridini bees (rosa & ramalho, 2011; aguiar, unpubl. data). nevertheless, although b. sericea occurs in the urban matrix, it is more abundant in the close atlantic rainforest fragments 1,200 m distant, a distance that the medium-large sized c. flavifrons can fly (zurbuchen et al., 2010) while s. paniculatum occurs abundantly in wastelands. these two species together accounted for 61% of the collected pollen. fragmentation, habitat degradation, and modern agricultural practices have been affecting the biota by the elimination of resources needed for successful reproduction such as nesting sites and pollen and nectar sources and are responsible for the decline of many bee species populations (müller et al., 2006; andrieu et al., 2009). urban areas are an extreme case of habitat fragmentation; therefore, it is relevant to know the adaptations of the foraging behavior of pollinators in these areas (andrieu et al., 2009). nevertheless, bee species, according to ecological traits associated with nesting and dietary breadth, may respond differently to urban habitat fragmentation in the arizona desert (cane et al., 2006). for instance, oligolectic ground-nesting species were underrepresented in smaller fragments and less abundant in the smaller and older fragments, while decline of polilectic ground-nesting species was weak and insignificant, and cavitynesting bees were overrepresented in the habitat fragments, probably due to enhanced nesting opportunities available in the urban matrix (cane et al., 2006). thus, this knowledge is likely to become crucial to conserve and manage populations of bees in the future. our study shows the importance of wastelands and natural areas to c. flavifrons, and probably other bee species populations in urban areas. wastelands are important areas for ruderal plant species that provide pollen and nectar to maintain crop pollinators in periods of floral resource shortage (cruz & martins, 2015). furthermore, it has been suggested that a close neighborhood of nesting and foraging habitat within few hundred meters is crucial to maintaining populations of most bee species (zurbuchen et al., 2010), which strengthens the proposal of maintaining these habitats in urban areas. concluding remarks using palynological analysis our study showed that the polilectic ground-nesting bee species c. flavifrons actually may rely on few plant species for brood rearing, and that females showed flexibility to replace the primary pollen source in different breeding seasons, or in the same season, probably according to availability and spatial distribution of pollen and oil sources. four pollen types, byrsonima sericea type, solanum paniculatum type, cestrum type, and myrcia type 1 together accounted for most of the pollen grains (>90%) foraged by females. thus, the plant species related to these pollen types are key pollen sources for the maintenance of this bee population. some plant species occur in a forest fragment relatively close to the study area but others grow in vacant lots inside the city, highlighting the importance of keeping natural areas and wastelands inside urban areas for keeping this, and likely other bee species and animals. in the future the planning of cities should consider the use of natural areas including areas that can be used for entertaining, human well-being, and at same time for keeping flora and fauna in general. therefore, more studies, especially long term approaches, arenecessary to understand the population dynamicsof c. flavifrons and its interactions with plant species in natural and urban areas. sociobiology 64(2): 166-173 (june, 2017) 171 acknowledgments we thank carolina liberal, jerônimo kahn villas bôas, liedson tavares carneiro, marcella pereira peixoto, renata marinho cruz, rita p. lima and vaneide lopes do nascimento, for the help in field work; 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(2010). maximum foraging ranges in solitary bees: only few individuals have the capability to cover long foraging distances. biological conservation, 143: 669-676. doi: 10.1016/j.biocon.2009.12.003 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5935sociobiology 68(1): e5935 (march, 2021) introduction maternal effects have been recognized in animals and plants for a long time (bernardo 1996; roach & wulff, 1987). maternal effects are that the phenotype of offspring is influenced by maternal phenotype rather than its own genotype (van dooren et al., 2016; schwabl & groothuis, 2019). vertebrate females can adjust their investment in eggs to get higher quality offspring (cunningham & russell, 2000). insects can also adaptively vary investment in their eggs (passera, 1980). honeybees are typical eusocial insect species, and the queen is the only female individual amongsterile workers (winston, 1991). previous studies showed that female larvae fed with richer nutritional diet develop into queens, whereas lower nutritional diet results in the development of workers (haydak, 1970). the space in abstract queen-worker caste dimorphism is a typical trait for honeybees (apis mellifera). we previously showed a maternal effect on caste differentiation and queen development, where queens emerged from queen-cell eggs (qe) had higher quality than queens developed from worker cell eggs (we). in this study, newly-emerged queens were reared from qe, we, and 2-day worker larvae (2l). the thorax size and dna methylation levels of queens were measured. we found that queens emerging from qe had significantly larger thorax length and width than we and 2l. epigenetic analysis showed that qe/2l comparison had the most different methylated genes (dmgs, 612) followed by we/2l (473), and qe/we (371). interestingly, a great number of dmgs (42) were in genes belonging to mtor, mapk, wnt, notch, hedgehog, foxo, and hippo signaling pathways that are involved in regulating caste differentiation, reproduction and longevity. this study proved that honeybee maternal effect causes epigenetic alteration regulating caste differentiation and queen development. sociobiology an international journal on social insects xu jiang he1,3, hao wei1, wu jun jiang2, yi bo liu1, xiao bo wu1, zhi jiang zeng1,3 article history edited by pilar de la rua, univ. de murcia, spain received 03 november 2020 initial acceptance 02 december 2020 final acceptance 14 january 2021 publication date 30 march 2021 keywords honeybees; maternal effect; development; caste differentiation; dna methylation. corresponding author prof. zhi jiang zeng honeybee research institute jiangxi agricultural university nanchang 330045, p.r. of china. e-mail: bees1965@sina.com which larvae develop within the cell also contributes to the queen-worker differentiation (shi et al., 2011). however, our previous study showed a maternal effect on honeybee caste differentiation, resulting in high quality queens. queens lay larger eggs into queen cells compared to worker cells, which results in queens with heavier body, more ovarioles and different gene expression patterns (wei et al., 2019). this maternal effect likely depends on the nutritional content of the fertilized queen cell egg, and not on genomic differences between queen-cell eggs and worker cell eggs. however, the underlying epigenetic mechanism for this maternal effect remains unclear. more importantly, the quality of the queen has strong effects on the fitness of a colony (winston, 1991; gilley et al., 2003; amiri et al., 2017). in recent years, honeybee colony loss has been frequently reported in public media, 1 honeybee research institute, jiangxi agricultural university, nanchang, jiangxi, p. r. of china 2 honeybee research institute of jiangxi province, nanchang, jiangxi, p. r. of china 3 jiangxi key laboratory of honeybee biology and bee keeping, nanchang, jiangxi, p. r. of china research article bees honeybee (apis mellifera) maternal effect causes alternation of dna methylation regulating queen development file:///c:/users/ane%20c%c3%a9lia/desktop/sociobiology%2068(1)_mar%c3%a7o2021/artigos-word/../../../../../../../../dell/appdata/local/youdao/dict/application/6.3.69.8341/resultui/frame/javascript:void(0); file:///c:/users/ane%20c%c3%a9lia/desktop/sociobiology%2068(1)_mar%c3%a7o2021/artigos-word/../../../../../../../../dell/appdata/local/youdao/dict/application/6.3.69.8341/resultui/frame/javascript:void(0); xu jiang he, hao wei, wu jun jiang, yi bo liu, xiao bo wu, zhi jiang zeng – dna methylation and honeybee maternal effect2 and the farming industry has been significantly impacted by the death of honeybees (michael et al., 2009; steinhauer et al., 2013; antúnez et al., 2016). for example, scientists showed a high rate of honeybee winter colony loss over 13 years from 2006-2019 (milius, 2019). the poor quality of queens was considered as one of main factors for honeybee winter loss (vanengelsdorp et al., 2010; delaney et al., 2011). rudolf steiner argued in 1923 that honeybees would become extinct within 100 years due to the weakening of queens in the commercial queen rearing industry (thomas, 1998). commercial beekeepers rear new queens by transplanting young worker larvae into queen cells, altering the larva’s diet and depriving the maternal effect from the queen (doolittle, 1888; büchler et al., 2013). there are many concerns about the adverse consequences of this queen rearing technology for queen quality and colony health. woyke (1971) showed that queens reared from young worker larvae were smaller and had a smaller spermatheca and fewer ovarioles compared to queens from worker eggs. rangel et al. (2013) reported that queens reared from old worker larvae produced fewer workers and drone combs and less honey than colonies with queens reared from young worker larvae. he et al. (2017) and wei et al. (2019) found that queens developed from queen cells are better than queens reared from worker cell eggs and young larvae. a recent study clearly showed that honeybee queens have an ability to alter egg size in response to both genetic and environmental factors (amiri et al., 2020). therefore, honeybee maternal effects may potentially influence the quality of the queen. dna methylation plays an important role in regulating honeybee queen development (kucharski et al., 2008; shi et al., 2011; maleszka, 2008). honeybee queens have lower genome-wide methylation levels than workers (shi et al., 2013). the dna methyltransferase dnmt3 profound shifts honeybee reproductive status (kucharski et al., 2008). libyarlay et al. (2013) showed that knocking down dna methyltransferase 3 (dnmt3) changes gene alternative splicing in honeybees. we previously found that transplanting younger larvae from worker cells result in better queens with lower dna methylation levels (he et al., 2017). cardoso-júnior et al. (2018) reported that dna methylation altered queen lifespan by regulating the expression of vitellogenin gene. therefore, we used honeybee eggs laid in queen cells to rear queens and compare dna methylation among queens reared from queen-cell eggs (qe), worker-cell eggs (we) and 2-day worker larvae (2l). our results showed that maternal effect caused dna methylation alternation in honeybee queens. materials and methods insects three honey bee colonies (apis mellifera) were used throughout this study. virgin queens were sisters reared from the same mother queen in order to minimize differences in genetic background. each colony had nine frames with approximately 30,000 workers and a single drone inseminated queen (sdi). these colonies were kept at the honeybee research institute, jiangxi agricultural university, nanchang, china (28.46 un, 115.49 ue). queens were collected from the same colonies as in a previous study (wei et al., 2019). queen rearing queens were caged in a plastic queen-cell frame for 6 hrs, and then transferred into a plastic worker-cell frame to lay worker-cell eggs following methods in wei et al., (2019). plastic queen cells frames were arranged horizontally. generally, 20-50 eggs were harvested from queen cells after 6 hrs laying. some worker-cell eggs were placed into plastic queen cells by transferring the base of each cell (pan et al., 2013). the rest of the worker-cell eggs were kept in their native colonies until they reached 2d worker larvae stage. these 2d worker larvae were removed and placed into plastic queen cells. cells from qe, we and 2l were mixed together and placed into the same colony to rear queens. newly emerged queens were harvested immediately for morphological measurement and dna methylation sequencing. morphological measurements thoraxes of queens were collected and placed under a zoom-stereo microscope system (panasonic co., ltd., accuracy: 0.01 mm). the width and length were measured according to the manufacturer’s instructions. each queen group had seven biological replicates, therefore, totally 21 queens were used for morphological measurements. dna methylation sequencing newly emerged queens were placed into liquid nitrogen, and heads and thoraxes were collected for total dna extraction. each sample contained tissue from two queens, and three biological replicates were conducted for each rearing condition. the qe group had two biological replicates since the sequencing of the third sample failed. total dna was extracted using universal genomic dna extraction kit (takara, dv811a) following the manufacturer’s protocol. concentrations of all samples were measured and adjusted to the same level, and dna samples (300 ng dna for each sample) were used for dna methylation sequencing by illumina hiseq™ 2500 (illumina inc., ca, usa). the detailed methods are listed in he et al. (2017). briefly, dna was sheared into 200-300 bp insert size targets using covaris ultrasonicator (life technology) and then purified using ampure xp beads and end repaired. a single ‘a’ nucleotide was added to the 3’ends of the blunt fragments followed by ligation to methylated adapter with a t overhang. insert size targets (200-300 bp) were purified by 2% agarose gel electrophoresis. a zymo ez dna methylation-goldtm kit (zymo, irvine, ca, usa) was used to conduct bisulfite conversion. bisulfite libraries were generated by pcr https://www.sciencenews.org/author/susan-milius file:///c:/users/ane%20c%c3%a9lia/desktop/sociobiology%2068(1)_mar%c3%a7o2021/artigos-word/../../../../../../../../dell/appdata/local/youdao/dict/application/6.3.69.8341/resultui/frame/javascript:void(0); file:///c:/users/ane%20c%c3%a9lia/desktop/sociobiology%2068(1)_mar%c3%a7o2021/artigos-word/../../../../../../../../dell/appdata/local/youdao/dict/application/6.3.69.8341/resultui/frame/javascript:void(0); sociobiology 68(1): e5935 (march, 2021) 3 amplification and quantified by qpcr (agilent qpcr ngs library quantification kit). in total, eight libraries were prepared and sequenced. these processes were performed in beijing biomarker technology co., ltd (beijing, china). low quality reads (reads containing adapter sequences, reads containing unknown nucleotide “n” over 10 % and reads with a quality value lower than 10 occupying more than 50% of the whole read) from eight bisulfite libraries were filtered. these filtered genomic fragments were then mapped to the honeybee genome (apis mellifera. amel 4.5), transformed into bisulfite-converted versions of the sequences (c-to-t and g-to-a) and then assigned to a digital index using bowtie2 according to langmead and salzberg (2012). methylation sites were predicted using a bismark methylation extractor (krueger & andrews, 2011). only uniquely mapped reads (clean reads) were retained. the ratio of c to ct was used to measure methylation levels. the bismark package was used to test 5mc (same to krueger & andrews, 2011) and different methylation regions (dmrs) were identified using bisulfighter (yutaka et al., 2014), according to our previous study (he et al., 2017). dmr related genes (dmgs) were annotated by mapping the target regions [gene body region (from transcription start sites to transcription end sites) or promoter region (upstream 2kb from the transcription start sites)] to the honeybee genome (amel 4.5), since dna methylation data was used to compare with our previous rna-seq data (wei et al., 2019). data analysis for queen thorax length and width, data were analyzed using an analysis of variance (anova) followed by fisher’s plsd test in statview 5.01 package (sas, usa), where p < 0.05 was considered as significantly different. results qe were larger than we and 2l the thorax length and width of qe were significantly higher than that of we and 2l. in addition, the thorax size of we was significantly higher than 2l (fig 1, p < 0.05). quality of methylation sequencing in total, 43.44 g clean bases were detected from eight libraries. q30 values of samples were over 85 % and the average ratio of bisulfite conversion reached to 99 % (table s1). these values indicate a considerably high sequencing quality. dmrs and dmgs contribute to queen development there were hundreds of dmrs among three queen types (fig 2 and s1). dmrs (cg type) were mapped to all 16 chromosomes. most chromosomes, such as chr4, chr5, chr10 and chr14, had more dmrs in qe/2l when comparing to we/2l and qe/we (fig 2). the dmrs in chh type were less than the cg type but showed a similar pattern (fig s1). there were no dmrs of chg type could map to the chromosomes. fig 1. thorax length and width of newly emerged queens from qe, we and 2l. each bar shows mean ± se of thorax length or width. each group had seven biological replicates. data were analyzed by anova test followed by fisher’s plsd test. different characters on the top of bars represent significant difference (p < 0.05), same character indicates no difference (p > 0.05). xu jiang he, hao wei, wu jun jiang, yi bo liu, xiao bo wu, zhi jiang zeng – dna methylation and honeybee maternal effect4 fig 2. distribution of significantly differentially methylated regions (dmrs, mcg) from three comparisons for the 16 honeybee chromosomes. the dmrs of qe/2l, we/2l and qe/we comparisons are presented from outer to inner, respectively. red plots are upregulated dmrs and green plots are downregulated ones. chromosome name and scale are indicated on the outer rim. fig 3. a: significantly differentially methylated genes (dmgs) in three comparisons. white bars represent upregulated dmgs in each comparison (former compares to latter) and grey bars represent downregulated ones. detailed information of each dmg refers to table s2 to 4. b: the venn diagram of dmgs among qe,we and 2l. qe/2l had more dmgs (612) than we/2l (473) and qe/we (371) comparisons (fig 3, table s2-4). these dmgs were enriched in 72 kegg pathways which belong to four kegg categories including metabolism, gene information processing, environmental information processing, cellular processes and organismal systems (fig 4). moreover, 42 dmgs sociobiology 68(1): e5935 (march, 2021) 5 enriched in environmental information processing were genes belonging to important signaling pathways, such as mtor, mapk, notch, and wnt signaling (fig 5), which are involved in regulating queen development and reproduction (chen et al., 2012; chen et al., 2017). similarly, qe/2l comparison had more dmgs (21) of these 42 genes than we/2l (20) and qe/we (17) comparisons (fig 5). dna methylation had a low relationship with previous rnaseq data by comparing the dna methylation data to our previous rna-seq data from wei et al., (2019), only 2 (gb41428 and gb46222), 1 (gb41428) and 0 dmgs were mapped to degs in qe/2l, we/2l and qe/we comparisons respectively (fig s2). fig 4. enrichment of dmgs to kegg categories and pathways. all dmgs from qe/2l, we/2l and qe/we comparisons were mapped to 72 kegg pathways which belong to four categories (color marked, right side). bars indicate number of dmgs in each pathway. we also compared 35 immunity and development degs with our dna methylation data, and the result (fig 6) showed that the differences of gene expression and dna methylation of these genes both increased as the age of transplant of the worker larvae increased. however, there wasn’t a clear negative correlation between gene expression and dna methylation in these genes (fig 6). discussion environmental factors, such as larval diet and maternal effect, contribute towards honeybee caste differentiation and queen development (haydak, 1970; wei et al., 2019). in this study, queens developed from queen-cells had significantly larger thorax size than those from worker eggs and young xu jiang he, hao wei, wu jun jiang, yi bo liu, xiao bo wu, zhi jiang zeng – dna methylation and honeybee maternal effect6 worker larvae (fig 1). many studies have shown that queen weight and body size are strongly correlated with queen ovariole number which influences queen fecundity and quality (borodacheva, 1973; bilash et al., 1983; huang & zhi, 1985; he et al., 2017; wei et al., 2019). consequently, these results from the present study are consistent with our previous study (wei et al., 2019) where we showed that qe had significantly higher weight and ovariole number than both we and 2l. we conclude there is a strong maternal effect on honeybee queen development and potentially contributes to the queen quality. previous studies showed that queens have lower global dna methylation level than workers, and dna methylated genes such as genes involved in mtor pathways can control queen-worker dimorphism (kucharski et al., 2008; chen et al., 2012; shi et al., 2013). here, we found that qe/2l had the most dmrs and dmgs, followed by we/2l and qe/we (fig 2 and 3). in addition, 42 dmgs were involved in 11 signaling pathways such as mtor, mapk, wnt and notch pathways (fig 4 and 5) that are known to regulate honeybee caste differentiation and queen development (chen et al., 2012; chen et al., 2017). fig 5. dna methylation of 42 dmgs enriched in 11 castedifferentiation related kegg pathways among qe, we and 2l. blue indicates significant downregulation in each comparison, red indicates upregulation and green indicates no difference. left side is the gene id and kegg pathways. some genes were involved in two or more pathways; therefore they were marked with different color lines. these dmgs induced by maternal effect are strongly related to queen-worker differentiation, reflecting that honeybee maternal effect could cause various epigenetic alteration in caste differentiation and queen development. how is dna methylation regulated by maternal effects? different diet during larval stage alters dna methylation (wang et al., 2006; kucharski et al., 2008; shi et al., 2011; foret et al., 2012; maleszka, 2008). we previously showed that queens deposited larger eggs into queen cells (wei et al., 2019). during development, eggs absorb nutrients such as vitellin from the ovary epidermal cells for 21 days (torres, 1980; li & zhang, 2017). larger eggs found in queen cells may contain more nutrients than eggs in worker cells, therefore, the nutrients available to the developing embryo may alter dna methylation and promote queen development. however, the kind of nutrients in the eggs and how queens control egg size requires further investigations. moreover, dna methylation plays an important role in soft inheritance and animal evolution (bird, 2002; dickins & rahman, 2012; klironomos, 2013). maternal effect also dramatically contributes to the process of animal evolution (galloway et al., 2009; marshall & uller, 2007). the commercial queen rearing technology started in the 19th century (doolittle, 1888) artificially transplants young worker larvae into queen cells to rear queens rather uses queen cell eggs. the widely use of commercial queen rearing technology for over 100 years continuously deprives the investment from mother queens (namely maternal effect) and may consistently weakens the queen and honeybee colony via altering dna methylation. as a proximal remedy, rearing queens from queen-cell eggs may be a good strategy to yield high quality queens and healthy colonies. similar to our previous study (he et al., 2017), our dmgs had a low relationship with previous rna-seq results (wei et al., 2019). only 3 of 1456 dmgs were mapped to our previous degs data (fig s2). evidence showed that honeybee dna methylation has an association with alternative splicing and gene duplication (elango et al., 2009; dyson & goodisman, 2020), but there isn’t a completely direct link between dna methylation and gene expression. the present study and our previous study showed a same pattern that qe/2l comparison had more dmgs and degs than qe/ we and we/2l comparisons (see fig 3 from this study and fig 3 from wei et al., 2019). many dmgs and degs were involved in queen-worker differentiation, body development and immunity etc., however they did not show a clear negative or positive relationship with our dna methylation data (fig 6). how dna methylation regulating gene expression remains essentially unknown in honeybees. dna methylation has been shown to associate with chromosome structure and histone modifications (hunt et al., 2013; dmitrijeva et al., 2018). perhaps the alternative splicing, chromosome structure, histone modification and other undiscovered factors jointly contribute to the regulation on honeybee gene expression by dna methylation, which needs further investigations. sociobiology 68(1): e5935 (march, 2021) 7 in conclusion, this study firstly indicates that honeybee maternal effect causes dna methylation changes in queen rearing and potentially contributes to the queen development and colony health, due to the function of dna methylation in soft inheritance and animal evolution. since the poor quality has been frequently reported as a main factor for colony losses (vanengelsdorp et al., 2010; delaney et al., 2011), therefore, the maternal effect should be deeply considered and used in commercial queen rearing. acknowledgments this work was supported by the national natural science foundation of china (31702193 and 31960685), natural science foundation of jiangxi province (20171ba b214018), key research and development project of jiangxi province (20181bbf60019) and the earmarked fund for china agriculture research system (cars-44-kxj15). fig 6. gene expression and related dna methylation levels of 35 differentially expressed genes. gene expression ratios and dna methylation ratios (colour coded by scale bars) of selected degs with proposed functional roles in immunity and development. the left heat map is gene expression and the right is dna methylation. xu jiang he, hao wei, wu jun jiang, yi bo liu, 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(2014). bisulfighter: accurate detection of methylated cytosines and differentially methylated regions. nucleic acids research, 42: e45. doi: 10.1093/nar/gkt1373 https://doi.org/10.1093/nar/gkt1373 sociobiology 68(1): e5935 (march, 2021) 11 figure s2. the venn diagram of dmgs and degs from wei et al., 2019. figure s1. distribution of significantly differentially methylated regions (dmrs, mchh) from three comparisons for the 16 honeybee chromosomes. the dmrs of qe/2l, we/2l and qe/we comparisons are presented from outer to inner, respectively. red plots are upregulated dmrs and green plots are downregulated ones. chromosome name and scale are indicated on the outer rim. supplementary material samples clean reads clean bases unique mapped gc (%) q20 (%) q30 (%) conversion rate (%) qe-replicate 1 20,822,309 5,244,037,368 12,707,931 15.76 91.70 86.16 99.77 qe-replicate 2 21,869,019 5,509,562,460 14,424,345 14.99 96.95 91.40 99.44 we-replicate 1 21,190,334 5,338,626,350 12,532,053 14.60 90.80 85.30 99.66 we-replicate 2 20,298,721 5,113,801,644 13,916,498 16.39 96.95 91.34 99.47 we-replicate 3 20,104,750 5,063,976,252 11,664,300 17.65 90.70 85.05 99.75 2l-replicate 1 25,195,534 6,347,643,620 14,766,653 15.27 90.65 85.00 99.76 2l-replicate 2 21,978,784 5,537,649,298 14,005,146 20.60 95.87 88.84 99.42 2l-replicate 3 21,023,761 5,296,681,454 12,266,636 16.56 91.09 85.40 99.77 table s1. summary of sequencing data. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.6022sociobiology 68(3): e6022 (september, 2021) introduction one of the benefits of living in social groups is the availability of defense, and one of the most effective ways of group defense is attacking the presumed predator (siebenaler & caldwell, 1956; vogel & fuentes-jiménez, 2006). for example, wasp and bee colonies can group attack a potential predator, usually mobilized by volatile chemicals, such as the alarm pheromone (morse & laigo, 1969; breed et al., 1990, 2004). abstract in social insects, situations can arise that threaten an individual or an entire colony. when the call for help goes out, different behavioral responses are elicited by signals emitted from nestmates. in ants, the response can be one of redemptive behavior by the worker receiving it. however, little is known about the evolution of this behavior and in which group of ants it manifests. therefore, this study investigates whether workers of odontomachus brunneus patton can act as rescuers, able to detect and respond to calls for help from nestmates. laboratory experiments were carried out in which the legs of ants were trapped by tape, simulating capture by a predator. nearby were nestmates able to receive and respond to a request for help. two experiments were performed: 1. calls for help were made at different distances, in order to test the response latency. 2. evaluation of whether rescuers would respond differently to calls for help from nestmates, non-nestmates of the same species, and ants of another species. finally, evaluation was made of the behaviors of the rescuers when they responded to requests for help from nestmates and ants of another species. it could be concluded from the results that o. brunneus workers respond to signals emitted by workers who may have been captured by a potential predator, prompting the performance of behaviors related to rescue attempts. the signals involved appear to have an optimal range and are species-specific. when exposed to a capture situation, this species transmits audible signals by stridulation, so it is possible that this type of signal may be involved, in addition to chemical signaling. sociobiology an international journal on social insects luiz c santos-junior1,2, emerson p silva3, william f antonialli-junior2 article history edited by kleber del-claro, ufu, brazil received 24 november 2020 initial acceptance 24 march 2021 final acceptance 17 may 2021 publication date 13 august 2021 keywords poneromorph; rescuers, intraspecific recognition; rescue. corresponding author luiz c. santos junior programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados rodovia dourados/itahum, km 12 unidade ii, caixa postal: 364 cep: 79804-970 dourados-ms, brasil. e-mail: lc.santosjunior@yahoo.com.br in some groups of ants, the morphologically modified caste of larger workers has, among other functions, the role of group defense when the colony is put under possible threat from an invader (wilson, 1976; hölldobler & wilson, 1990; powell & clark, 2004; pepper & herron, 2008; powell, 2008; hou et al., 2010; strassmann & queller, 2010). ants defend their colonies using various structures, when threatened by an individual from outside of the colony. in this way, bites and stings are commonly used in coordinated attacks 1 programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil 2 laboratório de ecologia comportamental, universidade estadual de mato grosso do sul, dourados-ms, brazil 3 museu da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil research article ants do odontomachus brunneus nestmates request help and are taken care of when caught? luiz c santos-junior, emerson p silva, william f antonialli-junior – odontomachus brunneus nestmates request help2 (hölldobler & wilson, 1990), as a strategy for group defense of the colony. some ant species have developed a unique means of defending themselves (beponis et al., 2014). if an ant is caught, nestmates may exhibit rescue behavior to save it (czechowski et al., 2002; nowbahari et al., 2009; nowbahari & hollis, 2010; miler, 2016). this behavior can be exhibited by one or more workers, known as first responders, and is directed towards another individual (the victim), in order to rescue it from a predator situation (nowbahari & hollis, 2010; miler, 2016). such behavior can involve relatively simple digging around the victim, with pulling of its limbs, to more precise behaviors such as directly attacking and stinging the trapping animal or object (taylor & visvader, 2013; miler, 2016). independent of the species that can perform this type of behavior, there must be some exchange of signals between the ant that is requesting help and the rescuers. ants and other social hymenopterans can produce and release volatile substances in the form of pheromones (wheeler & blum, 1973; jafé & marcuse, 1983; lahav et al., 1999; morgan et al., 1999; lenoir et al., 2001; hernández et al., 2002; howard & blomquist, 2005; blomquist & bagnères, 2010; sainzborgo et al., 2011). they can also emit acoustic signals (markl, 1973; golden & hill, 2016), such as those produced by the stridulatory organ, located between the petiole and the gaster in ants (markl, 1973; taylor, 1978; stuart & bell, 1980; hölldobler & wilson, 1990; golden & hill, 2016). this organ emits a “beep” that has different roles in the functioning of the colony (markl & hölldobler, 1978; chiu et al., 2011). in the odontomachus genus, workers, in particular, produce sounds in the form of stridulation, when they feel threatened (markl, 1973; golden & hill, 2016). therefore, it is likely that ants of the species odontomachus brunneus patton, 1894 may emit this type of signaling as a way of recruiting their nestmates, when they are exposed to a dangerous situation. the signaling may involve chemicals, sound signals, or a combination of both methods. most of the studies on this topic have been performed for ants inhabiting sandy areas, with exposure to lion ant larvae (neuroptera: myrmeleontidae). this relationship seems to have contributed to the evolution of rescue behavioral patterns that prevail in ants, especially in species of the genera cataglyphis, formica, and lasius, all belonging to the subfamily formicinae (gotelli, 1996; czechowski et al., 2002; hollis & nowbahari, 2013a; miler, 2016; hollis, 2017). however, studies show that ants from other subfamilies, such as myrmicinae and ponerinae, are also capable of exhibiting some kind of rescue behavior (hollis & nowbahari, 2013a; frank et al., 2017, 2018). hence, the occurrence of rescue behavior in relatively unrelated ant species suggests that this behavior is not phylogenetically restricted and that many factors may contribute to its occurrence. unfortunately, little is known about rescue behavior in poneromorphic ants. therefore, the aim of this study was to evaluate whether workers of o. brunneus, when exposed to a capture situation, emit some kind of signal that provokes help in the form of attempted rescue by nestmates. materials and methods six colonies of o. brunneus were collected in the urban area of dourados, in mato grosso do sul state, brazil (22º13’16’’ s; 54º48’20’’ w), between september 2016 and september 2017. all the ants were collected from hollow trunks of caesalpinia pluviosa (fabaceae), using tweezers, and were placed in plastic pots. in the laboratory, the ants were housed in artificial nests constructed using plastic trays (10 x 20 cm). inside there were plaster molds that simulated the nest chambers, which were connected to a foraging arena, where food was offered to the colonies. the colonies were kept at a controlled temperature of around 25.0 ± 1 ◦c, relative humidity of 65 ± 5%, and 12-hour photoperiod, for a seven-day habituation period, and were fed ad libitum with water and honey on moistened cotton inside an eppendorf tube. as a protein source, last instar tenebrio molitor linnaeus, 1758 larvae were offered every five days. the behavioral tests were performed after the habituation period. latency time and call for help to nestmate rescuer ants tests were conducted in order to understand if o. brunneus first responders could respond to their nestmates call for help, by simulating capture at different distances from the first responders. a system of plastic chambers and connectors (15 x 10 x 8 cm) was constructed, allowing for the insertion of rescuers and a trapping chamber (10 x 8 cm) where an ant was attached to simulate its capture. these two sites were connected by a tube 2 cm in diameter and with different lengths (30, 60, and 90 cm), in order to assess whether the call for assistance might vary as a function of distance (fig 1). ten foraging workers were inserted in the arena as potential rescuers. in the trapping chamber, a forager from the same colony was secured by tape, following the modified methodology used in rescue behavior studies by artificial imprisonment in ants (nowbahari et al., 2009, 2012, 2016; hollis & nowbahari, 2013a; duhoo et al., 2017). prior to each test, the arena, the trapping chamber, and the connector tube were sterilized with an alcohol-soaked filter paper. the tests were conducted under laboratory conditions, at a constant temperature of 25.0 ± 1 ◦c and humidity of 65 ± 5%. for each of the three different connector distances, 30 different ant groups were tested, with each group including 10 rescuers and 1 trapped ant. in each test, the observation time was 15 min, from the moment the ant was immobilized in the trapping chamber and all the rescuers were released into the arena. at the end of each test, the number of rescuers entering the trapping chamber was counted, so it was possible to assess sociobiology 68(3): e6022 (september, 2021) 3 whether distance was a factor affecting the number of ants able to respond to the call for help. rescuer latency in responding to the call for aid was calculated as described by nowbahari et al. (2016). this was defined as the period between the time of entry of the first rescuer into the connector tube and the time of the first attempt at rescuing the trapped ant performed by that rescuer. as a control, the same parameters were measured under the same conditions and with the same group of ants used in each test, but without the ant in the trapping chamber. fig 1. scheme used for the latency tests of rescue behavior between o. brunneus nestmates. a: arena connected to the trapping chamber through a 30 cm connector tube; b: arena connected to the trapping chamber through a 60 cm connector tube; c: arena connected to the trapping chamber through a 90 cm connector tube. test to evaluate the specificity of the response to help requests three different help request situations were simulated, in order to evaluate whether workers from a particular colony might respond to a request for help from non-nestmate ants (nowbahari & hollis, 2010; miler, 2016; uy et al., 2018). the chamber system and connectors used in this test were adapted from yusuf et al. (2014). the system included an arena where 10 rescuers were inserted, connected to a tube 25 cm long and 2 cm in diameter, the end of which had a connector allowing bifurcation into two other tubes, each 5 cm long and with the same diameter. each led to a plastic trapping chamber (10 x 8 cm), where at least one of them had a nestmate attached by tape. then, either a rescue ant from the same colony was inserted, or an ant from another colony and/ or species was inserted (fig 2). in each test, the observation time was 15 min, from the moment the ants were trapped in the imprisonment chambers and all the rescuers were released into the arena. three types of experiments were conducted. in the first, rescuers were tested for their ability to receive the call for help and respond to it, using a y-maze system. for this, an o. brunneus nestmate worker was fixed in only one of the entrapment chambers (fig 2-a), while the other chamber contained a loose nestmate. in the second experiment, rescuers were tested for their ability to distinguish between the requests for help made by a nestmate and a non-nestmate. for this test, a nestmate was trapped in one imprisonment chamber, while the other chamber contained a trapped nonnestmate (fig 2-b). in the third experiment, a nestmate was immobilized in one entrapment chamber, while the other contained a worker of another species, in this case odontomachus chelifer, latreille, 1802 (fig 2-c). each of the three experimental designs was performed using thirty tests with different groups of rescuers and trapped workers. all the behaviors exhibited by the rescuers upon entering the trapping chamber were observed and described according to the methodology of nowbahari et al. (2016). to obtain the average frequency of each behavioral act performed by the rescuers, the behaviors at the end of all the tests were summed and divided by the sum of execution of all the behavioral acts. luiz c santos-junior, emerson p silva, william f antonialli-junior – odontomachus brunneus nestmates request help4 statistical analysis differences among the treatments and the controls were evaluated using the student’s t-test applied to the average values for the number of rescuers remaining in the trapping chamber in the tests performed with the connectors of three different lengths (distances). the same test was used to evaluate differences between the average latency times of the rescuers in answering the requests for help by the trapped workers in the tests performed with the different connectors, as well as when there was no trapped worker (controls). the kruskal-wallis test was applied to determine any significant differences among the average latency times in responding to the request for help from a trapped worker, for the three different distances. a t-test was applied to evaluate any significant differences among the mean values of the number of first responders that remained in the trapping chamber during the y-maze decision tests. results in all the tests, independent of distance, rescuers went to their nestmate and performed rescue behaviors, using bites and stings against the tape that held it. however, the number of rescuers reaching the trapping chamber was significantly higher when the distance was 30 cm. the average numbers of ants in the trapping chamber with the trapped ant and in the control situation are shown in table 1. the latency times showed that the rescuers took less time to respond to the call for help within 30 cm. at this distance, there was a significant difference between the times recorded with an ant trapped in the chamber and with no ant (table 2). in the tests to assess specificity in responding to requests for help, the rescue workers opted for a trapping chamber containing a trapped worker from the same colony, as opposed to a nestmate loose in a chamber and/or an ant of another species in the chamber. no significant differences were found between the numbers of times rescuers opted for a chamber fig 2. scheme used for the y-maze decision tube tests with the rescue workers in the arena and the workers in the trapping chambers. a: test with nestmate workers loose and trapped in the trapping chambers; b: test with a trapped nestmate worker in one chamber and a trapped non-nestmate worker in the other chamber; c: test with a trapped nestmate in one of the chambers and a trapped o. chelifer worker in the other chamber. sociobiology 68(3): e6022 (september, 2021) 5 distance 30 cm 60 cm 90 cm with ant without ant with ant without ant with ant without ant latency (minutes) 0:29 ± 0.01 2:43 ± 0.01 3:00 ± 0.02 3:03 ± 0.02 9:27 ± 0.21 8:52 ± 0.20 p-value <0.0001 0.732 0.955 t-test -2.5 0.34 0.05 decision test nestmate (trapped) nestmate (loose) nestmate non-nestmate nestmate o. chelifer average number of rescuers 8.1 ± 1.24 1.00 ± 1.31 5.33 ± 3.45 4.13 ± 3.61 7.60 ± 1.67 1.06 ± 1.17 p-value <0.001 0.35 <0.001 t-test 0.80 0.93 1.31 table 2. average latency times of rescue ants present in the capture chamber positioned at different distances. table 3. average numbers of rescue ants present in the trapping chambers when an ant was trapped on one side of the connector tube. with a nestmate, compared to a chamber with a non-nestmate (table 3). the behaviors exhibited by the rescuers when they arrived at the trapping chamber are described in table 4. the behaviors suggested that some kind of rescue was only performed to assist either a nestmate or a non-nestmate of the same species (table 4). discussion the range of signals involved was even more evident from analysis of the significant differences between the distances in terms of latency time. alarm pheromones are volatile chemical compounds used for communication by various social insects (crewe et al., 1972; traniello, 1982; blomquist & bagnères, 2010). these include various species of less derived ants (robertson, 1971; hölldobler & taylor, 1983; hölldobler & wilson, 1990), such as o. bauri (sainzborgo et al., 2011). hölldobler and wilson (1990) reported that these alarm pheromones are transmitted over short distances and are coded by workers able to respond in various ways, such as by attacking. the diffusion model of bossert and wilson (1963) predicts that the alarm pheromone emitted by ants can reach a radius of approximately 20cm, in the absence of a draft. therefore, in this study, if the ants responded only to chemical signals, the range of these compounds would be greater, since some response occurred even at a distance of 90 cm. however, it should be noted that the testing employed a chamber-and-tube system, which may have reduced dispersion and assisted the targeting of the volatile compounds. another consideration is that when immobilized, the workers of o. brunneus make an audible sound produced by stridulation. although this would need to be tested, it should be highlighted here that the workers could emit this complementary signal, in order to enlist help. this acoustic signal is produced by friction between the petiole and the gaster (markl, 1965; taylor, 1978; hölldobler & wilson, 1990; grasso et al., 2000), as observed previously in ants of this genus (markl, 1973; golden & hill, 2016). among other functions, these signals emitted by ants may be a call for help, as indicated in several ant rescue behavior studies (czechowski et al., 2002; nowbahari et al., 2009; nowbahari & hollis, 2010; miler, 2016; frank et al., 2017, 2018). therefore, it is possible that nestmates may have emitted a chemical and/or audible signal that was coded by rescuers, distance 30 cm 60 cm 90 cm with ant without ant with ant without ant with ant without ant average number of rescuers 8.73 ± 1.05 1.53 ± 1.66 3.53 ± 1.31 3.33 ± 1.49 3.03 ± 1.27 2.06 ± 1.54 p-value <0.001 0.54 0.16 t-test 21.81 0.61 1.43 table 1. average numbers of rescue ants attending nestmates trapped at different distances. luiz c santos-junior, emerson p silva, william f antonialli-junior – odontomachus brunneus nestmates request help6 enabling rescue behavior to be directed towards their nestmates. the evidence suggested that the first responders could have responded to the call for help at distances greater than reported in the literature (bossert & wilson, 1963). in contrast, the number of first responders responding to the request for help by the ant in the trapping chamber decreased significantly as the distance increased. recent work has evidenced rescue behavior among nestmates in poneromorphic ants (frank et al., 2017, 2018), but no available data could be found describing the maximum distance of such signaling. rescue behavior in ants was discussed by hollis and nowbahari (2013b), who demonstrated that a greater number of workers involved in the rescue attempt could improve the chances of success. group defensive behavior in social insects, especially ants, is a well-known phenomenon (hölldobler & wilson, 1990) and includes rescue behavior (czechowski et al., 2002; hollis & nowbahari, 2013a; hollis et al., 2015; frank et al., 2017). the present results suggested that distance could be a major factor determining the number of ants recruited for rescue, indicating that distance may be a key factor influencing rescue success. in this case, it appeared that the request for help would receive a better response if the captured ant was not more than 30 cm distant, corresponding to relatively close proximity to the entrance of the nest, if the ant was captured outside it. the emission of signals by the captured ant is a major determinant of rescue success. miler (2016) evaluated rescue behavior among nestmates of formica cinerea, comparing capture by the lion ant (myrmeleon bore) with artificial capture, from which it was concluded that the latency time was shorter for artificially captured ants. when ants are captured by a lion ant, they are anesthetized by the action of the chemical compound injected by the myrmeleontidae. for this reason, the ants are slow to emit a help signal, resulting in a longer latency time for rescue. it was also concluded that the longer latency time in the aid request resulted in a lower expectation of rescue, compared to ants who promptly issued the aid request (miler, 2016). the results also suggested that the signals involved are similar between colonies of the same species. the rescue workers responded to requests for help from nestmates and non-nestmates, without any significant difference. previous studies with ants have also reported this rescue behavior for non-nestmates (taylor et al., 2013; uy et al., 2018). however, in studies of such behavior in the ant oecophylla smaragdina fabricius, 1775, uy et al. (2018) concluded that colonies with greater similarity in odor models could present a recognition error, with non-nestmates likely to be confused with nestmates, consequently being rescued. in this way, colonies that are closer are more likely to be genetically related, resulting in greater tolerance among the nearest colonies, which increases the possibility of rescuing non-nestmates (errard et al., 2006; newey et al., 2010; uy et al., 2018). this provides an explanation for the fact that the o. brunneus first responders responded to requests for help from non-nestmates, as well as from nestmates, since the colonies were collected at relatively close distances. on the other hand, the rescue workers responded to the request for help from o. chelifer ants with aggressive behaviors, stinging and biting the immobilized ant (table 4). this was different from how they acted when helping the ants of the same species, when the rescuers delivered bites and stings to the duct tape, suggesting an attempt to free their nestmate. these results corroborated those of hollis and nowbahari (2013a), who investigated the requests for help using five different species of ants, finding that all of them rescued their co-specifics, but not ants of other species, in the latter case also being aggressive towards the immobilized ant. the evidence suggests that the signals involved in this type of behavior are specific, at least at the species level. however, further analysis is needed to assess the level of specificity, since rescue behavior is onerous for the ants that perform it, and rescuing an ant from another colony without a degree of kinship would not make sense from an evolutionary point of view. it could be concluded from the results obtained in this work that o. brunneus workers respond to signals emitted by a worker who may have been captured by a potential predator, leading to an attempt at rescue. the signals involved seem to have an optimal range, in addition to being species-specific. this species emits sound signals by stridulation, when exposed to a capture situation, suggesting that this type of signal may be involved, in addition to chemical signals. however, further experiments will be necessary to test this possibility. table 4. frequencies of behaviors exhibited during interactions between rescue workers and the trapped ant in the decision-making experiments. frequency during the meeting (%) behaviors of rescue workers nestmate (loose) nestmate (trapped) nestmate non-nestmate o. brunneus o. chelifer recognition of the trapped individual 0.00 40.32 36.25 33.87 49.56 56.25 pull the trapped individual with the jaws 0.00 0.00 0.00 0.00 0.00 0.00 bite or pull the tape with the jaws 0.00 13.16 19.37 23.38 17.98 6.25 sting or bite the trapped individual 0.00 0.00 0.00 0.00 0.00 34.37 sting the tape 0.00 46.50 44.37 42.74 32.45 3.12 sociobiology 68(3): e6022 (september, 2021) 7 acknowledgments the authors thank fundação de apoio ao desenvolvimento do ensino ciência e tecnologia do estado de mato grosso do sul (fundect) for a doctoral scholarship awarded to the third author (fundect n° 03/2014), coordenação de aperfeiçoamento de pessoal de nível superior (capes) for doctoral scholarships awarded to the first and second authors, and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for a productivity scholarship (wfaj, grant number 307998/2014-2). author contributions conceived and designed the experiments: santos-junior, l.c. & antonialli-junior, w.f.; performed the experiments: santos-junior, l.c. & silva, e.p.; analyzed the data: santosjunior, l.c., silva, e.p., & antonialli-junior, w.f.; wrote the paper: santos-junior, l.c. & antonialli-junior, w.f. references breed, m.d., guzmán-novoa, e. & hunt, g.j. 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(2014). olfactory detection of prey by the termite-raiding ant pachycondyla analis. journal of insect science, 14: 53 doi: 10.1093/jis/14.1.53 351 temperature influence on species co-occurrence patterns in treefall gap and dense forest ant communities in a terrafirme forest of central amazon, brazil by wesley dáttilo1* and thiago j. izzo1 abstract in this study we evaluated the influence of temperature and species co-occurrence on the structure of an ant community of treefall gaps and surrounding dense forests in a terra-firme forest of central amazon, brazil. for this, we collected ants at different hours, and at the time of each collection we measured the temperature of the environment. even with the difference in the temperature variation and variation throughout the day, there was no difference in the richness and abundance of ants among the environments. also, the ant species are distributed randomly and independently of one another in both studied environments in accordance with a null model (c-score). however, although not influenced by temperature, the ant composition of treefall gaps was different from the ant community of the surrounding dense forest. possibly the composition and ant foraging in environments of treefall gaps and surrounding dense forests are not only influenced by temperature, but also by the interaction of this factor with the structural complexity of vegetation in terms of sites available for nesting and feeding, and other microclimatic factors. this generates a difference in ant composition of both environments. in addition, the structuring of ant community in tropical rain forests may actually be stochastic or neutral within each environment. key words: tropical rain forest; formicidae; microclimatic factors; competition; diversity. introduction in the tropics, most small natural forest disturbances are caused by falling trees, forming natural treefall gaps (uhl et al. 1978, brokaw 1985, almeida 1 departament of ecolog y and botany, insect-plant interactions lab., universidade federal de mato grosso, 78060-900. cuiabá, mato grosso, brazil. *email: wdattilo@hotmail.com 352 sociobiolog y vol. 59, no. 2, 2012 1989). this stochastic event is part of the natural forest dynamic and acts as an additional factor in forest structure, changing the light intensity, temperature and humidity in these sites (whitmore 1978, brown 1993, allen & meyer 1998). when this new habitat is formed, several adapted organisms can colonize these sites, replacing one another in succession (probert 1993, ekstam & forseby 1999, andrade 2000). among these organisms, ants emerge as one of the most prominent groups (ward 2006). because of their extreme abundance and diversity, ants act in the process of nutrient cycling, seed dispersal and aeration in the soil surface, and interact in various ways with other parts of the ecosystem, influencing the pattern of distribution and abundance of other taxa along succession (hölldobler & wilson 1990, majer & camer-pescl 1991, dáttilo et al. 2009). in tropical rain forests, the ant diversity is extremely high, reaching approximately 500 species in only one collection site (wilson 1959, fisher 1999, vasconcelos & delabie 2000, longino et al. 2002). the success of these organisms is mainly due to several ecological and social adaptations, including generalized feeding and nesting habits (benson & harada 1988, hölldobler & wilson 1990, fowler et al. 1991). the high abundance and diversity of ants in tropical rain forests is also due to the high habitat complexity (abundance of places to nesting ) and the climate stability in tropical regions over space and time, creating an ideal environment where several species specialists can establish (benson & harada 1988, hölldobler & wilson 1990, reyes-lopes et al. 2003). however, little is known about the relationship between the natural dynamics of tropical rainforests and the ant communities inside forests. in many cases ant colonies are considered modular organisms and, in analog y, comparable to plants. this comparison is made because most ant species do not present migration behavior of the colony and their foraging is centered at a point (andersen 1991, lópez 1994, santos et al. 2006). thus, competition has been identified as an important factor in the structure of ant communities (andersen 1992, adams 1994, acosta et al. 1995, punttila et al. 1996). the distribution of ant mosaics proposed by room (1971) is defined as the distribution in patches of dominant ant species with similar ecological requirements, and therefore there is no overlap of their territories (gause 1934, macarthur & levins 1964, chesson 2000). thus, in small spatial scales is possible that competitive exclusion occurs with some ant species 353 dattilo, w. & t.j. izzo — ant species co-occurrence patterns in amazonian forests (andersen 1995, parr et al. 2000). however, beyond the competition, one of the factors that regulates the ant diversity and their foraging is the species’ preference for different abiotic conditions, and several studies indicate that soil temperature is directly related to the foraging of these organisms on the soil surface (porter & tschinkel 1987, marsh 1988, andersen 1992, wehner et al. 1992, cerdá et al. 1998). due to the fact that after the opening of a clearing there is an increase in daily temperature fluctuations on the soil surface (vázquez-yanes & orozcosegovia 1982), and that the ants are sensitive to small microclimatic changes in the environment which they live (brown 1997, cerdá et al. 1997, 1998, bestelmeye 2000), different adaptive strategies of ants that live in these two environments are expected (aguiar & monteza 1996). thus, in this study, we evaluated the influence of temperature and co-occurrence structuring the ant community of treefall gaps and surrounding dense forests in a terra-firme forest of central amazon, brazil. material and methods study area this study was conducted during june and july 2007 in the forest reserve #1501# (54º50'w and 02º25's), located approximately 80 km north of manaus city, amazonas state, brazil. the reserve area covers 10,000 ha of continuous forest, surrounded by a much larger area of undisturbed forest, and is part of the biological dynamics of forest fragments project, bdffp (inpa/smithsonian institution). characterized as a primary tropical rainforest and terra-firme, it has canopy trees ranging from 30-40m in height and some emergent trees which reach 50m in height. the understory is relatively open and contains several species of palm trees (lovejoy & bierregaard 1990). according to the köppen classification, the climate is tropical humid (afi) with average annual temperature of 26.7 ºc, 85% humidity and 2.200 mm of precipitation, with periods of rain between november and may and a dry season between june and october (ribeiro 1976, lovejoy & bierregaard 1990, laurance 2001). data collection five treefall gaps and five surrounding dense forests were randomly selected approximately 300-800m away from each other. all treefall gaps had an area 354 sociobiolog y vol. 59, no. 2, 2012 exceeding 400 m² and were opened between 5 and 10 years before the present study (t.j. izzo, pers. obs.). in each site three baits were distributed with 5g of sardines and guava in a plastic card 10 m apart from each other, forming a triangle. after an hour, all the ants present in the baits were collected. the collections were made at five different hours (7h, 10h, 14h 18h, 22h), and at the time of each sample we measured the temperature, always at a height of 5 cm above the ground. the ants collected were stored in 70% alcohol for further identification. all individuals collected were deposited in the entomological collection of instituto nacional de pesquisas da amazônia (ce-inpa). data analysis in order to not overestimate the ant species with more efficient systems for recruiting and / or those whose colonies are closer to the bait (romero & jaffé 1989, leal & lopes 1992, tavares et al. 2001, gotelli et al. 2011), all quantitative matrices used in this study were calculated based on the frequency of species occurrence in the bait and not based on the number of workers. initially, we tested the temperature variation among the times of the two environments using an analysis of variance with repeated measurements (anova repeated). subsequently, we used general linear models (glm) to assess the influence of temperature, the type of environment, and the interaction of both factors on the richness and abundance of ants, through the software systat 10.0.0 (wilkinson 1998). to summarize the composition of the ant community foraging in environments of treefall gaps and surrounding dense forests, we ordered the similarity between points using non-metric multidimensional scaling (nmds). this type of ordination analysis is one of the most robust and often summarizes more information in less axes than other techniques for direct ordering (legendre & legendre 1998). the ordinations analyses were performed from a distance matrix calculated from the sorensen’s dissimilarity index (qualitative data) and bray-curtis’s dissimilarity index (quantitative data). additionally, we tested the difference in the ant species composition of treefall gaps and surrounding dense forests through a permutation test (10.000 permutations) based on analysis of similarities (anosim) (clarke 1993). both the ordination and analysis of similarities were made through the software r development core 355 dattilo, w. & t.j. izzo — ant species co-occurrence patterns in amazonian forests team (version 2.13.1). finally, we use the first two axes of each ordination in a multivariate analysis of covariance (mancova) (mancova), in order to evaluate the influence of temperature and interaction between the environment and the temperature in the ants composition through the software systat 10.0.0 (wilkinson 1998). to test the hypothesis that the coexistence of ant species in treefall gaps and surrounding dense forests is determined by competition, we used the co-occurrence index (c-score) through the software ecosim 7.72 (gotelli & entsminger 2009). in a competitively structured community, the observed c-score index should be significantly higher than expected. when this index is less than expected competitive exclusion does not occur between the ant species (gotelli 2000, gotelli & entsminger 2009). we compared the observed c-score index with 5000 replicates generated by the null model where the species occurrences and sites are fixed. this null model assumes that the presence of a given ant species does not influence the occurrence of other species, i.e. there is no evidence for deterministic process influencing species distribution (ribas & schoereder 2002, gotelli & entsminger 2009). results in this study we collected 37 ant species representing eight genera and 4 subfamilies. the subfamily myrmicinae had the greatest number of taxa (81.1%), followed by formicinae (8.1%), ectatomminae (5.4%) and ponerinae (5.4%). we collected 14 ant species in treefall gaps and 21 ant species in surrounding dense forests, and only six species occurred in both environments. eight species were exclusive to treefall gaps and 17 species were exclusive to surrounding dense forests (table 1). however, there was no difference in the richness and abundance of ants by area between the environments (anova, p> 0.05), and both factors also did not vary with temperature and are not different between the environments (anova, p> 0.05) (table 2). the temperature range in dense forest (mean 26,1°c – min 24,0°c, max 28. 3°c) was lower than in the treefall gaps (mean 27,5°c – min 23,7°c, max 32.3°c), and there was a difference in temperature variation per hour between the two environments (f (1.137) = 33.126; p< 0.001). despite the overlap in some points showed by the ordination analysis, the ant community of dense forest differs from the treefall gaps both qualitatively 356 sociobiolog y vol. 59, no. 2, 2012 table 1. number of occurrences of ants collected in treefall gaps and surrounding dense forests environments in the months of june and july 2007 in a terra-firme forest in central amazon, manaus city, amazonas state, brazil. number of occurrences ant species treefall gaps surrounding dense forests camponotus femoratus (fabricius 1804) 19 crematogaster sp1 25 10 crematogaster sp2 2 4 crematogaster sp3 4 crematogaster sp4 13 ectatomma quadridens (fabricius 1793) 1 ectatomma tuberculatum (olivier 1792) 1 odontomachus haematodus (linnaeus 1758) 1 odontomachus scalptus brown 1978 1 paratrechina sp1 3 2 paratrechina sp2 1 pheidole sp1 3 pheidole sp2 3 pheidole sp3 7 pheidole sp4 2 pheidole sp5 1 2 pheidole sp6 3 pheidole sp7 5 pheidole sp8 2 pheidole sp9 1 pheidole sp10 2 pheidole sp11 1 pheidole sp12 15 3 pheidole sp 13 1 pheidole sp 14 3 pheidole sp 15 1 pheidole sp16 6 1 pheidole sp17 1 pheidole sp18 1 pheidole sp19 1 pheidole sp20 1 solenopsis geminata (forel 1893) 1 solenopsis sp1 1 4 solenopsis sp2 3 trachymyrmex cornetzi (forel 1912) 2 trachymyrmex sp1 1 trachymyrmex sp2 1 1 357 dattilo, w. & t.j. izzo — ant species co-occurrence patterns in amazonian forests fig. 1. non-metric multidimensional scaling (nmds) of ants collected in treefall gaps (triangles) and surrounding dense forests (squares) in june and july 2007 in a terra-firme forest in central amazon, manaus city, amazonas state, brazil. this ordination analysis was calculated from the (a) sorensen’s dissimilarity index (stress= 0.2631 ; axis 1 + axis 2= 49.5% of explanation) and (b) bray-curtis’s dissimilarity index (stress= 0.2635 ; axis 1 + axis 2= 49.3% of explanation). 358 sociobiolog y vol. 59, no. 2, 2012 (nmds, followed by anosim; p< 0.001) and quantitatively (nmds, followed by anosim; p< 0.001) (fig. 1). in qualitative data, the temperature did not influence the species composition of ants (mancova, pillai-trace: f (0.007) = 1.037 ; p= 0.363), and the composition did not change with temperature differences between the environments (mancova, pillaitrace : f (0.007) = 0.279 ; p= 0.345). for the quantitative matrix, there was no influence of temperature on the species composition of ants (mancova, pillai-trace: f (0.034) = 0.793 ; p= 0.459) and the composition did not change with temperature differences between the environments (mancova, pillai-trace : f (0.051) = 1.197 ; p= 0.312). our data indicate that competition does not seem to be the principal factor in structuring of ant communities in treefall gaps and surrounding dense forests. the simulations performed for the ant community associated with the treefall gaps showed that the observed c-score index is lower than expected (p= 0.001) if the competition was a factor structuring the ant community (fig. 2a). in ant community associated with dense forests, the simulations realized showed that the observed c-score index lay within the 95% limits of random frequency obtained from the null model (p = 0.75) (fig. 2b). table 2. general linear models (glm) to assess the influence of temperature, the type of environment (treefall gaps and surrounding dense forests), and the interaction of both factors on the richness and abundance of ants. response effect df mean squares f-ratio p-value richness environment 1 2.043 1.250 0.269 temperature 1 0.242 0.148 0.702 environment*temperature 1 2.452 1.500 0.227 error 46 1.634 abundance environment 1 2.492 1.885 0.176 temperature 1 1.269 0.960 0.332 environment*temperature 1 2.771 2.096 0.154 error 46 1.322 359 dattilo, w. & t.j. izzo — ant species co-occurrence patterns in amazonian forests fig.2. frequency distribution of the co-occurrence c-score index obtained from 5000 replicates generated by the null model in the ant community associated with a) treefall gaps and b) surrounding dense forests in june and july 2007 in a terra-firme forest in central amazon, manaus city, amazonas state, brazil. arrows represent the observed c-score index. 360 sociobiolog y vol. 59, no. 2, 2012 discussion many studies have shown the factors that determine the ant diversity in different spatial scales (hölldobler & wilson 1990, andersen 1997, cerdá 1997, gibb & hochuli 2002, ribas et al. 2003, floren & linsenmair 2005, kaspari 2005). these factors act differently in different spatial scales and vegetation types (santos et al. 2006). in this study, in a small spatial scale, even with the difference in the amplitude and the thermic variation between the treefall gaps and surrounding dense forests environments, we did not observe a modification of the ant community as a function of temperature, but just due to strong differences on habitat structure. some studies have shown that the composition and foraging ants in treefall gaps environments are not only influenced by microclimatic factors, but also by the interaction of these factors with the structural complexity of vegetation in terms of availability of food and nesting sites (greenslade & halliday 1983, andersen 1986, aguiar & monteza 1996, basu 1997). thus, many factors act together so that the composition of ant species differs between forest clearings and around tropical forests (aguiar &montez 1996), as observed in our results. in regions with large climatic variations throughout the day and year, the temperature can directly influence the foraging strateg y of ants, and change the hierarchical level between the dominant ant species (herbers 1989, hölldobler & wilson1990, cerdá et al. 1997, 1998). on a broader level, some studies have shown that competition is the only or most important factor regulating the ant diversity (room 1971, andersen 1992, majer et al. 1994, acosta et al, 1995, djieto-lordon & dejean 1999, delabie et al. 2000; albrecht & gotelli 2001). however, variations in environmental conditions, ecological, spatial, beyond random events can modulate the importance of competition and the ant community structure in one site (hölldobler & wilson 1990, morrison 1996, basu 1997). our work corroborates with other work available in the literature that there is not substantial evidence that competition influences ant community structure at different spatial scales (levings & traniello 1981, fellers 1987, ryti & case 1992, punttila et al. 1996; ribas & schoereder 2002). in treefall gap environments, there was a great co-occurrence of ant species, possibly due to the fact that most species in disturbed environments are resource and habitat 361 dattilo, w. & t.j. izzo — ant species co-occurrence patterns in amazonian forests generalists (fowler 1990, andersen 1995, king et al. 1998, vasconcelos 1999, silvestre et al. 2003, dáttilo et al. 2011). that means limitation of a particular resource that can be dominated by some ant species does not necessarily cause the elimination of other species. so, the ants associated with these environments may use different food resources and change, even temporarily, their diets, avoiding competition and facilitating the co-occurrence (herbers 1989, yanoviak & kaspari 2000, albrecht & gotelli 2001, santos et al. 2006). on the other hand, in surrounding dense forests, the results showed that the co-occurrence observed lay within the 95% limits of random frequency and suggest that it is not always the biological processes which determines the spatial distribution of ants. ribas & schoereder (2002) tested the hypothesis of competition structuring the ant community in 14 different tropical rain forests and also obtained through simulations by null models, and the results indicate that the ant mosaic theory of structuring the ant community does not apply to these regions. given the high availability and spatial distribution of food resources, beyond the climatic conditions equilibrium, our study suggests that the structure of ant communities in tropical rainforests may really be stochastic or neutral within each environment (floren & linsenmair 2000, floren et al. 2001, hubble 2001). finally, as individuals differ in the probability of colonizing a type of environment due to dispersal ability and the effects of variation between environments, we expect that the ant composition is different in treefall gaps and surrounding dense forest environments (olden et al. 2001, bruna et al. 2011). the foraging and colonization of the environment immediately after the opening of the one treefall gap can be determining factors in the establishment of the colony as we observed, once ant species that occur in treefall gaps are different from the surrounding dense forests (aguiar & monteza 1996). so, some generalist species colonize treefall gaps environments, while habitat specialists probably can not survive and/or establish nests. additionally, the natural regeneration that occurs within the treefall gaps influencing important parameters of composition and distribution of the ant species that live there (pearson et al. 2003). 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forest canopy and litter. oikos 89: 259-266. doi: 10.13102/sociobiology.v64i3.1219sociobiology 64(3): 276-283 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the role of parabiotic ants and environment on epiphyte composition and protection in ant gardens introduction mutualism is a ubiquitous interspecific interaction that occurs among a great diversity of living organisms (janzen, 1985). plants and insects, in particular, are frequently involved in three types of mutualisms: pollination, seed dispersal and protection against herbivores (bronstein, 1994; bronstein et al., 2006). in these interactions, plants attract and reward insects for their actions by offering shelter and/or food through specialized structures, while insects guarantee flower pollination, seed dispersal or protection against consumers (bronstein et al., 2006). although the majority of plant-insect mutualisms are generalized and facultative (stanton, 2003; bronstein et al., 2006), some interactions can be very specialized (see ricoabstract ant gardens (ags) are a multi-partner specialized ant-plant interaction involving several ant and epiphyte species. although studies on ags have reported possible roles for some species in this system, there are unanswered questions regarding the process of epiphyte incorporation in the ags and the role of less aggressive ant species in ag protection. in this study, we used ags in the brazilian amazon forest formed by two parabiotic ant species to test a set of hypothesis regarding two main questions: 1) how is ag plant community composition affected by the surrounding environment? 2) does crematogaster levior play a role in the chemical detection of herbivory in the ags? after identifying epiphytes occurring at ags at the forest edge and in the interior, we found that ant gardens in each environment exhibited different compositions, and that plant species bearing oil or extrafloral nectar glands were more frequent in ags located in the forest interior than in those at the forest edge. by performing experiments with volatile compounds emitted from injured epiphytes, we detected that only camponotus femoratus was responsive, responding almost eight times faster in response to plant extracts than water treatments. our results support the idea that environmental conditions affect ant preference for feeding resources provided by epiphytes and consequently shape the structure of the epiphyte community in ags. on the other hand, the role of c. levior in ags remains unknown, since it seems to play no direct or indirect role in ag protection. sociobiology an international journal on social insects lc leal¹², cc jakovac³, ped bobrowiec³, jlc camargo³, pec peixoto¹ article history edited by gilberto m. m. santos, uefs, brazil received 24 october 2016 initial acceptance 17 april 2017 final acceptance 25 june 2017 publication date 17 october 2017 keywords camponotus femoratus, crematogaster levior, epiphytes, protective mutualism, partner selection, amazon. corresponding author laura catarina leal laboratório de ecologia comportamental departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina, s/n, novo horizonte 44054-008, feira de santana-ba, brasil. e-mail: lacaleal@gmail.com gray & oliveira, 2007). one remarkable case of specialized interaction are ant gardens (ags), mutualistic associations between epiphyte plants and ant species in asian and neotropical rainforests (hölldobler & wilson, 1990). ant gardens are ant nests built on the branches of trees and on which aggregates of epiphyte species grow (ule, 1901). the nest can hold one or, more frequently, more than one ant species and several phylogenetically distant epiphyte plant species (orivel & leroy, 2011). when more than one ant species occurs in the same nest, they show a parabiotic behavior, in which the ants live in close association sharing foraging trials but do not exhibit obvious parasitic or exploitative interactions (davidson, 1988; forel, 1898; vantaux & leroy, 2007; orivel & leroy, 2011, but see menzel et al., 2015). 1 laboratório de ecologia comportamental, departamento de ciências biológicas, universidade estadual de feira de santana-ba, brazil 2 departamento de ecologia e biologia evolutiva, universidade federal de são paulo, diadema-sp, brazil 3 projeto dinâmica biológica de fragmentos florestais, instituto nacional de pesquisas da amazônia, manaus-am, brazil research article ants sociobiology 64(3): 276-283 (september, 2017) 277 seeds from most ags epiphytes are dispersed by the associated ants, which incorporate these seeds into carton nest continuously over the lifespan of the nest (orivel & leroy, 2011). although seeds from ag epiphytes commonly bear aril or elaiosome, seed selection by ants seem not determined by the quantity or quality of such appendages as patterns of seeds selection by ags ants remains the same after the removal of such seed structures (orivel & dejean, 1999). in fact, it seems that ag ant species are attracted by a set of specific volatile compounds released by the seed coat of some of epiphyte species commonly found on ags (youngstead et al., 2008). after germination, ants of at least one species protect plants against herbivores by patrolling on leaves (vantaux et al., 2007), while the roots and stems of epiphytes increase the stability and moisture of ant nests (yu, 1994). some epiphytes also provide feeding resources for ants through extrafloral nectaries (efn), oil glands and fruits (kleinfeldt, 1978; hölldobler & wilson, 1990). although previous studies has been able to identify the general benefits for both interacting sides (see orivel & leroy, and references therein), it has been very hard to identify the determinants of plant and ant species composition in ags, and also the roles played by different partner species in this multispecific interaction. for this reason, here, we investigate two main processes that remain unclear: 1) the process of epiphyte incorporation in the ags and 2) the role of less aggressive ant species in ag protection. it is known that epiphyte species are not randomly distributed on ags, but that instead there are some preferential associations between particular ants and some epiphyte species (orivel & leroy, 2011). these preferential associations can be explained by two non-mutually exclusive mechanisms: 1) preferential incorporation of seeds from epiphyte species that confer larger benefits to the ant colonies, e.g. plant species that provide food resources; and 2) niche filtering, i.e. local abiotic conditions determining the plant species capable to reach and establish in the nests. since different habitats may restrict the plant species capable of surviving at specific environmental conditions, niche filtering may be the main force affecting which epiphyte species colonizing ags that occur in habitats with contrasting environmental conditions. however, ant preferences for some plants may still play a secondary role. therefore, it is likely that variation in epyphite composition among ags is primarily driven by species ‘filtering’ as a response to local abiotic conditions and, secondarily, by preferential incorporation of particular epiphyte species by the ants on ags. such epiphytes should be the ones that provide the greater benefits to ants. consequently, it is possible that ags located in contrasting habitats shelter a different pool of epiphyte species, but that the most common epiphyte species found in the ags provide similar benefits (e.g. food resources), regardless of the environment. in order to unravel the complete scenario related to the presence of some specific epiphyte species on ags it is necessary to understand not only the incorporation process of epiphyte species on ags, but also the roles that different ant species plays in these plants. although it is often assumed that all ant species living on ags benefit the epiphytes, ant species seem to differ in the efficiency with which they protect the epiphytes against herbivores. for example, among the ant species most commonly present in amazonian ags, crematogaster levior longino (2003) (myrmicinae) and camponotus femoratus fabricius (1804) (formicinae), only c. femoratus seems to protect the epiphytes (vantaux et al., 2007; vicente et al., 2014). however, most ant responses regarding epiphyte protection were measured based only on ant recruitment (vicente et al., 2014) or after the simultaneous usage of physical and chemical stimuli to trigger their responses (vantaux et al., 2007). although ant recruitment may be a reliable measure of aggressiveness towards potential herbivores, it does not consider other behavioral responses that may occur between the initial stimulus and ant recruitment. for example, c. levior might be able to detect and communicate the presence of herbivores through chemical stimuli and consequently could improve the aggressive response exhibited by c. femoratus. therefore, the study of such responses may reveal previously undetected interactions between ant species in their protective response in ags. here we investigate ags constructed by ant species crematogaster levior and camponotus femoratus in the central brazilian amazon forest. we addressed two main questions: 1) how is ag plant community composition affected by the surrounding environment and plant traits? 2) does c. levior play a role in the chemical detection of herbivory in the ags? for the first question, we postulated two non-mutually exclusive hypotheses. first, the distribution of adult epiphyte species in ags is determined not only by ant behavior, but also by local environmental conditions. second, ants prefer incorporate seeds from epiphyte species that provide food resources. to evaluate these hypotheses, we used ags located in two habitats with contrasting abiotic conditions within tropical forests: forest edges and interior (see murcia, 1995). then, by comparing ags found in areas of forest edge and interior, we expect to find different epiphyte species associated to the ags (first hypothesis), but with the most common species in each environment presenting extrafloral or oil producing glands (second hypothesis). to answer the second question, we hypothesize that c. levior is able to recognize compounds released by injured epiphytic plants, although it does not react aggressively to them. consequently, we expect that c. levior and c. femoratus will show similar time responses to detect chemical stimuli related to herbivory, although only c. femoratus will exhibit recruitment behavior. methods study area this study was conducted at the “km 41” reserve of the biological dynamics of forest fragments project (a collaborative initiative between inpa and stri), located ca. lc leal et al.– ant influence on epiphyte composition and protection278 80 km north of manaus, central amazon, brazil (02º24’ s 59º22’ w). we carried out the experiments during august 2006 and august 2010 in a continuous tropical rain forest at the km 41 reserve and at its border along the access road. annual rainfall varies between 1900 and 2500 mm, with a moderately dry season (< 100 mm/month) from june to october (gascon & bierregard, 2001). the forest is a dense evergreen forest, with a 2-12 m tall understory, a sub-canopy between 15 and 25 m tall, a canopy about 30-37 m tall and emergent trees up to 45 m 55 m tall (camargo & kapos, 1995; laurance et al., 2011). epiphyte occurrence in ags we sampled all ags found up to 6m high along two 5 km transects, one located in the forest interior (more than 50 m from the forest edges) and the other transect at the edge of the same mature forest. the forest edge was created by the construction and maintenance of a 10 m wide dirt road that gives access to the study area. in each ag we collected and identified all plant species growing in it as well as the ant species present in the nests. the same researcher (jakovac, c.c.) performed all observations. for every plant species collected, we looked for the presence of glands that indicated the production of extrafloral nectar or oil (elias & gelband, 1976). other plant traits like the presence of fruit pulp or an elaiosome are also potential drivers of differential epiphyte recruitment to ags after seed dispersal. however, we considered only the presence of glands associated with extrafloral nectar or oil as a predictor because these structures are temporally predictable. the other food resources offered by plants are normally seasonal, which should reduce their importance as drivers of the plant composition patterns we investigated here. to evaluate if epiphyte species composition depends on the location of the ags (forest interior and forest edge), we performed an analysis of similarity (anosim) with 999 permutations and ordinated the species composition using a non-metric multidimensional scaling analysis (nmds) with a sørensen index. to evaluate if the frequency of epiphytes in ags is determined by the presence of extrafloral nectaries or oil producing glands, we fitted generalized linear models with poisson error distribution, checking for overdispersion of the data. for that, we used each epiphyte species as a sample unit and the number of ags where each epiphyte species occurred in each environment as the response variable. as explanatory variables, we considered the presence of extrafloral nectaries or oil producing glands (as binary variable) in each epiphyte species, the environment where each ag was found (forest edge or interior) and the interaction between the presence of glands and the environment. we predicted that, if ants select epiphytes that provide continuous food resources to compose their ags, the presence of glands will have an effect on the frequency of epiphytes in both environments. we also compared epiphyte richness in ags between both environments with a t-test. we performed all analyses with the base and the vegan (oksanen et al., 2014) packages in r 3.0.2 (r core team, 2014). ant response to epiphyte volatile compounds to analyze ant response to volatile compounds released by injured plants, we selected 20 ant gardens located up to 2 m high at the forest edge, and occupied simultaneously by the parabiotic ants camponotus femoratus and crematogaster levior. for each ag, we applied two treatments: (1) aqueous extract of leaves from peperomia macrostachya (piperaceae) (0.34 g/ml) (hereafter referred as extract treatment) and (2) water (control treatment). peperomia macrostachya is one of the six most abundant epiphyte species in neotropical ags (orivel & leroy, 2011) and occurred in all ag selected for this experiment. we prepared the extracts from p. macrostachya leaves collected from ags that were not used in the experiment. to avoid oxidation of the extracts, we prepared them immediately before conducting the experiments. we applied the extracts to pieces of cotton and held them at a short distance (approximately 5 cm) from p. macrostachya leaves located near one of the nest entrances and without ants patrolling. we refrained from touching the leaves with the cotton pieces to make sure any subsequent response would be triggered only by volatile compounds and not by any physical stimulus. for each ag we applied one treatment at a time with minimal intervals of 20 min between treatments. the order in which treatments were applied to each ag was randomly assigned beforehand. we quantified ant response to treatments using three descriptors: ant attraction (binary), ant response time and ant recruitment. we considered ants to have been attracted by the extract if they exhibited exploitative behavior such as moving toward the tip of the focal leaf followed by inspection with the antennae and/or worker recruitment. we measured ant response time as the interval between bringing the cotton pieces close to the leaf and ant recruitment occurring on the focal leaf (up to a maximum of 10 min), and counted the number of ants recruited during this10 min. period. we calculated ant recruitment by subtracting the number of ants initially present on the focal leaf from the number observed 10 min. after the treatments. we chose a 10 min. period because although ants still patrolled the leaves after this time interval, their number started to decline. to evaluate ant response to each treatment, we looked for differences in ant attraction, ant response time and number of ants recruited between control and plant extract treatments. we fitted a generalized linear model with binomial error distribution, checking data overdispersion, to analyze differences in ant attraction between treatments. to evaluate if response time and ant recruitment differ between treatments we performed a factorial anova. for every analysis, we included each sociobiology 64(3): 276-283 (september, 2017) 279 sampled ag as a grouping factor. for the analysis considering ant response time, we included all ags. for the ags in which we did not observe any ant response after 10 min observation, we considered the maximum time of observation (10 min) as the time until ant response. we performed all analyses using r3.0.2 (r core team 2014). results colonization of ags by epiphytes we sampled 26 ags, 17 at the forest interior and nine at the forest edge. all ags were occupied only by parabiotic ants c. femoratus and c. levior. in total, we identified 15 epiphyte species, eight of them bearing extrafloral nectaries or oil producing glands (table 1). the most frequent family was araceae, which was found in 42% of the ags, followed by gesneriaceae (28%) and piperaceae (24%). the number of epiphytic species ranged from 1 to 5 per ag and did not differ between environments (t = 0.25; df = 24; p = 0.80). on the other hand, the composition of epiphyte species was different between edge and forest interior (anosim, r=0.52, p=0.001; nmds, stress=0.09; fig 1). regarding epiphyte frequency distributions, ags in both environments exhibited the same structure: one or two species were common to more than 70% of the ags, while the other species were less frequent (fig 2). the epiphytes codonanthe calcarata table 1. relative frequency of epiphyte species occurring in association with ants in ant gardens (ag) in forestinterior and forest edge in central amazonian forest, brazil. epiphytic species acronym extrafloral nectaries florest interior (%) forest edge (%) aechmea sp. asp x 5.8 0 anthurium gracile agra 5.8 0 anthurium sp.1 ansp1 0.2 0 anthurium sp.2 ansp2 5.8 44.4 anthurium trinerve atri 23.5 77.8 codonanthe calcarata ccal x 35.3 77.8 codonanthe crassifolia ccra x 0 11.1 codonanthopsis sp. csp x 5.8 0 codonanthe opsisulei cule x 17.6 11.1 ficus sp. fsp 5.8 0 oedematopus sp. osp 5.8 0 peperomia macrosthachya pmac x 88.2 11.1 philodendron megalophillum pmeg x 35.3 0 philodendron sp. psp x 11.8 22.2 psychotria sp. pssp 5.8 0 (gesneriaceae) and anthurium trinerve (araceae) were both found in 77.8% (n = 7) of the ags at the forest edge but in only 35.3% (6) and 23.5% (4) of the ags in the forest interior, respectively (table 1). in the forest interior, the most common epiphyte species were peperomia macrostachya (piperaceae) and philodendron megalophillum (araceae), occurring in 88.2% (15) and 41.2% (7) of the ags, respectively (table 01). those species were rarely found at the forest edge (table 01). the presence of glands that indicated production of extrafloral nectar or oil did not by itself explain the high frequency of a few epiphyte species on ags in either environment (fig 3). however, we found that the frequency of epiphytes in the ags was determined by an interaction between the presence of glands and environment. the frequency of plants bearing glands capable of producing oil or extrafloral nectar was more than threefold higher in ags located in the forest interior than in forest edge. accordingly, epiphytes not bearing such glands were almost two times more frequent in ags located in the forest edge than in the forest interior (χ²glands*environment = 13.36, df= 17, p < 0.001; fig 3). fig 1. epiphyte species composition in ant gardens in forest edges sites (empty circles) and interior sites (black circles) in central amazonian forest. (forest edge, n = 9; forest interior, n=17). lc leal et al.– ant influence on epiphyte composition and protection280 ants response to epiphytes volatile compounds only c. femoratus workers were able to detect and respond to the leaf extract treatment. although c. levior workers were observed foraging during the experiment, they did not exhibit any exploitative behavior (approximation and/ or contact attempts using antennae, for example) and did not recruit workers in response to any treatments. the response of c. femoratus to volatile compounds released by p. macrostachya was greater than the response to water for every tested descriptor. camponotus femoratus were attracted by the extract in 95% of the cases in which it was offered, but were attracted by water in only 40% of the cases (χ2= 15.64;df=1; p < 0.001; fig 2). this same species responded four times faster (f(1,196)= 47.94 p < 0.001; fig 4) and recruited nine times more workers (f(1.19)= 71.28; p < 0.001; extract: 5.17 ± 0.6, water: 0.6 ± 0.16) in response to the plant extracts than to the water treatment. discussion our results indicate that ant gardens at the forest edge and in its interior are occupied by plant communities with different species compositions, but following a similar structure. in both environments, ag-epiphyte communities fig 2. frequency of epiphyte plant species in ant gardens at the forest edge (a) and in the forest interior (b) in central amazonian forest. (forest edge, n = 9; forest interior, n=17. fig 3. mean number of plant species with (grey bars) and without extrafloral nectaries (black bars) occurring in ant gardens in forest edge and interior sites in central amazonian forest. bars represent standard deviation. fig 4. mean response time of the ant camponotus femoratus to volatile compounds released by water (control) and extracts of peperomia macrostachia leaves offered to ant gardens in a region of amazon forest. bars represent standard deviation. sociobiology 64(3): 276-283 (september, 2017) 281 were characterized by a few common species present in most ags and several less frequent species. interestingly, we found that the frequency of species with extrafloral nectars or oil glands was higher at the forest interior than at the forest edge, indicating that the probability of plants offering such resources to occur in the ags change according to the environment. we also showed that epiphytes able to establish and develop on ags seem to benefit from c. femoratus protection against herbivores. although this effect has already been reported (vantaux et al., 2007; vicente et al., 2014), our results further suggest that c. levior does not play any role, even an indirect one, in epiphyte defense. our results suggest that the environment determines the composition of epiphyte species in ags, and may affect the selection of epiphytes by ants due to differential resource availability. nest carton is considered a very suitable substrate for epiphyte establishment because it is rich in organic matter and at the same time allows for good root aeration (orivel & leroy, 2011). therefore, many epiphyte species could potentially germinate and develop on ags. because extrafloral nectaries are especially prevalent in plants with high growth rates and with affinity for full-light habitats as the ones found in the forest edge (schupp & feener, 1991; blüthgen & reifenrath, 2003), it seems unlikely that the low occurrence of epiphytes in the ags in the forest edge was due to unsuitable local conditions for such plants. instead, because plants with nectar and oil glands are common at the forest edge, it should be easy for ants to find those resources outside of their ags. consequently, the relative benefit for ants of recruiting and caring plants with extra food resources to the ags should be relatively lower at the edge than in the forest interior. thus, epiphytes bearing foodproviding glands may not be preferentially incorporated by ants into ags located at the forest edge due to their lower relative importance to ants occurring in this habitat. however, because c. femoratus responded aggressively to epiphyte volatile stimulus in the ags located at the forest edge, it seems that any epiphyte will be defended once incorporated into the ag. perhaps this occur because they also provide additional benefits to ants such as nest support aid. our finding that c. levior do not participate in any step of the aggressive response is curious. crematogaster species are known to preferentially feed on carbohydrate rich resources such as those present in extrafloral nectaries (bluthgen & fiedler, 2004). hence, these ants consequently may benefit from the presence of epiphyte species bearing this trait in the ags. since individuals of c. femoratus defend the epiphyte species, c. levior may increase its benefits by not paying the costs associated to plant defense (see archetti et al., 2011). but, if this is true, why do individuals of c. femoratus allow the presence of individuals of c. levior? if colonies of c. femoratus do not obtain any benefits from this association, theory predicts that they should avoid interactions with c. leviour (edwards et al., 2010). it is important to note that, although workers of c. femoratus forage exclusively on the forest understory for short periods during the day (e.g. vantaux et al., 2007), c. levior individuals are able to forage both on the forest understory and on the forest ground, over larger distances from the nest and under more extreme weather conditions (such as at forest edges) in comparison with c. femoratus (vantaux et al., 2007). those differences in foraging habits might limit the extent of interspecific competition between the two ant species for resources provided by their shared ag. additionally, camponotus individuals can get food from crematogaster workers through trophallaxis (menzel et al., 2014). therefore, c. femoratus may co-occur with c. levior due to the additional resource input provided by the latter. in summary, it seems that c. femoratus is the most important partner for epiphytes due to its protective services in the ags. the plants, on the other hand, are important as resource providers to both ant species, at least in areas with low light availability. if c. femoratus, in fact, benefit from c. levior by increasing food acquisition, it may be that that the multipartner interaction found in the ags is maintained by a series of two-way mutualisms: c. femoratus and epiphyte plants; c. femoratus and c. levior and the indirect effect of c. levior on epiphyte plants through the maintenance of c. femoratus. it is important to note, however, that the identity of partners in these two-way interactions, especially the identity of epiphytes, might vary due to changes in local species communities. acknowledgments this work was developed as part of the activities of forest amazon ecology course (efa/2006 and 2010). authors are supported by coordenação de aperfeiçoamento de pessoal de níıvel superior (capes) grants. this is article no. 726 of the technical series of biological dynamics of forest fragments project (pdbff -inpa/stri). references agrawal, a. & dubin-thaler, b.j. 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(1994). the structural role of epiphytes in ant gardens. biotropica, 26: 222-226. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.5863sociobiology 68(2): e5863 (june, 2021) introduction bees are of vital importance for the maintenance of floristic ecosystems due to their efficiency as pollinating agents (michener, 2007; klein et al., 2006). the interaction between bees and flowers is due to the dependence of bees on floral resources to obtain their food and that of their young (roubik, 1989; proctor et al., 1996; michener, 2007). as a result of this intrinsic need and constant co-evolution with flowering plants, bees have evolved many behaviors that increase the efficiency of their foraging (van nest & moore, 2012). abstract bees feed on nectar and pollen, however, these resources are often available to floral visitors during restricted temporal windows. the presence of temporal memory is an advantage, as foragers can save energy by scheduling their flight activity to coincide with peaks of nectar secretion in the flowers or at times of higher sugar concentration in the nectar. thus, the objectives of this study were (i) to investigate whether melipona subnitida has temporal memory, and evaluate whether it becomes more accurate over the days, and (ii) to determine whether the behavior of anticipating the offered resource presents inter-individual consistency in the behavior of foragers. the visitation of the bees was high before and during the opening interval of the food resource, but rare after the closing, suggesting that m. subnitida has the ability to memorize the time of availability of the resource, increasing the accuracy over the days, with bees anticipating their visits in relation to the time they discovered the resource, and the opening time of the resource. there was individual consistency in the behavior of food-anticipatory activity, with the presence of bees that consistently anticipated in relation to the opening time of the resource (inspectors) and bees that consistently did not anticipate (reactivated forager). by anticipating the search for a resource, foragers allow the group to exploit it effectively, as they exploit it in the first hours of its opening, and foragers that never anticipate avoid unnecessary risks of predation and energy expenditure. sociobiology an international journal on social insects albeane g silva1,2,3, gracy ca carvalho2,3, ana c miranda2,5, felipe al contrera4, márcia mc rego1,2,3 article history edited by celso martins, ufpb, brazil received 23 september 2020 initial acceptance 04 february 2021 final acceptance 04 march 2021 publication date 31 may 2021 keywords food-anticipatory activity, food resource, inspectors, reactivated, individual consistency, foragers. corresponding author albeane guimarães silva universidade federal do maranhão av. dos portugueses, 1966 cep 65080-805, são luís ma, brasil. e-mail: albeaneguimaraes@hotmail.com the main food sources for bees are nectar and pollen, which are often available to them during restricted temporal windows (van doorn & van meeteren, 2003; matile, 2006; edge et al., 2012). therefore, the presence of a temporal memory constitutes an evolutionary advantage in this group, as bees can save energy, programming their flight activity to coincide with the peaks of daily rhythms of nectar secretion in flowers (corbet & delfosse, 1984; edge et al., 2012) or with the times of higher sugar concentration in the nectar (edge et al., 2012). the collective foraging patterns arise from the diverse activities of individual foragers that make foraging decisions 1 programa de pós-graduação em rede bionorte, universidade federal do maranhão, são luís-ma, brazil 2 programa de pós-graduação em biodiversidade e conservação, universidade federal do maranhão, são luís-ma, brazil 3 laboratório de estudos sobre abelhas, universidade federal do maranhão, são luís-ma, brazil 4 laboratório de biologia e ecologia de abelhas, instituto de ciências biológicas, universidade federal do pará, campus guamá, belém-pa, brazil 5 programa de pós-graduação em neurociências e comportamento, universidade federal do pará, campus guamá, belém-pa, brazil research article bees temporal memory in foraging of the stingless bee melipona subnitida (hymenoptera: apidae: meliponini) albeane g silva, gracy ca carvalho, ana c miranda, felipe al contrera, márcia mc rego – temporal memory in foraging of the stingless bee2 based on intrinsic information (spontaneous preferences and memory) and extrinsic information (e. g. colony nutritional needs, nestmates, and nestmates information in the field) (jarau et al., 2003; biesmeijer & slaa, 2004). individual temporal memory provides a solution when foraging conditions change over the hours (dornhaus & chittka, 2004), given that inspector bees act as the colony’s short-term memory (dornhaus & chittka, 2004; biesmeijer & vries, 2001), enabling the colony to react to rapid changes in its environment, using previously utilized food sources, once they become profitable again. thus, it can be said that the inspection activity performed by the inspector bees is considered an important mechanism for the reallocation of foragers when food sources are difficult to find (dornhaus & chittka, 2004). studies on temporal memory and food-anticipatory activity are already well understood in apis mellifera linnaeus, 1758 (moore & rankin, 1985; aschoff, 1986; von frisch & aschoff, 1987; gould, 1987; wagner et al., 2013). however, this subject remains little explored in stingless bees. although there are studies for trigona amalthea olivier, 1789 (breed et al., 2002), trigona fulviventris guérin, 1844 (murphy & breed, 2008), and melipona fasciculata smith 1854 (jesus et al., 2014), until today, these works carried out on stingless bees have studied temporal memory and food-anticipatory activity at the colony level, this subject never having been studied at the individual level. thus, the present study aimed to evaluate the existence and accuracy of temporal memory in melipona subnitida ducke, 1910 through the observation of the individual foodanticipatory activity of foragers, and to determine if the behavior of anticipating the offered resource presents interindividual consistency in the behavior of foragers. material and methods study area and study species the experiment was conducted with stingless bee melipona subnitida, at the meliponary out in the village of “ponta do mangue” (2º34’52”s; 42º47’44”w), a sandbank area located on the eastern coast of the state of maranhão, in brazil. this settlement is within the domains of the lençóis maranhenses national park (rios, 2001). m. subnitida (popularly known as jandaíra), is a typical bee of the brazilian tropical dry-forest (zanela, 2000), and is highly adapted to the high annual temperatures and the short and irregular rainy season characteristic of this semiarid region (maia-silva et al., 2015). however, it can also be found on the coast of maranhão in the restinga biome, an environment in which it has been extensively investigated for food resources, foraging behaviors, and population genetics (pinto et al., 2014; silva et al., 2014; barbosa et al., 2019; pinto et al., 2020; diniz et al., 2021). this work was carried out with three colonies, two in embrapa models of rational boxes (venturieri, 2008) and one arranged on a natural substrate (trunk); between december 2014 and january 2015, the lowest flowering period in the region and, consequently, the period with the least availability of natural food sources for species. experimental design as an artificial feeder, we used a set of 1.5 ml plastic tubes (eppendorf) containing a 2.5 m sugar and water solution (60% sugar and 40% water), radially arranged in a cylindrical container simulating an inflorescence (based on jesus et al., 2014). to increase the attractiveness of the food resource for the foragers, two drops of vanilla essence were added per liter of syrup (nieh, 2004). during the experiment (7:00am – 11:00am) the tubes were constantly refilled to ensure a constant supply of food. on the training day (1st day), a single tube with food was opened at 7:00 am, close to the colony entrance, so that foragers would be attracted to the feeder. as soon as the bees started the visits, they received a mark on the thorax region with a non-toxic acrylic paint. the feeder was then gradually moved to its final position, 150m from the nest. in this position all the eppendorf tubes were opened, and remained so until 11:00 am, simulating the period of anthesis of a flower. the marked bees that visited the feeder before it was placed in the final position were collected, and kept isolated in a closed wooden box with food until the end of the experiment. these captured bees were important, as they recruited companions from the nest to the food source (nieh, 2004). however, as they were already aware of the feeder from previous distances, they were considered experienced foragers for that resource, and for this reason they were excluded from the experiment. upon reaching the final 150m position, the first ten workers recruited at that point, that is, those who had never visited the feeder, were individually marked with a unique combination of colors, and the times of their first and subsequent visits to the feeder were recorded. these ten bees were used to evaluate the ability of foragers to memorize the time when food is offered. bees that reached the final position after the ten individually marked individuals were classified as recruits. the time of occurrence was recorded, and these recruits were captured and kept isolated to limit visits to the final position to only the first ten foragers. after training (1st day), observations were made between the 2nd and 5th day from 5:00 am to 17:00 pm, to verify the occurrence of inspection at the times when the feeder was closed. on those days, the visiting times of the ten foragers marked on the first day, and the visits of bees recruited by them to the same place where they were closed on the previous day were observed. from 5:00 am to 6:59 am, the feeder remained closed without offering the food resource. from 7:00 am to 11:00 am the tubes were opened and food was provided. from sociobiology 68(2): e5863 (june, 2021) 3 11:00 am to 17:00 pm the feeder was closed again, simulating an inflorescence that stopped offering resources. the feeder was removed each day at 17:00 pm and replaced again the next day in the same position. for the behavioral classification of foragers, the definitions of biesmeijer and vries (2001) were used, which describes a recruit as an “individual which uses external information to search for a previously unknown food resource”, an inspector as an “individual which spontaneously visits a previously known source of food”, a reactivated forager as an “individual which visits a source already known only if they receive information about its availability”, and the employed forager as an “individual which finds and exploits a profitable source of food”. to evaluate temporal memory, the behavior of anticipated visitation of the marked bees was observed, in relation to the times of discovery of the food and time of opening of the feeder. we verified whether or not they arrived at the feeder on the days following the discovery, at the same time, or whether the visiting hours deviated significantly from the discovery time. inspection behavior was considered in relation to the time when food was offered on training days (7:00am – 11:00am). thus, the inspection could take place before the feeder was open (from 5: 00am – 6: 59am), or after it was closed (11:01am – 17:00pm). only visits that occurred during the opening hours of the feeder (7:00 am – 11:00 am) were considered accurate, or up to an hour before the food was offered (moore & rankin, 1983). in general, in order to estimate the precision of the temporal memory of the bees, the percentage of the total number of foragers employed that arrived within the time of the food offer and within the period of one-hour prior was verified. the bees that arrived in this period were designated as “accurate”, and all the others were designated as “inaccurate”. data analysis the watson williams test (zar, 1999; jammalamadaka & sengupta, 2001) was performed to investigate the existence of differences between the different days (1st day to 5th day) (i) in the discovery times of the resource and (ii) in the times of the last visitation of foragers employed. to evaluate the existence of an anticipatory foraging activity, we evaluated the existence of differences in the proportion of bees that visited the feeder before and after the period of offering the resource, using the cochran q test. in the current test, only the times of the first visits between the third and the fifth day were used, considering that the bees on the second day did not yet know the opening period of the feeder. to evaluate the individual consistency in the time of anticipation of the bees and the number of inspection visits carried out before the opening of the resource, we calculated the adjusted repeatability (r) using the rpt function of the rptr package (nakagawa & schielzeth, 2010). the circular watson-williams tests were performed using oriana 4.0 software. the cochran q test was performed using statistica 7.0 software (statsoftinc, 2004), and the repeatability and mann whitney tests were performed using the r 3. 6. 3 program (r development core team, 2020). for all tests, a critical p value of 0.05 was adopted. results visits to the feeder, between the 2nd and 5th day, occurred almost entirely during the time the resource was offered, corresponding to 97.86% (n = 9584) of all visits, confirming the existence of temporal memory in m. subnitida. in relation to the training time, it increased every day (3rd day: 83.3%; 4th day: 92.6%; 5th day: 95.7%). on the first day of the experiment, all the foragers employed by m. subnitida discovered the location of the feeder on average at 8:35 ± 0:52 am. with respect to the time of discovery of the feeder between the 2nd and 5th day, all the foragers employed anticipated their visits in relation to the 1st day, with a significant difference between the days studied (watsonwilliams, f(134,4) = 37.022, p < 0.001), this being noticed between the 1st day and each of the subsequent days (table 1, fig 1). in addition, there was a reduction in the mean and circular standard deviation of the time of the first visits to the feeder over the days (fig 1). all the foragers employed anticipated their first visit to the feeder (2nd to 5th day) before the discovery time (1st day) and, of these, 53% anticipated the opening time of the eppendorf tubes on the third day (6:51 ± 0:40 am; fig 1). we found no differences in the proportion of bees that visited the feeder before and after the period of offering the resource (cochran, q (79, 2) = 3.818; p < 0.14). first visits last visits days f p value f p value 1st and 2nd 59.861 <0.001** 2.416 0.126 1st and 3rd 73.642 <0.001** 3.633 0.062 1st and 4th 73.996 <0.001** 2.423 0.125 1st and 5th 81.222 <0.001** 2.009 0.162 2nd and 3rd 0.63 0.43 0.546 0.463 2nd and 4th 0.263 0.61 6.815 0.012* 2nd and 5th 1.734 0.194 0.037 0.848 3rd and 4th 0.122 0.729 6.949 0.011* 3rd and 5th 0.253 0.617 0.21 0.649 4th and 5th 0.989 0.325 5.002 0.03* **p < 0.001; *p < 0.05 table 1 results of watson-williams f tests performed to show the differences between days and times of the first visits and the last inspections of melipona subnitida foragers to a source of artificial food. albeane g silva, gracy ca carvalho, ana c miranda, felipe al contrera, márcia mc rego – temporal memory in foraging of the stingless bee4 regarding the inspection behavior between days 3 and 5, we concluded that there was inter-individual consistency (repeatability) in the anticipation time (r = 0.21 ± 0.09; p = 0.001) and in the number of visits before the opening of the resource (r = 0.55; p < 0.001). in general, inspector bees represented approximately 50% of all foragers employed (fig 2). fig 1. time of the first visit of m. subnitida foragers to an artificial feeder on each experimental day. the length of the sections indicates the number of bees in the corresponding time range. the radial line indicates the average time of the first occurrence ( ) and the line perpendicular to this indicates the circular standard deviation (s). fig 2. individual time of the first visit of each forager of m. subnitida to an artificial feeder on each experimental day (the numbers correspond to the days that occurred or not food-anticipatory activity 3rd, 4th and 5th day) sociobiology 68(2): e5863 (june, 2021) 5 with regard to the times of the last visits, 42% of these occurred after closure of the feeder, differing significantly over the days (watson-williams, f(134,4) = 2.78, p = 0.029), being that the majority of these visits occurred in the first minutes after closing (70.8%). this difference was observed between the 2nd and the 4th day (watson-williams, f(54,1) = 6.81, p = 0.012), 3rd and 4th day (watson-williams, f(54,1) = 6.94, p = 0.011), and 4th and 5th day (watson-williams, f(54,1) = 5.002, p = 0.03). the other days did not differ from each other (table 1). regarding the closing time of the artificial feeder, important differences were found between visits that occurred before and after 11:00am (cochran, q(79, 3)= 17.20; p < 0.0006), being that 61% of the last visits occurred before the feeder was closed. however, we noticed that even after the feeder closed, some bees continued to inspect it (fig 3). during the days of the experiment, the foragers employed in the three colonies recruited 281 bees (µ= 93.7 ± 63.03) in the final position, with the highest concentration of recruited bees occurring in the first hours after opening of the feeder ( = 08:23± 0:54am). fig 3. time of the last visits of forages of m. subnitida to an artificial feeder on each experimental day. the length of the sections indicates the number of bees in the corresponding time range. the radial line indicates the average time of the last occurrence ( ) and the line perpendicular to this indicates the circular standard deviation (s). discussion our results point to the existence of temporal memory in m. subnitida, which is verified from the behavior of foodanticipatory activity and the end of the search for the resource when the availability of food has ceased, as seen in other stingless bees: t. amalthea, t. fulviventris, and m. fasciculata (breed et al., 2002; murphy & breed, 2008; jesus et al., 2014). in m. subnitida this temporal memory proved to be accurate, with bees anticipating visits on the days following their discovery, as observed in previous studies with stingless bees (breed et al., 2002; murphy & breed, 2008; jesus et al., 2014), and for a. mellifera (moore & doherty, 2009). in a. mellifera (moore & doherty, 2009) as in the current study, a trend was observed, in which the accuracy of temporal memory increases with the passing of days, allowing these individuals to explore the resources available in the environment as quickly and efficiently as possible. this temporal memory also allows many foragers not to start the day as novices, eliminating the need to spend energy unnecessarily to rediscover the same food resources (wagner et al., 2013). all the foragers employed by m. subnitida presented anticipation in the search for the resource in relation to the time they discovered the resource on the first day, and some also anticipated the opening time of the feeder. the existence of food-anticipatory activity is important to demonstrate the temporal notion of these bees, considering that the individuals who arrived before the presentation of the food were not being attracted by olfactory and/or visual cues, and were therefore using a memory of the time of availability of the resource albeane g silva, gracy ca carvalho, ana c miranda, felipe al contrera, márcia mc rego – temporal memory in foraging of the stingless bee6 in the environment (murphy & breed, 2008). anticipating the search for a resource is an advantageous behavior, as it increases the opportunity to monopolize a resource before any potential competitor arrives, minimizing competition with species that arrive later or have yet to find the resource (hubbell & johnson, 1978; jesus et al., 2014). in addition, the anticipation of resource exploitation may also be related to the rhythm of flower nectar production, which could be less accurate than the artificial feeder and, therefore, less predictable throughout the day (moore & rankin, 1983). the existence of food-anticipatory activity in melipona bees, can be considered an advantageous behavior, since it allows the avoidance of direct competition with species that are ecologically dominant and have mass recruitment, such as, for example, foragers of the genus trigona, which present rapid mobilization of workers when food is available (nieh et al., 2004; breed et al., 2002), unlike what happens in foragers of the genus melipona, where the decision to revisit a resource, in most cases, is individual (biesmeijer et al., 1998; biesmeijer & vries, 2001). similar to what happens with trigona fulviventris (murphy & breed, 2008) and a. mellifera (von frisch, 1967), about half of the foragers of m. subnitida did not memorize the time of availability of the resource, that is, they did not anticipate the search for the resource before it was available. for murphy and breed (2008) this difference between bees that memorize, and bees that do not memorize, could indicate the existence of variability in this capacity between individuals, or even that all the bees have the ability to memorize, but only a subset of foragers express the behavior. this variability can be proven in the current work, where through the repeatability analyses we observe an inter-individual consistency in the forager behavior. according to moore et al. (2011) inspecting foragers that leave the colony in search of the resource in anticipation of the time when the food is available, can also be considered “persistent bees” and those that visit the resource only after receiving information about its availability, “reticent bees” (moore et al., 2011; wagner et al., 2013). in social bees, colonial behavior emerges from the actions of the individuals (pinter-wollman, 2012), and the variation between individual consistency could be important in determining the difference observed between colonies. on average, 50% of the marked foragers were considered inspectors, differing from the observations made by moore et al. (2011) with apis, in which a surprisingly high percentage of foragers were inspectors (about 40%, 60%, and 80% of foragers with 1, 2, or 3 days of experience of a food resource). for m. subnitida, the number of inspector bees did not vary over the days as occurred with honey bees (moore et al., 2011), but this percentage is also considered to be very high, as few bees are needed to recruit companions in the nest (biesmeijer et al., 1998; hrncir et al., 2000; jarau, 2009). according to van nest and moore (2012), bees that do not inspect, that is, wait in the nest for communication about a profitable resource (reactivated foragers), vary between 4090% of the total forager population, this distribution being extremely important, as it saves time and effort, since these bees only return to a known food after being recruited by inspector bees. the recruitment in m. subnitida started as soon as the feeder was opened, with a peak in the first hours of opening of the resource, suggesting the existence of an efficient strategy in the exploitation of a food resource that has a restricted offer period, with recruits and experienced foragers exploiting the resource in the first hours of the nectar secretion. according to moore and rankin (1983) this behavior ensures that a maximum number of bees forages during the availability of the resource. regarding the inspections that took place after 11 am, when the feeder was closed, few inspections were verified. this may be related to the fact that bees can exchange an unfavorable food resource for a profitable one in a short period of time (wagner et al., 2013; maia-silva et al., 2015). studies with apis bees found that this rapid relocation is largely due to changes in the recruitment of foragers to a more profitable resource (seeley et al., 1991; granovskiy et al., 2012; wagner et al., 2013). however, it was observed that some bees, even after the feeder was closed, carried out sporadic inspections in the middle of the afternoon when the resource was closed. this is because foragers spend less energy inspecting a known resource than they would spend constantly looking for abundant new resources (jesus et al., 2014). on almost every day of the experiment, bees visited the feeder more at the beginning than at the end of the opening period of the resource, a result similar to that found by moore and rankin (1983) with apis. this is to be expected if the bees fail to visit at the beginning of the training period, or after several visits without reward, the tendency is for return visits to decrease. in these cases, foraging efficiency can be seen by the ability to predict when a nectar resource will no longer be available that day (moore & rankin, 1983). however, exploring a resource early in the morning (for plants that have anthesis in this period), can also be seen as a strategy to collect large amounts of resources in the first hours of exposure, when the resource may be more abundant (maiasilva et al., 2015). another explanation for the preference for the first hours of availability of the resource can be explained by the existence of adverse climatic conditions – for example in areas with high temperatures like the caatinga or restinga, bees may be exposed to the danger of overheating in the hours close to noon (willmer & corbet, 1981). the foraging pattern of melipona bees is characterized by peak pollen collection in the early morning, and nectar collection in the late morning/early afternoon (bruijn & sommeijer, 1997; pierrot & schlindwein, 2003; correia et al., 2017). these bees collect nectar and water throughout the day, intensifying in the late afternoon (from 4pm) due to the high sociobiology 68(2): e5863 (june, 2021) 7 temperatures observed in the period from 12am to 2pm, thus requiring more water to maintain the balance of temperature and relative humidity inside the colony (correia et al., 2017). therefore, the nectar collection in our artificial feeder with defined anthesis time (7am to 11am), the food-anticipatory activity and a few inspection visits in the afternoon confirm the existence of temporal memory in m. subnitida. the knowledge of the existence of temporal memory and individual consistency in foragers of m. subnitida, bees that live in environments with a hot and dry climate, where there is a shortage of resources throughout the year, are evidenced as strategies to maximize the success of foraging of this species. in fact, after finding a profitable resource, the temporal memory allows the resource to be revisited on the following days, through the experience of the previous day. in addition, by anticipating the search for a resource, foragers allow the group to exploit it effectively, as they exploit it in the first hours of opening, when it is most abundant. on the other hand, foragers that never anticipate (reticent foragers) and wait for information on the resource avoid unnecessary risks of predation and energy expenditure. these adaptive strategies can assist in the permanence of this species in their natural environments, allowing foragers to schedule their flights to plants that have a more favorable anthesis period, avoiding exposure to high temperatures and low humidity, typical of their natural environments. acknowledgements to the brazilian agricultural research corporation – embrapa/macroprogram for financing the project (cód. 02.11.01.029.00.00) and the maranhão research support foundation – fapema. to capes for the grant of the master’s scholarship. to the graduate program in biodiversity and conservation for the opportunity and teaching. to the owners of jandaíra’s meliponary: irene aguiar, mr. emídio aguiar (in memoriam), and dona maria do socorro aguiar. authors’ contribution ags: conceptualization, methodology, investigation, formal analysis, writing gcac: methodology, investigation acm: methodology, formal analysis, writing falc: conceptualization, methodology, witing mmcr: conceptualization, methodology, witing references aschoff, j. 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(1999). biostatistical analysis. prentice hall, new jersey. move473310842 move473311059 doi: 10.13102/sociobiology.v65i2.1017sociobiology 65(2): 208-214 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 weaver ant oecophylla longinoda latreille (hymenoptera: formicidae) performance in mango and cashew trees under different management regimes introduction ants represent one of the most abundant and ubiquitous arthropod groups on earth and play a major role in regulating the environment (hölldobler & wilson, 1994). in tropical regions, weaver ants, oecophylla spp (hymenoptera: formicidae) are dominant ants (lim, 2007) in a wide range of host plant species such as citrus, mango, cashew, coconut, cocoa, among others. they build their nests by weaving living leaves on their host plants together with silk from their larvae. the host plants, thus, provide a habitat for the ants and they also provide the ants with food via floral and extra-floral nectaries and honeydew excreted from associated hemipteran trophobionts abstract weaver ants are used for biological control of insect pests in plantation crops. to obtain proper pest control, ant densities need to be high. food availability and nesting facilities on host plants and management practices may affect ant performance. in the present study, we tested the effect of two host plant species (mango and cashew) and three different management practices (ants only, ants fed with sugar and ants combined with the soft chemical insecticide spinosad) on weaver ant performance. performance was assessed over a 22 month period, as an index value based on the number of ant trails per tree and as the number of ant nests per tree. a total of 216 trees (72 per treatment) were observed in each crop. in all treatments, the ants performed better in mango compared to cashew. using the index based performance measure, ants also performed better in the sugar treatment as compared to the two other treatments, whereas this was not the case in cashew. we conclude that sugar feeding can be used to increase ant populations in mango. we also found that the treatment with spinosad in combination with ants showed performance equal to the treatments with only ants, suggesting that spinosad did not negatively affect ant populations. we therefore conclude that spinosad is compatible with the use of weaver ants in integrated pest management programs. sociobiology an international journal on social insects rb wargui1, a adandonon2, a sinzogan1, fm anato1, jf vayssieres3, dk kossou1, j offenberg4 article history edited by evandro n. silva, uefs, brazil received 03 march 2016 initial acceptance 07 august 2016 final acceptance 07 december 2016 publication date 09 july 2018 keywords oecophylla ants, density index, nest number, mango, cashew. corresponding author rosine brisso wargui université d’abomey calavi 03 bp 2819 cotonou, république du bénin e-mail: detie11@yahoo.fr (way, 1963; kenne et al., 2003). although oecophylla ants are opportunistic in their choice of host plants (way & khoo, 1992), the choice of nest sites is not random (djiéto-lordon & dejean, 1999). nest site selection depends on innate selective attraction and on environmental factors (djiétolordon & dejean, 1999). for example, leaf flexibility, i.e. the degree to which leaves bends when pulled by the ants, determines nest site selection (hölldobler, 1983). therefore, weaver ants may have preference toward certain host plant species (dejean et al., 2008). for instance, they preferred the leaves of citrus and mango rather than the leaves of cocoa and guava (dejean et al., 2007) and lim (2007) observed that oecophylla smaragdina (hymenoptera: formicidae) showed 1 faculté des sciences agronomiques, université d’abomey calavi, cotonou, république du bénin 2 ecole de gestion et de production végétale et semencière, université nationale d'agriculture, cotonou, république du bénin 3 cirad, upr hortsys, montpellier, france 4 department of bioscience, aarhus university, dk-8000 aarhus c, denmark research article ants mailto:detie11@yahoo.fr sociobiology 65(2): 208-214 (june, 2018) 209 a strong preference for morinda citrifolia l. (rubiaceae) because its leaves were larger and appear more pliable than the leaves of other hosts plants assessed. moreover, according to offenberg et al. (2006) weaver ants has a preference to use first position leaves for nest building because these leaves are still expanding and therefore smaller and more flexible than the older leaves and also more likely to host trophobionts. the characteristics of host plant leaves are therefore an important factor in the choice of nesting site. in return for a nesting site and food, weaver ants may protect their hosts against insect pests (hölldobler & wilson, 1990; majer, 1993; djiéto-lordon & dejean, 1999). as biocontrol agents weaver ants have proven efficient in controlling several pests in plantations of mango (peng & christian, 2004; peng & christian, 2005a; peng & christian, 2005b ; peng & christian, 2006; peng & christian, 2007; peng & christian, 2008; davidson et al., 2007; adandonon et al., 2009; van mele et al., 2007), citrus (huang & yang, 1987; van mele & cuc, 2000; van mele et al., 2002; van mele, 2008), cashew (peng et al., 1995; peng et al., 1997; peng et al., 2008; dwomoh et al., 2009, olotu et al., 2013), coconut (way, 1954; vanderplank, 1960; varela, 1992; sporleder & rapp, 1998) and cocoa (ayenor et al., 2004; ayenor et al., 2007). for reviews see way and khoo (1992) and offenberg (2015). for the ants to be effective control agents they need to be present at high densities (peng et al., 2008), therefore in some cases they need to be managed by e.g. providing artificial food (abdullah et al., 2015; peng et al., 2008). in other cases the ants need to be supplemented with complementary control methods in integrated pest management (ipm) programs in order to achieve adequate host plant protection (peng & christian, 2006). however, this type of plant management action may have negative effect on ants. in northern benin, mango and cashew are the most important tree crops with high economic importance (vayssières et al., 2008; balogoun et al., 2014). recently, pest control by weaver ants has been observed in mango and cashew (adandonon et al., 2009; van mele et al., 2007; anato et al., 2015) and here the fruit fly bait gf-120 has been identified as an effective supplementary ipm component that in combination with weaver ants may lead to better control of pests such as fruit flies and thrips (vayssières et al., 2009; anato et al., 2015). at the same time gf-120 is an environmentally safe insecticide to use. therefore weaver ant combined with this insecticide may be used in an ipm approach for the biological control of pests in benin. to test the effect of host plant species, ant feeding, and the application if ipm components on ant performance we compared ant performance in cashew and mango trees where weavers ant were managed as either not fed, fed sugar or combined with gf 120. performance was assessed by counting ant trails and nest numbers in the trees. materials and methods study site and experimental design the study was conducted in one mango and one cashew orchards in the parakou area (09° 22’ 13”n / 02° 40’16”e) of benin from 2012 (august) to 2014 (may). both plantations are located in the same area with approximately 20 m distance between them. mango trees were between 30 and 32 years old and cashew trees between 20 and 25 years old. each orchard had an average of 100 trees/ha at 10 m × 10 m spacing and trees were homogeneous in height. in each orchard 216 trees were randomly divided into three treatments: (1) trees with weaver ants (ants) used as control, (2) trees with weaver ants where the ants were fed with sugar (ants feeding), and (3) trees where weaver ants were combined with the conventional insecticide gf-120 (ants + gf-120). in each crop there was a total of 72 trees per treatment and two rows of trees were used as a buffer zone between two consecutive treatments. buffer zones of this size have been used in previous studies to protect neighboring treatments against the effect of insecticides (peng & christian, 2005b; offenberg et al., 2013). treatments in all treatments, an artificial bridge made of twisted polystrings of approximately 3 mm diameter was used to connect all the trees belonging to the same weaver ant colony in order to facilitate ant communication (peng et al., 2008; vayssières, 2012), and interlocking tree branches between neighboring trees with different ant colonies were removed by pruning, to avoid ant fighting between colonies. in the ant + gf-120 treatment, a conventional insecticide gf-120 (nf naturalyte fruit fly bait, dow agrosciences llc, indianapolis, in) with 0.02% spinosad (ai) and 98.8% inert ingredients (water, sugars, and attractants) was used to spray the trees. spraying was done weekly from february to march in 2013 and again in 2014 in the cashew orchard and from february to april in 2013 and 2014 in the mango orchard. one square meter of each tree was sprayed as recommended at head height in an area without fruits. the recommended dose (1.5 l of gf-120/ha) was used for treatment (vayssières et al., 2009) at the ratio of 1 liter of gf-120 for 5 liters of water (dow agrosciences, 2001). the insecticide was applied as a foliar spot spray, using a manual sprayer (berthoud apollo 16af) with a conventional conical nozzle (1–2 mm aperture to deliver droplets of 2–6 mm). during the weekly applications, rotation around the tree was used to avoid phytotoxicity on previously treated surfaces (vayssières et al., 2009). in the ant feeding treatment, weaver ants were fed with a 30% sucrose solution provided in 60 ml plastic bottles plugged with cotton with one bottle per tree. the sugar feeders were placed upside down on a tree trunk with a busy ant trail and refilled once per week. also, water was provided ad libitum in a bottle on each of the trees in the ant feeding treatment. sugar and water were provided at the same time in mango and cashew plantations. rb wargui et al. – african weaver ant in mango and cashew trees210 weaver ant density on mango and cashew trees weaver ant density was assessed fortnightly in each tree from august 2012 to may 2014 (50 times during the study period) in both the mango and the cashew orchard. density was assessed using the “branch method” presented in offenberg and wiwatwitaya (2010). with this method the number of ant trails in a tree is used as a measure of ant density, resulting in an index value ranging between 0 and 100 %. the counting of ant trails on the trees was conducted between 9:30 and 13:30 which is within the most active period of o. longinoda in benin (vayssières et al., 2011). weaver ant colonization level was also assessed by counting the number of weaver ant nests per tree in each tree once a month (22 times during the study period). nest numbers only included naturally occurring nests as artificial movement of nests between trees was not used in the management of ants in these plantations. data analysis weaver ant performance for each particular tree was expressed as the mean value of (1) the weaver ant density indexes recorded over the seasons and, (2) as the mean number of weaver ant nests counted over the two seasons. log10 (x + 1) and arcsin √x transformations were used, respectively, on nest number and index values to stabilize variance and normalize the data before analysis. values obtained were then compared with two way anovas and tukey pairwise comparisons to test the effect of crops (mango and cashew) and treatments (ants, ants feeding and ants + gf-120) on weaver ant performance. the number of main branches on each tree was also assessed and compared between crops and treatments with a poisson distribution, as trunk numbers may in some cases affect the index values (wargui et al., 2015). all the statistical analyses were done with jmp 10.0.0. (sas, 1995). results weaver ant abundances were above 50 % on the two crops during the entire study period (fig 1a) and both index values and nest numbers differed significantly between crops and among treatments (fig 1, tables 1 and 2). using either of the two ant density measures, the ants performed significantly better on mango compared to cashew in all the three treatments (tables 1 and 2, fig 1). the weaver ant density index in mango was on average 15 % higher than in cashew (8 %, 25 % and 12 % in the ants, ants feeding and ipm treatments, respectively), whereas, nest numbers were on average 95 % higher in mango (79 %, 107 % and 98 % in ants, ants feeding and ipm treatments respectively). in mango, ants performed better in the ants feeding treatment compared to both the ant (index: t ratio = 10.80, p < 0.0001; nest numbers: t ratio = 5.09, p < 0.0001) and the ant + gf-120 treatments (index: t ratio = 9.35, p < 0.0001; nest number: t ratio = 2.95, p = 0.039). on the other hand, there was fig 1. average density of weaver ant tree-1(± se) (a) and average number of weaver ants nests tree-1 (± se) (b) on cashew and mango crops. n= 72 trees per treatment in each crop. df sum of squares f ratio p crops 1 0.76 121.4 < 0.0001 treatments 2 0,70 56.3 < 0.0001 crop* treatment 2 0.22 17.9 < 0.0001 no significant difference between the ant and the ant + gf120 treatments (index: t ratio = -1.45, p = 0.70; nest number: t ratio = -2.14, p = 0.27). when comparing the ant feeding treatment with the ant and ant gf-120 in cashew, there were only significant (or nearly significant) differences when comparing the index values (t ratio = 2.78, p = 0.063; t ratio = 2.98, p = 0.036, respectively), not when using nest numbers (t ratio = 1.99, p = 0.34; t ratio = 1.58, p = 0.61, respectively). also, in cashew, there were no differences between the ant and the ant + gf-120 treatments, table 1. two way anova test for comparison of weaver ant mean density index per tree between crops (mango and cashew) and treatments (ants, ants feeding and ants + gf-120). n = 432 trees. sociobiology 65(2): 208-214 (june, 2018) 211 neither with the index (t ratio = 0.20, p = 0.99) nor with nest numbers (t ratio = -0.41, p = 0.998). the number of main trunks on mango trees was significantly higher compared to cashew (chi-square = 52.7, df = 1, p < 0.0001), whereas no difference was observed between treatments within each crop (table 3). the number of main trunks on mango ranged from three to eight with a median of five, whereas they ranged from two to seven with a median of four, in cashew. discussion this study showed that weaver ants performed better on mango than on cashew, based on ant trail activity and nest numbers. it should be noted, though, that also the numbers of main trunks on trees, which may affect the index values, were significantly different between the crops. according to wargui et al. (2015) a higher number of trunks may reduce the ant trail index value on trees. in the present case that would mean that index values are underestimated in mango and therefore the difference from cashew is conservative. moreover, the fact that nest numbers, which are unaffected by the number of main trunks, were also higher in mango, further support the conclusion that o. longinoda perform better in this crop. nest numbers may also not be a precise measure of ant abundance, especially when comparing different crops, since nest sizes may differ between host plant species due to different leaf morphology. ouagoussounon et al., (personal communication) demonstrated that o. longinoda build smaller nest in cashew trees compared to mango trees, and therefore each nest probably hold fewer ants. but again, this supports our conclusion as we may then have overestimated ant performance in cashew and yet found performance to be higher in mango. that ants prefer or perform better on some plants compared to others is also supported by other studies. for instance, according to kenne et al. (2003), o. longinoda was more frequent on both citrus and mango trees compared to guava trees and dejan et al. (2007) mentioned that oecophylla spp founding queens preferred citrus and mango leaves rather than the leaves of cocoa and guava trees. also the ants camponotus acvapimensis (mayr) and paratrechina longicornis (latreille) have been recorded more frequent on citrus than on mango trees (kenne et al., 2003) in cameroon, and the same authors (kenne et al., 2009) demonstrated that the arboreal ant species atopomyrmex mocquerysi (e. andré), was frequently found on safoo trees and rarely on cocoa, avocado, guava, mango and citrus. thus, for several ant species, there is a preference of host plant species, probably influenced by plants characteristics. according to lim (2007), characteristics such as leaf size, leaf flexibility and food resources on the plants may be important. o. smaragdina has been reported to prefer host plant species with leaf sizes ranging from 5 x 8 cm and up to 20 x 20 cm and avoiding tough-leaved plant species (blüthgen & fiedler, 2002). as mango leaves are smaller and more pliable than the rather tough cashew leaves (rosine wargui, personal observations) this may explain the difference found in the present study. further, secretions of extra-floral and floral nectars by host plants and the presence of hemipteran trophobionts on these host plant may favor the development of the ants (kenne et al., 2003). mango trees in the sudanian area of benin were reported to harbor large numbers of scale insects (12 species) particularly on mango fruit petioles and on the fruits (germain et al., 2010). in contrast, vayssières (2012) recorded only two hemiptera species on cashew in northern benin. these factors together, may explain why o. longinoda was more abundant in mango. however, further studies are needed to clarify mango and cashew characteristics allowing better knowledge of weaver ant preference and performance. the implications of the different performance in mango and cashew is, for example, that when using weaver ant as biological control agents the ants may require more management attention in cashew than in mango plantations. indeed, management measures such as baiting competitive ants species, connecting trees within the same ant colony, removing interlocking branches between trees belonging to different colonies and feeding ants, may facilitate weaver ants and in this way contribute to boost ant densities in less favorable crops. such measures are required in plantations when weaver ant densities are below 50 % using the index developed by peng and christian (2004). it is important to examine different crop species effect on ants and to monitor their abundance in order to gain profit from their presence in plantations trees. a varying performance on different hosts also suggests that intercropping inferior host with more ant profitable crops may increase biocontrol effectiveness. this is especially the case when crops have different phenologies. in that case the ants can continuously migrate to more actively growing hosts as the season change. this would benefit ants, as food is more abundant on trees with growing plant tissues, and it will benefit crops as ants will aggregate on developing plant tissue (new leaves, flowers and fruits) which is most in need of protection. df sum of squares f ratio p crops 1 7.8 514.7 < 0.0001 treatments 2 0.4 12.9 < 0.0001 crop* treatment 2 0.07 2.4 0.09 table 2. two ways anova comparing the mean number of weaver ant nests per tree between crops (mango and cashew) and treatments (ants, ants feeding and ants + gf-120). n = 432 trees. df l-r chi squares p crops 1 52.7 < 0.0001 treatments 2 1.6 0.46 crops x treatment 2 2.0 0.37 table 3. poisson test for comparison of number of main trunk per tree between crops (mango and cashew) and treatments (ants, ants feeding and ants + gf-120). n = 432 trees. rb wargui et al. – african weaver ant in mango and cashew trees212 our study also showed that weaver ants were more abundant on trees with sugar feeders compared to the two other treatments, suggesting that ant feeding promoted the ant population and can be used as a management tool to facilitate biological control. this was the case in both crops if the index was used as a population measure. however, nest numbers were not significantly higher in cashew, only in mango. as mentioned in a previous study (offenberg & wiwatwitaya, 2010), sugar feeders may themselves increase the index value, as they are placed on the main trunks where also ant trails are assessed. thus, if the sugar feeders attract more ants to the trunk, than would otherwise be the case, the ant trail based index may become inflated. therefore, the result based on the index should be interpreted with caution. as a result we cannot conclude with certainty that sugar feeding improved ants in cashew, though it is likely. however in mango we observed higher nest numbers as well as higher index values in the ant feeding treatment, indicating that sugar feeding did increase the populations in this crop. we therefore suggest sugar feeding as a possible measure to increase weaver ant population in plantations. lastly we found not difference in population measures between treatments with only ants and the treatments where ants were combined with gf-120. this was the case for both crops and using both population measures. we therefore find it safe to conclude that spinosad in the form of gf-120 and applied as described here and in anato et al. (2015), is compatible with the use of weaver ant. this is of importance as spinosad have proven effective against fruit flies in mango (prokopy et al., 2003; wang et al., 2005; vayssières et al., 2009) and against thrips in cashew (anato et al., 2015) and since these species are important pest in west africa (vayssières et al., 2008; anato et al., 2015), the efficiency of pest control may be advanced by combining weaver ants with gf-120. this is in concordance with previous studies demonstrating that gf-120 had no detectable effect on beneficial insects such as the parasitoids aphytis spp (thomas & mangan, 2005) and fopius arisanus (sonan) (vargas et al., 2002). according to stark et al. (2004) parasitoids are less susceptible than fruit flies to spinosad (provesta protein bait spray). spinosad in the form of gf-120 can therefore be included in ipm programs with o. longinoda as a major component for the control of both mango and cashew major pests. acknowledgments we would like to thank m. zoumarou-wallis, the owner of the two orchards where this study was conducted at korobororou village for providing field facilities. we thank also gbetable gabin, jacques sogbossi and crépin aniwanou, for their technical assistance. grateful thanks to the project “increasing value of african mango and cashew production”. danida (dfc no. 10 – 025au) for financial support. references abdulla, n.r.; rwegasira, g.m.; jensen, k-m.v.; mwatawala, m.w. & offenberg, j. 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(2005). effects of spinosad-based fruit fly bait gf-120 on tephritid fruit fly and aphid parasitoids. biological control, 35: 155-162. wargui, r.; offenberg, j.; sinzogan, a.; adandonon, a.; kossou, d. & vayssières, j-f. (2015). comparing different methods to assess weaver ants abundance in plantations trees. asian myrmecology, 7: 159-170. way, m.j. (1954). studies of the life history and ecology of the ant, oecophylla longinoda latreille. bulletin of entomological research, 45: 93-112. way, m.j. (1963). mutualism between ants and honeydewproducing homoptera. annual review of entomology, 8:307344. way, m.j. & khoo, k.c. (1992). role of ants in pestmanagement. annual review of entomology, 37: 479-503. http://dx.doi.org/10.1093/jee/96.5.1485 http://dx.doi.org/10.1093/jee/97.3.911 http://dx.doi.org/10.1603/0022-0493-98.6.1950 http://dx.doi.org/10.1603/0022-0493-98.6.1950 http://dx.doi.org/10.1093/jee/100.3.695 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1261sociobiology 64(1): 111-118 (march, 2017) diversity and structure of social wasps community (hymenoptera: vespidae, polistinae) in neotropical dry forest introduction the several brazilian ecosystems have different characteristics resulting in specificity in certain animal groups, including wasps. in this context, stands out the tropical dry forests of northeastern brazilian semi-arid region, which is characterized by technical criteria as a region with the average annual rainfall of less than 800 mm, irregularly distributed in space and time, and the risk of increased drought in 60% (brazil, 2007). northeastern brazil is composed by 90% of semi-arid climate, where neotropical dry forests are found in areas with very specific characteristics called caatinga (brazil, 2007). this biome is characterized by tropical dry forest with an increase on resource supply in the rainy periods (pereira filho et al., 2013). the caatinga biodiversity is considered to be the lowest when compared to other areas of brazil (see ducke, 1907). lewinsohn (2000) emphasized the importance of surveys on the biodiversity for this biome. brandão et al. (2000) abstract social wasps are potential predators of agricultural pest species. the objective of this study was to conduct a survey of the community of social wasps (polistinae species) that inhabit the neotropical dry forest named caatinga of paraiba, northeast of south america (brazil), and to compare the richness and abundance of wasps located in areas of caatinga with those inhabiting areas of organic intercropping farming. the present study sampled 10 polistinae species distributed in six genera. two new species were registered for the state. the comparison between the community of social wasps of the caatinga showed that there was not statistical difference in the richness and abundance between the natural vegetation and organic intercropping. this means that agroecosystem attracted community of polistinae, promoting the maintenance of social wasps in anthropic environment and possibly favoring biological control. sociobiology an international journal on social insects t elisei¹, e valadares¹, fa albuquerque², cf martins¹ article history edited by gilberto m. m. santos, uefs, brazil received 28 november 2016 initial acceptance 12 december 2016 final acceptance 13 february 2017 publication date 29 may 2017 keywords biodiversity, survey, caatinga, semi-arid, intercropping. corresponding author thiago elisei universidade federal da paraíba programa de pós-graduação em ciências biológicas (zoologia)/ccen joão pessoa-pb, brasil e-mail: thiagoelisei@gmail.com revealed the lack of studies on invertebrates for the northeast region. recent studies in the caatinga have showed that the fauna and flora were underrated and also revealed important adaptations of its organisms to the irregular rainfall (leal et al., 2005). in addition, the knowledge of its diversity is very important to understanding the environmental changes caused by human activities or by natural factors, including climate changes (lawton et al., 1998). the caatinga is considered a very important biome in the discovery of new species and new records, once it is understudied and has a long history of deforestation and fragmentation, mainly by human activities (guérnard, 2014). social wasps surveys in brazilian tropical dry forests were made mainly in the bahia state, which is responsible for the majority of polistinae diversity found in the semi-arid region (aguiar & santos, 2007; santos et al., 2007; santos et al., 2009; andena & carpenter, 2014). in paraiba state no surveys of social wasps were carried out. the diversity of this group is known only by studies of associated entomofauna 1 universidade federal da paraíba, programa de pós-graduação em ciências biológicas (zoologia)/ccen, joão pessoa-pb, brazil 2 empresa brasileira de pesquisa agropecuária (embrapa), campina grande-pb, brazil research article wasps t elisei, e valadares, fa albuquerque, cf martins – social wasps in the neotropical dry forest of brazil112 and floral visitors. only nine species of polistinae were recorded for paraiba (andena & carpenter, 2014). the aim of this study was to accomplish a survey of the polistinae species in the paraiba dry forest, and compare the richness, abundance and composition between the natural vegetation and intercropping on organic farms. material and methods study area this study was carried out from 2013 to 2015, in the cities of sumé (7 ° 40 ‘18’ ‘s, 36 ° 52’ 54 ‘’w) and prata (7 ° 41’27 “s, 37 ° 4’48 “w), both located in the western cariri of paraiba, one of driest regions of the brazil, in the northeast of south america. at these areas low mechanization and intercropping on organic farms are stablished. the region is predominantly agricultural and goat breeding with patches of tropical dry forest, characterizing the caatinga biome. the rainy season occurs mainly between january to july (rainfall season), representing 78% of total annual precipitation (sena et al., 2012). methodology of survey the sample and transportation of specimens were authorized and certified by the brazilian institute of environment and renewable natural resources (ibama). social wasps were sampled through attractive pet bottle trap and active search, guided by three transects of 100 meters length, 20 meters away one of the other. these transects were repeated in the farm areas as well as in the caatinga area. the wasps were captured through 60 traps (30 in each area), containing passion fruit juice concentrate and neutral detergent, distributed 10 per transect, distant from each other by 10 meters. these traps were exposed in the environment for five days per sample. the active search was performed with hand net, in fixed periods of 20 minutes, walking close to the transects with attractive traps. the perpendicular distance of search on the track was limited to two meters on each side of the transects. social wasps specimens captured were placed in plastic pots containing 70% alcohol. in addition to the field survey, it was analyzed a material captured by embrapa cotton in caatinga vegetation and intercrops areas, at sumé city. embrapa data were sampled in 2011 using malaise traps. data analyses the relative frequency and constancy values were calculated from the data obtained for wasps species and abundance. the frequencies were determined by the proportion of individuals of a species on total of individuals of the sample, showed as a percentage. constancy was calculated from the percentage of samples in which one particular species was present. the species were classified by their constancy, being constant (c) as present in over 50% of the samples; accessory (aa) as present in 2550% of the samples; accidental (al) as present at less than 25% of the samples. analyses were performed in the excel program. the data was submitted to normality shapiro-wilk test (r-program). analysis of the communities of wasps was performed in estimates (version 9.1.0), in which the shannon wiener indices and richness estimators (chao 1) were obtained. the rarefaction curve was built in excel. the data of abundance and richness were used to check whether the caatinga and intercrop areas differ statistically. for this, the wilcoxon test and levene (r-program) were used. species identification the sampled material was identified with the support of taxonomic keys, comparison with material deposited in the entomological collection of the state university of feira de santana, and by the specialist dr. sergio andena, from this institution. the specimens were deposited in the entomological collection of department of systematics and ecology/ufpb. results and discussion social wasps from cariri, paraiba, brazil the survey resulted in 341 specimens of social wasps, from ten species in six genera (table 1). the richness found was similar to that shown in the study of santos et al., (2006) and aguiar and santos (2007) in the caatinga of bahia state. these authors reported, respectively, 12 and 13 species of social wasps as flower visitors. nevertheless, santos et al., (2009) registered a richness of 17 species in a survey of nests in caatinga and agricultural systems. however, the highest number of species was recorded in the rainy season, while in the dry season only 13 species were found. the study recorded two new species of social wasps to paraiba state: polistes simillimus and mischocyttarus cearensis, being also the first record of mischocytarini tribe (elisei et al., 2015). these two species had been reported to caatinga, and found in other studies realized on northeastern brazil states (santos et al., 2007; melo et al., 2015). andena and carpenter (2014) registered only nine species of social wasps in paraiba state, being the addition of those two new species a significant increase on the diversity of polistinae. the present study was carried out in a historic drought period (table 2). thus, richness may have been affected by this weather phenomenon, reducing diversity of social wasps, once they are influenced by environmental variations (richter, 2000). this is so because with the decrease of nutrients, associated with reduced rainfall, also occurs a decline in search activity for resources (jeanne, 1991; resende et al., 2001; lima & prezoto 2003; elisei et al., 2005; ribeiro jr et sociobiology 64(1): 111-118 (march, 2017) 113 al., 2006; elisei et al., 2013). santos et al., (2009) and souza et al., (2012) verified that the number of nests diminished with decreasing rainfall during the two seasons (dry and wet). on this way, rainfall is a very important factor on the polistinae community dynamic. data analysis revealed that the sample effort recorded most of the species present in the areas (s = 10; chao 1 = 10). the rarefaction curve almost reached the assintote, fact that can explained by needed more intensive surveys in region and by rare species captured (figure 1). when compared with other biomes, the caatinga has a reduced diversity of social wasps. diniz and kitayama (1994) identified 30 species in chapada dos guimarães (savannah), mato grosso state. silva et al. (2011) reported 31 species of social wasps in savannah, maranhão state. rodrigues and machado (1982) found 33 species in the são paulo state, in areas of savannah, eucalyptus and secondary forest. tanaka junior and noll (2011) collected 29 species in semideciduous seasonal forest fragments in são paulo state. marques et al., (1993) recorded 20 species for atlantic forest. in the pará state, silva and silveira (2009) found 65 species and in other areas of the amazon forests, as in roraima (raw, 1992; 46 species) and rondônia (raw, 1998; 36 species), the number of species was much higher than recorded in this present study and other brazilian ecosystems. the species with the highest abundance was polybia occidentalis (n = 112, f = 32.84%), followed by polybia ignobilis (n = 69, f = 20.23%) (table 1). melo et al., (2015) reported these species also had the major abundance and constancy in the caatinga, bahia state. other similar studies showed p. occidetalis and p. ignobilis as abundant and constant in tropical dry forests of brazil (aguiar & santos species n f (%) c% const brachygastra lecheguana 35 10.26 21.88 al polybia ignobilis 69 20.23 68.75 c polybia sericea 43 12.61 12.50 al polybia occidentalis 112 32.84 75.00 c polybia sp. grupo occidentalis 20 5.87 25.00 aa protopolybia exigua 21 6.16 28.13 aa polistes canadensis 34 9.97 34.38 aa polistes simillimus 1 0.29 3.13 al mischocyttarus cearensis 3 0.88 9.38 al protonectarina sylveirea 3 0.88 9.38 al total (n) 341 richness (s) 10 diversity (h’) 1.85 table 1. abundance (n), relative frequency (f), constancy value (c) and constancy category ( c = constant; aa = accessory; al = accidental) richness (s) and shannon diversity index (h ‘), for social wasps collected in the cities of sumé and prata, paraiba state. sumé observed (mm) expected (mm) deficit (mm) deficit (%) 2012 27 559.3 -532.3 -95.2 2013 254.4 559.3 -304.9 -54.5 2014 726,1 559,3 166,8 29,8 2015 220.4 559.3 -338.9 -60.6 prata 2012 152.8 745.6 -592.8 -79.5 2013 445.7 745.6 -299.9 -40.2 2014 603.9 745.6 -141.7 -19 2015 317.6 745.6 -428 -57.4 table 2. average annual pluviosity for sumé and prata. average observed and expected precipitation measured in millimeters* (mm). *data: executive agency for water management in the state of paraiba. fig 1. rarefaction curve of social wasp species collected in the caatinga, paraiba state, brazil (s = observed diversity, chao 1 = estimated diversity). 2007; santos et al., 2006; santos et al., 2007). polybia genus comprised 72% of the trapped samples (figure 2). this value may be related with the swarm founding behavior of this genus, which guaranteed the more success in foundation (jeanne, 1991). another characteristic is that the nests of polybia have a protective envelope, creating a better micro climate for the colony members inside (richard & richard, 1951). in addition, this genus has been reported as having a high storage capacity of protein resources and carbohydrates (ihering, 1896; machado, 1984), which confers resistance to periods of environmental stress, such as the long drought period in the semi-arid (jeanne, 1991). polistes canadensis was considered accessory, and was found in 38% of the samples. the studies conducted in caatinga by santos (2006), silva-pereira and santos (2007), santos et al. (2007) and santos et al. (2009) showed the same species with high abundance and significant constancy. the polistes genus is characterized by building nests without protective envelope. this absence may result in lower internal control of the nests in relation to the environmental variables (jeanne, 1991). in the period of the present study occurred a t elisei, e valadares, fa albuquerque, cf martins – social wasps in the neotropical dry forest of brazil114 deficit in rainfall of 50% below historical averages (aesa, 2016). the decrease in water supply and, as a result, the fall in the supply of nutrients, may have resulted in the decline of this group in the study area. this means that, because of sensitivity to environmental variations, social wasps can be used as bioindicators of environment quality. souza et al. (2010) verified preference by some species to different environmental and appoint someone’s as bioindicators. polistes simillimus had the lowest constancy in samples, because just one specimen was captured. mischocyttarus cearensis, polybia sericea, protonectarina sylveirea and brachygastra lecheguana were considered accidental species (table 1). however, in other research also conducted in areas of caatinga, some of these wasps were classified as accessory or constant, as b. lecheguana, p. sericea and p. sylveirea by santos et al. (2006); m. cearensis and p. simillimus by melo et al. (2015). the lack of previous surveys in the region affects discussions, for example a possible reduction on the wasps populations. santos et al. (2009) reported a higher number of active colonies of social wasps in the wet period of the year in the caatinga. polistinae populations are strongly influenced by environment, and those euriecias are most resistant to unfavorable changes than estenoecias (souza, 2010). the present research can strengthen the influence of drought on the dynamics of the captured polistinae community in caatinga, resulting in a reduced abundance of certain populations. the predictions indicate that global warming will result in an intensification of drought extremes in the brazilian northeast (marengo, 2006). thus, surveys of different groups in the region are very important to determine the loss of biodiversity difference between the abundances in the analyzed areas (wp-value = 0.4663) (figure 3, table 3). in addition, the levene homogeneity test revealed that caatinga and intercrop showed homocedasticity (lp-value = 0.6562), confirming the similarity between abundances. the results of this study were similar to those registered in santos et al. (2009). these authors verified no significant differences among richness of polistinae in caatinga and crop areas (cowpea, corn and beans). however, they found a greater number of social wasp nests founded in crop area, which can explain the high abundance in the cultivated area reported in the present study. it is known that heterogeneous environments, with higher diversity of plants, tend to have an elevate number of niches and, thus, to promote the highest number of coexisting species (latham & ricklefs, 1993; bragança et al., 1998). the caatinga is an environment more heterogeneous than monoculture crops. however, the plantation in the study areas is done with consortia of different types of vegetables (mainly: beans, corn, cotton, peanuts and sesame). thus, the intercropping may exercise more attraction to individuals than the natural environment area. altieri et al. (2003) highlighted the importance of biodiversity in plantations, especially in areas where it applies the integrated pest management, once biodiversity favors the maintenance of fig 3. abundance and richness of social wasps captured in caatinga (1) and intercropping (2), in the cities of sumé and prata, paraiba state, brazil. fig 2. abundance of social wasps genera captured in semi-arid, municipalities of sumé and prata, paraiba state, brazil. due this phenomenon (lawton et al., 1998). comparative analysis of social wasps communities in caatinga and organic intercropping there was no statistical difference of richness between the caatinga vegetations and intercropping areas (wp-value = 0.2994) (figure 3, table 3). intercropping areas showed a higher number of individuals although there was no significant 1 2 0. 0 0. 2 0. 4 0. 6 0. 8 abundance richness sociobiology 64(1): 111-118 (march, 2017) 115 natural enemy species of pests. the most abundant species was p. occidentalis, both in intercropping and in the caatinga, and was classified as constant in the two study areas (table 3). this species has been reported as floral visitor and predator of caterpillar, exerting an important role in crop production (gravena, 1983; gobbi et al., 1984; resende et al., 2001; santos et al., species caatinga intercropping n f (%) c% const n f (%) c% const brachygastra lecheguana 6 5.41 11.11 al 29 12.61 35.7 aa polybia ignobilis 24 21.62 55.56 c 45 19.57 85.7 c polybia sericea 8 7.21 11.11 al 35 15.22 14.3 al polybia occidentalis 39 35.14 77.78 c 73 31.74 71.4 c polybia sp. grupo occidentalis 8 7.21 16.67 al 12 5.22 35.7 aa protopolybia exigua 8 7.21 27.78 aa 13 5.65 28.6 aa polistes canadensis 16 14.41 38.89 aa 18 7.83 28.6 aa polistes simillimus 0 0.00 0.00 1 0.43 7.1 al mischocyttarus cearensis 1 0.90 5.56 al 2 0.87 14.3 al protonectarina sylveirea 1 0.90 5.56 al 2 0.87 14.3 al total (n) 111 230 richness (s) 9 10 diversity (h’) 1.76 1.85 table 3. abundance (n), relative frequency (f), constance (c) and category of constancy (const; c => 50%; aa = 25% 50%; al = <25%), richness (s) and shannon diversity index (h ‘) of social wasps collected in the caatinga and intercropping, sumé and prata cities, paraiba state, brazil. 2006; nadia et al., 2007). surveys registered lower diversity of social wasps in monocultures when compared to natural vegetation (santos, 1996; souza et al., 2011; silva et al., 2013.). hence, our results suggest that intercropping may have an important role in the maintaining of community polistinae in paraiba caatinga. social wasps are natural enemies of pest species, thus it is important to favor their populations in environments with organic agriculture (parra et al., 2002). conclusion caatinga has a neglected knowledge of its biodiversity, and requires more detailed studies about various groups, including social wasps. the data increased the number of polistinae species registered for the paraiba state. in addition, the comparison between the social wasps community of the natural environment (caatinga) and intercropping, reinforced the importance of diversity in crops, favoring the maintenance of the natural enemy species. acknowledgments we thank prof. dr. ricardo andena, from the state university of feira de santana for wasp identification, and the brazilian national council for scientific and technological development (cnpq) for the financial support. references agência executiva de gestão das águas do estado da paraíba (aesa). monitoramento de chuvas acumuladas. http://www. aesa.pb.gov.br/. 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(2011). diversity of social wasps on semideciduous seasonal forest fragments with different surrounding matrix in brazil. psyche: a journal of entomology. doi: 10.1155/2011/861747 doi: 10.13102/sociobiology.v64i4.1209sociobiology 64(4): 442-450 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 association of the occurrence of ant species (hymenoptera: formicidae) with soil attributes, vegetation, and climate in the brazilian savanna northeastern region introduction ants are the most widely distributed and abundant social insects. they are found in almost all earth habitats, except the earth poles (harada & ketelhut, 2009). ants can be found in canopies, litter, and soil. many species use the interface between soil and litter for foraging and building their nests (hölldobler & wilson, 1990; longino & nadkarni, 1990). ants have high economic and ecological value because they can significantly influence their environments by incorporating nutrients, aerating soils, and regulating populations of other organisms (nakano et al., 2013). identification and ecological abstract ants occur in all tropical forest strata by nesting, foraging, and interacting with plants and other residents from these habitats. most ants build their nests in the soil. selecting the proper soil for nesting depends on soil attributes and other factors. the question is: which of these attributes can affect more strongly the decision for nest installation? this study aims to discover if the occurrence of some species of ants from cerrado, in the northeastern state of maranhão, depends on attributes of soils and climatic factors. we found 48 species of ants, of which ten had the highest importance value. these are correlated with soil properties, litter biomass, basal area, humidity, and temperature by using the principal component analysis (pca). the soil properties, vegetation (basal area and dry mass of litter), temperature, and humidity had an impact on the occurrence of the ten studied species of ants. camponotus comatulus (mackay, 2010), ectatomma muticum (mayr, 1870), solenopsis substituta (santschi, 1925), pseudomyrmex termitarius (smith, 1855) and pheidole casta (wheeler 1908) were associated with sites of arbustive vegetation, poorly and acidic chemical and physical limited (dense, high micro-pores volume) soils. pseudomyrmex boopis (roger, 1863), dinoponera gigantea (perty, 1833), ochetomyrmex neopolitus (fernández, 2003) are found in fertile soils, covered by arboreal forest, while crematogaster cf acuta, solenopsis bruesi (creighton, 1930) was mainly found in saturated soil, and covered with palm trees. the species of ants recorded in this study are strongly associated with soil properties, as well as vegetation parameters, air temperature, and relative humidity at soil level. sociobiology an international journal on social insects ef silva1, je corá2, ay harada3, ibm sampaio4 article history edited by kleber del-claro, ufu, brasil received 07 october 2016 initial acceptance 13 january 2017 final acceptance 05 august 2017 publication date 27 december 2017 keywords cerrado, soil properties, multivariate analysis, ant nest. corresponding author edison fernandes da silva universidade federal do maranhão campus de chapadinha br 222 km 06, s/n boa vista cep 65500-000, chapadinha-ma, brasil e-mail: edibocaiuva@yahoo.com.br characterization of ant communities, as well as studies designed for controlling pest species, have been the subject of both basic and applied research in most of the brazilian ecosystems, particularly in savanna areas (cerrado biome). however, most studies carried out in the brazilian cerrado biome addressed the geographical distribution of ants, associated with the qualitative effects of vegetation, air temperature, and humidity (caldeira et al., 2005; campos et al., 2008; cantarelli et al., 2006; fonseca & dihel, 2004; lopes & vasconcelos, 2008). some papers analyzed the relation between ants and environmental factors, and many of them associate the 1 federal university of maranhão, campus chapadinha, ma, brazil 2 são paulo state university, campus jaboticabal, sp, brazil 3 emilio goeldi museum of pará, belém-pa, brazil 4 federal university of minas gerais, belo horizonte-mg, brazil research article ants sociobiology 64(4): 442-450 (december, 2017) 443 occurrence of ants with different environments. however, they do not present the environmental factors that affect the distribution pattern of ant species. the genus camponotus, for example, is usually associated with different vegetation types of phytophysiognomies in the cerrado biome, and the ants of ectatomma found in cerrado areas are poorly covered by plants (pacheco & vasconcelos, 2012). pseudomyrmex is a genus of large distribution and high diversity that usually builds nests on arboreal substrates and dead logs (ward & downie, 2005); but there are a few species of pseudomyrmex that nest on soils (dejean et al., 2014). the ants of the myrmicinae subfamily, like the pseudomyrmex, are found in the neotropical region, and the main studies with myrmicinae establish a relation between the geographic distribution and environmental factors with discrete discussions on specific environmental factors that affect the distribution of these ants, mainly the myrmicinae that nest underground. the myrmicinae is a megadiverse subfamily in tropical areas, especially the pheidole (rodríguez et al., 2011; selvarani & amutha, 2013). usually, the myrmicinae ants are opportunistic and capable of massive foraging (sant’ana et al., 2008), remarkably in warm and rich leaf-litter areas (theunis et al., 2005). they can occupy both natural and anthropogenic environments (calle et al. 2013; franklin, 2012). the majority of myrmicinae species build their nests in the soil where there is a high content of food, mainly proteins. many ant species nest on the soil (campos et al., 2008; jaime 2010), where they can have stable shelters and abundant food supply, more specifically, and protein sources with low c:n ratio. the latter plays an important role in the stratification and complexity of ant communities nesting on the ground or tree canopies (kaspari & yanoviak, 2001). studies about ant species that live on soil horizons are quite restricted, since the majority of studies that approach soil ants deal with species from epigeic substrates collected with pitfall traps, besides the methodological difficulties in obtaining samples of ant species on the soil. ryder-wilkie et al. (2007) found 47 ant species at a 50-cm soil depth in the ecuadorian amazon. these authors recorded the following genera: wasmannia forel (1893), strumigenys smith (1860), pachycondyla smith (1858), gnamptogenys roger (1853), hypoponera santschi (1938), and solenopsis westwood (1840). however, there was not a relation between ants and soil attributes, only 3.0% of the species were found in the three studied layers: leaf litter, soil, and trees (ryder-wilkie et al., 2010). vegetation and soil attributes like silt, organic matter, nitrogen and water content have a significant impact on the frequency of ant nest in a transitional steppe and desert area in southwestern china (li et al., 2011). therefore, soil attributes seem to influence the distribution of ants. however, on what level and intensity do they do it? thus, a hypothesis of the present paper is that the occurrence of ants is probably associated with soil attributes, vegetation, and climatic factors. hence, the present study aimed to identify the relation between ant community and soil attributes in a cerrado area from the northeastern region of brazil. material and methods description of sites the study sites were located in chapadinha county, (3º44’31 ‘s and 43 21’36’’w, datum), maranhão state, northeastern brazil, at an altitude of approximately 100 m. according to thornthwaite climate classification, the regional climate is tropical sub-humid b1wa’a, with an annual temperature of 29±1 °c, annual rainfall of about 1,600 to 2,000 mm, and distinct dry (july-november) and wet (decemberjune) seasons (nogueira et al., 2012). the experimental area is included in the cerrado biome (savanna-like vegetation), which is the second largest biome in brazil, covering around 25.0% of the brazilian total area (sano et al., 2008). based on the us soil taxonomy (soil survey staff, 2010), the soil vegetation, slope class, and slope position of each site selected for the present study are presented in table 1 and figure 1. sites soil slope position slope class drainage class vegetation s1 xanthic hapludox summit gently sloping poorly drained shrub s2 xanthic hapludox summit very gently sloping moderately well drained cerrado (shrub and trees) s3 oxyaquic udipsamments footslope depressional level poorly drained mesophilic forest on secondary growth (dense vegetation and leaf litter), s4 petroferric hapludox summit moderately sloping somewhat excessively drained mesophilic forest on secondary growth with less dense leaf-litter s5 arenic kanhapludults footslope depressional level very poorly drained babaçu palm forest orbignya phalerata mart s6 lithic hapludults summit strongly sloping somewhat excessively drained mesophilic forest on secondary growth with scattered babaçu palm trees table 1. soil types, slope position, slope class, drainage class, and vegetation of the sites (s1, s2, s3, s4, s5, and s6) at cerrado, in maranhão, northeastern region of brazil. ef silva et al. – ant species, soil attributes, vegetation, and climate in the brazilian northeastern444 ant field collection ants were collected in six sites, each with four plots (40 x 40 m). the ants were attracted using sets of protein and carbohydrate baits (sardine and honey, respectively), each had a distance of 0.2 m from one another. nine sets of baits with a 10-m space from one another were distributed into every plot (figure 1). each set of bait was individually monitored for one hour from 8 to 11 a.m. ants that ate from the baits were followed to their nest and then collected. collections of the ants were conducted from may to november of 2012. all plots were sampled together in each month. the collected ants were preserved in 70.0% alcohol in proportion (7.54:2.46 alcohol 92.8°: water, v:v). subsequently, they were identified in the laboratory at species level using stereomicroscope and available literature to be compared with the ant collection at museu paraense emílio goeldi. the abundance of ant species was considered as the number of nests observed. temperature and humidity at soil level were measured every 30 minutes in the center of each plot using a digital thermos hygrometer (icel ht-208). vegetation analysis the basal vegetation area was calculated for every living tree trunk or dead log with a circumference at breast height (cbh) > 0.15 m and height > 1.0 m. the vegetal basal area was defined as the area occupied by the plant divided by the total soil surface area. the total vegetation basal area of each plot for all sites was estimated using fitopac 2.0. (shepherd, 2008). additionally, leaf-litter biomass on the soil surface was obtained at three randomly chosen 1.0 m2 (1.0 x 1.0 m) in each plot of all sites. the material was placed in an oven at 60°c to dry until constant weight was reached. the dried leaf-litter materials were weighed to calculate the biomass (kg.ha-1). soil sampling and analysis damaged soil samples were obtained in each plot of all sites, varying from 0.0-0.1, 0.11-0.2, and 0.21-0.3 m depth. twenty-four samples were randomly collected and mixed for each soil layer in order to obtain a composite sample, in which the following were determined: ph (cacl2), organic matter (om), p (resin method), k+, ca2+, mg2+ (h + al), based on the procedures proposed by raij et al. (2001). also, sand, silt, and clay contents and water clay dispersion were determined according to gee and or (2002). the degree of clay flocculation was obtained by fd = (cdw – clay dispersion in water/total clay) x 100. furthermore, the aggregate stability index (asi) considering aggregate diameters between 1.0 and 2.0 mm was fig 1. distribution of the baits (honey and sardines) at the plots of 40 x 40 m. elevation profile and prevailing vegetation on sites s1, s2, s3, s4, s5, and s6. sociobiology 64(4): 442-450 (december, 2017) 445 determined in the 24 samples of each soil layer, as described by nimmo and perkins (2002). additionally, 16 undamaged samples were collected in volumetric rings (5 x 5 cm) in each plot from 0.0-0.1 m, 0.11-0.20 m, and 0.21-0.30 m depth – site 6 was not because undamaged samples were obtained only from 0.0-0.1 m depth due to rock fragments on the soil profile. in each undamaged sample, soil bulk (grossman & reinsch, 2002), total porosity, macroporosity and microporosity (claessen et al., 1997) were measured. statistical analysis vegetation data were analyzed using fitopac 2.0 (shepherd, 2008), which generates the basal area and importance value index (ivi). data of the relation between soil attributes, the occurrence of ant species, and vegetation was obtained through the pca using the infostat 2012 version (di rienzo et al., 2008). ten ant species were selected for the pca, based on the ivi, which is the sum of the relative frequency, density, and dominance of each ant species obtained using fitopac 2.0 (shepherd, 2008). in addition, the ten selected ant species were divided into two subgroups to keep 70.0% as minimum inertia in the pca. the two groups of ants were formed considering the phylogenetic and phylogeographic relation of ants and the preservation of at least 70.0% of system inertia. the species in each subgroup are as follows: subgroup a – crematogaster cf. acuta, pheidole casta, solenopsis bruesi and s. substituta; and subgroup b – camponotus comatulus, dinoponera gigantea, ectatomma muticum, ochetomyrmex neopolitus, pseudomyrmex boopis and p. termitarius. results the species in the subfamily myrmicinae (crematogaster cf. acuta, ochetomyrmex neopolitus, pheidole casta, solenopsis bruesei and solenopsis substitute) were found in specific sites (table 2). similarly, ectatomma muticum, camponotus comatulus, pseudomyrmex termitarius, species s1 s2 s3 s4 s5 s6 ivi dinoponera gigantea (perty, 1833) 8 3 15 20 15 17.30 camponotus comatulus (mackay, 2010) 41 19 5 16.37 pseudomyrmex termitarius (smith, 1855) 40 7 4 2 14.46 ectatomma muticum (mayr, 1870) 45 3 11.88 ochetomyrmex neopolitus (fernández, 2003) 1 7 10 4 8.47 pheidole casta (wheeler, 1908) 5 4 6 9 6.87 pseudomyrmex boopis (roger, 1863) 3 5 2 6.40 solenopsis bruesi (creighton, 1930) 8 1 6 6.04 crematogaster cf. acuta 1 17 1 5.68 solenopsis substituta (santschi, 1925) 11 1 5.63 table 2. number of nests, importance value index (ivi) of ants species from sites s1, s2, s3, s4, s5 and s6 at cerrado, maranhão, northeastern region of brazil. fig 2. principal component analysis of soil, vegetation and climate variables related to the occurrence of camponotus comatulus, ectatomma muticum, pseudomyrmex boopis, dinoponera gigantea and p. termitarius ants species collected in a domain of cerrado, northeastern region of maranhão, brazil. circle refers to ants and diamond refers to variables. om: organic matter; fd: flocculation degree; cdw: clay disperse water; asi: aggregate stability index. the numerical labels beside variables show the third axis in the three-dimensional system. 74.0% system inertia. ef silva et al. – ant species, soil attributes, vegetation, and climate in the brazilian northeastern446 pseudomyrmex boopis and dinoponera gigantea were more specific for habitat use. they nested under very specific conditions of soil attributes, vegetation, humidity, and temperature at the soil level. thus, those species are more demanding for habitat use. the species e. muticum, c. comatulus and p. termitarius found in s1 and s2 sites were positively associated with higher soil densities, soil air temperatures, soil micropores and lower soil ph (figure 2 and table 3). however, in this open area of the cerrado, p. termitarius was also quite common. its nesting was more observed in the s1, s2, s5 and s6 sites. pseudomyrmex boopis was found exclusively in shady places and was more abundant on s3, s4 and s6 sites (table 2), where the soils presented high clay, mg2+, ca2+ and om contents, as well as high ph values (table 3). positive associations among those variables and p. boopis are shown in figure 2. ochetomyrmex neopolitus and dinoponera gigantea nests were predominantly observed at s3, s4, and s6 sites, where leaf litter dry mass was higher, and soils presented high macroporosity and moisture (figures 2, 3 and table 3). the presence of d. gigantea at s1, s2, s3, s4 and s6 sites was associated with mild temperatures and high leaflitter biomass, possibly under woody vegetation (sites s3, s4, and s6), providing favorable conditions for d. gigantea occupation. dinoponera gigantea nests were not found at s5, where there is high soil water content, low leaf-litter biomass, and low soil macroporosity (figure 2, table 3). nests of crematogaster cf. acuta and solenopsis bruesi were observed at s5, where soils presented low clay and organic matter contents, high silt content, and low percentages of asi, cdw (figure 3, table 3). these conditions were mainly found in s5, which is located at a depressional area, which soil is very poorly drained (figure 1, table 1). pheidole casta nests were observed at s1, s3, s5, and s6. the pheidole occurrence showed a strong association with low basal area values, low soil micropores, and low soil ph (figure 3). spots with these features should also have high sand content, high-density values and high temperatures at soil level (figure 3, table 3). nests of solenopsis substituta were observed at s1 and s5. these sites present plants with low potential for shading and soils with high bulk density, low clay, organic matter, ca+2 and mg+2 contents, low soil macroporosity, and low leaf litter biomass, as well as the high temperature at soil level (table 3). myrmicinae species were observed at all sites, especially at s6, where the soil has a high amount of lateritic concretions, which provides stable galleries under a thick layer of leaf litter. discussion the occurrence of myrmicinae and other species of ants in specific habitats of the cerrado show the high capacity of these ants to live in different types of sites, with the possibility of building their nests under very specific conditions of soil attributes, vegetation, humidity, and temperature. the associations of these ants to different conditions found in cerrado can be an adjustment or response to its diversity of habitats because the selectivity of ants from such habitat described by pacheco and vasconcelos (2012) show that cerrado physiognomies are richer in ant species than in homogeneous forest formations. fig 3. principal component analysis of variables of soil, vegetation and climate factors related to the occurrence of crematogaster cf. acuta, solenopsis substituta, s. bruesi and pheidole casta ant species collected in a domain of cerrado, northeastern region of maranhão, brazil. circle refers to ants and diamond refers to variables. om: organic matter; fd: flocculation degree; cdw: clay disperse water; asi: aggregate stability index. the numerical labels besides variables show the third axis in the three-dimensional system. 71.0% system inertia. sociobiology 64(4): 442-450 (december, 2017) 447 the presence of e. muticum, c. comatulus and p. termitarius in acidic, dense soils with the high bulk of micropores shows the adherence of these ant species to soils of xerophytic environments. ectatomma species were observed in open areas under sandy soils with low ph (pacheco & vasconcelos, 2012). melo et al. (2014) and soares et al. (2003) highlighted the occurrence of e. muticum in damaged habitats, more specifically in xerophytic environments like caatinga and in open areas from cerrado. the nests of p. termitarius found in open areas from cerrado, mainly in soil, contradict the results found by dejean et al. (2014), ward and downie (2005). the predominance of most of the pseudomyrmex genus and p. boopis nesting typically occur on rotten wood or near the ground (antwiki). in the present study, the highest occurrence of p. termitarius was observed in soil and areas with lower basal area and scarce vegetation. the record of p. termitarius in sites with the high basal area only occurred in sites covered mainly by palm trees. the high basal area value of sites where the nests of p. termitarius was registered is the result of the dominance of palm trees. the monopodial canopy growth of palm trees concentrates the leaves around a central point, which reduces shading on the ground, recreating strong insolation conditions as occurs in sites with the lower basal area and scarce vegetation. the fact that p. termitarius has been observed in different soil conditions, but under similar plant shading conditions, suggests that plant shading conditions play a more important role for the colonization by this species than soil attributes. pseudomyrmex termitarius is smaller and darker in black and red pigments of the gaster and mesossoma. p. termitarius was the only active species throughout all days, despite the fact that the air temperature on ground level reached 59.4° c and humidity was smaller than 20.0%. the darkest black and red pigmentation and the smallest thermic inertia, due to small body size, probably, are the reason the p. termitarius is tolerant to high temperatures, compared to other species in the present study. for instance, a less pigmented ant – p. boopis – was observed only in more shaded conditions. the register in the present study of p. termitarius exclusively in open areas is supported in other works, such as in mil (1981) in brazil and jaff et al. (1985) in venezuela. both authors reported the occurrence of p. termitarius on less shaded conditions (scarce vegetation). besides these authors, kemp (1960) also mentions the occurrence of p. termitarius in sunny sites, including inside termite nests. opposite areas where p. boopis nests were found have soils rich in nutrients and arboreal vegetation with great soil shading potential. fertile and moist soils can support a greater number of woodland communities (haridassan, 2000), which provides more shadow and consequently a good condition not only for p. boopis nesting but also for other ants like myrmicinae and ponerinae. mic. mac. fd asi s. dens. cdw clay silt sand om ph k+ ca2+ mg2+ (h+al) t u litter ba n = 30 ...m3.m-3.... .......%.... kg.m-3 ...................g.kg-1.................... kcl ...........mmolc.kg -1............. °c % kg.ha-1 m2.ha-1 s1 (xanthic hapludox) mean 0.3 0.1 20 93 1790 176 220 117 662 14.6 3.95 0.3 1.92 1.49 31 37.37 28.9 2212 0.23 sd 0 0.01 7.9 1.1 0.02 30.8 29.8 5 30.2 1.96 0.06 0.1 0.3 0.5 2.42 2.71 5.16 15.01 0.26 s2 (xanthic hapludox) mean 0.2 0.11 24 89 1670 141 185 99 715 17.9 3.7 0.4 2.13 1.46 51.1 35.35 32.2 6430 0.95 sd 0 0.01 12 1.7 0 27.6 7.7 11 17.2 0.24 0.05 0.1 0.06 0.41 3.61 1.12 3.4 50.01 0.89 s3 (oxyaquic udipsamments) mean 0.3 0.21 38 92 1410 145 234 88 678 33.8 4.48 0.7 18.2 14.6 66.3 29.69 52.9 16644 2.42 sd 0 0.02 7.3 2.7 0.05 33.5 60.5 21 79.9 10.8 0.24 0.2 8.39 7.34 17.8 2.01 15.2 107.98 0.31 s4 (petroferric hapludox ) mean 0.3 0.19 27 92 1280 190 219 108 590 49.4 4.62 0.5 25.6 28.8 87.5 31.49 41.1 14815 2.19 sd 0 0.02 7.2 0.7 0.08 14.7 41 14 30.6 5.49 0.21 0.1 6.52 6.6 9.01 1.71 11.4 45.53 0.73 s5 (arenic kanhapludults) mean 0.3 0.08 26 63 1620 88 121 295 584 14.2 4.19 1.6 7.51 8.31 35.5 36.78 30.9 2033 3.63 sd 0 0.04 5.2 27 0.02 16.6 14.5 128 14.2 2.36 0.28 0.5 1.67 3.73 7.13 2.55 2.03 65.5 0.2 s6 (lithic hapludults ) mean 0.3 0.21 27 84 1430 115 160 145 695 38.1 4.43 1.5 10.2 10.8 49.4 32.07 35.6 5177 1.91 sd 0.1 0.02 4.1 11 0.09 16.9 30 35 65.4 9.94 0.27 0.4 3.12 3.36 19.8 1.86 6.84 23.91 0.94 table 3. litter biomass, basal area, humidity, and temperature at soil level and physical and chemical layer parameters of 0.0 to 0.3 m soil in sites s1, s2, s3, s4, s5 and s6 in a domain of cerrado, maranhão, northeastern region of brazil. footnotes table 2 absent species. footnotes table 3 n: number of observations; mic: microporosity; mac: macroporosity; fd: flocculation degree; asi: aggregate stability index; s. dens.: soil density; cdw: clay dispersed in water; om: organic matter; t: temperature; u: atmospheric humidity at soil level; ba: basal area; sd: standard deviation. ef silva et al. – ant species, soil attributes, vegetation, and climate in the brazilian northeastern448 soils under woodland habitats usually present high leaf-litter dry mass and low c:n ratio. arboreal habitats often have large leaf-litter supplies and therefore low c:n ratio, and high content of protein available in the soil (kaspari & yanoviak, 2001). thus, the preference of o. neopolitus for soils under woodland with high-protein concentration potential corroborates the observations of rodríguez et al. (2011), who argued that habitats with these features are suitable for opportunistic and generalist ants, as myrmicinae species. dinoponera gigantea was associated with mild temperatures and high leaf-litter biomass, which is usually found on woody vegetation. however, this ant was also seen in sunny sites. in such climate condition, the nests of d. gigantea were always observed at the bottom of the trunk of trees and shrubs, which is possibly a strategy that reduces the effects of high temperatures as argued by lenhart et al. (2013) and vasconcellos et al. (2004). despite the preference of d. gigantea for mild temperatures and humidity sites, the excess of humidity is a limitation for the occurrence of d. gigantea, because these ants were not found in sites where there is high soil water content (close to saturation). poorly drained and structured soils may limit the survival of large ants. those found in these soils build their nests in petioles and stems above the ground. nevertheless, these environments are suitable for small ants like c. cf. acuta. costa-milanez et al. (2014) emphasize the occurrence of c. cf. acuta in wetlands by showing the versatility of these species in different habitats, especially in wetlands. the occurrence of c. cf. acuta and s. bruesi on poorly drained and structured sites emphasizes the adaptive capacity of many myrmicinae subfamily ant species to live in habitats that have not been occupied by other ants (calle et al., 2013). the association of pheidole casta with extreme climatic and pedological conditions, such as high sand content, soil density values, and temperatures at soil level, show these ants and other flavens group species have high capacities of adjusting themselves to different environmental conditions. therefore, they are widely distributed and can be found from north carolina (usa) to argentina (wilson, 2003). ecological investigations addressing the possible relations between ants and soil attributes, vegetation, and climate are rather discrete, but some authors have made approaches with some of these parameters, such as: vasconcelos et al. (2008) that observed high density of s. substituta nests in amazonia’s cerrado; and trager (1991) that mentions its occurrence is usually associated with areas with sites of scarce vegetation and uncovered soil. myrmicinae species were observed in all sites, mainly in those where the soil presents high amounts of lateritic concretions, which provide stable galleries under a thick layer of leaf litter. according to theunis et al. (2005), this soil feature supports dense populations of myrmicinae that allow vertical migrations when conditions become unsuitable for ground nesting, particularly during the rainy season when such soils become too wet. myrmicinae is a dominant group of ants of hyperdiversity that is found in tropical areas (selvarani & amutha, 2013), especially the genus pheidole (rodríguez et al., 2011). franklin (2012) found myrmicinae nests in the mexican sonoran desert. the opportunism of the myrmicinae species was observed in the present study since myrmicinae nests were observed on all sites. nevertheless, this paper showed lower abundance and distribution of ecitoninae, ectatomminae, dolichoderinae, formicinae, ponerinae, and pseudomyrmicinae when compared to myrmicinae ant species. the occurrence of many ants species of the myrmicinae subfamily was defined by specific soil attributes, vegetation, and microclimate, as observed in the present study. conclusions the ants species registered herein are strongly associated with soil properties (bulk density, porosity, sand and silt content, aggregate stability index, mg2+ and ca2+ content, organic carbon, and ph), as well as vegetation parameters (basal area and dry mass of litter), air temperature and relative humidity at soil level. the soil, vegetation, and climate factors have an impact on the structure of ant communities because they define together the presence or absence of ants in specific sites. thereby, these ants may be considered good indicators of soil quality, vegetation, and climate. in this study, p. boopis was mentioned for the first time in brazil. acknowledgements we gratefully acknowledge the foundation for research and scientific and technological development of maranhão (fapema) for their support and jivanildo miranda and ricardo santos for their valuable assistance to write this manuscript. references antwiki.www.antwiki.org/wiki/pseudomyrmex_boopis (accessed in 7/09/2015). campos, r.i., lopes, c.t., magalhães, w.c.s., vasconcelos, h.l. 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(2005). the ant subfamily pseudomyrmicinae (hymenoptera: formicidae): phylogeny and evolution of big-eyed arboreal ants. systematic entomology, 30: 310-335. doi: 10. 1111/j.1365-3113.2004.00281.x wilson e.o. (2003). pheidole in the new world: a dominant, hyperdiverse ant genus. harvard university press. 794p. http://dx.doi.org/10.1371/journal.pone.0013146 http://dx.doi.org/10.1371/journal.pone.0013146 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1853sociobiology 64(4): 393-397 (december, 2017) seasonal cycle of the nest composition in the ponerineant cryptopone sauteri (hymenoptera: formicidae) introduction the annual life history is a basic and important factor in ecological studies on temperate ant species (bourke & franks, 1995), but those of only a small number of model species have been investigated in detail (hölldobler & wilson, 1990; peeters & molet, 2010). for example, “encyclopedia of japanese ants”, which was published in 2014, listed more than 290 species in japan and provided important taxonomic descriptions; however, the life histories and biological characteristics of the listed ant species have not yet been examined in detail (terayama et al., 2014). life history data are indispensable to establish molecular and chemical studies (schlick-steiner et al., 2005; katzerke et al., 2006; purcell & chapuisat, 2012). recently, the biology of ponerinae ants has been studied for many species (e.g. monnin & peeters, 2008; oliveira et al., 2011; murata et al., 2016), but little attention has been paid to their life history. in fact, the information abstract the annual life history is a basic and important factor in ecological studies on temperate ant species. the biology of ponerinae species has been studied for many species, but little attention has been paid to their life history. cryptopone sauteri is one of the most common ants in temperate regions of japan. however, there is no quantitative information on the life history of this species. we report seasonal changes in brood development, the emergence of reproductives and social structures of c. sauteri. additionally, we found that the nuptial flight of alate females and males occurs in the end of summer in this species. sociobiology an international journal on social insects y yamaguchi1, h yazawa2, s iwanishi3, k kudô2 article history edited by gilberto m. m. santos, uefs, brazil received 10 july 2017 initial acceptance 03 october 2017 final acceptance 02 november 2017 publication date 27 december 2017 keywords life history, nuptial flight, unmated queens. corresponding author yuki yamaguchi niigata science museum niigata 950-0948, japan. e-mail: y-yamaguchi@sciencemuseum.jp of life history with the sampling of enough nests have been available for only two species of ponerinae ants (gotoh & ito, 2008; hart & tschinkel, 2012). gotoh and ito (2008) reported all adults of brachyponera chinensis emerged once a year, and brood are not present in the nests during the winter. hart and tschinkel (2012) reported the colonies of odontomachus brunneus produce brood for 6 months and are broodless for 6 months that includes the winter in north florida. the genus cryptopone is a small group of ponerinae, including about 20 species, and is distributed in the neotropical, nearctic, palearctic, afrotropical, oriental, indo-australian and australian regions (bolton et al., 2012 referred from bharti & wachkoo, 2013). c. sauteri is distributed in all islands excluding hokkaido and one of the most common ants in temperate regions of japan (terayama et al., 2014). murata (1994) reported the occurrence of alate queens in the nests and described the number of stadia in the larval stages and feeding habitats. yamaguchi et al. (2016) compared body sizes, the number of ovarioles and 1 niigata science museum, niigata, japan 2 laboratory of insect ecology, faculty of education, niigata university, niigata, japan 3 hoshizaki green foundation, shimane, japan research article ants y yamaguchi, h yazawa, s iwanishi, k kudô – seasonal cycle of the nest composition in a ponerine ant394 ovariole development between queens and workers to show the reproductive system of c. sauteri. the authors also found that this species is commonly monogyny, but partially appears to exhibit functional monogyny. however, there is no quantitative information on the life history of this species. here, we reported seasonal changes in brood development, the emergence of reproductives and social structures of c. sauteri in temperate regions of japan. methods we studied c. sauteri at coppice forest (37◦05´n, 138◦37´e), niigata prefecture in japan. the area is dominated by deciduous (fagus spp. and quercus serrata) and coniferous trees (cryptomeria japonica) with an annual average temperature of approximately 11.7°c (maximum: 24.9°c in august; minimum: -0.2°c in february). insect activity in the area is limited from november to may due to snowfall during the winter. we collected dead branches that c. sauteri nested in every two weeks between may and november in 2008 and 2009 to investigate seasonal changes in the nest composition.we defined the nests as independent if the dead branches existed separately. we carefully removed all adults and brood from every dead branch in the laboratory and recorded the numbers of mated queens, unmated queens, workers, alate females, alate males, and immature at each development stage (eggs, larvae, and pupae).the alate queens were possibly unmated, because they shed their wings after nuptial flight. all the queens collected for two years were dissected under a binocular microscope (olympus, sz2-ilst) to assess their insemination status. however, data of two queens collected on may 28 2008 were not included in this study due to failures in dissection. results a total of 331 nests were collected in the two years (2008: 134 nests, 2009: 197 nests), 199 of which were queenless. a total of 170 queens were collected in two years, and approximately half of these (61.7%) had been mated. as a result, the nest type was classified into four types (figure 1). although the number of mated queens markedly increased toward the end of the season, unmated queens were observed throughout the years. figure 2 shows seasonal changes in the number of adults (alate females and males, mated queens, unmated queens, and workers) and brood (larvae and pupae) per nest. alate females (unmated individuals) and males were only collected between august and september, suggesting that the nuptial flight occurs in the end of summer in this species. this view is also supported by the fact that the average number of mated queens/nest in october and november was significantly larger than that between may and september (t-test, 2008: p<0.01, 2009: p<0.01). the number of workers also increased between august and september, suggesting that worker production occurs simultaneously with alate individuals. eggs were only detected three times in our collection (2008: 1 nest in september; 2009: 2 nests in august and september). although larvae were collected throughout the year, the numbers tended to decrease toward the end of the season. pupae were detected between july and september in both years. discussion we examined seasonal changes in the number of adults and brood in the ponerine ant c. sauteri and found that the nuptial flight of alate females and males occurs in the end of summer in this species. one of the most striking characteristics of c. sauteri is that pupal cocoons were only detected at the end of summer, which suggested that all adults including workers and alates emerged once a year. all queens had wings, suggesting that they exhibit the nuptial flight in that season. because eggs were detected only three times in our collection, we were not able to determine the activities of egg laying by queens. however, mated queens with a developed ovary were collected between may and november (yamaguchi et al., 2016), suggesting that they lay eggs over the months. there were not consistent results on the existence of overwinter larvae in poneroid ants. the lack of overwinter larvae in the nests were reported in some ponerine ants brachyponera chinensis and odontomachus brunneus (gotoh & ito, 2008; fig 1. seasonal changes in the frequency of the four nest types. the numbers in the parentheses indicate the number of nests collected. sociobiology 64(4): 393-397 (december, 2017) 395 fig 2. seasonal changes in the number of adults (alate females andmales, mated queens, unmated queens, and workers)/ nest and brood (larvae and pupae)/nest in 2008 and 2009.the error bars represent se. y yamaguchi, h yazawa, s iwanishi, k kudô – seasonal cycle of the nest composition in a ponerine ant396 hart & tschinkel, 2012), while talbot (2012) reported that ponera differed from amblyopone or proceratium in that it did not overwinter larvae. in c. sauteri, the developed larvae were collected even in the beginning of spring (may), strongly suggesting that larvae overwinter. however, it is not clear that detail of life history, such as larval duration and life span of adults. future studies will have to focus on detail of life history by rear this ant species. the number of unmated queens was high throughout the year. five hypotheses on the presence of unmated queens have been proposed to date: producing trophic eggs, producing male-destined eggs, working queens, failure of nuptial flight and postponement of reproduction (vargo 1993; bourke & franks, 1995; brown 1999; kikuchi & tsuji, 2005; johnson et al. 2007). yamaguchi et al. (2016) suggested that the two possibilities (failure of nuptial flight or postponement of reproduction) are plausible for explaining the presence of unmated queens in c. sauteri, because the unmated queens had neither egg laying nor labor. our study showed that the unmated queens increased from june to august in 2008, but no such tendency was observed in 2009. however, it is unknown how such difference occurred among the years. the seasonal change of unmated queen frequency need to be examined in future studies. approximately 60% of the c. sauteri nests collected in the present study were queenless, which suggested that this species may use a polydomous nesting system. polydomous ants use at least two spatially segregated nests that exchange workers and broods, and this has been reported previously in some ponerine species (hypoponera bondroiti: yamauchi et al., 1996; pachycondyla goeldii: denis et al., 2006). we further need to investigate whether c. sauteri exhibits polydomy by testing inter-nest aggressiveness or observation of internest network. acknowledgments we thank n kobayashi, k komatsu for their assistance in the field. we also thank the echigo-matsunoyama museum of natural science for the use of current study. references bharti, h. & wachkoo, a.a. 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(2016). differences in body sizes and physiological conditions among castes in the ponerineant cryptopone sauteri (hymenoptera: formicidae). entomological science, 19: 124-128. doi: 10.11 11/ens.12176. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1199sociobiology 64(1): 119-121 (march, 2017) kick out the ants: a novel and striking behavior in ant-wasp interactions mutualism is an interaction where two organisms obtain higher reproductive success when together than would if they lived without such relationship. mutualism is clearly profitable for both parts and it is known as an important interaction for survival (stefani et al., 2015; del-claro et al., 2016). there are many forms of mutualism in nature, for instance, the widely known case of the trophobiosis observed between ants and hemipterans (del-claro & oliveira, 1999; styrsky& eubanks., 2007). this relationship is based on the hymenopteran use of honeydew, a sweet flavored, carbohydrate rich substance that is excreted by sap sucking insects. the honeydew is exploited as a food resource by ants, which establish a mutualistic relationship with the hemipterans by offering protection in exchange (delclaro & oliveira, 2000). wasps can also use honeydew as a food resource (letourneau & choe, 1987). therefore, competition between ants and wasps for access to hemipterans may occur. wasps establish a foraging territory and exclude ants that attempt to disrupt this exploitative interaction (dejean & turillazzi, abstract trophobiosis between ants and hemiptera is widely known. nevertheless, this interaction can also happen between hemipterans and other animals in a non-mutualistic way. for instance, here we observed the wasp pseudopolybia vespiceps (hymenoptera: vespidae) collecting honeydew flickered by aethalion reticulatum (hemiptera: cicadomorpha) as an alternative food resource. field work was conducted during three consecutive days, when we made ad libitum behavioral observations using video footage. we noted the behavior of p. vespiceps when collecting honeydew from a. reticulatum. strikingly, our observations resulted in the description of novel wasp behavior. the wasps compete with ants for access to hemipterans, first by hovering above the ants, and kicking them out of the plant, until all the ants were removed, and start antennating the aethalionids to obtain honeydew. studies like this contribute to the understanding of trophic networks that depend on hemipteran honeydew. sociobiology an international journal on social insects ls ricioli, g mendes, r guillermo-ferreira article history edited by jean santos, ufu, brazil received 28 september 2016 initial acceptance 07 december 2016 final acceptance 27 january 2017 publication date 29 may 2017 keywords kleptoparasitism, mutualism, foraging, competition, savannah, brazil. corresponding author rhainer guillermo-ferreira. universidade federal de são carlos departamento de hidrobiologia rodovia washington luis, km 235 cep 13565-905, são carlos-sp, brasil e-mail: rhainerguillermo@gmail.com 1992). in this case, wasps are benefited with the resource exploration while there is no mutualistic relationship with the hemipterans. the opportunistic foraging, also known as kleptobiosis (or kleptoparasitism), is a behavior observed in social wasps, which steal the resource during foraging (grangier & lester, 2011; breed et al., 2012). in this study, we observed the interaction between camponotus crassus (hymenoptera: formicidae) and aethalion reticulatum (linnaeus, 1767) (hemiptera: cicadomorpha) on crecopiapa chystachya trécul (urticaceae), where ants collected the honeydew from the hemipterans in exchange for protection (oliveira & del-claro, 1999). more surprisingly, we observed the exploitation of this interaction by the wasp pseudopolybia vespiceps (de saussure, 1854) (hymenoptera: vespidae, polistinae), which, besides collecting the honeydew and not protecting the hemipteran, kick the ants out of the plant using their forelegs while hovering. field work was conducted in the federal university of são carlos campus in são carlos, são paulo, brazil, during department of hydrobiology, federal university of são carlos, são paulo, brazil short note ls ricioli, g mendes, r guillermo-ferreira – a striking behavior in a wasp-ant interaction120 three consecutive days in march 2016. we made ad libitum behavioral observations using video footage (fuji finepix hs10). we noted the behavior of p. vespiceps when collecting honeydew from a. reticulatum and excluding ant competitors. the observations were carried out between 10:00am and 02:00pm. while the ants were attending the hemipterans (fig 1a), p. vespiceps wasps hovered in circles around the ants and stroke them with the forelegs (supplementary video 1) and landed on the hemipterans (fig 1b). this behavior started when the wasps flew up, stretched the forelegs and flew down towards the ants, which fell from the plant (fig. 1c, n =12). moreover, the hemipterans did not remain inert. the hemipterans attempted to use their hind legs to hit and avoid the wasp, without success. when the wasps had removed most of the ants, the wasps landed on the hemipterans and started to antennate their abdomen (supplementary video 2). ants and wasps exhibit this tactile stimulus that leads the hemipterans to flick a droplet of honeydew (styrsky & eubanks, 2007), however, the wasp did not appear to focus on a specific area, antennating all along the hemipteran body. the wasp spent around 30 seconds hovering in circles and kicking the ants off the plant, and 26.5 seconds (n=2) antennating the hemipterans. the interactions occurred in the lower surface of the leaf, close to the petiole. this wasp-hemipteran interaction is usually considered non-mutualistic, but a parasitism. differently from the anthemipteran interaction, the wasps provide no protection to the hemipterans. thus, the wasps only parasitize the ant-hemipteran interaction and exploit the resource without further costs. furthermore, the wasps may imply costs to hemipterans because the interaction with ants is interrupted by force and because the honeydew is lost in a non-profitable interaction. however, although this interaction apparently only benefits wasp, another study suggests that wasps may defend a territory around hemipterans (dejean & turillazzi, 1992). in this case, although wasp protection may not be as effective as it would be with ants, wasps may ultimately provide an indirect benefit to hemipterans. the ant-hemipteran interaction is no novelty. the same mutualism is very usual in nature between ants and aphids (stadler & dixon, 2005) and lepidopteran larvae (kaminski et al., 2013). wasp-hemipteran interactions can also be commonly found (letorneau & choe, 1987; maccarroll & reeves, 2004), however, the interference behavior adopted by wasps is rarely reported (dejean & turillazzi, 1992). as a rare case, vespula vulgaris wasps pick ant workers with their mandibles, fly backwards and drop them far from the food resource (grangier & lester, 2011). the interaction between c. crassus and p. vespiceps can be considered an exploitative interaction, where wasps exclude ant competitors and exploit a coevolutionary trait of ant-hemipteran interactions. several insect species have been reported to collect honeydew from a. reticulatum, such as the stingless bees trigona branneri (barônio et al., 2012), t. spinipes (vieira et al., 2007), t. hyalinata (oda et al., 2009), oxytrigona tataira (hymenoptera: apidae, meliponini) (oda et al., 2014), and camponotus crassus ants (brown, 1975). here we show the case of p. vespiceps, which is a species considered an indicator of high conservation status of riparian forests (de souza et al., 2010). therefore, we suggest that hemipterahymenopteran interactions in the brazilian cerrado may be an important factor for insect conservation and maintenance of ecological processes (styrsky & eubanks, 2007). acknowledgments we thank ana paula guaratini, aline mandelli, diogo silva vilela and gustavo rincon mazão for valuable comments in the initial version of the manuscript. we thank fernando barbosa noll for wasp rgf thanks cnpq and fapesp for constant financial support. suppplementary material video 1 http://dx.doi.org/10.13.102/sociobiology.v64i1.1199.s1374 h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfilemetadata/1199/0/1374 vídeo 2 http://dx.doi.org/10.13.102/sociobiology.v64i1.1199.s1375 h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfilemetadata/1199/0/1375 fig 1. 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(2007). interação entre trigona spinipes fabricius, 1793 (hymenoptera: apidae) e aethalion reticulatum linnaeus, 1767 (hemiptera: aethalionidae) em mangifera indica (anacardiaceae). bioscience journal, 23: 10-13. doi: 10.13102/sociobiology.v60i3.306-316sociobiology 60(3): 306-316 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 interactions of the cerrado palms butia paraguayensis and syagrus petraea with parasitic and pollinating insects i silberbauer-gottsberger¹, sa vanin2, g gottsberger¹ introduction palms are prominent, characteristic and abundant components of most tropical and subtropical landscapes. they are known to attract and to shelter many insects (lepesmé, 1947, howard et al., 2001). therefore, palms are also hotspots of insect abundance and diversity in any vegetation type. responsible for this are, among others, the commonly large inflorescences and infructescences, which carry many flowers and fruits and thus provide abundant food for insects in form of pollen, nectar and tissue. the other reason for insect richness on palms are the many possibilities for them to hide between the hard or woody leaf bases and the large inflorescence structures, which themselves have an extended abstract the two cerrado palms butia paraguayensis and syagrus petraea were studied with regard to parasitizing and pollinating insects that visit their reproductive organs. at the study site in botucatu, são paulo state, both species occurred together, while at the study site in indianópolis, minas gerais state, only s. petraea was growing. bees of the subfamily meliponinae and halictinae were the main pollinators of b. paraguayensis and several beetles and flies were additional pollinators. the visiting beetles divided up in more exclusive parasites, such as the weevils tripusus leiospathae and petalochilus lineolatus, and the colydiidae bitoma palmarum, which bored and oviposited the closed spathe and whose larvae fed on the flower buds, as well as species of the weevil microstrates, which bred in staminate flowers. differently, the curculionids anchylorhynchus bicolor, parisoschoenus sp., dialomia sp., and species of mystrops (nitidulididae) behaved as parasites and pollinators. on the other hand, s. petraea shows more evident adaptations for pollination by beetles. this species has trigona spinipes as prominent bee pollinator, but otherwise beetles dominated as visitors, being them either parasites, pollinators or both. although several identical beetle genera occur on both palms, at the species level they are different, as for example anchylorhynchus camposi on s. petraea. at the two distant study sites of s. petraea in botucatu and indianópolis, several species, especially of the parasitic insects, are identical, indicating that parasitism is a stronger bond than pollination in this species which is characterized by a generalist pollination mode. sociobiology an international journal on social insects 1 universität ulm, ulm, germany. 2 university of são paulo usp, são paulo, brazil. research article bees article history edited by helena m. torezan-silingardi, ufu, brazil received 01 july 2013 initial acceptance 26 july 2013 final acceptance 12 august 2013 key words anchylorhynchus, microstrates, hustachea, phytotribus, mystrops, meliponinae, halictinae corresponding author ilse silberbauer-gottsberger botanischer garten und herbarium universität ulm, hans-krebs-weg 89081 ulm, germany e-mail: ilse.silberbauer@uni-ulm.de developmental time and thus provide ample possibilities for insects to oviposit and breeding. one of the early brazilian pioneers working on palms and their insects was gregorio bondar, who noted that a considerable entomological fauna of hemiptera, lepidoptera and coleoptera has evolved on the native palms of brazil, some of them damaging or destroying leaves, flowers and fruits. bondar (1964) elaborated that of coleoptera, especially hispinae (chrysomelidae) and curculionidae have segregated on american palms a large number of genera and species. “each genus of native palms, and at times, each species, has its own entomological fauna, and by means of the insects collected, one can determine the genus and species of the palm.” (bondar, 1964). palms are mainly pollinated by insects (henderson, sociobiology 60(3): 306-316 (2013) 307 1986) and many palm species are visited jointly by an array of different insects, which are of varying importance in pollination. however, often a predominance of one group of insects as pollinators can be observed (silberbauer-gottsberger, 1990, gottsberger & silberbauer-gottsberger, 2006b). certain morphological and physiological differentiation patterns of flowers or inflorescences and scent emissions are correlated with this predominance of pollinators. palms are frequently associated with beetles, which may take advantage of different vegetative and floral organs for mating and reproduction. several of these beetles are involved in pollination. they may act as additional, predominant or exclusive pollinators. in the world checklist of palms by govaerts and dransfield (2005), 2364 species in 190 genera are accepted. sixty-seven genera and 550 species are said to occur naturally in the americas (henderson et al., 1995), and of these, 210 species are native to brazil (lorenzi et al., 2004). based on the last mentioned authors, 20 species are native to the central brazilian cerrado sensu lato vegetation (see gottsberger & silberbauer-gottsberger, 2006a). the distribution of the cerrado species among its six genera is the following: acanthococos (1 species), allagoptera (2 spp.), astrocaryum (1 sp.), attalea (4 spp.), butia (4 spp.) and syagrus (8 spp.) (lorenzi et al., 2004). in this paper we compare two cerrado palms, butia paraguayensis and syagrus petraea, with respect to the insects visiting and feeding on still closed and already opened inflorescences, flower buds and open flowers, as well as immature and mature fruits. in a previous paper a detailed account of the phenology, the growth and duration of the inflorescences and infructescences, of anthesis, and the morphology and functioning of extrafloral nectaries of butia (under the old, now invalid name b. leiospatha) was given (silberbauergottsberger, 1973). it was also mentioned that principally bees, beetles and flies were the visitors and pollinators of this species, but most insects at that time were not identified to genera and species and observations on their behavior have been scarce. also s. petraea (under the synonym s. loefgrenii) was already shortly mentioned as being pollinated by bees and beetles (silberbauer-gottsberger, 1990), again without being able at that time to provide details about visitors and their importance as pollinators and parasites. one aspect of the present paper was to distinguish for both species between insects which are flower visitors without being pollinators and others which are effective pollinators. another aspect was to verify, by direct observation and, additionally, by information from the literature, the level of parasitism of certain insect species; which one is an exclusive harmful floral parasite, destroying floral tissue without contributing to pollination and which one is a parasite and an effective pollinator at the same time? at one study site, in the state of são paulo, both studied species occurred sympatrically, which provided the opportunity to verify similarities and differences in the attracted parasitic and pollinating insects. in another study site in the state of minas gerais, only s. petraea occurred. since the open, unspecialized palm flowers often have a generalist pollination system (gottsberger & silberbauergottsberger, 2006b), we hypothesized that in distant sites of the same palm species, the parasitic insects, living inside and nourishing themselves of floral or fruit tissue, should be more specific than the pollinating insects which mainly collect and feed only on pollen and nectar. material and methods the two species studied have a quite vast distribution. butia paraguayensis (barb. rodr.) l.h. bailey is distributed from the brazilian states of minas gerais, mato grosso do sul, são paulo, paraná and rio grande do sul, to paraguay, argentina, and northern uruguay. this species occurs mainly in cerrado s.l. vegetation on sandy soils, at 500-900 m elevation (henderson et al., 1995, lorenzi et al., 2004). syagrus petraea (mart.) becc. has an even more northern distribution, occurring from the brazilian states pará, maranhão, piauí, bahia, minas gerais, tocantins, goiás, rondônia, mato grosso, mato grosso do sul, and são paulo, to eastern bolivia and eastern paraguay. also this species grows mainly in cerrado s.l. vegetation, however, apparently in more open physiognomies than b. paraguayensis (pers. observation), on sandy to red clayey soils, but also in open, semideciduous forests on stony soils, at 600-1000 m elevation (henderson et al., 1995, lorenzi et al., 2004). there were principally two study sites for the two species, one was in the municipality of botucatu in são paulo state, where both species occurred together, and the other was in the municipality of indianópolis, minas gerais, where only s. petraea occurred. the climate of the botucatu region (city coordinates 22°53´9´´s, 48°26´42´´w, alt. 756 to 920 m) is characterized by an average yearly precipitation of about 1300 mm. the seasonal distribution of precipitation indicates that 37% of the total annual rainfall occurs in spring, 44% in summer, 10% in autumn and only 9% in winter, that is, a warm rainy season from october to march alternates with a cooler dry season from april to september (tubelis et al., 1975). the daily temperature means throughout the year ranged from about 16 to 25°c, with the lowest in july and the highest in february. the yearly average temperature is about 21°c. the exact place of the botucatu study site, treze de maio, where both species occurred, about 8 km northwest of the city of botucatu, consisted of a mixture of several, more closed and more open, cerrado s.l. physiognomies and lies at 650 m alt. the other study site of s. petraea was close to indianópolis (city coordinates 19°2´20´´s, 47°55´1´´w, alt. c. 800 m) at fazenda bela taanda and neighboring fazendas. the temperatures in the more northern indianópolis site are somewhat higher than in botucatu. in b. paraguayensis in botucatu, 20 flowering individui silberbauer-gottsberger, sa vanin, g gottsberger interactions between cerrado palms and insects308 als were marked in 1970/1971. these individuals served for studying the phenology, the duration of flowering of staminate and pistillate flowers, the fruit development and ripening, as well as the approach to and the behavior of insects at the inflorescences and infructescences. to verify the duration of development during the colder and drier versus the warmer and more humid periods of the year, inflorescence buds were weekly measured (length and circumference) from a length of 30 cm on until their opening. visitors were partly observed and collected during blocks of 30 minutes at different times of the day to obtain a more equilibrated picture of occurrence of insects on this palm during whole days. additional and complementing observations on this species occurred in the years 1977, 1980, 1983, 1989, 1990, 2007 and 2009. for studies of s. petraea in botucatu in 1980, 1981, 1983, 1985 and 1990, 35 flowering individuals were selected and investigated at the same site as for b. paraguayensis. at the other site in indianópolis, similar comparative studies of s. petraea were done on 26 flowering individuals in 1983, 1985 and 1990. voucher specimens of both species are deposited in the herbaria botucatu (botu), brasília (b) and ulm (ulm). the insects collected at the palms are deposited in the insect collection of sergio vanin at the department of zoology, university of são paulo (usp), in são paulo, and in the private collection of the authors in ulm. results flowering and anthesis adult individuals of butia paraguayensis in botucatu are erect palms and can attain a maximum stem height of 1.5 to 2 m (gottsberger & silberbauer-gottsberger, 2006b); individuals become already sexually mature having a stem height of only half a meter. on the other hand, syagrus petraea is a so-called “acaulous” short palm. it has subterranean ramified stems. the apical part of the shoot, were the leaves and inflorescences are formed, emerges from the soil in an inclined position and may grow to a height of 20-40 cm (gottsberger & silberbauer-gottsberger, 2006b). when growing in a larger population of ca. 30-50 plants, both species are flowering and fruiting practically year-round. the flowers are unisexual and the inflorescences are monoecious. the pistillate flowers together with staminate ones are arranged in triads more at the basal part of the rachillae (inflorescence ramification of the first order), while staminate flowers alone occupy the apical part. there are about 30-40 rachillae with about ten times more staminate than pistillate flowers. flower number is variable and apparently depends on the size of the inflorescence. one inflorescence of b. paraguayensis can carry about 1200-2500 staminate and 120200 pistillate flowers. s. petraea has smaller and less ramified inflorescences (up to 8 rachillae) and with about five times more staminate than pistillate flowers. one inflorescence can carry about 150-250 staminate and 30-50 pistillate flowers. some inflorescences at the indianópolis site had only staminate flowers. we detected a considerable seasonal difference in the rate of reproductive organ development in b. paraguayensis. development of inflorescences, flowers and fruits was faster during the warm rainy season than during the cool dry season. in 30 cm long inflorescence buds, the time until spathe opening decreased from june to december, this is from mid-dry season to mid-rainy season. also from september onwards, there was an increase in the number of inflorescences produced by an individual. flowering and fruiting time also accelerated. for example, inflorescences which opened in june, july or august took 111 days (on average) to produce ripe fruits, those which opened in september and october took 93 and those in november and december only 57 days. the inflorescences of each individual of b. paraguayensis and s. petraea developed one after the other. only very rarely are staminate and pistillate flowers, either within or between inflorescences of the same plant, simultaneously in a functional stage. for this reason, these palms are mostly functionally dioecious. anthesis in both species begins in the morning with the longitudinal splitting of the woody peduncular bract, the spathe, which can be up to 80 cm long in b. paraguayensis and ca. 20 cm long in s. petraea. with the opening of the spathe the inflorescence is released. the rachillae are spread. the staminate flowers are functioning first, viz. the inflorescence is protandrous. in butia about 1/3 of staminate flowers open at the first day, while this was 1/10 in syagrus petraea. in both species the staminate phase of an inflorescence lasts for about fifteen days (or even longer in the dry season), while one individual flower lasts for one or two days only. only after the last staminate flower has withered, followed by a non-flowering interval of 10 to 15 days, the pistillate flowers become receptive. in butia the stigmas are spread apart, while in syagrus they keep together. all the pistillate flowers in butia enter their receptive phase within two to three days, in s. petraea within 5 to 7 days, while an individual pistillate flower may be receptive for one or two days only. in the population of butia there existed individuals with grayish purple staminate and pistillate flowers, and others with yellow flowers. the flowers emit a faint, pleasant, sweet scent. the flowers of s. petraea are pale yellow, the pistillate ones turning to orange yellow when sexually active. both sexes emit a similar odor, which is faint and straw-like during the day, gradually becoming stronger in the afternoon, as well as gaining spermaticand mushroom-like notes; this stronger odor persists overnight. staminate flowers in both species have a reduced, sexually non-functional, central gynoeceum, known as pistillode. nectar is produced by septal nectaries in both staminate and pistillate flowers, less abundant in s. petraea than in b. paraguayensis. sociobiology 60(3): 306-316 (2013) 309 insects visiting butia paraguayensis the insects observed visiting the reproductive organs of b. paraguayensis, from unopened inflorescences to ripe fruits, belong to 6 orders. the more frequent visitors were of the orders hymenoptera, diptera and coleoptera, while the hemiptera, orthoptera and the enigmatic order strepsiptera were less represented (table 1). more than 45 different insect species, representing 15 families were found visiting the reproductive organs of b. paraguayensis. the most common insects visiting both staminate and pistillate flowers, and thus being the effective pollinators, were bees, especially meliponinae, but also halictidae and apinae. trigona hyalinata and t. spinipes were frequent and about twice as abundant at staminate than at pistillate flowers, whereas the more rare paratrigona lineata lineata and the introduced apis mellifera sometimes visited the pistillate flowers more frequently. other bee species that visited both the staminate and pistillate flowers were ceratalictus theius, and chloralictus cf. opacus. bee species of the genera augochlora, augochlorella, and augochloropsis were seen at staminate and sometimes also more frequently on pistillate flowers and therefore are also effective pollinators. a further, not identified chloralictus species was seen order, family, subfamily, tribe genus, species inflor. bud flowers fruits staminate pistillate unripe ripe coleoptera corylophidae indet. + colydiidae bitoma palmarum +/op? +/pf chrysomelidae galucerinae indet. +/pf/nf +/nf curculionidae anthonominae anthonomini anthonomus sp. 2 + baridinae indet. +/op? ++/pf centrinini sp. 2 ++ centrinaspis sp. +/pf dialomia sp. 1 + revena rubiginosa + +/op madarini angelocentris schubarti ++/pf/nf +/nf/op? tripusus leiospathae +/op? +++/pf parisoschoenus sp. +/pf +/op? microstrates rufus +/pf/op? microstrates sp. nov. ++/pf/op? gen. 1 + erirhininae derelomini anchylorhynchus bicolor +++/pf +/mop molytinae conotrachelini conotrachelus sp. 6 + petalochilinae petalochilus lineolatus + nitidulidae mystrops palmarum +/pf/op? + mystrops sp. 2 ++/pnf mystrops sp. 4 ++/pnf ++ mystrops sp. 6 ++/pnf scarabaeidae rutelinae antichira capucina group +/ff scarabaeinae ateuchus sp. +/op silvanidae silvanus sp. 1 ++/pf tenebrionidae alleculinae prostenus cyaneus +/pf table 1. insects on reproductive organs of butia paraguayensis in botucatu, sp. +++ very frequent, ++ frequent, + rare, pc pollen collecting, pf pollen feeding, nf nectar feeding, pnf pollen and nectar feeding, ff fruit feeding, m mating, op ovipositing. i silberbauer-gottsberger, sa vanin, g gottsberger interactions between cerrado palms and insects310 only on staminate flowers. in staminate flowers, bees principally collected pollen, but fed also a little on nectar, whereas in pistillate ones only nectar was available for them. additionally, several fly, wasp and ant species visited both types of flowers and therefore at least the flies and wasps have to be considered effective pollinators as well. at our study site in botucatu, the two trigona species were very abundant and aggressive, and other bees, even the often dominant apis mellifera, as well as occasionally also wasps and flies were attacked and chased away by them. a recently opened inflorescence was often inhabited and visited by many species and individuals of beetles. sometimes we counted up to ten species of beetles in one inflorescence represented by more than 40 individuals. half of them were often made up by very small beetles, different species of mystrops (nitidulidae) and microstrates (curculionidae) or by the somewhat larger (8 mm) weevil anchylorhynchus bicolor. the numerous individuals of mystrops and microstrates species were mostly feeding on pollen, sometimes took profit also of the nectar, also oviposited in the stamens and visited pistillate flowers only sporadically, thus not being of great importance in pollination. the frequent derelomini weevil anchylorhynchus bicolor (sometimes represented by up to 20 individuals in one inflorescence) also fed on pollen and on nectar but visited the pistillate flowers more frequently and were also pollinating. these beetles were always present in the inflorescences, hidden at the base of the spathe, when either staminate or pistillate flowers were active, respectively. they left the inflorescences in the “inactive” interval phase. however, besides pollinating, they parasitized the pistillate flowers after mating and ovipositing the ovary. the larvae developed inside the gynoeceum and were eating the ovule. the destroyed flowers fell to the ground, where the beetles continued their development to pupae and adults. about 30 to 50% of the pistillate flowers were attacked and did not develop to fruits. the more rare visitors of the pistillate flowers, angelocentris schubartii and parisoschoenus sp., were also pollinators and we suspect them to parasitize the pistillate flowers too. in the open inflorescences of butia, in some years, anchylorhynchus bicolor was represented by two morphs. there was a common form of one color (gray-magenta) and another one with black longitudinal stripes. in another year an additional third black morph with one yellowish line surrounding the wings appeared. in 19 inflorescences in the diptera 3 spp. (muscidae, calliphoridae) +/nf +/nf hemiptera cydnidae indet. +/nf + coreidae indet. +/nf hymenoptera apidae apinae apis mellifera +/pc +/nf meliponinae trigonini paratrigona lineata lineata +/pc/nf +/nf trigona hyalinata +++/pc/nf, ++/nf trigona spinipes +++/pc/nf ++/nf halictidae halictinae augochlorini augochlora sp. +/pc ++/nf augochlorella michaelis +/pc aurochloropsis aurifluens +/pc aurochloropsis cleopatra +/pc ceratalictus theius +/nf ++/nf lasioglossini chloralictus cf. opacus ++/nf chloralictus sp. 1 +/nf formicidae several spp. indet. +/nf +/nf vespidae indet. +/nf +/nf orthoptera proscopiidae + strepsiptera indet. + table 1. [continued]. insects on reproductive organs of butia paraguayensis in botucatu, sp. +++ very frequent, ++ frequent, + rare, pc pollen collecting, pf pollen feeding, nf nectar feeding, pnf pollen and nectar feeding, ff fruit feeding, m mating, op ovipositing. sociobiology 60(3): 306-316 (2013) 311 staminate stage, 109 individuals of the one colored morph, 42 striped ones and only one of the dark morphs were counted. in ten inflorescences in pistillate phase, 8 one colored, eleven striped individuals and one dark individual were present. the microstrates species oviposited in the stamens and their larvae fed on the anthers; they were only occasionally seen to visit the pistillate flowers. at midday the pollen became powdery, thus pollination by wind apparently occurs as well. before opening, the inflorescence bud often was parasitized by the small colydiidae bitoma palmarum (2 mm long), the weevil tripusus leiospathae (3.5 mm long and with a 2.5 mm long proboscis), and a not identified further weevil of the subfamily baridinae (5.5 mm long with a 1.2 mm long proboscis). they made holes into the bud spathe and layed their eggs into the spathe. these holes were always closed by the plant by a resinous substance. bitoma larvae developed in the spathe tissue, while tripusus and the baridinae larvae probably fed on staminate flower buds in the interior of the inflorescence. when the inflorescence bud had recently opened, beetle larvae were present in between the flowers, and often had destroyed and eaten many of them. unfortunately, we could not rear the larvae to adults. as these species were never seen on pistillate flowers, they are probably no order, family, subfamily, tribe genus, species botucatu flowers indianópolis flowersstaminate pistillate staminate pistillate coleoptera chrysomelidae cassidinae indet. + hispinae imatidium sp. + cleridae indet. + cucujidae indet. + colydiidae bitoma palmarum +/pf ++ curculionidae baridinae baridini cryptobaris sulcata + centrinini dialomia sp. 1 + ladustes speciosus + palmocentrinus cf. lucidulus + + madarini hustachea campestris +++ ++/tf +++/op? +++ microstrates cocos-campestris +++ microstrates sp. nov. +++/pf + +++/pf/op? + parisoschoenus plagiatus + cryptorhynchinae cryptorhynchini coelosternus sp. 1 + erirhininae derelomini anchylorhynchus camposi +++/pf ++ +++/pf +++/nf/m/op phytotribus sp. 1 +/pf ++/pf/m/op + phytotribus sp. 2 +/pf + molytinae hilobiini heilus sp. 1 + nitidulidae mystrops sp. 3 +/pnf/op +/nf mystrops sp. 5 +/pnf +/nf +/pnf/op +/nf silvanidae silvanus sp. 2 + + +/pnf +/nf diptera indet. +/nf phoridae indet. +/nf +/nf hymenoptera apidae apinae apis mellifera +/pc meliponinae trigonini trigonisca pseudogreaffii + paratrigona lineata lineata +/pc + trigona spinipes ++/pc +/search +++/pc +/search halictidae augochlorini indet. +/pc formicidae indet. +/nf +/nf blattodea indet. + table 2. insects on reproductive organs of syagrus petraea in botucatu, sp and indianópolis, mg. +++ very frequent, ++ frequent, + rare, pc pollen collecting, pf pollen feeding, nf nectar feeding, pnf pollen and nectar feeding, tf tissue feeding, m mating , op ovipositing, search searching on the flower. i silberbauer-gottsberger, sa vanin, g gottsberger interactions between cerrado palms and insects312 pollinators. also the maiority of the other insects found in the inflorescence visited only the staminate flowers. many of the observed insects just crawled around or were sitting on the flowers and it was not possible to obtain informations about their behavior or their specific importance for the plant. not only flowers but also young and ripe fruits of butia were parasitized. one parasite was the weevil revena rubiginosa which oviposited in very young fruits. the large green rutelid beetle antichira capucina was seen to feed on ripe fruits of butia. under the palm individuals holes in the soil were seen. when digged out, the scarabaeid ateuchus sp. was detected sitting on ripe fruits of butia. it was even possible to observe once an individual of ateuchus rolling a ripe fruit about half a meter away from the palm, digging a hole of about 10 cm depth and than burying a fruit. in some of the holes we found larvae eating the fleshy part of the fruit. the seed inside the hard shell of the inner fruit layer remained intact. insects visiting syagrus petraea about 30 insect species (from 13 families and 4 orders) were observed and collected on the comparatively smaller inflorescences of s. petraea, at the two study sites in botucatu and indianópolis together (table 2). the recently opened inflorescences sometimes sheltered up to hundred individuals of beetles, most of them belonging to small mystrops and microstrates species, followed by anchylorhynchus camposi, hustachea campestris, 2 species of phytotribus and one silvanus sp., which all visited staminate and pistillate flowers. they are all pollinators. the other beetle species visited the staminate and/or pistillate flowers only sporadically and were of no real importance in pollination. many of the species were already active, even copulating, in the early morning at 5:30 am, changing the inflorescences also during the day, and increasing their activity in the evening hours. they fed on pollen, on the small amounts of nectar and on flower tissue at the base of the petals of the staminate flowers and on the margins of petals of the pistillate ones. mystrops, microstrates and phytotribus copulated on the staminate flowers and oviposited them. at different subpopulations and in different years the damage caused by these beetles at staminate flowers could vary from zero to 90%. of 100 dropped staminate flowers from seven inflorescences counted in september 1990 in indianópolis, 44% were damaged. in many of these damaged flowers 1.2 mm long larvae were found. in these so infected flowers mostly the anthers were eaten, but also the petals showed gnawing marks on the nutritious tissues. anchylorhynchus camposi copulated on the pistillate flowers and oviposited the ovary. this species was present only in inflorescences with sexually active flowers and left the inflorescence during the interval between the staminate and pistillate phases. hustachea campestris was seen on staminate and pistillate flowers, but no copulation or oviposition was detected. although the spathe of the bud was sometimes damaged, we were unable to detect which one of the insects was responsible for entering the interior of the inflorescence buds. from the morning on, meliponinae bees, mainly trigona and paratrigona, frequently collected pollen and also visited the pistillate flowers, where they seemed to search for something but were leaving them soon. however, the bees only visited the flowers from 7:00 am to 3:00 pm. diptera and formicidae were seen on staminate and pistillate flowers in very small numbers. discussion butia paraguayensis and syagrus petraea are visited by numerous insects, mainly bees and beetles, which play different roles during the development of the inflorescence buds, the flowers and fruits. there are nearly exclusive parasitic beetles, which oviposit and destroy the tissue of the young spathe, as well as staminate flowers and unripe fruits. a second group of insects are parasites and pollinators, and a third group are pure pollinators which only feed on pollen and/or nectar. there is also a forth group of insects, which only visit the staminate flowers, nourishing themselves from pollen and nectar. examples of pure parasites in b. paraguayensis are the weevils tripusus leiospathae (the former name of this palm was cocos leiospatha!) and an unidentified baridinae, which bores the spathe and the larvae feed inside the closed inflorescence on staminate flowers. as in nearly all baridinae, the larvae after dropping of the flowers complete their development in the soil (costa lima, 1956). the colydiidae bitoma palmarum supposedly develops inside the spathe tissue; we found the adult beetle in newly opened inflorescences. bondar described this beetle as predator of attalea funifera and cocos nucifera (costa lima, 1953). also the weevil petalochilus lineolatus may develop in the interior of the closed spathe. other species of this genus are mentioned to develop in the inner spathes of the palms of the genera diplothemium, attalea and syagrus (vaurie, 1954). the larvae of the two microstrates (curculionidae) species develop in the interior of staminate flower buds, eating anthers and pollen. they complete their development in the fallen flowers at the ground. this was also observed by bondar for microstrates ypsilon on syagrus coronata and cocos nucifera, and other species, among them butia eriospatha parasitized by m. hatschbachi, and syagrus flexuosa (syn. cocos campestris) parasitized by m. cocoscampestris (bondar, 1941, costa lima, 1956). revena rubiginosa, which was seen to oviposit young fruits may also be an exclusive parasite. another species, r. vagans, was described by bondar to bore the young fruits of syagrus vagans, and the growing larva was eating and destroying the whole interior (lepesmé, 1947). sociobiology 60(3): 306-316 (2013) 313 one prominent and frequent beetle that is a parasite of the pistillate flowers and at the same time causes pollination in b. paraguayensis is anchylorhynchus bicolor (curculionidae, derelomini). as this beetle feeds on pollen and passes over several pistillate flowers before ovipositing, it is a relatively effective pollinator. there are many species of anchylorhynchus associated quite specifically with palms (bondar, 1943, vaurie, 1954). angelocentris schubarti and parisoschoenus sp. also visit staminate and pistillate flowers and seem to parasitize the pistillate ones. parisoschoenus obesulus is a pest on cocos nucifera, parasitizing the ovary (sánchez & nakano, 2003, moura et al., 2009). one unidentified dialomia species was found on staminate flowers, but it may belong to the group of parasitic and pollinating insects as well. larvae of dialomia polyphaga feed on petals and anthers and adult females may lay their eggs in pistillate flowers of palms (lepesmé, 1947). the nitidulid mystrops species which feed on pollen and nectar apparently parasitize the staminate flower buds. following bondar (1940), in mystrops palmarum the larval stage takes 5 to 6 days in the staminate buds and the pupae need 4 to 6 days for their development. the whole cycle of the beetle does not exceed 12 days. hymenoptera and diptera are non-parasitic pollinators and are the most abundant and effective ones in b. paraguayensis. the scarab ateuchus functions as a short distance disperser of the fruit. it rolls the ripe fruits a short distance away from the mother individuals and digs holes to bring the fruit underground to oviposit there. the intact fruit is in the right depth for germinating and at the same time it is protected from fire. the visitor spectrum of s. petraea is different from b. paraguayensis. beetles are the most abundant visitors of the inflorescences and flowers, while hymenoptera and diptera are less prominent. this species shows adaptations for beetle pollination. the specific floral scent, especially in the afternoon and evening attracts beetles. some of them are nourished by nutritious tissues on the petals of both the staminate and pistillate flowers. as the flowers produce very low amounts of nectar, they are not that attractive as b. paraguayensis for nectar-imbibing insects such as bees and flies. the only insect species which s. petraea were found to share with b. paraguayensis are the bees trigona spinipes, paratrigona lineata, and the beetles bitoma palmarum, dialomia sp. 1 and microstrates sp. nov. these species showed on s. petraea about the same behavior as on b. paraguayensis, with exception of the new microstrates species. in both palm species microstrates sp. nov. developed in the staminate flowers, but visited the pistillate ones only in s. petraea, being a pollinator for this species. otherwise, only the genera of beetles are the same at the two palm species as, for instance, anchylorhynchus, parisoschoenus, microstrates, mystrops and silvanus, but the species are different ones. the set of species in s. petraea are anchylorhynchus camposi, parisoschoenus plagiatus, microstrates cocoscampestris, mystrops sp. 3, sp. 5, and silvanus sp. 2., while anchylorhynchus bicolor, parisoschoenus sp., microstrates rufus, mystrops palmarum, mystrops sp. 2, sp. 4, sp. 6, and silvanus sp. 1 occur in butia paraguayensis. one of the important pollinating beetles of s. petraea, hustachea campestris, did not occur on b. paraguayensis. hustachea species appear to parasite staminate flowers; bondar provided a drawing of h. bondari, shown in lepesmé (1947), piercing and ovipositing the petals of a staminate bud of a palm, with the subsequent development of the larva in its interior. the two phytotribus species also only occurred on s. petraea. species 1 was found at the two sites, ovipositing the staminate flowers in indianópolis, whereas species 2 occurred only at the botucatu site. there are differences in s. petraea with regard to the occurrence of several beetle and bee species at the botucatu and indianópolis sites. but the parasitic species anchylorhynchus camposi, phytotribus sp. 1, microstrates sp. nov., hustachea campestris, mystrops sp. 5, and bitoma palmarum were found on flowers at both sites. anchylorhynchus camposi was found by bondar (1941) also on flowers of syagrus flexuosa. apparently, there are beetles that have a more restrict occurrence on a small group of palms. to this group certainly belongs the weevil genus anchylorhynchus, visiting the two palm species treated in this paper, and petalochilus, which was found only on b. paraguayensis. after the revision of anchylorhynchus by bondar (1943), voss (1943) and vaurie (1954), 16 species of this genus were recognized and five in petalochilus (vaurie, 1954). bondar stated that all species of anchylorhynchus develop in palms and as far as verified they breed in flowers of the former genus cocos, which is now subdivided in the monotypic genus cocos, as well as the genera butia and syagrus. butia is a monophyletic genus of nine species confined to cooler, drier areas in southern brazil, paraguay, uruguay and argentina. recently, martel et al. (2013) verified raphidcontaining idioplasts in all butia staminate flower petals sampled, which do not occur in the most closely related genus jubaea, further supporting the cohesive monophyly of butia. syagrus with its 31 species in its present concept apparently is polyphyletic and occurs from venezuela southwards to argentina with many species in the brazilian cerrados and campos rupestris (dransfield et al., 2008). butia paraguayensis and b. yatay are said to be closely related species and being perhaps not distinct from each other (henderson et al., 1995); at least the beetle breeding on them, in both cases anchylorhynchus bicolor, eventually confirms that the two species are identical. for syagrus, at present recognized as being polyphyletic, future studies may show the whole dimension of the association with anchylorhynchus. it has to be seen after the circumscription of a cohesive monophyletic syagrus, which one of the species still has associations with anchylorhynchus. meanwhile, besides occurring on species i silberbauer-gottsberger, sa vanin, g gottsberger interactions between cerrado palms and insects314 of butia and syagrus, anchylorhynchus species were detected also on species of oenocarpus in central amazonia (küchmeister, 1997) and in the colombian andes (núnez-avellaneda and rojas-robles, 2008). on the other side, there are beetles which associate with a multitude of palm genera. notorious are the genera mystrops (present also on b. paraguayensis and s. petraea) and phyllotrox, but also, celetes, hustachea, andranthobius, dialomia and others (see e.g., schmid, 1970, uhl and moore, 1977, beach, 1984, búrquez et al., 1987, barfod et al., 1987, anderson et al., 1988, scariot & lleras, 1991, bernal & ervik, 1996, ervik & feil, 1997, küchmeister, 1997, küchmeister et al., 1997, listabart, 1999, voeks, 2002, henderson, 2002). several of these beetle genera might have numerous (but badly known or undescribed) species which are more or less specific to one or a few palm species. we are beginning to understand that even close-by standing different species of palms often share few visitors and no pollinators because differences in floral scent composition may cause isolation (e.g., ervik et al., 1999, knudsen et al., 2001, núñez et al., 2005). in many cases palms are parasitized by beetles which afterwards function as pollinators. we then can even say that these palms are breeding their own pollinators. this is an elegant way to make those palms more or less independent for pollination from their surroundings and at the same time this breeding of pollinators shapes the specificity of the beetles for “their” palm species. the nocturnal, thermogenic attalea microcarpa, studied by küchmeister (1997) in manaus, provides a most elegant example of a palm breeding its own pollinators. this palm is visited by more than 30 species of coleoptera, hymenoptera, lepidoptera, diptera and others, but species of staphylinid beetles, mystrops (nitidulidae), groatus, celetes, phyllotrox and belopoeus (all curculionidae) are the main pollinators. they are attracted in enormously large numbers (up to 60,000 estimated individuals per inflorescence) during the evening hours by the strongly heated staminate inflorescences which have a fruitand yeast-like scent emission. the beetles´ activity after entering the semi-closed staminate flowers was pollen eating and oviposition. the larvae develop in the anthers and nourish themselves from pollen grains and anther tissue. fast growing hyphae of a fungus envolve the totality of the staminate flowers and prevent their dropping to the ground. the fungus holds the withered and loose staminate flowers at the inflorescence, which gives the larvae of the beetles´ time to finish their development in a flushwater-free space above the ground in the heavily raining environment of the palm. the emerging beetles transport pollen grains and fungus spores to other inflorescences. the pistillate flowers also are heating, they emit a similar scent as the staminate ones and attract the same beetle species that visit the staminate inflorescences, however in much lower number. in a. microcarpa three organisms or groups of organisms, the palm and its flowers, the beetles and finally a fungus work together to make parasitism and at the same time pollination effective processes. not only in a. microcarpa but in all palms observed up to now, parasitism by flower beetles is more or less in balance with the number of flowers and inflorescences formed. there always remain enough intact flowers within a palm population to guarantee successful pollination and reproduction. the two studied cerrado palms, b. paraguayensis and s. petraea, are visited by insects which are non-pollinators or only occasional pollinators, besides others which are regular and effective pollinators and at the same time parasites. several of these parasites are effective pollinators. also these two palms breed 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(1999). pollination studies of palm populations : a step toward the application of a biological species concept. in: a. henderson & f. borchsenius, (eds.), evolution, variation, and classification of palms (pp. 79-97). memoirs new york botanical garden 83. lorenzi, h., moreira de souza, h., coelho de cerqueira, l s., medeiros-costa, jt. &, ferreira, e. (2004). palmeiras brasileiras e exóticas cultivadas. nova odessa, instituto plantarum de estudos da flora ltda. martel, c., noblick, l. & stauffer, f.w. (2013). an anatomical character to support the cohesive unit of butia species. palms, 57: 30-35. moura, l.l., ferreira j.m.s., sgrillo, r.b., valle, r.r., almeida, a.f. de, cividanes, f.j. & delabie, j.h.c. (2009). parisoschoenus obesulus casey (coleoptera: curculionidae) não é praga de frutos novos do coqueiro. neotrop. entomol., 38: 251-253. núñez, l.a., bernal, r. & knudsen, j.t. (2005). diurnal palm pollination by mystropine beetles: is it weather-related? pl. syst. evol., 254: 149-171. núñez-avellaneda, l.a. & rojas-robles, r. (2008). biologia reprodutiva y ecología de la polinización de la palma milpesos (oenocarpus bataua) en los andes colombianos. caldasia, 30: 101-125. sanchez, s. & nakano, o. (2003). presença de parisoschoenus obesulus casey (coleoptera: curculionidae) na cultura de coqueiro do estado de são paulo, brasil. entomotropica, 18: 77-78. scariot, a.o. & lleras, e. (1991). reproductive biology of the palm acrocomia aculeata in central brazil. biotropica, 23: 12-22. schmid, r. (1970). notes on the reproductive biology of asterogyne martiana (palmae). principes, 14: 3-9, 39-49. silberbauer-gottsberger, i. (1973). blütenund fruchtbiologie von butia leiospatha (arecaceae). österr. bot., z. 121: 171-185. silberbauer-gottsberger, i. (1990). pollination and evolution in palms. phyton (horn, austria), 30: 213-233. tubelis, a., nascimento, f.j.l. & foloni, l.l. (1975). parâmetros climáticos de botucatu. botucatu, 1975: 14-15. uhl, n.w. & moore jr., h.e. (1977). correlations of inflorescence, flower structure and floral anatomy with pollination in some palms. biotropica, 9: 170-190. vaurie, p. (1954). revision of the genera anchylorhynchus i silberbauer-gottsberger, sa vanin, g gottsberger interactions between cerrado palms and insects316 and petalochilus of the petalochilinae (coleoptera, curculionidae). am. mus. novit., 1651: 1-58. voeks, r.a. (2002). reproductive ecology of the piassava palm (attalea funifera) of bahia, brazil. j. trop. ecol., 18: 121-136. voss, e. (1943). ein überblick über die bisher bekannt gewordenen arten der gattung ancylorrhynchus aus der unterfamilie der petalochilinae (coleoptera, curculionidae). entomol. blätter, 39: 60-64. sociobiology 60(2): 214-216 (2013) doi: 10.13102/sociobiology.v60i2.214-216 age polyethism in the swarm-founding wasp metapolybia miltoni (andena & carpenter) (hymenoptera: vespidae; polistinae, epiponini) l chavarría1; fb noll1,2 introduction division of labor characterizes insect societies. workers allocate tasks responding to internal and external conditions, and to individual workers decisions (sendova-franks & franks, 1999). there are two general patterns: worker age polyethism (task allocation correlated with age) and morphological polymorphisms (changes in size or shape related to task performance) (beshers & fewell, 2001). organization of labor in wasps of the tribe epiponini seems not to be related with morphological polymorphism because there is no morphological specialization or subcastes. division of labor could be associated with worker’s age, or with individual task specialization (jeanne, 1996; karsai & wenzel, 2000). according to jeanne (1991) epiponines have the most evident worker age polyethism of social vespidae. previous studies in polybia (lepeletier), protopolybia (ducke) and agelaia (ducke) (simões, 1977; forsyth, 1978; jeanne et al., 1988; jeanne et al., 1992) found that young and middle aged workers perform nest tasks (building, brood care, nest mainabstract in epiponini division of labor is associated with age polyethism and individual task specialization. we observed worker activities in three colonies of metapoybia miltoni in brazil. we analyzed differences of task allocation among age groups. old workers tend to forage more than young, but age polyethism was less evident in other tasks. age composition of population could be a determinant factor in task allocation. workers are probably allocating to perform tasks according to colony needs, and not to individual’s age. considering age population in studies of division of labor could help to understand how colonies respond to different situations. sociobiology an international journal on social insects 1 universidade de são paulo, ribeirão preto, são paulo, brazil 2 universidade estadual paulista júlio de mesquita filho, são josé do rio preto, são paulo, brazil short note article history edited by: sergio r. andena, uefs brazil received 22 december 2012 initial acceptance 18 march 2113 final acceptance 01 april 2013 keywords social wasps, workers, division of labor corresponding author laura chavarría ffclrp-usp av. bandeirantes 3900 bloco 9 ribeirão preto, são paulo, brazil 14040-901 e-mail: laurachp@usp.br tenance, defense), while old workers forage. nevertheless, the presence of workers specialized in a particular task is rare in most of social hymenoptera (robinson, 1992; sendovafranks & franks, 1999; o’ donnell, 1998; karsai & wenzel, 2000; beshers & fewell, 2001; johnson, 2003). within epiponini, karsai and wenzel (2000) did not find specialization in colonies of metapolybia aztecoides (richards) and m. mesoamericana (smethurst & carpenter). because organization and regulation of work is complex, conventional patterns are insufficient to explain the division of labor of several insect societies (beshers & fewell, 2001). for these reasons, we studied task allocation according to age in three colonies of metapolybia miltoni (andena & carpenter) in maranhão, brazil. we observed colonies for three days in 2008: colonies n-1 and n-2 on february, in reserva merck (s 02° 39' 7.8" and w 44° 09' 04.0"); and colony n-3 on march, in reserva das paineiras (s 03° 14' 35.4" w 43° 25' 28.7"). nest envelope was removed in order to perform video recordings (sony handycam dcr-sr42). we took a random sample of worksociobiology 60(2): 214-216 (2013) 215 ers to perform individual observations (n=15 for n-1, n=11 for n-2 and n=18 for n-3). all individuals were marked with quick-drying paint. video recordings included 197, 194 and 370 minutes for n-1, n-2 and n-3, respectively. we observed allocation of work in three tasks: cell inspection, construction (envelope and cells) and forage. in these cases, forager’s activities were directly observed due to difficulties to follow them in video. females were classified according to age, based on three categories for the coloration of the transverse apodeme (richards, 1971): light for younger, brown for middle age, and black for older females. to verify if the amount of workers that performs a determinate task varied according to age, and within colonies n-1 and n-3 (all observed workers of n-2 were young) a chi-square test was applied. our results indicate that colony cycle stage did not affect workers frequency performing tasks among colonies (cell inspection x2=0,854 df= 2 p>0.5; construction x2=0,381 df= 2 p>0.5; foraging x2=3,641 df= 2 p>0.5). colony n-1 was in a mature stage of colony cycle (eggs, larvae, pupae and low queen proportion), and most of workers were young (old= 1% middle= 8% young= 90%). colony n-2 was also in a mature stage, but in male production and most of the workers were young (young= 90% middle= 10%). colony n-3 was in a preemergence phase (only eggs and higher queen proportion), and most of workers were old (middle= 17% old= 83%). we observed a similar number of old and young workers perfoming nest tasks (cell inspection and construction) and foraging (young x2= 4.588 df= 2 p>0.05; middle x2= 1.333 df= 2 p>0.5; old x2= 0.839 df= 2 p>0.5). nevertheless, it is clear that young workers tended to forage less than olders (fig 1 colonies n-1, n-3), even in colony n-2 young workers tended to forage less. also, the amount of workers that perform tasks across different ages inside colonies n-1 and n-3 was similar (n-1 x2= 2,798 df= 2 p>0.1; n-3 x2= 0,802 df= 2 p>0.5). as mencioned before, previous studies of division of labor in the epiponini found that as workers get old they switch to perform tasks less related with brood care (jeanne, 1991). similarly, we observed that old workers tend to forage more than young individuals (fig 1). we did not find significant differences probably because of the small sample of foragers (n=24). nevertheless, even when old workers tend to forage more, young workers can also forage (fig 1). age polyethism was less evident in the other tasks: old workers as young ones can build and inspect cells (fig 1). age composition of worker population within colonies may be a determinant factor in task allocation. the observed colonies did not include individuals of different ages; workers of colonies n-1 and n-2 were mostly young, and workers of colony n-3 were mostly old. because colonies population has little variation in terms of age, workers must be allocated to perform tasks according to colony needs and not to individual’s age. on the other hand, in colonies with differently aged workers, polyethism could be more important to delimit tasks, as demonstrated in previous studies (jeanne et al., 1988; jeanne et al., 1992). as observed by karsai and wenzel (2000), we also found flexibility in activities performed by workers; young, middle and old individuals perform different tasks. workers of all insect societies retain some behavioral flexibility that helps to respond to changing conditions (robinson, 1992). caste flexibility is decisive for colony survival in swarm wasps because it allows colonies to respond efficiently to different situations that may arise. fig 1. percentage of workers of different age (ly= light yellow for younger, b= brown for middle age, bl= black for older females) that perform a task (ic= cell inspection, const= construction, forrg= foraging) throught the studied colonies of metapolybia miltoni. workers decisions are dependent of colony context, and workers would be allocated to perform certain tasks when necessary (karsai & wenzel, 2000). in conclusion, considering age composition of population and studying colonies exposed to different situations would help to understand how colonies allocate tasks. the division of labor of swarm wasps is more complex than previously thought; colonies do not organize labor in the same manner. in fact, as evidenced by noll & wenzel (2008), caste dimorphism may have evolved at least eight times and social organization probably derived directly from an ancestor with incipient caste dimorphism in most taxa. for this reason general patterns are not enough to understand different strategies across the tribe. acknowledgments we especially thank to organization of america states (oas), the incentive commission of ministerio de ciencia y tecnología (micit) and consejo nacional para investigaciones científicas y tecnológicas (conicit) from costa rica, for financial support to do this investigation. to conl chavarría, fb noll age polyethism in metapolybia miltoni216 jeanne, rl. (1996). regulation of nest construction behavior in polybia occidentalis. an. behav. 52: 473-488. doi: 10.1006/anbe.1996.0191 johnson, br. (2003). organization of work in the honeybee: a compromise between division of labor and behavioral flexibility. proc. r. soc. lond. 270: 147-152. doi: 10.1098/ rspb.2002.2207 karsai, i. & wenzel, jw. (2000). organization and regulation of nest construction behaviour in metapolybia wasp. j. insect behav. 13(1): 111-140. doi: 10.1023/a:1007771727503 noll, f.b. & wenzel, j.w. (2008). caste in the swarming wasps: “queenless” societies in highly social insects. biol. j. lin. soc. 93: 509-522. doi: 10.1111/j.1095-8312.2007.00899.x o’donnell, s. (1998). reproductive caste determination in eusocial wasps (hymenoptera: vespidae). annu. rev. entomol. 43: 323-346. doi: 10.1146/annurev.ento.43.1.323 richards, ow. (1971). the biology of the social wasps (hymenoptera, vespidae). biol. rev. 46: 483-528. doi: 10.1111/ j.1469-185x.1971.tb01054.x robinson, ge. (1992). regulation of division of labor in insect societies. annu. rev. entomol. 37: 637-665. doi: 10.1146/annurev.en.37.010192.003225 sendovafranks, ab & franks, nr. (1999). selfassembly, selforganization and division of labour. phil. trans. r. soc. lond. 1388 (354): 1395-1405. doi: 10.1098/rstb.1999.0487 simões, d. (1977). etologia e diferenciação de casta em algumas vespas sociais (hymenoptera, vespidae). tese de doutorado. ribeirão pretousp. 169pp. selho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação de amparo à pesquisa do estado de são paulo (fapesp) (2007/08633-1) from brazil to support part of the investigation. to gisele garcia for all the help and friendship offered during the stay in maranhão. to otávio augusto lima de oliveira for field assistance. to john wenzel and sergio jansen for their comments, suggestions and helpful ideas. references andena, sr. & carpenter, jm. (2011). a new species of metapolybia (hymenoptera: vespidae; polistinae, epiponini). entomol. am. 117(3/4): 117-120. doi: 10.1664/11-ra-003.1 beshers, sn. & fewell, jh. (2001). models of division of labor in social insects. annu. rev. entomol. 46: 413-440. doi: 10.1146/annurev.ento.46.1.413 forsyth, ab. (1978). studies on the behavioral ecology of polygynous social wasps. dissertation, harvard university. jeanne, rl., downing, ha. & post, dc. (1988). age polyethism and individual variation in polybia ocidentalis, and advance eusocial wasp. in rl. jeanne (ed). interindividual behavioral variability in social insects (pp. 323-357). boulder, colorado: westview press. jeanne, rl. (1991). polyethism. in kg. ross & rw. matthews (eds). the social biology of wasps. (pp. 389-425). ithaca, new york: cornell university press. jeanne, rl., william, nm. & yandell, bs. (1992). age polyethism and defense in tropical social wasps (hymenoptera: vespidae). j. insect behav. 5(2): 211-227. doi: 10.1007/ bf01049290 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1052sociobiology 63(3):925-940 (september, 2016) a quantitative baseline of ants and orchid bees in human-modified amazonian landscapes in paragominas, pará, brazil introduction tropical forest ecosystems are the richest ecosystems on earth (pimm and raven 2000), harboring up to two thirds of the planet biodiversity (gardner et al., 2009). yet, the region has been suffering intense human-impacts and is the among the most actives frontiers of land-cover changes in the world (malhi et al., 2008). in brazil, government efforts were yielding positive results, and deforestation rates have decreased from 2004 to 2014, although these rates have increased recently (prodes-inpe 2015). given this loss abstract the lack of effective biodiversity baselines is a major impairment to implement conservation plans. hence, constructing and updating species lists provides vital information about species distribution records. the sustainable amazon network (in portuguese rede amazônia sustentável; ras) is an interdisciplinary research initiative that aims to evaluate land-cover changes effects in eastern brazilian amazonia. within the scope of this project, we sampled ants and orchid bees and herein present a list of species collected in paragominas, pa, brazil; the most complete lists of species published to date of these groups for the eastern amazon. we sampled these insects across several land-cover types, from undisturbed forested habitats, through varyingly disturbed forested habitats and secondary forests to production areas (silviculture, pastures and arable fields). in total we recorded 285 species of ants and 36 species of orchid bees. species richness was higher in primary forests for both groups, followed by production areas. orchid bees reached their highest richness in secondary forests. for orchid bees, production areas were dominated by a few hyper-dominant species, such as eulaema nigrita. for future assessments if the aim is to make a complete inventory, we recommend the use of additional sampling methods. finally, we expect this study can be used as a baseline for understanding the effectiveness of ongoing changes in forest conservation and land management practices and in determining conservation status for several taxa described here. sociobiology an international journal on social insects rrc solar1,2,3, jcm chaul2, m maués4, jh schoereder2 article history edited by gilberto m. m. santos, uefs, brazil received 27 april 2016 initial acceptance 24 august 2016 final acceptance 18 september 2016 publication date 25 october 2016 keywords amazon rainforest, biodiversity baselines, conservation, land-use change, monitoring. corresponding author ricardo rc solar; icb/ufmg, departamento de biologia geral, sala i3-245 avenida antônio carlos, nº 6627 cep 31270-901, belo horizonte-mg, brasil e-mail: rrsolar@gmail.com and modification of amazonian forest habitats, it is critical to understand the ramifications for regional biodiversity in order to foster conservation plans and actions in the region. nevertheless, due to poor infrastructure and the vast size of the region, our knowledge about amazonian biodiversity, particularly non-vertebrates is limited and most species lists underestimate total biodiversity (barlow et al., 2011). given that habitat loss is the most serious threat facing tropical biodiversity (laurance et al., 2014), regional inventories represent an important step for the conservation of insect communities. understanding patterns of species occurrence in 1 instituto de ciências biológicas, universidade federal de minas gerais, belo horizonte-mg, brazil 2 programa de pós-graduação em ecologia, universidade federal de viçosa-mg, brazil 3 lancaster university, lancaster uk 4 embrapa amazônia oriental, belém-pr, brazil research article ants mailto:rrsolar@gmail.com rrc solar et al. – paragominas ants and orchid-bees checklist.926 space and time, as well as across human-modified landscapes is a valuable tool for studying population ecology and biodiversity responses to human impacts in order to judge which human activities are most affecting biodiversity (lach et al., 2010). the sustainable amazon network (in portuguese, rede amazônia sustentável, ras, gardner et al., 2013) assessed the biodiversity, ecosystem services and social aspects of human-modified forest landscapes in two frontier regions (paragominas and santarém-belterra) of the eastern brazilian amazon, in the state of pará. these regions have suffered from intense deforestation since the 1970’s (lindenmayer et al., 2004), although several governmental and social initiatives have been contributing to minimize and revert this process (viana et al., 2016). within the municipality of paragominas, ras fieldwork sampled trees and lianas (berenguer et al., 2014), birds (lees et al., 2012) and here we present data on a comprehensive survey of two terrestrial invertebrate groups: ants (hymenoptera: formicidae) and orchid bees (hymenoptera: apidae: euglossina) selected for their ecological importance. ants (hymenoptera: formicidae) are a ubiquitous group, being numerically and ecologically dominant in tropical forests (hölldobler & wilson 2009; lach et al., 2010). they play roles as seed dispersers (christianini et al., 2007), in moving nutrients across soil horizons (sousasouto et al., 2007) and in regulating populations of prey species (folgarait, 1998). ants are also easy to sample, have a relatively well established taxonomy and are numerically dominant nearly everywhere in the neotropics throughout the year (underwood & fisher, 2006). orchid bees encompasses around 250 species endemic to the neotropics (nemesio & rasmussen, 2011). one of the striking characteristics of this group is pollination of tightly associated plant species (janzen, 1971). given their sensitivity to environmental change (nemesio & vasconcelos, 2013) and ease with which they can be sampled makes them a costeffective ecological disturbance indicator group (gardner et al., 2008). here we present an annotated checklist of ants and orchid bees collected in paragominas, pa, brazil; the most complete list species list produced to date for the western amazon that, together with other efforts already conducted in the region (e.g. kempf, 1970; kalif et al., 2001; santos et al., 2008) establishes a crucial biodiversity baseline for ongoing environmental monitoring. methods study site we sampled the insects in paragominas, a 2 million ha amazonian municipality in pará state, northeastern brazil (fig 1). the region was originally almost entirely covered with evergreen forests and regional climate is am according to the köppen classification (alvares et al., 2013) with an average annual rainfall of 1800mm (andrade, 2011) and mean annual temperatures of 26.3ºc (pinto et al., 2009). we conducted all fieldwork between january-june 2011, during the rainy season. sampling was undertaken in 18 ca. 5.000 ha. catchments covering the entire municipality (2 million hectares), distributed along a gradient of deforestation from undisturbed primary forest through varyingly disturbed primary forests, secondary forests, pastures and mechanized agriculture, where we established from 8-12 transects (300m) in each catchment, in a density of 1 transect/400ha (fig 1). in total, we sampled 192 transects across the major land-use classes present in the region including undisturbed primary forests, varyingly disturbed (from logging and fire) primary and secondary forests and production areas (silviculture – eucalyptus and schyzolobium amazonicum, cattle pastures and arable fields). insect sampling within each transect we sampled both insect groups concurrently. ants were sampled with epigaeic pitfall traps, composed of plastic containers (8 cm diameter) half filled with a solution of water, salt (5%) and soap (5%) and baited with sardines and honey, both inaccessible to the ants. in each transect we installed six pitfall traps separated 50 m from one another. to sample orchid bees, we used four plastic bottles per transect, with one scent bait each (2l, 10 cm diameter, 35 cm height), tied to a tree trunk, 1.5 m above the ground. male orchid bees were attracted to four types of scent baits (eugenol, methyl salicylate, vanilla and eucalyptol), separated by 50 m from each other. in both cases traps remained in the field for 48h prior to removal, see fig 1 for a graphical representation of our sampling design. we processed and identified the ants to the most precise taxonomic level possible using genera taxonomic keys (fernández, 2003, baccaro et al., 2015), and the most upto-date taxonomic revisions of each taxa, directly comparing with the available type images on antweb (available from http://www.antweb.org. accessed in april/2016), and checked against the reference collection of the community ecology lab, affiliated with the museu regional de entomologia da universidade federal de viçosa (ufvb). we checked all the species in ant cat (bolton; http://antcat. org, accessed in april/2016) to confirm the validity of the nomenclature we used. we processed and identified orchid bees at embrapa – amazônia oriental, adapting available taxonomic keys (nemesio, 2009) and using the reference collection of embrapa – amazônia oriental. a taxonomist, dr andré nemésio, checked species identifications. we deposited voucher specimens of ants in the reference collection of the community ecology lab, universidade federal de viçosa. orchid bees are deposited on the reference collection in embrapa – amazônia oriental. sociobiology 63(3): 925-940 (september, 2016) 927 statistical analyses to assess our sampling sufficiency, we built sitebased species accumulation curves (colwell et al., 2004) and estimated the total number of species sampled in each taxon using the first order jackknife richness estimator. all analyses were performed using the r v.3.1.2 (r core team 2015), using the package vegan (oksanen et al., 2015). results for both groups, species richness was higher in forested areas (average ± standard deviation; ants 25.2 ± 7.3; bees 6.45 ± 3.8) and lower in production areas (average ± standard deviation; ants 16.9 ± 6.0; bees 3.8 ± 2.5). worth of highlighting, the richest assemblage of orchid bees was found in secondary forests. in table 1, we present how species richness of both groups was distributed in transects across the studied land-use classes. ants we sampled a total of 285 species of ants, in 60 genera, belonging to nine subfamilies. we assigned a name to all genera and among them, 132 were identified to specieslevel or very close to a given taxa, and 11 were assigned to fig 1. map of the sampling region within the brazilian contexts. circles represent each sampled transect and circle sizes represent relative species richness for each taxa. in colours are represented micro-catchments, where samplings were located and greener colours represent higher primary forest cover, while redder colours represent lower primary forest cover. rrc solar et al. – paragominas ants and orchid-bees checklist.928 species groups or complexes where exact species identification was impossible. the remaining 142 are identified and were assigned a morphospecies code that applies only to this study. a list of the species and morphospecies is given in table 2. at least two new species of the genera xenomyrmex (xenomyrmex pgm-01) and hylomyrma (hylomyrma pgm01) were sampled (l. do prado and m. ulysséa, personal communications, respectively) and these specimens are deposited at the museu de zoologia da universidade de são paulo. by comparing with the type photos (antweb available from http://www.antweb.org. accessed in march 2015) and/or original descriptions, we considered some species in the list of ants to be slightly different from the original species (e.g. strumigenys aff. perparva; pachycondyla aff. purpurascens) and are therefore treated as morphospecies very close to that particular species. it remains to be investigated whether each of them represent still undescribed sister species or a case of intraspecific variation. others were tentatively assigned to a given taxa but precise identification was not possible (e.g. sericomyrmex cf. parvulus). the regional species accumulation curve is not asymptotic but does flatten towards the end (fig 2a), and the 1st order jackknife estimator suggests that we sampled 77.5% of the total species richness. discussion our study is the most comprehensive sampling to date of ants and orchid bees for any area of the eastern amazon and we hope it both fosters future biodiversity studies in the region, and can be used as an evidence-baseline for future red listing classification exercises for invertebrates. we consider our sampling effort sufficient for both taxa at the regional scale, with at least 77% of the estimated diversity sampled for all taxa. for ants, the only previous study we are aware of in paragominas yielded only 74 species belonging to 30 genera (kalif et al., 2001). even so, by using a different sampling method (winkler extractors), this study sampled species not represented in our study, demonstrating the importance of implementing complementary methods of sampling to survey this region. exploring seldom-studied habitats such as the forest canopy (basset et al., 2012) or underground soil layers (wilkie et al., 2007; schmidt & solar, 2010; schmidt et al., 2014) also offers significant potential to increase the number of species described for the region. orchid-bees are poorly known in the amazonian region, our total of 36 species is of a similar magnitude as other studies in current and former terra-firme forest habitats in the region (oliveira & campos, 1996; rasmussen, 2009; storck-tonon et al., 2009; abrahamczyk et al., 2011). we sampled in a very diverse range of habitats and in a large area, however additional species are likely to be encountered using a greater range of bait types (nemesio & vasconcelos, 2013). for both taxa, we acknowledge that by sampling only during the rainy season, we might have missed some species that prefer dry climates. we believe this was not the case for ants, as they are colonial organisms and are know to be far more active during the rainy season (hölldobler & wilson, 2009), improving capture by pitfall traps. orchid bees do have marked seasonality (abrahamczyk et al., 2012; abrahamczyk et al., 2014), however the highest species richness for this group is found in the rainy season (abrahamczyk et al., 2011). in order to cover a larger area as possible, while capturing the maximum diversity, we opted to sample during the rainy season. unsurprisingly we found forests to be more species rich than non-forest habitats, as was the case for other taxonomic fig 2. species accumulation curves for both studied taxonomic groups. each curve was drawn after 10.000 randomisations of original data and the shaded area represents the standard deviation. in the x-axis we have number of sampled transects, in the y-axis, accumulated species richness. taxon land-use class ants orchid-bees undisturbed forest 28.56 (±3.4) 4.85 (±2.6) logged forest 25.72 (±2.1) 6.46 (±0.9) logged and burned forest 27.25 (±2.1) 6.46 (±1.1) secondary forest 23.09 (±2.9) 8.39 (±1.6) reforestation (eucalyptus) 16.50 (±4.1) 4.42 (±2.0) pasture 17.70 (±1.6) 4.22 (±0.7) agriculture 12.93 (±2.9) 2.53 (±0.9) table 1. average species richness ± confidence intervals for species richness of each taxon per transect in each land-use type. orchid bees we sampled 3,769 orchid bees of 36 species, belonging to four of the five known genera in the group. out of the total, 34 species could be identified to species level. only one species of eufriesea and one eulaema were assigned to morphospecies. the complete list of species is available in table 3. species accumulation curves were near asymptotic (fig 2b), and we sampled 87% of the total species richness estimated by 1st the order jackknife estimator. sociobiology 63(3): 925-940 (september, 2016) 929 groups in the same study region (e.g. moura et al., 2013). however, it is worth highlighting here that in a detailed analysis of ant responses to land-use changes and forest disturbance, we observed more subtle patterns of diversity within and between the major land-use types (solar et al., 2016). studies on ants and other groups have been showing that the recovery of species diversity in disturbed forests is not guaranteed, even when considering samples conducted relatively mature secondary forests on average (wilkie et al., 2009; barlow et al., 2016; solar et al., 2016). by contrast the orchid bees exhibited similar levels of richness in all forest types, with highest richness in secondary forests. this is an expected result, considering orchid bees have high vagility, being able to fly several kilometers a day (janzen, 1971), which in turn could cause high rates of spillover. on the other hand, they may rapidly colonize new habitats, and may persist in small forest patches (nemesio & silveira, 2007, 2010). nevertheless, orchid bees are seriously affected by deforestation and forest fragmentation (nemesio & silveira, 2010) and forest-dependent species are seriously threatened; see nemesio (2013) for case study in the atlantic forest. open areas are often the least hospitable environments (gascon et al., 1999), and are commonly dominated by generalist species. this is the case of the orchid bee eulaema nigrita lepeletier de saint fargeau, 1841. this species is an example of a non-forest species, which can be rarely recorded in forest fragments but is highly abundant in open areas. compared with eu. nigrita, all other species in pasture transects had a relative occurrence frequency of less than 1%. conservation implications enhanced documentation of local diversity patterns of insects and other organisms are invaluable in helping to assess conservation priorities and assess management effectiveness. indeed, it would be highly desirable to develop conservation strategies or conclusions taking into account a more comprehensive understanding of diversity and distribution of the major groups of organisms inhabiting a given locality. we hope this assessment provides the baseline for new community and population studies on these groups of insects in the region. paragominas is the flagship municipality in the state of para for the green municipalities program (in portuguese, programa municípios verdes – http://municipiosverdes.com.br/), an initiative aiming to stop deforestation and promote secondary forest recovery and sustainable land-use practices in the region (viana et al. 2016). we suggest therefore this study and the patterns of species distributions can be used as baselines for future studies of forest changes in that region to assess the conservation success of the program. acknowledgements we are indebted to the invaluable dedication of our field assistants and support received from the farmers and community of paragominas. we are grateful to dr. rodrigo feitosa, dr. andré nemésio; thalyane a. moura; lívia p. prado and mônica a. ulysséa for their invaluable help in species identification. we are grateful to alexander c. lees, ricardo campos, tathiana sobrinho and frederico neves, for their suggestions in the first draft of this manuscript. alexander c. lees revised english grammar and spelling. we received financial support from instituto nacional de ciência e tecnologia – biodiversidade e uso da terra na amazônia (cnpq 574008/2008-0), empresa brasileira de pesquisa agropecuária – embrapa (seg:02.08.06.005.00), the uk government darwin initiative (17-023), the nature conservancy, and natural environment research council (nerc) (ne/f01614x/1 and ne/g000816/1). rrcs is funded by capes/pnpd. jhs is funded by cnpq. this is the contribution number 51 of the sustainable amazon network (http://www.redeamazoniasustentavel.org). land-use type species author pfu pfl pflb sef ref pas agr amblyoponinae prionopelta antillana forel, 1909 1 dolichoderinae azteca alfari emery, 1893 1 1 azteca chartifex emery, 1896 1 azteca ovaticeps forel, 1904 2 azteca pgm-03 1 azteca aurita emery, 1893 1 dolichoderus bispinosus (olivier, 1792) 2 1 2 dolichoderus decollatus smith, 1858 2 dolichoderus gagates emery, 1890 1 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. http://www.redeamazoniasustentavel.org rrc solar et al. – paragominas ants and orchid-bees checklist.930 land-use type species author pfu pfl pflb sef ref pas agr dolichoderinae dolichoderus imitator emery, 1894 1 dolichoderus lutosus (smith, 1858) 1 dolichoderus varians mann, 1916 2 dorymyrmex cf. goeldii forel, 1904 6 3 dorymyrmex pgm-01 1 1 2 15 9 7 dorymyrmex pgm-02 1 1 1 dorymyrmex spurius santschi, 1929 2 1 3 9 15 12 forelius pgm-01 2 gracilidris pombero wild & cuezzo, 2006 1 1 6 41 3 linepithema neotropicum wild, 2007 2 12 1 tapinoma melanocephalum (fabricius, 1793) 1 4 2 2 1 1 tapinoma pgm-01 1 1 dorylynae acanthostichus laticornis forel, 1908 1 cerapachys splendens borgmeier, 1957 1 eciton burchellii (westwood, 1842) 1 1 1 eciton mexicanum roger, 1863 1 eciton rapax smith, 1855 1 labidus coecus (latreille, 1802) 1 5 9 2 4 18 labidus mars (forel, 1912) 1 2 labidus praedator (smith, 1858) 2 1 2 labidus spininodis (emery, 1890) 3 2 neivamyrmex gibbatus borgmeier, 1953 1 neivamyrmex pgm-01 1 neivamyrmex pgm-02 1 1 nomamyrmex esenbecki (westwood, 1842) 1 1 ectatomminae ectatomma brunneum smith, 1858 7 32 32 3 154 19 ectatomma edentatum roger, 1863 1 4 8 1 6 ectatomma lugens emery, 1894 29 88 88 13 1 ectatomma tuberculatum (olivier, 1792) 6 11 2 gnamptogenys acuminata (emery, 1896) 1 5 gnamptogenys haenschi (emery, 1902) 1 1 gnamptogenys horni (santschi, 1929) 1 1 gnamptogenys aff. mecotyle brown, 1958 1 gnamptogenys moelleri (forel, 1912) 5 18 11 1 gnamptogenys striatula mayr, 1884 1 16 13 1 gnamptogenys gr. striatula pgm-02 3 gnamptogenys sulcata (smith, 1858) 1 1 gnamptogenys tortuolosa (smith, 1858) 3 6 6 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) sociobiology 63(3): 925-940 (september, 2016) 931 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr formicinae acropyga goeldii forel, 1893 1 brachymyrmex pgm-01 1 1 3 brachymyrmex pgm-02 3 1 11 42 25 brachymyrmex pgm-03 1 brachymyrmex pgm-04 1 9 7 1 brachymyrmex pgm-05 1 1 brachymyrmex pgm-06 1 brachymyrmex pgm-07 1 camponotus ager (smith, 1858) 1 1 camponotus atriceps (smith, 1858) 5 36 39 21 camponotus aff. balzani (emery, 1894) 1 2 1 camponotus blandus (smith, 1858) 1 8 7 52 1 camponotus crassus mayr, 1862 1 2 camponotus femoratus (fabricius, 1804) 1 1 camponotus leydigi forel, 1866 1 3 1 camponotus novogranadensis mayr, 1870 2 1 4 7 0 camponotus renggeri emery, 1894 1 3 9 2 21 2 camponotus senex (smith, 1858) 1 5 1 2 58 4 camponotus sexguttatus (fabricius, 1793) 1 5 camponotus pgm-03 1 24 7 camponotus pgm-04 14 17 11 1 camponotus pgm-05 2 1 camponotus pgm-08 2 12 13 12 4 8 camponotus pgm-12 1 camponotus pgm-14 1 1 camponotus pgm-15 1 gigantiops destructor (fabricius, 1804) 5 11 1 nylanderia pgm-01 1 nylanderia pgm-02 1 22 24 12 4 5 nylanderia pgm-03 7 12 7 1 nylanderia pgm-04 2 1 6 3 55 3 nylanderia pgm-05 13 63 69 22 1 4 2 nylanderia pgm-06 3 nylanderia pgm-07 5 5 6 1 nylanderia pgm-08 3 2 2 3 2 nylanderia pgm-09 1 nylanderia pgm-10 1 1 nylanderia pgm-11 1 1 paratrechina longicornis (latreille, 1802) 2 3 rrc solar et al. – paragominas ants and orchid-bees checklist.932 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr myrmicinae acromyrmex coronatus (fabricius, 1804) 1 acromyrmex laticeps (emery, 1905) 1 apterostigma carinatum latke, 1997 2 6 5 2 1 apterostigma pgm-02 1 apterostigma pgm-03 1 1 atta cephalotes (linnaeus, 1758) 7 4 atta sexdens (linnaeus, 1758) 2 1 4 5 7 cardiocondyla emeryi forel, 1881 1 8 1 cardiocondyla minutior forel, 1899 1 1 3 carebara brevipilosa fernández, 2004 1 4 2 1 carebara inca fernández, 2004 1 carebara urichi (wheeler, 1922) 3 5 8 1 1 1 carebara escherichi complex pgm-03 2 2 4 4 carebara lignata complex pgm-01 2 1 3 cephalotes atratus (linnaeus, 1758) 2 1 cephalotes cordatus (smith, 1853) 1 cephalotes maculatus (smith, 1876) 1 cephalotes oculatus (spinola, 1851) 3 cephalotes pusillus (klug, 1824) 1 3 crematogaster brasiliensis mayr, 1878 1 28 43 4 2 crematogaster curvispinosa mayr, 1862 1 crematogaster erecta mayr, 1866 2 2 6 1 crematogaster flavosensitiva longino, 2003 1 6 1 crematogaster levior longino, 2003 4 crematogaster limata smith, 1858 1 5 15 7 1 crematogaster aff. victima smith, 1858 1 crematogaster sotobosque longino, 2003 2 2 2 crematogaster tenuicula forel, 1904 32 55 13 7 1 1 crematogaster pgm-01 1 crematogaster pgm-02 1 crematogaster pgm-03 3 5 6 17 98 25 crematogaster pgm-04 1 crematogaster pgm-05 1 1 5 3 11 crematogaster pgm-06 2 12 cyphomyrmex pgm-01 1 1 9 3 2 17 3 cyphomyrmex gr. rimosus pgm-02 2 1 1 cyphomyrmex gr. rimosus pgm-03 1 2 1 cyphomyrmex laevigatus weber, 1938 1 1 1 cyphomyrmex rimosus (spinola, 1851) 1 1 1 hylomyrma pgm-01* 1 sociobiology 63(3): 925-940 (september, 2016) 933 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr myrmicinae megalomyrmex gr. leoninus pgm-01 5 megalomyrmex gr. silvestrii pgm-02 1 1 megalomyrmex gr. silvestrii pgm-03 1 monomorium floricola (jerdon, 1851) 1 1 1 mycocepurus smithii (forel, 1893) 1 1 8 4 2 3 3 myrmicocrypta bucki sosa-calvo & schultz, 2010 1 myrmicocrypta foreli mann, 1916 2 1 nesomyrmex spininodis (mayr, 1887) 1 ochetomyrmex neopolitus fernández, 2003 2 1 1 3 ochetomyrmex semipolitus mayr, 1878 1 15 3 2 octostruma iheringi (emery, 1888) 2 octostruma balzani (emery, 1888) 1 oxyepoecus inquilinus (kusnezov, 1952) 2 pheidole pgm-01 2 35 33 3 14 97 28 pheidole pgm-02 2 9 3 11 38 15 pheidole pgm-03 2 1 1 pheidole pgm-04 1 4 9 1 1 pheidole pgm-05 3 1 13 pheidole pgm-06 11 17 5 6 1 3 pheidole pgm-07 2 12 13 9 3 5 1 pheidole pgm-08 5 5 7 9 27 11 pheidole pgm-09 1 pheidole pgm-10 1 2 1 1 pheidole pgm-11 1 14 11 2 pheidole pgm-12 4 7 pheidole pgm-13 1 14 26 7 7 6 14 pheidole pgm-14 2 7 3 pheidole pgm-15 8 21 16 6 1 pheidole pgm-16 5 6 2 6 1 pheidole pgm-17 1 2 1 4 pheidole pgm-18 1 pheidole pgm-19 3 11 7 pheidole pgm-20 2 18 6 5 1 pheidole pgm-21 1 pheidole pgm-22 3 2 pheidole pgm-23 1 2 pheidole pgm-24 1 2 3 pheidole pgm-25 6 7 9 2 1 pheidole pgm-26 2 pheidole pgm-27 3 13 1 4 rrc solar et al. – paragominas ants and orchid-bees checklist.934 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr myrmicinae pheidole pgm-28 1 1 pheidole pgm-29 1 1 pheidole pgm-30 3 13 3 3 pheidole pgm-31 3 4 3 pheidole pgm-32 6 14 32 8 2 2 pheidole pgm-33 8 34 63 26 11 23 13 pheidole pgm-34 23 1 9 27 7 9 3 pheidole pgm-35 2 pheidole pgm-36 1 pheidole pgm-37 2 pheidole pgm-38 2 pheidole pgm-39 1 pheidole pgm-40 1 2 2 2 16 6 pheidole pgm-41 1 1 pheidole pgm-42 1 pheidole pgm-43 1 1 9 4 2 18 5 pheidole pgm-44 1 pheidole pgm-45 1 pheidole pgm-46 1 pheidole pgm-47 2 1 pheidole pgm-48 1 pheidole pgm-49 1 6 21 13 8 19 8 pheidole pgm-50 3 5 1 1 pheidole pgm-51 2 2 pheidole pgm-52 1 7 3 3 7 1 pheidole pgm-53 1 pheidole pgm-54 1 3 1 pheidole pgm-55 1 2 1 pheidole pgm-56 1 1 pheidole pgm-57 1 pheidole pgm-58 9 5 15 pheidole pgm-59 3 3 pheidole pgm-60 1 pheidole pgm-61 1 1 2 pheidole pgm-62 1 pheidole pgm-63 3 3 1 pheidole pgm-64 1 2 pheidole pgm-65 1 pogonomyrmex naegelii emery, 1878 3 26 sericomyrmex cf. parvulus forel, 1912 2 6 3 6 sociobiology 63(3): 925-940 (september, 2016) 935 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr myrmicinae sericomyrmex pgm-01 2 17 45 9 1 sericomyrmex pgm-02 1 3 1 sericomyrmex pgm-03 1 2 1 solenopsis geminata (fabricius, 1804) 8 33 43 27 49 27 solenopsis globularia (smith, 1858) 1 2 5 3 15 61 33 solenopsis invicta buren, 1972 8 2 22 8 95 22 solenopsis succinea emery, 1890 1 solenopsis virulens (smith, 1858) 2 4 6 solenopsis pgm-01 2 solenopsis pgm-02 6 42 45 11 6 6 5 solenopsis pgm-03 2 3 2 1 44 11 solenopsis pgm-04 7 44 37 12 4 solenopsis pgm-05 2 1 4 solenopsis pgm-06 1 22 21 9 1 1 solenopsis pgm-07 3 8 24 6 solenopsis pgm-08 8 12 17 4 2 solenopsis pgm-09 5 22 37 18 3 26 3 solenopsis pgm-10 1 1 1 1 solenopsis pgm-11 4 4 1 solenopsis pgm-12 3 1 solenopsis pgm-13 4 44 31 6 4 3 solenopsis pgm-14 5 3 1 2 15 solenopsis pgm-16 3 5 12 3 3 5 5 solenopsis pgm-17 3 solenopsis pgm-19 1 13 15 6 2 5 solenopsis pgm-20 1 1 solenopsis pgm-21 3 solenopsis pgm-22 4 1 solenopsis pgm-23 1 strumigenys auctidens (brown, 1959) 2 2 2 strumigenys beebei (wheeler, 1915) 1 1 strumigenys carinithorax borgmeier, 1934 1 strumigenys denticulata mayr, 1887 3 4 2 3 strumigenys eggersi emery, 1894 1 1 1 strumigenys elongata roger, 1863 1 strumigenys epinotalis (weber, 1934) 1 strumigenys grytava (bolton, 2000) 2 2 1 3 1 strumigenys infidelis santschi, 1919 4 2 strumigenys louisianae roger, 1863 3 strumigenys aff. perparva brown, 1958 strumigenys subedentata mayr, 1887 1 strumigenys urrhobia (bolton, 2000) 1 trachymyrmex bugnioni (forel, 1912) 9 13 5 trachymyrmex pgm-01 5 43 34 5 2 rrc solar et al. – paragominas ants and orchid-bees checklist.936 table 2. list of ant species collected in this study. values represent number of records per pitfall traps of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. (continuation) land-use type species author pfu pfl pflb sef ref pas agr myrmicinae trachymyrmex pgm-02 1 7 12 3 trachymyrmex pgm-03 1 1 wasmannia auropunctata (roger, 1863) 12 52 9 36 11 81 8 xenomyrmex pgm-01* 1 paraponerinae paraponera clavata smith, 1858 2 ponerinae anochetus horridus kempf, 1964 1 1 anochetus diegensis forel, 1912 1 dinoponera gigantea (perty, 1833) 11 52 65 9 1 hypoponera pgm-01 1 2 1 hypoponera pgm-02 2 hypoponera pgm-03 1 hypoponera pgm-04 1 leptogenys gaigei wheeler, 1923 1 4 mayaponera constricta (mayr, 1884) 6 13 26 7 3 2 neoponera apicalis (latreille, 1802) 12 68 51 neoponera commutata (roger, 1860) 2 neoponera verenae (forel, 1922) 4 5 56 7 1 1 odontomachus bauri emery, 1892 7 21 2 7 11 2 odontomachus brunneus (patton, 1894) 2 4 8 1 1 2 odontomachus caelatus brown, 1976 1 odontomachus haematodus (linnaeus, 1758) 6 5 1 odontomachus meinerti forel, 1905 8 2 1 odontomachus yucatecus brown, 1976 1 odontomachus pgm-01 1 pachycondyla crassinoda (latreille, 1802) 19 48 39 5 3 pachycondyla harpax (fabricius, 1804) 2 65 68 12 1 1 pachycondyla impressa (roger, 1861) 5 pachycondyla aff. purpurascens forel, 1899 1 platythyrea sinuata (roger, 1860) 1 rasopone arhuaca (forel, 1901) 1 pseudomyrmecinae pseudomyrmex termitarius (smith, 1855) 2 4 11 7 133 9 pseudomyrmex gr. gracillis pgm-01 1 pseudomyrmex pgm-04 1 1 pseudomyrmex gr. ocullatus pgm-03 1 6 2 pseudomyrmex gr. pallidus pgm-02 2 * new species sociobiology 63(3): 925-940 (september, 2016) 937 land-use type species author pfu pfl pflb sef ref pas agr eufriesea auripes (gribodo, 1882) 2 1 eufriesea ornata (mocsáry, 1896) 6 eufriesea pulchra (smith, 1854) 1 4 1 3 eufriesea pgm-01 1 2 eufriesea surinamensis (linnaeus, 1758) 2 1 5 4 euglossa amazonica dressler, 1982 14 59 106 33 19 36 2 euglossa augaspis dressler, 1982 7 24 15 8 3 8 euglossa bidentata dressler, 1982 1 3 3 2 1 euglossa cordata friese, 1923 6 16 20 8 5 30 2 euglossa chalybeata friese, 1925 6 16 23 6 1 2 1 euglossa cognata moure, 1970 4 4 10 euglossa crassipunctata moure, 1968 3 7 3 1 1 1 euglossa decorata smith, 1874 1 euglossa despecta moure, 1968 1 6 30 24 1 17 euglossa ignita smith, 1874 15 23 1 1 1 euglossa imperialis cockerell, 1922 16 77 102 16 6 8 6 euglossa intersecta audouin, 1824 1 16 19 7 2 1 euglossa laevicincta dressler, 1982 1 1 euglossa liopoda dressler, 1982 6 20 21 4 16 6 euglossa aff. mixta friese, 1899 4 29 36 21 2 3 euglossa modestior dressler, 1982 6 12 17 12 6 40 6 euglossa orellana roubik, 2004 5 30 12 8 1 1 euglossa parvula dressler, 1982 2 17 1 euglossa pgm-01 1 3 4 1 euglossa townsendi cockerell, 1904 26 84 113 109 3 7 3 euglossa variabilis friese, 1899 10 2 1 5 eulaema bombiformis (packard, 1869 ) 20 26 28 14 6 10 2 eulaema cingulata (fabricius, 1804) 5 16 27 2 40 1 eulaema marcii nemésio, 2009 4 9 14 2 13 1 eulaema meriana (olivier, 1789) 19 83 65 38 6 44 21 eulaema mocsaryi (friese, 1899) 3 8 10 eulaema nigrita lepeletier de saint fargeau, 1841 24 16 29 35 1010 231 eulaema pseudocingulata oliveira, 2006 1 exaerete frontalis (guérin-méneville, 1844) 13 24 13 5 2 2 exaerete lepeletieri oliveira & nemésio, 2003 1 4 exaerete smaragdina (guérin-méneville, 1844) 1 11 15 2 3 table 3. list of orchid bees species collected in this study. values represent the number of individuals of each species in each land-use type: pfu – primary forest undisturbed, pfl – primary forest logged, pflb – primary forest logged and burnt, sef – secondary forest, ref – reforestation with commercial species, pas – pasture, agr – agricultural areas. rrc solar et al. – paragominas ants and orchid-bees checklist.938 references abrahamczyk, s., de vos, j.m., sedivy, c., gottleuber, p., and kessler, m. 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old nests can be important for the population dynamics of some species of plants that benefit from a more fertile soil for seed germination and seedling development (ackerman et al., 2007; traoré et al., 2008; beaudrot et al., 2011); ii) termite nests serve as shelter abstract termites play an important role as ecosystem engineers in many tropical environments, acting as herbivore-detritivore organisms and strongly influencing vegetation structure and composition by modifying soil properties, providing nutrients by recycling the organic matter, and direct feeding on plants, notably in the cerrado (savanna) of brazil. to evaluate the intensity of termite foraging on cerrado plants, we recorded plants higher than 25 cm, which exhibited termite activity along nine transects (2 x 50m), at the estação ecológica de pirapitinga (eep), in the state of minas gerais, brazil. we recorded the height, basal area, and identified the species of each plant. simultaneously, we used cellulose baits disposed at each 10 m along six transects of 100 m to sample termites in this area, which was protected from fire for at least 40 years. twelve species of termites were recorded. termite foraging on cerrado plant species varied considerably and it was influenced by several factors including plant height and host species. taller plants presented more termites than smaller plants, probably due to the amount of available resources (for nesting and feeding) for the termites. sociobiology an international journal on social insects gw fernandes1, sl murcia1, jc santos2, o desouza3, r constantino4, i haifig2 article history edited by paulo cristaldo, ufs, brazil received 22 june 2017 initial acceptance 31 august 2017 final acceptance 24 october 2017 publication date 30 march 2018 keywords plant-insect interactions, isoptera, herbivory, bauhinia brevipes. corresponding author geraldo wilson fernandes ecologia evolutiva & biodiversidade/dbg icb/universidade federal de minas gerais cp 486, cep 30161-970 belo horizonte, mg, brasil. e-mail: gw.fernandes@gmail.com and nesting sites for many species of animals, including other termite species (marins et al., 2016); iii) many animals depend on termites for nutrition (redford & dorea, 1984); and iv) several symbiont organisms occur associated only with certain termite species (spain & mcivor, 1988; whitford, 1991; lavelle, 1997; cristaldo et al., 2012). termites feed primarily on cellulose and substances derived from the degradation of the cellulose and lignin, found in a wide variety of dead or live plant tissues, complete or partially decomposed material, such as leaves, seeds, wood, roots, litter, humus and feces (reviewed in lima & costaleonardo, 2007). some species, however, may specialize on a certain subset of resources and may attack live plant parts (see sands, 1969; lee & wood, 1971). they feed on roots or dig galleries in the trunk, occasionally causing the death of the plant (rao et al., 2000; sileshi et al., 2005). frequently, an attack starts on the dead tissues and later expands to the 1 universidade federal de minas gerais, belo horizonte-mg, brazil 2 universidade federal de uberlândia, uberlândia-mg, brazil 3 universidade federal de viçosa, viçosa-mg, brazil 4 universidade de brasília, brasília-df, brazil research article termites mailto:gw.fernandes@gmail.com sociobiology 65(1): 48-58 (march, 2018) special issue 49 live plant parts (costa lima, 1938). attacks to plant stems may also be related to damage inflicted by fungi (wood et al., 1980). some studies have also established links between termite attacks on plants and low soil fertility (wardell, 1987) or water stress suffered by plants (rao et al., 2000; van den berg & riekert, 2003). the dynamics, ontogeny and architecture of the aerial plant parts play a crucial role on the abundance and structure of their associated insect communities (lawton, 1983; strong et al., 1984; fonseca et al., 2006). generally, larger trees support higher richness and abundance of insects than smaller trees (price, 1997; basset, 2001; barrios, 2003). although termite preferences for cerrado species varies widely, plant morphological traits such as size, height and trunk circumference are thought to play important roles on the insect activities (gonçalves et al., 2005; lima-ribeiro et al., 2006; araújo et al., 2010). specifically for constrictotermes cyphergaster (silvestri), the diameter and inclination of the stem, branching density, tree height, and soil type affect the colonization, establishment, nest shape and colony size in the cerrado (cunha, 2000; lima-ribeiro et al., 2006). here, we evaluated the effects of plant species and plant dimensions (measured as height and basal area) on the intensity of termite foraging and provided the first survey of termite species in the estação ecológica de pirapitinga (epp). we tested the hypothesis that there is a positive relationship between termite intensity of foraging and the dimension of the structures of trees in the eep. such hypothesis was based on the findings by gonçalves et al. (2005), lima-ribeiro et al. (2006), araújo et al. (2010) and leite et al. (2011) that have shown that larger trees may present higher quantity and availability of resources for their associated termite species. materials and methods study area this study was carried out in an area of cerrado vegetation belonging to the estação ecológica de pirapitinga (eep). the eep is an island formed by the creation of the dam of três marias in the são francisco river in 1965. the island has 1090 ha in area and it is located between 18º20’s 18º23’s and 45º17’w – 45º20’w, at an altitude of 570 to 630 m above sea level (azevedo et al., 1987), in the municipality of morada nova de minas, in the state of minas gerais, southeastern brazil. in some years, during the dry season, the eep may form a peninsula and be reconnected to the surrounding vegetation. the vegetation in the station can be assigned to three main cerrado physiognomies: campo sujo, cerrado sensu stricto, and cerradão (gonçalves-alvim & fernandes, 2001). this study has been conducted in an area of cerrado sensu stricto. such a physiognomy is characterized in the station by low stature trees (3-6 m height), that present thick bark and sclerophyllous leaves (oliveira & marquis, 2002). climate is tropical, fitting to aw type in köppengeiger’s classification, bearing a long dry season (up to four months) between may and september (gonçalves-alvim & fernandes, 2001) and a rainy season between october and april. average monthly temperature varies between 20.9 and 25.1oc, and the average annual rainfall is 1.222 mm. heavily clayish, dystrophic dark read, oxysols dominate the gently undulated topography. some areas, however, present dystrophic to moderate cambisols in undulated to strongly undulated topography (azevedo et al., 1987). termite sampling using baits to survey termite fauna in eep, termite sampling followed a method modified from la fage et al. (1973). six 100 m long transects were randomly allocated in the field, within which unscented toilet paper rolls (10 cm wide × 30 m long, forming a 10 cm ø roll) were regularly distributed at each 10 m. soil surface was brushed off before receiving the paper rolls, assuring full contact of the bait to the soil. baits were covered by a black polyethylene bag (20 × 33 cm) and held in place by an inverted “u” wire inserted through the bait until 15 cm deep in the soil. empty and washed pet (polyethylene terephthalate) bottles, 2 l in volume, cut in halves perpendicular to the main axis were placed covering the bait for improved protection. rolls thereby arranged had their bottom surface in full contact with the soil. in addition to termite sampling using baits, as mentioned above, samples in the interior of the trunk of five plant individuals subjected to strong attack by termites were performed to complement the species sampling. sampling was carried out from may to august 2005 (what corresponds to the dry period in the station). termites were collected 95 days after the beginning of the experiment. in a pilot assay with another set of baits, this period corresponded to the time needed to detect the total (100%) consumption of the first bait by termites. in case the bait was totally consumed, termite attack was identified by indirect evidence, such as dead corpses of termites. baits were taken to the laboratory to quantify termites by direct count of individuals, preservation in 70% ethanol, and identification to species. plant species survey plant sampling was carried out from may to september 2005. to evaluate the effect of total tree height, basal area, and plant species on intensity of attack by termites, nine transects (2 m wide × 50 m long) were randomly marked in the field. within each transect, the above parameters were measured in every plant above 25 cm high. assessment of termite foraging on living plants termite foraging was categorized into four levels considering the intensity of attack (ia0, ia1, ia2, and ia3; see table 1) through the colonization process and injuries inflicted gw fernandes et al. – effects of tree height on termite foraging50 to the plant (modified from anonymous, 1989). every plant was visually inspected to record presence/absence of termites or their signs (galleries or nests). stems having termites and bearing injuries clearly attributable to them were evaluated at several positions on the plant in order to record whether the injury was deeper than the bark thickness (fig 1). an additional survey of termites in heavily attacked plants was performed aiming to confirm termite attacks. statistical analyses in order to know whether sampling effort was enough to quantify plant species, we calculated the estimators chao1 (based on abundance) and chao2 (based on incidence) using estimates version 7.5.0 (colwell, 2005). plant basal area was estimated using the general equation for the area of a circle [a= (πd²)/4; where a is the basal area, π is the value of the constant pi, and d is the stem diameter at the point where the plant touches the soil surface]. to guarantee accurate estimation of effects within plant species, statistical analysis has been conducted using a sub-set fig 1. schematic illustration of the categories of intensity of attack (ia) by termites on plants of cerrado in the estação ecológica de pirapitinga minas gerais. ia1 presence of termites with no attack – termite nests constructed around a plant stem, but without evidence of attack or gallery construction. ia2 attack limited to the bark gallery construction on a branch. displaying the damage to the bark. ia3 severe attack transversal section of one trunk severely damaged in its internal structure and still with presence of termites. intensity of attack (ia) description ia0 : absence of termites plants do not present evidences of termites, without any constructed structures and/or damaged parts attributable to the attack of termites. ia1 : presence of termites without attack plants with presence of termites and/or active structures of termites, but without damaged parts attributable to their attack. ia2 : attack limited to the bark. plants with termites and superficial damage limited to the bark attributable to the attack of termites. ia3 : severe attack plants with termites and attack visibly crossing the bark and the central part of trunk (sapwood/ heartwood). they could present trunk and branches very damaged or deceased, and leaf withering. plants can still present new shoots coming out of the root. table 1. categories of attack intensity by termites on the cerrado plants in the estação ecológica de pirapitinga minas gerais. adapted from anonymous (1989). of the nine most abundant plant species, among the 51 recorded along the transects. each of these nine plant species presented a minimum of 20 individuals, and represented as a whole, 60.33 % of all plant individuals recorded. to check whether termite attack intensity (y-var) correlated with plant traits (x-vars: height, basal area and plant identity), we performed multiple regressions under the generalized linear modelling (r development core team, 2006). the full model, therefore, was: ia ~ species + height + basalarea + species:height + species:basalarea + species:height:basalarea; where ia = intensity of attack, species = plant species identity, height = plant height, basalarea = area of plant stem where it touches the soil surface. model simplification was achieved contrasting a complex model with an alternative simpler one built by removing variables and interactions, and merging levels within categorical variables, as long as such a simplification did not cause significant (p < 0.05) changes in the model. every time a simplification was accepted, the resulting model was then contrasted again with an even simpler model and hence consecutively until a minimal adequate model was achieved. this minimal model was composed only by significant terms. residuals were inspected to check for the suitability of the model and error distribution. the categorical variable species, initially composed of nine levels (each of the nine most abundant plant species included in the analysis), was grouped following recommendations by crawley (2007). after calculating parameter values for each of the species, we ordered these parameters from smaller to larger, creating a simpler model in which the variable species entered with its levels merged in groups according to the significance of their parameters. to do such a merging, the levels presenting adjacent parameter values were amalgamated in a single level and the resulting model was contrasted with the previous and more complex one. this was done successively until merging caused significant chances (p < 0.05) in the simpler model. at this point, a species group was defined. merging proceeded, creating a second grouping of plant species merging the subsequent levels according to proximity of their parameter values and contrasting the resulting models as before. this proceeded until no further grouping was possible without causing significant changes. sociobiology 65(1): 48-58 (march, 2018) special issue 51 results the termite survey revealed the presence of 12 termite species in the eep, distributed in two families (table 2). seven species were recorded on the toilet paper baits while eight species were recorded in the tree trunks. four termite species were only recorded on the baits while five species were only found by manually collection inside tree trunk samples of five plants arbitrarily selected and strongly attacked (table 2). nasutitermes aff. coxipoensis was the most abundant species collected on the toilet paper baits, composing more than half of the individuals collected, while heterotermes sulcatus was the second most abundant species, with 35% of the individuals collected (table 2). the distance between the observed accumulation curve for plant species and the curves for estimators chao1 and chao2 were, respectively, 7.2 and 3.6 (fig 2). such values indicate that 88 to 93% of the local arboreal and shrubby plant species were effectively recorded by the sampling procedure and that, as shown in fig 2, a considerable effort would be needed in order to improve this sampling. a total of 713 plant individuals belonging to 51 species and 28 families were surveyed (table 3). among plant individuals, 49% did not show signs of termite presence family/species feeding group collection number of individuals (%) percent of records in baitsbaits tree trunks rhinotermitidae heterotermes sulcatus mathews 3. xyl x 8,437 (34.7%) 19.0% termitidae subfamily nasutitermitinae constrictotermes cyphergaster (silvestri) spe* x 42 (0.2%) 1.7% nasutitermes aff. coxipoensis (holmgren) lit x x 13,363 (54.9%) 43.1% nasutitermes kemneri snyder & emerson xyl x parvitermes bacchanalis mathews lit x 768 (3.2%) 6.9% velocitermes sp. lit x x 1,049 (4.3%) 15.5% velocitermes cf. paucipilis mathews lit x subfamily syntermitinae cornitermes silvestrii emerson lit x 526 (2.2%) 12.1% rhynchotermes nasutissimus (silvestri) lit x silvestritermes euamignathus (silvestri) int x x 143 (0.6%) 1.7% subfamily termitinae microcerotermes strunckii (sörensen) xyl x termes nigritus (silvestri) int x total 7 8 24,328 (100%) 100% * according to our observations, c. cyphergaster forages on trees, scraping the surface and feeding mostly on tree barks, soft dead plant tissue, and lichens. it does not feed on wood and is not a litter-feeder. table 2. termite species found in the cerrado of the estação ecológica de pirapitinga and their relative abundance and frequency in baits and in tree trunks. feeding groups (according to constantino 2015): xyl = xylophagous, wood-feeder; lit = litter-feeder; int = intermediate (species that feeds on wood and litter); spe* = specialized feeder. fig 2. plant species accumulation according to chao1 and chao2 estimators and observed accumulation of species of plants in the cerrado of the estação ecológica de pirapitinga minas gerais (see text for details). axis y (species number) was interrupted in interval 60 245 for better visualization of the figure. gw fernandes et al. – effects of tree height on termite foraging52 family species ab height (m) basal area (cm2) annonaceae annona sp. 36 (5.0%) 2.65 ± 0.30 29.03 ± 7.57 duguetia furfuraceae 15 (2.1%) 1.43 ± 0.12 4.87 ± 1.33 rollinia sp. 8 (1.1%) 2.47 ± 0.25 6.77 ± 1.35 xylopia aromatica 28 (3.9%) 3.43 ± 0.57 31.82 ± 9.29 apocynaceae aspidosperma tomentosum 5 (0.7%) 2.35 ± 1.05 41.48 ± 35.47 araliaceae schefflera macrocarpa 11 (1.5%) 2.46 ± 0.53 27.23 ± 6.35 asteraceae piptocarpha cf. rotundifolia 1 (0.1%) 2.35 6.16 piptocarpha rotundifolia 7 (1.0%) 3.73 ± 0.69 25.74 ± 7.71 bignoniaceae tabebuia ochracea 3 (0.4%) 3.85 ± 2.98 11.58 ± 1.66 bombacaceae eriotheca gracilipes 14 (2.0%) 3.78 ± 0.86 40.83 ± 16.67 burseraceae protium cf. brasiliense 2 (0.3%) 1.70 ± 0.41 2.32 ± 0.55 clusiaceae kielmeyera coriacea 9 (1.3%) 3.35 ± 0.86 84.57 ± 56.10 dilleniaceae davilla rugosa 6 (0.8%) 1.17 ± 0.18 4.22 ± 1.82 ebenaceae diospyros sp. 8 (1.1%) 5.69 ± 0.80 60.50 ± 22.14 erythroxylaceae erythroxylum tortuosum 1 (0.1%) 2.30 27.34 fabaceae acosmium dasycarpum 9 (1.3%) 5.52 ± 1.75 91.20 ± 40.82 bauhinia brevipes 31 (4.3%) 2.32 ± 0.27 7.00 ± 0.67 dimorphandra mollis 19 (2.7%) 2.11 ± 0.55 11.32 ± 4.83 enterolobium gummiferum 1 (0.1%) 2.67 8.77 hymenaea stigonocarpa 5 (0.7%) 4.66 ± 1.37 60.39 ± 24.65 pterodon emarginatus 20 (2.8%) 5.23 ± 0.81 98.47 ± 43.13 stryphnodendron adstringens 8 (1.1%) 3.51 ± 0.96 41.30 ± 14.91 flacourtiaceae casearia cf. arborea 18 (2.5%) 1.70 ± 0.24 6.81 ± 1.47 loganiaceae strychnos sp. 2 (0.3%) 6.88 ± 5.13 13.78 ± 12.01 malpighiaceae banisteriopsis sp. 5 (0.7%) 3.13 ± 1.41 16.31 ± 9.51 byrsonima cf. sericea 7 (1.0%) 1.82 ± 0.32 14.69 ± 6.04 byrsonima sp. 1 (0.1%) 3.50 24.37 heteropteris sp. 5 (0.7%) 4.59 ± 1.05 34.87 ± 13.50 tetrapteris ramiflora 1 (0.1%) 8.00 111.91 melastomataceae miconia albicans 209 (29.3%) 1.92 ± 0.06 7.77 ± 0.87 miconia sp. 2 (0.3%) 1.88 ± 0.13 2.58 ± 0.57 monimiaceae siparuna guianensis 28 (3.9%) 2.45 ± 0.24 4.50 ± 1.25 moraceae ficus sp. 5 (0.7%) 1.31 ± 0.20 4.56 ± 1.96 nyctaginaceae neea theifera 2 (0.3%) 1.84 ± 0.17 13.02 ± 5.84 ochnaceae ouratea cf. castaneifolia 1 (0.1%) 0.75 1.99 ouratea cf. hexasperma 1 (0.1%) 3.40 13.45 ouratea semiserrata 13 (1.8%) 2.17 ± 0.39 16.47 ± 3.60 proteaceae roupala montana 9 (1.3%) 2.68 ± 0.50 39.01 ± 10.58 rubiaceae alibertia edulis 1 (0.1%) 2.50 0.97 coussarea sp. 21 (2.9%) 2.91 ± 0.49 10.76 ± 3.59 guettarda sp. 4 (0.6%) 6.13 ± 1.21 90.85 ± 39.24 tocoyena cf. formosa 11 (1.5%) 2.23 ± 0.60 14.15 ± 4.19 sapindaceae matayba mollis 16 (2.2%) 2.87 ± 0.47 18.92 ± 7.82 serjania sp. 4 (0.6%) 3.46 ± 1.45 47.29 ± 24.54 sapotaceae pouteria ramiflora 14 (2.0%) 2.22 ± 0.36 16.55 ± 4.19 pouteria torta 4 (0.6%) 4.69 ± 1.88 109.07 ± 53.02 vochysiaceae qualea multiflora 31 (4.3%) 2.52 ± 0.35 34.68 ± 9.31 qualea parviflora 6 (0.8%) 2.99 ± 0.45 21.58 ± 4.40 qualea grandiflora 26 (3.6%) 2.85 ± 0.47 58.67 ± 19.30 qualea sp1. 1 (0.1%) 3.00 55.88 qualea sp2. 18 (2.5%) 2.28 ± 0.26 22.59 ± 6.23 28 families 51 species 713 (100%) table 3. records of the plant species, with their respective abundance (ab), average height and basal area (± se) in the estação ecologica de pirapitinga minas gerais. sociobiology 65(1): 48-58 (march, 2018) special issue 53 species (family) ia0 ia1 ia2 ia3 total annona sp. (annonaceae) 10 (2.3%) 9 (2.1%) 14 (3.3%) 3 (0.7%) 36 (8.4%) pterodon emarginatus (fabaceae) 3 (0.7%) 7 (1.6%) 8 (1.9%) 2 (0.5%) 20 (4.6%) qualea grandiflora (vochysiaceae) 5 (1.2%) 7 (1.6%) 7 (1.6%) 7 (1.6%) 26 (6.0%) qualea multiflora (vochysiaceae) 3 (0.7%) 11 (2.6%) 12 (2.8%) 5 (1.2%) 31 (7.2%) bauhinia brevipes (fabaceae) 1 (0.2%) 2 (0.5%) 4 (0.9%) 24 (5.6%) 31 (7.2%) xylopia aromatica (annonaceae) 15 (3.5%) 8 (1.9%) 3 (0.7%) 2 (0.5%) 28 (6.5%) miconia albicans (melastomataceae) 179 (41.6%) 18 (4.2%) 11 (2.6%) 1 (0.2%) 209 (48.6%) siparuna guianensis (monimiaceae) 23 (5.3%) 4 (0.9%) 0 (0.0%) 1 (0.2%) 28 (6.5%) coussarea sp. (rubiaceae) 10 (2.3%) 9 (2.1%) 2 (0.5%) 0 (0.0%) 21 (4.9%) total 249 (57.9%) 75 (17.4%) 61 (14.2%) 45 (10.5%) 430 (100%) source of variation df deviance f p species group 2 231.46 238.312 <0.0001 (2.2e-16) tree height 1 19.20 38.529 <0.0001 (8.045e-10) species group: tree height 2 11.10 11.431 <0.0001 (1.462e-05) error 424 205.91 total 429 467.67 table 4. abundance and percentage of plant individuals found in the categories of termite attack intensity in the nine vegetal species represented by 20 or more individuals in the sampling in the estação ecológica de pirapitinga minas gerais. bold indicates the most observed levels of attack intensity in each group. or attack (ia = 0), 22% presented termites but did not show signs of attack (ia = 1), 16% presented signs of attack to the bark (ia = 2), and 13% presented severe attack (ia = 3) (fig 3). only nine plant species were represented by 20 or more individuals (table 3). these nine plant species were selected to perform the analyses and summed 430 individuals or 60.33% of the total of plant individuals sampled. among these, 57.91% did not show any sign of termite presence or their attack. the proportion of attacked plants decreased as attack intensity increased (table 4). the intensity of termite attack on these plants may be explained by plant height and identity. three groups of fig 3. number of individuals in each category of intensity of attack (ia) of termites in the vegetation of the cerrado of the estação ecológica de pirapitinga minas gerais. ia0: absence of termites; ia1: presence of termites without attack; ia2: attack limited to the bark; and ia3: severe attack. plants were distinguished according to the pattern of response of termite attack to plant height. these groups resulted from merging levels (plant species) within categorical variable species, as described in the material and methods section. group 1 comprised annona sp., pterodon emarginatus, qualea grandiflora and q. multiflora. group 2 was composed of solely bauhinia brevipes, and group 3 was composed of xylopia aromatica, miconia albicans, siparuna guianensis and coussarea sp. (table 5, fig 4). termite attack intensity increased as plant height increased in groups 1 and 3 while a negative trend was observed on group 2 solely formed by the shrub bauhinia brevipes. table 5. effect of height of the plant species on the intensity of attack by termites in the cerrado of the estação ecológica de pirapitinga minas gerais. the adjusted minimum model was: ia~species_group+tree_height+species_group:tree_height where species group contains three groups of species that responded differently in the presented intensity of attack: group 1 (annona sp., pterodon emarginatus, qualea grandiflora and qualea multiflora), group 2 (bauhinia brevipes), and group 3 (xylopia aromatica, miconia albicans, siparuna guianensis and coussarea sp.). df = degrees of freedom. discussion termite species the ecological station where we conducted this study was protected from fire and human disturbance for at least 40 years, but according to our results, the recorded termite fauna was similar to other cerrado physiognomies (constantino, 2005; nunes et al., 2016), including areas where fire has occurred with some frequency (desouza et al., 2003). termites feed generally on lignified parts of live plants, although roots may represent an important route to invasion of the host plant (lee & wood, 1971; waller & lafage, 1987). gw fernandes et al. – effects of tree height on termite foraging54 the xylophagy is maintained in most groups of termites, including many derived species from the subfamily nasutitermitinae (termitidae) and species of the genus heterotermes (rhinotermitidae) (mathews, 1977; engel & krishna, 2004; engel, 2011). many species of nasutitermes build their nests on host trees and have been associated with wood damages (thorne, 1980; santos, 1982; bandeira et al., 1998), but the most abundant species found in this study was n. aff. coxipoensis, which is a mound-building termite (laffont et al., 2012). it presents a wide range of food resources, including dead wood from trunks of living trees, which may be deeply excavated by these insects (laffont et al., 1998, 2012). the genus heterotermes comprises several termite species that have a status as pests, such as h. tenuis (pizano & fontes 1986; haifig et al., 2008), h. longiceps (pizano & fontes, 1986, calderon & constantino, 2007) and h. sulcatus (vasconcellos et al., 2002). this later species has been considered one of the most important xylophagous species in caatinga vegetation, where it feeds preferentially on dead wood (mélo & bandeira, 2007). in this latter environment, other important xylophagous species such as nasutitermes and microcerotermes are rare or missing (mélo & bandeira, 2004). in our study, h. sulcatus was the second most abundant termite species, therefore being responsible for a great amount of plant attack. the additional survey in the interior of plant trunks revealed eight species, and four of which were solely found there: microcerotermes strunckii, nasutitermes kemneri, rhynchotermes nasutissimus, and termes nigritus. casual observation in the site also revealed that syntermes nanus constantino, a syntermitinae, leave their subterranean nests to forage on grasses. these additional observations reinforced the relevance of the nasutitermitinae-syntermitinae group fig 4. effect of the size of plant species on the intensity of attack by termites in the cerrado of the estação ecológica de pirapitinga. the adjusted minimum model presents three distinct groups based on intensity of termite attack: group 1 – continuous line (annona sp., pterodon emarginatus, qualea grandiflora and qualea multiflora); group 2 dotted line (bauhinia brevipes); and group 3 hatched line (xylopia aromatic, miconia albicans, siparuna guianensis and coussarea sp.). in the cerrado, as these groups were particularly sampled in our studies. overall, many of the termite species may be associated to plant damage (murcia et al., unpublished data), indicating the need for more detailed studies on termite-plant association in the several cerrado physiognomies. termite-plant interactions plant height positively affected the intensity of termite attack in eight out of nine plant species, therefore corroborating the hypothesis that larger plants are more attacked than smaller ones. such a hypothesis was also corroborated by other studies that show a positive relationship between herbivore species richness and size of the host plant (e.g., lawton, 1983; strong et al., 1984; gonçalves et al., 2005). termite feeding preferences for some plant species in field studies were recorded by wood (1978), whereas gontijo (1991) and cunha (2000) provided some data on such relationships in the brazilian cerrado. our results also show a variation in termite attack to plant species that can be summarized into two general patterns: (i) for most plants, the larger they are the more they are attacked and (ii) for a single plant species, b. brevipes, the opposite trend was observed. larger plants are more attacked by termites than smaller plants possibly because: (i) they offer more food to termites (gonçalves et al., 2005), (ii) they present better possibilities for foraging activities (jones & gathorne-hardy, 1995; gonçalves et al., 2005), and/or (iii) they provide better opportunities to escape predators (neves et al., 2014; pequeno, 2017). furthermore, larger plants may constitute a better resource for tree-nesting termites. cunha (2000) showed that c. cyphergaster prefers to build its nest in trees with 71 to 286 cm2 of basal area. among the plants sampled here, only 20 individuals (p. emarginatus) presented such dimensions and, among these, 85% presented termites and 50% presented attack intensity of levels ia2 and ia3. taller plants also bear larger bark surfaces, which in consequence present greater food resources for termites (jones & gathorne-hardy, 1995). similarly, larger plants tend to bear more complex architecture, presenting more ramifications (gontijo, 1991) and more epiphytes. we suggest that both, ramifications and epiphytes, may increase the chance to accumulate dead organic matter in the form of ‘suspended litter’ which, at the end, represents more food to termites, as observed in other ecosystems (gonçalves et al., 2005). a study by roisin et al. (2006) showed that soilfeeder termites, for example, were absent from the canopy in a tropical rainforest because larger epiphytes were lacking. larger plants are more attacked than smaller plants due to larger supply of food and foraging sites, indicating that plant age is also an important variable to be discussed. it is expected that older plants presented larger trunks and stems, and more natural dead trunks, stems and roots, offering attractive resources to termites. on the other hand, this variable may be complicated to be measured in the field, because larger plants are not necessarily older. sociobiology 65(1): 48-58 (march, 2018) special issue 55 it is likely that plant encounter by termites may occur simply by chance, without any relationship to plant nutritional quality (nutting, 1969; souto et al., 1999). in such cases, taller plants would be found more easily, simply because they are more conspicuous (gonçalves et al., 2005; araújo et al., 2010). such a circumstance may define a random pattern for termite distribution on host plants. however, this pattern may not stay strictly in the random realm, because after finding the plant, termites may be subjected to other non-random factors (such as plant chemical traits), which may define whether or not termites would settle themselves on the plant found. the nutritional quality of the plants and their chemical defenses against the attack by insects could also exert a relevant role in the behavior of attack of termites and susceptibility of the plant. however, such studies on termites are scarce and need to be carried out at population, community, and ecosystem levels, before drawing any conclusion. by exposing internal parts of the plant, higher intensity of attack may lead to easier contamination by pathogens (sellschop, 1965). interestingly, several termite species prefer plant tissues partially altered by fungi action, including termite species which are able to attack sound wood (noirot & noirottimothée, 1969). in the present study, termite attack was observed to occur along with fungi on b. brevipes individuals, the only plant species where termite attack correlated negatively with plant size. we hypothesize that the concurrent action of termites and fungi may have promoted branch loss in this species, in such a way that attack could lead to smaller sizes. this would explain the observed negative correlation between termite attack and b. brevipes size. bauhinia brevipes is a shrub typical to cerrado, growing up to three meters height, and presenting a rich fauna of herbivore insects, including many galling species (fernandes, 1998; fernandes et al., 2000; cornelissen & fernandes, 2001; maia & fernandes, 2005). heavy activity of nasutitermes spp. was observed in the damaged stems of b. brevipes. that is, b. brevipes individuals presenting higher attack intensities (ia = 3) were also the shortest individuals in the population, because their size was reduced by the loss of main stems caused by termite attack. this would, then, produce the observed negative relationship between termite attack intensity and b. brevipes size. the system termite b. brevipes galling herbivores may represent an interesting scenario, yet to be explored, where termite attack may induce the host plant to continually produce new shoots (new meristems) which are then attacked by leaf galling insects. this may represent another avenue for research in this tropical and dynamic ecosystem where termites may exert an important, yet not well known, role by manipulating the resources used by herbivores. acknowledgements we thank d. negreiros, m.a.c. carneiro, f.a.o. silveira, t. gonçalves; a.p. viana for the plant identifications, m.a. feisella for the illustrations and icmbio (três mariasmg) for the logistical support at the estação ecológica de pirapitinga. this study was supported by grants provided by the cnpq and fapemig. ods holds a cnpq fellowship (307990/2017-3). this study was in partial fulfillment for the master degree of s.l.m. at the universidade federal de minas gerais. we also thank three anonymous reviewers for their comments and suggestions. authors contribution experimental design (g.w.f., s.l.m.), sampling data (s.l.m., j.c.s.), species identification, (r.c.), statistical analysis (s.l.m., j.c.s., o.d.), text review (g.w.f., s.l.m., j.c.s., o.d., r.c., i.h.). all authors discussed and revised the manuscript. references ackerman, i.l., teixeira, w.g., riha, s.j., lehmann, j. & fernandes, e.c.m. 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(1978). the role of termites in ecosystems. in: m.v. brian (ed.) production ecology of ants and termites (pp. 245-292). cambridge: cambridge university press. doi: 10.13102/sociobiology.v64i3.1623sociobiology 64(3): 244-255 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 natural history of ants: what we (do not) know about trophic and temporal niches of neotropical species introduction ants (hymenoptera: formicidae) are among the most abundant groups of invertebrates in terrestrial ecosystems, presenting a wide variety of feeding habits, nesting sites, and interactions with organisms from all trophic levels (kaspari, 2000). they are the subject of extensive and diversified research, in basic and applied science. despite this, in all tropical regions, the biology of most species is virtually unknown, due to a combination of high richness, taxonomic abstract our understanding of the natural history of neotropical ants is limited, due to lack of descriptive efforts and widespread use of morphospecies in literature. use of trophic resources and period of activity are two central niche aspects little explored for most species. this work aimed to broadly review the literature and provide empirical field data on these aspects for 23 species. the fieldwork was carried out in the atlantic forest of southern brazil. trophic and temporal niches were assessed with pitfall traps and seven kinds of bait representing natural resources. crushed insects were the preferred resource, whereas bird feces and living prey were less exploited. most species broadly used the resources, but pronounced quantitative differences were found. odontomachus chelifer (latreille, 1802) and pachycondyla striata smith, 1858 were relatively well studied and field data matched previous accounts. they were the only species that consistently used large prey, and avoided oligosaccharides. wasmannia auropunctata (roger, 1863) differed remarkably from previous studies, using feces as its sole trophic resource. the six pheidole species had no previous records and presented quantitative differences in resource use. most species had no strong preference for period of activity. camponotus zenon forel, 1912 was nocturnal and crematogaster nigropilosa mayr, 1870, linepithema iniquum (mayr, 1870) and linepithema pulex wild, 2007 were diurnal. complementary methods, context-dependence and descriptive studies have a central role in the understanding of ant natural history. community assessments can contribute significantly to this knowledge if researchers also pay attention to the individual species involved. sociobiology an international journal on social insects fb rosumek article history edited by ricardo r. c. solar, ufmg, brazil received 30 march 2017 initial acceptance 24 may 2017 final acceptance 05 june 2017 publication date 17 october 2017 keywords formicidae, atlantic forest, pheidole, wasmannia auropunctata, pachycondyla striata, odontomachus chelifer. corresponding author félix baumgarten rosumek ecological networks technical university of darmstadt schnittspahnstr 3, 64287 darmstadt, germany. e-mail: rosumek@hotmail.com uncertainty, lack of descriptive studies and widespread use of morphospecies in literature (krell, 2004; greene, 2005). two fundamental aspects that remain elusive for many tropical species are trophic and temporal niche. ants in general are regarded as omnivorous, feeding on a combination of live prey, dead animals, seeds and plant exudates, with some notorious specialized behaviors such as fungus cultivation and pollen consumption (kaspari, 2000; blüthgen & feldhaar, 2010). at genus or species level, they are sometimes classified in broad groups or guilds like “generalist predators” or simply ecological networks, technical university of darmstadt, darmstadt, germany department of ecology and zoology, federal university of santa catarina, brazil research article ants sociobiology 64(3): 244-255 (september, 2017) 245 “generalists” (brandão et al., 2012). these generalizations have an important role to understand communities, but also leave out the remarkable variation among species in nature. this specific information can be assessed from indirect sources such as body ratios of stable isotopes (blüthgen et al., 2003) and remains found in nests (lattke, 1990), and direct observation such as interaction with plant resources (byk & del-claro, 2010) and items carried to nests (medeiros & oliveira, 2009; raimundo et al., 2009). every approach has its focus and limitations, and adds a piece to the puzzle that is the niche of a species. in turn, studies on temporal niche of ants are common in open areas and/or temperate habitats, where the fluctuations in abiotic factors could have a stronger effect on ant activity. this variation is often linked with temporal niche partitioning and coexistence of competing species (lessard et al., 2009; anjos et al., 2016). less information is available for tropical forests (e.g. medeiros & oliveira, 2009; raimundo et al., 2009; feitosa et al., 2016). inside a forest, less variation would be expected, because daily changes in temperature, humidity and wind are smaller. however, temporal niche could still be affected by the existing fluctuations or by competitive pressure. in view of the importance of both use of trophic resources and period of activity, and considering the lack of information available for most neotropical species, this work aims to quantify use of trophic resources and period of activity of ground-dwelling ants from a neotropical forest. an extensive literature review was performed to assess how much is known about these individual species and compare results from the viewpoint of complementarity of methods and ecological context-dependence. these case studies highlight that descriptive studies are still fundamental for tropical faunas. in this sense, broader ecological assessments can give a significant contribution, if researchers also pay attention to the individual species involved. methods study area and sample design fieldwork was carried out in desterro conservation unit, florianópolis, south brazil (27o31’38’’ s, 48o30’15’’ w, altitude ca. 250 m), between december 2015 and january 2016. average annual temperature and precipitation is 20.5 ºc and 140 mm per month (data from meteorological station of epagri/ciram). vegetation consists of secondary atlantic forest sensu stricto (= ombrophilous dense forest) with at least 60 years of relatively undisturbed regeneration. this work was conducted in accordance with brazilian laws, under authorization sisbio number 51173-1. the sampling was based on the recent design of houadria et al. (2015) to assess community patterns on resource use and daily activity, but here its suitability to understand individual species is explored. four plots with 4 x 4 sample points (16 per plot) were set up, with distances from 30 to 50 m between plots. distance between sample points was 10 m. the baits were set up in transparent plastic boxes with diameter of 10 cm and ground-level slits that allowed the entrance of ants, and retrieved after 90 minutes. all individuals were collected from the baits in laboratory and stored in ethanol 70% for subsequent sorting. seven baits were offered as proxies for common resources available to ants (table 1; see supplementary material for details on bait display and rationale for their choice). in each round, only one bait was offered per sample point, and bait types were mixed among points, with a similar number of points receiving each type (8-9 per round). fourteen baiting rounds were performed, with only one period sampled each day, at daytime (around 13:00-15:30) or nighttime (around 21:00 – 23:30). in total, 896 baits were applied in the 64 sample points, with all seven baits being offered in each sample point two times (one at day and one at night). this design is suitable to assess multidimensional trophic niches, which are inferred from how often ants use each resource. hence, “preferences” means simply relative high use of certain resources. distance between each colony and the bait does not change from one resource to another, and the use of one resource does not affect the other. thus, it differs from a typical cafeteria experiment, which is designed to assess preferences through choices among different resources offered at the same time (krebs, 1999). an independent community assessment was performed with three rounds of pitfall trapping, alternated with bait rounds. the plastic cups were 6 cm wide and contained propylene glycol 50% and a small amount of neutral detergent. cups were buried previously and replaced after each round to avoid the digging-in effect. pitfalls stayed opened for 10 hours during the day and 9 hours during the night (due to short summer nights), then an extra 3-hour nocturnal round was performed. one nocturnal and one diurnal pitfall round were performed in sequence, separated by intervals to avoid dusk and dawn times. specimen processing and identification for each sample point, at least one individual per morphospecies was mounted. they were identified to genus level with baccaro et al. (2015) and to species level with taxonomic revisions, and comparison to identified specimens in collections and antweb images (antweb, 2016). the taxonomic sources used were: crematogaster – longino (2003); cyphomyrmex – kempf (1965) and snealling and longino (1992); gnamptogenys – lattke (1995); hylomyrma – kempf (1973); linepithema – wild (2007); odontomachus and pachycondyla – fernández (2008); pheidole – wilson (2003); wasmannia – longino and fernández (2007). camponotus and strumigenys were identified just by comparison with collections. the identifications were partially confirmed by taxonomists of the laboratory of ant systematics and biology, federal university of paraná, brazil (see acknowledgements). vouchers were deposited at http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1623/0 fb rosumek – natural history of neotropical ants246 the laboratory of ant biology, federal university of santa catarina, brazil, and at the ecological networks research group, technical university of darmstadt, germany. analysis for analysis and literature review of trophic niche, all species with at least 10 bait records were included, and for daily activity all species with at least 6 records on baits and pitfalls. the systematic literature review included the following sources: search for species names in google scholar; original descriptions; taxonomic revisions and references therein; references found in antwiki (antwiki, 2016). in case of species subject to name changes, older versions were also considered. taxonomic history and current nomenclature of species were checked with antcat (bolton, 2016). distribution records were retrieved from antmaps (janicki et al., 2016). a representative, species-specific, literature on trophic and temporal niche for these species was gathered. artificial breeding diets for laboratory colonies were not included, and use of generic baits (e.g. tuna, cookies) was considered just when relevant to discuss trophic niche. data is shown as proportions of records in each bait type/period relative to the total records for that species. for bait use, day and night records were not pooled. records for the pitfall replicas of each period were pooled for every point. differences were tested with two-tailed exact multinomial and binomial goodness-to-fit tests against a hypothesis of no preference, that is, equal proportions expected for each bait (1/7 or 0.14) or period (1/2 or 0.5). tests were run in r 3.3.0 (r core team, 2016). exact tests are the most appropriate for nominal variables with small sample sizes (mcdonald, 2014), but are limited nonetheless. a non-significant result could mean either low sample size or very generalist diet/activity. species with low number of records and non-significant results are discussed more briefly, since their results may not quantitatively represent their trophic niche. results seventy-six morphospecies were collected. it was possible to name 46 species, of which 23 had at least 6 records and 15 at least 10 (table 2; see supplementary material for records of the remaining identified species). a similar number of species was recorded in most bait types, but they differed greatly in the number of records and individuals attracted (table 1). crushed insects not only attracted ants more often, but also triggered larger recruitments. seeds were extensively used by many species, but no specialized granivory was detected. crickets attracted less species and were the resource less exploited overall. feces and termites also presented a lower number of records and small recruitments compared to other resources. almost all common species (= frequent in pitfalls and/ or in sample points) were well represented in baits (table 2, supplementary material). the only species conspicuously absent was pachycondyla harpax (fabricius, 1804) and, to a lesser extent, cyphomyrmex rimosus (spinola, 1851) and hylomyrma reitteri (mayr, 1887). the use of trophic resources is discussed in the following sections (fig 1). literature review, results and discussion are presented for every individual species or genus. period of activity is presented afterwards, for all species combined (fig 2). general aspects are explored in a final discussion section. gnamptogenys striatula mayr, 1884 (ectatomminae) this species (or species complex – arias, 2008; g. p. camacho, ufpr, personal communication) is a rare example of a neotropical ant extensively studied in the laboratory, covering many aspects of its biology (e.g. giraud et al., 2000; kaptein et al., 2005). however, the only information available about its trophic niche in the wild comes from lattke (1990), who reports remnants of several insect orders inside nests, and posteriorly called it “a generalist epigeic forager of humid forests” (lattke, 1995). a recent account recorded it rarely on experimental vertebrate carcasses left to rot in a forest, predating the larvae and pupae of necrophagous insects (paula et al., 2016). in accordance with this short background, the species was observed using termites frequently, but crushed insects, feces and sucrose were important as well (fig 1). hence, the species will scavenge and consume sugar when given the opportunity (but notice the lower use of melezitose, discussed in the next section). the relatively high use of feces, a less preferred resource overall, is a noteworthy feature that differentiates g. striatula from most other species of this study, particularly the two “generalist predators” discussed next. odontomachus chelifer (latreille, 1802) and pachycondyla striata smith, 1858 (ponerinae) these two widespread species radically differ in morphology, but are similar in many aspects, therefore is appropriate to discuss both together. they are one of the most conspicuous elements of the southern atlantic forest ground fauna, due to their abundance, solitary foraging mode and large size. also, they are two of the most well-known species included in this study, and several account showed a multitude of functional roles and a broad trophic niche for them. observation of nest entrances showed that 80-90% of the items carried by pa. striata were arthropod parts, mostly termites and other ants, the remaining consisting of plant material (giannotti & machado, 1991; medeiros & oliveira, 2009). through direct observation, medeiros and oliveira (2009) also showed that scavenging accounts for more than 80% of its foraging behavior. on the other hand, fowler (1980) reported o. chelifer preferences for certain termite species in laboratory, and qualitatively stated that in the field prey sociobiology 64(3): 244-255 (september, 2017) 247 consisted almost entirely of termites. in the atlantic forest, all items carried to nests were arthropods, mainly termites, but other animal groups accounted for 60% of them (raimundo et al., 2009). scavenging was also cited in this study, although not quantified. both o. chelifer and pa. striata were recorded on experimental carcasses predating the larvae and pupae of necrophagous insects (paula et al., 2016). other important items used by the two species are seeds with elaiosomes and other fallen diaspores rich in proteins and lipids, frequently collected from the ground in the atlantic forest (pizo & oliveira, 2000; passos & oliveira, 2002, 2004). field records on use of liquid sugars are scant and qualitative. odontomachus chelifer was not observed using extra-floral nectaries (efns) by raimundo et al. (2009), while there is one record for pa. striata (in cerrado, the brazilian savannah – byk & del-claro, 2010). the results presented here are mostly consistent with this broader picture (fig 1). both species used more frequently dead insects, sucrose and large prey. in fact, they were the only two species consistently recorded on crickets. the low frequency in termite baits is unexpected and probably represents a methodological artifact. these large solitary foragers were observed quickly collecting termites (even glued ones) and leaving the baits in a few minutes, contrary to ants of smaller species that were recruited to them. in cricket baits, however, the two species spent more time, trying to carry out the tied cricket or dismembering it. smaller ants frequently took advantage of this to grab the remains or lick spilled hemolymph. this largely contributed to the richness found in this bait (table 1) and could happen in nature, whenever predators kill prey too large to carry them out at once. besides predation, scavenging was a common behavior, and p. striata in particular would prioritize it whenever possible, in accordance with what was observed by medeiros and oliveira (2009). this could be result of their morphology, because the triangular mandibles may be more suited to chop large carcasses than the trap-jaws of o. chelifer. in addition, it could be an effect of competition. the two species were never found at the same bait, and co-occurred in just 10 of the 62 points where they were recorded (table 2). the two were previously reported to avoid each other, but, when agonistic interactions occur, pa. striata usually is the winner, and can steal the food or kill (and eat) o. chelifer (medeiros & oliveira, 2009; raimundo et al., 2009). thus, pa. striata could displace o. chelifer and maintain control of a valuable resource such as dead arthropods through tandem recruitment (medeiros & oliveira, 2009; silva-melo & giannotti, 2012), while cooperative foraging behavior was not observed in o. chelifer (raimundo et al., 2009). effectively, the average numbers of workers per bait was smaller for the latter species (o. chelifer = 1.4 ± 0.9; pa. striata = 2.5 ± 2; mann-whitney, z = -2.27, p = 0.02). both species used sucrose frequently. they were never observed foraging on trees or low vegetation in this study, which fits previous accounts (fowler, 1980; medeiros & oliveira 2009), so it is unlikely that they commonly use nectar as food source. this behavior also should limit honeydew use by them. effectively, the difference between use of sucrose and melezitose is remarkable here. ants differ in their sugar preferences/tolerances, and melezitose is highly attractive to some species, and less so for others (völkl et al., 1999; blüthgen & fiedler, 2004). in some insects, weak or negative effect of melezitose on fitness was observed (zoebelein, 1956; chen & fadamiro, 2006), and some evidence points out to reduced suitability of aphid oligosaccharides for predators (wäckers, 2000). the low number of records for these ant species, even when melezitose was readily available, suggests a physiological constraint to the use of complex sugars. while some congeneric species are known to visit efns or tend hemipterans (e.g. o. troglodytes – lachaud & dejean, 1991), the main source of sugars for these ground foragers in the atlantic forest is more likely to be fallen fruits rich on mono and disaccharides. wasmannia auropunctata (roger, 1863) (myrmicinae) this tiny species is native to the neotropics, but infamous as an unpleasant guest worldwide. it is an exotic invader on many continents and islands, and also an indoor exotic species in colder places (wetterer & porter, 2003). a large body of knowledge describes how w. auropunctata dominate habitats and displace other ants, which often happens table 1 – baits used to represent natural resources in this work, with total number of species (s), records (ba) and average number of individuals ± s.d. (in) recorded. bait resource represented s ba in living crickets larger and highly mobile prey 26 107 4 ± 8 living termites smaller and slower prey 31 203 4 ± 15 crushed insects dead arthropods 33 422 14 ± 38 chicken feces bird droppings 32 215 3 ± 5 seeds mixture seeds of diverse sizes and shapes, without elaiosomes 32 344 7 ± 10 melezitose oligossacharides produced by sap-sucking insects 34 327 6 ± 9 sucrose dissacharides present in extra-floral nectar and fleshy fruits 34 366 7 ± 15 fb rosumek – natural history of neotropical ants248 when the species is introduced or, within its native range, in crops and other open/disturbed areas. the species is portrayed feeding virtually on everything: scavenging; preying on small and large arthropods; collecting diversified plant parts; visiting extra-floral nectaries and tending honeydew-producing insects (creighton, 1950; kusnezov, 1952; smith, 1954; smith, 1965; fabres & brown, 1978; clark et al., 1982; deyrup et al., 2000; wetterer & porter, 2003; longino & fernández, 2007). some of these authors suggest that honeydew is their main resource, such as clark et al. (1982). a comparatively small amount of information suggests that, inside forests within its native range, the species is not nearly as dominant (majer & delabie, 1999; longino & fernández, 2007). very little is known about w. auropunctata habits in this context. using generic baits, orivel et al. (2009) showed a steep decline in bait use and nest density within a gradient from open areas to undisturbed forest. in atlantic forest, ca. 1400 km north of the present study site, santana et al. (2013) qualitatively showed it interacting with seven nonmyrmecophorous diaspores on the ground. in light of this literature record, it was really surprising to find the species to be a strict specialist in feces (fig 1). in fact, it was the only species in this study that used a single resource. it was a comparatively frequent species (table 2), but appeared always in low numbers and was not collected in pitfalls. this result differs from the widespread use of fleshy diaspores found in santana et al. (2013), and also from the use of baits in orivel et al. (2009). the latter authors suggested that abiotic factors play a role in the ecological shift of w. auropunctata from open to forest areas. a physiological constraint related to environmental conditions (e.g. temperature) could explain why the species has a limited role inside forests, and why, in a higher latitude, it shifts to a resource less preferred by other species. this intriguing behavior will be explored further and shows that there are open questions related to this important species, particularly outside the invasive context. wasmannia affinis santschi, 1929 (myrmicinae) as a small genus (11 species) with one outstandingly famous representative, it is not unexpected to find very little information on the other wasmannia (longino & fernández, 2007). that is the case for w. affinis, which has a geographic distribution apparently restricted to atlantic forests of south and southwest brazil. the single record about its feeding habits comes from bieber et al. (2013), who reported it as the ant species most frequently interacting with fallen fruits of psychotria suterella (rubiaceae). the results for this species were very distinct from w. auropunctata (fig 1). wasmannia affinis had a smaller incidence in the community, but used a broader range of resources. feces were not particularly important, and having more records on termites, seeds and melezitose would make it unique among species of this work, although it was not possible to statistically confirm this pattern. pheidole (myrmicinae) with over a thousand species described (bolton, 2016), pheidole usually is the most rich, frequent and abundant genus on the ground of tropical and subtropical forests. this was also the case here, with 17 species, eight of them fitting previously described species. after the literature review, all the previous knowledge on these species can be summarized in wilson’s (2003) words: “biology: unknown”. not surprising at all, taking into account its complicated taxonomic history that only recently began to be solved (wilson, 2003; longino 2009). however, identification is a time-consuming task, depends on the infrequently collected major workers, and it is still common to find new species, which could be the case for five morphospecies in the present work (a. c. ferreira, ufpr, personal communication; see supplementary material). even if often labeled as “generalists”, the little we know about pheidole species shows a diversity of habits and functional roles (wilson, 2003), which is expected for such a large genus. in accordance with this, differences were found among the six species with at least 10 records (fig 1). pheidole lucretii santschi, 1923, pheidole nesiota wilson, 2003, and pheidole sigillata (wilson, 2003) had similar species ba pf pt camponotus lespesii forel, 1886 9 1 4 camponotus zenon forel, 1912 14 0 10 crematogaster nigropilosa mayr, 1870 5 1 5 cyphomyrmex rimosus (spinola, 1851) 6 10 15 gnamptogenys striatula mayr, 1884 47 26 26 hylomyrma reitteri (mayr, 1887) 8 10 13 linepithema iniquum (mayr, 1870) 10 0 7 linepithema micans (forel, 1908) 16 1 6 linepithema pulex wild, 2007 14 1 5 odontomachus chelifer (latreille, 1802) 42 12 25 pachycondyla harpax (fabricius, 1804) 1 11 10 pachycondyla striata smith, 1858 88 58 47 pheidole angusta forel, 1908 6 1 4 pheidole aper forel, 1912 27 10 10 pheidole avia forel, 1908 9 2 5 pheidole lucretii santschi, 1923 50 10 13 pheidole nesiota wilson, 2003 89 14 19 pheidole risii forel, 1892 21 4 5 pheidole sarcina forel, 1912 51 13 12 pheidole sigillata wilson, 2003 91 25 35 strumigenys denticulata mayr, 1887 0 6 6 wasmannia auropunctata (roger, 1863) 19 0 16 wasmannia affinis santschi, 1929 20 3 6 table 2 – species analyzed in this work. ba = total records in baits. pf = total records in pitfalls. pt = total records in sample points, considering both methods. sociobiology 64(3): 244-255 (september, 2017) 249 patterns and broadly used the most attractive resources. pheidole sarcina forel, 1912 included more seeds and feces than the others. pheidole aper forel, 1912 occupies a distinct niche, being the only species in this study that distinctively used more melezitose over other resources. since honeydew is the only reliable source of this sugar in nature, interaction with sap-sucking insects should be important for this species. finally, there is pheidole risii forel, 1892, the very definition of a generalist, which used all resources indiscriminately. the higher occurrence on living baits set it apart from its congeneric species. several mechanisms are proposed to explain the coexistence of dozens of ant species in a community, through a complex interplay of habitat structure, interspecific interactions and species traits (cerdá et al., 2013). behavioral adaptations might be the main factor allowing coexistence among ph. lucretii, ph. nesiota and ph. sigillata. but overall, the results also suggest that species-specific multidimensional trophic niches, presenting quantitative rather than qualitative differences, could play a role in coexistence, even among related species of the same “generalist” group. linepithema (dolichoderinae) the case of linepithema is similar to wasmannia. besides the invasive and extensively studied linepithema humile (mayr, 1868), little is known about most species of the genus (wild, 2007). linepithema micans (forel, 1908) is common in south brazilian vineyards, strongly associated with sap-sucking insects (morandi filho et al., 2015). this fig 1 – use of trophic resources by ant species in southern brazil. values above bars are numbers of records. the expected proportions in case of no preference for baits (= 0.14). asterisks indicate statistical significant differences. fb rosumek – natural history of neotropical ants250 species certainly suffers from a misdiagnosed past, and nondillo et al. (2013) suggest that many previous records of l. humile in infested vineyards should be l. micans instead. more is known about linepithema iniquum (mayr, 1870), mainly because it also appears as an exotic indoor species in north america and europe. a few instances of honeydew and extra-floral nectaries use exist in the literature (wheeler, 1929; wild, 2007; schmid et al., 2010) and smith (1929) described it collecting arthropods, although without specifying if that meant scavenging or predation. wild (2007) describes it as a primary arboreal ant, but clearly it also forages on the ground (table 2). finally, linepithema pulex wild, 2007 is one of the smallest and less-known representatives of the genus. it was recorded occasionally on experimental carcasses, predating the larvae and pupae of necrophagous insects (paula et al., 2016). none of these species showed statistically significant preferences, due to low number of records and use of several resources (fig 1). l. micans and l. pulex seem to use resources more broadly than l. iniquum, which might descend to the ground mostly to scavenge animal resources. the small l. pulex may have stronger carnivorous tendencies and, in fact, twice they were able to recruit a few dozens of workers and predate crickets just by themselves, a remarkable feat considering its size. camponotus (formicinae) camponotus is the only ant genus that currently rivals pheidole in richness (bolton, 2016), but still lacks comprehensive revisions at genus level. accordingly, the biology of most tropical species remains unknown, such as the two recorded here. camponotus lespesii forel, 1886 is widespread in the neotropics. byk and del-claro (2010) recorded it qualitatively visiting extra-floral nectaries and paula et al. (2016) observed it on experimental carcasses, predating the larvae and pupae of necrophagous insects and feeding on the carcass itself. conversely, camponotus zenon forel, 1912 has its distribution apparently restricted to southernmost brazil and nothing is known about its biology. the number of records was low for both species, precluding clear statistical results, even if their resource use was restricted (fig 1; ca. lespesii is included only 9 records due to its marginally significant result). the few records for both species were quite similar, both concentrated on crushed insects and sucrose. daily activity in this work, most species have not displayed strong tendencies to be active at a particular time (fig 2). in south brazil, summer is both the warmest and wettest season, and any temporal preference that is linked to abiotic factors should be at its lowest. still, some species showed preferences. three species were exclusively, or almost exclusively, diurnal (crematogaster nigropilosa mayr, 1870, l. iniquum and l. pulex) and one nocturnal (ca. zenon). gnamptogenys striatula showed a moderate, statistically significant, preference for the night, and ph. nesiota for the day. not much previous information on daily activity for individual species was found. the ones with information available are discussed below. a single account of “mainly diurnal activity” exists for ca. lespesii (byk & del-claro, 2010). in this work, performed in the brazilian savannah, most camponotus species were qualitatively classified as diurnals. this is rather distinct from the atlantic forest, where ca. lespesii had more records at night (and ca. zenon displayed a truly nocturnal behavior). for cr. nigropilosa in costa rica, longino (2003) says “foragers may be found day or night”. however, in south brazil the species seems to be a diurnal specialist. for w. auropunctata, the lack of preference was the same as previously observed in the invasive context (clark et al., 1982). fig 2 – daily activity of ant species in southern brazil. the dashed line shows expected proportions in case of no preference for period (= 0.5). asterisks indicate statistical significant differences. sociobiology 64(3): 244-255 (september, 2017) 251 the studies in the atlantic forest with o. chelifer and pa. striata also assessed their period of activity (medeiros & oliveira, 2009; raimundo et al., 2009). in these studies, o. chelifer showed a strong preference for nocturnal activity, and the inverse was found for pa. striata. however, this clear pattern was not repeated in the present study. there was a slight inclination towards the same trends, but far lower than compared to the equivalent season in these studies. a response to variable weather conditions, community context or distinct behavioral adaptations to coexistence could generate such discrepancies. the interaction between these species inside and across communities is still an interesting and open topic for a detailed study. discussion the life history of a species involves many aspects and is the result of a complex set of external variables and species traits. to fully understand one single history is not a trivial task. methodology plays a key role on this, and results must be interpreted in the light of the advantages and caveats associated to every approach (birkhofer et al., 2017). since the bait method used in this study relies on proxies to broadly access resource use, the possibility of artificial, non-representative results must be considered. however, the results were consistent with previous accounts for two wellstudied species (o. chelifer and pa. striata), excluding the use of termites explained before. the unusual result for w. auropunctata is unlikely to be an artifact. the species proved to be relatively frequent in the community and, if it maintained its generalist habits, at least some records on other baits would have been expected. also, this method does not evaluate extensively the natural variations for each resource, therefore is less suitable for detecting specialized behaviors, but focuses instead on the oft-neglected generalist species, which represent most of the community. the ants rarely recorded in baits were also uncommon in pitfalls, and many present known specialized behaviors or forage mainly on vegetation or inside leaf-litter (table 2 and supplementary material). pachycondyla harpax is the most notorious absence, and this could be due to a preference for specific termite species (garcía-pérez et al., 1997), or also an artifact, as for the other two large ponerinae. finally, ants may be driven to more limited resources, instead of the ones they used more frequently. this may be particularly true for nitrogen-deprived arboreal ants (kaspari & yanoviak, 2001), and could be the reason behind the lack of melezitose use by l. iniquum. however, the two camponotus species, which forage both on vegetation and ground, used sucrose and crushed insects similarly. therefore, this deviation might be less relevant for ground-dwelling ants. in short, although with potential bias that must be considered, this bait method seems to appropriately assess the trophic niche of most species in the site and season in question, and the data it yields is useful to understand individual species. many methods to assess resource use cannot discriminate well between hunting and scavenging (e.g. nest excavation, observation of foragers on a nest entrance, barcoding of gut content, stable isotopes). on the other hand, this bait method assesses what species prefer to use, but not what they have available in the community. carcasses are a rich and easy to gather resource, but in their absence ants have to fight their prey. even taking this into account, and in light of the previous accounts (medeiros & oliveira, 2009; raimundo et al., 2009), it is likely that resource use plays a role in the coexistence of the more scavenger pa. striata and the more predator o. chelifer. this reduces niche overlap and may lead to distinct functional roles. on the other hand, species frequent on crushed insects, but not in living baits, probably are restricted to scavenging, independent of resource availability. that was the case for most linepithema, pheidole and camponotus. sugar consumption is more frequently studied through observation of interactions with efns or hemipterans in plants, due to their role as main attractors in ant-plant interactions (rosumek et al., 2009). interactions of ground foragers with plant diaspores are assumed to be more associated with lipidrich elaiosomes that mimic animal prey (hughes et al., 1994; giladi, 2006). however, the use of fallen fruits simply as sugar sources might be overlooked. this could partially explain the pronounced lack of use of carbohydrate resources by the “poneroid” clade observed by lanan (2014). few species in the present work were not attracted by sucrose, and some of them probably climb vegetation in search for efns (e.g. camponotus). however, fallen fruits might be an occasional but disputed resource for species restricted to the ground. preference for melezitose was less common, and it was conspicuously avoided by some species. among physiological constrains that could reduce suitability of certain sugars to a species, particularly insect-synthesized oligosaccharides, are low gustatory perception, digestibility and nutritional value (boevé & wäckers, 2003). these constraints could be another source of niche partitioning among ant species. other fundamental aspect underlying the results of this work is the context-dependence of the patterns and processes studied in ecology, including the interaction between organisms and resources (agrawal et al., 2007). the few well-studied species told different stories in a distinct context, like the trophic niche of w. auropunctata and daily activity of pa. striata and o. chelifer. it is likely that the other, lessknown species would exhibit such variation in different contexts, influenced by biotic interactions, abiotic factors and evolutionary history of the community. taking into account the lack of knowledge about most species, the complementary results given by different methodologies, and the variation under distinct contexts, it is clear that descriptive studies are still very much needed for tropical species, even if these studies are often relegated to second plane in modern science practice and funding (greene, fb rosumek – natural history of neotropical ants252 2005). in this way, studies at community or larger scales could bring a considerable amount of information for individual species, above all when they are virtually unknown. this could be achieved first by spending time and effort on the taxonomic stage, and avoid use of morphospecies whenever possible. second, researches should learn about their species to point out relevant findings, and prevent these to end up buried in a datasheet cell or the supplementary material. the large scale, pattern-driven enterprise is clearly important for the advancement of knowledge, but such basic aspects of natural sciences still are important. claims for this “old-fashioned” natural history are not new (jordan, 1916). a century later they remain valid, because we still have a lot to describe. supplementary material h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfiles/1623/0 http://dx.doi.org/10.13102/sociobiology.v64i3.1623.s1740 acknowledgements cristian klunk, frederico rottgers marcineiro and larissa zanette da silva for support in the fieldwork and sample sorting. rodrigo machado feitosa, alexandre casadei ferreira and thiago sanches ranzani da silva for taxonomic assistance. nico blüthgen, michael heethoff and florian menzel for supervision and advice on the study design. christoph von beeren, adrian brückner, florian menzel, jeanpaul lachaud and one anonymous reviewer for critical reading of early drafts. this work was funded by the brazilian national council of technological and scientific development (cnpq). references agrawal, a.a., ackerly, d.d., adler, f., arnold, a.e., cáceres, c., doak, d.f., post, e., hudson, p.j., maron, j., mooney, k.a. 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(1956). der honigtau als nahrung der insekten: teil i. zeitschrift für angewandte entomologie, 38: 369-416. doi: 10.13102/sociobiology.v65i2.1895sociobiology 65(2): 232-243 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 morphometric divergence of anatolian honeybees through loss of original traits: a dangerous outcome of turkish apiculture introduction twenty-seven subspecies of apis mellifera (l.) have been identified based on morphometric characteristics and grouped into four evolutionary lineages: african lineage (a), western and northern european lineage (m), southeastern european lineage (c), and near and middle eastern lineage (o) (garnery et al., 1993; sheppard & meixner, 2003; cánovas et al., 2008; miguel et al., 2011). these subspecies have also been described as ‘geographic races’ (ruttner, 1998 sheppard et al., 1997; engel, 1999; sheppard & meixner, 2003; de la rùa et al., 2005). at least four of these subspecies are known to be present naturally in anatolia (turkey) (ruttner, 1988; smith, 1997; kandemir et al., 2000; palmer et al., 2000; bodur et al., 2007). abstract five honeybee subspecies exist naturally in anatolia. unfortunately, owing largely to migratory beekeeping and lack of control mechanisms against requeening, the native honey bee subspecies located in anatolia are facing extinction. beekeeping activities, especially migratory beekeeping jeopardizes the presence of the naturally evolved indigenous subspecies of anatolia. the present study examined morphological deformation in three apis mellifera (l.) subspecies (a. m. caucasica, a. m. carnica, a. m. syriaca) and two ecotypes of a. m. anatoliaca (muğla and yığılca) that have been kept all together in a long-term breeding program at the common apiary. worker bee samples representing each honeybee subspecies and ecotype were collected from the common apiary, and also from their original locations. to demonstrate the potential hybridization effect on variations of the anatolian native honeybee subspecies and ecotypes, the geometric morphometric method was applied on the samples of honeybees that had been kept together in the same apiary since 2008. the findings showed that the honeybee population of the common apiary and those from their native settings formed two different configurations on the scatter plots. hybridization and promiscuous mating among the different honeybee races maintained in the common apiary may have led to the loss of a valuable combination of morphometric traits. hence, there is an urgent need for an active monitoring system and a ban on queen trading and migratory practices as well as for periodic testing of registered apiaries to identify ongoing variations in the gene pool. sociobiology an international journal on social insects m kekeçoğlu1,2 article history edited by kleber del-claro, ufu, brazil received 25 july 2017 initial acceptance 07 november 2017 final acceptance 07 december 2017 publication date 09 july 2018 keywords honeybee biodiversity, migratory beekeeping, geometric morphometric. corresponding author meral kekeçoğlu duzce university faculty of arts and sciences department of biology konuralp, duzce 81620, turkey. e-mail: meralkekecoglu@duzce.edu.tr preliminary studies on the distributional patterns of the anatolian honeybee population were conducted by buttelreepen (1906) along limited areas of the aegean and marmara regions. these were followed by bodenheimer (1942) who employed morphological characteristics to divide turkey into seven zones. maa (1953) was the first taxonomist to name the subgenus of a. m. anatoliaca using morphometric characteristics. findings similar to those of bodenheimer (1942) were reported by adam (1983), who identified four honeybee races and many ecotypes of anatolian honeybees. he stressed that there were morphological differences between the bee populations of the black sea coast and the mediterranean coast of anatolia (adam, 1983), and this finding also revealed the traits of the syrian bee (a. m. syriaca). however, ruttner (1988) determined that a. m. anatoliaca was found throughout 1 duzce university, faculty of arts and sciences, department of biology, konuralp, duzce, turkey 2 duzce university, beekeeping research development and application centre, yığılca, turkey research article bees mailto:meralkekecoglu@duzce.edu.tr sociobiology 65(2): 232-243 (june, 2018) 233 all of turkey including the european part, with the exception of the northeast and the southeast corners of the country, where a. m. caucasica and a. m. meda, respectively, were identified. ruttner’s analysis of honeybee samples from southeast anatolia showed that they were not a. m. syriaca as generally assumed previously, but were the iranian bee, a. m. meda. many surveys based on alloenzyme and mtdna variation that have reviewed the honeybee biodiversity of anatolia support the findings of ruttner (1988), kandemir and kence (1995) and kandemir et al. (2000, 2006). recent studies on thracian honeybees have revealed the existence of a. m. carnica in the european part of turkey (smith et al., 1997; palmer et al., 2000; kandemir et al., 2006) and of a. m. syriaca in hatay province (palmer et al., 2000; kandemir et al., 2006). both morphometric and mtdna study results have shown five subgenera of a. mellifera spread throughout turkey, including a. m. anatoliaca, a. m. caucasica, a. m. meda, a. m. carnica and a. m. syriaca (ruttner, 1988; smith et al., 1997; palmer et al., 2000; kandemir et al., 2006; özdil & i̇lhan, 2012). in addition to those subspecies, various local ecotypes, which have not been adequately studied to date, such as the muğla and yığılca bees have also been identified in some isolated local areas of turkey (kekeçoğlu, 2010; kekeçoğlu & soysal, 2010a; bouga et al, 2011). the muğla bee is an ecotype of a. m. anatoliaca which has adapted its life cycle by foraging on the scale insect marchalleina hellenica on pine trees (güler & kaftanoğlu, 1999a, b). the yığılca bee is also considered to belong to ecotypes marked by larger size and darker color, respectively, while it differs from other bee species with regard to length of wings and legs. it rears its brood in the total brood area in early spring. in addition, it has a greater capacity for honey production than a. m. caucasica or a. m. anatoliaca (kekeçoğlu, 2010; gösterit et al., 2012, 2016). turkey is a geographically disjunctive region covering three continents, asia, europe and africa. due to its rich topography and climatic variation, it presents a wide range of habitats in a relatively small geographical area. thanks to its geographical and biological diversity, a large amount of genetic variation can be observed in the honeybees of turkey. however, within the last 15-20 years, migratory beekeeping has become widespread in turkey (güler, 2010). the extensive practices of migratory beekeeping and commercial breeding can promote gene flow between bee races. as a consequence, genetic homogenization has emerged as a threat to the turkish honeybee populations. if protective policies are not enacted by the ministry of agriculture against intensive queen breeding and migratory beekeeping, the gene pool of the turkish honeybee populations will be homogenized, as has already occurred in greece (bouga et al., 2005). the recent anxiety over the loss of biodiversity requires an international effort in order to develop strategies for conservation and sustainable use affecting biodiversity. it must be recognized that each honeybee race has peculiar characteristics for dealing with environmental conditions, stress and diseases (tozkar et al., 2015). if one takes into consideration that global climate changes and the increasing use of pesticides provide unfavorable conditions to certain races, some races may cease to exist despite their unique traits. therefore, it is highly crucial to preserve the genetic resources of the anatolian honeybee races and ecotypes. four out of the five anatolian honeybee lineages found around the mediterranean region, in particular, carry different traits of production capacity and hygiene behavior as well as resistance against climate change, disease, and pesticide residues (garnery et al., 1993; cánovas et al., 2008; miguel et al., 2011; tozkaret al., 2015). nevertheless the genetic hybridization caused by migratory beekeeping modifies the genetic pool of the local honeybee populations, leading to the loss of their unique genetic and morphological traits. because of migratory beekeeping, and especially commercial breeding, turkish native honeybee subspecies and ecotypes have been exposed to introgressive hybridization with caucasian bees (güler, 2010). future honeybee conservation efforts should therefore be aimed at reducing the introduction of alien subspecies into isolated areas already occupied by native honeybees. the objective of this study was to prove the effect of the hybridization on honeybee biodiversity. to this aim, colonies representing all the native honeybee races of turkey, a. m. anatoliaca, a. m. caucasica, a. m. carnica and a. m. syriaca, were maintained all together over a period of seven years in common apiary in central anatolia. specimens from the common apiary were then compared with samples from isolated areas where mating takes place in yığılca, muğla, artvin, kırklareli and hatay, and where hybridization among the standing populations is avoided. material and methods the sampling areas and the five symbols representing the native honeybee subspecies of turkey are shown in fig 1. the private research apiary in central anatolia, was established approximately seven years ago. since 2008, colonies representing turkish native honeybee subspecies and ecotypes (a. m. caucasica, a. m. carnica, a. m. syriaca and the muğla and yığılca ecotypes of a. m. anatoliaca) have been maintained all together in that apiary. between april and june 2015, honeybee worker samples representing the native honeybee subspecies and ecotypes of turkey were collected from the common apiary and also from isolated locationsin artvin (a. m. caucasica), düzce (yığılca ecotype), muğla (muğla ecotype), kırklareli (a. m. carnica) and hatay (a. m. syriaca). in each apiary, ten worker bees were obtained from each colony. honeybee worker samples were collected from the colony by uncovering the top of the hive and shaking the honey combs through clean cloths. samples of adult workers were transported to the laboratory in small plastic vials containing 96% ethanol, and then stored at 4°c. the localities and geographical coordinates based on gps (magellan explorist 110) are given in table 1. m kekeçoğlu – the effect of migratory beekeeping on honeybee biodiversity234 fig 1. the distribution of native honeybee subspecies of turkey and sampled provinces indicated by different symbol of the body segments were stored in 70% lactic acid for 24 h in order to soften the tissues for better resolution. after the dissection process, the right forewings and legs were placed in petri dishes filled with distilled water, and then fixed on the surface of microscope slides fastened on a 7.5 × 2.5 cm slide frame with special transparent tape (3m scotch® magic™ mat tape, 19 mm × 33 m). geometric and standard morphometric techniques images of the wings were obtained using a video camera (bs200pro, bab imaging systems, bab ltd, 1993) mounted on a microscope (bab str45) with a 1 × objective. photographs were downloaded to the basic research picture program (bab bs200pro image analysis systems) and all files were saved. the wing images were scaled and rotated, and then19 landmarks located at vein intersections (fig 2) were digitized and the two-dimensional x and y cartesian coordinates were recorded using the bs200pro program. in addition to the cartesian coordinates of the 19 landmarks, mathematical parameters among the landmark coordinates (angle, length, and index) were evaluated. characteristics of the hind legs and proboscis were determined via measurements made with bab bs200pro software (kambur, 2017). sampling honeybee subspecies region city-provinces apiary n altitude (m) geographical coordinates is ol at ed n at iv e re gi on s artvin (a.m.caucasica) northeast anatolia (blacksea) artvin borçka karşıköy 20 598 45°89’57”n 37°07’27”e balcı-naznara 20 701 45°76’41”n 37°07’36”e macahel 20 460 41°10’14”n 41°04’35”e düzce-yığılca (yığılca ecotype of a. m. anatoliaca) northwest anatolia düzce (yigilca) hoşafoğlu 15 329 36°16’27”n 36°53’36”e kırık 15 317 45°30’ 63”n 36°50’41”e redifler 15 401 45°35’ 75”n 37°72’39”e muğla (mugla ecotype of a. m. anatoliaca) aegean muğla (bodrum) ortakent 15 18 40°29’51”n 35°31’40”e çömlekçi 15 54 41°12’48”n 35°35’42”e yalı-alazeytin 15 181 40°29’40”n 35°54’52”e kırklareli (a. m. carniaca) thrace region kırklareli igneada 20 300 41°52’34”n 27°59’ 28”e demirkoyşişlioba 25 203 41°58’45”n27°54’58”e hatay (a. m. syriaca) southeast anatolia hatay erzin 20 253 36°12’ 39”n 36°52’56”e antakya 25 205 30°09’ 28”n 36°13’30”e c om m on a pi ar y a. m. caucasica a. m. syriaca a. m. carniaca muğlaecotype and yığılcaecotype of a. m. anatoliaca central anatolia ankara center (common apiary) 210 871 39°54’56” n 32°52’10”e common apiary: all colonies represented five different honeybee subspecies of turkey have been maintained altogether in the common apiary since 2008. table 1. geographical coordinates of sampling locations. n = number of colonies sampled. extraction techniques all of the specimens were kept in 96% ethanol until dissection of the wings using forceps. the right forewings of the honeybees were cut at the wing base and then transferred to 70%, 50%, and 20% ethanol solutions, respectively, for gradual hydration. distilled water was used to flatten the wings. the head segments of the worker bees were kept in 30% alcohol for the proboscis measurements, while the rest https://www.google.com.tr/search?q=3m+scotch%c2%ae+magic%e2%84%a2+mat+tape,+19mm+%c3%97+33m&spell=1&sa=x&ved=0ahukewj2r8af7pzvahxhf5okhrzybdaqbqgkkaa https://www.google.com.tr/search?q=3m+scotch%c2%ae+magic%e2%84%a2+mat+tape,+19mm+%c3%97+33m&spell=1&sa=x&ved=0ahukewj2r8af7pzvahxhf5okhrzybdaqbqgkkaa sociobiology 65(2): 232-243 (june, 2018) 235 statistical analysis data obtained from the x and y coordinates were analyzed via geometric morphometrics and angle, length and index values via standard morphometrics using the spss 15.0.1 package (spss, 2005). stepwise discriminant function analysis (dfa) was performed based on the spectral decomposition of a covariance matrix of the pooled results of each locality. canonical variant analyses (cva) and cross validation tests were conducted to check the accuracy of the equations in identifying the colonies. a dendrogram showing the relationships among the honeybee populations was constructed according to the pooled results of each honeybee population via the upgma (unweighted pair group method with arithmetic mean) using the pyhlip 3.67 package (felsenstein, 1993). multivariate (manova) and univariate (anova) statistical analyses were applied on the dataset of the morphometric characters for comparison of the groups. results morphometric differences in native ancestor honeybee populations honeybee colonies from isolated native populations were evaluated by using the geometric morphometric method. the discrimination of five native honeybee populations, artvin (a. m. caucasica), muğla (muğla ecotype of a. m. anatoliaca), hatay (a. m. syriaca), kırklareli (a. m. carnica) and düzce (yığılca ecotype of a. m. anatoliaca) was carried out by discriminant function analyses (dfa). four statistically significant canonical vectors were extracted from the matrix. the scatter plot generated via canonical variant analysis (cva) revealed that the colonies from the native areas had formed non-overlapping distant clusters, except for the kırklareli population (fig 3). the cva indicated the existence of great and highly significant differences among the native honeybee subspecies (wilks’ lambda=0.06, f(4,236)= 47.437 p <0.0001). the first three vectors discounted 95.4% of the among-group variations together. the first and second axis explained 65.6% and 20.4% of total variation, respectively. the contribution of different variables to the canonical coordinates were assessed using the standardize coefficients, and x14 was the variable with the highest loading on the first canonical axis, whereas the second canonical axis included y1, y2, y3, y4, y5, y6, y7, y8, y9, y10 and y11 variables contributing to the separation of the groups (fig 3). fig 2. right forewing of anatolian honey bee with 19 landmarks plotted in the vein junctions. **locations (isolated area) artvin (a.m.caucasica) muğla (muğla ecotype) yığılca (yığılcaecotype) hatay (a.m.syriaca) kırklareli (a.m.carnica) total artvin (a.m.caucasica) 56 (93%) 4 (6.7 %) 2 (3.3 %) 0 0 60 muğla (muğla ecotype) 3 (6.7 %) 41 (91.1 %) 0 1 (2,2 %) 0 45 yığlca (yığılca ecotype) 0 0 45 (100%) 0 0 45 hatay (a.m.syriaca) 0 0 0 45 (100%) 0 45 kırklareli (a.m.carnica) 0 0 0 3 (6.7 %) 42 (93.3 %) 45 number of coloniesand percent classifications are in parentheses were given in each column. ** indicate collection sites which are isolated area 93.8% of original grouped cases correctly classified. table 2. predicted group membership of honeybee samples from isolated areas including native anatolian honeybee races. fig 3. the discrimination of native honeybee race and ecotypes from isolated area on two dimensional scatter plot by using geometric morphometric. m kekeçoğlu – the effect of migratory beekeeping on honeybee biodiversity236 cross validation tests correctly identified 93.8%of native colonies from isolated original locations using geometric morphometrics. if only the bee colonies from artvin are taken into consideration, four of 60 identifications were incorrectly identified as the muğla ecotype (6.7%) and two as the yığılca ecotype (3.3%) of a. m. anatoliaca. the most common errors in the identifications were made in the honeybee colonies from artvin and muğla provinces.cross validation tests were able to correctly identify 100% of the honeybees from the yığılca and hatay populations and 93.3% of the honeybee samples from kırklareli (table 2). geometric morphometric differences in common apiary honeybee populations discrimination of the honeybee populations of the common apiary was carried out based on dfa. higher eigenvalues for four canonical functions were extracted from the matrix resulting from the product of the among-group by the within-group covariance matrix. the first two canonical discriminant functions calculated using the data from the honeybee colonies of the common apiary were able to explain 87.5% of the among-group variation. the first and second canonical discriminant functions explained 59.8% and 27.7% of the total variation, respectively. function one included y0, y1, y2, y3, y4, y5, y6, y7, y8, y9, y10, y11 and y18, and function two included x0, x1, x2, x3, x4, x5, x6, x7, x9, x10, x11, x12, x13, x14, x15, x16, y14, y15 and y16. the cva scores of the right forewings of the common apiary honeybee colonies showed that there was more overlapping among the honeybee populations and thus, there were no clear borderlines among the groups. samples from the common apiary where all turkish honeybee subspecies had been kept together in the same area showed that significant alterations of wing formation and loss of homogenic characteristics had occurred and new heterogeneous groups had been formed (fig 4). the canonical discriminant function data generated via geometric morphometrics were able to identify and assign the honeybee populations of the common apiary to their original populations based on wing-shape data. as a result, 54.4% of the honeybee colonies were correctly classified. correct classification rates of the bee populations were: a. m. caucasica 51.1%, a. m. carnica 55.6%, a. m. syriaca 61.7% and yığılca ecotype of a. m. anatoliaca 50 % (table 3). the honeybee colonies from the common apiary were not easily assigned to groups, possibly because significant morphological detailswere fragmentary, damaged, or obscured. they created an overlapping homogeny group on the scatter plot and showed fewer differences (fig 4, table 3). in contrast, the differences between the native ancestor honeybee populations from isolated locations were found to be significant for all of the variables except x0 and y12 (p < 0.05). the discriminant function analysis of the two groups (native ancestor and common apiary population) was also supported by the upgma clustering based on the matrix of mahalanobis distances. as shown on the first upgma dendrogram (fig 5a), the native ancestor population of kırklareli (kir), naturally covering a. m. carnica,was grouped with muğla and hatay, whereas on the second upgma dendogram (fig 5b) of the common garden apiary, a. m. carnica (car) was clustered with a. m. caucasica (cau) subspecies in common apiary a.m.caucasica a.m.carnica muğla ecotype a.m.syriaca yığılca ecotype total a.m.caucasica 23 (51.1 %) 10 (22.2 %) 4 (8.9 %) 2 (4.4 %) 6 (13.3 %) 45 a.m.carnica 4 (8.9 %) 25 (55.6 %) 8 (17.8 %) 5 (11.1 %) 3 (6.7 %) 45 muğla ecotype 3 (10 %) 5 (16.7 %) 11 (36.7 %) 9 (30 %) 2 (6.7 %) 30 a.m.syriaca 2 (3.3 %) 5 (8.3 %) 13 (21.7 %) 37 (61.7 %) 3 (5 %) 60 yığılca ecotype 8 (26.7 %) 3 (10 %) 2 (6.7 %) 2 (6.7 %) 15 (50 %) 30 regarding geometric morphometric 54.4% of original grouped cases correctly classified. table 3. predicted group membership of honeybee colonies from common apiary covered all honeybee races of turkey. fig 4. the discrimination of honeybee colonies from common apiary on two dimensional scatter plot by using geometric morphometric. sociobiology 65(2): 232-243 (june, 2018) 237 and the yığılca ecotype of a. m. anatoliaca (yige). the configuration differentiation between the first and second upgma dendrograms showed alteration of the morphometric construction due to hybridization (figs 5a and b). in addition, cross validation tests were also performed to determine the group affinity of the colonies. the ancestors of the populations of yığılca, muğla, artvin, kırklareli and hatay (figs 6 and 7) were known, while the colonies of the common apiary were maintained in the same area and the honeybees could have come into contact with each other. interbreeding between the subspecies can cause hybridization, morphometric deformation and emergence of novel morphometric characteristics. the mahalanobis squared distance among the populations was used to construct a upgma dendogram based on the matrix of the mahalanobis distance. as shown on the dendrogram, the native ancestor honeybees from isolated areas and the common apiary honeybees formed two distinct main clusters. except for the native muğla ecotype of a. m. anatoliaca, all ancestor honeybee populations constituted first main clusters, while the honeybees of the common apiary were located on a second cluster. furthermore, the first main cluster was divided into two groups revealing artvin as one cluster and yiğilca, kirklareli, and hatay as a second cluster. muğla was distinguished from the first main cluster (a), and the second main cluster (b) included the common apiary honeybees, a. m. carnica, a. m. caucasica, a. m. syriaca,the yığılca and muğla ecotypes of a. m. anatoliaca and the native muğla honeybee population (fig 7). fig 5. upgma dendogram constructed from mahalonobis distance a: native area art: artvin, yig: yığılca, mug: muğla, hat: hatay, kir: kırklareli; b: common apiary where all native honeybee subspecies were maintained altogether; cau: a. m. caucasica, syr: a. m. syriaca, car: a. m. carnica, mug: muğla ecotype of a. m. anatoliaca and yig: yigilca ecotype of a. m. anatoliaca. fig 6. two dimensional clustering by discriminant function analysis of colony means of honeybee subspecies from 5 isolated native area, artvin and ardahan (a. m. caucasica), kirklareli (a. m. carnica), muğla (muğla ecotype of a. m. anatoliaca), hatay (a. m. syriaca), düzce (yığılca ecotype of a. m. anatoliaca) and common apiary (a. m. caucasica, a. m. syriaca, a. m. carnica, mugla and yigilca ecotype of a. m. anatoliaca). a: isolated native area, b: common apiary. comparison of honeybee populations in native areas with those in the common apiary honeybee colonies from both the native areas and the common apiary were evaluated together by dfa, cva analysis and cross validation tests. native honeybee populations from artvin, kırklareli, hatay and muğla formed distinct clusters completely separated from the samples of the common apiary along the first cv axis (lambda = 0.002, chisq. = 3600.428, df = 280, p< 0.000). the yığılca honeybee ecotype of a. m. anatoliaca and the common apiary were further separated along the second cv axis (wilks’lambda = 0.14, chisq = 2399.686, df = 243, p<0.000). three main clusters occurred in canonical variant analysis (cva) (fig 6). as shown in fig 6, all the common apiary honeybee populations formed a completely overlapping group on the plot, whereas they would normally be expected to coincide with their own native ancestor honeybee populations. fig 7. the classification of the honeybee population based on upgma dendogram. a: isolated native area, b: common garden apiary. standard morphometric in addition to geometric morphometrics, standard morphometric analyses were used to determine whether quantifiable morphometric alteration could have occurred in any wing characteristics of the pure honeybee populations due to hybridization as a result of interbreeding in the common apiary. a total of 19 landmarks consisting of nine lengths, 17 angles and four index characters were evaluated on the right forewing, leg and proboscis. the results of 37 morphometric characteristics were accurately determined and recorded (tables 4 and 5). findings for the forewing showed that there were significant differences between the native ancestors and the hybrid common apiary honeybees according to morphological characteristics, with the exception of wl, tu, tb, b, c and d length. although the highest discodial shift (1.71 mm) was seen in the native artvin honeybees (a. m. caucasica), after the native colonies had been relocated and kept all together in the common apiary, the discodial shift (1.65 mm) of a. m. caucasica in the common apiary was decreased a b m kekeçoğlu – the effect of migratory beekeeping on honeybee biodiversity238 due to the morphometric modifications occurring in the large and small spatial scales. it can also be seen that the n23 values of a. m. caucasica (87.30 mm) and the yığılca ecotype of a. m. anatoliaca (89.95 mm) in the common apiary were lower than those of the native ancestors in the yığılca and the other stationary populations. the a4 value of theyığılca ecotype in the common apiary was higher than that of the yığılca stationary population as well. the cubital index (ci) is an important wing characteristic for a. m. carnica. the highest ci value was found in kırklareli province, covering the native ancestors of the a. m. carnica race. the ci value of the native kırklareli population was greater than that of a. m. carnica hybridized in the common apiary. the lowest ci values were those of the native hatay population (table 4). isolated native area common apiary length and ındex characters abv. mean±std. dev. mean±std. dev. artvin caucasica (n=60) kırklareli carnica (n=45) muğla ecotype (n=45) hatay syriaca (n=45) yığılca ecotype (n=45) a.m. caucasica (n=45) a.m. carnica (n=45) muğla ecotype (n=30) a.m. syriaca (n=60) yığılca ecotype (n=30) wing length wl 9,81±0,30 9,57±0,21 9,51±0,32 9,54±0,29 9,74±0,27 9,86±0,20 9,68±0,19 9,39±0,18 9,39±0,23 9,81±0.15 wing width ww 3,24±0,14 3,15±0,07 3,18±0,15 3,17±0,09 3,31±0,07 3,27±0,08 3,23±0,07 3,16±0,09 3,14±0,11 3,34±0.06 femur length fl 3,21±0,15 3,18±0,11 3,15±0,11 3,23±0,13 3,21±0,13 3,32±0,13 3,25±0,12 3,25±0,10 3,21±0,10 3,33±0,07 tibia length tl 3,19±0,13 3,14±0,09 3,10±0,13 3,11±0,10 3,10±0,21 3,30±0,12 3,19±0,12 3,18±0,10 3,17±0,10 3,26±0,10 basit. length bl 2,09±0,08 2,04±0,08 2,01±0,09 2,01±0,09 2,05±0,09 2,13±0,09 2,06±0,08 2,01±0,08 1,98±0,09 2,15±0,09 basit. length bw 1,21±0,07 1,17±0,06 1,16±0,07 1,14±0,08 1,23±0,08 1,24±0,06 1,20±0,05 1,19±0,05 1,19±0,05 1,27±0,06 proboscis length pl 6,44±0,50 6,56±0,31 6,44±0,43 5,96±0,63 6,77±0,20 6,67±0,32 6,50±0,40 6,68±0,17 6,56±0,17 6,89±0,24 radial length radl 3,58±0,13 3,43±0,08 3,45±0,09 3,51±0,09 3,58±0,10 3,58±0,07 3,53±0,08 3,46±0,09 3,45±0,09 3,64±0,06 length a a 0,51±0,05 0,52±0,06 0,52±0,06 0,53±0,06 0,57±0,05 0,54±0,05 0,57±0,04 0,53±0,06 0,51±0,07 0,54±0,03 length b b 0,27±0,04 0,26±0,04 0,24±0,02 0,24±0,04 0,25±0,03 0,27±0,03 0,27±0,03 0,26±0,03 0,27±0,04 0,23±0,03 length c c 0,91±0,05 0,88±0,04 0,88±0,03 0,89±0,04 0,93±0,04 0,91±0,03 0,93±0,04 0,88±0,03 0,88±0,04 0,92±0,03 length d d 1,99±0,08 1,93±0,05 1,92±0,05 1,92±0,06 2,02±0,06 1,99±0,05 1,95±0,05 1,92±0,05 1,90±0,07 1,99±0,05 inner wing length iwl 4,56±0,15 4,44±0,10 4,43±0,0,9 4,45±0,11 4,62±0,11 4,57±0,10 4,51±0,09 4,40±0,10 4,41±0,14 4,56±0,08 inner wing width iww 2,07±0,08 1,99±0,05 1,95±0,05 1,98±0,06 2,05±0,06 2,06±0,06 2,04±0,05 1,99±0,06 1,98±0,07 2,08±0,05 cubital index ci 1,92±0,41 2,00±0,46 2,16±0,34 2,20±0,49 2,26±0,43 2,00±0,36 2,10±0,32 2,04±0,32 1,95±0,45 2,30±0,34 pre cubital index pci 2,77±0,14 2,71±0,16 2,82±0,18 2,78±0,14 2,80±0,14 2,74±0,13 2,74±0,17 2,70±0,18 2,71±0,17 2,73±0,12 dum bel index dumbi 0,86±0,08 1,01±0,11 0,92±0,08 0,97±0,10 0,95±0,08 0,92±0,07 0,98±0,08 0,95±0,09 0,96±0,10 0,83±0,06 radial index ri 1,73±0,07 1,67±0,05 1,64±0,05 1,68±0,05 1,73±0,05 1,74±0,04 1,73±0,05 1,68±0,04 1,69±0,05 1,74±0,04 discodial shift discs 0,34±0,10 0,23±0,11 0,28±0,08 0,29±0,12 0,26±0,17 0,15±0,12 0,21±0,11 0,29±0,14 0,34±0,13 0,25±0,13 disc length discsl 1,71±0,07 1,66±0,05 1,63±0,05 1,66±0,06 1,70±0,06 1,73±0,05 1,72±0,66 1,65±0,05 1,66±0,06 1,73±0,05 table 4. means ± stdoflengths and indexcharacters determined by standard morphometric method by using 19 landmark. discussion in this study, in order to evaluate the effect of hybridization on turkish honeybee biodiversity due to migratory beekeeping, honeybee colonies representing three turkish honeybee races and two ecotypes were placed in the common apiary where they were subjected to interaction studies. the hybridization effect was evaluated by examining the morphological deformation in the wings of worker bees. the results of cva showed that populations from düzce-yığılca, muğla, artvin, hatay and kırklareli, respectively, were not known ancestors of the common apiary populations, which included a. m. anatoliaca, a. m. meda, a. m. caucasica, a. m. syriaca and a. m. carnica races. in previous studies, the distribution of the subspecies of apis mellifera in the native areasof this study was found as: a. m. anatoliaca in muğla and yığılca, a. m. caucasica in artvin, a. m. carnica in kırklareli and a. m. syriaca in hatay (kence, 2006; bodur et al., 2007; kekeçoğlu et al., 2009; kekeçoğlu & soysal, 2010a).thus, it was expected that each honeybee subspecies of the common apiary would be grouped with its own parental native honeybee population on the scatter plot (fig 5); however, this was not the case. sociobiology 65(2): 232-243 (june, 2018) 239 is o l a t e d n a t iv e a r e a c o m m o n a p ia r y a ng le s a bv . m ea n± st d. d ev . m ea n± st d. d ev . a rt vi n (n =6 0) k ır kl ar el i ( n =4 5) m uğ la ( n =4 5) h at ay ( n =4 5) y ığ ılc a (n =4 5) a .m . c au ca si ca (n =4 5) a .m . c ar ni ca (n =4 5) m uğ la e co ty pe (n =3 0) a .m . s yr ia ca (n =6 0) y ığ ılc a ec ot yp e (n =3 0) a ng le a 1 a 1 22 ,3 2± 4, 24 23 ,6 8± 5, 17 22 ,3 7± 4, 02 20 ,5 2± 2, 60 20 ,6 3± 2, 98 23 ,1 7± 5, 61 24 ,3 8± 4, 98 21 ,5 2± 5, 40 21 ,0 9± 5, 52 24 ,9 1± 5, 86 a ng le a 4 a 4 35 ,5 0± 2, 94 33 ,2 0± 3, 14 32 ,4 6± 2, 44 33 ,0 2± 3, 23 32 ,5 4± 2, 25 35 ,5 1± 3, 04 33 ,1 5± 2, 77 35 ,1 3± 3, 51 34 ,5 4± 3, 43 35 ,3 0± 2, 92 a ng le b 3 b 3 80 ,9 6± 4, 06 79 ,4 3± 3, 80 79 ,6 9± 3, 92 79 ,0 8± 4, 77 78 ,8 6± 5, 43 79 ,6 8± 4, 26 79 ,3 6± 5, 04 78 ,1 6± 4, 64 79 ,5 7± 4, 76 81 ,2 7± 3, 65 a ng le b 4 b 4 98 ,6 3± 6, 15 10 2, 15 ±6 ,5 5 10 3, 36 ±6 ,4 8 10 3, 06 ±5 ,7 0 10 0, 67 ±8 ,1 5 96 ,1 7± 7, 43 97 ,7 3± 6, 63 96 ,6 1± 6, 55 99 ,3 6± 6, 55 99 ,9 1± 7, 13 a ng le d 7 d 7 10 1, 78 ±3 ,6 4 99 ,6 2± 3, 14 10 1, 69 ±3 ,2 9 99 ,9 6± 3, 87 10 0, 06 ±3 ,4 5 99 ,2 8± 3, 30 99 ,7 5± 3, 16 99 ,4 4± 3, 21 99 ,9 6± 4, 05 10 3, 28 ±2 ,8 2 a ng le e 9 e 9 19 ,0 7± 1, 98 19 ,9 2± 2, 20 18 ,4 0± 1, 67 18 ,6 8± 2, 09 18 ,2 4± 1, 65 17 ,6 9± 1, 86 18 ,6 7± 1, 87 18 ,3 9± 2, 08 18 ,8 3± 2, 31 19 ,4 5± 2, 17 a ng le g 7 g 7 25 ,6 9± 17 ,2 9 24 ,0 9± 1, 40 23 ,5 7± 1, 26 25 ,8 7± 15 ,6 9 28 ,6 0± 23 ,8 2 24 ,0 9± 1, 07 25 ,2 2± 1, 18 24 ,3 3± 1, 06 24 ,3 3± 1, 31 23 ,8 7± 0, 94 a ng le g 18 g 18 90 ,0 4± 4, 21 89 ,0 7± 4, 91 90 ,2 4± 3, 49 88 ,5 1± 4, 80 87 ,6 0± 3, 68 89 ,2 6± 4, 58 88 ,7 5± 5, 29 90 ,5 7± 6, 45 89 ,9 1± 5, 56 90 ,6 7± 4, 33 a ng le h 12 h 12 14 ,1 3± 2, 07 18 ,1 4± 2, 53 15 ,4 6± 2, 44 14 ,7 2± 2, 88 15 ,5 6± 2, 07 15 ,8 8± 3, 03 16 ,1 6± 3, 41 16 ,4 5± 3, 13 17 ,2 9± 3, 65 14 ,5 0± 2, 97 a ng le j 10 j1 0 51 ,3 6± 4, 91 53 ,7 4± 5, 50 51 ,6 5± 4, 11 51 ,5 9± 4, 69 52 ,3 7± 9, 95 51 ,3 1± 5, 18 50 ,1 6± 5, 00 52 ,3 9± 6, 69 49 ,9 1± 4, 85 52 ,8 7± 4, 66 a ng le j 16 j1 6 85 ,8 9± 6, 07 89 ,2 2± 6, 01 88 ,6 5± 3, 77 87 ,7 8± 5, 20 89 ,3 7± 5, 28 85 ,4 7± 4, 49 86 ,6 4± 5, 37 88 ,2 5± 6, 09 87 ,5 2± 6, 63 80 ,7 5± 3, 52 a ng le k 19 k 19 73 ,8 4± 3, 65 78 ,6 0± 5, 37 76 ,0 9± 4, 43 75 ,7 2± 5, 26 75 ,6 7± 3, 88 77 ,7 0± 5, 05 77 ,6 1± 5, 28 77 ,5 6± 5, 72 79 ,2 5± 5, 52 75 ,4 9± 4, 85 a ng le l 13 l 13 13 ,7 7± 1, 68 11 ,9 3± 1, 86 13 ,3 3± 1, 32 12 ,4 8± 1, 52 15 ,0 8± 15 ,9 6 24 ,2 2± 38 ,1 1 13 ,6 3± 1, 52 13 ,2 3± 2, 15 13 ,0 2± 1, 89 13 ,9 2± 1, 71 a ng le m 17 m 17 33 ,0 7± 5, 36 32 ,8 5± 4, 32 31 ,5 6± 3, 38 31 ,5 8± 4, 83 31 ,5 5± 4, 39 30 ,1 4± 5, 14 31 ,7 7± 5, 54 31 ,1 4± 5, 26 30 ,6 7± 5, 36 29 ,2 4± 2, 84 a ng le n 23 n 23 87 ,3 0± 5, 91 91 ,9 5± 5, 73 88 ,6 7± 3, 70 90 ,1 7± 4, 81 89 ,9 5± 5, 40 85 ,2 3± 5, 20 85 ,3 7± 5, 13 86 ,7 0± 5, 18 86 ,3 8± 7, 86 82 ,7 2± 3, 55 a ng le o 26 o 26 36 ,0 6± 5, 94 37 ,7 2± 7, 28 37 ,2 6± 5, 00 36 ,4 0± 5, 70 39 ,1 5± 5, 64 38 ,6 6± 4, 42 37 ,4 9± 5, 85 40 ,6 9± 8, 56 34 ,1 7± 7, 40 33 ,3 0± 3, 58 a ng le q 21 q 21 36 ,5 1± 1, 77 38 ,2 9± 17 ,0 6 36 ,4 4± 1, 76 37 ,9 6± 1, 87 35 ,7 0± 1, 75 40 ,6 7± 22 ,3 3 43 ,3 6± 26 ,3 7 47 ,4 6± 32 ,7 4 36 ,5 2± 3, 92 36 ,8 6± 1, 80 c om m on a pi ar y: a ll co lo ni es r ep re se nt ed fi ve d if fe re nt h on ey be e su bs pe ci es o f t ur ke y ha ve b ee n m ai nt ai ne d al to ge th er in c om m on a pi ar y si nc e 20 08 . t ab le 5 . m ea n ±s td of an gl e ch ar ac te rs d et er m in ed b y st an da rd m or ph om et ri c m et ho d by u si ng 1 9 la nd m ar ks . m kekeçoğlu – the effect of migratory beekeeping on honeybee biodiversity240 the upgma dendogram showed that allof the muğla native samples were classified in their real group, whereas the common apiary samples were not classified in their native ancestor groups although the samples originated from the native areas of artvin, hatay, kırklareli, yığılca and muğla. in addition, the common apiary samples had different morphological characteristics than the native ancestor samples regarding a1, a4, b4, d7, j16, k19, l13, m17, n23, m17, o26, radf, a, b, d, innl, dumb, radi and disshıft. all native honeybee races had unique morphometric characteristics. after maintaining these bee subspecies all together in the common apiary, the occurrence of morphometric deformations was made possible, depending on the random mating among the bee races in common apiary. for example, although the ci value was 2.00 for the native bees of kırklareli (a. m. carnica), for those in common apiary it was 2.10. the a4, a3 and b4 values of the common apiary samples were higher than those of the native bees, although the same samples from common apiary showed a similarity with the native samples. this variation was attributed to hybridization. consistent with this idea is the observation that in turkey, native honeybee subspecies and ecotypes have been exposed to introgressive hybridization with caucasian bees (güler, 2010). future honeybee conservation efforts should therefore be aimed at reducing the introduction of alien subspecies into isolated areas already occupied by native honeybees. by cross validation testing, the assignment of native honeybee colonies to their population of origin was 93.8%, whereas the assignment of the hybrid honeybee colonies in the common apiary was 54.4%. the overall success of group designation decreased when the common apiary populations were taken into account, as they overlapped to form one group. these results demonstrated that the low rates of correct classification of the common apiary colonies (table 3) and the overlapping of different honeybee subspecies on the scatter plot (fig 4) indicated that a gene flow between honeybee subspecies had occurred. because all colonies representing three different subspecies and two ecotypes in turkey had been maintained all together in the common apiary since 2008, the native subspecies had come into contact with each other resulting in different formations of new heterogeneous groups and loss of homogenic characteristics due to random mating of honeybee subspecies.the mating system of the honeybee is considered to lead to one of the most extreme form of panmixia in the animal kingdom because it is based on the aggregation of thousands of males from many colonies at drone congregation areas, which virgin queens visit in order to mate repeatedly with tens of drones. controlling queen mating is extremely difficult within a small area (koeniger & koeniger, 2000; de la rùa et al., 2009). gene flow between indigenous honeybee subspecies maintained in the same area is therefore probable. it is recognized that native honeybee subspecies and ecotypes might have lost their characteristics because of the hybridization caused by migratory beekeeping, commercial queen bee usage, and uncontrolled mating (ruttner, 1988; rinderer et al., 1990; moritz, 1991; kauhausen-keller et al., 1997). migratory beekeeping, in which beekeepers move from north to south and from east to west following the blooming of the target honey plants, is very common and predominant in turkey. this means that the hives have to be moved from one place to another with the goal of ultimately collecting goodquality honey in reasonable quantities. beekeepers transport their hives extensively during the year, up to distances of 2000-4000 km (sıralı, 2002). the natural distribution of honeybee races and ecotypes has been affected seriously by this migratory beekeeping practice. in many regions of turkey, the native honeybee races and local ecotypes have been destroyed or hybridized (de la rùa, 2009; güler, 2010). in order to solve this problem, the movement of migratory beekeepers into certain districts in the seven geographic regions of turkey should be legally regulated. yet, there is not one regulation or restriction related to the routes that the beekeepers use. urgent regulation of routes used for migratory populations and legal geographical boundaries related to migratory beekeeping should be determined in order to prevent the gene flow between migratory populations and local populations. this is a crucial issue for the conservation of the current biodiversity of honeybees. to preserve the current diversity and genetic structure as much as possible, extensive isolated areas should be established in order to reduce the effect of migratory beekeeping. moreover, rules on the commercial trade of queen bees should be determined in order to prevent the loss of genetic diversity and the homogenization of the genetic structure of the honeybee population. first of all, the most important guarantee for the preservation of the genetic resources of the turkish honeybee races or ecotypes having different genetic composition is the registration of all the distinct natural turkish honeybee races and ecotypes. at present, the only standard of identification and registration is that of the caucasian bee race (a. m. caucasica), recorded under the declaration of the registration of native animal races and ecotypes (official gazette, 2004). (no: 2004/39, official gazette no. 25668, 12 december 2004, http://rega.basbakanlik.gov.tr/eskiler/2004/12/20041212. htm. this registration standard includes biochemical and molecular markers of dna in addition to the morphological characteristics. unfortunately, to date, there have been no registration studies establishing standards forthe anatolian (a. m. anatoliaca) honeybee. some scientific research based on morphometric, biochemical, and genetic analyses carried out for this purpose has reported the identification of ecotypes in isolated areas and other regions (asal et al., 1995; kandemir & kence, 1995; güler & kaftanoğlu, 1999a,b; kandemir et al., 2000, 2006; kekeçoğlu, 2009, 2010a,b); however, legal protection has still not been established. naturally-evolved, region-specific species can be threatened with extinction (mooney & cleland, 2001) through genetic pollution, potentially causing uncontrolled http://rega.basbakanlik.gov.tr/eskiler/2004 /12/20041212.htm http://rega.basbakanlik.gov.tr/eskiler/2004 /12/20041212.htm sociobiology 65(2): 232-243 (june, 2018) 241 hybridization, introgression and genetic swamping. these processes can lead to homogenization or replacement of local genotypes as a result of either a numerical and/or fitness advantage of the introduced plant or animal (aubry et al., 2005). non-native species can threaten native plants and animals with extinction by hybridization and introgression, either through purposeful introduction by humans or through habitat modification, bringing previously isolated species into contact. these phenomena can be especially detrimental for rare species coming into contact with more abundant ones. interbreeding between the species can cause a ‘swamping’ of the rarer species’ gene pool, creating hybrids that supplant the native stock. the extent of this phenomenon is not always evident from outward appearance alone. although some degree of gene flow occurs in the course of normal evolution, hybridization with or without introgression may threaten a rare species’ existence (rhymer & simberloff, 1996). these patterns are in accordance with the findings of this study. morphometric, alloenzymatic and genetic studies have shown that there are five subgenera of a. mellifera throughout turkey, including a. m. anatoliaca, a. m. meda, a. m. caucasica, a. m. syriaca and a. m. carnica (ruttner, 1988; smith et al., 1997; palmer et al., 2000; kandemir et al., 2006). in the present study, geometric morphometrics correctly identified the majority of all native honeybees except the kırklareli natural a. m. carnica. the cva scatter plot showed that except for the kırklareli, all native honeybee populations formed non-overlapping distant clusters. in the cross-validation test, some colonies from the native kırklareli were assigned to the native hatay population. the cubital index is an important wing characteristic for a. m. carnica (rutner, 1988). in general, the highest ci value was found in kırklareli province in the native ancestors of the a. m. carnica race (table 4). although the sampling area in kırklareli was declared to be the origin of the a. m. carnica, some hybridization was observed due to lack of a sufficient legal regulatory mechanism. in previous studies, kırpık et al. (2010) and güler (2010) reported similar results for the native a. m. caucasica population. as for the native kırklareli population, opinions differ regarding identification of the thracian honeybees in the european part of turkey. some apicultural scientists have described thracian bees as ecotypes of apis mellifera anatoliaca and others as those of apis mellifera carnica (adam, 1983; ruttner, 1988; smith, 1997). bees, as the main pollinators of food crops, represent a critical natural resource which needs to be carefully exploited and managed. in recent years, however, hybridization caused by migratory beekeeping has modified the genetic pool of the local honeybee population, leading to the loss of their unique genetic and morphological traits (de la rùa et al., 2009; güler, 2010). the present study has emphasized the effect of this hybridization on the diversity of turkish honeybees throughout all specific regions. studies on the morphometric divergence of anatolian honeybees through loss of their original traits have been quite limited (kence, 2006; güler, 2010; kekeçoğlu & soysal, 2010b). hence, the findings of this study provide a significant contribution to the current program for the conservation of native turkish honeybee gene resources. acknowledgements authors would like to thanks to dr. nazife eroğlu yalçın, the scientific and research council of turkey, marmara research center, food institute, pk. 21, 41470, gebze, turkey, for the sampling of worker honeybees. and also i would like to thanks to babacan uğuz for construction of geometric morphometric software, bab bs200pro image analysis systems. references anonym, official gazette, (2004). 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(2015). metatranscriptomic analyses of honey bee colonies. frontiers in genetics, 6: 1-12. doi: 10.3389/fgene.2015.00100. https://link.springer.com/article/10.1051/apido/2010048 http://doi.org/10.1073%2fpnas.091093398 http://dx.doi.org/10.1080/00218839.1991.11101234 https://doi.org/10.1093/jhered/91.1.42 http://www.annualreviews.org/doi/10.1146/annurev.ecolsys.27.1.83 http://www.annualreviews.org/doi/10.1146/annurev.ecolsys.27.1.83 https://doi.org/10.1093/aesa/83.3.346 https://doi.org/10.1051/apido:19970503 https://doi.org/10.1051/apido:19970505 https://doi.org/10.1051/apido:19970505 https://doi.org/10.1051/apido:2003037 http://www.spss.com.tr https://doi.org/10.3389/fgene.2015.00100 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5905sociobiology 68(1): e5905 (march, 2021) introduction european honey bees (apis mellifera l.) are among the most investigated insects in turkey and globally, primarily because of their significant role in agriculture and the ecosystem and their high economic value. honey bees are important not only for the honey, propolis, pollen, and wax they produce, but also as vital pollinators of agricultural and horticultural crops. a highly adaptable species, their range stretches from the southern parts of scandinavia to central asia and throughout africa (sheppard & meixner, 2003). a broad diversity of microorganisms is associated with honey bees (engel et al., 2016). in recent years, severe losses of bees abstract apis mellifera, widely farmed around the world, is the most economically important species within the genus apis. while the microbiota of live honey bees have been extensively examined, bacteria found in deceased honey bees (which might indicate infection or opportunistic pathogens) is in contrast poorly studied. therefore, we decided to investigate the mesophilic bacterial flora of dead honey bees. so, in september 2013, dead adult worker honey bees were collected from 12 different cities, most of which were in the border provinces of turkey. we identified bacterial isolates at the species level by using different morphological, biochemical, physical and molecular methods, in conjunction with molecular phylogenetic analysis. we constructed phylogenetic trees for isolated bacteria with the mega 6.0 program and neighbor-joining trees were reconstructed based on 16s rdna gene sequences. the phylogenetic trees indicated that isolates de003, de007, de011, de001, de019 and de016, de029 could be new members of the genera erwinia, acidovorax, hydrogenophaga and bacillus genus, respectively. in the bioassay study results, we observed that de019 hydrogenophaga sp. (64.7%) and de004 klebsiella grimontii (73.3%) had lethal effects on the honey bees. the other mortalities ranged from 10% to 25% (p>0.05), and according to a one-way anova analysis de004 and de019 significantly affect the a. mellifera caucasia in adult worker honey bees. this study is the first report of hydrogenophaga as honey bee pathogen. sociobiology an international journal on social insects elif çil1, ömer ertürk2, kamil işik3 article history edited by evandro n. silva, uefs, brazil received 06 october 2020 initial acceptance 19 february 2021 final acceptance 07 march 2021 publication date 31 march 2021 keywords honey bee, vitek®2, mis, 16s rdna. corresponding author elif çil cumhuriyet mah. ordu university, faculty of education, department of math and science 52200 altınoru/ordu, turkey. e-mail: elifdemir80@gmail.com from beehives and a decline in bee populations have been reported by scientists (stathers, 2017; potts et al., 2016). it has been hyphothesised that the pollinators face numerous threats, including pathogenic microorganisms. honey bees proceed through embryonic, larval, pupal, and adult development stages that each comprises a distinct ecological niche for microbes. how and if microbes transition successfully through this phase is mostly unknown, even though most microbes have transcribed to infect only the adult stage of the honey bee (kwong & moran, 2016). honey bee gut microbiota is well known nowadays, but it’sthis notdoes thenot sameapply forto its opportunistic pathogens. for bacterial community analyses, scientists 1 department of math and science, ordu university, ordu, turkey 2 department of molecular biology and genetics, ordu university, ordu, turkey 3 department of biology, ondokuz mayıs university, 55200 samsun, turkey research article bees identification of mesophylic bacterial flora in deceased worker adults of apis mellifera caucasia (pollmann, 1889) id https://orcid.org/0000-0003-1420-8729 elif çil, ömer ertürk, kamil işık – mesophylic bacterial flora in deceased worker bees2 generally useduse full-length 16s rdna sequences, single-cell genomic sequencing, or metagenomic datasets (kwong & moran, 2016). the full-length 16s rdna sequence analyses can be used for culture-based bacteria identification with the combination of phenotypic, biochemical characterization (engel et al., 2016). here we aim to debate the present-day information on bacteria found in bees, so we isolated, cultivated, and characterised the isolates from dead adult honey bee workers from different border locations in turkey. materıal and methods the sample of honey bees was collected from twelve locations throughout the border provinces in turkey from october to november 2013. they were immediatelly transported to the laboratory in plastic boxes (20cm×20 cm) with punched lids by ordu apiculture research institute staff. we used small sugar cakes to feed the honey bees during transport. then we separated the dead adult bees and stored them at -20 °c (the locations were mentioned in fig 1). we obtained bacterial isolates from dead a. mellifera caucasia l. adults by homogenisation. they were individually surface sterilised by using 70% ethanol for 3 min and washed three times with sterile water, according to boğ et al. (2020). the deceased honey bee bodies were homogenised in a feeder medium using a glass tissue mill, immersed in to the nutrient agar, and incubated at 30 °c for a week. the residuary mixtures were incubated at 30 °c to increase the number of bacteria. first, bacterial isolates were distinguished based on colony colour and morphology. pure cultures of bacterial isolates were preserved in 20% glycerol (v/v) at -20 °c at ordu university, department of molecular biology and genetics, laboratory of microbiology. we counducted gram staining first. we then tried to collect data from vitek®2 and microbial identification system (mis, whole-cell fatty acid analysis by gas chromatography). for identification by vitek®2, the isolates were inoculated into the vitek gni (for gram-negative bacteria), gpi (for gram-positive bacteria), and cap (for rod-shaped bacillus bacteria) cards, incubated at 35 °c in the reader incubator module for 18 h, and automatically read hourly by the optical scanner according to the manufacturer recommendations by using bioliaison software. for the determination of fatty acid profiles of bacterial isolates, we preferred to use mis as described by kireçci and aktaş (2004). for bacillus-like organisms, we added crystal staining as described by sharif and alaeddinoğlu (1988). except for gram and crystal stainings, vitek®2, and mis tests, 37 phenotypic tests and growth temperature tests from 10°c to 60 °c at five-degree intervals were performed manually in the laboratory. for details of the tests in table 1, demir’s master thesis can be viewed (demir, 2005). bacterial genomic dna was extracted from the isolates (de001, de003, de004, de017, de019, de023, de024, de029) by the employment of exiprep plus bacteria genomic dna kit (the automated dna extraction) supplemented by the manufacturing company (bioneer/korea) and by using the exiprep 16 plus instrument (bioneer/korea). the others were extracted by using a method by modifying the “guanidine thiocyanate dna isolation method” of pitcher et al. (1989). the taxonomic positions of the bacterial isolates were determined by 16s rdna analysis. the 16s rrna genes (rdna) were amplified by using universal primers 27f (5aga gtt tga tcm tgg ctc ag-3) and 1525r (5-aag gag gtg wtc car cc-3) (lane, 1991). amplification was carried out in a thermocycler (eppendorf, mastercycler gradient, hamburg, germany) for 36 reaction cycles. fig 1. the locality of dead honey bees collected from different provinces of turkey province name, isolate number(s). sociobiology 68(1): e5905 (march, 2021) 3 the 16s rrna gene (rdna) products were run on an abi 3730 xl genetic analyzer (applied biosystems) with cycle sequencing kits (gen plaza biotechnology center, turkey). oligonucleotide primers 27f (previously described), 518f (5-ccagcagccgcggtaatac-3) (muyzer, 1993), 800r (5-taccagggtatctaatcc-3) (chun, 1995), 1492r (5-tacggytaccttgttacgactt-3) (gyobu & miyadoh, 2001). dna sequences were edited visually using mega 6.0 and aligned manually. almost-complete 16s rdna gene sequences of the isolates were deposited in the genbank. we preferred to use the current version of the ezbiocloud (http://ezbiocloud.net/; yoon et al., 2017) server. the sequence alignments were used for constructing a phylogenetic tree, which was generated for the neighbor-joining as described by jukes and cantor (1969) using the mega server 6 (tamura et al., 2013) package. the insecticidal potential of the bacterial isolates was tested under laboratory conditions. after macroscopic examination, we selected the healthy adult worker honey bees used in bioassay analyses at random. the obtained single colonies were inoculated into nutrient broth medium and incubated at 30°c overnight. some of the isolates were incubated at 30°c for two days due to their slow growth. after incubation, the bacterial density was measured with a biosan densitometer, which provided the opportunity to measure solution turbidity in wide range mcfarland units (ben-dov et al., 1995; moar et al., 1995). one ml 6.0 mcfarland bacterial suspension of each isolate was saturated into 50% glucose syrup and placed in square shape singular cages (10cm×10cm×10cm). each cage contained 40 healthy adult worker honey bees and glucose syrup suspension with a single bacterial isolate. the bacterial inocula were expected to enter the honey bee body via feeding. the control group was fed sterilised glucose syrup. we did not observe the feeding behaviour of honey bees during the experiment. each bioassay experiment was separately carried out at least twice in duplicate. mortality data were corrected by abbott’s formula (abbott, 1925). the final results were presented as mean and standard deviations. the bioassay experiment data were analysed by one-way anova, followed by tukey’s post-hoc test using paws. a value of p˂0.05 was considered significant. results in the present study, we found few bacteria isolated from dead a. mellifera caucasia adult workers and divided them into three groups by constructing three neighbour-joining trees (bacillus-brevibacillus group, non-bacillus grampositive group, and gram-negative group). on the other hand, bacterial isolates obtained from a. mellifera caucasia adults produced different mortality values compared to each other nitrojen sources tests (0.1 w/v) carbon sources tests (1% w/v) degredation tests l-proline (l-pro) d(-) arabinoz dna (10 % w/v) l-iso-leucine (l-ile) paraffine l-tyrosine (0.4% w/v) l-valine (l-val) d-mannitole hypoxanthine (0.4% w/v) l-methionine (l-met) rhamnose starch (0.1% w/v) l-cysteine (l-cys) glicose tween 20 (1% v/v) lalanine (l-ala) (+) galactose tween 40 (1% v/v) lactose tween 80 (1% v/v) other tests fructose casein* catalase activity§ d(+) xylose tween 20√ aerobic growth# tween 40√ salt concentration tolerance (5–10%, w/v) tween 80√ hemolysis on blood agar surface kligler iron agar (kia) methyl red indol citrat voges-proskauer h2s * crosley skim milk agar (oxoid) was used for casein degredation. √ their capacity to metabolize tween 20, 40 and 80 were examined on sierra medium. § catalase activity was determined by measurements of bubble production after the application of a 3% (v/v) hydrogen peroxide solution. # aerobic growth were conducted using tryptone-glucose-yeast extract (tgy) media. table 1. the microbiological analyses applied to the test microorganisms manually. elif çil, ömer ertürk, kamil işık – mesophylic bacterial flora in deceased worker bees4 and the control group. the insecticidal activity of isolates at 1.8 × 109 bacteria.ml-1 doses within seven days of application to a. mellifera. there was a statistically significant difference in the lethal toxicity of selected bacterial strains. homogeneity of variance 0.244 p>0.05 so the data is homogeneous. means for groups in homogeneous subsets were displayed in the same letter (tukey test, p<0.05). also the provinces from which the bacteria were isolated are given in parentheses. we discussed these results together by grouping organisms below. thermophilic rod-shaped, aerobic or facultatively anaerobic, endospore-forming organisms like bacillus that grow optimally over the temperature range of 45-60°c have been isolated from both thermophilic and mesophilic environments. currently, thermophilic bacilli are classified into seven genera which including bacillus and brevibacillus (bae et al., 2005). in this study, all bacillus isolates were gram-stain and catalase positive, motile, rod-shaped cell that produced endospor and also all strains was found to negative results for indole test. however, in the bacillus genus, some characters are variable such as degradation of tyrosine, nitrate reduction, hydrolysis of aesculin, etc. we selected some descriptive test characteristics for differentiation based on previously reported and proposed tests (table 2). in fig 2, the nearly complete 16s rrna gene sequences of eleven bacillus isolates were clustered together with those of closely related twelve type species. the phenotypic characters of the group (bacillus and brevibacillus isolates) were characterized following the minimal standards for aerobic, endospore-forming bacteria, recommended by logan et al. (2009) and a phylogenetic tree was constructed by neighbor-joining (fig 2). b. aryabhattai and b. aerius are aerobic endosporeforming bacteria, isolated first time from cryotubes used for collecting air from the upper atmosphere by shivaji et al. (2009). then khaled et al. (2018) isolated b. aryabhattai, b. laterosporus, b. techuilensis, and b. sonorensis strains from the digestive tract of healthy honey bees in saudi arabia. de014 (kırklareli), de023 (hakkari), de024 (hakkari), and de026 (kırklareli) colonies were white/cream opaque colour onto nutrient agar, and their endospore positions within the sporangia appeared to be generally central. all isolates in this clade grew well at 44°c and up to ph 10 but weak at 50°c and ph 5. the isolates could hydrolyze aesculin and could not hydrolysis tween 80. all four isolates were positive for the utilization of paraffin. none of the isolates produced acid from glucose, sucrose, and lactose and used l-tyrosine fig 2. neighbor-joining tree reconstructed based on 16s rdna gene sequences showing the phylogenetic relationship between bacillus isolates and closely related type strains. bootstrap values (500 replication) >50% are given at nodes. sociobiology 68(1): e5905 (march, 2021) 5 also de031’s insecticidal activity was 10±1f %. these results were the same as b. megaterium and b. aryabhattai. de014 displayed ß hemolysis, and the others in the clade displayed γ hemolysis on blood agar and also 16s rdna gene sequence similarity were given on table 2. we could say de026’s insecticidal activity was low (10±2f %). b. megaterium and b. aryabhattai are closely related to bacterial species, so this causes mismatched identifications only by commercial identification systems or 16s rdna analyses (strejcek et al., 2018). when all the results were evaluated, we concluded that de014 was b. aryabhattai, de026 was b. megaterium, and de023 and de024 could be new relatives of b. aryabhattai. bacillus frigoritolerans, a strictly aerobic, chemoorganotrophic member of the genus bacillus (formerly brevibacterium), was originally isolated from the arid soils of morocco and known as an entomopathogenic species (selvakumar et al., 2011). b. megaterium, b. simplex, and b. frigotolerans are closely related species. all isolates and type strains in this clade can not grow at 44°c but degraded dna and hypoxanthine. the isolates and type strains exhibited positive results for l-tyrosine degradation, hydrolysis of tween 80, but negative results for hydrolysis of arbutin, aesculin. nevertheless, we also had some results different from type strains in this clade (table 2). de030 (antalya) and de031 (i̇zmir) could not grow on macconkey agar as b. frigotolerans, but b. simplex could grow (beesley et al., 2010). de031 gave positive results to α-glucosidase but also gave negative results for ß glucosidase and n-acetyl-ß-glucosaminidase test like b. simplex and also could tolerate ph10 on agar medium like b. frigotolerans and also de031’s insecticidal activity was 10±1f %. de030 could be a member of b. simplex clade and de031 could be a strain of b. frigoritolerans. to better understand the phenotype differences between de022 (ordu) and de029 (niğde), we reviewed the literature of previously reported phenotypes (fritze 2004; aramideh et al., 2010; jiménez et al., 2013) and performed some additional tests. all isolates and related type strains’ aesculin, arbutin were positive and grew well at 10-40°c but not 4% nacl added medium. de022 and de029 displayed ß hemolysis on blood agar like b. cereus group as described before (fritze 2004). de022 could utilize d-mannose like b. thuringiensis atcc 10792t. however, some phenotypical characters were different from the nearest neighbors; for example, citrate and casein test results (table 2). also, de029 did not motile and gave a negative result for the l-tyrosine test. de022’s insecticidal activity was 15±1e%. we know that classifying the b. cereus group based on the synthesis of the parasporal crystals is so popular nowadays. nevertheless, these crystals generally coded by plasmids (cry genes), and b. toyonensis has lack of that plasmids. according to coomassie brilliant blue staining results, de022 and de029 could not synthesize of the parasporal crystal. the results are in good agreement with ohba et al. (2000) and jiménez et al. (2013) observations that b. cereus genomospecies and b. toyonensis with non-insecticidal parasporal inclusions are far more widely distributed in nature than those with insecticidal inclusions. our findings suggest that de022 was a strain of b. thuringiensis and de029 could be a novel species of bacillus genus. so, de029 needs to be evaluated taxonomically in more depth. de013 (edirne), de016 (niğde), and de027 (mersin) were grouped into the genus bacillus and also in the same clade, which also included b. aerius, b. paralicheniformis, and b. licheniformis. b. aerius, b. paralicheniformis, and b. sonorensis are very similar to b. licheniformis, but could not be distinguished from each other only phenotypic characteristics, mis or 16s rdna analyses (fig 2). the isolates could not be distinguished from the close relatives by fatty acid profiles, and the same results were reported previously (dunlap et al., 2015; palmisano et al., 2001). the isolates spore position within the sporangia appeared to be generally subterminal. all isolates in this clade were able to grow tryptone glucose yeast extract agar (tgy), tyrosine agar (isp 7) and mannitol salt agar (msa). the colonies grew well on isp 7 and tyg mediums. it appeared creamy white on isp 7 agar medium, mucoid on tgy at 37°c. none of the isolates and related type strains grew at ph 4. hydrolysis of aesculin and urea tests were positive. all isolates gave positive results for tween 20, tween 40, tween 80 degradation, and could tolerate 10% nacl in medium, like b. licheniformis atcc 14580t. however, l-alanine utilization test results were different from b. licheniformis, and these three isolates gave positive results like b. paralicheniformis kj-16t, b. sonorensis nbrc 101234t and b. aerius jcm 13348t. the isolates and type strains except for b. aerius jcm 13348t in this clade could tolerate 55 °c. de013, de016, and de027 had nitrate reductase enzyme, but de027 also had nitrite reductase enzyme too. de013 and de016 displayed ß hemolysis, but de027 displayed γ hemolysis on blood agar also de013’s insecticidal activity was 10.7±1.5f %. to further distinguish the isolates from its closely related bacillus species, 16s rdna gen sequences were analyzed (fig 2). according to the obtained results, we conclude that de013 and de027 were appeared to be closely related to the type strains b. paralicheniformis kj-16t. but de016 could be a novel species of the genus. in fig 3, the nearly complete 16s rrna gene sequences of three non-bacillus gram-positive isolates were clustered together with those of closely related nine type species. all isolates were non-spore forming, h2s and indole negative, methyl red positive bacteria. we selected some descriptive test characteristics for differentiation based on previously reported and proposed tests for non-spore forming grampositive bacteria (table 3). de009 (artvin) had a nearly identical 16s rdna gene sequence with staphylococcus edaphicus and s. saprophyticus type strains. de009 was aerobic cocci. β-hemolytic activity on blood agar. good growth at 10°c, 45c°, ph 10 and week growth in the presence of 15% nacl. mannitol fermentation, allantoin and hydrolysis of tween 20 tests were positive. elif çil, ömer ertürk, kamil işık – mesophylic bacterial flora in deceased worker bees6 p ro pe rt y is ol at es b. aryabhattai b. megaterium b. paralicheniformis b. toyonensis b. thuringiensis b. frigoritolerans b. simplex d e 01 4 d e 02 3 d e 02 4 d e 02 6 d e 03 0 d e 03 1 d e 02 2 d e 02 9 d e 01 3 d e 01 6 d e 02 7 m ot ile + + + + + + + + + + + + + h yd ro ly si s of c as ei n + + + + + + + + + + + + h yd ro ly si s of s ta rc h + + + + + + + + + + + + + + + g el at in as e + + + + + + + + + + + + + + + + u re as e + + + + + + + + + v og es p ro sk au er + + + + + + + + + c itr at e + + + m an ni to le + + + + + + + + + + + + + + + l -r ha m no se + + + + + + + + + + + + + + h 2s p ro du ct io n + ß ga la ct os id as e + + + + + + + + + + n itr at e re du ct io n + + + + + + + + + + v it e k ® 2 (s im ila ri ty % ) nd nd nd b. megaterium (91%) nd b. smithii (94%) b. laterasporus (90%) nd b. licheniformis (87%) nd nd m is (s im ila ri ty % ) b. megaterium (49%) b. megaterium (81%) b. megaterium (76%) b. megaterium (82%) b. viscosus (83%) b. megaterium (72%) nd nd nd nd nd 16 s rd n a si m ila ri ty w ith t he c lo se st re la tiv e ty pe s tra in % (n t d if fe re nc e) 10 0. 00 % (0 ) 99 .9 3% (1 ) 99 .9 3% (1 ) 99 .9 3% (1 ) 99 .8 6% (2 ) 99 .9 3% (1 ) 99 .8 6% (2 ) 99 .9 3% (1 ) 99 .9 2% (1 ) 99 .9 2% (1 ) 99 .9 2% (1 ) t ab le 2 . c om pa ri so ns o f t he is ol at es an d cl os el y re la te d b ac ill us ty pe s tr ai ns . c ha ra ct er is tic s ar e sc or ed a s: -, n eg at iv e re ac tio n; + , p os iti ve re ac tio n; n d, n ot d et ec te d. sociobiology 68(1): e5905 (march, 2021) 7 hydrolysis casein, esculin, l-tyrosine, dna and gelatine tests were negative. susceptible to polymyxin b (300 iu) but resistance to novobiocin (5μg). ß-galactosidase and ß-glucuronidase tests were essential to differentiate closely related staphylococcus speciesand test results were showned in table 3. de009 also showed different test results opposite to s. edaphicus that pyrrolidonyl arylamidase, tween 80 tests were negative and not resistance to bacitracin (0.2 iu). s. edaphicus showed δ-hemolytic activity on blood agar but de009 was different (β-hemolytic activity). data from the study demonstrate that although de009 are in the same clade with s. saprophyticus subsp. saprophyticus and s. edaphicus. this isolate could be a strain of s. saprophyticus subsp. saprophyticus. aerococcus had been isolated from air, soil, human, and different animal samples. cells of de018 (edirne) were coccoid. colonies were non-pigmented, circular, and greywhite on nutrient agar. also, cells produced a ß hemolytic reaction. dglucose, d-fructose, d-mannose and gelatine hydrolysis test results were positive and d-sorbitol, d-tagatose test results were negative like closely related type strains a. urinaeequi and a. viridans. aesculin (-) and trehalose (+) test results are the same as a. urinaeequi. a. urinaeequi is a non-motile organism, but de018 was motile. a. viridans caused alpha hemolysis on blood agar, but de018 caused ß hemolysis. de018’s insecticidal activity was 15±1e%. it is challenging to differentiate correctly a. urinaeequi from the closely related type strain a. viridans through only phenotypic methods or even using 16s rrna sequencing (zhou et al., 2016; rasmussen, 2016). but these results indicate that de018 was a strain of a. urinaeequi. exiguobacterium motile, rod-shaped, alkaliphilic bacterium. de017 (hakkari) yellowish-orange colony colour like e. acetylicum, e. indicum, and e. enclense. the colony property isolates s. e da ph ic us s. s ap ro ph yt ic us su bs p. s ap ro ph yt ic us a . u ri na ee qu i e . a ce ty lic um d e 00 9 d e 01 8 d e 01 7 motile + + catalase + + + + + urease + + + voges proskauer + + + + + mannitole + + + + + + + ß galactosidase + + + + ß glucuronidase + nitrate reduction + vitek®2 (similarity %) s. s ap ro ph yt ic us (9 9 % ) nd nd mis (similarity %) s. c oh ni i c oh ni i (8 0% ) nd nd 16s rdna similarity with the closest relative type strain % (nt difference) 99.86 % (1) 99.93% (1) 99.66% (5) fig 3. neighbor-joining tree reconstructed based on 16s rdna gene sequences showing the phylogenetic relationship between non-bacillus gram-positive isolates and closely related type strains. bootstrap values (500 replication) >50% are given at nodes. table 3. comparisons of the isolatesand closely related type strains. characteristics are scored as: -, negative reaction; +, positive reaction; nd, not detected. elif çil, ömer ertürk, kamil işık – mesophylic bacterial flora in deceased worker bees8 shape of de017, e. indicum, and e. enclense were round, but e. acetylicum colony shape was irregular. hydrolyze casein, gelatine, esculin, and methyl red tests were positive for de017 and e. acetylicum but negative for others. starch utilization of de017 was weakly positive. also dnase activity test was negative for de017 and e. indicum but positive for e. acetylicum. data from the study demonstrate that de017 could be a member of e. acetylicum. khan et al. (2017) reported that e. acetylicum is a gut microbiota species from riyadh, saudi arabia, and our results supported this data. in fig 4, the nearly complete 16s rrna gene sequences of seven rod-shaped, gram-negative non-spore-forming bacteria which were clustered together with those of closely related 15 type species. all strains were catalase and mannitole positive and h2s negative. we selected some descriptive test characteristics for differentiation based on previously reported and proposed tests for rod-shaped non-spore forming gram-negative bacteria (table 4). colonies of de006 (ardahan) were yellow, round, and convex like pantoea vagans. colour of p. brenneri and p. conspicua colonies were beige colour. all clade members utilized the following carbon sources at 37° c within 7 days: d-glucose, d-galactose, aesculin. however, 5-ketogluconate, lactose, and l–tyrosine test results were negative. all these test results are the same as p. vagans and p. deleyi. p. anthophila can use 5-ketogluconate weakly, and this test could be used for showing differentiation. also, tweens 40 test result was positive like closely related type organisms, but tween 80 and maltose test results, which were different from the other type strains, were negative. de006’s insecticidal activity was 20.7d±0.6. our findings suggest that de006 could be a strain of p. vagans. our first results revealed that de004 (balıkesir) was a member of k. oxytoca phylogroup. k. grimontii ‘s most closely related neighbors are k. oxytoca and k. michiganensis (fig 4). k. michiganensis and k. grimontii do not exist in the vitek® 2 databases. so, only 16s rdna gene sequence or vitek® 2 results were not enough to differentiate these isolates. so, we selected some phenotypic tests for differentiation. de004 colonies on the nutrient agar are smooth, circular, white, capsulated and glistening and grown readily on ordinary media commonly used to isolate enterobacteriaceae, e.g., nutrient agar, blood agar, and macconkey agar like k. grimontii. de004 is different from k. michiganensis because k. michiganensis’ colonies are opaque. de004 was capable of growing over a temperature fig 4. neighbor-joining tree reconstructed based on 16s rdna gene sequences showing the phylogenetic relationship between gram-negative isolates and closely related type strains. bootstrap values (500 replication) >50% are given at nodes. sociobiology 68(1): e5905 (march, 2021) 9 range of 10–45 °c with an optimum of 35 °c, vogeusproskauer and lysine decarboxylase tests were positive and ornithine decarboxylase negative as k. oxytoca, k. michiganensis, and k grimontii. melezitoze fermentation test has been used for differantiation k. grimontii from closely related type strains. de004 could not fermantiate melezitose as k. grimontii (passet & brisse, 2018). stephan et al. (2019) reported that some bacteria from a. mellifera microbiota are pathogenic and klebsiella could be one of them. de004’s a. mellifera caucasia mortality rate was 73.3a±0.6%. according to one-way anova analyses de004 which isolated from balıkesir province, had significantly affected the a. mellifera caucasia adult worker honey bees. this was the highest mortality rate among the isolates. k. grimontii is a pathogenic microorganism so this is normal and expected result. erwinia is a member of the family enterobacteriaceae like klebsiella. de003 (kırıkkale) colonies on nutrient agar were light-beige, circular, smooth. strains grew well on nutrient agar at 36-37 °c, and no diffusible pigment is observed. e. tasmaniensis is not able to grow at 36°c, but e. percinicus can grow at 36°c like de003. e. percinicus produce watersoluble pink pigment, but de003 strains do not. the indole and voges–proskauer tests are negative, but the citrate test is positively opposed to e. tasmaniensis and e. rhapontici. nitrate reduction test is an important test to distinguish the erwinia strains. nitrate reduction test result is positive, like e. rhapontici. but gelatine was liquefied, and the urease test was positively opposed to other erwinia type strains. thus, based on the phenotypic and molecular evaluation, we concluded that de003, this isolate could be a novel strain of the erwinia genus. property isolates p. v ag an s k .m ic hi ga ne ns is k . o xy to ca k . g ri m on tii e . p er si ci na h . i nt er m ed ia a . t em pe ra ns d e 00 6 d e 00 4 d e 00 3 d e 01 9 d e 00 1 d e 01 1 d e 00 7 motile + + + + + + + gelatinase + + + + urease + + + + + indole + + + + citrate + + + + + + + + + l-rhamnose + + + + + + + + + + + + d-mannose + + + + + + + + + + + + ß galactosidase + + + + + + + + + nitrate reduction + + + + + + + + + + vitek®2 (similarity %) p. a gg lo m er an s (8 1% ) k . o xy to ca (9 8% ) s. p au ci m ob ili s (9 2% ) s. p au ci m ob ili s (8 6% ) s. p au ci m ob ili s (9 6% ) nd nd mis (similarity %) p. a gg lo m er an s (9 1% ) s. c ho le ra es ui s ho ut en ae (8 8% ) nd s. o do ri fe ra (7 0% ) nd nd nd 16s rdna similarity with the closest relative type strain % (nt difference) 99.78% (3) 99.85% (2) 99.45 % (8) 99.02% (13) 99.07% (14) 98.64% (20) 98.25% (25) table 4. comparisons of the gram-negative isolatesand closely related type strains. characteristics are scored as: -, negative reaction; +, positive reaction; nd, not detected. elif çil, ömer ertürk, kamil işık – mesophylic bacterial flora in deceased worker bees10 de001 (ordu) and de019 (i̇zmir)’s partial 16s rdna gene sequence pairwise analysis result exhibited that hydrogenophaga intermedia s1t was the closest neighbor. we know that, in the vitek® 2 databases, hydrogenophaga intermedia does not exist. the genus hydrogenophaga consists of yellow-pigmented that is generally considered capable of using hydrogen as an energy source. de001 and de019 were slightly convex and smooth, pale yellow colonies on ypg agar. glucose fermentation, utilization of d-xylose, d-cellobiose, d-maltose, and l-histidine are negative; utilization of d-mannitol, gamma-glutamyl transpeptidase and lipase results were positive like h. intermedia (contzen et al., 2000). on the other hand, some phenotypical characters were different from the nearest neighbor (table 4). isolate de019’s mortality rate was 64.7b±1.2. %. according to oneway anova analyse, de019 which isolated from i̇zmir province, had significantly affected the a. mellifera caucasia adult worker honey bees. jin et al. (2018) raported that h. intermedia is a potential waterborne pathogen. our findings suggest that de001 (ordu) and de019 (i̇zmir) could be new members of this genus. de011 (artvin) and de007 (ardahan) isolates were closely related with acidovorax temperans. in the vitek® 2 databases, a. temperans does not exist like h. intermedia. all acidovorax strains can not utilize d-xylose but can degrade tween 80. de007 and de011 were grown well at 28-37°c on nutrient agar medium but not 40°c and up like a. temperans. a. delafieldii had not catalase activity but de007 and de011 had. all type strains should be gave negative results for urease and ß galactosidase tests but de007 gave positive results for both. on the other hand de011’s some phenotypic characters were different from de007 and the closest type strain. for example de007 was motile like a. temperans but de011 did not motile. it can be seen from the results in table 4 that de011 and de007 could be new members of this genus. discussion turkey has a wide variety of climatic conditions and geological structures that show significant regional differences. this has played an essential role in the evolution of many living species as it forms a natural bridge between africa, europe, and asia. honey bees are one of these species, and turkey is home to many honey bee subspecies, including a. mellifera caucasia (kence, 2006). while the microbiota of live bees have been extensively examined, bacteria found in deceased bees (which may be indicative of infection or opportunistic pathogens) is in contrast poorly studied. so, we attempted to highlight mesophilic microorganisms found in deceased a. mellifera caucasia and describe their characteristic properties in this study. the presence of bacterial isolates was revealed by culture-dependent approaches in this project. in the future, the culture-independent methodology should be added to the study program. most of the isolated bacteria were of environmental origin, which was expected. in healthy adult apis mellifera, five bacteria genus called “core” bacteria (most gram-negative proteobacteria and gram-positive firmiculates firmiculates) is found (khan et al., 2020). it also has been reported to contain multiple gut symbionts, including about 1% yeast-like microbes, 29% gram-positive bacteria such as bacillus, lactobacillus, bifidobacterium, corynebacterium, streptococcus, and clostridium, and 70% gram-negative or gram-variable bacteria such as achromobacter, citrobacter, enterobacter, erwinia, escherichia coli, flavobacterium, klebsiella, proteus, and pseudomonas (raymann & moran, 2018; khan et al., 2020). in the present study, we found the same bacteria genus, which isolated from apis cerana japonica larvae’s gut (yoshiyama & kimura, 2009). yoshiyama & kimura reported that erwinia, bacillus, and staphylococcus saprophyticus subsp. were in a. c. japonica’s gut which was the same as the european honey bee. khan et al. also reported that bacillus is one of the most frequently isolated bacteria genus from the honey bee gut for australian chalkbrood-inhibiting (khan et al., 2020). thus, it is not the first bacillus report in the honey bee studies; previously, it was reported from different parts of the world (wang et al., 2015; audisio, 2017; kačániová et al., 2020; anjum et al., 2018; boğ et al., 2020). on the other hand, bacillus is a diverse genus, including over three hundred strains and its predominance can be explained by its prevalence in environmental samples and its ability to survive in harsh conditions by forming endospores (lupan et al., 2014; luis et. al., 2020). the species within the b. subtilis complex cannot be reliably differentiated based on morphological, physiological, biochemical, or other phenotypic properties (rooney et al., 2009). according to logan et al., although 16s rrna gene sequence comparisons are valuable in the determination of approximate phylogenetic relationships at the subspecies and generic levels and higher levels for describing new taxa of aerobic, endospore-forming bacteria, they are not always appropriate for the classification of strains at the species level (logan et al., 2009). we also agree with strejcek et al. (2018) that the relationship between whole-cell mass spectra, the average nucleotide identity of orthologous genes, and biological reproducibility issues, need to be addressed in the future to maximise the benefits of similarity-based and reference-free approaches. we agree with shivaji et al. and others that bacillus is a heterogeneous genus, and 16s rrna gene sequence analysis is not enough to clarify the strains to species level (shivajii et al., 2009; jeyaram et al., 2011; owusu-darko et al., 2020). despite the high similarity in the 16s rna gene sequence, many bacillus species can differ in their phenotypic and chemotaxonomic characteristics. unconditionally a polyphasic approach should be adopted in the definition of bacillus isolates (logan et al., 2009; lana et al., 2020). according to previous studies, current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms by using a polyphasic approach, and nowadays, next-generation sequencing technologies and computational tools for analysis sociobiology 68(1): e5905 (march, 2021) 11 of the genomes are very popular among bacterial taxonomists (khan et al., 2017; porrini et al., 2017). however, unlike homogeneous geniuses such as bacillus, the correct identification of an isolate can be a bit difficult. despite the high degree of genetic similarity among the different bacillus strains, these strains show substantial phenotypic variability from mammal or entomopathogen strains from all over the world (maughan & van der auwera, 2011; patino-navarrete & sanchis, 2017). since 1987, microbiologists have generally prefered to classify microorganisms by using only one type of dna sequence (mahato et al., 2017). but now we know that it is not enough to classify taxa alone, especially environmentally variable strains. in recent years, an increasein magnitude in the number of species predicted could lead to the conclusion that we are vastly underestimating the number of ecologically distinct species of bacillus (maughan & van der auwera, 2011). on the other hand, this brings us back to the fundamental flaw of defining species based on their ecological traits, which may not be congruent with their phylogenomic structures. we are probably missing out on our microbiological richness and the incredible molecular and ecological diversity of bacillus by choosing incorrect or insufficient taxonomic criteria. according to munson and carroll, k. michiganensis is a new taxon that could conceivably transcend human clinical disease, so its pathogenic potential should be noted (munson & carroll, 2017). in the google scholar database, we saw that, so many researchers prefer to use vitek microbial identification systems. this system is so practiced, and also very useful for quick biochemical test results but its identity results are not reliable every time, especially for environmental isolates. that’s because, the system should be updated and new pathogenic strains, like k. michiganensis, should be added to the database. sung et al. (2000) reported that s. paucimobilis is the most misdiagnosed organism in the vitek automicrobic system, and this study has supported that data too. de001, de003, de017, and de019 were identified as s. paucimobilis by vitek® 2, but none of these identifications was correct. new and quicker methods (reagents, instruments, software, etc.) need to be developed to characterise an isolate appropriately. we conclude that the honey bee’s bacterial biodiversity could have an essential role in its health and genomic heritage. many studies show that turkey is a resource of genetic diversity of a. mellifera caucasia, and it is microflora. this brings with it the need to conserve genetic resources (kence, 2006; solorzano et al., 2009). acknowledgments we would like to thank the staff ordu apiculture research institute, who helped during the sampling of adult worker honey bees. this study was supported by the ordu university scientific research projects department (project number: bap ar-1357). authors contribution e. ç. performed all the experiments with the help of ö. e. and k. i. and wrote the manuscript. e. ç and ö. e. designed the experiment. all the authors contributed to the revision of the manuscript and approved the version to be published. references abbott, w.s. 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(hymenoptera: apidae) have been morphologically and physiologically studied due to their importance on the production of royal jelly (cruz-landim 2009; seehuus et al. 2007; gatehouse et al. 2004). however, few studies have evaluated the development of glands in africanized honeybees as a function of the diet offered to them. the hg, located inside the worker's head, produces a protein-based substance that is responsible for the differentiation among castes and also used to feed young larvae, drones and the queen (feng et al. 2009; kamakura 2011).the structure of this gland varies according to the age and function of workers within the hive. young honeybees that function as nurses have active hg with large acini, while in older honeybees that present foraging activity, the gland will tend to be atrophied (sasagawa et al.1989). several factors may affect the physiolog y of glands and consequently the honeybee's role, impacting hive internal condition or organization (huang & ottis 1989). one important factor is the nutritional state of the honeybees. protein sources are extremely important to physiological development, especially to the young workers, as pollen is the main source of protein to their development (zahra & talal 2008). the activity period of the hg and the time that honeybees spend as nurses may vary according to the number of larvae in the hive. apparently, the activation of the hg will occur with the presence of a protein based food, and the maintenance of the active glands will depend on the existence of larvae in the hive (huang & ottis 1989; alghamdi et al. 2011). during periods of the year characterized by pollen scarcity, other food sources rich in protein such as soybean extract and milk powder might be used to feed the hives (zahra and talal 2008; degrandi-hoffman et al. 2010; al-ghamdi et al. 2011). however, despite those diets' high protein contents, their protein types may not be digested or absorbed by the bees, 449 santana, w.c. et al. — nutritional effects on bee hypopharyngeal gland development resulting in a negative impact on the overall development of the hive (pernal & currie 2000). several factors should be considered regarding the types of food offered to the honeybees, being that food organoleptic characteristics are fundamental to acceptance and ingestion of the diet, while food nutrient composition is important to its digestion and assimilation. the role of the hg on the production of royal jelly provides motivation for improving the current knowledge on the development of adequate diets that stimulate the development of the glands with the aim of empowering the production of this apicultural product of great nutritional properties. in this context, the aim of this study was to evaluate the effect of different diets on the development of hg of africanized honeybees. material and methods the study was conducted at the central apiary of the federal university of viçosa (ufv), where young workers of africanized apis mellifera were collected directly from the combs. the workers were confined inside wood boxes (09 x 09 x 03 cm), covered with nylon screens, and fed with the diets and wter ad libitum. the experiment consisted of four treatments (diets): honey only (t1), a mixture of soybean extract and honey (4:1) (t2), a mixture of pollen and honey (4:1) (t3), and a 50% p/v solution of sucrose only(t4). twelve wood boxes, each containing 10 young workers were mounted; each treatment had three replicates. the boxes were kept inside environmentally controlled chambers at 34 ± 2 ºc and 70 ± 10% ru. the honeybees from each treatment were dissected after two different periods: at the 7th and 10th day of exposure to the diets. prior to being dissected, five honeybees were taken from each box and kept in a fixing solution of dietrich (30 ml of ethanol, 10 ml of formaldehyde 40%, 2 ml of acetic acid and 58 ml of distilled water) for a two day period, and then they were transferred to an 80% ethanol solution. after the fixing period, the honeybees were dissected, and their hgs were extracted for subsequent measurement of an area of 20 acini per honeybee. photographic images were taken and recorded with an optical microscope (100x magnification). the acini areas were determined with the software image pro plus®. the obtained data of acini areas from hg of honeybees fed with different types of diets after two different periods of exposure (7 and 10 days) were submitted to anova and the tukey test (p< 0.05). 450 sociobiolog y vol. 59, no. 2, 2012 results the type of diet offered to the honeybees did affect mean acini area values (f= 17.78; p<0.05). smaller acini areas were observed for honeybees fed with sucrose solution (t4) (2.5 µm²), followed by honeybees fed with honey + soybean extract (t2) (4.0 µm²). the time factor also had a significant effect on acini areas (f= 11.00; p<0.05), with negative correlation (table 1). honeybees that were fed with honey + soybean extract (t2) for 10 days presented smaller acini areas as compared to honeybees fed with the same feed for a 7 days period. acini areas of honeybees fed with pollen (t3) after 7 or 10 days of exposure were not statistically different. however, it was observed that the general morpholog y of the acini for the four treatments presented a poor development when compared to acini extracted from hg of nursing bees aging from 7 to 10 days, taken from hives with young larvae and queen (fig. 1). table 1 – acini area in hg in workers of africanized honeybees (µm²) feed time (days) 7 10 honey 5.6 ± 0.95aa 3.2± 0.50bb honey + soybean extract 5.2± 0.75aa 2.8± 0.40bb honey + pollen 5.7± 0.90aa 5.5± 0.90aa sucrose 2.0± 0.55ba 3.1± 0.55ba means followed by the same letters (capital letters for rows and small letters for columns) do not statistically differ among one another using the tukey test at 5% probability. mean ± standard error. discussion the secretion produced by the hg is the main constituent of the royal jelly, a substance rich in protein and other nutrients that feeds young larvae, helps in caste differentiation due to its morphogenetic properties and is also the exclusive nutrient source for the queen (michener 2007; kamakura 2011). however, the development or activation of this structure is linked to some factors such as protein availability and quantitative or qualitative variations of this resource (al-ghamdi et al. 2011). the results obtained from this study demonstrated that the type of diet does affect the development of the workers' 451 santana, w.c. et al. — nutritional effects on bee hypopharyngeal gland development fig. 1. acini extracted from apis mellifera fed with different diets, without the stimulating presence of young larvae. a) t1 – honey, 7 days; b) t1 – honey, 10 days; c) t2 – honey + soybean extract, 7 days; d) t2 – honey + soybean extract, 10 days; e) t3 – honey + pollen, 7 days; f) t3 – honey + pollen, 10 days; g) t4 –50%sucrose solution, 7 days; h) t4 –50%sucrose solution, 10 days. i) hypopharyngeal gland of a nursing bee kept inside the beehive containing larvae and queen. 452 sociobiolog y vol. 59, no. 2, 2012 glands, as the best results were observed for the group fed on treatment t3 (honey + pollen). protein availability affected the size of acini, as groups of honeybees fed with protein supplements presented acini with greater areas as compared to groups that were fed exclusively with pollen and/or honey (degrandi-hoffman et al. 2010). food palatability and absorption are factors that should be taken into account when providing honeybees with supplemental diets. supplements with greater protein content may not always be the most efficient (al-ghamdi et al. 2011; degrandi-hoffman et al. 2010). in other words, because they are not naturally part of a honeybee diet, foods based on soybean extract might be rejected or only consumed in small amounts for not presenting attractive organoleptic characteristics. when evaluating food protein concentration and the development of the hg in carniolan bees, al-ghamdi et al. (2011) verified a better development of the structure in those that were fed with apicultural pollen, even though other groups of the same type of bees were fed up on rations with higher protein contents and presented underdeveloped glands. al-ghamdi et al. (2011) also observed that the consumption of diets based on soybean extract, milk powder and brewer’s yeast was 56.63% smaller than the consumption of diets based on apicultural pollen. hence, the development of diets containing fractions of apicultural pollen in their composition might be a good solution to reduce aversion to diets and increase the nutrient absorption by the honeybees, resulting in better overall development of the hive and improved production of royal jelly. even though the results obtained from this study show significant differences among treatments, for all cases the hg presented as poorly developed, with small and apparently atrophied acini as compared to those belonging to the nursing bees. one factor that might explain this difference in development was the absence of young brood, which might act as a stimulus to the development and activation of the hg, as observed in natural conditions (huang & ottis 1989). these glands are significantly bigger in the presence of young brood and will tend to reduce in size a few days of their absence inside the hives. thus, even with adequate protein sources, the full development and maintenance of this structure will only occur under the presence of young brood (huang & ottis 1989; mohammedi et al. 1996). 453 santana, w.c. et al. — nutritional effects on bee hypopharyngeal gland development along with the presence of pupae and good sources of protein, nutritional supplementation may also affect the development of the hg. zahra and talal (2008) observed that the effect of supplemental feeding in hives of a. mellifera promoted an increase in mean acinus size and hg ducts length, being that higher values were obtained in hives supplemented with vitamin c, overcoming supplements based on soybean extracts. factors such as parasitism may also have a negative impact on glands development. pinto et al. (2011) observed a cell area decrease of 11% for hgs in africanized honeybees parasitized by the mite varroa destructor, as compared to healthy honeybees. role assignment within hives with a. mellifera is directly related to the age of the workers, and this fact is due to physiological changes triggered by hormones mediated by biogenic amines as the honeybees get older (otto 1955; huang & otis 1989).these physiological changes cause the young workers of a. mellifera not to present active or developed hg right after the bee's emergence, so that they will perform tasks as cleaning the hive; afterwards, when they reach 6 to 12 days of age, the glands reach their full development, and the bees will then act as nurses (otto 1955; huang & otis 1989). thus, along with the availability of the necessary nutrients to the development and activation of the glands in workers, the nursing behavior due to the presence of the brood is an important factor for the maintenance of the hg activity (huang & ottis 1989). significant decrease in acini areas was observed in honeybee groups fed with soybean extract and honey, while groups of bees from the same study that were fed either with pollen, honey or sucrose presented mean acini areas statistically of the same size. the decrease in acini areas observed in workers fed only with honey is due to the lack of enough protein resources for the maintenance of the gland activity associated with the lack of stimulus from the brood; for honeybees fed with soybean extract and honey, the results can be explained by the aversive behavior to the non-natural protein source combined with the lack of stimulus due to the absence of brood. pernal and currie (2000), when analyzing the effect of the quality of several types of pollen on the protein secretion of the hg of workers, observed a significant decrease in secretion amount between 7 and 10 days according to the nutritional quality and acceptance of the pollen product offered to the workers. 454 sociobiolog y vol. 59, no. 2, 2012 the group that was fed solely with a sucrose solution presented the smallest acini area values, as there were no significant difference for workers after either 7 or 10 days. this outcome is in agreement to what was presented by pernal and currie (2000), who did not observe differences, for the same age periods, in the amount of protein secretion from glands of honeybees fed on sources of inferior nutritional values. in contrast, the group in this study that was fed with a diet containing pollen and honey, which presented acini areas significantly bigger than those obtained from the other dietetic treatments, did not differ between the ages of 7 and 10 days. the best results for acini area was obtained from the honeybee group fed with their natural protein source (pollen), even though the bees in this treatment did not have their hg fully developed. this outcome may be valuable in the development of supplemental diets for hives during periods of natural food scarcity. in order to reduce the costs, diets that combine pollen with other protein sources should be tested in future studies to evaluate the development of the hg and other structures such as the mandibular glands. providing the hives with an adequate diet might also act as a means to prevent the occurrence of pathogens and epizootics such as ccd, which has been the cause of millions in losses to the apicultural business worldwide (brodschneider & crailsheim 2010). conclusion the results obtained from the study described in this document reinforce the hypothesis that feeding plays an important role on the initial development of hg, and that different diets will affect gland development to different degrees, according to diet nutritional values. however, regardless of the type of food, the glands of the honeybees used in this study presented themselves to be less active and tended to present smaller sizes during the entire experimental period due to the absence of brood, which are necessary and act as a stimulus towards full gland development and maintenance. acknowledgements the authors would like to thank gecelmino correia for helping with the colony management. we also thank fapemig, cnpq and capes for supporting this work. 455 santana, w.c. et al. — nutritional effects on bee hypopharyngeal gland development references al-ghamdi, a.a., a.m. al-khaibari & m.o. omar. 2011. consumption rate of some proteinic diets affecting hypopharyngeal glands development in honeybee workers. saudi journal of biological sciences 18:73–77. brodschneider, r. & k. crailsheim. 2010. nutrition and health in honey bees. apidologie 41: 278–294. cruz-landim, c. 2009. abelhas: morfologia e função de sistemas. são paulo: ed unesp, 416p. gatehouse, h.s., l.n. gatehouse, l.a. malone, s. hodges, e. tregidga & j. todd. 2004. amylase activity in honey bee hypopharyngeal glands reduced by rna interference. journal of apicultural research 43:9-13. degrandi-hoffman, g., y. chen, e. huang & m.h. huang. 2010. the effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (apis mellifera l.). journal of insect physiolog y 56:1184–1191. feng, m., y. fang & j. li. 2009. proteomic analysis of honeybee worker (apis mellifera) hypopharyngeal gland development. bmc genomics 10:645-657. kamakura, m. 2011. royalactin induces queen differentiation in honeybees. nature 473:478–483. huang, z.y. & z.w. otis. 1989. factors determining hypopharingeal gland activity of worker honey bees (apis mellifera l.). insectes sociaux 36:264–276. michener, c.d. 2007. the bees of the world. baltimore: the johns hopkins university press. 953 p. mohammedi a., d. crauser, a. paris & y. le conte. 1996. effect of a brood pheromone on honeybee hypopharyngeal glands. comptesrendus de l’académie des sciences – series iii 319:769–772. otto, v.d. 1955. die phar ynxdriise der honig biene (apis rnellifera l.) bei prowona=hele=nahrung als pollennersatz . archiv fur geffiig elrucht und kleintievkunde 4:209–240. pernal, s. f. & r.w. currie 2000. pollen quality in fresh and 1-year-old single pollen diets for worker honey bees (apis mellifera l.). apidologie 31:387–409. pinto, f.a, g.k. souza, m.a. sanches & j.e. serrão 2011. parasitic effects of varroa destructor (acari: varroidae) on hypopharyngeal glands of africanized apis mellifera (hymenoptera: apidae). sociobiolog y 58 (3): 769-778. sasagawa, h., m. sasaki & i. okada 1989. hormonal control of the division of labor in adult honeybees (apis mellifera l.). i. effect of methoprene on corpora allata and hypopharyngeal gland, and its α-glucosidase activity. applied entomolog yand zoolog y24:66-77. seehuus, s., k. norberg, t. krekling, k. fondrk & g.v. amdam 2007. immunogold localization of vitellogenin in the ovaries, hypopharyngeal glands and head fat bodies of honeybee workers, apis mellifera. journal of insect science 7:52-60. wcislo, w.t., & j.h. cane 1996. floral resource utilization by solitary bees (hymenoptera: 456 sociobiolog y vol. 59, no. 2, 2012 apoidea) and exploitation of their stored foods by natural enemies. annual review of entomolog y 41: 257–286. zahra a. & m. talal 2008. impact of pollen supplements and vitamins on the development of hypopharyngeal glands and brood area in honey bees. journal of apicultural science 52:5–12. 741 ant (formicidae) assemblages associated with piper spp. (piperaceae) in the undergrowth of an atlantic rainforest remnant in southeastern bahia, brazil by anderson muñoz1, vitor o. becker2 & jacques h. c. delabie3, 4 abstract we studied ant assemblages associated with plants of genus piper (piperaceae) in the undergrowth of a fragment of atlantic forest in southeastern of state of bahia. the study was conducted in serra bonita natural reserve, in camacan and pau-brazil municipalities. hand collections of ants were made on four species of piper during seven months; 56 ant species were collected. the genus camponotus, pheidole and crematogaster were the most abundant. we highlighted the occurrence of linepithema spp. given their scarce records in the region. few ants were observed simultaneously (up to five) per plant during each survey. rarefaction curves showed that collections were not exhaustive. the shannon index showed that piper caldense presented the largest ant diversity occurrence, perhaps due to its higher amount of resources available. the ant community in the undergrowth of the atlantic forest appears to be diverse, although the species found in piper constitute only a fraction of the diversity that really occurs in this stratum of the forest. keywords: diversity, linepithema, rppn serra bonita. introduction ants have numerous attributes and are an example of ideal organisms for diversity studies, for their high numerical diversity and biomass dominance in almost every terrestrial landscape of the world (brown 2000). these organisms are especially important as predators, prey, anti-herbivore agents, or as seed 1mestrado em zoologia, universidade estadual de santa cruz, ilhéus, bahia, brazil. email: ander083q@ gmail.com 2reserva serra bonita, camacan, bahia, brasil. becker.vitor@gmail.com 3laboratório de mirmecologia, cepec-ceplac, ilhéus, bahia, brazil. email: jacques.delabie@ gmail.com 4departamento de ciências agrárias e ambientais, universidade estadual de santa cruz, ilhéus, bahia, brazil 742 sociobiolog y vol. 59, no. 3, 2012 dispersers; the last aspect, for instance, has a relevant ecological function for the maintenance and regeneration of the forests (ferreira 1998). ants have a wide range of interactions with plants and other animals (hölldobler & wilson 1990, beattie 1985, jolivet 1996, rico-gray & oliveira 2007), with which they show several types of associations: casual, obligatory, mutualistic or antagonistic. an example of these associations with plants is the use of domatia for the construction of ant nests; another is nutritional substances secreted by specialized structures in plants (secretory trichomes, extrafloral nectaries, pearl bodies, etc) given as an award for ant protection against herbivory (freitas & oliveira 1996, gaume et al. 1997, delabie & ospina 2003, rico-gray & oliveira 2007). in addition to these associations, it is possible to find ants in virtually all tropical wet forest strata, from the litter and undergrowth, to the canopy of larger trees (espírito santo et al. 2007). the angiosperm family piperaceae is pantropical and typical of the undergrowth. it has approximately 2,000 species belonging to 10 genera. in the new world, species of piperaceae are found from southern mexico to argentina (figuereido & sazima 2000). in brazilian forests, the genus piper (l.) includes 265 species (silva et al. 2009). they are aromatic shrubs or small trees up to 10m high, with alternate leaves, fruit-shaped drupe and nodes in the articulations (guimarães & silva 2004). some species of the genus, such as p. cenocladum (c. d.c.) or p. fimbriulatum (c. d.c.), develop interactions with ants, in which the amount of secretor cells producing nutrients is directly related to the number of ants foraging on plants: a greater number of ants are related with a greater number of secretor structures (risch & rickson 1981, letorneau & dyer 1998, fischer et al. 2003). the shrub-herbaceous vegetation of the undergrowth stratus is important because it represents up to 50% of all plant species found in a tropical forest area (gentry & dodson 1987). this fact influences the diversity of ants and consequently the foraging strategies that they can present. the present study was developed in order to characterize the assembly of ants that live in the undergrowth stratum in a remaining area in the northern part of the brazilian atlantic forest. 743 muñoz, a. et al. — ants associated with piper spp. in southeastern brazil materials and methods the study was conducted at the private reserve of natural heritage (rppn) serra bonita (15˚23’28.5”s 39˚33’52.0”w), altitudes between 300 and 960 m (amorim et al. 2009). the study area is located in the municipalities of camacan and pau-brazil, in the state of bahia, brazil (fig. 1). this area comprises one of the last remaining regions of altitude forest of the atlantic forest central corridor (ministério do meio ambiente 2006), with 7,500 hectares. about 50% of the area is covered by primary forest and the remainder is formed by a mosaic of vegetation in various stages of succession, including secondary forest, cabruca agrosystem (agro-forestry, where the small vegetation is removed but the largest trees maintained to provide shade to the cacao trees, see schroth et al. 2011) and pastures. the vegetation of the area ranges from tropical rain forest with elements of semideciduous fig. 1. localization map: serra bonita rppn, state of bahia, brazil. 744 sociobiolog y vol. 59, no. 3, 2012 forest in the lower parts, to submontane rainforest (cloud forest) high in the mountains (amorim et al. 2009). 120 individuals of the plants piper caldence c. d.c., p. cernuum vell., p. hispidum sw. and p. umbellatum l. (30 individuals per species) were randomly selected and labeled. p. caldense is characterized by having plants up to three meters high, with leaves glabrous, bright, up to 18 cm in length; p. hispidum has leaves of similar size to p. caldence, but are distinguished by their opaque color and pubescence. in addition, plants of p. hispidum may grow up to five meters high. this species is able to expand laterally more than any other species studied. p. umbellatum grows up two meters high, and presents few umbeliform leaves (up to six leaves per plant); p. cernuum was the largest species in the population studied here, with up to 8 meters high, and the species presents larger leaves (up to 40 cm long ), with domatia at the base of the petiole, a feature found only in this species among those here studied. the plants were selected with an interval of 20 m between successive plants aiming to decrease the probability of ants collected in two neighboring plants belonging to the same colony. the plants were labeled with the purpose of making observations on the same plants during all sampling series. ant sampling field work was conducted monthly from september to december of 2010, and march to may of 2011. in each field trip we made surveys of four days and four nights, for a total of eight days per field trip. in each case, surveys began at 08:00 hour (day), or 18:00 (night). we collected only foraging ants found on plants manually. the ants were identified and vouchers were deposited in the collection of the myrmecolog y laboratory (cpdc) of the centro de pesquisa do cacau (ceplac) at ilhéus, bahia, brazil. data analysis we estimated the richness and diversity of ant communities on plants using the shannon and chao1 indexes (colwell 2006). the results were analyzed using the statistical software past 1.74 (hammer et al. 2001). in addition, we constructed rarefaction curves using mau tau, bootstrap and chao2 indexes, in order to estimate and compare the ant richness on the plants. the data were analyzed using the statistical program estimates 8.2 (colwell 2006). finally, 745 muñoz, a. et al. — ants associated with piper spp. in southeastern brazil we compared ant assemblages with a dendrogram of similarity of bray-curtis using past 1.74 (hammer et al. 2001). results we collected 56 species of ants on the 120 individuals of piper belonging to the four species sampled in this study. the ants collected are distributed into four subfamilies and 16 genera. myrmicinae showed the largest number of occurrences, followed by formicinae, dolichoderinae and ponerinae (table 1). p. caldense and p. hispidum showed the greatest richness with 48.2% and 44.6% respectively of the species collected (table 1). pheidole, camponotus and solenopsis were the genera with the larger numbers of species, corresponding to 19.6%, 17.9% and 12.5% of the ant diversity respectively. other significant genera were camponotus, pheidole, crematogaster and linepithema representing respectively 8.2%, 8.2%, 19.8% and 31.0% of individual occurrences. only a few ants were found nesting on the plants, except wasmannia auropunctata, linepithema iniquum and l. neotropicum, in domatia of p. cernuum; dolichoderus attelaboides was found establishing initial nests in the abaxial region of leaves of p. cernuum forming a concave structure where the ants nested. crematogaster longispina was found nesting in the abaxial region of the leaves of p. hispidum, but in this case without forming a concavity below the leaves. the dolichoderinae d. attelaboides and l. iniquum, and the myrmicinae w. auropunctata were common on the four species of piper. it must be observed that the occurrence of l. iniquum, l. leucomelas, l. neotropicum and l. pulex represents the first reports of these ants foraging in plants of the genus piper in the atlantic forest, as well as the first record of most of these species for the region. the shannon index showed higher diversity in p. caldense and p. hispidum, respectively (table 1). regarding the chao 1 index, p. hispidum and p. cernuum showed a very high number of expected species (187. 9), which is expected to be an artifact due to the large number of singletons obtained in the series of data. rarefaction curves obtained did not reach asymptotes for any of the plants studied (fig. 2). 746 sociobiolog y vol. 59, no. 3, 2012 table 1. ant species collected, and related ecological characteristics, on four species of piper, september 2010 to may 2011, serra bonita rppn, bahia, brazil. *probable artifacts due to the large proportion of singletons in the sampled series. taxon p. caldense p. hispidum p. cernuum p. umbellatum formicidae ponerinae odontomachus haematodus 0 0 0 1 odontomachus hastatus 0 1 8 1 pachycondyla crenata 4 1 0 0 pachycondyla schultzi 0 1 0 0 dolichoderinae dolichoderus attelaboides 5 1 5 6 linepithema iniquum 6 1 31 11 linepithema leucomelas 1 0 1 2 linepithema neotropicum 14 0 3 9 linepithema pulex 0 4 1 0 tapinoma sp.1 0 0 3 0 tapinoma sp.2 1 0 0 0 formicinae brachymyrmex sp.1 0 1 1 0 brachymyrmex heeri 3 3 4 0 crematogaster sp. gp. heeri 0 0 1 1 camponotus (myrmaphaenus) sp.3 2 0 0 0 camponotus (myrmaphaenus) sp.4 2 1 0 0 camponotus (myrmaphaenus) sp.5 0 0 0 1 camponotus (tanaemyrmex) sp.1 0 2 0 1 camponotus (tanaemyrmex) sp.2 1 1 0 0 camponotus agra 0 1 0 0 camponotus cingulatus 0 3 0 2 camponotus crassus 1 0 0 0 camponotus depressus 0 1 0 0 camponotus nidulans 3 0 0 0 myrmelachista sp. 0 0 0 1 nylanderia guatemalensis 0 0 1 1 paratrechina longicornis 0 0 2 0 myrmicinae cephalotes angustus 0 0 0 1 cephalotes pavonii 1 0 0 0 crematogaster brasiliensis 0 1 0 0 crematogaster (gr. limata) sp. 2 0 5 0 747 muñoz, a. et al. — ants associated with piper spp. in southeastern brazil table 1. ant species collected, and related ecological characteristics, on four species of piper, september 2010 to may 2011, serra bonita rppn, bahia, brazil. *probable artifacts due to the large proportion of singletons in the sampled series (continued). taxon p. caldense p. hispidum p. cernuum p. umbellatum crematogaster longispina 5 9 15 0 crematogaster nigropilosa 3 7 6 0 pheidole (gr. flavens) sp.1 2 1 0 0 pheidole (gr. fallax) sp.2 0 0 1 7 pheidole sp.4 1 0 0 0 pheidole (gr. fallax) sp.5 1 0 0 0 pheidole sp.6 0 1 0 0 pheidole sp.7 2 0 0 0 pheidole sp.8 0 1 0 0 pheidole sp.9 0 0 1 0 pheidole sp.10 0 0 1 0 pheidole sp.11 0 0 0 1 pheidole sp.12 1 0 1 0 procryptocerus goeldii 1 0 0 0 procryptocerus hylaeus 0 1 0 0 procryptocerus pictipes 0 1 0 0 procryptocerus sp. 3 4 1 0 solenopsis sp.1 0 0 0 1 solenopsis sp.2 0 0 1 0 solenopsis sp.3 0 1 0 0 solenopsis sp.4 1 0 0 0 solenopsis sp.5 1 0 0 0 solenopsis sp.6 0 0 1 0 solenopsis sp.7 1 0 0 0 wasmannia auropunctata 3 19 5 1 observed ant species / host plant 27 25 23 18 observed species compared with the whole study (%) 48.2 44.6 41.1 32.1 h’ 3.0 2.9 2.4 2.3 exclusive species 12 9 6 6 exclusive/observed species (%) 21.4 16.1 10.7 10.7 expected ant species (chao 1 )/ host plant 41 187* 95* 47 sampling exhaustivity (%) 66.0 13.4 26.3 38.3 748 sociobiolog y vol. 59, no. 3, 2012 although small differences among the studied plants were observed, fig. 3 shows that the composition of ant assemblages around the four piper species has a high degree of similarity, especially between p. caldense and p. cernuum. discussion in our study, we used only hand collection because the structure and size of plants (never higher than 2.50 m for those observed) allowed ant observation and collection, and evaluation of the whole plants, without the use of another kind of sampling methodolog y. in comparison with similar studies, such as espirito santo et al. (2007), sirqueira et al. (2006) or schütte et al. (2007), the observed diversity was lower. it is then rather possible that these differences are due to the methodolog y used, because these studies combined manual collection, chemical shock and/or entomological umbrella, increasing the number of sampled species. of the 56 ant species collected, 59% were found on a single plant species (table 1), 21.4% of these occurred only on p. caldense, and 16.1% only on fig. 2. rarefaction curves for the plants studied in the serra bonita rppn, bahia, brazil. september 2010-may 2011: a) p. caldense, b) p. hispidum, c) p. cernuum, d) p. umbellatum. 749 muñoz, a. et al. — ants associated with piper spp. in southeastern brazil p. hispidum. our results may be related to random foraging ants on plants at the time of collection or, possibly due to the occurrence of some kind of interaction between ants and plants, related with the greater occurrence of secretory structures (rickson & risch 1984) that would not be present in the other two plant species studied. repeated collections of the same species of ants in piper suggest a nonrandomized behavioral pattern, reflected in the development of ecological relationships with these plants. similar results were found by rickson & risch (1984) and fischer et al. (2002, 2003), where close relationships among piper cenocladum, p. fimbriulatum, p. obliquum and p. sagittifolium, and ants of the genus pheidole were found; plants provide shelter (domatia) and food (food bodies) (risch & rickson 1981), while, in exchange for those services, the ants protect the plant against predators. fig. 3. bray-curtis similarity dendrogram comparing the ant assemblages according with their occurrences on four species of the genus piper: p. caldense; p. hispidum; p. cernuum and p. umbellatum. serra bonita rppn, bahia, brazil, september 2010 – may 2011. 750 sociobiolog y vol. 59, no. 3, 2012 three species of ants were common to all plants (table 1): d. attelaboides, l. iniquum and w. auropunctata. the genera wasmannia and dolichoderus were reported by agosti et al. (2000) as generalists foragers present in most tropical and temperate regions. ants of the genus linepithema are common in the neotropical ant-fauna, and they can be found in a wide variety of environments such as grasslands and montane habitats along the neotropic region (wild 2007). l. iniquum (mayr), l. leucomelas (emery), l. neotropicum, and l. pulex were reported by wild (2007) as widely distributed generalist ants, but these ants are scarcely found in this region of brazil. we observed ants of the genus dolichoderus foraging in the infructescenses of p. umbellatum, but active behavior of seed dispersal was not detected. however, judging by the size of seeds, it is presumed that this interaction is possible, mainly by generalist species, such as d. attelaboides. on the other hand, the diversity of ants observed on the four piper species was not significantly different. this is due to the fact that plants of this genus prefer open habitats of the undergrowth, especially open areas or clearings inside the forest (risch & rickson 1981). the preferences for analogous environments may explain the similarity between the diversity of ants collected in four plant species studied here. the major difference between the diversity of ants was observed between p. caldense (h’=3) and p. umbellatum (h’=2.3). given that these species have similar sizes, the differences found could be explained by the greatest number of observations of occurrences of ants in the leaves of p. caldense where they seem to be collecting some unidentified substances, behavior which was not observed in p. umbellatum. some species of piper are known to possess secretor trichomes and pearl bodies that can provide nutrients (fisher et al. 2002) attractive to ants. according the bray-curtis dendrogram (fig. 3), there exists a greater similarity between p. caldense, p. cernuum tan between p. cernuum and p. hispidum. this fact highlights that characteristics such as the occurrence of domatia at the base of the petioles in p. cernuum, or the occurrence of food bodies in p. caldense and p. hispidum as well as in other species of piper (fisher et al. 2002) have a significant influence on time of foraging of ants in these species. as the rarefaction curves do not stabilize (fig. 2), the ant diversity found in the samples represents only a fraction of the total number of species that 751 muñoz, a. et al. — ants associated with piper spp. in southeastern brazil might be found on the plants. consequently, hand collection of ants is certainly not the appropriate methodolog y to quantify the diversity in this type of habitat (itô et al. 1998). the assemblage of ants associated with p. umbellatum possessed the lowest diversity, and the least similarity of the four species of piper. this could be associated with the reduced occurrence of differentiated structures for feeding ants, or for the presence of potentially toxic or repellent substances in the leaves of this plant (i.e. isoasarone) (checksum & fasihuddin 2002), that could cause ants to avoid foraging in this species. the active foraging behavior of ants observed on the four species of piper can be considered as a study case of ant-plant interactions in the undergrowth of a tropical forest. the ants of the undergrowth may be especially important to the regeneration, development and maintenance of the rainforest, given their ecologically important role as predators, prey and seed dispersers (see also dáttilo et al. 2009, leal 2005, rico-gray & oliveira 2007). acknowledgments we thank the staffs of the myrmecolog y laboratory and serra bonita rppn for their cooperation, and h.e. ramires-chaves, andré amorim and clemira sousa for technical and logistic support. this research was founded in part with the aid of the project pronex-cnpq fapesb pnx 00112009. a. muñoz q. thanks capes for granting his scholarship, and j.h.c. delabie thanks the cnpq for his research fellowship. references agosti, d.m. & l.s. alonso. 2000. 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de souza et al., 2012). these insects show a abstract predation of lepidoptera caterpillars including agricultural pest species is one of the main ways through which social wasps gather proteinaceous resources. the presence of social wasps was sampled through active search and bait traps through a sugarcane culture cycle, totaling 12 months. our aim was to record the presence of these insects during the sugarcane development cycle in order to obtain data to support alternative pest control strategies. a total of 1091 individuals in seven genera and 20 species of social wasps were collected, including the swarm-founding agelaia vicina and polybia sericea (hymenoptera: vespidae). social wasp richness and abundance were not correlated with climatic variables (temperature, humidity and precipitation). however, richness was negatively correlated to the sugarcane plants’ height (r= -0.4360, p= 0.05). the presence of social wasps during the plant’s cycle shows their potential as predators in sugarcane culture pest management. sociobiology an international journal on social insects bc barbosa1, njj silva1, jc zanuncio2, f prezoto1 article history edited by gilberto m. m. santos, uefs, brazil received 16 october 2017 initial acceptance 07 february 2018 final acceptance 26 april 2018 publication date 09 july 2018 keywords biological control, dynamic populations, natural enemies, predator. corresponding author bc barbosa laboratório de ecologia comportamental e bioacústica – labec universidade federal de juiz de fora campus universitário, bairro martelos cep 36036-900, juiz de fora-mg, brasil. e-mail: barbosa.bc@outlook.com generalist and opportunistic alimentary behavior in the search for carbohydrates and proteinaceous resources used in larvae nutrition (hunt, 2007; elisei et al., 2010; clemente et al., 2012). thus, they prey on sugarcane pest insects such as diatraea saccharalis (fabricius, 1794) (lepidoptera: crambidae), mocis latipes (guen, 1852) (lepidoptera: noctuidae) and spodoptera frugiperda (smith, 1797) (lepidoptera: noctuidae) and are important for integrated pest management programs (giannotti et al., 1995; prezoto et al., 2008; prezoto et al., 2016). social wasps have been understudied regarding the management of agricultural pests, in spite of polistes simillimus zikán, 1951 (hymenoptera: vespidae) has being shown to increase productivity in corn plantations infested with s. frugiperda caterpillars (prezoto & machado, 1999a) and polistes versicolor (olivier, 1791) being recorded foraging in eucalyptus leaf-eating caterpillars (elisei et al., 2010). the aim of this study was to record the presence of these insects during the sugarcane development cycle in order to obtain data to support alternative pest control strategies. 1 laboratório de ecologia comportamental e bioacústica, departamento de zoologia, universidade federal de juiz de fora, minas gerais, brazil 2 departamento de entomologia/bioagro, universidade federal de viçosa, minas gerais, brazil research article wasps mailto:barbosa.bc@outlook.com sociobiology 65(2): 320-324 (june, 2018) 321 material and methods the study was carried out in juiz de fora (21° 47’ 21.19” s, 43°25’ 34.58” w, altitude: 678 m), minas gerais state, brazil in a sugarcane culture with 1.5 ha (saccharum sp. variety rb867515) (fig 1) bordered by a pasture, an orchard and an atlantic forest fragment. this region has humid subtropical climate, type cwa (mesothermic with hot rainy summers) (sá júnior et al., 2012). wasps were collected at a monthly basis from july 2010 to june 2011 in 420 hours of field work. sampling was carried out through active search from 10:00 to 17:00 during five days in a row each month in random transects within and around the culture. bait traps were also used, consisting of two-liter polystyrene bottles with three triangular holes (2x2x2 cm) cut at 15 cm from the base, the baits were made from passion fruit juice, honey water or sardine broth (souza & prezoto, 2006; souza et al., 2015). thirty-six traps were set each month in two transects (east and west) with 18 bottles each (six with sardine broth, six with passion fruit juice and six with honey water). traps were set at a 1.5 m height from the ground and at 10 meters apart from each other for five days. captured wasps were separated in the study area and kept in 100 ml sampling vials with ethanol 70%. +3 was standardized as reference for samples. five sugarcane plants were randomly measured each sampling month and the calculated mean of these measures was used. temperature (°c), relative air humidity (%) and precipitation (mm) data were obtained at the laboratório de climatologia e análise ambiental (labcaa) in the departamento de geociências do institutos de ciências humanas of universidade federal de juiz de fora. maximum attainable species richness from each study site was estimated through the use of the 1st order jacknife non-parametric estimator, in the statistical software dives v 3.0. constancy was obtained through the formula: c=px100/n (bodemheimer, 1955). species that were present in more than 50% of the samples were considered constant, from 25% to 50% were considered as accessory and in less than 25% were considered accidental. correlation between species richness/ abundance and the variables temperature (°c), humidity (%), precipitation (mm) and sugarcane plant height was calculated through the use of the coefficients of spearman (rs) and pearson ®. fig 1. geographical representation of the sugarcane culture area in juiz de fora, minas gerais state, southeastern brazil. fig 2. social wasp species richness and abundancy (a) through the sugarcane development phases. phase 0 (α): sugarcane cut, 1st phase (▲): budding and establishment (duration of ≈20-30 days after planting), 2nd phase (●): tillering (duration of ≈20-30 days after the emergence of the primary stem), 3rd fase (■): stem growth (beginning ≈ 4 months after planting), 4th phase (□): maturation (beginning ≈ 10 after planting) and b: height variation in sugarcane plants (cm) per month in the city of juiz de fora, minas gerais, brazil. collected wasps were identified through keys for genera and species (richards, 1978), and comparisons with specimens from the entomological collection of the laboratório de ecologia comportamental e bioacústica (labec) of the universidade federal de juiz de for a in juizfor afora, minas gerais, brazil. the development phases of the sugarcane were identified through the plants’ height according to the kujiper system (van dillewijn, 1952), with the first top-to-bottom leaf presenting fully visible insertion in auricle (leaf collar) being designated as +1. leaves under it were numbered +2, +3, etc., and the ones above it were numbered as 0, -1, -2, -3, etc. the leaf bc barbosa, njj silva, jc zanuncio, f prezoto – social wasps in sugarcane culture 322 the chi-square test (x2) was used to verify the existence of differences between seasons (hot/rainy, cold/dry), wasp abundance and richness in the software bioestat 5.0. results a total of 1,091 social wasps were captured from 20 species in seven genera, with 861 individuals belonging to agelaia vicina (de saussure, 1854). thirty percent of the species were constant, 20% were accessory and 50% were accidental. half of the constant species were swarm-founding wasps. the 1st order jacknife index estimated 21 species for the studied area. this confirms the efficiency of the method used, given that 20 species were found (95% of the estimated total). social wasp richness was higher in the hot/rainy season (oct 2010 to apr 2011), peaking in october 2010 with 12 species. however, it didn’t correlate to temperature (r=0.2379, p= 0.4566), relative humidity (r= -0.5208, p= 0.0825) or precipitation (r= 0.0860, p= 0.7905). social wasps were collected through the whole sugarcane cycle, and species richness was correlated to the plants’ height (r= -0.4360, p= 0.05) (fig 2). wasp abundance was not correlated to temperature (rs= -0.3713, p= 0.2347), relative humidity (rs= -0.4476, p= 0.1445) or precipitation (rs= -0.3916, p= 0.2080). nests of mischocyttarus drewseni saussure, 1857, polistes simillimus zikán, 1951, polistes versicolor (olivier, 1791), polybia occidentalis olivier, 1791 and polybia platycephala richards 1951 (hymenoptera: vespidae) were found within the planted area. nests from the other 10 species were found up to 100 meters away from the culture. discussion social wasp species richness can be considered high in the studied environment, being greater than that found in orchards (santos 1996; silva et al., 2013), eucalyptus plantations (de souza et al., 2012) and silvipasture areas (auad et al., 2010), even with the last two showing higher plant diversity than sugarcane cultures. this can be explained by the environmental diversity around plantations, with a great variety of resource sites for herbivore insects and, consequently, their predators. table 1. species, percentage of individuals per species captured through active search (search) and through bait traps with sardine, passion fruit and honey water and constancy (const.) of social wasps in a sugarcane culture from july 2010 to june 2011. city of juiz de fora, minas gerais, brazil. species search sardine passion fruit honey water const. agelaia vicina* 178 (16,32%) 644 (59,03%) 36 (3,30%) 3 (0,27%) ▲ mischocyttarus drewseni*+ 28 (2,57%) ▲ mischocyttarus rotundicollis 16 (1,47%) ▲ polistes simillimus*+ 50 (4,58%) ▲ polybia ignobilis 24 (2,20%) 2 (0,18%) 2 (0,18%) ▲ polybia sericea 55 (5,04%) ▲ brachygastra lecheguana* 13 (1,19%) ■ mischocyttarus cassununga* 4 (0,37%) ■ polybia jurinei 1 (0,09%) 3 (0,27%) ■ polybia platycephala*+ 11 (1,01%) ■ agelaia multipicta 3 (0,27%) 1 (0,09%) ● apoica sp.* 1 (0,09%) ● polistes billardieri 1 (0,09%) ● polistes cinerascens 1 (0,09%) ● polistes subsericius 2 (0,18%) ● polistes versicolor* 1 (0,09%) ● polistes sp. 1 (0,09%) ● polybia occidentalis*+ 1 (0,09%) 1 (0,09%) ● polybia paulista* 3 (0,27%) ● protonectarina silveirae 2 (0.18%) 2 (0.18%) 1 (0.09%) ● total 392 (35.93%) 651 (59.67%) 45 (4.12%) 3 (0.27%) shannon wiener (h’) 0.79 0.03 0.36 0 homogeneity 0.61 0.02 0.28 0 heterogeneity 0.39 0.98 0.72 1 species with nests up to 100 meters away from the culture (*), species with nests within the culture (+), species frequency and constancy (▲constant (c>50%), ■ accessory (c de 25% a 50%), ● accidental (c<25%). table 1. species, percentage of individuals per species captured through active search (search) and through bait traps with sardine, passion fruit and honey water and constancy (const.) of social wasps in a sugarcane culture from july 2010 to june 2011. city of juiz de fora, minas gerais, brazil. sociobiology 65(2): 320-324 (june, 2018) 323 the great number of a. vicina individuals can be explained by the larger size of their colonies, which may reach one million adults each. this species’ nests are built in termitaries, hollow trunks and even inside buildings (zucchi et al., 1995; oliveira et al., 2010). this species can prey on insects from 10 different orders, including coleoptera, diptera, hymenoptera, hemiptera and lepidoptera. many of these may include pest species such as the corn leafhopper (oliveira et al., 2010). swarm-founding species such as polybia sericea (olivier, 1791), which were classified as constant in this study, have 75.4% of their protein forage composed of lepidoptera caterpillars, including s. frugiperda (bichara et al., 2009). the presence of p. simillimus colonies within the sugarcane plantations is valuable, since the species foraging behavior in corn plantations have shown the species’ potential for pest management and the translocation of their colonies to artificial shelters in cultures reduces s. frugiperda populations (prezoto et al., 1994; prezoto & machado 1999a, b). finding no relation between climatic factors and wasp abundance and richness contrasts with most published studies. however, it is clear that this particular situation occurs in anthopized environments, as shown by ribeiro-junior (2008), auad et al. (2010) and barbosa (2015). this might explain the results reported here, since our study also took place at an environment altered by man. despite social wasps being present through the whole sugarcane cycle, different plant stages modified the size of planted area, decreasing the area available for insect flight and influencing in their abundance and richness. besides air viscosity and wind speed, vegetation density can also influence on the flight of insects (unwin, 1980; hilário et al., 2007). aside from the continuous presence of wasp colonies both in the surrounding vegetated areas and within the sugarcane plantation, the generalist diet is an indicative of their potential for controlling pests such as d. saccharalis, m. latipes e s. frugiperda, responsible for losses in sugarcane culture. references auad, a. m., carvalho c.a., clemente m. a. & prezoto, f. (2010). diversity of social wasps in a silvipastoral system. sociobiology, 55: 627-636. barbosa, b. c. 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(2015). use of flight interception traps of malaise type and attractive traps for social wasps record (vespidae: polistinae). sociobiology, 62: 450-456. doi: 10.13102/ sociobiology.v62i3.708 togni, o. c., locher, g. a., giannotti, e. & silveira, o. t. (2014). the social wasp community in an area of atlantic forest, ubatuba, brazil. checklist, 10: 10-17. doi: 10.15560/10.1.10 unica união da indústria de cana-de-açúcar. 2017. setor sucroenergético histórico. unwin, d.m. (1980). microclimate measurement for ecologists. academic press inc., orlando, 97 pp. van dillewijn, c. (1952). botany sof sugarcane. waltham: ckonica botanica co., waltham, massachusetts, usa, 371 pp. zucchi, r., sakagami, s., noll, f. b., mechi, m. r., mateus, s., baio, m. v. & shima, s. n. (1995). agelaia vicina, a swarm-founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). entomology society, 103: 129-137. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1077sociobiology 63(4): 1015-1021 (december, 2016) phorid flies parasitizing leaf-cutting ants: their occurrence, parasitism rates, biology and the first account of multiparasitism introduction diptera phoridae represents a group of insects with large diversity of life styles during their immature stages, the majority of the larvae being parasitoids (disney, 1994). the greater part of parasitism by these flies occurs on ants; among the genera attacked are the leaf-cutters atta fabricius and acromyrmex mayr (feener & moss, 1990; disney, 1996; brown et al., 2010), which are considered to be the most important herbivores of the neotropical region (della lucia, 2011). both host and parasitoids occur in the american continents, from southern united states to argentina; where 28 species of attine ants are parasitized by at least 70 species of phorids belonging to 11 genera (delabie et al., 2011; bragança, 2011; uribe et al., 2014), the most common being apocephalus coquillett, myrmosicarius borgmeier and eibesfeldtphora abstract the leaf-cutting ants atta sexdens (linnaeus) and atta laevigata (smith) were parasitized by the following phorid flies: apocephalus attophilus borgmeier, apocephalus vicosae disney, myrmosicarius grandicornis borgmeier and species of eibesfeldtphora disney. the area of occurrence of phorids parasitizing a. sexdens was extended to include central brazil. the rate of parasitism on a. sexdens was three times lower than the rate found on a. laevigata; the most common flies were, respectively, m. grandicornis in a. sexdens and a. attophilus in a. laevigata. this last phorid showed the shortest life span but the higher percentage of emergence. multiparasitism on workers of a. sexdens and of a. laevigata involving three combinations of four phorid species was rare and is here related for the first time for leaf-cutting ants. sociobiology an international journal on social insects mal bragança1, fv arruda2, lrr souza1, hc martins1, tmc della lucia3 article history edited by gilberto m. m. santos, uefs, brazil received 25 may 2016 initial acceptance 24 august 2016 final acceptance 07 october 2016 publication date 13 january 2017 keywords savannah, host-parasite association, natural enemies, phoridae, atta sexdens, atta laevigata. corresponding author marcos antonio lima bragança universidade federal do tocantins, curso de ciências biológicas, 77500-000, porto nacional-to, brasil e-mail: marcosbr@uft.edu.br disney. this last genus was previously considered as a subgenus of neodohrniphora malloch, having being raised to the category of genus by disney et al. (2009). the flies usually follow and attack the leaf-cutting workers walking down their trails or in their foraging area, laying the eggs on the head or on the gaster of the host (erthal & tonhasca, 2000; tonhasca et al., 2001; bragança et al., 2002). the parasitoids may be either species-specific or may attack more than one attine species. hosts may be parasitized by two or more species of phoridae (brown, 2001; disney, 1996; brown et al., 2010; uribe et al., 2014). during an attack, that lasts about one second, the female seems to lay only one egg inside the hostbody where the larva will feed and develop (bragança, 2011). the level of parasitism of leaf-cutters by phorids generally ranges between 1 and 6% of the ants in the trails 1 universidade federal do tocantins, porto nacional-to, brazil 2 universidade estadual de goiás, anápolis-go, brazil 3 universidade federal de viçosa, viçosa-mg, brazil research article ants mal bragança et al. – multiparasitism of leaf-cutter ants by phorid flies1016 (bragança, 2011).the attacks by the flies seem to have a more significant impact on foraging behavior than on mortality of parasitized ants. real attacks or simply the flight of the flies over the ants lead to their interrupting cutting activities and fleeingto the nest, thus resulting in a reduction of available plant material as fungus substrate(bragança et al., 1998; tonhasca et al., 2001). the discovery of these adverse effects on ant work demonstrates a possible contribution of these flies to management of leaf-cutting ants. several studies were conducted during the last decade aimed at increasing the knowledge of the biodiversity, geographical distribution, and biological and ecological characteristics of the parasitoid phorids, especially those concerned with atta (disney et al., 2006; bragança et al., 2009a; brown et al., 2010; bragança, 2011;guillade&folgarait, 2011; uribe et al., 2014). the species atta sexdens (linnaeus) and atta laevigata (smith) show wide distribution in brazil and are responsible for large and significant losses to agriculture and forests (della lucia, 2011). they are hosts of at least 10 and seven species of phorids, respectively; some of the flies can attack both hosts (bragança, 2011). data from the present work are part of a comprehensive research that is conducted in the savannah of central brazil, in the state of tocantins, and deals with the parasitoids and the two ant species. the rate of natural parasitism was determined for both ant species. data on occurrence were also gathered, as well more information on fliesbiology. the occurrence of multiparasitism is reported for the first time. material and methods study area and sampling of ants from august 2010 to august 2012, samples of ant workers were collected from trails of six colonies of a. laevigata and of 10 of a. sexdens. all were found in the municipality of porto nacional (10° 43’ s, 48° 24’ w), state of tocantins. at the same time, samples were taken from three other colonies of a. sexdens, localized in the neighboring city of palmas (10° 10’ s, 48°24’w). one to two hundred ants were collected from one or two trails of each colony, at random, in intervals of 15 days, between 7:00pm and 10:00 pm, following bragança & medeiros (2006), although not all of the colonies were always sampled. rearing ants to get flies when in the laboratory, workers of each colony were placed in a plastic tray (26 cm long, 17 cm wide), provided with a perforated lid to facilitate air flow and fed a 10 % honey solution impregnating a cotton wad that was replaced every second day. the trays and the ants were kept in climate controlled (25 °± 1°c, 80 ±5% rh and in the dark) and daily scrutinized to remove dead ants. these were individually taken into test tubes (13 mm x 100 mm) sealed with cotton and also kept in the acclimatized chambers. this procedure was adhered to for up to 15 days, at the end of which the parasitized ants are expected to be all dead (bragança & medeiros, 2006; bragança et al., 2009a). in the same day of the ant’s death or at most in the following day, the parasitized ants were inspected to check for the presence of phorid pupae in different parts of the host or next to it. the parasitized ants were kept in the chambers until the emergence of adult phorids. those ants that did not show signs of parasitism were dissected to verify if phorid larvae were still inside their bodies. emerged flies and larvae found upon dissection were preserved in eppendorf tubes containing 70% alcohol and deposited in the collection of the course of biological science of the federal university of tocantins, in porto nacional, to, brazil. results and discussion occurrence and rates of parasitism a total of 66,226 workers of a. sexdens and 23,473 of a. laevigata was collected, of which 1,042 (1.57 %) and 1,258 (5.36 %) were parasitized, respectively. workers of a. laevigata were parasitized by four phorid species: eibesfeldtphora erthali (brown), myrmosicarius grandicornis borgmeier, apocephalus attophilus borgmeier and apocephalus vicosae disney. these last three species were also found in a. sexdens but at least two other morpho species of eibesfeldtphora emerged from this host (table 1). amongst the attacked workers, the highest rate of parasitism in a.laevigata was by a. attophilus; in a. sexdens it was by m. grandicornis whereas the least frequent parasitoids were m. grandicornis and a. vicosae, respectively (table 1). the dissection indicated that about 2 % of the parasitized ants showed phorid larvae in their head capsules that could not be identified. furthermore, five workers (0.42 % of all the parasitized ants) showed three combinations of multiparasitism by four species of flies, two species of the parasitoid emerging from each individual (table 1). one a. attophilus fly and one of a. vicosae emerged from each of three workers of a. laevigata while one a. attophilus fly and one eibesfeldtphora emerged from one worker of a. sexdens. besides, one specimen of a. vicosae and one of m. grandicornis emerged from a single worker of a. sexdens (table 1). most of the phorid species and of their hosts in this study can also be found in southeastern brazil (disney & bragança, 2000; erthal & tonhasca, 2000; brown, 2001; tonhascaet al., 2001). the flies a. vicosae, a. attophilus and e. erthali had already been found attacking a. laevigata in the region of porto nacional (bragança & medeiros, 2006), but this is the first time m. grandicornis has been found attacking that host in that zone. thus, this work expands the list of parasitic phorids present in the savannah region of brazil. the data here presented corroborate other findings that indicate that the average parasitism rates on leaf-cutters by phorids generally vary from 1 to 6 % of workers on the sociobiology 63(4): 1015-1021 (december, 2016) 1017 trails (erthal & tonhasca, 2000; bragança & medeiros, 2006; bragança, 2011), granting that it can be much higher (35%) (folgarait, 2013), depending on the host, the fly, the site, and the time of year. the rate of parasitism in a. laevigata (5.4 %) was over three times that found in a. sexdens. among parasitized a. laevigata workers, the genus apocephalus (a. attophilus and a. vicosae) accounted for 81% of them, while the genera myrmosicarius and eibesfeldtphora prevailed in 81.3 % of the parasitized workers of a. sexdens. during a 12-month evaluation of parasitism on a. laevigata in the same region (bragança & medeiros 2006), the leading phorid was a. vicosae (68 %), while a. attophilus was found in only 5.6 % of parasitized workers. the reasons for these differences may be related to the sampling occasion and to variations in parasitism between the colonies of a. laevigata assessed and between habitats (dissimilar sampling stations). there are no reports of similar studies conducted on a. sexdens for further consideration. biology the average survival time of ant workers parasitized by phorids is considerably smaller than that of non-parasitized ones (bragança & medeiros, 2006) but little is known concerning the development times of eggs and larvae. the methodology used in this work does not allow a precise determination of egg development time plus that of larval development since the date of oviposition was not known. however, the survival (in days) of the parasitized workers may offer a comparative hint of that period between the species. the survival of attacked workers, that is, the time lapse between their collection and their death, with the recognition of parasitism by discovering pupae on the host bodies or next to them (see below), amounted to only half for a. attophilus when compared to workers parasitized by other phorids (table 2), thus indicating that the development period was likely only half of that of the other species. atta sexdens atta laevigata parasitoid # parasitized workers parasitism rate (%)* proportional rate of parasitism (%)** parasitoid # parasitized workers parasitism rate (%)* proportional rate of parasitism (%)** eibesfeldtphora spp. 253 0.382 24.28 e. erthali 192 0.818 15.26 m. grandicornis 594 0.896 57.01 m. grandicornis 18 0.076 1.43 a. attophilus 123 0.185 11.81 a. attophilus 758 3.230 60.25 a. vicosae 47 0.071 4.51 a. vicosae 261 1.112 20.75 larvae (not identified) 23 0.034 2.21 larvae (not identified) 26 0.111 2.07 multiparasitism multiparasitism a. attophilus and eibesfeldtphora sp. a. vicosae and m. grandicornis 1 1 0.001 0.001 0.09 0.09 a. attophilus and a. vicosae 3 0.013 0.24 total 1,042 1.570 100.00 total 1,258 5.360 100.00 * regarding the number of collected ants. ** regarding the number of parasitized ants. table 1. numbers and percentages of workers of two atta leaf-cutter ant species parasitized by three genera of phoridae flies (eibesfeldtphora, apocephalus and myrmosicarius) in the central region of the state of tocantins, brazil. a total of 66,226 workers of a. sexdens and 23,473 of a. laevigata was collected. the larvae were found inside the bodies of the hosts during dissections. five workers revealed cases of multiparasitism. in general, the pupal period was for a. attophilus also shorter than that of other species. a higher emergence percentage was also found in a.attophilus in both hosts (table 2). researches conducted in laboratory under 25 ± 1oc and 80 ± 5% uh have already shown that the time lapse from oviposition to pupal formation amounted to 9.1 ± 1.2 days for m. grandicornis (tonhasca et al., 2001) and 9.6 ± 0.8 days for eibesfeldtphora elongata (brown) and eibesfeldtphora tonhascai (brown) together (bragança et al., 2008). similar data are not available for a. vicosae and a. attophilus, but the information from this study under similar conditions seems to indicate that this last species has the life cycle2-3 days shorter than of the phorids parasitizing both a. sexdens and a. laevigata. after the death of all parasitized workers by a. attophilus, one to 14 larvae that had developed inside their head capsule left through their mouth cavity and changed to pupae far from the host, either on the tube walls or on the cotton swab. some species of parasitic flies adjust their larval development period and only kill their host when approaching pupal formation. in this aspect, phorids of the mal bragança et al. – multiparasitism of leaf-cutter ants by phorid flies1018 genus apocephalus parasitizing other species of ants may use two distinct strategies: those having short larval period, around 4-5 days, lay their eggs on wounded ants, or on those dying or recently dead. phorids that infect healthy ants usually have a larval period of two weeks or more before pupae formation (feener & brown, 1997). apocephalus attophilus lays the eggs inside the mouth of living workers of a. laevigata while they are cutting the plant (erthal & tonhasca, 2000). several larvae of a. attophilus can developing in the ant head, and this could accelerate the consumption of the head content of the host. thus, decreasing the survival of the host, along with a smaller larval period of this phorid compared with other flies. minimum survival time among workers of a. sexdens and a. laevigata infected by any phorid species was only of one day (table 2). as larvae were observed up to 24 hours after the ant’s death, either inside or near the bodies, parasitized workers of a. sexdens and a. laevigata do stay alive until the end of larval development. bragança & medeiros (2006) have found that workers of a. laevigata infected by phorids kept transporting plant fragments the day before their death, thus indicating that the host does not suspend its activity of cutting when having one or more larvae inside its body. among other phorid genera, only one larva developed inside the body of the host. the larval development of a. vicosae took place inside the ant’s prothorax; the rupture between pronotum and propleura exposed the pupa, thus phorid ant survival (days) pupal period of parasitoid (days) % emergence of the fly a. sexdens a. laevigata a. sexdens a. laevigata a. sexdens a. laevigata apocephalus attophilus 2.4 ± 1.0 (45) 1-5 2.8 ± 0.9 (487) 1-7 16.1 ± 2.6 (45) 12-24 16.5 ± 2.2 (487) 11-30 96.8 90.0 apocephalus vicosae 5.3 ± 2.6 (22) 1-12 6.0 ± 1.7 (143) 1-12 19.1 ± 2.5 (22) 16-26 19.0 ± 2.3 (143) 13-30 46.8 54.8 myrmosicarius grandicornis 5.8 ± 2.8 (228) 1-14 5.5 ± 2.1 (15) 1-13 18.8 ± 2.4 (228) 15-32 16.0 ± 1.4 (15) 15-28 38.4 83.3 eibesfeldtphora erthali 4.5 ± 2.0 (79) 1-11 24.1 ± 4.8 (79) 8-34 41.1 eibesfeldtphora spp. 5.2 ± 2.7 (162) 1-11 22.9 ± 2.1 (162) 17-30 64.0 (-) no parasitic relationship allowing the adult emergence (bragança & medeiros, 2006). the behavior of this phorid during oviposition on a. sexdens and a. laevigata is as follows: the female fly walks between the workers transporting leaf fragments and jumps onto a fragment to lay the egg in the ant’s mouth cavity (unpublished data). this same behavior has been described by brown et al. (2010) as occurring when this parasitoid attacks atta vollenweideri (forel). the larva of a. vicosae, using a still unknown method, then migrated to the thorax to feed on the contents of the prothorax. larva of m. grandicornis consumes the head content of the ant host; the pupa is found at the bottom of the capsule, under the “tentorium” (tonhasca et al., 2001). myrmosicarius brandaoi disney and myrmosicarius gonzalezae disney, elizalde and folgarait, 2006 do the same when attacking a. vollenweideri and atta saltensis forel (guillade & folgarait, 2011). oviposition by m. grandicornis is directed always at the right side of the host’s head because of an asymmetry of the ovipositor (tonhasca et al., 2001). larvae of eibesfeldtphora spp. develop inside the head capsule as well, devouring all of its content and locating themselves between the mandibles to pupate. oviposition by eibesfeldtphora spp. that parasitize a. sexdens occurs on the posterior part of the worker’s head (tonhasca, 1996; bragança et al., 2009a) in contrast with e. erthali, that uses the gaster of a. laevigata (bragança et al., 2002), thus indicating that the larvae somehow migrates towards the head capsule of the ant. table 2. survival in laboratory of workers of the leaf-cutter ants atta sexdens and atta laevigata parasitized by different phorid species, the pupal duration of these species and the percent of emergence of these parasitoids. means are shown ± sd with number of individuals in parentheses followed by the range in the next line. multiparasitism from a total of 89,699 workers collected five (0.0055%) exhibited multiparasitism and the emergence of two parasitoid species (two individuals) per host (table 1). in the multiparasitism by a. attophilus and a. vicosae in a. laevigata and by a. vicosae and m. grandicornis in a. sexdens, the parasitoid larvae developed in different parts of the host body; thus indicating no competition among them for the food resource. however, the phorid larvae a. attophilus and eibesfeldtphora sp. which multiparasitized the worker of a. sexdens (table 1) occupied the host head capsule simultaneously, sharing the food resource and completing their development at the same time. eibesfeldtphora sp. was perhaps the first to lay its egg, seeing that its development average period is longer than that of a. attophilus (table 2). a number of phorid species show inclination for attacking workers at different sites along the trails or in the sociobiology 63(4): 1015-1021 (december, 2016) 1019 foraging area; they may also show distinct attack behavior (erthal & tonhasca, 2000; tonhasca et al., 2001; bragança et al., 2009a). phorid females seem to be attracted to their hosts by chemical and visual signals. brown (1991) and morrison & king (2004) offered evidence that phorids infecting other genera of ants use alarm pheromone clues to locate their hosts. on the other hand, visual stimuli from a. sexdens were sufficient to trigger eibesfeldtphora to detect and recognize a host, although the stimuli related to the presence of a trail pheromone increased the period spent inspecting the host, thus increasing the probability of successful attack (gazal et al., 2009). the procedure for discriminating between parasitized and non-parasitized ants by the fly may be imperfect, bearing in mind that super parasitism has already been described both in the field and in the laboratory. feener & brown (1993) found a 19% incidence of natural super parasitism in atta cephalotes (linnaeus) by eibesfeldtphora curvinervis (malloch); bragança et al. (2009b) reported rates of 29.4 %of “self superparasitism” and 49.5 % of “conspecific super parasitism” on a. laevigata by e. elongate in laboratory experiments. in both situations super parasitism was confirmed by atypical behavior during oviposition or by the presence of more than one egg or larva in the body of the host; however, only one fly emerged in these studies. the development of more than one fly of a. attophilus inside the host´s head, including a. sexdens and a. laevigata, seems to be not a true super parasitism because the larvae were all in the same stage and the flies appeared within nearly the same day, thus indicating that they were not product of independent ovipositions. the only known instance of multiparasitism by phorid on ant was reported by brown (1999). that author wrote that the phorids rhyncophoromyia maculineura borgmeier and a species of diocophora borgmeier emerged from the gaster and the leg, respectively, of a single individual of camponotus sericeiventris guérin. although several species of phorids may be commonly seen attacking at the same time a particular colony of atta (marcos bragança, personal observation), multiparasitism had yet not been described as occurring in leaf-cutters, be it by consecutive attacks of distinct phorids on a same ant or by watching two or more species of phorids emerge from the same ant. the low level of multiparasitism here described (0.0055% of all the collected ants) shows that this phenomenon is rare among leaf-cutters and denotes only the cases when two flies from different species come out from the same host. the rates of multiparasitism described between a. attophilus and eibesfeldtphora sp. (0.001%), between a. vicosae and m. grandicornis (0.001%) and between a. attophilus e a. vicosae (0.013%) (table 1) were lower than the probability of multiparasitism predicted to these three combinations (0.070%, 0.063% and 3.591%, respectively). the successful multispecies events should only occur when the larvae happen to mature at the same time. however, when multiparasitism is reckoned as the oviposition of eggs by more than one fly species on the same individual, the real incidenceof the event here described may be higher, if female flies make mistakes in host recognition (already infected or not) as frequently as in the case of super parasitism in atta (feener & brown, 1993; bragança et al., 2009b). hence, cases of more than one fly emerging from an ant should be interpreted as accidental events. the results from this work enlarge the range occupied by phorids that attack a. sexdens to include the savannah of south america.they also allow assessment to be made on parasitic behavior between a. sexdens and a. laevigata, the two most important species of leaf-cutting ants damaging forest and farms in brazil. the proportional percentage of parasitism of around 60% of a. sexden worker ants by m. grandicornis and of a. laevigata by a. attophilus suggests that these two parasitoids should be the main subject of future studies aimed at the use of phorids for the management of leaf-cutting ants in brazil. this is especially true for a. laevigata, with the highest parasitism rate and its primary parasitoid with the lowest larval development time and higher rate of emergence. the small occurrence of multiparasitism by phorids, here related for the first time as occurring in leafcutting ants, gives rise to questions having to do with the peculiarities of the host that have still to be clarified as to attraction, recognition and discrimination between hosts. acknowledgements malb, lrrs, hcm and tmcdl were given scholarships by the national research council (cnpq); fva obtained a scholarship by the coordination for the development of high level personnel (capes). the studies by malbr on leaf-cutters and phorids were sponsored by cnpq/bionorte and by an agreement between the federal university of tocantins and the state secretary of development, science, technology and innovation of tocantins (sedecti/to).we are grateful to kelly o. amaral, kezia a. santos and helen aguiar for helping to collect the ants, and ananonymous reviewer for valuable corrections and suggestions. references bragança, m.a.l. 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(2001). http://dx.doi.org/10.1155/2013/539780 http://dx.doi.org/10.1017/s0007485307005548 http://dx.doi.org/10.1603/ec10173 sociobiology 63(4): 1015-1021 (december, 2016) 1021 parasitism and biology of myrmosicarius grandicornis (diptera, phoridae) in relationship to its host, the leaf-cutting ant atta sexdens (hymenoptera: formicidae). insectes sociaux, 48: 154-158.doi: 10.1007/pl00001759 uribe, s., brown, b.v., bragança, m.a.l., queiroz, j.m. & nogueira, c.a. (2014). new species of eibesfeldtphora disney (diptera: phoridae) and a new key to the genus. zootaxa, 3814: 443-450.doi: 10.11646/zootaxa.3814.3.11 http://dx.doi.org/10.11646/zootaxa.3814.3.11 _goback doi: 10.13102/sociobiology.v60i2.174-182sociobiology 60(2): 174-182 (2013) arboreal ant assemblages respond differently to food source and vegetation physiognomies: a study in the brazilian atlantic rain forest jj resende1, pec peixoto1, en silva1 jhc delabie2,3 & gmm santos1 introduction the richness and composition of ant assemblages have been related to the different structural aspects or the level of habitat preservation (schoener 1971, greenslade & greenslade 1977, levings 1983 & andersen 1995). ants are frequently chosen for studies that focus on the understanding of the effects of repetitive events of man-made habitat or ecosystem simplification on biodiversity (matos et al. 1994, majer 1996, perfecto et al. 1997). several studies have shown significant correlations between ant assemblages and habitat structural complexity, particularly in the tropics (andersen & majer, 2004, delabie et al. 2006). for example, the richness of ants in the forest leaf litter has a strong correlation with plant diversity (pereira et al. 2005) and in coffee agrosystems, the diversity of twig dwelling ants increases in habitats with more diverse shade tree cover (armbrecht et al. 2004). human activities have often caused the simplification of natural environments, leading to local extinction of populaabstract this study aimed to analyze assemblages of arboreal ants in different vegetation physiognomies within the tropical moist forest (atlantic rain forest) domain. the study was carried out at the michelin ecological reserve, state of bahia, northeast of brazil. we used sardine (protein resource) and honey (carbohydrate resource) baits to collect ants foraging in three vegetation types: (1) preserved native forest, (2) forest in regeneration (capoeira) with many invasive plants and (3) a mixed agroystem of rubber and cocoa tree plantation. we recorded 69 ant species attracted to the baits, 21 of them exclusive to honey bait and 25 exclusive to the sardine baits. the vegetation physiognomies preserved forest and rubber/cacao agrosystem showed higher species richness in relation to the forest in regeneration (capoeira), suggesting that rubber tree plantations can be a good matrix for the maintenance of some ant species typical of the forest matrix. the type of resource used is important for the structuring of the arboreal ant assemblages. the ants that were attracted to protein resources showed a guild composition that is more differentiated between vegetation types that of ants attracted to glucose resources. sociobiology an international journal on social insects 1 universidade estadual de feira de santana, feira de santana, bahia, brazil 2 universidade estadual de santa cruz / ceplac/cepec, ilhéus-itabuna, bahia, brazil 3 ceplac/centro de pesquisas do cacau, itabuna, bahia brazil research article ants article history edited by: kleber del-claro, ufu – brazil received 13 may 2013 initial acceptance 11 june 2013 final acceptance 17 june 2013 keywords habitat preference, resource preference, matrix quality, formicidae, community ecology corresponding author janete jane resende departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina s/n novo horizonte feira de santana, bahia, brazil 44036-900 e-mail: antforjane@gmail.com tions and species and, consequently, could negatively impact important ecological processes such as nutrient cycling, seed dispersal and pollination (thomas 2000, de marco & coelho 2004). simplified environments often harbor a lower richness and diversity of ants, with an ant fauna consisting of generalist species (sobrinho & schoereder 2006), unlike forested habitats that harbor ant assemblages with higher levels of diversity, consistent with the characteristics and the complexity of the vegetation (majer 1996, pereira et al. 2007). different sampling methods have been used in surveys of ants and there is no direct means of comparison between different collection procedures (romero & jaffe 1989). there are several methods used for sampling ants such as oil sardine, carbohydrates, meat and cassava flour baits, winkler extractor, pitfall or manual collection, each one of them suited to select different classes of ants (soil dwelling, carnivorous, detritivorous, omnivorous) (bestelmeyer et al. 2000; freitas et al. 2004). each of the collection procedures sampled a different set of ant species (romero & jaffe 1989) and the foragsociobiology 60(2): 174-182 (2013) 175 ing activities may reflect or indicate the nutrients that are most limiting to in the respective nesting habitats, some ants prefer honey baits (carbohydrate) and other ants set prefer fish baits (nitrogenprotein) (hashimoto et al. 2010). ants provide an ideal system to test how macronutrient availability affects the costs and benefits of competitive dominance (grover et al., 2007). considerable evidence suggests that resource competition strongly influences population and community dynamics in ants (hölldobler & wilson 1990) this study aimed to analyze arboreal ant assemblages in different vegetation physiognomies within the atlantic rain forest domain. we tested two hypotheses. first, if the agroforestry system constitutes a good matrix for the maintenance of ant species, richness of ants would be expected to be similar to or even greater than that found in forest like physiognomies. second, since trophic groups of ants that are glycoside and protein consumers are generalist and specialist, respectively, the ant assemblages of generalist ants are expected to be more similar while the second ones would be more dissimilar between habitats. material and methods study area the study was conducted at the michelin ecological reserve (headquarters: 13º 50’s, 39º 10’ w) ituberá, state of bahia, brazil. the climate is of the type as according to the köppen classification, tropical, rainy, hot, characterized by rainfall concentrated in summer and autumn, and average temperatures are never below 20º c. the landscape of the region is characterized by the dominance of cacao agroforestry, mixed rubber/cacao plantations, pastures and forest fragments with natural vegetation comprising primary forest (a small percentage) and different stages of forest regeneration (with a large proportion of secondary forest). we collected ants in six different periods between october 2007 and september 2008. ant assemblies attracted to carbohydrate and protein baits were collected in three vegetation physiognomies: (i) conserved atlantic forest fragment (pf), a typical low land humid tropical forest measuring about 550 ha comprising blocks of native forest canopy of 15-25 m in height, with isolated trees reaching 30-40 m; (ii) forest regeneration fragment (capoeira sf), an early to intermediate stage of secondary succession tract of land, measuring about 10 ha of contiguous forest in regeneration, characterized by the presence of lianas, bromeliads, orchids, rocky ground, shrub vegetation and invasive plants and (iii) agrosystem of mixed rubber tree/cacao plantation (ag), with over 20 years of age and 12 ha of area. sampling methods the sampling consisted of 18 transects distributed among three vegetation types. in each vegetation type six transects of 400 m were established. in each transect there were 20 sampling points spaced at 20 m intervals. each sampling point consisted of a bait rich in proteins and lipids (sardine oil) and another bait rich in carbohydrates (honey) installed within the same tree at a height of 2 m and at least 20 cm away from one another. after installation, the baits remained on the plants for about 30 min. they were then collected and the ants present were fixed in alcohol 70%. in total, 120 baits were placed in each vegetation type. the sorting, assembly and morphospeciation of ant specimens occurred in the laboratory of entomology, at feira de santana state university (uefs). for the identification of ant species we used the classification of bolton (2003), except for the genus nylanderia (based on lapolla et al. 2010), and the genera strumigenys e basiceros (based on baroni-urbani & de andrade 2007). vouchers were deposited in the entomological collection prof. johann becker, museum of zoology uefs (mzfs) at feira de santana and the collection of the myrmecology laboratory of cepec/ceplac (cpdc) at ilhéus, state of bahia. data analyses the analyses of data took into consideration only presence or absence of the species as usual in ant community ecology studies (longino 2000). the observed species richness was calculated using the rarefaction curve (mao tau) (colwell et al. 2004). the total richness was estimated using the 1st order jackknife estimator based on 50 randomizations (heltshe & forrester, 1983), performed with the program estimates, version 7.5.2 (colwell, 2006). the dissimilarity of ant assemblages between vegetation types was assessed by performing a principal coordinate analysis (pcoa) using the program r (r development core team 2010). this analysis was preceded of the calculation of the jaccard similarity index for the pair-wise combination of data collected in all transects, considering as a transect the 20 samples collected by vegetation type and sampling date. therefore, the analyses were based on 18 transects (three vegetation types times six dates). results we recorded 69 species of ants attracted to the baits. out of this total, 21 species were exclusive to honey baits and 25 to sardine baits (table 1). with respect to vegetation types, 17 species were exclusive to conserved forest, 16 to the agrosystem and seven to the “capoeira”. regarding the frequency of species, solenopsis sp.2 was the most frequent on both sardine (31.6%) and honey (20%) baits in rubber/cacao tree agroforestry. the second most frequent species in this physiognomy was camponotus sp.5 which was recorded on 11.6% of sardine baits. in the conserved forest physiognomy, the most frejj resende, pec peixoto, en silva, jhc delabie & gmm santos vegetation structure and arboreal ant diversity176 quent ants on sardine baits were crematogaster sp.1 (9.1% of the baits) and ectatomma tuberculatum (8.3%). the same happened in the “capoeira” area with crematogaster sp.1 present on 10% of the baits and e. tuberculatum in 9.1%. for honey baits, strumigenys sp.9 (5.8%) was the most frequent species in conserved forest while crematogaster sp.1 (6.6%) was the most abundant in “capoeira”. among the most speciose genera, pheidole (14 species), pachycondyla (8), camponotus (7) and solenopsis (5) were the most rich. the proportion of species belonging to these genera present in both types of baits remained very close, except for camponotus which had two and seven species recorded on honey and sardine baits, respectively. the two axis extracted from the pcoa analyses with data from the two bait types grouped explained 34.3% of the total variation in ant composition among samples (18.6% of variation explained by axis 1 and 15.6% of variation explained by axis 2). according to this analysis, the species collected in sardine differed from the species collected in honey baits. considering only ants collected in sardine baits, there was also a very clear separation between vegetation types, indicating that the composition of ant species differs between vegetation types (17.6% of variation explained by axis 1 and 17.1% of variation explained by axis 2). on the other hand, the analysis including only ants collected in honey baits demonstrated a low distinction between vegetation types (18.4% of variation explained by axis 1 and 11.7% of variation explained by axis 2), indicating that the assemblage of species that visited the baits rich in carbohydrates were similar among the three habitats (fig. 1). the greatest observed and expected richness of ants were recorded in the preserved forest fragment and in the agrosystem of mixed rubber/cacao plantation (fig. 2, table 1). the observed richness curves (mao tau) showed no stabilization in any of phytophysiognomies (fig. 2). neverfig. 1. ordination by pcoa of species of ants collected in different phytophysiognomies, reserva ecológica da michelin, ituberá e igrapiúna municipalities bahia, brazil. pf – conserved forest; sf capoeira; ag – agrosystem of mixed rubber tree/cacao plantation. (a) ants collected in honey and sardine baits; (b) ants collected in sardine baits and (c) ants collected in honey baits. a b c fig. 2. expected (jack1) and observed (mao tau) richness curves for species of ants collected in a total of 240 samples. reserva ecológica da michelin, ituberá e igrapiúna municipalities, bahia, brazil. pf – conserved forest; sfcapoeira; ag – agrosystem of mixed rubber tree/cacao plantation. r ic hn es s of s pe ci es number of samples sociobiology 60(2): 174-182 (2013) 177 theless, the expected richness according to the species accumulation curve (jack1) showed a sharp increase in the number of species as a function of sample size, followed by an asymptote in the agrosystem habitat. the shape of the curve suggests that in the agrosystem the sampled fauna is more homogeneous than in “capoeira” and conserved forest. therefore the majority of the species is sampled with a lower number of samples. extrapolation of the curves also suggests that the number of total species in the agrosystem is lower than the two other habitats. discussion according to our results, the vegetation physiognomies of conserved forest and agrosystem presented species richness highest than “capoeira”, showing that agricultural habitats including the association between cacao and rubber trees forms a suitable matrix for the maintenance of ant species typical of forest environment. however, the juxtaposition of forest areas close to the agrosystem is an important point allowing to maintain the ant diversity (delabie et al. 2007). similarity between bait types and vegetation physiognomies the similarity between the ant assemblages sampled in sardine baits among the studied vegetation physiognomies, is probably a result of the shared occurrence of species of the genera azteca, camponotus, crematogaster, pheidole and solenopsis which are considered dominant or subdominant if considering the structure of arboreal ant fauna (wilson 1976; majer et al. 1994; brandão et al. 2009). some species of these genera can significantly influence the structure of the arthropod community, exercising strong predation, especially on larvae of lepidoptera and coleoptera (majer & delabie 1993, floren et al. 2002; philpott & armbrecht 2006). this same ecological context may apply to explain the great similarity between the ant assemblages attracted to honey and sardine baits within the agrosystem, because almost the same species were present in both types of baits. other species that were recorded in this physiognomy such as e. tuberculatum, pachycondyla venusta and odontomachus haematodus belong to genera typically considered as those of generalist predator species. except for p. venusta, these ants may supplement their diet with nectar exudates from plants and honoydew producer insects (hölldobler & wilson 1990, delabie 2001). an additional outcome supporting this explanation is the occurrence of the species tapinoma sp.1 in the agrosystem of mixed rubber/cacao plantation. this species has as main feature the generalist behavior, being undemanding in terms of habitat quality and shifting easily from one food source to another. the numerous species of ants visiting sardine baits in each vegetation physiognomy, such as those of the genera hypoponera, megalomyrmex, nylanderia, wasmannia in conserved forest, linepithema in capoeira and dorymyrmex in the agroystem, has an important ecological implication. in case where protein-based resources are scarce in these habitats, they will be almost exclusively used by specialist species that tend to defend this resource with aggressive behaviors, by exhibiting a rapid recruitment of workers and thus preventing access of other species. the species of the genus solenopsis, for instance, have aggressive behavior and are common in disturbed habitats. on the other hand, carbohydrate-based resources are more common and visited by generalist species, which do not have any preference to the types of bait used, such as those of the genera camponotus, pheidole and solenopsis (hölldobler & wilson 1990). although, in general, species richness correlates positively with habitat complexity, this correlation seems to depend on the habitat, because the agrosystem had a higher observed richness compared to “capoeira”, which is considered structurally more complex than the former. lassau & hochuli (2004) found similar results with a greater richness in less complex habitats, believing that the movement of ants may be more efficient in terms of energy, in less complex habitats. gomes et al (2010) demonstrates that ant fauna is more influenced by vegetation integrity than by fragment size, distance to edge or forest cover surrounding fragments. lopes et al (2012) shows that the species that compose the ant assemblies in different phytophysiognomies are a reflex of the environment, especially of the plant species, supporting the hypothesis that differences in the vegetational composition result in different position of the ant assembly. the higher number of species in the agrosystem in comparison to “capoeira” suggests the occurrence of occasional species with low occurrence, sometimes called “tourists”. these species are treated by the estimators of richness as singletons or doubletons and can boost the expected richness estimation. in spite of this, a greater number of species considered dominant (genera camponotus, crematogaster, solenopsis and pheidole) was recorded for the agrosystem in comparison to “capoeira”. it is important to remark that although the species accumulation curve based on jack1 reached an asymptote in the agrosystem, the estimated richness in the capoeira is in an ascending trend and could surpass the estimated richness of the agrosystem if the sampling effort were increased. this leaves the inference on the quality of the rubber/cacao agrosystem as a good matrix open, from the perspective of which habitat is richer. however, looking at the list of species in table 1, the following results are found. out of the 69 ant species collected in all habitats, 13 (18.84%) are common to both conserved forest and agrosystem. six species are protein consumers and seven carbohydrate consumers. furthermore, the relative frequencies of most of these species are low and very similar in forest and agrosystem habitats. thus, from the point of view of life history, we can infer that the agrosystem is a good qualjj resende, pec peixoto, en silva, jhc delabie & gmm santos vegetation structure and arboreal ant diversity178 ity matrix for at least some rare species that also occur in forest vegetation. the choice of food resource (honey or sardine baits) by the ant assemblages, clearly differentiate the three vegetation physiognomies. both proteins and carbohydrates are of paramount importance for maintenance of ant populations. hashimoto et al. (2010) demonstrated that different populations of ants forage more actively on these resources when they are scarce. on one hand, the proteic resources are fundamental for brood development and tissue synthesis (hölldobler & wilson 1990). on the other hand, carbohydrates are key resources for the maintenance of the ant activity (davidson, 1998). byk and delclaro (2011) demonstrated the benefits of the carbohydrate resources to ant populations. the glucidic foods influence ant species composition, abundance, number of individuals per colony, body weight, survivorship, growth, reproduction and interactions at the community level. bihn et al. (2008) examined bait preferences of litter ants along a successional gradient of forests in the atlantic forest of brazil and observed that ants preferred protein-based baits in secondary forests, and carbohydrate-based baits in old-growth forests. in addition, the ant preference for carbohydrate or protein is subject to change individually according a previous ingestion of one of this food source, extrafloral nectar or insect. this is the theory of ecological stoichiometry, which relates nutrient balance to ecological processes (sterner & elser 2002). such nutritional complexity can mediate patterns of ecological interactions (anderson et al. 2004). therefore, if an ant individual is consuming extrafloral nectar, it is more propable it attacks a herbivore on the host plant aiming to balance the ingestion of protein after too much ingestion of carbohydrate (grover et al., 2007). our data support our working hypothesis that the type of resource used by ant species is important in structuring the community. the distribution of ant species that are specialist and consumers of protein-based resources (sardine baits) differed more between the vegetation types than the distribution of ant species that are generalist and consumers of carbohydrate-based resources. the similarity of species collected in each vegetation physiognomy was higher among ant assemblages that use carbohydrate-based resources (fig. 1c) than among ant assemblages that use protein-based resources, in an exclusive manner (fig. 1b) or together with carbohydrate sources (fig. 1c). acknowledgements to cnpq (project 620021/2008-0 – edital no 16/2008 and pronex fapesb-cnpq project pnx 011-2009); to foundation for research support de in bahia state (fundação de amparo à pesquisa do estado da bahia fapesb (project 5577/2009) and the michelin ecological reserve (rem) supported this study. gmms and jhcd thanks to cnpq for productivity fellowship. to many colleagues that contributed to this study, specially allan rhalff gomes teixeira and emerson mota da silva for helped with field sampling. we would like to thank two anonymous reviewers provided their insightful comments on the manuscript. references andersen, a.n. & majer, j.d. 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(1976). which are the most prevalent ant genera? studia ent., 9: 1-4. sociobiology 60(2): 174-182 (2013) 181 table 1. number of records and relative frequency (%) of ants collected in honey and sardine baits in three vegetation physiognomies in the reserva ecológica da michelin. ituberá e igrapiúna municipalities, bahia, brazil pf – preserved forest; sf secondary forest (capoeira); ag – agrosystem of mixed rubber tree/cacao plantation. food preferences (c= primarily carbohydrate consumer, p= primarily protein consumer, c/p = both; sensu brandão et al. 2009). sampled species food preferences honey bait sardine bait pf sf ag pf sf ag amblyoponinae prionopelta sp.1 p 1 (0.8) ectatomminae ectatomma sp.1 p 2 (1.6) ectatomma brunneum f. smith, 1858 p 3 (2.5) ectatomma tuberculatum f. smith, 1858 p 4 (3.3) 8 (6.6) 10 (8.3) 11 (9.1) 7 (5.8) gnamptogenys sp.3 p 3 (2.5) dolichoderinae azteca sp.1 c/p 2 (1.6) 3 (2.5) 7 (5.8) 1 (0.8) 6 (5) azteca sp.28 c/p 4 (3.3) 3 (2.5) 2 (1.6) 3 (2.5) azteca sp.3 c/p 2 (1.6) azteca sp.4 c/p 3 (2.5) dorymyrmex sp.1 c 4 (3.3) linepithema sp.1 c 3 (2.5) 4 (3.3) 2 (1.6) linepithema sp.3 c 1 (0.8) tapinoma sp.1 1 (0.8) 2 (1.6) 4 (3.3) 1 (0.8) formicinae camponotus sp.2 c 5 (4.1) camponotus sp.3 c 1 (0.8) 4 (3.3) camponotus sp.4 c 3 (2.5) camponotus sp.5 c 3 (2.5) 14 (11.6) camponotus sp.6 c 5 (4.1) camponotus sp.7 c 3 (2.5) camponotus sp.8 c 1 (0.8) brachymyrmex sp.1 c/p 3 (2.5) 3 (2.5) brachymyrmex sp.4 c/p 3 (2.5) 1 (0.8) 1 (0.8) nylanderia sp.2 c/p 1 (0.8) nylanderia sp.3 c/p 1 (0.8) nylanderia sp.4 c/p 1 (0.8) 3 (2.5) myrmicinae cephalotes atratus (linneus, 1758) c 1 (0.8) cephalotes sp.1 c 3 (2.5) cephalotes sp.2 c 1 (0.8) crematogaster sp.1 c 8(6.6) 3 (2.5) 11 (9.1) 12 (10) 8 (6.6) crematogaster sp.2 c 1 (0.8) 2 (1.6) 1 (0.8) 1 (0.8) crematogaster sp.3 c 3 (2.5) 1 (0.8) 12 (10) crematogaster sp.5 c 2 (1.6) 1 (0.8) crematogaster sp.6 c 10 (8.3) pheidole sp.1 c/p 2 (1.6) 1 (0.8) pheidole sp.2 c/p 1 (0.8) jj resende, pec peixoto, en silva, jhc delabie & gmm santos vegetation structure and arboreal ant diversity182 table 1 (continued) pheidole sp.5 c/p 3 (2.5) pheidole sp.10 c/p 1 (0.8) 1 (0.8) 3 (2.5) 1 (0.8) pheidole sp.12 c/p 3 (2.5) pheidole sp.14 c/p 6 (5) 4 (3.3) pheidole sp.15 c/p 1 (0.8) 1 (0.8) pheidole sp.16 c/p 5 (4.1) 3 (2.5) pheidole sp.17 c/p 1 (0.8) 2 (1.6) pheidole sp.18 c/p 1 (0.8) 1 (0.8) pheidole sp.20 c/p 1 (0.8) 3 (2.5) pheidole sp.23 c/p 1 (0.8) 1 (0.8) pheidole sp.24 c/p 2 (1.6) 1 (0.8) 1 (0.8) pheidole sp.26 c/p 3 (2.5) 2 (1.6) carebara pilosa fernández, 2004 p 2 (1.6) megalomyrmex sp.1 p 2 (1.6) solenopsis sp.1 p 1 (0.8) solenopsis sp.2 p 5 (4.1) 24 (20) 2 (1.6) 4 (3.3) 38 (31.6) solenopsis sp.3 p 2 (1.6) 5 (4.1) solenopsis sp.4 p 2 (1.6) 2 (1.6) 4 (3.3) 7 (5.8) 1 (0.8) solenopsis sp.5 p 2 (1.6) 5 (4.1) strumigenys sp.8 p 1 (0.8) strumigenys sp.9 p 7 (5.8) wasmannia auropunctata (roger, 1863) c/p 1 (0.8) basiceros (octostruma) sp.2 p 1 (0.8) ponerinae hypoponera sp.1 p 1 (0.8) hypoponera sp.2 p 2 (1.6) pachycondyla apicalis (latreille, 1802) p 2 (1.6) pachycondyla constricta (mayr, 1884) p 1 (1.6) pachycondyla complexo villosa (fabricius, 1804) p 5 (4.1) pachycondyla harpax (fabricius, 1804) p 1 (0.8) pachycondyla venusta (forel, 1912) p 2 (1.6) 2 (1.6) 3 (2.5) 1 (0.8) pachycondyla villosa (fabricius, 1804) p 2 (1.6) pachycondyla sp.1 p 1 (0.8) doi: 10.13102/sociobiology.v65i2.2076sociobiology 65(2): 244-252 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the roles of bees and hoverflies in the pollination of jacquemontia evolvuloides (moric.) meisn. (convolvulaceae) in a semiarid region introduction many ruderal plants grow in urban and agricultural environments and are considered pests due to their invasive natures (souza & lorenzi, 2008), and are usually overlooked for pollination studies. but those plants often display striking floral advertisements (e.g., bright colors) and offer attractants (such as pollen and nectar) that help maintain native and non-native floral visitors, many of which are also important pollinators of local plants. ruderal species of convolvulaceae are common in anthropogenic environments, but rarely studied, abstract jacquemontia species are often found in disturbed areas, and their flowers are ephemeral and very attractive to insects. we investigated the interaction between the flowers of jacquemontia evolvuloides and its visitors, with emphasis on potential pollinators, considering morphological, temporal, and behavioral aspects, between september/2009 and august/2010 in an anthropized area in bahia state, northeastern brazil. jacquemontia evolvuloides flowers are diurnal, ephemeral (approximately five to six hours duration), are present throughout the year, and offer nectar and pollen rewards. because of the floral morphology of j. evolvuloides insect visitors have easy access to its resources, and its pollination could be considered generalized. while j. evolvuloides flowers are visited by several insects, only small bees (augochlora spp.) and syrphids (toxomerus spp.) appear to play a role in pollination of flowers. although bees have been considered efficient pollinators of that plant, the significant presence of syrphids (considered occasional pollinators but showing high frequency visitation), may indicate their role as potential pollinators, especially when bees are not abundant in the fall/winter. as such, even in pollination systems that are considered generalized (with flowers allowing easy access to various visitor groups), visitor size may be an important factor for efficient pollination, especially when associated with high visitation frequencies. sociobiology an international journal on social insects jrl paz1, cm pigozzo2, m gimenes3 article history edited by astrid kleinert, usp, brazil received 13 september 2017 initial acceptance 23 february 2018 final acceptance 25 march 2018 publication date 09 july 2018 keywords apoidea; augochlora; caatinga; ruderal plant; syrphidae; toxomerus. corresponding author miriam gimenes universidade estadual de feira de santana departamento de ciências biológicas laboratório de entomologia av. transnordestina, s/nº, novo horizonte cep 44031-460, feira de santana-ba, brasil. e-mail: mgimenes@uefs.br as is the case of weed species of jacquemontia choisy (simão-bianchini & pirani, 2005) with significant ornamental potential (judd et al., 2009). more research is needed to know which species of weeds are beneficial and use them to attract pollinators. these plant species also occur on the edges fields and are important for the maintenance of pollinators (nicholls & altieri, 2013; garibaldi et al., 2016). jacquemontia comprises approximately 120 species (staples & brummitt, 2007) found throughout the tropical americas, occurring as natives in several vegetation formations. jacquemontia evolvuloides (moric.) meisn. is widely distributed, 1 programa de pós-graduação em botânica, universidade estadual de feira de santana, departamento de ciências biológicas, laboratório de entomologia, feira de santana-ba, brazil 2 centro universitário jorge amado, coordenação da licenciatura e bacharelado em ciências biológicas, campus paralela, salvador-ba, brazil 3 universidade estadual de feira de santana, departamento de ciências biológicas, feira de santana-ba, brazil research article bees mailto:mgimenes@uefs.br sociobiology 65(2): 244-252 (june, 2018) 245 occurring mainly in the cerrado, atlantic forest, and caatinga phytophysiognomies (bianchini & ferreira, 2010). their flowers are very attractive to insects, mostly bees (piedadekiill & ranga, 2000; pinto-torres & koptur, 2009; rodriguez et al., 2010; silva et al., 2010; kiill & simão-bianchini, 2011), as well as to larger animals such as hummingbirds (machado, 2009). information about the reproductive strategies and the pollinators of jacquemontia species is important because, as a ruderal plant, it produces many flowers with colorful corollas and nectar and pollen resources that can contribute to the maintenance of native pollinators in anthropized and agricultural areas (piedade-kiill & ranga, 2000). jacquemontia flowers are ephemeral and last less than 10 hours (silva et al., 2010), limiting the window of time for collecting resources, a fact that must be taken into account in pollination studies. considering the importance of ruderal plants in urban areas for the maintenance of local pollination communities, we investigated the floral biology of j. evolvuloides, with emphasis on the morphological, behavioral, and temporal aspects of its floral visitors as related to pollinating efficiency. materials and methods the present study was carried out on the campus of the universidade estadual de feira de santana – uefs, in feira de santana, bahia state, northeastern brazil (12º11’56’’s, 38º58’07’’w). the original local vegetation of the area was dryland caatinga (thorny, deciduous, shrub/arboreal) vegetation, but it is presently supplemented with introduced and invasive plants due to anthropization (santana & santos, 1999). the local climate is classified as bsh or semiarid (köppen’s classification), with average monthly temperatures ranging between 20.7 and 26.8 ºc, and total annual rainfall between 500 and 800 mm (sei, 2011). observations were performed on a monthly basis from september to november/2009, january/2010, and from april to august/2010. climatic data (temperature and relative humidity) were recorded using a digital thermohygrometer at hourly intervals during all of the field observations following anthesis. rainfall, average temperatures, and relative humidity data were obtained from the uefs climatology station (fig 1). jacquemontia evolvuloides is a creeping, prostrate shrub that usually occurs in cultivated areas, vegetable gardens, and orchards. it forms wide carpets covering the ground that inhibit the growth of other plants (moreira & bragança, 2011). it occurs spontaneously in the study area at vegetation borders, in clearings, and along roadsides. due to the difficulty of individualizing the plants, we considered all of the individuals present in a 15 m x 13 m area (195 m²) during our field observations. flowering was recorded monthly by counting all open flowers in the sampling area. to verify any correlations between meteorological variables and the numbers of flowers, a simple correlation test was carried out using spss 8.0 software for windows. to control error accumulations in the confidence intervals for each variable, the bonferroni correction was used, dividing the level of significance by 4 (p=0.0125). for morphological studies, ten flowers from different individuals were fixed in 70% alcohol and subsequently measured using a digital caliper to determine corolla diameter and length, and the sizes and dispositions of the reproductive structures. these values were used for calculating flower size, which was classified according to machado and lopes (2004). flower anthesis and pollen availability were recorded (n = 10 flowers) for three consecutive days each month. stigmatic receptivity was determined by immersing the stigmas in 10% hydrogen peroxide (h2o2) (dafni & maués, 1998) at hourly intervals starting at the bud phase through closing. the presence of osmophores was determined by immersing the flowers in a 1% neutral red solution for 15 minutes (dafni et al., 2005). in order to verify flower odor, fresh flowers were stored in a closed recipient for one hour. ultraviolet pigments were detected in flowers by exposing them to ammonium hydroxide vapors (p.a.) for 30 seconds, and then examining them for color changes (adapted from scogin et al., 1977). these procedures were carried out with ten flowers throughout the flowering period. floral reproductive biology was analyzed through pollination experiments, using 15 flowers in the pre-anthesis stage in each treatment: apomixis, spontaneous and induced self-pollination, manual cross pollination (xenogamy), and control (natural conditions). the flowers used in the experiments were emasculated and bagged, when appropriate, and followed until fruit formation. pollination success was estimated based on the fruit formation rates of marked flowers. the chi-squared test (p<0.01) was used to determine if there were differences among the pollination experiments in terms of fruit formation. floral visitors were observed and collected during three days (two days for collecting of individuals and one for behavioral observations and visitor visit counts) from 05:00 to 16:00 h, at 30 min intervals during every hour of observation during the study months, at one area of 15 m x 13 m (195 m²). in study area the number of flowers observed varied during each month (82 flowers in november to 337 in june). the behaviors of the floral visitors were determined using direct observations, videos, and photographs. only the numbers of floral visits by the most abundant and constant insects were taken into consideration in the analyses of daily activities. daily activities were analyzed during the dry (austral spring: september, october, and november) and rainy (austral autumn/ winter: may, june, and july) trimesters. for visitor species of difficult identification, the numbers of visits were considered at the family level (as for example, halictidae). the spearman correlation test was used for the analyses of the numbers of open flowers, numbers of sampled visits, numbers of visitors collected, and climatic factors, using jrl paz, cm pigozzo, m gimenes – bees and hoverflies in the pollination of j. evolvuloides246 spss 8.0 software for windows, with a bonferroni correction (p<0.01). the normality of the data was verified using the kolmogorov-smirnov test (p<0.05), also run on spss 8.0 software for windows. the sizes of floral visitors were determined by measuring the total lengths (from the median ocellus to the end of the abdomen) and widths (intertegular distance) of ten individuals of each visiting species. flower visitors size classification was based on the parameters proposed by viana and kleinert (2005). visitor constancy was calculated using the formula: c = (number of samples with the species/ number of samples) x 100. the species were classified as c = constant (c > 50%), a = accessory (c = > 25% < 50%), or ac = accidental (c < 25%) (thomazini & thomazini, 2002). glycerol gelatin semi-permanent slides were made with pollen from the bodies of the most frequent and constant floral visitors for analysis. seven individuals of each visiting species were analyzed, and five slides were made from each individual. the criteria used for evaluating the pollinators were: frequency, constancy, behavior, daily activity, body dimensions compatible with floral dimensions, contact with the reproductive structures of the plant, and pollen load analysis. the floral visitors collected were deposited in the prof. johann becker entomological collection at the zoology museum of the universidade estadual de feira de santana (mzfs). the botanical voucher specimens were deposited in the uefs (huefs 159.600) and maria eneyda p. kauffmann fidalgo herbaria (sp 420.331). 0 20 40 60 80 100 120 140 160 180 sep/09 nov jan/10 mar may jul months p lu vi o si ty r h t em p er at u re 0 50 100 150 200 250 300 350 400 n u m b er f lo w er s pluviosity rh temp flow ers fig 1. number of flowers in jacquemontia evolvuloides (convolvulaceae), and rainfall (mm), relative humidity (%), and temperature (°c) in anthropized semiarid (area observed: 195 m2), of bahia state, northeastern brazil, between september/2009 and january 2010, and between april and august/2010. source: uefs climatology station. results jacquemontia evolvuloides flowering was continuous during the months of observation, but with variations in the number of flowers. the greatest number of flowers were observed in may and june/2010 (231 and 337 open flowers respectively); the lowest number of flowers (n = 14) was observed in january/2010 (fig 1). correlation tests performed showed no significant correlation between the number of flowers and the climatic variables of temperature (r = -0.711, p>0.0125) or rainfall (r = -0.38, p>0.0125), but showed a statistically significant correlation with relative humidity (r = 0.152, p<0.0125). the flowers of j. evolvuloides are held in simple axillary cyme inflorescences, presenting one to three open flowers daily, with approximately 18 flower buds in different stages of development. the shallow, funnel-shaped flowers are odorless to humans and do not show osmophores; ultravioletreflecting pigments were detected in the corolla and anthers. the corolla is 20.7 ± 1.3 mm wide (average ± sd) and 17.5 ± 1.9 mm deep, being large, conspicuous, with blue flowers with a lilac inner-central region (fig 2). the androecium is white and surrounds the pistil. the two largest stamens are 8.5 ± 0.2 mm long, while the three smaller stamens measure 7.1 ± 0.2 mm. the anthers are white, 1.5 ± 0.1 mm long, with outward rimose dehiscence in relation to the corolla tube. the gynoecium is white, the stigma 8.2 ± 0.7 mm long, with a discoid nectary surrounding the ovary. 0 20 40 60 80 100 120 140 160 180 sep/09 nov jan/10 mar may jul months p lu vi o si ty r h t em p er at u re 0 50 100 150 200 250 300 350 400 n u m b er f lo w er s pluviosity rh temp flow ersflowers sociobiology 65(2): 244-252 (june, 2018) 247 the anthesis of j. evolvuloides flowers occurs in the morning. pollen is available to flower visitors from the moment of opening, and the stigma is receptive immediately and remains so until senescence; the flowers last approximately five to six hours. the first flowers of j. evolvuloides open near 05:30 h, with full opening generally occurring between 06:40 and 07:00 h, with a longer delay during the winter months due to lower temperatures and later sunrises. the flowers close between 11:47 and 14:09 h (table 1). the flowers opened and closed later in june and july when temperatures were lower and the photoperiod shorter (later sunrises). fruit set in both manual and spontaneous selfpollination experiments suggest that j. evolvuloides is self-compatible (table 2), although there were significant statistical differences between the treatments (c2 = 26.558, p<0.01). fruit set was observed in both the manual and spontaneous self-pollination experiments, but the highest fruit set rates were observed in cross-pollination experiments and in the control group, and with no occurrence of apomictic fruits (table 2). the fruits of j. evolvuloides are dry capsules, usually containing four light-black, pubescent seeds, which are formed within 7 to 10 days. fig 2. (a) flower of jacquemontia evolvuloides (convolvulaceae). detail of the reproductive structures. (b) detail of a floral visitor bee (augochlora sp., halictidae). months fo ± sd t (oc) fc ± sd t (oc) fd sep-2009 06:40 00:09 22.6 12:20 00:19 33.5 05:39 oct-2009 06:54 00:17 25.3 12:56 00:46 28.5 06:02 nov-2009 07:00 00:17 26.8 12:21 00:30 34.4 05:19 jan-2010 06:57 00:18 26.7 11:52 00:09 31.7 04:55 apr-2010 07:00 00:09 26.2 11:47 00:24 30.6 04:45 may-2010 07:18 00:30 22.4 12:13 00:17 27.3 04:55 jun-2010 08:05 00:22 20.6 13:42 01:08 26.6 05:37 jul-2010 08:51 00:39 22.0 14:09 01:12 24.6 05:18 aug-2010 06:54 00:12 21.8 12:31 00:23 28.4 05:37 table 1. average values (± standard deviation, sd) of the opening and closing times of flowers of jacquemontia evolvuloides (convolvulaceae), and average temperatures (t) in an anthropized semiarid area of bahia state, northeastern brazil, during the months between september/2009 and january/2010, and between april and august/2010. flower opening = fo, flower closing = fc, flower duration = fd. the flowers of j. evolvuloides were visited by small (48.3%) and medium small-sized (16.1%) insects. a total of 159 insect individuals were sampled, and 543 visits to the flowers were recorded, distributed among 31 insect species. most of the floral visitors were classified as accidental (84.5%), 12.5% were considered accessory, and 3% were constant. among them, the most frequent were: toxomerus watsoni (syrphidae, diptera) with 50 individuals collected and 157 floral visits and halictidae (fig 2) (especially the genus augochlora), with 25 individuals collected and 175 floral visits. t. watsoni and augochlora spp. were responsible for treatment flowers (n) fruits (n) % apomixis 15 0 0 spontaneous self-pollination 15 2 13.3 hand self-pollination 15 5 33.3 hand cross-pollination 15 11 70.3 natural pollination (control) 15 10 66.7 table 2. results of the reproduction experiments carried out with jacquemontia evolvuloides (convolvulaceae) flowers, in an anthropized semiarid area of bahia state, northeastern brazil, during the months between september/2009 and january 2010, and april and august/2010. jrl paz, cm pigozzo, m gimenes – bees and hoverflies in the pollination of j. evolvuloides248 61% of the total floral visits, and both were also considered constant on the flowers (table 3). although there was no correlation between the number of floral visitors and climatic factors (p>0.01), some trends were detected. when comparing the two study trimesters in relation to insect visits, we observed that the rainiest trimester (may, june and july, austral autumn/winter) had the largest number of flowers (719), but the bees visited fewer flowers (41 visits) than dipterans (125 visits); butterflies visited 44 times and wasps did not appear. the driest trimester (september, october, november, austral spring) had the lowest number of flowers (302), and bees were observed in higher numbers insect visitors ni nv size rc ppb co coleoptera bruchidae sp. 1 3 3 s ne n ac diptera bombyliidae sp. 1 2 2 s ne n ac syrphidae pseudodorus clavatus (fabricius, 1794) 5 3 ms ne n a toxomerus productus (curran, 1930) 2 6 s ne o (head) ac toxomerus watsoni (curran, 1930) 50 157 s ne o (head) c hymenoptera apidae apis mellifera linnaeus 1758 8 16 mi ne n a trigona spinipes (fabricius, 1793) 3 1 s ne n ac halictidae 175 c augochlora sp. 1 3 -ms po/ne y (head, thorax) augochlora sp. 2 13 -s po/ne y (head, thorax) augochlora sp. 3 2 -s po/ne y (head, thorax) augochlora sp. 4 7 -s po/ne y (head, thorax) pseudaugochlora pandora (smith, 1853) 2 -ms po/ne y (thorax) vespidae polybia occidentalis (olivier, 1791) 3 92 ms ne n ac lepidoptera hesperiidae hesperiidae sp. 1 1 6 mr ne n ac hesperiidae sp. 2 1 2 mr ne n ac hesperiinae sp. 1 8 26 mi ne n ac pyrgus orcus (stoll, 1780) 8 26 s ne n ac pyrgus veturius plötz, 1884 24 15 s ne n ac pieridae phoebis sennae (linnaeus, 1758) 1 2 mr ne n ac table 3. numbers of insect visits (nv), numbers of insect individuals (ni), and the resources collected (rc) from jacquemontia evolvuloides (convolvulaceae) in an anthropized semiarid area of bahia state, northeastern brazil, during the months between september/2009 and january/2010, and between april and august/2010. (size: mr = medium robust, mi = medium intermediate, ms = medium small, s = small; resource collected (rc): ne = nectar, po = pollen. constancy (co): c = constant, a = accessory, ac = accidental). presence of pollen on the insect body (ppb: yes = y, no = n, o = occasionally). than dipterans (112 and 12 visits respectively); lepidoptera species visited 8 flowers, and wasps 79 (fig 3). individuals of the orders lepidoptera, hymenoptera, and diptera visited flowers to collect resources from the beginning of anthesis to flower senescence, sometimes arriving even before the flowers were fully open. their visits were more abundant in the interval between 07:00 and 11:00 h, decreasing along the day during both the spring and autumn/winter seasons. floral resource collection behavior varied among the different groups of insects visiting j. evolvuloides. lepidopterans collected nectar by landing on the corolla and inserting their proboscis into the nectary, quite close to the sociobiology 65(2): 244-252 (june, 2018) 249 corolla wall and relatively distant from the anthers. no contact was observed between those insects and the floral reproductive structures during nectar collection (which lasted about 35 seconds on the average), independent of their sizes. syrphidae dipterans hovered in front of the flowers before landing on the corolla. these insects were generally small and reached the nectary by simply moving through the center of the corolla; their visits lasted about 23 seconds. some individuals of toxomerus spp. were observed to have pollen grains adhering to their heads after their visits, indicating contact occasional with the reproductive structures of the flowers. halictidae bees (medium/small, but mostly small) landed on the corolla and then moved towards the nectary area, where they inserted their tongue during visits that lasted only between 2 and 4 seconds. during most of these nectar collections, contact was observed between the ventral region of the bee’s thorax and the reproductive structures of the flowers (fig 2). halictidae bees also collected pollen as soon as the flowers opened by manipulating the anthers with their legs, “embracing” them with circular movements (up to 180º) during visits that lasted between 10 and 45 seconds. during that time, the bees often came into contact with the fig 3. number of flower-visiting insects (diptera, lepidoptera and hymenoptera) in jacquemontia evolvuloides (convolvulaceae) per hour throughout the day, in anthropized semiarid (area observed: 195 m2), of bahia state, northeastern brazil. (a) between september and november/2009 (dry months). (b) between may and july/2010 (wet months). september, october, november 0 10 20 30 40 50 60 70 05:00 07:00 09:00 11:00 13:00 hour n um be r of v is its 5 10 15 20 25 30 35 te m pe ra tu re ( o c ) diptera lepidoptera hym (bees) hym (wasps) temperature may, june, july 0 10 20 30 40 50 60 05:00 07:00 09:00 11:00 13:00 hour n um be r of v is its 5 10 15 20 25 30 te m pe ra tu re ( o c ) diptera lepidoptera hymenoptera (bees) temperature (a) (b) jrl paz, cm pigozzo, m gimenes – bees and hoverflies in the pollination of j. evolvuloides250 reproductive structures of the flowers and many pollen grains remained adhered to the ventral portion of their bodies. the analysis of pollen loads of the ventral portion, showed the presence of four different pollen morphotypes, with j. evolvuloides pollen being predominant (approximately 75% of the pollen grains). only halictidae bees were observed collecting nectar and pollen from flowers. other bees observed on j. evolvuloides flowers, including apis mellifera and trigona spinipes, as well as wasps, collected nectar by landing on the flowers and inserting their tongues into the nectary – but without contacting the reproductive structures. due to the fragility of the petals, especially larger visitors would have difficulty landing on the flowers to collect floral resources. discussion flowering of j. evolvuloides occurred in almost every month of the study period, with the peak in may and june (autumn/winter), correlated with relative humidity. in other areas of the caatinga of northeast brazil (pe), jacquemontia species may show flowering peak during during of the rainy season [jacquemontia nodiflora (desr.) g. don (kiill & simão-bianchini, 2011)] and at the end of the rainy season [jacquemontia multiflora (choisy) hallier f. (piedade-kiill & ranga, 2000)]. jacquemontia evolvuloides flowers are ephemeral and remain open for less than 10 hours, which somewhat limits the resources offered to floral visitors. those ephemeral flowers showed regularity in terms of their opening and closing times throughout the observation period, although they opened and closed later during winter, probably due to lower temperatures and later sunrises at that time of the year. the ephemeral nature of jacquemontia flowers is one of the main features of that genus, with flowers opening early in the morning (especially in environments with high temperatures, such as the caatinga) (piedade-kiill & ranga, 2000; kiill & simão-bianchini, 2011). flowers usually large, showy, and colorful of j. evolvuloides stand out from the surrounding vegetation and attract floral visitors as was noted by authors studying different genera of the convolvulaceae (kiill & ranga, 2003; kiill & simão-bianchini, 2011; paz et al., 2013). the presence of uv-reflecting pigments in the corolla and anthers may also facilitate their location, especially among insects sensitive to those wavelengths, such as bees (roubik, 1989). reproductive tests indicated that j. evolvuloides population studied was self-compatible, although the greatest fruit production was obtained through cross-pollination, as observed with other species of the genus jacquemontia (piedade-kiill & ranga, 2000; pinto-torres & koptur, 2009). while animal-mediated pollination is very successful, its selfpollination capability favors the invasion and establishment in disturbed and/or anthropized environments such as cultivated areas, vegetable gardens, and orchards in northeastern brazil. jacquemontia evolvuloides shows morphological features that classify it as melittophilous according to faegri and van der pijl (1979) but, in addition to bees, wasps, dipterans, lepidopterans, and coleopterans also visited their flowers and collected nectar. a rigid concept of floral syndromes could not be considered in the present study with j. evolvuloides, especially because the frequencies of different types of floral visitors change during the year. hymenoptera species predominate in the spring (especially bees of the genus augochlora, and the wasp polybia occidentalis) while autumn/winter shows the largest numbers of flowers and a predominance of visits by diptera (especially t. watsoni) and lepidoptera. only augochlora spp. and t. watsoni were constant visitors to j. evolvuloides flowers. a combination of floral features of j. evolvuloides (especially the shallow funnel shape of its flowers which can also function as a landing platform) facilitate access by floral visitors of different insect orders, but only limited types of visitors were observed coming into contact with its reproductive structures, with consequent variations among them in terms of their pollination abilities. augochlora spp. showed morphologies and behavioral characteristics that resulted in its contact with floral reproductive structures, which was also attested by the presence of the pollen of j. evolvuloides on that bee’s body. these bees also synchronized their arrivals with floral opening, and are therefore considered relevant pollinators of j. evolvuloides, especially in the spring. interactions of bees with jacquemontia flowers have been documented in various studies. piedade-kiill and ranga (2000) observed augochlora sp. visiting flowers of j. multiflora in the caatinga. but even though these bees were observed making contact with the reproductive structures of the flowers, they were not considered the principal pollinator as they did not make frequent visits. then, in these flowers the bees a. mellifera and t. spinipes were considered effective pollinators due to their visitation frequencies and their behaviors within the flowers. silva et al. (2010) observed the pollination of jacquemontia montana (moric.) meisn. by halictidae bees, with a predominance of the genus dialictus robertson, 1902, in a rupestrian field. pinto-torres and koptur (2009) observed the pollination of jacquemontia reclinata house ex small by apidae, megachilidae, and especially halictidae bees in southeastern florida (usa). toxomerus spp. showed the highest frequency of visits to j. evolvuloides flowers in the autumn/winter (with the presence of large numbers of flowers), occasionally touching their reproductive structures. its role in pollination may therefore be relevant due to its high frequency of visits and by the synchronization of these visits with the opening times of the flowers, making it a potential pollinator. the constant presence of syrphids in j. evolvuloides may be associated with its floral morphology, which promotes easy access to the nectary, as those insects have high energy demands (marinoni et al., 2007). sociobiology 65(2): 244-252 (june, 2018) 251 the presence of syrphids on the flowers of j. evolvuloides may indicate their role as potential pollinators, especially during times when bee visitors are infrequent. freitas and sazima (2003) observed that syrphids frequently visited the flowers of sisyrinchium vaginatum spreng (iridaceae) in serra da bocaina (sp), which has a flowering peak in the winter. according to those authors, only small numbers of bees are present at high altitudes in the winter, and pollination by syrphids appears to be of great importance at that time. the syrphids presented synchronization of visitation with the opening hours of the flowers (freitas & sazima, 2003). the flowers of j. evolvuloides were visited by numerous other insects, but they did not appear to play any role in pollination. the wasp p. occidentalis, as well as hesperiidae lepidopterans were frequent, but not constant, visitors to j. evolvuloides and collected nectar without touching the reproductive structures of the flowers and did not pollinate them. piedade-kiill and ranga (2000) noted that j. montana flowers were visited by butterflies considered nectar robbers. butterflies have likewise been mentioned as floral visitors to other species of the genus, such as j. multiflora (piedade-kiill & ranga, 2000), j. reclinata (piedade-kiill & ranga, 2000), and jacquemontia velutina (choisy) (rodriguez et al., 2010), but they were considered only occasional pollinators or not effective pollinators of those plants. because of the easy access of insect visitors to the floral offerings of j. evolvuloides due to its floral morphology, pollination in this plant could be considered generalist. pintotorres and koptur (2009) studied the pollination of j. reclinata and observed that all of the visitors that collected nectar could act as potential pollinators (except ants), and that its pollination system could be regarded as generalist. while j. evolvuloides flowers are visited by several insects, only small bees (augochlora spp.) and small syrphids (t. watsoni) appear to play a role in pollination of flowers. although bees have been considered efficient pollinators of this plant, the significant presence of syrphids (frequent and constant visitors), may indicate their role as potential pollinators, especially when bees are not abundant in the fall/winter. as such, even in pollination systems that are considered generalist (with flowers allowing easy access to various visitor groups), visitor size may be an important factor for efficient pollination, especially when associated with high visitation frequencies. acknowledgements the authors thank r. simão-bianchini (ib-usp) for identifying the botanical specimens, f.f. de oliveira (ufba-ba), t.m.v.l. muniz (inpa-am), f.c.v. zanella (unila – pr) and s.r. andena (uefs-ba) for identifying hymenoptera species; a.v.l. freitas (unicamp-sp), c.s. motta (inpa-am), j.a. duarte júnior (ufpe-pe), v.o. becker (unb-df), and t.z.b. taumaturgo (ufpr-pr) for identifying the lepidoptera species; priscila p. lopes (uefsba) for identifying coleoptera species; carlos j. e. lamas (mzusp-sp), ivan f. castro (uefs-ba), and mirian n. morales (ufla-mg) for identifying the diptera species; and the fundação de amparo à pesquisa of bahia state (fapesb) for granting a master’s scholarship to the first author. references bianchini, r.s. & ferreira, p.p.a. 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(2016). mutually beneficial pollinator diversity and crop yield outcomes in small and large farms. science, 351(6271): 388-391. doi: 10.1126/science.aac7287. judd, w.s., campbell, c.s., kellogg, e.a.; stevens, p.f. & donoghue, m.j. (2009). sistemática vegetal: um enfoque filogenético. porto alegre, artmed, 464 p. kiill, l.h.p. & ranga, n.t. (2003). ecologia da polinização de ipomoea asarifolia ders. roem. & schult. convolvulaceae na região semi-árida de pernambuco. acta botanica brasílica, 17: 355-362. doi: 10.1590/s0102-33062003000300003 kiill, l.h.p. & simão-bianchini, r. (2011). biologia reprodutiva e polinização de jacquemontia nodiflora desr. g. don convolvulaceae em caatinga na região de petrolina, pe, brasil. hoehnea, 38: 511-520. doi: 10.1590/s223689062011000400001. machado, c.g. (2009). os beija-flores aves: trochilidae e seus recursos florais em área de caatinga da chapada diamantina, bahia, brasil. revista brasileira de zoologia, 26: 255-265. doi: 10.1590/s1984-46702009000200008. machado, i.c.s. & lopes, a.v. (2004). floral traits and pollination systems in the caatinga, a brazilian tropical dry forest. annals of botany, 94: 365-376. doi: 10.1093/aob/ mch152. //floradobrasil.jbrj.gov.br/2010/fb007076 jrl paz, cm pigozzo, m gimenes – bees and hoverflies in the pollination of j. evolvuloides252 marinoni, l., morales, m.n. & spaler, i. (2007). chave de identificação ilustrada para os gêneros de syrphinae diptera, syrphidae de ocorrência no sul do brasil. biota neotropica, 7: 145-160. doi: 10.1590/s1676-06032007000100019. moreira, h.j.c. & bragança, h.b.n. (2011). manual de identificação de plantas infestantes – hotifruti. são paulo, fmc agricultural products, 1017 p. nicholls, c.i. & altieri, m.a. 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(2002). diversidade de abelhas hymenoptera: apoidea em inflorescências de piper hispidinervum c.dc. neotropical entomology, 31: 2734. doi: 10.1590/s1519-566x2002000100004. http://www.sei.ba.gov.br open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1262sociobiology 64(3): 359-362 (september, 2017) first record of the stingless bee lestrimelitta rufa (friese) (hymenoptera: apidae: meliponini) in ne brazil and its cleptobiotic behavior the geographic distribution of the genus lestrimelitta friese, 1903, includes the neotropical region, with 21 species and part of the neartic region, with two species (camargo & pedro, 2013). the species were reviewed by schwarz (1948) and marchi and melo (2006), but the latter studied only species from brazil. there are records of only three species in the state of ceará: lestrimelitta limao (smith, 1863), lestrimelitta rufipes (friese, 1903) and lestrimelitta tropica marchi and melo, 2006 (camargo & pedro, 2013). according to marchi and melo (2006), lestrimelitta rufa (friese, 1903) is a species with records of occurrence from the south and west of the amazon basin to ecuador (figure 1). there is no record of the species from the brazilian northeast region. bees of the genus lestrimelitta are characterized for not having a corbicule or penicillium (schwarz, 1948; moure, 1951; silveira et al., 2002). because it does not present these abstract the aim of this study was to expand occurrence records of lestrimelitta rufa (friese, 1903) to the brazilian northeast and to document the cleptobiotic behavior of this robber species in colonies of melipona quinquefasciata lepeletier, 1836. two attacks were carried out in a meliponary in the county of barbalha (ceará, brazil), where pot and larval food were sacked. even with direct confrontation between invading and inquiline bees, there was a total loss of one of the attacked nests because robber workers remained insistently in search of resources of these nests during the attacks. sociobiology an international journal on social insects vm mascena1, ds nogueira2, cm silva3, bm freitas1 article history edited by evandro n. silva, uefs, brazil received 01 december 2016 initial acceptance 27 february 2017 final acceptance 01 may 2017 publication date 17 october 2017 keywords robber bee, melipona, quinquefasciata, ethology, inquiline bee. corresponding author valdenio mendes mascena universidade federal do ceará av. da universidade, 2853 benfica cep 60020-181 fortaleza-ce, brasil. e-mail: vmmabelhas@gmail.com morphological characteristics, the bee cannot collect pollen from flowers, assuming obligatory cleptobiotic behavior and robbing other bees’ nests to keep its own (roubik, 1989; marchi & melo, 2006; michener, 2007). invasive bees can rob all resources of an inquiline colony, causing the complete death of the nest, or stealing part of the resources, but not exterminating the invaded colony (sakagami & laroca, 1963; santana et al., 2004; rech et al., 2013). however, some species of bees have developed important features to neutralize attacks and defend its own colonies, such as the ability to recognize citral, resistance to the effect of this substance and defense systems, for example, duckeola ghilianii (spinola, 1853) and melipona fulva lepeletier, 1836, developed a gallery of protection to part of the resources, besides direct confrontation with invasive lestrimelitta bees through the perception of the citric odor (van zweden et al., 2011; grüter et al., 2012; rech et al., 2013). 1 universidade federal do ceará, fortaleza-ce, brazil 2 instituto nacional de pesquisas da amazônia, manaus-am, brazil 3 instituto federal de educação, ciência e tecnologia do ceará, crato-ce, brazil short note vm mascena, ds nogueira, cm silva, bm freitas – new geographical record of robber bee and its behavior360 the aim of this study is to expand the record of occurrence of this species to the brazilian northeast and to document the cleptobiotic behavior of l. rufa in colonies of melipona quinquefasciata lepeletier, 1836. lestrimelitta rufa was observed looting a meliponary near the casa de guarda da santa rita of the instituto chico mendes de conservação da biodiversidade (icmbio), in the chapada do araripe, within the permanent conservation area of the araripeapodi national forest, county of barbalha, ceará, brazil (7º23’01.92”s, 39º21’15.97”w), with altitude of 929 meters, and annual rainfall of 1000 mm (cpmr, 2011). the locality has a sedimentary tabular relief from the cretaceous age and diverse vegetation formations with areas covered by caatinga from the sedimentary, cerrado, cerradão and rain forest also from the sedimentary (moro et al., 2015). we believe the occurrence of l. rufa on highland forests in the state of ceará is due to the ecological similarity, including animal and plant species, to the amazonian ecosystem, where the species originates. according to andrade-lima (1982), the vegetation of high elevation forests in ne brazil is associated to the amazonian vegetation because climate variations during the pleistocene allowed contact of these forests before a subsequent separation after interglacial periods. this is the first record of this species in ne brazil. the meliponary had five wooden and five pottery hives inhabited by colonies of m. quinquefasciata. two attacks were observed; in the first one, there was a total loss of the inquiline bee colony. in the second attack, we had to intervene to prevent a complete attack and another colony loss. in both attacks, inquiline and looter specimens were collected for further identification. the first attack occurred between may 12 and 15, 2014, and the first sign of invasion by l. rufa was noted by the modification of m. quinquefasciata’s characteristic nest entrance for a thin wax tube with widened exit edge, as reported by some authors (sakagami et al., 1993; bego et al., 1991), that in cases of long-lasting attacks, the entrance of the victim colony is modified or a new tube is constructed with cerumen and resin, collected from the same colony nest. besides the nest entrance, it was noticed the presence of about five guard bees (figure 2), as already reported to happen in other cases of attack (sakagami & laroca, 1963; sakagami et al., 1993; rech et al., 2013). in addition, it was noticeable a strong odor of volatile compounds coming from this beehive. this odor is typical of species of this genus and is used to signal the site of attack to workers of the same colony and disorganize the inquiline bees (blum et al., 1970; sakagami & laroca, 1993; pompeu & silveira, 2005; rech et al., 2013; van zweden et al., 2011). when we opened the hive, we noticed that all pollen from pots and larval food had been looted, and there were many individuals of m. quinquefasciata dead on the hive floor, including the queen. the inquiline bees that still resisted were constantly attacked by the looter bees, and forid larvae (diptera: phoridae) were observed among the rest of food, brood cells and wax. the flies apparently attacked the nest, because it was already unstructured by attack of looter bees. the second attack occurred on june 2, 2014, at around 9 a.m.. about ten workers of l. rufa surrounded a hive of m. quinquefasciata and around 3 p.m. the same day, about 50 workers invaded the inquiline nest and the citric odor was perceptible even up to five meters away from site of attack. fig 1. map of recorded occurrences for lestrimelitta rufa (friese, 1903). circles (○) are occurrences recorded by marchi and melo (2006); triangle (∆) is the new geographic record. sociobiology 64(3): 359-362 (september, 2017) 361 after the invasive workers entered the colony, a guard bee of m. quinquefasciata was seen ventilating in the entrance orifice. the attack continued inside the hive and individuals of both species were in direct confrontation. a single individual of m. quinquefasciata was attacked by three to four individuals of l. rufa. the inquiline workers were apparently disoriented, fig 2. small wax tube constructed by lestrimelitta rufa (friese, 1903) at the entrance orifice of a nest of melipona quinquefasciata lepeletier, 1836. fig 3. large number of specimens of melipona quinquefasciata lepelletier, 1836 killed on the ground of hive, after the first attack observed. in detail physogastric queen. probably due to effect of citric pheromone, as well as the stress of being attacked. many inquiline bees searched for shelter beneath the involucrum of the brood comb area, demonstrating no effective response to the attack. from this moment we had to intervene to prevent the total loss of that m. quinquefasciata nest. we removed the invasive bees and closed the hive until the following day. during a twelve-month period, these two attacks were the only ones observed. because l. rufa is an obligatory cleptobiotic species, these bees probably found other nests to rob in the surroundings to remain in same area or moved somewhere else. however, the latter situation is less likely to occur because stingless bee queens develop large gasters after fecundation which unable them to fly and colonies do not migrate without their queens (michener, 1974; roubik, 1989). the attacks of l. rufa showed to be potentially fatal to colonies of m. quinquefasciata living in man-made hives. considering that m. quinquefasciata bees do not react to the attack of l. rufa, it is possible that its defense relies in the discrete nesting behavior of this bee species. in meliponaries, the number of hives and much greater bee movement makes their nests easier to be found by l. rufa. acknowledgments we thank coordenação de aperfeiçoamento de pessoal de nível superior (capes) for funding the scholarships, conselho nacional de desenvolvimento científico e tecnológico (cnpq) for a productivity in research sponsorship (#305126/2013-0) to bm freitas and instituto nacional de pesquisas da amazônia (inpa) for the structure necessary to identify the bees. vm mascena, ds nogueira, cm silva, bm freitas – new geographical record of robber bee and its behavior362 references andrade-lima, d. 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(2011). hovering guards of the stingless bee tetragonisca angustula increase colony defensive perimeter as shown by intraand inter-specific comparisons. behavioral ecology and sociobiology, 65: 1277-1282. http://link.springer.com/journal/265 http://link.springer.com/journal/265 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1228sociobiology 64(4): 373-380 (december, 2017) defining habitat use by the parabiotic ants camponotus femoratus (fabricius, 1804) and crematogaster levior longino, 2003 introduction ants are involved in many biological interactions with plants, microorganisms and other invertebrates (izzo & vasconcelos, 2009; dáttilo et al., 2012; vicente et al., 2012; sanders et al., 2014; koch et al., 2015; puker et al., 2015). among these interactions, ants can interact with other species of ants throughout their life history from mutualistic to parasitic relationship (sanhudo et al., 2008; gallego-ropero & feitosa, 2014; powell et al., 2014). an example of mutualist interaction maintained by neotropical ants camponotus femoratus (fabricius, 1804) (formicinae) and crematogaster abstract ant-garden ants have a strong relationship with epiphytes that need light to grow, for these reason, it has been previously documented in forest gaps. moreover, larger gaps have more available area for nesting and habitats for use as forage. thus we hypothesize that 1) canopy openness influence the presence of ant´s gardens in gaps, and 2) greater gaps will have more nests, and 3) both openness canopy and area determine the colony size in forest gaps. furthermore, it is known that parabiotic ants foraging on the ground and in vegetation, the nests are arboreal. so, we also hypothesize that 4) parabiotic ants are more often sampled in arboreal strata and 5) increasing vegetation connectivity and the volume of accumulated litter in the soil increase the foraging of the ants in vegetation and ground, respectively, with the increase in canopy openness increasing the activity of the two species in both strata. presence, number of ant-gardens, as colony size, was affected by area and locality, but not by canopy openness. nevertheless, there was not overall difference in the use of strata by camponotus femoratus, neither by crematogaster levior. on the other hand, frequency of c. femoratus on the ground decreases with canopy openness but is not affected by the vegetation connectivity. also, c. levior frequency on the ground also decreases with the increase of complexity of vegetation and canopy openness. in addition, neither vegetation connectivity, or canopy openness influence the frequency of foraging of these ants in understory. sociobiology an international journal on social insects re vicente1,2,3, tj izzo2,3 article history edited by kleber del-klaro, ufu, brazil received 04 november 2016 initial acceptance 12 january 2017 final acceptance 09 october 2017 publication date 27 december 2017 keywords canopy openness, gap size, habitat use, niche partitioning, vegetation connectivity, vertical habitat. corresponding author ricardo eduardo vicente cetam unemat campus ii av. perimetral rogério silva, s/nº jardim flamboyant cep 78580-000 alta floresta, mato grosso, brasil. e-mail: ricardomyrmex@gmail.com levior longino, 2003 (myrmicinae) are known as parabiosis (orivel & leroy, 2011). in this interaction, ants share the same foraging trails and their arboreal nests (swain, 1980). in the nests, they actively plant one or several epiphyte species, forming a complex nest, known as ant-gardens (hereafter ags) (davidson, 1988; longino, 2003; youngsteadt et al., 2009). this interaction includes nine species of ants (obligate ags inhabitant) however, ags with parabiotic association between c. femoratus and c. levior are among the most commonly found in the amazon (davidson, 1988; orivel & leroy, 2011). in exchange for support and humidity control for the nest and, in some cases, extrafloral nectar 1 laboratório de biologia vegetal (cetam), departamento de ciências biológicas, universidade do estado de mato grosso (unemat), campus ii, alta floresta-mt, brazil 2 laboratório de ecologia de comunidades do departamento de ecologia e botânica, instituto de biologia da universidade federal do mato grosso, cuiabá-mt, brazil 3 núcleo de estudos da biodiversidade da amazônia mato-grossense, universidade federal de mato grosso, sinop-mt, brazil research article ants re vicente, tj izzo – niche of parabiotic neotropical ants of ant-gardens374 (davidson, 1988; schmit-neuerburg & blüthgen, 2007), the ants offer dispersion and protection to their mutualistic epiphyte (vantaux et al., 2007; youngsteadt et al., 2008, 2009; vicente et al., 2014). although the complexity of this interaction and these ants occurring throughout the neotropical region (souza et al., 2007; ryder-wilkie et al., 2010; emery & tsutsui, 2013; vicente et al., 2014, 2016) the natural history of these species is virtually unknown. ant-garden ants have a strong relationship with epiphytes that need light to grow (leroy et al., 2016; orivel & leroy 2011), for this reason, they have been previously documented in tree-fall gaps, river banks and abandoned roads (dáttilo & izzo, 2012; vicente et al., 2014). moreover, larger gaps have more available area for the building of nests and habitats for use as forage. thus we hypothesize that 1) the openness canopy influences the presence of ant´s gardens in gaps, and 2) greater gaps will have more nests, and 3) both openness canopy and area determine the colony size in forest gaps. furthermore, even though the nests are essentially arboreal, it is known that parabiotic ants forage on the ground and in vegetation. so, we also hypothesize that 4) parabiotic ants are more often sampled in arboreal strata and 5) increasing connectivity of vegetation and the litter high on the soil increase the foraging of the ants in vegetation and ground, respectively, with the increase in canopy openness increasing the activity of the two species in both strata. material and methods study area the study was carried out on two municipalities of the southern amazonia as part of the research program in biodiversity (ppbio). the standard sampling protocol used in ppbio is the rapeld methodology (see costa & magnusson, 2010). in each of the two municipality a set of trails of 250m in length are installed at a minimum distance of 1 km each other, linked by access trails (henceforward module). in claudia municipality, three modules are installed close to each other (less than 20km), in a large fragment surrounded by plantations. the claudia modules are module i (11°34’s, 55°17’w) and module ii (11°35’ s, 55°17’w) that are broadly connected, and module iii (11°39’s, 55°04’w) that is an area with about 900ha almost completely surrounded by plantations and narrowly connected to the rest of the fragment. the area of these three modules had selective logging more than eight years ago. because of this, they have older gaps caused by the management of selective logging as wood storage yards and trails used for the dragging of cut trees (abandoned roads). one other area is the parque estadual do cristalino reserve, a continuous pristine forest in the municipality of novo mundo municipality, mato grosso states (9°28’s, 55°50’w). according to the köppen classification, the climate is tropical humid (am) with annual average of 25°c. precipitation is about 2.500 to 2.800 mm year in forest remnants of the municipality of claudia and 2.800 to 3.100 mm year in parque estadual do cristalino (alvares et al., 2013). in both, there are two well-defined seasons, a rainy season between november and april and a dry season between may and october (vicente et al., 2014). the local vegetation in claudia is classified as area of ecological tension between open ombrophylous forest and savanna. for the parque estadual do cristalino local vegetation is classified as open ombrophylous forest and ecological tension areas between ombrophylous forest and savanna, savanna and seasonal forest, seasonal forest and ombrophylous forest (instituto brasileiro de geografia e estatística, 2004). data collection gap-occupation we carried out the experiment about occupation of forest-gaps only in forest remnants in the three modules (i, ii, iii) of claudia municipality in april 2014. in these areas, we surveyed all access trails that link plots, totaling approximately 32km. in these trails, all forest-gaps, including the gaps caused by selective logging, within the range of 10 meters of each side of the trails (20 m wide) were selected and we recorded the presence or absence of ags, number of nests and colony size. furthermore, we measured the canopy openness and the length of the two central perpendicular axes of the forest gap. canopy openness was measured with concave spherical densiometer. it is considered a practical and cheap device that provides a reliable proxy as to the relative availability of light (baudry et al., 2014). in the center of each forest-gap, we recorded four measurements in the cardinal directions to calculate an average availability of light (dáttilo et al., 2013, 2014; baudry et al., 2014). the first two major perpendicular measures of gaps were used as length and width to calculate the gap area, using the formula of the ellipse area (runkle, 1981; arihafa & mack, 2013). in forest-gaps formed by abandoned roads, because of the difficulty in measuring their main axes, we considered a continuous habitat and we assigned an arbitrary value area, the double of our largest forest gap. vertical habitat use to access the information about the use of the vertical habitat by parabiotic ants thirty four (34) plots of 250m, allocate in the four areas previously mentioned were investigated. collections were made between november 2009 and february 2010 in claudia modules and in november 2012 and may 2013 at parque estadual do cristalino. in each plot, every 25 meters a collection of c. femoratus and c. levior ants on the ground and in vegetation was performed, totaling 680 samples (340 on ground and 340 in vegetation). for the collection of grounddwelling ants, we installed pitfall-traps buried at ground level containing water and detergent. pitfalls were on field for 48 hours. for sampling arboreal ants was used beating-tray. at sociobiology 64(4): 373-380 (december, 2017) 375 four points on the north, south, east and west, 2 meters away from each pitfall all vegetation within 1m² between 1 to 3 meters tall was sampled (more details: vicente et al., 2016). posteriorly we made comparisons with specimens deposited at the laboratório de ecologia de comunidades from the centro de biodiversidade da universidade federal de mato grosso (ufmt) and the ant collection from the laboratório de sistemática, evolução e biologia de hymenoptera from the museu de zoologia da universidade de são paulo (mzsp). vouchers were deposited in the collections mentioned above and in setor de entomologia da coleção zoológica da universidade federal de mato grosso, departamento de biologia e zoologia, cuiabá, mato grosso, brazil (cemt). in these points, measurements of vegetation connectivity and litter height were taken, summed up and made the averages. connectivity of the vegetation was accessed with a number of touches in the understory of a stem of 2 meters positioned one meter high, perpendicular to the ground. litter height was measured with a rule and canopy openness with concave spherical densiometer as explained earlier in the section gap-occupation. data analysis gap-occupation to test which characteristics determine the presence and abundance of ant-gardens in gaps, presence of ag and number of nests were the dependent variables and forestgap area and canopy openness average were the independent variables. we tested the correlation between independent variables with pearson correlation (pearson: 0.026). among the three sampled areas, module iii is almost totally isolated area and which has the least amount of gaps colonized by parabiotic ants (only 10.3%) while module i and module ii showed more than twice the number of colonized gaps (24.2% and 25.5% respectively). this is an indication of the intrinsic characteristics of this locality, as area and edge effects, affecting the population dynamics of this ant species. consequentely, the location was inserted into the glm analyses using orthogonal contrast established a priori, considering the principles described by gotelli & ellison (2011), being set up as contrast: module i (+1), module ii (+1) and module iii (-2). we used the r software to perform all analyzes (r core team, 2014). to test whether the colony size, which indirectly is represented by the volume of the nests, was related to independent variables a glm was performed. since ags are spherical, the variable colony size was calculated using the formula of the volume of spheres (v) (khattar, 1968), where r is the radius from the ant-garden nest and π is the constant pi: v = 4 * π * r3 3 the volume of each nest of each evaluated forest-gap was added, representing the size of the colony by forest-gap. finally, to test whether the colony size was related to the amount of nests one pearson correlation was made. vertical habitat use to confirm the hypothesis that c. femoratus and c. levior have different preferences about the use of ground or vegetation a test-t was performed for each species. in this analysis the frequency of each species per plot was the dependent variable and stratum was the independent factor. to access the information about what structural environmental variable affects the strata use of c. femoratus and c. levior a glm with negative binomial distribution was used. this error distribution is indicated when poisson error distribution shows overdispersion (hilbe, 2007; lindén & mäntyniemi, 2011). because a correlation between independent variables was detected with pearson correlation with bonferroni correction, we excluded litter height variable of analyses. results gap-occupation we recorded 27 colonies of ant-garden ants in all 142 forest-gaps (125 in tree-fall gaps and 17 in abandoned roads). regarding environmental variables of forest gaps, they had an average size of 215.125 m2 (sd: 130.947 values excluding the 880.44m2 value established for abandoned roads). the canopy openness had an average of 32.94% (sd: 14.61). presence of ant-gardens colony was affected by area (z1,138: 2.470, p: < 0.05 – table 1) and locality (z1,138: 2.269, p: < 0.05), but not by canopy openness (z1,138: -1.849, p: < 0.05). abundance of ant-gardens nests was also affected by area (z1,138: 2.090, p: < 0.05) and locality (z1,138: 2.570, p: 0. 010) and also not by canopy openness (z1,138: -1.583, p: 0.113 – table 1). ants colony size was also influenced by the gap area (z1,138: 3.494, p: < 0.05), the locality (z1,138: 2.422, p: < 0.05) z p-value presence of ags area 2.470 < 0.05* locality 2.269 < 0.05* canopy openness -1.849 0.064 abundance of ags area 2.090 < 0.05* locality 2.570 < 0. 05* canopy openness -1.583 0.113 colony size of ags ants area 3.494 < 0.05* locality 2.422 < 0.05 * canopy openness -1.230 0.221 * significative table 1. influence of factors in gap occupation by ant-gardens nests of parabiotic ants camponotus femoratus and crematogaster levior. re vicente, tj izzo – niche of parabiotic neotropical ants of ant-gardens376 and not the canopy opening (z1,138: -1.230, p: 0.221 – table 1). therefore, the abundance of ant-gardens and the colony size present a strong correlation (pearson: 0.883). vertical habitat use camponotus femoratus and crematogaster levior were sampled in 21 of the 34 plots studied. at least one of the two species was collected in 69 samples (30 on the ground and 39 on vegetation) of the 420 samples (210 on the ground and 210 on vegetation) corresponding to 21 plots where these ants were found. c. femoratus was collected in 49 samples of 18 plots (30 samples in soil and 19 on vegetation) and c. levior on 30 samples (15 times in each stratum). we assumed that in all cases both species are co-occurring in the same area, but with differences in abundance and, thus, in the probably of being collected. therefore, these species were collected in the same sample in 13 cases (8 on the ground and 5 on vegetation). regarding the environmental variables evaluated, they had an average of vegetation connectivity of 1.417 (sd: 0.645). the canopy openness had an average of 32.94% (sd: 14.61). nevertheless, there was no an overall difference in the use of vegetation and ground strata by camponotus femoratus (z1,66: -1.519, p: 0.129 – figure 1), nor by crematogaster levior (z1,66: 0.908, p: 0.364 – figure 1). c. femoratus occurs in average 1.67 (sd: 1.63) samples per plot in ground and 1.06 (sd: 1.47) on vegetation. c. levior occurs in average 1.25 (sd: 1.16) samples per plot in ground and 1.25 (sd: 1.23) on vegetation. on the other hand, the frequency of c. femoratus on the ground decreases with canopy openness (z1,31: -2.435, p: < 0.05 – table 2) but is not affected neither by the vegetation connectivity (z1,31: -1.407, p: 0.159) nor for interaction of two factor (z1,31: 1.771, p: < 0.05). also, c. levior frequency on the ground also decreases with vegetation connectivity (z1,31: -2.243, p: < 0.05 – table 2), but, in this case, a decrease was also noted in the frequency associated with canopy openness (z1,31: -1.977, p: < 0.05) and an interaction between these factors (z1,31: 2.265, p: < 0.05). in addition, neither vegetation connectivity (c. femoratus: z1,32: 0.200, p: 0.841; c. levior z1,31: -1.225, p: 0.221 – table 2), nor canopy openness (c. femoratus: z1,32: 0.580, p: 0.562; c. levior z1,32: 0.804, p: 0.421) influence the frequency of foraging of these ants in understory. discussion our results demonstrate that the size and local intrinsic characteristics of the forest gaps are determinant to the occupation by the parabiotic ants camponotus femoratus and crematogaster levior. these factors influence the presence as well as the quantity of ant-gardens and the ants colony size. also, larger gaps host more and larger ags than smaller forest gaps. this can be explained because large forest gaps remains available for longer once the time of full restoration of a large forest gap, from its formation till the canopy closure is probably much longer than in small gaps. in fact, fig 1. works frequency by trail in vertical stratum (ground and vegetation). a) camponotus femoratus (b crematogaster levior. camponotus femoratus crematogaster levior z p-value z p-value ground canopy openness -2.572 < 0.05* -1.977 < 0.05* vegetation connectivity -1.407 0.159 -2.243 < 0.05* interaction 1.771 0.077 2,265 < 0.05* vegetation canopy openness -0.700 0.484 -1.034 0.301 vegetation connectivity -1.006 0.315 -1.474 0.141 interaction 1.022 0.307 1.396 0.163 * significative table 2. factors that influence the frequency of use of each stratum by camponotus femoratus and crematogaster levior. sociobiology 64(4): 373-380 (december, 2017) 377 larger patchs implies in a higher probability of colonization and a lower risk of extinction (macarthur & wilson, 1967; donner et al., 2010; mccarthy & lindenmayer, 1999). thus, a colony of parabiotic ants has more time to colonize, grow and cultivate its mutualistic epiphytes in large gaps. consequently, larger gaps are probably a much more profitable resource to maintain c. femoratus and c. levior populations. as in gaps the incident light in the lower vegetation is higher, there is an associated increase in the total primary productivity, including a major production by extrafloral nectar (radhika et al., 2010; bixenmann et al., 2011; brenesarguedas et al., 2011). several studies also demonstrate that this increase in primary production attracts more herbivores (harrison, 1987; coley & barony, 1996; louda & rodman 1996; sipura & tahvanainen, 2000). the increase in both extrafloral nectar and herbivores means an increase in food resources for ants (swain, 1980; davidson, 1988; vantaux et al., 2007). however, canopy openness of forest gaps has not influenced on ags presence, neither quantity of ags or colony size. probably the lack of a relationship in the occupation of forest gaps and canopy openness can be explained by the low variation among the sampled gaps. furthermore, the size of the forest gaps induces changes that go beyond the light intensity, changing moisture and biological properties of soil are characteristics responsible for the forest recovery (muscolo et al., 2014). parabiotic ants do not show any preference for foraging in the understory plants or on the ground. in fact, these ants are widespread and found in the soil and in vegetation in the amazon (davidson, 1988; dejean et al., 2000; ryder wilkie et al., 2010; vicente et al., 2014, 2016). nevertheless, canopy openness was a determining factor on the habitat use by c. femoratus and c. levior. with the increase of canopy openness that results in an increase in primary productivity and consequently of herbivores (brenes-arguedas et al., 2011; coley & barony, 1996), that increase food resources in vegetation (as discussed above) could induce a preferential foraging by ants, there was a reduction in the parabiotic ants frequency foraging on the ground. however, the decrease of ants on soil is not associated to an increase in the frequency of this ant species on vegetation. although the increased vegetation connectivity does not influence the use of the arboreal strata by c. levior, there was a decrease in soil use. the increase of the vegetation connectivity means a denser understory, forming bridges among plants. thus, valuable resources for c. levior as extrafloral nectary (davidson, 1988) are easier to access with a vegetation connectivity increase. the increase in the vegetation connectivity can provide a greater diversity of substrates, which may facilitate access to the resource or run away from ant predators and competitors (clay et al., 2010). this reduction in the need of the ground use minimizes possible confrontations with other ant species, once the ground strata is naturally richer in ant species (vasconcelos & vilhena, 2006; neves et al., 2013; vicente et al., 2016). competition between species for c. levior is damaging since it lost its chemical defense, depending on the defensive ability of c. femoratus (longino, 2003) that show aggressive behavior (vicente et al., 2014) as well as other species of neotropical species of camponotus (yamamoto & del-claro, 2008; santos & del-claro, 2009; alves-silva & del-claro, 2014; anjos et al., 2016). thus, c. femoratus did not show influence of vegetation connectivity in use of strata, showing a partition in the niche between the two ant species. this niche partitioning should go beyond the influence of plant connectivity in resource exploration demonstrated in this work. davidson (1988) showed that c. levior accumulates more workers in sugary baits than c. femoratus. also, while workers of c. levior did not show any preference among plants with extrafloral nectaries, c. femoratus actively choose plant species with larger nectaries and with greater concentration of nectar (davidson, 1988). therefore, plant traits are probably not associated to the complexity of the habitat. our results demonstrate that although these parabiotic ants occupy the same sites sharing their nests and maintenance activities of the colonies, they have divergences in their niche with regards to the exploitation of habitat. the use of habitat by c. femoratus and c. levior differ among each other depending on the stratum and environmental characteristics. it also shows that these dominant ants, with different biological characteristics, can influence both soil and vegetation communities differently. therefore, the influence of both c. femoratus and c. levior on soil and vegetation ant communities should be investigated in order to understand whether these parabiotic ants alter the ant communities, where they occur and what the mechanisms involved in this alteration are. acknowledgements authors also thank wesley dáttilo, rodrigo s. m. feitosa, danielle stork-tonon, lúcia a. f. mateus, andré pansonato and wesley o. souza for reviewing and useful contribution over previous design of manuscript. we also would like to thank the national council for scientific and technological development, brazil (cnpq n° 479243/20123) and nebam for logistical and financial support. rev thanks enhancement coordination of personnel of superior level, brazil (capes) for doctoral fellowship and the programa de capacitação institucional (pci – mpeg/mctic nº 301081/2017-4) and desenvolvimento científico regional program (dcr – fapemat/cnpq n° 003/2016) for research fellowships. this work is part of the doctoral thesis of rev in programa de pós-graduação em ecologia e conservação da biodiversidade – ufmt and this is publication 60 in the nebam technical series. re vicente, tj izzo – niche of parabiotic neotropical ants of ant-gardens378 references alvares, c.a., stape, j.l., sentelhas, p.c., de-moraes-gonçalves, j.l. & sparovek, g. 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(drumond et al., 2002; leal et al., 2005). this climate may be reflected in adaptive changes in the regional biota, which most probably influences the population dynamics of insects, in particular social wasps (melo et al., 2015). social wasps (vespidae: polistinae) play a decisive role in the trophic balance of ecosystems due to their food duplicity, since they act as well as predators and as scavengers of insect larvae and smaller insects (carpenter & marques, 2001) and as collectors of nectar and pollen (sühs et al., 2009). in addition, it is a group with high species richness and populations and very common in several places of the neotropical region (carpenter & marques, 2001). however, information about vespidae in the brazilian northeastern region is precarious, with few studies and few described abstract this work recorded 593 specimens allocated in 11 genera and 23 species of social wasps were collected in ubajara national park and sete cidades national park, caatinga biome, northeast of brazil. chartergellus communis richards, 1978, chartergus globiventris de saussure, 1854, metapolybia docilis richards, 1975, polybia paulista (von ihering, 1896), p. rejecta (fabricius, 1798), protonectarina sylveirae (de saussure, 1854) and protopolybia exigua (de saussure, 1854) are new occurrence records for ceará state. apoica flavissima van der vecht, 1898, brachygastra augusti (de saussure, 1854), c. globiventris, metapolybia cingulata (fabricius, 1804), polybia chrysothorax (lichtenstein, 1796), p. paulista, p. scutellaris (white, 1841) and protopolybia chartergoides gribodo, 1891 are new occurrence records for piauí state. eighteen species were collected in the ubajara national park, being eight exclusive and fifthteen in the sete cidades national park, being five exclusive; ten species were collected in both parks. brachygastra augusti and m.docilis are new records for caatinga biome. sociobiology an international journal on social insects a somavilla1, ml oliveira2, ja rafael2 article history edited by gilberto m. m. santos, uefs, brazil received 09 march 2017 initial acceptance 29 march 2017 final acceptance 25 april 2017 publication date 17 october 2017 keywords distribution; diversity; inventory; northeast region; polistinae. corresponding author alexandre somavilla instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo nº 2936 cep 69067-375 manaus-am, brasil. e-mail: alexandresomavilla@gmail.com species, due to the conception that the diversity is low in the local ecosystems (andena & carpenter, 2014). as mentioned earlier, there is an erroneous view that arid ecosystems are poor in diversity. this view was perpetrated by ducke (1907) in his article on brazilian social wasps in which he quotes: “in the ceará state, center of the dry region northeast of brazil, is very poor hymenoptera diversity, characterized to the absence of many frequent species throughout the rest of the country”. the same autor, "still did not know about the fauna of the rio grande do norte, paraíba, pernambuco, alagoas and sergipe states, and only supposed it to be a continuation of the fauna of bahia, probably still impoverished by the absence of species linked to humid climate.” among the most comprehensive study reporting the social wasps’s diversity in northeastern brazil, five were developed in bahia state: one in three ecosystems (floresta tropical atlântica, restinga and manguezal) with 21 species (santos et al., 2007a); two in caatinga area, with 13 visiting flowers (aguiar & santos, 2007) and nine using cactus fruit for food resources (santos et al., 2007b); one in cerrado area, 19 1 instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, bolsista pdj, manaus-am, brazil 2 instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus-am, brazil research article wasps sociobiology 64(3): 334-338 (september, 2017) 335 wasp species (santos et al., 2009), and the last one in an area of campos rupestres, collecting 11 species visiting flowers (silva-pereira & santos, 2006). additionally, one study was made in maranhão state, referring to a cerrados area, with 31 species, the largest number of species so far recorded from an area in the northeastern region (souza et al., 2011), another study was performed in the state of rio grande do norte, with 20 species (virgínio et al., 2014) and the last in piauí state with 12 species (rocha & silveira, 2014). andena and carpenter (2014) compiled information from collections and bibliographies and reported the occurrence of 76 social wasp species from the brazilian semi-arid region, mainly for bahia. despite these efforts, most of the natural areas belonging to the caatinga biome have not yet been inventoried. consequently, here we present information about the social wasp species in two brazilian national parks from the caatinga biome. material and methods the wasps were obtained during two collecting expeditions to the study areas, from april 18th to 21st of 2012 and february 7th to 13th of 2013 at the sete cidades national park (np sete cidades, piripiri municipality, piauí state 4°5’57”s, 41°42’34”w) and from abril 22st to 25th of 2012 and february 14th to 20th of 2013 at the ubajara national park (np ubajara, ubajara municipality, ceará state 3°50’18”s, 40°53’54”w) (fig 01). although both parks are included in the caatinga biome, the areas sampled within these parks seem to represent islands dominated by cerrado in sete cidades and montane humid forest in pn ubajara. the two parks are distant by approximately 110 km. the following wasps traps were used: 6-meter intercept malaise traps (gressitt & gressitt, 1962), suspended intercept traps (rafael & gorayeb, 1982), light traps using a white sheet. guided manual collections were also performed throughout the excursion with entomological nets and active search for wasps’s colonies, in addition to the use of the spraying method spraying method with a solution of water, salt and sugar, in a transect of 1000 m (gomes & noll, 2010). the collection effort was standardized between the two national parks, with the same number and models of traps, with 11 consecutive days in each park in the two sampling periods. a better detail of the study area and the methods used, can be found in the study of takiya et al. (2016). wasps were pinned and some were preserved in ethanol, with their colonies, when they were collected. material collected was divided and deposited in two institutions: coleção zoológica do maranhão, universidade estadual do maranhão, caxias (czma) and coleção de invertebrados, instituto nacional de pesquisas da amazônia, manaus (inpa). fig 1. map of brazilian states colored by four major phytogeographical domains: amazon forest, cerrado, atlantic forest, and caatinga; the last one including sete cidades (green star) and ubajara (blue star) national parks (figure from takiya et al., 2016). a somavilla, ml oliveira, ja rafael – social wasps from caatinga biome, brazil336 results and discussion it was collected 593 specimens allocated in 11 genera and 23 species of polistinae social wasps. all species are listed in the table 01, according to the two study areas. 18 species were collected in the ubajara national park eight exclusive and 15 in the sete cidades national parkfive exclusive; ten species were collected in both national parks. polybia occidentalis (olivier, 1792) and agelaia pallipes (olivier, 1792) are the most abundant species and only p. occidentalis was collected with all the methods: malaise, suspended, light trap, spraying trap and nest. chartergellus communis richards, 1978, chartergus globiventris de saussure, 1854, metapolybia docilis richards, 1975, polybia paulista (von ihering, 1896), p. rejecta (fabricius, 1798), protonectarina sylveirae (de saussure, 1854) and protopolybia exigua (de saussure, 1854) are new occurrence records for ceará state. apoica flavissima van der vecht, 1898, brachygastra augusti (de saussure, 1854), c. globiventris, metapolybia cingulata (fabricius, 1804), polybia chrysothorax (lichtenstein, 1796), p. paulista, p. scutellaris (white, 1841) and protopolybia chartergoides gribodo, 1891 are new records for piauí state. in addition, brachygastra augusti and metapolybia docilis are new records for caatinga biome. the use of different methods and traps is an important way to sample wasps richness of an area, since different species generally have varied foraging behaviors. active search with entomological net using the spraying method was the best way to collect social wasps species in both areas. however the use of indirect methods, like interception and light traps, were important for collecting certain wasps groups; for example, the light trap used for nocturnal foraging wasps (apoica) and some species of polybia and small vespids (metapolybia and protopolybia smaller than 10 mm) collected with malaise and suspended traps. taxa method used for collect np ubajara ce np sete cidades pi epiponini agelaia pallipes (olivier, 1792) malaise, spraying, suspended 51 33 apoica pallens (fabricius, 1804) light trap 05 apoica flavissima van der vecht, 1898 light trap, malaise 01 55 brachygastra augusti (de saussure, 1854)* malaise, nest, spraying 12 brachygastra lecheguana (latreille, 1804) malaise, nest, spraying 01 12 chartergellus communis richards, 1978 nest, spraying 12 chartergus globiventris de saussure, 1854 light trap, nest, spraying 01 12 metapolybia cingulata (fabricius, 1804) malaise, spraying, suspended, nest 49 metapolybia docilis richards, 1975* nest, spraying 62 polybia chrysothorax (lichtenstein, 1796) light trap, malaise, nest, spraying 10 23 polybia occidentalis (olivier, 1792) light trap, malaise, spraying, nest, suspended 30 63 polybia ignobilis (haliday, 1836) malaise, spraying 06 05 polybia paulista (von ihering, 1896) spraying 01 02 polybia rejecta (fabricius, 1798) malaise, nest, spraying, suspended 06 19 polybia scuttelaris (white, 1841) malaise 01 polybia sericea (olivier, 1791) light trap, spraying 10 polybia sp.1 spraying 01 protonectarina sylveirae (de saussure, 1854) malaise 02 protopolybia chartergoides gribodo, 1891 malaise, spraying 02 protopolybia exigua (de saussure, 1854) nest, spraying 13 55 mischocyttarini mischocyttarus cerberus ducke, 1898 nest 26 mischocyttarus cearensis zikán, 1945 nest 09 polistini polistes canadensis (linnaeus, 1758) nest 03 total 241 353 table 1. social wasps collected in sete cidades national park (piauí state) and ubajara national park (ceará state) and methods used for collect each species: (* new record for caatinga biome). sociobiology 64(3): 334-338 (september, 2017) 337 one colony of the wasp p. rejecta was found associated with the nest of the ant azteca sp. in the sete cidades national park, this association was only registered for the brazilian amazon (somavilla et al., 2013) and in the transition area of the atlantic forest and caatinga in rio grande do norte (virgínio et al., 2015), probably this association is more common. a curious fact was the collection of just two mischocyttarus species and one polistes species only in np ubajara, close to human constructions. these genera were not collected in pn sete cidades, where only epiponini species were represented. polybia, due to its great species diversity and swarming nests foundation, was the genus with the greatest amount of species in both parks, with eight species reported. due to the deficiency of local vespidae studies, there is insufficient information to attribute “endemic” or “overhanging” conditions to any species collected at this site. therefore, these species should be considered as insufficiently known or rare in terms of geographic distribution. in fact most of the species recorded are widespread species, but this is common in social wasps, as many species have very widespread distributions, especialy epiponinae. on the other hand, polistes and mischocyttarus could show to have more area-restricted species. this results highlights the importance of preserving the caatinga sensu lato environment as a way to maintain the diversity of social wasps in this region. they also reinforce the need of more taxonomy-related research with this group of insects, broadening the geographic coverage of samples in order to increase the knowledge on richness and biodiversity of northeastern brazil, as well as in poorly known biomes, such as caatinga and its transition areas with other biomes. acknowledgements specimens were collected as part of a project coordinated by jar and financed by conselho nacional de desenvolvimento científico e tecnológico (cnpq, proc. 551. 991/2011-9) and instituto chico mendes de conservação da biodiversidade (icmbio) through the program “pesquisa em unidades de conservação do bioma caatinga”. local logistic support for collecting trips was given by franscisco limeirade-oliveira (universidade estadual do maranhão) and his students. collecting permits in both national parks were granted by icmbio (sisbio #32560). mlo for scholarship of cnpq, process 306100/2016-9. as for scholarship of cnpq, pdj process 50029/2017-9. references andena, s.r. & carpenter, j.m. 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(2014). novas contribuições para o conhecimento de vespas sociais (hymenoptera: vespidae) para o estado do rio grande do norte, brasil. entomotropica, 31: 221-226. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1181sociobiology 63(4): 1031-1037 (december, 2016) nesting biology and seasonality of long-horned bee eucera nigrilabris lepeletier (hymenoptera: apidae) introduction the main objective of the current contribution is documenting some biological and ecological aspects of the nesting of the genus eucera scopoli in the mediterranean region. the bees of the widespread tribe eucerini are notorious for their large numbers of similar species, with the distinctions even between genera being subtle and challenging for pollination biologist (alqarni et al., 2012). eucera is a large genus that is widespread in eurasia and the new world and is often abundant in its habitat (o’toole & raw 1999). eucera bee species are common and important pollinator in the mediterranean region (nachtigall, 1994; sapir et al., 2005). there are approximately 300 total species, long tongued, ground nesting solitary bees and having one generation per year (amiet et al., 2007). nests and larvae of eucera are unknown, burrows in the ground, each cell is at the end of a rather long lateral burrow, and the cells are vertical and elongate. they line abstract we provide information on the nesting behavior, seasonality and nest soli type characteristics of eucera nigrilabris lepeletier, 1841 in egypt. a nest was discovered in a canal bank in abbis village, alexandria, western egypt. the species is protandrous, univoltine, ground nesting species. the bees built deep nests about 85cm under the ground and consisted of lined, branched tunnel with many cells. the bees start fly by end of january until end of march and active in winter seasons. the soil of the nest has yellow color, sandy loam texture, low salinty and sodicity, and low calcium carbonate content. the bee distrbiution was influnced by the soil with high content of sodium carbonate. the bees forage on the wild flora of the family asteraceae carriyng a yellow pollen load. there is no any record of a cleptoparasitism around the nesting area. sociobiology an international journal on social insects ma shebl1, rm al aser2, a ibrahim1 article history edited by evandro nascimento silva, uefs, brazil received 09 august 2016 initial acceptance 07 december 2016 final acceptance 08 december 2016 publication date 13 january 2017 keywords eucerin bees, ground nesting, univoltine, cleptoparasites, protandrous. corresponding author mohamed shebl abd elfattah dept. of plant protection faculty of agriculture suez canal university ismailia 41522, egypt e-mail: mohamedshebl2002@hotmail.com their brood cells with a waxlike material that they secrete (michener, 2007). there are some few publications of eucera and tetralonia nesting biology in the world (malyshev, 1924; 1929; linsley et al., 1952; michener & lange 1958; rozen, 1969; 1974), (wafa & mohamed 1970; miliczky, 1985; popova, 1990). the bee distribution is influenced by the plant community, plant diversity, canopy cover, land use and nesting suitability. in particular soil properties can play an important role in the distribution and diversity of ground nesting bees (grundel et al., 2010). eucera nigrilabris lep. (eucerindae) is a common species in the mediterranean region (ne’eman et al., 2007). this species is important for pollination of some wild plants like ophrys tenthredinifera (kullenberg et al., 1984; glaubrecht, 2010) and alkanna strigosa (ne’eman et al., 2007). the species is well abundant in egypt distributed in fayiuom, cairo and north coast, the flight activity started from january to march and the nesting biology and behavior is unknown. 1 dept. of soil and irrigation sciences, faculty of agriculture, suez canal university, ismailia, egypt 2 apiculture research dept., sabahia research station, agriculture research center, alexandria, egypt research article bees ma shebl, rm al – aser nesting and seasonality of long-horned bee1032 we hypothesized that the bee diversity influences by not only climate and vegetation, but also soil type and characteristics. here we try to examine the nesting biology of eucera nigrilabris and their soil preference for building their nest. material and methods specimens collection and identification several specimens of e. nigrilabris were collected by sweep net from natural nests and wild flowers from abbis village, alexandria, nw egypt. bees were killed in normal cyanide jars, pinned and stored in wooden boxes at the dep. of plant protection, fac. of agriculture, suez canal university. labels containing the collecting time and date, area of collection and scientific name of the host plant were attached to the specimens. examinations of male genitalia were carried out. male terminalia were cleared with 10% koh (potassium hydroxide) for at least half a day then transferred to distilled water for dissection. the bee species identified based on a reference collection at ain shams university and the species identification confirmed by dr. nicolas j. vereecken liberal university of brussels, belgium. field nesting site the bee nest was discovered during field collection of bees around alexandria governorate (western part of egypt). the nest found at abbis i village (n45’’82’31’ e57’’23’29’) in the main high way between alexandria – cairo agricultural road. the nest was in a small canal bank surrounded by some blooming flowers like brassica napus l., urtica dioica l. and other wild plants. at the same nesting site some other bees had been found nesting very close to our nesting site like andrena vetula lepeletier, 1841 and andrena fuscosa erichson, 1835. weekly observations of the nest and the bees were conducted from february until end of march. nest excavation had been carried out by digging above the soil surface for observing the nest architecture. the seasonal and daily abundance of bees was recorded at three times of the day 11am, 1pm and 3pm. soil characteristic analysis the soli characteristics analysis was conducted at dept. of soil and irrigation, faculty of agriculture, suez canal university. 1hydraulic conductivity: saturated hydraulic conductivity was determined using darcy’s law in the form ks= ql/ δψat where q was the volume of fluid, that moves through a soil per unit cross-sectional area (a), and time (t), is directly proportional to the total potential gradient (δψ), which drives the fluid flow and indirectly proportional to the length (l) of the soil column through which the fluid moves, according to hill & james (1995). 2bulk density: bulk densities of the calcareous, alluvial and sandy soils were determined according to blake and ha rtge (1986). 3electrical conductivity: of the saturated soil paste extract expressed as (dsm-1) were measured using conductivity meter model jenway 3310 according to richards (1954). 5soil ph: the ph of soil samples was determined by bench type beckman glass electrode ph meter, in 1:2.5 soilwater suspensions according to page et al. (1982). 6soluble cations and anions: the saturated soil paste extract was analyzed for soluble anions and cations. sodium and k+ were determined flamephotometerically, ca2+, mg2+, were volumetrically determined by titration with ethylene diamine tetra acetic acid (versinate), clwas determined by titration with silver nitrate, hc o3was determined by titration with standard sulphuric acid according page et al. (1982). results and discussion nest description a nest of e. nigrilabris was found at abbis village, alexandria (western part of egypt). the species fly during winter season (january – march), the males were started flying before females. the length of the tunnel was very deep about 70 to 80cm (n3), the diameter of the cell entrance was ranged between 0.7-0.8 cm (n3) and diameter of the cell end was ranged between 0.8 to 1cm (n3) (fig 1). it seems that the whole subfamily of anthophorinea dig a deep nests and other species could be found with the same nest like eucera and tetralonia. a compound nest of tatrealoina has been discovered during 1976 with 70 cm soil surface combined with nomia sp. nest (ibrahim, 1976; malyshev, 1929). there were are a few exception of the subfamily building shallow cavity nesting such as anthophora waltoni cockerell (shebl et al., 2014). during the nest excavation some cells were found empty specially those on the first third of the tunnel during the searching for the eggs. the insect eggs were whitish laid over the pollen ball. each tunnel has 4 to 6 cells the first cells were empty or false cells, below the first cells some cells were found contains the old body of the laid females by then the main basic cells as shown in fig 1. the female could used more than one entrance because the tunnels were branched and connected with each other under the soil surface. the cell chambers of species of the genus eucera were constructed as short branches from the main burrow, often two or three cells per nest (amiet et al., 2007). so some females were used entrance but during excavating the nest some females found in another tunnel. the whole tunnel was lined by wax. eucera longicornis (linnaeus, 1758) used to nest in a large aggregations and constructed burrows in the ground that branches into up to seven polished brood chambers filled with liquid pollen mass in which the egg is laid. sometimes two nesting females share the same entrance of nest (westrich, 1989). sociobiology 63(4): 1031-1037 (december, 2016) 1033 mating behavior the males were emerged several days before the females, the males easily distinguished from the females by the long antennae and the yellowish clypeus on the head. the males were started flying during the third week of january for almost one moth until the third week of february. two shapes of male were recognized differing in color and activity. at the beginning the males were reddish with slow movements around the nesting sites without any flight, they moved their legs and antennae from time to time. they remained without flight activities for several hours. the other shape of male was grey with very active movements around the nests or on the resting sites. the reddish males were the immature males and once they became mature their color changed. moreover, more than two or three males were recognized following each other at the same tunnel entrance (old tunnels), they seems that they were working probably helping the female for emergence or defending the nests. however, the individual on the surface keep moving their abdomen until leaving the entrance for several minutes. they enter theentered their nests by head but sometimes got out by abdomen and other times by head. the female was emerged several days after the male and was remained active for almost two months until end of march. the new emerged female was remained inactive for one minute more or less then flying around the nest. the males were fighting with each other before mating. the mating took place over the nesting sites. the mating time took about 3 to 6 minutes or longer. the receptive female female did not accepted other males for another mating so the mating occurred only once a time during the whole lifecycle. therefore, it is expected that the mated females laid few eggs (fig 2). digging the new tunnel after the mating the female were started digging her new tunnel. the females were started digging the soil with her head and legs and building a branched and curved tunnels and the whole process remained for several days. the females were dug only one tunnel during the whole life cycle with four to six cells. then the female were started foraging and collecting pollen, the collected pollens are dry, yellow and the average weight during one trip was about 0.015gm. the number of cells varied from one to another species of ground nesting bees (fig. 1-2). most of soil burrowing bees makes only one nest and very few make several nests with very few cells (stephen et al., 1969; kamel, 1981; coville et al., 1983; norden, 1984; neff & simpson, 1992; semida, 2000; shebl et al., 2014). bee seasonal and daily abundance bees were started flying at the third week of january and remain until mid of march so the bees is protandrous and univoltine. the daily activity of the insect were started at 9 or 10am but the maximum activity of the bees was during the midday day hours 12-1pm. the males started flying before females few days for reaching their maturation. the bees were more active during midday hours around 11pm to 1pm and the bees were fewer active during early morning and late afternoon which was noticed in most solitary bee (fig 3) (shebl et al., 2014; shebl & farag, 2015). soil characteristics of the nest the soil of the nest is too hard from the surface (very dry seems like soft rock) and becomes more softer by going depper due to high moisture. the soil of the nest has a yellow color with sandy loam texture, low salinty and sodicity and low calcium carbonate content (table 1). fig 1. nest architecture of eucera nigrilabris lepeletier, 1841. ma shebl, rm al – aser nesting and seasonality of long-horned bee1034 the sand, silt and clay were 70.6, 22.3 and 8%, respectively. the ec, sar, and caco3 were 1.5 dsm -1, 2.52, and 3.14 %, respectively. the soluble ca2+, mg2+, na+ and k+ were 4.9, 3.1, 5.0 and 2.0 meq l-1, while soluble hco-3, cl and so42 were 3.5, 7.7 and 3.8 meq l-1, respectively (table 1). during another field survey of bees in canal region (shebl et al., 2013) e. nigrilabris were not collected from that area. the type of the soil at that area was sand mainly desertic areas. our assumption that the bees composition could be affected not only by their floral resources but also by their nesting resources suitability (pots et al., 2005; cane et al., 2007). so some species could have a limited distribution due to their nesting resources and the soil characteristics of that nest. bee community composition is related to plant richness, soil characteristics potentially related to nesting suitability, and canopy cover. suitability for nesting can be related to soil and soil cover characteristics for example percent of organic content, sand, silt, and clay in the soil (grundel et al., 2010). the amount of organic matter, organic carbon and bulk density of surface layers are important factors in selection of nesting sites by solitary bees. many species of ground nesting bees of colletes, andrena, halictus and osmia preferred well drained areas with a good surface flow and a plant stand of sparse to intermediate density (osgood, 1972). choosing the site of the nest by bees depend on several intrinsic and extrinsic factors such as morphology, mechanical structure, moisture, presence fig 2. a. nesting site area; b. nest entrance; c. mature male of the resting site; d. mating, e. nesting activities, f. the eggs. sociobiology 63(4): 1031-1037 (december, 2016) 1035 of food and physical properties of the soil (semida, 2000). the nest of e. nigrilabris was very deep and this could be related to the soil structure. the nest architecture is characterized of the species with different individual variations. some females dig the nest deep or quite near ground because of the soil conditions (stephen et al., 1991; semida 2000). imapct of human interefrenc on the e. nigrilabris the decline of plant pollinators particularly bees (hymenoptera: apoidea) is well known worldwide. there are many research papers indicated that many solitary bees are threatened by the human interference such as fragmentation of natural habitats, lack of floral resources and extensive use of pesticides (shebl et al., 2013). the whole nesting are of e. nigrilabris area was eliminated due to national project of covered drainage. the whole area is not longer active, the same case was noticed with a number of leafcutting bees (kamel, et al., 2007). such studies encourage conservation strategies for protection natural biodiversity resources which has a great impact on our environment. acknowledgments we are so grateful to dr. nicolas vereecken, evolutionary biology and ecology, free university of brussels for his help with the species identification. our sincere appreciation to the following persons: mohamed attia al aser, mohamed ramadan, and ahsraf gaber for their help during nest excavation. our deep thanks for prof dr. soliman kamel for his guidance and recommendation during the study. we are so highly appreciated for the research facilities supported by dept. of plant protection and dept. soil & irrigation sciences, fac. of agriculture, suez canal university. our deep thanks for the chief editor and two anonyms referee of the journal for their comments. refernces alqarni a. s., hanna, a. m & engel, s. m. 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(1989): die wildbienen baden-württembergs. teil 1: allgemeiner teil. ulmer verlag. http://dx.doi.org/10.2317/jkes131028.1 top open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 77-95 (2013) acoustic evaluation of trees for coptotermes formosanus shiraki (isoptera: rhinotermitidae) treated with imidacloprid and noviflumuron in historic jackson square, new orleans, louisiana w. osbrink1, m. cornelius2 introduction the formosan subterranean termite, coptotermes formosanus shiraki (fst), is native to asia (bouillon, 1970), but was introduced into the southern united states where they have become devastating pests (su & tamashiro, 1987). in addition to structural infestations, c. formosanus infestations of living trees are common in the new orleans, la area (osbrink et al., 1999; osbrink & lax, 2003; osbrink et al., 2011). total economic loss due to termites in the united states was estimated at $11 billion per year (su, 2002). control of termites is important to prevent the destruction of materials where it is undesirable. following implementation of an area wide termite control strategy, a definitive question is what happens to the termite populations (osbrink et al., 2011). in addition to reducing termite pressure in areas where structure and tree damage is undesirable, termite population elimination also increases the area available for the establishment and growth of new or suppressed termite populations (su, 2002; lax & abstract nine years of periodic acoustical monitoring of 93 trees active with formosan subterranean termite, coptotermes formosanus shiraki, were evaluated for imidacloprid tree foam and noviflumuron bait to reduce termite activity in trees. long term, imidacloprid suppressed but did not eliminate termite activity in treated trees. noviflumuron bait did not significantly reduce the proportion of trees with high termite activity but significantly increased the number of trees with no termite activity. noviflumuron changed termite distribution by possibly eliminating only some fraction of numerous colonies whereby surviving colonies avoided trees containing dead termites. sociobiology an international journal on social insects 1 usda-ars-spa knipling-bushland u.s., kerrville, texas, usa 2 usda-ars-barc, beltsville, maryland, usa research article termites article history edited by: evandro n. silva, uefs brazil received 29 november 2012 initial acceptance 02 january 2013 final acceptance 05 february 2013 keywords formosan termite, aed, reinvasion corresponding author weste osbrink usda-ars-spa knipling-bushland u.s. livestock insects research lab 2700, fredericksburg road, kerrville, texas, 78028 e-mail: weste.osbrink@ars.usda.gov osbrink, 2003; su & lees, 2009; guillot et al., 2010; osbrink et al., 2011; mullins et al., 2011). because of the affinity of the formosan termite for living trees, they cannot be ignored in area wide population suppression efforts as they may be a primary source of termites (osbrink et al., 1999; osbrink & lax, 2002b; osbrink & lax, 2003). non-invasive monitoring of termite activity is ideal for evaluation of the efficacy of control efforts because monitoring has no effect on population dynamics. invasive monitoring techniques can push termites away from the monitor creating an artifact of apparent control because of relocation of the termites. efforts to develop techniques for detecting hidden termite infestations have produced only a few successful alternatives to traditional visual inspection methods (lewis, 1997). alternatives include ground-based monitoring devices with sensors that detect acoustic emissions of termites in wood (fujii et al., 1990; lewis & lemaster, 1991; noguchi et al., 1991; robbins et al., 1991). acoustic emission sensors are successful because they are nondestructive and operate at high frequencies (ca. 40 khz) where there is w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus78 negligible background noise to interfere with detection and interpretation of insect sounds (lewis & lemaster, 1991; robbins et al., 1991). acoustic emission systems have been applied as research tools to estimate termite population levels (fujii et al., 1990; lewis & lemaster, 1991; scheffrahn et al., 1993; osbrink et al., 2011). acoustic emission systems are ideal for detection of termites in trees (osbrink et al., 1999; kramer, 2001; mankin et al., 2002; osbrink et al., 2011). understanding pest population dynamics in space and time post-treatment integrates into an effective pest management strategy. the objective of this research was to monitor formosan termites treated with imidacloprid and noviflumuron in an area wide termite control effort. to meet this objective, trees were monitored for c. formosanus with an acoustical emissions detector to quantify activity. these studies provide insight into the dynamics of an area wide termite management approach. materials and methods jackson square historic jackson square (js) is a ≈ 0.9-ha (92 x 96 m) green space in the french quarter, new orleans, la. a total of 93 js trees, comprised of ten different species, were periodically monitored for termite activity with an acoustical emission device (aed) for 9 years. js was divided into 5 topographic regions: q1 south-east quarter with trees 1-15; q2 north-east quarter with trees 16-29; q3 north-west quarter with trees 30-42; q4 south-west quarter with trees 42-62; and center (cent) with trees 63-94 (fig. 1). trees are identified in table 1, years and months sampled in table 2. acoustical emission detector (aed) an aed-2000 acoustical emissions detector (acoustical emissions consulting, inc fair oaks, ca) was used to quantify termite activity within 93 live trees in js. lag bolt wave guides (150 x 9 mm) were screwed horizontally into pre-drilled pilot holes in the north-west trunk of test trees ≈20 cm from the ground. acoustical emissions were detected with a model sp-1l probe with model dmh-30 high force magnetic accessory attachment (acoustic emission consulting, inc., fair oaks, ca). for each tree, aed counts were acquired for 60 s with accompanying software which converts termite sounds to counts per second saved in excel (microsoft, redmond, wa). only the numbers of counts in the first 10 s of the 60 s recording were used to represent each unique individual recording. if the first 10 s of recording was contaminated with interference noise (elevated spiked counts), the first 10 s of recording following the cessation of interference noise were used to represent the unique individual recording. previous research has determined that aed counts measure termite activity in trees (mankin et al., 2002; osbrink et al., 2011). figure 1. map of jackson square, new orleans indicating locations of trees. noviflumuron bait by 2006, pest management professionals (pmp) installed 84 commercial in-ground sentricontm monitoring stations (dow agro sciences llc, indianapolis, in) with untreated wood every ≈5 m around the js perimeter (22 west, 22 north, 21 east, and 19 south). pmp also initiated and maintained baiting with 0.5% noviflumuron bait tubes in monitors becoming positive with fst. the baiting program was terminated june 2011. imidacloprid tree foaming in 2000, 7 trees (t7, t10, t20, t21, t38, t49, and t57) with c. formosanus activity were drilled and foamed with 0.5% imidacloprid (premisetm sc, bayer, kansas city, mo) by pmp. fst mud tube survey on may, 2011, js trees were visually inspected for fresh fst mud tubes created for spring distribution flights. sociobiology 60(1): 77-95 (2013) 79 data analysis ten consecutive count values (10 s) were used to calculate mean (±se) counts per second to represent termite activity associated with each unique aed tree attachment. acoustical data were analyzed using one way anova with means separated with protected tukey test, p < 0.05 (systat, 2008). proportions were arcsine square root transformed before analysis and actual proportion reported in tables. tree readings were defined as high (h) termite activity when significantly > 0, which was qualitatively confirmed with earphones, connected to the aed. low (l) tree readings were defined by readings of 0 or an event which only occurred only once (1 s) in 10 s, also confirmed qualitatively as above. readings were defined as medium (m) when between h and l. m was qualitatively verified and indicates the presence of termites. results jackson square all 93 trees had termite activity, and a total of 25 (≈26.9%) trees were lost or removed. only 4 trees (4.3%), tree # 1 (t1), t11, t19, and t58, had combined h activity > m or l activity (table 1). tree t58 was removed (lost) after 2006. over the study, 17 (≈18.3%) trees had 0 h, 8 of which were lost (table 1). trees with m activity > h or l occurred in 84 trees (90.3%). only 5 trees had l > m, and 3 trees had overall 0 l activity (table 1). noviflumuron bait two monitors (2.4%) adjacent to t14 and t34 had fst on january 2010 (fig. 1), initiating noviflumuron baiting. all trees had significantly high termite activity at some time, but few trees had consistently high termite activity (table 2 and 3). three trees (t1, t11, and t19) had repeated significantly high termite activity (tables 2 and 4) no significant reduction in trees with h termite activity occurred in the post-treatment years of 2010 and 2011 (table 5). post-treatment 2010 m show significant decrease in m trees in march, april, may, july (except 2008), september (except 2009), and october (except 2005). posttreatment 2011 m show significant decrease in trees occurred march (except 2008), april, may, july (except 2008, 2009), september (except 2009), and october (except 2003, 2005). m 2011 were consistently greater than in 2010 and significantly higher for the months of july and october (table 5). post-treatment 2010 l show significant increase for march, april, may, july (except 2008), september (except 2009), and october (except 2005, 2009). post-treatment 2011 l show significant increase in april, may, july (except 2008, 2009), september (except 2009), and october (except 2003, 2005, 2009) (table 5). imidacloprid tree foaming the seven imidacloprid foamed trees had a lower but non-significant h readings than un-foamed trees with a mean percent (± se) of 2.8 ± 0.9 and 11.3 ± 1.2, respectively (f = 3.666; df = 1, 92; p = 0.059). h events occurred twice in 2003, once in 2006, 1x in 2007, 2x in 2008, and 2x in 2011 (table 6). there was no difference in m levels of termite activity in foamed and un-foamed trees with mean (± se) percent of 62.2 ± 3.0 and 61.2 ± 1.3, respectively (f = 0.0433; df = 1, 92; p = 0.836). there was no difference between mean percent l activity between foamed and un-foamed trees (mean ± se) 35.3 ± 3.5 and 28.3 ± 1.4, respectively (f = 1.952; df = 1, 92; p = 0.166). mud tube survey in may 2011, six trees (t4, t10, t21, t29, t38, and t46) were found with active fst mud tubing (table 6). all mud tube trees had 0% h in 2011. five of six trees (83.3%) with fst mud tubing had been drilled and treated with imidacloprid. discussion certain events can interfere with successful recording of termite activity including wind noise, trucks with squeaking breaks, generators, crowd noise, leaf flutter, etc. aed recordings do not distinguish termite events from unrelated sound events. aed termite activity has a unique sound resembling rain on a tin roof. aed recordings are qualitatively monitored with earphones and a log maintained allowing data spikes of non-termite origin to be excluded from data analysis. jackson square fst infestation of 100% of trees with termite activity is unprecedented though not unreasonable. guillot et al. (2010) reported fst in 1.5% of 3000 trees visually inspected in the french quarter neighborhood surrounding js and noted this level was surprisingly low. messenger and su (2005) reported ≈ 32% infested trees in armstrong park, new orleans. osbrink et al. (1999) used visual inspection and staking to determine tree infestation in a portion of new orleans city park with results varying from 0 to 30.6% depending on tree species. inspection of hurricane damaged trees in city park revealed 75% of 21 trees were infested with fst (osbrink et al., 1999). the number of trees infested with formosan w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus80 termite is much higher than indicated by current established monitoring techniques as revealed following incidents of heavy wind. this is confirmed by the number of living and externally healthy trees which break or fall revealing fst infestations. successful treatment of termite populations in trees will require the development of improved, nondestructive, monitoring techniques. js has had increasing fst populations for > 60 years resulting in a high probability contact with 100% of available trees. the proportion of l infestations under such circumstance reflects the limitation of detection capabilities not the foraging ability of fst. noviflumuron bait of 84 in-ground monitors 2.4% became infested. this is consistent with the 4.8% and 6.7% of wooden stakes found infested with by fst in city park and armstrong park, new orleans, respectively (osbrink et al., 1999, messenger & su, 2005). noviflumuron has been shown to eliminate those termite colonies that take the bait (smith et al., 2002; karr et al., 2004; getty et al., 2007; husseneder et al., 2007; austin et al., 2008; thoms et al., 2009; eger et al., 2012; lee et al., 2012). termites adjacent to js have had control pressure for years in historic structures such as the st. louise cathedral and cabildo (site of louisiana purchase), followed by a decade of federal termite control pressure with operation full stop (su et al., 2000; guillot et al., 2010). control pressure changes the fst population demography away from a few large alpha colonies controlling most of the space and resource (aluko &husseneder, 2007). alpha-colonies are surrounded by suppressed fst colonies surviving like the bonsai-tree with reduced resources. alpha-colony elimination allows bonsaicolony expansion into vacated territory (aluko &husseneder, 2007). expanding bonsai-colonies avoid baited areas initially because they avoid dead termites (su & tamashiro, 1987) and because they are excluded by competing bonsai-colonies. dense colony fst populations become functionally resistant to the bait because colony elimination removes only a fraction of resident termites (husseneder et al., 2007). consistent with this are the results of messenger et al. (2005) who used hexaflumuron to eliminate formosan termite colonies in armstrong park, new orleans, in three mo, but observed reinvasion almost immediately. three trees (t1, t11, and t19) had repeated significantly high termite activity (table 4) and may indicate fst carton nest locations (table 4; fig. 1). these putative fst colonies are well spaced by > 35 m (t1 to t11 ≈36 m and t11 to t19 ≈ 37 m). coptotermes frenchi hill locates their colony in a tree and forage to neighboring trees from the colony tree (hill, 1942), which provides a plausible explanation for the changes in h trees overtime. fst regularly changes areas of high activity (tables 2, 3, and 4). while studies indicate rapid (months) population suppression or colony elimination with csi as reviewed by su (2003) and su and scheffrahn (1998) there are often problems with continued, long term reinvasions. su (2003) summarized hexaflumuron performance as 98.5% successful colony elimination from 1,3691 sites, with 199 sites experiencing control problems. however, glenn and gold (2002) baited c. formosanus with hexaflumuron for two yr in beaumont, tx and found termites remained active in or around two of five structures. using hexaflumuron, su et al. (2002) continued to detect c. formosanus populations for about two yr after initiating an area wide community test. guillot et al. (2010) reported hexaflumuron treated areas in the french quarter, la, with 3-4% of independent monitors that remaining active for ca. five yr. thus, colonies can be eliminated rapidly in area-wide management, but termite populations may remain because they may not come into contact with treatments. imidacloprid tree foaming the lower (non-significant) mean percent h activity in the foamed trees may be an indication that imidacloprid reduced suitability of central tree lumen as a habitat for fst. recorded m termite activity may be due to termites occupying the untreated wood surrounding the foamed hollow. osbrink and lax (2003) found that independent monitors up to 46 m from treated trees showed imidacloprid intoxication resulting from the direct application of toxicant to the termites occupying the hollow of the tree. after six to 15 mo, there was complete recovery of fst populations in the independent monitors. this effect was not seen with imidacloprid soil applications which require the termites to dose themselves by moving through the treated substrate dispelling theories that imidacloprid acted like liquid bait (osbrink et al., 2005). imidacloprid has been shown to have a relatively short half life in soil and trees when compared to other termiticides (ring et al., 2002; mulrooney et al., 2006; saran & kambel, 2008). though not eliminating termites from trees, the putative extended suppression of activity may be attributed to increased residual activity when bound to the substrate inside the protected tree hollow. mud tube survey presence of mud tubes provides visual confirmation of survival of functional fst colonies. fst may prefer placement of their swarm tubes in areas with the least amount of termite activity which is a possible mechanism to avoid antagonistic interactions at a vulnerable time in fst life cycle. the c. formosanus populations appeared to be prisociobiology 60(1): 77-95 (2013) 81 marily centered in trees (ehrhorn, 1934; osbrink et al., 1999; osbrink & lax, 2002a). hill (1942) determined that all large coptotermes frenchi hill colonies are centered inside living trees and that colony developments in alternate locations do not achieve the size or the longevity of colonies nesting in trees. though c. formosanus has flexible nesting habits, available hardwood trees may be their definitive host as it provides ideal harborage, mechanical protection, moisture for survival of small young colonies, flood protection, antibiotic benefits, and food (osbrink et al., 1999; fromm et al., 2001; cornelius et al., 2007; osbrink et al., 2008; cornelius & osbrink, 2010, osbrink et al., 2011), with mutualistic tree benefits with de novo antibiotic production and nitrogen fixation, soil aeration, and relocation of micronutrients (janzen, 1976; burris, 1988; ohkuma et al., 1999; osbrink & lax, 2003; apolinario & martius, 2004; jayasimha & henderson, 2007, chouvenc et al., 2009). because of the affinity of the formosan termite for living trees, they cannot be ignored in area-wide population suppression efforts as they may be a primary source of termites (osbrink et al., 1999, osbrink &lax 2002b; osbrink & lax, 2003). annual reinvasion of suppressed areas with alates establishing new colonies and the expansion of bonsai-colonies eventually will lead to a resurgence of termite pressure. the resulting new populations will be independent of one another, different from the larger suppressed populations, potentially altering the performance of continuing bait treatments (husseneder et al., 2007). such a dynamic promotes the establishment of many separate populations, similar to disturbed landscapes studied by aluko and husseneder (2007), reducing the impact of baits over time. termite baits are more effective against a few larger mature populations as opposed to with numerous independent populations. thus, large numbers of independent termite populations established upon reinvasion provide a mechanism of demographic resistance to lessen the effects of baits on overall termite populations possibly responsible for control plateaus reported in other area wide control studies (guillot et al., 2010). similarities may exist in the proliferation of polygyne over mongyne fire ants solenopsis invicta buren accompanying area wide baiting with hydramethylnon (glancy et al., 1987). in conclusion, after about two yr of area-wide treatment, there were as many trees with high termite activity post-treatment. the reinvasion and establishment of new independent termite populations provides a mechanism over time to decrease the effectiveness of baits protecting the structures. thus, continuous reevaluation of changing circumstance becomes critical for implementation of the best control strategies, including tree evaluations, to protect structures from reinvading colonies. acknowledgements we thank j. goolsby, f. guerrero, k. lohmeyer, j.m. pound, and s. skoda for agreeing to review this manuscript and valuable improvements contributed by their reviews. we also thank two anonymous reviewers for their contributions. references aluko, g. and husseneder, c. 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(2008). effects of flooding on field populations of formosan subterranean termites (isoptera: rhinotermitidae) in new orleans, louisiana. j. econ. entomol. 101: 1367-1372. doi: 10.1603/0022-0493(2008)101[1367:eofofp]2.0.co;2 osbrink, w. l. a., cornelius, m. and lax, a. (2011). areawide field study on effects of three chitin synthesis inhibitor baits on populations of coptotermes formosanus and reticulitermes flavipes (isoptera: rhinotermitidae). j. econ. entomol. 104: 1009-1017. doi: 10.1603/ec10217 ring, d., henderson, g. and mccown, c. (2002). evaluation of the louisiana state program to treat trees infested with formosan subterranean termites (isoptera: rhinotermitidae) in louisiana, pp. 259-266. in s. c. jones, j. zhai, and w. h. robertson (eds.), proceedings of the 4th international congress on urban pests (pp. 259-266). blacksburg, va: pocahontas press. robbins, w. p., mueller r., schaal, t. and ebeling, t. (1991). characteristics of acoustic emission signals generated by termite activity in wood, in proceedings, ieee ultrasonics symposium (pp. 1047-1051). orlando, fl. conference publications. saran, r. and kamble, s. (2008). concentration-dependent degradation of three termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (isoptera: rhinotermitidae). j. econ. entomol. 101: 1373-1383. doi: 10.1603/0022-0493(2008)101[1373:cdotti]2.0.co;2 scheffrahn, r., robbins, w., busey, p., su, n_y. and mueller, r. (1993). evaluation of a novel, hand-held, acoustic emissions detector to monitor termites (isoptera: kalotermitidae, rhinotermitidae) in wood. j. econ. entomol. 86: 1720-1729. smith, m. s., karr, l., king, j., kline, w., sbragia, r., sheets, j. and tolley, m. (2002). noviflumuron activity in household and structural insect pests. in s. c. jones, j. zhai, and w. h. robertson (eds.), proceedings of the 4th international congress on urban pests (pp. 345-353). blacksburg, va: pocahontas press,. su, n.-y. (2002). novel technologies for subterranean termite control. sociobiology 40: 95-101. su, n.-y. (2003). baits as a tool for population control of the formosan subterranean termite. sociobiology 41: 177-192. su, n.-y. and lees, m. (2009). biological activities of a bait toxicant for population management of subterranean termites. in c.j. peterson and d. m. stout ii (eds), pesticides in household, structural and residential pest management. am. chem. soc. symp. ser. 1015 (pp. 87-96). washington, dc: american chemical society. su, n.-y. and tamashiro, m. (1987). an overview of the formosan subterranean termite (isoptera: rhinotermitidae) in the world. in m. tamashiro and n.-y. su (eds.), biology and control of the formosan subterranean termite. hawaii institute of tropical agriculture and human resources research extension series 083 (pp. 3-15). honolulu, hi: university of hawaii and manoa. su, n.-y., and scheffrahn, r. (1998). a review of subterranean termite control practices and prospects for integrated pest management programs. integrated pest manag. reviews 3: 1-13. su, n.-y., freytag, e., bordes, e. and dicus, r. 2000. control of the formosan subterranean termite infestations in historic presbytere and the creole house of the cabildo, french quarter, new orleans, using baits containing an insect growth regulator, hexaflumuron. studies in conservation 45: 30-38. su, n.-y., ban, p. and scheffrahn, r. (2002). use of a bait impact index to assess effects of bait application against populations of formosan subterranean termite. j. econ. entomol. 86: 1453-1457. doi: 10.1603/0022-0493-97.6.2029 systat software. (2008). sigmaplot users guide: statistics, version 11. systat software, inc. san jose, ca. thoms, e. m., eger, j., messenger, m., vargo, e., cabrew. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus84 ra, b., riegel, c., murphree, s., mauldin, j. and scherer, p. (2009). bugs, baits, and bureaucracy: completing the first termite bait efficacy trials (quarterly replenishment of noviflumuron) initiated after adoption of florida rule, chapter 5e-2.0311. am. entomol. 55: 29-39. this article reports the results of research only. mention of a proprietary product does not constitute an endorsement or recommendation by the usda for its use. usda is an equal opportunity provider and employer. sociobiology 60(1): 77-95 (2013) 85 table 1. trees of jackson square with cumulative % h, m, and l formosan termite activity tree common name scientific name h m l 1 sweet olive osmanthus fragrans lour., oleaceae 39.2 ± 14.1 38.6 ± 12.8 22.2 ± 9.6 2 redbud cercis canadensis l., leguminales 19.4 ± 10.0 52.8 ± 12.1 27.8 ± 14.7 3 sweet olive osmanthus fragrans lour., oleaceae 10.6 ± 5.3 68.0 ± 7.1 21.4 ± 8.9 4 im magnolia magnolia grandiflora l., magnoliaceae 4.8 ± 3.4 71.7 ± 9.3 23.5 ± 9.9 5 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 7.1 ± 5.6 49.9 ± 8.8 43.0 ± 11. 6 sweet olive osmanthus fragrans lour., oleaceae 25.1 ± 9.4 51.8 ± 10.9 23.0 ± 8.0 7 sweet olive osmanthus fragrans lour., oleaceae 10.3 ± 6.9 47.9 ± 9.4 41.8 ± 5.4 8 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 0.0 ± 0.0 64.6 ± 11.9 35.4 ± 11.9 9 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 3.2 ± 3.2 60.8 ± 12.4 36.0 ± 12.2 10 im live oak quercus virginiana miller, fagaceae 5.6 ± 5.6 63.2 ± 9.3 31.2 ± 9.9 11 x redbud cercis canadensis l., leguminales 49.4 ± 13.6 43.3 ± 12.0 19.8 ± 12.4 11 x redbud cercis canadensis l., leguminales 49.4 ± 13.6 43.3 ± 12.0 19.8 ± 12.4 12 live oak quercus virginiana miller, fagaceae 1.6 ± 1.6 52.5 ± 6.9 45.9 ± 7.2 13 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 3.7 ± 3.7 57.5 ± 10.5 38.7 ± 10.1 14 n sweet olive osmanthus fragrans lour., oleaceae 6.4 ± 4.8 54.9 ± 8.0 49.9 ± 9.8 15 redbud cercis canadensis l., leguminales 6.9 ± 3.9 65.7 ± 6.1 27.4 ± 6.8 16 x magnolia magnolia grandiflora l., magnoliaceae 0.0 ± 0.0 88.9 ± 11.1 11.1 ± 11.1 17 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 0.0 ± 0.0 62.2 ± 11.2 37.8 ± 11.2 18 nd nd nd nd nd 19 redbud cercis canadensis l., leguminales 52.6 ± 9.8 42.6 ± 10.6 3.2 ± 2.1 20 i live oak quercus virginiana miller, fagaceae 1.6 ± 1.6 69.2 ± 11.9 29.2± 12.3 21 im magnolia magnolia grandiflora l., magnoliaceae 0.0 ± 0.0 61.0 ± 10.8 40.6 ± 11.7 22 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 7.1 ± 5.6 45.8 ± 12.2 55.0 ± 11.4 23 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 11.6 ± 8.3 67.6 ± 8.5 28.7 ± 9.2 24 sweet olive osmanthus fragrans lour., oleaceae 4.4 ± 3.0 62.3 ± 9.3 33.3 ± 10.1 25 redbud cercis canadensis l., leguminales 14.9 ± 4.5 56.0 ± 9.5 29.1 ± 10.4 26 x redbud cercis canadensis l., leguminales 8.3 ± 8.3 70.9 ± 10.5 20.8 ± 12.5 27 redbud cercis canadensis l., leguminales 7.9 ± 4.8 64.5 ± 11.4 27.5 ± 11.6 28 sweet olive osmanthus fragrans lour., oleaceae 13.4 ± 7.3 63.1 ± 10.8 14.0 ± 7.2 29 m magnolia magnolia grandiflora l., magnoliaceae 7.1 ± 5.6 60.3 ± 8.8 32.5 ± 9.9 30 sweet olive osmanthus fragrans lour., oleaceae 15.9 ± 10.8 53.7 ± 11.6 30.4 ± 8.5 31 x magnolia magnolia grandiflora l., magnoliaceae 16.7 ± 16.7 55.6 ± 5.6 27.8 ± 14.7 32 sweet olive osmanthus fragrans lour., oleaceae 19.0 ± 9.2 64.6 ± 13.0 16.4 ± 6.2 33 x redbud cercis canadensis l., leguminales 0.0 ± 0.0 100.0 ± 0.0 0.0± 0.0 34 n sweet olive osmanthus fragrans lour., oleaceae 28.6 ± 12.1 61.4 ± 11.1 10.1 ± 4.5 35 x magnolia magnolia grandiflora l., magnoliaceae 11.7 ± 7.3 76.7 ± 14.5 11.7 ± 7.3 36 x savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 14.6 ± 8.6 85.4 ± 8.6 0.0 ± 0.0 37 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 4.8 ± 4.8 60.1 ± 9.8 35.2 ± 9.9 38 im live oak quercus virginiana miller, fagaceae 4.8 ± 4.8 56.1 ± 5.9 39.1 ± 6.5 39 orchid tree bauhinia purpurea l., fabaceae 6.9 ± 4.6 64.8 ± 9.0 28.3 ± 10.4 40 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 3.2 ± 3.2 67.6 ± 8.9 29.2 ± 9.6 41 live oak quercus virginiana miller, fagaceae 3.7 ± 3.7 56.1 ± 11.7 32.8 ± 11.5 42 x redbud cercis canadensis l., leguminales 10.0 ± 10.0 76.7 ± 14.5 13.3 ± 13.3 43 redbud cercis canadensis l., leguminales 24.9 ± 8.7 57.7 ± 14.1 17.5 ± 9.8 44 live oak quercus virginiana miller, fagaceae 1.6 ± 1.6 53.4 ± 12.2 45.0 ± 12.1 (table continues) w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus86 table 1. trees of jackson square with cumulative % h, m, and l formosan termite activity tree # common name scientific name h m l 45 mulberry morus spp. , moraceae 11.9 ± 5.8 55.3 ± 9.7 32.8 ± 9.7 46 m live oak quercus virginiana miller, fagaceae 0.0 ± 0.0 57.3 ± 9.8 42.7 ± 9.8 47 redbud cercis canadensis l., leguminales 24.9 ± 6.9 58.9 ± 9.2 16.3 ± 6.6 48 magnolia magnolia grandiflora l., magnoliaceae 1.6 ± 1.6 70.4 ± 10.5 28.1 ± 10.4 49 i savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 2.8 ± 2.8 65.9 ± 10.5 31.4 ± 10.4 50 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 1.9 ± 1.9 57.5 ± 11.8 40.6 ± 12.4 51 sweet olive osmanthus fragrans lour., oleaceae 4.8 ± 3.4 69.3 ± 11.5 25.9 ± 11.4 52 sweet olive osmanthus fragrans lour., oleaceae 1.6 ± 1.6 54.8 ± 12.3 43.7 ± 12.4 53 savannah holly ilex x attenuata l.’savannah’, aquifoliaceae 0.0 ± 0.0 49.3 ± 9.7 50.7 ± 9.7 54 sweet olive osmanthus fragrans lour., oleaceae 4.8 ± 2.4 65.3 ± 12.5 31.5 ± 11.9 55 sweet olive osmanthus fragrans lour., oleaceae 6.4 ± 4.8 51.9 ± 12.5 41.8 ± 11.2 56 sweet olive osmanthus fragrans lour., oleaceae 3.2 ± 2.1 72.1 ± 6.4 24.7 ± 6.7 57 i live oak quercus virginiana miller, fagaceae 0.0 ± 0.0 48.3 ± 10.9 51.7 ± 10.9 58 x sweet olive osmanthus fragrans lour., oleaceae 52.1 ± 22.2 35.4 ± 14.6 12.5 ± 12.5 59 sweet olive osmanthus fragrans lour., oleaceae 23.5 ± 11.2 47.6 ± 10.3 28.9 ± 11.5 60 magnolia magnolia grandiflora l., magnoliaceae 17.5 ± 8.8 54.9 ± 11.8 27.6 ± 12.2 61 x crepe myrtle lagerstroemia indica l., lythraceae 21.4 ± 11.1 78.6 ± 11.1 0.0 ± 0.0 62 x sweet olive osmanthus fragrans lour., oleaceae 8.3 ± 8.3 66.7 ± 23.6 8.3 ± 8.3 63 crepe myrtle lagerstroemia indica l., lythraceae 7.1 ± 5.6 62.2 ± 9.6 30.7 ± 10.0 64 x crepe myrtle lagerstroemia indica l., lythraceae 0.0 ± 0.0 93.3 ± 6.7 6.7 ± 6.7 65 crepe myrtle lagerstroemia indica l., lythraceae 0.0 ± 0.0 60.3 ± 13.5 39.7 ± 13.5 66 x crepe myrtle lagerstroemia indica l., lythraceae 0.0 ± 0.0 62.5 ± 23.9 37.5 ± 23.9 67 x crepe myrtle lagerstroemia indica l., lythraceae 12.5 ± 12.5 79.2 ± 12.5 8.3 ± 8.3 68 x crepe myrtle lagerstroemia indica l., lythraceae 25.0 ± 19.4 61.7 ± 19.7 13.3 ± 13.3 69 crepe myrtle lagerstroemia indica l., lythraceae 13.8 ± 8.2 49.5 ± 9.3 34.0 ± 10.8 70 crepe myrtle lagerstroemia indica l., lythraceae 8.7 ± 5.6 54.1 ± 11.7 37.2 ± 10.4 71 x crepe myrtle lagerstroemia indica l., lythraceae 16.7 ± 16.7 58.3 ± 22.1 25.0 ± 25.0 72 x crepe myrtle lagerstroemia indica l., lythraceae 0.0 ± 0.0 93.8 ± 6.3 6.3 ± 6.3 73 x crepe myrtle lagerstroemia indica l., lythraceae 33.3 ± 16.7 58.3 ± 8.3 8.3 ± 8.3 74 crepe myrtle lagerstroemia indica l., lythraceae 13.1 ± 5.5 53.8 ± 11.9 33.1 ± 10.7 75 crepe myrtle lagerstroemia indica l., lythraceae 5.3 ± 3.8 62.8 ± 7.9 27.4 ± 7.7 76 crepe myrtle lagerstroemia indica l., lythraceae 3.2 ± 2.1 68.8 ± 9.9 28.0 ± 9.4 77 crepe myrtle lagerstroemia indica l., lythraceae 1.6 ± 1.6 77.3 ± 9.9 21.2 ± 9.0 78 crepe myrtle lagerstroemia indica l., lythraceae 7.1 ± 5.6 58.9 ± 13.5 45.1 ± 14.4 79 x aristocrat pear pyrus calleryana decne, rosaceae 0.0 ± 0.0 68.8 ± 23.7 31.3 ± 23.7 80 x aristocrat pear pyrus calleryana decne, rosaceae 20.8 ± 12.5 41.7 ± 4.8 37.5 ± 14.2 81 x aristocrat pear pyrus calleryana decne, rosaceae 0.0 ± 0.0 80.6 ± 10.0 19.4 ± 10.0 82 x aristocrat pear pyrus calleryana decne, rosaceae 16.7 ± 16.7 58.3 ± 22.1 25.0 ± 25.0 83 x aristocrat pear pyrus calleryana decne, rosaceae 8.3 ± 8.3 62.5 ± 14.2 29.2 ± 10.5 84 x aristocrat pear pyrus calleryana decne, rosaceae 11.1 ± 11.1 58.3 ± 12.7 30.6 ± 19.5 85 x aristocrat pear pyrus calleryana decne, rosaceae 0.0 ± 0.0 75.0 ± 25.0 25.0 ± 25.0 86 x aristocrat pear pyrus calleryana decne, rosaceae 27.1 ± 10.4 39.6 ± 16.5 33.3 ± 11.8 87 mediterranean palm chamaerops humilis l., arecaceae 18.5 ± 11.1 53.3 ± 12.8 39.3 ± 11.2 88 mediterranean palm chamaerops humilis l., arecaceae 14.3 ± 10.9 70.9 ± 10.9 25.9 ± 10.1 89 mediterranean palm chamaerops humilis l., arecaceae 0.0 ± 0.0 58.1 ± 10.4 41.9 ± 10.4 90 mediterranean palm chamaerops humilis l., arecaceae 4.4 ± 3.0 68.1 ± 9.8 27.5 ± 10.5 (table continues) sociobiology 60(1): 77-95 (2013) 87 table 1. trees of jackson square with cumulative % h, m, and l formosan termite activity tree # common name scientific name h m l 91 mediterranean palm chamaerops humilis l., arecaceae 0.0 ± 0.0 48.3 ± 10.5 51.7 ± 10.5 92 mediterranean palm chamaerops humilis l., arecaceae 6.0 ± 3.2 50.7 ± 11.2 51.7 ± 10.5 93 mediterranean palm chamaerops humilis l., arecaceae 7.9 ± 6.4 56.1 ± 12.7 47.1 ± 14.1 94 mediterranean palm chamaerops humilis l., arecaceae 20.1 ± 12.4 47.5 ± 12.6 43.5 ± 11.8 x tree removed before end of study. i imidacloprid treatment in 2000. m mud tubing may 2011. n tree adjacent to noviflumuron station. table 2. mean number (± se) jackson square tree acoustical counts tree mar. april may july aug. sept. oct. (q1) 2003 4 poim nd 14.1 ± 4.1 nd nd nd nd 0.0 ± 0.0 5 nd 140.5 ± 14.5* nd nd nd nd 1.1 ± 1.0 6 nd 83.8 ± 12.1* nd nd nd nd 0.4 ± 0.3 7 nd 101.2 ± 44.9* nd nd nd nd 0.0 ± 0.0 10 poim nd 11.6 ± 8.0 nd nd nd nd 4.2 ± 1.9* 14 prn nd 29.4 ± 15.2 nd nd nd nd 0.0 ± 0.0 nd f= 6.758 nd nd nd nd f = 2.269 nd df = 14, 149 nd nd nd nd df = 13, 139 nd p < 0.001 nd nd nd nd p = 0.010 (q2) 2003 20 poi nd 1.3 ± 0.7 nd nd nd nd 0.9 ± 0.5 21 poim nd 9.7 ± 2.3 nd nd nd nd 1.8 ± 1.0 22 nd 127.1 ± 21.1* nd nd nd nd 0.0 ± 0.0 29 m nd 142.6 ± 45.2* nd nd nd nd 11.0 ± 7.2 nd f = 6.687 nd nd nd nd f = 2.432 nd df = 13, 139 nd nd nd nd df = 12, 129 nd p < 0.001 nd nd nd nd p = 0.007 (q3) 2003 31 x nd 66.3 ± 16.2* nd nd nd nd 6.1 ± 2.8 34 prn nd 71.9 ± 20.3* nd nd nd nd 13.5 ± 4.8* 38 poim nd 9.0 ± 7.7 nd nd nd nd 0.0 ± 0.0 42 x nd 11.1 ± 4.6 nd nd nd nd 18.3 ±5.3* nd f = 3.055 nd nd nd nd f = 5.206 nd df = 11, 119 nd nd nd nd df = 12, 129 nd p < 0.001 nd nd nd nd p < 0.001 (q4) 2003 45 nd 16.1 ± 10.9 nd nd nd nd 34.1 ± 10.3* 46 m nd 4.5 ± 2.4 nd nd nd nd 1.1 ± 0.6 49 poi nd 5.7 ± 2.2 nd nd nd nd 0.0 ± 0.0 57 poi nd 2.9 ± 1.4 nd nd nd nd 0.0 ± 0.0 59 nd 126.2 ± 25.4* nd nd nd nd 36.8 ± 8.6* 60 nd 4.3 ± 1.4 nd nd nd nd 22.1 ± 10.8* 62 nd 88.9 ± 34.1* nd nd nd nd 24.8 ± 5.4* nd f = 7.459 nd nd nd nd f = 7.928 nd df = 18, 189 nd nd nd nd df = 19, 199 (table continues) w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus88 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. nd p < 0.001 nd nd nd nd p < 0.001 (q1) 2004 2 x nd 59.2 ± 8.5* nd 196.4 ± 20.3* nd 729.2 ± 322.8* 66.4 ± 15.6* 4 poim nd 16.5 ± 4.1* nd 1.1 ± 0.6 nd 17.1 ± 1.5 6.4 ± 1.8 10 poim nd 10.9 ± 3.5 nd 0.2 ± 0.1 nd 0.9 ± 0.6 2.1 ± 1.0 10 poim nd 1.6 ± 0.9 nd 0.9 ± 0.4 nd 0.6 ± 0.5 0.7 ± 0.5 14 prn nd 9.3 ± 3.9 nd 0.2 ± 0.2 nd 1.3 ± 0.8 1.3 ± 0.8 nd p < 0.001 nd p < 0.001 nd p < 0.001 p < 0.001 (q2) 2004 19 nd 44.2 ± 26.7* nd 3.5 ± 1.5 nd 17.2 ± 5.4* 17.3 ± 10.7 20 pol nd 3.1 ± 2. nd 0.3 ± 0.3 nd 1.5 ± 1.2 1.2 ± 1.0 21 poim nd 0.6 ± 0.6 nd 3.4 ± 1.9 nd 0.1 ± 0.1 0.0 ± 0.0 24 nd 4.0 ± 2.3 nd 0.8 ± 0.5 nd 12.6 ± 5.5* 5.4 ± 2.9 28 nd 6.0 ± 1.6 nd 1.9 ± 0.7 nd 2.1 ± 2.9 22.2 ± 10.5* 29 m nd 0.9 ± 0.6 nd 0.3 ± 0.3 nd 0.3 ± 0.3 2.9 ± 1.8 nd f = 2.616 nd f = 4.025 nd f = 5.376 f = 2.592 nd df = 12, 129 nd df = 12, 129 nd df = 12, 129 df = 12, 129 nd p = 0.004 nd p < 0.001 nd p < 0.001 p = 0.004 (q3) 2004 34 prn nd 15.6 ± 5.6 nd 5.2 ± 1.3 nd 1.3 ± 1.1 nd 35 x nd 1.6 ± 0.9 nd 31.2 ± 9.1* nd 0.9 ± 0.8 0.0 ± 0.0 35 x nd 64.2 ± 30.0* nd 0.5 ± 0.5 nd 1.6 ± 1.0 2.9 ± 1.4 38 poim nd 2.1 ± 1.0 nd 1.0 ± 1.0 nd 0.6 ± 0.6 1.1 ± 0.7 nd f = 3.676 nd f = 7.092 nd f = 0.643 f = 2.614 nd df = 12, 129 nd df = 12, 129 nd df = 12, 129 df = 10, 109 nd p < 0.001 nd p < 0.001 nd p < 0.802 p < 0.007 (q4) 2004 46 m nd 0.7 ± 0.7 nd 0.4 ± 0.3 nd 3.9 ± 1.9 3.2 ± 1.2 49 pol nd 13.9 ± 17.6 nd 24.3 ± 11.7* nd 3.6 ± 3.5 0.0 ± 0.0 57 pol nd 19.2 ± 12.6 nd 0.0 ± 0.0 nd 0.5 ± 0.3 1.8 ± 0.9 58 x nd 1.0 ± 0.9 nd 48.6 ± 15.7* nd 188.8 ± 44.7* 100.1 ± 20.6* 60 nd 20.9 ± 4.1* nd 1.1 ± 1.0 nd 1.8 ± 1.0 44.0 ± 14.4* nd f = 3.004 nd f = 5.999 nd f = 16.331 f = 37.6 nd df = 19, 199 nd df = 19, 199 nd df = 19. 199 df = 19, 199 nd p < 0.001 nd p < 0.001 nd p < 0.001 p < 0.001 (q1) 2005 1 6.7 ± 2.9 nd nd 51.4 ± 6.1* nd nd 2.9 ± 1.1* 2 x 2.0 ± 1.0 nd nd 22.2 ± 5.4* nd nd 0.1 ± 0.1 3 1.3 ± 0.7 nd nd 27.4 ± 6.7* nd nd 1.2 ± 0.6 4 poim 9.4 ± 6.3 nd nd 6.3 ± 5.2 nd nd 0.0 ± 0.0 6 14.2 ± 5.2* nd nd 21.8 ± 4.7* nd nd 0.2 ± 0.2 10 poim 0.3 ± 0.2 nd nd 3.4 ± 2.7 nd nd 0.0 ± 0.0 11 9.3 ± 2.1 nd nd 19.8 ± 2.8* nd nd 0.8 ± 0.3 14 prn 0.0 ± 0.0 nd nd 5.7 ± 2.9 nd nd 0.2 ± 0.2 f = 9.295 nd nd f = 13.427 nd nd f = 4.001 df = 14, 149 nd nd df = 14, 149 nd nd df = 14, 149 (table continues) sociobiology 60(1): 77-95 (2013) 89 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. p < 0.001 nd nd p < 0.001 nd nd p < 0.001 (q2) 2005 19 2.5 ± 1.2 nd nd 35.1 ± 14.4* nd nd 2.2 ± 1.1 20 pol 0.6 ± 0.6 nd nd 0.8 ± 0.6 nd nd 0.1 ± 0.1 21 polm 3.2 ± 1.8 nd nd 25.5 ± 12.0 nd nd 0.1 ± 0.1 25 5.6 ± 1.7 nd nd 56.9 ± 11.2* nd nd 0.1 ± 0.1 29 m 1.8 ± 1.8 nd nd 1.5 ± 1.0 nd nd 1.0 ± 1.0 f = 2.170 nd nd f = 7.645 nd nd f = 1.096 df = 12, 129 nd nd df = 12, 129 nd nd df = 12, 129 p = 0.017 nd nd p < 0.001 nd nd p = 0.370 (q3) 2005 34 prn 10.2 ± 3.3 nd nd 4.3 ± 1.5 nd nd 0.1 ± 0.1 35 x 48.5 ± 11.3* nd nd 7.2 ± 5.6 nd nd 0.0 ± 0.0 38 pol 3.6 ± 2.2 nd nd 0.3 ± 0.2 nd nd 0.1 ± 0.1 f = 10.805 nd nd f = 0.784 nd nd f = 0.613 df = 10, 109 nd nd df = 11, 119 nd nd df = 9, 99 p < 0.001 nd nd p = 0.656 nd nd p = 0.613 (q4) 2005 43 0.8 ± 0.6 nd nd 53.9 ± 11.1* nd nd 0.6 ± 0.3 49 pol 13.9 ± 5.2 nd nd 4.7 ± 1.6 nd nd 0.0 ± 0.0 57 pol 1.7 ± 0.8 nd nd 1.3 ± 0.6 nd nd 0.1 ± 0.1 58 x 58.6 ± 30.4* nd nd 25.8 ± 6.8 nd nd 1.9 ± 0.6 59 33.1 ± 16.4 nd nd 46.2 ± 18.3* nd nd 0.8 ± 0.7 f = 3.264 nd nd f = 7.646 nd nd f = 37.6 df = 19, 199 nd nd df = 19, 199 nd nd df = 19, 199 p < 0.001 nd nd p < 0.001 nd nd p = 0.078 (q1) 2006 1 nd 107.8 ± 14.2* nd 147.9 ± 7.7* nd 63.4 ± 9.3* nd 3 nd 43.9 ± 12.2* nd 18.6 ± 11.9 nd 23.9 ± 4.1 nd 4 pol nd 2.9 ± 2.3 nd 1.2 ± 1.1 nd 3.5 ± 2.3 nd 10 polm nd 0.9 ± 0.6 nd 0.1 ± 0.1 nd 3.4 ± 1.5 nd 11 nd 72.0 ± 8.8* nd 227.7 ± 14.3* nd 58.7 ± 20.9* nd 14 prn nd 13.2 ± 4.0 nd 4.0 ± 3.4 nd 4.8 ± 2.1 nd 15 nd 50.8 ± 20.4* nd 2.0 ± 1.2 nd 0.7 ± 0.5 nd nd f = 17.648 nd f = 24.5 nd f = 10.519 nd nd df = 13, 139 nd df = 13, 139 nd df = 13, 139 nd nd p < 0.001 nd p < 0.001 nd p < 0.001 nd (q2) 2006 19 nd 1.7 ± 0.7 nd 74.5 ± 16.2* nd 21.0 ± 8.5 nd 20 pol nd 13.3 ± 6.3 nd 7.2 ± 42.4 nd 7.2 ± 3.1 nd 21 polm nd 1.8 ± 1.3 nd 19.1 ± 7.3 nd 6.8 ± 3.8 nd 23 nd 18.0 ± 6.0* nd 1.8 ± 0.9 nd 1.9 ± 1.3 nd 25 nd 0.3 ± 0.2 nd 5.2 ± 2.9 nd 69.8 ± 13.0* nd 26 x nd 14.1 ± 1.9 nd 9.0 ± 3.0 nd 74.1 ± 13.0* nd 29 m nd 0.1 ± 0.1 nd 2.8 ± 1.4 nd 3.6 ± 1.8 nd nd f = 4.287 nd f = 12.436 nd f = 28.9 nd (table continues) w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus90 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. nd df = 11, 119 nd df = 11, 119 nd df = 11, 19 nd nd p < 0.001 nd p < 0.001 nd p < 0.001 nd (q3) 2006 34 prn nd 2.6 ± 1.0 nd 0.6 ± 0.5 nd 7.9 ± 2.3 5.2±4.6 36 x nd 1.8 ± 0.8 nd 48.5 ± 12.8* nd 7.3 ± 2.9 nd 38 polm nd 0.0 ± 0.0 nd 5.7 ± 3.0 nd 2.3 ± 1.5 nd 39 nd 32.0 ± 7.7* nd 17.9 ± 5.6 nd 7.4 ± 5.0 nd 41 nd 0.9 ± 0.6 nd 5.6 ± 3.0 nd 40.3 ± 25.8* nd nd f = 11.210 nd f = 5.445 nd f= 28.9 nd nd df = 10, 109 nd df = 10, 109 nd df = 10, 109 nd nd p < 0.001 nd p < 0.001 nd p = 0.037 nd (q4) 2006 43 nd 15.2 ±3.9 nd 175.8 ± 12.7* nd 17.0 ± 5.6 nd 46 m nd 8.7± 1.4 nd nd nd 1.7 ± 1.2 nd 47 nd 153.5 ± 40.8* nd 23.8 ± 7.5 nd 22.1 ± 10.0 nd 49 pol nd 0.7 ± 0.5 nd 12.4 ± 5.5 nd 21.3 ± 13.6 nd 57 pol nd 10.4±6.2 nd 2.3 1.3 nd 3.6 2.1 nd 58 x nd 207.4±24.1 nd 394.2 74.5* nd nd nd 59 nd f=18.348 nd 41.6 21.8 nd nd nd nd df=19.199 nd df = 18, 189 nd df = 16, 169 nd nd p<0.001 nd p < 0.001 nd p = 0.048 nd (q1) 2007 1 17.4 ±6.6* 2.8 ± 2.3 22.5 ± 5.6 5.5 ± 4.5 6.7±2.4 2.8 ± 2.3 6.1 ± 3.0 3 19.5 ±6.9* 4.7 ± 3.0 17.8 ± 11.3 3.2 ± 2.0 4.1±1.7 34.7 ± 5.8* 8.2 ± 5.1 4 polm 4.1 ±3.2 5.3 ± 73.8 20.9 ± 7.7 32.1 ± 12.6* 2.2±1.7 31.5 ± 8.9* 11.2 ± 3.6 5 0.6 ± 0.5 33.6 ± 14.1* 18.2 ± 9.5 13.3 ± 4.1 3.0 ± 2.8 6.0 ± 3.2 18.1 ± 4.8 6 0.0 ± 0.0 4.0 ± 1.1 9.5 ± 5.3 5.2 ± 2.5 6.1 ± 2.8 2.1 ± 1.4 33.5 ± 19.3* 10 polm 2.5 ± 0.9 16.7 ± 8.6 8.2 ± 3.2 4.5 ± 2.3 10.5 ± 12.9 1.7 ± 1.7 2.8 ± 2.0 11 11.9 ± 1.7 41.9 ± 4.2* 36.7 ± 4.5* 46.6 ± 7.4* 86.9 ± 16.9* 37.5 ± 4.6* 28.8 ± 3.9 12 0.6 ± 0.6 0.7 ± 0.6 36.4 ± 4.4* 1.4 ± 1.0 10.5 ± 2.8 0.0 ± 0.0 3.6 ± 2.1 14 prn 0.0 ± 0.0 1.3 ± 1.0 1.6 ± 0.8 0.1 ± 0.1 2.9 ± 1.5 3.1 ± 1.0 22.5 ± 7.5 f = 3.776 f = 4.990 f = 2.729 f = 8.748 f = 19.999 f = 17.827 f = 2.527 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 p < 0.001 p < 0.001 p = 0.002 p < 0.001 p < 0.001 p < 0.001 p = 0.004 (q2) 2007 19 82.6 ± 27.7* 18.8 ± 9.4 34.4 ± 9.9* 63.9 ± 17.5* 43.6 ± 12.4* 54.3 ± 12.1 100.6 ± 20.7* 20 pol 6.1 ± 1.8 1.2 ± 1.1 2.5 ± 1.8 11.4 ± 5.1 25.5 ± 7.2 nd 18.7 ± 6.0 21 polm 0.7 ± 0.7 13.2 ± 7.4 0.8 ± 0.5 18.6 ± 9.9 30.3 ± 16.6 8.6 ± 4.3 5.8 ± 3.9 25 16.2 ± 5.8 9.8 ± 5.0 1.9 ± 1.5 63.9 ± 17.5* 2.8 ± 2.3 5.9 ± 2.6 7.4 ± 3.9 27 6.5 ± 4.9 6.2 ± 2.1 6.3 ± 2.7 8.1 ± 2.5 236.1 ± 13.2* 95.0 ± 6.0* 71.1 ± 6.3* 28 4.5 ± 2.1 7.4 ± 5.2 6.4 ± 2.7 15.1 ± 5.1 4.6 ± 1.5 15.1 ± 6.8 93.2 ± 5.9* 29 m 6.9 ± 3.2 4.3 ± 2.5 9.2 ± 4.4 6.0 ± 5.1 2.7 ± 1.7 15.6 ± 7.6 7.5 ± 4.4 f = 24.6 f = 1.314 f = 32.4 f = 7.768 f = 77.567 f = 20.832 f = 20.238 df = 10, 109 df = 10, 104 df = 10, 109 df = 10, 109 df = 10, 109 df = 9, 99 df = 10, 109 p < 0.001 p = 0.234 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q3) 2007 34 prn 12.0 ± 4.5* 18.4 ± 8.7 28.2 ± 5.0* 9.2 ± 5.7 11.5 ± 5.3 75.9 ± 9.9* 5.2 ± 4.6 (table continues) sociobiology 60(1): 77-95 (2013) 91 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. 38 polm 4.7 ± 1.5 1.1 ± 0.8 6.7 ± 2.3 5.2 ± 3.2 1.9 ± 0.9 1.1 ± 1.1 0.0 ± 0.0 39 13.1 ± 3.4* 27.3 ± 9.5 26.5 ± 10.2* 21.4 ± 7.6 14.0 ± 4.7 50.9 ± 16.8 7.8 ± 3.6 f = 4.327 f = 24.6 f = 5.092 f = 2.289 f = 2.440 f = 10.214 f = 0.629 df = 8, 89 df = 7, 79 df = 7, 29 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 p < 0.001 p = 0.138 p < 0.001 p = 0.037 p = 0.027 p < 0.001 p = 0.731 (q4) 2007 45 0.8 ± 0.6 3.2 ± 2.2 4.5 ± 2.3 27.2 ± 13.7 2.4 ± 1.7 1.2 ± 0.9 44.1 ± 18.6* 46 m 1.8 ± 1.4 0.4 ± 0.4 1.1 ± 1.1 2.2 ± 2.2 9.5 ± 5.7 3.3 ± 2.2 8.4 ± 5.4 47 30.6 ± 14.7* 33.6 ± 14.1* 15.4 ± 10.8 4.3 ± 2.7 11.0 ± 4.4 13.0 ± 8.6 29.9 ± 12.8 49 pol nd 2.1 ± 2.0 3.9 ± 3.2 2.4 ± 2.1 1.9 ± 1.1 3.5 ± 1.4 1.7 ± 1.7 50 nd 18.6 ± 9.2 0.4 ± 0.3 4.6 ± 2.9 2.7 ± 1.0 29.0 ± 13.0* 1.0 ± 0.9 51 6.5 ± 2.8 2.9 ± 1.0 9.9 ± 3.7 49.5 ± 16.0* 2.3 ± 1.3 3.9 ± 1.2 2.0 ± 1.4 54 1.0 ± 0.8 9.7 ± 5.0 2.1 ± 1.1 65.6 ± 16.9* 28.0 ± 19.0 2.9 ± 1.3 8.6 ± 4.1 55 46.4 ± 12.1* 15.3 ± 14.2 5.6 ± 2.9 0.5 ± 0.5 1.0 ± 0.6 1.5 ± 0.7 5.4 ± 2.9 56 2.3 ± 0.8 10.7 ± 2.4 3.0 ± 2.3 14.1 ± 6.8 52.0 ± 10.8* 21.2 ± 11.1 1.7 ± 0.8 57 pol 3.2 ± 1.4 0.4 ± 0.4 2.3 ± 1.7 3.2 ± 2.0 4.9 ± 2.9 1.5 ± 1.5 0.1 ± 0.1 59 36.9 ± 6.4* 4.9 ± 2.1 11.1 ± 3.8 2.4 ± 1.5 32.4 ± 9.8 0.1 ± 0.1 5.9 ± 4.2 61 8.5 ± 2.6 37.3 ± 13.1* 32.0 ± 9.6* 8.7 ± 3.5 42.4 ± 5.4* 66.1 ± 8.3* 152.9 ± 11.1* f = 6.597 f = 2.885 f = 2.439 f = 5.042 f = 23.992 f = 8.440 f = 19.854 df = 15, 159 df = 17, 179 df = 17, 179 df = 17, 179 df = 17, 179 df = 17, 179 df = 17, 179 p < 0.001 p < 0.001 p = 0.002 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q1) 2008 30.8 ± 11.4 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 2.7 ± 1.8 29.0 ± 8.8* 4 polm 1.6 ± 0.9 30.7 ± 13.0* 2.5 ± 1.2 9.7 ± 4.4 3.7 ± 2.0 4.3 ± 5.2 0.0 ± 0.0 10 polm 6.5 ± 4.4 2.2 ± 1.8 2.8 ± 1.3 1.7 ± 1.3 1.8 ± 1.7 4.7 ± 2.5 5.7 ± 5.7 11 60.2 ± 28.2* 25.9 ± 6.7* 33.2 ± 3.9* 13.8 ± 9.2 51.4 ± 9.4* 52.0 ± 21.1* 52.2 ± 18.1* 14 prn 0.0 ± 0.0 2.4 ± 1.3 20.0 ± 12.4 0.0 ± 0.0 1.0 ± 0.7 nd 0.2 ± 0.2 f = 4.087 f = 5.043 f = 4.493 f = 1.624 f = 7.594 f = 4.696 f = 5.544 df = 12, 129 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 11, 119 df = 13, 139 p < 0.001 p < 0.001 p < 0.001 p = 0.087 p < 0.001 p < 0.001 p < 0.001 (q2) 2008 19 27.7 ± 4.7* 17.2 ± 4.8 108.1 ± 13.0* 49.3 ± 19.2* 242.1 ± 10.6* 84.2 ± 11.4* 41.7 ± 16.0 20 pol 1.3 ± 0.8 1.2 ± 0.6 0.7 ± 0.5 0.2 ± 0.2 6.3 ± 5.1 3.0 ± 1.2 7.6 ± 5.7 21 polm 2.8 ± 1.9 5.7 ± 3.9 21.1 ± 13.8 0.0 ± 0.0 0.0 ± 0.0 2.2 ± 1.0 7.3 ± 3.7 25 19.4 ± 6.1* 0.0 ± 0.0 7.4 ± 6.7 0.2 ± 0.2 22.5 ± 15.8 1.9 ± 1.1 17.6 ± 7.2 28 50.5 ± 9.0* nd 56.0 ± 14.1* 58.5 ± 12.8* 154.9 ± 19.0* 9.2 ± 2.2 19.5 ± 3.9 29 m 2.7 ± 1.5 91.3 ± 17.7* 2.2 ± 1.1 0.2 ± 0.2 67.3 ± 127.3 4.2 ± 1.8 1.2 ± 1.1 f = 15.581 f = 13.893 f = 20.604 f = 9.607 f = 26.301 f = 36.590 f = 1.851 df = 10, 109 df = 9, 99 df = 10, 109 df = 10, 109 df = 10, 109 df = 9, 99 df = 10, 109 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.061 (q3) 2008 32 1.9 ± 0.9 11.0 ± 3.0 17.9 ± 6.4 0.0 ± 0.0 3.3 ± 1.8 41.4 ± 7.9* 41.9 ± 13.7* 34 prn 15.4 ± 7.1 13.5 ± 3.4 31.0 ± 9.0 0.1 ± 0.1 5.0 ± 2.9 8.9 ± 4.5 30.2 ± 17.6 37 2.0 ± 1.7 4.5 ± 2.2 52.1 ± 19.4* 0.9 ± 0.5* 41.2 ± 16.9* 9.4 ± 7.2 0.0 ± 0.0 38 poim 2.9 ± 1.0 5.1 ± 4.6 6.9 ± 4.6 0.0 ± 0.0 7.1 ± 5.3 4.6 ± 2.1 1.4 ± 0.5 f = 2.219 f = 1.453 f = 2.576 f = 2.615 f = 3.716 f = 4.827 f = 4.297 (table continues) w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus92 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 p = 0.042 p = 0.198 p = 0.020 p = 0.018 p = 0.002 p < 0.001 p < 0.001 (q4) 2008 43 46.1 ± 15.8* 7.9 ± 4.6 6.3 ± 4.3 8.9 ± 5.4 9.9 ± 5.2 4.3 ± 2.9 7.5 ± 4.3 45 3.8 ± 3.1 1.1 ± 1.1 0.0 ± 0.0 4.2 ± 2.5 56.1 ± 8.5* 8.8 ± 3.0 0.0 ± 0.0 46 m 0.0 ± 0.0 0.3 ± 0.3 5.6 ± 2.7 0.6 ± 0.5 0.0 ± 0.0 0.7 ± 0.7 24.2 ± 8.5 47 0.0 ± 0.0 14.2 ± 5.6 23.9 ± 14.0* 14.0 ± 5.6 11.2 ± 3.0 23.8 ± 6.6 39.7 ± 17.8* 49 pol 2.0 ± 1.0 6.2 ± 5.3 9.2 ± 5.9 1.2 ± 0.6 17.5 ± 8.3 20.7 ± 8.7 24.7 ± 8.4 57 pol 24.3 ± 6.5 1.0 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 7.3 ± 2.1 0.0 ± 0.0 59 4.2 ± 2.3 17.8 ± 9.2 0.0 ± 0.0 1.9 ± 1.1 2.0 ± 1.3 45.0 ± 8.7* 3.9 ± 1.3 61 13.3 ± 7.9 18.1 ± 6.6 8.8 ± 3.2 52.3 ± 12.2* 48.7 ± 9.5* 24.4 ± 9.1 9.5 ± 4.7 f = 3.729 f = 1.841 f = 1.866 f = 8.719 f = 14.077 f = 5.262 f = 3.596 df = 17, 179 df = 17, 179 df = 17, 179 df = 16, 169 df = 16, 169 df = 17, 179 df = 17, 179 p < 0.001 p = 0.027 p = 0.024 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q1) 2009 4 polm 3.8 ± 1.2 1.5 ± 01.4 0.0 ± 0.0 2.4 ± 1.6 3.9 ± 2.2 0.0 ± 0.0 0.6 ± 0.5 9 18.6 ± 5.8* 25.1 ± 10.7* 4.0 ± 2.1 1.1 ± 0.7 12.9 ± 5.4 0.8 ± 0.8 0.0 ± 0.0 10 polm 0.1 ± 0.1 0.2 ± 0.2 0.6 ± 0.6 3.0 ± 2.4 0.7 ± 0.7 0.4 ± 0.4 0.4 ± 0.4 11 23.6 ± 6.5* 13.1 ± 3.2 62.2 ± 22.2* 15.4 ± 6.3 138.6 ± 8.6* 268.8 ± 54.4* 75.3 ± 18.2* 14 prn 1.9 ± 1.1 0.7 ± 0.4 0.3 ± 0.2 79.5 ± 9.9* 56.5 ± 21.2* 7.6 ± 6.5 26.7 ± 6.3* 15 4.1 ± 1.9 1.6 ± 0.6 1.1 ± 0.5 12.6 ± 4.8 72.1 ± 17.4* 0.3 ± 0.3 0.1 ± 0.1 f= 6.618 f= 5.206 f= 7.215 f= 35.891 f= 22.590 f= 23.800 f= 15.376 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 df = 13, 139 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q2) 2009 19 106.8 ± 30.5* 123.5 ± 30.5* 108.6 ± 29.5* 33.1 ± 5.1* 151.9 ± 16.9* 24.3 ± 10.1* 26.8 ± 17.8* 20 pol 16.1 ± 4.8 32.1 ± 17.2 1.4 ± 1.2 14.9 ± 5.3 9.8 ± 3.4 27.9 ± 12.9* 0.6 ± 0.5 21 polm 0.1 ± 0.1 1.9 ± 1.3 1.6 ± 1.1 1.9 ± 0.8 0.2 ± 0.2 0.0 ± 0.0 0.8 ± 0.5 29 m 0.0 ± 0.0 2.1 ± 1.6 5.4 ± 1.8 0.8 ± 0.6 1.5 ± 1.4 0.0 ± 0.0 0.6 ± 0.3 f = 10.979 f = 12.247 f = 11.490 f = 6.681 f = 64.455 f = 4.298 f = 2.150 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 9, 99 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.033 (q3) 2009 32 4.1 ± 1.1 25.6 ± 12.0* 2.7 ± 1.2 0.2 ± 0.2 0.0 ± 0.0 1.0 ± 0.9 0.5 ± 0.5 34 prn 1.6 ± 1.1 5.1 ± 1.4 0.5 ± 0.3 5.9 ± 2.3 0.4 ± 0.4 17.6 ± 9.5* 10.1 ± 6.5 38 polm 9.7 ± 6.3 16.1 ± 3.2 4.4 ± 2.6 0.8 ± 0.8 20.2 ± 4.6* 22.5 ± 2.3* 60.5 ± 14.1* 40 1.0 ± 0.6 15.7 ± 4.6 7.4 ± 5.1 14.8 ± 3.6* 17.9 ± 7.5* 1.3 ± 0.7 0.3 ± 0.2 f = 2.095 f = 2.665 f = 1.419 f = 9.686 f = 7.052 f = 5.448 f = 14.725 df = 7, 79 df = 6, 69 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 p = 0.055 p = 0.023 p = 0.211 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q4) 2009 43 4.0 ± 1.6 0.6 ± 0.5 1.1 ± 0.8 2.7 ± 2.7 5.4 ± 3.6 31.7 ± 10.1* 0.5 ± 0.2 44 1.1 ± 0.8 3.1 ± 2.1 1.7 ± 1.2 14.7 ± 5.5* 0.0 ± 0.0 1.5 ± 1.5 0.0 ± 0.0 46 m nd 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.8 ± 1.8 1.2 ± 0.8 0.1 ± 0.1 47 5.1 ± 2.0 13.8 ± 8.1* nd 6.9 ± 3.8 10.9 ± 5.1* 3.4 ± 1.8 0.6 ± 0.6 48 0.0 ± 0.0 1.4 ± 1.0 2.5 ± 1.4 0.5 ± 0.4 14.8 ± 4.3* 8.5 ± 2.7 0.1 ± 0.1 (table continues) sociobiology 60(1): 77-95 (2013) 93 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. 49 pol 0.6 ± 0.6 2.4 ± 0.9 4.3 ± 2.6 1.4 ± 0.7 1.2 ± 1.0 0.8 ± 0.6 1.3 ± 1.1 54 1.9 ± 1.3 1.9 ± 0.7 11.0 ± 3.6* 0.5 ± 0.4 0.8 ± 0.5 0.0 ± 0.0 0.6 ± 0.6 57 pol 0.2 ± 0.1 0.9 ± 0.9 0.0 ± 0.0 0.4 ± 0.3 2.9 ± 2.2 0.0 ± 0.0 0.3 ± 0.3 61 25.2 ± 5.5* 3.8 ± 1.8 nd nd nd nd nd f = 14.125 f = 2.264 f = 3.780 f = 3.682 f = 4.340 f = 7.566 f = 0.506 df = 16, 169 df = 17, 179 df = 15, 159 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 p < 0.001 p = 0.004 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.941 (q1) 2010 1 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 87.6 ± 6.3* 89.2 ± 2.9* 14.4 ± 1.8* 47.5 ± 8.3* 4 polm 0.3 ± 0.3 0.3 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 3.9 ± 2.4 0.0 ± 0.0 0.0 ± 0.0 6 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.2 1.9 ± 1.1 111.8 ± 7.4* 1.2 ± 1.0 0.0 ± 0.0 7 0.0 ± 0.0 0.0 ± 0.0 7.8 ± 2.6* 23.9 ± 3.9* 26.3 ± 4.8* 0.6 ± 0.4 0.0 ± 0.0 10 polm 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.3 0.2 ± 0.1 0.0 ± 0.0 11 2.6 ± 1.9 0.2 ± 0.2 19.0 ± 3.3* nd nd nd nd 12 0.1 ± 0.1 0.3 ± 0.3 1.5 ± 0.9 2.0 ± 1.0 0.2 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 13 0.8 ± 0.7 0.0 ± 0.0 0.6 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 14 n 0.8 ± 0.8 0.0 ± 0.0 3.8 ± 1.3 16.2 ± 3.7* 10.7 ± 1.8 0.5 ± 0.5 0.0 ± 0.0 15 31.0 ± 15.5* 0.5 ± 0.4 0.4 ± 0.3 0.4 ± 0.4 0.7 ± 0.3 0.5 ± 0.3 0.0 ± 0.0 f = 3.814 f = 0.801 f = 18.781 f = 109.855 f = 184.080 f = 35.529 f = 32.665 df = 13, 139 df = 13, 139 df = 13, 139 df = 12, 129 df = 12, 129 df = 12, 129 df = 12, 129 p < 0.001 p = 0.695 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q2) 2010 19 0.3 ± 0.3 0.4 ± 0.3 3.1 ± 1.9* 20.8 ± 10.4* 14.3 ± 4.0* 14.7 ± 2.6* 14.4 ± 6.2* 20 pol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 21 polm 0.0 ± 0.0 1.1 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 29 0.0 ± 0.0 0.8 ± 0.8 1.1 ± 0.9 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 f = 0.750 f = 1.255 f = 2.390 f = 3.876 f = 12.770 f = 31.982 f = 5.519 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 p = 0.676 p = 0.267 p = 0.014 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q3) 2010 30 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 193.0 ± 15.6* 234.2 ± 4.7* 376.7 ± 14.9* 229.0 ± 15.8* 32 0.0 ± 0.0 0.4 ± 0.4 0.0 ± 0.0 54.8 ± 4.7* 92.1 ± 2.9* 179.9 ± 18.8* 94.7 ± 8.8* 34 n 0.0 ± 0.0 0.2 ± 0.2 3.9 ± 3.2 26.5 ± 4.8* 20.6 ± 2.6* 15.3 ± 5.3 18.0 ± 3.0 37 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.5 1.2 ± 0.8 0.0 ± 0.0 38 polm 0.1 ± 0.1 0.0 ± 0.0 1.2 ±1.1 0.1 ±0.1 1.5 ±1.0 3.7 ±2.1 0.0 ±0.0 39 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 40 0.4 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3.8 ± 3.1 0.0 ± 0.0 0.0 ± 0.0 f=0.857 f = 0.905 f=1.456 f=125.154 f=1139.984 f=249.817 f = 160.770 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 p=0.544 p = 0.507 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q4) 43 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 25.5 ± 5.4* 17.4 ± 3.9* 88.6 ± 8.0* 3.8 ± 1.5* 46 m 0.9 ± 0.9 0.0 ± 0.0 0.3 ± 0.2 0.0 ± 0.0 0.4 ± 0.3 0.0 ± 0.0 nd 47 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 12.9 ± 2.4* 20.4 ± 2.9* 2.6 ± 1.5* 49 pol 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 54 0.0 ± 0.0 0.3 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 24.0 ± 9.2 0.0 ± 0.0 (table continues) w. osbrink, m. cornelius acoustic evaluation of trees for c. formosanus94 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. 57 pol 0.3 ± 0.3 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 f = 0.645 f = 0.673 f = 2.002 f = 21.858 f = 20.275 f = 45.598 f = 4.496 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 15, 159 p = 0.843 p = 0.817 p = 0.016 p < 0.001 p < 0.001 p < 0.001 p < 0.001 (q1) 2011 4mpol 0.4 ± 0.3 2.0 ± 1.3 1.0 ± 0.8 m 0.4 ± 0.2 1.3 ± 0.8 5.0 ± 3.9 0.3 ± 0.2 6 0.7 ± 0.5 0.3 ± 0.3 0.0 ± 0.0 0.3 ± 0.3 4.2 ± 1.7* 0.8 ± 0.8 0.0 ± 0.0 10 mpol 0.2 ± 0.1 0.0 ± 0.0 0.3 ± 0.2 m 3.3 ± 2.3 0.0 ± 0.0 0.6 ± 0.6 0.5 ± 0.5 12 0.9 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.7 ± 0.7 0.0 ± 0.0 0.1 ± 0.1 13 0.0 ± 0.0 1.7 ± 1.4 1.2 ± 0.9 0.2 ± 0.2 0.0 ± 0.0 0.1 ± 0.1 0.0 ± 0.0 14 n 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.3 0.3 ± 0.3 1.9 ± 1.2 0.2 ± 0.2 15 0.1 ± 0.1 0.2 ± 0.2 2.0 ± 1.8 0.2 ± 0.2 1.8 ± 1.6 1.9 ± 1.3 1.5 ± 1.4 f = 1.798 f = 1.164 f = 0.932 f = 1.504 f = 2.626 f = 1.258 f = 0.895 df = 12, 129 df = 12, 129 df = 12, 129 df = 12, 129 df = 12, 129 df = 12, 129 df = 12, 129 p = 0.056 p = 0.317 p = 0.518 p = 0.132 p = 0.004 p = 0.253 p = 0.554 (q2) 2011 17 0.1 ± 0.1 0.6 ± 0.6 3.0 ± 2.1 0.1 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.4 19 14.9 ± 3.5* 10.8 ± 5.8* 0.0 ± 0.0 10.8 ± 2.5* 4.7 ± 2.1 6.4 ± 1.9 5.0 ± 2.2 20 pol 0.6 ± 0.4 0.0 ± 0.0 0.5 ± 0.5 1.4 ± 1.2 0.1 ± 0.1 2.8 ± 1.7 0.5 ± 0.5 21 mpol 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 m 1.8 ± 1.7 0.7 ± 0.7 5.7 ± 3.1 1.2 ± 1.2 22 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.5 13.7 ± 4.9* 1.1 ± 1.1 0.0 ± 0.0 24 0.2 ± 0.2 0.8 ± 0.8 0.9 ± 0.6 0.9 ± 0.6 9.6 ± 3.4 13.6 ± 4.9* 7.8 ± 5.5 25 0.3 ± 0.3 2.2 ± 1.3 0.0 ± 0. 0.0 ± 0.0 12.6 ± 3.8* 0.0 ± 0.0 0.5 ± 0.5 27 0.0 ± 0.0 0.0 ± 0.0 1.1 ± 0.5 16.8 ± 2.5* 6.1 ± 1.4 0.2 ± 0.2 0.1 ± 0.1 28 0.0 ± 0.0 0.5 ± 0.4 3.3 ± 1.5 0.6 ± 0.3 11.8 ± 3.1* 0.0 ± 0.0 0.2 ± 0.1 29 m 0.0 ± 0.0 2.4 ± 1.1 1.5 ± 0.8 m 4.8 ± 2.6 0.4 ± 0.2 3.2 ± 2.2 2.0 ± 1.1 f = 17.039 f = 2.942 f = 2.029 f = 12.361 f = 5.224 f = 3.276 f = 1.780 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 df = 10, 109 p < 0.001 p = 0.003 p = 0.038 p < 0.001 p < 0.001 p < 0.001 p = 0.074 (q3) 2011 30 55.3±5.2* 226.4±18.8* 27.1±3.8* 349.1±20.9* 585.8±16.8* 66.2±10.5* 1.5±1.3 32 59.4 ± 4.7* 203.3 ± 16.2* 20.7 ± 2.9* 474.4 ± 19.3* 60.7 ± 67.6* 0.2 ± 0.2 3.2 ± 2.6 34 n 2.5 ± 1.2 51.1 ± 9.9* 18.7 ± 3.1* 24.7 ± 3.9* 75.7 ± 8.3* 29.0 ± 5.7* 2.8 ± 5.8 38 mpol 0.9 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 0.0 ± 0.0 1.1 ± 1.1 1.5 ± 0.9 39 0.1 ± 0.1 0.0 ± 0.0 1.0 ± 0.7 m 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.3 0.0 ± 0.0 40 4.2 ± 1.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.4 ± 0.9 0.2 ± 0.6 0.2 ± 0.2 1.0 ± 0.8 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 1.4 ± 0.9 0.1 ± 0.3 f = 98.312 f = 105.701 f = 32.291 f = 356.160 f = 802.957 f = 32.263 f = 0.941 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 df = 7, 79 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.481 (q4) 2011 43 8.3 ± 1.8* 2.5 ± 0.9 18.8 ± 2.2* 20.0 ± 3.5* 66.4 ± 6.7* 37.3 ± 12.7* 0.1 ± 0.1 45 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.4 34.1 ± 5.5* 76.8 ± 10.7* 0.0 ± 0.0 46 m 0.3 ± 0.2 0.0 ± 0.0 0.3 ± 0.2 m 0.3 ± 0.2 0.0 ± 0.0 3.1 ± 2.9 0.3 ± 0.3 47 6.1 ± 2.8* 10.4 ± 3.7* 7.4 ± 2.0* 5.2 ± 1.9 6.3 ± 2.8 47.8 ± 9.5* 8.3 ± 3.3 49 pol 4.0 ± 1.0 0.0 ± 0.0 1.2 ± 0.9 2.4 ± 0.9 0.2 ± 0.2 3.4 ± 1.7 0.2 ± 0.2 (table continues) sociobiology 60(1): 77-95 (2013) 95 table 2. mean number (± se) jackson square tree acoustical counts. tree mar. april may july aug. sept. oct. 47 6.1 ± 2.8* 10.4 ± 3.7* 7.4 ± 2.0* 5.2 ± 1.9 6.3 ± 2.8 47.8 ± 9.5* 8.3 ± 3.3 49 pol 4.0 ± 1.0 0.0 ± 0.0 1.2 ± 0.9 2.4 ± 0.9 0.2 ± 0.2 3.4 ± 1.7 0.2 ± 0.2 51 12.2 ± 0.9* 0.0 ± 0.0 2.5 ± 1.7 0.0 ± 0.0 0.7 ± 0.4 3.8 ± 2.4 87.2 ± 18.6* 52 0.0 ± 0.0 1.2 ± 0.9 0.4 ± 0.3 30.5 ± 4.1 16.5 ± 3.9* 0.7 ± 0.4 0.0 ± 0.0 55 11.7 ± 3.2* 0.0 ± 0.0 0.1 ± 0.1 10.4 ± 2.9* 12.7 ± 1.8* 0.1 ± 0.1 0.5 ± 0.5 56 1.0 ± 1.0 0.0 ± 0.0 1.0 ± 0.9 7.1 ± 2.4 1.3 ± 3.0 33.5 ± 12.9* 4.0 ± 1.78 57 pol 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.3 0.2 ± 0.2 0.0 ± 0.0 f = 11.561 f = 7.136 f = 24.224 f = 26.148 f = 50.062 f = 15.512 f = 19.783 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 df = 16, 169 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 * means significantly > 0; protected tukey test (p < 0.05). poi post imidacloprid treatment. m mud tube present in may 2011. n tree adjacent to active noviflumuron bait station. nd no data. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.2090sociobiology 65(1): 101-107 (march, 2018) special issue the use of tympanic arena as an alternative for behavioral vibroacoustic essays in termites (blattodea: isoptera) introduction communication is an important life trait of organisms, allowing the exchange of information among intraspecific, and in some cases the interception of valuable information from interspecific (evans et al., 2009; cristaldo et al., 2016; mark & rufus, 2013; šobotník et al., 2010). in termites, communication occurs basically by mechanical and chemical channels (cristaldo et al., 2015; šobotník et al., 2010; costaleonardo & haifig, 2014; bagnères & hanus, 2015). the mechanical channel is transmitted by substrate-borne vibration. when producing vibroacoustic signals, termites perform vertical and longitudinal oscillatory movements (respectively called “drumming” and “shaking”), which transmit vibrations to abstract in termites, substrate-borne vibrations play an important role in communication among nestmates. the adaptive significance of such an ability has led to an ever-increasing number of studies aimed at improving knowledge on vibroacoustic communication in these insects. such studies are commonly carried out in laboratory arenas consisting of petri dishes made of plastic or glass. however, the rigidness of such materials may limit the transmission of vibrational waves impairing accurate records of the feeble vibrations produced by termites. this is one of the reasons why such experiments must be carried out under strictly controlled conditions, using extremely sensitive equipment, usually connected to amplifiers. if, instead, arenas bear a flexible floor (hence simulating a tympanum), vibrations might not be dampened or even easily amplified, thereby overcoming the need for such a specialized setup. here we test such a hypothesis, using an accelerometer to measure and record vibrations whose intensity was tailored to mimic the feeble vibrations of a small termite species, constrictotermes cyphergaster. results support the notion that tympanic arenas portray such vibrations far more accurately than arenas made of plastic or glass. we hence recommend this type of arena as a cheap, albeit accurate, alternative in studies of vibroacoustic behaviors of termites and other insects of comparable size, especially in situations where noise is minimally controlled. these arenas, then, can be useful in conducting such studies just after termite collection in remote regions where well-equipped labs are not available. in doing so, we minimize the stress involved in transporting termites over long distances. sociobiology an international journal on social insects lf nunes1, ja roxinol1, p cristaldo2, r marinho1, o desouza1 article history edited by kleber del claro, ufu, brazil received 23 september 2017 initial acceptance 13 october 2017 final acceptance 16 october 2017 publication date 30 march 2018 keywords mechanical communication, methods, cheap apparatus. corresponding author lívia fonseca nunes universidade federal de viçosa (ufv) avenida peter henry rolfs, s/nº cep 36570-900 viçosa, mg, brasil. e-mail: livfnunes@gmail.com the substrate when the individual hits the ground and/or ceiling with its head or abdomen (howse, 1964; stuart, 1963; hager & kirchner, 2013; cristaldo et al., 2015). because substrate vibrations disseminate information quickly (hunt & richard, 2013) this pathway of communication has been reported to be important alarm signals inside and outside termite colonies (howse, 1965; cristaldo et al., 2015). it has been also demonstrated that vibroacoustic cues can be used by termites to assess food items (evans et al., 2005) and to eavesdrop their competitors and predators (evans et al., 2009; oberst et al., 2017). the undeniable adaptiveness of such an ability has boosted the amount of studies on termite vibroacoustic behavior in recent years (costa-leonardo & haifig, 2014; bagnères & hanus, 2015). 1 universidade federal de viçosa, minas gerais, brazil 2 universidade federal de sergipe, são cristóvão-se, brazil research article termites mailto:livfnunes@gmail.com lf nunes, et al. – behavioral vibroacoustic essays in termites102 vibroacoustic bioassays involving termites are commonly performed using arenas consisting of glass or plastic petri dishes (howse, 1965; hager & kirchner, 2014; cristaldo et al., 2015; oberst et al., 2017). rigid materials such as plastic and glass, however, are known to limit the transmission of vibrational waves (joyce et al., 2008). it follows that vibroacoustic bioassays using such arenas need to be carried out on anechoic conditions, using high sensitivity accelerometers connected to amplifiers in order to reveal oscillatory sequences in full detail. this could be particularly true for bioassays involving some termites which, being small, produce feeble signals. very often, however, termite collections occur in remote regions, away from well-equipped laboratories. taking termites thousands of kilometers away to where sophisticated setup is available is not always feasible, due to both biological and bureaucratic constraints. additionally, legal permits must be obtained for the transportation of live specimens (sometimes across country borders) and, on top of that, termites may get too stressed and die before the bioassay is actually run. in the absence of a sophisticated setup, an arena which mimics a tympanum seems a suitable alternative. structures known as “tympanums” consist of a flexible membrane anchored to a solid frame and whose main function is to pick up waves and transmit them as vibratory stimuli (sosa et al., 2002; errobidart et al., 2014). as opposed to rigid materials with their low transmission of waves (joyce et al., 2008), tympanums are sensitive to the intensity and frequency of the waves coming from a stimulus being, hence, a suitable flooring for arenas used in vibroacoustic bioassays. here we test the hypothesis that the high sensitivity of tympanums would pick feeble vibratory signals – equivalent to those produced by small termites – even in environments where noise is only minimally avoided. ultimately, we aim to establish an alternative protocol for vibroacustic bioassays, using an arena flooring which portrays authentic vibratory signals even under rough conditions, e.g., out of an anechoic room. materials and methods overall rationale to test our hypothesis, we connected an accelerometer sensor to distinct arenas and subjected them to a known vibratory stimulus whose intensity was equivalent to that of termites performing typical vibratory behavior. this testing stimulus was inflicted on the inner surface of the arenas’ floor, right on the spot corresponding to the external place of attachment of the accelerometer’s sensor. readings thereby obtained were compared to those from this same stimulus inflicted directly on the accelerometer’s sensor. the arena whose readings better approximated the direct readings was taken as the most viable arena for the analysis of such behavior in this group of insects, in that condition. tested arenas consisted of plastic or glass petri dishes and a homemade tympanic arena, and all assays have been carried out in a normal lab room with only minimal noise control (details are given below). the experiment was conducted in two steps: (i) we first identified the best model object to simulate termite vibrations (fig 1) and then (ii) we used the stimulus produced by this model object to compare the arenas (fig 2). the use of such a model object guaranteed that every tested arena would receive precisely the same stimulus, at the same spot, and with the same intensity. this would not be possible if we had used actual termites as they move around the arena and perform vibrations at random spots with varying intensity. fig 1. the pilot test: comparing the vibrations produced by termites on a tympanic arena with those produced by (a) a styrofoam ball, (b) a wooden stick, (c) an entomological pin. vibrations were recorded by the an accelerometer’s sensor (s) attached to the under surface of the tympanum. of those objects, only styrofoam ball produced vibrations similar to those produced by termites. it was hence concluded that termites can be modeled by styrofoam balls in this type of bioassay. fig 5 presents these results more formally. after choosing the best arena, we performed an additional test subjecting it to the object which was found most dissimilar to termites in the step “(i)” above. in doing this we checked whether the other arenas could still be useful in assays involving stronger stimuli such as those produce by bigger termites (fig 3). pilot test: validating the model object in the search of a model object that best simulated termite vibrations we tested (i) a styrofoam ball (ø = 0.5 mm), (ii) an entomological pin (number 1, ø = 0.4 mm), and (iii) a wooden stick (25 cm long; ø = 4 mm). such test is detailed at fig 1. the test consisted in comparing the intensity of the readings produced by such objects with those produced by constrictotermes cyphergaster (termitidae: nasutitermitinae) termites on the floor of an arena specially built to combine rigid and tympanic elements. such an arena consisted of the lid of plastic petri dish (ø = 53 mm) covering a piece of tracing paper kept taut by a frame. the accelerometer’s sensor was attached to the lower (external) surface of the arena’s sociobiology 65(1): 101-107 (march, 2018) special issue 103 floor. vibroacoustic stimuli were produced on the arena’s floor, allowing termites to bang their heads or by dropping the testing objects from a height of nine centimeters onto this floor. a small hole was drilled on the center of the petri dish serving as a lid, to allow objects to be dropped onto the arena’s floor. we have chosen as the model object the one whose readings recorded by the accelerometer resembled closer those readings originated from termites. a total of 40 independent trials have been conducted in the pilot test, producing 20,480 readings. each of these trials produced one vibrational profile with 512 readings, similar to the one depicted at fig 4. these correspond to 10 independent trials for each of the three objects under test plus 10 trials for termites. each of these trials corresponded to a given profile with 512 readings, similar to the one depicted at fig 4. see “statistical analyses” below for details on such measurements. fig 2. the main test using styrofoam ball: comparing the vibrations produced when such a ball was dropped directly onto the accelerometer’s sensor (s) with the vibrations produced by this same ball on (a) a tympanic arena, (b) an arena consisting of a glass petri dish, (c) an arena consisting of a plastic petri dish, all these having an accelerometer’s sensor attached to their under surface. vibrations produced on the tympanic arena did not differ from those produced directly on the sensor. it was hence concluded that losses of stimulus’ intensity due to the substrate are insignificant for tympanic arenas, confirming their suitability for this type of bioassays. fig 6 presents these results more formally. fig 3. the main test using a styrofoam ball: comparing the vibrations produced when such a stick was dropped directly onto the accelerometer’s sensor (s) with the vibrations produced by this same stick on (a) a tympanic arena, (b) an arena consisting of a glass petri dish, (c) an arena consisting of a plastic petri dish, all these having an accelerometer’s sensor attached to their under surface. vibrations produced on the tympanic arena did not differ from those produced directly on the sensor. it was hence concluded that losses of stimulus’ intensity due to the substrate are insignificant for tympanic arenas, confirming their suitability for this type of bioassays. fig 7 presents these results more formally. fig 4. a typical vibratory profile, as recorded by an accelerometer’s sensor fixed underneath an experimental arena. in order to calculate the effect of the respective arena on the accelerometer’s readings, we first extracted from this profile all values within the range mean ± 3 standard deviations, as these are too affected by residual oscillations besides those due to the treatment alone. then we summed the amplitudes corresponding to the extreme upper and lower values (“effect”) remaining in the series, to be taken as the treatment effects in the analyses. the main test: comparing arenas after selecting the model object, we proceeded to the main test which actually compared arenas (fig 2). to do so, we used two types of rigid arenas and one type of tympanic arena, each of them having the accelerometer’s sensor attached to the lower (external) surface of their floor. rigid arenas consisted of the lower part of either a plastic or a glass petri dish. tympanic arenas consisted of an embroidery hoop lined with tracing paper. embroidery hoops consist of a pair of concentric circular wooden rings which hold taut a piece of fabric, thereby helping artisans in their activities of cutting and sewing. this closely resembles a tympanum or a shallow drum, being a readily available and cheap apparatus. lf nunes, et al. – behavioral vibroacoustic essays in termites104 the tympanic arenas here described had, as their floor, a piece of tracing paper held by the concentric rings of the embroidery hoop. diameters of arenas varied as follows: plastic arenas from 49.4 to 144.4 mm, glass arenas from 42.0 to 140.0 mm, and tympanic arenas of 150.0 and 200.0 mm. the test consisted of dropping the model object onto the floor of each arena (precisely on the spot below which the sensor was attached to) and comparing the amplitude of such readings with that of the readings produced by dropping this same object directly onto the accelerometer’s sensor. the model object was dropped at the height of nine centimeters from the arenas floor or from the sensor. the arena whose readings resembled closer the readings produced by the model object directly on the sensor was defined as the best (among those tested) for vibroacoustic studies using these insects in that condition. a total of 51 independent trials have been conducted in the main tests, producing 26,111 readings. each of these trials produced one vibrational profile with 512 readings, similar to the one depicted at fig 4. these correspond to 27 trials for the main test involving styrofoam balls, conducted with three repetitions for direct stimulus on the sensor, six repetitions for tympanic arena, eight repetitions for plastic arenas and ten repetitions for glass arenas. the remaining 24 trials have been conducted with a wooden stick, including two repetitions for direct stimulus on the sensor, four repetitions for tympanic arena, eight repetitions for plastic arenas and ten repetitions for glass arenas. technical specifications in order to minimize noise and vibrations from human trafficking and other activities in nearby laboratories, all testing setups were mounted into a wooden box lined with a five centimeters layer of glass wool. arenas were placed inside this box over a pair of egg crate foam strips laying on a styrofoam hollowed cubic structure. vibratory stimuli have been measured and recorded using an usb accelerometer (gulf coast data concepts, llc tm model x2-2 logger) equipped with a kionix kxrb5-2050 tm sensor at 2.5 volts, which results in a sensitivity factor of 500 mv/g. the sensor registers the readings in three axes (x, y and z) separately. we have used only the values recorded for vertical axis z. to facilitate the essays, the sensor was removed from the accelerometer’s case while keeping it connected to the recording unit by electric wires. in doing so, we could attach this sensor directly to the external bottom surface of the arenas. focal species we used soldier and workers of constrictotermes cyphergaster, a neotropical termite species common in brazil, paraguay, bolivia and northern argentina (mathews, 1977). vibroacoustic behavior is one of the alarm responses known to this species’ defense arsenal (cristaldo et al., 2015). it consists of vertical and longitudinal oscillatory movements performed by a termite individual which result in alternate banging of its head and abdomen on the substrate, thereby transmitting vibrations which are interpreted as alarm by the nestmates. both soldiers and workers exhibit this mechanical alarm behavior. statistical analyses statistical analyses proceeded in r using generalized linear modeling (glm) under normal errors. model simplification was performed by stepwise deletion with f tests, lumping together treatment levels as long as these did not provoke significant changes (p<0.05) in the model (crawley, 2005). residual analyses confirmed the choice of the error distribution. for both, pilot test and main test, the amplitude of the accelerometer’s readings in a given treatment was used as y-var. this value was obtained using the extreme values from a given reading after residual oscillations have been extracted from the corresponding oscillatory profile, as detailed in fig 4. for the pilot test, a categorical x-var representing the stimulus held four levels, each one corresponding to a given object under test (“styrofoam ball”, “pin”, “wooden stick”) or to termites themselves. we aimed here to determine which of these objects, when dropped on the arena’s floor, would produce amplitude values most similar to those produced by termites exhibiting vibroacoustic behavior. the object thereby selected was used in the main experiment. the full model for the pilot test was hence: amplitude ~ stimulus for the main experiment, a categorical x-var representing the substrate held four levels, each one corresponding to a given arena under test (“tympanum”, “plastic”, “glass”) or to the sensor of the accelerometer onto which the object was directly dropped. the amplitude of vibration is known to depend on the extension of the substrate’s free span: the larger the substrate span, the lower the amplitude. this happens because of the loss of energy during the propagation of vibratory waves along the surface, favoring small petri dishes (ø = 44 mm) over large embroidery hoops (ø = 200 mm). in order to account for such an effect, the diameter of the arenas was included as a co-variate in the model. we aimed here to determine which of these arenas would produce amplitude values most similar to those produced by the model object dropped directly onto the sensor. the arena thereby selected was defined as the best one for this type of study in that condition. the full model for the main experiment was hence: amplitude ~ substrate + diameter + substrate * diameter results pilot test: validating the model object in tests to validate the model object, termites produced the lowest average amplitude of vibrations (3.4 ± 0.93 units; mean ± s.e. ), being followed by the styrofoam ball (430.6 ± 61.07 units), the pin (11645.9 ± 641.7 units) and the wooden stick (15056.1 ± 598.2 units). a summary of these is given at figs 1 and 5. sociobiology 65(1): 101-107 (march, 2018) special issue 105 in such trials, the averaged amplitude of the vibrations produced by termites did not differ from the averaged amplitude produced by the styrofoam ball (f(1,37) = 0.118, p = 0.7332), but these differed from those produced by the pin (f(1,38) = 115.4, p < 0.0001) which in turn also differed from the amplitudes due to the wooden stick (f(1,38) = 7.7022, p = 0.0086). these results support the notion that styrofoam balls, but not the other objects, can be used as an object to model termites in the tests here conducted. sensor was affected by the type of arena (f(3,20) = 70.395, p < 8.59e-10) but not by their diameter (f(1,19) = 3.375, p = 0.084) nor by the interaction between arena type and diameter (f(2,17) = 0.263, p = 0.772). as summarized in table 1, the averaged amplitude of vibrations produced directly on the sensor did not differ from those produced on the tympanum (f(1,21) = 0.002, p = 0.963), but these differed from those produced on the plastic arena (f(1,21) = 58.503, p < 0.001) which in turn also differed from the amplitudes recorded in the glass arena (f(1,21) = 4.416, p = 0.049). in these trials, while the tympanums transmitted 100% of the stimulus, plastic arenas transmitted 38% and glass arenas transmitted 28%. these results are summarized at table 1 and fig 7, supporting the notion that the loss of a strong stimulus while negligible in tympanic arenas, was still significant when using plastic or glass arenas. in summary, considering losses of stimulus to be transmitted to the accelerometer’s sensor, tympanum is better than plastic which, in turn, is better than glass. this is true for both, weak or strong stimuli. discussion as predicted by our hypothesis, tympanic arenas have revealed themselves as an excellent alternative to either glass or plastic arenas for lab bioassays of vibroacoustic signals emitted by small termites in a condition where noise is minimally controlled. losses of such stimuli when using tympanic arenas were insignificant (table 1) to the point of not being distinguishable from stimuli inflicted directly on the accelerometer’s sensor (fig 6, table 1). the other arenas did absorb much of the stimulus, failing to transmit it accurately to the accelerometer’s sensor. it must be warned that this is not to imply that previous vibroacoustic studies on termites, using petri dishes and alike, fig 5. defining the model object to be used in the main test. vibrational amplitudes (as defined in fig 4) produced by styrofoam ball do not differ from those produced by termites, but differ from the amplitudes produced by the pin and the wooden stick. the main test in trials using styrofoam balls as model object, the average amplitude of the vibrations produced directly on the sensor was affected by the type of arena (f(3,23) = 25.289, p = 5.20e-07) but not by their diameter (f(1,22) = 1.206, p = 0.285) nor by the interaction between arena type and diameter (f(2,20) = 1.077, p = 0.360). as summarized in table 1, the averaged amplitude of vibrations produced directly on the sensor did not differ from those produced on the tympanum (f(1,24) = 0.136, p = 0.7162), but these differed from those produced on the plastic arena (f(1,24) = 15.696, p < 0.001) which in turn also differed from the amplitudes recorded in the glass arena (f(1,24) = 13.428, p = 0.0013). whereas the tympanums transmitted 94% of the stimulus, plastic arenas transmitted only 43% and glass arenas transmitted 1%. in other words, plastic and glass arenas severely dampened stimuli, hence underestimating the readings. these results are summarized at table 1 and fig 6, supporting the notion that the loss of a feeble stimulus when using tympanic arena was negligible but that was not so for plastic or glass arenas. in the additional trials involving wooden sticks, the average amplitude of the vibrations produced directly on the substrate reading % transmitted % absorbed statistical significance styrofoam ball: sensor 229.70 ± 44.2 100 0 tympanum 215.39 ± 41.5 94 6 n.s. plastic 97.72 ± 18.8 43 57 *** glass 2.14 ± 0.4 1 99 *** wooden stick: sensor 22551.5 ± 4603.3 100 0 tympanum 22645.0 ± 4622.4 100 0 n.s. plastic 8628.6 ± 1761.3 38 62 *** glass 6333.6 ± 1292.8 28 72 *** table 1. the percentage of stimulus transmitted or absorbed by the arenas’ flooring to the accelerometer’s sensor as compared to this same stimulus provoked directly onto the sensor. stimuli were produced dropping either a styrofoam ball or a wooden stick onto the sensor or onto the arenas. more statistical details are given at figs 6 and 7. lf nunes, et al. – behavioral vibroacoustic essays in termites106 would be invalid. in general, these have been conducted using a strictly controlled setup and highly sensitive equipment (e.g. cristaldo et al., 2015; oberst et al., 2017) which would certainly compensate for the rigidity of the experimental arena. what we want to show here is an alternative which, while cheap, is still highly suitable and accurate. the high sensitivity of the tympanic arena here reported seems to compensate for the absence of, e.g., an anechoic chamber or a high sensitive accelerometer. these results find support on theoretical expectations (cocroft et al., 2006; michelsen et al., 1982; miklas et al., fig 7. the main test using wooden stick: vibrations produced by a wooden stick dropped directly onto the accelerometer’s sensor did not differ from those produced onto a tympanic arena, but they did differ from those produced dropping the stick onto the floor of an arena consisting of a glass petri dish or of a plastic petri dish. fig 6. the main test using styrofoam ball: vibrations produced when such a ball was dropped directly onto the accelerometer’s sensor did not differ from those produced onto a tympanic arena, but they did differ from those produced dropping the ball onto the floor of an arena consisting of a glass petri dish or of a plastic petri dish. 2001) according to which the substrate material interferes on the transmission of the vibrational stimuli, mainly when these materials are rigid. in fact, joyce et al. (2008) found that the amplitudes of waves coming from vibrational stimuli are influenced by the substrate: in rigid materials as plastic and glass, the transmission of stimuli is lower than in flexible materials, as maize and bean leafs. because plastic and glass are denser and more rigid than tracing paper, the floor of the tympanic arena is more elastic than that of petri dish arenas. it is then expectable this latter to convey stimulus to the accelerometer’s sensor less accurately. this qualifies these tympanic arenas as a very suitable apparatus to the study of vibroacoustic signals in termites in an environment where noise is only minimally controlled. the fact, however, that these arenas were also more accurate in transmitting even stronger stimuli makes these arenas even more recommendable. as depicted in the lower panel of table 1, as well as in fig 3, vibrations produced by the wooden stick were also severely dampened by plastic (62% lost) and glass arenas (72%), but not by the tympanum (0%). this is highly surprising specially considering that the stimulus produced by this stick is about 100 times stronger than the stimulus produced by the styrofoam ball (from “sensor” lines in table 1: 22551.5/229.7=98.17). since the stimulus produced by the styrofoam ball was indistinguishable from that produced by termites (fig 1 and 5), it follows that tympanic arenas could be recommended for vibroacoustic studies even for termites much larger than c. cyphergaster. the suitability of the tympanic arenas here studied goes beyond their accuracy in transmitting stimuli to accelerometer’s sensor. being made out of embroidery hoops lined with tracing paper, these are readily available and relatively inexpensive. a single 150 mm glass petri dish would cost not less than us$ 8 while a set of five hoops, from 130 to 230 mm, can cost as little as us$ 10 (http://www. amazon.com, retrieved: 12 aug 2017). concluding, the tympanic arenas here describe may be a suitable alternative for vibrational studies on termites, especially in situations where the noise is only minimally controlled. this could be useful, for instance, to run such bioassays directly in field stations just after the termites have been collected, avoiding the stresses resulted from transporting termites over long distances to better equipped laboratories. acknowledgements many thanks to dr. octavio miramontes (institute of physics unam, mexico) for providing us the accelerometer and for alex koone (gulf coast data concepts engineer) who made the modifications in the accelerometer, necessary for the study development. we also thank dr. sidney g. alves (department of physics and mathematics ufsj) who came up with the idea of using an embroidery hoop as tympanum. this work was partially supported by cnpq, fapemig, capes. lf nunes holds a msc grant from cnpq (130781/2016sociobiology 65(1): 101-107 (march, 2018) special issue 107 9) and ja roxinol holds a phd grant from cnpq (142077/20160). o desouza holds a cnpq fellowship (305736/2013-2). this work is part of the iv symtermes community at https:// zenodo.org/communities/symtermes/?page=1&size=20, where it is identified by doi 10.5281/zenodo.969973. this is contribution # 72 from the lab of termitology at federal univ. of viçosa, brazil (http://www.isoptera.ufv.br). references bagnères, a.g. & hanus, r. 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(1963). studies on the communication of alarm in the termite zootermopsis nevadensis (hagen), isoptera. physiological zoology, 36: 85–96. doi: 10.1086/physzool. 36.1.30152740. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1035sociobiology 63(3): 889-893 (september, 2016) red imported fire ant invasion reduced the populations of two banana insect pests in south china introduction as a famous invasive pest, red imported fire ant solenopsis invicta preyed on many different kinds of invertebrates at different developmental stages including egg, larvae, pupae and adults (stiles & jones, 2001), and caused a sharply decline on populations and diversity of invertebrates and some epigeous vertebrates in its introduced areas (cook, 2003; morrison, 2002; wojcik et al., 2001). however, s. invicta also is an active and efficient predator of pests. 58%85% cotton boll weevil (fillman & sterling, 1983) and 85% tobacco budworm (mcdaniel et al., 1981) were consumed by s .invicta in investigated fields. vogt et al. (2001) showed that s. invicta preyed on about seven times more pest arthropods than beneficial arthropods in peanut field. in florida, s. invicta preyed on eggs and larvae of soybean armyworm (anticarsia gemmatalis). s. invicta also reduced population of diamondback moth and leaf beetles in collards (harvey & eubanks, 2004). accordingly, harvey and eubanks (2004) believed that s. abstract as a severe invasive pest, red imported fire ant (solenopsis invicta buren) had important effects on ecosystem of its infected areas. here, we surveyed the impact of s. invicta on populations of two banana insect pests, banana skipper (erionota torus evans) and banana lacebug (stephanitis typical distant). the results showed that influences of s. invicta on the populations of e. torus and s. typical depend on weed coverage degree of banana plantations. comparing to the areas without s. invicta, banana skipper population was reduced by 39.2%, 41.4% and 23.4% respectively, in high, moderate and low weed coverage of banana plantations with s. invicta invasion. banana lace bug population was reduced by 17.8%, 43.0% and 39.2% respectively, in high, moderate and low weed coverage of banana plantations with s. invicta invasion. sociobiology an international journal on social insects l wang, z wang, l zeng, yy lu article history edited by evandro nascimento silva, uefs, brazil received 29 march 2016 initial acceptance 07 august 2016 final acceptance 12 september 2016 publication date 25 october 2016 keywords solenopsis invicta, ecological effect, banana, erionota torus, stephanitis typical. corresponding author yongyue lu south china agricultural university 526 college of agriculture, 483 wushan road tianhe district, guangzhou, guangdong china, 510642 e-mail: luyongyue@scau.edu.cn invicta may be particularly important biological control agent in agroecosystems. banana is an important economically crop in south china. in china, the most economically damaging pests of banana are odoiporus longicollis olivier, pentalonia nigronervosa van der goot, erionota torus evans, and stephanitis typical (lu et al., 2002). e. torus is a phytophagous lepidopteran that feeds on banana plants leaves. e. torus curled up banana leaves and took them as pupation sites (lu et al., 2002). nymphs and adults of s. typical feed on phloem sap of banana plant and also are vectors of banana rosette dwarf disease which causes serious damage of banana production (zhou et al., 1993). in south china, 10-50% banana plants were infected by s. typical and e. torus in banana garden (lin et al., 2009; guo et al., 2012). although conventional pesticides still are the primarily method for these pests control, integrated pest management (ipm) and biological control are essential for the control of banana pests because many people are concerned about the pollution of the environment and food security issues. college of agriculture, south china agricultural university, guangzhou, p.r. china research article ants l wang, z wang, l zeng, yy lu – fire ant invasion reduces the populations of two banana insect pests890 since it was detected in mainland china at 2004 (zeng et al., 2005), s. invicta has been found in many habitats including banana plantation. harvey and eubanks (2004) indicated that increasing habitat complexity can enhance s. invicta efficacy and herbivore control. so, s. invicta may be a potential effective predator of banana pests because banana plants are often grown in complex, intercropping systems in china (liu et al., 2015; ke et al., 2012; chen et al., 2008). the goal of our study is to assess the effect of s. invicta on population of s. typical and e. torus. meanwhile how habitat complexity affects s. invicta predation of s. typical and e. torus was also evaluated. materials and methods experimental plot and habitat conditions the experimental plots located in the banana plantation of south china agricultural university ningxi teaching and study training center, guangzhou, china. the banana plantation (total area of 3.5 acres, and 1.3 acres selected for experiment) was newly established and was in good ventilation and less external disturbance. in this study, dwarf banana was planted which is a hybrids cultivated variety of genus musa planted widely in south china. based on weed coverage, experimental plots were divided into high weed coverage plots, moderate weed coverage plots, and low weed coverage plots. depend on our investigations (fig 1), the s. invicta infestation level of all three types of experimental plot are high degree due to the density of fire ant mounds was 7-10 per 100m2, and 100-200 workers in each bait trap in each experimental plot before experiment was conducted (wang et al., 2009). plots with high weed coverage and without s. invicta were taken as control. each type of plots was consisted of 6 small plots (2m width × 70m length), and they are separated by water drain way (fig 1). in this study, high weed coverage was defined as weed was allowed freely growth and more than 70% of plot area was covered. moderate coverage was defined as weed was partially removed periodically and 30-70% of plot area was covered. low coverage was defined as weed was rooted out often and less than 30% of plot area was covered. the microclimate, soil condition and ecological environment in all plots were basically similar, and main weed species in our study sites are cynodon dactylon, cyperus exaltatus and cirsium setosum. experimental methods s. invicta colonies in our study sites were polygyne. the abundance of s. invicta in experimental plots was surveyed once in the middle of each month during may to october in 2008 by bait traps (lu et al., 2012). meanwhile, the number of s. invicta nests was also counted in experimental plots. the survey was conducted at 10:00 am in the sunny day. the larvae and pupae of banana skipper e. torus and the living nymphs and adults of banana lace bug s. typica on all leaves of a banana plant were counted. five banana plants in each small plot were surveyed, and three of six small plots were chosen randomly in every time of investigation. the survey was conducted once in the middle of each month from may to october, 2006. the study was conducted from may to october because it is the major occurring period of both pests. it also is the period that banana grow from the later seedling stage to young fruit stage. data analyses all data were tested for normal distribution and homogeneity of variances by the shapiro-wilk test and levene’s test, respectively. since all data were normal distribution, the analysis of variance followed by lsd mean comparison at α = 0.05 was used to compare the number of fire ant mounds, trapped fire ant workers and populations of e. torus and s. typica among different plots and among different months using generalized linear model (glm). all statistical analyses were conducted using spss, version 18.0 (spss inc., chicago, il, usa). ditch ditch ditch ditch ditch 1 4 7 10 13 16 19 22 2 5 8 11 14 17 20 23 3 6 9 12 15 18 21 24 control high vegetation coverage middle vegetation coverage low vegetation coverage fig 1. schematic diagram about experimental site. app:ds:schematic app:ds:diagram sociobiology 63(3): 889-893 (september, 2016) 891 results dynamic of s. invicta in banana plantation the numbers of s. invicta workers varied from may to october in high, moderate and low weed coverage experimental plots (table 1, f = 184.0, df = 17, p < 0.001). the numbers of trapped workers increased after may and reach a peak in july. in july, 252 ants/trap, 275 ants/trap and 290 ants/ trap were collected in high, moderate and low weed coverage experimental plots, respectively. the numbers of trapped ants decreased in august and september and significantly increased again in october. the density of s. invicta nests also changed with time in different types of coverage experimental plots. the density of ant nests reached a peak in august in high weed coverage plots and reached the peak in june in moderate and low weed coverage plots (f = 148.2, df = 17, p < 0.001). effect of s. invicta on banana skipper population e. torus in our investigation, e. torus populations in s. invicta infected plots were less than those in control from may to october (table 2, f = 150.07, df = 8, p < 0.0001). e. torus population reduction in high and moderate weed coverage plots were significantly higher than that in low weed coverage plot, but there was no significant difference between the high and moderate weed coverage plots from may to october (f = 150.07, df = 8, p < 0.0001). the population of e. torus reduced by 21.6% 53.4%, with an average 39.2% in high weed coverage plots. in moderate weed coverage plots, it was reduced by 13.4% 52.9%, with an average 41.4%. the population of e. torus also was reduced from -5.4% to 33.5% in low weed coverage plots, with an average 19.2%. date (mm-yy) area of high vegetation coverage area of moderate vegetation coverage area of low vegetation coverage worker (ind./trap) mound (ind./cell) worker (ind./trap) mound (ind./cell) worker (ind./trap) mound (ind./cell) 05-2008 49.7±10.2 8.0±1.7 87.8±16.8 8.7±0.7 54.5±11.9 10.3±1.3 06-2008 110.1±25.3 17.0±1.5 119.9±23.9 17.3±1.9 74.0±24.2 17.0±0.6 07-2008 252.4±25.8 19.0±1.5 274.5±33.3 14.7±0.7 289.8±32.7 8.7±0.9 08-2008 182.6±25.8 20.3±2.2 169.2±14.3 11.0±1.5 201.3±36.0 9.3±0.9 09-2008 167.8±22.6 16.3±0.7 103.1±23.0 11.0±1.2 147.0±45.4 8.0±1.0 10-2008 378.2±27.4 15.3±1.2 219.8±48.1 16.1±1.8 361.0±32.4 13.0±0.4 table 1. dynamics of s. invicta population in the three types of banana plantations. effect of s. invicta on banana lacebug s. typica populatione population of s. typica in experimental plots was significantly lower than that in control plots from may to october (table 3, f = 257.29, df = 8, p < 0.0001), and reduction rate varied from 20.8% to 67.9%. reduction of s. typica population in the higher weed coverage plots was significantly lower than that in either moderate or lower banana skipper density (ind./trunk) reduction of banana skipper (%) date (mm-yy) control area high vegetation coverage moderate vegetation coverage low vegetation coverage high vegetation coverage moderate vegetation coverage low vegetation coverage 05-2008 8.6±1.3a 6.2±0.8b 6.9±1.2ab 7.1±1.4b 27.9 19.8 20.9 06-2008 11.9±0.4a 6.8±0.5c 10.3±0.4b 9.9±0.5b 42.9 13.4 16.8 07-2008 16.1±1.0a 7.5±0.4c 6.6±0.5c 10.7±0.6b 53.4 59.0 33.5 08-2008 3.7±0.3ab 2.9±0.2b 1.8±0.2c 3.9±0.3a 21.6 51.4 -5.4 09-2008 3.4±0.2a 1.9±0.1c 1.6±0.2c 2.6±0.2b 44.1 52.9 23.5 10-2008 3.1±0.1a 1.7±0.2c 1.5±0.2c 2.3±0.1b 45.2 51.6 25.8 average 39.2± 5.3 41.4± 8.7 19.2±5.4 note: same letters indicated there is no significant difference in the same line (p<0.05). table 2. effects of s. invicta invasion on population of banana skipper e. torus. weed coverage plots, and there are no significant difference in reduction of s. typica population between moderate and low weed coverage plots from may to october (f = 257.29, df = 8, p < 0.0001). the population of s. typica reduced significantly in may and september in high weed coverage plots. in july, population reduction rate of s. typica in moderate and low weed coverage plots reached the peak with 50.9% and 67.9%, respectively. l wang, z wang, l zeng, yy lu – fire ant invasion reduces the populations of two banana insect pests892 discussion ants are important predators in agroecosystems (risch & carroll, 1982; way & khoo, 1992) and have been used in pest management (huang & yang, 1987; olotu et al., 2013), including the combination of ants in banana pest management programs banana (sirjusingh et al., 1992; gold et al., 2001). our study provides evidence that s. invicta also can be an important biological control agent in banana plantation. 19.2% 41.4% of e. torus population and 17.8%-43.0% of s. typical population decreased when s. invicta appeared in three types of weed coverage banana plantations. our results are consistent with previous studies indicating that s. invicta are particularly effective predators in agroecosystems (ali et al., 1984; bessin & reagan, 1989; eubanks, 2001; harvey & eubanks, 2004). our investigation also gives support to the suggestion that s. invicta can play beneficial role in pests control although it was considered a serious pest in human health and crops (vinson, 1997). it showed the efficacy of s. invicta as biological control agent on e. torus was greater in the more complex habitat, it consistent with harvey and eubanks (2004)’s study. harvey and eubanks (2004) speculated that complex habitat resulted simpler plant architecture which can increase predator efficiency. it may be the same result to explain our result. however, controlling efficacy of s. invicta on s. typical is negative with the habitat complex. s. typical adults have wings and can fly, and nymphs are more actives than e. torus larvae and pupae. complex habitant provides more hidden places for s. typical when they are attacked by s. invicta. our results suggested that efficacy of s. invicta as a biological control agent was related to not only complexity degree of habitat, but also target pests. our results partially that s. invicta can be important and effective biological control agents. although this study found that increasing habitat complexity only enhanced the efficacy of s. invicta on some target pests, intercropping may give more shelters to native species and reduce the negative effects of s. invicta on arthropod community. future work should focus on the impact of s. invicta on banana plantation ecosystem and production since more data are needed to fully understand the ecological effects of the fire ant invasion, and the action of an effective biological control agent must result in decreased crop damage and increased yield (symondson et al., 2002). references ali, a.d., reagan, t.e. & flynn, j.l. 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(2012). investigation on the species of banana insect pest and their damages in yunan province. banana lacebug (ind./trunk) reduction of banana lacebug (%) date (mm-yy) control area high vegetation coverage moderate vegetation coverage low vegetation coverage high vegetation coverage moderate vegetation coverage low vegetation coverage 05-2008 79.0±5.3a 62.6±3.1b 62.5±3.4b 57.0±4.5b 20.8 20.9 27.8 06-2008 43.2±3.6a 36.6±3.2a 22.7±2.4b 20.7±2.2b 15.3 47.5 52.1 07-2008 5.3±0.4a 3.8±0.5b 2.6±0.3bc 1.7±0.3c 28.3 50.9 67.9 08-2008 5.7±0.6a 5.4±0.8a 2.9±0.4b 4.5±0.5ab 5.3 49.1 21.1 09-2008 15.5±1.6a 8.0±1.3b 8.1±1.0b 9.8±1.0b 48.4 47.7 36.8 10-2008 14.6±1.3a 16.3±0.9a 8.6±0.8b 10.3±1.1b -11.6 41.1 29.3 average 17.8±8.3 43.0±4.6 39.2±7.2 note: same letters indicated there is no significant difference in the same line (p<0.05). table 3. effects of s. invicta invasion on the population of banana lacebug s. typica. sociobiology 63(3): 889-893 (september, 2016) 893 chinese journal of tropical agriculture, 32: 42-45. harvey c.t. & eubanks, m.d. 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(2009). national standard: guidelines for quarantine surveillance of solenopsis invicta buren. beijing: standards press of china. way, m.j. & khoo, k.c. (1992). role of ants in pest management. annual review of entomology, 37: 479-503. wojcik, d.p., allen, c.r., brenner, r.j., forys, e.a., jouvenaz, d.p. & lutz, r.s. (2001). red imported fire ants: impact on biodiversity. american entomologist, 47: 16-23. zeng, l., lu, y.y., he, x.f., zhang, w.q. & liang, g.w. (2005). identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan, guangdong. chinese bulletin of entomology, 42: 144-148. zhou, s., chen, z., zhang, q., kong, x., zhong, y., lai, b., chen, s. & wei m. (1993). the study on the transmission of banana rosette dwarf disease by stephanitis typicus distant. journal of chinese electron microscopy society, 3: 238-241. doi: 10.13102/sociobiology.v63i4.1171sociobiology 63(4): 1022-1030 (december, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 lower ant diversity on earth mounds in a semi-arid ecosystem: natural variation or a sign of degradation? introduction biodiversity is typically distributed heterogenously among habitats; therefore, understanding the spatial aspect of biodiversity underpins ecosystem conservation and management (ricklefs, 2004). arid and semi-arid ecosystems often possess spatial heterogeneity in habitat over a scale of several meters to more than 100 m (berg, 2012). life in such regions is often restricted to patches of vegetation that are surrounded by more in hospitable rock and dry soil. such vegetation tussocks are found to be biodiversity hotspots when compared to the surrounding landscape (housman et al., 2007). vegetation and litter act as physical barriers to soil temperature fluctuations and surface erosion while providing greater soil moisture content during the dry season as compared to the surrounding areas (sayer, 2006). studies abstract natural earth mounds in many ecosystems harbor higher biodiversity than surroundings because they provide greater habitat heterogeneity. however, in the semi-arid caatinga ecosystem of ne brazil, natural mounds have much less vegetation and leaf litter with lower biodiversity as compared to the surrounding lowlands. the following hypotheses were tested: (i) low vegetation cover on the mounds results from highly compacted and leached soils as compared to adjacent lowlands and (ii) low vegetation cover reduce ant populations and diversity because of limited foraging and nesting resources. this study was carried out in four mound fields. at each mound field, 30 sampling were taken using pitfall traps.the high resistance of the mound soil to root penetration and low soil ph were the main reason for the difference in ant diversity between mound and adjacent lowlands. adjacent lowlands were found to have twice as many ant individuals as the mounds along with higher ant species richness and diversity. these results suggest that environmental degradation in the caatinga led to deforestation and thus compaction and leaching of soil mounds. sociobiology an international journal on social insects ks carvalho1, maf carneiro2, ic nascimento1, ak saha3, em bruna4 article history edited by gilberto m. m. santos, uefs, brazil received 21 july 2016 initial acceptance 25 september 2016 final acceptance 07 november 2016 publication date 13 january 2017 keywords ants, biodiversity, spatial heterogeneity, caatinga. corresponding author karine santana carvalho. uesb, departamento de ciências biológicas av. josé moreira sobrinho, s/n cep: 45206-190, jequiezinho jequié-ba, brasil e-mail: ksczool@yahoo.com.br in african savannas have shown that active termite mounds in the otherwise relatively flat homogeneous landscapes increased nitrogen availability by supporting nitrogenfixing plant species, resulting in significantly higher nutrient concentrations than those resulting from droppings of herbivorous ungulates (fox-dobbs et al., 2010). similarly, semi-arid environments in asia possess high spatial variability in soil nutrient and vegetation properties, where organic matter and nutrients were shown to contribute to the islands of fertility phenomenon (chen et al., 2006), what is also registered for desert ecosystems (ganer & steinberg, 1989). on the other hand, there are also local biodiversity ‘coldspots’ (kareiva & marvier, 2003) a situation contrary to the ‘islands of fertility’ concept, whereby certain features deter colonization by flora and fauna due to various reasons such as lack of moisture and nutrients (sandy soils) or allelopathy 1 universidade estadual do sudoeste da bahia (uesb), departamento de ciências biológicas, jequié-ba, brazil 2 universidade estadual do sudoeste da bahia (uesb), departamento de ciências naturais, programa de pós-graduação em genética, biodiversidade e conservação, vitória da conquista-ba, brazil 3macarthur agro-ecological research center, archbold biological station, lake placid-fl, eua 4 university of florida (uf), department of wild life ecology and conservation, gainesville-fl, eua research article ants mailto:ksczool@yahoo.com.br sociobiology 63(4): 1022-1030 (december, 2016) 1023 by a plant species. in the caatinga ecosystem that is one of the largest continuous area of seasonal dry tropical and thorn-scrub forest in north-eastern brazil (pennington et al., 2009), we have observed naturally occurring earth mounds that have much lower vegetation cover and plant diversity than the surrounding lowlands. lower vegetation cover and diversity could in turn severely limit habitat for invertebrate fauna, including ants that play an important role in nutrient cycling in an ecosystem (vasconcelos et al., 2008). several studies have suggested that ant species diversity rises with the increase in environmental structural complexity (jeanne, 1979; benson & harada, 1988; corrêa et al., 2006; pacheco et al., 2009) by offering a wider range of sites for nesting, feeding and various ecological interactions favouring ant activities (benson & harada, 1988; ribas et al., 2003). the caatinga is a semi-arid ecosystem in northeastern brazil that is characterized by high plant species diversity and endemism (ribeiro et al., 2015) in response to low rainfall and soil heterogeneity. the intensity of year-round solar radiation and high soil temperatures causes rapid breakdown and decomposition of organic matter (pacchioni et al., 2014). in general, soils in the caatinga are shallow and rich in minerals but poor in residual organic matter (eg. andradelima 1977, eiten, 1983). the low prevalence of vegetation and organic matter is even more pronounced on elevated mounds as compared to the surrounding lowlands. naturally occurring mound fields occupy large areas in the caatinga (antunes et al., 2012), with a density of about 35 mounds/ha (funch, 2015). these mounds are extremely hard sun-baked elevated structures about 1-3 m high with regular spacing that do not touch or overlap (funch, 2015) thus indicating a scaledependent feedback among them (rietkerk & van de koppel, 2008). the mounds are piles of soil or a type of micro relief in landscapes associated with poor drainage conditions (resende et al., 2002). their origin remains controversial (renard et al., 2012) with two competing hypotheses (silva et al., 2010) – a termite mound origin versus differential erosion over time. the termite mound hypothesis suggests successful colonization by successive generations of termites that thereby increases the diameter and height of these structures, which are further acted upon by earthworms and ants (resende et al., 2002). the differential erosion hypothesis focuses on geomorphology and suggests that mounds are reliefs resulting from differential erosive action (oliveira-filho, 1992a, b; midgley, 2010), with the softer lithography eroded away. regardless of their origin, the presence of mounds can change the dynamics of environmental functioning since spatial structures are important in regulating interactions between communities and ecosystem processes (rietkerk & van de koppel, 2008). the greater solar exposure and lower vegetation cover on caatinga mounds as compared to the surrounding xericforested lowlands can alter: 1) the microclimate 2) the chemical and physical properties of soil and 3) the distribution of organisms that live and/or forageon mounds. this study aims to measure ant diversity along with soil properties and vegetation cover on mounds and surrounding lowlands, in order to verify whether the mounds are indeed relatively impoverished in species abundance. results will then provide a basis for further investigations, whether the low diversity is a natural feature or is it reflective of ongoing anthropogenic change involving removal of vegetation and consequent soil alteration.in this context, the following hypotheses were tested: (i) mounds with low vegetation cover have more compacted and leached soils than the surrounding lowlands and (ii) this negatively affects the distribution and diversity of ant species due to the shortage of resources for foraging or nesting, as compared to the surrounding matrix. material and methods study area the study was conducted in four mound fields, i.e. fields with numerous regularly spaced mounds. two of the mound fields had white soil (quartz-rich podzols) while the two others had red soil (latossols with a high concentration of iron and aluminium oxides). the mound fields on white soil exhibited a diverse thorny plant community with predominance of cacti. the mound fields on red soil had a relatively homogeneous vegetation cover composed of both trees and shrubs. the mound fields were separated from each other by a minimum of 1 km and were located near the protected area of the floresta nacional (flona) contendas do sincorá, bahia, brazil (13055’14.4”s; 041006’54.9” w). the climatic classification of the region, according to koppen, is semi-arid with scarce and irregular rainfall, concentrated in the summer. the average annual rainfall is 596 mm. most of the national forest and buffer zone is inserted in argissolos classification (eutrophic red-yellow podzolic), being small part, formed by latosols (ibama & mma, 2006). sampling design at each moundfield, 30 sampling points were taken 20 m apart from each other along a transect. each mound was randomly chosen while sampling transect on the adjacent lowland was chosen in a random direction. in order to test whether the mounds locally affect the distribution of richness and diversity of ant species, collections were carried out from october to december 2014 (rainy season) at each sample point using pitfall traps. the traps were kept active for 48 hours in the field. at each sample point using (bestelmeyer et al., 2000). in total, 120 mound samples (30 samples per mound) and 120 adjacent ground samples were collected. collected ants were identified following the classification proposed by bolton (2016) and witness individuals were deposited in the collection from zoology of the universidade estadual do sudoeste da bahia and in the entomological collection from the myrmecology laboratory from the comissão executiva ks carvalho et al. – earth mounds and ant diversity in north-eastern brazil1024 de pesquisa da lavoura cacaueira (cepec/ceplac), in itabuna, bahia. three soil penetration resistance measurements were made with a penetrometer (solotest 2.10) in order to test whether the soil of the mounds is more compressed or compacted as compared to the surrounding soil. penetration tests were carried out on ten mounds randomly chosen and similarly, three measurements each at ten points on the adjacent lowland in each field. soil samples were also collected from each mound and adjacent lowland and sent to soil laboratory of the universidade estadual do sudoeste da bahia (uesb) for chemical analysis of macronutrients and organic matter. height and width measures were taken and calculated by using the cone area to infer the surface area of mounds. data analysis analysis of variance was performed (two-way anova) using systat 12.0 (2007) in order to test whether the soil compaction and nutrient availability (abiotic variables) and the abundance, richness and diversity of ant species (ecological descriptors) differed between mounds and adjacent lowlands by using (1) site: mound or lowland; and (2) mound field: white and red soil, as sources of variation in the model. the diversity of ant species between sites (mound and lowland) was compared using the shannon-wiener index (using estimates 9.1.0; colwell, 2006) since species richness data alone cannot express the dominance of species in a community. for the calculation of this index, the relative frequency of each species in a given sample was used instead of the abundance of individuals. this is used due to worker ant recruitment for foraging, displayed by the majority of ants, which can be more or less efficient in a species and therefore it may underor overestimate the values. the composition of ant species between mound and adjacent lowland and among mounds fields was compared with the values of jaccard similarity index (systat,2007). a principal components analysis (pca) was performed to visualize correlations of abiotic factors in mounds fields. then, in order to know how these factors relate to ecological descriptors (abundance, richness and diversity) a pearson correlation analysis was performed using past 3.0 (2016). results structure of mounds and physicochemical characteristics of the soil in the mounds fields the density of mounds was approximately 9 per 100m2. on average, the surface area ranged from 27 to approximately 39m2 while height ranged from 1.4 ± 0.4 to 1.8 ± 0.3 m. the volume of a mound ranged from 14.6 to 38.3 m3. out of the 40 mounds sampled, (20 in the mound fields on white soil and 20 in the mound field on red soil) had 50-75% vegetation cover while the others had much lower cover ranging from less than 50% to almost none (table 1). the penetrometer tests found that the soil on mounds was more compact or dense as compared to the soil on adjacent lowlands (f = 3.90; df = 1; p<0.049; n = 240; table. 2). on average, the pressure (in kg / m3 of soil) necessary for penetration on the mounds was 29.2 ± 3.0 units, whereas the adjacent lowlands required much less 1.67 ± 2.56. white soils offered significantly higher resistance to penetration as compared to red soils (f = 120,518; df = 1; p≤0.001; n = 240). there was no interaction between the two factors. interestingly, soil organic matter did not differ between mound and adjacent lowland but the mounds had a more acidic ph (5.30 ± 0.7) than the adjacent lowland (6.24 ± 0.82; f = 15,145; df = 1; p ≤ 0.001; n = 40; table. 2). soil ph varied significantly with soil color (f = 8.62, df = 1; p ≤ 0.006; n = 40) with white soil being less acidic than red soil. the interaction between the factors was not significant. aside from calcium (ca2+) and magnesium (mg2+) ions, macronutrients did not differ between mounds and adjacent lowland in any of the studied fields. the ca content of the mounds was 2.98 ± 1.16 cmol / dm3 and 1.82 ± 0.64 cmol / dm3 in the adjacent lowland (f = 36.739; df = 1; p≤0.001; n = 40; table 2). mg presented 1.29 ± 0.4 cmol / dm3 in mounds and 0.98 ±0.28 cmol / dm3in the adjacent lowland (f = 8.984; df = 1; p≤0.006; n = 40; table 2). there appeared to be an effect of soil type on the ca content (f = 18.496, df = 1; p≤0.001; n = 40) and the mg content (f = 4.654; df = 1; p≤0.038; n = 40). white soil presented higher concentration of both macronutrients as compared to red soil. similarly, phosphorus (p) and potassium (k) concentration were different accordint to soil color (p: f = 7.921; df = 1; p≤0.008; k: f = 6.369, df = 1; p≤0.016 n = 40), being the highest in white soil mounds fields. abundance, richness, diversity and composition of ants assembly or community in the mound fields. we identified 30 ant species belonging to 16 genera and six subfamilies living in mounds fields caatinga (table 3). overall, the average number of ant individuals in pitfall traps on adjacent lowlands (14.5 ± 20.7) was twice higher than those on the mounds (7.7 ± 9.4; f = 13.2; df = 1; p≤0.001; n = 240). ant abundance also varied significantly with soil colour (f = 14.1; df = 1; p≤0.001; n = 240). mound fields having white soil were found to have the highest abundance surface area (m2) height (m) volume (m3) vegetation cover (%) mound field rs 1 27.01±7.3 1.51±0.3 14.8±6.6 0-50 (57%) mound field rs 2 34.7±10.3 1.6±0.4 32.1±16.1 26-50 (79%) mound field ws 1 29.9±11.1 1.40±0.4 38.3±31.7 0-50 (80%) mound field ws 2 38.8±9.6 1.81±0.3 35.2±18.1 0-25 (80%) rs = red soil; ws = white soil table 1. structure of mounds in mounds fields from caatinga ecosystem, northeastern brazil. sociobiology 63(4): 1022-1030 (december, 2016) 1025 of ants. the interaction between the site (mound or adjacent lowland) and the soil color (white or red one) was statistically significant (f = 6.13, df = 1; p≤0.014; n = 240). the richness of ant species was also higher in the adjacent lowlands (3.6 ± 1.8) than on the mounds (2.6 ± 1.9; f = 17.9; df = 1; p≤0.001; n = 240). there was a significant effect of soil colour on the species richness (f = 31.4; df = 1; p≤0.001; n = 240) and the interaction between these factors (f = 6.87, df = 1; p≤0.009; n = 240). the highest species richness was found in the mound fields of white soil. ants species diversity was higher in the adjacent lowland (f = 27.4, df = 1; p≤0.001; n = 240) than on the mounds and higher in white soil fields (f = 58.6, df = 1, p≤0.001; n = 240) than in the red soil fields. the interaction between the sources of variation (mound/lowland and soil color) was significant (f = 10.5; df = 1; p≤0.001; n = 240). there was significant difference in the composition of ant species between mound and adjacent lowland. the jaccard index found a species overlap of 58% between the mounds and adjacent lowlands on white soils, and a lower ovelap (39%) table 2. physicochemical characteristics of the soil in the mounds fields from caatinga ecosystem, northeastern brazil. mound field site pressure for penetration ph macronutrients (kg / m3 of soil) ca2+ mg2+ red soil mound 0.28±1.63 5.2±0.7 2.12±0.70 1.16±0.44 adjacent lowland 0.22±0.12 5.7±0.6 1.32±0.41 0.88±0.29 white soil mound 4.09±3.21 5.5±0.6 3.84±0.85 1.42±0.33 adjacent lowland 2.97±2.98 6.7±0.7 2.32±0.39 1.08±0.23 statistical difference mounds vs. adjacent lowland p≤ 0.049 p≤ 0.001 p≤ 0.001 p≤ 0.006 red soil vc white soil p≤ 0.001 p≤ 0.006 p≤ 0.001 p≤ 0.038 between the two habitats on red soils. the species with the highest frequency of occurrence in samples and more frequent in the adjacent lowland were dinoponera quadriceps, pheidole radoszkowskii and ectotomma muticum (table 3). the specie camponotus blandus was relatively common in both places (mound and adjacent lowland; table 3) interactions between mounds, mounds fields and ant communities the axis 1 (pc1) of the principal components analysis (pca) revealed two distinct groupings between soil types (white soil and red soil) which are influenced positively by macronutrient content (calcium and phosphorus). axis 2 (pc2) separated mound and adjacent lowland which are positively correlated with soil resistance and negatively correlated with ph (fig 1). the pearson correlation matrix found that there is a significant linear relationship between the diversity of ant species and the component 2 of the pca (table 4). fig 1. main component analysis of macronutrients (ca2+, p), ph and soil resistance to root penetration. (colours: black = red soil, grey = white soil; square = mound; circle = adjacent lowland). ks carvalho et al. – earth mounds and ant diversity in north-eastern brazil1026 discussion the results of this study show that mounds have highly compacted soils, lower vegetation cover and locally reduced population and diversity of ant species as compared to the adjacent lowland. ant diversity is otherwise quite high in the caatinga ecosystem. this study shows that the mounds in the caatinga are definitely spots of lower ant abundance and diversity a finding contrary to the usual biodiversity hotspots on elevated mounds and tussocks in dry ecosystems (housman et al., 2007; berg, 2012). apart from calcium and magnesium, there was no difference in the concentration of other macronutrients between mounds and adjacent lowlands within a mound field. the similarity in nutrients across mounds and adjacent lowlands suggests that nutrient limitation is not the main reason for low vegetation cover on mounds, thus supporting the idea that the mounds’ inhospitability to life (compacted soil) may have resulted from anthropogenic removal of vegetation. removal of natural vegetation can lead to the erosion and hardening of soils through increased exposure to natural elements. further investigation can look into whether this low species diversity status and low life-support capacity of mounds has always been naturally so (because of the origin of the mounds), or is that an outcome of the ongoing degradation in the region. ant population size, diversity and spatial landform type (mounds and adjacent lowlands) as expected, the population size and species diversity of ant species were lower on mounds compared to the adjacent lowland. the relatively lower population size and species diversity is likely related to the greater soil compaction and lower ph of soils on mounds, both factors that discourage the establishment of vegetation and nesting sites of ants. this was seen to be true in both white and red soils. the greater physical compaction of soils on mounds implies much higher resistance to penetration by roots, thereby acting as a deterrent to plant establishment (e.g., kozlowski, 1999). the smooth surface on mounds discourages the retention of seeds. seed germination table 3. frequency of occurrence (%) of ant species in mounds fields (2 fields in red soil and 2 fields in white soil) from caatinga ecosystem, northeastern brazil. (al = adjacent lowland; m = mound). correlation p < 0.05 abundance richness diversity pc 1 pc 2 abundance 0.048 0.007 0.076 0.061 richness 0.315 0.008 0.464 0.368 diversity 0.420 0.414 0.104 0.004 pc 1 0.284 0.119 0.261 0.911 pc 2 -0.299 -0.146 -0.451 -0.018 subfamily/species red soil fields white soil fields al m al m dolichoderinae dorymyrmex brunneus forel, 1908 6.7 6.7 26.7 dorymyrmex sp.3 10 dorymyrmex thoraxicus gallardo, 1916 3.3 forelius brasiliensis (forel, 1908) 1.7 1.7 20 11.7 forelius maranhaoensis cuezzo, 2000 10 linepithema neotropicum wild, 2007 18.3 10 ectatomminae ectatoma suzanae almeida filho, 1986 16.7 11.7 ectatomma muticum mayr, 1870 10 13.3 43.3 15 gnamptogenys acuminata (emery, 1896) 3.3 gnamptogenys moelleri (forel, 1912) 5 formicinae camponotus blandus(smith 1858) 31.7 33.3 23.3 43.3 camponotus meloticus (emery, 1894) 11.7 camponotus novogranadensis mayr, 1870 3.3 campotonotus (myrmephaenus) sp. 1 1.7 myrmicinae atta sexdens rubropilosa forel, 1908 6.7 13.3 cephalotes pusillus (klug, 1824) 3.3 1.7 1 crematogaster erecta mayr, 1866 1.7 crematogaster distans mayr, 1870 3.3 11.7 1 nesomyrmex itinerans (kempf, 1959) 1.7 pheidole radoszkowskii mayr, 1884 8.3 13.3 53.3 41.7 pheidole sp.7 3.3 pheidole gp. tristis sp.18 1.7 pogonomyrmex naegelii emery, 1878 1 solenopsis globularia (smith, 1858) 13.3 8.3 36.7 20 solenopsis sp.2 3.3 1.7 8.3 solenopsis sp.5 1.7 ponerinae dinoponera quadriceps kempf, 1971 56.7 35 26.7 6.7 platythyrea pilosula (smith, 1858) 1.7 pseudomyrmecinae pseudomyrmex termitarius (smith, 1855) 5 13.3 pseudomyrmex gracilis (fabricius, 1804) 3.3 table 4. pearson correlation matrix among abundance, richness and diversity of species of ants and the values of the two axes of a principal components analysis (pca).the numbers represent the r values of linear correlation between variables. the p values indicate the level of significance of the correlation (p <0.05). sociobiology 63(4): 1022-1030 (december, 2016) 1027 would also be discouraged by the relatively inhospitable conditions prevailing on mounds –much higher exposure to strong sunlight that increases soil temperature and decreases soil moisture. in comparison the adjacent lowlands have higher vegetation cover by shrubs and occasional trees that shade the soil against drying out by solar radiation and wind as well as reduce diurnal soil temperature variations (e.g. sayer, 2006). the vegetation cover also generates a litter layer that conserves moisture and adds to spatial heterogeneity of habitats, thereby supporting larger and more diverse populations of ants as noted by carvalho et al. (2012) in areas of caatinga. some ants which are strongly associated with vegetation, as the species of the genus camponotus (brandão et al. 2009), were virtually absent in the red soil mound fields. the exception was camponotus blandus, present at a high frequency (> 20% of samples) in the field mounds and at both sites (mounds and adjacent lowlands). the nests of c. blandus are found in the soil, in preexisting cavities under rocks or termite mounds (gallego-ropero & feitosa, 2014), and it is considered a predatory specie, especially of termites (mendonça & resende, 1996; gallego-ropero & feitosa, 2014). the occurrence of c. blandus homogeneously may indicate that it is nesting on these structures and/or making predation on termite inside the mounds. although we have not conducted systematic collection of other arthropods, termite galleries were observed in murudus. higher soil acidity (lower ph) poses constraints for soil biota thereby lowering the ecological functions of soil biota in creating favorable conditions for plant establishment and growth, from soil organic matter decomposition, nutrient availability and mycorrhizal networks (whitford, 1996). soil biota composition has been seen to be very sensitive to soil degradation and restoration in the caatinga (araújo et al., 2014). however, contrary to our first hypothesis, the mounds were not more leached than the adjacent lowlands since they have higher calcium (ca) and magnesium (mg) content. this result is initially surprising, considering that the acidic soils usually have low base concentrations (especially ca and mg despite the high presence of these elements in parent material). termite nests and tunnels were evident upon excavation of mounds. in fact, termites act as ecosystem engineers (jones et al., 1994), through their transportation of soil nutrients both vertically and horizontally (sileshi et al., 2010). according lobry de bruyn and conacher (1990), termites can increase carbon and nutrient levels.the extent of the increases depends on the type of mound construction and the degree to which organic material is incorporated. more studies are needed to understand the role of termites in increased levels of calcium and magnesium in mounds of caatinga. in addition to the soil that influences vegetation, the physical structure of mounds should also be considered. in this study, the average surface area of a mound ranged from 27 39 m2, a considerable area in terms of energy required by an ant to climb and travel in search for resources, especially if the mound has little or no resources like living or dead organic matter. in ants, as well as other animals, the ecological success depends on the ability to maximize success foraging and quantidade of energy invested (stephens & krebs, 1971). the total volume of soil (15-38 m3) can also be a physical barrier to hypogaeic species (underground foragers), due to the absence of roots and microorganisms associated with them, among other features. perhaps other factors not measured in this study, such as the areal extent of bare soil patches and links between plant and ant species compositions could also explain the differences found in the abundance and ant species richness between mounds and adjacent lowlands. ant diversity and soil types – influence of vegetation and nutrients mound fields with white soil had a greater abundance of individuals and richness of ant species as compared to mound fields with red soil. this was true for both mounds and the surrounding matrix in each soil type. the red soil fields have relatively homogeneous shrub vegetation and therefore possess less microclimatic and microhabitat variation as compared to fields with white soil types. mound fields of white soil have a much higher diversity of vegetation, including high levels of endemism, more diversified rupestrian vegetation, with the presence of rocky outcrops, and the predominance of cacti. thus, the highest abundance, richness and diversity of ant species in white soil fields are probably related to their more complex vegetation structure. further studies would be needed to understand this relationship in greater detail. however, it is known that climate, microclimate and particularly microhabitat explain variations in biodiversity in a mesoscale ranged from few kilometers to few hundred kilometers (hutchings et al., 2003). in addition, insect abundance in the caatinga was also found to be much higher during and just after the brief rainy season largely spurred on by higher vegetation productivity and food/nutrient availability during that time of the year (vasconcelos et al., 2010). mound fields with white soil had significantly higher values of calcium, magnesium and phosphorus than mound fields with red soil. this study did not look at the differences in vegetation composition in mound fields between the two soil types; however the higher availability of nutrients on white soils could also leads to greater availability of niches that promote higher species diversity and endemism for which the caatinga is well known. low diversity on mounds natural or anthropogenic? the caatinga is a unique ecosystem, where the combination of water stress and soil nutrient variability has resulted in high floral and faunal species diversity, particularly remarkable for plants, birds and fishfor instance, over 1,200 species of vascular plants occur here, with 30% of them being endemic to the region (leal et al., 2003). over fifty percent of habitat has been altered due to agriculture ks carvalho et al. – earth mounds and ant diversity in north-eastern brazil1028 development or cattle grazing (smithsonian information sheet) while only around 1% of the region is protected under national parks (tnc & associação caatinga, 2004). unplanned agricultural expansion can accelerate soil erosion, lead to fires and seriously threaten the structure of caatinga plant communities (mamede & araujo, 2008). funch (2015) mentions cattle grazing as a common activity in mound fields in the caatinga and that in recent decades the clearing of the fields has revealed mounds that were originally hidden by low (≤4 m) but very dense vegetation. funch (2015) describes the mounds as places normally covered by vegetation, penetrated by roots and limited erosion by wind and rain an opposite description to the mounds in this study which were seen to be bare spots on the landscape. probably the contrasting finding in our study reflects the degree of degradation of the areas selected for study, which has witnessed human influence over recent decades, by removal of vegetation from mounds that has led to exposurerelated soil hardening, with attendant deterrence for vegetation recolonization. in fact, according to the sociocultural survey of the region described in the management plan of the flona, the history of the region shows fields for the planting of various products such as beans, corn, cotton and especially cassava from the slavery period in northeastern brazil (xviii century). the region also has other more drastic uses such as logging for railroad construction and asphalt (fences), magnesium mining and coal mining until about september 1999, when the flona was created (ibama & mma, 2006). if that is the case, then mounds can be considered as sentinels indicating the ongoing degradation of the caatinga biome. a recent study by ribeiro et al. (2015) showed that chronic anthropogenic disturbance caused by the high population density and livestock, among others, are driving the vegetation of caatinga towards species impoverishment. this is the first investigation to our knowledge of the (negative) effects of spatial heterogeneity created by mounds on ant communities. in this context, the following hypotheses were tested: (i) mounds with low vegetation cover have more compacted and leached soils than the surrounding lowlands; (ii) this negatively affects the distribution and diversity of ant species due to the shortage of resources for foraging or nesting, as compared to the surrounding matrix. it provides at least two major for questions biodiversity conservation in dry ecosystems such as the caatinga: 1) how far do the negative effects of mounds in the distribution and diversity of the ants (and other arthropods) reach? are these negative effects limited only to mound structure or mound formation as well? in this sense, 2) what would be the best scale to study the effects of heterogeneity of the mound fields on ant communities? a larger scale study including mounds with a chronosequence of human disturbance can investigate whether the hardened soils, low vegetation cover and low ant diversity on mounds are natural features or are happening as a consequence of human activities. such a determination would then enable mounds to be seen as sentinels or indicators of ongoing environmental change in the caatinga, and thereby be useful for assessing ecological conditions in conservation and restoration strategies. acknowledgements we thanks anselmo santos, bianca miranda, ramon almeida and michel cardoso for help during fieldwork, andré teixeira da silva for help in data analysis. financial support came from the scholarship of cordenação de pessoal de nível superiorcapes (bex nº 10839/13-5), universidade estadual do sudoeste da bahia uesb and programa de núcleo de excelênciapronex (0011/2009) of fundação de amaaro à pesquisa do estado da bahiafapesb and conselho nacional de desenvolvimento científico e tecnológicocnpq. references andrade-lima, d. 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(1996).the importance of the biodiversity of soil biota in arid ecosystems. biodiversity and conservation, 5: 185-195. doi: 10.1007/bf00055829 http://www.ncbi.nlm.nih.gov/pubmed/24706600 _goback ole_link1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.1938sociobiology 65(2): 155-161 (june, 2018) seed removal by ants in brazilian savanna: optimizing fieldwork introduction ecological functions, such as seed removal, are extremely important for preservation of ecosystem stability (naeem et al., 1999), due to regulatory processes of ecosystem functioning and the influence of several organisms in the ecosystem dynamics. however, such functions may be transformed or even lost due to landscape modifications caused by human interventions, which may even result in the complete loss of habitats, species and resources (armbrecht et al., 2006; ribas et al., 2012b; edwards et al., 2014.). aiming to observe and evaluate such environmental modifications and their effects on ecological functions, many studies have widely used bioindicator organisms (e.g.: braga et al., 2013; costa et al., 2016; koivula, 2011; siddiget al., 2016). abstract there has been an increase in the number of studies using seed removal by ants to evaluate ecosystem functioning; however, these studies encompassed varying time periods and used different types of seeds. therefore, our aim was to evaluate differences in the proportion of seeds removed by ants in impacted and non-impacted sites in brazilian savanna. furthermore, we evaluated seed removal (1) during the morning and after a 24h period of seed exposure and (2) using natural and artificial seeds (manipulated resource to resemble natural seeds). the proportion of seeds removed was higher after the 24h exposure period (artificial seeds) regardless of site status, and more artificial seeds were removed than natural seeds. our recommendations regarding sampling period depend on whether evaluating impacted or non-impacted sites. although seed removal was greater after 24h in both impacted and non-impacted sites, we suggest that research evaluating the proportion of seeds removed in non-impacted sites should be performed only in the morning period to optimize the sampling time (removal of 60% during this period). when the aim is to compare non-impacted and impacted sites, we suggest evaluating after 24h of exposure, since the impacted sites experienced a higher proportion of seed removal during the afternoon and/or night time periods. furthermore, we recommend the use of artificial seeds because they are easier to obtain and manipulate, and allow us to do comparisons between studies at different regions. we consider these findings an important first step towards standardizing future research on seed removal in brazilian savannas by facilitating fieldwork and allowing comparisons to be made among different studies. sociobiology an international journal on social insects ma angotti1, am rabello1,2, gs santiago1, cr ribas1 article history edited by kleber del-claro, ufu, brazil received 11 august 2017 initial acceptance 08 october 2017 final acceptance 02 february 2018 publication date 09 july 2018 keywords artificial seed, ecological function, formicidae, natural seed, sampling standardization. corresponding author marina angotti universidade federal de lavras departamento de entomologia laboratório de ecologia de formigas po box 3037, cep 37200-000 lavras, mg , brazil. e-mail: marina.a.angotti@gmail.com ants are frequently used as bioindicator organisms for evaluating different types of impacts, such as: agricultural practices (armbrecht & perfecto, 2003; philpott et al., 2008), habitat fragmentation (vasconcelos et al., 2006; leal et al., 2012), fire (parr et al., 2004; andersen et al., 2006; santos et al., 2008; anjos et al., 2017) and mining (dominguez-haydar & armbrecht, 2011; rabello et al., 2015). furthermore, ants are abundant in practically all environments, sensitive to environmental modifications and respond quickly to human disturbances (ribas et al., 2012a; schmidt et al., 2013). in addition, ants carry out important ecological functions and services (folgarait, 1998; philpott et al., 2010), such as ecosystem engineers (frouz & jilková, 2008), predation, biological control of agricultural pests (armbrecht & gallego, 2007; de la mora et al., 2015; morris et al., 2015) and seed 1 universidade federal de lavras, depto. de biologia, setor de ecologia e conservação, lab. de ecologia de formigas, lavras-mg, brazil 2 programa de pesquisa em biodiversidade rede comcerrado, universidade federal de minas gerais, belo horizonte-mg, brazil research article ants mailto:marina.a.angotti@gmail.com ma angotti, am rabello, gs santiago, cr ribas – seed removal by ants156 removal (lima et al., 2013; gallegos et al., 2014; griffiths et al., 2017). seed removal by ants has been used by some studies in tropical regions to evaluate different types of environmental impacts (e.g.: zelikova & breed, 2008; bieber et al., 2014; gallegos et al., 2014; leal et al., 2014); however, there is no standardized sampling method for such purposes. for example, the period of time evaluated and the seed type used to evaluate the effect of environmental impact on seed removal by ants vary widely (christianini et al., 2012; bieber et al., 2014; leal et al., 2014; rabello et al., 2015). sometimes, researchers may have similar aims and use different methodologies for evaluating this, so it would be interesting to standardize methodology when the aims are similar.this lack of a standardized methodology hampers the comparison of responses of seed removal by ants in different vegetation types, habitats and biomes and prevents further advances towards a broader understanding of patterns of ecosystem functioning in response to environmental impacts. some researchers have determined the proportion of seeds removed by ants only after a period of 22 or 24 hours (christianini et al., 2007; bieber et al., 2014) or 48 hours (rocha-ortega et al., 2017) or 96 hours of seed exposure (ferreira et al., 2011). other studies followed this process during the morning and/or afternoon, with different periods of time of observation (parr et al., 2007; christianini et al., 2012; gallegos et al., 2014; leal et al., 2014). in addition to varying time periods, seed removal experiments have also differed in the types of seeds used, with some using natural seeds (christianini et al., 2007; parr et al., 2007; christianini et al., 2012; leal et al., 2014), and others using artificial seeds (manipulated resource to resemble seeds or fruits), both with varying compositions (uehara-prado, 2005; bieber et al., 2014; rabello et al., 2015). thus, this is the first step towards a standardized methodology for the evaluation of seed removal by ants, facilitating the comparison of results among different ecological studies. the aim of this present study is to evaluate the proportion of seeds removed by ants at different sites of brazilian savanna, comparing the use of artificial or natural seeds and two different sampling periods. as a first step towards optimizing methods for evaluating seed removal by ants in savannas, we compared the proportion of seeds removed by ants between: (1) the morning (first hours of the day) and after a 24h period of exposure; and (2) between natural and artificial seeds. we predicted that the proportion of seeds removed by ants will be (1) higher after 24h of seed exposure since the seeds would be available on the ground for a longer period of time. we believe that seed removal occurs during different periods of the day, thus, after 24h of seed exposure may generate an additive effect of seed removal, probably because different ant species have foraging throughout all day. we also predicted that (2) there will be greater removal of artificial than natural seeds, since artificial seeds present a standard size and are composed of a greater variety of nutrients attractive to ants, thus enhancing seed removal by different ant species. material and methods study sites we performed this study in brazilian savannas within southern and northern metropolitan regions in the state of minas gerais, brazil. these regions are classified as tropical with dry winters (april to september) and rainy summers (october to march). sampling was carried out during january and march, period that corresponds to the rainy season, between the years 2012 and 2016. our data come from the combination of other studies to consider different savanna vegetation types, regions, and types of impact, enhancing the broadness of the study. we sampled ants in 37 sites with native vegetation, hereafter referred to as non-impacted sites, including the following vegetation types: cerradão, cerrado sensu stricto, campo limpo, compo rupestre and dry forest. we also sampled 47 sites with different types of environmental impact, hereafter referred to as impacted sites, including: eucalyptus (eucalyptus sp.) monoculture; post-mining rehabilitation with exotic grass (braquiaria decumbens and melinis minutiflora) and pigeon pea (cajanus cajan); and camping sites (characterized by sandy soil with scarce or no herbaceous or shrubby vegetation). evaluating the proportion of seed removal we established a single transect in each site containing at least five sampling points, with a minimum distance of 20 m between each of them. we provided at least ten seeds (natural or artificial) per sampling point, and excluded them from vertebrates using a metal mesh cage (henao-gallego et al., 2012; rabello et al., 2015). the variation in the number of seeds and sampling points is due to the use of different sampling design in different experiments that compose this manuscript. the natural seeds were of croton floribundus (euphorbiaceae), a non-myrmecochorous species, with an average length of 4.7 and width of 4.4 mm (paoli et al., 1995). the artificial seeds were 1.8 mm plastic beads weighing 0.03g and attached in an artificial paste attractive to ants (similar to raimundo et al., 2004 and rabello et al., 2015). although the natural seeds were larger than the artificial seeds, both were classified as of small size according to the classification proposed by pizo and oliveira (2001). to evaluate the influence of period of exposure (during the morning and for 24h) on seed removal proportion, we only used data from the studies with artificial seeds in order to standardize the type of seed. we counted the seeds removed after 4 h of exposition (seed removal during the morning period from 8 a.m. to 12 a.m.) and after 24 h of seed exposure in 24 sites: 11 impacted sites and 13 non-impacted sites. sociobiology 65(2): 155-161 (june, 2018) 157 to compare the seed removal proportion between natural and artificial seeds, we used data from 42 sites using natural seeds (27 impacted sites and 15 non-impacted sites) and 24 sites using artificial seeds (11 impacted sites and 13 non-impacted sites); the sites with artificial seeds were not the same as those with natural seeds. in this case we considered only data for the period of 24h seed exposure. statistical analysis in order to evaluate differences in seed removal proportion between the two seed exposure periods, we performed generalized linear mixed models (glmm) with binomial distribution using the package lme4 in the software r v.3.2.3 (r development core team 2015). seed removal proportion was the response variable, the exposure period (morning or 24 h) was the explanatory variable and the site (non-impacted or impacted) was the random variable. we used glmm because we had pseudo replication of sampling points (due to sampling the same site during the morning and after a 24h period). we used generalized linear model (glm) for the seed type evaluation because there was no pseudo replication. for the glm, seed removal proportion was the response variable and the type of seeds (natural or artificial) was the explanatory variable and we carry out the analyses separately for impacted and non-impacted sites. for both evaluations we used the average proportion of seeds removed per transect performing the analyses using the software r v.3.2.3 (r development core team 2015). results the proportion of seeds removed by ants was greater after seed exposure for 24 h than during the morning period in both non-impacted sites (χ2 = 318.68; p < 0.0001; df = 23) (fig 1a) and impacted sites (χ2 = 649.1; p < 0.0001; df = 19) (fig 1b). in non-impacted sites, the seed removal proportion during the morning period was 60%, which increased to 80% percent after the 24 h exposure period. in impacted sites, the seed removal proportion during the morning period was less than 20%, with an increase to 60% after the 24 h exposure period. seed removal proportion for artificial seeds was greater than for natural seeds, both in non-impacted sites (f = 21.83; p < 0.0001; df = 26) (fig 2a) and impacted sites (f = 13.81; p = 0.0007; df = 36) (fig 2b). fig 1. proportion of artificial seeds removed by ants during the morning period and after 24 h of seed exposure in non-impacted (a) and impacted (b) sites. discussion the present study evaluated the proportion of seeds removed by ants at different sites of brazilian savanna, comparing the use of artificial and natural seeds and two different sampling periods. we observed that the proportion of seeds removed by ants is influenced by the exposure period of the seeds, with it always being greater after a 24 h exposure period, regardless of the type of area (non-impacted or impacted). in addition, artificial seeds removal by ants was higher than natural seeds. the proportion of removed seeds was lower in the morning period than after 24 h of exposure in both impacted and non-impacted sites. this is expected because seeds have a shorter period of exposure. extending the seed exposure period to 24 h may have enabled ants with different diel foraging periods to exploit the resource, and an additive effect on seed removal by diurnal and nocturnal ants, may explain the observed higher seed removal proportion after the 24 h exposure period. however, in non-impacted sites, the highest proportion of seed removal was observed during the morning period (60% out of 80% in 24 h), while in impacted sites few seeds were removed during the morning period (20% out of 60% in 24h). thus, there seemed to be greater ant activity during the evening and/or night time period in these impacted sites, probably due to change in the composition of the ant community (bieber et al., 2014). the lower ant activity in the morning could be a result of our impacted sites possibly having lost some functional groups of ant species which forage during the morning period. thus, the higher seed removal proportion, in our study, during the afternoon would be due to different ant species composition between non-impacted sites which harbor ant species with different diel foraging (aranda-rickert & fracchia, 2012). furthermore, the greater complexity of vegetation in the non-impacted sites may support better microclimatic conditions for ants, including a decrease in soil exposure, which lessens the exposure of ants to dehydration fig 2. proportion of artificial and natural seeds removed by ants after 24 h of exposure in non-impacted (a) and impacted (b) sites. ma angotti, am rabello, gs santiago, cr ribas – seed removal by ants158 and predation, since they are sensitive to temperature and moisture (traniello, 1989; lima & antonialli-junior, 2013), thus, contributing to seed removal during the morning periods. indeed, according to our personal observations, the ground of impacted sites had greater exposure to solar incidence and a lack of herbaceous vegetation, which may also have contributed to the differential ant species composition and activity (williams et al., 2012). based on our results in savanna sites, we suggest that when the main aim of a study is to determine the seed removal proportion in non-impacted sites, sampling could only be performed during the morning period. we make this recommendation because the cost-benefit ratio is better in these sites, with 60% of seed removal occurring during the morning representing a significant sampling, and only 20% being removed during the remaining period. additionally, when the aim of the study is to determine only seed removal in non-impacted sites, a faster sampling (4 hours during the morning) is more feasible and less costly than a sampling performed over a period of 24h, which contributes only 20% to total seed removal. it is also possible to collect the seedremoving ant species during this period. however, if the aim of the study is to compare seed removal proportion within impacted sites, samplings at least after 24 h of seed exposure are recommended, since seed removal in these sites may occur intensively during the night time. however, we think a future research would be interesting to evaluate and compare other periods of seed removal exposure, in order to verify if higher seed removal exposition (i.e. 48 h and 96 h as done by rochaortega et al., 2017; ferreira et al., 2011 respectively) increases percentage of seed removal increasing the robustness of the study. besides, we also consider important to evaluate other parameters of seed removal process such as seed-removing ant composition in diurnal and nocturnal periods, as well as, comparisons of these parameters between non-impacted and impacted sites. species composition is important because it allows observing the presence of harvester (pirk & lopez-decasenave, 2006; belchior et al., 2012) and seed-disperser ants (leal et al., 2014) which can provide a different destination for the seed. we observed that in the afternoon period, seedremoving ants re-start their foraging activity between 15h and 16h as was previously documented by aranda-rickert and fracchia (2012). the period of exposure and type of seed may depend on the aim of the research, however, we consider really important to standardize the sampling methodology in ecological studies that evaluate different environmental disturbances and impacts on ecological function. the seed removal proportion was greater when using artificial than when using natural seeds. this difference may be the result of the composition of the artificial seeds (being near 80% lipids), since the lipid-content have been shown to be major determinants of seed removal by ants (pizo & oliveira, 2001). pizo and oliveira (2001) showed that ants interact for longer periods of time and more frequently with diaspores containing aril or pulp rich in lipids. it is likely that our natural seeds possessed a lower amount of lipid than artificial seeds since most seeds of plants of the genus croton have 34 to 40% lipids (adeyinka et al., 2013; bello et al., 2014). artificial fruits have been used to evaluate ant-diaspore interactions (henao-gallego et al., 2012; bieber et al., 2014; rabello et al., 2015), and are considered an alternative to using natural diaspores, due to their convenience and reliability. such artificial fruits can reduce problems with data collection due to shortage of natural fruits during experimental periods, the use of fruits that are less attractive to ants, and variation in chemical and morphological characteristics of fruits, which may differ geographically (raimundo et al., 2004; bieber et al., 2014). the use of artificial fruits may also help to standardize the methodology of determining the proportion of seeds removed by ants and facilitate comparisons among different studies from different regions (i.e., different biomes), which would not be possible using natural seeds. moreover, attractive artificial fruits can be used in studies that evaluate the rehabilitation of degraded sites, which, in many cases, are initially devoid of natural seeds (henao-gallego et al., 2012; rabello et al., 2015). in this way, artificial seed removal proportion and the presence of seedremoving ant species could determine, in the absence of natural seeds, whether the ecological function of seed removal is being recovered. finally, we consider artificial seeds to be an effective alternativeto natural seeds for studies aiming to evaluate and monitor this ecosystem function under different types of impacts. the attractiveness of artificial seeds is an important factor to consider, especially when the sampling period does not coincide with the fruiting season or when there is no prior access to the site or knowledge about the fruiting plant species present there. in order to standardize the attractiveness of artificial seeds, we suggest using the same composition used in the present study (see also rabello et al., 2015). this artificial seed composition was the same used by raimundo et al. (2004), only with a modification in the type of lipid resource used in our study, and who also reported it as efficiently attractive to seedremoving ants. our study suggests that standardizing the type of seed used and the period of sampling could maximize the acquisition of information regarding this important function of seed removal by ants. lastly, it is important to emphasize that our study was conducted in brazilian savanna, and so we recommend that future researchers carry out more comprehensive studies involving different phytophysiognomies to see how general these findings are. acknowledgments this research was partially supported by a scholarship from fapemig cra ppm 00243/14, under project fapemigcra-rdp-00123-10 “biodiversidade e funções ecológicas de formigas bioindicação de impactos ambientais e de recuperacão de áreas degradadas’’ and by the project apqsociobiology 65(2): 155-161 (june, 2018) 159 03593-12 “desenvolvimento de ferramenta para a priorização de descomissionamento de pequenas centrais hidrelétricas (pch) no estado de minas gerais e estudo de caso para a pch pandeiros”. we are thankful to the vale s.a. company, which allowed us to sample in mines and rehabilitation sites, to mr. ramon braga and mr. cássio mendanha for logistic support and to mr. anderson matos for helping us inside the sites. we also thank the members of laboratório de ecologia de formigas (lef) at the universidade federal de lavras, for their assistance during fieldwork and data collecting. to the laboratory of chemical, biochemical and food analysis (department of food engendering at universidade federal de lavras), for helping to develop the artificial seeds. we are thankful to anonymous reviewers for their critical reading and suggestions. we also thank capes, fapemig, cnpq for their financial support through projects and scholarships. references adeyinka, a. m., joseph, o. b. & amoo, i. a. 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(2008). effects of habitat disturbance on ant community composition and seed dispersal by ants in a tropical dry forest in costa rica. journal of tropical ecology, 24: 309-316. doi: 10.1017/s0266467408004999 395 influence of pollen on the development of africanized bee colonies (hymenoptera: apidae) by anna frida hatsue modro1*, luís carlos marchini2, augusta carolina de camargo carmello moreti3 & emanuel maia1 abstract this study aimed to assess the importance of quality and quantity of pollen on the development of colonies in different seasons. the field experiment was conducted at the apiary of the department of entomolog y and acarolog y of luiz de queiroz college of agriculture, using five beehives of a. mellifera. in order to characterize the quality of pollen, researchers considered measures of total dry mass (g ) and the physical-chemical and pollen composition of pollen load and bee bread samples. the development of hives was assessed according to the area covered by pollen, honey, and brood population in the hive (cm²), as well as fluctuating asymmetry of worker bee wings. spearman’s correlation was calculated among the assessed parameters. the value of ether extract of pollen loads was the only component that was related to the development of hives, its value increased as the area occupied by pollen in the hive decreased (-0.3200*), and as the difference of the number of hamuli of right and left hind wings increased (0.3317*). there was a positive relationship between the wealth (0.3150*) and evenness (0.3019*) of pollen composition and the size of brood population inside the hive. it was concluded that the development of the colony, mainly considering the area occupied by brood, is more successful with increased quantity, wealth, and evenness of collected pollen. key words: apis mellifera, fluctuating asymmetry, physical-chemical composition, bee nutrition, bee pollen. 1universidade federal de rondônia, departamento de engenharia florestal, av. norte sul, 7300, nova morada, cep 76940-000-rolim de moura, ro, brazil 2escola superior de agricultura ‘luiz de queiroz’, departamento de entomologia e acarologia, caixa postal 9, cep 13418-900-piracicaba, sp, brazil 3secretaria de agricultura e abastecimento, apta, instituto de zootecnia, caixa postal 60, cep 13460-000nova odessa, sp, brazil *corresponding author: fridamodro@gmail.com. 396 sociobiolog y vol. 59, no. 2, 2012 introduction pollen is removed from the anthers of flowers and carried by the forager bees to the hive inside their corbicula or pollen baskets, located on the hind legs of the workers. the corbicula is surrounded by a fringe of hair into which pollen is placed to form the load (michener 1974). pollen loads, retained by collectors installed at the entrance of the hive or stored in honeycomb cells (bee bread), indicate period of production and botanical and geographical origins of the product (vanderhuck 1995, marques-souza 1996, carvalho et al. 1999). pollen is used in the feeding of larvae, and also adult bees, soon after their emergence until the beginning of the preparation of the synthesis of royal jelly for feeding the brood and the queen (crailsheim 1992). it is known that the amount and composition of pollen can change the behavior of adult bees, fecundity, life span of bees, the size of the brood stock and honey, colony population, the development of bees, and larval food quality (waller et al. 1981, schmidt et al. 1987, sagili et al. 2005, toth et al. 2005, hoover et al. 2006, mattila & otis 2007, human et al. 2007). it is noteworthy that ioirich (1986) mentions that, in periods of protein resource shortage, and with no other source of protein, the colony may be extinct. considering the factors discussed above, this study aimed to assess the relevance of pollen quality for the development of africanized bee (apis mellifera) colonies in an area of atlantic forest in the city of piracicaba, sp. materials and methods in order to characterize the quality of pollen, researchers considered as parameters: total dry mass (g ), some nutritional components, and the plant origin of pollen load and bee bread samples. to assess the development of hives it was considered the size of the area covered by pollen, honey, and brood population in the hive, as well as fluctuating asymmetry of wings (module of difference between right and left wings) of active worker bees inside the hive. the field experiment was conducted at the apiary of the department of entomolog y and acarolog y of luiz de queiroz college of agriculture, university of são paulo, piracicaba, são paulo, where five beehives of a. mellifera (l.) were installed with a front pollen collector having mean retention rate of 45 ± 1.19 grains (± mean standard error). 397 modro, a.f.h. et al. — influence of pollen on africanized bee development samples were collected in the fall (april 02, 2008 to may 28, 2008), winter ( july 09, 2008 to sept.03, 2008), spring (oct.07, 2008 to dec.02, 2008) and summer ( jan 09, 2009 to march 06, 2009), in a total period of 57 days for each season. pollen loads intercepted in the hives’ entrances were collected every two days, cleaned by picking, and dried at 45°c for 48 hours as suggested by funari et al. (1998a), the bee bread was collected every 15 days in a sorted manner within each hive. all samples were separated by fifteens and stored for further physical-chemical and pollen analyses. melissopalinological analysis of the samples was conducted with the use of acetolysis (erdtman 1960) and the form of subsampling was recommended for the determination of the main pollen types occurring in a sample of heterospecific pollen (modro et al. 2009). the identification of pollen types was based mainly on reference collection of microscope slides from the palynolog y library of the insects laboratory at the department of entomolog y and acarolog y of the luiz de queiroz college of agriculture, as well as specialized catalogs in pollen morpholog y of several species of plants. approximately 900 pollen grains per sample were identified and counted. the values of the physical-chemical composition, in samples of pollen loads and bee bread, were obtained from tests carried out according to silva & queiroz (2002) in the bromatolog y laboratory of the institute of animal science in nova odessa-sp. for pollen load samples, the percentage of dry matter, ether extract, mineral matter, crude protein, and total carbohydrates were measured. for the bee bread, percentages of dry matter and crude protein were determined. in samples with less than 2g, the value of dry matter was calculated from the other samples’ mean moisture. within intervals of 15 days, during the collection period, the development of a. mellifera hives was assessed by measuring the area (cm²) of brood, honey and pollen using the technique of al-tikrity et al. (1971). ten active worker bees inside each hive were collected every two weeks and fixed in 70% alcohol. temporary measuring slides were mounted, where fore and hind, right and left wings were detached and rectilinearly placed between two histological slides. the following left and right hind wing morphological characters were measured, according to silveira et al. (2002): radial subcostal vein, width and length of marginal cell, maximum total length and width 398 sociobiolog y vol. 59, no. 2, 2012 of wing, length of cubital medial vein, length of cubital vein, length of anal vein, width of the 2nd medial cell. the total number of hamuli of the right and left fore wings was counted. measurements were made using a leica stereomicroscope, containing an ocular micrometer, and the results were converted to millimeter units. spearman correlation was carried out among the areas occupied with brood, honey, and pollen, fluctuating asymmetry of wings, values of physical-chemical composition and diversity indexes of shannon-wiever (h’), evenness expressed by pielou ( j’) index, and wealth, calculated by pollen composition. statistical analyses were performed with the help of sas statistical software (sas institute 2003) and the tests were applied to the 5% level of probability. results and discussion the largest area occupied with brood and honey was during the fall, and summer had the largest area with pollen (table 1). sequentially analyzing from the beginning of field assessments, one can observe a marked reduction in occupied area inside the hive between fall and winter, remaining low during spring with an increase in area of pollen in the summer. the occupation of a larger area with pollen in the summer associated with the availability of trophic resources are perhaps two of the factors responsible for increased area of brood and honey in the fall, based on a study conducted by al-tikrity et al. (1972). the size of the bees showed similar absolute values among seasons, which confirms the results obtained by herbert jr. (1988) who found that nutritional stress did not influence the morphometric measurements of the bees, however, it is observed that the measures were slightly larger and more symmetrical in the fall (table 2). although the size of bees is a highly inheritable characteristic (oldroyd et al. 1991), the size associated with greater measures of wind symmetry denote a more stable development of bees, whose subjects have bilateral symmetry. according to woods et al. (1998), if the expression table 1. mean area (cm²) occupied by brood, honey and pollen by beehive of africanized bees (apis mellifera) located in piracicaba, sp, 2008-2009. season resource (cm2) brood honey pollen fall 341.49 389.33 104.00 winter 189.87 126.08 46.14 spring 256.87 90.33 42.94 summer 265.14 82.75 128.86 total (cm2) 1,052.79 688.48 321.94 399 modro, a.f.h. et al. — influence of pollen on africanized bee development of a bilateral character is produced by the same genome, then any asymmetry of the sides is a result of environmental disturbance. by comparing table 1's data with those from table 2 it is possible to observe that the fall was the season that had largest occupied area in the hive and also greater symmetry and size of bees. however, the summer, which was the season with the lowest and smallest area showed low levels of symmetry when compared to bees collected in other seasons, a characteristic table 2. mean values of the asymmetry measurements and float to the morphological characteristics of africanized bees (apis mellifera), piracicaba, sp, 2008-2009. characteristics season* fall winter spring summer hamuli n(1) 21.59±0.112 21.11±0.132 21.18±0.132 20.90±0.118 a(2) 1.179±0.071 1.448±0.102 1.256±0.074 1.284±0.089 total length t(3) 7.91±0.013 7.88±0.014 7.90±0.015 7.86±0.013 a 0.057±0.003 0.059±0.004 0.055±0.003 0.058±0.004 total width t 2.91±0.006 2.89±0.006 2.88±0.008 2.85±0.006 a 0.038±0.002 0.045±0.003 0.044±0.003 0.045±0.004 radial subcostal vein t 3.80±0.007 3.78±0.009 3.78±0.009 3.78±0.009 a 0.075±0.004 0.094±0.006 0.100±0.006 0.087±0.006 cubital medial vein t 2.08±0.005 2.06±0.005 2.05±0.006 2.05±0.006 a 0.056±0.003 0.055±0.003 0.053±0.003 0.047±0.003 cubital vein t 1.77±0.004 1.78±0.004 1.79±0.004 1.78±0.004 a 0.025±0.001 0.025±0.001 0.026±0.002 0.023±0.001 anal vein t 3.88±0.007 3.86±0.007 3.86±0.008 3.85±0.008 a 0.040±0.002 0.047±0.003 0.043±0.003 0.045±0.003 length of marginal cell t 2.97±0.007 2.95±0.006 2.96±0.006 2.94±0.007 a 0.036±0.002 0.037±0.002 0.034±0.002 0.037±0.002 width of marginal cell t 0.45±0.002 0.44±0.002 0.44±0.002 0.44±0.002 a 0.028±0.001 0.030±0.002 0.028±0.002 0.029±0.002 width of 2nd medial cell t 0.81±0.002 0.80±0.003 0.80±0.003 0.78±0.003 a 0.027±0.001 0.029±0.002 0.030±0.002 0.029±0.002 (1)n: mean number of hamuli per wing,(2)a: fluctuating asymmetry calculated from the module difference among the measures of right and left wings,(3)t: total size of the characteristic, measured in millimeters *mean ± mean standard error. 400 sociobiolog y vol. 59, no. 2, 2012 that denotes deviations from perfect symmetry of subjects with bilateral symmetry (soulé 1979). these results are strengthened by the assertion that, fluctuating asymmetry and size of subjects may reflect the instability development caused by stress, which in this study may be due to the amount of food available (scheiner et al. 1991, clarke 1995, nosil & reimchen 2001, kanegae & lomônaco 2003). the area occupied with brood decreased with time of hive exposure to the pollen collector (n = 45, p-value <1% r s = -0.38854). while pereira et al. (2006), attributed the reproductive diapause in hives with collectors to environmental stress, discarding the nutritional factor, the same trend also presented for the areas of pollen and honey, in this study, seems to support the assertion that the use of collectors in africanized bee hives interferes with the development of hives (mclellan 1974, duff & furgala 1986a, 1986b, shawer 1987, funari et al. 1998b). the value of ether extract of pollen loads was the only component that was related, significantly, with the development of hives, with greater value found with reduced area occupied with pollen in the hive, and with increased gap of the number of left and right hind wing hamuli (table 3). pollen with higher percentages of ether extract, when collected, may have been consumed more quickly than that with low percentages of such component, due to its phagostimulant function (singh et al. 1999). regarding the effect on hamuli fluctuation asymmetry, parra (2007) mentions that the presence of linoleic acid, one of the essential fatty acids, is important in the formation of anagasta kuehniella (lepidotera) wings. however, this study cannot come to conclusions regarding the effect of ether extract composition on the development of bees (table 3). the larger the area of brood, the greater the amount of pollen collected by bees (table 3) and the larger the area with pollen in the hive (n = 45, p-value <1% rs = 0.4086). this result reinforces the hypothesis that the presence of brood in the hive (pankiw et al. 1998, dreller & tarpy 2000), as well as the lack of stored pollen (hellmich & rothenbuhler 1986) stimulate forager bees to collect pollen. the pheromone of larvae or contact with the areas occupied by brood and stored pollen indicate the nutritional needs of the 401 modro, a.f.h. et al. — influence of pollen on africanized bee development hive and determine foraging behavior of bees, for the collection of pollen or nectar, even in the case of africanized bees. the area occupied with honey however, was not associated with the area of pollen (n = 45, p-value> 5%; r s = 0.1882) nor with the area of brood (n = 45, p-value> 5% r s = 0.0163), and that suggests that the search for sources of nectar occurs even in the absence of brood or with stored pollen, as proposed by free (1967). there was little relation between quality of pollen and development of the hive, which can be justified based on studies like the one conducted by human et al. (2007), who noted the imbalance of the protein: carbohydrate ratio, which in the opinion of schmidt (1985) would be more logical, in the case of a bee species that is extremely pollen oriented, in the use of resources and that feeds on pollen from a great diversity of plant species, so the cumulative effect of many compounds are responsible for the development and table 3. spearman correlation (n = 45) between the quality and quantity of pollen collected and the development of africanized bee (apis mellifera l.) hives, in piracicaba, sp, 2008-2009. development measures of hive bee bread pollen loads physical-chemical quality (%) ms(g )(1) pb pb ee mm ct area occupied in the hive (cm2) po 0.0429 0.2584 -0.3200* 0.2633 -0.2275 0.2234 honey -0.0555 -0.0448 -0.1175 -0.1768 0.1123 -0.0212 cr -0.1121 -0.0579 0.0165 -0.1033 0.0023 0.5675*** wind fluctuation asymmetry (µm)(2) ha -0.2547 -0.1955 0.3317* -0.2470 0.1147 0.0102 sc+r 0.1034 -0.0775 0.0977 0.1417 0.0129 0.0584 cm -0.1139 -0.1735 -0.0726 -0.1423 0.2388 0.0329 co 0.0727 -0.0441 -0.1109 0.1991 0.0663 0.1170 m+cu 0.0210 -0.0087 0.0780 0.1137 -0.0175 -0.0584 cu -0.0525 -0.0591 -0.1049 0.0855 0.0384 -0.0106 an 0.1150 -0.1181 -0.0522 -0.0723 0.1089 0.1585 la 0.1458 0.1023 -0.1641 0.2301 -0.0638 -0.0548 lm -0.1038 -0.1707 -0.1124 0.0877 0.1787 0.1093 2m 0.2135 0.1147 0.0114 0.0064 -0.1427 0.2198 notes:pollen (po), brood (cr), crude protein (pb), ether extract (ee), mineral matter (mm), total carbohydrates (ct) and dry matter (ms) (1)total amount of dry matter (g ) of pollen loads collected in a 15-day period, (2) fluctuating asymmetry in right and left hind wings, measure in micrometers (µm): radial subcostal vein (sc + r), length of marginal cell (cm) total length of wing (co); cubital medial vein (m + cu); cubital vein (cu); anal vein (an), full width of the wing (la), width of marginal cell (lm), width of the 2nd medial cell (2m). right and left fore wings: number of hamuli (ha), conventional signs used: * significant at 5%, ***significant at less than 0.01%. 402 sociobiolog y vol. 59, no. 2, 2012 behavior of bees, and not some specific compounds, which usually occurs in monophagous species. in accordance with this assertion, table 4 shows a positive relationship between wealth and evenness of pollen composition and the size of the area occupied by brood in the hive. although some studies show that the nutritional quality of pollen may determine the amount to be collected in foraging behavior, with an influential evoluting factor in food preference of bees, according to the needs of the hive (pernal & currie 2001, 2002), in this work, the amount of intercepted pollen in traps did not relate to its physical and chemical quality (n = 45, p-value> 5%). free (1967) and sagili & pankiw (2007) also found no relationship between the amount of pollen collected and its values of carbohydrates and crude protein, respectively. conclusions brood and honey occupied a largest area in hives during the fall, and summer had the largest area with pollen. the size of bees did not vary significantly among seasons. the development of the colony, measured primarily by the size of the area occupied with brood, is more successful with increased amount of pollen collected, wealth, and evenness of pollen composition and less successful with increased time of exposure to pollen collectors. the amount of pollen intercepted in traps shows no relationship to its physical and chemical quality. table 4. spearman coefficient (n = 56) between pollen composition and area occupied in hives by africanized bees (a. mellifera) in piracicaba, sp, 2008-2009. pollen composition area occupied in the hive (cm²) pollen honey bee springs bee bread wealth 0.2361 0.0408 0.3150* diversity 0.0113 -0.0686 0.2174 evenness 0.0871 -0.0831 0.3019* pollen loads wealth 0.0631 -0.0347 0.2484 diversity -0.0187 0.0130 0.0682 evenness -0.0149 0.0286 0.0068 conventional signs used: *significant at 5%. 403 modro, a.f.h. et al. — 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http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1786sociobiology 65(1): 1-9 (march, 2018) special issue the scaling of growth, reproduction and defense in colonies of amazonian termites introduction organisms often need to allocate resources simultaneously to growth, maintenance and reproduction. however, investing in one function often limits investment into other functions, so that selection optimizes phenotypes under the constraint of life-history tradeoffs (reznick, 2014). in eusocial organisms such as ants and termites, individuals differentiate into specialized castes that combine into highly cooperative colonies, suggesting that selection can optimize collective phenotypes (hölldobler & wilson, 2009). thus, tradeoffs may arise at the colony level, shaping resource allocation to different functions and, possibly, to different castes (oster & wilson, 1978; lepage & darlington, 2000; poitrineau et al., 2009). resource limitation abstract phenotypes can evolve through life-history tradeoffs. termites have been the first eusocial insects on earth, prompting life history evolution at the colony level. despite this, termite life-history allocation strategies are poorly known. here, we addressed this issue using novel data on three common species from the diverse, yet understudied amazonian termite fauna: neocapritermes braziliensis, labiotermes labralis and anoplotermes banksi. using oster and wilson’s optimal caste ratio theory and higashi et al.’s termite caste allocation theory as frameworks, we assessed how termite colonies should invest in growth (immatures), reproduction (alates) and defense (soldiers) as they accumulate workers. we also examined whether soldier loss in soilfeeding apicotermitinae (a. banksi) may have affected allocation strategies. we found that: (1) the scaling of immature number was isometric in the three species, contrary to the leveling off expected under resource limitation; (2) colonies of all sizes were equally likely to produce any number of alates, rather than having a size threshold for reproduction; (3) the scaling of soldier number was unrelated to alate production, but varied from isometry in n. braziliensis to negative allometry in l. labralis despite their similar defense strategies; (4) a. banksi had more immatures per worker and a higher maximum alate number per worker than the other species, suggesting that soldier loss may have allowed higher relative investment in colony growth and, possibly, reproduction. termites can provide novel insights into life-history allocation strategies and their relation to social evolution, and should be better incorporated into sociobiological theory. sociobiology an international journal on social insects pacl pequeno1,2, e franklin2 article history edited by og desouza, ufv, brazil received 30 september 2017 initial acceptance 30 october 2017 final acceptance 12 december 2017 publication date 30 march 2018 keywords adaptive demography, caste allocation, isoptera, optimal caste ratio, resource limitation, social insect. corresponding author ecology graduate program, national institute for amazonia research av. andré araújo, 2936, petrópolis cep 69067-375, manaus-am, brasil. e-mail: pacolipe@gmail.com could occur due to depletion of local resources, or due to the need of foragers to travel increasingly larger distances and for a longer time. in any case, foraging efficiency should decline as colonies grow, thus limiting colony productivity (naug & wenzel, 2006; poitrineau et al., 2009). this predicts that the number of newborns in a colony should level off as the number of workers increases (thomas, 2003; kramer et al., 2014). assuming resource limitation, optimal caste ratio theory predicts general patterns of caste allocation in eusocial colonies. the theory postulates that colony fitness is maximized by first producing workers until the colony reaches a certain population size (ergonomic phase), and then investing in sexuals (reproductive phase) (oster & wilson, 1978; poitrineau et al., 2009). this is because producing and maintaining a swarm of 1 ecology graduate program, national institute for amazonia research, manaus, brazil 2 laboratory of systematics and ecology of terrestrial arthropods, national institute for amazonia research, manaus, brazil research article termites pacl pequeno, e franklin – colony life history in termites2 alates (which are often larger than workers and do not forage) is costly and, thus requires a large workforce. thus, the probability of colony reproduction should increase suddenly beyond a threshold colony size. further, many eusocial species have evolved a caste solely committed to colony defense, the soldiers (tian & zhou, 2014). optimal caste ratio theory predicts that soldier number should increase in breeding relative to non-breeding colonies, with a concomitant shift in the way soldier number scales with worker number (oster & wilson, 1978). in non-breeding colonies, mortality should be less costly to larger colonies relative to smaller ones, as the former can partly dispose with their workforce. thus, soldier number should scale sublinearly with worker number (i.e. negative allometry), reflecting a relatively lower investment in defense by larger colonies. conversely, in breeding colonies, the larger commodity represented by sexuals should increase the fitness costs of mortality, particularly in larger colonies. thus, soldier number should scale superlinearly with worker number (i.e. positive allometry), reflecting a relatively higher investment in defense by larger colonies (oster & wilson, 1978). empirical tests of the above predictions have provided mixed results: productivity does not always level off as colonies grow (poitrineau et al., 2009; dornhaus et al., 2012; kramer et al., 2014), colony reproductive effort is often independent of colony size (cole, 2009; dornhaus et al., 2012), and soldier allocation strategy only partially matches theoretical expectations (walker & stamps, 1986; kaspari & byrne, 1995; dornhaus et al., 2012). however, such conclusions are heavily biased towards hymenopterans, particularly ants. termites are the oldest known eusocial organisms and the first to have evolved a soldier caste ever (engel et al., 2016), but their life-history strategies are much less known (lepage & darlington, 2000; pequeno et al., 2017; korb & thorne, 2017). despite this, higashi et al. (2000) offered a quantitative theory of termite caste allocation. within a colony, individuals are assumed to differentiate into particular castes so as to maximize their inclusive fitness, i.e. the propagation of their own genes plus those shared with colony mates. as the colony grows, the conditions affecting which castes fulfill this criterion change, thus shifting the pattern of caste allocation (higashi et al., 2000). two main predictions arise. first, and like optimal caste ratio theory, the probability of producing alates should increase with worker number. second, soldier number should be proportional to extrinsic mortality rate. in species that forage outside the nest (82% of all living species; krishna et al., 2013), this is assumed to reflect forager population size. assuming that foragers compose a constant fraction of the worker population, soldier number is then predicted to be proportional to worker number (i.e. isometry). yet, this theory remains largely untested. moreover, a diverse clade of soil-feeding termites, the apicotermitinae, lost the soldier caste (bourguignon et al., 2016), but the life-history consequences of this event are unknown. here, we assessed colony life-history allocation strategies using novel data on three species from the diverse, yet understudied amazonian termite fauna: neocapritermes braziliensis snyder (termitidae: termitinae), labiotermes labralis holmgren (termitidae: syntermitinae), and the soldier-less anoplotermes banksi emerson (termitidae: apicotermitinae). these are among the most common nestbuilding termites in central amazonia (pequeno et al., 2013; 2015; table 1). we evaluated the following theoretical expectations (fig 1): (1) immature number should scale sublinearly with worker number, assuming that colonies are resource-limited; (2) alate number or, alternatively, the probability of producing alates should increase with worker number, assuming that colonies need to grow to a certain size to reproduce; (3) in the species with soldiers, soldier number should be higher in breeding relative to non-breeding colonies, and scale sublinearly with worker number in the latter but superlinearly in the former, as predicted by optimal caste ratio theory; (4) alternatively, soldier number should scale isometrically with worker number, as predicted by termite caste allocation theory; (5) relative numbers (i.e. per worker) of immatures and alates should be higher in the soldier-less a. banksi than in the other species, assuming that soldier loss frees resources to invest in colony growth and reproduction. trait n. braziliensis l. labralis a. banksi soldier type asm ssm soldier-less worker body mass (mg) 3.0 4.3 0.8 mean colony population 111,332 172,105 19,765 food source fallen dead wood mineral soil organic soil main nest material soil + carton fine soil coarse soil preferred habitat sandy bottomlands clayish uplands mixed-soil slopes nest density (nests/ha) 14.3 3.3 43.3 colony breeding system likely monogamous monogamous monogamous *asm: asymmetrical snapping mandibles; **ssm: symmetrical slicing mandibles. table 1. summary of known life history and ecological traits of the amazonian termite species neocapritermes braziliensis, labiotermes labralis and anoplotermes banksi. data from dupont et al. (2009), pequeno et al. (2013, 2015), bourguignon et al. (2016), and the current study. for species pictures, see pequeno et al. (2015). sociobiology 65(1): 1-9 (march, 2018) special issue 3 materials and methods termite sampling termite colonies were sampled on different occasions in the surroundings of manaus, northern brazil (92 m a.s.l.; 3°06’07’’ s and 60°01’30’’ w). local vegetation is lowland tropical rainforest, with mean annual rainfall of 2479 mm (research coordination in climate and hydric resources, national institute for amazonia research). the terrain is rugged, alternating between clayish plateaus and sandy valleys connected through a dense drainage system. colonies of n. braziliensis (n = 16) were sampled by p. a. c. l. pequeno in may 2010 and february 2011 in the experimental farm of the federal university of amazonas (pequeno et al., 2013). the epigeal nests were removed from the ground and had their dimensions (i.e. height, width and thickness) measured with a tape measure, which were used to estimate nest volume according to a hemiellipsoidal shape: volume = π×height×width×thickness. in casual inspections of whole nests, we observed that individuals (including reproductives) tended to occur at the bottom of nests. thus, a cylindrical soil corer (5 cm in height and diameter) was used to collect standardized nest pieces from all over the nest surface, including the bottom. for each nest, one core was extracted for each 15 cm in nest height, so that sampling effort was proportional to nest size and accounted for heterogeneities in the distribution of castes within the nest. on average, 3.56 cores were extracted per nest, ranging from 2 to 6. termites were manually extracted from cores and preserved in alcohol 75%. this material was transported to fig 1. theoretical scaling relationships of growth, reproduction and defense in colonies of eusocial insects. (a) if colony growth rate declines as colonies grow, the number of immatures should be a decelerating function of the number of workers. (b) if colony reproduction requires a minimum workforce, the probability of reproducing should be a sigmoid function of worker number. (c) optimal caste ratio theory (oster & wilson 1978) predicts that the number of soldiers should be higher in breeding than in non-breeding colonies, and that the relationship between soldier and worker number should be a concave-up curve in the former and a concave-down curve in the latter. (d) termite caste allocation theory (higashi et al. 2000) predicts that soldier number should be proportional to worker number, assuming that workers are exposed to extrinsic mortality outside the nest. pacl pequeno, e franklin – colony life history in termites4 laboratory, where castes were sorted and counted, as follows: workers, soldiers (including presoldiers), alates (both winged nymphs and imagoes) and immatures (i.e. undifferentiated nymphs). whole-colony caste populations were estimated by extrapolating average counts per nest core to the volume of the whole nests. colonies of l. labralis (n = 18) were sampled by j. a. ribeiro in october 1993 and january 1995. colonies were collected from two sites: half in ducke reserve, and half in a reserve of the biological dynamics of forest fragments project (pdbff). the arboreal nests were measured (i.e. height, width and thickness) with a tape measure, and nest volume was estimated as before. likewise, cores with standardized volume were taken randomly from the surface of each nest with a cylindrical soil corer (10 cm in height, 3 cm in diameter), with one core for each 3.5 cm in nest height. this resulted in an average of 31 cores per nest, ranging from 16 to 62. termite castes were extracted, sorted, counted and had their wholecolony populations estimated as before. colonies of a. banksi (n = 17) were also sampled in the two aforementioned sites in two occasions: by c. martius and j. a. ribeiro from may to october 1993 and march 1994 (n = 7) (martius & ribeiro, 1996), and by f. apolinário from october 1996 to january 1997 (n = 10). in both cases, whole nests were taken to the laboratory and termites were exhaustively extracted either manually or using berlese-tullgren funnels, until no further termites were found. then, castes were sorted as described previously, and their populations were counted. statistical analyses for each species, whole-colony estimates of investment in growth, reproduction and defense were defined as the numbers of immatures, alates and soldiers, respectively (except for a. banksi, which is soldier-less). then, the scaling of these traits (dependent variables) was assessed by determining how they changed with the number of workers (independent variable), which comprise the bulk of the colony and supply the resources allocated to all castes. estimates of caste numbers obtained by extrapolation were rounded down, so that they could be modelled statistically as counts (which assumes integers). unless otherwise stated, analyses assumed a standard scaling function (y = a×xb), which was linearized by taking logs of both sides (logy = loga + b×logx). this allows the use of standard linear statistical models for inference. accordingly, each dependent variable was analyzed as a function of log-transformed number of workers using a generalized linear model (glm), assuming log link and poisson-distributed errors corrected for overdispersion. in the case of reproduction, we also analyzed (1) the scaling of alate number only among colonies containing alates (as such relationship may only be evident during the reproductive season), and (2) the relationship between presence-absence of alates and worker number, as an indicator of probability of reproduction. in the latter case, we assumed logit link and binomial-distributed errors. to test the predictions of optimal caste ratio theory on soldier allocation, we followed previous studies (walker & stamps, 1986; kaspari & byrnes 1995) and used the presenceabsence of alates as a covariate to represent the reproductive status of the colony (i.e. breeding vs. non-breeding). as the model predicts an independent effect of reproductive status on soldier number plus an interaction between this variable and worker number (fig 1c), we compared a series of nested models using deviance-based f tests, in the following order: a model including an interaction term against a model including independent effects only, and the latter against a model including number of workers as the sole predictor. to test for deviations from isometry (i.e. b ≠ 1) in the scalings of growth and defense, we determined whether 95% confidence intervals (95% ci) of estimated scaling exponents included 1. predictive power (r2) was calculated as the proportion of deviance accounted for by the fitted glm relative to the intercept-only model. lastly, we looked for interspecific differences in lifehistory allocation strategy by comparing the numbers of immatures, alates and soldiers per worker among species. for each dependent variable, we used a glm assuming inverse link and gamma-distributed errors; pairwise comparisons among species were performed with tukey’s test. a single n. braziliensis colony contained no immatures; this observation was excluded when comparing immature:worker ratios to meet distributional assumptions. further, because the proportion of colonies producing alates varied between species, alate: worker ratios were compared only among breeding colonies. all analyzes were performed in r 3.3.2 (r development core team, 2016), with support of package “multcomp” (hothorn et al., 2008). results species showed either similar or contrasting life-history allocation patterns, depending on the caste considered. first, there was evidence that species differed in the way they invested in immatures and soldiers across colony sizes. in n. braziliensis, the number of immatures scaled superlinearly with that of workers (t = 2.52, p = 0.02) (fig 2a), whereas it scaled sublinearly in l. labralis (t = 6.19, p < 0.001) (fig 2b) and a. banksi (t = 4.28, p < 0.001) (fig 2c). yet, scaling exponents were not statistically different from 1, so that isometry could not be conclusively rejected in any of the three species (table 2). second, reproduction was unrelated to worker number, both in terms of alate number (n. braziliensis: t = 1.18, p = 0.25; l. labralis: t = 0.78, p = 0.44; a. banksi: t = 0.81, p = 0.42) (table 2, fig 3a,b,c) and probability of producing alates (n. braziliensis: z = 1.04, p = 0.29; l. labralis: z = 1.30, p sociobiology 65(1): 1-9 (march, 2018) special issue 5 = 0.19; a. banksi: z = 0.44, p = 0.65) (table 2, fig 3d,e,f). considering the relationship between numbers of alates and workers only among breeding colonies revealed the same result (n. braziliensis: t = 0.85, p = 0.45; l. labralis: t = 0.37, p = 0.71; a. banksi: t = 0.85, p = 0.42). third, there was evidence for different scalings of soldier number between species. in n. braziliensis, there was no support for an interaction between worker number and breeding status (f = 2.09, p = 0.17), nor for an independent effect of the latter (f = 0.01, p = 0.89); rather, soldier number was proportional to worker number (t = 4.93, p < 0.001) (table 2, fig 4a). in l. labralis, breeding status had no effect on soldier number as well (interaction model: f = 1.33, p = 0.26; independent effect model: f = 1.66, p = 0.21), but soldier number scaled with worker number with an exponent significantly lower than 1 (t = 2.30, p = 0.03) (table 2, fig 4b). the mean number of immatures per worker was highest in a. banksi (1.60 ± 9.34, mean ± se) and lowest in n. braziliensis (0.15 ± 0.05), with l. labralis in the middle (0.66 ± 7.52); all pairwise differences were statistically significant (z > 3.0 and p < 0.01 in all cases). fewer colonies were breeding in n. braziliensis (five colonies out of 16, or 31%) than in l. labralis (nine colonies out of 18, or 50%) and a. banksi (nine colonies out of 17, or 52%). moreover, imagoes only occurred in l. labralis and a. banksi; breeding colonies of n. braziliensis contained winged nymphs only. still, considering only breeding colonies, there was no difference in mean number of alates per worker among species (|z| > 0.96 and p > 0.26 for all comparisons), with colonies averaging 0.03 ± 0.05 alates per worker. likewise, there was no difference in mean number of soldiers per worker between n. braziliensis and l. labralis (t = -0.55, p = 0.58), with both species averaging 0.05 ± 0.38 soldiers per worker. fig 2. whole-colony scalings of growth in three amazonian termite species. points represent colonies. solid lines represent statistically significant glm fits at the 0.05 level, as indicated in table 2. response species intercept slope r2 number of immatures n. braziliensis -5.58 (-19.9 – 4.87) 1.32 (0.41 – 2.52) 0.40 l. labralis 1.12 (-2.23 – 4.22) 0.86 (0.60 – 1.14) 0.74 a. banksi 2.24 (-1.25 – 5.38) 0.79 (0.44 – 1.17) 0.60 number of alates n. braziliensis 5.1 (2.31 – 6.83) 0.00 (0.00 – 0.00) 0.00 l. labralis -3.49 (-36.03 – 15.75) 0.78 (-0.93 – 3.48) 0.00 a. banksi -0.77 (-22.64 – 11.94) 0.43 (-0.75 – 3.05) 0.00 prob(alate) n. braziliensis -9.58 (-29.36 – 5.62) 0.78 (-0.57 – 2.50) 0.00 l. labralis -9.12 (-24.66 – 3.73) 0.81 (-0.32 – 2.18) 0.00 a. banksi -2.12 (-11.99 – 6.77) 0.23 (-0.80 – 1.37) 0.00 number of soldiers n. braziliensis -2.32 (-6.97 – 1.85) 0.94 (0.58 – 1.34) 0.70 l. labralis 2.75 (-2.20 – 7.25) 0.47 (0.08 – 0.90) 0.27 table 2. summary of glm results on the scaling of caste numbers with worker number in three amazonian termite species. numbers of immatures, alates and soldiers were modeled assuming poisson-distributed errors (corrected for overdispersion) and log link. prob(alate), or the probability of producing alates, was modeled using alate presence-absence and assuming binomial errors and logit link. worker number was log-transformed in all analyzes. numbers in parentheses are 95% confidence limits; numbers in bold are slopes significantly different from zero. n. braziliensis: neocarpitermes braziliensis; l. labralis: labiotermes labralis; a. banksi: anoplotermes banksi. pacl pequeno, e franklin – colony life history in termites6 discussion optimal caste ratio theory (oster & wilson, 1978) and termite caste allocation theory (higashi et al., 2000) make several predictions about how eusocial colonies should allocate resources to growth, reproduction and defense as their workforce increases (fig 1). using data on three common amazonian termite species, we found that most such predictions were not supported, suggesting that neither theory provides a generally valid explanation for life-history allocation strategies in termites. the fact that immature number did not level off as worker number increased suggests that colonies were not resource-limited in the studied species (kaspari, 1996; kramer et al., 2014). in line with this, the nest density of the three termite species was unrelated to the density of their respective food sources across the landscape (pequeno et al., 2015). it is also possible that extrinsic mortality rates are so high that colonies die before their growth saturates, as suggested for litter ants (kaspari, 1996). there are few data on termite colony demography, but bourguignon et al. (2011) estimated that around a third (and up to a half) of the nests of a. banksi within 1 ha of rainforest in french guiana died each year. whether this is high enough to prevent colonies from reaching their limiting size is unclear. yet, a non-saturating increase in colony productivity with colony size has also been reported for many ants and other hymenopterans (kaspari & byrne, 1995; kaspari, 1996; billick, 2001; bouwma et al., 2006; mcglynn, 2006; smith et al., 2007). taken together, these results suggest that, under natural conditions, colony growth is less constrained than usually assumed. the independence between reproduction (either measured as number of alates or as probability of producing alates) and worker number contradicts both optimal caste ratio theory and termite caste allocation theory (higashi et al., 2000; oster & wilson, 1978; poitrineau et al., 2009). this result also contrasts with the suggestion of a colony size threshold for reproduction in termites, albeit this is based on very few species (lepage & darlington, 2000). in the better studied hymenoptera, the uncoupling between reproduction and colony size seems to be at least as common as size-dependent reproduction within species (kaspari & byrne, 1995; beekman et al., 1998; cole, 2009). importantly, we found that alate number was independent of worker number even when considering breeding colonies only, so that variation in breeding status among colonies cannot account for this result. this may reflect the finding that, contrary to theory assumption, colonies did not appear to be resource-limited. if resources are abundant enough to allow production of alates early in the colony life cycle, there may be no fitness advantage in postponing reproduction until some threshold colony size. optimal caste ratio theory was also refuted by the observed patterns of soldier allocation in n. braziliensis and l. labralis, as soldier number was independent of colony breeding status. in parallel, termite caste allocation theory was only partially supported: while soldier number was predicted to be proportional to worker number in both species, this was fig 3. whole-colony scalings of reproduction in three amazonian termite species. points represent colonies. dashed lines represent statistically non-significant glm fits at the 0.05 level, as indicated in table 2. prob(alate): probability of producing alates. sociobiology 65(1): 1-9 (march, 2018) special issue 7 only the case in n. braziliensis; in l. labralis, soldier number scaled sublinearly with worker number. while the reason for this is unclear, our data hint at an apparent association between the scaling exponents of immatures and soldiers across species: although the scaling of immature number did not differ significantly from isometry in any species, both exponents were lower in l. labralis than in n. braziliensis (table 2). thus, the species seemingly experiencing stronger growth limitation (l. labralis) also appeared to invest relatively less in soldiers as colonies grew. as soldiers are generally costly to produce and maintain (tian & zhou, 2014), stronger resource limitation might constrain soldier allocation. while the scalings of growth and reproduction largely coincided among species, relative investment in the former differed markedly: a. banksi colonies produced 2.4 and 10.6 times more immatures per worker than l. labralis and n. braziliensis, respectively. moreover, although there were no consistent interspecific differences in relative investment in alates, the maximum number of alates per worker was much higher in a. banksi (0.45) than in the other species (ca. 0.027). this is consistent with the idea that dispensing with soldiers has allowed greater investment in other colony functions such as growth and reproduction, by freeing energy previously used in soldier production and maintenance. other species traits may account for this difference, though. for instance, across ant species, mean alate number increases at a decelerating rate with mean worker number, so that species with smaller colonies tend to invest on a relatively larger number of smaller alates (shik, 2008). if the same applies to termites, a. banski may achieve a higher relative reproductive investment simply by having relatively small colonies (table 1). discriminating between these alternative hypotheses will require a broad comparative analysis of termite life-history allocation strategies. our analysis did not cover one important aspect of termite caste allocation, the number of reproductives. several termite species are known to produce varying numbers of secondary reproductives (korb & thorne, 2017), which may affect caste allocation patterns (thorne, 1984). however, secondary reproductives were not found in any of the colonies used in this study; whenever reproductives were found, there was always a single pair (table 1). for a. banksi and l. labralis, this has been confirmed by experimental orphaning of colonies (bourguingon et al., 2016) and genetic data (dupont et al., 2009), respectively. our data on number of reproductives in n. braziliensis and l. labralis colonies is not conclusive as nests of these species were subsampled. yet, for a. banksi (whose entire nests were collected), only 2 nests in 17 (ca. 12%) did not had a queen, so that queen number was essentially held constant in the analyses of this species. therefore, it is unlikely that our results were affected by variation in number of reproductives. in conclusion, this study showed that optimal caste ratio theory and termite caste allocation theory generally failed to predict life-history allocation patterns of three common amazonian termite species. yet, we provide evidence that dispensing with a soldier caste may allow for higher investment in colony growth and reproduction. we propose that the level of resource limitation experienced by colonies (as measured by the scaling between immature and worker number) may partly account for interspecific variation in the scaling of soldier number. in turn, resource limitation may itself depend on other species traits, such as foraging distance, food quality and degree of susceptibility to the effects of competitors and/or predators during foraging (araújo et al., 2017). yet, we stress that current theory largely focuses on scaling exponents between caste numbers, and other factors could also influence caste allocation patterns. in fact, r2 varied fig 4. whole-colony scalings of defense in two amazonian termite species. points represent colonies. solid lines represent statistically significant glm fits at the 0.05 level, as indicated in table 2. pacl pequeno, e franklin – colony life history in termites8 between 0.00 a 0.74 in this study, indicating that theoretical relationships may account for small fractions of variance in caste numbers. moreover, sociobiological theory has largely developed from studies on hymenopterans, even though there is increasing evidence that termite life history evolution has differed from this paradigm in important ways (pequeno et al., 2017). a broad comparative analysis of termite lifehistory allocation strategies will likely provide new insights into social evolution and help refine sociobiological theory. acknowledgements we are grateful to the staff of the experimental farm of the federal university of amazonas, jonatha pereira da silva, rosinaldo conceição nascimento, pedro josé dos santos fernandes, and ana paula porto for their support during fieldwork. we also thank joana d’arc ribeiro, fabiano apolinário and christopher martius for providing data on termite colony composition. the first author received a posgrad scholarship from the foundation for research support of amazonas state (fapeam) and financial support from the national council of scientific and technological development (cnpq) (grants: 470375/2006-0; 558318/20096) during fieldwork, and a scholarship from the brazilian coordination for training of higher education personnel (capes) during the preparation of this manuscript. this study is dedicated to the memory of joana d’arc ribeiro (1953 – 2006), whose work contributed pioneering data on colony life history of amazonian termites. authors’ contribution pacl pequeno and e franklin conceived the study and wrote the manuscript; pacl pequeno assembled and analyzed the data. references araújo, a.p.a., cristaldo, p.f., florencio, d.f., araújo, f.s. & desouza, o. 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(1986). a test of optimal caste ratio theory using the ant camponotus (colobopsis) impressus. ecology, 67: 1052–1062. doi: 10.2307/1939828 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.405-412sociobiology 60(4): 405-412 (2013) temporal variation in the abundance of orchid bees (hymenoptera: apidae) in a neotropical hygrophilous forest mmn castro1, ca garófalo1, jc serrano1, ci silva1,2 introduction bees of the tribe euglossini, also known as orchid bees, are characterized by a relatively long tongue, highly modified hind tibias in males where they can store fragrances and, in most cases, a bright metallic tegument (cruz landim et al., 1965). euglossines occur in the neotropics and are found from southern north america (minckley & reyes, 1996) and northern mexico to northern argentina (dressler, 1982), at altitudes that vary from sea level to 1,500 and 1,600 m a.s.l.; euglossines are rare above 2,000 m a.s.l. (dressler, 1982). the fast flight, characteristic of this bee group and the rarity of finding them at flowers (nemésio & silveira, 2007), probably explains why these bees have been underestimated in studies on apoidea communities in the past. however, the accidental discovery of male attraction (lopez, 1963) to synthetic terpenoids and aromatic hydrocarbons, mimetic of chemical products found in floral fragrances (rebêlo & caabstract although bees are important pollinators in several ecosystems around the world, studies on bee diversity in hygrophilous forests are scarce. this type of vegetation is restricted to permanently wet soils and, therefore, has particular floristic, structure and physiognomy. the goal of the present study was to inventory and analyze the temporal variation of the euglossine bees that occur in a neotropical hygrophilous forest. in order to sample male bees we used four chemical baits, eucalyptol, eugenol, vanillin, and methyl salicylate. the captures were made once a month, from march 2010 to february 2011, from 9:00 am to 12:00 pm. we captured 113 individuals of three genera and five species (in decreasing order of abundance): eulaema nigrita lepeletier, 1841 (n=52), euglossa pleosticta dressler, 1982 (34), exaerete smaragdina (guérin-méneville, 1844) (12), euglossa carolina nemésio, 2009 (11), and euglossa fimbriata rebêlo & moure, 1968 (4). the most attractive bait was eucalyptol (n=98), followed by vanillin (11), and eugenol (4). both temperature and rainfall affected significantly the distribution of the number of males throughout the year. the highest number of euglossini species and individuals was sampled in the warm and rainy season, with activity peaks varying among species. sociobiology an international journal on social insects 1universidade de são paulo (ffclrp-usp), ribeirão preto, são paulo, brazil. 2universidade federal do ceará (ufce), fortaleza, ceará, brazil. research article bees article history edited by: candida m l aguiar, uefs, brazil received 19 june 2013 initial acceptance 19 july 2013 final acceptance 29 july 2013 keywords richness, abundance, euglossini, chemical baits, temporal fluctuation. corresponding author cláudia inês da silva faculdade de filosofia, ciências e letras de ribeirão preto – usp avenida bandeirantes 3900 14040-901 ribeirão preto, sp, brazil e-mail: claudiainess@gmail.com bral, 1997), allowed to quickly advance the knowledge about the ecology of these bees. in addition to floral fragrances, euglossini males are also attracted to a variety of non-floral fragrances, which are produced in wood, fungi, tree wounds, fruits (ackerman, 1983; whitten et al., 1993), and feces (eltz et al., 2007). the studies carried out by roubik and hanson (2004) and zimmermann et al. (2006) confirmed that the fragrances collected by males attract conspecific males, supporting the idea that those fragrances are analogous to pheromones and probably involved in mate recognition and choice (zimmermann et al., 2009). although the development of fragrances in the past 40 years allowed a large increase in the sampling of euglossini communities in several brazilian ecosystems, hygrophilous forests have been poorly studied. also known as seasonal semi-deciduous riparian forests with permanent fluvial influence (rodrigues, 2004), broadleaf hygrophilous forests, swamp forests (leitão filho, 1982), or just hygrophilous forests (toniato et al., 1998), this vegetation is characterized by mmn castro, ca garófalo, jc serrano, ci silva orchid bees of a hygrophilous forest406 a permanently flooded soil, contrary to periodically flooded riparian forests (toniato et al., 1998), and has the important function of protecting water sources (joly, 1992; marques, 1994; teixeira et al., 2008). currently, hygrophilous forests are extremely vulnerable to human impacts (teixeira et al., 2008). this kind of vegetation has been largely devastated in the state of são paulo (torres et al., 1994), and so has progressively disappeared before being studied (gomes et al., 2006). until the present study no information on euglossini communities of hygrophilous forests was available. these bees are important pollinators in several brazilian biomes and have close relationships with several plants (rocha-filho et al., 2012; silva et al., 2012) of different families (apocynaceae, bignoniaceae, clusiaceae, commelinaceae, convolvulaceae, cucurbitaceae, leguminosae, melastomataceae, myrtaceae, rubiaceae, solanaceae and verbenaceae) that also occur in hygrophilous forests (castro, personal observation). hence, the objective of the present study was to describe the richness, abundance and the diversity of euglossini bees that occur in a fragment of hygrophilous forest, as well as to assess the efficiency of odor baits in the attraction of males in this environment and to test for an effect of climate on the distribution and temporal fluctuations in the abundance of euglossini bees throughout the year. material and methods study area the study was carried out in reserva toca da paca (hereafter rtp), classified as a private reserve of natural heritage by the brazilian environmental law. this private reserve is within the boundaries of boa vista farm, on the margins of the mogi-guaçu river (21º27’30” s 48º05’12” w) at 510m a.s.l., in the city of guatapará, são paulo state, southeastern brazil (fig 1). the 187ha of rtp’s area has a predominant vegetation of hygrophilous forest which, despite being situated in a transition area of the cerrado and semideciduous forest vegetation types, is currently surrounded by agricultural crops, especially sugarcane. the regional climate type is aw (köppen’s classification), with two well-defined seasons: a dry and cold season, from april to september (autumn-winter), with the average temperature varying between 19.3 and 22.9 ºc and 201.5mm of total rainfall; and a rainy and warm season, from october to march (spring-summer), with the average temperature varying between 23.8 and 25.2ºc and 982.1mm of total rainfall (data from centro de pesquisas meteorológicas e climáticas aplicadas a agricultura unicamp). sampling of male bees we sampled bees monthly from march 2010 to february 2011 on a 50m long transect, inside the fragment and parallel to its edge. the distance between transect and the edge of the fragment was 80m and the nearest crop sugarcane was 200m (fig 1). we placed on this transect four odor baits with eucalyptol, eugenol, vanillin, and methyl salicylate, which proved to be efficient for attracting euglossine male bees (rebêlo & garófalo, 1991; sofia & suzuki, 2004). we prepared the baits using a wad of paper soaked with only one fragrance, which was tied with a string and attached to the vegetation on fig 1. location of the area where the euglossini bees were sampled in guatapará, são paulo state, brazil. the thicker gray line marks the edge of reserva toca da paca natural vegetation. a: distance of 80m from the sample point to the edge of reserva toca da paca’s natural vegetation. b: distance of 200m from the sample point to the edge the sugarcane plantation. sociobiology 60(4): 405-412 (2013) 407 a shaded place 1.5m above the ground and 10m away from other odor baits. sampling was always carried out at the same sites on a transect and each sampling session lasted three hours, from 9:00am to 12:00pm. this interval corresponds to the period of highest activity of males in the field in northeastern são paulo state (rebêlo & garófalo, 1991). at each half an hour we replaced the essences to compensate the loss of volatile compounds, as suggested by sofia and suzuki (2004). we captured the bees attracted to the baits with an insect net and stored them individually in 10ml falcon tubes, where they were killed with ethyl acetate vapor released by a paper soaked with the substance and attached to the cap. the specimens were deposited in the collection of bees and solitary wasps at the department of biology, ffclrp-usp. data analysis correlations between species richness, the number of individuals collected each month and average temperature and rainfall were made by pearson coefficient (r), following zar (1999). the software used for these calculations was program r version 2.15.2 (r development core team, 2012). to test the monthly distribution of the males activities in the range of one year, we used the rayleigh test of uniformity (z) and ran the calculations in the software oriana 4.0 (kovach computing services, 2012), in which months were converted to angles, beginning at 30º, which corresponds to january, and ending at 330º, which corresponds to december, in intervals of 30º. after the conversion, we calculated the mean date (a) of the capture frequency of males and the concentration (r) of this event around the mean angle. in the histogram, the vector indicates the mean angle, which corresponds to the mean date of occurrence of the event. the hypotheses were: h0= the males of each species are evenly distributed throughout the year and, hence, there is no temporal variation; h1= the males of each species are unevenly distributed throughout the year and, hence, there is temporal variation. in case h1 is true, i.e., (p<0.05), the concentration of male captures around the mean angle, denoted by (r), may be considered a measurement of temporal variation. according to morellato et al. (2000), r=0 when the distribution is even throughout the year, and r=1 when the distribution is concentrated around a single month. in discussion, to compare the euglossini richness of rtp with other areas of the same region in northeastern são paulo state, a rarefaction curve was made to equalize the differences in sampling effort. the diversity of bees were calculated by shannon-wiener index, and the values of the indices were compared using hutcheson’s t-test (hutcheson, 1970). uniformity and dominance were quantified following pielou index and berger-parker index (magurran, 2004) respectively. we also applied sørensen similarity coefficient (sørensen, 1948) to compare species composition among areas. since this coefficient does not consider abundance, it reduces the chances of errors caused by studies with different sampling effort. all of the aforementioned statistical analyses were done using the software past 2.17c (hammer et al., 2001). results we collected 113 males of five species and three genera in the studied hygrophilous forest. among the species studied, the most abundant was eulaema nigrita lepeletier, 1941, with 46% of all collected individuals, followed by euglossa pleosticta dressler, 1982 (30.1%), exaerete smaragdina (guérin-méneville, 1844) (10.6%), euglossa carolina nemésio, 2009 (9.7%), and euglossa fimbriata rebêlo & moure, 1968 (3.5%) (table 1). among the four odor baits used, eucalyptol attracted males of all species and also the highest number of males (n=98). except for eg. carolina, and eg. fimbriata, whose males were attracted exclusively to eucalyptol, the other species were attracted to at least two different kinds of odor baits (table 1). methyl salicylate did not attract any males. during the study, the average monthly temperature table 1. number of euglossini bees collected with eucalyptol (ec), vanillin (va), eugenol (eg) and methyl salicylate (ms) in reserva toca da paca, guatapará, brazil, from march 2010 to february 2011. varied from 18 to 26 ºc and monthly rainfall varied from 0 to 296 mm (fig 2). we observed the highest rainfall and temperature values between november and march, when the highest number of specimens was sampled (fig 2). species richness showed a significant correlation with temperature (r=0.85; p<0.01) and rainfall (r=0.83; p<0.01); male abundance also showed a significant correlation with temperature (r=0.92; p<0.01) and rainfall (r=0.81; p<0.01). the rayleigh test indicated a temporal variation in the occurrence of bees in rtp (r=0.50; z=28.35; p<0.01) (fig 3a), with concentration of individuals around january. we also observed temporal variation in different species separately, except for eg. fimbriata (r=0.69; z=1.91; p=1.15) (fig 3c), which was not sampled in december; this species, though, has a marked occurrence period between october and february. euglossa carolina (fig 3b) and eg. pleosticta (fig 3d) showed activity peaks in january, and el. nigrita (fig 3e) was the only species sampled throughout the year, species n % aromatic baits ec va eg ms eulaema nigrita 52 46 47 5 0 0 euglossa pleosticta 34 30.1 29 2 3 0 exaerete smaragdina 12 10.6 7 4 1 0 euglossa carolina 11 9.7 11 0 0 0 euglossa fimbriata 4 3.5 4 0 0 0 abunda nce 113 100 98 11 4 0 richness 5 5 3 2 0 mmn castro, ca garófalo, jc serrano, ci silva orchid bees of a hygrophilous forest408 with a peak in december (table 2 and fig 3e). el. nigrita, as well as the other species, had no males attracted to the baits in august. discussion the euglossine bees sampled in the studied fragment of hygrophilous forest have also been observed in other environments of northeastern são paulo state, brazil. (table 3). this similarity in bees species occurrence may be explained by the fact that the hygrophilous forest is composed of plant species that are also found in other vegetation types that occur in this region, such as the semi-deciduous forest and the cerrado. in addition, the geographic closeness and the relative similar altitudes of the areas in the northeastern são paulo state region may influence the share of the species composition of this tribe of bees, as has been observed in other studies (brosi, 2009; cordeiro et al., 2013). in contrast, species richness, expressed by the rarefacfig 2. monthly rainfall (mm), average temperature (oc), and number of euglossini males collected each month in reserva toca da paca, guatapará, brazil. from march 2010 to february 2011. fig 3. temporal variation of the total abundance and for each euglossini species sampled in reserva toca da paca, guatapará, brazil, from march 2010 to february 2011. the line at the top of the vector stands for standard deviation. except for eg. fimbriata with p=0.151, the remaining species presented p<0.01. the test values corresponding to these distributions are shown in table 2. e u glo s s a fim bria ta j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 e u glo s s a ple o s tic ta j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 e u la e m a n igrita j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 e x a e re te s m a ra gdin a j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 a f b c d e t ota l numbe r of indiv idua ls j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 e u glo s s a c a ro lin a j a nua ry f e brua ry m a rch a pril m a y j unej uly a ugus t s e pte mbe r o ctobe r n ov e mbe r de ce mbe r 20 20 20 20 10 10 10 10 table 2. results of circular statistic analyses testing for the occurrence of temporal variation in the abundance of euglossini species sampled in reserva toca da paca, guatapará, brazil, from march 2010 to february 2011. rayleigh test was performed for significance of the mean angle. eulaema nigrita euglossa pleosticta exaerete smaragdina euglossa carolina euglossa fimbriata total number of individuals number of males (n) 52 34 12 11 4 113 mean angle 354.43° 28.62° 41.45° 356.31° 345° 12.47° mean date (a) december january february december december january concentration (r) 0.39 0.64 0.68 0.64 0.69 0.50 rayleigh test (z) 7.95 14.08 5.56 4.51 1.90 28.35 rayleigh test (p) <0.01 <0.01 <0.01 <0.01 0.151 < 0.01 tion curves (fig 4), indicates a lower number of species than observed in other areas of northeastern são paulo state. this reflects in the sørensen similarity coefficient presented by cluster analysis in which the rtp is the least similar to other areas of same region (fig 5). the euglossini richness in areas of semi-deciduous forest varies from eight to 14 species (rebêlo & garófalo, 1997; jesus & garófalo, 2000). also in a semi-deciduous forest, but very close to the ecotone with cerrado, silveira et al. (2011) sampled 13 species, whereas hirotsu et al. (2010) in a cerrado and rebêlo and garófalo (1991) in a second-growth area (capoeira) sampled eight species each. the difference in euglossini richness found between rtp and other areas studied by rebêlo and garófalo (1991, 1997) and jesus and garófalo (2000) may be due to the size of the areas studied, since the fragments studied by these authors were significantly larger than rtp. another reason for the smaller number of species observed in the present study, compared to the one made by silveira et al. (2011), could be a difference in sampling effort, which in this latter were analyzed two simultaneous sampling points against one of the rtp. storck-tonon et al. (2013) reported that those differences is also caused by differences in the index of edge in the fragment, the connectivity and the landscape structure, or as observed by miletpinheiro and schlindwein (2005) affected by the surrounding matrix of the studied area. the abundance of euglossini bees in the rtp was also lower than other studied areas in the region, except the area studied by hirotsu et al. (2010) (table 3). eulaema nigrita and eg. pleostica were by far the most frequent species throughsociobiology 60(4): 405-412 (2013) 409 out the year and also the most abundant species, representing together 76% of all individuals collected in the present study. this was expected, since these species were also the most common in all other studies carried out in the same region of são paulo (rebêlo & garófalo, 1991, 1997; jesus & garófalo, 2000; hirotsu et al., 2010; silveira et al., 2011). however, while in rtp and the area studied by rebêlo and garófalo (1997) el. nigrita was the most abundant bee, being responsible for a dominance of 0.46 and 0.27 respectively, in the other studied areas eg. pleosticta was the most numerous species, reaching a 0.72 dominance in rebêlo and garófalo (1997) studied area (table 3). the 1.302 diversity index of rtp was very similar to that obtained by jesus and garófalo (2000), not been significantly different (t=0.216; p>0.05) and only higher than the one presented by rebêlo and garófalo (1997), which was influenced by its high dominance value (table 3). eucalyptol, or 1,8-cineol, was the most attractive bait for euglossini males in rtp, which corroborates the efficiency of that compound as attractive scent bait for a large number of species and individuals, as emphasized by dressler (1982) and observed in other studies carried out in several neotropical regions (e.g., janzen et al., 1982; ackerman, 1983; pearson & dressler, 1985; rebêlo & garófalo, 1991, 1997; cordeiro et al. 2013; rocha-filho & garófalo, 2013). as reported by alvarenga et al. (2007) and neves and viana (1999) in their study areas, methyl salicylate was also not attractive for males in the rtp. despite the significant efficiency of odor baits for the study of euglossini communities, two species that nested in the study area, euglossa townsendi cockerell, 1904 and euglossa truncata rebêlo & moure, 1996, were not sampled with the baits used in the present study (castro, personal observation). similar observations were reported by rebêlo and garófalo (1991), who found nests of eufriesea auriceps friese, 1899 and eg. townsendi in semi-deciduous forests, but did not collect males of these species with odor baits. recently, knoll and penatti (2012) reported that six fragrances used as odor baits, for 77 months, did not attract eg. townsendi males, which were captured only on flowers. these observations reveal a lack of attraction of males of those species by the baits used in those studies or, in the case of eg. townsendi, a weak association, because even when they were attracted by the baits they were always in small numbers (janzen et al., 1982; ackerman, 1989; rebêlo & garófalo, 1997; nemésio & silveira, 2007; abrahamczyk et al., 2011; silva, 2012). such occurrences show the importance of using alternative methods to sample euglossine bees, such as trap nests (garófalo et al., 1993) and direct collection on flowers fig 4. rarefaction curve for euglossini species richness in areas of northeastern são paulo state, brazil. aguatapará (present study), bsanta rita do passa quatro (hirotsu et al. 2010), csertãozinho (rebêlo & garófalo, 1997), dmatão (jesus & garófalo 2000), epatrocínio paulista (silveira et al. 2011), fcajuru (rebêlo & garófalo, 1991, 1997). with insect nets (rocha-filho et al., 2012), in order to obtain representative samples of the species composition. rebêlo (2001) pointed out that males have different preferences for aromatic fragrances depending on locality and season. regarding temporal variation in bee abundance, in the present study, temperature and rainfall affected significantly the distribution of males throughout the year. both the number of species and number of individuals were higher in the warm table 3. number of euglossini bees sampled in areas of the northeastern são paulo state. gua=guatapará (present study), sta=santa rita do passa quatro (hirotsu et al., 2010), ser=sertãzinho (rebêlo & garófalo, 1997), mat=matão (jesus & garófalo, 2000), pat=patrocínio paulista (silveira et al., 2011), caj=cajuru (rebêlo & garófalo, 1991, 1997). 1eg. cordata is treated as eg. carolina by nemésio, 2009. mmn castro, ca garófalo, jc serrano, ci silva orchid bees of a hygrophilous forest410 and rainy months with activity peaks varying among species. these results are consistent with other studies carried out in different ecosystems (e.g., rebêlo & garófalo, 1991, 1997; rebêlo & cabral, 1997; ramalho et al., 2009; cordeiro et al., 2013; silva, 2012) suggesting that the peaks in number of individuals and species are probably related to a higher availability of floral resources (rebêlo & garófalo, 1997), since the flowering in tropical environments is cued by the rains and many of the orchid bees may also use these rains as a cue larity in species composition with other regional vegetation types contrasts with the very low number of individuals sampled and suggests a strong effect of area and spatial isolation by sugarcane culture and deforestation. other studies should be carried out in other hygrophilous forests areas in order to increase the species list and also to advance the knowledge about community composition of these bees in this kind of vegetation, which is still poorly known. acknowledgements we thank daniela de azevedo souza defina, for allowing us to sample in her farm; the guatapará municipal government (through júlio yoji takaki) for providing the meteorological data; fapesp (process #2010/10285-4) and capes-pnpd (process #02958/09-0), for financial support, capes-demanda social for granted maurício m. n. castro a scholarship and research center on biodiversity and computing (biocomp). we thank three anonymous reviewers for comments that helped to improve this article. references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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(2006). speciesspecific attraction to pheromonal analogues in orchid bees. behav. ecol. sociobiol., 60: 833-843. doi: 10.1007/s00265006-0227-8 doi: 10.13102/sociobiology.v63i1.1025sociobiology 63(1): 682-687 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 does atta laevigata (smith, 1858) act as solanum lycocarpum seed dispersers? introduction seeds dispersal consist in a displacement process of these propagules from the parent plant to different locations, fundamental to the cycle of life of plant species (cordeiro & howe, 2003), especially in tropical environments (howe & miriti, 2004). regarding the zoochorous dispersal, ants are considered the main invertebrate seed dispersers in terrestrial ecosystems, a process known as myrmecochory (stiles, 1980; beattie, 1985). acting as primary (courtenay, 1994) and also secondary (roberts & heithaus, 1986; kaspari, 1993) dispersers, ants can markedly change the seeds distribution, affecting not only the reproductive success, but also the spatial structure of plant populations. studies reported that ants are capable of carrying a large number of seeds, and the dispersion distance provided by the ant fauna is considered a potential benefit to the plants abstract ants can act as seed dispersers, modifying their distribution, affecting the reproductive success and the vegetation spatial structure. the leaf-cutting ants function, as dispersers of non-myrmecochorous plants, is little known. this work aimed to evaluate descriptively the atta laevigata interaction with solanum lycocarpum diaspores. the observations were carried out, throughout 10 days, in a secondary fragment of semidecidual seasonal forest in ivinhema, ms. to determine the removal rate, 500 seeds were taken from ripe fruits, dried, labeled and distributed in groups ranged from five to 50 seeds, totaling 100 seeds per foraging trail. groups of 30 seeds with pulp were also distributed every 1.0 m on the trails. individuals of different sizes presented different interactions to the fruits and seeds, smaller workers carried pulp or seeds separately, medium workers carried seeds with pulp or cleaned them before carry to the nest and the largest workers carried the seeds to the nest. atta laevigata acted primarily as predators, with few seeds discarded. their actions may interfere in the native vegetation regeneration, with a significant role in removing s. lycocarpum seeds, a pioneer species, and in population control for this species by the severe predation of seeds. however, the remaining 1.6% of intact seeds allows germination, with a. laevigata acting as a seed disperser over short distances for this species, favoring the s. lycocarpum dispersion. sociobiology an international journal on social insects pra tavares1, vv alves-junior1, ga morais2 article history edited by kleber del-claro, ufu, brazil received 19 may 2015 initial acceptance 12 march 2016 final acceptance 14 march 2016 publication date 29 april 2016 keywords myrmecochory, diaspore, lobeira, leafcutter ant. corresponding author paulo roberto de abreu tavares laboratório de apicultura univ. federal da grande dourados rodovia dourados, itahum, km 12 cidade universitária, cep: 79.804-970 dourados-ms, brazil e-mail: paulo_robertoivi@hotmail.com (andersen, 1998). however, some authors report that this process is carried out mainly by a small group of species in the ant community (santos, 2007; zelikova & breed, 2008). harper et al. (1970) and dalling & hubbell (2002) emphasize that the dispersion advantage only becomes effective when the seed is deposited into a suitable microenvironment for the establishment of new plant. moreover, the dispersion of seeds can provide, for plants species, reduced parasites and predators attack sand intraspecific competition after germination (janzen, 1970), the colonization of new habitats (howe & smallwood, 1982) and the influence on recruitment patterns of plant species in tropical ecosystems (farji-brener & silva, 1996; böhning-gaese et al., 1999; passos & oliveira, 2002). in this matter, moutinho et al. (2003) and passos & oliveira (2004) emphasize the importance of ants to carry the seeds to places known as more favorable for germination, such as their nests. 1 universidade federal da grande dourados (ufgd), dourados-ms, brazil 3universidade estadual de mato grosso do sul (uems), ivinhema-ms, brazil research article ants sociobiology 63(1): 682-687 (march, 2016) 683 behavioral and ecological studies on ants foraging contribute to the understanding of seed dispersal by them in the environment (endringer, 2011). aspects, such as abundance, deposition place and seed characteristics (weight and presence of pulp or elaiosome), are also important in studies that seek to investigate the consumption activities and removal of seeds by those species (reader, 1993; edwards & crawley, 1999). diaspores dispersal by ants is a relevant process also for non-myrmecochorous plants, without pulp or elaiossome (farnese et al., 2011), but the leaf-cutting ants function as seed dispersal agents of these species, is still little known (leal & oliveira, 1998; smith et al., 2007; bieber et al. 2011). it is known that leaf-cutting ants play an important role in secondary seed dispersal of solanum lycocarpum (courtenay, 1994), but these ants act as primary dispersers of the diaspore of this plant at short distances? based on the hypothesis the existence of this interaction, it was verified that researches showing this dispersion behavior are lacking. neither is it known if seeds with pulp are more removed than seeds without pulp. thus, the aim of this work was to describe the interaction role of atta laevigata (smith, 1858) with the diaspores of s. lycocarpum. material and methods solanum lycocarpum is a shrubby species, with ramified stems, cylindrical, woody, fistulous branches, a little crooked and covered with dense starry hair (corrêa, 1984). it presents a continuous bloom pattern, according to the newstrom et al. (1994) classification, producing few flowers per individual during the year, but at flowering peak, there is a production increase of these structures (oliveira filho & oliveira, 1988). the fruit is a berry, greenish even when mature and produce numerous dark gray, reniform, flat seeds (almeida et al., 1998). the fruit production is early, the amount produced varies depending on the plant age and each fruit produces an average 1.200 seeds. the most of the seeds has an average of 6.00 to 7.00mm in length and 5.58 to 5.08 in width 5.08mm and 1.50 to 2.10mm thick (castelani et al., 2008). this work was carried out at the edge of a secondary fragment of semidecidual seasonal forest, with typical formation of biome transition vegetation, cerrado-atlantic forest, located a rural area in ivinhema, state of mato grosso do sul, brazil (22º16’43’’s, 53º48’47”w). the fragment consisted of vegetation resulting from regeneration processes, once the primary vegetation was removed for eucalyptus planting, which was afterwards removed for marketing, approximately 15 years before this work, thus, most trees from this vegetation, had height around 5 meters. in the study period, the area was being cleared again to be subdivided into small farms. at early may 2013, fifteen days before sampling, this fragment underwent a burning process by anthropic action, thus creeping plants and small bushes that lined the fragment edges were destroyed. for ant nest location, some of the individuals of a. laevigata foraging in s. lycocarpum bushes were followed while they move in the trails. the straightdistance between bushes and nests1, 2, 3, 4 and 5 was 0.96 (under a bush); 5.94; 6.10; 10.47 and 19.47 meters, respectively. to test the hypothesis that the ants act as primary dispersers at short distances of s. lycocarpum diaspores, direct observations of interaction behavioral of ants in naturally fallen fruits were carried out for 10 days, totaling 30 hours, divided into 15 hours of nighttime observations between 17h00 and 20h00, and 15 daytime observations between 5h00 and 8h00. during the nighttime observations, a white light flashlight was used to assist the ant behavioral assessments. given the large number of workers interacting with the fruits, not all could be followed in its movement to the nests. to determine the removal rate of seeds to the nests, 500 seeds were taken from five ripe fruits, washed in a sieve with running water to remove the pulp, and put to dry on filter paper. after drying, they were stained with white waterbased ink and distributed at sundown. it was used 100 seeds per foraging trail, as a way of intensifying the sampling of interactions, in groups ranging from five to 50 seeds, every 1.0 meter, along their entire length depending on the length of each trail. to verify preference in the removal of seeds, another extracted seeds, were left with the pulp, and distributed in groups of about 30 seeds every 1.0 m along the foraging trail of each colony. due to the bush-nest short distance in nest1 (0.96 m), which was under a s. lycocarpum individual, the 100 seeds were placed in two steps of 50 seeds each, with an hour interval. the seed groups (with and without pulp) were observed simultaneously for three hours along the foraging trails, registering the interaction behavior with the seeds, the final destination of transported seeds and seeds lost in transport were collected again. worker ants that were along the foraging trail to wards each of the five nests were collected, killed in a chamber containing ethyl acetate and classified into three distinct size classes, proposed in this study, regarding the individuals length, as follows: small, between 3 and 4.9 mm; medium between 5 and 8.9 mm and large between 9 and 12.9 mm. some specimens were prepared with an entomological pin, identified by an expert and deposited at the biodiversity museum (mubio) of the biological and environmental sciences college (fcba), grande dourados federal university (ufgd). results and discussion when the large worker ants found a fallen ripe fruit under the parent plant, they climbed and moved on the fruit, intensely moving their antennas, and other workers quickly approached and climbed the fruit as well. once on the fruit, some individuals attached their mandibles in the pericarp and spun, making circular moves until remove portions of this layer. this behavior resulted in small openings used by other workers, which continued piercing the pericarp until transpose it fully. pra tavares, vv alves-junior, ga morais – atta laevigata as solanum lycocarpum seed dispersers684 as the larger ants pierced the exocarp, smaller workers entered the fruit through the opened passage and collected both the mesocarp and the seeds. some of these workers removed the pulp from the seeds leaving them completely clean, while others transported it with parts of this material. small workers, in most cases, carried only small pieces of pulp, taken directly from the fruit. this behavior occurred with workers from all studied colonies. pulp removal can positively influence the seeds germination rate by decreasing fungal attack, as reported by oliveira et al. (1995) and leal & oliveira (1998). pinto (1998) reported that soldier ants of a. laevigata cut the pericarp of s. lycocarpum fruits, and that different sizes of workers withdrew pulp while only the largest workers and soldiers carried the seeds to the nest. however, in this work, the presence of a. laevigata soldiers interacting with fruits and seeds was not observed, only workers from the different sizes proposed. regarding their behavior towards the seeds arranged along the trails, those closest to the nest entrance were perceived in a few seconds by workers, which became very agitated when examining them and carried immediately into the nest. however, sometimes small and medium workers gathered and carried a whole mass of seeds to the nest. these results are according to statements of costa et al. (2007) and zelikova & breed (2008) that larger size ants have ease to carry seeds individually, while the smaller need help from others. however, it was observed that the largest workers had some difficulty to collect the seeds due to the larger size of their mandibles compared to the seeds. therefore, they grabbed in the middle region of the seeds with their mandibles, curved the body and pressing the seed against the sternum thorax to, apparently, put it in a better position, and only than carry it to the nest. according to gorb & gorb (1999), the ants shape and size determine the way they carry the seeds, reaffirming observations described in this work. smaller workers (4.17±1,01mm) carried pulp or seeds separately, while the medium workers (6.79±1,12mm) carried seeds with pulp or cleaned them before carry to the nest. medium workers were observed discarding intact seeds, without pulp, out of the nest. this behavior was observed in all colonies, suggesting that pulpe would be the reward for ant in this interaction. the largest workers (10.02±1.09 mm) constantly carried the seeds to the nest, but no discarding was observed. these observations allows to state that different sizes of a. laevigata individuals have different behaviors when interacting with the fruits and seeds of s. lycocarpum, and that the size of the collecting workers influence the behavior and the resource type collected directly from the fruit.in atta sexdens l., 1758, wilson (1980a, 1980b) also found the existence of division of labor, depending on body size and age, among workers. one of the five nests studied, was under a s. lycocarpum individual and the workers of this colony concentrated their activities close to the bush, while other colonies foragedat greater distances from the nest entrance. in general, they collected fruits parts, including dried fruit and leaves of s. lycocarpum in the litter fall, but they also collected leaves directly from small bushes of other plant species that were around the nests. the foraging activity of workers decreases as the sunlight increases at the dawn, when few workers were seen collecting resources. hölldobler and wilson (1990) report that leaf cutting ants prefer foraging during the night period when the temperature is cooler, which may explain this pattern of foraging. even with distance between bushes and nests varying between 0.96 and 19.47 meters, the ants were able to collect and carry the seeds through this interval, indicating the possibility of influencing the subpopulations maintenance of plant species, as reported by schupp et al. (2010) regarding the role of dispersers at short distances. dalling & wirth (1998) found that individuals from the genus atta are capable of carry seeds for over a hundred meters, but according to andersen (1988), in environments of high density of ants acting as seed dispersers, they may rather move shorter distances for removing the seeds. therefore, it was found that a. laevigata acts primarily in the removing and transport of s. lycocarpum seeds at short distances. although it is an experimental situation, all distributed seeds (100%) throughout the foraging trail were collected and carried to the nests. this high removal rate may be related to the lack of alternative resources, since the vegetation in the studied area had under gone significant damage by fire. wenny (2001) suggests that plants have a better chance to succeed in recruiting new dispersers when a greater amount of seeds is directed to appropriate places. some studies also report the relevance of propagules transport to the nests interior, which can provide safe places for seedlings development, since it is rich in nutrients (heithaus, 1981; culver & beattie, 1983; andersen, 1998). courtenay (1994) observed throughout a week, about 40% of seeds of s. lycocarpum discarded in the ants refuse, however, in the present study, it was found 1.6% of the marked seeds in the refuse material around the nest, excluding the seeds with pulp, which could not be identified in the refuse material. this fact, plus the observation that, some ants dropped the seeds along the trail, but then collected it again, indicates that the a. laevigata species behaved mainly as a predator of s. lycocarpum seeds. also, despite the ants have carried the seeds away from the parent plant, what, according to janzen (1970),would influence the dispersion process because they move away from the greatest competition areas during and after germination, they kept them inside their nests. therefore, they might be using them as a substrate for fungi cultivation from which the ants feed, what would prevent them to germinate and originate a new individual of s. lycocarpum. another factor to consider is the lack of elaiosome in the s. lycocarpum seeds, an attractive structure for many ant species due its high content of lipids, proteins and sugars (handel & beattie, 1990). seeds with this resource are collected and carried to the nests, where they are discarded after the removal of this structure (passos & oliveira, 2004). sociobiology 63(1): 682-687 (march, 2016) 685 on the other hand, the elaiosome is an attractive structure for ants from the genus atta (pizo & oliveira, 2001), reinforcing the proposition of the interest of this ant species to the studied seed. this interest could be related to the chemical composition seed (e.g., high lipids composition) (marshall et al., 1979; skidmore e heithaus, 1988; brew et al., 1989). in addition, it was observed no other organism contributing to the removal and dispersal of seeds of s. lycocarpum during assessments. that may be a consequence of the fire in the studied area reducing the populations that would contribute to this function, or because they are missing in the area. zelikova & breed (2008) stated that, when mainly a small number of species in an environment perform the removal of seeds, the removal rate might decrease, by the loss of these species or their density reduction. according to cordeiro et al. (2009), the loss of seed disperser organisms can endanger the plant species that depend on these for the dispersion process, or for the seeds germination, hence the establishment of new individuals and populations of this species. thus the a. laevigata foraging becomes important for s. lycocarpum seed dispersal, even in small proportions and the short distances. ants from the genus atta, on the other hand, build their nests deep and with an architecture that stabilizes the temperature and humidity fluctuations (roces & kleineidam, 2000; farji-brener, 2000), therefore, resistant to fire, enabling them to multiply and establish quickly in disturbed habitats (rao, 2000; santos et al., 2008). that could explain the permanency of these colonies in the area even after the fire, what increased the significance of the ants role as dispersers of s. lycocarpum seeds. although in this work had been demonstrated the a. laevigata as predators with few seeds discarded (1.6%), it is believed that the ant species activities in this studied area suggest an positive effect on the regeneration of the native vegetation. atta laevigata act in population control of s. lycocarpum, a pioneer plant species, by predation on their seeds, in addition to have a role in primary removing of s. lycocarpum seeds, favoring the dispersion at short distances. thus, the ant a. laevigata would be acting as disperser of this plant species in natural regeneration areas. acknowledgements the authors would like to thank the capes for the scholarship concession for the first author. references almeida, s. p.; proença, c. e. b.; sano, s. m. & ribeiro, j. f. 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(2008). effects of habitat disturbance on ant community composition and seed dispersal by ants in a tropical dry forest in costa rica. journal of tropical ecology, 24: 309-316. doi: 10.13102/sociobiology.v64i4.1820sociobiology 64(4): 484-487 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a note on the karyotype and morphology of the ant platythyrea sinuata (roger, 1860) (formicidae, ponerinae, platythyreini) the platythyreini emery, 1901 includes the genus platythyrea roger, 1863. this pantropical genus is composed of 38 species, 13 of which are neotropical, including four fossil species (bolton, 2017). identifying species and characterizing the diversity of platythyrea is not a simple task, because it is a genus with homogeneous morphological characters and it has a relatively high phenotypic plasticity (brown, 1975). despite the revision of brown (1975), there is still much to be clarified about this genus. platythyrea sinuata (roger, 1860) has suriname as typelocality, and is found in with a wide geographic distribution in forests of south america. according to brown (1975), p. sinuata has two synonyms, platythyrea meinerti forel, 1905 and platythyrea meinerti boliviana santschi, 1921, due to its phenotypic variation. however, the taxonomic description of p. sinuata still leaves doubts and needs more information. the precise identification of this and other species of this abstract taxonomy of the genus platythyrea is confusing because of high phenotypic plasticity, possible synonymies and morphological proximity to other species of this genus. the correct identification of platythyrea sinuata can be achieved using integrative taxonomy including both morphological and genetic components. here we describe the karyotype of p. sinuata, which has 2k=24m+20a with cma 3 +/dapion a single chromosome of the haploid set. these data will help to clarify the taxonomic status of this species. sociobiology an international journal on social insects pnc silva-rocha1, jpso correia1, csf mariano1,2, jhc delabie2, ma costa1 article history edited by gilberto m. m. santos, uefs, brazil received 22 june 2017 inicial acceptance 15 august 2017 final acceptance 20 august 2017 publication date 27 december 2017 keywords cytotaxonomy; cma 3 /dapi; chromosomal heteromorphism; phenotype. corresponding author patricia nayara caldas silva rocha departamento de ciências biológicas universidade estadual de santa cruz km 16 rodovia ilhéus-itabuna cep 45650-000, ilhéus-ba, brasil. e-mail: patinay9@gmail.com genus would be facilitated by integrative studies combining morphology, molecular and cytogenetic data, given the limitations of classical taxonomic characters (schlick-steiner et al., 2010). karyotype characters are useful for taxonomic and evolutionary studies. to date, more than 125 morphospecies of ponerinae ants had their karyotype data studied, with chromosome numbers varying from 2n = 8 to 120 (lorite & palomeque, 2010; mariano et al., 2015; correia et al., 2016). however, cytogenetic studies with platythyrea are restricted and only four species had their chromosomal numbers identified: platythyrea quadridenta donisthorpe, 1941, 2n (n) = 18 (9) and platythyrea tricuspidata emery, 1900, 2n = 92-96 (imai et al., 1983; 1990) from malaysia, platythyrea pilosula (smith, 1858), 2n = 40 (mariano et al., 2015) from french guiana and platythyrea punctata (smith, 1858), 2n = 84 from central america (schilder, 1999). 1universidade estadual de santa cruz (uesc), ilhéus-ba, brazil 2laboratório de mirmecologia, centro de pesquisas do cacau (cepec/ceplac), itabuna-ba, brazil short note sociobiology 64(4): 484-487 (december, 2017) 485 in this context we present a cytogenetic description and notes on the morphology of p. sinuata carried out in samples of a colony found in the brazilian atlantic forest. it is noteworthy that this relatively common species in the amazon region is surprisingly very rare in the atlantic forest, as well as two other species of this genus (delabie, 2001). our data will be useful for understanding the taxonomic status and evolution of this species in further integrative studies on platythyrea. the p. sinuata colony was collected in cabruca area (name given to cocoa plantation in native forest understory), between rotted trunk and soil in ilhéus, bahia state, brazil (14°47’51”s, 39°02’13”w). adult workers were identified following brown (1975). vouchers were deposited in the collection of the laboratory of mirmecology, at the centro de pesquisa do cacau, cepec-ceplac, in ilhéus, bahia, brazil. specimens were photographed under a leica m165c stereomicroscope. morphometric measurements were performed to an accuracy of 0.01 mm following the terminology of brown (1975). the colony was maintained in a b.o.d. incubator at 28°c to obtain larvae for cytogenetic analysis. metaphases were prepared from prepupae cerebral ganglion, following imai et al. (1988), after 40 minutes treatment in colchicine (0.005%). the slides were then stained with 3% giemsa solution and analyzed under an olympus cx41 equipped with a digital camera. a minimum of 10 metaphases was analyzed for each specimen. chromosomes were classified following levan et al. (1964) and the karyograms were organized using adobe photoshop® 7. chromomycin a3 (cma3) and 4’6-diamidino-2phenylindole (dapi) staining for the characterization of chromosomal segments rich in cg and at base pairs, respectively, followed guerra and souza (2002). metaphases images were captured with software image pro plus ® version 4.1 (media cybernetics) in an olympus bx51 microscope. the specimens had predominantly brown opaque coloration (fig 1a-f); moderately large eyes 0.31-0.34 (0.33 mm) (fig 1a, c-d); in lateral view, a curved groove (directed from the dorsal to lateral margin) not developed, present at the base of the mandible (fig 1d); in side view, long mesosome with weber length 2.89-3.01 (2.99 mm); in side view, a slope of propodeum with a tooth on the front side; in dorsal view relatively long and narrow petiole, forming a tooth in the postero-medial region; femur of the first leg about twice as wide as the femur of the 2nd leg 0.43-0.46 (0.45 mm) (fig 1a). the identification key for platythyrea of the new world by brown (1975) comprises few diagnostic characters considering the high variation in size, shape and color patterns in the species of this group. platythyrea sinuata exhibits a great morphological similarity with platythyrea angusta forel, 1901, p. pilosula and p. punctata making its correct identification difficult (brown, 1975). in formicidae discrete phenotypic variations observed are not always to be considered as useful characteristics for the separation of populations into distinct species, and may be only the reflection of geographic or ecological variations (lucas et al., 2002; fedoseeva, 2011). as p. sinuata has a wide geographical distribution and explores different habitats the best way to correctly identify possible cryptic species would be to analyze the greatest amount of data available (seifert, 2009; heled & drummond, 2010), mainly due to its phenotypic plasticity, which hinders classical taxonomic approaches. platythyrea sinuata had 2n=44 chromosomes, with karyotypic formula 2k=24m+20a (fig 2). a length heteromorphism occurs in the first chromosome pair. imai et al. (1983) reported a large difference in the chromosome number of two malaysian species p. quadridenta and p. tricuspidata showed, 2n=18 and 2n=96, respectively. in the neotropics (french guiana), mariano et al. (2015), reported fig 1. platythyrea sinuata: (a) side view; (b) dorsal view; (c) head in front view; (d) head in side view; (e) mesosome in lateral view; (f) petiole in lateral view. pnc silva-rocha et al. – karyotype and morphology of the ant platythyrea sinuata486 the chromosome number 2n=40 for p. pilosula, while p. punctata of central america, showed 2n=84 (schilder, 1999). this variation is significant considering the small number of karyotypes known for platythyrea to date. although the greatest variation was found among malaysian species (2n=18, 2n=96) high and intermediate numbers were reported in central america and south america, respectively. platythyreini (platythyrea) comprises a monophyletic group according to schmidt (2013), with low chromosome numbered p. quadridenta positioned basally compared to p. punctata (2n = 84). the inclusion of cytogenetic data from more taxa will allow drawing important conclusions about the genus karyotypic evolution. the fluorochrome staining showed a single marking, cma3 +/dapiin a single chromosome of the 1st pair, which coincides with a secondary constriction and length heteromorphism (fig 3). secondary constriction and cma3 +/ dapimarkings, consistently coincided with the nucleolar organizer region (nors) in several other species, and indicates that the heteromorphism of p. sinuata is related to the variation in nors length. length heteromorphism was observed in other ants such as pheidole westwood, 1839, and in temnothorax rugatulus (emery, 1895) (taber & cokendolpher, 1988). the morphological and cytogenetic characterization of platythyrea sinuata presented here will be useful for future taxonomic revision of the genus and in evolutionary studies, involving the establishment of phylogenetic relationships among species. acknowledgments the authors thank josé raimundo maia dos santos and josé crispim soares do carmo for their help in field work and fapesb for the study grant of the first author (pncsr). csfm, jhcd and mac are cnpq productivity fellows. references bolton, b. 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(1839). an introduction to the modern classification of insects; founded on the natural habits and corresponding organisation of the different families. volume 2. part xi. london: longman, orme, brown, green and longmans, pp. 193-224. https://doi.org/10.1093/molbev/msp274 https://doi.org/10.1146/annurev-ento-112408-085432 https://doi.org/10.1146/annurev-ento-112408-085432 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.115-118sociobiology 61(1): 115-118 (march, 2014) congregation sites and sleeping roost of male stingless bees (hymenoptera: apidae: meliponini) cf santos12*, c menezes3,4, a vollet-neto3, vl imperatriz-fonseca1,5 introduction male bees (hymenoptera: apoidea) patrol nests containing receptive females or aggregate at rendezvous sites in order to find sexual partners (alcock et al., 1978; eickwort & ginsberg, 1980; paxton, 2005). particularly in the eusocial bees, i.e., honeybees (apidae: apini) and stingless bees (apidae: meliponini), males can form mating swarms at specific areas, where they wait for virgin queens (nogueiraneto, 1954; kerr et al., 1962; zmarlicki & morse 1963; sommeijer & de bruijn, 1995; koeniger & koeniger, 2000). male stingless bees reach sexual maturity about two to three weeks after eclosure, after which they permanently leave their nests (engels & engels, 1984; van veen et al., 1997). they then join mating swarms close to nests containing virgin queens or nests in the process of being founded (nogueiraneto, 1954; kerr et al., 1962; nogueira-ferreira & soares, abstract very little is known about stingless bee reproductive biology or male behaviour. in this note we provide the first observations on the male aggregations (congregation sites and roosting sites) of some stingless bee species. our observations show that males of two stingless bee species can congregate on the same site. we also report for the first time the substrates used by stingless bee males for resting at night, that at least one species forms large sleeping roosts composed of hundreds of individuals, and that sleeping roost locations are not reused on subsequent nights. sociobiology an international journal on social insects 1 laboratório de abelhas, instituto de biociências, universidade de são paulo, são paulo, sp, brazil 2 laboratório de entomologia, pontifícia universidade católica do rio grande do sul, porto alegre, rs, brazil 3 embrapa amazônia oriental, empresa brasileira de pesquisa agropecuária, belém, pa, brazil 4 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, ribeirão preto, sp, brazil 5 universidade federal rural do semi-árido, mossoró, rn, brazil article history edited by: anne zillikens, univ. of tuebingen, germany received 14 february 2013 initial acceptance 13 may 2013 final acceptance 11 december 2013 keywords males, mating swarm, roosting sites, social bees, reproductive strategies corresponding author charles fernando dos santos laboratório de entomologia pontifícia universidade católica do rio grande do sul 90619-900 porto alegre, rs, brazil e-mail: charles.santos@pucrs.br 1998; van veen & sommeijer, 2000). still, in some stingless bee species (melipona), males can congregate in non-nest associated congregation sites such as concrete retaining wall or beekeepers cabin which are periodically visited by workers and queens (sommeijer & de bruijn, 1995; sommeijer et al., 2003; cortopassi-laurino, 2007). apart from these facts little is known about the reproductive behaviour of male stingless bees. nothing is known about where males rest overnight after visiting such reproductive aggregations, although males of solitary bees are known to frequently spend the night on plants, alone or in groups under leaves (kaiser, 1995; alcock, 1998; alves-dos-santos et al., 2009). furthermore, we know that solitary bee males may exhibit site fidelity by returning to a particular sleeping roost on the successive nights (kaiser, 1995; alcock, 1998; alves-dos-santos, 1999; oliveira & castro, 2002; alves-dos-santos et al., 2009). the present short note contributes to the knowledge short note cf santos et al male congregation and sleeping roost of stingless bees116 of reproductive behaviour (mating swarms) and sleeping roosts of males of some stingless bee species. as observations of such events are rare and have low predictability, we put together several sporadic observations we carried out during the course of other studies on stingless bee biology. non-nest associated congregation sites in a meliponary located in the city of pilar do sul, são paulo state, in october 2009, males of melipona quadrifasciata quadrifasciata lepeletier and plebeia droryana (friese) were observed aggregating at a congregation site, the leaves and twigs of an oriental raisin tree hovenia dulcis (rhamnaceae), approximately 4-5 m above ground. this congregation of males was observed on four consecutive days. males of both species periodically joined and departed the congregation site between about 08:00h and about 17:00 h. the congregation was formed of dozens of males of both m. q. quadrifasciata and p. droryana. however, it is unclear what the role of the frequent joining and leaving of individual males was. near the congregation site several phorid flies (diptera: phoridae) were sitting on or near the branch used by m. q. quadrifasciata males. such parasitic flies may have an important role in the mortality of stingless bee males (simões et al., 1980; brown, 1997; sommeijer et al., 2003). the visit of some m. quadrifasciata workers carrying white resin on their corbiculae was noted, but no interactions between them and the males were observed. no virgin queens were observed at the congregation site. it remains uncertain why males of m. q. quadrifasciata and p. droryana chose to congregate at the same site. another male congregation site was observed in the city of mossoró, state of rio grande do norte, for one day in march 2009. melipona subnitida (ducke) males gathered on a branch of a cashew tree anacardium occidentale (anacardiaceae). this congregation occurred in a meliponary containing approximately 90 hives of this species, and was observed between 10:00 and 17:00h. the males were either sitting in the congregation site or flying close to it (figure 1a) and received visits from a wasp (polybia sp.) with which they performed trophallaxis (figure 1b). one virgin queen with a distended abdomen was also observed at the congregation. however, this queen did not attract any males during her stay. the queen remained on the branch but in a very agitated manner, tightly circling a small point on the branch for approximately 20 minutes, and then flew away. the reason for the presence of virgin queens in non-nest associated congregation sites is unclear because their presence does not result in copulation attempts by the males (sommeijer & de bruijn, 1995; sommeijer et al., 2003). occasionally, worker bees were observed flying over the congregation site, landing briefly and disappearing afterwards. the presence of workers at some congregation sites of stingless bee males appears to be common; they perform trophallaxis with the males (cortopassi-laurino, 2007) or carry resins on their corbiculae to be distributed over the congregation site (sommeijer et al., 2003). however, the adaptive significance of the behaviour of such workers at the congregation site remains unclear. sleeping roosts overnight aggregations of bee males have been described previously for solitary bee species that exhibit site fidelity by returning to a particular sleeping roost on the successive nights (kaiser, 1995; alcock, 1998; alves-dossantos, 1999; oliveira & castro, 2002; alves-dos-santos et al., 2009). at ribeirão preto, the meliponary garden of the ffclrp was daily inspected from june 03 to september 05 2010 for sleeping roosts between 17:00 h and 07:00 h. this meliponary contained approximately 150 colonies, including scaptotrigona depilis (moure), tetragonisca angustula (latreille), frieseomelitta varia (lepeletier), m. scutellaris (latreille), m. quadrifasciata and nannotrigona testaceicornis (lepeletier). we searched for sleeping roosts on branches below 2 m. when a sleeping roost was found, the arrangement of the individuals was recorded and the location fig. 1 (a) congregation site of melipona subnitida males on a branch of anacardium occidentale (anacardiaceae) at mossoró, state of rio grande do norte; (b) polybia sp. wasp visiting the congregation. sociobiology 61(1): 115-118 (march, 2014) 117 was marked and monitored during subsequent nights so as to observe whether the males returned to it. we found s. depilis males at ffclrp sleeping on branches of ocimum basilicum (lamiaceae), citrus limonia (rutaceae) and cosmos sp. (asteraceae) (figure 2a). each of these plants was used as sleeping roosts by three to five males, but they remained separated from each other (table 1). none of roosts were reused in subsequent nights. a single m. scutellaris (latreille) male was also found sleeping on a daisy flower (montanoa pyramidata: asteraceae) (figure 2b). additionally, in a private meliponary approximately 1.2 km from the ffclrp-usp campus, a sleeping roost of f. varia males was observed in march 2011. there were three hives of this species on the site. the sleeping roost comprised approximately 250 f. varia males on any one night. the males were sitting on a metal wire (table 1, figure 2c) approximately 2 m from one of the three colonies of this species at the site. they were first observed at 22:00 h, and again at 01:30 h. the f. varia males remained densely grouped. occasionally, some males moved their hind legs or walked slowly on the wire and even over other males. they did not react to contact with entomological tweezers during the collection of some individuals. on the following morning at approximately 08:00 h, some males were still at the sleeping roost site, but they gradually left the site over the course of the morning. neither additional aggregations nor other sleeping roosts were reported thereafter. to our knowledge these are the first report of sleeping roosts of stingless bee males. the causes that led males of s. depilis, m. scutellaris and f. varia to choose their sleeping roosts remain unclear. there is still much to learn about the biology of stingless bee males and this report will help other researchers to find and study them. acknowledgements the authors thank dr. silvia r. m. pedro and dr. sidnei mateus (faculdade de filosofia, ciências e letras de ribeirão preto, usp) for the identification of plebeia droryana and and polybia sp., respectively. financial support received: cfs and avn from the coordenação de aperfeiçoamento de pessoal de nível superior (capes); cm from the fundação de amparo à pesquisa do estado de são paulo (fapesp, process 07–50218-1); vlif from the pvns scholarship from capes. the authors also thank the capes – proap 2010 program for field trip financial support for cfs. authors gratefully acknowledge rodolfo r. jaffé (beelab usp) for his critical reading of the manuscript and suggestions. references alcock, j., barrows, e.m., gordh, g., hubbard, l.j., kirkendall, l., pyle, d.w., ponder, t.l. & zalom, k.g. (1978). the ecology and evolution of male reproductive behaviour in the bees and wasps. zoological journal of the linnean. society, 64: 293-326. doi: 10.1111/j.1096-3642.1978.tb01075.x alcock, j. (1998). sleeping aggregations of the bee fig. 2 sleeping roost of males of: (a) scaptotrigona aff. depilis; (b) melipona scutellaris; (c) frieseomelitta varia. table 1. features of the sleeping roosts for male stingless bees (hymenoptera: meliponini). stingless bee species substrates plant species or locations number of males aggregation type scaptotrigona aff. depilis1 tree branch ocimum basilicum lamiaceae) 3 solitary tree branch citrus limonia (rutaceae) 5 solitary tree branch cosmos sp. (asteraceae) 3 solitary melipona scutellaris2 flower petal montanoa pyramidata (asteraceae) 1 solitary frieseomelitta varia3 metal wire metal wire ± 250 cluster 1 november 19, 2010; 2 june 03, 2010; 3 march 2011. cf santos et al male congregation and sleeping roost of stingless bees118 idiomelissodes duplocincta (cockerell) (hymenoptera: anthophorini) and their possible function. journal of the kansas entomological society, 71: 74–84. alves-dos-santos, i. (1999). aspectos morfológicos e comportamentais dos machos de ancyloscelis latreille (anthophoridae, apoidea). revista brasileira de zoologia, 16, (supl. 2): 37–43. doi: 10.1590/s0101-81751999000600005 alves-dos-santos, i., gaglianone, m.c., naxara, s.r.c., engel, m.s. (2009). male sleeping aggregations of solitary oil-collecting bees in brazil (centridini, tapinotaspidini, and tetrapediini; hymenoptera: apidae). genetics and molecular research, 8: 515-524. brown, b.v. 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(2005). male mating behaviour and mating systems of bees: an overview. apidologie, 36: 145-156. doi: 10.1051/apido:2005007 simões, d., bego, l.r., zucchi, r., sakagami, s.f. 1980. melaloncha sinistra borgmeier, an endoparasitic phorid fly attacking nannotrigona (scaptotrigona) postica latr. (hym., meliponinae). revista brasileira de entomologia, 24: 137142. sommeijer, m.j. & de bruijn, l.l.m. (1995). drone congregations apart from the nest in melipona favosa. insectes sociaux, 42: 123-127. doi: 10.1007/bf01242448 sommeijer, m.j., de bruijn, l.l.m., meeuwsen, f.j.a.j. (2003). behaviour of males, gynes and workers at drone congregation sites of the stingless bee melipona favosa (apidae: meliponini). entomologische berichten, 64: 10-15. van veen, j.w., sommeijer, m.j., meeuwsen, f. (1997). behaviour of drones in melipona (apidae, meliponinae). insectes sociaux, 44: 435–447. doi: 10.1007/s000400050063 van veen, j.w. & sommeijer, m.j. (2000). observations on gynes and drones around nuptial flights in the stingless bees tetragonisca angustula and melipona beecheii (hymenoptera, apidae, meliponinae). apidologie, 31: 47–54. doi: 10.1051/ apido:2000105 zmarlicki, c. & morse, r.a. (1963). drone congregation areas. journal of apicultural research., 2: 64-66. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5865sociobiology 68(3): e5865 (september, 2021) introduction euglossine bees or orchid bees are a diverse group of neotropical pollinators, assembling approximately 250 species, distributed in five genera (euglossa, eufriesea, eulaema, exaerete and aglae), belonging to the corbiculate bee clade (moure et al., 2012; saleh & ramírez, 2019; ascher & pickering, 2020). the group is best known for their metallic and shiny integument and the very long tongues of its representants, as well as by the males’ behavior of collecting fragrances from many orchid species to use during courtship (dressler, 1982; roubik & hanson, 2004; eltz et al., 2015). the group has become better known over the last five decades after the use of scent baits to attract males abstract in the last decades, the use of the trap-nest technique has helped to increase knowledge on the nest architecture of many orchid bee species. this study describes the nest architecture of eufriesea aff. auriceps constructed in trap-nests made of dried bamboo internodes (canes). the nests were placed in remnants of atlantic forest and in reforested areas next to forest remnants and monitored monthly from august 2015 to august 2016 and from august 2018 to august 2019 in southern brazil. the bamboo internodes occupied by bees varied in internal diameter from 1.0 cm to 2.0 cm ( = 1.7; sd = 0.3; n = 12) and in length from 11.0 cm to 28.0 cm ( = 19.5; sd = 4.8; n = 12). the total size of the nests inside the bamboo internodes ranged from 9.0 cm to 19.9 cm ( = 14.3; sd = 3.9; n = 12). the number of brood cells constructed per nest varied from 1 to 10 ( = 4.0; sd = 2.3; n = 15). the cells were built with small pieces of bark cemented with resin, linearly arranged along the bamboo tube. internally, the cell wall was lined with resin. the cells measured 1.5-3.0 cm ( = 2.3 ± 0.5; n = 48) in length and 1.4-1.7 cm ( = 1.5 ± 0.1; n = 17 cm) in width. the internal contour of the cells was elliptical. females of eufriesea aff. auriceps occupied trapnests in forest remnants and in areas undergoing restoration. sociobiology an international journal on social insects andré luiz gobatto1,2, amanda guimarães franciscon2, natalia uemura1,2, susanna mendes miranda2, giovanna gabriely cesar2, ana carolina oliveira-silva2, thais kotelok-diniz2, silvia helena sofia2 article history edited by candida aguiar, uefs, brazil received 26 september 2020 initial acceptance 09 april 2021 final acceptance 01 may 2021 publication date 02 september 2021 keywords orchid bee; euglossine; nest architecture; trap-nest; nest. corresponding author silvia helena sofia universidade estadual de londrina rodovia celso garcia cid, br 445 km 380, campus universitário cx. postal 10.011, cep 86.057-970 londrina, paraná, brasil. e-mail: shsofia@uel.br in several surveys carried out in different regions and ecosystems in central and south america (dressler, 1982; ramírez et al., 2002; roubik & hanson, 2004; nemésio, 2009). the scent baits are very efficient to attract males, who visit them searching for the synthetic chemicals which mimic the aromatic compounds produced by orchids (dodson et al., 1969; dressler, 1982). however, the use of this method in orchid bee surveys has some limitations, since only males visit the baits and males of some species are not or are only rarely attracted to baits (nemésio, 2012). as a consequence, as almost the totality of the ecological studies involving orchid bees are based on sampling male specimens attracted to scent baits (nemésio, 2012), the literature still needs more surveys and information concerning euglossine females. 1 programa de pós-graduação em ciências biológicas, universidade estadual de londrina, londrina-pr, brazil 2 universidade estadual de londrina, departamento de biologia geral, londrina-pr, brazil research article bees nests of eufriesea aff. auriceps (hymenoptera, apidae, euglossini) in remnants of atlantic forest and reforested areas silvia helena sofia – nests of eufriesea auriceps2 in the last three decades, information on euglossine females has been obtained mostly though the trap-nest technique (garófalo et al., 1993, 1998; augusto & garófalo, 2004), which is useful for collecting biological data on solitary bees nesting in preexisting cavities (garófalo et al., 2004). since female orchid bees rarely excavate their nests (solanobrenes et al., 2018) and several species build their nests in preexisting cavities in nature or even in human buildings (ramírez et al., 2002), the use of trap-nests for euglossine studies has provided, for instance, information on nesting biology of different species (garófalo et al., 1993, 1998; viana et al., 2001; augusto & garófalo, 2004). in fact, the use of trap-nests can be more effective than searching for euglossine nests in the field (garófalo et al., 1993). moreover, surveys with trap-nests have contributed by providing geographical records of those species for which males are only rarely or are not attracted to scent baits during inventories on orchid bees (see nemésio, 2012). despite this, the use of the trapnest method in surveys involving orchid bees is still scarce in the literature. recently, a study using chemical bait to attract males compared the euglossine assemblages from atlantic forest remnants and from reforested areas (ferronato et al., 2018). even though the occurrence of this species has been reported in the studied region (sofia et al., 2004), males of eufriesea auriceps (friese, 1889) were not attracted to baits in any of the studied areas surveyed by ferronato et al. (2018). the absence of males of this species is not surprising, since, with rare exceptions (e.g. andrade et al., 2012), only a few males have been sampled at scent baits in different surveys conducted in areas for which the occurrence of this species is known (e.g. sofia et al., 2004; rocha-filho & garófalo, 2013; viotti et al., 2013; martins et al., 2018; dec & alvesdos-santos, 2019). eufriesea auriceps (friese, 1899) is a solitary species of euglossine bees, widely distributed in brazilian territory (moure et al., 2012), with occurrences recorded for different ecosystems, including the cerrado biome (martins et al., 2018), campos rupestres (high-altitude rocky fields; viotti et al., 2013), and atlantic forest (sofia et al., 2004; nemésio, 2009; rocha-filho & garófalo, 2013; dec & alves-dossantos, 2019), from the northeastern to southern regions (moure et al., 2012; martins et al., 2018; dec & alves-dossantos, 2019). occurrences of e. auriceps have also been reported in paraguay and argentina. in this study, we describe the nest architecture of nests of eufriesea aff. auriceps sampled in trap-nests placed in forest remnants of the atlantic forest (af) and in reforested areas of this biome. although the structure of nests of this species has already been described elsewhere (garófalo et al., 1993), the current study aims both to add more information on the theme and to compare our findings concerning the preferences of e. auriceps in the use of trap-nests, with those reported by garófalo et al. (1993). moreover, we discuss our findings on the perspective of the reestablishment of orchid bee fauna in forest habitats under restoration processes, since studies on this subject are still very scarce in the literature (rasmussen, 2009; ferronato et al., 2018). material and methods study species considering that many species of the ‘auriceps’ group, to which e. auriceps belongs, are currently the focus of taxonomic discussion (nemésio 2009; moure et al., 2012), as well as which, the entire group needs a thorough revision (moure et al., 2012), the specimens of our study were identified as eufriesea aff. auriceps, by dr. gabriel a. r. melo (universidade federal do paraná, brazil). study areas and samplings the study was conducted in the north of the state of paraná, in southern brazil, in an area covered by seasonal semi-deciduous forest (ssf), a type of vegetation formation of af, often called matas de planalto (plateau forests), due to their distribution on the hinterland highlands (oliveira & fontes, 2000). the deforestation in this region occurred over a period of approximately a hundred years, with only 2-4% of native forests remaining, distributed in a fragmented landscape and embedded in agricultural areas (torezan, 2002). we conducted our surveys in three remnants of ssf and nine reforested areas, in both cases with variations in sizes (table 1). other relevant information regarding the study sites, such as the age of reforestation of each area, are also given in table 1. the samplings were conducted from august 2015 to augusto 2016 and from august 2018 to august 2019 (table 1). the methodology of the study was based on garófalo et al. (1993), with some modifications. in our study, the trapnests used to attract euglossine females, consisted only of bamboo internodes (canes), with diameters from 0.5 to 3.0 cm and variable lengths (mostly between 14 and 30 cm), which are cut so that the nodal septum closes one end of the internode (garófalo et al., 2004). twenty-five units of the bamboo canes were arranged in bundles inside a pet bottle cut open at one end. the bamboo bundles were placed horizontally on wooden platforms. each platform contained four bamboo bundles, totaling a hundred canes per platform. the platforms were fixed 1.5 m above the ground, attached to a wooden rod, sunk 30 cm deep into the soil. in the first year, the number of platforms per study area was two (1st year of study) and five (2nd year). a higher number of platforms was implemented in the second year of the study (august 2018 to august 2019) in an attempt to improve the efficiency of the surveys. in the first year of sampling, in each forest remnant and in restored areas, two platforms were placed, one 20 m from the edge and one 500 m from the edge, totaling 200 nests per study area. in the second year, in each forest remnant (a, b, and c) and sociobiology 68(3): e5865 (september, 2021) 3 in nine restored areas, five platforms were placed 20 m from the edge and 100 m apart from each other. in this latter case, distributed across a transect of 500 m towards the center of the fragment, totaling 500 trap-nests in each area. the inspections of the trap-nests in each study site were conducted once a month, at intervals of approximately 30 days. as in garófalo et al. (1993), the trap-nests occupied by females were marked and left in the location until the next inspection, when the entrance was closed with a stopper and they were moved to the laboratory. each inspection was performed with the aid of an otoscope. in the laboratory, nests were maintained in a cabinet and inspected regularly for adult emergences. when emergences did not occur, the trap-nests were opened for final inspections and measurements. for measurement procedures of brood cells and internal observation of the nests, the bamboo canes were opened along their lengths, resulting in two halves. all measures were taken with a digital caliper. we compared the similarity of both the diameter and length of traps used by e. auriceps between the current study and those obtained by garófalo et al. (1993, using the sorensen index). for this purpose, we clustered the different sizes of bamboo, as follows: into five classes of diameters (1.0-1.4; 1.5-1.9; 2.0-2.4; 2.5-2.9; 3.0-3.5 cm) and eight classes of lengths (11.0-13.9; 14.0-16.9; 17.0-19.9; 20.022.9; 23.0-25.9; 26.0-28.9; 29.0-31.9; 32.0-34.9 cm), which included all sizes of both measures of trap-nests occupied by females in our study and in the study of garófalo et al. (1993). table 1. municipality location, geographic coordinates, fragment size (ha), and forest stage (i.e., mature x secondary forests) for all study areas surveyed at northern region of the state of paraná (pr), southern brazil. in this table was also included the age of each reforested area. the mean altitude (m) for all areas is provided in the latter column. the vegetation type of all study areas is seasonal semi-deciduous forest. fr = forest remnant; r = restored areas. area code type of area municipality geographic coordinates size (ha) forest stage mean altitude (m) implantation date a fr alvorada do sul 22°49’1.89”s 51°11’25.77”w 137.3 secondary 350 b fr rancho alegre 23° 0’5.15”s 50°56’38.00”w 107.3 secondary 361.5 c fr sertanópolis 22°56’29.33”s 50°57’4.68”w 28.7 secondary 345.5 d r alvorada do sul 22°48’45.31”s 51°11’52.05”w 115.6 secondary 350 may 2004 e r rancho alegre 22°59’50.09”s 50°56’38.05”w 197.8 secondary 361.5 june 2003 f r sertanópolis 22°56’10.68”s 50°56’59.81”w 263.1 secondary 345.5 sept 2003 g r primeiro de maio 22°46’50.10”s 51°10’5.85”w 142.6 secondary 350 may 2003 h r alvorada do sul 22°45’8.58”s 51°13’29.93”w 115.30 secondary 350 nov 2003 i r primeiro de maio 22°54’33.57”s 50°57’5.70”w 125.1 secondary 337 august 2005 j r primeiro de maio 22°48’24.55”s 51° 5’35.04”w 150.4 secondary 335 nov 2004 k r alvorada do sul 22°49’16.07”s 51°18’28.31”w 151.9 secondary 340 october 2002 l r rancho alegre 23° 3’1.33”s 50°57’46.18”w 191 secondary 338 april 2004 results during the surveys, a total of 15 nests of e. aff. auriceps were obtained in the bamboo trap-nests. from these, four (26.6% of the total) were in restored areas (f and g), being three from f and one from g. out of the 11 nests of e. aff. auriceps constructed in bamboo canes in the three forest remnants, one was in fragment a, while 4 and 6 were in remnants b and c, respectively. table 2 contains information on the bamboo dimensions, nests, and brood cells of 12 nests. for three out of 15 nests obtained in the surveys, measures of nest dimensions and cells were not included in the analysis due to mistakes made during the measurement procedures. the bamboo internodes occupied by females varied in internal diameter from 1.2 cm to 2.0 cm ( = 1.7; sd = 0.3; n = 12) and lengths ranged from 11.0 cm to 28.0 cm ( = 19.5; sd = 4.8; n = 12). the total sizes of the nests inside the silvia helena sofia – nests of eufriesea auriceps4 bamboo internode ranged from 9.0 cm to 19.9 cm ( = 14.3; sd = 3.9; n = 12). the number of brood cells constructed per nest varied from 1 to 10 ( = 4.1; sd = 2.6; n = 12), (table 2). brood cells were built with pieces of vegetal bark and resin, linearly arranged along the bamboo tube (fig 1a-h). externally, the wall of the brood cells was covered by bark, arranged in variable positions, firmly fixed to each other with resin, which seemed to act as cement to join the pieces of bark (fig 1a-l). resin was also present between cells and the inner wall of the bamboo and in small deposits across the bamboo length (fig 1 e), probably to be used in the construction. internally, the cell wall was lined with resin and quite smooth (fig 1l). fig 1. a-e and i: nests of eufriesea aff. auriceps established in trap-nests of bamboo. c-d: nests of e. aff. auriceps and nests of the wasp auplopus sp (cells constructed with mud) in the same cavity. e: nests showing four brood cells and two males. f: detail of seven brood cells (closed). h: the same sevens cells (open) and larvae. i: nest with two cells and resin deposit in the trap-nest wall. j: detail of a pupa (male) and a cell. k: detail of an adult bee inside the cell; l: detail of a brood cell (open), showing its interior. sociobiology 68(3): e5865 (september, 2021) 5 the cells measured 1.5-3.0 cm ( = 2.3 ± 0.5; n = 48) in length and 1.4-1.7 ( = 1.5 ± 0.1; n = 17 cm) in width. the thickness of the cell ranged from 1.5 to 2.7 mm ( = 1.9 ± 0.3; n = 17). the cells were ovoid (externally) and elliptical in their internal form, clearly showing a higher axis (diagonal), demonstrated by the position of the bee inside the cell (fig 1k). although resin could be detected between cells, no conspicuous demarcation was detected between them. cells were closed with a plug made exclusively of resin. in some nests it was detected the presence of pieces of barks mixed with resin deposited between the last cell constructed by females and the nest entrance (figs 1d and 1g). two nests of e. aff. auriceps were also occupied by wasps of the genus auplopus sp (fig 1c-d). we recorded only few emergences of adults in december 2019, from nests collected in february and march 2019 (n = 8; table 2). in these cases, we observed that the cocoon was clear (beige) in most cases, although cocoons with a light brown shade were also found. on emergence from the cell, the bees opened a circular hole, smaller than the diameter of the cell plug. activity of e. aff. auriceps females occurred mainly during the warm-wet season, since most nests were surveyed from january to march. only three nests were collected in june and july, however they were probably out of activity (see table 2). the percentages of similarity obtained in the comparisons between the data from the current study and those reported by garófalo et al. (1993), considering the five classes of diameters and eight classes of length of trap-nests of bamboo occupied by females of e. auriceps, the estimates of similarity, given by sorensen index, were 50.0% and 80%, respectively. discussion information on nesting biology and nest structure of euglossine bees remains restricted to only about 20% of the species (ramírez et al., 2002; cameron, 2004; solano-brenes et al., 2018). in our study, we describe the nest architecture of the orchid bee e. aff auriceps, constructed in bamboo trapnests. the nest of this species has previously been described, in cavities of bamboo trap-nests, by garófalo et al. (1993). nest number* site fragment type data collection bamboo size (cm) bamboo ∅ (cm) total size of the nest (cm) no. of cells (no. of individuals in development) cell length (cm) additional informationvariation  ± sd 1 a fr 15/02/15 20.3 1.4 13.7 5 (5) 2.0-2.5 2.2 ± 0.2 no emergence 2 b fr 03/03/16 16.0 1.4 16.0 4 (4) 2.7-3.5 2.9 ± 0.5 no emergence 3 f r 03/03/16 22.0 1.9 19.1 5 (5) 2.2-2.7 2.4 ± 0.2 no emergence 4 b fr 17/03/16 15.5 1.5 15.5 1** (1) no emergence 8 c fr 25/02/19 28.0 1.7 9.00 4 (4) 1.9-2.3 2.1 ± 0.2 1 m and 3 fe (only 2 emerged in 12/12/19); with nest of auplopus sp 9 g r 10/07/19 11.0 1.5 9.00 1** (0) nest probably abandoned (only resin and bark) 10 c fr 26/03/19 26.9 2.0 19.9 10 (6) 1.5-2.8 2.1 ± 0.4 4 m and 2 fe emerged (in 18/12/19) 11 c fr 05/06/19 17.1 2.0 10.5 4 (2) 2.2-3.0 2.7 ± 0.4 2 fe (no emerged) 12 c fr 05/06/19 20.0 2.0 10.5 4 (0) 2.2-3.0 2.7 ± 0.4 no emergence 13 c fr 28/01/19 22.0 2.0 13.0 2 (0) 2.3-2.7 2.5 ± 0.3 larvae didn’t develop 14 c fr 26/03/19 18.1 1.4 18.1 3 (0) 2.0-3.0 2.5 ± 0.5 no emergence (attacked by ants); with nest of auplopus sp 15 f r 26/03/19 17.0 1.5 17.0 7 (7) 1.5-2.3 1.7 ± 0.3 no emergence table 2. identification of the nests and study areas, measures of bamboo trap-nest occupied by females of eufriesea aff. auriceps, number of brood cells per nest and measures of the main nest structures. a, b, c, d, f and g = study areas. fr = forest remnants of atlantic forest; r = reforested areas; m = male; fe = female. silvia helena sofia – nests of eufriesea auriceps6 while our results reveal remarkable similarities in most aspects of nesting habit of this species between both studies, some differences are pinpointed here. one of them is the fact that in all the nests we surveyed brood cells were in a conspicuous linear arrangement, while garófalo et al. (1993) reported that in 20% of the sampled nests of e. auriceps, cells were in an irregular cluster arrangement inside the bamboo cavity (garófalo et al., 1993). on the other hand, those authors speculated that the irregular cluster arrangement of the cells, in the two nests where this arrangement was found, was probably due to the wider diameters of the bamboo canes (≥2.2 cm). since in our study females of e. aff. auriceps did not use bamboo canes with diameters above 2.0 cm, the absence of brood cells in a cluster arrangement among the 15 nests could be because the females did not occupy wider cavities. furthermore, we can conclude that females of this species preferred bamboo canes with a diameter ≤ 2.0 cm, since although bamboo canes with a broader diameter were available, they were never used by the females. the eufriesea species are medium sized to quite large bees (14-26 mm long; dressler, 1982). eufriesea auriceps shows a total length ca. 18 mm (nemésio, 2009) and can be considered a large sized bee, a condition that could explain the preferential use of bamboo canes with widths between 1.5 and 2.0 cm and lengths, mostly, above 13.0 cm, both in our study and in that of garófalo et al. (1993). in fact, in the few studies involving eufriesea in which measures of brood cells were taken, the internal widths and lengths of the cells usually remained above 1.0 cm and 1.5 cm, respectively (garófalo et al., 1993; viana et al., 2001). only for eufriesea violacea (blanchard), the internal contour of cells did not reach 1.0 cm (sakagami & michener, 1965). moreover, the wall thickness of the cells, which are made from tree bark and resin in all species of eufriesea so far studied, contribute to increase the cell width (garófalo et al., 1993; viana et al., 2001; rochafilho et al., 2016; carvalho-filho & oliveira, 2017). all these conditions certainly explain the preference of e. auriceps for bamboo trap-nests herein described. many species of eufriesea are highly seasonal (dressler, 1982; garófalo et al., 1993; peruquetti & campos, 1997). our results, based on adult emergences and nesting activity of females, indicate that e. aff. auriceps has a univoltive life cycle, being active during the warm-wet season, which in the studied region ranges from october to march (sofia et al., 2004). our results also revealed that females of this species were active until the end of this season. with 67 species catalogued (moure et al., 2012) of the eufriesea genus, at most, the nests of a dozen species have been studied (sakagami & michener, 1965; kimsey, 1982; viana et al., 2001; ramírez et al., 2002; carvalho-silva & oliveira, 2017). our results on the nest architecture of e. aff. auriceps are in line with the few studies describing the nesting habit of eufriesea, which showed the use of tree bark associated with resin in cell construction (e.g. sakagami & michener, 1965; viana et al., 2001; carvalho-silva & oliveira, 2017; rocha-filho et al., 2017) and both the size and elliptical form of the brood cells (sakagami & michener, 1965; garófalo et al., 1993; viana et al., 2001). in this context, our results contribute to supporting the idea that no obvious taxonomic pattern exists among species of eufriesea for nesting behavior, at least not based on current recognition of species groups and available biological data (cameron, 2004). lastly, our findings add light on the strategy of using trap-nests for managing orchid bee fauna in forest fragments under anthropogenic interference. more than that, we showed here that the use of bamboo trap-nests can also help the colonization of forest areas undergoing restoration by orchid bee females, as observed for e. aff. auriceps. in a scenario where several studies have suggested that orchid bees or, at least some species of the group, are sensitive to environmental disturbances, such as habitat loss and forest fragmentation (tonhasca et al., 2002; brosi, 2009; giangarelli et al., 2009, nemésio, 2011, 2013), strategies to manage impacted areas become essential for the conservation of euglossine bees and their habitats. therefore, despite the colonization of nests in the restored areas studied herein, these accounted for only approximately a quarter of the nests sampled and were restricted to only two of the nine restored areas. thus, we recommend the continued use of trap-nests as a potential measure for managing the establishment of euglossine fauna in these areas. acknowledgments we thank robson rockembacher for his help in the field; to ibama and iap for permission to collect bees; and to the owners of the study areas for allowing us to conduct the study on their private properties. disclosure statement the authors declares that they have no conflict of interest. funding this study was financed in part by peld-manp (cnpq), by the coordenação de aperfeiçoamento de pessoal de nível superior – brazil (capes) – finance code 001, and cnpq. we are grateful to fundação araucária, capes, and cnpq. shs thank to cnpq for the scientific productivity fellowship (305343/2018-1). references andrade, a.c.r., nemésio, a., oliveira, f.f. & santos, f.n. 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(2002). nota sobre a vegetação da bacia do rio tibagi. in medri, m.e., e. bianchini, o.a. shibatta & j.a. pimenta (eds.), a bacia do rio tibagi (pp. 103-108), londrina: m.e. medri. viana, b.f., neves, e.l. & silva, f.o. (2001). aspectos da biologia de nidificação de euplusia mussitans (fabricius) (hymenoptera, apidae, euglossini). revista brsileira de zoologia, 18: 1081-1087. doi: 10.1590/s0101-81752001000 400006 viotti, m.a., moura, f.r. & lourenço, a.p. (2013). species diversity and temporal variation of the orchid-bee fauna (hymenoptera, apidae) in a conservation gradient of a rocky field area in the espinhaço range, state of minas gerais, southeastern brazil. neotropical entomology, 42: 565-575. doi: 10.1007/s13744-013-0164-y http://dx.doi.org/10.1590/s0073-47212004000200015 http://dx.doi.org/10.1007/s13744-013-0164-y 457 nidification of polybia platycephala and polistes versicolor (hymenoptera: vespidae) on plants of musa spp. in minas gerais state, brazil by f.a. rodríguez1, l.c. barros2, p. caroline2, m.m. souza1, j.e. serrão3 & j.c. zanuncio1* abstract social wasps are natural enemies of caterpillars and, therefore, they have potential to control insect pests in various crops. three colonies of polybia platycephala (richards) and one of polistes versicolor (olivier) (hymenoptera: vespidae) were found on plants of banana (musa spp.) in minas gerais state, brazil. these colonies were at 3.50 m high, under the leaves, which provide shelter from environmental stress. key words: banana, biological control, nest, pest, social wasps. introduction social wasps have many functions in ecosystems as pollinators, predators of insects, bioindicators and nutrient cycling (souza et al. 2010). social wasps are agents of biological control (prezoto & gobbi 2005; picanço et al. 2010), mainly of lepidopteran caterpillars (richter 2000; prezoto et al. 2006). polistes dominulus (christ) (eigenbrode et al. 2000); protonectarina sylveirae (de saussure), brachygastra lecheguana (latreille), polistes carnifex (fabricius), polistes melanosomes (de saussure), polistes versicolor (olivier), polybia ignobilis (haliday), polybia scutellaris (white), protopolybia exigua (de saussure) (desneux et al. 2010), polybia fastidosusculata (de saussare) prontonectarina sylveirae (de saussare) (moura et al. 2000), polistes erythrocephalus (latreille), polistes canadensis (linnaeus) and polybia sericea (olivier) 1 departamento de entomologia, universidade federal de viçosa, 36570-000 viçosa, minas gerais state, brazil, ingpachogro@hotmail.com,zanuncio@ufv.br. 2 departamento de biologia animal, universidade federal de viçosa, 36570-000 viçosa, minas gerais state, brazil, pricarola@gmail.com, lucasus@hotmail.com 3 departamento de biologia geral, universidade federal de viçosa, 36570-000 viçosa, minas gerais state, brazil. jeserrao@ufv.br *author for correspondence 458 sociobiolog y vol. 59, no. 2, 2012 (bellotti et al.1992) feed on larvae of forest and agricultural lepidopteran pests (zanuncio et al. 1993; leite et al. 2001). the study of these insects is important in food webs because they can feed on herbivores (gonring et al. 2003, weiss et al. 2004). insect defoliators of banana plants include calligo illioneus (cramer) (lepidoptera: nymphalidae), antichloris eriphia (fabricius) (lepidoptera: arctiidae) and opogona sacchari (bojer) (lepidoptera: lyonetiidae), which may decrease fruit production (watanabe 2007). wasps were collected in the campus of the federal university of viçosa (20°45's 42°52'w) in viçosa, minas gerais state, brazil on plants of banana (musa spp.) with entomological nets (souza & prezoto 2006). these insects were killed in ether vapor and preserved in 70% ethanol for identification. three colonies of polybia platycephala and one of p. versicolor were found at 3.50 m high under leaves of banana plants, which are long and wide, providing protection against adverse environmental conditions. fig. 1. nest of the social wasp polybia platycephala (hymenoptera: vespidae) on a musa spp. (banana) plant. 459 rodriguez, f.a. et al. — nidification of polistes sp. on musa spp. plants in brazil the occurrence of p. platycephala enlarges the geographic distribution of this wasp, which was reported in the brazilian states of amazonas, goiás, mato grosso, minas gerais, são paulo and rio de janeiro and also in peru and suriname. its nests have many horizontal combs built under leaves of perennial plants (richards 1978, lima & prezoto 2003, prezoto et al. 2005). the social wasp p. platycephala preys on different insects including spodoptera frugiperda ( j.e. smith) (lepidoptera: noctuidae), mocis latipes (guennée) (lepidoptera: noctuidae), alabama argilacea (hübner) (lepidoptera: noctuidae), sciara sp. (diptera) and psilla sp. (heteroptera) playing an important role as biological control agent of insect pests in the field and urban environments (prezoto et al. 2005). colonies of p. versicolor were reported on different substrates such as leaves, rocks, roots and abandoned nests of other wasps (de oliveira et al. 2010). polistes versicolor occurs from amazonas to rio grande do sul states in brazil and its nests are formed by a single comb attached to the substrate by a peduncle (richards 1978, prezoto et al. 2006). this wasp has been reported preying on larvae of the following lepidopterans: hedylepta indicata (fabricius), elasmopalpus lignosellus (zeller) (pyralidae), spodoptera frugiperda ( je smith), heliothis virescens (fabricius), pseudoplusia includens (walker) (noctuidae), chlosyne lacinia saundersii (doubleday & hewitson) (nymphalidae) and automeris sp. (saturniidae). management of p. versicolor colonies in artificial shelters may be an effective strateg y of pest control (prezoto et al. 2006). the presence of p. platycephala (fig. 1) on banana plants should be monitored, because this wasp may spread bacterial disease of banana and heliconia caused by ralstonia solanacearum (smith) yabuuchi et al. race 2, reported in belize, brazil, colombia, costa rica, ecuador, el salvador, grenada, guatemala, guyana, honduras, jamaica, mexico, nicaragua, panama, peru, suriname, trinidad and tobago, usa, venezuela (america), ethiopia, libya, malawi, nigeria, senegal (africa), india, philippines, indonesia, malaysia, thailand and vietnam (asia) (zoccoli et al. 2009). ralstonia solanacearum may infect plants by contact with infected tools, root-root contact, soil-root contact (zoccoli et al. 2009) or by flower visiting insects such as stingless bees trigona spp., wasps polybia spp. and fruit flies drosophila spp. (buddenhagen & kelman 1964). 460 sociobiolog y vol. 59, no. 2, 2012 wasp nests on plants of musa spp. can be dangerous to workers in this culture, mainly during banana harvesting. this is the first record of the social wasps p. platycephala and p. versicolor nesting on banana plants, suggesting that further studies should be focused on prey preference of these wasps in banana plantations to determine if these species can be used in programs of integrated pest management, mainly of defoliating caterpillars. acknowledgements the authors thank “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, coordenação de aperfeiçoamento de pessoal de nível superior (capes)” and “fundação de amparo a pesquisa do estado de minas gerais (fapemig)”. references buddenhagen, i. & a. kelman 1964. biological and physiological aspects of bacterial wilt caused by pseudomonas solanacearum. annual review of phytopatholog y 2(1): 203230. bellotti, a.c., v.b. arias & o.l. guzman 1992. biological control of the cassava hornworm 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(lepidoptera: pyralidae), in cucumber. biological agriculture and horticulture 20 (4): 365-380. leite, g.l.d., m. picanço, r.n.c. guedes & j.c. zanuncio 2001. role of plant age in the resistance of lycopersicon hirsutum f. glabratum to the tomato leafminer tuta absoluta (lepidoptera: gelechiidae). scientia horticulturae 89(2): 103-113. lima, m.a.p. & f. prezoto 2003. foraging activity rhythm in the neotropical swarm-founding wasp polybia platycephala sylvestris richards, 1978 (hymenoptera, vespidae) in different seasons of the year. sociobiolog y 42 (3): 754-752. 461 rodriguez, f.a. et al. — nidification of polistes sp. on musa spp. plants in brazil moura, f., m. picanço, a.h.r. gonring & c.h. bruckner 2000. seletividade de inseticidas a três vespidae predadores de dione juno juno (lepidoptera: heliconidae). pesquisa agropecuária brasileira 35(2): 251-257. picanço, m.c., i.r. oliveira, j.f. rosado, f.m. silva, p. c. gontijo & r.s. silva 2010. natural biological control of ascia monuste 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(heliconiaceae). neotropical entomolog y 36 (2): 312-313. weiss, m.r., e.e.wilson & i. castellanos 2004. predatory wasps learn to overcome the shelter defense of their larval prey. animal behaviour 68 (1): 45-54. zanuncio, j.c., j.b. alves, g.p. santos & w.o. campos 1993. levantamento e flutuação populacional de lepidópteros associados à eucaliptocultura: viregião de belo oriente, mina gerais. pesquisa agropecuária brasileira 28 (10): 1121-1127. zoccoli, m.d., k.c. tomita & h.c. uesugil 2009. ocorrência de murcha bacteriana em helicônias e musácea ornamental no distrito federal. tropical plant patholog y 34 (1): 045-046. doi: 10.13102/sociobiology.v64i2.1385sociobiology 64(2): 174-181 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 effects of sublethal concentrations of chlorpyrifos on olfactory learning and memory performances in two bee species, apis mellifera and apis cerana introduction insecticides are widely used worldwide and play an important role in protecting crops from damage caused by all kinds of pests, including aphids, sap-sucking insects and leaf-eating caterpillars, etc. (henry et al., 2012; mpumi et al., 2016). at the same time, a growing body of evidence shows that insecticides have inevitably caused adverse behavioral and physiological effects on individual bees and colonies (henry et al., 2012; di prisco et al., 2013). honey bees are important pollinating insects for wild plants and crops worldwide, and much attention has been paid to declines in pollinators. colony losses in some parts of the world are due to pathogens (neumann & carreck, 2010), insecticides (henry et al., 2012), weather, habitat loss (potts et al., 2010; vanengelsdorp & meixner, 2010), or interactions among these factors (goulson et al., 2015). abstract chlorpyrifos is a widely used organophosphorus insecticide. the acute oral 24 h median lethal dose (ld50) value of chlorpyrifos in apis mellifera and in apis cerana was estimated to assess differential acute chlorpyrifos toxicity in both bee species. the ld50 values of chlorpyrifos in a. mellifera and in a. cerana are 103.4 ng/bee and 81.8 ng/bee, respectively, which suggests that a. cerana bees are slightly more sensitive than a. mellifera bees to the toxicity of chlorpyrifos. doses half the acute ld50 of chlorpyrifos were selected to study behavioral changes in both bee species using proboscis extension response assay. a. mellifera foragers treated with chlorpyrifos showed significantly lower response to the 10% sucrose solution compared to control bees after 2, 24 and 48 h. chlorpyrifos significantly impaired the olfactory learning abilities and 2 h memory retention of forager bees regardless of honey bee species, which may affect the foraging success of bees exposed to chlorpyrifos. sociobiology an international journal on social insects zg li1, m li1, jn huang1, cs ma1, lc xiao1, q huang2, yz zhao3, hy nie1, sk su1 article history edited by evandro nascimento silva, uefs, brazil received 20 january 2017 initial acceptance 14 february 2017 final acceptance 28 february 2017 publication date 21 september 2017 keywords honey bees, chlorpyrifos, toxicity, olfactory learning, proboscis extension response. corresponding author songkun su college of bee science fujian agriculture and forestry university fuzhou, fujiang, 350002, china e-mail: susongkun@zju.edu.cn neonicotinoid insecticides induce chronic oral toxicity that alters the gustatory responsiveness of adult honey bees and impairs their olfactory learning performances (decourtye et al., 2003; goñalons & farina, 2015). both the type of insecticides exposure in honey bees and dose of insecticides administered to honey bees affect their behaviors based on proboscis extension response (per) (goñalons & farina, 2015). in addition, it was found that sublethal doses of neonicotinoid insecticides decrease the synaptic density of the mushroom body calyx of honey bees and therefore honey bees exposed to 0.04 ng imidacloprid per bee larvae in the larval stage exhibit an impaired olfactory associative behavior in the adult stage (yang et al., 2012; peng & yang, 2016). similar to neonicotinoid insecticides, organophosphorus pesticides, such as chlorpyrifos (cpf), are widely used to control pests infesting agricultural crops (qin et al., 2014). however, chlorpyrifos elicits toxic effects on beneficial insects, aquatic organisms and 1 fujian agriculture and forestry university, fuzhou, china 2 university of bern, bern, switzerland 3 chinese academy of agricultural sciences, beijing, china research article bees sociobiology 64(2): 174-181 (june, 2017) 175 amphibians by inhibiting the activities of acetylcholinesterase (ache) in their central nervous systems (pope, 1999; levin et al., 2004; dimitrie & sparling, 2014). chlorpyrifos is also ranked as the third most frequent pesticide residue detected in hives; this pesticide has also been detected in hive products, such as pollen, nectar, wax, and propolis (chauzat et al., 2006; mullin et al., 2010). the colony residue levels of chlorpyrifos has significantly increased larval mortality (zhu et al., 2014). apoptotic cells have also been found in the midgut of honey bee larvae treated with sublethal levels of chlorpyrifos compared with those in untreated larvae (gregorc & ellis, 2011). upon exposure to sublethal chlorpyrifos concentrations, adult honey bees suffer from impaired learning and recall performances (urlacher et al., 2016). in addition, few queens also emerge when larvae reared in colonies are exposed to sublethal chlorpyrifos levels, and emerged queens become infected with deformed wing virus; this finding indicates that sublethal chlorpyrifos levels impair the development of queens (degrandi-hoffman et al., 2013). olfactory learning and memory test based on per has been performed to investigate the cellular and molecular mechanisms of the learning and memory processes mainly in honey bees apis mellifera (menzel & muller, 1996). behavioral tests have also been conducted to evaluate the behavioral and physiological responses of honey bees to exogenous factors, such as chemicals and pathogens (decourtye et al., 2004; iqbal & mueller, 2007). this classic learning paradigm consists of two elements: an odor (conditioned stimulus, cs) and a sucrose solution (unconditioned stimulus, us) (menzel & muller, 1996). until recently the learning paradigm has also been applied to examine olfactory learning of apis cerana (wang & tan, 2014). a. cerana, a honey bee species native to asia, is an important crop and flora pollinator in the asian region. about two million a. cerana colonies are kept in hives in china (li et al., 2012), and these colonies are used to produce honey and pollinate crops, especially those in hilly and mountainous areas (hepburn & radloff, 2011). previous studies showed that subtheal doses of imidacloprid impair learning acquisition and decision-making abilities of a. cerana (tan et al., 2014; tan et al., 2015). however, although the acute toxicity and sublethal effects of insecticides on a. mellifera have been extensively explored, the toxic effects of pesticides on a. cerana have been rarely examined. it is therefore necessary to determine whether differences existed in the acute and chronic toxicity of chlorpyrifos to the two honey bee species. in our study, 50% oral lethal dose (ld50) was established for a. cerana, and differences in the acute toxicity of chlorpyrifos were compared between a. mellifera and a. cerana. the effects of sublethal chlorpyrifos concentrations on the sucrose responsiveness, olfactory learning, and memory performances of the two honey bee species were further evaluated on the basis of acute oral toxicity. our study provides evidence that a. cerana is more sensitive than a. mellifera to chlorpyrifos. the learning and memory performances of a. cerana treated with sublethal chlorpyrifos doses are more impaired than those of a. mellifera. material and methods acute oral toxicity tests acute oral toxicity testing in honey bees were performed following standard protocols (oecd 213 1998) and previous studies (suchail et al., 2000; iwasa et al., 2004). briefly, chlorpyrifos (sigma-aldrich co.) was dissolved with acetone (sinopharm chemical reagent co.) to prepare stock solutions (500 ng/µl). the stock solutions were stored at 4 °c and covered with tin foil to protect from light prior to testing. five test concentrations of the pesticide were obtained by diluting the stock solutions with 30% (w/v) sucrose solution to determine oral ld50 of chlorpyrifos at 24 h for a. mellifera and a. cerana. three a. mellifera colonies and three a. cerana colonies were used in our study. adult honey bees were randomly collected from hives and starved for 2 h before the toxicity testing in an incubator at 30 °c, 70% ± 5% rh. for each test concentration of chlorpyrifos, three cages, with each cage containing 20 bees, were used for the tests. sixty adult workers from each colony were subjected to acute oral toxicity test. the experiments were repeated thrice for all the colonies. using a 2.5 µl pipette, adult workers were fed with 2 µl of 30% sucrose solution with a specific dose of chlorpyrifos and were assigned as treatment groups. a. mellifera were fed with 72, 88, 104, 120, and 136 ng chlorpyrifos/bee. a. cerana were fed with 60, 70, 80, 90, and 100 ng chlorpyrifos/bee. the control adult workers were individually fed with 2 µl of 30% sucrose solution containing acetone. the treatment and control groups were kept in an incubator at 30 °c and 70% ± 5% rh and fed with 30% sucrose solution ad libitum. the number of dead honey bees was recorded, and different acute oral ld50 were determined 24 h after of the five test concentrations of chlorpyrifos were administered orally. sucrose responsiveness a. mellifera and a. cerana foragers collected at the entrance of the hives were exposed to sublethal doses of chlorpyrifos by using the same procedure to perform acute oral toxicity tests on the basis of the acute oral ld50 determined in this study. doses equal to ld50/2 were selected to assess the behavioral changes in the two honey bee species exposed to chlorpyrifos using the per assay. twenty foragers were captured at the hive entrance of each colony during the peak foraging time at day time. sixty foragers from three different colonies were used for sucrose responsiveness test. the honey bees were immobilized individually in an ice bath in a vial and were confined in a plastic tube with cloth adhesive tape. chlorpyrifos (52 ng/ bee) was administered orally to a. mellifera. chlorpyrifos (41 ng/bee) was also orally administered to a. cerana bees. the control bees were orally treated with acetone alone. the sucrose responsiveness test was performed at 2, 24, and 48 h after exposure in accordance with previously described zg li et al . – cpf impairs learning and memory in apis176 methods (li et al., 2013). harnessed bees were placed and kept in an incubator (30 °c, 70% ± 5% rh) until the bees were subjected to sucrose responsiveness test at each time point. serial concentrations of sucrose solution [0.1%, 0.3%, 1%, 3%, 10%, and 30% (wt/wt)] were used in the study. a wooden tooth pick was immersed in the sucrose solution and was placed in contact with the antenna of the confined bees. honey bees were tested by using increasing concentrations of sucrose and with two minute intervals between tests. the number of bees responding to each concentration was recorded. after the test, a drop of water was applied to touch the antenna of the bees between two consecutive tests of the sucrose solution and to prevent sensitization in honey bees as a result of repeated stimulation. the honey bees were further examined with 50% (wt/wt) sucrose solution, and the honey bees that were unresponsive to 50% sucrose solution were excluded from the analysis (li et al., 2013). olfactory learning and memory test the responsiveness to 30% sucrose solution, which was used to elicit per in honey bees during this test, was not significantly different between the treated honey bees and the control bees. forager bees were immobilized and harnessed in accordance with the same methods described above. about 80-90 foragers collected from three different colonies for each bee species and were used for olfactory learning and memory test. chlorpyrifos and acetone treated foragers were subjected to olfactory learning tests 2 and 24 h after the treatment was administered. the olfactory learning processes involved six training trials, and each training trial consisted of lemon odor (cs) paired with 30% sucrose solution (us). the interval between training trials was 10 min. the honey bees were acclimated for approximately 30 min in the conditioning site after they were removed from the incubator. we accurately controlled the timing and duration of cs by using a modified air pump device. the two branches of a y-shaped tube were connected via the two outlets of the air pump, and one of the two branches contained a round paper (2.5 cm in diameter) soaked with 10 µl of lemon oil. after the air pump was switched on, the common branch of the y-shaped tube constantly delivered lemon-scented air toward the antenna of the harnessed bees. the common branch of the y-shaped tube delivered a constant unscented airflow when the switch was off. odor associative conditioning learning was conducted in accordance with previously described methods (müller, 2002). after six training trials were completed, the retention capacities of the honey bees were examined at 2 and 24 h, respectively. the memory test was performed using lemon odor alone (felsenberg et al., 2011). the number of bees responding to the odor was recorded. the proboscis of the honey bees included in the analysis extended fully during the test; after the test was completed, the honey bees unresponsive to 50% sucrose solution were excluded from the analysis (felsenberg et al., 2011). statistical analysis ld50 of chlorpyrifos for a. mellifera and a. cerana were estimated through probit regression analysis of the survival data by using spss 16.0 software. sucrose responsiveness, olfactory learning, and memory performances were statistically compared between the chlorpyrifos-treated bees and the control bees by using fisher’s exact test. differences were considered significant at p <0.05. results differential acute toxicity of chlorpyrifos in a. mellifera and a. cerana the different oral acute toxic effects of chlorpyrifos on a. mellifera and a. cerana were observed in the study. the acute (24 h) oral ld50 of chlorpyrifos were 103.4 and 81.8 ng/bee for a. mellifera and a. cerana, respectively (table 1). a. cerana was more sensitive to chlorpyrifos than a. mellifera. table 1. the ld50 values and its 95% confidence limits of chlorpyrifos at 24 h in apis mellifera and apis cerana. bee species oral ld50 (ng/bee) 95% confidence limits apis mellifera 103.4 96.2-110.9 apis cerana 81.8 76.0-88.8 sucrose responsiveness of honey bees exposed to sublethal chlorpyrifos doses chlorpyrifos-treated bees and acetone-treated bees were compared for each concentration of sucrose solution separately. the responses to 10% sucrose solution of a. mellifera treated with chlorpyrifos were significantly lower (p <0.05) after 2, 24 and 48 h (fig 1a, 1b and 1c, respectively) than those of the control bees treated with acetone. the two groups did not significantly differ in terms of their responses to other concentrations of sucrose solution. the responses of chlorpyrifos-treated a. cerana and acetone-treated bees to serial concentrations of sucrose solution did not significantly differ after 2 (fig 1d), 24 (fig 1e), and 48 h (fig 1f) of exposure to sublethal chlorpyrifos doses. chlorpyrifos-induced impairment of learning and memory performances of the two honey bee species the tested bees did not show conditioned per in the first trial. the percentage of acquisition rate 2 h after exposure of the chlorpyrifos-treated a. mellifera ranged from 47.8% to 58.9% from the second trial to the sixth trial. by contrast, the percentage of acquisition rate of the corresponding control bees ranged from 74.4% to 84.4%. for each trial, the percentage of per significantly differed between the two groups (p<0.01; fig 2a). the memory retention at 2 and 24 h was significantly sociobiology 64(2): 174-181 (june, 2017) 177 lower in the chlorpyrifos-treated a. mellifera than in the control bees (p<0.01; fig 2c). the percentage of acquisition rate 24 h after exposure of the chlorpyrifos-treated a. mellifera ranged from 28.9% to 41.1% from the second trial to the sixth trial. the percentage of acquisition rate of the corresponding control bees ranged from 58.9% to 74.4%. for each trial, the percentage of per significantly differed between the two groups (p<0.01; fig 2b). memory retention at 2 and 24 h was significantly lower in the chlorpyrifos-treated a. mellifera than in the control bees (fig 2d). fig 1. analysis of sucrose responsiveness in honey bees exposed to sublethal doses of chlorpyrifos. response of honey bees (top: a. mellifera; bottom: a. cerana) treated with sublethal oral doses of chlorpyrifos to serial sucrose solutions after 2 h (a and d), 24 h (b and e) and 48 h (c and f) exposure. control bees received acetone. asterisks denote significant differences: one, p<0.05; two, p<0.01. fig 2. sublethal doses of chlorpyrifos impair learning and memory performances in a. mellifera. learning performances were significantly impaired in chlorpyrifos treated a. mellifera honey bees after 2 h (a), and 24 h (b) exposure. among honey bees exposed for 2 h, honey bees showed significantly impaired memory performances 2 and 24 h after six training trials (c); among honey bees exposed for 24 h, honey bees showed significantly impaired memory performances 2 and 24 h after six training trials as well (d). control bees received acetone. asterisks denote significant differences: two, p<0.01. zg li et al . – cpf impairs learning and memory in apis178 for a. cerana, the acquisition rates ranged from 8.0% to 46.6% in the chlorpyrifos-treated honey bees from the second trial to the sixth trial 2 h after exposure. by comparison, the acquisition rates ranged from 27.8% to 70.0% in the acetonetreated honey bees. similarly, the chlorpyrifos-treated honey bees significantly differed from the acetone-treated honey bees in each trial (p <0.01; fig 3a). the retention capacities were significantly higher in the acetone-treated honey bees than in the chlorpyrifos-treated honey bees 2 and 24 h after the training test was completed (p<0.01 and p<0.05, respectively; fig 3c). the acquisition rates ranged from 7.6% to 51.5% in the chlorpyrifos-treated honey bees from the second trial to the sixth trial 24 h after exposure. by contrast, the acquisition rates ranged from 33.8% to 74.6% in the acetone-treated honey bees. the chlorpyrifos-treated bees significantly differed from the acetone-treated honey bees for each trial (p<0.01) except in the fifth trial (p<0.05; fig 3b). the retention capacities were significantly higher in the acetone-treated honey bees than in the chlorpyrifostreated honey bees 2 h after the training test was completed (p<0.01). conversely, the retention capacities between the acetone-treated honey bees and the chlorpyrifos-treated honey bees were not significantly different 24 h after the test was completed (fig 3d). fig 3. sublethal doses of chlorpyrifos impair learning and memory performances in a. cerana. learning performances were significantly impaired in chlorpyrifos treated a. cerana honey bees after 2 h (a), and 24 h (b) exposure. among honey bees exposed for 2 h, honey bees showed significantly impaired memory performances 2 and 24 h after six training trials (c); among honey bees exposed for 24 h, honey bees showed significantly impaired memory performances 2 h, but not 24 h after six training trials (d). control bees received acetone. asterisks denote significant differences: one, p<0.05; two, p<0.01. discussion the sucrose responsiveness of the control a. mellifera and a. cerana revealed that the percentage of a. cerana showing per ranged from 45% to 88%. by contrast, the percentage of a. mellifera exhibiting per ranged from 68% to 100%. consistent with previous studies (yang et al., 2013), our study revealed that a. mellifera was more responsive to sucrose than a. cerana. in the six consecutive acquisition trials, both honey bee species were unresponsive to cs in the first acquisition trial, indicating that there were no sensitization responses in the honey bees. the acquisition rates of control a. mellifera ranged from 74% to 84%. by comparison, the acquisition rates of control a. cerana ranged from 28% to 70%. a. cerana possibly required more time to acclimate to the experimental confinement used in the per assay and thus might influence the behavioral motivation of a. cerana during the sucrose responsiveness test and olfactory learning test. the insecticide is one of the factors responsible for the impaired homing abilities, defective immune responses, delayed larval development, reduced adult bee longevity, and a. mellifera colony collapse (wu et al., 2011; henry et al., 2012; di prisco et al., 2013). honey bees were fed individually with test concentrations of chlorpyrifos, the real dose administration in each honey bee can therefore be obtained in the study. in our cage studies, a. cerana is more sensitive to oral acute toxicities of chlorpyrifos than a. mellifera. however, there are few reports of collapse due to applications sociobiology 64(2): 174-181 (june, 2017) 179 of insecticides in a. cerana colonies (park et al., 2015). this finding may be correlated with differential physiological and behavioral characteristics between the two honey bee species. the responses to higher or lower concentrations of sucrose solutions between chlorpyrifos-and acetone-treated honey bees were not significantly different. this finding suggested that sublethal chlorpyrifos concentrations may not damage the gustatory receptors of the antennae in honey bees. chlorpyrifostreated a. mellifera exhibited a significantly lower response to 10% sucrose solution than the corresponding control bees did. sublethal chlorpyrifos concentrations may induce a dynamic disorder in the normal function of the gustatory receptors of a. mellifera. therefore, this pesticide may elicit a dynamic response to a moderate sucrose concentration (10% sucrose solution). nicotinic acetylcholine receptors (nachrs) in the brain of honey bees participate in the olfactory learning and memory performances of honey bees (jones et al., 2006). acetylcholine (ach) is a potent neurotransmitter during this process (goldberg et al., 1999). chlorpyrifos, an important organophosphate, exert its mode of action by inhibiting ache activity (williamson et al., 2013). this enzyme cannot hydrolyze ach and terminate excitatory neurotransmission in the central nervous systems of forager bees exposed to sublethal chlorpyrifos concentrations (palmer et al., 2013). therefore, the acquisition and retention capacities of chlorpyrifos-treated forager bees were impaired. chlorpyrifos oxon is a chlorpyrifos metabolite more potent than the parent compound in terms of inhibiting ache activities (williamson et al., 2013). acquisition tests were performed 2 and 24 h after the honey bees were exposed to chlorpyrifos in the test. in our study, chlorpyrifos oxon possibly impaired the acquisition capacities of honey bees after 24 h of exposure. chlorpyrifos oxon may also elicit adverse effects on the recall process 24 h after the acquisition tests were completed. on the basis of these findings, we concluded that sublethal chlorpyrifos concentrations significantly impaired the olfactory learning abilities and 2 h memory retention of honey bees regardless of species. associative recall in honey bees can be used as an alternative strategy for navigation and successful search for food resources (reinhard et al., 2004). therefore, impaired associative recall performances may affect the successful foraging of honey bees exposed to chlorpyrifos and may eventually influence the growth and survival of honey bee colonies. in this study, the retention capacities of chlorpyrifos-treated a. cerana and acetone-treated bees did not significantly differ 24 h after the six trials which were performed 24 h after exposure to chlorpyrifos. this result is possibly due to the differential effects of chlorpyrifos on the physiological and molecular processes underlying the learning and memory functions of the two honey bee species. acknowledgements we thank huaifeng he, shaolei yang, and yifei liu for their assistance in research. this research was supported by the natural science foundation of fujian province (no. 2016j05063) and the 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(2014). four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. plos one, 9: e77547. doi: 10.1371/journal. pone.0077547 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.5938sociobiology 68(4): e5938 (december, 2021) introduction the social wasps of the subfamily polistinae are highly diversified in the neotropic region. a total of 381 species are known to occur in brazil, of which, more than 100 are endemic (somavilla et al., 2021). social wasps are an important component of tropical ecosystems due to their ubiquity and species diversity, as well as their complex relationships with other organisms (somavilla & fernandes, 2020). these wasps abstract social wasps are widely distributed in brazil, and their distribution is closely related to the type of habitat and vegetation structure. veredas (palm swamps) occur on moist soils and are characterized by an almost total predominance of buriti palms (mauritia flexuosa). the insect fauna of these environments is poorly known, especially in central brazil. some data on the diversity of the social wasps are available for the state of mato grosso, although its veredas have not been surveyed up to now. in the present study, we investigated the wasp species composition in six vereda environments over a 24-month sampling period, between august 2017 and july 2019. the specimens were collected using a sweep net, which was deployed along 200-m transects that were subdivided into 10 plots of 3 m2. to attract the wasps, we used a backpack sprayer to spray each plot with insect attractant made from five spoonfuls of granulated sugar and one spoonful of salt, dissolved in 5 liters of water. after spraying the plot, we waited 10 minutes before initiating the surveys, which lasted 10 minutes per plot, for a total of 200 minutes of sampling per palm swamp. a total of 1072 social wasp specimens were collected, representing 11 genera and 37 different species. the most abundant species were polybia cf. ruficeps xantops (richards, 1978), agelaia pallipes (olivier, 1792); polybia rejecta (lepeletier, 1836), and mischocyttarus mattogrossoensis zikán, 1935, which together accounted for 57% of the specimens collected over the 24 months of the study period. the total species richness estimated for the vereda environments was 39.88 ± 0.627, approximately 41% of the 88 wasp species of the subfamily polistinae found in the cerrado of eastern mato grosso. sociobiology an international journal on social insects lourivaldo a. castro1, sergio r. andena2, evandson josé a. silva3 article history edited by alexandre somavilla, inpa, brazil received 27 october 2020 initial acceptance 03 december 2020 final acceptance 27 september 2021 publication date 19 november 2021 keywords cerrado, buriti forest, social wasps, transitional zone. corresponding author lourivaldo a. castro departamento de biologia da faculdade de cièncias agrárias e sociais, universidade do estado de mato grosso, campus de nova xavantina, mato grosso, brasil. e-mail: louricastro@unemat.br are characterized by varying levels of eusociality, complex niche architecture, and aggressive nest defense behavior (carpenter, 1991; wenzel, 1998; pickett & carpenter, 2010; piekarsky et al., 2018). wasps are important pollinators, which overlap with bees in terms of the resources exploited and represent a meaningful component of the pollinator community (santos et al., 2010; clemente et al., 2012; brock et al., 2021). although the exact role of social wasps as predators and pollinators 1 departamento de biologia da faculdade de cièncias agrárias e sociais, universidade do estado de mato grosso, campus de nova xavantina, mato grosso, brazil 2 departamento de ciências biológicas, museu de zoologia, divisão entomologia, universidade estadual de feira de santana, bahia, brazil 3 faculdade de ciências agrárias e biológicas, universidade do estado de mato grosso, campus cárceres, mato grosso, brazil research article wasps assemblages of social wasps (hymenoptera, vespidae, polistinae) in the veredas of central brazil mailto:louricastro@unemat.br lourivaldo a castro, sergio r andena, evandson ja silva – social wasps in the veredas of central brazil2 is still poorly understood. some studies indicate they may contribute effectively to the control of the populations of organisms that are considered to be agricultural pests and also that they may contribute to pollination (prezoto et al., 2019; southon et al., 2019). given their abundance and ecological importance, social wasps have been and continue to be the subject of ecological studies in different regions and environments (diniz & kitayama, 1998; souza & prezoto, 2006; silva et al., 2011; da silva et al., 2019; clemente et al., 2020; ferreira et al., 2020). in brazil, these environments include the cerrado savanna (souza et al., 2012; souza et al., 2020; vicente et al., 2020), atlantic forest (grandinete & noll, 2013; togni et al., 2014; ribeiro et al., 2019; souza et al., 2021), the semi-arid northeast (santos et al., 2020), the amazon forest (silveira, 2002; silveira et al., 2012; somavilla & oliveira, 2017; graça & somavilla, 2018; somavilla et al., 2020; gomes et al., 2020), and the pantanal wetlands of mato grosso (almeida et al., 2014). in the most recent study of the social wasps of the brazilian state of mato grosso, ferreira et al. (2020) recorded 43 species. however, many regions or specific environments are still in need of research, as in the case of eastern mato grosso, where the most recent study was published 20 years ago (richard, 1978; carpenter & marques, 2001). more than 40 years ago, richards (1978) recorded a total of 88 species of social wasp in eastern mato grosso. while the region clearly has a high species richness, the number of species may be underestimated due to the lack of inventories in the region, in particular, in vereda environments. the vereda (palm swamp) is a phytophysiognomy of the central brazilian cerrado biome, which is characterized by a predominance of buriti palms (mauritia flexuosa) and an understory of shrubs and herbs, located on hydromorphic soils and in swampy or waterlogged areas, which typically form at the source of the region’s watercourses (ferreira, 2003). wetland environments, such as veredas, are important for the conservation of the biodiversity of the cerrado biome, mainly due to their ‘sponge effect’, buffering the effects of torrential local downpours, storing carbon in the soil, filtering allochthonous waste (e.g.: agricultural fertilizers and pesticides), and providing traditional communities with natural resources, such as fibers and fruit (rosolen et al., 2015). the veredas are important, sensitive environments that are protected under brazilian law (conama resolution number 303 of march 20th, 2002), although these palm swamps have nevertheless been suffering changes caused by different types of land use, such as cattle pasture, river impoundments, and cash cropping. these impacts cause the destruction of the natural vegetation, which affects hydrological parameters, increasing soil erosion, and changing the morphological and chemical properties of the soils of these environments (ferreira & troppmair, 2004). data on the biodiversity of the veredas are still very scant, in particular in the case of the invertebrates, a scenario exacerbated by the fact that anthropogenic shifts in natural environments have negative impacts on the distribution and diversity of the resident organisms (guimarães et al., 2002). in this context, the present study inventoried the social wasp species found in six veredas of the eastern extreme of the brazilian state of mato grosso. material and methods study area the study area comprises six veredas located in the municipality of nova xavantina in mato grosso state, in the araguaia-tocantins river basin of central brazil (table 1; figures 1, 2). these veredas are located within the transition zone between the amazon and cerrado biomes, which means that, while the vegetation associated with the veredas is composed of species commonly found in the cerrado, plant species typical of the amazon biome are also common in some areas, forming distinct patches within the savanna landscape (marques et al., 2019). the region’s climate is aw in the köppen classification system (peel et al., 2008), with two well-defined seasons, dry and rainy. vereda latitude (s) longitude (w) altitude area (ha) va 14◦45’52” 52◦33’02” 490 m 6.0 vb 14º45’41’’ 52º33’58’’ 490 m 6.2 vc 14º49’03’’ 52º34’35’’ 540 m 5.5 vd 14º50’47’’ 52º30’52’’ 550 m 4.0 ve 14º49’42’’ 52º30’59’’ 550 m 5.0 vf 14º47’09’’ 52º36’19’’ 390 m 3.0 table 1. veredas and code: va = botina stream, vd and ve = chupador stream, vc = areal stream, vf = coronel vanique stream, and vb = queixada stream. geographic coordinates, altitude, and area of the veredas surveyed in eastern mato grosso, brazil. sampling of social wasps the samples were collected in six veredas over a 24-month period from august 2017 to july 2019. each vereda was sampled on a total of 24 days, with 2 hours of sampling per day, i.e., 48 hours per vereda, and an overall total of 288 hours. the specimens were collected using a sweep net, which was deployed along transects of 200 m in length, which were subdivided into 10 plots of 3 m2. to attract the wasps, we used a backpack sprayer to spray each plot with insect attractant (5 spoonfuls of granulated sugar plus one spoonful of salt, dissolved in 5 liters of water). we waited for 10 minutes after this spraying before initiating the surveys, which lasted 10 minutes per plot, for a total of 200 minutes of sampling per vereda. this collection protocol was modified from noll and gomes (2009). sociobiology 68(4): e5938 (december, 2021) 3 preparation of the wasp specimens the wasp specimens were sorted, pinned, packed in entomological boxes, and identified using dichotomous keys (carpenter & marques, 2001; somavilla & carpenter, 2021). samples of each potential species were sent to dr. sérgio r. andena, a specialist in the taxonomy of the social wasps, to confirm their identification. the wasp specimens were deposited in the bee and wasp collection of the laboratory of neotropical bees and wasps, on the cáceres campus of mato grosso state university (unemat), under the curation of dr. evandson j. anjos silva. data analysis to evaluate sampling efficiency, we compiled a species accumulation curve using the nonparametric firstorder jackknife estimator, run in the estimateswin7.5.0 (colwell, 2013) software. the constancy (c) of each species was calculated by: c = px (100 / n), where c = constancy and p = the number of samples in which a given species was collected, and n = total number of samples collected. the sampling unit in this study were the 24 months of fieldwork. the species were classified as constant (present in > 50% of the samples), auxiliary (25–50%) or accidental, when < 25% (silveira-neto et al., 1976, 1995; dajoz, 1983). fig 1. location of the six study veredas in the cerrado–amazon transition zone in the municipality of nova xavantina, in mato grosso state, central brazil. the veredas are identified according to the catchment in which they are located. botina stream, and chupador stream i and ii, areal stream, coronel vanique stream, and queixada stream. results during the present study, we recorded 37 species of wasp of the subfamily polistinae (table 2). the species richness of the six veredas estimated by the jackknife procedure was s = 38.88, sd = 1.59, and ci = 0.627, and the general index of wasp diversity was h ‘= 3.319. the most abundant species were polybia cf. ruficeps xantops (richards, 1978), agelaia pallipes (olivier, 1792); polybia rejecta (lepeletier, 1836), and mischocyttarus mattogrossoensis zikán, 1935 which together accounted for 57% of all the specimens collected during the 24 months of the study period. the estimated species richness for the vereda environments was 38.88 ± 0.627, approximately 41% of the 88 species of polistine wasps known to occur in the cerrado of eastern mato grosso. based on the analysis of the frequency of records (table 2), as proposed by silveira-neto et al. (1976, 1995), eight (21.62% of the total) of the species were classified as constant, being the most abundant and widespread in the samples. a further 13 species (35.13%) had intermediate abundance, and were thus classified as accessory species. the remaining 16 species (43.24% of the total) were the least abundant, with between one and seven specimens being collected in no more than three samples. these species were classified as accidental. lourivaldo a castro, sergio r andena, evandson ja silva – social wasps in the veredas of central brazil4 the species accumulation curve (figure 3) indicates that the sampling efficiency of the present study provided a reliable estimate of the social wasp species richness found in the palm swamps of eastern mato grosso. specifically, the observed species richness represented 93% of that estimated by the jackknife procedure. discussion the social wasp species richness observed and estimated in this study in the veredas of central brazil, just one of the vegetation types of the cerrado biome, are highly similar to the previous estimates for the cerrado as a whole botina stream queixada stream areal stream chupador stream i chupador stream ii coronel vanique stream fig 2. landscape features of the six study veredas in the cerrado–amazon transition zone in the municipality of nova xavantina, in mato grosso state, central brazil. botina stream, chupador stream i and ii, areal stream, coronel vanique stream, and queixada stream. n = 36, 38, 19, 31, 38, 33 successively according to the authors (diniz & kitayama, 1998; souza & prezoto, 2006; santos et al., 2009; silva et al., 2011; souza et al., 2014; vicente et al., 2020). the species richness registered in the veredas of mato grosso is higher than the richness registered in the pantanal of poconé (northern pantanal), that has (n=15) species recorded (almeida et al., 2014). ferreira et al. (2020) recorded 43 wasp species representing 10 genera in areas of forest, the cerrado/amazon transition zone, and the cerrado savanna, based on specimens collected at 42 sampling points in eight municipalities in the state of mato grosso. however, in the present study in only six points in a single phytophysiognomy of the cerrado biome, sociobiology 68(4): e5938 (december, 2021) 5 taxon / vereda va vd ve vf vb vc n ct % agelaia agelaia pallipes (olivier, 1792) 37 70 32 15 0 2 156 c brachygastra brachygastra augusti (de saussure, 1854) 5 1 1 13 3 20 43 c brachygastra bilineolata (latreille, 1824) 1 0 0 0 0 2 3 ad brachygastra moebiana (de saussure, 1867) 1 1 3 2 6 4 17 ac brachygastra smithii (de saussure, 1853) 4 0 0 2 2 1 9 ac brachygastra sp.1 3 0 1 1 2 1 8 ac chartergellus chartergellus communis (richards, 1978) 9 0 0 0 0 4 13 ac chartergus chartergus globiventris (de saussure, 1853) 0 0 0 1 0 0 1 ad mischocyttarus mischocyttarus collares ducke, 1904 3 6 13 1 3 0 26 c mischocyttarus cerberus ducke, 1898 1 0 0 0 0 0 1 ad mischocyttarus mattogrossoensis zikán, 1935 29 5 14 49 0 42 139 c mischocyttarus (megacanthopus) 0 0 4 0 3 0 7 ad mischocyttarus (kappa) 0 0 2 0 0 1 3 ad mischocyttarus (mischocyttarus) 0 0 1 0 0 0 1 ad mischocyttarus drewseni de saussure, 1857 0 1 0 0 0 0 1 ad mischocyttarus sp.1 0 0 2 0 0 2 4 ad parachartergus parachartergus fraternus (gribodo, 1892) 0 7 3 9 0 1 20 ac polistes polistes canadensis (linnaeus, 1854) 0 0 0 1 0 2 3 ad polistes geminatus fox, 1898 0 0 4 3 0 1 8 ac polistes satan bequaert, 1940 1 0 0 0 0 7 8 ad polistes subsericeus (de saussure, 1854) 1 0 0 13 2 20 36 c polybia polybia cf ruficeps xantops (richards, 1978) 137 1 9 5 2 62 216 c polybia erythrothorax richards, 1978 0 0 0 0 1 4 5 ad polybia ignobilis (haliday, 1836) 0 6 0 1 13 5 25 ac polybia jurinei (haliday, 1836) 0 0 1 2 0 0 3 ad polybia liliacea (fabricius, 1804) 0 1 0 3 1 0 5 ad polybia occidentalis (olivier, 1791) 3 48 15 18 6 15 105 c polybia rejecta (lepeletier, 1836) 6 49 23 2 13 0 93 c polybia sericea (olivier, 1792) 2 0 0 4 0 5 11 ac polybia belemensis richards, 1970 4 2 1 1 4 10 22 ac polybia parvulina richards, 1970 0 6 2 1 0 3 12 ac polybia rufitarsis ducke, 1904 0 1 0 0 0 0 1 ad protopolybia protopolybia chartergoides gribodo, 1891 14 0 0 0 0 6 20 ac protopolybia sedula (de saussure, 1854) 2 0 0 1 13 8 24 ac protopolybia sp.1 0 0 0 3 0 0 3 ad pseudopolybia pseudopolybia vespiceps de saussure, 1864 0 2 0 0 0 0 2 ad synoeca synoeca surinama (lepeletier, 1836) 3 1 0 3 9 0 16 ac total 266 208 133 154 83 228 1072 table 2. abundance of the social wasp species of the subfamily polistinae recorded between august 2017 and july 2019 in the six study veredas in the cerrado–amazon transition zone in the municipality of nova xavantina, in mato grosso state, central brazil. va = botina stream, vd and ve = chupador stream, vc = areal stream, vf = coronel vanique stream, and vb = queixada stream, ct% constancy. lourivaldo a castro, sergio r andena, evandson ja silva – social wasps in the veredas of central brazil6 we sampled 37 species from 11 genera, six species less, but one more genus. that is, the vereda, which indicates clearly that these environments are rich in social wasp species. it is important to note, however, that only 15 species were found in common between the two studies, which may in part be related to the differences in the geographic distributions of the species. the predominance of species of the tribe epiponini was expected, given that this tribe has at least 246 species distributed among its 19 genera, which are almost exclusively endemic to the neotropical region (somavilla et al., 2021). epiponini species are widely distributed throughout brazil, where they have been recorded in all geographic regions (locher et al., 2014; somavilla et al., 2015; aragão & andena, 2016; somavilla et al., 2021). biogeographic reconstructions indicate that the amazon region is the principal source of epiponini diversity (menezes et al., 2020). according to marques et al. (2019) recent redefinition of the limits of the cerrado and amazon biomes, the veredas surveyed in the present study are located within the amazon/cerrado transition zone, which may have contributed to the epiponini diversity recorded in this study. the greater abundance of some of the epiponini species recorded in the present study may be related to the size of their colonies, which may include thousands of individuals, and the more diurnal habits of some taxa, in addition to more random factors, such as the presence of nests of two of the four most abundant species in the vicinity of some of the transects, which may have facilitated the collection of specimens during foraging. epiponini species provided more than 70% of the specimens collected in many other brazilian studies (diniz & kitayama, 1998; carpenter & marques, 2001; elpinocampos et al., 2007; santos et al., 2009; silva et al., 2011, togni et al., 2014; somavilla et al., 2021). however, some epiponini species (almeida et al., 2014) were less abundant in our study, with the number of individuals ranging from one to nine, an abundance similar to that recorded by elpinocampos et al. (2007), lima et al. (2010), simões et al. (2012), and locher (2014). the least abundant species belonged to the tribes mischocyttarini and polistini, as observed in previous studies (diniz & kitayama, 1994; souza & prezoto, 2005; elpinocampos et al., 2007; santos et al., 2009; simões et al., 2012; silveira et al., 2008; silva & silveira, 2009; almeida et al., 2014). the reduced abundance of these two tribes was expected, given that their species build nests that contain no more than a few dozen or hundreds of individuals (carpenter & marques, 2001: o’donnell, 2020; somavilla et al., 2021). the most abundant species in our study was polybia cf. ruficeps xanthops (n = 216 individuals), although more than 80% of the individuals were collected from only two of the six study veredas. we believe that the abundance of the species at these two sites may have been related to the proximity of nests to the study plots in these two veredas. a much lower abundance of this species has been recorded in previous studies of social wasps (richards 1978; diniz & kitayama, 1998; elpinocampos et al., 2007; gomes & noll, 2009; tanaka-junior & noll, 2011). fig 3. species accumulation curve of the social wasps and the estimated species richness of the six sstudy veredas, in the cerrado–amazon transition zone, estimated from the 60 plots sampled monthly between august 2017 and july 2019, in the municipality of nova xavantina, in eastern mato grosso state, brazil. sociobiology 68(4): e5938 (december, 2021) 7 the results of the present study indicate that social wasps of all three tribes, i.e., epiponini, mischocyttarini, and polistini, can be classified as constant, accessory or accidental, which is consistent with previous studies (togni et al., 2014; souza et al., 2014; souza et al., 2016; santos et al., 2020). the accidental species were the most numerous overall, followed by the accessory taxa, with the constant species being the least common. these findings contrast with those of togni et al. (2014), who recorded relatively more constant species and fewer accessory taxa. drawing on the conclusions of santos et al. (2007), togni et al. (2014) suggested that the greater abundance of constant species may be accounted for by the complexity of the dense rainforest habitats they surveyed. in this case, the difference found in the present study may be accounted for by the distinct characteristics and the area of the veredas surveyed, which varied in size from three to 10 hectares, and were divided into three distinct vegetation zones – seasonally and permanently flooded grassland, and a central zone dominated by buriti palms, mauritia flexuosa l.f. (santos & munhoz, 2012). this implies that the classification of almost half of the species collected in the veredas as accidental may reflect their sporadic forays into theses swamps in search of some specific resource that is abundant in the vereda, most likely, water. auad et al. (2010) and souza et al. (2014) obtained results similar to those of the present study, with a predominance of accidental species, which they attributed to the type of environment, habitat use, and the size of the areas sampled. the present study is the first systematic, standardized, and long-term inventory of the social wasp communities of the veredas of central brazil. the data are an important contribution to the understanding of the geographic distribution of brazilian wasps. acknowledgements xxx is a doctoral student at the doctorate program of the bionorte network – biodiversity and biotechnology of the legal amazon, and all the authors are grateful to this program for making this study possible. authors' contribution lourivaldo a. castro, main author, performed all steps; project idealization, proposal formatting and methodology, field steps, data analysis, writing and submission of the text in a journal. evandson josé a. silva, co-author, participated in project formatting, methodology and manuscript. sergio r. andena, co-author, participated in the design of the project, methodology and species identification. references almeida, s.m., andena, s.r. & anjos-silva, e.j. 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(2014). the social wasp community (hymenoptera, vespidae) in an área of atlantic florest, ubatuba, brazil: check list, 10: 10-17. vicente, l.o., jacques, g.c., souza, m.m. & corrêa, b.s. (2020). species richness of the social wasps (hymenoptera: vespidae) of the cerrado of southeastern brazil. nature and conservation, 13: 1-11. doi: 10.6008/cbpc2318-2881.2020. 004.0001 wenzel, j.w. (1998). a generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae: vespinae, polistinae). american museum novitates, 3224: 1-39. _hlk78827438 _hlk78789031 _hlk27402393 _hlk56590936 _hlk45027656 _hlk41472497 _hlk27232808 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 96-100 (2013) effect of habitat disturbance on colony productivity of the social wasp mischocyttarus consimilis zikán (hymenoptera, vespidae) kb michelutti1, ts montagna2, wf antonialli-junior1,2 introduction anthropogenic disturbance of natural habitats is one of the main factors that contribute to reduction of the biodiversity in tropical environments (reviewed in samways, 2005; 2007). the effect of habitat on the colony dynamics of social insects has been examined in several studies (santos & gobbi, 1998; inagawa et al., 2001; gamboa et al., 2005). however, few studies have evaluated the effect of habitat modification by humans on the development of colonies in a tropical environment (penna et al., 2007; montagna et al., 2010). evidence that human presence alters habitat quality has been reviewed recently by raupp et al. (2010) and schowalter (2012). the modification of natural habitats is due mainly to the progress of agriculture and livestock raising, as well as to urbanization (abensperg-traun & smith, 2000; reviewed in new, 2005; reviewed in raupp et al., 2010). abstract social wasps are important elements of the fauna in a variety of environments, including human-modified environments. evidence indicates that habitat quality affects the growth of colonies of social wasps in urban environments. this study investigated whether the colony productivity of the social wasp mischocyttarus consimilis zikán is affected by loss of habitat quality in a human-occupied environment. nests of m. consimilis were collected in forest and urban environments between january 2010 and june 2011. only nests that reached the declining stage were sampled. as productivity parameters, we measured the total number of cells constructed, the total number of adults produced and dry mass of the nests. productivity was significantly lower in urban than in forest environments for all parameters analyzed. habitat quality is probably the principal factor that contributed to the lower productivity in the urban environments. in this type of environment, particularly where the study was conducted, the vegetation adjacent to the nesting sites was composed predominantly of grasses. such habitats may have limited available resources, especially those used by wasps for feeding the larvae, such as immatures of other insects. this result suggests that human degradation of habitats negatively affects the final productivity of colonies of social wasps. sociobiology an international journal on social insects 1 universidade estadual de mato grosso do sul, dourados-ms, brazil 2 universidade federal da grande dourados, dourados-ms, brazil research article wasps article history edited by: sergio r. andena, uefs, brazil. received 25 october 2012 initial acceptance 21 november 2012 final acceptance 13 february 2013 keywords social wasp, synanthropism, independent foundation, functional ecology, conservation biology corresponding author kamylla b. michelutti programa de pós-graduação em recursos naturais univ. estadual de mato grosso do sul dourados-ms, brazil, 79804-970 e-mail: kamylla_michelutti@yahoo.com.br social wasps are important elements of the fauna in many environments and important predators of other arthropods in tropical regions (gould & jeanne, 1984; gobbi & machado, 1986; prezoto et al., 2005). in the tropics, many social wasps are facultatively synanthropic, occurring abundantly in both forest and urban environments (fowler, 1983; curtis & stamp, 2006; de oliveira et al., 2010). a recent approach suggests that nesting behavior in social insects is influenced by several ecological variables, including competition, foraging efficiency, microclimate, nest deterioration, nest quality, parasitism, predation, seasonality, and colony growth (reviewed in mcglynn, 2012). the wide range of nesting sites on human constructions and low interspecies competition may partly explain the facultative occurrence of some social wasp species in urban environments (giannotti & mansur, 1993; prezoto et al., 2007; de oliveira et al., 2010). however, synanthropic colonies of social wasps are directly exposed to the effects of human interference in the sociobiology 60(1): 96-100 (2013) 97 habitat (curtis & stamp, 2006). habitat quality is one of the principal factors that influence the colony dynamics of social wasps (inagawa et al., 2001; gamboa et al., 2005; d’adamo & lozada, 2007). it has been suggested that habitat quality is reduced drastically with increased urbanization (reviewed in raupp et al., 2010; reviewed in schowalter, 2012). therefore, it is expected that the low habitat quality in urban environments negatively affects the development of the social insect colonies. in the urban environment, as a direct consequence of the humancaused habitat degradation, the foraging areas surrounding the colony consist mainly of grasses, suggesting that this type of habitat offers less resources compared to sites where the original vegetation is preserved (gould & jeanne, 1984). for example, naug and wenzel (2006) demonstrated that the supply of resources in the surroundings of the nests was the main limiting factor on colony growth in ropalidia marginata (lepeletier). similarly, mead and pratte (2002) demonstrated that differences in local resource availability among populations of the social wasp polistes dominula (christ) resulted in considerable differences in terms of nest growth and production of offspring. mischocyttarus consimilis zikán is a neotropical social wasp with a distribution restricted to paraguay and the southern region of the state of mato grosso do sul (richards, 1978; montagna et al., 2009). colonies of m. consimilis are usually established by a single female, and the nests are composed of a single, uncovered comb that is attached to the substratum by a single, central petiole (montagna et al., 2010). colonies of this species found and abandon their colonies throughout the year (torres et al., 2011). this species can be considered facultatively synanthropic, since it is abundant in locations affected by intense movement of people, as well as in forest environments (montagna et al., 2010). this study investigated the effect of habitat disturbance on colony productivity of the social wasp m. consimilis. material and methods data collection and field procedures to evaluate the effects of the human-caused habitat disturbance on colony development, we compared the final productivity of colonies of m. consimilis located in a forest environment (conserved habitat) and an urban environment (disturbed habitat), in the municipality of dourados (22º13’16’’s; 54º48’20’’w) in the state of mato grosso do sul. we collected 11 abandoned nests in each environment during january 2010 to june 2011. colonies in the selected areas were monitored weekly to determine the end of the colony cycle. only nests that reached the declining stage (widespread presence of empty cells in the comb and nest abandonment), as defined by jeanne (1972), were used in the sample. as productivity parameters we measured the number of constructed cells, number of adults produced, and dry mass of the nest. the number of adults produced was estimated by counting the number of layers of meconium in each cell of the comb. the meconium layer is formed on the floor of the productive cells as a result of the elimination of feces by the last-instar larva, just before pupation (gobbi & zucchi, 1985; giannotti, 1999). the meconium layer was removed in the laboratory with the aid of tweezers, sectioning each cell of the comb. for analysis of the nest dry mass, the nest was placed in a separate petri dish, and then dried in a vacuum chamber for 24 h and immediately weighed on a precision balance. study sites the forest environment selected in this study belongs to an environmental preservation area known as the “reserva do coqueiro”, located 10 km from the urban perimeter of the city of dourados. the reserva do coqueiro is composed predominantly by a preserved forest, traversed by a road that leads to approximately a dozen camp-houses adjacent to the forest. the camp-houses are used mainly for recreational events and remain closed most of the year. human traffic in that area is sparse. all the nests collected in the reserve were located on the edges of the camp-houses. the contrasting urban environment is in a residential area known as the “cidade universitária”, near the campus of the universidade federal da grande dourados. this area is undergoing rapid development, with extensive new construction in progress. the areas adjacent to the buildings are predominantly grassed or covered by sidewalks or asphalt, and human traffic is intense year-round. all the nests collected in this environment were located on the edges of occupied houses and university buildings. statistical analysis the t-test for two independent samples was used to evaluate possible differences in colony productivity parameters between the two wasp populations. we performed a correlation analysis between the number of individuals produced and the number of cells constructed, to evaluate possible differences in strategies of comb use between the two populations. for all analyses, the variable was considered when the resulting regression coefficient was significant at the 0.05 level. results for nests in the forest environment, the mean values (± se; n=11) were: number of adults produced, 250.6 ± 6.62; number of constructed cells, 215 ± 22.8; nest dry mass, 1.11 ± 5.66 g; proportion of productive cells, 67.4 ± 39.6%; prokb michelutti, ts montagna, wf antonialli-junior effect of habitat disturbance on social wasp colony productivity98 portion of reused cells, 37.3 ± 6.85%; and number of adults produced per cell, 1.09 ± 0.13 (table 1). in this environment we found a significant positive correlation between the number of cells constructed and the number of adults produced (r=0.84; p<0.01; n=11) (fig. 1). for nests in the urban environment, the mean values (± se; n=11) were: number of adults produced, 131.9 ± 4.69; number of constructed cells, 142.2 ± 13.4; nest dry mass, 0.59 ± 5.70 g; proportion of productive cells, 56.2 ± 20.2%; proportion of reused cells, 39.3 ± 5.91%; and number of adults produced per cell, 0.98 ± 0.09 (table 1). in this environment, the correlation analysis was significantly positive between the number of constructed cells and the number of adults produced (r=0.64; p=0.03; n=11) (fig. 1). productivity in the urban environment was significantly lower than in the forest environment with respect to the number of adults produced (t=-2.35; df=13.69; p<0.05), number of cells constructed (t=-3.08; df=17.80; p<0.01), and dry mass of nests (t=-3.51; df=15.16; p<0.01) (table 1). in contrast, the proportion of productive cells (t=-1.59; df=19.73; p=0.12), proportion of reused cells (t=0.25; df=19.93; p=0.8), and number of adults produced per cell (t=-1.04; df=17.68; p=0.3) did not show significant differences between the two environments (table 1). discussion colony productivity for m. consimilis was determined by the size of the nest in both environments, with larger nests being more productive. however, nests collected in the urban environment were less productive than nests collected in the forest environment, as estimated by the number of cells constructed, number of adults produced, and dry mass of nests. montagna et al. (2010), studying colonies of m. consimilis in an urban environment, found a mean productivity of 72.9 ± 10.5 and 40.7 ± 14.0, for the number of cells constructed and adults produced, respectively. the productivity found by montagna and coworkers is lower than in our study; however, the authors evaluated only colonies in the post-emergence stage. our results and those of montagna and coworkers suggest that colony productivity in tropical social wasps is affected by the habitat quality, and an increase in urbanization negatively influences the development of the colonies. particularly in the study locale, the vegetation adjacent to the nesting sites was composed predominantly of grasses. studies demonstrate that habitats with those characteristics have low availability of resources, especially prey used by figure 1. correlation between the number of cells constructed and adults produced per colony of the social wasp mischocyttarus consimilis nesting in conserved and disturbed habitat. table 1. comparison of the colony productivity of the social wasp mischocyttarus consimilis nesting in conserved and disturbed habitat. parameter conserved habitat disturbed habitat t p n mean se n mean se built cells 11 215 22.8 11 142.2 13.4 -3.08 0.006 produced adults 11 250.6 6.62 11 131.9 4.69 -2.35 0.03 nests dry mass (g) 11 1.11 5.66 11 0.59 5.7 -3.51 0.003 productive cells (%) 11 67.4 39.6 11 56.2 20.2 -1.59 0.12 reused cells (%) 11 37.3 6.85 11 39.3 5.91 0.25 0.8 adults produced/cell 11 1.09 0.13 11 0.98 0.09 -1.04 0.3 n = nest number; se = standard error. sociobiology 60(1): 96-100 (2013) 99 social wasps to feed the larvae (gould & jeanne, 1984; raw, 1998; nadeau & stamp, 2003). mead and pratte (2002) suggested that the low availability of prey in a disturbed habitat negatively affects the food allocation rates for the colony, leading to deficient feeding of the larvae. poorly fed larvae have longer development periods, thus reducing the capacity to reuse the comb to produce more adults. a reduction of the colony cycle in wasp nesting in an urban environment is another factor that may have contributed to the lower productivity in this environment. although it was not possible to quantify this parameter, field observations during the study period indicated that colonies in the urban environment generally had shorter cycles than colonies in forest environments. torres et al. (2011) noted that the colony cycle of m. consimilis in a urban environment lasts approximately eight months, but can exceed one year in forest environments. the various human effects on the habitat can cause disturbances that could, on average, shorten the colony cycle. this appears to be a likely reason for the shortening of the colony cycle in m. consimilis, since in tropical regions, wasp colonies can ordinarily remain active year-round because climatic variables do not impose restrictions on the colony’s activities (gobbi & zucchi, 1980; giannotti, 1997; torres et al., 2011). there was no evidence that the different wasp populations use different strategies with respect to the use of the comb. the number of adults produced per cell as well as the proportions of reused and productive cells did not differ between the two populations. this result eliminates a possible strategy of a difference in use of the comb between the two wasp populations. montagna et al. (2010), studying this same species, demonstrated that old cells in the comb were more often used to produce adults. similarly, inagawa et al. (2001) observed an association between the proportion of productive cells and adult production, and did not find evidence for differentiated use of the comb among different populations of the social wasp polistes snelleni (saussure). despite the considerable reduction in the colony productivity, the social wasp m. consimilis usually nested in urban environments. the availability of nesting sites on human constructions, especially edges of houses and building may help to explain the optional occurrence of this species in this type of environment (clapperton, 2000; mead & pratte, 2002). in the same way, synanthropic nesting would be selected if the predation rate by vertebrates and interspecific competition are lower in environments occupied by humans compared to forest environments (judd, 1998; reviewed in mcglynn, 2012). acknowledgments the authors thank janet w. reid (jwr associates) for the revision of the english text and orlando t. silveira (museu paraense emílio goeldi) for the identification of the species. uems provided a graduation fellowship to the first author and capes provided a doctoral fellowship to the second author, and wfaj acknowledges his research grants from the cnpq. references abensperg-traun, m. & smith, g.t. 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(2011). colony cycle of the social wasp mischocyttarus consimilis zikán (hymenoptera, vespidae). rev. bras. entomol. 55: 247–252. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 41-49 (2013) spatial connectivity of aquatic macrophytes and flood cycle influence species richness of an ant community of a brazilian floodplain mc pereira1, jhc delabie2,3, yr súarez, wf antonialli junior1 introduction floodplain environments with flooding regime harbor a high biological diversity (welcomme, 1985; lowemcconnell, 1999) as a result of the spatial and dynamic complexity existing primarily in habitats with these characteristics (ward et al., 1999; robinson et al., 2002). junk et al. (1989) proposed the “flood pulse concept”, which considers the hydrological aspects along with the geomorphological aspects, responsible for spikes in floods and droughts, with different amplitudes and periods along the hydrographic basin and consider this seasonality the greatest driving force of biota composition in the rivers of the floodplain. the initial step of knowledge of available natural resources in a region corresponds to the collection and taxonomic identification of species that make up the fauna and flora, in order to facilitate the acquisition of subsidies for more detailed studies of ecological characteristics of their habitats. furthermore, these studies may eventually lead to abstract despite the environmental and economic importance of pantanal, there are few studies quantifying the influence of sazonality and spatial variation on biological diversity in this ecosystem. in this context, the present work aimed to study the assemblage of ants associated with macrophytes during the flood and dry period of paraguay river, in marginal environments in the pantanal of porto murtinho, mato grosso do sul. we observed a wide variation in the temporal distribution of the diversity of the assemblages of ants, since from 37 species, 36 occurred in the dry and 20 in the flood period. of the total of macrophyte species observed, only in 12.5% we found a more specific correlation with ants that were nesting in spaces provided by plants representing a total of 10.52% of the species analyzed. sociobiology an international journal on social insects 1 universidade estadual de mato grosso do sul, dourados-ms, brazil 2ceplac itabuna-ba, brazil 3 santa cruz state university, ilhéus-ba, brazil research article ants article history edited by: gilberto m.m. santos, uefs brazil received 11 october 2012 inicial acceptance 31 october 2012 final acceptance 21 november 2012 keywords diversity, flood pulse, foraging habits, formicidae, pantanal. corresponding author márlon césar pereira centro integ. de análise e monitoramento ambiental, universidade estadual de mato grosso do sul rodovia dourados/itahum, km 12,5 caixa postal 351, dourados-ms, brazil, 79804-970 e-mail: marloncesarp@yahoo.com.br a body of knowledge that allows the rational exploitation of biotic resources and adaptation to abiotic conditions of the studied environment (prado, 1980). according to lewinsohn et al. (2005) pantanal is one of the most unknown biomes of brazil, and the functional role of invertebrates in this ecosystem is a strong attribute for its conservation. among these organisms, the ant fauna is one of the most successful group of insects, dominant in number and biomass in several environments (harada & adis, 1997; santos et al., 2003; battirola et al., 2005), and considered abundant, easy to collect and identify as well as relatively quick to respond to changes in their habitats (ribas et al., 2003). in addition to its relatively high local abundance, they are especially rich in species and diverse in terms of foraging habits, nesting, among other ecological functions (blüthgen & feldhaar, 2010). in tropical ecosystems, ants constitute more than 15% of the total animal biomass (beattie & hughes, 2002). most species are predators and their structuring role in armc pereira, jhc delabie, yr súarez, wf antonialli jr ant assemblages respond to aquatic macrophytes and flood cycle42 thropod community is highlighted in several studies (e.g., carroll & janzen, 1973; jeanne, 1979; wilson, 1987; hölldobler & wilson, 1990). in addition, when foraging over the vegetation, they can decrease the rate of herbivory and increase the reproductive success of plants (oliveira et al., 1999). in this sense, there are mutualistic interactions between ants and plants in which the latest provide nesting site and food for ants in exchange for its defensive activity (beattie, 1985; styrsky & eubanks, 2010). despite the environmental importance of pantanal, which plays an important role in biological diversity due to the variety of natural habitats, opportunities for feeding and reproductive niches, and essential ecosystem services including carbon storage, flooding control, fish production, and aquifer recharge (alho, 2005; alho & gonçalves, 2005; alho et al., 1988; keddy & fraser, 2005), we can still say that there are few studies quantifying the influence of seasonality and spatial variation on its biological diversity. the few ecological studies are concentrated in some places, like the plain of cuiabá river, paraguay river in the region of corumbá, miranda river and negro river and refer mainly to communities of aquatic plants, fish, phyto and zooplankton, except for the work of raizer and amaral (2001) on spiders associated with aquatic macrophytes and alves-dos-santos (1999) on the pollination of pontederiaceae. in this context, this study aimed to evaluate the ant assemblages that occur during the change in the disposition of macrophytes during the season of flood and drought in marginal environments of the paraguay river in porto murtinho pantanal , mato grosso do sul , brazil. material and methods study area the samples were collected during the day period from march/2009 to march/2010 in porto murtinho, state of mato grosso do sul, brazil (fig. 1), in paraguay (21°41’40.86”s and 57°53’10”w) and amonguijá rivers (21°41’10.39” s and 57°52’51.81”w), criminosa (21°40’27”s and 57°53’30.6”w) and flores lakes (21°45’56”s and 57°54’58.67”w). the altitude of these areas is around 75 m, and the vegetation ranges from grassy to sparse shrub areas. data sampling the sampling sites, in each water body described above, were randomly chosen. in each sampling site, the ants were searched and collected in macrophyte beds at littoral zones. we developed our ant samplings at the emerged portions of aquatic macrophytes, using entomological nets and tweezers. the ant samplings were carried out on a monthly basis, at least in five plants in each sampling site throughout the study. the sampled ants were fixed at 70% ethanol in labeled vials, and identified by dr. jacques c. h. delabie, using by reference the coleção do laboratório de mirmecologia do centro de pesquisas do cacau (cepec), following the nomenclature adopted by bolton (1994; 1995b and 2003). voucher specimens were deposited at number 5602 in the collection previously cited. the ants were collected with mma – sisbio permission (17487 – 1). sampled macrophytes were collected and pressed to posterior identification at herbário da ufms in campo grande/ms by dr. arnildo pott, dr. vali joana pott and gabriela serra, with taxonomic keys proposed by scremindias (1999), pott &pott (2000) and amaral et al. (2008) and voucher specimens were deposited in herbário da ufms (cgms) in campo grande/ms. to facilitate the visualization of the dynamics of ant assemblages that occurred in macrophytes, we divided the year of collection in steps of dry season (september-february) and flood season (march-august), based on the level of the paraguay river every month (fig. 2). the total species richness was estimated using the bootstrap method (smith & van belle, 1984), with a confidence interval (α = 0.05), using presence/absence data for all samples. this method was selected for its robustness, with relatively large sample sizes (hellmann & fowler, 1999). we used pearson`s correlation between ant richness and environmental variables obtained from the meteorological station of porto murtinho (rainfall) and from field data (temperature and river level) (fig. 3). to assess whether or not there was specificity between species of ants and macrophytes, the data referring to all ants figure 1. map of the study area. porto murtinho, state of mato grosso do sul, brazil. sociobiology 60(1): 41-49 (2013) 43 and macrophytes were submitted to cluster analysis, through the methods of jaccard and upgma as linkage method, using the statistical program r (r development core team, 2011). to assess whether the generated dendrogram reflects the similarity matrix in species distribution in the plants analyzed we used the cophenetic correlation coefficient, defining the minimum value of 0.75 as a measure of dendrogram adjustment quality. figure 2. accumulated of rainfall and level of the paraguay river during the year, in the region of porto murtinho – ms. results and discussion we collected 582 ant individuals, belonging to six sub-families, 17 genera and 37 species that occurred in 36 species of aquatic and paludal macrophytes belonging to 14 orders and 18 families (table 1). through the boostrap method we estimate that 41 species of ants occur associated with these species of macrophytes (ic = 37 to 46) so, this result suggests that 92.7% of species of ants were sampled, indicating good sample size sufficiency. the higher proportion of ant species was represented by the subfamily formicinae with thirteen species (35.1%), followed by myrmicinae with ten species (27%) and dolichoderinae and pseudomyrmicinae with five species (13.5%) each. these results are not consistent with other studies about ant fauna (longino & nadkarni, 1990; johnson & ward, 2002; diehl et al., 2005) which the authors found more species of the subfamily myrmicinae in different biomes. moreover, the results differ from the findings of corrêa et al. (2006) “capões” of pantanal ms, which follows the pattern described above. however, the great proportion of formicinae found in our survey could be explained by the fact that our samples were accomplished in open areas, as in the investigations of marinho et al. (2002), leal (2002, 2003). the natural habitat of formicinae is vegetation (hölldobler & wilson, 1990; brühl et al., 1998), therefigure 3. person`s correlation analyses (a) ants species richness and temperature, (b) ants species richness and rainfall, (c) ants species richness and river level. fore they can be frequently found in this place (marques & del-claro, 2006). as for the correlation analyses, rainfall (fig. 3a) was the variable that most explained the ant’s richness, followed by temperature (fig. 3b) and river level (fig. 3c). the highmc pereira, jhc delabie, yr súarez, wf antonialli jr ant assemblages respond to aquatic macrophytes and flood cycle44 macrophyte species code ant species visited macrophytes species order alismatales azteca sp (15) family alismataceae dorymyrmex sp. 1 (4 33 echinodorus tenellus 1 dorymyrmex sp. 2 (40) order asterales dorymyrmex sp. 3 (330 family asteraceae linepithema humile (1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 28 29 30 31 32 33 34 35 36) enydra radicans 2 subfamily ectatomminae melanthera latifolia 3 ectatomma brunneum (8 11 12 14 15 22 28 33) pacourina edulis 4 subfamily formicinae order caryophyllales brachymyrmex patagonicus (15) family amaranthaceae camponotus (myrmaephaenus) sp 1 (11) alternanthera aquática 5 camponotus (myrmaephaenus) sp 2 (5 8 14 15 21 33) family polygonaceae camponotus (myrmaephaenus) sp 3 (2 14 22 28 33) polygonum acuminatum 6 camponotus crassus (2 3 7 8 10 11 14 15 19 22 23 28 29 32 33 35) polygonum ferrugineum 7 camponotus leydigi (2 22 28) polygonum punctatum 8 camponotus melanoticus (14 22 30 33) family portulacaceae camponotus novogranadensis (4 8 10 11 12 14 22 32 33) portulaca grandiflora 9 camponotus rufipes (4 11 14 16 22 33) order commelinales camponotus sexguttatus (4 14 32 33 35) family pontederiaceae camponotus vittatus (14) eichhornia azurea 10 nylanderia sp1 (4 5 7 14 29 33 36) eichhornia crassipes 11 nylanderia sp2 (33 35) pontederia rotundifolia 12 subfamily myrmicinae order curcubitales acromyrmex balzani (4 8 10 11 14 30 33) family curcubitaceae atta sexdens rubropilosa (2 4 10 11 22 35) cyclanthera hystrix 13 cephalotes minutus (4 10 11 20 33 35) order fabales cephalotes pavonii (4 14 15 28 33) family fabaceae crematogaster sp (8 10 11 12 35) aeschynomene sensitiva 14 crematogaster victima (33) bauhinia bauhinioides 15 cyphomyrmex transversus (33) mimosa pigra 16 pheidole obscurithorax (4 6 8 10 11 14 16 29 30 33 35) neptunia plena 17 solenopsis invicta (3 7 11 14 15 19 22 28 29 32 33 35 36) senna aculeata 18 solenopsis sp (4 36) senna alata 10 subfamily ponerinae senna occidentalis 20 hypoponera opaciceps (30) sesbania virgata 21 hypoponera sp. (11 32) vigna lasiocarpa 22 odontomachus haematodus (10 16 33 35) order malpighiales subfamily pseudomyrmecinae family euphorbiaceae pseudomyrmex denticollis (11 9 29 30 32) caperonia castaneifolia 23 pseudomyrmex gracilis (3 21 22 29 33 35) order malvales pseudomyrmex simplex (11 28 29 33 34) family malvaceae pseudomyrmex sp (pr. palidus) (19 26 27 33) hibiscus striatus 24 pseudomyrmex termitarius (2 8 15 22 33) pavonia laetevirens 25 order myrtales family onagraceae ludwigia grandiflora 26 ludwigia sedoides 27 order poales family poaceae setaria paucifolia 28 urochloa subquadripara 29 order salviniales family azollaceae azolla filiculoides 30 family marsileaceae marsilea crotophora 31 family salviniaceae salvinia auriculata 32 order solanales family convolvulaceae ipomoea alba 33 ipomoea chiliantha 34 order vitales family vitaceae cissus spinosa 35 order zingiberales family marantaceae thalia geniculata 36 table 1. list of ant species and macrophytes species sampled in marginal environments of the paraguay and amonguijá rivers and criminosa and flores lakes, in the region of porto murtinho, mato grosso do sul, brazil, from march/2009 to march/2010. sociobiology 60(1): 41-49 (2013) 45 est richness was observed in july, probably because in this month there is the flood peak of the region, which makes the ants that are foraging to look for fixation in the substrate, since the macrophytes that are more inside the river become subject to being dragged by the flood. as for the cluster analysis performed, in figure 4 we can observe 6 distinct groups. group 1 (fig. 4) in which the plants were used by the ants as a means of connection to other plants, such as azolla filiculoides (lam.), or the plants presented little floral cycles during the work, mimosa pigra (willd). group 2 (fig. 4) less common plants or plants with well-defined cycles of flowering, so that ants were only found in them when flowering occurred. the macrophytes of group 3 , all showing in common the fact of being without connection with other plants during the full sampling period, becoming “islands”, in which some species of ants were found isolated from other areas and/or species, as in the case of senna alata (roxb.) in which we found, in some instances, entire colonies of solenopsis invicta (buren 1972) adhered to the plant, possibly reaching it by boating floating (tschinkel, 1988). in group 4, are the macrophytes specie in which occurred the higher frequency of ant species throughout the study period. in common, they have as features, besides being abundant, the provision of important resources such as flowers, domatias and nectaries during most part of the year (pott & pott, 2000). in group 5 is the macrophyte pontederia rotundifolia (l. f.), which occurred usually in the center of large groups of macrophytes of pontederiaceae family, which hindered the access of ants to these plants. and the group 6 with a single member, salvinia auriculata (aubl.) which is used as a means of connection, as a. filiculoides, but presented large distribution during samplings and possesses bristles that hinders the passage of small ants. the plant species with the highest number of visiting ants species was ipomoea alba (l.), in which 25 species were recorded, probably due to the fact that this plant ia shrubby and amphibious, providing attractiveness to ants throughout the year. eichhornia crassipes (mart solms) was another species with a high relative number of visits, with 15 species recorded, and high abundance throughout the study period. for the same reason, and also because during the entire year the plant species pacourina edulis (aubl.), cissus spinosa (cambess.) and vigna lasiocarpa (benth. verdcourt) flourish, in these species we recorded 13, 11 and 11 species, respectively. figure 4. cluster analysis of the species of macrophytes according to the species of visitor ants. g1= macrophytes used as a means of transition by the ants, g2 = macrophytes that flourished during a time of year, g3 = islands macrophytes, g4 = macrophytes with high frequency of visits, g5 = connection bridges abundant at sampling area, and g6 = macrophyte isolated in pontederiaceae group. mc pereira, jhc delabie, yr súarez, wf antonialli jr ant assemblages respond to aquatic macrophytes and flood cycle46 the ant species linepithema humile (mayr 1868) is an indicator of 4 groups of plants (fig. 4) and occurred in 91.6% of plant species. however, it was more frequent in ipomoea chiliantha (hallier). this ant species is invasive in several environments, mainly in europe, united states, australia and brazil and as such, end up being opportunistic in environments newly colonized (suarez et al., 2001; holway et al., 2002). because they are aggressive in environments in which they settle, they can monopolize food sources of many native ants by the high recruitment capacity that this species presents (majer, 1994; gómez & oliveiras, 2003; touyama et al., 2003). in addition to this, we found colonies of l. humile nesting inside p. edulis and indeed the domatias offered by this macrophyte have already been described (pott & pott, 2000). the genus camponotus (emery 1889) was the most rich , with 11 ant species (29.7% of species richness), probably because it is one of the most abundant genera worldwide, with varied habits and an excellent foraging system (holldobler & wilson, 1990), reflecting the prevalence described by wilson (1976) and bolton (1995a). according to them, camponotus, pheidole (westwood 1839), solenopsis (smith 1858) and crematogaster (lund 1831) are the genera with the highest diversity of species and adaptations, greatest geographical distribution extension and greatest local abundance, and, therefore, are considered the most prevalent genera on a global scale. in fact, this genus is always very frequent in inventories of ants as can be seen in works such as those of majer and delabie (1994) studying the community of ants of the amazon, soares et al. (1998) in eucalyptus plantations and secondary atlantic forests, verhaagh and rosciszewski (1994) in different biomes in bolivia, and in open areas such as restinga (gonçalves & nunes, 1984), cerrado (marinho et al., 2002), caatinga (leal, 2002, 2003) and also associated with vegetation (wilson, 1976). in this group of ants is the species camponotus novogranadensis (mayr 1870) which has been shown to be an indicator of 3 groups of plants (fig. 4): group 2, probably because it is an abundant group and occurs throughout the year; group 5, due to the fact that the plant is difficult to reach and the ant is an opportunistic forager; and group 6, because the plant is widely distributed throughout the year as the group 2. the most common species of the genus camponotus was c. crassus (mayr 1862), occurring in 16 macrophyte species. only two species of the genus solenopsis were found (table 1), however, occurred in 14 species of macrophytes, this is probably related to physiological and behavioral aspects of these species, which may spend long periods of food shortage and compete with other species of ants or other groups of animals since they present an efficient bulk recruitment strategy (fowler et al., 1991). in both species the boating floating behavior was observed, this behavior (tschinkel, 1988) probably indicates that such colonies built their nests on the banks of rivers and streams during the dry season, and when those areas were flooded, they floated, waiting for the opportunity to restore their colony in some other appropriate place. those ants demonstrate the strong association of biological traits of such ant species with hydrological systems in general. the genus pseudomyrmex (lund 1831), frequently described as arboreal (ward, 1989), was found in 55.55% of the plants near the river bank, probably using the plants as substrate in search for prey, presented five species and one of them registered for the first time in brazil (pseudomyrmex denticollis emery 1890). however, its distribution was noted at the central region of paraguay, approximately 350km far from our sampling area in porto murtinho. considering, that the region where p. denticollis was registered in paraguay has the same vegetal formation of porto murtinho (dry chaco). the least frequent species of ants were found, most part of the time foraging, in the leaves, trunk or flowers, or even using the plant as a means of connecting to another which was providing resources at that moment. a. filiculoides and s. auriculata, for example, are species associated with floating macrophytes abundant in the region, which are commonly used as a means of connection so that these ants can forage in other plants. most species of ants visited more than one species of macrophyte, suggesting that in general they do not establish dependency relationship with these plants, what is probably related to the fact that this environment is very unstable throughout the year, mainly, due to the fast change in the level of the river during flood season (fig. 2). we can observe a large variation in the temporal distribution of diversity in the studied ant assemblage. from 37 species observed, 36 occurred in the dry season and 20 in the flood. this variation occurs probably because there is a variation in the availability of macrophyte banks throughout the year since, during the flood, the greater volume of water that generates greater transport capacity, carries along its course the majority of macrophytes in which the ants were foraging in the previous season. a greater diversity of species during the dry season, probably because during the months of flood, macrophytes decrease in number as they will gradually falling off, being dragged and changing position by the force of the currents and wind (tur, 1972). this dynamics, for sure, causes a considerable decrease in the foraging substrate for ants during this period. in the analysis of similarity among the ant species (fig. 5) we can see three main groups: group 1 contains the species that were uncommon in a few species of macrophytes. this group fits, for example, camponotus vittatus (forel 1904), which was isolated by having occurred only in aeschynomene sensitiva (sw.) during the full season (fig. 5). the low frequency is probably explained by the commonness of this species in urban areas (soares et al., 2006), not in rural areas. sociobiology 60(1): 41-49 (2013) 47 the species hypoponera opaciceps (mayr 1887), hypoponera sp. and p. denticollis foraged only in a. filiculoides, s. auriculata and e. crassipes. in these cases, the low frequency is probably explained by their trophic nature as generalist predators (santschi, 1938), specially hypoponera (taylor 1967). these ants must use these plants as substrate to capture prey that forage in search of different resources in these plants. in group 2, we also found species that occupied spaces offered by the plants, i. e. domatias (holldobler & wilson, 1990). this was the case of dorymyrmex sp 2, crematogaster victima (smith 1858) and cyphomyrmex transversus (emery 1894) found nesting in p. edulis and in young plants of i. alba. finally, belonging to group 3, are the ants that occurred in most plants and sampling months, which we considered opportunistic foragers of several resources. s. invicta represents such an example of taking advantage of plants, using them as resources when water flooded their nests and they used the mechanism of boating floating (tschinkel, 1988) to reach the closest plants, as discussed earlier. in plants where we found s. invicta no other ant species was found. most likely, this occurs because of the size and aggressiveness of s. invicta colonies (delabie & fowler, 1995). another species similar in occurrence was c. crassus, the most common ant of the genus camponotus, and l. humile, the argentine invasive ant (majer, 1994; suarez et al., 2001). thus, we can conclude that the several species of macrophytes are important resources for ants in pantanal, either as substrate where foragers find preys, nectaries, or other resources or as places to establish their colonies, finding therefore, besides food resources, shelter. acknowledgments the authors thank to pantanal research center, universidade estadual de mato grosso do sul and fundect for financial support. we thank gabriela serra do vale duarte for the plant species identification and ingrid de carvalho guimarães for the revision of the english text. we thank two anonymous reviewers for valuable comments that improved this article. references alho, c.j.r. 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(1987). the little things that run the world: the importance and conservation of invertebrates. conserv. biol., 1: 344-346. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2162sociobiology 65(2): 337-339 (june, 2018) simarouba versicolor (simaroubaceae) dispersal by the leaf-cutter ant atta sexdens simarouba versicolor is a semi-deciduous tree belonging to the family simaroubaceae, which is a heliophite and a selective xerophytic pioneer, and occurs in cerrado and caatinga biomes, preferably in open areas and welldrained soils. in brazil, it is found from the northeast region to the state of são paulo and in some parts of the states of pará and mato grosso do sul (lorenzi & flora, 2002). rapid growth, efficient germination, and survival, in direct seeding in the field, are characteristics that make it important in forest restoration (filho & sartorelli, 2015). its dispersal is zoocorical, since its drupe-type fruits serve as food for birds, fishes, and bats. so far, there have been no reports of myrmecochory, i.e., ants aiding its dispersal. ants can play an important role as secondary seed dispersers (gallegos et al., 2014). besides dispersal, ants also are able to deposit seeds in sites favorable to their germination and development (culver & beattie, 1983; oliveira & koptur, 2017), which helps to avoid seed consumption by predators (heithaus, 1981; tanaka et al., 2015), decrease interspecific and intraspecific competition between seedlings under the mother abstract the importance of simarouba versicolor st. hil. fruit dispersal by the leafcutting ant atta sexdens (l.) was studied in the cerrado, tocantins, brazil. the trees and nests were located between a forest area and a brachiaria decumbens stapf pasture. seeds were collected in october 2015 along foraging trails and on the anthill of an a. sexdens colony. germination of three groups of seeds was tested: (1) seeds with the tegument removed by the ants; (2) seeds without tegument, cleaned manually, and (3) seeds with tegument. the germination rates for the three treatments were similar; however, it was verified that the seeds cleaned by ants germinated faster. in addition, it was verified that the ants dispersed the seeds by at least 20 meters in the study area. simarouba versicolor is a plant studied for its insecticidal properties, and this is the first study to our knowledge reporting its dispersal by ants. sociobiology an international journal on social insects drs lopes, rc tavares, kom batista, pb souza, mo nascimento, dj souza article history edited by evandro nascimento silva, uefs, brazil received 13 october 2017 initial acceptance 12 february 2018 final acceptance 12 february 2018 publication date 09 july 2018 keywords myrmecochory, cerrado, forest fragmentation. corresponding author danival josé de souza federal university of tocantins (uft) campus of gurupi rua badejós, lote 7, chácaras 69/72, caixa postal 66, cep 77402-970, gurupi-to, brasil. e-mail: danival@uft.edu.br plant (leal et al., 2015; giliomee 1986), and protect seeds from fire (bond & slingsby, 1984; leal et al., 2015; new, 2014). the objective of the present study was to evaluate s. versicolor fruit dispersal by the leaf-cutter ant atta sexdens, and its potential benefits to the plant at the edges of a cerradotype forest remnant, in the southern state of tocantins. the study was carried out near the urban area of the municipality of gurupi to (11°44'42.4"s 49°00'53.1"w) in a cerrado area near a pasture composed primarily of brachiaria decumbens. the climate of the region is classified as tropical humid (aw) according to köppen’s climate classification (alvares et al., 2013), with rainy summers and dry winters, and an average temperature of 25 °c. annual precipitation varies from 1,327 to 1,798 mm (marcuzzo et al., 2012). in september 2015, seeds of s. versicolor, clean and with tegument, were observed along a. sexdens trails at the edges of the forest. simarouba versicolor has a fleshy drupe-type fruit with a single seed, which can only be mechanically separated. some fruits dry in the plant, whereas those that fall may be transported to ant nests where they are federal university of tocantins (uft), campus of gurupi, tocantins, brazil short note drs lopes et al. – simarouba versicolor seed dispersal338 cleaned and removed. seeds collected for the germination tests came from an individual s. versicolor located 21 m from the a. sexdens nest. the seeds of s. versicolor were collected from the soil, along the trail, under the plant and in the deposit formed near the anthill. more seeds cleaned by the ants (drupes in which the tegument and pulp were removed) were collected near the anthill than along the trail, whereas non-cleaned seeds (dried drupe still containing the pericarp) were found scattered under the mother tree and along the ant-foraging trail. germination tests were performed on 9 cm diameter petri dishes, properly sterilized and lined with two sheets of filter paper, moistened with 5 ml of distilled water. seeds that presented physical damage, such as perforations, were excluded from the germination tests. the treatments consisted of eight replicates (plates) containing eight seeds, with a total of 64 seeds for each of the three treatments: (t1) seeds cleaned by ants, (t2) seeds cleaned manually, and (t3) non-cleaned seeds. the seeds were incubated in a germination chamber at 25 °c and photoperiod of 12 h, and were observed every 2 d in the morning for a maximum period of 36 d. seeds were considered germinated when they had a root length ≥ 3 mm. based on these results, the average germination percentage and germination speed index were obtained at 36 d after sowing. the germination percentage was calculated by the following formula: g = (n/100) x 100, where: n = number of seeds germinated at the end of the test. the germination speed index (gsi) was calculated based on the methodology proposed by maguire (1962) using the formula: gsi = σ (ni /ti), where: ni = number of seeds germinating at time ‘i’; ti = time after test installation; i = 1 → 36 days. unit: dimensionless. the data of germination percentage and speed index did not present a normal distribution. therefore, the values were transformed using the following formulas: for the average germination percentage (2 × arc sine √ (y/100) + 0.5)) and for the germination rate (logarithm (y) +1). subsequently, they were submitted to analysis of variance using the f-test and compared by tukey’s test at 5% significance level. all analyses were performed using the software statistica 7.0. regarding the average germination percentage of s. versicolor at the end of the 36 d of observation, it was verified that there was no significant difference between the treatments (anova, f2.21 = 1.36, p = 0.28), i.e., the leaf-cutting ant cleaning did not change the chances of germination. the treatments showed the following germination rates (g): g = 37.5% for t1 (seeds cleaned by ants), g = 25% for t2 (seeds cleaned manually), and g = 29.7% for t3 (non-cleaned seeds). a significant difference was observed in the germination rate between the three treatments f2.21 = 7.24, p = .004, and the seeds cleaned by the ants had a higher average gsi than those in the other two treatments (fig 1, tukey’s test, α = 5%). the ants did not reduce the germination rate of the seeds by consuming the fruit pulp, which characterizes the positive effects of dispersion by the ants. in the study site, fruit dispersal was observed for distances up to 21 m from the mother plant. it should also be noted that the foraging distance of a. sexdens workers may exceed this distance by many meters depending on several factors, such as the size of the colony and availability of plant resources (della lucia, 2011). after a period of drought, which may begin in may, the first rains appear at the beginning of october in the southern tocantins. therefore, the fruits of s. versicolor and other species, although occasional, are important items in the survival of the leaf-cutter ant, particularly in regions and periods in which dry conditions prevail. at the same time that we observed the interaction of a. sexdens with s. versicolor, we also observed seeds of the following forest species: hirtella glandulosa (chrysobalanaceae), diospyros hispida (ebenaceae), and copaifera langsdorffii (fabaceae) in the trails and anthills. germination tests were not performed because of the small amount of seeds collected; however, these observations reinforce the importance of these fruits to leaf-cutting ants at this time of year. fig 1. germination speed index of t1, t2, and t3 treatments, where t1: seeds cleaned by ants (free of the pericarp, without pulp, and endocarp); t2: aryl-free seeds, cleaned manually; and t3: seeds with aryl. means followed by the same letters between columns do not differ from each other based on the tukey’s test at 5%. regarding the differences in speed germination, the myrmecochory may be an important ecological advantage for s. versicolor in the study area, since the rainy period, favorable for the development of the plants, is more restricted than that of other regions, and the presence of grasses and other common invasive species at this edge of the forest may inhibit the development of native species (machado et al., 2013). in this situation, the seedlings would have a longer time to develop and settle before the dry season. in the present study, it was possible to observe that some cleaned and non-cleaned seeds of s. versicolor remained on the foraging trail. the seed cleaning process occurred along the trail, but most seeds were cleaned inside the nests and sociobiology 65(2): 337-339 (june, 2018) 339 then removed and deposited at the anthills. thus, the colony did not suffer from the toxic effects of substances present in the seeds, including insecticides and fungicides (alves et al., 2014), and seeds maintained their chances of germination, since no physical damage was observed. therefore, it is concluded that s. versicolor seed cleaning by a. sexdens ants does not alter the germination percentage, but increases the germination speed. the role of ants is important in the dispersal of this arboreal species. acknowledgments we thank the anonymous reviewers for their careful reading of our manuscript and their many insightful comments and suggestions. this work was supported by grants of cnpq (project 403708-2013-3) and research funds of federal university of tocantins for dj souza. references alvares, c. a., stape, j. l., sentelhas, p. c., de moraes gonçalves, j. l. & sparovek, g. 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(2017). ant-plant interactions: impacts of humans on terrestrial ecosystems. cambridge, cambridge university press, 452 p. tanaka, k., ogata, k., mukai, h., yamawo, a. & tokuda, m. (2015). adaptive advantage of myrmecochory in the antdispersed herb lamium amplexicaule (lamiaceae): predation avoidance through the deterrence of post-dispersal seed predators. plos one, 10: e0133677. doi: 10.1371/journal. pone.0133677 doi: 10.13102/sociobiology.v65i2.2014sociobiology 65(2): 170-176 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 volatile component analysis of michelia alba leaves and their effect on fumigation activity and worker behavior of solenopsis invicta introduction red imported fire ant (rifa), or solenopsis invicta, named by buren in 1972, was first introduced to southern alabama in the 1930s (buren et al., 1974) and then rapidly spread to australia (moloney & vanderwoude, 2002; mccubbin & weiner, 2002), new zealand (ministry of agriculture and forestry of new zealand, 2006), malaysia (na & lee, 2001), taiwan (chen et al., 2006, zhang et al., 2007), and mainland china (zhang et al., 2007; zeng et al., 2005; ligon et al., 2012; wang et al., 2015; gutrich et al., 2007). red imported fire ant sting domestic animals and people. they cause significant economic losses in agriculture and other industries, and they also severely affect biodiversity (vinson, 1997; williams et al., 2001; wojcik et al., 2001; gutrich et al., 2007). prior to the 1980s, attempts to eradicate abstract volatile compounds from mashed (fresh, fallen, and dried) leaves of michelia alba were collected via solid-phase microextraction and were then identified via gas chromatography-mass spectrometry. the results showed that linalool was the dominant component in different leaves, together with caryophyllene, β-elemene, and selinene, the contents of which vary across the samples. the fumigation bioassay results showed that the volatiles from m. alba leaves exhibited insecticidal activity against red imported fire ant workers, and the mortality of workers could reach up to 100% after the fallen leaves were treated for 16 h. mashed fresh leaves could effectively reduce the aggregation and drinking ability of workers. the volatile substances released from the mashed leaves might kill the ants, or affect their behavior and weaken the activity by interfering transmit information between ants. a comprehensive consideration of the economic and ecological value of m. alba shows that fallen leaves might be a good resource to control red imported fire ant. sociobiology an international journal on social insects dq qin1, rl huang1, zh li1, sy wang1, dm cheng1,3, zx zhang1,2 article history edited by kleber del-claro, ufu, brazil received 02 october 2016 initial acceptance 20 february 2017 final acceptance 11 february 2018 publication date 09 july 2018 keywords solenopsis invicta, michelia alba, volatiles, toxicity. corresponding author zhixiang zhang key lab. of nat. pesticide and chemical biology, ministry of education south china agricultural university guangzhou, china, 510642. e-mail: zdsys@scau.edu.cn fire ants by using early insecticides and formulations have failed (drees et al., 2013). environmental pollution from traditional insecticides, such as pyrethroid or organophosphate, has led to the development of natural, safe, and nonpolluting insecticides to control these ants (appel et al., 2004; vogt et al., 2002; cheng et al., 2004; cheng et al., 2008). botanicals might be a good candidate for developing these insecticides. numerous studies have indicated that essential oils can be used to control red imported fire ant. clove powder applied at 3 mg/cm2 and 12 mg/cm2 resulted in 100% ant mortality within 6 h and repelled 99% of ants within 3h (kafle & shih, 2013). essential oils of mint (appel et al., 2004), artemisia annua (wang et al., 2014), camphor, eucalyptus, mugwort, and wintergreen all showed strong insecticidal, fumigation, or repellent activity against red imported fire ant (chen, 2009; tang et al., 2013). 1 key lab. of nat. pesticide and chemical biology, ministry of education, south china agricultural university, guangzhou, china, 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 department of plant protection, zhongkai university of agriculture and engineering, guangzhou, china research article ants mailto:zdsys@scau.edu.cn http://www.ncbi.nlm.nih.gov/pubmed/?term=kafle l%5bauthor%5d&cauthor=true&cauthor_uid=23448024 http://www.ncbi.nlm.nih.gov/pubmed/?term=shih cj%5bauthor%5d&cauthor=true&cauthor_uid=23448024 sociobiology 65(2): 170-176 (june, 2018) 171 michelia alba (magnoliaceae), named by dc in 1818, is an evergreen tree that is mainly distributed in the west and southeast of china (zhu et al., 1982; chen et al., 2008a). previous studies have reported the chemical compositions of essential oils from flowers, leaves, and stems of m. alba (huang et al., 2009; qin et al., 1999; chen et al., 2008b; gu et al., 2014). these reports mostly focused on the seasoning properties and application of the said tree in the perfume industry. in fact, spices are also valued for their bioactive efficacy as bacteriostatics, fungicides, antioxidants, and nutrients (hirasa & takemasa, 1984). jiang (2002) reported the insecticidal activity of m. alba essential oil to musca domestica l; however, the effect of which against other insects has not been studied. this study investigates the effects of volatile components from m. alba leaves on the fumigation activity and red imported fire ant worker behavior. the study aims to provide a new method for controlling ants. materials and methods insects: s. invicta colonies were collected from a suburb in guangzhou. the ants were reared with mealworms (tenebrio molitor). the water source was a test tube (ф25 mm × 200 mm) that was partially filled with water and plugged with cotton. the ants were maintained in the laboratory at 25±2 ºc and 75% relative humidity. plant materials: five m. alba trees were planted in the insecticidal botanical garden at south china agricultural university. the height and stem diameter were approximately 14.52 ± 4.39 m and 0.41 ± 0.1 m, respectively. three kinds of m. alba leaves were used in the test: fresh, fallen, and dried. the fresh leaves were immediately sent to the laboratory once collected. fallen leaves without dirt on the surface were selected. the fresh leaves were dried at 40 °c in an oven to a constant weight to obtain the dried leaves. fumigation toxicity bioassay: the fresh, fallen, and dried leaves of m. alba were mashed using a high-speed organization stamp mill for 3 min and then separately stored in a sealing bag at 4 °c for the application. coating fluon emulsion outside the vertical wall of a 100 ml beaker, then placed it in a 1000 ml beaker coated with fluon emulsion inside a vertical wall. a total of 30 large workers (body length of about 4.52 ± 0.01 mm) or 30 small workers (body length of about 3.03 ± 0.02 mm) in the same colony were confined in the larger beaker and outside the small one. approximately 30 g of mashed leaves were placed in the uncovered small beaker, and the 1000 ml beaker was sealed with plastic film. the deaths of worker ants were recorded after 6, 16, 40, and 64 h. a test tube (ф10.5 mm × 75 mm), which was partially filled with water and plugged with cotton, was used as a water source. the workers were maintained at 25 ± 2 °c and 65% ± 5% relative humidity. all treatments were replicated thrice. the same operation was done in the control group without the mashed leaves. the following equation was used to calculate the mortality of ants: nam(%) = x 100 nt where m is the mortality, nd is the number of dead ants, and nt is the total number of ants. behavior observation on aggregation of workers: the test method was the same as described in the preceding section, more than two workers gathered together, and the distance between each worker was less than 0.5 cm, which is defined as aggregation, the aggregating level was observed after 6, 16, 40, and 64 h. (depickère et al., 2004a; depickère et al., 2004 b; devigne et al., 2011). the following equation was used to calculate the aggregating level: ngg(%) = x 100 nt where g is the percentage of ants gathering together, namely the aggregating level, ng is the number of ants aggregating, and nt is the total number of ants. behavior observation on drinking ability of workers: the test method was the same as described previously. a water-soaked cotton ball (1 g) and 30 worker ants (the distance between the cotton ball and the ants was 25 cm) were placed on the midcourt line of a porcelain tray (20 cm × 30 cm × 5 cm) whose vertical wall was coated with a fluon emulsion (the same as below). worker ants were regarded as having water recognition ability if they continuously touched the cotton with its mouth for more than 10 s was regarded as drinking water (zhang et al., 2013a). at each time point, the observation time was controlled within 30 min. the following equation was used to calculate the drinking water rate: nww(%) = x 100 nt where w is the percentage of ants that can drink water, nw is the number of ants drank water, and nt is the total number of ants. extraction of volatiles via solid-phase micro extraction (spme). before the plant volatiles were extracted, 30 g of mashed fresh, fallen, and dried leaves were individually placed into a 250 ml conical flask sealed with tinfoil. the manual spme device (supelco, usa) with a fiber precoated with a 100 µm thick layer of polydimethylsiloxane was used to extract the volatiles. the fiber was cleaned before use at the conditioning temperature (250 °c) for 30 min. the fiber was exposed to the headspace of the material for 60 min at room temperature after being pushed into the conical flask containing the mashed leaves. the fiber was then inserted into the injection port of the gc-ms where it was heated at 250 °c for 3 min, and the adsorbed volatile compounds were rapidly thermally desorbed into a capillary gc column for analysis. dq qin, rl huang, zh li, sy wang, dm cheng, zx zhang – fumigation activity and worker behavior of solenopsis invicta172 chemical analysis via gas chromatography-mass spectrometry (gc-ms).the sample was detected directly using an agilent 6890 gas chromatograph coupled with an agilent mass spectrometer detector. a db-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm) was used to separate the compounds. the injection temperature was 230 °c. the oven temperature was initially set at 50 °c for 1 min to 200 °c (3 °c/min) for 2 min, then to 230 °c (10 °c/min) for 2 min. the detector was operated at 280 °c. helium was used as a carrier gas at a flow rate of 1 ml/min. the compounds were identified via mass spectroscopy and the spectra were then compared with those from computer mass libraries. statistical analysis the mortality, aggregating level, and drinking ability were analyzed with duncan′s multiple-range test for anova. the data were expressed as means ± sd and evaluated using spss17.0. differences at p<0.05 were considered significant. the figures were drawn using microsoft office excel 2007. results results of anova: table 1 shows that the aggregation and drinking ability of workers varied in terms of plant material (m), exposure time (t), and worker size (s) (p<0.05), and the mortality of workers markedly changed at different plant materials and exposure times (p<0.05), except for worker size (p=0.4970). however, no difference was observed reading the effect of interaction t×s on mortality (p=0.9759), aggregation (p=0.7988), and drinking ability (p=0.2842) of the workers, as well as that of interaction m×t×s, respectively (p=0.9173, 0.0786, 0.2226). factors df mortality aggregation drinking ability f values p values f values p values f values p values m 3 155.7554 0.0001 265.5776 0.0001 334.5175 0.0001 t 3 49.5313 0.0001 51.6354 0.0001 10.0814 0.0001 s 1 0.4666 0.4970 31.5410 0.0001 11.9353 0.0010 m×t 9 18.5101 0.0001 10.7687 0.0001 3.1519 0.0033 m×s 3 7.7240 0.0002 6.6313 0.0006 3.1400 0.0313 t×s 3 0.0697 0.9759 0.3367 0.7988 1.2936 0.2842 m×t×s 9 0.4245 0.9173 1.8361 0.0786 1.3654 0.2226 m: plant material, t: exposure time, s: worker size (p=0.05) (duncan′s multiple-range test, spss17.0) table 1. anova for the main factors of the fumigation test affecting the behaviors of red imported fire ant. fumigation toxicity bioassay: from fig 1, the exposure of both large and small workers to volatiles from mashed leaves of m. alba showed increasing mortality over time. the mortality of workers treated with fallen leaves could reach up to 100% after 16 h, which was significantly higher than those treated with dried and fresh leaves (f = 223.05, df = 3, p< 0.05). the mortality increased from 6.67% to 46.67% and 10.00% to 81.67% for large and small workers exposed fig 1. mortalities of workers (l: large workers, s: small workers) after treated with different mashed leaves of m. alba (duncan′s multiple-range test, spss17.0). to fresh leaves from 6 h to 64 h. the mortality rates were significantly higher than those of workers exposed to dried leaves, which were from 3.33% to 20.00% and from 1.67% to 11.67% for large and small workers(f = 223.05, df = 23, p< 0.05). however, the mortality of the ants treated with dried leaves was not significantly different from that of the control group (f = 223.05, df = 23, p> 0.05). fig 2. effects on aggregation level of workers (l: large workers, s: small workers) after treated with different mashed leaves of m. alba (duncan′s multiple-range test, spss17.0). sociobiology 65(2): 170-176 (june, 2018) 173 aggregating level and drinking ability: compared to the mortality, the aggregating level and drinking ability of red imported fire ant decreased over time after exposure to mashed leaves (figures 2 and 3). almost or more than 90.00% of workers in the control group always preferred to drink water and to aggregate. fig 3. effects on drinking ability of workers (l: large workers, s: small workers) after treated with different mashed leaves of m. alba (duncan′s multiple-range test, spss17.0). fresh leaves showed the highest inhibitory activity on drinking and aggregation of red imported fire ant. the aggregating level respectively decreased from 60.00% and 28.33% at 6 h to 0.00% and 0.00% at 64 h for major and minor workers treated with the fresh mashed leaves. the drinking ability also decreased from 23.33% and 5.00% to 3.33% and fig 4. gc-ms total ion chromatograms of mashed fresh (r), fallen (f), and dried (d) leaves of m. alba. 1.67%. the aggregation levels of workers exposed to dried leaves decreased over time with no significant difference (f = 26.02, df = 23, p> 0.05), while always higher than those treated with fallen leaves. similarly, after treated with dried leaves, the percentage of ants that drank water decreased from 83.33% and 86.67% at 6 h to 66.67% and 73.33% at 64 h for large and small ants, respectively. after 40 h exposure to fallen leaves, the drinking ability almost remained constant with time, which was observed among only 18.33% of minor workers after 64 h of treatment. chemical compositions of mashed leaves of m. alba: fig 4 shows the gc–ms total ion chromatograms of the spme extracts of fresh, fallen, and dried leaves of m. alba. the content of linalool was highest in volatiles from fresh, fallen, and dried leaves, which was 26.10%, 40.52%, and 36.52%, respectively. other compounds like isocaryophyllene, aromadendrene, α-caryophyllene and (-)-γ-cadinene etc, which account for more than 40% of all the volatiles detected from fresh leaves. volatiles from fallen leaves mainly contained linalool (40.52%), β-elemene (11.94%), β-caryophyllene (10.78%) and so on. similarly, β-elemene, β-caryophyllene, α-selinene, α-cubebene, and 36.52% of linalool, which accounted for 70% of the volatiles from dried leaves. discussion the results of the gc-ms analysis showed that in fresh, fallen, or dried leaves of m. alba, linalool was always the dominant component, together with common aromatic substances such as caryophyllene, β-elemene, selinene, and other alkene and terpenoids. the results were in accordance with previous findings (qin et al., 1999; li et al., 2000; liu et al., 2001). these compounds, especially those in fallen leaves, might kill the ants. zhang et al. (2014) reported that linalool could absolutely kill minor workers at 5 mg/centrifuge tube within 24 h and 3 mg/centrifuge tube (48 h) after treatment. few studies have reported on the insecticidal activity of caryophyllene. caryophyllene oxide exhibits insecticidal and antifeed ant activity (bettarini et al., 1993; liu et al., 2012) and could function as a nerve poison for pests via sodium channel modulators (balar & nakum, 2010). in addition, β-elemene is a kind of structural isomer of elemenes that are used as pheromones by particular insects because of the floral aromas of particular plants. aggregation behavior is crucial in many aspects of animal life (reproduction, defense, and alimentation) (depickère et al., 2008) and also regulates collective activities in nest digging (rasse & deneubourg, 2001). in addition, aggregation behavior is a strategy for workers to respond to adverse environments (wang k., et al., 2014). faced with stress factors such as floods or sudden changes in temperature, workers prefer to gather together for survival (mlot et al., 2011; hassall et al., 2010; wang l., et al., 2014). pheromones between individuals (brown et al., 2006; durieux et al., 2012) could affect the dq qin, rl huang, zh li, sy wang, dm cheng, zx zhang – fumigation activity and worker behavior of solenopsis invicta174 aggregation of insects; thus, the release of aromatic substances might interfere with the signals or pheromones between individual workers. the present study proved that the volatiles in m. alba leaves could effectively weaken the aggregation of red imported fire ant. thus, the capacity response of the workers to their external environment is reduced. under the condition of low aggregation rate, the ants’ daily behavior cannot be carried out normally. for example, the food and water transport rate will be reduced, which will seriously affect the survival of ants and lead to the reduction of ants population. which is conducive to control of ants. in this study, volatiles from m. alba leaves showed an inhibitory effect on the drinking ability of the workers, and the order of effectiveness from high to low was fresh leaves > fallen leaves > dried leaves > control treatment. after treatment with fresh leaves for 6 h, almost all of the small ants lost their drinking ability, but the drinking rate in the control group always remained at approximately 90%. during foraging, the workers would receive odor signals or distinguish directions by constantly swinging their antennas (xu et al., 2007). however, at extreme conditions, workers would have no food in their nest and no ability to move and recognize their surroundings. thus, their survival rate is almost zero (zhang et al., 2013a, b). the aromaticity of the volatiles might disturb the diffusion of odor signals between the individuals and food as well as weaken the water recognition or drinking rate of workers, which might indirectly threaten the survival of the species. according to test results, more workers died after exposure to fallen leaves in a closed system. however, fresh leaves significantly affected the aggregation and drinking ability of the ants. fallen leaves might be a better resource to control the ants and are also more cost-effective. thus, a good method to control red imported fire ant is directly incorporating mashed fallen leaves m. alba into the soil where ants build their nest or stay for prolonged periods. thus, the volatile substances could be constantly released from the leaves to affect the ants. in this way, people could avoid extracting essential oil through the complicated process, and also turn “waste” into wealth by collecting and reuse the leaves fallen from the tree, which reflect the economic and ecological value better. in the future , more and more plant materials, such as m. alba leaves, instead of chemical agents will be used to control red imported fire ant. and it’s environmentally to develop fumigants to control red imported fire ant, in order to achieve green control. acknowledgment the study was funded by the talent development program on joint training of postgraduates demonstration base in guangdong province, china (2013jdxm11), science roject of guangdong province (2014a020208114), science and technology planning and scientific research project of guangzhou city (201510010299). the authors thank haifeng wei for his assistance with gc-ms. the authors are also grateful to the reviewers for their constructive comments. references appel, a.g., gehret, m.j. & tanley, m.j. 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(1982). a preliminary study on the chemical constituents of the essential oil of michelia alba dc. acta botanica sinica, 24: 355-359. http://www.medsci.cn/sci/submit.do?id=66da963 http://www.medsci.cn/sci/submit.do?id=66da963 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1629sociobiology 64(3): 237-243 (september, 2017) seasonal patterns of the foraging ecology of myrmelachista arthuri forel, 1903 (formicidae: formicinae) introduction ants form an abundant and diverse insect group that is essential for ecosystem structure, especially in the tropics (del-claro et al., 2002; del-claro & torezan-silingardi, 2009; mertl et al., 2009). each species has inherent foraging dynamics, which are essential for colony success (hölldobler & wilson, 1990). these dynamics are particularly important in the context of ecosystems, because they directly influence trophic relationships (dátillo et al., 2009; elisei et al., 2012). foraging success is influenced primarily by temperature, but also by predation, interand intraspecific competition, colony development stage, resource availability, spatial orientation, and relative humidity (carroll & janzen, 1973; fowler et abstract temporal dynamics of foraging activity, diet and habitat are key for understanding the bioecology of ants. these patterns are poorly studied in many neotropical species, such as those belonging to genus myrmelachista. in the present work, we investigate the foraging behavior and diet of m. arthuri and describe aspects of their food-searching behavior. we recorded the dynamics of workers exiting and entering nests built in the twigs of native atlantic forest trees during the cold/ dry and warm/wet seasons. food items carried by workers were also counted and identified. myrmelachista arthuri foraged throughout the day, but worker activity became more intense as temperature increased and moisture declined, regardless of the season, and especially in the afternoon. this species had a generalist diet: 92% of the food items were live or dead arthropods or their remains, and a small proportion consisted of plant materials, such as seeds. arthropod fragments, mostly of m. arthuri workers, represented the largest proportion of the diet, followed by whole collembola individuals. food items did not vary between seasons, the number of items was higher in the cold/dry season. the results of this work contribute to the understanding of m. arthuri biology, especially related to foraging dynamics. sociobiology an international journal on social insects ghp castro1, dy kayano1, rf souza1, aws hilsdorf2, rm feitosa3, msc morini1 article history edited by gilberto m. m. santos, uefs, brazil received 31 march 2017 initial acceptance 17 july 2017 final acceptance 28 july 2017 publication date 12 october 2017 keywords collembola, arboreal ant, diet, alchornea triplinervia, mimosa bimucronata. corresponding author maria santina de castro morini universidade de mogi das cruzes núcleo de ciências ambientais laboratório de mirmecologia av. dr. cândido xavier de almeida souza, 200, cep 08780-911 mogi das cruzes-sp, brasil. e-mail: mscmorini@gmail.com al., 1991; belchior et al., 2012; brito et al., 2012; lima & antonialli-junior, 2013). the ant genus myrmelachista is exclusively neotropical and arboreal (kempf, 1972; longino, 2006), including 56 species and 13 subspecies (antwiki, 2017). ants in this genus are rarely observed on plants or in the leaf litter, despite being diurnal and abundant in forests (silvestre et al., 2003; longino, 2006). myrmelachista ants are generally small, but are specialized in terms of resource exploitation and niche colonization (silvestre et al., 2003). some species are associated with myrmecophytes. these ants use plants as shelter and feed on extrafloral nectaries; in return, the host plant receives protection against herbivores and parasites (frederickson & gordon, 2009). ants in this genus may 1 laboratório de mirmecologia, universidade de mogi das cruzes, são paulo, brazil 2 laboratório de genética de organismos aquáticos e aquicultura, universidade de mogi das cruzes, são paulo, brazil 3 universidade federal do paraná, curitiba, brazil research article ants ghp castro et al. – seasonal foraging ecology of myrmelachista arthuri238 also feed onanimal-derived proteins (nakano et al., 2013) or honeydew from scale insects (longino, 2006). myrmelachista colonies are usually big, polydomous and/or polygynous and occupy dead branches or live tree stems (frederickson, 2005; longino, 2006; frederickson & gordon, 2009; nakano et al., 2012; nakano et al., 2013), making it difficult to study the biology of the species. in this work, we describe how m. arthuri foraging patterns and diet vary between different seasons of the year, in addition to reporting other aspects of its natural history. material and methods collection area, nests and species the study was conducted in parque municipal leon feffer (23°31’49” s; 46°13’26” w). the park is located within the urban matrix of mogi das cruzes (são paulo state, brazil) (fig 1), but it has patches of native atlantic forest within an exotic vegetation matrix (pmmc, 2017). according to köppen’s classification, the climate is mesothermal with a dry winter (cwb). annual precipitation is between 1300 and 1700 mm (minuzzi et al., 2007; pagani, 2012). observations were carried out in an area of approximately 2784 m², where 12 nests were found: 11 of these were inside live stems of two locally common tree species, alchornea triplinervia (spreng) (9 nests) and mimosa bimucronata (de candolle, o. kuntze) (2 nests); the last nest was in a partially suspended dead stem. this nest was chosen as a reference distance from other nests (fig 1). twigs were collected from the leaf litter in the study area to search for colonies of this species. workers from each marked m.arthuri nest were molecularly characterized (genbank accession noky212125). voucher specimens were deposited in the entomological collection padre jesus santiago moure at federal university of paraná (pr, brazil) and university of mogi das cruzes (sp, brazil). worker foraging activity worker foraging activity was observed between october 2014 and april 2016 during the cold/dry (april-july) and warm/wet (january-october) seasons (minuzzi et al., 2007). worker activity was monitored in four nests, weekly, from 7:00 to 17:00, for a total of 16 observation days (i.e., 160 hours, or 80 hours per season).every hour, the number of workers leaving and entering the nest was recorded with a manual counter for 10 minutes. temperature and relative air humidity at each observation period were also recorded. diet the diet of m. arthuri was studied in 12 nests in the cold/dry (august and september) and warm/wet (february and march) seasons. once every 15 days, six nests were monitored, to avoid disrupting nest development. observations were carried out by a single person for one hour, after 12:00, for a total of 120 hours. food items carried to the nest by workers were manually collected and preserved in 80% ethanol. these items were sorted according to origin (plant or animal). animalderived items were identified to order using triplehorn and johnson (2011). if the food item was an ant, it was identified to species using suguituru et al. (2015). fig 1.map showing the location of parque municipal leon feffer (plf) in the municipality of mogi das cruzes (são paulo state, brazil) and approximate spatial distribution of m. arthuri nests (i-xii), including the distance (in meters) to the reference nest (i, nest in dead stem). illustration by l. menino. sociobiology 64(3): 237-243 (september, 2017) 239 fig 2. a. nest in a dead m. bimucronatastem; b. foraging trail on a live a. triplinerviastem; c. foraging trail on a live m. bimucronatastem. arrows indicate worker entrance and exit holes. identification of plant species by r.j. almeida-scabbia. fig 3.average number of m. arthuriworkers leaving and returning to the nest at different times of day and seasons. vertical lines: standard deviations. data analysis pearson correlation tests were used to evaluate the correlation between foraging frequency (worker exit and entrance) and temperature and humidity. foraging activity was compared between seasons and times of day using student’s t-tests. a mann-whitney test was used to analyze the variation in food item abundance between seasons. a g test was used to compare the two food sources (animalvs. plant-derived) between seasons. all analyses were preceded by the lilliefors test to verify data normality. the software bioestat 5.0 (ayres et al., 2007) was used for all of the tests, and the significance level was set at 0.05. results workers constantly exited and entered m. arthuri nests through holes along tree stems (fig 2a). the trails used to walk on the surface of stems were well-marked (fig 2b) and had a clear and distinctive color on live trees (fig 2c). workers used these trails to reach the leaf litter, but no nests were found in fallen twigs. no alates were observed during the study. only one mtdna haplotype was found in workers from each of the 12 nests through dna sequencing. foraging occurred throughout the day and was more intense in the afternoon (entering nest: t = 10.92, df = 1;p< 0.0001; leaving nest: t = 11.01, df =1; p< 0.0001) and during the cold/dry season (fig 3). foraging was also correlated with temperature and relative air humidity in the cold/dry season (temperature: r = 0.52; p < 0.0001; humidity: r = -0.55; p < 0.0001) and in the warm/wet season (temperature: r = 0.31; p < 0.0001; humidity: r = -0.13; p = 0.0132) (fig 4). by the end of the observation period, workers had collected a total of 336 food items, 92% of animal origin and 8% of plant origin. regardless of season, intraspecific predation was the most frequent food source (47%), followed by transportation of arthropod remains (24.1%), fragmented plant material (5.1%), and whole individuals of collembola (4.2%) and diptera (3.6%) (fig 5). in the cold/dry season, ants carried 19 types of food, while 17 types were carried in the warm/wet season. the number of collected resources differed between seasons (u = 24.50, df = 1, p= 0.003). in the cold/dry season, 222 items were collected, compared to 114 in the warm/wet season (table 1). the most frequently collected items in the cold/dry season were m. arthuriworkers, arthropod remains, whole springtails (collembola) and fragmented plant material. collection of plantderived items increased in the warm/wet season compared to the cold/dry season (g = 6.73, df = 1, p = 0.0094) (fig 6). there was also a drastic reduction in the capture of whole springtails in the warm/wet season. discussion the nesting strategy of m. arthuri is similar to the strategy used by other species of this genus, as reported by longino (2006), since nests were observed in live or dead tree stems. however, there were no nests inside fallen twigs in the leaf litter. similarly, in a large study of myrmelachista ghp castro et al. – seasonal foraging ecology of myrmelachista arthuri240 species, nakano et al. (2012, 2013) did not find m. arthuri nests in fallen twigs; these authors were only able to find this species in live tree stems or in recently fallen tree stems. according to longino (2006), myrmelachista workers rarely leave the nest, and there are no external signs of their presence. in contrast, we observed intense foraging on tree stems by m. arthuri, which formed clearly visible trails on the stems where the nests were found. workers used these trails throughout fig 4. daily and seasonal variation of the foraging activity of m. arthuri. vertical lines: standard deviations. the day to forage for resources in the leaf litter, mostly in the afternoon. arboreal ants, such as m. arthuri, exploit resources that are rare in the arboreal environment (delabie et al., 2015), especially resources rich in nitrogen (begon et al., 2006). many species, such as pogonomyrmex naegelii forel, 1878 and camponotus sericeiventris guérin, 1838, are more active outside the nest at the warmest times of day (yamamoto & del claro, 2008; belchior et al., 2012). myrmelachista fig 5. food items collected by m. arthuri during foraging. (a) flower bud exudate (b) collembola (c) remains of lepidoptera (d) remains of diptera. images a, c and d by j.p.s. rosa; b. illustration by d.y. kayano. sociobiology 64(3): 237-243 (september, 2017) 241 arthuri followed this pattern regardless of the season, although foraging was more intense in the cold/dry season. in this case, the activity pattern was influenced by season and probably related to low food availability, which causes the workers to leave the nest more often (fowler et al., 1991; yamamoto & del-claro, 2008; belchior et al., 2012; lima, 2013).workers foraged less frequently in the warm/wet season, despite higher energy requirements due to increases in temperature (lima, 2013) and a late production (nakano et al., 2013) and greater food availability in the environment (belchior et al., 2012). most plants flower in the warm/wet season (yamamoto & del-claro, 2008), since workers collected more plant material in this season. so, we can assume they provide better energy gains compared to the other food items. however, the observed reduction in worker activity during the warm/wet season is not common for ants. in pogonomyrmex naegelii, for instance, colony activity is higher after it rains, because workers remove soil particles from the nest and it is easier to capture dead arthropods in the surroundings (belchior et al., 2012). in addition to transporting more plant-derived items type of food item cold/dry season warm/wet season total % quantity % quantity % plant seeds 3 1.4 8 7.0 11 3.3 fragments* 7 3.2 10 8.8 17 5.1 animal acari 2 0.9 2 0.6 araneae 1 0.5 2 1.8 3 0.9 coleoptera 6 2.7 3 2.6 9 2.7 collembola 13 5.9 1 0.9 14 4.2 diptera 5 2.3 7 6.1 12 3.6 hemiptera 1 0.5 1 0.9 2 0.6 hymenoptera micro-hymenoptera 2 0.9 1 0.9 3 0.9 atta sexdens 4 1.8 1 0.9 5 1.5 camponotus rufipes 1 0.9 1 0.3 cephalotes atratus 1 0.9 1 0.3 cephalotes pusillus 1 0.5 1 0.9 2 0.6 crematogaster sp. 1 0.5 1 0.3 myrmelachista arthuri 114 51.4 44 38.6 158 47.0 nylanderia sp. 1 0.9 1 0.3 pachycondyla striata 1 0.5 1 0.3 pseudomyrmex sp. 1 0.5 1 0.3 solenopsis sp. 1 0.9 1 0.3 isopoda 5 2.3 3 2.6 8 2.4 isoptera 1 0.5 1 0.3 neuroptera 1 0.5 1 0.3 arthropoda fragments 53 23.9 28 24.6 81 24.1 total 222 100 114 100 336 100 *flowers, fruits and seeds table 1. types of food collected by m. arthuri workers along a 120-hour observation period splitbetween two seasons. fig 6. seasonal variation of the proportion of food types collected by m. arthuri workers. vertical lines: standard errors. in the warm/wet season, the number of collected items, in general, indicated seasonal shifts of the foraging habits of m. arthuri. this is probably related to resource availability and energy content, which vary seasonally (fowler et al., 1991; carvalho, 2004; yamamoto & del-claro, 2008). ghp castro et al. – seasonal foraging ecology of myrmelachista arthuri242 myrmelachista ants feed on plant exudates (silvestre et al., 2003), but they may also use seeds and arthropod fragments encountered in the foraging trails, as observed for m. arthuri. nakano et al. (2013) observed workers of this species carrying pieces of cockroaches and termites to the nest, corroborating our observations. however, the high proportion of whole springtail individuals in their diet was unexpected, because m. arthuri workers do not have the jaws of ants that specialize on this type of prey (brandão et al., 2009). based on the food items transported to the nest, we can conclude that their diet is generalist. because of diversification, the colony is able to fulfill its nutritional requirements, despite resource scarcity, competition and seasonality (fowler et al., 1991). many ant species, including m. catharinae and m. ruszkii, expand their colonies to twigs in the leaf litter (nakano et al., 2012), forming satellite nests. these nests promote territory defense, increase protection of the host plant and improve survival of the colony itself, which has a higher risk of being eliminated by predation or other disturbances when it is concentrated in one spot (krebs & davies, 1993; santos & del-claro, 2002; santos & del-claro, 2009). however, although expanding the colony to twigs in the leaf litter is structurally simple and demands small worker investment for colonization, it does not seem to be a strategy adopted by m. arthuri (mcglynn et al., 2012). despite many collection expeditions by carvalho and vasconcelos (2002), nakano et al. (2012), fernandes et al. (2012) and souza et al. (2012), this species has never been found nesting in this microhabitat. however, these ants do colonize surrounding trees, which was corroborated by the finding of a single mitochondrial haplotype in all studied colonies. this is the first study to collect information on the foraging activity and diet of an atlantic forest myrmelachista species, in addition to describing other behavioral aspects of m. arthuri, such as the formation of trails on trees and predation on springtails. this work also expands the list of myrmelachista host plant species (reviewed by nakano et al., 2013). based on our data, we can infer that this genus is generalist in relation to the occupation of dead or live tree stems, despite its complex mutualistic interactions with the host tree (longino, 2006).the knowledge of life history of organisms is fundamental to make appropriate decisions for management and biodiversity preservation. this information helps fill the gaps in knowledge about the biology of an exclusively neotropical genus with complex behavior. acknowledgments the authors would like to thank renata j. almeidascabbia, leonardo menino, joão paulo s. rosa and nathália s. silva.we also thank fapesp (grant no. 2013/16861-5) and faep. rmf was supported by the brazilian council of research and scientific development (cnpq grant 130 642/2016-9). references antwiki. 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(2012). serra do itapeti: aspectos históricos, sociais e naturalísticos. bauru: canal 6. nakano, m.a., feitosa, r.m., moraes, c.o., adriano, l.d.c., hengles, e.p., longui, e.l. & morini, m.s.c. (2012). assembly of myrmelachista roger (formicidae: formicinae) in twigs fallen on the leaf litter of brazilianatlantic forest. journal of natural history, 46: 1-13. doi: 10.1080/00222933.2012.707247 nakano, m.a., miranda, v.f., souza, d.r., feitosa, r.m. & morini, m.s.c. (2013). occurrence and natural history of myrmelachista roger (formicidae: formicinae) in the atlantic forest of southeastern brazil. revista chilena de história natural, 86: 169-179. doi: 10.4067/s0716078x2013000200006 pagani, m.i. (2012).preservação da serra do itapeti. in: morini, m.s.c. & miranda, v.f.o. serra do itapeti aspectos históricos, sociais e naturalísticos. bauru: canal 6, 45-58 p. pmmc – prefeitura municipal de mogi das cruzes (2017). retrieved from: http://visitemogi.com/places/ecologico/parqueleon-feffer/ silvestre, r., brandão, c. r. f & silva, r.r. (2003). grupos funcionales de hormigas: el caso de los grêmios del cerrado, pp. 113-148. in: f. fernández (ed.), introducción a las hormigas de la región neotropical. instituto de investigación de recursos biológicos alexander von humboldt, bogotá, colômbia. schmolke, a. (2009). benefits of dispersed central-place foraging: an individual-based modelo of a polydomous ant colony. the american naturalist, 6: 772-778. doi: 10.1086/598493 souza, d.r.; fernandes, t.t.; nascimento, j.r.o.; suguituru. s.s. & morini, m.s.c. (2012). characterization of ant communities (hymenoptera: formicidae) in twigs in the leaf litter of the atlantic rainforest and eucalyptus trees in the southeast region of brazil. psyche, 12: 1-12, doi: 10.1155/2012/532768 suguituru, s.s., morini, m.s.c., feitosa, r.m. & silva, r.r. (2015). formigas do alto tietê. bauru: canal 6, 456 p triplehorn, c.a.; johnson, n.f. (2011). estudo dos insetos. (tradução da 7. ed. de borror and delong’s: introduction to the study of insects). são paulo: cengage learning, 809 p. yamamoto, m. & del-claro, k. (2008). natural history and foraging behavior of the carpenter ant camponotus sericeiventris guérin, 1838 (formicinae, campotonini) in the brazilian tropical savanna. acta ethology, 11: 55-65. http://dx.doi.org/10.4067/s0716-078x2013000200006 http://dx.doi.org/10.4067/s0716-078x2013000200006 http://dx.doi.org/10.1155/2012/532768 sociobiology 67(3): 335-336 september (2020) doi: 10.13102/sociobiology.v67i3.5873 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 editorial bringing innovations to set up a better scientific journal for publication of your research sociobiology is a scientific journal dedicated to the study of social insects since 1976. its 45th anniversary is coming up and the board of editors is pleased to celebrate this milestone with the announcement of a set of changes that will leverage the journal to modern and innovative editorial standards. aiming to simplify the process of manuscript submission, effective immediately, we are accepting the initial submissions in the format of authors’ preference. this should allow authors, editors, and referees to focus on the quality of the science being reported, saving valuable time of everyone involved. the editors will access this version and decide whether its standards justify further analysis by the referees. only after a manuscript is accepted and enters the editing stage authors will be required to present a version in sociobiology's format. for more information we invite you to access the new authors' guidelines. starting on january 2021, articles will be published in continuous flow, immediately after the final acceptance. thus, we expect to reduce the time from submission to publication to four months in average (working on a minimum of two and a maximum of six months). as a commitment to provide a richer view of the journal visibility and performance, sociobiology is now a signatory of dora, the san francisco declaration of research assessment[1]. effective 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published content, effective for the next published issue, the editorial team will work with authors to prepare graphical abstracts suited for publication in social medias, either by the editorial team or the authors themselves. thus, the published content will reach more visibility and use by the scientific community. it is worth reassuring that all these improvements will not change our innovative open science model, in which publication is free of charge for both the author and the reader. we like to call it “diamond open access”, as opposed to “gold” or “green” open science models. this is only possible because sociobiology is financially sociobiology an international journal on social insects sociobiology 67(3): 335-336 september (2020) doi: 10.13102/sociobiology.v67i3.5873 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 supported by a public state university which receives endowments from the state treasure. costs of editing the journal are entirely covered by public money. furthermore, editors and editorial office staff are public employees spending part of their work hours in the journal management. hence, if the financial support comes from public funds, the authors and readers must benefit from this. the board of editors considers that scientific knowledge financed by public money cannot be appropriated in a private fashion at any step of the editorial business chain. we are totally committed to open access publishing and the promotion of free content aiming to support a greater global exchange of knowledge. evandro n. silva, og desouza, kleber del-claro and gilberto marcos m. santos the associate editors [1] https://sfdora.org/read/ accessed on september 20, 2020. [2] https://publicationethics.org/guidance/guidelines acessed on september 20, 2020. [3] brand, a., allen, l., altman, m., hlava, m. and scott, j. (2015). beyond authorship: attribution, contribution, collaboration, and credit. learned publishing, 28: 151-155. doi: 10.1087/20150211. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1263sociobiology 63(4): 1069-1072 (december, 2016) rediscovery of the morphologically remarkable social parasite pheidole acutidens (santschi, 1922), with the first records for brazil pheidole westwood, 1839 (formicidae: myrmicinae) is considered one of the largest ant genera, with more than 1,000 valid names (bolton, 2016). the species are generally known for being prevalent in all environmental strata making it a group of primordial ecological importance, especially in tropical forests (wilson, 2003). amongst the reproductive strategies found in pheidole, social parasitism is certainly the most remarkable. social parasites are exquisitely adapted to exploit their hosts, totally depending on them for food and brood care, since they do not produce workers (wilson, 1971; hölldobler & wilson, 1990; buschinger, 2009). we currently recognize nine species of social parasites in pheidole: p. acutidens (santschi, 1922), p. argentina (bruch, 1932), p. symbiotica (kusnezov, 1951), p. elecebra (wheeler, 1973), p. inquilina (wheeler, 1965), p. lanuginosa wilson (1984), p. neokholi wilson (1984), p. parasitica wilson (1984), and p. kosnezovi wilson (2003). like other social parasites p. acutidens presents a morphological syndrome that includes reduced size, abstract pheidole acutidens is a social parasite of the congeneric species p. nitidula. since its description, the species was considered native to argentina. in this paper we report the first records of p. acutidens for brazil in the southern states of santa catarina and rio grande do sul. these records extend the known distribution of the species at least 1,000 km to the north. we suggest that the scarce representation of this species in entomological collections is due to its peculiar reproductive strategy, which renders the species inconspicuous and limits its dispersion. sociobiology an international journal on social insects a casadei ferreira, mfo martins, rm feitosa article history edited by gilberto m. m. santos, uefs, brazil received 05 december 2016 initial acceptance 12 december 2016 final acceptance 13 december 2016 publication date 13 january 2017 keywords geographical distribution, myrmicine, native ant, taxonomic problem. corresponding author alexandre casadei ferreira laboratório de sistemática e biologia de formigas, departamento de zoologia universidade federal do paraná, curitiba-pr, brasil e-mail: acf.casadei@gmail.com lengthening of antennal scapes, reduction of the mandibles, smooth and shining body surface, and broadening of the postpetiole (wilson, 1971). also, queens of this species have a rounded head, falcate toothless mandibles ending in an extremely acute angle, antennae with 9-11 articles, a single pair of wings without venation, and a globose gaster, (santschi, 1922; bruch, 1931; wilson, 1984) (fig. 1). male morphology is even more bizarre. presenting a rounded head, 12 segmented antennae, and physiogastry. also, according to wilson (2003) the male also has vestigial or absent falcate mandibles, and pupiform body. in addition, males of p. acutidens are brachypterous (bruch, 1931; wilson, 1984). the taxonomic history of pheidole acutidens reflects its unique behavior and morphology. santschi (1922) described the monotypic genus bruchomyrma with bruchomyrma acutidens as its type species based on a single dealate queen collected by carlos bruch (1869-1943) in 1916. he also characterized bruchomyrma through an extensive diagnosis, and mentioned its morphological similarity to anergatides universidade federal do paraná, curitiba-pr, brazil short note a ferreira, mfo martins, rm feitosa – rediscovery of the social parasite pheidole acutidens1070 kohli wasmann, 1915, despite the “easily recognizable differential characters”. he also suggested that b. acutidens could be a social parasite of pheidole, as reported for a. kohli. wilson (1984) performed a comprehensive study on social parasites of pheidole and combined several genera under this genus, including anergatides and bruchomyrma. the combination made by wilson for p. acutidens was mostly based on evidence mentioned by brown (1973), which assumed that the different parasitic species of pheidole evolved independently, with the parasitic syndrome arising convergently. in this context, the nomenclatural act proposed by wilson (1984) may also be justified considering “emery’s rule” (emery, 1909) which states that social parasites are closely related to their host species. pheidole acutidens is currently known as a social parasite of p. nitidula. so far, the species distribution is restricted to argentina, specifically to las flores (35°57’49.5”s 58°56’20.4’’w) (buenos aires) and alta gracia (31°39’25.6’’s 64°26’05.1’’w) (córdoba) (bruch, 1931; wilson, 2003). a hundred years after its discovery (bruch, 1931), we rediscovered the species and present the first records for brazil. we examined two specimens, both queens. the first specimen is alate and was collected in caxias do sul (29°10’05’s 51°10’46’’w), state of rio grande do sul, on 21 march, 1988. there is no mention about the sampling method. the specimen is deposited in the myrmecological collection of the museu de zoologia da universidade de são paulo (mzsp), são paulo, brazil. the second specimen is a dealate queen from a pitfall sample collected in the municipality of otacílio costa (27°29’03.9’’s 49°54’14.4’’w), state of santa catarina, between december 2011 and january 2012 (bartz et al., 2014; rosa et al., 2015). the specimen was deposited in the coleção entomológica padre jesus santiago moure (dzup) of the universidade federal de paraná, curitiba, brazil. the distribution map was generated by the program qgis v.2.16.3 using data from geographic coordinates in google earth pro 7.1.1.1580. images of specimens were acquired with stereomicroscope leica m205c coupled to a camera leica dfc295 at laboratório de sistemática, evolução e biologia de hymenoptera of mzsp. the images of the layers were aligned and combined in program zerene stacker v. 1.04, and posteriorly treated in adobe photoshop cs6 for brightness and contrast corrections. these new records suggest the apparently restricted distribution of the species may be an artifact from lack of adequate sampling. besides this collecting bias, the unique morphology and reproductive biology of p. acutidens may also explain the considerably low number of specimens in ant collections. queens present an extremely enlarged mesosoma and have lost the hindwings, while males show extreme reduction in wing size and venation (brachyptery). fig 1. pheidole acutidens queens from caxias do sul (a, c) and otácilio costa (b, d). a, b: full-face view; c, d: body in lateral view. scale bars: 0,5mm. sociobiology 63(4): 1069-1072 (december, 2016) 1071 these unusual morphological features suggest limited flight capability and restricted dispersion rates in p. acutidens. as described by bruch (1931), p. acutidens queens are only accepted and adopted by p. nitidula workers in queenless nests. this limitation probably also restricts their dispersion, due the difficulty of finding suitable nests without host queens. however, once established, queens of p. acutidens can produce approximately 300 eggs and complete their life cycle in about three months (bruch, 1931). this mass production of individuals may be linked to the trial and error way of infestation, with a great number of founding queens searching for queenless colonies of p. nitidula. the intrinsic relationship between p. acutidens and p. nitidula can also be evidenced by the dependence of newly-hatched p. acutidens queens on p. nitidula workers to remove their cocoon and unfold their rudimentary wings after emergence. in addition, when invading a host colony, mated queens of p. acutidens rely entirely on the workers of p. nitidula to remove their wings and establish themselves in the new colony (bruch, 1931). considering the intrinsic association between both species, p. acutidens probably occurs in the same range as p. nitidula recorded from argentina to the states of rio de janeiro, southeastern brazil (wilson, 2003). in this scenario, the records presented here represent the first step towards a more complete mapping of p. acutidens distribution. acknowledgements we would like to thank dr. carlos roberto f. brandão for permission to access the myrmecological collection of the mzsp and for providing the equipment for acquiring highresolution images. brendon boudinot identified the specimen from caxias do sul. dr. john lattke and thiago s. r. silva made important suggestions in a previous version of this paper. acf (140260/2016-1) and mfom (130387/20150) thanks the fellowships granted by conselho nacional de desenvolvimento científico e tecnológico (cnpq). the fundação de amparo à pesquisa e inovação do estado de santa catarina (fapesc) (process 6.309/2011-6/fapesc) and cnpq (process 563251/2010-7) for the financial support. fig 2. distribution of pheidole acutidens in argentina and brazil. a ferreira, mfo martins, rm feitosa – rediscovery of the social parasite pheidole acutidens1072 references bartz, m. c.; brown, g. g.; rosa, m. g.; klauberg filho, o.; james, s. w.; decaëns, t. & baretta, d. 2014. earthworm richness in land-use systems in santa catarina, brazil. applied soil ecology. doi: 10.1016/j.apsoil.2014.03.003 bolton, b. 2016. an online catalog of the ants of the world. available from http://antcat.org. acess october 2016. brown, w. l. 1973. hylean and congo-west african rain forest ant faunas, in: b.j. meggers, e.s. ayensu, w.d. duckworth (editors): tropical forest ecosystems in africa and south america: a comparative review. smithsonian institution press. washington, d.c. p. 161-185. bruch, c. 1931. notas biologicas y sistematicas acerca de bruchomyrma acutidens. revista museo de la plata, tomo xxxiii: 31-55. buschinger, a. 2009. social parasitism among ants: a review. (hymenoptera: formicidae). myrmecological news, 12: 219-235. emery, c. 1909. über den ursprung der dulotischen, parasitischen und myrmekophilen ameisen. biologisches centralbatt, 29: 352-362. hölldobler, b. & wilson, e. o. 1990. the ants. cambridge, mass.: harvard university press, xii + 732 pp. rosa, m. g.; klauberg filho, o.; bartz, m. c. l.; mafra, a. l.; sousa, j. p. f. a. & baretta, d. 2015. macrofauna edáfica e atributos físicos e químicos em sistemas de uso do solo no planalto catarinense. revista brasileira de ciências do solo, 39: 1544-1553. santschi, f. 1922. description de nouvelles fourmis de l’argentine et payslimitrophes. anales de la sociedad cientifica argentina, 94:241-262. wasmann, e. 1915. anergatides kohli, eineneuearbeiterlose schmarotzer ameise vom oberen kongo (hymenoptera: formicidae). entomol. mitt., 4: 279-288. wilson, e. o. 1971. the insect societies. cambridge, mass.: harvard university press, x + 548pp wilson, e. o. 1984. tropical social parasites in the ant genus pheidole, with an analysis of the anatomical parasitic syndrome (hymenoptera: formicidae). insectes sociaux, 31:316-334. wilson, e. o. 2003. pheidole in the new world. harvard university press, cambridge, ma, usa. _goback doi: 10.13102/sociobiology.v65i2.1204sociobiology 65(2): 130-137 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant community in neotropical agrosystems: a four-year study in conventional and no-tillage systems introduction agriculture is a major cause of biodiversity loss around the world (mclaughlin & mineau, 1995; urrutia-escobar & armbrecht, 2013). while agricultural expansion has increased the productivity of farmland, the number of animal and plant species has decreased (isselstein et al., 1991). this scenario does not occur only in countries with extensive cultivated areas, but also in small countries with limited farmland (mclaughlin & mineau, 1995; büchs, 2003). the global scenario of transformation of the natural environment in agrosystems has stimulated researchers to look for alternatives, to minimize the negative effects of agriculture on biodiversity. in this sense, agricultural practices that have a reduced impact on nature, but are economically competitive, abstract studies comparing agricultural practi ces that maintain a better quality and a healthy soil fauna consider the no-tillage farming as the most effective practice when compared to other planting techniques. in order to evaluate the influence of the no-tillage and conventional tillage methods (with and without manipulation of the soil before planting, respectively) on ant communities, we monitored two areas with these two types of practice (conventional and no-tillage) over the period of four years. we collected ants once per month along 10 transects randomly distributed using three pitfall traps in each area. in addition, we collected the dead plant biomass present at each point sampled as a parameter for measuring the environmental complexity of the areas. in total, we captured 27,480 individuals belonging to 26 species in the no-tillage area and 24,570 individuals belonging to 24 species in the conventional tillage area. the generalised linear model analysis showed that the no-tillage system had the highest abundance of individuals, as well as richness and diversity of species, during most of the study period, as compared to conventional tillage areas. we also found a significant positive correlation between species richness and dead plant biomass. thus, it is possible to infer that the no-tillage area is a more complex environment with a greater diversity of ants and, therefore, a more sustainable agrosystem as compared to conventional tillage areas. sociobiology an international journal on social insects wd fernandes1, d lange2, jm pereira3, j raizer1 article history edited by inge armbrecht, university del valle, colombia received 02 october 2016 initial acceptance 20 november 2016 final acceptance 11 february 2018 publication date 09 july 2018 keywords agrosystem, biodiversity, community structure, soil use. corresponding author denise lange universidade tecnológica federal do paraná, campus santa helena rua cerejeira, s/n, cep 85892-000 santa helena-pr, brasil. e-mail: deniselange@yahoo.com.br has been the goal of sustainable agriculture in recent decades (tilman et al., 2002; sani, 2011). in searching for sustainable agricultural practices that are able to maintain the soil quality and the diversity of fauna, the no-tillage farming practice is currently been considered as the most efficient method (sani, 2011). unlike conventional tillage, in which the dry plant biomass of the previous crop is incorporated to the soil by a mechanization process, the no-tillage is a reduced form of soil preparation, where essentially no manipulation of the soil is performed before planting (stinner & house, 1990). in no-tillage systems, the dry plant biomass on soil after harvest provides protection against soil degradation and improves microclimate conditions for survival of the edaphic fauna (doran et al., 1994; hawksworth, 1991). 1 faculdade de ciências biológicas e ambientais, universidade federal da grande dourados, dourados-ms, brazil 2 universidade tecnológica federal do paraná, santa helena-pr, brazil 3 faculdade de matemática, universidade federal de uberlândia-mg, brazil research article ants mailto:deniselange@yahoo.com.br sociobiology 65(2): 130-137 (june, 2018) 131 several studies have evaluated the effect of tillage on soil fauna, using groups of arthropods, such as beetles (stinner & house, 1990; bohac, 1999, ashford et al., 2013), collembolans and spiders (stinner & house, 1990; ashford et al., 2013) and ants (stinner & house, 1990; booij & noorlander, 1992; harvey & eubank, 2004; sharley et al., 2008; ashford et al., 2013). other studies have also investigated communities of bacteria, fungi, mites and nematodes (behanpelletier, 1999; treonis et al., 2010). all of these organisms are affected by the type of soil management and have been used as bioindicators (ashford et al., 2013). in addition to their role as bioindicators of environmental change, most ant species are efficient predators of other insects, contributing to pest control in crops (perfecto, 1991; agarwal et al., 2007; rico-gray & oliveira, 2007; lange et al., 2008). according to booij and noorlander (1992), ants, beetles and spiders are the most effective generalist predators in agrosystems. several authors have shown that more complex environments exhibit higher predation rates by ants (harvey & eubanks, 2004; philpott & armbrecht, 2006; lange et al., 2008). according to mansfield et al. (2003), effective biological pest control is one of the goals of sustainable agriculture. during the present study, ants were monitored over the period of four years in two areas treated with different soil management practices, one with mechanized conventional tillage and the other with no-tillage. we hypothesised that the area with no-tillage will have a more diverse ant community when compared to the area with conventional tillage. this result should be associated with the greater structural complexity of the no-tillage system, because of greater availability of food, less competition between ant species, and therefore greater diversity. studies on ants communities in agrosystems such as this is aimed to understand the effect of tillage systems on edaphic fauna and inform stakeholders that aim to implement agricultural practices that have a lower impact on biodiversity. material and methods study area we carried out the study in dourados, mato grosso do sul, brazil (22°13’16” s and 54°48’20” w) from november 1999 to december 2003. the region is part of the cerrado biome, with an altitude ranging between 449 and 477 m. the climate, according to köppen, is cwa (humid mesothermal), which is characterized by the presence of two seasons: rainy (september-february) and dry (marchaugust), average annual rainfall of 1,500 mm and the annual average temperature of 22°c (± 2.3). the soil of the region is the type dystroferric hapludox clayey with flat topography (embrapa, 1999). we monitored the ant communities of two contiguous areas of 1.5 ha each, both belonging to the universidade federal da grande dourados. the two areas were kept under conventional tillage from 1997 until the beginning of this study (1999), when a no-tillage method was established in one of the areas. during the study, three annual harvests were carried out in both areas. the summer crops, sown in october or november and harvested in february or march, were soybeans and corn; in the winter, corn and wheat sown after the harvest of summer and harvested in june or july. the sorghum, oats, sunflower and turnip crops were sown in august or september to preserve the plant cover on the ground, without harvesting them. all seeded crops, as well as planting dates, were the same in both areas evaluated (tillage and no-tillage system) throughout the four years of study. data collection we used pit-fall traps in containers of 80 ml and seven centimetres of diameter, baited with sardines and honey (15 g and 5 g, respectively) to capture the ants. on the sides of each container, fluon-sliding liquid kept the ants in the trap. we installed 10 linear transects with three pitfall traps keeping a distance of three meters between traps of the same transect and 15 m between transects. the location of transects within the areas was randomised on a monthly basis, maintaining a distance of 10 m from the edge of areas previously sampled. the traps remained exposed in the field for 24 hours. after captured, we placed the ants in 70% alcohol and returned to the laboratory for identification. for taxonomic identification, we used dichotomous keys from bolton (2006), as well as comparisons with specimens from the reference collection of the insects ecology laboratory of the faculdade de ciências biológicas e ambientais of universidade federal da grande dourados. in addition to ants, to represent the presence or not of soil management, we also collected the dead biomass on soil in areas of 1 m² in the same location and time of removal of the ant traps. we put the dead biomass into paper bags and kept it in an oven at 40°c for 48 h. after this period, we weighted it using a balance that is accurate to 0.01 g. data analysis to describe and compare the ant communities of the two areas evaluated, we used data of individual abundance, species richness and diversity (shannon-wiener index). we used the data of dry plant biomass for inference about the structural complexity of the areas. to compare the ant abundance and the amount of dry plant biomass between the tillage systems and during the four years after implementation of each tillage system, we used the average values of the three sampled points in each transect, totalling 10 independent monthly samples in each area. for comparisons of the richness and diversity between areas and among the years after the implementation of each tillage system, we used one monthly value from the 10 monthly samples. in these later comparisons, each year had 24 samples (corresponding to 12 months for each area), with the exception of the fourth year, with 28 samples (14 months for each area). wd fernandes, d lange, jm pereira, j raizer –ant community in neotropical agrosystems132 for these comparisons, we used generalized linear models (glm). we chose this approach because the assumptions of the model related to a completely randomised design with two factors (type of planting and year of implementation), in a split plot design, were not attended. in this study, we modelled continuous variables, dry plant biomass and diversity, from the gamma distribution with the logarithmic link function, and discrete variables (abundance and richness) from the poisson distribution with the logarithmic link function. we defined “linear” for this study’s glm as a completely randomised design with two factors (area and year of implementation) in a split-plot design predictor. for comparison among means, we used the wald test at a significance level of 5%. we conducted these analyses using the software spss 17.0. in addition, we related the monthly values of abundance, richness and diversity with the monthly values of dry plant biomass, using the spearman correlation. in order to verify the similarity of the composition of ant species throughout the months (n = 100; comprising the 50-month study in both areas), we performed the nonmetric multidimensional scaling (nmds), and later, the “joint plot”. then, we related these sorts of scores, through pearson’s correlation, to the data regarding the monthly mean biomass. values were considered significant only if subfamilies/species relative frequency (%) rank of species by year no-tillage conventional nt c 1 2 3 4 1 2 3 4 formicinae brachymyrmex sp1 1.64 0.95 13 14 12 7 13 9 12 camponotus sp1 0.82 0.47 14 12 15 12 13 12 13 camponotus sp2 0.50 0.16 13 12 12 14 camponotus sp3 0.57 0.24 16 16 11 13 12 14 myrmicinae atta sp1 3.15 3.16 8 8 11 5 10 9 11 5 crematogaster sp1 1.14 1.18 11 11 10 9 12 12 12 pheidole sp1 15.14 15.01 2 3 2 2 2 2 3 2 pheidole sp2 8.20 6.48 4 4 6 6 5 4 7 7 pheidole sp3 6.12 5.77 5 5 8 9 6 5 5 6 pheidole sp4 0.82 0.95 14 15 13 11 10 9 pheidole sp5 5.55 4.27 7 7 5 6 8 6 6 10 solenopsis sp1 23.22 30.73 1 1 1 1 1 1 1 1 solenopsis sp2 2.21 0.95 9 10 10 8 11 12 12 11 mycocepurus goeldii (forel, 1893) 0.95 0.47 14 13 13 11 13 12 10 14 hylomyrma sp1 1.20 0.32 12 14 17 8 13 14 carebara sp1 0.06 13 ectatomminae ectatomma planidens borgmeier, 1939 13.19 12.09 3 2 3 3 3 3 4 3 ectatomma tuberculatum (olivier, 1792) 4.29 3.32 6 9 7 9 7 8 8 8 ponerinae neoponera obscuricornis emery, 1890 0.44 0.08 15 16 15 13 13 odontomachus haematodus (linnaeus, 1758) 0.50 0.24 13 14 12 pseudomyrmicinae pseudomyrmex sp1 0.32 15 17 11 dorylinae nomamyrmex sp1 0.63 0.95 15 14 15 12 12 12 neivamyrmex sp1 0.06 0.24 17 10 dolichoderinae dorymyrmex sp1 6.88 10.35 7 6 4 4 4 7 2 4 linepithema sp1 2.33 1.50 10 8 9 10 13 11 8 12 tapinoma sp1 0.06 0.16 17 13 12 species richness 26 24 21 20 24 23 20 18 17 19 table 1. relative frequency of ant species and rank of species by year in areas of no-tillage (nt) and conventional tillage (c) (n = 500 per area) during the period from november 1999 to december 2003 in dourados, ms, brazil. numbers from 1 to 4 represent the four years evaluated. species with the same frequency have the same hierarchical placement in the rank. sociobiology 65(2): 130-137 (june, 2018) 133 r2 ≥ 0.3, according to mccune and mefford (2006). the bray-curtis’ similarity index was used in this analysis, from an array of values with a monthly relative frequency of species in the samples. for these analyses, we used 250 interactions, and the significance was tested with the proportion of randomisations, with stress less than or equal to the observed stress. for this analysis, the software pcord 5.10 was used (mccune & mefford, 2006). results a total of 52,050 individuals were collected (27,480 in no-tillage and 24,570 in conventional tillage). these individuals represented 26 species, 18 genera and seven subfamilies located in the two areas evaluated during the four years of this study. the number of species found in the two areas were similar (26 in no-tillage, and 24 species in conventional) for most of the months evaluated (table 1). however, some species, such as oligomyrmex sp1 and pseudomyrmex sp1, were found only at the no-tillage site. other species, such as pachycondyla obscuricornis (emery, 1890) and odontomachus haematodus (linnaeus, 1758), initially found in two areas, were only present in the area with notillage after the second year of evaluation. according to the rank of species shown in table 1, we found a predominance of species of the subfamily myrmicinae during the four years; among them, the genera solenopsis and pheidole were the most abundant and frequent in both areas (table 1). due to the interaction between factors, the type of planting and the year of implementation were significant for the variables dead plant biomass and ant abundance. as a result, we evaluated the interaction effect by unfolding the type of planting within the year of implementation and viceversa (table 2). from the results shown in table 2, using the wald test and a significance level of 5%, it is possible to verify that the mean biomass in no-tillage sites was higher than the found in conventional tillage, with the exception of the first year of implementation, which showed no significant difference between areas (fig 1 and 2). the mean abundance fig 1. monthly variation of mean dead plant biomass during the four years of study in the area of no-tillage and conventional tillage in dourados, ms, brazil. the black balls and solid lines indicate notillage, and the white balls and dashed lines indicate conventional tillage. the arrows indicate the months when the cultures were harvested. fig 2. monthly variation, mean abundance of ants (a), species richness (b) and diversity index (c) during the four years of study in the area of no-tillage and conventional tillage in dourados, ms, brazil. the black balls and solid lines indicate no-tillage, and the white balls and dashed lines indicate conventional tillage. the arrows indicate the months when the cultures were harvested. of individuals was higher in the no-tillage area every year, except the last year. the highest values of abundance of individuals for conventional tillage were found in the first and third years, respectively; and for no-tillage also, the first and third years had highest abundance of individuals. due to the interaction between factors, the type of planting and year of implementation were not significant for the variables species richness and diversity; we evaluated the effect of the factors of type of planting and year of implementation in an isolated way. the monthly mean richness and diversity of species in conventional tillage were lower than those of no-tillage, and the mean monthly richness in the third year of implementation was higher for both types of planting (table 3). moreover, there is no significant difference between the mean richness for the first, second and fourth years of a b c wd fernandes, d lange, jm pereira, j raizer –ant community in neotropical agrosystems134 implementation. for diversity, the mean value was higher in the third year of implementation and lower in the fourth year, without any significant difference between diversity values for the first and second years of implementation. when analysing the correlation between the abundance of individuals and dry plant biomass, there was no correlation observed (spearman’s r = -0.003; p = 0.97; n = 100). however, a significant positive correlation was found between species richness and dry plant biomass (spearman’s r = 0.39; p <0.01; n = 100). we also observed that the highest values for dead plant biomass, total abundance, species richness and diversity were not concentrated at, or near, the harvest period (fig 1 and 2). we did not find relationships between the distribution of monthly samples formed by the composition and frequency of ant species (fig 3) and the mean monthly total quantity of dead plant biomass (pearson’s r = 0.041; p =0.045 for axis 1 and r = 0.074; p = 0.69; n = 100 for axis 2). discussion community composition the composition of the two communities evaluated in this study was similar. the subfamily myrmicinae (12 species) was the most abundant and frequent in all sampled months. according to hölldobler and wilson (1990), this group is the largest subfamily of formicidae in number of species and variety of food and nesting habits, with wide adaptation to disturbed environments. furthermore, many authors have shown that most myrmicinae species are aggressive and they have efficient and massive recruitment, being of ten sampled in agrosystems (castro & queiroz, 1987; perfecto, 1991; lange et al., 2008; falcão et al.,2015).they are considered to be effective agents in the biological control of pests in crops (see fernandes et al.,1994, 2012). in this study, species of the genera solenopsis and pheidole were most frequent in both areas, occupying 38.36% of the samples in no-tillage and 45.74% in conventional farming. these two genera may be considered as dominant in this study. agrosystems, exhibiting lower structural complexity than natural environments, may be predisposed (castro & queiroz, 1987) because of their vulnerability to colonization by more generalist and aggressive species, such as species of the genera pheidole, solenopsis and ectatomma. on the other hand, the low frequency of species of the genera nomamyrmex and neivamyrmex may be explained by their nomadic behaviour (hölldobler & wilson, 1990). the genus brachymyrmex, which was also found in low frequencies, is common in neotropical forests, and their nests may be found in small cavities under epiphytes, or even in leaf litter. individuals of brachymyrmex have small body sizes and are hardly dominant species, but they can get resources because even in the presence of larger species, they show an opportunistic behavior (agosti et al., 2000). species of the genera camponotus, crematogaster and pseudomyrmex are commonly found foraging on vegetation where they can nest and establish mutualistic associations with their host (rico-gray & oliveira, 2007). as the herbaceous cropping system employed in this study is not considered an appropriate environment for these species, they were also response variable planting type year since implementation first second third fourth dead plant biomass conventional no-tillage 170.40aa 146.38abb 84.83cb 12325bb 202.24ca 403.42aa 269.39ba 373.07aa ant abundance conventional no-tillage 54.24ab 35.71cb 53.52ab 44.64ba 70.79ba 38.01ca 73.09aa 38.23cb table 2. mean of dead plant biomass and ant abundance based on planting type and year since implementation of no-tillage system. lower case letters represent differences in years and capital letters represent differences between planting types. wald test at 5% significance. response variable factor factor level richness planting type conventional no-tillage 2.52b 3.16a year since implementation first second third fourth 2.78b 2.76b 3.21a 2.58b diversity planting type conventional no-tillage 0.76b 0.87a year since implementation first second third fourth 0.81b 0.82b 0.91a 0.73c table 3. mean of species richness and diversity on planting type and year since implementation for no-tillage system. lower case letters represent differences between years and capital letters represent differences between planting types. wald test at the 5% significance. fig 3. ordination in two dimensions (axis 1 indicate dimension 1, and axis 2 dimension 2) of monthly samples using the nonmetric multidimensional scaling (nmds) of bray-curtis distances at relative frequencies of ants species. the black balls indicate conventional tillage and the white balls indicate the no-tillage. the 50 monthly observations of each area were numbered in chronological order, from november 1999 to december 2003. sociobiology 65(2): 130-137 (june, 2018) 135 found in small number in this study. other genera, such as pachycondyla and odontomachus, have solitary foraging, and exhibit nests with few individuals (hölldobler & wilson, 1990). therefore, these species are often found at low frequencies in studies that use traps. in addition, they were found only in the first year in both areas, and posteriorly only in the no-tillage. this fact may be related directly to the increased availability of food offered by the no-tillage. because of the solitary strategy, foraging individuals of these genera hardly dominate the resources and are considered to be opportunistic (silvestre et al., 2003). ant communities and habitat complexity the high similarity of the two ant communities evaluated in this study may be related to the fact that before the implementation of no-tillage, the two areas were conventional tillage. however, with the implementation of no-tillage, and increasing the amount of dry plant biomass in the soil in the no-tillage area, the properties of the communities (abundance of individuals and species richness) were being changed from the second year of implementation, becoming significantly differences in the third year of the study. the highest total abundance of individuals was found in the area with no-tillage. according to kladivko (2001), there is a wider variety of species with higher abundance on dead plant biomass in no-tillage than in conventional methods. the same authors showed that most organisms are sensitive to the soil preparation, due to physical changes and the amount of water that result from the incorporation of residues from the previous crop into the soil. even though the abundance of ants is not a metric used in studies about community structure due to variation in species behaviour (longino et al., 2002), in this study, this metric (abundance of ants) should be taken into consideration because the impact of soil management directly affects the survival of colonies and individuals. thus, the abundance of ants, as well as other organisms, relates to the impact of soil management on the fauna in agrosystems. this fact may be evidenced in this study during the first year. however, when we observe the abundance of ants in the traps over the months evaluated, it is impossible to demonstrate a clear difference between areas. this result may be related to the enormous biological variation of the species and their seasonal variations in resource foraging (see lach et al., 2009). however, when we compare the richness and diversity of species between the two areas, we observe a clear pattern of higher values for the no-tillage area. initially, both areas had similar richness, which were differentiated after the first year of study, reaching significant values in the third year. this result supports our hypothesis that dry plant biomass present in the soil, derived from the previous cultures, provides a more complex habitat when compared to conventional tillage. the relationship between the diversity of ant species and the structural complexity of the habitat was also evidenced in many studies (lassau & hochuli, 2004; ribas et al., 2003; dáttilo & izzo, 2012; see also rico-gray & oliveira, 2007). the main factors influencing the increase in ant diversity include a variety of nesting sites, the amount of food available, the foraging area and the competitive interactions (kaspari et al., 2000; ashford et al., 2013). the increased availability and variety of food enables the establishment of the richest ant communities and with more co-occurrence among species (i.e., less competition) (sharley et al., 2008; lange et al., 2008). this result was shown in this study based on the data on ant abundance and richness found in the samples. in a broader scenario, there are good reasons to relate the abundance and diversity of predatory insects, such as ants, to the natural conservation in agrosystems (see cividanes & yamamoto, 2002; kladivko, 2001; mas & verdú, 2003). this is because the greater abundance and richness of predators decrease populations of pest insects (hooper et al., 2005; christiaans et al., 2007) and reduces the use of pesticides (booij & noorlander, 1992; french et al., 1998). the greater the number of predator species with different diet and diverse foraging strategies, stronger will be the effects on herbivores and plants (e.g. hooper et al., 2005; naeem & li, 1998; schmitz & suttle, 2001). a meta-analysis of studies in natural and agricultural systems showed that the greater the diversity of predators, the lower the herbivore community, and consequently, the growth and reproduction of plants increase (schmitz et al., 2000). besides affecting the community of herbivores, ants may also reduce fungal pathogens, eliminating spores (de la fuente & marquis, 1999) or restricting the interactions between plants and disease vectors (khoo & ho, 1992). the increase in dry plant biomass in the soil led to a gradual increase in the richness and abundance of ants in no-tillage area when compared to conventional tillage. our study brings important information on ant communities in two cropping systems over a long period, providing interesting results that have not yet been reported in other studies. we showed that the no-tillage system results in an environment that promotes the greater diversity of ant species when compared to conventional tillage. additional similar studies should be conducted in order to better understand the dynamics of communities, both in agrosystems and natural systems. in addition, more focus should be given to the areas with agrosystems, since they are often left out, making studies in protected areas (natural) more common (martin et al., 2012). acknowledgements we thank f. r. marcelino, o. s. gossler, s. á. pereira, m. v. sant’ana, v. r. regiani and r. a. p. faria for help in field, w. dáttilo, e. alves-silva, and a. a. vilela by suggestions. we also thanks the fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect) for financial support. references agarwal, v.m., rastogi, n.s. & raju, v.s. 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(2013). effect of two agroecological management strategies on ant (hymenoptera: formicidae) diversity on coffee plantations in southwestern colombia. environmental entomology, 42: 194-203. doi: 10.1603/en11084 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1142sociobiology 64(1):105-110 (march, 2017) opportunistic strategies for capture and storage of prey of two species of social wasps of the genus polybia lepeletier (vespidae: polistinae: epiponini) introduction foraging activity is considered one of the most important and complex behaviors exhibited by social wasps (lima & prezoto, 2003), and it is dependent on the ability of the insects to interact with the environment, as well as the availability of essential resources to support the colony (gomes et al., 2007). most species of predatory social insects obtain the necessary protein resources for the development of the colony by capturing other insects, especially immature beetles, termites, and lepidopterans (rabb & lawson, 1957). social wasps collect nectar and pollen in flowers, and prey on other arthropods, especially lepidopteran caterpillars (prezoto et al., 2007). the essential skills required to capture the prey during foraging activity include the ability to recognize the target, capture and transport it. these attributes in social wasps have abstract foraging activity in social wasps is a complex behavior that involves the ability to locate and transport the necessary resources for the colony. this activity is opportunistic and generalist, sometimes adapting to the availability of resources when colonies are exposed to critical environmental conditions. this study aimed at evaluating the opportunistic behavior of two species of polybia wasps for prey capture and storage in the form of flights of winged individuals. five colonies of polybia occidentalis (olivier) and two colonies of polybia paulista (l.) were analyzed. six of them contained stored winged individuals of termites, and one contained winged specimens of ants. the results indicated that these two species practice an opportunistic foraging strategy and they are able to store large quantities of protein resources in their colonies. sociobiology an international journal on social insects kb michelutti1, erp soares1,, f prezoto2, wf antonialli-junior1 article history edited by marcel g. hermes, ufpr, brazil received 07 june 2016 initial acceptance 24 october 2016 final acceptance 14 december 2016 publication date 29 may 2017 keywords foraging, nectar storage, prey, swarming wasps. corresponding author kamylla balbuena michelutti programa de pós graduação em recursos naturais laboratório de ecologia comportamental (labeco) universidade estadual de mato grosso do sul cep: 79804-970, dourados-ms, brasil e-mail: kamylla_michelutti@yahoo.com.br been investigated in several studies, cited in the review of richter (2000). foraging activities present significant risks to the workers, due to the high energy costs and the greater exposure to predators that ultimately affects longevity (o’donnell & jeanne, 1995). workers of mischocyttarus mastigophorus (richards) are responsible for the execution of riskier or more stressful tasks, at higher rates, compared to the dominant insects (o’donnell, 1998). hence, when social wasps find an abundant resource they predominantly forage on this food source (santos et al., 2007). an abundant resource is available when termite and ant colonies release their winged forms, and there is documented evidence of social wasp foragers attacking termites during the flight of the winged individuals (mill, 1983). this foraging activity in social insects involves the recruitment of nest mates to an area where a higher concentration of workers is required, optimizing the acquisition of resources for the 1 laboratório de ecologia comportamental (labeco), universidade estadual de mato grosso do sul, dourados, mato grosso do sul, brazil 2 laboratório de ecologia comportamental e bioacústica (labec), universidade federal de juiz de fora, minas gerais, brazil research article wasps kb michelutti et al. – opportunistic strategies of social wasps polybia106 colony by increasing the number of foragers exploiting a particular resource (wilson, 1971). social wasps employ a solitary opportunistic foraging strategy (jeanne et al., 1995), in which foragers often return to forage in a fruitful location (richter, 2000). in order to optimize this form of foraging, signals can be exchanged between nest mates to facilitate the collection of resources to the colony (taylor et al., 2011). this behavior has been reported in colonies of polybia occidentalis (olivier) (hrncir et al., 2007; schueller et al., 2010). therefore it is evident that social wasps exhibit opportunistic generalist behavior, and that they can forage according to their previous experiences (richter, 2000). after locating and capturing prey, workers cut them into pieces, then chew the materials and distribute them to offspring (gomes et al., 2007). in some cases, they can store food source, such as nectar, together with parts of prey (silveira et al., 2005). according to ross and hunt (1988), nectar storage can be performed in two ways: i) storage in empty cells that can generally be observed during the cold season, as it has been described in colonies of polybia paulista (von lhering) (machado, 1984); ii) storage in cells occupied by eggs or small larvae, as observed in colonies of mischocyttarus drewseni (saussure) (jeanne, 1972), polistes simillimus (zikán) (prezoto & gobbi, 2003), and mischocyttarus cassununga (von ihering) (guimarães et al., 2008). the capture and storage of prey and other materials in social wasp nests is still a relatively unexplored subject, with only a few reported studies (machado, 1977; mill, 1983; gobbi et al., 1984; prezoto et al., 2005; taylor et al., 2011), so the aim of this study is to describe an opportunistic strategy of prey capture and storage in two species of social wasps of the genus polybia. material and methods seven colonies of two species of social wasps were collected and analyzed: five colonies of p. occidentalis and two colonies of p. paulista. the number of colonies and sampling sites are provided in table 1. the colonies were collected after 19:00 to ensure that all individuals were in the nest, using the method by machado (1984). all members of the colony were recorded following sacrifice by storage in a freezer (-20 ºc) for 24 hours. to identify the type, quantity, and location of prey, the nests were dissected layer by layer. evaluation of the influence of nest size on the manner and location of storage was performed by calculating the area of each layer using the formula a=π*a*b, where a and b are the semi-major and semi-minor axes of an ellipse, since the nests of this species can be regarded as being ellipsoidal. the number of foragers was estimated for each colony, using as a defining parameter the fact that these are workers between intermediate and older ages, as proposed by giannotti (1997). the relative ages of the workers were confirmed using the progressive pigmentation of the apodeme of the fifth gastral sternite, which according to richards (1971) provides an indication of age progression. after confirmation of the workers age, the total number of foragers was estimated for each colony. in two colonies of p. occidentalis it was only possible to identify the types of prey captured, because the nest structure was lost due to problems at the time of collection. the number of preys captured by each forager was estimated by calculating the total number of prey divided by the total number of foragers in each colony. a linear correlation test was used to evaluate if there was a relationship between the number of foragers in the colony and the number of stored prey. results and discussion stored winged termites were found in four colonies of p. occidentalis and two colonies of p. paulista, while winged ants were only found in one colony of p. occidentalis (table 1). this showed that these wasps employed an opportunistic strategy, taking advantage of the moments when colonies of termites and ants released winged individuals, in accordance with mill (1983), who documented social wasps preying on winged social insects in flight. winged termites of the subfamily apicotermitinae were found in all the colonies, with five termite species in colonies 1 and 5, four species in colony 6, two species in colony 2, three species in colony 3, and one species in colony 4. a species of nasutitermitinae was found in colony 3. winged ants were only found in colony 7 and could not be identified because only males were found. the preys were mainly stored in the periphery of the different layers of the combs, inside and outside of empty cells, with rare exceptions in the central regions. in most cases, one prey was stored per cell, although up to three preys could be found in a single cell in the comb. in some cases, they were stored mixed with nectar, both singly and in sets with more than two individuals (figures 1a and 1c). preys were also found stored in the form of combined masses of winged and nectar, table 1. species and collection date and location of colonies with stored termite and ant winged. colony specie date location 01* polybia occidentalis 08/11/13 ribeirão preto/sp 02* polybia occidentalis 16/11/13 nova alvorada do sul/ms 03 polybia occidentalis 26/11/13 dourados/ms 04 polybia paulista 01/01/14 ponta porã/ms 05 polybia paulista 04/02/14 dourados/ms 06 polybia occidentalis 07/02/14 dourados/ms 07 polybia occidentalis 09/03/14 ponta porã/ms *colony in which it was not possible to quantify the stored material. sociobiology 64(1): 105-110 (march, 2017) 107 located in the periphery of the nest (figure 1b). molan (1999) reported that chemical compounds present in the nectar, such as flavonoids and aromatic acids, can inhibit microorganisms responsible for decomposition. hence, this form of storage could reduce proliferation of pathogenic microorganisms within the colony. this would favor the opportunistic strategy, with collection of a relatively high number of preys that would not be consumed immediately, and in the long term could reduce the pressure to forage for proteins. most of the preys were lacking various body parts such as wings, head, legs, and antennae (figure 1d), suggesting that preys were processed by workers, or alternatively it might have been a consequence of the process of capture and transport of the prey, as noted in polybia sericea (olivier) by machado et al. (1988). the total area of the nest, the number of adults, the estimated load capacity of each foraging action, and the number of stored prey are presented in table 2. based on these data, it can be inferred that the weight of the prey was not responsible for its selection for capture, because workers of p. occidentalis are supposedly able to carry up to 75% of their own weight (hunt et al., 1987). it appears, therefore, that in this case, the main factor involved in prey selection was the opportunistic strategy. the winged were the most abundant prey at that moment, and for this reason the most captured and stored. this was supported by the load capacity (table 2), since hunt et al. (1987) noted that although foragers of p. occidentalis captured larvae of lepidoptera in most of their travels, at certain times of the year other items were abundant and were also brought to the colony. fig 1. termite winged stored inside cells (a), and in a mass mixed with nectar (b). termite winged stored inside the cells with nectar (c), and termite winged with parts of its body extracted by foragers (d). colony area (cm2) adults foragers prey number of prey by forager 03 2200.6 1291 665 20410 30.70 04 962.4 564 290 5070 17.50 05 4815.2 2825 1455 3510 2.40 06 2561.5 1503 774 700 1.00 07 350.3 205.5 106 1214 11.50 table 2. nest area, number of adults, number of foragers, number of stored prey, and load capacity of each forager. there was no significant correlation between the number of foragers and the number of prey stored in the colony (p=0.96, r=0.03); in other words, a colony with more foragers did not collect and store a greater number of prey. it is possible that the foraging activity could be related to the colony stage, with foragers taking fewer risks outside and performing more tasks in the nest during certain periods (machado et al., 1988). on the other hand, in mature colonies, where there are more individuals, the demand for resources is higher and the number of workers engaged in foraging is also higher. however, the results showed that relatively small colonies could capture and store more prey than larger ones. this might be related to the requirements of the colony, or the availability of winged prey in the vicinity of the colony. studies with epiponini colonies have shown that seasonal differences in foraging rates are associated with the stage of colony development (gobbi & machado, 1985, 1986; paula et al., 2003). a major factor that has not been evaluated in this study is the number of immature of each nest collected, since prey provide protein for the development of the offspring, so the amount of prey captured by foragers is an indirect measure of the number of immature and, consequently, the colony’s demand for protein (canevazzi & noll, 2011). however, we believe that since this material can be stored in the colony to meet future demand, maybe the number of preys stockpiled does not represent a relation with the number of immature present in the nest at that moment. kb michelutti et al. – opportunistic strategies of social wasps polybia108 it therefore appears that colonies of these social wasps adopt an opportunistic strategy, without any direct synchronicity with the swarming periods of the termites and ants. mill (1983) found that the most dramatic of all forms of termite predation was the opportunistic predation of winged when they left the nest for bridal or swarm flights. indeed taylor et al. (2011) suggested that p. occidentalis might display this type of opportunistic foraging strategy. besides, it was reported by johnson (1983) that an opportunistic strategy enabled stingless bee foragers to find and collect resources more efficiently. social wasps are sensitive to environmental changes and alter their behavior accordingly (o’donnell & jeanne, 1992). as shown in table 1, colonies of wasps with stocked material were collected in warmer and humid times of the year, which is an ideal condition for termite colonies to release winged (mill, 1983; medeiros et al., 1999; costa-leonardo, 2002). indeed, studies with colonies of p. paulista show that their foraging activity is mainly influenced by variation of temperature and relative humidity (canevazzi & noll, 2011). similarly, in termites, the triggers for the release of winged on reproductive flights include, temperature, humidity, soil moisture and atmospheric pressure (costa-lima, 1938), as well as seasonality, meteorological and social changes, colony size, and physiological factors (costa-leonardo, 2002). in p. paulista colonies, machado (1984) noted that the period of reproductive swarms occurred in the rainiest and hottest months, as observed in this study (table 1), since the months in which the colonies were collected were the hottest and most humid months in the state of mato grosso do sul (zavatini, 1990). foraging habits of social wasps are generally diurnal (jeanne, 1972; rocha & giannotti, 2007), while winged are typically released at dusk or at night (costa-leonardo, 2002), which could be a strategy to protect against aerial predators (mill, 1983). this highlights the nature of the opportunism displayed by the social wasps, because the predominantly diurnal wasps needed to adjust to unusual circumstances and extend their period of activity in order to take advantage of the availability of this protein source. our results indicate that p. occidentalis and p. paulista both adopted an opportunistic foraging strategy. they were capable of storing large quantities of protein resources in their colonies. these wasps seem to use nectar as an inhibitor of the proliferation of microorganisms responsible for decomposition, enabling protein resources to be stored in the nests for longer periods and thereby taking advantage of the relatively short period during which a specific food resource is available in large abundance. acknowledgments the authors are grateful to dr. danilo oliveira for identification of the termite subfamily and the number of species. financial support was provided by fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect); coordenação de aperfeiçoamento de pessoal de nível superior (capes) provided masters fellowships to the first and second authors; authors fp and wfaj acknowledges research grants from conselho nacional de desenvolvimento científico e tecnológico (cnpq). references canevazzi, n. c. de s. & noll, f. b. 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(1990). a dinâmica atmosférica e a distribuição das chuvas no mato grosso do sul. ph.d. dissertation, fflch/ usp, são paulo. doi: 10.13102/sociobiology.v64i4.1857sociobiology 64(4): 466-476 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 intraspecific variation of the composition of linear alkanes in social wasp mischocyttarus consimilis introduction colonies of social insects that carry out various tasks requires interactions between individuals and the availability of mechanisms of nestmates recognition (singer et al., 1998). the ability to recognize nestmates is extremely important to the success of these insects, so it prevents exploitation by individuals from other colonies (hefetz, 2007). during the evolution, the social insects developed a signals mechanism which is based on the variation of the cuticular chemical composition on its exoskeletons that also protect and support its bodies (ross & matthews, 1991). these compounds, besides the primary function of protection against microorganisms, toxins and desiccation (lenoir et al., abstract an important attribute of the evolution and maintenance of sociality in insects is their ability to distinguish members of their own colonies by means of chemical signals during their interactions. from this type of signals, the cuticular hydrocarbons, is responsible for intraspecific recognition stand out, among other functions. linear alkanes are indicated as the class of compounds that would be most involved in water retention in the body of insects, however, some studies have investigated their role as mediators of interactions. thus it is possible that there is significant intraspecific variation of its composition, so the objective of this study was to test the hypothesis that linear alkanes vary significantly among populations, castes and sexes, as well as between newly emerged females of different ages of the mischocyttarus consimilis zikán 1949 wasp. the samples were analyzed by gas chromatography with flame ionization detector and gas chromatography coupled to mass spectrometry. the results show that there is a significant variation between the chemical cuticular profiles of samples from different populations, as well as between castes, sex and newly emergent workers of different ages. therefore, it is possible to infer that this class of compounds may vary according to genetic differences between populations, but also by different environmental conditions. the differences between castes, sex and ages suggest that these compounds may also be involved in mediating interactions between nestmates. sociobiology an international journal on social insects ac ferreira1,4, ef neves2, ts montagna3, a mendonça3,5, cal cardoso3, wf antonialli junior3 article history edited by gilberto m. m. santos, uefs, brazil received 12 july 2017 initial acceptance 09 august 2017 final acceptance 19 august 2017 publication date 27 december 2017 keywords chemical signature; castes; cuticular hydrocarbons; gas chromatography; population variation. corresponding author programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados rodovia dourados-itahum, km 12 cidade universitária, c.p. 533 cep 79804-970, dourados-ms, brasil. e-mail: angel_bio1@yahoo.com.br 2001), also play a determining role in inter and intraspecific recognition as already investigated in social bees (nunes et al., 2009; abdalla et al., 2003); ants (antonialli-junior et al., 2007, 2008) and social wasps (butts et al., 1995; sledge et al., 2001; ferreira et al., 2012; neves et al., 2012, 2013). thus, in social insects within the same species, the qualitative profile of chs is similar among colonies, varying only in their relative proportions according to their origin (bonavita-cougourda et al., 1987), which functions as a “chemistry signature” colonial. thus, through the chemical signals and due to the contact not only with the adult individuals between themselves and with the off-spring, but also with the nest substrate, the social insects can identify their nestmates from others (gamboa, 1996). 1 universidade federal do triângulo mineiro, uberaba-mg, brazil 2 instituto federal de mato grosso do sul, naviraí-ms, brazil 3 laboratório de ecologia comportamental (labeco), centro de estudos em recursos naturais (cerna), universidade estadual de mato grosso do sul, dourados-ms, brazil 4 programa de pós-graduação em recursos naturais, universidade federal da grande dourados, dourados-ms, brazil 5 programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil research article wasps sociobiology 64(4): 466-476 (december, 2017) 467 ruther et al. (2002) supported the hypothesis that colony specific cuticular hydrocarbon profiles are involved in the phenomenon of nestmate recognition among social insects. according to shellman and gamboa (1982), newly emerged females acquire this ability trough the exposition with its own nest material, and espelie et al. (1990) noticed that the nest substrate is rich in hydrocarbon compounds produced by colony’s members, so to gamboa (1996), the nest odor is like a colonial signature to be a reference to nestmate recognition. lorenzi et al. (1999) observed that newly wasps of polistes biglumis (linnaeus, 1758) do not yet have the colonial odor after their emergence. panek et al. (2001) noticed that the cuticular hydrocarbons present at the polistes fuscatus (fabricius, 1793) newly emerged wasps differ in composition from those present at the older wasps with 3 days of emergence, the same was observed by lorenzi et al. (2004) in polistes dominula (christ, 1791) wasps. the cuticular surface of p. dominula is most susceptible to acquire chemical components from the environment at the initial stage after emergence and this ability is reduced by the time, what means that the older wasps do not incorporate new chemical substance in their cuticles, so their colonial signature can not be altered (lorenzi et al., 2004). in this context, social insects recently emerged can be transferred with success between co-specified colonies (gamboa, 2004). polybia occidentalis olivier, 1791 with 24 hours of emergence are easily accepted by unknown colonies (o’donnell & jeanne, 1992). the newly emerged wasps are treated as nestmates by members of the others colonies because they do not have colonial odor from their original colony (gamboa, 2004). studies such as those by dani et al. (1996) and lorenzi et al. (2014) investigated the importance of branched alkanes among other cuticle compounds as the main responsible for signaling intraspecific interactions. however, although studies have identified linear alkanes as those most involved in the waterproofing of the cuticle to avoid insect dehydration, tannurenascimento et al. (2007) verified in their study with polistes satan bequaert, 1940 that linear alkanes had a greater abundance in relation to the other compounds to mediate the interactions. therefore, since there are few studies that have already investigated the intraspecific variation of linear alkanes in social wasps, the objective of this study was to test the hypothesis that linear alkanes vary significantly among populations, caste and sex, as well as among newly emerged females of different ages of the species mischocyttarus consimilis material and methods sample collection for the analysis of population variation of m. consimilis were collected 12 colonies from 6 different populations sampled, in different types of environment in the state of mato grosso do sul, brazil (fig 1 and table 1). all colonies were collected with the aid of plastic bags wrapped around the nest were then detached from substrates. after collect, all wasps were sacrificed and preserved by freezing, thus avoiding the use of any type of fixative or chemical preservative which could react with the chemicals of the cuticle. to evaluate the variation of the profile of linear alkanes between sex and caste were used eight queens, 19 males and 77 workers of different colonies. at a later time, there was an analysis to determine the chemical profile of the cuticle fig 1 map of brazil highlighting the municipalities of mato grosso do sul where populations of mischocyttarus consimilis wasps were collected. ac ferreira et al. – composition of linear alkanes in wasp mischocyttarus consimilis468 according to the worker caste and gynes, and the workers were separated into two groups; workers 01 (pre-emergency) which were the workers first in the colony; and workers 02 that were produced in the middle of the colony development (phase known as ergonomic) and gynes that are produced in the final stage of the colony; so were collected 13 females of the group workers 1; 12 individuals of the group workers 2 and 13 gynes, all collected in a single municipality, at the case dourados-ms, to minimize environmental differences. to evaluate when the composition of linear alkanes is completed after emergence were evaluated 10 workers of different age, from newly emerged up to 5 days after emergence, based on the results achieved by panek et al. (2001). to minimize the possible environmental variations all the workers were collected from different colony nesting in the city of dourados, ms. to determine wasps’ age, the colonies of m. consimilis were daily mapped according to the methodology proposed by giannotti (1998). the newly emerged wasps were marked on the thorax with non-toxic ink in order to monitor their age. during these daily observations was also possible to see which individuals presented behaviors of dominance and subordination, according to the study by torres et al (2012) with division of labor in this species. chemical and statistical analyses all the insects used for both analyses were anesthetized, killed, and preserved by freezing. for the analyses of the cuticular hydrocarbon profile, the gaster was extracted from each individual, individual, it was standardized using the gaster, since the intention of the study was to evaluate the variations between samples and not describe the compounds. the cuticular hydrocarbons of gaster were extracted in 2 ml hexane (hplc grade, vetec química fina ltda., rio de janeiro, brazil) in an ultrasonic bath for 30 min. this process was performed in duplicate. in sequence, the 2 fractions were united and the solvent dried. the dried extract was dissolved in 100 μl hexane for analysis using gas chromatography with flame ionization detector (focus, gc, thermo scientific san jose, usa). the gas chromatography with flame ionization detector (gc–fid) was equipped with an ov-5 capillary column of ohio mark (ohio, usa) with a composition of 5% phenyldimethyl-polysiloxane in a fused silica capillary with dimensions of 30 m in length x 0.25 mm in diameter x 0.25 μm film thickness. nitrogen was used as the carrier gas at a constant pressure of 0.8 bar. the analyses were performed in splitless mode with 1μl injection, injector temperature population sample collection geographical coordinates type of environment 01 dourados 22º13’6”s, 54º48’20”w urban areas with rural surroundings 02 sidrolândia 20º55’55”s, 54º57’41”w urban areas with rural surroundings 03 eldorado 23º47’13”s, 54º17’01”w urban areas with rural surroundings 04 bodoquena 20º32’19”s, 56º42’54”w rural area farm 05 maracaju 21º36’52”s, 55º10’06”w forest area 06 batayporã 22º17’96”s, 53º77’02”w urban area table 1. collection sites of mischocyttarus consimilis, indicating the geographical coordinates and types of environment. of 250°c, and detector temperature of 320°c. the oven was programmed to reach a final temperature of 320°c, starting from an initial temperature of 50°c and rising at a rate of 5°c/ min to 85°c, then rising at a rate of 8°c/min to 300°c, which was held for 15 min, and then raised to 320°c at a rate of 10°c/min and held for 35 min. the chromatograms were registered using the chrom quest 5.0 program and analyzed with the workstation chrom data review program. the chemical compounds were identified based on linear hydrocarbon standards of c7 to c40 (4 μg/ml in hexane) through comparison with retention times. the concentration of each compound was calculated using relative area, in percentage, referring to the peak areas of alkanes c7 to c40. the analyses of linear alkanes present on the m. consimilis cuticle were performed on the cg-fid and the differentiation analysis to these studies was completed using the peak areas of the corresponding c15 to c30 hydrocarbons, which are common to all of the individuals. fig 2. scatter plot using the peak areas obtained from linear alkanes by gc-fid present in the cuticle of the species of wasps mischocyttarus consimilis in colonies of different populations. sociobiology 64(4): 466-476 (december, 2017) 469 for the statistical analyzes the values of the percentage relative area of linear alkanes, analyzed by gc-fid, were subjected to stepwise discriminant function analysis of statistica 7 program and indicated by selecting a set of variables if the analyzed groups differ or not, the wilk’s lambda statistic is used as a measurement of the difference between the groups, in which values close to 0 indicate that the groups do not overlap, whereas values close to 1 indicate high overlap among groups and a lack of significant difference (quinn & keough, 2002). results the discriminant analysis shows that there are significant differences in the composition of linear alkanes among populations with wilk’s lambda = 0.003, f = 70.313 and p<0.0001, the first canonical root explains 52.25% of the results and the second canonical root 90.90%. linear alkanes are most significant for this discriminant analysis are shown in table 2. the compounds significant for separation of populations on the scatter plot were: n-octadecane for population 01; n-pentadecane and n-octacosane for the population 02; n-nonadecane to population 03; n-tricosano to population 04; n-hexacosane to population 5 and n-heptadecane for population 6 (table 2). the results show that there are significant differences between the composition of linear alkanes between the sex and castes of this species (fig 3 and table 3), the analyzes were significant with wilk’s lambda = 0.065, f = 18.606, p<0.001, canonical root 1 of 83.4% and canonical root 2 of 16.6%. the linear alkanes responsible for that are c15, c17, c19, c21, c23, c27, c28, c29 and c30 (table 3). fig 3. scatter plot using the peak areas obtained from linear alkanes by gc-fid present in the cuticle of the species of wasps mischocyttarus consimilis for caste and sex. and the canonical root 2 14%. the linear alkanes responsible for the separation among the gynes and the workers can be see in table 4. the discriminant analysis in function of the values found on linear alkanes on wasps with 1 to 5 days after emergence showed wilk’s lambda=0.029 and f=5.525 significant differences between the hydrocarbon profiles (p<0.0001), with canonical root 1 explaining 72% and along with root 2, 94% of the data. the fig 5 shows the 3 separated groups of individuals, one with 1 to 3 days and the others with 4 and 5 days after emergence. the table 5 shows the statistical values of each linear alkane evaluated and the responsible ones for this distinction among the newly wasps. fig 4. scatter plot using the peak areas of linear alkanes obtained by gc-fid present in the cuticle of wasps of the species mischocyttarus consimilis identified for gynes and workers. discussion the results show that there is significant variation in the composition of linear alkanes between the different populations (fig 2 and table 2). to explain the variations found, the authors (liang & silverman 2000; sorvari et al., 2008; zweden et al., 2009) discuss the importance of genetic factors, but above all the environmental to compose the cuticle of these insects. ridley (2006), in fact, discusses that the geographic variation exists in all species and tend to be caused by adaptations to the environment, which suggests that environmental conditions are relevant for analyzing population differences. therefore, in addition to genetic factors determine part of the chemical profile, exogenous factors also play an important role. according to the results (fig 2), in some cases the grouping appears to be geographic distance, but on the other hand, others may be due to the sharing of similar nidification environments. therefore, as already discussed in other studies (gamboa et al., 1986; singer et al., 1998; dapporto et al., 2004a; dapporto et al., 2004b), it appears that both genetic and environmental factors are important to explain the variation of these compounds in these samples. there are significant differences between the composition of linear alkanes of cuticle of different categories of females (fig 4) with wilks’s lambda = 0.000, f = 32.40, p<0.0001, with the canonical root 1 explaining 86% of results ac ferreira et al. – composition of linear alkanes in wasp mischocyttarus consimilis470 daportto et al. (2004a) observed quantitative and qualitative differences between the cuticular chemical profiles between three populations of the species p. dominula; this study also found greater similarity between these compounds in colony nesting nearest and attributed these results to both environmental components and genetic similarities. gamboa et al. (1986), studying p. fuscatus, discusses the importance of exogenous factors to determine the chs profile, especially the diet. furthermore, studies performed with ants show that the quantity and quality of food is also responsible for the changes of chs, since influences the chemical recognition during intraspecific interactions in ants (le moli et al., 1992; richard et al., 2004; liang & silverman, 2000; sorvari et al., 2008), suggesting that diet is one of the factors that certainly contribute to the construction of the chemical signature of the colony. bernardi et al. (2014) also observed a significant influence of external factors on the variation of chs among ants under natural diet and controlled in the specie ectatomma brunneum smith, 1858. ch wilk’s lambda p< canonical coefficients % relative area root 1 root 2 queens males workers c15 0.074 0.001 -0.563 0.111 0.084 ± 0.043 0.092 ± 0.053 0.159 ± 0.112 c16 0.065 ns c17 0.105 0.001 1.561 -0.972 0.319 ±0 .135 0.550 ± 0.159 0.562 ± 0.371 c18 0.068 ns c19 0.113 0.001 -1.350 0.151 0.233 ± 0.149 0.270 ± 0.068 0.420 ± 0.338 c21 0.070 0.028 -0.253 -0.296 0.930 ± 0.261 12.675 ± 10.921 5.028 ± 9.651 c23 0.223 0.001 1.235 0.314 1.706 ± 2.284 36.416 ± 8.423 6.592 ± 10.506 c24 0.069 ns c25 0.066 ns c26 0.068 ns c27 0.101 0.001 -0.433 1.778 38.108 ± 3.528 17.441 ± 7.510 27.55 ± 13.12 c28 0.095 0.001 1.351 -3.832 2.148 ± 0.800 1.379 ± 0.731 3.736 ± 2.258 c29 0.070 0.021 0.211 0.659 47.563 ± 4.545 17.741 ± 5.772 43.737 ± 16.601 c30 0.084 0.001 -1.945 0.968 0.829 ± 0.522 0.456 ± 0.157 1.979 ± 1.885 ch = cuticle hydrocarbon; ns = not significant. table 3. statistical values of discriminant analysis of the peak relative areas of linear alkanes obtained by gc-fid presents on the cuticle of mischocyttarus consimilis wasps identified by castes and sex. ch wilk’s lambda p< canonical coefficients % relative area root 1 root 2 population 01 population 02 population 03 population 04 population 05 population 06 c15 0.004 0.000 -0.176 0.592 0.123 ±0.085 0.180 ±0.060 0.095 ±0.035 0.132 ±0.039 0.197 ±0.090 0.120 ±0.117 c16 0.003 ns c17 0.007 0.000 0.612 1.888 0.384 ±0.182 0.456 ±0.152 0.423 ±0.241 0.506 ±0.187 0.608 ±0.319 0.643 ±0.279 c18 0.004 0.000 0.837 -6.321 0.550 ±1.053 0.162 ±0.057 0.205 ±0.110 1.900 ±4.215 0.681 ±1.424 0.182 ±0.085 c19 0.005 0.000 -0.667 -0.601 0.322 ±0.274 0.403 ±0.143 0.683 ±0.416 0.261 ±0.155 0.456 ±0.244 0.421 ±0.201 c21 0.003 ns c23 0.016 0.000 -1.321 0.266 6.853 ±10.487 12.845 ±16.340 34.616 ±9.759 42.742 ±3.262 2.527 ±2.517 1.270 ±0.707 c24 0.003 ns c25 0.003 ns c26 0.004 0.000 2.840 -0.646 1.470 ±1.331 0.415 ±0.260 0.592 ±0.284 2.065 ±4.669 2.117 ±2.313 1.579 ±1.421 c27 0.003 ns c28 0.004 0.000 -2.221 0.715 3.579 ±1.527 4.052 ±2.018 1.075 ±0.512 2.757 ±5.977 4.708 ±1.404 3.089 ±1.848 c29 0.003 ns c30 0.003 ns ch = cuticle hydrocarbon; ns = not significant. table 2. statistical values of the discriminant analysis and mean concentration values in % relative area for the linear alkanes that distinguishing populations mischocyttarus consimilis. sociobiology 64(4): 466-476 (december, 2017) 471 another aspect to be taken into account are the nest compounds that help to compose the chemical signature of the colonies (singer et al., 1992; singer & espelie, 1996; lenoir et al., 2009). thus, as part of the composition of the nest depends on plant material collected by the wasps in plants around the nest, it is expected that there would be a variation of these compounds depending on the material provided by the different plant materials available in each type of environment. specifically as the variation of linear alkanes, according to other studies, these compounds appear to have a significant role in water loss prevention in the insects (armold & regnier, 1975; gibbs, 1998, 2002). therefore, the composition of linear alkanes is directly related to prevailing conditions, as for example, in hot and dry climates there is an increased production of long-chain n-alkanes, which prevents desiccation (chapman et al., 1995; wagner et al., 2001). in general, the alkanes have a function more directed the prevention of desiccation, due to their high melting points, different from alkenes which are more related to chemical communication (gibbs, 2002). thus, it is also possible that the differences and similarities found in our results are at least in part due to the small climatic differences between the microhabitats of the nesting sites. the results show that there are significant differences between the composition of linear alkanes between workers, queens and males (fig 3), therefore, there is a variation according to the sex, but also according function that they realize in their colony. the variation according to gender is probably because females originate from fertilized eggs while the males originate from unfertilized eggs being haploid. the compound responsible for the separation of males in the dispersion diagram, occurring in higher contents is the n-tricosane (table 3). this linear alkane was also found in a higher concentration in the cuticle of the bee males melipona quadrifasciata lepeletier, 1836 being one of the compounds responsible for the separation of the groups, and as in our results this compound appeared in a lower concentration in the queen’s cuticle (borges et al., 2012). in fact, there should be sex flags in colonies of social wasps, since cotoneschi et al. (2009) observed the capability of workers to distinguish the sex of their nest mates through cuticular hydrocarbons in p. dominula. kudô and zucchi (2006) studying colonies of p. paulista assessed that newly emerged males were accepted in other colonies, until a certain age, when they begin to acquire specific chemical odors from their own colony. the compounds responsible for distinguishing queens and workers foram n-heptacosane (c27) and n-nonacosane (c29), which are more abundant in the cuticle of queens (table 3). these compounds were also found by hora et al. (2008) in the cuticle of ectatomma tuberculatum (olivier, 1792) and the authors suggested that the content of these compounds may signal the insemination of queens. in fact, van zweden et al. (2013) found the n-heptacosane compound in the highest concentration in the cuticle of the wasps of the dolichovespula saxonic (fabricius, 1793) wasp. the differences between these chemical signals in the cuticle of the two castes are directly related to the physiological and behavioral differences. the queens differ from the workers in the ovarian activity and in the repertoire of tasks realizing in the colony (billen & morgan, 1998), such as physical dominance, ch wilk’s lambda p< canonical coefficients % relative area root 1 root 2 gynes workers 02 workers 01 c15 0.000002 0.293970 -0.030468 -0.290215 0.71923±0.09768 1.6525±0.10001 0.46077±0.10332 c16 0.000003 0.013018 -0.132770 -0.181498 1.46615±0.09751 1.51167±0.12386 0.23231±0.044 c17 0.000002 0.270816 0.016698 -0.057446 0.88538±0.04446 1.22333±0.09059 1.09538±0.17309 c18 0.000002 0.022139 0.050672 0.025981 1.02231±0.0744 1.36667±0.22876 2.26769±0.34562 c19 0.000003 0.013462 0.004956 0.016801 2.03462±0.08412 1.91417±0.30318 2.18154±0.43437 c20 0.000002 0.322367 -0.054703 0.050502 2.63077±0.10444 1.64417±0.09913 1.36923±0.35323 c21 0.000002 0.062814 0.037402 0.175244 1.69154±0.07559 1.11167±0.10232 2.12±0.16921 c22 0.000002 0.034956 0.059754 0.084648 2.14769±0.10826 2.09917±0.15078 3.02385±0.17624 c23 0.000004 0.000646 0.013099 0.112682 4.26846±0.22248 2.82667±0.17068 4.72692±0.63617 c24 0.000003 0.008305 0.039653 0.064805 5.85385±0.39536 5.58833±0.3865 7.79231±0.64778 c25 0.000008 0.000000 -0.378256 0.085745 9.57385±0.13295 5.66917±0.16071 2.34385±0.24602 c26 0.000002 0.217125 0.011529 -0.046340 2.31±0.08803 3.03417±0.52153 2.71462±0.21046 c27 0.000003 0.011908 -0.044572 0.020945 8.44385±0.29202 6.42667±0.74986 5.21769±0.92065 c28 0.000002 0.220214 0.032369 -0.098701 5.55385±0.53285 8.24583±0.26834 7.36615±0.66523 c29 0.000002 0.032951 0.012388 0.108955 7.08923±0.37682 5.33667±0.38382 7.63±0.69082 c30 0.000002 0.120229 0.020553 0.080349 6.72615±0.5637 5.5475±0.40779 7.87±0.73228 ch = cuticle hydrocarbon. table 4 statistical values of discriminant analysis of the peak relative areas of linear alkanes obtained by gc-fid presents on the cuticle of mischocyttarus consimilis wasps identified by gynes and workers. https://pt.wikipedia.org/wiki/lepeletier https://pt.wikipedia.org/wiki/1836 https://en.wikipedia.org/wiki/johan_christian_fabricius ac ferreira et al. – composition of linear alkanes in wasp mischocyttarus consimilis472 food solicitation and oviposition realized by the queens, adultadult trofhallaxis, alarm, foraging realized by the workers, so the queens remain more time in the nest developing the activities of dominance hierarchy and oviposition while the workers were engaged in maintenance activities, defense and success of the colonies (montagna et al., 2009; torres et al., 2012). sledge (2001) noted that when removed the dominant females of p. dominula colonies, the subordinate assumed this position and, consequently, had changes in their ovarian developing and in their cuticular hydrocarbons what became similar to those removed, whose chemical profiles were rich in alkanes and metyl-alkanes. gynes and workers also showed significant differences between the composition of linear alkanes your cuticles. gynes and workers 01 presented higher contents of n-tricosane compared to workers 02, (fig 4 and table 4). as there are also differences between the categories of workers, since the n-docosane compound occurs in a higher concentration in the workers 01, whereas the n-pentadecane and n-hexadecane compounds appear in higher concentration in the cuticle of the workers 02 (fig 4 and table 4), probably for the same reason, since their physiological conditions and their functions in the colony may be distinct, although here we have not performed physiological and behavioral analyzes. according to dapporto et al. (2004a) in p. dominula there are variations qualitative and quantitative between the cuticular composition of the alphas founding compared at subordinate, probably due to the same reasons discussed above. therefore, the cuticular linear alkanes are important compounds used for the identification of individuals (singer et al., 1998), which can vary according to their tasks in the colony (leal, 2005). fig 5. scatter plot using the peak areas of linear alkanes obtained by gc-fid present in the cuticle of wasps of the species mischocyttarus consimilis with 1 to 5 days after emergence. ch wilk’s lambda p< canonical coefficients % relative area root 1 root 2 1 day 2 days 3 days 4 days 5 days c15 0.01 0.000 -0.60 -2.50 0.08 ± 0.03 0.11 ± 0.06 0.15 ± 0.07 0.19 ± 0.05 0.06 ± 0.02 c16 0.00 ns c17 0.00 ns c18 0.00 ns c19 0.00 ns c20 0.01 0.00 1.18 0.03 7.63 ± 7.68 3.46 ± 1.58 3.65 ± 1.08 19.90 ± 5.25 47.91 ± 9.51 c21 0.00 ns c23 0.01 0.03 0.75 1.28 37.21 ± 7.27 35.26 ± 10.26 42.14 ± 12.56 24.70 ± 11.74 18.49 ± 12.45 c24 0.01 0.01 0.52 2.04 0.71 ± 0.16 0.77 ± 0.41 0.91 ± 0.27 0.58 ± 0.27 0.18 ± 0.05 c25 0.01 0.01 -0.57 -2.70 10.61 ± 2.25 10.81 ± 2.03 10.67 ± 1.08 10.42 ± 1.33 5.86 ± 1.97 c26 0.00 ns c27 0.01 0.01 0.96 1.31 11.00 ± 4.53 13.92 ± 6.15 14.90 ± 5.63 17.37 ± 5.69 14.06 ± 6.00 c28 0.00 ns c29 0.00 ns c30 0.00 ns ch = cuticle hydrocarbon; ns = not significant. table 5. statistical values of discriminant analysis of the peak relative areas of linear alkanes obtained by gc-fid presents on the cuticle of mischocyttarus consimilis wasps with 1 to 5 days after emergence. however, despite our results show that linear alkanes too may signal the different caste and sex, lommelen et al. (2006) reports that branched alkanes appear to be more involved in signaling during interspecific interactions, as this class has a high complexity molecular, showing a high potential to encode information (leconte & hefetz, 2008; blomquist & bagnères, 2010), and linear alkanes would be more involved in building a barrier to prevent water loss (armold & regnier, 1975). according to fig 5, it is possible to affirm that the emerged wasp of the genus m. consimilis acquires more complete chemical composition of linear alkanes after the fourth day after the emergence. the compounds which differ sociobiology 64(4): 466-476 (december, 2017) 473 the age groups are n-pentadecane (c15); n-eicosane (c20); n-tricosane (c23); n-tetracosane (c24); n-pentacosane (c25) and n-heptacosane (c27). at the firth day emerged, the cuticular hydrocarbon profile of the wasp is distinct, showing in higher concentration of the compound n-eicosane (c20) and in lower concentration of the alkanes c23, c24 and c25 than the others age-groups (table 5). neves et al. (2012) had shown that the workers of this specie take the same time to complete a cuticular chemical composition, however, they used another technique, ftir-pas (fourier transform infrared-photoacoustic spectroscopy), which evaluates in general the variation all compounds found in the cuticle of insects. so, part of this significant variation between workers of different ages also refer to linear alkanes. in addition, kudô 2007 suggests that in colonies of p. paulista the newly emerged workers acquire chemical signatures specific to their colonies within a couple of days after emergence and, in experiments of transfer of individuals between colonies, the acceptance rate of young wasps depends on the stage of the receiver colony (kudô et al. 2010). in the same species, workers are able to distinguish larvae from their own colony from intruders, suggesting that even larvae recognition by workers might be mediated by specific colonial signals (kudô et al., 2017). in fact, while the colonial signature was not yet defined, the wasps can probably be accepted by members of others colonies, as observed with p. fuscatus wasps which not rejected strange newly individuals, but threw the foreign older ones with 3 days of emergence out their nests (panek et al., 2001). the same was noticed with the genus ropalidia marginata lepeletier, 1836, but with the age of 6 days after their emergence, more than that the individuals were rejected (arathi et al., 1997). the change in composition of linear alkanes, in this sense, demonstrates again how are relevant, as well as other compounds to mediate the interactions of social wasps’ colonies. thus, our results allow us to confirm the hypothesis that there is a significant variation between the composition of linear alkanes among populations, castes and sexes, as well as between newly emerged females of different ages. part of this variation may be a function of genetic factors, such as in the case of samples from different populations, between the sexes and the environment, as well as in the case of populations. the differences between the composition of females of different castes and categories, as well as ages, are probably related to the physiological differences and tasks they perform in their colonies. therefore, in these cases, the results allow to infer that this category of compound may also be involved in the mediations of the interactions between nestmates. acknowledgments the authors are grateful the coordenação de aperfeiçoamento de pessoal de nível superior (capes), the fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect). the authors also thank conselho nacional de desenvolvimento científico e tecnológico (cnpq) (wfaj grant number 3079 98/2014-2), (calc grant number 310801/2015-0). references abdalla, f. c., jones, g. r., morgan, e. d. & cruz-landim, c. 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(2009). disentangling environmental and heritable nestmate recognition cues in a carpenter ant. journal of insect physiology, 55: 158-163. doi: 10.1016/j.jinsphys.2008.11.001 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1053sociobiology 63(3): 941-949 (september, 2016) seasonal analysis of taxonomic and functional diversity of poneromorph ant assemblages in the amazon forest introduction the interaction between environmental conditions (e.g., temperature and humidity) and resource availability influences ecological communities (townsend et al., 2006). more studies are needed to understand how spatial and temporal variations in environmental factors affect communities of different types of organisms. although tropical rainforests are not considered restrictive environments in terms of conditions and resources (townsend et al., 2006), variations in geology, relief, or climate can influence communities (peroni & hernandez, 2011). a phenomenon is considered seasonal when it occurs in a predictable way at the same time every year (wolda, 1988). although climate seasonality is lower at low latitudes, the brazilian amazon has two climate types with marked abstract the present study aimed at assessing the effects of climate seasonality on poneromorph ants in the brazilian amazon, by studying variations in composition, richness, and taxonomic and functional diversity. the study was carried out in the tapirapé-aquiri national forest, southeastern pará state. we collected poneromorph ants in three areas of native forest with pitfall traps and sardine baits on the ground and vegetation, in two dry and rainy seasons. we collected 46 species of poneromorph ants, which belong to two subfamilies and eleven genera. the species composition, richness and taxonomic diversity did not vary significantly between seasons. there was no significant difference in the frequency of species of functional groups between dry and rainy seasons. there was no significant difference in the average richness and average diversity of functional groups between the dry and rainy seasons. in our study we found no seasonal differences in composition, taxonomic and functional richness and diversity of poneromorph ants in the amazon, which is useful for future studies that aim at using those ants as bioindicators. in addition, the identification of the species made in the present study has special relevance as it contributes to advance the knowledge of poneromorph ant diversity in the amazon. sociobiology an international journal on social insects lpc pereira1, fs almeida2, ab vargas3, ms araújo4, aj mayhé-nunes1, jm queiroz1 article history edited by gilberto m. m. santos, uefs, brazil received 28 april 2016 initial acceptance 26 may 2016 final acceptance 02 august 2016 publication date 25 october 2016 keywords biodiversity, formicidae, protected area. corresponding author jarbas marçal de queiroz depto. de ciências ambientais instituto de florestas/ufrrj br 465, km 7, seropédica-rj, brasil e-mail: jarquiz@gmail.com seasonality (souza filhoet al., 2005; alvares et al., 2013). variations in rainfall throughout the year may influence organisms, but little is known about those effects, as most studies involve sampling restricted to a single season. this information might help understand how ecological communities respond to environmental changes (leivas & carneiro, 2012; schmitz et al., 2014). in the amazon, ants and termites represent threequarters of the biomass of the soil fauna (fittkau & klinge, 1973) and play important ecological roles in the functioning of this ecosystem (hölldobler & wilson, 1990; fowler et al., 1991; gotwald, 1995). although poneromorph ants are not a monophyletic group, they are ecologically similar enough to be analyzed together (jimenez et al., 2008). in spite of being frequently called hunting ants (jimenez et al., 2008), 1 universidade federal rural do rio de janeiro, seropédica-rj, brazil 2 universidade federal rural do rio de janeiro, três rios-rj, brazil 3 centro universitário de volta redonda (unifoa), volta redonda-rj, brazil 4 universidade estadual paulista, são josé do rio preto-sp, brazil research article ants lpc pereira et al. – taxonomic and functional diversity of poneromorph ant assemblages942 poneromorph ants have a diversified diet, which includes live and dead prey, fruits, seeds, and liquids produced by plants or obtained from other insects (medeiros, 1997; lattke, 2003; jimenez et al., 2008). some species of poneromorph ants are important seed dispersers (passos & oliveira, 2002, 2004; almeida et al., 2013). they also have diversified nesting habits, including species that build nests in the soil, leaf litter, and trees (lattke, 2003; jimenez et al., 2008; wilkie et al., 2010). previous studies carried out in the amazon tested the effect of factors, such as the amount of leaf litter and competition, on the ant community (bastos & harada, 2011; baccaro et al., 2012), but the influence of climate is still poorly known. seasonal variations in climate affect insects directly and indirectly (wolda, 1988). climate conditions affect ant foraging and their development speed (hölldobler & wilson, 1990; vogt et al., 2003; almeida et al., 2007). indirectly, climate factors affect resource availability to ants as they interfere with primary productivity (silva et al., 2014). low resource availability can lead to higher interspecific competition and species exclusion, and is one of the regulating factors of ant communities (lach, 2005; carpintero & reyes-lópez, 2008). few studies have assessed the effects of environmental factors on the functional diversity of ants (e.g. groc et al., 2014) and most of them were carried out by sampling the community at a single season. hence, the present study aimed at assessing the effects of climate seasonality on poneromorph ants in the brazilian amazon, by studying variations in composition, richness, and taxonomic and functional diversity. material and methods study area the present study was carried out in the tapirapéaquiri national forest, which has an area of 196,351.42 ha and is located within the municipalities of marabá and são félix do xingú, southeastern of pará state. the climate is classified as tropical rainy type awi in the köppen system, with monthly temperatures above 18ºc, annual rainfall from 2,000 to 2,400 mm, and a dry season in winter (rolim et al., 2006). the rainy season occurs from december to april and the dry season from june to october (mma, 2006). the average monthly temperature in marabá and são félix do xingú from 1973 and 1990 varied from 24.3 to 26.9°c and from 24.6 to 25.4 °c, respectively (embrapa, 2015). the average monthly rainfall varied from 21 (driest month) to 387 mm (rainiest month) in marabá and from 17 to 322 mm in são félix do xingú (embrapa, 2015). sampling we collected poneromorph ants in three areas of native forest (a1, a2, and a3) in the dry seasons of 2009 (dry season i) and 2010 (dry season ii), and the rainy seasons of 2010 (rainy season i) and 2011 (rainy season ii). in the areas a1 (05º49’s, 50º30’w) and a2 (05º49’s, 50º29’w) we determined four sampling sites inside the forest, at distances of 0, 100, 500, and 1,000 m from the edge. in the area a3 (05º46’s, 50º31’w), we determined only one sampling site inside the forest at 500 m from the edge. at each distance we marked a 10 x 90 m plot to arrange the traps. during the sampling session of january 2011 (rainy season ii), it was not possible to sample the area a1, due to access problems caused by the seasonal rains; therefore, the sampling effort was lower in that season. in each sampling site, we distributed pairs of sardine baits on pieces of white paper on the ground and on the vegetation at approximately 1 m above the ground. the pairs of baits were arranged on the edges of the plots, 10 m away from one another, in a total of 20 baits on the ground and 20 baits on the vegetation per plot. the baits remained in the field for 1 h and after that each paper containing baits was individually transferred to plastic bags for sorting of the material in the laboratory. in the same plots, after sampling with baits, we installed pitfall traps, at every 10 m, which were made of 300-ml plastic cups containing 100 ml of alcohol 80%, in a total of 20 traps per plot. fortyeight hours later we removed the traps, placed their contents in plastic recipients, and took them to the laboratory for sorting and replacing of the fixing liquid with alcohol 70%. we identified ant genera following the key of bolton (1994) and classified subfamilies following bolton (2003). we also followed the classification proposed by schmidt & shattuck (2014). we identified ants to the species following available dichotomic keys published in taxonomic revisions. in addition, we compared the collected ants with identified specimens deposited in the costa lima entomological collection (cecl) at the institute of biology of the federal rural university of rio de janeiro. all vouchers were deposited in the same collection. we obtained from the literature information on diet and nesting sites of the species collected. we classified each species as omnivorous, predator, or detritivorous. we observed that poneromorph ants make nests in the underground (in the soil or under rocks), in the leaf litter (nesting on the soil in wood, branches, seeds, fruits or among leaves), or on the vegetation. however, it was not possible to determine the diet or nesting site of some species. we classified the species in functional groups, based on the literature (delabie et al., 2000; silvestre et al., 2003; silva & brandão, 2010; macedo et al., 2011; groc et al., 2014). functional groups are species sets that show similar characteristics and influence on ecosystem processes (diaz & cabido, 2001; silva & brandão, 2010). we classified poneromorph ant species in the following functional groups, according to diet and nesting habit: omnivores inhabiting the soil, omnivores inhabiting the soil and plants, omnivores inhabiting plants, detritivores inhabiting the soil, generalist predators inhabiting the soil, generalist predators inhabiting the soil and plants, generalist predators inhabiting plants, specialist predators inhabiting the soil. for the species whose sociobiology 63(3): 941-949 (september, 2016) 943 information on diet or nesting habits was not available, we used the information on the genus to allocate the ants into a functional group. data analysis we built species accumulation curves using the maotau method with a confidence interval of 95% in the program estimateswin8.0 (colwell, 2006). we considered an overlap in the confidence intervals of two species accumulation curves as evidence that they do not differ from one another (gomes et al., 2013). we used the analysis of variance and the tukey test to test for differences in taxonomic and functional richness and diversity between the dry and rainy seasons. average values were calculated per plot, and we pooled all species collected with the three methods (baits on the soil, baits on the vegetation, and pitfalls). we used a chi-square test (χ2) to compare relative richness and relative abundance (obtained by the frequency of species in samples) of the functional groups between the dry and rainy seasons. we also used a non-metric multidimensional scaling (nmds) and an anosim to compare species composition of the poneromorph ants between the dry and rainy seasons, based on the bray-curtis distance. this analysis was carried out in the program past (hammer et al., 2001). results and discussion we collected 46 species of poneromorph ants, which belong to two subfamilies and eleven genera (table 1). gnamptogenys was the genus with the highest species richness (nine species), followed by odontomachus (seven species), neoponera (six species), and hypoponera (five species). approximately 450 species of poneromorph ants are known for the neotropics, which represent 13% of the ant fauna of the region (bolton, 1995; lattke, 2003; fernández & sendoya, 2004). in tropical forests, some ant groups are highly diverse (hölldobler& wilson, 1990; vasconcelos et al., 2003) and the identification of their species is difficult due to taxonomic controversies (lyal & weitzman, 2004). however, these controversies are minor for other groups, such as poneromorphs, which allow studies to identify most ants to the species, which makes them more detailed and precise. such studies are useful in the definition of priorities for conservation, as they allow comparisons of species inventories between different localities, as the sampling effort and techniques used vary between studies (bestelmeyer et al., 2000). ectatomma lugens emery, 1894 was the species with the highest frequency of occurrence and was present in all seasons. next, the species with high frequencies were pachycondyla crassinoda (latreille, 1802) and gnamptogenys concinna (fr. smith, 1858), which were also present in all seasons. the species e. lugens has generalistic habits and broad distribution in tropical forests (fernandez, 1991), which could explain its high frequency. among the species identified, 39% nest only in the leaf litter, 32% make underground nests or nest in the leaf litter, 11% have only underground nests, and 7% nest only on the vegetation. two species can have subterraneous nests, nests in the leaf litter, or on the vegetation, whereas one species has already been observed nesting in the leaf litter or on the vegetation. it was not possible to obtain information on the nesting habits of two species. in terms of diet, 66% of the species identified are predators, 27% are omnivores, and only one species was classified as detritivore. the feeding habits of two species are unknown. as in other studies (e.g. lattke, 2003; fernández & arias-penna, 2008; jimenez et al., 2008), most poneromorph ants found use the soil or the leaf litter for nesting and are predators. in addition, wilkie et al. (2010) also observed that a relatively small number of ant species use more than one vertical stratum of the forest. however, it is important to highlight that in the present study the sampling effort was lower on the vegetation than in the soil. thirteen ant species were collected in a single sample (28% of the total of species), four species in two samples (9% of the total of species), and four species in three samples. the low frequency of occurrence observed in several ant species is common in inventories carried out in brazil (braga et al., 2010; vargas, 2011). species accumulation curves indicated no significant difference in species richness between seasons (fig 1 and fig 2). the fact that many ant species are infrequent creates a need for using a larger sampling effort. however, in the present study, even with a large number of samples the species accumulation curve did not reach an asymptote. this is partially explained by the high diversity of the group (silva & silvestre, 2000). the average species richness did not vary significantly between seasons (table 2, anova, f = 0.68, p = 0.57) and the same occurred with species diversity (anova, f = 1.56, p = 0.22). twenty-seven ant species were collected in at least one dry and one rainy season (59% of the total of species). hence, there was no significant difference between ant communities collected in different seasons (r = 0.035; p = 0.24). the functional group with the highest species richness was generalist predators inhabiting the soil, followed by the group of omnivores inhabiting the soil (table 3). however, the group of the omnivores inhabiting the soil was the most abundant, followed by the generalist predators inhabiting the soil. species with a generalist diet, mainly omnivores, can reach higher abundance as they may use different kinds of food. there was no significant difference in the frequency of species of functional groups between dry and rainy seasons (table 3, chi-squared test, χ2 = 7.15, gl = 21; p = 0.99). the same occurred with the abundance of functional groups (chi-squared test, χ2 = 28.85, gl = 21,p = 0.12). there was no significant difference in the average richness (table 4, anova, f = 0.338, p = 0.80) and average diversity of functional groups between the dry and rainy seasons (anova, f = 1.01, p = 0.37). lpc pereira et al. – taxonomic and functional diversity of poneromorph ant assemblages944 table 1. species list of poneromorph ants and their frequency of occurrence in the dry and rainy seasons. t = total frequency of occurrence of each species. nesting: sub = subterraneous nests, lit = leaf litter nests, veg = nests on live vegetation, u = unknown. diet: o = omnivorous, p = predator, d = detritivorous, u = unknown. subfamilies/species dry season i dry season ii rainy season i rainy season ii t nesting diet ectatomminae ectatomma edentatum roger, 1863 14 13 17 6 50 sub o ectatomma lugens emery, 1894 115 131 103 75 424 sub o ectatomma tuberculatum (olivier, 1791) 14 19 16 21 70 sub o ectatomma sp. 4 1 1 x x gnamptogeny sacuminata emery, 1896 3 2 2 4 11 lit p gnamptogeny sannulata mayr, 1887 2 2 lit/veg p gnamptogeny sconcinna (fr. smith, 1858) 47 48 49 14 158 veg o gnamptogeny shaenschi emery, 1902 1 1 sub/lit d gnamptogeny shorni santschi, 1929 16 19 11 11 57 sub/lit/veg p gnamptogeny slanei kempf, 1960 1 1 d p gnamptogenys moelleri (forel, 1912) 20 41 45 13 119 lit o gnamptogenys striatula mayr, 1883 1 5 3 2 11 sub/lit p gnamptogenys sulcata (fr. smith, 1858) 1 1 lit o typhlomyrmex pusillus emery, 1894 1 1 lit p ponerinae anochetus diegensis forel, 1912 1 1 lit p anochetus horridus kempf, 1964 1 1 lit p anochetus mayri emery, 1884 1 1 lit p anochetus targionii emery, 1894 1 1 2 lit p hypoponera argentina (santschi, 1922) 3 2 5 lit p hypoponera distinguenda (emery, 1890) 1 1 lit o hypoponera schmalzi (emery, 1896) 2 4 1 7 lit p hypoponera trigona (mayr, 1887) 2 1 3 lit p hypoponera sp. 1 1 2 4 7 x x leptogenys famelica emery, 1896 2 1 3 sub/lit p leptogenys gaigei wheeler, 1923 3 3 5 11 sub/lit p leptogenys guianensis wheeler, 1923 2 2 sub/lit p leptogenys langi wheeler, 1923 1 1 sub/lit p mayaponera constricta (mayr, 1884) 21 15 7 7 50 sub/lit p neoponera apicalis (latreille, 1802) 28 39 20 8 95 sub/lit/veg o neoponera commutata (roger, 1860) 1 1 1 3 sub p neoponera laevigata (fr. smith, 1858) 2 1 3 sub/lit p neoponera procidua emery, 1890 2 2 d u neoponera unidentata (mayr, 1862) 1 3 4 veg p neoponera prox. obscuricornis (emery, 1890) 35 36 25 13 109 lit p odontoma chusbauri emery, 1891 3 3 3 2 11 lit p odontoma chusbrunneus (patton, 1894) 16 24 20 3 63 sub/lit p odontoma chuscaelatus brown jr., 1976 1 3 4 sub p odontoma chushaematodus (linnaeus, 1758) 15 10 13 1 39 lit p odontoma chushastatus (fabricius, 1804) 1 1 veg p odontoma chusmeinerti forel, 1905 6 12 13 6 37 lit p odontoma chus prox. yucatecus brown jr., 1976 6 9 5 2 22 lit p pachycondy lacrassinoda (latreille, 1802) 64 70 51 29 214 sub/lit o pachycondy laharpax (fabricius, 1804) 26 51 25 9 111 sub/lit o pachycondy lastriata fr. smith, 1858 5 1 2 8 sub/lit o rasopone arhuaca (forel, 1901) 1 1 sub/lit o rasopone ferruginea (fr. smith, 1858) 1 1 sub/lit u sociobiology 63(3): 941-949 (september, 2016) 945 the temperature and rainfall are among the environmental factors that influence the ants. in the study region, temperature does not vary much throughout the year (embrapa, 2015). this fact can explain the lack of differences in species composition and taxonomic and functional diversity between seasons. rainfall, though, varies largely throughout the year (embrapa, 2015). however, even with such variation in rainfall, the fauna of poneromorph ants did not vary between the rainy and dry seasons. other authors observed variation in species richness or diversity between the dry and rainy seasons in the atlantic forest (coelho & ribeiro, 2006; vargas et al., 2007), but the results can be explained by higher average temperature in the rainy season. a seasonal increase in resource availability can be connected to an increase in taxonomic and functional diversity, as it allows ant populations to grow and, consequently, the number of foraging worker ants also grows (silva et al., 2014). on the other hand, in other studies in the atlantic forest the effects of climate seasonality on ant species richness or diversity were not observed (neves et al., 2010; montine et al., 2014). the lack of differences in community composition and functional groups can be related to the small number of specialist species. populations of generalist species can probably remain stable throughout the year, as they have higher food availability. fig 1.species accumulation curves (mao-tau) and 95% confidence interval for the fauna of poneromorph ants collected in the dry and rainy seasons. fig 2. species accumulation curves (mao-tau) and 95% confidence interval for the fauna of poneromorph ants collected in the dry and rainy seasons. variable dry season i dry season ii rainy season i rainy season ii total richness 30 31 31 25 total diversity 2.63 2.64 2.67 2.48 average richness (± sd) 14.67 ± 2.74 15.56 ± 1.67 14.56 ± 2.51 13.80 ± 1.92 average diversity (± sd) 2.29 ± 0.17 2.37 ± 0.12 2.31 ± 0.22 2.17 ± 0.12 table 2. species richness and shannon diversity ofponeromorph ants collected in the dry and rainy seasons in the tapirapé-aquirí national forest, pará, brazil. lpc pereira et al. – taxonomic and functional diversity of poneromorph ant assemblages946 the lack of response of the ant fauna to climate seasonality can also indicate that there are no expressive variations in resource availability throughout the year or that the variations in climate in the region are not strong enough to impact the fauna of poneromorph ants. in addition, taxonomic and functional variations in leaf litter ant communities in the amazon can occur due to changes in environmental heterogeneity, which affects abiotic variables (groc et al., 2014). hence, these ant species can be more influenced by the microclimate, which is more homogeneous throughout the year within the forest, than by the regional climate. given the present results, it is important to keep on searching for other factors, such as microclimate, ecological interactions, and evolutionary history, which may help understand the most important structuring processes for poneromorph ants in the study region. it is important to highlight that the environmental heterogeneity is cited as an important regulator of the ant fauna (vargas et al., 2007; martins et al., 2011; gomes et al., 2013). as most species sampled use the leaf litter for nesting or searching for food, new studies can assess the influence of the leaf litter amount and composition on poneromorph ants in the amazon. ants are considered a bioindicator group (silva & brandão, 1999). however, assessing the whole ant fauna can be difficult, as the species of some genera are hard to identify. this problem can be minimized by restricting the study to a taxonomically well-known group of ants, such as poneromorphs. in this way, more complete inventories can be compiled, and the results of the studies will be more precise and comparable between localities. in our study we found no seasonal differences in composition and in taxonomic and functional diversity of poneromorph ants in the amazon, which is useful for future studies that aim at using those ants as bioindicators. in addition, the identification of the species made in the present study has special relevance as it contributes to advance the knowledge of poneromorph ant diversity in the amazon. acknowledgments to faperj (proc.101.472/2010) for a research fellowship to jmq, and to habtec for the logistic support. we also thank rodrigo m. feitosa and an anonymous referee for their helpful comments on an early version of the manuscript. functional groups dry season i dry season ii rainy season i rainy season ii relative richness (%) omnivores inhabiting the soil 27.6 25.8 22.6 36.0 omnivores inhabiting the soil and plants 3.4 3.2 3.2 4.0 omnivores inhabiting plants 3.4 3.2 3.2 4.0 detritivores inhabiting thesoil 0.0 3.2 0.0 0.0 generalist predators inhabiting the soil 48.3 54.8 61.3 44.0 generalist predators inhabiting the soil and plants 6.9 3.2 3.2 4.0 specialist predators inhabiting the soil 6.9 3.2 3.2 8.0 generalist predators inhabiting plants 3.4 3.2 3.2 0.0 relative abundance (%) omnivores inhabiting the soil 54.0 57.5 57.1 66.5 omnivores inhabiting the soil and plants 6.0 6.9 4.4 3.4 omnivores inhabiting plants 10.2 8.4 10.8 5.9 detritivores inhabiting the soil 0.0 0.2 0.0 0.0 generalist predators inhabiting the soil 24.8 23.4 24.3 18.6 generalist predators inhabiting the soil and plants 3.9 3.3 2.4 4.7 specialist predators inhabiting the soil 0.9 0.2 0.2 0.8 generalist predators inhabiting plants 0.2 0.2 0.7 0.0 variable dry season i dry season ii rainy season i rainy season ii total richness 7 8 7 6 total diversity 1.26 1.20 1.18 1.05 average richness (± sd) 5.33 ± 0.50 5.22 ± 0.67 5.00 ± 0.87 5.20 ± 0.84 average diversity (± sd) 1.18 ± 0.14 1.15 ± 0.14 1.10 ± 0.25 1.01 ± 0.16 table 4. species richness and shannon diversity of functional groups of poneromorph ants collected in the dry and rainy seasons in the tapirapé-aquirí national forest, pará, brazil. table 3. relative richness and abundance of functional groups of poneromorph ants collected in the dry and rainy seasons in the tapirapéaquirí national forest, pará, brazil. sociobiology 63(3): 941-949 (september, 2016) 947 references almeida, f.s., queiroz, j.m. & mayhé-nunes, a.j. 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(1988). insect seasonality: why? annual review of ecology and systematics, 19: 1-18. doi: 10.1146/annurev. es.19.110188.000245 _goback open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.5906sociobiology 68(4): e5906 (december, 2021) introduction the pollination process depends on the adjustment of floral and of visitor traits. among these traits, morphology and behavior are related to the contact of visitors with the anthers and stigmas of flowers during the resource collection, and this has consequences for the pollination efficiency (pick & schlindwein 2011; paz et al., 2013; santos & gimenes 2016; paz et al., 2018; araujo et al., 2018). besides these adjustments, several studies already highlighted the importance of the temporal adjustments between floral visitors and flower and, in particular, associating the daily activity pattern of the pollinators to the temporal aspects of the plant (gimenes et al., 1993, 1996; paz et al., 2013). in this context, the time of the day of foraging activity of floral visitors is also an aspect that abstract pollination is an ecological process that relies on the matching traits of flower visitors and flowers. morphology, behavior, and temporal patterns play essential roles in mediating the interactions between plants and floral visitors. this study analyzed the temporal aspects of visitors and flowers interaction and the possible adjustment between both organisms. we used ipomoea bahiensis and its flower visitors as a model system. we evaluated the visitor frequency on the flowers throughout the day, flower opening and closing times, pollen availability and stigma receptivity. we also evaluated the highest fruit production time during the flower longevity, and the time of highest pollinator activity, related to climatic factors. among the floral visitors, bees, especially melitoma spp., apis mellifera, and pseudaugochlora pandora were the most frequent visitors, presenting regular visits synchronized with the flower opening and closing times, which were also regular. this system was influenced mainly by light intensity. besides, these bees were very active during the times of the highest fruit production. these data indicate the presence of temporal patterns for both the bees and the visited plants, and synchronization between them, being the light intensity as a modulator of the rhythms of bees and plant, confirming the importance of the temporal adjustments for pollination efficiency. sociobiology an international journal on social insects miriam gimenes1, laene s araujo2, anderson m medina3 article history edited by gilberto m. m. santos, uefs, brazil received 06 october 2020 initial acceptance 25 march 2021 final acceptance 02 august 2021 publication date 19 november 2021 keywords daily activity pattern, apis mellifera, melitoma, pseudaugochlora pandora, pollination. corresponding author miriam gimenes departamento de biologia universidade estadual de feira de santana (uefs) av. transnordestina s/nº, novo horizonte, feira de santana-ba, brasil. e-mail: mgimenes@uefs.br may affect the occurrence and quality of pollination (gimenes et al., 1993, 1996; paz et al., 2013; bloch et al., 2017). several plant reproductive events happen at specific times of the day, which may be the most propitious moment for pollination to occur. examples of such events include opening and closing times of flowers and anthers, stigma receptivity, production and concentration of nectar and odor (van doorn & van meeteren, 2003; terada et al., 2005; matile, 2006; silva et al., 2010; edge et al., 2012; paz & pigozzo, 2013; paz et al., 2013; van doorn & kamdee, 2014). these patterns observed in organisms throughout the day can be triggered by climatic clues that vary during the day, such as temperature, relative humidity, and light intensity. van doorn and van meeteren (2003) as well as van doorn and kamdee (2014) demonstrated the influence 1 departamento de biologia, universidade estadual de feira de santana, bahia, brazil 2 programa de pós graduação em ecologia e evolução, universidade estadual de feira de santana, bahia, brazil 3 instituto de biologia, universidade federal da bahia, salvador, bahia, brazil research article bees the light intensity mediates the pollination efficacy of a caatinga morning glory ipomoea bahiensis (convolvulaceae) mailto:mgimenes@uefs.br miriam gimenes, laene s araujo, anderson m medina – light intensity mediates the pollination efficacy2 of these factors by reporting several examples of how climatic variation (especially light and temperature) can have different outcomes of flower opening and closing for many plant species. in parallel, floral visitors may also change their activity patterns in response to different microclimatic conditions throughout the day (lutz, 1931; gimenes et al., 1993, 1996; torres-díaz et al., 2007). therefore, these factors may favor the adjustment of daily activity patterns of visitors and flowers (gimenes et al., 1993; gimenes et al., 1996). daily activity patterns of plant-pollinator interactions can be of particular interest when plants have ephemeral flowers (e.g., that last less than 12 hours) because the short exposure time of the reproductive structures limits the floral visitor activity time (terada et al., 2005; pick & schlindwein, 2011, paz et al., 2013, 2018, santos & gimenes, 2016; araujo et al., 2018). some plants have ephemeral flowers like those of the genus ipomoea (convolvulaceae) whose most common pollination syndrome is melitophilia. in particular, most pollinators of ipomoea are diurnal bees specialized in pollen harvesting from this genus of morning glories (wcislo & cane, 1996; martins, 2002). examples of specialized pollinators of ipomoea are the oligolectic bees of the genus melitoma (terada et al., 2005; pick & schlindwein, 2011; paz & pigozzo, 2013; araujo et al., 2018). however, most studies above were based on morphological and behavioral aspects, disregarding the relative importance of daily temporal patterns of the foraging activity of floral visitors. given the importance of temporal adjustments between plants and pollinators, this study aims to analyze the daily temporal pattern of the interaction between ephemeral flowers of ipomoea bahiensis (convolvulaceae) and the most frequent bees. furthermore, the influence of climatic factors on the flower opening and closing and the forage activities of bees will be verified. material and methods the study took place at campus of the universidade estadual de feira de santana (12º11’s, 38º58’w) in feira de santana city, state of bahia, brazil. the climate of the region is classified as bsh or semiarid (köppen & geiger, 1928). the campus has a total area of approximately 1.2 km2, and the original vegetation was composed of caatinga, but today most of the area has been converted in anthropic vegetation, with non-native and invasive plants (santana & santos, 1999). the study was performed between july and october/ 2014, period when ipomoea flowering peak occurred in this area. the samplings and observations were performed in four plots (20 x 20 m each) spaced at least 200 m apart. this species presents a vine habit. the flower and visitor were counted in plots and not on individual plants. floral biology to evaluate the stigma receptivity time throughout the flower duration (from 4:00 to 15:00 h), we immersed ten plant pistils from different plots in petri dishes containing hydrogen peroxide (h2o2) at each hour of the flower duration and observed them in the field with the help of a hand-held magnifier (20 x). bubble formation occurs when pistils are receptive and enter in contact with h2o2; therefore, bubbles represented a positive result for the stigma receptivity (dafni & maués, 1998). we evaluated the opening and closing of flowers by counting the number of flowers at each 15-minute interval while collecting climatic information. first, open flowers were counted from when the first flower opened until when all flowers were open in the sample plot. second, a similar method was used to determine the numbers of closed flowers, starting when the first flower closed until all the flowers in the sample were closed. third, the temperature (°c), light intensity, and relative air humidity were recorded simultaneously at each 15 minute interval. this procedure was repeated four times in each observation months (july, august, september, and october/14), each time made upon a different plot. to determine which climatic predictor was the best explanation for the events of flower opening and closing, we constructed two sets of generalized linear mixed models with poisson distribution (zuur et al., 2009). one set of models was built to explain the flower opening while the other was built to explain the flowers closing. both sets of candidate models consisted of a model with only temperature, relative humidity, or light intensity as predictor variables. also, we included sampling dates and time of day as random variables. six models were built using the lme4 package (bates et al., 2014) for each set of models (flower opening or flower closening). each set of models were compared among them using the akaike information criterion with correction for small sample size (akaike information criterion – aicc) to select the most parsimonious model (burnham & anderson, 2002) using the bbmle package (bolker & r development core team, 2014). to verify the time of most significant fruit production by i. bahiensis, we conducted a manipulation experiment where plants’ access to pollination was restricted to periods of one hour. we selected and bagged 200 buds before flower openings. beginning at 04:00 h, we exposed ten flowers to visitation by removing their bags, and after one hour, we bagged the same flowers. we repeated this procedure on another set of ten flowers every hour until 15:00 h. after this exposure, the flowers were bagged and labelled to monitoring fruit development and the fruits formed. this procedure was performed every sampling month, during four days, a day per sampling plot of 20 m², and varied according to the flowers available in the samples. daily bee foraging activity we selected the most frequent floral visitors (species with a frequency greater than 10% during the study), to assess the daily foraging activity in the flowers. these visitors were: melitoma spp. [melitoma segmentaria (fabricius, 1804), sociobiology 68(4): e5906 (december, 2021) 3 melitoma sp. 1, melitoma sp. 2)] (relative frequency = 25%), apis mellifera linnaeus, 1758 (23%), pseudaugochlora pandora (smith, 1853) (12%). araujo et al. (2018) provide detailed information regarding the number of visits and body size of floral visitors in i. bahiensis, in this area. the number of visits realized by the floral visitors was recorded in the four sampling plots from july to october. all plots were sampled monthly, and the sampling order that each plot was randomly established. sampling started 30 minutes before the flower opening and ended 30 minutes after the flower closing. during this period, the number of visits was obtained for 15 minutes. counts were made of the visits of each species that landed on the flower for resource collection (as described in araujo et al., 2018). concomitantly, we recorded the microclimatic data of temperature, relative humidity, and light intensity every 15 minutes. to determine which climatic predictor was the best explanation to the variation on visitor frequency, we constructed generalized linear mixed models with poisson distribution (zuur et al., 2009), using the number of visits (abundance) as response variable and temperature, relative humidity, and light intensity as predictor variables. in addition, we included sampling dates and time of day as random variables. these models were developed using the lme4 package (bates et al., 2014). the models were compared among them using the akaike information criterion with correction for small sample size (akaike information criterion – aicc) to select the most parsimonious model (burnham & anderson, 2002) using the bbmle package (bolker & r development core team 2014). all statistical analyses were performed in the r environment (r core team, 2014), and explanatory variables with high correlation (r > 0.7) were not included in the same model to avoid multicollinearity effects. the specimens of bees sampled were deposited in the entomological collection “prof. johann becker” of the museu de zoologia da universidade estadual de feira de santana (mzfs). flower-visitor interaction a generalized linear mixed model (glmm) with a binomial distribution (zuur et al., 2009) was used to evaluate the time interval of the most massive fruit production, using the time of day as the predictor variable and fruit production (binary) as the response variable, and also include sampling dates as the random variable. in this analysis, each flower was considered a sampling unit. the period between 4:00 and 5:00 h, and between 15:00 and 16:00 h was excluded from the analyses due to the absence of fruit production. this choice could bias the model estimates due to the presence of zeros. this analysis was performed in the lme4 package (bates et al., 2014). after establishing the model, we compared the fruit development at each hour with the control. in a scenario where development time was similar to the control counterpart, it would indicate a crucial time for fruit development. this analysis of comparisons was performed using the glht function of the multcomp package (hothorn et al., 2008). results ipomoea bahiensis flowers had average floral longevity of seven hours and thirty minutes (± 37 minutes). the flowers opened between 04:30 and 07:30 h. it can be observed a difference in the opening hours of the flowers in 3 plots (plot 1, 2 and 3) and also in two different days in the same plot (plot 3-a and plot 3-b, figure 1). this difference in opening times depends of the area where the plants were located, and also of the day on which the data collection was performed, fig 1. number of the open flowers in ipomoea bahiensis (convolvulaceae), per hour interval on the campus of universidade estadual de feira de santana, ba, brazil between july and october 2014. 23 0 10 20 30 40 50 60 04:30 05:00 05:30 06:00 06:30 07:00 07:30 hour o pe n fl ow er s (n ) plot 2_1 plot 3_2 plot_1 plot 3_1 fig 1 miriam gimenes, laene s araujo, anderson m medina – light intensity mediates the pollination efficacy4 with flowers opening later in the more shaded area. the microclimate factors of the plots displayed small differences one to the other, even when these were at a distance of just 200 m one from the other, especially concerning light intensity and temperature. furthermore, the flowers remained closed at temperatures below 18°c, even with an increase in light intensity. the flowers closed between 9:45 and 14:15 h. climatic factors played a role both on the flower opening and on the closing processes of i. bahiensis. regarding flower opening, the most parsimonious model included light intensity and temperature as climatic predictors in which the opening of i. bahiensis flowers occurred more frequently with increasing temperature and light intensity (χ2 = 65.85, gl = 2, p <0.001; β light intensity = 0.40; β temperature = 0.67) (table 1, figure 2). on the other hand, the best model for flower closing revealed that the closure of i. bahiensis flowers occurred more frequently with increasing air temperature and humidity and decreasing light intensity, simultaneously (χ2 = 69.80, gl = 3, p <0.001; β light intensity -1.00; β temperature = 0.34; β humidity 0.07) (table 1). the stigma remained receptive throughout the floral longevity but presented a higher amount of stigmas with bubbles from 8:00 to 12:00 h. table 2. models describing the probability of microclimatic variables that influenced the abundance of visits of the most frequent bees on the flowers of ipomoea bahiensis. comparisons of the different models with sample size correction (aicc), aicc differences (δ), number of variables (k). the models are classified in the ascending order of aicc. response model aicc δ k (i) log (light intensity+1) + temperature 712.2 0 5 log (light intensity+1) + temperature + relative humidity 714.6 2.5 6 temperature 720.0 7.8 4 temperature + relative humidity 722.4 10.2 5 log (light intensity+1) + relative humidity 741.4 29.2 5 log (light intensity+1) 750.5 38.3 4 relative humidity 762.5 50.3 4 intercept 773.4 61.2 3 (ii) log (light intensity+1) + temperature + relative humidity 465.6 0 6 temperature + relative humidity 472.9 7.3 5 log (light intensity+1) + temperature 506.3 40.7 5 temperature 509.6 44.0 4 relative humidity 524.9 59.3 4 log (light intensity+1) + relative humidity 525.2 59.6 5 log (light intensity+1) 527.7 62.1 4 intercept 527.8 62.2 3 table 1. models describing the probability of microclimatic variables that influenced the flower opening (i) and closing (ii) processes of ipomoea bahiensis (convolvulaceae). comparisons of the different models with sample size correction (aicc), aicc differences (δ), number of variables (k). the models are classified in the ascending order of aicc. daily activity of potential pollinators the most frequent bees (melitoma spp., and pseudaugochlora pandora, native bees, and apis mellifera, introduced bee) were observed throughout the floral longevity of i. bahiensis. potential pollinators began their activities at 05:00 h, which matched with the opening of the flowers. solitary native bees concentrated their highest number visits in the interval between 08:00 and 10:00 h (figure 3) while apis mellifera had their peak of visitation between 09:00 and 11:00 h. the peak of the visitation of both groups of potential pollinators happened when all the flowers were already open. the most parsimonious model for the abundance of potential pollinators on the flowers included the luminous intensity, and this model indicated that the number of visits was more response model aicc δ k bees log (light intensity+1) 2176,4 0 4 intercept 2231,9 55,5 3 temperature 2232,6 56,2 4 relative humidity of air 2233,7 57,3 4 significant with the increase in light intensity (χ² = 57.52; gl = 1, p < 0.001, table 2). sociobiology 68(4): e5906 (december, 2021) 5 fruit formation was higher between 08:00 and 09:00 h. this interval showed a result similar to that obtained in the control experiment (flowers exposed to visitors throughout the floral longevity) and also presented twice the probability of fruit formation when compared to other times intervals (figure 4; χ2 = 107.78; gl = 10; p < 0.001). at this time, the bees a. mellifera, fig 2. effect of luminosity (the top) and temperature (the bottom) on the number of ipomoea bahiensis open flowers. the predicted line is based on the best-fitted model using aic. fig 3. number of visits of the most frequent bees (melitoma spp, apis mellifera, and pseudaugochlora pandora) on flowers of ipomoea bahiensis (convolvulaceae), per hour interval throughout the day, on the campus of universidade estadual de feira de santana, ba, brazil between july and october 2014. 25 bee (number) and fruit (% ) 0 50 100 150 200 250 300 350 400 450 500 04:00 06:00 08:00 10:00 12:00 14:00 hora n um be r of v is its 0 5 10 15 20 25 30 35 40 45 50 fr ui ts (% ) pseudaugochlora pandora apis mellifera melitoma spp. fruits (%) figure 3 melitoma spp, p. pandora and ancyloscelis apiformes and the butterflies morys compta compta, nyctelius nyctelius nyctelius, phoebis sennae marcellina and synale hylaspes were visiting the flower of i. bahiensis (figure 5). among these bees, melitoma spp., were the most frequent in the flowers and more coincident with the time of greatest fruit production. miriam gimenes, laene s araujo, anderson m medina – light intensity mediates the pollination efficacy6 discussion the flowers of ipomoea bahiensis were studied in an area whose original vegetation was caatinga, and became anthropized area, where this species occurs frequently. the flowers displayed morphological features that classify them as melitophilous. among the floral visitor, the bees were more frequent, especially those of the genus melitoma. these bees are solitary and have already been quoted in the literature as associated with flowers of species of the genus ipomoea (pick & schlindwein, 2011). araujo et al. (2018) studied the same area and observed the bees melitoma spp., p. pandora, and a. mellifera as the most frequents on the flowers and considered them as potential pollinators. these authors also observed that these bees species had an average size that was compatible with the morphological features of the flowers. the size of the bees, and the proper behavior, allowed the contact between the bee’s body and the reproductive features of the plant. paz et al. (2013) also considered the medium-sized bees as potential pollinators of the ipomoea carnea flowers. besides, araujo et al. (2018) claimed that the visits of butterflies and small-sized bees did not produce fruits in the efficiency pollination tests in i. bahiensis. even the acknowledged relevance of the morphological and behavioral aspects in the pollination activities of these bees, just these adjustments do not guarantee efficient pollination. besides the aspects mentioned above, also the timing of the interaction is relevant: the flower visitors need to synchronize their activities with those of the flowers (opening and closing times of the flowers, pollen release, stigma receptivity, production of the nectar, and others) so that pollination can occur, and consequently a higher production of fruits (gimenes et al., 1993; 1996). the literature already described the behavioral and morphological adjustments between visitors and flowers, but few studies have considered the fig 4. effect of time of day on fruit formation probability. squares are mean for each period, and the continuous line is the control mean (treatment exposed all the time). the dashed line is 95% confidence intervals. fig 5. abundance of visits of species in ipomoea bahiensis (convolvulaceae) between 8:00 and 9:00 h, in feira de santana (ba). species represented: (a) ancyloscelis apiformes; (b) apis mellifera; (c) melitoma spp.; (d) morys compta compta; (e) nyctelius nyctelius nyctelius; (f) phoebis sennae marcellina; (g) pseudaugochlora pandora; (h) synale hylaspes. (n > 10 visits) (complete table with all visitors in araujo et al., 2018). species n um be r o f v is its sociobiology 68(4): e5906 (december, 2021) 7 timing adjustments until now. if the flowers’ timing (most especially as to the opening and closing) does not overlap with the feeding activities of their visitors, pollination may not occur. this timing is crucial, especially in plants with ephemeral flower (lasting less than 12 hours), as observed in the genus ipomea. in this case, the pollinator-flower synchronization must occur during the short flowers’ opening. ipomoea flowers remain open for about seven hours, and displayed similar hours of opening and closing along the flowering months, suggesting a daily pattern of the flowers’ opening and closing. time patterns of flowers ‘opening and closing have already been described in species of the convolvulaceae (van doorn & van meeteren, 2003, van doorn & kamdee, 2014) and other plants’ genera (kaihara & takimoto 1980, tanaka et al., 1989, ichimura & suto, 1998). the bees that most frequently visited these flowers must follow these time standards of the flowers’ opening (gimenes et al., 1993; 1996). the most frequent visiting bees probably follow these patterns of opening and closing time of the flowers (gimenes et al., 1993; 1996). therefore, these visits should occur at times where there is the greatest condition for the formation of fruit. so, how do we know what those times are? this work adopted a method that allowed to verify that hour interval of the best fruit development, and at this hour interval it was also observed the highest frequency of forage activity of melitoma sp. p. pandora and a. mellifera. these visiting bees were medium-sized and this characteristic favored contact with the reproductive structures of the flowers. in the literature, several reports have already been described of the presence of biological rhythm in the daily activities of bees, most especially focusing on the eusocial bees such as apis mellifera (moore & rankin, 1985; moore & rankin, 1993; moore, 2001) and sting-less bees (bellusci & marques, 2001). few studies have addressed on the relationship between the forage activities of bees and flowers, with a focus on temporal adaptations. some studies on this topic were carried out with solitary bee species and the flowers visited (gimenes et al., 1993, 1996; gottlieb et al., 2005). solitary and social bees visited the i. bahiensis flowers at similar hours, synchronized with the times of the highest fruit production. the presence of different bees’ species in the ipomoea flowers, displaying similar size, behavior and visiting times, favoring the pollination of the visited plant, can be characterized as functional redundancy. this redundancy can improve the stability of a community (blüthgen & klein, 2011). different visiting species, with similar behavioral and morphological features, may act as potential pollinators of the same plant, as in i. bahiensis. besides the morphological and behavioral features highlighted by blüthgen and klein (2011), our study also observed the timing features, where the time of the highest fruit production was coordinated to the activity of the potential pollinators bees of this plant. the occurrence of temporal events in plants and bees may be influenced by environmental and climatic events, which may cause the temporal adjustment (synchronization) of both organisms (koukkari & sothern, 2006). the luminous intensity and the temperature (as to the opening of the flowers) influenced the interaction between i. bahiensis and the visiting bees. besides this, the opening of the i. bahiensis flowers occurred synchronized with the sun rising and, in this period occurred also the most intense modifications of the light intensity and temperature. the relative humidity besides the light intensity and the temperature influenced the closing of the flowers. studies in controlled experiments highlighted the necessity of the exposition to specific temperatures and light conditions to start the opening process in the flowers of ipomea (kaihara & takimoto, 1979), and also in morning flowers of other genera (tanaka et al., 1989, ichimura & suto, 1998, van doorn & van meeteren, 2003, van doorn & kamdee, 2014). ichimura and suto (1998) studied the rhythm of opening and closing of flowers of hybrids of portulaca and pointed out that these rhythms are influenced by the joint action of the high temperature (30-35ºc) and light. bai and kawabata (2015) observed that the flowers of eustoma grandiflorum shinn. (gentianaceae) opened in the morning and closed at the afternoon’s end, and concluded that their rhythms were synchronized by the cycles of light/dark and the direct effects of light intensity. the climate also influences the daily activities of the bees visiting the i. bahiensis flowers. the light intensity is the factor that mostly explained these activities. lutz (1931) considered the light intensity as the possible signal of the opening time of the nests of eusocial bee and also as an agent of the adjustment of the biological rhythm. kilkenny and galloway (2008) observed that the environmental light has a direct and positive influence on the abundance of the visits of the pollinating insects. according to polatto et al. (2014), the beginning of the flowers’ visitor activity is regulated by light intensity, finishing at the end of the light phase, or with the depletion of the flowers’ resources. these factors might have regulated the frequency of the foraging activities of the bees during the day, signalizing the best moment for the foraging on the flower. prasad and hodge (2013) studied the bee braunsapis puangensis (cockerell) that visited the creeping plant sphagneticola trilobata (l.) pruski in the fiji islands. the authors observed the high positive correlation between the daily foraging activities of the bee and light intensity and a strong negative correlation with the relative humidity. as it is well known in the chronobiological concepts, environmental and climatic factors may influence the synchronization and biological rhythms of plants and insects (koukkari and sothern, 2006). the time adjustment between the visitors and the flowers may determine the pollinator and its efficiency (gimenes et al., 1993, 1996; paz et al., 2013). in conclusion, this study aimed to understand the different aspects that influence efficient pollination. for a long time, the researches only considered the morphological miriam gimenes, laene s araujo, anderson m medina – light intensity mediates the pollination efficacy8 and behavioral aspects of the flowers’ visitors to explain the efficiency of the plants’ pollination. the research observed the influence of both the morphological and behavioral aspects in the bees visiting i. bahiensis, but it also highlighted that the timing aspects are crucial for efficient pollination. the interaction between the visiting bees and the flowers highlighted that the flowers’ opening displayed a periodicity of about 24 hours, and the highest probability of fruit production at a specific time of the day. these times coincided with the daily activities of the most frequent bees considered potential pollinators of the flowers, mainly influenced by the light intensity. the light intensity is an external factor that would adjust the biological rhythms of the organisms, promoting the encounter between bee and plant, resulting in a higher pollination efficiency. acknowledgments the authors would like to thank fernando silveira (ufmg) for the identification of the bee species sampled; dr. marlon paluch (ufrb/ba) for the identification of the lepidoptera species; dr efigênia de melo (uefs/ba) for identifying of plant species. this study was financed by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. authors’ contribution mg: supervision, writing. lsa: investigation, data curation. amm: formal analisys, ilustrations, writing. references araujo, l. s., medina, a. m. & gimenes, m. 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(2003). flower opening and closure: a review. journal of experimental botany, 389: 1801-1812. van doorn, w.g. & kamdee, c. (2014). flower opening and closure: an update. journal of experimental botany, 65: 5749-5757. doi: 10.1093/jxb/eru327. wcislo w.t., cane j.h. (1996). floral resource utilization by solitary bees (hymenoptera: apoidea) and exploitation of their stored foods by natural enemies. annual review of entomology, 41: 257-86. zuur a.f., leno e.n., walker n., saveliev a.a. & smith g.m. (2009). mixed effects models and extensions in ecology with r. springer, berlin, heidelberg: 524 p. http://www.sciencedirect.com/science/article/pii/s0367253013000194 http://www.sciencedirect.com/science/article/pii/s0367253013000194 ole_link1 ole_link2 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2077sociobiology 65(2): 253-258 (june, 2018) orchid bees (hymenoptera: apidae: euglossini) in seasonally dry tropical forest (caatinga) in brazil introduction euglossini bees are also known as “orchid bees” due to the intimate interactions with orchidaceae (dressler, 1982), whose flowers are used by males to collect fragrances that they probably use for the court (dodson et al., 1969; eltz et al., 2005). other floral sources are exploited to collect fragrances, resins, pollen and nectar (whitten et al., 1993; cortopassi-laurino et al., 2009; perger, 2015). these bees are pollinators of plants included in approximately 40 botanical families (ramírez et al., 2002). this taxon is distributed in the neotropical region (dressler, 1982; moure et al., 2012), and higher species richness is found in tropical rainforests (roubik & hanson, 2004), but there are populations settled in arid areas, as in the seasonally dry tropical forest (caatinga) (lopes et al., 2007; andrade-silva et al., 2012). studies focusing on euglossini assemblages in the caatingas’ dominium are scarce and geographically restricted. some works were carried out in abstract euglossini bees are important neotropical pollinators, but there is a lack in the knowledge about this fauna in dry tropical environments. the aims of this study were to evaluate the richness and abundance of euglossine bees in two fragments of seasonally dry tropical forest (caatinga), as well as to assess the distribution of euglossine species richness in the caatinga environment. males were collected along 12 consecutive months, using traps with aromatic baits. the species richness (s=5) was lower than in rainforests and savannas. euglossa cordata (l.) was the dominant species in the assemblage, representing 70% of the individuals. the highest abundance occurred in the rainy season. euglossini fauna presents low local species richness in caatinga areas, however the beta diversity is higher, since assemblages in different habitats have differences in species composition. sociobiology an international journal on social insects ls carneiro1, wm aguiar1,2, cml aguiar1 & gmm santos1 article history edited by kleber del-claro, ufu, brazil received 13 september 2017 initial acceptance 15 december 2017 final acceptance 25 february 2018 publication date 09 july 2018 keywords bee assemblages, insect community, species richness, arid environments, caatinga. corresponding author lázaro da s. carneiro departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina, s/nº, novo horizonte cep 44036-900, feira de santana-ba, brasil. e-mail: lazarocarneiro16@gmail.com different phytophysiognomies located near the caatinga vegetation, such as riparian forests of the são francisco river (neves & viana, 1999; moura & schlindwein, 2009), while few studies focused on the euglossine assemblages in the typical xerophytic vegetation of caatinga (lopes et al., 2007; andrade-silva et al., 2012). habitat fragmentation has been recognized as one of the main causes of biodiversity loss (fahrig, 2003; krauss et al., 2010; haddad et al., 2015), while changes in abundance and species richness related to land use pressures also have been observed (laurence et al., 2011; newbold et al., 2015). the caatinga is a xerophilous vegetation in the northeast of brazil that occupies an area of approximately 750.000 km², reduced to almost half of its original area (ibge, 2015). the fragmentation these ecosystems is related to the proximity of cities and road construction (santos & tabarelli, 2002), livestock, slash and burn agriculture and use of wood for coal production (leal et al., 2005; holanda et al., 2015). the scarcity of knowledge on euglossine assemblages in 1 departamento de ciências biológicas, universidade estadual de feira de santana, bahia, brazil 2 instituto do meio ambiente e recursos hídricos, salvador-ba, brazil research article bees mailto:lazarocarneiro16@gmail.com ls carneiro, wm aguiar, cml aguiar & gmm santos – occurrence of euglossini bees in caatinga254 dry environments, and the high level of degradation of the seasonally dry tropical forest (caatinga) (leal et al., 2005; ibge, 2015) motivated the planning of this study, which aimed to characterize the euglossine assemblage in two fragments of caatinga, as well as to evaluate the distribution of euglossini species richness in the caatinga. material and methods study area the study was carried out in two fragments of caatinga located in the municipality of pé de serra, state of bahia, brazil. these fragments are 4 km apart and are surrounded by a predominant pasture matrix. fragment i (11°54’ s; 39°31’ w) has approximately 50 ha, consisting of a shrub caatinga vegetation (sema, 2014) in regeneration, and there is some goat and sheep farming in this site. fragment ii (11° 57’ s 39° 32’ w) has approximately 220 ha, is covered by arboreal and shrub caatinga (sema, 2014). the local vegetation is generally dense, although the site has already suffered burnings. the area of study has dry climate of type bsh according to classification of köppen. the rainy season lasting from november to march and annual rainfall around 658 mm (sei, 2014). annual rainfall was 495 mm during this study, which is below the expected average. rainfall data were obtained from the instituto estadual do meio ambiente (inema). methods males were collected monthly from november 2015 to october 2016, totalizing 216 h of sampling effort per site. sampling was carried out on sunny days and without rain. in each fragment, a sampling point was selected, where seven bait traps were hung from a height of 1.5 m above the ground, at least 2 m from each other. seven aromatic compounds (benzyl acetate, β-ionone, methyl cinnamate, eucalyptol, eugenol, methyl salicate and vanillin) were soaked in cotton and inserted into traps made from plastic bottles, in which three lateral openings were made to insert funnels, following a model proposed by aguiar and gaglianone (2008).traps were installed between 7:30 a.m. and 8:00 a.m., remaining exposed for two days. the bees caught on the first day of sampling were removed from the traps at the beginning of the second day, immediately before reapplication of the baits. the specimens were identified according to the taxonomic keys (nemésio, 2009; faria jr & melo, 2012) and confirmed by bee taxonomy specialist. we followed the moure’s bee catalog (moure et al., 2012) for species nomenclature. data analysis we have used the rank-abundance plot, where the relative abundance of species is determined in descending order (whittaker, 1965), to evaluate the distribution of species abundance in each fragment. additionally, the shannonwiener index (h’) was used to calculate diversity of species, following the algorithm h’= σpi x ln pi, where pi is the proportion of individuals of the species i represented in the sample and ln = neperian logarithm (magurran, 2004). pielou index (j’) was used to calculate the uniformity of abundance, following the algorithm j’= h’ / log2s, where h’= shannon diversity index and log2s = logarithm in base two of species richness (magurran, 2004). dominance was evaluated using berger-parker index (d), defined as d = nmax/n, where nmax = number of individuals of the most abundant species, n = total sample number. a linear correlation of sperman’s was performed to investigate the relation between abundance and rainfall. these indexes were calculated in the past 2.17c program (hammer et al., 2001). in order to evaluate if the sampling effort was enough to sample the species richness in each fragment, we used richness estimators (chao 1 and jackknife 1), calculated in the estimates 9.1 software (cowell, 2015). to analyze the distribution of euglossini species richness in different areas of the caatinga, we reviewed data published in studies on euglossini assemblages in which aromatic baits were used to attract males, totalizing nine sites sampled: 1. site parque estadual pedra da boca (zanella & martins, 2005); 2. site cacimba de dentro (zanella & martins, 2005); 3. site 1 (lopes et al., 2007); 4. site 2 (lopes et al., 2007); 5. site 3 (lopes et al., 2007); 6. site 4 (lopes et al., 2007); 7. site pindoba (andrade-silva et al., 2012); 8. site cambuí (andrade-silva et al., 2012); 9. site braúna (andrade-silva et al., 2012). from these data, we elaborated a table presenting the distribution of species by caatinga phytophysiognomy (arboreal or shrub) and species accumulation curves in function of the sample effort, given by the number of localities sampled. results the euglossini assemblage sampled was composed by five species (table 1). the species richness expected was the same as that observed in fragment i (chao1= 2.00±0.17; jackknife1=2.92±0.92) while in fragment ii, the species richness expected was higher than that observed (chao1=6.09±2.41; jackknife=6.86±1.72). abundance was also higher in fragment ii than in fragment i. euglossa cordata (linnaeus) was the most abundant species, representing 70% of the males collected, contributing to the highest relative abundance in both fragments (fig 1). three euglossa species were recorded only in fragment ii. although diversity was higher in fragment ii (h’= 0.977) compared to fragment i (h’= 0.636), the values did not differ significantly (t= -1.311, p= 0.245). the uniformity was higher in fragment i (j’= 0.92) than in fragment ii (j’= 0.607) (table 1). the highest abundance of euglossini was found in january (fig 2), a rainy month, influenced by the abundance of eg. cordata. in fragment ii, a number of males was in activity in march/2016, a month with low precipitation (fig 2). sociobiology 65(2): 253-258 (june, 2018) 255 eg. cordata was recorded in fragment i only in months with the highest rainfall (january and october), but in fragment ii it occurred in both rainy and dry months. eulaema nigrita lepeletier was recorded only in january, the rainiest month. there was no correlation between monthly abundance and rainfall (r = 0.411, p = 0.183). comparing euglossini species richness in different areas of caatinga, we observed that arboreal caatinga have a richer euglossini fauna (up to 14 species per assemblage) than shrub caatinga, in which only two species have been recorded (el. nigrita and eg. cordata) (table 2). the number of species registered in the site 9 (braúnas) (s = 14) was significantly higher than in the other sites (mean = 4.45; standard deviation = 3.70), and this assemblage had four exclusive species. species accumulation curves did not stabilize, including or excluding site 9 from the analysis (fig 3). species fragment i fragment ii total euglossa cordata (linnaeus) 2 26 28 euglossa securigera dressler 0 1 1 euglossa fimbriata moure 0 2 2 euglossa sp. 0 3 3 eulaema nigrita lepeletier 1 5 6 abundance 3 37 40 species richness 2 5 5 diversity (h’) 0.636 0.977 0.970 uniformity (j’) 0.918 0.607 0.603 dominance (d) 0.666 0.702 0.700 table 1. composition, species richness, diversity, uniformity and dominance in euglossini assemblages in two fragments of caatinga. fig 1. rank abundance plot for euglossini species in two fragments of caatinga (1 = eg. cordata; 2 = el. nigrita; 3 = euglossa sp; 4 = eg. fimbrita; 5 = eg. securigera). fig 2. rainfall and monthly flutuaction in abundance of euglossini bees in fragments of caatinga. fig 3. species accumulation curves in function of the number of localities sampled in caatinga vegetation. the solid line represents the curve based on all the sites (n = 11) sampled in the caatinga, and the dotted line the curve excluding the site 9 (braúnas). 1. site parque estadual pedra da boca (zanella & martins, 2005); 2. site cacimba de dentro (zanella & martins, 2005); 3. site 1 (lopes et al., 2007); 4. site 2 (lopes et al., 2007); 5. site 3 (lopes et al., 2007); 6.site 4 (lopes et al., 2007); 7. site pindoba (andrade-silva et al., 2012); 8. site cambuí (andrade-silva et al., 2012); 9. site braúna (andrade-silva et al., 2012); 10. fragment i (this study); 11. fragment ii (this study). discussion the species richness and abundance of euglossine bees found were similar to that observed in most areas covered by seasonally dry tropical forest (caatinga). the euglossine assemblages in the caatinga are commonly composed of two to five species (lopes et al., 2007; zanella & martins, 2005; this study), but there are some assemblages with higher species richness. a single assemblage in arboreal caatinga was composed of more than eight species (andrade-silva et al., 2012). the assemblages with the highest species richness were found in arboreal caatinga with canopy 5 to 12 m in high, located in areas with higher humidity, in the chapada diamantina highlands, a region characterized by a mosaic of different types of vegetation (semideciduous forest, riparian forests, savanna and caatinga), located at relatively close distances between each other, forming a complex landscape (juncá et al., 2005). the total number of euglossini species recorded in caatinga vegetation is fifteen, with a single endemic species, eufriesea nordestina (moure) (zanella & martins, 2005) and one undescribed species of eulaema (andrade-silva et al., ls carneiro, wm aguiar, cml aguiar & gmm santos – occurrence of euglossini bees in caatinga256 2012). however, due to the small number of studies, it is still difficult to measure the gamma diversity of euglossini bees. the species accumulation curves did not have tendency towards stabilization, indicating that sampling at other sites should reveal other species of euglossini bees living in the caatinga. the change from shaded vegetation to open vegetation seems to influence euglossini assemblages, leading to a decrease in the species richness, both in the brazilian savanna (cerrado) (nemésio & faria jr., 2004; alvarenga et al., 2007; silveira et al., 2015), and caatinga (neves & viana, 1999; lopes et al., 2007; andrade-silva et al., 2012). andradesilva et al. (2012) showed that temperature was the most important environmental variable to explain variations on composition and abundance of euglossini in three caatinga fragments. moura and schlindwein (2009) pointed out that although some species of euglossini bees from the atlantic forest use riparian forests of the são francisco river as habitat corridors, there is a decrease in the number of species towards the interior of the caatinga biome. additionally, the change from arboreal to shrubby vegetation in the caatinga seems to limit the occurrence of most species of euglossini. several species of euglossini bees appear to have restricted geographical distribution in the caatinga, while few species, such as eg. cordata and el. nigrita has greater ecological valence and wider distribution in these xeric areas. these species are tolerant to dry and open environments, and under anthropic influence (peruquetti et al., 1999; tonhasca et al., 2002), which may explain its occurrence in xerophilous caatinga environments. the uniformity and dominance indexes reflected differences in species abundance, since that a single dominant species (eg. cordata) was represented by many individuals in the assemblage, as reported in other euglossini assemblages (alvarenga et al., 2007; aguiar & gaglianone, 2008; rocha-filho & garófalo, 2013; silveira et al., 2015; vilhena et al., 2017). this study reinforces the idea of low species richness of orchid bees in xerophilous caatinga vegetation. additional studies focusing on the influence of habitat loss and fragmentation on these bee assemblages in the seasonally dry tropical forest are required to plan conservation strategies for these bee populations, since there is a high level of degradation of the caatinga environment. acknowledgements we thank to gabriel a. r. melo (ufpr) for the identification of the reference specimens, es aniceto for language revision, and the owners of the farm for allowing the collections. ls carneiro and gmm santos received a scientific initiation and research productivity fellowships, respectively, from cnpq (conselho nacional de desenv. científico e tecnológico). arboreal caatinga shrub caatinga 1 2 3 6 7 8 9 11 4 5 10 eufriesea nordestina (moure) _ x _ _ _ _ _ _ _ _ _ eufriesea auriceps (friese) _ _ _ _ x x x _ _ _ _ euglossa townsendi¹ cockerell _ _ _ _ _ _ x _ _ _ _ euglossa cordata² (linnaeus) x x x x x x x x x x x euglossa fimbriata moure _ _ _ _ _ _ x x _ _ _ euglossa leucotricha rebêlo & moure _ _ _ _ x _ x _ _ _ _ euglossa melanotricha moure _ _ _ _ x x x _ _ _ _ euglossa pleosticta dressler _ _ _ _ _ x x _ _ _ _ euglossa securigera dressler _ _ x _ x x x x _ _ _ euglossa sp. x _ _ _ _ _ _ _ _ _ _ euglossa sp.1 _ _ _ _ _ _ _ x _ _ _ euglossa stellfeldi moure _ _ _ _ _ _ x _ _ _ _ euglossa truncata rebêlo & moure _ _ _ _ _ _ x _ _ _ _ eulaema cingulata3 (fabricius) _ _ _ _ x x x _ _ _ _ eulaema nigrita lepeletier x _ x x x x x x x _ x eulaema sp. _ _ _ _ _ x x _ _ _ _ exaerete dentata (linnaeus) _ _ _ _ _ _ x _ _ _ _ 1cited as euglossa aratingae by andradesilva et al. (2012); 2cited as euglossa carolina by andradesilva et al. (2012); 3cited as eulaema marcii by andrade-lima et al. (2012). table 2. composition and species richness in euglossini assemblages in different caatinga phytophysiognomies. 1. site parque estadual pedra da boca (zanella & martins, 2005); 2. site cacimba de dentro (zanella & martins, 2005); 3. site 1 (lopes et al., 2007); 4. site 2 (lopes et al., 2007); 5. site 3 (lopes et al., 2007); 6. site 4 (lopes et al., 2007); 7. site pindoba (andrade-silva et al., 2012); 8. site cambuí (andradesilva et al., 2012); 9. site braúna (andrade-silva et al., 2012); 10. fragment i (this study); 11. fragment ii (this study). sociobiology 65(2): 253-258 (june, 2018) 257 references aguiar, w.m. & gaglianone, m.c. 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(eds.), análise das variações da biodiversidade do bioma caatinga: suporte a estratégias regionais de conservação, (pp. 379-391). brasília: ministério do meio ambiente http://www.moure.cria.org.br/catalogue/ https://www.nature.com/articles/nature14324#auth-1 https://www.nature.com/articles/nature14324#auth-2 https://www.nature.com/articles/nature14324#auth-3 https://www.nature.com/articles/nature14324#auth-4 https://www.nature.com/articles/nature14324#auth-5 https://www.nature.com/articles/nature14324#auth-6 https://www.nature.com/articles/nature14324#auth-7 https://www.nature.com/articles/nature14324#auth-8 https://www.nature.com/articles/nature14324#auth-9 https://www.nature.com/articles/nature14324#auth-10 https://www.nature.com/articles/nature14324#auth-11 https://www.nature.com/articles/nature14324#auth-13 https://www.nature.com/articles/nature14324#auth-14 https://www.nature.com/articles/nature14324#auth-15 https://www.nature.com/articles/nature14324#auth-16 https://www.nature.com/articles/nature14324#auth-17 https://www.nature.com/articles/nature14324#auth-18 https://www.nature.com/articles/nature14324#auth-19 https://www.nature.com/articles/nature14324#auth-19 https://www.nature.com/articles/nature14324#auth-20 https://www.nature.com/articles/nature14324#auth-21 https://www.nature.com/articles/nature14324#auth-22 https://www.nature.com/articles/nature14324#auth-23 https://www.nature.com/articles/nature14324#auth-24 https://www.nature.com/articles/nature14324#auth-24 https://www.nature.com/articles/nature14324#auth-25 https://www.nature.com/articles/nature14324#auth-26 https://www.nature.com/articles/nature14324#auth-27 https://www.nature.com/articles/nature14324#auth-28 https://www.nature.com/articles/nature14324#auth-28 https://www.nature.com/articles/nature14324#auth-29 https://www.nature.com/articles/nature14324#auth-30 https://www.nature.com/articles/nature14324#auth-31 https://www.nature.com/articles/nature14324#auth-32 https://www.nature.com/articles/nature14324#auth-33 https://www.nature.com/articles/nature14324#auth-34 https://www.nature.com/articles/nature14324#auth-35 https://www.nature.com/articles/nature14324#auth-36 https://www.nature.com/articles/nature14324#auth-37 https://www.nature.com/articles/nature14324#auth-38 https://www.nature.com/articles/nature14324#auth-39 https://www.nature.com/articles/nature14324#auth-40 https://www.nature.com/articles/nature14324#auth-41 http://dx.doi.org/10.1038/nature14324 http://www.inema.ba.gov.br/wp-content/files/mtematicos_vegetacao.pdf/ http://www.inema.ba.gov.br/wp-content/files/mtematicos_vegetacao.pdf/ doi: 10.13102/sociobiology.v60i3.222-228sociobiology 60(3): 222-228 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant fauna on cecropia pachystachya trécul (urticaceae) trees in an atlantic forest area, southeastern brazil pcj reis, wd darocha, lad falcão, tj guerra & fs neves introduction cecropia genus comprises pioneer trees which are characteristic elements of forest borders and gaps in the neotropics (vasconcelos & casimiro, 1997; sposito & santos, 2001). most species are inhabited by mutualistic ants that nest inside hollow internodes where they store eggs, larvae and pupae (harada & benson, 1988), and feed on glycogenrich müllerian bodies called trichilium located at the base of leaf petioles (yu & davidson, 1997). although cecropia can host a variety of resident ant genera, azteca spp are the most common ant inhabitants (longino, 1991a). usually only one mature azteca colony inhabits a cecropia tree, however up to five azteca species may be found in a cecropia population (longino, 1991a). in addition to a resident azteca colony, other ant species can be found living or foraging on the same cecropia tree (vieira et al., 2010). however, the diversity of non-azteca ants living on cecropia trees or using them as foraging substrates is still poorly known, as well as the factors that might affect ant richness and abundance on these trees. abstract cecropia are pioneer successional trees frequently associated with ants. generally a single dominant colony of azteca ant inhabits each mature cecropia tree, but other ant species may be found living or foraging on the same tree. in this study, we assessed the diversity of ant species on cecropia pachystachya trees in two sites in the brazilian atlantic forest: a dust-free roadside and a dusty roadside. we also investigated the influence of tree architecture on ant species richness. we found a total of 24 ant species distributed in 11 genera and five subfamilies on c. pachystachya trees; 18 in the dust-free roadside and 14 in the dusty roadside. we found up to five ant species on a single tree, but only azteca alfari was frequently encountered. ant species richness per tree did not differ significantly between sites and was related to tree architectural traits. on the other hand, ant species composition on trees differed significantly between sites. our study indicates that heavy dust deposition on cecropia trees may affect associated ant communities, not by changing ant species richness, but by causing different species to live and forage on trees under different dust exposure. sociobiology an international journal on social insects universidade federal de minas gerais (ufmg), belo horizonte, minas gerais, brazil. research article ants article history edited by kleber del-claro, ufu, brazil received 07 june 2013 initial acceptance 29 june 2013 final acceptance 25 july 2013 keywords azteca, road dust, roadside, plant architecture corresponding author paula campos junqueira reis prog. de pós-graduação em ecologia, cons. e manejo da vida silvestre universidade federal de minas gerais belo horizonte, mg, brazil, 36580-000 e-mail: paulacjr@gmail.com complex tree architecture is an indicative of resource diversity and availability, such as nesting sites, sites for oviposition, and food (lawton, 1983). therefore, tree architecture might be an important factor affecting the diversity of ant fauna associated to trees as reported for other insects (haysom & coulson, 1998; espírito-santo et al., 2007; neves et al., 2013). cecropia trees are known for their great variation in size and architectural traits (sposito & santos, 2001), but the way this variation affects the associated ants remains unexplored. distribution of ants on cecropia trees is also likely influenced by external factors such as changes of environmental conditions caused by human activities, which can directly or indirectly affect both plants and ants. the brazilian atlantic forest range largely coincides with the most populated areas of brazil (tabareli et al., 2005), thus this forest ecosystem suffers with impacts of a dense road network. roads are known to have many ecological effects on surrounding biota (spellerberg, 1998), mainly by altering landscape spatial patterns and processes and modifying abiotic and biotic conditions (forman & sociobiology 60(3): 222-228 (2013) 223 alexander, 1998; trombulak & frissell, 2000; barbosa et al., 2010). besides, production of dust either by cars exhausts or road surfaces may have important potential impacts on vegetation in roadsides including plant mortality (spellerberg, 1998), especially on unpaved roads where dust production is higher than on paved roads (farmer, 1993). dust deposition on leaves may increase water loss, cause leaf injuries, also leading to changes in associated invertebrate communities (farmer, 1993). nevertheless, the effects of roadside dust on ant fauna associated to cecropia trees have never been studied so far. in this context, our aim was to assess the diversity of ant fauna on cecropia pachystachya trécul (urticaceae) trees in a brazilian atlantic forest area in southeastern brazil. we evaluated ant species richness and composition on trees located in two roadside forest sites: one where plants were heavily covered by road dust and another where dust on vegetation was negligible. we also evaluated the relationship between ant species richness and the architectural traits of c. pachystachya trees. we hypothesized that ant species richness on trees should be positively related to higher complexity in plant architecture and should be lower in plants disturbed by heavy dust deposition. we also hypothesized that contrasting dust deposition should affect the composition of ant fauna associated to c. pachystachya trees. material and methods study area this study was conducted in the rio doce state park (parque estadual do rio doce-perd hereafter) (19°40’s; 42°33’w) and its surroundings in minas gerais state, southeastern brazil (fig 1). perd is the largest continuous remnant of atlantic forest in the state (c. 36,000 ha) (instituto estadual de florestas de minas gerais [ief-mg], 2012) and thus has a major importance to the regional conservation of this biome. it is surrounded by agriculture, pasturelands, large areas of eucalyptus plantations, metallurgical plants, and a substantial road network (brito et al., 1997). cecropia trees are abundant in early successional stages of forest both inside and outside the park, and at the roadsides of unpaved roads cecropia trees are often heavily covered by dust (fig 2). fig 1. rio doce state park in southeastern brazil. the circle shows the dust-free sampling area inside the park. adapted from ibge/ brasil topographical map by philippe maillard instituto de geociências-igc, universidade federal de minas gerais. fig 2. cecropia pachystachya leaves. a inside rio doce state park, not covered by road dust. b covered by unpaved road dust in the surroundings of the park. pcj reis, wd da rocha, lad falcão, tj guerra & fs neves ant fauna on cecropia pachystachya trees224 sampling data were collected in july 2012 in two atlantic forest early successional sites: a dust-free roadside nearby lake carioca inside perd and a dusty roadside in the surroundings of the park. although both sites are roadsides, the one inside perd is located in a rarely used airplane landing field while the road outside the park connects small municipalities and is frequently used by cars. to sample the associated ant fauna, we selected 25 mature cecropia pachystachya trees (circumference at breast height (cbh) ≥ 20 cm) at least 30 m away from each other along a transect in each site. ants found on leaves and trunks were manually captured with the assistance of a ladder and a pole pruner, and additional arboreal pitfall traps with sardine as bait were used to catch ants that we did not capture manually (bestelmeyer et al., 2000; ribas et al., 2003). pitfalls were set up at approximately 1.50 m height in c. pachystachya trunks and were removed 48 hours later. in order to avoid that ground-living ants were caught, we used grease in the trunks below pitfall traps. all ants caught were taken to the myrmecology laboratory of cepec/ceplac in ilhéus, bahia. in this laboratory, ant species were identified after preparation and deposited in the collection referred by cpdc acronym (#5687). for each c. pachystachya tree we measured the following architectural parameters: cbh, total height, number of branches and leaves, and first branch height (fbh) following sposito & santos (2001). we used ground-based methods of access (von matter et al., 2010) because of the difficulty of sampling very tall and fragile trees. total height of trees was measured with a 12 m pole, and branches and leaves were counted by eye. statistical analyses in order to evaluate the effectiveness of our sampling effort, we constructed rarefaction curves for each site using mao tao observed richness based on 100 randomizations. a generalized linear model (glm) was constructed to investigate the effect of area (dusty and dust-free roadsides; explanatory variable) on ant richness (response variable). difference in ant species composition between sites was tested using non-metric multidimensional scaling (nmds) with raup-crick index for incidence data and analysis of similarities (anosim). anosim provides a way to check whether there is a significant difference between groups or not (clarke, 1993). similarity percentage (simper) analysis was used to determine which ant species mostly contributed to differentiation between groups (dust-free and dusty sites). in order to determine which architectural variables (cbh, total height, number of branches and leaves, or fbh) affected ant species richness, we used hierarchical partitioning. then, generalized linear models (glms) were built to estimate the effect of these pre-selected architectural variables (explanatory variables) on ant species richness (response variable). residual analyses were conducted to check data adequacy to the probability distribution used as well as error distribution (crawley, 2007). species rarefaction curves were constructed on estimates 8.0 software (colwell, 2006); nmds, anosim and simper were carried out on past (paleontological statistics) version 2.15 (hammer et al., 2001); hierarchical partitioning and glms were performed using software r 2.6.2 (r development core team, 2008). results and discussion a total of 24 ant species distributed in 11 genera and five subfamilies were found on cecropia pachystachya trees (table 1). the absence of stabilization of the rarefaction curves (fig 3) indicated that ant species richness associated to c. pachystachya trees is probably underestimated in both study sites. therefore, additional studies are needed to determine more accurately the richness of ant species associated to these trees. formicinae and myrmicinae were the most representative subfamilies with eight and nine species respectively (71% of all ant species found); and camponotus mayr, 1861 was the most representative genus with seven species. although more than one azteca species can inhabit a single population of cecropia trees (longino, 1991a), we only found azteca alfari emery, 1893 on the trees we analyzed. azteca alfari is known to inhabit cecropia trees located in open and disturbed habitats such as the early successional sites we studied while other azteca species such as azteca constructor and azteca xanthochroa are more common in older forest stages (longino, 1991b). the presence of various non-azteca ant genera such as camponotus mayr, 1861, crematogaster lund, 1831 and pseudomyrmex lund, 1831 (table 1) on trees of cecropia was also reported by longino (1991a) and their presence indicates low aggressiveness of the azteca resident colony. as we observed on the field, some c. pachystachya trees inhabited by a. alfari also housed other ant genera nests (e.g. cephalotes species). indeed, a. alfari along with azteca ovaticeps forel, 1904 are the least aggressive species among all cecropia-inhabiting azteca ants (longino, 1991a). eighteen ant species were found on c. pachystachya trees in the dust-free site while 14 were found on the road dust covered trees. only eight ant species were common to both sites, six species were found exclusively in the dusty roadside and 10 were exclusive of the dust-free roadside inside the park (table 1). azteca alfari emery, 1893 and camponotus senex (smith, f., 1858) were the most frequent species on c. pachystachya trees in the dusty roadside, while more species were frequent in the dust-free area e.g. camponotus balzani emery, 1894. unlike the expected, each c. pachystachya tree under heavy dust deposition held an average number of ant species similar to each tree in the dust-free roadside (dusty roadside: 2.12±0.24; dust-free roadside: 2.25±0.21; deviance1,47 = 0.094; p = 0.687). sociobiology 60(3): 222-228 (2013) 225 to cbh (fig 5; p = 0.001). low fbh and large cbh are indicatives of structural complexity, since low fbh trees usually have long and dense crowns, and high cbh trees are generally large. larger crowns and trees probably support higher resource availability and higher diversity of conditions than smaller trees (campos et al., 2006) allowing more ant species to live or forage on the same tree. in fact, other studies found similar results of increase in ant and/or other insects’ richness with increase in tree or crown size (e.g. campos et al., 2006; costa et al., 2011; neves et al., 2013). table 1. ant species captured on cecropia pachystachya trees in rio doce state park and surroundings, southeastern brazil. sampled sites were a dusty roadside (in the surroundings of the park) and a dust-free roadside (inside the park). fig 3. rarefaction curves of ant species captured on cecropia pachystachya trees in the dusty roadside and in the dust-free roadside in rio doce state park and surroundings, southeastern brazil. bars denote 95% ci. among the architectural variables evaluated, only first branch height (fbh) and circumference at breast height (cbh) were significantly related to ant species richness. ant species richness on trees ranged from zero to five, being negatively related to fbh (fig 4; p = 0.02) and positively related fig 4. effect of first branch height (fbh) of cecropia pachystachya trees on ant species richness in rio doce state park and surroundings, southeastern brazil. composition of ant species found on c. pachystachya trees was significantly different between sampling sites (ranosim = 0.039; p = 0.04; fig. 6). simper analysis revealed that camponotus senex (smith, f., 1858), crematogaster curvispinosa mayr, 1862, camponotus balzani emery, 1894 and azteca alfari emery, 1893 were the species that mostly contributed to differentiation of groups and together explained 40.21% of the dissimilarity between sites (table 2). these ant genera (table 2) are commonly associated with myrmecophytes and are classified as dominant arboreal ants (brandão et al., 2011). therefore, ant fauna on c. pachystachya trees differed between sampling sites mainly due to dominant species that live on the trees, while opportunistic ants had a smaller contribution to differentiation between dusty and dust-free roadsides. camponotus senex is conspicuous and can occur both in mature and disturbed forests, nesting in high regions of canopy including dead branches of cecropia trees (longino, 2002). the greater frequency of c. senex in the dusty roadside (table 1) might be related to the abundance of dead branches on c. pachystachya trees in this site, as observed by us in the field. in turn, the abundance of dead branches in the dusty roadside could possibly be caused by road dust effects on c. pachystachya trees such as increased water loss. the second species that most contributed to differentiation between sites crematogaster curvispinosa (myrmicinae) is a very common but inconspicuous ant that feeds on extrafloral nectaries and inhabits initial secondary forests as well as highly disturbed areas such as roadsides (longino, 2003). subfamily/species number of c. pachystachya trees with given ant species dusty roadside (n=25) dust-free roadside (n=25) dolichoderinae azteca alfari emery, 1893 24 22 dolichoderus diversus emery, 1894 1 dolichoderus lutosus (smith, f., 1858) 1 ectatomminae ectatomma tuberculatum (olivier, 1791) 3 formicinae brachymyrmex sp.01 1 camponotus balzani emery, 1894 2 4 camponotus cingulatus mayr, 1862 1 camponotus gp. fastigatus sp.01 2 camponotus renggeri emery, 1894 3 3 camponotus rufipes (fabricius, 1775) 2 camponotus senex (smith, f., 1858) 9 3 camponotus vittatus forel, 1904 1 myrmicinae atta rubripilosa forel, 1908 1 cephalotes goeldii (forel, 1912) 1 cephalotes minutus (fabricius, 1804) 1 cephalotes pusillus (klug, 1824) 1 4 crematogaster brasiliensis mayr, 1878 1 crematogaster curvispinosa mayr, 1862 2 4 nesomyrmex costatus (emery, 1896) 1 solenopsis sp.01 2 2 solenopsis sp.02 1 pseudomyrmecinae pseudomyrmex gracilis (fabricius, 1804) 3 1 pseudomyrmex oculatus (smith, f., 1855) 1 pseudomyrmex schuppi (forel, 1901) 1 total species 14 18 pcj reis, wd da rocha, lad falcão, tj guerra & fs neves ant fauna on cecropia pachystachya trees226 it can tolerate other ant species nests nearby it (e.g. camponotus and cephalotes) and its colonies can occupy cecropia internodes that are located between resident azteca nests (longino, 2003). for instance, or it could indirectly affect ants through effects on vegetation that generate avoidance of dusty trees by some ant species and preference by others (e.g. dead branches for camponotus senex). cecropia pachystachya trees under heavy dust deposition may also have impaired development and growth which may reduce the quantity and quality of essential resources for resident and opportunistic ants. in addition, dust may have effects on associate invertebrate communities and these changes likely affect ant fauna especially through competition for resources. finally, our results indicated that effects of road dust might transcend impacts on vegetation and might have important impacts on animal associate communities especially ants. taxon av. dissim. contrib. % cumulative % mean abund. 1 mean abund. 2 camponotus senex 8.202 15.51 15.51 0.36 0.125 crematogaster curvispinosa 4.816 9.105 24.61 0.08 0.167 camponotus balzani 4.205 7.949 32.56 0.08 0.167 azteca alfari 4.047 7.651 40.21 0.96 0.917 table 2. similarity percentage (simper) analysis outcome. av. dissim.: average dissimilarity of taxon between sites; contrib. %: relative contribution of taxon to dissimilarity between sites; cumulative %: cumulative contribution of taxon to dissimilarity between sites; mean adund. 1: mean abundance of taxon in the dusty roadside; mean abund. 2: mean abundance of taxon in the dust-free roadside. despite its high tolerance in disturbed areas, c. curvispinosa was more abundant inside the park possibly because of the abundance of shrubs in the dust-free roadside in contrast to the scarcity of these in dusty site. abundant shrubs are indicatives of higher availability of extrafloral nectaries which are important food sources to c. curvispinosa. dust deposition might limit the abundance of shrubs outside the park through negative effects on plants as well as block access to the remaining extrafloral nectaries in the dusty roadside. as shown by our results, composition of ant communities on cecropia pachystachya trees differed between sites with distinct exposure to dust. the substantial difference in fig 5. effect of circumference at breast height (cbh) of cecropia pachystachya trees on ant species richness in rio doce state park and surroundings, southeastern brazil. acknowledgments this study was conducted during the ecology field course fully supported by the graduate program in ecologia, conservação e manejo da vida silvestre (ufmg) and ief-mg (instituto estadual de florestas de minas gerais). pcjr and wddr were granted cnpq (conselho nacional de desenvolvimento científico e tecnológico) scholarships and ladf was granted a capes (coordenação de aperfeiçoamento de pessoal de nível superior) scholarship. we thank two other anonymous referees for their comments and criticisms on the manuscript. references barbosa, n.p.u., fernandes, g. w., carneiro, m. a. a. & carlos, l. a. 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(1997). experimental studies of speciesspecificity in cecropia-ant relationships. ecol. monogr., 67: 273-294. doi: 10.2307/2963456 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1684sociobiology 64(4): 451-455 (december, 2017) two more social wasp species (vespidae, polistinae) collect mullerian bodies from cecropia trees (urticaceae) introduction associations between plants and insects have long been known to science and may be among the oldest coevolved systems on our planet (bronstein et al., 2006; mishra et al., 2015). the evolution of both specific and generalist interactions has certainly facilitated great diversification among both plants and insects (kasting & catling, 2003). the neotropical genus cecropia loefling (cecropieae, urticaceae) is commonly a participant in plant-insect mutualistic associations (weiblen & treiber 2015), with a large majority of its species (ca. 70) being myrmecophytic and developing specialized structures for sheltering ants (mostly the genus azteca roger; dolichoderinae) (treiber et al., 2016). cecropia trees provide nesting domatia and food bodies (mullerian bodies mbds) produced by “trichilia” (sing. trichilium; pad-like organs located on the base of the leaf petiole) (oliveira et al., 2015), and on the leaves (gonçalves-sousa & paiva, 2016). in exchange, ants provide nutrients to the plants and help protect them against herbivory. abstract the interactive behaviors of two species of social wasps, protopolybia chartergoides (gribodo) and charterginus fulvus fox with cecropia obtusifolia (cecropiaceae) are described. the use of cecropia müllerian bodies by both wasps is also described, as well as the distribution of this trait in protopolybia and charterginus. sociobiology an international journal on social insects sps felizardo, rc borges, jna santos, ot silveira article history edited by gilberto m. m. santos, uefs, brazil received 02 may 2017 initial acceptance 28 july 2017 final acceptance 04 october 2017 publication date 27 december 2017 keywords protopolybia chartergoides, charterginus fulvus, insect-plant interaction, herbivory. corresponding author orlando t silveira museu paraense emílio goeldi (mpeg-mcti) coordenação de zoologia av. perimetral, 1901, campus de pesquisa cep 66077-830, belém-pa, brasil. e-mail: orlando@museu-goeldi.br three social wasp species have been reported to interact with cecropia-ant associations. the first case was described by richards (1978), who collected protopolybia chartergoides (gribodo) on the trichilia pads of a cecropia sp. tree, and suggested that its trichomes were used in nest construction by the wasp. lapierre et al. (2007) made intensive observations of charterginus xanthura (de saussure) and c. nevermanni bequaert collecting mullerian bodies (mbds) on different cecropia species in french guiana and costa rica. these wasps can collect mbds from trichilia pads while individually foraging (although they are susceptible to aggressive encounters by patrolling ants), or they may use a group strategy (c. xanthura) in which one of the wasps faces a domatia entrance and prevents any ants from leaving the nest, while other wasps collect mbds from the trichilium. the present article originated from our observations of workers of charterginus fulvus fox bringing aggregates of minute whitish ovoid bodies to their nests. after consulting the paper by lapierre et al. (2007) where use of mbds by charterginus species is reported, the origin of this material was museu paraense emilio goeldi (mpeg), belém-pa, brazil research article wasps sps felizardo, rc borges, jna santos, ot silveira – social wasp species collect mullerian bodies452 confirmed by observations of c. fulvus and p. chartergoides visiting the trichilia of cecropia trees at the goeldi museum campus, in belém, brazil. both wasps are epiponini swarming species, with largely amazonian distributions (richards, 1978). we have observed those wasps collecting trichomes and mullerian bodies; their use of trichomes in nest construction is treated in another paper (borges et al., 2017). to better describe the interactions between these wasps and cecropia trees and ants, we made additional observations of their activities (mainly on cecropia obtusifolia bertol. trees) in the city of belém as well as other nearby localities in pará state, brazil. the following aspects were addressed: (1) frequency and associative aspects of the occurrence of wasps on mbd producing trees; (2) the wasps’ foraging behaviors while on the trees; (3) interactions between wasps and ants. methods observations were carried out mainly at the museu paraense emílio goeldi research campus (mpeg) (1°27’52”s x 48°26’36”w), belém, pará state, brazil. at the mpeg campus, data were collected on working days, during fifteen months, from september/2015 to december/2016, focusing on a set of seven cecropia trees, generally 2 m tall, spatially arranged in four groups, up to 350 m from each other, in an approximately 3.5 ha triangular area in the campus. however, not all of the trees were inspected during the entire 15-month observation period, for reasons varying from pruning by the campus administration (two cases), temporary inaccessibility because of a building reform (one case), or flooding (three cases). in general, all of the trees were observed for at least three months; three of them were observed for six months; and one tree was accompanied during the full observation period. the trees were inspected three times along each working day in a week (early in the morning, around midday, and late in the afternoon). each observation event lasted for approximately one hour, during which time all accessible plants were inspected. the times of mbd formation, as well as wasp foraging activities, and ant and wasp interactions were registered. the pruning of some of the trees eventually turned out to be a convenient circumstance because it “reset” the plantant interaction systems, and because observations became easier on lower trees. being a pioneer species, cecropia trees rapidly initiated regrowth (with some exceptions), with subsequent development of myrmecophytic structures for recolonization by ants. weekly inspections were performed during the initial phases of regrowth to accompany tree and trichilia development and reveal the presence of colonizing ants. regular observations reinitiated once the trees began to produce mullerian bodies. wasps and ants were collected directly on the plants (using entomological forceps) and subsequently identified in the laboratory using the keys published by richards (1978) for wasps, and by longino (2010) for ants. plant identifications were made by dr. mario jardim, of the botany department of the goeldi museum. photographs documenting wasp and ant activities on trees were taken using a canon 600d digital camera. additional observations were made on trees in other localities in belém and other cities in pará state (abaetetuba, acará, and barcarena), and in são josé de ribamar, maranhão state. observations were carried out for only one day in those areas. results wasps´ visits to the trees visits by the two vespid wasps to c. obtusifolia were largely limited to trees still unoccupied by azteca ants, with both wasp species being reasonably common in those cases; the visits occurred even during moderate rains. visitation frequencies could be very high (ca 80% of inspected trees) before tree colonization by the ants, and at the times of day when mbd production was most intense (mainly between 11:30 and 13:30). mature mbds were not commonly found in the early morning and late afternoon, and wasps were present in lower numbers at those times (mostly c. fulvus collecting trichomes). p. chartergoides was by far the most frequent vespid species, and the exclusive wasp in ca. 87% of the observed visits. in 10% of the cases, both p. chartergoides and c. fulvus were observed foraging together on trees (rarely on the same trichilium), and c. fulvus was the sole visiting wasp species in only 3% of cases. ants the ant species camponotus sp. and nylanderia sp. (formicinae), solenopsis cf invicta and crematogaster erecta mayr (myrmicinae), and azteca alfari emery (dolichoderinae) were commonly observed on focal trees. ants other than azteca species did not form resident colonies. protopolybia chartergoides behavior before occupation by azteca ants, p. chartergoides wasp foragers (from only a few, up to seven individuals) were commonly seen on cecropia trees (on nearby leaves) just before the appearance of mbds. the wasps remained largely inactive before the mbds matured, only sporadically inspecting the trichilia pads for mbd availability (fig 1). after the emergence of mbds on any given trichilium, individual foraging wasps would collect the corpuscles and then fly away. after a successful mbd harvesting event, the number of visiting wasps on a tree commonly increased, and two wasps could collect mbds from a single trichilium (fig 2). p. chartergoides wasps visiting a productive trichilium would quickly collect and swallow a small number of mature mbds (estimated between five and ten). the wasp would then regurgitate the material and masticate it for some time, without leaving the plant; they would then swallow it again and fly away. sociobiology 64(4): 451-455 (december, 2017) 453 charterginus fulvus behavior most c. fulvus foragers were observed alone on the trees. they could occasionally be seen in small groups of up to three foragers, but they would forage independently on different trichilia. unlike p. chartergoides, c. fulvus foragers were not observed to remain inactive and wait on a plant for the emergence of mature mbds. they would arrive and then immediately search the trichilia, collecting either trichomes or mbds, if the latter were available. to harvest the mbds, c. fulvus foragers would remove them with their mandibles and mash them into a ball using their mouthparts and forelegs. they were never observed swallowing mbd aggregates. observations on material brought by foragers to a c. fulvus nest on the mpeg campus (see borges et al., 2017), indicated that mbd masses were shared with workers in the nest, in the same way as other collected materials. wasp – ant encounters (non-azteca) before a. alfari colonies were established, other ant species were commonly observed on cecropia trees, camponotus sp. being the most common. encounters between vespid wasps and camponotus ants were clearly antagonistic, with ants consistently being chased away by the wasps. the wasps would typically flap their wings vigorously while facing the ants, but without leaving the trichilium (figs 3 and 4). resident ants after the trees had fully developed myrmecophytic structures, a. alfari ants became more frequent and camponotus ants became scarcer, eventually disappearing. when the domatia were open, azteca ants patrolled the entire plant and collected mbds on trichillia. once a. alfari had established their nests on the trees, wasp visitation frequencies diminished, until they completely stopped. no significant interactions were observed between wasps and a. alfari individuals. observations of plants in different stages of development, and the “resetting” produced by pruning the trees on the mpeg campus, showed that the interrelated phenomena of tree development and colonization by a. alfari limit the use of trichilia resources by the wasp species studied. while mbds are known to be mutualistic rewards for azteca ants, during the early stages of plant development those ants were absent from the trees and p. chartergoides and c. fulvus could freely exploit the trichilia mbds and trichomes. figs 1–4. protopolybia chartergoides and camponotus sp. on cecropia obtusifolia. 1-4, p. chartergoides individuals collecting mullerian bodies; 3-4, interaction between wasp and ant near a trichilium, with evasive behavior of ant. (scales in photographies are similar but not identical) sps felizardo, rc borges, jna santos, ot silveira – social wasp species collect mullerian bodies454 observations in other areas the same social wasp species were observed using cecropia tree resources in all of the other study locations, except maranhão state, as c. fulvus was not observed in são josé de ribamar. actually, this species has not been recorded from that state (see andena et al., 2009; richards, 1978). observations in these places also focused on relatively young trees (less than three meters tall, with diameters ranging from 1 to 10 cm). as observed in the mpeg campus, the wasps in other areas collected both mbds and trichomes, and trees with resident and patrolling ants showed reduced numbers of visiting wasps. discussion our observations showed that at least two more social wasp species harvest mullerian bodies on cecropia trees in the neotropics, one of them being a member of the genus protopolybia (with no previously published reports on that behavior). p. chartergoides was a far more frequent visitor to cecropia than c. fulvus, and its mbd collecting behavior was more elaborate, involving sequential acts of swallowingregurgitating-masticating and then swallowing the material again, to achieve a full load to transport to the nest. c. fulvus transported mbds externally (as aggregates) held between their mouthparts and legs. different from c. xanthura (lapierre, 2007), neither of these species was a frequent visitor, nor were they successful at harvesting mbds during later plant developmental stages when colonies of azteca ants became well-established; likewise, they did not demonstrate group strategies for coping with ant defenses. at least in that studied part of northeastern amazonia, visiting by the two focal social wasps to cecropia trees is certainly far from being occasional. to the contrary, based on the regularity of their visits, it appears that mbds constitute a significant proportion of the foraging items of those wasps, and undoubtedly represent a very important food resource for their colonies. in that respect, it is important to note that during parallel observations of a colony of c. fulvus at the goeldi museum campus, a large proportion of the observed foragers returned to the nest carrying trichomes and mbds. further investigation showed that the observed nest was entirely constructed with trichomes collected on trichilia of cecropia trees (borges et al., 2017). quantitative measurement of the relative importance of mbds as forage items should be undertaken in future studies of the foraging activities of those wasps. field observations by one of the present authors (ots) in the eastern amazonia region suggest that both wasp species studied here preferentially occupy habitats at rain forest edges (or in clearings), even in sites experiencing moderate anthropogenic pressure (but in those cases, always in areas with access to reasonably preserved forest patches; e.g., the goeldi museum campus). ecological information published earlier (lapierre et al., 2007), and now here, present a possible specific functional reason for the association of those wasp species with the early successional vegetation typical of edge habitats – given the strong character of cecropia species as pioneer plants in the neotropics. one might ask if foraging for cecropia mbds is a trait inherited from some common ancestor (to both p. chartergoides and the charterginus species) at some level in the polistinae phylogeny, or a convergent feature in those taxa. relationships between charterginus and protopolybia are probably “close”, in the sense that their respective branches in published trees have often appeared in relatively nearby positions. carpenter (1991) placed charterginus as a sister group to a component (arrangedasa polytomy) that included protopolybia and several other epiponine genera. more recently, in a study based on both morphological and molecular characters (pickett & carpenter, 2010), the species c. xanthura was found to be sister to a group formed by three protopolybia species, and brachygastra lecheguana latreille and protonectarina sylveirae (de saussure), the whole clade embedded within a large epiponine component (composed of 34 species from 12 of the total 19 genera). phylogenetic relationships in the genus charterginus, as studied by andena et al. (2009), showed two main components – one formed by c. fulvus and c. xanthura as sister taxa, and another formed by four remaining species, with the pattern c. weyrauchi (c. nevermanni (c. carinatus + c. zavattari)). the currently known distribution for the trait “mbd collecting” in fulvus, xanthura, and nevermanni indicates that it is possibly a primitive feature in the genus (expected to occur also in the other three species of the larger weyrauchi-zavattari clade). the situation in the larger genus protopolybia is quite different, with “mbd collecting” behavior by p. chartergoides being the only known occurrence of that trait in a genus with a slightly more than 30 species. p. chartergoides together with four other species form a small reasonably differentiated group formerly classified as the genus pseudochartergus ducke, 1905 (synonymized to protopolybia by carpenter & wenzel, 1989). a preliminary phylogenetic study of protopolybia by santos et al. (2015), suggested that the “pseudochartergus” group is probably monophyletic, although its relationships with other species-groups in protopolybia are still uncertain. however, in none of the alternative most parsimonious phylogenetic hypotheses evaluated by santos et al. (2015) the “pseudochartergus” group appeared as a basal (or primitive) lineage, which, taken with current evidence on the distribution of “mbd collecting” behavior, indicates that this trait would not be part of the ground plan of protopolybia. so, at present, this behavior would best be considered a convergent trait in p. chartergoides, independent of any close relationship between protopolybia and charterginus. it is interesting to note, in respect of possibility of independent origins of the “mbd collecting” trait, that sociobiology 64(4): 451-455 (december, 2017) 455 charterginus and some protopolybia species (especially those of the “pseudochartergus” group) share a peculiar (and certainly derived in epiponini) feature related to head morphology, with an extremely narrow gena (the lateral part of the head behind the composite eyes) (see andena et al., 2009; richards, 1978). the inner side of genal wall, jointly with the occipital and post occipital areas, form the attachment surfaces for the mandible musculature, principally the large adductor muscles (duncan, 1939; silveira & santos, 2011). additionally, in respect of the female mandible morphology, the species c. xanthura, c. fulvus, p. chartergoides, p. fuscatus, p. duckeianus, p. sedula, and several other of the p. exiguagroup studied by silveira and santos (2011) have very similar morphologies, with well-developed apical teeth, but considerably reduced mesial (inner) denticles. it therefore seems reasonable to speculate whether these particular morphologies (i.e. regarding to reduced head wall surfaces, and attenuated mandible mesial relief) may be adaptively related to a biological role of collecting relatively soft materials such as plant trichomes for nest construction as well as mullerian bodies as food items (see sarmiento, 2004). acknowledgements the first three authors received scholarships from “conselho nacional de desenvolvimento científico e tecnológico do brasil” (cnpq) (process numbers 141794/2015-1; 140080/2015-5, and 131289/2015-2). the authors thank dr. mario jardim (mpeg), for identification of cecropia species. references andena, s.r., carpenter, j.m., pickett, k.m. 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(ed.). mutualism (pp. 37-56). oxford: oxford university press. http://www.mapress.com/zootaxa/2010/f/zt02720p058.pdf http://www.mapress.com/zootaxa/2010/f/zt02720p058.pdf http://www.mapress.com/zootaxa/2010/f/zt02720p058.pdf https://en.wikipedia.org/wiki/zootaxa doi: 10.13102/sociobiology.v61i1.28-34sociobiology 61(1): 28-34 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 insecticidal effect of volatile compounds from fresh plant materials of tephrosia vogelii against solenopsis invicta workers ws li 1, y zhou 1, h li 1, k wang 1, dm cheng 1, 3, zx zhang 1, 2* introduction the red imported fire ant (rifa), solenopsis invicta, is one of over 280 species in the widespread genus solenopsis. although rifa is native to south america, it has become a pest in the southern united states, australia, thailand, taiwan, the philippines, hong kong, and the southern chinese provinces of guangdong, guangxi and fujian. there are also reports of ant hills in macau. rifa are known to have a strong, painful and persistent irritating sting that often lead a pustule on the skin (laura & rudolf, 2001). the ant stings humans, pets, farm animals and wildlife, as well as damaging farm, electrical equipments and irrigation systems. moreover, besides destroying crops and fruits directly or indirectly, they negatively affect the local biodiversity and cause approximately us$5 billion losses in urban and agricultural areas yearly in the usa (cheng et al., 2008). many botanical insecticidal abstract the effect of volatile compounds from the mashed fresh bean pods (b) as well as the branches and leaves (l) of tephrosia vogelii on the behavior of solenopsis invicta workers was investigated by fumigation toxicity bioassay. gas chromatography–mass spectrometry analysis was used to identify and quantify the volatile compounds. α-pinene, thujene, caryophyllene, and d-limonene were identified as major components of the volatile compounds, which were found toxic to workers when applied by fumigation. responses varied according to worker size, exposure time, and plant material. an increase in exposure time from 1h to 12h led to increases in mortality from 18.33% to 100.00% (b) and 13.33% to 100.00% (l) in minor workers as well as increases from 1.67% to 95.00% (b) and 15.00% to 98.33% (l) in major workers. the volatile compounds were also found to exert a behavioral effect against s. invicta in an a4 paper test. walking and grasping abilities decreased at exposure times ranging from 40 min to 280 min. these findings suggest that the volatile compounds of t. vogelii can be used to control s. invicta. sociobiology an international journal on social insects 1. key laboratory of natural pesticide and chemical biology, ministry of education, south china agricultural university, guangzhou, china, 510642 2. state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china, 510642 3. department of plant protection, zhongkai university of agriculture and engineering, guangzhou, china, 510225 article history edited by evandro n silva, uefs, brazil received 22 june 2013 initial acceptance 06 november 2013 final acceptance 19 december 2013 keywords volatile compounds, red imported fire ant corresponding author zhixiang zhang key laboratory of natural pesticide and chemical biology, ministry of education south china agricultural university guangzhou, china, 510642 e-mail: zdsys@scau.edu.cn compounds possessed good toxicity against rifa, and it is potential for applying botanical insecticides to control rifa. tephrosia vogelii is native to west africa, but is found in india, asia and other tropical regions (dalziel, 1937; lambert et al., 1993). it is widespread in tropical africa from sierra leone and ethiopia southwards to angola, the flora zambesiaca area and the comoro islands, also from assam to indonesia. it was introduced into china by 1986 and planted over a large area in guangdong province. it is a shrubby plant used as a fallow plant to improve soil fertility and to reduce erosion, particularly in higher areas. the leaf macerate is purgative and emetic (walker, 1961; burkill, 1995). powders of t. vogelii are effectively used in the congo against the stored ground nut pest caryedon serratus (delobel, 1987). it is also applied directly to treat head lice, fleas, scabies and other ectoparasites (klaassen, 1996; nwude, 1997). the water extract of the dried leaf possessed molluscicidal activity research article ants sociobiology 61(1): 28-34 (march, 2014) 29 against bulinus globosus (chiotha, 1986). water extract of oven dried stem, leaf and seed showed weak molluscicidal activity against biomphalaria pfeifferi (kloos, 1987). acetone extract of the leaf showed feeding deterrent activity against the insect pieris rapae (shin, 1989). this study is the first to report on the toxicity of the volatile compounds released from the mashed fresh bean pods (b) as well as the branches and leaves (l) of t. vogelii against rifa workers. in addition, this study aims to investigate the effects of the volatile compounds from the fresh plant materials of t. vogelii on the rifa workers and analyze the volatile compounds by gas chromatography– mass spectrometry (gc–ms). materials and methods plant materials t. vogelii (family: legume, papilionacea; genus: tephrosia) was planted in the sample region of insecticidal plants at south china agricultural university in april 2010. the b and l were cut off in april 2013 and immediately sent to the laboratory. insects s. invicta colonies were obtained from a suburb in guangzhou and maintained in the laboratory for bioassays in plastic containers under the following conditions: 25 ± 2 °c, 65%±5% relative humidity and constant darkness. colonies were fed with a mixture of live insects (tenebrio molitor) and 10% honey. a test tube (25mm × 200 mm) partially filled with water and plugged with cotton was used as the water source. experiments were performed under similar environmental conditions. fumigation toxicity bioassay indoor test of the toxicity of mashed fresh bean pods (b) as well as branches and leaves (l) of t. vogelii against rifa workers b (500 g) and l (500 g) t. vogelii were mashed with a high-speed organization stamp mill for 5 min. mashed plant materials (5 g) were weighed and placed at the bottom of the beaker (1000 ml). red imported fire ants were classified into minor workers (body length = 2.8 mm to 3.0 mm, head width = 0.6 mm to 0.7 mm) and major workers (body length = 4.3 mm to 4.5 mm, head width = 1.0 mm to 1.1 mm). twenty workers settled on the bottom of the beaker (100 ml), which had an inside vertical wall coated with fluon emulsion that was allowed to dry for 24 h to prevent the ants from escaping. the 100 ml beaker was allowed to settle on the bottom of the 1000 ml beaker without covering the mashed plant materials. the 1000 ml beaker was sealed with a plastic wrap. the worker was maintained at 25±2 °c and 65%±5% relative humidity. experiments 1 (fumigant toxicity bioassay), 2 (walking ability test), and 3 (grasping ability test) were conducted. all treatments were replicated thrice. the contrast was the absence of mashed plant materials in the 1000ml beaker. outdoor test of the toxicity of live bean pods (b) as well as branches and leaves (l) of t. vogelii against rifa workers twenty workers settled on the bottom of the beaker (100ml) sealed with gauze. one live branch of t. vogelii (not cut off from the shrub) with about 10 bean pods or 100 leaves was slipped into a transparent plastic bag (40cm × 29 cm × 10 cm) with two open ends. the beaker (100 ml) was tied upside down in the bag, and the other open end was sealed. experiments 1 (fumigant toxicity bioassay), 2 (walking ability test), and 3 (grasping ability test) were conducted. the average temperature was set between 25°c and 30°c, average humidity was 70% to 90%, and average wind velocity was <0.5m/s during the test. physiological index observation of rifa workers the cumulative mortalities of fumigant toxicity were determined 1, 2, 4, 6, 8, 10, and 12h after testing. behavioral observation on the walking ability of the workers was determined 40, 80, 120, 160, 200, 240, and 280min after testing. the workers were placed on an a4 paper. if the workers could walk continuously for 10 cm without falling, these workers were regarded to possess walking ability. we used the following equation: walking rate = (number of workers possessing walking ability/number of workers per replicate) × 100. behavioral observation on the grasping ability of the workers was determined 40, 80, 120, 160, 200, 240, and 280 min after testing. the workers were placed on an a4 paper, which was softly rotated in 180° after 10s. the workers that did not fall from the a4 paper were regarded to possess grasping ability. we used the following equation: grasping rate = (number of workers possessing grasping ability/number of workers per replicate) × 100. chemical analysis of the volatile compounds isolation, identification, and quantification of the component of the volatile compounds from the mashed fresh plant materials of t. vogelii were analyzed using gc–ms. the indoor test method was conducted following the procedure in 2.3; however, the 20 workers were replaced with a 2g adsorbents. the adsorbents were silicone (200 mesh to 300 mesh). the adsorbent was inserted into the injector (length = 20cm, diameter = 0.5cm) 6h after the treatment. the volatile ws li et al insecticidal effect of tephrosia vogelii against solenopsis invicta workers30 compounds were absorbed by the silicone, which was eluted with 5ml petroleum ether or acetone. the analytical conditions of the gc–ms are shown in table 1. most compounds were identified based on gc–ms retention times, kovats indices, and mass spectra. statistical analysis data were transformed into arcsine square root values for a three-way analysis of variance (anova) to determine the significance of the effects of plant material, exposure time, and worker size on the mortalities, walking rate, and grasping rate of the minor and major workers as well as various interactions. the differences in the data were also assessed using duncan’s multiple range test, with p < 0.05 considered statistically significant. the figures were generated using the microsoft office excel 2007 program. results indoor test when b and l of t. vogelii were bioassayed, the mortalities, walking rate, and grasping rate of minor and major workers varied significantly according to plant material, exposure time, and worker size (anova, p < 0.05).the results reveal that the three main effects and the partial interactions were significant (table 2). marked variations in the mortality of fumigant toxicity as well as the walking and grasping abilities were observed at different plant material, exposure time, and worker size (table 3). the mortality of rifa was significant at different plant material (f = 10.272, p =0.0022), exposure time(f = 235.652, p < 0.0001), and worker size(f = 54.450, p = 0.0001). however, there was no difference in the percentage of mortality between the interaction exposure time×worker size (f = 1.341, p =0.2547) and the interaction plant material×exposure time×worker size (f = 1.230, p = 0.3052). in addition, significant differences were found at walking rate and grasping rate of rifa: developing from different exposure time (f = 312.108, p < 0.0001; f = 286.326, < 0.0001) and worker size (f = 49.371, p = 0.0001; f = 9.496, p =0.0032). fumigant toxicity the indoor test of the mashed b and l of t. vogelii showed effective fumigant activity against the rifa workers; by contrast, the outdoor test showed no such activity (fig. 1). the volatile compounds released from the live b and l of t. vogelii caused no mortality in the fumigant toxicity assay, even after 12h exposure,there were no dead individuals. however, in all cases of the indoor test, a marked variation in insect mortalities was observed with the increase in exposure time. a significant difference in the mortalities between the minor and the major workers was indicated, with the volatile compound of the b and l of t. vogelii being significantly more toxic to the rifa minors than the majors. after a 1h exposure, the volatile compounds of b and l resulted in 13.33% (b) and 18.33% (l) mortality rates in minor workers as well as gas chromatography hp 6890n-5957 series plus (agilent) carrier gas helium injector temp 280℃ detector temp 230℃ capillary column hp-5ms (agilent) or db-5 (j&w) head pressure 100 kpa oven program initial temp 60℃ initial time 1 min rate final temp. final time (℃/min) (℃) (min) 25 210 5 10 280 10 mass spectrometer hp 5972 (agilent) ei voltage 70 ev mass spectrum database nbsli-brary table1. the analytical conditions of gc-ms factors mortality walking ability grasping ability df f values p values df f values p values df f values p values a 1 10.272 0.0022 1 0.610 0.4383 1 4.845 0.0319 b 6 235.652 0.0001 6 312.108 0.0001 6 286.326 0.0001 c 1 54.450 0.0001 1 49.371 0.0001 1 9.496 0.0032 a×b 6 3.000 0.0130 6 0.876 0.5183 6 1.390 0.2347 a×c 1 5.339 0.0246 1 0.343 0.5605 1 0.628 0.4315 b×c 6 1.341 0.2547 6 2.660 0.0243 6 0.796 0.5772 a×b×c 6 1.230 0.3052 6 0.521 0.7902 6 1.008 0.4296 a: plant material, b: exposure time, c: worker size (p=0.05) table 2. anova for the main factors of the indoor test affecting the behaviors of rifa sociobiology 61(1): 28-34 (march, 2014) 31 1.67% (b) and 15.00% (l) mortality rates in major workers. however, the mortality rates were 100% (b) and 100.00% (l) for the minors as well as 95.00% (b) and 98.33% (l) for the majors at 12h of treatment. walking and grasping abilities the volatile compound from the mashed b and l exhibited good fumigant activity observed at different exposure times against the rifa workers. however, the volatile compounds released from the live b and l of t. vogelii caused no reduction in the walking and grasping abilities of the rifa workers (figs. 2 and 3). the minor and major workers showed good walking and grasping abilities with the live plant materials in outdoor test. and the walking and grasping abilities of the rifa workers decreased with longer exposure in indoor test (table 3). at exposure times ranging from 40 min to 280 min, the walking abilities decreased from 98.33% to 20.00% (b) and 96.67% to 21.67% (l) for the major workers and from 96.67% to 13.33% (b) and 95.00% to 16.67% (l) for the minor workers; the grasping abilities was reduced from 96.67% to 15.00% (b) and 95.00% to 13.33% (l) for the major workers and from 91.67% to 15.00% (b) and 96.67% to 15.00% (l) for the minor workers (table 3). chemical components of the volatile compounds the data from the gc–ms analysis demonstrated that the volatile compound from the mashed b and l of t. vogelii contains 17 and 14 major constituents, respectively (table 4). the major components comprising 88.46% (b) and 75.37% (l) of the total volatile compound were identified as α-pinene, thujene, caryophyllene, and d-limonene. other major components of the volatile compound included eucalyptol, α-cubebene, β-pinene, sabinene, and α-guaiene. α-pinene is the major component, accounting for 63.44%(b) and 65.82%(l). discussion t. vogelii is a shrubby plant used as a fallow plant in southern china. our data show that the volatile compounds released from live b and l show no toxicity against rifa workers and can not reduce the walking and grasping abilities of the rifa workers despite abundant insecticidal contents. however, the volatile compounds released from the mashed b and l exhibited high toxicity against the rifa workers and evidently reduced the walking and grasping abilities of the rifa workers. this reduction indicated that the volatile material worker size mortality walking ability grasping ability time (h) percentage time (min) percentage time (min) percentage b major 1 1.67±1.67b 40 98.33 ±1.67a 40 96.67±1.67ab minor 13.33±1.67a 96.67±1.67a 91.67±1.67 b l major 15.00±2.89a 96.67±1.67a 95.00±2.89ab minor 18.33±1.67a 95.00±2.89a 96.67±3.33ab b major 2 6.67±1.67b 80 91.67±1.67b 80 90.00±2.89ab minor 30.00±5.77a 86.67±1.67bc 83.33±4.41b l major 30.00±7.64a 88.33±3.33bc 85.00±5.77b minor 35.00±2.89a 81.67 ±3.33c 81.67±3.33b b major 4 16.67±4.41b 120 80.00±2.89bc 120 76.67±4.41b minor 40.00±5.77a 73.33±1.67bc 71.67±1.67b l major 35.00±7.64a 81.67 ±4.41b 80.00±5.00b minor 45.00±2.89a 71.67 ±3.33c 73.33±4.41b b major 6 36.67±4.41b 160 65.00 ±5.77b 160 60.00±2.89b minor 61.67±4.41a 58.33 ±3.33b 58.33±3.33b l major 51.67±4.41a 70.00 ±5.77b 70.00±5.77b minor 60.00±5.77a 61.67 ±4.41b 61.67±4.41b b major 8 66.67±4.41a 200 60.00 ±2.89b 200 43.33±4.41bc minor 76.67±6.67a 40.00 ±2.89c 41.67±4.41c l major 63.33±4.41a 60.00±5.77b 53.33±3.33b minor 76.67±3.33a 45.00±2.89c 48.33±1.67bc b major 10 85.00±5.77ab 240 41.67 ±1.67b 240 23.33±1.67c minor 95.00±2.89a 23.33±4.41d 21.67±4.41c l major 76.67±1.67b 40.00±2.89b 36.67±1.67b minor 91.67±4.41a 31.67 ±1.67c 20.00±2.89c b major 12 95.00±2.89ab 280 20.00±2.89b 280 15.00±2.89b minor 100.00±0.00a 13.33±1.67c 15.00±2.89b l major 98.33±1.67ab 21.67±1.67b 13.33±3.33b minor 100.00±0.00a 16.67±1.67bc 15.00±2.89b values sharing same letters means they are not significantly different from each other (p>0.05, duncan’s multiple range test). table 3. influences of mashed b and l of t. vogelii on mortality, walking and grasping abilities of workers (mean ± se, %) ws li et al insecticidal effect of tephrosia vogelii against solenopsis invicta workers32 ta bl e 4. t he v ol at ile c om po un ds fr om m as he d b o r l (5 g ) o f t . v og el ii ad so rb ed o n si lic on (2 g) o f i nd oo r t es t. t he v ol at ile c om po un ds fr om 5 g m as he d fr es h be an p od s( b ) t he v ol at ile c om po un ds 5 g m as he d fr es h br an ch es a nd le av es (l ) v ol at ile c om po un d r e te n ti o n tim e (m in ) r el at iv e co nt en t ( % ) v ol at ile c om po un d r et en tio n tim e (m in ) r el at iv e co nt en t ( % ) th uj en e 3. 37 9 0. 88 1r -α -p in en e 3. 45 9 65 .8 2 1r -α -p in en e 3. 45 4 63 .4 4 βte rp in en e 3. 74 6 0. 23 βph el la nd re ne 3. 74 1 2. 21 2pr op yl p yr id in e 3. 82 7 0. 69 4m et hy le ne -1 -( 1m et hy le th yl )cy cl oh ex en e 3. 78 4 2. 56 dlim on en e 4. 14 5 0. 74 βpi ne ne 3. 82 2 5. 66 eu ca ly pt ol 4. 17 8 1. 28 dlim on en e 4. 14 6 0. 96 de ca m et hy lc yc lo pe nt as ilo xa ne 4. 84 2 3. 99 tr id ec an e 5. 79 9 0. 17 2, 3di hy dr o5m et hy l1h -i nd en e 4. 94 5 0. 50 cy cl oh ex en e 6. 12 3 0. 39 az ul en e 5. 28 5 0. 41 αcu be be ne 6. 37 1 1. 59 co pa en e 6. 36 6 0. 21 ar is to le ne 6. 43 6 1. 07 α. -g ua ie ne 6. 43 1 0. 11 ()is oc ar yo ph yl le ne 6. 56 0 0. 44 ca ry op hy lle ne 6. 63 6 0. 69 ca ry op hy lle ne 6. 64 1 5. 77 ca la re ne 6. 67 4 0. 16 1h -c yc lo pe nt a[ 1, 3] cy cl op ro pa [1 ,2 ] b en ze ne ,o ct ah yd ro 7m et hy l3m et hy le ne -4 -( 1m et hy le th yl )[ 3a s( 3a .α ,3 b. β, 4. β, 7. α, 7a s* )] 6. 67 9 0. 64 ge rm ac re ne 6. 95 5 0. 39 ()gca di ne ne 6. 82 0 0. 79 he xa de ca ne 7. 37 6 0. 15 βse sq ui ph el la nd re ne 6. 95 5 1. 30 ge rm ac re ne 7. 03 6 0. 31 10 s, 11 shi m ac ha la -3 (1 2) ,4 -d ie ne 7. 06 3 0. 28 sociobiology 61(1): 28-34 (march, 2014) 33 compounds could produce and release insecticidal compounds that could be defensive compounds, with functions varying from those in the b and l. the volatile compounds can be used to ward off pests when branches and leaves were harmed.the fumigant activity as well as walking and grasping abilities were influenced by the worker size, exposure time, and plant material. to the best of our knowledge, no previous studies have been reported regarding the effects of volatile compounds from fresh plant materials of t. vogelii on the fumigant activity as well as the walking and grasping abilities of rifa workers. however, earlier studies indicated that the insecticidal activity of the acetone extract of the leaf showed feeding deterrent activity against the insect pieris rapae (shin, 1989). plant-derived essential oils may contain hundreds of fig. 1 mortalities of workers after treated with fresh b or l of t. vogelii (indoor test5g mashed fresh plant material, outdoor test one live branch with about 10 bean pods or 100 leaves) fig. 2 the grasping abilities of workers after treated with fresh b or l of t. vogelii (indoor test: 5g mashed fresh plant materials, outdoor test: one live branch with about 10 bean pods or 100 leaves) fig. 3 the walking abilities of workers after treated with fresh b or l of t. vogelii (indoor test: 5g mashed fresh plant materials, outdoor test: one live branch with about 10 bean pods or 100 leaves) different constituents but only few components predominate (rajendran & sriranjini, 2008). the results of the gc–ms analysis showed thujene, α-pinene, caryophyllene, eucalyptol, and d-limonene were found in the compounds from the mashed b and l. eucalyptol, one of the monoterpenoids of the volatile compounds, is widely known for its high insecticidal property. α-pinene(64.63%)as its major components, is also found in other plants exhibiting biological activity against various insect species (ojimelukwe & alder, 1999; choi et al., 2006). the l of t. vogelii could have abundant insecticidal compounds and aboveground biomass (dry matter) produced by t. vogelii during the six-month growth period of 5.3 t/ ha with an l:stem ratio = 1:2 (venant, 1999). this finding indicates that t. vogelii can potentially control rifa. the dry matter of t. vogelii is often prepared in the production of rotenone-based insecticides. however, this study suggests that the mashed b and l of t. vogelii are more suitable than dry matter for producing insecticides. the volatile compounds from the mashed b and l of t. vogelii possess high toxicity against the rifa workers and can reduce the 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(1999). biomass production and nutrient accumulation by tephrosia vogelii (hemsley) a. gray and tithonia diversifolia hook f. fallows during the six-month growth period at maseno, western kenya. biotechnology, agronomy, society and environment, 3: 237-246. doi: 10.13102/sociobiology.v63i4.1026sociobiology 63(4): 1046-1050 (december, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 efficacy of 1% fipronil dust of activated carbon against subterranean termite coptotermes formosanus shiraki in laboratory conditions introduction many termite species are distributed in latitude 40 degrees south of the region in china and accounts for about 40% of the whole country. the main disaster area is located in the south of the yangtze river. su et al. (2012) reported that the economic losses caused by termites amounted to more than 200 million rmb in 2004. for a long time, the termites control mainly relied on chemical pesticides, such as chlordane, mirex. the production of the two above mentioned pesticides reached more than 100 tons from 2000 to 2003 (anonymous, 2005). however, controlling of termites is facing a great challenge after the stockholm convention on pops (persistent abstract toxicity and transmission of 1% fipronil dust of activated carbon were measured using the subterranean termite coptotermes formosanus shiraki in laboratory conditions. 1% fipronil dust of activated carbon has delayed toxicity towards c. formosanus compared with 0.5% fipronil dust of talcum powder; knockdown times kt 50 and kt 90 were delayed by >9 and >15 h respectively. furthermore, 1% fipronil dust of activated carbon showed excellent primary and secondary transfer levels. in primary transfer, recipient mortalities reached 100% by 24, 48 and 72 h at donorrecipient ratios of 1:1, 1:5 and 1:10, respectively. high transfer efficacies were also found if donor-recipient ratios were greatly increased: mortality reached 100% at 9 d at ratio 1:25 and >90% at 12 d at 1:50. in secondary transfer, the toxicant transmitting ability of c. formosanus was greater when the primary transfer ratio was lower, and the highest transfer efficacy was found with a donor-recipient ratio of 1:1 recipient mortalities reached 100% at 5 d and 11 d, respectively. application of 1% fipronil dust of activated carbon overcomes the problem that too high a concentration kills termites before they can contaminate their nestmates, while a lower concentration may not supply a sufficient dose for effective transfer from treated to untreated termites. the results showed that 1% fipronil dust of activated carbon was non-repellent and readily transferred from treated to untreated termites. as the post-exposure time and doses increased, the mortalities of both donors and recipients increased. and it has delayed toxicity to control c. formosanus. sociobiology an international journal on social insects qf li1, qf zhu2, sj fu2, dm zhang1, jb huang1, jz yuan1 article history edited by evandro nascimento silva, uefs, brazil received 15 march 2016 initial acceptance 01 august 2016 final acceptance 07 december 2016 publication date 13 january 2017 keywords coptotermes formosanus, fipronil, delayed activity, activated carbon, transfer level. corresponding author jianzhong yuan institute of plant physiology and ecology shanghai institutes for biological sciences the chinese academy of sciences china. fenglin road 300, shanghai e-mail: yuangang188@126.com organic pollutants), which the development of termiticides with high efficiency, low toxicity and environmentally friendly is imperative, especially development and improvement in the formulation. currently, termites are controlled by applying three formulations of termiticides: non-repellent liquid, dust and bait in china. insecticidal dusts against termites has been practiced for decades (madden et al., 2000). dust formulations (such as avermectin dust, borate dust and fipronil dust) are candidates for successful localized treatment to eliminate termite colonies (esenther 1985; lin et al., 2011; grace, 1991; zhao et al., 2012). however, 0.5% fipronil dust is the only powderform termiticide that is registered for use in china; it has high activity and excellent transfer efficacy (i.e. from treated to 1 institute of plant physiology and ecology, shanghai institutes for biological sciences, the chinese academy of sciences, china, shanghai 2 shanghai municipal property management affairs center, china, shanghai research article termites mailto:yuangang188@126.com sociobiology 63(4): 1046-1050 (december, 2016) 1047 untreated termites) (ibrahim et al., 2003; mao et al., 2011; shelton & grace, 2003; remmen & su, 2005; gautam et al., 2012; song & hu, 2006; ma et al., 2015; gautam et al., 2014). in 2008, a new dust of microllose-base formulated with 0.5% fipronil (termidorâ dry) was developed by basf, which was highly effective to kill c. formosanus and had excellent transfer level (basf. 2008). because of high toxicity and rapid effect on termites, only low concentrations of fipronil dust can be used for termite control. ma et al. (2015) reported that the recommended dose of fipronil dust is ≤0.5%. otherwise, termites will be killed rapidly and may not have sufficient time to carry and transfer a lethal dose to nestmates before they die. it can be difficult to obtain good transmission results away from the treatment site. low concentrations of fipronil dust have some control effect on termites, but may not supply a sufficient dose for contaminated termites to transfer a lethal dose to unexposed termites. shelton and grace (2003) emphasized the importance in dose reporting of the need to consider lethal transfer of fipronil and imidacloprid from donors to recipients. ma et al. (2015) reported that the transfer efficacy of low concentrations of fipronil dust are relatively poor compared with high concentrations. the combination of these effects greatly limits the further use and promotion of fipronil dust in the control of termites. the key to improving transmission of the insecticide is increasing the concentration of fipronil dust while prolonging the time it takes to kill termites. by screening many experimental materials, we found that 1% fipronil dust of activated carbon (i.e. activated carbon powder infused with 1% fipronil) meets these conditions. the present paper reports on the insecticidal activity towards c. formosanus and potential for transmission of 1% fipronil dust of activated carbon in laboratory conditions. materials and methods termites two colonies were used: field colony was collected from the campus of shanghai jiao tong university by using underground traps as previously described (hu & appel, 2004) and were kept in the laboratory for <1 month before testing at a temperature of 26 ± 1°c and 85 ± 10% relative humidity. laboratory colony was maintained in the laboratory for 10 years without exposure to any insecticides. 1% fipronil dust of activated carbon activated carbon (a kind of absorptive particle, which has strong adsorption capacity) and talcum powder (a kind of lubricant, which is helpful to the dispersion of solid pesticide) were purchased from sinopharm chemical reagent (shanghai) co., ltd. and shanghai junqian chemical co., ltd., respectively. both materials were sieved through 200 mesh and then sterilized in an oven at 100°c for 24 h. 1 g activated carbon or 0.5 g talcum powder was added to 4 ml 0.25% fipronil acetone solution (1 g activated carbon or 0.5 g talcum powder was added to 4 ml acetone solution as control), the mixture was vigorously vortexed and then incubated at 25 ± 1°c or 50 ± 1°c, respectively, for 2 h in 50 ml tubes. lids of the tubes were opened and they were placed under a fume hood for 2 d to completely evaporate the acetone. lethal concentration bioassay bioassays were performed using filter paper method as described (su et al., 1987) with slight modifications. briefly, 0.8 ml insecticide-acetone solutions of different concentration were pipetted onto whatman no. 1 filter papers that were placed in petri dishes (9 cm diameter) after evaporating the acetone completely (or 0.8 ml acetone as control). thirty worker termites were placed on each treated filter paper. experimental mortality was recorded after 24, 48 and 72 h. at least three independent replicates were tested for each colony. fipronil dust bioassay thirty workers were placed in a petri dish (9 cm diameter) containing 15 mg 1% fipronil dust of activated carbon (15 mg activated carbon as control) or 0.5% fipronil dust of talcum powder (15 mg talcum powder as control) evenly spread (by gently shaking back and forth) at the bottom of the dish. exposure was for 15 min. termites were then transferred to a clean petri dish containing a filter paper moistened with 0.2 ml distilled water. the number of termite deaths was recorded every 1 h until they were all dead. at least five independent replicates were tested for each colony. transfer bioassay before the experiment, untreated workers were marked by feeding filter papers dyed with 0.1% nile blue a (aldrich, milwaukee, wi) for 24 h. blue termites (healthy) were carefully collected using soft brushes and put in petri dishes in preparation for transfer bioassays for each colony. (1) primary transfer: to produce donors, thirty workers were allowed to be freely active for 15 min in a petri dish (9 cm diameter) containing 15 mg 1% fipronil dust of activated carbon spread evenly at the bottom of the dish. treated termites (donors) were then transferred to a clean petri dish and allowed to walk for 3 min. donors were then added to a petri dish containing a filter paper moistened with 0.2 ml distilled water and untreated blue termites (recipients) at donor-recipient ratios of 1:1, 1:5, 1:10, 1:25 and 1:50. the number of termites in each dish was 50 ± 5. the number of termite deaths was recorded every 8 h for 24 h and then at 1 d intervals for 12 d. (2) secondary transfer: treated non-dyed workers (as primary donors) were placed in a petri dish containing untreated blue termites (as primary recipients) at donor-recipient ratios of qf li et al. – 1% fipronil activated carbon dust as a termiticide1048 1:5 and 1:10. after 8 h (active), the blue termites (as secondary donors) were placed in a petri dish containing untreated nondyed workers (as secondary recipients) at donor-recipient ratios of 1:1, 1:5 and 1:10, respectively. the number of termites in each dish was 50 ± 5. the number of termite deaths was recorded every 8 h for 24 h and then at 1 d intervals for 12 d. all experimental termites were kept in polyethylene containers (30 × 30 × 20 cm) and incubated at 25 ± 1°c and 85 ± 10% relative humidity in total darkness. at least five independent replicates were tested. there were 10% soldiers in each dish. statistical analysis all data are expressed as the mean value ± se of independent experiments. analysis of all data used spss 13.0 software and significant differences of mortality between activated carbon and talcum powder and between 25°c and 50°c were determined by tukey’s honestly significant difference test following anova. mortality was corrected using abbott’s formula (abbott, 1925). results table 1 showed toxicity of fipronil to c. formosanus. lc50 and lc90 values were 11.05, 6.18, 4.46 mg/ml and 29.54, 17.13, 10.31 mg/ml after exposure for 24, 48 and 72 h, respectively. fipronil exhibited moderate to high toxic to c. formosanus. fig 1. average knockdown time (± se) of c. formosanus by 0.5% fipronil dust of talcum powder and 1% fipronil dust of activated carbon with 25°c and 50°c incubation. means with different lowercase letters show significant differences (p < 0.05). 0.5% fipronil dust of talcum powder has a quick knockdown time for c. formosanus: kt50 and kt90 were 1.57 and 2.16 h at 25°c and 1.56 and 2.10 h at 50°c, respectively. termites did not appear poisoning symptoms within 6 h after treatment of 1% fipronil dust of activated carbon. less termites (about 10%) have decreased activity capacity, such as slow-moving, dying kicks after 8 h, and mortality rate reached 100% in 23-24 h. kt50 and kt90 of 1% fipronil dust of activated carbon were both significantly prolonged compared with 0.5% fipronil dust of talcum powder (p < 0.05); the kt50 and kt90 were delayed by >9 h (25°c, f = 116.73, df = 1, p < 0.0001; 50°c, f = 178.11, df = 1, p < 0.0001) and >15 h (25°c, f = 333.33, df = 1, p < 0.0001; 50°c, f = 378.90, df = 1, p < 0.0001), respectively, at both temperatures (fig 1). there were no significant effects of temperature on the capacity for adsorption of fipronil applied via activated carbon (kt50, f = 0.14, df = 1, p =0.74; kt90, f = 0.21, df = 1, p =0.67) or talcum powder (kt50, f = 0.09, df = 1, p =0.75; kt90, f = 0.17, df = 1, p =0.66). for each colony, the results showed that 1% activated carbon and 0.5% talcum powder had no toxic to termites. for each colony, 1% fipronil dust of activated carbon was excellent in primary transfer. recipient mortalities reached 100% at 24, 48 and 72 h at donor-recipient ratios of 1:1, 1:5 and 1:10, respectively. interestingly, high transfer efficacies were also found as the donor-recipient ratio was greatly increased: mortality reached 100% at 9 d at ratio 1:25 and >90% at 12 d at a 1:50 ratio (fig 2). in the secondary transfer experiment, the toxicant (1% fipronil dust of activated carbon) transmitting ability of c. formosanus was greater with a primary transfer donorrecipient ratio of 1:5 than with a primary transfer ratio 1:10 at secondary donor-recipient ratios 1:1, 1:5 and 1:10 (fig 3). the highest transfer efficacy was found with a donor-recipient ratio of 1:1 recipient mortalities reached 100% at 5 d and 11 d, respectively. recipient mortalities were significantly reduced as donor-recipient ratios increased to 1:5 and 1:10 (58% , 29% and 27% , 19% at 12 d for ratios 1:5 and 1:10 in the primary transfer, respectively). mortality of recipients negatively correlated with donor-recipient ratios. fig 2. average mortalities (± se) of recipient c. formosanus at different donor-recipient ratios in the primary transfer of 1% fipronil dust of activated carbon. discussion in the present study, mortalities reached 100% at 3 h after treatment of c. formosanus with 0.5% fipronil dust of talcum powder (fig 1). it is evident that 0.5% fipronil dust of talcum powder kills c. formosanus too quickly and so cannot be used to eliminate or suppress the nest of termites. we found that fipronil exhibited moderate to high toxic to c. formosanus (table 1). these results are consistent with previous reports (ibrahim et al., 2003; mao et al., 2011). saran and rust (2007) found that c. formosanus became less mobile 1 h after exposure to fipronil-treated sand and transfer was limited to the vicinity of the treated sites. similarly, in a laboratory study of c. formosanus, they did not walk more than 5 m after sociobiology 63(4): 1046-1050 (december, 2016) 1049 contacting fipronil-treated soil (su 2005), whereas saran and rust (2007) found that termites exposed to lethal amounts of fipronil did not walk more than 1.5 m from the treated zone. quarcoo et al. (2012) reported that tunneling and working ability of intoxicated termites are important factors that affect their potential to transfer toxicants to untreated nestmates, along with the insecticide exposure duration and dose, the donor-recipient ratio (ibrahim et al., 2003; saran & rust, 2007; shelton & grace, 2003; hu 2005; song & hu, 2006), the temperature (spomer et al., 2008), substrate type and caste structure (gautam & henderson, 2011). however, 1% fipronil dust of activated carbon makes up for this shortcoming since the kt50 value is delayed by >9 h compared with 0.5% fipronil dust of talcum powder; i.e., this preparation greatly delayed the death time of c. formosanus. 1% fipronil dust of activated carbon has an excellent effect on c. formosanus. when the primary donor-recipient ratios were 1:1, 1:5 and 1:10, the recipient mortalities were 100% within 24-72 h. shelton and grace (2003) reported that untreated termites had a significantly greater mortality after a 15-day interaction at a high donor-recipient ratio (1:19). we found a similar situation in our experiments: mortalities reached 100% at 9 d at donor-recipient ratio 1:25 and >90% at 12 d for a 1:50 ratio. there are few previous reports about the secondary transfer level of fipronil to c. formosanus. in the present study, the mortalities of recipients (secondary mortality of recipient) were determined at different donorrecipient ratios in the secondary transfer. recipient mortalities (secondary donor-recipient ratio 1:1) reached 100% at 5 d and 11 d after primary transfer at donor-recipient ratios of 1:5 and 1:10, respectively. there was a negative correlation between recipient mortality and the donor-recipient ratio for both primary and secondary transfer. in our experiments, we used 200 mesh sieved activated carbon, i.e. fine particles. such particles adhere easily to the surface of termites. because of the strong adsorption of activated carbon, much of the toxic agent is absorbed in the particles, so treated termites are not killed immediately but instead are able to transfer a lethal dose to other (untreated) termites before they die. this may impact upon the entire colony. in addition, we observed a black substance in the intestines of donor termites, which may be due to the grooming ritual and the intake of activated carbon following primary transfer; this phenomenon is not obvious in recipients. the transfer of fipronil among termites can be caused by body contract, mutual grooming and trophallaxis (song and hu 2006; saran and rust 2007). the transmission mechanism of 1% fipronil dust of activated carbon is unknown and requires further study. acknowledgments we thank prof. l. m. tao (school of pharmacy, east china university of science and technology) for his editorial assistance. this study was supported by shanghai municipal property management affairs center. references abbott, m.s. 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(2012). ivermectin dust for the control of coptotermes formosanus in residential areas. sociobiology, 59: 1365-1373. _enref_1 _enref_2 _enref_3 _enref_4 _enref_5 _enref_6 _enref_7 _enref_8 _enref_9 _enref_10 _enref_11 _enref_12 _enref_13 _enref_14 _enref_15 _enref_16 _enref_17 _enref_18 _enref_19 _enref_20 _enref_21 _enref_22 _enref_23 _enref_24 _enref_25 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1037sociobiology 63(3): 991-997 (september, 2016) the influence of habitat and species attributes on the density and nest spacing of a stingless bee (meliponini) in the atlantic rainforest introduction the stingless bees occupy multiple habitat types but are particularly diverse and abundant in tropical rainforests. they depend mainly on nest structure and building materials for thermoregulation, which likely constrains their distribution to the hot tropical-subtropical zone (sakagami, 1982; roubik, 1989). they live in sessile, perennial and eusocial colonies, most species depend on pre-existing cavities as nesting sites, and very few species build exposed nests (hubbell & johnson, 1977; nogueira-neto, 1997; camargo, 2007). because of nesting habit, some authors suggest that the density and spatial distribution of colonies should be influenced by the availability of nesting cavities, however this hypothesis has abstract trigona spinipes (fabricius, 1793) lives in perennial eusocial colonies and builds nests exposed on tree branches. the reason why this generalist habitat with exposed nests cannot nest successfully in forest habitats is intriguing. this study explores the hypothesis that this species reaches higher densities in vegetation with open canopies and the subsidiary shading hypothesis, assuming the failure of large exposed nests in closed canopy rainforest. comparative field data on nest density in open canopy vegetation (this study) and adjacent closed canopy forest (previous data) are used to test this hypothesis. at random 18 nests distributed in 40 20x200m plots in rubber groves with a density of 1.1 nests/ha, were recorded. this is a high nest density for a single species of stingless bee and correspond to 36 times the very low density in adjacent rainforest. the high density in the rubber groves is also associated with a regular spatial distribution of nests and it suggests territorial patrolling mechanism operating at short distances (60 to 70m). we conclude that: (a) this species faces powerful nesting constraints in the ever green and closed canopy of rainforest, because the external structure of the nest remain wet for prolonged periods due to exposion to heavy rains and high shading; (b) the high availability of sunny sites for nesting within the deciduous and open canopy of rubber trees favours rapid nest drying and the high nest density of t. spinipes and realeasing its wide spread distribution where this agroforestry system dominates. sociobiology an international journal on social insects md silva1,2 , m ramalho1 article history edited by evandro nascimento silva, uefs, brazil received 02 april 2016 initial acceptance 11 june 2016 final acceptance 01 august 2016 publication date 25 october 2016 keywords interference competition, patrolling mechanism, exposed nest constraint, territoriality, rubber groves. corresponding author marília silva instituto federal de educação, ciência e tecnologia baiano campus governador mangabeira rua waldemar mascarenhas, s/n portão cep 44350000 governador mangabeira-ba, brasil e-mail: marilia.silva@gm.ifbaiano.edu.br been refuted by several studies in tropical forests (hubbell & johnson, 1977; eltz et al., 2002; silva et al., 2014). on the other hand, species building exposed nests should be less influenced by the availability of nesting sites, however this simple assumption has not been tested yet. intra and inter-specific interactions, flowering pattern, and colony attibutes, such as population sizes, are also assumed to play a role in regulating density and spacing of nests in local populations and communities of stingless bees (hubbell & johnson, 1977; johnson, 1987; eltz et al., 2003; biesmeijer & slaa, 2006). here, trigona spinipes (fabricius, 1793) is used as a model to investigate factors affecting nest density and nesting spatial pattern of stingless bees, because it builds huge 1universidade federal da bahia, salvador-ba, brazil 2instituto federal de educação, ciência e tecnologia baiano, governador mangabeira-ba, brazil research article bees mailto:marilia.silva@gm.ifbaiano.edu.br md silva, m ramalho – density and nest spacing of a stingless bee992 exposed nests with very populous colonies (rough estimates from 5,000-180,000 individuals; e.g. almeida & laroca, 1988; camargo, 2007; see also biesmeijer & slaa, 2006), with high demands for food sources throughout the year. moreover, their workers are extremely aggressive against potential intruders in the vicinity of the colony (shakleton et al., 2014) and they are refered to be territorial, without a clear evidence on the role of such aggressiveness on nest spacing. t. spinipes is widely distributed in the tropics and subtropics of south america (almeida & laroca, 1988; silveira et al., 2002; camargo, 2007) and it visits the flowers of many plant species in open vegetation habitats (cortopassilaurino & ramalho, 1988; almeida & laroca, 1988; knoll, 1990; martins, 1994; silveira & campos, 1995; aguiar & martins, 1997; carvalho & marchini, 1999; viana, 1999), but barely in the flowerings of closed canopy forest (ramalho, 2004). in some nest census of t. spinipes (taura & laroca, 1991; henriques, 1997; batista et al., 2003; pioker-hara, 2011; silva et al., 2013), the lowest density was observed in the closed canopy forest (silva et al., 2013), and the highest density in open canopy habitats. (roubik, 1989; henriques, 1997). the hypothesis put forth in this study is that t. spinipes faces powerful nesting constraints in the ever green and closed canopy of tropical rainforest, throughout its wide ecological distribution. it builds exposed nests on tree branches, that probably provides adequate heat up take to thermoregulation (zucchi & sakagami, 1972; almeida & laroca, 1988) under subtropical conditions. due to its apparent reliance on exposure of the nest to direct sunlight, we assume that the main constraint on its nesting success relates to the degree of canopy shading. in the tropical forest, nest humidity is probably a constraint to huge exposed nests, because the more closed the canopy, the more slowly the nests will dry, especially in high rainfall evergreen forest. this hypothesis is tested by using comparative data on density and spacing of nests in open canopy habitat (rubber groves) and adjacent rainforest closed canopy habitat (rainforest). the understanding of the role of wood grooves with open canopy (such as rubber and eucalyptus) on the ecological expansion of t.spinipes might be important to management of this species in rural areas spreading on previous forested areas; first, because it is part of a group of stingless bees with extensive record of floral theft and damage to flowers both in natural habitats and orchards (renner, 1983; roubik, 1989); second, due to the increasing deforestation of tropical forests (e.g. brazilian atlantic forest) . material and methods the study landscape is located in the northern portion of the central corridor of the brazilian atlantic forest within the limits of ‘reserva ecológica michelin’ (rem) (13°50’ s and 39°15’ w). the rem protects three remnants of closed canopy dense tropical evergreen rainforest (140, 550, 625 ha) at altitudes ranging from 40-330m (flesher, 2006), under af climate type (köppen classification 1948), with average monthly temperatures ranging from 18 to 30°c, relative humidity between 80% and 85%, and annual rainfalls of around 2,000mm. rubber (hevea brasiliensis muell. arg), and rubber/ cacao plantations lie to the south and east of the forest remnants covering >5000 ha with many small forest fragments (<10ha) of pioneer forest on rocky outcrops and along the water ways. in the plantation the rubber trees are spaced in 3m lines and 7m between lines, resulting in a density of around 500 adult trees per hectare (flesher, 2006), with a basal diameter greater than 14cm (circumference at breast height above 160cm) and an upper canopy 8-18m high. it is common for the inter-row vegetation to be cut back once every 6-12 months. rubber is a deciduous tree, changing all of its leaves once a year, mostly during the cooler wetter months (july and august) which creates a profound physiognomy and microclimate change, with the entire canopy exposed to full sunlight and increased sunlight reaching the understory. nest sampling the nests were sampled in two habitats that represents the two extremes of canopy exposure to sunlight: the more open deciduous rubber grove canopies that are exposed to direct sunlight for 1-2 months each year; and the dense closed evergreen canopies of the rainforest. we used data on nest density of stingless bees in the rainforest of the rem (silva et al., 2013) to compare with a density measure collected in the rubber plantation. t. spinipes builds exposed nests on medium and large tree branches and in vacant arboreal termite mounds (roubik, 1989; zucchi et al., 1993; vieira et al., 2007; camargo, 2007) which makes is easy to detect these large and conspicuous nests (fig.1) by visual searching. the nests were sampled in 40 20 x 200m plots (totalling 16ha), which were randomly distributed in 2,000 ha of rubber groves. the plots were sampled monthly for five consecutive days between august and december 2010. the nests were georeferenced (gps garmin 60cs) and some bee specimens were collected checking in order to ensure that the species inhabiting the nests were t. spinipes. bee specimens were identified by dr. favizia freitas de oliveira of the insect bionomics, biogeography, and systematics laboratory (biosis) at federal university of bahia ufba, and they were deposited in the zoology museum at ufba (mzufba) and in the pollination ecology laboratory (ecopol-ufba). data analysis the permutation t-test (10.000 permutations) were used to test for the equality of densities in both habitats (anderson, 2001), considering random samples with excess zero-count data (very small probability of occurrence per sociobiology 63(3): 991-997 (september, 2016) 993 plot unit) and likely the poisson-distribution of data. the spatial distribution of the nests was evaluated with the nearest neighbour (r) dispersion index (clark & evans, 1954), using the software ecological methodology, 2nd edition (kenney & krebs, 2000). the dispersion index is the ratio between the observed average distance from nearest neighbour and the expected average distance from the nearest neighbour if the nests were randomly distributed (aggregated distribution r <0, random r = 1, and uniform r> 1). results and discussion 18 nests of t. spinipes were recorded in the 16 hectare of rubber groves sampled, with a density of 1.1 nests/ha (table 1). this high density for a species of stingless bee contrasts to rarity of its nests in adjacent forest habitats (silva et al., 2013) where a very low frequency of individuals (3.2%) was also observed foraging on attractive baits (silva et al., 2008). independent sampling of nests (table 1) and bees visiting flowers in many habitat types showed that t. spinipes is common in open vegetation habitats and uncommon in forest (knoll, 1990; martins, 1994; silveira & campos, 1995; aguiar & martins, 1997; carvalho & marchini, 1999; viana, fig 1. trigona spinipes (fabricius, 1793) exposed nest in a rubber tree at michelin ecological reserve (mer).general view and close (righ corner). habitat type density (nest/ha) spatial distribution nesting height references anthropic (urban) 0,096 undetermined 10m freitas, 2001 anthropic (urban) 0,12 undetermined 11,1m souza et al., 2005 anthropic (urban) 0,18 undetermined 10m taura & laroca 1991 anthropic (urban) 0,023 undetermined sousa et al., 2002 anthropic (urban) 0,033 undetermined 14m silva & ramalho, in press savannah/steppe (semi-arid caatinga) 0,4 undetermined 6m teixeira & viana, 2005 tropical savannah (brazilian cerrados) 0,3 undetermined henriques, 1997 tropical savannah (brazilian cerrados) 0,56 random and clumped 2,2 pioker-hara, 2011 tropical rain forest (brazilian atlantic forest) 0,265 undetermined 16,8m batista et al., 2003 tropical forest (brazilian atlantic forest) 0,025 undetermined mateus et al., 2009 tropical forest (brazilian atlantic forest) 0,039 undetermined siqueira & ferreira, 2007 tropical forest (brazilian atlantic forest) 0,014 undetermined werneck et al., 2007 tropical rain forest (brazilian atlantic forest) 0,031 undetermined 12m silva et al., 2013 rubber tree plantation 1,1 uniform 10m this study table 1. density of nests of eusocial bee trigona spinipes in different habitats in brazil. undetermined spatial distribution is related to very low nest densities and then to large and unaccessed distances between neighbor nests. md silva, m ramalho – density and nest spacing of a stingless bee994 1999; ramalho, 2004). an apparent exception would be the moderate nest density observed by batista et al., (2003) in a very small and narrow patch (100-200m wide) of disturbed forest bordering a water resorvoir in a suburban landscape, where the nest density were likely influenced by a strong edge effect. on the rubber trees, the large exposed nests of t. spinipes were built around branches between 6 and 16m height (10.2m ±3.2m). in general, this species builds nests on the upper canopy of trees, and nests at low heights are only recorded in absence of large trees (table 1). assuming its tight association with tree canopy (e.g. wille & michener, 1973; roubik, 2006), the high availability of nesting sites in both the rubber groves and the forest does not explain the significant variation in the observed density between these two habitats (p = 0.0001, permutation t test; see also silva et al., 2013). the much higher density of t. spinipes in the rubber groves than in the adjacent forest could not be explained by availability of flower sources either, because there are fewer flower resources throughout the year in the rubber groves than in the forest. since other stingless bee species with high food demands reached high nest densities (3.5 nests/ha) in the rem forest (silva et al., 2013), the availability of floral resources or flowering pattern (e.g. eltz et al., 2002) per se does not provide a satisfactory explanation for the low nest density of t. spinipes in this habitat, either. moreover, tropical lowland forests often support a high diversity and abundance of stingless bees, which depend on the continuous and abundant floral resources to maintain their populous perennial colonies (eg. roubik, 1993; ramalho, 2004). on the other hand, cortopassi-laurino & imperatrizfonseca (2009) observed that the nests of t. spinipes were often associated with deciduous trees that loose their leaves during the drier and colder season (e.g. chorisia speciosa st.-hil.), thus increasing exposure to direct sunlight and heat absorption assumed to be necessary for thermoregulation (zucchi & sakagami, 1972; almeida & laroca, 1988). in the rem, the rubber trees loose their leaves seasonally and mainly during june and august, when the rainfall is more frequent and the temperature drops by up to 19˚ c. these favorable physical conditions whith in this open canopy are likely responsible for the high density of nests in the rubber groves. in contrast, the closed shaded canopy in the adjacent rainforest provides poor nesting habitat. however, it is unlikely that restrictions on direct absorption of heat from sunlight play a role in thermoregulation of such populous colonies, under moderate air temperatures (around 20 ° c), in the whole range of tropical lowland forests. exposed to heavy rains, the external structure of its huge nest remain wet for prolonged and we suggest t. spinipes depends on direct insolation to keep its nests dry and fungus-free. in the tropical forest, nest humidity is probably a constraint to huge exposed nests, because the more closed the canopy, the more slowly the nests will dry, especially in high rainfall evergreen forest. in contrast, the high availability of sunny sites for nesting within the deciduous and open canopy of rubber trees favours rapid drying of external nest structures and realeasing the high nest density and wide spread distribution of t. spinipes where this agroforestry system dominates. the results of this study in the rem show that the large difference in nest density of t. spinipes between adjacent habitats with very distinctive canopy shading supports the argument that shading is a powerful constraint in the selection of appropriate nesting sites. whether for thermoregulation or reducing nest moisture after heavy rains, the dependence on direct insolation is likely the the principal habitat variable that constrains the use by t. spinipes of the closed canopy tropical rainforests. the rubber groves, with upper tree canopies exposed to the full sun, favor the occupation of this extensive agroforestry landscape by this stingless bee. as nest density rises and approaches the density threshold, the species’patrolling mechanism becomes effective and regulates nest densities resulting in the uniform distribution of t. spinipes colonies in the rubber groves. this study in the rubber plantations had the highest t. spinipes density observed to date (table 1). most previous studies has detected low nest density, and undetermined spatial pattern due to nonaccessed distances between nearest neighbor nests (>90m <400m based on nest densities in table 1). at intermediate density (0.56 nests/ha), pioker-hara (2011) recorded random or clumped nest spacing in the tropical savannah. at low or moderate densities (table 1), the spacing of t.spinipes nests (e.g. > 90m) should be poorly influenced by inter-colonial interactions because the degree of agonistic behavior (shakleton et al., 2014 ) likely depends on the distances between nests and home range of colonies (hubbell & johnson, 1977). in contrast, in the rubber groves the spatial distribution of nests is even (r = 2.02) (fig 2) probably because the density of t.spinipes is high enough to make the patrolling by workers an effective territorial mechanism of spacing in this habitat. assuming the home range of colonies of stingless bees could be estimated by the inverse density of nests (hubbell & johnson, 1977), nest densities of 1.0 and 0.6 nest/ha indicates a patrolling distance threshold between 60m and 70m to produce even nest spacing in t. spinipes. therefore, even for such a huge nest population, patrolling could prevent the establishment of new nests only at short distances. besides scalate aggressiviness towards co-specifics (hubbel & johnson, 1977), and very large woker population, such patrolling mechanism is likely supported by the ability of t. spinipes to use directional olfactory stimuli to fast attraction of nest-mates to very specific sites (nieh et al., 2004) and at the very beginning of swarming process of competing colonies. in synthesis, use of aggression to control nest density is likely a very exceptional territorial mechanisms in stingless bee that could be effectively used at short distances. sociobiology 63(3): 991-997 (september, 2016) 995 fig 2. spatial distribution of t. spinipes nests (black circles) in the ‘reserva ecológica michelin’ (rem). the dark grey areas are rainforest remnants (brazilian atlantic forest). the white areas are mainly covered by rubber tree groves intercropped with cacao 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(eds c. cruzlandim, n.j. hebling, e. lello & c.s. takahashi), pp. 301309. unesp, rio claro. tx open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1789sociobiology 64(3): 339-346 (september, 2017) updating the geographic records of social wasps (vespidae: polistinae) in roraima state introduction many areas of brazil lack the most basic biodiversity studies, particularly in the case of invertebrates. in order to develop any effective conservation proposals, it is first of all necessary to acquire knowledge of the species that occur in a particular area (melo et al. 2005). this taxonomic baseline is obtained by conducting biodiversity inventories. vespidae is a family of wasps that includes over 5.018 valid species worldwide and despite the worldwide distribution of some species they are found in high abundance in the tropical region (pickett & carpenter, 2010). polistinae social wasps comprise 26 genera and 958 species (pickett & carpenter, 2010); the subfamily is divided in four tribes: ropalidiini, polistini, myschocyttarini and epiponini except for ropalidiini, the other tribes are represented in brazil (carpenter & marques, 2001). polistes latreille, mischocyttarus de saussure and the 19 genera of epiponini compound the brazilian fauna abstract the roraima state in brazil is part of northern amazon, an area harboring high biodiversity and high degree of endemism. nevertheless, there are few studies on diversity of social wasps occurring in this region. this study presents a list of social wasps (vespidae: polistinae) collected actively and using malaise, suspended and light trap in six localities in roraima state. a total of 85 species of 14 genera were collected. fourty-five of these species are new distribution records to roraima state, some species are not common found in the collections and lists of species, and some are recorded for the second time to brazil or the amazon region. this increase may be an indication that the polistinae richness is probably higher in the regions studied and that roraima may well contain a number of additional (as yet unrecorded) social wasp species. more comprehensive studies are needed in order to increase the knowledge of wasp species in roraima, contributing to increased knowledge of the diversity in northern brazil. sociobiology an international journal on social insects pcs barroso1, a somavilla2, r boldrini1 article history edited by gilberto m. m. santos, uefs, brazil received 12 june 2017 initial acceptance 09 july 2017 final acceptance 02 august 2017 publication date 17 october 2017 keywords new records; north amazon of brazil; paper wasps; survey. corresponding author alexandre somavilla instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo nº 2936 cep 69067-375, manaus-am, brasil. e-mail: alexandresomavilla@gmail.com of wasps totalizing about 300 species, in brazil is among the richest in the world, with 321 species (carpenter & marques, 2001; hermes et al., 2017). brazilian amazon rainforest has one of the greatest biodiversity in world, being registered the higher diversity of social wasps in this biome 20 genera and more than 200 species were recorded, representing about 70% of the brazilian fauna of social wasps (silveira, 2002; somavilla et al., 2014; barbosa et al., 2016). recently, some studies have been carried out in the brazilian amazon in acre state (morato et al., 2008), amapá state (silveira et al., 2008), amazonas state (silveira et al., 2008; somavilla et al., 2014, somavilla et al., 2015; somavilla & de oliveira, 2017), maranhão state (somavilla et al., 2014), pará state (silveira, 2002; silva & silveira, 2009) and roraima state (raw, 1998). despite the constant effort in studies about social wasps conducted in brazil, little is known regarding their diversity and distribution in the amazon region. 1 universidade federal de roraima, centro de estudos da biodiversidade (cbio), boa vista, roraima, brazil 2 instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, bolsista pdj, manaus, amazonas, brazil research article wasps mailto:alexandresomavilla@gmail.com pcs barroso, a somavilla, r boldrini – social wasps from roraima state340 furthermore, for roraima there are only one work about social wasp in estação ecológica de maracá (raw, 1998) in addition to the previous records from richards (1978), they reporting 40 species for this state. moreover, it is an area close to venezuela and guiana, regions known for presenting endemic species for different groups of organisms, but about the diversity of social wasps in this part of amazon is very little known, generating a gap in knowledge about the species occurring in north amazon of brazil. the purpose of this work was to present an updated about the geographic records of social wasps’ fauna in roraima state. material and methods characteristic of the roraima state roraima state, located in the center-north from the amazon region, it has an area of ~224.300 km², represents 3% inserted in the field of amazonian ecosystem and 2.6% of the brazilian territory (iteraima, 2005). there is the largest continuous block of savannas in the brazilian amazon (barbosa et al., 2005). roraima has the “lavrado” and open areas of the south of the state, with 43,281 km² and 17,500 km² respectively, occupying ~ 27% of the territory. the region north and northwest of roraima cover a mountainous system with strong tectonic structural contact between the parima and pacaraima. that can reach 1.100 m (serra do tepequém) or near to 1.700 m (serra da mocidade) (barbosa, 1997; carvalho et al., 2016). the climatic type of the region defined as aw with well-defined dry period, but of greater intensity in the months of december to march (northeast of roraima), af with a high annual rainfall > 2.000 mm (southern region) and am with intermediate between the other climatic types, with not so defined dry period and medium annual rainfall > 1.700 mm (mountainous system) (barbosa, 1997). the data were collected from five municipalities in roraima state: amajari serra do tepequém (3°46’13”n, 61°43’57”w, 626 m); boa vista universidade federal de roraima (2°48’41’’n, 60°41’08”w, 83 m); caracaraí parque nacional serra da mocidade (1°42’ n, 61°47’w, 1.050 m); and parque nacional do viruá (1º29’23”n, 61º00’12”w, 44 m); pacaraima (4°28’30’’n, 61°09’44’’w, 900 m) and rorainópolis (00°56’46” n, 60°25’05’’w, 98 m) (fig. 01). sampling method and species preservation were used 6-meter intercept malaise traps (gressitt & gressitt, 1962), 2-meter intercept malaise traps (townes model) for collect wasps in understory; intercept suspended traps (rafael & gorayeb, 1982) for collect wasps in canopy tree and light traps (a white sheet attached to a white light). additionally, guided manual collections were also performed throughout the excursion with entomological nets and active search for wasps’ colonies along trails, such as margins of “igarapés” and surroundings of houses, and they were listed just to add the data. all material collected was fixed in combustive alcohol 96% and after pinned. the identification of the specimens fig 1. map with five municipalities and six localities in roraima state: amajari (serra do tepequém), boa vista (universidade federal de roraima), caracaraí (parque nacional do viruá and parque nacional serra da mocidade), pacaraima and rorainópolis. sociobiology 64(3): 339-346 (september, 2017) 341 followed the keys proposed by richards (1978) and carpenter (2004). voucher specimens were deposited at the entomological laboratory of universidade federal de roraima (ufrr) and zoological collection of invertebrates at the instituto nacional de pesquisas da amazônia (inpa) in manaus, brazil. results in this work, we listed the occurrence of 85 species of social wasps for roraima state in 14 genera; we identified 77 species in our collects and add other eight species from literature, did not collected in this study (richards, 1978; raw, 1998) (tab. 01). more than 60% of the collected species belong to three genera: polybia lepeletier (25 species), mischocyttarus de saussure (14 species) and agelaia lepeletier (13 species). in the polistes genera nine species and two subspecies were collected, apoica lepeletier (six species), protopolybia ducke (five species), brachygastra perty (three species), angiopolybia araujo, metapolybia ducke and synoeca de saussure (two species) and chatergus lepeletier, leipomeles moebius, parachartergus r. von ihering and pseudopolybia de saussure one species, complete the list. from 85 species sampled, around half of them (n=45) represent new records for roraima state, which are indicated (*) in table 01. most of the species were captured actively with entomological net, 57 species. however, some species were captured by indirect capture methods such as intercept malaise trap (n=41), intercept suspended trap (n=07) and light trap (n=08) (table 01). discussion data collected and presented here aggregate information on the diversity of social wasps in the north amazon, contributing to the elucidation of the gap of knowledge for this region. from 85 species sampled, 45 represent new records for roraima state: agelaia centralis, ag. constructor, ag. flavipennis, ag. hamiltoni, ag. lobipleura, ag. myrmecophila, angiopolybia paraensis, apoica albimaculata, ap. pallens, ap. strigata, brachygastra bilineolata, leipomeles spilogastra, metapolybia cingulata, mischocyttarus cerberus, m. collaris, m. flavicans, m. lecointei, m. smithii, m. tomentosus, parachartergus fraternus, polistes carnifex, p. deceptor, p. pacificus, p. subsericeus, p. testaceicolor, polybia bicyttarela, po. bifasciata, po. bistriata, po. chrysothorax, po. gorytoides, po. incerta, po. jurinei, po. lugubris, po. micans, po. minarum, po. platycephala, po. signata, po. singularis, po. spinifex, po. striata, po. tinctipennis, protopolybia acutiscutis, pr. chartergoides, pr. emortualis and pr. rugulosa. richards (1978) recorded the occurrence of 18 species in roraima state, and four species of this study agelaia multipicta (haliday, 1836), brachygastra smithii (de saussure, 1854), polistes billardieri fabricius, 1804 and polistes brevifissus richards, 1978 were not collected in this present study. raw (1998) recorded the occurrence of 36 species in ilha de maracároraima state and just four species not collected in our work: mischocyttarus alboniger richards, 1978, mischocyttarus carbonarius (de saussure, 1854), mischocyttarus prominulus richards, 1941 and polybia dimorpha richards 1978 species not collected in our work. most species are widely distributed in the amazon, except for some species of mischocyttarus and polybia. the other 77 specie are recorded in ours collects. however, some species are not common found in the collections and lists of species for the region. this is the case of species such as a. flavipennis, a. hamiltoni, a. lobipleura, l. spilogastra, po. bicyttarela, po. bifasciata, po. gorytoides, po. incerta, po. lugubris, po. roraimae, po. signata, po. spinifex, pr. acutiscutis and pr. emortualis which they have rarely been collected, and some even being recorded for the second time to brazil or the amazon region. the subspecies polistes carnifex carnifex and p. carnifex rufipennis, p. versicolor versicolor and p. versicolor kaieteurensis, polybia gorytoides sculpturata and polybia occidentalis occidentalis have been identified as subspecies, since they are morphologically different, and as variations have a more restricted distribution, already being registered for venezuela, some now for the roraima state. two species were incorrectly cited for the roraima state. richards (1978) present incorrect information about p. gorytoides sculpturata, this species is not indicated in the examined material, but it is indicated in identification key for roraima state. raw (1998) cited the occurrence of ap. pallens for the roraima state according richards (1978), but this species was not record in the original work. the two species were confirmed in this work and considered as new occurrences. this study corroborates the results of silva and silveira (2009) and somavilla et al. (2014), who considered active search the most efficient method for collecting social wasps, mainly close to the forest ground and in the understory; in this study, we collected 59 species in active search in roraima state. but the addition of different collection methods is an important tool for sampling of the richness of social wasps in an area, as in general the species have a varied foraging behavior. for exemple, 41 species were collected using intercept malaise trap, demonstrating the efficiency of this method for this taxon. additionally, seven species were collected using intercept suspended trap and eight species were collected using light trap, apoica mainly. these last two techniques are not used in collections. amazon region has the highest diversity of polistinae (richards, 1978; carpenter & marques, 2001; barbosa et al., 2016). in brazilian amazon 20 genera and more than 200 species have been recorded, which represents about 2/3 of the brazilian fauna (silveira, 2002), with 125 species recorded only for the state of amazonas (barbosa et al., 2016). nevertheless, this impressive number surely does not yet represent the region’s mega diversity, since there were pcs barroso, a somavilla, r boldrini – social wasps from roraima state342 only four studies carried out on this state (barbosa et al., 2016). this statement becomes clear when we see the state of roraima: with reports of richards (1978) and raw (1998) work 40 species. this study presents new occurrences of 45 social wasp species in roraima. our findings extend the species distributions and increase the number of species recorded in roraima to 85 and more two subspecies for polistes. this increase may be an indication that the richness is probably higher in the regions studied and that roraima may well contain a number of additional (as yet unrecorded) social wasp species. more comprehensive studies are needed in order to increase the knowledge of wasp species in roraima, contributing to increased knowledge of the diversity in north brazil. acknowledgements specimens were collected as part of a project rede de biodiversidade de insecta na amazônia coordinated by josé albertino rafael (inpa) and financed by conselho nacional de desenvolvimento científico e tecnológico (cnpq, process: 407623/2013-2). we also thank to marcio luiz de oliveira (inpa) for the lab support and ismael barreto de oliveira (ufrr) for help in roraima map. as for scholarship of cnpq, pdj process 50029/2017-9. taxon richards (1978) raw (1998) present work locality collected method agelaia angulata (fabricius, 1804) x x amajari: serra do tepequém caracaraí: serra da mocidade malaise active agelaia cajenennsis (fabricius, 1798) x amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade active, malaise malaise active agelaia centralis (cameron, 1907)* amajari: serra do tepequém caracaraí: parque nacional viruá active, malaise malaise agelaia constructor (de saussure, 1854)* rorainópolis active agelaia flavipennis (ducke, 1905)* amajari: serra do tepequém malaise, suspended agelaia fulvofasciata (degeer, 1773) x x amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade pacaraima malaise, suspended malaise active active agelaia hamiltoni (richards, 1978)* caracaraí: parque nacional viruá malaise agelaia lobipleura (richards, 1978)* amajari: serra do tepequém active agelaia multipicta (haliday, 1836) x x agelaia myrmecophila (ducke, 1905)* caracaraí: parque nacional viruá malaise agelaia ornata (ducke, 1905) x amajari: serra do tepequém caracaraí: serra da mocidade active, malaise active agelaia pallipes (olivier, 1791) x caracaraí: parque nacional viruá caracaraí: serra da mocidade rorainópolis malaise active active agelaia testacea (fabricius, 1804) x x amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade active, malaise, suspended malaise active angiopolybia pallens (lepeletier, 1836) x amajari: serra do tepequém caracaraí: serra da mocidade active, malaise active angiopolybia paraensis (spinola, 1851)* amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade malaise active active apoica albimacula (fabricius, 1804)* caracaraí: serra da mocidade light apoica flavissima van der vecht, 1972 x caracaraí: serra da mocidade light apoica pallens (fabricius, 1804)* amajari: serra do tepequém caracaraí: serra da mocidade rorainópolis active, malaise light malaise apoica pallida olivier, 1791 x x amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade pacaraima rorainópolis light light, malaise light light active, light table 01. species from roraima state, listed for richards (1978), raw (1998) and in present work with six localities: amajari: serra do tepequém; boa vista campus; caracaraí: serra da mocidade; caracaraí: parque nacional viruá-national park; pacaraima and rorainópolis in br174 close city, with different collected methods: active with entomological nets; intercept malaise trap (2 and 6 meters); intercept suspended trap and light trap. note: the method line is the same for each locality. sociobiology 64(3): 339-346 (september, 2017) 343 taxon richards (1978) raw (1998) present work locality collected method apoica strigata richards, 1978* caracaraí: serra da mocidade light apoica thoracica du buysson, 1906 x amajari: serra do tepequém caracaraí: parque nacional viruá pacaraima rorainópolis light light light light brachygastra bilineolata spinola, 1841* boa vista – campus active brachygastra lecheguana (latreille, 1824) x caracaraí: serra da mocidaderorainópolis active active brachygastra smithii (de saussure, 1854) x x chartergus artifex (christ, 1791) x rorainópolis active leipomeles spilogastra (cameron, 1912)* amajari: serra do tepequém active metapolybia cingulata (fabricius, 1804)* rorainópolis active metapolybia unilineata (r. von ihering, 1904) x amajari: serra do tepequém rorainópolis active active mischocyttarus alboniger richards, 1978 x mischocyttarus carbonarius (de saussure, 1854) x mischocyttarus cerberus ducke, 1918* amajari: serra do tepequém malaise mischocyttarus collaris ducke, 1904* rorainópolis active mischocyttarus flavicans (fabricius, 1804)* caracaraí: serra da mocidade active mischocyttarus injucundus (de saussure, 1854) x rorainópolis active mischocyttarus labiatus (fabricius, 1804) x amajari: serra do tepequém ro-rainópolis malaise active mischocyttarus lecointei ducke, 1904* caracaraí: serra da mocidade active mischocyttarus maracaensis raw, 1999 x amajari: serra do tepequém active mischocyttarus metathoracicus de saussure, 1854 x amajari: serra do tepequém malaise mischocyttarus prominulus richards, 1941 x mischocyttarus smithii de saussure, 1853* caracaraí: serra da mocidade active mischocyttarus surinamensis de saussure, 1854 x amajari: serra do tepequém malaise mischocyttarus tomentosus zikán, 1935* amajari: serra do tepequém malaise parachartergus fraternus (gribodo, 1892)* amajari: serra do tepequém ro-rainópolis malaise active polistes billardieri fabricius, 1804 x polistes brevifissus richards, 1978 x x polistes canadensis (linnaeus, 1758) x amajari: serra do tepequém pacaraima rorainópolis active, light active active polistes carnifex carnifex (fabricius, 1775)* pacaraima active polistes carnifex rufipennis latreille, 1817* caracaraí active polistes deceptor schulz, 1905* amajari: serra do tepequémpacaraima malaise active polistes pacificus fabricius, 1804* rorainópolis active polistes subsericeus de saussure, 1854* rorainópolis active polistes testaceicolor bequaert, 1937* amajari: serra do tepequémcaracaraí: parque nacional viruá malaise malaise polistes versicolor kaieteurensis bequaert, 1934 x caracaraí: parque nacional viruá active polistes versicolor versicolor (olivier, 1792) x x amajari: serra do tepequém active polybia belemensis richards, 1970 x amajari: serra do tepequém malaise polybia bicyttarella richards, 1951* rorainópolis active table 01. species from roraima state, listed for richards (1978), raw (1998) and in present work with six localities: amajari: serra do tepequém; boa vista campus; caracaraí: serra da mocidade; caracaraí: parque nacional viruá-national park; pacaraima and rorainópolis in br174 close city, with different collected methods: active with entomological nets; intercept malaise trap (2 and 6 meters); intercept suspended trap and light trap. note: the method line is the same for each locality. (continuation) pcs barroso, a somavilla, r boldrini – social wasps from roraima state344 table 01. species from roraima state, listed for richards (1978), raw (1998) and in present work with six localities: amajari: serra do tepequém; boa vista campus; caracaraí: serra da mocidade; caracaraí: parque nacional viruá-national park; pacaraima and rorainópolis in br174 close city, with different collected methods: active with entomological nets; intercept malaise trap (2 and 6 meters); intercept suspended trap and light trap. note: the method line is the same for each locality. (continuation) taxon richards (1978) raw (1998) present work locality collected method polybia bifasciata de saussure, 1854* amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade malaise malaise active polybia bistriata (fabricius, 1804)* amajari: serra do tepequém ro-rainópolis malaise active polybia chrysothorax (lichtenstein, 1796)* amajari: serra do tepequémrorainópolis active active polybia dimidiata (olivier, 1791) x x amajari: serra do tepequémrorainópolis malaise active polybia dimorpha richards, 1978 x polybia gorytoides sculpturata ducke, 1904* caracaraí: parque nacional viruá malaise polybia ignobilis (haliday, 1836) x x boa vista active polybia incerta ducke, 1907* amajari: serra do tepequémcaracaraí: parque nacional viruá malaise malaise polybia jurinei de saussure, 1854* caracaraí: serra da mocidaderorainópolis active active polybia liliacea (fabricius, 1804) x amajari: serra do tepequém caracaraí: parque nacional viruá pacaraima active, malaise active, malaise, light active polybia lugubris (curtis, 1844)* amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade malaise malaise active polybia micans ducke, 1904* amajari: serra do tepequémcaracaraí: parque nacional viruá active, malaise malaise polybia minarum ducke, 1906* amajari: serra do tepequémcaracaraí: serra da mocidade suspended active polybia occidentalis occidentalis (olivier, 1792) x x amajari: serra do tepequém boa vista caracaraí: parque nacional viruá pacaraima rorainópolis active active active active active polybia platycephala richards, 1951* amajari: serra do tepequém caracaraí: serra da mocidade rorainópolis malaise active active polybia rejecta (fabricius, 1798) x amajari: serra do tepequém caracaraí: parque nacional viruá caracaraí: serra da mocidade pacaraima rorainópolis active, malaise, suspended active, malaise active active active polybia roraimae raw, 1999 x amajari: serra do tepequém malaise polybia sericea (olivier, 1792) x x amajari: serra do tepequém boa vista caracaraí: parque nacional viruá active active active polybia signata ducke, 1905* caracaraí: serra da mocidade active polybia singularis ducke, 1909* caracaraí: parque nacional viruá malaise polybia spinifex richards, 1978* amajari: serra do tepequém suspended polybia striata (fabricius, 1787)* amajari: serra do tepequém caracaraí: parque nacional viruá rorainópolis malaise active active polybia tinctipennis fox, 1898* amajari: serra do tepequém malaise protopolybia acutiscutis (cameron, 1906)* rorainópolis active sociobiology 64(3): 339-346 (september, 2017) 345 * new record from roraima state. table 01. species from roraima state, listed for richards (1978), raw (1998) and in present work with six localities: amajari: serra do tepequém; boa vista campus; caracaraí: serra da mocidade; caracaraí: parque nacional viruá-national park; pacaraima and rorainópolis in br174 close city, with different collected methods: active with entomological nets; intercept malaise trap (2 and 6 meters); intercept suspended trap and light trap. note: the method line is the same for each locality. (continuation) taxon richards (1978) raw (1998) present work locality collected method protopolybia chartergoides (gribodo, 1892)* rorainópolis active protopolybia emortualis de saussure, 1855* caracaraí: parque nacional viruá malaise protopolybia exigua (de saussure, 1854) x x amajari: serra do tepequém active, malaise protopolybia rugulosa ducke, 1905* rorainópolis active pseudopolybia vespiceps (de saussure, 1863) x caracaraí: parque nacional viruá active synoeca surinama (linnaeus, 1767) x amajari: serra do tepequém caracaraí: parque nacional viruá rorainópolis active, malaise, suspended active, malaise active synoeca virginea (fabricius, 1804) x amajari: serra do tepequémcaracaraí: parque nacional viruá active active, malaise references barbosa, b.c., detoni, m., maciel, t.t. & prezoto, f. 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(2014). vespas sociais (vespidae: polistinae) em uma área de floresta ombrófila densa amazônica no estado do maranhão, brasil. entomobrasilis, 7: 183-187. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.397-404sociobiology 60(4): 397-404 (2013) parasitoids of acromyrmex (hymenoptera: formicidae) leaf-cutting ants in continuous and fragmented atlantic forest l elizalde1, jm queiroz2 introduction fragmentation of the habitat is a major threat to global biodiversity (kruess & tscharntke, 1994; foley et al., 2005). habitat fragmentation due to anthropogenic activity results in landscapes composed of a mosaic of remnant habitats surrounded by a more or less hostile agricultural or pasture land for cattle, called the matrix. the atlantic forest, once the largest forest in america, is an ecosystem severely affected by habitat fragmentation. most of the remaining atlantic forest exists as small fragments (<100 ha; ranta et al., 1998), that are composed of second-growth forests in early to intermediate stages of succession (metzger et al., 2009), and the few large fragments are located in steep terrain that made human occupation difficult (silva et al., 2007). most of the original forest is fragmented and 80% of the fragments are <50 ha, and together with intermediate secondary forests, correspond to approximately 32–40% of what remains (ribeiro et al., 2009). abstract fragmentation of the habitat is a major threat to biodiversity in the atlantic forest. parasitoids seem to be particularly susceptible to habitat fragmentation. this study evaluated whether habitat fragmentation affected the interactions between phorid parasitoids and their acromyrmex leaf-cutting ant host. host density, and parasitoid species richness, abundance and proportion of nests with phorids were compared for a fragmented landscape and a well-preserved continuous forest in the atlantic forest of southeast brazil. five acromyrmex species and seven species of phorid parasitoids were found, most of them attacking exclusively acromyrmex niger (smith). host nest density was similar in continuous and fragmented forests, and host species density was higher in fragmented forest. parasitoid species richness, abundance and proportion of ant nests with phorids were higher in the continuous forest. this work showed for the first time the negative effect that forest fragmentation has on parasitoid species of acromyrmex ants, apparently due to phorid inability to reach fragments. however, even when phorid abundance was considerably reduced in forest fragments, phorids of some species were able to parasitize ants there. in addition, the quantitative interactions among acromyrmex ants and their parasitoids in atlantic forest are described for the first time. sociobiology an international journal on social insects 1 universidad nacional del comahue, inibioma–conicet, pasaje gutiérrez , bariloche, argentina. 2 universidade federal rural do rio de janeiro, seropédica, rio de janeiro, brazil. research article ants article history edited by: qiuying huang, hau, china received 29 july 2013 initial acceptance 18 august 2013 final acceptance 17 october 2013 keywords habitat fragmentation, phoridae, species richness, host-parasitoid interactions, apocephalus, myrmosicarius corresponding author luciana elizalde laboratorio ecotono crub–univ. nacional del comahue inibioma–conicet, pasaje gutiérrez 1125, 8407, bariloche, argentina e-mail: luelizalde@gmail.com in fragmented landscapes, population structure can be altered by several endogenous and exogenous processes (tscharntke et al., 2002), and species are differentially affected, with some being very sensitive while others can even increase their population sizes (see fahrig, 2003 for a review). because community structure, interspecific interactions, and ecological functions are also affected, the magnitude and direction of the changes due to habitat fragmentation are sometimes unpredictable, especially if there is not enough information about natural history of the taxa in question. some general responses to habitat fragmentation had been elucidated, however (tscharntke et al., 2002; ewers & didham, 2006). one general response is that the effect of habitat fragmentation is more severe for species in higher trophic levels (kruess & tscharntke, 2000; tscharntke et al., 2002; montoya et al., 2006). parasitoids, insects that deposit eggs inside a host from which a carnivorous larva emerges and feeds on it, occupy higher trophic levels in food webs. several studies have shown the negative effect that habitat l elizalde, jm queiroz habitat fragmentation in phorid–leaf-cutting ant interactions398 fragmentation has on parasitoids, both in tropical and nontropical areas (see tscharntke et al., 2002 for a review; cagnolo et al., 2009). in addition, parasitoids will be more susceptible to habitat fragmentation because of their narrow host range, as most of them are specialist, developing on few hosts only (godfray, 1994). it has been proposed that species with narrower niches are more affected than generalists by fragmentation of their habitat (henle et al., 2004; cagnolo et al., 2009). leaf-cutting ants in the genera acromyrmex and atta are emblematic organisms in the atlantic forest. these ants are generalist herbivores, and cut leaves of a wide array of plants that carry back to their nests to feed a fungus (hölldobler & wilson, 1990). while they are outside their nests, workers are attacked by dipteran parasitoids that belong to the family phoridae. these parasitoids oviposit in adult workers of leaf-cutting ants while they are foraging, cutting leaves or removing wastes (elizalde & folgarait, 2012). two lines of evidence suggest that these phorid parasitoids may be negatively affected by habitat fragmentation. first, many of these phorids are specialists (elizalde & folgarait, 2011). second, phorid flies that attack leaf-cutting ants have small body sizes (elizalde & folgarait, 2011). because body size correlates positively with dispersal abilities (blackburn et al., 1999) and adult phorid parasitoids, which are the dispersal stage, do not have special long-dispersal means, it is possible that dispersal throughout a hostile matrix might imply a risky task. there is some evidence that phorid parasitoids of atta leafcutting ants, are negatively affected by habitat fragmentation. in fact, release of these natural enemies has been proposed as one of the explanations of the high abundance of some species of atta ants in small forest fragments and forest edges (rao, 2000; almeida et al., 2008). however, the focus of this work is on acromyrmex ant hosts, which do not share phorid parasitoid species with atta (elizalde & folgarait, 2011). this study aims to provide evidence on whether habitat fragmentation is a negative factor affecting the interactions between phorid parasitoids and their acromyrmex leafcutting ant hosts. we first tested whether acromyrmex species are affected by forest fragmentation in the atlantic forest of southeast brazil. then, we compared parasitoid species richness, abundance and proportion of nests with parasitoids in fragmented and well-preserved forests. we also described for the first time the quantitative interactions among acromyrmex ants and their parasitoids in atlantic forest. materials and methods study sites. the study was conducted in two areas within the atlantic forest of rio de janeiro state, southeast brazil, which were chosen based on their different degree of fragmentation (fig. 1). the vassouras-barra do piraí figure 1. sampled areas in rio de janeiro state. insets show forest areas in black. vassouras-barra do piraí fragmented landscape is in the inset at the bottom, and regua continuous forest on the top, both have the same scale. sociobiology 60(4): 397-404 (2013) 399 area (22º 24’s, 43º 40’w) was selected as the highly fragmented forest. in the area, forest remains as small–medium sized fragments surrounded by a deforested matrix, which is mainly used as pasture for cattle ranching, but once was used for coffee plantations (francelino et al., 2012). forest remnants in the atlantic forest of rio de janeiro represent only a 15.7% of the vegetation cover of the area, and are mainly secondary lowland and submontane semi-deciduous forests (oliveira-filho & fontes, 2000; francelino et al., 2012). mean annual temperature is 20°c, and annual rainfall is 1,196 mm (francelino et al., 2012). the continuous forest is located in a ngo reserve, reserva ecológica de guapiaçu—regua (22º 25’s, 42º 44’w), located in the municipality of cachoeiras de macacu. the area is a well preserved forest and vegetation type can be classified as a lowland and submontane rain forest of the coastal mountain range of the rio de janeiro state (oliveirafilho & fontes, 2000). late successional forest was selected to sample in this area (fig. 1). the mean annual temperature is 20°c, and annual rainfall is 2,010 mm (massera da hora & gonçalves costa, 2010). the regional climate is characterized by a hot and rainy season from october to march and a cooler and drier season from april to september (francelino et al., 2012). thus, we sampled during the wet season, in february and march 2012, to reduce differences in precipitation between areas (170 vs. 193 mm mean monthly precipitation in february for the fragmented forest and the continuous forest, respectively; record-meteo, 2007; massera da hora & gonçalves costa, 2010), although leaf-cutting ant phorids do not seem to be especially susceptible to low precipitation (bragança et al., 2006). the areas were 100 km apart. although the first site is a semi-deciduous forest and the other is a rain forest, they had similar elevation, distance to the ocean, mean temperature, ant species composition and ant nest density (2 more taxa were found in the fragmented forest, see results). host diversity was a more important factor than environmental variables to account for leaf-cutting ant parasitoid species richness (elizalde & folgarait, 2010). in the vassouras-barra do piraí area, 11 fragments were selected, with a range of size variation of 0.1-10.6 km2 (mean 3.4 km2), spanned over an area of 726 km2 (22º 13’–22º 30’ latitude, 43º 38’–43º 51’ longitude; fig. 1). in the regua reserve two areas of the continuous forest were selected, separated by 2 km, and covering an area of 9 km2 (fig. 1). ant and parasitoid sampling. in each selected fragment in vassouras-barra do piraí area and in the two areas in the continuous forest in regua reserve, acromyrmex nests were searched for while walking along transects. because nests of some acromyrmex species in the area are subterranean and not easily found (gonçalves, 1961), the only evidence for nest presence are foraging trails. thus, we walked the transects during periods of ant activity to also record foraging trails. we followed each foraging trail until it became subterranean and we counted it as a nest (unless two trail openings of the same ant species were closer than 30 m, in which case they were considered as belonging to the same nest). transect length ranged from 250 m in the smallest forest fragments to 4km in continuous forest (mean length = 1.29 km, se = 0.40, n = 13). transect length in the fragmented area depended on fragment size because we extended each transect as to reach the center of the fragment. in continuous forest, transect length depended on trails already established. when a nest was found, we collected 5-10 major ants for later identification. phorids attacking ants were collected using an aspirator for five minutes in a section of 2 m length of the foraging trail, near nest entrance, where most phorid parasitoids look for leaf-cutting ants hosts (elizalde & folgarait, 2012). in the few instances when nest entrance was not located, parasitoids were searched for in the most proximal portion of the foraging trail to the nest where it was possible to access, also in a section of 2 m of the trail. ants and parasitoids collected at the same nest were kept in vials appropriately labeled as to later associate ants and phorids with each nest. insects were identified with the available keys (for ants: gonçalves, 1961; for parasitoids: brown, 1997; disney et al., 2006; 2008; 2009; brown et al., 2010), and reference specimens were deposited in the museum de zoologia, universidade de são paulo, brazil. we determined ant species density by dividing the number of ant species in each transect by its area. ant nest density was calculated dividing the number of nests from all acromyrmex species. we compared ant species density and nest density per transect in fragmented and continuous forests using mann-whitney tests. to evaluate the effect of forest fragmentation on parasitoids, interactions involving only a. niger were used. the dependent variables measured were phorid species richness, phorid abundance over each nest, and the proportion of nests with parasitoids. because more a. niger nests were sampled in the continuous forest, parasitoid species richness and abundance were rarified to the same number of nests sampled in the fragmented forest using estimates v. 8.2 (colwell, 2006). the effect of forest type, i.e. continuous or fragmented, on parasitoid abundance over nests was compared using kruskal-wallis tests, and the proportion of nests with phorids was compared with χ2-tests (with yates correction for continuity). results acromyrmex-parasitoid interactions. we found five acromyrmex species. acromyrmex niger was by far the most abundant species (table 1). the other species collected were a. subterraneus (forel), with three subspecies (a. s. brunneus, a. s. molestans, a. s. subterral elizalde, jm queiroz habitat fragmentation in phorid–leaf-cutting ant interactions400 neus), a. coronatus (fabricius), a. aspersus (smith), and a. disciger (mayr) (table 1). table 1. mean (se) relative abundance (%) for acromyrmex taxa in fragmented and continuous forests in rio de janeiro, brazil. number of transects and total transect length for each forest type are indicated in square brackets. in the last column, total number of nests sampled for phorids, pooling all acromyrmex species (and for a. niger only between parenthesis). taxa forest type fragmented [11; 8018 m] continuous [2; 8370 m] a. niger 54.31 (11.19) 82.92 (0.42) a. subterraneus 29.96 (5.70) 12.20 (0.30) a.s. brunneus 15.61 (4.69) 9.82 (2.68) a. s. subterraneus 4.03 (3.62) 2.38 (2.28) a. s.molestans 3.12 (2.13) 0 a. disciger 6.83 (4.71) 0 a. coronatus 3.86 (2.80) 3.69 (1.31) a. aspersus 0.40 (0.40) 1.19 (1.19) number of nests sampled 111 (59) 82 (68) seven species of phorid parasitoids were found, with five of them attacking exclusively a. niger (table 2). only one phorid species was attacking a. disciger and another was attacking a. coronatus only (table 2). none of the subspecies of a. subterraneus nor a. aspersus were found to be attacked by phorids. the parasitoids apocephalus lutheihalteratus borgmeier and myrmosicarius catharinensis borgmeier were the most abundant species, and they used a. niger as host. the other parasitoid species had very low abundance (table 2). only five nests in the continuous forest, all belonging to a. niger, had more than one phorid species attacking at the same time, with ap. lutheihalteratus and m. catharinensis found together in four nests and in the other nest ap. lutheihalteratus and neodorhniphora similis prado were attacking ants in the same trail. effect of forest fragmentation on ant-phorid interactions all acromyrmex species were present in the fragmented forest when all fragments were pooled, and only a. disciger and a. s. molestans were not collected in the continuous forest (table 1). in fact, ant species density was higher in the fragmented forest (u = 20, p = 0.03; mean = 4.6 x 10-4 species/m2, se = 0.7 x 10-4, n = 11 for fragmented forest; mean = 0.9 x 10-4 species/m2, se = 0.1 x 10-4, n = 2 for continuous forest). however, nest density of all acromyrmex species did not differ between forest types, and neither did nest density of a. niger (u = 18, p = 0.12; mean = 1.5 x 10-3 acromyrmex nests/m2, se =0.2 x 10-3, n = 11 for fragmented forest; mean = 0.8 x 10-3 acromyrmex nests/m2, se =0.1 x 10-3, n = 2 for continuous forest; u = 12, p = 0.75; mean = 0.7 x 10-3 a. niger nests/m2, se =0.2 x 10-3, n = 11 for fragmented forest; mean = 0.6 x 10-3 a. niger nests/m2, se =0.1 x 10-3, n = 2 for continuous forest). in only 37.5% of the fragments where a. niger was present (n = 8) were there phorids attacking it (considering all acromyrmex there were 45% of the 11 fragments with phorids). the rarefied phorid species richness in continuous forest was 4.81 (lower 95% confidence interval = 3.51), higher than the three species gathered over a. niger in the fragmented forest (table 3). in addition, parasitoid abundance over nests was higher than that found in fragmented forest (kruskal-wallis χ21 = 12.9, p = 0.0003; table 3), and the same pattern was shown by rarefied total parasitoid abundance (51.5 and 5 phorids in the continuous vs. fragmented forest, respectively). continuous forests also had a higher proportion of a. niger nests with parasitoids than fragmented forest (χ21 = 7.06, p = 0.008; table 3). in fact, phorids attacking ants were collected only in 3 out of 8 forest fragments sampled, where a. niger was present. only two phorid species were abundant enough for comparisons at phorid species level, ap. lutheihalteratus and m. catharinensis. the first species was not present in the fragmented forest, and in the continuous forest 17.3% of the table 2. interactions between acromyrmex and phorid parasitoid species in two localities (+: presence; -: absence), percentage relative abundance for parasitoid species over each host (between parentheses, pooled number of parasitoids gathered in both forest types). ant species phorid species forest type % relative abundance over each hostfragmented continuous acromyrmex coronatus myrmosicarius persecutor + + 100 (2) acromyrmex disciger neodohrniphora acromyrmecis + 100 (1) acromyrmex niger apocephalus lutheihalteratus + 32 (21) myrmosicarius catharinensis + + 47 (31) myrmosicarius simplex + 3 (2) myrmosicarius tarsipennis + + 11 (7) neodohrniphora similis + + 7 (5) sociobiology 60(4): 397-404 (2013) 401 a. niger nests sampled had this species attacking, with most of these nests having only one ap. lutheihalteratus attacking (except one nest that had three and another where 11 phorids were attacking). meanwhile, m. catharinensis in the continuous forest was present in 26.9% of the a. niger nests sampled, and typically two phorids were attacking per trail (two phorids in median, range 1-6); however, in the fragmented forest only 5.9% of the nests had phorids of this species and in all cases only one individual was attacking ants. discussion habitat fragmentation at the atlantic forest of southeast brazil had a negative effect on phorids that are parasitoids of acromyrmex, mainly affecting abundance. however, their ant hosts were not noticeably affected by forest fragmentation. the effect on phorids was particularly evident for the number of adult parasitoids attacking ants and the proportion of nests with parasitoids. although species richness was also lower in the fragmented forest, the difference was due to two species, one of them with low abundance (myrmosicarius simplex). there is no clear information on whether historically higher anthropogenic use in the fragmented forest area, which is a covariate always present in forest fragmentation processes, would have an important influence in our data. however, across the atlantic forest recent history, the human intervention is very similar, with conversion of forests to crop fields and pastures after (dean, 1997). thus, our results adds to the body of knowledge that posit parasitoids as very susceptible animals to this type disturbance (kruess & tscharntke, 2000; cagnolo et al., 2009). two differences between these parasitoids and their hosts seem to make phorids more affected by habitat fragmentation than their ant hosts. the first one is related to dispersal abilities. leaf-cutting ants disperse mainly during the nuptial flight, when male and female alates fly out their parental nest to find mates, and later establish a new nest (hölldobler & wilson, 1990). although it is not known how long the acromyrmex studied here can disperse, atta species can disperse for several kilometers (helmkampf et al., 2008). this exceeds the distance among neighbor forest fragments in the study area (fig. 1). meanwhile phorids are not known to disperse much from their hosts’ nests. nothing is known of the dispersion ability of leaf-cutting ants’ phorids, but other phorids that attack fire ant are able to fly up to 650 m from their hosts nests (morrison et al., 1999) and phorid activity was much lower in areas without nearby hosts (philpott et al., 2009). thus, it seems plausible that flying through a matrix with pasture where hosts are rarely present (personal observation) may complicate parasitoid arrival to forest fragments. this is supported by the fact that only 37.5% of the fragments had phorids attacking a. niger. the second trait that can theoretically increase susceptibility to habitat fragmentation is specialization. leafcutting ants are generalist herbivores that can use several food resources, while phorids collected here were only attacking one host species. specialists may be at greater risk in fragmented habitats mainly because resources will become scarcer and difficult to find (tscharntke at al., 2002). because we did not find differences in resource availability for phorids, i.e. host nest density, specializing in one host species for these phorids did not mediate the abundance reduction detected in fragmented forest. in addition, most phorid species collected during this study are reported to attack other acromyrmex species (borgmeier, 1928; 1929; brown, 1997), which were not present in the area sampled. this suggests that even when these parasitoids were acting locally as specialists, they might have the flexibility to use other hosts in the case that they need to. two previous studies reported a negative effect of habitat fragmentation on the abundance of phorid parasitoids of atta leaf-cutting ants (rao, 2000; almeida et al., 2008). one showed that the proportion of nests with phorids was lower in small forested islands compared to large islands or the mainland with continuous forest (rao, 2000). the other study showed lower phorid abundance and attack rates in nests that were located on the edge of the fragment compared to nests that were more than 200 m from the edge (almeida et al., 2008). this negative effect was explained by unfavorable climatic conditions at the edge forest, such as higher temperature, lower humidity and greater variation of these variables (almeida et al., 2008). however, phorid species that are parasitoids of ants differ in their climatic tolerances and microclimate preferences (folgarait et al., 2005; 2007; elizalde & folgarait, 2010, gomes et al., 2013). moreover, in other host-parasitoid system, some parasitoid species showed a higher population size in continuous forest area, but other species were more abundant in the fragmented forest (roland & taylor, 1997). these facts highlight the importance of testing the effect of habitat fragmentation at the species level. in our study, the two most frequently collected phorid species were less abundant or not collected at all in the fragmented forest. therefore, the negative effect of habitat fragmentation seems to be general for phorid species attacking acromyrmex niger in the atlantic forest of rio de janeiro. phorid parasitoids attacking atta are well-known, estable 3. parasitoid species richness, total (and average) abundance over nests, and the proportion of acromyrmex niger nests with phorids attacking in fragmented vs. continuous forests. forest type fragmented continuous species richness 3 5 abundance 5 (1) 61 (2.17) % of nests with phorids 6 26 l elizalde, jm queiroz habitat fragmentation in phorid–leaf-cutting ant interactions402 pecially in brazil (tonhasca, 1996; erthal & tonhasca, 2000; tonhasca et al., 2001; bragança et al., 2002; 2003; 2009; bragança & medeiros, 2006). however, parasitoids attacking acromyrmex are poorly studied, and all work done remounts to studies of frey borgmeier around 1930 (borgmeier, 1928; borgmeier, 1929) and studies in argentina (elizalde & folgarait, 2011; 2012). because phorid species attacking atta are different from those attacking acromyrmex (elizalde & folgarait, 2011), the information gathered for atta–phorid system cannot be extrapolated to acromyrmex–phorid system. thus, this study contributes to the knowledge of phorids using acromyrmex hosts. seven phorid species were found attacking acromyrmex, most of them over a. niger. in the continuous forest, a. niger had a very high parasitoid load compared to what is frequently found in leaf-cutting ant-phorid parasitoid assemblages (elizalde & folgarait, 2011; elizalde et al., in rev.). all phorid species reported to attack acromyrmex in the region (borgmeier, 1928; 1929) were collected during this work, suggesting that our sampling was enough. the only exception was ap. lamellatus, which was recorded in association with a. niger (borgmeier, 1926; as cited in brown, 1997), although it is not known whether this is a parasitoid of that ant species. few nests had two or more phorid species attacking ants at the same time, and that only occurred in the continuous forest. this low overlap of phorid species in nests, coupled with the very low abundance of phorids attacking ants in each nest in forest fragments, ruled out an effect of interactions among phorid species influencing the observed phorid abundance pattern. our work also showed that even when phorid abundance is considerably reduced in forest fragments, possibly because of their inability to reach fragments, phorids of some species are able to live there. because leaf-cutting ants are agricultural pests, and agricultural activities are among the main drivers of habitat fragmentation (foley et al., 2005), it is necessary to understand how leaf-cutting ants’ natural enemies such as phorids are affected by forest fragmentation. an important management tool to control pests in agricultural croplands is to leave fragments of native forest around crops, which function as refuges for natural enemies (bragança et al., 1998; zanetti et al., 2000). since some phorid parasitoids were able to live on fragments, more studies on the population dynamics of these phorids on fragmented landscapes are necessary to understand if and how forest fragments could function as reservoirs for parasitoids of leaf-cutting ants. acknowledgments we are grateful to fábio souto almeida and luana priscila de carvalho pereira for their help in the fieldwork. special thanks to nicholas and raquel locke, from regua, for providing invaluable logistical support. farmers from vassouras-barra do piraí region kindly authorized our fieldwork in the forest fragments. faperj granted jmq a scholarship (proc.101.472/2010). le thanks fundación bunge y born (argentina). ants and parasitoids were collected under permit number sisbio-14674. references almeida, w.r.; wirth, r. & leal, i.r. 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(2000). efeito da densidade e do tamanho de sauveiros sobre a produção de madeira em eucaliptais. an. soc. entomol. bras., 29: 105–112. doi: 10.1590/s030180592000000100013 1535 types of antennal sensilla of three pseudacteon species (diptera: phoridae) females that parasitize red imported fire ants (solenopsis invicta) ( hymenoptera: formicidae) by yong-yue lu*, xiao-qing li**, ling zeng, xiao-ling fan abstract while antenna is the main organ for insect to accept the external chemical signals, the antennal sensilla that are diverse in structure and function form the insect receptors in chemical communication. since a variety of pseudacteon species are important natural enemies of the red imported fire ant, solenopsis invicta buren (. invicta), to elucidate the types of pseudacteon sensilla will promote the study and understanding of the selection behavior of pseudacteon in parasitizing . invicta. this study has used scanning electron microscope (sem) to observe and investigate the female’s antennal sensilla of three pseudacteon species, the pseudacteon (p.) litoralis, p. obtusus, and p. tricuspis, and demonstrated that there are four types of sensilla, the trichoid, basiconic, coeloconic, and chaetic sensilla, on their antennal flagellum. among them, the former three are common in all three species, with trichoid sensillum as mostly abundant, while the chaetic sensillum exists only in the antennae of p. obtusus. the trichoid sensilla exhibit significant interspecies variations and are further classified into two subtypes based on the presence or absence of protrusions, the surface of which contains different shades of groove-like or irregular punctate structures. the basiconic sensilla resemble short spines with densely porous structures on the surface and are in the length of 7.3-9.8 μm and the width of 1.3-1.6 μm, upright or slightly bent. the coeloconic sensilla are irregularly formed in the middle and base of the flagellum, without surface pores; each coeloconic sensillum has eight finger-like folds in unequal lengths, while the end of the fold resembles a blunt cone. the chaetic sensilla enlarge at the base, possess multiple fold-like structures and fine-tipped ends, and are approximately 5 μm in length. red imported fire ant research center, south china agricultural university, guangzhou 510642, p.r. china *corresponding authoryong yue lu email: luyong yue@scau.edu.cn, insectlu@163.com 1536 sociobiolog y vol. 59, no. 4, 2012 keywords: fir ant; natural enemy; solenopsis invicta; pseudacteon; antennal sensilla introduction . invicta is one of the internationally recognized and dangerous invasive species. it originally distributes in the parana river basin of south america but later on continuously spreads to and jeopardizes other countries including united states, australia, china, etc. (zeng et al. 2005a, zeng et al. 2005b, callcott 2002, callcott & collins 1996, nattrass & vanderwoude 2001). because this pest threatens the local agriculture and forestry, public safety, human health, and biodiversity, the affected countries and regions have paid serious attentions to its prevention and control (lofgren et al. 1975, porter & savignano 1990, tschinkel et al. 1995, wojcik et al. 2001). an important factor to restrain the . invicta population is their original natural enemies in south america, among which pseudacteon is the most potential parasitoid and has received the most in-depth studies (borgmeier & prado 1975, fowler et al. 1995, orr et al. 1995, porter 1998a, porter 1998b, porter 2000). fire ant-attacking pseudacteon exhibits specificity (borgmeier 1962, porter 1998a, porter & alonso 1999, porter 2000). since the fly is small in size, and the oviposition is quick, it is difficult to investigate the relationship between the ovipositor and the host-locating mechanism. pseudacteon locates fire ants likely by pursuing their chemical odors (borgmeier 1922, porter et al. 1995). therefore, to study the pseudacteon’s antennal sensilla may help answering this question. each pseudacteon species usually parasitizes ant of certain fixed range of body size (morrison et al. 1997). the present study has investigated three pseudacteon species, the p. tricuspis, p. litoralis, and p. obtusus. the former two have already been used for field release to control the fire ants, while the latter one is also under preparation to be released to the field (lu et al. 2008; gilbert & patrock 2002). this study has observed and investigated the female antennal sensilla of these three pseudacteon species, aiming to provide theoretical basis for better understanding of the insect chemosensory system, the relationship between insect antennal sensilla and their behavioral responses, as well as the chemical links between insects and natural enemy, and to provide scientific basis for the development of new measures for . invicta prevention, as well 1537 y lu, y-y. et al. — antennal sensilla of three pseudacteon species as technical support for future morphological screening of other pseudacteon species to apply in the biological control of . invicta. materials and methods the insect samples the alcohol-impregnated specimens of p. tricuspis, p. litoralis, and p. obtusus were generous gifts from dr. sanford d. porter (center for medical, agricultural, and veterinary entomolog y, usa), and were kept in the refrigerator with -18℃ for test. specimen preparation for scanning electron microscopy (sem) female fly was singled out with a fine brush, rinsed three times in 0.1 mol/l phosphate buffer (ph 7.0), 20 min each time, added with mixed solution of 2.5% glutaraldehyde and 2% polyformaldehyde, and incubated at 4 °c for 24 h. the specimen was then rinsed three times in phosphate buffer for 30 min each time, added with 1% osmium tetroxide solution, and incubated at 4 °c for 24 h. the sample was rinsed three times with phosphate buffer for 10 min each time, dehydrated in 30% (v/v), 50%, 70%, 80%, and 90% alcohol, respectively, each for 10 min, and then dehydrated in absolute ethanol three times for 10 min each time. the specimen was finally treated with tert-butanol for three times, 10 min each time and freeze-dried in a freeze dryer ( jfd-310 freeze drying device). the gold/palladium was sputtered using an jfc-1600 auto fine coater. the specimen was fitted with conductive adhesive under stereoscope and observed with sem ( jms-6360, japan electron optics laboratory co., ltd, japan). the sem micrographs of the antennae and sensilla of the female flies were taken and observed. the antennal sensilla types among the females of three species phorid flies were compared. terminolog y in this study, the antennal morpholog y and sensilla types were described and classified according to the terminologies and the snodgrass classification presented by schneider (1964) and zacharuk (1985). 1538 sociobiolog y vol. 59, no. 4, 2012 fig.1 sem micrographs of heads and antenna of the decapitating phorid flies females p. litoralis, p. obtusus and p. tricuspis (a) p.litoralis head; (b) p.litoralis antenna; (c) p. obtusus head; (d) p. obtusus antenna; (e) p. tricuspis head; (f) p. tricuspis antenna. an, antenna; ce, compound eyes; mp, maxillary palp; ar, arista; pe, pedicel; fl, flagellum; sc, scape. scale bars: (a, c, and e) 100 μm; (b, d, and f) 50 μm. 1539 y lu, y-y. et al. — antennal sensilla of three pseudacteon species results antennal morphology of female pseudacteon the pseudacteon antennae present at the front end of the two compound eyes, with a length of 198-337 μm (figure 1a, c, and e). each antenna comprises three segments, the scape, the pedicel, and the flagellum. among them, the shortest one is pedicel, hiding under the flagellum. the flagellum enlarges in the female pseudacteon, and its end has an arista in the length of ~170 μm. scape and pedicel there are no sensilla on the scape and pedicel in pseudacteon. the scape is in a shape of flat cylinder, covered by dense micro-hairs, while the pedicel is relatively small with its end buried into the depression of the flagellum (figure 1b, d, and f). flagellum the flagellum forms the most important segment of pseudacteon antenna fig.2 sem micrographs of antennal sensilla of the decapitating phorid fly p. litoralis female (a and b) antennal sensilla on flagellum: (ba) sensilla basiconica, (co) sensilla coeloconica, and (tr) sensilla trichodea; (c) sensilla trichodea; (d) sensilla basiconic; (e) sensilla coeloconica. scale bars: (a , b and d) 5μm; (c and e) 1μm. 1540 sociobiolog y vol. 59, no. 4, 2012 and is covered by large amount of sensilla. the length of sensilla increases gradually from the flagellar base to its end. the sensillum directions are all towards the end, which makes the flagellum looks very supple. there are four types of sensilla observed in these three pseudacteon species. p. tricuspis and p. litoralis share three types of sensilla, the trichoid, basiconic, and coeloconic sensilla, while all four types of sensilla are found in p. obtusus, i.e., the trichoid, basiconic, coeloconic, and chaetic sensilla. however, the morpholog y of trichoid sensilla is significantly differentiated and varies evidently among the three species. the chaetic sensillum exists only in p. obtusus (figure 3i and j). trichoid sensillum the trichoid sensillum is the mostly abundant sensillum in these three observed pseudacteon species. they densely cover almost the end of the antennal flagellum, with the diameter of 1.1-1.3 μm (fig.2 a, b and c, fig.3a and b, fig.4 a). under high magnification, it is observed that the surface of sensilla trichodea has different shades of groove-like structure or irregular punctate structures( fig.3 c and d, fig.4 b). basiconic sensillum sem results show that the number of basiconic sensilla ranks second right after that of trichoid sensilla. the shape of basiconic sensillum resembles a short spine, and its surface is densely covered by pore-like structures, which stand vertical or slightly curved on the flagellum (fig. 2 d, 3 e and f, and 4 e and f). the basiconic sensillum has a length of 7.3-9.8 μm and a width of 1.3-1.6 μm. coeloconic sensillum among the four types of sensilla, the morpholog y of coeloconic sensilla manifests least variations among the three species. on the antennal flagellum of the female flies, coeloconic sensilla distribute randomly at the base and in the middle; there are no coeloconic sensilla observed at the end of flagellum. the coeloconic sensillum rises from a circular cavity, with a finger-like fold structure, in the length of 2.7-3.4 μm and the diameter of 0.8-1.1 μm. sem indicates that each coeloconic sensillum possesses eight finger-like folds, with 1541 y lu, y-y. et al. — antennal sensilla of three pseudacteon species unequal lengths, the diameters of which are not tapering along the growth direction of the sensilla (figure 2 e, 3 g and h, 4 c and d). chaetic sensillum the chaetic sensillum is only observed on the flagellum of p. obtusus (figure 3 i and j). the chaetic sensilla stand upright like thorns, and their base also has multiple fold structures. however, these folds are not as obvious as those of coeloconic sensilla. they only exist at the base of the sensilla and disappear fig.3 sem micrographs of antennal sensilla of female decapitating phorid fly p. obtusus (a and b) antennal sensilla on flagellum: (ba) sensilla basiconica, (ch) sensilla chaetica, (co) sensilla coeloconica, (tr) sensilla trichodea; (c and d) sensilla trichodea; (e and f) sensilla basiconica; (g and h) sensilla coeloconica; (i and j) sensilla chaetica. scale bars: (a and b) 5μm; (c, d, e, f, g, h, i, and j) 1μm. 1542 sociobiolog y vol. 59, no. 4, 2012 along the growth direction above the base portion. the sensillum chaeticum is about 5 μm long and tapered gradually towards the end. arista the end of antennal flagellum of female pseudacteon tapers gradually and forms a wheat awn-like arista. each aristae comprises three segments. the two segments close to the base are relatively thick, densely covered with microhairs and spines, and with irregular long grooves. the third segment is a long thin strip with dense micro-hairs and the similar grooves (fig. 5 b). discussion sensillum is a part of specialized epidermis on the insect body wall and a important structure to respond to the surrounding environment and a variety fig.4. sem micrographs of antennal sensilla of female decapitating phorid fly p. tricuspis (a) antennal sensilla on flagellum: (ba) sensilla basiconica, (co) sensilla coeloconica, and (tr) sensilla trichodea; (b) sensilla trichodea; (c and d) sensilla coeloconica; (e and f) sensilla basiconica. scale bars: (a) 5μm; (b, c, d, e, and f) 1μm. 1543 y lu, y-y. et al. — antennal sensilla of three pseudacteon species of internal stimuli. it is the information receiving device for insect body to perceive the internal and external environments, to make chemical communication, and in company with the nervous system to control and regulate the insect behavior (yu 2007). in consistence with the observation of chen & fadamiro (2008), the antennal sensilla of female pseudacteon present only on the flagellum. sensilla trichodea are the dominant type of antennal sensilla in both quantity and distribution. the trichoid sensilla were classified into short, medium, and long three subtypes according to their lengths (chen & fadamiro 2008). however, our sem results indicate that the length of trichoid sensilla varies greatly, and it is difficult to divide them into subtypes based on mere length. according to yu (2007), this type of sensilla may have dual functions to act as both mechanical and sense of touch receptors. pseudacteon hovers over the . invicta colony, locks the target, and quickly dives and inserts an egg into the worker ant’s thorax region. during this process, the trichoid sensilla likely play an important role. the speed of female pseudacteon’s oviposition attack is quick, merely lasting 0.1 to 1.0 second (borgmeier 1922, porter et al. 1995). therefore, female pseudacteon needs more sensitive and abundant mechanical as well as sense of touch receptors. the structure of trichoid sensilla in these three pseudacteon species is less variable and only forms two subtypes fig.5. sem micrographs of antennal arista of female decapitating phorid fly p. litoralis (a) antennal arista; (b) two coxae and one apical segment of antennal arista. scale bars:(a) 10μm; (b) 5μm. 1544 sociobiolog y vol. 59, no. 4, 2012 according to the presence of surface protrusions, which may be evolved from the fly’s long parasitic life and may have increased the area of receptors and hence facilitated the perception of host information. the surface of pseudacteon basiconic sensilla is porous. this type of sensilla had olfactory function and, sometimes, mechanoreceptive function, too (na et al. 2008). basiconic sensillum with such structure is also called type ii basiconic sensillum. their walls are thin and rich in nerve cells, mainly stimulated by general odorant from the outside world (e.g., the plant odor, and the odor from natural enemies) (na et al. 2008). the number of basiconic sensilla is only after that of trichoid sensilla. they may function, in assistance to the trichoid sensilla, to identify and determine the host information, via the perception of chemical odor. coeloconic sensilla are the least in female pseudacteon. studies have shown that this type of sensilla is located in the abdomen and outer side of the antennal flagellum, and stimulated by carbon dioxide, humidity, plant odors, etc (na et al. 2008). therefore, pseudacteon’s coeloconic sensilla may perform the similar functions. the chaetic sensillum only exists on the flagellum of p. obtusus. its length (~5 μm) is only longer than that of coeloconic sensilla. each pseudacteon species generally parasitizes ants in certain range of body size. p. tricuspis attacks medium to large fire ant workers, and p. litoralis attacks medium-large to large fire ant workers. no chaetic sensilla are observed on both of these species, implying that the function of chaetic sensilla is related to the identification of body type of the host, which warrants further studies. the arista observed in this study are all located in the end of antennal flagellum, which differs from the reported aristae location in diptera flies (sukontason et al. 2004, sukontason et al. 2007). there are no sensilla observed on the arista of these three pseudacteon species, implying that arista may not relate to the chemical responses. in summary, this study has observed and compared the differences in morpholog y, number, and distribution of female antennal sensilla in three pseudacteon species. the two major types of sensilla, the trichoid and basiconic sensilla, are most likely related to the perception of chemical information, suggesting a close relationship between the chemical odor and the parasitic behavior of pseudacteon. the results of this study provide theoretical basis for 1545 y lu, y-y. et al. — antennal sensilla of three pseudacteon species further exploring the relationship between the sensillum types of parasitic pseudacteon and the mechanism of locating host . invicta, and for further in-depth understanding of the neurobehavior of these important . invictaparasitic pseudacteon species, as well as provide scientific basis for developing new means to biologically control . invicta. acknowledgments we would like to thank sanford d porter for kindly furnishing the specimen. our study was supported by national basic research program of china (award# 2009cb119200) and national natural science foundation of china (award# 30900942). references borgmeier, t. 1922 (1921). zur lebensweise von pseudacteon borgmeieri schmitz (in litt.) (diptera: phoridae). the kunst sao paulo: zs of deut ver wiss 2:239-248. borgmeier, t. 1962. cinco espécies novas do genero pseudacteon coquillett. arq mus nac 52:27-30. borgmeier,t. & a.p. prado 1975. new or little known neotropical phorid flies, which description of eight new genera (diptera: phoridae). stud entomol 18:3-90. callcott, a.m. & h.l. collins 1996. invasion and range expansion of red imported fire ant (hymenoptera: formicidae) in north america from 1918~1995. flor entomol 79:240-251. callcott, a.m. 2002. range expansion of the imported fire ant 1918~2001. in: diffie s k. 2002 annual imported fire ant research conference. athens, georgia. chen, l. & h.y. fadamiro 2008. antennal sensilla of the decapitating phorid fly, pseudacteon tricuspis (diptera: phoridae). micron 39:517-525. fowler, h.g., m.a. pesquero, s. campiolo & s.d. porter 1995. seasonal activity of species of pseudacteon (diptera: phoridae) parasitoids of fire ants (solenopsis saevissima) (hymenoptera: formicidae) in brazil. cientifica 23: 367-371. gilbert, l.e. & r.j.w. patrock 2002. phorid flies for the biological suppression of imported fire ants in texas: region specific challenges, recent advances and future prospects. south west entomol (suppl) 25:7-17. lofgren, c.s., w.a. banks & b.m. glancey 1975. biolog y and control of imported fire ants. ann rev entomol 20:1-30. lu, y.y., l. zeng, y.j. xu & g.w. liang 2008. chapter 20. biological control of red imported fire ant. in wan f: biological invasions: biological control. beijing : science press 9: 442-477. morrison, l.w., c.g. dall’agilo-holvorcem & l.e. gilbert 1997. oviposition behavior and development of pseudacteon flies (diptera: phoridae), parasitoids of solenopsis fire ants (hymenoptera: formicidae). environ entomol 26:716-724. 1546 sociobiolog y vol. 59, no. 4, 2012 na, j., w.x. yu, y.p. li, x. dong & j. jiao 2008. types of insect sensilla and their physiological and ecological significance. j anhui agricul univ 26:213-215. nattrass, r. & c. vanderwoude 2001. a preliminary investigation of the ecological effects red imported fire ants (solenopsis invicta) in brisbane. ecol manag restor 2:521-523. orr, m.r., s.h. seike & w.w. benson & l.e. gilbert 1995. flies suppress fire ants. nature, 373:292-293. porter, s.d. & d.a. savignano 1990. invasion of polyg yne fire ants decimates native ants and disrupts arthropod community. ecol 71:2095-2016. porter, s.d. & l.e. alonso 1999. host specificity of fire ant decapitating flies (diptera: phoridae) in laboratory oviposition tests. j econ entomol 92:110-114. porter, s.d. 1998a. host-specific attraction of pseudacteon flies (diptera: phoridae) to fire ant colonies in brazil. flor entomol 81:423-429. porter, s.d. 1998b. biolog y and behavior of pseudacteon decapitating flies (diptera: phoridae) that parasitize solenopisis fire ants (hymenoptera: formicidae). flor entomol 81:292309. porter, s.d. 2000. host specificity and risk assessment of releasing the decapitating fly pseudacteon curvatus as a classical biocontrol agent for imported fire ants. biol contr 19:35-47. porter, s.d., h.g. fowler, s. campiolo & m.a. pesquero 1995. host specificity of several pseudacteon (diptera: phoridae) parasites of fire ants (hymenoptera: formicidae) in south america. flor entomol 78:70-75. schneider, d. 1964. insect antennae. ann rev ent 9:103-122. sukontason, k., k.l. sukontason, s. piangjai, n. boonchu, t. chaiwong, r. ngernklun, d. sripakdee, r.c .vogtsberger & j.k. olson 2004. antennal sensilla of some forensically important flies in families calliphoridae, sarcophagidae and muscidae. micron 35(8):671-679. sukontason, k., r. methanitikorn, t. chaiwong, h. kurahashi, r.c. vogtsberger & k.l. sukontason 2007. sensilla of the antenna and palp of hydrotaea chalcogaster (diptera: muscidae). micron 38:218-223. tschinkel w.r., e.s. adams & t. macom 1995. territory area and colony size in the fire ant solenopsis invicta. j anim ecol 64:473-480. wojcik, d.p., c.r. allen, r.j. brenner, e.a. forys, d.p. jouvenaz & r.s. lutz 2001. red imported fire ant impact on biodiversity. amer entomol 47:16-23. yu, h. 2007. insect sensilla research progress. anhui agricul sci 35:4238-4240. zacharuk, r.y. 1985. antennal sensilla. in: kerkut, g.a., gilbert, l.i. (eds.),comparative insect physiolog y, biochemistry and pharmacolog y, vol. 6. pergamon press, oxford, pp. 1–69. zeng, l., y.y. lu & z.n. chen 2005. management and surveillance of red imported fire ant. guangdong science & technique press, guangzhou, china, 106pp. zeng, l., y.y. lu, x.f. he, w.q. zhang & g.w. liang 2005. identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan, guangdong. chin bul ent 42(2):144-148. doi: 10.13102/sociobiology.v61i1.78-81sociobiology 61(1): 78-81 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 methodology for internal damage percentage assessment by subterranean termites in urban trees fj zorzenon, ae de c campos introduction problems associated to urban trees are common and are mostly caused by inadequate management (jim, 2001; rodrigues et al., 2002; nowak & dwyer, 2007). lack of planning to choose suitable plant species to the site, lack of knowledge on plant biology and physiology, lack of care when transplanting the trees, lack of space for plant growth, drastic pruning, infestations by wood boring, sucking and defoliating insects, termites, ants, besides plant diseases, and negligence are some of the problems that make urban trees die early (zorzenon & potenza, 2006). tree survival in different environments depends on the mechanical reliability of its structures, and problems in the trunk may cause falling risks followed by serious or fatal accidents (niklas, 1992, mattheck & breloer, 1997; pereira et al., 2007). infestations by subterranean termites are common in brazil (constantino, 2002) and promote damping off, especially when attacks occur in street trees. abstract one of the most important problems in urban trees is termite infestation. simple observations of damages on outside trunks or dead branches and leaves do not always confirm infestations. several trees may present severe termite damage internally that can only be observed through drilling. this paper presents a methodology to evaluate estimated percentages of internal damage caused by termites in urban trees. tests were made on 1,477 plants in a neighborhood in the city of são paulo, brazil and 27% of them were infested by subterranean termites. the results showed that the methodology is simple to use, fast and inexpensive, and it allows assessment of termite internal damage which may help in making decisions on tree management. the trees did not show any phytosanitary problems along the 9 year study after being submitted to the new technique. sociobiology an international journal on social insects instituto biológico, unidade laboratorial de referência em pragas urbanas, são paulo, sp, brazil. article history edited by evandro n silva, uefs, brazil received 19 july 2013 initial acceptance 21 august 2013 final acceptance 02 octuber 2013 keywords isoptera, urban forest, tree management, falling risk, inspection corresponding author ana eugênia de carvalho campos instituto biológico unidade laboratorial de referência em pragas urbanas av. conselheiro rodrigues alves, 1252 são paulo, sp, brazil 04014-002 e-mail: anaefari@biologico.sp.gov.br studies have evaluated methods for assessing termite infestations in trees. mattheck and breloer (1994) developed a method called visual tree assessment (vta). if symptoms are detected the related defect has to be confirmed and measured by deeper inspection, which includes measuring the speed of a sound wave traveling through a cross-section on the trunk and by drilling methods. the strength of the remaining healthy wood is determined with a fractometer. lax and osbrink (2003) refers to other nondestructive methods using a resistograph. nicolotti et al. (2003) reported application of electric, ultrasonic, and georadar tomography for detection of decay in trees and their comparison with the penetrometer. mankin et al. (2002) used a portable, low-frequency acoustic system to detect termite infestations in urban trees. the likelihood of infestation was rated independently by a computer program and an experienced listener that distinguished insect sounds from background noises. mankin and benshemesh (2006) used a geophone system to monitor activity of subterranean termites and ants in a desert environment with low vibration noise. research article termites sociobiology 61(1): 78-81 (march, 2014) 79 therefore, the methodology must be used in quiet environments due to its sensibility and it is not adequate to urban environments. pereira et al. (2007) reported that nondestructive techniques have been developed by tomography investigations, where the impulse tomography enables transversal section reconstructions of the whole tree, through the energy that flows through the wood. however, they advise that the technique is still under development and needs further study. osbrink and cornelius (2013a) as well as osbrink and cornelius (2013b) on the other hand used an acoustic emission detector (aed) to evaluate the presence of termite infestations in trees and could determine the presence of the insects. since termite damages are common, pest control operators should have a range of methods which can be chosen for each condition. this paper proposes a simpler methodology for tree assessment to estimate the percentage of internal damages caused by subterranean termites in urban trees that could be used worldwide. material and methods the study was conducted from january 2004 to january 2013, in a neighborhood with 1,609 plant specimens, including trees and palm trees along eight kilometers of streets and avenues, in the city of são paulo, brazil (s 23º 35’ 34.72” / w 46º 41’ 57.60”). we identified 1,339 trees and 138 palm trees, totaling 1,477 plant specimens to test the proposed methodology. steel drills, measuring 6 mm diameter by 200 mm in length, and 10mm diameter by 320 to 400mm in length, normally used to drill wooden stakes and fiber cement tiles, were used with single or triple ends. a professional hammer driller (bosch gsb 19-2 model 650-watt) and an electric/gasoline generator (branco model bt2 950) were also used. small drills were used on trees with the circumference at breast height (cbh) smaller than or equal to 40cm and the larger ones for the trees with cbh greater than 40cm. the cbh was measured at 1.30m height from the base of the plant (daniel, 2006). all plants were georeferenced. after perforation, holes were painted with bordeaux mixture (copper sulfate, hydrated lime and water), and sealed with silicone rubber to prevent penetration of moisture and the entry of plant pathogens. a borescope, which is a micro camera attached to a 6mm flexible stem joined to a lcd monitor, was used to visualize internal damage caused by termites, and to check for the presence of live termites inside the trunk of the trees. to determine the size and exact location of the termite damage in the inner region of the trunk, three holes (n) were drilled at a 45 degree angle into the base of the trees to establish triangulation points (fig. 1 – wider arrows). the intention was to reach the deepest regions of the heartwood for inner exploration to confirm termite infestations that are not externally visible, and to estimate the percentage of damage. the percentage of internal damage ranged from 0% (no damage) to 90% (extensive damage); this range was used to determine the correlation between cbh and percentage of internal damage. figure 1 schematic cross section of tree trunk showing dbh, r, dr, and mp90º. holes drilled are indicated by wide arrows. termite assessments were conducted in 2004. infested trees and palm trees were controlled, and plants were individually inspected each month during the nine year study to determine if the technique caused any other damage to the trees. estimated percentage of internal damage to obtain the estimated percentage of internal termite damage a methodology was developed for assessing and adapting trigonometric formulas for converting values into percentage estimates of internal damage. percentage estimates were obtained by measuring the depths penetrated by the drills, to evaluate the difference in strength of a healthy tissue of the trunk (high resistance) to a termite-damaged tissue (low resistance) or hollow (no resistance). the estimated average was obtained from the sum of the three depth measurements. once the angle of the drill relative to the tree trunk is 45 degrees (dp45°), the mean obtained values from the three holes were converted to the following formula to obtain mp90° (average measured depth of the drill at 90 degrees): mp90º = mp90º = average measured depth of the drill at 90 degrees dp45º = diagonal depth of the drill at 45 degrees n = hole number for those results of depth measurements in which the drill did not hit any cavity, the number to be considered in the r mp 90º d b h dr 2 º45       σ n dp fj zorzenon, ae de c campos methodology for assessing subterranean termite damages in urban trees80 sum of dp45 o should be a value obtained through r. 2 . for practical purposes it was assumed that the cross section of the trunk was a circular shape. it was also assumed that the transverse extent of internal damage was circular in shape. therefore, to make the calculations of estimated percentages of internal damage, it is necessary to first obtain the trunk diameter: cbh = circumference at breast height (1.3 m) dbh = diameter at breast height (1.3 m) π = 3,1416 r = radius of the circumference pd = % of internal damages dr = trunk damaged region for obtaining the diameter: subsequently determine the radius of the circle: 2 dbh r = subtracting the mp90º (average measured depth of the drill at 90 degrees) of r (radius of the circumference), an estimated value of the injured area is obtained, where: dr = r mp90º thus, to obtain the estimated percentage of the injured area (pd): pd = results and discussion according to juttner (1997) unique observations of external damage, such as tunnels, do not correspond to real termite infestations on urban trees. numerous trees, apparently healthy, have serious internal damage that can only be determined after drilling. the survey on the 1,477 plants revealed 27% (399) trees infested by subterranean termites. the practical methodology presented here proved to be feasible both in operational terms, regarding the use of low cost tools, and reliability when compared to the reported difficulties using other methods (lax & osbrink, 2003). tomography, for example, is a noninvasive way to assess internal injuries caused by termites in trees (pereira et al., 2007). although technologically superior, the equipment has high cost (ca. us$ 12,500.00) and must be calibrated according to the density of the wood, what makes operating it in the field difficult and time consuming. the methodology used in this work costs approximately us$ 550.00. the procedure used during this nine year study did not promote significant lesions on the assessed trees, and there were no interferences due to phytosanitary aspects. the injuries due to the holes drilled in the trees did not lead to diseases, and there was satisfactory healing after some months. it was not observed any tree death or impoverishment due to the adopted procedure. it is suggested that once the termite infestations are found trees must be treated, but trees with more than 60% of damages must be cut off, once they have risk of falling down. the mathematical formulas for data conversion and obtention of internal damage estimated percentages were developed to be simple and easy to understand. they have the advantage of being easy to use by properly trained technicians. acknowlegments the authors thank dr. william h. robinson for revising the english of the manuscript. we thank two other anonymous referees for their comments and criticisms on the manuscript. references constantino, r. (2002). the pest termites of south america: taxonomy, distribution and status. journal of applied entomo-journal of applied entomology, 126: 355-365. doi: 10.1046/j.1439-0418.2002.00670.x daniel, o. (2006). silvicultura. apostila cultivo de árvores. universidade federal da grande dourados. http://pt.scribd. com/doc/70003302/silvicultura-%e2%80%93-omar-daniel-apostila-cultivo-de-arvores-universidade-federalda-grande-dourados-faculdade-de-cienciasagrarias-dedourados-ms-br. (accessed date: 5 april, 2013). jim, c.y. (2001). managing urban trees and their soil envelopes in a contiguously developed city environment. environmental management, 28: 819-832. doi: 10.1007/s002670010264 juttner, a.s. (1997). termite control drill n treat form control of formosan termite in tree. arbor age. http://www. greenmediaonline.com/aa/1997/1097/1097dri.html (accessed date: 15 june, 2004). lax, a.r., osbrink, w.l.a. (2003). united states department of agriculture—agriculture research service research on targeted management of the formosan subterranean termite coptotermes formosanus shiraki (isoptera: rhinotermitidae). pest management science, 59:788-800. doi: 10.1002/ps.721 mankin, r.w., osbrink, w.l., oi, f.m., anderson, j.b. (2002). acoustic detection of termite infestations in urban trees. journal of economic entomology, 95: 981-988. doi: 10.1603/0022-0493-95.5.981 mankin, r.w., benshemesh, j. (2006). geophone detection of subterranean termite and ant activity. journal of economic entomology, 99: 244-250. π cbh dbh = r dr 100. sociobiology 61(1): 78-81 (march, 2014) 81 mattheck, c., breloer, h. (1994). field guide for visual tree assessment (vta). journal of arboriculture, 18: 1-23. mattheck, c., breloer, h. (1997). the body language of trees: a handbook for failure analysis. london: her majesty´s stationery office, 260p. nicolotti, g., socco, i.v., martinis, r., godio, a., sambuelli, l. (2003). application and comparison of three tomographic techniques for detection of decay in trees. journal of arboriculture, 29: 66-78. niklas, k. (1992). plant biomechanics: an engineering approach to plant form and function. university of chicago press, chicago, il. 622 p. nowak, d.j., dwyer, j.f. (2007). understanding the benefits and costs of urban forest ecosystems. in kuser, j.e. (ed.), urban and community forestry in the northeast (pp. 25-46). springer netherlands. doi: 10.1007/978-1-4020-4289-8_2. osbrink,w., cornelius, m. (2013a). utility of acoustical detection of coptotermes formosanus (isoptera: rhinotermitidae). sociobiology, 60: 69-76. doi: 10.13102/sociobiology.v60i1 .69-76 osbrink,w., cornelius, m. (2013b). acoustic evaluation of trees for coptotermes formosanus shiraki (isoptera: rhinotermitidae) treated with imidacloprid and noviflumuron in historic jackson square, new orleans, louisiana. sociobio-sociobiology, 60: 77-95. doi: 10.13102/sociobiology.v60i1.77-95 pereira, l.c., silva filho, d.f., tomazello filho, m., couto, h.t.z., moreira, j.m.m.a.p., polizel, j.l. (2007). tomografia de impulso para avaliação do interior do lenho de árvores revista da sociedade brasileira de arborização urbana, 2: 65-75. rodrigues, c.a.g., bezerra, b.c., ishii, i.h., cardoso, e.l., soriano, b.m.a., oliveira, h. (2002). arborização urbana e produção de mudas de essências florestais nativas em corumbá, ms. corumbá (ms): embrapa. zorzenon, f.j., potenza, m.r. (2006). cupins: pragas em áreas urbanas. boletim técnico, instituto biológico, brazil. 18. doi: 10.13102/sociobiology.v65i2.1939sociobiology 65(2): 162-169 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the influence of extrafloral nectaries on arboreal ant species richness in tree communities introduction the resource availability hypothesis presumes a bottomup process in which species richness is limited by the amount of food resources in the next lower trophic level. in consequence, higher species richness is expected in areas with higher food resource availability (ferger et al., 2014). several studies have attempted to understand biodiversity patterns using resource availability as the main explanation to variation in species richness (ferger et al., 2014; hurlbert & stegen, 2014; rabosky & hurlbert, 2015). in this sense, species richness is positively correlated to amount and resource variety although abstract studies investigating the role of resource availability in the species richness patterns can elucidate ecological processes and contribute to conservation strategies. in this study, we test two hypotheses: i) arboreal ant species richness increases with abundance of extrafloral nectaries-bearing trees; and ii) arboreal ant species richness increases with the diversity of extrafloral nectaries-bearing trees. we used data of ant sampling and tree inventories from 30 plots of brazilian cerrado. arboreal ant species richness was positively influenced by the proportional abundance of extrafloral nectaries-bearing trees, total tree density and total tree diversity. there was no effect of species richness of extrafloral nectaries-bearing trees. coefficient of determination of proportional abundance of extrafloral nectaries-bearing trees was larger when compared to coefficient obtained using tree density as explanatory variable. these results suggest that variation in arboreal ant species richness is better explained by extrafloral nectaries-bearing tree abundance than total tree density. generalist foraging behavior of sampled ant species may explain their association with proportional abundance of extrafloral nectariesbearing trees and their non-significant relation with proportional richness of extrafloral nectaries-bearing tree species. extrafloral nectaries-bearing trees abundance may be a specific estimate of the amount of food resource available in plots. thus, this is a more specific way to quantify which resources may explain variation of the arboreal ant species richness in tree communities. we hope these results will be helpful to understanding the local variation in ant species richness and as criteria to biodiversity conservation. sociobiology an international journal on social insects ms madureira1, tg sobrinho2, jh schoereder3 article history edited by jean santos, ufu, brazil received 12 august 2017 initial acceptance 29 november 2017 final acceptance 19 february 2018 publication date 09 july 2018 keywords resource availability, generalist foraging, cerrado. corresponding author marcelo silva madureira universidade do estado da bahia departamento de educação av. kaikan s/n, cep 45.995-000 teixeira de freitas-ba, brasil. e-mail: mmadureira@uneb.br some studies have related absence and negative effect of resource availability (dáttilo et al., 2014a, staab et al., 2016). an example of the role of resource availability in species diversity is the positive relationship between arboreal ant species richness with tree density and species richness in cerrado habitats, as reported by ribas et al. (2003). the authors considered the density and diversity of trees as surrogates of amount and variety of resources, respectively. such correlations have been explained by changes in habitat conditions and by the increase in total resource availability, which allows a higher ant species coexistence, thus increasing ant species richness. in this sense, a greater amount of 1 universidade do estado da bahia (uneb), departamento de educação-campus x, teixeira de freitas-ba, brazil; universidade federal de viçosa (ufv), departamento de entomologia, viçosa, brazil 2 universidade federal de viçosa (ufv), departamento de entomologia, viçosa, brazil; universidade federal do espírito santo (ufes), ceunes, departamento de ciências agrárias e biológicas, são mateus-es, brazil 3 universidade federal de viçosa (ufv), departamento de biologia geral, viçosa, brazil research article ants mailto:mmadureira@uneb.br sociobiology 65(2): 162-169 (june, 2018) 163 resources would allow the coexistence of more generalist species, increasing the ant diversity at a local scale. in contrast, competitively superior ant species can monopolize resources diminishing ant species richness as suggested by dáttilo et al. (2014a). additionally, sites with higher resource variety would support more ant species, because there would be greater coexistence of a higher number of specialist species. however, tree density and diversity as explanation to ant species richness are too general. trees may provide several types of resources to ants, such as nesting sites (yasuda & koike, 2009; fagundes et al., 2015), foraging area for prey and honeydew (blüthgen et al., 2000; oliveira & del-claro, 2005), fungal hyphae and other microorganisms (davidson et al., 2003) and extrafloral nectaries (blüthgen et al., 2000; schoereder et al., 2010; fagundes et al., 2015). considering this, the study of relationships between species richness and specific resources may provide more informative explanations linked to ant species diversity. extrafloral nectaries (efns) are plant structures usually located on petioles and leaf blades, which produce a liquid substance rich in carbohydrates, amino acids and lipids (gonzálesteuber & heil, 2009) used as food by several animal groups (beatie, 1985; rosumek et al., 2009; melo et al., 2010). ants visit efn-bearing plants for nectar consumption and some authors emphasize the possible role of efns in secondary defence against herbivory, because these ants frequently attack and/or drive away herbivore insects (rico-gray & oliveira, 2007, and references therein). in these cases, the presence of ants is correlated to increases of the plant reproductive fitness (nascimento & del-claro 2010; lange & del-claro, 2014). in addition, ants regulate the abundance of associated insects in efn-bearing plants in different ecosystems (mody & linsenmair 2004; fernandes et al., 2005; mathews et al., 2009; rosumek et al., 2009). on the other hand, the presence of ants on efn-bearing plants may not necessarily be beneficial to the plants (cembrowski et al., 2014; le van et al., 2014; alma et al., 2015). for ants, the consumption of extrafloral nectar enhances their aggressiveness, change predatory behaviour, increases survival rates and number of individuals per colony (heil, 2015 and references therein). other extrafloral nectar consumers are distributed among several groups of arthropods such as spiders, wasps, beetles, bees, bugs and mites (heil, 2015). as for ants, extrafloral nectar consumption enhanced the survivorship of spiders and wasps (pfannenstiel & patt, 2005; géneau et al., 2013) and egg production in crab spiders (wu et al. 2011). despite many studies, the function of efns remains as open issue with four current hypotheses as following: 1) protective: efns attracts predators which attack herbivores, 2) exploitation: efns is secreted as excess of carbohydrates, 3) flowerdistraction: efns keep ants away from flowers optimizing pollination and 4) antdistracting: efns avoid ants from attending hemipterans reducing damages to the host plant (del-claro et al 2016 and included references). plants with efns are present in various habitats, and the cerrado (brazilian savannah) harbours a large availability of this plant types (rico-gray & oliveira, 2007). in this biome, efn-bearing plants represent 15 to 25% of the plant diversity and comprise locally up to 31% of individuals (oliveira & freitas, 2004). ants are the insects that most commonly exploit efns (oliveira et al., 1987) and one plant species can harbour up to 34 species of visiting ants (oliveira & brandão, 1991). in the cerrado, extrafloral nectar secretion by different plant species is influenced by their phenology and can occur along whole year attracting several ant species (vilela et al., 2014). thus, extrafloral nectar might be an important food resource for arboreal ants in these environments (byk & del claro, 2011). considering the wide availability of extrafloral nectar, ant dominance on the efn-secreting plants, and dynamic network involving ants and efn-bearing plants (dáttilo et al., 2014a; dáttilo et al., 2014b), efns availability can enhance arboreal ant species richness in cerrado tree communities. efns may affect the ant diversity by at least two different reasons. firstly, ant diversity may be affected by the amount of efn-bearing plants, due to the higher food resource availability. in this case, a higher abundance of efnbearing trees would allow the coexistence of more generalist ant species, i.e, those ant species visiting any efn-bearing tree species (dáttilo et al., 2013; díaz-castelazo et al., 2013; dáttilo et al., 2014a). secondly, ant diversity may respond to the diversity of efn-bearing plants, because efns location, their density and chemical composition of nectar varies among plant species (rios et al., 2008; gonzáles-teuber & heil, 2009; rosumek et al., 2009). moreover, a same plant can vary its nectar quantity and quality along the day (heil et al., 2000; falcão et al., 2014) influencing the rate of ant foraging (falcão et al., 2014). then, there are reasons to expected different plants in a site attracting different ant species (blüthgen et al., 2000), thus increasing local ant diversity. studies investigating the role of resource availability in the species richness patterns can elucidate ecological processes and contribute to conservation strategies, for example, providing criteria for selecting protected areas. based on extrafloral nectaries as an important food resource to ants, the positive relationships between arboreal ant species richness and tree density and diversity found in cerrado (ribas et al., 2003) needs to be further analysed in order to understand the explanations of such correlations. in the present study, we addressed the following question: why are there positive relationships between ant species richness and tree density and diversity? we tested here two hypotheses: i) arboreal ant species richness increases with the abundance of efn-bearing plants; and ii) arboreal ant species richness increases with the diversity of efn-bearing plants. methods study area and data collection the study was carried out in the central portion of the cerrado, in the gama-cabeça de veado reserve, distrito ms madureira, tg sobrinho, jh schoereder – arboreal ant species richness164 federal (15°55’15°57’s; 47°55’47°57’w), brazil, covering an area of approximately 10,000 ha, with an average altitude of 1,100 m a.s.l. the cerrado is characterized by marked rainy (november-may) and dry seasons (june-october), with an average annual rainfall of 1,500 mm. the plant formations range from grasslands to forest areas, with trees that reach up to 12m height. there is no unified classification to cerrado physiognomies and several criteria are used to this purpose (ribeiro & walter 1998; oliveira-filho & ratter, 2002). here, we choose a classification according to tree density because this criterion will be used as explanatory variable to ant species richness. in this sense, four vegetation physiognomies are recognized: “campo sujo” (up to 1,000 trees / ha), “campo cerrado” (more than 1,400 trees / ha), “cerrado sensu stricto” (more than 2,000 trees / ha) and “cerradão” with more than 3,000 trees per hectare (oliveira-filho & ratter, 2002). to test the premises and hypotheses (see below), we used data available in ribas et al. (2003) and schoereder et al. (2010), in which full details of ant and tree sampling may be obtained. the ant sampling was conducted from january to march 2000. we used ant relative abundance, i.e. percentage of arboreal pitfall traps in which each species was caught (lindsey & skinner, 2001), ant species richness, tree species richness and abundance, species richness and abundance of efn-bearing trees from 30 20x50 m plots, 15 located in campo sujo areas and 15 in campo cerrado areas. we arbitrarily chose these physiognomies to increase the likelihood of achieving greater variation in the values of tree abundance and diversity. statistical analyses our hypotheses assume that plots with greater tree abundance and species richness offer more resources to arboreal ants, represented here by efns. therefore, before testing the hypotheses of this study, we tested two premises related to this assumption: i) efn-bearing tree abundance is positively related to total abundance of trees; and ii) species richness of efn-bearing trees is positively related to total tree species richness. although these may look like obvious probabilistic relationships, we tested these two premises to avoid a potential paralogism (renon, 2010), once diversity and abundance of trees with efns are used as estimates of resource availability. in order to test the premises, we performed two regression analyses. in the first, the abundance of efn-bearing trees was our response variable and the explanatory variable was the total abundance of trees from each plot. in the second, we used species richness of efn-bearing trees and total number of tree species from each plot as response and explanatory variable, respectively. we used proportional abundance and richness of efnbearing trees in relation to the total of trees in each sampled plot as estimates of quantity and variety of resources, respectively. since trees represent a broad spectrum of resource to ants, we used the proportion of efn-bearing tree individuals and species richness because these parameters estimate how much extrafloral nectaries represent of the total tree resource available in each plot. we obtained the proportional abundance of efn-bearing trees by dividing the number of efn-bearing trees by the total tree individuals per plot. likewise, we calculated the proportional species richness of efn-bearing trees by dividing the number of efn-bearing tree species by the total tree species per plot. to test the hypotheses that abundance and diversity of efn-bearing trees increase arboreal ant species richness, we carried out a multiple regression analysis in which the response variable was the number of ant species within plots. our explanatory variables were proportional abundance and richness of efn-bearing trees and the interaction between these variables. we expect positive correlations between arboreal ant species richness and proportional abundance and diversity of efn-bearing trees. however, the number of ant species may be correlated to total trees as well as efn-bearing trees. then, it is needed to distinguish the relationship of ant species richness with efn-bearing trees from ant species richness with overall trees. for this purpose, we used the coefficients of determination (r2) to quantify the goodness-of-fit of fixed models as a measure of variance explained. coefficients of determination were obtained from regression analyses using arboreal ant species richness as response variable. tree density and tree species richness were used as explanatory variables likewise ribas et al. (2003). then, we compared the coefficients of determination to evaluate what variables are better associated to arboreal ant species richness: tree density versus proportional abundance of efn-bearing trees, and total tree species richness versus proportional species richness of efn-bearing trees. our models followed poisson distribution corrected for over dispersion (crawley, 2007). we adjusted the complete model and the minimal adequate model was obtained by removing non-significant explanatory variables. coefficients of determination were calculated using maximum likelihood of the full and null models for generalized linear models (nagelkerke, 1991; menard, 2000). we carried out all analyses under r environment (r development core team 2017). the analyses were followed by residual analyses to check for the suitability of model and distribution employed. results efn-bearing trees represented 25% of total plant species richness (table s1 in the supplementary) and their proportional abundance varied from 50 to 90% in sampled plots. the most representative efn-bearing plant species were ouratea hexasperma (st. hil.) baill. (468 individuals), qualea grandiflora (mart.) (165), qualea parviflora (mart.) (124), and cariocar brasilisensis (camb.) (113). both premises postulated sociobiology 65(2): 162-169 (june, 2018) 165 were corroborated. there was a positive correlation between efn-bearing tree abundance and total tree abundance (f1, 28=158.5; p=0.0001). equally, efn-bearing tree richness and total tree richness were positively correlated (f1, 28 =18.9; p=0.001). sixty-three ant species were collected in 300 arboreal pitfall traps. the ten most frequent species were camponotus genatus (santschi) (54.5%), camponotus punctulatus (mayr) (34.1%), camponotus crassus (mayr) (31.1%), camponotus atriceps (smith) (27.1%), camponotus melanoticus (emery) (25.8%), cephalotes minutus (fabricius) (25.4%), cephalotes betoi (andrade) (22.1%), solenopsis sp1 (19.1%), azteca instabilis (smith) (17.4%) and cephalotes grandinosus (smith) (12%). the complete list of ant species sampled by ribas et. al. (2003) is available in table s2 as supplementary material. arboreal ant species richness and proportional abundance of efn-bearing trees were positively related (f3,26=5.54; p=0.027; fig 1) but the relation with the proportional richness of efn-bearing trees (f3,26=0.43; p=0.51) and the interaction term were not significant (f3,26=0.27; p=0.60). as expected, coefficient of determination of proportional abundance of efn-bearing trees was greater (0.156) when compared to tree density (0.014). as the relationship between ant species richness and proportional efn-bearing tree species richness was not significant, we did not compare the r2 values of the total plant species richness and proportional efn-bearing tree species richness (table 1). discussion proportional abundance of efn-bearing trees may be a more specific estimate of the amount of resources relative to tree density. thus, we argue that ant species richness may be better associated to proportional abundance of efnbearing trees because this one shows a higher coefficient of determination (table 1). our results differ from dáttilo et al. (2014a) in which ant species richness was not correlated to number of efns from three plant species analyzed separately. these authors explain their results based on dominance of efns by competitively superior ants, resulting in the coexistence of only a few other ant species (blüthgen & fiedler, 2004a; blüthgen & fiedler, 2004b). in contrast, we estimated the availability of efns at a larger scale, i.e., in each sampled plot. in this way, a higher proportion of efn-bearing trees could reduce the interspecific competition allowing a higher coexistence of ant species in the sampled plots. taken together, these findings suggest that extrafloral nectar availability do not affect ant species richness on tree individuals, in spite of this, the positive correlation between proportional abundance of efns-bearing trees and arboreal ant species richness may indicate the role of extrafloral nectar at a larger scale. in other words, in tree communities the ant species richness may be associated to the amount of efn-bearing trees but not necessarily explained by efns explanatory variable r2 p-value proportional abundance of efn-bearing trees 0.156 0.027 total density of trees 0.014 0.046* proportional species richness of efn-bearing trees ---0.421 total species richness of trees ---0.007* table 1. coefficients of determination and p-values of explanatory variables used in linear regression analyses. highlighted (*) p-values are according to ribas et al. (2003). as the relationship between ant species richness and proportional efn-bearing tree species richness was not significant, we did not compare the r2 values of the total plant species richness and proportional efn-bearing tree species richness. fig 1. influence of the proportional abundance of efn-bearing trees on arboreal ant species richness (f3,26=5.54; p=0.027). number on tree individuals. plant phenology is an important trait related to efn activity (lange et al., 2013). for plant species in cerrado the highest extrafloral nectar production occurs simultaneously to flowering period resulting in an associated ant and herbivore insects fauna (vilela et al., 2014; muniz et al., 2012). in addition, several plant species have a sequential phenological development in which individuals of different species bloom in sequence (torenzan-silingardi 2007; mendes et al. 2011) followed by highest extrafloral nectar production and ants moving from plant species to plant species along whole year (del-claro et al., 2016; vilela et al., 2014). based on this, one cannot exclude the possibility that proportion of efn-bearing trees explains arboreal ant species richness because some abundant tree species sampled were in their flowering period, for example qualea grandiflora and q. parviflora which bloom from january to february (silvério & lenza, 2010). in contrast, two abundant tree species sampled (ouratea hexasperma and caryocar brasiliense) bloom from august to november (muniz et al., 2012). in spite of this, even though not all plants were flowering and producing extrafloral nectar, ms madureira, tg sobrinho, jh schoereder – arboreal ant species richness166 the positive relationship between arboreal ant species richness and proportional abundance of efn-bearing trees highlights the importance of extrafloral nectar as food resource to ant communities in cerrado. as there was a positive correlation between the species richness of efn-bearing trees and total number of the tree species, with arboreal ant species richness not being associated with proportional richness of efn-bearing trees, we suppose that generalist foraging on extrafloral nectaries may have caused such result. hereafter, we present two arguments to generalist foraging of the ants: variation of extrafloral nectaries phenology and foraging on myrmecophylic plants. sugar and amino acids are known as components of extrafloral nectar that are attractive to ants (lanza et al., 1993; blüthgen et al., 2004) and their different concentrations and compositions have previously explained the different ant faunas on efn-bearing plants (rios et al., 2008). however, if there is a high generalist ant species richness foraging on several different nectar compositions, the expected effect of the proportional richness of efn-bearing trees on ant species richness would disappear. ants can explore different efnbearing plant species generating complex ecological networks and recent studies have found groups of generalist ants visiting extrafloral nectaries (dáttilo et al., 2013; díaz-castelazo et al., 2013; dáttilo et al., 2014a; lange & del-claro, 2014). this pattern is stable over time and exhibits more significant level of generalisation at rainy season when extrafloral nectar is more abundant (lange et al., 2013). in our study, the ants were sampled at the rainy season (january to march 2000). in this sense, despite the variation in proportional richness of efn-bearing trees between plots, the high availability of extrafloral nectar could attract equal groups of ants feeding on different efn-bearing tree species, nullifying the expected effect of nectar heterogeneity on the ant fauna. also, ants that forage on myrmecophylic plants (without domatia) are usually more generalist than ants that forage on myrmecophytic plants (blüthgen et al., 2000; gonzales-teuber & heil, 2009). because trees sampled in our study did not have domatia, we expected a higher number of generalist ant species, which in turn may be influenced by extrafloral nectar availability. thus, the generalist foraging of ant species may explain their association with proportional abundance of trees and their non-significant relation with proportional richness of efnbearing tree species. studies analysing the ant community on efn-bearing plants in cerrado have reported a core of generalist species being these ones the most abundant (lange et al., 2013; dáttilo et al., 2014a; lange & del-claro, 2014). for example, azteca genus, camponotus crassus, campontus melanoticus and cephalotes sp. are pointed as generalist and the most frequent in three ecological network studies (lange et al., 2013; dáttilo et al., 2014a, lange & del-claro, 2014). in our data, these ant groups are among the most abundant sampled species (e.g., c. crassus, c. melanoticus). in this sense, we have an additional evidence to explain the positive relationship between ant species richness and proportional abundance of efn-bearing trees. these results highlight the importance of the resource availability (efns) and its association with generalist species as explanation to arboreal ant species richness in cerrado. our results support the idea that abundance of efnbearing trees may be more important than its diversity to explain variation in ant species richness. several studies have reported the role of efns as defence structures for plants (rosumek et al., 2009 and included references). other studies have reported how extrafloral nectaries affect ant species richness on plant individuals, by comparing ant fauna on sampled trees (blüthgen et al., 2000; goítia & jaffé, 2009). here, we reported how the proportion of efn-bearing trees influences the arboreal ant species richness in tree communities, reinforcing the idea linked to the resource availability hypothesis in which areas with higher food resource amount have higher species richness. we hope these results will be helpful to understanding the local variation in ant species richness and as criterion to biodiversity conservation. for example, since extrafloral nectar is an important food to several arthropod species, plant communities harbouring high abundance of efn-bearing plants would be selected as protected areas to conserving biodiversity in cerrado. supplementary material doi: 10.13102/sociobiology.v65i2.1939.s1653 http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1939/0 acknowledgements we are grateful to mireille pic for providing analyzed data. thanks to carlos f. sperber, renata b.f campos, ricardo i. campos and ricardo r.c. solar for comments on the early version of the paper. m.s.m is supported by fapesb (fundação de amparo à pesquisa do estado da bahia), j.h.s and t.g.s are supported by grants from cnpq (conselho nacional de desenvolvimento científico e tecnológico). references alma, a. m., pol, r. g., pacheco, l. f. & vázquez, d. p. 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(2009). the contribution of the bark of isolated trees as habitat for ants in an urban landscape. landscape and urban planning, 92: 276-281. doi: 10.1016/j. landurbplan.2009.05.008 http://apps.isiknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=4&sid=2eh75dhalfdcdn@96ma&page=1&doc=1 http://apps.isiknowledge.com/full_record.do?product=wos&search_mode=generalsearch&qid=4&sid=2eh75dhalfdcdn@96ma&page=1&doc=1 http://dx.doi.org/10.1016/j.landurbplan.2009.05.008 http://dx.doi.org/10.1016/j.landurbplan.2009.05.008 doi: 10.13102/sociobiology.v67i4.5841sociobiology 67(4): 554-565 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction bee populations are at risk in several parts of the world. the removal of native vegetation to expand agricultural areas has been one of the factors with a significant contribution to the fragmentation and reduction of natural and semi-natural habitats. deforestation reduces the diversity of plants used as food resources, nesting substrates, and sources of materials necessary for bee nesting (freitas et al., 2009; kevan, 2018). knowledge about food plants used by bees is important to support habitat restoration programs and the conservation of bee populations, as well as to subsidize beekeeping (maiasilva et al., 2020). in addition, it is necessary to produce a scientific knowledge basis capable of subsidizing friendly agricultural practices to pollinators. therefore, efforts must be made to identify the resources necessary for the persistence of bee populations in these habitats. abstract in this study, we investigated the group of floral resources that support bee populations in a savanna area and how bee species use these food resources, with an emphasis on the breadth and overlap of trophic niches. the interactions between 75 bee species and 62 plant species were recorded on a brazilian savanna area. the bee species explored a diverse set of plant species, but concentrated the collection of resources in a few species. the trophic niche breadth of the eusocial bees ranged from 0.77 to 2.59, while in non-eusocial bees the variation was from 0.35 to 1.99. the distribution of the samples over a long period favored a robust characterization of the food niche of the bee populations. byrsonima sericea, serjania faveolata, and stigmaphyllon paralias were the plant species with the highest number of links with bees. in general, the trophic niche overlap was low, with 75% of pairs of bee species having a niche overlap (no) less than 0.33. only four pairs showed high overlap (no>0.70) and all cases were related to the exploitation of floral resources provided by b. sericea, a key resource for the maintenance of the local bee fauna, an oil and pollen provider. sociobiology an international journal on social insects co santos1, cml aguiar2, cf martins3, eb santana4, f frança2, e melo2, gmm santos2 article history edited by denise alves, usp, brazil received 09 september 2020 initial acceptance 10 november 2020 final acceptance 08 december 2020 publication date 28 december 2020 keywords anthophila, malpighiaceae, trophic niche, niche breadth, niche overlap, plantpollinator network. corresponding author cândida m. l. aguiar https://orcid.org/0000-0002-2220-5387 departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina s/nº, novo horizonte 44036-900, feira de santana-ba, brasil. e-mail: candida.aguiar@gmail.com vizentin-bugoni et al. (2018) pointed out that there is a notable gap in plant-pollinator network studies in central neotropical savanna and that studies should be conducted in these areas of geographical gaps so that the spatial variation in plant-pollinator networks is better understood. the available database on the food plants exploited by bees is best known in the most southern part of the brazilian savanna (= cerrado) (19o to 24o s) (pedro & camargo, 1991; carvalho & bego, 1997; andena et al., 2005; 2012; biesmeijer & slaa, 2006), however in the northern and middle portions of the cerrado domain studies are scarce (martins, 1995; pacheco-filho et al., 2015; souza et al., 2018). in this study, we investigated bee-plant interactions, focusing on which plants are most important for maintaining bee populations in an area in the middle portion of the brazilian cerrado domain, which has undergone rapid agricultural expansion in the past two decades. this is an area of high 1 ppg ciências agrárias, universidade federal do recôncavo da bahia (ufrb), cruz das almas, bahia, brazil 2 universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil 3 universidade federal da paraíba (ufpb), joão pessoa, paraíba, brazil 4 ppg ecologia & evolução, universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil research article bees food niche of solitary and social bees (hymenoptera: apoidea) in a neotropical savanna sociobiology 67(4): 554-565 (december, 2020) 555 interest for biodiversity conservation because it is located in the buffer zone of the chapada diamantina national park (cdnp). the aim of this study was to investigate the resources utilization by bee populations, i.e, measuring their realized niches, which are delimited by interspecific interactions (chesson, 2000; biesmeijer & slaa, 2006). we seek to use niche analysis tools to assess the interactions between bee species and between them and the associated flora. our hypothesis is that bee species with similar requirements use a similar set of resources. for example, oil-collecting bees depend on a specific floral resource (neff & simpson, 2017), which is produced by a small subset of the melitophilous flora. eusocial bees have similar requirements, as they continually depend on pollen and nectar sources to maintain their perennial colonies (roubik, 1989). our prediction is that regardless of the richness of flowering plant species, some plant species will be primarily exploited by bees, determining that bee species with similar requirements have higher rates of overlap with each other, than with species with different requirements. material and methods study area in this region there is a mosaic of phytophysiognomies, such as campo rupestre (rupestrian fields, sandstone outcrop vegetation), cerrado (brazilian savanna), caatinga (seasonally dry forest), sub-montane to montane semi-deciduous seasonal forests, sub-montane to montane evergreen riparian forests, wetlands and capitinga (harley, 1995; funch et al., 2009), sometimes separated by only a few kilometers. the climate is tropical humid, characterized by a marked seasonality. the rainiest period usually occurs from december to april while august to november is the driest period (jesus et al., 1983). the mean annual rainfall in the area varies from 600 to 1000 mm, with a mean temperature of 22° c (cei, 1994). three sites, 900 to 1,500 m apart, located in the buffer zone of the chapada diamantina national park (cdnp; 12° 20’ 12° 25’ s; 41° 35’ 41° 15’ w), municipality of palmeiras, bahia state, brazil, were sampled. the local vegetation is a cerrado type with phytophysiognomy of herbaceous−shrubby field, with small, scattered trees. the species byrsonima sericea dc and byrsonima cydoniifolia a. juss. (malpighiaceae) were very abundant plant species in the three locations (aguiar et al., 2017a). sampling we collected bees that visited plants to gather floral resources in 2013 (october, november, and december), 2014 (january, february, march, september, and november) 2015 (february, march, april, august, october) and 2016 (january, march, may and july). in each of the 17 collection expeditions, bee-plant interactions were recorded over two consecutive days, from 8:00 to 16:00, over three transects (1,500 x 6m each). each bee collected on a flower was considered a sampling unit. the bees were captured with entomological nets, without choice, for 5 to 10 minutes in each flowering plant, according to sakagami et al. (1967). fertile material from the plants visited by the bees was collected and herborized. data analysis to evaluate the trophic niche breadth, we used the shannon diversity index (h´) (shannon, 1948). additionally, we used the pielou equitability index (j´) as an indicator of how plant diversity was used by each bee species (ludwig & reynolds, 1988). the level of niche overlap (noih) between each pair of bee species was assessed using the schoener index (1968), noih = 1 1/2 σk | pik phk |, where: i and h are the bee species compared, pik and phk are the proportions of individuals, respectively of the bee species i and h collected in the plant species k. the pik value was obtained by dividing the number of individuals of species i collected in plant k by the total number of individuals of species i collected in all plants. the schoener index is symmetrical and varies from 0 to 1. only bee species represented by eight or more individuals were included in the analysis. the overlap between each bee speciespair (noih) was analyzed in 105 possible combinations of pairs formed by 15 bee species. following aguiar (2003) and aguiar et al. (2017b), the overlap of the trophic niche was considered low when the noih value was less than or equal to 0.30, it was moderate when the value was greater than 0.30 and equal to or less than 0.70, and high if the noih values were greater than 0.70. the data of the bee species (i) in each plant species (j) were used to build a pij incidence matrix and calculate the connectance, which is the relationship between the actual number of interactions found and the theoretical number of possible interactions. to draw our network, we order the matrix in decrease frequencies of interactions between plants and bees and then we used the function plotweb from bipartite package (dormann et al., 2009) in r program (r core team, 2020). results the interactions between 75 bee species of bees and 62 plant species were recorded on this cerrado site (fig 1, table 1, supplementary material 1). byrsonima sericea, serjania faveolata radlk., stigmaphyllon paralias a. juss and pityrocarpa moniliformis (benth.) luckow & r.w. jobson were the plants with greatest number of links with bee species. additionally, these plants received together almost half of the total visits (table 1). the guild of oil-collecting bees, here composed of the species included in the centridini, tetrapediini and tapinotaspidini tribes, showed high diversity and many interactions with b. sericea and s. paralias (malpighiaceae) (supplementary material 1). the bee-plant network showed connectance = 4.67%, with 242 interactions found out of 4,650 theoretically possible interactions. considering only the guild of oil-collecting bees (26 species) and oil-plants (8 species), 75 interactions were recorded out of 208 theoretically possible and connectance was 16.8%. co santos, cml aguiar, cf martins, eb santana, f frança, e melo, gmm santos – food niche of solitary and social bees556 fig 1 plant-bee network in a cerrado area. plant species: p01 anemopaegma sp. 1, p02 banisteriopsis harleyi, p03 borreria verticillata , p04 bowdichia virgilioides, p05 byrsonima correifolia, p06 byrsonima cydoniifolia, p07 byrsonima dealbata, p08 byrsonima sericea, p09 centrosema coriaceum, p10 chamaecrista mucronata, p11 cordia rufescens, p12 croton sp. 1, p13 cuphea sessiliflora, p14 dalechampia brasiliensis, p15 diplopterys pubipetala, p16 eremanthus capitatus, p17 erythroxylum loefgrenii, p18 eugenia cf. punicifolia, p19 eugenia excelsa, p20 eugenia pistaciifolia, p21 evolvulus sp. 1, p22 fridericia cinerea, p23 gymneia sp.1, p24 herissantia crispa, p25 ipomoea incarnata, p26 ipomoea sp. 1, p27 jacquemontia sp. 1, p28 lasiolaena lychnophorioides, p29 lepidaploa chalybaea, p30 lippia sp. 1, p31 lippia sp. 2, p32 malpighiaceae sp. 1, p33 manihot sp. 1, p34 microstachys corniculata, p35 mikania elliptica, p36 mimosa somnians, p37 mimosa sp.1, p38 moquiniastrum blanchetianum, p39 myrtaceae sp. 1, p40 myrtaceae sp. 2, p41 passiflora edmundoi, p42 passiflora edulis, p43 periandra mediterranea, p44 piriqueta sidifolia, p45 pityrocarpa moniliformis, p46 rhaphiodon echinus, p47 senegalia langsdorffii, p48 senna acuruensis, p49 senna macranthera, p50 senna macranthera, p51 serjania faveolata, p52 serjania lethalis, p53 serjania sp. 1, p54 serjania sp. 2, p55 serjania sp. 3, p56 simarouba amara, p57 stachytarpheta crassifolia, p58 stigmaphyllon paralias, p59 stylosanthes scabra, p60 turnera sp. 1, p61 urochloa decumbens, p62 waltheria cf. indica. bee species: b01 acanthopus excellens, b02 apis melífera, b03 augochlora (augochlora) sp. 5, b04 augochlora (oxystoglossella) sp. 2, b05 augochlora (oxystoglossella) sp. 3, b06 augochloropsis sp. 3, b07 augochloropsis sp. 4, b08 augochloropsis sp. 5, b09 augochloropsis sp. 6, b10 augochloropsis sp. 7, b11 augochloropsis sp. 8, b12 bombus morio, b13 centris varia, b14 centris moerens, b15 centris tetrazona, b16 centris lutea, b17 centris aenea, b18 centris caxienses, b19 centris cf. spilopoda, b20 centris decolorata, b21 centris nitens, b22 centris perforator, b23 centris sp. 1, b24 centris sp. 3, b25 centris sp. 6, b26 centris tarsata, b27 ceratina (crewella) sp.1, b28 ceratina (crewella) sp.2, b29 ceratina (crewella) sp.3, b30 colletes sp.1, b31 diadasia sp.1, b32 dialictus opacus, b33 dicranthidium sp. 1, b34 epicharis analis, b35 epicharis bicolor, b36 epicharis cockerelli, b37 epicharis flava, b38 euglossa cordata, b39 eulaema nigrita, b40 exomalopsis (exomalopsis) sp. 2, b41 exomalopsis (phanomalopsis) sp. 1, b42 florilegus sp. 1, b43 frieseomelitta francoi, b44 geotrigona mombuca, b45 leiopodus abnormis, b46 lophopedia nigrispinis, b47 megachile (pseudocentron) sp. 3, b48 megachile sp. 8, b49 melipona quadrifasciata, b50 melitoma sp. 1, b51 melitomella grisescens, b52 mesoplia friesei, b53 mesoplia rufipes, b54 monoeca affs. moure, b55 nannotrigona testaceicornis, b56 oxaea flavescens, b57 paratrigona incerta, b58 partamona combinata, b59 pseudaugochlora pandora, b60 pseudaugochlora sp. 1, b61 scaptotrigona aff. postica, b62 tapinotaspoides sp.1, b63 temnosoma cf. metallicum, b64 tetragonisca sp. 1, b65 tetrapedia amplitarsis, b66 tetrapedia diversipes, b67 trigona hyalinata, b68 trigona spinipes, b69 tropidopedia nigrocarinata, b70 urbanapsis diamantina, b71 xanthopedia sp., b72 xylocopa subcyanea, b73 xylocopa cearensis, b74 xylocopa sp. 2, b75 xylocopa frontalis. sociobiology 67(4): 554-565 (december, 2020) 557 fifteen bee species were included in the niche analyzes. the eusocial species trigona spinipes (fabricius) and apis mellifera l. exploited the largest plant spectrum (fig 1; supplementary material 1) and showed high equitability in the distribution of visits to the plants, with broader trophic niches (h’>2.00) (table 2). epicharis bicolor smith, centris aenea lepeletier and melitoma sp.1 presented the narrowest trophic niches, and the first two showed a foraging concentration in b. sericea, causing low equitability of visits to the spectrum of exploited plants, which decreased the value of the h’ index downwards (table 2; supplementary material 1). family plant species code n1 n2 asteraceae eremanthus capitatus (spreng.) macleish p16 2 3 asteraceae lasiolaena lychnophorioides roque et al. p28 1 1 asteraceae lepidaploa chalybaea (mart. ex dc.) h.rob. p29 3 3 asteraceae mikania elliptica dc. p35 2 3 asteraceae moquiniastrum blanchetianum (dc.) g. sancho p38 5 11 bignoniaceae anemopaegma sp. 1 p01 2 2 bignoniaceae fridericia cinerea (bureau ex k.schum.) l.g.lohmann p22 1 2 boraginaceae cordia rufescens a.dc. p11 1 1 convolvulaceae evolvulus sp. 1 p21 1 1 convolvulaceae ipomoea incarnata (vahl) choisy p25 2 2 convolvulaceae ipomoea sp. 1 p26 2 4 convolvulaceae jacquemontia sp1. choisy p27 3 3 erythroxylaceae erythroxylum loefgrenii diogo p17 2 2 euphorbiaceae croton sp. 1 p12 1 1 euphorbiaceae dalechampia brasiliensis lam. p14 1 1 euphorbiaceae manihot sp. 1 p33 2 20 euphorbiaceae microstachys corniculata (vahl) griseb. p34 1 3 fabaceae bowdichia virgilioides kunth p04 4 5 fabaceae centrosema coriaceum benth. p09 5 7 fabaceae chamaecrista mucronata (spreng.) h.s.irwin & barneby p10 2 5 fabaceae mimosa somnians humb. & bonpl. ex willd. p36 1 2 fabaceae mimosa sp.1 p37 1 1 fabaceae periandra mediterranea (vell.) taub. p43 5 12 fabaceae pityrocarpa moniliformis (benth.) luckow & r.w.jobson p45 12 36 fabaceae senegalia langsdorffii (benth.) seigler & ebinger p47 6 15 fabaceae senna acuruensis (benth.) h.s.irwin & barneby p48 1 1 fabaceae senna macranthera (dc. ex collad.) h.s.irwin & barneby p49 2 9 fabaceae senna macranthera var. micans (nees) h.s.irwin & barneby p50 2 3 fabaceae stylosanthes scabra vogel p59 3 3 lamiaceae gymneia sp. 1 p23 1 1 lamiaceae rhaphiodon echinus schauer p46 1 1 lythraceae cuphea sessiliflora a.st.-hil. p13 1 1 malpighiaceae banisteriopsis harleyi b.gates p02 2 4 table 1. plant species exploited by bee species in a cerrado area in the chapada diamantina, bahia, brazil. n1: number of bee species visiting each plant species. n2: number of individual bees collected from each plant species. co santos, cml aguiar, cf martins, eb santana, f frança, e melo, gmm santos – food niche of solitary and social bees558 family plant species code n1 n2 malpighiaceae byrsonima correifolia a.juss. p05 2 4 malpighiaceae byrsonima cydoniifolia a.juss. p06 6 9 malpighiaceae byrsonima dealbata griseb. p07 4 7 malpighiaceae byrsonima sericea dc. p08 20 232 malpighiaceae diplopterys pubipetala (a.juss.) w.r.anderson & c.c.davis p15 2 2 malphigiaceae malpighiaceae sp. 1 p32 1 1 malpighiaceae stigmaphyllon paralias a.juss. p58 13 58 malvaceae herissantia crispa (l.) brizicky p24 2 21 malvaceae waltheria cf. indica l. p62 2 2 myrtaceae eugenia cf. punicifolia (kunth) dc. p18 2 7 myrtaceae eugenia excelsa o.berg p19 1 1 myrtaceae eugenia pistaciifolia dc. p20 1 1 myrtaceae myrtaceae sp. 1 p39 4 5 myrtaceae myrtaceae sp. 2 p40 1 3 passifloraceae passiflora edmundoi sacco p41 1 1 passifloraceae passiflora edulis sims p42 6 7 poaceae urochloa decumbens (stapf) r.d.webster p61 1 1 rubiaceae borreria verticillata (l.) g.mey p03 1 1 sapindaceae serjania faveolata radlk. p51 18 78 sapindaceae serjania lethalis a.st.-hil. p52 6 39 sapindaceae serjania sp. 1 p53 1 3 sapindaceae serjania sp. 2 p54 3 14 sapindaceae serjania sp. 3 p55 9 41 simaroubaceae simarouba amara aubl. p56 6 14 turneraceae piriqueta sidifolia (cambess.) urb. p44 5 9 turneraceae turnera sp. 1 p60 1 2 verbenaceae lippia sp. 1 p30 3 25 verbenaceae lippia sp. 2 p31 5 21 verbenaceae stachytarpheta crassifolia schrad. p57 1 1 table 1. plant species exploited by bee species in a cerrado area in the chapada diamantina, bahia, brazil. n1: number of bee species visiting each plant species. n2: number of individual bees collected from each plant species. (continuation) the trophic niche overlap between each bee species pair ranged from 0.01 to 0.87, being higher between e. bicolor and c. aenea, and between trigona hyalinata (lepeletier) and e. bicolor (table 2). the vast majority of species pairs analyzed (~75%) showed low overlap of the trophic niche (no<0.33), and approximately half of them had a very low level of overlap (no<0.1). only four pairs showed high overlap (no>0.7): e. bicolor/c. aenea; t. hyalinata/e. bicolor; urbanapsis diamantina aguiar and melo/t. hyalinata; t. hyalinata/c. aenea. the high level of overlap found in these pairs was mainly influenced by the exploitation of resources from b. sericea (p08), floral oil and/or pollen (supplementary material 1). a. mellifera showed a low overlap of the trophic niche with the other bee species (table 2), due mainly to scattered foraging in 23 plant species. however, this exotic species heavily exploited s. faveolata, a food plant visited by many native bee species (supplementary material 1). discussion bees exploited a diverse flora, however a small set of plant species, either due to their abundance or by providing specific resources, can be considered as key species for the maintenance of bee populations in this community. sociobiology 67(4): 554-565 (december, 2020) 559 b02 b14 b17 b18 b19 b22 b27 b34 b35 b50 b61 b67 b68 b69 b70 b14 0.11 b17 0.11 0.26 b18 0.14 0.25 0.43 b19 0.05 0.25 0.50 0.39 b22 0.18 0.24 0.28 0.18 0.18 b27 0.02 0.00 0.08 0.06 0.00 0.20 b34 0.06 0.25 0.44 0.37 0.44 0.29 0.10 b35 0.07 0.25 0.87 0.45 0.50 0.21 0.03 0.40 b50 0.01 0.00 0.00 0.11 0.00 0.00 0.00 0.00 0.06 b61 0.14 0.00 0.02 0.10 0.00 0.01 0.03 0.01 0.07 0.10 b67 0.06 0.25 0.72 0.50 0.50 0.18 0.00 0.37 0.78 0.19 0.20 b68 0.21 0.09 0.07 0.21 0.05 0.12 0.01 0.05 0.11 0.28 0.21 0.24 b69 0.30 0.22 0.22 0.33 0.22 0.29 0.00 0.22 0.28 0.11 0.13 0.33 0.32 b70 0.16 0.25 0.56 0.50 0.50 0.29 0.00 0.37 0.61 0.33 0.10 0.74 0.40 0.44 h’ 2.45 1.91 0.69 1.72 0.97 1.99 1.75 0.89 0.35 0.66 1.89 0.77 2.59 1.15 0.94 j’ 0.78 0.98 0.31 0.88 0.89 0.96 0.90 0.81 0.32 0.95 0.79 0.70 0.85 0.83 0.85 spl 23 7 9 7 3 8 7 3 3 2 11 3 21 4 3 nab 168 8 121 18 8 17 10 27 34 8 96 32 78 9 9 table 2. trophic niche overlap among bee species in a cerrado area in chapada diamantina, bahia, brazil. h’: niche breadth. j’: equitability index. spl: number of plant species visited by each bee species. nab: number of individuals of each bee species. b02 apis mellifera, b14 centris moerens, b17 centris aenea, b18 centris caxiensis, b19 centris cf. spilopoda, b22 centris perforator, b27 -ceratina (crewella) sp.1, b34 epicharis analis, b35 epicharis bicolor, b50 melitoma sp.1, b61 scaptotrigona aff. postica, b67 trigona hyalinata, b68 trigona spinipes, b69 tropidopedia nigrocarinata, b70 urbanapsis diamantina b. sericea and s. paralias are plants that produce floral oil. in addition, b. sericea pollen is collected by several distinct bee groups (teixeira & machado, 2000), which increases its attractiveness for both oil-bees and non-oil-bees. another aspect that increases the possibilities of b. sericea interactions with bees is the great local abundance of this plant species (aguiar et al., 2017a). the serjania genus comprises nectar-producing species (matos & santos, 2017), and p. moniliformis is a nectar (santos et al., 2018) and pollen source for bees (maiasilva et al., 2012). the visits of oil-bees to oil-plant flowers are not optional as these bees demand floral oil to complete their reproductive cycle (alves-dos-santos et al., 2007; neff & simpson, 2017). oil-bees have, at least in part, the same requirements, all of which demand floral oil, which explains the high overlap of trophic niche. it is expected that species with similar ecological requirements will show some redundancy in the use of resources and high overlap in their niches. in fact, the connectance in the oil-bees guild and oil-plants is approximately four times greater than the connectance found in the entire community. in the small world formed by the oilbees and oil-plants, species share evolutionary histories and mutually specialized structures (bezerra et al., 2009), forming modules in which interactions have higher intimacy than in the network as a whole. hembry et al. (2018) demonstrated that the level of interaction intimacy affects the structure of communities. the most abundant oil-bee species in this assemblage was c. aenea, who exploited floral resources of a diversity of non-phylogenetically related plant species, that is, a tendency to a generalist foraging behavior, previously pointed out in other habitats (aguiar & gaglianone, 2003; mello et al., 2013). however, its niche breadth, measured by the h’ index, showed one of the lowest values among these bee species, because of the high concentration of nesting females on the oil-plant b. sericea. the strong mutualistic interaction between c. aenea and b. sericea has been reported in many habitats (mello et al., 2013), as well as the behavior of this oil-bee to nest in aggregations near this food plant (aguiar & gaglianone, 2003). b. sericea played a central role in the trophic niche of c. aenea and other oil-bees, since the exploitation of the floral resources of this plant explains all the cases in which there was a high overlap of trophic niche (no>0.70), even when one of the species involved was not an oil-bee, such as t. hyalinata (meliponini), whose visits to b. sericea were probably for pollen foraging. among the eusocial species, t. spinipes showed the broader trophic niche, as expected based on its supergeneralist foraging behavior (biesmeijer & slaa, 2006; giannini et al., 2015; pacheco-filho et al., 2015). its trophic niche breadth in this habitat was influenced both by the richness of plant species visited and by the distribution of the foragers in many floral resources. this scattered foraging in several plant co santos, cml aguiar, cf martins, eb santana, f frança, e melo, gmm santos – food niche of solitary and social bees560 species contributed to the low level of niche overlap of this species with the other bee species, including a. mellifera and the congeneric species t. hyalinata. biesmeijer and slaa (2006) highlighted that these two congeneric species, despite having aggressive group forager behavior (nieh et al., 2003, 2005; slaa et al., 2003), in general present different diets and probably do not interact regularly during foraging. surprisingly, t. spinipes and a. mellifera, species that have a strong association with each other in the use of floral resources (e.g. biesmeijer & slaa, 2006) and are supergeneralists species in bee-plant networks (giannini et al., 2015), also showed low overlap level in our study, which was influenced by the allocation of many honeybee foragers in three plant species that received few visits of t. spinipes. these two supergeneralist species have been considered fundamental to the maintenance of the bee-plant networks, although they have different effects on network structure, with a. mellifera having a strong effect on nestedness, whereas t. spinipes has a main effect on the niche overlap of the bees (giannini et al., 2015). the low overlap of the trophic niche of a. mellifera with the other bee species was also caused by scattering of foragers in many plant species. however, some of these plant species, such as s. faveolata, were important in the diet of other bees. the distribution of the samples over a long period (almost 4 years), as well as the large sampling effort, favored a more robust characterization of the food niche of the bee populations, as it allows recording of bee-plant interactions in different periods of flowering of the melitophilous plants, as well as the registration of different generations of bee species. on the other hand, it probably contributed to the very low values of niche overlap found, which may be related to the differences between flowering periods of the melitophilous plants and between periods of nesting activity of different species of solitary bees. additionally, the low levels of niche overlap found may be, at least in part, influenced by the sampling method, collection of bees during foraging, as previously discussed by ranta and lundberg (1981). these authors compared the overlapping levels of the food niche between bombus species using three sampling methods, and found that the mean niche overlap values were significantly lower when calculated using direct observations of flower visits data than when using analyses of pollen contents in pollen loads and in nectar loads. according to the authors, these differences in overlapping levels would be largely explained by differences in the contribution of different food plants in number of pollen grains for the pollen loads, since only a few plant species were dominant, as they are represented by many pollen grains in the pollen loads, resulting in an increase in the niche overlap values. finally, we emphasize that the method of collecting bees on flowers is more viable for the analysis of the food niche in tropical bee assemblages, even although only a few species can be evaluated according to their abundance. acknowledgments we are grateful to the bee specialists who identified some bee species collected in this research (felipe vivallo, antonio aguiar, fernando zanella, cristina gaglianone and willian aguiar), and to emanuelle brito for assistance with the figures. we also thank the national council for scientific and technological development, brazil (cnpq, no. 558228/2009-7, peld; 403774/2012-8, peld; 474065/2013-8) for financial support for this project. cnpq granted research fellowship to c.f. martins and g.m.m. santos. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nivel superior brasil (capes) finance code 001. capes granted co santos and eb santana. references aguiar, c.m.l. 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(2018). plant-pollinator networks in the tropics: a review. in, w. dáttilo & v. rico-gray (eds.), ecological networks in the tropics, (pp. 73-91). dordrecht, the netherlands: springer. doi:10.1007/978-3-319-68228-0_6 sociobiology 67(4): 554-565 (december, 2020) 563 bee species bee code nab plant species andrenidae oxaeini oxaea flavescens klug, 1807 b56 2 p1(1), p49(1) apidae apini apis mellifera linnaeus, 1758 b02 168 p6(1), p8(8), p17(1), p18(6), p24(20), p28(1), p30(23), p31(11), p32(1), p35(2), p36(2), p39(2), p45(2), p47(10), p51(42), p52(1), p53(3), p54(10), p55(18), p56(1), p58(1), p59(1), p62(1) bombini bombus morio (swederus, 1787) b12 4 p9(1), p10(1), p22(2) centridini centris aenea lepeletier, 1841 b17 121 p4(1), p6(1), p8(102), p15(1), p18(1), p31(4), p42(1), p45(9), p56(1) centris caxienses ducke, 1907 b18 18 p5(3), p6(2), p8(7), p20(1), p31(2), p39(1), p58(2) centris decolorata lepeletier, 1841 b20 2 p8(1), p42(1) centris nitens lepeletier, 1841 b21 1 p8(1) centris varia (erichson, 1849) b13 1 p8(1) centris tarsata smith, 1874 b26 3 p8(1), p13(1), p31(1) centris moerens (perty, 1833) b14 8 p1(1), p8(2), p42(1), p47(1), p48(1), p50(1), p57(1) centris lutea friese, 1899 b16 1 p2(1) centris perforator (smith, 1874) b22 17 p4(2), p8(3), p9(2), p10(4), p15(1), p42(1), p45(2), p51(2) centris cf. spilopoda moure, 1969 b19 8 p7(3), p8(4), p19(1) centris tetrazona moure & seabra, 1962 b15 5 p8(1), p42(2), p45(1), p56(1) centris sp. 1 b23 4 p6(1), p8(3) centris sp. 3 b24 1 p8(1) centris sp. 6 b25 1 p45(1) epicharis analis lepeletier, 1841 b34 27 p7(2), p8(10), p45(15) epicharis bicolor smith, 1854 b35 34 p8(31), p45(1), p58(2) epicharis cockerelli friese, 1900 b36 4 p8(4) epicharis flava friese, 1900 b37 1 p8(1) emphorini diadasia sp. 1 b31 1 p23(1) melitoma sp. 1 b50 8 p26(3), p58(5) melitomella grisescens (ducke, 1907) b51 2 p16(2) ericrocidini acanthopus excellens schrottky, 1902 b01 1 p55 (1) mesoplia friesei (ducke, 1902) b52 2 p14(1), p46(1) mesoplia rufipes (perty, 1833) b53 4 p4(1), p31(3) eucerini florilegus sp. 1 b42 1 p29(1) euglossini euglossa cordata (linnaeus, 1758) b38 2 p8(1), p35(1) eulaema nigrita lepeletier, 1841 b39 5 p9(1), p30(1), p42(1), p51(2) exomalopsini exomalopsis (phanomalopsis) sp. 1 b41 1 p55(1) exomalopsis (exomalopsis) sp. 2 b40 1 p55(1) supplementary material 1. species of bees and plants visited in a cerrado area in the chapada diamantina, bahia, brazil. nab: number of individuals of each bee species. within the brackets the number of individuals of each bee species collected in each plant species is presented. the plant species codes according to table 1. co santos, cml aguiar, cf martins, eb santana, f frança, e melo, gmm santos – food niche of solitary and social bees564 bee species bee code nab plant species meliponini frieseomelitta francoi (moure, 1946) b43 1 p44(1) geotrigona mombuca (smith, 1863) b44 2 p45(1), p55(1) melipona quadrifasciata lepeletier, 1836 b49 2 p45(1), p47(1) nannotrigona testaceicornis (lepeletier, 1836) b55 4 p38(1), p44(1), p52(1), p55(1) paratrigona incerta camargo & moure, 1994 b57 7 p7(1), p8(4), p38(1), p55(1) partamona combinata pedro & camargo, 2003 b58 2 p47(1), p55(1) scaptotrigona aff. postica (latreille, 1807) b61 96 p16(1), p17(1), p33(17), p38(7), p43(2), p44(2), p45(1), p52(32), p55(16), p56(7), p58(10) tetragonisca sp. 1 b64 1 p43(1) trigona hyalinata (lepeletier, 1836) b67 32 p8(23), p52(3), p58(6) trigona spinipes (fabricius, 1793) b68 78 p2(3), p6(3), p8(4), p25(1), p27(1), p30(1), p33(3), p34(3), p38(1), p39(1), p40(3), p41(1), p43(7), p47(1), p49(8), p50(2), p51(5), p54(3), p56(3), p58(22), p60(2) protepeolini leiopodus abnormis (jörgensen, 1912) b45 1 p27(1) tapinotaspidini lophopedia nigrispinis (vachal, 1909) b46 2 p45(1), p51(1) monoeca affs.moure aguiar, 2012 b54 1 p58(1) tapinotaspoides sp. 1 b62 1 p5(1) tropidopedia nigrocarinata aguiar & melo, 2007 b69 9 p8(2), p43(1), p51(5), p58(1) urbanapsis diamantina aguiar & melo, 2007 b70 9 p8(5), p51(1), p58(3) xanthopedia sp. b71 5 p8(4), p51(1) tetrapediini tetrapedia amplitarsis friese, 1899 b65 3 p8(3) tetrapedia diversipes klug, 1810 b66 4 p43(1), p51(2), p61(1) xylocopini ceratina (crewella) sp.1 b27 10 p4(1), p11(1), p12(1), p37(1), p39(1), p44(4), p45(1) ceratina (crewella) sp.2 b28 1 p29(1) ceratina (crewella) sp.3 b29 1 p26(1) xylocopa cearensis ducke, 1910 b73 4 p9(2), p51(2) xylocopa frontalis (olivier, 1789) b75 1 p54(1) xylocopa subcyanea pérez, 1901 b72 1 p51(1) xylocopa sp. 2 b74 1 p9(1) colletidae colletes sp.1 b30 5 p51(5) halictidae augochlorini augochlora (oxystoglossella) sp. 2 b04 1 p56(1) augochlora (oxystoglossella) sp. 3 b05 5 p25(1), p51(2), p58(1), p59(1) augochlora (augochlora) sp. 5 b03 1 p52(1) augochloropsis sp. 3 b06 1 p47(1) augochloropsis sp. 4 b07 1 p38(1) augochloropsis sp. 5 b08 7 p8(3), p21(1), p24(1), p27(1), p59(1) augochloropsis sp. 6 b09 1 p7(1) augochloropsis sp. 7 b10 6 p6(1), p8(1), p51(3), p58(1) augochloropsis sp. 8 b11 2 p51(1), p52(1) pseudaugochlora pandora (smith, 1853) b59 1 p51(1) pseudaugochlora sp. 1 b60 1 p44(1) sociobiology 67(4): 554-565 (december, 2020) 565 plant species code anemopaegma sp. 1 mart. ex meisn. p01 banisteriopsis harleyi b. gates p02 borreria verticillata (l.) g. mey p03 bowdichia virgilioides kunth p04 byrsonima correifolia a.juss. p05 byrsonima cydoniifolia a.juss. p06 byrsonima dealbata griseb. p07 byrsonima sericea dc. p08 centrosema coriaceum benth. p09 chamaecrista mucronata (spreng.) h.s.irwin & barneby p10 cordia rufescens a.dc. p11 croton sp.1 p12 cuphea sessiliflora a.st.-hil. p13 dalechampia brasiliensis lam. p14 diplopterys pubipetala (a.juss.) w.r.anderson & c.c.davis p15 eremanthus capitatus (spreng.) macleish p16 erythroxylum loefgrenii diogo p17 eugenia cf. punicifolia (kunth) dc. p18 eugenia excelsa o.berg p19 eugenia pistaciifolia dc. p20 evolvulus sp.1 p21 fridericia cinerea (bureau ex k.schum.) l.g.lohmann p22 gymneia sp. 1 (benth.) harley & j.f.b.pastore p23 herissantia crispa (l.) brizicky p24 ipomoea incarnata (vahl) choisy p25 ipomoea sp.1 p26 jacquemontia sp1. choisy p27 lasiolaena lychnophorioides roque et al. p28 lepidaploa chalybaea (mart. ex dc.) h.rob. p29 lippia sp.1 p30 lippia sp.2 p31 plant species code malpighiaceae sp.1 p32 manihot sp.1 p33 microstachys corniculata (vahl) griseb. p34 mikania elliptica dc. p35 mimosa somnians humb. & bonpl. ex willd. p36 mimosa sp.1 p37 moquiniastrum blanchetianum (dc.) g. sancho p38 myrtaceae sp.1 p39 myrtaceae sp.2 p40 passiflora edmundoi sacco p41 passiflora edulis sims p42 periandra mediterranea (vell.) taub. p43 piriqueta sidifolia (cambess.) urb. p44 pityrocarpa moniliformis (benth.) luckow & r.w.jobson p45 rhaphiodon echinus schauer p46 senegalia langsdorffii (benth.) seigler & ebinger p47 senna acuruensis (benth.) h.s.irwin & barneby p48 senna macranthera (dc. ex collad.) h.s.irwin & barneby p49 senna macranthera var. micans (nees) h.s.irwin & barneby p50 serjania faveolata radlk. p51 serjania lethalis a.st.-hil. p52 serjania sp. 1 p53 serjania sp. 2 p54 serjania sp. 3 p55 simarouba amara aubl. p56 stachytarpheta crassifolia schrad. p57 stigmaphyllon paralias a.juss. p58 stylosanthes scabra vogel p59 turnera sp. 1 p60 urochloa decumbens (stapf) r.d.webster p61 waltheria cf. indica l. p62 bee species bee code nab plant species temnosoma cf. metallicum smith, 1853 b63 1 p62(1) halictini dialictus opacus (moure, 1940) b32 5 p8(1), p51(1), p58(3) megachilidae anthidiini dicranthidium sp. 1 b33 1 p3(1) megachilini megachile (pseudocentron) sp. 3 b47 1 p29(1) megachile sp. 8 b48 1 p51(1) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.35-42sociobiology 61(1): 35-42 (march, 2014) trophic guild structure of a canopy ants community in a mexican tropical deciduous forest gabriela castaño-meneses1,2 introduction in tropical regions, ants constitute one of the most abundant and diverse groups in canopies (tobin, 1995; davidson et al., 2003; lach et al., 2010). in peruvian forest canopy, wilson (1987) found 135 ant species while in budongo forest, uganda, schulz and wagner (2002) recorded 161 species; in the south part of australia, ants represented about 48% of the total of arboricolous fauna (andersen & yen, abstract seasonality in tropical dry forest shows extreme changes in the physiognomy of forest as well the available resources in each season, thus, the composition and diversity of fauna inhabiting in that ecosystem show seasonal variations in answer to that changes. the ants constitute a very important element in the canopies of tropical forests, and there is few information about their communities in dry forest. in most of ecosystems, the general patterns of ant distribution show increase of their abundance during the wet season, but according with the characteristics of chamela tropical dry forest in the pacific cost of mexico, the great amount of epiphytes in the area can be an important resource to the ants, and the canopy can be an environment visited for different species of ants during the driest month. in order to study the seasonal variations in species richness, composition and diversity of ant canopy community in a tropical deciduous forest, seven fogging were performed in a watershed of the chamela biological station, jalisco state, mexico, including dry and rainy season. a total of 5 563 ant specimens were collected belong to 46 morphospecies from 17 genera. the most species richness genera were camponotus and cephalotes, with 13 and 6 species respectively, and the most abundant ants were species of crematogaster, tapinoma, cephalotes and camponotus genera. nevertheless dominant species were present during all the study, abundance show a great seasonality, with highest values during the dry season. the dominant guild in the canopy was the omnivorous in all study, but differences in guild trophic composition were recorded in each fogging. the ant community in the canopy of chamela shows important seasonal variations in the composition and trophic guilds dominance, due conditions of this forest, that differences can be result of variations in the exploitation of resources along the year, and vertical migrations of ant species from soil and shrub layer to canopy in the tropical deciduous forest. sociobiology an international journal on social insects 1 ecología y sistemática de microartrópodos, depto de ecología y recursos naturales, facultad de ciencias, universidad nacional autónoma de méxico, méxico, df. 2 current address: unidad multidisciplinaria de docencia e investigación, facultad de ciencias, universidad nacional autónoma de méxico, campus juriquilla, querétaro, méxico. article history edited by: jacques h. c. delabie, uesc, brazil received 27 july 2013 initial acceptance 01 november 2013 final acceptance 14 november 2013 keywords diversity, chamela, fogging, species richness corresponding author gabriela castaño-meneses ecología y sistemática de microartrópodos deptamiento ecología y recursos naturales, facultad de ciencias, unam ciudad universitaria, 04510 méxico, d. f. phone: (52) 55 56 22 49 02 e-mail: gabycast99@hotmail.com 1992), while in borneo constituted near 18% (stork, 1987) and in new caledonia close to 7% (guilbert et al., 1995). those variations are related with the habitat heterogeneity and the availability of resources, factors that affect the species richness and abundance of the communities, and in the case of tropical ants, they can exploit a great variety of resources that are provided directly or indirectly by several tree species with different phenologies (ribas et al., 2003; armbrecht et al., 2004). research article ants g castaño-meneses trophic structure of canopy ants from mexican deciduous forest 36 the seasonality is a very important component of the ant communities structure in tropical forests (basset et al., 2003), and in the tropical deciduous forest its importance increases considerably (dirzo et al., 2011), due to the changes in the availability of resources and the ability of the organisms to use them according to their feeding habits. thus, species composition of trophic guilds shows changes according to the season, due to trophic guilds that answer differently to environmental conditions modifiing the interactions between species and the composition of community (meyer et al., 2010; cook et al., 2011). into the tropical forest areas, the tropical dry forest ecosystems comprise more than 40% of surface area in the world, but these ecosystems show an important loss of forest area in recent years as a result of accelerated anthropogenic disturbance (trejo & dirzo, 2000; dirzo et al., 2011). there are few information about the importance of seasonality on the ant activity and structure of communities in the tropical dry forest (neves et al., 2010), and in mexico there are only records for the atlantic coast (gove et al., 2005), but there are no studies in the pacific coast. the study of ant communities in canopies from mexico is still unexplored, with a few notes about their abundance (palacios-vargas et al., 1999) or their importance as indicators of perturbation (gove et al., 2005). thus, in the present work, the temporal variation and the trophic guild distribution in the canopy ant community of chamela, jalisco, in the pacific cost of mexico were analyzed in order to study the seasonal pattern shown by ants in this vegetation. material and methods the study was carried out in the chamela biological station (chbs) of the instituto de biología of universidad nacional autónoma de méxico (unam). this is a natural reserve located at the pacific coast of mexico, in the state of jalisco (19°83’00”n 105°80’30”w; 150 m elevation). the rainy season, according to bullock (1986), lasts four months, from july to october, with more than 50% of precipitation during september and october (garcía-oliva et al., 2002). mean annual precipitation and temperature are 788 mm and 24.68ºc, respectively (1977–2000; garcía-oliva et al., 2002). details of physical and biological parameters of the reserve have been compiled by bullock (1988) and noguera et al. (2002). the flora and vegetation structure of the forest have been described (lott, 1985; lott et al., 1987; balvanera et al., 2002). a total of seven fumigations were performed in order to sample the canopy. fogging sessions were made using a dyna fog machine. the sampling was performed during rainy and dry seasons from 1992 to 1994, in august and september 1992 (rainy season); may (dry season), july (rainy) and november (dry) 1993; and february and may 1994 (dry). sampling sites were located in the watershed named 4a (cervantes et al., 1988), where the tree layer was about 25 m tall. dominant species in the area are guapira macrocarpa (miranda), celaenodendron mexicanum standl., lonchocarpus eriocarinalis micheli, lonchocarpus constrictus pittier, bursera instabilis mcvaugh & rzed., tabebuia impetiginosa (mart.) standl. and caesalpinia eriostachys benth., and tree density is about 2,686 ± 84 ind. ha-1 (maass et al., 2002a). the average number of trees sampled in each plot was 30 ± 7 ind. in each occasion, a new plot of 100 m2 was delimited and 50 plastic funnels with 50 cm of diameter were hung randomly in the shrub layer at 50 cm above floor forest at intervals of 50 cm. according to the number and area of the funnels, the biological material retained in each fogging comprises an area of 9.82m2, and in total were sampled 68.7m2 in the seven foggings. the average net primary productivity in the forest is 3.2 mg ha-1y-1 (martínez-yrízar et al., 1996). the application of insecticide was between 04:00 and 06:00, using a solution of 3% of resmethrin in kerosene solution. a total of 6l of solution was used in each application. after 5h of the insecticide application, the funnels were washed with 80% ethanol, in order to collect the specimens fell in them. the material was stored in plastic bottles with 1l capacity. the ant specimens obtained were isolated, quantified and identified as morphospecies. the specimens were deposited in colección de hormigas del ecología y sistemática de microartrópodos (lesm), facultad de ciencias, unam. only workers and soldiers were considered, because they are a better reference to sample canopy habitats (wilson, 1987). according to their feeding habit preferences, the species were classified as omnivorous, predators, herbivorous, granivorous and nectarivorous (considered the consumption of extrafloral nectaries and hemipteran secretions; byk & del-claro 2010, 2011). the diversity index per fumigation was estimated by shannon diversity index. species richness, pielou’s evenness, and simpson’s dominance indices were also calculated for the study (ludwing & reynolds, 1988). the effect of the season on the ant abundance was evaluated by a nested anova test, nesting month collection within the corresponding season and significant differences were tested by post hoc tukey’s test (zar, 1984). the spearman correlation between precipitation and temperature and the diversity indices were calculated, as well as the correlation between ant density and the precipitation and temperature. climatic data were obtained from the meteorological station of chamela (http://www.ibiologia.unam.mx/ebchamela/www/ clima.html). the analyses were performed using statistica version 9.0 software (statsoft, 2009). seasonal variations in the ant community composition were analyzed by ordination analysis through nonmetric dimensional scaling (nmds), and similarity between groups was tested by a similarity analysis (anosim), using 1000 permutations, following clarke (1993) and clarke and green (1988). analyses were performed using past software (hammer et al., 2001). sociobiology 61(1): 35-42 (march, 2014) 37 results a total of 5,563 specimens belonging to 46 morphospecies belonging to 17 genera of ants were collected during the seven fumigations (appendix 1). the average density of ants in the canopy was 81 ind/m2, while the species ant density was 26 species/m2. the myrmicinae subfamily was the most diverse, represented by 21 morphospecies grouped in nine genera. the most abundant genus in the sampling was crematogaster, with crematogaster crinosa mayr as the most abundant species, shown an average density of 26 ind/m2 in the canopy, representing the 32% of the total. this genus is considered predominantly arboriculous. the genus camponotus was the most rich in morphospecies number, with 13 species, followed by pseudomyrmex and cephalotes (five each one). all of them are considered predominantly arboricolous. the calculated diversity indices to the ant community in canopy show values relatively high, compared with other studies, and there are variations between fumigations (table 1). the highest diversity values were found during the rainy months (august, september), except july, where the diversity and species richness recorded were the lowest during the study. the dominant ants were different in each fumigation, as show by simpson’s dominance index (table 1; fig 1). the species better represented in the canopy along the study were c. crinosa, c. sumichrasti, tapinoma melanocephalum (fabricius) and forelius kieferi wheeler (fig 2), represent 77% of the total. the variation in the species richness, diversity and evenness show that there are high temporal variation in the ant canopy community in chamela. the nmds analysis shows the aggregation of september and july, nevertheless the other rainy month, august is located in the same quadrant with november, because both were the months with more species richness. there is an important note that during november, atypical rains fell in chamela, with a higher amount of precipitation than in rainy months of the same year and in other years (http://www.ibiologia.unam.mx/ebchamela/www/ clima.html). the two samplings from may are grouped, showing similar composition. the anosim test indicated that the observed community in the sampling months was significantly different (global r = 0.64). the date of collection is an important factor in the structure of the community of ants in the canopy of chamela. the nested anova test showed a significant effect of the season on the ant density in the canopy (f = 8.45; df = 5, 343; p<0.005), and post hoc tukey’s test showed differences between the fumigations performed in july and may in relation to the other months (p<0.05). a possible reason for which july showed a difference with the other rainy months is that it is the month of the begining of the rains, and the conditions can differ regarding august and september, in the middle of the rainy season. the ant density in the canopy was higher during the dry months’ fumigations (may and february). density average during rainy months it was 73 ind/m2, while in the dry months was 92 ind/m2. nevertheless there was not a significant correlation between density of ants and the precipitation recorded during the months of fumigation (r = -0.35, df=5; p>0.05), fig 1 temporal variation in canopy ant community composition in chamela, jalisco. numbers on the lines indicate the number of species included in others in each sampling. table 1. parameters of the ant canopy community in chamela, jalisco, mexico. s = species richness; h’= shannon diversity index; j´= pielou’s evenness; 1/d = simpson’s dominance index fumigation s h’ j´ 1/d august-1992 26 2.02 0.62 4.2 september1992 17 2.13 0.75 6.3 may-1993 17 1.68 0.59 3.4 july-1993 16 1.16 0.42 1.8 november-1993 29 1.58 0.47 2.8 february-1994 19 1.59 0.54 1.5 may-1994 22 1.34 0.43 2.5 total 46 1.99 0.52 4.8 fig 2 non-metric multidimensional scaling ordination (nmds) in two-dimensions of the canopy ant community inhabiting in a tropical dry forests in chamela, jalisco, mexico. ordination was based on bray-curtis dissimilarity index. stress = 17%. g castaño-meneses trophic structure of canopy ants from mexican deciduous forest 38 neither with the temperature (r =-0.17, df = 5; p >0.05; table 2). in relation to the trophic guilds of ants found in canopy of chamela, five guilds were recorded: omnivorous, predators, herbivorous, granivorous and nectarivorous. omnivorous was the dominant guild in the canopy, representing about 60% of the species founded, followed by granivorous and predators (fig 3). the trophic guilds distribution was different during the fumigations, and some guilds were found only during the fumigations performed in rainy months, as the case of the herbivorous. predators increased its abundance during the rainy months while omnivorous increased during the dry months (fig 3). that pattern produce differences in ant composition along the year, due the variations of feeding habits and the capability of the ant species to use different resources. discussion according to the results of palacios-vargas et al. (1999), ants represent the 0.5% of the total arthropods collected by fogging in chamela, and the pattern of abundance differs of the observed in other groups, as springtails, where the highest abundances were found during rainy season, while ants showed higher abundances in the dry season. the canopy of chamela showed a particular phenology, because leaves of tree species fall during dry season, but there are many tree species that are in flowering in this season (bullock & solís-magallanes, 1990; bullock, 2002), and that constitute important resources for many arthropods, including ants. furthermore, there is a high density and diversity of epipythes in the area (lott & atkinson, 2006). these epiphytes can be exploited by ants, and constitute an important refuge during the dry season, due to their capacity to accumulate detritus and water. in epiphytes and branches of trees in chamela there are important accumulation of organic matter, with amounts higher in the canopy than in the soil (maass et al., 2002b), and an important phase of the decomposition cycle is developed in the canopy, and many groups of invertebrates can live in that environment, such as collembola (palacios-vargas & gómez-anaya, 1993; palacios-vargas et al., 1998; palaciosvargas & castaño-meneses, 2003) that can be potential preys to some groups of ants as strumigenys and neivamyrmex (brown, 1959; bolton 1999). in the present study the most abundant genus in the canopy was crematogaster, an ant genus considered as arboricolous and is frequently found in high populations in rainy forest, different studies show that it represents more than 44% of the total collected arthropods (basset et al., 1992), and in thailand is the genus with the highest species richness in the canopy of dominant deciduous tree elateriospermum tapos blume (jantarit et al., 2009). the two species of the genus crematogaster found in chamela (c. crinosa and c. sumichrasti mayr) are probably not competitors, nevertheless both are considered as arboricolous, even the first though can be found in the shrub layer and soil in the forest (castañomeneses, 2008; castaño-meneses et al., 2009). this species has been recorded as very abundant in the upland forest in peru (wilson, 1987). genera as cephalotes, pseudomyrmex and camponotus were also abundant and species richness in canopy of chamela. dominance of that genera has been recorded in different tropical canopies around the world (wilson, 1987; guilbert & casevitz-weulersse, 1997; watanasit et al., 2005), thus the composition and dominance in tropical dry forest is similar to the found in tropical rainy forest. the presence and abundance of t. melanocephalus in canopy of chamela is remarkable, because this is considered as tramp ant species or invasive. in mexico, t. melanocephalus has been reported in canopies of rain forest in chiapas, associated to orchids (damon & pérez-soriano, 2005), as well in epiphytes from panama rain forest (stuntz et al., 2003). this species has one of the widest distribution ranges for any ant species, and its origin has been discussed (wilson & taylor, 1967), but, in general. consensus suggests t. melanocephalum origin in the old world tropics, and recent studies indicate that it is most probably originated in the indo-pacific (wetterer, 2009). nevertheless ,there are some evidence that support the hypothesis of the neotropical origin of t. melanocephalum (wetterer, 2009), and the presence of alated females in samples of canopy of chamela, as well the similar seasonal pattern of this species with the other abundant species considered as arboricolous, can be an evidence to support this hypothesis, or well suggest the success of this ant colonizing the forest. table 2. spearman correlation coefficient between the diversity index with precipitation and temperature monthly average (n = 7) in the canopy of chamela biological station, chamela, jalisco, mexico. h’= shannon diversity index, s = species richness, j’ = pielou’s evenness index, ns= no significant at α=0.05. index precipitation temperature h’ 0.23 ns 0.38 ns s -0.34 ns 0.23 ns j’ 0.27 ns 0.32 ns 0 200 400 600 800 1000 1200 1400 augt-92 sep-1992 may-93 july-93 nov-93 feb-94 may-94 a b u n d an ce crematogaster crinosa crematogaster sumichrasti tapinoma melanocephalum forelius kieferi cephalotes sp. 1 camponotus sp. 8 temnothorax sp. 5 others rainy season dry seasondry season rainy season 20 spp. 10 spp. 10 spp. 9 spp. 22 spp. 14 spp. 16 spp. fig 3 temporal variation of trophic guilds of ants from the canopy of chamela, jalisco, mexico. percentage of ant species from canopy of chamela included in each trophic guild. n=46. average percent of each guild: omnivorous: 60%; granivorous: 17%; herviborous: 2%; predators: 14%; nectarivorous: 7%. sociobiology 61(1): 35-42 (march, 2014) 39 studies developed in new caledonia using fogging, recorded 27 species and 14 genera of ants in the canopy (guilbert & casevitz-weulersse, 1997). in the tropical rain forest of peru, wilson (1987) recorded 135 species and 40 genera of ants, while in australia there is a great variation, with records of 37 species in the north region (majer, 1990), to 102 species in the south (andersen & yen, 1992). species richness recorded in the canopy of chamela shown values between rainy forest and temperate forest, according with different studied performed in that canopies vegetation, nevertheless different sampling methods has been used (wilson, 1959; schonberg et al., 2004; bos et al., 2007; jaffe et al., 2007). the genera composition recorded in all canopies is similar in different studies. the forest canopy can support high populations of organisms which had been recorded in tropical rain forest (longino & nadkarni, 1990; paoletti et al., 1991). it has been proposed that the arboricolous fauna estimation can give a good estimation of the total species in the world (erwin, 1983; ødegaard, 2000; longino et al., 2002). ants can exploit a great variety of resources due the diversity of their feeding habits. the distribution of trophic guilds was different during the fumigations. that produced differences in ant composition along the year, due the variations of feeding habits and the capability of the ant species to use different resources. the domination of omnivorous is frequent in many ecosystems, including the canopy, and in environments with limited resources (rojas 2001), due to the specialized feeding habits characteristic of environments with diversity of resources (lévieux, 1977). although no significant correlations were found in the study between abundance, species richness and diversity with precipitation and temperature, these results must be viwed with caution, because the data were collected in atypical climatic conditions recorded during the studied period, as the great precipitation amount in november. the results show that the ant community in the canopy of chamela is diverse and have important changes in functional composition along the time. the diversity of trophic guilds showed that the ecosystem present a high productivity supporting different trophic levels in communities of ants and other faunistic groups. acknowledgements this project was supported by in2078/91 dgapa-unam coordinated by dr. josé g. palacios-vargas (science faculty, unam). the field work was developed with the help of alfonso pescador rubio, josé antonio gómez anaya, alex cadena carrión and alicia rodríguez palafox (†). drs. josé g. palacios-vargas, betty benrey, alfonso pescador, zenón cano, francisco villalobos, norma garcía-calderón, victor rico-gray and patricia rojas gave invaluable suggestions on a first draft of manuscript. two anonymous reviewers and dr. jacques delabie gave invaluable suggestions to improve the manuscript. references andersen, a.n. & yen, a.y. 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(1984). biostatistical analysis. second edition. new jersey: prentice hall, 718 p. g castaño-meneses trophic structure of canopy ants from mexican deciduous forest 42 appendix 1 monthly and total abundance of ants collected by fogging at the canopy of biological station chamela. 1 = august 1992, 2 = september 1992, 3 = may 1993, 4 = july 1993, 5 = november 1993, 6= february 1994, 7 = may 1994. subfamily species 1 2 3 4 5 6 7 total amblyoponinae stigmatomma sp. 1 1 dolichoderinae forelius keferi wheeler, 1934 12 30 160 3 50 255 tapinoma melanocephalum (fabricius, 1793) 20 30 110 40 84 255 550 1071 ecitoninae neivamyrmex chamelensis watkins, 1986 5 4 1 10 formicinae brachymyrmex sp. 1 10 15 25 brachymyrmex sp. 2 1 1 camponotus sp. 1 7 3 13 23 camponotus sp. 2 1 2 3 camponotus sp. 4 14 10 2 10 20 4 6 64 camponotus sp. 5 3 2 5 camponotus sp. 6 2 3 4 9 camponotus sp. 8 28 10 6 2 10 14 70 camponotus sp. 9 1 2 3 camponotus sp. 10 4 4 camponotus sp. 12 10 2 12 camponotus sp. 13 2 2 camponotus sp. 14 20 10 13 10 53 camponotus sp. 15 13 1 14 camponotus sp. 16 3 3 myrmicinae acromyrmex sp. 1 1 2 carebara sp. 1 1 cephalotes sp. 1 15 5 9 8 30 39 1 107 cephalotes sp. 2 1 7 5 13 cephalotes sp. 3 14 14 cephalotes sp. 4 1 7 11 8 27 cephalotes sp. 5 1 1 cephalotes sp. 7 2 2 crematogaster crinosa mayr, 1862 236 60 280 383 326 200 258 1743 crematogaster sumichrasti mayr, 1870 189 70 60 20 730 481 10 1560 temnothorax sp. 2 4 7 5 16 temnothorax sp. 3 4 4 temnothorax sp. 4 13 1 7 12 5 38 temnothorax sp. 5 90 1 2 3 2 98 pheidole sp. 1 6 1 1 3 11 pheidole sp. 5 5 5 pheidole sp. 6 3 2 5 pheidole sp. 7 12 1 1 14 solenopsis geminata (fabricius, 1804) 22 30 22 30 49 153 strumigenys sp. 2 1 2 3 strumigenys sp. 3 14 30 43 ponerinae pachycondyla sp. 2 2 13 2 2 21 pseudomyrmecinae pseudomyrmex sp. 1 3 3 1 2 9 pseudomyrmex sp. 2 4 2 2 2 10 pseudomyrmex sp. 3 6 5 2 1 12 6 32 pseudomyrmex sp. 4 6 2 8 pseudomyrmex sp. 5 2 1 3 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.243-249sociobiology 61(3): 243-249 (september 2014) first-year nest growth in the leaf-cutting ants atta bisphaerica and atta sexdens rubropilosa srs cardoso1,2, lc forti1, ns nagamoto1, rs camargo1 introduction most ant species construct their nests underground, which promotes protection for the colony and aids in the maintenance of environmental conditions, facilitating the development of immature and adult individuals (hölldobler & wilson, 1990). however, nest construction requires intensive labor by the ants to excavate the soil (halley et al., 2005). in the genus atta f. 1805 (hymenoptera: formicidae) the adult nests are formed by numerous interconnected chambers, reaching several meters in depth, with deposition of soil on the surface, forming a pile of loose soil (gonçalves, 1960; araújo & della lucia, 1997). such chambers may present variations in format, localization and dimensions depending on the species and function (fungus, waste and abstract most ants, as leaf cutting ants, construct nests underground to maintain environmental conditions favorable to the development of immature and adult individuals. but there was little works about this, especially doing comparison of nest growth among leafcutting ant species. thus, we studied the growth of nests of the leaf-cutting ants atta bisphaerica and atta sexdens rubropilosa from nest foundation until the appearance of a second chamber. to this end, we verified the measurements of the chamber recently constructed by the queen and monitored its growth in the initial phase of nest development. the nests were marked immediately after nuptial flight, with 40 nests of each species being dug at 45, 90, 135, 180, and 225 days afterwards. as a result, it was found a first time statistical demonstration that an ellipsoid chamber shape was verified in both species in the initial chamber at 45 days, and that, after, these chamber became spherical. in general, chamber size increased and format change was found in both species. the depth of the first chamber was found to increase significantly only in a. bisphaerica; this result means that this chamber growths downside rather than upside. the occurrence of a second chamber was verified from six months after nest foundation, in both species. our study contributes to knowledge of the colony development for up to 1 year, by performing comparison of two leaf-cutting ants species. sociobiology an international journal on social insects 1 universidade estadual paulista (unesp), botucatu, são paulo, brazil 2 instituto federal do tocantins, araguatins, tocantins, brazil article history edited by evandro n. silva, uefs, brazil received 15 july 2013 initial acceptance 09 august 2013 final acceptance 08 july 2014 keywords initial chamber, leaf-cutting ant, nest architecture, nest foundation, excavation corresponding author nilson satoru nagamoto depto. proteção vegetal, fca, unesp po box 237, botucatu, sp, brazil 18.610-307 tel. +55 14 38117167 fax: +55 14 38117206e e-mail: nsnagamoto@yahoo.com soil) (moreira, 2001). despite such knowledge, little is known about the initial growth of nests formed by this genus. in most social insects the size of the nest is a function of its population; however the dynamic of construction and enlargement of underground nests is not fully known (rasse & deneubourg, 2001). in atta, the work of nest foundation is performed by a single recently fecundated queen that constructs the first chamber (autuori, 1942; ribeiro, 1995; camargo et al., 2011; fröhle & roces, 2012). after the first worker ants initiate foraging, they assume activities within and outside of the nest, including the excavation of new tunnels and chambers (autuori, 1942; amante, 1972); so, the nest enlargement is thus the responsibility of adult offspring. furthermore, the initial chamber is also enlarged each day by the excavation effort of the worker ants (jacoby, 1943). some research article ants srs cardoso et al. nest growth in the leaf-cutting ants244 studies indicate that for several species of ants the nest size is adjusted to the population (rasse & deneubourg, 2001; buhl et al., 2004; mikheyev & tschinkel, 2004; camargo & forti, 2014; römer & roces, 2014). although the genus atta presents the ability to construct deep nests, the initial chamber is based in the topsoil layer at a depth of about 8.5 to 15 cm (autuori, 1941), and is subject not only to variations in temperature and humidity, but also to the influence of strong and abundant rains that are typical of the period in which nest foundation occurs (autuori, 1941; bento et al., 1991). therefore, one of the most important aspects in the study of atta is undoubtedly the knowledge of its initial development, which can be externally monitored through measurements of the nest openings and the soil mound (mariconi, 1970) and internally by means of excavations, to ascertain, for example, the depth and size of the first chambers constructed. although there have already been reports on the chamber format and initial chamber depth of atta (autuori, 1942; jacoby, 1943, 1950; mariconi, 1970), the literature on the foundation and initial development of atta nests remains highly limited. due to the scarcity of biological information on nests of atta species in their first year of life, the present paper studied the growth and morphology of nests in two species which exhibit contrasting adaptations: atta bisphaerica forel 1908, which nests in full sun and foraging predominantly grasses, and atta sexdens rubropilosa forel 1908, which nests in shaded places and forages dicots (mariconi, 1970; fowler et al., 1986; nagamoto et al., 2009). material and methods the work was conducted in the city of botucatu, sao paulo state, brazil with the a. bisphaerica nests studied in a pasture area of santana farm (22o50.720’ s and 48o26.155’ w), and the a. sexdens rubropilosa nests being selected in an area of eucalyptus plantations belonging to fca/unesp, botucatu, sp (22o50.833’ s and 48o26.476’ w). both areas present the oxisol soil type (dark red latosol). in each area 500 nests were randomly selected at the moment of nest foundation by the queen in october 2007. the nests were marked by labeled wire stakes. the excavations were performed with using digging tools (such as shovels, picks and spatulas) by opening a trench at the side of the nest, carefully exposing the channel and chamber as described by autuori (1942), pereira-dasilva (1979) and pretto (1996), and then the biomass (fungus garden, ants and brood) was discarded. forty nests of each species were excavated at 45, 90, 135, 180 and 225 days after nest foundation (a total of 400 nests). the measurements, including depth, length, width, and height were obtained for each excavated nest (figure 1). format of the initial chamber based on the geometric aspect, some authors have defined the format of the chamber for the cultivation of fungi, as typically spherical or ellipsoid (jacoby, 1950; silva junior et al., 2013). here we purpose, for the first time (to our best knowledge), to classify the leaf cutting ant chamber statistically, taking into account that the ellipse and sphere posses well known coordinates: if the cartesian coordinates x, y and z are equal, it is a sphere; if there are differences, is ellipsoid. for this, we compared length (l), width (w) and height (h), at each excavation period, using analysis of variance (anova) (α=0.05). volume of the chamber the volume of the chamber was monitored through comparison of each chamber measurements (l, w, h) among the periods studied: volume = 3/4πlwh (ellipsoid volume). for this, only the measurements of nests with alive queen were taken into account. the volume data among periods among a species, and in each period between these species, were submitted to analysis of variance (anova) (α=0.05), figure 1. the measurements of depth (d), length (l), width (w) and height (h) at a nest of atta bisphaerica, that was cement-moulded (as in moreira et al., 2004) to enable a better view. sociobiology 61(3): 243-249 (september 2014) 245 with the means compared by bonferroni’s test. to evaluate the difference in the depth of the chamber between the two species, their measurements in each period were compared and the data obtained were submitted to student’s t test (α=0.05). results format of the initial chamber the initial chamber presented two distinct formats: first ellipsoid and spherical in the last periods of evaluation. a. bisphaerica showed an ellipsoid format until 90 days after foundation, with the dimensions of chamber differing significantly in 45 days (p<0.001) and 90 days (p=0.006) (table 1). from 135 to 225 days after the foundation, the measurements did not differ from each other, indicating a spherical format. in a. sexdens rubropilosa, it was verified a difference among the dimensions of chamber in 45 days (p<0.001). the change in format occurred after 90 days because ceased to exist significant differences between length, width and height (p>0.050) (table 1). volume the size of the initial chamber was growing significantly in both species (fig. 2). in general, the mean measurements of the initial chamber were greater for a. bisphaerica than for a. sexdens rubropilosa, with volume differing significantly in many periods evaluated (table 2). at 45, 135 and 225 days after foundation the volume was greater in a. bisphaerica than a. sexdens rubropilosa, significantly differing, p<0.001, p=0.014 and p=0.035, respectively (table 2). depth the depth of the initial chamber was growing significantly for a. bisphaerica (fig. 3), but not in a. sexdens rubropilosa. in general, chamber depth significantly differed between the ant species (table 3), with a. bisphaerica showing deeper chambers (fig. 3). second chamber from 180 days, a new tunnel emanating down from the first chamber, it was always detected. the occurrence of a second chamber was verified from six months after nest foundation, in both species. for a. bisphaerica the mean measurements of the second chamber were: depth of 104.3 + 35.0 cm, vol. of 29.4 + 28.7 cm3. for a. sexdens rubropilosa, a second chamber occurred in only two nests, with the first day atta bisphaerica atta sexdens rubropilosa p value f df p value f df 45 <0.001* 40.363 98 <0.001* 15.338 80 90 0.006* 5.523 65 0.060ns 3.085 32 135 0.137ns 2.040 83 0.226ns 1.596 23 180 0.120ns 2.263 35 0.830ns 0.189 14 225 0.631ns 0.470 26 0.366ns 1.127 11 table 1. summary statistic for values of anova of the initial fungus chambers measurements (length, width and height), classification of the leaf-cutting ant initial chamber, statistically, into ellipsoid or spherical: if the cartesian coordinates x, y and z are not significantly different, it is spherical; if there are significant differences, is ellipsoid. comparisons in each excavation period (day) for each species. *significant (= ellipsoid chambers); nsnon significant (= spherical chambers) day atta bisphaerica vs atta sexdens rupropilosa p value t df 45 <0,001* 5,372 58 90 0,002* 3,463 31 135 0,009* 2,751 34 180 <0,001* 4,831 15 225 0,062 ns 2,081 11 table 3. students’s t test summary statistic for values of the initial fungus chamber depth between leaf-cutting ant species. day atta bisphaerica vs atta sexdens rupropilosa p value t df 45 <0,001* 4,249 58 90 0,133 ns 1,541 31 135 0,014* 2,601 34 180 0,589 ns -0,552 15 225 0,035* 2,411 11 table 2. students’s t test summary statistic for values of the initial fungus chamber volume between leaf-cutting ant species. *significant difference in t test; ns non significant difference in t test. *significant difference in t test; ns non significant difference in t test. in a. bisphaerica, at 45 days, the dimensions are (cm + sd): the length (4.28 + 0.65) differed from width (3.04 + 0.37) and height (3.33 + 0.68). for day 90, the length (5.14 + 1.21) differed only from width (3.95 + 0.93). later, the highest growth in width and height in relation to height, resulted in no significant differences between these measurements (p>0.050). in a. sexdens rubropilosa, for day 45, the length (3.66 + 0.57) differed from width (2.89 + 0.80) and height (2.76 + 0.52). then, for both species, the width and height was the chamber measurements that grew most from 45 to 225 days. srs cardoso et al. nest growth in the leaf-cutting ants246 presenting the following measurements: depth of 39 cm, and vol. of 73.8 cm3. in the second nest, the depth was found to be less than 150 cm (an accurate measurement was not made due to some excavation difficulties). discussion format of the initial chamber in the present study the occurrence of two shapes, ellipsoid and spherical, was observed in both studied species. the chamber founded by the queen initially presented an ellipsoid format, with changing to a spherical shape proceeded by the excavation by the worker ants. these workers excavated the chamber in all dimensions, but more in width and height than in depth. the queen body is longer than wider or higher (moser et al., 2004), so the chamber may also be optimized in this format to just accommodate it and to avoid unnecessary lost energy in digging process by the queen (camargo et al., 2011). later, it turns into spherical format, which would be more appropriate (optimized) for growing the fungus by the workers (römer & roces, 2014). day 45 90 135 180 225 d ep th ( cm ) 0 5 10 15 20 25 30 a a ab bc bc c day 45 90 135 180 225 d ep ht ( cm ) 0 5 10 15 20 25 b a a a a a day 45 90 135 180 225 v ol um e (c m 3) 0 50 100 150 200 250 300 ab b ab b b b day 45 90 135 180 225 v ol um e (c m 3) 0 50 100 150 200 bc c a b b a fig 2. mean volumes of the initial chamber of atta bisphaerica (a) and atta sexdens rubropilosa (b) as a function of time since foundation. means followed by the same letter are not significantly different (bonferroni test). fig 3. mean chamber depths of the initial chamber of atta bisphaerica (a) and atta sexdens rubropilosa (b) as a function of time since foundation. means followed by the same letter are not significantly different (bonferroni test). sociobiology 61(3): 243-249 (september 2014) 247 growth in both species, the size of the initial chamber increased as a function of time until digging of the second chamber started, approximately 180 days after nest foundation. the enlargement of the chamber probably occurred because of colony population growth, which can explain why a. sexdens rubropilosa chambers grew less: although not quantified, a. sexdens rubropilosa had fewer ants and smaller symbiotic fungus amounts in each period studied compared to a. bisphaerica. these results corroborate buhl et al. (2004), who verified that the digging activity and volume increase in the initial chamber in messor sanctar emery were adjusted to colony population size. rasse and deneubourg (2001) also verified that both the volume and the maximum rate of excavation are related to the size of the group, and that the digging activity diminished when a lasius niger l. nest reaches a particular volume. similarly, mikheyev and tschinkel (2004) confirmed that total nest volume is highly correlated with the number of worker ants in formica pallidefulva wheeler. in leaf cutting ants, it is known that the fungus garden acts as a three-dimensional pattern for the final size of the chamber, that is, according to its growth, the ants increase the size of the chambers, as discussed by fröhle and roces (2009), camargo et al. (2013) and camargo and forti (2014), for acromyrmex lundi and a. sexdens rubropilosa, respectively. depth the initial chamber depth significantly differed between the ant species, with a. bisphaerica showing deeper chambers than a. sexdens rubropilosa. it is known that atta bisphaerica forel 1908, presents nests in full sun and foraging predominantly grasses, and atta sexdens rubropilosa forel 1908 presents nests in shaded places and forages dicots (mariconi, 1970; fowler et al., 1986; nagamoto et al., 2009), therefore differing in nesting habits and foraging strategies. probably, the differences in nest depth between species are correlated to soil temperature, because shading alters soil temperature regimes by locally diminishing soil temperature (rosenberg et al., 1983). in this perspective, we can hypothesize that nest exposed in grassland should have deeper fungus chamber than nest under shade of trees or inside the woods, given that soil temperature is negatively correlated with soil depth (rosenberg et al., 1983). for leaf cutting ants, is indicated that soil moisture and temperature acts together: (i) bollazzi et al. (2008) verified that workers’ thermopreferences lead to the construction of superficial nests in cold soils, and subterranean ones in hot soils; and (ii) pielström & roces (2014) verified that soil moisture also varies according to soil depth, and demonstrably affects the digging behavior of leaf cutting ants. second chamber the excavation of the second chamber by the worker ants occurred 6 months after the nest foundation in both species. this result differs from mariconi (1970) who describes a 15-month period since foundation for digging the new channel and second chamber of atta capiguara. however, the results approximate those of jacoby (1943) who verified in a. sexdens rubropilosa the construction of a second chamber at 4 and 5 months after nest foundation. at 180 days of age, despite the presence of a second chamber, symbiotic fungus, workers and queens were still found in the first chamber. for this reason, it is suggested that in this period the new chamber had not been completely constructed, since according to jacoby (1943), the fungus is established only in finalized chambers. in addition, camargo and forti (2013) argued that the structural growth of the nests occurred vertically, without lateral expansion of tunnels and chambers in the first year. the 3-month-old nests had a chamber with an average depth of 15 cm. in the course of 1 year, the nests expanded to have three to four chambers and were 3–4 m deep. the lateral expansion occurred after 1 year, when the nests grew laterally and reached large dimensions when adult ants were 3 years old. for example, adult nests of a. laevigata are 7 m deep, with a number of chambers ranging from 1149 to 7864 and a volume from 0.03 to 51 liters (moreira et al., 2004). conclusion it may be suggested from the present study that the worker ants actively dig the initial chamber, causing an increase in its size and influencing its depth to favor the chances of establishment of the colony. although the adult nests of a. sexdens rubropilosa present different depth, chamber number and nest structure from a. bisphaerica, the initial nests are similar in shape as well as the time for construction of the second chamber. however, the initial chambers of a. bisphaerica present greater size and depth than those of a. sexdens rubropilosa. besides, the chamber depth of a. bisphaerica is deeper than a. sexdens rubropilosa. acknowledgments we wish to thank fapema for the fellowship granted to the first author and fapesp for financial support (2008/07032-7). l.c. forti gratefully acknowledges the support of cnpq (472671/2008-1). references amante, e. 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(2014). soil moisture and excavation behaviour in the chaco leaf-cutting ant (atta vollenweideri): digging performance and prevention of water inflow into the nest. plos one 9(4): e95658. doi:10.1371/journal. pone.0095658 pretto, d.r. (1996). arquitetura dos túneis de forrageamento e do ninho de atta sexdens rubropilosa forel, 1908 (hymenoptera formicidae), dispersão de substrato e dinâmica do inseticida na colônia. m.sc. dissertation, faculdade de ciências agronômicas, unesp. botucatu, sp, brazil. rasse, p.h. & deneubourg, j.l. (2001). dynamics of nest excavation and nest size regulation of lasius niger (hymenoptera: formicidae). journal of insect behavior, 14 (4): 433-449. doi: 10.1023/a:1011163804217 ribeiro, f.j.l. (1995). a escavação do solo pela fêmea da saúva (atta sexdens rubropilosa). psicologia-usp, 6: 75-93. römer, d. & roces, f. (2014). nest enlargement in leafcutting ants: relocated brood and fungus trigger the excavation of new chambers. plos one, 9 (5): e97872. doi:10.1371/ journal.pone.0097872 rosenberg, n.j., blad, b.l., & verma, s.b. (1983). microclimate-the biological environment. new york: wiley. silva junior, m.r., castellani, m.a., moreira, a.a., d’esquivel, m., forti, l.c. & lacau, s. (2013). spatial distribution and architecture of acromyrmex landolti forel (hymenoptera, formicidae) nests in pastures of southwestern bahia, brazil. sociobiology, 60: 20-29. doi: 10.13102/sociobiology.v64i1.1206sociobiology 64(1): 122-124 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 first data on the host ant usage of large blue from the carpathian basin maculinea van eecke (1915) (lepidoptera: lycaenidae) species are endangered and protected in europe (gimenez dixon, 1996; munguira & martín, 1999; settele et al., 2005). they have an extraordinary life cycle, where larvae start their development on specific host plants. after feeding on the developing seeds of these plants, the fourth instar caterpillars then complete their development in myrmica latreille (1804) (hymenoptera: formicidae) ant nests (thomas et al., 1989). different maculinea populations are adopted to different myrmica species (als et al., 2002; tartally, 2008), which means that their protection can only be successfully managed with knowledge of the local host ant species (munguira & martín, 1999; settele et al., 2005). furthermore, such knowledge is interesting both biogeographically and evolutionarily (nash et al., 2008). the large blue butterfly, maculinea arion (linnaeus, 1758), is a well-studied maculinea species, especially from its conservation aspect (thomas, 1995). however, our knowledge abstract the protected maculinea arion is an obligate myrmecophilous butterfly (lepidoptera, lycaenidae). fourth instar larvae and pupae develop in myrmica (hymenoptera: formicidae) ant nests. host ant specificity varies geographically, and knowledge of the local host ant species is important to understand the biogeography and evolution of this species, and vital for its conservation. here we report the first data on the host ant usage of m. arion in the carpathian basin, one prepupal caterpillar from a myrmica specioides and one pupa from a m. scabrinodis nest. myrmica specioides is a new host ant species of m. arion. it is important to collect further data on the host ant usage of m. arion, despite the difficulties of data collection. sociobiology an international journal on social insects a tartally1, jp tóth2, a váradi1, j bereczki1,2 article history edited by jean c. santos, ufu, brazil received 10 october 2016 initial acceptance 07 december 2016 final acceptance 16 january 2017 publication date 29 may 2017 keywords social parasitism, maculinea arion, phengaris, myrmica specioides, myrmica scabrinodis, hungary. corresponding author andrás tartally department of evolutionary zoology and human biology university of debrecen, egyetem tér 1, h-4032 debrecen, hungary e-mail: tartally.andras@science.unideb.hu about its host ant specificity is lower than is the case for the other european species (settele et al., 2005; hayes, 2015), because it is very difficult to find m. arion caterpillars in myrmica nests (sielezniew et al., 2010a). hence, every observation of its host ant usage is important, and especially from the areas where m. arion exists in two phenological forms (’spring’ and ‘summer’ types, based on their flight periods, see bereczki et al. 2011, 2014, 2015 for more details). the carpathian basin is such a region, where data have not been available about the host ant specificity of m. arion. to get such data, myrmica nests were carefully opened and the presence of m. arion caterpillars was checked at 12 m. arion sites (table 1 of supplementary file) since the spring of 2000. after excavation the ground and vegetation were restored as close to the original conditions as possible. only myrmica nests found ~2 m within around the initial host plants (thymus spp. and origanum vulgare: munguira 1 department of evolutionary zoology and human biology, university of debrecen, hungary 2 mta-de “lendület” behavioural ecology research group, dept. of evolutionary zoology and human biology, university of debrecen, hungary short note sociobiology 64(1): 122-124 (march, 2017) 123 & martín, 1999) of the butterfly were checked, as this is the approximate foraging range of myrmica workers (elmes et al., 1998). excavations were carried out just before, or at the beginning of, the usual flying periods of the different populations. search periods earlier in the life cycle are less suitable because ant colonies adopting young fourth-instar caterpillars may later kill them (typically around winter because of starving: elmes et al., 2004). five to ten workers were collected from each myrmica nest and preserved in ethanol for identification in the laboratory (according to: seifert, 1988; radchenko & elmes, 2010). altogether 289 nests of nine myrmica species [m. lobicornis nylander, 1846; m. lonae finzi, 1926; m. rubra (linnaeus, 1758); m. ruginodis nylander, 1846; m. sabuleti meinert, 1861; m. scabrinodis nylander, 1846; m. schencki viereck, 1903; m. specioides bondroit, 1918; m. vandeli bondroit, 1920] were opened and only two (i.e. less than 1%) were infected with m. arion, at two different sites. one spring arion pupa was recorded in a m. scabrinodis nest, and a summer arion prepupa with myrmica specioides (see table 1 and fig 1 of supplementary file). the identification of phenological forms was carried out based on collection date (see table 1 of supplementary file for details). the specific identification of these pre-adult stages was confirmed using the coi genetic barcoding gene (see table 1 of supplementary file for the accession numbers). myrmica scabrinodis has already been recorded as a host of m. arion from western europe, although just in a few cases (thomas et al., 1989; elmes et al., 1998). however, as far as we know, this is the first record of m. specioides as a host ant of m. arion. the known host ant species of m. arion are: (i) myrmica sulcinodis nylander (1846) and m. lonae from italy (sielezniew et al., 2010b; casacci et al., 2011); (ii) m. hellenica finzi (1926); m. lobicornis; m. lonae; m. rugulosa nylander (1849); m. sabuleti; m. scabrinodis and m. schencki from poland (sielezniew & stankiewicz, 2008; sielezniew et al., 2010b; sielezniew et al., 2010c; sielezniew et al., 2010a) (iii); m. scabrinodis and a m. specioides from the carpathian basin (this paper); and (iv) m. sabuleti, as the well-proved main host, from western europe (england, france and sweden: thomas et al., 1989; elmes et al., 1998; nielsen, 2012) where m. arion is also recorded from m. scabrinodis and m. lonae nests, but only in a few cases. according to these observations, it seems that m. arion shows less host ant specificity in central than in western european regions (similarly to the other european maculinea species: tartally, 2008). however, this does not exclude local adaptations to some host ant species in central europe, such as the m. schencki using polish populations (sielezniew et al., 2010c). this phenomenon could be explained by the geographic mosaic of coevolution between the butterflies and their host ants (nash et al., 2008). because m. arion can sometimes be found with myrmica species, which are not suitable to maintain populations (thomas, 1980), we should be cautious not to place too much emphasis on single host ant records. on the other hand, it is very important to publish all host ant records, including single observations, because the greater the available data about host ant usage, the greater is our understanding of the biology and conservation potential of this endangered butterfly. at the same time, it is important to emphasize that finding myrmica nests infected with m. arion is extremely difficult, and requires dedicated and systematic surveys (sielezniew et al., 2010a). therefore, building up detailed knowledge about the host ant usage of this butterfly across its range would need a much more intensive research involving numerous competent people. acknowledgements to: dr nash and two anonymous reviewers (revising), wp pfliegler (supplementary file: fig 1b), “macman” rtd project (evk2-ct-2001-00126), hungarian scientific research fund (otka k109223 and k84071), ‘indifferant’ marie curie intra european fellowship (at), ‘antlab’ marie curie career integration grant (at), jános bolyai scholarship of the hungarian academy of sciences (at and jb). supplementary material http://dx.doi.org/10.13102/sociobiology.v64i1.1206.s1572 http://periodicos.uefs.br/index.php/sociobiology/rt/suppfilemetadata/1206/0/1572 references als, t.d., nash, d.r. & boomsma, j.j. 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(1995). the ecology and conservation of maculinea arion and other european species of large blue butterfly. in a.s. pullin (ed), ecology and conservation of butterflies (pp. 180-197). london: chapman & hall thomas, j.a., elmes, g.w., wardlaw, j.c. & woyciechowski, m. (1989). host specificity among maculinea butterflies in myrmica ant nests. oecologia, 79: 452-457. doi: 10.1007/ bf00378660. tóth, j.p., bereczki, j. & varga, z. (2014). a nagypettyes hangyaboglárka (maculinea arion) és a magyar tarkalepke (melitaea ornata kovacsi) (lepidoptera) az aggteleki nemzeti park területén. [large blue (maculinea arion) and eastern knapweed fritillary (melitaea ornata kovacsi) in aggtelek national. in v. tóth (ed.), kutatások az aggteleki nemzeti parkban ii. [researches in aggtelek national park and biosphere reserve ii.] anp füzete (pp. 119-132). jósvafő: aggteleki nemzeti park igazgatóság. varga, z. (2010). magyarország nagylepkéi [macrolepidoptera of hungary]. budapest: heterocera press, 124 pp open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.6030sociobiology 68(1): e-6030 (march, 2021) introduction foraging requires appropriate decisions related to the time spent in exploration and choosing the appropriate time to maximize net amount of energy gained, foraging efficiency, and survival, or to minimize the risk of starvation (emlen, 1966; macarthur & pianka, 1966; ydenberg, 2007). these decisions are subject to constraints that limit viable alternatives, such as time spent and the energy cost of the activity (emlen, 1966; macarthur & pianka, 1966; perry & pianka, 1997; ydenberg, 2007). decisions may be related abstract dinoponera lucida is a poneromorph ant endemic to the atlantic forest of brazil. the species is classified as endangered in brazil’s red list due to its peculiar reproductive biology and high habitat fragmentation. herein, we characterize d. lucida foraging activity and response to litter surface temperature in a lowland forest remnant in south-eastern brazil. the mean flow of workers at nest openings was 3.8 ± 0.6 per hour, mean foraging trip was 14.2 ± 2.2 min, and mean foraging distance was 3.8 ± 0.4 m. the time spent per foraging trip and litter surface temperature were positively correlated. flow of workers at nest openings was higher with mean temperature of litter surface between 21.0 and 27.0 °c. our results show that d. lucida has a diurnal foraging activity related to habitat temperature. our data contribute to the knowledge about the ecology of d. lucida and support the hypothesis of optimal food foraging regulated by habitat temperature. in addition, the better understanding of d. lucida activity patterns can assist on conservation planning of this endangered and endemic ant. sociobiology an international journal on social insects flávio curbani1,2,3, cássio zocca1,4,5, rodrigo b ferreira4,5,6, cecilia waichert1,5,6, tathiana guerra sobrinho7, ana carolina srbek-araujo1,8,9 article history edited by evandro n. silva, uefs, brazil received 24 november 2020 initial acceptance 18 january 2021 final acceptance 05 february 2021 publication date 17 march 2021 keywords optimal foraging, poneromorph ants, litter surface, microclimate, diet. corresponding author flávio curbani universidade federal do espírito santo centro tecnológico departamento de tecnologia industrial av. fernando ferrari, 514, goiabeiras cep 29.060-970, vitória-es, brasil. e-mail: flavio.curbani@ufes.br to the time spent searching for food, affecting foraging efficiency, when the increase in time spent does not result in an increase in the amount of food obtained (macarthur & pianka, 1966; norberg, 1977; pyke, 1984; perry & pianka, 1997). the trade-off between costs and benefits does not depend only on the forager’s intrinsic factors; external factors (e.g. environmental conditions and prey density) can alter the foraging activity dynamics (norberg, 1977; pyke, 1984). the eight species of dinoponera roger, 1861 (hymenoptera: formicidae: ponerinae) are endemics to south america (kempf, 1971; lenhart et al., 2013), and 1 programa de pós-graduação em ecologia de ecossistemas, universidade vila velha, vila velha-es, brazil 2 departamento de tecnologia industrial, universidade federal do espírito santo, vitória-es, brazil 3 projeto caiman, instituto marcos daniel, vitória-es, brazil 4 instituto nacional da mata atlântica, santa teresa-es, brazil 5 projeto bromeligenous, instituto marcos daniel, vitória-es, brazil 6 programa de pós-graduação em biologia animal, universidade federal do espírito santo, vitória-es, brazil 7 departamento de ciências agrárias e biológicas, universidade federal do espírito santo, são mateus-es, brazil 8 programa de pós-graduação em ciência animal, universidade vila velha, vila velha-es, brazil 9 instituto serradical de pesquisa e conservação, belo horizonte-mg, brazil research article ants litter surface temperature: a driving factor affecting foraging activity in dinoponera lucida (hymenoptera: formicidae) mailto:flavio.curbani@ufes.br f curbani, c zocca, rb ferreira, c waichert, tg sobrinho, ac srbek-araujo – temperature and foraging activity of dinoponera lucida2 called giant ants because reach up to 40 mm (kempf, 1971; lenhart et al., 2013; escárraga et al., 2017). among these species, dinoponera lucida emery, 1901 is endemic to brazil’s atlantic forest from the states of espírito santo, eastern minas gerais and southern bahia (peixoto et al., 2010; simon et al., 2020). currently, only 12% of the area originally covered by atlantic forest has forest remnants, mostly small and isolated across the human-altered matrix (fundação sos mata atlântica & instituto nacional de pesquisas espaciais, 2019). dinoponera lucida is a forest-specialist and currently recorded only in forest remnants. the area of geographic range of the d. lucida was estimated at 156 km2 (instituto chico mendes de conservação da biodiversidade & ministério do meio ambiente, 2018). habitat fragmentation causes isolation of populations, since the species is unable to colonize over long distances, leading to a clumped distribution pattern over the geographic range (instituto chico mendes de conservação da biodiversidade & ministério do meio ambiente, 2018). new colonies of d. lucida are generated by fission of extant colonies, increasing inbreeding and reducing genetic diversity, which has led to local extinctions (mariano et al., 2008; campiolo et al., 2015), a genetically fragmented distribution (mariano et al., 2008; resende et al., 2010; simon et al., 2016), and endangered (en) species status on the brazilian red list (ministério do meio ambiente, 2014; instituto chico mendes de conservação da biodiversidade & ministério do meio ambiente, 2018). thereby d. lucida conservation is intrinsically linked to the preservation and recovery of the atlantic forest and the connection between its forest remnants. how microclimate affects the ecology of d. lucida remains poorly understood. for instance, d. lucida responds to regional temperature and rainfall pattern (simon et al., 2020); populations settle mainly in areas with higher annual mean temperatures, lower annual temperature range and less precipitation during the driest month (simon et al., 2020). microclimatic variables such as temperature, rainfall, and humidity are known to influence the realized niche of insects, and can do so by influencing behavior and foraging strategy (willmer, 1982; colinet et al., 2015; pincebourde & suppo, 2016; simon et al., 2020; welch et al., 2020). dinoponera lucida increase foraging activity in mild temperature associated with high elevations (peixoto et al., 2010). the pattern of foraging activity of d. lucida has an optimal temperature range in areas surrounding the nest (peixoto et al., 2010). inside the forest, microclimatic variables are influenced by canopy cover, edge effects, evapotranspiration and other factors related to vegetation and habitat heterogeneity (magnago et al., 2015; stangler et al., 2015; boehnke et al., 2017). thus, an appropriate approach to assess the foraging activity of d. lucida should consider microclimate conditions. dinoponera lucida commonly prey upon epigeic ectotherm taxa, and these prey respond to temperature, which alters its density and availability (may, 1979; colinet et al., 2015; pincebourde & suppo, 2016; welch et al., 2020). we expect that d. lucida will adapt foraging time to maintain an optimal strategy (norberg, 1977; bernstein, 1979; azevedo et al., 2014; medeiros et al., 2014). herein, we characterized the foraging activity and the response to litter surface temperature by d. lucida. we expected that daily variation in air and litter surface temperatures to influence the foraging activity of d. lucida. also, we expected more workers to forage during the optimal temperature range, due to physiological constraints and prey availability. materials and methods study site we carried out this study in the vale natural reserve (vnr; 22,711 ha; 19º 09’ 05” s, 40º 04’ 15” w), linhares, northern espírito santo state, south-eastern brazil. vnr has flat relief and altitude between 28 to 65 m (peixoto et al., 1995). vnr is an important remnant of the tabuleiro forest, which is a specific formation of the atlantic forest (kierulff et al., 2014). vnr region has tropical and humid climate with two dry months (alvares et al., 2014). the annual mean temperature is 24.3 °c and the annual mean rainfall is 1,214 mm (kierulff et al., 2014). sampling we conducted active visual survey in a 30 m × 30 m plot to locate d. lucida workers (19º 09’ 14.5” s, 40º 04’ 14.0” w) from september 29 to october 2, 2017. we used sardines as bait along the worker’s route (silvestre et al., 2015). we located three nests by following workers carrying the bait back to the nests. we estimated height, width of the nest openings and canopy coverage over the nest openings with the aid of photos analyzed at imagej 1.52p software (schindelin et al., 2012; schneider et al., 2012). we neither disturbed the workers nor excavated the nests. in the center of the sampling area, we measured air temperature (ta) and relative humidity (rh) at 1.5 m high using a kestrel portable weather device (model 3000); and litter surface temperature (ts) using a tekpower digital infrared thermometer (model dt-8380). ta, rh and ts were recorded every 30 min from 6 h to 18 h, which is the expected foraging period of d. lucida (peixoto et al., 2010). it rained from 6 h to 9 h on the third day of sampling. flow of workers ceased during the rain. the data affected by rain were not considered for the quantitative analyzes. we observed foraging workers exiting the nests and searching for food in the surrounding litter. we recorded inflow and outflow of workers for 15 min in each nest per hour (peixoto et al., 2010). we considered inflow (fin) as the number of workers entering the nest and outflow (fout) as the number of workers exiting nests to forage. we considered sociobiology 68(1): e-6030 (march, 2021) 3 total flow (ft) in the nest openings as the sum of flows of workers. total flow is colinear with fin and fout. using the 15 min measured data, we estimated ft per hour in each nest. we used focal-animal sampling (altmann, 1974) and followed the workers individually during foraging activities from the time workers exited until they returned to the nest. we remained at least 2 m away from the observed worker to avoid interference of behavior. we recorded duration of foraging trip (tf) as the time spent searching for food items from their exit to their return to the nests. we used tf as a proxy to estimate foraging efficiency (η ~ tf -1). we considered a returning worker those that were observed collecting a food item (successful trip) or those that started moving toward the nest even without food item (unsuccessful trip). the efficiency rate of foraging was calculated as number of workers returning to the nest with food divided by total number of workers returning to the nest (giannotti & machado, 1991; medeiros et al., 2014). we defined reached distance (df) as the maximum distance reached by a worker during a foraging trip, measured as a straight line from the nest opening to the returning point. we identified collected food items visually or from zoomed photos to the lowest possible taxonomic level. data analysis first, we perform pearson correlation analyzes between environment variables (ts, ta, and rh), tf ts, and tf df. because ta is positively correlated with ts, we used ts for description of the behavioral patterns studied, since ts represents the microclimate variable related to the microhabitat of d. lucida workers. we established the curves with the variation of the foraging activity (ft) as function of ts and time of day. a fourth-order polynomial fit was used in the nonlinear regression model to describe the bimodal pattern between ft and time of day. we tested if there was a relationship between ft and ts applying a pearson’s chisquared test (χ2) from a six-categories of ts (from 19 to 31 ºc, with regular intervals of 2 ºc, as showed in fig 2b) defining a contingency table. the relative frequencies of ft (rf) were calculated for each hour of the day and each ts category as a proportion of ft in each hour sampled or ts range category divided by the total ft of all hours or all ts categories, as described by equation 1: to calculate the relative frequencies of ft, we calculated the means of ft of all nests to represent the foraging activity in the sampling area. we applied linear regression analysis to describe the relationship between tf and ts. we used student’s t test to assess differences for each ts category between successful and unsuccessful trips. we used graphpad prism 7.00 (graphpad software, 2017) to perform statistical analyses. a p-value ≤ 0.05 was considered significant. we presented the variables in terms of mean ± standard error of the mean (se). results characteristics of the sampling area and nest openings the three monitored nests were beneath understory vegetation roots in a forested area (900 m2) with ground totally covered by leaf litter. the nest density was 33 nests/ha. the mean distance between nests was 11.3 ± 1.4 m, showing a triangular shaping on the ground (nest 1 to 2: 8.5 m, nest 1 to 3: 12.5 m and nest 2 to 3: 13.0 m). mean canopy cover on the nest’s openings was 79 ± 4 % (nest 1: 74%, nest 2: 86% and nest 3: 78%), which characterizes the sampling area as a mosaic of light and shade. the nests had an elliptical entrance with mean height of 20.4 ± 1.0 mm (nest 1: 18.5 mm, nest 2: 21.5 mm and nest 3: 21.3 mm) and mean width of 41.1 ± 1. mm (nest 1: 43.1 mm, nest 2: 39.7 mm and nest 3: 40.5 mm). environment variables the ta during the foraging activity was 27.2 ± 0.5 ºc and ts was 24.5 ± 0.6 °c (table 1). ta and ts were positively correlated (r = 0.89, p < 0.01, n = 26). highest temperatures were recorded in the middle of the day (fig 1). the rh during the foraging activity was 82.6 ± 2.1 %, with lower rh in the same hours of higher temperatures (fig 1). ta and rh were negatively correlated (r = -0.80, p < 0.01, n = 26). ts and rh were also negatively correlated (r = -0.81, p < 0.01, n = 26). fig 1. mean air temperature (ta, left y axis, red dotted line), litter surface temperature (ts, left y axis, black solid line), and relative humidity (rh, right y axis, blue dotted line) at each hour of observation. data sampled at vale natural reserve, linhares, state of espírito santo, brazil. nest flow, foraging activity time, distance, and influence of temperature dinoponera lucida foraging activities were done between 7 h and 17 h. dinoponera lucida exhibited a bimodal pattern of foraging activity (r2 = 0.26, p < 0.01, aicc = 209.6, df = 58) (fig 2a). the first ft peak was between 8 h and 10 h. the second ft peak was recorded between 14 h and 17 h. f curbani, c zocca, rb ferreira, c waichert, tg sobrinho, ac srbek-araujo – temperature and foraging activity of dinoponera lucida4 flow of workers at nest openings was higher (74%) between 21.0 and 27.0 ºc (χ2 = 22.41, df = 5, p < 0.01) (fig 2b), and lower (26%) between 27.0 and 31.0 ºc. we did not observe foraging activity under rainfall, even when ts reached the values related to the highest ft values. the mean ft at nest openings was 3.8 ± 0.6 h-1 (table 1). the mean df of foraging workers was 3.8 ± 0.4 m. some workers from nest 2 travelled 10.1 m away from the nest entrance, which was the maximum df observed. the mean tf in foraging activity was 14.2 ± 2.2 min. the tf were independent of df (r = 0.18, p = 0.15, n = 24). the values of tf were positively correlated to ts values (r = 0.76, p < 0.01, n = 24), with linear regression model (r² = 0.57, f1, 22 = 29.60, p < 0.01, tf = 3.614ts – 75.227) (fig 3). fig 2. flow of workers at openings of dinoponera lucida nests. (a) total flow of workers (ft, left y axis, black circles), fourth-order polynomial fit used in the nonlinear regression model of ft as a function of the time of day (left y axis, red solid line), and relative frequency of ft at different time of day (right y axis, blue solid line). (b) total flow of workers (ft, left y axis, grey bars), and relative frequency of ft (right y axis, blue solid line) at each litter surface temperature category. data sampled at vale natural reserve, linhares, state of espírito santo, brazil. error bars represent standard errors. variable nest minimum maximum mean se n df (m) 1 1.0 4.0 2.6 0.4 10 2 1.0 10.1 4.5 0.6 19 3 2.0 5.5 3.5 0.4 8 t 1.0 10.1 3.8 0.4 37 tf (min) 1 2.0 22.0 9.8 3.7 6 2 2.0 20.0 11.8 1.7 12 3 5.0 45.0 23.3 6.1 6 t 2.0 45.0 14.2 2.2 24 ft (h -1) 1 0.0 20.0 2.8 1.0 26 2 0.0 28.0 5.7 1.0 33 3 0.0 16.0 2.2 0.9 24 t 0.0 28.0 3.8 0.6 83 fin (h -1) 1 0.0 8.0 1.7 0.5 26 2 0.0 16.0 2.7 0.6 33 3 0.0 12.0 1.0 0.6 24 t 0.0 16.0 1.9 0.3 83 fout (h -1) 1 0.0 12.0 1.1 0.5 26 2 0.0 12.0 3.0 0.6 33 3 0.0 8.0 1.2 0.5 24 t 0.0 12.0 1.9 0.3 83 ts (ºc) 19.5 30.3 24.5 0.6 26 ta (ºc) 21.8 30.9 27.2 0.5 26 rh (%) 64.7 97.5 82.6 2.1 26 table 1. descriptive variables of the foraging activity of dinoponera lucida and environmental conditions at vale natural reserve, linhares, state of espírito santo, brazil. the values are presented for each sampling nest and values marked with t indicate a summary of all observations of each variable in the sampling area. se: standard error of the mean, n: number of observations, df: distance reached during foraging activity, tf: time spent in foraging trip, ft, fin and fout: total flow, inflow and outflow of workers in the nest openings, ts: litter surface temperature, ta: air temperature, and rh: relative humidity. we recorded 17 successful trips (ts = 24.6 ± 0.4 ºc) and seven unsuccessful trips (ts = 26.8 ± 0.8 ºc). the ts was lower during successful trips (t = 2.62, p = 0.01). we recorded 37 returns to the nests (29 with food) resulting in an efficiency rate of foraging of 0.78 ± 0.06 (nest 1: 0.70, nest 2: 0.78, nest 3: 0.89). collected food items we recorded 48 food items collected by d. lucida workers, of which, 94% were animals and 6% vegetation items (table 2). all food items were solid. we observed workers capturing macroinvertebrates with high mobility (such as spiders, grasshoppers, 78%), low mobility (insect larvae and gastropods, 13%), and no mobility (insect pupae, 9%). these percentages about mobility of macroinvertebrates consider only identified animal food items. sociobiology 68(1): e-6030 (march, 2021) 5 discussion dinoponera lucida presented bimodal cycle on daily foraging activity with a higher peak in the morning. the foraging activity was higher in a range of litter surface temperature between 21.0 and 27.0 ºc, and successful foraging trips were mostly during lower litter surface temperatures (24.6 ± 0.4 ºc) in comparison to unsuccessful trips (26.8 ± 0.8 ºc). similar patterns were found for d. lucida and dinoponera quadriceps kempf, 1971, both showing bimodal foraging cycles and higher peak in the morning than in the afternoon (peixoto et al., 2010; medeiros et al., 2014). in contrast, dinoponera longipes emery, 1901 seems to be mainly nocturnal, but workers are also active during the day (morgan, 1993). in addition, dinoponera gigantea (perty, 1833) also has a bimodal pattern of foraging activity, but with higher peaks at dawn and dusk (fourcassié & oliveira, 2002). finally, d. lucida likely reduces foraging in response to temperature cues to minimize foraging activity during times of low prey availability (norberg, 1977; bernstein, 1979). dinoponera lucida prey mainly on macroinvertebrates which are also influenced by litter temperature (bernstein, 1979; may, 1979; willmer, 1982; colinet et al., 2015; pincebourde & suppo, 2016; welch et al., 2020). several epigeic ectotherm taxa, which are potential preys for d. lucida, tend to migrate to deeper layers of litter to forage or for refuge (willmer, 1982; colinet et al., 2015; pincebourde & suppo, 2016). the foraging activity of d. quadriceps also is positively related to availability of potential prey (medeiros et al., 2014). our data suggest that d. lucida limits foraging according to the activity pattern of prey, suggesting an optimization strategy driven by prey availability (emlen, 1966; macarthur & pianka, 1966; norberg, 1977; perry & pianka, 1997), with litter temperature serving as a proxy for successful foraging likelihood rather than being a direct influence on d. lucida foraging behavior. foraging attempts had no relationship with time spent or distance reached, suggesting that the foraging success depends on choosing the appropriate time. it seems that these ants optimize their foraging behavior by relying on the chances to find a food resource instead of covering large areas or traveled distance during the foraging trip. our data on patterns of flow of workers of d. lucida in foraging was similar to other populations (peixoto et al., 2010). the increase of time spent in foraging trip could be related to search and capture efficiency, supporting the hypothesis of optimal food searching (emlen, 1966; macarthur & pianka, 1966; norberg, 1977; perry & pianka, 1997). the positive correlation between time spent in foraging trip and litter surface temperature suggests the existence of an optimal fig 3. scatterplot and linear regression line (blue solid line) of the time spent in foraging trip (tf) of dinoponera lucida workers as a function of the litter surface temperature (ts) at vale natural reserve, linhares, state of espírito santo, brazil. the filled circle with the error bars represents the bivariate means and standard errors. kingdom taxon item n (%) animalia arachnida: araneae spider 5 (10.4) arachnida: scorpiones scorpion 1 (2.1) blattodea cockroach 1 (2.1) coleoptera beetle 1 (2.1) hemiptera: pentatomidae shield bug 1 (2.1) hymenoptera: apoidea bee 1 (2.1) insecta insect larva, likely lepidoptera 2 (4.2) insecta insect pupa 2 (4.2) blattodea: isoptera termite 4 (8.3) mollusca: gastropoda gastropod 1 (2.1) orthoptera: caelifera grasshopper 4 (8.3) squamata: serpentes snake scale 1 (2.1) vertebrata fecal pellet 1 (2.1) unidentified macroinvertebrate 20 (41.7) plantae swartzia myrtifolia var. elegans (schott) r.s.cowan seed 1 (2.1) cordia magnoliifolia cham. fruit 1 (2.1) angiospermae tree bark 1 (2.1) table 2. food items collected by dinoponera lucida workers in foraging activity at vale natural reserve, linhares, state of espírito santo, brazil. n: number of records. percentages presented in parentheses are relative to the total of items. f curbani, c zocca, rb ferreira, c waichert, tg sobrinho, ac srbek-araujo – temperature and foraging activity of dinoponera lucida6 temperature range for the foraging activity. however, it is not clear that the workers use temperature as a proxy. it seems that temperature is an effective proxy for prey availability. in addition, as temperature is highly correlated with time of day, it may be that they simply use time of day as a proxy for prey availability. it also seems possibly that foraging activity is directly related to prey availability, with ants foraging more when preys are abundant and less as prey availability decreases. as d. lucida is an eusocial species, some decisions about the time to forage could be related to communication between the nestmates (leonhardt et al., 2016). further studies could assess these questions assertively with prey density data combined with the other sampled data. according to our data d. lucida was predominantly carnivorous and might be categorized as a hunting and collecting species (almeida & queiroz, 2015). we did not observe predation of other ant species, even when they were abundant near a worker in the foraging activity. similarly, ants were not in the diet of d. quadriceps (araújo & rodrigues, 2006). ants, however, have been observed in low frequency in the diet of other species of dinoponera (fourcassié & oliveira, 2002; peixoto et al., 2010). considering the number of prey items, ants comprise 2% of d. lucida diet (peixoto et al., 2010) and 4% of d. gigantea diet (fourcassié & oliveira, 2002). the capture of animals with high mobility (78% of the total macroinvertebrate items) indicates that d. lucida performs active hunting besides collection of food items. this performance was also observed in other species of dinoponera, including capture of relative larger prey such as small lizards (sousa & freire, 2010; ribeiro et al., 2011; carvalho et al., 2012). dinoponera lucida seems to have a more diverse diet than we recorded, which may be related to our short sampling period. specifically, we identified a relatively low frequency of plant items (6%) when compared to 16% from other population of d. lucida (peixoto et al., 2010), 22% from d. gigantea (fourcassié & oliveira, 2002) and 30% from d. quadriceps (araújo & rodrigues, 2006). workers of d. quadriceps (araújo & rodrigues, 2006) and d. gigantea (fourcassié & oliveira, 2002) collect mainly animal food items, mostly arthropods, as we observed for d. lucida. it is possible that the diet of d. lucida and others dinoponera vary seasonally according to the availability of food resources in their territories. therefore, additional studies on the diet composition of d. lucida may be useful to better describe its ecological role in the atlantic forest. dinoponera lucida and congeners use underground nests in closed-canopy forest (paiva & brandão, 1995; peixoto et al., 2010). dinoponera as well as the other ponerinae ants avoid foraging with high insolation (willmer & corbet, 1981; willmer, 1982). it is likely underground and closed-canopy nests prevent water loss (chown et al., 2011) and regulate nests temperatures (willmer, 1982; jones & oldroyd, 2006). our results were only focused on three nests. we recommend further study to assess populations level responses to microclimatic factors across the species range. the density recorded herein, however, was similar to the reported by peixoto et al. (2010), but far from describing a continuous and homogeneous distribution of d. lucida nests in the forest. nests of d. lucida occur in an aggregate distribution (peixoto et al., 2010), a consequence of its reproduction mode by colony fission (mariano et al., 2008; campiolo et al., 2015). during the active search for nests, we found vast areas within the atlantic forest in vnr without the presence of d. lucida workers. this is aligned to the endangered conservation status of the species by brazilian red list (ministério do meio ambiente, 2014; instituto chico mendes de conservação da biodiversidade & ministério do meio ambiente, 2018), which does not seem to be particularly abundant, even where adequate conditions and resources exist. our data support the hypothesis of optimal foraging regulated by habitat temperature. it would be a direct response by prey species and an indirect response by d. lucida as they may respond more directly to prey availability. time spent in foraging trip and litter temperature were positively correlated. flow of workers was higher in an optimal litter surface temperature range, with more successful foraging trips and more food items (mainly macroinvertebrates) collected in the same temperature range. the better understanding about d. lucida activity patterns can assist in the planning of study activities, such as monitoring and inventory of this endangered and endemic species. acknowledgements we are grateful to josé simplício dos santos for the assistance during location of the d. lucida nests and sampling area set-up. we thank vale natural reserve for the support in field activities. czz thanks coordenação de aperfeiçoamento pessoal de nível superior (capes) for scholarship and conselho nacional de desenvolvimento científico e tecnológico (cnpq) (001/1700071 and 301362/2021-1). acsa thanks universidade vila velha (uvv) and fundação de amparo à pesquisa e inovação do espírito santo (fapes) (0607/2015 and 510/2016). cw thanks fapes (#85320846) and cnpq (#435045/2018-0). author contributions fc: conceptualization, methodology, investigation, formal analysis and writing cz: conceptualization, methodology, investigation and writing rbf: conceptualization, methodology and writing acsa: conceptualization, methodology and writing tgs: formal analysis and writing cw: formal analysis and writing declarations conflict of interest the authors declare that they have no conflict of interest to disclose. sociobiology 68(1): e-6030 (march, 2021) 7 compliance and ethical approval all applicable institutional and/or national guidelines for the care and use of animals were followed. references almeida, f.s. & queiroz, j.m. 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(1981). temporal and microclimatic partitioning of the floral resources of justicia aurea amongst a concourse of pollen vectors and nectar robbers. oecologia, 51: 67–78. doi: 10.1007/bf00344655 ydenberg, r.c. (2007). provisioning. in: foraging: behavior and ecology (eds. stephens, d.w., brown, j.s. & ydenberg, r.c.). the university of chicago press, chicago, pp. 273-303. _hlk62214149 __fieldmark__2691_416698021 __fieldmark__2644_416698021 __fieldmark__2655_416698021 __fieldmark__2681_416698021 _hlk62224894 __fieldmark__3132_416698021 __fieldmark__3157_416698021 _hlk62240518 __fieldmark__4202_416698021 doi: 10.13102/sociobiology.v60i4.436-440sociobiology 60(4): 436-440 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 pollen content in honey of apis mellifera linnaeus (hymenoptera, apidae) in an atlantic forest fragment in the municipality of piracicaba, são paulo state, brazil dfd araujo1, accc moreti2, ta silveira1, lc marchini1, ip otsuk2 introduction the population growth of a bee colony depends entirely on the quality and quantity of nectar and pollen sources collected by workers, since honey and pollen, a carbohydrate and a protein source, respectively, are essential for the nutrition of larvae and adults of apis mellifera (l.) (zerbo et al., 2001). the composition and quality of food sources as well as other factors that affect the colony growth may vary according to the region and year season (funari et al., 2003; marchini et al., 2006). to obtain an adequate food storage and high population growth rate, colonies need to build an optimal food inventory. the climatic conditions and food availability in the region directly affects the productive and reproductive characteristics of bee colonies. therefore, food storage (honey and pollen), oviposition and occupation of combs are subject to seasonal variations (modro et al., 2011). abstract the productive and reproductive characteristics of apis mellifera l. bees are directly affected by climatic conditions and food availability in the region where the bees are reared or kept. therefore, food storage (honey and pollen), oviposition and population growth of these bees are subject to seasonal variations. these variations lead the bees to constantly search for food, making exploratory trips, called “foraging”. this study investigated the botanical origin of nectar sources collected by a. mellifera bees for six consecutive months, from october/2011 to march/2012 in six bee colonies. the study was carried out in the experimental apiary of the entomology and acarology department of the college of agriculture “luiz de queiroz”, from the university of são paulo, in the municipality of piracicaba, são paulo state. the study site has a predominant vegetation of semideciduous forest (atlantic forest). in each sampling month, we analyzed the pollen types in the honey samples. we used the acetolysis method to prepare the samples for melissopalynology. we carried out the quantitative analysis by successive count of 900 sample grains. the samples were grouped in terms of botanical species, families and/or pollen types. the results show that bees used several plants from the region as a nectar source. however, the arecaceae, fabaceae/mimosoideae and myrtaceae families were predominant throughout the sampling period. the occurrence of these plant species was significant and essential for the maintenance of the bee colonies. sociobiology an international journal on social insects 1 universidade de são paulo, esalq /usp, piracicaba-são paulo, brazil. 2 secretaria de agricultura e abastecimento, nova odessa, são paulo, brazil. research article bees article history edited by celso f. martins, ufpb, brazil received 13 may 2013 initial acceptance 26 june 2013 final acceptance 18 october 2013 keywords melissopalynology; nectar; foraging; apiculture; food source. corresponding author: diogo feliciano dias araujo departo. de entomologia e acarologia universidade de são paulo campus “luiz de queiroz” avenida pádua dias, 11, são dimas, 13418-900, piracicaba, são paulo sp, brazil e-mail: diogofdaraujo@gmail.com the biological characterization of the honey plays an important role in ecological and commercial aspects, contributing to the process for quality control of the honey and even to the standardization to honey products used in food and pharmaceutical industries (lins et al., 2005; neves et al., 2009). melissopalynology is the science that deals with the morphological characteristics of pollen grains and spores, as well as their dispersion and use (barth, 1989). it is a very important tool to identify plant species visited by bees. this science branch contributes to research by providing information on the food source location and allows to identify the range that bees travel to collect food in the year seasons. this study investigated the pollen in honey produced by a. mellifera bees during six consecutive months, between october 2011 and march 2012. sociobiology 60(4): 436-440 (2013) 437 material and methods study site we carried out the experiment at the experimental apiary of the laboratory of beneficial insects from the entomology and acarology department of the college of agriculture “luiz de queiroz”, at the university of são paulo. the research facility is located in the municipality of piracicaba, são paulo state at 22º 42’ 02” s / 47º 37’ 35.18” o, at 539 meters above sea level. the site has predominant vegetation of semideciduous forest (atlantic forest). honey bee colonies we installed six colonies of africanized bees (a. mellifera) in langstroth hives. initially, we set up a nest for oviposition and pollen and nectar storage for colony maintenance. during the sampling period, we added supers to collect honey. ten days before each collection, the supers were added to the colonies for a complete clean and reconstruction of alveolus and later honey storage. sampling the samples were collected on a monthly basis from october 2011 to march 2012. afterwards, we added the supers to the nests, each with ten plots, and carried out inspections every15 days. in case there was honey deposited into the supers, we removed it, bagged it individually in sterilized plastic bags, centrifuged it, stored about 200g in sterile plastic flasks and placed in acclimatized chambers of bod (biologic oxygen demand) at constant temperature of 20 °c, with the respective identification of each colony. melissopalynological analyses the melissopalynological analyses of the samples were carried out according to the standard methodology using acetolysis (erdtman, 1952). the identification of the pollen types was based mainly on the reference collection of microscopy blades from the pollen database of the laboratory of beneficial insects of the entomology and acarology department of college of agriculture “luiz de queiroz”. we also used specialized catalogs of pollen morphology of several floral species (barth, 1989; roubik & moreno, 1991; carreira et al., 1996; colinvaux et al., 1999; moreti et al., 2002; carreira & barth, 2003; santos, 2006). the term used for identification of pollen types is not related to the international code for botanical nomenclature; however, it gives an approximate identification of the samples with an existing taxonomic group (joosten & klerk, 2002). the scientific nomenclature used follows the norms proposed by apg ii (angiosperm phylogeny group ii), according to souza and lorenzi (2008), and the nomenclature is in accordance to tropicos.org (2011). we identified and counted about 900 pollen grains per sample and each pollen grain was photomicrographed using a zeiss photomicroscope. after counting the pollen grains, we grouped them according to the following international criteria: predominant pollen (pp) – more than 45% of total pollen grains counted; accessory pollen (ap) – from 16 to 45%; isolate pollen (ip) – up to 15%, subdivided into: important isolate pollen (iip): 3 to 15% and occasional isolate pollen (oip): less than 3% (louveaux et al., 1978). data analysis the experimental design was completely randomized with six replications (represented by the colonies) and data were repeated to study the families and pollen types of the plants visited by the bees during the six months of study. after classification, the data were analyzed through the mixed procedure of the sas (statistical analysis system), sas institute (2001) to determine structure of matrix for variance and covariance at 5% probability. the data were converted to square root of (x+1) to obtain homocedasticidity in the analysis of variance. results and discussion we identified 26 pollen types in the honey samples (table 1) belonging to 15 botanical families and the families arecaceae, fabaceae and myrtaceae (figure 1) were the main food sources identified. comparing our findings to results obtained by modro (2011) and silveira et al. (2012), carried out in the same region to identify pollen types collected by a. mellifera bees for one year, in our study, we observed a smaller number of pollen types collected by this bee species. the authors identified between 60 and 80 pollen types; however, despite the smaller number of pollen identified, the melissopalynological analysis in study was carried out in two seasons, showing an even greater diversity of botanical species visited by the bees during the experiment. the variation in foraging of the botanical sources used by the bees, regardless the source collected (honey or pollen), can vary according to several factors such as flowering, climate and competition with other bee species (gonzalez et al., 1995; villanueva & roubik 2004; webby, 2004; keller et al., 2005). the pollen type from the arecaceae family was found in the months of november and december of 2011 and january of 2012. however, its occurrence was ap only in november and december of 2011, remained as ap and ip in the other months. this is surprising because species of this family normally have greater occurrence during palynological studies on pollen types collected by the bees. the arecaceae family shows a high pollen production rate due to the pollination syndrome occurring in most species (barfod et al., 2011). however, bees collect dfd araujo et al melissopalynology of a. mellifera in an atlantic forest438 nectar from arecaceae because, in addition to great pollen production, its flowers are used in nectar production (küchmeister et al., 1997; mantovani & morellato, 2000; venturieri, 2008; kidyoo & mckey, 2012). the arecaceae species are also important pollen sources for bees. this can contaminate honey and mask the significance of their relative high occurrence in honey samples. similarly, many mimosa species provide pollen grains that are identified in the melissopalynological analysis. despite its importance for honey characterization, mimosa is not the main nectar source collected for honey production. this fact may explain the occurrence of pollen types of the arecaceae family during the palynological studies in honey samples. the arecaceae family is an important food source for bees in the study area. the pollen types of the fabaceae family, such as that of anadenanthera sp., showed greater occurrence in october/2011, and did not show significance in the other months. the mimosa scabrella type was significant only in october/2011 and december/2011. in the same family, mimosa caesalpinifolia was pp and ap in the months of february and march/2012, respectively, presenting itself as an important food source for all colonies, given that in february, this species was pp in 83.33% of the colonies, according to the classification of louveaux et al. (1978). the same condition was found in march. in the myrtaceae family, we observed two pollen types, eucalyptus sp. and myrcia. the myrcia type presented itself as an important food source for the bees, because this family showed significant occurrence (pp and pa) from october/2011 to january/2012, and can be considered one of the main species used for colony maintenance. eucalyptus sp. showed occurrence after january/2012 and also considered an important food source for bees during the period. other studies in the region showed that the fabaceae/ mimosoideae and myrtaceae families are the main food sources for a. mellifera bees, showing the importance of this species for the colonies, both as nectar and pollen sources during certain periods of the year (modro et al., 2011). we identified species that were relevant as food sources based on the pollen types found, such as plants with potential use in management programs of apiculture in pasture. despite the occurrence of other pollen types, bees showed intensive collecting activities of these plant species. in a study on geographic location of food sources used by bees, the pollen considered as isolated occurrence can also serve as an important indicative to monitor the site, even showing low relevance in terms of quantity of nectar collected. conclusion although poorly used as a nectar source, species from the arecaceae family are important food sources for bees during some seasons in the year. species from the families arecaceae, fabaceae/mimosoideae and myrtaceae are the most indicated for the implementation or improvement of apiculture pastures in the region. the family myrtaceae, represented by the pollen type myrcia, which encompasses the genera psidium, eugenia, myrcia and myrciaria), show great importance as food source during the productive period of bees in the region. acknowledgments the authors wish to thank cnpq for granting a scholarship to the first author. references barfod, a.s., hagen, m.,borchsenius, f. 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(2011) missouri botanical garden. <http:// www.tropicos.org>. venturieri, g. (2008) floral biology and management of stingless bees to pollinate assai palm (euterpeoleracea mart., arecaceae) in eastern amazon. in: alvarez, c.a.b; landeiro, m. pollinators management in brazil. brasília: ministério do meioambiente, p. 10-13. villanueva, g.r. & roubik, d.w. (2004) why are african honey bees and not european bees invasive? pollen diet diversity in community experiments. apidologie, 35: 481–491. webby, r. (2004) floral origin and seasonal variation of beecollected pollens from individual colonies in new zealand. j. apic. res., 43: 83-92. zerbo, a.c.,moraes, r.l.m.s. & brochetto-braga, m.r. (2001) protein requirements in larvae and adults of scaptotrigona postica (hymenoptera: apidia, meliponinae) midgutproteolytic activity and pollen digestion. comp. biochem. physiol., 129, p. 139 -147. dfd araujo et al melissopalynology of a. mellifera in an atlantic forest440 table 1. mean rate (%) of pollen types in honey samples of apis mellifera collected from fragments of the atlantic forest in the municipality of piracicaba, são paulo state, brazil, from october 2011 to march 2012. means followed by different uppercase letters in columns differ in the tukey-kramer test (p<0.05). 1 means between parenthesis are converted to root (x+1). #statistics referring to converted data. predominant pollen (pp); accessory pollen (ap); isolate pollen (ip), subdivided into: important isolate pollen (iip) and occasional isolate pollen (oip). botany family/ pollen type sampling months oct. nov. dec. jan. feb. mar. alismataceae/ alismataceae type 0,73±2,22 oip (1.25±0.27)bc amarantaceae/ alternanthera ficoidea 4,17±1,82 iip (2,11±0,22)bcd 0,l7±1,82 oip (1,07±0,22)c amarantaceae/ amarantus sp. 0,40±1,99 oip (1,16±0,24)c anacardiaceae/ anacardiaceae type 0,20±1,99 oip (1.08±0.24)cd 0,20±1,99 oip (1.08±0.24)c araceae/ araceae type 1.00±1,82 oip (1.38±0.22)bc 16.83±1.82 ap (3.67±0.22)b 8.83±1.82 iip (2.99±0.22)c arecaceae/ arecaceae type 1.17±1.82 oip (1.44±0.22)cd 37.83±1.82 ap (6.21±0.22)a 34.33±1.82 ap (5.71±0.22)a 21.67±1.82 ap (4.47±0.22)b 13.83±1.82 iip (3.78±0.22)bc 12.00±1.82 iip (3.49±0.22)b bombacaceae/ pachira aquatica 0.33±1.82 oip (1.14±0.22)c 0.17±1.82 oip (1.07±0.22)c compositae/ mikania laevigata 0.17±1.82 oip (1.07±0.22)e compositae/ parthenium sp. 0.17±1.82 oip (1.07±0.22)c cruciferae/ raphanus sp. 0.33±1.82 oip (1.12±0.22)d 2.67±1.82 iip (1.58±0.22)d fabaceae/ caesalpinia pelthophoroides 0.67±1.82 oip (1.23±0.22)cd 0.17±1.82 oip (1.07±0.22)c 0.17±1.82 oip (1.07±0.22)c fabaceae / caesalpinia sp. 4.50±1.82 iip (2.29±0.22)cd 2.50±1.82 oip (1.71±0.22)cd 1.83±1.82 oip (1.59±0.22)bc 0.17±1.82 oip (1.07±0.22)c fabaceae/ cassia sp. 1.83±1.82 oip (1.62±0.22)cd 0,17±1,82 oip (1,07±0,22)c fabaceae/ centrosema sp. 3.17±1.82 iip (1.96±0.22)cd 3.50±1.82 iip (2.10±0.22)bcd 2.67±1.82 oip (1.89±0.22)bc 0.50±1.82 oip (1.21±0.22)c fabaceae/ anadenanthera sp. 17.50±1.82 ap (3.90±0.22)b 4.50±1.82 iip (2.31±0.22)bcd 4.17±1.82 iip (2.23±0.22)bc 3.67±1.82 iip (2.05±0.22)c 0.17±1.82 oip (1.07±0.22)d fabaceae / leucaena sp. 3.83±1.82 iip (1.99±0.22)cd 5.67±1.82 iip (2.53±0.22)bc 2.00±1.82 iip (1.70±0.22)bc 1.50±1.82 oip (1.57±0.22)c 0.50±1.82 oip (1.21±0.22)d fabaceae / mimosa caesalpinifolia 1.33±1.82 oip (1.44±0.22)c 55.00±1.82 pp (7.47±0.22)a 28.50±1.82 ap (5.37±0.22)a fabaceae / mimosa scabrella type 5.00±1.82 iip (1.96±0.22)cd 10.33±1.82 iip (2.65±0.22)b 2.50±1.82 oip (1.72±0.22)c 6.83±1.82 iip (2.76±0.22)cd 1.67±1.82 oip (1.53±0.22)d fabaceae/ macroptilium sp. 0.33±1.82 oip (1.14±0.22)c lamiaceae/ hyptis sp. 0.67±1.82 oip (1.26±0.22)d malvaceae/ dombeya sp. 0.40±1.99 oip (1.15±0.24)cd moraceae/ morus sp. 0.17±1.82 oip (1.07±0.22)d 0.17±1.82 oip (1.07±0.22)d 0.17±1.82 oip (1.07±0.22)c myrtaceae/ myrcia type 54.17±1.82 pp (7.07±0.22)a 27.50±1.82 ap (5.31±0.22)a 34.67±1.82 ap (5.93±0.22)a 36.33±1.82 ap (6.03±0.22)a 2.00±1.82 oip (1.68±0.22)de 1.33±1.82 oip (1.44±0.22)d myrtaceae / eucalyptus sp. 7.83±1.82 iip (2.80±0.22)bc 9.17±1.82 iip (3.15±0.22)b 6.83±1.82 iip (2.72±0.22)b 13.50±1.82 iip (3.78±0.22)b 21.83±1.82 ap (4.77±0.22)b 42.67±1.82 ap (6.58±0.22)a rubiaceae/ rubiaceae type 0.50±2.22 oip (1.21±0.27)cd rutaceae/ citrus sp. 0.17±1.82 oip (1.08±0.22)d 0.17±1.82 oip (1.07±0.22)d 0.50±1.82 oip (1.19±0.22)c 0.33±1.82 oip (1.14±0.22)c open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1266sociobiology 64(3): 369-372 (september, 2017) solenopsis saevissima (smith) (hymenoptera: formicidae) activity delays vertebrate carcass decomposition in addition to climatic conditions, such as temperature and rainfall, the presence of and ecological interactions between insect guilds play a key role in the decomposition rate of vertebrate carcasses (simmons et al. 2010; santos & alves, 2016). ants (hymenoptera: formicidae) are among the insects involved in decomposition processes, and exhibit both predatory and necrophagous behavior (carvalho et al. 2004; cruz & vasconcelos, 2006). furthermore, they interact with other arthropods around decomposing bodies (rámon & donoso, 2015). due to their aggressive behavior, ants from the genus solenopsis can reduce the number of eggs and larvae in cadaver-occupying species, impacting carcass colonization (stoker et al., 1995) and either accelerating or delaying the rate of decomposition (andrade-silva et al. 2015). therefore, these ants may potentially confound interpretation of the postmortem interval (pmi). abstract ants are among the insects involved in cadaveric decomposition processes, as they exhibit predatory and necrophagous behavior. fire ants exhibit an aggressive behavior, which impacts carcass colonization, accelerates or delays the decomposition rates and potentially confounds interpretation of the postmortem interval. here, we estimated the effects of solenopsis saevissima (smith) activity on the decomposition rate in domestic pig carcasses. we placed two pig carcasses close to s. saevissima nests, and two other pig carcasses (controls) in other locations 50 m away from the nest. decomposition processes were delayed by at least three days for carcasses on the nest compared to those without direct exposure to ants. our results showed that predatory activity of s. saevissima interfered with carcass colonization by scavenger insects, functioning as an ecological barrier to the establishment of immature diptera. such results highlight the importance of considering ecological processes that may interfere with mechanisms determining post-mortem intervals. sociobiology an international journal on social insects ekc pereira1, j andrade-silva 1,2, o silva1, clc santos1,3, ls moraes1, mca bandeira1,2, crr silva1, jmm rebêlo1,2 article history edited by evandro do nascimento silva, uefs, brazil received: 09 december 2016 initial acceptance 01 may 2017 final acceptance 20 june 2017 publication date 17 october 2017 keywords fire ants, decomposition rate, forensic entomology, forensic taphonomy corresponding author joudellys andrade-silva universidade federal do maranhão laboratório de entomologia e vetores departamento de biologia av. dos portugueses s/n, 65085-580 são luís, maranhão, brasil e-mail: andradejoudellys@yahoo.com.br despite their relevance in forensic entomology, little is known about the effects of solenopsis spp. on the process of cadaveric decomposition. most studies conclude that these ants alter insect succession patterns and decomposition processes, but do not indicate specifically how these factors are altered (zara & caetano, 2010; andrade-silva et al. 2015; maciel et al. 2015). here, we analyzed qualitative effects of solenopsis saevissima (smith) presence on decomposition rates of domestic pig (sus scrofa l.) carcasses. also, we sought to better understand the ant interactions with other insects, as well as the presence of post-mortem lesions throughout the decomposition process. this information may help to increase the accuracy of pmi estimates, thereby improving the quality of crime evidence based on forensic entomology. the study was conducted between january and february 2013 at the beginning of the rainy season, in an urban remnant of amazon forest located within the universidade short note 1laboratório de entomologia e vetores, universidade federal do maranhão, são luís–ma, brasil. 2programa de pós-graduação em biodiversidade e conservação, universidade federal do maranhão, são luís–ma, brasil. 3programa de pós-graduação em ecologia e conservação, universidade federal de mato grosso do sul, campo grande–ms, brasil. ekc pereira et al. solenopsis saevissima influences carcass decomposition370 federal do maranhão (02° 33' 36" s, 44° 18' 33" w). this area has an altitude of less than 100 m a.s.l., climate tropical hot and humid, annual rainfall between 1,900 and 2,200 mm and an average annual temperature above 26 °c (alvares et al., 2013). the environmental characteristics indicate high degree of anthropogenic intervention in the original vegetation (icmbio, 2014), which has been reduced to low capoeira due to the expansion of the university’s campus. four domestic pigs weighing between 20 and 25 kg were sacrificed from close range shot (<30cm; pistol .40) to the head (carried out by a criminal examiner). we placed two carcasses (10 m apart) close to s. saevissima nests, and the other two (without exposure) were placed about 50 m from the other carcasses to guarantee the independence of the samples. carcasses were observed daily between the hours of 12:00 and 13:00. decomposition phase was determined following classifications by payne (1965): fresh (s1), bloated (s2), active decay (s3), advanced decay (s4), and dry/remains (s5). throughout the study, the carcasses were protected from large animals by metal cages surrounded by wire mesh (5 cm diameter) installed into the ground. the ants were obtained from carcasses by active collection. identification of subfamily and genera followed baccaro et al (2015) and species were confirmed by the laboratório de mirmecologia (cpdc) at the centro de pesquisa da lavoura cacaueira (ceplac) in bahia, where voucher specimens were deposited. during stage s1, we found little odor caused by the decomposition of the carcasses and observed a low number of ants in the first hours of decomposition, mainly for the carcasses unexposed to the nests. in s2, we observed marks on the carcasses resulting from the activity of s. saevissima, which also presented eggs and small and punctual (mouth and muzzle) masses of diptera larvae. the s3 phase was characterized by masses of large larvae throughout the carcass, except for those exposed to the ant nests, where mass was very reduced. in addition to the strong odor of decomposition at this stage, liquids resulting from this process were perceptible around the carcasses, especially for the carcasses not exposed to the s. saevissima nests. the s4 phase was characterized by a great reduction in the resource offered by the carcass and the beginning of its skeletonization. there was also a decrease in the size of larval mass seen in the previous phase. in the s5 phase we observed an absence of larvae mass, and completely dry and fragmented carcasses, with exposed bones. we recorded differences in the duration of the decomposition phases between carcasses exposed to ant nests and controls. the ant-exposed carcasses lasted three additional days in the s3 and s4 phases, and more than 10 days in the final phase (fig 1). solenopsis saevissima displayed the following behaviors on the carcasses: production of tissue lesions visible in early decomposition (fig 2-a); predation on dipteran eggs, larvae, and adults (figs 2 b-d); and feeding on exudates released by the carcass. these ant behaviors likely led to differences in fly larval biomass between experimental (carcasses on the ant nest) and control groups (figs 2e-f). although in very small densities, other ant species such as camponotus rufipes (fabricius), dorymyrmex brunneus forel, ectatomma brunneum smith and pheidole synarmata wilson, were collected from the carcasses. carcasses are a limited and ecologically expensive resource, because many organisms (e.g. flies, beetles, bees, ants, and others) compete for them (noll et al., 1996; alves et al., 2014; santos et al., 2014). the presence of s. saevissima reduces fly larval populations and makes oviposition by adults more difficult, which reduces colonization and can decrease associated entomofauna, therefore, decreasing the rate of decomposition (wells & greenberg, 1994; moura et al., 2005). when acting as necrophages, ants, such solenopsis, cause lesions that can lead to errors in criminal investigations (patel, 1994). in the short term these lesions can darken, which may lead to the diagnosis of pre-mortem mutilations, thereby compromising interpretations by crime experts. the higher larval biomass from the control carcasses suggests that the end of the s3 phase is strongly influenced by ecological factors, specifically by the activity of fly larvae. consequently, factors that prevent or delay oviposition by adult flies or hatching of immatures can alter the timing of carcass decomposition. some studies have emphasized the possibility of variation in decomposition rates and insect succession patterns in the same location (shean et al., 1993; joy et al., 2006; sharanowski et al., 2008; santos & alves, 2016), as well as visible lesions in the first few weeks of decomposition (campobasso et al., 2009). although we did not observe them here, two genera of ants, camponotus and pheidole, have been known to remove eggs, larvae, and pupae fig 1 average duration (number of days) of decomposition phases for domestic pig carcasses with and without exposure to solenopsis saevissima nest in an urban amazon forest remnant in são luís, maranhão, brazil. s1: fresh; s2: bloated; s3: active decay; s4: advanced decay; s5: dry and remains. sociobiology 64(3): 369-372 (september, 2017) 371 from decomposing carcasses (barros et al., 2008). our results showed that the predatory activity of s. saevissima interfered with carcass colonization of flies, by effectively functioning as an ecological barrier for the establishment of immatures. as a consequence, the first stages of decomposition were extended by three days, when ants were present on carcasses. we emphasize the importance of considering ecological processes (e.g. interspecific interactions) and abiotic factors on decomposition when estimating the postmortem interval. studies of this nature will limit underestimation and improve the accuracy of pmi estimates, as well as reinforce the importance of investigating ant behavior on carcasses within the field of forensic entomology. acknowledgements we would like to thank fundação de amparo à pesquisa e ao desenvolvimento científico e tecnológico do maranhão (fapema) for the financial support and scholarship of scientific initiation for ekc pereira. the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for providing a master’s fellowship to j andrade-silva. we are grateful to jacques hc delabie (uesc/ceplac, ilhéusba) for ant species identification. we also thank emília z de albuquerque, lívia p prado, rony ps almeida (mpeg) and anonymous reviewers for considerations and improvements to this manuscript. this project was approved by the animal research ethics committee of the universidade federal do maranhão (protocol n°. 23115.004032/2012-34). fig 2 marks (a), predation to eggs (b), larvae (c) and adults (d) of diptera caused by solenopsis saevissima activity and difference in mass of fly larvae present in sus scrofa carcasses with (e) and without (f) exposure to solenopsis saevissima nest. ekc pereira et al. solenopsis saevissima influences carcass decomposition372 references alvares, c.a., stape, j.l., sentelhas, p.c., gonçalves, j.l.m., sparovek, g. 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(pp. 237-269). rio de janeiro: technical books doi: 10.13102/sociobiology.v64i1.1183sociobiology 64(1): 42-49 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 honey bee (apis mellifera) health in stationary and migratory apiaries introduction in migratory beekeeping, honey bee (apis mellifera) colonies are transported between locations so that they can pollinate crops, a practice that considerably increases agricultural production (souza et al., 2007). however, the stress to honey bees due to the confinement they undergo for long periods and distances during migration, pathogens and pests, changing environments, intensive management, abstract the practice of migratory beekeeping is based on moving honey bee (apis mellifera) colonies between different locations to intensify agricultural production through improved pollination services. however, due to stress caused by exposure of bee hives to different environments, migratory beekeeping activities can lead colonies to greater susceptibility of these insects to pathogens and pests, thus leading to population decline and mortality. the aim of this study was to evaluate the health profile of apiaries that adopt two types of management (stationary and migratory), located in the central-eastern region of são paulo state, brazil, during two sampling periods, one in spring (october 2010), and one in autumn (may 2011). we collected 474 samples of honeycomb from the brood area, combs containing capped brood, adult bees that covered the brood area, and foraging bees, to evaluate the presence and prevalence of paenibacillus larvae, varroa destructor, nosema apis and n. ceranae. seasonality was identified as a determining factor in the health condition of africanized a. mellifera colonies, causing a stronger effect on health than the type of management employed (stationary vs migratory beekeeping). the infection rates of n. ceranae were higher during the autumn in relation to the spring (387 ± 554 spores per bee in the spring and 1,167 ± 1,202 spores per bee in the autumn in stationary apiaries and 361 ± 687 spores per bee in the spring and 1,082 ± 1,277 spores per bee in the autumn in migratory apiaries). the same pattern was found for infestation rates of v. destructor (2.83 ± 1.97 in the spring and 9.48 ± 6.15 in the autumn in stationary apiaries and 3.25 ± 2.32 in the spring and 6.34 ± 6.58 in the autumn in migratory apiaries). these results demonstrate that the seasonality affects the health of a. mellifera colonies, but it does not depend on the type of management adopted (stationary or migratory). sociobiology an international journal on social insects l guimarães-cestaro1; mltmf alves2; d message3; mvgb silva4; ew teixeira2 article history edited by denise a alves, esalq-usp, brazil received 12 august 2016 initial acceptance 05 december 2016 final acceptance 10 march 2017 publication date 29 may 2017 keywords honey bee health, apis mellifera, varroa destructor, paenibacillus larvae, nosema spp. corresponding author lubiane guimarães cestaro federal university of viçosa (ufv) viçosa-mg, brasil e-mail: lubi.guimaraes@gmail.com poor nutrition, among other factors, have been linked to the weakening of the bees’ immune system, making colonies more susceptible to disease and potential collapse (naug et al., 2009; bacandritsos et al., 2010). honey bees can be affected by various pathogens and parasites including the the bacterium paenibacillus larvae, microsporidian species in the genus nosema, and the ectoparasitic mite varroa destructor (medina and martin, 1999; fries et al., 2010; genersch et al., 2010). 1 federal university of viçosa (ufv), viçosa-mg, brazil. 2 honey bee health laboratory (lasa), são paulo state agribusiness technology agency (apta-saa), pindamonhangaba-sp, brazil 3 federal rural university of the semiarid (ufersa), mossoró-rn, brazil 4 bioinformatics and animal genomics laboratory, embrapa dairy cattle, juiz de fora-mg, brazil research article bees sociobiology 64(1): 42-49 (march, 2017) 43 the bacterium p. larvae is responsible for causing american foulbrood (afb), considered the main cause of bee mortality in the world (funfhaus et al., 2013). the contamination of bees with afb occurs during feeding, when the larvae ingest food contaminated with bacterial spores (garrido-bailón et al., 2013). in brazil, american foulbrood was first detected in 2001 in the municipality of candelaria, rio grande do sul, where samples of adult bees, honey, pollen and brood combs were collected and analyzed for afb (schuch et al., 2003), detecting the presence of bacterial spores in adult bees and honeycomb, as well as in imported honey and pollen samples. in 2006, clinical signs of the disease were reported in samples of the municipality of quatro barras, paraná (mapa, 2006). nosemosis, or disease caused by nosema spp., can be caused by the microsporidia n. apis and n. ceranae after ingestion of spores from contaminated food or during the cleaning or foraging process (higes et al., 2010; oie, 2014). the infection affects intestinal epithelial cells (fries et al., 2010) causing disorders in the digestive system, as well as decreased worker longevity, therefore reducing the population of bees in the colony (eiri et al., 2015). nosemosis can be found on five continents (klee et al., 2007) and acts synergistically with pesticide contamination to lower bee health, a factor also considered responsible for the declining population of honey bees (pettis et al., 2012). in brazil, n. ceranae has been present since the 1970s (teixeira et al., 2013), but the disease has not caused major problems in the country so far. just like nosemosis, infestations by the ectoparasitic mite v. destructor can also harm honey bee colonies, especially by transmitting different types of viruses (le conte et al., 2010). the mite feeds on honey bee hemolymph in its larval and adult stage, interfering with the flight performance and leading to a reduced lifespan (rosenkranz et al., 2010), accounting for severe colony losses to beekeepers in many countries (genersch et al., 2010; martin et al., 2012). preventive monitoring to learn the health profile of honey bee colonies in brazil is important, especially for commercial beekeeping operations .this study evaluated the presence and prevalence of p. larvae, n. apis, n. ceranae and v. destructor, in colonies from the east-central region of são paulo state, brazil, in two different seasons and under two different management systems (stationary vs. migratory). material and methods sampling bees for analysis of the presence and prevalence of p. larvae, n. apis, n. ceranae and v. destructor in stationary and migratory honey bee colonies was based on teixeira and message (2010). we collected a total of 474 samples: 124 samples of forager bees collected at the entrance of the hive; 110 honeycomb samples from the brood area; 118 samples of comb containing capped brood; and 122 samples of adult bees present in the brood area. samples were collected during two different sampling periods, one in spring (collection 1 october 2010) and one in autumn (collection 2 may 2011) in stationary and migratory apiaries in the east-central region of são paulo state. five municipalities were included in the study: rio claro (22º 24’ s 47º 33’ w), ipeúna (22º 26’ s 47º 43’ w), araras (22º 21’ s 47º 23’ w), pirassununga (21º 59’ s 47º 25’ w) and descalvado (21º 54’ s 47º 37’ w). apiaries that were considered migratory referred to those that had just returned to the municipalities mentioned above from nearby municipalities (corumbataí analândia, botucatu, são simão, tambaú, luís antônio, among others) after fulfilling pollination services in those locations. molecular analyses were performed to identify the bacterium p. larvae and microsporidia of the genus nosema. for bacterial analysis, 20 ml of honey was diluted in 40 ml of distilled water and centrifuged at 2,518 x g for 40 min. the supernatant was discarded and the pellet was resuspended in 1 ml of sterile distilled water, with subsequent homogenization and centrifugation at 10,000 × g for 20 min. the supernatant was again discarded and 600 µl of pellet was inoculated on agar pla (schuch et al., 2001). the remaining 400 µl of the honey solution (pellet) was used for dna extraction, with the qiagen dneasy plant mini kit® following the manufacturer’s recommendations. after extraction of genetic material, pcr was performed with a final volume of 20 µl using the following primer sequences (piccini et al., 2002): f 5´cga cgc gac ctt gtg ttt cc– 3´ r 5´tca gtt ata ggc cag aaa gc – 3´ the program used for pcr consisted of an initial denaturation step at 94 °c for 2 min, followed by 35 cycles at 94 °c for 30 s, 58 °c for 30 s and 72 °c for 1 min, and a final extension at 72 °c for 5 min (puker, 2011). to identify the species of microsporidia, thirty forager honey bees were pooled and macerated, using 1 ml of sterile distilled water per bee. the macerate was filtered and after constant agitation of the filtrate, a micropipette was used to remove a small aliquot of the suspension (10µl), which was deposited in a neubauer chamber. the spore suspension was analyzed under light microscopy at 400x (cantwell, 1970), to count the spores. the remainder of the spore suspension was centrifuged at 2,518g for 40 min at room temperature. the supernatant was discarded and the pellet was resuspended in 1 ml of sterile water. this suspension was centrifuged at 10,000g for 5 min, after which the supernatant was discarded and the pellet was submitted for dna extraction employing a qiagen dneasy® plant mini kit, according to the manufacturer’s recommendations. after extraction of genetic material, the pcr reactions were performed with a final volume of 20 µl, using the primers presented by martin-hernandez et al. (2007): nosema ceranae (218 pb): f 5´cgg cga cga tgt gat atg aaa ata tta a –3´ r 5´ccc ggt cat tct caa aca aaa aac cg – 3´ nosema apis (321 pb): f 5´ggg ggc atg tct ttg acg tac tat gta – 3´ l guimarães-cestaro – honey bee health in stationary and migratory apiaries44 r 5´ggg ggg cgt tta aaa tgt gaa aca act atg – 3´ the program used for pcr consisted initially of a denaturation step at 94 °c for 2 min, 35 cycles of 94 °c for 30 s, 57 °c for 30 s, 72 °c for 50 s and extension at 72 °c for 5 min. after the pcr reaction, 5 µl was subjected to electrophoresis in 2% (w/v) agarose gel, stained with sybr safe® in 1x tbe buffer (89mm tris base, 89 mm boric acid and 2 mm edta). after electrophoresis, the gel was visualized with an e-gel imager (life technologies). the 100pb marker (invitrogen) was used as a reference to determine the size of the fragments of interest. the infestation rate of the mite v. destructor on adult honey bees was measured according to de jong et al. (1982): approximately 200 adult honey bees were in plastic pots containing 70% ethanol, with enough volume to cover them. subsequently, the containers were shaken in order to promote the detachment of possible parasites attached to the bees, which, when presented, are easily visible in the white plastic tray. considering the number of mites as well as the number of bees per sample were obtained the infestation rates. to know the infestation rate of the mite v. destructor on comb containing capped brood was utilized the method presented by medina and martin (1999) and dietemann et al. (2013): 100 pupae with dark eyes were removed and evaluated for the presence of adults and / or offspring of mite per cell. the average number of adult female mites, infested cells, cells infested with offspring, viable offspring (females possibly become adults until the emergence of honey bee) and reproductive potential (viable offspring / number of adult females and males) were measured. the data were submitted to analysis of variance according to the statistical model represented by ijkjiijk ecly +++= µ , where: yijk = dependent variables; μ = general average; li = effect of the ith site, cj = effect of j-th collection and eijk = random error effect. the degrees of freedom relating to the sources of variation studied were decomposed into contrasts and evaluated by the f-test at 1% significance level. for comparing the infection rate by nosema spp. and the infestation rate by v. destructor during the two collection periods, the analyses were performed using the glm procedure of the sas statistical package (1990). all analyses were carried out in the honey bee health laboratory (lasa) of the são paulo state agribusiness technology agency (apta/saa). results neither the bacterium p. larvae nor the microsporidian nosema apis was detected by molecular analysis in any of the samples examined. only n. ceranae was detected in the analyzed samples (figure 1). fig 1. agarose gel (2%) showing duplex nosema apis and n. ceranae pcr products. m: 100 bp marker. column 16: samples showing n. ceranae amplification. column c+: positive control – nosema ceranae (218 bp) and nosema apis (321 bp). column c-: negative control. table 1. average number of spores of nosema ceranae x 103 per bee (or per milliliter) collected in entrance of the hives, and percentage of infestation of varroa destructor mites in adult honeybees collected in brood area in two collection periods in stationary and migratory apiaries. collection 1 (october) collection 2 (may) stationary apiaries (n=33) migratory apiaries (n=39) stationary apiaries (n=29) migratory apiaries (n=23) spores of n. ceranae (x 103) 387±554a 361±687a 1.167±1.202b 1.082±1.277b v. destructor (%) 2.83±1.97a 3.54±2.32a 9.48±6.15b 6.34±6.58b means followed by different letters differ statistically (p< 0.01). the number of spores of n. ceranae per bee was significantly higher in the autumn compared to the spring. we did not find a statistical significance in the number of n. ceranae spores based on the type of management adopted (stationary vs migratory) (table 1 and 3). the levels of v. destructor infestation of adult bees covering the brood area, the average quantities of infested brood cells of workers, adult female mites per cell and cells containing descendants of the mite were significantly higher in spring of 2010 than in autumn of 2011 (table 1, 2 and 3). sociobiology 64(1): 42-49 (march, 2017) 45 however, when comparing the number of descendants, number of viable offspring per cell, and the reproductive potential, no significant differences were observed between the collection collection 1 (october) collection 2 (may) stationary apiaries (n=33) migratory apiaries (n=37) stationary apiaries (n=26) migratory apiaries (n=22) adult females (total) 1.68±2.28a 1.31±1.97a 3.58±3.40b 2.87±4.03b descendants (total) .24±4.36c 1.42±2.62d 5.15±6.19c 2.52±3.96d infested cells (%) 1.91±2.31e 1.27±1.73e 4.23±3.29f 2.59±3.51f infested cells with offspring (%) 1.18±1.51g 0.75±1.32g 2.69±2.74h 1.63±2.27h viable offspring (total) 1.94±3.01i 0.83±1.54j 3.34±4.46bi 1.00±1.57j reproductive potential 1.08±2.37k 0.17±0.35l 0.78±0.79k 0.22±0.33l means followed by different letters differ statistically (p< 0.01). table 2. average number of adult females, descendants, infested brood cells (%), infested cells with descendants (%), total viable offspring and reproductive potential of the mite varroa destructor in brood combs, in two collections, in stationary and migratory apiaries. means followed by different letters differ statistically (p< 0.01). collection type of management p value f value df p value f value df nosema ceranae 0.0002 15.21 1 0.9049 0.01 1 v. destructor in adult honey bees 0.0026 9.58 1 0.3772 0.79 1 adult female mites 0.0016 10.50 1 0.0934 2.86 1 descendants 0.0325 4.68 1 0.0042 8.54 1 infested cells 0.0004 13.17 1 0.0144 6.17 1 infested cells with offspring 0.0014 10.66 1 0.0346 4.57 1 viable offspring 0.0904 2.92 1 0.0010 11.43 1 reproductive potential 0.6467 0.21 1 <.0001 16.99 1 table 3. statistical results of infection of n. ceranae in adults honey bees and infestation of v. destructor in adult honey bees and brood cells. periods (table 2 and 3). furthermore, the reproduction of the mites was significantly higher in stationary hives compared to migratory apiaries. discussion although the movement of hives between different locations is cited as one of the possible causes for the population decline of honey bee colonies, our results showed no statistically significant differences in infestation rates of pathogens analyzed in stationary and migratory apiaries. our results are similar to those inferred by stokstad (2007). based on data from the united states, where this practice has been adopted for many years, the author infers that the movement of hives alone cannot explain the recent hive collapse problem (van engelsdorp et al., 2013). the difference (table 1) observed in intensity of infection of n. ceranae between collections in october (spring) and may (autumn), both in stationary and migratory apiaries with a significant increase in the intensity of infection in the second collection, corroborates our earlier findings in the vale do paraíba region, where a higher intensity of infection was observed during the fall (santos et al., 2014). martinhernandez et al. (2009), studying bees in temperate regions, justified the higher intensities of infection found in the autumn based on the temperature drop, which leads to a prolonged period during which bees are confined in hives, increasing the risk of horizontal transmission of pathogens. higes et al. (2008) also observed higher n. ceranae infection intensity in the cooler months and lower levels in early spring. analysis of european and africanized bees has shown considerable prevalence of the species n. ceranae (paxton et al., 2007; teixeira et al., 2013), strengthening the hypothesis that n. ceranae overlap n. apis in infestation of honey bee colonies. the same was observed in this study, as in other recent studies (santos et al., 2014; guimarães-cestaro et al., 2016). another analysis of the presence of honey bee pathogens in turkey, tozkar et al. (2015) showed also observed a higher prevalence of the species n. ceranae in relation to n. apis. in the same study, unlike the results obtained here, the authors noted higher levels of infection by n. ceranae in migratory colonies than stationary colonies. l guimarães-cestaro – honey bee health in stationary and migratory apiaries46 according to the authors, it seems likely that the practice of migratory beekeeping allows the spread of disease, constituting a major threat to these insects. regarding the assessment of the infestation levels of the mite v. destructor on adult bees, there were considerably lower levels of varroa in both sampling periods and types of management (p> 0.01), comparing with other studies conducted in similar climatic conditions (de jong & gonçalves, 1998; lamb et al., 2014; santos et al., 2014; guimarães-cestaro et al., 2016). several factors can explain the significant increase in varroa infestation rates in autumn. dooremalen et al. (2012), measured infestation rates in a temperate region and concluded that losses of colonies due to mites are higher during the colder months. moretto et al. (1991) stated that the type of climate has a strong influence on the mite infestation rate, which is higher in colder regions. in that study, the authors found infestation rates of 3.5%, 5.11% and 11.37% in municipalities with respective average annual temperatures of 21 ºc (ribeirão preto, sp), 18 ºc (rio do sul, sc) and 13 ºc (são joaquim, sc). carneiro et al. (2014), in a yearlong evaluation of apiaries in blumenau (sc), observed significant differences in mean values of mite infestation, with the highest rate occurring in winter. moreover, the shortage of rain, decrease in the amount of pollen and consequent lack of brood in colonies should also be considered as causes of increased mite infestation in the autumn months (moretto, 1997). furthermore, defense mechanisms such as grooming behavior tend to increase as the brood number declines (junkes et al., 2007). despite the differences observed in varroa levels between the sampling periods (autumn and spring) and the management types (stationary and migratory), the rates of infestation by adult female mites can be considered low compared with the result of 19.8% obtained by zhang et al. (2000) in new zealand, or by martin et al. (1994, 2001), ranging from 18 to 49% in bulgaria, 15 to 40% england and 6 to 42% in the united kingdom. according to van engelsdorp et al. (2013), migratory colonies have higher risk of mortality and morbidity due to queen-related events (losses/replacement) and low brood quality. the migratory hives from which we obtained samples had just returned from several locations where they had been intensively managed for honey production. as such, these hives had not been kept in conditions as satisfactory as the stationary apiaries with respect to the brood quantity and quality. this condition may have negatively influenced the reproductive capacity of the mite, explaining the smaller infestation rate in brood comb samples observed in migratory hives. as argued by correia-marques et al. (2003), it is unfortunately virtually impossible to make an objective comparison with the various studies on reproduction of mites due to a lack of uniformity of the variables analyzed. according to carneiro et al. (2014), despite the increase in reproductive capacity, infestation levels remain low, which probably means that defense mechanisms, such as grooming and hygienic behavior, have allowed colonies of africanized honey bees maintain low infestation rates in brazil (de jong, 1997; carneiro et al., 2014.). according to van engelsdorp et al. (2013), migratory colonies have higher risk of mortality and morbidity due to queen-related events (losses/replacement) and low brood quality. the migratory hives from which we obtained samples had just returned from several locations where they had been intensively managed for honey production. as such, these hives had not been kept in conditions as satisfactory as the stationary apiaries with respect to the brood quantity and quality. this condition may have negatively influenced the reproductive capacity of the mite, explaining the smaller infestation rate in brood comb samples observed in migratory hives. in relation to infestation rates of mite v. destructor, the results obtained here corroborate with the inferences made by correia-marques et al. (2003). according the author, it is unfortunately virtually impossible to make an objective comparison with the various studies on reproduction of mites due to a lack of uniformity of the variables analyzed. in brazil, the infestation rates of mites are considered low, even though an increase in their reproductive capacity has been observed (carneiro et al., 2014). here we also observed low infection rates, as seen by santos et al. (2014) and guimarães-cestaro et al. (2016) in the region of vale do paraíba and vale do ribeira, respectively and carneiro et al. (2014) in blumenau. these results may be related to defense mechanisms performed by africanized honey bees, such as hygienic behavior (de jong et al., 1997; carneiro et al., 2014). the parameters evaluated in this study, in the region and periods studied, indicate that seasonality affects the health of colonies of africanized a. mellifera but does not depend on the type of management adopted (stationary or migratory). the intensity of infection by n. ceranae and infestation by v. destructor were higher during the fall compared to the spring. acknowledgments we thank carmen l. monteiro for assistance in the analyses, cnpq for specific funding (mapa / cnpq, process 2008-0, coordinated by erica w. teixeira) and apta-sp for institutional support. references anderson, d. l. & trueman, j. w. h. 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(2000). notes on varroa destructor (acari: varroidae) parasitic on honeybees in new zealand. systematic & applied acarology special publications 5: 9-14. doi: 10.11158/saasp.5.1.2. 317 two new species of stenamma (hymenoptera: formicidae) from indian himalaya with a revised key to the palaearctic and oriental species by himender bharti, irfan gul & yash paul sharma abstract two new species of genus stenamma viz stenamma wilsoni sp. nov. and stenamma jhitingriense sp. nov. are described from indian himalaya. this adds two more species to the genus from indian himalaya, with only stenamma kashmirense baroni urbani, 1977 described earlier. a revised key to 26 species from palaearctic and oriental is provided here with. key words: stenamma, taxonomy, myrmicinae, ants, new species, key, indian himalaya introduction genus stenamma westwood 1839 is widely distributed with 47 extant species (bolton 2011; liu & xu 2011). recently liu & xu (2011) described three new species of this genus and provided a revised key for the known species of palaearctic and oriental regions. based on molecular evidence branstetter (2009) redefined stenamma as monophyletic genus. earlier dubois (1998) had revised the palaearctic and oriental species of this genus. however, the representation of this genus has been poor from indian himalaya, as only one species, stenamma kashmirense, has been reported by baroni urbani (1977). during the course of present study two new species have been recorded and these differ considerably from already described species of this genus. a revised key (modified after liu & xu 2011) which includes 26 known species from palaearctic and oriental regions has been provided. materials and methods the specimens were collected by the handpicking method. digital color images were prepared by michael branstetter (california academy of scidepartment of zoolog y and environmental sciences, punjabi university, patiala147002, india e-mail: himenderbharti@gmail.com; irfangulhhh@gmail.com; yashraina007@gmail.com 318 sociobiolog y vol. 59, no. 2, 2012 ences) vide specimen number casent 0126224 (stenamma wilsoni sp. nov.) and casent 0126222 (stenamma jhitingriense sp. nov.). taxonomic analysis was conducted using a nikon smz 1500 stereo zoom microscope. morphological terminolog y for measurements and indices (given in millimeters) includes: hlhead length: the length of the head capsule excluding the mandibles, measured in full face view in a straight line from the midpoint of the anterior clypeal margin to the mid-point of the occipital margin. hwhead width: the maximum width of the head in full face view, excluding the eyes. slscape length: the maximum straight line length of the scape, excluding the basal constriction or neck that occurs just distal of the condylar bulb. pwpronotal width: the maximum width of the pronotum in dorsal view. mlmesosomal length: the diagonal length of the mesosoma in lateral view from the point at which the pronotum meets the cervical shield to the posterior basal angle of the metapleuron. edeye diameter: the maximum diameter of the eye. plpetiole length: maximum length of petiole, measured from the juncture with propodeum to the juncture with postpetiole. phpetiole height: the perpendicularly maximum height of the petiole, measured from the apex of the node to venter of petiole. dpwdorsal petiole width: maximum width of petiole, measured across node in dorsal view. pplpostpetiole length: maximum length of postpetiole, measured from the juncture with petiole to the juncture with gaster. pphpostpetiole height: the perpendicularly maximum height of the postpetiole, measured from the apex of the postpetiolar node to the venter of postpetiole. ppwpostpetiole width: maximum width of postpetiole, measured across the postpetiolar node in dorsal view. gl: gaster length: length of the gaster in lateral view from the anteriormost point of first gastral segment to the posterior-most point. tltotal length: hl+ml+pl+ppl+gl. cicephalic index: hw×100/hl. 319 bharti, h. et al. — two new species of stenamma from indian himalaya siscape index: sl×100/hw. pipetiole index: ph×100/pl. ppipostpetiole index: pph×100/ppl. description stenamma wilsoni sp. nov. (figs. 1-3) holotype worker: india, himachal pradesh, reckongpeo, 31.540432n, 78.272352e, 2050m above msl, 02.ix.2008, hand picking. paratypes; 3 workers, same data as holotype (coll. irfan gul and yash paul sharma). depository: pupac, punjabi university patiala ant collection, patiala, india. description of worker (figs. 1-3): worker measurements: tl 3.58-3.72(3.61); hl 0.78-0.83(0.78) ; hw 0.66-0.71(0.66); sl 0.58-0.62(0.58); pw 0.42-0.46(0.42); ml 0.981.04(0.98); ed 0.07-0.08(0.07); pl 0.40(0.40); ph 0.21-0.22(0.22); dpw 0.14-0.15(0.14); ppl 0.27-0.30(0.27); pph 0.21-0.22(0.21); ppw 0.210.23(0.21); gl 1.11-1.18(1.18); ci 84.62-86.25(84.62); si 86.96-87.9(87.9); pi 181.82-190.5(190.5); ppi 73.33-77.78(77.78) (4 individuals measured) head: head distinctly rectangular, longer than broad in full face view; occipital margin straight; occipital corners less distinct, moderately round; lateral sides almost parallel; anterior clypeal margin convex, slightly concave in the middle; eyes small, with 5 facets in their greatest diameters, located below the mid points of the lateral sides of head; mandibles triangular, the masticator y border with 3 prominent apical teeth, and 5 less distinct basal teeth; antennae short, 12-segmented, scape stout, falling short by about 1/6 of its length to reach the occipital corners, club 4-segemented. mesosoma, petiole and postpetiole: in profile view promesonotum high and convex nearly arched, promesonotal suture less distinct; fig. 1. stenamma wilsoni sp. nov.; head, dorsal view. 320 sociobiolog y vol. 59, no. 2, 2012 figs. 2-3. stenamma wilsoni sp. nov.; 2: body, lateral view, 3: body, dorsal view. 321 bharti, h. et al. — two new species of stenamma from indian himalaya mesometanotum suture well marked making a wide groove; propodeum distinctly lower than promesonotum, convex from side to side, forms a gentle slope towards apex; propodeal spines short, as long as 1/3 the range of their bases; propodeal plates broad, as long as is the length of propodeal spines, posterodorsal corner bluntly angled, posteroventral corner rounded; petiole long, petiolar node approximately as long as anterior peduncle, anteroventral face slightly convex, posterioventral face slightly depressed, anteroventral corner of petiole bluntly angled; postpetiolar dorsum round, ventral face weakly concave, anteroventral corner slightly extruding, tooth like. gaster: gaster ovate, smooth and shining all over. sculpture: head retirugose, except longitudinal irregular rugae below eyes and rugae in between the frontal carinae which run to the occiput; mandibles with less distinct striations; clypeus smooth; promesonotal dorsum rugose, rugae sparse, the central rugae longitudinal, sides of pronotum with indistinct rugae; propodeum and sides of mesonotum retirugose; propodeal declivity smooth; dorsum of petiolar peduncle smooth without longitudinal carina; petiole and postpetiole interweaved with fine, less distinct longitudinal rugae; gaster smooth and shining except a few short rugae at base. pilosity: body clothed with suberect to erect hairs, more abundant on head and gaster; on mesosomal surface hairs erect, sparse and scattered; shorter subdecumbent hairs on antennae and legs; decumbent pubescence on antennae and legs, more dense on antennal funiculus. color: head, mesosoma, petiole, postpetiole and middle of gaster reddish brown; mandibles, antennae, legs, and remaining part of gaster yellowish brown; eyes black; pilosity yellowish white. distribution and habitat: the species has been collected from a single locality of north-west region of indian himalaya. the specimen has been collected by hand picking, from a rotten log of wood, in open woodland on a hill slope. etymology: the species is named in the honour of prof. e. o. wilson. remarks: the species is significantly different from already described species of this genus. the species has a very distinct sculpture of head and mesosoma, differs in shape and size of propodeal plates, in propodeal spines and petiole. due to petiolar node as long as its anterior peduncle and scape not reaching to the occipital corners, this species can be easily separated from 322 sociobiolog y vol. 59, no. 2, 2012 stenamma kashmirense baroni urbani, 1977 (the only species reported from indian himalaya hitherto). however stenamma wilsoni sp. nov. is somewhat allied to stenamma jeriorum dubois, 1998 and stenamma lippulum nylander, 1849, but can be easily separated from former by parallel sides of head and propodeal spines as long as are the propodeal plates, where as lateral sides of head are strongly convex in stenamma jeriorum and propodeal spines shorter than propodeal plates. from stenamma lippulum this new species is considerably distinct on the basis of the sculpture of head, where the rugae make concentric loop like structures above eyes, whereas in this new species sculpture of head is retirugose. stenamma jhitingriense sp. nov. (figs 4-6) holotype worker: india, himachal pradesh, jhitingri, 32.006475n, 76.839237e, 1750m above msl, 27.vi.2010, hand picking. no paratypes. (coll. yash paul sharma and irfan gul). depository: pupac, punjabi university patiala ant collection, patiala, india. description of worker (figs. 4-6): worker measurements: tl (3.44); hl (0.77); hw (0.66); sl (0.57); pw (0.43); ml (1.0); ed (0.07); pl (0.40); ph (0.20); dpw (0.15); ppl (0.29); pph (0.21); ppw (0.21); gl (0.98); ci (85.71); si (86.36); pi (200); ppi (72.41) (1individual measured). head: head rectangular, longer than broad in full face view; occipital margin straight; occipital corners less distinct, more round; lateral sides almost parallel; anterior clypeal margin convex, emarginate in the middle; eyes small, with 4 less distinct facets in their greatest diameters, located below the mid points of the lateral sides of head; mandibles triangular, the masticatory border with 3 prominent apical fig. 4. stenamma jhitingriense sp. nov.; head, dorsal view. 323 bharti, h. et al. — two new species of stenamma from indian himalaya figs. 5-6. stenamma jhitingriense sp. nov.; 5: body, lateral view, 6: body, dorsal view. 324 sociobiolog y vol. 59, no. 2, 2012 teeth, and 5 less distinct basal teeth; antennae short, 12-segmented, scape stout, falling short by nearly 1/6 of its length to reach the occipital corners, club 4-segmented. mesosoma, petiole and postpetiole: in profile view promesonotum high and convex nearly arched, promesonotal suture less distinct; mesometanotum suture well marked making a deep and wide groove; propodeum distinctly lower than promesonotum, dorsum more flat, forms a gentle slope towards apex; propodeal spines short, acute, as long as 2/5 the range of their bases; propodeal plates broad, roughly rectangular, slightly shorter than propodeal spines, bluntly angled on posterodorsal and posteroventral corners; petiole long, petiolar node approximately as long as anterior peduncle, anteroventral face straight, posteroventral face slightly concave, anteroventral corner of petiole bluntly angled; postpetiolar dorsum round, ventral face weakly concave, anteroventral corner strongly extruding. gaster: gaster ovate, smooth and shining all over. sculpture: head retirugose, except longitudinal irregular rugae in between the frontal carinae; mandibles indistinctly striate; clypeus smooth; promesonotal dorsum with distinct retirugose sculpture, sides of pronotum with indistinct rugae; propodeum and sides of mesonotum retirugose; propodeal declivity smooth; dorsum of petiolar peduncle with a fine longitudinal central carina; petiole and postpetiole interweaved with fine, less distinct longitudinal rugae; gaster smooth and shining except a few short rugae at base. pilosity: body clothed with sub-erect to erect hairs, more abundant on the head and gaster; on mesosomal surface hairs erect, sparse and scattered; shorter subdecumbent hairs on antennae and legs; decumbent pubescence on antennae and legs, more dense on antennal funiculus. color: head, mesosoma, petiole, postpetiole and middle of gaster blackish brown; mandibles, antennae, legs, and remaining part of gaster yellowish brown; eyes black; pilosity yellowish white. distribution and habitat: the specimen has been collected from a forest area of jhitingri, himachal pradesh in north-west himalayas. the specimen has been collected from leaf litter by handpicking. etymology: the species is named after the type locality, jhitingri. remarks: stenamma jhitingriense sp. nov. is significantly different from previously reported species of this genus due to the following combination 325 bharti, h. et al. — two new species of stenamma from indian himalaya of characters: blunt anteroventral corner of petiole, scape not reaching occipital corners, equal length of petiole and peduncle, longer propodeal spines than propodeal plates and blackish brown color of body. however it shows few affinities with stenamma gurkhale dubois, 1998, stenamma punctiventre emery, 1908 and stenamma koreanense lyu, dubois & cho, 2002. in stenamma gurkhale and stenamma koreanense the petiolar anteroventral corner is acutely toothed, stenamma punctiventre has an anteroventral corner of petiole extended and finger-like, but in stenamma jhitingriense the anteroventral corner of the petiole is blunt. it is easily separated from stenamma kashmirense baroni urbani, 1977, as in stenamma kashmirense the peduncle is less than the half the length of petiole, and the scape reaches the occipital corners distinctly. a revised key to the known palaearctic and oriental species of stenamma based on the worker caste (modified after liu & xu 2011) 1. in full face view, antennal scapes distinctly surpassed occipital corners ..2 in full face view, antennal scapes reached to or not reached to occipital corners ...................................................................................................................9 2. in profile view, petiolar node distinctly shorter than anterior peduncle ....3 in profile view, petiolar node as long as or longer than anterior peduncle .................................................................................................................................4 3. in full face view, occipital corners rounded. in profile view, metanotal groove deeply depressed. propodeal plates narrow, nearly triangular (dubois, 1998: figs. 200-202). (distribution: japan)………………………………………. ..............................................................s. nipponense yasumatsu & murakami in full face view, occipital corners roundly prominent. in profile view, metanotal groove shallowly depressed. propodeal plates broad, nearly trapezoid (figs. 1-3). (distribution: china (yunnan province)) ………… …………………………………………............... .....................s. ailaoense liu & xu 4. in full face view, occipital margin roundly convex ......................................5 in full face view, occipital margin nearly straight .........................................6 5. in profile view, dorsum of promesonotum evenly convex. propodeal dorsum straight. propodeal plates nearly trapezoid, truncated at apices (dubois, 1998: figs. 187-190). (distribution: algeria, morocco, tunisia) .............. 326 sociobiolog y vol. 59, no. 2, 2012 .................................................................................................. s. msilanum forel in profile view, dorsum of promesonotum nearly straight. propodeal dorsum weakly convex. propodeal plates nearly semicircular, rounded at apices (dubois, 1998: figs. 240-242). (distribution: southern europe, mostly france and italy) ............................................................... s. petiolatum emery 6. in full face view, head broadest at back, narrowed forward, lateral sides relatively straight .................................................................................................7 in full face view, head broadest in the middle, lateral sides evenly convex .................................................................................................................................8 7. mandibles with 7 teeth. in profile view, anteroventral corner of petiole extruding and forming a rightly angled tooth (dubois, 1998: figs. 328330). (distribution: england, belgium) ..............s. westwoodii westwood mandibles with 9 teeth. in profile view, anteroventral corner of petiole weakly convex, not forming a tooth (dubois, 1998: figs. 184-186). (distribution:russia) ................................................... .s. lippulum nylander 8. in profile view, dorsum of promesonotum evenly convex. dorsum of petiolar node roundly prominent (dubois, 1998: figs. 280-282). (distribution:spain) ........................................................... s. sardoum emery in profile view, dorsum of promesonotum nearly straight. dorsum of petiolar node rounded (dubois, 1998: figs. 277-279). (distribution: sardinia) .. .................................................................................................. s. sardoum emery 9. in full face view, antennal scapes distinctly not reached to occipital corners ..................................................................................................................... 10 in full face view, antennal scapes reached to occipital corners ................ 16 10. in profile view, petiolar node distinctly longer than anterior peduncle (dubois, 1998: figs. 310-314). (distribution: eastern russia) .................. .............................................................................................s. ussuriense arnol’di in profile view, petiolar node approximately as long as anterior peduncle .............................................................................................................................. 11 11. in profile view, anteroventral corner of petiole extended and finger-like (dubois, 1998: figs. 269-271). (distribution: morocco) ........................ ........................................................................................... s. punctiventre emery in profile view, anteroventral corner of petiole prominent, tooth-like or bluntly angled ................................................................................................... 12 12. in profile view, anteroventral corner of petiole acutely toothed (lyu etal., 327 bharti, h. et al. — two new species of stenamma from indian himalaya 2002: figs. 1-4). (distribution: korea) ........................................................... ................................................................... s. koreanense lyu, dubois & cho in profile view, anteroventral corner of petiole bluntly angled ............... 13 13. in full face view, lateral sides of head strongly convex. in profile view, propodeal spines shorter than propodeal plates (dubois, 1998: figs. 148152). (distribution: pakistan) ......................................... s. jeriorum dubois in full face view, lateral sides of head weakly convex or parallel. in profile view, propodeal spines as long as or longer than propodeal plates ........ 14 14. in full face view, lateral sides of head weakly convex. in profile view, propodeal spines as long as or longer than propodeal plates. head dorsum above eyes with rugae forming concentric loop like structures (dubois, 1998: figs. 170-177). (distribution: azerbaijan, georgia, southern russia) ...... ............................................................................................ s. lippulum nylander in full face view, lateral sides of head parallel. in profile view, propodeal spines longer or equal to propodeal plates. head dorsum above eyes with rugae not forming loop like structures. (distribution: himalaya) ........ 15 15. dorsum of petiolar peduncle smooth without a central longitudinal carina. postpetiolar anteroventral corner slightly extruding, tooth like. propodeal plates broad, as long as is the length of propodeal spines ............................ ....................................................................................................s. wilsoni sp. nov. dorsum of petiolar peduncle with a central longitudinal carina. postpetiolar anteroventral corner strongly extruding. propodeal plates broad, roughly rectangular, slightly shorter than propodeal spines ...................................... .......................................................................................... s. jhitingriense sp. nov. 16. in profile view, petiolar node distinctly shorter than anterior peduncle . .............................................................................................................................. 17 in profile view, petiolar node as long as or longer than anterior peduncle .............................................................................................................................. 18 17. eyes with 8-9 ommatidia in the maximum diameter. mesopleura retirugose (dubois, 1998: figs. 226-228). (distribution: japan, china (sichuan province) ............................................................................. s. owstoni wheeler eyes with 4 ommatidia in the maximum diameter. mesopleura longitudinally rugose (figs. 4-6 ). (distribution: china (yunnan province)) .......... ..................................................................................... s. wumengense liu & xu 18. in profile view, petiolar node distinctly longer than anterior peduncle .. 328 sociobiolog y vol. 59, no. 2, 2012 .............................................................................................................................. 19 in profile view, petiolar node about as long as anterior peduncle ........... 21 19. in profile view, propodeal spines longer than propodeal plates. propodeal plates triangular, bluntly angled at apices (dubois, 1998: figs. 191-193). (distribution: algeria, morocco, tunisia) .................... s. msilanum forel in profile view, propodeal spines shorter than propodeal plates. propodeal plates nearly trapezoid, truncated at apices ................................................ 20 20. in profile view, dorsum of promesonotum roundly convex. propodeal spines posteriorly curved. dorsum of petiolar node narrowly prominent (dubois, 1998: figs. 131-135). (distribution: tajikstan) ............................ ....................................................................................... s. hissarianum arnol’di in profile view, dorsum of promesonotum relatively straight. propodeal spines not posteriorly curved. dorsum of petiolar node broadly rounded (dubois, 1998: figs. 251-255). (distribution: kazakhstan, kirghizia) .. .................................................................................. s. picetojuglandeti arnol’di 21. in profile view, anteroventral corner of petiole acutely toothed. petiolar node lower, with dorsum rounded (dubois, 1998: figs. 126-129). (distribution: nepal) ................................................................ s. gurkhale dubois in profile view, anteroventral corner of petiole bluntly angled or roundly prominent. petiolar node higher, with dorsum roundly or narrowly prominent ..................................................................................................................... 22 22. in full face view, head distinctly narrowed forward ............................... 23 in full face view, head not distinctly narrowed forward ........................ 25 23. in full face view, occipital margin weakly convex. in profile view, dorsum of petiolar node broadly rounded, both anterior and posterior faces convex (dubois, 1998: figs. 290-295). (distribution: from spain to turkey) … ...............................................................................................s. striatulum emery in full face view, occipital margin straight. in profile view, dorsum of petiolar node narrowly prominent, both anterior and posterior faces relatively straight ............................................................................................................... 24 24. in full face view, occipital corners roundly prominent. in profile view, dorsum of promesonotum weakly convex (dubois, 1998: figs. 237-239). (distribution: southern europe, mostly france and italy) .......................... .............................................................................................. s. petiolatum emery in full face view, occipital corners rounded. in profile view, dorsum of 329 bharti, h. et al. — two new species of stenamma from indian himalaya promesonotum strongly convex (dubois, 1998: figs. 111-119). (distribution: georgia, southern russia) .......................................s. georgii arnol’di 25. in full face view, occipital margin evenly roundly convex (dubois, 1998: figs. 100-102). (distribution: throughout europe) .. s. debile (foerster) in full face view, occipital margin straight or weakly concave in the middle ........................................................................................................................ 26 26. in profile view, propodeal dorsum nearly horizontal .............................. 27 in profile view, propodeal dorsum slope down backward ....................... 28 27. in profile view, dorsum of promesonotum roundly convex. propodeal dorsum weakly convex (dubois, 1998: figs. 214-217). (distribution: france (corsica), italy (sardinia), spain) ..s. orousseti casevitz-weulersse in profile view dorsum of promesonotum nearly straight. propodeal dorsum straight (dubois, 1998: figs. 163-168). (distribution: russia (kuril) ... ..............................................................................................s. kurilense arnol’di 28. in profile view, propodeal declivity roundly concave. anterior face of petiolar node formed a strong depression with peduncle (dubois, 1998: figs. 284-288). (distribution: uzebekistan) ........... s. sogdianum arnol’di in profile view, propodeal declivity straight. anterior face of petiolar node formed a weak depression with peduncle .................................................. 29 29. in profile view, propodeal spines shorter than propodeal plates. propodeal plates truncated at apices (dubois, 1998: figs. 154-161). (distribution: india (kashimir), pakistan) ......................... s. kashmirense baroni urbani in profile view, propodeal spines as long as propodeal plates. propodeal plates rounded at apices (figs. 7-9). (distribution: china (tibet, sichuan province) ................................................................. s. yaluzangbum liu & xu acknowledgments sincere thanks to michael branstetter (california academy of sciences) for getting this species digitized. financial assistance rendered by department of science and technolog y (grant no. sr/so/as-65/2007), govt. of india, new delhi is gratefully acknowledged. references baroni urbani, c. 1977. ergebnisse der bhutan-expedition 1972 des naturhiswrischen museums in basel. hymenoptera: fam. formicidae. genus stenamma, con una nuova species del kashmir. enwmologica basiliensia 2: 415-422. 330 sociobiolog y vol. 59, no. 2, 2012 bolton, b. 2011. bolton’s catalogue and synopsis, in http://gap.entclub.org/ “ version: 3 january 2011. branstetter, m.g. 2009. the ant genus stenamma westwood (hymenoptera: formicidae) redefined, with a description of a new genus propodilobus. zootaxa 2221: 41-57. dubois, m. b. 1998. a revision of the ant genus stenamma in the palaearctic and oriental regions. sociobiolog y 29: 193-4·03. liu, x. & z.h. xu 2011. three new species of the ant genus stenamma (hymenoptera: formicidae) from himalaya and the hengduan mountains with a revised key to the known species of the palaearctic and oriental regions. sociobiolog y 58: 733747. westwood, j.o. 1839. an introduction to the modern classification of insects; founded on the natural habits and corresponding organisation of the different families 2 (part 11), pp. 193224. london. doi: 10.13102/sociobiology.v60i4.484-486sociobiology 60(4): 484-486 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 laboratory rearing and niche resources of pseudacteon spp. coquillett (diptera: phoridae) parasitoids of solenopsis saevissima (smith) (hymenoptera: formicidae) ma pesquero, apa vaz, fv arruda the fire ants solenopsis invicta buren and solenopsis saevissima (smith) have wide distribution in south america (trager, 1991). the workers of these species are aggressive and have a painful sting, with the colonies located on the surface of the soil, thus increasing the chances of accidents with people. after the inadvertent introduction in the united states, s. invicta has victimized thousands of people, including records of deaths from anaphylactic shock (caldwell et al., 1999). the geographical distribution in the central region of south america may have reduced the risk of dispersal of s. saevissima to other countries via sea transport, as was probably the case with s. invicta from northern argentina in the united states (caldera et al., 2008). however, urban areas infested with s. saevissima were reported in the amazon region (brazil, 2008), suggesting the possibility of this species becoming a pest, and previous efforts to establish control strategies are important in preventing damage, whether it be economic, ecological or health related. in brazil, nine parasitoids species of pseudacteon coquillett are associated with s. saevissima (pesquero & dias, 2011). confirmation of parasitism by these species is important for future biological abstract solenopsis saevissima (smith) is associated with a group of nine pseudacteon coquillett species in brazil. adult female flies of pseudacteon affinis borgmeier, pseudacteon dentiger borgmeier and pseudacteon disneyi pesquero were created in a laboratory from parasitized workers of s. saevissima. the initial development of pseudacteon cultellatus borgmeier was faster than the other phorid species, taking 12 days to kill the host workers. similar to the group of phorid species parasitizing solenopsis invicta buren, the daily period of activity and body size are important factors to be considered in the use of these natural enemies in future programs of biological control of s. saevissima. sociobiology an international journal on social insects universidade estadual de goiás, morrinhos, goiás, brazil short note article history edited by: evandro n silva, uefs, brazil received 26 july 2013 initial acceptance 07 september 2013 final acceptance 02 october 2013 keywords community, biological control, fire ant brazil corresponding author marcos antônio pesquero universidade estadual de goiás unidade universitária de morrinhos rua 14, 625. jardim américa. morrinhos, goiás, brazil cep 75.650-000 e-mail: mapesq@ueg.br control programs of s. saevissima, but it still remains unknown for many pseudacteon species. thus, the aim of this study was to describe seasonality and body size distribution for understanding the pseudacteon community structure associated with s. saevissima and to prove the parasitism in a laboratory. this study was conducted in the municipalities of goiânia and morrinhos, goiás, brazil, in the region originally occupied by cerrado and dominated by cattle and soybean. data on the abundance of parasitoids and air temperature were obtained monthly in one-hour intervals throughout the day period, from sep/2010 to aug/2011, in eight sites surrounding the urban area. all the mounds of the fire ants were counted and disturbed to attract parasitoids which were collected, identified (porter & pesquero, 2001) and transferred for closed trays containing 5g of workers of s. saevissima maintained in quarantine for 45 days. a maximum of five individuals of the same species were transferred per tray for two hours, totaling up to 30 individuals of each pseudacteon species. these trays with parasitized ants were later taken to the laboratory and kept in a climatic chamber under consociobiology 60(4): 484-486 (2013) 493 trolled temperature (24 °c), photoperiod (12:12 h) and humidity (40-60%). the trays were checked daily to record the development of parasitoids. a maximum of 30 individuals of each parasitoid species were sacrificed for size measurements (length of hind tibia). the density of fire ant mounds, all identified as s. saevissima, was 37.5 ± 3.0 mounds/ha (n = 8). we captured 314 female flies on the 56 fire ant mounds identified as p. affinis (26.75%), p. cultellatus (24.52%), p. disneyi (16.56%), p. dentiger (14.65%), p. nudicornis (7%), the small biotype p. tricuspis (4.14%), p. fowleri (1.91%) and p. lenkoi (1.27%). ten individuals (3.18%) of p. solenopsidis were observed attacking workers on fire ant trails. the five most abundant species occurred in the eight sites of the two municipalities; p. lenkoi and p. solenopsidis occurred in two sites of the two municipalities; p. fowleri occurred in one site of the two municipalities and p. tricuspis small biotype occurred in three sites of goiânia. the four most abundant species were seasonally constant throughout the year. with the exception of p. dentiger that has been active in the hottest hours of the day (32.44 ± 0.41 °c, n = 46) (χ2 = 19.58, df = 2, p < 0.00001), p. affinis and p. disneyi were more active during the last hours of the day (χ2 = 23.74 and χ2 = 23.65, df = 2, p < 0.00001, respectively), and p. cultellatus was more active during the first hours of the day (χ2 = 33.26, df = 2, p < 0.000001) (fig 1). daily activity patterns have been previously described (pesquero et al., 1996), suggesting the importance that physical factors have on the niche dimension and coexistence of this group of insects. similarly, the gradient body size of worker ants seems to be an important resource to be shared among pseudacteon species (fowler, 1997). the body size of the four most abundant species differ significantly (table 1), and p. affinis parasitized larger workers ants compared with p. disneyi (mann-whitney test, z = -4.54, p < 0.00001) and p. cultellatus (mann-whitney test, z = -3.06, p < 0.005) (table 1). however, the worker ants parasitized by p. disneyi and p. cultellatus did not differ statistically. the low relative abundance and the small body size of p. tricuspis (table 1) differ from the observations of fowler (1977) in southeastern brazil, and these differences suggest the presence of two different species (kronforst et al., 2007). with the exception of p. solenopsidis all species of parasitoids found in the study sites attacked workers of s. saevissima in the trays. however, only one individual of p. affinis, p. dentiger and p. disneyi completed the development in the laboratory resulting in adults. it is likely that the high mortality of larvae and pupae was a result of the low rearing conditions (temperature of 24°c and humidity of 40-60%) compared with other studies (folgarait et al., 2005). the larval development time from the adult phorid flies attack to the ant host death was two days faster for p. cultellatus (12 days) compared with p. affinis (mann-whitney test, z = -3.40, p < 0.001) and p. disneyi (mann-whitney test, z = -3.72, p < 0.001) (table 1). the larval development period observed for the latter two species (14 days) is similar to that observed for p. obtusus on s. richteri (14,6 days) (folgarait et al., 2005). evidence of parasitism for only three of the nine species of pseudacteon associated with s. saevissima is a technical problem. the elevation of temperature and hu pseudacteon tibia length (mm) mean±sd n head width of parasitized ants (mm) mean ± sd n lifetime ant after parasitoid attack (days) n tricuspis * 0.266 ± 0.0223 12 nudicornis 0.296 ± 0.0202 17 disneyi 0.296 ± 0.0293 a 30 0.810 ± 0.101 31 14.161 ± 2.238 31 cultellatus 0.339 ± 0.0401 b 30 0.841 ± 0.116 29 12.310 ± 1.366 29 fowleri 0.392 ± 0.0314 06 affinis 0.426 ± 0.0561 c 30 0.967 ± 0.184 48 14.104 ± 3.019 48 solenopsidis 0.433 ± 0.0258 10 dentiger 0.471 ± 0.0403 d 30 lenkoi 0.477 ± 0.0252 04 * small biotype table 1. tibia length of pseudacteon species; head width of solenopsis saevissima and lifetime worker ant after parasitoid attack. only species with n > 30 were used in the analysis. means followed by the same letter are not statistically different (f = 121.82, df = 168, p < 0.01. tukey, p < 0.01). fig 1. daily seasonality of the four most abundant pseudacteon species associated with solenopsis saevissima. ma pesquero, apa vaz, fv arruda laboratory rearing and niche resources of pseudacteon spp.494 midity in the chambers of creation should result in greater reproductive success and extend the proof of parasitism for other species as well. similar to the group of phorid species parasitizing s. invicta, the daily period of activity and body size are important factors to be considered in the use of these natural enemies in future programs of biological control of s. saevissima. references brazil. (2008). empresa brasil de comunicação. formigas carnívoras atacam cidade do amazonas. brasília, 14/01/2008. available in: http://agenciabrasil.ebc.com.br/noticia/200801-14/formigas-carnivoras-atacam-cidade-do-amazonas. accessed: 10/09/2013. caldera, e.j., ross, k.g., deheer, c.j., shoemaker, d.d. (2008). putative native source of the invasive fire ant solenopsis invicta in the usa. biol. invasions, 10: 1457-1479. doi: 10.1007/s10530-008-9219-0. caldwell, s.t., s.h. schuman & w.m. simpson, jr. (1999). fire ants: a continuing community health threat in south carolina. j. s. c. med. assoc., 90: 231-235. folgarait, p.j., chirino, m.g., patrok, r.j.w. & gilbert, l.e. (2005). development of pseudacteon obtusus (diptera: phoridae) on solenopsis invicta and solenopsis richteri fire ants (hymenoptera: formicidae). environ. entomol., 34: 308-316. doi: dx.doi.org/10.1603/0046-225x-34.2.308. fowler, h.g. (1997). morphological prediction of worker size discrimination and relative abundance of sympatric species of pseudacteon (dipt., phoridae) parasitoids of the fire ant, solenopsis saevissima (hym., formicidae) in brazil. j. appl. entomol., 121(1-5): 37-40. doi: 10.1111/j.14390418.1997.tb01367.x. kronforst, m.r., folgarait, p.j., patrock, r.j.w., gilbert, l.e. (2007). genetic differentiation between body size biotypes of the parasitoid fly pseudacteon obtusus (diptera: phoridae). mol. phylogenet. evol., 43: 1178-1184. doi:10.1016/j. ympev.2006.09.003. pesquero, m.a., campiolo, s., fowler, h.g. & porter, s.d. (1996). diurnal pattern of ovipositional activity in two pseudacteon fly parasitoids (diptera: phoridae) of solenopsis fire ants (hymenoptera: formicidae). fla. entomol., 79: 455457. pesquero, m.a. & dias, a.m.p.m. (2011). geographical transition zone of solenopsis fire ants (hymenoptera: formicidae) and pseudacteon fly parasitoids (diptera: phoridae) in the state of são paulo, brazil. neotrop. entomol., 40: 647652. doi: dx.doi.org/10.1590/s1519-566x2011000600003. porter, s.d. & pesquero, m.a. (2001). illustrated key to pseudacteon decapitating flies (diptera: phoridae) that attack solenopsis saevissima complex fire ants in south america. fla. entomol., 84: 691-699. trager, j.c. (1991). a revision of the fire ants, solenopsis geminata group (hymenoptera: formicidae: myrmicinae). j. n. y. entomol. soc., 99: 141-198. 1547 performance of termidor® he high-efficiency termiticide co-pack with termidor® he technology against eastern subterranean termites reticulitermes flavipes and formosan subterranean termite coptotermes formosanus in laboratory trials and field applications by t. chris keefer1,2, robert t. puckett1, & roger e. gold1 abstract two laboratory and one field study were performed utilizing fipronil (c 12 h 4 cl l2 f 6 n 4 os), in the form of termidor® he co-pack with termidor® he technolog y. laboratory studies included glass tube bioassays and collateral transfer effect trials. glass tube bioassay results indicated that this formulation of termidor® he co-pack was efficacious against reticulitermes flavipes and coptotermes formosanus caused 100% mortality of both species by 72 h post-exposure. collateral transfer effect trials also showed efficacy of termidor® he co-pack with termidor® he technolog y against both r. flavipes and c. formosanus by causing 100% mortality by 96 h. termidor® he co-pack with termidor® he technolog y was also applied to structures as a post-construction treatment for remediation of subterranean termite infestations. in field trials, 12 termite structures were treated with termidor® he co-pack with termidor® he technolog y (hereafter referred to as termidor® he) and no subterranean termites were detected through 36 months post-treatment. key words: termidor® he co-pack, reticulitermes flavipes, coptotermes formosanus introduction fipronil (c 12 h 4 cl 2 f 6 n 4 os) is a phenylpyrazole (tingle et al. 2003) insec1department of entomolog y, texas a&m university, 2143 tamu, college station, tx 778432143. 1*corresponding author e-mail: tckeef@tamu.edu 1548 sociobiolog y vol. 59, no. 4, 2012 ticide first discovered in 1987 by rhone-poluenc agro (tomlin 2000). it was first introduced and registered in the united states in 1996 (bobe et al. 1998, ware 2000) for use against piercing, sucking chewing insect pests (bostian et al.1996). the mode of action for fipronil is interference with the passage of chloride ions through the gamma-aminobutyric acid-regulated chloride channel which disrupts the arthropods central nervous system (gant et al. 1998). fipronil has a wide range of uses on many different pests worldwide (yanese and andoh 1998). this compound is considered a broad-spectrum insecticide and has been demonstrated to be effacacious against a variety of insects including termites in urban environments (tomlin 2000). fipronil, as a liquid termiticide is sold astermidor® by basf corporation (research triangle park, nc). it is a non-repellent termiticide that can be applied as a soil barrier to protect structures from subterranean termite invasion. subterranean termites cause damage to structures throughout the united states, and cost estimates to control and repair damage caused by these insects are as high as $11 billion annually (su and scheffrahn 1998, su 2002). compounds that are non-repellent, have a delayed reaction on subterranean termites as compared to repellent products. it has been reported that fipronil can be transferred through the colony from individual to individual by trophallaxis, grooming, and contact with contaminated soil (kard 2003). methods and materials laboratory study i the objective of this work was to evaluate the efficacy of fipronil, in the form of termidor® he 0.06% ai and termidor® he 0.12% ai in glass tube bioassays against r. flavipes and c. formosanus subterranean termites. arenas consisted of glass tubes measuring 15 x 1.5 cm which were capped on both ends with aluminum foil (fig. 1) (gold et al. 1994). the following treatments were utilized in this study; termidor® he (0.06%) and termidor® he(0.12%), and untreated controls (water only). treatments to soil were made according to the manufacturer’s label, and untreated controls were identically ‘treated’, but with water only. equal volumes of termidor® he and the he technolog y additive were thoroughly mixed and diluted at a rate equivalent to 5.75 ml (0.06%) solution and 11.75 ml (0.12%) solution in 1549 keefer, c. et al. — performance of termidor® he high-efficiency termiticide fig. 1. schematic showing components of glass-tube arenas used in fst feeding cessation experiment (.waare (2000). 946 ml of water. soil was treated with the equivalent rate of 4 l per 3 m per 0.3 m of depth. twenty worker termites and two soldiers of either r. flavipes or c. formosanus were added to each arena after assembly. soldier termites were added to stimulate colony dynamics and stimulate termite acticity. posttreatment obser vations were made daily for 7 d. four replications of each treatment and untreated controls were constructed for this trial. data collected included distance tunneled and mortality, and these data were analyzed via analysis of variance (anova) at p < 0.05 and means separated using tukey’s hsd (honest significant difference) test at p < 0.05 (spss for windows, v. 18.0). laboratory study ii the objective of this work was to determine collateral transfer effects, if any, of termidor® he 0.125% ai treated soil utilizing varying ratios (donor:recipient) of r. flavipes and c. formosanus subterranean termite species through time. recently collected termites were utilized in these trials and they were allowed to adjust to the laboratory conditions for at least 48 hrs before beginning study. worker termites were placed in labeled petri dishes that corresponded to a final untreated arena to allow observation of collateral effects. these worker termites of both species were marked using methods similar to forschler 1994 with rust-o-leum® (vernon hills, il) orange fluorescent paint. marked termites were allowed to adjust to laboratory environment for 48 h. treatments to the soil were made with the following products; termidor® sc (bas 350 95 i) lot no. 1219502fi and termidor® he technolog y additive (bas 270 00 s) lot no. 502012. these products were used to create the termidor he co-pack and were mixed just prior to study initiation. treatments to soil were made according to the manufacturer’s 1550 sociobiolog y vol. 59, no. 4, 2012 label, and were identical to treatments in the laboratory study i (described above). untreated controls were ‘treated’ with water only. both the treated and the untreated control soils were allowed to dry for 24 h prior to exposing of termites to the soils. treatment arenas consisted of petri dishes measuring 9.0 x 2.5 cm. approximately 34 g of treated soil was placed in each petri dish. worker termites of either r. flavipes or c. formosanus were added to each arena after assembly, and were allowed to contact treated soil or untreated control sand for 30 min. after the contact period elapsed, these termites (donors), were placed in an untreated, clean petri dish (9.0 x 2.5 cm) with moistened #4 whatman® (maidstone, england) filter paper, and unexposed termites (recipients). four replications of each of five donor:recipient ratios and untreated controls were constructed for this trial. the following ratios of donor:recipient termite cohorts were utilized in these trials 20:0, 15:5, 10:10, 5:15, 1:19, 0:20 (untreated marked control), and 0:20 (untreated unmarked control). two soldier termites of corresponding species were placed in all arenas to simulate colony dynamics and stimulate worker termite activity. observations of worker termite mortality (donors and recipients) were made at 1, 4 and 24 hours after donor:recipient mixing, then daily until 100% mortality was reached in each treatment. data were analyzed statistically via analysis of variance (anova) at p < 0.05 and means separated using tukey’s hsd (honest significant difference) test at p < 0.05 (spss for windows, v. 18.0). field study the objective of this field study was to determine effectiveness, if any, of termidor® he against two species of subterranean termites when applied as a post-construction treatment to infested structures. eleven structures infested with r. flavipes and one infested with c. formosanus were utilized in this field study. all structures were located in the houston/galveston, tx area. soldier termites were collected from all 12 structures, identified with termite identification keys (scheffrahn and hope 1996), and stored in 100% ethanol as voucher specimens. structure owners were interviewed to verify that none of the 12 structures had received a subterranean termite treatment within the past 12 months prior to the termidor® he treatments. four of the structures were pier and beam construction, which included one 1551 keefer, c. et al. — performance of termidor® he high-efficiency termiticide structure infested with c. formosanus, the other eight structures consisted of monolithic slab construction. all 12 structures had at least one active subterranean termite mud tube leading from the soil into the structure that was associated with an exterior wall. a diagram was made of each structure prior to treatment to include all known points of subterranean termite infestation. all subterranean termite mud tubes were documented and marked relative to a permanent benchmark (ie. corner of the foundation). under the supervision of personnel from the center for urban and structural entomolog y and basf, all termidor® he applications were made by a licensed pest management professional. all termiticide applications were made following the termidor® sc exterior perimeter/localized interior directions for use according to the label with the following exceptions: all exterior drilling was done on 46 cm centers and all trenches were 5 x 10 cm. volume and concentration of finished dilution applied varied according to the treatment specifications described below. at each of the structures, one half of the desired volume of water was added to the tank and then the appropriate volume of termidor® he was introduced to the tank, and the remaining volume of water was then added to ensure thorough mixing of the dilution. in setting up for the study, the linear length for each structure was measured prior to the treatment to ensure the proper volume of finished dilution was applied. six structures each received one of the following treatments; 1. termidor® he 0.06% ai applied at 7.5 l of finished dilution/3 linear m /0.30 m of depth. 2. termidor® he 0.12% ai applied at 7.5 l of finished dilution/3 linear m /0.30 m of depth. there were no interior applications made at any of the 12 structures. a great plains industry inc. 01n series (wichita, ks) digital flow meter was utilized during this study to measure volumes of termiticide applied at each structure. all structures were treated between august 10 and august 13, 2009. post-treatment inspections were made on or about 1, 3, 6, 12, 24, and 36 months. post-treatment inspections included visual assessment as well as the use of mechanical tools such as a termatrac®, borescope, and/or infrared camera. 1552 sociobiolog y vol. 59, no. 4, 2012 results laboratory study i termidor® he 0.06% and termidor he® 0.12% both caused 100% mortality against both species of termites by 72 h post-treatment (table 1). mean mortality associated with the untreated controls was significantly less than (p <0.01) from all three treatments at the end of the trials (figs. 2 and 3), and was < 10% in both species. in the termidor® he 0.06% treatment c. formosanus tunneled a mean distance of 10.0±7.6 mm and r. flavipes tunneled 7.8±2.2 mm (table 1 and figs. 4 and 5). this tunneling distances in the termidor® he 0.12% ai treatment were c. formosanus 16.0±3.9 mm for c. formosanus and 8.3±4.6 mm fig. 2. mean percent mortality of coptotermes formosanus through time when exposed to soils treated with termidor® he. 1553 keefer, c. et al. — performance of termidor® he high-efficiency termiticide r. flavipes (table 1 and figs. 4 and 5). both termite species in the untreated control groups tunneled the maximum distance of 50 mm, and there were significant differences (p <0.05) in the distance tunneled by both species of termites in the treatments compared to the untreated controls at the end of the trial. laboratory study ii coptotermes formosanusat the 1 h observation period for c. formosanus, the 10:10 donor:recipient cohort was the only one that demonstrated mortality, but it was not significantly different from the other treatments or untreated controls (table 2 and figs. 6 and 7). there were significant differences (p <0.05) in total (donors:recipients) mortality beginning at the 4 h observafig. 3. mean percent mortality of reticulitermes flavipes through time when exposed to soils treated with termidor® he. 1554 sociobiolog y vol. 59, no. 4, 2012 tion period, the 10:10 cohort showed a significant difference (p<0.01) from the rest of the treatments and the untreated controls (table 2 and figs. 6 and 7). starting at the 72 h observation, all of the effects of the treatments were significantly different (p <0.05) from the untreated controls (table 2 and figs. 6 and 7). at the 1 and 4 h observation periods, the only donor populations with any mortality were in the 10:10 cohort (fig. 6). at the 72 h observation, all donor populations in the treatments had 100% mortality and the untreated controls had less than 10% mortality (fig. 6). at the 1 h observation, there was no mortality in the recipient populations in the treatments (fig. 7). at the 96 h observation, all recipient populations in the treatments had 100% mortality and were significantly different (p <0.05) from the untreated controls, which had less than 10% mortality throughout the study (fig. 7). reticulitermes flavipes at the 1h observation period, there were no significant differences in mortality between the treatments and the untreated controls (table 3). at the 4 h observation period the 20:0 cohort showed the greatest mortality followed by the 15:5 and then the 10:10 cohorts, respectively (table 3 and figs. 8 and 9). these three cohorts were significantly different (p <0.01) table 1. mean percent mortality and distance tunneled by coptotermes formosanus and reticulitermes flavipes in soils treated with termidor® he co-pack (two concentrations). treatment % mortality distance tunneled (mm)*** c . f o r m o sanus* r. flavipes** c. formosanus r. flavipes termidor he 0.06% 100.0 a 100.0 a 10.0 b 7.8 b termidor he 0.12% 100.0 a 100.0 a 16.0 b 8.3 b untreated control 0.0 b 5.0 b 50.0 a 50.0 a p-values <0.01 <0.01 <0.01 <0.01 f-stats 1521.00 1574.33 101.79 197.30 df 15 15 15 15 *all treatments caused 100% mortality in the c. formosanus by 120 h post-treatment. **all treatments caused 100% mortality in the r. flavipes by 96 h post-treatment. ***maximum distance tunneled could not exceed 50 mm. means followed by the same letter in the same column were not significantly different (p=0.05) as per tukey’s honest significant difference (hsd). 1555 keefer, c. et al. — performance of termidor® he high-efficiency termiticide from all of the other cohorts, and the untreated controls. by the 48 h observation, all of the treatments were significantly different (p <0.05) from the untreated controls (table 3 and figs. 8 and 9). at the 1 h observation period, there was no mortality in the donor populations in any of the treatments (fig.8). at the 48 h observation all of the donor populations in all the treatments showed 100% mortality, and were significantly different (p <0.01) from the untreated controls (fig. 8). at the 1 h observation period there was no mortality in the recipient population in any of the treatments (fig. 9). at the 48 h observation period the recipient populations in the 15:5 cohort was 100%, and at the 72 h observation period mean mortality of all recipient populations in the treatments was 100%. at the 72 h observation, all of the recipient populations were significantly different (p <0.01) from fig. 4. mean distance tunneled (mm) by coptotermes formosanus in a glass tube bioassay through time in soils treated with termidor® he (maximum distance tunneled could not exceed 50 mm). 1556 sociobiolog y vol. 59, no. 4, 2012 the untreated controls, which had less than 10% mortality throughout the study (fig. 9). field study the mean perimeter length of structures in this test was 57.8±18.6 m. the mean number of pre-trial termite mud tubes per structure was 2.3±8. the mean volume of finished solution applied to each structure was 162.7±52.3 l. there was no detection of subterranean termite activity on any of the 12 structures at any time post-treatment through 36 months. discussion exposure of r. flavipes and c. formosanus to termidor® he treated soils fig. 5. mean distance tunneled (mm) by reticulitermes flavipes in a glass tube bioassay through time in soils treated with termidor® he (maximum distance tunneled could not exceed 50 mm). 1557 keefer, c. et al. — performance of termidor® he high-efficiency termiticide resulted in significantly greater mortality than untreated soils. the distance tunneled by both termite species was also statistically less in treated soils than in untreated control soils. the results show that termidor® he was efficacious against r. flavipes and c. formosanus. termidor® he 0.06% and 0.12% caused 100% mortality to both r. flavipes and in c. formosanus at 72 hrs post-treatment, respectively. coptotermes formosanus had a slower response to the treatments than r. flavipes at 24 and 48 hrs. the treatments caused 100% mortality, while the untreated controls caused less than 10% mortality for the duration of the study. there was a positive correlation between increased donor numbers and recipient mortality, which provided evidence that there was transfer of active ingredient among nestmates. this was evident based on the fact that the treatments caused 100% mortality of donor and recipient termites, while the untreated controls caused less than 10% mortality (p <0.05) for the duration of the study. whether the transfer in this study was by grooming, trophallaxis, movement of contaminated sand, or simply contact, could not be determined table 2. total mean percent mortality (both donors and recipients combined) of different cohorts of donor:recipient coptotermes formosanus when donors were exposed to soil treated with termidor® he at 0.12% ai through time. hours post-treatment cohort 1 4 24 48 72 96 0:20* 0.00 a 0.00 b 0.00 b 0.00 c 1.25 b 2.50 b 1:19 0.00 a 0.00 b 1.25 b 5.00 c 87.50 a 100.00 a 5:15 0.00 a 0.00 b 1.25 b 45.00 b 90.00 a 100.00 a 10:10 2.50 a 5.00 a 13.75 b 92.50 a 100.00 a 100.00 a 15:5 0.00 a 0.00 b 75.00 a 96.25 a 100.00 a 100.00 20:0 0.00 a 0.00 b 80.00 a 100.00 a 100.00 a 100.00 a 0:20** 0.00 a 0.00 b 0.00 b 2.50 c 2.50 b 5.00 b p-values 0.28 <0.01 <0.01 <0.01 <0.01 <0.01 f-stats 3.00 6.00 53.47 201.76 246.94 1482.60 df 27 27 27 27 27 27 *untreated unmarked control **untreated marked control means followed by the same letter in the same column are not significantly different p=0.05. 1558 sociobiolog y vol. 59, no. 4, 2012 fig. 6. mean % mortality of donor populations in different cohorts of donor:recipient coptotermes formosanus termites through time when exposed for 30 minutes to soil treated with termidor® he at 0.12% ai. table 3. total mean percent mortality of different cohorts of donor:recipient reticulitermes flavipes when donors were exposed to soil treated with termidor® he at 0.12% ai through time. hours post-treatment cohort 1 4 24 48 72 0:20* 0.00 a 0.00 a 1.25 a 1.25 a 6.25 a 1:19 0.00 a 0.00 a 20.00 a 78.75 b 100.00 b 5:15 0.00 a 0.00 a 53.75 b 97.50 c 100.00 b 10:10 0.00 a 30.00 b 95.00 c 98.75 c 100.00 b 15:5 0.00 a 43.75 bc 100.00 c 100.00 c 100.00 b 20:0 0.00 a 57.50 c 100.00 c 100.00 c 100.00 b 0:20** 0.00 a 0.00 a 0.00 a 1.25 a 3.75 a p-values <0.01 <0.01 <0.01 4814.50 f-stats 19.37 42.65 150.38 <0.01 df 27 27 27 27 27 *untreated unmarked control **untreated marked control means followed by the same letter in the same column are not significantly different p=0.05. 1559 keefer, c. et al. — performance of termidor® he high-efficiency termiticide and was outside of the scope of this work. all of the treatments, excluding the untreated controls, in this study caused 100% mortality in all the different ratios of donor:recipient termites in both species by 96 h post-exposure. coptotermes formosanus had a slower response to the treatments than did r. flavipes. the higher donor:recipient ratios (20:0 and 15:5) in both species of termite were the fastest to reach 100% mortality. in the field study, the application methods were performed by a licensed pest management professional (pmp) and were done so by the normal practices of a pmp. the application of fipronil in and around the structures was done so that applications were made on, or as close as possible to, subterranean termite entry points. this was critical when applying termiticide for control of the subterranean termites. there has been no detection of subterranean termite fig. 7. mean % mortality of recipient populations in different cohorts of donor:recipient coptotermes formosanus termites through time when exposed for 30 minutes to soil treated with termidor® he at 0.12% ai. 1560 sociobiolog y vol. 59, no. 4, 2012 activity on any of the 12 structures through 36 months post-treatment. the results of these trials demonstrate that at either concentration, fipronil in the form of termidor® he is effective at causing significant mortality of c. formosanus and r. flavipes upon contact. additionally, collateral transfer of termidor® he was demonstrated among both subterranean termite species investigated in these studies. thus, termidor® he is not only transferred among termite nestmates, contact with the compound results in death of the insects. given these laboratory findings, it is not surprising that when applied to termite infested structures, termidor® he successfully remediated all infestations and prevented re-infestation for a period of at least three years when applied at 7.5 l per 3 m per 0.3 m depth. fig. 8. mean % mortality of donor populations in varying cohorts of donor:recipient reticulitermes flavipes termites through time when donors were exposed for 30 minutes to soil treated with termidor® he at 0.12% ai. 1561 keefer, c. et al. — performance of termidor® he high-efficiency termiticide references bobe, a., j. cooper, c.m. coste, and m.a. muller. 1998. behavior of fipronil in soil under sahelian plain field conditions. pestic. sci. 52, 275-281. bostian, a. l., s. swasdichai, and l. darus. 1996. activity of fipronil on diamondback moth. proceedings of the third international workshop: the management of the diamondback moth and other crucifer pests, kuala lumpur, maylasia, 195-198. forschler, b.t. 1994. fluorescent spray paint as a topical maker on subterranean termites (isoptera: rhinotermitidae). socio. 24 (1): 27-38. gant, d.b., a. e. chalmers, m. a. wolff, h. b. hoffman and d.f. bushey 1998. fipronil: action at the gaba receptor. review in toxicolog y 2: 147-156. gold, r.e. , a.a. collins, b. m. pawson, & h.n. howell, jr. 1994. termiticide technolog y the isofenphos dilemma. j. of the franklin institute, vol. 331a, 189-198. kard, b. m. (2003). integrated pest management of subterranean termites (isoptera). j. of entomol. sci. 38: 200-224. fig. 9. mean % mortality of recipient populations in varying cohorts of donor:recipient reticulitermes flavipes termites through time when exposed for 30 minutes to soil treated with termidor® he at 0.12% ai. 1562 sociobiolog y vol. 59, no. 4, 2012 scheffrahn r. h., and p.w. hope. 1996. key to termite soldiers. university of florida institute of food and agriculture, rec research report ftc 96-2, gainsville, fl. spss inc. 2009. pasw statistics for windows, version 18.0, chicago, il. su, n.-y. & r . h. scheffrahn. 1998. foraging populations and territor y of the formosansubterranean termite (isoptera: rhinotermitidae) in an urban environment. sociobiolog y 14: 353-359. su, n.-y. 2002. novel technologies for subterranean termite control. socibiolog y 40(1): 95-101. tingle, c. c., j. a. rother, c. f. dewhust, s. lauer, & w. j. king. 2003. fipronil: environmental fate, ecotoxicolog y, and human health concerns. rev. environ. contam. toxicol.176: 1-66. tomlin, c. d. 2000. the pesticide manual-incorporating the agrochemical handbook. 12th ed. (crop protection publications: farnham, england). ware, g. w. 2000. the pesticide book. 5th ed. thomson publications. fresno, ca. yanese, d. & a andoh. 1998. porphyrin synthesis involvement in diphyenyl ether-like mode of action on tnpp-ethyl, a novel phenylpyrazoles herbicide. pestic. biochem. physiol. 35: 70. doi: 10.13102/sociobiology.v63i3.1057sociobiology 63(3): 950-955 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant-fungus interactions: laboulbenia camponoti batra in italy and a new host for l. formicarum thaxter (fungi: ascomycota, laboulbeniales) introduction laboulbeniales, with over 2000 species, are obligate ectoparasites living attached to the cuticle of arthropod hosts (tavares, 1985). most are known to parasitize coleoptera, although diptera, dermaptera, dyctioptera, acari and diplopoda are also known to harbour specific laboulbeniales (weir & hammond, 1997). only six species of laboulbeniales parasitize hymenoptera, and all attach to ants (formicidae). ant laboulbeniales are globally distributed, being known from the palaearctic, nearctic, neotropical and indo-malayan regions, with the afrotropical and australasian regions still lacking any register (santamaria & espadaler, 2015). here we deal with two species from this last, and small, group of laboulbeniales. material and methods a total of 35 localities (8 in france and 27 in italy) were haphazardly sampled during a road trip in october 2015. most of the sampled locations habitats were urban or ruderal. abstract one laboulbenia species is added to the checklist of italian fungi. laboulbenia camponoti was detected on the ant camponotus aethiops. additionally, l. formicarum was found on a new host (lasius niger) in france. an updated map of world distribution for the two laboulbenia is presented. based on present knowledge, l. camponoti shows a much higher structural and phylogenetic host specificity than l. formicarum. sociobiology an international journal on social insects k gómez1, x espadaler2, s santamaria3 article history edited by gilberto m. m. santos, uefs, brazil received 03 may 2016 initial acceptance 01 august 2016 final acceptance 26 september 2016 publication date 25 october 2016 keywords laboulbenia, camponotus aethiops, lasius niger, formicidae. corresponding author xavier espadaler unitat d’ecologia i creaf universitat autònoma de barcelona cerdanyola del vallès, barcelona 08193, spain e-mail: xavierespadaler@gmail.com ants were directly preserved in alcohol and examined under a dissecting microscope. all ant samples were scrutinised with a leica smz16 with magnifications ranging from 40x to 115x. special attention was given to femora and tibiae as those parts use to be the surfaces were fungi are most visible. due to the non-systematic sampling method, our results have to be taken more as descriptive of the scarcity of fungi rather than a quantitative measure of infestation prevalence in a given area. permanent slides were prepared following benjamin (1971) and are kept in the bcb mycotheca of universitat autònoma de barcelona (bcb slides). ant specimens with attached fungi are kept in the private collection of k. gómez (kgac). although the database is meagre, we explore host specificity of the two antparasite laboulbenia, which accumulate 61 world records. following poulin et al. (2011), we use simpson and shannon indices as metrics for host structural diversity. indices were obtained using estimates (colwell, 2013). distribution maps were created with cartodb (www. cartodb.com) 1 castelldefels, barcelona, spain 2 creaf universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain 3 unitat de botànica, departament de biologia animal, de biologia vegetal i d'ecologia, facultat de biociències, universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain research article ants sociobiology 63(3): 950-955 (september, 2016) 951 results a total of 145 camponotus workers of six species and 322 lasius (233 workers, 65 queens, 24 males) of five species were searched for parasites, and only three workers (one camponotus and two lasius; 0.6% prevalence in workers) were positive for laboulbenia species. two out of 33 camponotus or lasius samples were infested (6% prevalence in samples). table 1 summarizes the localities where lasius or camponotus species were collected. laboulbenia camponoti s.w.t. batra, 1963 (fig 1a, 1b) on camponotus (tanaemyrmex) aethiops (latreille). one out of eight collected workers was infested with the fungus. italy: toscana: montopoli 43º 40.4’n 10º 44.98’e; 50m leg: gómez, k. 03/10/2015. garden with acer sp., pinus sp. and olea europaea. foraging on pinus trees. [kg03133]. the fact that the worker was captured among other non infested workers seems to reinforce the notion that ant species 1 2 3 4 5 6 7 8 9 10 11 france bouches-du-rhône: bus station (arles) 43º 40,92’n 4º 37,88’e 215m x bouches-du-rhône: cathedral square (arles) 43º 40,92’n 4º 37,88’e 215m l languedoc-roussillon: gruissan pond (aude) 43º 7,02’n 3º 4,74’e 10m x x var: chemin des costettes 43º 23,91’n 6º 20,64’e 200m x x italy lazio: camping (lughezza) 41º 55,92’n 12º 42’e 60m x liguria: balzi-rossi caves (2) (grimaldi) 43º 47,15’n 7º 31,86’e 5m x liguria: camping il giglio (monterosso al mare) 44º 9,3’n 9º 39,6’e 250m x x x liguria: marinella di sarzana (carrara) 44º 2,59’n 10º 1,46’e 5m x x liguria: ruta 9 a monterosso (1) (monterosso al mare) 44º 9,25’n 9º 39,47’e 200m x liguria: ruta 9 a monterosso (2) (monterosso al mare) 44º 9,34’n 9º 39,63’e 310m x liguria: torre di santamaria square (san bartolomeo al mare) 43º 55,21’n 8º 6,19’e 10m x toscana: meadow close to parking lot (vinci) 43º 40,4’n 10º 45,12’e 60m x x toscana: montopoli 43º 40,4’n 10º 44,98’e 50m l x x toscana: montopoli 43º 40,45’n 10º 44,74’e 40m x toscana: parking lot (montepoli) 43º 40,4’n 10º 45,12’e 60m x x x toscana: pkg decathlon (cascina) 43º 40,53’n 10º 28,29’e 10m x toscana: via scandicci 221 (florence) 43º 45,75’n 11º 12,53’e 50m x umbria: castle garden (orvieto) 42º 43,36’n 12º 7,22’e 275m x x umbria: catedral square (orvieto) 42º 42,99’n 12º 6,8’e 320m x x x x table 1. mediterranean localities with camponotus or lasius species. 1: camponotus aethiops (latreille); 2: camponotus cruentatus (latreille); 3: camponotus lateralis (olivier); 4: camponotus piceus (leach); 5: camponotus sylvaticus (olivier); 6: camponotus vagus (scopoli); 7: lasius lasioides (emery); 8: lasius myops forel; 9: lasius niger (linné); 10: lasius paralienus seifert. x: species present. l: species infested with laboulbenia. k gómez, x espadaler, s santamaria – laboulbenia camponoti in italy and new l. formicarum ant host 952 laboulbenia fungi does not seem to be a strong handicap for infested ants while foraging. l. camponoti was previously known from 19 world records, from six countries (fig 2): india loc. typ. (batra, 1963), austria (báthori et al. 2014), bulgaria (lapeva-gjonova & santamaria, 2011), romania (báthory et al., 2014), spain (balazuc et al., 1982; espadaler & blasco, 1991), turkey (espadaler & lodos, 1983). laboulbenia formicarum thaxter, 1902 (fig 1c, 1d, 1e) on lasius (s.str.) niger (linnaeus). 2 out of 2 collected workers were infested, both drowned in a fountain. this species is a new natural host for the fungus, although it had already been proved to be susceptible using experimental infestations in the laboratory (tragust et al. 2015). france: bouches-durhône. cathedral square (arles) 43º 40.92’n 4º 37.88’e 215m. leg: gómez, k. 28/09/2015. urban environment. [kg03116]. curiously enough, this site matches the pattern of coastal localities where this fungus has been collected −4 out of 5 localities− in europe. l. formicarum was previously known from 41 world records, from five countries (fig 3): u.s.a. loc. typ. (thaxter, 1902), canada (judd & benjamin, 1958), france (espadaler et al., 2011), portugal (espadaler & santamaria, 2003), spain (herraiz & espadaler, 2007). other ant genera collected (# of species) in france (cataglyphis (2), crematogaster (1), hypoponera (1), messor (2), plagiolepis (1), pheidole (1), tapinoma (1), tetramorium (1)) or italy (aphaenogaster (2), crematogaster (1), formica (1), hypoponera (1), linepithema (1), messor (1), pheidole (1), plagiolepis (2), solenopsis (1), tapinoma (1), temnothorax(1), tetramorium (1)) were not infested. fig 1. a, b: laboulbenia camponoti batra. a, mature specimen, b, slightly immature specimen. darkened foot was broken in slide mounting; c, d, e: laboubenia formicarum thaxter. c, d, mature female specimens. in e, paired thalli, male at left and female at right. scales: 50 µm. sociobiology 63(3): 950-955 (september, 2016) 953 discussion one hundred years have elapsed since the first instance of an ant-infesting laboulbenial was collected in italy. rickia wasmannii cavara was detected by spegazzini (1914) on workers of myrmica scabrinodis nylander from conegliano (veneto). we are unaware of any other register of antlaboulbenial interaction in italy. the two laboulbenia species here discussed seem to be limited to the northern hemisphere. the genus camponotus has a world distribution. instead, the diverse ant genera known as hosts for l. formicarum (see below) have a northern hemisphere distribution (http://www.antwiki.org/wiki/category :genus_distribution_map accessed march 2016). the absence of data for ant laboulbeniales from central and eastern eurasia calls for a dedicated search. it is perhaps noteworthy the contrasting host range of both species. for laboulbenia camponoti, 17 out of 19 known hosts belong in six species of camponotus (tanaemyrmex). subgenus host identity for the two citations from india remains unknown. instead, for laboulbenia formicarum, known host belong in 25 ant species from five genera (several subgenera): (lasius (s.str.), lasius (acanthomyops), formica (neoformica), formica (raptiformica), formica (serviformica), myrmecocystus, prenolepis, polyergus) (espadaler & santamaria, 2012), in two tribes (ward et al. 2016). host specificity encompasses several components: 1) structural (=basic) specificity or the number, and proportion, of host use; 2) phylogenetic specificity, the range of phylogenetic spectrum of hosts; 3) geographic specificity, the consistency of host use across the parasite geographical distribution. depending on the quantity and quality of available knowledge, those different components may be singly explored, or in their interactions (poulin et al. 2011). a simple analysis of the structural and phylogenetic components of host specificity indicates a lower specificity in l. formicarum (table 2). the low host phylogenetic specificity in l. formicarum – sensu poulin et al. 2011 – is the exception, rather than the rule, with host-laboulbeniales relationships (tavares, 1985; weir & hammond, 1997). geographic specificity is likely low in l. camponoti as shows its detection on three camponotus (c. aethiops, c. pilicornis, c. sylvaticus) at a single organic citrus grove in la selva del camp (tarragona, spain ; 41º13’07”n, 01º08’35”e) (unpub. obs.). fig 3. known distribution for laboulbenia formicarum thaxter, up to february 2016 (blue dots, known distribution; orange dot, new record). fig 2. known distribution for laboulbenia camponoti batra, up to february 2016 (blue dots, known distribution; orange dot, new record). http://www.antwiki.org/wiki/category:genus_distribution_map http://www.antwiki.org/wiki/category:genus_distribution_map k gómez, x espadaler, s santamaria – laboulbenia camponoti in italy and new l. formicarum ant host 954 a similar example of extremely low phylogenetic specificity is exhibited by the ant parasitic fungus myrmicinosporidium hölldobler. known from the palaearctic, the nearctic and a single location in the southern hemisphere, its spores have been detected in a wide host range of 38 species, 17 genera, and three ant subfamilies (gonçalves et al., 2012; lapeva-gjonova, 2014; giehr et al. 2015). furthermore, geographic specificity seems to be also very low in myrmicinosporidium since it was documented on seven ant hosts, belonging to seven genera, from five tribes, and three subfamilies at a single olive grove from póvoa de são miguel (portugal) (gonçalves et al., 2012). with this note we add one more laboulbenial to the list of the italian mycoflora and prove right experimental, cross-infection laboratory results by tragust et al. (2015) in the wild for laboulbenia formicarum infecting lasius niger in france. the ant genera camponotus (subg. tanaemyrmex) with eight species in italy, and lasius (s.str.) with nine species (baroni urbani, 1971; poldi et al., 1995) offer ample host opportunities for the fungi. the genus messor, with eight species in italy (op. cit.), provides also possible hosts for the recently described rickia lenoirii santam. from greece (santamaria & espadaler, 2015). thus, it can be only expected an enlargement of the database for those interesting antfungus interactions when a dedicated search for is undertaken, either in the field as in this paper, or in museum collections (suarez & tsutsui, 2004; báthori et al., 2014, 2015). ant genera myrmica, camponotus (tanaemyrmex), lasius (s.str.) and messor should be specifically focused. references balazuc, j., espadaler, x. & girbal, j. (1982). laboulbenials (ascomicets) ibèriques. collectanea bot. barcelona 13: 403-421. baroni urbani, c. (1971). catalogo delle specie di formicidae d’italia (studi sulla mirmecofauna d’italia x). memorie della società entomologica italiana, 50: 5-287. báthori, f., pflieger, w.p. & tartally, a. (2014). first records of the myrmecophilous fungus laboulbenia camponoti batra (ascomycetes, laboulbeniales) from the carpathian bassin. sociobiology, 61: 338-340. doi: 10.13102/sociobiology.v61 i3.338-340 báthori, f., pfliegler, w.p. & tartally, a. (2015). first records of the recently described ectoparasitic rickia lenoirii santam. (ascomycota: laboulbeniales) in the carpathian basin. sociobiology, 62: 620-622. doi: 10.13102/sociobiology.v62i4.901 benjamin r.k. (1971). introduction and supplement to roland thaxter’s contribution towards a monograph of the laboulbeniaceae. bibliotheca mycologica, 30: 1-155. bharti, h., guénard, b., bharti, m. & economo, e.m. (2016). an updated checklist of the ants of india with their specific distributions in indian states (hymenoptera, formicidae). zookeys, 551: 1-83. doi: 10.3897/zookeys.551.6767 colwell, r. k. (2013). estimates: statistical estimation of species richness and shared species from samples. version 9. user’s guide and application. http://purl.oclc.org/estimates. espadaler, x. & blasco, j. (1991). laboulbenia camponoti batra, 1963 (fungi, ascomycotina) en aragón. lucas mallada, 2: 13-23. espadaler, x. & lodos, n. (1983). camponotus baldaccii emery (hym., formicidae) parasitized by laboulbenia camponoti batra (ascomycetes) in turkey. turkish journal of plant protection, 7: 217-219. espadaler, x., lebas, c., wagenknecht, j. & tragust, s. (2011). laboulbenia formicarum (ascomycota, laboulbeniales), an exotic parasitic fungus, on an exotic ant in france. vie et milieu, 61: 41-44. espadaler, x. & santamaria, s. (2003). laboulbenia formicarum crosses the atlantic. orsis, 18: 97-101. espadaler, x. & santamaria, s. (2012). ectoand endoparasitic fungi on ants from the holarctic region. psyche, e168478. doi: 10.1155/2012/168478 giehr, j., heinze, j. & schrempf, a. (2015). the ant cardiocondyla elegans as host of the enigmatic endoparasitic fungus myrmicinosporidium durum. psyche, 2015: 364967. doi: 10.1155/2015/364967 gonçalves, c., patanita, i. & espadaler, x. (2012). substantial, and significant, expansion of ant hosts range for myrmicinosporidium hölldobler, 1933 (fungi). insectes sociaux, 59: 395-399. doi: 10.1007/s00040-012-0232-z shannon diversity exponential shannon diversity ( hill’s n1) simpson diversity (hill’s n2) (inverse form) tribes genera subgenera species # global records l. camponoti 1.66 5.26 3.77 1 1 1 7-8 20 l. formicarum 3 20.16 16.00 2 5 >5 25 41 although a mere six camponotus species are known from the state of delhi, the genus has 83 species in india (bharti et al. 2016). we take the conservative approach to consider the two registers of l. camponoti from india to belong in two different host species. table 2. ant host structural and phylogenetic diversity of two laboulbenia species. sociobiology 63(3): 950-955 (september, 2016) 955 herraiz, j.a. & espadaler, x. (2007). laboulbenia formicarum (ascomycota: laboulbeniales) reaches the mediterranean. sociobiology, 50: 449-455. judd, w.w. & benjamin, r.k. 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(2016). a revised phylogenetic classification of the ant subfamily formicinae (hymenoptera: formicidae), with resurrection of the genera colobopsis and dinomyrmex. zootaxa, 4072: 343-357. doi: 10.11646/zootaxa.4072.3.4 weir, a. & hammond, p.m. (1997). laboulbeniales on beetles: host utilization patterns and species richness of the parasites. biodiversity and conservation, 6: 701-719. doi: 10.1023/a:10 18318320019. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.277-282sociobiology 60(3): 277-282 (2013) foraging behavior of scaptotrigona depilis (hymenoptera, apidae, meliponini) and its relationship with temporal and abiotic factors g figueiredo-mecca, lr bego, fs nascimento introduction it is known the importance of bees as pollinators and visiting of flowers in the most world’s ecosystems and the special role that stingless bees play in the tropical areas (roubik, 1989; biesmeijer & slaa, 2006). bees are also known to be efficient pollinators of many crops as about 30% of human food is derived frombee-pollinated crops (reviewed in slaa et al., 2006). unpredictable environmental changes affect foraging activities in terms of timing and location of food. there are two main features regulating departure of foraging bees: individual memory and threshold response to react to the foraging stimuli (internal factors), and environmental and colony conditions which determine the level of exposure to stimuli associated with decision to forage (external factors) (biesmeijer & de vries, 2001). colonies of honey bees and stingless bees can allocate more foragers to collect nectar and pollen in response to the amount of food in storage and abstract stingless bees play an important role in tropical and subtropical ecosystems as pollinators of many plants. the aim of this study was to characterize the pattern of flight activity and foraging for pollen by scaptotrigona depilis workers and their relation with climatic and temporal factors. we observed flight activity from july 2001 to june 2002 and pollen collection by scoring the flow of workers entering and exiting the colonies. we tested whether environmental (temperature, relative humidity, pluviosity and luminosity) and temporal predictors (month and time of day) affected bees’ activities. the study was conducted during one year and the records of observations showed that during the dry season when the length of light is longer, the external activity was more intensive, while during cold months with shorter light period, foraging activity was reduced. pollen collection showed a peak in february, but in general this activity was regulated by temperature, humidity and daily luminosity. sociobiology an international journal on social insects universidade de são paulo (ffclrp-usp), ribeirão preto, são paulo, brazil. article history edited by kleber del-claro, ufu, brazil received 13 june 2013 initial acceptance 11 july 2013 final acceptance 14 august 2013 key words flight activity, pollen collection, stingless bees, climatic factors corresponding author gláucya de figueredo mecca lab. de comportamento e ecologia de insetos sociais departamento de biologia faculdade de filosofia, ciências e letras de ribeirão preto (ffclrp), universidade de são paulo av. bandeirantes, 3900. cep 14040-901 ribeirão preto, são paulo, brazil e-mailglaucyafm@usp.br availability of resources in the field (seeley, 1995; biemeijeret al., 1999, fewell &winston, 1992). the stingless bee scaptotrigona depilis has an efficient communication by pheromones trails and presents a highly eusocial pattern in which the oldest workers perform basically foraging activities while younger workers are engaged in intra nest activities such as maintenance, brood care and defense (nogueira-neto, 1970; roubik, 1989; engels et al., 1997; michener, 2000). the colonies of this species are estimated around 2.000 to 50.000 individuals (referred as s. postica in lindauer & kerr, 1960). the nests, built with wax, resin and cerumen, occur in tree cavities and present an elaborated architecture that include an entrance tube that communicates with the brood cells, pollen and honey pots as a food storage in the sides of the nest and horizontal brood combs with vertical cells and involucrum to provide protection (nogueira-neto, 1970; michener, 1974; kerr, 1996). although the forager bees collect different products such as resins, rotting wood, seeds, leaves, trichomes, fraresearch article bees g figueiredo-mecca, lr bego, fs nascimento scaptotrigona depilis foraging related to environmental factors278 grances, spores, waxes, feces and animal hairs, the principal materials collected are pollen and nectar used for feeding bees (roubik, 1989). according to michener (1974) colony development depends to the success of the foraging flight and the ability to bring resources to the nest from carefully chosen flowers. the interaction between the bees’ activity and the environmental factors has been described by several authors. the mode that the bees perceive and react to these factors reflected in the flight activity pattern as described by nelson and jay (1967), szabo (1972) for apis mellifera, szabo and smith (1972) for megachile rotundata, and shiag and abrol (1986) for apis florae. especially in meliponini the pioneering works on foraging activities were in the following species: plebeia saiqui and p. droryana (oliveira, 1973), tetragonisca angustula (iwama, 1977), p. emerina (kleinert-giovanini, 1982), p. remota (imperatriz-fonseca et al., 1985) and in two subspecies of melipona marginata (kleinert-giovanini & imperatriz-fonseca, 1986). the aim of this study was to test whether the pattern of flight activity and foraging for pollen in s. depilis is affected by environmental and temporal effects. we predicted that in high-sized populated stingless bee colonies such as s. depilis environmental factors have not significant effects compared to temporal or seasonal variables. as seasonality is a limiting trait for plants, nectar and pollen availability may represent a key constraint for tropical stingless bees. materials and methods scaptotrigona depilis occurs from the southeast of mato grosso state to the west of são paulo state (camargo & pedro, 2012). the estudy site was the meliponary at the university of são paulo, campus of ribeirão preto in são paulo, brazil. we used four colonies of scaptotrigona depilis housed inside the wooden boxes. the observations were made from july 2001 to june 2002, and were carried out twice a month with intervals of approximately fifteen days. each observation was made with intervals of 1 hour and 15 minutes from 7:00 am to 18:15 pm. for each colony the pattern of flight activity and pollen collected was scored by counting the number of workers entering and exiting the colonies during three minutes. later, we recorded the number of workers that entered the colonies with pollen during two minutes. these data were obtained using manual counters and a chronometer. the environmental predictors (temperature, relative humidity, pluviosity and luminosity) were recorded at each interval of time using digital thermo-hygrometer and a luximeter. the data were analysed with a general linear model (glm) where colonies, season and time of day were entered into the analysis as the independent variables and number of bees entering or exiting as the dependent variables (neter et al., 1996). a regression test was also used to estimate the relationship between abiotic, colony and the frequency of departures. all analyses were made using statistica 7.0 (statsoft inc.). results general activities our data showed that foraging activities were ruled out by the typical seasonal pattern found in the southern of brazil: the external activity increased during the wet season in warm months (august /march) and decreased during the cold and driest months (april/july). the general results showed that in lower temperature periods bees began the flight activity later and the traffic was less intense, while in high temperature values the bees began the flight activity earlier and more intensively. the onset of the external movement occurred only at temperatures above 15°c. the end of the activity was determined by the decreasing of light intensity more than under the temperature effect. however, in warmer months, when the days are longer, the bees flew even in low luminous intensity in both the beginning and ending of the activity. the foraging activity occurred during all year but it presented peaks at different months. although the pollen collection occurred throughout the year, there was a conspicuous decline in july/2001 and from march to june/2002. in february/2002 there was a large increment in all activities, but mainly for the pollen collection (fig. 1). general linear models showed that foraging activities were significantly affected by almost all parameters tested (table 1). variance between colonies was significant, meaning that thenumber of foraging departures and returns between the four colonies was different. concerning the temporal variables, the general linear models showed that month (season), time of day and state of colony presented a significant influence on the flight departure (table 1). abiotic variables had a smaller influence on the bees’ departures compared to the temporal variables. only luminosity and pluviosity were significantly important to regulate foraging departure. multiple regression analysis revealed a low, but significant interaction between all abiotic variables taken altogether and exiting of foragers (r2 = 0.04; f = 9.73; p < 0.0001). pollen collection foraging for pollen was regular during almost all period of study, presenting a conspicuous peak during february, rainy season (fig. 2a). multiple regression analysis revealed a low but still significant influence of climatic factors on the pollen collection (r2=0.17; f = 40.83; p<0.001; (fig. 2b). the most important parameters explaining the influence of abiotic factors on pollen collection were temperature (r = 0.17; p < 0.05), luminosity (r = 0.19; p < 0.05) and humidity (r = -0.21; p < 0.05). sociobiology 60(3): 277-282 (2013) 279 discussion this study showed that in this species flight activity starts with a few number of bees and the external movement increases gradually according to the raising of the luminosity and temperature. however, the peak of entrance and exit do not occur at times of the day at higher temperatures, as also verified by heard and hendrikz (1993), in trigona carbonaria and hilário et al. (2001) in p. pugnax. in cold days, the flight activity was less intense and the beginning of the flights occurs later as reported by hilário et al. (2012) for p. remota as a “more compact flight activity”. on the other hand, in warmer days the traffic of bees was more intense and began earlier, therefore the period of the external activity was longer as observed by hilário et al. (2000). in late afternoon the luminosity seems to determine the end of the activity as also observed for tetragonisca angustula (iwama, 1977). however, in days with higher temperatures, both the first flights that occurs approximately 21°c and the last between 27° to 30°c were maintained even in significantly lower luminosity conditions. therefore, there was a significant interaction between temperature and luminosity determining the activity of flight. during heavy rain or stronger wind, the entrance of workers became more intense and the departures almost stopped, decreasing practically all the traffic, just returning to regular flow right after the rain, as also reported for p. pugnax (hilário et al., 2001). hilário et al. (2007b) observed that p. remota foragers guided most of their flights to certain wind directions that matched with the local where the floral resources were. on cloudy drizzled days, the external movement was finished at 5:00 p.m. despite the high temperatures. however on cloudy days preceded by rainy night, even with high levels of light and temperature, the external activity became greatly reduced. in days of weather instability, with a few moments of rain and variations of temperature and luminosity, the external activity occurred irregularly. it demonstrated clearly the influence of climatic factors on flight activity of bees. bees began the foraging for pollen soon after the start of flight activity and arrived with pollen during all period of observation. the peaks of collection occurred at different times on each day, so it was not possible to establish a pattern of activity for pollen. in february, there was a significant increase in the external activity, especially for pollen collecting. it may be related to the climatic factors, although the interactions between these factors and the some changes in the availability of flower resources. in fact, faria et al. (2012) verified that the foraging for pollen was concentrated on a few sources, despite of all the plants species visited fig 1. mean±s.e. of scaptotrigona depilis entries (black bars) and exits (white bars) related to temporal variables and season from july 2001 to june 2002. table 1. general linear models results of the departure frequency of scaptotrigona depilis foragers related to temporal and abiotic variables. d.f. meansquared f p temporal effects intercept 1 345765.6 684.70 <0.0001 month 11 5273.0 10.44 <0.0001 timeofday 9 6238.7 12.35 <0.0001 month*time 99 1071.7 2.12 <0.0001 error 478 505.0 abioticeffects intercept 1 11242.29 3.13 0.077 luminosity (lux) 1 51158.03 14.25 <0.0001 pluviosity (pluv) 1 20888.02 5.82 0.016 temperature (temp) 1 276.40 0.07 0.781 humidity (humid) 1 6863.24 1.91 0.167 lux*temp*humid*pluv 1 12455.51 3.47 0.063 error 752 3588.96 fr eq ue nc e fr eq ue nc e g figueiredo-mecca, lr bego, fs nascimento scaptotrigona depilis foraging related to environmental factors280 for s. depilis. according to this study, both the availability and localization of floral resources affect the choice of the pollen sources in this species. in m. bicolor, pollen forager ratios varied significantly between seasons and this variation was related to both the production of reproductives and the number of individuals in the colonies (hilário & imperatrizfonseca, 2009). quezada-euán et al (2011) also reported that the worker’s body size of nannotrigona perilampoides may change according to the protein and sugar concentration in larval food. as pollen is the main protein source of larval food, so flower phenology and the seasonal availability of different pollen types may influence brood development in this species. on the days of intense rain, bees reduced the external activity, so restarting quickly after the rain ceased probably to maintain the food storage, also described by hilário (2007a) for p. remota. veiga et al. (2013) observed in melipona flavolineata foraging decline on rainy season followed by a decrease of food stores and the subsequent change in the size of the worker, which suggested that the climate is a key determinant of foraging activity. so, the foraging “explosion” occurred on february could be explained by the highest rates of rainfall recorded from october to march, especially on february, when there were consecutive rainy days before the observation. this increase in pollen foraging may be influenced by the resource availability, probably acting together with climatic factors, as observed by other authors. according to pereboom and biesmeijer (2003) stingless bees has a differentiated capability to gain and losing heat due the size and coloration of the body, and it can be explained by biophysical principles that correspond to differential thermo regulatory capabilities. so, for these authors the thermal characteristic scan influence the pattern of foraging according to the morphology and anatomy of the bees. bellusci and marques (2001) observed that even if exiting to outside is restricted, activity of the foragers follow a circadian rhythm, synchronized by the light/dark cycle and probably influenced by other environmental cycles as temperature and the availability of food sources.thus, all these factors should be considered interactively in determining the external activity of bees. based on the present and previous studies we can state that s. depilis due to its large colonies and number of individuals tolerate climate changes such as higher temperatures, high and low relative humidity values and variations of light intensity. there was recorded no flights activity on temperatures below 15°c so the only factor that can limit the flight activity was the low luminosity. on the other hand, the pollen collectionis probably influenced by climatic factors associated with availability of the food resources. fig. 2.a average number of scaptotrigona depilis workers that arrived with pollen, and b. average luminosity, relative humidity and temperature from july/2001 to june/2002. humidity in july and august were not registered. a b acknowledgments we thank to dr sidnei mateus for assistance in manipulation and maintenance of the colonies and two anonymous reviewers for the important suggestions on the manuscript. references bellusci, s. & marques, m.d. 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(2013) the bigger, the smaller: relationship between body size and food stores in the stingless bee melipona flavolineata. apidologie, 44: 324-333. doi: 10.13102/sociobiology.v64i2.1386sociobiology 64(2): 146-154 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a preliminary list of the ant fauna in northeastern sahara of algeria (hymenoptera: formicidae) introduction in algeria, as in countries where it is not too cold, the ants have the advantage of being abundant. they exist everywhere, in the forest as in open areas, along the water as in dry areas, on clay as on rocks (cagniant, 1973). comparing with other countries of the north of africa, cagniant (2009) evaluate the moroccan myrmecofauna approximately to 220 species, while both of algeria and tunisia include 180 in total (cagniant, 2006). the myrmecofauna of algeria is known through some fragmentary studies done by: (e.g. forel, 1890; 1894; 1902; sanchi, 1915; 1929a; 1929b; bernard, 1955; 1963; 1973; 1977; 1982; cagniant, 1966a; 1966b; 1967; 1968a; 1968b; 1969; 1970a; barech et al., 2011; 2015). unfortunately, abstract we present here a preliminary list of ant fauna of some study sites in the northeastern sahara of algeria using two methodologies, quadrat and pitfall traps (barber-pots) methods. this work was conducted in wild and agricultural ecosystems in the basin of ouargla, el-oued region and djamaa region. we record a total of 26 species of 12 genera belonging to three subfamilies dolichoderinae, formicinae, and myrmicinae. species of the myrmicinae and formicinae were the most abundant with 62.96% and 29.63% respectively. the most diverse genus was monomorium mayr, 1855 (6 species), followed by messor forel, 1890 (5). the highest diversity of ants was in djamaa region (24 species), followed by ouargla (18) and el-oued (13). moreover, this work shows the first record of the species strumigenys membranifera emery, 1869 for the country. finally, we observed a variation in the distribution of ant species between study sites, for why, ecological determinants such as soil need to be studied deeply to explain their influence on the repartition and richness of the saharan myrmycofauna of algeria. sociobiology an international journal on social insects a chemala1*, m benhamacha1, dm ould el hadj2, f marniche3, s daoudi1 article history edited by gilberto m. m. santos, uefs, brazil received 19 january 2017 initial acceptance 29 march 2017 final acceptance 16 may 2017 publication date 21 september 2017 keywords naturel areas, cultivated areas, strumigenys membranifera, sahara, myrmecofauna. corresponding author abdellatif chemala departement of forest and agricultural zoology national high school of agricultural sciences avenue hassan badi, el harrach algiers, es1603, algeria. e-mail: a.chemala@yahoo.fr no serious studies have been made on repartition of these microfauna in the algerian territory, excepting those of (cagniant, 1968c; 1970b; 1970c) who have established a list of the forest ants of algeria with a total of 120 species. recently, djioua and sadoudi-ali ahmed (2015) listed the richness of ant fauna in some forest and agricultural areas of kabylia. barech et al. (2016) added a checklist of the myrmecological fauna of a saline lake area, chott el hodna. in northern sahara of algeria, the ant fauna has a significant percentage among the arthropods that inhabit this region by adapting to the conditions of the arid environment. however, the saharan species were poorly studied. the objective of this study is to evaluate the diversity of myrmecofauna in wild and agricultural ecosystems of the northeastern sahara of algeria. 1 department of forest and agricultural zoology, national high school of agricultural sciences, algiers, algeria 2 department of naturel science, kasdi merbah university, ouargla, algeria 3 laboratory of zoology, national high school of veterinary, algiers, algeria research article ants sociobiology 64(2): 146-154 (june, 2017) 147 material and methods study sites this study was conducted in wild and agricultural ecosystems owns of several areas of the northeastern sahara of algeria. for that, three study sites were selected because of the high variation in their flora (appendix i), represented by the basin of ouargla, el-oued region and djamaa region (table 1). to represent the physiognomy of the vegetation of sampling sites environment type of areas geographic coordinates provided in the study djamaa region agricultural ecosystems market gardening: semiopen 33º28’85’’ n, 6º03’75’’ e palmary: semiopen 33º38’45’’ n, 5º59’36’’ e wild ecosystem open and exposed 33º33’92’’ n, 5º59’78’’ e el-oued region agricultural ecosystems market gardening : semiopen and sunny 33º26’48’’ n, 6º57’42’’ e palmary: open and exposed 33º26’93’’ n, 6º57’70’’ e wild ecosystem open and exposed 32º26’85’’ n, 6º57’59’’ e ouargla region agricultural ecosystems market gardening: open 32º02’54’’ n, 5º21’42’’ e palmary: semiopen and sunny 31°58’16’’ n, 5º20’96’’ e wild ecosystem open and exposed 32º02’66’’ n, 5º19’81’’ e table 1. sites where ants were sampled in the northern-east sahara of algeria. each study site, a transect of 500 m2 (10m x 50m) was carried out. it consists to produce an inventory of plant species found there and represent them graphically next two figures, one in vertical projection onto a plane and the other in profile. the projected orthogonally on a plane representation allows specifying the structure of the plant community and the recovery rate. however, the profile representation provides indications on physiognomy of the milieu, showing if it is open, semi-open or closed. sampling methods data sampling was carried out on a monthly basis, between april 2012 and march 2013. for that, two standard ant collecting methods were used. ants for quantitative study were collected using quadrat methods which consist to a manual capture onto a delimited surface, during a known time interval. in this study, ants were sampled from a surface of 10 m x 10 m, during 3min. this method allows the observation of nests and the enumeration of population of each ant species (berville et al., 2015). a second method was added for complementary data on ant diversity by using pitfall trap (barber pots). these interceptions traps consisted of a metallic container (7.4 cm diameter × 10.5 cm long), placed at the same level as the surrounding ground, filled one-third with a solution of water and a drop of liquid dishwashing soap to break the surface tension. each pitfall trap distant from the other by 5 m along a transect of 50 m2 (10 traps) for each study site. the pitfall traps were left running during 24 hours before being gathered and emptied of their contents. this method was used by several authors such as (hernández-ruiz & castaño-meneses, 2006; berville et al., 2015). data analysis ant diversity in the study sites was expressed as species richness, abundance and proportion. moreover, factorial correspondence analysis (fca) was carried out using xlstat in order to provide a factorial map of the repartition of ant species between the three regions. hoffman and franke (1986) defined the correspondence analysis as an exploratory data analysis technique for the graphical display of contingency tables and multivariate categorical data. results and discussion richness and abundance we recorded 26 ant species of 12 genera and three subfamilies. myrmicinae was the most abundant subfamily (62.96 %), followed by formicinae (29.63%) and dolichoderinae (7.41%). the most diverse genus was monomorium and messor (5 species each ones), followed by cataglyphis (4). tapinoma, componotus and tetramorium, each is represented by two species. however, the genera lepisiota, plagiolepis, cardiocandyla, crematogaster, pheidole and strumigenys are presented by one species each one. the genus strumigenys is recorded for the first time from algeria by the species s. membranifera emery, 1869. the region of djamaa was the most diversify with 24 ant species, followed by ouargla (18) and el-oued (13). similarly, djamaa region included the high number of restricted ant species, which are represented by: tapinoma simrothi, cataglyphis albicans, plagiolepis barbara, crematogaster inermis, messor sanctus and tetramorium sericeiventre. nevertheless, both of el-oued region and ouargla region include one restricted ant species each ones, represented by messor aegyptiacus tunetinus and s. membranifera respectively. according to deyrup (1997), this species often occurs in disturbed open areas, such as lawns and pastures. effectively, one sample of this species, identified as a queen was collected from the wild ecosystem of chemala et al. – ants from northeastern sahara of algeria148 ouargla region. in saudi arabia, it was recorded from an area of date palm trees cultivation (sharaf et al., 2014). wetterer (2011) considered s. membranifera as a cosmopolitan ant species which has spread around the world through human commerce, in all zoogeographical regions (bolton, 1983; bolton, 2000). but it was probably suggested that it has an african origin (brown & wilson, 1959). in the mediterranean region, it occurs in: france, greece, italy, malta, spain, egypt and tunisia (wetterer, 2011). the absence of record of this species in the adjacent countries such as morocco, libya, niger and mali allowed us to suggest a hypothesis of being introduced from tunisia because of its localization near el oued region and their similar ecosystems. although its known that s. membranifera and s.emmae are a successful invasive species (wetterer 2011), sharaf et al. (2014) thought that s. membranifera reaches more temperate areas. so, the sahara of algeria presents a good area for their invasion. the analyze of the data of table 2 shows that some ant species are common in all study sites. for example: monomorium salomonis obscuriceps and pheidole pallidula are the only species which occurred in all study stations. this result confirms the view of (hernández-ruiz & castañomeneses, 2006) which stated that populations of the genus monomorium are frequent and occupy environments with different conditions of soil (humid or dry), because they are not so highly affected by micro weather changes. in other hand, the species p. pallidula has spread through the mediterranean regions (bernard, 1956). the study of the abundance of ant species in different ecosystems of the northeastern sahara of algeria is given for the first time. among the identified species, the most abundant ant species was lepisiota frauenfeldi atlantis (34.02%) for djamaa region, messor aegyptiacus tunetinus (28.24%) for el-oued region and tapinoma nigerrimum (23.58%) for ouargla region (tables 3, 4, 5). by analyzing subfamilies species djamaa region el-oued region ouargla region we ae we ae we ae dolichoderinae tapinoma nigerrimum (nylander, 1856) + + tapinoma simrothi (krausse, 1911) + formicinae camponotus barbaricus (emery, 1905) + + + + camponotus thoracicus (fabricius, 1804) + + + + + cataglyphis albicans (roger, 1859) + cataglyphis bicolor (fabricius, 1793) + + + + + cataglyphis bombycina (roger, 1859) + + + + cataglyphis rubra (forel, 1903) + + lepisiota frauenfeldi atlantis (santschi, 1917) + + + + plagiolepis barbara (santschi, 1911) + + myrmicinae cardiocondyla batesii (forel, 1894) + + + + + crematogaster inermis (mayr, 1862) + messor arenarius (fabricius, 1787) + + + + messor aegyptiacus tunetinus (santschi, 1923) + + messor foreli (santschi, 1923) + + + messor medioruber sublaeviceps (santschi, 1910) + + + messor sanctus (emery, 1921) + + monomorium areniphilum (santschi, 1911) + + + + + monomorium destructor (jerdon, 1851) + + + monomorium salomonis obscuratum (linnaeus, 1758) + + + monomorium salomonis obscuriceps (santschi, 1921) + + + + + + monomorium subopacum (smith, 1858) + + + + pheidole pallidula (nylander, 1849) + + + + + + strumigenys membranifera (emery, 1869) + tetramorium biskrense (forel, 1904) + + + tetramorium sericeiventre (emery, 1877) + total/area 19 18 7 12 15 11 total/ region 24 13 18 we: wild ecosystem; ae: agricultural ecosystem table 2. occurrence and distribution of ant species collected in the northeastern sahara of algeria. sociobiology 64(2): 146-154 (june, 2017) 149 species wild ecosystem agricultural ecosystem total a p (%) a p (%) a p (%) tapinoma nigerrimum (nylander, 1856) 0 0 1779 18.26 1779 11.92 tapinoma simrothi (krausse, 1911) 0 0 76 0.78 76 0.51 camponotus barbaricus (emery, 1905) 0 0 43 0.44 43 0.29 camponotus thoracicus (fabricius, 1804) 40 0.77 16 0.16 56 0.38 cataglyphis albicans (roger, 1859) 0 0 27 0.28 27 0.18 cataglyphis bicolor (fabricius, 1793) 81 1.56 651 6.68 732 4.90 cataglyphis bombycina (roger, 1859) 80 1.54 0 0.00 80 0.54 cataglyphis rubra (forel, 1903) 15 0.29 0 0.00 15 0.10 lepisiota frauenfeldi atlantis (santschi, 1917) 3110 59.96 1968 20.21 5078 34.02 plagiolepis barbara (santschi, 1911) 18 0.35 26 0.27 44 0.29 cardiocondyla batesii (forel, 1894) 16 0.31 20 0.21 36 0.24 crematogaster inermis (mayr, 1862) 0 0 40 0.41 40 0.27 messor arenarius (fabricius, 1787) 9 0.17 0 0.00 9 0.06 messor foreli (santschi, 1923) 972 18.74 0 0.00 972 6.51 messor medioruber sublaeviceps (santschi, 1910) 6 0.12 2020 20.74 2026 13.57 messor sanctus (emery, 1921) 30 0.58 44 0.45 74 0.50 monomorium areniphilum (santschi, 1911) 268 5.17 843 8.66 1111 7.44 monomorium destructor (jerdon, 1851) 13 0.25 0 0.00 13 0.09 monomorium salomonis obscuratum (linnaeus, 1758) 10 0.19 2 0.02 12 0.08 monomorium salomonis obscuriceps (santschi, 1921) 57 1.10 1354 13.90 1411 9.45 monomorium subopacum (smith, 1858) 42 0.81 338 3.47 380 2.55 pheidole pallidula (nylander, 1849) 394 7.60 438 4.50 832 5.57 tetramorium biskrense (forel, 1904) 1 0.02 55 0.56 56 0.38 tetramorium sericeiventre (emery, 1877) 25 0.48 0 0.00 25 0.17 total 5187 100 9740 100 14927 100 a: abundance, p: proportion table 3. abundance and proportion of ant species recorded in djamaa region. the data of tables (3, 4, 5), we observe that most species of the genus cataglyphis was found more abundant in the wild ecosystems. however, c. albicans was restricted to the agricultural areas. this genus is occured in open areas such as clearing and steppe in the north of africa from the seaside to 2800m in hoggar (cagniant, 2009). also, cataglyphis bombycina was the most abundant. its high abundance is explained by their common existence in sand-dune and other sandy areas of the south of morocco and algeria (cagniant, 2009). similarly, l. frauenfeldi atlantis occured greatly on arid zone (cagniant, 2006). the lowest abundance is reported in the genus cardiocondyla. according to (cagniant, 2009), this genus appears discretely in open habitat. generally, there is a difference and variation in the proportion of each ant species between different study sites and habitats. some species are found in wild and agricultural ecosystems. in other hand, some species are collected just from the wild ecosystem such as, cataglyphis rubra, tetramorium sericeiventre and s. membranifera. cagniant (1997) confirmed the presence of the species t. sericeiventre in the region of biskra which is located near the region of djamaa. in opposition, the species, tapinoma nigerrimum, t. simrothi and crematogaster inermis were collected only from agricultural areas. t. nigerrimum was found in pasturage milieu from the north of algeria into the saharan atlas (cagniant, 1970b). whereas, t. simrothi is more common in pasture areas but it suffers if exposed to cold. the same author confirmed its presence in the saharan atlas of algeria above 1300 m. this variation in abundance and distribution between the two species is explained by (cagniant, 1966a) who wrote that t. nigerrimum resists better cold, this capacity allowed it to be maintained in the north of algeria. for the crematogaster genus, cagniant (2005) found c. inermis on tree (fruit and tamarix) in morocco and include the other species of this group to the arboreal insect, most of the time. chemala et al. – ants from northeastern sahara of algeria150 factorial correspondence analysis (fca) the factorial map displays a total inertia of 100 % for both axes, f1 (60. 62%) and f2 (39. 38%) (fig 1). the chart shows the existence of six grouping (a, b, c, d, e, f) distribute between the study regions. the groups a, c and e contain exclusive species for djamaa, el-oued and ouargla region respectively, which are already mentioned in the text above. the group b contains one species shared between djamaa and el-oued region, represented by t. biskrense. the group d contains 11 species shared between the three regions represented by: camponotus barbaricus, camponotus thoracicus, cataglyphis bicolor, c. bombycina, cardiocondyla batesii, messor arenarius, messor foreli, monomorium areniphilum, m. salomonis obscuriceps, monomorium subopacum, p. pallidula. the last group f contains 6 species shared between the region of ouargla and the region of djamaa. this group groups the species t. nigerrimum, c. rubra, l. frauenfeldi atlantis, messor medioruber sublaeviceps, monomorium destructor, monomorium salomonis obscuratum. species wild ecosystem agricultural ecosystem total a p (%) a p (%) a p (%) camponotus barbaricus (emery, 1905) 0 0.00 93 2.60 93 2.25 camponotus thoracicus (fabricius, 1804) 0 0.00 3 0.08 3 0.07 cataglyphis bicolor (fabricius, 1793) 0 0.00 13 0.36 13 0.32 cataglyphis bombycina (roger, 1859) 163 29.80 277 7.74 440 10.67 cardiocondyla batesii (forel, 1894) 0 0.00 98 2.74 98 2.38 messor arenarius (fabricius, 1787) 157 28.70 569 15.90 726 17.60 messor aegyptiacus tunetinus (santschi, 1923) 98 17.92 1067 29.82 1165 28.24 messor foreli (santschi, 1923) 0 0 190 5.31 190 4.61 monomorium areniphilum (santschi, 1911) 53 9.69 512 14.31 565 13.70 monomorium salomonis obscuriceps (santschi, 1921) 20 3.66 16 0.45 36 0.87 monomorium subopacum (smith, 1858) 4 0.73 0 0.00 4 0.10 pheidole pallidula (nylander, 1849) 52 9.51 698 19.51 750 18.18 tetramorium biskrense (forel, 1904) 0 0.00 42 1.17 42 1 .02 total 547 100 3578 100 4125 100 table 4. abundance and proportion of ant species recorded in el-oued region. species wild ecosystem agricultural ecosystem total a p (%) a p (%) a p (%) tapinoma nigerrimum (nylander, 1856) 0 0.00 1142 34.04 1142 23.58 camponotus barbaricus (emery, 1905) 3 0.20 7 0.21 10 0.21 camponotus thoracicus (fabricius, 1804) 3 0.20 71 2.12 74 1.53 cataglyphis bicolor (fabricius, 1793) 50 3.36 200 5.96 250 5.16 cataglyphis bombycina (roger, 1859) 683 45.87 0 0.00 683 14.10 cataglyphis rubra (forel, 1903) 20 1.34 0 0.00 20 0.41 lepisiota frauenfeldi atlantis (santschi, 1917) 24 1.61 953 28.41 977 20.17 cardiocondyla batesii (forel, 1894) 2 0.13 29 0.86 31 0.64 messor arenarius (fabricius, 1787) 3 0.20 0 0.00 3 0.06 messor foreli (santschi, 1923) 482 32.37 0 0.00 482 9.95 messor medioruber sublaeviceps (santschi, 1910) 2 0.13 0 0.00 2 0.04 monomorium areniphilum (santschi, 1911) 0.00 0 217 6.47 217 4.48 monomorium destructor (jerdon, 1851) 10 0.67 9 0.27 19 0.39 monomorium salomonis obscuratum (linnaeus, 1758) 0 0.00 9 0.27 9 0.19 monomorium salomonis obscuriceps (santschi, 1921) 129 8.66 31 0.92 160 3.30 monomorium subopacum (smith, 1858) 1 0.07 0 0.00 1 0.02 pheidole pallidula (nylander, 1849) 76 5.10 687 20.48 763 15.75 strumigenys membranifera (emery, 1869) 1 0.07 0 0.00 1 0.02 total 1489 100 3355 100 4844 100 table 5. abundance and proportion of ant species recorded in ouargla region. sociobiology 64(2): 146-154 (june, 2017) 151 for a better understanding of the variation in the distribution of ants species, others researches must be made in the future. this last is consistent with the study of the influence of ecological determinants such as soil parameters and vegetation cover on ant distribution. bardgett et al. (2005) wrote that soil characteristics are well known with their strong effects on spatial distribution of soil communities. meanwhile, boulton et al. (2005) concluded that overall ant species richness and abundance are more consistently associated with soil chemistry and texture than plants. additionally, a particular change in environmental conditions may increase the diversity of one subset of organisms within a community, while decreasing the diversity of a different group of organisms (semida et al., 2001). also, there is a relationship between ant community abundance and altitude. taheri and reyes-lo´pez (2015) stated that some approach followed in the study of myrmecofauna such as season, altitude, aridity, geology and vegetation types and animal farming practices give a better picture of the composition and distribution of the species. this work is a contribution for the study of algerian myrmecofauna of the northeastern sahara. data on ant fauna from the occidental and central sahara stay unknown and need a great effort in sampling methods to dispatch richness and abundance of species. finally, it is certain that other survey expanded in space and in time will enrich more this list. fig 1. chart represents factorial map of the repartition of ant species in the study region cas: cataglyphis albicans, cba: cataglyphis bombycina, cbi: cardiocondyla batesii, cbr: cataglyphis bicolor, cis: crematogaster inermis, cra: cataglyphis rubra, cts: camponotus thoracicus, lfa: lepisiota frauenfeldi atlantis, mam : monomorium areniphilum, mas: messor arenarius, mdr: monomorium destructor, mfi: messor foreli, mms: messor medioruber sublaeviceps, msm: monomorium subopacum, mso: monomorium salomonis obscuratum, msob: monomorium salomonis obscuriceps, mss: messor sanctus, pba: plagiolepis barbara, ppa: pheidole pallidula, tse: tetramorium sericeiventre. chemala et al. – ants from northeastern sahara of algeria152 acknowledgments we would like to express our gratitude to the professor h. cagniant who helped us to confirm identification of some ant species. references bardgett, r.d., usher, m.b. & hopkins, d.w. 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(2011). worldwide spread of the membraniferous dacetine ant, strumigenys membranifera (hymenoptera: formicidae). myrmecological news, 14: 129-135. chemala et al. – ants from northeastern sahara of algeria154 appendix i. vegetation found in different environment of the study sites. sampling sites environment vegetation djamaa region agricultural ecosystem lactuca sativa l., allium cepa l., allium sativum l., mentha pulegium l., phoenix dactylifera l., medicago sativa l. wild ecosystem zygophyllum album l., tamarix gallica l. el-oued region agricultural ecosystem solanum tuberosum l., cynodon glabratus l., phoenix dactylifera wild ecosystem stipa tenacissima l. ouargla region agricultural ecosystem lactuca sativa l., phoenix dactylifera l., prunus armeniaca l., allium sativum l., medicago sativa l., phragmites communis l. wild ecosystem reseda sp l., zygophyllum album l. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2214sociobiology 65(2): 185-190 (june, 2018) cytogenetic studies in trachymyrmex holmgreni wheeler, 1925 (formicidae: myrmicinae) by conventional and molecular methods introduction fungus-growing ants are found exclusively in the new world, primarily in the neotropical region (mayhé-nunes & jaffé, 1998; schultz & meier, 1995), and are suggested to have originated around 50 mya (chapela et al., 1994; schultz & brady, 2008). these ants comprise nearly 300 described species divided into 15 genera (brandão et al., 2011). trachymyrmex forel, 1983, currently includes 47 species (bolton, 2017), and is suspected to be the sister group of the leafcutter ants, atta and acromyrmex genera. it is therefore a key group for understanding the relationship among “higher attine” (brandão et al., 2011). the trachymyrmex septentrionalis group, a clade of north american species, is abstract over the past several decades, ant cytogenetic studies have focused on chromosome number and morphology; however, recently, additional information concerning heterochromatin composition and 45s rdna location has become accessible. the fungus-growing ants are a peculiar ant group that cultivates fungus for food, and trachymyrmex is suspected to be the sister group of leafcutter ants. cytogenetic data are so far available for sixn trachymyrmex species. the present study aimed to increase the knowledge about trachymyrmex cytogenetics by the chromosomal characterization of trachymyrmex holmgreni including the karyotyping, fluorochromes staining, 18s rdna, and microsatellite (ga) 15 fluorescence in situ hybridization (fish). karyotyped samples from four ant colonies showed 2n = 20 metacentric chromosomes. centromeric heterochromatin rich in gc base pairs was detected in all chromosomes. fish revealed the presence of rdna clusters on the fourth chromosome pair, and an intense spreading of the microsatellite (ga) 15 including exclusively euchromatic areas of the chromosomes. the gc-rich heterochromatin observed in different ant species may have a common origin and, thus, phylogenetic implication that needs to be further investigated. to the best of our knowledge, this study is the first report of the use of chromosomal physical location of repetitive dna sequences by means of microsatellite probes in formicidae. sociobiology an international journal on social insects lac barros1,2, gateixeira2, hjac aguiar1, dm lopes3, sg pompolo2,3 article history edited by marco costa, uesc, brazil received 16 october 2017 initial acceptance 05 february 2018 final acceptance 20 march 2018 publication date 09 july 2018 keywords heterochromatin, fish, ribosomal genes, microsatellite repeats, ant. corresponding author luísa antônia campos barros universidade federal do amapá campus binacional oiapoque, amapá, brasil. e-mail: luufv@yahoo.com.br considered by schultz and brady (2008) to be closely related to the leafcutter ants. trachymyrmex is possibly a paraphyletic group (schultz & brady, 2008; mehdiabadi & schultz, 2010) and taxonomic uncertainties remain. ant cytogenetics has drawn the attention of myrmecologists (delabie et al., 2012) and is useful in phylogenetic, taxonomic, evolutionary and conservation applications (e.g., cristiano et al., 2013; barros et al., 2015; santos et al., 2016; aguiar et al., 2017). cytogenetic studies in fungus-growing ants are scarce, especially for the so called “lower attine” group (reviewed in barros et al., 2011). cytogenetic data in trachymyrmex are available for six species (table 1), and the chromosome numbers range from 2n = 12 to 2n = 22 chromosomes. trachymyrmex septentrionalis (mccook 1881), 1 universidade federal do amapá, campus binacional, oiapoque, amapá, brazil 2 programa de pós-graduação em genética e melhoramento, universidade federal de viçosa, minas gerais, brazil 3 departamento de biologia geral, universidade federal de viçosa, minas gerais, brazil research article ants lac barros et al. – cytogenetics of the fungus-growing ant trachymyrmex holmgreni186 included in the t. septentrionalis group, is closely related to leafcutter ants (schultz & brady 2008), and presents 2n=20 chromosomes (murakami et al., 1998). this chromosome number is similar to that observed in atta spp. already studied, and also ac. striatus, with 2n = 22, although minor chromosome morphology differences are observed between these two group species. the other leafcutter clade, with the other acromyrmex, present 2n = 38, meaning, a derived karyotype was originated by centric fissions, according to barros et al. (2016). studies concerning heterochromatin information are valuable in cytogenetic analysis of ants owing to the central role of heterochromatin in the chromosome evolution of the group (imai et al., 1994). the cytogenetic data available for fungus-growing ants show heterochromatin distribution mainly at the centromeric and/or pericentromeric regions for species with low chromosome numbers n ≤ 12 (sensu imai et al., 1988), such as mycocepurus goeldii (forel, 1893) (barros et al., 2010), trachymyrmex relictus borgmeier, 1934 (barros et al., 2013), trachymyrmex fuscus emery, 1934 (barros et al., 2014a), and atta spp. (barros et al., 2014b, 2015). this pattern is expected in species with a low chromosome number, according to the minimum interaction theory (imai et al., 1994). other cytogenetic techniques including fluorescent in situ hybridization (fish) using rdna probes have been used to detect differences among fungus-growing ants (barros et al., 2016; teixeira et al., 2017). the detection of heterochromatic gc-rich regions using the fluorochrome cma3 pointed to single bands for most species of the neotropical region, including the leafcutter ants of the genus atta (barros et al., 2014b, 2015) and acromyrmex (barros et al., 2016). however, some fungus-growing ants showed multiple gcrich regions, which correspond to heterochromatic bands such as those observed in m. goeldii (barros et al., 2010), t. fuscus (barros et al., 2014a), and acromyrmex striatus (roger, 1863) (cristiano et al., 2013). in ants from the neotropical region, the reports of 18s or 45s physical location are available for 25 species (mariano et al., 2008; aguiar et al., 2011; santos et al., 2010, 2016; barros et al., 2012, 2015, 2016; teixeira et al., 2017; aguiar et al., 2017). the majority of these species present the gc-rich regions coinciding with the nucleolus organizer regions (nors). the exceptions are m. goeldii (barros et al., 2010, 2012), acromyrmex niger (smith, 1858) (barros et al., 2016), dolichoderus lutosus (smith, 1858), dolichoderus voraginosus mackay, 1993, dolichoderus bidens( linnaeus, 1758) (santos et al., 2016), and ac. striatus (cristiano et al., 2013; teixeira et al., 2017), which presented multiple gcrich heterochromatic bands and a single pair of nors. recently, the detection of specific microsatellites as landmarks has been used in different organisms including insects such as orthopterans (milani & cabral-de-mello, 2014, palacios-gimenez et al. 2015, palacios-gimenez & cabral-de-mello, 2015).these microsatellites can be useful markers in evolutionary studies. microsatellites distribution is highly variable in the species genomes, and can be found in specific regions or dispersed throughout the chromosomes (sumner, 2003). many of the studied species present a scattered distribution pattern of microsatellites along the chromosomes. regarding the phylogenetic position of trachymyrmex within the “higher attine” group, and the absence of previous physical mapping of rdna genes data in this genus, this study aimed to describe the fungus-growing ant t. holmgreni, wheeler 1925, included in the iheringi group (reviewed in mayhé-nunes & brandão, 2005) by means of classical and molecular cytogenetics. material and methods four colonies of t. holmgreni were collected in itutinga, state of minas gerais, brazil (21º 17’ s; 44º 39’ w), on july 29th, 2012. sample collection was done under the authorization of the instituto chico mendes de conservação da biodiversidade (icmbio) for the collection of biological material issued to luísa antônia campos barros (sisbio accession number 32459). ant vouchers (workers) were identified by jacques hubert charles delabie and deposited in the reference collection at the laboratório de mirmecologia, centro de pesquisas do cacau (cpdc/brazil) under the record #5725. the metaphases were obtained from cerebral ganglia of the larvae after meconium elimination, according to imai et al. (1988). to determine the morphology of the chromosomes, a total of 10 metaphases were measured and chromosomes were classified according to levan et al. (1964). corel photopaint x3® and image pro plus® were the softwares used for mounting the chromosomal karyotype and measurements, respectively. for subsequent techniques, at least 15 metaphases were analyzed. at least five individuals per colony were analyzed. c-banding was performed according to sumner (1972) with minor adaptations as suggested by barros et al. (2013). specific gcand at-rich regions were detected using sequential staining with the fluorochrome cromomicin a3 (cma3) and 4’6diamidino-2-phenylindole (dapi) following schweizer (1980). ribosomal 18s genes were detected by fish, following pinkel et al. (1986). the 18s rdna probe was obtained via pcr (polymerase chain reaction) amplification employing the primers rdna 18sf1 (5’-gtc ata gct ttg tct caa aga-3’) and 18sr1.1 (5’-cgc aaa tga aac ttt ttt aat ct-3’) designed for the bee melipona quinquefasciata (pereira, 2006). total dna of the ant camponotus rufipes was used as template in the pcr reactions. 18s rdna probes were labeled maintaining the conditions for pcr amplification (pereira, 2006) by an indirect method using digoxigenin-11dutp (roche, mannheim, germany), and the fish signals were detected with anti-digoxigenin-rhodamine (roche applied science), following the manufacturer’s protocol. sociobiology 65(2): 185-190 (june, 2018) 187 microsatellite (ga)15 was used as probe in the physical location of repetitive dna. the sequence of this probe was directly labeled with cyanine-3 (cy3) in the 5’ terminal during synthesis by sigma (st. louis, mo, usa). the microsatellite hybridization procedures were performed according to kubat et al. (2008), with the modifications of cioffi et al. (2010). the metaphases were observed and documented using a fluorescence microscope olympus bx 60, coupled with capture system q-color3 olympus® images, using the software q capture® with the filters wb (450-480 nm), wu (330-385 nm) and wg (510-550 nm) for analyzing cma3, dapi and rhodamine, respectively. the metaphases labeled with the microsatellite (ga)15 probe were observed using a microscope olympus bx 53f coupled with an olympus mx10 camera and the image software cellsens® with the filter wg (510-550 nm) for the probe rich in cy3 and wu forthe chromosomes (330-385 nm). results t. holmgreni presented 2n = 20 chromosomes, all of them metacentric (fig 1a; table 1). centromeric heterochromatin (fig 1d) rich in gc-base pairs (fig 1b) was observed in all chromosomes. differentially, at base pairs rich regions were not found (fig 1c), but only negative regions complementing the fluorochrome cma3 (fig 1d). the detection of ribosomal 18s genes by fish analysis showed bands in the centromeric region of the fourth pair of metacentric chromosomes (fig 1e). the results indicated an intense spreading of the dinucleotide microsatellite (ga)15 in the t. holmgreni genome including only euchromatic areas of the chromosomes (fig 1f). discussion the karyotype presented by t. holmgreni is similar in number and morphology to that of t. septentrionalis (murakami et al., 1998), a species closely related to leafcutter ants (schultz & brady 2008), and t. relictus (barros et al., 2013). the predominance of metacentric chromosomes is a karyotypic characteristic of the species of trachymyrmex studied so far (murakami et al., 1988; barros et al., 2013, 2014a). the heterochromatin pattern observed in t. holmgreni is similar to that previously described in t. fuscus (2n = 18) (barros et al., 2014a), with centromeric and pericentromeric bands that coincided with gc-rich regions (cma3 +). fig 1. conventional and molecular cytogenetics of mitotic cells of trachymyrmex holmgreni. a) diploid karyotype arranged in descending order of size (2n = 20), b) cma3 and c) dapi staining for the detection of gc and at rich blocks, respectively, d) c-banding for heterochromatin detection, e) fish analysis for 18s rdna, and f) fish analysis for dinucleotide microsatellites repeats (ga)15. lac barros et al. – cytogenetics of the fungus-growing ant trachymyrmex holmgreni188 chromosomal fusion hypothesis was suggested for the taxa with 2n = 12 owing to the low chromosome number and the presence of interstitial heterochromatic blocks (murakami et al., 1998). considering the chromosome number available for trachymyrmex spp. (table 1), associated with the location and composition of heterochromatin for the studied species (murakami et al., 1998; barros et al., 2013, 2014, present study), centric fusion rearrangements seems to have occurred during the chromosomal evolution of this genus. further cytogenetic studies will enable more robust inferences. although t. holmgreni had presented multiple gcrich bands, only a single pair of nor-bearing chromosomes was observed, demonstrating that most of the gc-rich heterochromatin bands in this species do not correspond to the 18s ribosomal genes. this was also observed in other fungusgrowing ants such as m. goeldii (barros et al., 2010, 2012), a. niger (barros et al., 2016) and ac. striatus (cristiano et al., 2013, teixeira et al. 2017). cytogenetic data of t. holmgreni in the present study showed predominance of metacentric chromosomes, multiple gc-rich heterochromatic bands and a single 18s rdna pair: similar chromosomal traits that are observed in ac. striatus (cristiano et al., 2013). however, most acromyrmex (2n = 38) and all atta species already studied have a single pair of 18s rdna rich in gc (barros et al., 2014b, 2015, 2016; teixeira et al., 2017). it is suggested that ac. striatus is the sister group of the remaining leafcutter ants (cristiano et al., 2013), and the gc-rich patterns observed in ac. striatus which are also found in t. holmgreni and t. fuscus may have a common origin and, thus, a phylogenetic implication. this must be further investigated in other trachymyrmex and other fungus-growing ant groups. trachymyrmex holmgreni (2n = 20), as well as ac. striatus (2n = 22) and atta spp. (2n = 22), presented 18s rdna located in metacentric chromosomes (barros et al., 2015; teixeira et al. 2017), thus differing from acromyrmex spp. which presented these genes in the terminal region of the larger subtelocentric chromosome pair (barros et al., 2016; teixeira et al., 2017). considering the phylogenetic relationships of these species (schultz & brady, 2008; cristiano et al., 2013), the 18s rdna locations in metacentric chromosomes seem to be the ancestral condition of the group. usually nors are rich in gc base pairs (sumner, 2003). another peculiar observation was described in d. voraginosus in which gc-rich regions did not correspond with rdna 45s clusters (santos et al. 2016), indicating the importance of the extension of rdna mapping in formicidae. regions with differential staining with dapi, indicative of regions rich in at base pairs, were not observed in t. holmgreni, a similar pattern to that observed in other ants such as t. fuscus emery, 1934 (barros et al. 2014a), atta spp. (barros et al. 2014b, 2015), dolichoderus (santos et al., 2016), and pseudoponera (correia et al., 2016). the repetitive probe (ga)15 presented dispersed distribution in the euchromatic regions of the chromosomes. the dispersed pattern of the (ga)15 microsatellite differs from that observed in the orthopteran abracris flavolineata (de geer, 1773),which presented specific euchromatic and heterochromatic bands (milani & cabral de mello, 2014). in formicidae, there are no data of chromosomal physical location using repetitive dna sequences for comparisons. however, initial descriptive data can generate new insights into the comprehension of the ant genome. these data open new possibilities for population and evolutionary studies and the use of additional probes. species 2n morphology locality reference trachymyrmex fuscus 18 16m + 2a paraopeba mg brazil barros et al., 2014a trachymyrmex holmgreni 20 20m itutinga mg brazil present study trachymyrmex relictus 20 20m viçosa mg brazil barros et al., 2013 trachymyrmex septentrionalis 20 20m barro colorado panama murakami et al., 1998 trachymyrmex sp. 1 12 12m barro colorado panama murakami et al., 1998 trachymyrmex sp. 2 18 18m barro colorado panama murakami et al., 1998 trachymyrmex sp. 22 18m + 4sm viçosa -mg brazil barros et al., 2013 table 1 – trachymyrmex species studied cytogenetically. chromosome number (2n). chromosome morphology. sampling sites (mg = minas gerais state). references of the data acknowledgments the authors are grateful to jacques hubert charles delabie for species identification, júlio cézar mário chaul and francisko de moraes rezende for valuable assistance in field work, and laboratório de sistemática molecular beagle of the universidade federal de viçosa for providing the microscope for microsatellite analysis. we also acknowledge conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the scholarship grant provided to lacb and gat. compliance with ethical standards: all the authors declare that they have no conflict of interest. references aguiar, h. j. a. c., barros, l. a. c., mariano, c. s. f., delabie, j. h. c., pompolo, s. g. 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(2017) comparative physical mapping of 18s rdna in the karyotypes of six leafcutter ant species of the genera atta and acromyrmex (formicidae: myrmicinae). genetica, 145: 351-357. doi: 10.1007/s10709-017-9970-1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5907sociobiology 68(3): e5907 (september, 2021) introduction pakistan has a total area of 882000 km2 with a world biogeographic realm, including indo-malayan, palearctic, and afro-tropical (cox & moore, 1993). the endemism rate is meager; about 7% flowering plants and 3% for mammals have been documented (gop, 1999). pakistan’s ant fauna is highly diverse, with mountainous and desert areas with a low degree of endemism. the worldwide transfer of various organisms through humans is well documented. due to the development of commerce and improvements in transportations, more species are being transported to their nonnative range of occurrence (ivanov, 2016). many agro-ecological activities are performed by ants such as predators of other arthropods, role in mineralization of nutrients, herbivores, seed dispersal, and increasing soil fertility (pfeiffer et al., 2013). abstract ants of the genus cardiocondyla are considered omnivorous in nature. this genus is native to afrotropical, australasia, indomalaya, malagasy, oceania, and palearctic regions. a total of 72 valid species and 2 valid subspecies has been described worldwide. however, little is known about these ants in pakistan. as a result of a survey of different sites in the gatwala park of district faisalabad during 2018, we collected individuals of the genus cardiocondyla. collected specimens were identified using the most recent and available literature. prior to current work, only two species of this genus were reported for pakistan. in this study we added the first record of cardiocondyla obscurior for pakistan, followed by a brief description, distribution, and identification key. sociobiology an international journal on social insects waqar majeed1, elmo ba koch2, naureen rana1, rimsha naseem1 article history edited by evandro nascimento silva, uefs, brazil received 06 october 2020 initial acceptance 26 february 2021 final acceptance 28 march 2021 publication date 13 august 2021 keywords new record, cardiocondyla; faisalabad, gatwala park. corresponding author elmo b.a. koch laboratory of myrmecology cocoa research center – cepec/ceplac caixa postal 7, cep: 45600-970 itabuna, bahia, brasil. e-mail: elmoborges@gmail.com emery originally described the genus cardiocondyla in 1969. according to seifert (2003), this is native to afrotropical, australasia, indomalaya, malagasy, oceania, and palearctic regions. a total of 72 valid species and two valid subspecies has been described worldwide (antweb, 2020). ants of this genus are commonly found in naturally disturbed sites, wood margin, sand dunes, near the roadside, and often in an environment with less water availability (seifert, 2003). workers can be diagnosed based on the following characters; small to minute in size, mandibles with five teeth (variable in size). clypeus flattened with prominent lateral projection. the antennal segment usually 11-12 with the distinct apical club (3-segmented). mesosoma forming single connectivity in dorsal view. the propodeal declivity is unarmed to bi-spinose. pilosity very sparse or absent dorsally (seifert, 2003). 1 department of zoology, wildlife and fisheries, university of agriculture faisalabad, pakistan 2 myrmecology laboratory, cocoa research center, cepec/ceplac, ilhéus-ba, brazil short note first record of cardiocondyla obscurior wheeler, 1929 (hymenoptera: formicidae: myrmicinae) for pakistan id mailto:elmoborges@gmail.com https://orcid.org/0000-0002-2022-4066 waqar majeed, elmo ba koch, naureen rana, rimsha naseem – first record of cardiocondyla obscurior for pakistan2 a remarkable contribution to this genus includes emery (1869); andré (1883); chapman and capco (1951); radchenko (1995); mackay (1995); rigato (2002); seifert (2003). seifert et al. (2017) provided the revisionary work on the genus cardiocondyla. however, little is known about these ants from pakistan (menozzi, 1939; rasheed et al., 2019). in the present work, we added c. obscurior as a new record for pakistan’s ant fauna. materials and methods as a result of a survey of different sites in the gatwala park of district faisalabad during 2018, we collected individuals of genus cardiocondyla manually using a mouth aspirator. the collected material was identified using an n2gg zoom stereo microscope and available literature: bingham (1903) and seifert (2003). identified specimens are housed in department of zoology, university of agriculture faisalabad (31°26′2.18″ n, 73°3′53.6″ e). results cardiocondyla obscurior wheeler, 1929 cardiocondyla wroughtoni var. obscurior wheeler. 1929: 44 (worker and queen) taiwan. raised to species and senior synonym of bicolor: seifert, 2003: 271. cardiocondyla bicolor donisthorpe, 1930b: 366 (worker) israel. junior synonym of wroughtonii: kugler, 1984: 6; obscurior: seifert, 2003: 271. material examined pakistan: 2 workers, 16.v.2018 and 1 worker, 29.v.2018 respectively, in pakistan, faisalabad, gatwala park, 31°25′49.1″ n, 73°02′06.9″ e, 186 m a.s.l., (fig 1) coll. w. majeed. we deposited the identified material (voucher number: bl01uaf) in the biodiversity laboratory, department of zoology, wildlife and fisheries, university of agriculture, faisalabad. description (worker) head shorter, anterior margin narrower while posterior margin slightly broader, sub-rectangular in fullface view, color light brown-dark brownish, antennae yellow, gaster black and highly polished. metanotal grove present, propodeal spines of moderate size and slightly turned inward at outer face. antennae 12-segmented with 3 segmented club. eyes placed just below the middle of head, with smaller post ocular distance. clypeal margin angulates. mandibles smaller with minute tooth, apical tooth prominent. antennal scape shorter, not reaching beyond the vertex; carinae prominent, slightly divergent laterally. pilosity: head, mesosoma, petiole and postpetiole with minute, whitish and decumbent hairs in profile view. head, mesosoma, and waist brightly yellowish or yellowish brown. key to the pakistani species of the ant genus cardiocondyla based on worker caste 1. body yellow-light yellowish brown; body length < 2 mm; propodeal declivity with short and acute spines.................…2 body light brown-black brown; body length > 2 mm; fig 1. geographical record of c. obscurior in gatwala faisalabad, pakistan. https://antcat.org/references/122253 https://antcat.org/references/123583 https://antcat.org/references/127013 https://antcat.org/references/132139 sociobiology 68(3): e5907 (september, 2021) 3 propodeal declivity with tubercles in lateral.…..cardiocondyla mauritanica 2. petiole trapezoidal dorsally; mesosoma with fine sculptured, the area near the propodeal spines having minute and irregular sculptures in dorsal………...…...cardiocondyla wroughtonii petiole globose; mesosoma with irregular and minute sculptured throughout in dorsal..….cardiocondyla obscurior ecology the c. obscurior individuals were observed on small and large shrubs and herbs, shrubs, and the leaf litter for nesting. however, few workers were observed while they were foraging on small grasses and flowers. global distribution canary island, germany, israel, kenya, india, nepal, japan, taiwan, hawai, mariana island, brazil, puerto rico, virgin island, florida (seifert, 2003). discussion c. obscurior is considered as cosmopolitan species of indomalayan region. some of the species of cardiocondyla genus were recorded previously as mentioned above in results (rasheed et al., 2019) from pakistan, but the genus cardiocondyla and other species of the formicidae family were published with a major extension of species record from india (bharti, 2016; bharti & kumar, 2017). this species mostly found in urban areas/outdoors localities (sanchezgarcia & espadaler, 2015; trigos-peral & reyes-lopez, 2016; ivanov, 2016), while seifert (2003), boer et al. (2018), as well as espadaler and nilo (2019) found these species as indoor in france, the netherlands and spain, respectively. many types of tramp ants have been dispersed worldwide, found in our plant materials, packing materials, construction supplies and heavy machines like logging. in certain places they invaded, some of these animals had large population disturbances, which caused severe ecological and economic challenges (wetterer, 2012). the species is considered an accomplished tramp ant (heinze et al., 2006) and has established populations across the world, including northern europe (rasplus et al., 2010; seifert, 2003), but it is not considered a pest nor known to affect native ecosystems adversely. moreover, it seemed like this species has a social system similar to unicoloniality and is non-informative either in one-to-one experiments (heinze et al., 2006). the restriction of the two non-tramp ants in the c. wroughtonii group to india and borneo suggests the native range of c. obscurior to be southeast asia. c. obscurior is polygynous and often founds new colonies by nest splitting (seifert, 2003). according to the present study results, we believe that the occurrence of c. obscurior in pakistan should not be trivial. the lack of records in the literature is probably associated with the lack of studies in the group, and probably more records for the species should be pointed out with the development of future research. acknowledgment the authors would like to thank the university of agriculture for conducting and supporting this research. we also acknowledge muhammad tariq rasheed (department of entomology, pmas arid agriculture university, rawalpindi) for helping in identification and manuscript revision. authors’ contribution wm: conceptualization, methodology, investigation, material identification, writing & revision. ebak: conceptualization, methodology, writing & revision. nr: conceptualization, methodology, writing & revision rn: conceptualization, methodology, material identification, writing & revision. conflict of interests the authors declare that they have no conflict of interest. references andré, e. 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(1929). ants collected by professor f. silvestri in formosa, the malay peninsula and the philippines. bollettino del laboratorio di zoologia generale e agraria della reale scuola superiore d’ agricoltura portici, 24: 27-64. _30j0zll _1fob9te _hlk67833665 _hlk41950441 _hlk41950471 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1232sociobiology 64(2): 191-201 (june, 2017) male orchid bees (hymenoptera: apidae: euglossini) in canopy and understory of amazon várzea floodplain forest. i. microclimatic, seasonal and faunal aspects introduction studies have shown significant differences in the diversity and community composition of insects across distinct arboreal strata, such as canopy and understory, regardless of the taxon and type of forest analyzed (smith, 1973; dulmen, 2001; giovanni et al., 2015). these differences may be related to abiotic conditions and biotic resources, such as light availability, temperature, wind exposure, forest structure and composition, food and nesting sites, which differ in intensity and availability between strata (ulyshen, 2011). for some groups of insects these differences are fairly important so that there are species restricted to specific strata (erwin, 1983). as emphasized by ulyshen et al. (2010), few studies have been conducted to determine the occurrence of the vertical distribution of bees. among such studies, bawa et al. (1985) abstract floodplain forests are important ecosystems of the amazon basin. the aim of the present study was to investigate whether male orchid bees have preference for arboreal strata in a forest with a dynamic understory. traps with aromatic scents to attract bees were placed at heights of 25 m and 1.5 m. data loggers were used to register temperature and relative humidity at both heights. a total of 835 individuals belonging to 28 species were sampled, 645 males of 23 species in the understory and 190 males of 24 species in the canopy. the temperature was 1.4 0c higher in the canopy and the air relative humidity was 11.58% higher in the understory. temperature, relative humidity and winds were the most important abiotic variables influencing the assemblage in the canopy. the abundance increased in the understory and decreased in the canopy in the less rainy season. euglossa cognata moure, eulaema meriana olivier and euglossa ignita smith were species closely associated with strata and seasons. the present study revealed higher richness and abundance in the understory all months of the year, reinforcing the likelihood that orchid bees forage preferably in the lower layer of rainforests. sociobiology an international journal on social insects ps vilhena¹, lj rocha², ca garófalo¹ article history edited by og de souza, ufv, brazil received 01 november 2016 initial acceptance 07 march 2017 final acceptance 01 may 2017 publication date 21 september 2017 keywords amapá, vertical stratification, diversity, euglossine bees. corresponding author patricia dos santos vilhena laboratório de ecologia e evolução de abelhas e vespas av. bandeirantes, 3900, monte alegre cep 14040-901, ribeirão preto são paulo, brasil. e-mail: vilhena.p.s@gmail.com found that tree species pollinated by small bees were especially common in the understory, whereas species pollinated by medium-sized to large bees were predominant in the canopy of a tropical forest. roubik (1993) performed systematic collections of bees from the canopy and understory in two forests in panama and found that two species of nocturnal bees were conspicuously abundant in the canopy, despite of that most of the species did not have a preference for a particular stratum. sobek et al. (2009) reported spatial stratification in the abundance of cavity-nesting bees and wasps and their parasitoids as well as higher parasitism rates in the canopy than the understory in temperate deciduous forests in germany. in a study on the vertical distribution of trapnesting bees along a fragmentation gradient of secondary forest remnants in costa rica, stangler et al. (2016) found that bees were more abundant in the canopy and understory in comparison to an intermediate height but the diversity was higher in the canopy. 1 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo (usp), ribeirão preto-sp, brazil 2 universidade federal do amapá, macapá-ap, brazil research article bees ps vilhena, lj rocha, ca garófalo – male orchid bees in strata of várzea floodplain forest192 some authors have investigated the occurrence of vertical stratification in populations of orchid bees. roubik (1993) found that large euglossine bees demonstrated a tendency to forage high, which was directly related to their capacity for heat loss during flight, compared to smaller euglossine bees. oliveira and campos (1996), in two areas of terra firme forest in the amazon, found similar values of richness and abundance between canopy and understory, but higher diversity in the canopy; the authors also reported that some species were more associated with the canopy and others were more common in the understory. in areas of the atlantic rainforest (coastal brazil), martins and souza (2005) found that the abundance and richness of euglossine bees were higher in absolute numbers in the understory, but the diversity was higher in the canopy. studying population fluctuations of euglossine bees in a remnant of cerrado scrubland in the state of mato grosso do sul, brazil, ferreira et al. (2011) found no differences in species richness or diversity between strata, but the abundance of males was higher in the canopy than the understory. all these studies carried out with orchid bees had focused on differences and similarities in the richness, abundance and diversity between canopy and understory, but none of them assessed the influence of abiotic conditions on the community and distribution of species across the different strata. therefore, the aim of the present study was to investigate how the assemblage of male orchid bees is structured in canopy and understory of a várzea floodplain forest in the brazilian amazon, taking into consideration that floodplains have a more dynamic understory than non-flooded forests, which could favor faunal differences in both strata. microclimatic differences regarding temperature and air relative humidity were analyzed in each stratum and the influence of such abiotic variables, together with others as rainfall, wind speed and tidal height, was investigated on the assemblage. material and methods study area this study was conducted in the protected area of fazendinha, category v of the iucn list (dudley, 2008), which is located in the southeastern portion of the state of amapá, eastern amazon, brazil (00º03’02”s 051º07’51” w). the area encompasses 137 ha comprised mostly of dense tidal várzea forest (prance, 1979), a kind of floodplain subject to floods by white-water rivers, which is different of igapó, another kind of amazonian floodplain subject to floods by black or clean-water rivers (prance, 1979). these forests represent about 27% of the amazon basin and differ one from each other regarding many aspects, as forest structure and composition, number of arboreal strata, height and architecture of the canopies, density coverage, soil composition, and so on (pires & prance, 1985; piedade et al., 2001; carim, 2017). large species of trees adapted to flood regimes are present in the area, such as rubber tree (hevea brasiliensis muell. arg., euphorbiaceae), andiroba nut (carapa guianensis aubl., meliaceae), pracuúba (mora paraensis ducke, leguminosae-caesalpinioideae), palm species, such as açaí (euterpe oleracea mart., arecaceae) and others (prance, 1979; carim et al., 2017). this vegetation gives to the forest an over-20-m canopy in height and a clear understory that is flooded up to 2 m by tides mainly from the amazon river and some creeks (igarapés), such as paxicú and fortaleza, that flow throughout the forest (drummond et al., 2008). the climate is typical of tropical forest, af according to köppen’s classification (peel at al., 2007), with high rainfall throughout most of the year. mean annual precipitation is approximately 2100 mm. a rainier season extends from january to july and a less rainy season extends from august to december. mean annual temperature is about 27 ºc and mean annual humidity is higher than 78% (drummond et al., 2008). sampling the fieldwork was carried out once a month, from january 2011 to march 2012, in an open natural area 500 m from the edge of the forest. twenty-six traps were used to capture the bees. each trap consisted of a 2-l plastic bottle with four holes measuring 3 cm in diameter spaced 5 cm apart and 17 cm up from the bottom of the bottle. transparent hoses measuring 4 cm in length were cut in half longitudinally and attached to the holes to facilitate the entrance of the males. three cotton swabs with extra cotton (ball diameter: 2 cm) were contained in each trap and impregnated with an aromatic scent. thirteen scents were used: 1.8-cineole, eugenol, vanillin, benzyl acetate, methyl salicylate, methyl cinnamate, β-ionone, benzyl benzoate, β-myrcene, ethyl butyrate, linalool, methyl benzoate and 2-phenyletanol. one pair of traps with the same scent was securely fixed, to avoid wind shaking, on the same tree at the height of 25 m (canopy) and 1.5 m above the ground (understory). the trees were approximately 10 m one from each other, encompassing an area of approximately 150 m². the traps were monitored from 8:00 a.m. to 6:00 p.m., being replenished every four hours, when the attracted males were removed. abiotic variables general abiotic data of the area: rainfall (mm), temperature (ºc), air relative humidity (%) and wind speed (m/s) were obtained from the núcleo de hidrometereologia e energias renováveis/ instituto de pesquisas científicas e tecnológicas do estado do amapá (nhmet/ iepa). microclimatic data of each arboreal stratum [temperature (ºc) and air relative humidity (%)] were obtained from two climatic data loggers (hobo® pro series) placed at 25 m and 1.5 m in height to record data on an hourly basis. these loggers were used from april 2011 to march 2012 only on sampling days. data on tidal flows [height (m)] were obtained from the capitania dos portos do amapá/ marinha do brasil. sociobiology 64(2): 191-201 (june, 2017) 193 data analysis the kolmogorov-smirnov test was used to determine the normality of the data of richness and abundance. depending on the results, either the wilcoxon test (w) or paired t-test (t) were used to determine the significance of such data comparing between strata and seasons (zar, 2014). principal component analysis (pca) was performed to determine how species were associated with each stratum and season. pearson’s correlation coefficients (r) were calculated to determine the strength of relationships between faunal data (richness and abundance) and abiotic variables in each stratum. similarity coefficients were used to compare the strata with regard to diversity (whittaker βw), number of shared species (sørensen ss) and exclusive species (jaccard sj). pielou evenness index (j’), berger-parker dominance (d) and simpson index (used its reciprocal form 1-d, in which the assemblage becomes more even when the value rises (magurran, 2004) were calculated for each stratum to determine how individuals were distributed per species. shannon-wiener index (h’) was calculated to determine diversity in each stratum regarding the presence of rare species. hutcheson’s t-test was used to determine the significance of differences in the shannon-wiener diversity index (magurran, 2004). results a total of 835 individuals belonging to 28 species was sampled (table 1). the abundance was significantly higher in the understory considering the accumulated number in each stratum (nund. = 645 (77.25%), ncan. = 190 (22.75%); w = 168.0, z = 2.265, p = 0.024) and the relative abundance in each stratum per month (t = -5.587, df = 14, p = <0.001). the richness was similar in both strata considering the accumulated number (sund. = 23, scan. = 24; w = -5.00, z = -0.333, p = 0.820), but it was significantly higher in the understory considering the relative richness in each stratum per month (t = -5.565, df = 14, p = <0.001) (fig 1). microclimatic differences between strata the temperature was 1.4 ºc higher in the canopy (range: 25.60 to 30.83; mean: 28.62 ± 1.79) in comparison to the understory (range: 24.89 to 29.27; mean: 27.22 ± 1.56), representing a statistically significant difference between the strata (t = 13.083, df = 11, p = <0.001). the air relative humidity was higher 11.58% in the understory (range: 80.73 to 104.10; mean: 93.53 ± 9.14) than the canopy (range: 68.32 to 99.20; mean: 81.95 ± 11.68) being a statistically significant difference (t = -9.790, df = 11, p = <0.001). from april to june/2011 and february to march/2012 the air relative humidity of the understory was higher than 100%, revealing a supersaturated environment, which coincided with the highest levels of tides and rainfall and lowest levels of winds and temperatures (fig 1, supple. mat. 1). correlation with abiotic variables in the canopy, the number of males was negatively correlated with the temperature (rcan.temp. = -0.78, p = <0.01) and positively correlated with the air relative humidity (rboth = 0.66, p = 0.02) (supple. mat. 1). the richness in the canopy was negatively correlated with the wind speed (r = -0.64, p = 0.001) and positively correlated with the tidal height (r = 0.53, p = 0.044). in the understory, neither richness nor abundance were significantly correlated with the abiotic variables analyzed (supple. mat. 1). seasonal aspects considering accumulated numbers per season, richness (w = -55.0, z = -3.162, p = 0.002) and abundance (w = -281.0, z = -3.792, p = <0.001) were higher in the rainier season (table 1), when tides, rainfall and air relative humidity were higher (fig 1). accordingly, fewer individuals and species occurred fig 1. monthly fluctuation of abiotic variables, abundance and richness of male euglossine bees sampled in canopy (25 m) and understory (1.5 m) of amazon várzea floodplain forest. can_temp. (ºc) = mean temperature (ºc) in canopy; und_ temp. (ºc) = mean temperature (ºc) in understory; can_rh (%) = relative humidity (%) in canopy; und_ rh (%) = relative humidity (%) in understory. obs.: rainfall (mm) values divided by 100 to fit of image. ps vilhena, lj rocha, ca garófalo – male orchid bees in strata of várzea floodplain forest194 in the less rainy season (table 1), when temperatures were higher and winds were stronger (fig 1). the analysis of each stratum per season revealed a significant difference between canopy and understory with regard to the abundance of males (table 1). in the less rainy season, the abundance was six-fold higher in the understory (w = 110, z = 2.847, p = 0.003) while in the rainier season it was twofold higher comparing to the canopy (w = 158.0, z = 2.263, p = 0.025). the richness did not differ significantly between the strata neither in the rainier (w = 0.00, z = 0.00, p = 1.000) nor in the less rainy season (w = 9.000, z = 0.707, p = 0.547) (table 1). faunal composition more species and individuals of the genus eufriesea cockerell were recorded in the canopy (table 2). euglossa latreille, eulaema lepeletier and exaerete hoffmannsegg were more abundant in the understory but were quite similar in both strata with regard to richness (table 2). the genera occurred with higher richness and abundance in the rainier season, except eulaema, which had the same species present in both seasons (table 2). eufriesea auripes, e. superba, e. surinamensis, euglossa augaspis and exaerete dentata were sampled species of euglossine bees rainier (↑) season less rainy (↓) season total per season total per strata total can. und. can. und total ↑rain total ↓rain total can. total und. eufriesea auripes (gribodo, 1882) 4 0 1 0 4 1 5 0 5 eufriesea concava (friese, 1899) 5 1 0 0 6 0 5 1 6 eufriesea flaviventris (friese, 1899) 1 1 0 0 2 0 1 1 2 eufriesea superba (hoffmannsegg, 1817) 1 0 0 0 1 0 1 0 1 eufriesea surinamensis (linnaeus, 1758) 1 0 0 0 1 0 1 0 1 eufriesea vidua (moure, 1976) 0 1 0 0 1 0 0 1 1 euglossa (euglossa) amazonica dressler, 1982 4 4 1 1 8 2 5 5 10 euglossa (euglossa) avicula dressler, 1982 0 4 0 1 4 1 0 5 5 euglossa (euglossa) bidentata dressler, 1982 0 1 0 0 1 0 0 1 1 euglossa (euglossa) cordata (linnaeus, 1758) 10 35 13 29 45 42 23 64 87 euglossa (euglossa) cognata moure, 1970 47 24 0 0 71 0 47 24 71 euglossa (euglossa) gaianii dressler, 1982 2 1 2 0 3 2 4 1 5 euglossa (euglossa) modestior dressler, 1982 3 10 1 12 13 13 4 22 26 euglossa (euglossa) mourei dressler, 1982 1 1 0 0 2 0 1 1 2 euglossa (euglossa) sp 3 5 3 0 8 3 6 5 11 euglossa (glossura) allosticta moure, 1969 2 2 0 4 4 4 2 6 8 euglossa (glossura) ignita smith, 1874 5 59 5 98 64 103 10 157 167 euglossa (glossura) viridifrons dressler, 1982 4 6 1 2 10 3 5 8 13 euglossa (glossurella) augaspis dressler, 1982 5 0 0 0 5 0 5 0 5 euglossa (glossuropoda) intersecta latreille, 1817 1 23 0 6 24 6 1 29 30 eulaema (apeulaema) nigrita lepeletier, 1841 7 14 4 11 21 15 11 25 36 eulaema (apeulaema) pseudocingulata oliveira, 2006 24 70 4 15 94 19 28 85 113 eulaema (eulaema) bombiformis (packard, 1869) 0 4 1 1 4 2 1 5 6 eulaema (eulaema) meriana (olivier, 1789) 13 101 3 59 114 62 16 160 176 eulaema (eulaema) polyzona (mocsáry, 1897) 0 3 0 2 3 2 0 5 5 exaerete dentata (linnaeus, 1758) 1 0 0 0 1 0 1 0 1 exaerete lepeletieri oliveira & nemésio, 2003 4 10 1 13 14 14 5 23 28 exaerete smaragdina (guérin, 1844) 2 8 0 3 10 3 2 11 13 number of individuals 150 388 40 257 538 297 190 645 835 number of species 23 23 13 15 28 18 24 23 28 table 1. species of male euglossine bees sampled in canopy (can. [25 m]) and understory (und. [1.5 m]) in rainier (↑rain) and less rainy (↓rain) seasons. amazon várzea floodplain forest, state of amapá, brazil, jan. 2011 to mar. 2012 sociobiology 64(2): 191-201 (june, 2017) 195 exclusively in the canopy, whereas e. vidua, e. avicula, e. bidentata and e. polyzona were only sampled in the understory (table 1). however, these exclusive species were represented per few individuals, up to 5 males. nineteen species (67.86% of the total) were present in both strata. the most abundant species in the canopy were e. cognata, e. pseudocingulata and e. cordata, whereas e. meriana, e. ignita, e. pseudocingulata and e. cordata were the most abundant in the understory (table 1). diagnostic species for strata. euglossa cognata was closely related to the canopy, whereas e. meriana, e. ignita, e. intersecta, e. modestior and e. lepeletieri were closely related to the understory, as demonstrated by the pca (fig 2). euglossa cordata, e. nigrita and e. pseudocingulata were related to both strata (fig 2). diagnostic species for seasons. eulaema meriana, e. pseudocingulata, e. cognata and e. intersecta were strongly associated with the rainier season, whereas e. ignita was the only associated with the less rainy season, according to the pca (fig 3). moreover, we add the species of eufriesea as related to the rainier season (table 1). euglossa cordata, e. modestior, e. nigrita and e. lepeletieri were related to both seasons (table 1 and fig 3). the ecological analysis demonstrated higher diversity in the canopy based on the presence of rare species, according to the shannon-wiener index, and greater evenness in the distribution of individuals per species, according to the pielou index (table 3). these results were supported by the simpson index, which demonstrated higher diversity in the canopy also with regard to abundant species and evenness (table 3). according to the berger-parker index, the dominance of species was similar in both strata (table 3). there was high similarity between canopy and understory with regard to the number of species shared between the strata, according to the sørensen coefficient. high similarity was found considering the composition and diversity in each stratum, according to the whittaker coefficient. moderate similarity was found regard to the number of exclusive species in each stratum, according to the jaccard coefficient (table 3). genus number of individuals number of species can. und. ↑rain ↓rain can. und. ↑rain ↓rain eufriesea 13 3 15 1 5 3 6 1 euglossa 113 328 262 179 12 13 14 10 eulaema 56 280 236 100 4 5 5 5 exaerete 8 34 25 17 3 2 3 2 total 190 645 538 297 24 23 28 18 table 2. abundance and richness of males per genus of euglossine bees in canopy (can.) and understory (und.) in rainier (↑rain) and less rainy (↓rain) seasons. amazon várzea floodplain forest, state of amapá, brazil, jan. 2011 to mar. 2012 fig 2. principal component analysis (pca) between species and strata. ellipse indicates 95% confidence interval. species farther from origin of axis are more representative of each stratum. species names: 1. e. auripes; 2. e. concava; 3. e. flaviventris; 4. e. superba; 5. e. surinamensis; 6. e. vidua; 7. e. allosticta; 8. e. amazonica; 9. e. augaspis; 10. e. avicula; 11. e. bidentata; 12. e. cognata; 13. e. cordata; 14. e. gaianii; 15. e. ignita; 16. e. intersecta; 17. e. sp; 18. e. modestior; 19. e. mourei; 20. e. viridifrons; 21. e. bombiformis; 22. e. meriana; 23. e. nigrita; 24. e. polyzona; 25. e. pseudocingulata; 26. e. dentata; 27. e. lepeletieri; 28. e. smaragdina. ps vilhena, lj rocha, ca garófalo – male orchid bees in strata of várzea floodplain forest196 discussion analyze the differences between strata considering only accumulated numbers of richness and abundance in each of them, as reported by oliveira and campos (1996) and ferreira et al. (2011), may lead to erroneous interpretations regarding the real distribution or preference of species for strata. this assumption is supported by the present results regarding richness, which revealed to be significantly different between the strata only when the data were analyzed per sampling month. martins and souza (2005), studying the vertical stratification of orchid bees in area of atlantic rainforest, reported that although there have been higher total abundance in the understory, no difference was found between the strata when the monthly averages were analyzed. these results reinforce the supposed above and therefore we consider that if species or individuals are much more present in one stratum throughout all or most months of the year, this should be interpreted as the preferred stratum or that more favorable to foraging. thus, although in our study the accumulated richness have been similar in both strata, we thought the understory as the stratum used by most of the species of orchid bees because along all months of the year more species and individuals were foraging therein than in the canopy. roubik (1993) and otero and sallenave (2003) found the highest richness and abundance of orchid bees in the understory, reinforcing the likelihood that males forage preferably in the lower layer of rainforests. this preference is supported by some features regarding the biology of these bees, for instance males collect scents from sources usually found in the understory, as the surface of rotting wood, fruits and seeds, leaf litter and terrestrial mushrooms (whitten et al., 1993; cappellari & harter-marques, 2010). moreover, their courtship behavior is carried out few meters from the ground (stern, 1991; eltz et al., 2003), where they are less exposed to predators, such as birds (roubik, 1993). in the understory there are more substrates for females build nests, such as large cavities in trunks which can be used by species with large body sizes, as eulaema spp. (bennet, 1965; cameron & ramírez, 2001), and those with communal or primitively social life habit (roberts & dodson, 1967; santos & garófalo, 1994). the cavities can also be found in the canopy, but they decline in size with height (ulyshen, 2011). fig 3. principal components analysis (pca) between species and seasons. ellipse indicates 95% confidence interval. species farther from origin of axis are more representative of each season. species names: 1. e. auripes; 2. e. concava; 3. e. flaviventris; 4. e. superba; 5. e. surinamensis; 6. e. vidua; 7. e. allosticta; 8. e. amazonica; 9. e. augaspis; 10. e. avicula; 11. e. bidentata; 12. e. cognata; 13. e. cordata; 14. e. gaianii; 15. e. ignita; 16. e. intersecta; 17. e. sp; 18. e. modestior; 19. e. mourei; 20. e. viridifrons; 21. e. bombiformis; 22. e. meriana; 23. e. nigrita; 24. e. polyzona; 25. e. pseudocingulata; 26. e. dentata; 27. e. lepeletieri; 28. e. smaragdina. index canopy (25 m) understory (1.5 m) diversity h’ = 1.1061 h’ = 0.9659 t = 3.7569, df: 321, p=0.01 evenness j’ = 0.8014 j’ = 0.7093 dominance 1-d = 0.8828 d = 0.2474 1-d = 0.8438 d = 0.2481 similarity ss = 0.81; sj = 0.68; βw = 0.1915 table 3. ecological indices for assemblage of male euglossine bees in each arboreal stratum. h’ = shannon-wiener; t = hutcheson t-test; j’ = pielou; 1-d = simpson; d = berger-parker; ss = sørensen; sj = jaccard; βw = whittaker; range of similarity indices: 0 to 1 (0 = similar, 1 = different) sociobiology 64(2): 191-201 (june, 2017) 197 microclimatic differences between strata studies carried out in tropical forests of the panama (read, 1977) and costa rica (fetcher et al., 1985) reported wider ranges of temperature in canopy and understory, with at least 14 ºc of difference between the minimum and maximum temperatures of each stratum. this was different of the observed in the present study, in which the ranges were approximately of 5 ºc, due the absence of low temperatures in the brazilian amazon. regardless of this, in all these studies the higher values of temperature were found in the canopy and the difference between the strata was from 1 to 2 ºc, which must be the difference expected between canopy and understory of undisturbed rainforests. in two areas of terra firme forest in the amazon, the males were active in the field when the temperature ranged between 24.5 and 27 ºc, with minimal or nonexistent records of males outside these limits (oliveira, 1999). in the present study, average temperatures within that range occurred only during three months in the canopy and during seven months in the understory. in the remaining months, the average temperatures were higher than that range in both strata, suggesting that males demonstrate greater tolerance to this climatic variable in várzea floodplain forest. the high humidity of understories was reported by read (1977), when analyzing the potential evaporation ratio in canopy and understory it was accounted a rate of 60% to the canopy and of 45% to the understory, suggesting “an excess of the water needs of the forest”. fetcher et al. (1985) reported higher values of vapor pressure deficit in the canopy and lower in the understory, suggesting that low values implies in saturated air, which also represents a lower potential of evaporation and, thus, a moister air. these results lay support to the present study and, besides that, we reported that in some months of the year the understory becomes a supersaturated environment and it could represent less capacity of evapotranspiration to plants, warming of air and animals (read, 1977; andrews, 2010). the tides have an important role in this process of supersaturation because they increase the amount of water vapor and gases in the understory (bartlett et al., 1990). further, the intense rainfall and the already high humidity of the region also contribute to more vapors. the low temperatures, low wind flows and the canopy coverage difficult the evaporation and the air remains supersaturated in the understory in some days or months of the year. this combination allows an air relative humidity higher than 100% (read, 1977; andrews, 2010) and it could be particularly adverse to small bees, such as euglossa, which have lower thermoregulation capacity (may & casey, 1983). abiotic and seasonal aspects the fact that only the canopy assemblage was significantly affected by abiotic variables suggests that the understory is a more stable environment for the males, offering protection against adverse weather conditions related mainly to rainfall, temperature and winds. this hypothesis is supported by read (1977) who reported that canopies intercept approximately 48% of the rains, which does not reach the understory under weak rains, further they have solar radiation 57% higher than the understories. such conditions significantly increase the energetic costs for living in the canopy, since the bees are required to perform greater thermoregulation activity and exert greater power output of flight muscles to tolerate high temperatures and strong winds many months of the year (inouye, 1975; may & casey, 1983). this was corroborated in the less rainy season of the present study, since the peaks of wind and temperature did coincide with the lowest richness and abundance in the canopy and with the highest abundance of males in the understory. roubik (1993) reported a general tendency of orchid bees to favor lower traps in the dry season in panama, except for one species (e. nigrita). this author also reported greater difference between canopy and understory in the dry season in comparison to the wet season, supporting our results that in the less rainy season the difference between the strata is more accentuate than in the rainier season. the period with higher tidal levels did coincide with the rainier season when some species exclusive to this period occurred, such as e. cognata, all species of eufriesea and others less abundant species. we consider that the correlation between tidal height and canopy richness was related with the arising of these seasonal species, which were strictly abundant in the canopy. cameron (1976), comparing insect populations in salt marshes environments during samplings with and without floods, observed that seasonal species remained in the vegetation even when the tidal level was high. this permanence occurred because it was better to stay in that situation than go out whenever the tides rose, therefore they would maximize their lifetime and reproductive success, being regulated primarily by biological rather than physical factors. these results support our assumption that tides would not exert direct effects on the euglossine assemblage in várzea forest. nonetheless, our sampling was not conducted to compare days with and without floods, some of them randomly occurred during few sampling months. thus, it is necessary studies comparing both situations of the tidal regime to better understand whether this variable have some influence on the orchid bees in floodplains. faunal composition the major difference between the fauna observed in the studied area and that reported in studies carried out in non-flooded amazonian forests (oliveira & campos, 1995; storck-tonon et al., 2009) is related to the high abundance of large-sized species in várzea forest, such as eulaema, exaerete and large euglossa species, as e. intersecta. such observations are corroborated by pearson and dressler (1985), ps vilhena, lj rocha, ca garófalo – male orchid bees in strata of várzea floodplain forest198 who reported the occurrence of large orchid bees as e. meriana, e. bombiformis, e. smaragdina, as more abundant in floodplain forest while e. augaspis, e. bidentata and others small species were significantly more abundant in terra firme forest. moreover, dulmen (2001), in the colombian amazon, reported that large bees were the primary pollinators in flooded forest while small bees were in upland forests. roubik (1993) reported that species of eulaema and exaerete had greater tendency to forage in the canopy while euglossa species were more common in the understory, suggesting that large bees are better fit to forage in the upper layers due to their efficient thermoregulation capacity (may & casey, 1983). we assume this hypothesis to support the prevalence of large bees in the understory of the várzea forests, because the high air relative humidity present in this stratum increases the sensation of heat and requires species with more thermoregulation capacity, especially in the supersaturated months. the occurrence of most of the species and individuals of eufriesea in the canopy was also reported by oliveira and campos (1996) and ferreira et al. (2011), laying support about the preference of this genus to canopies. furthermore, the seasonal habits of these species have been known for a long time, which is attributed to their prepupal diapause (dressler 1982; garófalo et al., 1993; kamke et al., 2008). because of these aspects, in the present study, eufriesea species were considered diagnostic for stratum and season, despite they have not been highlighted in the pca due to low abundance values. if foraging in the canopy requires higher energy costs and greater exposure to predators, there should be a biological trade-off (kneitel & chase, 2004) for seasonal and univoltine species, such as e. cognata and eufriesea species, since the costs experienced in the canopy is trade-off by a habitat with less competition and with higher likelihood to get quick success in a short lifetime, for instance finding a mate, copulating and finding resources to build and provisioning the nest. this assumption may also be suggested for e. nigrita in environments in which it has a seasonal behavior, mainly related to cool periods (rebêlo & garófalo, 1997, rocha-filho & garófalo, 2014). this seasonality would explain the abundance of this species in the forest canopy found in the studies of roubik (1993) and ferreira et al. (2011). similar to reported by ackerman (1983), in the present study e. nigrita did not exhibit seasonality. in this case, behaving like a multivoltine species, it is better to live in the understory and avoid additional costs unless other constraints, such as high population density, conspecific competition, natural enemies, and others, lead them to seek another stratum, as observed in e. cordata, e. nigrita and e. pseudocingulata, species sharing both strata. oliveira and campos (1996), martins and souza (2005) and the present study showed that the canopy is the arboreal stratum with greater diversity and evenness. furthermore, the studies on the vertical stratification of orchid bees (roubik, 1993; oliveira & campos 1996; martins & souza, 2005; ferreira et al., 2011) agree that the faunal composition of both strata are similar, since the forests share many species between strata, which could be called as permanent species, according to magurran (2004). further, there is a low number of exclusive species in each stratum, most of them being singletons and doubletons, or called as occasional species (magurran, 2004). the permanent species are important pollinators to both strata, especially to the canopy when the seasonal species disappear. although várzea forest has a dynamic understory, this was the arboreal stratum used by most of the species and individuals of the orchid bees. in the canopy prevailed seasonal species and others without preference for strata. the fauna was constituted especially by large bees, most of them was found in both strata while few were exclusive in each one. despite of that, it was possible to suggest diagnostic species to strata and seasons. in the less rainy season there was greater difference between the strata, and the abundance increased in the understory and decreased in the canopy. the microclimatic differences between the strata were significant, being temperature, air relative humidity and winds the most important abiotic variables influencing the canopy assemblage. the understory was not significantly affected by the abiotic variables analyzed, proving that it is a more stable environment to the establishment of the orchid bees, having milder microclimatic conditions most of the months of de year, albeit in some months it becomes a supersaturated environment. acknowledgments the authors are grateful to márcio luiz de oliveira (inpa) for helping with the taxonomic identification of the euglossine species, to the anonymous referees for their contributions to the work, and to the brazilian fostering agency cnpq for awarding a master’s degree scholarship to the first author. references ackerman, j.d. 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(2014). biostatistical analysis. 5 ed. england: pearson. 960 p. sociobiology 64(2): 191-201 (june, 2017) 201 su pp le m en ta ry m at er ia l 1 . p ea rs on ’s c or re la tio n co ef fic ie nt s am on g da ta o n fa un a (r ic hn es s an d ab un da nc e) a nd a bi ot ic v ar ia bl es a na ly ze d in a m az on v ár ze a flo od pl ai n fo re st , s ta te o f a m ap á, b ra zi l. n .t ot : t ot al n um be r of in di vi du al s in a re a; n .c an : n um be r of in di vi du al s in c an op y; n .u nd : n um be r of in di vi du al s in u nd er st or y; s .c an : n um be r of s pe ci es in c an op y; s .u nd : n um be r of sp ec ie s in u nd er st or y; t em p. c an : m ea n te m pe ra tu re (º c ) i n ca no py ; t em p. u nd : m ea n te m pe ra tu re (º c ) i n un de rs to ry ; r h. c an : r el at iv e hu m id ity (% ) i n ca no py ; r h. u nd : r el at iv e hu m id ity (% )i n un de rs to ry ; r ai n. t ot : r ai nf al l ( m m ) i n ar ea ; r h. t ot : r el at iv e hu m id ity (% ) i n ar ea ; t em p. t ot : m ea n te m pe ra tu re in a re a; t id es (m ): ti da l h ei gh t ( m ); w in ds (m /s ): w in d sp ee d (m /s ). n .t ot n .c an n .u nd s. c an s. u nd te m p. c an te m p. u nd r h. c an r h. u nd r ai n. to t r h. to t te m p. to t t id es (m ) w in ds (m /s ) s. to t 0. 19 6 0. 21 6 0. 14 0. 58 7 0. 87 6 -0 .1 54 -0 .2 18 0. 23 8 0. 39 3 -0 .1 49 0. 13 4 0. 09 02 0. 12 4 -0 .2 14 0. 48 5 0. 43 8 0. 61 9 0. 02 14 1. 88 e -0 5 0. 63 3 0. 49 7 0. 45 6 0. 20 6 0. 59 6 0. 63 4 0. 74 9 0. 66 0. 44 4 n .t ot 0. 38 3 0. 95 3 0. 25 2 0. 21 7 -0 .0 59 2 -0 .0 15 9 0. 07 19 -0 .0 06 93 -0 .1 42 -0 .2 51 0. 28 3 -0 .0 19 6 -0 .0 09 53 0. 15 9 4. 12 e 08 0. 36 4 0. 43 8 0. 85 5 0. 96 1 0. 82 4 0. 98 3 0. 61 3 0. 36 6 0. 30 6 0. 94 5 0. 97 3 n .c an 0. 08 5 0. 32 2 0. 02 53 -0 .7 84 -0 .7 59 0. 66 2 0. 65 7 0. 43 2 0. 39 6 -0 .4 66 -0 .0 20 5 -0 .3 77 0. 76 3 0. 24 2 0. 92 9 0. 00 25 2 0. 00 42 3 0. 01 9 0. 02 03 0. 10 8 0. 14 4 0. 08 02 0. 94 2 0. 16 6 n .u nd 0. 16 7 0. 22 5 0. 23 3 0. 26 8 -0 .1 74 -0 .2 54 -0 .2 95 -0 .4 01 0. 45 8 -0 .0 14 4 0. 11 3 0. 55 3 0. 42 0. 46 6 0. 39 9 0. 58 8 0. 42 6 0. 28 6 0. 13 9 0. 08 58 0. 95 9 0. 68 8 s. c an 0. 23 5 -0 .3 24 -0 .3 47 0. 49 2 0. 51 1 0. 08 61 0. 37 9 -0 .0 58 3 0. 52 7 -0 .6 36 0. 39 8 0. 30 5 0. 27 0. 10 4 0. 08 93 0. 76 0. 16 3 0. 83 7 0. 04 37 0. 01 08 s. u nd 0. 09 99 0. 03 01 -0 .0 89 3 0. 09 26 -0 .1 97 -0 .0 56 3 0. 20 2 -0 .0 31 6 0. 01 83 0. 75 7 0. 92 6 0. 78 3 0. 77 5 0. 48 3 0. 84 2 0. 47 1 0. 91 1 0. 94 8 te m p. c an 0. 98 6 -0 .9 08 -0 .9 17 -0 .8 66 -0 .9 02 0. 95 8 -0 .4 64 0. 73 7 4. 3e -0 9 4. 55 e -0 5 2. 64 e -0 5 0. 00 02 67 6. 15 e -0 5 9. 48 e -0 7 0. 12 9 0. 00 62 9 te m p. u nd -0 .9 01 -0 .9 17 -0 .8 71 -0 .9 22 0. 95 6 -0 .4 45 0. 75 2 6. 38 e -0 5 2. 68 e -0 5 0. 00 02 29 1. 97 e -0 5 1. 22 e -0 6 0. 14 7 0. 00 48 r h. c an 0. 95 2 0. 80 9 0. 92 9 -0 .8 47 0. 70 2 -0 .8 38 1. 92 e -0 6 0. 00 14 4 1. 26 e -0 5 0. 00 05 0. 01 09 0. 00 06 61 r h. u nd 0. 82 8 0. 95 5 -0 .8 69 0. 66 4 -0 .8 61 0. 00 08 85 1. 41 e -0 6 0. 00 02 43 0. 01 86 0. 00 03 22 r ai n. to t 0. 87 4 -0 .8 81 0. 5 -0 .7 75 2. 02 e -0 5 1. 43 e -0 5 0. 05 78 0. 00 06 84 r h. to t -0 .9 04 0. 63 9 -0 .8 83 3. 81 e -0 6 0. 01 03 1. 31 e -0 5 te m p. to t -0 .3 44 0. 64 9 0. 20 9 0. 00 88 2 t id es (m ) -0 .8 12 0. 00 02 35 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.610sociobiology 62(4): 583-592 (december, 2015) network of bee-plant interactions and recognition of key species in semideciduous forest introduction the scanty information about species richness, diversity, taxonomy, distribution and population dynamics, along with the impact of human activities on most bee species represent the major environmental issues that hinder the conservation of native areas in latin america (freitas et al., 2009). determining the diversity of both the fauna and flora of many ecosystems is the starting point for specific investigations, such as assessing the role of pollinators in maintaining the flora (anacleto & marchini, 2005). the importance of this matter arises therefrom. studies about networks of interactions between bees and plants are fundamental for the conservation and management of native pollinators habitat (biesmeijer et al., 2005). network approaches to ecological research emphasize the pattern of interactions among species, i.e., how the links (interactions) are systematized within the network, rather than only providing the identity of the species that constitute a community (bascompte, 2009). abstract bees are the most effective pollinators of native plants, contributing to the maintenance of many ecosystems, including forests. studies about networks of bee-plant interactions are critical for conservation and habitat management of native pollinators. this study aimed to determine the richness of flower-visiting bees in a fragment of the semideciduous forest (sf) in uberlândia-mg, identify the plants visited by them and build the bee-plant interactions network in this fragment. the study was conducted between october 2010 and september 2011 along a transect of 200 meters on the edge of the forest, with monthly collections performed from 8:00 am to 2:30 pm. we yielded the bee-plant interaction networks and calculated the nodf index and betweenness centrality. we collected 70 bee species and 25 plant species. the network of interactions was nested (nodf = 10.97, p = 0.03). apis mellifera linnaeus and merremia macrocalyx (ruiz & pav.) o’donell showed the highest centrality. the nestedness of the bee-plant interaction confers stability to the network, demonstrating its importance for the fragment conservation. it is likely that m. macrocalyx is a key species in this network since this plant attracted the highest number of bee species and showed the highest centrality, contributing to network cohesion. sociobiology an international journal on social insects if aidar, bf bartelli, fh nogueira-ferreira article history edited by gilberto m. m. santos, uefs, brazil received 26 september 2014 initial acceptance 31 october 2014 final acceptance 08 august 2015 keywords inventory, bees, fragment, nestedness. corresponding author fernanda helena nogueira ferreira universidade federal de uberlândia instituto de biologia ceará st., umuarama, 38400-902 uberlândia-mg, brazil e-mail: ferferre@inbio.ufu.br thus, investigations of bee-plant interaction networks can provide relevant information about the community, such as the identification of key species (martín gonzalez et al., 2010) and coexistence and stability of species in the system (bascompte & jordano, 2007). consequently, the identification of key species in a community can be helpful in setting priorities and conservation goals (jordán, 2009). inventories of the bee fauna are crucial for the knowledge of composition, geographic distribution patterns and relationships between fauna and flora. several factors influence the determination of the richness and abundance of local fauna of bees. for example, the amount of food and diversity of available food sources, which favor the existence of large populations and greater consumer species richness, respectively (silveira et al., 2002). several surveys of bee species have been conducted in the cerrado (carvalho & bego, 1996; andena et al., 2009; siqueira et al., 2012). the conservation status of this biome is becoming increasingly alarming because its vegetation is being hastily replaced research article bees universidade federal de uberlândia, uberlândia, minas gerais, brazil if aidar, bf bartelli, fh nogueira-ferreira – bee-plant interactions and key species in semideciduous forest584 by pasture and crops (freitas et al., 2009). about 50% of the 2 million km2 of the cerrado original biome have been transformed into agricultural areas and pastures, besides other types of use, such as planted forests and urban areas (silva et al., 2006). as a result of this fragmentation process, plant and animal populations have been lost and extinctions may occur. the semideciduous forests (sf) are among the most threatened and fragmented ecosystems in the world. the history of disturbance in brazilian sf reduced these forests to small scattered fragments and, consequently, led to biodiversity loss in these environments (santos et al., 2009). few studies on bee communities have been conducted in sf and studies on beeplant interactions are also rare in this phytophysiognomy. due to anthropic activities, such as habitat destruction for agriculture and excessive use of pesticides, bee populations have been drastically reduced (michener, 2000). the impoverishment of pollinators in forest fragments can cause changes in gene flow within and among plant populations and directly affect natural regeneration (engel et al., 1998). bees are the main pollinators of most species of tropical forest trees (michener, 2000), playing an important role in the conservation of plants and animals that depend on them. this study aimed to deepen our knowledge about the richness and composition of flower-visiting bees in a fragment of semideciduous forest, analyzing the patterns of interactions between bees and visited plants. material and methods study area this study was conducted in a fragment of semideciduous forest (18º 51’ 36’’ s and 48º 13’ 53’’ w) located in the legal reserve of the “fazenda são josé”, in uberlândia, minas gerais, brazil. the fragment has 22 hectares. its interior shows evidence of selective logging and cattle trails and the surrounding matrix is composed of monoculture eucalyptus plantations, annual crops and pastures (prado júnior et al., 2011). the regional climate presents two distinct seasons, a rainy season that extends from october to march and a dry season, from april to september. the annual rainfall ranges from 1160 to 1460mm/year and the average annual temperature varies between 23 and 25°c, and is uniform throughout the year (alves & rosa, 2008). data collection the study was conducted between october 2010 and september 2011, totalizing one year of sampling. bees were collected with entomological nets during their visits to flowers, and eventually during flight, along a 200 metre transect at the forest edge, the most representative section in number of flowering plants. there were no collections within the fragment due to the difficulty in simultaneously observing and collecting, since the flowers were several feet high, preferably in the canopy. bees were collected from 8:00 to 14:30, the time of highest foraging activity. the collection effort involved the work of two collectors during 30 minutes every hour, totalizing 84 hours of sampling. at each time, the temperature and relative humidity were recorded using a digital thermo-hygrometer. only the composition of bee species was sampled. the abundance of these insects was not quantified. bees were sacrificed in a chamber with ethyl acetate. after undergoing pinpricking, they were deposited in the “coleção do museu de biodiversidade do cerrado (mbc)”, located in the “laboratório de ecologia e comportamento de abelhas (leca)” of the “instituto de biologia (inbio)” at the “universidade federal de uberlândia (ufu)”. the plants visited by bees were marked, and three flowering branches of each type were collected and deposited in the “herbarium uberlandense (hufu)” of ufu. plants and bees were subsequently identified using identification keys. we had the help from experts, when necessary. data analysis to evaluate sampling sufficiency, we elaborated the rarefaction curve using the program estimates 8.2.0 (colwell, 2006). the nonparametric estimator jack 1 was used to verify if the species richness value found corresponded to the estimated value. this estimator was chosen because it is one of the most accurate richness estimators (palmer, 1990; krebs, 1999). bee-plant interactions were represented in a bipartite graph (network) generated by the bipartite package (dormann et al., 2008) in r 3.0.1 software (r development core team http://www.r-project.org). the centrality of species was calculated by the program pajek (program for large network analysis) (batagelj & mrvar, 1998). the nestedness index nodf (nestedness metric based on overlap and decreasing fill) was calculated using aninhado 3.0.3 software (guimarães & guimarães, 2006). we used a null model which assumes that probability of each cell being occupied is the average of probabilities of occupancy of its row and column. biologically, this means that probability of drawing an interaction is proportional to the level of generalization (degree) of both the animal and the plant species (bascompte et al., 2003). the higher the value of this index, the greater the nestedness network (almeidaneto et al., 2008). nested networks are characterized by interactions between generalists, specialists interacting with generalists and absent or rare interactions between specialists (guimarães et al., 2006). for the calculation of centrality, the index of betweenness centrality was chosen. this index shows the importance of a species as a connector between different parts of the network and evaluates the contribution of each species to its cohesion (borgatti & everrett, 2006). sociobiology 62(4): 583-592 (december, 2015) 585 results throughout the study period, 74 bee species distributed in five families were collected (table 1). four species were caught only in flight (anthidioctes megachiloides, augochlora sp.5, eulaema nigrita and lorocanthidium sp.). apidae was the most represented family with 48 species, followed by halictidae and megachilidae. andrenidae and colletidae were the less representative families. meliponina was the group with the highest richness, with 15 species sampled. the rarefaction curve showed no tendency to stabilization. the value found for the richness estimator jack 1 was 114.51, suggesting that approximately 65% of the species in the area were sampled (figure 1). fig 1. rarefaction curve representing the cumulative number of species sampled in function of the quantity of samples in semideciduous forest of "fazenda são josé", uberlândia-mg, in 2010 and 2011. family subfamily tribe subtribe species voucher number andrenidae oxeinae oxaea flavescens klug mbc-372 panurginae calliopsini acamptopoeum prinii (holmberg) mbc-361 protandrenini cephalurgus anomalus moure & lucas de oliveira mbc-230 apidae apinae apini apina apis mellifera linnaeus mbc-70 bombina bombus morio (swederus) mbc-653 euglossina euglossa (euglossa) sp. mbc-284 euglossa imperialis cockerell mbc-286 euglossa pleosticta dressler mbc-285 eulaema nigrita lepeletier mbc-288 meliponina cephalotrigona capitata (smith) mbc-337 leurotrigona muelleri (friese) 378 melipona rufiventris lepeletier mbc-228 oxytrigona cf. tataira smith mbc-01 paratrigona lineata (lepeletier) mbc-340 partamona ailyae camargo 440 partamona combinata pedro & camargo 317 plebeia droryana (friese) 231 scaptotrigona aff. depilis (moure) mbc-139 scaptotrigona sp. 459 tetragona clavipes (fabricius) mbc-174 tetragonisca angustula (latreille) mbc-199 trigona cilipes (fabricius) 463 trigona hyalinata (lepeletier) mbc-57 trigona spinipes (fabricius) mbc-111 centridini centris (centris) aenea lepeletier 434 centris (melacentris) collaris lepeletier mbc-274 centris (trachina) sp. mbc-371 centris tarsata smith mbc-370 epicharis (hoplepicharis) affinis mbc-271 epicharis (epicharana) flava (friese) mbc-262 table 1. bee species collected on the edge of the semideciduous forest of "fazenda são josé", uberlândia-mg, in 2010 and 2011. classification based on silveira et al. (2002). if aidar, bf bartelli, fh nogueira-ferreira – bee-plant interactions and key species in semideciduous forest586 family subfamily tribe subtribe species voucher number apidae apinae emphorini ancyloscelis apiformis (fabricius) 395 exomalopsini exomalopsis auropilosa spinola 260 exomalopsis fulvofasciata smith mbc-275 tapinotaspidini chalepogenus sp. mbc-295 monoeca cf. brasiliensis lepeletier & serville mbc-315 monoeca planaltina aguiar 423 monoeca sp. 460 paratetrapedia cf. flaveola aguiar & melo mbc-289 paratetrapedia cf. lugubris (cresson) mbc-304 paratetrapedia connexa (vachal) 176 paratetrapedia punctata aguiar & melo mbc-313 tropidopedia flavolineata aguiar & melo mbc-871 tetrapediini tetrapedia cf. diversipes klug mbc-310 tetrapedia cf. ornata (spinola) 418 tetrapedia sp. 414 xylocopinae ceratinini ceratina (calloceratina) chloris (fabricius) 217 ceratina (crewella) sp.1 mbc-650 ceratina (crewella) sp.2 242 ceratina (crewella) sp.3 223 ceratina sp.1 221 xylocopini xylocopa (neoxylocopa) suspecta moure & camargo mbc-339 colletidae hylaeinae hylaeus cf. nasutus (vachal) 220 hylaeus cf. transversus (vachal) 135 hylaeus sp.1 146 hylaeus sp.2 148 augochlora sp.1 augochlora sp.1 mbc-367 augochlora sp.2 305 augochlora sp.3 132 augochlora sp.4 137 augochlora sp.5 155 augochloropsis cf. aurifluens (vachal) mbc-216 augochloropsis cf. hebescens (smith) mbc-225 augochloropsis cf. patens (vachal) mbc-223 augochloropsis sp.1 mbc-364 augochloropsis sp.2 mbc-365 augochloropsis sp.3 mbc-366 pseudaugochlora graminea (fabricius) mbc-369 thectoclora sp. 149 temnosoma sp. mbc-368 halictini dialictus sp. mbc-322 megachilidae megachilinae anthidiini anthodioctes megachiloides holmberg mbc-316 larocanthidium sp. mbc-318 lithurgini lithurgus huberi ducke mbc-320 megachilini megachile (moureapis) sp. mbc-317 table 1. bee species collected on the edge of the semideciduous forest of "fazenda são josé", uberlândia-mg, in 2010 and 2011. classification based on silveira et al. (2002) (continuation). sociobiology 62(4): 583-592 (december, 2015) 587 bees visited flowers of 25 plant species belonging to 14 families (supplementary material). the most representative families were sapindaceae, with 4 species of visited plants, followed by fabaceae and bignoniaceae, with 3 species each. the network of interactions consisted of 70 species of bees and 25 species of plants and presented a nested pattern, nodf = 10.97 (p = 0.03) (figure 2). merremia macrocalyx fig 2. bipartite graph of bee-plant interaction’s network sampled in the semideciduous forest of “fazenda são josé”, uberlândia-mg, in 2010 and 2011. bees are represented on the left side of the graph and plants on the right side. table 2. values of the betwenness centrality index for species of the network of bee-plant interaction sampled in the semidecidous forest of “fazenda são josé”, uberlândia-mg, in 2010 and 2011. bee species index plant index apis mellifera 0.305 merremia macrocalyx 0.319 paratrigona lineata 0.108 coccoloba mollis 0.152 tetragona clavipes 0.105 banisteriopsis argyrophylla 0.174 paratetrapedia cf. lugubris 0.097 trema micrantha 0.107 scaptotrigona aff. depilis 0.040 serjania lethalis 0.100 trigona spinipes 0.053 arrabidaea florida 0.084 augochloropsis cf. aurifluens 0.016 bauhinia brevipes 0.101 augochloropsis cf. patens 0.049 bidens gardneri 0.058 exomalopsis fulvofasciata 0.039 celtis iguanae 0.035 paratetrapedia punctata 0.079 heteropterys cf. campestris 0.068 epicharis flava 0.028 machaerium aculeatum 0.056 paratetrapedia cf. flaveola 0.010 matayba guianensis 0.083 bombus morio 0.010 oxalis grisea 0.052 chalepogenus sp. 0.008 sida rhombifolia 0.052 dialictus sp. 0.002 solanum lycocarpum 0.045 e. (hoplepicharis) affinis 0.012 brachiaria decumbens 0.009 euglossa imperialis 0.041 prestonia coalita 0.022 melipona rufiventris 0.013 elephantopus cf. mollis 0.021 oxytrigona cf. tataira 0.0003 ipomoea tubata 0.021 paratetrapedia connexa 0.015 luehea divaricata 0.003 partamona ailyae 0.013 terminalia argentea 0.001 temnosoma sp. 0.009 memora axillaris 0.000 tetragonisca angustula 0.006 senna ocidentalis 0.000 tetrapedia sp. 0.018 serjania mansiana 0.000 trigona hyalinata 0.011 zeyheria montana 0.000 note: bee species that were not presented in the table obtained index equal to zero. was the plant with the highest betweenness centrality index, followed by coccoloba mollis and banisteriopsis argyrophylla (table 2). bees that had the highest centrality were apis mellifera, paratrigona lineata and paratetrapedia lugubris, respectively (table 2). bees presented the highest richness in may and september, both with 19 species (figure 3). the greatest richness of flowering plants visited by bees occurred in april, with nine species (figure 3). if aidar, bf bartelli, fh nogueira-ferreira – bee-plant interactions and key species in semideciduous forest588 fig 3. richness of bees and plants and averages of abiotic data collected throughout the year of study at “fazenda são josé”, uberlândia-mg. (a) average temperature. (b) average relative humidity. discussion the network was essential for the identification of keyspecies in the studied area. the nestedness pattern found in this study corroborates other researches in other types of vegetation. one of the most important factors associated with the richness of bees is environmental heterogeneity. the more varied the floristic composition of the site, the greater the possibility of available niches providing a larger number of species living in the same area (andena et al., 2009). regarding bee fauna in any particular location, the greater the diversity of plants, the greater the variety of bees, since they are their potential pollinators (michener, 2000). thus, a high diversity of plants contributes to a high diversity of pollinators, which are one of the key components responsible for the maintenance of plant diversity (michener, 2000). for the maintenance of potential pollinators in an area, the preservation of nesting sites for bees, such as rotten tree trunks and bounds in the case of solitary bees are essential. trees with hollows and abandoned ant and termite nests should be preserved to be used for social bee nesting sites (cortopassi-laurino et al., 2009). thus, the conservation of the studied area subject of this research is extremely relevant. its significance lies in the fact that it is a fragment located near an urban area that maintains important and diverse plant species, offering a possible refuge, and providing food resources for many bee species. regarding the bee richness, it was expected that the rarefaction curve was not stabilized, since samplings including all the species present in an area is not common in insect inventories due to the high diversity of this group (gotelli & colwell, 2001; brosi et al., 2007). the extreme incidence of the apidae family seems to be a characteristic pattern in the brazilian cerrado biome, since similar results have been found in other surveys of different types of vegetation in the cerrado biome (carvalho & bego, 1996; antonini & martins, 2003; anacleto & marchini, 2005; andena et al., 2012). apidae is one of the most diverse families widely distributed in brazil and worldwide. it occurs in different biomes and under different environmental characteristics (michener, 2000), which may explain the major representativeness of this family in this study and in others. the halictidae family, the second most representative in this study, has a worldwide distribution. however, it is more diverse in temperate regions, despite possessing some unique neotropical genera (michener, 1979). in ecosystems with disturbances, there is a trend towards increased species of halictidae (roubik, 1989), which may indicate that the studied semideciduous forest is a disturbed area. the andrenidae and colletidae families had low occurrence in this study, and are poorly represented in the neotropical region (silveira & campos, 1995), which may have contributed to the results. colletidae is an australian family, although some genera occur in south america (silveira et al., 2002). silveira & campos (1995) found that bees of the cerrado biome compared to the fauna from other brazilian biomes are characterized by the high representativeness of the tribes that collect oil (centridini, tapinotaspidini and tetrapediini). for example, the species centris tarsata, epicharis flava, paratetrapedia flaveola, tetrapedia diversipes, and bee species of the subtribe meliponina, have high representativeness, as we verified in this study. the high centrality of apis mellifera shows that this species is essential for the maintenance of the interactions network studied. apis mellifera was the species that had the broadest niche, as it interacted with the largest number of plant species in the present study. similar results were found in an area of cerrado biome in the state of são paulo (andena et al., 2012) and in a review of inventories carried out in several regions of brazil (kleinert & giannini, 2012). this fact may be related to its long daily and annual periods of foraging, high population density and sophisticated communication system (roubik, 1989), allowing for a large number of plant species to be visited by these bees, resulting in a broader niche (andena et al., 2012). a. mellifera is an exotic species scattered throughout various biomes, well adapted to different climatic conditions and presents generalist behaviour (kleinert & giannini, 2012). it is among the most important pollinators of natural environments and crops (potts et al., 2010). amidst the most generalist bees sampled in this study, there were several species of meliponina, such as paratrigona lineata, tetragona clavipes, scaptotrigona depilis and trigona spinipes. other surveys carried out in the cerrado biome have also reported large amplitude niches for some of these species sociobiology 62(4): 583-592 (december, 2015) 589 (antonini & martins, 2003; nogueira-ferreira & augusto, 2007). the niche size may be related to their eusocial group behavior, perenniality of their colonies and generalized foraging and recruitment habits (roubik, 1989). in general, social species are more generalists and, therefore, have broader niches than solitary bees (biesmeijer et al., 2005). the nestedness of the network observed in this study means that plants with few interactions are associated with generalist animals. however, specialist animals are associated with plants with many interactions, and generalists of one group interact with generalists of the other group, forming a dense core of interactions (lewinsohn et al., 2006). nested networks of bee-plant interactions were also found in studies in the caatinga in the state of bahia (pigozzo & viana, 2010) and in another area of cerrado sensu stricto in the state of são paulo (andena et al., 2012). nested networks are thoroughly cohesive, present a dense mass of interactions that extend throughout the community. they also have heterogeneity in their distribution of connections and possess possible alternative routes in response to environmental perturbations (bascompte et al. 2003; bascompte & jordano, 2007). thus, nested networks are asymmetric, with generalist species interacting with specialists, providing pathways for rare species to withstand environmental adversities and alternative routes for system responses to perturbations, such as deletion of a mutualist (jordano, 1987). these characteristics exert fundamental influence on the network stability (bascompte et al., 2006; santos et al., 2010) and maintenance of biodiversity (bascompte et al., 2006). thus, the network of bee-plant interactions found in this study has important properties for the maintenance of the community of bees and plants. in pollination systems, the most generalist species are usually key species in the network (martín gonzález et al., 2010). these species are vital to the network structure, functioning and resilience, playing a pivotal role in community cohesion (martín gonzález et al., 2010; kleinert & giannini, 2012). not all nodes (species) are equally important for the dynamics and stability of the system (jordán, 2009; martín gonzález et al., 2010). the importance of a node can be quantified by centrality indices (freeman, 1979). in the present study, the plants that had the highest centrality indices compared to other species were merremia macrocalyx, coccoloba mollis and banisteriopsis argyrophylla, which were also the plant species visited by more bee species. these plants are essential to the structure and stability of the network (martín gonzález et al., 2010), in other words, they are indispensable to the maintenance of the community, and suppression of these species can quickly affect other species (jordán, 2006). m. macrocalyx presents the highest centrality index and can be considered a key species in the obtained network of interactions. it is extremely relevant for conservation in the studied fragment since it contributes to the maintenance of the existing bee community. as a climbing plant, m. macrocalyx is a substantial element in the structure of the forest, helping maintain optimal microclimate conditions for germination and contributes to increased resistance to wind on the edges of the fragment (engel et al., 1998). the considerable number of bee species foraging on m. macrocalyx observed in this study may be associated with the supply of available resources offered by its flowers, as corroborated by neves et al. (2006). they reported that pollen remains available from the time of anthesis, early in the morning, and decreases after 15h. nectar secretion is intensive in the morning with a decreasing tendency during the day. coccoloba mollis, the second most important species in our study, has inflorescences in terminal panicles, with many small and fragrant flowers with nectaries (lorenzi, 1992). according to lorenzi (1992), the flowers of this species are considered apicultural, which explains the high number of bee species collected visiting its flowers. the greatest richness of bees occurred in may and september coinciding with the flowering periods of m. macrocalyx and c. mollis, respectively, which attracted a large proportion of species sampled in these months. the network of bee-plant interactions presented is nested. this aspect can be positive in case of disturbances since nested networks offer alternative routes for the maintenance of rare species. m. macrocalyx can be considered a key species in the studied area since it attracted a large number of bee species and presented the highest centrality in the network of interactions. this plant species is essential to the cohesion of the network as a whole, and greatly relevant to the conservation of the cerrado biome. acknowledgements we thank dr. fernando amaral da silveira and his students rodolfo césar costa arantes, igor rismo coelho e josé eustáquio santos júnior from the “laboratório de sistemática e ecologia de abelhas” of “universidade federal de minas gerais (ufmg)” for identification of bees, dr. silvia regina pedro menezes and dr. sidnei mateus from the “faculdade de filosofia, ciência e letras de ribeirão preto (usp-ffclrp)” for identifying bees of the meliponina group, dr. ivan schiavini, dr. glein monteiro araújo, dr. ana angélica barbosa, ms. betânia da cunha vargas, ms. danilo marques, ms. ana isa marquez rocha machado and ms. rodrigo andrade pacheco for identifying plant species, and “conselho nacional de desenvolvimento científico e tecnológico (cnpq)” for financial support. references almeida-neto m., guimarães p., guimarães p. r. jr., loyola r. d. & ulrich w. a. 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(2002). abelhas brasileiras: sistemática e identificação. belo horizonte: ministério do meio ambiente, probio – pnud, fundação araucária. siqueira, e. n. l., bartelli, b. f., nascimento, a. r. t. & nogueira-ferreira, f. h. (2012). diversity and nesting substrates of stingless bees (hymenoptera, meliponina) in a forest remnant. psyche, 2012: 1-9. doi: 10.1155/2012/370895. if aidar, bf bartelli, fh nogueira-ferreira – bee-plant interactions and key species in semideciduous forest592 supplementary material. plant species visited by bees in the semideciduous forest of “fazenda são josé”, uberlândia-mg, in 2010 and 2011. family species voucher number apocynaceae prestonia coalita (vell.) woodson 60686 asteraceae bidens gardneri baker 60836 elephantopus mollis kunth 60835 bignoniaceae arrabidaea florida dc. pl18 memora axillaris k. schum. 60688 zeyheria montana mart. pl38 cannabaceae celtis iguanae (jacq.) sarg. 58978 trema micrantha (l.) blume 58977 combretaceae terminalia argentea mart. pl45 convolvulaceae ipomoea tubata nees pl26 merremia macrocalyx (ruiz & pav.) o’donell 60685 fabaceae bauhinia brevipes vogel pl41 machaerium aculeatum raddi 60690 senna occidentalis (l.) link pl27 malvaceae luehea divaricata mart. pl42 sida rhombifolia l. pl23 malpighiaceae banisteriopsis argyrophylla (a. juss.) b. gates 60166 heteropterys campestris a. juss. 60164 oxalidaceae oxalis grisea a. st.-hil. & naudin pl12 poaceae brachiaria decumbens stapf pl15 polygonaceae coccoloba mollis casar. 58980 sapindaceae matayba guianensis aubl. pl6 serjania lethalis a. st.-hil. 58972 serjania mansiana mart. 60687 solanaceae solanum lycocarpum a. st.-hil. pl8 doi: 10.13102/sociobiology.v64i3.1686sociobiology 64(3): 356-358 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 resource storage in the neotropical social wasp mischocyttarus socialis (saussure, 1854) (vespidae: polistini) food provisioning in social hymenoptera colonies is described for honeybees, taking place in honeycombs (seeley, 1989), and for many groups of ants who can store large quantities of vegetal material inside their nests (taber, 1999). social wasps (vespidae: polistinae) may also present food storage behaviors, although there are few reports and studies approaching this phenomenon for the group, especially when considering its diversity (richards & richards, 1951; machado et al., 1977; gobbi et al., 1984; prezoto & gobbi, 2003; guimarães et al., 2008 michleutti et al., 2017). the wasps may store liquid resources, such as sugary substances (often called “wasp honey” or just “honey”) (garcía, 1978; strassman, 1979; guimarães et al., 2008) or solid prey fragments (prezoto et al., 2005; rocha, 2011; michleutti et al., 2017), which are deposited inside cells in their nest; both types of resources mayoccasionally be stored together in abstract like other hymenoptera, the social wasps may store both liquid and solid resources inside the cells of their nests as reserve for periods of food shortage. this study describes storage of honey in colonies of the neotropical independentfounding wasp mischocyttarus socialis. during august 2016, in the dry season, 15 colonies in the post-emergence phase were recorded at the botanical garden of universidade federal de juiz de fora in southwestern of brazil. five of the colonies showed honey stored inside empty and immature (eggs) cells. the presence of these two patterns for this species suggests that the storage behavior may be a short-term strategy to feed larvae as soon as they emerge and also a long-term storage to complement population diet during food shortages. sociobiology an international journal on social insects bc barbosa, m detoni, tt maciel, f prezoto article history edited by gilberto m. m. santos, uefs, brazil received 03 may 2017 initial acceptance 24 may 2017 final acceptance 01 july 2017 publication date 17 october 2017 keywords anthropic influence, disturbance, honey, polistinae, social wasps. corresponding author bruno corrêa barbosa laboratório de ecologiacomportamental e bioacústica universidade federal de juiz de fora campus universitário bairro martelos, cep 36036-900 juiz de fora, minas gerais, brasil. e-mail: barbosa.bc@outlook.com the same cell. resource storage in social wasps is seen as a reflex of the group’s opportunistic foraging habit in response to sporadically abundant food sources (richards & richards, 1951); stored food is an important complement to the colony’s diet, especially during dry seasons, when resources are usually scarce (hunt et al., 1987). mischocyttarus socialis (saussure, 1854) is an independent founding social wasp species whose nests consist of an exposed comb, attached to the surface through a peduncle (jeanne, 1975). despite the species’ reported presence in various diversity studies (barbosa et al., 2016), there is almost no data on its behavior or basic biology, and it is unknown whether the species presents food storing behaviors such as other mischocyttarus species (guimarães et al., 2008). aiming to fulfill this lack of information, our objective in this study was to report the storage of honey in m. socialis colonies. laboratório de ecologia comportamental e bioacústica (labec), universidade federal de juiz de fora, minas gerais, brazil short note sociobiology 64(3): 356-358 (september, 2017) 357 the observations took place during august of 2016, in the dry season, at the botanical garden of universidade federal de juiz de fora in the municipality of juiz de fora, minas gerais state (21 ° 43’28 “s 43 ° 16’47” w 800m asl), southwestern of brazil. the area’s climate is classified as cwa according to köppen (sá-júnior et al., 2012) and its 84 hectares extension has a floristic heterogeneity, with presence of endangered species and a predominance of pioneer plant species. there is also a considerable presence of exotic plant species, making up a novel ecosystem (santiago et al., 2014; maciel & barbosa, 2015). we recorded 15 m. socialis colonies in post-emergence phase, from which five showed honey storage in the form of droplets deposited inside the nests’ cells. we observed the two storage patterns described for mischocyttarus cassununga (von ihering) (guimarães et al., 2008): honey was found both inside empty cells and in immature (eggs) cells (fig 1; table 1). we didn’t find any storage in larvae cells, contrary to the observations gathered fromother independentfounding speciessuch as polistes simillimus zikan, 1951 and m. cassununga (prezoto & gobbi, 2003; guimarães et al., 2008). the presence of both patterns for this species suggests that the storage behavior can be used for more than one end, since storing honey in immature cells is explained as a shortterm strategy to feed larvae as soon as they emerge (jeanne, 1972), while honey in empty cells suggests a long-term storage to complement population diet during food shortages (strassman, 1979; hunt et al., 1987). surverys carried out from february 2011 to february 2014, recorded 55 coloniesin that area, but we did not observe resource storage in these nests. this period represents the transition from pre-implementation of the botanical garden, which by then meant a much smaller impact on the local vegetation due to anthropic disturbance, such as the traffic of people and vehicles. on the other hand, the botanical garden was at the final constructions stage in 2016, which was when resource storage was observed in m. socialis nests. these observations suggest that storing resources may not only be a response to the climatic season (hunt et al., 1987), but also to a resource shortage related to vegetal loss caused by a long-term disturbance process, granting the colony’s survival during harsh periods. recording the storage behavior for m. socialis increases the number of species known to show this behavior and may reveal important information on its ecology, as well as for the whole genus. it may also help understanding how these groups react to different environmental pressure events such as food shortage in harsh periods given the asynchrony of the neotropical social wasps’ colonial cycle. acknowledgments the authors thank the national council for scientific and technological development (cnpq) (f. prezoto 310713 /2013-7) for financial support. references barbosa bc, detoni m, maciel tt, prezoto f (2016). studies of social wasp diversity in brazil: over 30 years of research, advancements and priorities. sociobiology, 63: 858-880. doi: 10.13102/sociobiology.v63i3.1031 fig 1. honey storage inside the cells of a mischocyttarus socialis nest in post-emergence phase: (a) storage in empty cells and (b) storage in egg cells. table 1. composition of the five colonies of mischocyttarus socialis found with storage of honey at the botanical garden of universidade federal de juiz de fora and the percentage of cells used for the storage. colony total number of cells number of adults number of empty cells with storage number of egg cells with storage number of larvae/ pupae cells with storage percentage of cells with storage 1 26 6 1 1 2 7.7 2 24 5 4 5 9 37.5 3 82 9 2 6 8 9.7 4 39 6 1 3 4 10.2 5 16 2 2 0 2 12.5 total 187 28 10 15 25 13.5 bc barbosa, m detoni, tt maciel, f prezoto – resource storage in the neotropical social wasp358 gobbi n, machado vll, tavares-filho ja (1984). sazonalidade das presas utilizadas na alimentação de polybia occidentalis (olivier, 1791) (hymenoptera, vespidae). anais da sociedade entomológica do brasil, 13: 63-69. guimarães dl, castro mm, prezoto f (2008). patterns of honey storage in colonies of the social wasp mischocyttarus cassununga (hymenoptera, vespidae). sociobiology, 51: 655-660. hunt jh, jeanne rl, baker i & grogan de (1987). nutrient dynamics of a swarm-founding social wasp species, polybia occidentalis (hymenoptera: vespidae). ethology, 75: 291-305. garcía r (1978). cuatro estúdios sobre a vispas sociales del perú (hymenoptera: vespidae) revista peruana de entomología, 21: 1-22. jeanne r.l. (1972). social biology of the neotropical wasp mischocyttarus drewseni. bulletin of the museum of comparative zoologyof harvard, 144: 63-150. jeanne r.l. (1975). the adaptiveness of social wasp nest architecture. the quarterly review of biology, 50: 267-287. doi: 10.1086/408564 machado vll (1977). estudos biológicos de polybia occidentali soccidentalis (olivier, 1791) (hym., vespidae). anais da sociedade entomológica do brasil, 6: 7-24. maciel tt & barbosa bc (2015). áreas verdes urbanas: história, conceitos e importância ecológica. ces revista, 29: 30-42. michelutti kb, soares erp, prezoto f, antonialli-junior wf (2017). opportunistic strategies for capture and storage of prey of two species of social wasps of the genus polybia lepeletier (vespidae: polistinae: epiponini). sociobiology, 64:105-110. doi: 10.13102/sociobiology.v64i1.1142 prezoto f, gobbi n (2003). patterns of honey storage in nests of the neotropical paper wasp polistes simillimuszikan, 1951 (hymenoptera, vespidae). sociobiology, 41: 437-442. prezoto f, lima ma, machado vl (2005). survey of preys captured and used by polybia platycephala (richards) (hym.: vespidae, epiponini). neotropical entomology, 34: 849-851. richards ow, richards mj (1951). observations on the social wasps of south america (hymenoptera, vespidae). transactions of the royal entomological society of london, 102: 1-170. rocha mp (2011). ecologia comportamental de polybia patycephala (richards, 1978) (hym., vespidae, epiponini). m. sc dissertation universidade federal de juiz de fora, juiz de fora, minas gerais, brazil, pp 77. sá júnior a, carvalho lg, silva ff, carvalho alves m (2012). application of the köppen classification for climatic zoning in the state of minas gerais, brazil. theoretical and applied climatology, 108: 1-7. doi: 10.1007/s00704-011-0507-8 santiago ds, fonseca cr, carvalho fa (2014). fitossociologia da regeneração natural de um fragmento urbano de floresta estacional semidecidual (juiz de fora, mg). revista brasileira de ciências agrárias, 9: 117-123. seeley td (1989). the honey bee colony as a superorganism. american scientist, 77: 546-553. strassmann je. 1979. honey caches help female paper wasps (polistes annularis) survive texas winters. science, 204: 207-209. taber sw (1999). the world of the harvester ants. texas a & m university press: austin, texas. 213 pp. doi: 10.13102/sociobiology.v64i2.1348sociobiology 64(2): 182-190 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the orchid bee communities in different phytophysiognomies in the atlantic forest: from lowland to montane rainforests introduction euglossini bees, also known as orchid bees, have high biological relevance as pollinators of native and some cultivated plants (ramírez et al., 2002; roubik & hanson, 2004; singer, 2004; farias et al., 2007). several studies as tonhasca et al. (2002a), ramalho et al. (2009), rosa et al. (2015) and aguiar et al. (2015) have reported the sensitivity of these bees to environmental degradation. for this reason, some species have been suggested as bioindicators of habitat quality, such as euglossa marianae nemésio, euglossa iopoecila dressler, and euglossa imperialis cockerell (tonhasca et al., 2002; nemésio 2009; ramalho et al., 2009) orchid bees are diverse in tropical rainforests. the atlantic forest is the second richest biome in orchid bee abstract to investigate species composition, diversity, richness, and monthly abundance of orchid bees in different types of dense rainforest in different altitudes, the orchid bees were collected for 11 months in three vegetation types: dense montane ombrophilous forest (dmof), dense submontane ombrophilous forest (dsof_1 and dsof_2 in different altitudes), and dense lowland ombrophilous forest (dlof). using aromatic traps 1,945 males of 20 species were collected. the dmof showed the highest species richness (19), and diversity (h’ = 2.42), whereas the dsof_1 showed the highest abundance (775). the dlof which is the largest continuous forest remnant sampled (15,300 ha), showed the lowest species richness (11) and diversity index (1.63), additionally presented the highest dominance (0,49). euglossa cordata (linnaeus) was the most abundant species in dlof, dsof_1 and dmof, while euglossa ignita smith was the most abundant species in the dsof_2. abiotic factors (temperature and humidity) were correlated with abundance of species; already the altitude had correlation with dominance and diversity parameters. our results demonstrate that phytophysiognomy influences the richness and diversity of orchid bees. the results obtained in dlof did not corroborate what was expected, we attributed these results to the management and extractivism activities of the palm tree attalea funifera martius. finally, our study reveals the importance of preserving different phytophysiognomies within the same biome to conserve orchid bees. sociobiology an international journal on social insects rls medeiros1, wm aguiar1, cml aguiar1, igm borges2 article history edited by celso f. martins, ufpb, brazil received 20 december 2016 initial acceptance 14 february 2017 final acceptance 06 march 2017 publication date 21 september 2017 keywords euglossini, bee fauna, bee community, species richness, orchid pollinators, odor baits. corresponding author willian moura de aguiar departamento de ciências biológicas laboratório de entomologia universidade estadual de feira de santana — uefs av. transnordestina, s/nº novo horizonte, 44036-900 feira de santana-ba, brasil e-mail: wmag26@yahoo.com.br species in brazil, with over 60 species recorded. among those species, 27 are endemic (nemésio & silveira, 2007; nemésio, 2009; moure et al., 2012). the environmental heterogeneity of this forest, together with climatic variations, contribute to the high species diversity and endemism (silva & casteleti, 2003; galindo-leal & câmara, 2003). however, this high biodiversity for the orchid bees is susceptible to the effects of habitat fragmentation (brosi et al., 2007; brosi, 2009; aguiar & gaglianone, 2012; aguiar et al., 2015). in addition to these factors, the differences in climate and local phytophysionomic features can interfere in the distribution patterns of the orchid bees (roubik & hanson, 2004). according to nemésio and silveira (2007), differences in the composition species in the atlantic forest domain may be associated with floristic differences between dense and semi-deciduous 1 pós-graduação em ecologia e evolução, universidade estadual de feira de santana, bahia, brazil 2 pós-graduação em modelagem em ciências da terra e do ambiente, universidade estadual de feira de santana, bahia, brazil research article bees sociobiology 64(2): 182-190 (june, 2017) 183 ombrophylous forests. the altitude is another factor that has shown a strong influence on the geographic distribution of these bees (ramirez et al., 2002). in the brazilian atlantic forest, three regions are considered to be biodiversity corridors: northeastern brazil (the pernambuco corridor), southern bahia and espírito santo state (the central corridor), and the serra do mar corridor (fonseca et al., 2004; galindo-leal & câmara, 2005). one of the last remnants of atlantic forest in the central corridor lies in southern bahia (franke et al., 2005; saatchi et al., 2001). the region is known as an important endemism area for several taxonomic groups, and has been considered one of the most important in the atlantic forest (franke et al., 2005; nemésio, 2013a). the orchid bee fauna of the central atlantic forest corridor was studied in the southern portion of bahia (nemésio, 2013a; nemésio, 2013b; nemésio, 2013c; nemésio, 2014) and in a region near to our study area (rosa et al., 2015). however, large variations have been observed in orchid bee communities, even in neighboring regions, which could be related to habitat heterogeneity (tonhasca et al., 2002b). the objective of the present study was to investigate species composition, diversity, richness, and monthly abundance of orchid bees in different types of dense rainforest in different altitudes. material and methods study area the present study was carried out in atlantic forest remnants located in the pratigi environmental protection area, southern bahia, northeastern brazil. the reserve has an area of approximately 85,686 ha, covered by three vegetation types: montane, submontane, and lowland rainforests, at different conservation status (lopes et al., 2011). the annual rainfall is of approximately 2,000 mm; the average relative humidity varies between 80% and 90%; and the average temperature, between 21 ºc and 25 ºc (fischer, 2007). for the inventory of euglossini bees, four sampling sites were selected. each sampling site had two sampling points, 500 m apart from one another. data sampled in each of these sampling-point pairs were lumped in a single sample. the site covered by dense lowland ombrophilous forest (dlof, 13º41'07" s; 39º05'18" w) is located at 70 m a.s.l. it is characterized as a transition zone from dense rainforest to sandbank “(restinga)” and mangrove. the first area of dense submontane ombrophilous forest (dsof_1, 13º50'36" s; 39º17'21" w) is located at 250 m a.s.l. it is an agroforestry system producing cacao, clove, and peach-palm, and it is also a rubber tapping pole. the second area of dense submontane ombrophilous forest (dsof_2, 13º54'50" s; 39º27'24" w) is located at 470 m a.s.l. in this sampling site, the hills form mountain ranges interconnected to one another by ecological microcorridors. the sampling point in the vegetation type by dense montane ombrophilous forest (dmof) is located around 2,5 kilometers nearby dsof_2, but the sampling site was defined (13º53'52" s, 39º27'45" w) at a higher altitude (690 m a.s.l.). sampling and taxonomic identification the males of the orchid bees were sampled monthly, from july 2012 to may 2013, from 09:00 a.m. to 3 p.m. strong rains hindered sampling in april 2013, when only dsof_2 and dmof were sampled. these bees were captured when attracted by seven aromatic baits (eugenol, methyl cinnamate, vanillin, eucalyptol, benzyl acetate, methyl salicylate, and beta-ionone). attractive baits were imbibed in cotton ball inside traps placed at 1.5 m above the ground with a minimum distance of 2 m between baits (aguiar et al., 2014). the captured specimens were deposited in the entomological collection of the laboratory of environmental studies (laboratório de estudos ambientais lea) of the universidade estadual de feira de santana. digital thermo-hygrometer was employed to measure humidity and temperature, and remained in the sampling sites during the entire study, recording daily minimum and maximum values throughout the sampling period. daily rainfall data were obtained from the estação meteorológica do vale juliana. data analysis the shannon species diversity index (h’) was calculated, using the formula h’ = σ (pi) (log2pi), where, pi = proportion of individuals of species i and log2pi = logarithm base 2 of pi (magurran, 2003). the berger-parker index was calculated to species dominance using the formula d = nmax/ntotal, where: nmax is the number of individuals of the most abundant species and ntotal is the total of individuals sampled. evenness (j’) was calculated with the pielou index (magurran, 2003): j’ = h’/ log2s, where: j’ = pielou evenness index, h’ = shannon diversity index, and log2s = logarithm base 2 of the species richness. to evaluate the similarity between areas, was applied the morisita index, which analyzes abundance data of the species in the community. to test for relationships between community parameters (abundance, richness, diversity, and dominance) and environmental variables (temperature, humidity, rainfall, and altitude), spearman linear correlations were performed. the rarefaction curve was estimated with 1,000 randomizations, following magurran (2003). all analyses were carried out in the program past 1.91 (hammer et al., 2001). bee identification was based on taxonomical keys and other studies (nemésio, 2009; nemésio & engel, 2012; hinojosa-diaz et al., 2012; faria jr & melo, 2012), and later confirmed by dr. gabriel a. r. melo (universidade federal do paraná). rls medeiros et al. – orchid bee communities in the atlantic rain forest184 results in the four sites we captured 1,945 male orchid bees of 20 species. the site with the highest abundance was a submontane forest area (dsof_1) with 775 individuals of 17 species. dmof showed the highest species richness (19 species). species diversity (h’) in the four areas varied from 1.63 (dlof) to 2.41 (dmof). evenness (j’) varied from 0.68 to 0.82 (table 1). the species rarefaction curves built for the four study sites (fig 1) suggest that sampling was complete in all areas, as the curve tended to stabilization between 200 and 300 individuals (fig 1). the composition of species among the sites sampled showed important differences, for example: euglossa amazonica dressler, e. clausi nemésio and engel, e. imperialis cockerell, e. marianae nemésio, and e. iopoecila dressler were not registered in the dlof. on the other hand, euglossa pepei nemésio and engel occurred only in dsof_1 and dmof, and e. botocuda faria and melo sampling only in the dsof_2 and dmof, whereas e. ioprosopa dressler was exclusive to the dmof (table 1). the morisita index showed two clusters (70% similarity), one corresponding to the dsof_2 and dmof with 88% similarity and the other formed by dsof_1 and dlof with similarity of 95% (fig 2). fig 1. rarefaction curves for four areas in the brazilian atlantic forest. dlof: dense lowland ombrophilous forest, dsof_1: dense submontane ombrophilous forest, area 1, dsof_2: dense submontane ombrophilous forest, area 2, dmof: dense montane ombrophilous forest. fig 2. similarity index (jaccard) for species composition of four areas in the brazilian atlantic forest. dlof: dense lowland ombrophilous forest), dsof_1: dense submontane ombrophilous forest, area 1, dsof_2: dense submontane ombrophilous forest, area 2, dmof: dense montane ombrophilous forest. the dominant species showed different abundance distribution patterns in the four areas. euglossa cordata (linnaeus) was dominant in all areas except for the dsof_2, where e. ignita smith was the most abundant species. euglossa milenae bembé was the second most abundant species in the dsof_2 and dmof (table 1, fig 3). the highest peaks of abundance occurred in the period of moderate rainfall, from november to january (fig 4). the monthly variation in euglossini abundance was mainly influenced by the two most abundant species, e. cordata and e. ignita. the abiotic variables (temperature, humidity, rainfall, and altitude) had no influence on species richness (p > 0.05). on the other hand, abundance was slightly influenced by temperature (r = 0.35; p = 0.03), slightly negatively influenced by humidity (r = -0.40; p = 0.02) and not influenced by rainfall and altitude (p > 0.05). diversity and dominance were only affected by altitude (r = 0.36; p = 0.03; and r = -0.37; p = 0.03, respectively). fig 3. rank abundance plot for euglossini species in four areas in the brazilian atlantic forest. dlof: dense lowland ombrophilous forest), dsof_1: dense submontane ombrophilous forest, area 1, dsof_2: dense submontane ombrophilous forest, area 2, dmof: dense montane ombrophilous forest. 1: euglossa cordata 2: euglossa ignita 3: eulaema atleticana 4: euglossa imperialis; 5: euglossa milenae; 6: eulaema nigrita. sociobiology 64(2): 182-190 (june, 2017) 185 discussion the orchid bee richness recorded in the pratigi environmental protection area was similar to that in an area within the atlantic forest, in the serra do conduru state park, where 22 species were reported (nemésio, 2011a); and in the michelin ecological reserve, where 21 species were registered, both localized in the southern bahia, brazil (rosa et al., 2015) however, the species richness found was lower than that reported for the monte pascoal national park, located 360 km away from the pratigi environmental protection area (36 species) (nemésio, 2013b). according to souza et al. (2005), variation in of orchid-bee richness can be influenced by differences factors, including in collection method, types of fragrances used and the sampling effort. additionality, species dlof dsof_1 dsof_2 dmof total euglossa amazonica dressler 5 1 2 8 euglossa augaspis dressler 1 2 2 5 euglossa cordata (linnaeus) 151 287 52 85 575 euglossa clausi nemésio & engel 8 6 23 37 euglossa despecta moure 11 32 5 18 66 euglossa ignita smith 54 102 93 55 304 euglossa imperialis cockerell 79 27 35 141 euglossa iopoecila dressler 4 4 20 28 euglossa ioprosopa dressler 2 2 euglossa botocuda faria & melo 1 4 5 euglossa liopoda dressler 6 8 3 2 19 euglossa marianae nemésio 12 11 3 26 euglossa milenae bembé 22 63 56 71 212 euglossa pepei nemésio & engel 1 1 2 euglossa securigera dressler 2 2 9 13 eulaema atleticana nemésio 31 99 32 55 217 eulaema cingulata (fabricius) 10 32 31 25 98 eulaema nigrita lepeletier 14 24 32 62 132 eulaema niveofasciata (friese) 4 13 25 9 51 exaerete sp. 4 4 total abundance 306 775 381 484 1.945 species richness 11 17 16 19 20 diversity (h’) 1.63 2.04 2.25 2.41 2.22 dominance (d) 0.49 0.37 0.24 0.18 0.30 evenness (j’) 0.68 0.72 0.81 0.82 0.74 table 1. species composition, abundance, richness, diversity and evenness in euglossini bee communities in different phytophysiognomies of the atlantic forest in southern bahia, brazil. dlof: dense lowland ombrophilous forest), dsof_1: dense submontane ombrophilous forest, area 1, dsof_2: dense submontane ombrophilous forest, area 2, dmof: dense montane ombrophilous forest. fig 4. rainfall (a) and monthly fluctuations in the abundance of euglossini bees (b) in four areas in the brazilian atlantic forest, from june 2012 to may 2013. dlof: dense lowland ombrophilous forest), dsof_1: dense submontane ombrophilous forest, area 1, dsof_2: dense submontane ombrophilous forest, area 2, dmof: dense montane ombrophilous forest. rls medeiros et al. – orchid bee communities in the atlantic rain forest186 studies suggest that differences in local floristic composition, resource availability, and habitat heterogeneity may influence richness, abundance, and species composition of orchid bees (armbruster, 1993; souza et al., 2005; tonhasca et al., 2002b). considering our data observed in the four sampling sites located in the atlantic forest, from lowland to montane rainforests it demonstrated differences in their orchid bee community. differences related to phyto physiognomy influenced species richness and diversity. variations in species richness can also reflect differences in floristic composition and resource availability for feeding, nesting and reproduction, since euglossa species, for example, require resin and preexisting cavities for nesting (roubik & hanson, 2004). the dense montane ombrophilous forest (dmof) showed the highest richness and diversity. on the other hand, the dense lowland rainforest (dlof), which is the largest continuous forest remnant sampled (15,300 ha), showed the lowest species richness and a lower diversity index than the other areas. in this lowland rainforest, there is extractivism of the palm tree attalea funifera martius (piassava), an endemic plant used in handicraft (barreto, 2009). the habitat underwent some changes resulting from this extractive activity, as there is the removal of surrounding plants to favor the palm tree growth (r.l.s. medeiros, personal observation). this practice apparently contributes to the lower plant diversity in the dlof area, resulting in a less heterogeneous habitat. in this case, our study shows that the low abundance, richness and diversity of species seems to be more related to the conservation status of the dlof area than to the phytophysiognomic type and altitude, however, new efforts must be applied to prove this perspective. an important aspect to be considered is that some euglossini species are sensitive to environmental changes (powell & powell, 1987), which seems to be the case of e. marianae (= euglossa analis westood; see nemésio, 2011a), absent in the lowland rainforest (dlof) and present in the other areas. this species is considered a bioindicator of habitat quality due to its sensitivity to environmental change (tonhasca et al., 2002; ramalho et al., 2009). euglossa amazonica, e. clausi, and e. imperialis were not recorded in the dlof. in spite of being recorded in the other three areas, e. amazonica was not an abundant species, as other authors have already pointed out (nemésio, 2011b, 2013a, b; pires et al., 2013, mentioned as euglossa aratingae nemésio). according to nemésio (2009), this species has a broad distribution, but it is rarely sampled. euglossa clausi has been sampled in well preserved areas in the atlantic forest (mentioned as euglossa sapphirina moure) (tonhasca et al., 2002a; nemésio & silveira, 2006; ramalho et al., 2009; nemesio, 2013b). its absence in the dlof can be related to the conservation status of this area, . alternatively, its abundance can be locally low, and therefore, this species may not have been sampled. it was recorded in low frequency in other areas, as in the michelin ecological reserve, approximately 25 km away (ramalho et al., 2013), which is included in the pratigi environmental protection area. euglossa imperialis showed expressive abundance in three of our four study areas. rosa et al. (2015) suggested that e. imperialis is sensitive to changes in forest cover. our results corroborate rosa et al. (2015), as the abundance of this species in the lowland rainforest area seems to be related to the conservation status of the area. the high abundance of e. cordata in the dlof was responsible for the low local evenness and high dominance (berger-parker index). studies carried out in other atlantic forest areas also pointed out to the dominance of this species (aguiar & gaglianone, 2008, 2012; ramalho et al., 2009; ramalho et al., 2013; nemésio, 2013a, 2013b). euglossa cordata has been frequently considered a bioindicator of environmental disturbances, as this species if favored by dry and/or perturbed environments (peruquetti et al., 1999; silva & rebelo, 2002; aguiar & gaglianone, 2008; rocha-filho & garófalo, 2013). however, e. cordata has also been recorded in well preserved areas (tonhasca et al., 2002a; ramalho et al., 2009, aguiar & gaglianone, 2008, 2012; nemésio, 2013a, 2013b), as observed in our study. hence, our data, like those of ramalho et al. (2009) and aguiar et al. (2014), suggest that e. cordata occurs in high abundance in areas at different conservation status. the two clusters of similarity (70%) for the orchid bee community are apparently related to the altitude and phytophysiognomic type, since it the occurrence of some elements of the euglossini fauna varied among the areas. euglossa ioprosopa dressler was exclusive to the montane rainforest (dmof). this species occurs in the amazon and atlantic forest (nemésio & silveira, 2007), and it has been associated with well-preserved environments (nemésio, 2009; ramalho et al., 2009; rocha-filho & garófalo, 2013). the highest richness and diversity at dmof suggests that this area has a better conservation status than the other areas analyzed. we observed two abundance peaks, one in the period of lowest rainfall, which was influenced mainly by e. cordata and e. ignita, and another in the beginning of the rainy season, influenced by e. cordata. the trends in monthly variation in abundance found in the studied areas were similar to those found by aguiar & gaglianone, (2008) and aguiar et al. (2014) in the semideciduous seasonal forest and dense ombrophilous forest in the rio de janeiro state, in the region with altitudes between 40 and 1000 m a.s.l . these authors observed two abundance peaks, one in the dry season and another in the rainy season in atlantic forest areas. rebelo and cabral (1997) made similar reports for the brazilian savanna, and oliveira (1999) for the amazon forest. orchid bees abundance peaks in the rainy season have been recorded in several types of vegetation, such as atlantic forest (rebêlo & garófalo, 1997; ramalho et al., 2009), savanna (brito & rego, 2001), sandbank coastal vegetation (“restinga”) (viana et al., 2002; silva et al., 2009), and xerophilous vegetation (“caatinga”) (andrade-silva et al., 2012). sociobiology 64(2): 182-190 (june, 2017) 187 abiotic variables did not influence species richness, but temperature influenced abundance. other studies in the atlantic forest (bezerra & martins, 2001) and savanna (carvalho et al., 2006; mendes et al., 2008) also reported a positive correlation between abundance and temperature. the correlation between euglossini abundance and relative humidity is controversial. as in this study, carvalho et al. (2006) observed higher activity of orchid bees in lower air relative humidity in atlantic forest areas. however, other studies reported higher abundance of orchid bees in periods of higher air relative humidity in the atlantic forest (aguiar & gaglianone, 2012; andrade-silva et al., 2012) and savanna (mendes et al., 2008). this suggests that air relative humidity alone does not explain orchid bees abundance. nemésio and silveira, (2006) argued that, variations in the abundance of orchid bees may represent responses to small changes in light incidence, temperature and humidity, and other variables difficult to measure, including the dispersal of scents inside the forest. in the case of the studied areas, the humidity is relatively high throughout the year (80-90%), with little variation due to the high rainfall rates in the region (2000 mm) (fisher, 2007), so other factors such as specific periods of nesting and availability of resources should control the patterns of abundance and occurrence of the species. apart from phytophysiognomic and climate factors, altitude variation is among the main factors that influence the geographic distribution of orchid bees (ramirez et al., 2002). we did not find a correlation between species abundance and richness and altitude (70-690 m a.s.l. variation). on the other hand, diversity showed a positive correlation, and dominance showed a negative correlation with altitude, which suggests a more equitable distribution of orchid bee communities at higher altitudes. aguiar and gaglianone (2012) found a positive relationship between abundance of orchid bees and altitude in a study carried out in atlantic forest fragments with an altitudinal range similar to our study (between 40 and 825 m a.s.l.). other studies on the influence of altitude on the distribution of those bees observed a decrease in abundance with altitude, but not richness, another hand, nemésio (2008) observed that bee species richness decreases markedly with altitude, in the range between 850 and 1,350 m a.s.l., however the range analyzed differs from that studied in this work. dias (unpublished data) observed differences in species composition of orchid bees along a broad altitudinal gradient (125 m – 2,150 m). we investigated whether the altitude influenced richness and abundance of orchid bees along a narrower altitudinal gradient than those analyzed in other studies that found a correlation between those variables the results obtained in the present study suggest that differences in phytophysiognomy affect the richness and abundance of orchid bees in the atlantic forest. differences in species composition and abundance of these bees between close areas in the same region highlight the importance of conserving different portions of the habitat to assure the conservation of orchid bees in different phytophysiognomies within the same ecosystem. acknowledgments the authors are grateful to dr. gabriel a. r. de melo for his help in bee taxonomic identification, to ‘organização para conservação de terras do baixo sul da bahia’ (oct) and ‘associação guardiã da apa do pratigi’ (agir) for funding this study and supporting field work. we also thank the brazilian environment ministry (mma), brazilian institute for the environment and renewable natural resources (ibama) and the chico mendes institute for biodiversity conservation (icmbio) for issuing the permit to capture bee populations. c.m.l. aguiar received a research fellowship from brazilian council for scientific and technological development (cnpq) (process 306403/2012-9), and r.l.m. santos received a grant from fundação de amparo à pesquisa do estado da bahia (fapesb). references aguiar, w.m. & gaglianone, m.c. 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(2002a). abundance and diversity of euglossine bees in the fragmented landscape of the brazilian atlantic forest. biotropica, 34: 416-422. doi: 10.1646/0006-3606(2002)034 [0416:aadoeb]2.0.co;2 rls medeiros et al. – orchid bee communities in the atlantic rain forest190 tonhasca, jr.a., blackmer, j.l. & albuquerque, g.s. (2002b). within-hábitat heterogeneity of euglossine bee populations: a re-evaluation of the evidence. journal of tropical ecology, 18: 929-933. doi: 10.1017/s0266467402002602. viana, b.f., kleinert, a.m.p. & neves, e.l. (2002). comunidade de euglossini (hymenoptera, apidae) das dunas litorâneas do abaeté, salvador, bahia, brasil. revista brasileira de entomologia, 46: 539-545. doi: 10.1590/s0085-56262002000400008 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1793sociobiology 64(3): 363-365 (september, 2017) first record of the occurrence of partamona ailyae camargo (hymenoptera: apidae) in atlantic forest the tribe meliponini clusters 417 valid species of stingless bees in the neotropical region (camargo & pedro, 2003). recent data demonstrate 244 species described and another 89 undescribed species for brazil (pedro, 2014). stingless bees have broad geographic distribution, a large number of individuals per colony and generalist feeding habits (roubik, 1989). species of the genus partamona schwarz are distributed over a large geographic range, with records from central mexico to southern brazil (i.e. roubik, 2006). these stingless bees are found in forests, savannas, semiarid regions and locations with an altitude of more than 2000 m above sea level (pedro & camargo, 2003; camargo & pedro, 2003). they use a wide variety of substrates for nest building, but most nest in termite mounds (camargo, 1980; camargo & pedro, 2003). thirty-three valid species have been described for the genus partamona (camargo & pedro, 2003). twentythree of these species occur exclusively in brazil (pedro, abstract this is the first record of the stingless bee partamona ailyae camargo in an area of the atlantic forest in brazil. the morphological identification and coi and 12s sequences indicated that the samples collected in the atlantic forest and other areas of the range of distribution belong to the same biological species. the data revealed low intraspecific variation and high interspecific divergence, with no overlap of the taxa compared. sociobiology an international journal on social insects map andrade¹, srm pedro2, pfm cardoso3, ea miranda3,4, ma del lama3, m silva1 article history edited by cândida aguiar, uefs, brazil received 20 may 2017 initial acceptance 22 august 2017 final acceptance 01 september 2017 publication date 17 october 2017 keywords dna barcoding, coi and 12s haplotypes, gene variation, geographic distribution, meliponini. corresponding author faculdade de tecnologia e ciências rua itapetinga, quadra 05, lote 67, casa 03 loteamento jardim brasília, cep 41100-240 pernambués, salvador-ba, brasil. e-mail: msilva_santos@outlook.com 2014), three of which have previously been recorded in areas of the atlantic forest (hereafter af): partamona criptica pedro and camargo, partamona helleri friese and partamona sooretamae pedro and camargo (camargo & pedro, 2003). partamona ailyae camargo is known to nest in epigeal termite mounds. this bee has broad distribution, occurring from the rainforest in southeastern amazonia to savannas in central brazil and semiarid areas of the state of piauí in the northeastern region of brazil (camargo, 1980; camargo & pedro, 2003). however, there are no previous records of this species in af areas. this paper reports the first record of p. ailyae in the atlantic forest, broadening its distribution to the atlantic portion of southeastern brazil. twelve p. ailyae nests were located in the upper portion of the south of the state of bahia, in an area of approximately 100,000 m² in af (13°27’14’’ s and 39°25’15’’ 1 faculdade de tecnologia e ciências (ftc), salvador-ba, brazil 2 universidade de são paulo (ffclrp-usp), ribeirão preto-sp, brazil 3 universidade federal de são carlos (ufscar), são paulo, brazil 4 universidade estadual de santa cruz (uesc), bahia, brazil short note map andrade et al. – first record of partamonaailyae in atlantic forest364 w), within the limits of the bom jesus, jacarandá and riacho do louro farms (tancredo neves municipality); additionally, in the southern region of this state, two nests were located in rural areas of the municipalities of uruçuca and una (table 1, fig 1). the regions have characteristic secondary atlantic forest vegetation in different stages of regeneration: low vegetation, high vegetation and secondary forest. according to the köppen classification (veloso et al., 1991), the climate is af (humid tropical or equatorial), with the temperature ranging between approximately 22 and 31º c. mean annual precipitation in af is 1600 mm, with rains distributed throughout the year. the first observation of a specimen of p. ailyae in the region occurred in 2004 during a bee sampling from flowers with an insect net. in 2008, the first colony was found using an artificial feeder containing sugar water. the localization of the colony was possible due to successive movements of the feeder along a straight line, approximately five meters at a time, following the flight path of the worker bees. subsequently, other nests were located through active searches and georeferenced. thirteen p. ailyae nests (12 in presidente tancredo neves and one in uruçuca) were found on clay soil in epigeal termite mounds of the subfamily apicotermitinae and one nest was found in una at the base of a tree. specimens from each colony were sent to the laboratório de abelhas solitárias e ecologia de ecossistemas da faculdade de tecnologia e ciências (labee/ftc), pinned, labeled and dried. the morphological identification of p. ailyae was performed by dr. silvia regina de menezes pedro (ffclrp-usp). voucher specimens were deposited in the zoology museum of the faculty of technology and science (mz/ftc) and prof. j.m.f. camargo collection (rpsp), (ffclrp-usp). specimens from 10 out the 14 p. ailyae nests were analyzed through molecular markers for the confirmation of their taxonomic status. dna extraction, pcr amplification, amplicon purification, sequencing of the gene regions cytochrome c oxidase (coi) and 12s ribosomal dna, as well as the edition and analysis of the sequences were performed according to previous reports (miranda et al., 2016). nest localities farm coordinates termite nest height x diameter (cm) h 1 ptn bom jesus 13º26’57.2” s, 39º30”26.4” w 93 x 203 37 2 jacarandá nc 72 x 175 29 3 bom jesus 13º26’28.3” s, 39º29’57.3” w 80 x 210 38 4 bom jesus 13º26’57.2” s, 39º30’26.4” w 84 x 198 43 5 jacarandá 13º26’40.5” s, 39º30”35.0” w 60 x 170 17 6 jacarandá 13º26’37.3” s, 39º30”36.0” w 81 x 208 50 7 riacho do louro nc 52 x 140 8 8 bom jesus nc 83 x 196 10 9 jacarandá 13º26’57.2” s, 39º30”26.7” w 70 x 150 20 10 jacarandá 13º26’28.2” s, 39º29”27.3” w 73 x 202 13 11 jacarandá nc 75 x 195 15 12 jacarandá nc 85 x 208 42 13 uru nc 14°37’51.6”s, 39°16’48.9”w nc nc 14 una nc 15°11’41.1”s, 39°03’00.6”w nc nc table 1. partamona ailyae camargo nests sampled in municipality of presidente tancredo neves (nests 1-12) in south lowlands, state of bahia, and municipalities of uruçuca and una (nests 13-14) in southern portion of state of bahia. some nest characteristics are presented. “h” = height of nest entrance in the termite nest. nc = non-collected datum. samples from the atlantic forest and other areas of distribution were compared through an analysis of 629 base pairs of the coi gene. the optimum threshold (ot) was estimated considering 12 haplotypes of p. ailyae [three obtained from the af samples and nine from samples of the known distribution area, as well as nine haplotypes from partamona rustica pedro and camargo and five haplotypes from partamona cupira smith as controls]. using the “local minima” function of the spider (species identity and evolution in r) package, the estimated ot was 1.95% between intraspecific and interspecific variation. the kimura twoparameter model of the mega 7 program demonstrated lower intraspecific and higher interspecific variation values for all combined pairs in comparison to the ot, indicating that the three species are well differentiated. moreover, the 12s haplotypes of the p. ailyae af samples revealed the same insertion of five base pairs beginning at position 25 of the sequence (417 bp) previously described by cardoso (personal communication, september 13, 2016) in samples of p. ailyae from geographically distinct origins. sociobiology 64(3): 363-365 (september, 2017) 365 thus, the molecular analyses confirmed the morphological identification of the samples. it is very unlikely that p. ailyae arrived to the atlantic forest in bahia by human hands. we believe that partamona species cannot be maintained by beekeepers or common people for three main reasons: i) these species do not produce much honey and it is inappropriate for human consumption (some partamona species collect feces); ii) they can hardly be kept in rational boxes because most of them are termitophilic species and their nests are hosted by specific termite species, and they are not managed by humans yet; iii) p. ailyae has a strong defensive behavior and bites hard when its nests are opened. within the meliponini tribe, partamona is an excellent study model for population genetics and biogeographic studies because their colonies are rarely manipulated. our findings demonstrate the occurrence of p. ailyae in an area of af in brazil, broadening the known area of the distribution of the species (fig 1).this new record makes this species particularly interesting for future phylogeographic studies, as it is found in the amazon forest, savannas, semiarid regions and af in brazil. such studies could contribute toward clarifying evolutionary relations in the biota of different biomes of the neotropical region. acknowledgements we are grateful to profa. dra. maria avany bezerra gusmão (campina grande state university) for identifying the thermita subfamily and to fundação de amparo à pesquisa do estado de são paulo (fapesp) for financial support (process 2011/21501-2, 2012/23342-1) and national council for scientific and technological development, brazil (cnpq) granted scholarship scientific initiation to andrade map and granted scholarship to miranda ea (process 154912/2016-6). references camargo, jmf. (1980). o grupo partamona (partamona) testacea (klung): suas espécies, distribuição e diferenciação geográfica (meliponinae, apidae, hymenoptera). acta amazonica, 10:1-175. doi: 10.1590/1809-43921980104s005 camargo, jmf., pedro, srm. (2003). meliponini neotropicais: o gênero partamona schwarz, 1939 (hymenoptera, apidae, apinae) – bionomia e biogeografia. revista brasileira de entomologia, 47: 311-372. doi: 10.1590/s0085-56262003 000300001. miranda, ea., batalha-filho, h., congrains, c., carvalho, af., ferreira, km., del lama, ma. (2016). phylogeography of partamona rustica (hymenoptera, apidae), an endemic stingless bee from the neotropical dry forest diagonal. plos one, 11(10): e164441. doi: 10.1371/journal.pone.0164441 pedro, srm., camargo, jmf. (2003). meliponini neotropicais: o gênero partamona schwarz, 1939 (hymenoptera, apidae). revista brasileira de entomologia, 47: 1-117 (http://www. scielo.br/pdf/rbent/v47s1/16453.pdf) pedro, srm. (2014). the stingless bee fauna in brazil (hymenoptera: apidae). sociobiology, 61: 348-354. roubik, dw. (2006). stingless bee nesting biology. apidologie, 37: 124-143. doi: 10.1051/apido:2006026 roubik, dw. (1989). ecology and natural history of bees. cambridge university press, new york, p.1-515. doi: 10.11 26/science.248.4958.1026 veloso, hp., rangel filho, al., lima, jca. (1991). classificação da vegetação brasileira, adaptada a um sistema universal. ibge, rio de janeiro, p 124.fig 1. new records of occurrence of partamona ailyae in atlantic forest (diagonal lines). ptn = municipality of presidente tancredo neves in upper portion of the south of the state of bahia; uru = municipality of uruçuca and una = municipality of una, both located in southern portion of state of bahia. these records broaden the known distribution of the species (in yellow) proposed by camargo and pedro (2003). http://www.scielo.br/readcube/epdf.php?doi=10.1590/1809-43921980104s005&pid=s0044-59671980000800005&pdf_path=aa/v10n4s1/1809-4392-aa-10-4-s1-0005.pdf&lang=pt http://dx.doi.org/10.1590/s0085-56262003000300001 http://dx.doi.org/10.1590/s0085-56262003000300001 https://doi.org/10.1371/journal.pone.0164441 http://www.scielo.br/pdf/rbent/v47s1/16453.pdf http://www.scielo.br/pdf/rbent/v47s1/16453.pdf doi: 10.13102/sociobiology.v64i1.1143sociobiology 64(1): 26-32 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nesting substrate characteristics of partamona seridoensis pedro & camargo (hymenoptera: apidae) in areas of dry forest in brazil introduction stingless bees (meliponini) comprise more than 500 described species and possibly 100 more as yet undescribed (michener, 2013), the real number is unknown because there are many cryptic species (michener, 2007). the species are pantropical and southern subtropical, make honey as the closely related stinging honey bees, and live in perennial eusocial colonies of a few dozen to more than 100,000 workers (michener, 2007). nesting biology is highly diverse in stingless bees, unknown in many species, and closely related to the biology and behavior of each species (roubik, 2006). most species nest in pre-existing cavities in living trees or trunks, although abstract for the first time the association between partamona seridoensis and constrictotermes cyphergaster is described. partamona seridoensis occurs in xeric areas of northeastern brazil, and it is a termitophile species as its nests are built in active and inactive arboreal termite nests of the species c. cyphergaster. this study aimed to verify the characteristics of the nesting substrate used by p. seridoensis in two areas of dry forest (caatinga) in cariri region, paraíba state. it has been found that the vertical distribution of termites that contained colonies of p. seridoensis varied from 10 cm to 3.60 m, while the height of the nest entrance varied from 20 cm to 3.70 m. commiphora leptophloeos, popularly known as imburana, was the support tree of 22 (43.1%) from 51 observed termite nests that harbored bee colonies. most (44; 86.2%) of the host termites colonies were active. most of the colonies showed the nest entrances not directed to the east/southeast. all colonies located were housed in large termite nests, whose volumes exceed 30 liters. in the two areas surveyed, frequently the entrances of the nests were directed to other nearby colonies, suggesting a parental relationship that should be further investigated. sociobiology an international journal on social insects fernandes crm1, almeida ab2, del lama ma3, martins cf4 article history edited by vera lúcia imperatriz-fonseca, usp, brazil received 07 june 2016 initial acceptance 31 january 2017 final acceptance 03 february 2017 publication date 29 may 2017 keywords stingless bees, meliponini, caatinga, constrictotermes cyphergaster, isoptera, termitophiles. corresponding author celso feitosa martins departamento de sistemática e ecologia/ ccen, universidade federal da paraíba cidade universitária s/n, castelo branco cep 58051-900, joão pessoa-pb, brasil e-mail: cmartins@dse.ufpb.br some species establish their colonies in crevices in rocks, aerial nests, underground cavities, roots and plants and active or not nests of ants and termites (nogueira-neto, 1997; roubik, 2006). the genus partamona is a neotropical stingless bee comprising 33 described species, many of which are cryptic, with aggressive bees that nest in a wide variety of substrates, and many species are obligatory termitophiles (camargo & pedro, 2003; pedro & camargo, 2003). because they present similar morphological traits, nesting behavior and the nest entrance structure are useful traits for species identification (camargo & pedro, 2003). partamona seridoensis pedro and camargo occurs mainly in the dry forest biome, called caatinga and nests 1 união de ensino superior de campina grande (unesc), campina grande-pb, brazil 2 universidade estadual da paraíba (uepb), campina grande-pb, brazil 3 universidade federal de são carlos (ufscar), são carlos-sp, brazil 4 universidade federal da paraíba (ufpb), joão pessoa-pb, brazil research article bees sociobiology 64(1): 26-32 (march, 2017) 27 only inside termite nests (camargo & pedro, 2003). there are few data on the ecology of species of partamona (barreto & castro, 2007; miranda et al., 2015), and in general the genetics of these species is poorly known (fernandes et al., 2012). however, p. seridoensis gene variation and the degree of differentiation between two populations from cariri region in the state of paraíba, brazil, have been studied studied through allozymic and microsatellite analyses (fernandes et al., 2012). furthermore, this species was sampled in studies about the relations of melittophilous plants and bee species at the same region (aguiar et al. 1995; aguiar & martins, 1997 in both cited as partamona sp., martins et al., 2003). in addition, p. seridoensis was the most abundant species in association with pig carcasses exposed in a forensic entomological study in the same area (santos et al., 2014), and lorenzon et al. (1999) has preliminarily studied the association of p. seridoensis and its host termite constrictotermes cyphergaster (silvestri) (referred by authors as p. aff. nigrior and constrictotermes sp.). as lorenzon et al. (1999) did not identify both species, carrijo et al. (2012) in a recent review of published records of bee species as guests in termite nests stated that the p. seridoensis host is unknown. although in a recent short note oliveira et al. (2016) cited the association between p. seridoensis and c. cyphergaster, however the citations referred in the note, as reported above, did not identify the species. furthermore, many aspects of the association between this stingless bee species and its termite host remain unknown. the objective of this study was to improve our understanding of the nesting substrate characteristics of p. seridoensis in areas of dry forest in northeastern brazil with emphasis on size, volume, height and substrate of the termite nests, interaction between the bees and termites and the orientation of the nest entrances. material and methods the study was carried out at two farms located in the dry forest of northeastern brazil, called caatinga, in the cariri region of paraíba state. the caatinga is a xeric biome (andrade-lima, 1981) that comprises xeric vegetation, ranging from shrub lands to small forest-like savannas (prado, 2000). the almas farm, comprising a particular natural heritage reserve rppn (7º28`23.7”s; 36º54`17.1”w), is located at the municipalities of são josé dos cordeiros and sumé, paraíba, whose vegetation ranges from open shrubby arboreal and dense shrubby arboreal vegetation between flagstones with typical vegetation, with annual average rainfall of 400-800mm/year (lima & heckendorff, 1985). the moreiras farm (7º23`36.3”s; 36º24`53.9”w) is located at the municipality of são joão do cariri, paraíba, which has an open shrubby arboreal vegetation, annual average rainfall of 386.6 mm, relative humidity 50%, temperature between 28.5 and 35 °c, and a dry irregular period of eight months, including the months of june to february (atlas geográfico da paraiba, 1985). data collection was carried out at monthly intervals between march 2008 to june 2009, and every two months from july 2009 to december 2010. in each area transect lines, measuring 10 m wide by 200 m long each = 2,000 m2) were plotted and inspected in four consecutive days (two at each area) from 5:00h to 17:00h. in each transect line, the p. seridoensis colonies identified inside the arboreal termite nests of c. cyphergaster were counted, georeferenced and had these information recorded: the presence or absence of termites (at the time of the colony discovery and throughout the study period); the tree species supporting the termite nest; the height of termite nest and of the colony entrance (relative to ground, using a measuring tape), the colony entrance position (in relation to the cardinal points using the compass of an etrex® summitgps); the termite nest size, measuring with a wooden ruler the height of the termite nest, the largest diameter, and the smallest diameter. the volumes were obtained using the formula v= 2/3.π.a.b.c/1000, where a = the height of termites; b = ½ the largest diameters; c = ½ smaller diameters (fontes, 1980). from each colony, some specimens were collected and placed in lethal chambers, later they were mounted on entomological pins and deposited in the entomological collection of the department of systematics and ecology of the federal university of paraíba (ufpb). termites were collected, fixed in 70% ethanol for identification by experts, and subsequently deposited in the entomological collection of the department of systematics and ecology of the federal university of paraíba (ufpb). normality and test t were calculated using statistica v7.0 and the homogeneity of the distribution of the orientation of the nest entrances in relation to the cardinal and collateral points for each studied area, and for the total, were tested with rayleigh test using bioestat v5. results a total of 51 colonies of p. seridoensis were found in the study areas (29 at almas farm and 22 at moreiras farm) all of them inside nests of c. cyphergaster. among these, almost 61% were in termite nests supported by only two tree species: 43.1% (22) in termite nests supported by commiphora leptophloeos (imburana), and 17.6% (9) in poincianella pyramidalis (catingueira) trees (table 1). two other colonies were housed in termite nests supported by fences made with dry sticks, while another was on the floor. this last one had been removed probably by bee hunters and was outside from their place of origin. the vertical distribution of the termite nests that hosted colonies of p. seridoensis in the studied areas varied from 10 cm to 3.60 m, considering the height of the base of termites to the ground and was significantly different in both areas (t= 5.235; p<0.0001; df=47; fig. 1). no bee colonies were observed in termite nests supported directly on the ground or on stones in any of the studied areas. the height of the bees nest entrance varied from 20 cm to 3.70 m (mean = 1.75 m to 0.80 m in almas and moreiras farms, respectively), as the entrances were located from 0 to 20 cm above the base of the termite nests. fernandes crm et al. – nesting substrate characteristics of partamona seridoensis 28 the presence of termites was recorded in 44 (86.2%) of the host termite colonies of p. seridoensis. only six (11.8%) of the host termite colonies were dead. in one colony at almas farm, it was not possible to verify the presence or absence of termites because it had been opened by local inhabitants, probably to collect honey and pollen, and was damaged with very few bees. agonistic behaviors between the termites and the bees were not observed. the rayleigh’s test showed that the orientation of the nest entrances of the bees was not uniformly distributed, with a significant concentration around the mean angle of 205.5° in almas farm (south, r= 10.2426, p<0.05, n=28) and the mean angle of 211.4° (southwest, r=13.3206, p<0.05, n=50) in total sample (figs. 3a,c). at moreiras farm, the rayleigh’s test showed no significant difference regarding the orientation of the nest entrances of the bees (r= 3.3059, p>0.05, n=22), although southwest was also the direction with the highest number of entrances directed (fig. 3b). it is noteworthy that in moreiras farm there is a small mountain blocking the east side. plant family plant species common name number of bee colonies burseraceae commiphora leptophloeos (mart.) j. b. gillett imburana 22 caesalpinioideae poincianella pyramidalis tul catingueira 9 cactaceae cereus jamacaru dc. mandacaru 4 anacardiaceae spondias tuberosa arruda umbuzeiro 3 euphorbiaceae manihot glaziovii müll.arg. maniçoba 2 cactaceae pilosocereus gounellei (f. a. c. weber) byles & g. d. rowley xique-xique 2 mimosoideae mimosa tenuiflora (willd.) poir. jurema-preta 2 apocynaceae aspidosperma pyrifolium mart. pereiro 1 anacardiaceae myracrodruon urundeuva allemão aroeira 1 anacardiaceae schinopsis brasiliensis engl baraúna 1 apocynaceae caesalpinioideae aspidosperma pyrifolium mart. poincianella pyramidalis tul pereiro catingueira 1 1 1one nest supported by two tree species. table 1. plant species used by constrictotermes cyphergaster to support their nests where colonies of partamona seridoensis were found from march 2008 to december 2010. fig 1. box plot showing the height (meters) of termitaria nested by partamona seridoensis in almas (n= 28) and moreiras farms (n= 21). the volume of the termite nests in which the bee colonies were hosted varied from 33.8 liters to 233 liters (mean = 98.2 ± 45.88 liters; fig 2). fig 2. frequency distribution of the number of termite nests inhabited by bee colonies by volume (liters). the line shows the observed normal distribution of the size of large termite nests occupied by bee colonies (kolmogorov-smirnov d=0.09902, chi-square=3.53640, df=3, p=0.31607, n=45). mean mean±sd mean±1.96*sd almas moreiras -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 h ei gh t (m et er s) 0 20 40 60 80 100 120 140 160 180 200 220 240 260 volume (liters) 0 2 4 6 8 10 n o. o f t er m ite n es ts sociobiology 64(1): 26-32 (march, 2017) 29 discussion the results record formally for the first time the nesting of p. seridoensis in termite nests of c. cyphergaster. although lorenzon et al. (1999) observed this relationship, they did not identify the species and referred them as p. aff. nigrior and constrictotermes sp. in a recent short note, oliveira et al. (2016) cited the relation between p. seridoensis and c. cyphergaster however using the same citations referred above. thus, here it is the first time that this specific relationship is formally recorded. furthermore, p. seridoensis is considered a termitophile species sensu stricto (sensu carrijo et al., 2012) because all the nests were found exclusively in termite nests. in addition, the distribution area of p. seridoensis is endemic to caatinga (camargo & pedro, 2003) and inserted in the distribution area of c. cyphergaster. c. cyphergaster is also commonly found in the central savanna of brazil, paraguay, bolivia, and northern argentina (constantino, 1998), and is reported in the caatinga as the main species of termite that builds conspicuous nests in this biome (godinho et al., 1989; melo & bandeira, 2004). however, it is noteworthy that recently oliveira et al. (2016) found in the extreme northeast of the caatinga p. seridoensis colonies in association with termite nests of the genus microcerotermes (14 bee colonies, 13 associated with microcerotermes indistinctus mathews and one with microcerotermes strunckii sörensen). as these authors did not find c. cyphergaster in the studied area, results showed that the occurrence of p. seridoensis is not at all restricted to the local availability of c. cyphergaster, and that the bee may occupy opportunistically nests of other termite species available in the environment, at least in the absence of c. cyphergaster. our data show that the termite nests hosting bees were supported by the larger species of trees in the area (mostly commiphora leptophloeos and poincianella pyramidalis = caesalpinia pyramidalis). barreto and castro (2007) also observed in an arboreal caatinga other partamona species (p. rustica pedro & camargo, and p. cupira (smith)) nesting in c. cyphergaster termitaria supported mainly by commiphora leptophloeos, psidium sp., and poincianella pyramidalis (= caesalpinia pyramidalis) trees. commiphora leptophloeos and poincianella pyramidalis were recorded among the most common and phytosociologically important tree species at almas farm and são joão do cariri (barbosa et al. 2007). furthermore, bezerra-gusmão et al. (2013) demonstrated that in são joão do cariri, c. cyphergaster nests in the plant species with the higher values of importance, cover, volume, basal area, and density, poincianella pyramidalis (= caesalpinia pyramidalis), which was the species with the highest index of importance value and also that supported the greatest number of large-sized nests. this is related to the observation in this study that p. seridoensis nested only in large termite nests (as defined by mélo & bandeira, 2004), above 30 liters, which need large trees for support and comprise between 7-14% of total nests (bezerra-gusmão et al., 2013). another important observation is that p. seridoensis nests in active termite nests as noted by camargo and pedro (2003). all bee colonies found by oliveira et al. (2016) were also nesting in active arboreal nests of termites of the genus microcerotermes. however, in são joão do cariri, among 22 termite nests examined, lorenzon et al. (1999) recorded 12 p. seridoensis nests (cited by authors as p. aff. nigrior and constrictotermes sp.), half of them in inactive termite nests. lorenzon et al. (1999) suggested that the occupation of the termite nests by the bees could cause the abandonment of the nest by the termites because the bees occupy the central position of the nests and they observed termite soldiers attacking the bees when the separation between the nests was destroyed. although this needs to be tested, the low (6%) proportion of inactive termite nests with p. seridoensis nests, fig 3. number of bee nests (percent) sampled at almas and moreiras farms accordingly to orientation of entrances to cardinal and collateral points, a almas farm, b moreiras farm, and c total. fernandes crm et al. – nesting substrate characteristics of partamona seridoensis 30 and the absence of agonistic behavior between the species, when the nests are not disturbed, suggests that both species live for a long time in association. it is noteworthy that c. cyphergaster produces seasonal polycalic nests, satellite nests connected to the large or “mother” nest by tunnels, thus, when the bees occupy the central position of the termite nests, the reproductives could stay in the other nest. in addition, we observed bee colonies that died after the death of termites, and inactive termite nests begin to crumble due to lack of maintenance. thus, because they have perennial colonies, it is expected that the bees have been selected for nesting in enduring structures (roubik, 1989, 2006). partamona rustica and p. cupira were also observed nesting in c. cyphergaster termitaria (barreto & castro, 2007; miranda et al., 2015). in addition, barreto and castro (2007) observed that the nesting cavities were initially constructed by a parakeet called “jandaia” or “gangarra” (eupsittula cactorum (kuhl) = aratinga cactorum (kuhl) for reproduction. despite e. cactorum occurs in almas farm (araujo et al., 2012) it was not observed their reproductive nesting behavior in c. cyphergaster termitaria. miranda et al. (2015) report that although termite nests with hollows made by this bird were found, no nesting activity of p. rustica in such cavities was observed. it is interesting to note that the number of large termite nests observed in the two study areas was higher than the number of colonies of bees, suggesting that the local availability of nesting sites is not a limiting factor in the area (e.g. hubbell & johnson, 1977; eltz et al., 2002). however, the behavior of the parakeet or other agent that makes the cavities in the termite nests can be a limiting factor and deserves further investigation. the results showed that the nesting features of p. seridoensis are similar in the two areas. in almas farm the height of the termite nests with bee colonies were higher and this is related to the better conservation state of the particular natural heritage reserve of almas farm, and, consequently, higher vertical distribution of this area when compared to moreiras farm. in our study, p. seridoensis presented the entrance less directed to the east/southeast which are the predominant wind directions in cariri region (inmet, 2016). preference for nest entrances non-oriented to the prevailing wind directions have also been observed in another stingless bee species, melipona bicolor schencki gribodo (witter et al., 2010) and in solitary bees and wasps nesting in pre-existing cavities in trap nests (martins et al., 2012). it is possible that the entrances protected from wind also have a relation with the rain and luminosity. one interesting observation was that frequently the entrances of the nests were directed to other nearby colonies. within the transect lines many neighboring colonies were observed that each colony successively had the entrance of its nest directed to the nearest colony, suggesting they maybe sister colonies. further genetic analysis may confirm this fact. recently, four colonies of melipona subnitida ducke were observed nesting in c. cyphergaster termitaria (carvalho et al., 2014) in the caatinga. because this species nests in hollows of live trees (martins et al., 2004), carvalho et al. (op. cit.) discuss whether or not this opportunistic nesting habit of m. subnitida is an adaptation to shortage of pre-existing cavities in trees locally. caatinga ecosystem is one of the most vulnerable and threatened environments in brazil. in addition to the severe climate conditions, deforestation, inadequate land use practices, and the use of firewood and charcoal, have increased the risk of desertification. the natural vegetation remains at only 54% of the original area, and the average rate of deforestation is 2,700 km2 per year (mma 2011). nevertheless, miranda et al. 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(2010). ninhos da abelha guaraipo (melipona bicolor schencki), espécie ameaçada, em remanescente de mata com araucária no rio grande do sul. porto alegre, série técnica fepagro, 5: 37p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1827sociobiology 65(1): 67-78 (march, 2018) special issue landscape factors associated with subterranean termite (isoptera: rhinotermitidae) treatments and colony structure in residential subdivisions introduction urban development alters natural processes including hydrologic regimes, soil chemistry and fertility, overall plant distribution, and fragmentation of the landscape (mcdonnell & pickett, 1990; turner et al., 2001). animal and plant populations may respond to these changes by expanding or declining in numbers, migrating, or becoming extirpated from altered landscapes (lomolino et al., 2006). arthropod populations in urban landscapes are potentially relicts from abstract subterranean termites (isoptera: reticulitermes) are common structural pests, but it is not well known how landscape factors are associated with urban colonization. this study examined patterns of subterranean termite colonization in 13 midmissouri residential subdivisions. tenand 20-year-old homes built on historically agricultural and forested landscapes were inspected for treatment by termiticide application or bait stations. contemporary and historical aerial imagery was analyzed using gis software, and patterns of colonization were compared among subdivisions. the genetic structure of termite colonies collected in undeveloped landscapes and residential subdivisions was compared using microsatellite dna. twenty-year-old subdivisions had significantly higher treatment proportions than 10-year-old subdivisions. at year 10, historically forested subdivisions had a higher treatment proportion than historically agricultural subdivisions. by year 20, there was no significant difference in treatment proportion between historical landscape types, indicating that subdivisions built on agricultural landscapes eventually catch up to subdivisions built on forest landscapes. although there was not strong statistical support, treated homes in historically agricultural subdivisions tended to be close to forest patches, but there was less of an association in historically forested subdivisions. colonies in undeveloped landscapes were more inbred compared to colonies in residential subdivisions, indicating that colonies sampled in subdivisions had fewer secondary reproductives and were potentially younger than those sampled in undeveloped landscapes. this study provides some correlative support for the role of dispersing alates as urban colonizers because treatments were often located at relatively long distances from undisturbed forest patches in historically agricultural subdivisions. sociobiology an international journal on social insects ps botch1,2, rm houseman1 article history edited by paulo cristaldo, ufs, brazil received 27 june 2017 initial acceptance 19 august 2017 final acceptance 20 september 2017 publication date 30 march 2018 keywords colonization, termiticide, landscape ecology, microsatellites, reticulitermes. corresponding author paul steven botch department of entomology michigan state university 288 farm lane, room 46 east lansing, mi 48824, usa. e-mail: botchpau@msu.edu pre-urban landscapes but may also colonize via dispersal from external habitats or anthropogenic introductions (sattler et al., 2011). despite their abundance in urban landscapes, little research has focused on the response of many arthropod groups to urban development (mcintyre, 2000). subterranean termites (isoptera: reticulitermes) occur in a variety of habitat types, including undisturbed forests and urban landscapes (weesner, 1965). in forests, termites are beneficial because they break down woody debris, which contributes to nutrient cycling and soil fertility (waller & lafage, 1 division of plant sciences, university of missouri, columbia, mo, usa 2 department of entomology, michigan state university, east lansing, mi, usa research article termites mailto:botchpau@msu.edu ps botch, rm houseman – landscape patterns of termite treatment and colony structure68 1987). however, termites are economically destructive pests in urban landscapes when they feed on structural wood. repair and prevention costs associated with termite structural infestations have been estimated to reach $40 billion per year worldwide (rust & su, 2012). despite their economic importance, it is not well known how termite populations respond to urban development or what factors may be associated with subsequent urban colonization. subterranean termite activity occurs almost exclusively underground. a branching network of tunnels connects subterranean nests with cellulose food resources on the soil surface, where most of the foraging activity tends to concentrate (traniello & leuthold, 2000). because subterranean termite life history is inherently connected to soil conditions, it is likely that soil disturbances accompanying urban development would have negative effects on subterranean colonies. during urban development, topsoil is highly altered by removing or filling in areas to prepare landscapes for construction (capachi, 1978; petschek, 2008). soil alterations may also remove subterranean termite populations locally from areas of disturbance. for example, soil alterations were reported to physically remove the majority of soil-dwelling ant species in lots prior to home construction (buczkowski & richmond, 2012). reticulitermes spp. in missouri are also likely removed locally when soil is heavily disturbed by construction processes (botch & houseman, 2016). if subterranean termites are locally removed by subdivision construction processes, infested homes are presumably colonized from dispersing source populations outside of the disturbed area, or by accidental human introductions. subterranean termites can disperse to new areas by tunneling underground or through swarming by reproductive adults (alates). reticulitermes spp. have been estimated to tunnel at generally short linear distances (< 10 m) based on population analyses using molecular techniques (vargo & husseneder, 2009). it is not known how far alates can disperse from parent nests, but termites are recognized as poor fliers with trajectories often influenced by wind (weesner, 1965). however, thousands of individuals are produced during swarming season, some of which could be capable of establishing incipient colonies at long distances from the parent colonies. the goals of this study were to examine patterns of colonization in residential subdivisions of different ages and historical vegetation. the first objective was to identify landscape patterns of colonization over time by inspecting homes for evidence of subterranean termite treatment. in missouri, subterranean termite management usually consists of either a post-construction application of soil termiticide, or use of underground bait stations. post-construction applications of soil termiticide involve trenching into the ground around the outside perimeter of the home and injecting the chemical into the soil to form a continuous treated zone around the house (su & scheffrahn, 1990; su & scheffrahn, 1998; houseman, 2007). concrete slabs adjoining the building, such as porches or driveways, require that holes are drilled through the concrete to treat the soil beneath. the injection holes are later filled with mortar, leaving distinct, evenlyspaced markings in the concrete. unlike southern states with higher termite pressure, there are no laws in missouri requiring homes be treated for subterranean termites prior to or during construction. consequently, most homes are not pretreated. according to the suggested guidelines for completing the wood destroying insect inspection report npma33, termite control treatment should only be recommended by an inspector when there are either live termites in or on the structure, or there is evidence of infestation in or on a structure (houseman, 2007). due to these guidelines, and because subterranean termite treatments are expensive, it is likely that very few post-construction treatments are the result of preventative measures, and therefore most treated homes in missouri are due to previous termite activity. we hypothesized that treatments would be correlated with historical and contemporary landscape features such as forest patches from which termites may disperse, and subdivision age, which can predict treatment frequency (houseman 2010). understanding how historic and contemporary landscape factors influence subterranean termite colonization could improve the ability to predict the risk of homes to infestation and lead to better management strategies. the second objective was to determine the potential effects subdivision development has on subterranean termites by examining colony structure. direct observation of subterranean colonization and breeding is inherently challenging due to their cryptic nature; however molecular tools can be used to infer genetic relationships of colonies (vargo & husseneder, 2009). our goal was to use these techniques to evaluate the breeding structure of termites in undisturbed forests and subdivisions to compare relative colony maturities. the offspring of colonies headed by a monogamous pair of primary reproductive (king and queen) have genotypes conforming to mendelian ratios and are termed simple families (vargo, 2003b; vargo & husseneder, 2009). offspring are reproductively suppressed by a pheromone produced by the primary queen (matsuura et al., 2010). when the pheromone is lacking or diminishes in the colony, due to either queen death or spatial isolation from the queen, worker termites may differentiate into neotenics in high numbers leading to increased colony growth (thorne et al., 1999). because neotenics do not leave the nest, these colonies (termed extended families) experience generations of inbreeding and can be identified by genotype frequencies that deviate significantly from mendelian ratios (vargo, 2003b; vargo & husseneder, 2009; thorne et al., 1999). we hypothesized that undeveloped forest landscapes would contain a higher proportion of extended families and higher levels of inbreeding because these areas lack anthropogenic disturbances. conversely, we expected residential subdivisions to contain a higher proportion of simple families and lower sociobiology 65(1): 67-78 (march, 2018) special issue 69 levels of inbreeding due to construction, termite management, or other anthropogenic disturbances. methods three undeveloped forest landscapes and thirteen subdivisions in columbia, missouri were used as study areas for this project. residential subdivisions were chosen based on the following classifications: 1) 10-year-old subdivisions built on historically forested landscapes (3 subdivisions surveyed); 2) 10-year-old subdivisions built on historically agricultural landscapes (4 subdivisions surveyed); 3) 20-yearold subdivisions built on historically forested landscapes (3 subdivisions surveyed); or 4) 20-year-old subdivisions built on historically agricultural landscapes (3 subdivisions surveyed). subdivisions were defined as historically forested or agricultural when over 60% of the area consisted of the corresponding land cover prior to construction. the landscapes were classified and verified using gis and land use land cover (lulc) data and aerial photography from the missouri spatial data information service (http://msdis.missouri. edu), the center for applied research and environmental systems (http://www.cares.missouri.edu/), the boone county assessor’s office online parcel information viewer (http:// www.showmeboone.com/assessor/), and google earth. overall landscape composition was digitized and quantified using arcmap v.10.0. remnant forest patches were also delineated by comparing historical imagery of forested areas prior to subdivision construction with contemporary forest patches. the historical land cover for each home was defined at two spatial scales: 1) at the overall subdivision level, and 2) for individual home lots (fig 1) using gis datasets. at the overall subdivision level, the historical landscape was classified as forest or agricultural based on the dominant landscape type prior to construction of the subdivision. historical land cover for individual lots was classified after aerial photography was georeferenced and digitized. home ages for each property were determined using the boone county assessor’s office online parcel information viewer. homes that were eight to twelve years-old were grouped into the 10-year age class, and homes that were 18 to 22 years old were grouped into the 20year age class. fig 1. historical landscapes were classified at two spatial scales: 1) for the overall subdivision and 2) for individual lots. the dominant historical landscape-type for this 10-year-old subdivision was agricultural. woodlot patches that existed prior to construction were digitized (shaded in green) from georeferenced historical aerial imagery to determine the pre-construction vegetation for individual home lots. thus, although the overwhelming majority of homes were built on agricultural landscape, some homes were built on woodlot patches, which were cleared before construction. uncleared remnant patches of woodlot may serve as refuges for relict subterranean termite populations and sources of future colonization. ps botch, rm houseman – landscape patterns of termite treatment and colony structure70 individual homes in each subdivision were inspected for subterranean termite treatment. homes were considered treated when termiticide treatment marks were identified in concrete slabs adjoining homes, including driveways and porches, or if termite bait stations were observed in yards or mulch beds. treatment proportions were compared between homes of different ages and historical landscape types using the glimmix procedure (proc glimmix, sas institute inc., 2011) at both spatial scales. the overall proportion of woodlot area for each subdivision-type was compared using a t-test. patch isolation between woodlots was calculated using the euclidean nearest neighbor (enn) statistic in fragstats 3.3 (mcgarigal et al., 2002). distances (m) between homes and their nearest forest patches were calculated in arcmap and compared between treated and untreated homes in all subdivisions. significant differences were assessed using the kruskal-wallis one-way analysis of variance (proc npar1way, sas institute inc., 2011). subterranean termites were sampled from undeveloped forest landscapes and untreated homes in 10and 20-year-old previously forested subdivisions. sampling was conducted using monitor units consisting of two adjacent 9” pine stakes with a steel nail driven into the top of the stakes so they could be found with a metal detector. the monitoring units were placed into holes excavated by an auger and then covered with soil. at each home, monitors were buried at: 1) the front of the property near the sidewalk; 2) near the front porch; 3) near the back porch; and 4) toward the rear property line. in forested landscapes, grids containing 25 m x 50 m plots were overlaid onto geo-referenced aerial landscape photography using arcmap 10.0, to simulate subdivision plats. monitoring units were buried 12 meters apart in linear transects of randomly selected plats. monitors were examined for termites over two consecutive years. termites were also sampled from logs, landscaping timbers, and other woody debris found within individual lots. sampled termites were preserved in 90-100% ethanol and stored in a -25°c freezer. pcr-rflp methods developed by szalanski et al. (2003) and described in botch and houseman (2016) were used to identify species of subterranean termites collected. only colonies identified as r. flavipes were analyzed in this study. fourteen microsatellite loci were screened for polymorphisms among termite populations by amplifying dna from 10 workers collected at 10 different locations. the reaction mixture consisted of 1.0 µl template dna, 2.5 µl of 5x reaction buffer, 2.0 µl mgcl2, 0.5 µl 10mm dntp mix, 1.0 µl bsa, 1.0 µl each of forward and reverse primer, 0.2 µl gotaq flexi dna polymerase, and 15.8 µl ddh20 to a final volume of 25.0 µl. the pcr reactions were conducted on an eppendorf mastercycler pro using following program: initial denaturation step at 94 °c (30 s), followed by 30 cycles at 94 °c (30 s), 55-57 °c (30 s), 72 °c (30 s), with a final extension step at 72 °c (5 min). the pcr product was separated by 6% agarose gel electrophoresis, stained with ethidium bromide, and photographed using a syngene g:box imaging system. microsatellite loci were considered polymorphic when gels produced bands of different sizes, indicating allelic variation among populations. from these tests, eight loci rf1-3, rf11-1, rf15-2, and rf21-1 (vargo, 2000), and rs16, rs43, rs68, and rs85 (dronnet et al., 2004) were considered polymorphic and appropriate for analyzing populations. ten r. flavipes colonies were randomly chosen from undeveloped forested landscapes, 10-year-old subdivisions, and 20-year-old subdivisions for a total of 30 analyzed colonies. dna was isolated from the heads of 20 worker termites from most colonies (in two colonies, only 13 or 11 workers were available). based on dna fragment length (bp) and optimal annealing temperatures, the loci were grouped into two multiplex reactions using the software program multiplex manager© v 1.2. loci in each multiplex reaction were assigned one of four fluorescent dyes: 6-fam (blue), vic (green), ned (yellow), or pet (red). the first multiplex contained loci rf21-1 (6-fam), rf15-2 (vic), rs43 (ned), and rf1-3(pet), and the second multiplex contained loci rf11-1 (6-fam), rs85 (vic), rs16 (ned), and rs68 (pet). each termite was genotyped by amplifying dna in both multiplex reactions using abi platinum® multiplex pcr master mix according to the manufacturer’s recommendations. individual reactions contained 1.00 µl template dna, 3.90 µl master mix, 0.93 µl gc enhancer, 0.89 µl diluted primer mix, and 1.28 µl ddh20 to a final volume of 8.0 µl. the pcr reactions were conducted on an eppendorf mastercycler pro, using following program: initial denaturation step at 95 °c (2 min), followed by 28 cycles at 95 °c (30 s), 57 °c (1:30 min), 72 °c (1 min), with a final extension step at 60 °c (30 min). the amplified product was diluted to 1:100 and sent to the university of missouri’s dna core facility for fragment analysis using the abi 3730xl dna analyzer. the software program peak scannertm v 1.0 (thermo fisher scientific, 2012) was used to score allele sizes of microsatellite loci for each individual termite at all loci. the genotype at each locus was determined to be homozygous if only one allele size was detected and heterozygous if two allele sizes were detected. the genotype frequency within each colony was used to classify r. flavipes colonies as simple, extended, or mixed families. simple families contain genotypes consistent with a colony headed by a monogamous pair of reproductives. these colonies contain four or fewer allelic combinations at each locus, or genotype frequencies that do not significantly deviate from expected frequencies based on a g-test combined over all loci (vargo, 2003b). a g-value for each locus was assessed using the following equation: g=2∑[o*ln(o/e)], where o = the observed number for a genotype and e = the expected number for a genotype (sokal & rohlf, 2012). the observed and expected values for each genotype were calculated using the program fstat v 2.9.3.2 (goudet, 2002). because the g-test is additive, g-values are added across all loci for each colony sociobiology 65(1): 67-78 (march, 2018) special issue 71 to determine an overall g-value. the alpha level 0.05 was adjusted with a bonferroni correction based on the number of loci used in the g-test. extended families contain either more than four genotypes at a given locus, or genotype frequencies that significantly deviate from expected frequencies based on a g-test (vargo, 2003b). unlike simple or extended families, mixed families can be identified by the presence of five or more alleles at one or more loci. five or more alleles at a given locus is not possible among colony members descended from a single reproductive pair, and therefore, mixed families are formed by two or more unrelated reproductive lines, possibly as a result of colony fusion (vargo, 2003b, deheer & vargo, 2004). inbreeding and relatedness was further examined by using f-statistics (weir & cockerham, 1984) to infer breeding structure within colonies (thorne et al., 1999). f-statistics calculate genetic differentiation at three levels: 1) fis, which is the level of inbreeding of individuals relative to the subpopulation; 2) fst, which is the level of inbreeding among subpopulations; and 3) fit, which is the overall inbreeding coefficient of individuals relative to the total population (freeland, 2005). a unique f-statistic notation is given to social insects, whereas: 1) fic is the level of inbreeding of individuals relative to the colony; 2) fct is the level of inbreeding among colonies; and 3) fit is the level of inbreeding of individuals relative to the total population (thorne et al., 1999). fic is particularly useful in determining breeding structure in subterranean termite colonies because it is expected to increase in value relative to the number of secondary reproductives mating within a colony (thorne et al., 1999; bulmer et al., 2001; vargo, 2003b). strongly negative values are indicative of low levels of inbreeding associated with simple families headed by monogamous reproductives. fic values that are less negative, approach zero, or are positive are associated with extended families containing inbreeding by many secondary reproductives (thorne et al., 1999; bulmer et al., 2001; vargo, 2003b). all f-statistics and confidence intervals (95%) were calculated using fstat. results a total of 1,728 homes were surveyed and 913 homes fell within the 10and 20-year-old age classes. of these, 109 homes had been previously treated for subterranean termites (table 1). mean treatment proportions for subdivisions according to overall landscape type were: 10-year-old historically agricultural 2.64%, std = 1.84; 10-yearold historically forested 7.66%, std = 3.19; 20-year-old historically agricultural 19.93%, std = 4.45; and 20-yearold forested 23.40%, std = 7.20. treatment proportion was significantly lower in 10-year-old subdivisions than 20-year-old subdivisions for both historically agricultural (α ≤ 0.05; p = 0.0001) and historically forested (α ≤ 0.05; p = 0.0069) subdivisions (fig 2). ten-year-old historically forested subdivisions had a significantly higher treatment proportion than 10-year-old historically agricultural subdivisions (α ≤ 0.05; p = 0.0246). there was no significant difference in treatment proportion between historically forested and agricultural subdivisions at year 20 (α ≤ 0.05; p = 0.5616), however (fig 2). mean treatment proportions based on the historical vegetation at individual home lots were: 10-year-old historically agricultural lots 4.09%, std = 6.09; 10-yearsubdivision type no. subdivisions total homes total treated homes treated homes in 5-yr window 10 yo subdivision previous forest 3 344 22 16 10 yo subdivision previously agricultural 4 619 17 11 20 yo subdivision previously forest 3 408 88 22 20 yo subdivision previously agricultural 3 357 68 60 total 13 1728 195 109 table 1. summary of subdivision homes and subterranean termite (isoptera: reticulitermes) treatment totals in historically agricultural 10-year-old subdivisions, historically forested 10-year-old subdivisions, historically agricultural 20-year-old subdivisions, and historically forested 20-year-old subdivisions in columbia, mo. fig 2. mean proportions and standard deviations for subterranean termite (isoptera: reticulitermes) treatments in historically agricultural 10-year-old subdivisions, historically forested 10-yearold subdivisions, historically agricultural 20-year-old subdivisions, and historically forested 20-year-old subdivisions. there was a significantly higher treatment proportion in 20-year-old subdivisions than 10-year-old subdivisions that were historically agricultural (α ≤ 0.05; p = 0.0001) and forested (α ≤ 0.05; p = 0.0069). historically forested 10-year-old subdivisions had a significantly higher treatment proportion than historically agricultural 10-year-old subdivisions (α ≤ 0.05; p = 0.0246); however historically forested 20-year-old subdivisions did not have a significantly higher treatment proportion than historically agricultural subdivisions (α ≤ 0.05; p = 0.5616). ps botch, rm houseman – landscape patterns of termite treatment and colony structure72 old historically forested lots 4.63%, std = 3.36; 20-yearold historically agricultural lots 19.13%, std = 9.36; and 20-year-old historically forested lots 23.03%, std = 6.44. treatment proportions were significantly higher for 20-yearold homes than 10-year-old homes for historically agricultural lots (α ≤ 0.05; p = 0.0005) and forested lots (α ≤ 0.05; p = 0.0007). there was no significant difference in treatment proportions between homes built on historically agricultural or historically forested lots at either 10 (α ≤ 0.05; p = 0.7269) or 20 years (α ≤ 0.05; p = 0.4226) (fig 3). in 20-year-old historically forested subdivisions, treated homes had a shorter mean distance to contemporary forest patches (12.96 m) than untreated homes (15.45 m) (table 2). proportions of forested area within these subdivisions were previously reported in botch & houseman (2016) and were also used to evaluate how they were associated with treatment proportion for this study. mean proportion of contemporary forest area for subdivisions was 5.25% for 10-year-old historically agricultural, 29.30% for 10-yearold historically forest, 17.16% for 20-year-old historically agricultural, and 43.29% for 20-year-old historically forest (fig 4). at year 10, historically forest subdivisions had significantly more contemporary forest area than historically agricultural subdivisions (p = 0.0177; α ≤ 0.05; t-test). however, at year 20, there was no significant difference in contemporary forest area between historically agricultural and historically forest subdivisions (p = 0.0701; α ≤ 0.05; t-test). forest patches in 10-year-old historically agricultural subdivisions were more isolated than in other subdivision types and had the highest patchiness value (enn = 4617.8), compared to 10-year-old historically forested subdivisions (enn = 2575.2), 20-year-old historically agricultural subdivisions (enn = 2225.2), or 20-year-old historically forested subdivisions (enn = 2681.7). non-treated treated subdivision nn nt mean distance to forest stdev mean distance to forest stdev p 10yo ag n = 406 n = 11 66.71 42.04 52.26 36.55 0.23 10yo for n = 192 n = 16 39.68 42.17 55.54 49.18 0.30 20yo ag n = 241 n = 60 32.18 23.90 24.45 21.22 0.01 20yo for n = 74 n = 22 15.45 9.23 12.96 3.65 0.69 fig 3. mean proportions and standard deviations for subterranean termite (isoptera: reticulitermes) treatments for individual home lots that were historically agricultural or historically forested at years 10 and 20. treatment proportions were significantly different between 10-year-old homes and 20-year-old homes built on agricultural land (α ≤ 0.05; p = 0.0005) and forested land (α ≤ 0.05; p = 0.0007), but there were no significant differences between treatment proportions when historical landscape conditions of individual home lots were compared at 10 years (α ≤ 0.05; p = 0.7269) or 20 years (α ≤ 0.05; p = 0.4226). table 2. mean distance (m) to nearest forest patch for untreated homes (nu) and treated homes (nt) in historically agricultural 10-year-old subdivisions, historically forested 10-year-old subdivisions, historically agricultural 20-year-old subdivisions, and historically forested 20-year-old subdivisions in columbia, mo. in historically agricultural subdivisions, treated homes had a shorter mean distance to contemporary forest patches (52.26 m) than untreated homes (66.71 m) at year ten (table 2). twenty-year-old historically agricultural subdivisions showed a similar trend where treated homes had a significantly shorter (α ≤ 0.05) mean distance (24.45 m) to contemporary forest patches than untreated homes (32.18 m). ten-year-old historically forested subdivisions showed the opposite trend where treated homes had a greater distance to contemporary forest patches (55.54 m) than untreated homes (39.68). fig 4. mean proportion of contemporary forest area in a) 10-yearold historically agricultural and forested subdivisions, and b) 20-year-old historically agricultural and forested subdivisions in columbia, mo. there was a significant difference in forest area between subdivisions built on different historical landscapes at year 10 (p=0.0177; α ≤ 0.05; t-test), but not at year 20 (p=0.0701; α ≤ 0.05; t-test). significant differences are indicated by an asterisk above standard deviation bars. sociobiology 65(1): 67-78 (march, 2018) special issue 73 six of the eight microsatellite loci used in this study were determined to be polymorphic among colonies that were sampled. the overall number of detected alleles ranged between 2 and 14 at loci rf21-1, rf1-3, rf11-1, rs85, rs16, and rs68 (table 3). loci rf15-2 and rs43 showed no variation among colonies, and therefore were not used to interpret breeding structure. no colonies contained more than four alleles at a given locus, and therefore there was no evidence of mixed families. colonies that deviated from mendelian rules of inheritance and from expected genotype frequencies using a g-test were determined to be extended families. this included 6 of 10 colonies from undeveloped forested areas, 5 of 10 colonies from 10-year-old subdivisions, and 5 of 10 from 20-year-old subdivisions (table 4). the overall fct value was 0.345 (table 4), and therefore there was distinct genetic variation between colonies. the fit, or overall inbreeding coefficient of individuals relative to the entire population, was highest in undeveloped forested areas (fit = 0.302, 95% ci = 0.1600.451, table 4). this value is over twice as high as 10-yearold subdivisions (fit = 0.125, 95% ci = 0.054 0.179) or 20-year-old subdivisions (fit = 0.116, 95% ci = -0.083 0.199), though not significantly higher due to overlapping ci’s. fic values are expected to be strongly negative for simple families and approach zero in extended families due to an increase in secondary reproductives within the colony. the overall fic value for undeveloped forests is -0.081 (95% ci = -0.149 to -0.023). partitioning this value among family types gives values of -0.126 (95% ci = -0.324 to -0.002) for simple families and -0.061 (95% ci = -0.149 to -0.023) for extended families. the value for extended families is closer to zero, which is expected. fic values for simple and extended families deviated from these expectations, however. in 10-year-old subdivisions, simple families had an fic value of -0.177 (95% ci = -0.251 to -0.087) and extended families had an fic value of -0.308 (95% ci = -0.382 to -0.213). in 20-year-old subdivisions, simple families had an fic value of -0.265 (95% ci = -0.304 to -0.218) and extended families had an fic value of -0.411 (95% ci = -0.524 to -0.324) (table 4). discussion we found that subterranean termite treatment proportion was positively associated with subdivision age, as well as historical landscape-type for newer subdivisions. treatment proportion was significantly higher in 20-year-old subdivisions than in 10-year-old subdivisions, indicating that termite infestations become more common as homes age. treatment proportion was also significantly higher in 10-yearold subdivisions built on historically forested landscapes than in 10-year-old subdivisions built on historically agricultural landscapes. there was no difference between treatment proportions in 20-year-old subdivisions built on historically forested or agricultural landscapes. treatment proportion for allele size all_w all_uw rf21-1 210 0.209 0.214 213 0.293 0.291 216 0.019 0.019 225 0.044 0.041 229 0.128 0.122 231 0.011 0.01 237 0.016 0.016 240 0.004 0.004 244 0.012 0.011 247 0.12 0.126 250 0.015 0.014 256 0.105 0.103 259 0.013 0.018 286 0.012 0.011 rf1-3 195 0.595 0.596 210 0.003 0.003 212 0.371 0.371 213 0.032 0.031 rf11-1 221 0.007 0.007 222 0.028 0.03 223 0.015 0.014 226 0.022 0.022 230 0.372 0.361 232 0.295 0.306 235 0.262 0.259 rs85 236 0.011 0.01 240 0.989 0.99 rs16 286 0.904 0.902 288 0.096 0.098 rs68 159 0.161 0.157 163 0.833 0.833 167 0.006 0.011 rf15-2 213 1 1 rs43 215 1 1 table 3. overall allele frequencies for each locus in a microsatellite analysis of eight loci. the size (bp) and frequency of alleles at each locus are given. all_w is the estimated allele frequency weighted by sample size, while all_uw is the unweighted allele frequency among colonies. 20-year-old subdivisions built on either forests or agricultural landscapes was approximately 20%. this supports the 20-20 rule for treatment rates in central missouri previously reported by houseman (2010).thus, historical landscape conditions appear to influence subterranean termite infestations in younger subdivisions, but the influence declines as subdivisions age. treatment proportion in historically agricultural subdivisions ps botch, rm houseman – landscape patterns of termite treatment and colony structure74 eventually catches up to treatments in historically forested subdivisions. although the overall increase in treatment proportion in older subdivisions is likely to be due in part to degradation of homes over time, it may also be influenced by landscape composition. we found that remnant forest patches tended to increase in size and fill in around homes as historically agricultural subdivisions age. the increase in termite infestations by year 20 may also be influenced by increased available undeveloped habitat and woody debris, in addition to home age. remnant forest patches in subdivision landscapes that are not altered potentially contain relict termite colonies. tenyear-old historically forested landscapes tended to have more remnant patches than 10-year-old historically agricultural landscapes, which had the fewest treatments and the least amount of forested area. this may explain why treatment proportions were significantly different between historical landscape types at year 10. this also provides further evidence that the presence of forest cover may be related to infestation proportion (houseman, 2010). few studies have evaluated termite colonization following landscape perturbation. in uganda forests, okwakol (2000) reported that termite species richness was significantly reduced following clear-cutting, and species richness was further diminished following land cultivation. in southern cameroon, experimental plots were disturbed by removing different combinations of epigeal termite mounds, surrounding litter, and soil to examine how termite community reassembly occurs following different degrees of disturbance. it was reported that communities reassembled quicker when significant dead wood was left on the ground (davies et al. 1999). the vegetative history of the subdivision at the landscape-level may be an important predictor of future infestations due to nearby remnant woodlot patches and woody debris that potentially serve as sources of future subterranean termite colonizers. although treatment proportions were associated with dominant historical vegetation for the overall subdivision, treatments did not seem to be influenced by the historical vegetation of individual home lots. twenty-year-old homes had a higher treatment proportion than 10-year-old homes, regardless of historical vegetation on individual lots. this demonstrates the importance of home age as a predictor of treatment proportion. however, there was no difference in treatment proportion between individual lots of the same age that were previously forested or agricultural. pre-construction soil grading has been shown to directly remove ants from areas of soil disturbance (buczkowski & richmond, 2012), and there is correlative evidence that subterranean termites fit fct fic r all colonies 0.203 0.345 -0.218 0.574 (n = 30) (0.148 0.258) (0.307 0.371) (-0.26 0.155) (0.526 0.600) undeveloped forest colonies 0.302 0.354 -0.081 0.544 (n = 10) (0.160 0.451) (0.259 0.495) (-0.149 0.023) (0.444 0.684) simple families 0.374 0.444 -0.126 0.646 (n = 4) (0.187 0.474) (0.368 0.512) (-0.324 0.002) (0.586 0.718) extended families 0.294 0.334 -0.061 0.516 (n = 6) (0.148 0.470) (0.217 0.481) (-0.114 0.013) (0.378 0.659) 10-yo subdivision colonies 0.125 0.299 -0.248 0.531 (n = 10) (0.054 0.179) (0.228 0.337) (-0.290 0.199) (0.432 0.582) simple families 0.28 0.388 -0.177 0.607 (n = 5) (0.093 0.476) (0.248 0.525) (-0.251 0.087) (0.453 0.713) extended families -0.006 0.231 -0.308 0.465 (n = 5) (-0.172 0.084) (0.133 0.277) (-0.382 0.213) (0.317 0.519) 20-yo subdivision colonies 0.116 0.344 -0.346 0.616 (n = 10) (-0.083 0.199) (0.233 0.397) (-0.422 0.276) (0.502 0.666) simple families 0.28 0.431 -0.265 0.674 (n = 5) (0.118 0.394) (0.312 0.522) (-0.304 0.218) (0.559 0.749) extended families -0.017 0.279 -0.411 0.569 (n = 5) (-0.296 0.117) (0.154 0.335) (-0.524 0.324) (0.437 0.603) table 4. f-statistics and relatedness coefficients for reticulitermes flavipes (kollar) colonies in undeveloped forests, 10-yearold subdivisions, and 20-year-old subdivisions. fit is the level of inbreeding of individuals relative to the total population. fct is the level of inbreeding among colonies. fic is the level of inbreeding of individuals relative to the colony. the relatedness r is a measure of average relatedness of individuals within colonies relative to the entire population. 95% confidence intervals (ci) are given in parentheses under each value. 95% confidence intervals (ci) are given in parentheses under each value. sociobiology 65(1): 67-78 (march, 2018) special issue 75 are similarly removed from areas of disturbance (botch & houseman, 2016). vegetation and top soil is also removed or highly altered by grading (capachi, 1978; petschek, 2008), which may reset soil ecological conditions. thus, the vegetative history of individual lots may not be an important predictor of future subterranean termite infestations because soil environmental conditions have been so heavily altered. we predicted that treated homes would be found in relative close proximity to forest patches that potentially serve as colonization sources; however, there was less statistical support for this prediction than we expected. a greater sample size of treated homes may have helped us to better understand this relationship, as adjacency to forest patches was found to be an important predictor of treatment proportion in another study in missouri (houseman, 2010). in both 10and 20-yearold historically agricultural subdivisions, treated homes were on average closer to forest patches than untreated homes. this is expected if subterranean termites colonize from forest patches via tunneling activity or dispersal flights. interestingly, 10-year-old historically forested subdivisions showed a more ambiguous pattern, where treated homes were on average farther away from forest patches at year 10, but not at year 20. these subdivisions had the lowest forest patch isolation (enn value) and the greatest proportion of forested area near homes. since densities of mature termite colonies can be very high in large forested areas (vargo & husseneder, 2009), this may increase the likelihood of swarming termites dispersing further into subdivisions than they would by tunneling. consequently, they might infest homes at greater distances, which would complicate our ability to predict infestations based on proximity to forest patches. spatial patterns and rates of subterranean termite colonization are likely to be species-specific, which we were not able to differentiate in this termiticide treatment survey. in a companion study to this survey, where wooden stake monitors were used to monitor homes not been previously treated for subterranean termites, we found that reticulitermes flavipes (kollar) was overwhelmingly the most common species found near homes (botch & houseman, 2016). r. flavipes is the most widely distributed subterranean termite in north america (snyder, 1954) and has often been reported to be the most common colonizer of urban areas (austin et al., 2004a; austin et al., 2004b; wang et al., 2009; vargo, 2003a; parman & vargo, 2008; pinzon & houseman, 2009). in our companion study, the subterranean termite species reticulitermes hageni banks was found in subdivisions less often, and tended to be located at the back of property lines, near or within woodlots (botch & houseman, 2016). studies have reported that r. hageni is also associated with forested areas in oklahoma (austin et al., 2004a; brown et al., 2004). thus, it is likely that most of the homes in this survey were treated for r. flavipes infestations, and r. hageni infestations are more likely to have occurred in subdivisions containing large patches of undeveloped forest. breeding structure in undeveloped forested areas was consistent with extended families in 60% of the colonies sampled (6 of 10). there was a 50:50 ratio of simple and extended families in both 10and 20-year-old subdivisions. therefore, a slightly higher proportion of extended families were found in forested landscapes than in urban subdivisions, potentially due to absence of anthropogenic disturbances. the overall inbreeding coefficient of individuals relative to the entire population (fit) was highest in forested areas, so it can be inferred that termites in forested areas are more inbred overall than termites in urban subdivisions. extended families had an fic value close to zero, which is expected when secondary reproductives are present (thorne et al., 1999; vargo, 2003b). fic values in 10and 20-year-old subdivisions were all strongly negative however, suggesting that these colonies contain few supplementary reproductives. it is possible that some colonies in residential subdivisions may have been misidentified as extended families due to low allelic diversity at two loci. this is supported by the fact that few colonies in 10and 20-year-old subdivisions contained more than four genotypes at a given loci, compared to forested landscapes where 40% of the colonies contained five or more genotypes among one to three loci. deheer et al. (2005) concluded that 20 worker termites may not provide enough statistical power for distinguishing between simple and extended families when allelic variation is extremely low at any one locus. in this study, the loci rs18 and rs85 contained only two alleles among all colonies. in particular, rs85 was only polymorphic in colonies from forested areas, and there was no variability in urban areas. thus, it is possible that subdivisions contained a greater proportion of simple families than we were able to report genotyping only 20 workers per colony. however, inbreeding coefficients indicated that termite colonies in subdivisions had fewer supplementary reproductives, and thus were likely more recently established than colonies in forests. presumably, undisturbed forests provide an abundance of available food resources and favorable conditions for longterm colony growth and maturity, which may eventually lead to supplementary reproductives, increased fecundity, and inbreeding among colony members (thorne et al., 1999). we found high levels of inbreeding in r. flavipes colonies collected in forests that supports this. deheer et al. (2005) reported populations of r. grassei in young forests contained relatively few supplementary reproductives and noted that there may be a correlation between colony age and forest age, because older forests contain more woody debris.in addition to resource availability, colony structure may also be affected by disturbance frequency. aluko and husseneder (2007) reported that extended families of coptotermes formosanus replaced simple families in a frequently disturbed urban area in new orleans over time and noted a transition of one colony from a simple to extended family. it is not clear whether low levels of inbreeding found in colonies we sampled in subdivisions was associated with human disturbance or new colonization ps botch, rm houseman – landscape patterns of termite treatment and colony structure76 events; however, it seems likely that an overall increase in treatment applications over time would likely suppress colony transition from simple to extended by killing colonies before they can produce supplementary reproductives. this study may also provide evidence of the role of swarming into subdivisions. in 10-year-old historically agricultural subdivisions, treated homes were often found at distances exceeding 50 m from forest patches. this exceeds typical subterranean linear foraging distances for reticulitermes spp. (vargo & husseneder, 2009). when mature subterranean termite colonies swarm from adjacent large forest patches into subdivisions, they can presumably infest homes that are further away sooner than colonies that colonize via tunneling. if home infestations in 10-year-old historically agricultural subdivisions did not result from movement of wood and timber, then it is likely that termites colonized homes by dispersal flights rather than tunneling. future studies of swarming patterns in subdivisions would provide important information about colonization dynamics and subsequent home infestation by subterranean termites. acknowledgements we thank deborah finke, bruce barrett, and mark cowell for feedback and advice with this project. thank you to lori eggert and emily puckett for valuable help with molecular assays. support was provided by the national institute of food and agriculture, u.s. department of agriculture-hatch project and the university of missouri research council. references aluko, g.a., & husseneder, c. 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(1984). estimating f-statistics for the analysis of population structure. evolution, 38: 13581370. doi: 10.2307/2408641 doi: 10.13102/sociobiology.v64i4.1928sociobiology 64(4): 488-491 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 notes on a nest of megachile (moureapis) apicipennis schrottky (megachilidae) constructed in an abandoned gallery of xylocopa frontalis (olivier) (apidae) leafcutting bees (megachile latreille) comprise a cosmopolitan, morphologically diverse genus with 1524 recognized species distributed in more than 50 subgenera (michener, 2007; ascher & pickering, 2017). nesting habits are diversified, including use of pre-existing cavities in the ground, wood, stems, arboreal termite nests or man-made substrates (krombein, 1967; eickwort et al., 1981; michener, 2007). most megachile females cut pieces of fresh leaves or petals to build their brood cells but mud and resin can also be used as nest-building materials (krombein, 1967; michener, 2007). this habit of using foreign material in nest construction may have driven much increase in species diversity in megachilid bees (litman et al., 2011). the neotropical subgenus megachile (moureapis) raw encompasses 28 species that range from tamaulipas state, mexico to buenos aires province, argentina (moure et al., abstract notes on a nest of the leafcutting bee megachile (moureapis) apicipennis schrottky (megachilidae) found in an abandoned gallery excavated by the carpenter bee xylocopa frontalis (olivier) (apidae) are presented. a total of three linear series of three cells each and one solitary cell were found at the end of the gallery. brood cells were lined with imbricate pieces of leaves of centrosema virginianum (l.) benth. (fabaceae). four males and two females of m. apicipennis emerged as well as individuals of three species of natural enemies: coelioxys otomita cresson (megachilidae), brachymeria paraguayensis (brèthes) (chalcididae) and melittobia australica girault (eulophidae). our results were similar to the data obtained from other species of megachile (moureapis) raw. sociobiology an international journal on social insects lc rocha-filho1, ac martins2, p marchi3 article history edited by cândida aguiar, uefs, brazil received 01 august 2017 initial acceptance 25 september 2017 final acceptance 13 october 2017 publication date 27 december 2017 keywords carpenter bee, centrosema leaves, natural enemies, leafcutting bee. corresponding author léo correia rocha filho departamento de biologia faculdade de filosofia, ciências e letras de ribeirão preto ffclrp universidade de são paulo (usp) av. bandeirantes, 3900, cidade universitária cep 14040-901 ribeirão preto-sp, brasil e-mail: correiadarocha@yahoo.com.br 2012). despite its high richness and wide distribution, only a few studies have reported the nesting biology of species from this subgenus (ihering, 1904; laroca, 1991; teixeira et al., 2011; cardoso & silveira, 2012; sabino & antonini, 2017). here we provide notes on a nest of megachile (moureapis) apicipennis schrottky (megachilidae) built in an abandoned nest of xylocopa frontalis (olivier) (apidae). the study was conducted at the edge of a secondary forest at instituto agronômico do paraná – (iapar) (25º30’33’’s; 48°48’30’’w; 64 m) in morretes, paraná state, southern brazil. the area is located in the atlantic forest biome domain, one of the world’s biodiversity hotspots, characterized by the outstanding species richness and level of endemism and by being threatened by anthropogenic changes (myers et al., 2000). a small crop area of yellow passion fruit, passiflora edulis sims (passifloraceae), is located at the study site 1 faculdade de filosofia, ciências e letras de ribeirão preto-ffclrp, universidade de são paulo (usp), ribeirão preto-sp, brazil 2 universidade federal do paraná (ufpr), curitiba-pr, brazil 3 universidade de são paulo (usp), são paulo-sp, brazil short note mailto:correiadarocha@yahoo.com.br sociobiology 64(4): 488-491 (december, 2017) 489 approximately 100 m from a shelter that consisted of a tiled roof supported by wooden pillars. in this construction, several nests of x. frontalis were established in dead tree trunks attached with wire to the rafter and to the wooden columns. on 11 january 2006 around 17h00, four females of m. apicipennis were recorded simultaneously entering the nest entrance on the tree trunk. females of m. apicipennis were also observed cutting rounded pieces from the leaves of centrosema virginianum (l.) benth. (fabaceae) plants (figs 1a, b) that were located just 4 m distant from the nest. the leaf pieces were then folded between the legs and transported to the nest by the females (fig 1c). the movements of females with leaves was observed on the first day, when the nest was discovered, whereas on the second day we only observed females carrying pollen on the ventral scopae. no further observations on the nest activities were performed. after the end of the nest activities, on 17 january 2006, the nest was removed from the trunk cavity and taken to the laboratory for study. no sign of nesting activity of x. frontalis was observed within the gallery. a total of three linear series of three cells each and one solitary cell were found at the end of the gallery (fig 1d). the cell series were parallel to each other and lined with imbricately arranged leaf pieces and closed basally with rounded small leaf fragments. imagines of m. apicipennis (four males and two females) emerged 38 to 49 days, respectively, after the end of nesting activities. mortality of 40% was caused by the attack of natural enemies (30%) and unknown factors (10%). three brood cells had been attacked by the cleptoparasitic bee coelioxys otomita cresson (megachilidae), the parasitic wasp brachymeria paraguayensis (brèthes) (chalcididae) and the gregarious parasitoid melittobia australica girault (eulophidae). the nest architecture registered herein was also observed in other studies for megachile (moureapis) spp. (laroca, 1991; teixeira et al., 2011; cardoso & silveira, 2012; sabino & antonini, 2017) except for ihering (1904). according to this author, the brood cell walls of a m. apicipennis nest were constructed with mud rather than with pieces of leaves. as pointed out by cardoso and silveira (2012) that nest was probably constructed by a species belonging to another subgenus such as m. (chrysosarus) mitchell. on the other hand, laroca (1991) also described a nest of m. apicipennis (cited as pseudocentron apicipennis) with cells arranged in linear series and built with leaf fragments as observed in the present study. fig 1. nest building by females of megachile apicipennis. (a) female of megachile apicipennis cutting a piece of a leaf of centrosema virginianum, (b) specimen of centrosema virginianum (four meter distant from the nest) showing damage to leaves done by leafcutting bees, (c) female of megachile apicipennis entering the nest with a leaf piece of centrosema virginianum, and (d) three cell series of megachile apicipennis located at the end of the abandoned gallery of xylocopa frontalis (d). lc rocha-filho, ac martins, p marchi – nest of megachile apicipennis in a gallery of xylocopa frontalis490 the use of leaves of leguminous species (fabaceae) to construct brood cells was also recorded in another m. (moureapis) species. sabino and antonini (2017) identified leaves of senna pendula (willd.) h.s.irwin and barneby and dalbergia miscolobium benth. in cells built by females of m. maculata smith. other leafcutting bee species belonging to different subgenera, in contrast, collect leaves from a high diversity of plant species ranging from eight to 20 plant families (macivor, 2016). the author reported that females of megachile (eutricharaea) rotundata (fabricius) preferred leaves from fabaceae to the other nine families whereas rosaceae species were more used by both megachile (megachile) centuncularis (l.) and megachile (sayapis) pugnata say. in spite of the high richness of plant species used by those three megachile spp., macivor (2016) pointed out that almost all plant species identified from nest samples had antimicrobial properties, a feature that might inform selection among leaf types. four females of m. apicipennis were recorded entering the nest either carrying pieces of leaves or pollen without leaving the nest. however, given the lack of data on the ovary dissection of females it is not possible to conclude that the nest was communal but it is presumable that each female has constructed her own nest. as megachile (moureapis) species select wide cavities to nest (laroca, 1991; teixeira et al., 2011) it is likely to assume that communal nests among these bees may occur. cardoso and silveira (2012) emphasized that a single bamboo cane used as trap nest could host multiple nests built by different females. in megachilidae, communal nests were described for megachile (callomegachile) pluto smith, microthurge corumbae (cockerell) and afranthidium repetitum (schulz) (michener, 1968; messer, 1984; garófalo et al., 1992). three species of natural enemies were reared from the nest of m. apicipennis. the cuckoo bee coelioxys otomita was also registered as a cleptoparasite in m. benigna nests by teixeira et al. (2011). the only host record for brachymeria paraguayensis was provided by noyes (2003) for an unidentified megachile species. the generalist parasitoid melittobia australica has been recorded as a natural enemy of several bee species, including five in the genus megachile, as m. maculata (sabino & antonini, 2017), as well as flies (calliphoridae, sarcophagidae) and species of crabronidae, formicidae, pompilidae, sphecidae, and vespidae (noyes, 2003). species of coelioxys latreille and melittobia westwood were also reported parasitizing brood cells of other m. (moureapis) species (teixeira et al., 2011; cardoso & silveira, 2012; sabino & antonini, 2017). acknowledgements the authors are grateful to the staff of iapar for allowing our access to the study area and all taxonomists who have identified the studied species: gabriel a. r. melo (ufpr) (bee species); marcelo t. tavares (ufes) (brachymeria); jorge m. gonzález (texas a & m university) (melittobia) and osmar dos santos ribas (mbm) (centrosema). all authors were supported by scholarships from “conselho nacional de desenvolvimento científico e tecnológico cnpq”: 130784/2005-2 (first author), 182060/2006-3 (second author) and 140087/2004-4 (third author). references ascher, j.s., pickering, j. (2017). discover life bee species guide and world checklist (hymenoptera: apoidea: anthophila). http://www.discoverlife.org/mp/20q?guide=apoidea_species& flags=has. (accessed date: 1 august, 2017) cardoso, c.f., silveira, f.a. (2012). nesting biology of two species of megachile (moureapis) (hymenoptera: megachilidae) in a semideciduous forest reserve in southeastern brazil. apidologie, 43: 71-81. doi: 10.1007/ s13592-011-0091-z eickwort, g.c., matthews, r.w., carpenter, j. (1981). observation on the nesting behavior of megachile rubi and m. texana with a discussion of the significance of soil nesting in the evolution of megachilid bees (hymenoptera: megachilidae). journal of the kansas entomological society, 54: 557-570. garófalo, c.a., camillo, e., campos, m.j.o., serrano, j.c. (1992). nest re-use and communal nesting in microthurge corumbae (hymenoptera, megachilidae), with special reference to nest defense. insectes sociaux, 39: 301-311. ihering, r. (1904). biologia das abelhas solitárias do brazil. revista do museu paulista, 6: 461-481. krombein, k.v. (1967). trap-nesting wasp and bees: life histories nest and associates. washington, dc: smithsonian press, 570 p. laroca, s. (1991). euglossa stellfeldi: arquitetura do ninho e coexistência com pseudocentron apicipennis em uma mesma cavidade. acta biologica paranaense, 20: 103-108. litman, j.r., danforth, b.n., eardley, c.d., praz, c.j. (2011). why do leafcutter bees cut leaves? new insights into the early evolution of bees. proceedings of the royal society of london b, 278: 3593-3600. doi: 10.1098/rspb.2011.0365 macivor, j.s. (2016). dna barcoding to identify leaf preference of leafcutting bees. royal society open science, 3: 150623. doi: 10.1098/rsos.150623 messer, a.c. (1984). chalicodoma pluto: the world’s largest bee rediscovered living communally in termite nests. journal of the kansas entomological society, 57: 165-168. michener, c.d. (1968). nests of some african megachilid bees, with description of a new hoplitis. journal of the entomological society of southern africa, 31: 337-359. http://www.discoverlife.org/mp/20q?guide=apoidea_species&flags=has http://www.discoverlife.org/mp/20q?guide=apoidea_species&flags=has sociobiology 64(4): 488-491 (december, 2017) 491 michener, c.d. (2007). the bees of the world. 2nd ed. baltimore, md: the johns hopkins university press, 992 p. moure, j.s., melo, g.a.r., dalmolin, a. (2012). megachilini latreille, 1802. in j.s. moure, d. urban & g.a.r. melo (orgs.), catalogue of bees (hymenoptera, apoidea) in the neotropical region online version. http://www.moure.cria. org.br/catalogue. (accessed on: 18 may, 2016) myers, n., mittermeier, r.a., mittermeier, c.g., fonseca, g.a.b., kent, j. (2000). biodiversity hotspots for conservation priorities. nature, 403: 853-858. doi:10.1038/35002501 noyes, j.s. (2003). universal chalcidoidea database. http:// www.nhm.ac.uk/our-science/data/chalcidoids/database/. (accessed on: 18 may, 2016) sabino, w.o., antonini, y. (2017). nest architecture, life cycle, and natural enemies of the neotropical leafcutting bee megachile (moureapis) maculata (hymenoptera: megachilidae) in a montane forest. apidologie, 48: 450-460. doi: 10.1007/ s13592-016-0488-9 teixeira, f.m., schwartz, t.a.c., gaglianone, m.c. (2011). biologia da nidificação de megachile (moureapis) benigna mitchell. entomobrasilis, 4: 92-99. http://www.moure.cria.org.br/catalogue http://www.moure.cria.org.br/catalogue http://www.nhm.ac.uk/our-science/data/chalcidoids/database/ http://www.nhm.ac.uk/our-science/data/chalcidoids/database/ open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.229-235sociobiology 60(3): 229-235 (2013) the importance of poneromorph ants for seed dispersal in altered environments fs almeida1, aj mayhé-nunes2, jm queiroz 1,2 introduction seed dispersal is important for plants, as it reduces mortality by seed parasites and predators and decreases intraspecific competition (janzen, 1970; howe & smallwood, 1982). seed dispersal by animals, also known as zoochory, can be carried out by ants that nest in the ground and remove seeds to their nest or its surroundings (pizo et al., 2005). the habit of feeding on seeds (granivory) or of collecting seeds to remove their appendages occurs in several species (hölldobler & wilson, 1990). seed consumption rate and removal distance vary according to ant behavior and morphology (ness et al., 2004; zelikova & breed, 2008). in south american tropical forests, poneromorph ants pachycondyla striata fr. smith and odontomachus chelifer (latreille) feed on the seeds of a wide variety of native plants (pizo & oliveira, 1998; passos & oliveira, 2003). these ants have a relatively large size and can remove diaspores up to abstract changes in species composition and an increase in the probability of local or regional extinctions alone are considered alarming consequences of human disturbances. however, these changes bring other damages that have passed unnoticed by scientists, such as the loss of ecological interactions. in the present study, we assessed fragments of secondary forest and pastures focusing on two aspects of the seed dispersal process: removal rate and dispersal distance. we collected data in forest fragments named forest 1 (6 ha), forest 2 (36 ha), and forest 3 (780 ha), and in a pasture in the municipality of vassouras, rio de janeiro, southeastern brazil. in each site, we established 40 observation stations containing six seeds of carica papaya l. (papaya) and monitored seed removal for 2 h at each station. seventeen ant species removed a total of 316 seeds (32.92% of the seeds). the species that removed the highest number of seeds was pachycondyla striata fr. smith, followed by odontomachus chelifer (latreille). the seed removal rate was significantly higher (p < 0.05, tukey test) in the forest fragment where larger species were more frequent. the average removal distance was significantly longer in two out of three forest fragments (p < 0.05, tukey test). larger ants removed more seeds and for longer distances. hence, seed dispersal was increased by the presence of large-bodied ant species and their high frequency in forest fragments seed dispersal. sociobiology an international journal on social insects 1 universidade federal rural do rio de janeiro, três rios, rio de janeiro, brazil. 2 universidade federal rural do rio de janeiro, seropédica, rio de janeiro, brazil. research article ants article history edited by kleber del-claro, ufu, brazil received 25 june 2013 initial acceptance 22 july 2013 final acceptance 10 august 2013 key words biodiversity, communities, diaspores, myrmecochory, human disturbances corresponding author jarbas m. queiroz depto. de ciências ambientais instituto de florestas/ufrrj br 465, km 7, seropédica, rj, brazil e-mail: jarquiz@gmail.com 13 m (passos & oliveira, 2003). small ants, mainly of the genera pheidole and solenopsis, are frequently found consuming diaspores in tropical forests. these ants explore the resource where they find it, instead of taking it to the nest, as observed in solenopsis ants (pizo & oliveira, 1998; passos & oliveira, 2003; leal et al., 2007). in general, large-bodied species forage at larger distances than small-bodied ants (gomez & espadaler, 1998) and can more easily carry seeds individually (zelikova & breed, 2008). forest fragmentation may change the composition of ant communities, since small fragments may be invaded by generalists ants and parasites of mutualistic systems (puntilla et al., 1994; gibb & hochuli, 2002; ness et al., 2004; schoereder et al. 2004). in many cases, forest fragments are more susceptible to biological invasions (ness et al., 2004). although these changes in community composition caused by environmental disturbances are relatively well documented, the consequences of these alterations for ecological processes fs almeida, aj mayhé-nunes, jm queiroz seed dispersal by poneromorph ants230 are still poorly known. the impact of environmental changes on seed-dispersing ants may compromise the seed dispersal process and plant species that depend on animals for this process (guimarães-jr & cogni, 2002; ness et al., 2004; cordeiro et al., 2009). starting from the assumption that environmental changes, for instance forest fragmentation, affect the composition of ant communities, it is relevant to analyze the relationship between the guild of ants that consume seeds and the seed dispersal process in altered environments. in the present study, we assessed fragments of secondary forest and a pasture, and monitored two aspects of the seed dispersal process: removal rate and dispersal distance. we aimed at testing the following hypotheses: (1) changes in ant community structure cause changes in the rate and distance of seed dispersal; (2) the ant body size affects the number of seeds removed and the removal distance. material and methods study area we collected data in the municipality of vassouras, state of rio de janeiro, southeastern brazil. the region is located within the domain of the atlantic forest and the vegetation is classified as semi-deciduous seasonal forest. in the municipality of vassouras, red-yellow argisols predominate and the landscape is broadly dominated by pastures (60.2%) and also by secondary forests (35.3%) (francelino et al., 2012). according to the köppen system, the climate of the region is humid mesothermal (cwa) (francelino et al., 2012), with average annual rainfall of 1,280 mm; rainfall is higher in summer than in winter. the average temperature in the coldest month may be below 18 ºc (july) and in the warmest month, above 23 ºc (february) (embrapa, 2011). to measure seed removal rate, dispersal distance, ant body size, and the guild of ants that consume seeds, we chose three secondary forest fragments of different sizes and a pasture. the smallest fragment (forest 1) had an area of 6 ha and a perimeter of 1,056 m (22° 27' 41" s; 43° 38' 57" w); the intermediate fragment (forest 2) had an area of 36 ha and a perimeter of 4,464 m (22° 27' 3" s; 43° 38' 40" w), and the largest fragment (forest 3) had an area of 780 ha and a perimeter of 37,571 m (22° 27' 11" s; 43° 39' 47" w). the pasture (pasture) (22º 27’ 30’’ s; 43º 39’ 29’’ w), used for raising bovine cattle, was covered by brachiaria decumbens stapf. and was located at approximately 200 m from the largest fragment (forest 3). the altitude in the region is approximately 600 m a.s.l. assessment of seed removal by ants to quantify the rate and distance of seed removal by ants, we used seeds of carica papaya l. (papaya) (zelikova & breed, 2008). although seed size and weight affect removal by ants (pizo & oliveira, 2001), the characteristics of the papaya seeds used in the present study can be considered intermediate within the range of native seeds used by ants in tropical forests (passos & oliveira, 2003). the average weight of fresh papaya seeds was 0.09 ± 0.01 g, the average length was 0.63 ± 0.06 cm and the average width was 0.47 ± 0.05 cm (n = 100 seeds). to avoid the introduction of papaya in the study sites, we pierced the seeds used in the experiment with a pin, making three perforations to make them unviable. to confirm that the seeds were unviable, we placed 100 pierced seeds and 100 non-pierced seeds in trays with sterile moist sand. after 60 days of observation no pierced seed germinated, but 48% of the non-pierced seeds germinated, a high value considering that no treatment to break dormancy was applied (tokuhisa et al., 2007). in october and november 2009, we installed four groups of 10 observation stations in each environment. the distance between stations was 10 m and between groups, 30 m. each station consisted of a 10 x 10 cm white paper on which we placed six papaya seeds. each station was observed for 120 min, between 07:00 and 17:00 h. the ants were monitored until they dropped the seeds; we collected the ants that carried the seeds for more than 5 cm, which was considered a removal event (zelikova & breed, 2008). whenever possible, we observed whether the seeds were taken to inside the nest or nest surroundings (at least 20 cm from the nest entrance). we placed the ants collected in identified plastic vials containing alcohol 70%. we deposited voucher specimens in the entomological collection coleção entomológica ângelo moreira da costa lima (cecl), instituto de biologia/ ufrrj, and the numbers used to refer to morphospecies are the same used in cecl. as a measurement of ant size we used head width (kaspari & weiser, 1999), measured in up to six workers of each species. next, we calculated the average size of each species. the measurements were taken in a light chamber coupled to a stereomicroscope. data analysis we described the guild of ant species that removed the experimental seeds in each study site in terms of seeds removed, removal distance, and ant body size. we classified the ant species collected in each environment by body size and counted for each size class the number of stations where the ants removed seeds. we used simple linear regressions to test for a relationship between ant size, number of seeds removed, and removal distance. in the regression between ant size and removal distance we considered only species that removed four or more seeds. we used a chi-squared test (zar, 1999) to compare the proportion of stations with at least one seed removed among the three forest environments. sociobiology 60(3): 229-235 (2013) 231 species number of seeds removed pasture forest 1 forest 2 forest 3 total pachycondyla striata fr. smith 3 11 78 92 odontomachus chelifer (latreille) 20 21 38 79 pheidole fallax mayr 37 37 ectatomma edentatum roger 14 8 10 32 pheidole radoszkowskii mayr 17 17 pheidole sp.1 7 6 13 pheidole sp.53 12 12 pheidole sp.52 11 11 solenopsis sp.12 10 10 pheidole gertrudae forel 3 1 4 pachycondyla verenae (forel) 2 2 trachymyrmex prox. oetkeri 2 2 camponotus rufipes (fabricius) 1 1 crematogaster sp.11 1 1 pheidole sensitiva borgmeier 1 1 pheidole sp.49 1 1 pheidole sp.51 1 1 total 68 53 50 145 316 number of species 6 8 5 6 17 to compare seed removal rate and average removal distance in different environments we used an analysis of variance (anova) and a tukey test. results seventeen ant species removed seeds in our experiment; pheidole was the ant genus with the largest number of species (9). we collected eight species in forest 1, six species in the pasture and in forest 3, and five species in forest 2. the ant species that removed seeds in forest fragments did not occur in the pasture and vice-versa (table 1). in total, 316 seeds were removed, which represents a removal rate of 32.9%. among the five species that most removed the experimental seeds, three occurred in forest fragments: p. striata (29.1% of all seeds removed), o. chelifer (25%), and ectatomma edentatum roger (10.1%); and two in the pasture: pheidole fallax mayr (11.7%) and pheidole radoszkowskii mayr (5.4%). 0.60; p = 0.01; figure 3). more than half of the seeds in the pasture were removed by p. fallax. for the three forest environments, the proportion of stations with at least one seed removed by e. edentatum or o. chelifer in each forest did not differ from the proportion expected by chance (e. edentatum: χ2 = 2.00; df = 2; p = 0.37 and o. chelifer: χ2 = 0.74; df = 2; p = 0.69), but p. striata was more frequent in the largest fragment (χ2 = 19.19; df = 2; p < 0.01). the average rate of seed removal differed only between the largest fragment and the other environments (tukey test, p < 0.05; figure 4). however, the average removal distance was significantly longer in forest 2 and forest 3 than in the other environments (tukey test, p < 0.05; figure 5). of all seeds removed by p. fallax, 86.5% were transported to the nest surroundings. the percentage of seeds taken to the nest surroundings was 84.4% for e. edentatum, 89.9% for o. chelifer, and 87.0% for p. striata. in the pasture and in forest 1, most stations were visited by small ants. the number of stations visited by large ants was higher in forest 3 than in the other environments (figure 1). the longest seed removal distances were observed for o. chelifer, followed by p. striata. these two species had also larger body size (table 2). larger ants removed more seeds (r2 = 0.44; p = 0.003; figure 2) and for longer distances (r2 = table 2. average and maximum removal distance (± se) of papaya seeds by each ant species, including information on average head width of ants. figure 1. number of stations containing papaya seeds where ants, classified according to head width categories, removed seeds in different environments. species removal distance (m) head width (mm) average maximum odontomachus chelifer 2.35 ± 1.17 5.20 2.77 pachycondyla striata 1.69 ± 0.93 4.31 2.67 trachymyrmex prox. oetkeri 1.25 ± 0.38 1.60 1.17 pheidole gertrudae 1.23 ± 0.37 1.60 0.61 pheidole sensitiva 1.10 ± 0.00 1.10 0.55 pheidole fallax 1.03 ± 0.07 1.60 0.53 pheidole sp.1 0.98 ± 0.08 1.25 0.50 crematogaster sp.11 0.96 ± 0.00 0.96 0.65 camponotus rufipes 0.90 ± 0.00 0.90 2.31 pachycondyla verenae 0.84 ± 0.46 1.30 1.85 pheidole radoszkowskii 0.82 ± 0.04 1.05 0.49 ectatomma edentatum 0.74 ± 0.37 1.62 1.53 pheidole sp.53 0.74 ± 0.40 1.20 0.54 pheidole sp.49 0.35 ± 0.00 0.35 0.43 pheidole sp.52 0.30 ± 0.03 0.47 0.61 pheidole sp.51 0.26 ± 0.00 0.26 0.42 solenopsis sp.12 0.12 ± 0.02 0.21 0.65 table 1. number of papaya seeds removed by ants in different environments of vassouras, rio de janeiro, southeastern brazil. fs almeida, aj mayhé-nunes, jm queiroz seed dispersal by poneromorph ants232 discussion previous studies on ant-seed interactions in forests of southeastern brazil focused mainly on large tracts of atlantic rain forest (e.g. passos & oliveira, 2002; passos & oliveira, 2004; bottcher 2006; costa et al. 2007, but see guimarães & cogni, 2002). our focus in this study was the fragments of second-growth semi-deciduous forest. the original cover of semi-deciduous forest in southeastern brazil has been drastically reduced, but the remaining or second-growth fragments still harbor high biodiversity and endangered species (ribeiro et al., 2009; dan et al., 2010). in the fragments of secondary forest monitored in the present study, the species p. striata, o. chelifer, and e. edentatum stood out in seed removal. these species are common in semi-deciduous seasonal forests (santos et al., 2006; castilho et al., 2011), as well as in other forest types of south america (antonialli jr. & giannotti, 2001; schütte et al., 2007; silva-melo, 2008) and central america (kempf, 1972). the species p. fallax and p. radoszkowskii, which removed most seeds in the pasture, occur in several habitat types in south and central america, mainly altered environments, and their interaction with seeds have already been studied by other authors (hölldobler & wilson, 1990; zelikova & breed, 2008, lobo et al., 2011). the highest seed removal rate found in forest 3 may be explained by the presence of large ants in most stations, mainly p. striata. large ants are able to carry seeds individually, whereas small ants need the help of other ants (pizo & oliveira, 1998; zelikova & breed, 2008). however, behavioral characteristics of ants should also be taken into account to assess seed dispersal (zelikova & breed, 2008). the removal distance was also longer in environments where more visits by large-bodied ants occurred. indeed there was a positive relationship between ant size and removal distance. this result contrasts with zelikova & breed (2008), who did not find a relationship between ant size and removal distance. this difference in results is related to ant behavior. ectatomma ruidum, the large species that removed most seeds in the study by zelikova & breed (2008), removes seeds at a smaller distance than pheidole fallax, a small ant. on the other hand, p. striata and o. chelifer, the largest species found in the present study, removed seeds at greater distances than p. fallax, which was found in figure 2. relationship between ant size and number of experimental seeds of caryca papaya removed. the pasture. ectatomma edentatum, which is also a large ant, removed seeds at a distance similar to e. ruidum. hence, we conclude that, although ectatomma species are large-bodied, they are not as good dispersers as p. striata and o. chelifer. the highest removal rates and distances found in the largest fragment are mainly related to a higher frequency of interactions involving p. striata. in several tropical forests of south america, the species o. chelifer and p. striata seem to play important roles in seed dispersal. in addition, the activities of o. chelifer and p. striata increase nutrient concentration in the surroundings of the nest and potentialize the recruitment of dispersed plants (passos & oliveira, 2002; bottcher, 2006). hence, these ants are ecosystem engineers (sensu jones et al., 1994) and may contribute to the maintenance of biodiversity in the forests where they occur, which is very important to threatened ecosystems, such as semi-deciduous seasonal forests (dean 2002; mma, 2007). however, we suggest that the effect of ants may depend not only on occurrence, but also on abunfigure 3. relationship between removal distance of papaya seeds (average and maximum) and head width of ant species (n = 10). note: we considered only species that removed at least four seeds. sociobiology 60(3): 229-235 (2013) 233 dance. hence, a greater effort is required to understand which mechanisms are responsible for the differences in abundance between ant species in forests. since seed removal was performed mainly by a small number of species in each environment, local extinctions or decreases in population size may decrease the seed removal rate. this may put at risk plant species that depend on zoochory for germination and establishment. taking into account that forest fragmentation influences the composition and structure of ant communities (gibb & hochuli, 2002, schoereder et al., 2004), we can also assume that it affects seed dispersal by ants too. aknowledgments we thank flavia rocha, inara leal and an anonymous referee for their valuable comments on an earlier version of the manuscript. cnpq and faperj funded our study (processes 471061/2008-5 and e-26/112.121/2008, respectively). fsa received a scholarship from capes and jmq received a scholarship from faperj (proc. e-26/101.472/2010). jacques h. c. delabie helped in ant species identification. references antonialli jr., w.f. & giannotti, e. 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(1999). biostatistical analysis. 4ed. new jersey: prentice hall. zelikova, t.j. & breed, m.d. (2008). effects of habitat disturbance on ant community composition and seed dispersal by ants in a tropical dry forest in costa rica. j. trop. ecol., 24: 309-316. doi:10.1017/s0266467408004999 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.5942sociobiology 68(4): e5942 (december, 2021) the complexity and diversity of life rely strongly on the interactions of the organisms and the specializations that result from such interactions (thompson, 1994). indeed, some interactions are so robust that at some point a phylogenetic step on one side, is closely followed by a phylogenetic step on the other side, and vice versa: a phenomenon called coevolution (janzen, 1980). this process leads to tangled trees, and relations worthy of investigation. an interesting case, yet poorly explored, is that of the association between termites and other organisms, the termitophily. the rove beetles of the subfamily aleocharinae, comprising the most diverse group of the family staphylinidae (irmler et al., 2018), is also the group that most evolved towards the termitophilic habit (parker et al., 2018), resulting in a variety of termitophilous lineages with seemingly abstract termites have a tight interaction with their social parasitic corotocini beetles. this association is thought to be mainly host-specific, despite some host-switch events. by analyzing the taxonomic partition between species and genera of corotocini, we propose the hypothesis that the main driver of the diversity of these termitophiles is coevolution. sociobiology an international journal on social insects igor eloi1, carlos m. pires-silva2, bruno zilberman2 article history edited by og desouza, ufv, brazil received 03 november 2020 initial acceptance 04 february 2021 final acceptance 29 september 2021 publication date 24 november 2021 keywords corotocini, termitophily, evolution, biodiversity scaling. corresponding author igor eloi programa de pós-graduação em ecologia e conservação universidade estadual da paraíba r. baraúnas, 351 universitário cep: 58429-500, campina grande, paraíba, brasil. e-mail: eloi.igor@yandex.com parasitic habits and extravagant adaptations (seevers, 1957). within aleocharinae, the most specialized and diverse group is the tribe corotocini (jacobson et al., 1986), which represents a mostly pantropical group with a total of 214 physogastric species sorted into 64 genera (eloi et al., 2020). scenarios of the relationship and diversification of parasites and hosts are constructed by historical events occurring along with the coevolutionary processes. these events are not always quite apparent and require investigation to ensure their presence during the codiversifications. they are often a mixture of cospeciation, host-switching, independent speciation, and lineage sorting (page, 2003). the common line of thought is that the current corotocini assemblages are the result of the inheritance of ancestral associations, especially cospeciation and independent speciation, rather than frequent 1 programa de pós-graduação em ecologia e conservação, universidade estadual da paraíba, campina grande, paraíba, brazil 2 programa de pós-graduação em sistemática, taxonomia animal e biodiversidade, museu de zoologia da universidade de são paulo, são paulo, brazil taxonomic partition suggests a high degree of coevolution between termites and their termitophiles short note mailto:eloi.igor@yandex.com igor eloi, carlos m. pires-silva, bruno zilberman – taxonomic partition clarifies corotocini diversification2 host transfers (jacobson et al., 1986). but within the tribe, we observe some host-relationship disjunctions, sometimes even between different termite subfamilies, suggesting that host-switching could very well be a reality for the tribe. here, we sought to contribute to the solution of this problem by analyzing the degree of taxonomic partition of these termitophiles. we expected the existence of two alternative scenarios: (i) the diversity of corotocini is the outcome of specialization processes in synchrony with their hosts, resulting in species arising from the same ancestral lineages; or (ii) the diversity of corotocini is mainly the result of corotocini lineages in constantly host-switching along the coevolutionary process, resulting in species arising from independent lineages. in order to test it, we sampled from the literature which termite species had corotocini tenants associated with their nests and the number of different genera and species of beetles associated with each host (data available at https:// doi.org/10.5281/zenodo.4568772). then, we fitted a log-log linear model with the number of genera (g) per host species as a response to the number of species (s) per host species and used the regression to compute the scaling coefficient (slope). this model linearization derives from the allometric power scaling (pagel & harvey, 1989) and was used by enquist et al. (2002), to test for biomass partitioning in tree assemblages and by mouillot and poulin (2004), to test for macroecological patterns in the partitioning of parasite assemblages. here we follow the latter in our logic: a slope that is closer to zero indicates that s grows faster than g, therefore suggesting that several species belong to the same genera, indicating that they share a common lineage (scenario i), while a slope that is really close to one, would indicate a similar growth rate between g and s, suggesting that each species had an independent origin, and therefore that host-switching is probably the main driver of diversification (scenario ii). the regression (fig 1) pointed out that the number of genera is moderately modulated by the number of species found in association with a host species (r2 = 0.53; p < 0.001), and the computed power function for species is low (g = s 0.45 (ci = 0.38 – 0.52)), indicating the existence of high levels of endemism in these beetle assemblages as a function of their specific hosts and highlighting that they are likely the result of cladogenesis within the same branches (see mouillot & poulin (2004) for comparison between parasites populations). our data suggest that the majority of the hosts are known to harbor only one species of termitophile, that was generally attributed to an individual genus. but 21.7% of the data is made of host species that house congeners. not coincidentally, this is the situation of the termites that are traditional biological models, such as nasutitermes corniger (motschulsky, 1855) and constrictotermes cyphergaster (silvestri, 1901), and of organisms that were well sampled over the years, such as the case of the termitogaster species associated to nasutitermes spp. or the genera of termitophiles associated with trinervitermes spp. similar scenarios are likely to increase in frequency as we acquire more information about other termite species and their termitophiles. in general, our results suggest that the composition of the termitophilic assemblages is likely the result of codiversification with specific host lineages throughout evolution rather than host-switching, with the latter possibly occurring with some periodicity (reflected by the moderate coefficient of determination r2 = 0.53). the common thought is that the relationship between termites and aleocharines started about 99mya, soon after the arising of eusociality in modern termites (cai et al., 2017a; 2017b). since the rise of the first termitophiles, several lineages of aleocharinae evolved the same lifestyle (seevers, 1957), indicating a clear case of evolvability towards termitophily, and we suggest that the results found in corotocini could potentially be generalized for other taxa of termitophilous aleocharinae, since termitophilous beetles accompanied termites throughout the major events in their evolutionary history, such as the development of truly sterile functional castes, the beginning of fungal culture, the colonization of the world, and the emergence of the higher termites and their niche occupation (bourguignon et al., 2014). during this millennial symbiosis, the termitophiles turned out to become extremely specialized, and have accumulated adaptations according to their hosts’ ecology, rather than a generalized “way of living”. we indeed often observe traits that hint at niche specialization within corotocini, demanded from these close interactions, reflected in extravagant strategies to reproduction (oliveira et al., 2018) and specific ways to acquire food from their hosts. fig 1. scatter plot between the number of genera and the number of species per host for 214 species of corotocini beetles. the tendency line was estimated using the least square linear regression. the data points were jittered due to superposition. sociobiology 68(4): e5942 (december, 2021) 3 despite the degree of synchrony between termites and their associates, we must highlight that the making of specific inferences regarding specific coevolutionary processes requires more robust methods, and needs at least phylogenetic topologies for which coevolution is hypothesized. however, they demand much more time and resources, which make them less pragmatic. the method we applied is useful in the process of raising hypotheses (gotelli, 2002), and the results can be tested in conjunction with future studies on coevolution. thus, this study shows that the biodiversity of corotocini is likely the result of an old association plus niche specialization, pointing to a testable scenario, usually through cophylogeny (page, 2003). acknowledgments we thank two anonymous referees, og de souza and paulo cristaldo for carefully reading and for giving constructive suggestions. this manuscript has benefited from discussions with mario cupello and vinicius ferreira at the coleoptera meeting (2021). the authors thank the coordenação de aperfeiçoamento de nível superior (capes) for the financial support. authors' contribution all authors contributed equally. references bourguignon, t., lo, n., cameron, s.l., šobotník, j., hayashi, y., shigenobu, s., watanabe, d., roisin, y., miura, t. & evans, t.a. 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(1994). the coevolutionary process. university of chicago press, 383. _1fob9te _bdf9vlgxyuo8 _xgeldi9ohw52 _rjq977vnh5fp _qp7fk3t93j9x _t3dwv6jl9f00 _phzxqipgmmni 407 trigona branneri (hymenoptera: apidae) as a collector of honeydew from aethalion reticulatum (hemiptera: aethalionidae) on bauhinia forficata (fabaceae: caesalpinoideae) in a brazilian savanna by gudryan jackson barônio1, ana carolina vieira pires² & camila aoki³ abstract the presence of aggregates of a. reticulatum on bauhinia has been reported, but the insects were mainly attended by ants of the genus camponotus, and stingless bees were not regularly recorded in aggregations. we observed a colony of thetreehopper a. reticulatum and stingless bees, trigona branneri, interacting on bauhinia forficata (fabaceae). agonistic behavior was observed in bees when another individual of the same species or ants approached. although this is not proof that the interaction between stingless bees and treehoppers is mutualistic, the interactions between ants and this insect are common and mutualistic. thus, if t. branneri effectively provides protection for the aphids, a new mutualism can be the focus of future research to determine if the bee-aphid interactions have same ecological functions as the ant-aphid interactions. key words: ant-aphid interaction, camponotus, trophobiosis, stingless bees, hemipterans introduction the interactions between species that coexist in time and space have an important role for biodiversity in the communities (del-claro 2004). among 1 programa de pós graduação em ecologia e conservação de recursos naturais, universidade federal de uberlândia. av. pará, s/n, uberlândia, mg, cep 38400902, instituto de biologia, campus umuarama, bloco 2d. ² laboratório de ecologia evolutiva e biodiversidade, universidade federal de minas gerais. av. presidente antônio carlos, 6627, belo horizonte, mg, cep 30161970, departamento de biologia geral, bloco i3. ³ programa de pós-graduação em ecologia e conservação, universidade federal de mato grosso do sul. av. costa e silva, s/n, campo grande, ms, cep 79070900, centro de ciências biológicas e da saúde. 1 author for correspondence: gudryan jackson barônio (gudryan@gmail.com) 408 sociobiolog y vol. 59, no. 2, 2012 the types of interactions, mutualisms are important processes for the structure and composition of communities (way 1963; stadler & dixon 2005; bascompte & jordano 2007). symbiotic interactions have been reported involving aethalion reticulatum linnaeus 1767 with wasps or ants (letourneau & choe 1987). there are other species associated with this treehopper, such as synoeca septentrionalis (vespidae) on piper aduncum (ramoni-perazzi et al. 2006), and polistes erythrocephalus on solanaceae in peru (maccarroll & reeves 2004), and even stingless bees of the genus trigona, which take advantage of treehoppers' sugary excretions (castro 1975; vieira et al. 2007; oda et al. 2009). bees of the genus trigona jurine 1807, popularly known as xupé, mombuca and arapuá, are common in the neotropics (michener 2000) and very important pollinators of the brazilian cerrado species (almeida & laroca 1988, nogueira-neto 1997, silva et al. 2007), amazon (maués & couturier 2002) and cultivated species (lorenzon et al. 1993; silva et al. 1997). some of these species have the behavior of robbing resources (nectar thiefs), negatively affecting the relationship between plants and pollinators (murphy & breed 2008; santos & absy 2010). bauhinia forficata link., belonging to the fabaceae family, generally known as pata-de-vaca, is a thorny plant of 5 to 9 meters in height, and is heliophytic, deciduous or semi-deciduous (lorenzi 2002). it is found in the northern (pernambuco, bahia, alagoas), southern (minas gerais, espírito santo, são paulo, rio de janeiro) and south (paraná, santa catarina, rio grande do sul) regions of brazil (lorenzi 2002; vaz 2010). this species is commonly used as a shade tree in urban areas (fowler 1992), which may explain their presence in other regions of the country that are not within the cerrado. the presence of a. reticulatum aggregations in bauhinia has already been observed by fowler (1992), but the insects were mainly attended by ants of the camponotus genus, and no stingless bees or wasps were regularly recorded at the aggregations. the goal of this work is show the interaction between the stingless bee trigona branneri cockreall, 1912 and the treehopper a. reticulatum. material and methods we observed interactions between a. reticulatum and t. branneri three times daily for four consecutive days in august and september 2009 in one 409 baronio, g.j. et al. — trigona branneri collecting honeydew from aethalion reticulatum individual of bauhinia forficata in parque nacional das emas, go, brazil. our observations were at 6:00, 12:00 and 18:00 hours, lasting at least 60 minutes each. the treehopper a. reticulatum is a small (< 10 mm) rusty brown sucking insect which lives in colonies made up of young wingless nymphs and winged adults (santana et al. 2005). individuals feed on by sucking the content of leaf tissues from host plants and releasing a carbohydrate-rich solution known as honeydew (gallo et al. 2002). drops of this sweet substance are consumed by ants, bees and other insects (brown 1976). through continuous sap sucking, treehoppers negatively affect the growth and development of host plants to the extent of killing the plant in the most severe cases (gallo et al. 2002; santana et al. 2005). results we detected the presence of 19 individuals of trigona banneri collecting honeydew exudates of eight individual a. reticulatum adults and 167 nymphs of the same species. on b. forficata, these stingless bees touched primarily the proximal upper abdomen of a. reticulatum with their front legs and antennas. the bees repeated the stimulus toward the distal part of the abdomen where the exudate droplet was collected with the first pair of legs and inserted into the proboscis. this behavior of the bees was recorded in both adult treehoppers and their nymphs, although there was always at least one adult on the same branch as the nymphs. the behavior was clearly observed in individuals in nymphal stages. however, the stimulus in adults across the back of the a. reticulatum abdomen was not observed with the same frequency as in the nymphs (fig. 1). in the early hours of the morning and late afternoon there were no bees around the aggregations and the presence of at most three camponotus ants patrolling on insects were observed, but without collecting honeydew. we did not find ant nests in proximity to host plant. agonistic behavior was observed in bees when touched by another individual of the same species or by very close ants. after they raised their wings partially as a warning sign, they started a short flight over the a. reticulatum colony (fig. 2). 410 sociobiolog y vol. 59, no. 2, 2012 fig. 1. antennal stimulation by t. branneri on the distal part of a. reticulatum nymphs' abdomen, next to a flower of b. forficata in parque nacional das emas, goiás, brazil (a). note the number of trigona bees on the colony of a. reticulatum (b). 411 baronio, g.j. et al. — trigona branneri collecting honeydew from aethalion reticulatum discussion t. branneri has been registered eating and storing exudates of terminalia argentea, and foraging in groups (boff et al. 2008). however, bittrich & amaral (1996) showed it as a nectar-thief in symphonia globulifera. although the interaction between the stingless bee trigona hyalinata lepeletier, 1836 and aethalion reticulatum and has already been observed (oda et al. 2009), this paper is the first record of interaction between t. branneri and a. reticulatum. in the interaction of a. reticulatum and t. spinipes on mangifera indica, an exotic fruit species very common in brazil, the meliponines excite a. reticulatum individuals by walking over them. initially the bees stay on treehoppers touching their antenna to the head of the insects and the first two pairs of legs on the back of the abdomen, after that beating the antennas on the distal part of the abdomen and quickly sucking the droplet released after stimulation (vieira et al. 2007). in the same study the researchers also observed agonistic behavior in relation to other bees or ants of the camponotus genus, although fig. 2. t. branneri making a short flight over the a. reticulatum colony. 412 sociobiolog y vol. 59, no. 2, 2012 the insects were less frequent in the period of greatest bee activity. these interactions are determining factors for both species and community (blüthgen et al. 2000; wimp & whitham 2001; styrsky & eubanks 2007). wimp & whitham (2001) showed that aphids indirectly influence community structure by reducing the diversity of arthropods found in habitats where there is a mutualism with ants. in the same study, they experimentally showed that there is an effect of predators (top-down control) well as an effect of host plants (bottom-up) in aphid abundance. the association between aphids and ants is essential for survival in view of the negative top-down effects, and protection from predators the most important service provided by ants to the aphids (wimp & whitham 2001). the authors also demonstrated that there is a dependence on ants for the establishment of aphids, as the number of aphids decreased as the distance from the nests of ants increased. although in our observations there is evidence that stingless bees could protect a. reticulatum we cannot say this with certainty, but in these kinds of interactions is common for the ants to protect the aphids in order to preserve the resource (honeydew) that is released by the aphids (wimp & whitham 2001; styrsky & eubanks 2007; blüthgen & chung 2008). we showed two interacting species possibly mutualistic, on b. forficata a host species commonly encountered in brazilian regions. thus, if t. branneri effectively provides protection for the aphids, this mutualism may be deserving of future studies to determine whether the bee-aphid interactions provide the same patterns or ecological functions as the ant-aphid interactions do (styrsky & eubanks 2007). acknowledgments we thank gabriel melo and helcio gil santana for their help in insect identification, the staff of parque nacional das emas for logistical support and estevão alves da silva for comments on the manuscript. references almeida, m.c. & s. laroca 1988. trigona spinipes (apidae, meliponinae): taxonomia, bionomia e relações tróficas em áreas restritas. acta biologica paranaense 17(14):67–108. bascompte, j. & p. jordano 2007. plant-animal mutualistic networks: the architecture of biodiversity. annual review of entomolog y 38(1):567–593. 413 baronio, g.j. et al. — trigona branneri collecting honeydew from aethalion reticulatum boff, s., g. graciolli, a.g. boaretto & m.r. marques 2008. insetos visitantes de gomas exsudadas por terminalia argentea mart & zucc (combretaceae). revista brasileira de entomologia 52(3):477–479. blüthgen, n., m. verhaagh, w. goitía, k. jaffé, w. morawetz & w. barthlott 2000. how plants shape the ant community in the amazonian rainforest canopy: the key role of extrafloral nectaries and homopteran honeydew. oecologia 125(1):229–240. blüthgen, n. & a.y.c. chung 2008. ants in a bornean rainforest: aggressive resource defence and a surprising case of tolerance. antenna 32(1):38–47. brown, r .l. 1976. behavioral obser vations on aethalion reticulatum (hemiptera, aethalionidae) and associated ants. insect sociaux 23(1):99–108. castro, p.r.c. 1975. mutualismo entre trigona spinipes (fabricius, 1793) e aethalion reticulatum (l., 1767) em cajanus indicus spreng. na presença de camponotus spp. ciência e cultura 27(1):537–539. del-claro, k. 2004. mulitrphic relationships, conditional mutualisms, and the study of interaction biodiversity in tropical savannas. neotropical entomolog y 33(6):665672 fowler, h.g. 1992. aethalionidae: functional equivalents of extrafloral nectaries in bauhinia (caesalpinoideae). anales de biologia 18(1):155–159. gallo, d., o. nakano, s. silveira neto, r.p.l carvalho, g.c. baptista, e. berti filho, j.r.p. parra, r.a. zucchi, s.b. alves, j.d. vendramim, l.c. marchini, j.r.s. lopes & c. omoto. 2002. insetos úteis. in: gallo, d. 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2005; lassau et al., 2005). in general, species diversity of terrestrial arthropod communities has been found to be positively associated with habitat complexity (lassau & hochuli, 2005 2007; lassau et al., 2005). it has been proposed that community composition is strongly influenced by differences in species pool sizes (the number of species in the species pool) and hence by factors that vary at broad spatial scales and trickle down to local scales (kraft et al., 2011). such factors include habitat area, the evolutionary history of lineages and regions (whittaker et al. 2001; kraft et al., 2011; lessard et al., 2012), the cumulative effects of stochastic variation or sampling constraints (chao et al., 2006; tuomisto, 2010, a;b). orchid bee communities have been widely sampled in different neotropical ecosystems in recent decades, however, few abstract here we investigated responses of orchid bee assemblages to habitat complexity, with the aim of assessing complexity as a useful surrogate for species diversity of this group. for the purposes of our study, we defined habitat complexity as the heterogeneity in the arrangement in physical structure of habitat (vegetation), although there is a large range of operational definitions in the literature. we test the following hypotheses: (i) there is a greater species richness and abundance of orchid bee in sites with high habitat complexity than lower habitat complexity; (ii) high habitat complexity sites have a different species composition of orchid bee than lower habitat complexity sites. in fact, orchid bee species richness was higher in high complexity areas while community composition was not affected by habitat complexity, due to the effect of dominant species. habitat complexity, measured as a function of differences in multiple strata in forests, may be of great worth as a surrogate for the diversity of a range of arthropod groups including orchid bees. sociobiology an international journal on social insects y antonini1, ra silveira1, ml oliveira2, c martins1, r oliveira1 article history edited by evandro nascimento silva, uefs, brazil received 06 april 2016 initial acceptance 17 may 2016 final acceptance 29 may 2016 publication date 15 july 2016 keywords orchid bee assemblage, habitat complexity, species composition. corresponding author yasmine antonini laboratório de biodiversidade universidade federal de ouro preto campus morro do cruzeiro, s/n 35400-000, ouro preto-mg, brazil e-mail: antonini.y@gmail.com included the savanna (cerrados) of central brazil (nemésio & faria, 2004; alvarenga et al., 2007; faria & silveira, 2011; viotii et al., 2013). these studies have shown structural differences in bee communities from distinct biogeographical regions, particularly in relation to composition, richness and patterns of dominance. usually the differences have been attributed mainly to historical factors, although regional differences in community structure at less encompassing spatial scales can be analyzed based on current ecological characteristics related to climatic, geomorphological, and/or vegetational parameters (sydney et al., 2010; nemésio & vasconcelos, 2013) recently, nemésio and vasconcelos (2013) evaluated the beta diversity of euglossine in the atlantic forest and noted that climate variations explain twice as much variation in the species data than the spatial variation in species distribution. nevertheless, part of the observed latitudinal changes in community composition appears to be explained by a concomitant seasonal gradient of precipitation. similarly, low temperatures and a seasonal rainfall may help explain the 1universidade federal de ouro preto, ouro preto-mg, brazil 2instituto nacional de pesquisas da amazônia (inpa), manaus-am, brazil research article bees y antonini et al. – orchid bee and habitat complexity820 relative specificity of the fauna of some of the most western atlantic forest. orchid bee seems to be an excellent taxon for examining species composition changes in low and high complexity habitats. first, relatively small forest patches, even in urban areas, can sustain viable populations of at least some species of these insects (bezerra & martins, 2001; storti et al., 2013). second, as vagile, long-flighted organisms (wikelski et al., 2010), orchid bees are able to fly many kilometers daily in search for food and other resources (wikelski et al., 2010), what theoretically presumes a relatively high ability to colonize different kinds of environments and finally, their males are easily attracted to synthetic compounds that mimic floral fragrances, making field studies easy to conduct. since a greater distance can be covered by air for an equal energy cost, we expect orchid bee species to be highly ‘spatially mobile’, and the effects of habitat complexity (if any) to be less pronounced (chust et al., 2004). to our knowledge, there have been no comparative studies describing orchid bee community patterns in relation to habitat complexity on a landscape scale. our aim was to assess differences in orchid bee assemblages between low and high complexity habitats, within the same vegetation type, considering that habitat complexity may affect orchid bee fauna at various levels of vegetation strata (aguiar et al., 2014). in this study, we test the following hypotheses: (i) there is a greater species richness and abundance of orchid bee in sites with high habitat complexity than low habitat complexity; (ii) high habitat complexity sites have a different species composition of orchid bee than low habitat complexity sites. for the purposes of our study, we defined habitat complexity as the heterogeneity in the arrangement in physical structure of habitat (vegetation), although there are a large range of operational definitions in the literature. methods study area the vegetation of espinhaço mountain range is a mosaic of savanna vegetation, patches of semideciduous atlantic forest and outcrop fields (menezes & giulietti, 2000). for the study we selected 15 sampling sites, in this “mosaic” located in the conservation unity “parque estadual do rio preto” (perpreto), in the municipality of são gonçalo do rio preto, minas gerais, brazil (6758200 e-7990420 s) (figure 1). the climate in perpreto is classified as cwa köppen (alvares et al., 2013), with dry winter and hot summer (with an average total annual rainfall of approximately 1350 mm). during the study year, the total precipitation was 1300 mm, with temperatures varying from 18º c to 20º c. sampling male euglossine bees were sampled once a month from july, 2011 to july, 2012. at each collecting day, fragrance baits (methyl cinnamate, vanillin, cineole, benzyl acetate, methyl salicylate, beta-ionone and eugenol) were exposed from 08:00 a.m. to 03:00 p.m. , using traps following bezerra and martins (2001) totalizing 252 sampling hours (seven traps by each sampling plot) were placed 1.5 m from the fig 1. map showing collecting localities of euglossine bee at the parque estadual do rio preto, são gonçalo do rio preto municipality, state of minas gerais, southeastern of brazil. source of map: google maps pro. sociobiology 63(2): 819-825 (june, 2016) 821 ground and 2 m apart each other. sampling was consistently undertaken on sunny days, and never during periods of atypical low temperatures. orchid bees were identified by márcio oliveira. all voucher specimens are deposited in the entomological collection of the laboratory of biodiversity at the federal university of ouro preto and in the collection of invertebrates of instituto de pesquisas da amazonia (inpa). each sampling area was selected in habitats of riparian forest, savannah and outcrop fields so that it contained high and low complexity plots. this resulted in 15 sampling plots in 3 areas over the perpreto. the minimum distance between each sampling point in each sampling sites was 500 m and the the distance between each sampling site was of approximately 5 km. each of the 15 plots was characterized for habitat complexity (in the end of the sampling period), using scores between 0 and 3 for three habitat variables (table 1). this is a modified version of the technique used by coops and catling (1997) and lassau and hochuli (2005). the scoring of sampling points, in each sampling site, results in 6 plots being categorized as low complexity (lch), after scoring 2-5 (of a total of 9), and 9 as high complexity (hch), scoring 6 or greater on the habitat complexity scores. the differences in habitat complexity did not appear to reflect a different successional state, but rather the patchy mosaic of different physiognomies. we assessed differences in composition of orchid bee assemblages between areas of low and high habitat complexity using permanova. we constructed a braycurtis dissimilarity matrix of the data from our orchid species using a fourth root transformation to allow a more equal contribution of rare species (clarke, 1993). non-standardized data were used, since throughout the study all collection sites were treated with equal importance. the differences in the abundance of the genus of orchid bee fauna in low and high complexity habitats were accessed using a t test. results a total of 1,833 male euglossine bees were collected, belonging to tree genera and 12 species. euglossa melanotricha and e. leucotricha were the dominant species, representing more than 60% of the individuals collected (table 2). in the hch habitat 1,276 individuals belonging to 11 species were collected, while 557 individuals belonging to 12 species were collected in the lch (table 2). structure score 0 1 2 3 tree canopy (% cover) 0 <30 30-70 >70 shrub canopy 0 <30 30-70 >70 ground flora (height in m) sparse* (<0,5 m) sparse (>0.5m) dense** (<0.5m) dense (>0.5m) *sparse ground flora refers to grasses covering < 50% of a study site; ** dense ground flora refers to grasses covering > 50% of a study site. table 1. visual method for scoring habitat complexity (modified from coops and catling 1997). statistical comparisons we examined differences in the species richness and abundance of orchid bees at high and low habitat complexity sites using single-factor analysis of variance (anova). we created individual-based rarefaction curves within each treatment using estimates (colwell et al., 2012). rarefaction was used to ensure that any responses we detected were not a product of sampling bias (krebs, 1989), caused by trapping methods in habitat with varying structure (melbourne, 1999). correlations between orchid bee species richness and individual habitat variable scores were tested using spearman rank correlation (src). we also tested correlations between habitat variables using srcs. orchid bee species hch lch euglossa melanotricha 439 162 euglossa leucotricha 175 106 euglossa securigera 142 83 eulaema nigrita 100 72 euglossa imperialis 120 44 eulaema cingulata 126 13 euglossa fimbriata 80 58 eufriesea nigrohirta 63 2 euglossa truncata 13 12 euglossa annectans 13 1 eufriesea auriceps 0 2 euglossa violaceifrons 5 2 table 2. abundance of orchid bee species in sampling sites with high complexity (hch) and low complexity (lch) at perpreto, mg, brazil. effects of habitat complexity on orchid bee assemblage the species richness and abundance of orchid bee were higher in more complex habitat types (anova f1,14= 33.01, p < 0.001) however, the species abundance was not significantly different (figure 2). average abundance was higher in more complex habitat types only for euglossa species (t = 7.25, p < 0.001) (figure 3). individual based rarefaction curves suggest that the orchid bee species richness may be higher in high complexity habitats (figure 4). the species composition of orchid bee fauna was not different in habitats of high and low complexity (permanova r = 0.27, p = <0.2). y antonini et al. – orchid bee and habitat complexity822 the species richness of orchid bees was positively associated to the total score of habitat variables (spearman rho = 0.62, n = 15, p = 0.012). the species richness of orchid bees was positively associated with the shrub canopy cover (spearman rho = 0.45, n = 15, p = 0.034), and ground flora (spearman rho = 0.47, n = 15, p = 0.042). among habitat variables, tree canopy cover was negatively associated with ground flora (spearman rho = -0.43, n = 15, p < 0.001) and shrub canopy cover negatively associated with ground herb cover (spearman rho = -0.40, n = 15, p < 0.05). discussion the results of the present study, plus those obtained by viotti et al. (2013), justino and augusto (2012) faria and silveira (2011), alvarenga et al. 2007 and nemésio and faria (2004), in other areas of savanna (cerrado) in minas gerais state, revealed a total of 16 orchid bee species for this region. so far there is no list for the orchid bee fauna in the cerrado of minas gerais. considering the result of the rarefaction curves and richness estimators, the orchid bee fauna in cerrado areas was underestimated and the real number of species may be higher. the species richness in parque do rio preto is higher to those found in other inventories carried out in the cerrado domain (e.g nemésio & faria, 2004; alvarenga et al., 2007; justino & augusto, 2010; faria & silveira, 2011; viotti et al., 2013) and in fragments of the semideciduous “low mountain rain forest”, at elevations of 300-900 m, in the atlantic forest domain (rebêlo & garófalo, 1997; sofia & suzuki, 2004; nemésio & silveira, 2010; silveira et al., 2011; ferreira et al., 2013). these forests are characterized by relatively open canopy, 15-25 m tall, and occur under climates with two well defined seasons, rainy and dry. this suggests that the species richness of local faunas in the cerrado is comparable to those more complex forests, in the so-called “inland forests” or semideciduous forests within the atlantic forest domain. such species richness, however, are much smaller than those found in coastal atlantic forest sites (perennial forests) in southeastern brazil (e.g. tonhasca-jr. et al., 2002; nemésio & vasconcelos, 2013). despite faria and silveira (2011) explain the low richness and abundance of orchid bees in cerrado areas, as a result from relative food scarcity, which, in turn, would be a consequence of a combination of low soil fertility and relatively low seasonal precipitation, our results are going in an opposite way. the cerrado is a very rich floristic region with many different habitat types (joly et al., 1999) and that habitat heterogeneity may provide the necessary resources for orchid bee species. it should be considered that the orchid fig 2. orchid bee species richness (a) and abundance (b) in high and low complexity habitats. (±se) ** anova p<0.05. fig 4. rarefaction curves of orchid bee fauna sampled in high (hch) and low (lch) complexity habitats perpreto, mg, brazil. fig 3. orchid bee genus abudance in high and low complexity habitats (±se) sociobiology 63(2): 819-825 (june, 2016) 823 bees present in the cerrado domain are able to explore both high and low complexity habitats (the savannic and the forest environments). it is important to notice that the species composition of orchid bee, in our study, was not different in habitats of high and low complexity. it is possible, then, that orchid bees vague through both environments, exploiting whatever resources they can find in each of them. for example, riparian forests would be important for orchid bees, offering them nesting sites, food resources and protection against intense solar radiation and winds (neves & viana 1999, wikelski et al., 2010). habitat complexity the higher average species richness in more complex habitat was expected and is in agreement with our findings since the individual rarefaction curves shown that the expected richness of hch was always higher than that in lch. nevertheless, for abundance, the result was the opposite since the “more common” orchid bee species (e. melanotricha and e. leucotricha) present higher abundance both in high and less complex habitat types. shrub canopy cover and ground flora cover all had significant positive associations with species richness of orchid bees. the interpretation of this result is somewhat complicated by the negatively correlations between tree canopy cover and ground flora and shrub canopy cover negatively associated with ground herb cover. for the three genera of orchid bee, habitat complexity may not be important in determining bee richness but abundance. for the most rich genera – euglossa – there was no difference in richness but the abundance. it should be notice that the abundance of the three more abundant euglossa species (e. melanotricha, e. leucotricha and e. securigera) was also higher in sites with lch when compared with the other species. for e. melanotricha and e. leucotricha, previous studies had demonstrated that these species are much abundant in mosaic of cerrado and outcrop fields as well as in a mosaic of cerrado and eucalyptus plantation (see faria and silveira, 2011; viotti et al., 2013). it should also be considered that mosaic of physiognomies may be favoring the occurrence of orchid bees, as demonstrated by aguiar et al. (2014). recently, nemésio and vasconcelos (2013) evaluated the beta diversity of orchid bees in the atlantic forest and noted that climate variations explain twice as much variation in the species data than the spatial (latitudinal) variation in species distribution. nevertheless, part of the observed latitudinal changes in com munity composition appears to be explained by a concomitant seasonal gradient of precipitation. overall, our results agree with a growing body of evidence (e.g. myster, 2009; sydney et al., 2010; vasconcelos et al., 2010) showing that environmental gradients affect the turnover of animal and plant species in tropical forests more strongly than geographic gradients. our study reinforces the importance of habitat complexity to preserve high local and regional species richness, as the composition of orchid bees communities, as well as their patterns of abundance and species dominance have been found to differ among habitat with high and low complexity. the mechanisms driving associations between habitat complexity and patterns in orchid bee communities may also provide a basis for maintenance of ecological services and the genetic diversity of their host plant populations, and attention should be given to studies focusing on these points. acknowledgements we thank capes and fapemig fellowships to the first and last author. cnpq for the pq scholarships to ya capes for pd scholarships to ro. ief, antonio tonhão de almeida and parque do rio preto staff, for logistic support. erik wild for english revision. references aguiar, w.m., melo, g.a.r. & gaglianone, m.c., (2014). does forest physiognomy affect the structure of orchid bee (hymenoptera, apidae, euglossini) communities? a study in the atlantic forest of rio de janeiro state, brazil. sociobiology, 61: 68–77. alvarenga, p.e.f., freitas, r.f. & augusto, s.c. 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(2010). large-range movements of neotropical orchid bees observed via radio telemetry. plos one. doi: 10.1371/ journal.pone.0010738 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 69-76 (2013) utility of acoustical detection of coptotermes formosanus (isoptera: rhinotermitidae) w. osbrink,1 m. cornelius2 introduction total economic loss due to termites in the united states has been estimated at $11 billion per year, and where they occur, the formosan termite, coptotermes formosanus shiraki (fst), is the most devastating termite pest in the world (su, 2002). the fst was introduced into the united states from asia when troops and equipment returned from world war ii (su & tamashiro, 1987). in addition to structural infestations, c. formosanus infestations of living trees are common (osbrink et al., 1999; osbrink & lax, 2002; ring et al., 2002; osbrink & lax, 2003). development of techniques for detecting hidden termite infestations have produced only a few successful alternatives to traditional visual inspection methods (lewis, 1997). efficient non-invasive detection of termite activity can provide timely location of an infestation thereby reducing economic impact. non-invasive detection is also ideal for evaluating the efficacy of control efforts because noninvasive monitoring has no effect on population dynamics. abstract the aed 2000 and 2010 are extremely sensitive listening devices which can effectively detect and monitor termite activity through a wave guide (e.g. bolt) both qualitatively and quantitatively. experiments conducted with one to ten thousand termites from differing colonies infesting wood in buckets demonstrated that acoustical emission detector readings significantly increased when number of termites increased. termites were also detected in infested trees with the installation of several wave guides into their trunks. these devices can detect termites and changes in termite activity caused by changes in termite numbers, making it an effective pest management professional and research tool for finding and evaluating termite infestations and efficacy of treatments in specific locations. sociobiology an international journal on social insects 1 usda-ars-spa, kerrville, texas, usa. 2 usda-ars-barc, beltsville, maryland, usa. research article termites article history edited by: evandro n. silva, uefs brazil received 29 november 2012 initial acceptance 02 january 2013 final acceptance 14 february 2013 keywords aed 2000, aed 2010, formosan subterranean termite, tree, monitoring, pest control corresponding author weste osbrink usda-ars-spa knipling-bushland u.s. livestock insects research lab 2700 fredericksburg road, kerrville, texas 78028 phone: 504.330.41326 e-mail: weste.osbrink@ars.usda.gov conversely, invasive monitoring techniques can drive termites away from the monitor, creating an artifact of apparent control because of relocation of the termites (aluko & husseneder, 2007). alternatives to visual inspection include monitoring devices with sensors that detect acoustic emissions of termites in wood (fujii et al., 1990; lewis & lemaster, 1991; noguchi et al., 1991; robbins et al., 1991). acoustic emission sensors are successful because they are nondestructive and operate at high frequencies (ca. 40 khz) where there is negligible background noise to interfere with detection and interpretation of insect sounds (lewis & lemaster, 1991; robbins et al., 1991). acoustic emission systems have been applied as research tools to estimate termite population levels (fujii et al., 1990, lewis & lemaster, 1991; scheffrahn et al., 1993; osbrink et al., 2011). acoustic emission systems are also ideal for detection of termites in trees (osbrink et al., 1999; kramer, 2001; mankin et al., 2002; osbrink et al., 2011). understanding the efficacy and dynamics of acoustical detection is critical to it being successfully integrated w. osbrink, m. cornelius acoustical detection of coptotermes formosanus70 into an effective pest management strategy. the central objective of this research was to determine the efficacy of using the aed 2000 acoustical emissions detector (acoustical emissions consulting, inc fair oaks, ca) to detect and quantify termite infestations. to meet this objective, studies were conducted to monitor c. formosanus through acoustical emission detection both in the laboratory and in trees outdoors. these studies provide evidence that aed significant potential for application in termite management efforts. materials and methods acoustical emission detector (aed) an aed 2000 acoustical emissions detector (acoustical emissions consulting, inc fair oaks, ca) was used to quantify termite activity. lag bolt wave guides (76.2 or 150 x 9 mm) were screwed horizontally into pre-drilled pilot holes in wood substrates. acoustical emissions were detected with a sensor probe (model sp-1l with model dmh-30 high force magnetic accessory attachment, acoustic emission consulting, inc). aed counts were acquired for 60 s with accompanying software, which converts termite sounds to counts per second and enters them into excel (microsoft, redmond wa). only the numbers of counts in the first 10 s of the 60 s recording were used to represent each unique individual recording. if the first 10 s of recording was contaminated with interference noise (elevated spiked counts), the first 10 s of recording following the cessation of interference noise were used to represent the unique individual recording. comparisons also were made between the aed 2000 and the more recently manufactured aed 2010 (acoustical emissions consulting, inc fair oaks, ca). laboratory bucket tests a vertically oriented section of spruce (picea sp.) 38 x 89 mm (2x4 inch) dimensional lumber 17 cm in length was attached to the inner side of a lidded plastic buckets (3.79 l, 18.5 cm height x 20 cm diam.) with 2 horizontally applied drywall screws (high and low) and central lag bolt wave guide (76.2 x 9 mm). the head of the lag bolt was accessible from the outside of the lidded bucket (fig. 1). the bucket was filled to within 4 cm of top with a moist (≈ 20% water wt/wt) mixture of sand and vermiculite (50:50 by volume). ten holes were created in the sand-vermiculite substrate with a 5 ml pipette to increase surface area and accelerate acclimatization of termites. four buckets were prepared for each of 4 termite densities (0, 1,000, 5,000, and 10,000 termites per bucket), in which each density level represents 4 distinct colonies (a, b, c, and d), one colony per bucket. in laboratory bucket tests there were 4 replicates (bucket a, b, c, d), each replicate consisting of a 10 s recording from a specific bucket. termites were obtained from bucket trap monitors (su & scheffrahn, 1986) and termite numbers determined by weight. soldier proportions were about 10%, unchanged from when collected. termite density response at 7 and 14 d. formosan termites were placed in buckets on day 0 (0 d) as described above. buckets were held in the laboratory (≈ 26.7º c). on 7 d and 14 d aed readings were taken from each bucket. in laboratory bucket tests there were 4 replicates (bucket a, b, c, d), each replicate consisting of a 10 s recording from a specific bucket. termite density response at three temperatures after completion of readings at 14 d for dose response at 1 temperature, buckets were placed in 3 incubators stabilized at 15, 20, and 25º c, respectively, and evaluated according to the schedule indicated in table 1. after readings, buckets were rotated to a new temperature (incubator) and allowed 24 h to acclimate before acoustic readings were again taken. incubators space limitations required the d samples to be split to fit the 12 buckets into three incubators. in laboratory bucket tests there were 4 replicates (bucket a, b, c, d), each replicate consisting of a 10 s recording from a specific bucket. disturbance test aed recordings were taken before and after the application of three sharp strikes with a screwdriver to the high density laboratory buckets. in laboratory bucket tests there were 4 replicates (bucket a, b, c, d), each replicate consisting of a 10 s recording from a specific bucket. field test on trees nine wave guides in the form of lag bolts (150 x 9 mm) were screwed horizontally into pre-drilled pilot holes in the trunk of test trees facing north, east, south, and west (fig 1). four wave guides were installed at ground level, four at 20 cm above ground level, and one into the east side of the trunk at a height of approximately 122 cm from the ground. test trees consisted of four southern live oak trees (quercus virginiana philip miller) with a diameter at breast height (dbh) of ≈ 90 cm, adjacent to su-bucket-trap-monitors active with c. formosanus (su & scheffrahn 1986) located on the city park campus of the southern regional research center, new orleans, la. in field tests on trees, only the numbers of counts in the first 10 s of the 60 s recording were used to represent each unique individual recording. if the first 10 s of recording was contaminated with interference noise (elevated spiked counts), the first 10 s of recording sociobiology 60(1): 69-76 (2013) 71 following the cessation of interference noise were used to represent the unique individual recording. in field tests on trees, ten consecutive counts (10 s) were used to calculate mean (± se) counts per second to quantify termite activity associated with each unique aed tree bolt attachment. comparison of aed 2000 with aed 2010. eight different recordings from trees were conducted with each model of acoustical emissions detector and results were compared between the aed 2000 and the aed 2010. in field tests on trees, only the numbers of counts in the first 10 s of the 60 s recording were used to represent each unique individual recording. if the first 10 s of recording was contaminated with interference noise (elevated spiked counts), the first 10 s of recording following the cessation of interference noise were used to represent the unique individual recording. in field tests on trees, ten consecutive counts (10 s) were used to calculate mean (± se) counts per second to quantify termite activity associated with each unique aed tree bolt attachment. data analysis. ten consecutive count values (10 s) were used to represent termite activity associated with each unique aed attachment. in laboratory bucket tests there were 4 replicates (bucket a, b, c, d), each replicate consisting of a 10 s recording from a specific bucket. in field tests on trees, ten consecutive counts (10 s) were used to calculate mean (± se) counts per second to quantify termite activity associated with each unique aed tree bolt attachment. acoustical data were analyzed using one way analysis of variance (anova) with means separated using the protected tukey test, p < 0.05 (systat, 2008). results termite density response at 7 and 14 d. buckets with no termites produced aed readings of zero (control). there were highly significant differences in termite activity between termite colonies, and acoustical emission activity increased concomitantly with increased termite density (table 2). the highest density always had significantly greater activity than the lowest. overall, there was no consistent change in termite acuity between 7 d and 14 d, however at the lowest density there was non-significant but numerically consistent increase in activity (table 2). termite density response at three temperatures. buckets with no termites produced aed readings of zero (control). at low termite density, there was no significant difference in inter-colony activity at all 3 temp (table 3), but there was a significant increase in termite activity at the highest density with two colonies and combined colonies (table 4). at 20 and 25º c there was always a significant activity dose response except with colony d which did not statistically but did numerically separate 5k from 10k (table 3). combined colonies demonstrated highly significant density dose response at all temps. at the highest density there was always significantly less termite activity at the lowest temp (table 4). at lower density this temperature separation was not as clearly defined. disturbance test three of the four colonies displayed a significant decrease in termite activity, and one colony had a numerical but non-significant increase in recorded activity (table 5). qualitatively, termite activity could be heard though the earphones to increase for a brief time before the recording began. field test on trees out of the nine bolts per tree, generally only one or two had significantly high termite activity, with the remainder of the bolts displaying low termite activity (table 6). comparison of aed 2000 with aed 2010 of eight different recordings of trees, there was little difference observed between the aed 2000 and aed 2010. the aed 2010 had consistently higher readings that may indicate that it may be slightly more sensitive (table 7). table 1. incubator temperature and rotation of termite densities. temp (º c) colony # termites 10 15 20 25 0 1000 a 5000 day1 day 2 day 3 10000 0 1000 b 5000 day 3 day 1 day 2 10000 0 1000 c 5000 day 2 day 3 day 1 10000 0 d day 1 day 2 day 3 1000 5000 d day 3 day1 day 2 10000 w. osbrink, m. cornelius acoustical detection of coptotermes formosanus72 discussion termite density response at 7 and 14 d. having highly significant differences in termite activity between colonies is consistent with the generally accepted understanding that there can be profound inter-colony differences. these findings support the suggestion of su and la fage (1984) to use multiple colonies when conducting bioassays. a possible explanation of the non-significant but numerically consistent increase in activity at the lowest density is that it takes longer for fewer termites to create a gallery system in the wood. increased size of galleries increases the surface area occupied by termites creating an opportunity for increased generation of acoustical emission. termite density response at three temperatures. dose responses to density and temperature were demonstrated most clearly with the combined colony data due to the increased number of samples. these results demonstrate the efficacy of using an acoustical emission detector to detect and monitor termite activity. because there were significant differences in the aed readings based on termite density, the detector can be useful not only in detecting the presence of termites but also in estimating population density in infested trees or structures. disturbance test. though a post-disturbance decrease in activity occurred, a substantial amount of termite activity remained (table 5). qualitatively, earphone monitoring indicated an immediate, brief increase in termite sounds in all instances that is consistent with absconding. unpublished video has shown fst to cease feeding and abscond following a disturbance, and soldiers (incapable of chewing wood) produce characteristic termite sounds monitored with the aed 2000 (wo personal observation). additionally, the presence of red imported fire ant colonies, solenopsis invicta buren, at tree study sites have produced sounds similar to termites (wo personal observation). thus, results indicate that aed 2000 recordings are created by termite movement and not feeding activity, possibly a result tarsal claw-substrate interaction. this is inconsistent with reports of scheffrahn et al. (1993) and fujii et al. (1990) who attribute signals detected by their devices specifically to termite feeding. this difference in interpretation of results may reflect differences in the nature of the disturbance or in the specifics of the detection mechanism. field test on trees. of the nine bolts per tree, generally only one or two transmitted high termite activity while the remainder of the bolts displayed low termite activity (table 6). energy attenuates much more rapidly horizontally across the trunk than vertically up and down the trunk (mankin et al., 2002), suggesting that termite activity is oriented vertical to the bolt. thus, a single bolt, or readings from a single point cannot determine that a tree is not infested with termites. limitations of aed 2000 and aed 2010. certain events can interfere with successful recording of termite activity including wind noise, trucks with squeaking breaks, generators, crowd noise, etc. wind speeds > 14 km/h interfere with recording activity in trees because of leaf flutter, and excel recordings do not distinguish termite events from unrelated sound events, therefore maintaining a log with qualitative notes is advised. elevated wind can be a common cause for cancellation of field tests, and demands flexibility in scheduling. radio interference can also become an issue that may be mitigated by incorporating ferrite chokes and the shortest cord possible. in conclusion, the aed 2000 and 2010 are extremely sensitive devises which can detect termite activity channeled through a wave guide. because of the significant increases in aed readings with increasing group size, a trained pest management professional would be able to use the acoustical detectors to estimate the severity of an infestation, in addition to merely determining the presence or absence of termites. use of this technology may be quite valuable in specific applications such as preand post-treatment evaluations of termite activity. in applications where multiple locations are to be evaluated, interference from external noise can become an issue. acknowledgements we thank r. davey, k. lohmeyer, j.m. pound, and s. skoda for agreeing to review this manuscript and valuable improvements contributed by their reviews. references aluko, g. & husseneder, c. 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(1984) differences in survival and feeding activity among colonies of the formosan subterranean termite (isoptera: rhinotermitidae). z. angew. entomol., 94: 134-138. su, n.-y. & scheffrahn, r. (1986) a method to access, trap, and monitor field populations of the formosan subterranean termite (isoptera: rhinotermitidae) in the urban environment. sociobiology, 12: 299-304. su, n.-y. & tamashiro, m. (1987) an overview of the formosan subterranean termite (isoptera: rhinotermitidae) in the world. in: m. tamashiro & n.-y. su (eds.), biology and figure 1. aed 2000 attached to bucket and tree. w. osbrink, m. cornelius acoustical detection of coptotermes formosanus74 control of the formosan subterranean termite. hawaii institute of tropical agriculture and human resources research extension series 083 (pp. 3-15). honolulu, hi: university of hawaii and manoa. systat software. (2008) sigmaplot users guide: statistics, version 11. systat software, inc. san jose, ca. table 2. acoustical emission dose response (mean ± se) by termites (10 s). number termites (x1,000) 7 d 14 d _____________________________________ _______________________________________ colony 1 5 10 1 5 10 a 23.8 ± 4.4cb 189.9 ±25.5 aa 235.6 ± 12.3ab 65.8 ± 1.3ca 105.0 ± 8.9bc 129.5 ± 12.4bb f = 34.703 df = 5, 59 p < 0.001 b 13.3 ± 2.7cbc 96.0 ± 11.9bb 146.8 ± 29.9bc 24.0 ± 3.0cb 152.7 ± 14.2aba 210.8 ± 14.7ac f = 26.757 df = 5, 59 p < 0.001 c 6.5 ± 1.9dc 39.1 ± 7.4cdb 124.0 ± 16.2bc 19.7 ± 2.6db 56.9 ± 7.3cb 176.0 ± 16.4abc f = 40.423 df = 5, 59 p < 0.001 d 46.4 ± 4.1da 166.0 ± 9.5ca 744.3 ± 23.4aa 60.7 ± 6.7da 149.7 ± 16.0cac 320.2 ± 29.1ba f = 229.179 df = 5, 59 p < 0.001 f=25.790 f=20.158 f=195.680 f=36.223 f=13.753 f=17.787 df=3.39 df=3.39 df=3.39 df=3.39 df=3.39 df=3.39 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 means within a row (lower case) or column (upper case.) with same letter are not significantly different, protected tukey test (p > 0.05). table 3. varied temperature with aed dose response (mean ± se) by termites (10 s) . colony temp. # termites (º c) (x1,000) a b c d combined 15 1000 1.5 ± 0.5ab 3.2 ± 0.7ab 2.9 ± 0.8ab 2.9 ± 0.9ab 2.6 ± 0.4ac f = 0.808 df = 4, 79 p = 0.524 5000 17.4 ± 2.8abac 28.0 ± 4.6aac 2.7 ± 0.6cb 10.4 ± 2.7bcb 14.6 ± 2.1bb f = 6.463 df = 4, 79 p < 0.001 10000 15.5 ± 3.4bc 18.4 ± 2.3bc 34.3 ± 6.0aba 57.3 ± 6.0aa 31.3 ± 3.5ba f = 7.678 df = 4, 79 p < 0.001 f = 11.7 f = 17.418 f = 26.681 f = 59.908 f = 37.647 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 119 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 20 1000 3.0 ± 1.2ac 2.1 ± 0.7ac 1.1 ± 0.4ac 2.2 ± 0.4ab 2.1 ± 0.4ac f = 0.767 df = 4, 79 p = 0.550 5000 25.2 ± 6.1abb 28.1 ± 5.1abb 23.9 ± 4.1bb 51.1 ± 9.4aa 32.1 ± 3.6abb f = 2.623 df = 4, 79 p = 0.041 10000 45.3 ± 5.4aa 57.8 ± 6.2aa 54.1 ± 4.9aa 63.9 ± 7.6aa 55.3 ± 3.1aa f = 1.204 df = 4, 79 p = 0.316 f = 19.643 f = 36.039 f = 57.422 f = 21.872 f = 93.620 df=2.29 df=2.29 df=2.29 df=2.29 df=2.29 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 25 1000 17.0 ± 5.2ac 2.4 ± 0.6ac 14.1 ± 3.9ac 5.8 ± 1.7ac 13.5 ± 2.2ac f = 2.751 df = 4, 79 p = 0.034 5000 53.4 ± 4.6ab 42.7 ± 5.6abb 30.9 ± 4.3bb 32.3 ± 4.5bb 42.5 ± 2.8abb f = 3.195 df = 4, 79 p = 0.018 10000 159.3 ± 12.9aa 65.9 ± 6.2bca 69.7 ± 4.9bca 46.3 ± 3.3ca 108.7 ± 9.4ba f = 10.226 df = 4, 79 p < 0.001 f = 76.815 f = 44.548 f = 42.584 f = 37.628 f = 70.673 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 means within a row (lower case) or column (upper case) with same letter are not significantly different, protected tukey test (p > 0.05). sociobiology 60(1): 69-76 (2013) 75 table 4. temperature and termite density; mean (± se) number of aed counts (10 s). colony temp. number termites ________________________________________________________________ (º c) (x1,000) a b c d combined 15 10 15.5 ± 3.3c 18.4 ± 2.3b 34.3 ± 6.0b 57.3 ± 6.0a 31.4 ± 3.5c 20 10 45.3 ± 5.4b 57.8 ± 6.2a 57.8 ± 5.1a 63.9 ± 7.6a 55.3 ± 3.1b 25 10 159.3 ± 12.9a 65.9 ± 6.2a 69.7 ± 4.9a 46.3 ± 3.3a 108.7 ± 9.4a f = 83.805 f = 23.678 f = 26.681 f = 2.290 f = 42.547 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 119 p < 0.001 p < 0.001 p < 0.001 p = 0.121 p < 0.001 15 5 17.4 ± 2.8a 28.0 ± 4.6a 2.7 ± 0.6b 10.4 ± 2.7b 14.6 ± 2.1c 20 5 3.0 ± 1.2b 28.1 ± 5.1a 23.9 ± 4.1a 51.1 ± 9.4a 32.1± 3.6b 25 5 17.0 ± 5.2a 42.7 ± 5.6a 30.9 ± 4.3a 32.3 ± 4.5a 42.5 ± 2.8a f = 5.566 f = 2.754 f = 18.182 f = 10.796 f = 23.928 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 119 p = 0.009 p = 0.082 p < 0.001 p < 0.001 p < 0.001 15 1 1.5 ± 0.5b 3.2 ± 0.7a 2.9 ± 0.9b 2.9 ± 0.9a 2.6 ± 0.4b 20 1 3.0 ± 1.2b 2.1 ± 0.7a 1.1 ± 0.4b 2.2 ± 0.4a 2.1 ± 0.4b 25 1 17.0 ± 5.2a 2.4 ± 0.6a 14.1 ± 3.9a 5.8 ± 1.7a 13.5 ± 2.2a f = 7.659 f = 0.699 f = 9.171 f = 2.859 f = 24.819 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 29 df = 2, 119 p = 0.002 p = 0.506 p < 0.001 p = 0.075 p < 0.001 means within a row with same letter not significantly different, protected tukey test (p > 0.05). table 5. pre and post disturbance mean (± se) number aed counts (10 s) of termites. colony termite disturbance ___________________ pre posta 126.0 ± 9.5a 222.8 ± 109.5a f = 0.776 df = 1, 19 p = 0.390 b 141.0 ± 8.3a 70.6 ± 10.5b f = 27.742 df = 1, 19 p < 0.001 c 255.3 ± 17.4a 180.8 ± 27.9b f = 5.137 df = 1, 19 p = 0.036 d 272.5 ± 19.1a 91.4 ± 18.9b f = 45.558 df = 1, 19 p < 0.001 means within a row with same letter not significantly different, protected tukey test (p > 0.05). w. osbrink, m. cornelius acoustical detection of coptotermes formosanus76 table 6. aed counts (10 s) of termites in trees (mean ± se). wave guide location on tree oak trees north east south west ______________________ ______________________________________ ______________________ __________________________ base 20 cm base 20 cm 122 cm base 20 cm base 20 cm 1 3.0 ± 0.1c 4.2 ± 0.8c 2.8 ± 0.5c 14.3 ± 4.4bc 2.8 ± 2.8c 23.5 ± 5.7b 2.1±0.4c 3.5±0.9c 58.2±5.2a f = 37.895 df = 8, 89 p < 0.001 2 3.7 ± 0.1b 3.2 ± 0.1b 1.9 ± 0.5b 2.4 ± 0.6b 4.2 ± 0.6b 3.2 ± 0.7b 354.6±55.4a 35.1±6.1b 4.1±1.3b f = 39.070 df = 8, 89 p < 0.001 3 276.0 ± 9.1a 4.3 ± 1.0b 3.9 ± 0.9b 5.0 ± 0.5b 5.0 ± 0.7b 8.9 ± 2.7b 3.2±0.5b 4.3±0.9b 1.2±0.9b f = 777.511 df = 8, 89 p < 0.001 4 25.6 ± 4.1b 0.0 ± 0.0b 3.9 ± 0.6b 149.7 ± 19.1a 5.9 ± 0.8b 3.3 ± 0.9b 6.7±1.2b 0.0±0.0b 4.6±0.8b f = 54.887 df = 8, 89 p < 0.001 means within a row with same letter not significantly different, protected tukey test (p > 0.05). table 7. comparison of aed 2000 with aed 2010 with aed counts (10 s) of termites in trees (mean ± se). wave guide location on tree oak trees north east south west 6 aed 2010 2.6 ± 0.9a 8.9 ± 2.0a 17.9 ± 3.3a 3.5 ± 1.5a aed 2000 0.3 ± 0.2b 1.9 ± 0.8b 12.7 ± 2.5b 0.9 ± 0.6a f = 6.065 f = 10.357 f = 1.535 f = 2.500 df = 1, 19 df = 1, 19 df = 1, 19 df = 1, 19 p = 0.024 p = 0.005 p = 0.231 p = 0.131 south (march) south (june) 350 aed 2010 1.9 ± 1.0a 0.5 ± 0.3a 13.7 ± 3.6a 47.2± 6.0a aed 2000 0.9 ± 0.4a 0.0 ± 0.0a 8.2 ± 1.3a 27.2 ± 2.3b f = 0.820 f = 2.143 f = 2.041 f = 9.571 df = 1, 19 df = 1, 19 df = 1, 19 df = 1, 19 p = 0.377 p = 0.160 p = 0.170 p = 0.006 means within a row with same letter not significantly different protected, tukey test (p > 0.05). doi: 10.13102/sociobiology.v65i3.2228sociobiology 65(3): 524-526 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a fatal nest construction: man-mixed cement used by mud-daubing wasps species sometimes make mistakes and choose a poor habitat quality or adopt an erroneous behavior in response to analog environmental cues (e.g. ecological traps: kokko & sutherland, 2001; schlaepfer et al., 2002; robertson & hutton, 2006).the use of unsuitable materials for nesting is one of this type of errors since bad choices can have fatal consequences for individuals (rodríguez-estrella et al.,1995; schaedelin & taborsky, 2009; votier et al., 2011). among mud-daubing wasps, sceliphron jamaicense (fabricius, 1775) is a spider hunter species commonly associated with human buildings where it establishes its nests (bohart & menke, 1976;coville, 1987; jiménez et al., 1992; starr et al., 2018). until now, no previous reports have documented a wasp behavior of taking man-made cement to construct the nest. in this work, we discuss potential causes and consequences of this maladaptive behavior for this species around house constructions in urban environments. a survey to collect nests of s. jamaicense was conducted abstract some sphecid wasps apparently show tolerance to urban habitats. however, resilience to man-made environments may have harmful consequences when behavioral errors can lead to ecological traps. we report failures in nesting construction of sceliphron jamaicense by erroneous choosing of building material (i.e. mud). we found a proportion of nests (1.26%)where the wasps used both mud and concrete to seal the nests. consequently, the brood was unable to emerge through the hardened material. it seems that the discrimination between building materials appears to be poor in these hymenopterans. such ecological traps could have long term negative consequences around urban environments. sociobiology an international journal on social insects a falcón-brindis1*, r rodríguez-estrella1-2, ml jiménez1 article history edited by gilberto m. m. santos, uefs, brazil received 17 october 2017 initial acceptance 19 july 2018 final acceptance 07 august 2018 publication date 02 october 2018 keywords sphecidae, sceliphron jamaicense, nesting behavior, brood mortality. corresponding author armando falcón-brindis centro de investigaciones biológicas del noroeste, av. instituto politécnico nacional 195 col. playa palo de santa rita sur 23096 la paz, baja california sur, mexico. email: armandofalcon123@gmail.com in four oases of the cape region of baja california sur, mexico: a) el triunfo, 23°48’n, 110°06’w, b) san bartolo, 23°45’n, 109°51’w, c) las cuevas, 23°32’n, 109°41’w, and d) santiago, 23°29’n, 109°43’w. these localities are small suburban towns with at most 800 inhabitants where most nests of s. jamaicense (≈ 90%) occur in human buildings (jiménez et al., 1992). the vegetation surrounding these communities is low deciduous tropical dry forest (axelrod, 1978), dominated by prosopis spp., washingtonia spp., and several exotic plants (rodríguez-estrella et al., 2010). we collected 316 nests of s. jamaicense that contained 2,224 cells. more than 90% of the nests were collected from man-made constructions, especially those places that were highly protected from sun and rain. remarkably, at el triunfo town we found four nests (1.26%) built using a mud-cement mix. females of the wasp s. jamaicense applied outer layers of recently-mixed concrete to cover their nests. this action affected 24 brood cells, causing mortality to two dozen 1centro de investigaciones biológicas del noroeste, col. playa palo de santa rita sur, baja california sur, mexico. 2university of arizona, school of natural resources and the environment, tucson, az, usa. short note sociobiology 65(3): 524-526 (september, 2018) 525 potential progeny, which represented the 5.05% of cells samples at el triunfo. the offspring reached adulthood, but they could not emerge because these wasps were not able chew the hardened layer with their mandibles to emerge from the nest (fig 1). it could be that this was a random or even an exceptional error due to an opportunistic behavior. however, most mistakes can lead in the long-term to ecological traps that negatively affect an unknown number of individuals (battin, 2004). choosing the wrong habitat can be attributed to analog environmental cues that may modify the instinct and learning critical in making decisions (dukas, 2008). adaptation to urban landscapes has costs and benefits for colonizing species (king & buckney, 2000; mckinney, 2008; ordeñana et al.,2010). for example, 40% of female nesting wasps sceliphron spirifex l. inhabiting urban zones omit the last layer of mud of the nest apparently because the urban environment overprotects nests (white, 1962). previously, s. jamaicense was defined as a generalist predator fig 1. a. transversal view. fully developed female of s. jamaicense (right cell) tried to emerge from the pupa. b. oblique view. note the pattern in which small spheres of concrete were applied (solid arrow) and how the wasp intended to apply another mud layer (dashed arrows). that broadly accepts human residences and takes advantage of any possible shelter (jimenez et al.,1992). in our study, more than 90% of the nests were found in man-made structures. this wasp seems to benefit from human settlements, finding shelter and probably less predation. however, a potential cost could be utilization of foreign resources, especially when materials with analogous texture to mud (e.g. fresh cement) are available, with fatal results for the offspring, as we found. it could be interesting to evaluate if other mud-daubing wasp species (e.g. sceliphron caementarium (drury) and s. spirifex l.) gather wrong materials for construction of nests as well. mud selection by females of s. jamaicense may rely on moisture content and texture, rather than chemical composition, color or scent of the substrate. the mortality of s. jamaicense affected by concrete usage could be small compared to natural failure and parasitism rates (starr et al., 2018). nevertheless, more research is needed to determine the cues utilized in the choice of building materials and to quantify the potential impact on wasp populations of cement usage under highly urbanized conditions. acknowledgments the authors thank to c. palacios cardiel for field assistance. funding was provided by the cibnor. afb received a phd. scholarship (273254) from conacyt, mexico. rre received a conacyt sabbatical grant, during the writing of this manuscript at the university of arizona. references axelrod, d. i. 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(sphecidae). journal of animal ecology, 31: 317-329. doi: 10.13102/sociobiology.v64i4.1586sociobiology 64(4): 430-436 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nest emigration behavior of the asian needle ant, brachyponera chinensis emery (hymenoptera: formicidae) introduction the process of nest emigration is essential to the propagation and establishment of ant colonies. many ant colonies are labile with members changing nest location in response to drought, flooding, predation, competition, budding, and nest disturbance (fowler, 1981, tay & lee, 2015). although emigration is a familiar process, recruitment methods during emigration differ between and within ant species (hölldobler & wilson, 1990; planque et al., 2010). recruitment methods include pheromone trail following, tandem running, and physical adult transport (maschwitz et al., 1986, beckers et al., 1989). a single species may employ a variety of recruitment methods during an emigration event. for example, bothroponera tesseronoda (emery) uses tandem running and trail following to complete emigration (jessen & abstract ant colonies change nest location in response to physical disturbance, climate fluctuation, and resource availability. during the emigration process, worker recruitment is vital to ensuring that individual colony members are moved to the new nest site. recruitment methods used during emigration differ between ant species. in a laboratory study, we investigated the recruitment behaviors of the invasive asian needle ant, brachyponera (=pachycondyla) chinensis (emery), during nest emigration. subsets of b. chinensis worker ants were subjected to physical nest disturbance, and the recruitment methods and associated behaviors were recorded. before recruitment to the new nest location began, b. chinensis ants organized into three distinctive groups: queen-tending, broodtending, and scouting. once the new nest site was identified, scout ants began physically transporting nestmates into the new harborage. transport rates increased with time in the first 30 minutes and did not change during the 30 to 55 minute interval when brood was transported. however, adult transport rate increased again after brood transport was completed and decreased after 90 minutes. these studies are the first to identify the recruitment methods, division of labor, and social organization behavior of b. chinensis during nest emigration. sociobiology an international journal on social insects hr allen, pa zungoli, ep benson, p gerard article history edited by ricardo castro solar, ufmg, brazil received 06 march 2017 initial acceptance 24 may 2017 final acceptance 25 september 2017 publication date 27 december 2017 keywords invasive biology, asian needle ant, foraging, ponerinae corresponding author patricia zungoli department of plant and environmental sciences clemson university 130 mcginty court 171 poole agricultural center clemson, sc 29631, usa. e-mail: pzngl@clemson.edu maschwitz, 1986), whereas tandem running is preceded by adult transport in neoponera obscuricornis (emery) (traniello & hölldobler, 1984) and leptothorax albipennis (curtis) (pratt et al., 2002). adult transport by ants was initially characterized solely as a nest emigration recruitment method (haskins and haskins 1950). however, guénard and silverman (2011) redescribed the physical transport methods of the invasive asian needle ant, brachyponera chinensis (emery), during foraging expeditions (takimoto, 1988). the foraging recruitment method, tandem carrying, is a newly described context-dependent recruitment behavior, occurring three to ten times more often when a food item is too large to be moved by a single worker. in addition to being a new foraging strategy, tandem carrying was further distinguished from other forms of adult transport because the carrying posture differed from previously described clemson university, clemson, sc, usa research article ants http://www.researchgate.net/profile/benoit_guenard sociobiology 64(4): 430-436 (december, 2017) 431 transport postures (guénard & silverman, 2011). in other ant species, worker ants are grasped and/or transported by the mandibles, the neck, or on their side (möglich & hölldobler, 1974), whereas ants being transported during tandem carrying are grasped between their first and second pairs of legs on the ventral mesothorax. guénard and silverman (2011) described this region as the mesometasternum but this designation is incorrect, as the sternites on ants are internal invaginations and not external features. research on b. chinensis has increased due to its potential as a public health threat (cho et al., 2002; nelder et al., 2006; lee et al., 2009), negative impact on native and introduced ant species populations (guénard & dunn, 2010; bednar et al., 2013; spicer-rice & silverman, 2013), spread in the southeastern u.s., and potential spread into areas outside its current range (bertelsmeier et al., 2013). b. chinensis typically nests in downed timber or under objects in contact with the soil, usually in areas where termites are abundant, contributing to their success (bednar & silverman, 2011). identifying and determining the potential for geographic spread and nesting behavior are central to understanding the invasive ecology of b. chinensis. during field and laboratory observations, b chinensis workers engaged in adult transport. the observed transport took place in the absence of immovable food items. the ants were physically transporting nestmates to various locations in space but we did not identify any subsequent tasks. because adult transport occurred outside of foraging recruitment and is commonly associated with nest emigration, we hypothesize that b. chinensis employ adult transport to complete nest emigration using the posture assumed during tandem carrying reported by guénard and silverman (2011). in the laboratory study reported here, field-collected colony fragments were subjected to identical nest emigration trials elicited by physical disturbance to determine the recruitment methods of b. chinensis during emigration. also, in a separate study, ants were marked with paint and subsequently subjected to nest emigration trials to determine the extent of task allocation during nest emigration in b. chinenesis. materials and methods twenty queen-right b. chinensis nests were collected from different locations within the clemson experimental forest, a mixed hardwood-pine forest in pickens county, sc (34043’55.018” n,82051’6.654” w) between augustoctober 2012 and april-june 2013. each nest used in the study contained at least one queen, brood, and 200 worker ants. laboratory trails were conducted between septembernovember 2012 and april-june 2013. collected nests were subjected to experimental trials within one week of collection. ants nests were housed 20 gallon uncovered plastic tubs (40.89 cm x 39.06 cm) and provided with a glass test-tube (250 mm x 25 mm) wrapped in red transparent cellophane with a piece of moistened cellulose sponge inside as a water source (figure 1). test-tube harborages were used to mimic b. chinensis galleries in fallen timber. colonies were fed reticulitermes sp. workers, and tenebrio molitor (linnaeus) larvae ad libitum, and maintained at 21°c at a 12:12 l/d cycle with 70-80% rh. all studies were conducted in arenas consisting of a plastic container (60 cm x 42 cm x 16 cm) divided into equal halves by a 37-cm acrylic glass insert (figure 1). before testing began, an uninhabited glass test-tube harborage was placed in one half of the arena, and the other half remained empty. because the acrylic glass insert was not completely flush with the bottom of the arena, 37-cm strips of playdoh® (hasbro corporation, pawtucket, ri) were used to seal openings. worker ants inspected play-doh® strips but did not attempt to feed on or remove the inserts. colony emigration studies were conducted concurrently in separate treatment (n = 10) and control (n = 10) arenas. overall, ten colonies were used per treatment. on the day of experimentation, a colony subset consisting of 200 workers, one queen, and 20 brood items (eggs, larvae, or pupae) were manually removed from two separate colonies and transferred to two different glass test-tubes (250 mm x 25 mm) using featherweight forceps and/or a small paintbrush. each testtube, wrapped in red transparent cellophane, contained a piece of moistened cellulose sponge as a water source (figure 1). after removal, the test subjects were allowed to acclimate to the new environment for two hours. treatments consisted of a physical nest disturbance, defined as manually removing all nest members and associated materials from a test tube harborage. specifically, during disturbance, the sponge insert was removed and the test tube was lightly shaken to dislodge any ants remaining in the test tube. any ants latching onto the sponge insert were removed using a paintbrush. testtube harborages were not physically disturbed in control treatments. in each round of experimental trials two colonies were selected. selected colonies were randomly designated as “treatment” or “control” and were used once during the study. in both treatments, the play-doh® barrier and plexiglass inserts were removed from the arenas after a one-hour acclimation period, permitting unrestricted ant movement. data collection began after the first successful carrying event was observed. successful transport consisted of the carried individual being released inside the new harborage. all carrying events were visually observed, time of carry was recorded, as well as the total number of successful carries. data collection ceased after 90 minutes. preliminary data indicate, after this time, the interval between carrying events was greater than five minutes. a one-way wilcoxon test was conducted to determine differences between total number of successful transports occurring in treatment and control arenas during nest emigration trials (jmp® pro 10, sas institute, inc. 2012. cary, nc). a p-value of < 0.05 indicated statistical significance. the difference in number of hr allen et al. – asian needle ant nest emigration432 ants being carried in 15-minute intervals was compared using a repeated measures manova in sas 9.3 (sas institute, inc. 2012. cary, nc). a p-value of < 0.05 indicated statistical significance. all raw data provided as supplementary material. trails were taken with a sony cybershot® camera (sony corporation of america, new york, ny). all images provided as supplementary materials. results brachyponera chinensis workers used adult transport as a recruitment method during nest emigration. in total, we observed 396 successful transport events in treatment arenas and 42 in control arenas. the mean number of successful transports occurring in treatment arenas (39.6 ± 6.94 sd) was significantly more than the number of successful transports observed (4.2 ± 2.2 sd) in control arenas (see supplementary materials). treatment had a significant effect on the number of successful transports occurring during the study (wilcoxon test, x2 = 14.35, p = 0.0002). on average, 19% of ants were physically transported inside the new harborages. the remaining 81% of ants walked into the new harborage without worker assistance. we observed workers in treatment arenas organizing into task-associated groups before acrylic glassinserts were removed. groups consisted of brood retrievers, members of the queen’s tending group, and scouts (see supplementary materials). queen-tending ants surrounded the queen, remaining with the group until the queen walked into the new harborage. scouting groups consisted of transporting and non-transporting worker ants. non-transporting scouts moved around the arena but not in a particular pattern. after the insert was removed, ants either remained in groups or began walking into or under the new test tube harborage. adult transport began after several scouts explored the new test tube harborage 12-15 minutes after the trial began (figure 2). the number of successful transports occurring every fifteen minutes in treatment arenas was compared. results indicate that the number of transports did change with time (f= 62.58,p = 0.0002). once initiated, transport continued at a steady rate for 30 min. (figure 2). transport rates remained constant from 35 to 50 minutes. during this period, retrieval and placement of brood from the open arena into the new harborage became the focus of non-transporting ants. queen ants were also moved during the 35-55 minute period. after 55 minutes, the number of successful transports increased again for 15 minutes on average. when transporting scouts encounter other ants, the pair interacts by drumming their antennae together. in each successful case, the ant being transported lowers its head, allowing the transporting ant to grasp it by the mandibles. from this position, as defined by guénard and silverman (2011), the carried ant assumes a pharate pupal posture and eventually is grasped on its venter. the pair then walked toward the harborage; however, the path to the new nest site may not be direct. once the pair reaches the new nest, the carrier ant releases its nest mate within the harborage. the carrier may remain inside the nest or return to the arena after releasing its nestmate. task allocation to determine if b. chinensis workers performed repeated adult transport episodes during emigration, worker ants were marked with paint and subjected to physical disturbance. the ants used in the current study were obtained from four of the nests used in the previous study. as part of the study, two hundred worker ants were removed from a colony along with one queen and brood. queen ants and brood items were not marked with paint during trials. before marking, five ants were selected, placed into a 1.5 ml plastic medicine cup, and transferred to a freezer (-18 0c) for 2 minutes. after removal from the freezer, the ants were transferred from the medicine cup to onto a chilled metal panel. to mark an ant, a single ant was placed onto a foam platform with a single strand of hair taped down as a loop. the hair loop served as a restraint for the ants. worker ants were marked on the head, abdomen, or thorax with testors® modeling paint (testors,vernon hills, il) to distinguish workers. after marking, each ant was placed into a plastic container (60 cm x 42 cm x 16 cm). the queen ant and brood items were transferred to the arena after the worker ants were distributed. a glass test 250 mm x 25 mm) wrapped in red transparent cellophane with a piece of moistened cellulose sponge inside as a water source was added to the arena to elicit emigration. observational data was recorded but statistical analyses were not performed. pictures of worker and queen assemblages during nest emigration fig 1. brachyponera chinensis nest emigration arena (60.9 cm x 42.6 cm x 16.7 cm) with test tube (250 mm x 25 mm) harborages, plexiglass insert, and play-doh® lining. test tubes are wrapped with red cellophane paper with a moistened 8-cm sponge inserted. during experimental trials, the plexiglass insert, play-doh®, and one harborage were removed allowing the ants to freely move in the arena. http://www.researchgate.net/profile/benoit_guenard sociobiology 64(4): 430-436 (december, 2017) 433 the marking experiments were not successful but one ant was observed performing two consecutive transports. the marked ant picked up a nestmate dropped her off inside of the harborage and immediately entered the arena to retrieve another nestmate. consecutive transporting was not observed in any of the other trials. discussion social carrying in b. chinensis was initially characterized as a context-dependent behavior performed only to recruit nestmates to food items too large to be carried by individual ants, a process known as tandem carrying (guénard & silverman, 2011). the data presented in the current study indicate that b. chinensis workers also employ adult transport during nest emigration. in some ant species, adult transport during emigration is sometimes preceded by tandem running (möglich, 1978; traniello & hölldobler, 1984; pratt, 2005). brachyponera chinensis, however, do not employ multiple methods during emigration. on average in this study, 19% of workers were physically transported into the new harborages during trials and the remaining 81% traveled alone to the nest site. consequently, the question of how non-transported ants locate new nest sites remains unanswered. non-transported ants were observed walking directly into the harborage without exhibiting tandem-running or trail-laying behavior. however, for a worker ant to locate the new nest site, directional cues must be present. guénard and silverman (2011) attempted to determine if b. chinensis use trail pheromones during tandem carries, but their experimental results were inconclusive. however, the use of trail pheromones should not be discounted. to exclude pheromones as a contributing factor, gaster positions during emigration must be analyzed and extractions of glands commonly associated with pheromone production, such as the dufour’s gland or pygidial gland should be made (holldobler et al., 1982). in addition to chemical signaling, tactile and visual signaling should also be evaluated as directional cues used by b. chinensis during emigration. although adult transport is an effective recruitment strategy for b. chinensis, each carrying attempt is not successful. the “transporting” ant always initiated the process in this experiment, but the “transported” ant may resist. resisting ants pull away from the transporter or place their thorax/abdomen on the floor of the arena, preventing the other ant from gaining the leverage needed for carrying. this observation lies in accordance with langridge et al. (2008) who documented comparable behaviors in temnothorax albipennis (curtis) during colony emigration.an unsuccessful transport could be the result of a “transporter” ant encountering an individual that previously experienced visiting the new nest, another “transporter”, or encountering an ant responsible for protecting brood or the queen. before initiating transport, b. chinensis worker ants organized into groups. groups consisted of scouts, brood tenders, and the queens’ retinue. scouting ants spent time exploring the foraging arena and were the first to locate the new nest. it appeared that worker ants were protecting the queen during emigration. queen protective behavior during emigration was also observed in oecophylla longinoda latrielle, the weaver ant (holldobler & wilson, 1983). oecophylla longinoda queen sexude exocrine gland produced pheromones that attract workers to the queen during emigrations, allow workers to produce trophic eggs, fig 2. pooled total number of successful carries by b. chinensis workers occurring at 5-minute intervals during treatment nest emigration trials (n = 10 colonies). worker ants organized into groups (grp org) from 0-13 min. adult transport (at) began at the 12 min and continued for 90 min. queens and brood were transported from 35-55 min. the number of successful transports performed by b. chinensis workers decreased during queen and brood movement. transport activities increased after the queen and brood were moved to the new harborage. hr allen et al. – asian needle ant nest emigration434 but prevent workers from producing viable developing eggs. the loss of worker egg production ensures that queens are the sole producer of life within a colony making her a vital asset to the longevity of the colony. worker ants of b. chinensis (ito and ohkawara, 1994) and its sister species b. nakasujii do not possess ovarioles (gotoh & ito, 2008). as a result, b. chinensis colonies solely depend on the queen for egg production. the presence of queen retinue in b. chinensis and absence of ovarioles in b. chinensis worker ants may be linked to queen produced pheromones. brood items and the queen were transported during the middle (35-50 min.) of the study. queen movement during this period is consistent with pezon et al. (2005) in which n. obscuricornis queens were transported mid-way through emigration presumably to optimize their protection. the number of potential transporters was lower than the number of potential transportees suggesting that a small proportion of the total workforce is allocated towards adult transport. previous studies show similar results (langridge et al., 2008, sendova-franks & franks, 1995) but studies attempting to identify if carrying behavior was relegated to a specific group of ants during emigration have been inconclusive (sendova-franks & franks, 1995). however, physical marking caused workers to devote more time to grooming than to colony tasks. yet, in one marking trial, we documented one marked ant carrying thirteen nestmates into the new harborage. in view of repeat transports, we anticipate that b. chinensis workers are capable of performing multiple carries, but additional marking studies are needed to determine the worker antcarrying frequency. colony duties may also be associated with a worker ant’s age, a phenomenon known as temporal polyethism (robinson et al. 1994, sendova-franks and franks 1993). in some studies, younger ants tend to work within the nest whereas older ants usually take on tasks outside the nest, such as colony defense, foraging, and recruitment. during trials, we observed workers transporting dusky-yellow colored, callow workers into the nest, but callow workers never behaved as transporters. abraham and pasteels (1980) reported similar behavior in myrmica rubra (l.). recognition of this phenomenon raises the possibility that temporal polyethism also may play a role in task allocation during adult transport in b. chinensis. as b. chinensis continues to increase its geographic range (guénard & dunn, 2010), thorough documentation of colony movements will be more important. the dispersal abilities of invasive ant species are affected by dispersal type. after establishment, invasive ants may naturally increase their range by mating flights (markin et al., 1971) or increase their foraging range through an emigration process known as budding (holway et al., 2002). during budding a portion of a colony leaves the original nest to found a new nest a few meters away. brachyponera use adult transport during foraging (guénard & silverman& 2011) and during nest emigrations (current study) so it is possible to suggest that ants also use adult transport to increase their range. our studies serve as the first to provide insight into the nest emigration recruitment behaviors of b. chinensis workers. in this laboratory study, b. chinensis employed adult transport during emigration to move colony members to new nesting locations, but only a subset of the entire colony was relocated in this manner. these results suggest that b. chinensis may disperse through budding. however, zungoli and benson (2008) collected winged males and females in a light trapping study, suggesting that mating flights may also occur; although, males alates were trapped more frequently than females (19:1). future studies of b. chinensis emigration should attempt to address colony propagation and task allocation during emigrations. studies of this nature will help us understand dispersal factors contributing to the invasive success of b. chinensis in the united states. acknowledgements funding and support was provided by pi chi omega national pest control fraternity. we also thank two anonymous reviewers for providing valuable editorial assistance. lastly, we would like to thank members of the zungoli/benson lab for assistance with fieldwork. supplementary material http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1586/0 doi: 10.13102/sociobiology.v64i4.1586.s1853 references abraham m, pasteels jm (1980). social behavior during nest moving in the ant myrmica rubra. insectes sociaux, 27: 127147. doi: 10.1007/bf02229249 beckers r, goss s, deneubourg jl, pasteels jm (1989). colony size, communication and ant foraging 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(1988). carrying behavior for the recruitment of workers in brachyponera chinensis. ari, 16: 21-22. tay jw, lee cy (2015). induced disturbances cause monomorium pharaonis (hymenoptera: formicidae) nest relocation. journal of economic entomology, 108: 1237-1242. doi: 10.1093/jee/tov079 traniello jf, hölldobler b (1984). chemical communication during tandem running in pachycondyla obscuricornis (hymenoptera: formicidae). journal of chemical ecology, 10: 783-794. doi: 10.1007/bf00988543 zungoli pa, benson ep (2008). seasonal occurrence of swarming activity and worker abundance of pachycondyla chinensis. proc 6th internat conf urban pests 51-57. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.2092sociobiology 64(4): 417-422 (december, 2017) independent colony foundation in paraponera clavata (hymenoptera: formicidae): first workers lay trophic eggs to feed queen’s larvae introduction paraponera clavata smith is a conspicuous, 18-30 mm long inhabitant of wet forests in the neotropics. its notoriously painful sting underlies a variety of common names (‘24 hour’ ant, ‘bullet ant’) and even native initiation rites. it nests in soil next to the base of trees and forages in the canopy. such arboreal habits are found in only few poneroid ants (gobin et al., 1998a). colonies of p. clavata are monogynous with a few hundred workers, reaching up to 2326 workers (breed & harrison, 1988). paraponera is the only genus in the poneroid subfamily paraponerinae (bolton, 2003). its morphology includes a mixture of primitive traits shared with poneroid subfamilies and derived traits found among formicoids (keller, 2011). the latter include the presence of well-developed frontal lobes and antennal scrobes, and a clypeus broadly inserted between the antennal sockets. also, unusual for a poneroid is the fusion of the promesonotal junction, as well as the abstract paraponera clavata smith is a large, notorious, and widely distributed ant, yet its colony founding behavior is poorly known. in the laboratory, a dealate queen collected from peru reared a first generation of ten adult workers over 18 months; eight cocoons and several larvae failed. food was obtained outside the nest and given to larvae. it took five and six months before the first two workers emerged, and they were smaller than average (i.e.‘nanitic’). at q+4, trophic eggs were laid by workers and given directly to medium and mature larvae on three occasions. six workers were dissected immediately after the queen’s death, and five had yolky oocytes in their ovaries. queen foraging is known from anecdotal field observations, despite the prothorax (and corresponding neck muscles) being smaller than in other poneroid queens. sociobiology an international journal on social insects c peeters article history edited by gilberto m. m. santos, uefs, brazil received 25 september 2017 initial acceptance 02 november 2017 final acceptance 12 november 2017 publication date 27 december 2017 keywords caste, founding behavior, prothorax, neck muscles, functional morphology. corresponding author christian peeters sorbonne université upmc institute of ecology and environmental sciences (iees) 4 place jussieu, paris 75005, france e-mail: christian.peeters@upmc.fr tergosternal plates of the petiole (keller, 2011). the second gaster segment shows a well-marked constriction anteriorly, but without forming a true postpetiole. this combination of traits was long considered evidence of paraponera’s affinities with ectatomminae (now considered as formicoids), although paraponera was always kept separate. molecular studies support the view that paraponera is a unique lineage that evolved in isolation from other ant groups (e.g. moreau et al., 2006). independent colony foundation (icf) implies risks as the queen is not assisted by workers. mortality is high in species with queens that forage outside the nest (termed ‘nonclautral’ icf) (peeters, 1997). two factors determine icf in poneroids (peeters & molet, 2010): (1) queens and workers exhibit a very limited dimorphism in body size, so a foundress must provide a lot of food to each larva; (2) founding queens have small wing muscles (compared to formicoid queens; keller et al., 2014) as well as limited fat and protein reserves in the gaster, hence additional food is needed for the first larvae. when foundresses hunt outside, more foraging trips institute of ecology and environmental sciences (iees), sorbonne université upmc, paris, france research article ants c peeters – independent colony foundation in paraponera ants 418 means more risks. it is crucial that the first workers quickly forage outside instead of the foundress, and new data in p. clavata show they also feed larvae with trophic eggs. material and methods a few winged queens of p. clavata were walking on the ground during ant course 2013, held in estación biológica villa carmen (12°53’25” s, 71°24’39” w, 545m altitude), madre de dios province, peru. on 13 august, i collected a lone dealate queen close to the base of a small tree. it was brought back to paris and kept in a plaster nest with 2 small chambers (7 x 7 x 1 cm) under a glass roof, with a separate foraging arena. temperature was controlled at 25°c, and plaster was moistened irregularly. freshly cut-up mealworms and small crickets were put in the arena at regular intervals, as well as pieces of fruits and honey diluted in water. mites were intermittently removed from inside the nest. most first workers were color-marked to keep approximate age records and monitor individual activities; to keep disturbance to a minimum, marking was usually done when an individual was active in the foraging arena. gaps between successive emergence of workers were sufficiently long to facilitate keeping track of the order of emergence. records of behavior (photos and videos) were compiled using an iphone 4. freshly dead workers were dissected to determine ovarian activity, and all six surviving individuals were dissected at the end. prior to this, these six workers were weighed with an electronic balance. head width (hw; broadest point of the head capsule) was measured with a binocular eyepiece. results queen behavior the foundress was able to rear the first generation of workers, and this includes foraging and cutting up prey for larvae (fig 1). eggs were laid regularly in small numbers (table 1); only towards the end were there more than 10 eggs. counts of queen eggs over short periods (e.g. 8-18 nov. 2014) suggest some were eaten by larvae. queen-laid eggs acquired a stiff golden chorion after a few days (figs 1 and 2), thus oophagy must occur before. mature larvae were buried with debris by the queen to help them spin a cocoon. spinning took several hours and several larvae frequently died at this stage. the meconium (from voiding of the gut; ejected at one extremity of the cocoon) appeared after 4-5 days. the first two cocoons were cut open by the queen, and the pupae discarded outside. slightly over 5 months elapsed before the first adult worker emerged (fig 2). noteworthy is an eccentric behavior of the foundress: it remained highly alert inside the brood chamber, darting around at the slightest disturbance. i have never observed such wary behavior in the workers of this or other poneroid species. after a few workers had emerged, the queen stopped foraging and became very quiet (fig 3). colony growth another five pupae were discarded outside and a few larvae failed to produce a cocoon, consequently only ten workers reached the adult stage over 18 months (table 1). one reason for this mortality may have been infestation by mites, which settled on the neck region of larvae and were included in cocoons. newly emerged (‘callow’) workers retained lightly pigmented leg extremities for a few days (fig 3). two workers (one was callow) were taken from inside the nest for color-marking and then released in the arena, and older workers carried them back inside (grabbing their head induced a pupal posture). the first two workers were nanitic (see below), and died five months (worker #1, denoting order of emergence) and seven months (#2) after emergence (table 1). workers #2 and #3 were marked outside when five and three months-old, respectively. worker #4 was also three months-old when first seen outside. fig 1. founding queen together with first brood. a medium larva is busy feeding on a piece of mealworm (arrow). one yellowish egg can be seen. fig 2. founding queen together with worker #1 and brood. two large larvae are feeding. yellowish eggs lie singly on the ground next to a small larva (arrow). sociobiology 64(4): 417-422 (december, 2017) 419 worker #2 was six months-old at this time, and had been foraging three weeks earlier. another worker (yet unmarked, thus young) also laid a trophic egg. upon the queen’s death, all six remaining workers were promptly dissected, revealing that all but the oldest (8 monthsold) had large yolky oocytes. this suggests that trophic eggs could be laid regularly, but because they are directly consumed by larvae, this behavior was only rarely observed. ovaries consisted of 14 ovarioles in both queens and workers. workers #1 and #2 were distinctly smaller (hw= 3.7 mm and 3.9 mm, respectively) and thus nanitic. other workers had head widths 4.2-4.7 mm, with size roughly increasing with order of emergence. for comparison, the queen’s head was 4.8 mm wide. fresh weights of workers ranged 120-160mg, while the queen weighed 185mg (she lacked yolky oocytes). discussion the incipient colony of p. clavata studied here took about one year to produce the first five workers, even though food was provided ad libitum in the foraging arena. during independent colony foundation (icf) in ants, the success rate of first larvae to develop as adult workers depends almost entirely on the queen’s ability to provide them with adequate food. this is harder in poneroid species because workers are almost as big as their mother, hence more food is needed before each larva can pupate (as opposed to most formicoids where workers are much smaller than queens; peeters & ito, 2015). not only foundresses must take big risks to forage outside, but they need to continue this for a few months, which presumably causes a very high failure rate. the prolonged development of poneroid brood contributes to the difficulty date first workers cocoons larvae eggs 26-9-2013 7 29-10 4 9 8-11 5 4 15-11 5 2 18-11 5 1 20-12 3 3 4-1-2014 2 1 4 20-01 1 + 2 dead 1 4 21-02 2 4 2 7-03 worker #1 2 3 4 31-03 q + 1w 1 + 1 dead 3 7 9-04 worker #2 1 1 +1spin § 8 16-04 q + 2 1 dead 3 7 9-05 q + 2 0 2 9 28-05 q + 2 1 3 4 10-06 q + 2 1 3 5 23-06 #3 1 3 8 ?-07 #4 4-08 #5; dead#1 1 dead n/a n/a 4-09 q + 4 1 5 6 15-09 q + 4 4 2 9 25-09 q + 4* 3 + 1 dead 2 +1spin § 8 29-09 q + 4 3 1 +1spin § 8 15-10 #6 3 3 7 27-10 #7 1 +1 dead 3 n/a 4-11 #8; dead#2 0 2 +1spin 4 7-11 q + 6 0 3 5 12-12 dead #6 + #7 2 2 5 30-12 q + 4 2 1 8 16-2-2015 #9 + #10 0 4 17 24-02 q + 6 queen dies 1 6 15 * 3 workers active outside § failed table 1. demographic data for an incipient colony of paraponera clavata monitored in the laboratory for 18 months. fig 3. founding queen with workers and brood on 4 november 2014. the oldest worker (#3) is guarding the entrance. one mature larva (next to worker #4) has reached final size and started spinning. individuals #4 and #5 have the same colour dot with different shapes. the two recently emerged workers were not marked, but the youngest (#8) can be recognized by its lightly pigmented hind tarsi (arrow). small and large larvae were kept alive for two days after the queen died and all workers had been dissected; a small larva molted its skin (head capsule and hind parts) which remained uneaten. this larval skin was seen to be eaten by one worker before, indicating that the adults benefit from this recyclable resource. production of trophic eggs at stage q+4, three larvae (medium as well as large) fed on white eggs that were smaller than queen eggs. worker #2 was observed with the sting extruded, an egg was laid and immediately placed on a nearby larva who quickly consumed it. the whole sequence lasted less than one minute. c peeters – independent colony foundation in paraponera ants 420 of icf. brood development times are difficult to determine in ants because larvae do not grow in fixed cells, unlike social wasps and bees. thus, individuals cannot be monitored over time, and estimates must be based on demographic data. although complicated by oophagy and larval mortality, data in this colony of p. clavata suggest the following: egg-larva 4 weeks; larva-cocoon = 6-8 weeks (n=2): cocoon-adult 6-7 weeks (n=3). these very long development times are somewhat higher than those recorded for other poneroids: 2-3 months in total (peeters & ito, 2015). this may be linked to the large size of paraponera workers. in sharp contrast, many formicoids develop considerably quicker, e.g. 28 days for major workers of solenopsis invicta (tschinkel, 2006), 40 days for soldiers of pheidole bicarinata (wheeler, 1982), 54 days for camponotus kiusiuensis (ito et al., 1988). shorter developmental times seem mostly due to a striking reduction in the body size of formicoid workers. producing nanitic workers is an adaptation to reduce the time taken before the first workers can forage in replacement of the founding queen. nanitics are common in the formicoid subfamilies myrmicinae and formicinae, but there are only very few anecdotal reports in ponerinae (peeters & molet, 2010). in p. clavata, only two workers were conspicuously nanitic, however the first two cocoons that failed may have enclosed nanitics also. workers emerging later were larger, although below the upper range of head widths (5.4 mm) recorded in a mature colony by breed and harrison (1988). nanitic workers result from a novel modification of the endocrine regulation of larval development: although the queen controls access to pieces of prey (and she was regularly observed to take prey away from larvae busy feeding), the spinning behavior is initiated by the larvae. this prompts the queen or workers to bury pupating larvae with debris, thus providing a scaffold that is crucial for the attachment of silk strands. once a few adult offspring were present in the colony, the foundress stopped going outside as workers started to forage. the latter also groomed the brood and distributed prey pieces. limited observations indicated that first workers help to feed the queen’s larvae with trophic eggs. larval oophagy is known during icf in attini (fernández-marín et al., 2004) and some other lineages (e.g. rhytidoponera confusa; c. peeters, unpublished data) but involves eggs laid by founding queens only. production of trophic eggs by first workers has never been reported. in many ant species, trophic eggs cannot develop as they derive from unviable oocytes (e.g. gobin et al., 1998b). such yolk packets are a valuable protein source that facilitates the development of larvae, and fills the gaps while hunting is unsuccessful. breed and harrison (1988) found that many young workers collected inside established colonies of p. clavata had large yolky oocytes; these could be laid as trophic eggs. p. clavata collects both nectar and insect prey (tillberg & breed, 2004), however nectar is likely to be less important for larval development. pseudotrophallaxis (i.e. carrying a drop of liquid between open mandibles) was observed infrequently, but there was also intimate contact between the mouths of some workers and larvae, suggesting trophallaxis. this may function to distribute nectar, while trophic eggs increase the flow of proteins to larvae. the founding behavior of paraponera queens has not been investigated in the field, but the following anecdotal observations suggest icf is non-claustral. in the field, dealate queens hunt arthropod preys and collect nectar, moreover nest entrances of newly founded colonies remained open (m. breed, personal communication, april 3, 2012). hölldobler and wilson (1990) collected queens foraging on the lower trunks of pentaclethra trees; most paraponera colonies nest at the base of such trees in costa rica. haskins and enzmann (1937) stated that founding queens need to forage. similarly, in the laboratory context described here, a foundress regularly went outside to get food. non-claustral icf is generally associated with small differences in body size between queens and workers, together with limited reserves accumulated by queens before aerial dispersal (peeters & molet, 2010). queen foraging also depends on worker-like neck muscles, and the thorax of p. clavata queens can give information about this (see below). all ant workers have a large prothorax (t1) that houses powerful muscles connecting with the head, essential to use the mandibles as tools (keller et al., 2014). in flying insects, the second thoracic segment (t2) is typically large with a predominant mesonotum for the attachment of wing muscles. formicoid queens generally have a large mesonotum that overhangs a much smaller prothorax, hence the neck muscles are reduced in size and this is consistent with lack of foraging during claustral icf. in ponerinae, flying queens have a pronotum intermediate in size to workers’, reflecting a distinct trade-off between the size of muscles that power the head and wing muscles, thus allowing queens to hunt and carry prey during colony foundation (keller et al., 2014). surprisingly among poneroids, p. clavata queens have a small prothorax (fig 4), more similar to that of queens in myrmicinae. in tatuidris tatusia, belonging to a poneroid subfamily (agroecomyrmecinae) closely related to paraponerinae, winged queens also have a small prothorax (donoso, 2012). this derived trait shared by paraponera and tatuidris distinguish them from queens in ponerinae, and suggests an evolutionary trend towards larger wing muscles that are used as metabolic reserves. in attine (fungus-growing) ants, most genera are non-claustral except atta (fernández-marín et al., 2004). attini queens were not included in the comparative study of keller et al. (2014), but visual examination of the thorax in different attine genera (antweb.org) points a trend to a small prothorax even in nonclaustral genera. observations of icf are generally scarce in the ant literature, and there are no published records for many widespread genera. thorax morphology can be used to predict the founding behavior of queens (keller et al., 2014), but predictions need to be tested empirically. sociobiology 64(4): 417-422 (december, 2017) 421 besides various morphological peculiarities (see introduction), what other differences separate ponerinae from paraponerinae? paraponera exhibits an omnivorous diet, the beginnings of trophallaxis, and a reduced prothorax reflecting modifications in flight morphology. the latter may be necessary to carry a heavier abdomen filled with fat and protein reserves (see helms & kaspari, 2014), which is characteristic of most formicoids, i.e. over 80% of ant species. paraponera is one of several scattered experiments in gigantism because the majority of ants evolved dwarf workers and larger colonies (peeters & ito, 2015). breed, m. & harrison, j.f. (1988). worker size, ovary development and division of labor in the giant tropical ant, paraponera clavata (hymenoptera: formicidae). journal of the kansas entomological society, 61: 285-290. donoso, d.a. (2012). additions to the taxonomy of the armadillo ants (hymenoptera, formicidae, tatuidris). zootaxa, 3503: 61-81. fernández-marín, h., zimmerman, j. k. & wcislo, w. t. (2004). ecological traits and evolutionary sequence of nest establishment in fungus-growing ants (hymenoptera, formicidae, attini). biological journal of the linnean society, 81: 39-48. doi: 10.1111/j.1095-8312.2004.00268.x gobin, b., peeters, c. & billen, j. (1998a). colony reproduction and arboreal life in the ponerine ant gnamptogenys menadensis (hymenoptera: formicidae). netherlands journal of zoology, 48: 53-63. doi: 10.1046/j.1365-3032.1998.234102.x gobin, b., peeters, c. & billen, j. (1998b). production of trophic eggs by virgin workers in the ponerine ant gnamptogenys menadensis. physiological entomology, 23: 329-336. haskins, c.p. & enzmann, e.v. (1937). studies of certain sociological and physiological features in the formicidae. annals of the new york academy of sciences, 37: 97-162. helms, j.a. & kaspari, m. (2014). found or fly: nutrient loading of dispersing ant queens decreases metrics of flight ability (hymenoptera: formicidae). myrmecological news, 19: 85-91. hölldobler, b. & wilson, e.o. (1990). host tree selection by the neotropical ant paraponera clavata (hymenoptera: formicidae). biotropica, 22: 213-214. ito, f., higashi, s. & maeta, y. (1988). growth and development of camponotus (paramyrmamblys) kiusiuensis santschi colonies (hym. formicidae). insectes sociaux, 35: 251-261. keller r.a. (2011). a phylogenetic analysis of ant morphology (hymenoptera: formicidae) with special reference to the poneromorph subfamilies. bulletin of the american museum of natural history, 355: 1-90. doi: 10.1206/355.1 keller, r.a., peeters, c. & beldade, p. (2014). evolution of thorax architecture in ant castes highlights trade-off between flight and ground behaviors. elife, 3:e01539. doi: 10.7554/elife. 01539.001 moreau, c.s., bell, c.d., vila, r., archibald, s.b. & pierce, n.e. (2006). phylogeny of the ants: diversification in the age of angiosperms. science, 312: 101-104. doi: 10.1126/ science.1124891 peeters, c. (1997). morphologically “primitive” ants: comparative review of social characters, and the importance of queenworker dimorphism. in j. choe & b. crespi (eds.), the evolution of social behavior in insects and arachnids (pp. 372–391). cambridge university press. doi: 10.13140/rg.2. 1.2001.8646 fig 4. dealate queen and worker of paraponera clavata, showing differences in thorax segmentation. in queens (top), pronotum (dorsum of prothorax, t1) is small relative to the mesonotum (t2), reflecting a trade-off between neck muscles and wing muscles. posterior segments of queen gaster are missing because of ovarian dissection. photos by mônica ulysséa acknowledgments this research was made possible through funding from ant course 2013, and a collecting permit to b.l. fisher. i thank romain péronnet for help with caring for live ants (and monitoring them during my absences), roberto keller for comments on the systematics of paraponera, mônica antunes ulysséa for automontage photos (fig 4), and thibaud monnin for useful comments on a draft. funded by the french national research agency (antevo anr-12-jsv7-0003-01). references bolton, b. (2003). synopsis and classification of formicidae. memoirs of the american entomological institute, 71: 1-370. file://localhost/doi/ 10.1126:science.1124891 file://localhost/doi/ 10.1126:science.1124891 c peeters – independent colony foundation in paraponera ants 422 peeters, c. & ito, f. (2015). wingless and dwarf workers underlie the ecological success of ants (hymenoptera: formicidae). myrmecological news, 21: 117-130. doi: 10.52 81/zenodo.845751 peeters, c. & molet, m. (2010). colonial reproduction and life histories. in: l. lach, c. parr & k. abbott (eds.), ant ecology (pp. 159–176). oxford university press. doi: 10.1093/ acprof:oso/9780199544639.003.0009 tillberg, c.v. & breed, m. (2004). placing an omnivore in a complex food web: dietary contributions to adult biomass of an ant. biotropica, 36: 266-271. doi: 10.1111/j.1744-7429. 2004.tb00318.x tschinkel, w.r. (2006). the fire ants. the belknap press of harvard university press, cambridge, ma, 723 pp. wheeler, d.e. (1982). soldier determination in the ant, pheidole bicarinata. ph.d. dissertation, duke university usa. doi: 10.13102/sociobiology.v61i4.566-569sociobiology 61(4): 566-569 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 oxytrigona tataira (smith) (hymenoptera: apidae: meliponini) as a collector of honeydew from erechtia carinata (funkhouser) (hemiptera: membracidae) on caryocar brasiliense cambessèdes (malpighiales: caryocaraceae) in the brazilian savanna fh oda1, af oliveira2, c aoki3 mutualisms are interspecific interactions where individuals of two species experience higher fitness when they occur together than when they occur alone (bronstein, 1998). this interaction is an important process for the structure and composition of communities (bascompte & jordano, 2007; lange & del-claro, 2014). many species of phytophagous hemipterans (e.g. aphids, coccids and treehoppers) are mutualistically associated with ants (flatt & weisser, 2000; offenberg, 2001; fagundes et al., 2013). in these relationships, ants protect their hemipteran partners against predators and parasites (schultz & mcglynn, 2000; fagundes et al., 2013) and often rely on honeydew as one of their most important food source (davidson et al., 2003), besides extrafloral nectar (del-claro et al., 2013) and lepidopteran secretions (alves-silva et al., 2013). abstract trophobiont insects are of general interest to behavioral ecology due to the fact that the outcomes of their interactions with hymenopterans can result in strong facultative mutualisms. in this paper we present observations of oxytrigona tataira (smith) collecting honeydew from erechtia carinata (funkhouser) on caryocar brasiliense cambessèdes in a cerrado sensu stricto fragment from brazilian savanna. our observations showed that these bees stimulate e. carinata individuals touching them with the antennas in the gaster to collect honeydew. o. tataira has not been previously recorded collecting honeydew on e. carinata. thus, we present these observations as a novel bee-treehopper interaction. sociobiology an international journal on social insects 1 universidade estadual de maringá (uem), maringá, pr, brazil 2 universidade federal de mato grosso do sul (ufms), campo grande, ms, brazil 3 universidade federal de mato grosso do sul (ufms), aquidauana, ms, brazil article history edited by cândida maria l. aguiar, uefs – brazil received: 22 september 2014 initial acceptance: 29 october 2014 final acceptance: 18 november 2014 keywords hemipterans, exudate, feeding behavior, "pequi", stingless bee corresponding author fabrício hiroiuki oda laboratório de ictioparasitologia núcleo de pesquisas em limnologia ictiologia e aqüicultura universidade estadual de maringá bloco g-90 avenida colombo, 5.790 87020-900, maringá, pr, brazil e-mail: fabricio_oda@hotmail.com although ants are the most common hymenopteran interacting with treehoppers, bees and wasps can also be associated with them (lin, 2006). mutualistic interactions have been reported involving the aetalionid treehopper aetalion reticulatum (linnaeus) with wasps or ants (letourneau & choe, 1987; ramoni-perazzi et al., 2006), and even trigona jurine, a stingless bee, which take advantage of treehoppers sugary excretions, the honeydew (castro, 1975; vieira et al., 2007; oda et al., 2009; barônio et al., 2012). godoy et al. (2006) reported eight associations between membracids and bees, including the interaction between potnia stål and oxytrigona tataira (smith). azevedo et al. (2007) reported the associations between trigona spinipes (fabricius) and five membracids species, and observed that the bees repelled some insects that approached the membracids, although with low aggressiveness. short note sociobiology 61(4): 566-569 (december 2014) 567 this kind of interaction has not been reported between the stingless bee o. tataira (apidae: meliponini) and the treehopper erechtia carinata (funkhouser) (auchenorrhyncha: membracidae). this membracid belongs to the tribe talipedini and it was recently reassessed, and trinarea goding is considered a junior synonym of erechtia walker (sakakibara, 2012). oxytrigona tataira has a wide distribution, occurring in eight brazilian states (silveira et al., 2002; lima et al., 2013). oxytrigona cockerell is commonly known in brazil as “tataíra” or “cospe-fogo” (fire spitting), due to caustic substance (formic acid) produced by worker bees for defense. these bees are highly aggressive, and also cleptobiotic, being colony’s robbers of other meliponine species (roubik et al., 1987; roubik, 1992). caryocar brasiliense cambessèdes has a wide distribution, occurring in 15 brazilian states and paraguay (lopes et al., 2006). it is commonly known as "pequi", found from shrubs to trees of 1.5 to 11 meters high, with semi-deciduous behavior of foliar change (lopes et al., 2006). although the use of secretions from sucking insects (hemiptera) by species of trigona and oxytrigona has been reported before (cortopassi-laurino, 1977; letourneau & choe, 1987), herein we present first observations of interaction between o. tataira and e. carinata on c. brasiliense in the brazilian savanna. during three non-consecutive days in january 2012, individuals of o. tataira were observed collecting honeydew from e. carinata in a cerrado sensu stricto fragment (17º 21’ s, 53º 18’ w) at alto araguaia municipality, state of mato grosso, mid-western brazil. the observations were carried out once per day, between 7:00 and 10:00 h. each observation lasted 15 to 20 minutes. voucher specimens were collected, processed and given to specialists for identification and deposition in the entomological collection padre jesus s. moure (dzup) of the department of zoology at the universidade federal do paraná (ufpr). we observed variable numbers of individuals of o. tataira (mean = 55 individuals ± 42.7 sd) collecting honeydew of approximately 130 nymphs and one adult of e. carinata. the number of o. tataira was lower during the second day of observations after heavy rain. the bees stimulated e. carinata individuals by primarily touching the treehoppers at the proximal upper abdomen with their front legs and antennas. the bees repeated the stimulus toward the distal part of the abdomen where the exudate droplet was collected with aid of the first pair of legs and quickly sucked. this behavior of the bees was recorded mainly in the treehoppers nymphs because only one adult was with the nymphs on the branch (fig 1). as a result of our presence the bees defended the food source by beating their wings as a warning sign (fig 2), and some individuals started a short flight over the e. carinata colony. in the first day of observations, inattentive researchers were attacked by the bees. we did not record the presence of ants patrolling the treehoppers. the behavior of e. carinata stimulation by o. tataira resembles that observed in species of trigona in association with a. reticulatum: the bees touch their antennas to the head of the treehoppers, and then the first two pairs of legs on the back of the abdomen, after that touching the antennas on the distal part of the abdomen and quickly sucking the honeydew droplet released after stimulation (vieira et al., 2007; oda et al., 2009; barônio et al., 2012). this behavior is also similar to that reported between ants and honeydew-producing hemipterans, including membracids, in which the ants actively touch the body of the insects with their antennae to stimulate the releasing of honeydew droplets (stefani et al., 2000; pfeiffer & linsenmair, 2007; guerra et al., 2011; gjonov & gjonova-lapeva, 2013). previous studies suggest that this possibly facultative mutualistic association is beneficial for both species (vieira et al., 2007; oda et al., 2009; barônio et al., 2012), promoting a protection against natural enemies of the treehoppers, and supplying part of the bees diet with the honeydew rich in carbohydrates (way, 1963; fagundes et al., 2013). additionally, the association with the bees can reduce fungus attack because of the removal of the contaminant honeydew (way, 1963; buckley, 1987), decreasing the chances of local extinction of these hemipterans (buckley, 1987). on ant-tending, the mortality risks of the tended insects can be considerably reduced due to protection against predators and parasitoids (flatt & weisser, 2000; fagundes et al., 2013). however, we did not test it. thus, it is important to emphasize the necessity of future studies, which are essential to know the true nature of this interaction. fig 1. oxytrigona tataira (smith) (hymenoptera: apidae: meliponini) collecting honeydew from erechtia carinata (funkhouser) (hemiptera: membracidae) on branch of caryocar brasiliense cambessèdes (malpighiales: caryocaraceae). fh oda, af oliveira, c aoki a bee-treehopper interaction 568 acknowledgements we are grateful to albino m. sakakibara and sebastião laroca for the identification of treehopper and bee species, respectively. to editor and two anonymous reviewers for their constructive comments, that helped us to improve the manuscript. guilherme h. barbosa, frederico pereira, vinícius cerqueira and patrícia g. ferreira for field assistance. mariana f. felismino reviewed the english language. the biocev provided logistical support during fieldwork. references alves-silva, e., bächtold, a., barônio, g. j. & del-claro, k. 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(1963). mutualism between ants and honeydewproducing homoptera. annu. rev. entomot., 8: 307-344. doi: 10.1146/annurev.en.08.010163.001515 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1360sociobiology 63(4): 1073-1075 (december, 2016) new records of social wasps around brasília (hymenoptera: vespidae: polistinae) from 1976 to 1998, social wasps were collected at various locations in the federal district (an area of 5,822 km2) at altitudes of 900 to 1,100 m. additional species in surrounding parts of goiás state might also be present in the federal district or might move there, so localities within 20 km of the state border were also surveyed. only a standard entomological net was used to collect the wasps. the specimens are in the collections of the universidade de brasília and of universidade federal de minas gerais. a total of 75 species in 18 genera have been recorded in and around brasília. of the 56 species of social wasps in 16 genera collected in the federal district (table 1), 9 are new records and some greatly extend the species ranges; some > 500 km. some species are geographically widespread, some have restricted and some disjunct distributions. a further 19 species and 2 genera were found in neighbouring parts of goiás state (table 2). the species richness of the social wasps of the federal district is substantially higher than has been recorded elsewhere abstract the aim of the present work was to discover how many species inhabit the environs of brasília. being an approximate rectangle in the middle of the cerrado biome, the federal district of brasília is a representative “quadrat” to sample the biome’s fauna. sociobiology an international journal on social insects a raw article history edited by gilberto m. m. santos, uefs, brazil received 27 september 2016 initial acceptance 20 october 2016 final acceptance 19 november 2016 publication date 13 january 2017 keywords paper wasps, swarm founding wasps, species substitution. corresponding author anthony raw departamento de ciências biológicas universidade estadual de santa cruz cep 45650-662 ilhéus-ba, brasil e-mail: anthonyraw2@gmail.com in the region (diniz & kitayama, 1998; grandinete & noll, 2013; elpino-campos et al., 2007; pereira & antonialli-junior, 2011). presumably, this greater species richness recorded from the federal district is a result of two factors. first, the region surveyed comprises various types of habitat. there are gallery forests, forest margins, dry forests, cerrados and suburban gardens. secondly, the survey was conducted, albeit with interruptions, over a period of more than twenty years. the prolonged collecting period substantially increased the previous species count. the present work adds nine species to the list of species known from the federal district. previous records are cited in four publications (richards, 1978; raw 1985, 1992, 1998). two species, m. campestris and m. giffordi were new to science (raw, 1985). a species cited as epipona quadrituberculata (gribodo, 1892) is now known to be epipona media cooper (2002). some species occurring in central brazil have broad geographical distributions (richards, 1978), but polistes satan, universidade de brasília, departamento de zoologia, brasília-df, brazil short note a raw – social wasps around brasília1074 polistes billardieri (f) brachygastra augusti (de saussure) cinerascens de saussure lecheguana (latreille) davillae richards moebiana (de saussure) erythrocephalus latreille chartergellus communis richards ferreri de saussure clypearia angustior ducke geminatus fox epipona media cooper goeldii (ducke) tatua (olivier) occipitalis ducke metapolybia cingulata (f) satan bequaert parachartergus fraternus (gribodo) subsericeus de saussure polybia chrysothorax (lichtenstein) versicolor (olivier) dimidiata (olivier) mischocyttarus annulatus richards emaciata ducke campestris raw fastidiosuscula de saussure cassanunga (von ihering) flavifrons smith cerberus richards ignobilis (haliday) drewseni (de saussure) jurinei de saussure giffordi raw liliacea (f) goyanus zikán occidentalis (olivier) latior (fox) paulista von ihering lecointei (ducke) rejecta (f) marginatus (fox) ruficeps schrottky mattogrossoensis zikán scrobalis richards melanoxanthus richards sericea (olivier) rotundicollis (cameron) protonectarina sylveirae (de saussure) agelaia pallipes (olivier) protopolybia exigua (de saussure) angiopolybia pallens (lepeletier) sedula (de saussure) apoica pallens (f) pseudopolybia vespiceps (de saussure) thoracica du buysson synoeca surinama (l) table 1. list of the 56 species of social wasps recorded in and around the federal district of brasília. mischocyttarus giffordi and m. goyanus seem to be restricted to the higher altitudes of goiás and minas gerais states and the federal district of brasília. extensions of geographical range of several species are recorded. the closest that polistes goeldii and m. annulatus had been recorded to brasília is at “base camp”, mato grosso, 540 km to the north-west (richards, 1978). polistes occipitalis is known from the states of pará, espírito santo, rio de janeiro and são paulo (op. cit.) so the closest previous known locality is at least 500 km away. apparently, the distribution of epipona media is disjunct; it has been found just 33 km south of the present brasília record (menezes et al., 2010). mischocyttarus campestris was found recently on the serra de bodoquena, mato grosso do sul (auko, 2015). after creating so many permanently green areas in the federal district over the past decades, some species have become more common in brasília. despite innumerable collections from 1976 to 1986,clypearia angustior was never found in the region, but in 1987 it was locally common in the residential area of lago sul. it is known from pará and são paulo states and an undetermined locality in minas gerais state (richards, 1978). polistes carnifex (f) metapolybia suffusa (fox) melanosoma de saussure parachartergus smithii (de saussure) mischocyttarus frontalis (fox) polybia bifasciata de saussure agelaia flavipennis (ducke) erythrothorax richards multipicta (haliday) quadricincta de saussure vicina (de saussure) striata (f) apoica flavissima van der vecht tinctipennis fox chartergus artifex christ protopolybia chartergoides (gribodo) globiventris de saussure pseudopolybia compressa (de saussure) charterginus fulvus fox table 2. list of the 19 species of social wasps recorded in goiás state within 20 kilometres of the federal district of brasília. sociobiology 63(4): 1073-1075 (december, 2016) 1075 acknowledgements the assistance of the late professor o. w. richards was invaluable in the identifications. the university of brasília provided me with the facilities to conduct this survey. dr alain dejean, université de toulouse; dr christopher k. starr, university of the west indies, trinidad and tobago and dr fernando noll, universidade estadual paulista (unesp), são josé do rio preto, são paulo, provided invaluable comments on an earlier version of the manuscript. references auko, t. h. (2015). diversidade local e regional de vespas (hymenoptera: vespoidea) no mato grosso do sul: com notas sobre a biologia de espécies solitárias. universidade federal da grande dourados, dourados, mato grosso do sul, brazil. unpublished thesis. diniz, i. r. and k. kitayama. (1998). seasonality of vespid species (hymenoptera: vespidae) in a centralbrazilian cerrado.revista de biologia tropical, 46: 109-114. elpino-campos, a., k. del-claro and f. prezoto. (2007). diversity of social wasps (hymenoptera: vespidae) in cerrado fragments of uberlândia, minas gerais state, brazil. neotropical entomology, 36: 685-692. grandinete, y. c. and f. b. noll.(2013). checklist of social (polistinae) and solitary (eumeninae) wasps from a fragment of cerrado “campo sujo” in the state of mato grosso do sul. sociobiology, 60: 101-106. pereira, m. g. c. and w. f. antonialli-junior. (2011). social wasps in riparian forest in bataiporã, mato grosso do sul, brazil. sociobiology, 57: 153-163. raw, a. (1985). two new species of mischocyttarus (vespidae, hymenoptera) from brazil. revista brasileira de entomologia, 29: 107-112. raw, a.(1992). the forest-savanna boundary and habitat selection by brazilian social wasps (hymenoptera: vespidae):499-511. in p.a.furley, j.a.ratter and j. proctor (eds.)the nature and dynamics of the forest-savanna boundary. chapman & hall, london, xxi + 616 pp. raw, a.(1998). population densities and biomass of neotropical social wasps (hymenoptera, vespidae) related to colony size, hunting range and wasp size. revista brasileira de zoologia, 15: 815-822. richards, o. w.(1978). the social wasps of the americas. british museum (natural history), london, 580 pp. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1027sociobiology 63(4): 1005-1014 (december, 2016) lipid mobilization and acyl-coenzyme a dehydrogenase abundance in the postpharyngeal gland of a leaf-cutting ant introduction the atta and acromyrimex genera are popularly known as leaf-cutting ants, the most economically important ants. these ant species cut large amounts of fresh plant material to feed the symbiotic fungus leucoagaricus gongylophorus singer (m.), which in turn provides food for the larvae and in part for the adult (hölldobler & wilson, 1990). the fatty acids may come either from feeding fungi or sap. fungi are rich in carbohydrates and protein, and low in lipids (martin et al., 1969). sap may be ingested by workers while they are cutting and trimming leaves and, therefore, take lipids directly (quinlan & cherrett, 1979). therefore, studies have shown that the postpharyngeal glands (ppgs) are involved in lipid accumulation. ppg anatomy and lipid content were first described by peregrine et al. (1972, 1973), peregrine and mudd (1974), delage-darchen (1976), and vieira and bueno (2015). abstract the postpharyngeal gland (ppg) in ants has a central role in lipid accumulation. our objective was to investigate whether the ppg of workers of the leaf-cutting ant atta sexdens rubropilosa (f.) performs lipid mobilization to the hemolymph, and to verify whether lipids in gland cells are in part oxidized to acetyl-coa. increased levels of fatty acids were observed in both the ppg and the hemolymph after 48 h of lipid supplementation. in contrast, after 120 h of lipid supplementation, fatty acid levels were reduced in the ppg and significantly increased in the hemolymph. the gland cells of the ppg of workers supplemented with lipids exhibited a high amount of acylcoa dehydrogenase, and the lipids were in part β-oxidized to acetylcoa. these findings suggest that the ppg of a. sexdens rubropilosa workers mobilize fatty acids from their gland cells to the hemolymph. further work will be needed to elucidate lipid transport from the hemolymph to other organs in this organism. sociobiology an international journal on social insects as vieira, oc bueno article history edited by evandro nascimento silva, uefs, brazil received 14 march 2016 initial acceptance 08 june 2016 final acceptance 15 december 2016 publication date 13 january 2017 keywords attini, confocal microscopy, hemolymph, insects, gc-ms. corresponding author alexsandro santana vieira centro de estudos de insetos sociais instituto de biociências unesp av. 24a, nº. 1515, cx. postal 199 cep: 13506-900, rio claro-sp, brasil e-mail: alexsvieira@yahoo.com.br ppgs, the salivary system of ants, occur in pairs and are located dorsally in the transition between the pharynx and the esophagus regardless of sex, caste, or lifestyle (hölldobler & wilson, 1990; caetano, 1998). ppgs are of ectodermal origin and form during post-embryonic development (late prepupal stage) from two dorsal evaginations of the pharyngeal epithelium (gama, 1985; janet, 1905). until recently, ppgs were thought to be exclusive to formicidae (delage-darchen, 1976; hölldobler & wilson, 1990), but herzner et al. (2007) found them in the solitary wasps philanthus triangulum (f.), ampulex compressa (f.), trachypus boharti (r.-e.), and trachypus elongatus (f.) (herzner et al., 2011; 2013). ppgs also play a role in lipid metabolism (peregrine et al., 1972, 1973; peregrine & mudd 1974; delage-darchen, 1976; decio et al., 2016). in insects, the fat body is the central organ for lipid metabolism, synthesizing several proteins and also acting as the storage site for carbohydrates, proteins, and universidade estadual paulista (unesp), rio claro-sp, brazil research article ants as vieira; oc bueno – lipid and acyl-coa dehydrogenase in ppg of leaf-cutting ants1006 mainly lipids (arrese & soulages, 2010). however, the source of lipids in the ppg is controversial. one possibility is that lipids originate from secretions of the glandular epithelium itself. another possibility is that it is taken up from the diet. delage-darchen (1976) proposed three hypotheses about the accumulation of lipid in ppgs: 1) transcellular route: lipids would penetrate the gland cells and be metabolized; 2) extraoral route: lipids would be regurgitated directly from the glands to other members of the colony; and 3) digestive route: lipids would flow from the glands to the crop and then to the midgut. using labeled isotopes, phillips and vinson (1980) observed that, in solenopsis invicta (b.), fatty acids and triacylglycerides injected into the hemolymph were not transferred to the ppgs, suggesting that lipids in this gland would be derived from food. delage-darchen (1976) and forbes and macfarlane (1961) hypothesized that ppgs would be responsible for the digestion of ingested fats based on the presence of digestive enzymes such as lipases in the gland lumen (peregrine et al., 1973; vinson et al., 1980). the digestive route hypothesis proposed by delage-darchen (1976) was tested by bueno (2005), which demonstrated that atta sexdens rubropilosa workers were capable of separating, in the final portion of the pharynx, lipid from non-lipid compounds present in the ingested food. on a lipid-rich diet, these compounds would reach the ppgs and the non-lipid components (e.g., carbohydrates) would move to the crop, which would allow adults to use lipids available in nature (bueno et al., 2008). under extreme conditions, in which a large amount of lipid is offered to ants, some of it reaches the crop; however, sometime later, they return through the esophagus and enter the ppg (phillips & vinson, 1980; wheeler, 1994; jesus, 2006; bueno et al., 2008). moreover, using autoradiography, liquid scintillation counting, and tritiated oleic acid, bueno (2005) showed that lipids are mobilized into the lumen of glands of a. sexdens rubropilosa workers and queens, with subsequent absorption by the gland epithelium. however, little is known about the presence of fatty acids in the hemolymph. therefore, the transcellular route hypothesis of delage-darchen (1976) should be tested: is the ppg of workers of the leaf-cutting ant a. sexdens rubropilosa capable of mobilizing lipids to the hemolymph? in this way, the present study aimed to investigate whether the ppg of workers of the leaf-cutting ant a. sexdens rubropilosa performs lipid mobilization to the hemolymph. workers of a. sexdens rubropilosa ants supplemented with lipids showed an increase in the amount of mitochondria and peroxisomes within ppg cells compared to those without supplement (vieira & bueno, 2015) so, another aimed to verify whether lipids are in part oxidized to acetyl-coa in the mitochondria of gland cells. material and methods part i – gas chromatography/mass spectroscopy – lipid mobilization to the hemolymph bioassays i medium workers of a. sexdens rubropilosa were divided into three groups. group bls (before lipid supplement) received a pure diet and groups als (after lipid supplement) received pure diet with lipid supplementation for 48 h and 120 h. the pure diet consisted of 5 % glucose, 1 % casein peptone, 0.1 % brewer’s yeast extract, and 1.5 % bacteriological agar. the diet of groups als consisted of 0.3 ml of soybean oil in 30 ml of pure diet. each group contained 50 individuals that were placed in five petri dishes (10 ants in each dish), and 0.5 g of supplementation was added daily to each dish for 25 days. the dishes were kept in a chamber incubator (bod, biochemistry oxygen demand) at 25 ± 1 °c and at a relative humidity of 70 %. five replicates were performed for each experimental group. chromatographic analysis was performed using medium workers of the group bls, als 48 h and als 120 h. a total of 114 workers were used for this analysis, or 57 ants for each extraction (hemolymph and ppg): 18 ants for group bls, 18 for group als 48 h, and 18 for group als 120 h. the hemolymph of the workers was extracted by capillarity. with a microcapillary tube, 0.5 to 0.75 µl of hemolymph was extracted from the neck region of each ant and placed in vials. each vial contained hemolymph from three ants, totaling six vials per group. after extraction, 0.5 ml of acetone was added to each vial. ppgs from ants fed with and without lipid supplementation were extracted and three glands were placed in each vial, resulting in six vials per group; 0.5 ml of acetone was added to each vial and the ppgs were then macerated. fischer esterification (methylation) fischer esterification is a special type of esterification by refluxing a carboxylic acid and an alcohol in the presence of an acid catalyst (fischer & speier, 1895). in our study, the hydrolysis of lipids through esterification was performed to obtain the corresponding fatty acid ester and thus facilitate its movement through the injector and chromatography column in the gas phase chromatography. due to the esterification process, the fatty acids were identified in their ester form. samples underwent fischer esterification prior to chromatographic analysis. the vials containing the hemolymph and the macerated ppgs were left half open to allow evaporation of acetone in an infrared weighing-machine at 60 °c (for approximately 20 min). a saponification reaction was carried out by adding 0.3 ml of 0.5 n koh in anhydrous methanol, and the mixture was then homogenized and incubated at 60 °c for 20 min. the esterification reaction was initiated by the addition of 0.5 ml of 0.9 n h2so4 in anhydrous methanol, and the mixture was homogenized and incubated at 60 °c for 20 min. next, 1 ml of hexane was added for spectrometry and the mixture homogenized. a biphasic solution was obtained, and 50 µl were taken from sociobiology 63(4): 1005-1014 (december, 2016) 1007 the upper (aqueous) phase and placed in a new vial. the contents were then diluted with 450 µl of hexane, and 1 µl was injected into the gas chromatograph. gas phase chromatography mass spectrometry chemical analyses were conducted at the laboratory of apicultural products (laboratório de produtos apícolas), center for studies of social insects (centro de estudos de insetos sociais ceis), using a gas chromatograph gc-210 (shimadzu scientific instruments, japan) equipped with an rtx ms capillary column (30 m × 0.25 mm id, 0.25-μm thick film) coupled to a mass-selective detector (shimadzu gcmsqp2010 plus). the gas chromatograph was connected to a computer and the data were processed using gcmssolution software version 2.5. elution was carried out with helium at 1 ml/min. samples were injected using the splitless mode (port temperature: 250 °c) and held for 1 min. the temperature of the column was set to start at 160 °c for 5 min, increase to 200 °c at 5 °c/min, remain at 200 °c for 3 min, increase to 280 °c at 8 °c/min, and remain at 280 °c for 6 min. the mass spectrometer was operated in the electron ionization mode at 70 ev, with a scanning range of 40 to 500 m/z. to allow sample identification and quantification, a calibration curve was obtained using fatty acid standards (lauric, myristic, palmitic, linoleic, oleic, stearic, and erucic acid) from sigmaaldrich at the following concentrations: 0.5, 1, 2, 3, and 5 ppm. statistics after testing the data for normality, analysis of variance (anova) was conducted with a significance level of 5 % (0.05) between group bls and groups als 48 h and als 120 h. when significant differences were observed, a pairwise tukey test was performed to check which groups were different from each other using statistical software version 7. part ii confocal microscopy β-oxidation of fatty acids bioassays ii medium workers were divided into three groups of 24 individuals: two groups were fed a diet without lipid supplementation and starved for 48 h (fast 48 h) and 120 h (fast 120 h), whereas two groups were fed a diet supplemented with 2 µl soybean oil placed in the mouthparts and starved for 48 h (lipid 48 h). workers were placed individually in plastic containers (200 ml) and kept in a bod incubator under controlled conditions (25 ± 1 °c; relative humidity of 70 %). workers were anesthetized by cooling (4 °c) for a few min, their heads were removed and placed in petri dishes, and ppgs were dissected in insect saline solution (0.128 m nacl, 0.016 m na2hpo4, and 0.019 m kh2po4, ph 7.2) under a stereomicroscope using fine-tipped tweezers and surgical microscissors. confocal laser scanning microscopy – immunofluorescence of the enzyme acyl-coa dehydrogenase β-oxidation of fatty acids ppgs from groups fast 48 h, fast 120 h, and lipid 48 h were used. after dissection, 24 glands were fixed in 4 % paraformaldehyde for 30 min and then permeabilized with 0.1 % triton® x-100 for 30 min. the glands were incubated at 4 °c for 16 h with 10 μg/ml of anti-acyl-coa dehydrogenase primary mouse monoclonal antibody (very long-chain acyl-coa dehydrogenase, vlcad, monoclonal antibody, molecular probes). cy5-conjugated goat anti-mouse igg (h+l) (molecular probes) diluted 1:1,000 and incubated for 1 h was used as secondary antibody. at all stages, 10 % goat serum was used as a blocking agent. cell nuclei were counterstained with dapi (4 ‘,6-diamino-2-fenilindol). immunofluorescence from vlcad and nuclei (dapi) in the ppgs were recorded in a confocal laser scanning microscope lsm780-nlo (zeiss). fluorescent images were obtained using lasers with 405 nm and 633 nm excitation wavelengths. optical sections were acquired in a suitable z-axis sectioning step (0.63 µm and 0.67 µm). photography parameters were the same for all groups to quantify the emitted fluorescence intensity and to acquire images of the total gland area. imagej software and different modules of the zeiss lsm780-nlo software were used for image analysis, including maximum projection (join all z-axis sectioning step sizes). fluorescence emission of ppg without incubation with primary antibody but only with cy5 secondary antibody was used as negative control. statistical analysis fluorescence emitted (grayscale) by immunofluorescence of the acyl-coa dehydrogenase enzyme was quantified using imagej software. using bioestat statistical software, the shapiro-wilk test was carried out to assess normal distribution of data, and then the one-way anova parametric statistical test was performed. results lipid mobilization to the hemolymph overall, four fatty acids (c16, c18:2, c18:1, and c18) were present in both the hemolymph and ppg of ants from groups bls and als. only two fatty acids, c16 and c18, were found in group bls (table 1, fig 1a, 1b, and 1c). fatty acids bls hemolymph (ppm) hemolymph (%) ppg (ppm) ppg (%) palmitic acid 0.04716 53.43 0.06840 49.99 linoleic acid 0 0 0 0 oleic acid 0 0 0 0 stearic acid 0.04110 46.57 0.06844 50.01 total 0.08826 100 0.13685 100 table 1. fatty acid quantification in the hemolymph and postpharyngeal glands (ppg) of atta sexdens rubropilosa workers before lipid supplementation (bls). as vieira; oc bueno – lipid and acyl-coa dehydrogenase in ppg of leaf-cutting ants1008 in addition to c16 and c18, two other fatty acids, c18:2 and c18:1, were found in the hemolymph and ppg in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). fatty acid content in the hemolymph increased considerably in groups als 48 h and als 120 h. interestingly, fatty acid content increased in the ppg in the first 48 h of lipid fig 1. chromatographic analysis of fatty acids in the hemolymph and postpharyngeal glands (ppg) of atta sexdens rubropilosa workers. a. group of workers before lipid supplementation (bls). b. group of workers after lipid supplementation for 48 h (als 48 h). c. group of workers after lipid supplementation for 120 h (als 120 h). 1, palmitic acid; 2, stearic acid. arrows indicate a saturated hydrocarbon (alkane) with different retention times. sociobiology 63(4): 1005-1014 (december, 2016) 1009 supplementation, but decreased after 120 h (tables 1 to 3). palmitic acid c16. c16 was the predominant fatty acid in the hemolymph in group bls (table 1; fig 1a), whereas in groups als 48 h and als 120 h it corresponded to the second and third most abundant fatty acid, respectively (tables 2 and 3; figs 1b and 1c). c16 was the second most abundant fatty acid in the ppg in group bls (table 1; fig 1a), and the third most abundant in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). linoleic acid (c18:2). c18:2 was absent from the hemolymph in group bls (table 1; fig 1a), whereas it was the predominant fatty acid in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and c). c18:2 was also absent from the ppg in group bls (table 1; fig 1a), but it was the predominant fatty acid in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). oleic acid (c18:1). c18:1 was absent from the hemolymph in group bls (table 1; fig 1a). on the other hand, this fatty acid was the third and second most abundant in groups als 48 h and als 120 h, respectively (tables 2 and 3; fig 1b and 1c). c18:1 was also absent from the ppg in group bls (table 1; fig 1a), but it was the second most abundant fatty acid in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). stearic acid (c18). ants of group bls showed c18 as the second most abundant fatty acid in the hemolymph (table 1; fig 1a). c18 was the fourth most abundant fatty acid in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). c18 was the predominant fatty acid in the ppg in group bls (table 1; fig 1a), but it was only the fourth most abundant in groups als 48 h and als 120 h (tables 2 and 3; fig 1b and 1c). hydrocarbons (hc). as can be seen in figure 1a, two hcs identified as alkanes were detected in the ppg in group bls. hcs were also observed in ants of groups als 48 h and als 120 h, although with a smaller area than that in group bls (fig 1b and 1c). one-way anova revealed a statistically significant difference in fatty acid content in the hemolymph of groups als 48 h and als 120 h (f = 6.9161, p = 0.0151) in comparison with group bls (fig 2). a posteriori tukey test revealed a statistically significant difference between groups fatty acids als 48 h hemolymph (ppm) hemolymph (%) ppg (ppm) ppg (%) palmitic acid 0.03918 21.22 0.50576 9.36 linoleic acid 0.07802 42.25 3.03704 56.18 oleic acid 0.03816 20.67 1.55904 28.84 stearic acid 0.02929 15.86 0.30375 5.62 total 0.18465 100 5.40559 100 table 2. fatty acid quantification in the hemolymph and postpharyngeal glands (ppg) of atta sexdens rubropilosa workers after lipid supplementation for 48 h (als 48 h). fatty acids als 120 h hemolymph (ppm) hemolymph (%) ppg (ppm) ppg (%) palmitic acid 0.12077 19.94 0.45538 9.59 linoleic acid 0.26897 44.41 2.65249 55.85 oleic acid 0.14476 23.90 1.35750 28.58 stearic acid 0.07113 11.75 0.28409 5.98 total 0.60563 100 4.74946 100 table 3. fatty acid quantification in the hemolymph and postpharyngeal glands (ppg) of atta sexdens rubropilosa workers after lipid supplementation for 120 h (als 120 h). fig 2. mean concentration (ppm) and standard deviation of fatty acid content in the hemolymph in groups of workers before lipid supplementation (bls) and after lipid supplementation (als 48 h and als 120 h). bars marked with the same letter show no statistically significant difference (p > 0.05); bars marked with different letters show statistically significant differences (p < 0.05). p values below 5 % were considered significant. a statistically significant difference in fatty acid content was found in ppgs of ants in group bls compared to that in groups als 48 h and als 120 h (f = 13.6607; p = 0.0001). a posteriori tukey test revealed a statistically significant difference between group bls and groups als 48 h and als 120 h (p < 0.01), but not between group als 48 h and group als 120 h (fig 3). the same pattern was found for individual fatty acids (table 4). a schematic representation of lipid mobilization from the ppg to the hemolymph after lipid supplementation in leaf-cutting ants is shown in fig 4. β-oxidation of fatty acids the ppgs of a. sexdens rubropilosa medium workers have two lobes (named left and right). these lobes have digitiform prolongations formed by a simple epithelium composed of epithelial cells with large, round nuclei (shown in fig 5 in blue). indirect immunofluorescence analysis of ppgs bls and als 120 h (p < 0.05), as well as between groups als 48 h and als 120 h (p < 0.05). although fatty acid content increased from group bls to group als 48 h, no statistically significant difference was found between these groups (fig 2). as vieira; oc bueno – lipid and acyl-coa dehydrogenase in ppg of leaf-cutting ants1010 fig 4. schematic representation of lipid mobilization from the postpharyngeal gland (ppg) to the hemolymph after lipid supplementation in the leaf-cutting ant atta sexdens rubropilosa. lipids from the diet are forwarded (white arrow) to the lumen (l) of a digitiform prolongation in the ppg and arrive at the secretory cell (sc) after crossing the cuticle (cut). once inside the cell, lipids are mobilized (black arrow) to the hemolymph. fatty acids identified by gc-ms are shown as colored ovals: c16, green; c18, yellow; c18:1, blue; c18:2, black. oval size is proportional to the amount of fatty acid detected. fatty acids ppg anova tukey bls and als 48 h bls and als 120 h als 48 h and als 120 h palmitic acid f(2.15) = 26.952, p < 0.01 p < 0.01 p < 0.01 p > 0.05 linoleic acid f(2.15) = 24.426, p < 0.01 p < 0.01 p < 0.01 p > 0.05 oleic acid f(2.15) = 33.718, p < 0.01 p < 0.01 p < 0.01 p > 0.05 stearic acid f(2.15) = 32.302, p < 0.01 p < 0.01 p < 0.01 p > 0.05 p values below 5 % were considered significant. table 4. one-way anova of fatty acid content in the postpharyngeal gland (ppg) of a. sexdens rubropilosa workers between groups before lipid supplementation (bls) and after lipid supplementation (als) for 48 and 120 h. p values below 5 % were considered significant. of a. sexdens rubropilosa workers revealed a low fluorescence intensity, and therefore a low acyl-coa dehydrogenase abundance, in cells of the digitiform prolongation in the ppgs in groups fast 48 h and fast 120 h (fig 5a to 5d, red). in contrast, a strong fluorescence signal was detected in the ppgs of group lipid 48 h, indicating an abundance of acylcoa dehydrogenase in these cells (fig 5e and 5f, red). after quantification of the emitted fluorescence intensity, statistical analysis was performed. a statistically significant difference was found between groups with (lipid 48 h) and without (fast 48 h and fast 120 h) lipid supplementation (f = 7.978; p = 0.0065). a posteriori pairwise tukey test revealed a significant difference between groups fast 48 h and lipid 48 h (p < 0.05), as well as between groups fast 120 h and lipid 48 h (p < 0.01) (fig 6). p values below 5 % were considered significant. fig 3. mean concentration (ppm) and standard deviation of the fatty acid content in the postpharyngeal gland (ppg) in groups before lipid supplementation (bls) and after lipid supplementation (als 48 h and als 120 h). bars marked with the same letter show no statistically significant difference (p> 0.05); bars marked with different letters show statistically significant differences (p < 0.01). p values below 5 % were considered significant. fig 6. one-way anova analysis of fluorescence intensity (values in grey scale) reflecting acyl-coa dehydrogenase abundance in the measured areas of a gland cell of postpharyngeal glands of atta sexdens rubropilosa workers in groups without (fast 48 and fast 120 h) and with (lipid 48 h) lipid supplementation. bars marked with the same letter show no statistically significant difference (p > 0.05); bars marked with different letters show statistically significant differences (p < 0.05). p values below 5 % were considered significant. sociobiology 63(4): 1005-1014 (december, 2016) 1011 discussion the presence of lipid inside ppgs has been observed in several ant species, and its origin is controversial. one possibility is that lipid would originate from secretions of the glandular epithelium itself; others believe it comes from food. our study has shown that a. sexdens rubropilosa workers from group bls contained two fatty acids (c16 and c18) both in the ppg and hemolymph. fatty acids may come either from feeding the fungi or from sap. fungi are rich in carbohydrates and protein, but poor in lipids (martin et al., 1969). workers may ingest sap during cutting and trimming fig 5. confocal microscopy analysis of postpharyngeal glands (ppg) of atta sexdens rubropilosa workers showing the fluorescence emitted (cy5) by immunofluorescence of acyl-coa dehydrogenase (red) and stained (dapi) nuclei (blue). a-b. digitiform prolongation of the ppg of a worker from group fast 48 h showing nuclei and the low abundance of acyl-coa dehydrogenase in gland cells. c-d. digitiform prolongation of the ppg of a worker from group fast 120 h showing nuclei and the low abundance of acylcoa dehydrogenase in gland cells. e-f. digitiform prolongation of the ppg of a worker from group lipid 48 h showing nuclei and the high abundance of acyl-coa dehydrogenase in gland cells. as vieira; oc bueno – lipid and acyl-coa dehydrogenase in ppg of leaf-cutting ants1012 leaves, and, therefore, lipids may be taken directly (quinlan & cherrett, 1979). the two fatty acids (c16 and c18) we have detected in the ppg may thus be obtained either from fungal food or from cutting leaves. in previous studies using cytochemical ultrastructural analysis, vieira and bueno (2015) observed lipid droplets in the basal plasma membrane near the hemolymph. our study demonstrated that when ants were supplemented with lipids (groups als 48 h and als 120 h), two additional fatty acids (18:2 and c18:1) were found both in the ppg and hemolymph. these results are in agreement with the transcellular route hypothesis of delage-darchen (1976), in which lipids go from the lumen to the gland cells and are then metabolized. phillips and vinson (1980) observed in s. invicta that fatty acids and triacylglycerides injected into the hemolymph did not reach the interior of the ppgs. according to arrese and soulages (2010), in insects, lipids from food reach the digestive tract and are then transported by lipoproteins to the fat body for storage. after metabolization of these lipids according to nutritional needs, they are transported to the hemolymph and from there distributed to the tissues. we suggest that lipids reach the hemolymph from the ppg after lipid supplementation. the ppg would therefore be capable of storing lipids and of secreting them into the hemolymph, from which these molecules can be stored in the fat body. the entry of lipids into the ppgs and subsequent mobilization to the hemolymph requires some type of hydrolysis. the hydrolysis of triacylglycerol to free fatty acids may initially involve lipases (alberts et al., 2010). lipases have not been found in the ppgs of acromyrmex octospinosus (f.) (febvay & kermarrec, 1986), and α-glucosidase and esterase were detected at low levels in the ppg. the authors suggested that this gland does not have a digestive function, as hypothesized by delage-darchen (1976). it has been shown that lipids from the diet go straight to the ppg and that, when large amounts of lipids are offered to ants, some of it reaches the crop, but not the midgut (bueno et al., 2008). erthal et al. (2004) detected α-glucosidase as the most abundant enzyme in the midgut of leaf-cutting ants, indicating that it is probably involved in glucose assimilation from nutrient sources such as maltose and sucrose present in plant material. decio et al. (2016) showed that the ppg is an organ specialized for lipid nutrition in leaf-cutting ants, and this organ is responsible for lipid metabolism in a. sexdens rubropilosa. furthermore, two proteins responsible for lipid transport in ppg cells, long-chain fatty acid transport protein and fatty acid binding protein, were shown to be present in higher amounts in ants fed with lipid supplementation than in ants fed without lipid supplementation (decio et al., 2016). these proteins act as carriers and direct the mobilization of lipids into or out of the cell (börchers & spener, 1994; schaffera & lodish, 1994). based on our results, we believe that the ppg of leaf-cutting ants is also specialized in lipid mobilization to the hemolymph. statistical analysis showed that lipid supplementation caused fatty acid accumulation in the ppg during the first 48 h, with moderate mobilization of individual fatty acids to the hemolymph. however, after 120 hours of lipid supplementation, mobilization of fatty acids to the hemolymph was greatly increased, with a concomitant decrease in fatty acid content in the ppg. these results are in agreement with those described by bueno (2005), in which a. sexdens rubropilosa workers fed on tritiated oleic acid showed a decrease in lipid content in the ppg after 120 h. therefore, our results indicate that lipid mobilization from the ppg to the hemolymph occurs after some time, and that this delay reflects the storage capacity of its gland cells. since the abundance of different fatty acids in the ppg and the hemolymph vary between groups bls and als, we believe that lipid mobilization from the ppg to the hemolymph may depend on both the nutritional needs and the resources available to a. sexdens rubropilosa workers. furthermore, c18:1 and c18:2 in group als may be used as vehicles for active ingredients in the chemical control of leaf-cutting ants. corroborating this idea, we detected c18:2 as the most abundant fatty acid in both the hemolymph and ppg in groups als 48 and als 120 h, whereas c18:1 was the second most abundant. thus, after lipid supplementation, c18:2 becomes the main fatty acid in the ppg and hemolymph of a. sexdens rubropilosa workers. toxicological bioassays conducted with hydramethylnon diluted in soybean oil and a. sexdens rubropilosa (sumida, 2007; decio et al., 2013) showed a possible alternative route of ingestion of this active ingredient in the ppgs of leaf-cutting ants with increased pesticide action. the presence of hydrocarbons (alkanes) in the ppgs of groups bls, als 48 h, and als 120 h may result from allogrooming or from the cuticle. soroker et al. (1998) and morgan (2008) showed that hydrocarbon mobilization to the ppg could occur by allogrooming. a study on hydrocarbon biosynthesis in cataglyphis niger (a.) showed that some lipids, but not hydrocarbons, are synthesized in the gland (soroker & hefetz, 2000). still, bagnères and morgan (1991) demonstrated that, in several ant species, the ppgs contain the same substances that are found in the cuticle, i.e., mainly long-chain acids and esters. it is possible that the presence of hydrocarbons in the ppg of a. sexdens rubropilosa reflects the fact that this organ is of ectodermal origin, i.e., it has a cuticle, and that these alkanes originate from the cuticle. the most abundant organelles in ppgs are mitochondria, distributed throughout the cytoplasm. in the ppg of camponotus rufipes (f.), dinoponera australis (e.), and dinoponera quadriceps (k.), mitochondria are elongated and show different sizes (falco, 1992; schoeters & billen, 1997). recent studies by vieira and bueno (2015) have found an increase in the number of peroxisomes and mitochondria in gland cells of the ppg of a. sexdens rubropilosa workers when subjected to lipid supplementation. the mitochondrial organelle is responsible for the β-oxidation of fatty acids to acetyl-coa, which then enters the citric acid cycle to be further oxidized and generate atp (alberts et al., 2010; sociobiology 63(4): 1005-1014 (december, 2016) 1013 nelson & cox, 2014). decio et al. (2016) showed that part of the lipids in the ppg of mated females are used as substrate for the synthesis of acetyl-coa. based on these findings, we investigated the β-oxidation of fatty acids in the ppg of workers and the results were surprising: they show a low abundance of acyl-coa dehydrogenase in ppg cells of workers in groups fast 48 h and fast 120 h, and a high abundance in group lipid 48 h. ppgs of both the mated females (decio et al., 2016) and the a. sexdens rubropilosa workers in our study metabolized part of the lipids by β-oxidation. we hypothesize that the energy produced in gland cells by β-oxidation is necessary for lipid mobilization between the ppg and the hemolymph. finally, we conclude that the ppg of a. sexdens rubropilosa workers mobilize fatty acids from their gland cells to the hemolymph. therefore, in addition to performing lipid metabolism (decio et al., 2016), the ppg is also an organ specialized in lipid mobilization to the hemolymph. further work will be needed to elucidate lipid transport from the hemolymph to other organs in this organism. acknowledgements we thank technician sebastião zanão from the gas chromatography mass spectrometry laboratory (unesp) for his help with the protocol and handling of the equipment and technician mariana ozello baratti from the confocal microscopy laboratory infabic-unicamp, campinas, sp, brazil. we also thank the são paulo research foundation (fapesp) (grant 2012/12541-3) and the national council for scientific and technological development (cnpq) (grant 157837/2015-7) for financial support. references alberts, b., johnson, a. & walter, p. 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(ed.). nourishment & evolution in insect societies. boulder, colorado: westview press. p. 245-278. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1067sociobiology 64(1): 1-6 (march, 2017) insecticidal activity of the soil in the rhizosphere of viburnum odoratissimum against solenopsis invicta (hymenoptera: formicidae) introduction the red imported fire ant (rifa), solenopsis invicta buren, is an important invasive species native to south america. rifas are more aggressive than other ant species and have been identified to disrupt the balance of natural ecosystems (porter, 1990). rifas also cause considerable harm in infested urban and agricultural areas. in the city, rifa colonies are commonly found in gardens, lawns, parks, schoolyards, and golf courses. these insects are a threat to people’s health because of their sting, which may cause inflammation, vesicles, sterile pustules, allergic phenomena, and even anaphylactic shock (haddad et al., 2015). the venom of the rifa is composed of various alkaloids, which can negatively affect the cardiovascular and central nervous system of vertebrates (howell et al., 2005). the rifa has abstract methyl salicylate produced by viburnum odoratissimum is known to exert lethal or sublethal effects on insects. replacing conventional pesticides with insecticidal plants is necessary for environmental protection. we evaluated the behavioral and toxicological responses of the red imported fire ant (rifa solenopsis invicta, buren) (hymenoptera: formicidae) at different soil depths in the rhizosphere of v. odoratissimum. results of insecticidal activity bioassays indicated that the mortality for minor and major ants in soil at depths of 0-10 cm at days 11 and 12 both ranged from 68.75% to 100.00%, with repellency rates of 83.54%-100.00% and 85.31%-100.00%, respectively. in behavioral ability tests, 85.45%-100.00% of minor ants and 86.74%-94.85% of major ants lost their ability to grasp after nine days, with crawling rates at only 0.00%29.25% and 0.00%-55.77%, respectively. therefore, we conclude from the result that the soil under v. odoratissimum at depths of 0-10 cm exhibited excellent insecticidal effect in controlling rifa. sociobiology an international journal on social insects y zhang1, j t fu1, c l huang1, d m cheng1,3, r l huang1, z x zhang1,2,3 article history edited by evandro nascimento silva, uefs, brazil received 10 may 2016 initial acceptance 08 january 2017 final acceptance 27 february 2017 publication date 29 may 2017 keywords mortality, repellency, soil, leaf, methyl salicylate. corresponding author zx zhang south china agricultural university key laboratory of natural pesticide & chemical biology ministry of education, guangzhou 510642, china. e-mail: zdsys@scau.edu.cn spread over a number of countries and regions; it was detected in taiwan in 2003 (zhang et al., 2007) and mainland china in 2004 (zhang et al., 2013). at present, the species can be found in south china and occupies an area of at least 71 km2 (lai et al., 2015). rifas are usually controlled by applying traditional insecticides, such as richlorphon or clofenotane; both are harmful to humans and the environment. thus, the search for natural, safe and non-polluting methods began to attract considerable attention, and many efforts have focused on exploring biological control (appel et al., 2004; vogt et al., 2002). methyl salicylate is an organic ester that is naturally produced by viburnum odoratissimum (wei et al., 2013). it is a volatile oil with a characteristic wintergreen odour and taste, and is used as a flavouring agent and as a topical counterirritant for muscle pain (liu et al., 2013). the compound was first 1 south china agricultural university, key laboratory of natural pesticide & chemical biology, ministry of education, guangzhou, china 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 zhongkai university of agriculture and engineering, guangzhou, china research article ants y zhang et al. – insecticidal activity of the soil in the rhizosphere 2 registered as a pesticide in 1972 for use as an animal repellent in impregnated twist tabs hung on plants to repel dogs and cats from flower gardens. in 1996, the hygienic standards for uses of food additives approved methyl salicylate as an insect repellent to be used as a constituent of food and feed packaging material to repel insects in stored commodities. in this study, methyl salicylate from v. odoratissimum leaves and soil were analyzed by using high-performance liquid chromatography (hplc), and soil toxicity was determined to evaluate the effectiveness of the soil in the rhizosphere of v. odoratissimum in controlling the rifas. materials and methods plant v. odoratissimum used in the study was planted in the insecticidal botanical garden at south china agricultural university. the height of the trees was approximately 2 m, and the basal stem diameter was approximately 3 cm. grasses and other small plants within 5 m of the basal stem were cleared artificially within 30 days. all plants were cleared artificially within 30 days in a ck field (20 m2), which is located approximately 30 m from the field of v. odoratissimum. chemicals methyl salicylate was purchased from jingchun chemical reagent co., ltd., shanghai, china. hplc-grade acetonitrile and methanol, as well as analytically pure phosphoric acid, were purchased from guangzhou chemical reagent factory. insects adult workers of s. invicta colonies were obtained directly from nests in south china agricultural university. workers were reared in plastic cases coated with teflon emulsion on the top, in which a test tube (25 mm × 200 mm) filled with water and plugged with cotton was used to provide water. a petri dish (8.5 cm × 1.5 cm) that contained individuals of tenebrio molitor was placed in containers for the food source. individuals of t. molitor was purchased from the insect/fish market in guangzhou, fed with wheat bran, and kept in a dry indoor environment at 25 ± 2 °c until use. small worker ants were approximately 3 mm in length, whereas large worker ants applied in the experiment were approximately 6 mm in length (cheng et al., 2008; zhou et al., 2013; zhang et al., 2013; tang et al., 2013). collection of soil and leaf samples the soil samples were collected at five points/tree, 20 cm from the basal stem, at depths of 0-5, 5-10, 10-15 and 15-20 cm. soil samples from the same depth were combined and ground, and then sifted with a 40 mesh sieve. each sample was replicated thrice, and a tree was used in each replication. soil weight/replicate was set at >500 g. the soil samples were collected randomly at five points at the same depth. for the leaf samples, young and old green leaves, as well as yellow leaves, were picked from each tree. leaf weight/replicate was obtained at >200 g. half of the leaves and soil samples were placed in a dryer to dry for 12 h at 100 °c. the collected leaf and soil samples were stored in the refrigerator at -4 °c for analysis. hplc analysis graded soil (20 g) or mashed leaves (10 g) were mixed with 100 ml methanol in a beaker (500 ml) and then subjected to ultrasonic extraction for 30 min. this procedure was replicated thrice. the extract solutions were combined, and the soil or leaves were removed through filtration. methyl salicylate contents in the extract solution were determined by hplc. hplc was performed with an agilent tc-c18 column (250 mm × 4.6 mm, 5 μm) by using a mobile phase of acetonitrile 0.01% phosphoric acid solution (40:60), with a flow rate of 1.0 ml/min, column temperature of 30 °c, detection wavelength of 302 nm, and injection volume of 10 µl. toxicity bioassay toxicity bioassay was determined by using the method proposed by zhang et al. (2013) with some modifications. graded soil (10 g) was placed in a 200 ml beaker, with the vertical wall inside each glass coated with fluon emulsion, and allowed to dry for 24 h to prevent the ants from escaping. twenty individuals of each minor and major workers were laid at the bottom of a bunsen beaker. mortalities, walking rate, and grasping rate were observed after 5 h, 1 day, and daily thereafter until the 12th day after treatment. the treatments were replicated thrice. the ants were kept in the laboratory at 25 ± 2 °c and relative humidity of 80%. the mortality statistics were determined as follows: mortality was assessed after 5 h, 1 day, and daily thereafter until the 11th day after testing with different soils. all treatments were replicated three times. the following formula was adopted: mortality = (number of dead ants / number of total ants) × 100% corrected mortality = (the mortality of treatment group − the mortality of control group) / (1 the mortality of control group) × 100% the determination of repellency rate statistics is given as follows: the rationale for the bioassay is that the ants will always exhibit digging behavior whenever an adequate digging substrate, such as soil, is available. however, the ants do not dig or dig less when the substrate contains a repellent. therefore, repellency was defined as suppression of ant digging behavior. soil samples (20 g) were placed at the bottom of a 500 ml beaker with a vertical wall inside each glass coated with fluon emulsion. then, 20 g of control sociobiology 64(1): 1-6 (march, 2017) 3 soil covered the upper part of the beaker, and 10 individuals of each minor and major workers were selected and placed in a beaker. in the experiment, four treatment groups and one control group were adopted. each treatment was replicated thrice. we found that the ants preferred to inhabit the soil without repellent over the soil with repellent. the following equation was used to determine the repellency: repellency rate = (number of ants in the control group − number of ants in the treatment group) / number of ants in the control group × 100. behavioral observation on the ants’ walking ability was conducted as follows: observations on the walking ability of the workers were recorded after 5 h, 1 day, and daily thereafter until the 12th day after testing. afterward, the workers were placed on a piece of a4 paper. the workers were observed to possess walking ability when they walked continuously for 10 cm without falling. walking rate was obtained using the following formula: walking rate = (number of workers possessing walking ability/number of workers per replicate) × 100. behavioral observation on the grasping ability of ants was conducted as follows: the grasping ability of the workers was recorded after 5 h, 1 day, and daily thereafter until the 12th day after testing. the workers were placed on an a4 paper, which was gently rotated 180° after 10 s. the workers that did not fall from the a4 paper were considered to possess grasping ability. we used the following formula: grasping rate = (number of workers possessing grasping ability / number of workers per replicate) × 100. statistical analysis the mortality and repellency rates were analyzed and transformed to arcsine square root values for anova, followed by duncan’s multiple range test. all data were expressed as means ± standard error. statistical analyses were carried out using spss 13.0 (spss inc., chicago). differences between the values were considered significant when p < 0.05. results methyl salicylate content in the soil and leaf a methyl salicylate standard was quantified with an external standard curve between 0.01 and 5 mg l-1 (y = 36289x, r2 = 0.9991). methyl salicylate content in fresh soil ranged from 0.13 to 4.88 mg/kg and 0.16 to 5.97 mg/kg in dry soil; a significant difference in contents was observed among depths of 0-5, 5-10, 10-15, and 15-20 cm (f = 28.36, p < 0.01). methyl salicylate content (4.88 mg/kg) in the soil at depths of 0-5 cm was significantly higher than at other depths (f = 14.56, p < 0.01). the methyl salicylate contents in the fresh and dry soil showed little difference in every depth (fig 1). the methyl salicylate contents in the fresh leaves were 133.3, 150.8 and 106.3 mg/kg, whereas those in the dry leaves were 486.6, 401.2, and 193.9 mg/kg. the methyl salicylate contents in the fresh yellow leaves (106.3 mg/kg) and in the dry yellow leaves (193.9 mg/kg) were significantly lower than fig 1. the content of methyl salicylate in soil in different depth. fig 2. the content of methyl salicylate in v. odoratissimum leaves (a=younger green leaves b=older green leaves c=yellow leaves). those of others (f = 8.41, p < 0.05) (fig 2). insecticidal toxicity the major ants were erradicated completely with the soil at 0-5 cm, whereas 30.75% were killed with the soil at 15-20 cm depth after 12 days. this finding indicates that the soil exerted toxicity against rifa (fig 4). for the minor ants (fig 3), the soil at 0-5 cm depth required 11 days to achieve complete minor ant death. soil samples at 15-20 cm depth fig 3. mortality of minor red imported fire ants after open exposure to soil in different depth under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. y zhang et al. – insecticidal activity of the soil in the rhizosphere 4 caused 34.75% minor ant death. we also found that in all the treatments, no ants died on the first three days, but the mortality of both minor and major workers increased with exposure time ranging from 3-11 days. mortality of the major and minor ants almost overlapped as the exposure interval was prolonged. no significant difference (f = 1.58, p ˃ 0.05) in the mortalities between minor and major workers was found. the ability to walk after 12 days. meanwhile, the values were 100.00% (0-5 cm), 85.72% (5-10 cm), 41.35% (10-15 cm), and 27.23% (15-20 cm) for the minor ants. all the major and minor ants lost walking abilities at the treated soil depths of 0-5 cm (figs 7-8), which was significantly higher than the others (f = 11.26, p < 0.01). fig 5. repellent rate of minor ants after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. fig 4. mortality of major red imported tire ants after open exposure to soil in different depth under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. repellent activity bioassay when the major ants were placed in a beaker with soil samples at 0-5, 5-10, 10-15, and 15-20 cm depths, the repellency against major ants reached 100.00% (0-5 cm), 86.32% (5-10 cm), 55.56% (10-15 cm), and 35.31% (15-20 cm) after 10 days (fig 6). at the 9th day of treatment, the repellency against the minor ants was 100%, 82.72%, 41.29%, and 29.54% (fig 5). both soils caused significant repellency against the major and minor ants. walking ability bioassay we calculated walking abilities in accordance with figs 7 and 8. among the major ants, 100.00% (0-5 cm), 78.75% (5-10 cm), 48.75% (10-15 cm), and 30.00% (15-20 cm) lost fig 7. the rate of minor ants which lose walk ability after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. fig 6. repellent rate of major ants after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. fig 8. the rate of major ants which lose walk ability after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. sociobiology 64(1): 1-6 (march, 2017) 5 grasping ability bioassay the soil in the rhizosphere of v. odoratissimum indicated a considerable grasping suppression (figs 9-10). among the major ants, 95.85% (0–5 cm), 70.75% (5-10 cm), 39.64% (105 cm), and 26.74% (15-20 cm) lost the ability to grasp after nine days. meanwhile, when the minor ants were subjected to the test sequentially, the values were 100% (0-5 cm), 84.00% (5-10 cm), 38.29% (10-15 cm), and 26.45% (15-20 cm). however, almost none of the ants lost grasping ability on the first three days. drop across all four seasons, and numerous fallen leaves cover the ground every year. methyl salicylate could be introduced into the soil in the natural decomposition process of fallen leaves. therefore, the chemicals can be relatively stable in the soil. this investigation also revealed that soil at different depths had different toxicities and repellent activity against s. invicta workers. in this study, the soil at 0-5 cm depth, which contained higher methyl salicylate levels than other samples, showed more effective insecticidal and repellent activities against rifa than the soil at other depths. the difference in mortality values may be attributed to the content of methyl salicylate. insecticidal and repellent activity this study also showed that the soil in the rhizosphere of v. odoratissimum containing the methyl salicylate compounds showed effective insecticidal and repellent activity against rifa. as a fumigant, methyl salicylate is potent against many arthropods, such as mosquitoes and mites (delaplane et al., 1992). the importance of toxic and repellent influence of the soil may provide good practical significance if such effects can also be observed when applied to urban greening. the use of chemicals from natural products in insect pest management is generally considered a safer alternative to using synthetic contact insecticides. some components in plants have long been known as insect repellents. for example, camphor has been used as a repellent against many insect species (abivardi et al., 1984). eucalyptol has also been found repel various insects, such as american cockroaches (periplaneta americana linn.) (maugh et al., 1982), mosquitoes (aedes aegypti), and colorado potato beetles (leptinotarsa decemlineata say) (scheaper et al., 1984). repellency of some natural products has been tested against the rifas, such as mint oil and water suspensions of pine needle (chenet et al., 2008). however, the use of the plant itself to control pests was rarely reported. v. odoratissimum is an important garden landscape plant. given its resilience, the plant has also been applied in forest fire prevention and urban greening. based on its long history of topical application by humans, v. odoratissimum is considered safe for humans and other non-target organisms in its use in many scenarios for fire ant prevention. the dose-response relationships reported in this study provide a foundation for future investigations of v. odoratissimum as toxicant and repellent plant against adult rifas. the potential use of these selective and fully biodegradable materials in the management of invasive insects is promising. however, the success of a fire ant toxic and repellent plant depends heavily on its ecosystem. therefore, additional knowledge is needed to use v. odoratissimum successfully. acknwledgement this work was supported by natural science foundation of guangdong province (2016a030313387), and guangdong provincial science and technology program (2014a020208114). fig 10. the rate of major ants which lose grasp ability after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. fig 9. the rate of minor ants which lose grasp ability after open exposure to soil under v. odoratissimum. each data point represents mean ± se of three replicate beakers each containing 20 ants. discussion methyl salicylate content in the leaves and soil the leaf and soil both contain a certain amount of methyl salicylate, and the content in the fresh and dry leaves increased initially and then decreased. this result was achieved potentially because the leaf samples were in different growing stages. hence, the methyl salicylate in plants may serve different functions in different growth stages. however, methyl salicylate was also obtained in the soil under trees. this finding may have been achieved because of fallen leaves. v. odoratissimum leaves y zhang et al. – insecticidal activity of the soil in the rhizosphere 6 reference abivardi, c. & benz g. 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(2013). effects of hematoporphyrin monomethyl ether (hmme) on worker behavior of red imported fire ant, solenopsis invcita. sociobiology, 60: 169-173. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5910sociobiology 68(3): e5910 (september, 2021) bombus terrestris is the most abundant and widespread bumblebee in the west palearctic where it has differentiated into eight subspecies that differ in morphology and genetic characters, behaviour and phenological traits (rasmont et al., 2008). this species has a great economic and ecological importance because several b. terrestris subspecies have been massively reared and exported worldwide for crop pollination (velthuis & van doorn, 2004). this commercialization has resulted in reproductive interference with native species (tsuchida et al., 2019) or hybridization with native subspecies (cejas et al., 2018, 2020; seabra et al., 2019; bartomeus et al., 2020), multiple invasions with competitive displacement of native taxa (matsumura et al., 2004; ings et al., 2006; inoue et al., 2008; nagamitsu et al., 2010; morales et al., 2013) and possibly pathogen spillover to local bumblebees (goka et al., 2001; arbetman et al., 2013; schmid-hempel et al., 2014). abstract the taxonomic status of bombus terrestris subspecies is complex and has deep implications in the management of commercial bumblebees for crop pollination as well as in the establishment of appropriate conservation plans. herein, the complete mitogenome of the endemic canary islands subspecies bombus terrestris canariensis is newly sequenced and compared with available mitochondrial sequences in order to shed light into its taxonomic status. the obtained sequence of the mitochondrial genome was 17,300 bp in length and contained 37 genes, including 13 protein-coding genes (pcgs), two rrnas, and 22 trnas and a partial sequence of the at rich control region. the phylogenetic analysis of pcgs of the mitogenome was congruent with its subspecies status and a close relationship with the north african subspecies africanus as previously suggested. the sequencing of the mitogenome of b. t. canariensis provides useful genetic information to study the conservation genetics and genetic diversity of these island bumblebee populations. sociobiology an international journal on social insects carlos ruiz1, diego cejas2, irene muñoz3, pilar de la rua4 article history edited by marco antonio costa, uesc, brazil received 08 october 2020 initial acceptance 07 february 2021 final acceptance 16 may 2021 publication date 13 august 2021 keywords pollinators, mitochondrial dna, conservation, phylogeny, bombus terrestris canariensis, insularity. corresponding author pilar de la rua university of murcia calle campus universitario, 11 30100 murcia, espanha. e-mail: pdelarua@um.es given these problems, the trade of the different commercial b. terrestris subspecies has been restricted in few places such as the israel, norway, turkey, the united kingdom and the canary islands, where only the local subspecies b. terrestris canariensis is allowed to be commercialized (velthuis & van doorn, 2006; lecocq et al., 2016). molecular, morphological and pheromone markers have been used to clarify the taxonomy of the terrestris subspecies complex. in particular, the endemic taxon of the canary island was firstly described as a b. terrestris subspecies (perez, 1895) and later elevated to species status by erlandsson (1979) based on its distinct coloration pattern and geographic isolation. several mtdna and nuclear markers have supported both species (widmer, 1998) and subspecies status (estoup et al., 1996) or remain unclear (bertsch, 2010). integrative taxonomic approaches combining independent 1 dpto. biología animal, edafología y geología, facultad de ciencias, universidad de la laguna. la laguna, 38206 tenerife, espanha 2 université de mons, laboratoire de zoologie. place du parc 23, 7000 mons, belgique 3 dpto. de zoología y antropología física, facultad de veterinaria, universidad de murcia. 30100 murcia, espanha 4 university of murcia. 30100 murcia, espanha short note characterizing the mitogenome of the endemic bumblebee subspecies from the canary islands for conservation purposes carlos ruiz, diego cejas, irene muñoz, pilar de la rua – mitogenome sequence of endemic bumblebee subspecies2 markers such as pheromones, morphological and molecular data generated contrasting results supporting both species (de meulemeester, 2012) and subspecies status (lecocq et al., 2016). furthermore, crossing experiments showed that canariensis interbreed with other b. terrestris subspecies leading to fertile offspring (van den eijnde & de ruijter, 2000), what has been used as an evidence of its subspecies status (velthuis & van doorn, 2006), although interspecific mating cannot be rule out (kondo et al., 2009; yoon et al., 2009). the taxonomy of b. terrestris subspecies has deep implications also in the establishment of appropriate conservation plans (lecocq et al., 2016). therefore, a solid taxonomic status must be achieved using novel molecular approaches. mitogenome sequence combined with other molecular markers has successfully resolved phylogenetic and taxonomic issues in several taxa, resulting in an effective tool for bee phylogeny and conservation genetics (nishimoto et al., 2018; du et al., 2016; lin et al., 2019). here we have sequenced the complete mitogenome of the canariensis taxon and compared it with available mitochondrial sequences to shed light into its taxonomic status. to ensure an adequate amount of mitochondrial dna for sequencing (cejas et al., 2020), muscle tissue was dissected from each of ten b. t. canariensis individuals collected from flowers at a single locality on la gomera (canary islands, spain) and pooled. dna extraction and mtdna enrichment was done using the minipred kit of qiagen (hilden, germany). sequencing was performed on a hiseq2000 (illumina) using an illumina truseq nano dna library prep kit for 350 base pairs (bp) (macrogen, south korea). an agilent technologies 2100 bioanalyzer with a dna 1000 chip was used to measure the size of the raw library reads. reads were filtered with the software fastqc (babraham bioinformatics 2012) before assembling them into contigs and scaffolds as in cejas et al. (2020). annotation of pcgs, transfer rna (trna) genes and ribosomal rna (rrna) genes for the consensus sequence was obtained by comparison with previous annotated genomes in the web server mitos (bernt et al., 2013). in order to resolve the phylogenetic position of canariensis, ten mitogenomes of related species and subspecies were obtained from genbank (supplementary material 1). two mitochondrial markers (cox1 and cytb) comprising 1,307 bp were used for all the subspecies with no mitogenome available (except for africanus with only sequence data of cox1). the phylogenetic position of canariensis was estimated from a concatenated dataset including the 13 protein coding genes (pcgs) using the software mrbayes 3.2.6 (ronquist & huelsenbeck, 2003). the best-fitting substitution model was assessed with iq-tree. a phylogenetic tree was constructed using b. consobrinus (mf995069) as outgroup. fig 1. physical map of the obtained sequence mitochondrial genome of bombus terrestris canariensis in absence of the complete a+t control region. protein coding genes (pcg) are indicated in light blue, trna genes in green, and rrna genes in dark blue. the gc content is indicated in black. sociobiology 68(3): e5910 (september, 2021) 3 the a+t control region was not completely sequenced, owing to its extreme variability and the pooling approach followed. thus, the mitogenome of b. t. canariensis, was sequenced to a length of 17,300 bp (genbank accession number mw959771) (fig 1). the sequenced part varies in length in comparison to other b. terrestris subspecies (b. t. terrestris: 17,232 bp; b. t. lusitanicus 17,049 pb) mainly due to the presence of indels in intergenic regions. gene order was consistent with published data (cejas et al., 2020). it contained 13 protein-coding genes (pcgs), two rrnas, and 22 trnas and a partial sequence of the at rich control region. the average a+t content was 86.4%, slightly higher than that in other b. terrestris subspecies (b. t. lusitanicus and b. t. terrestris 86%). this a+t content was higher in non pcgs (87.5%) than in the pcgs region (83.7%). most of the variation observed in the available mitogenomes of terrestris subspecies occurred in non pcgs. pcgs of b. t. canariensis showed 193 snps, especially in nad4 (34), nad5 (33) and cox1 (27) genes. transitions (83%) where more frequent than transversions in the snps. no indels were found within the pcgs whereas a 6 bp deletion was observed in the large rna gene sequence. fig 2. bayesian phylogenetic tree showing the relationship between bombus terrestris canariensis (in red) and other eight b. terrestris subspecies. an arrow indicates the b. t. canariensis mitogenome sequenced in this study and bold names the mitogenomes used. empty and filled circles indicates posterior probabilities of 0.90-0.95 and >0.95 respectively. carlos ruiz, diego cejas, irene muñoz, pilar de la rua – mitogenome sequence of endemic bumblebee subspecies4 the combined analysis of concatenated pcgs of mitogenomes and available markers of the different subspecies showed high support for all the basal nodes and low support for the nodes within terrestris clade (fig 2). b. terrestris appeared within the subgenus bombus, related with species such as b. lucorum, b. hypocrita and b. cryptarum. all the studied terrestris subspecies including canariensis appeared as monophyletic in a supported clade, thus reinforcing the subspecies status of canariensis. however, other taxon such as xanthopus that has been previously elevated to species (lecocq et al., 2015) appeared within the b. terrestris clade. these results should be taken with caution as only three mitogenomes were available for the terrestris subspecies and only partial mitochondrial data (1,307 bp) have been analysed for the remaining subspecies. given its importance as a commercial species, further mitogenomes of the remaining subspecies will establish a solid taxonomy of the group. the sequencing of the b. t. canariensis genome itself provides additional genetic information useful for studying the conservation genetics of these island bumblebee populations, creating a framework for establishing conservation programs for pollination networks of b. terrestris subspecies with locally endemic flora. acknowledgments this work was supported by the projects e-rta201400003-c03 (inia, spain; 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(2009). interspecific hybridization of the bumblebees bombus ignitus and b. terrestris. international journal of industrial entomology, 18: 41-48 electronic supplementary material supplementary file 1: sampling information of the individuals used for the phylogenetic analysis. 1427 performance of altriset™ (chlorantraniliprole) termiticide against formosan subterranean termites, coptotermes formosanus shiraki, in laboratory feeding cessation and collateral transfer trials, and field applications by robert t. puckett1, t. chris keefer1, & roger e. gold1* abstract chlorantraniliprole represents the first compound to be registered as a termiticide by the environmental protection agency (epa) in over a decade. this novel termiticide is currently registered as a ‘reduced-risk pesticide’ by the epa. laboratory and field trials were conducted to quantify mortality of formosan subterranean termites (fst), coptotermes formosanus shiraki resulting from chlorantraniliprole treated soil, the degree to which the termites curtail feeding intensity post-exposure to chlorantraniliprole treated soil, collateral transfer of chlorantraniliprole among nest mates, and the effectiveness of chlorantraniliprole as a remedial treatment against structural infestations of fst. termites which were exposed to chlorantraniliprole treated soil consumed significantly less paper than unexposed fst. the mean percent mortality of those termites exposed to chlorantraniliprole treated soil was significantly greater than that of unexposed fst. depending on donor:recipient ratios, the mean mortality of recipients ranged from 14.65 – 90.00 % in the collateral transfer trials. there was a positive correlation between increased donor density and recipient mortality. through 24 mo post-treatment, 27.3% of the structures which were treated in field trials were observed to have infestations of termites that required re-treatment; however, no active fst were observed to be infesting any of the structures during the 30 and 36 month post-treatment inspections. additionally, a novel scoring rubric was developed that will allow standardization of field study sites with respect to dissimilarity in site variables, and will allow for more consistent comparison of results 1department of entomology, texas a&m university, 2143 tamu, college station, tx 778432143. 1*corresponding author e-mail: r-gold@tamu.edu 1428 sociobiology vol. 59, no. 4, 2012 across disparate field experiments. an explanation for the lack of successful remediation of many of the structures involved in the field trial is proposed and is based on our novel scoring system. key words: coptotermes, chlorantraniliprole, termiticide, reducedrisk, invasive species introduction formosan subterranean termites (fst) coptotermes formosanus shiraki (isoptera: rhinotermitidae), are an invasive insect pest in the united states and elsewhere. while this termite species is endemic to mainland china (kistner 1985), their introduction into the hawaiian archipelago is believed to have been the result of maritime activity originating from the island of taiwan (formerly formosa), with subsequent introductions to continental united states originating from hawaii (su and scheffrahn 1998; cabrera et al. 2000; hawthorne et al. 2000; howell et al. 2000; scheffrahn et al. 2001; jenkins et al. 2002). however, austin et al. 2006 suggests two distinct fst introductions to hawaii, and then the continental united states, both originating from mainland china. fst represent one of the most economically important pest insects in the united states. estimates indicate that the cost associated with monitoring, control, and repair of damages caused by fst exceeds $1 billion per year (paudel et al. 2010). fst control methods include physical and chemical barriers to prevent termites from gaining access to structures, biological controls (including nematodes, bacteria, fungi, and botanical extracts), chemical treatment of soil and wood, and baits (verma et al. 2009). the general public is educated regarding many of these termite control tactics, but when surveyed they cite (in descending order of acceptability) perimeter treatment with a liquid termiticide, bait treatment, and liquid + bait treatments at the most preferred methods of termite control (paudel et al. 2010). however, there is an increasing emphasis towards the development of least-risk chemical treatments, as well as non-chemical tactics for termite control (lewis 1997). belonging to a new class of chemical insecticides, chlorantraniliprole (altriset™) was recently developed and marketed by dupont crop protection. chlorantraniliprole is currently classified as a ‘reduced-risk pesticide’ 1429 puckett, r.t. et al. — performance of altrisettm by the environmental protection agency (epa, 2008). the compound is an anthranilic diamide, and exhibits a novel mode of action in which insect ryanodine receptors are activated, resulting in rapid paralytic muscle dysfunction (hannig et al. 2009, cordova et al. 2006, cordova et al. 2007, lahm et al. 2005, and lahm et al. 2007). regulation of the release of internal cell calcium is affected by activation of ryanodine receptors. the downstream physiological effect of this disruption of calcium homeostasis results in feeding cessation, and eventual death of the insect (teixeira et al. 2008). the effectiveness of this compound has been demonstrated in mortality trials against a variety of insect species belonging to the orders coleoptera, diptera, and hemiptera (kuhar et al. 2008, palumbo 2008, and schuster 2007). we designed laboratory trials to study the efficacy of chlorantraniliprole on fst feeding rates, collateral transfer of chlorantraniliprole among fst nestmates, and field trials to determine the effectiveness of chlorantraniliprole to control infestations of fst in structures and to protect those structures from reinvasion through time. gautam and henderson (2011) described the effects of chlorantraniliprole on fst in laboratory trials. our work represents a synthesis of data related to the effectiveness of this new compound on fst in field applications correlated with laboratory trials. material and methods laboratory trials: termite feeding cessation fst were field-collected from beaumont, tx approximately 2 weeks prior to the initiation of this study. sandy-loam soil was prepared for these trials by treating it with chlorantraniliprole using the following procedures. a 1,000 ppm stock solution of chlorantraniliprole and water was made by adding 0.10 g of technical grade chlorantraniliprole to 100 ml of deionized water. next, a serial dilution of the stock solution was accomplished by adding 15 ml of deionized water to 15 ml of stock solution. this final solution was added to 270 g of soil and distributed by mixing the soil with a stir rod within a 750 ml plastic beaker. the treated soil was allowed to rest for a period of 24 hrs. glass test-tubes (10 cm in length and open at 1430 sociobiology vol. 59, no. 4, 2012 both ends) served as arenas for this experiment and followed the design of gold et al. 1996 (fig. 1). after preparation, the tubes were stored vertically in test-tube racks (untreated-agar end at top). tubes were stored at 25˚c overnight to allow moisture to become uniformly distributed in the matrix. there were 10 replications of each treatment and untreated control groups. after the 24 hr period had elapsed, 20 fst workers and 5 soldiers per replication were randomly selected from laboratory stock and introduced into the “untreated-agar end” of the test-tube arenas, and the introduction time was recorded. the time at which the tunneling termites reached the treated soil was recorded as the exposure ‘start’ time. cohorts of termites fig. 1. schematic shows components of glass-tube arenas used in fst feeding cessation experiment. 1431 puckett, r.t. et al. — performance of altrisettm were allowed to tunnel in the soil for four different time periods (1, 2, 4, & 8 hr). these four time periods represent four distinct treatments. after the termites had tunneled for the pre-determined time period, the rubber stoppers were removed and termites and soil were carefully tapped out through the untreated end of the arena into a clean petri-dish. to measure feeding intensity, pre-feeding digital images of 5 x 5 cm pieces of brown paper towel (cormatic-georgia pacific, atlanta, ga) were taken for comparison to post-feeding images at the end of the trial. using soft forceps, the termites were then carefully moved to a smaller petri dish containing the paper towel, half of which was covered by 50 g of play sand, moistened with 10 ml of water. this arrangement of paper towel and moistened sand provided ambient humidity within the arenas, as well as constantly moist paper towel, on which the termites fed. petri dishes were sealed with parafilm®, transferred to an environmental chamber, and maintained at 25 ± 5° c and 85 ± 5% rh. untreated control tubes were constructed in the same manner as the treatment tubes, but the soil remained untreated. termite cohorts were allowed to tunnel for the same time as the treatment groups periods (1, 2, 4, and 8 h). termites were transferred to identical feeding dishes, and feeding rates were then calculated in the same manner as in the treatment groups. these control groups are referred to as ‘tunneling controls’ (tc). an additional set of controls which were not allowed to tunnel, were established in feeding dishes (as above) to compare the amount of feeding for the same time period as the treatment and tc groups. twenty termite workers were introduced directly into these petri dishes without subjecting them to tunneling, and observation periods were identical to the treatment and untreated control groups. this control group is referred to as ‘feeding controls’ (fc). termite mortality was recorded daily for 12 d post-exposure. at the end of the trial, the remaining paper towel was allowed to dry in the laboratory, after which, digital images of the paper were taken with a canon eos 50d 15.1 megapixel digital camera fitted with a 28-135 mm lens (canon u.s.a., inc. lake success, ny). pre-feeding and post-feeding images were then compared using sigmascan pro v.5.0 photo-editing software, and the surface area (cm2) differential was calculated and statistically analyzed 1432 sociobiology vol. 59, no. 4, 2012 (calibration of the photography technique was made for each image prior to area measurement). collateral transfer as in the feeding cessation study detailed above, termites used in this study were field-collected from beaumont, tx approximately 2 weeks prior to the initiation of the trial. ten replications of each treatment (donor:recipient ratios), and untreated controls were conducted for this trial. arenas consisted of a 15 cm petri dish, each with a a 7.6 x 7.6 cm piece of brown paper towel (cormatic-georgia pacific, atlanta, ga) placed on the floor of the petri dish and moistened with 8-10 droplets of water from a 25 ml samco scientific corporation pipette (san fernando, ca). the paper served as food and harborage for termites. donor:recipient ratios in this trial included 0:20 (untreated controls), 1:19, 5:15, 10:10, 15:5, 19:1, and 20:0. donor fst were marked using orange rust-oleum marking paint (vernon hills, il). the methodology used to mark the the donors was similar to that described by forschler 1994. a stock solution of 50 ppm was made using formulated chlorantraniliprole. donor fst were treated on the thorax with 0.3 µl of 50 ppm chlorantraniliprole using a hamilton 700 series micro syringe pipette (reno, nv). two untreated fst soldiers were added to each arena. additionally, two sets of post-treatment observations were made to observe mortality at 4 h then daily through 7 d. feeding intensity was measured in these trials by taking pre-feeding images of the 7.6 x 7.6 cm brown paper towel and post-feeding images at the end of these trials (7 d after treatment). as described above, preand postfeeding images were compared using sigmascan pro v.5.0 photo-editing software, and the surface area (cm2) differential was the metric used for statistical analysis. calibration of photography was made for each image prior to area measurement. a separate trial was established simultaneously to determine if marking had a significant deleterious effect on the termites. this trial included ten replications each of untreated-unmarked controls and untreated-marked controls. field trials: for the purposes of this field trial, center for urban and structural entomology (cuse) and dupont personnel jointly inspected and agreed 1433 puckett, r.t. et al. — performance of altrisettm upon 11 structures with monolithic slabs or pier and beam construction, each of which had at least one active fst shelter tube on the exterior of the structure. the minimum area of any structure included in the trial was 78.97 m2. pre-treatment inspections also included the interior of structures and bath traps. a diagram of each structure was prepared by cuse personnel, who measured the size and recorded the shape of the foundation, and areas of termite activity. live termites were collected from each structure, preserved in 100% ethanol, and stored as voucher specimens. all of these structures were located in southeast texas. chlorantraniliprole treatments were made by a pest management professional at each property according to the manufacturer’s label directions. these treatments were overseen by cuse personnel. all structures were treated between may and july 2008. nine of the structures were of monolithic slab construction, and the two were pier and beam. post-treatment exterior (and interior when possible) inspections were made on or about 2 wk, and then at 1, 3, 6, 12, 18, 24, 30, and 36 months. this monitoring schedule is more robust than that which would generally be incorporated into professional pest management protocols. if active termites were discovered during post-treatment inspections, termite samples were collected and preserved in 100% ethanol as voucher specimens. dupont authorized personnel were notified of any post-treatment activity before any supplemental treatments (st) or re-treatments (rt) were performed. a supplemental treatment (st) in this study is defined as: (a). chlorantraniliprole spot treatments to active termite areas that were not originally treated at the initial application. this is not a failure of chlorantraniliprole, since the termiticide was not applied to that area, and termites penetrated through an untreated zone. (b). treatment of infested elements of construction where termites survived due to conducive conditions, such as leaking pipes, unusual construction elements that did not allow application to reach the infested area, or an isolated above ground colony with no soil contact. (c). treatment where a conducive condition existed which contributed to or allowed termites to remain active and penetrate the treated zone. thus, it was not considered a chlorantraniliprole failure. 1434 sociobiology vol. 59, no. 4, 2012 a re-treatment (rt) in this study was defined as: the application of chlorantraniliprole as a spot treatment to active termite areas that were originally treated at the initial application. this was considered a failure of chlorantraniliprole. that is, termites were able to penetrate through the treated zone; however, if that area of penetration had conducive conditions (or other issues as above) that allowed termites to penetrate, and the condition was not corrected, then this was considered a supplemental treatment (st) as described above. in this study, all structures were ranked based on several parameters related to the difficulty of the structure-specific treatment procedures. the parameters used to populate the ranking rubric included: termite species fig. 2. mean paper consumed (cm2) by fst through 12 d after exposure to chlorantraniliprole treated soil, untreated soil, or no soil. treatments were replicated 10 times. bars with the same letter are not significantly different using analysis of variance (anova) and tukey’s hsd mean separation test at p < 0.05. 1435 puckett, r.t. et al. — performance of altrisettm (1-desert termite, 2drywood termite, 3-reticulitermes, and 6-coptotermes); number of mud tubes and location; the number of conducive conditions present at each structure; and, construction type (1 monolithic slabs, 2 pier and beam, and 3 floating slabs). the total difficulty score (tds) of each structure was calculated by summing all points assigned for each category. it is presumed that difficulty to control termites at structures is positively correlated with higher tds. results termite feeding cessation consumption: all treatment cohorts that were exposed to chlorantraniliprole treated soil (regardless of exposure time) consumed significantly less paper (f = 21.37; df = 8,89; p < 0.01) than the ‘tunneling controls’ (tc) and ‘feeding controls’ (fc) (fig. 2). additionally, the mean amount of paper consumed by fst after exposure to chlorantraniliprole treated soil was negatively correlated with time of exposure (fig. 2). a similar trend was not observed in the tc (fig. 2). mortality: with the exception of the 1 and 4 hr tunneling control groups, the mean % mortality of fst remained below 10% in the unfig. 3. mean accumulated fst % mortality through 12 d of observation. 1436 sociobiology vol. 59, no. 4, 2012 treated controls through 11 d after exposure to untreated soil (fig. 3). at and beyond 3 d post exposure, the mean % mortality of all treatment cohorts that were exposed to chlorantraniliprole treated soil (regardless of exposure time) was significantly greater (f = 9.64; df = 8,89; p < 0.01) than the tc and fc (fig. 3). additionally, with the exception of the 12 d observation period, the mean % mortality of treatment cohorts that were exposed to chlorantraniliprole treated soil was positively correlated with time of exposure. collateral transfer mortality percent donor mortality in all of the treatment ratios was significantly different (f = 17.73; df = 8,89; p < 0.01) than that of the 0:20 donor:recipient ratio (untreated controls) at 7 d post-treatment (table 1). however, there were no significant differences in donor mortality between the different treatment ratios at the 7 d observation period, and mean mortality ranged from 50.00 – 76.00 % (table 1). there were significant differences (f = 17.73; df = 8,89; p < 0.01) in recipient mortality levels among the different treatment ratios at 7 d post-treatment, and mean mortality ranged from 14.65 – 90.00 % (table 1). regarding total mortality (donors and recipients) for each donor:recipient ratio, there were signifitable 1. mean percent mortality at 7 d post-treatment of fst donors and recipients when donors were treated with 50 ppm chlorantraniliprole. % mortality total ratio donor recipient % mortality 0:20 (untreated control) 0.00 (a) 42.50 (bcd) 42.50 (abcd) 1:19 70.00 (b) 48.93 (cd) 47.50 (bcd) 5:15 76.00 (b) 14.65 (abc) 29.50 (abc) 10:10 68.00 (b) 62.00 (de) 65.00 (cd) 15:5 69.98 (b) 88.00 (e) 74.50 (d) 19:1 65.76 (b) 90.00 (e) 67.50 (bcd) 20:0 50.00 (b) 0.00 (a) 50.00 (bcd ) *means followed by the same letter(s) in the same column are not significantly different (tukey’s hsd, p=0.05) 1437 puckett, r.t. et al. — performance of altrisettm cant differences (f = 7.58; df = 8,89; p < 0.01) at the 7 d post-treatment observation period (table 1). the greatest percent total mortality (74.5 %) occurred in the 15:5 ratio at 7 d, followed by 67.5% mortality in the 19:1 donor:recipient ratio (table 1). mortality in the untreated and unmarked group was 9.00 %, and was not significantly different from that of the untreated and marked group (21.00 %) starting at 24 h thru 120 h posttreatment (f = 0.067, df=19, p = 0.31). fig. 4. mean amount of consumption of substrate (cm2) by fst (donors and recipients) in each donor:recipient ratio through 7 d post-treatment. bars with the same letter are not significantly different using analysis of variance (anova) and tukey’s hsd mean separation test at p < 0.05. consumptionthere were significant differences in consumption of paper among the different treatment groups and the 0:20 (untreated control) donor:recipient group (f = 15.33; df = 8,89; p < 0.01) (fig. 4). the greatest consumption occurred in the 0:20 ratio, followed by the 5:15, and then the 10:10 (fig. 4). field trial the mean number of pre-trial exterior termite mud tubes per structure was 3.91, and ranged from 1 – 10 (table 2). the mean volume of finished 1438 sociobiology vol. 59, no. 4, 2012 solution applied to structure exteriors was 302.15 l, and 10.33 l on the interiors (table 2). during the first 24 mo of the 36 mo trial duration, 27.27% of the structures were infested with fst and required re-treatment (rt), and 18.18% required supplemental treatment (st). this includes structure #3 in which fst were not completely controlled until a re-treatment (rt) was made after the 1 mo inspection, when fst were discovered on the exterior at the original site of infestation (fig. 5 and table 3). the retreatment (rt) was performed with 15.14 l of chlorantraniliprole. due to damage caused by hurricane ike, access to eight of the structures was limited at the 3 mo post-treatment inspection, and only three of the eleven structures were inspected. no termite activity was found at those three structures. at the 6 mo inspection, two structures were found to have termite activity (fig. 5 and table 3), including structure #2, which had active termites on the exterior and received a re-treatment (rt) with 15.14 l of chlorantraniliprole. additionally during the 6 mo inspection, active termites were found swarming from an interior wall within structure #9. after further investigation of the swarm, a previously unknown cold joint was discovered and this structure received a supplemental treatment (st) with 37.85 l of chlorantraniliprole. at the 12 mo inspection, structure #8 had active fst swarming from a previously treated bath trap. upon further examination, it was determined that there was a water leak in the bath trap area, this leak was repaired, and the area received a supplemental treatment (st) using 22.71 l of chlorantraniliprole. no subterranean termite activity was found during the 18 mo inspections. at 22 mo post-treatment, structure #9 homeowners notified us of fst swarming again from the table 2. mean, standard deviation, and range of exterior and interior mud tubes, perimeter length, and amount of chlorantraniliprole applied to the exterior and interior of structures associated with the field study during initial observations and treatment. exterior mud tubes interior mud tubes perimeter l eng th (m) product applied to exterior (l) product applied to interior (l) m e a n a n d s.d. 3.91 (3.28) 0.18 (0.40) 57.05 (55.90) 302.15 (25.37) 10.33 (1.62) range 1 10 0 1 30.48 – 91.44 151.42 454.25 0.00 -18.93 1439 puckett, r.t. et al. — performance of altrisettm same cold joint, but in a different area. this area received a supplemental treatment (st) with 189.27 l of chlorantraniliprole. this st is included in the 24 mo post-treatment observation period on fig. 5. also during the 24 mo inspection, active fst were discovered at structures # 2 and 5 (fig. 5 and table 3). both structures had active termites on the exterior, and both were re-treated (rt) with 15.14 l of chlorantraniliprole. no subterranean termite activity was found on any of these structures at the 30 or 36 mo inspection. after ranking, using the rubric described in the material and methods section, the mean total difficulty score (tds) of the structures in this study was 12.36, and ranged from 9 – 18 (table 3). at least one st was required in 18.18% of the structures, and all were fig. 5. mean % of re-treated (rt) structures infested with fst after a post-construction treatment with chlorantraniliprole through 36 mo post-treatment. nomenclature above bar: individual structure number and rt (re-treatment). note: active termites were found at structure #3 at 2 weeks post-treatment, but no corrective action was taken until 1 month post-treatment. 1440 sociobiology vol. 59, no. 4, 2012 made to structures that ranked below the mean tds. of the re-treated structures (rt), all but one occurred in structures which ranked above, or just below the mean tds (table 3). discussion in laboratory trials, exposure of fst to chlorantraniliprole treated soil resulted in significantly greater mortality and significantly less consumption of food than either; 1) fst exposed to untreated soil, and 2) fst which were not exposed to soil at all. these data suggest that chlorantraniliprole could be considered an appropriate soil barrier-treatment for use against fst. however, it should be noted that the minimum termite exposure time investigated in these trials was 1 hr. it is not known if it would be realistic to presume that foraging termites would remain in a ‘treatment zone’ for 1441 puckett, r.t. et al. — performance of altrisettm this period of time in actual situations in which chlorantraniliprole is used to provide perimeter protection to a structure against subterranean termite infestations. thus, we intend to initiate further investigations to determine the minimum temporal exposure threshold required for significant mortality and consumption cessation. the significantly greater mortality of chlorantraniliprole-exposed termites relative to that of the tc and fc is a noteworthy aspect of this study. however, it is important to note that mortality of termites which were exposed to chlorantraniliprole ranged from a minimum of 51.31% (1 hr of exposure) to a maximum of 85.51% (4 hrs of exposure) after 12 d of post-treatment observation. it is likely that the mortality exhibited by these termites was partially the result of deprivation of nutrients resulting from feeding cessation. this effect of treatment is significant, and demonstrates a potential mode of action of chlorantraniliprole, and subsequent cascade of physiological alterations in termites that is more nuanced than that of many other classes of termiticides. no attempt was made in these trials to determine the minimum concentration required to illicit the observed feeding inhibitory effects. we intend to design experiments to investigate this aspect of chlorantraniliprole. additionally, the effects of chlorantraniliprole on other important subterranean termite species should likewise be investigated. the positive correlation (with exception of 5:15 donor:recipient ratio) between increased donor numbers and recipient mortality provides evidence of the transfer effect of chlorantraniliprole (altriset™) among fst nest-mates, and suggests that this compound could affect mortality of fst in field applications via insect-to-insect transfer. this density-dependent effect is dependent on the density of chlorantraniliprole-exposed insects (donors) relative to the density of recipients. this of course calls into question the feasibility and efficiency of reliance on this insect-to-insect transfer effect in order for the use of this product to provide effective remedial or prophylactic treatments on fst populations. that is, the density of chlorantraniliprole-exposed foragers (donors) in field treated colonies would likely not reach a level which would be analogous to the donor:recipient ratios which were required in these trials to significantly effect the density of recipients, and thus the colony. interestingly, mortality 1442 sociobiology vol. 59, no. 4, 2012 of the donor population remained lower than expected and, on average (66.62 % overall mean mortality of donors) was similar to that observed in fst after exposure to chlorantraniliprole treated soil for 4 and 8 hrs in the feeding cessation trial. when considered in their totality, these results suggest that the chlorantraniliprole concentration to which fst were exposed in these trials (50 ppm) may be less than that required for acceptable termite control in field applications. however, altriset™ is labeled for application at 500 ppm, or 10 times the concentration used in this laboratory trial. evidence of the slow acting nature of chlorantraniliprole when applied to infestations of fst is provided by the fact that while several of the structures (27.27%) which were treated and monitored over the course of these trials were observed to have continuing infestations of fst. these structures required re-treatment (rt) during the first 24 mo of the monitoring phase of the study. in one case, rt became necessary twice within the same structure. however, by 30 months post-treatment, no fst activity was observed at any of the structures. this suggests that while chlorantraniliprole effectively controlled fst at structures in these trials, the compound requires a longer period of time than alternative termiticides to reduce populations of structure-infesting fst, but the end result of structure protection is the same. comparisons of field experiment results (such as those discussed in the field trial section of this document) across treatment environments are greatly complicated by the myriad variations associated with treatment scenarios and structure variables (french 1988; french and ahmed 2005). differences in treatment sites such as termite species, degree and location of infestation, degree and location of conditions conducive to termite infestation, foundation type, construction materials, and soil type result in great difficulty in standardization of the finite number of treatment environments that are available to researchers. we have proposed a rubric for scoring treatment environments which allows for enhanced ability to compare and assess data related to field experimentation such as that documented herein. careful examination of the results of the utilization of this rubric in this work revealed an interesting bifurcation of the mean total difficulty scores (tds) of those structures that required either a supplemental treat1443 puckett, r.t. et al. — performance of altrisettm ment (st) or re-treatment (rt). that is, the tds of all st’s fell below the mean of 12.37, and a trend was noted in which rt’s scored greater than or just below the mean tds. this more thorough characterization and comparison of our treatment environments provides a refined and more complete picture of the challenges involved with treatment for fst in all study structures, and presents explanatory variables that we believe led to the lack of successful remediation and protection of rt structures with regards to fst. it is our hope that others will attempt to use this system in future work and that after subsequent model refinement, eventually this will provide a consistent method to compare results across disparate, but related field research involving subterranean termites. acknowledgments we thank mrs. laura nelson for her helpful reviews and comments on earlier drafts of this document. additionally, we would like to thank mr. bryan springer (bevis pest control, texas city, tx) for his technical assistance with treatment of structures. the center for urban and structural entomology also thanks dr. clay scherer (dupont) for his support of this work. references austin, j.w., a.l. szalanski, r.h. scheffrahn, m.t. messenger, j.a. mckern, and r.e. gold. 2006. genetic evidence for two introductions of the formosan subterranean termite, coptotermes formosanus (isoptera: rhinotermitidae), to the united states fla. entomol. 89:183-193. cabrera, b.j., p.g. koehler, f.m. oi, r.h. scheffrahn, and n.-y. su. 2000. the formosan subterranean termite. eny-126, florida cooperative extension service, ifas, university of florida. 7pp. cordova d., e.a. benner, 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hasan et al., 2006; park et al., 2003; melani et al., 2016). sweet flag is native to asia and europe and can now be found in most countries as well. in ancient asian medicine it is used as a “rejuvenation” for the brain and nervous system, and as a remedy for digestive disorders (balakumbahan et al., 2010). the red imported fire ant (rifa), solenopsis invicta buren, native to south america is also distributed in new zealand, australia, china, taiwan and many other countries (ascunce et al., 2011; kafle et al., 2011). after practicing abstract due to public health and environmental issues, alternatives of synthetic pesticides have been researched for a long time. we evaluated the toxicity and repellency of the sweet flag (acorus calamus l.) powder and two bioactive compounds (α-asarone and β-asarone) against workers of the red imported fire ant (rifa), solenopsis invicta buren under laboratory conditions. sweet flag powder applied at 1 mg/cm2 or more provided 100% ant mortality within 18 hours, and repelled almost 97% of ants within one hour. βeta-asarone was the faster acting compound against rifa compared to α-asarone and sweet flag powders. the lt 50 values inclined exponentially with the increase in the application rate of the test items. on the other hand, repellency did not increase with the application rate of the test items, but did increase with the exposure time. based upon the results of this study, α-asarone and β-asarone, as well as sweet flag powders could be another alternative tool to control the rifa. sociobiology an international journal on social insects l kafle1, cj shih2 article history edited by gilberto m. m. santos, uefs, brazil received 22 july 2017 initial acceptance 03 october 2017 final acceptance 11 october 2017 publication date 27 december 2017 keywords mortality, repellency, α-asarone, β-asarone, toxicity, lt 50 . corresponding author cheng-jen shih national taiwan university department of entomology chong-fei building room no. 308, 3f, no. 86, joushan rd. taipei 106, taiwan e-mail: shihcj@ntu.edu. chemical control of rifa for decades, now usa is moving towards the integrated pest management (ipm) of rifa. the ipm treatment consisted of the release of two biological control agents, the decapitating phorid fly, pseudacteon tricuspis borgneier, and a pathogen of fire ants, thelohania solenopsae knell, allen and hazard including the application of granular fipronil. ipm practices have resulted in the significant control of rifa over a time (oi et al., 2008). while searching for the alternative chemical control of rifa, many studies has been conducted. the indigenous cinnamon (cinnamomum osmophloeum kanehira) leaf essential oil and trans-cinnamaldehyde had an excellent inhibitory effect in controlling rifa (cheng et al., 2008). similarly, the toxicity and repellency of the clove powder, clove oil and bioactive compounds of clove oil (syzygium aromaticum (l.) merrill & perry) reported that eugenol, eugenol acetate, as well as β-caryophyllene and clove oil were very effective against rifa (kafle & shih, 2013). 1 national pingtung university of science and technology, department of tropical agriculture and international cooperation, pingtung, taiwan 2 national taiwan university, department of entomology, taipei, taiwan research article ants sociobiology 64(4): 398-403 (december, 2017) 399 essential oil from mint oil and wintergreen are also reported as being toxic to fire ants (appel et al., 2004; tang et al., 2013). bioactive compounds from tephrosia vogelii hook. f. (α-pinene, thujene, d-limonene), artemisia annua l. (cineole, d-camphor, α-terpineol, l (-)-borneol) and pronephrium megacuspe (bak.) holtt. (phenol-3-o-beta-d-glucoside) are reported as repellents and toxicants to fire ants (li et al., 2014; zhang et al., 2014; huang et al., 2016). similarly, essential oils of cymbopogon nardus (l.) rendle, cinnamomum osmophloeum, ilex purpurea hassk., capsicum annum l., mentha longifolia (l.) huds., cedrus deodara (roxb.) g. don, cinnamomum camphora (l.) j. presl., artemisia annua, eucalyptus globulus labill. and artemisia argyi h. lév. and vaniot are also reported as repellents against fire ants (wang et al., 2012; tang et al., 2013; wang et al., 2014). although a number of nature-based products could be toxic or repellent to rifa, there are no reports on the repellency and mortality of sweet flag powder and the bioactive compounds of sweet flag against the rifa. therefore, a series of studies were conducted to determine the toxicity and repellency of sweet flag and two bioactive compounds (α-asarone and β-asarone) from sweet flag against rifa under laboratory conditions. materials and methods source of s. invicta solenopsis invicta polygyne colonies were collected from hsinchu county, taiwan. the field collected ants were separated from soil using the methods reported by chen (2007) and reared under laboratory conditions for at least one week prior to conducting the experiments at ambient temperature and relative humidity, 27 ± 1°c and 50 ± 3% rh, respectively, under a 14:10 ll:dd photoperiod (kafle et al., 2010; kafle & shih, 2013). this study was conducted in laboratory of extension entomology and science education, department of entomology, national taiwan university, taipei, taiwan. sweet flag powder and bioactive compounds dried sweet flag tubers were bought from local market in butwal, nepal and washed with tap water and dried under room conditions for 48 h and then ground to a fine powder (< 0.8 mm). alpha-asarone and β-asaroneare are the most common bioactive compounds of the sweet flag (lee et al., 2002; yao et al., 2007; liu et al., 2013) and were purchased from sigma-aldrich (sigma-aldrich china, inc., china). a 10% stock solution of each bioactive compounds was prepared separately using methyl alcohol as solvent and stored at 5ºc for future uses. toxicity tests to determine the toxicity of the sweet flag powder, α-asarone and β-asarone against the rifa, methods reported by cheng et al. (2008) and kafle and shih (2013) were applied with some modifications. finely grounded sweet flag powder was used during this study. beakers (9 cm dia.) were used for the toxicity tests of the test materials. the inside vertical wall of each beaker was coated with a teflon emulsion to prevent the rifa from escaping (kafle & shih, 2013). sweet flag powder was dusted on the bottom surface inside the beaker and then 20 rifa workers were transferred into each beaker. the 0.021 g, 0.064 g, 0.191 g, 0.573 g and 0.785 g of sweet flag powder was sprayed on the beaker those were equals to the five concentrations (0.33, 1, 3, 9 and 12 mg/cm2). for the control treatment, the beaker only contained ants but no sweet flag powder. to determine the toxicity of α-asarone and β-asarone, compounds were sprayed according to the tests on the inside bottom surface of the beaker. then kept under an exhaust hood until the sprayed chemical had dried. then, 20 rifa workers were transferred into each beaker containing a test chemical. the 0.21 ml, 0.64 ml and 1.91 ml of stock solution of α-asarone and β-asarone applied separately on each beaker those were equal to the three concentrations (0.33, 1 and 3 ml/cm2). the beakers for the control treatment were only sprayed with solvent. ant mortality was determined by counting and removing dead ants every 30 min for 24 h. during the tests, only water was provided to the fire ants. repellency tests to determine the repellency of sweet flag powder, α-asarone and β-asarone against rifa, methods reported by appel et al. (2004) and kafle and shih (2013) were applied with some modifications. the repellency of each test item was determined using 9 cm dia. glass petri dishes. the inside of upper vertical wall of each petri dish was coated with a teflon emulsion to prevent the ants from escaping. the sweet flag powder was distributed uniformly over one-half of the bottom of each petri dish, and the other bottom half remained untreated. the sweet flag powder was tested at four concentrations (0, 0.33, 1 and 3 mg/cm2) to get those concentrations, 0 g, 0.012 g, 0.032 g and 0.095 g of sweet flag powder was applied separately per petri dish. and ten worker ants were tested in each treatment. the ants were able to move freely between the treated and untreated surfaces. each petri dish containing ants were then uncovered and exposed to the air. the number of ants on each side (sweet flag powder treated and the untreated glass) of each petri dish were counted every 10 min for 60 min after which the ants were released into the dishes. for the repellency evaluation of the bioactive compounds, the same process as for sweet flag powder was followed, but the test compounds were sprayed on the bottom surface inside the petri dish using pipettes and dried under the exhaust hood before 10 ants were transferred to the 9 cm petri dish containing the test compound. since the mortality of https://en.wikipedia.org/wiki/hassk. l kafle, cj shih – efficacy of bioactive compounds from sweet flag to fire ants 400 α-asarone and β-asarone at 1 ml/cm2 were significantly higher than 0.33 ml/cm2 but statistically similar to 3 ml/cm2, therefore only the repellency of 1 ml/cm2 was evaluated for both test items against fire ants. to get the 1 ml/cm2 concentration of test compounds for this test 0.32 ml of 10% stock solution sprayed on half of the petri dish (9 cm dia.). the petri dishes for the control study were only sprayed with solvent. the toxicity tests of sweet flag powder were replicated four times, and the toxicity and repellency of the bioactive compounds (α-asarone and β-asarone) were replicated three and five times, respectively each time using ants from different colonies. total 20 worker ants were used for toxicity tests and 10 worker ants were used for repellency tests. the studies were conducted under ambient temperature and relative humidity, averaging 27 ± 1°c and 50 ± 3% rh, respectively, under a 14:10 ll:dd photoperiod. data analysis means were compared using tukey’s hsd test, and the lethal time (lt50), which is the time (hours) required for 50% of the ants to die, were estimated by probit analysis (sas, 2015). results toxicity of sweet flag powder five different concentrations of sweet flag powder were evaluated against fire ants. at 24 h after treatment (hat), all the treatments had killed a significantly higher number of ants than the control (f = 154.62, df = 5, p = 0.001). beakers containing 12 mg/cm2 of sweet flag powder had the lowest lt50 values among all the treatments. when the amount of sweet flag powder was increased from 0.33 mg/cm2 to 12 mg/ cm2, the lt50 value was reduced 7.7 folds and mortality of the ants increased from 87.5% to 100% at 24 hat (table 1). repellency of sweet flag powder when three different concentrations of sweet flag powder were evaluated for 1 h for its repellency against fire ants, significantly more ants were observed on the untreated half of the petri dishes than the sweet flag powder-treated half of the petri dishes at all the concentrations. however, at the control treatments, the numbers of ants on both halves were not significantly different (f = 33.39, df = 3, p = 0.001). the mean repellency of ants ranged from 97.1% to 98.8% when the application rate of sweet flag powder was increased from 0.33 mg/cm2 to 3 mg/cm2 (table 2). table 1. toxicity of sweet flag powder against solenopsis invicta workers. application rates no. of ants died (mean ± se)1 lt50 2 (h) 0.33 mg/cm2 17.5 ± 1.5a 15.61 1 mg/cm2 20 ± 0a 7.99 3 mg/cm2 20 ± 0a 5.53 9 mg/cm2 20 ± 0a 2.66 12 mg/cm2 20 ± 0a 2.03 0 mg/cm2 0.75 ± 0.25b 1 means within the same column followed by the same letter are not significantly different (p < 0.05) (tukey’s hsd test, sas, 2015). 2 lt50 values (h) were determined by probit analysis (sas, 2015). application rates no. of ants (mean ± se)1 treated area non-treated area 0.33 mg/cm2 0.13 ± 0.13bb 9.87 ± 0.13aa 1 mg/cm2 0.29 ± 0.29bb 9.71 ± 0.29aa 3 mg/cm2 0.29 ± 0.17bb 9.71 ± 0.17aa 0 mg/ cm2 4.46 ± 0.64aa 5.54 ± 0.64ba 1 means within the same column (lower case) and same row (upper case) followed by the same letter are not significantly different (p < 0.05) (tukey’s hsd test, sas, 2015). table 2. repellency of sweet flag powder to the solenopsis invicta workers. toxicity of the bioactive compounds from sweet flag three different concentrations of α-asarone were evaluated against fire ants. at 24 hat, all treatments had killed a significantly higher number of ants than the control (f = 31.05, df = 3, p = 0.001). beakers containing 3 ml/cm2 α-asarone had the lowest lt50 values among all the treatments. when the amount of α-asarone was increased from 0.33 ml/cm2 to 3 ml/cm2, the lt50 value was reduced 3.1 folds and mortality of the ants increased from 50% to 100% at 24 hat (table 3). three different concentrations of β-asarone were evaluated against fire ants. at 24 hat, all the treatments had killed a significantly higher number of ants than the control (f = 26.02, df = 3, p = 0.002). beakers containing 3 ml/cm2 of β-asarone had the lowest lt50 values among all the treatments. when the amount of β-asarone was increased from 0.33 mg/cm2 to 3 ml/cm2, the lt50 value was reduced 2.9 folds and mortality of the ants increased from 80% to 100% at 24 hat (table 3). when the mortality of fire ants for α-asarone and β-asarone were compared for the 0.33 ml/cm2 at 24 hat, β-asarone killed a significantly higher number of ants than the α-asarone (f = 13.50, df = 1, p = 0.021). the application of 0.33 ml/cm2 of α-asarone and β-asarone killed 50% and 80% of the ants, respectively and β-asarone’s ant killing speed was 10% faster rate than the α-asarone (table 3). when the mortality of fire ants for α-asarone and β-asarone were compared for the 1 ml/cm2 of α-asarone and β-asarone at 24 hat, number of ants killed by α-asarone and β-asarone were not significantly different to each other (f = 0.47, df = 1, p = 0.53). the application of 1 ml/cm2 of α-asarone and β-asarone killed 88.4 % and 80% of the ants, respectively and β-asarone’s ant killing speed was faster 16% rate than the α-asarone (table 3). sociobiology 64(4): 398-403 (december, 2017) 401 when the mortality of fire ants for α-asarone and β-asarone were compared for the 3 ml/cm2 of α-asarone and β-asarone at 24 hat, 100% ants were killed by both tests items and β-asarone’s ants killing speed was 6% faster rate than the α-asarone (table 3). repellency of the bioactive compounds in sweet flag when the application rate of 1 ml/cm2 of the compounds (α-asarone and β-asarone) were evaluated against fire ants for 1 h, the mean repellency of ants were 92.7% and 96.7% for α-asarone and β-asarone, respectively. however, in the control treatments, the number of ants in both halves was not significantly different (f = 18.21, df = 1, p = 0.002) (table 4). 2010; patil & chavani, 2010; kumar et al., 2015). all these studies have proved that sweet flag powder is an effective tool to control different insect pests. the application of only 0.33 mg/cm2 sweet flag powder could control almost 88% of fire ants and if the application rate was increased to 1 mg/cm2 the fire ant control rate reached up to 100% within 24 hat. the lt50 values showed that the rate of application of sweet flag powder and the numbers of ants killed were directly correlated (table 1). during this study, we also found that applying only 0.33 ml/cm2 of α-asarone and β-asarone could control 5080% % of fire ants and if the application rate increased to 3 ml/cm2 the fire ant control rate also increased to 100% within 24 hat. the lt50 values showed that the rate of application of α-asarone and β-asarone and the numbers of ants killed were directly correlated. this study proved that bioactive compounds from sweet flag are toxic to fire ants. limited studies have been reported on efficacy of sweet flag powder, oil or its bioactive compounds against ants. most of the studies were focused on determining the efficacy of sweet flag powder, oil or its bioactive compounds against stored insect pests. schmidt and streloke (1994) reported that treatment with only 0.01% sweet flag oil could reduce grain feeding by prostephunus truncutus (horn) by 50%. similarly, when melani et al. (2016) used sweet flag oil against third instar larvae of s. litura, the toxicity and antifeedant activity values were 92.5% and 79.3%, respectively, with an lc50 value 586.96 ppm. moreover, when hasan et al. (2006) applied sweet flag oil vapors against grubs of trogaderma granarium (everts), they observed that the exposure period is the most important factor affecting the toxic effect of the sweet flag oil rather than the dosage. tang et al. (2013) reported that the application rate of essential oils and mortality of fire ants were directly correlated. higher the application rates of essential oils, higher the mortality rate of fire ants during their study conducted. that similarity was also observed during this study. huang et al. (2016) and zhang et al. (2014) also found the similar effects on mortality of s. invicta by different bioactive compounds or essential oils during their study. both α-asarone and β-asarone are lipophilic in nature (shenvi et al., 2011). kafle and shih (2013) hypothesized that lipophilic compounds were absorbed into the cuticular lipids of the fire ants and then slowly entered into the hemocoel and nervous system. besides that, it was also assumed that these compounds were absorbed into the tracheal system (appel et al., 2004; cheng et al., 2008; kafle & shih, 2013). paneru et al. (1997) reported that both α-asarone and β-asarone were genotoxic at high concentrations in cultured rat hepatocytes and hepatocarcinogenic in pre-weaning mice. hasan et al. (2006) reported that β-asarone served as a contact and stomach poison. oh et al. (2004) confirmed that sweet flag extracts inhibit acetylcholinesterase, while melani et al. (2016) further explained that β-asarone is a contact poison that penetrates the insect body through the cuticle layer towards hemolymph, table 4. repellency of sweet flag powder to the solenopsis invicta workers. application rates no. of ants died (mean ± se)1 lt50 2 (h) α-asarone β-asarone α-asarone β-asarone 0.33 ml/cm2 10 ± 2.65 b 16 ± 1.53 a 8.45 7.63 1 ml/cm2 16.00 ± 0.58 a 17.67 ±2.33 a 6.98 4.05 3 ml/cm2 20 ±0 a 20 ±0 a 2.77 2.61 0 mg/cm2 3.33±0.88 c 0.33±0.88 b 1 means within the same column followed by the same letter are not significantly different (p < 0.05) (tukey’s hsd test, sas 2015). 2 lt50 values (h) were determined by probit analysis (sas, 2015). test items (1 ml/cm2) no. of ants (mean ± se)1 treated area non-treated area α-asarone 0.93±0.27bb 9.27±0.27aa β-asarone 0.33±0.15bb 9.67±0.15aa control 5.93±1.23aa 4.07±1.23ab 1 means within the same column (lower case) and same row (upper case) followed by the same letter are not significantly different (p < 0.05) (tukey’s hsd test, sas, 2015). table 3. toxicity of α-asarone and β-asarone against solenopsis invicta workers. discussion many plant-based products were reported as being effective against fire ants (appel et al., 2004; cheng et al., 2008; wang et al., 2012; tang et al., 2013; li et al., 2014; wang et al., 2014; zhang et al., 2014; huang et al., 2016). the present study demonstrated that sweet flag powder is also an effective plant-based product that not only kills ants, but also repels them. sweet flag powder and its two bioactive compounds also repelled fire ants even at a very low application rate. during this study, the level of repellency of all test items increased with the exposure time. the sweet flag powder is not only toxic to the fire ants but also for many insect pests as well (balakumbahan et al., l kafle, cj shih – efficacy of bioactive compounds from sweet flag to fire ants 402 affecting the nervous system by targeting acetylcholinesterase. due to a series of physiological and chemical actions and reactions the insect becomes spastic and paralyzed, and then eventually dies. sharma et al. (2008) reported that when ingested, β-asarone can damage the insect’s intestinal wall which can lead to death. beta-asarone penetrates and disrupts the function of the mesenteron a tissue layer composed of epithelial cells that absorb nutrients and secrete digestive enzymes in insects. both α-asarone and β-asarone displayed delayed toxicity having lt50 value at least 2.6 h even for highest concentrations. αlpha-asarone, β-asarone and sweet flag powder have low mammalian toxicity and are nature-based substances. the repellency and toxicity of α-asarone, β-asarone and sweet flag powder to the fire ants could be potentially useful in a comprehensive integrated pest management program. acknowledgements we would like to thank national taiwan university, department of entomology, laboratory of extension entomology and science education, taipei, taiwan for providing financial support and laboratory facilities for this study. references appel, a.g., gehret, m.j. & tanley, m.j. 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(2014). insecticidal, fumigant, and repellent activities of sweet wormwood oil and its individual components against red imported fire ant workers (hymenoptera: formicidae). journal of insect science, 14: 1-6. doi: 10.1093/jisesa/ieu103. https://doi.org/10.1016/s0022-474x(02)00027-9 https://doi.org/10.1016/j.micron.2007.07.005 463 aggregation behavior in spiderlings: a strategy for increasing life expectancy in latrodectus geometricus (araneae: theridiidae) by ingrid de carvalho guimarães1; hermano marques da silva & william fernando antonialli junior1 abstract studies on the biolog y of latrodectus geometricus are scarce, especially on the behavior and life expectancy of the species. in this study we investigated the importance of the aggregation behavior of juveniles on life expectancy and longevity of the species. egg sacs were collected in the urban area of douradosms and transferred to the laboratory. the spiderlings hatched were separated into two groups: spiderlings aggregated and isolated, kept in the presence of luminosity. the same tests were run with egg sacs deprived of luminosity. a calculation of entropy was performed for all cases. individuals grouped held under light exposure showed 14.3 days of life expectancy and 46 maximum longevity and 14.8 days of life expectancy and 32 days of longevity when isolated. for individuals grouped and deprived of light life expectancy was 29.8 days and maximum longevity 85 days and 19.3 days of life expectancy and 26 days of maximum longevity when isolated. the entropy of individuals kept in the presence of light, when grouped was h=0.692 and when isolated h=0.377. for individuals deprived of light, the entropy was h = 0.628 when kept grouped and h = 0.143 when isolated. therefore it is concluded that the aggregation behavior and luminosity influence the longevity and life expectancy of spiderlings. cannibalism must be a strateg y to acquire food reserves contributing to the survival of a small number of individuals. keywords: hourglass spider, cannibalism, survival. introduction the genus latrodectus (walckenaer 1805) (araneae: theridiidae), which includes the well-known black-widows, currently comprises 31 species (platnick 1laboratory of ecolog y, center of integrated analysis and environmental monitoring, state university of mato grosso do sul, rodovia dourados / itahum km 12, mailbox 351, 79804-907, dourados – ms, brazil. guimaraes_ingrid@yahoo.com.br 464 sociobiolog y vol. 59, no. 2, 2012 2007), which are widespread, occurring on multiple continents and islands (garb et al. 2004). these spiders have nocturnal habits and several members of the genus are synanthropic and can be frequently found in new locations (andrade 2003; garb et al. 2004). they are generalist predators, known to feed on insects, crustaceans, other arachnids and even small vertebrates, including lizards and young rats (forster 1995; hódar & sánchez-piñero 2002). they have a round abdomen and thin legs; females reach up to 2 cm in length, while males may achieve only 4 mm (cardoso & lucas 2003). the egg hatching occurs after approximately 20 days and the females remain near the egg sacs during the first days after hatching, a period in which the spiderlings remain grouped in the natal web and the index of juvenile cannibalism is high (baerg 1923; kaston 1968, 1970). after this period, males and females leave the web, the males in search of females for mating and the females seek to disperse from the natal web, living usually alone in their own webs (kasumovic & andrade 2004). the species l. geometricus (c. l. koch ), commonly called the brown widow, has the least toxic venom of the genus. it is found virtually throughout the brazilian territory, including human constructions: around and inside houses, gardens, barns and also in agricultural areas (muller 1993; eicktedt 1999). these spiders have a varied coloration since greenish to grayish, with orange spots on the dorsal face and an hourglass in the ventral face, usually orange or yellowish, which is why they are also called “hourglass spiders” (eicktedt 1999; lucas 2003). brown widows can often be found with their egg sacs, which are easily identified by having projections along its entire length (abalos 1962; forster & kingsford 1983). works on l. geometricus are scarce probably due to the low number of accidents involving the species (levi & levi 2002). most studies focus on biochemical analysis of the toxin of these spiders and their geographical distribution (garb et al. 2004; albuquerque et al. 2005; brown et al. 2008), while studies on the basic biolog y are rare. however, the knowledge of aspects related to the basic biolog y and behavior of these spiders are relevant, because the study of these aspects is essential by being the biological key to many issues, and also provide support for further works (bury 2006). the study of behavior, for example, is important because it serves as a link between the molecular and physiological aspects of biolog y and ecolog y. 465 guimarães, i.c. et al. — aggregation behavior in spiderlings thus, the behavior is one of the most important properties of animal life, because it represents the aspect of an organism with which it interacts with the environment (snowdon 1999). a relatively common behavior in spiders (busker et al. 1984; elgar 1992; elgar & schneider 2004) that can ensure greater reproductive success is sexual cannibalism. the most studied case involving complicity of males is the widow spider latrodectus hasselti (thorell 1870), whose popular name is “australian redback”, in which males place their abdomen in front of the female chelicerae (somersault behavior) and are consumed during copulation, doubling their reproductive success compared to males who do not perform such behavior (andrade 1996). according to segoli et al. (2008) brown widows also have shown this behavior, however more studies are needed to help the understanding of the evolution of this extreme form of male monogamy. sexual cannibalism is not the only form of cannibalism in spiders. many studies demonstrate that spiders, in general, cannibalize according to food resource limitation, relatedness, population density, size differences and habitat complexity (wagner & wise 1996, 1997; samu et al. 1999; rickers & scheu 2005; wise 2006). the study by johnson et al. (2010) shows that for l. hesperus (chamberlin & ivie 1935) the family origin influences the propensity to cannibalism and that this behavior can be a heritable trait, although there are no genetic data that prove the heredity of cannibalism in any spider taxa. in this context, this study investigated the importance of the aggregation behavior of juveniles on the life expectancy and longevity of this species, perhaps leading to an increase in their reproductive success. material and methods to obtain spiderlings and adults for experiments, thirty-two egg sacs of l. geometricus and their females were collected in the urban area of douradosms (22°14’s 54°49’w). the egg sacs were used to identify the specimens, because they have a peculiar conformation that facilitates the identification of the species (abalos 1962). for greater reliability, the identification was confirmed by professor jerome rovner, from the university of ohio (usa). individuals were transferred to the laboratory where they were individualized in plastic transparent 15x5cm containers. the egg sacs collected in natural environments and those produced in the laboratory, totaling 32 egg 466 sociobiolog y vol. 59, no. 2, 2012 sacs, were measured and then individualized in plastic circular pots of 7x5cm in airy locations. the relative humidity was controlled by moistened cotton placed at the bottom of each pot, as a way to preserve the elasticity of the silk of egg sacs, which may be affected if the habitat is too dry (work & morosoff 1982, work & young 1987; edmonds & vollrath 1992). data of temperature and relative humidity were registered in two periods of the day, always at 12:00 and 21:00 hours, remaining on average 23.9 ± 3.6° c and relative humidity of 56.3 ± 10%. for the experiments, the spiderlings hatched from 10 egg sacs were separated into two groups: 100 spiderlings, from five egg sacs, were kept aggregated in groups of 20, and 100 spiderlings from other five egg sacs were isolated. both groups were maintained in presence of luminosity. to assess whether light exposure influences the time of hatching of the spiderlings, as well as the life expectancy, 11 egg sacs were kept in the total absence of light. after hatching the spiderlings were divided again into two situations; 3 groups of 20 spiderlings, from 3 egg sacs, were kept aggregated (n=60) and 60 spiderlings, from the other egg sacs were kept isolated. both groups were maintained in total absence of light. monitoring of spiderlings was done every two days, in order to register the molts, as well as determine the mortality rate of egg sacs in all cases studied. to build the life table we applied the parameter of carey (1993), estimated by: where tx= total number of days at age x of survivors until the last possible day of life; x = age interval; nx = number of survivors at the beginning of each age interval; w = maximum age in days reached by the last surviving individual; lx = average of probability of survivors between successive ages, calculated as lx = lx-(dx)/2; lx = proportion of survivors from emergence in relation to age interval; dx = proportion of individuals that died between age x and x+1, calculated as dx = lx-lx+1; dx = number of deaths that occurred during each age interval; qx = probability of death at age x, calculated as qx = d x /l x ; px= probability of survivors; px =1-qx; ex = remaining life expectancy 467 guimarães, i.c. et al. — aggregation behavior in spiderlings of individuals at the beginning of each age interval, calculated as ex = tx/lx. life expectancy was calculated to an interval of a day. the entropy was calculated by the equation: h = σ ex dx / e0x = 0 w h = σ ex dx / e0x = 0 w carey (1993) uses the entropy as an estimator of the standard survival in experimental populations, quantitative distribution of mortality according to age. according to him, the value of the entropy curve i is 0.0, the curve ii is 0.5 and the iv curve is 1.0. if all individuals die at exactly the same age, the proportion of survivors lx according to age x would be characterized by a rectangular curve and h = 0, indicating a hypothetical situation in which all individuals reach the maximum physiological longevity for the species. on the other hand, if all individuals have equal probability of death for every age x (e. g. if all values of px are identical), the curve lx would decrease linearly and matches h = 0.5, so mortality would not depend on the age. when h = 1.0, the pattern of survival would be characterized by high mortality in the initial interval and life expectancy would decrease with age of survival. in this case, survival would decline exponentially with age. results and discussion the egg sacs of l. geometricus have a pyriform aspect, resembling the basic structure of a virus or an armor, and are whitish when just constructed, becoming yellowish with time (abalos 1962). the egg sacs collected presented diameters between 6.8 and 8.6 mm. thus, compared with other species of the genus latrodectus the brown widow has one of the smallest egg sacs. l. variegatus (nicolet 1849) and l. corallinus (abalos 1980) present egg sacs between 14 and 18 mm, l. antheratus (badcock 1932) between 7 and 10 mm, l. mirabilis (holmberg 1876) and l. diaguita (carcavallo 1960) between 15 and 25 mm (gonzález et al. 1998). on average around 105± 54 spiderlings were produced per egg sac, less than in l. variegatus which presents a number of births around 183 per egg sac (gonzález et al. 1998). the development time and hatching of the spiderlings from egg sacs submitted to the absence of light was 30±11days, higher than those kept exposed to light which took 25±9 days to hatch, demonstrating that the luminosity is a determining factor for the development time and hatching. the hatch468 sociobiolog y vol. 59, no. 2, 2012 fig. 2. life expectancy of spiderlings of l. geometricus kept isolated and in the presence of luminosity. life expectancy of 14.8 days and maximum longevity of 32 days. fig. 1. life expectancy of spiderlings of l. geometricus kept grouped and in the presence of luminosity. life expectancy of 14.3 days and maximum longevity of 46 days. ing time of the spiderlings, exposed to light, in this species was lower than in other species of the genus. in l. atritus (urquhart 1890) the time is 40 days, l. katipo (powell 1871), 30 days and l. hasselti about 43 days (forster & kingsford 1983). it was possible to distinguish between males and females by observations on the differentiation of the embolus, in the case of males, and the differentiation of the genital opening in females (epig ynum), because these are characteristics that guarantee the precise distinction between the sexes to the naked eye (levi 1959). of the total number of spiderlings used in the experiments, the distinction between males and females was possible only in 45 individuals. this low number is due to the fact that it was not possible to identify the sex of individuals who died before the start of the differentiation of the external reproductive organs, because before that, males and females 469 guimarães, i.c. et al. — aggregation behavior in spiderlings fig. 3. life expectancy of spiderlings of l. geometricus kept grouped and deprived of light. life expectancy of 29.8 days and maximum longevity of 85 days. fig. 4. life expectancy of spiderlings of l. geometricus kept isolated and deprived of light. life expectancy of 19.3 days and maximum longevity of 26 days. are virtually identical. individuals kept under light exposure showed 14.3 days of life expectancy and 46 days of maximum longevity when kept grouped (fig. 1) and 14.8 days of life expectancy and longevity of 32 days when isolated (fig. 2). for individuals deprived of light life expectancy was 29.8 days and maximum longevity of 85 days when kept grouped (fig. 3) and 19.3 days of life expectancy and 26 days of maximum longevity when kept isolated (fig. 4). it is noted that when the spiderlings were kept together, both in the presence and absence of light, the rate of cannibalism was high, and although life expectancy decreased in the early days, the longevity of the few survivors wass greater than those that had been individualized, demonstrating that the grouping, which occurs naturally in the first days of life should be an important strateg y for the reproductive success of the species (baerg 1923; 470 sociobiolog y vol. 59, no. 2, 2012 fig. 5. entropy analysis for the spiderlings of l. geometricus kept grouped and in the presence of light: h=0.692. fig. 6. entropy analysis for the spiderlings of l. geometricus kept isolated and in the presence of light:h=0.377. fig. 7. entropy analysis for the spiderlings of l. geometricus kept grouped and deprived of light: h=0.628. 471 guimarães, i.c. et al. — aggregation behavior in spiderlings fig. 8. entropy analysis for the spiderlings of l. geometricus kept isolated and deprived of light: h=0.143. kaston 1968). the entropy analysis for individuals kept under light exposure showed that when grouped the value of h was 0.692 (fig. 5) and when isolated individuals presented h = 0.377 (fig. 6). for individuals deprived of light, the entropy was h = 0.628 when kept grouped (fig. 7) and h=0.143 when isolated (fig. 8). these results show that when spiderlings were kept together, both in the presence and absence of light, the values found, h=0.692 and h=0.628 respectively, stood between the theoretical values of h = 0.5 and h = 1.0, indicating that there was a relatively high mortality rate during the first days of life for both cases. consequently, the survival curve has a convex aspect during the early days and there is a gradual increase in deaths after this period. however, when isolated, the values found of h = 0.377 in the presence of light and h = 0.143 deprived of luminosity, stood between the theoretical values of h = 0 and h = 0.5 indicating that the mortality rate in the early days was lower than in previous treatments, however there was a considerable decrease in life expectancy and longevity for these individuals, creating a relatively concave survival curve pattern. even under experimental conditions, the results corroborate other studies, which underscore the importance of cannibalism among spiderlings of latrodectus to the reproductive success of the species, even when there is food availability (baerg 1923; kaston 1968). therefore, in contrast with some authors who suggest that cannibalism can be an accidental consequence as a result of 472 sociobiolog y vol. 59, no. 2, 2012 competition for prey (forster 1995) perhaps cannibalizing individuals is an additional strateg y to acquire food reserves and thus contribute to survival of at least a small number of individuals, a common strateg y by several animals (polis 1981 and references cited there; belles & fitzgerald 1993; giray et al. 2001; johnson 2001). in a study with l. hesperus, johnson et al. (2010), suggested that cannibalism among spiderlings may be a genetically heritable character, acting as an adaptive strateg y of foraging, developed by spiderlings from egg sacs that share a poorly provisioned maternal environment. as noticed, luminosity, apart from the time of hatching, also affects the development of the spiderlings. individuals maintained grouped presented the highest median longevity (figs. 1 and 3), however, those who were kept in the absence of light reached higher longevity (fig. 3). few studies have investigated the influence of luminosity during development of spiderlings on longevity, although it is known that the molting process can be influenced by sexual, nutritional, hormonal and genetic characteristics (forster & kingsford 1983; nentwig 1987). thus, under the conditions evaluated in this work, it can be inferred that the aggregation behavior inducing spiderlings to cannibalism and also the incidence of light influence the longevity and life expectancy 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the amino acid compositions of major and minor ampullate silks of certain orb-web building spiders (araneae, araneidae).the journal of arachnolog y 15:65-80. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2044sociobiology 65(2): 177-184 (june, 2018) the habitat affects the ecological interactions between azteca forel (hymenoptera: formicidae) and cecropia loefl. (urticaceae juss.) introduction the genus cecropia loefl. (1758) represents an important and abundant group of pioneering plants (berg et al., 2005) with a characteristic morphological aspect (vasconcelos & casimiro, 1997; sposito & santos, 2001), which presents a large number of species structurally adapted to shelter ants with which establishes mutualistic relations (janzen, 1969). ants nest within their trunks (harada & benson, 1988) and feed on a glycogen-rich substance produced by the müllerian bodies located at the base of leaf petioles (yu & davidson, 1997). these species, known as myrmecophytes, abstract in order to understand the effects of human impacts on structure and functioning of tropical forests, we should consider studies on animal-plant interactions such as antplant mutualistic interactions.we investigated the mutualistic interactions between ants (azteca genera) and cecropia plants in habitats of secondary forest and pasture used as cattle fields. we tested for the following hyphothesis: (i) cecropia from pasture are more susceptible to foliar herbivory than the cecropia from the forest, and (ii) the defense promoted by ants of azteca genus is less efficient in the pasture when compared to the forested areas. we selected four areas inserted in atlantic rain forest domain surrounded by secondary forest and by cattle pastures. the herbivory was more intense in the pasture than in the secondary forest. the presence of azteca species diminished foliar herbivory only in the forested areas, where we observed a significant increase in herbivory after the removal of a. alfari colony. we argue that the greater herbivory in pasture occurs probably due the lack of other plant resource, being cecropia paschystachya trécul and c. glaziovii snethl., isolated in a “sea of grass” without connection with other tree vegetation, opposite scenario observed in forested habitats. the defense of azteca only in the secondary forest, leading us to suppose that: 1) not even the your aggressive behavior is able to reduce the intense herbivory in the pasture; 2) the your behavioral pattern in forest is not the same in deforested environments and / or 3) mutualism may be undergoing changes due to abiotic effects on pasture. sociobiology an international journal on social insects maf carneiro1, al gaglioti2, ks carvalho1, ic nascimento1, j zina1 article history edited by kleber del-claro, ufu, brazil received 28 august 2017 initial acceptance 07 november 2017 final acceptance 24 november 2017 publication date 09 july 2018 keywords mutualism, atlantic forest, herbivory, ant-plant ecology, conservation. corresponding author marcos augusto ferraz carneiro caixa postal 61, cep 45200-970 jequié-ba, brasil. e-mail: afc.marcos@yahoo.com.br are commonly colonized by ants of the genus azteca forel, which presents up to five species occupying the same plant (longino, 1991). this mutualistic relation can act as an environment of biotic defense against herbivory (longino, 1989; berg et al., 2005; davidson, 2005) and in natural environment, cecropia occupied by ants suffers less herbivorous attacks than those not occupied (jolivet, 1990). this is an extremely important event, especially if we consider the high herbivory rates of cecropia (12 to 18% according to coley, 1983) that are considered significantly higher than those presented for other myrmecophytes (4 to 12% according to frederickson, 2005). 1 universidade estadual do sudoeste da bahia (uesb), programa de pós-graduação em genética, biodiversidade e conservação – ppggbc, departamento de ciências biológicas – dcb, campus jequié, bahia, brazil 2 universidade estadual do centro-oeste (unicentro), programa de pós-graduação em biologia evolutiva – ppgbe, laboratório de biologia molecular e genética em plantas, guarapuava-pr, brazil research article ants mailto:afc.marcos@yahoo.com.br maf carneiro, al gaglioti, ks carvalho, ic nascimento, j zina – mutualism azteca-cecropia in secondary forest and pasture 178 despite ant protection against herbivory, a classic study by janzen (1969) shows that it is so common to find cecropia plants occupied by ants with foliage devoured by insects, as it is common to find unoccupied and healthy plants. it evidencing that the intensity and / or quality of the mutualistic relationship can be extremely variable between habitats or plants. moreover, it perhaps a punctual condition, responds for momentary differences of these relations. the myrmecophytes, in fact, can vary between the different species of plans and between populations of the same species along one gradient (longino, 1989; berg et al., 2005). thus, studies of the ecological relationships between herbivores and plants of wide geographic distribution such as cecropia (from mexico to the south of brazil according to berg et al., 2005) can elucidate some gaps in the insect-plant evolution process. this knowledge becomes especially important in a scenario of constant anthropic aggression and expansion of the agricultural frontier, the main cause of deforestation and conversion of primary forests to secondary habitats (geist & lambin, 2002; sanchez-azofeifa & portillo-quintero, 2011). in this scenario, forest loss favors insect herbivory by undermining the bottom-up control and by improving the conditions required for herbivores proliferation (morantefilho et al., 2016). in this context, the present study investigated azteca-cecropia mutualistic interactions in secondary forest and pasture habitats. in particular, we try to answer the following question: what is the relationship between different habitat types and herbivory in ant-plant mutualistic systems? this question was based on the assumption that vegetational diversity can decrease or increase the likelihood of damage to a focal plant (tahvanainen & root, 1972; letourneau et al., 2011; kim, 2017) so we predict that more simplified and/or homogeneous environments, such as pastures, offer less resources and may lead to a higher feeding pressure of herbivores on cecropia plants compared to forests. in this sense, we formulate the following hypotheses: (i) cecropia plants established in pasture habitats present more foliar herbivory than plants established in the secondary forest, and (ii) the defense against herbivory performed by azteca is less efficient in cecropia plants of pasture than in secondary forest plants. material and methods study area the study was carried out from january to june of 2016, in four areas located along the highways margins of the municipalities of jequié and ubatã, state of bahia, brazil. we selected the areas of secondary forest (area 1: 14°00’55.9”s, 39°55’53.6”w; area 2: 13°59’11.2”s, 39°56’20.1”w) and two pasture areas (area 1:14°11’45.7”s, 39°37’43.0”w; area 2: 14°12’38.3”s 39°34’43.6”w). according to the surrounding vegetation, all areas were immersed in ombrophilous atlantic forest. experimental design and determination of cecropia and azteca species we established a minimum distance of one kilometer between the four sample areas, to ensure data independence, and in each area, we randomly chose 20 plants (totaling 80 plants). in each area, we determined the minimum distance of 5 meters between the selected plants, in order to ensure the aerial isolation of the colonies located in each plant, in which we performed morphological measures (plant height, cap circumference at breast height and total number of leaves) to homogenize the samples. for the determination of the species of cecropia, we collected samples of leaves and reproductive material for the preparation of exsiccates that were identified by the specialists: dr. andré luiz gaglioti and dr. sergio romaniuc neto of the instituto de botânica, são paulo, brazil (registration number: sp 489810/sp 489811c. pachystachya and sp 489812/ sp 489813c. glaziovii). specimens of formicidae colonizer (azteca spp.) collected manually at the entrance of the nest (prostoma) were identified by dr. jacques hubert charles delabie, at cepec/ ceplac laboratory of myrmecology in ilhéus, bahia, brazil, where they are deposited (registration number: 5823). determination of herbivory levels and leaf damage in order to determine the herbivory rates of the plants, we selected, in each area, ten cecropia plants (treatment) that were submitted to the experimental removal (in situ) of the colonies of azteca, and ten plants of cecropia (control) with the presence of colonies of azteca. in each plant, we selected three leaves in an initial state of development, which we individually marked with the use of plastic clamps. leaf shoots were under the same conditions/position in the branch, indicating a similar age. we monitored the selected plants weekly, and at the end of 45 days, we removed the leaves marked for the determination of the foliar area that suffered herbivory. in order to determine the total area of leaf damage by herbivory by plants, we adopted the average area (cm2) of leaf consumed in each individual of cecropia (treatment and control). to measure the area of leaf damage, we removed the marked leaves and submitted them individually to digitalization by photographic method for image generation with a resolution of 15mp (standardized with scaling, tripod and canon 50d camera). we used imagej software version 1.49s (wayne rasband national institutes of health, usa) to measure the leaf damaged area in the images. to describe the patterns of damage, we adopted the categories described in delunardo et al. (2010), as: marginal cut; simple drilling; sequential perforation, following rib; scraping and leaves totally consumed (removed). sociobiology 65(2): 177-184 (june, 2018) 179 experiments of removal and elimination of colonies in order to verify the influence of mutualism on herbivory, we removed ant nests using a contact insecticide (dimy ddvp, dimy prod. jard. ltda. cajamar, sp, brazil reg. ms 33994.0004 / 001-2, formerly dimmyt , serv-san). we applied it abundantly with a syringe in each prosthesis of the plant by the prostome, until the visual verification of the internodes filling, with the transhipment during the application. we carried out the removal of the colonies in ten plants (treatment) and left ten plants intact (control), without removal in all areas. we monitored the treatment plants weekly, to avoid recolonization and whenever the presence of azteca was detected, the insecticide was reapplied. the insecticide applied is non-residual and action by direct contact with insect, not affecting the action of herbivores in the plants with colonies removed (izzo & vasconcelos, 2002). data analysis we used the mann-whitney test to evaluate differences in herbivory levels between plants of both habitats (secondary forest and pasture). we evaluated the effects of mutualism on herbivory levels by means of analysis of variance (two-way anova) using habitat (secondary forest and pasture) and ant colony (present or excluded) in the model, as explanatory variables. we used shapiro-wilk (zar, 1996) to test the assumptions of normality and residual homogeneity of variance. the analyses were performed in the statistical program systat version 12.0 (2007) and in all the tests, we adopted the level of significance of p <0.05. results patterns of foliar damage by herbivory found in leaves of cecropia in the secondary forest, simple drilling (fig 1b) was the predominant foliar damage, while in the pasture, simple drilling (fig 1b) and marginal cutting (fig 1a) occurred similarly. in general, the pattern of sequential drilling (fig 1d) was more pronounced in the pasture (25%), when compared to the secondary forest (2.5%). a complete leaf removal (fig 1f) was registered only on pasture. establishment and characterization of cecropia and its mutual association with azteca ants in habitats of secondary forest and pasture two species of cecropia are established in the studied areas: cecropia pachystachya trécul (1847) and c. glaziovii snethl. (1923), being the first one more frequent in both habitats (pasture: 92% and secondary forest: 78%). two species of azteca ants were associated with cecropia plants: azteca alfari emery, 1893 and a. ovaticeps forel, 1904. the azteca alfari species had the highest occurrence frequency in cecropia pachystachya, in both habitats in a similar proportion (pasture: 63.2.% and secondary forest: 60%). cecropia foliar herbivory in secondary forest and pasture habitats the average of leaf herbivory per plant was higher in pasture (104.4 cm2; sd ± 248.4, n = 40) than in the secondary forest (14.05 cm2; sd ± 20.05; mann-whitney = 4647.500; d.f = 1; p = 0.024; n = 40) (fig 2). fig 1. patterns of leaf damage caused by herbivory in cecropia, following the classification adopted by delunardo et al. (2010); a) marginal cut; b) drilling; c) scraping; d) sequential perforations; e) in the direction of the rib; f) total removal of the limbus. these standards were registered from february to june 2016 in secondary forest and pasture (mata atlântica domain), bahia, brazil. fig 2. leaf herbivory (cm2) in cecropia plants established in the pasture and secondary forest habitat registered during 45 days between february and june 2016in secondary forest and pasture (mata atlântica domain), bahia, brazil. pa stu re se co nd ar y f or est 0 200 400 600 800 1000 1200 l ea f h er bi vo ry (c m 2) maf carneiro, al gaglioti, ks carvalho, ic nascimento, j zina – mutualism azteca-cecropia in secondary forest and pasture 180 in the secondary forest, leaf herbivory was higher in the plants that had the ant colony removed (treatment) (19.36 cm2; sd ± 26.4) compared to plants where the colonies were present (control) (8.74 cm2; sd ± 8.22; mann-whitney = 2158.500; d.f = 1; p =0.005; n = 40) (fig 3a). there was no difference between the treatment (101.8 cm2, sd ± 220.5) and control in the pasture (107.3 cm2, sd ± 283.3, mann-whitney = 1667.500; d.f = 1; p=0.411, n = 40) (fig 3b). influences of azteca ant colonies on cecropia leaf herbivory in secondary forest and pasture habitats in the secondary forest, the azteca ant species influenced the herbivory levels (f = 6.553; df = 1; p=0.015; n = 40) in the host plant. the presence of ant species azteca alfari showed a decrease in foliar herbivory levels in cecropia compared to a. ovaticeps. neither the species of cecropia (f = 3.060; df = 1; p=0.089; n = 40) nor the interaction between cecropia and azteca species (f = 2,523; df = 1; p=0,121; n = 40) explained variations in herbivory in the secondary forest. in the pasture, where the herbivory was larger in relation to the secondary forest habitat, there was no difference between the treatments, neither the ant species (f = 0.090; df = 1; p=0.766; n = 40), nor the species of (f = 0.227; df = 1; p=0.637; n = 40) or the interaction between these two factors (f = 0.086; df = 1; p=0.771; n = 40) influenced herbivory levels. discussion foliar herbivory as expected, foliar herbivory in cecropia plants established in the pasture was higher than in the secondary forest, probably due to the greater abundance of herbivorous insects present in this habitat. secondary forest, a forested habitat and visually more heterogeneous than a local pasture with a predominance of grasses, offers a greater supply of resources for herbivorous insects and possibly exerts an herbivory dilution effect (tahvanainen & root, 1972; letourneau et al., 2011; kim, 2017). arnold and asquith (2002) point out that herbivory patterns are sensitive to fragmentation and that they depend on a number of factors, including the plant establishment place. in addition to high levels of herbivory, when we compare the patterns of foliar damage, between the two habitats, only the pasture presented total leaf removal, the most extreme type of herbivory. this result, according arnold and asquith (2002) can indicate various ecological and behavioral changes in abundance, diversity or herbivores composition and variations in their oviposition in this local, when we compared to less degraded environments such as secondary or primary forests. in fact, in degraded environments can occur the exclusion of specialized species and/or low dispersal of some species to the detriment of others (terborgh et al., 1997; shahabuddin & terborgh, 1999). caterpillar species often seen under cecropia leaves in pasture (personal observation). it is a specialized herbivore and it is considered as one of the main responsible for the high rates of herbivory in the tropics (basset et al., 2001; neves et al., 2013). also in the tropics, the especialized herbivores (monophagous or oligophagous) account for 40-100% of foliar damage whereas the general herbivores play a minimal role (barone, 1998). only in the pasture, we saw active nests of leaf-cutting ants near the studied plants. leaf-cutting ants are prominent herbivores in a) secondary forest not removed removed ant colonies 0 20 40 60 80 100 120 l ea f h er bi vo ry (c m 2) b) pasture not removed removed ant colonies 0 200 400 600 800 1000 1200 l ea f h er bi vo ry (c m 2) fig 3. leaf herbivory (cm2) in cecropia sp. with the removal of colonies of azteca sp. and without removal established in secondary forest habitat (a) and pasture (b). these values refer to a period of 45 days, from the experimental phase of removal of colonies with insecticide treatment plants, between the months of february and june 2016 in secondary forest and pasture (mata atlântica domain), bahia, brazil. a) secondary forest not removed removed ant colonies 0 20 40 60 80 100 120 l ea f h er bi vo ry (c m 2) b) pasture not removed removed ant colonies 0 200 400 600 800 1000 1200 l ea f h er bi vo ry (c m 2) sociobiology 65(2): 177-184 (june, 2018) 181 the neotropics (cherrett, 1986) and their population increases in disturbed habitats (rao, 2000). there are many mechanisms or factores that may direct herbivory patterns in environments. in our study, probably the conversion of forest to pasture has favored herbivory since this event simplifies and homogenizes the habitat. although it is not a consensus, several studies show, in fact, that the habitat loss and degradation by human impacts increase herbivory levels (guimarães et al., 2014, peter et al., 2015; morantefilho et al., 2016). this increase, according to morante-filho et al. (2016), may occur due to both by increasing in the abundance of herbivores and by simplifying the vegetation structure of habitat. also for them, that habitat loss increases local pressure of herbivory and reinforces the idea a pervasive threat to biodiversity. in natural environments, pioneer plants such as cecropia, usually occur as temporary staining of herbivorous insect resources and temporal patterns in local insect abundance can be regulated, for example, by bottom-up (hunter et al., 1992, power, 1992). in this sense, besides the conversion of forests to landscapes by human activity cause species destruction (fahrig 2013), populations loss (clavel et al. 2011, tabarelli et al. 2012), it changes ecological interactions such as regulation of herbivore abundance by bottom-up. ultimately, besides anthropic disturbances favor the herbivory, according lôbo et al. (2011), act on the tree flora both evolutionarily, favoring phenotypes adapted to the conditions imposed, and ecologically, exerting effects on populations, communities and ecosystems that can be revealed from a local to a regional scale mutualism removal of azteca colonies in cecropia plants significantly increased herbivory levels in the secondary forest. in the pasture, there was no difference, confirming the hypothesis that the herbivorous defense exerted by these ants is less efficient in this habitat than in the secondary forest. one of the factors that can explain this result is the similarity of pasture with the hostile environment of forest edges (murcia, 1995). forest boundaries present environmental changes such as increased exposure to wind (which increases tree mortality) and sunlight (which increases the temperature), factors that cause the proliferation of plants that invest in rapid growth (laurance et al., 2006, 2007). these plant species have little defense against herbivores, which can be abundant in these sites (coley & barone, 1996). in fact, there is a consensus in the literature that the action of herbivores is greater in border environment (barbosa et al., 2005; urbas et al., 2007). although all the individuals of cecropia studied were established at the edge of highways, in a large clearing or linear border, this effect can be enhanced if the plants are as in “islands” surrounded by grasses. like in pasture habitats. so it is reasonable to assume that mutualistic ants fail to effectively protect plants against herbivores in such a disturbed and modified environment. the damage caused by herbivores triggers several strategies of physical, chemical and biological defenses in plants (coley & barone, 2001; ohata et al., 2010). of these, the biological occurs by the presence of other mutualistic organisms attracted to the plant, by the plant itself, by some kind of compensatory resources (heil & mckey, 2003). in fact, biological defense exerted by ants is considered one of the most effective (rosumek et al., 2009, llandres et al., 2011), especially when it comes to the cecropia-azteca mutualistic association (janzen, 1969; davidson, 2005), where all the ants of this genus present highly aggressive behavior (yu & davidson, 1997). according to bruna et al. (2004), the aid of alarm pheromones released by the ants at the time of herbivorous attack accelerates the defense response to herbivory. although herbivory is considered an important factor in the induction of ant recruitment this mechanism varies according to ant species. in our study, only a. alfari reduced herbivory levels in secondary forest, while the other species (a. ovaticeps) did not show differences with the removal of their colonies. it evidencing that the effectiveness of the defense actually varies between ant species. different levels of worker aggressiveness may directly relate to nestmate recognition ability, as in myrmecophyte ant, pseudomyrmex concolor (pacheco & del-claro, 2015). the species a. alfari is considered to be the least aggressive of all the azteca inhabitants of cecropia (longino, 2005). nevertheless, it has been shown to be more efficient in protecting cecropia plants in the secondary forest, when compared to the congener and sympatric a. ovaticeps. on the other hand, for vasconcelos and casimiro (1997) a. alfari is considered efficient in removing at least some types of herbivorous insects from its host plants. in this way, we need to rethink the mutualism of the cecropia-azteca system as originally proposed by longino (1991), in the context of “when” and “where”. other factors may explain the effectiveness of a. alfari in repelling herbivores. perhaps the simple presence of ant workers may discourage the action of herbivores or your constant vigilance in the host plant. this behavior, which is connected to the production of substances by the host plant, can be related to several factors (e.g.temperature and environmental humidity), which in turn can exert different patterns among ants of the same taxon, as proposed by yamamoto and del-claro (2008). perhaps this explains the lack of biological defense shown by a. ovaticeps. based on our findings, we can affirm that the secondary forests can still preserve the mutuality of the cecropia-azteca system, unlike the pasture, where the presence of the ants did not reveal any difference in the action of the herbivores. so, perhaps the biggest implication for this outcome is the direction which cecropia-azteca maf carneiro, al gaglioti, ks carvalho, ic nascimento, j zina – mutualism azteca-cecropia in secondary forest and pasture 182 mutualism is taking. because, in a global scenario of climatic changes with increasingly pronounced dry seasons, it can be assumed that the synergy between deforestation, conversion of forests to simpler habitats and drier climate, can modify the herbivory patterns. it, consequently, modify the efficiency of the azteca defense in mutualism with cecropia in places such as the studied pastures. the maintenance of preserved areas is important not only to protect specific organisms but also to avoid the loss of important mutual associations for the ecological system. conclusion and implications the biological defense exerted by azteca was effective only for the cecropia plants that occurred in the secondary forest. this leads us to suppose that perhaps even the aggressive behavior of the azteca species is not able to reduce herbivory damage, compared to many herbivores in the pasture environment. or yet, the pattern of behavior expected by this ant in the forest environment is not the same in deforested environments as is the case of pasture. moreover, cecropia-azteca mutualism may be suffering modifications due to abiotic effects. for example, the production of plant resources (eg, müllerian bodies) in exchange for protection against herbivores may be insufficient to supply the colonies and maintain an efficient patrol over the plants in the pasture habitat. if this is true, there may also be a change in the size of the colonies in this environment (due to a lower resource supply) being smaller than those in the secondary forest. in this context, many hypotheses could be tested in the non-forest pasture system versus secondary forest, especially considering that the great majority of studies of the cecropiaazteca relationship have been conducted primarily in primary forests of the amazon and central america (davidson, 2005). in fact, in natural systems, plants usually invest in defenses by reducing the intensity of damage caused by herbivores, consequently by reducing the negative effects of herbivory (coley & barone, 2001). but in environments under constant disturbance, such as the studied pasture, is this true? based on the results of the present study, such research is worthwhile. ultimately, it is necessary to consider that the pasture habitat is very different from that in which the association cecropia-azteca evolved (clearings of forests and riverbank) as punctuated by fáveri and vasconcelos (2004). in this environment, the presence of azteca, didn´t decrease the action of herbivores, despite the known positive association of cecropia-azteca mutualism. acknowledgements we are grateful to: anselmo santos, iran silva, iracema santos, and silas a. f. carneiro for help during field work. dr. sergio romaniuc neto of the botany institute of são paulo, sp, brazil, for the identification of cecropia plants. to dr. jacques delabie of cepec/ceplac, ilhéus, ba, brazil, for the aid in the identification of the ants azteca sp. to dr. heraldo l. vasconcelos of the universidade federal de uberlândia, mg, brazil, for the assistance and collaboration during the project phases. adriano r. da silva, chemist responsible for dimy prod. contact us cajamar, sp, brazil, for the supply and technical guidelines for application of the insecticide. dra. guadalupe l. de macedo and the team of the herbarium of the universidade estadual do sudoeste da bahia (huesb), for the preparation of the plant material for identification. to the coordenação de pessoal de nível superior (capes) for the scholarship; to the programa de pós-graduação em genética, biodiversidade e conservação (ppggbc) and universidade estadual do sudoeste da bahia uesb, jequié campus, ba, brazil, for logistical support. grants to this research were obtained from: cnpq/capes/ protax (proc. 440502/2015-2; proc. 88882. 156814/201701) and fapesp/ap.r (proc. 2016/50385-4). references arnold, a.e. & asquith, n.m. 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(1996). biostatiscal analysis. third edition. prentice hall. upper saddle river, nj, usa. 662p. https://doi.org/10.1016/s0169-5347(00)88977-6 http://www.systatsoftware.com http://www.systatsoftware.com open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.367-373sociobiology 60(4): 367-373 (2013) microhabitat characteristics that regulate ant richness patterns: the importance of leaf litter for epigaeic ants acm queiroz, cr ribas, fm frança introduction understanding the relationships between species diversity and environmental conditions is one major goal of community ecology, although some relationships are not clearly understood yet (ricklefs, 2004). higher structural complexity favors the diversity of ways to exploit resources and a higher number of niches, which in turn increases species richness (bazzaz, 1975). however, the perception of structural complexity for some species can occur at different spatial scales. for example, both a higher density of patches in a landscape, or tree species richness in a forest may influence the community structure of different groups (flick et al., 2012; wenninger & inouye, 2008; wardhaugh et al., 2012). tropical forests harbor most of the earth’s biodiversity (myers et al., 2000; nageswara-rao, 2012). a large part of this biodiversity in these ecosystems is attributed to soil abstract we assessed the effect of conditions and resources on the species richness of epigaeic ants in a cloud forest of the ibitipoca state park, brazil. we hypothesized that the characteristics that are best related with the epigaeic microhabitat affects ant richness more closely than other characteristics. at each sampling site (36 in total) we set up an epigaeic pitfall to capture ants and measured the following environmental variables (conditions and resources): tree density, tree height, circumference at breast height, density of the herbaceous and shrubby vegetation, weight and heterogeneity of the leaf litter, and canopy cover. we built general linear models and used a function that classifies alternative models according to the second-order akaike information criterion (aicc), in order to assess which environmental variables affect ant species richness. we collected 37 ant species and the models that better explained the variations in ant richness were: (1) null model; (2) heterogeneity of the leaf litter; and (3) weight of the leaf litter. these two environmental variables are positively related to ant richness. it is possible that epigaeic ants are influenced by the high quantity and quality of food and nesting sites provided by a heterogeneous and abundant leaf litter. relationships between species richness and environmental characteristics should consider different predictor variables related to the microhabitat, since each microhabitat may have a specific pattern and predictor variable. thus, the understanding of the effects of the microhabitat variables on species richness could help predicting the consequences of anthropogenic impacts. sociobiology an international journal on social insects universidade federal de lavras. lavras, minas gerais, brazil research article ants article history edited by gilberto m.m. santos, uefs, brazil received 13 may 2013 initial acceptance 11 june 2013 final acceptance 02 august 2013 keywords environmental heterogeneity, aicc, leaf litter, model selection, cloud forest corresponding author antonio cesar medeiros de queiroz universidade federal de lavras ufla dbi, setor de ecologia e conservação laboratório de ecologia de formigas caixa postal 3037, cep 37200-000 lavras, mg, brazil e-mail: queirozacm@gmail.com arthropods, mainly social insects, which contribute with a large number of species as well as with a high abundance and biomass (wilson, 1990). in these environments, ants are one of the dominant groups in terms of richness and abundance (hölldobler & wilson, 1990; fisher, 2012) and therefore may influence the ecosystem as a whole, as they participate in several processes of the ecosystem such as seed dispersal, biological control, and nutrient cycling (hölldobler & wilson, 1990; lach et al., 2010). the distribution of ants can be attributed to biotic or abiotic factors, human impacts or habitat structure (armbrecht et al., 2004; philpott et al., 2010;wittman et al., 2010). on a local scale, however, to have a better understanding of the relationship between ants and their environment is necessary to know about specific microhabitat features because ants have a great diversity of habits, diets and nesting sites (blüthgen & feldhaar, 2010). thus, each microhabitat characteristic may acm queiroz et al factors affecting the richnes of ants368 affect differently ants of distinct strata. for example, the diversity of arboreal ants can be closely linked to the presence, quality and quantity of resources from the trees, as extrafloral nectaries or presence of trophobiont insects and other nitrogen-rich resources (yanoviak & kaspari, 2000; schoereder et al., 2010). therefore, it is possible to infer that this stratum will be the first to suffer with changes in vegetation structure. epigaeic ants are closely related to the soil surface resources, especially in the litter, in tropical ecosystems (yanoviak & kaspari, 2000). the litter is a complex two-dimensional location covered by leaves, twigs and other components (kaspari & weiser, 1999). these more complex habitats can provide a greater number of resources, such as food resources (e.g. springtails, termites and other scavengers insects abundant in the litter) (brühl et al., 1999), as well as sites for nesting, as trunks. on the other hand, subterranean ant species appear to be more related to physical characteristics of their microhabitat, such as soil density (schmidt et al., 2013). studies on ant communities and their relationships with the environment are relatively common in tropical forests (e.g. vasconcelos, 1999; bihn et al., 2008; neves et al., 2010; teodoro et al., 2010). however, with the imminent threat to forests, further studies on ant communities may help predict the biota’s responses to environmental changes. if we are able to understand the effects of these variables and the responses of ant communities to them, besides the particularities of each microhabitat, we can predict the consequences of the anthropogenic impacts on biota. since ants are important biological indicators, and studies in preserved habitats are needed to uncover richness patterns and help in conservation strategies (hölldobler & wilson, 1990; gardner et al., 2009; leal et al., 2010; ribas et al., 2012), in the present study, we assessed the influence of environmental characteristics, which represent conditions and resources for ants, on ant richness in a cloud forest. we hypothesized that the characteristics that are best related with the epigaeic microhabitat affects ant richness more closely than other characteristics. material and methods study area we carried out the present study in october 2011 in mata grande, a cloud forest in ibitipoca state park, zona da mata, state of minas gerais, southeastern brazil (21º40’–21º44’s and 43º52’–43º54’w). the altitude in the park varies from 1,000 to 1,700 meters. the local climate is characterized by dry winters, from april to september, and rainy summers, from october to march, with an annual average temperature of 14.8 ºc and annual rainfall of 1,544 mm. the cloud forest is a type of atlantic forest with trees that reach from 15 to 25 m in height (aragona & seitz, 2001). in the park, the cloud forest covers 90 ha, is surrounded by campos rupestres (rupestrian grasslands), and harbors several epiphytes and lichens. some characteristics, such as frequent mist, wind, and sunlight incidence exert a strong influence on this forest (carvalho et al., 2000). the most common plant families in this environment are myrtaceae, melastomataceae, lauraceae, rubiaceae, and fabaceae (oliveira-filho & fontes, 2000). we can define cloud forests as a tropical forest that occurs at high altitudes. this vegetation type has great conservation importance because it is naturally distributed in patches and harbors several endemic species (merlin & juvik, 1993). its well-defined boundaries and dissimilarity to surrounding environments can result in peculiar communities (ricklefs, 2004). furthermore, this forest is considered one of the most threatened vegetation types due to environmental changes and human disturbance through changes in the habitat and conversion of these forests to different land use regimes (loope & giambelluca, 1998; schonberg et al., 2004). sampling we delimited 36 sampling sites with a minimum distance of 20 m between them. for sampling ants we set up epigaeic pitfalls without bait at each site, and left them opened for 48 h. then we identified the ant genera with taxonomic keys (bolton, 1994; palacio & fernández, 2003), and calculated the species richness in each trap based on morphospecies. specimens of each morphospecies were deposited in the collection of the laboratory of ant ecology, universidade federal de lavras. to measure environmental variables, we delimited a 6 x 6 m plot around each trap and measured the following variables: tree density (of trees with circumference at breast height above 15 cm) (dtree), tree height (htree), tree circumference at breast height (cbhtree), density of herbs and shrubs (dhs), weight (wll) and heterogeneity of the leaf litter (hll), and canopy cover (ccan). these variables (related to habitat structure, habitat heterogeneity, resource availability, and microclimatic variation) represent conditions and resources for ants. we counted all trees within the plot in order to calculate tree density, and we estimated their height and measured their circumference at breast height. to measure the density of herbs and shrubs we took standardized photographs at the four directions of the trap in each sampling site. the photographs were taken with a 100 x 100 cm white background, 3 m away from the background and at 1 m above the ground (nobis, 2005). we calculated the density of herbs and shrubs using the option global analysis in the software sidelook (nobis, 2005). to calculate density, we used the following formula: vegetation density = background area* (black pixels / white pixels) background length. we collected leaf litter inside a 25 x 25 cm square, counted the items present in it, and calculated leaf litter hetsociobiology 60(4): 367-373 (2013) 369 erogeneity with the shannon index, a new approach suggested by lucas paolucci (personal communication, october 28, 2011). after this procedure, we dried out the leaf litter in an oven for 96 h to measure its dry weight, and weighted it on a precision balance. to estimate canopy cover, we made a digital hemispherical photograph of the canopy using a camera equipped with a 0.20 x fisheye objective lens. the camera was positioned at 1.5 m above the ground and adjacent to the pitfall. with these photographs it was possible to indirectly calculate canopy cover (engelbrecht & herz, 2001). we analyzed photographs in the software gap light analyzer 2.0 (gla), which calculates canopy cover (frazer et al., 1999). data analysis we used general linear models (glm) to test which environmental variables affect ant species richness within a theoretical approach based on the second-order akaike information criterion (aicc burnham & anderson, 2002). we tested the data normality with a shapiro-wilk test (w = 0.9635, p = 0.2758) and ran the model (based on the normal distribution) with r software 2.14 (r development core team 2011) using the aiccmodavg package (mazerolle, 2013). we tested if there is a correlation between tree height and tree circumference at breast height because both represent the tree size (spearman, p = 0.0003, r2 = 0.56). because there is a positive correlation between the variables, we chose the tree circumference because it was the more accurate measure, since tree height was visually estimated. we build models with four combinations of variables (hll+wll, dtree+dhs, dtree+ccan, cbhtree+ccan), and the null model. since we do not have enough sampling points to run all possible models, we use the above combinations of variables, believing they could be related between themselves, as litter variables or vegetation structure variables, and as being the better predictors of ant richness. posteriorly, we used the function “aictab” to test which variables affect ant species richness the most. this function classifies the models according to the aicc (burnham & anderson, 2002). we considered as acceptable the models with the smallest aicc-values and with a delta value (difference between aicc-values) smaller than two (burnham & andersen, 2002). after this, we used the function “importance” for the considered models (delta < 2) to calculate the importance values (w+). results ant fauna and environmental variables we collected 37 epigeic ant species of four subfamilies (table 1). the most frequent species was acromyrmex sp.1, the most common genera were pheidole and hypoponera, and the most frequent subfamilies were myrmicinae and ponerinae. the average (± standart deviation, sd) ant richness was 4.11 ± 2.05 species per pitfall. the average tree density was 0.22 ± 0.07 trees/m2 and the average cbh was 38.85 ± 10.63 cm. the average herb-shrub density was 31.7 ± 38.56. the average diversity of the leaf litter was 2.39 ± 0.19 and its average dry weight was 46.54 ± 22.50 g. the average canopy cover was 82.45 ± 3.24%. model selection the null model had the smallest aicc-value, i.e., with no environmental variables affecting ant richness (table 2). table 1. species of epigeic ants sampled in the cloud forest of ibitipoca state park. subfamilies genus species ectatomminae gnamptogenys 1 formicinae brachymyrmex 2 camponotus 2 myrmicinae acromyrmex 1 basiceros 2 eurhopalotrix 2 hylomyrma 2 pheidole 9 sericomyrmex 2 solenopsis 2 strumigenys 1 trachymyrmex 2 x sp. 1 ponerinae hypoponera 4 leptogenys 2 pachycondyla 2 total 37 table 2. model selection based on the second-order akaike information criterion (aicc) for richness of epigeic ants in the cloud forest of ibitipoca state park. the general linear model was built with the following explanatory variables: tree density (dtree), weight (wll) and heterogeneity of the leaf litter (hll), and canopy cover (ccan). we considered only the models with delta-values equal or larger than 2. number of predictor variables (k), differences in aicc-values (δ), and akaike weight (ω). ranking model k aicc δ ω ω accumulated log likelihood 1 intercept 2 157.3 0.00 0.25 0.25 -76.47 2 hll 3 158.3 0.97 0.16 0.41 -75.76 3 wll 3 158.8 1.53 0.12 0.52 -76.05 4 dtree 3 159.3 2.01 0.09 0.62 -76.29 5 ccan 3 159.7 2.34 0.08 0.69 -76.45 acm queiroz et al factors affecting the richnes of ants370 however, two other models also showed small aicc-values, and could also explain the variation in species richness. these models pointed to leaf litter heterogeneity (w+ = 0.39) and weight of leaf litter (w+ = 0.32) as the most important correlates of ant richness. leaf litter heterogeneity and weight positively affected ant species richness. only the models with up to 70% of the accumulated value of akaike weights (ω accumulated) are presented. discussion the environmental characteristics that best explained the variation in species richness of epigeic ants were leaf litter heterogeneity and weight. these variables represent the availability of space, food and nesting resources, as well as microhabitat complexity. ant fauna the number of species observed in our samples was similar to other studies on leaf litter ants in cloud forests (longino & nadkarni, 1990; patrick et al., 2012). likewise, the most common subfamilies in our study, myrmicinae, ponerinae, and formicinae, are the most frequent in cloud forests (longino & nadkarni, 1990). furthermore, as in longino and nadkarni (1990), the subfamily dolichoderinae, which is common in the neotropics, was not found in the present study. the number of ant species in the present study was smaller than in other atlantic forest areas in brazil (61 species in leal et al., 1993; 146 species in gomes et al., 2010), but the most representative families were the same. the small number of species in cloud forests may be explained by long-term isolation, which makes it more difficult for ants to colonize this environment and thus reduces its species richness. ecological isolation may also make local species more vulnerable to extinction (gillespie & roderick, 2002). model selection the first selected model did not explain the variation in richness of epigaeic ants, as in other studies, in which relationships between habitat heterogeneity and ant richness were not found (corrêa et al., 2006; ribas & schoereder, 2007; muscardi et al., 2008). this shows that the ant species richness can also be linked to unmeasured characteristics of the environment or be distributed randomly. although other studies present similar variation in some of these predictors (ribas et al., 2003; neves et al., 2013) the variation of these variables in this study may not have been sufficient to detect stronger relationships with species richness. however, other models, which comprised variables that represent environmental conditions and resources for ants, pointed to positive relationships between leaf litter heterogeneity, leaf litter weight and richness of epigaeic ants. this is probably due to a higher availability of space, heterogeneity, food and nesting resources leading to a larger number of ant species. leaf litter parameters regulate the species richness of soil and leaf litter ants (mezger & pfeiffer, 2011). the richness of soil arthropods is related to factors that operate at small spatial scales, such as differences in nutrient concentrations among layers of organic matter, which suggests that habitat quality is one of the most important correlates of species diversity (sayer et al., 2010). leaf litter heterogeneity may indirectly indicate higher environmental quality, explaining the higher richness of epigaeic ants in our study. the natural dynamics of the leaf litter may also alter the heterogeneity of this stratum, creating new sites for colonization and a higher variety of food resources and thereby increasing the richness of this group (campos et al., 2007). the relationship between leaf litter weight and ant richness shows that resource availability may be a limiting factor for epigaeic ants in cloud forests (soares & schoereder, 2001). the richness of epigeic ants is higher in microhabitats with a large number of nesting sites and a high availability of food (paolucci et al., 2010). these arthropods may be more abundant in sites with large amounts of leaf litter, as space is also an important predictor of the species abundance of soil arthropods (sayer et al., 2010). other factors that operate at different spatial scales and may affect the structure of epigeic ant communities, such as topographic variation (gunawardene et al., 2012) and soil humidity (lassau & hochuli, 2004), were not considered in the present study and should be assessed in future studies. probably, characteristics that are directly related to the vegetation (e. g. habitat structure) or to a large temporal scale (e.g. area size) may strongly affect arboreal ants and other groups of epigaeic ants, which are sensitive to variations in environmental heterogeneity and resource availability (ribas et al., 2003; campos et al., 2006; costa et al., 2011). this has also been found in cloud forests (schonberg et al., 2004). it is known that the combination of fragment area and habitat structure is an important predictor of species and functional group richness, and that habitat structure affects some functional groups such as arboreal ants in fragments of atlantic forest (leal et al., 2012). together with our results these findings corroborate our hypothesis that ants are strongly related to microhabitat characteristics (e.g., heterogeneity and weight of leaf litter for epigaeic ants, and richness and density of trees for arboreal ants). alternatively, characteristics of habitat structure have been also considered important predictors of species richness in studies that assessed environmental impacts on ant communities. other common factors in the montane forest of ibitipoca that could indirectly affect ant richness, are tree fall and, consequently, clearing opening (carvalho et al., 2000; patrick et al., 2012). this kind of disturbance is the main factor responsible for increasing sunlight incidence and improved sociobiology 60(4): 367-373 (2013) 371 establishment success of some plant species. the canopy cover variation can negatively affect the ant richness, because it alters microclimatic conditions, quality and quantity of resources (neves et al., 2013). nevertheless, our results do not support that canopy cover may affect the community of epigaeic ants in the studied cloud forest, since this model was not selected in the results. in our case, we found a small variation in the studied area that could be not enough to influence ant species richness. leaf litter heterogeneity and weight of litter were the best predictors of the species richness of epigaeic ants. as we hypothesized, ant richness can be better explained by the specific characteristics of the microhabitat in which ants live or forage. relationships between species richness and environmental characteristics should consider different predictor variables related to the microhabitat, since each microhabitat may have a specific pattern and predictor variable. in this study we detect a relationship between ant species and its microhabitat, and the modification of habitat by anthropogenic impacts can cause a loss of this relationship and affect the biological communities negatively. thus, the conservation of habitats and their species richness patterns are essential to the ecosystem functioning. bioindicators studies that evaluate the effects of anthropogenic impacts should focus on the specific characterization of the microhabitats to better detect community changes since the understanding of the effects of the microhabitats variables on species richness could help predicting the consequences of anthropogenic impacts. acknowledgments this study was carried out during the courses field ecology i (pec 506), field ecology ii (pec 521), and scientific publication (pec 527) of the graduate program in applied ecology of universidade federal de lavras. we thank r.m. ribeiro and j. tuller for their help in the field. we thank k. malves, h. griffiths and t. walker, and two other anonymous referees for their comments and criticisms on the manuscript. instituto 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(1990). success and dominance in ecosystems. orlendorf/luhe: ecology institute. wittman, s.e., sanders, n.j., ellison, a.m., jules, e.j., ratchford, j.s. & gotelli, n.j.(2010). species interactions and thermal constraints on ant community structure. oikos, 119: 551–559. doi: 10.1111/j.1600-0706.2009.17792.x. yanoviak, s.p. & kaspari, m. (2000). community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 259-266. doi: 10.1034/j.1600-0706 .2000.890206.x. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2078sociobiology 65(2): 259-270 (june, 2018) pollen storage by stingless bees as an environmental marker for metal contamination: spatial and temporal distribution of metal elements introduction since the middle of the 20th century, growing industrialization, urbanization, transportation and agriculture has led to overall ecosystem contamination and major modifications to landscape structure and composition. the presence of metals in the environment can be the result of external sources such as industrial smelter pollution, emissions from ferrous metallurgy, external mining activities and busy highway traffic (bilandžić et al., 2011; lambert et al., 2012). mining activities represent a major source of environmental contamination by metal residues (freedman & hutchinson, 1981; perugini et al., 2011). in the brazilian state of minas gerais, mine exploitation in the iron quadrangle reached its peak towards the middle of last century and the activity is still intense (meneses et al., 2011). one of the consequences of this industry is the daily release of huge amounts of dust into the atmosphere (meneses et al., 2011). abstract since the middle of the 20th century, human activities have led to overall ecosystem contamination and to major modifications in landscape structure and composition. mining activities represent a major source of environmental contamination by metal residues. the objective of our study was to evaluate the presence of heavy metals and other elements on stingless bee pollen, and compare them to samples of suspended particulate material (spm) in five points a mineral province, in brazil. more than 50 elements were identified by icp-oes and icp-ms, after microwave digestion. overall, we found a strong relation among elements present on pollen and spm. samples from the four areas exhibited higher levels of minerals compared to the reference site. mineral levels varied widely within the two seasonal periods. some elements, like pb, cd, as, cu, zn, and fe were found at levels considered potentially toxic to human health. pollen stored by stingless bees was a successful bioindicator, and demonstrated the value of quantitative ecological information for detecting air pollution. sociobiology an international journal on social insects no nascimento1, ha nalini2, f ataide2, at abreu2, y antonini1 article history edited by celso martins, ufpb, brazil received 13 november 2017 initial acceptance 08 december 2017 final acceptance 21 january 2018 publication date 09 july 2018 keywords bee; pollen; suspended particulate material; mining activities; iron quadrangle. corresponding author yasmine antonini universidade federal de ouro preto departamento de biodiversidade evolução e meio ambiente instituto de ciências exatas e biológicas (iceb) campus morro do cruzeiro, s/nº, bauxita ouro preto, minas gerais, brasil. e-mail: antonini.y@gmail.com the rocks (or soils) of iron quadrangle naturally have high levels of metals that can be considered toxic for human health (rapini et al., 2008; azevedo et al., 2012; messias et al., 2013; carvalho-filho et al., 2010). on the other hand, these regions shelter a relatively high variety of very poorly studied metallophilic plants, living in a harsh environment with high concentrations of metals in the soil and subject to atmospheric deposition of those toxic elements, due to the mining activities. studies carried by valim (2012) and baêta (2012) for example, found that relevant quantities of elements such as al, ba, ca, fe, k, na, mg, mn, s, sr, and zn are deposited annually on the soil surface and vegetation via wet and dry deposition. according to these authors, this deposition depends on the geochemical nature of the environments that may be affected by weathering and anthropic activities, being sources of input of both water and nutrients and even pollutants for plants and animals. 1 universidade federal de ouro preto, departamento de biodiversidade evolução e meio ambiente, instituto de ciências exatas e biológicas, ouro preto, minas gerais, brazil 2 universidade federal de ouro preto, departamento de geologia, escola de minas, ouro preto, minas gerais, brazil research article bees mailto:antonini.y@gmail.com no nascimento, ha nalini, f ataide, at abreu, y antonini – stingless bees as an environmental marker for metal contamination260 due to their enormous diversity (species richness and abundance) and role in ecosystem functioning, the use of insects as bioindicators has been increasing (mcgeoch, 2007; lambert et al., 2012). the functions and use of bioindicators are better depicted if we take a look at the bees. the idea of employing bees in environmental monitoring is not a new one. since 1970, honeybees have been increasingly employed to monitor heavy metals in territorial and urban areas, pesticides in rural areas and radio nuclides (see pohl, 2009 for a revision; oliveira et al., 2017). besides honeybees, their products have been employed in studies aiming to determine levels of metal pollution in different ecosystems (agricultural, urban and industrial; e.g. bratu & beorgescu, 2005; bilandžić et al., 2011; morgano et al., 2012; formick et al., 2013; lambert et al., 2012; leita et al., 1996). in many countries, there are no reports regarding the use of stingless bees to measure pollen contamination by toxic elements or as environmental markers in mining areas. apparently, mines, steelworks, industrial and urban areas, and highways in or near bee foraging areas can result in an increase in the concentrations of certain metals (e.g., al, ba, ca, cd, cu, mg, mn, ni, pb, pd or zn) in bee matrices (honey and pollen) due to pollution from chemical wastes and exhaust fumes (wayne, 1983; bilandžić et al., 2011; formick et al., 2013). the fundamental difference between heavy metals and other pollutants, like pesticides, is their mode of introduction and their environmental fate. pesticides are scattered both in time and space and, depending on the type of chemical compound, are degraded by various environmental processes over longer or shorter periods of time. heavy metals, on the other hand, are emitted in a continuous manner by various natural and anthropic sources and, since they are not degraded, are continuously kept “in play”, thus entering into physical and biological cycles (matina et al., 2016). in this scenario, we propose the use of stingless bees as active environmental markers in mining areas; they have been mostly used in standard exploration procedures of mining companies as efficient mineral prospectors. stingless bees, like other bee species, are exposed to atmospheric pollutants during their foraging activities. their hairy bodies easily hold residues, and they may be exposed to contaminants via contaminated food resources such as nectar, pollen or water (pohl. 2009). heavy metals present in the atmosphere can be deposited on these hairy bodies and be brought back to the hive with pollen, or they may be absorbed together with nectar, water or honeydew (caroli et al., 1999; matei et al., 2004; rodriguez garcia et al., 2006; bogdanov, 2006). furthermore, almost all environments (soil, vegetation, water and air) are sampled by stingless bees, and thus provide numerous indicators (through foraging) for each season. finally, a variety of potentially contaminated materials are brought into the hive (nectar, pollen, honeydew, propolis and water) and stored accordingly. a number of variables need to be considered when using bees, or beehive products such as pollen, to monitor heavy metals in the environment. these include flight distance, proximity to the contamination source, weather (rain and wind can clean the atmosphere or transfer heavy metals to other environmental sectors), season (nectar flow, which is usually greater in spring than in summer and autumn, could dilute pollutants) and botanical origin of the honey (nectar of flowers with an open morphology and honeydew are much more exposed to pollutants). the present study reports the first effort to use stingless bees as ecological indicators. our purpose was to investigate whether stingless bees are an effective environmental marker of contamination for monitoring environment pollution caused by mining activities through the analysis of pollen matrices over two climatic periods (dry and wet season) by comparing their contaminant content with suspended particulate matter (spm). the study areas were located in mining sites that are susceptible to various levels of contamination due to different kinds of minerals present in the rocks. materials and methods sampling sites and biological matrices sampling of pollen matrices of tetragonisca angustula (latreille) beehives and suspended particulate matter (spm) were performed from february to april (wet period) and august to september (dry period) of 2013 in five areas of the iron quadrangle, minas gerais, brazil (figure 1). we avoid sampling during periods of intense rainfall, in wet period. suspended particulate matter (spm) is here considered as finely divided solids that may be dispersed through the air from mining activities. this area is known worldwide as one of the most important iron and gold producers of the world consequently undergo environmental contamination (cprm 2014, figueiredo et al., 2000). the substrates in this region are rich in iron, manganese and aluminum, and have given rise to the development of rupestrian fields (campos rupestres), also called ferruginous rupestrian fields, ferruginous rocky outcrops, or “canga” vegetation (schaefer et al., 2016; silveira et al, 2016). four sites were located in mining sites of iron (sm), limestone (bm), gravel (im) and soapstone (vm) mines, and one control site (ct). besides the past intensive mining activity in a radius of 3 km, the ct site is today a natural protected area, covered by forest. three samples in each period (dry and wet) were taken from the bee hives of tetragosnica angustula (3 colonies in each sampling site, totaling 90 samples) installed one km distance to the main mining pit. the distance of 1km is twice a maximum distance that t. angustula workers can fly (nogueira-neto, 1997). the samples were collected from the same colonies throughout the survey, with the exception that dead colonies were replaced in order to maintain the number of hives sampled. pollen was collected directly from uncapped pollen combs in order to assure fresh pollen was collected. sociobiology 65(2): 259-270 (june, 2018) 261 these field-collected pools were immediately placed on ice and then stored at 22ºc until analysis. colonies used for the experiment were obtained from natural areas, outside of iron quadrangle. with a final volume of 50 ml. chemical element content of the solutions was measured by icp-oes (optical emission spectrometry with inductively coupled plasma) and icp-ms (mass spectrometry with inductively coupled plasma). instruments and equipment analyses were carried out using a microwave milestone ethos s1, icp-ms (model agilent 7700x) and icpoes (model agilent 725). analyses employed the following procedure: optimization of the instrument, calibration with standard solutions, analysis of the sample blank consisting of 2% ultra-pure nitric acid and analysis of the reference materials (after every 10 samples). the analyses were performed at the laboratory of geochemistry of the geological department of the federal university of ouro preto (brazil). assurance of analytical quality method precision was evaluated by recovery tests conducted at two concentration levels covering the concentration ranges in the samples. these solutions were analyzed after every ten samples and the coefficient of variation was determined for 3 repetitions due to the heterogeneity of the pollen samples. because of the unavailability of certified reference material for bee pollen, a certified reference material (apple leaf, nist srm 1515) was evaluated to assess method accuracy for the recovery of elements. limit of detection (lod) and limit of quantification (loq) values were calculated as suggested by mermet and poussel (1995): lod = (3 rsdxbec)/100 and loq = 5 x lod, where rsd is the relative standard deviation of blank samples and bec is the background equivalent concentration, determined experimentally (n = 10). limits of quantification (loq) are provided in table 1. fig 1. sampling areas of the iron quadrangle, minas gerais, brazil. bm (bemil), ct (control area), im (irmãos machado), sm(safm), vm (viamar). sample preparation a simple system made from a high-density polyethylene (hdpe) bottle connected to a hdpe funnel was used for total spm collection, following azimi et al. (2003) (suplementary material figure 1). these collectors were arranged in a northsouth-east-west direction, relative to the bee’s nest with a collector next to it in a central position. the five collectors per sampling area were arranged distant about 50 meters from the central. collection bottles were filled in advance with 100 ml of a solution of 10% nitric acid (65% suprapur nitric acid, merck) and mili-q water in order to dissolve particulate matter and to avoid trace metal adsorption by bottle walls. the collection bottles were filled with concentrated nitric acid after the sampling period (7 days) to reach a final ph of 1, and samples were kept in a dark room at 5°c for one month in order to dissolve most of the particles. after storage for onemonth, samples were filtered under a class 100 laminar hood with 0.45 μm porosity filters (sartorius, cellulose nitrate). the sub-samples obtained were kept at 5 °c until analysis. materials were washed following the procedures of azimi et al. (2003). sample preparation was performed according to previously described methods (miller & miller, 2005; vinas et al., 2000). triplicates of 0.5 g of each samples of pollen were incinerated in a microwave oven (step1: ramp time 10 min until 200°c; step 2: hold time 15 min at 200 ºc; step 3: cooling 30 min) after acid digestion with 7 ml 65% m/m nitric acid (suprapur, merck, darmstadt, germany) and 1 ml 30% m/m hydrogen peroxide (suprapur, merck). all solutions were prepared with deionized water obtained by passing distilled water through a millipore milli-q water purification system (waters corporation, milford, ma, usa) table 1. limits of quantification (loq) for major, minor and trace elements determined by icp-oes and icp-ms. element spm (µg/l) pollen (*µg/kg; **mg/kg) al 8.920 3.674** as 104.000 0.0328* ba 0.480 0.121* cd 6.640 0.00733* co 26.300 0.00766* cr 17.100 0.142* cu 5.370 0.896** fe 7.040 3.068** mn 2.280 0.382** ni 37.000 0.214* pb 181.000 0.0639* sb 0.0296* y 2.740 0.044** zn 6.210 0.634** no nascimento, ha nalini, f ataide, at abreu, y antonini – stingless bees as an environmental marker for metal contamination262 statistical analysis in total, 54 pollen and 60 spm samples were analysed. for the statistical analyses, the site sm had to be excluded – the colonies perished due to high levels of dust deposited outside the nests. the statistical parameters for concentrations (mean, median and standard deviation) were calculated from all the analyzed samples of each matrix, and not only from samples for which residues were detected or quantified. when a compound was not quantified (< loq), the concentration used for statistical analysis was loq/2 (hewett & ganser, 2007). statistical analyses were performed only for pollen and spm residues that were detected or quantified at least once. comparisons of the means of element concentrations in pollen between climatic periods (dry and wet) were performed using the friedman test (non-parametric test for paired samples). the effects of climatic period and site location on metal concentrations were evaluated using a general linear mixed effect model (glm). to be valid, a linear mixed effects model must exhibit independent and normally distributed residuals, which were assessed for each model through diagnostic plots using r-packages (plots of deviance residuals versus fitted values and normal quartile plot of pearson residuals; plots not shown). contrast analyses comparing metal concentrations among sites using metal as the explanatory variable were then been performed using r software (r development core team 2010). aglm analysis was used to test if the atmospheric deposition (spm), as explanatory variable, was the primary factor for metal concentrations in pollen. principal component analysis (pca) was used to investigate the multivariate structure of the dataset and to highlight possible trends among the data. principal component analysis reduces the number of dimensions of a dataset by determining a linear combination of initial variables that maximizes the information content of a group of data. this is achieved by decomposition of the correlation matrix into eigenvalues and eigenvectors, with the eigenvectors representing the linear combination of coefficients and the corresponding eigenvalues representing the variance described by each linear combination. since the eigenvalues are in decreasing order, the first linear components account for the largest amount of variance. once derived, the principal components (pcs) can be used for further analyses to visualize groupings of the data, to verify the presence of outlier values and to determine the variables that discriminate among groups. for all these statistical tests, a significant effect was considered to have a type-i error risk of p < 0.05. results forty-seven chemical elements were detected and quantified from the pollen samples and 34 elements from the spm samples. mean contents, standard deviations and concentration ranges obtained for the elements of the 54 dehydrated bee pollen samples and 60 spm samples from the four sites in the iron quadrangle are shown in tables 2 and 3, respectively. sampling sites ct im vm bm element average±sd min/max al 78.18±11.69 470±381 318.7±114.5 140.5±27.4 68.3/101 102/836 207/461 116/179 as 24.01±10.12 104.99±9.88 118.5±71.7 38.68±11.54 11.87/35.33 94.51/114.14 57.2/242.7 29.69/55.85 ba 6552*±206 11590±4287 2690±681 461.1*±65.2 6315/6692 6095/15686 1821/3431 415/507.2 cd <loq <loq <loq <loq < loq <loq <loq <loq co 59.2±32.3 160.8±114 135.7±76.6 61.89±15.17 29.4/106.1 33.8/273.1 62.7/213.3 46.42/76.51 cr 864±207.5 211.5±172.1 3925±2038 1591±649 717.9/1277 33.4/412.9 1976/5997 876/2560 cu 4544±4950 3454±1773 2559*±725 8070±1715 109/10145 1399/5734 1878/3321 6369/10934 fe 351.3±122.3 603±392 318.5±123.7 534.3±54 213/482 218/973 205/454 463/607 mn 73.5±27.2 31.63±6.96 34.41±5.13 63.87±12.98 47.247.2 24.55/39.58 28.32/39.31 49.99/79.58 ni 562.3±96.6 158.5±169.6 2655±494 775.1±136.9 438.5/695.6 18.1/114.1 2094/3222 590.7/916.9 pb <loq 363.2±157.1 <loq <loq <loq 129.9/472.1 <loq <loq sb 210.3±28.6 286.8±117.8 216.1±27 277.4±92.9 182.2/255.2 186.8/503.7 187.4/262.3 199.3/426.9 y 9.027±2.35 285±251 28.16±11.21 32.9±27.2 6.907/13.091 43/535 13.05/42 6.2/61.8 zn 33.16±5.51 29.98±2.93 36.2±2.5 40.71±6.83 26.78/38.66 27.02/33.25 32.82/40.24 33.67/50.91 table 2. mineral contents in µg/kg and mg/kg for 54 dehydrated bee pollen samples collected from four sites in the iron quadrangle, mg, brazil. (*) average of concentration no samples with <loq. samples read in icp-oes: mg/kg (al, fe, mn and zn). samples read in icp-ms: µg/kg (as, ba, cd, co, cr, cs, cu, ni, pb, sb and y). codes for areas are as in figure 1. spatial and temporal distribution of metal elements for comparisons among sites and seasons we chose to analyze the elements here classified as heavy metals due their toxicity and potential to cause damage to the environment: cr, co, ni, cu, as, y, sb, cs, ba, cd, pb, al, fe, mn and zn. mean concentration of each of these 15 elements in pollen samples are shown in table 2 and in spm samples in table 3. in general, wide within-site and among-sites concentration ranges were obtained for almost all elements both in pollen and spm samples (table 4). three element groups can be sociobiology 65(2): 259-270 (june, 2018) 263 table 3. mineral contents in µg/l for 60 suspended particulate material (smp) samples collected from four sites in the iron quadrangle, mg, brazil (*) average of concentration no samples with <loq. codes for the areas are as in figure 1. sampling sites ct im vm bm elements average±sd min/max al 138.7±94.9 744±559 322.1±235.3 346±429 39.4/286 90/1838 70.5/754 17/1454 as 0.1267±0.0635 0.1042±0.0458 0.2359±0.1666 0.927±1.997 0.051/0.282 0.045/0.185 0.085/0.619 0.072/5.837 ba 4.73±3.57 135.4±297.9 8.32±4.9 12.22±11.56 1.99/13.23 6.2/976 3.57/16.11 2.28/41.71 cd 0.02028±0.01607 0.0958±0.1925 0.0618±0.1173 0.0138±0.00898 0.00078/0.0623 0.0075/0.6371 0.0117/0.3924 0.00078/0.0292 co 0.593±1.345 0.516±0.2887 0.4308±0.2581 0.4319±0.2129 0.059/4.409 0.173/0.871 0.185/0.89 0.396/0.707 cr 8.61±7.71 4.9±5.13 12.11±11.29 5.36±7.14 1.21/21.77 0.95/13.45 1.53/29.38 0.51/20.76 cu 3.98±3.77 6.81±6.52 6.85±5.87 2.776±3.009 0.73/12.86 0.51/16.66 0.37/19.41 0.15/10.51 fe 108.6±60.6 1328±1112 315.2±255.2 2751±3518 50.9/241 144/3147 101/818 169/12030 mn 101.1±101.9 330±677 65.2±80.7 779±848 11.2/344 21/2244 12.7/275 39/2918 ni 2.169±2.656 0.6039±0.204 5.61±4.43 1.083±0.471 0.291/7.11 0.323/0.886 1.15/13.42 0.46/1.76 pb 0.643±0.523 1.125±0.658 1.718±2.193 0.5332±0.2472 0.259/2.03 0.697/2.93 0.344/7.5 0.188/0.864 sb 2.562±0.6 1.827±0.376 2.738±1.341 1.38±0.601 1.92/4.07 1.21/2.26 1.95/6.5 0.287/2.29 y <loq 3.125*±0.233 <loq 8.38±10.59 2.96/3.29 2.97/31.82 zn 106.7±170.5 151.3±66.1 121.8±97 100.4±55.7 18.8/586 96.6/314 55.8/373 58.4/247 differentiated: elements that are very abundant – present in the four sites in high concentrations (cr,cu, ni,fe, ba); elements in moderate concentration; and trace elements (pb, cd, cs, y and zn). metal concentration in pollen was expressed in µg/ kg(cr, co, ni, as, y, sb, cs e ba, cd, pb) or mg/kg (cu, al, fe, mn, zn) and for spm in µg/l (cr, co, ni, cu, as, y, sb, cs, ba, cd, pb, al, fe, mn and z). samples coming from the sample site ct differed significantly (p<0.01) from those found in samples coming from mining areas. pollen from site ct had, in general, lower concentrations of elements than the other sites with the exception of ba, cu, mn and ni elements. for spm the concentrations of most of the elements in ct were highly variable in relation to the mining areas. the fe concentration was very similar in the pollen samples of the four sampling sites, however, for spm samples, values of fe from ct fig 2. relationship between mean concentration of metals on pollen and in suspended particulate material (smp). no nascimento, ha nalini, f ataide, at abreu, y antonini – stingless bees as an environmental marker for metal contamination264 ta bl e 4. t -t es t v al ue s f or th e co m pa ri so n of e le m en t c on ce nt ra tio ns in th e dr y an d w et se as on s f or b ot h fo r po lle n an d sp m sa m pl es . ( *) sa m pl e < l o q ; b ol d nu m be rs p < 0. 05 ; p = p v al u; t = tte st v al ue . c od es fo r ar ea s ar e as in f ig ur e 1. q e sa m pl in g si te s c t im v m b m po lle n sp m po lle n sp m po lle n sp m po lle n sp m p t p t p t p t p t p t p t p t a l 0. 09 9 -1 .9 5 0. 40 5 1. 05 0. 00 3 -4 .9 8 0 -3 1. 4 0. 03 2 2. 93 0. 02 4 -6 .3 2 0. 13 7 -1 .8 5 0. 89 2 -0 .1 5 a s 0. 69 5 -0 .4 2 0. 00 6 -6 .9 1 0. 00 3 -4 .5 8 * * 0. 74 5 0. 34 0. 08 7 -3 .1 6 0. 45 1 0. 83 0. 97 8 0. 03 b a 0. 12 5 -1 .9 4 * * 0. 36 7 1. 02 0. 17 9 -2 .0 4 0. 17 3 1. 55 0. 00 5 -7 .5 5 0. 08 4 -2 .2 9 * * c d 0. 62 5 -0 .5 3 * * 0. 38 0 0. 99 * * 0. 29 8 -1 .1 9 * * 0. 22 1 1. 34 * * c o 0. 36 1 -1 .0 3 0. 02 9 -5 .7 3 0 -7 .7 4 0. 01 8 -7 .4 3 0. 08 2 2. 17 0. 00 1 -2 6. 34 -1 .9 60 -1 .9 6 0 -2 4. 79 c r 0. 00 6 5. 42 0. 45 3 0. 92 0. 02 6 3. 43 0. 01 -9 .9 3 0. 00 4 6. 16 0. 00 1 -2 8. 61 0. 10 7 2. 08 0. 39 9 0. 98 c u 0. 29 9 -1 .1 6 * * 0. 34 5 1. 01 0. 01 1 5. 66 0. 07 5 -2 .3 9 * * 0. 51 4 0. 72 0. 37 9 -1 .1 2 f e 0. 05 0 -2 .5 7 0. 00 1 -1 1. 68 0 -9 .2 2 0. 00 1 -2 8. 84 0. 05 3 2. 72 0. 01 0 -1 0. 18 0. 17 4 -1 .6 5 0. 03 5 -3 .6 9 m n 0. 32 7 -1 .0 9 0. 00 4 -1 6. 00 0. 51 1 0. 72 0. 00 2 -1 1. 05 0. 30 0 -1 .1 9 0. 00 7 -1 2. 23 0. 08 9 -2 .1 1 0. 00 5 -7 .6 0 n i 0. 25 2 -1 .3 4 0. 01 9 4. 64 0. 08 5 2. 00 0. 96 7 -0 .0 5 0. 44 5 0. 81 0. 00 8 -6 .4 3 0. 28 6 -1 .1 7 0. 01 0 -5 .8 6 p b 0. 52 7 -0 .6 9 * * 0. 40 2 0. 94 * * 0. 71 4 -0 .3 9 * * 0. 78 3 0. 29 * * sb 0. 58 7 -0 .5 9 0. 16 4 -2 .1 6 0. 52 0 0. 68 0. 13 2 -2 .4 7 0. 36 7 -1 .0 2 0. 43 7 -0 .9 6 0. 56 0 0. 62 0. 10 3 -2 .8 7 y * * 0. 79 2 -0 .3 0 * * 0 -2 9. 2 * * 0. 02 8 -3 .9 8 0. 71 0 0. 40 0. 00 1 -1 2. 34 z n 0. 24 3 -1 .3 7 0. 00 6 12 .3 6 0. 76 8 0. 31 0. 65 4 -0 .5 0. 53 1 -0 .6 7 0. 14 0 1. 99 0. 51 4 0. 72 0. 06 8 -3 .6 3 sociobiology 65(2): 259-270 (june, 2018) 265 and vmwere much lower. samples of spm did not differ significantly in sb and cd concentration among the sites. we find a relation among elements presence on pollen and spm samples in our sites (f=4.92, p=0,03) (figure 2). pronounced variation within-sites and among-sites was observed in the levels of all elements during the two climatic periods both in pollen and in spm samples (tables 5 and 6). for pollen, the highest mean concentration was found in samples collected in the dry season, and all 15 elements differed significantly between the dry and wet seasons (table 4 and 5). the concentration of sb did not differ significantly between the two seasonal sampling periods for the four sampling sites, whereas site vm had the greatest differences in concentrations between dry and wet season (table 4 and 5). for the spm samples, the concentrations of most of elements did not differ significantly between wet and dry period; only cr and al at sites ct, im and vm, and co, as and fe at site im differed significantly between climatic periods (table 4 and 6). the grouping of samples based on their similarities through pca (figure 3 and 4) indicates which chemical elements present in the pollen and spm samples are responsible for clustering in both climatic periods. for pollen samples, the cca explained 64.7% of the metal concentration variation between wet and dry climatic period. samples from im had higher concentrations of cd, sb, ba, cs, fe, pb, y,fe, al, and mn in the dry season (figure 3a and b), while those from vm had higher concentrations of as, co and al in the dry climatic period. for spm samples, only 45.6% of the variation was explained by metal concentration in wet and dry climatic periods. but we found differences only for site sm, that had higher concentrations of co, mn, al and fe (figure 4a and b). discussion our results showed that various quantities of toxic elements had accumulated in the stingless bee pollen samples, as expected. the level of accumulation depended on the type of rock being mined near where the pollen samples originated, as well as the sampling season. in the present study, pollen load was found to be the more contaminated matrix since we were able to detect and quantify more elements there than in spm, maybe because bees fly in many directions searching for food, gathering “contamined” pollen from a broader area. it must be noted that this pollen matrix has not been widely studied in other monitoring programs (borgdanov, 2006; chauzat et al., 2011; oliveira et al., 2017). the samples from pollen loads from t. angustula proved to be a good indicator of the presence of metal released in the atmosphere and subsequently throughout the environment, exhibiting differences in the composition of metals among the sampling sites. among the 15 chosen elements, the highest concentrations were found for barium, chrome, copper, iron and nickel, both in pollen and spm, independent of the region from which the samples originated. these elements are widespread throughout the iron quadrangle since they are abundant components of its rock and soil (costa et al., 2015; oliveira et al., 2017). even though it is not possible to quantitatively compare our results with those of other monitoring programs, since only a few have been published and they considered different chemical elements, some qualitative comparisons can be made. our findings were in line with other published studies in finding many different elements present in bee matrices in industrial areas. high heavy element concentrations, as we found, has been reported previously for pollen and honey coming from areas with heavy industrial activities or busy table 5. mineral contents in µg/kg and mg/kg for 54 dehydrated bee pollen samples collected during wet (w) and dry (d) season, from four sites in the iron quadrangle, mg, brazil. (*) average of concentration no samples with <loq. samples read in icp-oes: mg/kg (al, fe, mn and zn). samples read in icp-ms: µg/kg (as, ba, cd, co, cr, cs, cu, ni, pb, sb and y). codes for areas are as in figure 1. bm-w bm-d ct-w ct-d im-w im-d vm-w vm-d al 138.6667 142.3333 83.1333 73.2333 122.6667 816.6667 219.0000 418.3333 as 38.8373 38.5166 15.1382 32.8802 1.2119 104.9892 63.2459 173.8340 ba 36.9478 319.8446 35.6247 6551.5150 8797.5120 14383.4800 2089.0270 3290.3260 cd 3.8774 3.9120 3.7385 3.8832 3.8453 3.8776 3.7891 3.803702 co 48.0900 75.6960 31.3442 87.1070 60.267 261.3072 66.0247 205.4273 cr 1851.7450 1330.2330 943.3929 784.6034 57.4582 365.4744 2068.8300 5780.761 cu 7305.7980 8834.9580 8978.2190 110.0410 4979.3780 1927.9390 107.3742 2558.859 fe 491.0000 577.6667 241.3333 461.3333 246.0000 960.6667 207.6667 429.3333 mn 52.4100 75.3367 48.9000 98.1600 25.3833 37.8833 29.7900 39.02667 ni 656.8879 893.3705 643.2998 481.3357 110.4292 206.6642 2224.7640 3085.544 pb 9.8713 9.9571 9.5179 9.8863 49.8360 440.9491 9.6466 9.683775 sb 207.7850 347.0570 191.1601 229.4160 203.2694 370.2805 205.3813 226.7653 y 8.3697 57.4485 8.7082 9.3452 56.1115 513.9255 19.0124 37.30830 zn 35.2533 46.1733 38.1233 28.1868 29.3366 30.6233 37.8100 34.59333 no nascimento, ha nalini, f ataide, at abreu, y antonini – stingless bees as an environmental marker for metal contamination266 highways (d’ambrosio & marchesini, 1982; gajek et al., 1987; oliveira et al., 2017). the spatial distribution of metal elements was directly related to the region/mining type, since almost all five sampling areas had a high degree of similarity in the presence and concentration of metals, including, to our surprise, the control area. thus, the elements detected in the pollen samples of this study are a “mirror” of the rock type as well as the spm composition. it is important to notice that the most similar sampling sites were those with the most similar mining rock, table 6. mineral contents in µg/l for 160 suspended particulate material (smp) samples collected during wet (w) and dry (d) season, from four sites in the iron quadrangle, mg, brazil (*) average of concentration no samples with <loq. codes for the areas are as in figure 1. bm-w bm-d ct-w ct-d im-w im-d vm-w vm-d al 150.5860 541.4000 145.2520 132.0880 651.4000 835.9880 451.0060 193.1060 as 13.5000 0.1344 0.1080 0.1454 0.0980 0.1104 0.2356 0.2362 ba 68.1200 176.3000 4.8480 4.6160 239.5120 31.2060 10.6280 6.0120 cd 0.0132 0.0092 0.0132 0.0274 0.1634 0.0290 0.0946 0.0290 co 0.2781 0.4994 10.0120 0.1850 0.5070 0.5250 0.5484 0.3132 cr 649.9200 42.9920 94.8280 7.7300 60.3760 37.65400 19.2420 4.9740 cu 347.2000 20.8080 2.3160 5.6340 10.7560 2.8700 3.9920 9.7120 fe 1109.8000 4391.6000 100.9400 116.1680 1106.2000 1.5490 453.0000 177.4000 mn 346.9760 1210.6000 109.7600 92.4160 530.5260 129.486 37.7220 92.7480 ni 0.6324 1.3220 21.6260 2.1760 0.7606 0.4472 86.6380 2.5580 pb 0.5090 0.4522 0.3956 0.8900 13.0960 0.9410 27.9580 0.6408 sb 14.6140 1.2980 2.6820 2.4420 1.8440 1.8100 2.3020 3.1740 y 77.9760 418.9580 0.4315 0.4315 0.4315 151.0407 0.4315 0.4315 zn 110.5300 90.364 57.206 156.122 167.2 135.314 148.244 95.272 fig 3. loading plot (a) and score plot (b) principal components 1 and 2 obtained from mean composition of mineral elements in 40 dehydrated bee pollen samples from four areas in the iron quadrangle in dry (d) and wet (w) seasons. fig 4. loading plot (a) and score plot (b) principal components 1 and 2 obtained from mean composition of mineral elements in 60 spm samples from four areas in the iron quadrangle in dry (d) and wet (w) seasons. sociobiology 65(2): 259-270 (june, 2018) 267 like bm and im (ore for crushed stone), which had a high degree of similarity, while sf (iron ore) was more similar to ct. besides ct is a natural protected area, it is in a region with many abandoned or reclaimed iron ore mining. according to navarro et al. (2008) residues from mining and metallurgical operations are often dispersed by wind and/or water after their disposal. according to costa et al. (2015), the iron quadrangle possesses some signature minerals that represent the main rock or soil. this also could explain the similarities in metal presence among ct site and sf (iron ore), for example. in our sampling sites, as was quantified in both pollen and spm, although in low concentrations. the significant presence of as in this lithotype is corroborated by its occurrence in arsenopyrite, loellingite, realgar and arseniferous pyrite and the local gold mineralization. furthermore, the geochemical map of the iron quadrangle showed the highest density of positive as, cd, cu, ni, cr and zn anomalies (costa et al., 2015) according to these authors, these elevated concentrations were due to the interaction between geology and human interference. for cd, however, we found concentrations above that recommended by brazilian regulation only in spm. also, according to costa et al. (2015), the iron quadrangle possesses a natural liberation of trace elements, such as cd, from rocks and soils, which may explain the higher level of this element in spm samples. lead was detected in spm samples of the four studied sites, however, for pollen samples it was quantified only in samples from im. despite occurring in low concentrations, the amount of pb in spm samples was detected and quantified for all sampling sites. again, according to costa et al. (2015), pb distribution in the iron quadrangle is characterized by low concentrations. it is important to note, however, that the highest pb concentrations were found in areas heavily impacted by mining activities, and related to rock types such as granites and gneisses, as are found at the im mining site. perugini et al. (2011) reported similar results, with a significant difference in pb concentration in bees among stations located in urban areas and stations located in natural reserves; the highest mean concentrations were detected in honeybees collected from hives with surroundings characterized by intense air traffic and motor vehicle circulation. zinc distribution and concentration was found to be very similar among sampling sites, both for pollen and spm samples, however, the concentrations were low. this finding is corroborated by the fact that a large part of the iron quadrangle possesses zn values below the limits of intervention established by environmental legislation (pereira et al. 2007). climatic seasons the low level of variation observed in the concentration of almost all chemical elements within each of the two seasonal periods, even though the highest mean concentration of some elements was found in samples collected in dry climatic period, only a few elements had significantly different concentrations than in the wet period. although this variation presents a challenge to the appraisal of geographic effects, it also can reflect the influence of climate on the concentration of metal in the environment. it should be also considered that in iron quadrangle the wet period (october to march) are marked for days of intense rainfall, followed by weeks with no rain, what could explain the similarities in metal concentration among the climatic periods. for pollen samples, greater differences between climatic periods were found for al, co, cr and fe. pollen seemed to be more sensitive to climatic influences than spm in the present study, which was expected since mining activity does not stop, even during the rainy season. foraging bees must search for food on flowering plants that, in the case of mining areas, only exist inside the remaining small patches of forest. nonetheless, these plants still continuously receive high volumes of spm deposition on their flowers. however, for spm pronounced differences were found between seasons, with higher concentrations of more elements during the dry season. in the iron quadrangle, there is very little rainfall in the dry season and the amount of dust in the atmosphere is very high. therefore, it seems likely that this temporal distribution of contamination levels is linked to meteorological conditions and/or to the different types of plants foraged by bees (peruginni et al., 2011). azimi et al. (2003), also found high levels of variation in the concentration of elements (pb, cd and cu) among studies conducted in different years and seasons. lambert et al. (2012) also compared the concentration of pb among dry and wet seasons and found higher values for the wet season. past studies (magalhaes, 2007; oliveira et al., 2017) have shown that differences in contamination levels might be related to the landscape, but they have also revealed a relationship between urbanization, industrialization or anthropogenic activities and high contaminations in matrices. in our study, the general trends were influenced by certain contaminants. some chemical elements have already been quantified in bee pollen samples from several countries: potassium (k), phosphorus (p), magnesium (mg), calcium (ca), sodium (na), sulfur (s), iron (fe), copper (cu), manganese (mn), zinc (zn), chromium (cr), nickel (ni) and selenium (se) (e.g. azimi et al., 2013; bilandžić et al., 2011, bratu & beorgescu, 2005; chauzat et al., 2011). the number of samples analyzed, however, is limited, varying from one to fifty per study. two of these were studies in brazil (magalhaes, 2007; oliveira et al., 2017) and involved 24 samples collected from only one sampling site in the state of são paulo, and 36 samples from southern brazil. there is no benchmark for the maximum concentration of heavy metal allowed in pollen, as there is for honey, although, for pollen, there is for few individual elements like as (0.30 µg/kg), pb (0.30 µg/kg) and cd (0.10 µg/kg) no nascimento, ha nalini, f ataide, at abreu, y antonini – stingless bees as an environmental marker for metal contamination268 (anvisa 1998). for as in pollen samples, for example, we found concentrations many times higher than those established by regulations. for samples of pb, from spm, at the site im, the concentrations were also much higher than those established by regulations for honey. it is important to note that the concentrations of other elements, without benchmark values for pollen and honey, were also was very high. other chemical elements that have a limit established for food in general, or for food other than honey and pollen, are: cr (0.10 mg/kg), cu (10.0 mg/kg), ni (5.0 mg/kg), sb (2.0 mg/kg), zn (50.0 mg/kg) (anvisa1998), al (7.0 mg/kg), fe (found no tolerance limit established for human consumption, but found a stipulated maximum value of 0.8 mg/kg body weight), mn (between 780 – 930 mg/kg) (anvisa1998). due to the deficiency of regulations, it can be inferred that many of the samples had anomalous values exceeding stipulated limits when considering the few regulations that do exist for human consumption as mentioned above. we assumed that the contribution of chemicals elements on pollen, caused by translocation via soil, may be much lower than that of atmospheric deposition. more than 600 plant species were identified in the iron quadrangle, but information about metal concentrations in leaves and other plant parts is available for only 14 species (porto & silva, 1989; ribeiro et al., 2016). in a more recent effort, foliar metal concentrations, specifically al, fe and mn, were determined for 55 plant species (31 botanical families) (antonella schettini pers. comm.), with the ability of accumulating at least one of these metals in the leaves seemingly present in most of them, however, varying from minimum to maximum concentration by a factor of 47 for mn, 50 for fe and 314 for al. according to ribeiro et al. (2016) three plant species visited by t. angustula (eremanthus incanus, e. erytropappus and baccharis sp.) have the ability to translocate metals such as pb, cd, cu, cr, co, y, ni, ba, fe, al, zn, and mn to leaves, but not to flowers. wide within-area and among-areas variation was found in the mineral composition of dehydrated bee pollen, and fe, ba, al and mg were the major chemicals elements present. the five areas exhibited differences in chemical composition. in terms of mean values, bee pollen may be considered a rich source of information as a bioindicator for biomonitoring. it should be mentioned that pollen matrices from stingless bees proved to be an efficient environmental marker for metal contamination of the environment since we found significant differences in the quality and quantity of metals among the four sampling sites. since the sampling sites were associated with different types of mining, the metal composition present likely reflects the mineral composition of the dust generated by mining activities, which accumulates on flowers and consequently is carried to nests by stingless bee workers. acknowledgments the authors gratefully acknowledge cnpq for scholarships to ya and hanj, capes for the scholarship to fa, and capes for the scholarship to non. many thanks also to the following mining companies for the permition: safm, viamar, pedreira irmãos machado and bemil. we also like to thank adam roman and john trumble for the suggestions made in an earlier version of the manuscript. references anvisa agência nacional de vigilância sanitária; brazilian ministry of health. 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(1998). keys to soil taxonomy. united states department of agriculture, new york. valim, e.a.r. (2012). ciclagem de nutrientes no sistema atmosfera-solo-planta em formação campestre e florestal sobre canga em minas gerais. dissertação (mestrado) instituto de ciências exatas e biológicas, universidade federal de ouro preto, minas gerais vinas, p., pardo-martinez, m. & hernandez-cordoba, m. (2000). rapid determination of selenium, lead, and cadmium in baby food samples using electrothermal atomic absorption spectrometry and slurry atomization. analytica chimica acta, 412: 121-130. wayne, l. (1983). bee miners join british columbia god hunt. american bee journal, 3: 635-637. doi: 10.13102/sociobiology.v64i1.1184sociobiology 64(1): 92-100 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 termite (isoptera) diversity in a gallery forest relict in the colombian eastern plains introduction gallery forests along streams and rivers are important elements of the complex natural savanna ecosystems found in the colombian orinoco region. the region is currently experiencing accelerated changes in land use, mainly in the eastern plains (romero-ruiz et al., 2012), that will have unpredictable consequences for its ecological integrity (lavelle et al., 2014). termites are a key group for the dynamics of tropical forests where they are primary decomposers and drivers of nutrient cycling (ackerman et al., 2009; bandeira & vasconcelos, 2003) but research on termites in general in colombia is scarce and particularly in forest of the orinoco region (jiménez & decaëns, 2006); few records exist for this group in the region (lasso et al., 2010; pinzon et al., 2012). the available studies comprise a comparison of the density of soil macrofauna, including termites, in landscapes with abstract termites are known to influence soil nutrient cycle and decomposition processes, but its diversity and ecology in colombian gallery forests have been little studied. richness and relative abundance of termites found in gallery forest fragments protected within commercial forest plantations were quantified. sampling was conducted at three sites surrounding the creeks huerta la grande, claro and los micos, along 50 m long linear transects divided into five plots (10 × 2 m); in total, there were nine transects and 45 plots. termite sampling involved the examination of fallen branches, pieces of wood in contact with the soil, arboreal termitaria, epigeal nests and soil samples of 20 cm deep (four per plot). thirty-eight species, from the families termitidae (apicotermitinae, termitinae, nasutitermitinae, syntermitinae) and rhinotermitidae (heterotermitinae), were found. no differences regarding termite species, abundance or feeding guilds were detected among sampling sites; neither, association between richness or termite abundance, and tree size, or accumulation depth of leaf litter on the soil or canopy light. the termite diversity found in fragments of gallery forest highlights the importance of maintaining this type of vegetation to preserve biodiversity and the ecosystem services derived from the biological activity of termites. sociobiology an international journal on social insects op pinzon florian, ls baquero carvajal, ma beltran diaz article history edited by paulo cristsaldo, ufs, brazil received 12 august 2016 initial acceptance 18 september 2016 final acceptance 04 march 2017 publication date 29 may 2017 keywords conservation, orinoco, colombian eastern plains, macrofauna. corresponding author olga patricia pinzon florian universidad distrital francisco josé de caldas cra. 5e 3 15-82 bogotá, d.e. colombia e-mail: opatriciap@udistrital.edu.co different degrees of intensification (decaëns et al., 1994), and an analysis of the spatial distribution of nutrients in epigeal termite mounds of nasutitermes in a gallery forest (jiménez & decaëns, 2006). termites are sensitive to habitat fragmentation and changes in soil use (bandeira & vasconcellos, 2003; constantino & schlemmermeyer, 2000; ackerman et al., 2009; moura et al., 2009). therefore, their diversity may serve as an indicator of conservation status, for example in forest fragments (alves et al., 2011, oliveira et al., 2013, almeida et al., 2016). termites are the most abundant organisms in the soil macrofauna of natural ecosystems and semi-cultivated and cultivated areas in the eastern plains of colombia, where they can form more than 50% of the total abundance (decaëns et al., 1994; lavelle et al., 2014). termite activity influences the physical, chemical and biological activity of soils in this region (galvis et al., 1978; decaëns et al., 1994; jiménez & decaëns, 2006). universidad distrital francisco josé de caldas bogotá, colombia research article termites sociobiology 64(1): 92-100 (march, 2017) 93 the aim of this study was to characterize termite diversity in fragments of gallery forests conserved within a landscape matrix of pastures and commercial forest plantations in the eastern plains of colombia, in the orinoco region. we hypothesized that characteristics of the forest (e.g. tree size, canopy light and organic matter accumulation) may explain termite species composition found in the areas. materials and methods sampling was carried out in gallery forests surrounding the creeks known as huerta la grande, claro and los micos, in the basin of the upia river in the municipality of villanueva, department of casanare, colombia, within the geological formation known as mesa de san pedro 4°39.386´ n 72°55.185´ w. the three sites are 2 – 5 km apart and differ in their accessibility for humans, vegetation structure and development. the gallery forest sites have been protected since 1980, and are within commercial pine and eucalyptus plantations. the area is located at approx. 360 m elevation, has an annual average rainfall of approx. 2900 mm and distinct wet and dry seasons. rain occurs mostly from april to november; the dry season is from december to march. the annual average temperature in the area is 25.8 °c. the area is within the tropical rainforest life zone, and the vegetation diversity and structure of the sampling sites were characterized by fernandez et al. (2012). at each site, termite samples were obtained using the sampling protocol described by jones and eggleton (2000) with some modifications: along three linear transects, each 50m long and divided into five plots (10 × 2 m), resulting in 15 plots per site and 45 plots in total. there were at least 50 m from the edge of the forest to each transect and 50m of distance between them, following the linear shape of the forest that surround the creeks. sampling took 60 minutes per plot; the total sampling effort was 45 hours. sampling involved examination of and direct manual capture of termites from epigeal nests, fallen branches, pieces of wood in contact with the soil, superficial organic soil layer (four samples per plot: 20 x 20 cm [length x width] and 20 cm deep), and arboreal termitaria up to 3 m high. sampling took place at the beginning of the wet season (april 2013) in the huerta la grande site, and at the end of the wet season (november 2013) in the remaining two sites. to describe the structural and environmental characteristics of each sampling site, the height and trunk diameter of trees located on the main line of each transect were measured using an ultrasound measurement instrument (haglof vertex iv) and diametric tape, respectively. also, the percentage of canopy cover was estimated from photographs taken using a fisheye lens adapted to a camera compatible with winscanopy software. in addition, leaf litter accumulation depth was measured at three points per plot, and the average used for statistical purposes. also, one soil sample was taken per site to determine soil moisture content, ph and organic c. identification at the genus and species levels was conducted using appropriate keys (bourguignon et al., 2010, 2013, 2016; carrijo et al., 2011; constantino, 2002; cuezzo & cancello, 2009; rocha et al., 2012), and by comparison with some specimens kept at the collection (cefud) that had been identified by dr. reginaldo constantino. nasutitermes and heterotermes samples were reviewed by dr. tiago fernandes carrijo. in addition, specimens for which it was not possible to confirm the species were named and numbered as morphospecies. samples of apicotermitinae containing at least ten individuals were separated at the species and morphospecies levels by dissecting the enteric valve and comparing its morphology with that described in the literature (bourguignon et al., 2010, 2013, 2016). termite samples are kept in 2 ml vials containing 85% ethanol, in the colección entomològica forestal (cefud) at universidad distrital francisco jose de caldas in bogota d.c. termites were assigned to the feeding guilds described in the literature (donovan et al., 2001) using available information for the genus/species (de souza & brown, 1994, constantino, 1999, bourguignon et al., 2016). feeding guilds included xylophages (feed on wood), humivores (feed on humus and soil organic matter), intermediate feeders (feed at the soil/ wood interface and do not fit into the categories mentioned above; collected under decomposed fallen logs) and litter feeders (feed on plant material deposited on the surface of the forest floor, including grasses, leaf material, litter). the relative abundance of each termite species (expressed as the number of encounters) was determined by recording the presence of each species only once in each plot up to a maximum of five per transect (15 per site). richness was calculated as the number of species and morphospecies found at each site. termite diversity was characterized using estimated species number based on coverage rarefaction (chao & joost, 2012). we obtained estimated richness (q0) and the estimators shannon (q=1) and simpson (q=2) with a 95% confidence level. to test the hypothesis of differences in termite species composition among sites, we performed a permutacional analysis of variance (permanova; α:0,05) based on bray– curtis dissimilarity (anderson, 2005). all the analyses were performed with the software r (r development core team, 2014), using the vegan package (oksanen et al., 2012) and inext (hsiech et al., 2016). to test the hypothesis of differences in termite abundance or richness among sites regarding feeding guilds and to compared tree size, canopy light and organic matter accumulation among sites we performed a one-way anova using spss (ibm spss version 20). results termites from 24 genera and approximately 38 species were found in a total of 278 samples (table 1) and 199 encounters. most species and morphospecies (81.2%) belonged to the family termitidae (and to the subfamilies op pinzon florian, ls baquero carvajal, ma beltran diaz – termite diversity in a gallery forest relict94 taxon claro huerta la grande los micos total feeding group feeding habits references rhinotermitidae 12 10 11 33 coptotermitinae 0 0 1 1 coptotermes testaceus (linnaeus, 1758) 0 0 1 1 x constantino, 1999; donovan et al. 2001 heterotermitinae 11 9 10 30 heterotermes tenuis (hagen, 1858) 7 8 7 22 x constantino, 1999; de souza & brown, 1994 heterotermes convexinotatus (snyder, 1924d) 4 1 3 8 x constantino, 1999; de souza & brown, 1994 rhinotermitinae 1 1 0 2 rhinotermes marginalis (linnaeus, 1758) 1 1 0 2 x constantino, 1999; de souza & brown, 1994 termitidae 60 39 67 166 apicotermitinae 7 8 19 34 anoplotermes parvus snyder,1923 2 0 1 3 h constantino, 1999; de souza & brown, 1994 apicotermitinae sp. 1 1 0 1 2 h constantino, 1999; de souza & brown, 1994 apicotermitinae sp. 2 0 1 1 2 h constantino, 1999; de souza & brown, 1994 apicotermitinae sp. 3 0 2 1 3 h constantino, 1999; de souza & brown, 1994 apicotermitinae sp. 4 0 3 4 7 h constantino, 1999; de souza & brown, 1994 apicotermitinae sp. 5 3 1 1 5 h constantino, 1999; de souza & brown, 1994 patawatermes turricola (silvestri, 1901) 1 1 7 9 h bourguignon et al., 2016 ruptitermes sp. 1 0 0 2 2 h constantino, 1999; de souza & brown, 1994 ruptitermes sp. 2 0 0 1 1 h constantino, 1999; de souza & brown, 1994 nasutitermitinae 26 17 29 72 angularitermes nasutissimus (emerson, 1925) 0 0 1 1 i constantino, 1999 atlantitermes raripilus (emerson, 1925) 1 0 0 1 i constantino, 1999 coatitermes clevelandi (snyder, 1926,d) 1 2 0 3 i constantino, 1999 nasutitermes banksi emerson, 1925 5 4 6 15 x constantino, 1999; de souza & brown, 1994 nasutitermes cf. ephratae 6 3 3 12 x constantino, 1999; de souza & brown, 1994 nasutitermes cf. gaigei 0 0 1 1 x constantino, 1999; de souza & brown, 1994 nasutitermes cf. guayanae 5 4 7 16 x constantino, 1999; de souza & brown, 1994 nasutitermes cf. similis 7 3 8 18 x constantino, 1999; de souza & brown, 1994 nasutitermes cf. surinamensis 0 1 0 1 x constantino, 1999; de souza & brown, 1994 rotunditermes bragantinus (roonwal & rathore) 0 0 3 3 x constantino, 1999 subulitermes sp.1 1 0 0 1 h constantino, 1999; de souza & brown, 1994 table 1. termite diversity, relative abundance, and feeding guilds of termites found in gallery forests. x: xylophagous, h: humivorous, l: litter feeder, i: intermediate; c: caño claro, hlg: caño huerta la grande, m: caño los micos. sociobiology 64(1): 92-100 (march, 2017) 95 taxon claro huerta la grande los micos total feeding group feeding habits references syntermitinae 11 2 10 23 cyrilliotermes angulariceps (mathews,1977) 1 0 0 1 h constantino, 1999 embiratermes neotenicus (holmgren,1906) 2 1 6 9 i constantino, 1999 labiotermes labralis (holmgren,1906) 0 0 3 3 h constantino, 1999; donovan et al. 2001 mapinguaritermes cf. grandidens (silvestri,1901) 1 0 0 1 i constantino, 1999 silvestritermes cf. euamignathus (silvestri,1901) 3 0 0 3 i constantino, 1999 syntermes sp.1 4 1 1 6 l constantino, 1999; donovan et al. 2001 termitinae 16 12 9 37 crepititermes verruculosus emerson, 1925 1 0 0 1 h constantino, 1999 cylindrotermes flangiatus (holmgren, 1906) 1 1 1 3 x constantino, 1999 cylindrotermes parvignathus (holmgren, 1906) 3 0 1 4 x constantino, 1999 microcerotermes cf. exiguus (hagen,1858) 5 5 4 14 x constantino, 1999 neocapritermes cf. pumilis constantino,1991c 1 0 0 1 x constantino, 1999 neocapritermes cf. talpoides krishna & araujo, 1968 0 1 0 1 x constantino, 1999 neocapritermes cf. taracua krishna & araujo, 1968 0 0 1 1 x constantino, 1999 termes sp.1 5 5 2 12 i constantino, 1999; de souza & brown, 1994 total morphospecies 25 20 27 38 relative abundance 72 49 78 199 table 1. termite diversity, relative abundance, and feeding guilds of termites found in gallery forests. x: xylophagous, h: humivorous, l: litter feeder, i: intermediate; c: caño claro, hlg: caño huerta la grande, m: caño los micos. (continuation) apicotermitinae [23.7%], nasutitermitinae [31.4%], termitinae [21.0%], and syntermitinae [15.8%]); the remaining (11.4%) belonged to the family rhinotermitidae. termites were found in 98% of the 45 plots and in 17% of the 180 soil samples examined. seventy-five per cent of the 198 encounters were with termites of the genera nasutitermes, heterotermes, anoplotermes, microcerotermes, termes and embiratermes. the most abundant species found was heterotermes tenuis followed by nasutitermes similis, n. cf. guayanae and n. banksi. approximately 30% of the morphospecies were found only once. in the three sampling sites, richness ranged from 20 (52.6% of the total number of morphospecies) to 27 (71%), and abundance (number of encounters) ranged from 49 (24.6% ) fig 1. estimated termite species richness by coverage based rarefaction in caño claro, huerta la grande and los micos gallery forests. estimates are based on 0.86 coverage. error bars correspond to 95% confidence intervals. collection substrate claro los micos huerta la grande total arboreal 3.3 0.7 2.9 6.9 dead wood 28.7 17.1 30.9 76.7 mound 1.8 1.5 2.9 5.5 soil sample 2.2 4.0 4.7 10.9 total 36.0 23.3 41.5 100 table 2. relative abundance (%) of termite samples according to foraging substrate. op pinzon florian, ls baquero carvajal, ma beltran diaz – termite diversity in a gallery forest relict96 of the total number of encounters to 78 (39.2%). the maximum percentage of total morphospecies and species found in each site was 70%; about 30% of the species we sampled were singletons or doubletons. although, at the huerta la grande numerically lower number of species and encounters were found, estimated species richness (q0) based on coverage rarefaction including 95% confidence intervals indicates no significant differences among sampling sites, therefore, neither significant differences were estimated regarding shanon (q1) or simpsom (q2). as observed in figure 1, confidence intervals overlap among sampling sites. sampling coverage curve including interpolation and extrapolation values for each sampling site, averaged 82% of completeness, and indicates a trend towards stabilization, suggesting the sampling appropriately represent the termite diversity in the area (figure 2). the 38 termite species and morphospecies found were assigned to four feeding guilds as follows: xylophages 42.47%, humivores 34.2%, intermediate feeders 18.4% and litter feeders 2.6%. xylophagous dominated (61.8% on average) the encounters per site (figure 2). most termites (76.7%) were collected foraging in deadwood (table 3). the sampling sites were also similar regarding termite richness or abundance of feeding guilds (figure 3). fig 2. sample coverage curve as indicative of sampling completeness at caño claro, huerta la grande and los micos gallery forests. site trees per transect dap (cm) tree height (m) organic matter accumulation (cm) canopy light (%) organic carbon (%) ph soil moisture (%) claro 8 ± 2.5 24.2 ± 8.4 a 15.3 ± 3.3 a 3.7 ± 1.4 a,b 13.7 ± 5.6 a 3.0 4.4 29 huerta la grande 23 ± 7.2 13.6 ± 3.4 b 10.2 ± 4.7 b 4.6 ± 1.6 b 16.0 ± 5.3 a 1.2 4.5 9 los micos 18 ± 6.1 11.7 ± 6.3 b 11.1 ± 2.5 b 3.1 ± 1.3 a 15.8 ± 6.0 a 4.3 4.3 22.3 df. 2 2 2 2 p 0,000 0.001 0,018 0,46 f *** *** table 3. summary of one-way anova of structural and environmental variables. fig 3. richness (a) and abundance (b) of termites by trophic guild in caño claro, huerta la grande and los micos. relative number of species (top left), relative number of encounters (below, left.) size of the trees (expressed by height and diameter) was higher in caño claro (table 4). however, we did not find any structural variable related with diversity as indicated by the permanova analysis based on bray-curtis dissimilarity and including the structural variables (table 5). discussion in this study, angularitermes, crepititermes, mapinguaritermes and subulitermes were recorded for the first time in colombia, and rhinotermes, coptotermes, sociobiology 64(1): 92-100 (march, 2017) 97 cyrilliotermes, coatitermes and termes were recorded for the first time in the orinoco region of colombia. consequently, our findings increase to 27, the number of genera and to 40 the number of (morpho) species of termites known to occur in the orinoco region (vargas-niño et al., 2005; morales-castaño & medina, 2009; pinzón et al., 2012). unfortunately, was impossible to compare the diversity and abundance found in our study with those of other colombian studies because there have been no previous reports of inventories from colombian gallery forests. however, unpublished data (pinzon personal communication) found only 21 morphospecies from 18 genera in a gallery forests and 10 genera in a commercial rubber plantation (pinzon et al., 2012) in pto. lòpez, a nearby location to the study site from the present study. sampled sites in the current study kept 40% more species. although primary amazonian forests may substantially differ from the forest of our study, only in order to have a reference, a recent study, using the sampling protocol, revealed eighteen more species than our study in a primary no flooding forest in central amazonia (ackerman et al., 2009). the same study reported humivorous to account for 50% of the encounters (we found 20%), and the dominance of nasutitermitinae, as well as nasutitermes. in another study in tropical forests, jones et al. (2003) found fewer number of species than reported here, in a primary forest (34 species), with substantial dropping of termite diversity and encounters when progressive degree of land use disturbance was compared; a trend that appear to be consistent when natural forest is replaced by agricultural activities. seventeen species of termites (61.8%) belonging to the xylophagous feeding guild were found in the areas. as indicated in a study of a gallery forest in the cerrado (oliveira et al., 2013), we also expected to find more representatives of this guild than the others, given the easy availability of wood and cellulosic resources derived from fallen branches, palm leaves and mulch at the sites sampled in this study (most of the xylophagous were collected in dead wood pieces in the forest floor). species of heterotermes and nasutitermes were the main removers of wood and leaf litter in the study areas. contrary to oliveira et al. (2013), xylophage species in our study were found foraging within the niches explored in this sampling method and predominated in the samples, while humivorous where the second more important group. structural and environmental variables of forests have been used to attempt to explain termite diversity (davies et al., 2003; apolinario & martius, 2004; vasconcellos et al., 2010; dahlsjö et al., 2015). in particular, leaf litter accumulation depth and the basal area of woody plants may correlate with termite abundance (gillison et al., 2013), and tree size may correlate with the abundance of some termitidae, particularly those that build arboreal termitaria (gillison, 2003; jones et al., 2003; gonçalves et al., 2005). in this study, the site claro had more well-developed trees (with greater trunk diameter and height) than los micos and huerta la grande, however, these structural differences in the habitat did not influenced the termite diversity; neither the abundance or richness of any feeding group differed significantly between sites as have been reported in some tropical forest (jones et al., 2003). our results are similar to recent observations in some brazilian forest fragments (oliveira et al., 2013; almeida et al., 2016), in which termite diversity is not directly explained by the structural/environmental variables of the vegetation included in those studies. besides vegetation structure and environmental conditions, understanding termite plasticity and habitat requirements as well as quality and availability of food (kirton et al,. 1999; araujo et al., 2011; almeida et al., 2016) or predation (dambros et al., 2016) may contribute to better explain termite diversity association in these particular habitats. the low soil moisture content (9%) at huerta la grande, due to the fact that sampling was done in a relatively drier period at this site, may explain the lower numerical values of richness and abundance of termites at this site compared to the other two sites, which were sampled in a more humid period. it is known how the frequency of encounters, especially with subterranean termites, decreases during dry times. under these conditions, termites migrate to deeper soil layers, because high temperatures and low humidity in the surface layers of wood strongly limit superficial foraging (nobre et al., 2009). decreases in termite abundance and richness in rubber tree plantations during the dry period have been reported in areas close to our sampling site (pinzón et al., 2012). despite the fact that the gallery forest relicts included in this study are subject of anthropocentric pressure derived from the surrounding forested and pasture matrix, diversity of termites found in this study are considerable, and besides adding to our knowledge about local and regional termite fauna sources of variation df ss mean ss f model r2** p value dap (cm) 1 0,288 0,288 0,741 0,017 0,795 tree height (m) 1 0,390 0,390 1,003 0,023 0,443 canopy light (%) 1 0,290 0,290 0,747 0,017 0,768 organic matter acum. (cm) 1 0,313 0,313 0,804 0,018 0,679 residuals 39 15,169 0,388 0,909 total 44 16,685 1 ** variance explained by each source of variation. table 4. summary of permanova of termite composition including sampled sites and forest structural variables. op pinzon florian, ls baquero carvajal, ma beltran diaz – termite diversity in a gallery forest relict98 in colombia, also highlights the importance of gallery forest fragments as reservoirs for these key arthropods. termites are of clear conservation interest; therefore, other forest fragments with lower or no human influence in the orinoco region should be surveyed to obtain a better representation of the termite diversity of the region. acknowledgements we are grateful to guido gasca and mónica sarmiento and refocosta s.a.s for providing logistic support and access to sampling sites. laboratory and field assistants: natalia arias, daniel castro, jeffer vega and eulises vanegas and yulieth toro. dr. tiago carrijo revised samples of heterotermes, nasutitermes and some apicotermitinae. dr. jaime pinzon and msc. walter garcia provided support for community data analysis. we are indebted to anonymous reviewers for providing comments and insights of an earlier version of this paper. fieldwork, field assistance and publication were supported by centro de investigaciones científicas, universidad distrital francisco josé de caldas and colciencias contrato 415 de 2013. opp wrote the proposal, participated in field collecting, did the identification to species and morphospecies, analyzed data and wrote the paper; lbc and mbd participated in field collecting, laboratory sorting and genus taxonomic determination. references ackerman, i. l., constantino, r., gauch jr, h. g., lehmann, j., riha, s. j. & fernandes, e. 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unsolved, is “why have aleocharines, as opposed to almost any other insect group, been so successful at invading colonies of social insects?” (yamamoto et al., 2016). it is being argued by parker (2016) that preadaptation such as a predatory diet, physically or chemically defensive morphology and small body size would have acted in synergy to allow myrmecophily and termitophily in this group. abstract inter-specific symbiotic links are often reinforced by morphological, physiological, or behavioral trait modification undergone by the associated species. in some cases, such as in physogastric termitophile staphylinids, such modifications do facilitate the social interaction. here we inspect chemical traits of the physogastric staphylinid corotoca melantho (insecta: coleoptera) and its termite host constrictotermes cyphergaster (insecta: blattodea: isoptera), aiming to verify whether staphylinids resemble their host. first, we compared chc profiles of hosts and guests within and among termitaria, to gather evidence on the origin of such profiles in guests. then, we examined nitrogen and carbon isotopic signatures of these cohabitants to inspect whether chemical disguise is achieved by predation of host workers by staphylinids. beetles presented chc more similar to the chc of their cohabiting termites than to (i) their conspecifics and (ii) termites from another nest, thereby favouring the hypothesis on chc acquisition by guests. isotopic signatures revealed that such similarities could not be majorly determined by share nutrition between these cohabitants. in general, our results evidenced that chemical disguise in termitophiles may function as a strategy for social integration in morphological mimics. sociobiology an international journal on social insects cs rosa1,2, pf cristaldo1,3, df florencio1,4, a marins1, er lima1, o desouza1 article history edited by alexandre vasconcellos, ufpb, brazil received 13 august 2017 initial acceptance 18 november 2017 final acceptance 18 november 2017 publication date 30 march 2018 keywords cohabitation, corotoca melantho, symbiosis, constrictotermes cyphergaster. corresponding author paulo felipe cristaldo universidade federal de sergipe são cristóvão-se, brasil. e-mail: pfellipec@gmail.com specifically for termitophilic aleocharines, physically defensive morphologies have been reported as important facilitators of their social integration. these can be recognized in two types of body plan: a limuloid and a disguising form kistner (1982). in the limuloid (horseshoe crab) body form, expansions in the lateral margins of the body conceal vulnerable appendages, protecting them from host attacks. in the disguising form, a swollen thorax or –more commonly– a grotesquely expanded abdomen provides striking resemblance to the host (cunha et al., 2015) hence avoiding detection of the beetle as a non-nestmate. recent studies (kanao et al., 2016; yamamoto et al., 2016) on the evolution of the limuloid body of the aleocharinae have shed revealing light on the reasons for the success of this subfamily as social parasites. as for physogastry, we still need more efforts to give continuity to the seminal works by seevers (seevers, 1957) and kistner (kistner, 1979, 1982). 1 pós-graduação em entomologia, departamento de entomologia, universidade federal de viçosa, minas gerais, brazil 2 universidade federal do triângulo mineiro, iturama-mg, brazil 3 universidade federal de sergipe, são cristóvão-se, brazil 4 departamento de agrotecnologia e ciências sociais, universidade federal rural do semi-árido, mossoró-rn, brazil research article termites mailto:pfellipec@gmail.com sociobiology 65(1): 38-47 (march, 2018) special issue 39 physogastry in rove beetles is notable for its apparent termitophilic role: morphometrical congruence between the hypertrophic abdomens of hosts and guests (cunha et al., 2015) can be reasonably interpreted as tactile (or “wasmannian mimicry” sensu rettenmeyer, 1970). in fact, morphological similarity has been long considered a disguising trait allowing termitophiles to pass host worker’s inspection, as these latter cannot distinguish mimics from models based on their body constitution and surface structure (sands & lamb, 1975). succeeding inspection means that the termitophile is recognized as a nestmate and this would not only avoid aggression but could also ease allogrooming and trophallactic exchanges with their host (kistner, 1979). more extremely, such a disguise could help termitophiles to approach and prey upon termites, their brood, and their eggs. the integrity of social insect colonies, however, does not seem to majorly rely on morphological inspection (in fact, a lot of social insect symbionts do not resemble their hosts). rather, a sophisticated recognition system, based on chemical cues (nash & boomsma, 2008) notably from cuticular hydrocarbons (chc) (blomquist & bagneres, 2010), helps detecting alien species. chc allow colony members to distinguish nestmates from non-nestmates through comparison of their own chc template. morphological disguise, therefore, should work properly when in consonance with some form of chemical disguise. a range of chemical disguise strategies can be used by invaders, such as (i) biosynthesize disguising chc profiles (’innate chemical mimicry’), (ii) biosynthesize some specific chc compounds of their host and acquiring other disguising compounds after getting in touch with termites and/or their nest walls (’acquired chemical mimicry’) and (iii) displaying no or a weak quantity of recognition cues allowing them to keep unnoticed into the host colonies (’chemical insignificance’). in addition to these strategies, host can not able to detect the mimic as a different organism (’chemical crypsis’) and host can detect and recognize invaders as an uninteresting organism (’chemical masquerade’) (for more details see von beeren et al., 2012). here we investigated the chemical disguise strategy used by corotoca melantho shiødte, 1853, one of the most striking physogastric staphylinidae: aleocharinae beetles, which is an obligatory termitophile of constrictotermes cyphergaster (silvestre, 1901) termites and has previously been observed engaging physical contacts with their host (grassé, 1986). we start off by comparing chc profiles of hosts and guests, within and among termitaria. if guests acquire chc, one should expect their profiles to match colony-specific chc profiles of termites. alternatively, if guests only biosynthesize disguising chc, the similarity between beetles and termites would be most obvious at the speciesrather than at the colony-level. finally, we also examine nitrogen and carbon isotopic signatures of these cohabitants, to inspect whether chemical disguise is acquired by predation of termite workers by staphylinid beetles. material and methods ethics statement. all necessary permits were obtained for the described study, which complied with all relevant regulations of brazil. this includes collecting and transportation permits from ibama (the brazilian institute for the environment and renewable natural resources), and permission from embrapa (the brazilian enterprise for agricultural research) to conduct the study on their site. tacit approval from the brazilian federal government is implied by hiring the authors as scientific researchers. no protected species were sampled. terms definition. the terms ‘‘termitarium’’ (plural: termitaria), ‘‘mound’’, or ‘‘nest’’ denote the physical structure built by termites. ‘‘colony’’ denotes the assemblage of termite individuals living and cooperating intra-specifically within a nest. ‘‘coexistence’’ and ‘‘cohabitation’’ are used as synonyms and refer to the simultaneous occurrence of termites and other arthropods within a given termitarium, without implication of reciprocal positive or negative influences. these nontermite cohabitants are referred to as “termitophiles”, that is, “[...] arthropods which live in the same nests as their host or have some other obligatory relationship with them” (kistner, 1979). the terms “guest”, “resident”, and “intruder” may also be used to refer to termitophiles simply because they establish themselves –not necessarily by force– in nests which had not been built by/for them in the first place. study system: the termite host and its termitophile. constrictotermes cyphergaster (blattodea: isoptera: termitidae: nasutitermitinae) is a common termite species in brazilian savannas (“cerrado”) (mathews, 1977) and dry scrub (“caatinga”) (vasconcellos et al., 2007). nests of this species are frequently built on trees (desouza et al., 2016; moura et al., 2006; vasconcellos et al., 2007), harbouring many organisms, including other termite species –so called “inquilines”– and a number of termitophiles, mainly staphylinidae (insecta: coleoptera) (cunha et al., 2003; mathews, 1977; seevers, 1957). among these latter, corotoca melantho shiødte, 1853 (staphylinidae: aleocharinae: corotocini) is the most frequent and abundant obligatory cohabitant. in the studied site, this termitophile has been recorded in 83% of all c. cyphergaster nests as long as they are bigger than 2.2 litres (cristaldo et al., 2012). under natural conditions, corotoca melantho is reported to live in close contact with their termite host with no aggressive behavior from soldiers and workers of host colonies (costa & vanin, 2010; cunha et al., 2015). stomodeal trophallaxis is assumed (but not explicitly observed) to occur based on their unusual mouthparts: differently from other aleocharinae, members of the corotocini tribe have the mentum fused to the submentum forming a shield-like sclerite (figure 18q in seevers, 1957). this stationary condition of the mentum has led seevers (op. cit) to suppose that these beetles receive liquid aliment from their hosts. apart from records (grassé, 1986) on termites licking cs rosa et al. – chemical disguise of a rove beetle40 their cohabiting c. melantho, additional explicit information on the specific behavior of this termitophile is not available in the literature. termitophile staphylinids (but not free-living or myrmecophile ones), often present hypertrophic abdomen (i.e., “physogastric”) folded over the thorax, making them visually similar to their host. among them, the aleocharinae: corotocini comprise the most pronounced cases (fig 1) and among corotocini, c. melantho’s physogastry is so marked as to make it morphometrically indistinguishable from its host (cunha et al., 2015). this pronounced abdominal hypertrophy is compatible with their ovoviviparity. study area and sampling c. cyphergaster nests (n=11) were collected in brazilian savanna (“cerrado”), near the town of sete lagoas (19°27’ s, 44°14’ w), state of minas gerais, south-eastern brazil. in the study area, altitude varies from 800 to 900 m above sea level and climate can be characterized as equatorial with dry winter (aw) according to köppen’s classification. epigeous and arboreal nests with no signal of damage were sampled in an area of 114 m². collections were performed in july 2008 and may 2011. in the laboratory, nests were completely dissected and inspected to collect, using entomological soft forceps, a sample of individuals (soldiers and workers) from termite colonies and all termitophilous staphylinidae. specimens to be used for identification were preserved in 80% alcohol, labelled, and subsequently identified to the lowest possible taxonomic level. termite hosts were identified to species following mathews (1977) and by comparison with the samples in the termite section of the entomological museum of the federal university of viçosa (meuv), where voucher specimens were deposited (# ufv8735, ufv8736, ufv8737). the staphylinidae were sent to a specialist for identification. specimens used for chemical analyses (chc and stable isotopes) were prepared as described below. each type of analysis always involved both staphylinids and termites from the same nest, but not all nests provided enough material for all analyses. hence, out of the 11 nests studied, four provided enough guest-host pairs for chc and isotope analyses, five nests provided material just for chc analyses and three nests provided material for isotope analyses only. in summary, staphylinids and termites were probed in nine nests for chc and in seven nests for isotopes. cuticular hydrocarbons: extraction and analysis. in order to assess the degree to which chc profiles of c. melantho resembled those of its termite hosts, individuals of each of species were washed in solvent and extracts thereby obtained where then analysed by gas chromatography coupled with mass spectrometry (gc–ms). for chc extraction, initially one sample composed of 10 termite workers and another sample composed of 10 beetles from each nest were stored separately in glass vials, and then freeze-killed. thereafter, samples were submerged in 100 μl of n-hexane (hplc grade) for 10 min, following the procedure described by haverty et al. (1996) and marten et al. (2009). chc extractions were conducted without agitation of the glass vials. extracts were then transferred to a fresh vial and the solvent was allowed to evaporate at room temperature. extracts were redissolved with 10 μl of n-hexane per sample. extractions for each of the cohabiting species from a single nest were carried out independently, to avoid chc mixing. gc-ms analyses were performed using a gcms-qp2010 se shimadzu equipped with rtx®-5ms, with samples (1 μl) injected under 220°c, in splitless mode. a fused silica column (30 m 0.25 mm id; 0.25 μm) was used for separation. helium was used as a carrier gas at a constant flow rate of 0.87 ml/min. the temperature program was: 130°c to 290°c at 7°c/min and held for 15 min. scans of mass spectrometer were performed from 40 to 500 m/z using the electron impact mode (70 ev). samples were injected in no particular order, i.e. randomly. the peaks were classified based on their mass spectra and non-isothermal kovats retention indices (ki) (using definition of van den dool & dec. kratz, 1963) evaluated on the basis of n-alkane series (carlson et al., 1998). stable isotopes analysis. in order to inspect trophic relationships between guests and hosts, we analysed the nitrogen and carbon stable isotope concentrations of both c. melantho beetles and their host c. cyphergaster termite workers. such isotopic concentrations, here also referred to as “isotopic signatures”, were obtained by measuring 13c/12c and 15n/14n ratios (hereafter δ13c and δ15n, respectively) from the bodies of the beetles and termite individuals under study. termite workers of each species in the termitaria were sorted, when possible, into 10 subsamples, each with a sufficient number of individuals to obtain a dry biomass of 1.5 μg for full-body isotopic analysis. this allowed to characterize these species by a set of δ13c and δ15n pairs obtained from several individuals from the same colony, this set circumscribing a bidimensional space in a cartesian plot whose axes represent the concentration of such isotopes in the bodies of the individuals. the δ13c and δ15n ratios in the body of an organism reflect the food consumed and assimilated during its lifespan (deniro & epstein, 1978; eggers & jones, 2000). full-body isotopic analysis has been recently used to infer the diet (florencio et al., 2013) and the trophic relationships (de visser et al., 2008) of termitaria cohabitants. such inferences are possible because, while δ13c changes little as carbon moves through the food web, δ15n enriched by 3–4‰ each trophic step (post, 2002). hence, two organisms positioned at two adjacent trophic steps (e.g., a consumer and its prey or its donor of exudates) will show roughly no change in their δ13c body content but will differ by 3‰ in δ15n. such differences in δ15n accumulate across trophic levels (tayassu et al., 1997). we have, hence, used δ13c and δ15n ratios to infer whether diets of c. melantho beetles could be based on termite-derived materials. sociobiology 65(1): 38-47 (march, 2018) special issue 41 samples for stable isotope analysis were prepared according to florencio et al. (2011) (field procedure) and florencio et al. (2013) (lab procedure). δ13c and δ15n concentrations were measured using an isotope ratio mass spectrometer (irms, anca-gls 20-20, sercon, uk) from the stable isotopes laboratory at federal university of viçosa (ufv). the instrument’s analytical precision was estimated to be ±0.1‰ for carbon and ±0.2‰ for nitrogen. the natural abundance of the heavy δ13c and δ15n relative to the light 12c and 15n, typically corresponding to the rare and abundant isotopes, is expressed as per thousand (‰) deviation from an international standard (belemnite of the pee dee formation in south carolina, usa (pdb) for carbon and atmospheric nitrogen (air) for nitrogen). statistical analyses all statistical analyses were conducted in the r statistical computing environment (r development core team, 2015). bray-curtis dissimilarities in chcs profiles between staphylinids and their termite host were compared using multivariate analyses with the “vegan” package (oksanen et al., 2015). dissimilarities were calculated for quantitative (proportion of the compound relative to the total amount of compounds in a run) and qualitative (presence/absence of the compound) data. for quantitative data, squared-root transformation and standardization was applied before calculating bray-curtis dissimilarities. compounds whose peaks contributed to less than 1% of the total area of peaks in a run were not used in the quantitative nor in the qualitative analysis. non-metric multidimensional scaling (nmds) was used to plot dissimilarities among samples. permutational multivariate analysis of variance (permanova), performed via “adonis” routine of the “vegan” package based on the bray– curtis dissimilarity and 999 permutations, was conducted to test the existence of significant variations in the chcs profiles between species and among nests, similarly to what was done by proffit et al. (2011) for chc profiles of moths ovipositing in tomato leaves. isotopic diet limits were statistically defined as bayesian standard ellipses plotted around pairs of δ13c and δ15n points representative of termites’ or beetles’ diet space, such ellipses being to bivariate data as standard deviation is to univariate data. because these ellipses define the statistical limits for the dimensions of each diet, non-overlapping ellipses indicate statistically distinct diet spaces. ellipses and associated metrics were calculated using the “siber” routines jackson et al. (2011) from the “siar” parnell et al. (2010) package. results a total of 199 c. melantho beetles (fig 1) were collected from the c. cyphergaster nests. the host colonies contained between three to 36 of these beetles, with an average of 19.9±1.4 (mean±se). besides c. melantho, three other staphylinidae species were also found living in some but not all of these nests: termitocola silvestrii (wasmann 1902) (aleocharinae: termitonannini), spirachtha eurymedusa schiødte, 1853 (aleocharinae: corotocini) and termitoiceus sp. (aleocharinae: corotocini), all of which occurring in lower abundance than c. melantho. chc profiles. in total, we identified 67 gc peaks in the beetles and their termite host species (supplementary materuial table sm01), corresponding to 14 to 20 compounds in c. melantho beetles and 15 to 23 compounds fig 1. morphological congruence between corotoca melantho beetles and their termite host, constrictotermes cyphergaster. (a) dorsal view of termite worker, (b) dorsal view and (c) lateral view of termitophile beetle. scale bar = 0.5 mm. cs rosa et al. – chemical disguise of a rove beetle42 that is, chc profiles of beetles and their termite host species are similar chemical composition (the presence of compounds) and the relative abundance of compounds. beetles presented chc more similar to the chc of their cohabiting termites than to (i) their conspecifics from another nest and (ii) termites from another nest. this pattern was consistent for both quantitative and qualitative chc analyses table 1. permutational analysis of variance confirmed the above patterns (table 2): differences in chc profiles between termitophiles and termites could not be attributed to their specific identity (p=0.124 for compound relative abundance and p=0.141 for compounds presence). rather, these differences were explained only by their nest of origin (p=0.001 for compound relative abundance and p=0.007 for compound presence), with termitophiles differing less from their heterospecific cohabitants than from their con-specifics found in distinct nests. fig 2. typical cuticular hidrocarbon profiles detected for constrictotermes cyphergaster (blattodea: isoptera: termitidae) and their termitophile corotoca melantho (coleoptera: staphylinidae) inhabiting a single nest (n14) in a cerrado environment at the municipality of sete lagoas, mg, south-eastern brazil. fig 3. the chemical congruence between cuticular hidrocarbon profiles from staphylinid beetles, corotoca melantho, and their termite host, constrictotermes cyphergaster, as revealed by non-metric multidimensional scaling (mnds) based on bray-curtis dissimilarities. (a) dissimilarities estimated using the relative abundance of compounds in the chc profiles. (b) dissimilarities estimated using presence/ absence of compounds in the chc profiles. red symbols: staphylinid beetles; black symbols: termite host. similar symbols indicate beetles and termite sampled in the same nest. comparison qualitative quantitative c. melantho × c. cyphergaster same nest 0.40±0.08 0.54±0.08 c. melantho × c. cyphergaster different nest 0.55±0.02 0.72±0.02 c. melantho × c. different nest 0.53±0.02 0.70±0.03 table 1. bray-curtis dissimilarities (mean ± se) between cuticular hidrocarbon (chc) profiles of (i) termitophile staphylinids corotoca melantho and their cohabiting host termites constrictotermes cyphergaster, (ii) these same beetle and termite species inhabiting distinct termitaria, and (iii) con-specific staphylinids inhabiting distinct termitaria. dissimilarities were calculated based on the presence/absence (“qualitative”) or the relative abundance (“quantitative) of compounds in the chc profiles. in their termite hosts. these profiles were rather congruent between guests and hosts (fig 2), such that nmds ordination did not reveal any clear distinction between them, in both quantitative (fig 3a) and qualitative analysis (fig 3b). sociobiology 65(1): 38-47 (march, 2018) special issue 43 isotopic signatures placed cohabitant termitophiles and termite workers apart in diet spaces (fig 4). δ13c ratios indicated that termitophiles and termite workers did not feed nor did they assimilate from the same carbon source. accordingly, δ15n ratios indicated that staphylinids fed either df sums of sqs mean sqs pseudo-f r pr(>f) presence of compounds nest 8 1.75898 0.21987 2.3490 0.65566 0.001 *** species 1 0.17495 0.17495 1.8691 0.06521 0.141 residuals 8 0.74883 0.09360 0.27913 total 17 2.68277 1.00000 relative abundance of compounds nest 8 3.0172 0.37716 2.3243 0.65947 0.001 *** species 1 0.2599 0.25988 1.6015 0.05680 0.124 residuals 8 1.2982 0.16227 0.28373 total 17 4.5753 1.00000 table 2. permutational multivariate analysis of variance using bray-curtis distance matrices under “vegan” package. bray-curties dissimilarities were calculated based on the presence/absence (“qualitative”) or the relative abundance (“quantitative”) of compounds in the chc profiles. on highly decomposed materials or preyed upon a wide variety of prey, as suggested for termitophile and myrmecophile staphylinids by costa-lima (1952, p. 315). either of these diets, however, cannot be said to derive from termites, as δ13c ratios are not coincidental among guests and hosts. discussion adaptations to integrate into colonies of ants and termites abound in staphylinid beetles (kistner, 1982). among these, c. melantho would stand as archetypical termitophiles (seevers, 1957) on top of their striking physogastry (see fig 1 and section “introduction”), these beetles are obligatory cohabitants of c. cyphergaster termites, reproduce by ovoviviparity within these hosts’ termitaria, and present wings reduced to membranous pads hence limiting mobility and favouring a confined lifestyle. our findings extend to chemical disguise the list of c. melantho adaptations to termitophily. previous work has been showed that c. melantho also have morphological disguise (cunha et al, 2015). chemical disguise provided by chc profiles built on similar host-specific compounds have been reported as the primary mechanism of invertebrate integration into nests of ants (akino et al., 1999; elgar & allan, 2006; elmes et al., 1999; guillem et al., 2014; schönrogge et al., 2008), bees (kather et al., 2015), and wasps (van oystaeyen et al., 2015). as for termites, extreme congruence at the species level in guest-host chc profiles has been already detected for limuloid staphylinidae (howard et al., 1980). here, we found chc profiles of c. melantho to bear close resemblance to their hosts’ profiles (figs 2 and 3), but this similarity, rather than broadly defined at the species level, was established at the level of the colony (tables 1 and 2). such a result supports the notion that these termitophiles would acquire chc compounds after invade their host nests (‘acquired chemical mimicry’ sensu von-beeren et al. (2012)). in the ’acquired chemical mimicry’, invaders can (i) biosynthesize some specific recognition cues of their host and, after invasion, acquired other recognition cues after getting in touch with termites or their nests or (ii) acquire disguising recognition cues only after getting in touch with termites or their nest. this consists in a major distinction between limuloid and physogastric termitophilic rove beetles as, in the former, mimetic chc can only be biosynthesized (howard et al., 1980) (i.e., ’innate chemical mimicry’). such a scenario is entirely in accordance with previous records on the chemical invisibility of obligate myrmecophiles (lenoir et al., 2001) and termitophile aleocharinae beetles (sands & lamb, 1975) from the same corotocini tribe as c. melantho here reported. these are known to profit from being “chemically insignificant” (odourless) to enter the host colony, subsequently acquiring the gestalt colony odour by physical contact. the likelihood of such distinct chc origins in limuloid versus physogastric termitophiles is reinforced by their opposing rank at the continuum of degrees of interactions between guests and hosts (lenoir et al., 2001): while limuloids avoid contacts, physogastrics engage constant interactions with their hosts (kistner, 1982). it is plausible to suspect a major role of physogastry in facilitating such acquisition by promoting intimate (tactile, feeding) interactions between guests and hosts, such that chemical and physical traits act synergistically to allow termitophily, as posited by parker (2016). after all, chc compounds can be transferred interspecifically by interindividual physical and feeding interactions (elgar & allan, 2004; liang & silverman, 2000). results from our isotopic signatures analysis (fig 4) revealed that these staphylinids and termites were positioned apart, mainly, in δ15n axes. in six out of seven studied nests, staphylinids where located from two to seven trophic steps above their hosts, indicating highly variable and in many cases highly decomposed diets. cs rosa et al. – chemical disguise of a rove beetle44 in a single case guests were only one trophic step above hosts (fig 4, nest “n14”), indicating direct ingestion of termite via predation. the high variation in δ15n axis between beetles and host is somehow stranger. however, such high variations may have occurred due to the methodological procedures in the isotopic analyses (i.e. analysis with gut contents, which can have plant or fungi material) or by predation of life stages not measure by us (i.e., larve and/or eggs). two other non-dietary and non-exclusive routes seem more likely: (i) tactile interactions (e.g. allogrooming) between guests and hosts or (ii) direct rubbing by the guests on internal nest lining. these routes remain hypothetical, as the evaluation of their relative importance to the acquisition of hosts’ chc by guests is outside the scope of this paper. in the event that interindividual tactile interactions are proven relevant for mimetic chc transfer in this system, then parker’s hypothesis (parker, 2016) on the synergistic action between physical and chemical termitophilic traits gains additional support. future studies must investigate trophallaxis behavior between guest and host, in order to clarify this unrevealed association. in summary, evidence indicates that chemical (present work) and morphological (cunha et al., 2015) disguises in termitophiles may function as a strategy for social integration in c. cyphergaster colonies. acknowledgements we thank e. caron for the identification of staphylinidae species, i.r. silva and laboratory technicians for their help in the stable isotopic analysis, j.m. waquil from embrapa for the logistic support. this work was partially funded by brazilian council for research (cnpq), minas gerais state agency for research support (fapemig) and coordination for the improvement of higher education fig 4. isotopic signatures of termites and staphylinid beetles coexisting in termitaria, delimited by bayesian standard ellipses. each panel represents a single termitarium. axes represent the ratio of stable carbon and nitrogen isotopes in termite bodies. each dot refers to a sample weighing ≥1.5 mg from either a group of termite workers (hollowed dots) or staphylinids (cross-marked dots). termites are constrictotermes cyphergaster and beetles are corotoca melantho. dotted lines parallel to x-axis define 3 intervals thought to correspond to trophically distinct positions. sociobiology 65(1): 38-47 (march, 2018) special issue 45 personnel (capes). ods holds a cnpq fellowship (pq 305736/2013-2), pfc is supported by cnpq/fapitec-se (dcr 302246/2014-2). while deriving from the phd thesis by csr (#150362/2012-9), this work is contribution #71 of the termitology lab at federal university of viçosa, brazil (http://www.isoptera.ufv.br). supplementary material http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1942/0 http://dx.doi.org/10.13102/sociobiology.v65i1.1942.s1936 references akino, t. k., knapp, j. j., thomas, j. a. & elmes, g. w. 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(2016). evidence for social parasitism of early insect societies by cretaceous rove beetles. nature communications, 7: 13658. doi: 10.1038/ ncomms13658 doi: 10.13102/sociobiology.v64i1.1250sociobiology 64(1): 78-84 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 exploitation of floral resources and niche overlap within an oil-collecting bee guild (hymenoptera: apidae) in a neotropical savannah introduction bee populations usually share food items of adults and larvae diets (quiroz-garcia et al., 2001; müller et al., 2006; roubik & villanueva, 2009; vilhena et al., 2012; andena et al., 2012). they may also use the same nest-building materials, as well as the substrates for nesting (frankie et al., 1993; thiele, 2005; aguiar et al., 2005; martins et al., 2012), among other resources. additionally, these populations also overlap each other to some level in the exploitation of these resources throughout the day and/or year (santos et al., 2013). therefore, bee populations face varying levels of overlap in several dimensions of their niches. research on the dynamics of niches overlap is a promising approach for understanding the organization of bees communities, the local patterns of populations abundance, and its temporal fluctuations. however, the effects of trophic niches overlap among bee species have been little discussed (wilms et al., 1996; roubik & villanueva, 2009). abstract oil bees exploit host plants for resources to feed the adults and offspring, as well as for the construction of their nests. the aim of the current study is to investigate how the species in this guild distribute their foraging effort, and the range in their niche overlap levels. our hypothesis is that niche overlap levels are strongly affected by the exploitation of some key plants, those profitable and locally abundant oil-plants. the bees were sampled for six months, during their visits to the flowers in a savannah (cerrado). these oil-bee species explored the floral resources provided by 13 plant species. the trophic niche of the most abundant species, centris aenea lepeletier, was relatively narrow, similarly to those of epicharis species. low overlap of trophic niches (trno≤30%) was most commonly found. the distribution of bee visits to the host plants revealed redundancy in the floral resource exploitation. however, the foraging concentration levels in some key plants were different for distinct oil-bee species, and it contributed to the low overlap of niches between many pairs of species. sociobiology an international journal on social insects cml aguiar¹, j. caramés², f frança, e melo¹ article history edited by kleber del-claro, ufu, brazil received 18 november 2016 initial acceptance 08 march 2017 final acceptance 08 march 2017 publication date 29 may 2017 keywords bee-plant interactions, host plants, diet, oil-bees, cerrado, solitary bees. corresponding author cândida m. l. aguiar departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina, s/n, novo horizonte 44036-900, feira de santana-ba, brazil e-mail: candida.aguiar @gmail.com studies on niche overlap among bee species have mostly addressed the trophic dimension, while few have investigated the overlap in the temporal dimension of the niches (santos et al., 2013; barônio & torezan-silingardi, 2016). these studies have focused on a small portion of bee communities, the eusocial species (e.g. camillo & garófalo, 1989; wilms et al., 1996; wilms & wieschers, 1997). in spite of non-eusocial bee species being the largest portion of the neotropical communities, they are rarely included in niches overlap studies (steffan-dewenter & tscharntke, 2000; aguiar, 2003b; aguiar et al., 2013; andena et al., 2012; carvalho et al., 2013; rabêlo et al. 2014). the guild of oil-collecting bees is composed of species showing specialized morphology and behavior concerning floral oil gathering (neff & simpson, 1981; vogel & machado, 1991; coccuci et al., 2000), which is a fundamental resource for the establishment of these bee populations in the habitat, since the floral oils are essential for nest construction, as well 1 – universidade estadual de feira de santana, bahia, brazil 2 – pós-graduação em zoologia, universidade estadual de feira de santana, bahia, brazil research article bees sociobiology 64(1): 78-84 (march, 2017) 79 as larval food in several species (jesus & garofalo, 2000; vinson et al., 1996; pereira et al., 1999; aguiar & garofalo, 2004). therefore, progeny production by these bees strongly depends on the exploitation of oil-producing plants as well as of pollen-source plants. the oil-bee/oil-plant ecological relationships are generally not species-specific (sigrist & sazima, 2004; rosa & ramalho, 2011; mello et al., 2013). thus, some of the oilproducing plants are visited by most oil-bee species in each habitat (gaglianone, 2003; mello et al., 2013). in the current study we investigated how the species belonging to a guild of oil-collecting bees distribute their foraging efforts in the floral resource sources – including oil-producing and non-oilproducing plants and how the overlap levels of trophic and temporal niches vary. since oil-collecting bees have a strong dependence on floral oils, we hypothesized that this guild has a structure that is driven by the bottom-up principle. our hypothesis is that niche overlap levels are strongly affected by the exploitation of some key plants, those profitable and locally abundant oil-plants. materials and methods study area the field work was carried out in an area located in chapada diamantina region, where a long-term ecological research has been developed. the sampled vegetation is of cerrado type, and it is located in palmeiras municipality (12°25’s /41°29’w), bahia state, brazil. the mean annual temperature is 22o c and the annual rainfall ranges from 600 to 1,000 mm (centro de estatística e informações, 1994). sampling three sampling sites 900-1,500m distant from each other were sampled in this area, and three transects (1,000 m length and 20 m width) were chosen in each site. sampling was monthly carried out for three consecutive days, from october 2013 to march 2014 period that includes the end of the dry season and all the rainy season, which began in november. a transect of each site was randomly selected for sampling every collection day. the daily sampling effort was of 8 hours (from 08:00 a.m. to 04:00 p.m.), thus totaling a sampling effort of 144 hours distributed in 18 sampling days. bees were captured on the flowers using entomological nets, without choosing bee or plant species (adapted from sakagami et al., 1967). fertile botanical material was collected for exsiccates. we collected data on the two locally most abundant oil-plants byrsonima sericea and byrsonima cydoniifolia. we used data from 30 individuals of b. sericea and b. cydoniifolia (10 individual plants of each species from each site) to determine the flower production period and to estimate their flowering intensity. regarding each individual plant, a 25 x 25 cm “square box” (height ranging from 1.40 m to 1.70 m) was placed on the canopy and the inflorescences found in the canopy volume delimited by the “square box” were counted. flower buds and open flowers were counted in at least 10 inflorescences in order to calculate the mean number of flowers per inflorescence. the radius and height of each individual plant were measured to calculate the volume of the flowering canopy. these measurements were used to estimate the inflorescence density of each plant by multiplying the number of inflorescences by the flowering canopy volume/”square box” volume ratio. after estimating the inflorescence density per individual plant and obtaining the mean number of flowers per inflorescence, we estimated the amount of flowers monthly produced by each byrsonima plant. data analysis we used the shannon diversity index (h ‘= σ pk x ln pk) (shannon, 1948) to calculate the niche breadth of bee species, where pk is the proportion of individuals of the bee species collected in the plant species “k” and, ln pk is the neperian logarithm of pk. we used the schoener index (no = 1 ½ σk | pik phk |) (schoener, 1968) to measure the niche overlap between each pair of oil-bee species, where pik and phk are the proportions of individuals of the bee species “i” and “h”collected in each species of plant (“ k “) (to calculate the trophic niche overlap) or in each month (“k”) (to calculate the temporal niche overlap). this overlap index varies from 0 to 1. bee species represented by five or more individuals were selected to analyze the width and the overlap of trophic and temporal niches. this abundance threshold is lower than that used in previous studies (e.g. aguiar, 2003b; aguiar et al., 2013; santos et al., 2013). however, it has already been adopted in other niche overlap analyses (e.g. andena et al., 2012; carvalho et al., 2013). the main reason for using this lower threshold lies on the fact that most oil-collecting bee species occur on flowers in low frequency in natural habitats (aguiar 2003a; 2003b; aguiar & zanella, 2005; andena et al., 2005; 2012; santos et al., 2013; silva et al., 2015). consequently, oil-collecting bees are rarely included in niche analyses. abundance influences the species ability to occupy the niches, and is influenced by the multiple dimensions of the niche, which regulate the size of the population (begon et al., 2006). thus, it is worth analyzing the niche of infrequent species to look for evidences of how the interactions among them and other species in the guild during resource exploitation may affect their local abundance. results twenty species were recorded in this guild of oilcollecting bees (table 1). these bee species explored floral resources provided by 13 plant species. among them, only three species were oil-producing plants (b. sericea, b. cydoniifolia and stigmaphyllon paralias, malpighiaceae). b. sericea and pityrocarpa moniliformis (fabaceae) were the most important food resources to these bees (table 1). during the late dry cml aguiar, j. caramés, f frança, e melo – oil-bee trophic niche80 season and the early rainy season (oct/ nov), these bees explored food resources on few plant species (1 and 4) (fig. 1). in this period, bee frequencies on flowers were lower than in the rainiest months (table 1). two byrsonima species produced flowers during the rainy season, from december on (fig. 2a, b), resulting in increased floral oil and pollen supply in the habitat, since these plants show high local abundance. a bee abundance peak was recorded in february, and it was strongly influenced by the high abundance of centris aenea lepeletier (table 1). these bees explored floral resources from 1 to 7 plant species. the two most abundant species of centris (c. aenea and c. fuscata) differed sharply in their trophic niche width. centris fuscata lepeletier showed the widest trophic niche (h’1= 1.58). in contrast, c. aenea, the most abundant species, had a trophic niche (h’1 = 0.60) narrower than c. fuscata and other species of centris (table 1). although c. aenea explored more plant species than the other oil-collecting bees, its preferential foraging on b. sericea strongly influenced the trophic niche width decrease. this trend to a narrow trophic niche became even more pronounced when this species was abundant on flowers (h’1february = 0.13 in; h’1march = 0.32). the very different levels of foraging on b. sericea flowers by the two most abundant epicharis species also resulted in a strong difference in the width of their trophic niches, with moderate overlap (table 1). c. aenea, epicharis analis lepeletier and c. fuscata showed the longest activity period (table 1). among the most abundant species, c. fuscata and e. analis showed the highest temporal niches width (h’2 = 1.27; h’2 = 1.07, respectively) (table 1). although c. aenea showed the longest activity period among the bees in this guild, its temporal niche was not so wide (h’2=0.51), because the h’ index suffered strong influence from a peak of abundance concentrated in february (table 1). bee species n h’1 plants visited h’2 oct nov dec jan feb mar centridini centris aenea lepeletier 94 0.60 bv (1)/pe (1)/bc (1)/bs (80)/ ep(1)/ pi (9)/ sa (1) 0.51 1 1 1 81 10 centris moerens perty 3 1.09 bs (1)/pe (1)/sc (1) 0.63 1 2 centris sponsa smith 5 1.33 pe (2)/bs (1)/ pi (1)/sa (1) 0.10 1 3 1 centris caxiensis ducke 6 1.32 bc (2)/ bs (1)/ my (1)/sp (2) 1.01 2 3 2 centris leprieuri spinola 2 0.69 pe (1)/bs (1) * 2 centris nitens lepeletier 1 * bs (1) * 1 centris fuscata lepeletier 9 1.58 bv (2)/pe (1)/sf (2)/cc (2)/ pi (2) 1.27 4 2 1 2 centris sp.1 4 0.56 bc (1)/bs (3) 0.56 1 3 centris sp.3 1 * bs (1) * 1 centris sp. 6 1 * pi (1) * 1 epicharis analis lepeletier 24 0.64 bs (9)/pi (15) 1.07 1 4 5 14 epicharis bicolor smith 33 0.36 bs (30)/pi (1)/ sp (2) 0.80 18 14 1 epicharis cockerelli friese 4 * bs (4) 1.03 1 1 2 tapinotaspidini lophopedia nigrispinis vachal 2 0.69 sf (1)/ pi (1) 0.69 1 1 monoeca affs. mourei aguiar 1 * sp (1) * 1 tropidopedia nigrocarinata aguiar & melo 9 1.14 sf (5)/ bs (2)/ pm (1)/ sp (1) 0.32 8 1 xanthopedia sp.1 5 0.50 sf (1)/ bs (4) 0.50 1 4 urbanapsis diamantina aguiar & melo 7 0.79 bs (4)/ sf (1)/ sp(2) 0.79 1 5 1 tetrapediini tetrapedia amplitarsis friese 3 * bs (3) * 3 tetrapedia diversipes klug 3 0.63 pm (1)/ sf (2) * 3 total 217 9 4 19 36 119 31 table 1. abundance of oil-collecting bee species (n), trophic niche width (h’1) (shannon index), temporal niche width (h’2), and plant species visited for floral resources in a savannah area in the chapada diamantina, brazil. number of flower-visiting bees captured in each plant species in brackets. * bee species collected in a single plant or month. bv: bowdichia virgilioides, cc: centrosema coriaceum, pm: periandra mediterranea, pi: pityrocarpa moniliformis (fabaceae)/ bc: byrsonima cydoniifolia, bs: byrsonima sericea, sp: stigmaphyllon paralias (malpighiaceae)/ ep: eugenia cf. punicifolia (myrtaceae), my: myrtaceae sp.1/ pe: passiflora edulis (passifloraceae)/ sf: serjania faveolata (sapindaceae)/ sa: simarouba amara (simaroubaceae)/ sc: stachytarpheta crassifolia (verbenaceae). sociobiology 64(1): 78-84 (march, 2017) 81 fig 1. plant and bee species richness in a savannah area in the chapada diamantina, bahia, brazil, from oct, 2013 to mar, 2014. bee species cc cf cs ea eb tn xa ud centris aenea (ca) 0.17 0.11 0.31 0.47 0.88 0.22 0.60 0.71 centris caxiensis (cc) 0.12 0.16 0.16 0.22 0.27 0.36 0.30 centris fuscata (cf) 0.00 0.25 0.03 0.12 0.12 0.12 centris sponsa (cs) 0.40 0.43 0.20 0.20 0.20 epicharis analis (ea) 0.40 0.22 0.37 0.37 epicharis bicolor (eb) 0.28 0.66 0.77 tropidopedia nigrocarinata (tn) 0.53 0.47 xanthopedia sp.1 (xa) 0.88 urbanapsis diamantina (ud) table 2. trophic niche overlap (trno) (schoener index) between oil-collecting bees in a savannah area in the chapada diamantina, bahia, brazil. bee species cc cf cs ea eb tn xa ud centris aenea (ca) 0.18 0.23 0.71 0.32 0.45 0.01 0.81 0.12 centris caxiensis (cc) 0.33 0.17 0.37 0.66 0.44 0.36 0.64 centris fuscata (cf) 0.31 0.37 0.14 0.22 0.31 0.28 centris sponsa (cs) 0.40 0.45 0.00 0.60 0.14 epicharis analis (ea) 0.40 0.15 0.25 0.35 epicharis bicolor (eb) 0.11 0.42 0.58 tropidopedia nigrocarinata (tn) 0.20 0.25 xanthopedia sp.1 (xa) 0.14 urbanapsis diamantina (ud) fig 2. (a) estimated number of flowers produced by two species of byrsonima (malpighiaceae) in a savannah area in the chapada diamantina, bahia, brazil. (b) monthly rainfall in the municipality of lençóis, bahia, brazil, from oct, 2013 to mar, 2014. (instituto nacional de meteorologia, www.inmet.gov.br) table 3. temporal niche overlap (teno) (schoener index) between oil-collecting bees in a savannah area in the chapada diamantina, bahia, brazil. a b the overlap of trophic niches (trno) between each pair of oil-collecting bee species ranged from very low (less than 10%) to very high (close to 90%) (table 2). c. aenea and epicharis bicolor smith showed the highest overlap (trno =0.88) (table 2), which resulted from their preferential foraging in b. sericea (table 1). the trophic niche of c. aenea showed higher similarity to epicharis species niches, and to the small bees tapinotaspidini, than to other centris. e. bicolor also showed high overlap with tapinotaspidini species (table 2). on the other hand, the niche overlap between the two most abundant epicharis species was moderate, and it resulted from the differential distribution of individuals on the most visited plants. low overlap of trophic niches (trno≤30%) was the most common situation found in this guild of oil-collecting bees, and it was recorded in 20 among the 36 pairs of species analyzed. moderate overlap (30%<trno≤70%) was found in 12 pairs. only four pairs of species showed high trno (> 70%). three of these pairs involved urbanapsis iamantine aguiar & melo, whose sample was small. the temporal niche overlap (teno) between the pairs of bee species ranged between 0 and 0.81 (table 3). fortyfour percent of the pairs showed low temporal niche overlap (teno < 30%) and only two pairs showed high temporal niche overlap (teno >70%). discussion our hypothesis was confirmed, since the variations in the levels of overlap between species in the oil-collecting guild cml aguiar, j. caramés, f frança, e melo – oil-bee trophic niche82 were affected mainly by the intensity of exploitation of b. sericea. this oil-plant is locally abundant and produces a high amount of floral oil (aguiar et al, accepted for publication on sociobiology). additionally, it also supplies many bee species with pollen (teixeira & machado, 2000). thus, b. sericea is a key resource to the maintenance of oil bee populations in this savannah. several oil-collecting bee showed relatively narrow trophic niches (h’1<0.8), compared to other bee species in other phytophysiognomies (aguiar, 2003b; aguiar et al., 2013). similarly, narrow trophic niches were recorded for centris and epicharis species (h’<0.9) in another brazilian cerrado site, where these species also used a small set of host plants, and mainly explored byrsonima intermedia, another malpighiaceae oil-plant (andena et al., 2012). on the other hand, it is known that some centris and epicharis species use many plant species in their diet (gaglianone, 2003; aguiar et al., 2003; vilhena et al., 2012; santos et al., 2013). it means that some of them can replace the sources of floral resources in different habitats. the number of plant species exploited by c. fuscata and c. aenea suggested that these bee species probably exploit a diverse set of food sources. this finding agrees with previous records of the c. fuscata and c. aenea host plants (> 30 species), which were compiled in a number of areas covered by xerophilous vegetation (caatinga) (aguiar et al., 2003). in the studied savannah, c. aenea showed a trophic niche narrower than that recorded in the xerophilous vegetation (caatinga), where its niche width was approximately two times wider (h’=1.59) (aguiar & santos, 2007). previous studies have indicated that this species exploits many host plants (aguiar et al., 2003; aguiar & gaglianone, 2003), but we found that females can show floral fidelity to abundant resources, such as byrsonima. this c. aenea population seems to be particularly favored in periods of high availability of the floral resources provided by b. sericea and it shows high dominance in the guild of oil-collecting bees. this bee species may form large nesting aggregations in places showing high density of byrsonima trees (aguiar & gaglianone, 2003), and this strategy leads to reduction in the costs and time required to collect resources for brood cell building and provisioning. c. aenea and c. fuscata showed very low trophic niche overlap, mainly due to the non-use of the host plant b. sericea by c. fuscata. the overlap was also low in the time axis, because the highest abundance of each occurred in different months. thus, we found a niche-separation trend in both analyzed axes (diet and time). large differences in these two populations size may influence the distribution of individuals in the host plants. consequently, these differences may affect the observed overlap, since c. aenea was approximately 10 times more abundant than c. fuscata. apparently, neither the differences between body size (robust vs. small bees) nor differences in abundance explain the overlap levels in the trophic niches. these findings suggest that, in the studied community, the foraging effort allocation of each oil-bee species to explore b. sericea resources is a more important factor to determine the niche overlap levels than the phylogenetic proximity among the species. similarly, andena et al. (2012) found that bee species of the same genus, including centris, use different food resources, thus they show trophic niches more similar to those of species from other genera than to those of congeneric species. although bee visits to the host plants revealed redundancy in the exploitation of floral resources, the foraging concentration on some key plants were different among oil-bee species, that contributed to the low niche overlap in many of the comparisons made between each set of two species (each pair of species). in addition, interspecific differences in the length of nesting periods and/or in the number of generations per breeding season may have some effect on the lowering of both temporal and trophic niche overlap, since individuals of different generations can forage on different host plants blooming at different periods throughout the nesting season of each bee species. acknowledgments the authors are grateful dr. antonio aguiar and dr. cristina gaglianone for the bee species taxonomic identification, to dr. gilberto m. m. santos (uefs) and maise silva (ftc) for suggestions, and to the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for funding this study (processes 474065/2013 and 403774/20128, peld). c.m.l. aguiar received a research fellowship from cnpq (process 306403/2012-9), and j. c. duarte received a grant from coordenadoria de aperfeiçoamento de pessoal de nível superior (capes). references aguiar, c.m.l. 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(1997). floral resource partitioning between native melipona bees and the introduced africanized honey bee in the brazilian atlantic rain forest. apidologie, 28: 339-355. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 114-122 (2013) division of labor in stable social hierarchy of the independent-founding wasp mischocyttarus (monocyttarus) cassununga, von ihering (hymenoptera, vespidae) asn murakami, ic desuó, sn shima introduction division of labor is important to the colonial organization of social insects and the resulting castes in the colonies are basically the reproductive and the worker castes (wilson, 1971; naug & gadagkar, 1998). most studies regarding labor distribution in these insects have focused on polyethism, i.e. the rules regulating how each individual worker is assigned to specific tasks from the whole repertoire of activities of the colony (naug, 2001). according to jeanne (1991), this behavioral specialization of the colony members is one of the most striking features of eusocial insects. in independent-founding wasps there is no morphological differentiation between different castes, thus queens abstract in colonies of independent-founding wasps, social organization is characterized by a division of labor wherein the most dominant female (the queen) spends more time on the nest comb during its development, while the subordinates (workers) perform most of the foraging. the present study aimed at describing how tasks are assigned among the members of colonies of mischocyttarus cassununga (von ihering) during the postemergence phase. for this, we daily tape recorded the behavioral repertoire of thirteen colonies in the field, later analyzing the results of each colonial subphase with statistic analysis (principal component analysis, clustering analysis and anova tukey-kramer t-test p<0.05). our results showed that the assignment of tasks in m. cassununga generally reflects the dominance hierarchy. despite of the behavioral flexibility of basal eusocial wasps (or absence of a well-defined temporal polyethism), it was possible to identify different groups of subordinate females (workers) in the colony. the division of labor during the pre-male subphase was delineated with four defined groups, whereas the post-male subphase with three groups. pre-male: g1 a dominant group of reproductive females, g2 a group of higher hierarchically subordinate females (these can sometimes perform tasks very similar to the queens), g3 a subordinate group of forager females, and g4 a subordinate group of inactive and principally young wasps. post-male: g1, g2 and g3 (similar to groups during pre-male subphase), but without g4. we think these analyses suggest a novel view of the importance of the behavioral repertoire of higher hierarchically subordinate females in wasp nests, at least in the genus mischocyttarus. sociobiology an international journal on social insects universidade estadual paulista "júlio de mesquista filho", unesp, rio claro, sp, brazil. research article wasps article history edited by: marcel hermes, ufpr brazil received 21 january 2013 initial acceptance 19 february 2013 final acceptance 22 february 2013 keywords social biology; dominance hierarchy; behavioral repertoire; polistinae; mischocyttarini. corresponding author andré sunao nishiuchi murakami departamento de zoologia instituto de biociências, unesp 24-a avenue 1515, rio claro, sp, brazil, 13506-900. e-mail: sunamigobio@yahoo.com.br and workers have to be discerned by behavioral characteristics (jeanne, 1986; giannotti, 1992). the reproductive division of labor derives from the polygynic association among foundresses, as well as between these females and the new workers in a same colony (haplometrosis) (jeanne, 1991). as a rule, the dominant female (queen) remains most of the time performing intranidal tasks, whereas the subordinates are mainly responsible for the foraging activities (westeberhard, 1969; richards, 1971; gobbi, 1977; torres et al., 2009, 2012). therefore, dominance interactions take place among all members of the colony, and affect how tasks are assigned within the colony (pardi, 1948; jeanne, 1972; chandrashekara & gadagkar, 1991; o’donnell, 1998a, b). moreover, in this basal group, the hierarchical position of a wasp is directly correlated with its reproductive success sociobiology 60(1): 114-122 (2013) 115 (litte, 1977, 1981; pratte, 1989; chandrashekara & gadagkar, 1991; tindo & dejean, 1998). in the present study we aimed at evaluating polyethism in the social organization of developing colonies of the independent-founding wasp mischocyttarus cassununga (von ihering) in the field, focusing on the pre-male and postmale substages of the post-emergence phase of the colony cycle. materials and methods twelve colonies were chosen for observation on the field in order to assess the dynamics of labor division in m. cassununga, consequently also evaluating social regulation and organization during the ontogeny of the colonies: eight colonies undergoing pre-male subphase of post-emergence (a1, a2, a3, a4, a6, a7, a8 and a9), and four in the postmale subphase of post-emergence (b1, b2, b3 and b5). all of these colonies were located inside the university campus of unesp, rio claro, sp, brazil (22o24’26”s, 47o33’36”w). the colonial cycle phases were determined based on the immature forms found, and by counting the number of adults in the nests. every adult wasp was individually marked with a combination of colors (airplane modelling acrylic paint) applied over their mesoscutum, and was observed from the day of its emergence until the end of the experiment, or until it eventually disappeared from the colony. based on this, we were able to determine the relative age of some wasps, except for those which already were in the nests when our observations began. all behaviors in the colonies were tape-recorded with a sony-dcr-trv17 video camera during one hour each day, resulting in approximately 10 hours of observation per colony. the age of each female was determined from the day when the young individual emerged from the colony and in case of the females that were present at beginning of field study, the age was estimated based on the total time of observation (pre-male subphase: ± 100 days and post-male subphase: ± 50 days). the hierarchical position of the females in each colony was determined by the frequency of the aggressive behaviors (d = dominance and s = subordination) and the method of linearization (id = dominance index) of premnath et al. (1990). once a social hierarchy was determined in the colonies, we started to record the following behaviors (the frequency each individual wasp performed every behavior): cell inspection (ci): when a wasp surveys the cells for eggs, larvae, or pupae with its antennae; foraging for food (f): when a wasp returns with food (either nectar or a macerated prey) to the nest between its mandibles and shares it with other females (frequently dominants); ingesting food (if): a part of the regurgitated material (prey) to a larva is rapidly incorporated from the crop to nourish other body regions of the adult wasps (hunt, 1984). this happens during the provisioning behavior, when a wasp malaxates food given by another wasp, before feeding larvae; feeding larvae (fl): when the wasp inserts the head into a cell to give the malaxated prey to a larva. in our previous studies, the behaviors that were selected had a high importance for discrimination between dominant and subordinate wasps in the colony. all behavior variables (d, s, ci, f, if and fl) were standardized for a better analysis, and then, these six components were submitted to a principal components analysis (biplot analysis) and clustering analysis (zar, 1999). the first analysis helped us to obtain the visualization and the distribution pattern (biplot analysis) of our data, while table 1. values of id = dominance index during the establishment of social hierarchy in m. cassununga. sb col rank in the dominance hierarchy 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th a 1 1 0.46 0.41 0.35 0.14 0.04 0.03 0.01 0 0 0 0 2 1 0.42 0.18 0.08 0 3 1.05 0.38 0.18 0.11 0.07 0.05 0.02 4 1 0.22 0.14 0 0 0 6 1.04 0.42 0.24 0.22 7 1.02 0.74 0.37 8 1.07 0.12 0.50 0.33 9 1 0.50 0.38 0.22 b 1 1.09 0.98 0.88 0.58 0.51 2 1 0.30 0.21 0 3 1.02 0.71 0.55 0.33 0.18 0.13 0.04 5 1 0.60 0.56 0.41 0.38 0.12 0.09 0.07 sb = subphase; a = pre-male subphase; b = post-male subphase; col = colony. x = hierarchical position not occupied. asn murakami, ic desuó, sn shima division of labour in m. cassununga116 the second allowed us to identify groups of individuals that behave similarly or showed similar characteristics (clustering analysis). finally, we used tukey-kramer method with anova to test the significance among all groups of individuals by comparison. the data were analysed using statistica 8.0, excel 14.0 and sas softwares, according to the colony cycle (preor postmale subphases). results dominance index and the social hierarchy in the colonies the study of dominance-subordinate relationship among all members from each colony resulted in a defined linear hierarchy according to dominance index (id) (table 1). it also shows that the queen is clearly the most aggressive individual in the colony of m. cassununga, that is, there is a direct relation between aggressiveness and position of social ranking. considering the pca (figure 1) and the clustering analysis (figure 2), it is clearly noted that, in these colonies, the dominant females (11, 21, 31, 41, 91, 61, 71 and 81) were grouped (g1) in a position distinct from all subordinates (on the right bottom side of the graph), because of the dominant characters (figure 1): dominance (d) (=aggressiveness) and especially cell inspection (ci). these females were also the oldest individuals in the colonies, as they were present in the nests at the beginning of the observation (age: more than 100 days). in addition, it is possible to differentiate the subordinates in three basic groups (g2, g3 and g4). the subordinate females of g2 (12, 13, 14, 15, 16, 24, 32, 33, 37, 43, 44, 52, 62, 72 and 82) are generally relatively old females (age: 72 ± 52.21 days) of higher hierarchical position that perform mainly on-nest tasks together with the dominant female (figure 1: if and fl). however, they are constantly dominated by the dominant at high rate (as demonstrated in table 1). in turn, the wasps of g3 (18, 19, 25, 34 and 45; age: 56.66 ± 32.93 days) were characterized by a more intense foraging activity in comparison to the other groups, whereas the individuals of g4 (the rest of individuals) were the youngest wasps in the nest (age: 6.75 ± 2.22 days) and relatively the most inactive individuals (figures 1 and 2). in the pre-male subphase, the results of anova significance (tukey-kramer t-test p<0.05) showed that, through the analysed behaviors, it is possible to differentiate and group individuals into four categories in the division of labor of the colony (g1, g2, g3 and g4) (table 3). table 3 demonstrates the relative percentage of behaviors among defined groups (g1: reproductively dominant females; g2: other females of higher hierarchically position; g3: foragers; and g4: more inactive females). statistical component analysis division of labor during the post-male subphase of the post-emergence phase during the post-male subphase in the post-emergence phase, the principal component analysis of colonies yielded table 2. eigenvector coefficients from principal components analysis of colonies a1, a2, a3, a4, a6, a7, a8 and a9 in the pre-male subphase (post-emergence phase) of m. cassununga. hierarchical position/ behaviours vector 1 vector 2 vector 3 d dominance 0.564 -0.307 0.180 s subordination 0.067 0.687 -0.362 if ingesting food 0.477 0.130 -0.170 fl feeding larvae 0.352 0.448 -0.109 f foraging for food 0.041 0.422 0.892 ci cell inspection 0.570 -0.193 0.011 eigenvalues 2.823 1.501 0.937 cumulative % 47.100 72.100 87.700 figure 1. principal component analysis of 6 components and the distribution of individuals from colonies a1, a2, a3, a4, a6, a7, a8 and a9 in the pre-male subphase (post-emergence phase) of m. cassununga in relation to vectors 1 and 2 (1 = cell inspection and 2 = subordination). statistical component analysis division of labor during the pre-male subphase of the post-emergence phase the principal components analysis applied during this subphase yielded three vectors that explained 87.7% of variance in the behavioral data (table 2). thus, the first principal component (pc) was most heavily weighted by individuals that inspected the content of cells and that showed high frequency of dominance, whereas the second pc was most heavily weighted by individuals that showed high frequency of subordination and that fed the larvae, and the third pc was most heavily weighted by individuals that foraged during most of the observation time (table 2). sociobiology 60(1): 114-122 (2013) 117 three vectors that together explained 86.5% of the overall variance of behaviors (table 4). similarly, the first principal component (pc) was most heavily weighted by individuals that inspected the content of cells and showed high frequency of dominance, whereas the second pc was most heavily weighted by individuals that showed high frequency of subordination and fed the larvae (table 4). finally, the third pc was most heavily weighted by individuals that were responsible for the foraging activity on the colonies. according to the pca (figure 3) and clustering analysis (figure 4), individuals of colonies during the postmale subphase can be distributed into three groups (g1, g2 and g3) (table 5). the first group (g1) is constituted by the most dominant and the oldest females (age: more than 50 days), whereas the second group (g2) was formed by figure 2. hierarchical clustering analysis. cluster of individuals from colonies in the pre-male subphase (post-emergence phase) of m. cassununga. data from fig.1 were used. *others individuals of undetermined age: presence at the beginning of the observation. individuals of higher hierarchical position (age: 30.5 ± 4.95 days) that remain most of the time on the nest, performing tasks together with the dominant female. the third group (g3) was constituted by young subordinate females (age: 23.75 ± 10.21 days) that mainly perform the foraging activity (figures 4 and 5). a fourth group was not identified in this subphase. in turn, in this subphase, the results (tukey-kramer t-test p<0.05) showed that, through observing some behaviors, it is possible to differentiate and group individuals into three categories in the division of labor of the colony (g1, g2 and g3) (table 5). according to the relative percentage of behaviors in table 5, the defined groups are represented by g1: reproductively dominant females, g2: other females of higher hierarchically position and g3: foragers. semi – parcial r-squared asn murakami, ic desuó, sn shima division of labour in m. cassununga118 table 3. comparison among four groups derived from clustering analysis according to all behavioral categories (means±sd) in the pre-male subphase (post-emergence phase) of m. cassununga. group of females means±sd (frequency of behavior) d s if fl f ci g1 (n=08) 6.94±5.16 0.03±0.09 0.81±0.43 2.36±2.26 0.41±0.51 4.47±5.04 g2 (n=13) 2.17±2.34 4.30±2.84 0.68±0.50 2.30±3.25 0.37±0.40 2.48±2.49 g3 (n=05) 0.10±0.22 0.72±0.61 0.03±0.06 1.00±0.61 0.90±0.32 1.33±0.18 g4 (n=18) 0.52±0.82 1.15±1.23 0.21±0.24 0.93±1.19 0.17±0.18 0.35±0.63 post-hoc comparisons g1 vs g2 * * ns ns ns ns g1 vs g3 * * * ns * * g1 vs g4 * * * * ns * g2 vs g3 * * * ns * * g2 vs g4 * * * ns ns * g3 vs g4 ns ns ns ns * ns d dominance, s – subordination, if ingesting food, fl feeding larvae, f foraging for food, ci cell inspection. * = anova significance (tukey-kramer t-test p<0.05), ns – no statistical significance. table 4. eigenvector coefficients from principal components analysis of colonies b1, b2, b3 and b5 in the pre-male subphase (post-emergence phase) of m. cassununga. hierarchical position/ behaviors vector 1 vector 2 vector 3 d dominance 0.551 -0.351 -0.019 s subordination -0.104 0.575 -0.433 if ingesting food 0.457 0.370 0.239 fl feeding larvae 0.173 0.542 0.596 f foraging for food -0.351 -0.282 0.630 ci cell inspection 0.569 -0.191 -0.046 eigenvalues 3.026 1.245 0.918 cumulative % 50.400 71.200 86.500 table 5. comparison among three groups derived from clustering analysis according to all behavioral categories (means±sd) in the post-male subphase (post-emergence phase) of m. cassununga. group of females means±sd (frequency of behaviors) d s if fl f ci g1 (n=08) 5.73±1.78 0.29±0.58 1.30±1.02 1.53±2.10 0.03±0.07 4.45±5.24 g2 (n=13) 1.83±1.27 2.82±1.70 1.05±0.50 2.13±1.45 0.13±0.14 2.00±2.50 g3 (n=05) 0.51±0.90 1.85±1.56 0.22±0.27 1.45±1.50 0.78±0.80 0.71±0.96 post-hoc comparisons g1 vs g2 * * ns ns ns ns g1 vs g3 * * * ns * * g2 vs g3 * ns * ns * ns d dominance, s – subordination, if ingesting food, fl feeding larvae, f foraging for food, ci cell inspection. * = anova significance (tukey-kramer t-test p<0.05), ns – no statistical significance. sociobiology 60(1): 114-122 (2013) 119 discussion our results illustrate that dominant females (g1) in m. cassununga tend to be distinguished from the subordinates (g2, g3 and g4 in the pre-male subphase and g2 and g3 in the post-male subphase) mainly by aggressiveness (d) and, to a less extent, by inspecting cells (ci). these behaviors are characteristic of the reproductive dominant wasp and contribute to the social status. according to the studies of pardi (1948) with polistes dominulus (=gallicus) (christ), of jeanne (1972) with mischocyttarus drewseni (saussure), and of o’donnell (1998b) and markiewicz and o’donnell (2001) with mischocyttarus mastigophorus (richards), the dominant wasp in the nests tends to avoid energy-expensive tasks (like foraging), as to invest more energy in social control and reproductive performance. the only difference between g1 and g2 is in the dominance characteristic for both subphases (tables 3 and 5). in the genus polistes, apart from behaviors that are typical of dominant wasps (initiating cells and oviposition), other frequent behaviours like dominance, inspecting cells and oophagy were also performed by the dominant females of the nests (pardi, 1948; west-eberhard, 1969; richards, 1971; gobbi, 1977; torres et al., 2009). a new interesting finding is that some females of higher hierarchical positions (g2) engaged in more on-nest activities, meaning that these females of the g2 group remain on the nest, taking care of the brood together with the dominant wasp. the results show that these females also feed the larvae (fl), ingest food (if) and inspect the cells (ci) in higher frequencies (tables 3 and 5). according to reeve and gamboa (1983), in nests with more females, the queens of polistes fuscatus (fabricius) intensify their activities and focus their attention onto some females for maintaining the reproductive monopolisation. these individuals that receive more attention by the queens occupy high hierarchical positions, thus regulating activities of other groups and having a chance to have a share in the reproductive yield of the colony. moreover, the g2 individuals are able to lay 2n eggs (ep), because they are inseminated, as demonstrated by murakami and shima (2009) for the same species of this study. hence, we can suppose that the main reason for the wasps to spend much time in the nest would be different for these two hierarchical classes (reproductively dominant females = “queens” and females of hierarchically higher position): the queen can directly manipulate the reproductive dynamics during its stay in the nest, while the females of higher hierarchical positions can increment their nutritional reserves and, increase their reproductive potential. according to murakami and shima (2006), the existence of a hierarchy related to the food quantity consumed by the adult individuals of m. cassununga was demonstrated. furthermore, the experimental manipulation in this study showed that adult females of hierarchically higher positions can replace the dominant wasp after her retreat. in mischocyttarus cerberus styx (richards), costa-filho et al. (2011) observed that these females in higher positions may be saving energy, which would otherwise be primarily spent on foraging trips. thus, these individuals would have a much higher chance of assuming the position of an egg layer than a regular forager. additionally, considering the age of these females (the second oldest individuals in the nest), our results indicate that the age is an important point during the reproductive competition inside the colony. it is more advantageous for an old female to stay on the nest and have the opportunity for reproduction or substitution of the dominant female, instead of abandoning the nest to start a new cycle of a colony. on the other hand, the females of the g3 group are responsible for foraging for food (ff), and consequently, also for feeding larvae after partially distribute the food for other wasps (especially for dominant wasps) on the nest (tables 3 and 5). thus, we considered in our study, that these females are typical foragers, and remain on the nest for less time. regarding the behavioral repertoire of independentfounding wasps, the foraging activity is more significant for the subordinate females (pardi, 1948; jeanne, 1972; giannotti, 1999; torres et al., 2009). differently from the post-male subphase, a fourth group (g4) can be identified during the pre-male subphase (table 3 and 5). this group is constituted by relatively more inactive and principally younger wasps which remain more time on the back of the nest. it may be noted that some individuals of hierarchically higher position or foragers (a2: 22 and 23; a4: 42; a9: 92 and 93) are grouped together here, and this can be explained by differences in data collection of the total frequency of behaviors observed in the same period of time (total of 10 hours). once they perform their characteristic activities in their respective colonies, this may influence the analysis of the total frequency of each behavior for this group and lead to a misinterpretation. in the post-male figure 3. principal component analysis of 6 components and the distribution of individuals from colonies a1, a2, a3, a4, a6, a7, a8 and a9 in the post-male subphase (post-emergence phase) of m. cassununga in relation to vectors 1 and 2 (1 = cell inspection and 2 = subordination). asn murakami, ic desuó, sn shima division of labour in m. cassununga120 subphase this group was not identified, probably by the reducing number of individuals, which is characteristic of the final period of this subphase. despite the flexibility of each independent wasp, some studies demonstrated that it is possible to separate the individuals of the colony in task groups. through the pca analysis in m. drewseni, araújo (1980) obtained a social division in queens, intermediates, and workers that constructed new cells and foraged for food. studying m. cerberus styx, noda et al. (2001) demonstrated the existence of three well defined hierarchical groups in post-emergence: a dominant group composed of the three wasps of higher hierarchical positions, a neutral group including the young and the new reproductive individuals who defend the colony, and a subordinate group of lower hierarchical posts, probably all strict foragers. a similar organization was noted by pietrobon (2005) in polistes versicolor (olivier) through principal components analysis, but in addition to these three groups (dominant, neutral and subordinate groups), there was another group of subordinates actively performing nearly all of the internal maintenance tasks, and responsible for expanding the colony. based on statistical analysis (pca, clustering and anova with tukey-kramer t-test), our results indicate that during the post-emergence phase, the division of labor in m. cassununga is structured, being basically defined in the groups: (g1) a dominant group of reproductive individuals; (g2) an intermediate group of females in higher hierarchical positions, which are, together with the dominant female, responsible for most internal nest tasks, and are potential substitute of the dominant wasp; (g3) a primary subordinate group constituted by typical foragers, and (g4) a secondary subordinate group composed of basically inactive and predominantly young individuals. concluding, the present study shows that the distribution of tasks in colonies of m. cassununga reflects the dominance hierarchy. moreover, in spite of the presence of a behavioral flexibility (or absence of a well-defined temporal polyethism) in this basal eusocial wasp, it is possible to identify groups of subordinate females engaged in different functions (work) in the colony. internal tasks performed by the dominant female (the oldest female in the colony) of m. cassununga are of high adaptive value, since the dominant is frequently capable of maintaining the social control during the development of the colony. staying in the nest, laying eggs and feeding the brood with the help of females of higher hierarchical positions are bound to secure high inclusive fitness. this way, we think our analyses suggest a novel view about the importance of the behavioral repertoire of females in wasp nests, at least in the genus mischocyttarus, which is endemic to the americas. we think that this new scenario might explain why m. cassununga is characterized by a stable linear social hierarchy (murakami & shima, 2010) and why dominants of this species eat more protein than the subordinates of the nest (murakami & shima, 2006). acknowledgements we thank dr. sidnei mateus (university of são paulo-usp) and dr. nivar gobbi (são paulo state universityunesp) for their suggestions to this study, and msc. eduardo fox for english language review on the manuscript. this work was supported by cnpq (national counsel of technological and scientific development). figure 4. hierarchical clustering analysis. cluster of individuals from colonies in the post-male subphase (post-emergence phase) of m. cassununga. data from fig.2 were used. *others individuals of undetermined age: presence at the beginning of the observation. sociobiology 60(1): 114-122 (2013) 121 references araújo, c.z.d. 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(1999). biostatistical analysis, 4th ed. upper saddle river, prentice-hall international, new jersey. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1270sociobiology 64(3): 301-309 (september, 2017) the influence of fire and deforestation on the floral symmetry and fitness of adenocalymma nodosun (bignoniaceae) introduction burning and deforestation are disturbances that affect the most diverse ecological processes by dramatically influencing the structure, composition and nutrient cycling of terrestrial ecosystems (coutinho, 1990; smithwick et al., 2005). these disturbances are common in neotropical savanna areas, and brazilian savanna or cerrado is the most diverse of them (gottsberger & silberbauer-gottsberger, 2008). the abundance of dead dry biomass, mainly from grass, results in bushfires (bond & keely, 2005) able to alter the ecological relationships between animals and plants in this system during the cold season and drought (del-claro & marquis, 2015; fagundes et al., 2015). adjustments to the presence of the fire were imposed to the plants (coutinho, 1990; gottsberger & silberbauer-gottsberger, 2008; simon et al., 2009; silva & batalha, 2010a, b) due to constant burns in the cerrado abstract burnings and deforestation are severe disturbances to plants and may represent a stressful situation for plant growth, and they can also affect plant-pollinator interactions and the reproductive success of plants. in this study, we verified the variation in floral symmetry of adenocalymma nodosum (bignoniacea) in two areas, one post-fire and other after deforestation. we also verified the effects on plant-pollinator interactions and fruit set production. results showed that a. nodosum flowers were more asymmetric in mowing areas than in fire areas. asymmetrical flowers presented low nectar concentration and bee visitation rates. although mowed environments produce fewer fruits and seeds than areas affected by fire, the change was not significant. soil from the burnt area showed higher nutrient and organic matter concentration and less aluminum than that of mowed areas. our results showed that a. nodosum flowers in the deforestation area are more asymmetric than those in the post-fire area. this result suggest that cerrado plants may be less adapted to deforestation than to fire, since they have been facing fire events for thousand years in this biome. we suggest that the effects of environmental stress on the development and fitness of plants may provide an important breakthrough to the understanding of insect-plant interactions in cerrado savanna, where burnings and deforestation are frequent anthropogenic effects. sociobiology an international journal on social insects v stefani1, d lange2, aa vilela1, ca ferreria1, k del-claro1 article history edited by gilberto m. m. santos, uefs, brazil received 12 december 2016 initial acceptance 13 february 2017 final acceptance 14 february 2017 publication date 17 october 2017 keywords cerrado, pollinator-plant, analysis of the soil. corresponding author vanessa stefani laboratório de ecologia comportamental e de interações universidade federal de uberlândia av. pará, 1720 cep 38400-902 uberlândia-mg, brasil. e-mail: vastefani@hotmail.com that date back to times prior to the human occupation of the continent (hoffmann & moreira, 2002; miranda, 2002). in addition, the fire is a negative disturbance that causes structural and physiological stress and it alters the performance and development of plants (schmidt et al., 2005). deforestation has a significant effect on species and ecosystems. the death of species is inevitable in mowed natural areas and in areas replaced by urban or agricultural sites. it affects all the destructed environments and causes local erosion, climate changes and soil impoverishment (hajabbasi et al., 1997; christian et al., 2008). thus, plants that sprout in a cerrado area completely changed by deforestation may suffer significant effects on their development and survival (rizzini, 1962; hoffmann, 1998). the analysis of symmetry was the tool used to check the impact of disturbances, whether of biotic or abiotic origin, on the development of plants. for example, the fluctuating 1 laboratório de ecologia, comportamento & interações, universidade federal de uberlândia, minas gerais, brazil 2 universidade tecnológica federal do paraná, campus santa helena, paraná, brazil research article bees mailto:vastefani@hotmail.com v stefani et al. – fire and deforestation on the floral symmetry302 asymmetry (fa) consists of verifying small random variations or the instability in the right and left sides of bilateral-character bodies, which deviate from perfect symmetry (cornelissen & stiling, 2005; graham et al., 2010). these instabilities in symmetry result from the genotype inability to produce the same phenotype in a particular environment or under certain conditions (møller & swaddle, 1997). it indicates an interference in the stability of character development due to genetic or environmental stress (polak, 2003; graham et al., 2010; cuevas-reyes et al., 2011). these disturbances affect not only the bilateral growth patterns of the body, but they can also negatively affect plant fitness (díaz et al., 2004; graham et al., 2010; alves-silva & del-claro, 2015). plant-pollinator interactions select the flowers that will be visited and depend on the quality of the provided remedy (møller & eriksson, 1994; ferreira & torezan-silingardi, 2013), this floral quality will directly affect the reproduction success of plants. thus, the shape, size, color of the floral structure and the quality of reward most likely result from coevolution characters between plants and their pollinators (møller, 1995; torezan-silingardi, 2011; pellegrino, 2015). accordingly, recent studies are seeking to understand the relationship between the intraspecific variation in the morphology of the flowers and their reflection on the fitness of the plant, whereas pollinating insects exhibit preferences for visiting symmetrical flowers (milligan et al., 2008; frey & bukoski, 2013; daloso, 2014). therefore, the symmetry may be indicative of a floral of good quality and it would affect the attractiveness and the pollinator visitation rates, as well as increase the reproductive potential of plants (møller & eriksson, 1995; frey & bukoski, 2013). the overall aim of the present study is to verify whether the floral symmetry of a pioneer species typical of the cerrado, adenocalymma nodosum (silva manso) l.g. lohmann (bignoniaceae), is more affected by fire or by mowing. we formulated three questions to address such aim: (1) are flowers produced by plants that sprouted after a deforestation process more asymmetric those flowers from burned areas? (2) do these flowers have similar quality resources and are, consequently, attractive to pollinators? (3) is there difference in the fruit and seed production of plants of these two areas? our hypothesis is that flowers from both the burnt and the mowing areas will be asymmetric; however, the degree of variation in the symmetry will be smaller in the burnt area, because plants are less adapted to deforestation than to fire. consequently, asymmetrical flowers will be less attractive to pollinators and; therefore, less visited; thus, they will negatively influence the fruit and seed production of these plants. materials and methods study and sampling area the study was conducted next to the clube caça e pesca itororó ecological reserve, (18°58’00” s and 48°17’30” w ccpiu), located in uberlândia, minas gerais state, brazil. the region is located in cerrado biome with dirty field gradients and the presence of tree and shrub plants (from 10 to 60 % coverage) in an area dominated by grasses (eiten, 1972). adenocalymma nodosum is a caespitose shrub with flexible branches up to 2 m high (tresvenzol et al., 2010). its leaves are petiolate, bi or tripinnate and imparipinnate, its leaflets are narrow and lanceolate, sessile when simple, petiolate when they are compound leaflets and the flowers arise in the axils of bracts (sampaio et al., 2016). the plant species studied is important for the reproductive pollinating insects (sampaio et al., 2016). two groups of a. nodosum that have suffered different types of environmental degradation were used in the present study: a) post-fire area, re-growth group with 30 plants, located in a fire recovery area, distributed over approximately 2.1 hectares; b) postdeforestation area (four years without fire event), regrowth group with 30 plants, located in a recovery area, mechanical clearing close to the ground, spread over approximately 1.9 hectare. adenocalymma nodosum individuals were randomly selected in november 2013, one month after the areas were burnt or cleared. the study was conducted during the plant-breeding season, which occurred between january and april 2015. the subjects from the two populations were approximately 1.5m tall and had the same phenological state, approximately the same number of branches, leaves and flower bud formations. floral resources fifteen flower buds were bagged with voile in the late afternoon in the day before the collections were made in order to assess the volume and concentration of nectar produced in the two populations. the following morning, from 8:00 am on, data on nectar volume and sucrose concentration were collected using 10μl microcapillary and graduated refractometer eclipse®), respectively. after the nectar was measured, the flowers in anthesis were removed, packed in styrofoam box with ice gel, and taken to the laboratory so the grains could be counted and the viability analysis could be done. a. nodosum flowers have two fillets with different heights (a larger and a smaller one). therefore, an anther was selected from each fillet on the left side of the flower to prepare the slides according to the staining method using acetic carmine (radford et al., 1974). the counting of viable and non-viable pollen grains was conducted in optical microscope, the reddish grains were considered viable and the non-stained ones were considered non-viable. the differences in the volume and concentration of nectar, as well as the amount of viable and non-viable pollen from flowers from both areas were obtained through t-test, through log10 processing, whenever necessary. all the statistical analyses were conducted in systat software 12.0 and in graphpad prism. sociobiology 64(3): 301-309 (september, 2017) 303 floral visitors the monitoring of pollinators’ behavior was done through visual observations in situ. initially, the flower buds from the same 30 individuals selected in both areas were previously bagged. the observations were made in the same measurements flower symmetry -, i.e., for 2 days in each area from 7:00 am to 03:00 pm, 10-minute sessions per hour, in each flower. the samples were accompanied since flower opening to flower fall. the branches were bagged in voile bags and observed throughout fruit development. after 64 days, the formed fruits were collected, dried in an oven at 60°c for 72 hours, weighed, and the number of seeds was counted. data describing the richness and frequency of bee visitations were recorded for further comparison between areas. specimens were collected using netting and traps (baitodors) specific for each pollinator group. the traps consisted of perforated plastic bottles containing cotton and eucalyptol essence. they were hung at approximately 1.5m from the soil and remained exposed for eight hours (krug & alves-dossantos, 2008). after this time, the traps were closed and the visitors were taken to the laboratory for identification. we used the chi-square test of independence with yates’ correction to compare the total number of visitations to the flowers between the areas. we just considered the visitors that entered the flowers. we used spearman’s correlation to verify the association between fa (as length and width of flowers) and the number of bee visitations to the flowers. all the analyses were performed in the biostat 5.0 software. symmetry test in flowers each individual (n = 30 individuals from each study area) had a bud at the bagged pre-anthesis to investigate the relationship between the change in floral symmetry and pollination. the bud was released the following day and it was kept full opened thereof. observations were made during two days, in each area, from 7:00 am to 03:00pm, 10-minute session per hour in each flower. the samples were observed until the time of flower fall. after flower fall, every flower that was observed for floral visitors had their corollas collected to be measured through fluctuating asymmetry techniques (fa). the corolla was placed on a gridded surface and pressed with a glass to remain completely open. length and width measurements on the right and left sides of the petals were taken using a digital caliper. the length of the petal was defined as the central length of the petal, from the lower part of the corolla. the width was defined as the greatest width presented by the petal perpendicularly to the length (fig 1). although the flowers were tubular, they had bilateral symmetry and the fa value was determined as the difference in length and width of the right hand side petals (rw) and the left hand side petals (lw), according to møller and eriksson (1994). according to potts and strobeck (1986), it is necessary to firstly discriminate the fa of other types of asymmetry, such as the directional asymmetry and the anti-symmetry. the fa indicates random and small symmetry deviations of a character and the frequency distribution of differences between the left and right sides (rw-lw); zero mean and normal distribution (bell-shaped curve). the directional asymmetry occurs whenever the development of a character in one side of the symmetry plane is greater than in the other side (van valen, 1962), most asymmetric individuals are in the same direction (palmer, 2004) and have frequency distribution graph moved to the left or to the right. since antisymmetry reflects lack of symmetry, but there is no specific displacement direction; thus, it results in a bimodal frequency plot (sanseverino & nessimian, 2008). the main ecological importance among asymmetry types is that fa is caused by environmental and biotic factors that can be measured and analyzed. such measurements enable conclusions about how some variables affect the development of organisms. the fluctuating asymmetry in plants is often measured in sheets by considering the midrib as the axis of symmetry (møller, 1995; cornelissen & stiling, 2005; alves-silva & del-claro, 2013). the fa of the sexual characters more strongly respond to environmental and genetic disorders in the analysis of specificity related to fluctuating asymmetry and to the character of sexual ornament developments such as flowers (møller, 1995). initially, it was necessary to test the absence of directional asymmetry and anti-symmetry, which would represent the presence of fluctuating asymmetry (fa). thus, the t-test and the lilliefors normality test were performed to measure the directional asymmetry and antisymmetry, respectively, through the absolute difference between the right and left sides to the length and width. the differences between the left and right length and the width of the flower was not significantly deviated from zero in the post-mowing sample, respectively (t = 0.03, df = 56, p> 0.05 t = 0.219, df = 54; p> 0.05), as well as in the post-fire one (t = 0.139, df = 58, p> 0.05 and t = 0.132, df = 58, p> 0.05). all the data presented normality in the distribution (p> 0.05; lilliefors test) and it validated the fa hypothesis. fig 1. shrub adenocalymma nodosum, a) final portion of the bush illustrating the flowers and their flower buds; b) representative illustration of how a flower had the right (rw) and left (rw) width and the right (ri) and left (li) length measured in the petals. measurements were always carried out in the central length and in the greatest width of the petal perpendicular to such length. v stefani et al. – fire and deforestation on the floral symmetry304 we used the difference module between the left and the right side of the width and length (fa = average | e-d |) to examine whether the different populations have different fa levels in their flowers. the comparison of the fa level means was performed through nonparametric mann-whitney. soil analysis an abiotic measure was done to distinguish the stresses suffered by the two evaluated environments. two soil samples were collected 50 cm from each sampled plant, one sample at 10 cm and another one at 20 cm depth, thus totaling 60 samples from each study area. the soil samples collected in the field were placed in plastic bags and sent to laboratório de análises de solos e calcários da universidade federal de uberlândia in order to determine the following parameters: water ph (1:2,5), k (mg.dm-3), ca (cmol.dm-3), mg (cmol.dm-3), al (cmol.dm-3) , organic matter m.o. (g.kg-1), b (mg.dm-3), fe (mg.dm-3), mn (mg.dm-3). the mann-whitney test was used to compare each parameter in the distinct areas except for fe (iron), which was measured through students t-test. results the total length of the a. nodosum corolla in both areas was similar (t =0.452; df = 54; p = 0.115), as well as the width of the right (u = 336.5; p = 0.135) and left petals (u = 410.5; p = 0.715). nevertheless, the flowers in the post-mowing area were significantly more asymmetrical than those of the postfire area (u = 174.0; df = 15; p < 0.001 for width of petals; u = 193.0; df = 15; p < 0.001 for length of petals) (fig 2). we recorded bee species as floral visitors in both studied areas: bombus pauloensis friese, 1913, apis mellifera linnaeus, 1758, euglossa melanotricha moure, 1967, epicharis flava friese, 1900, trigona spinipes fabricius, 1793 and some species were not collected from ceratinini and emphorini tribes. we recorded 204 bee visitations in flowers from the post-fire area, which corresponds to 0.75 visitations per hour, on average. we recorded 62 bee visitations in the flowers from the post-mowing area, which corresponds to 0.30 visitations per hour, on average. we detected variations in the total rate of visitations between areas (x2 = 74.74; df = 1; p < 0.001). the number of visitations to flowers in the post-fire area was higher throughout the day than that of the post-mowing area (fig 3). we recorded a similar visitation peak pattern and the subsequent visitation decline in the early afternoon, after 02:00 pm. fig 2. fluctuating asymmetry (cm) of the width and length of adenocalymma nodosun petals on post-fire and post-mowing areas in february/march 2013 in uberlândia, brazil. the asterisks mean significant difference between areas (mann-whitney test). fig 3. number of bee visitations to adenocalymma nodosum flowers in post-fire and post-mowing areas in uberlândia, brazil. the volume of nectar was similar between the areas (t = 1.76; df = 28; p = 0.08), as well as the total amount of produced (u = 110.0; df = 28; p = 0.93) and unfeasible pollen (t = 1.185; df = 28; p = 0.24). we just observed differences in the concentration of sugar in the nectar, and higher values of it in the post-fire area. (t = 3.352; df = 27; p < 0.05). the evaluated plants produced fruits in both areas. although the burnt area has produced more fruits than the mowed area (33% and 20%, respectively), the difference was not significant. (χ2 = 1.20; df = 1; p = 0.273). the fruit weight was also similar between areas (t = 0.721; df = 6; p = 0.493), as well as the average number of seeds per fruit (t = 1.07; df = 6; p = 0.07; 7.5 seed per fruit in the burnt area and 4.43 seed per fruit in the mowed area). there was no relation between the width of petals and of the number of bee visitations to flowers in both areas (rs = 0.058; t = 0.305; p=0.76 in the mowed area; rs = 0.008; t = 0.004; p = 0.996 in the burnt area). we also found no relation between the length of the petals and the number of bee visitations to flowers in the burnt area (rs = 0.294; t = 1.637; p = 0.136). however, there was significant positive relation between the length of the petals and the number of visitations to flowers in the mowed area (rs = 0.401; t = 2.275; p < 0.05 – fig. 4). the assessed soil parameters were significantly different between areas (see table 1), just ph, at the depth 20 cm, were similar in both areas. sociobiology 64(3): 301-309 (september, 2017) 305 discussion our results corroborate the initial hypothesis that a. nodosum flowers in the deforestation area are more asymmetric than those from the burned area. this result is an evidence that cerrado plants may be less adapted to deforestation than to fire, since they have been facing fire events for thousand years in this biome. according to ledru (2002), the oldest fossil record of fire actions in the cerrado dates back to 32 thousand years ago. this period is prior to the arrival of humans to the americas (cooke, 1998). however, it is most likely that fire has occurred more often in the last 10,000 years due to indigenous practices such as hunting and gardening (miranda et al., 2002, 2009). according to simon et al. (2009), fire is a key driver of natural cerrado plant selection in brazil. it is evidenced by the sprouting ability of post-fire plants (gottsberger & silberbauer-gottsberger, 2008). this ability is a very common adaptive strategy of savanna species (bond & midgley, 2001). however, the sprouting ability of post-fire plants may be limited by the annual burnings, which decrease the height and diameter of the sprouts and, consequently, increase the mortality rate (medeiros & miranda, 2005). annual burnings are common in cerrado areas where fire is used to turn the natural environment into farmland and to renew pastures (miranda et al., 2002). on the other hand, deforestation in brazil was boosted in the early 1970s due to political incentive to productive occupations in cerrado areas, mainly to agricultural production. this fact resulted in increased environmental degradation and in significant biodiversity losses (myers et al., 2000; oliveira & marquis, 2002). currently, cerrado fragments play a vital role in preserving the local endemic fauna and flora and they are considered an important biodiversity conservation area on the planet (hotspot sensu myers et al., 2000). thus, deforestation may be considered a recent event in the evolutionary history of cerrado plants, whereas fire, and its impact on plant sprouting, may be higher due to the plants that are not yet adapted to this stress. we suggest, that the sprouting of a. nodosum individuals after environmental stress (fire and deforestation) could have caused fa in flowers, and it corroborates the study by alves-silva and del-claro (2013), who found fa in post-fire banisteriopsis campestris (malpighiaceae) leaves. however, plants often have high degree of phenotypic plasticity associated with high fa levels (schlichting, 1986) and stresses that generate faster plant growth may be more efficiently genetically corrected through the repairing or decreasing of fa (møller & pomiankowski, 1993). it might have occurred in the plants from the post-fire area in the current study. fire and pruning can also indirectly act on the formation of plants in the affected areas through changes in soil chemistry. our results show that the soil in the burnt area has concentrations of some nutrients in the upper layers higher than that of the soil in the deforested area. according to kauffman et al. (1994), in the short term, fire becomes a mineralizing agent of organic matter through the increased availability of nutrients used for plant growth, especially at depths shorter than 0.5 cm, due to ashes with high p, k and ca fig 4. relation (spearman’s correlation rs = 0.40; t = 2.27; p < 0.05) between the number of bee visitations and the fluctuating asymmetry length (cm) of the flowers in post-mowing area in uberlândia, brazil. parameters 10 cm 20 cm post-fire x ± ep (n=30) post-mowing x ± ep (n=30) test value p post-fire x ± ep (n=30) post-mowing x ± ep (n=30) test value p ph h2o (1:2,5) 5.95±0.06 5.52±0.03 119.5 < 0.001 6.9±0.64 5.41±0.23 341.5 0.108 k (mg.dm-3) 0.26±0.01 0.18±0.009 63.00 < 0.001 0.15±0,024 0.075±0.004 109.5 < 0.001 ca (cmol.dm-3) 2.05±0.011 0.48±0.03 1.500 < 0.001 1.16±0.14 0.27±0.01 11.50 < 0.001 al (cmol.dm-3) 0.007±0.005 0.33±0.035 64.00 < 0.001 0.08±0.03 0.5±0.02 42.00 < 0.001 o. m. (g.kg-1) 5.39±0.021 2.85±0.07 30.00 < 0.001 4.08±0.14 2.01±0.05 0.000 < 0.001 p(mg.dm-3) 0.4±0.08 0.141±0.01 28.00 < 0.001 0.23±0.14 0.1±0.003 19.00 < 0.001 table 1. soil chemical features in the post-fire and post-mowing areas, in two different depths in uberlândia, brazil. we used the mannwhitney test in all the comparisons (k = potassium; ca = calcium; al = aluminum; o.m.= organic matter, p = phosphorus). v stefani et al. – fire and deforestation on the floral symmetry306 concentrations. the mineralization of organic matter causes faster plant growth in the burned area than in other areas because the nutrients provide quick benefit to pioneer plants, such as a. nodosum. besides soil enrichment, there is decrease in the al content shortly after the fire, and it can be understood as beneficial because this component can be toxic, especially if the soil ph is low (coutinho, 1990). ashes are usually rich in soluble oxide bases and carbonates, which become able to neutralize the ph of the soil (oliveira & silva, 1994) by reducing the aluminum toxicity. this result was also showed in the current study because we found low aluminum rates in the soil of the post-fire area. different processes occur in deforested areas, because the deposited plant matter is slowly decomposed, and it gradually provides nutrients to the plants, and hinders the rapid sprouting in most of the species (zech et al., 1997). however, all these aspects will depend on the intensity, frequency, type of soil and climate and vegetation features before the fire, age and on the affected plant species (type of sprouting), as well as on the period of the year when the stress event occurred (see catry et al., 2010). we also detected a significantly higher concentration of sugar in the nectar of less asymmetric plants in burned area. several studies have shown that fa may be featured as a quantitative and quality indicator of available resources of flowers pollinators (wignall et al., 2006; marazzi & endress, 2008; wolowski & freitas, 2010; potts, 2015). with more symmetrical flowers in the same species as a reliable sign of good quality resources to its visitors (wignall et al. 2006). according to cornelissen and stiling (2005), fa may also directly influence plant metabolism and the production of chemical compounds such as floral nectar and møller and eriksson (1995) confirms the increased production of nectar and preference of pollinating insects for more symmetrical flowers. other studies describe the recognition and preference of pollinators for better resource offers (weiss, 1991; gumbert, 2000; chittka & thomson, 2004; miller et al., 2011). in addition, our results support the idea of pollinators’ preference for less asymmetrical flowers and a higher production of fruit (although not significant) in burned area than in the area of pruning. sampaio et al. (2016) mentions that the species a. nodusum has naturally low reproductive success, which is usually related to the limitation of pollen or ovule. in this sense, the presence of asymmetry could further reduce productivity. based on an evolutionary scale viewpoint, many plants were positively selected to increase the capacity of surviving the different types of fire regimes and resprouting systems in cerrado (simon et al., 2009). the same type of natural selection may not have been effective to plants under pruning stress. therefore, in respect to plasticity and natural selection, it is possible that a. nodosum individuals from the burned area in the current study may be undergoing a selection process more advanced than that of individuals from the deforested area. innovative studies, which assess the effects of environmental stresses on the development and fitness of plants, provide an important advance to the understanding of insect-plant interactions and of their co-evolutionary aspects, especially in cerrado areas where burning and deforestation are frequently performed by humans. the fa was widely used in these studies as a way to provide relevant information to biology conservation. acknowledgements the authors thank the staff of clube de caça e pesca itororó de uberlândia, where the study was carried out. we are grateful to solange cristina augusto for their invaluable help in species identification. this work was financial supported by the national post-doctoral plan (coordenação de aperfeiçoamento de pessoal de nível superior – plano nacional de pós-doutorado; capes-pnpd/ 1533482 pós-graduação em ecologia e conservação ufu) for vs; thanks to cnpq (pq 301605/2013-0); cnpq (473055/2012-0) for kdc and fapemig for awarding fellowships to aav. references alves-silva, e. & del-claro, k. 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(1997). factors controlling humification and mineralization of soil organic matter in the tropics. geoderma, 79: 117-161. doi: 10.13102/sociobiology.v63i3.1043sociobiology 63(3): 894-908 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 amazon rainforest ant-fauna of parque estadual do cristalino: understory and grounddwelling ants introduction ants are ecologically dominant, playing several ecosystem services in the environment (del toro et al., 2012) and occupying the most varied habitats (bruhl et al., 1998; dejean et al., 2015; rocha et al. 2015; pape et al., 2016). ants strongly influence several terrestrial communities due to their abundance and potential relationships with various biological groups. the interactions of ants can be positive or negative, ranging from interactions with microorganisms (hanshew et al., 2014; sanders et al., 2014), plants (izzo & vasconcelos, 2009; vicente et al., 2012, 2014; koch et al., 2015) and other invertebrates (dáttilo et al., 2012a; freitas & rossi, 2015; puker et al., 2015), even other ants (adams et al., 2007; sanhudo et al., 2008; gallego-ropero & feitosa, 2014). therefore, ants have been used as a model to understand the various ecological patterns and as valuable bio-indicators of abstract ants are ecologically dominant and have been used as valuable bio-indicators of environmental change or disturbance being used in monitoring inventories. however, the majority of inventories have concentrated on ground-dwelling ant fauna disregarding arboreal fauna. this paper aimed to list the ant species collected both on the ground and in the vegetation of the parque estadual do cristalino, an important protected site in the center of the southern amazon. moreover, we compared the composition of the ground dwelling and vegetation foraging ants. two hundred and three (203) species distributed among 23 genera and eight subfamilies were sampled, wherein 34 species had not yet been reported in the literature for mato grosso state. as expected, the abundance and richness of ants was higher on the ground than in the understory. also, the composition of the ant assemblages was different between these habitats (with only 20% occurring in both) indicating that complementary methods which include arboreal and terrestrial ants are indicated for efficient inventory. this study provides an inventory of the arboreal and ground ant fauna contributing to the knowledge and conservation of amazonian ant fauna. sociobiology an international journal on social insects re vicente1, lp prado2, tj izzo1 article history edited by frederico s. neves, ufmg, brazil received 12 april 2016 initial acceptance 28 may 2016 final acceptance 22 july 2016 publication date 25 october 2016 keywords arboreal ants, conservation, diversity, formicidae, inventory. corresponding author ricardo eduardo vicente laboratório de ecologia de comunidades do instituto de biologia universidade federal de mato grosso cuiabá-mt, brasil e-mail: ricardomyrmex@gmail.com environmental change or disturbance (andersen et al., 2002; bruna et al., 2014; falcão et al., 2015). regardless of their ecological importance and diversity, studies on brazilian ant fauna were usually concentrated in the atlantic forest (feitosa & ribeiro, 2005; figueiredo et al., 2013; silva et al., 2007), the cerrado (dáttilo et al., 2012b; silva & brandão, 2014; camacho & vasconcelos, 2015), and pantanal (ribas & schoereder, 2007; silva et al., 2013; meurer et al., 2015). although the amazon is one of the largest and heterogeneous tropical forests remaining, the panorama is not different. the studies of amazonian ants in brazil are generally concentrated in small parts of the central region (e.g. vasconcelos et al., 2006, 2010; santos et al., 2007; baccaro et al., 2013; souza et al., 2012). studies in the southern amazon region are few, and because they focus on local ecological patterns, the list of species therein usually does not have confirmation of specialists and comprises a 1 universidade federal de mato grosso, cuiabá-mt, brazil 2 museu de zoologia da universidade de são paulo, são paulo-sp, brazil research article ants sociobiology 63(3): 894-908 (september, 2016) 895 large number of morphospecies (for example: dáttilo et al., 2013; falcão et al., 2015). the absence of surveys in this area is particularly problematic to the knowledge of the distribution of ant diversity in the tropics. more than being a frontier between the amazonia and cerrado biomes, the south of amazonia is a frontier of fast agricultural expansion, being subject to enormous rates of deforestation (fearnside, 2005). in such a scenario, several species could become, and probably some already have become extinct without being known by science. this is worrying in a region that has many species with unknown distribution (vicente et al., 2011, 2012, 2015, 2016; prado et al., 2016). whereas inventories are fundamental to promote conservation of tropical forest remnants and the ants are important components of biodiversity, this work aims to contribute to the knowledge of the amazonian ground-dwelling and arboreal ant fauna. the reserve is a terra firme tropical rainforest located in the southern amazon, but with little known diversity of various taxa. in this study, we list the fauna of ground-dwelling ants and the assemblages of ants foraging on lower vegetation in the same site. we compared the difference between the strata in order to demonstrate the increase in local biodiversity by using a simple additional method. material and methods study area the surveys were conducted in an amazonian research program station in biodiversity in the brazilian parque estadual do cristalino (henceforward pec), with rapeld methodology (see magnusson et al., 2005). the park is a large protected area defined as "priority for conservation site" (ministério do meio ambiente [mma], 2001) that hosts an area of 184,900 ha, situated in the middle of an area called “arc of deforestation” (fearnside, 2005), comprising the brazilian municipality of alta floresta and novo mundo, state of mato grosso bordering on pará state. the research study was carried in a station installed in the north of the park (9° 32’47 “s, 55° 47’38” w) (fig 1) in the municipality of novo mundo. the vegetation is characterized as transition zones between ombrophilous and seasonal forest, seasonal forest and savanna (cerrado), and ombrophilous and savanna (cerrado) (institituto brasileiro de geografia e estatística [ibge], 2004). ant sampling the inventory of ants was conducted between november 2012 (begining of the rainy season with sporadic rains) and may 2013 (end of the rainy season with sporadic rains) in 11 trails 250m long, with a distance between sites of at least 1 km. in each trail, ants were collected every 25m, resulting in 20 samples per trail (10 samples of the ground-dwelling ants and 10 samples of the arboreal ants) and totaling 220 samples (110 samples of ground-dwelling ants and 110 samples of arboreal ants). the collection of the ground ant assemblages was made using a single pitfall trap that remains installed for 48 hours in each sample point. for sampling arboreal ants a beating-tray method was used in vegetation of understory. in each of these points, we selected the four treelets equidistant of about 2 m of the pitfall. under each treelet, a white canvas was installed to prevent some ants from jumping and getting away from the sampling (dáttilo et fig 1. location map of the parque estadual do cristalino (filled circle). re vicente, lp prado, tj izzo – amazon rainforest ant-fauna of parque estadual do cristalino896 al., 2013), and about one meter from the ground to prevent ants from walking on the ground and climbing on the canvas. then all vegetation in each treelet within 1m² with between 1-3 meters in height was steadily shaken and the ants that fell on the canvas were properly collected. the sampling of ground and arboreal ants was carried out concomitantly, so the sampling of arboreal ants was performed between 9 am and 3 pm on the same day that the pitfall was removed. ant identification ants collected initially were identified using dichotomous key for subfamilies and genera available in baccaro et al. (2015). so as to separate into morphospecies and identify to a specific level we used several taxonomic keys (brandão, 1990; fernández, 2003; longino, 2003a; mackay & mackay, 2010; fernandes et al., 2014). posteriorly we made comparisons with specimens deposited at the laboratório de ecologia de comunidades from the centro de biodiversidade da universidade federal de mato grosso (ufmt) and the ant collection from the laboratório de sistemática, evolução e biologia de hymenoptera from the museu de zoologia da universidade de são paulo (mzsp). we also consulted specialists to confirm species identification (see acknowledgments). vouchers were deposited in the collections mentioned above. after the identification and confirmation by specialists we consulted the distribution of each species using the antmaps (janicki et al., 2016), a new interactive tool recently used by taxonomists and ecologists (gérnard & economo, 2015; vicente et al., 2015; santos-silva et al., 2016; wepfer et al., 2016). this tool comprises the geographic distributions of more than 15,000 species/subspecies in over 1.7 million records of about 8,650 publications, museum collections and specimen databases (janicki et al., 2016). furthermore, we researched in bibliographic references related to taxonomy of species or lists of ants from region. data analysis we carried out a t-test to access the difference of ant abundance and richness patterns between ground and understory. because ants are social insects, the sampled abundance of workers in pitfalls may be strongly related to the proximity to the nest (gotelli et al., 2011) or workers’ number in a colony, which varies greatly between species (baccaro et al., 2015). to minimize this effect, we treated the abundance as sample-based occurrences (gotelli et al., 2011) as is commonly done in studies with ants (ryderwilkie et al., 2010; dáttilo & izzo, 2012; baccaro et al., 2013). to access the distribution patterns of the ant assemblages we performed an ordenation with principal coordinate analysis (pcoa) technique based on a matrix of raup-crick dissimilarity measures calculated in a binary matrix with presence and absence date. raup-crick was used because it is a robust index that calculates how different pairwise samples are than expected by chance implementing null models which consider the variation in the number of local species and alpha diversity (chase et al., 2011). this index is frequently used to compare the dissimilarity of invertebrate communities between different habitats (ryderwilkie et al., 2010; ribas et al., 2012; reis et al., 2013). we utilized the first two pcoa axis (which represented 66.76 % of explication) in a multivariate analysis of variance (manova) as dependent variables and vertical habitat (ground and understory) as a factor. all analysis were performed with r-software (r core team, 2015) using the vegan-package (oksanen et al., 2015). results we recorded 1,581 occurrences of ants in the 220 samples. the ants collected belong to 203 species, 45 genera and eight subfamilies. subfamilies with greater richness were myrmicinae with 23 genera and 113 species, ponerinae with eight genera and 23 species and formicinae with five genera and 27 species. the genus with greater richness was pheidole (37 species), camponotus (17), neoponera and crematogaster (both 10 species). both abundance (p < 0.001) and richness (p < 0.001) of ants were different between the vertical strata (table 1 – fig 2), being greater on the ground than on the understory. we collected almost 63.4% of ant occurrences (total: 1,002, mean: 89.1, sd: ±26.3 per sample) and 65% of ant species (total: 143 species, mean: 47.91, sd: ±10.26) on the ground stratum. the understory showed a total of 579 occurrences (mean: 51±, sd: 15.1) and a total of 100 species (mean: 26.6, ±sd: 6.67). of these 203 species, 164 species were restricted to a vertical stratum with 104 species collected only on the ground and 60 only on the understory, and just 39 species collected in both strata (table 2). although on average 41.09 species by trail (±sd: 9.07) were exclusive to the ground, there is a mean increase of 19.73 species by trail (±sd: 5.85) by adding beating tray in the ant collection. only one subfamily (amblyoponinae) was sampled exclusively on the ground. consequently, the species composition was different between the habitats (manova pillai-trace: 0.852, f1,20: 54.873, p < 0.001 – fig 3). among the species sampled, 34 were recorded for the first time for mato grosso state, brazil (table 2). table 1. t-test measures for difference in abundance and richness between ground and arboreal ant assemblages. parameters t df p-value abundance 4.1599 15.979 <0.001 richness 5.7656 17.167 <0.001 sociobiology 63(3): 894-908 (september, 2016) 897 discussion of the inventories on ant assemblages conducted in the amazon, the major part of the studies focused just on grounddwelling ants (miranda et al., 2012; souza et al., 2012, 2015; baccaro et al., 2013) and few comprised both terrestrial and arboreal ant fauna detailing the strata in which each species was collected (e.g.: vasconcelos & vilhena, 2006; ryder-wilkie et al., 2010). these few studies carried out in the amazon embracing both soil and vegetation ants have demonstrated extremely different ant assemblages between these habitats (vasconcelos & vilhena, 2006; ryder-wilkie et al., 2010). the methods employed here are very different. the both methods capture active foraging ants, however the pitfall traps were active during day and night whereas the beating tray method was used only during the day. this can explain the small number of species collected on vegetation, preventing strong inferences towards the patterns of species richness. however we observed a strong turnover on ant composition, since only about 20% of all collected species were sampled both on the ground and on vegetation. hence, even missing several nocturnal species on vegetation strata, the species collected just on vegetation were certainly absent on ground strata. therefore, the vertical stratification in the ant fauna is a robust ecological pattern found in both the amazon forest and in other biomes (bruhl et al., 1998; vasconcelos & vilhena, 2006; neves et al., 2013; camacho & vasconcelos, 2015). additionally our results indicate that, using an additional simple method focusing on vegetation foraging ants, as beating-tray, one can increase the number of collected species. therefore, besides the vegetation foraging ant community can show different ecological patterns they contribute to our understanding of the real ant biodiversity in an area and, as well as the knowledge of biogeographical patterns. the amblyoponinae subfamily, considered as a basal group in the evolution of ants (saux et al., 2004), as well as the 18 other genera were sampled exclusively on the ground. among these genera collected only in soil are the fungusgrowing ants atta, mycetarotes, mycocepurus, myrmicocrypta and sericomyrmex. these ants nest directly on the ground or in rotten wood in contact with the soil (baccaro et al., 2015). except for the atta species which climbs into vegetation to cut fresh vegetable matter, the other abovementioned species take advantage of organic matter found in litter such as fallen leaves, fruits, flowers, feces and corpses (leal & oliveira, 2000; mayhé-nunes & brandão, 2006). hylomyrma immanis, octostruma balzani, rogeria scobinata and three unidentified hypoponera species also were collected exclusively in the soil. these genera have species with cryptic behavior (lapolla fig 2. (a) abundance mean of ants and (b) richness of ant species in vertical strata (ground and understory). fig 3. composition of ground (filled circle) and understory (filled circle) ant community of the parque estadual do cristalino. principal coordinate analysis axis represented 71.6% of total ordination. (a) (b) re vicente, lp prado, tj izzo – amazon rainforest ant-fauna of parque estadual do cristalino898 & sosa-calvo, 2006; longino, 2013b). the sampling of only one daceton armigerum worker on the ground should be considered as an accidental record, since species of this genus have adaptations such as hook-shaped claws which provide remarkable adhesion to the arboreal substrate (billen et al., 2016) where they nest in tree branches (azorsa & sosa-calvo, 2008; vicente et al., 2011) and forage (dejean et al., 2012). despite their hypogeal origin, ants diversify occupying other habitats (lucky et al., 2013) and the occupation of the arboreal environment resulted in morphological and behavioral adaptations both to get around and feed on available resources in vegetation (orivel & dejean, 1999; dejean et al., 2005). these adaptations were observed in the various neoponera species sampled. neoponera is composed mainly by arboreal behavior species (schmidt & shattuck, 2014) that live or forage on vegetation, and have morphological adaptations regarding modifications on claws, shape and adhesion (orivel et al., 2001). four other genera were restricted to vegetation: azteca, myrmelachista, nesomyrmex and tapinoma. species of these genera usually nest on plants (longino, 2007; nakano et al., 2013; longino, 2004). cephalotes was another genus with a greater number of arboreal species, with just c. atratus being collected in both strata. the cephalotes ant genus has as their main features a diet based largely on pollen and nectar and nesting in pre-existing plant cavities (byk & del-claro, 2010), however, c. atratus can often be found foraging on the ground (corn, 1980). although some subfamilies as pseudomyrmecinae and dolichoderinae had a large proportion of species collected in the vegetation, no subfamily has been sampled exclusively to only this vertical strata. in pseudomyrmecinae, only pseudomyrmex tenuis was collected in both soil (one worker) and vegetation (seven workers). p. tenuis is a widespread ant species, frequently sampled foraging in all strata as epigaeic, arboreal and canopy stratum (vasconcelos & vilhena, 2006; neves et al., 2013; camacho & vasconcelos, 2015; souza et al., 2015). also, in a dolichoderinae subfamily with 18 ant species, only five species of dolichoderus genus, occurred also on the ground. camponotus genus was particularly representative (17 species; 84 samples) in relation to the total number of collected species (5.6%). in two areas of central amazon, both in amazonas state, brazil, camponotus species comprises 2.7% and 2.3% of total species collect on the ground (souza et al., 2015). in addition, in the central south amazon this proportion ranges from 1.99% to 4.62% of ground ants in preserved areas of rondônia state (souza et al., 2015). on the other hand, in samples made in ground and understory of patches of forest in southern amazon in rondonia state and central east amazon in pará state, both in brazil, there was a ratio of 9.33% and 7.09%, respectively (santos-silva et al. 2016; vasconcelos et al., 2006). therefore, this high proportion of camponotus found in the parque estadual do cristalino confirm that simple methodologies could sampled a greater number of considered arboreal ant species, such as camponotus. mato grosso state is the third largest state in brazil, hosting three different biomes and the many new records in an inventory (18 species collected in the soil, seven in vegetation and 10 in both strata) demonstrating a gap or shortcoming related mainly to the lack of surveys throughout the south american territory (but see: kempf 1972; brandão 1991; silva et al., 2013; meurer et al., 2015). although there are a number of studies on the ecology and distribution of ants in the amazon in northern mato grosso (dáttilo et al., 2013; falcão et al., 2015; vicente et al., 2011, 2012, 2014, 2015) this is the first list of ant species for the region, as well as being the most complete list published until now for the southern amazon. in summary, this work demonstrates that inventories should consider ants of both ground and vegetation for a better sampling of local ant diversity. in addition, the number of new records of ant fauna (35 species) for mato grosso state and the number of unnamed species (97 morphospecies), show that the ant fauna of the amazon in general are not common or are extremely unknown. the tropical forests are being increasingly threatened by anthropic activities and mato grosso state in particular because they are located in a region with a historically strong pressure for disturbance, being an agricultural frontier. therefore, efforts should be made to understand ant diversity distribution patterns and propose conservation strategies in this region. acknowledgments we thank the researchers vinicius soares, mariana silva, bruna e. valério and bruno carvalho for assistance in field data collection. rev and lpp thanks coordenação de aperfeiçoamento de pessoal de nível superior [capes] to their phd and master scholarship (respectively). tji thanks capes (bex 2548-14-3) to financial support. furthermore, we thanks conselho nacional de pesquisas (cnpq 479243/2012–3, 558225/2009-8, 501408/2009-6 e 457466/2012-0) for fund research and secretária estadual de meio ambiente and universidade federal de mato grosso for logistical support. we also thank to alexandre ferreira [ufpr], gabriela camacho [ufpr], itanna fernandes [inpa], jorge souza [inpa], lina hernández [iavh], mônica ulysséa [mzsp] and thiago silva [ufpr] for their help with the identification of the ant species and antônio queiroz (ufla) and two anonymous reviewers for the critical review of the manuscript. this work is publication 53 in the nebam technical series. references adams, r.m.m. & longino, j. 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(2015). expanding the distribution of the remarkable ant gnamptogenys vriesi brandão & lattke (formicidae: ectatomminae): first record from brazil. sociobiology, 62: 615-619. doi: 10.13102/ sociobiology.v62i4.920 wepfer, p., guénard, b. & economo, e.p. (2016). influences of climate and historical land connectivity on ant beta diversity in east asia. journal of biogeography. doi: 10.1111/jbi.12762 sociobiology 63(3): 894-908 (september, 2016) 903 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total amblyoponinae prionopelta prionopelta punctulata mayr, 1866* 4 4 dolichoderinae azteca azteca alfari emery, 1893 4 4 azteca prox.aurita emery, 1893 3 3 azteca inpa01 19 19 azteca tji02 5 5 azteca tji04 2 2 azteca tji05 1 1 azteca tji07 4 4 azteca tji08 2 2 azteca tji09 3 3 dolichoderus dolichoderus abruptus (smith, 1858)* 1 1 dolichoderus attelaboides (fabricius, 1775)* 2 3 5 dolichoderus bidens (linnaeus, 1758)* 1 2 3 dolichoderus bispinosus (oliver, 1792) 1 1 dolichoderus gagates emery, 1890 1 5 6 dolichoderus ghilianii emery, 1894 1 1 dolichoderus imitator emery, 1894 3 2 5 tapinoma tapinoma cr1 12 12 tapinoma cr2 5 5 dorylinae eciton eciton burchellii (westwood, 1842) 3 3 neivamyrmex neivamyrmex prox. pilosus (smith, 1858) 1 1 neivamyrmex inpa02 1 1 ectatomminae ectatomma ectatomma edentatum roger, 1863 14 14 ectatomma lugens emery, 1894* 65 65 ectatomma tuberculatum (olivier, 1792) 3 31 34 gnamptogenys gnamptogenys prox. ericae (forel, 1912) 5 5 gnamptogenys horni (santschi, 1929)* 17 17 gnamptogenys kempfi lenko, 1964* 1 1 gnamptogenys moelleri (forel, 1912)* 10 10 gnamptogenys pleurodon (emery, 1896) 1 1 gnamptogenys striatula mayr, 1884 5 13 18 gnamptogenys prox. sulcata (smith, 1858) 1 1 gnamptogenys tji4 1 1 re vicente, lp prado, tj izzo – amazon rainforest ant-fauna of parque estadual do cristalino904 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. (continuation) subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total formicinae acropyga acropyga tji1 3 3 brachymyrmex brachymyrmex cr1 4 41 45 brachymyrmex cr2 1 1 brachymyrmex cr3 13 13 camponotus camponotus atriceps (smith, 1858) 1 1 2 camponotus diversipalpus santschi, 1922 3 3 camponotus femoratus (fabricius, 1804) 13 10 23 camponotus latangulus roger, 1863 8 8 camponotus mus roger, 1863 1 1 camponotus nidulans (smith, 1860) 5 5 camponotus novogranadensis mayr, 1870 1 1 camponotus scissus mayr, 1887* 1 1 camponotus trapezoideus mayr, 1870 5 5 camponotus cr02 2 2 camponotus cr06 1 1 camponotus cr07 2 2 camponotus cr11 8 8 camponotus cr16 3 2 5 camponotus cr19 2 2 camponotus tji01 3 3 camponotus tji04 10 2 12 gigantiops gigantiops destructor (fabricius 1804) 2 1 3 nylanderia nylanderia prox. caeciliae (forel, 1899) 15 11 26 nylanderia prox. fulva (mayr, 1862) 4 1 5 nylanderia cr1 26 16 42 nylanderia cr5 1 1 nylanderia tji2 17 3 20 myrmicinae apterostigma apterostigma urichii forel, 1893 9 9 apterostigma cr1 2 2 apterostigma inpa01 4 4 apterostigma inpa04 5 5 atta atta cephalotes (linnaeus, 1758) 13 13 atta sexdens (linnaeus, 1758) 1 1 carebara carebara urichi (wheeler, 1922) 13 13 carebara jti1 2 2 sociobiology 63(3): 894-908 (september, 2016) 905 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. (continuation) subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total cephalotes cephalotes atratus (linnaeus, 1758) 3 3 6 cephalotes oculatus (spinola, 1851)* 5 5 cephalotes pallens (klug, 1824) 1 1 cephalotes cr1 2 2 cephalotes cr3 1 1 crematogaster crematogaster arcuata forel, 1899 2 2 crematogaster brasiliensis mayr, 1878 14 14 crematogaster carinata mayr, 1862* 1 1 crematogaster curvispinosa mayr, 1862* 1 1 crematogaster erecta mayr, 1866 3 8 11 crematogaster levior longino 2003* 8 7 15 crematogaster limata smith, 1858* 6 23 29 crematogaster nigropilosa mayr, 1870* 3 26 29 crematogaster stollii forel, 1885 1 1 crematogaster tenuicula forel, 1904* 32 24 56 cyphomyrmex cyphomyrmex laevigatus weber, 1938* 7 7 cyphomyrmex peltatus kempf, 1966 5 5 cyphomyrmex prox. rimosus 3 3 cyphomyrmex cr7 1 1 cyphomyrmex tji02 1 1 cyphomyrmex tji03 9 9 cyphomyrmex tji06 1 1 daceton daceton armigerum (latreille, 1802) 1 1 hylomyrma hylomyrma immanis kempf, 1973* 8 8 megalomyrmex megalomyrmex ayri brandão, 1990 16 16 megalomyrmex cuatiara brandão, 1990* 1 1 megalomyrmex drifti kempf, 1961 2 2 megalomyrmex cr3 1 1 mycetarotes mycetarotes cr1 1 1 mycocepurus mycocepurus smithii (forel, 1893) 4 4 myrmelachista myrmelachista tji01 1 1 myrmicocrypta myrmicocrypta inpa01 2 2 myrmicocrypta inpa02 1 1 re vicente, lp prado, tj izzo – amazon rainforest ant-fauna of parque estadual do cristalino906 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. (continuation) subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total nesomyrmex nesomyrmex prox. asper (mayr, 1887) 1 1 nesomyrmex prox. pleuriticus (kempf, 1959) 1 1 nesomyrmex tonsuratus (kempf, 1959) 1 1 nesomyrmex cr1 1 1 nesomyrmex tji2 1 1 ochetomyrmex ochetomyrmex neopolitus fernández, 2003 18 8 26 ochetomyrmex semipolitus mayr, 1878 3 56 59 octostruma octostruma balzani (emery, 1894) 1 1 pheidole pheidole biconstricta mayr, 1870* 4 4 8 pheidole bufo wilson, 2003 1 1 pheidole gertrudae forel, 1886* 1 1 pheidole prox. gilva wilson, 2003 2 2 pheidole nitella wilson, 2003* 22 22 pheidole radoszkowskii mayr, 1884* 17 7 24 pheidole transversostriata mayr, 1887* 65 2 67 pheidole vorax (fabricius, 1804)* 1 1 pheidole cr16 22 22 pheidole cr44 2 2 pheidole inpa008 2 2 4 pheidole inpa019 6 6 pheidole inpa020 4 4 pheidole inpa025 1 1 pheidole inpa026 1 1 pheidole inpa037 5 5 pheidole inpa045 3 2 5 pheidole inpa048 8 8 pheidole inpa049 2 2 pheidole inpa051 13 13 pheidole tji02 16 1 17 pheidole tji07 1 1 pheidole tji09 10 1 11 pheidole tji10 2 2 4 pheidole tji17 6 6 pheidole tji21 22 22 pheidole tji22 8 8 pheidole tji23 2 2 pheidole tji24 3 3 pheidole tji26 3 3 pheidole tji28 5 5 pheidole tji29 6 6 pheidole tji30 4 4 pheidole tji31 4 4 pheidole tji33 3 3 pheidole tji40 1 1 pheidole tji41 1 1 sociobiology 63(3): 894-908 (september, 2016) 907 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. (continuation) subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total rogeria rogeria scobinata kugler, 1994 3 3 sericomyrmex sericomyrmex inpa001 14 14 solenopsis solenopsis prox. geminata (fabricius, 1804) 4 4 solenopsis cr1 2 44 46 solenopsis cr6 18 18 solenopsis cr7 6 6 solenopsis cr8 8 8 solenopsis cr9 6 6 solenopsis tji2 22 6 28 solenopsis tji4 1 1 strumigenys strumigenys alberti forel, 1893 1 1 strumigenys beebei (wheeler, 1915)* 2 2 strumigenys elongata roger, 1863 3 3 strumigenys fairchildi brown, 1961 1 1 strumigenys trinidadensis wheeler, 1922 3 3 strumigenys vilhenai bolton, 2000* 8 8 strumigenys inpa03 1 1 strumigenys tji8 1 1 trachymyrmex trachymyrmex inpa03 8 8 trachymyrmex inpa05 6 6 trachymyrmex inpa10 5 5 trachymyrmex tji1 33 1 34 trachymyrmex tji4 7 7 trachymyrmex sp. 3 3 wasmannia wasmannia auropunctata (roger, 1893) 22 9 31 wasmannia rochai forel, 1912* 2 2 wasmannia scrobifera kempf, 1961* 24 4 28 ponerinae anochetus anochetus diegensis forel, 1912* 1 1 anochetus horridus kempf, 1964* 1 1 dinoponera dinoponera quadriceps kempf, 1971* 4 4 hypoponera hypoponera tji1 1 1 hypoponera tji2 2 2 hypoponera tji3 2 2 leptogenys leptogenys inpa02 2 2 re vicente, lp prado, tj izzo – amazon rainforest ant-fauna of parque estadual do cristalino908 appendix. list of species recorded at the parque estadual do cristalino. numbers represent the total number of occurrences in strata. *first record in literature from mato grosso state. (continuation) subfamily/genus/species ground (pitfall trap) understory (beating-tray method) total mayaponera mayaponera constricta (mayr, 1884) 10 1 11 neoponera neoponera apicalis (latreille, 1802) 4 4 neoponera commutata (roger, 1860) 2 2 neoponera crenata (roger, 1861) 2 2 neoponera globularia (mackay & mackay, 2010)* 1 1 neoponera inversa (smith, 1858) 1 3 4 neoponera striatinodis (emery, 1890)* 2 2 neoponera unidentata (mayr, 1862) 4 4 neoponera verenae forel, 1922* 14 14 neoponera villosa (fabricius, 1804) 1 1 neoponera tji8 4 4 odontomachus odontomachus haematodus (linnaeus, 1758) 1 1 odontomachus meinerti forel, 1905 2 2 odontomachus tji1 3 3 pachycondyla pachycondyla crassinoda (latreille, 1802) 31 31 pachycondyla harpax (fabricius, 1804) 6 6 pseudomyrmecinae pseudomyrmex pseudomyrmex oculatus (smith, 1855) 2 2 pseudomyrmex tenuis (fabricius, 1804) 1 7 8 pseudomyrmex tenuissimus (emery, 1906) 2 2 pseudomyrmex unicolor (smith, 1855) 1 1 pseudomyrmex inpa001 1 1 pseudomyrmex tji3 3 3 pseudomyrmex tji8 1 1 total occurrences 1002 579 1581 doi: 10.13102/sociobiology.v65i3.1640sociobiology 65(3): 357-348 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sustainable management of acromyrmex octospinosus (reich): how botanical extracts should promote an ecofriendly control strategy introduction the synthetic pesticides used to manage insect pests cause damage to ecosystems, enhance resistance to insecticides in agricultural pests, adversely affect non-target organisms, cause environmental pollution, and have negative side effects on human health. these facts suggest a clear need for alternatives and have led to a renewed interest in biopesticides. botanical insecticides represent an ecofriendly alternative for pest management because of their biodegradability, which results from the choice of solvent such as water (boulogne et al 2012c), and their potential to reduce the evolution of resistance because plant extracts that contain a mixture of active phytochemicals should reduce the rate of evolution of resistance compared to the selective pressure exerted by single pure toxins (arnason et al 1993). abstract the leaf-cutting ant acromyrmex octospinosus (reich) causes serious damage to crops and protected areas due to its foraging activity. the main method of control of this species consists of the use of synthetic insecticides that can lead to environmental damage and negative side effects on human health. consequently, alternative strategies, such as biopesticides, are needed. insecticide evaluation by ingestion assays was performed using a. octospinosus in vitro bioassay and laboratory nests. chemical analyses were also performed to know the contents of plant extracts. this study showed that mammea americana l. is the most promising insecticidal plant extract in the control of a. octospinosus. indeed, the lethal concentrations (lc 50 and lc 99 ) an the lethal dose (ld 99 ) of the m. americana extract (51.31 mg.ml-1, 131.92 mg.ml-1, and 17.36 mg/g of ant respectively) were the closest to those of fipronil, 0.03 g/kg, the commercial insecticide used as positive control. sociobiology an international journal on social insects i boulogne1,2,3*, l desfontaines2, h ozier-lafontaine2, g loranger-merciris1,2 article history edited by gilberto m. m. santos uefs, brazil received 04 april 2017 initial acceptance 28 march 2018 final acceptance 07 august 2018 publication date 01 october 2018 keywords attini, mammea americana, chemical analysis, lethal concentrations, lethal doses, lethal times. corresponding author isabelle boulogne normandie université, unirouen laboratoire glyco-mev ea 4358 fédération de recherche normandie végétal fed 4277, f-76821 mont saint aignan, france email: isabelle.boulogne@univ-rouen.fr the leaf-cutting ants [genera atta and acromyrmex (hymenoptera: formicidae: attini)] in general and acromyrmex octospinosus (reich) in particular, cause serious damage on fields crops, pastures and plantations due to their foraging activities for its symbiotic fungus cultivation (pérez et al 2011). estimated damage was, for example, several million dollars per year in usa and brazil (cameron & riggs 1985). a. octospinosus is native to south and central america and exotic to guadeloupe. this species was introduced in guadeloupe in 1954 and progressively colonized the entire territory (boulogne et al 2014). tremendous losses have also been observed in agricultural and protected areas. indeed, a regional federation of defense against pests (fredon) survey carried out in 2008 indicated that vegetable and fruit crops account for 90.9 % of attacks. the 1995 cyclone favored ant invasion in natural areas where some plant species, such as the arborescent ferns 1 université des antilles et de la guyane, ufr sciences exactes et naturelles, pointe-à-pitre cedex (guadeloupe), france. 2 inra, ur1321, astro agrosystèmes tropicaux, 97170, petit-bourg (guadeloupe), france. 3 normandie université, unirouen, laboratoire glyco-mev ea 4358, fédération de recherche normandie végétal fed 4277, f-76821 mont saint aignan, france. research article ants sociobiology 65(3): 357-348 (september, 2018) 349 of the genus cyathea, are now threatened and might completely disappear due to their endemism (boulogne et al 2014). the united states department of agriculture (usda) classifies this ant among the most serious pests of tropical and subtropical america (pollard 1982). the main leaf-cutting ant control method is the application of granulated toxic baits, which are basically attractive citric matrices that contain a synthetic active ingredient that exerts a delayed action on workers (i.e., dechlorane, fipronil, sulfluramid gx071hb, or sulfluramid gx439) (nagamoto et al 2007). previous studies explored the activities of active ingredients (allelochemicals) from plants. these studies have shown that plant extracts cited by tramil ethnopharmacological surveys have insecticidal potential for the control of the leafcutting ant a. octospinosus. specifically, four plant extracts have ingestion toxicity [mammea americana l. (calophyllaceae), nicotiana tabacum l. (solanaceae), and two extracts of nerium oleander l. (apocynaceae)] (boulogne et al 2012a). some others have fungicidal potential to control the symbiotic fungus leucoagaricus gongylophorus (singer) möller (agaricales, basidiomycota), particularly a foliage extract of senna alata (l.) roxb. (fabaceae) (boulogne et al 2012b). an exhaustive literature search was conducted to identify the published papers related to insecticidal and fungicidal chemical compounds that stem from plant species. this meta-analysis revealed that alkaloids, phenolics, and terpenoids are the three main chemical classes that are most often cited for insecticidal and fungicidal activities (boulogne et al 2012c). to date, very few studies have used artificial nest bioassays (hebling et al 1996, 2000) or described the lethal doses, concentrations and times of botanical pesticides that are used for the control of leaf-cutting ants. therefore, the aims of this study were i) to determine the lethal doses, concentrations and times of four preselected insecticidal plant extracts, ii) to manage a preliminary artificial nest bioassay, and iii) to characterize their biochemical contents in terms of alkaloids, phenolics, and terpenoids, in order to determine the most promising insecticidal and fungicidal plant extracts for use in controlling entire colonies. material and methods rearing conditions two adult a. octospinosus nests (all casts, queen and symbiotic fungi) were collected from the field in different locations in guadeloupe (french west indies) and were bred in the laboratory by housing each colony in an artificial nest. the colonies were maintained for one month in the lab after collection before the initiation of the experiments and were supplied with flowers, leaves, sugarcane, corn flakes, and water daily. the workers used in the ingestion assays were collected from these laboratory nests. some worker vouchers were deposited in the astro lab at the national institute of agronomic research (inra) of the french west indies and guiana. extracts: source and preparation the plants used for extract preparation were collected in different locations in guadeloupe fwi (16°22’44.36”n -61°29’14.60”w, 16°12’25.01”n-61°29’49.42”w, 16°16’48.41”n-61°30’13.30”w, 16°11’20.65”n 61°35’39.54w, 15°59’53.35”n-61°43’33.71”w), identified by voucher number (boulogne,gd,1,uag/inra; boulogne,gd,2,uag/inra; boulogne,gd,3,uag/inra), deposited at the herbarium of the santo domingo botanical garden and identified by a botanist of this herbarium, mr. brigido peguero. the m. americana seed maceration, the two extracts with leaves of n. oleander and the decoction of dried leaves of n. tabacum were prepared as previously described in boulogne et al (2012a). all the aqueous plant extracts obtained and fresh leaves of s. alata were freeze dried, ground with a coffee mill, and sieved at 0.5 mm. the residues represented 99, 139, 56, 216 and 295 grams per kilogram of fresh plants of m. americana, n. oleander (crushed), n. oleander (decoction), n. tabacum, and s. alata, respectively. statistical analyses lethal concentrations (lc50 and lc99), lethal dose values (ld50 and ld99) and lethal times (lt50 and lt99) (concentrations, dose and time that killed 50% and 99% of ants) and their 95% confidence intervals were calculated with logistic regression by probit analysis. for in vitro insecticidal and preliminary artificial nests bioassays (records three times daily during 28 days for each nest), non-parametric analyses were performed with the friedman test with multiple comparison method of nemenyi. for chemical analysis, non-parametric analysis was performed with the kruskal-wallis test, multiple comparisons were performed with the dunn method, and bonferroni corrections. all these tests were made with xlstat® software. ingestion bioassay regarding control methods used against leaf-cutting ants, our objective was to find a toxic and attractive extract that exerted a delayed action on the workers. with this objective, ingestion bioassays were performed with groups of ten ant workers, which belong to the same colony (the bioassays were then repeated once again with an other colony). the ants were placed in 30 jam bottles (volume 324 ml, diameter 82 mm). six bottles were used for each of the concentrations of the lyophilized plant extracts (i.e., 1, 5 and 10 mg.ml-1), 6 bottles were negative controls, and 6 bottles containing blitz® commercial insecticide (granular bait, fipronil 0.03 g/ kg, 06/08/2001, 9800377) were used as positive controls. the ants were fed daily over a period of 21 days with an autoclaved artificial diet placed in plastic caps. this artificial diet was composed of glucose (50 mg.ml-1), peptone (10 mg.ml-1), i boulogne et al. sustainable management of acromyrmex octospinosus (reich)350 yeast extract (1 mg.ml-1), agar (15 mg.ml-1) (bigi et al 2004), and the freeze-dried plant extracts, distilled water (negative control), or fipronil (0.03 g/kg) q.s. to 1 l. the experiments were performed at 25 °c and 70-80% relative humidity on a 12:12 h light:dark photoperiod. each day, the number of dead ants in each jam bottle was recorded, the caps were removed and weighed, and new caps with fresh artificial diet were offered. the amounts of food eaten daily in g were corrected for the numbers of live ants. mortality was analyzed on the based on the percentages of dead ants corrected by means of abbott formula. in all insecticidal bioassays, the concentrations used for concentration response estimates were 1, 5 and 10 mg.ml-1. the delayed action of an extract was defined according to camargo et al (2006) as follows: a weak action occurred when 50% of the ants died before 48 h, a medium action when 50% of ants died between 48 h and 72 h, and a high action when 50% of ants died after 72 h. results the most toxic lyophilized extract was that of the n. tabacum dried leaf decoction with an lc50 = 1.33 mg.ml -1 and an lc99 = 3.93 mg.ml -1 after 24 h. the lethal concentrations (lc50 and lc99) of the m. americana extract (51.31 mg.ml -1 and 131.92 mg.ml-1, respectively) were the closest to those of fipronil, 0.03 g/kg (78.85 mg.ml-1 and 196.95 mg.ml-1, respectively, table 1). the ld50 of the n. tabacum extract (0.87 mg/g of ant) was the closest to that of fipronil, 0.03 g/kg (1.48 mg/g of ant), whereas the ld99 of the m. americana extract (17.36 mg/g of ant) was the closest to that of fipronil, 0.03 g/kg (5.05 mg/g of ant, table 1). the lt50 of the n. oleander extracts at 10 mg.ml -1 (47.2 and 69.98 hours) were the closest to those of fipronil, 0.03 g/ kg at 5 and 10 mg.ml-1(52.78 and 62.8 hours), whereas the lt50 of the m. americana extract at 5 and 10 mg.ml-1 (102.21 and 122.95 hours) were the closest to that of fipronil, 0.03 g/kg at 1 mg.ml-1 (111.94 hours) (table 2). the lt99 of the m. americana extract at 10 mg.ml-1 (311.96 hours) were the closest to that of fipronil, 0.03 g/kg at 1 mg.ml-1 (220.31 hours) (table 2). moreover, linear regressions (with r2 and equations) between mortalities of the ants and concentrations of the plant extracts and the positive control at 24h, 48h and 72h, (figure 1) indicate a high delayed action for the m. americana extracts, a weak delayed action for most of the n. oleander extracts, a weak delayed action for n. tabacum and a “medium to high” delayed action for fipronil, 0.03 g/kg (positive control). the quantities of artificial food eaten each day by the ants during the insecticidal bioassays with all of the extracts and the positive control (fipronil 0.03 g/kg, blitz©) at 10 mg.ml-1 and most of extracts at 5 mg.ml-1 did not differ from the quantities consumed in the negative control condition (without extracts) with values between 0.2 and 0.3 g. the extracts at 1 mg.ml-1 were more attractive than the control with values between 0.4 and 0.45 g (figure 2). data on table 3 demontrate that the toxicity/appetency and delayed action characteristics of the m. americana seed extract were the most similar to those of fipronil, 0.03 g/kg. this extract was thus chosen for the preliminary artificial nest bioassay. although the extract of n. tabacum seemed to have interesting characteristics, its lack of delayed action and some toxic properties on mammals do not lead us to choose it. indeed, its well known alkaloid (nicotine) is known for its high level of toxicity in mammals in miming the activity of acetylcholine and binding to post-synaptic receptors to cause stimulation and subsequent depression of the central nervous system, autonomic nervous system, and muscular nerve endings (philogène et al 2008). n lc50 (mg.ml -1) 95% confidence intervals lc99 (mg.ml -1) 95% confidence intervals chi2 lr (p value) mammea americana 60 5.13 1.07 – 7.09 13.19 12.97 – 17.83 1.32 (<0.0001) nerium oleander (crushed) 60 1.72 1.23 – 3.59 4.43 2.95 – 10.29 1.26 (<0.0001) nerium oleander (decoction) 60 2.78 1.62 – 9.49 6.58 3.57 – 25.19 0.96 (<0.0001) nicotiana tabacum 60 1.33 0.99 – 2.26 3.93 2.75 – 7.549 1.45 (<0.0001) fipronil, 0.03 g/kg 60 7.88 4.74 – 10.12 19.69 10.81 – 24.86 1.38 (<0.0001) n ld50 (mg/g) 95% confidence intervals ld99 (mg/g) 95% confidence intervals chi2 lr (p value) mammea americana 60 4.03 3.24 – 5.54 17.36 15.24 – 20.49 1.32 (<0.0001) nerium oleander (crushed) 60 3.66 1.32 – 5.54 40.2 34.82 – 48.15 1.26 (<0.0001) nerium oleander (decoction) 60 8.47 7.41 – 9.52 30.8 27.58 – 35.2 0.96 (<0.0001) nicotiana tabacum 60 0.87 0.77 – 2.47 25.19 20.98 – 32.24 1.45 (<0.0001) fipronil, 0.03 g/kg 60 1.48 1.14 – 1.82 5.05 4.12 – 6.88 1.38 (<0.0001) table 1lethal concentrations (lc50 and lc99) and lethal doses (ld50 and ld99) after 24 h with number of ants exposed, the 95% confidence intervals, likehood ratio chi2, associated p values and degrees of freedom in the laboratory insecticidal bioassays of the mammea americana extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, decoction of nicotiana tabacum dried leaves and the positive control (blitz©). sociobiology 65(3): 357-348 (september, 2018) 351 n lt50 (hours) 95% confidence intervals chi2 lr (p value) mammea americana 1mg.ml-1 60 396.58 370.92 427.85 1.74 (<0.0001) mammea americana 5mg.ml-1 60 122.95 115.67 129.89 1.13 (< 0.0001) mammea americana 10mg.ml-1 60 102.21 87.52 115.28 0.58 (< 0.0001) nerium oleander (crushed) 1mg.ml-1 60 778.72 573.30 1580.29 0.10 (0.001) nerium oleander (crushed) 5mg.ml-1 60 82.39 36.99 116.88 1.26 (< 0.0001) nerium oleander (crushed) 10mg.ml-1 60 47.20 22.04 68.31 0.36 (< 0.0001) nerium oleander (decoction) 1mg.ml-1 60 938.23 770.46 1266.19 0.35 (< 0.0001) nerium oleander (decoction) 5mg.ml-1 60 261.57 233.78 289.15 0.10 (< 0.0001) nerium oleander (decoction) 10mg.ml-1 60 69.98 47.43 89.20 0.38 (< 0.0001) nicotiana tabacum 1mg.ml-1 60 172.57 124.81 209.42 0.66 (< 0.0001) nicotiana tabacum 5mg.ml-1 60 30.40 26.96 72.18 0.11 (< 0.0001) nicotiana tabacum 10mg.ml-1 60 33.88 27.94 66.29 0.15 (< 0.0001) fipronil, 0.03 g/kg 1mg.ml-1 60 111.94 106.97 116.91 1.43 (< 0.0001) fipronil, 0.03 g/kg 5mg.ml-1 60 62.80 59.34 66.24 1.04 (< 0.0001) fipronil, 0.03 g/kg 10mg.ml-1 60 52.78 49.79 55.76 0.93 (< 0.0001) n lt99 (hours) 95% confidence intervals chi2 lr (p value) mammea americana 1mg.ml-1 60 1283.84 1150.01 1464.90 0.17 (<0.0001) mammea americana 5mg.ml-1 60 479.16 451.70 512.00 0.58 (< 0.0001) mammea americana 10mg.ml-1 60 311.96 296.46 330.28 1.13 (< 0.0001) nerium oleander (crushed) 1mg.ml-1 60 4513.30 2908.31 11067.41 0.10 (0.001) nerium oleander (crushed) 5mg.ml-1 60 1124.38 990.54 1315.08 0.12 (< 0.0001) nerium oleander (crushed) 10mg.ml-1 60 557.48 519.13 604.91 0.36 (< 0.0001) nerium oleander (decoction) 1mg.ml-1 60 2704.99 2102.63 3899.17 0.35 (< 0.0001) nerium oleander (decoction) 5mg.ml-1 60 1423.15 1236.86 1695.78 0.11 (< 0.0001) nerium oleander (decoction) 10mg.ml-1 60 576.69 538.19 624.03 0.38 (< 0.0001) nicotiana tabacum 1mg.ml-1 60 1660.83 1386.44 2109.04 0.66 (< 0.0001) nicotiana tabacum 5mg.ml-1 60 1041.08 907.23 1237.89 0.10 (< 0.0001) nicotiana tabacum 10mg.ml-1 60 657.88 589.80 751.42 0.15 (< 0.0001) fipronil, 0.03 g/kg 1mg.ml-1 60 220.31 209.28 233.57 1.43 (< 0.0001) fipronil, 0.03 g/kg 5mg.ml-1 60 115.30 107.99 124.83 1.04 (< 0.0001) fipronil, 0.03 g/kg 10mg.ml-1 60 91.70 85.72 99.68 0.93 (< 0.0001) table 2lethal times (lt50 and lt99) with number of ants exposed, the 95% confidence intervals, likehood ratio chi2, associated p values and degrees of freedom in the laboratory insecticidal bioassays of the mammea americana extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, decoction of nicotiana tabacum dried leaves and the positive control (blitz©). lc50 and lc99 ld50 ld99 appetency delayed action mammea americana + ++ +++ ++ +++ nerium oleander (crushed) +++ ++ + ++ ++ nerium oleander (decoction) ++ + ++ + ++ nicotiana tabacum +++ +++ ++ ++ + fipronil, 0.03 g/kg + +++ +++ ++ ++ table 3 summary table showing lethal concentrations, lethal doses, appetencies and delayed actions of mammea americana extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, decoction of nicotiana tabacum dried leaves and the positive control (blitz©). the lethal concentrations (lc50) are indicated as follows: +, lc50 > 50 mg.ml-1; ++, lc50 between 50 mg.ml-1 and 25 mg.ml-1; and +++, lc50 < 25 mg.ml-1. the lethal concentration (lc99) are indicated as follows: +, lc99 > 100 mg.ml-1; ++, lc99 between 100 mg.ml-1 and 50 mg.ml-1; and +++, cl99 < 50 mg.ml-1. the lethal doses (ld50) are indicated as follows: +, ld50 > 5 mg/g; ++, ld50 between 5 mg/g and 2 mg/g; and +++, dl50 < 2 mg/g. the lethal doses (ld99) are indicated as follows: +, ld99 > 30 mg/g; ++, ld99 between 20 mg/g and 30 mg/g; and +++, ld99 < 20 mg/g. the appetencies are indicated as follows: +, daily consumed quantity < 0.20 g; and ++, daily consumed quantity > 0.20 g. the delayed actions are indicated as follows: +, 50% of the ants died before 48 h; ++, 50% of ants died between 48 h and 72 h; and +++, 50% of ants died after 72 h for the highest concentration (according to lt50 showed in table 2). i boulogne et al. sustainable management of acromyrmex octospinosus (reich)352 preliminary artificial nest bioassay to determine whether the effects of our selected extracts could be efficient against entire colonies, preliminary artificial nest bioassays were conducted. for this purpose, ten colonies were observed in field conditions in order to select the 5 most active extracts for use in the preliminary artificial nest bioassays. these colonies were placed in artificial nests and supplied with flowers, leaves, sugarcane, and corn flakes daily prior to the initiation of the bioassays. each artificial nest was composed of 3 plastic boxes (60 ´ 40 ´ 40 cm) that were linked together by short hoses. the central box was non-transparent and hence suitable for the placement of the colony. the other two boxes on either side were transparent to simulate the outdoor environment, and the daily rations were placed in these boxes. the nests were exposed to each of the following treatments prepared in artificial diet (one nest by treatment): m. americana extract at 10 mg.ml-1, s. alata extract at 2 mg.ml-1, m. americana extract combined with s. alata extract at 10 and 2 mg.ml-1, respectively, a positive control (fipronil, 0.03 g/kg), and a negative control (no extracts). the artificial diet was the same as that used in the laboratory ingestion bioassays with the addition of the lyophilized plant extracts, distilled water (negative control) or fipronil, 0.03 g/kg. dried citrus sinensis (l.) osbeck (rutaceae) peels and dioscorea alata l. (dioscoreaceae) leaves (ground with a coffee mill and sieved at 0.5 mm) were added to this artificial food at 10 g.l-1 because of the natural appetency and attractiveness of these components to leaf-cutting ants (verza et al 2006). this attractive mixture has previously been tested, and its lack of insecticidal and fungicidal activities has been verified according to established protocols of ingestion bioassay and antifungal test (boulogne et al 2012b). the diets were placed in plastic caps and offered to the ants daily. before and after the treatments, the nests were supplied daily with flowers and leaves for 7 days. during the treatments, the nests were supplied daily for 14 days exclusively with plastic caps containing the artificial diet with or without the active extracts. the foraging activities (number figure 1 linear regressions (with r2 and equations) between mortalities of the ants (%) and concentrations (mg.ml-1) in the insecticidal bioassays of the mammea americana seed extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, decoction of nicotiana tabacum dried leaves, and the positive control (blitz©) at 24h (a), 48h (b) and 72h (c). figure 2 quantities of artificial food eaten by the ants daily during the laboratory insecticidal bioassays of the mammea americana extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, decoction of nicotiana tabacum dried leaves, the positive control (fipronil, 0.03 g/kg) at 1, 5 and 10 mg.ml-1 and the negative control (no extracts). the bars represent the medians, and the error bars are the 25% and 75% quartiles. the treatments without common letters differed significantly based on friedman test with multiple comparison method of nemenyi. the comparisons between specific concentrations are reported with small letters, and the comparisons between all treatments are reported with capital letters. sociobiology 65(3): 357-348 (september, 2018) 353 of ants incoming the nest with plant material or artificial diet during ten minutes) and quantities of food eaten were recorded three times daily at the same time for each nest before, during, and after the treatments (lopez & orduz 2003). only the nests treated with m. americana extract and fipronil, 0.03 g/kg exhibited significant reductions in foraging activity (by minute) after the treatments. similarly, only these nests exhibited significant reductions in the quantities of artificial food eaten daily in grams after the treatments. the quantities of artificial food eaten daily by these ants were significantly lower during the treatments than before or after the treatments with the exception of the nest treated with s. alata (figures 3-a and b). notably, 6 weeks after the treatment, all of the nests died out with the exception of the negative control nest (which lasted more than 2 months after this last observation). the nests treated with fipronil, 0.03 g/kg and the s. alata extract contained no surviving ants or fungus gardens. the nest treated with the m. americana extract exhibited no surviving ants, and its fungus garden was infested with fungal competitors. the nest treated with the m. americana extract combined with the s. alata extract contained no ants and no fungus garden and was infested with fungal competitors (figure 3-c). chemical analysis the extracts were submitted to phytochemical analyses for plant secondary metabolites that are known for their potential insecticidal activities, i.e., alkaloids, phenolic figure 3 foraging activity by minute (a) and quantities of artificial food eaten (in g) daily by ants (b) in artificial before, during and after treatments (records three times daily during 28 days) of mammea americana extract (ma), senna alata extract (sa), m. americana extract combinated with s. alata extract, positive control (fipronil, 0.03 g/kg) and negative control (without extracts). treatments without common letters differ significantly based friedman test with multiple comparison method of nemenyi. the bars represent the medians, and the error bars are the 25% and 75% quartiles. (c) acromyrmex octospinosus artificial nests 6 weeks after treatments with mammea americana extract (ma), senna alata extract (sa), mammea americana extract combinated with senna alata extract (ma+sa), blitz© (positive control) and control (without extracts). i boulogne et al. sustainable management of acromyrmex octospinosus (reich)354 compounds, and terpenoids. the quantity of each compound was determined using a spectrophotometer according to the following quantitative methods: alkaloids were determined using marquis’s reagent with a colorimetric method (szabo et al 2003), total phenolic compounds were determined using the folin-ciocalteau colorimetric method (heilerová et al 2003), and terpenoids were determined using the iron (iii) chloride-o-phosphoric acid-sulfuric acid colorimetric method (zak et al 1956). all values are expressed in micrograms of standard per g of freeze-dried fresh plant sample and three replications were performed. the n. oleander extracts contained the greatest quantities of total alkaloids, the n. tabacum and n. oleander decoctions contained the greatest quantities of total phenolic compounds and the n. oleander extracts contained the most total terpenoids. terpenoids were the most abundant compounds in all of the extracts (between 550 and 1400 mg of standard/g of freeze-dried plant sample) ( figure 4). m. americana we showed that the m. americana seed lyophilized extract induced insecticidal toxicity following ingestion. there are few data available regarding the toxicities of m. americana, on attini (boulogne et al 2012a), but seeds of this plant showed insecticidal effects in other studies. indeed, m. americana seeds exhibited activity against cerotoma ruficornis (oliver) (coleoptera: chrysomelidae) (dev & koul 1997) and aedes aegypti (l.) (diptera: culicidae) (sievers et al 1949). these seeds are also larvicidal against laphygma frugiperda (smith and abbot) (lepidoptera: noctuidae) and plutella maculipennis (curt.) (lepidoptera: acrolepiidae) (plank 1944). our preliminary chemical analysis revealed that the m. americana seed extract contained alkaloids, phenolics and terpenoids. analysis of the literature revealed that the compounds responsible for the insecticidal activities of m. americana are well-known phenolic compounds such as coumarins and particularly mammein (duke 1989). methanol and ethyl acetate extractions and hplc or lc-ms analyses have revealed 4.8 mg of coumarins per gram of seed (yang et al 2006). the m. americana seed extract exhibited an lc50 of 51.31 mg.ml-1 and an ld50 of 706.65 mg/g of insect. to compare our results with existing data we found a study of the toxicities of aqueous and ethanolic extracts of m. americana on artemia salina l. (anostraca: artemiidae). this study revealed an lc50 greater than 10 mg.ml-1 for an aqueous extract and an lc50 of 197 μg/ml for the ethanolic extract (bussmann et al 2011). another study of coumarins in m. americana revealed an ld50 of 4 μg/insect against phaedon cochleariae fab. (coleoptera; chrysomelidae) (perez et al 2010). these results might suggest that ethanolic extracts and bioguided fractionations of m. americana seed extracts might increase the toxicity (coumarins are less miscible in water than in ethanol). s. alata the nests treated with the s. alata extract contained no fungus garden after 6 weeks of treatment. this effect was consistent with our previous study, which showed that the s. alata foliar extract was fungicidal against l. gongylophorus (boulogne et al 2012b). we also reported a preliminary chemical analysis, which revealed that s. alata foliage contains alkaloids, phenolic compounds and terpenoids. previous studies exhibited fungicidal activity against several other fungi and showed that the fungicidal activity of s. alata figure 4 quantitation of the alkaloid (a), phenolic compound (b), and terpenoid (c) contents (milligrams of standard per gram of freeze-dried plant material) of the mammea americana extract, crushed extract of nerium oleander dried leaves, decoction of n. oleander fresh leaves, and decoction of nicotiana tabacum dried leaves. the quantities without common letters differed significantly based on kruskal-wallis tests with dunn's multiple comparison tests and bonferroni corrections (n=6 and df=3). the bars represent the medians, and the error bars are the 25% and 75% quartiles. sociobiology 65(3): 357-348 (september, 2018) 355 foliar extracts can be attributed to phenolic compounds such as anthraquinones (somchit et al 2003) like chrysophanol (palanichamy & nagarajan 1990). artificial nests bioassay we were aware that our artificial nest bioassay was really preliminary and all results of differences among extracts should be treated as preliminary and with caution. indeed, the experimental design of the test had no replicates to the treatments and it could bias the results (differences might be attributed to differences in nests themselves and not to treatments). however, this preliminary assay is crucial and important to this study line since they show that these extracts are attractive to ants and consumed by them. concerning the length of treatment, similar bioassays revealed that atta sexdens rubropilosa nests that are supplied daily with a diet containing ricinus communis leaves exhibit gradual decreases in fungus gardens and substantial worker mortality after 6 weeks of treatment (hebling et al 1996). another study of atta sexdens nests that were fed daily with a diet containing canavalia ensiformis l. (leguminosae) leaves reported complete nest extinction after 11 weeks of treatments (hebling et al 2000). ant baits in field conditions regarding delayed action, the seed of m. americana seemed to naturally possess this property. it would be interesting to increase this delayed action of the substance with a digestible polymer using microencapsulation techniques (benita 2005). microcapsules could be made to contain a freeze-dried ethanolic or aqueous extracts of m. americana seeds and placed in a mix of citrus sp. pulp and dried dioscorea alata leaves to increase the appetency based on our laboratory results and existing data (verza et al 2006). these granular baits (microcapsules and attractant mixtures compressed in granular form easy to apply) might be protected in biodegradable and compostable plastics to preserve them against sunlight and adverse weather conditions. thus, like others well known commercial formulation containing botanical extracts (e.g. pyrethrins, rotenone, sabadilla, ryania, nicotine, azadirachtins or limonene) (weinzierl 2000), a field study should be conducted with these kinds of baits to improve our work in field conditions. however compared to these previous studies, the force of our argument is to use mixtures of active phytochemicals to reduce conventional resistance compared with the selection pressure exerted by single pure molecule (arnason et al 1993) and make the choice of extraction type and solvent with the greatest sustainability (water or eventually ethanol extractions) (boulogne et al 2012c). as the example of m. americana extract, toxicity may be increased and transformed in granular baits (microcapsules and attractant mixture) protected in a plastic. as the same way, to improve our fungicidal results (in vitro and with artificial nest) in field conditions, s. alata foliar extracts may also be transformed in granular baits. our results might be useful in the control of a. octospinosus and might be applicable to the control of other leaf-cutting ant species. however, we need to keep in mind that, in guadeloupe (fwi), this species is exotic. this ant might behave different in its exotic range than in its native range (e.g. the case of linepithema humile (human & gordon 1996), and the results found might only apply for non-native populations. thus, these results should be treated with caution before their generalization. our study is the first report of the toxicities of m. americana seed, n. oleander leaf, and n. tabacum leaf freezedried extracts due to ingestion by attini. it allowed us to determine the lethal doses, concentrations and times of four insecticidal plant extracts that were selected for examination based on previous studies. this study revealed that the m. americana seed extract was the most similar in terms of toxicity/appetency and delayed action to the commercial bait fipronil, 0.03 g/kg. our analyses also revealed that these extracts contained alkaloids, phenolics, and terpenoids. the preliminary artificial nest bioassays showed that the most promising insecticidal (m. americana) and fungicidal (s. alata) plant extracts might be useful in the control of a. octospinosus in guadeloupe and where the species is exotic. further studies should be conducted to optimize the toxicities of these extracts against a. octospinosus, to verify that they lack toxicity against non-targeted organisms and to confirm their activities in entire nests in natural conditions. acknowledgements the authors thank cécilia delag, fred burner, andève mulciba, guy gougougnan, michèle salles, and hervé mauléon for their technical assistance. references arnason, j.t., mackinnon, s., durst, a., philogene, b.j.r., hasbun, c., sanchez, p., poveda, l., san roman, l., isman, m.b., satasook, c., towers, g.h.n., wiriyachitra, p., maclaughlin, j.l. 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(2006). comparative analyses of bioactive mammea coumarins from seven parts of mammea americana by hplc-pda with lc-ms. journal of agricultural and food chemistry, 54: 4114-4120. doi: 10.1021/jf0532462 zak, b., luz, d., fisher, m. (1956). determination of serum cholesterol. the american journal of medical technology, 23: 283-287. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1838sociobiology 65(1): 59-66 (march, 2018) special issue changes in the contribution of termites to mass loss of dead wood among three tree species during 23 months in a lowland tropical rainforest introduction dead wood has a critical impact on the carbon and nutrient cycles in terrestrial ecosystems with slow decomposition rates, owing to its large size, limited nitrogen content, decay-resistant structure, and recalcitrant compounds (cornwell et al., 2009; kim et al., 2015). dead wood is mostly decomposed by fungi and invertebrates, and invertebrates contribute to approximately 10-20% loss of dead wood in terrestrial ecosystems (ulyshen, 2016). especially in tropical rainforests, termites are abundant and diverse (eggleton, 2000), and the activities of termites control the turnover of dead wood (bradford et al., 2014). although termites decompose large amounts of dead wood, they can feed on only the accessible and digestible abstract this study investigated the contribution of termites to mass loss of dead wood (macaranga bancana, elateriospermum tapos, and dillenia beccariana) in a lowland tropical rainforest, brunei darussalam. mesh bag method was used to exclude termites, and the mass remaining was monitored after 3, 7, 13, and 23 months. c/n ratio of the samples was analyzed after 13 and 23 months. initial wood density was 0.63, 0.92, and 1.02 g/cm3 for m. bancana, e. tapos, and d. beccariana, respectively, and the termite contribution to mass loss (%) was an average (range) of 13.05±5.68 (4.17-29.59), 3.48±1.13 (2.20-6.49), and 3.40±1.92 (0.74-10.78), respectively. until 7 months, termites contributed highly to mass loss, given the low initial wood density, and interaction effect of species and treatment was significant. after 7 months, the contribution decreased in m. bancana and e. tapos, whereas it increased consistently in d. beccariana. the interaction effect was not significant, whereas differences in c/n ratio among the species were significant, with a lower c/n ratio in m. bancana and e. tapos than in d. beccariana. after 23 months, the differences in c/n ratio were not significant, and ants were present at 40% of control samples in m. bancana and e. tapos. our results suggest that the contribution of termites to mass loss varies by dead wood species and is temporally variable. initial wood traits could affect the termite feeding in the beginning, however, termites thereafter could forage in response to the varying c/n ratio among species and predators. sociobiology an international journal on social insects y roh1, s lee1, g li1, s kim1, j lee1, sh han1, h chang1, ka salim2, y son1 article history edited by og desouza, ufv, brazil received 01 july 2017 initial acceptance 14 august 2017 final acceptance 02 october 2017 publication date 30 march 2017 keywords dillenia beccariana, elateriospermum tapos, invertebrate, macaranga bancana, mesh bag method. corresponding author yowhan son department of environmental science and ecological engineering graduate school, korea university seoul 02841, republic of korea. e-mail: yson@korea.ac.kr lingo-cellulose part of dead wood, which vary across species (bultman & southwell, 1976). when termites feed on dead wood, the mass loss of dead wood is accelerated (stoklosa et al., 2016). termites prefer certain tropical tree species, thereby, accelerating more rapid mass loss in these species (gentry & whitford, 1982). however, termite preference on major tree species of lowland tropical rainforests in southeast asia has rarely been investigated. the contribution of termites to the mass loss of dead wood could vary over time, because of changes in termite activities in response to wood traits and predators. in the beginning, dead wood has certain specific traits. as termites mechanically attack dead wood with their mandibles, their feeding could be affected by dead wood traits such as density, which disturbs action of mandible (gentry & whitford, 1982). 1 department of environmental science and ecological engineering, graduate school, korea university, seoul, republic of korea 2 environmental and life sciences, faculty of science, universiti brunei darussalam, bandar seri begawan, brunei darussalam research article termites y roh et al. – effect of termites on dead wood decomposition60 also, as termites prefer the relatively labile and nutritious parts of woods (e.g. nitrogen-rich cambium, or the softest rings of spring wood), they can selectively forage for new dead wood after consuming the parts (shellman-reeve, 1994; stoklosa et al., 2016; traniello & leuthod, 2000; ulyshen et al., 2014). finally, as dead wood loses its specific traits during the decomposition, the termite activity could be suppressed because of the invasion of non-tree specific termite predators on dead wood. besides, tunnels made on dead wood by termite mechanical attack also facilitate the invasion (cornwell et al., 2009). nevertheless, quantification on the changes in the termite contribution to dead wood decomposition has rarely been identified, and the contribution was considered as constant over time (ulyshen et al., 2016). the aim of this study was to investigate the changes in the contribution of termites to mass loss among major tropical tree species over time in a lowland tropical rainforest of southeast asia. the following hypotheses were examined: (i) termites would accelerate the mass loss of dead wood, (ii) termite contribution to the mass loss of dead wood would differ by species, and (iii) the contribution would change over time. materials and methods the study site was located in a lowland tropical rainforest, at the kuala belalong field studies centre, brunei darussalam (04°63′50.3″n, 115°22′79.1″e). the forest is classified as an old-growth mixed dipterocarp forest, with a mean annual temperate of 26.5 °c, and a mean annual precipitation of approximately 5203 mm, without a distinct dry season. the topology consists of ridges with steep slope on ultisol soils (anderson-teixeira et al., 2015; ashton & hall, 1992; small et al., 2004). three species were selected for the study: macaranga bancana (euphorbiaceae), elateriospermum tapos (euphorbiaceae), and dillenia beccariana (dilleniaceae). they are common in the lowland tropical rainforest at kuala belalong, and have distinct traits. m. bancana is a myrmecophytic tree species, which has an obligate mutualism with ants via development of a hollow stem (domatia) and food body (heil et al., 2004). e. tapos is a non-myrmecophytic tree species; however, it has a facultative mutualism with various invertebrates. this species not only has extrafloral nectaries to attract invertebrates but also produces soft resin to defend the stem from the invertebrates. the pith of this species is soft (fiala & maschwitz, 1992). d. beccariana is a non-myrmecophytic tree species without mutualism, and has a solid stem. trees with similar diameter were selected and logged, and the logs were cut into 10-cm pieces. the mean diameter (± sd) of the samples was 6.96 (0.77), 6.88 (0.40), and 6.98 (0.59) cm for m. bancana, e. tapos, and d. beccariana, respectively, and the mean initial air-dried density (± sd) of the samples was 0.63 (0.05), 0.92 (0.05), and 1.02 (0.07) g/ cm3, respectively. the mesh bag method was used to physically block the termite access. a nylon mesh with 1.4 mm openings was used, and each sample of dead wood was tightly wrapped with the mesh twice (fig 1a). control (without mesh) samples were also prepared to measure the mass remaining of dead wood without blocking the termite access (fig 1b). a 20 m ⨯ 20 m plot was established on a slope. the plot was subdivided into three 4 m ⨯ 13 m subplots. the samples were laid on the soil at the distance of 1 m, along three transects (figs 1c & 1d). in jan., 2015, a total of 108 samples (3 subplots ⨯ 3 species ⨯ 2 treatments (mesh bag & control) ⨯ 6 samples) were laid in the site. the samples were retrieved after 3 (108 samples), 7 (101 samples), 13 (97 fig 1. photographs of dillenia beccariana installed as (a) a mesh bag sample, and (b) a control sample in jul., 2016 (after 18 months), and (c) a picture of the study site, and (d) a diagram of an experimental design of each subplot in jan., 2015. species: m = m. bancana; e = e. tapos; d = d. beccariana. treatment: white box = control; black box = mesh bag. sociobiology 65(1): 59-66 (march, 2018) special issue 61 samples) and 23 (43 samples) months from the beginning of the experiment. after the mesh was removed, the retrieved samples were air-dried for 48 h (figs 2a & 2b), and the sprouts were removed using a scissors (fig 2c). termiteimported soils were also found in samples with termites as ulyshen & wagner (2013) stated (fig 2d). therefore, the soils were removed from the samples using forceps after air-drying, and then the surface of the dead wood samples was cleaned carefully using small brushes (fig 2e). the mass remaining [%; (air-dried weight ⨯ 100)/initial air-dried weight] was determined. the difference in mass remaining between the treatments (control & mesh bag) was regarded as the contribution of termites to the mass loss (%). because of limited electricity and time, and to keep the integrity of samples, an air-drying method was conducted, which might cause an error on the wood weight. however, the study site has relatively constant temperature and precipitation (andersonteixeira et al., 2015), and all of the samples were air-dried at the same time (collins, 1981). thereby, we expected that the results of the termite contribution to mass loss of dead wood might not be distorted. when other invertebrates were found on samples, the time of occurrence was recorded. the meshes were checked at the time of measurements. subsequently, the samples were laid again in the study site. sampling was conducted after 13 months (27 samples) and 23 months (20 samples) for carbon (c) and nitrogen (n) analyses. the collected samples were ground, and ovendried at 103 °c. to determine the c/n ratio, an elemental analyzer (vario macro chn, elementar analysensystem gmbh, germany) was used. to assess the effects of species, treatment, and their interaction, the mass remaining was analyzed using a two-way analysis of variance (anova) at the time of measurements. to assess the differences in mass remaining between treatments, and the differences in c/n ratio of control among the species, a one-way anova was conducted. all statistical tests were carried out using sas 9.4 software (sas system, cary, usa). results during the study period, termites were found in a few of mesh bag samples, because the use of 1.4 mm opening was not enough to block all of the termite access. to deal with this issue, all of the mesh bag samples were checked before measuring airdried weight, and then the mesh bags having termites or traces of termites were excluded from the data analyses. throughout the study period, species had a significant effect on the mass remaining (table 1). when termites were not excluded, the mass loss of dead wood increased by 4.1729.59% in m. bancana, 2.20-6.49% in e. tapos, and 0.7410.78% in d. beccariana (figs 3a, 3b & 3c). the difference in mass remaining between treatments was highest after 7 months in m. bancana and e. tapos, whereas it was highest after 23 months in d. beccariana (fig 3d). resprouting occurred only in d. beccariana after 7 months, and the sprouts were not found afterward (fig 2c). until 7 months, the interaction effect of species and treatment was significant (table 1). during this period, the contribution of termites to mass loss increased the highest in m. bancana, followed by e. tapos, and then d. beccariana, i.e., fig 2. photographs of an air-dried (a) mesh bag samples and (b) control samples, and (c) a resprout in a control sample, and control samples (d) before removing termite-imported soils, and (e) after removing the soils. y roh et al. – effect of termites on dead wood decomposition62 in the order of species with lowest initial wood densities (fig 3d). after 7 months, the interaction effect was not significant anymore (table 1). the contribution of termites to mass loss began to decline in m. bancana and e. tapos, whereas it still increased in d. beccariana (fig 3d). especially between 13 and 23 months, the mass remaining of control samples did not decrease in m. bancana, whereas it showed a greater decrease than before in d. beccariana (figs 3a & 3c). during this period, the control samples of m. bancana and e. tapos had a significantly lower c/n ratio than those of d. beccariana (n = 9, f = 18.36, p < 0.05; fig 4). after 23 months, termite contribution still decreased in m. bancana and e. tapos, whereas it increased in d. beccariana, without table 1. two-way anova of the mass remaining on the effect of species, treatment, and their interaction after 3, 7, 13, and 23 months.the number of samples was 108, 108, 104, and 50 for 3, 7, 13, and 23 months, respectively. the values highlighted in bold indicate statistical significance (p < 0.05). effect df 3 months 7 months 13 months 23 months f p f p f p f p species (s) 2 51.78 <.0001 5.42 0.0059 6.57 0.0022 3.49 0.0409 treatment (t) 1 15.24 0.0002 13.3 0.0004 5.27 0.0240 1.32 0.2585 s ⨯ t 2 3.45 0.0359 5.88 0.0039 2.28 0.1082 0.33 0.7245 × = interaction effect fig 3. mean mass remaining for (a) m. bancana, (b) e. tapos, and (c) d. beccariana, and (d) the contribution of termites during the 23 months. contribution of termites = differences in mass remaining between treatments. vertical bars represent the standard error. asterisks indicate significant differences (*p < 0.1, ** p < 0.05, *** p < 0.01). a significant difference in c/n ratio among the species (n = 9, f = 0.83, p > 0.05; fig 4). simultaneously, ant colonies were found at 40% of the control samples of m. bancana and e. tapos. the coexistence of termites and ants on dead wood was not observed. discussion termites contributed to the mass loss of dead wood differently among the species. this result might be due to the differences in wood density. the wood density is a representative property, indicating resistance from fungi and pathogens. previous studies have found that termites sociobiology 65(1): 59-66 (march, 2018) special issue 63 preferentially feed on dead wood with low wood density (gentry & whitford, 1982; takamura et al., 2001). another possible explanation is the difference in chemical composition of stems among species. species having high resprouting potential, such as d. beccariana, is known to have several strategies to protect their stems against decomposers (just et al., 2017; poorter et al., 2010). especially, stems of d. beccariana might contain high lignin or secondary metabolites such as phenols, which possibly hamper termite feeding (freschet et al., 2012; guérard et al., 2007; just et al., 2017). the termites might feed more on d. beccariana after these metabolites were sufficiently degraded by fungi (ulyshen, 2016). there were changes in the contribution of termites to the mass loss of dead wood over time. until 7 months, the termite feeding might depend on the initial traits of dead wood, such as initial wood density and secondary metabolites. after 7 months, the termites might forage from m. bancana and e. tapos to d. beccariana, in response to the differences in c/n ratio among the species. mortality of termites, which feed on sound wood, increases with a decreasing c/n ratio of the substrate, owing to an imbalanced symbiotic system with gut microbes or the accumulation of ammonia in the guts (majeed et al., 2015). after 23 months, the decreasing trend of termite foraging might be caused by appearance of ants, which would have accelerated termite emigration. ants are known termite predators and non-tree specific invertebrates that nest on small dead wood (levings & franks, 1982). when ants are present on dead wood, the decomposition activities of termites and fungi decrease (warren & bradford, 2012). wang et al. (2003) also found ant colonies in the branches hollowed out by termites in a temperate forest. overall, termites accelerated the mass loss of dead wood, and the acceleration depended on the dead wood species. these results are in accordance with the findings of liu et al. (2015), who reported the role of termites in enhancing the effect of wood traits on decomposition. moreover, root foraging occurs more on dead wood hollowed by termites, thereby, accelerating nutrient cycling (lu et al., 2013). based on these previous findings, we expected that the effects of dead wood on c and nutrient cycles in forest ecosystems would be enhanced by termites, and the effects would be different depending on the dominant dead wood species. in this study site, termites contributed to 0.74-29.59% mass loss of dead wood within 23 months. gentry and whitford (1982) also reported a wide range of termite-driven mass loss in dead wood (3-20%) in savanna. these wide ranges in these results suggest that there is an uncertainty in quantifying the contribution of termites to the mass loss of dead wood in terrestrial ecosystems. three explanations are possible for this uncertainty. first, termite feeding is highly affected by dead wood species, which have developed various defense strategies against attacks from herbivores and pathogens, such as mutualism or secondary metabolites. these various traits differently affect the termites, thereby, reducing or enhancing termite feeding (verma et al., 2009). second, ants are also abundant in tropical rainforests (levings & franks, 1982). there is a possibility that the amount of termite-driven mass loss in dead wood is overestimated without considering the predator effects. it could lead to incorrect extrapolation if the measurement was conducted once or stopped in the middle of mass loss process. the last possibility might be the different accessibility of termite among the dead wood samples. the magnitude of wood decomposition could vary with the number and type of termite colonies near the dead wood, because these factors could control accessibility of termites and influence mass loss caused by termite feeding accordingly (ulyshen et al., 2016). this study demonstrated that termites contribute to mass loss of dead wood, thereby, affecting c and nutrient cycles in forest ecosystems. in addition, the contribution varies by not only species but changing factors such as wood traits and predators. therefore, the changes should be considered to estimate the termite contribution to dead wood decomposition precisely. fig 4. mean c/n ratio across the species after 13 months and 23 months. vertical bars indicate the standard error. asterisks indicate significant differences (* p < 0.05). c /n r at io y roh et al. – effect of termites on dead wood decomposition64 acknowledgment we thank the staffs at ubd and kbfsc for their support during the study. we also thank ubd and ctfs for allowing us to conduct this study at the site. this study was supported by research grants from korea forest service (s121315l130110) and national research foundation of korea (2015r1d1a1a01057124). author contribution yowhan son and kamariah abu salim conceived and designed the experiments; yujin roh, sohye lee, guanlin li, seongjun kim, seung hyun han, and jongyeol lee were responsible for the collection of data from the study site; yujin roh, guanlin li and hanna chang analyzed the data; all authors participated in the discussion of results; yujin roh wrote the manuscript. references anderson-teixeira, k.j., et al. 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(2003). abundance and distribution of subterranean termites in southern mississippi forests (isoptera: rhinotermitidae). sociobiology, 42: 533542. doi: 10.1.1.495.4859. warren, r. & bradford, m. (2012). ant colonization and coarse woody debris decomposition in temperate forests. insectes sociaux, 59: 215-221. doi: 10.1007/s00040-011-0208-4. http://dx.doi.org/10.1016/j.apsoil.2016.03.017 http://dx.doi.org/10.1016/j.apsoil.2016.03.017 y roh et al. – effect of termites on dead wood decomposition66 supplement table s1. mean mass remaining (± sd; %) of m. bancana, e. tapos, and d. beccariana during the 23 months. n = the number of samples. table s2. mean contribution of termites (± sd; %) for m. bancana, e. tapos, and d. beccariana during the 23 months. contribution of termites = differences in mass remaining between treatments. n = the number of samples. species treatment 3 months 7 months 13 months 23 months mean n mean n mean n mean n m. bancana control 102.47 (2.52) 18 77.76 (7.55) 18 56.86 (8.40) 16 57.01 (7.84) 5 mesh bag 110.88 (1.81) 16 107.35 (6.26) 16 79.94 (9.49) 15 61.18 (9.83) 5 e. tapos control 89.46 (1.20) 18 76.08 (2.38) 18 64.61 (1.94) 17 47.47 (4.50) 10 mesh bag 94.52 (1.26) 14 82.57 (1.59) 14 68.28 (3.34) 14 49.68 (8.93) 5 d. beccariana control 95.58 (0.27) 18 89.62 (0.64) 18 82.11 (1.87) 18 57.89 (3.40) 9 mesh bag 96.33 (0.33) 17 92.06 (0.96) 17 85.16 (1.90) 17 68.67 (4.63) 9 species 3 months 7 months 13 months 23 months mean n mean n mean n mean n m. bancana 8.51 (4.46) 3 29.84 (8.04) 3 23.27 (7.66) 3 6.39 (6.77) 3 e. tapos 5.11 (1.10) 3 6.29 (1.24) 3 4.14 (4.44) 3 3.92 (8.89) 3 d. beccariana 0.71 (0.41) 3 2.37 (0.97) 3 2.91 (3.91) 3 11.88 (6.04) 3 331 gaoligongidris planodorsa, a new genus and species of the ant subfamily myrmicinae from china with a key to the genera of stenammini of the world (hymenoptera: formicidae) by zheng-hui xu1 abstract a new genus and species of the ant subfamily myrmicinae collected from the gaoligong mountain nature reserve of the hengduan mountains, southwestern china, is described. the new genus, gaoligongidris gen. nov., is close to lasiomyrma terayama & yamane, but with anterior clypeal margin not angled, metanotal groove deeply impressed, propodeal spiracles large, propodeal spines long and slender, and petiolar peduncle longer than the node. the new genus is distributed in the oriental region and belongs to the tribe stenammini of myrmicinae. a key to the known genera of stenammini of the world based on worker and queen castes is provided. key words: hymenoptera, formicidae, myrmicinae, gaoligongidris, new genus, china. introduction after the publication of the identification guide to the ant genera of the world (bolton 1994), 14 new living genera of myrmicinae have been established in the world. of these, 12 genera have been recognized as valid ones, and 3 genera (lasiomyrma terayama & yamane, 2000; austromorium shattuck, 2009; propodilobus branstetter, 2009) belong to the tribe stenammini (bolton 2011). a new genus, gaoligongidris gen. nov., was discovered in the ant diversity investigation of gaoligong mountain nature reserve, southwestern china. the type species, g. planodorsa sp. nov., is the only known species of the genus. the new genus is distributed in the oriental region and belongs to the tribe stenammini of myrmicinae. currently, 20 genera of stenammini 1 key laboratory of forest disaster warning and control in yunnan province, college of forestry, southwest forestry university, kunming , yunnan province 650224, china, e-mail: xuzhenghui1962@163.com 332 sociobiolog y vol. 59, no. 2, 2012 are recognized in the world (bolton 1995, 2011). in order to understand the differentiation between the new genus and other ones, a key to the known genera of stenammini is provided. materials and methods the worker caste of the type species, g. planodorsa sp. nov., was collected by the sample-plot method. descriptions and measurements were made under a xtb-1 stereo microscope with a micrometer. illustrations were made under a motic-700z stereo microscope with illustrative equipment. standard measurements and indices are as defined in bolton (1987), in addition, ed is supplemented: tl-total length: the total outstretched length of the ant from the mandibular apex to the gastral apex. hl-head length: the length of the head proper, excluding the mandibles, measured in a straight line from the mid-point of the anterior clypeal margin to the mid-point of the occipital margin, in full-face view. in species where the occipital margin or the clypeal margin is concave, the measurement is taken from the mid-point of a transverse line spanning the anteriormost or posteriormost projecting points, respectively. hw-head width: the maximum width of the head in full face view, excluding the eyes. ci-cephalic index = hw×100 / hl. sl-scape length: the maximum straight line length of the antennal scape excluding the basal constriction or neck close to the condylar bulb. si-scape index = sl×100 / hw. ed-eye diameter: the maximum diameter of the eye. pw-pronotal width: the maximum width of the pronotum in dorsal view. al-alitrunk length: the diagonal length of the alitrunk in profile view from the point at which the pronotum meets the cervical shield to the posterior base of the metapleuron. all measurements are expressed in millimeters. the type specimens are deposited in the insect collection, southwest forestry university (swfu), kunming, yunnan province, china. 333 xu, z.-h. — a new genus and species of myrmicinae from china key to known genera of stenammini of the world based on worker and queen castes 1 antennae 9-segmented (malaysia) ........ rostromyrmex rosciszewski, 1994 antennae 11or 12-segmented .........................................................................2 2 antennae 11-segmented......................................................................................3 antennae 12-segmented .................................................................................. 10 3 antennal scrobes present ....................................................................................4 antennal scrobes absent ......................................................................................7 4 ventral surfaces of petiole and postpetiole with spongiform appendages (indo-australian) .................................. dacetinops brown & wilson, 1957 ventral surfaces of petiole and postpetiole without spongiform appendages .........................................................................................................................5 5 anterior margin of clypeus with a pair of fork-like teeth in the middle (afrotropical) ............................................................. dicroaspis emery, 1908 anterior margin of clypeus without a pair of fork-like teeth in the middle .................................................................................................................................6 6 counting from the apex, the third antennal segment long, about as long as the second one (afrotropical .................................. cyphoidris weber, 1952 counting from the apex, the third antennal segment short, about 1/2 length of the second one (neotropical) ................ lachnomyrmex wheeler, 1910 7 ventral surfaces of petiole and postpetiole with spongiform appendages (borneo) ................................................................ tetheamyrma bolton, 1991 ventral surfaces of petiole and postpetiole without spongiform appendages .........................................................................................................................8 8 metanotal groove absent. counting from the apex, the third antennal segment long, about as long as the second one (india) ....................................... ..................................................................................... indomyrma brown, 1986 metanotal groove present. counting from the apex, the third antennal segment short, distinctly shorter than the second one ................................9 9 anterior margin of clypeus nearly straight in the middle portion. metanotal groove widely deeply impressed. propodeum with a pair of long slender spines. propodeal spiracle large. petiolar peduncle longer than the node (oriental) (figs. 1-7) .................................................gaoligongidris gen. nov. anterior margin of clypeus bluntly angled. metanotal groove narrowly 334 sociobiolog y vol. 59, no. 2, 2012 notched. propodeum with a pair of short triangular teeth. propodeal spiracle small. petiolar peduncle not longer than the node (indo-australian) (figs. 8-12) ............................................... lasiomyrma terayama & yamane, 2000 10 petiole sessile (old world tropics and subtropics except africa) ............. ........................................................................................ vollenhovia mayr, 1865 petiole pedunculate ......................................................................................... 11 11 anterior margin of clypeus with a pair of fork-like teeth in the middle (old world tropics) ...................................... calyptomyrmex emery, 1887 anterior margin of clypeus without a pair of fork-like teeth in the middle ........................................................................................................................ 12 12 metanotal groove absent................................................................................ 13 metanotal groove present .............................................................................. 14 13 posterior clypeal extension narrower than frontal lobes. propodeal spiracle large, its diameter greater than or about as long as the distance from spiracle to the declivity margin (u.s.a., neotropical, indo-australian) ................. ............................................................................................. rogeria emery, 1894 posterior clypeal extension broader than frontal lobes. propodeal spiracle small, its diameter about 1/3 of the distance from spiracle to the declivity margin (australia) .......................................... austromorium shattuck, 2009 14 propodeal spines anterolaterally curved and hook-like. petiole with a pair of posteriorly curved spines (new guinea) ...... ancyridris wheeler, 1935 propodeal spines straight and not hook-like. petiole without spines ... 15 15 dorsum of alitrunk without any standing hairs ....................................... 16 dorsum of alitrunk with sparse to abundant standing hairs .................. 17 16 frontal carinae present. in profile view, dorsum of alitrunk with 5 hornlike processes (indo-australian) ......................................proatta forel, 1912 frontal carinae absent. in profile view, dorsum of alitrunk with 3 bluntly convex tubercles (oriental) ....................................... dacatria rigato, 1994 17 clypeus without a pair of longitudinal carinae. propodeal lobes strongly developed, elongate and broad (borneo) ......................................................... ........................................................................... propodilobus branstetter, 2009 clypeus with a pair of longitudinal carinae. propodeal lobes weakly developed, either short or narrow ..................................................................... 18 18 frontal carinae absent (neotropical, holarctic, oriental, indo-australian) ...................................................................... stenamma westwood, 1839 335 xu, z.-h. — a new genus and species of myrmicinae from china frontal carinae present................................................................................... 19 19 petiolar peduncle longer than the node (venezuela) .................................... ....................................................................................bariamyrma lattke, 1990 petiolar peduncle as long as or shorter than the node (oriental, indoaustralian, australasian) ..................................... lordomyrma emery, 1914 descriptions of new genus and species gaoligongidris gen. nov. (figs. 1-7) type-species: g. planodorsa sp. nov. gender: feminine. etymology: the new genus is named after the type locality “gaoligong” plus the common suffix “-idris”. diagnosis of worker: monomorphic terrestrial myrmicine ants with the following combination of characters. head square, occipital margin nearly straight. palp formula 2, 2 (2 individuals dissected). mandible triangular, with 6 sharp teeth which decrease in size from apex to base. median portion of clypeus longitudinally bicarinate, the posterior portion broadly inserted between the frontal lobes. anterior clypeal margin extruding, nearly straight in the middle portion. anterior clypeal margin with a row of long setae, but lacking an isolated median seta. frontal lobes as broad as the posterior portion of clypeus which is inserted between them. frontal carinae and antennal scrobes absent. antennae 11-segmented; scapes short, apices failed to reach occipital corners; antennal clubs 3-segmented, the third segment of the club counting from apex reduced in volume and much shorter than the second one. eyes moderately large, drawn out anteroventrally, located before the midpoints of lateral sides of the head. promesonotum forming a high plateau, without teeth or prominences. promesonotal suture absent, metanotal groove deeply impressed. 336 sociobiolog y vol. 59, no. 2, 2012 propodeum low, with a pair of long slender spines. propodeal spiracles large and circular, well before the declivity margin and high up on the sides. propodeal lobes small and bluntly angled at apex. metapleural gland bullae large and close to the propodeal spiracles. tibial spurs absent from middle and hind legs. petiole with long anterior peduncle, the spiracles located about at the midlength of the peduncle, subpetiolar process absent. postpetiolar dorsum roundly convex, subpostpetiolar process absent. cuticle thick, sculptures strongly developed on the head, alitrunk, petiole, and postpetiole. pilosity abundant. female and male: unknown. comparison: the new genus is close to the indo-australian genus lasiomyrma terayama & yamane (figs. 8-12) in the tribe stenammini, but with anterior clypeal margin not angled, metanotal groove deeply impressed, propodeal spiracles large, propodeal spines long and slender, petiolar peduncle longer than the node. systematic position: myrmicinae: stenammini. geographical range: oriental. discussion: at first glance, the new genus is somewhat similar to lophomyrmex emery of the tribe pheidolini by the following characters: 11-segmented antennae, long propodeal spines, large propodeal spiracles, and long petiolar peduncle. however, the genus obviously belongs to the tribe stenammini by the following characters: median portion of clypeus longitudinally bicarinate, anterior clypeal margin nearly straight in the middle portion; masticatory margins of mandibles with only 6 teeth; antennae short, apices of the scapes failed to reach occipital corners, the third segment of antennal club counting from apex distinctly reduced in volume and much shorter than the second one; pronotum without teeth, prominences, or lateral margins; head and alitrunk strongly sculptured. gaoligongidris planodorsa sp. nov. (figs. 1-7) 337 xu, z.-h. — a new genus and species of myrmicinae from china figs. 1-7: worker of gaoligongidris planodorsa sp. nov.; 1. head and body in profile view; 2. head in full face view; 3. mandible in dorsal view; 4. antenna in dorsal view; 5. body in dorsal view; 6. maxilla and maxillary palp in ventral view; 7. labium and labial palp in ventral view. 338 sociobiolog y vol. 59, no. 2, 2012 figs. 8-12:worker of lasiomyrma gedensis terayama & yamane; 8. head and body in profile view; 9. head in full face view; 10. anterior clypeal margin in dorsal view; 11. mandible in dorsal view; 12. alitrunk in dorsal view. (cited from terayama & yamane 2000). 339 xu, z.-h. — a new genus and species of myrmicinae from china holotype worker: tl 2.6, hl 0.67, hw 0.65, ci 98, sl 0.43, si 67, ed 0.10, pw 0.42, al 0.73. head square, as broad as long. occipital margin nearly straight, occipital corners roundly prominent. lateral sides weakly convex. mandibles subtriangular, masticatory margin with 6 teeth, which decrease in size from apex to base. median portion of clypeus bicarinate and extruding forward, anterior margin nearly straight. posterior clypeal extension about as broad as the frontal lobes. frontal carinae and antennal scrobes absent. antennae short, 11-segmented, apex of scape reached to 3/4 of the distance from antennal socket to occipital corner. the apical 3 segments form the antennal club, apical segment 2.5 times as long as the preceding one; the third segment counting from apex weakly enlarged, about 1/2 length of the second one. eyes moderately large, situated in front of the midpoints of the lateral sides of the head, with about 12 ommatidia in the maximum diameter. in profile view, pronotum and mesonotum form a high plateau which gently slopes down backward, posterodorsal corner of mesonotum bluntly angled and steeply slopes down to the metanotal groove. promesonotal suture absent. metanotal groove deeply impressed. propodeum low, dorsum short and straight. propodeal spines long, sharp, and straight, laterally compressed, about as long as propodeal dorsum. declivity weakly concave, lateral sides marginate. propodeal spiracles large and circular, well before the declivity margins, and high up on the sides. propodeal lobes small and bluntly angled at apex. metapleural gland bullae large and roughly triangular. petiolar node triangular, anterior face straight, posterior face weakly convex, anterior peduncle longer than the node, ventral face weakly concave under the node, and weakly convex before the concavity, subpetiolar process absent. postpetiolar dorsum roundly convex, slightly lower than petiolar node, ventral face with 2 small convexities. in dorsal view, promesonotum nearly triangular, narrowed backward. lateral sides of mesonotum marginate, lateroposterior corners rightly angled. postpetiolar node about 1.4 times as broad as petiolar node, lateral sides of postpetiole roundly convex. mandibles densely longitudinally striate. head densely reticulate, but the vertex longitudinally striate, interfaces finely punctured. alitrunk densely coarsely punctured, but pronotal dorsum densely reticulate. petiole and postpetiole densely finely punctured, but dorsum of postpetiolar node finely 340 sociobiolog y vol. 59, no. 2, 2012 longitudinally striate. gaster smooth and shining, but the first tergite with short basal costulae, which distinctly shorter than the postpetiolar node. dorsum of head with dense erect short hairs. dorsum of alitrunk with sparse erect to suberect longer hairs and abundant decumbent pubescence. petiole, postpetiole, and gaster with abundant suberect hairs and decumbent pubescence. scapes and tibiae with dense subdecumbent to decumbent short hairs. color yellowish brown, but dorsum of head and middle portion of gaster blackish brown. paratype workers: tl 2.3-2.6, hl 0.60-0.67, hw 0.57-0.65, ci 89-100, sl 0.37-0.43, si 63-67, ed 0.10-0.12, pw 0.37-0.42, al 0.70-0.80 (10 individuals measured). as holotype, but color brownish yellow to yellowish brown. holotype: worker, china: yunnan province, tengchong county, jietou town, datang village, 2000m, collected from a soil sample in the subalpine moist evergreen broadleaf forest on the west slope of gaoligong mountain, 1999.v.1, ji-guai li leg., no.a99-195. paratypes: 20 workers, with the same data as holotype; 3 workers, china: yunnan province, tengchong county, jietou town, datang village, 1750m, collected from a soil sample in the monsoon evergreen broadleaf forest, 1999.v.2, qi-zhen long leg., no.a99-215; 3 workers, china: yunnan province, tengchong county, shang ying town, cuanlong village, 1500m, collected from a soil sample in the monsoon evergreen broadleaf forest, 1999.iv.29, qi-zhen long leg., no.a99-176; 6 workers, china: yunnan province, tengchong county, jietou town, shaba village, 2000m, collected from a ground sample in the shrub, 1999.v.3, qi-zhen long leg., no.a99-320; 3 workers, with the same data as no.a99-320, but no.a99326, and collected from a soil sample; 1 worker, china: yunnan province, tengchong county, qushi town, youtu village, 2000m, collected from a ground sample in the subalpine moist evergreen broadleaf forest, 1999.v.4, ji-guai li leg., no.a99-367; 2 workers, with the same data as no.a99-367, but nos.a99-369, a99-376, and collected from soil samples; 2 workers, china: yunnan province, tengchong county, qushi town, youtu village, 1900m, collected from a ground sample in the subalpine moist evergreen broadleaf forest, 1999.v.4, zheng-hui xu leg., no.a99-447; 1 worker, china: yunnan province, baoshan city, bawan town, pumanshao village, 2000m, 341 xu, z.-h. — a new genus and species of myrmicinae from china collected from a ground sample in the subalpine moist evergreen broadleaf forest, 1998.viii.13, lei fu leg., no.a98-2101; 1 worker, with the same data as no.a98-2101, but no.a98-2110 and collected from a soil sample. etymology: the name of the new species is descriptive of the “plane” promesonotal “dorsum” of alitrunk in profile view. remarks: currently, g. planodorsa sp. nov. is the only species in the new genus. according to the rich collections, the new species is mainly found in subalpine moist evergreen broadleaf forest, sometimes habitats in the monsoon evergreen broadleaf forest and shrubs between the altitudes of 1500-2000m. the species is obviously nesting in the soil, and foraging on the ground. acknowledgments this study is supported by the applied and basic research foundation of yunnan province (no. 97c006g), the national natural science foundation of china (no. 30870333), and the key subject of forest protection of yunnan province. i thank miss qi-zhen long, mr. ji-guai li, and mr. lei fu (students of forest protection class 95-1, southwest forestry university, kunming ) for collecting the type specimens with me. references bolton, b. 1987. a review of the solenopsis genus-group and revision of afrotropical monomorium mayr. bulletin of the british museum (natural history) (entomolog y) 54: 263-452. bolton, b. 1991. new myrmicine ant genera from the oriental region. systematic entomology 16: 1-13. bolton, b. 1994. identification guide to the ant genera of the world. harvard university press, 222 pp. cambridge, massachusetts. bolton, b. 1995. a new general catalogue of the ants of the world. harvard university press, 504 pp. cambridge, massachusetts. bolton, b. 2011. an online catalog of the ants of the world. http://www.antcat.org/. branstetter, m.g. 2009. the ant genus stenamma westwood redefined, with a description of a new genus propodilobus. zootaxa 2221: 41-57. brown, w.l., jr. 1986. indomyrma dasypyx, new genus and species, a myrmicine ant from peninsular india. israel journal of entomolog y 19 (1985): 37-49. brown, w.l., jr. & e.o. wilson 1957. dacetinops, a new ant genus from new guinea. breviora 77: 1-7. 342 sociobiolog y vol. 59, no. 2, 2012 emery, c. 1887. catalogo delle formiche esistenti nelle collezioni del museo civico di genova. parte terza. formiche della regione indo-malese e dell’australia (continuazione e fine). annali del museo civico di storia naturale di genova (2) 5 [25]: 427-473. emery, c. 1894. studi sulle formiche della fauna neotropica. bullettino della società entomologica italiana 26: 137-241. emery, c. 1908. descriptions d’un genre nouveau et de plusiers formes nouvelles de fourmis du congo. annales de la société entomologique de belgique 52: 184-189. emery, c. 1914. les fourmis de la nouvelle-calédonie et des îles loyalty. in sarasin, f. & roux, j. nova caledonia zoologie 1: 393-437. wiesbaden. forel, a. 1912. descriptions provisoires de genres, sous-genres et espèces de formicides des indes orientales. revue suisse de zoologie 20: 761-774. lattke, j.e. 1990. a new genus of myrmicine ants from venezuela. entomologica scandinavica 21: 173-178. mayr, g. 1865. reise der österreichischen fregatte novara um die erde in den jahren 1857, 1858, 1859, unter den befehlen des commodore b. von wüllerstorf-urbair. zoologischer theil. formicidae: 119 pp. wien. rigato, f. 1994. dacatria templaris gen. n., sp. n. a new myrmicine ant from the republic of korea. deutsche entomologische zeitschrift (n. f.) 1: 155-162. rosciszewski, k. 1994. rostromyrmex, a new genus of myrmicine ants from peninsular malaysia. entomologica scandinavica 25: 159-168. shattuck, s.o. 2009. austromorium, a new myrmicine ant genus from australia. zootaxa 2193: 62-68. terayama, m. & s. yamane 2000. lasiomyrma, a new stenammine ant genus from southeast asia. entomological science 3: 523-527. weber, n.a. 1952. studies on african myrmicinae, 1. american museum novitates 1548: 1-32. westwood, j.o. 1839. an introduction to the modern classification of insects; founded on the natural habits and corresponding organisation of the different families 2 (part 11): 193-224. london. wheeler, w.m. 1935. two new genera of myrmicine ants from papua and the philippines. proceedings of the new england zoological club 15: 1-9. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.5922sociobiology 68(2): e5922 (june, 2021) leaf-cutting ants of the genus atta fabricius, 1805 (hymenoptera: formicidae) are eusocial insects found exclusively in the neotropical region (hölldobler & wilson, 1990). these ants build stable and long-lasting nests that protect themselves against abiotic and biotic factors, besides growing the fungus leucocoprinus gongylophorus heim, 1957, on which they feed (sudd, 1982; hölldobler & wilson, 1990; rocha et al., 2020). however, due to favorable conditions, like temperature and humidity, that these nests offer (camargo et al., 2016), they also become favorable habitats for a multitude of inquilines, from invertebrates such as beetles, cockroaches, mites, crickets, dipterans and springtails (waller & moser, 1990; dekoninck et al., 2007; forti et al., 2007), to vertebrates such as frogs, lizards and snakes (hölldobler & wilson, 1990; baer et al., 2009; lima barros et al., 2016). according to hölldobler and wilson (1990), abstract cyphoderus innominatus mills, 1938 (collembola: paronellidae) was first observed in early colonies of atta sexdens leaf-cutting ants (hymenoptera: formicidae). the colonies were collected on february 6, 2019, from a transition area between the atlantic forest and the cerrado, located in the municipality of botucatu, são paulo, brazil. a total of four colonies collected had an average population density of 227 ± 212 c. innominatus individuals, and most of the latter were found in peripheral areas inside the fungusgarden-growing chamber of the colony. in addition, we observed a possible defensive behavior on the part of workers when c. innominatus individuals were present in the fungus garden chamber. thus, this is the first record of c. innominatus living in association with early colonies of a. sexdens. sociobiology an international journal on social insects tarcísio marcos m mota filho1, kátia kaelly andrade sousa1, roberto s camargo1, joão victor lemos cavalcante oliveira2, nadia caldato1, douglas zeppelini2, luiz carlos forti1 article history edited by paulo cristaldo, ufrpe, brazil received 20 october 2020 initial acceptance 28 december 2020 final acceptance 23 february 2021 publication date 20 may 2021 keywords attini, cyphoderus, myrmecophily, cyphoderinae. corresponding author tarcísio marcos macedo mota filho laboratory of social insects-pests plant protection department school of agronomic sciences são paulo state university botucatu, são paulo, brasil. e-mail: tarcisio972010@hotmail.com these ant guests are commonly known as myrmecophiles and may be present in ant nests temporarily engaging in predation or commensalism, or may also depend on such nests for a while or throughout their life cycle. information about their ecology, behavior, life cycles and dispersion is scarce for most of these myrmecophilous species because they are difficult to observe and can only be found when specific studies are conducted (dekoninck et al., 2007). nonetheless, springtails are inquilines commonly reported and studied in nests of fungus-growing ants (castañomeneses et al., 2017). even a specific behavior known as jigging, performed by cyphomyrmex costatus workers, mann, 1922, and myrmicocrypta buenzlii workers, borgmeier, 1934, (formicidae: myrmicinae), has been reported as a defense strategy to keep springtails away from the fungus-gardengrowing chamber (weber, 1957, 1972; kweskin, 2004). 1 laboratory of social insects-pests, plant protection department, school of agronomic sciences, são paulo state university, botucatu-sp, brazil 2 laboratory of collembola systematics and conservation, biology department, center for applied biological and social sciences, paraiba state university, campus v, joão pessoa-pb, brazil first record of cyphoderus innominatus mills, 1938 (collembola: paronellidae) in early colonies of the leaf-cutting ant atta sexdens short note id mailto:tarcisio972010@hotmail.com https://orcid.org/0000-0002-2157-261x tarcísio mm mota filho et al. – cyphoderus innominatus in early colonies of atta sexdens2 in this study, we identify, quantify and report the first species of springtails of the genus cyphoderus living in early colonies of a. sexdens. in addition, we provide information on a possible defensive behavior performed by workers when springtails are present in their fungus garden chamber. the study design relied on sampling, six hundred colonies of a. sexdens (hymenoptera: formicidae), aged approximately four months old, were collected on february 6, 2019, in a transition area between the atlantic forest and the cerrado, located in the municipality of botucatu (22° 48’ 23.5” s 48° 25’ 52.8” w), são paulo, brazil. in the laboratory, a high population density of springtails was observed in four of the 600 collected colonies. these colonies were separated, and then, with the aid of an aspirator, all springtails present were removed by suction and stored in 98% alcohol. afterwards, population density was quantified, and samples were taken for species identification. the identification was carried out by the laboratory of collembola systematics and conservation of the state university of paraíba (uepb). the methodology adopted for identification consisted of clearing and mounting of the specimens, following the procedures described by jordana et al. (1997), with clearing being done using nesbitt’s solution and a phase-contrast microscope (zeiss axioskop). then, to identify the species, the classification key provided by oliveira et al. (2017) was used. the specimens are deposited in the uepb’s soil fauna reference collection [coleção de referência da fauna de solo] (crfs). cyphoderus innominatus mills, 1938 (collembola: paronellidae) was the species of springtails found living in association with early colonies of a. sexdens (fig 1). cyphoderus innominatus were found in four (0.67%) of the 600 collected colonies, with an average population density of 227 ± 212 individuals (table 1). most of the individuals were present in peripheral zones inside the fungus garden chamber of the colony, where some debris accumulated, but the springtails did not make direct contact with the ants (fig 1b and video: https://youtu.be/eobszykzs30). the video showed that the springtails (https://youtu. be/eobszykzs30) were initially present in the fungus garden chamber of the ants, apparently without causing any disturbance. until a medium worker (ant with head width of approximately 2.2 mm), between the 38th and 46th seconds, presented a defensive behavior against the springtails. with its jaws open, it jumped three consecutive times towards some springtails. however, the attacks were unsuccessful, and they managed to escape. the present study recorded, for the first time, the occurrence of c. innominatus living in association with early colonies of a. sexdens leaf-cutting ants collected in a transition area between the atlantic forest and the cerrado. previously, in paraguay, springtails of the genus cyphoderus had been reported living in colonies of a. sexdens subjected to laboratory conditions (fowler, 1981). however, despite the occurrence of this genus being recorded, no species was identified. in addition, three other species of springtails had already been found in association with fungus growing carried out by leaf-cutting ants of the genera atta and acromyrmex, namely: cyphoderus inaequalis with acromyrmex octospinosus; pseudosinella violenta (lepidocyrtidae) with atta texana; and seira edmanni (seiridae) with a. sexdens (eidmann, 1937; weber, 1958; kistner, 1982; waller & moser, 1990; castaño-meneses et al., 2017). the presence of springtails of the genus cyphoderus in early colonies of fungus-growing ants can be explained by the large amount of food found in these colonies; being myrmecophilous species, they feed on the mycelium of the fungus grown by the ants (kistner, 1982). moreover, colonies of fungus-growing ants also indirectly provide favorable conditions, such as temperature, moisture and protection from predators, to enable the growth, development and multiplication of the tenant population (hughes et al., 2008). the average density of c. innominatus individuals found in the early colonies of a. sexdens was 227 ± 212 individuals. it is a relatively high density compared to the population density of workers (small, medium and large) from an early nest (up to 4 months) of a. sexdens, which, according to camargo and forti (2013), comprises approximately 121 individuals. according to kweskin (2004), a high population density of springtails ends up consuming a large amount of the mycelium grown in the fungus garden, consequently disturbing the colony (kweskin, 2004). some behavioral acts performed by ants against these disturbances have been reported. among said acts, jigging is a peculiar rhythmic swinging behavior, and there are two hypotheses as to why it is performed: alerting mates about disturbances, or expelling competitors (weber, 1957;1972). however, this behavior was not observed in our study. on the other hand, the behavioral act also performed by ants in the presence of springtails in their fungus gardens, and which was observed in our study, is jumping, when ants jump and try to capture springtails with their jaws and legs. according to kweskin (2004), this type of behavior seems to be exclusively meant for defense against invaders, and the same behavior was also performed by c. costatus workers when springtails approached their fungus garden. the actual reason and the efficiency of this behavioral act against these disturbances are still not well understood. in light of our findings, future studies should be carried out to elucidate the effect of the presence of c. innominatus in fungus gardens of a. sexdens, and whether their presence causes disturbances and induces defensive behaviors. thus, our study provides important information about a species of myrmecophilous springtails that live in association with early colonies of fungus-growing ants, since we reported here, for the first time, the occurrence of c. innominatus in early colonies of a. sexdens. furthermore, we provided information about a possible defensive behavior performed by a. sexdens workers when springtails are present in their fungus garden chamber. https://youtu https://youtu sociobiology 68(2): e5922 (june, 2021) 3 colony number of individuals of cyphoderus innominatus mills, 1938 1 539 2 133 3 165 4 70 mean ± standard deviation 227 ± 212 table 1. number of individuals of cyphoderus innominatus mills, 1938 (collembola: paronellidae) found in early colonies of atta sexdens (hymenoptera: formicidae). fig 1. (a) early nest of atta sexdens (hymenoptera: formicidae); (b) cyphoderus innominatus mills, 1938 (collembola: paronellidae) present in peripheral zone inside the fungus-garden-growing chamber of a. sexdens fungus in laboratory condition [temperature of 22 ± 2 ºc, relative humidity of 70 ± 10% and photoperiod of 14:10 h (l: d)]; (c) side view of c. innominatus in 98% alcohol; (d) slide view with zeiss axioskop microscope. acknowledgments tmm mota filho and kka sousa thank the support of the coordination for the improvement of higher education personnel [coordenação de aperfeiçoamento de pessoal de nível superior] – brazil (capes) – finance code 001. lc forti gratefully acknowledges the support of the national council for scientific and technological development [conselho nacional de desenvolvimento científico e tecnológico] (cnpqpq) (donation no. 301938/2017-2). conflict of interest the authors declare no conflicts of interest. authors’ contributions tmm mota filho, kka sousa, rs camargo, n caldato and forti l. c. conceptualization, investigation, data curation, writing. jvc oliveira and d zeppelini carried out the identification of the species. references baer, b., den boer, s.p.a., kronauer, d.j.c., nash, d.r. & boomsma, j.j. (2009). fungus gardens of the leafcutter ant atta colombica function as egg nurseries for the snake leptodeira annulata. insectes sociaux, 56: 289-291. doi: 10.1007/s00040-009-0026-0. camargo, r.s. & forti, l.c. (2013). queen lipid content and nest growth in the leaf cutting ant (atta sexdens rubropilosa) (hymenoptera: formicidae). journal of natural history, 47: 65-73. doi: 10.1080/00222933.2012.738836. tarcísio mm mota filho et al. – cyphoderus innominatus in early colonies of atta sexdens4 camargo, r.s., forti, l.c., matos, c.a.o., caldato, n. & fonseca, o.s. (2016). is the initial nest depth adapted to favorable conditions for the incipient colony in leaf-cutting ants? sociobiology, 63: 792-799. doi: 10.13102/sociobiology. v63i2.976. castaño-meneses, g., palacios-vargas, j.g., delabie, j.h.c., zeppelini, d. & mariano, c.s.f. 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(1972). gardening ants, the attines. the american philosophical society, philadelpha, penn. 146 p. https://doi.org/10.1016/j.tree.2008.07.011 https://doi.org/10.1016/j.tree.2008.07.011 https://doi.org/10.11646/zootaxa.4350.1.2 https://doi.org/10.2307/1929893 https://doi.org/10.2307/1929893 doi: 10.13102/sociobiology.v65i2.1207sociobiology 65(2): 138-148 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a comparison between time of exposure, number of pitfall traps and the sampling cost to capture ground-dwelling poneromorph ants (hymenoptera: formicidae) introduction assessing the effects of environmental changes on biodiversity is expected to increase with the increasing evidence of biodiversity loss. additionally, information gathered through biodiversity assessment can play an important role in conservation plans. nevertheless, conservation planning needs to be based fundamentally on biodiversity data, which requires taxonomic knowledge (fisher, 2005). however, in order to transfer this important scientific knowledge to decision makers biodiversity assessment is required to be more than abstract using effective survey protocols to address the effects of environmental change are key to saving time, resources and costs. although exhaustive sampling in any location has been shown as impractical, biodiversity sampling projects must capture sufficient information to show how species assemblages change with the environmental variables. this study investigated time of exposure in the field and the number of pitfall traps that efficiently sampled poneromorph ants in 30 250 m long plots across an area of 25 km2 of tropical rain forest in brazil. the treatments used for the surveys included two days and 300 traps, 14 days and 300 traps, 14 days and 750 traps, and were considered the minimum, intermediate and maximum sampling efforts, respectively. we characterized each assemblage of ants in relation to a gradient of soil texture, terrain slope and leaf and branch litter volume, and then tested whether the ecological relationships observed with the maximum effort were comparable to data on intermediate and minimum sampling efforts. we also estimated the cost-effectiveness of using the protocols in survey programs. the assemblage of species sampled during 14 days was similar to the assemblage captured during two days, indicating that the number of days influenced the assemblage similarity more than the number of sampling traps. all ecological patterns detected with the maximum effort were also captured with lesser sampling efforts. overall, both the intermediate and minimum sampling efforts represented savings around 26˗40% of total project costs and 43˗45% of time to process the samples. we recommend that two days of trapping time combined with 300 pitfall traps is a highly effective shortcut for monitoring assessment, which can be applied to large-scale biodiversity surveys in tropical forests. sociobiology an international journal on social insects cb gomes1,3, jlp souza2,3,4, e franklin2,3 article history edited by frederico neves, ufmg, brazil received 14 october 2016 initial acceptance 01 march 2017 final acceptance 18 march 2018 publication date 09 july 2018 keywords amazon, ant survey, bio-monitoring, cost-effectiveness, sampling protocol. corresponding author camila de brito gomes national institute for amazonian research – inpa avenida andré araújo, 2936, petrópolis cep 69080-971 manaus-am, brazil e-mail: camilabritogomes@gmail.com just species lists, but surveys which highlight the relationships among the community and the changing environmental variables (landeiro et al., 2010; franklin et al., 2013). in addition, the serious lack of funding for ongoing taxonomical monitoring (tahseen, 2014) dictates that the costs of the survey should not exceed the potential economic benefits resulting from the investigations (evans & viengkham, 2001). thus, independent of their study aims, researchers often face a conflict of interest to decide between area sampled and sampling intensity in initial or sequential environmental assessment (souza et al., 2012; pos et al., 2014). 1 entomology graduate program, national institute for amazonian research, inpa, manaus, amazonas, brazil 2 biodiversity coordination, inpa, manaus, amazonas, brazil 3 center for integrated studies of the amazonian biodiversity, cenbam, manaus, amazonas, brazil 4 graduate program in science and technology for amazonian resources, institute of exact sciences and technology, icet, itacoatiara-am, brazil research article ants mailto:camilabritogomes@gmail.com sociobiology 65(2): 138-148 (june, 2018) 139 ants are a mega-diverse invertebrate group with a wide geographical distribution and functional importance at various trophic levels, providing several advantages for indicating environmental changes (folgarait, 1998; andersen & majer, 2004; silva & brandão, 1999; osborn et al., 1999). they can be easily surveyed in large numbers in most ecosystems using several sampling methods and are relatively easily distinguished at morphospecies level (bestelmeyer et al., 2000; souza et al., 2012; 2016). because ants are widely used as bio-indicators in land management and monitoring, shortcuts are necessary to improve the efficiency of the survey (andersen & majer, 2004; souza et al., 2016). there are about 14,000 species of ants described worldwide with approximately 30% of these found in the neotropics (agosti & johnson, 2005; baccaro et al., 2015). the poneromorph ants have currently 71 genera and are defined as an informal (not monophyletic) group (bolton, 2003), which remains primitive in some of their general characteristics (schmidt & shattuck, 2014). these ants are distributed throughout the world’s zoogeographic regions and occupy a great diversity of ecological niches, from both small and cryptic, to large and remarkable (ouellette et al., 2006). the poneromorph ants have a great ecological importance, occupying different trophic levels (brandão et al., 2011), thus participating in mutualistic plant associations (pereira et al., 2013), facilitating seed dispersal (leal et al., 2014). this group of insect also plays a prominent role inbioindicators of anthropic impacts such as the use of pastures (dias et al., 2008), fires (endanger et al., 2008), and mining (ribas et al., 2012). several studies have evaluated the efficiency of pitfall traps in catching ground-dwelling ants in comparison with other techniques (wang et al., 2001; ivanov & keiper 2009; souza et al., 2012). traditionally, pitfall traps remain in the field for 48 hours in studies with ground-dwelling ants (pik et al., 1999; agosti & alonso 2000; bestelmeyer et al., 2000; vasconcelos et al., 2003; oliveira et al., 2009; souza et al., 2009; baccaro et al., 2012, 2013; souza et al., 2012). nevertheless, it is argued that the permanence of the traps for longer periods could capture more diversity (bestelmeyer et al., 2000). although the shortest period of 48 hours has been proven adequate to show the representative assemblage of ants (borgelt & new, 2006), longer periods are recommended in order to capture rare species (borgelt & new, 2006; schirmel et al., 2010). the efficiency of sampling with pitfall traps is also affected by the number of samples collected (borgelt & new, 2006; schirmel et al., 2010) and trap spacing (wang et al., 2001). inventories of invertebrates involve many hours of processing samples and sampling of soil from an extensive area, and this process is hampered due to limitations of time and money (jiménez-valverde & lobo, 2006), which in turn limits work to a few years (danielsen et al., 2003). the major constraint to the expansion of existing ant surveys in tropical forests is the labor-intensive laboratory effort in sorting and identifying these invertebrates (lawton et al., 1998; purvis & hector, 2000; souza et al., 2009, 2012). the biggest challenge to investigate a more efficient use of resources is to ensure that sufficient ecological gradients are captured in relation to changing environmental variables (souza et al., 2009). tropical amazonian rainforests contain mosaic textures, which are correlated with forest architecture and linked to the altitudinal gradient (guillaumet, 1987; costa & magnusson, 2010). these combinations of environmental variables account for the variation associated with ant’s assemblage structure across tropical landscapes (vasconcelos et al., 2003; oliveira et al., 2009). investigating these types of relationships between ants and environmental variables is of more practical importance than simply showing correlations between the numbers of taxonomic entities (souza et al., 2016). in fact, some studies have shown how environmental variables can be used to evaluate decisions about sampling techniques (souza et al., 2012) or the use of genus as a surrogate for species (souza et al., 2016) to optimize ant surveys. using poneropmoph ants, we compared two trapping times and the number of pitfall traps to test the efficiency of three sampling efforts. we first checked for variations in poneropmoph ant composition in each sampling combination. secondly, we tested whether the ecological patterns observed with the maximum effort could be retrieved from data on reduced efforts. we investigated the influence of biotic (leaf and branch litter volume) and abiotic factors (terrain slope and soil clay content) on the poneropmoph ant species composition, , and compared with the results obtained with different trapping times and number of pitfall traps. lastly, we checked the consequent gain in terms of time and costs, and the loss of information of each effort. we hypothesize that fewer sampling days and a smaller number of traps would reduce the catch, while still maintaining enough information to capture the relationship with environmental variables, thus improving the efficacy of the survey. materials and methods study site the ducke reserve is located 26 km on the manausitacoatiara highway (3o00’s, 59o55’w), near to manaus city, amazonas state, brazil. the reserve is covered by a terrafirma evergreen forest along a moderately uneven terrain (30– 140 m a.s.l.) covering 10,000 hectares (ribeiro et al., 1999). the nutrient-poor soils are classified as yellow clay latosols (xan-thic hapludox) in the higher, well drained, flat plateaus, grading to clay-sand (typic epiaquods) on the slopes, and sandy soils (typic endoaquods) in the wet or temporarily floodplain (chauvel et al., 1987; santos et al., 2006).the climate is characterized by a rainy season from november to may, with a relatively dry season (less than 100 mm of monthly rainfall) occurring from july to september (luizão et al., 2004). mean daily air humidity and mean daily temperature between 2008 and 2011 were 77.7 percent and 25.7ºc, respectively (coordination of environmental dynamics, inpa). cb gomes, jlp souza, e franklin – a comparison between time, number and cost of pitfall traps 140 ant sampling the ducke reserve contains a grid of six regularly spaced north-south and six east-west trails. each trail is 5 km long, forming a 5 × 5 km grid (https://ppbio.inpa.gov.br/ sitios/ducke; fig 1). the east-west trails have five 250m long plots that follow terrain contours to minimize the variation in soil features (rapeld method, magnusson et al., 2005). the grid allows access to 30, 250m long plots, located 1 km apart from each other along the trails. the ants were sampled. environmental variable data included the soil clay content, leaf and branch litter volume and terrain slope (available in: https://ppbio.inpa.gov.br/repositorio/dados). to determine soil clay content and terrain slope, we sampled and combined six soil subsamples to a depth of 5 cm and at least 50 m distant from each other for each plot and analyzed them at the soil laboratory of the agronomy department at inpa. litter volume was collected in the same 30 plots and period where the ground dwelling ants were sampled. about 1 m2 of litter was put on a graduated plastic bucket to measure the volume (in liters). in each plot, 10 subsamples were collected and pooled to derive an average litter volume per plot. the environmental datasets and the descriptions of each sampling protocol (metadata) for each variable are available in the ppbio web site (http:// ppbio.inpa.gov.br/). there were no significant interaction between soil clay content and terrain slope (pearson’s product-moment correlation t = -0.994, df = 28, p-value = 0.329, cor:-0.185), clay content and litter and branch volume (pearson’s product-moment correlation t = -0.174, df = 28, p-value = 0.863, cor:-0.033), and terrain slope and litter and branch volume (pearson’s product-moment correlation t = -0.453, df = 28, p-value = 0.654, cor:-0.085). the environmental variable were used merely to show whether an effect significant or not detected in one analysis can still be detected in a subsequent analysis based on reduced-effort sampling. data analysis the presence and absence data was used to reduce the influence of social behavior, foraging and mass distribution of the ant nests in leaf litter (hölldobler &wilson, 1990). the effectiveness of sampling technique depends on sampling intensity, and results of comparative analyses can be biased by variation in sampling intensity between efforts (souza et al., 2012). to assess how many samples are needed to record a comparable number of species, the average species-accumulation curves from 1000 randomizations were used. the time and number of pitfall-traps could affect the composition of species in the area, so we used the permutational multivariate analysis of variance (np-manova, anderson, 2001) to test for differences in ant assemblages between time of exposure and the number of samples. we tested hypothesis considering that the composition of the poneromorph ant assemblages in each sampling effort were different. the congruence between ant assemblages by each sampling effort was tested. the composition of the assemblages were reduced with the technique of non-metric multidimensional scaling (nmds, minchin, 1987), applied to an association matrix using the sørensen index for qualitative data. the congruence between the nmds ordinations for each sampling effort was quantified by procrustean superimposition with 1000 monte carlo permutations to test for statistical significance (peres-neto & jackson, 2001). the assemblages of ants and their relationship to environmental variables were compared for the maximum, fig 1. location of the 25 km2 sampling grid on reserva ducke in the brazilian amazon. points indicate 1-km equidistant sample plots. between july and september 2006, poneromorph ants were sampled in the 30 plots using pitfall traps, which is the most widely used sampling technique for arthropods, such as ants (schirmel etal., 2010).the traps consist of 500 ml plastic containers (6.5 cm diameter; 8 cm depth; 200 ml volume), buried with the rim at ground level and partially filled with the killing/preservative agent, and remaining open during 14 days (336 hours). the invertebrates that were active on the surface and fell into the trap were preserved in 70% alcohol. in total 750 samples were taken from 25 pitfall traps installed at 10 m intervals in each plot. we also used data from oliveira et al. (2009), which used the same pitfall traps in the same period and area but with a different sampling effort, using a total 300 samples taken from 10 pitfall traps installed at 25 m intervals in each plot and left open for 48 hours. thus, using data from both surveys, we are analyzing three sampling efforts in the 30 plots: two days and 300 traps, 14 days and 300 traps, 14 days and 750 traps were considered as the minimum, intermediate and maximum effort, respectively. all ants were identified to genus using the taxonomic keys provide by baccaro et al. (2015). after that, the ants were sorted to morphospecies, and whenever possible were identified to a species level (sensu bolton et al., 2005), using the available taxonomic keys or by comparison with specimens in collections previously identified by experts. vouchers are deposited in national institute for amazonian research (inpa) entomological collection. environmental variables data for independent variables that are recognized as important for ants were measured at the same 30 plots where sociobiology 65(2): 138-148 (june, 2018) 141 intermediate and minimum sampling efforts. multiple regression test was used to verify that the environmental variables volume of litter, clay content and terrain slope affected the distribution of species (nmds axes). pearson’s correlation was used to prevent errors of collinearity between variables to avoid putting correlated variables in the same regression model. all analyses were run in the r environment for statistical computing (r core team, 2017, version 4.3), using vegan package 2.4-6 (oksanen et al., 2018). time and monetary costs for the all-sampling efforts were considered in relation to the maximum effort and the fractions of these costs were calculated for each reduced effort. the costs were based on the acquisition of material, maintenance, field sampling and laboratory activities (mainly salary and scholarships). the laboratory costs were those associated with species sorting, mounting, identifying, and chemicals for the conservation of voucher species. as recommended by gardner et al. (2008), capital costs, which vary greatly among projects, such as non-perishable laboratory equipment (e.g. microscopes) and accommodation buildings for field staff, were not included. the time to carry out the work was the sum of the time spent to collect samples in the field and the time spent sorting and identifying ants in the laboratory. sampling effort taxon 14 days 2 days 750 sub-samples 300 sub-samples 300 sub-samples abundance frequency abundance frequency abundance frequency acanthostichus bentoni mackay, 1996 13 6 1 1 4 2 anochetus diegenis forel, 1912 3 1 0 0 6 4 anochetus emarginatus (fabricius, 1804) 1 1 0 0 1 1 anochetus horridus kempf, 1964 12 6 8 4 4 3 centromyrmex gigas forel, 1911 2 2 0 0 1 1 ectatomma edentatum roger, 1863 145 23 94 19 70 17 ectatomma lugens emery, 1894 243 21 104 18 117 21 ectatomma tuberculatum (olivier, 1792) 7 4 2 2 0 0 gnamptogenys horni (santschi, 1929) 52 19 17 13 21 13 gnamptogenys lineolata brown, 1993 8 5 6 3 0 0 gnamptogenys moelleri (forel, 1912) 28 1 0 0 20 1 gnamptogenys relicta (mann, 1916) 9 4 2 1 0 0 gnamptogenys sp. 02 5 3 1 1 3 2 gnamptogenys sp. 04 0 0 0 0 1 1 gnamptogenys sp. 07 1 1 0 0 0 0 gnamptogenys minuta (emery, 1896) 1 1 1 1 0 0 gnamptogenys striatula mayr, 1884 1 1 2 2 0 0 gnamptogenys tortuolosa (smith, 1858) 33 11 9 7 13 6 hypoponera sp. 01 9 1 0 0 8 2 hypoponera sp. 02 0 0 0 0 2 2 hypoponera sp. 03 1 1 0 0 2 1 hypoponera sp. 04 4 2 3 2 10 5 results sixty-four species/morphospecies of poneromorph ants were recorded combining the results of the maximum, intermediate and minimum efforts. using the maximum effort (14 days with 750 samples), a total of 944 poneromorph ants distributed in 58 species/morphospecies and 16 genera were recorded (table 1). using the minimum effort (2 days with 300 samples), 440 individuals distributed in 39 species and 14 genera were recorded. ectatomma spp. and pachycondyla spp. were the most abundant genera, independent of the applied effort. hypoponera spp. was the most diverse with 12 species. thirteen species were collected only once in 30 plots. fourteen genera were also captured in both reduced efforts. only two singletons genera (platythyrea sp. and typhlomyrmex sp.) were sampled with longer sampling period (14 days).the number of individuals caught decreased to more than a half (~53%) with a reduced number of pitfall traps, but the number of species/ morphospecies still represented about 67% (minimum effort) and 74% (intermediate effort) of the maximum effort. the regression line trends for the species accumulation curves were similar for all efforts (fig 2). the slope for the intermediate effort increased more rapidly compared to the table 1. abundance and frequency of poneromorph ant species (sensu bolton et al., 2005), sampled with pitfall traps in 30 250-m long plots in an amazonian rain forest. this data is deposited in the digital repository of the brazilian biodiversity research program (ppbio). cb gomes, jlp souza, e franklin – a comparison between time, number and cost of pitfall traps 142 sampling effort taxon 14 days 2 days 750 sub-samples 300 sub-samples 300 sub-samples abundance frequency abundance frequency abundance frequency hypoponera sp. 05 0 0 0 0 1 1 hypoponera sp. 06 10 7 4 3 1 1 hypoponera sp. 07 3 3 2 2 1 1 hypoponera sp. 08 0 0 0 0 1 1 hypoponera sp. 09 1 1 0 0 0 0 hypoponera sp. 10 1 1 1 1 0 0 hypoponera sp. 11 4 4 2 2 0 0 hypoponera sp. 12 1 1 0 0 0 0 leptogenys gaigei wheeler, 1923 16 9 7 6 5 4 leptogenys pusilla (emery, 1890) 1 1 1 1 1 1 leptogenys wheeleri forel, 1901 1 1 0 0 3 2 leptogenys sp. 03 2 2 2 2 0 0 leptogenys sp. 04 3 3 2 2 0 0 leptogenys sp. 05 1 1 1 1 0 0 mayaponera constricta (mayr, 1884) 109 20 51 16 29 14 neoponera apicalis (latreille, 1802) 15 7 10 5 2 2 neoponera commutata (roger, 1860) 10 4 5 2 1 1 neoponera laevigata (smith, 1858) 1 1 0 0 0 0 neoponera marginata (roger, 1861) 2 2 0 0 0 0 neoponera unidentata (mayr, 1862) 1 1 0 0 0 0 neoponera verenae (forel, 1922) 1 1 1 1 0 0 neoponera villosa (fabricius, 1804) 1 1 1 1 0 0 odontomachus bauri emery, 1892 2 2 1 1 0 0 odontomachus brunneus (patton, 1894) 0 0 0 0 1 1 odontomachus caelatus brown, 1976 16 7 12 5 10 3 odontomachus haematodus (linnaeus, 1758) 9 3 5 2 2 2 odontomachus laticeps roger, 1861 0 0 0 0 5 1 odontomachus meinerti forel, 1905 6 3 5 2 3 1 odontomachus opaciventris forel, 1899 2 1 2 1 9 4 odontomachus scalptus brown, 1978 12 5 2 1 2 1 odontomachus sp. 01 6 5 1 1 0 0 pachycondyla crassinoda (latreille, 1802) 94 18 39 13 54 17 pachycondyla harpax (fabricius, 1804) 22 14 16 10 19 13 pachycondyla impressa (roger, 1861) 3 2 3 2 0 0 paraponera clavata (fabricius, 1775) 2 2 1 1 1 1 platythyrea pilosula (smith, 1858) 1 1 1 1 0 0 prionopelta punctulata mayr, 1866 10 6 4 2 3 3 pseudoponera stigma (fabricius, 1804) 1 1 0 0 2 2 rasopone arhuaca (forel, 1901) 14 5 6 3 1 1 rasopone lunaris (emery, 1896) 2 1 2 1 0 0 typhlomyrmex rogenhoferi mayr, 1862 1 1 0 0 0 0 total number of individuals 944 441 440 total number of species 58 43 39 table 1. abundance and frequency of poneromorph ant species (sensu bolton et al., 2005), sampled with pitfall traps in 30 250-m long plots in an amazonian rain forest. this data is deposited in the digital repository of the brazilian biodiversity research program (ppbio). (cont.) sociobiology 65(2): 138-148 (june, 2018) 143 other treatments, although there was no significant difference amongst treatments. in general, all efforts are equivalent, since their confidence intervals overlap. the species composition of poneromorph ants (fig 3) differed for period of time and number of samples (np-manova: f2, 81= 83.07; r 2 = 0.664; p ≤ 0.001). the congruence between assemblages was affected by the time the pitfall traps were operating in the field. the three efforts had moderate similarity (r > 0.40). assemblages sampled during longer periods (14 days) were more similar to each other (r ~ 0.66) than when compared with the assemblage captured during two days (r ~ 0.43), regardless of the number of sampling traps used (table 2). multivariate regressions analysis indicated that the ecological pattern captured with the maximum effort was still captured in the two reduced effort treatments. in all tested efforts, soil clay content significantly affected the composition of the ground-dwelling ants. in addition, a consistent pattern of no significant relationship was captured for the terrain slope and volume of litter across all effort treatments (table 3). along the clay concentration gradient, some species that were mostly singletons and doubletons were restricted to the extremes of the gradient. species with a wider distribution tended to occupy more uniformly the entire gradient (fig 4). the relative monetary and time costs differed among efforts (table 4). the maximum effort was the most expensive. reduced efforts accounted to 60˗74% of the total costs and 55˗57% of the total time. discussion although trapping time influenced the similarity of the assemblages more than the number of sampling traps, the minimum effort (two trapping days using 300 pitfall traps) was still more effective at capturing ants compared with the 0 200 400 600 800 1000 0 20 40 60 80 number of individuals n um be r of s pe ci es pitfall traps − time 750 − 14 days 300 − 14 days 300 − 2 days fig 2. accumulation curves for poneromorph ant species assemblages captured in 30, 250 m long plots in the amazonian rain forest. dotted lines mark the 95% confidence intervals. −1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 nmds axis 1 n m d s a xi s 2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● time − pitfall traps 2 days (300) 14 days (300) 14 days (750) fig 3. an nmds ordination (stress = 0.148) plot indicating the congruence in poneromorph ant species associations among period of time and number of sampling traps in 30, 250-m long plots in an amazonian rain forest. sampling effort maximum effort treatment medium effort treatment minimum effort treatment maximum effort treatment 1 medium effort treatment 0.657** 1 minimum effort treatment 0.498** 0.427* 1 table 2. congruence between the poneromorph ant datasets sampled by the minimum (two days and 300 sampling traps), intermediate (14 days and 300 sampling traps), and maximum (14 days and 750 sampling traps) effort sampled along 30 250 m long plots in an amazonian rain forest. congruence was evaluated by symmetric procrustean rotations using sørensen dissimilarity index calculated for poneromorph ant species presence/absence data. p values were estimated by 999 monte carlo permutations. significance levels: *p ≤ 0.01; **p ≤ 0.001. sampling effort environmental variables 14 days 2 days 750 sampling traps 300 sampling traps 300 sampling traps clay content (partial) 0.0093** 0.0252* 0.0093** litter volume (partial) 0.6145 0.4243 0.6145 terrain slope (partial) 0.1634 0.1026 0.1634 r2 0.3188 0.3435 0.3188 p 0.0172* 0.0196* 0.0172* table 3. proportion of variance on the poneromorph ant species, assemblage composition explained by the environmental variables in multivariate regression models. significance levels: *p ≤ 0.01; **p ≤ 0.001. cb gomes, jlp souza, e franklin – a comparison between time, number and cost of pitfall traps 144 intermediate and maximum efforts. the sampling effort using 48 hours of pitfall-trap exposure has already been reported in previous studies (olson, 1991; bestelmeyr et al., 2000), as well as other studies that have evaluated the exposure time of the sampling traps (delabie et al., 2000; borgelt & new, 2006; schirmel et al., 2010). the 48 hours sampling table 4. summary of the relative effort (cost and time) required to process the material for poneromorph antspecies assemblages captured in two and 14 days in 30, 250-m long plots in an amazonian rain forest. sampling effort cost time % 14 days/750 sampling traps 100 100 14 days/300 sampling traps 74 57 2 days/300 sampling traps 60 55 fig 4. distribution of poneromorph ant species along clay content gradient in 30, 250m long plots at the ducke reserve. time protocol in this study did reduce the abundance of ants in traps to more than a half but was robust enough in describing the diversity of ground-dwelling ants, revealing the presence of more than 60% of the poneromorph ant species present in the surveyed area. the ecological patterns of ground-dwelling ants detected with the maximum effort treatments were also captured with reduced effort treatments, indicating that this pattern can be detected faster and cheaper, both in the field and in the laboratory. pitfall trapping time depends on the objective of work (delabie et al., 2000) and from the survey and monitoring standpoint. thus, reducing the number of pitfall traps from 750 to only 300 units and from 14 to only two days will greatly improve surveys effectiveness. this reduced time and trap sampling protocol will save about 40% of the cost and 45% of the resources used by the maximum effort treatment while maintaining the integrity of the ecological information collected. sociobiology 65(2): 138-148 (june, 2018) 145 most studies evaluating ant-sampling techniques usually test the taxonomic aspect, i.e. they use metrics related to species richness or composition (fisher, 1999; delabie et al., 2000; tista & fildler, 2010). as a result, most of the investigations on ant sampling protocols were also based on these metrics (olson, 1991; bestelmeyr et al., 2000; lopes & vasconcelos, 2008). more recent sampling protocol approaches have included ecological responses and environmental variables to evaluate the efficiency of the collection techniques (souza et al., 2009; 2012). this way, our analyses provide insights into which environmental variable is explaining the pattern of ant distribution. although it was expected that the reduced effort treatment may have incurred a loss of ecological information, all assemblages of grounddwelling ants captured within each one of the three treatments have their spatial distributions influenced by soil clay content. the ecological pattern of non-significant results for the effect of terrain and volume of litter also was maintained across all efforts. these results reveal that the use of reduced efforts is efficient in representing the ants sampling with maximum effort in both taxonomic and ecological approaches. changes in soil characteristics are related to the topography in the amazon basin (chauvel et al., 1987), the highest clay percentage is concentrated on the uplands, and the soils along stream valleys are particularly poorly drained (ranzani, 1980). in tropical rainforest, soil clay content has also been shown to affect the distribution of palms (costa et al., 2009), frogs (menin et al., 2007), oribatid mites (franklin et al., 2013) and ants (oliveira et al., 2009; vasconcelos et al., 2003; souza et al., 2007; 2012; 2016). the effect of each environmental factor varies depending on the scale of analysis (total area surveyed and the number of sampling units). due to the broad scale of this survey and the number of pitfall traps, the detected trends of significant and non-significant values of the environmental variables would be consistent even with efforts higher than those that we used here. especially in the amazon, financial costs limit the extent of biodiversity studies (costa and magnusson, 2010). the cost (time and money) of environmental monitoring is a crucial factor for the viability of biodiversity studies (margules & pressey, 2000), and any reliable alternative should be used to increase the area covered or the duration of the time series. in 2010, approximately us $ 0.01 per hectare was invested in monitoring actions in the brazilian amazon, and no increase is expected in the near future (magnusson et al., 2013). thus, we must consider that the financial resources saved both in the field and in the laboratory using lesser efforts can be used for biodiversity surveys in other areas. we suggest that only two days of exposure in the field and smaller numbers of pitfall traps is sufficient to capture a representative amount of individuals and species, and is effective in describing basic ecological attributes of poneromorph ants. the rarest species in this study were located at the extremes of clay content gradient, which are either valley areas with low soil clay content or high lands with a high concentration of clay. therefore, these extremes are peculiar and important environments for the maintenance of rare species. we believe that due to the ecological plasticity of ants, as well as their association with other organisms and their use as bioindicators, the results found here are likely to be used in general studies with ants, especially in situations where the time and cost of financing are limiting. consequently, investigators can process a smaller volume of biological material in the laboratory, and give faster results. effective monitoring is a vital tool for use in almost any program aiming at measure changing in the ecosystem. survey managers may want to apply the results of this study to extrapolate to another sampling universe. the consequence of a substantial reduction in exposure time and the number of sampling traps in the field, can decrease the time required to process samples in the laboratory, but can be crucial in the appropriate sampling of taxa or groups of taxa for quick monitoring and inventory of biodiversity. acknowledgements we thank everaldo pereira, juliana s. araújo and pollyana y.o. cavalcante, for their help in sampling ants. itanna o. fernandes confirmed the species identifications for this study. cristian s. dambros and victor l. landeiro developed the poncho and generico functions respectively, which allowed the construction of the species distribution figure along the environmental gradient. j.l.p.s. was supported by cnpq and fapeam post-doctoral scholarship, c.b.g. was supported by a master scholarship. financial support was provided by projects pipt/fapeam 1750/08, pnpd/capes 03017/19-05, fapeam 062.01325/2014, 062.00674/2015, cnpq, program for biodiversity research (ppbio) and the national institute for amazonian biodiversity (cenbam). data are maintained by ppbio and cenbam. references agosti, d. & alonso, l.e. 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(2013) noted that the ants most frequently involved abstract three compound nests of formica lemani bondroit, 1917 and lasius flavus (fabricius, 1782) are reported from lygra, western norway. this is the first plesiobiontic relationship reported for f. lemani and the 9th for l. flavus. behavioural and landscape ecological traits associated with plesiobiosis are discussed. sociobiology an international journal on social insects gm kvifte1, ta legøy2, j soulé2 article history edited by evandro n. silva, uefs, brazil rodrigo feitosa, ufpr, brazil received 23 march 2016 initial acceptance 09 may 2016 final acceptance 01 may 2017 publication date 17 october 2017 keywords plesiobiosis, interactions, mixtobiosis, commensalism, mixed colonies. corresponding author gunnar mikalsen kvifte dept. of natural history, university of bergen university museum of bergen p.o. box 7800 n-5040 bergen, norway e-mail: gunnar.kvifte@uib.no in plesiobiontic relationships tend to be comparatively less aggressive ants, and that plesiobiont partners tend to show little overlap in foraging strategies. the most frequently plesiobiont species in the holarctic region is formica fusca l., 1758; which forms part of more than 60% of observed plesiobiontic relationships (kanizsai et al., 2013). despite the two species formica lemani bondroit, 1917 and f. gagatoides ruzsky, 1904 being overall very similar to f. fusca in behaviour and biology (collingwood, 1979), no plesiobiontic associations of either species were listed by kanizsai et al. (2013). in this note we document the first record of plesiobiosis in the ant species formica lemani, from a nest shared with lasius flavus (fabricius, 1782) in a heathland ecosystem in norway. the observations contribute the 50th observed plesiobiont species and the 49th observed species association pair in holarctic ants. 1 department of natural history, university museum of bergen, university of bergen, norway 2 institute of biology, university of bergen, norway short note sociobiology 64(3): 366-368 (september, 2017) 367 material and methods ant nests were searched for under rocks and roots and in bryophytes on august 18th and 26th, 2015, at lyngheisenteret, lygra, western norway. the area is part of an open heathland landscape which is grazed by sheep through the year and managed by controlled burnings approximately once a decade. a total of nine people were involved in the search, which spanned around 100 m2 of area surrounding 60.700736° n, 5.100393° e. three different compound nests and one single-species nest were found, all under rocks, and voucher specimens of pupae and workers from each compound nest were collected for subsequent identification. specimens were identified using douwes et al. (2012). adult and pupal voucher specimens from two nests are stored in alcohol in the entomological collections at the university museum of bergen (zmub, collection numbers a-47723–a-47728, see table 1 for details). fig 1. detail of ant nest after removal of stone. a, lasius flavus pupae, b, lasius flavus worker, c, formica lemani worker, d, formica lemani pupae. museum id nest # species number of specimens collection date a-47723 1 lasius flavus 2 workers, 5 pupae 18.viii.2015 a-47724 1 formica lemani 7 workers 18.viii.2015 a-47725 2 lasius flavus 3 workers, 3 pupae 18.viii.2015 a-47726 2 formica lemani 1 worker, 1 pupa 18.viii.2015 a-47727 2 lasius flavus 6 alates, 5 workers, 1 pupa 26.viii.2015 a-47728 2 formica lemani 1 worker, 1 pupa 26.viii.2015 table 1. voucher material preserved in this study. results and discussion all three compound nests were found under rocks and contained workers and pupae of both lasius flavus and formica lemani. in one of the nests, six alate l. flavus were collected as well. in each nest, pupae of the different species were found in different clusters, separated by a few centimeters (fig 1). when the nests were uncovered, worker ants evacuated the pupae via apparently different systems of soil corridors. apart from this small separation, no difference in microhabitat use could be observed. adult workers could be observed among pupal clusters of different species, however this may be due to our disturbance of the colonies (fig 1). our observations are the first of f. lemani in a plesiobiontic relationship with another ant species. however, some of morley’s (1945) observations of plesiobiontic f. fusca may represent misidentified f. lemani since the observations were made prior to yarrow’s (1954) revision. nevertheless our observations represent the first confirmed case of f. lemani in a plesiobiontic relationship with another ant species, providing further evidence for collingwood’s (1979) claim that the habits of f. lemani are similar to f. fusca – the most frequently recorded plesiobiont in the palearctic region (kaniszai et al., 2013). workers of formica lemani and lasius flavus differ markedly in size and foraging behaviour. whereas f. lemani is a free-living and active predacious, aphidicolous and nectarivorous species, l. flavus is mostly subterranean and feeds on smaller arthropods and honeydew from root feeding aphids (collingwood, 1979; douwes et al., 2012). the resources exploited by each species thus show little overlap, permitting coexistence without competition. this follows the general pattern outlined for plesiobiontic relationships by kanizsai et al. (2013). colony sizes of the two species are listed in the literature as a few hundred to a few thousand for f. lemani and up to 100 000 workers for l. flavus (douwes et al., 2012). on the landscape level at the study site at lygra, our observation matches well with czechowski’s (2004) suggestion that plesiobiontic nests develop when the limiting factor controlling ant abundances is nesting site availability rather than food resources. our study area is dominated by calluna vulgaris heathlands with scattered moorlands, and all ant colonies we found were limited to a small rocky outcrop. it is thus likely that plesiobiontic relationships are common in the area, and further plesiobiont associations may well be observed here in the future. gm kvifte, ta legøy, j soulé – plesiobiosis between formica lemani and lasius flavus368 acknowledgements the fieldwork happened during a student field course arranged by the university of bergen, lead by johnarvid grytnes. we are indebted to him for encouragement and advice, and to our bio102 students for helping us searching for ant nests in the field. finally, we are grateful to frode ødegaard, norwegian institute for nature research, trondheim, for controlling our identifications of formica lemani. comments from two anonymous reviewers helped improve the manuscript. references collingwood, c.a. 1979: the formicidae (hymenoptera) of fennoscandia and denmark. fauna entomologica scandinavica, 8: 1-174. czechowski, w. 2004: scarcity of sites suitable for nesting promotes plesiobiosis in ants (hymenoptera: formicidae). entomologica fennica, 15: 211-218. retrieved from http://www. entomologicafennica.org/volume15/ef_15_4/3czechowski. pdf douwes, p., abenius, k., cederberg, b., wahlstedt, u., hall, k., starkenberg, m., reisborg, c. & östman, t. 2012: nationalnyckeln till sveriges flora och fauna. steklar: myrorgetingar. hymenoptera: formicidae-vespidae. uppsala: artdatabanken, slu, 382 pp. kanizsai, o., lörinczi, g. & gallé, l. 2013: nesting associations without interdependence: a preliminary review of plesiobiosis in ants. psyche, 2013: 238602. doi: 10.1155/2013/238602 morley, d.w. 1945: observations on some plesiobiotic colonies of ants (hymenoptera), with notes on some other mixtobiotic colonies. proceedings of the royal entomological society of london, 20: 1-4. doi:10.1111/j.1365-3032.1945.tb01041.x yarrow, i.h.h. 1954: the british ants allied to formica fusca l. (hym., formicidae). transactions of the society for british entomology, 11: 229-244. doi: 10.13102/sociobiology.v64i2.1396sociobiology 64(2): 202-211 (june, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 multiple aggressions among nestmates lead to weak dominance hampering primitively eusocial behaviour in an orchid bee introduction to understand the aspects of how bee sociality evolved from solitary individuals to group living is often the aim of evolutionary biologists (paxton et al., 2002; schwarz et al., 2007; kocher & paxton 2014; rehan et al., 2014; andrade et al., 2016). several insect societies rely on hierarchies to establish social organization. in eusocial insects for example the queen is the absolute reproductive dominant female while the workers (non-reproductive) are subordinated within welldefined activities organized in different castes (michener, 1974). despite the eusocial insects the species with plastic behaviour seem to be prone to better access social evolution. these species may offer cues of how the transition from abstract reproductive conflict expressed as aggression is common in social hymenoptera. in eusocial species, as in honeybees, several mechanisms however alleviate the conflicts and reduce aggressive interactions. unlike their sister group, the orchid bees do not exhibit eusocial behaviour. instead, some of the species presents a primitively eusocial behaviour, with a single dominant female and fertile subordinate females participating on egg laying activities. in the current study we investigated the aggressive interactions of euglossa annectans dressler females through five generations of phylopatry and reuse of the natal nest. although network analysis indicates that central individuals, those with more interactions, were more commonly the aggressors and others were more commonly the recipients, multiple attacks and several potential dominant females within the nest indicated a labile sociality. this suggests that there is an unstable social hierarchy in the species. euglossa annectans, despite having overlapping generations, during which several individuals share a nest, there is no division of labour into reproductive females and interactions are often competitive. aggressive behaviours conducted by multiple fertile females were often followed by egg, larvae or pupae replacement. sociobiology an international journal on social insects s boff1, ca saito2, ia santos1,2 article history edited by cândida aguiar, uefs, brazil received 29 january 2017 initial acceptance 24 march 2017 final acceptance 08 may 2017 publication date 21 september 2017 keywords euglossa annectans, female bias sex ratio, offspring replacement, reproductive conflict, social evolution. corresponding author samuel boff general biology and bioprospecting program, faculty of biology and environmental sciences federal university of grande dourados rodovia dourados-itahum, km 12cidade universitaria-unidade ii, cx postal 364 zipcode: 79804970, dourados, brasil. e-mail: samboff@gmail.com solitary to social or vice versa evolved (schwarz et al., 2007). thus, studies on species whose individuals exhibit more alternative or facultative social behaviour (compared to species with predetermined caste differentiation) may help to provide more clear evidences of how advanced forms of sociality may have evolved (dunn & richards, 2003; peso & richards, 2010; prager, 2014; may-itzá et al., 2014; rehan et al., 2014; andrade et al., 2016). the establishment of societies usually involves several behavioural traits which promote group living. the behavioural traits reflecting the interactions between reproductive conspecific females, for example, are often a key factor (ratnieks et al., 2006; rehan & richards, 2013; dolezal et al., 2014; andrade et al., 2016). in addition to 1 programa de pós-graduação em entomologia, faculdade de filosofia ciências e letras de ribeirão preto, universidade de são paulo (usp), ribeirão preto-sp, brazil 2 departamento ecologia, instituto de biociências, universidade de são paulo (usp), são paulo-sp, brazil research article bees sociobiology 64(2): 202-211 (june, 2017) 203 cooperative behaviour commonly seen between conspecifics in shared nests (wilson, 1971; schwarz et al., 1998; rehan et al., 2014) there can be reproductive conflict and aggression (breed et al., 1978; wcislo, 1997; rehan & richards, 2013) which are often negatively correlated with genetic relatedness (langer et al., 2004). thus, aggression arises because of, or is highly correlated with, genetic relatedness, or is highly correlated with selfishness. if on one hand higher degree of altruism is found in genetically related individuals on the other hand when resources at stake is important/rare genetically unrelated societies may display equal, if not more, degree of altruism and cooperation (andrade et al., 2016). reproductive conflict is wide spread in apidae bee species (crozier & pamilo, 1996; schwarz et al., 2007). although it is minimal in eusocial species, it has been described for small carpenter bees (rehan & richards, 2013), bumble bees (amsalem et al., 2009; zanette et al., 2012), stingless bees (peters et al., 1999; wenseleers et al., 2003) and orchid bees (augusto & garófalo, 2004; 2009; 2010; andrade-silva & nascimento, 2015). the latter group, the tribe euglossini, is sister to the apini (cardinal & danforth, 2011). in apini all species are eusocial. euglossini however do not exhibit eusocial species but are rather primitively eusocial with overlapping generation, subordination but lacks non-reproductive workers (kocher & paxton, 2014). in communal euglossini species, as it was described previously (garófalo et al. 1998; cameron 2004; otero et al. 2008), individuals share the same nest cavity with no interaction or alloparental care between the nestmates (da silva et al., 2016; dew et al., 2016). yet primitively eusocial orchid bee species show overlapping generations, some division of labour, dominance behaviour, offspring control through oophagy (cocom-pech et al., 2008; augusto & garofalo, 2009; 2010) and may present subordinate female specialization in guarding the nest entrance (boff et al., 2015). in parallel to eusocial species in which only the queen lays most of the eggs, worker policing behaviour, oophagy and social contracts helps to reduce potential reproductive conflicts (ratnieks, 1988; ratnieks et al., 2006; andrade et al., 2016). however, in the course of evolution some species may have selected traits in order to mitigate direct conflicts. physiological traits (e.g. body size, ovary development) and social experience were potentially traits selected to avoid aggressive encounters (wcislo, 1997; rehan & richards, 2010). in some species pheromones seem to play an important role controlling ovary development, making non-dominant females fail in reproduction and become loyal helpers (dor et al. 2005). in some primitively eusocial orchid bee species, egg-laying is performed by dominant females as well as by subordinate females increasing for instance the aggression ratio between nestmates (augusto & garófalo, 2009; 2010). in some bee societies, including orchid bees, totipotent individuals have plastic behaviour and can transit from one hierarchical condition to another (see crespi & yanega, 1995; schwarz et al., 2007; may-itzá et al., 2014). in the majority of species with totipotent individuals, reproductive dominance (queen-like vs worker-like females) and behavioural dominance (aggressive vs less aggressive individuals) can be predicted as it is usually the older or the bigger individual in the colony (schwarz, 1994). nevertheless it is not always easy to assign hierarchies for totipotent females since they can present a cryptic hierarchy (bang & gadagkar, 2012). furthermore in many totipotent social hymenoptera species, both queen-like and worker-like females are breeders (andrade-silva & nascimento, 2012; schwarz et al., 2007) and they both mate and produce offspring (sons and daughters) hence as the relatedness in the colony decreases, nestmates face more reproductive competition (langer et al., 2004). thus, competition and aggression between nestmates is often a mechanism used to achieve dominance on hierarchical societies (bang & gadagkar, 2015). in the current study, we investigated social interactions analysing the aggression exchanged between nestmates of euglossa annectans dressler a species previously described as communal (garófalo et al., 1998). moreover, we established levels of attacks displayed between co-specifics and we tested if they were different among categories. besides that, we tested if aggressive attacks towards nestmates could predict nest dominance. finally, we tested if the number of nestmates present in the nest predicts the number of brood cells in a given generation. material and methods nest development the study was conducted in the bee laboratory at the university of são paulo (23° 33’ s, 46° 43’ w), são paulo, brazil. the nest, previously occupied by tetragonisca angustula latreille (apidae: meliponini), was found in april 2010 in the garden of the bee laboratory with five brood cells with juxtaposed walls and only one active female of e. annectans called the founder female. the founder female disappeared from the nest before offspring emergences. at the end of the five brood emergences and during the sororal association of two females (parental phase) the observations ceased for a period of ca. 45 days, at the end of which there were 12 cells inside the nest with no female activity. at this point the nest was transferred inside the laboratory and access to the field was offered via a short entrance/exit rubber hose tube. the nest consisted of a wooden box 20 x 30 x 8cm with a glass lid in the top which facilitated observations. adult females present were manually removed from the nest and colour marked after placing them for two to five minutes in the fridge at 4 °c. for all generations, the nest was opened and adult females present in the nest marked only once. after marking the females, they were all delivered back to nest and their interactions were quantified. we recorded diurnal intranidal activities along two years from september 2010 (1st generation) to march 2012 (5th generation). the total of observation covered approximately s boff, ca saito, ia santos – labile society in neotropical orchid bee204 400 hours which were not evenly distributed across days of observation. we recorded data of their nest biology and of interactions between nestmates. daily observation time varied between 30 minutes and 6 hours. in the course of the study we recorded five generations, at least two generations per year, with overlap of generations during three generations (fig 1). the number of operculated cells was recorded directly by mapping non-sealed and sealed cells. sex ratio was assigned and tested through a non-parametric chi-square test. to visualize the position of the females in a central network analysis. according to the theory of central networks the most central individual is the one with the most interactions with the other nestmates (freeman, 1978). thus, using this approach, we were able to visualize the interactions among the females and indicate potential dominant (s) female (s), based on its centrality. aggression events in distinct generations we recorded aggressive interactions observing females from four different generations (from 2nd to 5th) which were easily distinguished based on the coloured mark on their thorax (n = 20). thus, in the 2nd and 3rd generation the observations were made on seven and eight nestmates, respectively and on two and three females, in the 4th and 5th generations, respectively. non-marked individuals (which emerged after the marking day) did not have the aggressive interaction recorded. we present the results of aggressive interactions in terms of frequency. we also investigated the presence of potential dominant (pd) female(s) according to the following index of dominance: pd=na-nra, where the potential dominant (pd) is assigned when the difference between the number of attacks (na) and the number of received attacks (nra) for the same female was positive (pd>0). by consequence a potential subordinate female is designated when the number of received attacks (injury) from the nestmate(s) is bigger than the number of attacks she made toward her nestmate(s) (pd<0). through this approach, with bee behaviour recorded individually, we analysed if the total number of attacks was different from the total number of received attacks using a paired t-test. out of these 20 females from four different generations, 10 attacked other females more than they were aggressed (potential dominant-pd, i.e. pd>0, see above explanation for potential dominant) and the other 10 females received more attacks than they initiated (potential subordinates-ps, i.e. pd<0). we first tested whether the number of attacks started by potential dominant and potential subordinate were different from the number of injures the same group received. then we tested whether the number of attacks of the potential dominant females was different from the number of attacks initiated by potential subordinated females. we also tested if the number of received attacks for the potential subordinated females was higher than the number of received attacks for the potential dominant females. we used non-parametric mann-whitney for all paired tests. moreover, during our observations, we recorded number of eggs laid in a given generation and the number of females that take part in the nest reactivation in the same generation. we then tested if both variables were correlated (pearson correlation). we also counted the frequency of egg replacement (conspecific brood parasitism) as well as the number of oophagy events carried out by the studied females. the statistical analyses were all conducted using ibm spss 19. fig 1. nest succession of euglossa annectans for two years between 2010-2012. during this time we recorded five generations. the horizontal bars show the length of time between a given generation when the first bee emerged until the period when the last bee from the same generation left the nest. the numbers over the horizontal bars followed by the symbol ♀ (female) indicate the number of active female during the generation. the light grey areas highlight the period of sororal (daughter-daughter) associations within one generation. overlapping generations were observed among all generations. previous accounts of this species reported no interactions among nestmates (garófalo et al. 1998). however as conflicts may be expected from reproductive conspecific females living together, we observed in the first generation and recorded (in the following generations) the number and duration of antagonistic interactions when body contact between nestmates was evident. the quantified aggressions were categorized according to its aggressive intensity (least aggressive “1” to most aggressive “3”) as follow: 1) females push another nestmate with brief body contact; 2) female attacks another nestmate using mandibles, bites her opponent, and the opponent turns away, and 3) females attacks another nestmate using her mandibles, bites her opponent, chases her and pushes the injured female until she has left the brood cluster. we tested whether the number of quantified aggressions was different within each category using a non-parametric chi-square test. aggression between distinct nestmates during the second generation, we tracked 150 pairwise intranidal agonistic interactions (attack vs. injury) between seven nestmates across 14 non-consecutive days. two females from this generation (which emerged after the marking day) were not colour marked. thus, they did not have their interaction recorded. we also investigated the interactions using the software social network visualizer (kalamaras, socnet v) in order sociobiology 64(2): 202-211 (june, 2017) 205 results nest development the number of eggs laid (counted by operculated cells), either in a new cell or in a reactivated one, was quite variable during the five generations. the highest number of operculations occurred during warmer season in the 5th generation followed by the 2nd generation. the lowest number of operculations occurred during the parental generation (table 1). the number of females which engaged in reactivating the nest along five generations was positively correlated with the number of operculated cells inside the nest (r = 0.987, p = 0.002). the number of days in the nest considering females that stayed longer than 5 days was 37.6 ± 33.88 dias (n = 34 females). one female remained in the nest for 134 days. we did not observe the entire number of brood emergences, except for the brood from the first generation when all the individuals (n = 12) developed into females. however, based on the total number of cells and recorded emergences, a significant female-biased sex ratio was recorded. even when the individuals that emerged and disappeared without been sexed and were theoretically included as males, there was still a bias to female production (x2 = 22.349, df = 1, p <0.001; fig 2). cells with juxtaposed walls, creating an single active cellular brood cluster, similar to a comb, although with brood cells in both, vertical and inclined directions (fig 3). in this context all females were laying eggs and operculating their cells in very close proximity to each other, leading to frequent encounters followed by aggressive behaviour. we recorded a total of 446 aggressive interactions a long four generations. taking into account only number of attacks towards a given female (n = 251), the most frequent attack registered was that belonging to category “3” (n = 143) followed by “2” (n=91). the least frequent was category “1” (n = 17). these results show that there were overall and pairwise significant difference between categories of attack (x2 overall= 239.086, df = 2, p <0.005; x2 1 vs. 2 = 50.704, df = 1, p <0.005; x 2 1 vs. 3 = 204.313, df = 1, p <0.005; x 2 2 vs. 3 = 79.854, df= 1, p = <0.005). the aggression with intensity “3” had the longest mean duration (4.2s), followed by intensity “2” (2.1s) and intensity “1” (1s). intranidal dynamics generation i ii iii iv v females emerging from previous generation 3 12 38 17 20 females that started reactivation 2 9 7 6 11 operculated cells 12 46 30 29 55 females emerging from current generation 12 38 17 20 30 table 1. a summary of sororal actvities across five generations of euglossa annectans. along the parental generation we did observe the mother leaving the nest but it did not bring any resources (eg. pollen or resin) before its complete disappearance after 30 days of nest sharing. except for the first generation, two remaining females from previous generations were still foraging and laying eggs when the first of their brood emerged. thus, in the beginning of the reproductive season females from previous generation (n = 2) and recent emergent female (s) took part in the production of a generation. the young females initiate a domestic work replacing resin inside the nest. they used to carry resin from the nest entrance to brood cells or to any other cavity in the nest or from cavities to brood cells or to the nest entrance. aggressive interactions we observed attacks of different levels (from “1” to “3”, see material and methods) between nestmates. a female was attacked when it occupied the brood cell area (particular area where a female lays her eggs) of one of its nestmates. all females of e. annectans in our observation nest built brood fig 2. number of females, males and unobserved emergences during five generations of euglossa annectans based on the observed number of brood cells except for those of the first generation, not all emerging bees were observed and sexed. the sex ratio was biased to females even when all non-sexed individuals (dotted bar) were theoretically considered as males (dotted bar + black bar = ♂ vs. grey bar = ♀; x2 = 22.349, df = 1, p <0.001). fig 3. nest of euglossa annectans in september 2010. during this phase three females were sharing the nest (the third female is missing in the picture). the picture shows females in a matrifilial association. the mother (on the left) is observing her daughter reactivating a brood cell. s boff, ca saito, ia santos – labile society in neotropical orchid bee206 network analysis between seven nestmates from the second generation identified the most central individual, tagged as female “1” (fig 4). the 150 recorded interactions of aggression among these nestmates showed female “1” to be responsible for the majority of attacks (44%) towards its nestmates, followed by female “2” (19.3%). the majority of attacks that female “2” (75%) received was made by female “1”. female “6” attacked the least (only female 1 and 7) and suffered attacks from all other nestmates (fig 4). female “1” was also the oldest, followed by females “2”, “3”, “4”, “5”, “6” and “7”. only female “1” and female “7” attacked more than they were attacked (pd >0, see table 2), although the latter interacted less than all other females. the other five (2-6) females (potential subordinated) were attacked more frequently than they initiated an attack (pd < 0, see table 2). across all five generations we did not observe a bee younger than this female “7” (1-3 days old) initiating a fight, but they were attacked if they walked over the brood cells of conspecific nestmates. although we did not track the number of eggs laid and the emergence of all females during the entire study, female “1” at this time had the highest reproductive investment since it laid more eggs (n = 6) than its nestmates. aggressive events from multiple generations out of 20 colour marked females of four generations (n = 7♀, 2nd generation; n =8 ♀, 3rd generation; n = 2♀, 4th generation; n = 3♀, 5th generation) 10 females (n = 2, 2nd generation; n = 6, 3rd generation; n=1, 4th; n = 1, 5th generation) conducted more attacks (potential dominant) towards other females compared to the number of attacks they received. the other 10 females (n = 5, 2nd generation; n=2, 3rd generation; n = 1, 4th generation; n = 2, 5th generation) were more frequently attacked (potential subordinated) compared to the number of attacks they initiated (fig 5). fig 4. results of the network analysis for seven females tracked in the second generation of euglossa annectans. in the graph of the network, a link between two nodes (two females) reports the interaction. the arrows (black) departing from one node (e.g. female “1”) to another node (female “5”) indicate the direction of an interaction, i.e. female “1” attacking female “2”. the absence of an arrow from one node to another node or a complete absence of a link indicates absence of an attack or an interaction, respectively. if two individuals exchange mutual attacks, two arrows (white) in opposite directions are attached to the link. the thickness of the link (an interaction) indicates how many interactions between two given nestmates were observed, and the size of the node indicates how much a given bee was attacked. thus, bigger nodes indicate a given female that received a greater number of attacks. thus the most central bee (female “1”) was interacting more than others and also more frequently initiated an aggressive event. every individual has a concentric circle showing the spatial place of each individual in the network. na female 1 2 3 4 5 6 7 total nra nra 1 13 3 1 0 4 3 24 2 33 11 0 0 0 0 44 3 18 7 0 0 0 0 25 4 6 0 0 2 0 3 11 5 5 6 0 2 0 7 20 6 3 3 3 4 3 3 19 7 1 0 0 0 4 2 7 total na 66 29 17 7 9 6 16 pi 0.44 0.193 0.113 0.046 0.006 0.004 0.106 pd=na-nra 42 -15 -8 -4 -11 -13 9 table 2. agonistic interactions of seven nestmates in a sororal association of euglossa annectans. na = number of attacks, nra = number of received a attacks, pi = percentage of interaction, pd = potential dominant. sociobiology 64(2): 202-211 (june, 2017) 207 the mean number of attacks initiated by marked females (potential dominant + potential subordinated) was not significantly different from the mean number of injuries the same group of females received (mann whitney u = 190.500, n1 = n2 = 20, p = 0.799, 1 tailed). nonetheless the number of attacks started by potential dominants was significantly higher when compared to the number of attacks started by potential subordinates (mann whitney u = 10.500, n1 = n2 = 10, p = 0.002, 1tailed). albeit slightly higher, the number of received attacks for potential subordinated females was not significantly different from the number of attacks received for potential dominant females (mann whitney u = 33.500, n1 = n2 = 10, p = 0.218, 1 tailed). we observed that all females (either potential dominant or potential subordinated) behaved as foraging, egg laying females. immature offspring replacement in the course of our observation, we recorded 60 events of two types of offspring replacement which occurred in general mainly during the wet season (55%).twenty eight of these replacements (1st case) occurred with immatures in advanced stages of larval or pupal development stage. after having its cells opened these injured juvenile-stage bees were removed either by pd or ps from their cells before they could reach adulthood. they were left outside their cell but inside the nest, where they died. after removing the immature bee adult female often worked in the brood cell and filled it with food provision, before using it to lay her own egg inside. the frequency of larval and pupal replacement occurred mainly during the dry season (n = 18) in comparison to wet season (n = 10). fig 5. agonistic interactions observed for 20 females of euglossa annectans across four generations. the interactions were noted for seven females from the second generation, eight females from third generation, two females from the fourth and three females from the fifth generation. the dark grey bars show the number of attacks started by a given female (indicated from 1 to 20). the light grey bars indicate when the same female was attacked by another female. we found no significant difference when both events were compared, attack vs injury; paired t test (mann whitney u=190.500, n1=n2=20, p=0.799, 1 tailed). the symbol “*” indicates a potential dominant female and the bees with no symbol indicate a potential subordinate female (mann whitney u = 10.500, n1= n2=10 p = 0.002, 1tailed). the other 32 (2nd case) offspring replacements were the result of oophagy.the majority of the observed oophagy events (60%) were made by a female which, during her lifetime, emptied the cell of another female only once. in 33% of the cases a female replaced the egg of another female twice. only one female was observed replacing three different eggs from three different brood cells. only in one case was an egg replacement observed up to three times in the same cell. egg replacement was more often recorded during the wet season (n = 23) than in the dry season (n = 9). during the study the mean number of oophagies per female, considering 18 females across all 5 generations, was 1.44 (±0.61). the oophagies were performed by females from different generation as well as by females of the same generation. after oophagy females were observed bringing fresh pollen and added it to the pollen uneaten by the previous immature bees. all 60 events of replacement happened while the female that layed the initial egg was not inside the nest, suggesting that her presence in the nest is a very efficient deterrent. discussion the females of the orchid bee e. annectans studied here are highly aggressive and the conflict between nestmates seems to be part of the intranidal female offspring control. several females from the same and different generations were laying eggs (which developed into females) at the same time but live in a conspicuous reproductive conflict. although we did find a clear pattern of dominance in the 2nd generation, we are aware that it was found in a reduced dataset. considering s boff, ca saito, ia santos – labile society in neotropical orchid bee208 the entire data set (four different generations) the patterns of aggressive interactions (non directional but instead with multiple directions) may suggest that dominance itself is not concentrated by only one female. the fact that the potential dominant was often attacked shows how loose her control actually was. unlike previous assumptions made for this orchid bee species, mentioned as communal since nestmates did not interact socially and only shared nest cavity, the females of e. annectans that were studied here exhibit an advanced or a transitional case of communal social organization to primitively eusocial. there were some social interactions (e.g. following behaviour, mutual attack, defense against intruders) between mother and daughters (matrifilial association) and also between females of the same generation (sororal association). although we have found a clear dominance hierarchy, the presence of only one female which adressess unidirectional attacks towards her subordinates, as reported for euglossa fimbriata moure, 1968 and euglossa melanotricha moure, 1967 (augusto & garófalo, 2009; andrade-silva & nascimento, 2015) is lacking in e. annectans. instead several females attacked each other as well as multiple females replaced offspring. those traits may broaden the complexity of more egalitarian societies since it would help to control reproductive skew. we observed some criteria of social behaviour, according to michener (1974): overlapping generations, aggression and egg replacement between nestmates, group nest defense (when the nest was invaded by non-identified ants and swarming individuals of stingless bees) and cooperative work between young females (1-3 days old) which replaced resin within the nest. this common behaviour trait developed by young bees seems to maximize resin use inside the nest. in contrast to primitively eusocial orchid bees there was no evidence for the presence of a single dominant female as seen for euglossa atroveneta dressler, 1978 (ramirez-arriaga et al., 1996), e. fimbriata and euglossa cordata linnaeus, 1758 (augusto & garófalo, 2009; 2010, respectively) and e. melanotricha (andrade-silva & nascimento, 2012; 2015; andrade et al., 2016) neither for the presence of a subordinated female specialized in guarding the nest entrance as reported for euglossa viridissima friese, 1899 (boff et al., 2015). it is known that age plays an important role in dominance and by consequence, aggression (augusto & garófalo, 2009; rehan & richards, 2013; andrade-silva & nascimento, 2015). the agonistic behaviour recorded in our study was frequently started by the same, oldest female. during the tracked interactions, we observed a presumed dominance hierarchy. the observed aggressions were mainly made by only one female (oldest female) followed by a two days younger female which received 20% of all registered aggression. this hierarchy reflected the distribution of eggs and brood cells: the brood cluster of the oldest female (1) was the largest (six cells) compared to the other tracked females whose brood cluster varied from one to five cells. brood cluster size and aggression between nestmates has been correlated in different bee societies (batra, 1978; kukuk, 1992; moritz & neumann, 2004; augusto & garófalo, 2010; rehan & richards, 2013). in the current study as all females behaved as foragers and they also layed eggs, the total costs per nest involved to produced an offspring might be higher when compared to eusocial species in which the dominant female is far less prone to be predated, since it rarely leaves the nest. thus, the more eggs a female lays the more aggressive it may become because of its higher reproductive investment. in the social network analysis we found that the most central position is occupied by female “1”. according to the centrality theory, more central individuals are more closely connected by interaction with all other individuals in the network (freeman, 1978). in our analysis, centrality indicates potential dominance since female “1” interacted and aggressed more than all the other females. she also had the largest brood cluster. the frequency of attacks by female “2” was lower than the frequency of attacks by female “1”. moreover, the former was more frequently attacked by female “1” than by any other female. although there was no significant evidence for a single dominant female, these bees seem to be in a hierarchical tug-of-war (langer et al., 2004), when multiple females display dominant behaviour and do not stop trying to take the dominant position. however, it may be the case that the older individuals are more prone to be aggressive towards other females as a consequence of their greater reproductive investment in the nest. thus, oophagy or immature replacement seems to be an opportunistic behaviour either as an element of tug-of-war or parental parasitism. although centrality seems to be a robust approach for assigning dominance it needs to be considered with caution since network interactions resulted from only one generation. the parental parasitism hypothesis introduced by charnov (1978) and adjusted for dominant females replacing the eggs of subordinated females (field, 1992) proposes that daughters or younger females are submissive towards an older nestmate. thus, unlike of what has been described for e. cordata, e. fimbriata, e. melanotricha, and e. viridissima in which the egg replacement occurs largely by one dominant female, in e. annectans the egg replacements were made by several females occupying the nest at the same time. this occurrence of multiple oophagies and/or the replacement of non-emerged bees better supports the tug-of-war hypothesis than the parental parasitism hypothesis. however the former hypothesis must be carefully interpreted here because the tug-of-war hypothesis originally also explains reproductive skew, an adaptative trait that describes the unequal sharing of reproduction within the group (trubenová & hager, 2012). thus, the lack of a single dominant female may hampers e. annectans from becoming primitively eusocial. we did not notice any female replacing its own egg, which seems to indicate that females were able to recognise their own brood cells and the eggs of their nestmates. thus aggression between nestmates in e. annectans might be an adaptive response to the multiple oophagies in the nest. in the sociobiology 64(2): 202-211 (june, 2017) 209 primitively eusocial orchid bee e. fimbriata and e. cordata species, only one female has been detected replacing eggs of her nestmates (augusto & garófalo, 2009; 2010, respectively). however in the primitively eusocial e. viridissima daughters were observed replacing eggs from mothers (cocom-pech et al., 2008). in the eusocial species apis mellifera linnaeus, female workers are responsible for oophagy of the eggs laid by other workers (policing behaviour) and are also able to eliminate some individuals, e.g. diploid males, but they do not replace eggs (ratnieks 1998). in additon to egg replacement the behaviour of removing larvae/pupae from its nestmate`s cell was similar to the behaviour of hoplostelis bilineolata (spinola, 1841) (hymenoptera, megachilidae), in nests of e. cordata (augusto & garófalo, 1998). in e. annectans after the immature was dragged out from its brood cell to the nest floor it was completely ignored by any other nestmate within the nest while h. bilineolata places resin on the larvae of e. cordata (augusto & garófalo, 1998). in previous studies regarding social interactions among individuals of euglossini females, there was no evidence for attacks made by more then one female in a given nest. the exception was the communal euglossa nigropilosa moure, 1965. although in this species multiple individuals attack each other, interactions do not seem to be associated with dominance but are related to the presence of territories in the nest (otero et al., 2008). understanding the mechanisms of the major evolutionary transition from solitary to group living including their losses and benefits is one of the challenges for evolutionary biologists. the studies of species showing advanced levels of eusociality like the honeybee a. mellifera have contributed to knowledge about caste differentiation, division of labour and several conflicts involving workers and queens. the studies on primitively eusocial species have contributed to understand not only the transition from solitary to social but the adaptations, costs and benefits for sociality (schwarz et al., 1998; schwarz et al., 2007; kocher & paxton, 2014; prager, 2014; rehan et al., 2014; andrade et al., 2016). this study shows that e. annectans has a unique social structure that exhibits aspects of communal and primitively eusocial societies where multiple females are prone to attack each other and multiple female are performing oophagy. this behaviour leads to a weak dominance in e. annectans different from e. cordata whose dominant females performed oophagy and replaced all the eggs of the subordinate with her own (freiria et al., 2017). albeit e. annectans is in a close phylogenetic relationship to honey bees and other socially advanced apid species, it shows a less advanced social organization (romiguier et al., 2015). our find showed the importance of long term nest observations, making e. annecatns an important system to study female totipotency and reproductive hierarchy formation in the apidae. topics that motivates further exploration which may provide 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(1971). the insect societies. belknap press of harvard university press, cambridge, mass. zanette, l. r. s., miller, s. d. l., faria, c. m. a., almond, e. j., huggins, t. j., jordan, w. c. & bourke, a. f. g. (2012). reproductive conflict in bumblebees and the evolution of worker policing. evolution, 66: 3765-3777. doi: 10.1111/j.15585646.2012.01709.x doi: 10.13102/sociobiology.v65i1.2095sociobiology 65(1): 108-111 (march, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 suitable light regimes for filming termites in laboratory bioassays introduction laboratory bioassays require methods to keep animals in conditions as close as possible to their natural ambiance, so that to minimize stress and keep individuals alive as long as the test demands. however, some experimental procedures do require unnatural, potentially stressful conditions, such as the illumination demanded for video recordings of termite behavior. after all, by living inside nests and galleries and foraging only inside tunnels, most termites are better thought as accustomed to dark than to light conditions. illumination normally needed for video recordings would, hence, be a potential source of stress for these insects. in fact, termite workers and soldiers – the castes normally used in bioassays – show some aversion to open air conditions, ducking to darker places when their mound is opened (williams, 1959) or when exposed to incandescent light in experimental arenas (park & raina, 2005). being eyeless, these termites would not be thought to be bothered by light, escaping to darken areas for reasons other than typical photophobia. further studies, however, have shown that ultraviolet light percolating abstract laboratory bioassays require strategies to minimize stress and keep animals alive as long as the test demands. sometimes, however, experimental procedures seem notoriously stressful as, for instance, when exposing termites to the illumination needed for video recordings. being a condition opposed to what termites naturally experience, light might easily stress such insects or might not affect them at all, as they are blind. here we check for the effects of distinct light regimes on the survival of termites confined in a typical bioassay setup involving footage. the survival of cornitermes cumulans (termitidae: syntermitinae) workers kept in the dark, or subjected to infrared or to cold white light was compared, finding no statistical difference in their survival in these three treatments. while pointing directions for further research on the reasons for such results, we conclude that video recordings of c. cumulans termite workers can be conducted under infrared or cold white leds, as these light regimes do not affect the survival of tested individuals. sociobiology an international journal on social insects yc carvalho, lo clemente, mp guimarães, o desouza article history edited by gilberto m. m. santos, uefs, brazil received 26 september 2017 initial acceptance 06 january 2018 final acceptance 06 january 2018 publication date 30 march 2018 keywords cornitermes cumulans, infrared, led, isoptera. corresponding author og de souza lab of termitology depto entomology federal university of viçosa cep 36570-900, viçosa-mg, brazil e-mail: og.souza@ufv.br through the unpigmented cuticle of reticulitermes spp. can harmfully react with an alkaloid norharmane present in their hemolymph, impairing these termites’ survival (siderhurst et al., 2006) whether or not unpigmented termites other than reticulitermes would equally be harmed by uv-light exposure, remains to be tested. after all, norharmane is produced by endosymbionts (siderhurst et al., 2005) whose species composition varies across termites (ohkuma, 2008). it also remains to be tested whether other electromagnetic wavelengths could harm termites as uv-light does. on the other hand, even species such as coptotermes formosanus, presenting low norharmane amounts (itakura et al., 2008), do show aversion to light (park & raina, 2005), reinforcing the idea of light as a potential source of stress, irrespective of norhamane content, at least for the so called “lower termites”. on their turn, many “higher termites”, belonging to the family termitidae, present unpigmented workers and cryptic habits similar to those of reticulitermes and coptotermes, being hence potentially affected by light exposure. among lab of termitology, department of entomology, federal university of viçosa, viçosa-mg, brazil research article termites mailto:og.souza@ufv.br sociobiology 65(1): 108-111 (march, 2018) special issue 109 these, the neotropical cornitermes cumulans, would seem to deserve attention. being easily captured from conspicuous and populous nests, this species large individuals allow video recording without macro lenses, and are hence an obvious choice for lab bioassays footage. miramontes et al. (2014), for instance, studying patterns of spatial exploratory behavior in individual termites, video-recorded the movement of c. cumulans workers continuously for 5-6 hours. in order to accurately track every termite cartesian location during the assay, the arena was constantly illuminated by a cold white fluorescent lamp. here we intend to contribute to this type of study, testing the effects of distinct light regimes on the survival of c. cumulans workers confined in arenas, as typically done in many lab bioassays. three light regimes were tested: no lights (i.e. darkness), infrared, and cold white lights. infrared illumination is the closest to darkness an affordable camera could record whereas cold white illumination is suitable to footage by any type of camera. if c. cumulans is harmed by illumination (as in reticulitermes affected by uv-lights), we expect their survival to differ between these light regimes, no difference in survival being found otherwise. materials and methods focal species c. cumulans live inside epigeous nests and feed on living and dead grasses and herbs, which they reach through subterranean tunnels, occasionally foraging under a fine layer of soil-sheeting (de negret & redford, 1982). their mounds are abundant in grasslands in the neotropics, and are found in pastures, monocultures, savannas (araujo, 1970) and even in gardens in urban areas. this species builds mounds with a very hard outer shell of soil which surrounds a soft inner core of carton (fecal material, comminuted plant material and bits of soil) (de negret & redford, 1982). c. cumulans is a key species in grasslands, providing stable and predictable shelter for other termites, invertebrates, and vertebrates (redford, 1984; campbell et al., 2016). data collection the study was carried out in viçosa, in the state of minas gerais state, southeastern brazil, located at 20º45’ s 42º51’ w, in the facilities of the lab of termitology, federal university of viçosa (‘ufv’ in brazilian acronym). the experiment aimed to verify the effect of distinct illumination regimes on the survival of c. cumulans workers kept in petri dishes in the lab. termites were collected from colonies found in the ufv’s campus on three occasions: 30 may, 2nd june and 5th june 2017. fragments from three different nests were collected per date, totaling nine nests. each nest fragment was taken to the lab, where 20 workers (third instar and beyond) were extracted to form an experimental unit confined in a closed petri dish (59 mm internal diameter). a total of nine experimental units were formed, three for each illumination regime: (i) dark; (ii) cold white led (ourolux™12w; color temperature: 6500k; luminous flux: 1200lm; luminous efficacy: 100lm/w); (iii) infrared light (ils, oslon ir 1 powerstar ir star led, peak wavelength: 850nm, radiant flux 1070mw, radiance angle: ±45, 2-pin, smd, using current=1.12a;voltage=15v). experimental units were then taken to bod incubator, under 25ºc±1, within which one of the three types of illumination regimes was set up. no food or water was provided in the petri dishes. since group size can affect survival in termites, and this survival is associated with the spatial density termites are subjected to, we kept each experimental group at a density of 0.14 (the ratio between the total area occupied by termites and the total area of the arena’s floor). this is within the range 0.10 to 0.19 considered to favor survival in termites (miramontes & desouza, 1996; desouza et al., 2001; desouza & miramontes, 2004) counting of dead and live individuals proceeded every hour in each petri dish for the first 12 h after which it was halted for the next 8 hours. then the hourly tally was resumed until all individuals were dead. data analysis data were subjected to censored survival analysis under weibull distribution, similar to what desouza et al. (2009) have done, using survival package in r (r core team, 2015). survival analysis is the statistical analysis of data where the response of interest is the time, t, from a well-defined time origin to the occurrence of some given event (end-point) (martinussen & scheike, 2006). in our case, time origin is when we put the petri dish inside the bod incubator and the end-point is when we record the death of a termite worker in this arena. the general model for this analysis follows the equation: loge s(t)=μ α t α where s(t) is the accumulated proportion of dead termites until time, t; the mean time μ is the time elapsed until 50% of termites are dead in a given treatment; and α is the shape parameter for the survival curve. when α=1, the probability of a termite dying does not change as time elapses. if α<1 this probability reduces as time elapses, the opposite happening when α>1. statistical analysis was used to check whether the light regime would affect the mean time, μ, until termites are found dead in a given treatment. under the null hypothesis, the mean time, μ, does not differ between light regimes, hence, time, t, alone explains s(t). that is, under the null hypothesis, termite deaths are not speeded up nor delayed by the light regime. alternatively, if the light regime affects the time at which a termite dies, a typical μ can be calculated for each regime. modelling proceeded by building a full model with a single term, light regime, represented by a categorical variable with three levels: darkness, infrared light, and white light. model simplification was performed removing this term from the full model and inspecting the consequent change yc carvalho et al. – suitable lights for filming termites in laboratory assays110 in deviance. in case of significant changes (i.e., p≤0.05), the term would have been returned to the model to proceed further simplification amalgamating its levels. results cornitermes cumulans termite workers confined in petri dishes in a bod incubator kept at 25ºc did not show any obvious quantitative changes in behavior along the whole experiment. within each of these experimental arenas, termites circulated normally, stopping to interact or simply passing by when meeting a given conspecific. interactions involved antennations, allogrooming, mouth-to-mouth contacts (presumably, oral trophallaxis), and occasional mouth-to-anus contacts (presumably, anal trophallaxis). survival curves presented a shape parameter α>1 (α=3.369), implying that mortality tended to be more expressive towards the end of the experiment. in fact, after 500 min had elapsed, more than 80% of the termites were still alive in all treatments. mortality crossed the 50% threshold more than 1000 min after the beginning of the experiment (fig 1). light intensity experienced by termites in the experimental units varied from 0 lux (darkness) to 5 lux (infrared) to 672 lux (white led light). this has led to an average survival time of 1085 min for termites kept in the dark, 1065 min for termites kept under white led light and 1102 min for termites exposed to infrared light. these timings and hence their respective light regimes, however, did not differ statistically (table 1, p=0.31). discussion we did not observe any effect of the distinct light regimes on the mortality of c. cumulans workers in this work: such termites presented similar survival patterns when confined in darkness, under cold white light, or under infrared light (fig 1, table 1). while arising intriguing theoretical issues, these results have important practical connotations. on the theoretical front, these results would evoke the question of why these termites, as opposed to reticulitermes, were not harmed by the light regimes under test? among plausible hypotheses, one has to consider the possibility that the light regime of highest intensity here applied (672 lux) was still below any harmful potential. this hypothesis, still to be tested, gains significance from the fact that daylight illuminance under bright sun attains not less than 100,000 lux, or 20,000 lux in the shade. an alternative hypothesis would point to the absence of phototoxic compounds in the hemolymph of c. cumulans. in order to answer it, one would have to inspect the presence of compounds such as norhamane, which is responsible for phototoxic effects in other termites (siderhurst et al., 2006). clearly, these hypotheses are beyond the aims of this work but we present them here in an attempt to sign the research avenues they could open. on the practical front, our results validate the use of infrared and cold white lights in studies that demand video recording with c. cumulans, as none of these light regimes affected workers survival. being closest to dark that we can record with a video camera, infrared would seem the best choice for such assays. as a drawback, not any camera can record video under infrared, and those which can, are normally more expensive (albeit many are still within the affordable price range). compared to infrared lights, cold white leds are cheaper and easier to find. better still, all cameras can record under this type of light. as a word of caution, it must be considered that our test regards survival only. despite not observing any obvious behavioral changes while counting dead and alive individuals for our test, no quantitative behavioral parameter was inspected (and this is, incidentally, another possible development from the current work). we conclude, therefore, that video recordings of c. cumulans termite workers can be conducted under infrared or cold white leds, as these light regimes do not affect the survival of tested individuals. acknowledgements we thank helder hugo dos santos for providing us the infrared lights used in this study. this work was partially supported by cnpq, fapemig, capes. ycc, loc, mpg hold msc grants from capes. ods holds a cnpq fellowship (#307990/20176). this work is part of the iv symtermes community at https://zenodo.org/communities/symtermes/?page=1&size=20, where it is identified by doi 10.5281/zenodo.894421. this is contribution # 71 from the lab of termitology at federal univ. of viçosa, brazil (http://www.isoptera.ufv.br). fig 1. survival of c. cumulans workers confined in petri dishes in the dark, exposed to white lights, or exposed to infrared lights, in a bod incubator kept at 25ºc. curves do not differ statistically (p=0.31). df deviance resid. df -2*ll pr (> χ²) null 178.00 2660.90 light regime 2.00 2.31 176.00 2658.59 0.31 table 1. analysis of deviance for the effects of light regime on the survival of c. cumulans termite workers confined in petri dishes. three light regimes were applied continuously to the experimental arenas: darkness, infrared light, and cold white light. see material & methods for more detail. “ll” = log-likelihood. sociobiology 65(1): 108-111 (march, 2018) special issue 111 references araujo, r.l. (1970). termites of the neotropical region. in k. krishna & f.m. weesner (eds.), biology of termites, vol ii (pp. 527–576). new york: academic press. campbell, c., russo, l., marins, a., desouza, o., schönrogge, k., mortensen, d., tooker, j., albert, r. & shea, k. (2016). top-down network analysis characterizes hidden termitetermite interactions. ecology and evolution. doi: 10.1002/ ece3.2313. de negret, h.r.c. & redford, k.h. (1982). the biology of nine termite species (isoptera: termitidae) from the cerrado of central brazil. psyche, 89: 81–106. desouza, o., araújo, a. & reis-jr, r. (2009). trophic controls delaying foraging by termites: reasons for the ground being brown? bulletin of entomological research, 99: 603–609. desouza, o. & miramontes, o. (2004). non-asymptotic trends in the social facilitated survival of termites (isoptera). sociobiology, 44: 527–538. desouza, o., miramontes, o., santos, c. & bernardo, d. 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(2008). symbioses of flagellates and prokaryotes in the gut of lower termites. trends in microbiology, 16: 345– 352. doi: 10.1016/j.tim.2008.04.004. park, y. & raina, a. (2005). light sensitivity in workers and soldiers of the formosan subterranean termite, coptotermes formosanus (isoptera: rhinotermitidae). sociobiology, 45: 367-376. r core team (2015). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url http://www.r-project.org/. redford, k.h. (1984). the termitaria of cornitermes cumulans (isoptera, termitidae) and their role in determining a potential keystone species. biotropica, (pp. 112–119). siderhurst, m.s., james, d.m. & bjostad, l.b. (2006). ultraviolet light induced autophototoxicity and negative phototaxis in reticulitermes termites (isoptera: rhinotermitidae). sociobiology, 48: 27–49. siderhurst, m.s., james, d.m., blunt, t.d. & bjostad, l.b. (2005). endosymbiont biosynthesis of norharmane in reticulitermes termites (isoptera: rhinotermitidae). sociobiology, 45: 687–705. williams, r. (1959). flight and colony foundation in two cubitermes species (isoptera: termitidae). insectes sociaux, 6: 203–218. http://www.r-project.org/ open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 50-55 (2013) exotic spread of solenopsis invicta buren (hymenoptera: formicidae) beyond north america jk wetterer introduction more than 100 years ago, forel (1911) compiled a list of 15 tramp ant species, spread by human commerce, which had achieved or were in the process of achieving cosmopolitan distributions. eight of these have become major ecological, agricultural, and/or household pests: anoplolepis gracilipes (smith), linepithema humile (mayr), monomorium destructor (jerdon), monomorium pharaonis (l.), paratrechina longicornis (latreille), pheidole megacephala (fabricius), solenopsis geminata (fabricius), and tapinoma melanocephalum (fabricius) (wetterer 2005, 2007, 2008, 2009a, b, 2011, wetterer et al. 2009). over the past 100 years, many additional ant species, not on forel’s (1911) list, have begun to spread around the world. the most notorious of these is the fire ant solenopsis invicta buren. abstract the south america fire ant solenopsis invicta buren arrived in mobile, alabama by ship sometime before 1945. since then, s. invicta has spread in north america across the southern us and northeastern mexico. more recently, s. invicta has invaded the west indies and parts of the old world. here, i examine this more recent exotic spread of s. invicta beyond north america, reporting new west indian records and questioning some asian records. in 1981, s. invicta was first found in the west indies, on puerto rico. with my new records from vieques, aruba, and jamaica, s. invicta is now known from 28 west indian islands. in 2001, the first old world populations of s. invicta were discovered in new zealand and australia. nascent populations of s. invicta in new zealand have been exterminated and australia populations have been kept in check through intensive control efforts. populations of s. invicta in taiwan and china first found in 2003-2004, however, have spread broadly. published reports of s. invicta from malaysia and singapore were based on misidentifications, presumably of the more widespread neotropical fire ant, solenopsis geminata (fabricius). reports of s. invicta from india and the philippines seem questionable and need confirmation. where s. invicta has invaded, it has displaced s. geminata in open habitats, leaving remnant s. geminata populations, primarily in forested areas. in working to limit the spread and impacts of fire ants, it will be important to differentiate among the species, and recognize their similarities and their differences. sociobiology an international journal on social insects florida atlantic university, jupiter, florida, usa research article ants article history edited by: kleber del-claro, ufu brazil received 07 november 2012 initial acceptance 03 december 2012 final acceptance 17 december 2012 keywords biological invasion, exotic species, invasive species, stinging ants corresponding author james k. wetterer wilkes honors college florida atlantic university 5353, parkside drive, jupiter, fl 33458, usa e-mail: wetterer@fau.edu solenopsis invicta is well known for its painful sting, which in humans causes a burning sensation, usually followed in a day or two by the appearance of a white pustule. the venom can cause severe allergic responses and result in secondary infections, sepsis, anaphylactic shock, and even death (prahlow & barnard 1998). solenopsis invicta poses a threat to wildlife (allen et al. 2004). for example, s. invicta attacks and kills hatchling sea turtles (allen et al. 2001, parris et al. 2002, krahe et al. 2003, krahe 2005). originally from the grasslands of south america, s. invicta arrived in north america by ship at the port of mobile, alabama apparently between 1933 and 1945 (buren et al. 1974) and has spread across the southern us and northeastern mexico, particularly in open disturbed areas, causing ecological and economic damage. many studies have documented the current and potential range of s. invicta within sociobiology 60(1): 50-55 (2013) 51 north america (e.g., callcott & collins 1996, korzukhin et al. 2001). this dreaded ant is now spreading through the west indies (wetterer & davis 2010) and has recently arrived in new zealand, australia, and parts of asia (chen et al. 2005, zhang et al. 2007, ascunce et al. 2011). climate tolerance models predict that s. invicta should be able to successfully invade many additional regions in both the new world and old world (morrison et al. 2004, sutherst & maywald 2005). here, i examine this recent exotic spread of s. invicta beyond north america, reporting new records for the west indies and questioning some published records from asia. methods using published and unpublished records, i documented the worldwide range of s. invicta. i obtained unpublished site records from museum specimens in the collections of archbold biological station (abs, identified by m. deyrup) and the museum of comparative zoology (mcz, identified by s. cover). in addition, i used on-line databases with collection information on specimens by antweb (www. antweb.org), and the global biodiversity information facility (www.gbif.org). geographic coordinates for collection sites came from published references, specimen labels, maps, or geography web sites (e.g., earth.google.com, www.tageo.com, and www.fallingrain.com). if a site record listed a geographic region rather than a “point locale,” and i had no other record for this region, i used the coordinates of the largest town within the region or, in the case of small islands and natural areas, the center of the region. i made one exception, for peru. trager (1991) and pitts (2002) both listed s. invicta from peru with no site, but peru is a large country, so i mapped the record to esperanza, the city in peru closest to a known s. invicta population (in brazil). taber (2000) presented a range map with s. invicta only in this part of peru. in total, i plotted >1600 site records for s. invicta (fig. 1). results i collected solenopsis invicta in the southeastern us and on west indian islands (vouchers deposited in harvard university’s museum of comparative zoology). i documented s. invicta records from 28 west indian islands (table 1), including its first records from vieques (seven sites: colonia lujan, trees by pasture, 18.116n, 65.440w, 13 october 2005; montealta, forest patch, 18.143n, 65.454w, 13 october 2005; west of puerto martineau, route 200 at 4 km mark, 18.141n, 65.479w, 18 october 2005; east of airport, route 200 at 5 km mark, 18.137n, 65.487w, 18 october 2005; pier turn-off, route 200, 18.130n, 65.513w, 18 october 2005; mosquito pier, acacia near end, 18.148n, 65.513w, 7 june 2006; santa maria, baseball field, 18.154, 65.432w, 8 june 2006), aruba (two sites: malmok, tierra del sol golf course, 12.607n, 70.043w, 3 august 2007; oranjestad, divi village golf course, 12.538n, 70.058w, 3 august 2007), and jamaica (one site: cinnamon hill, cinnamon hill golf course, 18.517n, 77.813w, 20 december 2010). the issg (2010) website reported records of s. invicta from three of the cayman islands (table 1), citing “burton, 2003 in varnham, 2006,” though varnham (2006) gives no specific island records. f. burton (pers. comm.) could not recall the original source of the s. invicta records, however, in 2008, i collected s. invicta on grand cayman. it would be useful to confirm records from cayman brac and little cayman. for published old world records of s. invicta, site records and species identity are well documented for exotic populations in new zealand, australia, taiwan, and china (table 2). there have been three successive populations of s. invicta discovered in new zealand, and each has been successfully exterminated (maf biosecurity 2010). i was unable to verify reports of s. invicta from malaysia, singapore, india, and the philippines (na & lee 2001, rajagopal et al. 2005, vanderwoude et al. 2006, sarty 2007, kuo 2008, wikipedia 2012). an and lee (2001) reported s. invicta from malaysia, sarty (2007) listed s. invicta from singapore, and vanderwoude et al. (2006) mentioned s. invicta from both malaysia and singapore. i e-mailed c.y lee, m. sarty, and c. vanderwoude asking about the bases of these records. c.y. lee (pers. comm.) wrote: “s. invicta is not found in malaysia and singapore so far. it was a mistake in identification in the na & lee (2001) paper.” c. vanderwoude (pers. comm.) sent me copies of na & lee (2001) and sarty (2007) and wrote “i’m scratching around for a better ref for rifa [s. invicta] in singapore but can’t seem to lay my hands on it.” rajagopal et al. (2005) surveyed ants in sattur taluk, tamil nadu, india, reporting s. invicta from all three habitats studied: riverine, cultivated, and industrial areas. it seems improbable to find high saturation of s. invicta in this one small area, but no s. invicta anywhere else in india. i e-mailed t. rajagopal and r. gadagkar (whom rajagopal et al. 2005 credited with ant identification). r. gadagkar (pers. comm.) replied: “i have now confirmed from my colleague dr. thresiamma varghese that she did identify some ant specimens for rajagopal et al but solenopsis invicta was certainly not one of them.” kuo (2008) listed s. invicta from the philippines and wikipedia (2012) included a report of s. invicta in the philippines, which has been paraphrased on many other web sites: “there have also been reports of colonies in metro manila and the province of cavite in the philippines since july 2005; however, since early 2007, they have spread now as far as the bicol region.” i could not find an e-mail contact for k.c. kuo, nor the source of the wikipedia reports, jk wetterer exotic spread of solenopsis invicta beyond north america52 which were posted anonymously from two ip addresses in houston, texas. g. alpert and d. general do not include s. invicta on their list of ants of the philippines on antweb. org. d. general (pers. comm.), who worked with s. invicta for six years in arkansas, has been actively searching for s. invicta in the philippines, but so far has not found any. although i cannot be certain, it seems likely that reports of s. invicta from india and the philippines were based on misidentifications. discussion sometime before 1945, s. invicta arrived in alabama from south america. solenopsis invicta has since become widespread across the southern us and into northeastern mexico, with scattered indoor records in more temperate areas (fig. 1). starting about 1980, s. invicta began spreading through the west indies, with the earliest records from puerto rico and the us virgin islands (table 1, wetterer & snelling 2006). solenopsis invicta is now known from 28 west indian islands (table 1). the most recent first records, from aruba and jamaica, all come from golf courses. cinnamon hill golf course in jamaica, where i found s. invicta, imports sod from florida. such importation of sod from florida may be an important mode of spreading s. invicta in the west indies. solenopsis invicta was first recorded in the old world in 2001 (nattrass & vanderwoude 2001, harris 2001). nascent populations of s. invicta have been exterminated in table 1. earliest known records of solenopsis invicta from west indian islands. mcz = museum of comparative zoology. + = no previously published records. earliest record puerto rico 1981 (buren 1982) st croix, usvi 1988 (wetterer & snelling 2006) trinidad 1991 (g.l. white, pers. comm.): several sites san salvador, bahamas 1993 (deyrup 1994) new providence, bahamas 1995 (deyrup et al. 1998) north andros, bahamas 1996 (deyrup et al. 1998) guana island, bvi 1996 (davis et al. 2001) gorda cay, bahamas 1997 (davis et al. 2001) antigua 2000 (davis et al. 2001) abaco, bahamas 2000 (davis et al. 2001) grand bahama, bahamas 2000 (davis et al. 2001) providenciales, tci 2001 (davis et al. 2001) grand cayman ≤2003 (issg 2010) cayman brac ≤2003 (issg 2010) little cayman ≤2003 (issg 2010) berry islands, bahamas 2005 (wetterer & snelling 2006) st thomas, usvi 2005 (wetterer & snelling 2006) st john, usvi 2005 (wetterer & snelling 2006) tortola, bvi 2005 (wetterer & snelling 2006) +vieques, pr 2005 (j.k. wetterer, mcz): colonia lujan anguilla 2006 (wetterer & davis 2010) st martin 2006 (wetterer & davis 2010) barbuda 2007 (wetterer & davis 2010) montserrat 2007 (wetterer & davis 2010) st kitts 2007 (wetterer & davis 2010) nevis 2007 (wetterer & davis 2010) +aruba 2007 (j.k. wetterer, mcz): tierra del sol golf course +jamaica 2010 (j.k. wetterer, mcz): cinnamon hill golf course table 2. earliest known records of solenopsis invicta from the old world. * = needs confirmation. earliest record new zealand 2001 (harris 2001) australia 2001 (nattrass & vanderwoude 2001) *india 2001-2002 (rajagopal et al. 2005) taiwan 2003 (chen et al. 2005) guangdong province, china 2004 (zeng et al. 2005) hong kong 2004 (zeng et al. 2005) fujian province, china 2005 (zhang et al. 2007) guangxi province, china 2005 (zhang et al. 2007) hunan province, china 2005 (zhang et al. 2007) macau 2005 (zhang et al. 2007) *philippines 2005 (kuo 2008) sociobiology 60(1): 50-55 (2013) 53 new zealand (maf biosecurity 2010) and have been largely kept in check in australia through intensive control efforts (vanderwoude et al. 2004). however, s. invicta populations in taiwan and china, first identified in 2003 and 2004 respectively, have spread broadly (fig. 1, table 2). lu et al. (2008) estimated that, based on its geographic spread, s. invicta first arrived in china around 1995. there seems to be no geographic or climatic barriers to prevent s. invicta from spreading throughout tropical and subtropical asia (morrison et al. 2004, sutherst & maywald 2005). published reports of s. invicta from malaysia and singapore were based on misidentifications (see above), presumably of the much more widespread fire ant, solenopsis geminata (fabricius), which invaded asia from south america more than 160 years ago (jerdon 1851). reports of s. invicta from india (rajagopal et al. 2005) and the philippines (kuo 2008) are questionable. it would be prudent to try to confirm whether or not s. invicta has actually invaded these countries. ascunce et al. (2011) analyzed dna from s. invicta populations in australia, taiwan, and china and found they all originated from exotic s. invicta populations in the us. it seems most likely that s. invicta populations in the west indies also originated in the us, imported on plant products, such as grass sod grown in florida. despite its continued spread, s. invicta is still much less widespread globally than is s. geminata (see wetterer 2011). when s. invicta invades, it typically displaces s. geminata, particularly in habitats preferred by both species: open, grassy areas. as a result, s. geminata has largely disappeared from much of its former exotic range in north america (wojcik et al. 1976). in florida, porter (1992) found s. geminata at 83% of roadside sites where s. invicta were absent, but only 7% of sites where s. invicta was present. models predict that s. invicta should be able to invade many tropical and subtropical regions now occupied by s. geminata (morrison et al. 2004, sutherst & maywald 2005). in working to mitigate the spread and negative impact of s. invicta, s. geminata, and other fire ants around the world, it will be important to distinguish between these ants, and recognize both their similarities and their differences. acknowledgments i thank m. wetterer for comments on this manuscript, s. cover and l. davis for ant identification, s. cover (mcz) and m. deyrup (abs) for help with their respective ant collections, d. general his search for s. invicta in the philippines, c. vanderwoude, c.y lee, d. general, r. gadagkar, and f. burton for their personal communications, w. o’brien for gis help, d.p. wojcik and s.d. porter for compiling their formis bibliography, c. scheid and r. pasos of the fau library for processing so many interlibrary loans, florida atlantic university, the national science foundation (deb-0515648) for financial support. figure 1. worldwide distribution records of solenopsis invicta. records from india and the philippines are questionable and need to be confirmed (see text). jk wetterer exotic spread of solenopsis invicta beyond north america54 references allen, c.r., epperson, d.m. & garmestani, a.s. 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(2007) an overview of the red imported fire ant (hymenoptera: formicidae) in mainland china. florida entomol. 90:723-731. doi: 10.13102/sociobiology.v60i3.236-241sociobiology 60(3): 236-241 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the ecological effects of ant-aphid mutualism on plants at a large spatial scale s zhang, y zhang, k ma introduction mutualism has been increasingly considered to play an important role in shaping community structure, diversity and ecological functions (bronstein, 1994; bruno et al., 2003; bascompte and jordano, 2007). the role of mutualistic interactions in nature has been considered as one of the “key gaps in population and community ecology” (agrawal et al., 2007). ant-hemipteran interaction is one of the most common mutualistic interactions in nature; in the interaction ants take the honeydew excreted by hemipterans as food and in return, they protect those insects from natural enemies (del-claro, 2004; moreira & del-claro, 2005). for a long time, much of the attention has been paid for the effect of the interaction on each other, especially the impacts of ants on hemipteran (way, 1963; buckley, 1983; stadler & dixon, 2005). more recent studies show that this mutualistic interaction has a wider range of ecological effects, especially for the host plants and related arthropods on foliage (wimp & whitham, 2001; kaplan & eubanks, 2005; styrsky & eubanks, 2007). abstract the protective ant-plant interaction has been considered as a model system in studying mutualistic interactions, but we know little about the ecological effects of the mutualism at relatively larger spatial scales. in this study, by excluding an aphid-tending ant species (lasius fuliginosus) from all host oak trees (quercus liaotungensis) in 20x20 m plots, we evaluated the effects of ants on herbivory, fruit production and leaf toughness of the host tree. through a two years study, we found that ants have a significant anti-herbivory effect on the host tree, with no effects on fruit production. at the end of the growing season, leaf toughness for plants without ants increased significantly. this suggests that ants are reliable and effective bodyguards for plants at larger spatial scales. for plants, the possible tradeoff between different defensive strategies at larger scale should be focused in further works. sociobiology an international journal on social insects chinese academy of sciences, beijing, p. r. china. research article ants article history edited by kleber del-claro, ufu, brazil received 25 june 2013 initial acceptance 22 july 2013 final acceptance 19 august 2013 key words ant defense, ant-plant interaction, herbivory, exclusion experiment corresponding author yuxin zhang state key laboratory of urban and regional ecology, research center for eco-environmental sciences chinese academy of sciences beijing 100085, p. r. china e-mail: yxzhang@rcees.ac.cn the honeydew-collecting ants often benefit plants through their attack and expel on herbivores (moreira & delclaro, 2005; chamberlain & holland, 2009; rosumek et al., 2009; trager et al., 2010; romero & koricheva, 2011; zhang et al,. 2012b). ants can decrease herbivory through their negative effect on the abundances of herbivores on plants (zhang et al. 2012b). an important problem to be declared is that the possible effect of spatial scale on the ecological effects of ant-hemipteran interaction has long been ignored. many biotic interactions can be scale dependent, such as pollination (leiss & klinkhamer, 2005; westphal et al., 2006); herbivory (wallisdevries et al., 1999), frugivory (garcia et al,. 2011) and seed predation (curran & webb, 2000). but to our knowledge, most of the studies on ant-plants interactions conducted at the level of branches or individual plants (chamberlain & holland, 2009; zhang et al., 2012b), the ecological effects of this interaction at larger spatial scales are poorly known. considering the complexity of biotic interactions in nature, the conclusions and predictions drawn from a smaller spatial scale may be inconsistent with that got from larger sociobiology 60(3): 236-241 (2013) 237 spatial scale. therefore studies conducted at even larger spatial scales are needed to fully understanding the ecological effects of the ant-hemipteran interaction in nature. theoretical models argue that plant defense should not be redundant (stamp, 2003). in ant-plant interactions, it has been long assumed that there is a tradeoff between ant defense and the defense of plant itself (such as chemical or physical defense), especially in obligate ant-plant interactions (janzen, 1966). mixed evidences for the hypothesis have been found for obligate ant-plant interactions (heil et al,. 2002, frederickson et al., 2013). but few studies have tested the hypothesis in the facultative ant-plant interactions mediated by hemipteran. base on the finding that the honeydew collecting ants have significant anti-herbivory effects for plants, we argue that the tradeoff among different defensive strategies can also be existence in the ant-hemipteran-plant system. the exclusion of ants at a relative large scale can facilitate the tradeoff to be shown. in this study, we evaluated the impacts of aphid-tending ants lasius fuliginosus on the host oak tree quercus liaotungensis by experimentally excluding ants from all oak trees in a plot (20*20 m). we hypothesized that 1) the aphidtending ants lasius fuliginosus have protective effect on plants at the plot scale 2) the physical defense of plants (leaf toughness) should be stronger when ants were excluded. material and methods the study area is located in the beijing forest ecosystem research station (30°57′29n, 115°25′33e, altitude 1,200-1,400m), a member of the chinese ecological research network (cern), about 100 km northwest of beijing city, china. this area typically has a warm temperate continental monsoon climate with average annual precipitation of 500650 mm. the mean annual temperature is 5-10°c. it is an oak (q. liaotungensis) dominated, 80-year-old secondary forest with a few birches (betula spp.), maples (acer mono), and shrubs (e.g., prunus spp., vitex negundo var. hetertophylla). we conducted this experiment during two consecutive growing seasons (2009, 2010) of the oak tree q. liaotungensis, which is the dominant tree species in the study area (zhang et al., 2006). we selected a slope in a small watershed to conduct the experiment. we chose this area because the previous pitfall trap sampling found that the ant lasius fuliginosus was the only active ant species with high abundance in this area. l. fuliginosus is a typical honeydew-feeding ant that has mutualistic relationships with some aphid species (hopkins & thacker 1999). in the study area, l. fuliginosus was attracted by aphids lachnus tropicalis and tuberculatus sp. in the canopy and stomaphis japonica on the trunk of q. liaotungensis. the aphid was the key factor attracting ants in the canopy of q. liaotungensis in the study site. in 2009, we set up four pairs of plots (20x20 m) (in 2010, three pairs) with a distance of at least 50 m between the adjacent pairs. for each pair, one of them was set as the ant exclusion plot and the other as the control plot, with a distance of more than 15 m between each other. in april of each year (before the growing season), an adhesive ring was smeared around the trunk (about 1 m above the ground, and 5 cm in width) on all trees in the treatment plot to impede the access of ants to aphids on the canopy. the adhesive was made of a polymer resin mixture (beijing nonghaha s & t co. ltd) and was nontoxic, harmless to plants, and non-attractive to insects. the adhesive was re-smeared every two months during the growing season until the end of the study, it worked effectively through our study. any bridges that could allow ants to climb onto trees were cut off throughout the study. the differences between tree densities, leaf area index (lai), and canopy coverage in the treated and control plots were insignificant (table 1). from late may to september, the percentage of leafarea loss was calculated monthly. in each month, we randomly chose ten trees in a plot to evaluate plant herbivory and leaf toughness. the percentage of leaves damaged by herbivores was used as an indicator of plant herbivory. for each tree, one randomly chosen twig (about 4-5 year variable treatment (mean, se) control (mean, se) p value 2009 lai 1.83 (0.09) 1.75 (0.09) 0.69 cover 79.2% (1.5%) 77.8% (1.5%) 0.68 tree density 38.0 (5.5) 30.5 (4.5) 0.25 2010 lai 1.64 (0.11) 1.65 (0.12) 0.49 cover 75.6% (1.8%) 75.4% (2.3%) 0.55 tree density 33.33 (5.5) 24.33 (1.5) 0.13 table 1 the leaf area index (lai), cover and tree densities in the treated and control plots. m high) was cut off. for each twig, from the tip, the first to sixth leaves were collected. all the leaves were scanned by epson perfection 4870 photo (epson america, inc., usa) and then used to calculate herbivory. for each leaf, the herbivore-damaged parts were repaired using the adobe photoshop cs2 (adobe systems inc., usa), according to the expected shape. the original (a) and repaired (b) areas of leaves were calculated with winfolia basic 2004a (regent instruments inc., australia). the percentage of leaf-area loss was calculated as l= (b-a)/b*100%. in each plot, we randomly chose ten trees to test leaf toughness. for each tree, a twig 4-5 m high above ground was cut off. three randomly chosen leaves were used to test toughness using a puncher immediately after the leaves were cut off. three holes were punched for each leaf. the weight needed to punch the leaf was recorded as the indicator of toughness. this experiment was conducted only in 2010. in late september (only in 2010), the fruiting season of q.liaotungensis, fruit numbers were recorded by counting the fruit within five 1x1 m small plots in each 20x20 m plot. s zhang, y zhang, k ma ant-aphid mutualism on plants238 data analysis each pair of the plots was treated as a block in data analysis. a mixed effect model was used to test the treatment and year on plant herbivory at first. in this model, treatment, year and their interaction were set as fixed effect; block was set as random effect. different months were treated as repeated measures, and the type of the covariance structure was selected using the akaike information criterion (aic). if the difference for the effect of the two-year was insignificant, data of different years were pooled together for analysis, otherwise the data were analyzed separately. then, for each year, a mixed effect model was used to test the effects of ants on herbivory. treatment, month and their interaction were set as fixed effects; the block was set as random factor. different months were treated as repeated measures, and the type of the covariance structure was selected using the akaike information criterion (aic). this model was also used for evaluating the effects of ants on herbivory. a poisson regression model was used to test the effect of treatment on fruit production. all the analyses were performed with sas 9.2 with the mixed and genmod procedure (sas institute 2008). p<0.0001) and the interaction between treatment and month (f=7.90, p<0.0001). in 2010, the herbivory for plants with and without ants were 6.7% (n=907, se=0.02%) and 8.8% (n=907, se=8.8%) respectively, with significant differences (f=32.59, p<0.0001). plant herbivory also showed significant monthly variation in 2010 (f=22.48, p<0.0001), but the interaction between month and treatment on herbivory was not significant (f=0.73, p=0.5685). further analysis show that in 2009, the anti-herbivory effect of ants was significant only at the earlier of the growth season (may, jun) (fig.1), but in 2010, the effect was significant through the growth season except in july (fig.1). treatment had significant positive effect on leaf toughness (f=11.04, p=0.0009). month and it’s interaction between treatment also showed significant effect on leaf toughness (month, f=731.75, p<0.0001; the interaction between month and treatment, f=6.36, p<0.0001). further analysis showed that the effect of treatment on leaf toughness was only significant at the end of the growing season (september) (fig.2).the fruit number in ant exclusive plots (mean=40.67/m2, se=6.46, n=15) seemed to be higher than that in control plots (mean=28.73/m2, se=4.22, n=15), but the difference between the two groups was insignificant (χ2=2.44, p=0.1184) (fig.3). fig. 1. the monthly variation of herbivory in ant-excluded and control plots (mean, se). fig. 2. the monthly variation of leaf toughness in ant-exclued and control plots (mean, se). results in total, 4234 leaves of q. liaotungensis were analyzed for herbivory. for plants without ants, 10.1% (n=2105, se=0.2%) of the leaf area were eaten by herbivores, for plants with ants, the value was 8.5% (n=2129, se=0.2%), the difference between the two group was significant (f=24.73, p<0.0001). plant herbivory in 2009 and 2010 were 10.5% (n=2420, se=0.2%) and 7.7% (n=1814, se=0.2%) respectively, with significant differences between the two years (f=5.28, p=0.0216). therefore, the data of herbivory for the two years were analyzed separately. in 2009, plant herbivory in treatment plot (mean=11.1%, n=1198, se=3%) was significantly higher than that of plants with ants (mean=10.0%, n=1222, se=4%, f=5.35, p=0.0208). in 2009, plant herbivory was also influenced by month (f=9.84, sociobiology 60(3): 236-241 (2013) 239 discussion recent meta-analyses found that in general the mutualistic interaction between ants and aphids can benefit plants (styrsky & eubanks, 2007, zhang et al., 2012b), but all the studies used in these meta-analysis were conducted at the individual plant or smaller scale (such as branches or leaves). whether the conclusions drawn from those smaller scales can still be solid at larger scales is unknown. here through an experimental treatment at the 20x20 m plot scale, we confirmed the beneficial effect of the aphid-tending ants on plants. the results show that leaf toughness can be an induced defensive trait at a larger scale. these findings are essential for us to evaluate the ecological effect of mutualism in natural communities. our studies found that the ecological effect of antaphid mutualism is significant for plants beyond the scale of individual plants. therefore ants can be a reliable bodyguard across different spatial scales. we found that the significant anti-herbivory effect of ants at the scale of individual trees as well as branches in previous work (zhang et al., 2012a). the strength of the anti-herbivory effect for ants was 1.6% at the plot scale; this value is also within the variation range of the anti-herbivory effect (from 1.38 to 2.96%) at lower scales (zhang et al., 2012a). although biotic interactions are assumed to be scale depended (wallisdevries et al., 1999; leiss & klinkhamer, 2005; westphal et al., 2006; garcia et al.. 2011), this study indicates that from the point of anti-herbivory, the effect of ants on plants can keep consistent at a wide range of spatial scales. in our study site, both the activity of ants and herbivores varied with the process of the growth season (especially in 2009), this can lead to the variation of the anti-herbivory effects of ants on plants. for different years, conditional outcomes of ant-plant interaction depending on climatic should be considered (del-claro & oliveira, 2000). the climatic variation can lead to the differences of caterpillars as well as herbivory in the two different years. a noticeable result is that the antiherbivory effect of ants kept significant at the earlier period of the growth season both in 2009 and 2010. at this period, the q. liaotungensis are expending their leaves. considering those young leaves are especially valuable for plants in photosynthesis (harper. 1989; pringle et al., 2011), the protective effects of ants at this period can have deep effects on plant growth. ants showed significant protective effects for plants but not for the fruit production; this result is also consistent with experiment conducted at smaller scales (moreira & del-claro, 2005; zhang et al., 2012a). however, there was a trend that plants with ants tended to produce fewer fruits in this work (fig. 3); the possible negative effects of ants on the flowering of oak should be paid more attention in future researches. leaf toughness is an important factor that affects herbivory (onoda et al., 2011). a recent study found that in obligate ant-plant interactions, there is a tradeoff between ant defense and leaf toughness (frederickson et al., 2013), but in the facultative ant-plant interactions such as our study system, such examples are rare (but see korndörfer & delclaro, 2006). our study indicates in facultative ant-plant interaction, if plants lost ants at a larger spatial scale, they can also increase their leaf toughness to resist herbivore damages. in this study, we found that the leaf toughness in treatment plots was significantly higher than that in control plots only at the end of the growth season. the reason for this monthly variation pattern is unclear, but it is possible that the induced defensive traits has the time lag effects (agrawal, 2007; agrawal, 2011). further studies should pay more attention to the inducible plant defensive (both physical and chemical defense) beyond the scale of individual plants. in conclusion, this study confirmed that the antiherbivory effect of the aphid tending ants can also function at a relatively large scale, not limited at the level of branches or individual plants. this suggests that ants are reliable and effective bodyguard for plants regardless across different spatial scales. for plants, the possible tradeoff among different defensive strategies at larger scale should 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(2012b). the ecological effects of the ant-hemipteran mutualism: a meta-analysis. basic appl. ecol., 13: 116-124. zhang, y. x., k. m. ma, m. anand, and b. j. fu. 2006. do generalized scaling laws exist for species abundance distribution in mountains? oikos 115:81-88. doi: 10.13102/sociobiology.v60i3.252-258sociobiology 60(3): 252-258 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 an herbivore’s thermal tolerance is higher than that of the ant defenders in a desert protection mutualism g fitzpatrick, g davidowitz, jl bronstein introduction plants exhibit a variety of defenses against the constant threat of damage from herbivores. mutualistic association with ant species is one such defense. in ant-plant protection mutualisms, ants are employed as guards against herbivores in exchange for rewards such as food and shelter (beattie, 1985; heil & mckey, 2003; marazzi et al., 2013). mutualisms between ants and plants are common across a variety of habitats (rico-gray & oliveira, 2007; oliver et al., 2008). plants tended by ant defenders often have fewer herbivores, less damage from herbivory, and higher reproduction (rudgers, 2004; kersch & fonseca, 2005; chamberlain & holland, 2009; trager et al., 2010). plants and insects experience a variety of biotic and abstract in north american deserts, many species of cactus attract ants to their extrafloral nectaries; the ants actively defend the food source, and hence the plant, against herbivores. in thermally extreme environments, however, networks of positive and negative interactions like these are likely to be sensitive to the thermal limitations of each of the interacting species. we compared the thermal tolerance of a common phytophagous cactus bug, narnia pallidicornis (hemiptera: coreidae), to that of the ants that defend the cactus ferocactus wislizeni in the sonoran desert, usa. we used flow-through respirometry to experimentally determine the thermal limit of the herbivore and compared this to the thermal limits of the ant defenders, determined previously. in the field, we recorded herbivore frequency (proportion of plants with n. pallidicornis) and abundance (the number of n. pallidicornis per plant) in relation to ambient temperature, ant species presence and identity, and fruit production. we show that n. pallidicornis has a higher thermal tolerance than the four most common ant mutualists, and in the laboratory can survive very high temperatures, up to 43°c. herbivore frequency and abundance in the field were not related to the daily high temperatures observed. plants that were not defended by ants were occupied by more n. pallidicornis, although they showed no reduction in fruit set. therefore, herbivory is likely to continue on fishhook barrel cacti even at high temperatures, especially those temperatures beyond the thermal tolerance of the ant defenders. the consequences of increased herbivory, however, remain unclear. mutualisms are essential for ecosystem functioning; it is important to understand the thermal sensitivity of these interactions, especially in light of expected increases in global temperature regimes. sociobiology an international journal on social insects university of arizona, tucson, arizona, usa. article history edited by kleber del-klaro, ufu, brazil received 15 july 2013 initial acceptance 07 august 2013 final acceptance 21 august 2013 key words thermal ecology, herbivory, ant-plant interaction, narnia, temperature corresponding author ginny fitzpatrick department of ecology & evolutionary biology university of arizona po box 210088, tucson, az 85721 usa e-mail: ginfitz@email.arizona.edu abiotic stresses depending on the environment in which they interact. these include intense competition in the tropics, widely fluctuating seasonality in temperate zones, and extreme temperatures in deserts and polar regions (chamberlain & holland, 2009). a commonly measured physiological response to temperature is thermal tolerance, the temperature beyond which an organism cannot perform life functions or dies. depending on the environment, the focus may be on low (sinclair et al., 2003) or high (e.g., huey et al., 1992) thermal limits. thermal tolerance has been frequently measured in insects (sinclair et al., 2003) and plants (e.g., nobel, 1982; nobel, 2003), as well as in fishes (becker & genoway, 1979, reviewed by beitinger et al., 2000), marine organisms such as bivalves (e.g., peck et al., 2004) and corals (e.g., berkelmans & van oppen, 2006), and reptiles (e.g., angilresearch article ants sociobiology 60(3): 252-258 (2013) 253 letta et al., 2009). however, there is scant evidence for how interactions among species, as opposed to species considered in isolation, are affected by extreme temperatures. such studies are imperative to better understand how networks of species interactions, especially those in extreme environments, will respond to increasing temperatures expected as a consequence of global climate change. in this study, we consider the relative thermal tolerances of ants and herbivores that interact within an antplant protection mutualism in the sonoran desert, usa. the fishhook barrel cactus, ferocactus wislizeni, is protected by four common ant mutualist species. typically, only one ant species occupies any given plant for a period of days and even months (morris et al., 2005). there is a hierarchy of mutualist effectiveness among the ant species (ness et al., 2006). the least-effective mutualistic ant species, forelius pruinosis, is the most thermally tolerant and occupies hotter plants relative to those occupied by other ant species (fitzpatrick et al. in review). thus, rising temperatures might increase the frequency with which plants interact with this ant. because mutualist effectiveness (as measured by success at herbivore removal) is positively associated with increased production of fruits and seeds (ness et al., 2006), higher temperatures could have negative reproductive consequences for the fishhook barrel cactus, as well as for other extrafloral nectary-bearing plants in the sonoran desert that rely on herbivore protection from this same group of generalized ant defenders. however, this scenario does not take into account whether or not herbivores themselves are responsive to temperature. if they are more tolerant of high temperatures than are the mutualistic ant defenders, then the consequences for plants may well be severe. however, if herbivores are less tolerant of high temperatures than are the ants, then the effects of rising temperatures might be mitigated: although less-mutualistic ants would dominate in a warmer world, herbivores would be less abundant there. to distinguish between these two possibilities, it is necessary to have data on thermal tolerances of both ant mutualists and the herbivores they deter. we are unaware of the existence of such data for any ant protection system. we chose to focus on one abundant herbivore, the cactus bug narnia pallidicornis (hemiptera: coreidae), which feeds upon the reproductive tissues of f. wislizeni. we asked: (1) how does narnia thermal tolerance compare to that of f. wislizeni’s ant mutualists, and (2) how does narnia abundance and frequency change in response to environmental factors, including ant mutualist identity? materials and methods study system the fishhook barrel cactus, ferocactus wislizeni, is found between 0 and 1500 m in elevation throughout the sonoran and chihuahuan deserts, from mexico into arizona, new mexico, and texas, usa. flowering begins in late july and continues through mid-october, with a peak in late august (mcintosh 2002). the extrafloral nectaries are positioned around the crown of the plant surrounding the fruit. cacti can live more than 40 years, and individuals produce extrafloral nectar year-round from the time they are 1-2 years old (fitzpatrick unpublished data); this creates a reliable, longterm resource for foraging ants (lanan & bronstein 2013). this study was conducted at the desert laboratory in tucson, arizona (32°13’ n, 111°05’ w), a 370 ha reserve consisting of sonoran desert scrub. we used two study plots within which the mutualism between f. wislizeni and its ant mutualists has been extensively studied (ness et al., 2006; ness et al., 2009; morris et al., 2005; lanan & bronstein, 2013; fitzpatrick et al. in review; fitzpatrick & bronstein unpublished data). observational data were collected in the field once a month from january through september 2011. laboratory experiments were conducted from august-october 2012. august-october is the time of year when f. wislizeni produces buds and flowers and should be most vulnerable to attack by herbivores that attack these water-rich, delicate tissues. average high temperatures from august-october range from 29 to 36°c, with extreme temperatures above 44°c (for tucson, 1981 to 2010; national oceanic and atmospheric association). this time overlaps with the end of the monsoon season, with average monthly rainfall of 3-5 cm. four native ant species make up >90% of ant occupants of f. wislizeni at this study site: forelius pruinosus, solenopsis aurea, solenopsis xyloni, and crematogaster opuntiae. typically, a single ant species occupies a given plant at a given time. a percentage of the plants are naturally unoccupied by ants, which changes with the season. there is predictable seasonal turnover in the most abundant species across the site (morris et al. 2005). crematogaster opuntiae is most frequent on f. wislizeni (measured as the proportion of plants on which a species is active) during winter and spring months. solenopsis aurea, s. xyloni, and f. pruinosus are most frequent during summer and autumn (morris et al. 2005, unpublished data). all four ant species were observed frequently on cacti during this study. there is a clear hierarchy of mutualistic effectiveness among the ant species (ness et al. 2006). solenopsis xyloni is most effective at removing herbivores (measured as per capita aggressiveness towards surrogate herbivores intentionally placed on the plant), more so than c. opuntiae and s. aurea, which are equivalently effective. all three are more effective than f. pruinosus. forelius pruinosus was also the least dominant species, as measured by colley dominance rankings, which reflect the proportion of staged confrontations won (lanan et al. in review). the dominance hierarchy was: s. xyloni > s. aurea > c. opuntiae > f. pruinosus. the most frequently observed herbivores on the plants are cactus bugs, narnia pallidicornis (hemiptera: coreidae; subsequently, narnia), phytophagous insects that specialize on the phloem of cacti, preferring to feed on reproductive g fitzpatrick, g davidowitz, jl bronstein herbivore’s thermal tolerance is higher than ant mutualists254 structures such as fruits (miller et al. 2006; miller 2007). narnia are iteroparous, with two generations per year. adults overwinter in debris at the base of plants (miller et al. 2006). juveniles are flightless and develop on a single host plant. on f. wislizeni, narnia are often observed in the ribs of the cactus and are attacked by ants when they approach the extrafloral nectaries where the ants are foraging (fitzpatrick, personal observation). herbivore thermal tolerance individual narnia were collected from the study plots on the day of the trial in which they were used in october of 2012. three individuals were collected each day for three days. individuals were weighed before and after the experiment using an analytical balance (mettler toledo newclassic ml). mass ranged from 64.6-128.0 mg prior to the experiment and from 28.7-76.9 mg after death. individuals were placed in 10 ml syringes in a climate-controlled chamber (pelt 5, sable systems international) and allowed to acclimate for 15 min. the rate of co2 production (vco2) was measured using flow-through respirometry (foxbox, sable systems international). air was scrubbed of co2 and water vapor with a flow rate of 100 ml/min. data were collected and analyzed using expedata (sable systems international). all values of metabolic rate were mass-specific and used individual mass measured prior to the experiment, as above. the temperature of the climate chamber (32, 36, 40, 42, 44, 46, 47, and 48°c) was increased at 60-min intervals, with 5 min of baseline and 15 min of recording for three individuals at each temperature. one of the syringes in the chamber did not record properly, so data for only two individuals per trial were collected. we decreased the temperature interval at higher temperatures to attempt to capture the exact temperature at which individuals ceased co2 production. the experimental temperature gradient simulated typical temperatures experienced in the hottest months in the field. narnia frequency and abundance narnia abundance in the field was measured as the number of individuals observed on long-term study plants throughout 2011. approximately 182 cacti were regularly included in a census carried out monthly, although the number fell slightly over the study period due to mortality. observations were typically made between 0600-1100, when herbivores and ants were known to be most active (fitzpatrick unpublished data). herbivore frequency was calculated as the proportion of cacti that had at least one narnia present. thermal tolerance comparison to compare the thermal tolerance of narnia to that of the four ant mutualists, we calculated the predicted critical thermal maximum from the recorded surface temperature of cacti using the linear relationship between ambient and surface temperature in the field. the critical thermal maximum temperatures of the ant species were determined using a water bath by fitzpatrick et al. (in review) as the temperature above which 50% of ants died. this commonly used method is described by cerda and retana (e.g. 1997; 2000). the water bath method for determining thermal tolerance could not be used for narnia because of the low abundance of individuals in the field; the minimum number of individuals necessary for this method was not available for the experiment. thus, flowthrough respirometry was more appropriate for measuring the thermal tolerance of narnia. plant surface temperature and ambient temperature were recorded in the field using an omega 871a digital thermometer with type k thermocouple. we then calculated the positive linear relationship between plant surface temperature and ambient temperature by comparing the observed temperatures recorded. statistical analysis the thermal tolerance curve for narnia was plotted using the means and standard deviations of vco2 during the 15-min sampling interval averaged across individuals for each temperature. sample sizes ranged from 2-6 individuals at the different temperatures, for a total of 35 measurements. the relationship between ambient temperature and plant surface temperature was determined using previously recorded temperatures in the field. to determine narnia frequency and abundance in response to biotic and abiotic environmental factors in the field (time of year, daily high temperature, fruit production, and ant species attendance), we performed an anova to compare values across months. we performed a simple linear regression to determine the relationship between the number of narnia per plant and the number of fruit per plant. these data were log (base 10) transformed to meet the assumptions of linear models. results herbivore thermal tolerance there was a curvilinear relationship between mean vco2 of narnia and experimental temperatures, peaking at 43°c, then decreasing sharply with further temperature increases (fig. 1). co2 production ceased in all individuals at 48°c. after correcting for the difference between ambient temperature (ta; based on the method for measuring herbivore thermal tolerance) and surface temperature (pst; based on the method for measuring ant thermal tolerances), and using the relationship between the two as observed in the field (pst = 1.49ta – 8.16), narnia thermal tolerance was higher than for any of the four ant mutualist species (table 1). sociobiology 60(3): 252-258 (2013) 255 herbivore frequency and abundance in the field, herbivore frequency (p=0.049) and abundance (p=0.013) were both higher on plants that were not defended by ants (fig. 2; anova; r2 = 0.446, f8,71 = 1.570, p=0.049). after accounting for the effect of the number of fruits per plant, herbivore frequency was not related to ant species identity (anova; r2 = 0.446, f4,71 = 1.099, p=0.366, log-transformed proportion of plants with herbivores). overall, however, narnia were rare, and the majority of plants had none present during any given observation. frequency of narnia (i.e., the percentage of plants with at least one individual) ranged from 0% in june to 9% in september. narnia abundance (number per plant) ranged from 0-5. daily high temperatures in the field ranged from 18.3 to 43.9°c; there was no relationship between temperature and either frequency (regression; r2 = 0.014, f1,71 = 1.011, p=0.318) or abundance (r2 = 0.0001, f1,71 = 0.010, p=0.922) of narnia. surprisingly, narnia frequency (regression; r2 = 0.446, f1,71 = 71.789, p<0.0001) and abundance (r 2 = 0.437, f1,71 = 56.301, p<0.0001) were both positively correlated with the number of fruits produced per plant (fig. 3). fig 1. the thermal tolerance curve of narnia pallidicornis. error bars are averages of the average standard deviation across individuals and in some cases are smaller than the symbol. table 1. thermal tolerance of four common ant mutualists and one herbivore of ferocactus wislizeni. mutualist effectiveness rank (high, moderate, low) is based on the hierarchy as determined by ness et al. (2006). mean plant surface temperature (pst ) was calculated from measurements in the field. critical thermal maximum values were determined using laboratory experiments and then transformed for comparison using the relationship between ambient temperature (ta) and plant surface temperature (pst) as determined in the field (pst = 1.49ta – 8.16). asterisks indicate values that were calculated using this formula describing the relationship between ta and pst. fig 2. change in observed frequency and abundance data over time (jan-sept 2011). a) frequency of herbivores measured as the proportion of plants with at least one herbivore observed (anova; r2 = 0.196, f8,71 = 1.920, p=0.072). b) frequency of defended plants measured as the proportion of plants on which at least one ant was observed (r2 = 0.963, f8,14 = 19.343, p=0.001). c) abundance of herbivores measured as the mean number of herbivores per plant (r2 = 0.219, f8,71 = 2.213, p=0.038). d) abundance of fruit measured as the mean number of fruit per plant (r2 = 0.218, f8,71 = 2.219, p=0.039). discussion herbivores threaten the survival and reproduction of plants by consuming and damaging plant tissue. in this study, we documented that the herbivore narnia pallidicornis, a common phytophagous insect that specializes on cacti, had a higher thermal tolerance than any of the four ant species that defend its host, the fishhook barrel cactus ferocactus wislizeni (table 1). as we also found that undefended plants had more herbivores, these results suggest that herbivory may become an increasing threat for f. wislizeni as temperatures rise. however, at least at the low frequencies of narnia present at our study site, there was no evidence that the presence of narnia inflicted a fitness cost on f. wislizeni. indeed, fruits were more numerous on plants with narnia. it is possible that initially, cacti with more flowers, buds, and fruit attract more herbivores, producing a positive correlation between fruit number and narnia abundance. on the tree cholla cactus (opuntia imbricata), miller (2008) found that population dynamics of narnia pallidicornis are determined largely by host plant quality, and that increased allocation to reproduction resulted in more attack by narnia and a higher rate of flower abortion. our study terminated in september, a month when fruit are in the early stages of formation. if the study had persisted through january, it is possible that the correlation between narnia abundance and fruit number would have reversed, as damage from herbivory might cause a reduction species mutualism role mean ta termal max. oc mean pst termal max. oc (se) mean pst. oc (se) s. xyloni high *37.7 48.0 (0.54) 43.3 (1.6) c. opuntiae modera te *38.8 49.6 (0.48) 38.9 (1.6) s. aurea modera te *38.0 48.5 (0.54) 26.4 (1.8) f. pruinosus low *40.4 52.9 (0.48) 44.4 (1.6) n, pallidicornis herbivore 43.0 55.9 g fitzpatrick, g davidowitz, jl bronstein herbivore’s thermal tolerance is higher than ant mutualists256 in fruit maturation and an increase in fruit abortion. also, miller (2008) observed much higher numbers of narnia on cacti than in this study, which may account for the difference in findings. future studies that measure seed number and recruitment in relation to herbivory are necessary to accurately estimate the consequences of narnia on cacti reproduction, especially at high temperatures when plants are undefended by ants. no study to our knowledge has investigated herbivore activity and damage at high temperatures in relation to the presence and identity of ant defenders. in general, warmer temperatures tend to increase insect development rate, which could result in a higher frequency and abundance of herbivores (ayres & lombardero, 2000) and increased interaction with the plants on which they feed (o’connor 2009), as well as with the ants that defend the plants; however, interaction with ant defenders would be lower at high temperatures beyond the tolerance of the ants. barton (2011) found no direct effect of temperature on grasshoppers, but at high temperatures their predators were forced to seek thermal refuge, which reduced spider predation risk. barton (2011) also found that the predators from warmer study sites tolerated higher temperatures. in our study system, the least effective mutualistic ant species is a heat specialist that occupies plants that are on average hotter than those occupied by ant species that are more effective at removing and deterring herbivores (fitzpatrick et al. in review). thus, plants that are hotter will be defended by a less effective ant defender and consequently be vulnerable to increased abundance of herbivores. during this study, narnia frequency (the proportion of plants that had at least one individual present) and abundance did not vary predictably with daily high temperatures, although no herbivores were present on plants in the hottest month of june, and there was very low frequency in july. we note that we did not measure narnia’s activity or feeding rate as a function of temperature; it is possible that it behaves differently on hotter days. also, we studied thermal tolerance in the laboratory, and field studies are needed to confirm narnia’s response to temperature in nature. if temperatures continue to rise in southwestern north america, as projected by the ipcc (2007), temperatures in the sonoran desert could come to exceed narnia’s thermal tolerance. such high temperatures would also cause mortality in the ant and plant species. however, evidence suggests that in general, species will not be directly threatened by higher temperatures, but will instead adapt or disperse (berg et al. 2010). this suggests that species interactions are likely to change in unforeseen ways. for this reason, understanding the thermal ecology of species interactions is essential. given that the consequences of warming at the community level will likely be determined by the complicated interplay of species’ life history traits, biotic interactions, and abiotic responses (berg et al. 2010), more studies that investigate the direct and indirect effects of temperature on interaction networks such as the one that links plants, herbivores, and mutualistic ant defenders are necessary to predict changes in response to rising temperatures. acknowledgements we thank blake pellman for assistance in the field, bryan helm and cristina francois for research assistance and intellectual input, and kristopher h. pruitt for comments. thank you to michele c. lanan, william f. morris, and joshua h. ness for intellectual input and for access to extensive long-term data recorded for this system. field data for this work were collected at the university of arizona desert laboratory, on tumamoc hill in tucson, arizona. this material is based upon work supported by the national science foundation graduate research program under grant no. dge-1143953 to g.f. and nsf grant ios-1053318 to g.d.. fig 3. linear log-log relationship between number of fruit per plant and a) frequency of herbivores (f1,71 = 71.789, p < 0.0001) and b) abundance of herbivores (f1,71 = 56.301, p < 0.0001). sociobiology 60(3): 252-258 (2013) 257 references angilletta, m.j. jr. 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(2008) global change and species interactions in terrestrial ecosystems. ecol. lett., 11: 1351-1363. doi: 10.1111/ j.1461-0248.2008.01250.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1039sociobiology 64(3): 327-333 (september, 2017) linear alkanes and reproductive status of polistes versicolor (hymenoptera: vespidae) females in winter aggregates introduction in social wasps, winter aggregates are composed of hibernating females, originated from colonies abandoned at the beginning of the cold season (ihering, 1904). they usually occur when atmospheric conditions are not adequate to support their colonies (tannure & nascimento, 1999; gonzález et al., 2002; gobbi et al., 2006). females remain in this state for a variable period between one to four months, and later they disperse and found new colonies (gonzález et al., 2002; gobbi et al., 2006, 2009). the types of interactions involved in winter aggregates of social wasps were well investigated in species of temperate climate (strassmann, 1991; dapporto et al., 2004; dapporto & palagi, 2006). in polistes dominula christ 1791, social abstract female wasps such as polistes versicolor can form aggregates to face weather conditions that are not suitable to sustain their colonies. the interactions between individuals in these aggregates, just as in other associations, are probably facilitated by chemical signals. of these compounds some of the most efficient during social interactions of insects are those called contact pheromones or superficial pheromones. this special type of pheromones, known as cuticular hydrocarbons, can be found in insects cuticle. they facilitate the differentiation of caste, species and nestmates, and may be important indicators of dominance as well as fertility. some studies indicate that linear alkanes are important cuticular compounds for intraspecific recognition and discrimination. the aim of this study was to evaluate the relationship between reproductive physiologic condition and the linear alkanes present in the cuticle of females of p. versicolor in aggregates employing gas chromatography with flame ionization detector (gc-fid). females from distinct aggregates were differentiated by the chemical composition of their cuticle. in each aggregate, there was difference in cuticular chemical composition between females with different ovarian development degrees, allowing the distinction between inseminated and non-inseminated females. sociobiology an international journal on social insects jhs brito1,2, wf antonialli-junior1, ts montagna1, a mendonça1,3, d sguarizi-antonio1,2, yr súarez1, cal cardoso1 article history edited by sergio ricardo andena, uefs, brazil received 06 april 2016 initial acceptance 12 september 2016 final acceptance 12 september 2017 publication date 17 october 2017 keywords social insects, cuticular compounds, wasp, chemical signature, chromatography. corresponding author denise sguarizi antonio universidade estadual do mato grosso do sul, cidade universitária rodovia dourados/itahum, km 12, cep 79804-970, dourados-ms, brasil. e-mail: denisesguarizi@hotmail.com interactions were observed with wasps performing external tasks, feeding the others by trophallaxis (dapporto et al., 2005). however, few studies have explored these aspects in wasps that inhabit tropical regions. in a study by gobbi et al. (2006) with p. versicolor aggregates, potential queens (females inseminated and with well-developed ovary) had a significantly larger size, a fact that was associated by the authors with greater accumulation of adipose tissue (gobbi & zucchi, 1985), so that the wasps can go through an unfavorable period. in fact, the particular conditions of these females aggregates even in tropical regions is an adaptation that allows females to survive until more favorable environmental conditions for founding new colonies (gobbi et al., 2006). although few interactions occur in these aggregates, it is important for the females that these associations are 1 laboratório de ecologia comportamental (labeco), centro de estudos em recursos naturais (cerna), universidade estadual de mato grosso do sul, dourados-ms, brazil 2 programa de pós graduação em recursos naturais, universidade estadual de mato grosso do sul, dourados-ms, brazil 3 programa de pós graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, mato grosso do sul, brazil research article wasps jhs brito et al. – linear alkanes in aggregates of wasp polistes versicolor328 maintained. the coherence of these aggregates might be facilitated by chemical compounds, which are the main signals exchanged during social interactions in colonies of social wasps (wilson, 1971). indeed, social insects present a complex organization system that is mediated by several chemical compounds called pheromones (billen, 2006). among these pheromones, there is a particular group called contact pheromones that are found in the insects’ cuticle (lockey, 1988). these compounds are the so-called cuticular hydrocarbons (chcs). their primary function is to prevent water loss, acting as a protective coating for insects (blomquist & bagnères, 2010). additionally, chcs act as contact or surface pheromones (abdalla et al., 2003; ginzel, 2010; neves et al., 2012; olaniran et al., 2013) that are involved in the identification of co-specifics. furthermore they facilitate the distinction of individuals according to their function in the colony, physiological status and hierarchical rank (provost et al., 2008), thus acting as a chemical signature of the colony (neves et al., 2012). among the cuticular compounds there are basically 3 categories, linear alkanes, branched alkanes and alkenes (devigne & biseau, 2012; olaniran et al., 2013). while several studies have indicated branched alkanes as the most important compounds for intraspecific recognition (bonavitacougourdan et al., 1991; dani et al., 1996; lorenzi et al., 1997; blomquist & bagnères, 2010; richard & hunt, 2013), others demonstrated the role of linear alkanes for the same purpose (lorenzi et al., 2004: p. dominula; tannure-nascimento et al., 2007: p. satan bequaert 1940). in addition to nestmate identity, the chcs may signal the fertility and physiological condition of females (van oystaeyen et al., 2014; oi et al., 2015a; oi et al., 2015b). here, in particular, linear alkanes of queens have been proposed to signal their condition within the colony and further induce the workers to remain sterile (van oystaeyen et al., 2014). therefore, despite the knowledge of the strong correlation between physiological condition and cuticular composition of females in colonies of social hymenoptera, few studies have explored this subject in depth in social wasps, especially using linear alkanes as signs to distinguish members of the same species. in this sense, the aim of this study was to evaluate the relationship between reproductive physiology and the cuticular composition of linear alkanes in females of p. versicolor olivier 1791 in winter aggregates. materials and methods wasp samples sixty nine females of the wasp p. versicolor were evaluated in two winter aggregates, both sampled in dourados, mato grosso do sul, brazil (22 º 12 ‘43’’ s, 54 º 54’ 53’’ w) in may/2013. the aggregates were approximately 30 km apart, with aggregate 1 (a1) located on a bridge and aggregate 2 (a2) on a roof. all individuals from the aggregates were collected at dusk, wrapping them with a sterile plastic container. in the laboratory, all subjects were anesthetized in freezer (4 °c) for 30 min. subsequently, with the aid of gloves and tweezers, the mesosome and metasome were removed and stored separately in sterile eppendorf vials. for the analysis of physiological conditions of females, the metasomes were fixed in absolute ethanol (vetec, 99.8%), kept in eppendorf vials and stored for 6 months at 4º c for further analysis of ovarian development and insemination. the cuticular hydrocarbons were extracted from the mesosome immediately after the collection (see below for details) and analyzed by gas chromatography – flame ionization detector (gc-fid). analysis of reproductive physiology for the analysis of ovarian development, the metasomes of females were dissected, and the ovarian development stages were analyzed using a stereomicroscope zeiss stemi 2000-c, equipped with an ocular micrometer and digital camera. the ovarian development stage classification was performed according to the developmental degree of ovarioles (adapted from baio et al., 2004). ovary type 1: presence of filamentous ovarioles (first stage); ovary type 2: ovarioles slightly swollen and some mature oocytes (second stage) (gobbi et al., 2006; oliveira et al., 2007); ovary type 3: large ovarioles with well developed oocytes (third stage) (gobbi et al., 2006). to assess whether females were inseminated, basic fuchsin colorimetric reagent was applied to the spermatheca, which acquires opaque or pearly staining in the presence of sperm (noda et al., 2003). cuticular hydrocarbons: extraction and gc-fid analysis the mesosomes were submitted to the extraction process for subsequent analysis by gc-fid. the cuticular constituents of each sample were extracted with 1 ml hexane (hplc grade, tedia) for 2 minutes. after filtration, the solvent was removed in a laminar flow exhaustion chapel. each extract was dissolved in 50 µl of hexane for gc-fid analysis. the samples and a standard mixture of linear alkanes (c7-c31, sigma aldrich with purity ≥ 90%) were analyzed using a gas chromatograph with flame ionization detector (thermo scientific focus gc, san jose, ca, usa) equipped with an ov-5 capillary column (5% phenyl dimethylpolysiloxane, 30 m long x 0.25 mm id x 0.25 µm film thickness, ohio valley specialty company, marietta, oh, usa). the injector and detector were kept at 280ºc using n2 as the carrier gas with a flow rate of 1.0 ml/min). the oven temperature was kept at 50 ºc for three minutes, reaching 280°c at a rate of 8ºc/ min, and remaining at 280 °c for 15 minutes. the injection was in splitless mode, and 1.0 µl of sample was injected. the chromatograms were recorded using the software chrom quest 5.0, and analyzed with the software workstation chrom data review. the linear alkanes analyzed by gc-fid were identified by comparison with the retention times of the standards c7-c31 alkenes. sociobiology 64(3): 327-333 (september, 2017) 329 we used the values of the relative percentual area of the linear alkanes, obtained by gc-fid. to assess whether there are significant differences among females from different aggregates and physiological status a canonical discriminant analysis was performed. results three degrees of ovarian development were identified in females of p. versicolor from the two aggregates analyzed (fig 1, table 1). in aggregate 1 (a1) 15% of the females fig 1. different ovarian development degrees found in two wasps of polistes versicolor. a) first stage of ovarian development, ovary type 1; b) second stage of ovarian development, ovary type 2; c) third stage of ovarian development, ovary type 3. were inseminated, 33.3% of which showed ovary type 1 (fig 1a), 50% ovary type 2 (fig 1b) and 16.7% ovary type 3 (fig 1c). while in aggregate 2 (a2), 17.2% of the females were inseminated, 80% of which presented ovary type 1 and 20% ovary type 2 (figs 1a and 1b). 25 linear alkanes were identified ranging from c7 to c31. there was significant difference in linear alkanes composition between females of aggregate 1 (wilks’ lambda = 0.07, f = 49.02; p > 0.05), based on their ovarian development degrees. the first canonical root explained 72% of these results (fig 2a). aggregate stage of ovarian development (sod) spermatheca total of females first second third inseminated non-inseminated a1 52.5 40.0 7.5 15.0 85.0 40 a2 48.3 51.7 17.2 82.8 29 table 1. percentage of polistes versicolor of each different ovarian development stage and spermatheca insemination in two winter aggregates. fig 2. discriminant analysis of linear alkanes present in polistes versicolor females from two aggregates and with different ovarian development degrees obtained by gc-fid. a) aggregate 1, b) aggregate 2, 1º sod=first stage of ovarian development, 2º sod= second stage of ovarian development, 3º sod =third stage of ovarian development. jhs brito et al. – linear alkanes in aggregates of wasp polistes versicolor330 significant differences were also found between females of aggregate 2 (wilks’ lambda = 0.32, f = 9.31; p > 0.05), with the first canonical root explaining 84% of results (fig 2b). the same type of analysis showed significant differences in cuticular linear alkanes composition between inseminated and non-inseminated females (wilks’ lambda = 0.09, f = 47.02; p < 0.05), and the first canonical root explains 76% of results (fig 3). discussion females of p. versicolor from aggregate 1 presented 3 degrees of ovarian development and females from aggregate 2 presented 2 degrees of ovarian development, the proportion of linear alkanes differed significantly between females with different ovarian development degrees and also between inseminated and non-inseminated females. females with different ovarian development degrees had significantly different linear alkane composition (fig 2a and b), which indicates that these category of compounds may be important to signal the reproductive status of these females. this is in agreement with other studies on hymenoptera females that reported a variation in cuticular chemical composition due to the fertility (cuvillier-hot et al., 2001; sledge et al., 2001; biseau et al., 2004; will et al., 2012; van oystaeyen et al., 2014). hence, fig 3. discriminant analysis of linear alkanes present in inseminated and non-inseminated females from two aggregates of polistes versicolor obtained by gc-fid. a1= aggregate 1, a2= aggregate 2, i= inseminated females and ni= non-inseminated females. there is apparently a strong correlation between reproductive status and cuticular chemical composition in females of social insects colonies (sledge et al., 2001; monnin, 2006). dapporto et al. (2004) studying colonies of p. dominula found qualitative differences in cuticular chemical composition between wasps with different reproductive status. the same species was investigated by sledge et al. (2001, 2004) who found similar results. likewise, bonckaert et al. (2012), investigating colonies of vespula vulgaris (linnaeus), also found significant differences between cuticular composition among females with different physiological conditions. studies with other social hymenoptera, as in colonies of the ant temnothorax unifasciatus (latreille) (brunner et al., 2009), also found significant differences between the cuticular chemical composition of females of differing reproductive status. likewise, nunes et al. (2009) found similar results in the bee schwarziana quadripunctata (lepeletier). however, it should be noted that these studies evaluated all cuticular compounds. despite studies have shown that, indeed, cuticular chemical compounds, especially branched alkanes and alkenes, may signal the physiological status of females, tannurenascimento (2007) assessed that in colonies of polistes satan linear alkanes can also play this role. therefore, despite these compounds being more involved with the ability of the cuticle to prevent desiccation (blomquist, & bagnères, 2010), our results demonstrate that the functions of these compounds, as well as of others might have been underestimated. according to our results, insemination also leads to significant differences between linear alkanes composition of females (fig 3). torres et al. (2014) studying the relationship between cuticular chemical profile and physiological condition of p. versicolor females, found correlation between ovarian development and spermatheca condition. soares et al. (2014) also found similar results studying colonies of polistes ferreri saussure 1853. studies with other social hymenoptera, such as ants of the species cardiocondyla obscurior wheeler 1929, in which inseminated and non-inseminated females showed quantitative differences between some linear alkanes, provide evidence of these compounds importance for signaling reproductive status (will et al., 2012). biseau et al. (2004) found qualitative differences in linear and branched alkanes between inseminated and non-inseminated females of the ant linepithema humile mayr 1868. other studies such as those by oettler et al. (2008) with crematogaster smithi creighton 1950 and oppelt and heinze (2009) with leptothorax gredleri mayr 1855 also reported that linear alkanes may be important for signaling the reproductive status of females in their colonies. in aggregates, in particular, these signals can be used during interactions between females or serve to identify which females are already inseminated allowing males to identify them and, thus, invest their efforts in females that have not been inseminated, when they may still be associated with males. indeed, according to blomquist and bagnères (2010) sociobiology 64(3): 327-333 (september, 2017) 331 social pheromones of queens have probably evolved from preexisting fertility signals targeted at males, for example, in the context of choice or partner attraction. thus, linear alkanes seem to be as important as other compounds involved in intraspecific signaling. therefore, females of p. versicolor in aggregates may have three different degrees of ovarian development. all these females can be identified by the proportion of linear alkanes in their cuticle, corroborating the results of other studies that investigated the importance of this category of compounds for signaling reproductive status and, therefore, also be responsible for mediating social interactions in wasps. acknowledgments agência brasileira de inovação (finep), coordenação de aperfeiçoamento de pessoal de nível superior (capes), fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect) and conselho nacional de desenvolvimento científico e tecnológico (cnpq), and productivity grants of wfaj and calc. references abdalla, f.c., jones, g.r., morgan, e.d. & cruz-landim, c. 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(1971). the insect societies. belknap, cambridge, 548p. doi: 10.13102/sociobiology.v64i3.1692sociobiology 64(3): 352-355 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 isolation and characterization of polymorphic microsatellite markers for two subterranean termites subterranean termites are the most common and economically important species (nobre et al., 2006; pinzon et al., 2009). many termite species have become successfully established outside their native ranges, and cause environmental and economic damages to the invaded areas (perdereau et al., 2011). reticulitermes aculabialis and r. labralis are two important subterranean termites, particularly in the middle and eastern china, where they are considered invasive (su et al., 2016; wang et al., 2016). although the population status and the breeding structure of several termite species have been analyzed by microsatellite loci (vargo & carlson, 2006; vargo et al., 2013; huang et al., 2013; perdereau et al., 2015), highly polymorphic microsatellite markers used for analysis of reticulitermes termites are still insufficient. to facilitate the research of termite genetic diversity and population structure, we have screened 147 abstract we isolated 15 and 18 highly polymorphic genomic microsatellite markers from two subterranean termites, reticulitermes aculabialis and r. labralis, respectively. a total of 53 alleles were detected in 15 microsatellite loci of r. aculabialis, and the alleles were 3.533±1.302 (mean±sd), while the corresponding data of r. labralis were 115 detected alleles in 18 microsatellite loci with 6.389±1.754 alleles. the observed and expected heterozygosity was 0.496±0.236 and 0.564±0.125 in r. aculabialis, and 0.368±0.263 and 0.702±0.115 in r. labralis, respectively. seven loci were highly polymorphic (pic>0.5) in r. aculabialis, and 15 loci were highly polymorphic (pic>0.5) in r. labralis. all loci showed hardy–weinberg equilibrium. these polymorphic markers provide useful tools for population genetic and breeding system studies of subterranean termites. sociobiology an international journal on social insects yl dang1 2 3, hg zhang1 2 3, yf meng1 2 3, m zhang1 2 3, s zhao1 2 3, p you4, xh su1 2 3, lx xing1 2 3 article history edited by qiuying huang, huazhong agricultural university, china received 08 may 2017 initial acceptance 09 september 2017 final acceptance 12 september 2017 publication date 17 october 2017 keywords subterranean termites, reticulitermes, genetic diversity, microsatellite loci. corresponding author lian-xi xing college of life sciences, northwest university nº 229, north taibai rd., xi’an, shaanxi province, 710069, p. r. china. e-mail: lxxing@nwu.edu.cn microsatellites and identified 15 and 18 highly polymorphic loci for r. aculabialis and r. labralis, respectively. the samples of r. aculabialis and r. labralis were both collected from eight locations of xi’an, china. for each location, two worker samples were randomly selected to detect the polymorphism. in order to exclude the intestinal microbes in the termite abdomen, we extracted dna from the head of the termite. the genomic sequence library and microsatellite library were designed and set up according to the dna of r. aculabialis. the constructed library obtained a number of 100 bp short sequence fragments by illumina hiseq 2000 sequencing. we used default parameter settings of clc genomics workbench v7.5 software to trim the fragments with excellent quality and de novo assemble them. only the consensus sequences were used for microsatellite scanning. by scanning the microsatellite loci of above consensus sequences short note 1 shaanxi key laboratory for animal conservation, northwest university, xi’an, china 2 key laboratory of resource biology and biotechnology in western china, ministry of education, northwest university, xi’an, china 3 college of life sciences, northwest university, xi’an, china 4 college of life sciences, shaanxi normal university, xi’an,china sociobiology 64(3): 352-355 (september, 2017) 353 with sciroko v3.4 software (kofler et al., 2007), a total of 594 microsatellite loci were found, distributed on 571 consensus sequences. 147 consensus sequences were randomly selected among the above 571 sequences and designed by primer v3.0 (rozen & skaletsky, 2000). the primers were synthesized by genedigger (xi’an) technology co., ltd. dna was extracted using tianamp genomic dna kit, and the extracted dna were amplified with a thermocycler (mastercycler nexus gradient) in 5 µl reactions containing 2×taq pcr mix 2.5 μl, ddh2o 1.1 μl,0.2 µl of each primer and 1 μl dna. the amplification program consisted of an initial denaturation at 94°c for 5 min followed by 30 cycles at 94°c for 30 sec, annealing temperature (ta in table1and table 2) for 30 sec and 72°c for 30 sec, final extension at 72°c for 10 min. amplification products were analyzed with polyacrylamide gel electrophoresis and genotyped using quantity quantity one software. the number of alleles (k), observed heterozygosity (ho), expected heterozygosity (he) and hardy-weinberg equilibrium (hwe) test were performed by genalex v6.501, and polymorphism information content (pic) were estimated by cervus v3.0 (kalinowsk etal., 2007). all loci met with hardy–weinberg equilibrium. the 147 loci were screened both in r. aculabialis and r. labralis, with an isolation of 22 pairs and 21 pairs microsatellite primers in these two species, respectively. all these primers yielded stable and effective amplification bands. then, 15 highly polymorphic microsatellite loci in r. aculabialis and 18 highly polymorphic microsatellite loci in r. labralis were identified. for the 15 polymorphic loci of r. aculabialis, 8 are moderately polymorphic (0.25<pic<0.5), 7 are highly polymorphic (pic>0.5) (table1). for the 18 polymorphic loci of r. labralis, 3 are moderately polymorphic (0.25<pic<0.5), 15 are highly polymorphic (pic>0.5) (table2). we calculated the genetic diversity per locus, i.e number of alleles (k), observed heterozygosity (ho), expected heterozygosity (he) and pic. number of alleles (k) for each locus ranged between 2 and 6 in r. aculabialis. the mean alleles were 3.533±1.302 (mean±sd). whereas the observed and expected heterozygosity table 1. highly polymorphic microsatellite primers in reticulitermes aculabialis. locus primer sequence (5’–3’) k size motif ta ho he pic ra014 f:cgtactgcgggaagtactga r:tgttgtgctttagtgctggc 4 259-284 (aattc)5 51.8 1.000 0.711 0.658 ra022 f:acagatcagacgcaaggctc r:agatgatgatgctgggctct 3 213-235 (aggc)6 55.6 0.938 0.607 0.539 ra024 f:agaaggactctgctgcatgg r:cgttgtaaccacatgccaag 2 180-184 (agtt)10 55.6 0.500 0.492 0.371 ra050 f:tccagttgtcacttcgacaga r:gtcaaggtcccgtcctgtta 4 107-119 (atgt)15 50.3 0.438 0.525 0.486 ra070 f:tacagagctttcatggcacg r:aaacctcgaaatgaggaggc 3 150-156 (cta)12 58.2 0.500 0.607 0.530 ra079 f:taccctgtggagaactcgct r:aatgaccttcttgggcgttt 3 200-208 (gaat)9 55.6 0.250 0.508 0.428 ra095 f:ctgctaggaagcaacgaacc r:aagacctcggaaagaggagg 3 160-163 (taa)9 59.1 0.375 0.389 0.334 ra096 f:tcgtacatacagacggacgtg r:gcttctcaagaaggactgtgc 3 212-232 (taac)10 59.1 0.250 0.406 0.371 ra098 f:acagcttacgccgctgtatc r:ctcaagaaggactctgctcca 2 233-237 (taac)6 59.1 0.313 0.451 0.349 ra103 f:tgcctgtttcgttgatgaag r:atccaatcctacttgcgtgg 2 241-245 (taca)11 50.3 0.438 0.498 0.374 ra116 f:tcgaccgactcagtagcctt r:aaagatggagggacgaggtt 6 201-219 (tct)11+(cct)6 59.1 0.500 0.766 0.727 ra128 f:gtctcgtcaaattgttggca r:atcaccgttggttcaagagg 4 105-114 (tta)10 51.8 0.438 0.443 0.402 ra130 f:aaagaggaggcaagaggagg r:catctctgcggtgatgagag 5 225-246 (tta)11 56.9 0.313 0.717 0.676 ra132 f:gattggtttcctccgaatca r:aaagactactgccaccggg 3 201-213 (tta)14 58.2 0.375 0.602 0.516 ra144 f:caaatagagctccgtgtttcg r:ccatagaaacctccgaaagg 6 146-186 (ttag)7 56.9 0.813 0.736 0.698 ta the annealing temperature, size approximately product size, k number of alleles, ho observed heterozygosity, he expected heterozygosity, pic polymorphism information content. yl dang et al. – polymorphic microsatellite markers for reticulitermes termites354 ranged between 0.250 to 1.000 and 0.389 to 0.766, respectively. the mean observed and expected heterozygosity were 0.496±0.236 and 0.564±0.125, respectively. the pic varied from 0.334 to 0.727 with a mean of 0.497±0.137. in addition, number of alleles (k) ranged from 3 to 9 in r. labralis. the mean alleles were 6.389±1.754. whereas the observed and expected heterozygosity ranged between 0.063 to 1.000 and 0.447 to 0.859, respectively. the mean observed and expected heterozygosity were 0.368±0.263 and 0.702±0.115, respectively. the pic ranged from 0.371 to 0.843 with a mean of 0.663±0.127. these results showed that the genetic variation of r. aculabialis and r. labralis were very high, suggesting that these microsatellite markers are essential for estimating the genetic diversity and population genetic of reticulitermes termites. acknowledgements this study was funded by the national science foundation of china (31170363, 31370428) and opening foundation of key laboratory of resource biology and biotechnology in western china (northwest university), ministry of education. we would like to thank ms hui-min li (college of life sciences, northwest university) for her support of software analysis. disclosure the authors declare no conflict of interest. locus primer sequence (5’–3’) k size motif ta ho he pic ra014 f:cgtactgcgggaagtactga r:tgttgtgctttagtgctggc 7 260-290 (aattc)5 51.8 0.438 0.635 0.603 ra022 f:acagatcagacgcaaggctc r:agatgatgatgctgggctct 6 212-244 (aggc)6 55.6 0.500 0.590 0.548 ra024 f:agaaggactctgctgcatgg r:cgttgtaaccacatgccaag 6 156-184 (agtt)10 55.6 0.313 0.721 0.677 ra050 f:tccagttgtcacttcgacaga r:gtcaaggtcccgtcctgtta 9 160-232 (atgt)15 50.3 0.500 0.859 0.843 ra070 f:tacagagctttcatggcacg r:aaacctcgaaatgaggaggc 7 144-174 (cta)12 58.2 0.313 0.723 0.695 ra071 f:gaacaatggtcatccagcct r:ttggctattcagtcagcaca 8 252-285 (cta)8 59.1 0.313 0.760 0.728 ra079 f:taccctgtggagaactcgct r:aatgaccttcttgggcgttt 6 184-212 (gaat)9 55.6 0.063 0.666 0.635 ra091 f:aacttcctttgaatgcgctc r:gcataccagaggtcctgcat 9 233-261 (taa)5 56.9 0.188 0.846 0.828 ra095 f:ctgctaggaagcaacgaacc r:aagacctcggaaagaggagg 4 153-162 (taa)9 59.1 0.313 0.580 0.493 ra096 f:tcgtacatacagacggacgtg r:gcttctcaagaaggactgtgc 5 196-232 (taac)10 59.1 0.250 0.500 0.474 ra097 f:cactgcaagacgcaaagtgt r:gcttctcaagaaggactgtgc 4 252-264 (taac)5 59.7 0.375 0.729 0.677 ra103 f:tgcctgtttcgttgatgaag r:atccaatcctacttgcgtgg 5 232-252 (taca)11 50.3 0.125 0.729 0.682 ra116 f:tcgaccgactcagtagcctt r:aaagatggagggacgaggtt 8 177-219 (tct)11+(cct)6 59.1 1.000 0.752 0.721 ra126 f:gtgccgttagtttgccattt r:agtgggagccgagttgttc 3 216-224 (tgtc)7 59.1 0.063 0.447 0.371 ra128 f:gtctcgtcaaattgttggca r:atcaccgttggttcaagagg 6 115-139 (tta)10 51.8 0.125 0.695 0.644 ra130 f:aaagaggaggcaagaggagg r:catctctgcggtgatgagag 8 216-249 (tta)11 56.9 0.563 0.789 0.759 ra141 f:cacatttgaggttcgcaaga r:gccagaaggccaattacaga 6 165-210 (tta)8 56.9 0.250 0.813 0.785 ra144 f:caaatagagctccgtgtttcg r:ccatagaaacctccgaaagg 8 148-184 (ttag)7 56.9 0.938 0.805 0.779 abbreviations as in table 1. table 2. highly polymorphic microsatellite primers in reticulitermes labralis. sociobiology 64(3): 352-355 (september, 2017) 355 references huang, q.y., li, g.h., claudia, h. & lei, c.l. 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(2016). complete mitochondrial genome of a parthenogenetic subterranean termite, reticulitermes aculabialis tsai et hwang (isoptera: rhinotermitidae). mitochondrial dna part a: dna mapping, sequencing and analysis, 5: 3133-3134. doi: 10.3109/19401736.2015.1007299 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.1795sociobiology 64(4): 381-392 (december, 2017) ground-dwelling and vegetation ant fauna in southern brazilian grasslands introduction non-forest ecosystems, as grasslands, savannas, shrublands and open woodlands, cover large extensions of land in four of the six brazilian biomes (overbeck et al., 2015). such ecosystems host high levels of unique biodiversity that provisions ecosystem services but are severely neglected concerning conservation policies and protection when compared to forest ecosystems (andrade et al., 2015). suitable conservation strategies of non-forest ecosystems often demand different perceptions related to land management (e.g. the role of grazing and fire in grasslands) and offers great opportunities to conciliation with sustainable economic use (overbeck et al., 2007). considering the high habitat conversion rates of these ecosystems to other land uses, ecological restoration is a highly necessary component abstract non-forest ecosystems, as natural grasslands from southern brazil, are still neglected in conservation policies. measuring their biodiversity is one of the main steps to generate management strategies for these habitats. this study aims to (i) describe grassland ant richness and composition in rio grande do sul state, and (ii) compare ant communities sampled on the ground and in grassland vegetation, adding to our knowledge of habitat use patterns and vegetation associated species. six sites were sampled, three belonging to the pampa biome and three in highland region from the atlantic forest biome. ant fauna was collected once per year in summer during four years in each site with pitfalls traps and sweeping nets. overall, 29,812 ant individuals were sampled belonging to eight subfamilies, 30 genera e 106 species. the grasslands of pampa accumulated 91 species and 45 exclusive species, while highland grasslands summed up 61 species and only 15 exclusive species. species composition differs between biomes as well as between sampling methods. ant communities sampled from vegetation represented a clear subset of the fauna sampled with pitfall traps, and indication analysis showed only two species associated with this stratum: myrmelachista gallicola and pseudomyrmex nr. flavidulus. this study highlights the importance of southern brazilian grasslands and the need for specific conservation strategies for the natural grasslands from each biome. sociobiology an international journal on social insects w dröse1, lr podgaiski1, a cavalleri2, rm feitosa3, ms mendonça jr1 article history edited by ricardo solar, ufmg, brazil received 14 june 2017 initial acceptance 19 september 2017 final acceptance 25 september 2017 publication date 27 december 2017 keywords campos sulinos, pampa biome, atlantic forest biome, formicidae, lter. corresponding author william dröse programa de pós-graduação em biologia animal, universidade federal do rio grande do sul cep 91501-970 porto alegre-rs, brasil. e-mail: william_drose@hotmail.com of their conservation (overbeck et al., 2013). nevertheless, to define suitable conservation and restoration strategies it is first necessary to better understand species diversity patterns and composition. in south brazil, grasslands are naturally widespread over the states of rio grande do sul, santa catarina and paraná, where they are known as campos. the southern part of campos ecosystems embraces the pampa biome, which are among the most species-rich grasslands in the world, extending to argentina and uruguay (rio de la plata grasslands) (bilenca & miñarro, 2004; overbeck et al., 2007). the northern part of campos comprises highland grasslands (altitude at about 800 to 1,000 m, with highest peaks up to 1,800 m) that belong to the atlantic forest biome where it forms mosaics with araucaria forests (andrade et al., 2016). grassland physiognomy and structure varies greatly depending 1 universidade federal do rio grande do sul, porto alegre-rs, brazil 2 universidade federal do rio grande, rio grande-rs, brazil 3 universidade federal do paraná, curitiba-pr, brazil research article ants w dröse et al. – ant fauna in south brazilian grasslands382 on the region, the altitude, and mostly on the management it receives, ranging from very short vegetation in highly grazed systems, to very tall and complex vegetation with shrub and treelet species under low management (overbeck et al., 2007). pampa grasslands are more intensively grazed than the highland grasslands of atlantic forest, presenting typically a high dominance of prostate plant species. on the other hand, highland grasslands receive frequent burnings (i.e. every one or two years) in the end of the winter, and its physiognomy is dominated by highly fire-resilient grass tussock species (boldrini, 2009). although grassland plant diversity is relatively well known in south brazilian grasslands, diversity patterns of invertebrate groups are barely studied at all. for example, there is a huge information gap regarding ant fauna when compared to other brazilian biomes. ants are abundant and diverse organisms that present very special roles as ecosystem engineering and in provision of ecosystem services (hölldobler & wilson, 1990). worldwide, ant communities have been largely used as a bioindicator group for land use changes and disturbance analysis (e.g. underwood & fisher, 2006; nemec, 2014), showing very positive contributions to rangeland systems monitoring (e.g. hoffmann, 2010), and evaluation of habitat restoration success (e.g. andersen & sparling, 1997). until now, only a few ant studies were conducted and published specifically in campos ecosystems. among them, albuquerque and diehl (2009) present an ant survey on highland grasslands, pinheiro et al. (2010) analyze edge effects in grassland-forest transitions also at this region, and rosado et al. (2012) compare ant fauna from vineyards and adjacent grassland ecosystems in the pampa biome. in northeastern argentina, calcaterra et al. (2010) and calcaterra et al. (2014) studied ground-foraging ant responses to grazing and fire in grasslands and savannas in the iberá nature reserve. ants explore different resources in a variety of microhabitats (hölldobler & wilson, 1990), occupying from forest canopies to subterranean layers. the differential use of a specific strata or microhabitat is commonly found in ant communities in several systems (vasconcelos & vilhena, 2006; schmidt & solar, 2010; wilkie et al., 2010), including non-forest ecosystems as savannas (e.g. cerrado: campos et al., 2008). similarly to the habitat heterogeneity hypothesis (sarty et al., 2006), habitat vertical partition commonly increases species diversity in the ecosystems by reducing competition by resources and allowing coexistence of more species. it is still unknown how ant communities are structured between ground and vegetation layers in grassland ecosystems of south brazil (e.g. species foraging patterns and microhabitat use), and whether habitat partition can actually occur. this study describes the ant fauna of sites in a longterm ecological research program in south brazilian grasslands (lter/peld campos sulinos cnpq), including six natural grassland ecosystems both in pampa and atlantic forest biomes, sampled with two different methods. the aims of this study were to describe grassland ant community richness and composition (i) from different sites and regions of the campos ecosystems, and (ii) from ground and vegetation strata, pointing out habitat use patterns and vegetation associated species. material and methods study area the study was undertaken in six natural grasslands under traditional cattle grazing in the state of rio grande do sul, brazil. three sites were located within private properties in the pampa biome: aceguá (31º38’55”s, 54º09’26”w), alegrete (30º04’11”s, 55º59’34”w) and lavras do sul municipalities (30º42’02”s, 53º58’53”w). the other three sites were located within conservation units in the atlantic forest biome, in the highland region: cambará do sul (29º08’19”s, 50º09’27”w; aparados da serra national park), jaquirana (29º05’43”s, 50º22’02”w; tainhas state park) and são francisco de paula municipalities (29º23’35”s, 50º14’26”w; aratinga ecological station) (fig 1). climate in rs is temperate, wet, with hot summers and no dry season (nimer, 1979). according to the köppen climate classification, the largest area of rs is classified as cfb climate, with cfa restricted to regions with high altitudes in the pampa and in the highland region of the atlantic forest (kuinchtner & buriol, 2001). sites sampled in the pampa biome have annual mean temperatures of 18ºc, annual mean precipitation of 1423 mm and mean altitude of 224 m. however, sites in the atlantic forest have annual mean temperature of 15.3ºc, annual mean precipitation of 1935 mm and mean altitude of 931 m (climate data, 2016). sampling design at each grassland site, a homogeneous area of approximately 14.700 m² with traditional grazing was chosen fig 1. study sites of long-term ecological research program (lter/peld campos sulinos) in rio grande do sul state, brazil (numbers 1-6). light gray area represents pampa grasslands original area, and dark gray area the atlantic forest biome. sociobiology 64(4): 381-392 (december, 2017) 383 where 3 plots of 70 x 70 m were settled. by chance, one experimental plot was completely excluded from grazing, another received a conservative grazing management, and the third remained with the local traditional grazing regime. for the present study, treatment information from these three plots was not considered; the differences in ant fauna regarding the grazing treatments will be addressed in a further study. ants were sampled once per year in november/ december during four years at each site. all samples were carried out from 2011 to 2014; except for cambará do sul where they occurred from 2012 to 2015. two sampling methods were employed: pitfall traps for ground-dwelling ants and sweeping net for ants from grassland vegetation. at each plot eight pitfall traps were installed (24 per site) at least 15 m far from each other. the trap consisted in a 500 ml transparent plastic jar (10 cm diameter, 12 cm deep) filled with 150 ml of formalin (3% formaldehyde), which remained open during seven days. to reduce the evaporation rate of formalin and to protect the traps from direct rainfall, green plastic dishes sustained by wooden sticks were used as rain guards. the ants from vegetation were sampled with sweep net (50 cm wide; sampling area of 0.1 m²) along four parallel transects in each plot, all pooled together in a unique sample. vegetation was swept during two different occasions (before pitfall installation and just before their removal) per year, totaling six samples per site (3 plots x 2 occasions). the ant specimens were previously stored in a plastic bag with ethyl acetate. all ant individuals were preserved in ethanol 80% and stored in the laboratório de ecologia de interações (lein) in universidade federal do rio grande do sul (ufrgs). ants were assigned to genera based on dichotomic keys (baccaro et al., 2015). for species classification, specific literature was used and comparisons were done with material in scientific ant collections in lein and the entomological collection padre jesus santiago moure of the universidade federal do paraná (dzup). all species/morphospecies names follow a standard number from the ant collection of lein to standardize different studies and further publications. vouchers are deposited in ant collections of lein and dzup. data analysis to compare species richness among the different study sites, sample-based species rarefaction curves were calculated for each site with 9999 bootstraps with the inext online tool (hsieh et al., 2016). separated curves were built for the different sampling methodologies as well. for that, a matrix with ant incidence data considering all the records of the species in all plots and years pooled together was used; for vegetation ants, incidence was the number of times the species was sampled by sweeping the vegetation (e.g. maximum 24 times per site), and for the ground it was the number of pitfall traps that the species was found in (e.g. maximum 96 pitfalls per site). non-metric multidimensional scaling (nmds) was used to represent the ordination of species composition in the sites within the biomes, considering both methods of sampling. a species absence/presence matrix was built containing the ant species from vegetation and ground in the columns and the plots per site in the rows (18 sampling units), compared with jaccard similarity index. to test whether there were significant differences in species composition between biomes and between sampling methods, an analysis of similarity (twoway anosim) was employed, with 9999 permutations. nmds and anosim analysis were performed with past software (hammer et al., 2001). to determine ant species association to a specific stratum (vegetation or ground), the indicator value (indval) method of dufrêne and legendre (1997) was used. this method combines measures of specificity of a species to a habitat type and its fidelity within that group. species with values of 100 would mean perfect indication. here a value of 70 or higher was considered sufficient for indication of a special relationship between a species and a habitat (nakamura et al., 2007; chen et al., 2011; verdu et al., 2011). a matrix was arranged containing ant species composition in columns and the 18 sampling units (3 plots per site) in rows, in two different groups (vegetation and ground). as the different strata were sampled with different methods and sampling efforts, the matrix was standardized by only considering the incidence data of the species per year in each strata, varying from 0 (no incidence) to 4 (incidence in all years) in each plot. the indicator value was calculated for each species using the “multipatt” function of the r package “indicspecies” (r development core team, 2016), based on 9999 permutations. results overall, 29,812 ant individuals from eight subfamilies, 30 genera and 106 species were sampled (appendix 1). myrmicinae was the richest subfamily, with 58 species, followed by formicinae (16), ponerinae (13), dolichoderinae (10), dorylinae (4), ectatomminae (3) and heteroponerinae fig 2. venn diagrams showing the number and percentage of exclusive and shared ant species for pampa (red) and highland (green) grasslands (atlantic forest biome), and their respective contribution to differences between vegetation and ground samples. w dröse et al. – ant fauna in south brazilian grasslands384 and pseudomyrmecinae (one species each). the richest genera were pheidole (18 species), solenopsis (13), hypoponera (10) and camponotus (8). the most frequent genera were pheidole (1,694 occurrences), solenopsis (864), camponotus (514) and brachymyrmex (457). overall, the grasslands from pampa biome accumulated 91 species, while the highland grassland summed up 61 species. in addition, pampa grasslands had more exclusive species (45 species) than highland ones (15 species), with 46 shared species (fig 2). rarefaction curves showed lavras do sul and alegrete municipalities to present significantly higher ant species richness for both sampling methods (fig 3). yet, aceguá and jaquirana municipalities presented an intermediate richness when considering the ground ant fauna, accumulating more species than cambará do sul and são francisco de paula sites. the ant species composition showed clear differences between biomes (anosim: r=0.55, p<0.001), as well as between ground and vegetation sampling (anosim: r=0.68, p<0.001) (fig 4). pitfall traps sampled a total of 100 ant species, and sweeping net 55 species, with 49 species (46%) shared between sampling methods. the proportion of shared species between methods was similar between biomes (fig 2). overall, six species occurred exclusively in samples from the vegetation (5% of the total species richness), but the indicator analysis (indval) showed only two species particularly associated to this stratum: myrmelachista gallicola mayr, 1887 and pseudomyrmex nr. flavidulus (smith, 1858) (appendix 2). fifty-one species (48% of the total species richness) were only sampled from the ground, and the indicator analysis revealed 17 species strictly associated to the ground stratum (appendix 2). for grassland vegetation sampling, 13 ant species were fig 3. incidence-based rarefaction for vegetation (24 sampling units by sweep net) and ground samples (96 pitfall traps) in six grassland sites in rio grande do sul state, brazil, under a multinomial model, with 95% unconditional confidence intervals (shaded area, bootstrap with 9999 replications). alegrete, lavras do sul and aceguá belong to pampa grasslands (red) and cambará do sul, são francisco de paula and jaquirana to highland grasslands (atlantic forest biome) (green). considered dominant (more than 20% frequent in samples) in pampa grasslands, while only four species did so in highland grasslands (fig 5). the most dominant species in vegetation in both biomes were camponotus punctulatus mayr, 1868, brachymyrmex sp. 1 and camponotus sp. 2, but their level of dominance changed considering the biome, e.g. they comprised more than 50% of all ants in pampa and less than 30% in highland grasslands (fig 5). for ground sampling, 16 ant species were considered dominant in pampa and 10 in highland grasslands (fig 5). solenopsis invicta buren, 1972 was the most frequent species in traps from pampa (62%), followed by cyphomyrmex gr. rimosus sp. 1 (54%). in highland grasslands, pheidole obtusopilosa mayr, 1887 was highly dominant, present in fig 4. nmds ordination of grassland sampling sites ant species compositions (presence/absence) with jaccard similarity index. pampa grasslands (red): aceguá (circle), alegrete (square), lavras do sul (triangle); highland grasslands (green, atlantic forest biome): cambará do sul (triangle), jaquirana (plus sign), são francisco de paula (diamond). sociobiology 64(4): 381-392 (december, 2017) 385 51% of all pitfall traps (fig 5). discussion this study represents the first attempt to characterize the ant fauna from south brazilian grasslands reaching sites distributed in two biomes sampled long-term in two target microhabitats. the survey presents the highest ant richness already recorded for these ecosystems, and also adds two new ant species records to rio grande do sul state (based on diehl et al., 2014 and specialized literature): trachymyrmex pruinosus (emery, 1906) and wasmannia sulcaticeps emery, 1894 and a new record to brazil: pheidole pampana santschi, 1929 (ant maps, 2016; ant wiki, 2016). furthermore, a new species was found (acanthoponera sp. n., rmf unpublished data). in summary, this study reveals differences in ant community structure occurring in grasslands from pampa and atlantic forest biomes, singling out the grasslands of pampa as very rich ant spots. ant fauna sampled from vegetation by sweeping net appeared to be a subset of the ant fauna sampled from the ground by pitfall trapping. altogether, the results presented here may provide useful information for future studies and conservation efforts. few, if any, ant studies so far have managed to sample for longer times and used widely spaced sampling sites for fig 5. most frequent ant species in pampa and highland grasslands (atlantic forest biome) from vegetation and ground samples. only species with >20% presence across all sampling units are shown. w dröse et al. – ant fauna in south brazilian grasslands386 campos ecosystems. for example, albuquerque and diehl (2009) surveyed 32 ant species along eight grassland sites in cambará do sul municipality at the highland region. pinheiro et al. (2010) recorded 31 morphospecies in grassland-forest ecotones also at this region. rosado et al. (2012) related 72 ant species to vineyards and adjacent grassland habitats at the campanha region in pampa biome. in northeastern argentina, which represents an extension of the campos ecosystems, calcaterra et al. (2010) evaluated the effect of grazing on 50 ant species in savanna and grassland, while calcaterra et al. (2014) studied fire effects on 67 grassland ant species, both at iberá nature reserve. in comparison to these studies, our survey shows an expressive ant species richness (106 species) that could be clearly explained by (i) the longer sampling duration (i.e. 4 years) increasing the likelihood of finding rare or eventual species, and (ii) the broader geographic scale attained, which incorporates a greater variety of environments and management situations. variation in site characteristics, such as latitude, altitude, soil types, climate, land management (e.g. grazing intensity and fire frequency) and vegetation physiognomy, are generally correlated to a greater variation in ant species composition, likely enhancing gamma diversity (i.e. total species richness; schoeman & foord, 2012). this explanation seems to be also useful to elucidate why we sampled more ant richness (i.e. 91 spp.) in pampa grasslands than in the highland region (i.e. 61 spp.). since the geographic area in pampa is larger and the sampled sites were spatially further apart, there is indeed more heterogeneity of associated habitat conditions (e.g. soil types, vegetation physiognomies, plant richness; streck et al., 2008; ferreira et al., unpublished data) and thus a higher probability of finding a richer associated ant community in the pampa. ant fauna composition was also singular between these grassland regions (sharing only 43% of the total richness), and community structure based on dominant species also shifts regarding number of dominant species and their identity. a pattern that could be draw is that ant communities from highland grasslands seemed to be more even in terms of species incidence. altitude could certainly have an important contribution explaining ant fauna differences between the two biomes (szewczyk & mccain, 2016). highland grasslands in the atlantic forest biome are situated at altitudes from 800 to 1,000 m and have lower mean and minimal annual temperatures. several temperature-based hypothesis emerge to explain the decline in species diversity from low to higher altitudes, for example, relating colder temperatures to decreased food resources, reduced foraging periods, and lower metabolic rates (sanders et al., 2007; malsch et al., 2008). broad-scale diversity patterns in ants are likely to be supported by multiple entangled drivers, including interspecific competition, not yet comprehensively understood (szewczyk & mccain, 2016), thus our explanations here are tentative. considering samplings in the ground, s. invicta and c. gr. rimosus sp. 1 were dominant species in pampa, while p. obtusopilosa was dominant in highlands. solenopsis invicta is a generalist ant, known for its high competitive ability and one of the main species of invasive ants elsewhere. native from south america, this species was first introduced to southern united states and later to other regions of the world (ascunce et al., 2011). despite being responsible for damages in urban, agricultural and natural environments in non-native regions, their occurrence as the most abundant and/or frequent species is also reported in studies carried out in grassland ecosystems of argentina (calcaterra et al., 2010; calcaterra et al., 2014). cyphomyrmex is a fungus-farming ant. species in this group usually nest in the ground, leaf litter and rotten logs (mackay & serna, 2010). however, the rimosus group is known by the morphological complexity of its species, and a comprehensive taxonomic revision is currently under preparation (e. z. albuquerque & c. r. f. brandão, unpublished data). ants in the genus pheidole are usually generalist. they are widely distributed both global and locally and may occur from the vegetation canopy to the soil of open areas and forests (wilson, 2003). little is known about the biology of p. obtusopilosa, and its distribution is recorded from argentina, uruguay and brazil (rio grande do sul state) (ant maps, 2016; ant wiki, 2016). forty-nine ant species were found using both ground and grassland vegetation strata. although ant nests are predominantly established in the ground for grassland ecosystems, ant use of the local vegetation depends on the foraging behavior of each species (blüthgen & feldhaar, 2010). habitat structure (i.e. biomass, plant height, plant richness, and their spatial heterogeneity), and resource availability (i.e. presence of flowers, fruits, seeds, plants with extrafloral nectaries) could attract ant communities to forage and nest in the vegetation (campos et al., 2008), which in its turn depends on the management employed on each site (overbeck et al., 2007; overbeck et al., 2016). looking at the ant composition ordination diagram, a greater dispersion among vegetation samples is detected when compared to ground samples. this might be explained by the variability in vegetation structure and resources found between plots and sites. one species of brachymyrmex and two species of camponotus were the most frequent species foraging at vegetation in both biomes; these genera belong to subfamily formicinae and can present arboreal habits (baccaro et al., 2015). marques and del-claro (2006) found formicinae as dominant on the vegetation in either open or closed areas of cerrado, especially camponotus species. two ant species showed association with vegetation stratum (m. gallicola and p. nr. flavidulus). myrmelachista is considered an exclusively arboreal genus (longino, 2006), nesting in cavities and dry twigs of living trees, and is rarely found foraging on the ground (nakano et al., 2013; baccaro et al., 2015). species in this genus may also develop associations with host plants, extrafloral nectaries or associated aphids. pseudomyrmex is also a predominantly arboreal genus; it builds nests on tree twigs or hollow trunk cavities, and forages sociobiology 64(4): 381-392 (december, 2017) 387 predominantly in the vegetation (baccaro et al., 2015). both m. gallicola and p. nr. flavidulus were found in different plots at sites from pampa and highland region (appendix 1). a high number of plant species associated to the grass matrix, including shrub, treelet and even pioneer woody species, could be hosting ant populations of m. gallicola and p. nr. flavidulus. as our sweeping net sampling included all vegetation found within the plots, further studies should specifically investigate details on host plants, and a possible relation to plot grazing management applied. this study contributes with the overall description of ant diversity and composition from different sites and biomes in the south brazilian grasslands. the dynamics of the grassland ant communities along time (i.e. four years), which is a significant dimension of ecological studies contributing to consolidation of diversity patterns, and the ant responses to different grassland managements (i.e. grazing exclusion, traditional grazing and conservative grazing) applied to our study sites will be approached in details in a further manuscript. the conservation planning of biodiversity encompasses a variety of knowledges; one of the first and more fundamental aspects is surveying biodiversity, providing spatially consistent information on surrogate taxa and habitats. information gained from this study could be used in future research, and may help design a regional plan for grassland conservation and restoration, for example, helping definitions of areas to be protected or serving as reference sites for restoration. we emphasize that grassland biodiversity conservation efforts should consider different strategies for each biome, in order to maximize biodiversity conservation. furthermore, the creation of conservation units in the pampa biome is urgently needed, since the current conservation units in the atlantic forest biome cannot preserve all biodiversity associated to south brazilian grasslands. acknowledgments thanks to landowners and sema for research permissions and valério d. pillar for coordinating the campos sulinos lter/ peld program. thanks also to alexandre ferreira for confirming p. pampana species identification and all students that helped out in field and lab work. wd received a master scholarship from capes, brazil, and lrp received a research scholarship (dti-a) from cnpq, brazil. rmf was supported by the brazilian council of research and scientific development (cnpq grant 302462/2016-3). mmj thanks cnpq for a productivity scholarship (309616/2015-8). the project was funded by grants from cnpq to valério d. pillar (558282/2009-1). references albuquerque, e.d. & diehl, e. 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(2003). pheidole in the new world: a dominant, hyperdiverse ant genus. cambridge: harvard university press, 794 p w dröse et al. – ant fauna in south brazilian grasslands390 appendix 1. list of species recorded in sites of long-term ecological research program (lter/peld campos sulinos) in rio grande do sul state, brazil (1 aceguá; 2 alegrete; 3 lavras do sul; 4 cambará do sul; 5 são francisco de paula; 6 jaquirana). numbers represent the total number of occurrences in the two biomes (pampa and atlantic forest) and strata (g ground; v vegetation). ant species composition pampa biome atlantic forest biome sites 1 2 3 4 5 6 strata g v g v g v g v g v g v dolichoderinae dorymyrmex pyramicus (roger, 1863) 0 0 11 4 3 1 1 0 0 0 3 2 dorymyrmex sp. 1 0 0 0 0 0 0 0 0 0 0 1 0 dorymyrmex sp. 2 0 0 0 0 1 0 0 0 0 0 0 0 dorymyrmex sp. 4 0 0 0 5 0 1 0 0 0 0 0 0 dorymyrmex sp. 5 0 0 1 0 0 0 0 0 0 0 0 0 dorymyrmex sp. 6 0 0 0 1 0 0 0 0 0 0 0 0 gracilidris pombero wild & cuezzo, 2006 12 0 0 0 2 0 0 0 0 0 0 0 linepithema micans (forel, 1908) 2 0 39 6 16 3 48 13 11 1 24 3 linepithema sp. 2 0 0 0 1 4 10 0 0 0 0 0 0 tapinoma sp. 1 6 0 49 1 0 1 0 0 1 0 0 0 dorylinae acanthostichus quadratus emery, 1895 0 0 0 0 0 0 0 0 0 0 1 0 neivamyrmex sp. 4 0 0 0 0 1 0 0 0 2 0 3 0 neivamyrmex sp. 5 0 0 0 0 1 0 0 0 0 0 0 0 neivamyrmex sp. 6 0 0 0 0 0 0 0 0 0 0 1 0 ectatomminae ectatomma edentatum roger, 1863 5 0 0 0 5 0 0 0 0 0 6 0 gnamptogenys rastrata (mayr, 1866) 0 0 1 0 6 0 0 0 2 0 1 0 gnamptogenys striatula mayr, 1884 1 0 0 0 34 0 2 0 0 0 18 0 formicinae brachymyrmex coactus mayr, 1887 0 0 18 12 10 5 0 0 0 0 0 0 brachymyrmex sp. 1 42 10 62 23 36 15 43 6 17 13 1 2 brachymyrmex sp. 2 11 5 15 3 29 9 15 3 10 1 4 1 brachymyrmex sp. 3 0 0 7 10 6 6 0 0 0 0 0 0 brachymyrmex sp. 4 0 0 4 1 1 0 0 0 0 0 0 0 brachymyrmex sp. 5 0 0 1 0 0 0 0 0 0 0 0 0 camponotus koseritzi emery, 1888 0 0 0 0 21 15 0 0 0 0 0 0 camponotus punctulatus mayr, 1868 58 19 23 17 25 15 5 7 11 10 8 4 camponotus rufipes (fabricius, 1775) 0 0 3 0 0 0 0 0 0 0 0 0 camponotus sp. 1 11 2 23 16 22 4 4 0 2 0 23 10 camponotus sp. 2 3 3 21 22 36 17 0 0 0 0 24 21 camponotus sp. 4 0 0 0 0 2 0 0 0 0 0 0 0 camponotus sp. 5 0 0 0 0 1 0 0 0 2 0 0 0 camponotus sp. 6 0 0 0 1 0 1 0 0 0 0 0 2 myrmelachista gallicola mayr, 1887 0 0 0 2 0 11 0 1 0 0 0 3 nylanderia fulva (mayr, 1862) 11 0 0 0 31 1 0 0 0 0 6 0 heteroponerinae acanthoponera sp. n. 1 0 0 0 0 0 0 0 0 0 0 2 1 myrmicinae acromyrmex ambiguus (emery, 1888) 8 2 0 0 17 5 0 0 0 0 0 0 acromyrmex coronatus (fabricius, 1804) 2 0 0 0 0 0 4 0 7 0 0 1 acromyrmex heyeri (forel, 1899) 21 6 1 1 1 0 1 2 3 2 0 0 sociobiology 64(4): 381-392 (december, 2017) 391 appendix 1. list of species recorded in sites of long-term ecological research program (lter/peld campos sulinos) in rio grande do sul state, brazil (1 aceguá; 2 alegrete; 3 lavras do sul; 4 cambará do sul; 5 são francisco de paula; 6 jaquirana). numbers represent the total number of occurrences in the two biomes (pampa and atlantic forest) and strata (g ground; v vegetation). (continuation) ant species composition pampa biome atlantic forest biome sites 1 2 3 4 5 6 strata g v g v g v g v g v g v acromyrmex landolti (forel, 1885) 0 0 0 0 0 0 0 0 0 0 1 0 acromyrmex lobicornis (emery, 1888) 14 1 2 0 0 0 0 0 0 0 0 0 cephalotes incertus (emery, 1906) 0 0 0 0 0 2 0 0 0 0 0 0 crematogaster quadriformis roger, 1863 6 1 36 16 4 2 0 0 0 0 0 0 crematogaster sp. 1 1 2 0 0 0 0 0 0 0 0 0 0 crematogaster sp. 2 0 0 1 0 8 1 0 0 0 0 0 0 crematogaster sp. 3 0 0 0 1 0 0 0 0 0 0 0 0 crematogaster sp. 4 0 0 1 0 0 0 0 0 0 0 0 0 cyphomyrmex gr. rimosus sp. 1 55 0 33 1 69 0 5 0 16 0 31 0 cyphomyrmex transversus emery, 1894 0 0 39 0 0 0 0 0 0 0 0 0 megalomyrmex gr. silvestrii sp. 1 0 0 1 0 0 0 0 0 0 0 0 0 megalomyrmex sp. 2 1 0 0 0 0 0 0 0 0 0 0 0 mycetophylax nr. lilloanus (kusnezov,1949) 0 0 1 0 0 0 0 0 0 0 0 0 pheidole gr. fallax sp. 1 2 0 3 0 0 0 0 0 0 0 0 0 pheidole gr. tristis sp. 1 75 4 32 1 25 3 41 6 56 2 2 0 pheidole gr. tristis sp. 2 0 0 4 0 0 0 0 0 0 0 0 0 pheidole aberrans mayr, 1868 35 0 28 1 17 0 4 0 1 0 2 0 pheidole breviseta santschi, 1919 2 0 6 1 46 0 19 1 2 0 10 0 pheidole cavifrons emery, 1906 0 0 17 0 31 0 23 0 8 0 23 0 pheidole nr. jelskii mayr, 1884 0 0 1 0 8 0 2 0 13 0 52 1 pheidole nubila emery, 1906 25 0 21 0 22 1 0 0 0 0 0 0 pheidole obtusopilosa mayr, 1887 43 5 32 1 50 11 48 3 73 6 26 2 pheidole pampana santschi, 1929 33 0 22 2 35 4 19 1 10 0 23 0 pheidole radoszkowskii mayr, 1884 29 1 53 7 53 7 12 0 16 0 17 1 pheidole nr. rufipilis forel, 1908 0 0 0 0 0 0 0 0 0 0 1 0 pheidole spininods mayr, 1887 1 0 54 1 47 0 0 0 0 0 0 0 pheidole sp. 1 0 0 0 0 38 0 17 0 35 0 2 0 pheidole sp. 2 1 0 0 0 36 0 30 1 44 1 5 0 pheidole sp. 3 0 0 10 1 0 0 0 0 0 0 36 1 pheidole sp. 4 1 0 1 0 5 0 0 0 0 0 0 0 pheidole sp. 13 0 0 0 0 0 0 0 0 0 0 1 0 pogonomyrmex naegelii emery, 1878 0 0 0 0 0 0 0 0 1 0 15 0 solenopsis invicta buren, 1972 52 2 57 2 72 0 19 0 71 6 5 1 solenopsis sp. 2 4 3 8 10 22 5 7 2 9 0 0 0 solenopsis sp. 3 0 0 0 0 0 0 5 0 0 4 1 5 solenopsis sp. 4 7 0 46 4 9 1 0 0 0 0 4 0 solenopsis sp. 5 8 0 27 0 29 0 33 0 16 0 18 2 solenopsis sp. 6 0 0 0 0 0 0 0 0 0 0 1 0 solenopsis sp. 7 13 0 3 0 0 0 0 0 0 0 0 0 solenopsis sp. 8 1 0 16 0 0 0 0 0 0 0 0 0 solenopsis sp. 9 0 0 1 0 6 0 3 0 1 0 2 0 solenopsis sp. 10 13 0 29 0 0 0 0 0 0 0 0 0 w dröse et al. – ant fauna in south brazilian grasslands392 appendix 2. results from indicator value (indval) analysis showing ant associations to either vegetation or ground strata. were considered indval of 70 or higher as important. for all indval presented, p<0.001. species stratum indval pseudomyrmex nr. flavidulus vegetation 73.1 myrmelachista gallicola vegetation 70.7 solenopsis sp. 5 ground 98.3 cyphomyrmex gr. rimosus sp. 1 ground 96.4 pheidole pampana ground 95.5 pheidole cavifrons ground 91.3 pheidole radoszkowskii ground 88.4 solenopsis sp. 11 ground 88 solenopsis invicta ground 87.7 pheidole aberrans ground 87 solenopsis sp. 11 23 3 27 3 68 2 41 1 1 1 21 0 solenopsis sp. 12 0 0 1 1 2 0 0 0 1 0 2 0 solenopsis sp. 13 0 0 1 0 0 0 0 0 0 0 0 0 strumigenys emiliae forel, 1907 0 0 0 0 1 0 0 0 0 0 0 0 strumigenys louisianae roger, 1863 0 0 0 0 18 2 15 1 6 0 0 0 trachymyrmex gr. urich sp. 1 4 0 5 1 13 0 19 0 3 0 0 0 trachymyrmex holmgreni wheeler, 1925 0 0 0 0 0 0 0 0 0 0 5 0 trachymyrmex kempf fowler, 1982 4 0 16 1 14 0 0 0 0 0 0 0 trachymyrmex pruinosus (emery, 1906) 0 0 0 0 0 0 11 0 0 0 0 wasmannia auropunctata (roger, 1863) 14 0 1 0 61 5 26 1 27 2 23 0 wasmannia sulcaticeps emery, 1894 6 0 11 0 5 0 0 0 2 1 0 0 wasmannia nr. sulcaticeps emery, 1894 0 0 0 0 5 0 8 0 0 0 0 0 wasmannia williamsoni kusnezov, 1952 10 0 1 0 12 1 0 0 0 0 0 0 ponerinae anochetus neglectus emery, 1894 6 0 7 0 3 0 0 0 0 0 0 0 hypoponera sp. 1 0 0 1 0 1 1 2 0 0 0 0 0 hypoponera sp. 2 0 0 3 0 0 0 0 0 2 0 2 0 hypoponera sp. 3 0 0 2 0 0 0 0 0 0 0 0 0 hypoponera sp. 4 0 0 2 0 0 0 0 0 0 0 0 0 hypoponera sp. 5 1 0 0 0 0 0 0 0 0 0 0 0 hypoponera sp. 6 4 0 1 0 0 0 0 0 0 0 0 0 hypoponera sp. 7 0 0 0 0 3 0 0 0 1 0 0 0 hypoponera sp. 8 0 0 2 0 0 0 0 0 0 0 0 0 hypoponera sp. 9 0 0 0 0 0 0 0 0 0 0 1 0 hypoponera sp. 10 0 0 0 0 0 0 1 0 0 0 0 0 neoponera bucki (borgmeier, 1927) 0 0 0 0 3 0 0 0 0 0 0 0 pachycondyla striata smith, 1858 0 0 0 0 0 0 0 0 23 0 1 0 pseudomyrmecinae pseudomyrmex nr. flavidulus (smith, 1858) 0 1 1 12 0 11 0 0 0 0 0 10 pheidole breviseta ground 85.9 wasmannia auropunctata ground 82.6 pheidole nr. jelskii ground 80.3 trachymyrmex gr. urich sp. 1 ground 80.2 pheidole sp. 2 ground 79.7 pheidole sp. 1 ground 78.2 wasmannia sulcaticeps ground 76.3 solenopsis sp. 4 ground 73.1 gnamptogenys striatula ground 70.7 species stratum indval doi: 10.13102/sociobiology.v64i3.1070sociobiology 64(3): 256-260 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 effects of diafenthiuron in toxic baits on colonies of leaf-cutting ants (hymenoptera: formicidae) introduction the leaf-cutting ants are considered important pest insects in agricultural and forestry crops by causing economic damage in areas of the neotropical region, mainly in brazil (reis et al., 2010). the main challenge in controlling leafcutting ants is the search for insecticides which are less harmful to agricultural ecosystems, but still effective when formulated as toxic granular baits (forti et al., 2007). some insecticides have already been tested for control of leaf-cutting ants, such as chlorpyriphos (forti et al., 2003), diflubenzuron and dechlorane (nagamoto et al., 2007), but without success in manufacturing and marketing them as formicide baits. however, diafenthiuron (c23h32n2os) is presented as a promising formicide; its activity against other groups of insects and mites is very well known (ishaaya et al., abstract dianfenthiuron is a pre-insecticide that can be activated by photolysis, and may be a promising formicide. this study evaluated the effect of diafenthiuron after photolysis in colonies of atta sexdens rubropilosa forel, 1908. the experiment was conducted in a completely randomized design with five treatments and five replications: control (no active ingredient), sulfluramid (standard formicide), diafenthiuron (no exposure to uv), diafenthiuron (2h exposure to uv) and diafenthiuron (6h exposure to uv). toxic baits were applied at a rate of 0.5 g per colony, and we observed the transport and incorporation of the baits into the colonies. a grading scale was used (0 to 4) to measure the cutting of acalypha l. (euphorbiaceae) leaves by workers at 2, 7, 14 and 21 days after application (daa) and we also measured the garden mass (fungus + adult + brood) at 21 daa in order to check for growth of the fungus culture. total loading and incorporation occurred one hour after application of the baits. colonies that received sulfluramid did not transport leaves at 2 daa. workers that received baits with d2h showed an average of 20% transport and 55% incorporation of leaves at 21 daa. the grading scale indicated that treatments d2h and d6h had the lowest averages, 0.80 and 2.00, respectively. the treatments d2h and d6h reduced cutting of leaves and fungus garden mass, but did not kill the colonies of a. sexdens rubropilosa. sociobiology an international journal on social insects ms barbosa1, lc forti2, rt fujihara3, cg raetano2 article history edited by evandro n. silva, uefs, brazil received 16 may 2016 initial acceptance 08 january 2017 final acceptance 07 may 2017 publication date 17 october 2017 keywords atta, chemical control, ultraviolet light, carbodiimide. corresponding author marcílio souza barbosa laboratório de entomologia e acarologia universidade federal de alagoas campus de arapiraca, p.o. box 61 cep 57309-005 arapiraca-al, brasil. e-mail: agromss@hotmail.com 2007). moreover, as it affects cellular respiration (dekeyser, 2005), its mechanism of action in insects is similar to that of sulfluramid, which is the main active ingredient in toxic baits. diafenthiuron is a pre-insecticide that, after photolysis, is converted into carbodiimide, becoming a potent insecticide (ruder & kayser, 1993). this physical-chemical characteristic can be manipulated in the laboratory. in this context, the present study evaluated the effect of diafenthiuron after photolysis, when supplied in formulation of toxic bait to colonies of atta sexdens rubropilosa forel, 1908. material and methods study site in 2012, young colonies (8 months old) from the laboratory of social pest insects, fca/unesp, botucatu, são 1 universidade federal de alagoas (ufal), arapiraca-al, brazil 2 faculdade de ciências agronômicas (fca/unesp), botucatu-sp, brazil 3 universidade federal de são carlos (cca/ufscar), araras-sp, brazil research article ants sociobiology 64(3): 256-260 (september, 2017) 257 paulo, brazil, were maintained under controlled temperature and humidity (24°c ± 2 and 70 ± 5%) in plastic containers with a capacity of 395 ml volume for the fungus chamber and 250 ml for the waste and foraging chambers. at the fungus chamber, a 1.0 cm plaster layer was placed at the base (maintenance of humidity). colonies were given leaves of acalypha l. (euphorbiaceae) as vegetable substrate, which were offered in a natural state or dehydrated, according to humidity conditions. preparation and application of treatments the baits applied to the colonies of a. sexdens rubropilosa had the following treatments: ctl control (no active ingredient); sul sulfluramid (standard formicide 0.3% of the active ingredient mirex-s®); dne diafenthiuron (no exposure to ultraviolet light); d2h diafenthiuron [2h exposure to ultraviolet light (uv)] and; d6h diafenthiuron [6h exposure to ultraviolet light (uv)]. the use of sulfluramid as formicide is already known (zanetti et al., 2003; nagamoto et al., 2004), and so it was used as a standard comparative to diafenthiuron. treatments with diafenthiuron contained the following ingredients: vegetable soybean oil (soya®) (5%), dehydrated citrus pulp (94%), active ingredient (1%) and distilled water. the treatments d2h and d6h were mixed with a solution of acetone + distilled water (3:1) and then exposed to ultraviolet light with a peak wavelength of 253nm (tuv-30w t8, philips electronics brasil®) (adapted and modified from keum et al., 2002) during the predetermined periods (2h and 6h). afterwards, they remained in the absence of light for 24 hours for the decomposition into bioactive forms (mainly carbodiimide). we applied 0.5g baits/colony, the equivalent of 22 pellets of 0.5 cm in length each. the baits were offered in the foraging chamber only once, when it was observed the transport and the incorporation to the colony. evaluations and data analysis a grading scale was used [(grading cutting % leaf cutting area cm2): 0-0-0.00; 1-25-17.38; 2-50-34.76; 3-7552.14; 4-100-69.52] to evaluate the cutting of leaves of acalypha by workers after the treatment with baits at 2, 7, 14 and 21 days after application (daa). this scale was designed based on the average sampling of acalypha leaves supplied daily (1 whole leaf of average size for each colony) after analysis of leaf area by the equipment li-cor® biosciences (li-3100c area meter). the mass of the garden (fungus + adult + brood) was also measured at 21 daa, to check for growth of the fungus culture. the experiment was conducted in a completely randomized design with five treatments (bait) and five replications, each plot was represented by a colony. to assess the cutting of acalypha leaves (grading scale), it was used a factorial arrangement (5x4), where the first factor was the group of treatments with baits, and the second, the days after application of baits (daa), comparing the means by tukey’s test (p <0.05). data of transport and incorporation of the leaves were evaluated in percentage. differences in the growth of the fungus culture (mass) were tested by analysis of variance with comparison of means of treatments with baits by tukey’s test (p <0.05). the assumption of normality was tested by shapirowilk test (p <0.05). the statistical analysis was performed in the sisvar 5.3 software (ferreira, 2011). results and discussion total transport and incorporation of baits were observed in all treatments, one hour after application. although young, the colonies exhibited significant foraging activity throughout the rearing period. however, at 2 daa, the workers that received sulfluramid no longer transported leaves (fig 1-a) and showed visible symptoms of poisoning, such as slow motion, stretched and paralyzed hind legs (nagamoto et al., 2007), reducing or even stopping the execution of the task. at 7 daa, only the colonies that received the treatment ctl continued to transport leaves in a way similar to that observed before the beginning of the experiment. the treatments d2h and d6h have already demonstrated reduced transport of leaves (fig 1-a). in the treatment ctl, 100% of the transported leaves were incorporated, and the percentages were higher than 90% in dne and d6h. on the other hand, at 7 daa, there was no incorporation in sul, due to the mortality of workers and in d2h, the average incorporation of leaves was reduced by 55% at 21 daa (fig 1-b). the grading scale for the cutting of leaves of acalypha by foragers (f = 115.94; p = 0.0001) (table 1) assigned the highest score to the treatment ctl, and then to dne. the treatments d2h and d6h reached intermediate scores because the workers made the cut, but carried few fragments to the fungus chamber. on the other hand, sulfluramid prevented the cutting of leaves by the workers, yielding a score of zero (no cutting). this score refers to the disruption of the activity, because ants stop performing it after contact with the bait containing this active ingredient. however, the average of the grading scale was obtained regardless of the period of observation of the cutting behavior, because the worker can reduce, maintain or increase this activity when under the effect of active ingredients. thus, the evaluation of cutting of leaves was based on the significant interaction between treatments (bait) and daa (f = 3.39; p = 0.0005) breaking down the treatments within each day and using the grading scale in the period from 7 to 21 daa (table 1). thus, ctl colonies maintained an average of about 4.00 in all periods analyzed. in dne, the cutting of leaves decreased over time (3.00) and d2h showed a marked decrease (0.80). ms barbosa, lc forti, rt fujihara, cg raetano – effects of diafenthiuron on leaf-cutting ants258 to assess the general development of the colony, we analyzed the growth based on the mass of the fungus chamber = queen, workers and brood (f = 25.75, p = 0.0001) (table 2). at 21 daa, the mass of a. sexdens rubropilosa was higher in ctl due to lack of active ingredient, followed by dne, d2h and d6h. there was no assessment in sul, given the total mortality of the colony. aspects of poisoning of workers (symptoms), presence of dead ants, growth of contaminant yeast (mainly escovopsis muchovej & della lucia, ascomycetes) and survival of the colony at the end of the study are shown in table 3. the effect of baits in the colonies of a. sexdens rubropilosa was only detected when baits contained the active ingredient sulfluramid. this result was remarkable in this treatment for all the variables cited, however, the baits applied with dne, d2h and d6h caused symptoms of poisoning, reduced mobility (slowness) during the first daa, but somehow ants managed to regain mobility without affecting survival of the colony, only changing the natural state of development. a general analysis of diafenthiuron efficiency as formicide leads to some questions before assessing its actual effect on colonies of a. sexdens rubropilosa: the bait was attractive? when preparing a formicide, the attractiveness can directly affect the efficiency, because it is essential to encourage the contact and ingestion with the active ingredient, thus allowing the contamination by the insecticide. however, the transport by the workers was 100% one hour after application of baits, proving the attractiveness of citrus pulp, which has been already recognized (brugger et al., 2008). were the colonies in constant foraging? another problem is the activity of the colonies prior to experimentation (rearing stage), because according to delabie et al. (2000), it is important to make sure that nests used in experiments of attractiveness are active. these should have good transport of leaves and large size workers (head width greater than 2 mm). although some of these steps are determined for experimentation in the field, they must also be taken in the laboratory (nagamoto et al., 2004), since they can mask the efficiency of promising insecticides. was there a simple rejection (selection) of the leaves of acalypha after application of baits? in figure 1-a, treatments with diafenthiuron reduced the transport of leaves, reaching only 20% with d2h. in this case, it would hardly be possible, because from the beginning of rearing it has been offered, cut and transported the same plant material, preventing confusion in the selection due to the conditioning of foragers workers and scouts to remain with this odor, making it a characteristic sign of the substrate, as mentioned by brugger et al. (2008). once clarified these questions they may be disregarded, because all colonies transported the baits efficiently and did not discharge them in the waste chamber, or in the foraging chamber, in observations held 24 hours after application. what is the mechanism of action of diafenthiuron? sulfluramid is a chemical compound that acts directly on the mitochondria by inhibiting the atp synthesis (adenosine triphosphate). the de-ethylated metabolite of sulfluramid is produced by the metabolism of cytochrome p450, a potent electron uncoupler of the mitochondrial respiratory chain, interrupting the production of energy. this disruption and subsequent loss of atp results in inactivity, paralysis and death of the insect exposed to sulfluramid (heong et al., 2011). diafenthiuron also has a mechanism of action similar to sulfluramid in insects and mites (ruder & kayser, 1993), which could be enhanced when subjected to uv, as it is a preinsecticide, which needs to turn into a secondary metabolite, such as the carbodiimide, to become a potent insecticide (kayser & eilinger, 2001; dekeyser, 2005). table 1. grading scale (0 to 4) for the cutting of acalypha sp. leaves by atta sexdens rubropilosa workers after the treatment with baits at 2, 7, 14 and 21 daa (days after application). treatment daa 2 7 14 21 ctl 3.80 ± 0.20 ca 3.80 ± 0.20 da 3.60 ± 0.24 da 4.00 ± 0.00 ca sul 0.00 ± 0.00 aa 0.00 ± 0.00 aa 0.00 ± 0.00 aa 0.00 ± 0.00 aa dne 3.80 ± 0.20 ca 3.40 ± 0.40 cda 3.40 ± 0.24 cda 3.00 ± 0.00 bca d2h 3.80 ± 0.20 cc 2.40 ± 0.60 bcb 1.60 ± 0.24 bab 0.80 ± 0.37 aa d6h 2.04 ± 0.24 ba 2.00 ± 0.32 ba 2.20 ± 0.37 bca 2.00 ± 0.32 ba means, within a column/row, followed by different small/capital letters are different by tukey test (p<0,05). treatments: ctl (control); sul (sulfluramid); dne (diafenthiuron no exposure to ultraviolet light), d2h (diafenthiuron with 2h exposure to ultraviolet light); d6h (diafenthiuron with 6h exposure to ultraviolet light). d6h (diafenthiuron with 6h exposure to ultraviolet light). daa: days after application. treatment fungus garden + adults + offspring (g) ctl 30.38 ± 1.46 c dne 26.00 ± 1.22 b d2h 22.85 ± 0.24 b d6h 17.35 ± 0.97 a coefficient of variation (%) 10,00 means followed by different letters are significantly different by tukey test (p<0,05). treatments: ctl (control); dne (diafenthiuron no exposure to ultraviolet light), d2h (diafenthiuron with 2h exposure to ultraviolet light); d6h (diafenthiuron with 6h exposure to ultraviolet light). table 2. mass of the fungus of atta sexdens rubropilosa colonies 21 daa (days after application). sociobiology 64(3): 256-260 (september, 2017) 259 diafenthiuron was initially developed to control pests, such as mites, white flies and aphids (kayser & eilinger, 2001). given the low toxicity to some species of insects, it has been reported as a promising insecticide when applied at recommended doses, and with potential to be used in integrated pest management (ipm), as it is not harmful to beneficial insects, especially the biological control agents of insect pests (preetha et al., 2009). among these, stands out the predator of aphids chrysoperla carnea (stephens, 1836) (neuroptera: chrysopidae), beetles of the family coccinellidae and trichogramma spp. (hymenoptera: trichogrammatidae) wasps. for c. carnea, for instance, the application of a high dose of diafenthiuron (3.2 g.l-1) caused 53% of adult mortality, and a low dose (0.8 g.l-1), only 3%, showing low effect on this species, as the increase in diafenthiuron by up to four times only increased by 50% the adult mortality (preetha et al., 2009). in leaf-cutting ants, the low toxicity of diafenthiuron, when ingested, can be explained by two ways: (i) due to the possibility of low dose application to the treated colonies, which reduces the effect of the compound, and (ii) due to the resistance of workers to the insecticide, which may have presented mechanisms of detoxification in relation to diafenthiuron or its secondary metabolite, although it had affected the execution of activities by workers. at last, treatments containing d2h and d6h reduced the cutting of leaves and fungus garden mass, but did not kill the colonies of a. sexdens rubropilosa. bueno (2013) also studied the toxicity of active ingredients (diafenthiuron and tolfenpyrad) in a. sexdens rubropilosa workers and the results showed significant reduction in the survival of workers when compared with controls of each ingredient, particularly when they were dissolved in soybean oil. however, baits containing the active ingredients were not effective in controlling colonies of a. sexdens rubropilosa. diafenthiuron did not kill the colonies of a. sexdens rubropilosa on the doses studied, however it should be tested in other dosages of ultraviolet light and in different formulations or in consortium with other chemicals compounds before being totally discarded as formicide. after photolysis by uv, diafenthiuron interfered with the cutting and transport of leaves to the colony, but was not efficient against young colonies of a. sexdens rubropilosa. the colonies of leaf-cutting ants that received toxic baits with diafenthiuron (6h exposure to uv) developed less mass of the fungus 21 days after application. acknowledgments the first author would like to thank coordenadoria de aperfeiçoamento de pessoal de nível superior (capes) for a granted scholarship. references brugger, m. s., fernandes, m. a. c., hallack, n. m. r. & lopes, j. f. s. 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(2000). protocolo de experimentação para avaliar a atratividade fig 1. transport of acalypha leaves (a) and substrate incorporation (b) by a. sexdens rubropilosa workers after application of baits. treatments: ctl (control); sul (sulfluramid); dne (diafenthiuron no exposure to ultraviolet light), d2h (diafenthiuron with 2h exposure to ultraviolet light); d6h (diafenthiuron with 6h exposure to ultraviolet light). daa (days after application). treatment state of colonies intoxication dead ants contaminant fungus dead colonies ctl dno nm dno dno sul 1 daa tm 3 daa 5 daa dne ofdaa nm dno dno d2h ofdaa nm dno dno d6h ofdaa nm dno dno treatments: ctl (control); sul (sulfluramid); dne (diafenthiuron no exposure to ultraviolet light), d2h (diafenthiuron with 2h exposure to ultraviolet light); d6h (diafenthiuron with 6h exposure to ultraviolet light). dno: do not occurred. daa: days after application. ofdaa: occurred in the first days after application. nm: natural mortality. tm: total mortality. table 3. general state of atta sexdens rubropilosa colonies after application of baits. ms barbosa, lc forti, rt fujihara, cg raetano – effects of diafenthiuron on leaf-cutting ants260 de novas formulações de iscas granuladas utilizadas no controle das formigas cortadeiras acromyrmex spp. e atta spp. (hymenoptera: formicidae: myrmicinae: attini) no campo. anais da sociedade entomológica do brasil, 29: 843-848. doi: 10.1590/s0301-80592000000400029 ferreira, d.f. 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(2003). eficiência de isca formicida aplicada sobre o monte de terra solta de ninhos de atta sexdens rubropilosa (hymenoptera: formicidae). revista árvore, 27: 410-407. doi: 10.1590/s0100-67622003000300019 doi: 10.13102/sociobiology.v64i1.1251sociobiology 64(1): 130-132 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 “empty spaces ‘where’ we are living for” – first record of dinoponera quadriceps reusing nests of atta sexdens the queenless ant dinoponera quadriceps (formicidae: ponerinae) is distributed throughout northeastern brazil (paiva & brandão, 1995; lenhart et al., 2013) particularly in the caatinga biome. this environment, which covers approximately 800.000 km2 (santos et al., 2011), is characterized by dry and nutrient poor soil (menezes et al., 2012). dinoponera quadriceps are predominantly predators, but solitary individuals also forage small fruits (araújo & rodrigues, 2006). their body size can vary from 3-4cm (paiva & brandão, 1995). active nests can be identified by twigs around and/or above the nest entrance (paiva & brandão, 1995; personal observation). here we report d. quadriceps colonizing empty atta sexdens nests and using them to raise brood. the hot and arid conditions found in the natural range of d. quadriceps require them to dig deep nests, often over 3m in depth, with as many as 16 chambers below, but with a single entrance (vasconcelos et al., 2004). nests in caatinga abstract the reuse of nests by the same or different species can save a colony energy and resources. furthermore, it can increase colony growth and the production of brood. the queenless ant dinoponera quadriceps builds deep nests in caatinga to escape from the dry and hot environment. the reuse of deep nests from other species can provide d. quadriceps with protection from high temperature, whilst saving on the energy required to build new nests. here, we present the first finding of d. quadriceps reusing the nest of atta sexdens species. sociobiology an international journal on social insects ds assis¹, s morris², fs nascimento¹ article history edited by evandro nascimento silva, uefs, brazil received 18 november 2016 initial acceptance 06 january 2017 final acceptance 18 january 2017 publication date 29 may 2017 keywords nesting behavior, ants, queenless ants. corresponding author diego santana assis laboratório de ecologia e comportamento de insetos sociais universidade de são paulo/ffclrp avenida bandeirantes nº 3900, cep 14040-901 ribeirão preto-sp, brasil e-mail: diegoassis@usp.br are deeper than in atlantic forest, possibly because of the hotter temperatures and drier air found in this biome. the foundation of a new nest occurs via colony fission (paiva & brandão, 1995; monnin & peeters, 1998), as the new gamergate and several workers search for a suitable location to establish a new nest (medeiros & araújo, 2014). nests of a. sexdens can reach up to seven meters of depth (moreira et al., 2004), again to protect against desiccation from the high temperatures. (camargo et al., 2011). it is plausible that the new founding members may cohabit their new nest with atta workers (personal observation). nests are energetically expensive to produce in a natural environment (hansell, 1993); hence reusing the nests of hetero or conspecifics may confer a benefit in reduced energy and time expenditure (jimenez-franco et al., 2014). for instance, new queens of the social wasp polistes dominula can reuse an old nest, accelerating the process of brood production (nakar et al., 2015). 1 laboratório de ecologia e comportamento de insetos sociais, universidade de são paulo/ffclrp, são paulo, brazil 2 university of bristol, united kingdom short note sociobiology 64(1): 130-132 (march, 2017) 131 we sampled d. quadriceps’ nests in caatinga biome in campo formoso, bahia state in brazil (10° 30’ 32” s, 40° 19’ 15” w) (fig 1). a total of 24 d. quadriceps colonies were located in the field, from which five (20.84% of all nests found) had utilized abandoned (or nests with a low population) nests belonging to a. sexdens (fig 2). these nests were dispersed across a field which had previously been used for cattle grazing. in conclusion, we propose that d. quadriceps can facultatively use the pre-dug nests of particular atta species as their own, in order to save energy expenditure. it would fig 1. sampled nests, in blue nests of atta sexdens used by dinoponera quadriceps. seem likely that this may be the case for other atta species with similarly deep nests – indeed, it would be surprising if this was not more commonly observed in other species, given the likely vast energy savings gained from nest-reuse. acknowledgments we thank to fapesp for financial support of d. s. assis for this study (2015/17358-0) and f. s. nascimento (2015/253019-9), also we thank to evandro silva for his comments on the text. fig 2. nest of atta used by dinoponera quadriceps. a) atta entrance; b) dinoponera quadriceps entrance; c) and d) the entrances sight above. ds assis, s morris, fs nascimento – reuse of atta sexdens nest by dinoponera quadriceps132 references araújo, a., & rodrigues, z. (2006). foraging behavior of the queenless ant dinoponera quadriceps santschi (hymenoptera: formicidae). neotropical entomology, 35: 159-164. bakar, n. a. a., baracchi, d., & turillazzi, s. (2016). reuse of old nests by the european paper wasp polistes dominula (hymenoptera vespidae). redia, 98: 21-24. camargo, r. s., forti, l. c., fujihara, r. t., & roces, f. (2011). digging effort in leaf-cutting ant queens (atta sexdens rubropilosa) and its effects on survival and colony growth during the claustral phase. insectes sociaux, 58: 17-22. hansell, m. h. (1993). the ecological impact of animal nests and burrows. functional ecology, 7: 5-12. jiménez-franco, m. v., martínez, j. e., & calvo, j. f. (2014). patterns of nest reuse in forest raptors and their effects on reproductive output. journal of zoology, 292: 64-70. lenhart, p., dash, s. t., & mackay, w. p. (2013). a revision of the giant amazonian ants of the genus dinoponera (hymenoptera: formicidae). journal of hymenoptera research, 31: 119-164. medeiros, j., & araújo, a. (2014). workers’ extra-nest behavioral changes during colony fission in dinoponera quadriceps (santschi). neotropical entomology, 43, 115-121. menezes, r. s. c., sampaio, e. v. s. b., giongo, v., & pérezmarin, a. m. (2012). biogeochemical cycling in terrestrial ecosystems of the caatinga biome. brazilian journal of biology, 72: 643-653. monnin, t., & teeters, c. (1998). monogyny and regulation of worker mating in the queenless ant dinoponera quadriceps. animal behaviour, 55: 299-306. moreira, a., forti, l. c., andrade, a. p., boaretto, m. a., & lopes, j. (2004). nest architecture of atta laevigata (f. smith, 1858) (hymenoptera: formicidae). studies on neotropical fauna and environment, 39: 109-116. paiva, r. v. s., & brandão, c. r. f. (1995). nests, worker population, and reproductive status of workers, in the giant queenless ponerine ant dinoponera roger (hymenoptera: formicidae). ethology, ecology and evolution, 7: 297-312. santos, j. c., leal, i. r., almeida-cortez, j. s., fernandes, g. w., & tabarelli, m. (2011). caatinga: the scientific negligence experienced by a dry tropical forest. tropical conservation science, 4: 276-286. vasconcellos, a., santana, g. g., & souza, a. k. (2004). nest spacing and architecture, and swarming of males of dinoponera quadriceps (hymenoptera: formicidae) in a remnant of the atlantic forest in northeast brazil. brazilian journal of biology, 64: 357-362. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5898sociobiology 68(1): e5898 (march, 2021) introduction the white-footed ant, technomyrmex brunneus forel, 1895, is widespread in south, southeast, and east asia, which includes india, sri lanka to indochina, indonesia, malaysia, new guinea, taiwan, china, the korean peninsula, and japan. in japan, the species has been recorded in the ryukyus (including the daito islands), ogasawara islands (chichi-jima, ototo-jima and ani-jima), volcano islands (iwo-jima), minamitori-shima, kyushu (kagoshima prefecture, aoshima), shikoku (southern part), izu islands (hachijo-jima), and kanagawa prefecture (yokohama: naka-ku: kamome-cho) (terayama, 2020; terayama et al., 2014, 2018). additionally, it has been found indoors in shimoda city, shizuoka pref. (greenhouse); futtsu city, chiba pref. (greenhouse); and taitoku, tokyo (ueno zoo: vivarium of amphibians and reptiles) (sakamoto et al., 2011). abstract the white-footed ant, technomyrmex brunneus, was newly introduced and established in a remote island of japan and has caused unacceptable damage to the daily life of residents. to establish proper control measures, the present study investigated whether t. brunneus is effectively attracted to commercially available poison baits used to exterminate common household pest ants and the argentine ant in japan. cafeteria experiments using three types of nontoxic baits and eight types of commercial poison baits for ants were conducted in the field, and the attractiveness was compared among the baits. the liquid poison bait “arimetsu,” which consists of 42.6% water, 55.4% sugar, and 2.0% borate, and nontoxic 10% (w/v) sucrose water showed the highest attractiveness. on the other hand, other commercial poison baits were not as attractive. therefore, sucrose liquid is the most effective attractive component to use in poison baits for t. brunneus. sociobiology an international journal on social insects mamoru terayama1, eiriki sunamura1,2, ryota fujimaki3, takashi ono3, katsuyuki eguchi1 article history edited by evandro nascimento silva, uefs, brazil received 01 october 2020 initial acceptance 25 february 2020 final acceptance 26 february 2020 publication date 26 march 2021 keywords invasive species; pest; formicidae; japan; control. corresponding author katsuyuki eguchi graduate school of science tokyo metropolitan university 1-1, minami-osawa, hachioji-shi, tokyo 192-0397, japan. e-mail: antist@tmu.ac.jp the japanese populations of this species (japanese common name: ashijiro-hirafushi-ari) had been referred to as technomyrmex albipes (f. smith, 1861) for a long time. however, in his world revision of the genus technomyrmex, bolton (2007) revived the status of “t. albipes subsp. brunneus” as a full species, and discriminated it morphologically from t. albipes. by following his revision, “ashijiro-hirafushi-ari” or “technomyrmex albipes” studied previously in japan are likely to be reidentified as technomyrmex brunneus (terayama et al., 2014; yamane et al., 2018). recently, the range of t. brunneus has expanded in japan. in kyushu, it was recorded in aoshima (miyazaki prefecture) before world war ii (teranishi, 1929), and the population was confirmed to be established later (ogata, 1995). the southern part of kagoshima city had been recognized as the northern limit of the t. brunneus range in mainland kyushu. however, around 2001, the southern kagoshima 1 graduate school of science, tokyo metropolitan university, tokyo, japan 2 forestry and forest products research institute, ibaraki, japan 3 hachijo town hall, tokyo, japan research article ants a surprisingly non-attractiveness of commercial poison baits to newly established population of white-footed ant, technomyrmex brunneus (hymenoptera: formicidae), in a remote island of japan mamoru terayama, eiriki sunamura, ryota fujimaki, takashi ono, katsuyuki eguchi – non-attractiveness of poison baits to technomyrmex brunneus2 population suddenly began to expand northward, and as of 2007, t. brunneus was established in the lowlands of northwestern kagoshima prefecture (shimana & yamane, 2007). in the ogasawara islands, t. brunneus was established on chichi-jima island (sugiura, 2008; misidentified as t. albipes), and recently, the species was also discovered on ototo-jima island (shimano et al., 2018). in the izu islands, ogura et al. (2017) formally reported the establishment of a t. brunneus population on hachijo-jima island. the authors found that the population density in four of five villages was quite high, and pointed out that a single “super colony” occupied the island. on the other hand, around 2011, the islanders noticed the “outbreak” and the damage inflicted by this species (hachijo town hall: residents’ section, unpublished). colonies of t. brunneus nested around houses in the villages and often invaded resident homes to build nests. as a result, not only did the invasions cause discomfort to the residents but the ants also damaged and raided food items. the repeated invasions forced residents to incur heavy expenses to exterminate the ants using commercial pesticides. furthermore, colonies nesting in electrical devices, such as switchboards and air conditioners, have caused mechanical damage in various parts of the island and have forced owners to suffer expensive repair costs (hachijo town hall: residents section, unpublished data). therefore, hachijo town decided to implement control methods for t. brunneus beginning in the 2020 fiscal year. in several ant species, nutrient exchange among individuals is performed via mouth-to-mouth transfer (trophallaxis); thus, poison bait methods utilizing this behavior are generally used to control invasive ants (hoffmann et al., 2016). foraging worker ants carry poison bait (a mixture of the attractant and the insecticidal component) to their nests and pass it to other individuals (workers, queens, and larvae) directly or via trophallaxis. as a result, the insecticide spreads throughout the colony and kills the entire colony. however, t. brunneus has a combination of ecological features that are not seen in other notorious pest ant species such as the fire ant and the argentine ant. the characteristics of t. brunneus are as follows: they are highly polydomous, nesting on the ground and in trees; they are highly polygynous, with many fertilized permanently wingless worker-like queens, so-called ergatoid queens, in a colony (yamauchi et al., 1991; tsuji et al., 1991; tsuji & yamauchi, 1994); and nutrient transfer from adults to other colony members (workers, queens, and larvae) is achieved exclusively by specialized trophic eggs (yamauchi et al., 1991). therefore, to establish proper control measures for t. brunneus, some issues need to be considered because of their ecological features. in addition, knowledge of related species may not always apply to this species. for example, there is no literature on t. brunneus food preferences. the present study aims to confirm whether the hachijojima population of t. brunneus is effectively attracted to commercially available poison baits that are used to exterminate common native pest ants, as well as the invasive argentine ant that occurs in japan. materials and methods study sites hachijo-jima island has five villages (mitsune, okago, kashitate, nakanogo, and sueyoshi), where most residents live. the first trial of the cafeteria experiment (trial 1) was conducted on june 20, 2020, at nine sites: two sites in mitsune, three sites in okago, one site in kashitate, two sites in nakanogo, and one site in sueyoshi (filled circles in fig 1). the second trial (trial 2) was conducted on july 11, 2020, at nine sites: one site in mitsune, three sites in okago, two sites in kashitate, one site in nakanogo, and two sites in sueyoshi (unfilled circles in fig 1). cafeteria experiment one bait series was set at each of the nine sites. each bait was placed on a sheet of white drawing paper (100 mm in diameter), and the papers were set approximately 100 mm apart from each other. in the first trial conducted on june 20 (hereafter referred to as “trial 1”), the bait series consisted of four commercial poison baits [fumakilla ultra suno ari fumakilla (usf), kincho ariyou combat (ac), earth garden hyper arinosu korori (hak), and arimetsu (am)] and three non-poison baits [i.e., 10% (w/v) sucrose water (sw), 10% (w/v) honey water (hw), and peanut cream (pc; https://www.sonton.co.jp/products/familycup/)]; filtered tap water (ftw) was used as a negative control (table 1). am (liquid poison bait), 10% sucrose water, 10% honey water, and filtered tap water were soaked separately in 40 × 40-mm absorbent cotton squares and placed on the 100-mm-diameter sheets of paper. in the second trial conducted on july 11 (hereafter referred to as “trial 2”), four other commercial poison baits [ariatol house sugoto taiji (ast), earth super arinosu korori (sak), intice gelanimo ant bait (gab), and advion ant gel (aag)] were set together with the three non-poison baits and filtered tap water (table 1). gel-type baits, i.e., gab and aag, were injected separately into 40 × 40-mm square plastic containers (sc environmental science co., ltd.; https://www.sumika-env-sci.jp/items/detail/2/76/), and placed on the 100-mm-diameter sheets of paper. bait series for trials 1 and 2 were set at 09:00 am, and the 100-mm-diameter sheets of paper were photographed 1 hour and 6 hours later in trial 1, and 1 hour and 3 hours later in trial 2. ants found on each 100-mm-diameter paper were considered to be attracted, and the number of attracted ants was counted for each bait based on photographs. the total number of attracted ants at the nine sites (for the eight different baits at the two different time windows) were compiled for each trial (tables 2 and 3). sociobiology 68(1): e5898 (march, 2021) 3 fig 1. the location of hachijo-jima island (in gray color) in the izu islands (a), and the locations of the 11 sites where cafeteria experiments were conducted (b). the filled circles represent the sites of trial 1 and the unfilled circles represent the sites of trial 2. product name url insecticidal component form abbreviation trial 1 fumakilla ultra suno ari fumakilla fipronil paste usf https://fumakilla.jp/insecticide/272/ kincho ariyou combat fipronil paste ac https://www.kincho.co.jp/seihin/insecticide/ari/combat_alpha/index.html earth garden hyper arinosu korori fipronil paste hak https://www.earth.jp/products/earth-garden-ari-korori/index.html arimetsu borate liquid am https://www.yokohamaueki.co.jp/arimetsu/ trial 2 ariatol house sugoto taiji fipronil paste ast https://www.sc-engei.co.jp/guide/detail/1481.html pyriproxyfen earth super arinosu korori hydramethylnon granule sak https://www.earth.jp/products/ari-korori-super/ dinotefuran gel intice gelanimo ant bait borate gel gab https://www.rockwelllabs.com/inticegelanimo.html advion ant gel indoxacarb gel aag https://www.syngentappm.com/advionrant-gel table 1. details of the commercial poison baits for ants. statistical analyses two-way analysis of variance (two-way anova) tests were performed separately for the trial 1 and trial 2 datasets (tables 2 and 3) to compare the numbers of attracted individuals for each bait and for each time window (tables 4 and 5). furthermore, significant main effects were examined using holm’s multiple comparison tests (srb). additionally, oneway anova tests were performed as needed. all anova and srb tests were performed using the software js-star 9.8.6j (tanaka & nakano, 2018). https://fumakilla.jp/insecticide/272/ https://www.kincho.co.jp/seihin/insecticide/ari/combat_alpha/index.html https://www.earth.jp/products/earth-garden-ari-korori/index.html https://www.yokohamaueki.co.jp/arimetsu/ https://www.sc-engei.co.jp/guide/detail/1481.html https://www.earth.jp/products/ari-korori-super/ https://www.rockwelllabs.com/inticegelanimo.html https://www.syngentappm.com/advionrant-gel mamoru terayama, eiriki sunamura, ryota fujimaki, takashi ono, katsuyuki eguchi – non-attractiveness of poison baits to technomyrmex brunneus4 results trial 1 table 2 shows the number of attracted individuals at the two different time windows, i.e., 1 hour (1 ha) and 6 hours (6 ha) after setting the bait series. monomorium chinense (mc) and pristomyrmex punctatus (pp), as well as t. brunneus, were attracted to the bait series. of the three ant species, t. brunneus is extremely dominant in all locations, while the number of attracted individuals varied widely depending on the locations and time windows; the standard deviation was large for each bait (table 3). the results of the two-way anova showed no significant difference in the number of attracted t. brunneus individuals between the time windows at the 5% level (f(1, 7) = 4.36, 0.10 > p > 0.05), but there were significant differences among the baits at the 1% level (f(7, 7) = 7.33, p < 0.01). significant differences in the mean number of attracted t. brunneus individuals were observed between the following combinations at the 5% level: usf < am, ac < am, hw < am, pc < am, ftw < am, and ac < sucrose water (sw). sites time window usf ac hak am sw hw pc ftw mitsune-a 1 ha 0 1 7 157 96 89 10 4 6 ha 0 0 2 121 33 284 231 10 mitsune-b 1 ha 0 0 0 0 41 0 0 0 6 ha 1 3 2 31 173 9 0 0 okago-a 1 ha 0 0 7 15 50 34 0 1 6 ha 8 1 7 174 52 14 118 0 okago-b 1 ha 0 0 2 5 0 1 0 0 6 ha 1 2 2 225 26 20 78 1 okago-c 1 ha 0 0 1 37 (mc 30) 18 30 5 1 (pp 8) 6 ha 2 0 1 83 55 38 46 1 kashitate-a 1 ha 2 0 7 377 89 41 4 7 6 ha 11 1 6 238 499 141 73 1 nakanogo-a 1 ha 0 0 0 29 14 4 3 1 6 ha 0 1 1 38 36 109 4 5 nakanogo-b 1 ha 0 0 0 5 4 0 2 0 6 ha 0 0 0 87 18 1 50 0 sueyoshi-a 1 ha 0 0 0 146 207 87 1 3 6 ha 79 8 82 729 66 62 19 19 table 2. number of attracted individuals in trial 1. abbreviations of the commercial poison baits are given in table 1; sw, 10% (w/v) sucrose water; hw, 10% (w/v) honey water; pc, peanut cream; ftw, filtered tap water. abbreviations of the scientific names of non-technomyrmex brunneus species are as follows: mc, monomorium chinense santschi, 1925; pp, pristomyrmex punctatus (f. smith, 1860). abbreviations of the time windows are as follows: 1 ha, 1 hour after setting the bait series; 6 ha, 6 hours after setting the bait series. for site locations, see fig 1. baits 1 ha 6 ha usf b 0.22 a ± 0.63 (2) 11.33 a ± 34.12 (102) ac a, b 0.11 a ± 0.31 (1) 1.78 a ± 2.39 (16) hak b 2.67 a ± 3.13 (24) 11.45 a ± 25.04 (103) am c 85.67 b ± 117.63 (771) 191.78 b ± 202.62 (1726) sw b, c 57.67 a ± 62.16 (519) 106.45 a ± 145.46 (958) hw b 31.78 a ± 33.59 (286) 75.33 a ± 86.48 (678) pc b 2.78 a ± 3.20 (25) 68.78 a ± 67.59 (619) ftw b 1.89 a ± 2.23 (17) 4.11 a ± 6.12 (37) anova results f 4.22 4.01 df 7 7 p <0.01 <0.01 table 3. the mean and standard error of the number of attracted individuals of technomyrmex brunneus in trial 1. the total number of individuals attracted to each bait is given in parentheses. abbreviations of the baits and time windows are given in tables 1 and 2. the superscripts (a, b, and c) of the baits indicate significant differences at the 5% level by holm’s multiple comparison tests following analysis by two-way anova. sociobiology 68(1): e5898 (march, 2021) 5 sites time window ast sak gab aag sw hw pc ftw mitsune-a 1 ha 0 10 0 0 1 0 0 0 3 ha 0 1 0 0 1 0 0 2 okago-a 1 ha 2 0 2 0 117 60 21 3 3 ha 2 9 80 19 416 486 5 5 okago-b 1 ha 0 0 0 1 0 0 0 (mc 1) 0 3 ha 1 14 60 34 50 4 1 0 okago-c 1 ha 0 0 0 0 0 1 0 0 3 ha 10 37 76 54 46 12 17 (ps 1) 4 kashitate-a 1 ha 0 4 18 0 78 28 1 (tb 1) 0 3 ha 2 2 20 21 206 102 0 (tb 52) 0 kashitate-b 1 ha 7 4 1 1 35 6 0 0 3 ha 12 5 33 45 27 4 0 0 nakanogo-a 1 ha 0 0 (ps 1) 0 0 0 (pp 1) 0 0 (mc 4) 0 3 ha 1 0 (pp 1) 1 (pp 2) 0 18 (mc 36) 1 (pp 1) 0 (mc 66) 0 sueyoshi-a 1 ha 0 19 12 55 210 61 1 0 3 ha 1 50 170 57 282 45 0 0 sueyoshi-b 1 ha 0 (cv 1) 0 (pp 4) 0 0 0 0 0 1 3 ha 0 0 (pp 68) 0 (pp 1) 0 13 14 0 0 table 4. number of attracted individuals in trial 2. abbreviations of the commercial poison and non-poison baits are given in tables 1 and 2. abbreviations of the scientific names of non-technomyrmex brunneus species are as follows: tb, tetramorium bicarinatum; ps, paraparatrechina sakurae; cv, camponotus vitiosus (for others, see table 2). abbreviations of the time windows are as follows: 1 ha, 1 hour after setting the bait series; 3 ha, 3 hours after setting the bait series. for site locations, see fig 1. in addition, a × b, which indicates the interaction between factor a (time window) and factor b (bait), was not significantly different at the 5% level (f (7, 7) = 1.06, n.s.). attractiveness was tested with anova after dividing the trial 1 dataset into two sub-datasets by the two different time windows. the results showed significant differences at the 1% level in both the sub-datasets [1 ha: ss = 67,647.875, df = 7, ms = 9664.125, f = 4.22 (p < 0.01); 6 ha: ss = 279,785.097, df = 7, ms = 39,696.300, f = 4.01 (p < 0.01)]. the combinations in which a significant difference was observed at the 5% level were usf < am, ac < am, hak < am, pc < am, pw < am in 1 ha, and usf < am, ac < am, hak < am, pw < am in 6 ha. from the above results, the bait that showed the highest attractiveness in trial 1 was arimetsu (am); 10% sw also showed a similar attractiveness. in contrast, the attractiveness of the paste-form poison baits (usf, ac, and hak) was low, and when compared with the mean number of attracted t. brunneus individuals, those were less than 5% of the attractiveness of arimetsu. trial 2 table 3 shows the number of attracted individuals at the two different time windows, i.e., 1 hour (1 ha) and 3 hours (3 ha) after setting the bait series. tetramorium bicarinatum (nylander, 1846) (tb), m. chinense (mc), p. punctatus (pp), mamoru terayama, eiriki sunamura, ryota fujimaki, takashi ono, katsuyuki eguchi – non-attractiveness of poison baits to technomyrmex brunneus6 paraparatrechina sakurae (ito, 1914) (ps), and camponotus vitiosus (f. smith, 1874) (cv), as well as t. brunneus, were attracted to the bait series. of the six ant species, t. brunneus is extremely dominant in all locations, while the number of t. brunneus individuals varied widely depending on the locations and time windows; the standard deviation was large for each bait (table 5). from the above results, no bait exceeded the attractiveness of 10% sw in trial 2. in contrast, the attractiveness of the gel-form poison baits (gab and aag) was low, and when compared with the mean number of attracted t. brunneus individuals, those were less than 32% of the attractiveness of sw. discussion the results of the two trials of the cafeteria experiment conducted on hachijo-jima island revealed that sw effectively attracts t. brunneus. among the commercial poison baits for ants, only arimetsu showed a strong attractiveness, equivalent to or more than that of sw. arimetsu consists of 42.6% water, 55.4% sugar (attracting component), and 2.0% borate (insecticidal component). technomyrmex difficilis forel, 1892, belonging to the t. albipes species group in north america (florida), is known to be attracted strongly to sugar-rich baits, but is less attracted to proteinand lipid-rich baits (klotz et al., 2008). based on indoor experiments conducted by warner and scheffrahn (2005) and warner et al. (2005), sucroseenhanced liquid baits are recommended to effectively attract t. difficilis (misidentified as t. albipes in the paper). warner and scheffrahn (2005) reported that t. difficilis is attracted most strongly to 25%–40% sw. on the other hand, the present study revealed that paste-form poison baits containing fipronil, which is often used to exterminate common native ants, as well as fire ants and the argentine ant in japan, cannot effectively attract t. brunneus. therefore, from the viewpoint of attractiveness to t. brunneus, the substrate of the bait suitable for t. brunneus would be a liquid composed mainly of sugar (sucrose), or a biodegradable sponge or hydrogel containing such liquid. the insecticidal component of arimetsu is borate. however, in experiments with t. difficilis, warner and scheffrahn (2005) found that thiamethoxam and imidacloprid of neonicotinoid showed a higher insecticidal effect than fipronil and borate when sucrose solution or a similar liquid was used as the substrate. additionally, there are no successful cases in which borate was used to eradicate highly invasive ant populations (hoffmann et al., 2016). the effectiveness of insecticidal components is known to differ depending on the ant species. for example, among the insecticide components used commonly, hydramethylnon controls monomorium pharaonis (linnaeus, 1758) and linepithema humile (mayr, 1868) effectively, but controls tapinoma melanocephalum (fabricius, 1793) less effectively (klotz et al., 1996; ulloachacón & jaramillo, 2003). therefore, the confirmation of an insecticidal component suitable for t. brunneus will be an important issue in future studies. as t. brunneus individuals exchange nutrients with each other exclusively through specialized trophic eggs (yamauchi et al., 1991), it is also quite important to know how this behavioral feature influences the diffusion efficiency of insecticidal components in the nest. baits 1 ha 3 ha ast a 1.00 a ± 2.21 (9) 3.00 a ± 3.80 (27) sak a 4.11 a ± 6.15 (37) 13.11 a ± 17.09 (118) gab a 3.67 a ± 6.25 (33) 37.78 a ± 32.18 (340) aag a 6.33 a ± 17.21 (57) 23.33 a ± 23.11 (210) sw b 49.00 b ± 69.51 (441) 117.67 b ± 139.94 (1059) hw a, b 17.33 a ± 24.58 (156) 74.22 a ± 148.85 (668) pc a 2.56 a ± 6.53 (23) 2.56 a ± 5.34 (23) ftw a 0.44 a ± 0.96 (4) 1.22 a ± 1.87 (11) anova results f 3.67 3.02 df 7 7 p <0.01 <0.05 table 5. the mean and standard error of the number of attracted individuals of technomyrmex brunneus in trial 2. the total number of individuals attracted to each bait is given in parentheses. abbreviations of the baits and time windows are given in tables 1, 2, and 4. the superscripts (a and b) of the baits indicate significant differences at the 5% level by holm’s multiple comparison tests following analysis by two-way anova. the results of the two-way anova showed no significant difference in the number of attracted t. brunneus individuals between the time windows at the 5% level (f(1, 7) = 2.77, n.s.), but there were significant differences among the baits at the 1% level (f(7, 7) = 4.98, p < 0.01). significant differences in the mean number of attracted t. brunneus individuals were observed between the following combinations at the 5% level: ast < sw, sak < sw, gab < sw, aag < sw, pc < sw, and ftw < sw. in addition, a × b, which indicates the interaction between factor a (time windows) and factor b (baits), was not significantly different at the 5% level (f(7, 7) = 1.19, n.s.). the attractiveness was tested by anova after dividing the trial 2 dataset into two sub-datasets by the two different time windows. the results showed significant differences at the 1% level in the 1 ha sub-dataset and at the 5% level in the 3 ha sub-datasets [1 ha: ss = 16,994.444, df = 7, ms = 2427.777, f = 3.67 (p < 0.01); 3 ha: ss = 110,984.388, df = 7, ms = 15,854.912, f = 3.02 (p < 0.05)]. the combinations in which a significant difference was observed at the 5% level were ast < sw, sak < sw, gab < sw, aag < sw, pc < sw, and ftw < sw in 1 ha, and ast < sw, sak < sw, pc < sw, and ftw < sw in 3 ha. sociobiology 68(1): e5898 (march, 2021) 7 thus, from the viewpoint of the attractant and insecticidal bait components, it is clear that conventional extermination protocols used for fire and argentine ants cannot be applied directly to t. brunneus. along with the development of baits suitable for t. brunneus, it is necessary to establish a comprehensive control protocol specialized for the hachijojima population, such as the establishment of bait installation methods, environmental improvement methods, and local quarantine measures, according to the characteristics of the t. brunneus life history under the local bioclimate and other environmental conditions on hachijo-jima island. acknowledgments we would like to thank hisashi sasaki, rika kikuchi, nanae hijikata, miho osawa, kisako wada, sakura hirano, ai okiyama, yuta okiyama, and syuhei kono (residents’ section of the hachijo town hall, tokyo, japan) for their contributions to the cafeteria experiment. we also extend our thanks to the handling editor and two anonymous reviewers for their valuable comments and suggestions. we would also like to thank dr. kouichi goka (national institute for environmental studies, japan), dr. hitoshi onishi (kanto regional environment office, ministry of the environment, saitama, japan), and mr. yasuhiro tomioka (technical research laboratory, ikari shodoku co., ltd., narashinoshi, chiba pref., japan) for their valuable comments and for providing research articles. this research was conducted using funding from hachijo town, tokyo. additionally, part of the expenses for research planning and publishing was paid by the tokyo metropolitan university fund for tmu strategic research (leader: noriaki murakami; 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(2018). taiwanese species of the ant genus technomyrmex (formicidae: dolichoderinae). zootaxa, 4410: 35-56. doi: 10.11646/ zootaxa.4410.1.2 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5911sociobiology 68(1): e5911 (june, 2021) introduction the subterranean termite globitermes sulphureus is commonly found in malaysia, singapore, thailand, and vietnam (ahmad, 1965; bordereau et al., 1997; kuswanto et al., 2015; lee et al., 2007; ngee & lee, 2002). this termite belongs to higher group termites which possess only bacteria and archaea in their gut (bujang et al., 2014). as a wood feeder termite, this species has been reported to infest premises’ wood structures (ab majid & ahmad, 2009; neoh et al., 2011). moreover, it was also reported as the primary pest in agricultural sectors such as coconut and oil palm plantations (lee et al., 2003). g. sulphureus is recognized as a pest of significant economic importance in southeast asia (rust & su, 2012). abstract the subterranean higher termite globitermes sulphureus (blattodea: termitidae) is a peridomestic forager and regarded as a significant pest in southeast asia. in this study, populations of g. sulphureus from the usm main campus area were investigated based on partial sequences of the mitochondrial coii gene. the genetic diversity was determined using dnasp v5 software, while the phylogenetic relationship was defined using neighbor-joining (nj) and maximum likelihood (ml) methods using molecular evolutionary genetics analysis (mega 7) software. a total of 2 haplotypes were detected among 5 sample sequences distinguished through two variable sites. also, both phylogenetic trees gave similar topology and supporting the results from haplotype diversity. based on the haplotype diversity and molecular phylogeny, it is proposed that geographic isolation and lack of human activities have contributed to the neutral genetic diversity of g. sulphureus. sociobiology an international journal on social insects nurul akmar hussin, abdul hafiz ab majid article history edited by qiuying huang, hzau, china received 09 october 2020 initial acceptance 13 february 2021 final acceptance 11 march 2021 publication date 11 june 2021 keywords genetic diversity, phylogenetic, termite, globitermes sulphureus, coii. corresponding author abdul hafiz ab majid household & structural urban entomology laboratory vector control research unit, school of biological sciences universiti sains malaysia penang, 11800 minden, malaysia. e-mail: abdhafiz@usm.my most of the currently available data for g. sulphureus distribution in malaysia is based on samples gathered from home infestations, disturbed forest, forest reserve, and rural areas (ab majid & ahmad, 2009; aiman hanis et al., 2014; khizam & ab majid, 2019; lee et al., 2004). however, its invasion biology, geographical distribution, and the pattern of introducing this species are still less understood. the phylogenetic relationships among populations and the impact of introductions to new habitats on genetic diversity and colony structure of g. sulphureus are relatively unknown (khizam & ab majid, 2021). previous researches focused on the genetic diversity and phylogenetics of the lower termites, especially in the genus reticulitermes and coptotermes, and dry wood termites genus incisitermes, cryptotermes, kalotermes, neotermes, household & structural urban entomology laboratory, vector control research unit, school of biological sciences, universiti sains malaysia, penang, malaysia research article termites genetic diversity and phylogenetic relationship of higher termite globitermes sulphureus (haviland) (blattodea: termitidae) nurul a hussin, abdul ha majid – genetic diversity and phylogenetic of globitermes sulphureus2 and mastotermes isolated from multiple locations (austin et al., 2012; husseneder et al., 2012; leniaud et al., 2010; pinzon & houseman, 2009; szalanski et al., 2008; thompson et al., 2000; yeap et al., 2007). the genetic diversity and phylogenetics of higher group termites have also been widely studied, such as genus odontotermes, microtermes, microcerotermes, macrotermes, labiotermes, and nasutitermes (dupont et al., 2009; ab majid et al., 2018; murthy et al., 2015; ohkuma et al., 2004; singla et al., 2016; singla et al., 2015). most of the studies mentioned above use coii gene sequences as molecular markers in the genetic diversity and phylogenetic relationship. the coii is a subunit of cytochrome c oxidase in the mitochondria (frati et al., 1997). coii is considered the fastest evolving gene compared to 12s and 16s genes (yeap et al., 2007). the rapid evolution of these genes has been proven helpful for deducing phylogenetic relationships between closely related insect species due to the relatively high degree of variation at the 3’ end of this gene (aly et al., 2012; singla et al., 2016). g. sulphureus is a peridomestic forager and moundbuilding termite (lee et al., 2003). its mound is easily identified based on a dome shape and a dark brown color (ahmad, 1965). g. sulphureus is easily identified since the soldiers possess a bright-yellow colored body (ab majid & ahmad, 2011; hussin & ab majid, 2017; hussin et al., 2018; khizam & ab majid, 2019). the goal of this study is to determine the genetic diversity and phylogenetic relationship among local populations of g. sulphureus in universiti sains malaysia (usm) main campus, penang by using partial sequences of the mitochondrial coii gene. there may also be differences in the termite genome from several mounds of g. sulphureus in response to local ecological conditions. materials and methods termites collection termite specimens were collected from the termite mounds. there are five accessible termite mounds identified in universiti sains malaysia (fig 1). the five colonies are colony 1: durian valley (dv), colony 2: indah kembara (ik), colony 3: padang minden (pm), colony 4: tasik harapan (th), colony 5: bakti permai (bp) (table 1). the termites were collected in a universal bottle containing 10 ml of 90% ethanol. fig 1. map of the usm main campus, penang showing the sites of g. sulphureus populations being collected. the abbreviation details are listed in table 1. the map was edited and retrieved from meng et al., (2002). table 1. collection sites, isolated code, source, and gps coordinate of each g. sulphureus in this study. isolated code collection site source lat_lon dv durian valley mound 5˚21’34.9” n 100˚18’19.3” e ik indah kembara mound 5˚21’20.0” n 100˚17’35.2” e pm padang minden mound 5˚21’34.6” n 100˚18’29.4” e th tasik harapan mound 5˚21’14.5” n 100˚18’01.1” e bp bakti permai mound 5˚21’30.7” n 100˚18’04.9” e sociobiology 68(1): e5911 (june, 2021) 3 dna extraction and pcr amplification ten worker termites from each collection site were rinsed in sterile distilled water and dried with a paper towel. the workers’ heads were cut off from the body using sterile dissecting scissors for dna extraction. genomic dna extraction was performed using the dneasy blood & tissues kit (qiagen, germany) for coii gene pcr amplification. the coii gene was amplified using a pair of primer, forward (5’-cattgcacccgcaatcatcc-3’) and reverse (5’-gaatctgtggtttgctcctccgc-3’). the pcr reaction mixture (50 μl) contains 25 μl of 2x toptaq master mix (qiagen, germany), producing a final concentration of 1.5 mm mgcl2, 1.25 units toptaq dna polymerase, 1× pcr buffer, and 200 µm of each dntp, together with 1 μl (10 μm) of each primer, 5 μl (10x) coralload concentrate (as a substitute to loading dye), 10-100 ng of a bulk dna template, and sterile distilled water. the pcr reaction profile comprises an initial denaturation at 94 °c for 2 min, followed by 40 cycles of denaturation at 94 °c for 45 seconds, primer annealing at 50 °c for 45 seconds, the first extension step at 72 °c for 60 seconds, and the final extension step at 72 °c for 5 min. the pcr product was purified with megaquick-spintm total fragment dna purification kit (intron biotechnology, south korea). sequencing and analysis of coii gene sequences the purified pcr products were sequenced using the sanger sequencing machine at first base laboratories sdn. bhd., malaysia. raw data obtained were extracted using finchtv 1.4 (www.geospiza.com). then, the forward and reverse sequences were aligned using t-coffee (notredame et al., 2000). the aligned sequences were edited manually to remove the low-quality bases before converting the files to fasta format. the fasta files were blastn search at the ncbi database (https://blast.ncbi.nlm.nih.gov) to compare with all coii gene sequences available in the genbank. the species identified are based on ≥ 99% sequences similarity with the ncbi database. the partial coii sequences were submitted to the genbank. genetic diversity and phylogenetic analysis all sequences were aligned automatically using the multiple alignment algorithm in clustalx v2.1 (larkin et al., 2007; thompson et al., 1997) with default settings. genetic characteristics such as haplotype diversity, nucleotide diversity, and the total number of mutations were calculated using dnasp v5 (librado & rozas, 2009). the pairwise genetic distance was calculated using the p-distance method in mega 7. the phylogenetic relationship was carried out using the molecular evolutionary genetics analysis (mega) version 7 (kumar et al., 2016). the phylogenetic trees were constructed using the neighbor-joining (nj) (saitou & nei, 1987) and maximum likelihood (ml) method based on p-distance (nei & kumar, 2000) and hasegawakishino-yano model (hasegawa et al., 1985), respectively. the c. gestroi (lower termite) with an accession number gu931692.1 was used as an outgroup. bootstrap analysis with 1000 resamplings was used to establish the nj and ml trees (fellsenstein, 1985). results five coii gene sequences were used with an average amplicon size of 420 base pairs after editing and removing the low-quality bases (bp). blastn results confirm the termite species is g. sulphureus, and the accession number for submitted sequences is written in table 2. the edited sequences are used for genetic diversity and phylogenetic analysis. isolated code collection sites haplotype hd pi accession no. th tasik harapan 1 mf997559.1 ik indah kembara 1 mf997563.1 bp bakti permai 1 0.400 0.00192 mf997545.1 pm padang minden 1 mf997577.1 dv durian valley 2 mf997568.1 nucleotide analysis the average base frequencies for five coii nucleotide sequences are a = 35.5%, t = 22.9%, g = 16.1%, and c = 25.5%. the total content of a+t is 58.4%, much higher than table 2. haplotype, haplotype diversity (hd), nucleotide diversity (pi), and accession g. sulphureus from five sample locations. c+g = 41.6%. the nucleotide diversity, pi, is 0.00192 (table 2). two polymorphic or singleton sites are observed in the coii nucleotide sequence of dv samples at positions 252 and 255 (table 3). the pairwise genetic distance between partial coii gene sequences is 0.000 to 0.005 (table 4). https://blast.ncbi.nlm.nih.gov nurul a hussin, abdul ha majid – genetic diversity and phylogenetic of globitermes sulphureus4 haplotype analysis we observed two haplotypes, with haplotype 1 consisting of four colonies and haplotype 2 consisting of only one colony (table 2), distinguished by two variable sites (table 3). the haplotype diversity (hd) of the five samples is 0.400 (table 2). the relationship between haplotypes is further confirmed upon employing nj and ml methods as implemented in mega 7 (fig 2 and fig 3). phylogenetic relationship inferred from coii genes the phylogenetic relationship of g. sulphureus from five locations is analyzed using two approaches; distance matrix: nj (fig 2) and character-based method: ml (fig 3). both trees show a monophyletic group among five samples but with three separate clades corresponding to the outgroup species, c. gestroi cg003tw. the first clade comprises haplotype 1, while the second clade comprises haplotype 2. the third clade shows the separation of termitidae from the rhinotermitidae family. the trees are also supported by pairwise genetic distance values (table 4). colony dv has a pairwise genetic distance value of 0.005 between other colonies, causing it to be located in the second clade. meanwhile, all colonies in haplotype 1 have 0.000 value of the pairwise genetic distance between them. nucleotide position samples 248 249 250 251 252 253 254 255 256 257 258 th c a a t c c g g t t a ik . . . . . . . . . . . bp . . . . . . . . . . . pm . . . . . . . . . . . dv . . . . t . . a . . . table 3. aligned haplotype sequences showing polymorphic sites (highlighted in green color). 1 2 3 4 5 1. th 2. ik 0.000 3. bp 0.000 0.000 4. pm 0.000 0.000 0.000 5. dv 0.005 0.005 0.005 0.005 table 4. pairwise genetic distance (p-distance) in species under study. fig 2. neighbor-joining tree built on the partial coii gene sequences of g. sulphureus from five different mounds. c. gestroi is used as an outgroup. the number at the branch refers to the bootstrap value. the evolutionary distances are computed using the p-distance method. bar = 0.02 substitutions per nucleotide position. sociobiology 68(1): e5911 (june, 2021) 5 discussion there are five accessible mounds of g. sulphureus identified on the usm main campus. colony dv is located at durian valley, known as natural forest/habitat in the usm main campus area. this location is protected to preserve flora and fauna there (meng et al., 2002). colony pm is located at the bamboo trees near the minden field, where students actively play football. colony bp is located closer to the hostel’s area, while colony ik is near the main roadside. lastly, colony th is located between hostels and the lake. globitermus sulphureus is regarded as a peridomestic forager and previously found in a disturbed forest area rather than a natural forest area (aiman hanis et al., 2014). a disturbed forest area is a place that had been cleared and developed for eco-tourism activities where various wooden structures and facilities were built. g. sulphureus is also commonly found in rural and urban areas (lee et al., 2007; lee et al., 2003; ab majid & ahmad, 2009). urbanization of plantation areas attracted this termite, causing infestation at the door and window frames of houses (ngee & lee, 2002). therefore, the encounter of this species in the usm area is not surprising. partial coii gene sequences of g. sulphureus isolated from five colonies are used for genetic diversity and phylogenetic analysis. the composition of different nucleotides in coii gene sequences from the colonies is calculated. from the results, the coii gene sequences show a high percentage of a+t (58.4%) than g+c (41.6%). adenine and thymine bias in nucleotide sequences is consistent with the data on coii mitochondrial genes of other higher group termites species (genus microtermes, microcerotermes, and odontotermes) and lower termites (genus coptotermes) (singla et al., 2016; yeap et al., 2007). high a+t content is typical in insect mitochondrial dna (keller et al., 2007). however, the adenine-thymine percentage recorded in this study is lower than reported by those studies (59.87 62.0%). this result might occur because partial sequences of the coii gene are used in this study instead of full sequences (675 680 bp). the pairwise genetic distance for this study ranges from 0.000 to 0.005. this value is lower than reported previously regarding the genetic distance comparison between different genus of odontotermes (ranged from 0.025 to 0.072) and genus microtermes (ranged from 0.028 to 0.229) (singla et al., 2016), and higher than the pairwise genetic distance between macrotermes carbonarius populations (0.003) (ab majid et al., 2018). this result suggests that populations within a species have smaller genetic distances than populations within a genus because intraspecific populations are more closely related and have a recent common ancestor. from the haplotype analysis, two haplotypes are formed with dv colony solely in haplotype 2. the dv colony has two variable sites causing the pairwise genetic distance value to be 0.005 between other colonies. this result might occur due to the biased nature of natural forest zones (dv) compared to urban zones (ik, pm, bp, and th). in a previous study, szalanski et al. (2008) demonstrated that the genus reticulitermes isolated from lake wedington (national forest) has a higher frequency of rare haplotypes than reticulitermes isolated from urban areas. geographic fig 3. maximum likelihood tree inferred from the partial coii gene sequences of g. sulphureus from five different mounds. c. gestroi is used as an outgroup. the number at the branch refers to the bootstrap value. the evolutionary history is computed using the hasegawa-kishino-yano model. bar = 0.02 substitutions per nucleotide position. nurul a hussin, abdul ha majid – genetic diversity and phylogenetic of globitermes sulphureus6 isolation and the lack of human involvement in forested regions contributed to the rare haplotype of reticulitermes species. besides, demographic fluctuations and natural selections can affect any particular species’ neutral genetic diversity (ellegren & galtier, 2016). two variable sites are detected in this study. this is contrary to other higher group termite (m. carbonarius) isolated from usm main campus, which has only one variable site (ab majid et al., 2018). both variable sites are silent mutations since changes in the nucleotide bases do not affect amino acid sequences. silent mutations in this study can be regarded as synonymous mutations since the nucleotide bases only change at the codon’s third position. synonymous mutations usually occur by neutral selection and later become fixed (fouks & lattorff, 2016). it is long thought to be without phenotypic consequences but is currently recognized as critical in shaping gene expression, protein folding, cellular function, and the organism’s fitness (plotkin & kudla, 2011; zwart et al., 2018). however, the consequences of synonymous mutations in termites fitness remained understood. the phylogenetic trees supported genetic diversity results where two clusters are formed regarding the haplotypes. both trees (nj and ml) show monophyletic of all five colonies. bootstrap values (> 80%) show the significance of both trees. in the phylogenetic tree, closely related organisms are group together according to the order, family, subfamily, genus, and species (ab majid et al., 2018; bourguignon et al., 2014; singla et al., 2015; yeap et al., 2007). since there is no parsimony-informative site detected in this study, parsimony analysis is excluded. more sampling sites and analysis are needed to confirm the geographic distribution and the native and invasive termite g. sulphureus in usm main campus and malaysia. genotype analysis may explain the breeding pattern at the microsatellite level and reveal the nature of haplotype variability within g. sulphureus. in conclusion, this study demonstrated the coii gene’s ability to differentiate between g. sulphureus populations from a few different usm main campus locations. the genetic diversity analysis shows nucleotide divergence between isolated populations. phylogenetic analysis supports the haplotype relationship of g. sulphureus. however, geographical area influences on the species’ genetic diversity require more sampling sites and further analysis such as microsatellite genotyping. acknowledgement this research is supported by research university grant (rui) (1001 / pbiologi / 8011104) funded by universiti sains malaysia . author contributions nah: conceptualization, methodology, investigation, data curation, visualization, formal analysis, writing-original draft. aham: supervision, conceptualization, methodology, visualization, validation, project administration, resources, funding acquisition, writing -review & editing declaration – conflict of interest the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. data availability statement the data that support the findings of this study are openly available in [ncbi] at [https://www.ncbi.nlm. nih.gov/], reference number mf997559.1, mf997563.1, mf997545.1, mf997577.1, mf997568.1]. references ab majid, a.h. & ahmad, a.h. 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(2018). unraveling the causes of adaptive benefits of synonymous mutations in tem-1 β-lactamase. heredity, 121: 406-421. doi: 10.1038/s41437018-0104-z _hlk67661769 doi: 10.13102/sociobiology.v64i3.1611sociobiology 64(3): 292-300 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 floral resource partitioning between centris (heterocentris) analis (fabricius, 1804) and centris (heterocentris) terminata smith, 1874 (hymenoptera, apidae, centridini), in an urban fragment of the atlantic forest introduction bees of the genus centris fabricius, 1804 are widely distributed in the americas (michener, 2007). centris analis (fabricius, 1804) occurs from mexico to brazil and centris terminata smith, 1874 occurs from ecuador to brazil (moure et al., 2012). these two species belong to the subgenus heterocentris cockerell, 1899. females of c. analis and c. terminata build nests in preexisting cavities, such as the abandoned nests of other bees or wasps, galleries dug by other insects and trap-nests (michener & lange, 1958; coville et al., 1983). as occurs with other bees, centris females visit flowers for the purpose of collecting pollen and nectar to supply their own energy needs and provision brood cells. these bees also use the floral oil to feed larvae and/or build nests abstract the knowledge on plant species used for the collection of floral resources is crucial to understanding interactions between plants and bees. the aim of the present study was to identify floral resources used by centris analis and centris terminata to provision brood cells and determine the niche breadth and overlap of these two species in a fragment of the atlantic forest in brazil. this study was conducted at the universidade federal da bahia and parque zoobotânico getúlio vargas, both of which are located in urban areas of the city of salvador in the state of bahia. in general, twelve and eight pollen types were identified in c. analis and c. terminata nests, respectively. the most frequent pollen types were from species of malpighiaceae and fabaceae. a larger trophic niche breadth was found in the parque zoobotânico getúlio vargas for c. analis and in the universidade federal da bahia for c. terminata. pianka’s index demonstrated trophic niche overlap between c. analis and c. terminata, which was greater in the parque zoobotânico getúlio vargas. this study is the first to provide data on plants used as food sources by species of the genus centris in a fragment of the atlantic forest situated within urban areas. sociobiology an international journal on social insects r lima1, mj ferreira-caliman1, mc dórea2, ct garcia3, far santos2, ff de oliveira3, ca garófalo1 article history edited by celso f. martins, ufpb, brazil received 21 march 2017 initial acceptance 10 may 2017 final acceptance 09 june 2017 publication date 17 october 2017 keywords pollen analysis, oil-collecting bees, solitary bees, trophic niche, urban areas. corresponding author reinanda lima usp/faculdade de filosofia, ciências e letras de ribeirão preto av. bandeirantes nº 3900, cep 14040-901 ribeirão preto, são paulo, brasil. e-mail: reinanda-09lima@usp.br (vogel, 1974; neff & simpson, 1981; vieira-de-jesus & garófalo, 2000; alves-dos-santos et al., 2007). species of the genus are efficient pollinators of native and cultivated plants with economic importance as, for example, c. analis being a effective pollinators of malpighia emarginata sessé and moc ex dc (vilhena & augusto, 2007; oliveira & schlindwein, 2009), byrsonima sericea d.c (ramalho & silva, 2002) and c. terminata being an known pollinator of passiflora alata curtis (mello et al., 2014). in general, centris bees are considered generalists due to the behavior of collecting resources from different botanical families (quiroz-garcia et al., 2001; aguiar et al., 2003; dórea et al., 2010; santos et al., 2013). however, according to gonçalves et al. (2012), females of centris tarsata showed an oligolectic behavior, collecting pollen from solanaceae species for larval provisioning in a region of southern brazil. 1 universidade de são paulo (usp), faculdade de filosofia, ciências e letras de ribeirão preto, são paulo, brazil 2 universidade estadual de feira de santana (uefs), bahia, brazil 3 universidade federal da bahia (ufba), salvador-ba, brazil research article bees sociobiology 64(3): 292-300 (september, 2017) 293 studies involving the analysis of residual pollen from centris nests show that species of the family malpighiaceae are the most frequently used, followed by fabaceae and solanaceae. several studies showed the preferences of pollen types used by females of centris to feed their larvae, as well, centris flavifrons (fabricius, 1775) (rêgo et al., 2006), centris maranhensis ducke, 1910 (ramos et al., 2007), centris caxienses (ducke, 1907) (ribeiro et al., 2008), centris tarsata smith, 1874 (mendes & rêgo, 2007; dórea et al., 2009; gonçalves et al., 2012; cruz et al., 2015), centris analis (fabricius, 1804) (quirózgarcia et al., 2001; oliveira & schlindwein, 2009; roubik & villanueva-gutiérrez, 2009; dórea et al., 2010; rabelo et al., 2012; santos et al., 2013), centris trigonoides lepeletier, 1841 (quirózgarcia et al., 2001; dórea et al., 2013) and centris flavofasciata friese, 1899 (quiroz-garcia & de la arreguin-sánchez, 2006). the number of species investigated thus far is less than 10% of the total of the genus known by science. for c. terminata (one of the two species analyzed in the present study), there are no previous records of the resources collected by females to provision nests. direct observations in the field can provide information on resource utilization by centris bees. moreover, an analysis of pollen stored in nests enables a detailed account of the number of plant species used as resources (cane & sipes, 2006). knowledge of food sources is essential to understanding associations among bees and plants and provides valuable information for the development of management and conservation plans. therefore, the aim of the present study was to identify floral resources used by c. analis and c. terminata females to provision brood cells and assess the niche breadth and overlap of these species in an urban fragment of the atlantic forest in the city of salvador, brazil. material and methods study area this study was carried out in an urban fragment of the atlantic forest in the city of salvador, state of bahia, brazil (13º01’ s and 38º31’ w). based on the köppen typology, the climate in salvador is af – tropical hot and wet. mean annual temperature is 25.3°c and mean annual rainfall is 1800 mm (conder, 1994). the study sites were the parque zoobotânico getúlio vargas (pzbgv) and the campus of universidade federal da bahia (ufba). the pzbgv covers a green area of about 240,000 m2 and is structurally characterized by sparse tree-shrub vegetation, with several species of fabaceae, solanaceae, malpighiaceae, myrtaceae, bignoniaceae and melastomataceae (n. roque, personal information). the ufba campus occupies an area of 302,000 m2 and is composed of small fragments of heterogeneous herbaceous vegetation, with a predominance of grasses and secondary shrubbyarboreal atlantic forest. several species of botanical families are scattered around the campus, such as anacardiaceae, arecaceae, bignoniaceae, fabaceae, malvaceae, malpighiaceae, meliaceae, moraceae and myrtaceae (carvalho et al., 2007). there are also squares, buildings and gardens with ornamental plants (carvalho et al., 2007). data collection and pollen analysis the pollen samples were obtained from nests established in trap-nests. the trap-nests were installed in the field in april 2014 and inspected once a month from may 2014 to april 2015. once established, the closed nests were taken to the laboratory and stored at a room temperature until the emergence of individuals. the pollen samples of c. analis were obtained from 53 nests (ufba= 27 and pzbgv=26) and those of c. terminata from 20 nests (ufba= 7 and pzbgv=13). all c. analis nests were established in black cardboard trap-nests with inner diameters of 0.6 and 0.8 cm. the nests of c. terminata were established in bamboo canes (n = 11 nests) with inner diameters of 0.6 and 1.0 cm, and wooden blocks (n = 9 nests) with an inner diameter of 1.0 cm. after the emergence of the individuals, residual pollen from the inner wall of the brood cells was removed for analysis. in brood cells containing dead eggs or larvae, all food present in the cell was collected for analysis. the pollen material of each nest was transferred to test tubes containing 50 ml of an aqueous ethanol solution (70%). after 24 hours, the ethanol was discarded and the samples were placed in 4 ml of glacial acetic acid for 24 hours (silva et al., 2010). subsequently, the pollen material was submitted to the acetolysis process following the method described by erdtman (1960). the pollen was then put in an aqueous glycerine solution (50%). after 24 hours, small amounts of pollen were removed with cubes of glycerine jelly for the preparation of three microscopic slides per nest. photomicrographs were taken and pollen types were identified by comparisons with the specialized bibliography and reference slides from the collection of the plant micromorphology laboratory, universidade estadual de feira de santana. quantitative analyses were based on the first 1000 pollen grains counted per sample, as proposed by vergeron (1964). data analysis based on santos et al. (2013), the monthly relative frequency (rfm) and total relative frequency (rft) of each pollen type were calculated by the ratio between the number of grains of each registered pollen type (monthly and during the nesting period) and the total number of grains counted each month and during the entire nesting period, respectively. trophic niche breadth was calculated using shannon’s index (h’) (pielou, 1975) and compared using hutcheson’s t-test (zar, 1984). pielou’s index (j’) (pielou, 1966) was used to calculate the evenness of the use of floral resources by c. analis and c. terminata. pianka’s index (ojk) (pianka, 1973) was used to calculate trophic niche overlap between c. analis and c. terminata: ojk= r lima et al. – resource partitioning between oil-collecting bees294 where ojk= corresponds to the measure of overlap of the trophic niche of pianka between species j and species k; pij = is the occurrence of pollen type i in the diet of the bee species j; pik= is the occurrence of pollen type j in the diet of the bee species k and ∑ = is sum that considers the values related to pollen type 1 to pollen type s (richness).the index varies between 0 (no overlap) and 1 (complete overlap).the analysis of diet breadth was performed using the past statistical software, version 2.17 (hammer et al., 2001). the analysis of trophic niche overlap was performed using the r statistical software, version 2.01 (r development core team, 2011). fig 1. pollen types found in nests of centris analis (fabricius, 1804) and centris terminata smith, 1874 sampled from urban fragment of atlantic forest in salvador, bahia. a-b: malpighia emarginata (malpighiaceae). c-d: stigmaphyllon cavernulosum (malpighiaceae). e-f: cestrum axillare (solanaceae). g-h: handroanthus chrysotrichus (bignoniaceae). i-j: byrsonima sericea (malpighiaceae). k-l: aeschynomene paucifolia (fabaceae). m-n: dioclea grandiflora (fabaceae).o: serjania type (sapindaceae). p: delonix regia (fabaceae). q: securidaca diversifolia (polygalaceae). r: amphilophium crucigerum (bignoniaceae). s: elaeis guineensis (arecaceae). (bar scale = 25µm). results pollen types used by centris analis we described the pollen types used by c. analis as larval food sources per month in both studied areas. the nesting activity of the females was registered to the months of may, october, november, and december (2014), january, february, march and april (2015). centris analis females used 12 pollen types belonging to seven botanical families to provision nests established at ufba (table 1 and figure 1a-s), whereas 11 pollen types belonging to six botanical sociobiology 64(3): 292-300 (september, 2017) 295 p ol le n ty pe s 1 r f t ( % ) 2 n n m on th ly r el at iv e fr eq ue nc y of p ol le n ty pe s (3 r f m ) ( % ) m ay /1 4 (n =4 ) o ct /1 4 (n =2 ) n ov /1 4 (n =2 ) d ec /1 4 (n =1 ) ja n/ 15 (n =5 ) f eb /1 5 (n =2 ) m ar /1 5 (n =5 ) a pr /1 5 (n =5 ) m al pi gh ia ce ae m al pi gh ia em ar gi na ta . 69 .1 5 25 62 .1 5 99 .0 7 91 .7 7 78 .4 7 39 .2 8 98 .9 9 64 .3 5 73 .4 0 st ig m ap hy llo n ca ve rn ul os um 14 .2 21 19 .7 1 0. 04 5. 6 21 .5 3 14 .1 8 0. 41 31 .5 7 8. 0 by rs on im a se ric ea . 13 .6 5 15 17 .8 0 0. 00 4 1. 81 41 .0 3 0. 01 13 .5 2 fa ba ce ae d el on ix re gi a 0. 00 9 1 0. 00 06 ae sc hy no m en e p au ci fo lia 1. 49 10 0. 00 03 5. 2 2. 06 0. 32 d io cl ea g ra nd ifl or a 1. 3 8 0. 00 1 0. 00 08 0. 6 0. 11 0. 41 0. 47 0. 03 b ig no ni ac ea e h an dr oa nt hu s c hr ys ot ric hu s 1. 3 15 0. 00 1 0. 11 0. 18 1. 53 4. 7 am ph ilo ph iu m cr uc ig er um 0. 01 6 1 0. 00 04 0. 37 0. 01 0. 03 so la na ce ae c es tru m a xi lla re 0. 00 9 1 0. 04 a re ca ce ae el ae is gu in ee ns is 0. 00 3 1 0. 01 po ly ga la ce ae se cu rid ac a di ve rs ifo lia 0. 00 9 1 0. 1 sa pi nd ac ea e se rja ni a 0. 00 3 1 0. 01 1 r ft = t ot al re la tiv e fr eq ue nc y of e ac h po lle n ty pe u se d fo r l ar va l f oo d in n es ts b ui lt th ro ug ho ut n es tin g pe ri od a t u fb a . 2 r f m = m on th ly re la tiv e fr eq ue nc y of e ac h po lle n ty pe u se d fo r l ar va l f oo d in n es ts b ui lt th ro ug ho ut n es tin g pe ri od a t u fb a . 3 n n = n um be r o f n es ts c on ta in in g ea ch p ol le n ty pe re co rd ed th ro ug ho ut n es tin g pe ri od . t ab le 1 . r el at iv e fr eq ue nc ie s of p ol le n ty pe s in la rv al f oo d fr om 2 6 ne st s of c en tr is a na lis ( fa br ic iu s, 1 80 4) c ol le ct ed in si de th e ca m pu s of th e u ni ve rs id ad e fe de ra l d a b ah ia ( u fb a ) in u rb an fr ag m en t o f a tla nt ic f or es t i n sa lv ad or , b ah ia , b ra zi l. r lima et al. – resource partitioning between oil-collecting bees296 families were identified from nests built at pzbgv (table 2 and figure 1 a-p, r-s). pollen types from the family malpighiaceae were the most abundant types used by c. analis females in both study areas. at ufba, the most common pollen type was malpighia emarginata, with frequencies varying from 39 to 99%, in the months of may, october, november, december 2014 and january, february, march and april 2015 (table 1). also, byrsonima sericea. showed high frequencies (41%) in january 2015. at pzbgv, the pollen type with the highest relative frequency was stigmaphyllon cavernulosum in nests of may (51.65%) and december 2014 (89.70%). byrsonima sericea was most frequent pollen in the nests of january 2015 (72.63%). malpighiae emarginata pollen was the most abundant source in the nests of february, march and april 2015 (table 2). the trophic niche breadth of c. analis was significantly higher for nests established at pzbgv (h’pzbgv= 1.32) than for nests built at ufba (h’ufba= 0.94) (t = 4.16; p > 0.05). the equitability indices were j’ufba = 0.21 and j’ pzbgv = 0.36). pollen types used by centris terminata pollen types 1rft (%) 2nn monthly relative frequency of pollen types (3rfm) (%) may/14 (n=2) dec/14 (n=1) jan/15 (n=5) feb/15 (n=9) mar/15 (n=5) apr/15 (n=5) malpighiaceae malpighia emarginata 25.65 20 44.58 10.52 32.63 60.23 69.74 stigmaphyllon cavernulosum 25.71 24 51.65 89.70 0.83 27.16 36.34 7.06 byrsonima sericea 38.81 19 0.60 0.27 72.63 32.45 0.19 19.98 fabaceae delonix regia 0.02 4 0.03 0.08 aeschynomene paucifolia 6.45 14 0.90 15.06 3.770 2.03 0.86 dioclea grandiflora 3.25 16 2.73 9.13 0.96 4.02 0.08 1.80 bignoniaceae handroanthus chrysotrichus 0.04 5 0.35 0.009 0.82 0.54 amphilophium crucigerum 0.04 4 0.21 0.01 solanaceae cestrum axillare 0.01 5 0.04 0.009 0.01 arecaceae elaeis guineensis 0.01 1 0.04 sapindaceae serjania type 0.01 1 0.01 1rft = total relative frequency of each pollen type used for larval food in nests built throughout nesting period at pzbgv. 2rfm = monthly relative frequency of each pollen type used for larval food in nests built throughout nesting period at pzbgv. 3nn = number of nests containing each pollen type recorded throughout nesting period. table 2. relative frequencies of pollen types in larval food from 27 nests of centris analis (fabricius, 1804) collected from parque zoobotânico getúlio vargas (pzbgv) in urban fragment of atlantic forest in salvador, bahia, brazil. nesting activities of c. terminata were restricted to the months of may (2014), january, february, and march (2015). at ufba, the females used seven pollen types belonging to four botanical families (table 3 and figure 1 a-n) while at pzbgv, they used six pollen types belonging to three botanical families (table 4 and figure 1 a-d, i-n, r). pollen types from the families malpighiaceae and fabaceae were the most abundant in c. terminata nests in both areas. at ufba, the pollen with the highest monthly relative frequency was m. emarginata (94.87%), in may 2014, followed by aeschynomene paucifolia (73.58%), in february 2015, and b. sericea (64.05%), in march 2015 (table 3). at pzbgv, the pollen type with the highest relative frequency was b. sericea (61.37%), in january 2015, and m. emarginata type (38.02%) was the most common source in the nests of march 2015 (table 4). no significant difference (t = 1.39; p < 0.05) was found between the trophic niche breadth of nests established at ufba (h’ufba=1.21) and those established at pzbgv (h’pzbgv =1.07). the equitability indices were j’ufba= 0.48 and j’ pzbgv = 0.42. trophic niche overlap between centris analis and centris terminata among the 12 pollen types found in the nests of c. analis and c. terminata, eight types were common to both species (tables 1-4). four pollen types were exclusively found in c. analis nests: delonix regia, securidaca diversifolia, sociobiology 64(3): 292-300 (september, 2017) 297 elaeis guineensis. and serjania type. based on the similarity of the use of the pollen types, trophic niche overlap between c. analis and c. terminata was ojk=0.03 at ufba and ojk=0.99 at pzbgv. discussion the present study showed that the most frequent pollen types collected by females of c. analis and c. terminata in the urban fragment of the atlantic forest were m. emarginata, b. sericea, s. cavernulosum and a. paucifolia,which indicates the importance of these species as sources of food for immature bees. the association between species of the tribe centridini and species of malpighiaceae results in a very specialized mutualism, with flowers depending on bees for pollination and the larvae of those bees depending on pollen and flower oils for nourishment (vogel, 1974), although some species of bees use nectar rather than floral oils (aguiar & garófalo, 2004). moreover, these bees use the oil as construction material for nests (vogel, 1974; vinson & frankie, 2000; vieira-de-jesus & garófalo, 2000; aguiar et al., 2003; dórea et al., 2009, 2010ab, 2013; rabelo et al., 2012). studies performed in different biomes and orchards report these same three genera of malpighiaceae as the main sources of pollen and oil for many species of centris (gaglianone, 2001; ramalho & silva, 2002; rêgo et al., 2006; vilhena & augusto, 2007; ramos et al., 2007; mendes & rêgo, 2007; dórea et al., 2009, 2013; mello et al., 2013 and references therein; santos et al.,2013 pollen types 1rft (%) 2nn monthly relative frequency of pollen types (3rfm) (%) may/14 (n=1) feb/15 (n=5) mar/15 (n=1) malpighiaceae malpighia emarginata 23.29 5 94.87 17.37 stigmaphyllon cavernulosum 7.46 5 4.79 0.85 32.71 byrsonima sericea 13.5 5 0.17 2.88 64.05 fabaceae aeschynomene paucifolia 66.12 7 0.17 73.58 0.07 dioclea grandiflora 1.24 3 1.39 3.17 bignoniaceae handroanthus chrysotrichus 0.29 3 0.64 solanaceae cestrum axillare 1.5 2 3.30 1rft = total relative frequency of each pollen type used for larval food in nests built throughout nesting period at ufba. 2rfm = monthly relative frequency of each pollen type used for larval food in nests built throughout nesting period at ufba. 3nn = number of nests containing each pollen type recorded throughout nesting period. table 3. relative frequencies of pollen types in the larval food from 7 nests of centris terminata smith, 1874 collected from campus of universidade federal dabahia (ufba) in urban fragment of atlantic forest in salvador, bahia, brazil. pollen types 1rft (%) 2nn monthly relative frequency of pollen types (3rfm) (%) jan/15 (n=8) mar/15 (n=5) malpighiaceae malpighia emarginata 16.85 8 4.54 38.02 stigmaphyllon cavernulosum 21.72 13 19.29 25.63 byrsonima sericea 47.45 11 61.37 23.80 fabaceae aeschynomene paucifolia 12.57 9 13.90 10.28 dioclea grandiflora 1.18 10 0.89 1.66 bignoniaceae amphilophium crucigerum 0.15 3 0.38 1rft = total relative frequency of each pollen type used for larval food in nests built throughout nesting period at pzbgv. 2rfm = monthly relative frequency of each pollen type used for larval food in nests built throughout nesting period at pzbgv. 3nn = number of nests containing each pollen type recorded throughout nesting period. table 4. relative frequencies of pollen types in larval food from 13 nests of centris terminata smith, 1874 collected from parque zoobotânico getúlio (pzbgv) in urban fragment of atlantic forest in salvador, bahia, brazil. r lima et al. – resource partitioning between oil-collecting bees298 and references therein; rabelo et al., 2015). fabaceae was the second most important botanical family found in the larval diet of c. analis and c. terminata. we observed a high frequency of occurrence of a. paucifolia in the nests of c. terminata established at ufba in february 2015. the species from genus aeschynomene seem to be an important source of pollen in the larval diet of the centris species, once the pollen of aeschynomene martii benth was found in highest frequencies in c. tarsata nests in the semiarid caatinga biome (cruz et al., 2015). on the other hand, considering the two areas studied herein, a. paucifolia was found at low frequencies in some nests of c. terminata and c. analis. this data suggest that a. paucifolia can be used as a source of nectar, as reported in some studies with c. tarsata and c. trigonoides from caatinga biome (dórea et al., 2009; dórea et al., 2010a; dórea et al., 2013). regarding the pollen types found at low frequencies in the larval food of both bee species, handroanthus chrysotrichus (bignoniaceae), amphilophium crucigerum (bignoniaceae) and dioclea grandiflora (fabaceae) can be considered sources of nectar, whereas cestrum axillare (solanaceae), due to its floral morphology (small, long, tubular flowers), may have been used as a source of pollen for both species of centris. on the other hand, the low frequency of occurrence per nest (<1%) of pollen from e. guineenses (arecaceae), serjania (sapindaceae), d. regia (fabaceae) and s. diversifolia (polygalaceae) makes it difficult to determine the type of resources collected by c. analis females. concerning the trophic niche of the studied species, the breadth of the niche of c. analis at pzbgv differed from that found at ufba while for c. terminata, on the contrary, the breadths of the niches were similar. as reported by rabelo et al. (2012), such results are directly related to the frequencies of the utilization of each food source by the species. niche overlap refers to the utilization of some of the same resource type by two or more species; thus, the greater the number of resources common to both species, the greater the overlap (abrams, 1980). some authors have emphasized that the niche overlap between bee species can be influenced by the availability temporal of resources (carvalho et al., 2013; santos et al., 2013), by the resource abundance (aguiar et al., 2013), and by the body size of the individuals exploiting the same resources (rabelo et al., 2015). in the present study, the low value of the niche overlap between c. analis and c. terminata at ufba can be explained to the high frequency of the use of a. paucifolia (fabaceae) by c. terminata in the nests established in february (2015). on the other hand, the high niche overlap observed at pzbgv between those species can be due to the body size, as reported by rabelo et al. (2015) for some oil-collecting species. centris analis and c. terminata collected resources in several plant species to provision their cells. similar behaviorwas reported for c. analis by quiroz-garcia et al. (2001), roubik & villanueva-gutiérrez (2009) and santos et al.(2013). studies on the floral resources used by bees in urban areas can contribute to the establishment of conservation measures and management plans for these important pollinators. moreover, urban environments are considered important refuges for bee fauna, especially those that nest in preexisting cavities. acknowledgments the authors are grateful to the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for providing a scholarship to the first and second authors; josafá santos and taniele santana of the laboratório de bionomia, biogeografia e sistemática de insetos (biosis ufba) for assistance in the data collection; dr. nádia roque for the information on the flora of the ufba and parque zoobotânico getúlio vargas. the authors also thank the anonymous reviewers that contributed to improving the manuscript. authors’ contributions r lima and ca garófalo conceived the study, analyzed the results and contributed to the writing of the manuscript. r lima and ct garcia performed the field collection of nests. mj ferreira-caliman, mc dórea and far santos conducted the palynological analysis, analyzed the results and contributed to the writing of the manuscript. ff oliveira identified the bees and contributed to the writing of the manuscript. references abrams, p.a. 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(2000). nest selection, usurpation, and a function for the nest entrance plug of centris bicornuta (hymenoptera: apidae). annals of the entomological society of america, 93: 254-260. doi:10.1603/0013-8746(2000)093%5b0 254:nsuaaf%5d2.0.co%3b2. vogel, s. (1974). ölblumen und ölsammelnde bienen. tropische und subtropische pflanzenwelt 7:285-547. zar, j.h. (1984). biostatistical analysis. prentice hall, new jersey, usa, 718 p. doi: 10.13102/sociobiology.v65i2.1272sociobiology 65(2): 330-332 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 fortress with sticky moats: the functional role of small particles around tetragonisca angustula latreille (apidae: hymenoptera) nest entrance species are usually defined by the physical attributes of their individuals, even though none of them is able to exist without the interactions they establish with other organisms and the environment they live in (queiroz et al., 2007). these relationships may enlarge the species range or increase its persistence probability over time, as it happens in mutualisms, or the opposite in antagonistic interactions (stanton-geddes & anderson, 2011). bees and flowers have a longstanding mutualistic relationship established over the evolutionary time; however, by storing pollen and honey provisions, bees create attractive scenarios to antagonist thieves. other bee species as well as other groups such as ants or vertebrates (roubik, 1989; breed et al., 2012) are among the bees antagonists. some bee species may use their poisoning sting to protect the stored resources and the brood (roubik, 1989). moreover, bees from the meliponini tribe do not have stings. therefore, they use other defensive strategies to protect themselves against intruders (rech et al., 2013 and references therein). abstract many bee species are able to defend themselves against pollen or honey thievery. we herein report the functional role of small sticky particles deposited by tetragonisca angustula latreille in its nest entrance external side. this strategy was very effective to prevent ants from invading the bees’ nest. we reported many dead ants attached to the nest entrance and different ant species easily immobilized after being moved onto the entrance tube containing sticky particles. this is the first description of the functional role played by the nest entrance sticky particles under natural conditions. sociobiology an international journal on social insects a alves1, sf sendoya2, ar rech3 article history edited by evandro nascimento silva, uefs, brazil received 12 december 2016 initial acceptance 26 january 2018 final acceptance 13 february 2018 publication date 09 july 2018 keywords defensive behaviour, stingless bees, ant, nest invasion. corresponding author andré r. rech, universidade federal dos vales do jequitinhonha e mucuri, campus jk rodovia mgt 367 km 583 nº 5000 alto da jacuba cep 39100-000 diamantina-mg, brasil. e-mail: andrerodrigorech@gmail.com ants are considered the greatest predators in the tropics (hölldobler & wilson, 1990) and a threat to bee keeping in this region (lehmberg et al., 2008; rech et al., 2013). some bee species are protected against ant predation by the chemical repellence on their bodies surface (lehmberg et al., 2008). moreover, it was already shown that, under lab conditions, trigona bees are able to immobilize attacking ants by gluing small sticky particles (supposedly resin) on them (roubik, 2006; grüteret al., 2011). the same small particles are found on the external side of stingless bees’ nest entrances. however, their functional role under natural conditions is still unknown (wittmann, 1985; roubik, 2006). “jatay” bees (tetragonisca angustula latreille, 1811) are known for showing efficient defensive behaviour against flying intruders (wittmann, 1985). there are soldiers among t. angustula that protect the colonies either by flying in front of the nest entrance or by controlling the entrance standing inside tube (kärcher & ratnieks, 2009; grüteret al., 2011). 1 sqn 308 bloco h ap. 406, cep 70747-080, brasília, distrito federal, brazil 2 departamento de ecologia, zoologia e genética, instituto de biologia, universidade federal de pelotas, rio grande do sul, brazil 3 curso de licenciatura em educação do campo (lec), universidade federal dos vales do jequitinhonha e mucuri, diamantina-mg, brazil short note mailto:andrerodrigorech@gmail.com sociobiology 65(2): 330-332 (june, 2018) 331 these soldier bees show particular behaviour, morphology and longevity, thus differing from the other workers (grüter et al., 2012). the present study increases the set of nest defence strategies known in t. angustula by describing, under natural conditions, the functional role played by the small sticky particles deposited on the external side of the nest entrance. our observations were performed at a private residential area in são bartolomeu, a newly urbanized area from brasília (brazilian capital). the local vegetation is a typical neotropical savannah (cerrado) from the central brazil with the predominance of the locally called cerradão physiognomy (15˚50’47.70” s; 47˚47’09.10w). the defensive strategy herein described was first observed on august 8th 2014 and recorded in pictures. the observed t. angustula colony was located inside a qualea grandiflora mart. (vochysiaceae) tree trunk, 20cm above the ground level (fig 1a). the first ants found stuck to the t. angustula nest entrance were cephalotes clypeatus (fabricius, 1804) dead workers covered by thin layers of the same sticky material used to build the nest entrance (fig 1b). fig 1. functional role of sticky particles deposited on the external side of tetragonisca angustula nest entrance. a. entrance tube as it was found by the authors in august 2014, check on the dead ants covered by thin layers of the same material used in the nest entrance, b. close view of cephalotes clypeatus stuck in the nest entrance found in the field, c. ants (c. clypeatus) being experimentally moved to the nest entrance tube for monitoring purpose, d. fighting behaviour between t. angustula and atta sexdens experimentally moved to nearby the nest entrance and the resulting dead ant with a bee attached to its antenna, e. non-fighting behaviour related to a large ant (odontomachus bauri) partially immobilized by the entrance tube sticky particles. a alves, sf sendoya, ar rech – the functional meaning of a new defensive strategy in bees332 two hypotheses were formulated to explain the ants’ death: 1. the bees have killed the ants while they were partially immobilized by the sticky particles, or 2. the ants slowly died due to complete immobilization. on march 5th 2015,aiming at investigating which of these hypothesis was the most plausible, we collected c. clypeatus (the most frequent species found attached to the nest entrance) healthy workers from the same tree the bee nest was located in and manually moved them to the nest entrance (fig 1c). we then monitored and recorded in video what happened next. subsequently, in order to check if the bee response was constant to any species of ant, we manually glued workers of odontomachus bauri emery, 1892 and atta sexdens (linnaeus, 1758) to the nest entrance observing and describing what happened. behaviour observation in the field revealed that bees completely ignored the living ants artificially stuck in the entrance tube. this behaviour differs from that of honeybees, which are able to recognize ants’ odour and promote a ritualized defence (spangler & taber, 1970). however, it was clear that the ants were completely immobilized by the sticky particles (fig 1d). a similar nest entrance of t. angustula with dead individuals of camponotus sp. (formicidae) was also seen in ribeirão preto são paulo (barbara rodrigues, phd student at usp personal communication), indicating that this behaviour may be more widespread than previously thought. moreover, we used odontomachus bauri emery, 1892 ant workers to check the bee strategy efficiency against larger ants and they were also completely immobilized by the sticky particles (fig 1e). similar trials using atta sexdens workers (linnaeus, 1758) showed that besides the fact that these ants were stuck in the nest entrances, they were actively attacked by bees (fig 1f). it suggests that the bees’ defensive behaviour may vary according to the ants’ identity or traits (e.g. size or chemical components of the ant’s cuticle) (spangler & taber, 1970). further research should be performed both on the chemical composition of the sticky particles (to clarify what determines, if there is any, toxic effect on ants) and on the variation in the entrances features according to local conditions (e.g. threat levels posed by local ants). the current study described the defensive role played by the sticky particles in the nest entrances of stingless bees, thus confirming the hypothesis assumed by previous studies. the use of sticky particles by tetragonisca angustula workers is an efficient strategy to avoid nest invasion by walking intruders and increase the array of behavioural defensive strategies known in this species and also in social bees. references breed, m.d., cook, c. & krasnec, m.o. 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(1990). the ants. cambridge: harvard university press,732 p roubik, d.w. (1989). ecology and natural history of tropical bees. new york:cambridge university press, 514 p spangler, h.g. & taber, s. (1970). defensive behavior of honeybees toward ants. psyche: a journal of entomology,1: 184-189. doi: 10.1155/1970/49131 grüter, c., kärcher, m.h. & ratnieks, f.l.w. (2011). the natural history of nest defence in a stingless bee, tetragonisca angustula (latreille) (hymenoptera: apidae), with two distinct types of entrance guards. neotropical entomology, 40: 55-61. doi: 10.1590/s1519-566x2011000100008 grüter, c., menezes, c., imperatriz-fonseca, v.l. & ratnieks, f.l.w. (2012). a morphologically specialized soldier caste improves colony defense in a neotropical eusocial bee. proceedings of the national academy of sciences, usa, 109: 1182-1186. doi: 10.1073/pnas.1113398109 kärcher, m.h. & ratnieks, f.l.w. (2009). standing and hovering guards of the stingless bee tetragonisca angustula complement each other in entrance guarding and intruder recognition. journal of apicultural research, 48: 209-214. doi: 10.3896/ibra.1.48.3.10 rech, a.r., schwade, m.a. & schwade, m.r. (2013). abelhassem-ferrão amazônicas defendem meliponários contra saques de outras abelhas. acta amazonica, 43: 389-394. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1532sociobiology 64(2): 155-158 (june, 2017) first record of an epizoic laboulbenia (fungi: laboulbeniales) on ants (hymenoptera: formicidae) in africa introduction laboulbeniales is an order of ascomycetous fungi consisting of over 2000 species that have obligate association with arthropods, mostly insects, especially coleoptera and diptera, and on a few ant species, mites, thrips and millipedes (tavares, 1985). those fungi show a rather high level of host specificity. their study remains in a challenging interface, between entomologists and mycologists and, consequently, clearly belong in the domain of a very few specialists in the world. ants are known to harbor six species of laboulbeniales: two species of the genus rickia cavara (on myrmica latreille or messor forel hosts), one dimorphomyces thaxter (on nylanderia emery) and three species of laboulbenia thaxter (on camponotus mayr, lasius fabricius, formica linnaeus, polyergus latreille, myrmecocystus wesmael, prenolepis mayr, eciton latreille). the global database of published antabstract the first ant-laboulbenial interaction in africa is recorded. the fungus laboulbenia camponoti batra was detected over the body of workers camponotus oasium forel collected in senegal. this is a huge range extension of its eurasian previously known distribution, to the afrotropical region. we present the updated global database for l. camponoti. sociobiology an international journal on social insects k gómez 1, x espadaler 2, s santamaria3 article history edited by gilberto m. m. santos, uefs, brazil received 02 march 2017 initial acceptance 02 may 2017 final acceptance 26 june 2017 publication date 21 september 2017 keywords camponotus oasium; host; laboulbenia camponoti; senegal. corresponding author xavier espadaler unitat d’ecologia i creaf universitat autònoma de barcelona cerdanyola del vallès barcelona 08193, spain. e-mail: xavierespadaler@gmail.com laboulbeniales interactions contains 125 instances, from 95 localities and 28 countries (unpub. data). in the splendid work on the ants of the congo basin (wheeler, 1922), bequaert writes about general ant-fungus relationships, and mentions laboulbeniales noting “... they are inconspicuous and, when examined in situ on the host insect, appear in general like minute, usually dark-colored or yellowish bristles or bushy hairs, projecting from its chitinous integument either singly or in pairs, more commonly scattered, but often densely crowded over certain areas on which they form a furry coating.”. the thallus attaches to the insect cuticle with a darkened foot. there are no penetrating hyphae or rhizoids into the ant body cavity in laboulbenia formicarum thaxter and l. camponoti batra, or rickia wasmannii cavara and r. lenoirii santamaria (tragust et al., 2016). therefore, the energy source(s) for growing and reproducing in ant-laboulbeniales is unknown. this is an intriguing and fundamental issue to unravel in the biology of laboulbeniales. 1 castelldefels, barcelona, spain 2 creaf universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain 3 unitat de botànica, departament de biologia animal, de biologia vegetal i d’ecologia, facultat de biociències, universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain research article ants k gómez, x espadaler, s santamaria – laboulbenia camponoti in senegal156 material and methods the camponotus sample was collected during a general ant sampling done close to the n-1 road in senegal (14º6.11’n 15º41.12’w). the habitat was an open savannah, mainly agricultural, with scattered acaciae trees. according to tappan et al. (2004), the zone belongs to the west central agricultural region close to the saloum agricultural region. several mature and immature thalli were scratched off the third leg of one camponotus worker. permanent slides were prepared following previously described methods (benjamin, 1971) and are kept in the bcb mycotheca of universitat autònoma de barcelona (bcb-slides). ant specimens with attached fungi are kept in the private collections of k.g. and x.e. photomicrographs were made with a jenoptik progres 10 plus digital camera on a leica dmr microscope equipped with differential interference contrast optics (dic). images were processed with photoshop cs5 software and dpx view pro for its included feature of extended focus function. macroscopic images were taken using a nikon coolpix s3600 and a nikon binocular. retouched with microsoft office picture manager. the map was done using carto builder. results camponotus ants were found inside an abandoned termite nest under an acacia tree. six workers were collected: three minor workers of camponotus oasium forel were detected with laboulbenia camponoti thalli (fig 1) over nearly all body parts (clypeus, eyes, scape, mesosoma, gaster and legs (fig 2). small black spots, the point of insertion of the thallus, contrasting with the yellow integument, were also abundant at the gula and coxae but no thalli were present or had developed, likely because of the frequent frictional forces those body parts bear. fig 1. laboulbenia camponoti batra. senegal: kaolack, road between birkelane and kaffrine. two mature thalli showing the black foot, the perithecium with spores, and the elongated appendages. scale: 50 μm. fig 2. different views of laboulbenia.camponoti batra on the legs of camponotus oasium forel. senegal: kaolack, road between birkelane and kaffrine. mature thalli show a darkened body; immature thalli are still hyaline and have an uninflated perithecium. a) mature, blackish thalli on the third leg tibia. b) immature specimens at the proximal end of second tibia. c) one fully mature and one nearly mature thallus on the third tibia. another two recently hatched workers had signs of early infestation, as immature thalli were developed only in the meso and/or metatibiae, but the insertion points were not so developed and the general aspect is that of long, twisted white setae. one recently hatched worker showed no signs of infestation at all. senegal: kaolack, road between birkelane and kaffrine. 14º6.11’n 15º41.12’w 55 m. 25/09/2016. (gómez, k. leg.). savannah, sandy terrain. abandoned termite nest sociobiology 64(2): 155-158 (june, 2017) 157 [kg03265b-1], [kg03265b-2]. the spot is 120 km distant from the sea and 2.7 km from a small river. mean annual temperature and precipitation at kaffrine (14 km from the spot) is 28.5 ºc and 704.5 mm. other species collected at the spot where brachyponera sennaarensis (mayr), tapinoma demissum bolton, tapinolepis simulans (santschi), messor galla (mayr), monomorium bicolor emery and trichomyrmex sp. discussion the discovery of l. formicarum on c. (tanaemyrmex) oasium is the first mention of any ant-laboulbenial in africa. at its description, this fungus was described from india, from the indomalayan region. later it was recorded from europe, from the palaearctic region. now it is recorded from the afrotropical region, separated by the sahara from the palaearctic. this is a nice example that poor research about the distribution of a species can be misleading about its area. senegal is the eighth country known for the fungus. other countries (discovery publication year) are: india (1963; original description), spain (1982), turkey (1983), bulgaria (2011), austria and romania (2014) and italy (2016). for an updated world database –21 world records, including four new ant-fungus records from spain– for l. camponoti, see table 1. all african camponotus, especially those belonging in the subgenus tanaemyrmex, are candidates to harbour l. camponoti. a rough appraisal of the number of tanaemyrmex species according to emery (1925) and wheeler (1922; as subgenus myrmoturba forel, 1912) is >130 in africa south of sahara. thus, there are many possible hosts to search for l. camponoti. other ant genera in the subfamilies formicinae, dorylinae, myrmicinae might be also infected with laboulbeniales. for a summary of worldwide knowledge of ant-laboulbenial interactions, see santamaria and espadaler (2015: table 2 and fig 2). the enormous range of the known localities for l. camponoti (some ten thousand km from delhi (india) to dakar (senegal) (fig 3) fits well the notion that, among fungi, those that are parasites on animals tend to have more wide-ranging geographical distribution than all other soil fungi functional groups (tedersoo et al., 2014). one interesting future aim would be to compare the genetics of the different populations from the various regions. the majority of data concerning the small group of ant-infesting laboulbeniales come from europe and northamerica. mesoand south america have four registers and asia has a few examples restrained to the delhi region (india). nothing is recorded from the australian region or from the enormous extensions between japan and the ural mountains, arabian peninsula and indonesia. the absence of data for antlaboulbeniales in the australian region and asia (santamaria & espadaler (2015: fig 2) calls for a geographically focused consideration. host country (localities) reference 1 camponotus sp. india (1) 1 2 c. pilicornis (roger) spain (1) 2 3 c. baldaccii emery turkey (1) 3 4 c. pilicornis (roger) spain (1) 4 5 c. sylvaticus (olivier) spain (1) 4 6 camponotus sp. india (1) 5 7-11 c. aethiops (latreille) bulgaria (5) 6 12 c. universitatis forel bulgaria (1) 6 13 c. sp. (as pilicornis) bulgaria (1) 6 14 c. aethiops (latreille) austria (1) 7 15 c. aethiops (latreille) rumania (1) 7 16 c. aethiops (latreille) italy (1) 8 17 c. pilicornis (roger) puebla de azaba, spaina this paper 18 c. pilicornis (roger) la selva del camp, spainb this paper 19 c. aethiops (latreille) la selva del camp, spainb this paper 20 c. sylvaticus (olivier) la selva del camp, spainb this paper 21 c. oasium forel senegal this paper reference: 1: batra (1963). 2: balazuc et al. 1983. 3: espadaler & lodos (1983). 4: espadaler & blasco (1991). 5: kaur & mukerji (1995). 6: lapevagjonova & santamaria (2011). 7: báthori et al. (2014). 8: gómez et al. (2016). a. one worker. 2006. l. gonzález leg. b. 13 workers c. aethiops, 2 workers c. pilicornis and 11 workers c. sylvaticus collected in pitfall traps set 1-3 july 2011 and 6-8 september 2012 in organic citrus grove. table 1. world records of laboulbenia camponoti batra. updated to february, 2017. all specific host identifications belong in the subgenus tanaemyrmex ashmead. we request the attention of entomologists dealing with african fauna, towards those small and interesting epizoic fungi. our finding suggests that l. camponoti may be more widespread geographically than previously thought. one positive circumstance for the study of laboulbeniales is the possibility of its detection either in alcohol-preserved or in dry-preserved specimens, also from rather old collections, rendering a new value to museum collections (suarez & tsutsui, 2004). we cannot but expect a growth of information about the antlaboulbeniales interaction in africa, if proper attention is dedicated to those epizoic fungi on ants. k gómez, x espadaler, s santamaria – laboulbenia camponoti in senegal158 acknowledgments to an unknown reviewer for his/her corrections and useful comments and insights, some of which we have included in the revised version. references balazuc, j., girbal, j. & espadaler, x. (1982). laboulbenials (ascomicets) ibèriques. collectanea botanica, 13: 403-421. báthori, f., pflieger, w.p. & tartally, a. (2014). first records of the myrmecophilous fungus laboulbenia camponoti batra (ascomycetes, laboulbeniales) from the carpathian basin. sociobiology, 61: 338-340. doi: 10.13102/sociobiology. v61i3. 338-340 benjamin r.k. (1971). introduction and supplement to roland thaxter’s contribution towards a monograph of the laboulbeniaceae. bibliotheca mycologica, 30: 1-155. carto. https://carto.com/ accessed january 2017. emery, c. (1925). hymenoptera. fam. formicidae. subfam. formicinae. genera insectorum, 183: 1-302. espadaler, x. & blasco, j. (1991). laboulbenia camponoti batra, 1963 (fungi, ascomycotina) en aragón. lucas mallada, 2: 13-23. espadaler, x. & lodos, n. (1983). camponotus baldaccii emery (hymenoptera, formicidae) parasitized by laboulbenia camponoti batra (ascomycetes) in turkey. turkish journal of plant protection, 7: 217-219. gómez, k., espadaler, x., santamaria, s. (2016). ant-fungus interactions: laboulbenia camponoti batra in italy and a new host for l. formicarum thaxter (fungi: ascomycota, laboulbeniales). sociobiology, 63: 950-955. doi: 10.13102/ sociobiology.v63i3.1057 kaur, s. & mukerji, k.g. (1995). studies on indian laboulbeniales iv: three species of laboulbenia. mycoscience, 36: 311-314. doi: 10.1007/bf02268606 lapeva-gjonova, a. & santamaria, s. (2011). first record of laboulbeniales (ascomycota) on ants (hymenoptera: formicidae) in bulgaria. zoonotes, 22: 1-6. santamaria, s. & espadaler, x. (2015). rickia lenoirii, a new ectoparasitic species, with comments on world laboulbeniales associated with ants. mycoscience, 56: 224-229. doi: 10.1016/j. myc.2014.06.006 suarez, a.v. & tsutsui, n.d. (2004). the value of museum collections for research and society. bioscience, 54: 66-74. doi: 1/0006-3568(2004)054[0066:tvomcf]2.0.co;2 tappan, g.g., sall, m., wood, e. c. & cushing, m. (2004). ecoregions and land cover trends in senegal. journal of arid environments, 59: 427-462. tavares, i.i. (1985). laboulbeniales (fungi, ascomycetes). mycologia memoir, 9: 1-627. tedersoo, l. et al. (57 authors). global diversity and geography of soil fungi. science, 346 (6213): 1256688. doi: 10.1126/ science.1256688 tragust, s., tartally, a., espadaler, x. & billen, j. (2016). histopathology of laboulbeniales (ascomycota: laboulbeniales): ectoparasitic fungi on ants (hymenoptera: formicidae). myrmecological news, 23: 81-89. wheeler, w.m. (1922). ants of the american museum congo expedition. a contribution to the myrmecology of africa. viii. a synonymic list of the ants of the ethiopian region. bulletin of the american museum of natural history, 45: 711-1004. fig 3. known distribution for laboulbenia camponoti batra, up to february 2017 (blue dots, known distribution; orange dot, new record). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1188sociobiology 64(2): 133-137 (june, 2017) urn:lsid:zoobank.org:pub:5cae8d6c-a1d8-4f61-b1ce-351a7d80f774 discovery of remarkable new ant species of the genus pseudolasius emery (hymenoptera: formicidae) from western ghats of india introduction the genus pseudolasius emery, 1887 is distributed in australasia, indomalayan, palearctic regions and it is currently represented by 49 species and 15 subspecies (bolton, 2017). from india the genus is represented by six species (bharti et al., 2016). global taxonomic revision still eludes the genus, however recent significant contributions to the genus include those of wu and wang (1995), xu (1997), zhou (2001), lapolla (2004), lapolla et al. (2010), bharti et al. (2012), wachkoo and bharti (2014). here we present description of the new species pseudolasius zamrood sp. n., collected from thanikkudy region of periyar tiger reserve. this also confirms the first report of the genus from south india. the species appears to be distinct among regional species of the genus, with its extremely reduced eyes size both in major and minor workers. this character state is also exhibited by some pseudolasius species from afrotropical and indo-australian regions. these species represent rare hypogaeic evolutionary lineages and are quite different abstract pseudolasius zamrood sp. n., is described based upon the worker and the sexual caste. the new species marks the first record of the genus from south india and represents only the seventh species of the genus to be reported from india. the species appears distinct with its extremely reduced eyes. the new species is closest to p. typhlos wheeler, 1912 from philippines. however the new species has truncated anterior clypeal margin at the middle lobe, scapes relatively short and propodeum has relatively narrow dorsum in lateral view. sociobiology an international journal on social insects sa akbar, h bharti, aa wachkoo article history edited by jacques delabie, uesc, brazil received 02 september 2016 initial acceptance 01 may 2017 final acceptance 07 may 2017 publication date 21 september 2017 keywords ants, pseudolasius, india, western ghats, south india. corresponding author shahid ali akbar 117 pomposh colony natipora, srinagar, jammu and kashmir india, 190015. e-mail: kingakbarali@gmail.com from the more common, larger, presumably epigaeic species (lapolla, 2004). the species is the first of its kind among the indian species of the genus and easily diagnosed from the other known similar hypogaeic species by its truncate anterior clypeal margin at the middle lobe, relatively short scapes and propodeum with relatively narrow dorsum in lateral view. materials and methods the specimens were collected by hand picking method. taxonomic analysis was conducted using nikon smz 1500 stereo zoom microscope. for digital images, an mp evolution digital camera was used on the same microscope with automontage (syncroscopy, division of synoptics, ltd.) software. later, the images were cleaned as per requirement with adobe photoshop cs6. holotype and 13 paratypes of the new species have been deposited in puac, punjabi university ant collection, patiala. four paratypes will be deposited in bmnh, natural history museum, london, united kingdom. morphological terminology for measurements (in millimeters) department of zoology & environmental sciences, punjabi university, patiala, punjab, india research article ants sa akbar, h bharti, aa wachkoo – discovery of remarkable new ant species from india134 and indices are as follows: tl: total out stretched length of the specimen, measured along lateral view, from mandibular apex to the tip of the abdomen. hl: maximum length of head in full-face view, measured in straight line from the anteriormost point of the median clypeal margin to a line drawn across the posterior margin from its highest points (to accommodate the concave posterior margin). hw: maximum width of head in full-face view (excluding the portion of eyes that extends past the lateral margins of the head). sl: maximum length of the scape excluding the basal neck and condyle. pw: maximum width of the pronotum in dorsal view. wl: weber’s length measured from the anterior surface of the pronotum proper (excluding the collar) to the posteriormost point of the propodeal lobes. gl: maximum length of the gaster in lateral view from the anteriormost point of first gastral segment to the posteriormost point of the last segment. prfl: maximum length of the profemur from its margin with the trochanter to its margin with the tibia. prfw: maximum width of the profemur. ci: cephalic index: hw/hl ×100. si: scape index: sl/hw×100. results description of new species pseudolasius zamrood sp. n. (figures 1-10) urn:lsid:zoobank.org:act:2a6de69a-071f-45aa-8750-6e6e473b51a5 type material holotype (major worker): india: kerala, periyar tiger reserve, thanikkudy, 9o.30`n, 77o.16`e, 1003m, 15 oct 2011, hand picking (coll. shahid a. akbar). paratypes: 12 workers (5 major and 7 minor workers), 1 gyne and 4 males (one with damaged abdomen), with same data as holotype. holotype and 13 paratypes deposited in puac, 4 paratypes will be deposited in bmnh. diagnosis. workers (majors and minors) body with profuse pilosity; numerous erect hairs on head, scape, mesosoma, and legs, with appressed pubescence underneath; extremely reduced eyes; short scapes; anterior clypeal margin truncate medially; mandibles with four distinct teeth; gyne with subquadrate head having broadly emarginate posterior margin; males with oval head; parameres paddle shaped with rounded tips; long and tubular cuspis; weakly anvil shaped digitus. worker measurements (holotype in brackets): hl (0.81) 0.71-0.83; hw (0.77) 0.69-0.77; sl (0.50) 0.50-0.55; pw (0.51) 0.42-0.55; prfl (0.67) 0.59-0.71; prfw (0.17) 0.14-0.20; wl (1.25) 0.77-1.31; gl (1.13) 1.01-2.04; tl (3.18) 2.49-4.18. indices: ci (95) 92.77-97.18; si (64) 71.4272.46 (n=10). roughly rectangular head with strongly emarginate posterior margin in the middle; rounded postero-lateral corners; lateral sides sub-parallel and gently converging anteriorly; frontal carinae, nearly parallel with lateral sides of head; figures 1-3. pseudolasius zamrood sp. n.: holotype worker (major): 1. head in full-face view; 2. body in profile view; 3. body in dorsal view. sociobiology 64(2): 133-137 (june, 2017) 135 almost rectangular clypeus, protruding in the middle; bluntly toothed anterolateral corner; antennae 12-segmented; short scape, reaching 3/4th of occipital margin of head; mandibles with four distinct teeth, extremely reduced eyes, rudimentary represented by small pigmented dots both in major and in minor workers. in lateral view, relatively flat promesonotum; strongly developed metanotal groove; distinct metanotal area, well developed and depressed; slightly elevated propodeum, convex and narrow dorsal face, with sides diverging basally; declivity steep, about two times as long as dorsal face; spiracle rounded propodeal; low scale like petiole, with longer posterior face than anterior face, transverse dorsum, ventral projection prominent and inclined forward; first gastral segment with transverse anterior face. body covered with abundant pilosity throughout; head and gaster with abundant appressed pubescence, mesosomal dorsum with several, erect hairs sparced throughout; mandible with 4-5 short curved setae near masticatory border; anterior clypeal margin with 3-5 erect setae, anteriorly directed; fringe of short 1-2 setae also present towards mandibular base; gaster with erect hairs throughout the surface. body with a prominent yellow opaque cuticle with micro-reticulate, superficial sculpture. body uniformly dark yellowish brown in colour. minor workers similar to major workers with the following differences; subquadrate head with posterior margin having less distinct strongly emargination in the middle; longer antennal scapes reaching 4/5th of occipital margin of the head; in lateral view, convex promesonotum. although entire next series of the new species was collected under the stone, without any further specimens available at that time, there is still possibility that maximum sized major workers may still be discovered. the larger available specimens collected are here treated as major workers. the distinction of castes among worker specimens in the genus is difficult to make with blending of the major and minor castes (lapolla, 2004). gyne measurements: hl 1.36, hw 1.24, sl 0.77, el 0.22, pw 1.37, prfl 1.07, prfw 0.20, wl 1.88. indices: ci 91; si 62 (n=1). gyne similar to worker with usual differences for the caste, including three ocelli, complete thoracic structure and wings; sub-quadrate head with broadly emarginate posterior margin; anterior clypeal margin transverse; scape surpass posterior occipital margin by about 1/5th its length; indistinct propodeum; feebler body sculpture than in worker; darker colouration; pubescence abundant on all the body; gyne damaged without abdomen. male measurements: hl 0.58-0.61, hw 0.55-0.58, sl 0.44-0.46, el 0.19, pnw 0.63-0.65, pfl 0.58-0.61, pfw 0.13-0.15, gl 1.21-125, wl 1.01-1.20, tl 2.08. indices: ci 98.21-100; si 79.31-80.00 (n=3). oval head, as long as wide excluding large compound eyes; sub-globulose eyes, bulging, projecting well beyond figures 4–6. pseudolasius zamrood sp. n.: minor worker: 4. head in full-face view; 5. body in profile view; 6. body in dorsal view. head outline in full-face view; three prominent ocelli present; transverse anterior clypeal margin; antennae 13-segmented, filiform; long scape, surpass posterior occipital margin by about three-tenths their length; slender mandible; reduced dentition, with prominent apical and apico-basal teeth and 1-2 denticles in-between. sa akbar, h bharti, aa wachkoo – discovery of remarkable new ant species from india136 broad mesosoma; in lateral view, strongly convex mesoscutum while the mesoscutellum is weakly convex; distinct propodeum; petiole as in worker; elongate gaster; parameres paddle-shaped with rounded tips, turning slightly inwards toward midline of body posteriorly; long setae extending off parameres; long and tubular cuspis, bent toward digitus; weakly anvil-shaped digitus; curved outward and covered with short peg-like teeth; penis valves projecting forward. body mostly smooth and shiny; erect setae shorter and sparse. colour light yellow, head mostly brownish. figures 7–8. pseudolasius zamrood sp. n. (gyne): 7. head in fullface view; 8. body in profile. ecology the specimens of this rare species were handpicked from thanikkudy region of periyar tiger reserve; a primary, undisturbed tropical moist evergreen forest. the specimens were collected by dislodging a big stone near a river side. the area is situated at 1003 meters elevation. it is a shady place with little sunlight penetration. etymology the species epithet is arabic for ‘precious stone’ in reference to habitat of the new species. remarks the genus pseudolasius is represented by 22 species from south and east asia. these include; (p. bidenticlypeus xu, 1997; p. binghami emery, 1911; p. butteli forel, 1913; p. cibdelus wu & wang, 1992; p. diversus wachkoo and bharti, 2014; p. emeryi forel, 1911; p. familiaris (smith, 1860); p. hummeli stitz, 1934; p. isabellae forel, 1908; p. lasioides wheeler, 1927; p. longiscapus wang & zhao, 2009; p. machhediensis bharti, gul & sharma, 2012; p. martini forel, 1911; p. polymorphicus wachkoo and bharti, 2014; p. pygmaeus forel, 1913; p. risii forel, 1894; p. salvazai santschi, 1920; p. sauteri forel, 1913; p. silvestrii wheeler, 1927; p. similus zhou, 2001; p. streesemanni viehmeyer, 1914; p. typhlops wheeler, 1935. from india the genus is represented by six species. these include; p. binghami; p. diversus; p. emeryi; p. familiaris; p. machhediensis and p. polymorphicus). among the known species of the genus from south and east asia pseudolasius zamrood n. sp. is closest to p. typhlos wheeler, 1912 from philippines, both have strongly reduced eyes. however, the new species has truncated anterior clypeal margin at the middle lobe, relatively short scapes just reaching to 3/4th of occipital margin of head, lighter body coloration and propodeum has relatively narrow dorsum in lateral view. key to species of genus pseudolasius of india based on major worker caste 1 eyes reduced, rudimentary represented by small dots without any distinct ommatidia..............................p. zamrood n. sp. eyes distinct consisting of prominent ommatidia...................2 2 cephalic dorsum with an inverted “y” shaped brown spot ... ....................................................................… p. emeryi forel cephalic dorsum without an inverted “y” shaped brown spot.........................................................................................3 3 antennal segments 3-9 longer than wide; frontal carinae parallel…..............................................……………………..4 antennal segments 3-9 wider than long; frontal carinae divergent posteriorly…...p. polymorphicus wachkoo and bharti sociobiology 64(2): 133-137 (june, 2017) 137 references bharti, h., guénard, b., bharti, m. & economo e.p. (2016). an updated checklist of the ants of india with their specific distributions in indian states (hymenoptera: formicidae). zookeys, 551: 1-83. doi: 10.3897/zookeys.551.6767. bharti, h., gul, i. & sharma, y.p. (2012). pseudolasius machhediensis, a new ant species from indian himalaya (hymenoptera: formicidae). sociobiology, 59(3): 805-813. doi: 10.13102/sociobiology.v59i3.548. bolton, b. (2017). an online catalog of the ants of the world. available from: http://antcat.org (accessed on 10-07-2017). lapolla, j.s. (2004). taxonomic review of the ant genus pseudolasius in the afrotropical region. journal of the new york entomological society, 112: 97-105. doi: 10.1664/0028 7199(2004)112[0097:trotag]2.0.co;2. lapolla, j.s., brady, s.g. & shattuck, s.o. (2010). phylogeny and taxonomy of the prenolepis genus-group of ants. systematic entomology, 35: 118-131. doi: 10.1111/j.13653113.2009.00492.x. wachkoo, a.a. & bharti, h. (2014). two new species of pseudolasius (hymenoptera: formicidae) from india. sociobiology, 61: 274-280. doi: 10.13102/sociobiology. v61i3.274-280. wheeler, w.m. (1935). new ants from the philippines. psyche (cambridge), 42:38-52. wu, j. & wang, c. (1995). the ants of china. china forestry publishing house, beijing, 214 p. xu, z. (1997). a taxonomic study of the ant genus pseudolasius emery in china. zoological research, 18: 1-6. zhou, s.y. (2001). ants of guangxi. guangxi normal university press, guilin, china, 255 p. figures 9-10. pseudolasius zamrood sp. n. (male): 9. head in full-face view; 10. body in profile. 4 scapes easily surpass the posterior margin of head ............5 scapes never surpass the posterior margin of head............... .......................................... p. diversus wachkoo and bharti 5 posterior margin of head strongly emarginate in the middle; mandibles with basal two teeth..............................…….……6 posterior margin of head weekly emarginate in the middle; mandibles without basal two teeth combined….p. binghami emery 6 mandibles armed with 8 teeth........ p. familiaris (smith, f.) mandibles armed with 6 teeth................................................ ...................p. machhediensis bharti, gul & sharma, 2012 acknowledgements we thank prof. zhenghui xu (college of forestry, southwest forestry university, yunnan province, china) for comparing and confirming the identity of new species. we also thank anonymous reviewers for helpful comments and suggestions about the manuscript. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1364sociobiology 64(2): 217-224 (june, 2017) social wasp guild (hymenoptera: vespidae) visiting flowers in two of the phytophysiognomic formations: riparian forest and campos rupestres introduction social wasps belong to the order hymenoptera and the family vespidae, which is divided into six subfamilies. polistinae contains 26 genera (carpenter, 2004) and is the only eusocial subfamily, with occurrence throughout brazil (carpenter, 1993). social wasp surveys have been carried out in riparian forest (souza et al., 2010; pereira & aantonialli-junior, 2011; locher et al., 2014) and campos rupestres (silva-pereira & santos, 2006). however, studies that focus on the use of floral resources by wasps in the neotropical region are scarce, particularly in these habitats. social wasps constitute part of the floral visitor guild, and overlap with the bees in the exploitation of floral resources; they are thus a representative of the foragers (heithaus, 1977 a, b; santos et al., 2010; melo abstract social wasps are part of the floral visitor guild. in this study, we investigated the distribution of flower-visiting wasp species in two phytophysiognomies in the state park of ibitipoca, minas gerais, brazil. we inspected flowering plants with visiting wasps along a 1 km transect in riparian forest and another 1 km transect in campos rupestres over the course of one year, for a total sampling effort of 240 hours. we found a total of 103 individuals with 15 species distributed among 7 genera, the most common belonging to the erythroxylaceae (n = 10) and asteraceae (n = 10) families. asteraceae had the highest abundance (n = 55). the diversity and evenness of these insects was higher in riparian forest (h ‘= 0.78 and j’ = 0.75) than in campos rupestres (h ‘= 0.30, j’ = 0.39). all species visited flowers of 19 plant species in riparian forest, and eight wasp species visited 11 plant species in campos rupestres. these polistinae acted as regular floral visitors, thus, conservation programs in these areas may be relevant for the maintenance of social wasp diversity. sociobiology an international journal on social insects ¹ma clemente, ³nr campos, ¹km vieira, ² k del-claro, ¹f prezoto article history edited by gilberto m. m. santos, uefs, brazil received 17 january 2017 initial acceptance 02 march 2017 final acceptance 02 march 2017 publication date 21 september 2017 keywords trophic niche; pollinators; richness; abundance. corresponding author: mateus aparecido clemete universidade estadual paulista instituto de biociências departamento de zoologia avenida 24 a, nº 1515 13506-900, rio claro -sp, brasil e-mail: mateus1981@gmail.com et al., 2011). male wasps, principally social wasps, are regular floral visitors that consume nectar and pollen, in addition to storing nectar for colonies (gadagkar, 1991). riparian forest and campos rupestres are vegetation formations of extreme importance for conservation. the riparian forest study site is considered a permanent preservation area (conselho nacional do meio ambiente – conama, 2002) for protecting water resources and maintaining their quality in equilibrium with local flora and fauna (lima & leopold, 1999). the campos rupestres site is protected due to high species richness (with many in the process of extinction) and endemism (giulietti et al., 1997; menezes & giulietti, 2000). an accurate assessment of social wasp species acting as floral visitors and possible pollinators is fundamental for our knowledge of plant reproductive cycles in the study areas, and has implications for conservation of both plants and wasps. 1 programa de pós-graduacão em ciências biológicas, universidade federal de juiz de fora, minas gerais, brazil 2 laboratório de ecologia comportamental e de interações, instituto de biologia, universidade federal de uberlândia, minas gerais, brazil 3 departamento de biodiversidade, evolução e meio ambiente instituto de ciencias exatas e biologia ufop (debio-iceb_ufop), mg, brazil research article wasps ma clemente, nr campos, km vieira, k del-claro, f prezoto – social wasp guild visting flowers218 in this study we estimated the richness, diversity, abundance, niche amplitude, and evenness of social wasp floral visitors in riparian forest and campos rupestres in ibitipoca state park, minas gerais, brazil. material and methods the state park of ibitipoca has an area of 1,923 hectares and is located in the serra de ibitipoca in southeastern minas gerais, brazil. the climate is characterized as cwb according to the köppen classification (humid mesothermic with dry winters and mild summers). the mean annual precipitation is approximately 1,532 mm and the mean annual temperature is 18.9 °c (cetec, 1983). social wasp collections were carried out in two of the phytophysiognomic formations present in ibitipoca state park: riparian forest and campos rupestres (sand land). riparian forestthe phytophysiognomic profile of this area is transition between cerrado de altitude and ombrophilic forest, which consists of a vegetational gradient that shifts from arbustive-arboreal physiognomy to predominantly arboreal (rodela, 1998). the main plant families of the ibitipoca state park are orchdaceae, myrsinaceae, melastomataceae, bromeliaceae, rubiaceae, labiatae, piperacea, araceae, passifloracea, gesneriaceae, polypodiaceae, compositeae and erythroxylaceae (rodela, 1998). campos rupestresthese areas present a xeromorphic physiognomy composed of a great diversity of herbaceous species, primarily represented by the families orchidaceae, poaceae, asteraceae and bromeliaceae, and shrub species, represented mainly by the velloziaceae, asteraceae, melastomataceae and asclepiadaceae, distributed across quartzitic outcrops (rodela, 1998). the study was carried out from november 2007 to october 2008, with one collection day per month per site taking place from 07:00 am to 05:00 pm. total sampling effort was 240 hours. one transect of approximately one kilometer was established in each area, along which two observers surveyed plants on collection days. when wasps were observed foraging in the flowers, observers collected them using an entomological net and remained near the plant for ten minutes in case of arrival of new individuals (aguiar, 2003; lorenzon et al., 2003). collected specimens were fixed in 70% alcohol and transported to the laboratório de ecologia comportamental (labec) at the universidade federal de juiz de fora in minas gerais, brazil. specimens were identified using taxonomic keys for genus and species (carpenter, 2004; cooper, 1997; carpenter & marques, 2001; pickett & wenzel, 2007). the plants visited by the wasps were described in the field according to floral characteristics, such as type of flower, color, and size. date and collection location were recorded. exsiccates from plant species visited by wasps were identified using keys for family and genus, and reviewed by professor fátima regina gonçalves salimena. we prepared a species list in alphabetical order of families and genera based on the angiosperm phylogeny group iii system (apg, 2009). species nomenclature confirmed using the brazilian plant species list (jardim botânico do rio de janeiro, 2012), the international plant names index sites (inpi, 2016), and electronic databases of the missouri botanical garden (mobot, 2016), the latter being also used to confirm nomenclature of exotic species. the diversity and evenness of visiting social wasps in the hot-humid and cold-dry periods were calculated using the shannon-wiener index (h’), (shannon, 1948) in the past program (hammer et al., 2001). shannon-wiener index (h’) and evenness values were compared using a mann-whitney in bioestat 4.0 (freeware). niche amplitude was calculated using the shannon-wiener index (h’) with the following formula: h’ = -σ pk x ln where pk is the proportion of individuals collected in plant k, and ln is the logarithm of the pk value. plant species visitation evenness for each wasp species was calculated using the following formula: j’ = h’ / h’max where h’max is the natural logarithm of the total number of plant species visited by wasp species. the values found for the shannon-wiener index (h’) and evenness were compared using a mann-whitney test in bioestat 4.0 (freeware). results and discussion a total of 103 individuals were collected, including 15 species distributed among seven genera. wasps visited 27 plant species distributed among 14 families. the most important plant families for wasps were erythroxylaceae (n = 10), asteraceae (n = 10) and rubiaceae (n = 4), malastomataceae (n = 3), and myrtaceae (n = 3). the highest abundance of visiting social wasps was observed in asteraceae with 55 individuals out 103 visiting this family, followed by erythroxylaceae (n = 14), rubiaceae (n = 10), melastomataceae (n = 5), cunoniaceae, myrtaceae (n = 4), apocynaceae (n = 3) and velloziaceae (n = 2). the families with only one visitor were orquidaceae, lythraceae, fabaceae, theaceae, poaceae, and ochnaceae, accounting for 55% of the foraging species during the entire collection period (tables 1 and 2). the diversity and evenness values for social wasp visits to flowers were higher in riparian forest (h’ = 0.78, j’ = 0.75) than in campos rupestres (h’ = 0.30, j’ = 0.39), however the shannon-wiener indices for the two sites did not significantly differ (z (u) = 1.84 (p) = 0.064). there were 19 plant species visited by wasps in riparian forest, and all wasp visitor species were observed visiting all 19 plant species. the most abundant wasp species were mischocyttarus confusus zikán 1935 (n = 16), polybia occidentalis (oliver) (n = 14) sociobiology 64(2): 217-224 (june, 2017) 219 and mischocyttarus drewseni saussure 1857 (n = 11), which were found foraging in eight, five, and seven plant species, respectively (table 1). in the campos rupestres site eight social wasp species visited 11 plant species, with p. occidentalis (n = 7) and m. drewseni (n = 6) having highest abundance and greatest diversity in resource use (visiting three and five plant species, respectively) (table 2). the diversity and evenness were higher in the hot-humid period (october-march) in riparian forest (h’ = 0.83, j’ = 0.73) and campos rupestres (h’ = 0.73, j’ = 0, 62) than in the cold-dry period (april-september) (h’= 0.31 and h’ = 0.21, respectively), however shannon-weiner index values for hothumid versus cold-dry seasons did not differ in riparian forest (z (u) = 0.2402 (p) = 0.8102) or campos rupestres (z (u) = 0.080 (p) = 0.936). the amplitude of the trophic niche (h’) varied from 0.63 to 2.34 (table 3), with m. drewseni and m. confusus having highest values (h’ = 2.34 and h’ = 1, respectively). plant family/species social wasp species total amu apa ble mco mdr pbi pci pfe pac pfa pig ppa pse poc psy apocynaceae mandevilla sellowii 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 minaria acerosa 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 2 asteraceae inulopsis scaposa 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 mikania acuminata 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 l. linearifolius* 0 0 0 0 0 2 0 0 0 0 1 0 0 0 0 3 baccharis crispa 0 0 0 1 4 0 0 0 1 0 0 1 0 3 5 15 senecio brasiliensis 0 0 0 6 2 0 0 0 0 0 0 6 0 6 0 20 baccharis rufidula 1 0 0 1 1 0 0 0 0 0 0 3 0 1 0 7 cunoniaceae w. paulliniifolia* 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 4 erythroxylaceae e. gonucladum* 0 0 0 1 1 1 0 2 1 1 2 1 1 3 0 14 fabaceae p. mediterranea* 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 melastomataceae trembleya, parviflora 0 2 0 0 0 1 1 0 0 0 0 0 0 0 0 4 myrtaceae eugenia acutata 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 orquidaceae p. pachysepala* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 poaceae tristachya chrysothrix 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 rubiaceae posoqueria latifolia 0 0 0 3 0 0 0 0 0 0 0 0 0 0 1 4 theaceae laplacea fructicosa 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 velloziaceae barbacenia flava 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 vellozia albiflora 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 abundance 1 2 1 16 11 4 1 2 2 1 3 16 2 14 7 83 abbreviations used for social wasp species. amu= agelaia multipicta, apa= apoica pallens, ble= brachgastra lecheguana mco=myschocyttarus confusos, mdr= mischocyttarus drewseni, pbi= polistes billardieri pci= polistes cinerascens, pfe= polistes ferreri, pac= polistes actaeon, pfa= polybia fastidiosuscula, pig= polybia ignobilis, ppa= polybia paulista, pse= polybia sericea, poc= polybia occidentalis, psy= protonectarina sylveirae; *lessingianthus linearifolius; *weinmannia paulliniifolia; *erythroxylum gonucladum; *periantra mediterranea; *prosthechea pachysepala. table 1: numbers of social wasps collected while visiting plant species at the ibitipoca state park in riparian forest phytophysiognomy at the ibitipoca state park, minas gerais, brazil. collections took place from november 2007 to october 2008. ma clemente, nr campos, km vieira, k del-claro, f prezoto – social wasp guild visting flowers220 polistes ferreri, apoica pallens (fabricius, 1804), and polybia ignobilis had the lowest value (h’ = 0.63). m. drewseni visited the greatest number of plant species (n = 12), but did not concentrate foraging on any particular species. polybia occidentalis (h’ = 1.78) also visited a significant number of plant species (n = 7) and had the highest abundance (n = 21) during the collection period. we note that half of all plant species sampled in this study (n = 13) received a low number of individual visitors (from one to three). nine wasp species (60%) had low abundance in flowers, with only one to three individuals found in each study site. the evenness of wasp visits ranged from 0.77 to 0.96, with the lowest value found for protonectarina sylveirae saussure, 1854; 62.5% of all floral visits by this species were to baccharia crispa (asteraceae). the higher species richness of social wasps visiting asteraceae and erythroxylaceae is partially explained by the fact that these botanical families are, according to rodela (1998), widely distributed in riparian forests and campos rupestres at the ibitipoca state park. wasps display opportunistic behavior, foraging in the most abundant resources to meet population resource demand (raveret-richter, 2000). further, asteraceae have a large number of small compressed flowers, called “brushlike” flowers, which provides easy access to floral resources (proctor & lack, 1996; cerana, 2004). these flowers produce a high abundance of nectar which is located only a few millimeters deep, and present shiny, yellow and white flowers that tend to attract insects (faegri & pijl, 1979). granja and barros (1998) in a study in open cerrado at the reserva ecológica da fazenda experimental of the university of brasilia, found 14 species of wasps using erythroxylaceae floral resources. the authors, as well as amaral jr. (1980), suggest that some polybia and polistes are effective pollinators of erythroxylaceae due to their efficiency in contacting the stigmas. in the present study, polistes billardieri fabricius, 1804, polybia ignobilis (haliday, 1836), and polybia sericea (oliver, 1791) were found in erythroxylum gonucladum, the only representative of the family erythroxylaceae in either study area. niche amplitude values for p. ferreri, a. pallens (fabricius) and p. ignobilis were low, indicating a narrow niche. this is reflected in the small number of floral resources used by these species and the small number of individuals. variation in niche amplitude values among social wasps may be associated with factors that influence foraging activity (e.g., large differences in biomass), where greater values may indicate higher foraging capacity (santos et al., 1998). plant family/species social wasp species total apa mco mdr pbi pfe ppa poc psy asteraceae chromolaena decumbens 0 0 0 1 0 0 0 0 1 lessingianthus linearifolius 0 0 1 0 0 0 0 0 1 mikania microdonta 0 0 0 0 0 0 0 1 1 baccharis crispa 0 0 0 0 0 0 1 0 1 eremanthus glomerulatus 0 0 0 0 1 0 2 0 3 lythraceae cuphea carthagenensis 0 0 1 0 0 0 0 0 1 melastomataceae tibouchina hieracioides 0 0 1 0 0 0 0 0 1 myrtaceae myrcia eriocalyx 0 1 2 0 0 0 0 0 3 ochnaceae ouratea semiserrata 1 0 0 0 0 0 0 0 1 orchidaceae prosthechea pachysepala 0 0 1 0 0 0 0 0 1 rubiaceae borreria capitata 0 0 0 0 0 2 4 0 6 abundance 1 1 6 1 1 2 7 1 20 abbreviations used for social wasp species. amu= agelaia multipicta, apa= apoica pallens, ble= brachigastra lecheguana mco=myschocyttarus confusos, mdr= mischocyttarus drewseni, pbi= polistes billardieri pci= polistes cinerascens, pfe= polistes ferreri, pac= polistes actaeon, pfa= polybia fastidiosuscula, pig= polybia ignobilis, ppa= polybia paulista, pse= polybia sericea, poc= polybia occidentalis, psy= protonectarina sylveirae. table 2: numbers of social wasps collected while visiting plant species in campos rupestres phytophysiognomy at the ibitipoca state park, minas gerais, brazil. collections took place from november 2007 to october 2008. sociobiology 64(2): 217-224 (june, 2017) 221 populations with higher biomass also use a greater amount of food resources (santos et al., 2006). for example, in the current study polybia occidentalis had the highest abundance of visiting individuals (n = 21) and displayed a broad foraging spectrum, visiting nine plant species. swarming wasps build large colonies that host many individuals, and the size of the colony can be the determining factor for resource consumption. this may affect foraging amplitude of these species through the influence of colony productivity on foraging activity (spradbery, 1973). the amplitude of a species trophic niche is an index subject to temporal and spatial changes, and may influence the degree of specialization (camillo & garófalo, 1989). in costa rica, heitaus (1977b) observed such variation in wasp species both among habitats and in the same habitat in different seasons. this author emphasizes the influence of plant flowering phenology on the abundance of floral resources and the strategy of resource exploration by visitors, which involves substitution of resources based on the quantity of food available. flexibility in foraging behavior makes the amplitude of the trophic niche a dynamic variable in ecosystems. wasp species with the highest evenness values with respect to plant visits frequently explored only one or two plant species, but also occurred in other species (table 3). this generally agrees with the results of heithaus (1977a) and santos et al. (2006), who found that social wasp species are infrequent floral visitors, and that many plant species were visited by a small number of individuals. social wasp diversity was higher in the hot-humid period in both phytophysiognomies due to the increased biomass of plants and other insects (e.g., lepidopteran larvae), which provides more abundant supply of food (souza & prezoto, 2006; auad et al., 2010). the greater number of social wasp species visiting flowers in the riparian forest can be explained by vegetational heterogeneity in this environment, which generates higher niche complexity. this forest formation in ibitipoca is characterized by a sequence of shrub-tree species (predominantly trees) located near waterways (rodela, 1998), an ideal location for wasp colony founding. in a study by clemente et al. (2013) in the same site, riparian forest fostered a greater number of interactions between social wasp and plant species, providing support for the idea that vegetation complexity is positively correlated with social wasp diversity. the campos rupestres site showed lower species richness of social wasp floral visitors, likely due to having a less complex vegetation structure composed predominantly by herbaceous plants, grasses, and shrubs that are distributed among quartzite outcrops (rodela, 1998). this area also has strong winds and a higher light incidence, which can lead to strong variation in microclimate (elpino-campos et al., 2007). wasp-flower interactions in campos rupestres tend to be more specialized (clemente et al., 2013), a situation that increases risk of species co-extinctions (hernandes et al., 2004). protonectarina sylveirae saussure, 1854, p. sericea, p. paulista, a. pallens, b. lecheguana, were among the social wasp species found in this environment. the occurrence of these species is unsurprising, as they are known to have high ecological tolerance (clemente et al., 2013) and to be generally dominant in open ecosystems, thriving under severe environmental conditions. these and other highly tolerant species may thus be important for plant and insect community structure in harsh ecosystems such as campos rupestres. santos et al. (2007) evaluated social wasp communities in mangrove, atlantic forest and restinga, and found that wasp diversity was significantly (positively) correlated with plant diversity in each environment. the tropical atlantic forest had the highest species individuals plant species visited niche amplitude (h’) evenness (j’) mischocyttarus drewseni saussure, 1857 17 12 2.34 0.94 mischocyttarus confusus zikán, 1935 17 9 1.92 0.87 polybia occidentalis (oliver, 1791) 21 9 1.78 0.91 polybia paulista (von. ihering, 1896) 18 7 1.72 0.88 polistes billardieri fabricius, 1804 5 4 1.33 0.96 protonectarina sylveirae saussure, 1854 8 4 1.07 0.77 polistes actaeon haliday, 1836 2 2 0.69 1 polybia sericea (oliver, 1791) 2 2 0.69 1 apoica pallens (fabricius, 1804) 3 2 0.63 0.91 polistes ferreri saussure, 1853 3 2 0.63 0.91 polybia ignobilis (haliday, 1836) 3 2 0.63 0.91 agelaia multipicta haliday, 1836 1 1 brachgastra lecheguana (latreille, 1824) 1 1 polistes cinerascens (saussure, 1854) 1 1 polybia fastidiosuscula saussure, 1854 1 1 table 3: amplitude and evenness of the trophic niche in social wasps collected in the ibitipoca state park, lima duarte, minas gerais, brazil. ma clemente, nr campos, km vieira, k del-claro, f prezoto – social wasp guild visting flowers222 wasp richness (18 species), followed by restinga (16 species), and mangrove (eight species). mangrove forest presents some ecological restrictions (i.e., salinity, temperature, and aridity) that can influence the diversity of wasps. floristic composition, and vegetation structure and complexity are determinants of social wasp community composition and structure, directly influencing their fundamental and realized niches (santos et al., 2007). further, vegetation provides nesting substrate (santos et al., 2006), carbohydrate resources (santos et al., 1998), and materials for nest construction such as plant fibers (rodrigues & machado, 1982; marques & carvalho, 1993), and prey foraging area (santos et al., 1998). some wasp species only nest under certain structural conditions (e.g., open or closed physiognomies), and depend on specific plant morphological parameters (e.g., leaf shape and size, trunk diameter, presence of spines) (henriques et al., 1992, santos & gobbi, 1998; cruz et al., 2006). conclusion the ibitipoca state park contains various discrete vegetational profiles, and is composed by semi-deciduous forest and cerrado species as well as endemic campos rupestres vegetation, typical of the rare and unique brazilian montane savanna ecoregion. the flora of ibitipoca provide diverse resources, favoring a rich fauna of social wasps. although considered to be less effective compared to other pollinators in natural ecosystems, wasps did regularly visit flowers in the study areas. thus, conservation of these areas may be relevant for the maintenance of social wasp diversity. acknowledgments we would like to thank cnpq for the financial support (authors fp and kd), profa. dra. fátima salimena for identification of botanical material, ief mg for providing the license and structural support. the universidade federal de juiz de fora provided financial assistance through a scholarship, and ibama icmbio provided the license for research. references aguiar, c. m. 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(1954). blütenbiologische typen als elemente der sippengliederung dargestellt anhand der flora südafrikas. jena, gustav fischer verlag, 338 p. 415 a new case of ants nesting within branches of a fig tree: the case of ficus subpisocarpa in taiwan by a. bain1, 2, b. chantarasuwan3, l.s. chou1, m. hossaert mckey2, b. schatz2 & f. kjellberg2* abstract ficus is one of many plant genera involved in interactions with ants. the interaction is however little documented. we show here that ants, belonging mainly to the genus crematogaster, nest in hollow internodes of young branches of ficus subpisocarpa, a monoecious fig species studied in taiwan. the ants feed on the mutualistic fig-pollinating wasps as well as on parasitic non-pollinating fig wasps. nevertheless fig-wasps may not constitute a sufficient food source to ensure permanent presence of ants on the tree as the ants were observed to be frequently associated with hemipterans such as coccids and aphids. fig wasps seem to constitute a reliable and sufficient food source on some dioecious ficus species. on the contrary, in monoecious ficus species, resident ants have always been observed to tend homopteran in addition to feeding on fig wasps. frequent fruiting, prolonged fruit ripening period, ramiflory and rapid growth could constitute traits facilitating strong association based on fig-wasps' consumption of the monoecious f. subpisocarpa with ants. despite these traits, ants were observed to tend hemipterans, and f. subpisocarpa does not seem to have evolved specialized morphological traits to facilitate the association. key words: ant-plant interaction, community ecolog y, asian biodiversity, myrmecophytism, ant foraging. 1 institute of ecolog y and evolutionary biolog y, college of life sciences, national taiwan university, 1, sec. 4, roosevelt rd., taipei, 10617 taiwan. 2centre d’ecologie fonctionnelle et evolutive cefe, umr 5175 cnrs, 1919 route de mende, 34293 montpellier france 3netherlands centre for biodiversity naturalis (section nhn), leiden university, p.o. box 9514, 2300 ra leiden, the netherlands and thailand natural history museum, national science museum, pathum thani, 12120, thailand. * correspondence: finn.kjellberg@cefe.cnrs.fr 416 sociobiolog y vol. 59, no. 2, 2012 introduction a major challenge of ecolog y is the assessment of species' communities and the understanding of the determinants of their structure and organization (fortuna & bascompte 2006; bascompte & jordano 2007; proffit et al. 2007; bascompte 2009; cavender-bares et al. 2009; ings et al. 2009; vázquez et al. 2009). various groups of insects have inspired the development of theories in community ecolog y. they have long been recognized as convenient models for testing hypotheses in this domain (heil & mckey 2003). among them, ants (hymenoptera: formicidae) can be central for such perspectives, as they often play a major role in interaction networks as predators (lach et al. 2009), seed dispersers (giladi 2006), pollinators (rico-gray & oliveira 2007), mutualistic defenders of their host-plants (heil & mckey 2003; heil 2007) and exploiters of various mutualisms (schatz et al. 2006, 2008). more specifically, ants have been widely studied for their interactions with plants. ant-plant protection mutualisms have been reported in 100 plant genera and 40 ant genera, mainly in tropical regions (davidson & mckey 1993; speight et al. 2008). the high diversity of this type of interaction even within genus (webber et al. 2007) is explained by easy development of mutualistic interactions. indeed, the strong potential protection against herbivores provided by ant colonies may substantially increase plant fitness (rosumek et al. 2009). specialized ant-plant interactions have been used as ecological and evolutionary models to understand selective factors affecting plants (heil & mckey 2003). some of these specialized interactions have been shown to be structured by chemical mediation (ranganathan & borges 2009; schatz et al. 2010) and support complex networks of plurispecific interactions involving herbivores, parasites, predators and secondary mutualists (palmer et al. 2008; schatz et al., 2008, 2010; blatrix et al. 2009; goheen & palmer 2010). among the numerous genera of plants associated with ants, the interaction between ficus and ants has been poorly studied. ficus is one of the 100 plant genera presenting what has been interpreted as structures specifically evolved to host ant nests (speight et al. 2008), with only a single case hitherto reported for this genus (maschwitz et al. 1996). fig species are known for their obligate species-specific nursery pollination mutualisms with fig wasps ( janzen 1979; kjellberg et al. 2005). specificity is reinforced by a physical filter (matching 417 bain, a. et al. — a new case of ants nesting in fig trees between pollinator head shape and ostiole structure; van noort & compton 1996) and chemical mediation (emission of volatile compounds responsible for pollinating wasp attraction by receptive figs; grison et al. 1999; proffit et al. 2007, 2008, hossaert-mckey et al. 2010). ficus are increasingly investigated for their role in supporting a complex network of interactions involving a community of species-specific parasitic chalcid wasps (regionally up to 25 parasitic species for a single ficus species, bouček et al. 1981), but also several species of ants, all of them capable of detecting and using chemical signals emitted by figs (chen et al. 1999; kjellberg et al. 2005; proffit et al. 2007; ranganathan et al. 2010; schatz et al. 2008, 2010). within that network, ants have been shown to severely reduce the prevalence of parasites and to be a structuring factor for various other insect species (other predators, herbivores, visitors) (schatz & hossaert-mckey 2003; schatz et al. 2006, 2008, wei et al. 2005). ants have often been reported to be predators of fig wasps. they have also been observed to participate in fig-seed dispersion (kaufmann et al. 1991; roberts & heithaus 1986; laman 1995, 1996) and they are involved in ant-homopteran-fig tree tritrophic interactions (compton & robertson 1988, 1991; dejean et al. 1997, ranganathan et al. 2010). fig wasps may constitute a major source of food for ants. indeed, schatz et al. (2008) demonstrated on dioecious ficus species that fig wasps on male trees provided a sufficiently abundant and reliable resource to allow continuous presence of dominant ants on the trees. however, whether fig wasps may constitute a sufficient resource in monoecious ficus species is still an open question. indeed, these species produce figs less frequently than dioecious ones and often present more synchronized crops (e.g. shanahan et al. 2001). hemipterans could constitute an alternative resource that could allow the continuous presence of tending ants on monoecious fig trees (schatz et al. 2008). however, the presence of hemipterans and tending ants on monoecious fig species has been mainly reported from africa and madagascar (f. sur in south africa [compton & robertson 1988, 1991; zachariades 1994]; f. vallis-choudae in cameroon [dejean et al. 1997], and several fig species in madagascar, malawi, south africa, zambia, and zimbabwe [cushman et al. 1998]) while the most convincing data on fig wasps providing sufficient food for ants in dioecious figs stems from borneo (schatz et al. 2008). hence, to confirm that continuous ant presence on monoecious fig trees is systematically 418 sociobiolog y vol. 59, no. 2, 2012 associated with the presence of hemipterans, we need to record whether in other parts of the world, ants on monoecious ficus are systematically associated with hemipterans. for ants, a fig tree may constitute an appropriate support presenting dispersed feeding sites, located exclusively in the arboreal stratum. optimization of fig wasp capture efficiency could lead ants to inhabit this stratum in order to reduce distance between their nest and this food source in accordance with central place foraging theory (stephens & krebs 1986; bell 1991; schatz et al. 2008). diverse reports of arboreal ant nests within fig trees support this hypothesis, and suggest that ants effectively find sufficient food sources within fig trees. examples include nest location among the leaves of f. fistulosa for oecophylla smaragdina and of f. sur for o. longinoda (thomas 1988; schatz et al. 2008), between grouped figs of the cauliflorous f. botryoides for unidentified formicidae (dalecky et al. 2003) and cauliflorous figs of f. fistulosa for crematogaster sp. (schatz et al. 2008), within figs of f. sycomorus by cardiocondyla wroughtoni (lupo & galil 1985), in large persistent stipules (up to 80 x 20 mm) of f. paracamptophylla often tenanted by ants (corner 1976), in ant gardens on f. paraensis and f. trigona for camponotus femoratus and azteca cf. traili (davidson 1988; benzing 1991) and within the dead parts of branches on f. carica by crematogaster scutellaris (schatz & hossaert-mckey 2003). moreover, one species of ficus presents what appear to be structures specifically evolved to host ant nests (maschwitz et al. 1996). this study showed that 64% of the opened structures they called domatia of ficus obscura var. borneensis (included in ficus pisifera sensu berg & corner 2005) were inhabited by ants, belonging to eight species distributed into five genera (camponotus, cardiocondyla, cataulacus, crematogaster and tetramorium). despite lack of description of ant behavior on this species, the presence of extrafloral nectaries and structures sheltering opportunistic ants on f. obscura var. borneensis suggests existence of a non-specific protection mutualism. interestingly, in their field survey, maschwitz et al. failed to detect similar structures in ficus species belonging to over 20 other malaysian fig species and they also failed to detect such structures in a survey of herbarium samples from 37 australasian fig species. in this study conducted in the island of taiwan, we focused on the monoecious ficus subpisocarpa. we describe here the presence of hollow structures 419 bain, a. et al. — a new case of ants nesting in fig trees inhabited by ants in a fig tree associated with an plant-ant interaction. we determined the frequency of the hollow structures and the ant occupancy and we identified the observed ants to genus and describe traits of their presence. we also correlate ant presence and presence of hemipterans. we then discuss the insights into ant-fig interactions brought about by this discovery of a new case of ants inhabiting hollow structures in fig trees. materials & methods ficus subpisocarpa gagnep. is a monoecious fig tree belonging to subsection urostigma (sensu berg & corner 2005). it is a hemiepyphitic or terrestrial tree which grows up to 7m in height distributed in south asia from the south of japan to taiwan and to the malay peninsula and the maluku islands (berg and corner 2005). this species produces abundant crops, 2-4 times per year, of small cauliflorous figs, ripening over a 1-3 week period (corlett 2006; bain pers. obs. for taiwan). as most species of subsection urostigma, f. subpisocarpa is a deciduous tree presenting rhythmic growth (berg & corner 2005). hollowed stems hosting ants were incidentally noted on individual trees growing in taiwan when inspecting trees for ant presence. to better describe and quantify this trait we examined branches from a series of individual trees. branches were collected on eight trees: six in taipei (national taiwan university campus and fuzhoushan park), one 25 km north, at bitou cape on the north coast of taiwan and one 350 km south, in kenting in the extreme south of the island. five to 16 apical branches were sampled for each tree at their junction with a main trunk. indeed such branches usually grow directly from the trunk or from main branches and usually wither without reaching more 60 cm (bain pers. obs.). for each branch we measured length, diameter of the branch at the cutting level, diameter at the middle of the apical internode and we counted the number of ramifications. then the branches were split lengthwise, and the length of each hollow section (hereafter called cavity) was measured. branch diameter was measured at the middle of the cavity as well as the distance from the branch apex. when ants were observed in the cavity, the inner diameter of the cavity was measured and the number of exit holes noted. the cavities were characterized as described in fig. 1 into six categories: young cavities empty or attacked 420 sociobiolog y vol. 59, no. 2, 2012 by insect larvae, inhabited and previously inhabited cavities, mature cavities, and old cavities. the sequence of the cavities from the apex was noted and for trees 02, 03, 04 and 06, the precise internodes, numbered from the apex, where the structures were observed were also noted. the insect content of inhabited cavities was collected, identified and counted. ants were identified to genus using bolton (1994) and lin & wu (2003). fig. 1. the different types of cavities. a) young cavity: these cavities are characterized by their young tissues. only the central part is hollowed, the inner pith has not dried out yet. b) young attacked cavity: the structure of the cavity is the same as for young cavities but insect larvae are feeding inside. c) inhabited cavity: ants are observed inside. all pith tissue has been removed. d) entry holes of an inhabited cavity. e) formerly inhabited cavity: all the pith has been removed but no ant live inside. often fungi or mould have grown on the inner surface. f) mature cavity: the inner pith has dried out all along the internode. g) old cavity: secondary growth is beginning to compress the cavity 421 bain, a. et al. — a new case of ants nesting in fig trees an additional set of trees were examined for the presence of ants and of hemipterans to determine whether there was a frequent association. one hundred ten branches distributed on 11 trees located in taipei were examined. additionally herbarium samples originating from japan, vietnam and thailand were examined for the presence of similar hollow structures. they included samples of ficus subpisocarpa subpisocarpa, f. subpisocarpa pubipoda c.c. berg and the closely related f. geniculata. the herbarium samples examined were: ficus subpisocarpa gagnep., japan: kagoshima pref., yaku is., 20 may 1984, t. yahara, t. nagamasu & t. kawahara 10340 (l); ryukyu islands, misato-mura, matsumo to-aza, 7 august 1951, e.h. walker, s. tawada, t. amano 6441 (l); vietnam: sai wong mo shan (sai vong mo leng ), lomg ngong village, dam-ha, tonkin, july 18 – sept 9, 1940, w.t. tsang 30348 (l). ficus subpisocarpa gagnep. subsp. pubipoda c.c. berg. thailand: ranong, kaper, laem sohn national park, sea level, 30 november 1996, j.f. maxwell 96 – 1568 (l); chiang mai, muang, doi sutep – pui national park, elevation 1350m, 26 march 2002, j.f. maxwell 02 – 103 (l); chaiyaphum, between nam phrom and thungkamang, alt. 800m, 13 dec 1971, c.f. van beusekom, r. geesink, c. phengkhlai & b. wongwan 4218 (l). ficus geniculata kurz. vietnam: cana province de phanrang, 25 oct 1925, m. poilane 12491 (p); ka rom pr. phanrang, 7 mar 1924, m. poilane 9964 (p); thailand: lampang, jae home, doi jae sawn national park, alt. 425 m, 7 jan 1992, j.f. maxwell 92 – 16 (e); tak, mueang, rd. no. 105, km 19 – 20, alt. 400 m, 21 nov 2005, r. pooma, c.c. berg & m. poopath 5734 (l). results occurrence of cavities sampled branch length ranged from 21 cm to 156 cm. ten percent of the studied branches presented no cavity, 36% presented a single cavity and 54% presented several cavities (table 1). the average length and diameter (at cutting level) of branches did not differ between branches with and without cavity (mann-whitney u test, ns). 422 sociobiolog y vol. 59, no. 2, 2012 on the 86 analyzed branches, a total of 204 cavities were observed (2.4 ± 2.0 cavities per branch or 4.2 ± 3.1 cavities per meter). the number of cavities per branch varied significantly among trees from 0.90 to 5.0 (one-way anova f 6,79 = 11.85; p<0.001) (table 1). the different types of cavities we identified six kinds of cavities in f. subpisocarpa branches (fig. 1): young cavities, intact or attacked by insect larvae, cavities inhabited by ants and cavities formerly inhabited by ants, mature cavities, old cavities. the most frequent hollow structures were mature cavities (45%), followed by young cavities (30%, 17% intact and 13% attacked by insect larvae) and inhabited cavities (15%). young and mature cavities were observed on all trees. cavities inhabited by ants were observed in six of the trees (75%), and a formerly inhabited cavity was observed on an additional tree. two-third of the inhabited cavities sheltered ant pupae or larvae. out of the 32 inhabited cavities, 7 were located on branches that presented a young cavity attacked by insect larvae (22% of the inhabited cavities): protection, if any, is not perfect. length of cavities the length of most cavities (75) ranged from 2 to 14cm (fig. 2). four cavities measured less than 2cm and seven were longer than 22cm. young cavities table 1. cavity data per tree. cavity types are defined according to fig. 1. the two undetermined cavities correspond to cavities that were damaged during sampling. all trees located in taipei, except for tree 7 located at bitou cape and tree 8 located at kenting. tree 1 2 3 4 5 6 7 8 proportion number of branches 13 10 10 16 12 10 10 5 young healthy cavity 11 6 4 3 4 3 3 1 0.172 young attacked cavity 3 6 5 0 6 4 1 1 0.127 inhabited cavity 3 4 0 0 16 7 1 1 0.152 formerly inhabited cavity 0 0 0 1 9 1 1 0 0.059 mature unused cavity 7 17 3 22 24 15 2 2 0.451 old cavity 0 3 0 0 3 0 0 0 0.029 undetermined 0 0 0 0 0 0 1 1 0.010 total 24 36 12 26 61 30 9 6 423 bain, a. et al. — a new case of ants nesting in fig trees were most frequent (46%) in the short length classes (l<4cm) and were overall significantly shorter than the other ones (one-way anova: f 1,233 = 12.72; p<0.001; average: 7.1±4.3cm). mature cavities were most frequent in the seven following length classes (4cm<l<18cm). only few cavities measured more than 18cm. among these longer cavities, inhabited and formerly inhabited cavities represented 7 out of 16 cavities. indeed inhabited cavities were overall significantly longer than other types of cavities (one-way anova: f 1,233 = 35.61; p<0.001; 14.1±7.0cm versus 8.8±5.5cm). location of the different cavity types on the branches fig.3 a, b, c and d illustrates the pattern of branch growth in diameter and the distribution of cavity types according to distance from the apex. diameter remained constant at about 7mm for the first 10cm from the branch apex, then from 10cm to 60cm from the apex the diameter progressively increased to about 12mm. finally from 60cm to 130cm the branch diameter remained constant at about 12mm. fig. 2. cavity length as a function of cavity type. inhabited cavities are longer than average. 424 sociobiolog y vol. 59, no. 2, 2012 young cavities (empty or attacked by insect larvae) seem to constitute a homogeneous class. they were mainly concentrated in the first portion up to 10 cm from the apex (respectively 55 and 56% of them), but also in the second portion up to 20 cm and they were present, though in lower numbers, up to 40 cm from the apex (respectively 94% of intact ones and 86% of attacked ones were located less 40 cm from the apex). they were significantly closer to the apex than other cavity types (one-way anova: f 1,221 = 32.37; p<0.001). as a result, branch diameter at the level of young cavities was on average smaller than for other cavity types (one-way anova: f 1,237 = 48.91; p<0.001), fig. 3. a. distance of the different types of cavities from the apex and branch diameter. b, c & d: detailed views of portions of graph a. note that young cavities are only observed up to 40 cm from the apex and inhabited cavities are mainly observed up to 50 cm from the apex. 425 bain, a. et al. — a new case of ants nesting in fig trees inhabited cavities and formerly inhabited cavities seem to form a second homogeneous class. they were present from the apical internode and concentrated in the first 35cm from the apex although some were observed further away from the apex. mature cavities seem to constitute a third class. they were observed at all distances from the apex. finally only few cavities were in the process of closing due to secondary growth and they were located from 26cm from the apex to 68cm from the apex. statistically, mature and old cavities were grouped as the furthest from the apex (one-way anova: f 4,218 = 13.74; p<0.001). the young cavities (intact and attacked) were mainly the most apical cavity when the branches presented more than one cavity. young cavities attacked by insect larvae were frequent at the most apical internode; they represented 33% of the cavities at this internode. on the other hand old cavities were observed only from the fourth to the eleventh internode from the branch apex. mature cavities were the most common for all the ranks, and they were observed up to the eleventh internode. inhabited cavities were only observed in the four apical internodes, with a peak frequency at the third internode (50% of the inhabited cavities were located on the third internode). when a single cavity was noted for a branch, the cavity was in 67% of the cases a young cavity (intact or attacked). ant species present within cavities four ant genera were observed within the cavities of f. subpisocarpa branches: crematogaster (myrmicinae), myrmica (myrmicinae), technomyrmex (dolichorinae) and prenolepis (formicinae). on the six trees presenting inhabited cavities, 32 inhabited cavities were observed on 21 branches. crematogaster ants occupied 25 cavities (78% of the cavities), the three others genera occupied each two cavities. for the six branches presenting more than one occupied cavity (five branches with two occupied cavities and one with three), all were occupied by crematogaster. one structure was shared by two species (crematogaster sp. and prenolepis sp.). this was in one of the few naturally open cavities, and ants from the two genera were living on each side of the open structure. the branches from trees 07 and 08 (table 1) presented only one inhabited cavity each and the ants belonged both to 426 sociobiolog y vol. 59, no. 2, 2012 genus technomyrmex. all other trees hosted a mix of crematogaster ants and other genera. patterns of ant presence when crematogaster ants were present, workers were observed in all the inhabited structures and their number ranged from eight to 320 individuals. larvae and pupae were present in 10 of the 25 occupied cavities (40%), winged crematogaster adults in nine of them (36%) and queens in four (16%). in the prenolepis inhabited cavities, the nest containing most workers (25 individuals) also contained larvae and pupae. the second one was the one shared with crematogaster ants and sheltered only workers (16 individuals). one of the two cavities containing technomyrmex contained 135 workers, nine queens, 51 pupae, 36 larvae but no winged adults, and the second one contained 37 workers, two queens, six pupae, three larvae and seven winged adults. finally the two cavities inhabited by myrmica ants presented fewer workers than for the other species (nine and 12 individuals), no winged males and both of them had two queens and winged females. in four cavities inhabited by crematogaster, other ant species were also collected. in tree 05, all inhabited cavities contained crematogaster ants but in two of them technomyrmex ants were also collected inside the nest (one and six individuals). moreover one prenolepis ant was found in a cavity on tree 01, and this tree sheltered a sampled prenolepis nest. similarly, a myrmica ant was found in a cavity inhabited by crematogaster in tree 06, and that tree presented a cavity inhabited by myrmica ants. crematogaster ants tended to inhabit longer and wider cavities than the other species (fig. 4). nevertheless, cavity diameter was independent of fig. 4. worker distribution according to cavity inner diameter (left) and to cavity length (right). cavity diameter was measured at its mid point. cavity diameter was independent of colony size. 427 bain, a. et al. — a new case of ants nesting in fig trees branch diameter: all crematogaster ants were living in similar-width nests. the ants of this genus were occupying cavities from very close to the apex to more than one meter away from the apex (fig. 4). presence of other insects within cavities fig wasps (captured by ants) were found in six inhabited cavities. both technomyrmex nests contained fig wasps (the first one, one f. subpisocarpa female pollinating wasp (platyscapa ischiana) and three wingless males and the second one, one p. ischiana female wasp and two female non-pollinating fig-wasps of genus sycoscapter). then one crematogaster cavity contained two non-pollinating female wasps otitesella ako (a genus whose precise oviposition period is species dependent, compton 1993) while a second one contained a female non-pollinating fig wasp from genus sycoscapter (a genus ovipositing after the pollinator in the fig development sequence; tzeng et al. 2008). finally in the cavity inhabited by two ant species two female platyscapa ischiana were collected and 18 female sycoscapter sp. the antennae and the ovipositors of the collected fig wasps had been cut off by the ants. a scale insect (hemiptera: coccoidea) and an aphid (hemiptera: aphidoidea) were found in two distinct crematogaster inhabited cavities. the larvae attacking young cavities were coleopteran larvae. finally, curculionidae beetles (coleoptera) were often observed on f. subpisocarpa branches (bain, pers. obs.). correlation between presence of ants and presence of hemipterans we frequently observed sap sucking insects on different vegetative parts of f. subpisocarpa. scale insects were often observed on leaves and in shelters built by crematogaster ants between figs or at the base of ramifications, but also occurred on figs, while aphids were almost only observed on figs. ants were observed tending both scale insects and aphids. ants, coccids and aphids were observed on the branches. in the set of 110 branches that were specifically monitored for ant and hemipteran presences, ants were observed on 24 branches and were distributed into 3 genera: crematogaster (23 branches), prenolepis (1 branch, on which crematogaster was also present) and myrmica (1 branch). the branch presenting myrmica ants presented no hemipterans, so in all cases when ants 428 sociobiolog y vol. 59, no. 2, 2012 were associated with hemipterans, crematogaster ants were present on the branch. we observed a correlation between the presence of crematogaster ants and the presence of hemipterans on the 110 branches examined. indeed, crematogaster were observed on 23 branches and were associated with hemipterans in 20 cases, while hemipterans were present on 33 branches (correlation test on the cumulative observations: kendall tau=0.940, p<0.001). aphids were mainly observed on figs (10 cases, often in association with coccids) as opposed to on the leaves (3 cases out of which two corresponded to winged individuals). in 8 out of 10 cases aphids were observed on figs, they were associated with crematogaster ants. coccids seemed to be much less specialised as the coccids were observed only in the cavities (2 branches), only on the leaves (12 branches), or both in the cavities and on the leaves (2 cases), on leaves and figs (4 cases) and in all three locations (4 cases). comparison with cavities observed in herbarium samples cavities similar to those observed in taiwan were found in all herbarium samples from typical ficus subpisocarpa gagnep. collected in japan and vietnam, from ficus subpisocarpa gagnep. subsp. pubipoda c.c. berg collected in thailand and from ficus geniculata kurz collected in vietnam and thailand. discussion while the presence of a central cavity within the branches of fig trees is quite common, we demonstrated here that cavity traits in f. subpisocarpa allow a series of ant species to inhabit these cavities. after an analysis of occurrence of branch cavities in dioecious and monoecious fig species, we will examine how this trait may favor myrmecophytism, in conjunction with the different food sources exploited by ants on different ficus species. the presence of hollow internodes is well known though hardly investigated in ficus. it is frequent in dioecious species as exemplified by the reference in the species name to fistulose branches in ficus fistulosa reinw. ex blume, ficus fistulosa koord (= f. congesta roxb.) and ficus fistulosa kurz (=ficus schwarzii koord). corner (1967) stated that, in f. cristobalensis, twigs are 10-20 mm thick, with indistinct internodes and the wide pith is often hol429 bain, a. et al. — a new case of ants nesting in fig trees lowed by ants. nevertheless, quantitative reports on the presence of hollow internodes in live plant material and their use by ants in ficus are basically lacking. we provide here a first report on the presence of hollow internodes in a monoecious fig species and on the colonization of these hollow internodes by ants. ficus subpisocarpa presents hollow internodes all over its range, and hollow internodes are also present in its close relative f. geniculata. in f. subpisocarpa these hollow internodes are used by ants that make entry/exit holes to access the cavities. these occupied hollow internodes are located close to the branch apices. while hollow internodes could be rather frequent in dioecious figs, they seem to be less frequent in monoecious figs of subgenus urostigma. however, a characteristic trait of subsection urostigma (about 23 species) to which f. subpisocarpa belongs, is intermittent growth, usually seasonal and accompanied with deciduousness (berg & corner 2005), as opposed to continuous growth in many subgenus urostigma species. fast growing plants often produce twigs presenting more pith than species producing slowly growing twigs. we suggest that quick growth of the twig associated with intermittent growth is a major feature at the origin of the hollow internodes in f. subpisocarpa. hollow internodes are a feature that may facilitate the evolution of myrmecophytism. myrmecophytism also involves producing food (nectar, food bodies) that will stabilize the ant nests. ficus provide ants with an access to fig-wasps, pollinators and parasites. while fig-pollinating wasps are present on the trees for short spells of time, at fig receptivity and at wasp emergence, parasites are present on the fig surfaces for longer periods of time. schatz et al. (2008) suggested that cauliflory, the abundant production of figs on short specialized branches, allowed easier patrolling of figs by ants and thus could be conceived as a way of stabilizing ant nests in a tree and getting protection against parasitic wasps. while cauliflory is little represented in subgenus urostigma, f. subpisocarpa as a number of species of subsection urostigma is cauliflorous, producing numerous clusters of small figs on the branches. the species also fruits frequently producing 2-4 fig crops a year and a crop may ripen over a period of 3 weeks (corlett 2006, bain pers. obs.), two characters that increase the number and length of periods when fig-wasps can be used as a food source exploited by ants. this is all the more true as the ants, beyond pollinators, collected non-pollinating fig wasps ovipositing before 430 sociobiolog y vol. 59, no. 2, 2012 (otitesella) and after (sycoscapter) the pollinators, thus extending the period during which a crop provides food for ants. intermittent growth, cauliflory, frequent fruiting and prolonged fig maturation period may constitute a set of characters of f. subpisocarpa that facilitate the establishment of a strong interaction with ants. nevertheless, despite these features, in monoecious figs, fig wasps may still not provide sufficient resources for ants to establish stable nests within fig branches. we hypothesize that beyond capturing fig wasps during their presence on figs (oviposition period and the period of emergence from figs), ants may also depend on hemipterans when fig wasps are less abundant. several of our observations support this hypothesis. first, we observed hemipterans more frequently on branches colonized by ants, and we observed captured hemipterans (scale insects and aphids) within cavities inhabited by ants. hemipterans present in f. subpisocarpa could thus constitute a regular source of both honeydew and prey. ant-hemipterans interactions have previously been reported in two african monoecious fig species (dejean et al. 1997; zachariades et al. 2009), but we provide here the first observations of predation by ants in the fig context. more detailed studies will be required to investigate the contribution of hemipterans in the diet and maintenance of ants on fig trees. second, fig wasps were found in six nests of crematogaster, technomyrmex and prenolepis ants. on two dioecious fig species, ants (especially crematogaster species) are also known to feed on fig wasps (schatz et al. 2008), and to constitute strong non-pollinating wasp repellents (schatz & hossaert-mckey 2003). non-pollinating fig wasps with cut ovipositors discovered in f. subpisocarpa cavities suggests that crematogaster ants capture them while they are ovipositing with their ovipositor inserted through the fig wall, as observed on dioecious figs (schatz et al. 2006, 2008). more detailed studies are required to determine the impact of ants on fig wasps population. available data suggests that exploitation of both hemipterans and fig wasps as two permanent food sources may be sufficient to explain continuous ant presence in monoecious fig trees. we also observed on the monoecious f. subpisocarpa that ants seem to predate more non-pollinating fig wasps than pollinating ones. this observation suggests a positive role of ants for the fig-fig wasp mutualism, acting here as indirect mutualists (dawkins & krebs 1979; bronstein 1991; schatz et al. 2006, 431 bain, a. et al. — a new case of ants nesting in fig trees 2008). even if the impact of ants on the populations of hemipterans and fig wasps needs to be investigated in detail, we may surmise that the monoecious f. subpisocarpa may be selected to provide shelters for ant nests. nevertheless, the caulinary cavities in which the ants nested are hardly sophisticated and the dwellers need to excavate the inner pith to be able to inhabit them and they have to create openings to gain access. thus, the hollow structures of f. subpisocarpa branches sheltering ants should be considered as potential domatia precursors according to current terminolog y (webber et al. 2007). in addition, the predominance of crematogaster among ants inhabiting this fig species may appear as a trend towards specialization in this ant-plant interaction. because we observed similar structures in the related ficus geniculata, we may infer that the current level of association is quite old and that selective pressures are not strong enough to lead to the evolution of more specialized structures such as in the acacia or macaranga genera. such results certainly incite further investigation about insects-fig interactions, and reinforce the status of fig trees as supporting complex networks of interactions (compton and robertson 1988; zachariades 1994; schatz & hossaert-mckey 2003; schatz et al. 2006, 2010). acknowledgments many thanks to b. di giusto and florian massip for technical help in the field. this study was supported by anr 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xu & d.r. yang. 2005. impact of oecophylla smaragina on the percentage number of offspring of pollinator and non-pollinating wasps on ficus racemosa. zoological research 26:386-390. weiblen, g. 2002. how to be a fig wasp? annual review of entomolog y 47:299-330. zachariades, c., s.g. compton & b. schatz. 2009. honeydew as danegeld? ants (hymenoptera: formicidae) tending a honeydew-producing homopetran do not offer protection from its main natural enemies. sociobiolog y 54 471-488. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.595sociobiology 62(3): 459-460 (september, 2015) new distribution record of reticulitermes speratus kolbe (isoptera: rhinotermitidae) in the coldest highland in central japan the japanese subterranean termite reticulitermes speratus kolbe (isoptera: rhinotermitidae) is a very important ecological component in the cycling of organic matter via decomposition of litter and dead wood (donovan et al., 2001). in addition, it is a very important destructive pest, causing extensive damage to wooden structures (su, 2002). the termite is distributed in the korean peninsula, kyushu island, shikoku island, honshu island, and southwestern hokkaido island (austin et al., 2002; park et al., 2006; kim et al., 2012). although the northern limit of r. speratus distribution has been investigated (aoyama & murakami, 2003), little is known about an altitudinal limit. reticulitermes speratus has expanded its geographic range from southwestern into central hokkaido island (aoyama & murakami, 2003); however the termites found in central hokkaido nested and overwintered near places artificially kept warm in winter. in addition, the new distribution records of the termites in hokkaido were discontinuous, suggesting an anthropogenic origin of termite populations in central hokkaido were artificially introduced. sugadaira highland, ueda city, nagano prefecture, central japan lies at an altitude of approximately 1300 m and is one of the coldest areas in central japan. reticulitermes abstract the distribution of reticulitermes speratus kolbe was investigated in the sugadaira highland, the coldest area in central japan. we found r. speratus in an empty lot in june 2013. the following spring, we found the overwintered termites in the log, which had been naturally covered in snow. conversely, the population size in the empty lot had decreased from april through june 2014. moreover, r. speratus were not found in the forest. these suggest that the individuals found in the empty lot had been artificially introduced and the establishment might be difficult in the coldest areas in japan. sociobiology an international journal on social insects e takagi, t ogai article history edited by og de souza, ufv, brazil received 24 september 2014 initial acceptance 14 january 2015 final acceptance 12 february 2015 keywords cold tolerance, establishment, invasion, japanese termite. corresponding author etsuro takagi sugadaira montane research center university of tsukuba, sugadaira kogen, ueda 386-2204, nagano, japan e-mail: takagi@sugadaira.tsukuba.ac.jp speratus has not been found in the sugadaira highland or in the northernmost part of hokkaido. monthly temperatures in the sugadaira highland are lower than those in northernmost hokkaido and are slightly higher than those in central hokkaido (japan meteorological agency, 2014). we accordingly hypothesized that r. speratus could invade artificially, but the termites could not overwinter or be established in the coldest areas in central japan. we investigated the presence of r. speratus in the sugadaira highland. our objectives of this study were as follows: 1) to determine the presence of r. speratus in summer, and 2) to determine whether r. speratus overwinters and is established in the sugadaira highland. two study areas were selected in the sugadaira highland (36°31′n, 138°20′e). the first was an empty lot where four logs of japanese red pine, pinus densiflora siebold & zucc., three logs of nikko fir, abies homolepis siebold & zucc., and > 10 logs of japanese alder, alnus japonica (thunb.) steud. were piled and left away (> 20 m) from houses. to assess the presence of r. speratus in spring, we identified log species using bark characteristics. subsequently, on june 19, 2013, we dissected one, one, and three logs of japanese red pine, nikko fir, and japanese alder, respectively, and recorded the presence of r. speratus. short note sugadaira montane research center, university of tsukuba, nagano, japan e takagi, t ogai reticulitermes speratus overwinters in the coldest highland460 when the termites were found in the logs, the logs in the empty lot were experimentally left and the termites in the logs were allowed to overwinter and be covered in snow. to determine whether r. speratus had overwintered in the sugadaira highland, all japanese red pine and nikko fir logs, and 10 japanese alder logs were dissected, and the presence of r. speratus was recorded on april 22, 2014, immediately after the logs emerged from under snow. moreover, the logs were dissected, and the presence of r. speratus was recorded on june 18, 2014. the second study area was in the sugadaira montane research center, university of tsukuba, 1.2 km away from the empty lot. to assess the presence of r. speratus in early summer in the forest, we conducted a census in the site’s center. on july 24, 2014, all japanese red pine logs found along the observation route in the japanese red pine forest and in the japanese red pine and broad-leaved mixed forest were dissected, and the presence of r. speratus was recorded. to record under-snow temperatures, two temperature loggers (ibutton®, maxim integrated products, inc., california, usa) were placed in the center of the logs from december 22, 2013, through april 1, 2014. we found r. speratus nesting in a japanese red pine log in spring 2013. to the best of our knowledge, this is a new distribution record of r. speratus in the coldest area of central japan. no termites were found in nikko fir or japanese alder logs. reticulitermes speratus is not found in the northernmost part of hokkaido because of low winter temperatures (aoyama & murakami, 2003), but temperatures in the sugadaira highland are lower than those in the northernmost part of hokkaido (the japan meteorological agency, 2014). thus, our results suggest that r. speratus can damage wooden structures in early summer not only in the coldest areas in central japan, but also in northernmost hokkaido. we found r. speratus nesting in a japanese red pine log in spring 2014; however, only a few individuals were found in the logs in the following early summer. again, no termites were found in nikko fir or japanese alder logs. the results indicate that the overwintering of r. speratus might be difficult, although under-snow temperatures in the sugadaira highland ranged from −0.5°c to 0.5°c. in addition, termites or their tunnels were not found in 45 japanese red pine logs along the observation route in the forest, at the site’s center. this suggests that the individuals found in the empty lot had been artificially introduced. although r. speratus was not established in the study area, artificial introductions may temporarily affect the cycling of organic matter via decomposition of litter and dead wood in the sugadaira highland, as well as in northernmost and central hokkaido. acknowledgments we are grateful to dr. yuta mashimo and ms. michiyo matsushima (sugadaira montane research center, university of tsukuba) for providing information about the distribution of r. speratus in japan. we also thank mr. teruyoshi kimura (sugadaira kogen, ueda, nagano) for his assistance with field surveying. this study was supported by research and education funding for japanese alps inter-universities cooperative project, mext, japan. references aoyama, s. & murakami, t. (2003). the northernmost point of the japanese termite, reticulitermes sperarus in hokkaido. pestology 18: 59–63. (in japanese with english summary) austin, j.w., szalanski, a.l., uva, p., bagnéres, a-g. & kence, a. (2002). a comparative genetic analysis of the subterranean termite genus reticulitermes (isoptera: rhinotermitidae). annals of the entomological society of america, 95: 753–760. doi: 10.1603/0013-8746(2002)095[0753:acgaot]2.0.co;2 donovan, s.e., eggleton, p., dubbin, w.e., batchelder, m. & didog, l. (2001). the effect of a soil-feeding termite, cubitermes fungifaber (isoptera: termitidae) on soil properties: termites may be an important source of soil microhabitat heterogeneity in tropical forests. pedobiologia, 45: 1-11. doi: 10.1078/0031-4056-00063 holt, j.a. & lepage, m. (2000). termites and soil properties. in: abe, t., bignell, d.e. & higashi, m. (eds.) termites: evolution, sociality, symbioses, ecology (pp. 389–407). kluwer academic publishing, netherlands. japan meteorological agency (2014). climate statistics. http://www.data.jma.go.jp. (accessed data: 24 april, 2014) kim, m.-j., choi, y.-s., kim, j.-j. & kim, g.-h. (2012). molecular characteristics of subterranean termites of the genus reticulitermes (isoptera: rhinotermitidae) from korea. annals of the entomological society of america, 105: 97102. doi: 10.1603/an11078 park, y.c., kitade, o., schwarz, m., kim, j.p. & kim, w. (2006). intraspecific molecular phylogeny, genetic variation and phylogeography of reticulitermes speratus (isoptera: rhinotermitidae). molecules and cells, 21: 89-103. su, n.-y. (2002). novel technologies for subterranean termite control. sociobiology, 40: 95-101. sugimoto, a., bignell, d.e. & macdonald, j.a. (2000). global impact of termites on the carbon cycle and atmospheric trace gases. in: abe, t., bignell, d.e. & higashi, m. (eds.) termites: evolution, sociality, symbioses, ecology (pp. 409–435). kluwer academic publishing, netherlands. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1647sociobiology 64(3): 347-351 (september, 2017) description of the intramandibular gland ii in polybia emaciata lucas, 1879 (hymenoptera: vespidae) introduction exocrine glands have a major role in insect social life, because these glands regulate communication, social structuring, and nest construction of colonies, among other behaviors (billen & morgan, 1998). the characterization of exocrine glands has been an important research area dated back to the 17th century (heselhaus, 1922; billen & wilson, 2008). however, the discovery of new glands is ongoing with new reports published on a regular basis, particularly for social wasps for which studies are less frequent (samacá et al., 2013; da silva et al., 2015; penagos-arévalo et al., 2015). polybia emaciata lucas, 1879 is a medium-sized wasp (head width 2.18 mm, wing length 10 mm), with a color pattern that is primarily dark yellow with black or brown markings on several parts of the body. the wasp is a abstract the intramandibular gland ii in polybia emaciata lucas is described. this species is among the few that uses mud for nest construction, and their nests persist for a long time following abandonment. the intramandibular gland ii has been found in single representatives of the genera mischocyttarus, apoica and leipomeles, and this record is the second for the genus polybia. despite expectations derived from the nest characteristics of the species, gland dimensions such as cell diameter were well within the range observed for other species, with the cell number even comparatively small. gland function remains to be investigated. sociobiology an international journal on social insects c cely1, ap arévalo2, j billen3, ce sarmiento2 article history edited by marcel g. hermes, ufla, brazil received 06 april 2017 initial acceptance 25 july 2017 final acceptance 26 july 2017 publication date 17 october 2017 keywords exocrine glands, polistinae, mandibles. corresponding author carlos e. sarmiento laboratorio de sistemática y biología comparada de insectos instituto de ciencias naturales universidad nacional de colombia a. a. 7495 bogotá, colombia. e-mail: cesarmientom@unal.edu.co swarm-founding species found extensively from mexico to brazil, from sea level up to 1500 m.a.s.l., and is very common in farmlands. the colonies range from 100 to 500 individuals and include several queens (richards, 1978; strassman et al., 1992). a notorious characteristic of this species, shared only with four other congeneric species of the polistinae, is the use of mud for the nest; these nests persist for a long time even after being abandoned (rau, 1933; skutch, 1971; o´donnell & jeanne, 2007). this persistence may be attributed to the mudsaliva mixture used in nest construction (schremmer, 1984). given the results of schremmer (1984) on nest composition and characteristics, which require extensive chewing and processing behaviors from the wasps, we agreed with the proposed role of the head glands in determining the characteristics of the nest materials for this species. in addition to the salivary glands, palpal and mandibular glands also occur and may have a function 1 department of ecology and evolutionary biology, university of michigan, ann arbor, usa 2 instituto de ciencias naturales, universidad nacional de colombia, bogotá, colombia 3 zoological institute, katholieke universiteit leuven, leuven, belgium research article wasps mailto:cesarmientom@unal.edu.co c cely, ap arévalo, j billen, ce sarmiento – intramandibular gland ii in polybia emaciata348 associated with nest construction material characteristics; however, these structures have not been investigated. inside the mandibles of neotropical social wasps, three glands have been identified, the intramandibular gland i, the intramandibular gland ii, and the ectal mandibular gland (penagos-arévalo et al., 2015). the intramandibular gland i is a class 1 gland, whereas the other two are class 3 glands. class 1 glands are a lining of epithelial cells on the internal surface of the body wall, and class 3 glands are bicellular units consisting of a large secretory cell and a duct cell (noirot & quennedey, 1974). previous authors have identified the intramandibular gland ii in other hymenopterans (nedel, 1960; costa leonardo, 1978; schoeters & billen 1994). cely-ortiz observed this gland in polybia emaciata in his unpublished undergraduate work (2011). however, in vespids, penagos-arévalo et al., (2015) provide the formal record of this gland. these authors recorded the gland in four of 33 species studied, and although p. emaciata was included in their extensive study, they did not report the intramandibular gland ii in this species. here, we describe the intramandibular gland ii for polybia emaciata. materials and methods for our results to be more comparable with previous publications (penagos-arévalo et al., 2015) and because foragers are the caste involved in nest construction, foragers of polybia emaciata were net-collected from silvania, a farmland municipality in cundinamarca, colombia (4°24´19.7”n, 74° 22´30.9”w, 1475 m.a.s.l.). wasps were delicately handled with soft forceps. head section preparation followed standard protocols as described by billen (1998), samacá et al. (2013) and penagos-arévalo et al. (2015). forager heads were sectioned after the posterior part of the vertex was removed and were immediately submerged in 2% glutaraldehyde in na-cacodylate buffer for approximately 12 h at 4°c. the heads were then transferred to fresh buffer. tissues were then dehydrated in a graded acetone series and embedded in araldite. serial sections of 2 µm in thickness were obtained with a leica em-uc6 ultramicrotome (leica microsystems, vienna, austria), followed by staining with methylene blue and thionin. a zeiss jenaval optical microscope (carl zeiss, jena, germany) was used for morphological analysis and a canon d90 digital camera for photography (canon, japan). we described cell shape, cell distribution, and duct presence of the gland cells following herzner et al. (2007) and samacá et al. (2013). gland description included the following: location, cell type according to the classification provided by noirot and quennedey (1974), maximum dorsoventral or anteroposterior extension, and cell diameter (britto et al., 2004; billen, 2009; penagos-arévalo et al., 2015). results as described by penagos-arévalo et al. (2015) for other species, the intramandibular gland ii is a class 3 exocrine organ located inside the mandibles at the mesal and ectal sides close to the denticles. in p. emaciata, secretory cell clusters were formed by approximately 28 round-shaped well-defined cells, with an average diameter of 22.3 µm. the nuclear membrane was not clearly defined but between 4 and 5 nucleoli were observed per cell. ducts with an internal diameter of 0.5μm directed to the mesal part of the mandible exoskeleton were observed, although we did not find skeletal pores (fig 1). discussion contrary to our expectations, given the nature of the material used for nest construction and the potential role of head glands in processing this material, the gland characteristics observed in p. emaciata fit well within those observed for the other four species in which the gland has been observed: mischocyttarus angulatus richards, 1978; apoica gelida van der vecht, 1973; leipomeles spilogastra (cameron, 1912); and polybia liliacea (fabricius, 1804). the gland cell diameter of p. emaciata, 22.3 µm, was within the range of 10-70 µm for the other four species, as described by penagos-arévalo et al. (2015). the diameter was most similar to that observed for the distant species mischocyttarus angulatus (25 µm) and most different from that of the more closely related species polybia liliacea (70 µm). the number of glandular cells was much lower than the range of 50-150 cells observed in the other species, including in the congeneric p. liliacea (60) (penagos-arévalo et al., 2015). we did not observe any pattern for the number of cells and the species mischocyttarus angulatus and apoica gelida, show the extreme variations in cell number (50 and 150 cells, respectively). the low number of cells might be a consequence of the age stage at which wasps were captured. task partitioning is well known in vespids (jeanne, 1991), and several studies demonstrate a relationship between gland size and task frequency (downing, 1991; garcía & noll, 2013); however, studies have also debated this correlation (britto et al., 2004). we collected foragers of p. emaciata, and therefore, the gland was possibly not active in individuals at this stage. a planned sampling strategy is required to determine the relation in p. emaciata. the hypopharyngeal and clypeal glands of p. emaciata are not particularly different in gland cell diameter and cell number from other polistines of similar size or from congeneric species (penagos-arévalo et al., 2015). for example, the cell number for the hypopharyngeal gland of p. emaciata is within the range observed in other species of similar head-widths, such as brachygastra augusti (de saussure, 1854), angiopolybia pallens (lepeletier, 1836) and polybia occidentalis (olivier, 1791). for this gland in p. emaciata, the number of cells was also within the range of the number for seven other polybia species. the taxonomic distribution of this gland is somewhat challenging, because the gland has been found in only four phylogenetically dispersed genera, including mischocyttarus, sociobiology 64(3): 347-351 (september, 2017) 349 fig 1 a-b. intramandibular gland ii in forager of polybia emaciata lucas. a. general cross-sectional view of the mandibles; the inset indicates the location of gland cells at the base of the mandible. b. close-up view of framed part in a, showing gland cells (g) and ducts (d). scale bars: a=50 µm, b=20 µm. c cely, ap arévalo, j billen, ce sarmiento – intramandibular gland ii in polybia emaciata350 apoica, leipomeles and polybia itself; additionally, congeneric species of mischocyttarus and apoica that have been carefully studied previously do not present this organ (penagos-arévalo et al., 2015). within the genus polybia, we observe a similar situation, because the gland was reported for polybia liliacea (f. 1804) by penagos-arévalo et al. (2015), but not for six other species studied. in all species of neotropical polistinae studied, glands are found such as the hypopharyngeal gland, the periocular gland, and the intramandibular gland i (penagos-arévalo et al., 2015); however, either a reversal of a gland or a scattered occurrence in the polistinae has been previously reported. for example, following a more precise definition of richards´ gland, this gland apparently evolved in the epiponini but two reversals occurred (samacá et al., 2013). in the study by penagos-arévalo et al. (2015) conducted with 33 species of neotropical polistinae, the periocular gland, the posterobasal gland, the ocellar gland i, the ocellar gland ii, and the subantennal gland were reported in only a few taxa or absent in some species. clarifying the factors responsible for this lability in gland occurrence should be an interesting area of research that requires detailed information. schremmer (1984) conducted an interesting study on the nests of p. emaciata, and the results strongly indicate that organic compounds provided by the workers are the agents responsible for the physical characteristics of the structure. he hypothesized that the salivary glands could provide these molecules. although we did not directly study the salivary gland, our results and the comparative data provided by penagos-arévalo et al. (2015) for all the other head glands provide indirect support to that hypothesis, because none of the morphometric characteristics observed in these organs are significantly different from other species. a specific study on this organ will help to confirm this hypothesis. acknowledgements we are grateful to an vandoren, k.u. leuven, for her help in section preparation. we thank the referees for their helpful comments. references billen, j., & morgan, e.d. (1998). pheromone communication in social insects-sources and secretions. in r.k. vander meer, m.d. breed, m.l. winston, & k.e. espelie (eds.), pheromone communication in social insects: ants, wasps, bees, and termites. (pp. 3-33). boulder: westview press. billen, j. (2009).occurrence and structural organization of the exocrine glands in the legs of ants. arthropod structure and development, 38: 2-15. doi: 10.1016/j.asd.2008.08.002. billen, j., & wilson, e.o. 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(1992). demographic and genetic evidence for cyclical changes in queen number in a neotropical wasp, polybia emaciata. the american naturalist, 140: 363-372. doi: 10.10 86/285417. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.5654sociobiology 68(2): e5654 (june, 2021) introduction honeybees are typical social insects. a honeybee colony is usually made up of a queen, tens of thousands of workers, and hundreds of drones. the queen and workers are diploid females developed from fertilized eggs; the drones are haploid males developed from unfertilized eggs. as two female castes, the queens and workers differ significantly in morphology, physiology and behaviors (winston, 1987). thus, the differentiation of queen, worker, and drone contains two critical developmental processes: sex determination and caste differentiation. sex determination of honeybees is controlled by the cascade of csd > fem > amdsx (beye et al., 2003; cho et al., 2007; hasselmann et al., 2008; gempe et al., 2009). during the early embryonic stage, csd, as the primary signal, transmits the sex-determining signal to the downstream genes so that the embryo develops toward female (beye et al., 2003). fem is homologous to csd, and its function is to maintain and stabilize the female embryo development and abstract sex determination and caste differentiation are two crucial processes for morphology building in honeybees. it is unclear whether there is an interaction between these two processes. here, we investigated the expression of fem and amdsx genes between female castes of honeybees. we found that the expression of fem and amdsx is significantly higher in queens than in workers, and this expression was positively regulated by juvenile hormone (jh). our results suggest that sex-determining genes fem and amdsx are also involved in honeybee caste differentiation. sociobiology an international journal on social insects lu-xia pan1,2, fu-ping cheng1,2, zi-long wang1,2 article history edited by qiuying huang, huazhong agric. univ., china received 22 july 2020 initial acceptance 17 january 2021 final acceptance 13 february 2021 publication date 15 april 2021 keywords sex determination; caste differentiation; gene expression; quantitative rt-pcr; juvenile hormone. corresponding author zi-long wang honeybee research institute jiangxi agricultural university nanchang 330045, jiangxi, china. e-mail: wzlcqbb@126.com regulate the female-specific splicing of downstream gene amdsx (hasselmann et al., 2008; gempe et al., 2009). dsx is a conserved gene in insects located at the bottom of the sex determination cascade (gempe & beye, 2010). the pre-mrna of amdsx is sex-specifically spliced in males and females to produce maleand female-specific amdsx proteins to regulate sex differentiation of honeybees (cho et al., 2007). caste differentiation of female honeybees is regulated by many factors, such as nutrition (haydak, 1970; rembold & dietz, 1966; wittmann & engels, 1987), hormones (wirtz et al., 1972; asencot & lensky, 1976; rachinsky & beetsma, 1990), epigenetic modifications (kucharski et al., 2008; spannhoff et al., 2011; wojciechowski et al., 2018), etc. the queen larvae were fed royal jelly during the whole larvae stage, while the worker larvae were just fed worker jelly for three days and then were fed bee bread till pupation. factors such as sufficient food at the larvae stage, high titer of jh, and low level of genomic dna methylation in the larvae can promote the queen phenotype’s development. on the other hand, dicoumaric acid (mao et al., 2015) and 1honeybee research institute, jiangxi agricultural university, nanchang 330045, jiangxi, china 2 jiangxi province key laboratory of honeybee biology and beekeeping, nanchang 330045, jiangxi, china research article bees caste-biased expression of fem and amdsx genes in apis mellifera ligustica (hymenoptera: apidae) lu-xia pan, fu-ping cheng, zi-long wang – caste-biased expression of fem and amdsx genes in apis mellifera2 plant rnas (zhu et al., 2017) can induce worker phenotype development. studies confirmed that insulin/insulin-like growth factor 1 signaling (iis) pathway (wolschin et al., 2010; mutti et al., 2011; wang et al., 2013), the target of rapamycin (tor) signaling pathway (patel et al., 2007; mutti et al., 2011) and epidermal growth factor receptor (efgr) signaling pathways (kamakura, 2011) are involved in caste differentiation. these signaling pathways transmit the initial nutritional signals to jh, which plays a dominant role in caste differentiation. compared with worker larvae, the titer of jh in queen larvae is 26 times higher than that in worker larvae during the 4th to 5th instars (rachinsk & hartfelder, 1990). meanwhile, topical application of synthetic jh to worker larvae leads to the development of queen-like traits (rembold et al., 1974). although both sex determination and caste differentiation pathways are related to the development of honeybees’ morphological and physiological traits, the possible link between these two pathways is poorly understood. recent studies have shown that sex-determining genes may also play a role in caste differentiation (brito et al., 2015; klein et al., 2016; roth et al., 2019). in this study, we compared the expression of fem and amdsx between queen and worker castes and assessed the effect of jh on their expression. we found caste-biased expression of fem and amdsx, which indicates an interaction between sex determination pathway and caste differentiation pathway. materials and methods sample collection the honeybees used in this study were western honeybee, apis mellifera ligustica spinola, 1806. according to standard beekeeping techniques at the honeybee research institute, we raised colonies, jiangxi agricultural university, nanchang, china (28°46’ n, 115°49’ e). three colonies of a. m. ligustica with similar populations were selected. the queen of each colony was confined on a comb to lay eggs for six hours. then, we transferred the comb to a super box of the same colony. when the eggs hatched into larvae, some of the larvae were grafted to queen cells, and all the larvae continued to be bred in a super box. when the larvae reached 48 h, 84 h, and 120 h, the queen larvae and worker larvae were sampled respectively to analyze expression differences of fem and amdsx between them by qrt-pcr. at 48 h, every three larvae were collected as a sample; at 84 h and 120 h, each larva is a sample. during sampling, the larvae were washed three times with double distilled water. seven biological replicates were sampled for each time point. the head and stomach of newly emerged queens and workers with five biological replicates were sampled. during sampling, the stomach and intestines of bees were removed. all the samples were quickly frozen in liquid nitrogen and were stored at -80 °c. juvenile hormone treatment an a. m. ligustica colony was used in the jh treatment experiment. the queen was limited on a comb to lay eggs for six hours. when the eggs hatched, the 1-day old larvae were grafted to 24-well cell culture plates with 200 μl/hole artificial larval food (glucose 6%, fructose 6%, royal jelly 50%, yeast extract 1%, distilled water 37%). they were randomly divided into three groups. one group was topically applied with 1µl of jh iii (apexbio, purity ≥ 65.00%) solution diluted in ethanol (10 µg/µl). the second group was topically applied with 1µl of ethanol and was set as control. the third group was normal larvae without any treatment. larvae were reared in an incubator with a temperature of 34 °c and humidity of 85%. for the next two days, the jh treatment group and ethanol treatment group continued to be treated with jh iii or ethanol three times every day. twenty-four hours after the end of the treatment, larvae of the three groups were collected to detect mrna expression changes of fem and amdsx through qrt-pcr. for each group, seven biological replicates were sampled. after washing three times with double distilled water, the larvae were immediately frozen in liquid nitrogen and stored at -80 °c for rna extraction. quantitative real-time pcr total rna was extracted from the above samples according to the instructions of the transzol up plus rna kit (transgen biotech, beijing, china). cdna was obtained by reverse transcription following the instructions of the primescripttm rt reagent kit (takara, japan). the housekeeping gene amactin of a. mellifera was used as an internal reference gene. quantitative real-time pcr primers were designed based on mrna sequences of fem, amdsx, and amactin with primer premier 5.0 software. the sequences of primers are listed in table 1. table 1. primer sequences used for qrtpcr assays. gene accession number primer sequence (5’ to 3’) fem eu101388.1 forward: ccaatgcggggtcaagta reverse: tatctggaggaataaatcgtg amdsx eu236954.1 forward: ctcacactgcgatggtcac reverse: cgtctcactacttcttccg amactin nm_001185145.1 forward: gtattgtattggattcgggtg reverse: tgccatttcctgttcaaagtca sociobiology 68(2): e5654 (june, 2021) 3 amplification conditions of qrt-pcr: 50 °c for 2 min, predenaturation at 95 °c for 10 min, followed by 40 cycles of 95 °c for 10 s, (fem, 58.2 °c; amdsx, 52.9 °c and amactin, 52.9 °c) for 1 min. for each sample, the specificity of the pcr amplification was verified by melting curve analysis. the data were analyzed by the comparative ct method (2-δδct) (schmittgen & livak, 2008). the expression differences of fem and amdsx between samples were analyzed by t-test or one-way anova using spss17.0 software (spss inc., 2008). results fem and amdsx have expression differences between queen and worker the expression levels of fem and amdsx in queen larvae and worker larvae at 48 h, 84 h, and 120 hand in the head and stomach of adult queens and workers were detected by qrtpcr analysis. the results showed that: the expression levels of fem in queen larvae at 84 h and 120 h were significantly higher than those in worker larvae (84 h: t=3.99, df=4.23, p=0.0146; 120 h: t=6.25, df=8.00, p=0.0002) (fig 1), and there was no significant difference at 48 h (t=0.39, df=8.00, p=0.7053); the expression levels of amdsx in queen larvae at all the three stages were significantly higher than those in worker larvae (48 h: t=-3.46, df=8.00, p=0.0085; 84 h: t=-7.56, df=8.00, p<0.0001; 120 h: t=4.04, df=5.98, p=0.0069); both fem and amdsx expressed higher in stomach of queen than in that of worker (fem: t=-6.48, df=4.58, p=0.0018; amdsx: t=-9.30, df=8.00, p<0.0001) (fig 2), and there were no significant expression difference of fem and amdsx between the head of queen and worker (fem: t=-1.79, df=8.00, p= 0.1107; amdsx: t=-0.74, df=7.98, p= 0.4786). fig 1. expression differences of fem and amdsx between queen larvae and worker larvae. *represent significant difference (t-test, p<0.05). fig 2. expression differences of fem and amdsx between head and stomach of queen and that of workers. *represent significant difference (t-test, p<0.05). lu-xia pan, fu-ping cheng, zi-long wang – caste-biased expression of fem and amdsx genes in apis mellifera4 jh up-regulated fem and amdsx after treatment of larvae with jh, the expression levels of both fem and amdsx in jh treatment group were significantly up-regulated compared with the two control groups, and there was no significant difference between the two control groups (fem: jh vs. normal vs. ethanol, f(2, 18)=20, p<0.0001; jh vs. ethanol, p=0.0006; jh vs. normal, p<0.0001; ethanol vs. normal, p=0.0639. amdsx: jh vs normal vs. ethanol, f(2, 18)=20, p=0.0005; jh vs. ethanol, p=0.0003; jh vs. normal, p=0.0009; ethanol vs. normal, p=0.6773) (fig 3). fig 3. expression change of fem and amdsx after jh iii treatment. *represent significant difference (anova, p<0.05). discussion fem and amdsx are critical regulators in the sex determination cascade of honeybees. several studies have indicated that sex-determining genes may also be involved in caste differentiation (brito et al., 2015; klein et al., 2016; roth et al., 2019). a study in stingless bee melipona interrupta showed that the fem gene expressed higher in the queen than in the worker (brito et al., 2015). in ant cardiocondyla obscurior, dsx exhibits both sex and form-specific expression across life stages (klein et al., 2016). knocking out fem and amdsx using crispr/cas9, (roth et al., 2019) found that nutrition could affect the gonad size of honeybees through the fem gene. in this study, we also found that fem and amdsx had higher expression levels in the queen than in the worker. it suggests that fem and amdsx play a critical role in sex determination and are involved in caste differentiation of female bees. jh plays a vital role in the process of honeybee caste differentiation. the increase of jh can make female larvae develop into queens (de oliveira campos et al., 1975) and promote the increase in the number of ovarian tubes in workers (dorn, 1985). we investigated the relationship between fem, amdsx, and jh, and found that the expression levels of fem and amdsx in the larvae were significantly increased after jh iii treatment. this indicates that jh regulates the differential expression of fem and amdsx between queen and worker. the increase of fem and amdsx expression after jh treatment is associated with queen development. in conclusion, our study indicates that sex-determining genes fem and amdsx are also involved in honeybee caste differentiation, and they are positively regulated by jh. further studies are needed to clarify how these sexdetermining genes function in the caste differentiation process of honeybees. acknowledgments this work was supported by the national natural science foundation of china (no. 31402147, 31860686) and the natural science foundation for outstanding young scholars of jiangxi province (no. 2018acb21028). authors contribution zw: conceptualization, methodology lp: resources, invetigation, writing fc: investigation zw: writing references asencot, m. & lensky, y. 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(2010). insulin receptor substrate influences female caste development in honeybees. biology letters, 7: 112-115. doi: 10.1098/rsbl. 20100463. zhu, k., liu, m., fu, z., zhou, z., kong, y., liang, h., lin, z., luo, j., zheng, h., wan, p., zhang, j., zen, k., chen, j., hu, f., zhang, c.y., ren, j. & chen, x. (2017). plant micrornas in larval food regulate honeybee caste development. plos genetics, 13: e1006 946. doi: 10.1371/journal.pgen.1006946. ole_link39 ole_link40 ole_link41 ole_link1 ole_link42 ole_link44 ole_link2 ole_link6 ole_link7 _goback ole_link17 ole_link13 ole_link15 ole_link14 ole_link9 ole_link5 ole_link12 ole_link4 ole_link3 ole_link10 ole_link11 ole_link16 ole_link8 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1215sociobiology 64(1): 125-129 (march, 2017) social wasps (vespidae: polistinae) from an amazon rainforest fragment: ducke reserve the polistinae social wasps comprise 26 genera and 958 species, and brazilian social wasps fauna include the richest in the world, with 321 species (carpenter & marques, 2001). species in this subfamily belong to three tribes: mischocyttarini (mischocyttarus, with 117 species in brazil), polistini (polistes, with 38 species in brazil), and epiponini (20 genera, 166 species in brazil) (carpenter, 2004; pickett & carpenter, 2010). for the brazilian amazonian rainforest, 20 genera and more than 200 species were recorded, representing about 70% of the brazilian fauna of social wasps (silveira, 2002). recently, some works have been carried out in the brazilian amazon, as follow: maracá ecological station, roraima state with 36 species (raw, 1998), caxiuanã reserve, pará state with 79 and 65 species, respectively (silveira, 2002; silva & silveira, 2009), serra do divisor national park, acre state with 20 species (morato et al., 2008), lakes of amapá state with31 species(silveira et al., 2008), mamirauá and alvarães reserve, amazonas state with 46 and 42 species, abstract social wasps are common elements in neotropics, although even elementary data about this taxon in the amazon region is partially unknown. therefore the purpose of this work was to increase the knowledge of social wasp fauna at the ducke reserve, amazonas. one hundred and three species belonging to nineteen genera were recorded. the richest genera were polybia (28 species), agelaia (12) and mischocyttarus (12). seventy species were collected in active search, 42 species using malaise trap, 25 in suspended trap, 20 in attractive trap and nine in light trap. ducke reserve has one of the highest number of polistinae wasps in reserves or parks in the neotropic region. sociobiology an international journal on social insects a. somavilla1,2, m.l. de oliveira1 article history edited by gilberto m. m. santos, uefs, brazil received 16 october 2016 initial acceptance 13 february 2017 final acceptance 14 february 2017 publication date 29 may 2017 keywords agelaia, amazon rainforest, inpa, paper wasps, polybia. corresponding author alexandre somavilla instituto nacional de pesquisas da amazônia coordenação de biodiversidade avenida andré araújo, 2936 petrópolis, cep 69067-375 manaus, amazonas, brasil e-mail: alexandresomavilla@gmail.com respectively (silveira et al., 2008),gurupi biological reserve with 38 species (somavilla et al., 2014), jaú national park with 49 species (somavilla et al., 2015) and embrapa-manaus with 52 species (somavilla et al., 2016). despite the constant efforts in studies about social wasps conducted in brazil, little is known regarding the diversity and distribution of these in the amazon region. thus, this work aims to expand knowledge about the species of social wasps in ducke reserve, manaus, amazonas, brazil. the ducke reserve comprising an area of approximately 100 km2 of “terra firme” rainforest near manaus city, amazonas, brazil (02º55’ to 03º01’s and 59º53’ to 59º59.5’ w) (baccaro et al., 2008). the climate is humid tropical, with mean annual relative humidity of about 80% and mean annual precipitation of 1,750-2,500 mm and mean annual temperature is 26ºc. vegetation is lowland tropical rainforest, with fairly closed canopy and shady understory, characterized by the abundance of palm trees (ribeiro et al., 1999; costa et al., 2008). instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus, amazonas, brazil short note a. somavilla, m.l. de oliveira – social wasps from ducke reserve126 we analyze all the wasps deposited at the zoological collection of invertebrates at the instituto nacional de pesquisas da amazônia (inpa) in manaus, brazil. these wasps were collected anteriorly to the development period of this paper. identification of the specimens followed the keys proposed by richards (1978) and reference collections. wasps were collected with entomological net in the understory (active search); additionally, malaise traps (townes model); suspended trap on the heights of 1.5m; 10m; 15m; 20m; 30m and 45m (rafael & gorayeb model); light traps (a white sheet attached to a white light); meat attractive trap (rattus norvegicus berkenhoid, 1769) and fruit attractive trap (banana). we identified 103 social wasp species, in 19 genera (table 1). about 50% of the collected species belong to three genera: polybia lepeletier (28 species), agelaia ducke (12 species), and mischocyttarus de saussure (12 species). only asteloeca raw and protonectarina ducke were not collected in the ducke reserve. the scope of this inventory is similar to the silveira (2002) in the ferreira penna scientific station, in caxiuanã, pará, that the author collected 79 species in 18 genera in intensive work active search and different traps in an area predominantly constituted of primary forests. the similarity of results obtained in ducke reserve and caxiuanã surprising, considering the proximity of ducke with the city of manaus, unlike the preservation condition which is that of caxiuanã, fully isolated from any human action. many species of social wasps have preferences for particular types of habitat, in this way, the ambiguous character of the ducke reserve may have influenced the high species richness. for the part north of the reserve consists of preserved sites with high forest and closed canopy favoring the collection of taxa such as chartergellus, charterginus, chartergus, clypearia, epipona, leipomeles and nectarinella and some species of mischocyttarus, polybia, agelaia and angiopolybia which usually occur in more preserved sites. the part south of the reserve, with areas close to urban areas, most affected by human activities, having more open spaces and even degraded, favoring the collection of some species that are more tolerant to these types of environments and support such conditions, as agelaia pallipes, polybia rejecta, p. chrysothorax, p. sericea. these species are hardly collected in closed vegetation environments creating a network with low light. a few are common in built-up areas as polistes, metapolybia, some mischocyttarus’ species and polybia occidentalis. comparing to the different techniques used in the wasps collect, we considered active search the most efficient method for collecting vespidae, was collected in active search 70 species, 42 species using malaise trap, 25 using suspended trap, 20 attractive trap and only nine species using light trap. this study corroborates the results of silva and silveira (2009) and somavilla et al. (2014), who considered active search the most efficient method for collecting vespidae, mainly close to the forest ground and in the understory; in this study, we collected 70 species in active search in ducke reserve. but the addition of different collection methods is an important tool for sampling of the richness of social wasps in an area, as in general the species have a varied foraging behavior. fourty-two species were collected using malaise trap, demonstrating the efficiency of this method for this taxon. in fast inventories, the use of such a trap can be a good way to sample this hornet’s group, mainly those that tend to forage in the forest understory. however, the use of malaise trap as the only way of sampling for social wasps may underestimate the real number of species, mainly of polistes and mischocyttarus in a particular area (silveira, 2002). twenty-five species were collected using suspended interceptation traps, confirming that for the amazon, where the canopy forest is high, this technique is one of the most efficient to collect social wasps. apoica does its foraging at night, reducing the possibility of being captured during the day by active search. in this study, seven apoica species were only captured in light traps. still, for twenty species belonging mainly to agelaia and polybia the attractive trap was efficient; however, the use attractive traps as the only way for collecting social wasps may underestimate the real number of polistes, mischocyttarus and some epiponini taxa. in surveys conducted in the amazon rainforest, wasp’s diversity is generally higher than other biomes, but in ducke reserve, exceeds these indices, with 103 species. in amazonas state, the closest diversity number (only 50% of this value) was found in embrapa, and for others states of amazon region, the closest diversity (about 75% of this value)in caxiuanã, pará. however, this difference can make up the sampling effort of several years in ducke reserve. adding the new occurrences, together with the material already registered by somavilla et al. (2014), it is suggested that the ducke reserve has one of the highest number of social wasps’ species (polistinae) in reserves or parks in the neotropical region. acknowledgements we thank to conselho nacional de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado do amazonas (fapeam) and biodiversity research program (ppbio) for support. references baccaro, f.b., drucker, d.p., do vale, j.,de oliveira, m.l., magalhães, c., lepsch-cunha, n. & magnusson, w.e. (2008). a reserva ducke; pp. 11-20, in: de oliveira, m.l., baccaro, f.b., braga-neto, r. & magnusson, w.e. (eds.). reserva ducke, a biodiversidade amazônica através de uma grade. instituto nacional de pesquisas da amazônia, manaus: áttema design editorial, 166p. sociobiology 64(1): 125-129 (march, 2017) 127 carpenter, j.m. 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(2008). avaliação ecológica rápida da fauna de vespas (hymenoptera: aculeata) do parque nacional da serra do divisor, acre, brasil. acta amazonica, 38: 789-798. pickett, k.m. & carpenter, j.m. (2010). simultaneous analysis and the origin of eusociality in the vespidae (insecta: hymenoptera). arthropod systematics and phylogeny, 68: 3-33. raw, a. (1998). social wasps (hymenoptera: vespidae) of the ilha de maracá; pp. 307-321, in: j.a. ratter and w. milliken (eds.). maracá. biodiversity and environment of an amazonian rainforest. new york: chichester, john & sons. 508 p. ribeiro, j.e.l.s., hopkins, m.j.g.,vicentini, a., sothers, c.a., costa, m.a.s., brito, j.m., souza, m.a.d., martins, l.h.p., lohmann, l.g., assunção, p.a.c.l., pereira, e.c., silva, c.f., mesquita, m.r. & procópio, l.c. (1999). flora da reserva ducke: guia de identificação das plantas vasculares de uma floresta de terra-firme na amazônia central. instituto nacional de pesquisas da amazônia, manaus.799 p. silva, s.s. & silveira, o.t. (2009). vespas sociais (hymenoptera, vespidae, polistinae) de floresta pluvial amazônica de terra firme em caxiuanã, melgaço, pará. iheringia, série zoologia, 99: 317-323. silveira, o.t. (2002). surveying neotropical social wasps. an evaluation of methods in the “ferreira penna” research station (ecfpn), in caxiuanã, pa, brazil (hym., vespidae, polistinae). papéis avulsos de zoologia, 42: 299-323. somavilla, a., de oliveira, m.l. & silveira, o.t. (2014). diversity and aspects of the ecology of social wasps (vespidae, polistinae) in central amazonian “terra firme” forest. revista brasileira de entomologia, 58: 349-355. somavilla, a., andena, s.r. & de oliveira, m.l. (2015). social wasps (hymenoptera: vespidae: polistinae) of the jaú national park, amazonas, brazil. entomobrasilis, 8: 45-50. somavilla, a., marques, d.w.a., barbosa, e.a.s., junior, j. da s.p, de oliveira, m.l. (2014). vespas sociais (vespidae: polistinae) em uma área de floresta ombrófila densa amazônica no estado do maranhão, brasil. entomobrasilis, 7: 183-187. somavilla, a., schoeninger, k., castro, d.g.d., oliveira, m.l., krug, c. 2016. diversity of wasps (hymenoptera: vespidae) in conventional and organic guarana (paullinia cupana var. sorbilis) crops in the brazilian amazon. sociobiology, 63: 1051-1057. table 1. species of social wasps collected at ducke reserve, amazonas, brazil, as well as the method used to capture the species. active (active search with entomological net); malaise (malaise trap); suspended (suspended trap); light (light trap); meat (attractive meat trap); fruit (attractive fruit trap): taxa active malaise suspended light meat fruit polistinae epiponini agelaia acreana silveira & carpenter, 1995 x agelaia angulata (fabricius, 1804) x x x agelaia cajennensis fabricius, 1798 x x x x agelaia centralis (cameron, 1907) x agelaia constructor (de saussure, 1854) x x x x agelaia fulvofasciata (degeer, 1773) x x x x x agelaia hamiltoni richards, 1978 x agelaia myrmecophila (ducke, 1905) x x agelaia ornata (ducke, 1905) x agelaia pallidiventris richards, 1978 x agelaia pallipes (olivier, 1791) x x x x agelaia testacea (fabricius, 1804) x x x x x angiopolybia obidensis (ducke, 1904) x x x angiopolybia pallens (lepeletier, 1836) x x x x a. somavilla, m.l. de oliveira – social wasps from ducke reserve128 table 1. species of social wasps collected at ducke reserve, amazonas, brazil, as well as the method used to capture the species. active (active search with entomological net); malaise (malaise trap); suspended (suspended trap); light (light trap); meat (attractive meat trap); fruit (attractive fruit trap): (continuation) taxa active malaise suspended light meat fruit epiponini angiopolybia paraensis (spinola, 1851) x x x apoica albimacula (fabricius, 1804) x x apoica arborea de saussure, 1854 x x x apoica gelida van der vecht, 1973 x apoica pallens (fabricius, 1804) x x x apoica pallida (olivier, 1791) x apoica strigata richards, 1978 x apoica thoracica du buysson, 1906 x x x x x brachygastra billineolata spinola, 1841 x brachygastra scutellaris (fabricius, 1804) x chartergellus amazonicus richards, 1978 x x x chartergellus jeannei andena & soleman, 2015 x charterginus fulvus fox, 1898 x chartergus chartarius (olivier, 1791) x clypearia apicipennis (spinola, 1851) x clypearia duckei richards, 1978 x clypearia sulcata (de saussure, 1854) x x x x epipona tatua (cuvier, 1797) x leipomeles dorsata (fabricius, 1804) x leipomeles pussila (ducke, 1904) leipomeles spilogastra cameron, 1912 x x metapolybia nigra richards, 1978 x x metapolybia rufata richards, 1978 x metapolybia unilineata (r. von ihering, 1904) x nectarinella manauara silveira & nazareno jr, 2016 x parachartergus fasciipennis ducke, 1905 x parachartergus fraternus (gribodo, 1892) x x x x parachartergus griseus (fox, 1898) x parachartergus richardsi willink, 1951 x x parachartergus smithii de saussure, 1854 x polybia belemensis richards, 1970 x x x polybia bicyttarella richards, 1951 x polybia bifasciata de saussure, 1854 x polybia bistriata (fabricius, 1804) x x x polybia chrysothorax (lichtenstein, 1796) x polybia depressa (ducke, 1905) x x polybia dimidiata (olivier, 1791) x x x polybia dimorpha richards, 1978 x x polybia emaciata lucas, 1879 x polybia fastidiosuscula de saussure, 1854 x polybia gorytoides fox, 1898 x polybia ignobilis (haliday, 1836) x polybia jurinei de saussure, 1854 x x x polybia liliacea (fabricius, 1804) x x polybia occidentalis (olivier, 1791) x x sociobiology 64(1): 125-129 (march, 2017) 129 table 1. species of social wasps collected at ducke reserve, amazonas, brazil, as well as the method used to capture the species. active (active search with entomological net); malaise (malaise trap); suspended (suspended trap); light (light trap); meat (attractive meat trap); fruit (attractive fruit trap): (continuation) taxa active malaise suspended light meat fruit epiponini polybia signata ducke, 1905 x polybia singularis ducke, 1905 x polybia striata (fabricius, 1787) x polybia tinctipennis fox, 1898 x polybia velutina ducke, 1905 x protopolybia bituberculata silveira & carpenter, 1995 x protopolybia chartergoides gribodo, 1891 x x protopolybia duckeianus richards, 1978 x protopolybia emortualis (de saussure, 1855) x protopolybia holoxantha (ducke, 1904) x protopolybia minutissima (spinola, 1851) x protopolybia rugulosa ducke, 1907 x protopolybia sedula (de saussure, 1854) x pseudopolybia compressa (de saussure, 1854) x pseudopolybia langi bequaert, 1944 x pseudopolybia vespisceps (de saussure, 1863) x synoeca surinama (linnaeus, 1767) x x x synoeca virginea (fabricius, 1804) x x x mischocyttarini mischocyttarus atramentarius zikan, 1949 x mischocyttarus collaris (ducke, 1904) mischocyttarus flavicans (fabricius, 1804) x mischocyttarus imitator (ducke, 1904) x x mischocyttarus labiatus (fabricius, 1804) x mischocyttaruslecointei (ducke, 1904) x mischocyttarus metathoracicus (de saussure, 1854) x mischocyttarus prominulus richards, 1941 x mischocyttarus rotundicollis (cameron, 1912) x mischocyttarus surinamensis (de saussure) x mischocyttarus synoecus richards, 1940 x mischocyttarus helliconius group x polistini polistes canadensis (linnaeus, 1758) x polistes geminatus fox, 1898 x polistes goeldi ducke, 1904 x polistes pacificus fabricius, 1804 x polistes testaceicolor bequaert, 1798 x polistes versicolor olivier, 1792 x doi: 10.13102/sociobiology.v60i4.446-452sociobiology 60(4): 446-452 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 neotenic reproductives influence worker caste differentiation in the termite reticulitermes speratus (isoptera; rhinotermitidae) y hayashi1,2, h miyata2, o kitade2, n lo1 introduction a defining characteristic of social insect colonies is the presence of different castes. each caste has specialized behavior and morphology, and carries out specific tasks for colony development. such division of labor between castes accounts for the high ergonomic efficiency of insect societies and thus their ecological success (oster & wilson, 1978). because ergonomic efficiency critically depends on caste composition of the colony (oster & wilson, 1978), colonies are expected to adjust caste ratios in changing environments and during different developmental stages of the colony life cycle (wilson, 1971; schmid-hempel, 1992; helms cahan et al., 2010; lecoutey et al., 2011). mechanisms for controlling caste ratio are thus likely to evolve adaptively so that caste compositions can promptly reach their optima (schmidhempel, 1992; helms cahan et al., 2010). proximate factors that determine caste fates of individuals play an important abstract division of labor among castes in social insect colonies increases ergonomic efficiency and colony-level fitness, and has played a key role in the ecological success of social insects. knowledge of the factors that regulate castes is important for understanding adaptive social organization. our previous study on the termite reticulitermes speratus demonstrated that the presence of a pair of nymphoid reproductives during development affected offspring caste ratios. in the present study, we investigated further the influence of individual neotenics on offspring caste ratios. parthenogenetically-produced offspring were reared in worker-tended experimental colonies with the addition of different forms (nymphoid or ergatoid) and numbers of neotenics, and compared the caste ratios of the offspring between the different experimental treatments. we found that all offspring in worker-only tended colonies became nymphs, while a proportion of offspring in colonies with a single neotenic (with the exception of male ergatoids) differentiated into workers. these results show offspring caste ratios are influenced by the presence of single female ergatoids, single female and male nymphoids, while they remain unaffected by the presence of male ergatoids. sociobiology an international journal on social insects 1 the university of sydney, sydney, australia. 2 ibaraki university, mito, ibaraki, japan. research article termites article history edited by og de souza, ufv, brazil received 12 july 2013 initial acceptance 21 august 2013 final acceptance 11 september 2013 keywords caste determination, parental effect, parthenogenesis, social organization corresponding author: yoshinobu hayashi college of science, ibaraki university mito, ibaraki 310-8512, japan e-mail: yoshinobu.hayashi.2012@gmail.com role in the control of caste ratio (hughes & boomsma, 2008; smith et al., 2008a), and are key to understanding adaptive social organization (helms cahan et al., 2010). proximate factors influencing caste determination have been the subject of many studies over several decades. most of these studies demonstrated that environmental factors influence caste determination, and it became generally accepted that caste differentiation was controlled purely by environmental factors (wilson, 1971; wheeler, 2003). in termites, a major group of social insects, caste development is highly plastic and environmental signals are thought to be critical for caste determination (wilson, 1971; miura, 2001; miura et al., 2003; scharf et al., 2003; scharf et al., 2005; scharf et al., 2007; matsuura et al., 2010; hartke & baer, 2011). in recent years, however, genetic influences on caste determination have been reported in some social insects (reviewed in crozier & schluns, 2008; smith et al., 2008b; schwander et al., 2010). sociobiology 60(4): 446-452 (2013) 447 our previous studies on the japanese subterranean termites reticulitermes speratus (hayashi et al., 2007), r. okinawanus, r. kanmonensis, and r. yaeyamanus (kitade et al., 2011) provided evidence for genetic influences on caste determination of larvae. in reticulitermes, each individual becomes either a nymph or a worker after two larval instar stages (buchli, 1958; shimizu, 1970; takematsu, 1992). nymphs have wing buds and are able to eventually develop into alates (adults), which can establish new colonies and become kings/ queens. workers are functionally sterile and never develop wing buds. under some conditions (e.g. in the absence of reproductives under laboratory conditions), workers and nymphs can differentiate through special molts into neotenics, which are reproductive individuals retaining juvenile characteristics (watanabe & noda, 1991; thorne et al., 1999; miyata et al., 2004; leniaud et al., 2011). neotenics derived from workers are termed “ergatoids”, which are apterous, and those from nymphs are termed “nymphoids”, which have wing buds. studies of natural colonies have shown multiple nymphoids are typically present, and that they play a key role in reproduction (matsuura et al., 2009). ergatoids, on the other hand, have not yet been found in extensive surveys of natural colonies (matsuura et al., 2009; matsuura et al., 2010), although there is some evidence that they may be present (shimizu, 1970). in our previous study of r. speratus (hayashi et al., 2007) we carried out crossing with four possible combinations of ergatoids and nymphoids, and examined caste and sex of their offspring, which were reared under uniform environmental conditions. there were extreme differences in the proportions of caste and sex between offspring from different combinations of parents, indicating that genetic factors influenced the developmental commitment to the worker or nymph caste. we also showed that almost all of the offspring produced through parthenogenesis developed into nymphs when they were reared only with tending workers, while 24% of parthenogenetically-produced offspring differentiated into workers when reared with an additional pair of nymphoids (hayashi et al., 2007). this suggests that the existence of pairs of nymphoids can influence the developmental trajectory of larvae. in our previous study, however, the effects of individual neotenics were not surveyed. thus, in this study, we focused on examining whether individual ergatoids and nymphoids influence proportion of worker caste among offspring. we reared eggs produced through parthenogenesis in treatments with workers plus a single female or male ergatoid or nymphoid (fig 1). we also included a treatment with a pair of nymphoids [to replicate our previous study (hayashi et al., 2007)], and a control treatment (with workers only). to examine variation of the influence on offspring caste ratio among different sexes and forms (nymphoid/ergatoid) of neotenics within a colony, we made replication of the experiment with neotenics collected from a single field colony. materials and methods termites two r. speratus colonies (a and b) were collected from mito shinrin park, mito, ibaraki, japan (36°25′n, 140°22′e) with their nest woods at 7 jun and 25 july 2005 respectively. the colonies were transferred to the laboratory and maintained in large plastic boxes at room temperature until 1st aug when experiments were commenced. experiments to obtain parthenogenetic eggs for the experiments, we first made “ergatoid egg-source colonies” (n = 3) that consisted of three virgin ergatoids and 100 female workers, and “nymphoid egg-source colonies” (n = 3) that contained three virgin nymphoids and 100 female workers. male workers were not used for the care of virgin neotenics. this is because they are able to differentiate into male ergatoids, which are difficult to distinguish morphologically from workers (pichon et al., 2007; fujita & watanabe 2010), and male ergatoids may copulate with females and prevent us from obtaining parthenogenetic eggs. to obtain virgin ergatoids and nymphoids to be used for the egg-source colonies, we collected workers and nymphs from the field colony a. we then divided them fig 1. schematic diagram of the experiment. “e”, “n” and “w” represent ergatoid, nymphoid and worker, respectively. two kinds of egg-source colonies were established: “ergatoid egg-source colonies”, which contained 3 female ergatoids and 200 workers, and “nymphoid egg-source colonies”, which contained 3 female nymphoids and 200 workers. offspring-rearing colony w, ♀e, ♂e, ♀n, ♂n, and ♀n♂n consisted of 50 workers, a female ergatoid and 50 workers, a male ergatoids and 50 workers, a female nymphoid and 50 workers, a male nymphoid and 50 workers, and a female and a male nymphoid and 50 workers, respectively. female neotenics in egg-source colonies were obtained from field colony a and the other individuals were from field colony b. arrows show egg transfer from the egg-source colony to the offspring-rearing colony. y hayashi, h miyata, o kitade, n lo worker caste differentiation in reticulitermes speratus448 by sex on the basis of sternite morphology, as described in r. flavipes (thompson & snyder, 1920) and prepared an artificial colony that consisted only of female workers and female nymphs. in the colony some workers and nymphs began to differentiate into nymphoids and ergatoids within a month, as reported in (watanabe & noda, 1991; miyata et al., 2004). the female workers included with virgin neotenics were obtained from the field colony b. we then made six types of offspring-rearing colonies: (1) colony w, containing 50 workers, (2) colony ♀e, containing a female ergatoid and 50 workers, (3) colony ♂e, containing a male ergatoid and 50 workers, (4) colony ♀n, containing a female nymphoid and 50 workers, (5) colony ♂n, containing a male nymphoid and 50 workers, and (6) colony ♀n♂n, containing a pair of nymphoids and 50 workers (fig 1). because our previous study revealed that a pair of nymphoids influenced the offspring caste differentiation, we set up colony ♀n♂n as a positive control for the experiment. colony w was established as a negative control. the workers were randomly extracted from field colony b irrespective of their sex. the ergatoids and nymphoids of the offspringrearing colonies were obtained from artificially orphaned colonies that consisted of workers and nymphs of both sexes from field colony b. six replications were made for each of the six types of offspring-rearing colonies. eggs produced in each of the ergatoid egg-source colonies were reared in three of them, while those of the nymphoid egg-source colonies were reared in the other three. from the egg-source colonies, we collected welldeveloped eggs that had exhausted their yolk and transferred them equally into the six types of offspring-rearing colonies. some reticulitermes species, including r. speratus, do not distinctively eliminate eggs of the other congeneric species (matsuura et al., 2007), and also in this study eggs of the neotenics (derived from field colony a) were piled up and reared by the non-nestmate workers (from field colony b). because the positive control (i. e., colony ♀n♂n) was set up based on the result of our previous study (hayashi et al., 2007), in which eggs were transferred into colonies comprised of workers derived from a different field colony, we followed the method of the previous study and reared eggs with workers from a different colony also in the present experiment. we reared the offspring until they reached the third instar, and then identified their caste on the basis of wing bud development. young eggs laid by the neotenics in the offspringrearing colonies, which could be easily discriminated from the well-developed eggs transferred from egg-source colonies, were carefully removed. all of the above experimental treatments were carried out once every three days. all rearing experiments were carried out at 25°c under constant darkness. the termites were fed with a mixture of cellulose powder and sawdust of quercus woods (mixed food). we provided arbitrary amount of the mixed food when termites almost exhausted it. we continued the experiments for 360 days. we determined the castes of offspring at the third instar on the basis of presence/absence of wing buds. a small number of offspring (10 individuals) in the offspring-rearing colonies with neotenics were morphologically intermediate with respect to their wing buds; these were reared up to fourth or fifth instar to determine their caste, and 8 of 10 offspring were classified as nymph caste. we examined the number of offspring that differentiated into workers and nymphs, for each type of offspring-rearing colony, and then calculated proportions of each caste to the total number of third-instar offspring. survival rates of offspring were calculated by dividing the number of offspring that developed to the third instar by the number of eggs that were transferred into offspring-rearing colonies. statistical analyses for testing difference in survival rates between offspring produced by nymphoids and those by ergatoids of the egg-source colonies, logistic regression analysis was carried out. offspring survival status (i. e., death or survival), which is considered to follow the binomial distribution, was set as the dependent variable, and types of offspring-rearing colonies and forms (ergatoid/nymphoid) of the mother neotenics in egg-source colonies as the independent variables. interaction between those two dependent variables was not significant in the preliminary logistic regression analysis and we used the model without the interaction for the logistic regression. effect of the forms of the mother neotenics in eggsource colonies and forms and sexes of neotenics in the offspring-rearing colonies on offspring caste ratio was analyzed by logistic regression analysis. in the analysis, offspring caste (nymph or worker) was set as the dependent variable, and types of offspring-rearing colonies and forms of the mother neotenics in egg-source colonies as the independent variables. in the preliminary logistic regression analysis with the interaction term, because there were no significant effects of the interaction between the independent variables, we used the model without the interaction. we applied the logistic regression using the penalized maximum likelihood estimation (firth, 1993; kosmidis, 2007), because no offspring differentiated into workers in some offspring-rearing colonies and thus the logistic regression analysis included some zerocells in the contingency table of offspring caste status, which causes bias of the maximum likelihood estimates (allison et al., 2004). we carried out all of the logistic regression analyses in r2.13.1 (r development core team, 2011). in the logistic regression analyses, to examine effects of types of the offspring-rearing colonies, colony w was set as the reference level and compared with the other colony types. for the logistic regression with penalized method for maximum likelihood estimation the r function 'brglm' (kosmidis, 2008) was used. sociobiology 60(4): 446-452 (2013) 449 results we obtained 184, 194, and 267 eggs from three replicates of egg-source colonies with parthenogenetic ergatoids, and 341, 232, and 253 eggs from those with parthenogenetic nymphoids. number of eggs was not significantly different between nymphoids and ergatoids (student’s t test, t = -1.42, d. f. = 4, p = 0.23), although it is common in reticulitermes that nymphoids have higher fecundity than ergatoids (myles 1999). we divided those eggs almost equally into six types of offspring-rearing colonies. we obtained 101, 109 and 136 third-instar offspring from ergatoid egg-source colonies and 186, 119, and 133 from nymphoid egg-source colonies. the number of eggs reared and the number of offspring that developed to the third instar in each offspring-rearing colony are shown in table 1. cantly different between w and ♀e, w and ♂n, and w and ♀n♂n colonies (logistic regression analysis; wald χ2 = 5.313, p = 0.021; wald χ2 = 6.755, p = 0.009; wald χ2 = 7.166, p = 0.007; respectively). the difference between w and ♀n was marginally significant (p = 0.051). no significant differences in offspring caste ratios were found between w and ♂e (p = 0.985). the form of neotenics (ergatoid/nymphoid) that provided eggs had no significant effects on the survival rates of offspring (logistic regression analysis, wald χ2 = 0.019, p = 0.891; table 1). the result of the logistic regression analysis also showed no significant differences in offspring survival rates between w colonies and the other offspring-rearing colonies (w vs ♀e, wald χ2 = 1.445, p = 0.229; w vs ♂e, wald χ2 = 0.084, p = 0.773; w vs ♀n, wald χ2 = 1.362, p = 0.243; w vs ♂n, wald χ2 = 0.294, p = 0.588; w vs ♀n♂n, wald χ2 = 1.716, p = 0.190), suggesting that rearing conditions are similar among different colony types, and that differences in offspring caste ratios are purely a result of the presence/absence of reproductives. discussion our previous study (hayashi et al., 2007) showed that presence of a pair of female and male nymphoids resulted in increased worker rate of offspring that were produced parthenogenetically, when compared to offspring raised by workers alone. the current study demonstrates that presence table 1. number of eggs transferred from egg-source colonies to offspring-rearing colonies, number of offspring that reached third instar from the eggs and survival rate of offspring (mean ± se). offspringrearing colony no of eggs no of third instar offspring survival rate w 31, 32, 44 16, 16, 22 0.51 ± 0.005 57, 39, 42 25, 18, 22 0.47 ± 0.025 ♀e 30, 32, 44 18, 22, 27 0.63 ± 0.027 57, 38, 42 34, 18, 23 0.54 ± 0.036 ♂e 30, 32, 44 18, 18, 18 0.52 ± 0.058 56, 38, 42 30, 16, 23 0.50 ± 0.040 ♀n 31, 33, 45 14, 19, 22 0.51 ±0.037 57, 39, 43 40, 25, 24 0.63 ± 0.042 ♂n 31, 33, 45 16, 15, 18 0.46 ± 0.034 57, 39, 42 24, 19, 18 0.45 ± 0.021 ♀n♂n 31, 32, 45 19, 19, 29 0.62 ±0.015 57, 39, 42 33, 23, 23 0.57 ± 0.013 fig 2. worker proportions (mean + se) of offspring that were reared in the offspring-rearing colony w, ♀e, ♂e, ♀n, ♂n, and ♀n♂n. filled and open bars indicate the proportions of those produced through parthenogenesis of ergatoids and nymphoids, respectively. a logistic regression analysis indicated no significant difference in caste ratio between offspring produced by ergatoids and those by nymphoid (p = 0.317). significant differences in offspring caste ratio were found between w and ♀e, w and ♂n, and w and ♀n♂n colonies (p = 0.021, p = 0.009, and p = 0.007, respectively) by the logistic regression analysis. the upper and bottom rows show offspring numbers derived from egg-source colonies with ergatoids and those with nymphoids, respectively. in each row, except for the column for survival rates, numbers of offspring of three replicates are shown. for calculating survival rates the numbers of eggs transferred from eggsource colonies to offspring-rearing colonies were divided by the number of offspring that reached the third instar from the egg stage (n = 3). in w and ♂e colonies, all of the offspring differentiated into nymphs (fig 2). in contrast, some offspring differentiated into workers in ♀e colonies [proportions of workers derived from ergatoid and nymphoid egg-source colonies (mean ± se): 0.099 ± 0.065, and 0.160 ± 0.087, respectively], ♀n colonies (0.072 ± 0.011, 0.070 ± 0.047), ♂n colonies (0.176 ± 0.081, 0.177 ± 0.070), and ♀n♂n colonies (0.128 ± 0.022, 0.258 ± 0.069). the result of the logistic regression analysis showed no significant effect of egg-providing neotenic (i.e. ergatoid vs nymphoid) on offspring caste ratios (wald χ2 = 1.002, p = 0.317). the offspring caste ratios were signifiy hayashi, h miyata, o kitade, n lo worker caste differentiation in reticulitermes speratus450 of single female ergatoids and single female and male nymphoids also increased worker rate among offspring, and on the other hand, that of single male ergatoids did not. in the field, hundreds to thousands of neotenics, with various sex ratios, were found from single nests of reticulitermes and most of them were nymphoids (howard & haverty, 1980; matsuura et al., 2009; hu & forschler, 2012). thus, the result of the present study suggests that worker rates tend to be higher in the field colonies harboring a lot of neotenics. however, our study used neotenics derived from a single source colony. further studies involving neotenics from additional colonies are required to determine whether our results represent a widespread feature of the neotenic effect on the caste differentiation of this species. two possible mechanisms are possible for the control of worker rate. first, the caste developmental trajectory to nymph caste would have been converted to that to worker caste in some larvae that were reared in ♀e, ♀n, ♂n, and ♀n♂n colonies, which result in increased worker rates in those colonies. this is supported by the fact that a few of the offspring from ♀e, ♀n, ♂n, and ♀n♂n colonies were morphologically intermediate between nymphs and workers (cf. “experiments” in materials and methods): those intermediates had relatively smaller wing buds. in termites, morphologically intermediate individuals have also been found during artificial induction of caste differentiation with juvenile hormone analogs, which strongly induce workers to differentiate into presoldiers, (miura et al., 2003; tsuchiya et al., 2008; watanabe & maekawa, 2008). the second possible mechanism is that larvae destined to be workers were eliminated by attending workers in w and ♂e colonies. however, elimination of worker-destined larvae is unlikely because survival rates of offspring were not significantly different between w colonies and the other colonies. if worker-destined larvae were eliminated in w colonies, survival rates of offspring from the late stages of the egg phase to third instar in w colonies would be significantly lower than the other colonies. the survival rates from late egg stages to third instar were not significantly different between eggs produced parthenogenetically from nymphoids and ergatoids. in contrast, our previous study showed that survival rates from early stages of the egg phase to third instar were significantly lower in eggs parthenogenetically produced by ergatoids than in those produced parthenogenetically by nymphoids (hayashi et al., 2007). these data suggest that lethal genetic effects in eggs produced by ergatoids (hayashi et al., 2007) appeared only during the early stages of the egg phase. one potential proximate cause for worker differentiation in ♀e, ♀n, ♂n, and ♀n♂n colonies is the transfer of substances between neotenics, workers and larvae via grooming, and stomodeal and proctodeal trophallaxis. stomodeal trophallaxis is thought to be important in caste determination in other termite species (lüscher, 1961; korb et al., 2012). we observed stomodeal trophallaxis and grooming among workers, larvae, and all forms of neotenics with the exception of male ergatoids (data not shown). a recent study revealed the existence of queen-specific termite proteins (hanus et al., 2010); similar substances might be responsible for the effects on caste differentiation seen in this study. another potential cause of worker differentiation could be the action of volatile compounds. recent studies on r. speratus revealed that volatile pheromones influence the development of ergatoids (matsuura et al., 2010; matsuura & yamamoto, 2011). further experiments are needed to reveal the roles of direct behavioral interactions and volatile pheromones in induction of worker differentiation. our results revealed that the egg-stage is relatively insensitive to the induction of worker differentiation by neotenics. eggs were reared together with their parental neotenics until just before hatching, and only the eggs that were transferred to the w and ♂e developed solely into nymphs. further studies are needed to determine which stages of development are crucial for the induction of worker vs nymph development by reproductive individuals. in addition to nymph/worker caste ratio regulation demonstrated in the current and the previous study (hayashi et al., 2007), neotenics are known to suppress differentiation of new neotenics through pheromones in r. speratus (matsuura et al., 2010), and some other termite species (castle, 1934; 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(1971) the insect societies. cambridge, ma: belknap press of harvard university press. 369 analysis of mortality in africanized honey bee colonies with high levels of infestation by varroa destructor by igor médici de mattos¹* & josé chaud-netto¹ abstract the mite varroa destructor (anderson & treuman 2000) is one of the world’s most important plagues of apiculture. in brazil this mite does not encounter good conditions for parasitism because weather conditions are not ideal for its maintenance, and some strains of africanized honey bees are resistant to the parasite. this status is reflected in the low number of dead colonies caused by varroatosis and also the stability of infestation levels. the aim of this study was to evaluate the damage caused by mite infestations in hives with higher levels of infestation than the ones considered normal for brazilian apiaries. the level of infestation in each colony was determined and the mortality rates of parasitized bees during development were periodically recorded. the g test of independence and a test of proportions were used to compare the data. the rates of mortality of pupae and larvae were mostly proportional to the level of infestation in each colony. all colonies showed mortality rates significantly higher than the control one. in africanized honeybee colonies with high rates of infestation by varroa destructor mortality rates varied from 19.27% to 23.28% in pupae ( x = 21.27%) and from 15.71% to 16.15% in larvae ( x = 15.93%), against 3.85% and 3.74% in the control colony, respectively. in the parasitized colonies the average rates of mortality caused by the hurtful effects of the mite were, respectively, 5.52 and 4.26 times greater in those two developmental stages. thus it can be concluded that even in tropical regions, like brazil, it is necessary to give special attention to the levels of mite infestation (ir), particularly where the ir tends to be higher. keywords: africanized honeybees, apis mellifera, varroa destructor, mortality analysis, development. ¹departamento de biologia, instituto de biociências de rio claro, universidade estadual paulista –unesp, av. 24-a no 1515, cep 13506-900 rio claro, são paulo, brasil. *corresponding author. e-mail: igormmattos@yahoo.com.br 370 sociobiolog y vol. 59, no. 2, 2012 introduction varroa jacobsoni oudemans was described as an ectoparasitic mite of the eastern honeybee, apis cerana fabricius. this parasite is incapable of reproducing on apis mellifera brood (anderson 1994, anderson & sukarsih 1996). the mite that parasites the western honeybees (apis mellifera fabricius) presents size variations and is a different species, renamed as varroa destructor (anderson & trueman 2000). this new species started causing serious damages to these bees and consequently to apiculture (lfantidis & rosenkranz 1988, erickson et al. 1994, donze´ & guerin 1994, anderson & trueman 2000). the extensive damage caused by varroa destructor is often related to the brief period of coevolution between this parasite and the new host, since, except for african and africanized honeybees, the other races of apis mellifera did not develop an effective defense behavior against the mite (de jong 1984, de jong et al. 1984, moretto et al. 1993, de jong 1997, boecking & genersch 2008). the varroatosis, as the parasitism is called, causes serious harm to the developing bees as well as the adults (delfinado-baker et al. 1992, de jong 1997, beestma et al. 1999). as a consequence of parasitism during development, newly emerged honeybee workers present reduced weight, wing deformations and changes in several other appendices (mainly the legs), besides a significant decrease in the size of the abdomen. other signs, such as hypopharyngeal gland malformation and decreased life span (in adults) are also commonly found (de jong et al. 1982, schneider & drescher 1987, schatton-gadelmayer & engels 1988, beestma et al. 1989, bowen-walker & gunn 2001, romero-vera & otero-colina 2002, garedew et al. 2004, genersch 2005, kralj & fuchs 2006). furthermore, the mites act as a vector for the transmission of some viruses, for example, dwv (deformed wing virus) (ball 1988, martin 1998, bowen-walker et al. 1999, martin 2001, tentcheva et al. 2006). some researchers relate the varroa mite to ccd (colony collapse disorder) and van engelsdorp et al. (2009) suggested that this syndrome originates from an interaction between pathogens and other factors that cause stress to the colony, whereas the mite could suppress some immune responses of its host. according to le conte et al. (2010) the hypothesis that ccd is due to the parasitic mite is feasible and, indeed, is reinforced by studies carried 371 mattos, i.m. & j. chaud-netto — varroa destructor in africanized bee colonies out by van engelsdorp et al. (2009). ccd was first reported in colonies of a. mellifera in the u.s.a. interestingly, at the time of collapse, the infestation levels of varroa had not reached levels known to cause economic damage or declining populations (van engelsdorp et al. 2009). considering this information, we may conclude that varroa can play an important role as a cause of ccd even in brazil, where the infestation rates by the mite are not commonly high. considering this worldwide scenario of damages, controlling the mite has become necessary. but the use of acaricids has shown no satisfactory results because of important drawbacks such as contamination of bee products and resistant mite lineages (milani 1995, 1999, hillesheim et al. 1996, jacobs et al. 1997, elzen et al. 1998, 2000, wallner 1999, bogdanov 2006, martel et al. 2007). african honeybees and their hybrids (africanized honey bees ahb) have greater tolerance to varroa destructor infestations, with few reports of major damage (de jong et al. 1984, montiel & piola 1976, gusman-novoa et al. 1999). in brazil, where ahb are commonly used, the levels of infestation have remained stable since 1978 (between 2% and 3%, reaching 5% in some apiaries) (gonçalves 1986, rocha & almeida-lara 1994), but as shown by mattos & chaud-netto (2011) even at those levels of infestation the death rates of developing bees could be 2.28 times greater (for pupae) to 2.65 times greater (for larvae) than those found in less-infested colonies. so, it is apparent that new information and research about the relationship between the mite and its host, such as the harm caused by the parasite, are very important to the development of new methods of controlling and preventing varroatosis. the aim of this study was to quantify the damages caused by mites in colonies with high levels of infestation, in relation to those considered common in tropical areas of brazil (2-5%), by registering the loss of individuals during development. material and methods to quantify the level of infestation in each colony the method of stort et al. (1981) was used: frames covered by bees were removed from colonies 372 sociobiolog y vol. 59, no. 2, 2012 of the apiary from unesp campus at rio claro, and the workers swept with a brush into containers load with 150 ml of 96 % alcohol (volume corresponding to about 300 bees). next the samples were placed in a shaker for 30 minutes so that the mites still attached to the bees could be separated. the samples were then transferred to plastic containers fitted internally with a fine metallic net which separates bees and mites. all bees (nb) and mites (nm) were counted and the infestation rate (ir) was calculated: ir= (nm/nb) x 100. this experimental procedure was repeated three times with one week interval between samples collected. then an average of three data obtained was calculated in order to determine the final degree of parasitism in each colony, making possible the establishment of a rank of infestation. three colonies were chosen from the rank of degree of infestation, two of which had levels of infestation higher than values usually found in brazilian apiaries (between 2% and 5%). the colony named c1 presented ir=15.91%, while the colony c11 had ir=10.96%. the colony that showed the lowest degree of infestation (c20: 0.20%) was used as a control. the experiment began with the introduction of an empty comb with a demarcated area in each colony. that area contains approximately 500 brood cells. after the queens performed postures in the demarcated area, the combs were periodically inspected in order to follow the bees’ development. the technique described by garófalo (1977) was used to quantify the loss of developing bees. the results were analyzed using the software biostat 5.0 (ayres et al. 2007). the binomial statistical test (two proportions) and the g test of independence were used to compare the data. results and discussion the results showed that the rate of mortality of pupae was proportional to the degree of infestation in each colony, i.e., colonies with major rates of infestation presented higher frequencies of individuals died in the pupa stage (fig. 1). the g test of independence indicated a significant interaction between the mortality of pupae and the infestation rates (test g = 117. 33; p <0.0001; test-g (williams) = 117. 04; p <0.0001). in the colony used as control (c20) the experiment was performed during three complete cycles of development of apis mellifera workers (table 1; fig. 373 mattos, i.m. & j. chaud-netto — varroa destructor in africanized bee colonies 3). in this period 1220 pupae were observed, but 47 did not complete their development, which represents 3.85% loss. colony c11 was studied by a period of two cycles for worker brood (table 2; fig. 4). from 638 pupae observed in the experiment, 123 did not survive (19.27%). the test of proportions indicated a significant difference between the mortality rates of pupae obtained for the control colony (c20) and colony c11 (z = 10.95; p <0.0001). in colony c1, with the highest infestation rate, a proportional death rate for pupae was observed (table 3; fig. 5). during three cycles of development 1224 pupae were observed, and 285 of them did not complete the cycle (23.28%). a significant difference between the mortality rates of pupae obtained for the control colony (c20) and the colony c1 (z= 14.02; p<0.0001) was detected. the tests concerning the death of larvae showed a similar trend to those of pupae, i.e., the colony with the higher rate of infestation showed the higher frequency of dead larvae (fig. 2). the g test of independence confirmed the existence of a significant interaction between the mortality of larvae and the fig 1. number of dead pupae (grey) recorded in africanized honeybee colonies infested by varroa destructor. table 1. percentage survival during each immature stage in colony c20. 1º cycle 2º cycle 3º cycle days no. eggs 386 414 420 0-1 100% 100% 100% 1-2 98.7% 98.55% 100% 2-3 99.47% 94.85% 99.52% 3-4 99.2% 100% 99.76% survival 97.4% 93.47% 99.28% days larvae 376 387 417 0-1 100% 99.48% 100% 1-2 99.73% 97.4% 99.52% 2-3 96% 99.2% 98.79% 3-4 99.72% 99.46% 99.26% 4-5 100% 100% 99.50% survival 95.47% 95.6% 97.12% days pupae 359 370 405 0-12 95.26% 98.91% 94.09% no. adults produced 342 366 379 survival 88.6% 88.4% 93.58% 374 sociobiolog y vol. 59, no. 2, 2012 number of mites (test g = 73.85; p <0.0001; test-g (williams) = 73.64; p <0.0001). in colony c20 (control group), 1229 worker larvae were followed for three cycles of development (table 1; fig. 3). in this period, 46 dead lar vae were recorded, corresponding to a mortality rate of 3.74%. in c11 439 larvae were registered and 69 died before completing their development (15.71%). the test of proportions indicated a significant difference between the mortality rates of larvae obtained for c11 and c20 colonies (z = 7. 39; p <0. 0001). (table 2; fig. 4). in c1 984 larvae were observed (table 3; fig. 5), but 159 of them did not survive (16.15%). a significant difference between the mortality rates of larvae obtained for c1 and c20 colonies (z = 8. 72; p <0. 0001) was also detected. survival tables were made with basis on the total number of eggs laid in the observation area (garófalo 1977). tables 1, 2 and 3 show the percentage of survivors during the stages of development in each colony. in those tables day 3 – 4 was included in the egg stage and day 5 – 6 was included in the larval stage in order to show the mortality in these periods (considering that the morfig. 2. number of dead larvae (grey) recorded in colonies infested by varroa destructor. table 2. percentage sur vival during each immature stage in colony c11. 1º cycle 2º cycle days no. eggs 240 494 0-1 100% 100% 1-2 98.75% 99.79% 2-3 100% 99.59% 3-4 100% 99.79% survival 98.75% 99.19% days larvae 237 490 0-1 97.04% 100% 1-2 95.65% 97.75% 2-3 91.81% 99.58% 3-4 96.53% 98.95% 4-5 96.92% 99.36% survival 82.17% 95.71% days pupae 189 469 0-12 48.14% 94.66% no. adults produced 91 444 survival 37.91% 89.87% 375 mattos, i.m. & j. chaud-netto — varroa destructor in africanized bee colonies phological alterations to the next stage had not already occurred) (garófalo 1977). the duration of the pupae stage was considered to be 12 days, as garófalo (1977) said it is the most common duration. in all tables the frequency of survival in each period of 24 hours was calculated, so that a general survival frequency was calculated in each stage of development. at last the number of adults produced was compared with the number of eggs laid and the final frequency of survival was calculated. figs. 3, 4 and 5 show the survival curves during those stages, in each colony. the g test of independence was used to verify if there was table 3. percentage survival during each immature stage in colony c1. 1º cycle 2º cycle 3º cycle days no. eggs 487 373 364 0-1 98.35% 99.73% 100% 1-2 98.74% 100% 97.52% 2-3 99.15% 98.65% 99.15% 3-4 100% 99.18% 99.71% survival 96.3% 97.58% 96.42% days larvae 469 364 351 0-1 100% 99.17% 100% 1-2 89.97% 98.33% 97.43% 2-3 94.07% 96.05% 93.56% 3-4 98.99% 98.24% 97.18% 4-5 96.69% 99.7% 100% survival 81.02% 89.54% 88.6% days pupae 380 334 311 0-12 74.47% 78.14% 63.02% no. adults produced 283 261 196 survival 58.11% 69.97% 53.84% fig. 3. survival curves for immature stages in colony c20. 376 sociobiolog y vol. 59, no. 2, 2012 any relationship among the total number of dead larvae and the number of larval deaths that occurred between the 3rd and 4th days of larval life. this interval of days was characterized by laidlaw et al. (1956) as the period of main effects of inbreeding. the statistical analysis indicated no significant fig. 4. survival curves for immature stages in colony c11. fig. 5. survival curves for immature stages in colony c1 377 mattos, i.m. & j. chaud-netto — varroa destructor in africanized bee colonies interaction among the parameters tested (g test= 11.86, p= 0.1050; g test (williams) = 11.21, p= 0.1297). laidlaw et al. (1956) observed that in colonies with low genetic variability (major inbreeding ) the developing bees show higher rates of mortality on the 3rd and 4th days of larval life. mattos & chaud-netto (2011) have shown similar correlation on data obtained for lower ir of ahb (between 2% and 5%). in the present research the g test of independence did not indicate a significant difference between the number of dead larvae and the number of larvae which died on the 3rd and 4th days of life (inbreeding effect). so it can be concluded that the deaths occurred during the period cited by laidlaw et al. (1956) did not contribute significantly to the total number of larval deaths. conclusion the results obtained in this research revealed that in ahb colonies parasitized by varroa destructor mortality rates of larvae under conditions of high infestation ranged from 15.71% to 16.15% ( x = 15.93%). in the case of pupae, the frequencies of dead bees varied from 19.27% to 23.28% ( x = 21.27%). considering that in the control group the rate of dead bees was 3.74% in the larval stage and 3.85% in the pupal stage, it can be deduced that in the infested colonies the average rates of mortality caused by the harmful effects of the mite were, respectively, 4.26 times and 5.52 times greater in those two developmental stages. this implies a significant loss of developing bees in the colony and consequently a lower number of adults produced, which could be reflected directly on hive productivity. it proves that high ir could be significantly detrimental for beekeeping. thus it can be concluded that even in tropical regions, like brazil, it is necessary to devote special attention to the levels of mite infestation (ir), particularly when the ir tends to be higher such as in winter, or in the case of susceptibility to the mite or weakening of the colony (caused by any reason). acknowledgments the authors are grateful to capes (coordenação de aperfeiçoamento de pessoal de nível superior) for the financial support of this research. 378 sociobiolog y vol. 59, no. 2, 2012 references anderson, d.l. 1994. non-reproduction of varroa jacobsoni in apis mellifera colonies in papua newguinea and indonesia. apidologie 25: 412–421. anderson, d. & j. trueman 2000. varroa jacobsoni is more than one species. experimental and applied acarolog y 24:165–189. anderson, d.l. & sukarsih 1996. changed varroa jacobsoni reproduction in apis mellifera colonies in java. apidologie 27: 461–466. ayres, m., m. ayres jr, d. lima ayres & a. a. santos dos santos 2007. bioestat 5.0. belém– pa, brasil: sociedade civil mamirauá, 2007, 364 p. ball, b.v. 1988. the impact of secondary infections in honey-bee colonies infested with the parasitic mite varroa jacobsoni, pp. 457461. in: needham g.r., r.e. page, m. delfinado-baker & c.e. bowman (eds.), africanized honeybees and bee mites.1988. ellis horwood ltd., chichester, uk. beestma, j., w.j. boot & j. calis 1999. invasion of varroa jacobsoni oud. from bees into brood cells. apidologie 30: 125-140. beestma, j., r. de vries, b.e. yeganeh, m.e. tabrizi & v. bandpay 1989. effects of varroa jacobsoni on colony development, worker bee weight and longevity and brood mortality, pp 163-170. in: cavalloro r. 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30:235-248. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.337-344sociobiology 60(3): 337-344 (2013) social wasp-flower visiting guild interactions in less structurally complex habitats are more susceptible to local extinction ma clemente1, d lange2, w dáttilo3, k del-claro2, f prezoto4 introduction the importance of species interactions for the maintenance of biodiversity has been widely recognized (thompson, 2013; del-claro & torezan-silingardi, 2009). these interactions vary from mutualistic to antagonistic and affect populations and individuals in different ways, since all species establish ecological interaction at some point in their lifetime. it is also recognized that ecological interactions vary in time and space (thompson, 2013; del-claro et al., 2013) and that structurally more heterogeneous and complex environments have a higher species richness and diversity. alternatively, more homogeneous environments are simpler in their structure and less diverse (santos et al., 2007; tews et al., 2004). currently, network analysis has been widely used for ecological interaction studies. this tool enables conclusions concerning the structure, stability and robustness of ecologiabstract several studies have shown that habitat complexity is an important factor for the dynamic and stability of interacting species. however, it is not known how the habitat complexity may affect the tolerance of wasp-flower interactions to local extinction. based on this perspective, in this study, we aimed to compare the tolerance of wasp flower visiting guild to local extinction in two different types of vegetation (riparian forest and rocky grassland). through observations made during one year, we verified that the structure of the plant-wasp interaction network differed between the two areas, as well as that the robustness to cumulative extinctions had different patterns. the simulations of cumulative removal of species showed that the network in the riparian forest is more robust against the removal of both plants and wasps than that network in rocky grassland, since their extinction curves declined more slowly. therefore, in our study area, we demonstrate that social wasp-plant interactions in areas with lower structural complexity are less tolerant to extinction (i.e. more fragile). we therefore suggest that studies that aim at biodiversity conservation should focus not only in areas where diversity is high, but also in area with lower species richness for the conservation of ecological roles within communities. sociobiology an international journal on social insects 1 universidade estadual paulista, rio claro, são paulo, brazil 2 universidade federal de uberlândia, uberlândia, minas gerais, brazil 3 universidad veracruzana, xalapa, veracruz, mexico 4 universidade federal de juiz de fora. juiz de fora, minas gerais, brazil article history edited by gilberto m m santos, uefs, brazil received 08 june 2013 initial acceptance 12 july 2013 final acceptance 26 july 2013 keywords ecological networks, flower-visiting, species loss, habitat complexity, robustness corresponding author denise lange lab. de ecologia comportamental e interações instituto de biologia universidade federal de uberlândia uberlândia, minas gerais, cp 593 38400-058, brazil e-mail: deniselange@yahoo.com.br cal interactions involving two or more groups of organisms to be reached (montoya et al., 2006; thébault & fontaine, 2010; hernández-yáñez et al., 2013). our current understanding about how ecological networks might have different patterns in distinct habitats has, in general, been limited to few studies (see clemente et al., 2012; dáttilo et al., 2013a). from this, we can infer that structurally different habitats might have different patterns of ecological networks and consequently vary in their tolerance to species loss. studies on species extinction have been the focus of conservation ecologists who aim to preserve biodiversity, however, such studies approach only the loss of species, without considering the loss of the functional role that species exert (memmott et al., 2007; dyer et al., 2010). in natural systems, each species performs specialized functions or those complementary to its partners (blüthgen & klein, 2011). in interactions among species specialists (i.e. with a narrow research article wasps ma clemente, d lange, w dáttilo, k del-claro, f prezoto extinction of an ecological interaction338 niche breadth), each species is extremely important for interactive pair and the loss of one species results in the loss of the interaction and the extinction of one of its associates (blüthgen & klein, 2011). alternatively, the extinction of generalist species (i.e. with a wide niche), causes fewer effects on its associates, because complementary functions are provide by other species. however, regardless of the ecological function, the extinction of an interaction between two species can influence the structure of the ecological network within an entire community (olesen & jain, 1994). according to santos et al. (2007), interactions established among social wasps and plants (e.g. torezan-silingardi 2011) are a good system to study the issues of co-extinction in ecological networks and the effect of abiotic factors on them, since they have certain requirements from the environment, for example, the habitat might provide a nesting substrate (santos & gobbi, 1998; cruz et al., 2006), food resources (e.g. nectar) (santos et al., 1998; silva-pereira & santos, 2006; santos et al., 2006), material for nest building (e.g. plant fiber) (machado, 1982; marques & carvalho, 1993) and prey (santos et al., 1998). thus, the floristic composition, vegetation structure and complexity are determinants of the composition and community structure of social wasps (santos et al., 2007). additionally, some wasp species build their nests only under specific structural conditions of vegetation (sinzato et al., 2011), which can be open or closed landscapes, as well as morphological conditions of the plant species; the size and shape of leaves, stem diameter and/or the presence of thorns (henriques et al., 1992; santos & gobbi, 1998; cruz et al., 2006). thus, the complexity of the vegetation is determinant for the composition and community structure of social wasps and its fundamental and realized niche (santos et al., 2007; carvalho et al., 2013). therefore, environments with different complexities of vegetation might hypothetically have interaction networks with different compositions and topological properties, and consequently have different degrees of tolerance to species extinction. in this study, we compared the tolerance of the interaction network between flower-visiting social wasps and plants to extinction, in two distinct areas with different types of vegetation in the brazilian savanna (riparian forest and rocky grassland), to analyze in which vegetation physiognomy the wasp-plant interactions are more fragile. materials and methods we collected data in the parque estadual do ibitipoca (unidade de conservação) (peib 21º40’44”s and 43º52’55”w), southeastern brazil, from november 2007 to october 2008. the peib is managed by the forestry institute, organ of the environment agency of the state of minas gerais. moreover, the area is composed of several vegetation types that form different phytophysiognomies. riparian forest and rocky grassland are the most abundant phytophysiognomies of the peib. the riparian forest exhibits a profile of transition vegetation from high-altitude savannas to ombrophilous forests, with a physiognomy sequence from shrubby-arboreal to predominantly arboreal and a great heterogeneity of plant species (durigan et al., 2000), with a predominance of cloudiness (i.e. high moisture) (fontes, 1997). the rocky grassland exhibits a xeromorphic aspect, with a wide diversity of herbs and shrubs distributed over quartzite outcrops (rodela, 1998). this area is dominated by plants tolerant to water stress, due to the high incidence of light and wind (giulietti et al., 1997). at each of the two studied sites, which were 1,200 m distant from each other, we used one transect of 800 x 4 m, where we carried out monthly observations on two consecutive days between 7:00 17:00 h, during this period, we observed each plant that had flowers for 10 min and collected one individual of each wasp species that visited the flowers. capture was performed using entomological sweep net, according to the method of sakagami et al. (1967). data analysis initially, we built two quantitative wasp-plant adjacency matrices (one for each vegetation type), cij = number of interactions between wasp species i and plant species j inside in transects. in order not to overestimate the plant species with more number of flowers, we considered as interaction frequency only the interactions among individuals within transects. to test whether the composition of plants and wasps shifts along the core-periphery gradient of the networks in each area, we defined the core of generalist species according to dáttilo et al. (2013b). the species that exhibit a cp > 1 are species with a higher proportion of interactions in relation to other species of the same group and are therefore considered core species of the network. the species with a cp < 1 are species with a lower proportion of interactions and are considered periphery species. we calculated the robustness to extinction for both wasp-plant interaction networks (riparian forest and rocky grassland), based on the cumulative removal of species from the network at random (sensu burgos et al., 2007; dáttilo et al., 2012). we also calculated the area under the extinction curve (r) according to burgos et al. (2007), as a measure of the robustness of the networks, which varied from 1.0 (a more robust network) to 0 (a less robust network). we ran 100 randomizations for each network to simulate species removal and we chose the r index because it is more robust and is not sensitive to the shape of the curve, in contrast to the index proposed by memmott et al. (2004). we used the network 3d software (williams, 2010) to create the graphical representations of social wasp–plant networks, and the attack tolerance curve (atc) to calculate the r index. sociobiology 60(3): 337-344 (2013) 339 results in this study, we recorded 15 wasp species in associations with 27 plant species (or morphospecies) (table 1). a list of all wasp–plant interactions recorded can be viewed in the supplementary material (appendix 1). the social wasp-plant interaction networks of two cerrado physiognomies showed different structure (fig 1). the network in the riparian forest had a greater number of species (18 plant species and 15 wasp species) and a fourfold greater number amount of connections between them (83 associations) compared to the rocky grassland (11 plant species, eight wasp species, and 20 associations). the percentage of plant and wasp core species was also different between the two landscapes, with a greater number of wasp species belonging to the principal core in the riparian forest (16.6 % for plants and 20% for wasps in riparian forest, and 37.5% for plants and 9.1% for wasps in rocky grassland). the average degree of plant and wasp species was also different between areas (2.44 for plants and 2.93 for wasps in riparian forest, and 1.27 for plants and 1.75 for wasps in rocky grassland). the robustness to cumulative extinctions showed different pattern at each site (fig 2). the simulations of the cumulative removal of species showed that the network in the riparian forest is more robust against the removal of both plants and wasps than the network in the rocky grassland, since their extinction curves declined more slowly. the robustness of the network in the riparian forest was relatively high, both for plants (r = 0.914) and wasps (r = 0.706) (see fig 2c, d). in the rocky grassland network, the robustness was lower for plants (r = 0.799) and wasps (r = 0.658) (see fig 2a, b). discussion our results show that interactions between social wasps and flowers have a different tolerance to species extinction in habitats with a distinct vegetation structure. furthermore, we showed that in the brazilian cerrado, a more heterogeneous habitat exhibit plant-social wasp networks that are more tolerant to the extinction of interactions. table 1. code of plant and wasp species exhibited in figure 1. code plant species code wasp species mse mandevilla sellowii (müll. arg.) woodson amu agelaia multipicta (haliday, 1836) dla ditassa laevis mart. apa apoica pallens (fabricius, 1804) as1 asteraceae sp1 ble brachygastra lecheguana (latreille, 1824) as2 asteraceae sp2 com mischocyttarus confusus zikán, 1935 as3 asteraceae sp3 mdr mischocyttarus drewseni saussure, 1857 as4 asteraceae sp4 pbi polistes billardieri fabricius, 1804 as5 asteraceae sp5 pci polistes cinerascens saussure, 1854 as6 asteraceae sp6 pfe polistes ferreri saussure, 1853 ve1 vernonia sp1 po1 polistes sp1 bac baccharis sp1 pff polybia fastidiosuscula saussure, 1854 ver vanillosmopsis erythropappa (dc.) sch. bip. pig polybia ignobilis (haliday, 1836) wei weimmannia sp1 ppa polybia paulista (von. ihering, 1896) ego erythroxylum gonocladum (c. martius) o. e. schulz psc polybia sericea (oliver, 1791) per periandra sp1 pb1 polybia sp1 cup cuphea sp1 psy protonectarina sylveirae (saussure, 1854) mel melastomataceae sp1 tpa trembleya parviflora (d. don) cogn. mfa myrcia fallax (rich.) dc cco calyptranthes concinna dc. our ouratea sp1 pve prosthechea vespa (sw.) w.e.higgins tsp trachypogon spicatus (l. f.) kuntze pla posoqueria latifolia (rudge) schult. bar barreria sp1 gfr gordonia fruticosa (schrad.) h. keng bfl barbacenia flava mart. ex schult. f. val vellozia albiflora pohl ma clemente, d lange, w dáttilo, k del-claro, f prezoto extinction of an ecological interaction340 vegetation complexity is positively related to species diversity in many groups of organisms (see tews et al., 2004) including social wasps (see lawton, 1983; santos et al., 2000, 2007). according to santos et al. (2007), vegetation is the main substrate for the foundation of social wasp nests, so more heterogeneous environments increase the quantity and variety of sites for nesting, and consequently, the coexistence of wasp species in these locations becomes greater. in our study, this finding was confirmed in the riparian forest, which showed the greatest number of wasp and plant species, number of associations and interaction degree between plant-wasp species. a greater complexity in forest than in grassland areas has also been demonstrated in other studies (see review in tews et al., 2004). previous studies have shown that wasp-plant networks are highly generalists (mello et al., 2011; aguiar & santos, 2007). here, we show that that despite the generalization of these networks, this pattern can vary between habitats with different complexity. environments with a greater availability of resources (food or nesting sites) have lower levels of species competition (markwell et al., 1993; harris et al., 1994), mainly among wasps, where the competition for floral resources is rare (de souza et al., 2010). the lower competition, or lack thereof, contributes to the increase in species coexistence and local biodiversity (henriques et al., 1992; bastolla et al., 2009). although, the social interaction between wasps and flowers in the riparian forest is more generalist that in the rocky grassland, most wasp species of both networks established an association with only one plant species. this result might relate to limitations in resource collection by wasps from plants with a specific floral morphology (i.e. length corollas) (see heithaus, 1979a), because the mouthpart of wasps is short. several authors have suggested that this is one of the main factors limiting of the niche breadth in wasps (heithaus, 1979a; johnson & steiner, 2000; shuttleworth & johnson, 2006). nectar is an important resource for hymenoptera development and survivorship (e.g. byk & del-claro, 2011), and despite nectar being considered an accessory food in the diet of wasps (faegri & van der pijl, 1979), they tend to preferentially visit plants that produce higher amounts of nectar (gess & gess, 1993), which leads to an increase in the coexistence between of wasp species in a particular plant species. since the competition for food in wasps is rare (de souza et al., 2010), this factor poorly explains the specialization in this interaction, because the specialization observed in this study might be explained by a low abundance and richness of wasps and not by the coevolution of the associates. fig 2. robustness to cumulative species removal of plants (a) and wasps (b) in the rocky grassland, and plants (c) and wasps (d) in the riparian forest network. fig 1. graphical representation of social wasp–plant networks of two phytophysiognomies (a) rocky grassland and (b) riparian forest of a brazilian savanna in the period from november 2007 to october 2008. the red nodes represent different plant species, and the yellow nodes correspond to wasp species that interact with plants. lines represent wasp–plant interactions. plant and wasp species codes are presented in table 1. sociobiology 60(3): 337-344 (2013) 341 in addition to the aforementioned factors, abiotic components might also explain the variation in specialization of the social wasp-flower interaction. some social wasp species such as euriecias have a wide ecological valence, nesting in every type of habitat (wenzel, 1991; marques & carvalho, 1993; santos & gobbi, 1998). however, the species estenoecias exhibits a narrow limit of ecological valence, only nesting in specific locations (silva-pereira & santos, 2006; santos et al., 2007, souza et al., 2010), which might result in local extinction as an outcome of variations in habitat conditions. for example, wasp species such as angiopolybia pallens (lepeletier, 1836) and synoeca cyanea (fabricius, 1775) are only found in environments with specific substrate conditions (marques, 1996; santos et al., 2007), whereas polistes canadensis (linnaeus, 1758), and polybia ignobilis (haliday, 1836) can alter their nesting habits according to the available environmental conditions and substrates (santos & gobbi, 1998; santos et al., 2007). thus, the ecological valence of wasp species might influence species diversity in an environment and major change in its microclimate might cause the extinction or migration of species at higher rates than in an environment where climatic conditions are more stable (wilson, 1988). the factors mentioned above might have been decisive in the differentiation of the specialization and variation in the tolerance to extinction of the species interaction in the social wasp-flower networks studied. the rocky grassland area, which has less dense vegetation with strong winds and a high light incidence, might experience a large variation in microclimates, which might have contributed to a lower species diversity in this area (see elpino-campos et al., 2007). unlike the riparian forest, which has a greater availability of resources, species coexistence, and a more generalist social wasp-flower interaction network. in fact, the more generalist networks tend to be functionally redundant and more robust to species extinction (dáttilo et al., 2012). furthermore, the presence of trees in this area maintains the stability of the local microclimate, as shown by hernandes et al. (2004). the importance of interaction conservation between social wasps and flowers has been only recently recognized (shuttleworth & johnson, 2009), because wasps were previously considered as thieves of floral resources (hunt et al., 1991; elpino-campos et al., 2007). recent studies have shown that some species can contribute to pollination (sühs et al., 2009). according to heithaus (1979a, b), the guild of wasps that visit flowers surpasses that of bees in terms of exploitation of nectar in various ecosystems. some species, such as polistes versicolor (olivier, 1791), polistes simillimus zikán, 1951, polybia sericea (olivier, 1791), and polybia ignobilis (haliday, 1836), can be more representative in richness and abundance than bee species and are efficient pollinators (see sühs et al., 2009). despite that social wasp-plant interactions be considered as non-obligatory mutualistic associations and leans to generalization rather than to specialization (santos et al., 2010; mello et al., 2011), as other interactions involving plants and pollinators (see hernández-yáñez et al., 2013), the extinction of this interaction might result in the loss of part of the ecological function of pollination and cause damages to the community (see blüthgen & klein, 2011), especially in environments where this interaction is more specialized (olesen & jain, 1994). in these cases, the extinction of a pollinator (even an eventual pollinator) might trigger the loss of plant species and begin a ‘cascade of linked extinctions’ (myers, 1986). although studies containing greater number of networks are needed to confirm these results, our data show that social wasp-flower interactions in areas with low structural complexity might be less tolerant to extinction, (i.e. they are more fragile). thus, we suggest that studies that aim at biodiversity conservation should focus not only on areas where diversity is high, but also in areas with lower species richness, because the dependence among species partners is greater in these areas. acknowledgments we thank f. salimena and l. menini (leopoldo krieger herbarium) for support in the identification of the botanical material and e. giannotti for the identification of social wasp species and two anonymous referees for helpful remarks in an early version of this manuscript. we also wish to acknowledge the coordenação de aperfeiçoamento pessoal de nível superior (capes) (m.a. clemente: doctoral fellowship), and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) (d. lange 160012/2012-0; w. dáttilo 237339/2012-9; k. del-claro 476074/2008-8, 472046/2011-0, and 301248/2009-5) for financial support, and the instituto estadual de floresta de minas gerais (ief/ mg) for logistical support. references aguiar, c.m.l. & santos, g.m.m. 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(1988). biodiversity. washington: national academy press. this article has supplementary material that can be downloaded in electronic format (data set spreadsheet). appendix 1. interaction matrix between social wasps-flowers doi: 10.13102/sociobiology.v60i3.337-344.s183 url: http://periodicos.uefs.br/ojs/index.php/sociobiology/ rt/suppfiles/195/0 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.1397sociobiology 65(2): 299-304 (june, 2018) visitation of social wasps in arabica coffee crop (coffea arabica l.) intercropped with different tree species introduction brazil stands currently as the world’s largest coffee producer, having a coffee park of over 2.2 million hectares with a revenue of over 600 million dollars in 2015 (martins, 2008; conab, 2016). the crop yield is closely related to the rainfall in the producing regions and several biotic factors, among which we can mention the insect pests that cause huge yield losses (reis & souza, 2002). a lot of phytophagous insects cause damage to coffee plantations. two stand out as primary pests: the coffee leaf miner leucoptera coffeella (guérin-mèneville, 1842) (lepidoptera: lyonetiidae) and the coffee berry borer hypothenemus hampei (ferrari, 1867) (coleoptera: curculionidae, scolytinae), which cause large losses because of the injury they inflict (gallo et al., 2002; reis & souza, 2002). the leaf miner is a small whitish moth with nocturnal habits, whose caterpillars cause damage to the leaf parenchyma. the coffee berry borer is a tiny beetle, sleek black, which penetrates the fruit and the larvae destroy partially or totally the seed (reis et al., 2002). abstract brazil stands out for its coffee plantations for which the 2015 harvest yielded a revenue of over 600 million dollars. its production is closely related to biotic and abiotic factors, and insect pests are noted for reducing this production. however, those insects are highly influenced by biological control agents such as predator wasps. this study aimed to survey the wasps visiting intercropping coffee cultivation with different tree species. four plots of coffee intercropped with different tree species and coffee in full sun (control) were sampled for comparison. tree species were: teak (tectona grandis l.f), australian redcedar (toona ciliata m. roem.), mangium (acacia mangium willd.) and avocado (persea americana mill.). six hundred and thirty-nine individuals of social wasps were collected, with 20 species and 7 genera, and an overall diversity index of 1.14. the plot with avocado had the highest shannon diversity index (h') 1.23 and the lowest dominance according to the index berger-paker (dpb) 0.54. sociobiology an international journal on social insects vb tomazella1, gc jacques1,2, ac lira1, lcp silveira1 article history edited by evandro n. silva. uefs, brazil received 30 january 2017 initial acceptance 31 january 2018 final acceptance 17 march 2018 publication date 09 july 2018 keywords diversification, conservation biological control, avocado. corresponding author vitor barrile tomazella departamento de entomologia universidade federal de lavras cep 37200-000, lavras-mg, brasil. e-mail: vitorento@gmail.com the most common form of control of coffee pests is the conventional method, with large and frequent applications of chemicals to control, especially against the miner and the coffee berry borer, the main pests of the crop. the application of such products directly and indirectly affects the natural enemies, causing great loss of their diversity in the fields. in contrast, the diversification of farm agroecosystem contributes to an increase in the diversity of natural enemies (root, 1973; altieri & letourneau, 1982; altieri et al., 2003) due to several factors, such as alternative food supply for adults (e.g. nectar, pollen and sugary substances), and suitable microclimate, and the presence of preys and alternative hosts (root, 1973; andow, 1991). in coffee plantations, several species of predator wasps benefit from this diversification (fernandes, 2013) and the presence of trees promotes greater predation of leaf miners (leucoptera coffeella) by these wasps (hymenoptera: vespidae) (amaral et al., 2010). wasps are social insects belonging to order hymenoptera, family vespidae, and subfamilies stenogastrinae, polistinae and vespidae (carpenter, 1993; carpenter & marques, 2001). 1 departamente de entomologia, universidade federal de lavras, minas gerais, brazil 2 instituto federal de educação, ciência e tecnologia de minas gerais – campus bambuí, minas gerais, brazil research article wasps vb tomazella, gc jacques, ac lira , lcp silveira – visitation of social wasps in arabica coffee crop (coffea arabica l.)300 wasps of polistinae subfamily are the only ones that occur in brazil, belonging to three tribes, polistini, mischocyttarini and epiponini, with 23 genera and 319 species (carpenter & marques, 2001). these wasps forages, mainly, in hot periods of the day (picanço et al., 2010), to find water, nectar, pollen, material for construction of nests and prey for their larvae (lima & prezoto, 2003), being the main source of protein of social wasps in their first stages of development (evans & west-eberhard, 1970). the main preys of social wasps (around 90%) are insects from the order lepidoptera. it also preys on insects of the orders diptera, hemiptera and hymenoptera (gobbi & machado, 1986; prezoto et al., 2005; bichara-filho et al., 2009). several species of social wasps have been reported as effective predators of the coffee pests, such as agelaia pallipes (olivier, 1972), brachygastra lecheguana (latreille, 1824), polistes sp., polybia ignobilis (haliday, 1836), polybia occidentalis (olivier, 1791), polybia scutellaris (white, 1841), polybia sericea (olivier, 1971), protonectarina sylveirae (de saussure, 1854) and synoeca surinama cyanea (fabricius, 1775) (parra et al., 1977; reis & souza, 2002; perioto et al., 2011) accounting for approximately 70% of control in coffee plantations (reis & souza, 2002). thus, this study aimed to evaluate the influence of different tree species in the diversity of social wasps that visit coffee plantation (coffea arabica l.). material and methods this study was conducted at fazenda da lagoa, km 642 on the br 381, located in santo antônio do amparo municipality, minas gerais, brazil (20 ° 91’s / 44 ° 85’w / 1100m) in arabica coffee (coffea arabica l. ) catuaí 99 variety, in a two-year-old stand with spacing of 3.40m x 0.65m, conducted in the conventional cultivation system with total control of weeds, leaving the culture always in bare ground. four intercropping coffee plots were used with different tree species and coffee in full sun (control) for comparison (fig 1). tree species were: teak (tectona grandis l.f), australian redcedar (toona ciliata m. roem.), mangium (acacia mangium willd.) and avocado (persea americana mill.). wasp sampling was carried out monthly from november/2014 to october/2015 using two types of traps: oval yellow plastic traps, moericke type adapted, with 20 cm in greater diameter and 10 cm in the least, suspended 50 cm from the ground, attached to a bamboo stick, and filled in halfway with a saline solution of 10% nacl and 5 drops of detergent. the second type was an attractive trap, consisting of a pet bottle with a capacity of 2 litres, containing three triangular holes with 2 cm edges, approximately 5 cm from the bottom of the bottle (souza & prezoto, 2006). these were hung on a bamboo stake at 1.10 m from the ground, containing 200 ml of passion fruit juice (passiflora edulis f. flavicarpa deg. passifloraceae) as attractant, made with 1 kg of fruit pulp, 1 l of water and 150 g sugar. six attractive traps and 18 moerick adapted yellow trap were used for each treatment. three moerick composed a sample and both traps were left in the field for 48 hours. the insects collected were mounted on entomological pins for later identification with the aid of entomological keys (richards, 1978). diversity was calculated using shannon index (h') and the dominance with the berger-parker index (dpb) through past® program, also the comparative analysis of means (r development core team, 2015). results and discussion six hundred and thirty-nine subjects of social wasps were collected, with 20 species and 7 genera, with an overall diversity index of 1.14 (table 1). this diversity index was low due to the high dominance of agelaia vicina de saussure fig 1. aerial image of the sample area with separation of plots. santo antônio do amparo, mg. sociobiology 65(2): 299-304 (june, 2018) 301 (1854). this dominance is explained because some species of the genus agelaia (lepeletier, 1836) can build colonies with population estimated at up to one million adults (zucchi et al., 1995), what means higher foraging capacity by a greater number of wasps and increase of the chances of capturing individuals of this group (hunt et al., 2001). the high abundance of this genus was also reported in different ecosystems in brazil (gomes & noll, 2009; arab et al., 2010; jacques et al., 2012; jacques et al., 2015; gradinete & noll, 2013; locher et al., 2014). among the 20 species collected, the species brachygastra lecheguana (latreille, 1824), polybia ignobilis (haliday, 1836), polybia occidentalis (olivier,1791), polybia sericea (olivier, 1971) and protonectarina sylveirae (de saussure, 1854) have already been reported as effective predators of coffee pests (parra et al., 1977; reis & souza, 2002; perioto et al., 2011). even in low population levels, these predators contribute for the decrease of pests, lowering infestation peaks (debach, 1951). there was also the collection of two species of polistes, a genus that is associated with decreased damage caused by pests in different crops such as cotton (kirkton, 1970), tobacco (lawson et al., 1961), cabbage (gould & jeanne, 1984) and coffee (gravena, 1983), showing the importance of this genus for the study of biological control of pests. the introduction of wasps colonies of this genus in tobacco plantation reduced in 68% the damage caused by the caterpillar protoparce sexta (johan) (rabb & lawson, 1957). a single colony of polistes can prey on around 2,000 caterpillars of pieris rapae l., a kale pest, during its development cycle (morimoto, 1961). for instance, (prezoto & machado, 1999b) observed a reduction of 77.16% in the incidence of spodoptera frugiperda in corn plantations with the utilization of polistes simillimus zikan, 1951. the species richness (s) (table 2) between the treatments was very similar, what can be explained by the proximity of the treatments, because the shorter the distance between the sampling areas, the greater the probability of finding out a faunistic similarity (souza et al., 2015). moreover, there is also the fact that these insects have the habit of nesting in one place and forage in another (pereira & santos, 2006). the avocado treatment had the higher shannon index (h´) and the lowest berger-parker index (dpb) (table 2). the avocado trees were the most leafy and with a more closed canopy, which can justify this greater diversity index, because a better structured vegetation provides more substrate for nesting (santos & gobbi, 1998; cruz et al., 2006), glycidic resources ( santos & gobbi, 1998; pereira & santos, 2006), material for nest building (marques & carvalho, 1993), hunting area (santos & gobbi, 1998) and weather protection (altieri et al., 2003). furthermore, avocado trees were the only ones that bloomed during the experiment period, possibly attracting a higher diversity of wasps, because the adults feed on nectar (melo et al., 2011). traps species nº of individuals frequency (%) agelaia multipicta (haliday, 1836) 17 2,66% agelaia vicina de saussure, 1854 486 76,06% apoica pallens (fabricius, 1804) 10 1,56% brachygastra lecheguana (latreille, 1824) 1 0,16% parachartergus fraternus (griboldo, 1892) 1 0,16% polistes ferreri (saussure, 1853) 2 0,31% polistes versicolor (olivier, 1971) 15 2,35% polybia bifasciata saussure, 1854 4 0,63% polybia chrysothorax (lichtenstein, 1796) 2 0,31% polybia diguetana (buysson, 1905) 10 1,56% polybia fastidiosuscula (saussure, 1854) 39 6,10% polybia ignobilis (haliday, 1836) 17 2,66% polybia jurinei saussure, 1854 2 0,31% polybia occidentalis (olivier, 1971) 5 0,78% polybia platycephala (richards, 1978) 5 0,78% polybia punctata du buysson, 1907 1 0,16% polybia sericea (olivier, 1971) 1 0,16% polybia sp1 5 0,78% polybia sp2 7 1,10% protonectarina sylveirae (saussure, 1854) 9 1,41% total of individuals 639 specie richness (s`) 20 shannon-wiener index (h`) 1,14 berger-parker index (dpb) 0,76 table 1. total richness, diversity and dominance of species of social wasps collected in arabica coffee (coffea arabica l.). the diversification of coffee plantations with avocado threes contribute positively to diversity of social wasp’s species, and thus possibly contribute to a better control and stability of the plantation. vb tomazella, gc jacques, ac lira , lcp silveira – visitation of social wasps in arabica coffee crop (coffea arabica l.)302 acknowledgements we thank fapemig, cnpq, capes and embrapacafe for the financial resources and scholarships granted. references altieri, m.a. & letourneau, d.k. 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(1995). agelaia vicina, a swarm-founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137. doi: 10.13102/sociobiology.v63i3.1145sociobiology 63(3): 956-975 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 biogeography and ecology of myrmica species (formicidae: myrmicinae) in himalayan regions introduction the ant genus myrmica latreille, 1804 comprises of 164 valid species (including five fossil species from the european late eocene ambers) in the old world (radchenko & elmes, 2010; bharti & sharma, 2011a, b, c, 2013; bharti, 2012a, b; bharti et al., 2016a, b; chen et al., 2016). these species are widely distributed in the palearctic and southeast asian tropical and subtropical regions. the myrmica fauna of the central asian mountains, an area covering hindu kush, karakorum, southwestern slope of himalaya (afghanistan, pakistan, india, nepal and bhutan), corresponds to 43 known species, 41 of which are, up to date, endemic to this region (radchenko & elmes, 1998, 1999, 2001, 2003a, b, 2009, 2010; radchenko et al., 2007; bharti, 2008, 2012a, b, 2013; elmes & radchenko, 2009; bharti & sharma, 2011a, b, c, 2013; bharti et al., 2016a, b). the himalayan myrmica species are cold-hardy and survive well in the harsh high altitude conditions like paucity of resources, negligible nesting sites, short summers and long winters with below zero temperatures. abstract updated information on distribution and ecology of himalayan myrmica species is provided. altitudinal ranges for most of the myrmica species in himalaya are redefined. sociobiology an international journal on social insects h bharti1, s sasi1, a radchenko2 article history edited by rodrigo m. feitosa, ufpr, brazil received 18 june 2016 initial acceptance 18 july 2016 final acceptance 23 september 2016 publication date 25 october 2016 keywords ants, diversity, altitudinal distribution, ecology. corresponding author himender bharti, department of zoology and environmental sciences punjabi university patiala-147002, india e-mail: himenderbharti@gmail.com due to their adaptability, sheer dominance and exploitation of meagre resources, these species are the keystone players in the high altitude ecosystems of himalaya (bharti, 2013). however, not much is known about the ecology of these species, since most were described based on the already collected material deposited in various museums. thus, here we provide an updated distribution, notes on ecological aspects and redefined altitudinal ranges for most of the myrmica species from himalaya. materials and methods the updated information on distribution, altitudinal ranges and ecology is primarily based on the material recently collected by the first author and his team from himalaya, with input of already existing information in literature as well. for sake of convenience, the distribution cited from earlier data has been labelled as follows: (1) specimens at the puac punjabi university, patiala, ant collection. 1 department of zoology and environmental sciences, punjabi university, patiala, india 2 shmalhausen institute of zoology of the national academy of sciences of ukraine, kiev, ukraine research article ants mailto:himenderbharti@gmail.com sociobiology 63(3): 956-975 (september, 2016) 957 (2) radchenko and elmes, 2001. (3) radchenko and elmes, 2010. regarding the usage of term gyne and queen in the manuscript: “gyne” means alate young female, “queen” means dealate, most probably fertile, female (wilson, 1971; hölldobler & wilson, 1990). maps showing the known distribution of myrmica species were generated using diva-gis software version 7.5.0.0. map 1. geographic position of states of india (lowercase) and other countries (uppercase) where myrmica species are distributed. map 2. geographic divisions of indian himalaya. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species958 acronyms of depositories of myrmica material analysed for the manuscript: aspc: leilingen, germany (private collection of schulz) bmnh: natural history museum, london (collections of donisthorpe and bolton, and very rich material from the whole world) cac: leeds museum uk (collection of cedric collingwood) gepc: centre for ecology and hydrology, wallingford, uk (private collection of elmes) jmpc: university of mainz, germany (private collection of martens) mcz: museum of comparative zoology, harvard university, cambridge, massachusetts, usa (many types plus collections of wheeler, finzi and weber) mhng: muséum d’histoire naturelle, geneva, switzerland (collection of forel) mmpc: instituto di zoologia, roma, italy (private collection of mei) msng: museo civico di storia naturale “giacomo doria”, map 3. known distribution of myrmica adrijae, m. afghanica, m. aimonissabaudiae, m. alperti, m. boltoni. map 4. known distribution of myrmica brancuccii, m. collingwoodi, m. curvispinosa, m. elmesi, m. ereptrix. sociobiology 63(3): 956-975 (september, 2016) 959 genoa, italy (collection of emery and some type specimens of ruzsky) msnm: museo civico di storia naturale, milan, italy (collections of rigato and poldi) nhmb: naturhistorisches museum, basel, switzerland (collections of santschi and partly of forel) nhrs: naturhistoriska riksmuseet, stockholm, sweden pswc: university of california, usa (collection of ward) puac: punjabi university, patiala, ant collection sizk: schmalhausen institute of zoology of the ukrainian national academy of sciences, kiev, ukraine (collections of karawajew and radchenko) umo: hope entomological collections, university museum, oxford, england, uk (collections of rothney, f. smith, etc.) zisp: zoological institute of russian academy of sciences, st. petersburg, russia (part of material of ruzsky’s and arnoldi’s collections) zmhb: museum für naturkunde der humboldt universität, berlin, germany (collections of stitz and partly of mayr and viechmeyer) zmmu: zoological museum of moscow state university, moscow, russia (collections of arnoldi, dlussky, and partly of nasonov and ruzsky). zmuc: zoological museum of the university of copenhagen, denmark (collection of meinert) map 5. known distribution of myrmica foreliana, m. fortior, m. indica, m. inezae. map 6. known distribution of myrmica kothiensis, m. kozlovi, m. longisculpta, m. martensi, m. nefaria, m. nitida. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species960 results myrmica adrijae bharti, 2012 holotype: worker, “india, himachal pradesh, kothi, 2479 meters above m. s. l., 29.vi.1999” (coll. h. bharti, code = 193) (puac); paratypes: 5 workers, collected from the same nest. gps coordinates 32.1890°n–77.1170°e (bmnh; puac). distribution: india: himachal pradesh (1). ecology: myrmica adrijae was collected from a nest under stone on a mountain slope with a patchy cedrus forest having grass cover. the nest was small with 6 workers. the recorded nest temperature at the site was 20°c. map 7. known distribution of myrmica ordinaria, m. pachei, m. petita, m. pseudorugosa, m. rhytida, m. hecate. map 8. known distribution of myrmica religiosa, m. radchenkoi, m. rigatoi, m. rupestris, m. urbanii. sociobiology 63(3): 956-975 (september, 2016) 961 myrmica afghanica radchenko et elmes, 2003 holotype: worker, “afghanistan, pashki nuristan, 6.vi.1948, leg. k. paludan” (zmuc); paratypes: 2 workers, 1 gyne (specimen without postpetiole and gaster), “afghanistan, pashki nuristan, 6.vi.1948, leg. k. paludan” (zmuc). distribution: northeastern afghanistan (3). ecology: unknown. myrmica aimonissabaudiae menozzi, 1939 = myrmica dicaporiaccoi menozzi, 1939, synonymy by radchenko and elmes, 2001. lectotype of myrmica aimonissabaudiae (designated by radchenko & elmes, 2001): worker, “karakorum, gund, valle sind, 2080m, 9.iv.1929” (msnm); paralectotypes (designated by radchenko & elmes, 2001): 3 workers, “karakorum, gund, valle sind, 2080m, 9.iv.1929” (msnm); 1 gyne, “askol, braldo, 3100m, 10.viii.1929” (msnm); lectotype of m. dicaporiaccoi (designated by radchenko & elmes, 2001), worker, “sped. karakorum, shigar, 23.viii.29, 2200m” (msnm). map 10. known distribution of myrmica weberi, m. williamsi, m. wittmeri. map 9. known distribution of myrmica smythiesii, m. cachmiriensis, m. tenuispina, m. varisculpta, m. villosa. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species962 distribution (a) india: uttarakhand: mana 3200m (1), badrinath 3100m (1), flower valley 3500m (1), ghangria 3500m (1), chakrata 3000m (1); himachal pradesh: badhal 2090m (1), manali 1900-2063m (1, 2), kothi 2440-2538m (1), manikaran 1778m (1), naddi 1829m (1), nalivan 2280-2300m (1), narkanda 2710m (1), old manali 2243m (1), pattidhank 2440m (1), snaba 2440m (1), solang 2460-2730m (1), sissu 3200m (1), bhaggi 2700m (1), kharapathar 2700m (1), koholara 2200m (1), prounthi 2280m (1), khinang village 2940m (1), roggling 2740m (1), tingrit 3150m (1), tindi 2500m (1), trilokinath 2760m (1), mohri 3000-3200m (2), 3km e fagu, 20km e shimla 2300m (2), theong 25km e shimla 2400m (2), kullu valley, manali 2000m (2), kullu valley, 5-7km sw rottang la pass 2500-2900m (2), kullu valley vic. nagga, 20km sw manali 1500-1600m (2); jammu and kashmir: aharbal 2029m (1), aru 2453m (1), babarishi 2359m (1), dachigam 1678m (1), drang 2229-2231m (1), gulmarg 2650-3000m (1, 2), kargil 2650m (1), kokernag 1912m (1), kongdoori 2880m (1), mughal garden 1670m (1), pahalgam 21903100m (1, 2), patnitop 1973m (1, 2), sarthal 2390-2393m (1), seven springs 3077m (1, 2), shopian 2029m (1), tangmarg 2160m (1, 2), verinag 1875m (1), yusmarg 2380-2390m (1), daksum 2400-2700m (2), lidderwat 2459-2700m (1, 2), kulan-shrinagar 2100m (2), ladakh (chellong river 3360m (2), panikhar, suru riv. 3150m (2), leh 3450m (2), khalsi 2950m (2)), sonamarg 2700m (2), dahigam game sanctuary 1650m (2); arunachal pradesh: bomdila 2430m (1); west bengal: darjeeling (bharapabec lebong 1800-1900m (2)); sikkim: taungloo 3048m (2); meghalaya: shillong (2). map 12. known distribution of myrmica wardi. map 11. known distribution of myrmica latra, m. vittata, m. rugosa. sociobiology 63(3): 956-975 (september, 2016) 963 (b) nepal: lumleek (2), ghasa-tukhe, larjung, 16km sw jomosom 2550m (2). (c) bhutan: gogona 3100m (2), nobding, 41km o wangdi phodrang 2800m (2), wangdi phodrang 1300m (2). (d) pakistan: shagram, khagan valley 2200-2750m (2), kalam 2300m (2), changla gali, between muree and nathiagali 2200m (2), naran (2), maduglarht (2), shogram (2). (e) afghanistan: lalma (2), walang, salangtal 2550m (2), paghman (2). ecology: this species has a widespread distribution in himalaya, occupying a variety of habitats, under stones, rotten wood, near the foot of trees, riverine habitats and nests in open meadow mostly in pinus, cedrus and juniperus forests. the nests of this species are polydomous, with the internal temperature ranging from 14°c to 28°c, and thrive well in disturbed habitats with considerable anthropogenic activities. large nests were observed to contain more than 200 workers including alates (both males and gynes) were observed in june and as late up to first week of august. the altitudinal range for the species in himalaya is 1300m to 3500m above mean sea level. myrmica alperti elmes et radchenko, 2009 holotype: worker, “nepal, 3200m, thodung, 2-9.iv.1973 (leg. j. martens), coniferenwald”, “m. indica weber det. radchenko and elmes”, nhmb); paratypes: 14 workers “nepal, 3200m, thodung, 2-9.iv.1973 (leg. j. martens)”; 9 workers, “nepal, jiri-thodung, 28.v.1976 (leg. w. wittmer and с baroni urbani)”, “m. indica weber det. radchenko and elmes”; 1 worker, “nepal, shiralaybis, jiri-grat, 2200 m, 8.vi.1973 (leg. j. martens)”, “m. indica weber det. radchenko and elmes” (nhmb, sizk, gepc). distribution: nepal (3). ecology: virtually unknown, except that the type series of this species was found at altitudes between 2200 and 3200m above mean sea level. myrmica boltoni radchenko et elmes, 1998 holotype: worker, “nepal, dhorpantan, 3000m, 20.v.1973, leg. t. martens” (nhmb); paratypes: 15 workers “nepal, dhorpantan, 3000m, 20.v.1973, leg. t. martens” (probably same nest); 3 workers, “nepal umg. goropani, w. pokhara, zentral nepal, sept.-oct. 1971, leg. h. franz”; 1 worker, “nepal, 2 miles, s.e. sikha, 7800-8000fts., 21-22.v.1954, leg. j. quinlan”; 1 worker, “nepal, 18 km nne baglung, 28°24’n, 83°42’e, 2540m 29.xi.1988 no.9619 leg. ward”; 1 worker, “nepal-23, prov. kosi, distr. sankhuwasawa, vallee d’induwa koa, 2000m, 16.iv.1984, leg. lobl and a. smetana”; 1 worker, “nepal-140, prov. manang, marsyandi, 2550m, 14-17.iv.1980, leg. j. martens and ausobsky”; 2 workers, “nepal-161, prov. mustang, lethe, 2450-2600m, 30.iv.1980, leg. j. martens and ausobsky”; 1 worker, “nepal-233, prov. gorkha, chuing khola, meme kharka, 3300-3400m, leg. j. martens and w. schwaller” (nhmb, bmnh, sizk, gepc, pswc, jmpc, aspc). distribution: nepal (2). ecology: almost unknown, but one worker was collected under rotten wood in a quercus-rhododendron forest. all samples were collected at altitudes between 2000m and 3400m above mean sea level. map 13. species richness based on nominal myrmica species for the regions considered in the study. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species964 myrmica brancuccii radchenko, elmes et collingwood, 1999 holotype: worker, “nepal, utrot, 13.v.1983, leg. m. brancucci” (bmnh); paratypes: 5 workers, with the same label as holotype; 14 workers, “nepal, lawarai, 21.05.1983, leg. m. brancucci”; 1 worker, “nepal, lumie, vi.1988, leg. collingwood”; 9 workers, “pakistan, chitral v., between dir and lavari pass, 2400m, 11.viii.1994, leg. s. dakatra” (bmnh, nhmb, cac, msnm, sizk, gepc). distribution: nepal and ne pakistan (2). ecology: virtually unknown, except that one sample was collected at an altitude 2400m above mean sea level. myrmica cachmiriensis forel, 1904 = myrmica smythiesi var. lutescens menozzi, 1939 (first available use of myrmica smythiesii r. cachmiriensis var. lutescens forel, 1904) lectotype of m. cachmiriensis (designated by radchenko & elmes, 2001): “kashmir, sind valley, 9500ft (wroughton)”, “m.smythiesii v. kashmiriensis forel, type” (mhng); paralectotypes (designated by radchenko & elmes, 2001): 2 workers with same labels as lectotype (mhng); 1worker, “m.smythiesii for. var. kaschmiriensis w type sind valley, 7500’ kaschmir (wroughton) jh.6” (msng); lectotype of m. smythiesi var. lutescens (designated by radchenko & elmes, 2001): worker, “cachmire (smythies)”, “m. smythiesi for. r. cachmiriensis for. v. lutescens for., type” (mhng). distribution (a) india: jammu and kashmir: seven springs 3077m (1), patnitop 1973m (1), aru 2500m (2), pahalgam 2100-2190m (2), kulan 2100m (2), daksum 2400-2700m (2), gulmargtangmarg 2300-2650m (2), tusmarg 2300-2400m (2); himachal pradesh: naddi 1829m (1), solang 2480-2677m (1), kothi 2420-2441m (1), gulaba 2800m (1). (b) pakistan: kagan valley, shogran 2300-2600m (2), changlu gali between mure and nathia gali 2200m (2), sobodan gali (bagh) 2300m (2). ecology: this species forms small nests (with 10-50 workers) under stones in soil covered with scattered vegetation. however, few nests were found in dense forests in soil covered with leaf litter. these ants appear to prefer shady areas, as they were never observed in exposed habitats. nest temperature ranged from 14°c to 22°c and relative humidity 91%. alates were observed during july and august. altitudinal range for this species is 1829 to 3500m above mean sea level. myrmica collingwoodi radchenko et elmes, 1998 holotype: worker, “bhutan, dorjula, 3100m, 26.06.1972, nat. hist. museum basle bhutan expedition 1972, alk. no 56” (nhmb); paratypes: 9 workers, 1 queen, “bhutan, dorjula, 3100m, 26.06.1972, nat. hist. museum basle bhutan expedition 1972, alk. no 56”(probably same nest); 6 workers, “bhutan, dorjula, 3100m, 6.06.1972, nat. hist. museum basle bhutan expedition 1972, alk. no 56”; 6 workers, “dorjula, 2450-3100m, 6.06.1972, nat.-hist. museum basle bhutan expedition 1972, no 24-36” (nhmb, bmnh, sizk, gepc). distribution: bhutan (2). ecology: virtually unknown, except that this species was found at altitudes ranging 2450-3100m above mean sea level. myrmica curvispinosa bharti et sharma, 2013 holotype: worker, “india, himachal pradesh, shoja, 31.568 069°n, 77.372096°e, 2700m, 10.ix.2008” (puac); paratypes: 85 workers and 2 gynes, same colony as the holotype; 1 worker and 1 intercaste, “india, himachal pradesh, kothi, 32.319325°n, 77.197945°e, 2479m, 16.vi.2003” (puac). distribution: india: himachal pradesh (1). ecology: myrmica curvispinosa was manually collected from a nest under stones, found in a patchy cedrus forest located in a temperate region of the himalaya. the forest was surrounded by cultivated fields and an apple orchard. the temperature recorded at the site was 23°c. one paratype (intercaste) was collected from another locality (kothi), beneath a stone in moist soil, in a grass-covered area surrounded by a patchy cedrus forest. myrmica elmesi bharti et sharma, 2011 holotype: worker, “india, jammu and kashmir, machedi, 32.72364°n, 75.669464°e, 2000 meters, collected 3rd august, 2008” (puac); paratypes: 1 worker with same data as of holotype and 10 workers “india, jammu and kashmir, sarthal, 32.812947°n, 75.762503°e, 2200 metres” (puac). distribution: india: jammu & kashmir (1). ecology: the type series of this species was collected from leaf litter in both the habitats. the collection site at machedi has a patchy cedrus forest along with agricultural land surrounding the site; moreover, the area has lot of anthropogenic pressure (in terms of grazing and cropland management) with a dry type of environment (mean temperature during collection period 32°c, relative humidity 36.62%, annual rainfall 970mm and thickness of leaf litter 2.1cm). the collection site at sarthal has dense cedrus forest with abundant leaf litter, no agricultural land, and has very limited anthropogenic activities with only nomads visiting the area. the site is usually snow covered from november to beginning of march (mean temperature during collection period 22°c, relative humidity 66.38%, annual rainfall 1476mm and thickness of leaf litter 3.9cm). the area where this species is distributed is a transitional zone between subtemperate and temperate himalaya, penetrating into the palearctic zone (whose boundary in southern asia is largely altitudinal, where an altitude of 2000–2500 meters above mean sea level forms the boundary between palearctic and indo-malayan ecozones) (bharti & sharma, 2011a). myrmica ereptrix bolton, 1988 holotype: gyne “holotype female, india: kashmir, gulmarg, 20.vii.1986, 2800m, picea forest, leg. p.h. williams” (bmnh). distribution: india: jammu & kashmir (2). ecology: this species was collected from picea forest. sociobiology 63(3): 956-975 (september, 2016) 965 myrmica foreliana radchenko et elmes, 2001 (replacement name for myrmica smythiesii r. carbonaria forel, 1902) holotype: worker, “himalaya, pachmarhi, shurr, 30.vii.93, leg. rothney” (mhng). distribution: india: uttarakhand: chopta, 2900m (1). ecology: queen and workers were collected under a stone during the month of may, when temperature of chopta valley was 23°c and humidity 75%. this region has alpine vegetation, with orchids, rhododendron and other high-altitude vegetation. myrmica fortior forel, 1904 lectotype (designated by radchenko & elmes, 2001): worker, “india, sind valley, 6500 ft, kashmir (wroughton), var. fortior forel” (mhng); paralectotypes (designated by radchenko & elmes, 2001): 2 workers “india, sind valley, 6500 ft, kashmir (wroughton), var. fortior forel” (mhng); 1 worker, “smythiesi var. fortior sind valley” (msng). distribution: india: jammu & kashmir: pahalgam 21002202.5m (1, 2), yusmarg 2380m (1), babarishi 2359m (1), aru 2450-2500m (1, 2), shopian 2029m (1), seven springs (srinagar) 3077m (1), sonamarg 2700m (2), daksum 24002700m (2), lidderwatt 2700m (2); shankarachayra (2), sorale san, kangwan-do (2). ecology: this species was commonly observed nesting under stones in grasslands, pine forest and at the edge of sprucefir grooves. nesting sites had temperature ranging from 20°c in babarishi to 25°c in pahalgam and humidity from 51% in babarishi to 57% in yusmarg. average nest temperature was found to be 33°c. myrmica hecate weber, 1947 lectotype (designated by radchenko & elmes, 2001): worker: “india, himalaya, darjeeling, botanical gardens, 6900 ft, 7.viii.09, no.8609-19, pavia” (mcz); paralectotype (designated by radchenko & elmes, 2001): worker (damaged, without postpetiole and gaster), “darjeeling, 6000 ft, 24.x.1909. no.8607-19, brunetti” (mcz). distribution (a) india: himachal pradesh: solang 2610m (1), marhi 3334m (1), kothi 2400m (1), manali, oalti kumal (2), chopal-khangna nallah 2250m (2), kullu valley, 2-4km sw rothang la, pass 3400-3700m (2); jammu & kashmir: yusmarg 2300-2400m (2); uttarakhand: chakrata 3000m (1); west bengal: darjeeling (1, 2), happy valley 1880m (1), mirik 2080m (1), sukhia 2000m (1), kalingpong 1850m (1), tiger hills (2), bhara patee lebong 1800-1900m (2), ihepi 1300-1400m (2); sikkim: phadamchen 1710m (1) (east sikkim); arunachal pradesh: bomdila 2430m (1). (b) nepal: lumle (2), baira bali von katmandu (2), daman 2400m (2). ecology: the nests of this species were found under stones in grasslands and in soil covered with scarce to dense vegetation. nest temperature and humidity ranged from 24.4-28°c and 45% to 90%, respectively. large colonies contained more than 200 workers. alates of this species occasionally appear as late as in september in northeast himalaya. the altitudinal range for this species is 1300m to 3700m above mean sea level. myrmica indica weber, 1950 lectotype (designated by radchenko & elmes, 1998): worker, “tonglu, e. himalayas, 10,000 ft., 22.04.10 (c. w. beebe)” (mcz); paralectotype (designated by radchenko & elmes, 1998): 2 workers, with the same label as lectotype (mcz). distribution (a) india: west bengal: darjiling, tigerhills 2500m (2); arunachal pradesh: tawang 2660m (1). (b) nepal: chordung, jiri 2900m (2), thodung (2), shiralaybis, jiri-grat 2200m (2), jiri-thodung (2), phulchoki 2600m (2), zentral-nepal, zw.tare-pati, u.gasaikunde (2), taplejung, simbua khola, vicinity lassetham 3000-3150m (2), prov. sankhua, sabha, vic. phakhola 2600-2800m (2), maeva khola, sanghu (2), chautra, nanling lekh 2895m (2), phulkhola 2743m (2). (c) bhutan: thimphu (2), sampa-kotoka 1400m-2600m (2), nobding, 41km o wangdi 2800m (2). ecology: poorly known, this species lives in open forests at an altitude of about 2500m above mean sea level (radchenko & elmes, 2010). myrmica inezae forel, 1902 holotype: worker, “himalaya, pachmarhi (schurr)” (mhng). distribution india: uttarakhand: chourangi khaal 2300m (1), mussorie 1900m (2); himachal pradesh: solang 2590-2734m (1), manali 1826-3000m (2), chopal 2400-2750m (2), vic. theong 25km e shimla 2400m (2). ecology: colonies of myrmica inezae were found nesting under stones and in rotten logs at shady places. these areas are covered with cedrus and pinus trees with very dense ground vegetation. lowest and highest air temperature observed at the collection site was 21.4°c and 30°c, respectively, the nest temperature never exceeding 17°c. humidity of the collection site ranged from 54% to 79%. alates appear in june and july. the altitudinal range for this species is 1900-3000m above mean sea level. myrmica kothiensis bharti et sharma, 2013 holotype: worker, “india, himachal pradesh, kothi, 32.31 9325°n, 77.197945°e, 2479 m, 16.vi.2003” (puac); paratypes: 3 workers from the nest of holotype (puac). distribution: india: himachal pradesh (1). ecology: this species was collected from a grass-covered site with a patchy cedrus forest. the workers were found nesting under a stone in moist soil, on a shady mountain slope. the recorded nest temperature was 20°c. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species966 myrmica kozlovi ruzsky, 1915 = myrmica kozlovi subsp. mekongi ruzsky, 1915, synonymy by radchenko and elmes, 2010 = myrmica kozlovi subsp. subbrevispinosa ruzsky, 1915, synonymy by radchenko and elmes, 2010 = myrmica kozlovi subsp. subalpina ruzsky, 1915, synonymy by radchenko and elmes, 2010 = myrmica specularis donisthorpe, 1929, synonymy by radchenko and elmes, 2001 lectotype of m. kozlovi (designated by radchenko & elmes, 2010) 1 worker, “tributary of riv. dza-chju, kam, riv. yangtze, 12-13000’, leg. kozlov, beginning of iii.01” (original label in russian) (zisp); paralectotypes (designated by radchenko & elmes, 2010): 34 workers “tributary of riv. dza-chju, kam, riv. yangtze, 12-13000’, leg. kozlov, beginning of iii.01”; 27 workers, “valley of riv. yangtze, kam, tibet, leg. kozlov, iii.01”; 1 worker, “riv. dza-chju, 11000’, kam, basin of riv. yangtze, leg. kozlov, middle of iv.01”; 10 workers, “riv. dza-chju, riv. yangtze, 12-13000’, leg. kozlov, beginning of v.01” (all original labels in russian) (zisp, zmmu, sizk); lectotype of m. kozlovi subsp. mekongi, (designated by radchenko & elmes, 2010): worker, “riv. bar-chju, basin of riv. mekong, kam, end of ix.1900, leg. kozlov” (original label in russian), “myrm. kozlovi sub. mekongi n. sub. m. ruzsky” (zmmu); paralectotypes (designated by radchenko & elmes, 2010): 15 workers “riv. bar-chju, basin of riv. mekong, kam, end of ix.1900, leg. kozlov”; 1 worker, “riv. ba-chju 12,000’ kam, basin of riv. yangtze, leg. kozlov, 2-3.viii.00” (original label in russian) (zmmu, zisp, sizk); holotype of m. kozlovi subsp. subbrevispinosa, worker, “valley of riv. yangtze, kam, eastern tibet, leg. kozlov, iii.01” (original label in russian), “m. kozlovi v. subbrevispinosa n. var.” (zmmu); holotype of m. specularis, worker, “tibetan side of the mt. everest: tibet, gautsa, 13000 ft, 5.ìv.1924” (hingston) (bmnh); paratypes: 4 workers “tibetan side of the mt. everest: tibet, gautsa, 13000 ft, 5.ìv.1924” (bmnh). distribution (a) india: sikkim: khamba jong 4572-4876m (2); west bengal: darjiling, tigerhills 2500m (1); arunachal pradesh: tawang 2660m (1). (b) tibet: s e tibet, dzogang 2743-4267m (2). ecology: according to radchenko and elmes (2010), “the ecology of this species is poorly known. it has been found at the highest altitudes recorded for any myrmica species recorded worldwide (4800m) which accounts for it being the only myrmica species that has crossed the himalayan barrier to live on both the western and eastern slopes”. donisthorpe (1929) wrote that “according to major higston (the collector of m. specularis) the ants found at the higher elevations (in the himalaya) are exceedingly lethargic and sluggish in their movements”. myrmica latra bharti, radchenko et sasi, 2016 holotype: queen, “india, himachal pradesh: prounthi, 31.1043, 77.6487, 2260m, hand picking, 14 july 2013, joginder singh leg.” (puac); paratype: male (alate), “india, himachal pradesh, roggling, 32.5514, 76.9704, 2740m, 12 july 2015, pawanpreet kaur leg.” (puac). distribution: india: himachal pradesh (1). ecology: both queen and male were collected from nests of m. aimonissabaudiae built under stones. the ground was covered with low vegetation, and scattered pinus and cedrus trees. the recorded nest temperature and humidity at site one, where queen was collected was 18°c and 76%, whereas at site two where male was collected the recorded nest temperature was 19°c and humidity 66% (bharti et al., 2016b). myrmica longisculpta bharti et sharma, 2011 holotype: worker, “india, jammu and kashmir, sarthal, 32.812947°n, 75.762503°e, 2200m a.s.l., 15.vi. 2009, coll. sharma” (puac); paratypes: 4 workers “india, jammu and kashmir, sarthal, 32.812947°n, 75.762503°e, 2200m a.s.l., 15.vi. 2009, coll. sharma”, not from same nest; 1 worker, “india, jammu and kashmir, machedi, 32.72364°n, 75.669464°e, 2000 m a.s.l., 3.viii.2008 (coll. sharma)”, 1 worker, “india: jammu and kashmir: shopian, 33.668354°n, 74.779472°e, 3100 m a.s.l., 12.ix.2009, coll. sharma” (puac). distribution: india: jammu & kashmir (1). ecology: all the known type material of this species was hand-collected from two localities (sarthal, 32.812947°n,75.762503°e, 2200m a.s.l and shopian, 33.668354°n, 74.779472°e, 3100m a.s.l.) and from leaf litter submitted to winkler’s extractor at another locality (machedi, 32.72364°n, 75.669464°e, 2000m a.s.l.). the collection site at machedi has a patchy cedrus forest surrounded by agricultural land; moreover, the area has a lot of anthropogenic activities with dry type of environment (mean temperature during collection period 32°c, relative humidity 36.62% and thickness of leaf litter 2.1cm). the collection site at sarthal has dense cedrus forest with abundant leaf litter and no agricultural land. it remains snow clad from november to the beginning of march and has very limited anthropogenic activities with only nomads visiting the area (mean temperature during collection period 22°c, relative humidity 66.38%, thickness of leaf litter 3.9cm) with a comparatively wet environment. at the third collection site (shopian), specimens were collected under a stone. the area has scattered cedrus trees, as the forest has largely been cleared by human activities. the mean temperature and relative humidity recorded during collection period was 22°c and 54%, respectively. (bharti & sharma, 2011c). myrmica martensi radchenko et elmes, 1998 holotype: worker, “nepal, gosainkund, sing gyang, 3200 m, 26.04.1973, leg. j. martens” (basle); sociobiology 63(3): 956-975 (september, 2016) 967 paratypes: 3 workers, 1 queen, with same label (probably same nest) as holotype (nhmb). distribution: nepal (2). ecology: unknown. myrmica nefaria bharti, 2012 holotype: gyne, “india, himachal pradesh, solang valley (32.312° n, 77.1556° e), 2469 meters a.s.l., 20.vi.2010, leg. h. bharti” (puac); paratypes: 62 gynes, 5 workers, 4 males, all from the nest of holotype (puac). distribution: india: himachal pradesh (1). ecology: as per bharti (2012a), “the nest of the host species m. rupestris with m. nefaria was found under a stone, in open grassland with bushes, shrubs, broadleaf trees and widely scattered coniferous trees; the recorded air temperature was 30°c and the relative humidity was 68% at the site. topographically, this valley has two very distinct areas, which differ quite significantly in their environmental conditions. the area which is directly exposed to sun is rock-strewn, dry, with only patches of grass (without herbs or shrubs) and harbors maximum abundance of formica species. the other area is comparatively wet, shady, with ample vegetation, and has more myrmica species together with some patches occupied by temnothorax, lasius, and formica species. the region of solang valley represents the temperate zone of himalaya and remains snow covered from december to march. additionally, the region typifies the transitional zone between greater himalaya and trans himalaya and harbors a rich diversity of flora of both ecological conditions mentioned and is rich in endemic plants.” myrmica nitida radchenko et elmes, 1999 holotype: worker, “kashmir, 1km. n.e. yehmer pass, 34o13’n, 75o10’e, 3600m, 06.08.1978, leg p.ward, acc. no 3044: alpine vegetation, under stone” (bmnh); paratypes: 9 workers from the nest of holotype; 17 workers, 4 gynes, 2 males, “kashmir, sanang, 2600-2750 m, leg.w.wittmer” (bmnh, nhmb, pswc, sizk, gepc) distribution: india: jammu and kashmir & himachal pradesh: keylong 3100m (1). ecology: m. nitida is distributed in alpine zone of northwest himalaya. the nests are located under stones in grasslands with scattered juniper and rhododendron, with ground cover of herbs and shrubs. the recorded nest temperature and humidity was 23.6°c and 89%, respectively. the altitudinal range for this species is 2600-3600m above mean sea level. myrmica ordinaria radchenko et elmes, 1999 holotype: worker, “india: kashmir, seven springs, 29.07.86 leg. p.williams” (bmnh); paratypes: 23 workers, 1 queen, “india: kashmir, seven springs, 29.07.86 leg. p.williams” (probably same nest); 6 workers, “pakistan, kalam, 2300m, 12.07.94, leg. s.dakatra” (bmnh, nhmb, msnm, sizk, gepc). distribution: india and pakistan (known only from the type series) (2). ecology: unknown. myrmica pachei forel, 1906 lectotype (designated by radchenko & elmes, 2001): worker, “ne nepal, tseram, 3600 m, pache” (nhmg). paralectotypes (designated by radchenko & elmes, 2001): 2 workers (nhmg) and l worker “ne nepal, tseram, 3600 m, pache” (mcz); 1 worker, 1 male, “myrmica pachei n. nepal, himalaya 3600 m” (msng). distribution: (a)india: arunachal pradesh: jaswantgarh 3146-3180m (1). (b)nepal: prov. taplejung, upper simbu khola valley, vic. tseram 3250-3350m (2), prov.taplejung, dhara und alm lasea 3000-3300m (2). (c)bhutan: nobding, 41km o wangdi 2800m (2), kotokagogona 2600-3400m (2), doriula 3100m (2). ecology: this species was found nesting under stones in wet soil, with ground covered with grass, low vegetation and scattered pinus trees. the maximum altitude at which specimens were collected was 3180m (jaswantgarh in arunachal pradesh), where temperature ranged from 15°c to 24.2°c and humidity 65-85%. the colonies were small with up to 30 workers. alates were collected during the month of june and first week of october. myrmica petita radchenko et elmes, 1999 holotype: queen, “kashmir, yusmar 2300-2400m, 6.vii.1976, leg. w. wittmer” (nhmb). distribution: india: kashmir (2). ecology: unknown. myrmica pseudorugosa bharti, 2012 holotype: worker, “pakistan, kaghan valley, gittidas, 3600 meters a.s.l., 17.ix.2005 (seiki yamane coll. code = pk05sky-42)” (puac); paratypes: 3 workers, collected from the same nest (bmnh; puac). approximate gps coordinates 35.1167°n–73.9833°e. distribution: pakistan (1). ecology: this species has been collected at an altitude of 3600 meters a.s.l., which represents the trans-himalayan alpine zone, corresponding to a dry desert area above the timberline. myrmica radchenkoi bharti et sharma, 2011 holotype: worker, “india, jammu and kashmir, machedi, 32.72364°n, 75.669464°e, 2000 meters above msl, 3rd august, 2008” (puac); paratypes: 3 workers and 1 queen “india, jammu and kashmir, machedi, 32.72364°n, 75.669464°e, 2000 meters above msl, 3rd august, 2008” (puac). distribution: india: jammu & kashmir (1). ecology: this species was collected from leaf litter in a http://www.antweb.org/browse.do?genus=myrmica&species=pachei&rank=species&project=indiaants h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species968 patchy cedrus forest, and the area is a transitional zone between temperate and subtemperate himalaya. the ambient temperature at the collection site was 32°c and the nest temperature was 30°c (bharti & sharma, 2011b). myrmica religiosa bharti et sharma, 2013 holotype: worker, “india, uttarakhand, chourangikhaal, 30.683614°n, 78.432684°e, 2300 m, 02.vi.2010” (puac); paratypes: 20 workers from the nest of holotype (puac). distribution: india: uttarakhand (1). ecology: myrmica religiosa was manually collected living under stones in a dry forested area of chourangikhaal. the trees like cedrus, quercus, rhododendron and pinus predominate the area. the area has numerous anthropogenic activities. the recorded temperature and humidity at the collection site was 30°c and 65%, respectively. myrmica rhytida radchenko et elmes, 1999 holotype: worker, “kashmir, up. kainthal nar, 34o00’n, 75o45’e, 3750 m, no. 3061, 14.viii.1978, leg. p.ward” (bmnh). paratypes: 113 workers and 32 males from same nest of holotype; 3 gynes, 4 males, “kashmir, 3 km ne tar sar, 34o09’n, 75o11’e, 3300 m, no. 3037, 02.08.1978, leg. p. ward”; ~220 workers, 10 queens, 5 males, “kashmir, tarsar, 34o09’n, 75o09’e, 3950 m. no.3038, 3039, 3040, 3041, 3042, 4.viii.l978, leg. p. ward”; 25 workers, “kashmir, 1km ne yehmer pass, 34o13’n, 75o10’e, 3600 m, no.3043, 3045, 06.viii.1978, leg. p. ward”; 84 workers, 7 queens, 4 males, “kashmir, 4km s kulan, 34o14’n, 75o10’e, 3599 m, no.3046, 3048, 07.viii.1978, leg. p. ward”; 3 workers, “kashmir, sain nar, 34o06’n, 75o34’e, 3750 m, no.3054, 11.viii.1978 leg. p. ward”; 4 workers, 1 gyne, 4 males, “kashmir, wampet, 34o04’n, 75o37’e, 3700m, no.3057, 12.viii.l978, leg. p. ward”; 1 male, “kashmir, up. kaintal nar, oitto, 34o00’n, 75o45’e, 4200 m. no.3068, leg. p. ward”; 3 workers, “india, beastel, 20km s rhotang, 2800 m, no. j 23, 18.viii.1990, leg. j. heinze”. (bmnh, nhmb, izk, msnm, aspc, pswc, gepc, mmpc). distribution: india: jammu and kashmir & himachal pradesh: sagnam 3600m (1). ecology: m. rhytida is found at an altitudinal range of 2800m and 4200m in himalaya. nests are built in the soil (often under stones) and in birch logs. it inhabits alpine meadows often containing juniper species, dry alpine scrub and birch trees and at the edges of birch forests at an altitude where rhododendron begin to appear. the nest temperature at one of the collection site (sagnam) was 31°c and relative humidity 41%. it is primarily distributed above timberline in cold desert regions of himalaya, with low pluviosity. there are records of sympatry between m. rhytida and m. wardi, but m. rhytida tend to live at higher altitudes in comparison to the latter (mean of 3600m vs. 2700m). there are records of this species tending root aphids inside several of its nests. the foraging behaviour of m. rhytida appears to be quite cryptic, it stays close to the soil surface and was never found foraging on plants (radchenko & elmes, 2010). myrmica rigatoi radchenko et elmes, 1998 holotype: worker, “pakistan, changla gali (between marree and nathia gali), 2200 m, 16.08.1994, leg. s. dakatra” (msnm). distribution: pakistan: changla gali and hazara durgo gal. 2300m (2). ecology: unknown. myrmica rugosa mayr, 1865 lectotype (designated by radchenko & elmes, 2001): worker, “himalaya”, “m. rugosa mayr” (zmhb); paralectotypes (designated by radchenko & elmes, 2001): 1 worker, “himalaya”, “m. rugosa sp. n. mayr” (bmnh); 1 worker, “myrmica rugosa m. himal.”; 1 worker, “himalaya mayr е. с ej.” (msng); 2 workers, “himalaya”, “coll. mayr” (original labels in russian), “295” (zmmu). distribution: (a) india: jammu & kashmir: seven springs 3077m (1), bhadarwah 1600m (1), yusmarg 2390m (1), pahalgam 2202m (1), srinagar 1560m (2), lidderwat 2150m (2); uttarakhand: kedarnath 3300m (1), mana 3200m (1), ghangria 3500m (1); west bengal: darjeeling 1890m (1). (b) nepal: thakkhola, alt-marsa 3100-3200m (2). (c) bhutan: 20km s thimphu 2300-2440m (2), sampakotoka 1400-2600m (2), doriula 2900m (2), kotoka-gogona 2600-3400m (2), diechli paka 3300m (2), tanglu, 22km w thimphu 2600-2800m (2), para (2), passeling 2700-3100m (2). note: some of the material of “m. rugosa” has been cited to be collected in kyrgyzstan (jansen et al., 2010), well outside the limits of known geographic distribution of this species, which we consider doubtful (bharti et al., 2016b). ecology: myrmica rugosa nests were found under rotten wood at a forested site and under stones in grassland. nesting sites had temperature ranging from 15°c to 25°c and humidity between 42% and 70%. the species seems to be well distributed in subalpine zone of northwest himalaya (3200m), which is dominated by the himalayan maple (acer caesium), west himalayan fir (abies pindrow), himalayan white birch (betula utilis), and bell rhododendron (rhododendron campanulatum), with himalayan yew (taxus wallichiana), himalayan lilac (syringa emodi) and hairy rowan (sorbus lanata). some of the common herbs are jacquemont’s cobra lily (arisaema jacquemontii), boschniakia himalaica, kashmir corydalis (corydalis cashmeriana), himalayan jacob’s ladder (polemonium caeruleum), rampant tall weed (polygonum polystachyum), gigantic himalayan balsam (impatiens sulcata), wallich geranium (geranium wallichianum), cleavers (galium aparine), himalayan whorlflower (moringa longifolia), showy inula (inula grandiflora), yellow himalayan lily (nomocharis oxypetala), river anemone (anemone rivularis), lousewort (pedicularis pectinata), horned sociobiology 63(3): 956-975 (september, 2016) 969 lousewort (p. bicornuta), drumstick primrose (primula denticulate) and himalayan trillium (trillium govanianum). in this zone, myrmica rugosa is found to be sympatric with m. smythiesii. the altitudinal range for this species is 1400m to 3500m. m. rupestris forel, 1902 = myrmica rugosa var. debilior forel, 1902, synonymy by radchenko and elmes, 2001 = myrmica rugosa var. rugososmythiesi forel, 1902, synonymy by radchenko and elmes, 2001 = myrmica everesti donisthorpe, 1929, synonymy by radchenko and elmes, 2001 lectotype of m. rupestris (designated by radchenko & elmes, 2001), worker (upper specimen on the pin with 3 workers), “n-w himalaya, ekra peak, 4400 ft (smythies)”, “lxxxix/12, m. smythiesii for. v. rupestris for., typus” (mhng); paralectotypes: 2 workers on the same pin as lectotype (mhng); 1 worker, “w m. smythiesii for. var. rupestris forel, ekra peak himalaya” (msng); lectotype: worker of m. rugosa var. debilior (designated by radchenko & elmes, 2001), (upper specimen on the pin with 3 workers), “himalaya (smythies)”, “lix/4, m. rugosa r. debilior” (mhng); paralectotypes (designated by radchenko & elmes, 2001): 2 workers on the same pin as lectotype (mhng); 1 worker, with same labels as lectotype (umo); 2 workers, “m. rugososmythiesii forel w himalaya smythies, lx/4”, “var. debilior for.” (msng); 3 workers, “himalaya (smythies)”, “forel coll.”, “m.c.z. cotype no. 556” (originally labelled as m. rugosa; for details see radchenko & elmes, 2001) (mcz); holotype of m. everesti: worker, “himalaya, jelap la, tibetan side, 12,000 ft., l.iv.1924, hingston” (bmnh); paratypes: 2 workers (bmnh), 1 worker “himalaya, jelap la, tibetan side, 12,000 ft., l.iv.1924, hingston” (zmmu). distribution: (a) india: jammu & kashmir: sarthal 2390m (1), pahalgam (2), lidderwatt 2700m (2); himachal pradesh: badhal (jubbal) 2090m (1), sarthal 2050m (1), bharmour 2400m (1), old manali 2231m (1), solang 2640m (1), koholaro 2210m (1), sundli 2090m (1), sarhan 2300m (1), sarot (1), giriganga nr. jubbal (1), roggling 2720m (1), kothi 2400m (1), vic. kufri, 15km e shimla 2500-2700m (2); uttarakhand: mana 3200m (1), auli 3000m (1), chourangi khaal 2300m (1), chopta 2900m (1), gobind ghat 3000m (1) ghanghria 3500m (1), gangotri 3000m (1), khara pathhar 2700m (1), khanag 2300m (1); west bengal: darjeeling (sukhia 1880m (1), tiger hills 2400m (1), cheitry 2100m (1); arunachal pradesh: bomdila 2940m (1), jaswanthgarh 3146m (1); sikkim: phadamchen 1710m (1). (b) nepal: luhme (2), padmara-khari longa bumra 27503400m (2), thodung via those 3100m (2), phulchoki 2600m (2), daman 2400m (2), namsche bazar 3450m (2), 18km nne baglung 2540m (2), 16 km sw jomosom 2550m (2), 20km ssw jomosom 2300m (2), w. nepal, sigarhi-doti, lokondo 2133m (2), siklis 2133m (2), gurjakhani 2591m (2), tadopani, 3 km n pakhar 2700m (2), prov. taplejung, omje kharka, nw yamputh 2300-2500m (2), namde-jorsla 2890m (2). (c) bhutan: sampa-kotoka 2500m (2), 20km s thimphu 2400m (2), kotoka-gogona 2600-3400m (2), gogona 3100m (2), dechli paka 3300m (2), bumthang (2), thang-rudungla 24003500m (2), paesseling 3100-3400m (2), batbalitang(bumtang) 2600m (2). ecology: in the northwest himalaya, the species was collected under the stones in subtemperate forests of scattered cedrus, oak and some broad-leaved trees. few collection sites were under intense anthropogenic activities, surrounded by apple orchards. the mean temperature and humidity recorded at the nest was 31°c and 63% respectively. the temperature ranges in summer (march to june) from 8°c to 36°c and during winter (november to february) from 3°c to 27°c. in northeast himalaya, nests were observed under stone in grasslands and on ground covered with dense vegetation, mostly in wet soil. nesting sites had an average temperature of 15°c and relative humidity of 85%. alates were collected during the months of june to early august in northwest himalaya, and in september to october in northeast himalaya. altitudinal range of this species varies from 1341m at the ekra peak to 4084m in central himalaya. myrmica smythiesii forel, 1902 = myrmica smythiesi subsp. himalayana weber, 1947, synonymy by radchenko and elmes, 2001 lectotype of m. smythiesii (designated by radchenko & elmes, 2001): worker, “himalaya (smythies)”, “m. smythiesii forel”, “lx/1, lx/12” (mhng); paralectotypes (designated by radchenko & elmes, 2001): 2 workers “himalaya (smythies)”, “m. smythiesii forel”, “lx/1, lx/12” ; 1 worker, “m. smythiesi forel, himalaya, 7000-12,000 ft (smythies)”, “m.c.z type 20533” (mcz); 2 workers (upper with gaster and waist stuck separately; bottom without postpetiole and gaster), “m. smythiesii for. himalaya” (msng); lectotype of m. smythiesi subsp. himalayana (designated by radchenko & elmes, 2001): worker, “india, simla (wroughton)” (mcz); paralectotypes: (designated by radchenko & elmes, 2001): 2 workers “india, simla (wroughton)” (mcz). distribution: (a) india: himachal pradesh: jachli 2400m (1), pattidhank (25km above jubbal) 2440m (1), giriganga (28km from jubbal) 2700m (1), simla hills, kulala 2438m (2); jammu & kashmir: tangmarg 2160m (1), gulshan 3500m (1); uttarakhand: chopta, 2900m (1), chakrata 2100m (1), gobind ghat to ghanghria 3500m (1), flower valley 3500m (1), gangotri 2900m (1), yangti, almora 3749m (2). (b) nepal: gompa, bei tarahot 3400m (2). h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species970 ecology: myrmica smythiesii is distributed in forest composed of conifers (pinus, cedrus) and rhododendron species at the subtemperate zone of northwest himalaya. the nests were located under stones and in rotten wood covered with leaves. the temperature and humidity recorded at the collection site were 25°c and 67%, respectively, the nest temperature varied from 15°c to 20°c. alates appear in the months of may, june and july. at few localities, myrmica smythiesii is sympatric with m. rugosa. the altitudinal range for this species is 21333749m above mean sea level. myrmica tenuispina ruzsky, 1905 lectotype (designated by radchenko & elmes, 2001): worker (upper specimen on a pin with 4 workers), “tabi-dara zagyrdesht, e. bukhara, 17.vi.97, leg. kaznakov” (original label in russian), “myrmica rubra l. r. laevinodis var. tenuispina forel det, w, type” (zmmu); paralectotypes (designated by radchenko & elmes, 2001): 3 workers on a pin with lectotype; 3 workers with the same labels (zmmu); 8 workers with the same labels (zisp); 1 worker with the same labels (msng); 2 workers, “kala i khont, karategin, e bukhara, leg. kaznakov, 21.vi.97” (original label in russian); “myrmica rubra l. r. laevinodis var. tenuispina forel det, w, type” (zisp). distribution: (a) tadjikistan: additional localities: gissar range, anzobsky pass, 2800m (2); peter i range, djirgital, 3000-3400m (3); zeravshan range, near lake iskanderkul; zeravshan valley, guldara, 2400m (3). (b) uzbekistan: kashka-daria prov.: ishkent; khan-talhta; 3300-3400m (3). (c) afghanistan: badakshan, sarekanda, 4100m (2). note: previous records from kyrgyzstan (tarbinsky, 1976) and planes of middle asia (dlussky et al., 1990) were based on misidentifications. ecology: m. tenuispina is associated with the mountains of middle asia and ne afghanistan, and have been collected on subalpine meadows at altitudes between 2400 and 4100m above mean sea level and nests in the soil and under stones. myrmica urbanii radchenko et elmes 1998 holotype: worker, “india, shillong, mawphlang, east khasi, 3200ft, 2.05.86. leg. v. darlong” (bmnh); paratypes: 86 workers from the same nest as holotype; 2 workers, “india, mawphlang, megalaya, 1850m, 15.05.1976, leg. wittmer, baroni urbani”; 6 workers, “upper shillong, megalaya, 1900m, 13.05.1976, leg. wittmer, baroni urbani”; 3 workers, “cherranukh, india, 1961, leg. korovin” (bmnh, gepc, nhmb, sizk, zmmu) distribution: india: meghalaya (2). ecology: according to radchenko and elmes (2010), the ecology of this species is poorly known. it has been found at altitudes of 1000-1900m above mean sea level and one nest was found in soil beneath a log in a quercus forest. myrmica varisculpta radchenko et rigato, 2009 holotype: worker, “india, kashmir, leh, viii.1986 leg. c. canepari” (msnm) distribution: india: kashmir (3). ecology: unknown. myrmica villosa radchenko et elmes, 1999 holotype: worker, “dechli раkа, 5km o pélela, 3300m, 1920.06.1972 (natural-history museum basel bhutan expedition, 1972)” (nhmb); paratypes: 15 workers, 1 queen with the same label (nhmb, bmnh, sizk, gepc). distribution: bhutan (2). ecology: virtually unknown, except that the type series were found at the altitude 3300m above mean sea level. myrmica vittata radchenko et elmes, 1999 holotype: worker, “pakistan, bumburet, 24.05.1983, leg. m. brancucci” (bmnh); paratypes: 2 workers, “pakistan, bumburet, 24.05.1983, leg. m. brancucci”; 2 workers, “pakistan, kalam, 12.08.1994, leg. s. dakatra” (bmnh, msnm, sizk, gepc). distribution: pakistan (2). ecology: unknown. myrmica wardi radchenko et elmes, 1999 holotype: worker, “kashmir, ladakh, leh, 34o11’n, 77o35’e, 3450m, no.3094, 21.viii.1978 leg. p. ward” (bmnh). paratypes: 6 workers with same label as holotype; 6 workers, 1 gyne, “kashmir, pahalgam, 34o02’n, 75o19’e, 2190m, no.3003, 27.07.1978”; about l00 workers, 2 males, “kashmir, lidderwat, 34o09’n, 75ol5’e, 2700 m, no.3015, 3018c, 3023, 3029, 30.07.1978”; 1 worker, l gyne, “kashmir, kulan, 34ol6’n, 75o09’e, 2100 m, no.3050-10, 3050-12, 08.08. l978”; 29 workers, 1 gyne, “kashmir, ladakh. panikhar, suru r., 34o07’n, 75o57’e, no.3077, 3078, 17.08.1978”; 3 workers, “kashmir, ladakh, leh, 34o11’n, 77o35’e, 3450 m, no.3090, 21.08.l978”; 4 workers, “kashmir, sonamarg, 34ol8’n, 75o18’e, 2700 m, no.3102” (all leg. p.ward); 8 workers, “pakistan, chitral valley, between dir and lawari pass, 2400 m, 11.08.1994, leg. s. dacatra”; 3 workers, “indien, himachal pradesh, vic. theong, 25 km e shimla, 2400 m, no 394, 29.09.1996, leg. a. schulz and k. vock”; 3 workers, 2 gynes, “india, himachal pradesh, kullu valley, 5-7 km sw rothang la pass, 2500-2900 m, no. 432, 01.10.1996, leg. a. schulz and k. vock” (bmnh, nhmb, msnm, aspc, pswc, sizk, gepc, mmpc). distribution: (a) india: jammu & kashmir: kongdoori 2880m (1), seven springs 3077m (1), yusmarg 2380m (1), patnitop 1973m (1), gulmarg 2683m (1), mughal garden 1670m (1), pahalgham 2200m (1), kongdoori 2880m (1); himachal pradesh: khinang village 2940m (1), solang 2573m (1), gulaba 2700m (1), keylong 2940m (1), roggling 2740m (1), nalivan (22km from jubbal) 2300m (1), prounthi 2260m (1), sarthal 2200m (1), sociobiology 63(3): 956-975 (september, 2016) 971 sarchu (1); uttarakhand: gangotri 2900m (1). (b) pakistan: chitral valley, between dir and lawari pass, 2400m. ecology: according to the available information, myrmica wardi was found in meadows with sparse willow trees, prunus, acer and fir species among woody shrubs with rosa sp., and in stony grasslands. all recorded colonies were nesting in the soil. most specimens were collected while foraging over low vegetation displaying the same behaviour of the european myrmica rubra, which it could resemble in other habits (radchenko & elmes, 2010). however, according to the current information, this species was also collected at few places from open grasslands without any dense vegetation or forest cover, and the nests were not as deep as in m. rubra, the depth varied from 6-8 inches. additionally, a couple of nests were found in shady places, where it was found to be sympatric with myrmica cachmiriensis. the temperature of nesting site ranged from 15°c to 25°c and relative humidity from 39% to 69%. alates were collected during the months of july and august. altitudinal range for this species is 1670m-3450m above mean sea level. myrmica weberi elmes et radchenko, 2009 holotype: worker, “npl28 (no. 11elmes coll. label), nepal, sankhawalaya maghang, kharka, makalu barun conservation area, 27o36’18.5”n 87o7’30”e, 2634m, 7.xi.2005, leg. g. alpert, alonso and subedi, ck-3, yak meadow under rocks, under stones” (mcz). paratypes: 15 workers, 1 queen with the same label as holotype; 7 workers, “npl25, nepal, sankhawalava maghang, kharka, makalu barun conservation area, 27o35’36.6”n 87o7’20.7”e, 2548 m, 5.xi.2005, leg. d.emmett and subedi, mk 21-283, winkler trap”; 3 workers, “npl 31, nepal, sankhawalava makalu barun conservation area, mk-4, 27o 35’24.8”n 87o7’18.7”e, 2563 m, 3.xi.2005, leg. g. alpert, on rock under moss, large colony with 2q”; 3 workers “npl 32, nepal, sankhawalava makalu barun conservation area, mk-6, 27o35’24.8”n 87o7’18.7”e, 2563 m, 3.xi.2005, leg. g. alpert, open meadows under logs”; 7 workers “npl 33, nepal, sankhawalava, makalu barun conservation area, mk w17, 7.xi.05, winkler trap”; 1 worker, “india, darjeeling distr., tiger hill, 2500 m, 27.v.1975, leg. w. wittmer”, “m.indica weber det. radchenko and elmes”; 1 worker, “kosi, chauki, 2o11-12’n 87o27-28’e 2000-3000 m, 2224.vi.01, nhmb expd. npl. 2001”; 1 worker, “bhutan, nolding, 41km o wangdi, 2800 m, nat. hist. museum baselbhutan expedition, 1972”, “m. indica weber det radchenko and elmes”; 1 worker, “npl2.1, nepal, rigmo, h.tabata, 7.vi.1978”, “m. indica weber det radchenko and elmes”(nhmb, sizk, gepc, mcz). distribution: (a) india: west bengal (3). (b) nepal (3). (c) bhutan: sampa-kotoka, 1400-2600m (3). ecology: this species is probably restricted to the southern slopes of the central himalaya (nepal, india and bhutan), where it lives between 2000 and 3000m. the collection data suggests that myrmica weberi might be a rather reclusive species that nests under moss, dead wood and rocks, mostly foraging close to the ground and litter (radchenko & elmes, 2010). myrmica williamsi radchenko et elmes, 1999 holotype: worker, “india, kashmir, pantitop, 2000m, 6.09.86, leg. p. williams” (bmnh). distribution: india: jammu and kashmir: seven springs 30 77m (1); uttarakhand: ghangria 3500m (1), chopta 2900m (1). ecology: the nests were found under stones in open meadows of mountain slopes. temperature and humidity of the collection site ranged from 19-23°c and 63-75%. altitudinal range of this species varies from 2000m to 3500m above mean sea level. myrmica wittmeri radchenko et elmes, 1999 holotype: worker, “indien, him. prad. [himahal pradesh], mahri, 3000–3200 m, 15.05.1977, leg. wittmer et brancucci” (nhmb). paratypes: 3 workers (one missing head), “indien, him. prad. [himahal pradesh], mahri, 3000–3200 m, 15.05.1977, leg. wittmer et brancucci” ; 6 workers, “india, himachal pradesh, kullu valley, la pass, 3400-3700m, 2.10.1996, no.420, 422. leg. a.schulz & k.vock”; 3workers, “pakistan, kalam, 2300m, 12.07.1994. leg. s. dakatra” (nhmb, izk, msnm, aspc, gepc). distribution: (a) india: uttarakhand: chopta 2900m (1), gangotri 2900m (1), kedarnath 3300m (1); himachal pradesh: marhi 30903160m (1, 2), rekongpeo 2600m (1). (b) pakistan: kalam, 2300m. ecology: myrmica wittmeri seems to be well adapted to high altitude regions of himalaya above the timberline. the nests were observed under stone and in rotten wood, in ground covered with scarce vegetation mainly of rhododendron. nest temperature ranged from 15°c to 23°c and relative humidity varied from 43% to 72%. alates appear in the month of june and july. altitudinal range of this species is 2300m to 3300m. conclusion extending nearly 3000km from the east to west, the himalayan mountain range consists of complex topographical features, varied climate, which differs profoundly in the extreme eastern and western ends of himalaya and thus supports remarkable assemblage of vegetation types (mani, 1968, 1974; bharti, 2008). the dry climatic conditions of northwest have favoured the penetration of middle asian floral elements currently represented by species of juniperus, artemisia, eremurus, ferula, cedrus, etc. additionally, the moist climatic conditions of eastern region of himalaya has favoured the influx of elements originating from western china extending from yunnan, which include primula, magnolia h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species972 etc., with lineages of circaester agretis extending from china across tibet to himalaya. thus, the distributional patterns of present day plants in himalaya clearly indicate that the plants of eastern region show affinities with flora of western and northwestern china and of western region with flora of western, middle and north asian mountains (mani, 1974). however, there are few other plant species, which have extended distribution along the entire stretch of himalaya after their penetration either from east or west. based on the evidence generated by phyto-geographical affinities of himalayan elements, hooker (1906) and stearn (1960) consider 80° to 84° el (west of nepal) as a transitional zone “where climatic factors presumably limit the capacity of the plants suited to one provenance to compete with those of the other” (stearn, 1960). consequently, the west and middle asian mountain plant species adapted to comparatively dry conditions extend along the himalayan mountain chain from afghanistan to west nepal, whereas species originating from the moist regions of mountains of china extend as far as far west in eastern kumaon bordering nepal (stearn, 1960; rau, 1974). furthermore, the historical and geological facts also suggest that himalaya has served as primarily as a ‘route of emigration and colonization from east and northwest, secondarily of endemic development’ (stearn, 1960; rau, 1974). similar sort of distributional pattern has been noticed in some of the vertebrates and invertebrates (mani, 1974), however such cases have not been well documented. in any instance, the evolution of himalayan flora and fauna is of considerable significance, as the adjoining mountain regions of china, tibet and middle asia, which are much older in age as compared to himalaya, have considerable influence on himalayan life. later, in pleistocene, the changes in topography and climate of himalaya impacted by intermittent glacial and interglacial periods followed by post-pleistocene uplift might have influenced the animal and plant life (mani, 1974). a direct consequence of that glacial period was extinctions, survival of relic forms, migrations, formation of new refugia, exchange of elements, speciation etc., hence accounting for present day diversity, endemism and biogeographical affinities with adjoining regions. in accordance with the distributional data presented in the preceding text, myrmica lineages in himalaya depict a pattern quite similar to the other life forms occurring in himalaya. likewise, some of the myrmica species are restricted to northwestern ranges, some to northeastern region of indian himalaya, while few others have wider distribution extending throughout the himalayan range. these lineages are distributed within an altitudinal range of 1000-4800m (table 1), but the majority are restricted to the temperate zone. m. afghanica, m. tenuispina, m. rigatoi, m. pseudorugosa and m. vittata are restricted to northeastern afghanistan and pakistan, while m. cachmiriensis, m. ordinaria, m. wardi and m. wittmeri extend from northeastern pakistan to northwestern part of indian himalaya. nineteen of the species (m. adrijae, m. curvispinosa, m. elmesi, m. ereptrix, species altitudinal range 1 myrmica adrijae bharti, 2012 2479m 2 myrmica afghanica radchenko et elmes, 2003 3660m (approx.) 3 myrmica aimonissabaudiae menozzi, 1939 1300 3500m 4 myrmica alperti elmes et radchenko, 2009 2200 3200m 5 myrmica boltoni radchenko et elmes, 1998 2000 3400m 6 myrmica brancuccii radchenko, elmes et collingwood, 1999 2400m 7 myrmica cachmiriensis forel, 1904 1829 3500m 8 myrmica collingwoodi radchenko et elmes, 1998 2450 3100m 9 myrmica curvispinosa bharti et sharma, 2013 2479 2700m 10 myrmica elmesi bharti et sharma, 2011 2000 2200m 11 myrmica ereptrix bolton, 1988 2800m 12 myrmica foreliana radchenko et elmes, 2001 2900m 13 myrmica fortior forel, 1904 1981 2700m 14 myrmica hecate weber, 1947 1300 3700m 15 myrmica indica weber, 1950 1400 3150m 16 myrmica inezae forel, 1902 1900 3000m 17 myrmica kothiensis bharti et sharma, 2013 2479m 18 myrmica kozlovi ruzsky, 1915 2743 4800m 19 myrmica latra bharti, radchenko et sasi, 2016 2260 2740m 20 myrmica longisculpta bharti et sharma, 2011 2000 3100m 21 myrmica martensi radchenko et elmes, 1998 3200m 22 myrmica nefaria bharti, 2012 2469m 23 myrmica nitida radchenko et elmes, 1999 2600 3600m 24 myrmica ordinaria radchenko et elmes, 1999 2300 3077m 25 myrmica pachei forel, 1906 2600 3600m 26 myrmica petita radchenko et elmes, 1999 2300 2400m 27 myrmica pseudorugosa bharti, 2012 3600m 28 myrmica radchenkoi bharti et sharma, 2011 2000m 29 myrmica religiosa bharti et sharma, 2013 2300m 30 myrmica rhytida radchenko et elmes, 1999 2800 4200m 31 myrmica rigatoi radchenko et elmes, 1998 2200 2300m 32 myrmica rugosa mayr, 1865 1400 3500m 33 myrmica rupestris forel, 1902 1341 4084m 34 myrmica smythiesii forel, 1902 2133 3749m 35 myrmica tenuispina ruzsky, 1905 2400 4100m 36 myrmica urbanii radchenko et elmes, 1998 975 1900m 37 myrmica varisculpta radchenko et rigato, 2009 3350m 38 myrmica villosa radchenko et elmes, 1999 3300m 39 myrmica vittata radchenko et elmes, 1999 1690 3784m (approx.) 40 myrmica wardi radchenko et elmes,1999 1670 3450m 41 myrmica weberi elmes et radchenko, 2009 1400 -3000m 42 myrmica williamsi radchenko et elmes, 1999 2000 -3500m 43 myrmica wittmeri radchenko et elmes, 1999 2300 3300m table 1. list of myrmica species with their altitudinal range. http://www.antweb.org/browse.do?genus=myrmica&species=indica&rank=species&project=indiaants http://www.antweb.org/browse.do?genus=myrmica&species=pachei&rank=species&project=indiaants http://www.antweb.org/browse.do?genus=myrmica&species=petita&rank=species&project=indiaants sociobiology 63(3): 956-975 (september, 2016) 973 species name/locality jk hp uk ap wb mh sk np bt pk af myrmica adrijae bharti, 2012 + myrmica afghanica radchenko et elmes, 2003 + myrmica aimonissabaudiae menozzi, 1939 + + + + + + + + + + + myrmica alperti elmes et radchenko, 2009 + myrmica boltoni radchenko et elmes, 1998 + myrmica brancuccii radchenko, elmes et collingwood, 1999 + + myrmica cachmiriensis forel, 1904 + + + myrmica collingwoodi radchenko et elmes, 1998 + myrmica curvispinosa bharti et sharma, 2013 + myrmica elmesi bharti et sharma, 2011 + myrmica ereptrix bolton, 1988 + myrmica foreliana radchenko et elmes, 2001 + myrmica fortior forel, 1904 + myrmica hecate weber, 1947 + + + + + + + myrmica indica weber, 1950 + + + + myrmica inezae forel, 1902 + + myrmica kothiensis bharti et sharma, 2013 + myrmica kozlovi ruzsky, 1915 + + + myrmica latra bharti, radchenko et sasi, 2016 + myrmica longisculpta bharti et sharma, 2011 + myrmica martensi radchenko et elmes, 1998 + myrmica nefaria bharti, 2012 + myrmica nitida radchenko et elmes, 1999 + + myrmica ordinaria radchenko et elmes, 1999 + + myrmica pachei forel, 1906 + + + myrmica petita radchenko et elmes, 1999 + myrmica pseudorugosa bharti, 2012 + myrmica radchenkoi bharti et sharma, 2011 + myrmica religiosa bharti et sharma, 2013 + myrmica rhytida radchenko et elmes, 1999 + + myrmica rigatoi radchenko et elmes, 1998 + myrmica rugosa mayr, 1865 + + + + + myrmica rupestris forel, 1902 + + + + + + + + myrmica smythiesii forel, 1902 + + + + myrmica tenuispina ruzsky, 1905 + myrmica urbanii radchenko et elmes, 1998 + myrmica varisculpta radchenko et rigato, 2009 + myrmica villosa radchenko et elmes, 1999 + myrmica vittata radchenko et elmes, 1999 + myrmica wardi radchenko et elmes,1999 + + + + myrmica weberi elmes et radchenko, 2009 + + + myrmica williamsi radchenko et elmes, 1999 + + myrmica wittmeri radchenko et elmes, 1999 + + no. of species 18 15 11 6 7 2 4 12 8 9 3 *jkjammu & kashmir; hphimachal pradesh; ukuttarakhand; aparunachal pradesh; wbwest bengal; mhmeghalaya; sksikkim; npnepal; btbhutan; pkpakistan; afafghanistan table 2. known species diversity of genus myrmica in each state/country. h bharti, s sasi, a radchenko – biogeography and ecology of myrmica species974 m. foreliana, m. fortior, m. inezae, m. kothiensis, m. latra, m. longisculpta, m. nefaria, m. nitida, m. petita, m. radchenkoi, m. religiosa, m. rhytida, m. smythiesii, m. varisculpta and m. williamsi) are exclusively restricted to northwestern region of indian himalaya and do not extend much further; whereas some of the species (m. alperti, m. boltoni, m. martensi) are concentrated in central himalaya (nepal). similarly, m. collingwoodi and m. villosa are only restricted to bhutan. on contrary, m.urbanii, m. weberi, m. indica, m. kozlovi and m. pachei seem to have penetrated from northeastern region of himalaya and extend up to central himalaya and are not recorded hitherto from northwestern region. besides, m. aimonissabaudiae, m. rugosa, m. hecate, m. rupestris have wider distribution and extend throughout the himalayan range. based on the similarity of certain peculiar morphological features, radchenko and elmes (2010) have clustered himalayan myrmica species in to various species groups; inezae species group includes m. inezae, m. radchenkoi, m. kothiensis, m. religiosa, m. curvispinosa and m. rigatoi and this species group is exclusively restricted to northwestern and central himalaya and does not penetrate into northeastern himalaya. the same pattern has been observed in the case of rugosa species group (m. longisculpta, m. cachmiriensis, m. ordinaria, m. wardi, m. foreliana, m. afghanica, m. pseudorugosa) with the exclusion of m. aimonissabaudiae, m. rugosa, m. rupestris, m. hecate having wider distribution. a similar case is present by the smythiesii species group (m. smythiesii, m. adrijae, m. fortior, m. wittmeri, m. nefaria, m. ereptrix and m. latra) and few other species m. nitida, m. petita, m. rhytida, m. williamsi, which have not been placed in any of the species groups. ritae species group (m. alperti, m. boltoni, m. collingwoodi, m.indica, m.martensi, m.urbanii and m.weberi) and pachei species group (m. pachei and m.villosa) are restricted to northeastern and central himalaya. from the foregoing discussion, it seems quite probable that some of the ancestral lineages of rugosa species group after penetration from north in himalaya got isolated from rest of myrmica fauna, speciated and spread from northwest himalaya up to central himalaya (with exception of m. aimonissabaudiae, m. rugosa, m. rupestris and m. hecate, which extend throughout the himalayan range). almost similar is the distributional pattern of species belonging to inezae and smythiesii species groups with none of the species belonging to this group has penetration beyond central himalaya. on contrary, it seems quite logical that the other lineages currently represented by species of pachei and ritae species groups infiltrated from western china and diversified in himalaya, and spread from northeast himalaya up to central himalaya. this west-east, east-west spreading of lineages in himalaya is even validated by the absence of species belonging to rugosa species group in mountains of china, and by the presence of species belonging to ritae and pachei species groups in china and northeastern himalaya (as 50% of myrmica fauna occurring in mountains of china is represented by species belonging to ritae and pachei species groups). acknowledgements the grants received from department of science and technology (dst project no. sr/so/as-68/2011), ministry of science of science and technology, govt. of india for generating the present data are gratefully acknowledged. references bharti, h. 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(1971). the insect societies. cambridge, mass.: harvard university press, x + 548 p doi: 10.13102/sociobiology.v61i2.184-188sociobiology 61(2): 184-188 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 susceptibility of melipona scutellaris latreille, 1811 (hymenoptera: apidae) to beauveria bassiana (bals.) vuill. p de j conceição, cm de l neves, g da s sodré, cal de carvalho, av souza, gs ribeiro & r de c pereira introduction the improper use of agricultural chemicals has led to studies on alternative methods of sustainable pest control. among these methods biological control with entomopathogenic fungi stands out as a broadly used method of pest control in agroecosystems (messias 1989; marques et al. 2004). these fungi are highly viable, because they are able to preserve parasitoid, predator, and pollinator populations, and represent an important factor in integrated pest management (neves et al. 2001; oliveira 2008). however, some authors state that these fungi can be pathogenic to bees. espinosa-ortiz et al. (2011) studied the susceptibility of larva and adult honeybees (apis mellifera) to three types of isolates of entomopathogenic fungi and observed high mortality (90-100%) caused by the fungus beauveria bassiana, when applied at the dose of 1 x 107 conidia/ ml. bee mortality by entomopathogenic fungi was also recorded by butt et al. (1994, 1998). abstract entomopathogenic fungi are frequently used as an alternative method for insect pest control. however, only a few studies have focused on the effect of these fungi on bees and on the selectivity of fungi to beneficial organisms in agroecosystems. the objective of the present study was to assess the susceptibility of worker bees of the species melipona scutellaris (locally known as "uruçu") to the isolate (biofungi 1) of the entomopathogenic fungus beauveria bassiana. the experiment was carried through indirect contact between the fungal suspension and newlyemerged bees and topical application of the fungal suspension on the back of newly-emerged bees. the sampling design was completely randomized and comprised five treatments, which included four different concentrations of the fungus: 1 x 105, 1 x 106, 1 x 107, 1 x 108 conidia/ml, and a control composed of distilled water. each treatment had five replicates. the mortality data were subjected to an analysis of variance and a probit regression analysis, which provided an estimate of the lethal dose to 50% of the population (ld 50 ). the adjustment of the curves to the model was tested with a chi-squared test and differences between curves were tested with a test for parallelism. beauveria bassiana was virulent to uruçu bees, killing the bees at the lowest dose used. these findings may help minimize the impact of this entomopathogen and, therefore, contribute to the maintenance of natural populations of these insects. sociobiology an international journal on social insects federal university of recôncavo da bahia (ufrb), cruz das almas-ba, brazil. article history edited by gilberto m m santos uefs, brazil received 11 february 2013 initial acceptance 17 july 2013 final acceptance 11 september 2013 keywords uruçu bee, entomopathogenic fungi, biological control corresponding author rozimar de campos pereira center of agricultural, environmental and biological sciences federal university of recôncavo da bahia (ufrb) rua rui barbosa, 710 44380-000, cruz das almas-ba, brazil. e-mail: rozimarcp@uol.com.br al mazra’awia (2007) studied the impacts of b. bassiana on a. mellifera and concluded that bees exposed to high concentrations of this fungus show high mortality. however, he also affirmed that the beehives exposed to high densities of inoculum of the same pathogen had low mortality. according to hokkaner et al. (2003), the temperature is higher inside than outside the beehives, which makes them safer to fungal infection. when hokkenen et al. (2003) assessed the impacts of metarhizium and beauveria on bees they observed that different strategies for the application of these fungi should be considered due to the risks they could bring to insects in the natural ecosystem. the conservation of pollinators requires the attention of scientists due to the large number of problems faced by them in natural ecosystems, including death by pesticides (otterstatter & thomson 2008; freitas & pinheiro 2010; rocha 2012). there are few robust data on the effect of entomopathogenic fungi on social bees (nogueira-neto 1953; mcgregor research article bees sociobiology 61(2): 184-188 (june, 2014) 185 1976; ferraz et al. 2006; braga et al. 2010) and the impact of these fungi on beneficial insects associated to crops (espinosaortiz et al. 2011; kanga et al. 2002). ferraz et al. (2006) stated that, in spite of not having unequivocal data at hand, it is possible that the entomopathogenic fungi beauveria bassiana, metarhizium anisopliae, and metarhizium flavoviride cause the death of indigenous bees. the objective of the present study was to assess the susceptibility of worker bees of the species melipona scutellaris to b. bassiana isolates at different concentrations and contact forms. material and methods the experiment was conducted at the laboratory of the center for the study of insects (insecta), at the federal university of recôncavo da bahia (ufrb), with newly-emerged worker bees of the species m. scutellaris, locally known as uruçu. the bees were provided by the rearing facilities of insecta/ufrb. in the treatments we used the commercial isolate of the fungus b. bassiana (biofungi 1), produced in the laboratory of research and production of microorganisms/biofactory, state university of southeast bahia (uesb), vitória da conquista, state of bahia, where this pathogen has been successfully tested for the control of crop pests. collection and sampling brood combs of m. scutellaris were removed from the colonies of the rearing facilities at insecta and maintained in growth chambers of the b.o.d. type (biologic oxygen demand) at a temperature of 28 ± 2ºc, relative humidity of 70% ± 2%, and a photoperiod of 12h for a possible emergence of bees (espinosa-ortiz et al. 2011). preparation of different concentrations of conidia and application one-gram samples were randomly removed from the fungal substrate and added to 10 ml of sterilized water containing tween 80 adhesive spreader at 1% (v/v). to obtain a homogenized suspension, serial dilutions (102) were made, so that the conidia could be counted in a newbauer chamber under a microscope (100x). the preparation of the suspensions followed alves (1998b). the treatments included four fungal concentrations: 1 x 105, 1 x 106, 1 x 107, 1 x 108 conidia/ml-1 and composed of distilled water. newly-emerged bees were anaesthetized for 1min in a refrigerator at 16°c to facilitate the handling of worker bees. bees were exposed to the fungal suspension through topical application on the dorsum and indirect contact. in the topical application, 1µl of each treatment was applied to the dorsum of each bee with a sterile 10μl micro syringe (bd plastipak). in the exposure by indirect contact, the bees were placed on filter paper sheets slightly moistened with 1 ml of each treatment for 5 min (rother et al. 2009). after each treatment, the worker bees were placed in plastic containers (6.0 cm x 8.0 cm) and transferred to an acclimatized chamber. five worker bees were placed in each plastic container, totaling 125 individuals. the bees were fed with honey at 10% (100g/1000ml distilled water), placed on a sterilized wad of cotton to prevent contamination (rother et al. 2009). data analysis the experimental design was completely randomized. it was composed of five treatments and five replicates, totaling 25 plots. the mortality of worker bees was monitored at 24h intervals for 10 days. the results were corrected considering natural mortality in accordance with the abbott formula (alves 1998). the corrected mortality data were subjected to an analysis of variance. for this analysis, the data were transformed using the formula x’ = arcsin (xi / n), where x’ is the datum after the transformation, xi is the mortality observed in the replicate i and n is the total number of insects in the experimental plot. data normality was assessed with a kolmogorov-smirnov test and variance homogeneity was assessed with a levene test. mortality data from different treatments were subjected to a probit regression analysis (sokal 1958) in statistica (statsof inc.). this analysis provided an estimate of the lethal dose to 50% of the population (ld50). the adjustment of the curves to the model was assessed with a chi-squared test and differences between curves for the exposure methods were assessed with a test of parallelism (alves 1998). the mortality data at highest dose were used to build survival curves for the two exposure methods following the kaplan-meier method (blanford et al. 2005). based on these curves the time to mortality (or survival) of 50% of the insects (s50) was estimated. the curves were compared using a logrank test (p = 0.95) and a gehan-breslow-wilcoxon test (gbw) in graphpad prism 5.0 (motulsky 1995). results and discussion under the experimental conditions, there was no significant effect (p > 0.05) of the exposure methods on the mortality of uruçu bees (m. scutellaris). however, there was a significant interaction between exposure methods and the doses applied (df = 4.396; f = 17.68; p < 0.001). the corrected mortality caused by different doses of the fungal suspension was higher when applied on the dorsum of the bees than when bees got in indirect contact with the fungal suspension (figure 1). even at the lowest dose (105 conidia ml-1), the worker bees were affected: they lost mobility and had an average mortality of 56%. these results differ from those of butt et al. (1994), who tested the pathogenicity of metarhizium anisopliae to adult bees of a. mellifera and observed significant mortalipj conceição et al. susceptibility of melipona scutellaris to beauveria bassiana186 sion model (χ2 = 2.897; df = 2; p = 0.235). the isolate of b. bassiana used in the experiments was pathogenic to uruçu bees and caused high mortality even at low doses. based on the results obtained, a ld50 of 2.04x10 5 conidia ml-1 was estimated (7.95. 103; 3.70.105) (figure 2). the data obtained from the exposure method of indirect contact with the fungal suspension at different doses showed high variability among doses, and did not fit the probit regression model (χ2 = 26.811; df = 2; p < 0.001). with this result, it was not possible to estimate the ld50 or make a comparison between exposure methods through the assessment of the parallelism of curves. for the comparison between different exposure methods, the survival curve at the two highest doses was compared with mantel-cox and gbw tests. data analysis through the construction of survival curves using the kaplan-meier method made it possible to assess mortality details over time, and allowed the identification of differences in the survival curve between exposure methods. we observed that the indirect contact method resulted in a lower mortality rate at the lowest doses in the beginning of the observation period, but in the end of the observation period the total mortality was also high. figure 3 shows that the topical application of the fungal suspension of b. bassiana (108 ml-1) resulted in high mortality in the end of the experiment, with a survival of only 20.4% (± 5.1) of the bees; whereas the exposure method through indirect contact resulted in higher survival (69.1% ± 4.2). the survival curves obtained for different methods of exposure to the fungus were significantly different from the control and from one another when compared by mantel-cox (log rank test) and gbw tests (table 1). the mean survival value (s50) estimated for the topical application method was 216.0 days (table 1). for the config 1 – corrected mortality (%) of uruçu bees (m. scutellaris) ten days after exposure to fungal suspensions with increasing concentration of b. bassiana conidia by the methods of topical application and indirect contact. ties only at very high doses. in a similar study, kampongo et al. (2008) reported a mortality rate of 42 45% for bombus sp. bees exposed to high doses of b. bassiana (2x1011 conidia ml-1). espinosa-ortiz et al. (2011) tested the virulence of different commercial isolates of b. bassiana, m.anisopliae, and p. fumosoroseu on worker bees of the species a. mellifera and obtained a mortality rate of 90% 100% with a dose of 107 conidia ml-1 of b. bassiana. therefore, it is possible that the isolates exhibit specificity to bee species. the topical application of the fungal suspension on the dorsum of bees caused high mortality, with low variability between replicates and uniformity among treatments. the data obtained from this method adjusted well to the probit regresfig 2 – dose-response curve resulting from the methods of topical application of fungal suspensions containing b. bassiana conidia on uruçu bees (m. scutellaris). dotted lines represent the fiduciary limits of the estimated doses. fig 3 – kaplan-meier survival curves of uruçu bees (m. scutellaris) subjected to two exposure methods to fungal suspensions containing b. bassiana conidia (108 ml-1). the fungal suspensions were applied topically on the dorsum of bees or by indirect contact of the bees with a surface previously sprayed with the suspension. sociobiology 61(2): 184-188 (june, 2014) 187 trol treatment or application by indirect contact, the amount of s50 could not be estimated. for the control treatment or exposure method by indirect contact, s50 could not be estimated because bee mortality did not exceed 50%, and the use of the kaplan-meier model to calculate s50 is limited by the increased survival time of the individuals studied. the estimated value of hazard ratio (hr) between the two exposure methods was 0.35, between the topical application and the control it was 8.48, and between the indirect contact and the control it was 5.96 (table 1). the hazard ratio estimates the difference in mortality between treatments based on the slope of the respective survival curves. in this particular case, the average mortality estimated for the topical application exposure method was consistently 35% higher throughout the experiment in comparison with the indirect contact exposure method. table 1 – comparison of survival curves estimated for uruçu bees (m. scutellaris) exposed to topical application or indirect contact with the fungal suspension of b. bassiana conidia (108 ml-1). mantel-cox test (logrank) topical application x control 76.96**1 indirect contact x control 21.23** topical application x indirect contact 26.17** gehan-breslow-wilcoxon test (gbw) topical application x control 57.51** indirect contact x control 19.58** topical application x indirect contact 7.69** median survival (s50) topical application 216 indirect contact 192 control hazard ratio (hr) topical application x control 8.48 (5.26 to 13.67)2 indirect contact x control 5.86 (2.76 to 12.45) topical application x indirect contact 0.35 (0.22 to 0.61) 1 significant at p < 0.001; 2 confidence interval estimated (ci) delaplane & mayer (2005) reported that methods used to apply the compounds may interfere with the results of toxicity assessment of pesticides on non-target insects in the laboratory; there may be an interaction between the active ingredients and exposure methods. carvalho et al. (2009) tested four methods to assess the toxicity of pesticides to a. mellifera and found different responses according to the active ingredient used. thiamethoxam and methidathion were highly toxic, with low median lethal time (lt50) for topical application, supply of contaminated food, and indirect contact of bees to previously sprayed surfaces. abamectin showed lowest lt50 when provided in contaminated food, whereas deltamethrin showed highest toxicity when the insects were exposed to previously sprayed surfaces (carvalho et al. 2009). pest control with entomopathogenic agents has the advantage of leaving no toxic residues, and therefore can be used for long periods with low environmental impact (alves 1998). however, the susceptibility of stingless bees to other commercial isolates of entomopathogenic fungi should be tested in future studies, with special attention to the concentration. only by knowing the effects of entomopathogenic agents on bees, it will be possible to achieve greater efficiency in pest control with minimal impact on these beneficial insects. beauveria bassiana (biofungi 1) was highly virulent to uruçu bees (m. scutellaris), killing them at the lowest dose used. this information is important because the use of biological products for insect pest control has been growing, and these products require good management to avoid damage to beneficial insects. acknowledgments the authors thank the brazilian council for scientific and technological development (cnpq) and the brazilian coordination for the improvement of higher education personnel (capes) for the scholarships granted, the center for the study of insects (insecta/ufrb), dr. tiyoko nair hojo rebouças, researcher at the laboratory of research and production of microorganisms/ biofactory, state university of southeast bahia (uesb), for supplying the fungal material, and dr. carlos alberto tuão gava of embrapa semiárido (brazilian agricultural research corporation) for the help in the statistical analysis. we thank two anonymous reviewers for valuable comments. references al mazraawi, m.s. 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(1958). probit analysis. journal of entomology, 50: 738-739. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1044sociobiology 63(3): 909-918 (september, 2016) survey of ants in dry forests of southwestern ecuador (hymenoptera: formicidae) introduction the status of ecuador as hyperdiverse in terms of its biological heritage is well documented, but the coverage for studies supporting this reality is uneven as most studies are concentrated on the northeastern amazon region or the galapagos islands. considerable parts of the country have barely been sampled (guayasamin & bonaccorso, 2011) or remain unknown (brehm et al., 2008). the seasonally dry forests of southwestern ecuador are such an area, extending into peru and forming the tumbesian region with an extension of over 130,000 km2 (loaiza, 2013), they are considered the least well characterized of the seasonally dry forests in the american tropics (särkinen et al., 2011). the dry forests on the ecuadorean side are considered better preserved than in peru, yet at least 50% of the original cover has been lost (aguirre-mendoza & kvist, 2005). the dry forests on the ecuadorean side were declared part of the world network of biosphere reserves by the unesco in 2014. what little abstract two dry forests of southwestern ecuador separated 43 km from each other, one situated at 460 m above sea level and the other at 680 m, are surveyed for ants giving a total of 28 species collected manually and from pitfall traps. eleven species are shared between the sites whilst four are exclusive to one site and 13 to the other. differences in humidity, rainfall seasonality, and disturbance regimes may account for at least part of the differences observed between the ant communities of the two sites. dorymyrmex pyramicus peruvianum wheeler, 1919 and pseudomyrmex kuenckeli (emery, 1890) are reported from ecuador for the first time. cardiocondyla emeryi forel, 1881 and camponotus conspicuus zonatus emery, 1894 are reported from mainland ecuador for the first time. the genus dorymyrmex mayr, 1866 is recorded from ecuador for the first time. sociobiology an international journal on social insects je lattke1, m vélez2, n aguirre2 article history edited by evandro nascimento silva, uefs, brazil received 18 april 2016 initial acceptance 07 august 2016 final acceptance 24 august 2016 publication date 25 october 2016 keywords tumbesian forests, biodiversity, new records, endemism. corresponding author john e. lattke departamento de zoologia, universidade federal do paraná caixa postal 19020, cep 81531-980 curitiba-pr, brasil e-mail: piquihuye@gmail.com is known of them points to important endemism for plants and birds (linares-palomino et al., 2010). studies of insects in the seasonally dry forests of southern ecuador are scarce and more recent (domínguez et al., 2015), and for ants in particular, nonexistent (salazar et al., 2015). ants are an ecologically dominant and diverse group of insects that intervene in important processes for the maintenance of many ecosystems, particularly in the tropics (folgarait, 1998; del toro et al., 2012; guenard, 2013). they are sensitive to environmental change and easily sampled quantitatively, winning them an established role as biological indicators (underwood & fisher, 2006; majer et al., 2007, ribas et al., 2012). these and other traits have made for an ever increasing amount of studies on their diversity and the roles they play in ecosystems. given the increasing amount of interdisciplinary research in southern ecuador centering around conservation, biodiversity, and climate change (gradstein et al., 2008; bendix et al., 2013), the lack of knowledge about a key group of organisms, such as the region’s ants, is a major 1 universidade federal do paraná, curitiba-pr, brazil 2 universidad nacional de loja, loja, ecuador research article ants je lattke, m vélez, n aguirre – dry forest ants from southwestern ecuador910 hindrance towards greater understanding of the regional patterns and processes of biological diversity. an exploratory survey using both quantitative and qualitative collecting methods was undertaken to collect and identify at least the most common ants in the study area and establish a baseline of ant diversity information for steering further studies. the first results of this work are presented. materials and methods study sites sampling was carried out during the last days of september and first week of october of 2014 during the dry season in two dry forests of southwestern ecuador. both sites are located in loja province. site i. zapotillo canton. hacienda el chilco, 14 km nnw of zapotillo, –04.26306°, –80.26874°, 460 m. the site is owned and managed by the universidad nacional de loja as a place for field studies. the terrain is rolling hills with a sparse forest and shallow leaf litter layer where ceiba trichistandra bakh., eriotheca ruizii (k. schum.) a. robyns, and tabebuia chrysantha (jacq.) g. nicholson are amongst the dominant species (muñoz et al., 2014). the hacienda is regularly entered by herds of goats from neighboring lands where they browse at leisure. average annual precipitation between the years 2000 and 2010 in zapotillo was recorded as 884 mm, ranging from 243 mm in 2003 to 1506 mm in 2008. most rainfall is concentrated from january to april, and the remaining months are considered the dry season; temperatures averaged 25.6°c annually and relative humidity 75.2% during the same period (inamhi, 2000-2010). site ii. macará canton. reserva laipuna, 20 km nne macará, –04.21056°, –79.88682°, 680 m. the site is owned and managed by naturaleza y conservacion internacional, an ngo based in loja, ecuador and is part of a regional system of sites for long-term environmental monitoring (bendix et al., 2014). the reserve is on steep slopes facing the catamayo river. sierra et al. (1999) classifies the general area as a montane thorny scrub forest (espinar seco montano). the sampling site lacks significant leaf litter and, besides the presence of shrubs and trees, also had columnar cactus (not present in el chilco). published information dealing with the biota of the site is scarce, and apparently lacking in refereed journals. one unpublished thesis from a local university (ambuludí, in thesis) cites some of the dominant trees as eriotheca ruizii (k. schum.) a. robyns, acacia macracantha humb. & bonpl. ex willd., and bursera graveolens (kunth) triana & planch. precipitation between 2008 and 2009 averaged 617 mm annually, with most of it concentrated from january to march (peters & richter, 2011), implying very marked seasonality. for the same period the average annual temperature was 23.3°c and average annual relative humidity was 78%. the laipuna site is 42.7 km wsw from el chilco. the soil proved quite hard in both sites, necessitating the use of metallic chisels and a hammer to permit placement of pitfall traps. data collection field activities: ants for quantitative analysis were collected using a modified all protocol (bestelmeyer et al., 2000) whilst complementary data on ant diversity was obtained by manual collecting. the modification to the original all protocol was imposed by logistical constraints that imposed reducing the running time of the pitfall traps to 24 hours (instead of the standard 48 hours) before being gathered and emptied of their contents. leaf litter samples and pitfall traps were used in el chilco, but only pitfall traps were used in laipuna as the ground is mostly bare with scant, small fallen leaves, and a few dry branches scattered upon the surface. a linear transect of 220 m was set up at el chilco and 20 one m2 plots, separated 10 m from each other, were delimited. the litter was collected from each plot and sifted with a winkler sieve; the material was placed in cloth bags, one for each sample. strictly leaf litter was collected as the hard ground did not permit the scraping of topsoil or nonexistent humus. litter was sampled at mid-morning under the thin shade of the canopy and processed with mini-winkler extractors during 48 hours, with removal and refilling of the litter from each extractor at 24 h (guénard & lucky, 2011). pitfall traps were placed parallel to the litter transect, one pitfall 10 m distant from each plot. each pitfall trap consisted of a 10 ounce disposable plastic cup placed at the same level as the surrounding ground filled one-third to one-half with a solution of water and a drop of liquid dishwashing soap to break the surface tension. the pitfall traps were left running during 24 hours before being gathered and emptied of their contents. additional ants were collected manually at each site during 1.5 2 hours by five persons during two days. ants were manually collected at night until 23:00 h in the laipuna site. field activities were covered by permit 011-2014-ic-flo-dplma granted by the ecuadorean ministry of the environment. lab activities: ants were kept in vials with 90% ethanol. ants from each sample were put in petri dishes and sorted into morphospecies using a zeiss stemi 2000c stereo microscope. in the case of genera with numerous species, a fiche was kept for each species with sketches and diagnostic characters highlighted and defined. relevant taxonomic literature was consulted for species identifications as well as online resources such as antweb (2016a), and antwiki (2016a). for comparative purposes specimens from the ecuadorian ant reference collection (arc-e), housed in the instituto de ciencias biológicas of the escuela politécnica nacional in quito and curated by dr. david donoso, were also consulted. ants identified to morphospecies bear an arc-e morphospecies number (ec-##). arc-e serves as a national reference collection for all ant species of ecuador. only worker ants were considered for the quantitative analysis as solitary queens or males do not necessarily imply an established colony. solitary soldier ants, not associated with the worker caste, were also excluded from the analysis so as not to inflate the species number by misidentifying as http://www.antweb.org/ http://www.antwiki.org/ sociobiology 63(3): 909-918 (september, 2016) 911 different species, different castes of the same species, a hazard especially relevant for pheidole. solenopsis is a particularly rich genus in southern ecuador and there are indications that using morphology alone in determining species for the smaller “thief ants” group of this genus can be extremely misleading (delsinne et al., 2012). given such a degree of uncertainty, thief ants were excluded from the present study, nevertheless apparent morphospecies were mounted in repeated series. solenopsis species of the geminata group (trager, 1991) were included for the analysis. voucher specimens of all species and morphospecies (including thief ants) have been deposited in the arc-e collection. specimens of rare species and type specimens of new species to be described will be deposited in the quito catholic zoology museum, qcaz, pontificia universidad católica de ecuador. additional material is deposited in the insect collection of the universidad nacional de loja, loja, ecuador. specimens collected in zapotillo cover lattke collection numbers from 3598 to 3604 with 36041 to 3604-20 covering the pitfall trap transect. specimens collected in macará cover lattke collection numbers from 3605 to 3617 with 3611-1 to 3611-20 covering the pitfall transect. additional specimens were collected in both sites by m. vélez and three field assistants. towards the end of this project we were given the opportunity to have dna extracted from some of the specimens for barcoding by the international barcode of life consortium. the data may be accessed at the following url: http://dx.doi.org/10.5883/ds-dryloja. these results will be treated in a separate publication covering the barcoding of ants in additional dry forest sites in southern ecuador (domínguez et al., 2016). data analysis litter and pitfall data was organized as presence – abscence (incidence) of each morphospecies for each sample plot as ants are colonial organisms. mao tao species accumulation curves were calculated using estimates 9.1.0 (colwell, 2013). using the same software the following estimators were calculated after 100 random shufflings of the samples: ice, chao 2, and second order jackknife (jk2). beta diversity comparison uses the chao jaccard index, also calculated by estimates 9.1.0. the present functional groups (fgs) are conceptually based upon silvestre et al. (2003), anderson (2010), and narenda et al. (2010) but modified taking into account known natural history and phylogenetic affinities: (1) generalist epigeic hunters. predatory ants of medium to small size (body length 0.8 to 1.5 cm) that forage and hunt on the ground and leaf-litter. (examples: smaller species of odontomachus latreille and ectatomma smith). (2) nomadic hunters. ants that hunt as a group with temporary nests, all of the dorylinae. (3) fungus growing ants (detritus). attines that do not cut foliage for making their fungus gardens, but use miscellaneous organic matter instead (cyphomyrmex mayr). (4) fungus growing (foliage) ants that preferably gather fresh leaves (acromyrmex mayr). (5) epigeic omnivores. large to medium-sized ants that forage upon the ground and litter surface. (camponotus mayr, dorymyrmex mayr, solenopsis geminata (fabricius 1804), pheidole westwood, and some pseudomyrmex lund). (6) arboreal foragers. ants that nest and mainly forage in trees, shrubs, cacti, and epiphytes (cephalotes latreille, azteca forel, most pseudomyrmex). dissimilarity of species between functional groups (fg) was quantified by the complementarity index: cab=uab/sab, where a is the number of species in fg a, b the number of species in fg b, and c is the number of shared species between a and b. uab is the number of species unique to any of the two fgs, a+b-2c, and sab is the total richness from both fg, a+b-c (colwell and coddington 1994). if all species are different for each fg the value is one and it is zero if all are the same. results richness and abundance a total of 17 genera and 28 species from seven subfamilies were found (table 1). the richest subfamily is myrmicinae with 12 species from eight genera and the poorest are ectatomminae and ponerinae, both with only one species. the most species rich genera are pheidole, with five species, and camponotus, with four species. twelve genera have only a single species. apparently one solenopsis westwood thief ant species was found in el chilco and perhaps two to three in laipuna, the chilco species is apparently also present in laipuna. el chilco has a total of 16 species and nine genera from six subfamilies. ten of these species were sampled with pitfall traps whilst none were recovered from leaf-litter samples. two of the pitfall traps failed to capture any ants while the rest had from one to four species with an average of 1.6 species per trap for the whole transect. the most species rich subfamily is myrmicinae with five species, followed by formicinae with three species. laipuna has a total of 25 species and 15 genera from seven subfamilies. eleven of these species were sampled with pitfall traps. all pitfall traps had at least one species present, with an average of two and a maximum of five species per trap. the most species rich subfamily is myrmicinae with at least 12 species, followed by dolichoderinae, formicinae, and pseudomyrmicinae with four species each (table 1). the species accumulation curves for both sites are similar, climbing gently with nine species attained for the tenth sample in laipuna (fig 2) and eight for el chilco (fig 3), the laipuna curve showing more flattening towards the end. in contrast, the estimators show different behaviors between the two sites. estimators for laipuna converge upon the observed species curve with a difference of 86-98% from the sampled value, suggesting the presence of 12 species. estimators for el chilco are spread out, with differences of 7291% suggesting the presence of up to 14 species (table 2). in both sites manual sampling proved more effective in terms of http://dx.doi.org/10.5883/ds-dryloja je lattke, m vélez, n aguirre – dry forest ants from southwestern ecuador912 the number of collected species, with 24 species collected in laipuna and 11 in el chilco. this compares with 11 and nine species respectively for each site just using pitfall tramps. of 28 species only camponotus conspicuus zonatus (emery, 1894) and rogeria ec-06 were taken exclusively by pitfall traps. beta diversity of the 28 species collected, 11 species are shared between the two sites with four exclusive to el chilco and 13 exclusive to laipuna (figure 1, table 3). considering only taxa functional group chilco 460m laipuna 680m n freq sampling method freq sampling method dolichoderinae azteca ec-03 arboreal manual manual azteca ec-04 arboreal 2 2 (0.10) pf, manual manual dorymyrmex pyramicus peruvianum epi om manual tapinoma melanocephalum epi om manual dorylinae labidus coecus nom h manual neivamyrmex iridescens borgmeier, 1950 nom h manual ectatomminae ectatomma ruidum gen h 2 2 (1.0) pf, manual formicinae camponotus atriceps epi om 12 7 (3.5) pf, manual 5 (2.5) pf, manual camponotus conspicuus zonatus epi om 1 1 (0.05) pf camponotus ec-07 (cf. conspicuus zonatus) epi om 1 (0.05) pf, manual camponotus ec-08 epi om 5 4 (2.0) pf, manual 1 (0.05) pf, manual myrmicinae acromyrmex ec-02 (octospinosus gp.) fung l 5 2 (0.10) pf, manual 3 (1.5) pf, manual cardiocondyla emeryi epi om manual cephalotes maculatus arboreal manual crematogaster ec-06 (crinosa complex) arboreal 3 manual 3 (1.5) pf, manual cyphomyrmex ec-06 (rimosus complex) fung d manual pheidole ec-29 epi om 6 3 (1.50) pf 3 (1.5) pf, manual pheidole ec-30 epi om manual manual pheidole ec-31 epi om 15 4 (4.0) pf 11 (0.55) pf, manual pheidole ec-32 epi om 2 2 (1.0) pf, manual pheidole ec-33 epi om manual manual rogeria ec-06 1 1 (0.05) pf solenopsis geminata epi om 2 2 (1.0) pf, manual solenopsis ssp. 2 ssp ponerinae odontomachus bauri gen h 14 7 (3.5) pf, manual 7 (3.5) pf, manual pseudomyrmecinae pseudomyrmex boopis epi om 1 1 (0.05) pf, manual pseudomyrmex gracilis arboreal manual pseudomyrmex kuenckeli arboreal manual pseudomyrmex ec-03 arboreal manual total species 15 24 table 1. species collected at each locality. functional group abbreviations: arboreal = nest and forage in plants, fung d = fungus grower using detritus as substrate, fung l = fungus grower using fresh leaves as substrate, gen h = generalist hunter, epi om = epigeic omnivore, nom h = nomadic hunter. see more detailed explanation in text. n = total number of pitfall traps in which species is present. under “freq” whole numbers equal to number of pitfall traps where each species was recorded at each site and between parenthesis is the relative frequency of the species at each site according to pitfall samples only. pf = pitfall traps. manual = manual collection. sociobiology 63(3): 909-918 (september, 2016) 913 pitfall collected material the number of shared species are five, with three exclusive to el chilco and five exclusive to laipuna. of the 11 shared species, eight were collected both manually and with pitfall traps, and only three were collected manually. six (75%) of the shared species collected by both methods were found in at least five sample plots. fig 1. number of species collected for each subfamily at both sites. samples collected1 exclusives2 uniques3 duplicates4 ice5 chao 26 jk27 % range8 chilco 10 3 (30%) 3 (30%) 2 (20%) 12.21 10.95 13.84 72-91% laipuna 11 5 (45%) 2 (18%) 3 (27%) 12.08 11.24 12.14 86-98% table 2. pitfall trap alpha diversity for sampled sites. 1 total species collected in each transect, 2 species collected only in that transect, 3 species collected in only one quadrat (0.05) and their proportion relative the total number of species for the transect, 4 species collected in two quadrats (0.10) and their proportion relative the total number of species for the transect. species richness according to the following estimators: 5 ice, 6 chao 2, 7 jackknife 2; 8 minimum and maximum values of the proportion of observed species according to the estimators. functional groups the collected ants could be distributed amongst six functional groups (table 4), with the most species rich fg being generalist epigeic foragers with a total of 14 species, eight present in el chilco, 12 present in laipuna, with six shared species. the second most species rich fg is arboreal foragers with a total of seven species, of which three are present in el chilco, four in laipuna, with three shared species. only two species of generalist epigeic hunters were found, one in el chilco and both present in laipuna. a different species of nomadic hunter was found in each of the sites. the species of rogeria emery was not designated into any fg on account of the little information available on the biology of the genus (kugler, 1994), especially for those species in arid environments. selected species accounts acromyrmex ec-02 (octospinosus complex). the amount of nocturnal acromyrmex activity in laipuna, especially close to the station, is impressive, with many active foraging columns from different colonies relatively near to one another, particularly in areas with greater tree cover. many aggressive interactions were observed, with workers locked in combat, and a noticeable amount of dead and maimed workers piled throughout areas under greatest tree cover. in areas with sparse canopy the density seemed less and there were no noticeable “battlefields”. in fernández et al. (2015) the specimens key uneasily to a. octospinosus (reich, 1793). the species at hand does not correspond to the image of a syntype (casent0900490) of a. octospinosus in antweb (2016b) as its lateral propodeal spines are longer than the anterior mesonotal spines, and all are very elongate and slender the hairs issuing from the head, scapes, mesosoma, and gaster are all relatively longer than of the imaged type. camponotus atriceps (f. smith, 1858), camponotus conspicuus zonatus, and camponotus ec-07 [cf. conspicuus zonatus] were not seen foraging during the day, in contrast fig 3. species accumulation curve and estimator curves for the el chilco dry forest. fig 2. species accumulation curve and estimator curves for the laipuna reserve dry forest. transect 1 transect 2 shared ssp1 chao jaccard2 chao jacc est3 complement4 chilco laipuna 11 0.657 0.694 0.63 table 3. between site comparisons. 1 number of species shared by 2 sites, 2 chao jaccard similarity values, 3 chao jaccard estimator values, 4 dissimilarity or complementarity values. https://www.antweb.org/specimenimages.do?code=casent0900490 je lattke, m vélez, n aguirre – dry forest ants from southwestern ecuador914 with camponotus ec-08. camponotus conspicuus zonatus and c. ec-07 seem very closely related to each other on account of morphology. c. atriceps is widespread from mexico into most of south america, found in many habitats from savannahs to forests but usually in disturbed areas. it can also be a domestic pest. this is the first record of c. conspicuus zonatus from the ecuadorean mainland. cardiocondyla emeryi forel, 1881 is non-native globally distributed tramp ant, recorded from the galapagos (herrera, 2014) but here recorded for the first time from mainland ecuador. the same species has been reported in low densities in other dry forest sites in southern ecuador such as alamala, about 57 km ne from laipuna (d. donoso, personal communication, september 1, 2015). this species seems to have become a naturalized and discrete member of the local fauna. cephalotes maculatus (f. smith, 1876) is a widespread species found from southern mexico to northern argentina with two previous records from ecuador, both from lowland humid forested areas in the eastern part of the country (de andrade & baroni urbani, 1999; ryder et al., 2010). a striking contrast in vegetation and climate with the present record suggesting considerable ecological flexibility. dorymyrmex pyramicus peruvianus wheeler, 1919. this species was described from the peruvian province of piura, bordering with ecuador. the specimens at hand match well with images of a cotype in the usnm. the genus is reported as present in the country in antwiki (2016b), but not in antmaps (2016). we could not find a published record and neither could salazar et al. (2015). ectatomma ruidum (roger, 1860) is very flexible habitatwise, found not only in arid localities but also mesic forested areas, both disturbed and undisturbed, as well as urban parks and gardens throughout much of northern south america. recent work by nettel et al. (2015) strongly suggest what is presently known as e. ruidum includes more than one species. there is data to suggest the laipuna e. ruidum may not be a single species (domínguez et al. 2016). nocturnal trail walking permitted observing abundant individuals of e. ruidum foraging in laipuna, yet two pitfalls contained this species compared with 7 pitfalls, in each site, containing odontomachus bauri emery, 1892. in the field e. ruidum seems to have much slower movements than o. bauri, pausing intermittently or at the slightest disturbance, and this could partially explain the paucity of pitfall records for e. ruidum compared with o. bauri. e. ruidum was observed foraging after 16:30 h. labidus coecus (latreille, 1802). cephalic capsules of labidus coecus majors were found entangled in spider webs under rocks, and a single male was captured at a light one night. this is an extremely flexible, mostly subterranean ant, in its habitat preferences, ranging from lowland rainforests to paramo conditions at 3,000 m and geographically spread from southern texas to northern argentina (wetterer & gordon, 2015). odontomachus bauri emery is found throughout many types of habitats, from arid localities but also mesic forested areas, both disturbed and undisturbed, as well as urban parks and gardens at least throughout much of colombia and venezuela (fernández, 2008). its foraging activities were observed starting at approximately 18:30 h. pheidole ec-29, ec-30, and ec-31 all apparently belong to the diligens group. they are morphologically very similar and presumably quite closely related to each other, at least much more so than with pheidole ec-32 or ec-33. pseudomyrmex kuenckeli (emery, 1890) is widespread, though not necessarily common, in dry forests from mexico to paraguay and argentina (ward, 1999). in laipuna these ants were found nesting in a legume, apparently a species of tachigali aublet, though they generally do not nest in trees of this genus as they are associated with other members of the viduus group of pseudomyrmex (ward, 1999). they reacted very aggressively to our collecting efforts, forcing us to retreat from the vicinity of the trees. this is the first record of this species in ecuador. pseudomyrmex boopis (f. smith, 1855) is widespread from mexico throughout northwestern south american, but little is known of its biology other than a preference for nesting in the ground in relatively mesic forests, so its presence in this dry forest underscores our scant knowledge about its biology. this is the second record of this species in ecuador, the first being specimens studied by kempf (1960) from near río bamba. rogeria ec-06. the two captured specimens were compared with other specimens of the same genus from the utpl collection, images from antweb (2016c), and examined using the revision by (kugler, 1994) and the review by (lapolla & sosa-calvo, 2006). they do not seem to coincide with any of the known species. functional groups total species1 species chilco2 species laipuna2 shared species3 % shared species4 cab5 generalist epigeic hunters 2 1 2 1 50% 0.50 nomadic hunters 2 1 1 0 0 1.00 fungus growing (detritus) 1 0 1 0 0 1.00 fungus growing (fresh) 1 1 1 1 100% 0 epigeic omnivores 14 8 12 6 43% 0.57 arboreal foragers 7 3 7 3 43% 0.57 table 4. functional group diversity. 1total species for each group, 2species found only in each site, 3species shared amongst sites, 4 percentage of shared species, 5functional group complementarity values between each site. sociobiology 63(3): 909-918 (september, 2016) 915 tapinoma melanocephalum (fabricius, 1793) is another non-native tramp ant. it was found in the kitchen of the station house only. given this species is also known from other mainland localities in ecuador (donoso et al., 2014), it could be present in irrigated agricultural fields close to the station or habitats along the neighboring catamayo river. it has been recorded in dry forest habitats further south in lambayeque, peru (castro delgado et al., 2008). its absence in the sampling sites suggest the prevailing environmental conditions on the slopes are too extreme for its survival. the cacti, and some other plants, in laipuna had significant colonies of scale insects and these were frequented by ants, especially crematogaster, and both species of azteca. discussion the accumulation curves and estimators suggest the ground fauna was relatively well sampled (72-98%), especially in laipuna, where the estimators converge close to the observed value. the situation is different for zapotillo, with the curves spread apart and only the value of chao2 approaching that of the observed value. sampling in zapotillo could be considered more intense as both litter samples and pit fall traps were used compared with laipuna where only pitfalls were used. the increase in species richness may thus be in part due to the different methods used as winkler and pitfall traps are considered complementary, the larger ants with longer legs being taken more often with pitfall traps (donoso & ramon, 2009). comparing species diversity in our sites with those of dry forest in southwestern colombia we find the number of genera and species for southern ecuador much lower. (arcilacardona et al., 2004) sampled poneromorph ants from five sites and found 22 species from 10 genera. a rarefaction curve puts species richness from just 20 of his samples at approximately eight “poneromorphs” from forest fragments alone, compared with only two for both sites of the present study. (armbrecht & ulloa-chacon, 1999) found a total of 38-81 species of ants each in seven different secondary forest fragments. the sampled colombian dry forests have at least twice the annual rainfall as the ecuadorean sites. the number of species for el chilco and laipuna have values similiar to those in more arid, desert like conditions such as scrub forests. surveys of ants in arid environments of the venezuelan coast yielded more similar species richness such as 22 and 31 species respectively for two sites on the paria peninsula (pérez-sánchez et al., 2013) and 30 – 42 species for three sites on the paraguana peninsula (pérez-sánchez et al., 2012). the venezuelan sites averaged lesser annual precipitation than the ecuadorean sites, ranging from 410 – 244 mm. (castro et al., 2008) sampled ants manually and with pitfalls in the dry ferreñafe region of lambayeque in peru, finding 21 morphospecies in four sites between 600 and 810 m above sea level. despite the very arid conditions both of the present sites have some tree cover, particularly el chilco with trees over 20 m high, in contrast with the venezuelan sites. the greater diversity of ants present in laipuna vs. el chilco, despite the greater rainfall and presence of leaf litter for the latter site, was unexpected. the presence of foraging goats in el chilco as opposed to their abscence in laipuna might have an influence, as their disruptive effects on understorey vegetation might reduce resources for ants that forage on the ground and low herbacious vegetation. another underlying factor may be the slightly higher relative humidity and lower average temperature for laipuna, conditions more amenable to ants than those in el chilco. precipitation is also slightly more concentrated in el chilco with 98% falling from january to april while in laipuna 85% of the precipitation is concentrated from january to march. the two sites share approximately a little under one half of their species, especially evident for the two most speciose functional groups, epigeic omnivores and arboreal foragers, implying that an important number of species are restricted to each site. conclusions the number of new records, either for ecuador or the ecuadorean mainland, including the first published record for a relatively common and widespread genus such as dorymyrmex, highlight the poor knowledge of the tumbesian forest biota. we hope to point attention to this fact with the present evidence and encourage further surveys and other research on the ant fauna of this vast region of dry forests. the tumbesian dry forests are not homogenous but quite varied and more extensive collecting, taking into account both seasonal and successional changes are sure to reveal further new records, probable new species, and a better resolution of how ants are distributed in this region. acknowledgements we acknowledge to david donoso, cristina gómez, maría tuza, gabriela piedra, william mackay, benoit guénard, and josé ramirez for their assistance. m. vélez carried out his share of the work as part of the requirements for obtaining the degree of ingeniero ambiental from the universidad nacional de loja. d. donoso for making valuable suggestions to the manuscript. naturaleza y conservación internacional granted permission to carry out field work in the laipuna reserve. mayron escárraga helped with the mansucript. this research was mostly financed by the prometeo project of the secretary for higher education, science, technology, and innovation (senescyt) of the republic of ecuador. additional support was provided by the program for biodiversity, forests, and ecosystem services of the universidad nacional de loja. je lattke, m vélez, n aguirre – dry forest ants from southwestern ecuador916 references aguirre-mendoza, z. & kvist, l.p. 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(1999). systematics, biogeography and host plant associations of the pseudomyrmex viduus group (hymenoptera : formicidae), triplaris and tachigali inhabiting ants. zoological journal of the linnean society of london, 126: 451–540. wetterer, j.k. & snelling, g.c. (2015). geographic distribution of labidus coecus (latr.) (hymenoptera, formicidae), a subterranean army ant. journal of hymenoptera research, 44: 31-38. doi: 10.3897/jhr.44.4672 http://dx.doi.org/10.3897/jhr.44.4672 cite_print open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.5928sociobiology 68(2): e5928 (june, 2021) introduction pitfall trapping is one of the most frequently used methods to assess ground-active arthropods’ diversity and density (brown & matthews, 2016; greenslade 1964; southwood, 1978). its advantages and drawbacks have been the subject of discussion for a long time (adis, 1979, southwood & henderson, 2016). many attempts have been made to correct some of the most salient biases resulting from it (greenslade, 1964; hayes, 1970; gist & crossley, 1973; luff, 1975). sheikh et al. (2018) provide a detailed review on the use of pitfall trapping for ants worldwide. however, despite the many complaints about the method and the voluminous literature about the subject, the possibility that abstract pitfall trapping remains one of the most frequently used methods to assess ground-active arthropods’ diversity and density. yet, one of its main drawbacks, the possibility that repeated collecting may affect the study objects’ population, has not been formally tested. we studied the effect of a yearlong epigeal pitfall trapping exercise with 22 fortnightly capture events in four differently disturbed areas at the colombian pacific coast. a transect of 100 m length with ten equidistant pitfall traps was established in each area, and the traps were operated twice a month for 24 hours. using count data regression models, we find that trapping did not affect subsequent captures when we analyzed non-ant arthropods. for ants, regression estimates indicate that each subsequent trapping in highly-disturbed environments ended, on average, reducing all ants in between -3.8 and -4.1%, and ectatomma ruidum between -4.7 and -5.1%. we recommend bio-ecological aspects of the species under study be considered when interpreting results. this is important for future studies that rely on this method to deliver consistent estimates of population sizes or study their dynamics through time. at the same time, it is also a call for scientists to revise more carefully how species’ peculiar traits may limit the reliability of traditional methods. sociobiology an international journal on social insects josé m martinez1, rubilma tarazona1, bernhard l lohr1, consuelo a narvaez2 article history edited by eduardo calixto, university of florida, usa received 21 october 2020 initial acceptance 29 december 2020 final acceptance 24 april 2021 publication date 11 june 2021 keywords arthropods population, ectatomma ruidum, long-term trapping, pitfall trapping. corresponding author josé m. martinez corporación colombiana de investigación agropecuaria – agrosavia km 14 vía mosquera – bogotá código postal: 250047 colombia. e-mail: jose.martinez.rioja@gmail.com repeated epigeal collecting may affect the population size of the study objects (southwood, 1978) is a claim that has never been adequately tested. greenslade (1973) and joosse (1965) define a “diggingin effect” as the repeatedly found evidence of considerably larger captures right after pitfall traps have been installed when compared to the observed captures in subsequent catch counts. there are mainly four plausible causes for a systematically diminishing number of ant captures because of pitfall traps (greenslade, 1973), namely: (a) penetration of nest galleries while setting up the traps; (b) traps are coincidentally located between nests and/or permanent food sources, i.e., traps over food trails; (c) ants exploring new features within their usual territories, and (d) depletion of populations. joosse and 1 corporación colombiana de investigación agropecuaria – agrosavia, colombia 2 universidade federal de viçosa, department of entomology, viçosa, mg, brazil research article ants measuring the effect of long-term pitfall trapping on the prevalence of epigeal arthropods: a case study in the pacific coast of colombia mailto:jose.martinez.rioja@gmail.com jose m martinez, rubilma tarazona, bernhard l lohr, consuelo a narvaez – the effect of long-term pitfall trapping on the prevalence of epigeal arthropods2 kapteijn (1968) suggest an additional short-term diggingin effect, observed when epigeal traps are operated right after being installed. there is a systematic increase in the early captures of collembola (joosse & kapteijn, 1968) and some ant species (greenslade, 1973) since their locomotory activity also increases because of higher concentrations of co2. more recent studies have tested how the robustness of pitfall trapping is affected by habitat specificities (jiménez-carmona et al., 2019), the timing of pitfall opening after installation (lasmar et al., 2017), and length of the sampling interval (schimel et al., 2010). still, the hypothesis of a decrease in observed captures resulting from the reduction of ant populations caused by the pitfall traps, as proposed by jansen and metz (1977), has never been formally tested. we reanalyzed datasets generated by löhr and narváez (2021) during a year-long study in the pacific lowlands of colombia to identify surface-active predators that could be useful for controlling the oil palm root borer, sagalassa valida walker (lepidoptera: brachodidae), in four different environments. some of the datasets seemed to indicate that different dynamics were present in the environments and the possibility of a diminishing number of arthropod captures over time, even though the sampling design was low-intensity. we test whether there is a long-term trapping effect on the subsequent captures of all groups of recorded arthropods through plot-fixed effects and plot-level count data regression models. these methods differ from most studies that try to model the dynamics of catches from pitfall trapping, which usually rely on anova (or derivations of it) and/or loglinear regression analysis. we explain why count models that directly account for high-variance data better help capture the nature of the available information, hence better modeling the effect of pitfall trapping. we argue why the experiment’s structure helps correctly identifying a reduction of population effect, which is not confounded by other possible digging-in effects. hence, it allows us to statistically determine the impact of long-term pitfall trapping over the prevalence of ground-active arthropods. materials and methods the study area was within 564 ha of the el mira research center (1° 32′ 58″ n, 78° 41′ 21″ w) of the corporación colombiana de investigación agropecuaria [colombian corporation for agricultural research] (agrosavia), dedicated to research on oil palm (elaeis guineensis jacq.), peach palm (bactris gasipaes kunth), cacao (theobroma cacao l.) and non-timber forestry products. the center is located 38 km southeast of tumaco, in nariño department (colombia), at 16 m. a. s. l. (figure 1). the annual mean precipitation is 3,067 m.m., while the average temperature is 25.5ºc (reyes, 2012). an on-site weather station recorded hourly solar radiation and daily rainfall data. areawide flooding (i.e., more than 100 mm in the last 24 hrs.) occurred twice, and it was recorded to control for its possible effect on ground active arthropods. fig 1. geographic location of el mira research center, tumaco, pacific coast of colombia. sociobiology 68(2): e5928 (june, 2021) 3 the set-up of the study consisted of four transects, each a straight line of 100 m length with ten traps each at 10 m distance, located in plots with different levels of disturbance: a secondary forest regrowth with over ten years without interference (14 ha, transect parallel to and about 20 m from the forest edge); a 19 years old peach palm plantation (9.9 ha); a seven years old oil palm plantation (3 ha) and a three years old oil palm plantation of 14 ha. routine management practices (fortnightly harvesting, quarterly weeding, and halfyearly fertilization) were implemented in the palm plantations while the secondary forest remained without interference. traps consisted of 150 ml conical plastic cups (upper ø 60 mm, 55 mm height), two of which were interred with the rim of the upper cup flush with the soil surface. for the collections, the upper cups were exchanged for cups filled with 50 ml water and a drop of dishwashing liquid. the time of each exposure was 24 hours. between capture events, the upper cup was covered with a lid. collections were made fortnightly with two exceptions in august 2016 and february 2017, when only one collection could be made. the collected material was processed in the laboratory, where all ants were removed and stored for posterior counting and identification. all other arthropods were counted individually and identified to order or family level where possible and grouped as follows: hymenoptera, diptera, coleoptera, hemiptera, arachneae, acari, collembola, and others. to check for the effect of temperature and sunshine hours, we calculated the average values of the period before the capture. we added them as a covariate to model capture events via poisson and negative binomial type 2 regression. to assess flooding impacts, we included an index variable that accounts for whether there was such an event immediately before the concurrent count. before modeling the trends of captures across the four areas of interest, we test for statistical differences in captures between them for non-ant arthropods, ants, and ectatomma ruidum via manova (stevens, 2002). this is an f-test where the null hypothesis states that the three response variables are the same across the four different plots. we used count data regression models to model the effect of repeated trapping on the prevalence of arthropods across the plots (cameron & trivedi, 2005). we assumed that the observed counts of arthropods ( ) followed a poisson distribution with the usual probability mass function (1) where is the scale parameter or the expected value of . more specifically, we followed the standard exponential mean parametrization setting, where (2) or, simply put, a generalized linear model with a poisson error vector. for our study, the covariates set ( ) included the round of trapping when the count was observed ( ), and a binary variable indicating whether the capture event occurred right after a flooding event in the plots ( ). we ran an overall regression including fixed effects for three of the plots (using the secondary forest as the base category) and four additional regressions to test for plot-specific effects. all regressions were performed for arthropods, all ant species, and ectatomma ruidum only counts. namely, at the overall level, we assumed that the rate parameter for trap from plot at the moment was (2’) whereas for each plot would follow (2’’) . furthermore, for every regression, we also fitted a model including same-day total solar radiation (w/m2) and precipitation (mm), since evidence suggests that these variables directly affect the behavior of ground arthropods, especially ectatomma ruidum (santamaría et al., 2009). the data is available from a hydrometeorological station installed in the research center, within 200 m from the plots, so we assume the precision to be high. now, letting and be the notation for solar radiation and precipitation, respectively. the additional overall model is (3) whereas for each plot, it follows (3’) broadly, we were interested in testing the null hypothesis where the semi-elasticity . this is, we tested whether repeated trapping influences the observed counts of arthropods, all ant species, or e. ruidum. estimation of the parameters followed quasi-maximum likelihood (qml), and the standard errors were corrected for robustness due to the data’s high variance (cameron & trivedi, 2005; greene, 2012). since bias due to data overdispersion might not be overcome solely by using robust standard errors, we further extended our analysis by including a negative binomial (nb2) regression approach, which yields unbiased estimators even in the presence of overdispersion. the nb2 strategy suggests that the observed counts would again follow a poisson data generating process with scale parameter , where is a positive, independently and identically distributed shock so that the expected value of is still , the variance now follows1 (5) , so that high-order variance is now reflected as a quadratic function of the mean. results nearly a third of all captures through all trapping rounds (33%) were of formicidae, whereas collembola captures accounted for 35% of the total (fig 2). a complete list of jose m martinez, rubilma tarazona, bernhard l lohr, consuelo a narvaez – the effect of long-term pitfall trapping on the prevalence of epigeal arthropods4 the taxonomic groups, numbers collected, and land use influence is available in löhr and narváez (2021). the data distribution shows a strong positive asymmetry2 for overall captures and somehow less overall variance for the captures of ants (fig 3). also, we noticed how the overall captures are related to the distribution of captures of arthropods that are not ants, particularly collembola – i.e., the statistical distribution of overall captures is more similar to that of nonants. these initial findings further reassure our modeling strategy’s appropriateness, which relies on count-data nonfig 2. identified arthropod orders in repeated captures in pitfall traps. el mira research center, tumaco, pacific coast of colombia, 2017. fig 3. density of total captured individuals per major group of analysis. el mira research center, tumaco, pacific coast of colombia, 2017. sociobiology 68(2): e5928 (june, 2021) 5 linear regression models that separately account for the overdispersion across groups. since the experiment was carried across different environments, we further explored differences in the dynamics of captures. first, plotting the observed captures on each round of trapping revealed that the data carried an important degree of variance (statistical overdispersion). the environments reported different patterns and data concentrations. as an example, the total ectatomma ruidum captures were, on average, higher in both palm plots (4,616-6,697) than in the secondary forest (4,053) or the peach palm plantation (3,393) (see also fig 4). moreover, a multivariate analysis of variance (table 1) reveals significant statistical differences among the captures in the different environments. we reject the null hypothesis of no-differences between plots on every test, further strengthening our argument for modeling the effects fig 4. log-frequency of captured ants, ectatomma ruidum, and other arthropods in repeated captures in pitfall traps. el mira research center, tumaco, pacific coast of colombia, 2017. reported in tables 5, 6, and 7, providing details for non-ant arthropods, all ants, and ectatomma ruidum. it is important to highlight how implementing both approaches reveals the offset to some significance after controlling for the data’s high variance via the nb2 method. for example, if we had only followed the poisson method, table 2 would have suggested an apparent overall negative effect from trapping on all arthropods, and more specifically on the peach palm and seven y.o. oil-palm plantation. however, table 5 shows that the nb2 approach attenuates such results, hence avoiding that coefficient estimates are affected due to high-variance (i.e., helps increase the efficiency of inferences). therefore, our results suggest no effects of pitfall trapping over non-ant arthropods, neither at the aggregate level or plot-specific level. on the other hand, although reduced in absolute value from one method to another, results regarding ants hold their sign and statistical significance. initially, the overall effect table 1. statistics of manova for observed captures of non-ant arthropods, ants, and ectatomma ruidum across plots. statistic estimate df f[df1 ; df2] f-stat p-value wilk’s lambda 0.873 3 [9 ; 2107.8] 13.46 0.000*** pillai’s trace 0.129 [9 ; 2604.0] 12.99 0.000*** lawleyhotelling trace 0.143 [9 ; 2594.0] 13.8 0.000*** roy’s largest root 0.128 [3 ; 868.0] 37.16 0.000*** *** p<0.001; ** p<0.005; * p<0.01. of pitfall trapping both by including fixed effects and at the plot level. we summarize the results from poisson regressions on tables 2, 3, and 4, whereas those from nb2 models are jose m martinez, rubilma tarazona, bernhard l lohr, consuelo a narvaez – the effect of long-term pitfall trapping on the prevalence of epigeal arthropods6 seems to be inexistent after controlling for climatic covariates. nevertheless, tables 3-4 reveal that on a poisson setting, on average, each 24hrs trapping event implies a negative effect of -4.3% and -5.1% on subsequent captures of all ants and ectatomma ruidum, respectively, specifically in the younger oil-palm plantation. these effects decrease (in absolute value) to -3.8% and -4.7% in the nb2 model. after further controlling for same-day solar radiation and precipitation, these effects slightly decreased to -3.0% and -3.9% in poisson and -3.8% and -2.7% in the negative binomial specification. thus, these results are robust and hold at a 0.05 level of significance. response variable: total captured individuals of non-ant arthopods covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.012 -0.029*** -0.003 -0.011 -0.008 -0.032*** -0.027 -0.046** -0.004 -0.018 (0.01) (0.01) (0.01) (0.01) (0.01) (0.01) (0.01) (0.01) (0.02) (0.03) capture after flooding -0.937*** -0.425*** -0.752* -0.435 -0.766*** -0.111 -1.128*** -0.623** -1.143** -0.567 (0.14) (0.13) (0.28) (0.27) (0.22) (0.26) (0.22) (0.20) (0.37) (0.23) solar radiation (w/m2) -0.000*** 0.000 -0.000*** -0.000* -0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) -0.003*** -0.004*** -0.002 -0.003 -0.006 (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a poisson model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 table 2. estimated effect of pitfall trapping over non-ant arthropods by poisson regression model. table 3. estimated effect of pitfall trapping over all ants by poisson regression model. response variable: total captured individuals of all ants covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.023*** -0.012 -0.012 0.009 -0.015 -0.012 -0.019 -0.013 -0.043*** -0.030*** (0.01) (0.01) (0.01) (0.01) (0.02) (0.02) (0.01) (0.01) (0.01) (0.01) capture after flooding -0.162 -0.449*** -0.102 -0.606 -0.493 -0.604 -0.303 -0.495 0.122 -0.184 (0.10) (0.12) (0.23) (0.29) (0.28) (0.38) (0.17) (0.20) (0.13) (0.15) solar radiation (w/m2) 0.000 0.000** 0.000 0.000 0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) 0.002*** 0.003 0.001 0.001 0.002** (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a poisson model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 sociobiology 68(2): e5928 (june, 2021) 7 there are statistically significant differences between captures that occur after flooding events and those performed on average weather conditions, specifically for non-ant arthropods. we found no effects of this kind for ants. overall, non-ant pitfall catches after flooding events are expected to report about 39.1% of the captures that would have been found at normal conditions3 under a poisson distribution. after controlling for the observed solar radiation and daily precipitation on the series, the scale goes up to 65.3% (i.e., the effect is slightly half than that of the uncontrolled case). if a negative binomial distribution is assumed, these metrics change to 40.7% and 69.2%. nonetheless, such effect only holds at the plot level in 7 yeas old oil-palm plantations, regardless of the statistical method. conversely, flooding events do not seem to affect the expected number of antcaptures when analyzing the effect at the plot level. table 4. estimated effect of pitfall trapping over ectatomma ruidum by poisson regression model. response variable: total captured individuals of ectatomma ruidum covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.026*** -0.014 -0.013 0.006 -0.005 0.009 -0.023 -0.016 -0.051*** -0.039*** (0.01) (0.01) (0.01) (0.01) (0.02) (0.01) (0.01) (0.01) (0.01) (0.01) capture after flooding -0.142 -0.435*** -0.161 -0.480 -0.262 -0.598 -0.315 -0.532 0.107 -0.202 (0.10) (0.12) (0.23) (0.28) (0.28) (0.42) (0.18) (0.22) (0.14) (0.17) solar radiation (w/m2) 0.000* 0.000*** 0.000 0.000 0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) 0.002** 0.001 0.002 0.001 0.002** (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a poisson model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 table 5. estimated effect of pitfall trapping over non-ant arthropods by negative binomial (nb2) regression model. response variable: total captured individuals of non-ant arthopods covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.006 -0.020 0.006 -0.004 -0.003 -0.022 -0.038 -0.042 0.004 -0.010 (0.01) (0.01) (0.01) (0.01) (0.01) (0.01) (0.02) (0.02) (0.03) (0.03) capture after flooding -0.898*** -0.368* -0.738** -0.435 -0.735*** -0.049 -1.144*** -0.705** -1.118*** -0.457 (0.13) (0.14) (0.25) (0.25) (0.21) (0.26) (0.19) (0.24) (0.33) (0.23) solar radiation (w/m2) -0.000*** 0.000 -0.000*** -0.000* -0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) -0.003*** -0.005*** -0.002 -0.001 -0.003 (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a negative binomial (nb2) model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 jose m martinez, rubilma tarazona, bernhard l lohr, consuelo a narvaez – the effect of long-term pitfall trapping on the prevalence of epigeal arthropods8 response variable: total captured individuals of all ants covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.019*** -0.007 -0.010 0.015 -0.008 -0.005 -0.016 -0.011 -0.038*** -0.027** (0.01) (0.01) (0.01) (0.01) (0.01) (0.02) (0.01) (0.01) (0.01) (0.01) capture after flooding -0.122 -0.373*** -0.094 -0.578 -0.411 -0.451 -0.270 -0.391 0.156 -0.108 (0.09) (0.11) (0.23) (0.26) (0.23) (0.29) (0.15) (0.16) (0.12) (0.15) solar radiation (w/m2) 0.000 0.000** 0.000 0.000 0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) 0.002** 0.003 0.000 0.001 0.002 (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a negative binomial (nb2) model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 table 6. estimated effect of pitfall trapping over all ants by negative binomial (nb2) regression model. response variable: total captured individuals of ectatomma ruidum covariates (1)(a) (2) (3) (4) (5) (6) (7) (8) (9) (10) round of capture (cumulative) -0.020** -0.004 -0.010 0.011 -0.000 0.029 -0.020 -0.014 -0.047*** -0.038*** (0.01) (0.01) (0.01) (0.01) (0.02) (0.02) (0.01) (0.01) (0.01) (0.01) capture after flooding -0.077 -0.352** -0.137 -0.472 -0.241 -0.605 -0.269 -0.400 0.164 -0.081 (0.10) (0.12) (0.22) (0.27) (0.23) (0.30) (0.16) (0.18) (0.12) (0.16) solar radiation (w/m2) 0.000** 0.000*** 0.000 0.000 0.000 (0.00) (0.00) (0.00) (0.00) (0.00) precipitation (mm) 0.002** 0.001 0.002 0.001 0.002 (0.00) (0.00) (0.00) (0.00) (0.00) observations 872 220 219 217 216 method poisson poisson poisson poisson poisson plot fixed effects yes n/a n/a n/a n/a plot n/a secondary forest peach palm oil-palms (7 years) oil-palms (3 years) (a) each column presents the coefficients of regressing the response variable on the specific covariates, either including or excluding weather controls, under a negative binomial (nb2) model. columns 1-2 pool the data and include plot-fixed effects, whereas the remaining columns report regression coefficients based on data from specific plots (see bottom of each column). robust standard errors in parentheses. *** p<0.001, ** p<0.005, * p<0.01 table 7. estimated effect of pitfall trapping over ectatomma ruidum by negative binomial (nb2) regression model. discussion theory suggests epigeal pitfall trapping may significantly affect the populations of ground-active arthropods. greenslade (1973) warns about three specific digging-in effects that might influence captures when sampling ants and likely confound estimates of pitfall-trapping effects. these are: (a) penetration of nest galleries, (b) traps located between nests and food sources, and (c) ants exploring new features in their territories. using a dataset from a study in southwestern colombia, we circumvent the likelihood of such confounding effects to provide an unbiased and precise measure of pitfall trapping effects, specifically for ectatomma ruidum. we have two main arguments for this purpose. first, the largest share of captured ants was of e. ruidum (>87%), and traps were small in height (60 mm) and widely distributed across the plots. ectatomma ruidum nests are usually singleentrance vertical galleries (franz & wcislo, 2003), located sociobiology 68(2): e5928 (june, 2021) 9 at a medium depth of 35 cm (armbrecht & santamaría, personal communication, june 21, 2019). moreover, this species does not follow specific trails while foraging (franz & wcislo, 2003). thus, it is an acceptable assumption to consider that confounders (a) and (b) do not apply. second, when considering the duration of the study (one year) and the specifics of ectatomma ruidum behavior, it is simple to rule out any relation with confounder (c) and with the case of higher initial captures due to increased co2. whereas there are cases like slezák et al. (2010), which reported a drastic reduction of carabid and diplopod numbers in the second year after one year of pitfall sampling and attributed the difference to over-catching in the first year, their traps were installed and actively collected specimens throughout the season. this study’s sampling was limited to two periods of 24 hours of trapping per month, hence – third, and finally – ruling out short-term digging-in effects and making a stronger case of any detected impact to be defined as of depletion of populations. this also complies with the data, which shows that the decrease in captures is not systematically seen only at first capture events; conversely, it is observed only in the long-term. our results are made further robust by including weather controls (solar radiation and precipitation), as well as extreme variability shocks, namely floodings. the latter seems to affect non-ant arthropods’ captures systematically, but no effect is found either on all ants or ectatomma ruidum. as of why ectatomma ruidum is not affected by these flooding events, this likely follows from their vertical, single-entrance, deep nests, as well as their trait of distributing across several small colonies instead of a single nest (franz & wcislo, 2003; armbrecht & santamaría, personal communication, june 21, 2019). in brief, since the analysis relies on low-intensity trapping data with important exogenous controls, we consider this an estimate of a lower boundary of such an effect. therefore, if the effect is detected even with fortnightly events, this is a minimum effect attributed to pitfall trapping in the absence of confounding events. besides, there were important differences in total ant – and ectatomma ruidum – captures across the four different plots, representing different levels of disturbance. following the findings of jiménez-carmona et al. (2019), this is of importance. habitat specifics might influence differences in detected digging-in effects. the dominance of ectatomma ruidum in the oil palm plots can be explained as these are highly disturbed environments where scarcity of food sources is a common issue. ectatomma ruidum, as a generalist forager (franz & wcislo, 2003; lachaud, 1990), has a definitive advantage over more specialized ant species. however, as the data show, they are vulnerable to activities like pitfall trapping, which has measurably reduced their numbers with each subsequent capture. an explanation to this finding is the small size of the colonies, which are about 50-150 individuals (franz & wcislo, 2003). if 20% of the ants of any colony are engaged in foraging, each colony may have just 10 40 active foragers. yet, the average number of individuals caught per trapping occasion in the oil palm plots went up to 30 individuals. this could have had a significant impact on the amount of food gathered and, thus, on colony maintenance and growth. in summary, results suggest that although overall arthropods’ captures were stable through time, plot-level analyses indicate that, on average, each additional capture significantly reduced the overall population of ants – specifically ectatomma ruidum – in some plots. this species is characterized by numerous, small colonies with a limited number of foragers, which even low-intensity pitfall trapping can affect by repeated removal of a large proportion of the active foragers. even though this is a case study, this result might be important for future studies that rely on this method to deliver consistent estimates of their population sizes or their dynamics through time since the possibility of a biased estimation is latent by the factors here explored. disclaimer the authors agree with this article’s publication and declare no conflicts of interest that affect the results. note 1 cameron & trivedi (2005, 2009) show that setting the density of as , yields the negative binomial distribution that possesses a mixture density where . 2 we tested for non-normality (not included here) and found that there are strong differences in which the mean value is statistically larger than the median. for example, mean and median captures were (577; 489.5), (363.9; 274), and (213.1; 195) for all captures, non-ant captures, and ant captures, respectively. 3 due to the magnitude of the detected effect of captures-afterflooding, the best interpretation of this coefficient is the scale difference (cameron & trivedi, 2009). for small effects, the semi-elasticity interpretation holds since it is symmetric to the scale change. references adis, j. 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(2016). ecological methods, 4th ed. wiley-blackwell. hoboken, nj. https://doi.org/10.1023/a:1024880110189 https://doi.org/10.1093/ee/2.5.951 http://doi.org/10.2307/2632 https://doi.org/10.1007/bf02226087 https://doi.org/10.1007/bf02226087 http://doi.org/10.2307/1935388 https://doi.org/10.1007/bf00046807 https://doi.org/10.1007/bf00046807 http://doi.org/10.1111/eea.12834 https://doi.org/10.1007/bf00407477 https://doi.org/10.1007/bf00386692 https://doi.org/10.1007/s00040-017-0558-7 https://doi.org/10.1007/s00040-017-0558-7 https://doi.org/10.25100/socolen.v47i1.7640 http://doi.org/10.1007/bf00348110 http://doi.org/10.1007/bf00348110 https://doi.org/10.1111/j.1570-7458.2010.01020.x http://doi.org/10.5958/0974-4576.2018.00072.5 http://doi.org/10.5958/0974-4576.2018.00072.5 _hlk62887387 _hlk62926526 _hlk32828345 doi: 10.13102/sociobiology.v63i4.1041sociobiology 63(4): 1038-1045 (december, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 colony development and management of the stingless bee scaptotrigona aff. postica (apidae: meliponini) using different hive models introduction the keeping of stingless bees (tribe meliponini), also known as meliponiculture, is an ancient practice in the americas, carried out mainly by traditional communities (villanueva-g et al., 2005; cortopassi-laurino et al., 2006). generally, these communities still keep the stingless bees in a rudimentary way (villanueva-g et al., 2005; reyez-gonzález et al., 2014), although recently, there have been attempts to enhance general bee keeping techniques and practices (contrera et al., 2011; venturieri et al., 2012). it is possible to divide existing and the most frequently used hive models into two groups –horizontal and vertical hives. horizontal hives are the most traditionally used in brazil abstract meliponiculture, i.e. the management and propagation of stingless bees is an old activity throughout the americas, and is widely practiced in brazil. however, since stingless bee keeping is still not entirely standardized, studies are necessary to achieve a better practice. for example, the type of hive used in breeding must be designed for ease of colony management and be suitable for each species, as size, behavior, and nest architecture vary among the meliponines. this study tested the use of a vertical hive for breeding an undescribed species of the group scaptotrigona postica (latreille), scaptotrigona aff. postica, and evaluated characteristics such as colony development and management in comparison to colonies bred using traditional horizontal hives. ten colonies of the studied species were used, five transferred to each hive type tested. during six months, monthly observations of colony size, hive occupation and hive management were made. brood comb area, contrasting with the other parameters, was approximately twice as large in vertical hives compared to horizontal hives. no significant difference in hive occupation was found among the tested models. management differences were found among the two hive models, with vertical hives noted for having characteristics that facilitated colony inspection and division, causing only minor damage to the nest structures. thus, the vertical model hive has clear advantages for the keeping of the studied scaptotrigona species. sociobiology an international journal on social insects kl leão1,2, acm queiroz1, jc veiga1,2, fal contrera2, gc venturieri1 article history edited by cândida m. l. aguiar, uefs, brazil received 12 april 2016 initial acceptance 24 august 2016 final acceptance 16 november 2016 publication date 13 january 2017 keywords meliponiculture, standardized hives, stingless bee keeping, traditional hives. corresponding author kamila leão leão laboratório de biologia de abelhas instituto de ciências biológicas universidade federal do pará rua augusto corrêa, 01, campus básico guamá cep 66075-110 belém-pa, brasil e-mail: kamilabelha@gmail.com and mesoamerica (nogueira-neto, 1997; sommeijer, 1999; cortopassi-laurino et al., 2006). horizontal hives are somewhat similar to hollow logs, placed horizontally (vilamueva-g et al., 2005), and area common means of keeping stingless bee nests (i.e. “hobones”: quezada-euán & gonzalez-acereto, 1994, or “cortiços”: nogueira-neto, 1997). horizontal hives may be completely hollow, without any internal division, or more elaborate, with internal divisions to separate the nest from the honey storage space (nogueira-neto, 1997; sommeijer, 1999). the vertical, baiano or nordestino, uberlândia, pnn, the utob and the inpa hive models are widely used in brazil and in other countries as well, with or without local adaptations (quezada-euán & gonzalez-acereto, 1994; kerr et al., 1996; nogueira-neto, 1997; sommeijer, 1999).the 1 laboratório de botânica embrapa amazônia oriental, belém, brazil 2 universidade federal do pará, belém, brazil research article bees sociobiology 63(4): 1038-1045 (december, 2016) 1039 many hive models in the meliponiculture are directly linked to the vast diversity of stingless bee species present in these regions (venturieri et al., 2012), and also based on personal and/or cultural preferences, as many beekeepers tend to create new hive models on their own even when the more known models (i.e. inpa, embrapa, pnn, utob) are available. the number of beekeepers using vertical hives is growing despite the horizontal models still being more widely used (venturieri, 2008a). the vertical hive follows the natural brood comb pattern in tree trunks, divided into two main modules, the base chamber (to shelter the nest) and the upper chamber (to store honey and pollen; portugal-araújo, 1955). the creation of a specific honey storage compartment enables a faster and cleaner harvest, with minimum nest damage, the great advantage of this hive model (venturieri et al., 2003; venturieri, 2008a). however, actual comparisons on the advantages of each type of hive in regards to biological parameters, such as hive occupation and thermoregulation, as instance, are scarce, but in general point to that the type of hive that different species are bred result in clear differences in the health and development of the colonies (quezada-euán & gonzalez-acereto, 1994). several bee species, mostly of the genus melipona illiger, are used in meliponiculture in brazil and mesoamerica (nogueira-neto, 1997; reyez-gonzález et al., 2014). the genus scaptotrigona moure is also an important genus for stingless bee keeping; well known for its populous colonies, and production of good quality honey in large quantities, in comparison to other meliponines (cortopassi-laurino et al., 2006; reyez-gonzález et al., 2014). a scaptotrigona species from the group scaptotrigona postica latreille; thereafter called s. aff. postica (currently under review) that occurs in the state of pará (brazil) and commonly known as “canudo” (i.e. “straw”) is a commonly kept stingless bee species in the amazon region, as it has great honey production potential. in addition, this species may be of significant value for use in the pollination of native crop species (venturieri et al., 2012), due to its tolerance of handling, populous colonies and the possibility to be multiplied on a large scale (menezes et al., 2013). therefore, this study aimed to investigate the potential of vertical hives to breed s. aff. postica, assessing aspects such as biological adaptation and colony management, and comparing its performance to a horizontal hive model commonly used in the study region, as the optimization and standardization of management practices have the potential to increase productivity and income of beekeepers (venturieri et al., 2003; jaffé et al., 2015). material and methods study site and details of the population studied this study was carried out in the meliponary of the botanical department of embrapa amazônia oriental (1º26′11.52″s, 48°26′35.50″w), in the city of belém, pará, brazil, from august/2013 to january/2014. this sampling period comprised both dry (from august to november) and rainy periods (from december to january). the climate type in the area is af (tropical-rainforest) according to the updated köppen classification (peel et al., 2007). embrapa is located in an environmental protection area (utinga state park), where managed and secondary forests and crops of several tropical, native and introduced species can be found. scaptotrigona aff. postica colonies from the municipality of castanhal, inhangapi, state of pará, brazil (1°26’45.50”s 47°52’56.14”o) were used. these colonies were collected from tree hollows of areas used for agriculture and transferred to non-standardized hives by local beekeepers until they were transferred to the hives used in this study. the same care and procedures described by nogueira-neto (1997) were used during hive transfers. the bee colonies were fed monthly (about 30ml/colony/month) with sugar syrup (60%) offered in pots placed directly inside the nest, and arranged in individual shelters. the hives the vertical hive model proposed (embrapa model) was compared to the horizontal hive, and based on the hive model used in northern brazil to breed bees from genus melipona (venturieri, 2008b). the vertical hive model, in turn, is based on the portugal-araújo (1955) hive model, modified by venturieri (2008a) (fig 1, fig 2a).the vertical hive used in this study was made from wood and has square sections, measuring 25 x 25 x 34 cm and a 2.5 cm wide, segmented into four main parts: base chamber (25x25x8 cm= 3.2l), upper chamber (25x25x8 cm=3.2l), honey super (25x25x8 cm) and cover (25x25x2.5 cm), as follows: fig 1.vertical (embrapa) hive model developed for housing scaptotrigona aff. postica colonies. kl leão et al. – hives for scaptotrigona1040 base chamber: area destined for the brood combs, shelter and food pots (honey and pollen) for the basic colony sustenance. a hole of 20 mm diameter in the frontal region enables bee entrance and exit. in the proposed hive model, an entrance corridor (20 mm wide and 25 mm high) runs within the wooden wall in the hive floor level, where bees circulate, to replicate the natural entrance tube formation of the studied species (roubik, 2006). a hole of 31 mm diameter exists in the center of the base to enable air circulation within the hive. this orifice is sealed with a thin metal wire mesh to prevent the bees from making it a second entrance. upper chamber: segment destined for nest expansion. there are two thick split rods (3 cm wide, 1 cm thick and 20 cm long) on the base of the upper chamber, which helps support the brood combs and/or food pots and in the colony division process. six thin support laths (2 cm wide, 0.5 cm thick and 21 cm in length) are placed in the top to keep the brood combs from sticking on the lid or on the honey super. honey super: area intended to store food pots for future harvesting. the honey super has the total internal volume of 6.4 l, a 1 cm wide passage in its center bottom part for the access of the bees to the food pots. the honey super was not provided in this study, as the colonies were not used for honey production. cover: upper end of the hive that has two 31 mm diameter holes in the middle region, to enable air circulation and supplementary feeding with bottles filled with sugar solution. the horizontal (“cabocla”) hives that were compared with the vertical hives had external dimensions of 49.5 x 17 x 17 cm, 2.5 cm thick wall, internal volume of 6.4 l and no internal divisions. therefore, the two models had the same volume (6.4 l) and wall thickness, and varied only in regards to shape and internal divisions (i.e. the vertical hive did not include the supper; fig 2). both hive models were built using the timber of “louro-canela” (ocotea sp., lauraceae), which is used for hive building due to its resistance to high levels of humidity, common in the brazilian amazon. parameters analyzed ten colonies of s. aff. postica were used, where five were transferred to the vertical hive and five to the horizontal. the nests began being evaluated 30 days after the colonies were transferred, to enable adaptation of the bees to the new hive. the evaluations were carried out considering the following parameters: nest components: 1.1 entrance tube size: the size and diameter (in millimeters) of the entrance tube – a structure considered as indicative of colonial strength and defense capacity (couvillon et al., 2008) – were measured each month during six consecutive months for all colonies. for the analysis, in order to guarantee the independence of data, it was used the monthly difference of the measured values as independent variables. 1.2 – number of brood combs: the brood combs of each colony were counted in non-consecutive months (e.g.: august-october-december/2014) in order to guarantee the independence of data. since the brood combs develop over a 35days period, counting them in non-consecutive months ensured all brood combs would be new, and thus, considered independent in the analysis. 1.3 brood comb area: the first mature brood comb of each colony was measured with a caliper ruler, and its largest and smallest diameter (in millimeters) were recorded and multiplied to calculate the brood comb area (in mm2: highest diameter x smallest diameter). this procedure was adopted to avoid an excessive handling of the colony; previous attempts to measure all the brood combs resulted in extensive damages and the subsequent weakening of the colonies. the same procedures to guarantee the independence of data regarding the number of brood combs were used for the brood comb area estimates. fig 2. (a) vertical (embrapa) hive model.scale bar: 1:6.5cm. (b) horizontal (“cabocla”) hives used in the comparison with the vertical hive. scale bar: 1:4.5cm. sociobiology 63(4): 1038-1045 (december, 2016) 1041 hive occupation: the space occupied by each colony was estimated and later transformed into a percentage. the same procedure to guarantee the independence of data regarding the number of brood combs was used for the hive occupation. thus, we used the monthly difference on the hive occupation in our analysis. an independent-samples t-test with a 5% significance level was used to compare the nest components and hive occupation of s. aff. postica colonies housed in the two hive models. management: colony performance grades. the colonies were subjectively graded during the experimental period, based on the observations of a single observer, considering the criteria described below, and compared by using a general linear model with poisson residuals (poisson glm). analyses were performed in r 3.3.1using the lme4 package (r core team, 2013). grade 1 – very poor: colony with a small number of new, young and adult workers. brood combs with small size. nest without laying queen and without food pots and empty upper chamber. grade 2 – poor: colony with few young and adult workers. few new brood combs and few food pots and empty upper chamber. grade 3 – average: colony with a good number of workers, with some small brood combs and few entirely filled food pots and partially occupied upper chamber. grade 4 – strong: colony with an average number of worker bees, with some brood combs. large food pots (honey and pollen) occupy all the space surrounding the brood combs, filling the whole base chamber and upper chamber over half occupied. grade 5 – very strong: colony with a large number of workers, several brood combs, and food pots occupy all the space surrounding the brood combs. base and upper chambers filled. other parameters: the occurrence of phorid flies, cockroaches, lizards and beetles present, was registered to compare the different hive types. dvision-eight colonies (which reached grade 5) were divided and the time spent in division, and the advantages and disadvantages among the two hive models were recorded at the end of the experiment. results nest components and hive occupation brood comb area was approximately two times larger in vertical hives (5,491.6 mm2 higher on average) than in the horizontal hives (p= 0.001). there were no differences between the colonies placed in the different hive models considering all the other parameters (table 1). no differences were observed between the two tested hive models, regarding the percentage of occupation (horizontal: 78.17% ± 13.88 (sd) of occupation; vertical: 83.20% ± 22.34 (sd) of occupation; t=-0.485; df=48; p= 0.630). only two (40%) of the colonies housed in the horizontal hives completely occupied the hive space, whereas four colonies (80%) in vertical hives completely occupied the available hive space. nest components hive horizontal vertical t df p entrance tube – size* 5.00+13.63 7.80+22.96 0.52 48 0.602 entrance tube – diameter* 0.00+4.08 -0.20+4.53 0.16 48 0.870 number of brood combs† 10.07+2.40 9.60+2.32 0.54 28 0.593 brood comb area (mm²)† 7,856.6+2,905.72 13,348.2+4,897.55 -3.73 28 0.001 data presented as: *: mean monthly differences during the whole study period; †: means obtained in alternate months. table 1. comparisons (means ± sd) of the nest components of scaptotrigona aff. postica colonies housed in two different hive models: vertical and horizontal, august/2013 to january/2014. comparisons were made through an independent t-test. management the colony grades ranged from 2 to 5 for both hive models, and did not differ regarding the hive type (glm poisson: p= 0.86; fig 3). relevant differences regarding management activities when opening the hives were observed for the horizontal hive, which did not have the laths at the top of upper chamber. the brood combs were stuck to the cover of the hive, which were frequently damaged during hive inspection, and hindered the proper brood manipulation. the brood often remained stuck to the cover, forcing the inspector to remove them to avoid turning them upside down (fig 4), a harmful process for the immature bees (see discussion). in contrast, the brood combs were fixed to the support laths of the vertical hives, not to the cover. however, there was a greater accumulation of propolis (collected tree resin) in the space between laths and the cover, leading to a greater difficulty in opening the hives, especially in strong colonies. eight of the ten colonies were divided, as two did not develop sufficiently to allow division. time spent on the division process was not different between the hive types (horizontal hive: 14.35 ± 4.88 min; vertical hive: 8.90 ± 1.67 kl leão et al. – hives for scaptotrigona1042 min; t= 1.986; d.f.= 5; p= 0.103). however, brood combs and food jars were frequently ruptured in the horizontal hives during the division procedure. symbionts or predators were not observed in any hive during the experiment. phorids occurred only in october/2014 and in a single horizontal hive, although not in an abundance considered to be significant for colony health. discussion in our study, several parameters of colony development of a meliponine species (s. aff. postica) were compared between two hive models, a vertical model and a horizontal model. our results show that the colonies developed similarly in both hive models, although there were relevant differences to qualify in the adaptation of colonies to both tested models. although the number of brood combs was similar between the hive models, its area was about twice the size in the vertical hive relative to horizontal hives. a feasible explanation for this relies on the narrowness of the horizontal hive; this type of hive does not allow a colony of scaptotrigona to reach its maximum size, as in order for new combs to be added, the lower one must be of a minimum size to allow the construction of the upper comb. a consequence of this reduced brood comb area is that the populations of colonies housed in horizontal hives are expected to be smaller. a larger population (in vertical hives) guarantees an improved execution of nest maintenance activities, such as the collection of food resources and the resistance to natural enemies (couvillon et al., 2008), which are desirable characteristics of nests aimed to be used in production. there was no difference in entrance tube length or diameter between the two hive types. entrance tube length is perhaps more a consequence of the colony state and temporal food resource availability (seasonality), than the hive type. fig 3. variation of the subjective grades (1-very poor; 2-poor; 3-average; 4-strong; 5-very strong) of 10 scaptotrigona aff. postica colonies housed in horizontal and vertical hives. data presented as median and se. fig 4. brood combs of scaptotrigona aff. postica stuck to the lid of the horizontal hive, hindering management. a-cover; b-base nest. sociobiology 63(4): 1038-1045 (december, 2016) 1043 no difference in hive occupation was found between colonies in the two hive types, although there were clearly observed empty spaces in horizontal hives throughout the experiment. empty spaces are not good for bee development, as it allows the establishment of undesirable inquilines (nogueira-neto, 1997), and jeopardizes nest thermoregulation (roubik, 1989). the uniformity of the core where the immature individuals occur is important to stabilize and control the incubation temperature in stingless bee colonies (roubik, 1989). it is likely that in the horizontal hives there was a larger effort in thermoregulation, which reduced brood production (vollet-neto et al., 2009), contrasting with the vertical hives, in which the nest structures entirely occupied the hive. differences on the adaptation of colonies in different hive models are already reported by quezada-euán and gonzalez-acereto (1994), by comparing the inpa (vertical, 14.35 l), pnn (horizontal, 14.3 l), and a traditional horizontal (“hobone”, 10.06 l) hive models for another meliponine species (melipona beecheii bennett). the authors concluded that in the traditional hive the amount of brood comb was greater than in the others models and thus, recommended a reduction in the volume of the other hive designs to ease the thermoregulation of the colonies. although their experimental design (non-standardized hive volumes) was different from our study (i.e. equal hive volumes), the conclusion that the hives must be adequate to avoid the occurrence of empty spaces in order to ease thermoregulation is similar. we also showed that there were differences in the ease of colony management between the different hive models; the vertical hive model facilitated the inspection and division of the colonies, contrasted to the horizontal hive model. the main problem observed when comparing the hive models was during hive opening. the absence of support laths in the horizontal hive caused the brood combs to be stuck to the lid of the hive and consequently to be turned upside down during the management. thus, causing the death of hundreds of immature bees, as the eggs, after turned upside down, sink in the larval food and drown (jungnickel et al., 2001). even in brood combs with bees in pupal stages, the combs stuck in the cover leads to a greater time spent to release them. the support laths of the vertical hive avoided the loss of new brood combs since the brood pillars (roubik, 2006) were stuck to the laths and not to the cover. since it is possible to manually remove each lath, the impact of the manipulation is reduced in comparison to a colony without them. however, propolis accumulation between the laths and the cover hampered opening of vertical hives, although this problem may be easily controlled by increasing the space between the laths and the cover. the large amount of propolis adhered in the colonies indicates the potential of scaptotrigona to produce this material, another product of potentially high economic value derived from stingless bee colonies (sawaya et al., 2009). the subjective grades of colony strength were not statistically different among the hive models. still, it was important to evaluate the general colony development in both hive types, being useful to identify/characterize the state of the colonies. there were no differences in division time between the colonies sheltered in the two hive types. however, division method differed between hive models. the division of the colonies in the horizontal hives was often considered more problematic than the division of colonies housed in vertical hives, where the division process was simpler. the manipulation of the brood combs and the use of a cutting tool to separate the brood combs was necessary in the horizontal hive, requiring greater technical skill. brood comb manipulation may cause the mortality of the new individuals (e.g., by drowning in the larval food, as previously mentioned), and the rupture of the honey pots. broken honey pots caused honey to drain to the bottom of the hive, sometimes causing the death of individuals of the colony, and attracting predators such as ants, pillaging bees, and phorid flies. the division process was easily carried out in the vertical hive, once this hive model has subdivisions and split rods. brood comb separation occurred naturally in the vertical hive, without major interventions, because the use of sticks between the nest and the upper nest facilitated separation and minimized damage. this result had already been reported for the stingless bee species in hives similar to the model proposed (venturieri, 2008a). another advantage of the vertical hives is the facilitation of honey extraction. in vertical hives, the honey is stored in a separated compartment (the super), which can be easily separated from the hive without disturbing the colony (venturieri, 2008a). in regard to parasites, we observed no problems with phorids, a significant natural enemy of stingless bees, in either hive models. the absence of phorids was possibly because the period during which this study was carried out was unfavorable for phorid proliferation (oliveira et al., 2013), and due to a larger capacity of scaptotrigona to defend itself from natural enemies. we rarely found other animals living in the hives along with the bees in this study (e.g. parasites or symbionts, common in stingless bees; nogueira-neto, 1997), possibly due to the short time these bees were established in these hives. in order to enhance the stingless bee keeping practice, a standardization of procedures and methods is necessary, and the hive models are an important aspect to be considered. in our study, we showed that the vertical hive had some advantages compared with a horizontal (traditional) hive, and thus we recommend its use for housing this scaptotrigona species. however, the keeping of colonies in horizontal hives is not necessarily impractical, since colonies still develop relatively well in this kind in hive, but when we consider that the vertical types ease the management of colonies, especially during nest divisions, the vertical hives are better from this perspective. acknowledgments we would like to thank janete teixeira gomes, lorival juracy lucas and josé alves da rocha for their valuable kl leão et al. – hives for scaptotrigona1044 assistance in the field and rodolfo jaffé for statistical advice. we also thank alistair campbell for linguistic advice and two anonymous referees for insightful suggestions. we thank capes/embrapa (15/2014) for providing grants for kll and jcv. this research was funded by a fapespa grant (iccaf: 004/2012)/cnpq (554318/2010-5) through the bionorte project. references contrera, f.a.l., menezes, c. & venturieri, g.c. 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(2009). aquecimento de colméias de abelhas sem ferrão: vale a pena? mensagem doce, 103: 1-4. retrived from: http:// www.apacame.org.br/mensagemdoce/103/artigo4.htm http://www.apacame.org.br/mensagemdoce/103/artigo4.htm http://www.apacame.org.br/mensagemdoce/103/artigo4.htm 343 aspartame-based sweetener as a strong ant poison: falsifying an urban legend? by jouni sorvari* & marja-katariina haatanen abstract information about the usability of artificial sweeteners, mainly aspartame, for controlling pest ants has spread widely in the internet. with a laboratory experiment we tested the effect of an aspartame based sweetener on the mortality of the black garden ant lasius niger, a common pest ant in kitchens in europe. the aspartame-based sweetener was added to the laboratory jelly food of ants in the experimental group (16 colonies). the control group (14 colonies) received otherwise similar jelly but without the aspartame-based sweetener. during the 35 day period of experiment we did not find any signs of aspartame induced mortality in tested ants. in addition, 135 colony founding l. niger queens were submerged in a sweetener solution (artificial sweetener + distilled water) and 135 queens were submerged in distilled water (control). the overall mortality was very low (<1.5%) and no between-group differences in mortality were found within 24 and 96 hours. our results strongly oppose the rumors that aspartame sweeteners are effective as an ant poison, at least with a typical dose of household aspartame products. key words: ants, artificial sweetener, black garden ant, hoax, lasius niger, pest control introduction in ants, protein is used for egg formation and larval growth whereas carbohydrates are used as an energ y source for adult workers (brian 1983; dussutour & simpson 2009), and thus it may have indirect, but important role in the production of offspring (porter 1989). accordingly, carbohydrates increased sexual investment in the ant myrmica brevispinosa (bono & herbers 2003). department of biolog y, section of ecolog y, fi-20014 university of turku, finland *correspondence: j. sorvari, department of biolog y, section of ecolog y, fi-20014 university of turku, finland; e-mail: jouni.sorvari@utu.fi, 344 sociobiolog y vol. 59, no. 2, 2012 in the green-headed ants (rhytidoponera sp.), carbohydrates increased the survival of larvae (dussutour & simpson 2009). aspartame is an artificial sweetener used in sugar-free food and beverage products. aspartame is widely and frequently used and it was discovered by chance over 40 years ago (lajtha et al. 1994). it was at first used as a food additive over 20 years ago. numerous studies have been made on various aspects of this compound, and the extensive literature includes observations for example on its absorption, metabolism, taste, structural analogies and effects on nutrition. also several reviewers have raised concerns in regard to potential or anecdotal interactions. in the case of aspartame, concerns gives rise due to aspartame or its metabolic products (aspartic acid, phenylalanine, methanol) each of which may have effects on the nervous system in high concentrations in blood. information about the usability of artificial sweeteners, mainly aspartame, for controlling pest ants has spread widely in the internet. also many gardening websites recommend aspartame as a method for ant control. aspartame is paralleled as a neuropoison and is said to kill ants in a short period, e.g. within 24 hours. however, one internet site (anonymous 2010) claims that the information about aspartame as an ant poison is a hoax, because aspartame was not poisonous in three different experiments. these three experiments, however, had methodological problems or in one case, the experiment was not conducted in a scientific manner. in one of the experiments (anonymous 2010), ants that were provided aspartame diet were said to thrive. however, the study contained only one nest fed with an aspartame-containing diet and two other nests were fed with another kind of diet, thus, clearly not reaching the number of replicates needed for a scientific experiment. in addition, the duration of the experiment was not reported. the author of this experiment remained anonymous. the other experiments were made by exposing nest mounds of fire ants (solenopsis invicta buren, 1972) with dry aspartame sprinkled over nest mounds (brown 2007), and with mild aspartame solution (1 table spoon of artificial sweetener containing aspartame and 3.785 liters of water) (brown 2008). no differences were found between aspartame-treated mounds and untreated control plots in either year. in these experiments aspartame was applied to the nest mound and it is not sure that ants ate the aspartame at 345 sorvari, j. & m.-k. haatanen — aspartame-based sweetener as an ant poison all. in fact, tinti and nofre (2001) reported that ants are not attracted by aspartame. so the previous experiments can be doubted due to insufficient numbers of replicates or the fact that it is not known whether the ants consumed aspartame at all. therefore, a careful reliable experiment is needed to assess the toxicity of aspartame based sweetener on ants. with a realistic dose (typical household artificial sweetener) we tested the effect of aspartame (together with asesulfame-k) containing food in laboratory tests on the mortality of the black garden ant lasius niger (linnaeus, 1758), a common pest ant in kitchens in europe. materials and methods lasius niger is a common ant in europe (czechowski et al. 2002; seifert 2007). its colonies are monog ynous i.e. there is only one queen at a time, and colonies contain up to ten thousand workers (collingwood 1979; czechowski et al. 2002). in finland it is often called the “sugar ant”. as this name implies, l. niger is particularly attracted to sweet substances. in the presence of various species of aphids, it feeds aphid honey-dew (fischer et al. 2001; staedler & dixon 2008). lasius niger is abundant in human environments as lawns, gardens and households, and it is often regarded as nuisance. we reared 30 young small colonies of l. niger in laboratory in plastic boxes (10x8x10 cm). in the beginning of the experiment the nests contained a queen and approximately same number of workers and larvae (table 1). the ants nested in test tubes. humidity in test tubes was maintained by moist cotton. walls of the nest boxes were lined with fluon (ptfe) to prevent escapes. the treatment group got a standard laboratory food jelly (bhatkar & whitcomb 1970) enriched with artificial sweetener containing 2% of aspartame and 1% of asesulfame-k (hermesetas™, hermes sweeteners ltd, zürich, switzerland). the diet included one chicken egg, 62 ml honey, 3.5 g artificial sweetener (7 mg aspartame, 3.5 mg asesulfame-k), 1 g vitamins, 1 g minerals and salts, 5 g agar and 500 ml water. the control group (n = 14) got otherwise the same food but without the artificial sweetener. fresh food was offered daily ad libitum. we kept the experiment running for 35 days (13 may-16 june 2011) in a climate chamber (+25°c, 60% rh, 12:12 light:dark rhythm). the mortality 346 sociobiolog y vol. 59, no. 2, 2012 of workers and queens was checked in a daily basis. deceased workers were removed from nest boxes. the number of larvae was counted in the beginning and at the end of the experiment. it is not known whether aspartame and asesulfame-k are absorbed into the haemolymph in digestion in ants. it is possible that aspartame is absorbed into the haemolymph via tracheal openings and causes toxic effects. therefore we made a submerging experiment with 270 l. niger colony founding queens (21 july 2011). 135 queens were submerged briefly (approximately 1 second) in 10 ml solution containing 9 g of distilled water and 0.13 g of artificial sweetener (2.6 mg aspartame, 1.3 mg asesulfame-k). the control queens (n = 135) were dipped in a similar way in 10 ml of distilled water. in order to avoid the possible contamination of the solution by formicid acid sprayed by the alarmed queens the solution was renewed after every 10 queens. the mortality was checked after 24 and 96 hours. statistical analyses were performed using sas statistical software version 9.2 (sas institute, 2001). the difference in the numbers of workers and larvae in the beginning and at the end of the experiment were analyzed with t tests (the equality of variances were confirmed with levene’s tests; proc ttest). differences in mortality and the development of larval number was analyzed with generalized linear models (proc genmod) using binomial error term and logit link function. in the analyses of worker mortality the number of deceased workers was used as the dependent variable (events) with the number of all individuals (living and deceased) as the denominator (trials). the development of larval number was analysed in a similar way: the number of larvae at the end of the experiment was used as dependent event variable and the number of larvae in the beginning of the experiment as trial denominator. in the analysis of queen mortality the deceased queens were coded as 0 and live queens as 1 (binomial error term, logit link function). results colony rearing experiment numbers of workers and larvae were similar between the treatment and control colonies both in the beginning and at the end of the experiment (table 1). the average within colony mortality of adult workers was 10.9% ± sd 14.2 in aspartame treatment colonies and 11.7% ± sd 9.4 in the control colonies. 347 sorvari, j. & m.-k. haatanen — aspartame-based sweetener as an ant poison the mortality did not differ statistically (generalized linear model: df = 1, χ2 = 0.12, p = 0.72). during the experiment one queen died in both groups. the mortality of queens was similar between treatments (generalized linear model: df = 1, χ2 = 0.01, p = 0.92). we did not found dead larvae from any of the colonies at the end of the experiment. if larval mortality occurred the deceased larvae were consumed as protein food. however, it was possible to compare the development of larval number, i.e. the proportion of larvae at the end of the experiment from the larvae at the beginning of the experiment. no differences were found between the treatment and control colonies (generalized linear model: df = 1, χ2 = 0.01, p = 0.90). queen submerging experiment all of the queens survived the submerging treatment (checked immediately after the submerging ). one queen was found dead in the control group and no dead queens were found in the artificial sweetener treatment group after 24 hours (generalized linear model: df = 1, χ2 = 1.39, p = 0.24). the mortality was one in the control group and three in the sweetener treatment group 96 hours after treatment (generalized linear model: df = 1, χ2 = 1.06, p = 0.30). discussion previous studies did not sufficiently monitor the consumption of aspartame by the ants. this is important in the light of tinti and nofre’s (2001) choice table 1. mean numbers (± sd) of adult workers and larvae in the aspartame based sweetener colonies (n = 16) and in the control colonies (n = 14). the variances were equal in all tests (p = 0.46 –0.98). abs = aspartame-based sweetener colonies. workers ± sd larvae ± sd beginning end beginning end abs 11.4 ± 4.0 12.4 ± 5.3 5.9 ± 2.5 1.3 ± 1.9 control 10.1 ± 3.3 10.6 ± 5.3 5.9 ± 2.5 1.0 ± 1.6 t 1.0 1.0 1.35 0.5 p 0.32 0.34 0.19 0.63 348 sociobiolog y vol. 59, no. 2, 2012 experiment, where the ants (l. niger) were not interested in aspartame. we avoided this by giving the aspartame based sweetener as an un-separable part of the ants' jelly food, thus, the treatment ants were forced to eat it. the treatment group ants ate the aspartame containing food as readily as the control ants ate their non-aspartame containing, otherwise similar jelly food. in addition, we did not detect any signs of starvation (passivity, increased mortality of workers and larvae). many reports in the internet do not report in which form (e.g. liquid, hail, etc.) and how the aspartame exactly is given to the ants. if aspartame is spread on a nest as a sticky liquid (e.g. aspartame containing juice concentration), the ants may be killed not by the aspartame but by the stickiness of the product. in addition, observations that ant nests were “dead” soon after applying aspartame may be due to possible desertion of the nest after the nest material is contaminated by foreign substances. besides the amount, and how the aspartame is given to ants, aspartame may affect ants in time. according to the information spread on the web, aspartame killed ants in hours and days, not in weeks or months. in one web site (anonymous 2010), where aspartame had no effect, the length of observation period is not reported and the number of replicates was way below scientific standards (only one aspartame treatment colony). our study, with a sufficient number of colonies, lasted 35 days and no effects were discovered. in addition, in the submerging experiment the survival of high number of queens were monitored four days with no effects detected. our current results strongly oppose the rumor that aspartame based sweeteners are effective lethal poisons for ants. we used doses that are typical in household aspartame products and these doses were clearly sub-lethal. it is, of course, possible that higher doses can be toxic for ants. it must also be noted that we measured only the differences in mortality in a sterile laboratory environment. in theory it is possible that metabolic products of aspartame can disturb the immune system and cause increased mortality if the ants are exposed to pathogens at the same time. therefore, a further study with variable doses and pathogen exposure needs to be conducted. 349 sorvari, j. & m.-k. haatanen — aspartame-based sweetener as an ant poison acknowledgments salla-riikka vesterlund provided information on the use of aspartame as an ant poison. the study was funded by emil aaltonen’s foundation ( js) and by finnish cultural foundation (mkh). animal tests with insects do not violate any laws of finland. references anonymous 2006 [cited 2011 december 1]. the world’s best ant poison [internet]. available from: http://www.snopes.com/humor/iftrue/antpoison.asp bhatkar, a. & w.h. whitcomb 1970. artificial diet for rearing various species of ants. florida entomologist 53:229-232. bono, j.m. & j.m. herbers 2003. proximate and ultimate control of sex ratios in myrmica brevispinosa colonies. proceedings of the royal society of london b 270:811-817. brian, m.v. 1983. social insects: ecolog y and behavioural biolog y. london (uk): chapman and hall. 377 p. brown, e. 2007 [cited 2011 december 1]. evaluation of aspartame as a mound treatment for red imported fire ant management. integrated pest management – urban ipm program 2007. texas agrilife extension service, college station, texas, usa. pp. 65-67 [internet]. available from: http://fireant.tamu.edu/research/projects/pdf/ urbanipmhandbook2007edited.pdf brown, e. 2008 [cited 2011 december 1]. evaluation of watered in aspartame as a mound treatment for red imported fire ant management. integrated pest management – urban ipm program 2008. texas agrilife extension service, college station, texas, usa. pp. 33-35 [internet]. available from: http://fireant.tamu.edu/research/projects/pdf/ ipmmanual08a1.pdf collingwood, c.a. 1979. fauna entomologica scandinavica. volume 8, the formicidae (hymenoptera) of fennoscandia and denmark. klampenborg (denmark): scandinavian science press. 174 p. czechowski, w., a. radchenko & w. czechowska 2002. the ants (hymenoptera, formicidae) of poland. warsaw (poland): museum and institute of zoolog y pas. 200 p. dussutour, a. & s.j. simpson 2008. description of a simple synthetic diet for studying nutritional responses in ants. insectes sociaux 55:329-333. fischer, m.k., k.h. hoffmann & w. völkl 2001. competition for mutualists in an anthomopteran interaction mediated by hierarchies of ant-attendance. oikos 92:531541. lajtha, a., m.a. reilly & d.s. dunlop 1994. aspartame consumption: lack of effects on neural function. journal of nutritional biochemistry 5:266-283. porter, s.d. 1989. effects of diet on the growth of laboratory fire ant colonies (hymenoptera: formicidae). journal of the kansas entomological society 62:288-291. 350 sociobiolog y vol. 59, no. 2, 2012 seifert, b. 2007. die ameisen mittelund nordeuropas. tauer (germany): lutra verlags und vertriebsgesellschaft. 368 p. staedler, b. & a.f.g. dixon 2008. mutualism: ants and their insect partners. cambridge (uk): cambridge university press. 226 p. tinti, j.-m. & c. nofre 2001. responses of the ant lasius niger to various compounds perceived as sweet in humans: a structure-activity relationship study. chemical senses 26:231-237. doi: 10.13102/sociobiology.v64i4.1839sociobiology 64(4): 456-465 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 species composition, relative abundance and distribution of social wasps fauna on different ecosystems introduction the netropical region presents high biodiversity as a result of ecosystem heterogeneity that includes cerrado, campos rupestres, high-altitude fields, deciduous (dry) and semideciduous forest, atlantic forest, and caatinga (scolforo & carvalho, 2006). studies of various taxa in minas gerais have revealed highly rich plant (mendonça & lins, 2000), amphibian (feio et al., 2008), and insect communities, including social wasps (brunismann et al., 2016; prezoto et al., 2016). social wasps carry out numerous ecological functions, including floral visitation and pollination. several studies on socials waspsthat are floral visitors of different species of angiosperms have been conducted in brazilian ecosystems such as cerrado (mechi, 2005), araucária forest (hermes & kohler, 2006) and caatinga (santos et al., 2006), as well as in urban areas (silva-pereira & santos, 2006; hermes & kohler, 2006). abstract the state of minas gerais has high biodiversity, characterized by strong ecosystem heterogeneity that favors high richness of social wasps. there are currently 109 species known to occur in the state, however, there is lack of information concerning the distribution of these social insects among different ecosystems. the objective of this study was to evaluate social wasp species richness and distributions by ecosystem, thereby generating data for use in discerning relevant and priority environments for vespid conservation in minas gerais. we evaluated articles, theses, and dissertations published up to the year 2016 containing data on biodiversity of social wasps in natural and agricultural environments. we found 18 studies, in which the highest reported species richness was in semideciduous seasonal forest (n = 68), followed by cerrado (n = 53), ombrophilous forest (n = 39), deciduous seasonal forest, and campos rupestres (n = 35). the lowest richness was reported in mixed forest (n = 21) and high-altitude fields (n = 19). considering the high degree of landscape diversity of the state of minas gerais, more studies are needed to accurately assess social wasp diversity in these ecosystems, especially cerrado. ombrophilous and deciduous forests should be considered strongly relevant for these social insects, especially the rio doce state park and the rio pandeiros wildlife refuge. sociobiology an international journal on social insects mm souza1, ag brunismann1, ma clemente2 article history edited by gilberto m. m. santos, uefs, brazil received 30 june 2017 initial acceptance 15 july 2017 final acceptance 01 august 2017 publication date 27 december 2017 keywords polistinae, biodiversity, species inventories. corresponding author marcos magalhães de souza instituto federal de educação ciência e tecnologia do sul de minas gerais praça tiradentes nº 416, centro cep 37576-000, inconfidentes-mg, brasil. e-mail: marcos.souza@ifsuldeminas.edu.br although wasps are more frequently regarded as floral resource thieves (hunt et al., 1991; santos et al., 2010); recent papers show that wasps can also effectively contribute to pollination (shuttleworth & johnson, 2009). in a study with schinus terebinthifolius raddi (anacardiaceae), suhs et al. (2009) showed that social wasps, especially polistes versicolor (olivier, 1791), polybia sericea (olivier, 1791), polistes simillimus (zikán, 1951), and polybia ignobilis (haliday, 1836), were more representative in richness and abundance than bee species, being considered efficient pollinators. other studies, such as barros (1998) and hermes and kohler, (2006) also demonstrated the efficiency of the wasps as pollinators. these insects also play a role in controlling agricultural pests (prezoto et al., 2008; elisei et al., 2010), and are sensitive to environmental changes, making them useful as indicators of environmental quality (souza et al., 1 instituto federal de educação, ciência e tecnologia do sul de minas gerais, inconfidentes-mg, brazil 2 departamento de zoologia, instituto de biociências, universidade estadual paulista júlio de mesquita filho, rio claro-sp, brazil research article wasps mailto:marcos.souza@ifsuldeminas.edu.br sociobiology 64(4): 456-465 (december, 2017) 457 2010). minas gerais has been more frequently sampled than other brazilian states and regions (prezoto et al., 2016), and there are currently 109 social wasp species known to occur in the state (http://vespas.ifs.ifsuldeminas.edu.br/). however, there is currently a paucity of information concerning species distributions among ecosystems, and studies that discuss phytophysiognomy (santos et al., 2007; santos et al., 2009). the study of biological diversity, species distributions, and richness on social wasps are relevant because biodiversity directly influences natural and agricultural ecosystem function through effects on trophic network structure (clemente et al., 2012; clemente et al., 2013), pollination (suhs et al., 2009; barros, 2009; hermes & kohler, 2006), nutrient cycling (gomes et al., 2007), and ecological succession, among other processes (souza et al., 2010). it is estimated that ecosystems that still have low sampling may present a high unknown richness, and may be of considerable relevance for the conservation of social wasps. therefore, the objective of this study was to better understand the social wasp richness and species geographic distributions among ecosystems, with the purpose of identifying relevant and priority environments for vespid conservation. materials and methods we evaluated data of occurrence of social wasps in different ecosystems between coordinates north 14º13’52’’ and south 22º55’22’’, east 39º51’23’’ e a west 51º02’56’’. the data used in this study are derived from articles, theses, books, catalogues and dissertations published in 2006 up to 2016 with data on social wasp biodiversity in natural and agricultural environments, totaling 18 studies (table 01). we followed the species classifications proposed by oliveira filho (2006). we evaluated the social wasp richness and species distributions in seven distinct ecosystems: 1. seasonal semideciduous rain forest, 2. deciduous seasonal dry (forest, 3. ombrophilous forest, 4. mixed forest (all on the atlantic forest domain); 5. cerrado; 6. campos rupestres, and 7. high-altitude fields (figure 01). semideciduous rain forests are formed by arboreal species that have a deciduous rate of around 50% and a climate varying between tropical and tropical at altitude, whereas deciduous dry forests have around 90% and semiarid climate, and forests ombrophilous to deciduous is around 10% and tropical climate. the mixed forests are composed of araucaria angustifolia kuntze 1898 associated with species of semidecidual rainforest, tropical climate of altitude; cerrado shows variable vegetation, from rural, savanna and forest formations, tropical climate; campos rupestres areas are characterized by rocky outcrops of quartz, located in areas above 900 meters of altitude, endemic flora and tropical climate of altitude; and altitude fields show a predominance of species of the family poaceae and flora very similar to the patagonia fields, southern argentina, altitude above 1,000 meters and altitude topical climate (oliveira filho, 2006). author year ecosystem number of species souza & prezoto 2006 seasonal semideciduous rain forest 38 (45)* souza et al. 2010 seasonal semideciduous rain forest 36 souza et al. 2015 b seasonal semideciduous rain forest 34 albuquerque et al. 2015 seasonal semideciduous rain forest 34 jacques et al. 2012 seasonal semideciduous rain forest 26 barbosa 2015 seasonal semideciduous rain forest 36 auad et al. 2010 agricultural system inserted in seasonal semideciduous rain forest 13 freitas et al. 2015 agricultural system inserted in seasonal semideciduous rain forest 19 clemente 2009 ombrophilous forest 5 souza et al. 2012 ombrophilous forest 38 elpino-campos et al. 2007 cerrado 29 simões et al. 2012 cerrado 32 jacques et al. 2015 agricultural system inserted in cerrado 29 brunismann et al. 2016 seasonal semideciduous dry forest 37 prezoto & clemente 2010 campo rupestre 23 (24)* souza et al. 2008 campo rupestre 32 souza et al. 2015 a high-altitude fields and mixed forest 22 (23)* bruger 2014 seasonal semideciduous rain forest 23 *the sum of species recorded by the listed authors and those recorded since the study. table 01. biodiversity studies of social wasps carried out in the state of minas gerais by author, date, ecosystem, and number of species registered. mm souza, ag brunismann, ma clemente – social wasps fauna on different ecosystems458 results and discussion a total of 109 species of 16 genera of social wasps were recorded in seven distinct ecosystems (table 02). the exception three species mischocyttarus buysoni (ducke, 1906), polybia brunnea (curtis, 1844) e polybia emaciata (saussure, 1854) which has a record for the study area (richard, 1978), but there is no occurrence information per ecosystem. nine of the 18 studies (50%) carried out in our research were in semideciduous seasonal rain forest (hereafter ssrainfor), which together reported a total of 68 social wasp species, being 16 species of them exclusive to this forest type (table 02). semideciduous seasonal rain forests have the largest number of studies due to wide distribution in minas gerais. seasonal semideciduous forests are widely distributed in areas with a seasonal rainfall regime in the atlantic forest and cerrado domains. it is the predominant vegetation typology in these domains, and occurs in the form of enclaves and forests associated with permanent or intermittent bodies of water (scolforo & carvalho, 2006). about 50% of the territory of minas gerais occurs in the atlantic forest domain according to the atlas of evolution of forest remnants and associated ecosystems in the atlantic forest domain (sos mata atlântica foundation, 2002). social wasp richness in semideciduous seasonal rain forest is considerable, with social wasp richnes ranging from 13 to 45 species among the study sites in the evaluated scientific works. the lowest richness values for this biome were reported in freitas et al., (2015) (s = 19) and auad et al., (2010) (s = 13), both of which share the characteristic of being carried out in agricultural systems within forest areas. according to santos et al. (2009), despite the relative structural homogeneity of vegetation in environments with agricultural systems, there is high availability of resources such as nectar, prey, and water, which can support high numbers of social wasp species. however, lawton (1983) and santos et al. (2007) demonstrated that environments with higher structural complexity facilitate the establishment and survival of greater numbers of species. vegetation structure influences social wasp communities directly through provisioning of nesting and food resources, and indirectly through influencing variation in temperature and air humidity, as well as the amount of shade in the environment. some social wasp species nest only under certain conditions, selecting nest sites according to vegetation density and type, extent of canopy closure, and the shape and arrangement of leaves and other plant structures (santos & gobbi, 1998). this partly contributed to the higher richness in other sites compared to those situated in agrarian systems. clemente (2009) recorded only five species in an fig 01. map of records of species of social wasps in the state of minas gerais, brazil. sociobiology 64(4): 456-465 (december, 2017) 459 ombrophilous forest site, while souza et al. (2012) recorded 38 species; in this ecosystem 39 species of social wasps have been recorded, with 17 exclusive species (table 02). clemente (2009) justifies the low sample numbers in this forest fragment (dense ombrophilous forest) due to local floristic composition, which presents trees varying from 17 to 25 meters. this canopy type (i.e., height) restricts light penetration inside the forest, reducing visibility making it difficult to search for and capture wasps. the atlantic forest fragment in the current study contains clearings formed by tree fall (fontes, 1997), which facilitates active search and collection of social wasps due to higher visibility in those areas. for example, a study with synoeca cyanea demonstrated that light intensity is positively correlated with the number of foraging individuals (elisei et al., 2005). dense vegetation also makes it difficult to find species and colonies, which may be imperceptible in this type of environment (wenzel & carpenter, 1994). these data corroborate those of souza and prezoto, (2006) in a semi-deciduous forest which had lower richness (n=25) than cerrado fields (n=33). togni et al., (2014) also reported nine wasp species actively captured in anthropogenic and open forest areas, whereas in the closed forest (i..e, a site with a lower light intensity) only five species were found by active collection. in the amazon forest, silveira (2002) reported difficulty in capturing social wasps in the forest interior, and were only effective only near the edges. however, santos et al. (2007) reported higher wasp richness in atlantic forest (18 species) compared to restinga (16 species) and mangrove (eight species). this difference is richness may be associated with differences in environmental conditions (e.g., salinity, temperature, aridity) in sites with lower richness. in the study by souza et al. (2012) in ombrophilous forest, 38 social wasp species were recorded the rio doce state park (19º38’00.00” s and 42º31’00.00” w), which is greater than that reported in studies in são paulo (togni et al., 2014) and bahia (santos et al., 2007). this may be partially attributed to the greater degree of conservation and lower anthropogenic activity in the rio doce area. these factors can affect the distribution and diversity of these insects, because conserved or regenerating environments typically present a mosaic of vegetation which provides higher availability of food and nesting resources, as well as habitat for species with more restricted ecological needs. the large size of the rio doce area also increases the number of nesting sites and population gene flow relative to smaller sites (souza et al., 2012). most species were found at the forest edge, again likely due to the difficulty of capture in dense and closed forest area. fifteen species were found exclusively in the state park area, which justifies the creation of conservation plans for the rio doce state park for maintenance of social wasp diversity in minas gerais. the high number of exclusive species also justifies additional studies in the area (souza et al., 2012). three of the studies were carried out in cerrado of minas gerais, with a total of 53 social wasp species, seven exclusive (table 02). jacques et al., (2015) registered 29 species in an agricultural system inserted in the cerrado. this high richness can be explained by the fact that many species of social wasps present a degree of synanthropy (michulleti et al., 2013). the same study also has a very diverse and structurally heterogeneous environment, which may explain the considerable number of species collected. this is because more structurally complex sites can support the coexistence of a greater number of species, mainly due to higher availability of microhabitats, greater protection against predators, and greater availability and diversity of food resources and substrate for nesting (santos et al., 2007; souza et al., 2014). the second study in cerrado was conducted by elpinocampos et al. (2007) a portion of the ecological reserve of the itororó hunting and fishing club in uberlândia (15º57’46.57” s, 48º26’13.36” w), minas gerais (ccpiu), the authors registered 29 species of social wasps distributed among 10 genera. the authors report that the study areas are cerrado fragments that have been subject to anthropogenic (e.g., intentional fires, deforestation, cultivation) or natural alterations (fire). fire events are common in the cerrado and natural events are necessary for ecological balance of this ecosystem (oliveira & marquis, 2002). however, when they occur frequently as a result of anthropogenic activity, they can easily diminish diversity. the authors reported that three fires occurred during the study period, which may explain the low number of colonies found for most species collected, and perhaps generally the lower numbers of species recorded in the area. the third cerrado study, carried out by simões et al. (2012) in the unilavras/boqueirão biological reserve in ingaí (21º20’01.62” s, 44º59’01.41” w), recorded the greatest richness among the cerrados of minas gerais, with a total of 32 species and 10 genera. this was the first study carried out in the southern portion of minas gerais, and the high local diversity of social wasps stands out compared to studies carried out in other regions of the state. the study site is surrounded by pasture and agricultural areas, and thus may act as a refuge, protecting organisms from pesticides (which are used in most of the surrounding areas). according to the jacques et al. (2015) favorable climate conditions in the region may have also contributed to the high observed richness through effects on foraging activity, as demonstrated in several previous studies (giannotti et al., 1995; elpino-campos et al., 2007; auad et al., 2010). higher temperature and precipitation, in particular, may have influenced the observed species richness, as there is greater variation in these factors in cerrado than in forest areas. in minas gerais, the cerrado corresponds to 57% of the state territory, and three studies have indicated the area as containing the second highest number of social wasps in the state. this highlights the need to expand studies in this ecosystem, as richness is likely underestimated. the campos rupestres site presented 35 species, with one exclusive (table 02). souza et al. (2010) found 32 social wasp species at the apa são josé (21°05’00.77” s 44°10’03.70” w) in the central-southern region of minas gerais, an area mm souza, ag brunismann, ma clemente – social wasps fauna on different ecosystems460 with strong anthropogenic activity resulting in high incidence of fire. despite the human impact, this are produced the first state record for two species: polistes davillae, which was previously thought to occur only in mato grosso and amazonas states, and mischocyttarus ypiranguensis, which has been reported in são paulo state. another two species were reported for only the second time, including mischocyttarus marginatus, for which a single record exists for minas gerais found among the exemplar specimens deposited at the federal university of paraná (richards, 1978), and m. mirificus, which has a peculiar nesting pattern with colonies resembling roots hanging on rocky walls, which makes then highly inconspicuous. additional studies are needed to better elucidate the factors that influence such unique patterns of nest construction among social wasp species. souza et al. (2010) also reported that the in areas of campo rupestre (21°05’00.77” s 44°10’03.70” w) contain the largest number of vespid species in the country (also in campo rupestres), and suggest that conservation of these areas of campo rupestre is of paramount importance for the maintenance of social wasp diversity in brazil. clemente (2009) found 14 social wasp species in campos rupestres (21º40’00.67” s, 43º52’01.38” w). diversity was higher (h’=2.16) than in riparian forest (h’ = 1, 43) and atlantic forest (h ‘= 0.18) present in apa são josé. campos rupestres are composed of vegetation structure with a high diversity of grasses, herbs, and shrubs distributed along quartzite outcrops, and these areas are generally considered to be severe environments compared to riparian forest. however, wasp species with greater ecological tolerance such as protonectarina sylveirae saussure, 1854, polybia sericea, p. paulista, p. canadensis, p. ignobilis, apoica pallens, brachygastra. lecheguana, are generally dominant in open ecosystems such as campos rupestres (silva-pereira & santos, 2006). the only study of deciduous (dry) forest reported 35 species, six of which were exclusive (table 02) (brunismann, et al., 2016). dry forest occupies only 3.5% of the vegetation cover in minas gerais, and this ecosystem is unique to the state. the few studies carried out in dry forest revealed a rich fauna of insects (oliveira et al., 2011) and plants (sales et al. 2009). this foest type also hosts the highest number of social wasps genera in the state (n=14) ( brunismann et al. 2016), and, together with the rio doce state park (19º38’00.00” s e 42º31’00.00” w) presents the most distinct fauna in minas gerais, which makes this forest type a priority for the conservation of social vespids. mixed forests and high-altitude fields were evaluated by souza et al. (2015a) in the papagaio state park (22º12’18.22”s e 44º47’11.30”w), and yielded 21 and 19 species of social wasps, respectively, with one species exclusive to mixed forest (table 02). these richness values are the lowest reported among studies in the state. the high-altitude field ecosystem in souza et al. (2015a) is situated above 1,600 meters, and at this elevation abiotic factors may be quite different from those at other, lower-altitude sites. for example, low temperatures, among other factors, may negatively affect social wasp richness, as found in other studies of high-altitude fields (albuquerque et al., 2015). there is also a paucity of studies in this ecosystem type (only one), as well as in mixed forest, which was poorly sampled in terms of effort compared to the study in highaltitude fields (souza et al., 2015a). considering the complexity and diversity of ecosystems of the state of minas gerais, there is a strong need for additional studies of social wasp diversity. we suggest that ombrophilous and deciduous forests should be considered of high relevance for conservation of these social insects, especially sites within the rio doce state park (19º38’00.00” s e 42º31’00.00” w) and rio pandeiros wildlife refuge (15.50552°s, 044.75714°w). references auad, a. m., carvalho, c.a., clemente, m.a. & prezoto, f. 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(2013). social wasp-flower visiting guild interactions in less structurally complex habitats are more susceptible to local extinction. sociobiology, 60: 337-344. doi: 10.13102/ sociobiology.v60i3.337-344. clemente m. a, lange d., del–claro k, prezoto f., campos n. r., barbosa b. c.. 2012. flower-visiting social wasps http://dx.doi.org/10.5007/2175-7925.2015v28n4p131 sociobiology 64(4): 456-465 (december, 2017) 461 species of social wasps (109) ssf haf cr ce of srf cmf agelaia angulata (fabricius, 1804) 0 0 0 0 1 0 0 agelaia centralis (cameron, 1907) 0 0 0 1 1 0 0 agelaia myrmechophila (ducke, 1905) 0 0 0 0 1 0 0 agelaia multipicta (haliday, 1836) 1 1 1 1 0 1 1 agelaia pallipes (olivier, 1792) 0 0 0 1 0 1 0 agelaia vicina (de saussure, 1854) 1 1 1 1 1 1 1 total of 6 species 2 2 2 4 4 3 2 apoica gelida van der vecht 1972 1 0 0 1 0 1 0 apoica pallens (fabricius, 1804) 1 0 1 1 1 1 0 apoica thoracica dubuysson 1906 1 0 0 1 0 0 0 total of 3 species 3 0 1 3 1 2 0 brachygastra augusti (de saussure, 1854) 1 0 1 0 1 1 0 brachygastra lecheguana (latreille, 1804) 1 1 1 1 1 1 1 brachygastra moebiana (de saussure, 1867) 1 0 0 0 0 0 0 total of 3 species 3 1 2 1 2 2 1 chartergellus communis richards, 1978 1 0 0 1 0 0 0 total of 1 specie 1 0 0 1 0 0 0 chartergus globiventris de saussure, 1854 1 0 0 0 0 0 0 total of 1 specie 1 0 0 0 0 0 0 clypearia angustior ducke, 1906 1 0 0 0 1 1 0 total of 1 specie 1 0 0 0 1 1 0 epipona tatua (cuvier, 1797) 0 0 0 0 1 0 0 total of 1 specie 0 0 0 0 1 0 0 metapolybia cingulata (fabricius, 1804) 1 0 0 0 1 1 0 metapolybia docilis richards, 1978 0 0 0 0 0 1 0 total of 2 species 1 0 0 0 1 2 0 mischocyttarus artifex ducke, 1914 0 0 0 0 0 1 0 mischocyttarus annulatus richards, 1978 0 0 0 0 1 0 0 mischocyttarus atramentarius zikán, 1949 0 0 0 1 1 1 0 mischocyttarus bahiaensis zikán, 1949 0 0 0 0 1 0 0 mischocyttarus buysoni (ducke, 1906) 0* 0* 0* 0* 0* 0* 0* mischocyttarus cerberus ducke, 1898 1 0 0 1 0 1 0 mischocyttarus confusus zikán, 1935 0 0 0 1 0 1 0 mischocyttarus giffordi raw, 1985 0 0 0 0 0 0 1 mischocyttarus ignotus zikán, 1949 0 0 0 0 0 1 0 mischocyttarus ihering zikán, 1935 0 0 0 0 0 1 0 mischocyttarus mirificus zikán, 1935 0 0 1 0 0 1 0 mischocyttarus nomurae richards, 1978 1 0 0 0 0 0 0 mischocyttarus saussurei zikán, 1949 0 0 0 0 0 1 0 mischocyttarus tricolor richards, 1945 0 0 0 1 0 1 0 mischocyttarus ypiranguensis da fonseca, 1926 1 0 0 0 0 1 0 mischocyttarus bertonii (ducke) 1908 1 0 0 0 0 1 0 mischocyttarus flavoscutellatus zikán 1935 0 0 0 0 1 1 0 mischocyttarus frontalis (fox, 1898) 0 0 0 0 1 0 0 mischocyttarus funerulus zikán, 1949 0 0 0 0 0 1 0 mischocyttarus latior (fox, 1898) 0 0 1 1 0 1 0 table 02. species of social wasps recorded in different ecosystems: ssf (seciduous seasonal dry forest), haf – (high-altitude fields), cr (high-altitude fields), ce – (cerrado), of(high-altitude fields), srf – (seasonal semideciduous rain forest ), cmf – (complex mixed forest). (presence 1/absence 0). mm souza, ag brunismann, ma clemente – social wasps fauna on different ecosystems462 species of social wasps (109) ssf haf cr ce of srf cmf mischocyttarus drewseni de saussure, 1857 1 1 1 1 1 1 1 mischocyttarus labiatus (fabricius, 1804) 0 0 0 0 0 1 0 mischocyttarus mattogrossensis zikán, 1935 0 0 0 1 0 0 0 mischocyttarus rotundicollis (cameron, 1912) 1 1 1 1 0 1 1 mischocyttarus montei zikán 1949 1 0 0 0 0 0 0 mischocyttarus punctatus (ducke, 1904) 0 0 0 0 1 0 0 mischocyttarus araujoi zikán, 1949 0 0 0 0 0 1 0 mischocyttarus bahiae richards, 1945 0 0 0 0 1 0 0 mischocyttarus cassununga (r. von ihering, 1903) 1 1 1 1 0 1 1 mischocyttarus consimilis zikán, 1941 0 0 0 0 0 1 0 mischocyttarus fluminensis zikán, 1949 0 0 0 0 1 0 0 mischocyttarus marginatus (fox, 1898) 0 0 1 1 0 0 0 mischocyttarus mourei zikán, 1949 0 0 0 0 0 1 0 mischocyttarus nomurae richards, 1978 1 0 0 0 0 0 0 mischocyttarus paraguayensis zikán, 1935 0 0 0 0 0 1 0 mischocyttarus wagneri (du buysson, 1908) 0 0 1 0 1 1 0 mischocyttarus parallellogrammus zikán, 1935 0 0 0 0 0 1 0 total of 37 species 9 3 7 10 10 24 4 parachartergus fraternus (gribodo, 1892) 1 0 1 1 0 1 0 parachartergus pseudapicalis willink, 1959 0 0 0 1 0 0 0 parachartergus smithii (de saussure, 1854) 1 0 0 0 0 0 0 parachartergus wagneri du buysson, 1904 0 0 1 1 0 1 0 total of 4 species 2 0 2 3 0 2 0 polistes actaeon haliday, 1836 0 0 0 1 1 1 0 polistes billardieri fabricius, 1804 0 1 1 1 0 1 1 polistes canadensis (linnaeus, 1758) 0 0 0 0 1 0 0 polistes carnifex (fabricius, 1775) 0 0 0 0 1 0 0 polistes cavapytiformis richards, 1978 0 0 0 0 1 0 0 polistes cinerascens de saussure, 1854 0 1 1 1 0 1 1 polistes davillae richards, 1978 0 0 1 0 0 0 0 polistes ferreri de saussure, 1853 0 0 1 1 0 1 0 polistes geminatus fox, 1898 0 0 0 1 0 0 0 polistes goeldii ducke, 1904 0 0 0 1 0 1 0 polistes lanio (fabricius, 1775) 0 0 1 1 0 1 0 polistes melanossoma saussure, 1853 0 0 0 0 1 0 0 polistes occipitalis ducke, 1904 0 0 0 0 1 0 0 polistes pacificus pacificus fabricius, 1804 0 0 0 0 0 1 0 polistes pacificus flavopictus ducke, 1918 0 0 0 0 0 1 0 polistes satan bequaert, 1940 0 0 0 1 0 0 0 polistes simillimus zikán, 1948 1 1 1 1 0 1 1 polistes subsericeus de saussure, 1854 0 0 1 1 0 1 0 polistes versicolor (oliver, 1792) 1 0 1 1 1 1 0 total of 19 species 2 3 8 11 7 11 3 table 02. species of social wasps recorded in different ecosystems: ssf (seciduous seasonal dry forest), haf – (high-altitude fields), cr (high-altitude fields), ce – (cerrado), of(high-altitude fields), srf – (seasonal semideciduous rain forest ), cmf – (complex mixed forest). (presence 1/absence 0). (continuation) sociobiology 64(4): 456-465 (december, 2017) 463 species of social wasps (109) ssf haf cr ce of srf cmf polybia bifasciata de saussure, 1854 0 0 0 1 0 1 0 polybia brunnea (curtis, 1844)* 0* 0* 0* 0* 0* 0* 0* polybia emaciata (saussure, 1854)* 0* 0* 0* 0* 0* 0* 0* polybia signata ducke, 1910 0 0 0 0 1 0 0 polybia quadricincta de saussure, 1854 0 0 0 1 0 1 0 polybia jurinei de saussure, 1854 1 0 1 1 1 1 0 polybia dimidiata (olivier, 1792) 0 0 0 0 1 0 0 polybia rejecta (fabricius, 1798) 0 0 0 0 1 0 0 polybia bistriata (fabricius, 1804) 0 0 0 0 0 1 0 polybia erythrothorax richards, 1978 0 0 0 1 0 0 0 polybia fastidiosuscula de saussure, 1854 0 1 1 1 0 1 1 polybia flavifrons smith, 1857 0 0 0 1 0 0 0 polybia occidentalis (olivier, 1792) 1 0 1 1 1 1 0 polybia paulista h. von ihering, 1896 0 1 1 1 0 1 1 polybia platicephala richards, 1978 0 1 1 1 1 1 1 polybia ruficeps schrottky, 1902 1 0 0 1 0 0 0 polybia scutellaris (white, 1841) 0 1 1 1 0 1 1 polybia liliacea (fabricius, 1804) 0 0 0 0 1 1 0 polybia striata (fabricius, 1787) 0 0 0 1 1 0 0 polybia ignobilis (haliday, 1836) 1 1 1 1 1 1 1 polybia lugubris de saussure, 1854 0 0 0 0 0 1 0 polybia minarum ducke, 1906 0 1 1 1 0 1 1 polybia punctata dubuysson, 1908 1 1 1 0 0 1 1 polybia chrysothorax (lichtenstein, 1796) 0 1 1 1 1 1 1 polybia sericea (olivier, 1792) 1 1 1 1 1 1 1 total of 25 species 6 9 11 16 11 16 9 protonectarina silverae (de saussure) 1854 1 1 1 1 0 1 1 total of 1 specie 1 1 1 1 0 1 1 protopolybia exigua (de saussure, 1854) 1 0 0 0 0 1 0 protopolybia sedula (de saussure, 1854) 1 0 1 1 0 1 0 total of 2 species 2 0 1 1 0 2 0 pseudopolybia vespiceps (de saussure, 1863) 0 0 0 1 0 1 0 total of 1 specie 0 0 0 1 0 1 0 synoeca cyanea (fabricius, 1775) 0 0 0 1 1 1 1 synoeca surinama (linnaeus, 1767) 1 0 0 0 0 0 0 total of 2 species 1 0 0 1 1 1 1 total spp. 35 19 35 53 39 68 21 spp. exclusive 6 0 1 7 17 16 1 * species with occurrence registered in literature by without information by ecosystems table 02. species of social wasps recorded in different ecosystems: ssf (seciduous seasonal dry forest), haf – (high-altitude fields), cr (high-altitude fields), ce – (cerrado), of(high-altitude fields), srf – (seasonal semideciduous rain forest ), cmf – (complex mixed forest). 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(1994). comparing methods, adaptative traits and tests of adaptation. p. 79-101. in: p. eggleton; r. vane-wright (eds.) phylogeneticsand ecology. london, academic press. 616p. http://dx.doi.org/10.15560/10.1.10 doi: 10.13102/sociobiology.v61i1.60-67sociobiology 61(1): 60-67 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 colony performance of melipona quadrifasciata (hymenoptera, meliponina) in a greenhouse of lycopersicon esculentum (solanaceae) bf bartelli, aor santos, fh nogueira-ferreira introduction worldwide, there are approximately 20,000 described bee species (michener, 2007). among them, the stingless bees are highly social organisms that constitute an important group due to the ecological and economic roles they play. these bees belong to the subtribe meliponina, which consists of approximately 400 species grouped in about 50 genera, distributed across tropical and subtropical regions, in america, southeast asia, africa, madagascar and australia (silveira et al., 2002). stingless bees are responsible for the pollination of many native (michener, 2007) and cultivated (heard, 1999) plant species. as a result, since the 1990s, the number of studies involving the introduction of these bees in greenhouses to evaluate their pollination efficiency on different crops has grown steadily. particularly noteworthy among these are: eggplant pollination, using melipona quadrifasciata lepeletier, 1836 (bispo dos santos, 2008); basil pollination, tested with nannotrigona testaceicornis lepeletier, abstract the use of stingless bees in greenhouses has provided tremendous benefits to diverse crops in terms of productivity and fruit quality. however, knowledge about management techniques in these environments is still scarce. the present study aimed to evaluate colony performance of melipona quadrifasciata lepeletier, 1836 in a greenhouse of lycopersicon esculentum mill. and its potential use in pollinating this crop. six nests of m. quadrifasciata were introduced in a greenhouse in araguari, minas gerais state, brazil. the development of colonies inside the greenhouse was investigated and the foraging behavior of the workers was assessed before introduction, into the greenhouse and after the nests had been removed from the greenhouse. the vital activities of colony maintenance were performed unevenly throughout the day inside and outside the greenhouse, but with confinement the daily period of foraging decreased and bees started collecting pollen from the flowers after approximately six months. the difficulty in orienting to and identifying flowers by the workers was attributed to sunlight diffusion and blockage of ultraviolet radiation caused by the cover on the greenhouse. structural changes in the greenhouses, as well as improvements in management techniques, are required for better utilization of stingless bees for pollination of plant species grown in greenhouses. sociobiology an international journal on social insects universidade federal de uberlândia, instituto de biologia, uberlândia, minas gerais, brazil article history edited by: celso f martins, ufpb, brazil received 01 july 2013 initial acceptance 27 august 2013 final acceptance 29 october 2013 keywords stingless bees, management of bees, external activity, resource collection corresponding author fernanda helena nogueira-ferreira universidade federal de uberlândia instituto de biologia ceará st., umuarama uberlândia-mg, brazil, 38400-902 phone: (34) 3218-2243 fax: (34) 3218-2243 e-mail: ferferre@inbio.ufu.br 1836 (bispo dos santos, 2008); strawberry pollination, tested with tetragonisca angustula latreille, 1811 (malagodi-braga, 2002), scaptotrigona aff. depilis moure, 1942 (roselino et al., 2009) and n. testaceicornis (roselino et al., 2009); sweet pepper pollination, using m. subnitida ducke, 1910 (cruz et al., 2005), m. quadrifasciata anthidioides lepeletier, 1836 (roselino et al., 2010) and m. scutellaris latreille, 1811 (roselino et al., 2010); and tomato pollination, tested with n. perilampoides cresson, 1878 (cauich et al., 2004; palma et al., 2008b) and m. quadrifasciata (del sarto et al., 2005; bispo dos santos et al., 2009). tomato, lycopersicon esculentum mill. (solanaceae), is one of the most widespread vegetable crops in the world, being cultivated in almost all parts of the world under different cropping systems and various levels of management. it is a self-fertilizing plant and its flowers are bisexual, do not produce nectar and present poricidal anthers. therefore, in order to release the pollen, vibration of the anthers with consequent opening thereof is necessary (buchmann, 1983). pollination can be performed by the shaking of the anthers research article bees sociobiology 61(1): 60-67 (march, 2014) 61 by the wind, but cross-pollination is ensured through visits of bees that exhibit vibratory behavior or buzz-pollination (buchmann & hurley, 1978; heard, 1999; nunes-silva et al., 2010). tomato can be grown in open areas or in greenhouses. when grown in open areas, the released pollen is carried by the wind (mcgregor, 1976; free, 1993) and/or natural pollinators, especially bees, which have free access to the flowers. as for cultivation in greenhouses, pollination is normally performed by the mechanical method of vibrating the flowers, to compensate for the absence of wind and natural pollinators. however, studies involving the management of pollinators in greenhouses have indicated improvements in productivity and fruit quality (cruz & campos, 2009). among the pollinators of tomato in greenhouses, m. quadrifasciata proved to be efficient in pollinating the long-lived variety (del sarto et al., 2005), but studies testing colony performance and pollination efficiency of this bee on the grape variety, grown mostly in greenhouses, are yet to be performed. stingless bees are considered particularly promising for use as commercial pollinators (cruz & campos, 2009), given that they do not present a functional sting, are easy to handle (usually low aggressive-nonstinging), have populous and perennial nests, present a marked worker recruitment behavior and stock a large amount of food (heard, 1999; malagodi-braga et al., 2004). nevertheless, the lack of techniques for management and multiplication of nests has hindered the availability and use of these bees in agriculture on a large-scale (imperatrizfonseca et al., 2006). the present study sought to advance knowledge about management of stingless bees in greenhouses for use as commercial pollinators. we have specifically evaluated colony performance of m. quadrifasciata in a greenhouse of grape tomato, l. esculentum. material and methods study area the study was performed at the “meliponário da universidade federal de uberlândia (ufu)”, located at the “fazenda experimental do glória” (feg) (18°56’57”s 48°12’14”'w), uberlândia, minas gerais state, brazil, and in the “chácara paraíso” (cp) (18°39’3.55”s 48°11’7.51”w), located in the municipality of araguari, minas gerais state, brazil. at feg, agricultural and cattle raising activities are developed, but preserved fragments of cerrado and semideciduous forest can be found. cp consists of areas of cultivation and pasture, fragments of cerrado and 12 greenhouses where grape tomatoes are grown. one of these was used for the experiments. the climate of the regions of uberlândia and araguari is marked by two distinct seasons, the rainy season that extends from october to march and a dry season from april to september. annual rainfall varies between 1,160 and 1,460 mm/year and the average annual temperature is between 23 and 25°c, being uniform throughout the year (alves & rosa, 2008). the greenhouse used in the experiment comprised approximately 1,344 m2 (48 m x 28 m), being covered at the top with an extra long life (ell) diffuser antivirus plastic diffuser and fully enclosed on the sides with anti-aphid screens (fig 1a). the greenhouse presented 24 planting rows and each of these had an average of 112 tomato plants, adding up 2,688 plants. introduction of nests of m. quadrifasciata six nests of m. quadrifasciata with similar population sizes were introduced at the onset of flowering, in march 2012. the nests were kept in wooden boxes with an approximate size of 40x25x25cm (fig 1b). before introduction, due to the difficulty in bee orientation inside the greenhouse (bartelli, personal communication; del sarto, 2005), the population of old foragers was removed to avoid their loss in the enclosed space, following the methodology used by cauich et al. (2004). moreover, for the same reason, the nests were placed in the greenhouse after dark (cuypers, 1968). in order to allow a homogeneous distribution of the bees on the flowers, nests were arranged in the central region of the greenhouse (free, 1993) and supported by plastic boxes installed in eucalyptus logs located within the planting rows. to increase the number of reference points for the bees, since the uniformity of greenhouses may hinder orientation (dyer, 1994), the entrance to each nest was painted with different color patterns. containers with water, mud and cerumen (alternative source for plant resin) of t. angustula were placed on the plastic boxes (fig 1c). nests were sporadically fed with a. mellifera pollen macerated with sugar and water until the onset of flower visitation, and fed weekly with syrup (a mixture of honey of a. mellifera, sugar and water in the ratio 1:1:1) over the entire period of confinement. for pesticide application, a common management practice, the entrances of all nests were sealed with paper, the nests themselves were protected with plastic bags and the containers covered with cardboard boxes. these protections were removed only after the pesticide had dried; then, the entrances of the nests were unobstructed after dark. furthermore, to increase luminosity inside the greenhouse, another common management practice, the ceiling and sides of the greenhouse were washed with soap and water in late august. in order to facilitate bee orientation, one mercury vapour lamp and two mixed lamps (alternative sources of light and ultraviolet radiation) were installed inside the greenhouse in one of the corridors where some of the nests were placed (fig 1d). the lamps were connected to a digital timer, which was programmed to switch them on at 6 am and switch them bf bartelli et al. colony performance of m. quadrifasciata in greenhouse of l. esculentum62 off at 6 pm. they remained in the greenhouse from early june to mid-july. the lamps’ efficiency, as well as the influence of the diffuser film on solar radiation focused on the greenhouse, was evaluated by analysing the pattern of reflectance of the flowers. 10 flowers were photographed between 10 and 11 am in a white background with a sony cyber-shot dsc-h20 digital camera. each flower had its picture taken in three different circumstances: inside the greenhouse and next to the lamps; inside the greenhouse and 12m far from the lamps; and outside the greenhouse. photoshop software cs3 10.0 was used to quantify patterns of chromatic (rgb red, green and blue in the light spectrum) and achromatic (luminosity) saturation of flower corollas. colony performance of m. quadrifasciata to assess colony performance, the internal conditions of nests, behavior of workers in flight and on flowers, foraging activities and temperature inside and outside the greenhouse were analyzed. figure 2 outlines the arrangement of nests inside the greenhouse. nests d and f remained in the greenhouse for three months and then they were relocated to the outside area. internal conditions biweekly, from march to october 2012, we assessed qualitative features of nests, such as the presence of cells in construction (yes / no), number of workers (very low / low / medium / high) and presence of guards (yes / no). behavior of workers biweekly, from may to october 2012, the flight behavior of bees and the behavior of workers visiting tomato flowers were observed inside the greenhouse. these behaviors were assessed every 15 minutes during each hour (from 6 am to 6 pm). foraging activities foraging activities of bees were assessed through direct observations of the flow of workers from nests. the quantity and quality of resources (pollen, nectar/water or water, resin, mud and garbage) that entered and/or left the nests were recorded every 10 minutes during each hour (from 6 am to 6 pm). in order to compare the daily pattern of resource collection of workers inside and outside the greenhouse, the procedures described above were performed: before introduction in three nests (b, d and f, fig 2), over three non-consecutive days in late february 2012 at feg; inside the greenhouse in two nests (c and e, fig 2), biweekly from may to october 2012; and outside the greenhouse in one (nest f, fig 2) of the nests removed from the greenhouse at cp, biweekly from june to october 2012. temperature through the use of a digital thermohygrometer, the temperature inside and outside the greenhouse was measured every day at 8 am, 11 am and 3 pm from april to october 2012. statistical analyses to verify whether the patterns of reflectance of tomato flowers depended on the presence of the lamps and diffuser film or not, we conducted an analysis of variance (anova) followed by tukey's test, since there was no difference between blocks (flowers) for rgb (f = 1.118; df = 9; p = 0.399) and luminosity (f = 1.037; df = 9; p = 0.449) (zar, 2010). in order to evaluate the daily pattern of resource collection of bees inside and outside the greenhouse, circular analyses were performed using oriana 4.1 software (kovach, 2011), using the rayleigh test for the calculation of probabilities of distribution of bees throughout the day. to investigate whether the temperature differed inside and outside the greenhouse during the months the nests were confined, we conducted a paired t test (zar, 2010). fig 1. a) greenhouse used in the experiment at chácara paraíso in araguari; b) nest of melipona quadrifasciata installed inside the greenhouse; c) containers with water, mud and cerumen available inside the greenhouse; d) lamps installed inside the greenhouse. fig 2. diagram representing the arrangement of nests inside and outside the greenhouse. sociobiology 61(1): 60-67 (march, 2014) 63 results internal conditions two nests (a and b, fig 2) did not survive the conditions of confinement and died after about two months. to avoid their loss, after three months of confinement, two other nests (d and f, fig 2) were removed from the greenhouse and placed outside due to the absence of cells in construction, the low number of workers and the absence of guard workers. one of these nests (f, fig 2) survived and increased the number of workers to “high” after two months, but the other died (d, fig 2), attacked by phorids (diptera). nests c and e (fig 2) survived the conditions of confinement and remained in the greenhouse during the entire experiment, from march to october 2012, with cells in construction, guard workers and a high number of workers. behavior of workers inside the greenhouse, flight activities of bees began after 22 days of confinement. however, workers were limited only to the removal of garbage from the colonies and many of them clashed against the ceiling or sides of the greenhouse, where they remained and ended up dying. this flight behavior toward the ceiling and sides of the greenhouse decreased over time during confinement but did not cease, even after the beginning of collection activities in the flowers. the beginning of pollen collection from the tomato flowers occurred in late july, but there was only one record during that period and none in the following month. intensive visits of workers to flowers (six records of pollen collection per nest) occurred in early september, after nearly six months of confinement, and the ceiling and sides of the greenhouse had been washed with soap and water. bees landed on the anthers of a flower and bowed around or at the apex of the anthers cone to grab it (fig 3a). thus, they transmitted vibrations to the anthers through their thorax and legs to release the pollen. for the transfer of pollen from the body to the corbicula, some bees remained stuck to the base of the anthers cone by the legs and/or jaws, while others performed the transfer during flight or on other parts of the plant, such as the leaves and fruits (fig 3b). pattern of reflectance of the flowers the lamps installed inside the greenhouse did not alter the flight behavior of the bees and did not influence the rgb and luminosity of tomato flower corollas. however, the patterns of chromatic (f = 10.51; df = 2; p< 0.001) and achromatic (f = 8.44; df = 2; p = 0.001) saturation of the flowers were significantly different inside and outside the greenhouse (fig 4). foraging activities before introduction, at fazenda experimental do glória (feg), the bees began to forage at around 5:40 am. the number of forager workers coming in and out of nests was not uniform throughout the day and the period of highest activity occurred at 6-8 am. the relative frequency of collection per resource varied considerably between the nests. of the workers observed, 7.7-18.2% transported pollen, 17.0-81.4% nectar/ water, 3.6-55.7% resin and 0.2-11.1% mud. the resources were not obtained uniformly throughout the day. pollen collection occurred only in the morning, with a peak at 6-8 am. the highest mean frequency observed for nectar/water occurred between 6 and 7 am and the collection of this resource gradually decreased throughout the day. the peaks of collection of resin and mud were, respectively, at 8-10 am and at 8-11 am (fig 5, table 1). fig 3. a) melipona quadrifasciata worker collecting pollen in tomato flower; b) m. quadrifasciata worker transferring pollen from the body to corbicula on a fruit. fig 4. mean values (± standard error) of patterns of chromatic (or rgb a) and achromatic (or luminosity b) saturation of tomato flowers in the different treatments (near: inside the greenhouse and next to the lamps; far: inside the greenhouse and far from the lamps; and outside: outside the greenhouse) at chácara paraíso in araguari. distinct letters indicate significant differences between treatments. bf bartelli et al. colony performance of m. quadrifasciata in greenhouse of l. esculentum64 inside the greenhouse, the activity of bees began approximately at 7 am and was not uniform throughout the day, with the greatest movement of workers occurring between 5 and 6 pm for nest c, and between 12 and 1 pm for nest e. the relative frequency of collection per resource varied between the nests. from the workers observed over the entire period of confinement, 14.8-21.4% transported pollen, 37.5-60.7% water and 7.8-23.3% resin (cerumen). there was no collection of mud. workers obtained pollen and water in a heterogeneous way throughout the day and there was no defined peak of resin collection. pollen collection was limited to the morning, with a peak between 8 and 9 am. the highest mean frequency observed for water occurred between 5 and 6 pm (fig 5, table 1). after the nests were removed from the greenhouse and placed in the outer area, it was possible to observe workers carrying pollen in their corbiculas on the following day. the movement of bees was not uniform throughout the day and peak activity occurred between 6 and 7 am. of workers observed over the months, 31.4% carried pollen, 57.5% nectar/water and 7.0% resin. the resources were obtained in a heterogeneous way throughout the day, except resin. pollen collection, limited again to the morning, and nectar/water collection had peaks between 7 and 8 am, and between 6 and 7 am, respectively. there was no collection of mud (fig 5, table 1). temperature the temperature inside the greenhouse was significantly higher over the entire period of confinement of the nests of m. quadrifasciata (t = 6.99; df = 6; p< 0.001; fig 6). discussion the results of our experiments showed that acclimation of melipona quadrifasciata to conditions inside greenhouses are colony-dependent and, besides foraging activities varied a little inside and outside the greenhouse, the daily period of foraging into the greenhouse decreased and bees took a long time to visit flowers consistently for pollen collection. the foraging behavior of stingless bees is related both to factors intrinsic to the nest, including the ability to communicate and population size, and extrinsic factors, such as the abundance and distribution of resources in the environment and susceptibility to abiotic factors (fidalgo & kleinert, 2007). however, despite the methodological differences used in the introduction of the nests and the different conditions of confinement (size and structure of the greenhouses, crop type, etc), our results about the foraging behavior of m. quadrifasciata were similar to results found in other studies that evaluated the introduction of stingless bees in greenhouses (del sarto et al., 2005; nunes-silva et al., 2013). the workers concentrated pollen collection in the morning. inside the greenhouse, the foraging behavior of m. quadrifasciata showed little variation compared to the outside. however, stronger patterns (represented by smaller probability values, table 1) in the external activities of bees were observed at feg, before confinement. additionally, the daily period of foraging in the greenhouse decreased, as well as for n. perilampoides (cauich et al., 2004; palma et al., 2008a), and bees visited flowers consistently for pollen collection only after approximately six months of confinement. the time required for acclimation to protected environments table 1. mean time vector (with the number of observations (n) and probability values (p), according to rayleigh test) for resources collection and total external activity throughout the day by melipona quadrifasciata at fazenda experimental do glória (feg), in uberlândia, and inside and outside the greenhouse at chácara paraíso, in araguari. resource feg inside outside nest b nest d nest f nest c nest e nest f pollen 7:56 am (n = 31) (p < 0.001) 6:51 am (n = 60) (p < 0.001) 6:44 am (n = 67) (p < 0.001) 7:40 am (n = 3) (p = 0.036) 8:10 am (n = 6) (p < 0.001) 7:29 am (n = 10) (p < 0.001) nectar/water or water 7:18 am (n = 329) (p < 0.001) 7:44 am (n = 79) (p < 0.001) 7:52 am (n = 117) (p < 0.001) 4:00 pm (n = 3) (p = 0.042) 2:45 pm (n = 6) (p = 0.011) 7:33 am (n = 19) (p < 0.001) resin 10:41 am (n = 15) (p < 0.001) 9:50 am (n = 259) (p < 0.001) 9:39 am (n = 136) (p < 0.001) ----11:30 am (n = 2) (p = 0.144) 6:30 am (n = 2) (p = 0.144) mud 9:00 am (n = 1) (p = 0.512) 9:42 am (n = 50) (p < 0.001) 10:38 am (n = 23) (p < 0.001) ------------total external activity 7:32 am (n = 801) (p < 0.001) 9:00 am (n = 910) (p < 0.001) 8:36 am (n = 719) (p < 0.001) 11:43 am (n = 17) (p < 0.001) 11:28 am (n = 39) (p < 0.001) 7:45 am (n = 62) (p < 0.001) (-----) insufficient data for analysis or resource not collected sociobiology 61(1): 60-67 (march, 2014) 65 varies both between species and between colonies of the same species (malagodi-braga, 2002). however, this timing was probably determined by the presence of the diffuser film on the coverage of the greenhouse. as evidence of this, pollen collection observed in nests placed in the outer area started immediately after their removal from the greenhouse. due to the dispersant effect of plastic films used in greenhouses coverage, solar radiation is one of many environmental factors that can be changed by using protected crops (schwengber et al., 1996). this could be evidenced via the different reflectance patterns found on tomato flowers inside and outside the studied greenhouse. this dispersant effect is favorable to plants, since the fraction of diffuse solar radiation is more effective for photosynthesis (farias et al., 1993), but may have hindered bee orientation and identification of flowers by worker bees, affecting foraging activities inside the greenhouse and thereby delaying the beginning of pollen collection. in relation to the external environment, global solar radiation (measured by luminous flux density) and diffuse solar radiation (multidirectional) are respectively lower and higher inside the greenhouse as a result of reflection and absorption by the material of the plastic coverage. in turn, this reflection and absorption are determined, for example, by the conditions of coverage at the time of use and dust deposition (farias et al., 1993). this explains why visits to flowers by m. quadrifasciata workers intensified when the ceiling and sides of the greenhouse were washed with soap and water as part of the mana-gement of the protected tomato crop. for orientation, bees use elements like the sun, polarized light, visual cues present in the environment and ultraviolet radiation (kerr, 1973; dyer, 1994; briscoe & chittka, 2001). the presence of clouds and fog cause dispersion of light rays and reduces the polarization signal (shashar & cronin, 1998). thus, greenhouse cover can produce a similar effect to that promoted by clouds and restrict bee activity to a period in which light rays are less dispersed by the cover (malagodibraga, 2002). besides the dispersant effect, the film ell diffuser antivirus eliminates the entry of ultraviolet (uv) to hinder the vision of tomato pest insects (electro plastic, 2013). this could also have been an aggravating factor hindering the orientation of m. quadrifasciata forager workers inside the greenhouse. although this species present a wide distribution throughout brazil (camargo & pedro, 2012), foraging in places relatively poor in uv, such as under the canopies of dense tropical forests (briscoe & chittka, 2001), the nests were in an open environment at feg and were accustomed to high uv exposure before introduction to the greenhouse. in temperate areas, where bumble bees are largely used for greenhouse tomato pollination, showing high efficiency (banda & paxton, 1991; morandin et al., 2001), the structure of the greenhouses are similar to those used in this study. however, in the case of bumblebees, it seems that the reduction in uv light can be compensated and as a result their visit to flowers is not affected by the type of greenhouse coverage (dyer & chittka, 2004). in the present study, as in del sarto et al. (2005), the performance of m. quadrifasciata to conditions of confinefig 5. mean number of workers for each resource collected throughout the day by melipona quadrifasciata before introduction (a), at fazenda experimental do glória, in uberlândia, and inside (b) and outside (c) the greenhouse at chácara paraíso, in araguari. fig 6. mean values (± standard error) of temperature (°c) inside and outside (environment) the greenhouse during the period of confinement of nests of melipona quadrifasciata at chácara paraíso in araguari. distinct letters indicate significant differences for each month. bf bartelli et al. colony performance of m. quadrifasciata in greenhouse of l. esculentum66 ment proved to be colony-dependent. studies have shown that stingless are tolerant to high temperatures and capable of cooling the inside of their colony by ventilation generated by beating their wings in the nest entrance. however, we believe that the high temperatures inside the greenhouse may have been an aggravating factor for colony development, since the intense heat decreases the density of larval food, causing the eggs to sink and death of larvae by drowning (amano et al., 2000). from results obtained in this study and considering the benefits that the use of stingless bees has provided to diverse crops grown in greenhouses (cruz & campos, 2009), the control of temperature and humidity inside greenhouses would be an important step. another idea would be to exchange the plastic greenhouse coverings for materials that interfere less significantly in solar radiation, or, alternatively, a less drastic alternative would be to intercalate the plastic cover with such materials, allowing at least partial diffusion of solar radiation to occur in the greenhouse. for the farmer, taking into account the duration of the cycle of grape tomato in greenhouses, which is eight months, using stingless bees is not practical if these bees require a great deal of time to acclimate and initiate foraging activities. however, studies asses-sing the cost-benefit relationship of such structural changes in greenhouses must still be made and more information that will permit improvements in management techniques for stingless bees in greenhouses is needed. acknowledgements the authors are grateful to conselho nacional de desenvolvimento científico e tecnológico (cnpq), to fundação de amparo à pesquisa do estado de minas gerais (fapemig) and to coordenação de aperfeiçoamento de pessoal de nível superior (capes) for financial support, to the farmers edson, clóvis and joão, who allowed and gave full support to this study, to dr. paulo eugênio alves macedo de oliveira for suggestions given to this study and to the biologists isabel farias aidar and jaqueline eterna batista for contributions in the field. references alves, k.a. & rosa, r. 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(2010). biostatistical analysis fifth edition. new jersey: prentice-hall/pearson. doi: 10.13102/sociobiology.v63i2.1071sociobiology 63(2): 826-830 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 temporal variation of membracidae (hemiptera: auchenorrhyncha) composition in areas of caatinga with different vegetation structures introduction the caatinga, an exclusively brazilian biome, is one of the largest seasonal savannas in the world, comprising about 800,000 km2, and is represented by multiple types of forests, whose plants exhibit many structures adapted to the predominate climatic conditions (sampaio, 1995; prado, 2003). the caatinga is among the most degraded and least studied ecosystems in brazil. the causes of degradation are numerous; however, the most common include exploitation of wood for fuel and substitution of native vegetation with agricultural practices that have been inappropriate for hundreds of years (velloso et al., 2002). only about 30% of the caatinga is considered a non-altered region, and this area is a sum of islands dispersed throughout the interior of the biome (mma, 2002). striking weather patterns between seasons, mainly influenced by changes in rainfall and humidity, determine strong seasonality in the caatinga. rainfall varies from 240 abstract the present study investigated the diversity of membracids in different caatinga vegetation structures (preserved, intermediate and degraded) during dry and rainy seasons in 2006. we recorded 1,107 individuals, belonging to 13 species, mostly during the rainy season (693). melusinella nervosa, enchenopa gracilis and e. eunicea were the most abundant species, although this pattern varied in the three areas. m. nervosa and e. gracilis were the most abundant during the rainy and dry seasons, respectively. thrasymedes pallescens was the species least affected by seasonality, with 51.3% and 48.7% of the specimens collected in the rainy and dry seasons, respectively. a cluster analysis showed that membracids from preserved areas in the dry season were more related to the ones from rainy season. seasonality of membracids seems to be related to seasonal ecology and phenology of their host plants. furthermore, preserved areas are very important for maintenance of membracids diversity during the dry season. sociobiology an international journal on social insects aj creão-duarte1, mim hernández2, rrad rothéa1, we santos1 article history edited by kleber del-claro, ufu, brazil received 17 may 2016 initial acceptance 06 june 2016 final acceptance 10 june 2016 publication date 15 july 2016 keywords caatinga, host plants, treehoppers. corresponding author antonio josé creão-duarte programa de pós-graduação em ciências biológicas (zoologia) departamento de sistemática e ecologia universidade federal da paraíba – ufpb 58059-900, joão pessoa-pb, brazil e-mail: creaoduarte@yahoo.com.br mm to 1,500 mm with 50 to 70% of the total rain concentrated in three consecutive months (prado, 2003). during the rainy season there is a substantial increase in plant biomass that provides resources for many different groups of insects such as the hemiptera-auchenorrhyncha, which become abundant under these conditions (vasconcellos et al., 2010). phytophagous insects form bonds with their host plants (using them as site for reproduction, egg laying and food source) which are particularly vulnerable to fluctuations in variables such as humidity, nutrient availability, and plant cycle (lawton, 1983). changes in insect abundance indicate an increase or decrease in its primary resources or an alteration in habitat, or even in its natural enemies (brown, 1997). faunal diversity studies in the tropics and particularly for insects in the caatinga have indicated that taxa reach higher indicators of diversity in environments with more complex vegetation structure (lawton, 1983; leal, 2003). insect communities respond in different manners when inhabiting gradients of different proportions of 1 universidade federal da paraíba (ufpb), joão pessoa-pb, brazil 2 universidade federal de santa catarina (ufsc), florianópolis-sc, brazil research article social arthropods sociobiology 63(2): 826-830 (june, 2016) 827 pioneer plants, different physiognomies of the same vegetation and/or different special verticality limits (humphrey et al., 1999; leal, 2003). membracids are phytophagous insects found on the softer parts of plants, and parts with the most exposure to sunlight, such as branches, inflorescences and fruits (wood, 1993). from these parts, they suck plant sap and release honeydew, which attracts ants, with which they establish biological associations (moreira & del-claro, 2005; fagundes et al., 2013). they have varying degrees of host selectivity (creão-duarte et al., 2012), and some species have gregarious habits, and may form colonies with a significant number of nymphs and adults (wood, 1993). however, in general, in most degraded environments there are changes in abundance status, marked by strong dominance of a few species (lopes, 1995; creão-duarte et al., 2012). neotropical membracids have been predominantly studied from the taxonomic perspective and from the perspective of hymenopteras’ mutualistic relationships (moreira & del-claro, 2005; fagundes et al., 2013). however, in studies surveying entomofauna they are always present (yamamoto & gravena, 2000; hickel et al., 2001; favretto et al., 2013). colonial species are less active and easy to collect, their life cycles are closely related to the host plant, and they have a stable classification. these conditions make them potentially useful for conservation and monitoring of neotropical forests (wood, 1993; brown, 1997). in this context, the present study investigated the diversity and abundance exhibited by membracid assemblies in different types of semi-arid vegetation structures in the caatinga, during the dry and rainy seasons. we asked: (1) do membracids seasonally explore host plants in this environment? (2) how abundant and diverse were the membracids throughout preserved, intermediate and degraded areas? material and methods this study was conducted in the private reserve for the environmental inheritance “fazenda almas”, with an area of 3,500 ha in the municipality of são josé dos cordeiros, paraíba, brazil (7°28’45” s; 36°54’18” w). the reserve is located in the ecoregion “depressão sertaneja setentrional”, one of the areas most impacted by anthropic actions and characterized by irregular rainfall (historical average of 350 mm/year in the “cariri paraibano”) (velloso et al., 2002). the vegetation varies from open to dense arboreal caatinga, with a strong deciduous characteristic during the dry season. the soil is sandy and arid with irregular topography, with inselbergs and rocky outcrops (vasconcellos et al., 2010). three vegetation structures (preserved, intermediate and degraded) were defined for insect collection with bases on a phytosociological analysis of three 100 m2 plots located nearly 1 km from each other. preserved: n of trees = 23; height = 20.3±12.5 m; height at beginning of canopy = 5.2±1.8 m and canopy diameter = 2.7±1.2 m. intermediate: n of trees = 5.5; height = 12.4±5.4 m; height at beginning of canopy = 3.4±1.9 m and canopy diameter = 2.9±2.1 m. degraded: n of trees = 4.5; height = 2.8±0.3 m; height at beginning of canopy = 2.8±0.3 m and canopy diameter = 1.5±0.3 m. two collections were conducted within each vegetation structure, one during the rainy period (april 2006) and the other during the dry period (september 2006), with a significant variation in rainfall (fig 1), totaling six collections. the method of active collection (both sweeping and direct collection into collection bottle) was used to capture adults. random walking and observation of host plants parts (leaves, branches, inflorescences and fruits) were conducted for 6 hours per area during each period (rainy and dry), totaling 36 hours of observation. for species already represented within the collections we only registered the species and number of individuals. the collected membracids were incorporated into the entomological collection of the department of ecology and systematics (dsec) of the federal university of paraíba (ufpb). the cluster analysis calculates the abundance data for each species per area per period using upgma method, as well as bray-curtis similarity index, with the software past (hammer et al., 2001). fig 1. monthly rainfall variation (mm) and relative membracid abundance (%) collected in são josé dos cordeiros, paraíba in 2006 (source: aesa). results a total of 1,107 membracids were recorded, distributed into 11 genera and 13 species (table 1). melusinella nervosa (fairmaire, 1846) (43.18%), enchenopa gracilis (germar, 1821) (24.84%) and enchenopa euniceae rothéa & creão-duarte, 2006 (22.04%) were the most abundant species, while the other species together amounted to only 9.94% of the total (table 1, fig 1). the number of individuals collected during the rainy season (n=693 from 10 species) was 67.4% greater than the number of individuals collected during the dry period (n=414 from 8 species). m. nervosa and e. gracilis showed strong seasonality, the first more present during the rainy season and the second during the dry season. trasymedes pallescens (stål, 1896) was the species less subjected to seasonality with 51% and 49% of individuals collected during the rainy and dry period, respectively. e. euniceae was well represented in almost all aj creão-duarte – membracidae composition in caatinga828 areas during both seasons; the exception was in the degraded area during the dry season. melusinella nervosa was found in large quantities in the three vegetation profiles during the rainy season. on the other hand, e. gracilis was associated in large quantities only to intermediate and degraded areas during the dry season. according the cluster analysis, during the rainy season membracids from different vegetation structures are relatively similar (more than 60% similarity); on the other hand, during the dry season they are different, where the degraded and intermediate areas during this period have a very low similarity when compared with others (fig 2). discussion the seasonality of m. nervosa and e. gracilis was due to seasonal ecology and phenology of the species primary host plants, respectively, sida galheirensis ulbr. and indigofera suffruticosa mill. (creão-duarte et al., 2012), but also probably due to the reproductive cycle of the membracids (wallace & maloney, 2010). on the other hand, t. pallescens was the species less subjected because it is found exclusively on caesalpinia pyramidalis tul., arboreal plant that can remain green during periods of less rigorous droughts (creão-duarte et al., 2012). similarly, e. euniceae was well represented in both seasons due to the perenniality of its main host plants mimosa tenuiflora (wild.) poir. and mimosa ophthalmocentra mart. ex benth. (creão-duarte et al., 2012). the increase in plant biomass during the rainy season represents an increase in resources for many insects in the caatinga (vasconcellos et al., 2010). during these periods, plant diversity in the intermediate and degraded areas increases with the emergence of numerous herbaceous species within the open spaces (reis et al., 2006; santos et al., 2013) and, thus, increases the resource supply to be exploited by the membracid community. the effects of seasonality on entomofauna, principally rainfall and humidity as the main predictors of abundance has already been observed in isoptera (moura et al., 2006), diptera (alves et al., 2014), coleoptera (santos et al., 2014a), hymenoptera (santos et al. 2014b), lepidoptera (gusmão & creão-duarte, 2004) and other orders (vasconcellos et al., 2010). however, for membracids this pattern has not been directly observed since they heavily rely on the cycle of their host plants (wood, 1993; creão-duarte et al., 2012). the cluster analysis allows us to deduce that the climate is primarily responsible for the difference between the fig 2. similarity between areas/periods in function of the collected membracids in são josé dos cordeiros, paraíba in 2006 (area: p, preserved; i, intermediate; d, degraded; period: r, rainy; d, dry). species rainy period dry period total p i d total p i d total n % melusinella nervosa 167 234 77 478 478 43.18 enchenopa gracilis 1 1 3 150 121 274 275 24.84 enchenopa euniceae 11 117 32 160 31 51 2 84 244 22.04 thrasymedes pallescens 20 13 6 39 37 37 76 6.87 enchenopa minuta 2 2 4 11 15 17 1.54 sundarion flavum 2 3 5 1 1 6 0.54 erosne parvula 2 2 4 4 0.36 micrutalis binaria 2 2 2 0.18 ceresa ustulata 1 1 1 0.09 darnis olivacea 1 1 1 0.09 tolania sp. 1 1 1 0.09 amastris sp. 1 1 1 0.09 phylia sp. 1 1 1 0.09 individuals/area 204 367 122 693 77 212 125 414 1,107 100 species/area 7 5 6 10 6 3 4 8 table 1. membracids collected in three areas with different vegetation structures in the caatinga (p, preserved; i, intermediate; d, degraded) during the rainy and dry periods of 2006 in são josé dos cordeiros, paraíba, brazil. sociobiology 63(2): 826-830 (june, 2016) 829 assemblies of the various areas. however, it is interesting to note that the climate did not affect the preserved area as much, since membracids in this area during the dry season appear more related to those of the rainy season. thus, underscoring the importance of preserved areas for the conservation of membracid species (especially t. pallescens) during the dry period, which is a limiting factor for those assemblies in the semiarid region. during the dry season plants from intermediate and degraded areas suffer climate effects earlier, since the vegetation structure is composed mostly of herbaceous plants that soon lose their leaves and cease to house their guests, membracids. where as in the preserved areas plants are larger and take longer to relay the effects of the drought, and thus can accommodate membracids for a longer period of time (lima & rodal, 2010; creão-duarte et al., 2012). brown (1997) includes the membracidae in the group of insects that are potential indicators for conservation and monitoring, in order to promote sustainable use of tropical forests due to the strong association of membracids with their host plants (wood, 1993). these floras respond to environmental disturbances (andrade et al., 2005), as well as climate change (reis et al., 2006; lima & rodal, 2010). diversity analysis of these insects, as observed, can provide answers strongly related to the environment in the brazilian semiarid region. in this context, e. gracilis can be a good indicator of disturbance, since its main host plant, i. suffruticosa, is an invasive species with little demands that sprouts in any type of soil, and may also be common in dry and disturbed areas in northeastern brazil (salvador et al., 2010; creão-duarte et al., 2012). in conclusion, seasonality of membracids in caatinga seems to be closely related to seasonal life cycle of their host plants. furthermore, preserved areas kept a great richness during the dry season, highlighting the importance of these areas for maintenance of diversity of membracids. acknowledgements we are also grateful to dr. kleber del-claro (ufu) and to the anonymous reviewers for their comments on the manuscript. this study received support from the programa ecológico de longa duração (peld/caatinga) and the coordenação de aperfeiçoamento de pessoal de nível superior (capes). references alves, a.c.f., santos, w.e., farias, r.a.c.p. & creão-duarte, a.j. 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(2000). espécies e abundância de cigarrinhas e psilídeos (homoptera) em pomares cítricos. anais da sociedade entomológica do brasil, 29: 169-176. doi: 10.1590/s0301-80592000000100021. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.289-294sociobiology 60(3): 289-294 (2013) melittopalynology and trophic niche analysis of apis cerana and apis mellifera in yunnan province of southwest china yj liu1, 2, tr zhao1, xw zhang3, c liang3, fy zhao1 introduction honey can be produced from the nectar of plants (nectar honey), from secretions of living parts of plants or from the excretion of plant sucking insects (honeydew honey). however, honey analysis was focus on nectar honeys and identified the nectar plants in this region through the analysis of pollen of nectar honeys (caccavari, m. et al. 2010). this research method is generally recognized (behm et al., 1996). bees are generally regarded as beneficial insects for their role in pollination (goulson, 2003). so the species of honeybee apis mellifera linnaeus was introduced into china 100 years ago for their good production of honey. apis cerana fabricius is the most widely distributed native bee species before apis mellifera been introduced. however, its colonies become less and less in china now. apis cerana and apis mellifera are two different species; they have many differences in their body structure and habits. the average length of worker bees body of apis mellifera is 12.0 ~ 14.0 mm, and abstract qualitative melittopalynological data of 34 honey samples obtained from apis cerana and apis mellifera between 2011 and 2012 in mengzi (yunnan, china) are analyzed. and trophic niche of the two bee species on pollen food resources are also investigated in the present study. results show that a diverse spectrum of 17 pollen types was identified into 14 families, and the asteraceae and myrtaceae are the most frequent at non-nectar flow and the lythraceae are most frequent at main nectar flow of pomegranate in our study area. as for trophic niche analysis of apis cerana and apis mellifera, the niche breadth value is 0.65 and 0.57 at non-nectar flow and 0.41 and 0.24 at nectar flow, respectively. meanwhile, the value of trophic niche overlap index at nectar flow (0.68) is greater than that of non-nectar flow (0.61). and the value of interspecific competition index at nectar flow (0.92) is greater than non-nectar flow (0.77), too. these results may promote the development of local beekeeping and provide some reference for scholars to assess the influence of introducing exotic bees on chinese native bees. sociobiology an international journal on social insects 1 kunming university of science and technology, kunming, pr china. 2 south china agricultural university, guangzhou, pr china. 3 yunnan academy of agricultural sciences, mengzi, pr china. article history edited by kleber del-claro, ufu, brazil received 21 june 2013 initial acceptance 18 july 2013 final acceptance 07 august 2013 key words melittopalynology, apis cerana, apis mellifera, trophic niche analysis corresponding author feng-yun zhao college of chemistry and engineering kunming university of science and technology, kunming 650500 people’s republic of china e-mail: zhaofy@kmust.edu.cn that of apis cerana is 9.5 ~ 13.0 mm. they normally display different strategies in cooling hive temperature (yang et al., 2010) and guarding against invading bee viruses (sharm & dharam, 2005; ai et al., 2012) or parasitic mites (peng et al., 1987). different bee species show their preference for different plants (ramírez-arriaga & navarro-calvo, 2011), resulting in a variety of pollen types and special proportions of them in honeys. bees play a very important role in balancing the local ecosystem, especially for the pollination and reproduction of many plant groups (larkin et al., 2008). pollen analysis can get an effective assessment on ecological impacts of invasive bee species on native bees (stout & morales, 2009). the use of trophic niche analysis methods can effectively assess the impact of bees on the local ecological environment (santos & absy, 2010). in this research, we study the pollen types and proportion in samples of honey from two bee species, apis cerana and apis mellifera, at non-nectar flow and nectar flow two research article bees yj liu, tr zhao, xw zhang, c liang, fy zhao melittopalynology and trophic niche analysis290 periods, to assess the impact of introduced bees on native bees and compare the pollen date and analyze their trophic niche as well. materials and methods study area honey samples were collected from different colonies of apis cerana (n=17) and apis mellifera (n=17) between 2011 and 2012. different colonies were placed at two different apiaries (~300 m apart) around the city of mengzi (23°31′n;103°25′e). the region is located in the southeast of yunnan province, china. the climate is subtropical plateau monsoon, with an annual rainfall of 857 mm and an annual temperatures of 18.6. the altitude of apiariy is about 1288 m and the sunshine is sufficient with an annual 2234 hours. honey preparation and melissopalynological analysis honey samples produced by bees in different months and different main nectar flow were provided by beekeepers and centrifuged at first. the collecting time of honey samples is shown in table 1. qualitative melissopalynological analysis were conducted by the methods reported by louveaux et al. (1978) with slightly modification. in brief, 20 g of honey samples were dissolved in 40 ml distilled water and centrifuged (10 minutes, 4000 r/min). the supernatant was disposed and the residue washed again with 20 ml water. pollen sediment was mounted in glycerine-gelatine and sealed with paraffin to determine frequency classes by microscope. pollen types were trophic niche analysis niche is a functional relationship between a species and other population at the same time and space in the ecosystem. in a biome, niche overlap is a phenomenon of multiple species to feed on the same food, and the resulting competition between different species and increasing competition while having scarce food. in this paper, set up a framework to think about and estimate the difference between apis cerana and apis mellifera in terms of ecological niche. the ecological parameters were calculated using a niche breadth, trophic niche overlap index and interspecific competition index. niche breadth (b) were calculated by the equation (levins, 1968): (1) where b is niche breadth, s is the level of resource, pi is the proportion of the species take advantage of i-th level resources accounted in total resources. trophic niche overlap index (c) were calculated by the equation (colwell & futuyma, 1971): (2) where cij is the trophic niche overlap index and cij = cji, pjh and pjh are the proportion of the species i and species j take advantage of h resource sequence accounted in total resources, s is the grade of the resource sequence. interspecific competition index (a) were calculated by the equation (southwood, 1978): (3) where a is the interspecific competition index of species i and species j in a same resource. pjh and pjh are the proportion of the species i and species j in each resource sequence. interspecific competition is not fierce in the resource utilization when α≥1 and competition is fierce when α<1. statistical analysis for descriptive purposes, simple statistical analysis, was applied into our study using spss 17.0. the arithmetic means and standard deviations were obtained for variables measured in the present study. the pollen types number of honey samples from two kinds of bee species were compared by one-way analysis of variance (anova) and lsd for multiple comparison tests (p>0.05). data were assessed, such that statistical significance was based on p<0.05. table 1 serial number of honey samples from apis cerana and apis mellifera at different main nectar flow. identified by comparing them with a reference collection that was obtained from the plants grown in the area surrounding the beehives. pollen atlases of china woody plants pollen photo using scanning electron microscopy (2011) and nectariferous plant of china (1992) were also consulted. the pollen types were classified, according to frequency, into four categories: predominant pollen (≥ 45%), secondary pollen (16–45%), important minor pollen (3–15%) and minor pollen (≤3%) (louveaux et al., 1978). the frequency occurrence of pollen, expressed as a percentage, was calculated per melliferous area. jul/2011 (non-necta r flow) apr/2012 (necta r flow) a.cerana no.1-9 no.19-26 a. mellifera no.10-18 no.27-34 sociobiology 60(3): 289-294 (2013) 291 results honeys were sampled on july 2011(n=18) and april 2012(n=16). at july, there is not any nectar flow of plant in study area. april is nectar flow of punica granatum and bee activity for honey production. thus two different periods, covering the periods of non-nectar flow and main nectar flow of one plant, were representative of the two different species in trophic niche analysis of the study area. honey produced by apis cerana and apis mellifera in non-nectar flow showed no significant difference (p>0.05). honey from apis cerana and apis mellifera showed a significant difference in nectar flow of pomegranate flowers (p<0.05), and contained 8.13±1.46 and 6.00±1.77 (mean value ± standard deviation) pollen types, respectively. the number of pollen types are show in table 2. ta of myrtaceae is the most represented by apis cerana, the next is bidens pilosa. in nectar flow, the punica granatum of lythraceae is the main pollen in all samples from apis mellifera and apis cerana, the next is bidens pilosa (apis cerana) and eucalyptus robusta (apis mellifera). as for the percentage of honey samples produced by apis cerana and apis mellifera in different months (fig 1), the distribution of various pollen types percentage is balanced at non-nectar flow, the range of percentage (except minor pollen) is from 11.83% to 18.34% by apis cerana and from 5.61% to 35.91% by apis mellifera. in study areas, as soon as the pomegranate began flowering at the end of march, bees started to visit pomegranate flowers, thereby producing the pomegranate honey. at the nectar flow of punica granatum, the percentage of punica granatum pollen becomes important.however, there is still some other pollen types, like bidens pilosa (14.59%) and pisum sativum (12.43%) of fabaceae sampled by apis cerana, like eucalyptus robusta (10.08%) and rudbeckia laciniata (8.77%) of asteraceae sampled by apis mellifera. the range of percentage are from 3.24% to 40.32% by apis cerana and from 4.05% to 66.82% by apis mellifera. the distribution of pollen percentage is polarized (fig. 2). non-nectar flow nectar flow mean range s.d. mean range s.d. a. cerana 7.44a 5-10 1.42 8.13a 6-11 1.46 a.s mellifera 7.56a 4-8 1.42 6b 4-9 1.77 table 2 the number of pollen types in honey samples by apis cerana and apis mellifera in different main nectar flow. note: s.d.-standard deviation. different letters in the same line indicates significant difference (p<0.05) a total of 17 pollen types from 34 honey samples were identified to belong to 14 families (table 3). it is apparent that none of the pollen types in all samples. the most important families of pollen were lythraceae, asteraceae, fabaceae and myrtaceae according to their percentage frequency. in non-nectar flow, the bidens pilosa of asteraceae is the most represented in the pollen of honey by apis mellifera, the next is trifolium repens of fabaceae. and the eucalyptus robusfig 1. pollen type spectra in honey samples by apis cerana and apis mellifera in different main nectar flow. fig 2. frequency of appearance of pollen types in samples by apis cerana and apis mellifera. the pollen types were classified into four categories. the percentage of one same category is different between apis cerana and apis mellifera through data analysis of all honey samples. important minor pollen is the largest proportion in honey by apis cerana (63%) and apis mellifera (58%). comparing the honey produced by apis cerana and apis mellifera in other three pollen categories, the percentage is a wide difference. the percentages of four categories are show in fig 2. through statistical analysis of all pollen information and trophic niche analysis, the niche breadth, trophic niche overlap index and interspecific competition index were calculated to show the relationship of the two kinds of bees in ecology. the proportion of each resource sequence and calculated value are show in table 4. the niche breadth compared the different yj liu, tr zhao, xw zhang, c liang, fy zhao melittopalynology and trophic niche analysis292 bee species within the same period. at non-nectar flow, the niche breadth is 0.65 and 0.57, respectively. at nectar flow, the niche breadth is 0.41 and 0.24, respectively. the niche breadth of apis cerana is greater than apis mellifera at two different periods. the trophic niche overlap index and interspecific competition index compared the bees within two periods of non-nectar flow and nectar flow. the value of trophic niche overlap index at nectar flow (0.68) is greater than that of non-nectar flow (0.61) and the value of interspecific competition index at nectar flow (0.92) is also greater than non-nectar flow (0.77). through the two values described above, the two species show stronger interspecific competition at nectar flow. table 4. frequency of pollen types from samples of apis cerana and apis mellifera and trophic niche analysis. discussion the present study provides new insights of the ecological relationship between two bee species by pollen composition analysis. the honey samples used in this study were collected for drawing pollen atlases and knowing the main nectar plants at the study area. these studies will help the local beekeeping. the honey from the different bee species of non-nectar flow and nectar flow has their own characteristic. at non-nectar flow, they show 8 pollen types, but eucalyptus robust is the maximum frequency in samples from apis cerana, followed by bidens pilosa, ligustrum lucidum, trifolium repens and zea mays. bidens pilosa is the maximum frequency in samples from apis mellifera, followed by trifolium repens, agave americana and leonurus japonicas. at nectar flow, they also show 8 pollen types and they have punica granatum pollen as predominant pollen. the next is bidens pilosa (apis ceran) and eucalyptus robusta (apis mellifera), respectively. different bee species show their preference of plants, the reasons for which may be that pheromone attract produced by plant to two bee species is different (jennifer et al., 2009). as for the varying degrees, the number of pollen types in honey and the niche breadth of apis cerana is greater than apis mellifera. whether nectar flow or not, the trophic niche overlap index and interspecific competition index all are sho-ta bl e 3. p ol le n ty pe s id en tifi ed in 3 4 ho ne ys a na ly se d an d th ei r fr eq ue nc y cl as se s– r ef er ri ng to t ab le 1 . fa m ily po lle n ty pe 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 a ga va ce ae a ga ve a m er ic an a l. i m m i i i s i m i i i a st er ac ea e b id en s pi lo sa l. i i i s s s s s i p p s s s i s s s i i s i i s s i i i i i i r ud be ck ia la ci ni at a l. i i i i i i i i i i i i i ta ra xa cu m m on go lic um h an d. -m az z. m m fa ba ce ae tr if ol iu m re pe ns l. i i s i i i i i i i i s s p s i i i i i i m m m m i pi su m sa ti vu m l. i i s i s i i i m i i i i i i i g er an ia ce ae pe la rg on iu m ho rt or um b ai le y m i i la bi at ae el sh ol tz ia de ns a b en th . i i i s i i i i la m ia ce ae le on ur us ar te m is ia (l au r.) s . y. h u i i i i i s i s la ur ac ea e ci nn am om um gl an du lif er um (w al l.) n ee s i i i i m le gu m in os ae m el ilo tu s al ba m ed ic . i s s s i i i i i i m i i i s i i s i m m i ly th ra ce ae pu ni ca gr an at um l. s s s s s s s p p p p p i p p p m al va ce ae m al va si ne ns is ca v. m m m m m m m yr ta ce ae eu ca ly pt us ro bu st a sm it h s s s s s s s s i i i i i i m i i i i i i i i i i i i i i i i i o le ac ea e li gu st ru m lu ci du m a it . i i s s i s s i s i m m i i i s i i po ac ea e ze a m ay s l. s i i i i i i i i m m m i m i i s s m i m i i m m ve rb en ac ea e ve rb en a of fi ci na lis l. i i i m m family pi (non-nectar flow) pi (nectar flow) apis cerana apis mellifera apis cerana apis mellifera agavaceae agave americana l. 1.92 9.60 — — asteraceae bidens pilosa l. 18.34 35.91 14.59 4.05 rudbeckia laciniata l. — — 4.65 8.77 taraxacum mongolicum hand.-mazz. — — — 0.28 fabaceae trifolium repens l. 11.83 15.71 3.24 1.60 pisum sativum l. — — 12.43 6.97 geraniaceae pelargonium hortorum bailey 0.89 — — — labiatae elsholtzia densa benth. — — 10.38 — lamiaceae leonurus artemisia (laur.) s. y. hu — 9.60 — — lauraceae cinnamomum glanduliferum (wall.) nees 2.37 0.25 — — leguminosae melilotus alba medic. 12.87 9.10 1.19 — lythraceae punica granatum l. — — 40.32 66.82 malvaceae malva sinensis cav. — — 0.43 0.75 myrtaceae eucalyptus robusta smith 21.89 8.10 9.62 10.08 oleaceae ligustrum lucidum ait. 16.12 6.11 — — poaceae zea mays l. 11.83 5.61 2.92 0.66 verbenaceae verbena officinalis l. 1.92 — 0.22 — niche breadth 0.65 0.57 0.41 0.24 trophic niche overlap index 0.61 0.68 interspecific competition index 0.77 0.92 sociobiology 60(3): 289-294 (2013) 293 wed that two bee species exist in strong interspecific competition from each other. bees pollinate most of the world's wild plant species and provide economically valuable pollination services to crops. the western honeybee, apis mellifera, has been introduced to many parts of the world and sometimes purported to be detrimental to native bees because it reduces their food base(forup & memmott, 2005). except the competition in food, there are some more direct competition, for example, apis mellifera may stolen the honey from hives of apis cerana sometimes, and apis mellifera drone chase and attempt to mate with apis cerana virgin queen caused mating interferes to drone and virgin queen of apis cerana (wang et al., 2003). certainly, we have to face the fact that some plants which mainly depend on apis cerana for pollination is becoming less with the reduction of apis cerana. some researchers have started to pay attention to assess the ecological impact of alien bee species on native bee by building various niche models (villanueva and roubik, 2004; franco et al., 2009; villemant et al., 2011). the alien invasive species, some are not the hopes of the people but some are introduced because of its high production capacity. apis mellifera, which is really good at production of honey, have been introduced to china. however, consideration must be given to the potential impact that expanding populations of introduced bees could have on native flora and fauna (howlett & donovan, 2010). little is known of the potential coevolution of flowers and bees in changing, biodiverse environments (roubik & villanueva, 2009). therefore, these studies assist us to understand the pollen types in honey and main nectar plants at the study area and could better help in evaluating the effects of the presence of apis mellifera on the foraging of apis cerana in this habitat. acknowledgements we thank the numerous friends and colleagues who helped us collect honey and plant samples for analysis, especially weiting luo, yusheng yu, ynahui wang, wenfei song. this study was financially supported by grants from fundamental research project of yunnan, china (no.2011fz046). we also thank two anonymous referees for helpful remarks in an early version of this manuscript. references ai h.x., yan x., han r.c. 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(hymenoptera: vespidae) colonies j nadolski introduction the presence of the european hornet (vespa crabro, linnaeus 1758), the largest wasp in europe in different areas inhabited by humans is commonly known (haeseler, 1982; skibińska, 1982; ahrné, 2008) and it is observed , that their colonies more and more often are established in the cities (nadolski, 2001). unfortunately, it is a species that is troublesome for the people. their colonies always cause significant concerns among the people because sizes of hornets’ individuals and their societies are very large (nadolski, 2012) and furthermore many factors may cause the increasing aggression of these insects and their attack on the people. people often do not know how to behave in the vicinity of the colony of hornets and what to dress to avoid their attack. it has been shown that stinging of hornets is not more dangerous than bees and some other species of wasps (nadolski 2000). moreover outside of the nest area hornets never attack groundlessly. the main reason for the attack of wasps’ societies is defending their colonies. the each external factor, which could underabstract defensive behavior of the european hornet vespa crabro colonies were studied using the dummy and balloons in different colors. the strong aggressiveness level of hornet’s workers was caused by dark colors (black and brown) and orange. the colors white and green did not cause their attack. however, the strongest reactions of hornets were caused by dummy with the animal snout and dark hair on the head. thus people who are near wasp colonies should have clothes in white or green and hair obscured but when the whole nest is eliminate, they have to have a safe outfit properly constructed. sociobiology an international journal on social insects university of łódź, kilińskiego, łódź, poland research article wasps article history edited by: gilberto m. m. santos, uefs, brazil received 17 june 2013 initial acceptance 11 july 2013 final acceptance 02 august 2013 keywords: social wasps; defensive behavior; the european hornet corresponding author jerzy nadolski faculty of biology and environmental protection natural history museum university of łódź kilińskiego 101, 90–011 łódź, poland e-mail: nadolski@biol.uni.lodz.pl mine structure of the nest and threaten the whole colony or even a single individual, may lead to the uneasiness among these insects. thus, the seemingly trifling cause, as a shadow cast on the entrance of the nest, sudden or even delicate jolt due to closing or to opening of a window or a door, may induce an attack of hornets on their alleged aggressor. in the event of an attack on humans, the smell of the person, the type of clothes, and even sex can become a cause of aggression (notman & beggs, 1993) and the first stinging by the wasp causes the release the alarm pheromones that are contained in the venom (veith et al., 1984; moritz & bürgin, 1987) causing attack of the whole colony. ability to see colors and shapes are key features to enable orientation in space for insects. in studies of distinguishing colors by wasps a significant similarity between different species of social wasps were shown (chittka et al., 1992) and also that the temperature and light have a major influence on their speed (spievok & schmolz, 2006). these skills allow them to determine the direction of flight, the search for food, and proper assessment of the possible threat. individuals of v. sociobiology 60(4): 466-470 (2013) 467 crabro can to see in the dark (kelber et al., 2011) and often they arrive to light in the home at night. the purpose of this study was to assess the impact of colors and shapes on defensive behavior of urban colonies of v. crabro using very simple and uncomplicated test methods. these studied issues, can be certainly relevant for the safety of the human population, but the complete assessment of all risks arising from the presence of hornets in urban areas is beyond the scope of this short study. material and methods the study of defense behavior of hornet’s societies was carried out in 2006 -2009 in august and september, because in temperate climates colonies of v. crabro are the largest in these months. because there is the positive correlation between the number of attackers of hornets and the total number of adults (bichara filho et al. 2006) as well as between the size of the nest and the number of individuals in the colony (nadolski 2012), for these studies 8 colonies of hornets of similar size and at the same stage of development (7 combs) were used. in subsequent analysis of their nests it was confirmed. the aggressiveness level showed by the colonies of vespa crabro was tested using methodology described by stort (1974) expanded and adapted in order to be used in hornets for this study. as a safety measure, the observer wore special suits and mask every time he approached the nests. studied hornet’s nests were established in the same wooden nesting boxes for birds with dimensions of 13 x 13 x 36 cm with an entrance hole with diameter of 29 mm situated 29 cm above the bottom of the box. the same size of boxes ensured that the size of nests were similar. boxes were located in few areas in the city of łódź in poland inter alia: botanic and zoological garden (51045’n 19024’e (84 ha) located in the western part of the city and łagiewniki forest (51050n 19029’e) located n-e of łódź. these activities were correlated with the studies on breeding of birds (alabrudzińska et al. 2003, marciniak et al. 2007) and on structure of nests and sizes of wasps colonies (nadolski 2001, 2012). in studies of the influence of colors on defensive behavior, different balloons with a diameter of about 30 cm in various colors were used: blue, black, brown, red, yellow, orange, green and white, which were singly placed in the immediate vicinity (30-50 cm) of the exit of the box. the wasps were vexed by three vigorous strokes with a stick in the nesting box. the behavior and number of workers of hornet flying out from the nest was observed. the degree of defensive activity was assessed based on the number of the attacking individuals and the time of their attack (the time was counted from the moment of hitting with a stick until the moment when the balloon ceased to be of interest to hornets). the result was rounded to 1 minute. every experiment was conducted in the hours before noon and in the rainless days, with an air temperature of about 20 25° c, with breaks (2-5 days) between successive tests for the same societies, because too often disturbing the peace of colonies increases aggressiveness of wasps (author’s own observations). their response to a particular color was tested twice on the same colony. thus, a total of 16 tests were performed for each color. the following criteria for assessing the degree of defensive activity were accepted (table 1). for studies of the effect of the aggressor’s appearance on defensive behavior, similar methods were used which were based on the same criteria as in the previous experiment and with the use of hornet colonies also located in nesting boxes. however, instead of the balloons, the white dummy mounted on a stick with visible head devoid of any facial features were used, to which the wig with dark hair or the mask looks like an big animal muzzle could be assumed. graphics and statistical analyses were made using statistica 9 (statsoft, inc. 2009). results and discussion the results of conducted studies determining the influence of colors on the level of defensive activity of v. crabro workers were subjected to statistical assessments. figure 1 presents results of these analysis for the activity time of colonies (anova nonparametric kruskal-wallis test h6,112 = 86.8 p<0.0001). in figure 2 results of analysis for the number of attacking workers (anova nonparametric kruskal-wallis test h6,112 = 90.2 p<0.0001) are presented. from them it can be concluded that the strong defensive activity was induced by dark colors especially black and brown, and bright (especially orange). reactions of hornet’s workers on green and white colors were weak or were no response. the analysis of multiple comparisons for kruskalwallis test demonstrated that there are statistically significant table 1. the criteria for assessing the degree of defensive activity of vespa crabro colonies. number of attacking workers symbol time of attack (min) degree of defensive activity up to 5 e to 3 inaktivity 6-10 d 3-4 weak activity 11-15 c 4-5 average activity 16-20 b 5-6 high activity over 20 a more than 6 very high activity j nadolski defensive behavior of vespa crabro468 differences of rank for the activity time between analyses variables (p-values for multiple comparisons <0.05), except between the dark colors (black, brown) and orange as well as between red and yellow and between white and green (p=1). result of the study of the influence of the aggressor’s looks on the defensive activity of v. crabro colonies showed, that most intense attacks in colonies defense by hornets were caused when the colony was disturbed by the presence of an individual with dark hair. the same contour of animal muzzle without hair did not cause such a strong concern. these received results (table 2), could not be subjected to statistical analysis. in recent years greatly increased the scope of various activities for reducing the populations of social wasps especially in urban areas (nadolski, 2001). publicizing information on the risks arising from the presence of colonies of wasps causes that people massively want to destroy nests of wasps, but they are not prepared for this type of dangerous actions. in their opinion the main element to protect against stinging of wasps and hornets is plastic bags worn on the head and mosquito spray or deodorants, as effective wasps’ deterrents. such “heroic” acts usually end up in hospital, often in the medical icu. there were also cases of necessity and the evacuation of whole families from their homes after such “experiments”. simultaneously, different municipal departments including the fire brigade are very often involved to remove nests of bees or wasps. the scale of these phenomena is so serious, that in poland, the fire brigade headquarters had to develop special procedures to be used for these types of threats (łyszkiewicz & nadolski 2009). based on the information about the behavior of wasps, methods of removing colonies of wasps and bees as well as technical measures for these actions were developed. studies of v. crabro defensive behavior were helped in understanding simple reactions of hornets caused by the presence of potential aggressor. defensive behavior in social wasps is similar for different species and involves two steps (jeanne, 1981): first, in response to jarring of the nest, large numbers of adults are recruited rapidly to the outer surface of the envelope. second, a fraction of these wasps may fly out to attack the intruder. outside the nest, the odor of venom greatly reduces the threshold for release of attack behavior, but is not itself a releaser of attack. the release of attack behavior requires an appropriate visual stimulus. in v. crabro the attack behavior is similar (nadolski, unpubl.) and interpretations of studies results were relatively easy. among beekeepers there is widespread belief that the outfit used to work at the apiary should be white. the results obtained fully confirm the validity of this principle, figure 1. the influence of colors on the level of defensive activity of workers of vespa crabro measured by an activity time. figure 2. the influence of colors on the level of defensive activity of vespa crabro measured by the number of attacking workers. table 2. the effect of the look of a potential aggressor on the level of attack of vespa crabro workers. n. l.– no limitations – the attack lasted until the time of removal of dummy; a*the number of attacking of workers was too large and impossible to estimate the form of aggressor the number of workers symbol mean of the time (min.) white dummy, head without hair 7±4 d 3±1 white dummy, head with dark hair over 40 a* n. l. white dummy with animal muzzle, head without hair about 20-40 a 9±3 white dummy with animal muzzle, head with dark hair over 40 a* n. l. sociobiology 60(4): 466-470 (2013) 469 and points to also the green as an alternative color of such clothing. the adaptive value of the ability to distinguish colors and to respond to them also seems to be fairly obvious. in a moderate climate zone, the social wasps are active only in spring, summer and autumn, when the white color is not present in the surroundings and, what is more, the potential predators also are not white. the green is the color of the surrounding environment and this is not the color of the aggressor as well. the situation is quite different with dark colors, in shades of brown and black, and also with vivid, bright colors. natural selection should favor behavior that treats so colored an intruder as a potential predator. the results of these studies have become the basis for the development by the author of a project of special protective overalls to work with social aculeate, especially hornets. this type of outfit was accepted as the standard protective clothing recommended for the fire department sections involved in removing the ‘threats from swarms and nests of hymenopterans’. the result of observation of the defensive behavior conditioned by the intruder looks is equally interesting. the outline of the body shape does not cause too much concern in the colony of hornets. only the shape, which resembles animal snout and especially hairy head, causes aggression of insects. these results could not be analyzed statistically, however the overall picture of the experiment clearly points to mammals as the main enemies of wasps’ societies against whom the whole strategy of defense is directed. the social behavior of vespidae is dependent on their social structure (carpenter, 1991; smith et al., 2001). the social structure is higher, defensive behavior is more complex. in solitary wasps defensive behavior is directed against different types of arthropods especially other insects. in eusocial aculeate they are first of all directed against vertebrate. the studies on venom activity of aculaete also confirm this opinion. along with the development of social behaviors, venom composition underwent changes towards producing such ingredients that would be more and more effective at defending against such an attacker. the venom of solitary wasps have properties allowing to use it primarily for paralyzing and then killing the victims serving as food for its larvae (piek & spanjer, 1986). vespinae produce venoms, which by of its composition is effective in obtaining food as well as in defense (nadolski, 2000). however, in the defense aspect, the most effective is the venom of honey bee (apis mellifera l.) (o’connor, et al., 1967). the main component of this toxin is melittin, one of the most effective detergents that causing red blood cell haemolysis and damage cells of muscles (banks & shipolini, 1986; nabil et al., 1988). the v. crabro venom can be used not only to defend the colony and rarely to attack on other insects in order to acquire food, but it is also an effective tool to combat individuals of hornet from foreign societies (nadolski, 2013) in conclusion of these results, it is very important that people who are near wasp colonies should have clothes in white or green and hair obscured but when removing whole nests a man have to have a safe outfit properly constructed. acknowledgments i thank two anonymous referees for helpful remarks in an early version of this manuscript. references ahrné, k. 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(1984). 2-methyl3-butene-2-ol, a major component of the alarm pheromone of the hornet vespa crabro. naturwissenschaften, 7: 328-329. doi: 10.13102/sociobiology.v64i1.1216sociobiology 64(1): 14-17 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the use of light to enhance weaver ant oecophylla longinoda latreille (hymenoptera: formicidae) queen catches introduction weaver ants (o. longinoda and o. smaragdina) are increasingly being utilized for human applications as they perform multiple valuable ecosystem services. firstly, these ants are used as biological control agents against a number of pests in several tropical perennial crops (way & khoo, 1992; peng & christian, 2006), where they, in many cases, are as efficient in insect pest control as conventional chemical pesticides, and in some cases, are even more efficient (offenberg, 2015). secondly, they may also fertilize their host plants via their deposition of ant manure on the foliage; manure that contains n-rich urea that can be taken up by plants directly through the leaves (pinkalski et al., 2015; vidkjær et al., 2015; vidkjær et al., 2016). thirdly, the ants turn the pests and other prey they feed on into edible protein rich ant biomass which is easy to harvest and thus are being utilized as human food or animal abstract production of live weaver ant (oecophylla longinoda and o. smaragdina) colonies is being developed as the ants provide several ecosystem services in agriculture and as they are used in education and research laboratories. founding queens needed for colony production can be caught in artificial nests made of live leaves that are curled on trees. in this study we investigated if the catch rate of o. longinoda queens in artificial nests could be improved by attracting queens to trees with a light source (electric torches). we compared catch rates of 50 artificial nests on each of eight citrus trees, four of them with light and four without light. during two mating seasons covering 9 mating flights we caught a total of 178 queens. however, 3.8 times more queens were caught in the trees with light compared to trees without light. we conclude that queen catches can be highly improved by combining artificial nests with an attracting light source. sociobiology an international journal on social insects w nene¹, j offenberg², gm rwegasira¹, m mwatawala¹ article history edited by evandro nascimento silva, uefs, brazil received 18 october 2016 initial acceptance 08 january 2017 final acceptance 09 january 2017 publication date 29 may 2017 keywords light source, natural predator, founding queen, tanzania. corresponding author wilson a. nene sokoine university of agriculture department of crop science and production, box 3005 morogoro, tanzania e-mail: wilsoninene@gmail.com feed (césard, 2004; offenberg, 2011; van huis et al., 2013); a utilization that can be combined with the use of ants as biocontrol agents and thus provide double benefits (offenberg & wiwatwitaya, 2010). fourthly, weaver ants are increasingly being investigated by researchers and are utilized in science education as a laboratory model organism (van mele, 2008; offenberg, 2015). based on these applications there is an increasing needs to obtain founding weaver ant colonies. such colonies can be used to stock ant nurseries that are culturing ants to farmers in order to facilitate implementation of weaver ant biocontrol programs and weaver ant mini-livestock productions (ouagoussounon et al., 2013). further they can be used to supply laboratories with colonies used in research and education. this is especially important as it is difficult to detect the single nest with the maternal queen(s) in weaver ant colonies as queen nests are small, placed high in the canopy, 1 sokoine university of agriculture, department of crop science and production, box 3005, morogoro, tanzania 2 department of bioscience, aarhus university, aarhus, denmark research article ants sociobiology 64(1): 14-17 (march, 2017) 15 and are one out of up to several hundred nests per colony (peng et al., 1998; van itterbeeck et al., 2015) weaver ant queens disperse under their nuptial flight which takes place during rainy seasons (nene et al., 2015; nielsen et al., 2015; rwegasira et al., 2015a; rwegasira et al., 2015b). after mating they search for suitable nesting sites which under natural conditions are overlapping or curled leaves on host plants placed outside established weaver ant territories. during this process queen mortality is extremely high with probably less than 1 % of the queens surviving the first few days since good hiding places are in limited supply (offenberg unpublished data). to arrest queens and increase their survival the application of artificially curled leaves have been used to ease the collection of founding queens. by providing such nests, collection success can be increased up to 100-fold compared to simple visual searches of plant foliage, and even with less time expenditures (rwegasira et al., 2015a). during recent studies on weaver ant mating flights it was observed that o. longinoda performed their nuptial flights at sunset (nene et al., 2016). this is in contrast to o. smaragdina, which perform the flights at sunrise (nielsen et al., 2015). since o. longinoda perform flights just before nightfall, high numbers of queens are attracted to light sources right after the flight (rwegasira et al., 2015a). based on this, we hypothesized that a light source could be used to further increase the collection success of founding queens if combined with artificial nests. if trees with artificial nests are provided with a light source, queens may be attracted/lured to these trees where after it becomes easier for them to find and settle in the nests. in this study we compared queen catch frequencies in artificially nests on trees with and without a light source. methodology experiments were carried out at naliendele agricultural research institute, (100 21´ 22.49” s, 400 09´ 57.05” e and 140 meters above sea level) about 10 km from mtwara town in the southern part of tanzania. the mtwara region has a unimodal wet season, with regular rains from november/december to april/may. the annual rainfall ranges from 810 to 1090 mm. weaver ant queens were trapped using artificially folded leaves as described by peng et al., (2013). the study period covered two different mating seasons (december 2014 to february 2015 and december 2015 to february 2016). two different locations were selected, each with several small citrus trees (ranging between 3 and 5 m height) without weaver ants or other ant species in the canopy, in an area otherwise inhabited by mature o. longinoda colonies producing sexuals. on each site two pairs of the small ant-free trees were selected and 50 artificial nests were made in each tree. in each pair, one of the trees was mounted with two battery driven light torches (three volt each) attached to the canopy at a height between 2 and 3.5 m. the other tree in each pair was left without light. whenever a mating flight was observed during each of the mating periods, the torches were turned on, on the evening of the flight between 6.45 and 7.00 pm. the same night they were switched off again between 8.30 and 9.00 pm, allowing the queens to search for nests during the rest of the night. on each morning following a flight (between 6.30 and and 7.30 am) the presence of queens in the artificial nests was assessed and the number of queens caught in each tree was recorded. the caught queens were subsequently kept in match boxes for 60 days to test if they had mated before being attracted to the light. as successfully mated queens lay eggs that are able to hatch into larvae, queens were recorded as mated if they produced brood beyond the egg stage (nene et al., 2015). as queens caught with light traps potentially could be harmed by flying into the torches, queen mortality was also recorded after the 60 day period. to avoid pseudo-sampling the number of queens caught on each tree was summed for each season as more flights occurred per season. the effect of light on catch efficiency was tested by comparing the total number of queens caught per tree between trees with and without light with wilcoxon oneway tests (using jmp 11). mortality was compared between treatments with a g-test. furthermore, the total number of queen caught per tree per site in a season is illustrated in fig 1. fig 1. number of o. longinoda queen catches per tree from two sites in 2014/15 and 2015/16 at naliendele agricultural research institute, mtwara, tanzania. where: t1 and t2 are tree number 1 and 2 respectively. results a total of nine flights were observed during the study with five flights in the 2014/15 season and four flights in the 2015/16 season. in the first season two and three flights (one was common for both sites) were observed at the two sites, whereas two flights were observed per site in the second season. in the first season a total of 91 queens were caught, with 72 of them being caught on trees with light traps and the remaining 19 caught on trees without light (wilcoxon: w nene et al. – use of light to enhance weaver ant queen catches16 chi square =5.5, p = 0.019). five of the 72 queens in the light treatment died whereas 1 queen of the 19 queens from the trees without light died (g-test: g = 0.07, p = 0.80). in the second season a total of 87 queens were caught with 69 of them originating from trees with light and 18 from trees without light (wilcoxon: chisquare =5.4, p = 0.020). in this season all the queens survived. in total (pooling the two seasons) 3.8 times more queens were caught in the trees with light compared to the control trees without a light bulb. the average catch rate per tree (50 artificial nests) per mating flight was 7.8 (± 1.18 se) and 2.1 (± 0.33 se) queens in the light and control treatments, respectively. all the queens from both seasons and treatments produced eggs that hatched to larvae and were thus expected to have been mated successfully prior to settling in the artificial nests. discussion these results show that combining lights with artificial nests could successfully increase catch rates of o. longinoda queens almost four-fold compared to the use of artificial nests only. this may help researchers and ant nursery managers to reduce the costs associated to the collection of ant queens. also the increased catch efficiency means that collectors may catch queens even after small flights and in this way the season of positive catches may be extended. this means higher security for collectors. whether the method also works for o. smaragdina is questionable as this species make their nuptial flights in the morning (nielsen et al., 2015). thus, queens have most of the day available to find suitable nesting sites and are therefore likely to settle before nightfall. on the other hand, o. smaragdina queens have been caught at light sources in the night time in thailand (offenberg, unpublished data), suggesting that not all queens settle within the first day of the flight. as queen mortality did not differ between the two treatments it is unlikely that the queens damaged themselves when flying into the light sources, in this case electric torches. this potential drawback of the method, thus, seems to be of no concern. another concern was that the queens could be attracted to the light before having had a chance to mate. this, however, was also not a problem in the present study, as all queens that were caught produced viable offspring. this means that mating takes place at least before the lights were turned off at no later than 9.00 pm, and probably much sooner. we therefore conclude that the mating takes no longer than two and a half hours and probably considerably less. this is the time from the beginning of the flights that does not take place earlier than 6.25 pm (nene et al., 2016) until 9.00 pm, the latest at which lights were switched off. in comparison o. smaragdina in darwin, australia start their flights at sunrise and start to settle on foliage during the afternoon. however, when exactly their mating takes place and how long it takes is unknown (nielsen et al., 2015). acknowledgement we thank the naliendele agricultural research institute and farmers at naliendele village for providing the fields for the experiments. our sincere gratitude is to mr. hasan libubulu for his assistance in data collection. references césard, n. (2004) harvesting and commercialisation of kroto (oecophylla smaragdina) in the malingpeng area, west java, indonesia. forest products, livelihoods and conservation. case studies of non-timber product systems (eds k. kusters & b. belcher), pp. 61-77. center for international forestry research, bogor. nene, w. a., rwegasira, g. m., nielsen, m. g., mwatawala, m. & offenberg, j. (2016). nuptial flights behavior of the african weaver ant, oecophylla longinoda latreille (hymenoptera: formicidae) and weather factors triggering flights. insectes sociaux, 63: 243-248. doi: 10.1007/s00040-015-0456-9 nene, w. a., rwegasira, g. m., offenberg, j., mwatawala, m. & nielsen, m. g. (2015). mating behavior of the african weaver ant, oecophylla longinoda (latreille) (hymenoptera: formicidae). sociobiology, 62: 396-400. doi: 10.13102/ sociobiology.v62i3.650 nielsen, m.g., peng, r., offenberg, j. & birkmose, d. (2015). mating strategy of oecophylla smaragdina (hymenoptera: formicidae) in northern australia. austral entomology, 55: 261-267. doi: 10.1111/aen.12182 offenberg, j. (2011). oecophylla smaragdina food conversion efficiency: prospects for ant farming. journal of applied entomology, 135: 575-581. doi: 10.1111/j. 14390418. 2010.01588.x offenberg, j. (2015). ants as tools in sustainable agriculture. journal of applied ecology, 52: 1197-1205. doi: 10.1111/1365-2664.12496 offenberg, j. & wiwatwitaya, d. (2010). sustainable weaver ant (oecophylla smaragdina) farming: harvest yields and effects on worker ant density. asian myrmecology, 3: 55-62. doi: 10.20362/am.003008 ouagoussounon, i., sinzogan, a., offenberg, j., adandonon, a., vayssières, j. f. & kossou, d. (2013). pupae transplantation to boost early colony growth in the weaver ant oecophylla longinoda latreille (hymenoptera: formicidae) pupae transplantation to boost early colony growth in the weaver ant oecophylla longinoda latreille (hymenoptera: formicidae). sociobiology, 60: 374-379. doi: 10.13102/sociobiology.v60i4.374-379 peng, r., nielsen, m. g., offenberg, j. & birkmose, d. (2013). utilisation of multiple queens and pupae transplantation to boost early colony growth of weaver ants oecophylla smaragdina. asian myrmecology, 5: 177-184. sociobiology 64(1): 14-17 (march, 2017) 17 peng, r. k. & christian, k. (2006). effective control of jarvis’s fruit fly, bactrocera jarvisi (diptera : tephritidae), by the weaver ant, oecophylla smaragdina (hymenoptera : formicidae), in mango orchards in the northern territory of australia. international journal of pest management, 52: 275282. doi: 10.1080/09670870600795989 peng, r. k., christian, k. & gibb, k. (1998). locating queen ant nests in the green ant, oecophylla smaragdina (hymenoptera, formicidae). insectes sociaux, 45: 477-480. doi: 10.1007/s000400050103. pinkalski, c., damgaard, c., jensen, k. m.v., peng, r. & offenberg, j. (2015). quantification of ant manure deposition in a tropical agroecosystem: implications for host plant nitrogen acquisition. ecosystems, 18: 1373-1382. doi: 10.1007/s10021-015-9906-5 rwegasira, r. g., mwatawala, m., rwegasira, g. m., mogens, g. n. & offenberg, j. (2015a) . comparing different methods for trapping mated queens of weaver ants (oecophylla longinoda; hymenoptera: formicidae). biocontrol science and technology, 25: 503-512. doi: 10.1080/ 09583157.2014.992861 rwegasira, r. g., mwatawala, m., rwegasira, g. m. & offenberg, j. (2015b). occurrence of sexuals of african weaver ant (oecophylla longinoda latreille) (hymenoptera: formicidae) under a bimodal rainfall pattern in eastern tanzania. bulletin of entomological research, 105: 182-186. doi: 10.1017/s0007485314000868 van huis, a., van itterbeeck, j., klunder, h., mertens, e., halloran, a., muir, g. & vantomme, p. (2013). edible insects: future prospects for food and feed security. pp. 201. fao, rome. van itterbeeck, j., sivongxay, n., praxaysombath, b. & van huis, a. (2015). location and external characteristics of the oecophylla smaragdina queen nest. insectes sociaux, 62: 351-356. doi: 10.1007/s00040-015-0411-9 van mele, p. (2008). a historical review of research on the weaver ant oecophylla in biological control. agricultural and forest entomology, 10: 13-22. doi: 10.1111/j.1461-9563. 2007.00350.x vidkjær, n. h., wollenweber, b., gislum, r., jensen, k. m. v. & fomsgaard, i. s. (2015). are ant feces nutrients for plants? a metabolomics approach to elucidate the nutritional effects on plants hosting weaver ants. metabolomics, 11: 1013-1028. doi: 10.1007/s11306-014-0757-4 vidkjær, n. h., wollenweber, b., jensen, k. m.v., ambus, p. l., offenberg, j. & fomsgaard, i. s. (2016). urea in weaver ant feces: quantification and investigation of the uptake and translocation of urea in coffea arabica. journal of plant growth regulation, 35: 1-12. doi: 10.1007/s00344016-9586-1 way, m. j. & khoo, k. c. (1992). role of ants in pestmanagement. annual review of entomology, 37: 479-503. doi: 10.1146/annurev.en.37.010192.002403 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1256sociobiology 64(1): 85-91 (march, 2017) first characterization of sphingomyeline phosphodiesterase expression in the bumblebee, bombus lantschouensis introduction sphingomyeline phosphodiesterase (smase) is a hydrolytic enzyme that is involved in metabolic reactions of sphingolipids. smase generates ceramide through the hydrolysis of sphingomyelin to phosphocholine and ceramide to regulate the ceramide-mediated cell (ago et al., 2006; hofmann et al., 2000; kramer et al., 2015). sphingomyeline phosphodiesterase gene (smpd) was first cloned from bacillus cereus (yamada et al., 1988). smases are classified into three categories based on its ph activity profiles. for the optimal activity ph value, the acid smase (asmases) was reported to be 4.5~5 (brady et al., 1966; jenkins et al., 2009), abstract the bumblebee (bombus lantschouensis vogt) is an important pollinator of wild plants. sphingomyelin phosphodiesterase (smpd) is a hydrolase that plays a major role in sphingolipid metabolism reactions. we report the preparation and characterization of a polyclonal antibody for bumblebee smpd. we then use the polyclonal antiserum to detect the smpd protein at different development stages and in different tissues. our results showed that a 1228bp fragment homologous with the b. terrestris smpd gene was successfully amplified. the molecular weight of the fusion protein was about 70kda by sds-page. an effective polyclonal antibody against smpd was also obtained from mice and found to have a higher specificity for bumblebee smpd. western blotting detection showed that smpd was expressed at a high level in queen ovaries, although expression was lower in the midgut and venom gland. smpd expression decreased from the egg stage until the pbl pupal stage. we interpret our results as showing that the development of an effective polyclonal antiserum for the smpd protein of a bumblebee, which provides a tool for exploring the function of the smpd gene. in addition, the work has confirmed that smpd should be considered as an important enzyme during bumblebee egg and larval stages. sociobiology an international journal on social insects l han1, 2, s he2, j dong1, y wang1, j huang1, j wu1 article history edited by evandro nascimento silva, uefs, brazil received 24 november 2016 initial acceptance 27 february 2017 final acceptance 05 march 2017 publication date 29 may 2017 keywords bombus lantschouensis, smpdgene, gene clone, prokaryotic expression, polyclonal antibody, expression characteristics. corresponding author jie wu (jw); jiaxing huang (jh) key laboratory for insect-pollinator biology institute of apicultural research, caas beijing 100093, china e-mail: apis@vip.sina.com huangjiaxing@caas.cn neutral smase (nsmases) near ph 7 (sabourdy et al., 2008), and the alkaline smase is about ph 9 (kraut, 2011). among these sphingomyelinases, the acid smase was the most explored in the past few years (brady et al., 1966; jenkins et al., 2009; bartelsen et al., 1998). currently, it has been reported that smase is correlated to many human diseases, such as niemannpick, lung or liver diseases, neuronal disorders, sepsis organ failure, non-hemolytic enterotoxin, and regulation of immune response (ago et al., 2006; horinouchi et al., 1995; smith & schuchman 2008; doll et al., 2013). it has been demonstrated that the function of smase can inhibit colonic cancer and colitis (herterving et al., 1997; sjöqvist 1 key laboratory of insect-pollinator biology of the ministry of agriculture, institute of apicultural research, chinese academy of agricultural sciences, beijing, china 2 eastern bee research institute, yunnan agricultural university, kunming, china research article bees l han et al. – bumblebee development affected by smpd86 et al., 2002). moreover, increased secretion levels of smase could also lead to accelerated at herogenesis (truman et al., 2011). although the function of smase in human research has been explained clearly, little work has been done on insect smase, except in flies. based on the homology analysis of drosophila (diptera: drosophiloidade) genome, five paralogues of smpd gene were annotated and research explorations have demonstrated that they are highly expressed at flies embryonic development stages (renault et al., 2002). furthermore, based on microarray analysis, it was found that smpd expression levels decreased with the sugar regulation (zinke et al., 2002). interestingly, it was suggested that the silence of smpd gene by sirna has strong influence on pregnancy and progeny development in glossina morsitans morsitans westwood (diptera: glossinidade) (benoit et al., 2012). however, the function of smpd in bees hasn’t been explored. bombus lantschouensis vogt (hymenoptera: apidae) is a native bumblebee species that play an important role for wild plant pollination in china (peng et al., 2009; an et al., 2010). furthermore, it has been a candidate species for year round rearing and greenhouse crop pollination in recent years. the investigations of basic biology of b.lantschouensis are very important for its successful year round rearing. according to a comparative proteomics analysis, smpd gene was found to be significantly different in expressions between egg-laying and non-egg-laying queens (unpublished data). therefore, it was important to explore the basic biology of smpd in bumblebee. since smpd plays an important role in sphingolipid metabolic reactions, investigating smpd protein profileswill help us to understand its functions in bumblebees. in this study, we described the clone and expression of the recombinant protein with a partial fragment of smpd gene from b. lantschouensis. the fusion protein was purified as antigen for antiserum preparation against mice. furthermore, the specificity of antiserum was validated on his-tag expression fusion protein and original bumblebee protein. finally, the smpd expression profile was detected at the different tissues and development stages of b. lantschouensis by western blotting. this antiserum can be used to explore the function of smpd gene in further research. materials and methods bumblebees colonies of b. lantschouensis were obtained from department of insect pollination and ecology laboratory of institute of apicultural research, chinese academy of agricultural sciences. the colonies were supplied with pollen pellet and sugar syrup (1:1) every other day. the rearing boxes were kept at a temperature of 28 ± 2°c and 60 ± 10% relative humidity according to a method described by kwon (kwon et al., 2003). all samples were prepared and kept at -80°c until used. smpd amplification and fusion expression vector construction the total rna was isolated from the abdomen of b. lantschouensis workers through a technique that uses trizol (invitrogen, usa), according to the manufacturer’s instruction. the complementary dna was synthesized from 1μg of rna using a kit (takara, dalian, china) following manufacturer’s manual. the forward primer 5'-ccggaattcaacagtttgtggcgttgttcta-3' and reverse primer 5'aaatatgcggccgcgtgtttca ttctttgctgctg-3' with noti and ecori restriction enzyme sites were designed and used to amplify smpd coding gene sequence. at the same time, a pair of similar primers with ecori and saci restriction enzyme sites was synthesized for his-tag plasmid construction. pcr amplification reaction system contained1μl cdna template,0.5 μm of each primer, and 10 μl of the pcr mix and finally topped to 20 μl volume with nuclease-free water. pcr was performed under the following conditions: an initial denaturation at 94°c for 3 min followed by 35 cycles of amplification (with 30 sec of denaturation at 94°c, 30 sec of annealing at 56°c, 1.5 min of extension at 72°c, and 10 min of further elongation at 72°c). the pcr products amplified were then sequenced (sinogenomax, beijing) from both ends to validate the smpd amplicon. pcr products were purified and digested with noti and ecori restriction enzymes for pgex-6p-1 vector recombination and ecori and saci restriction enzymes for pcold-ii vector recombination. more specifically, the digested pcr products were gel purified and ligated into pgex-6p-1 and pcold-ii vectors using t4 dna ligase at 16°c. the ligated constructs were transformed into e. colibl21 (de3) competent cells and cultured overnight at 37°c. we selected an ampicillin-resistant clone and sub-cultured in 5ml lb liquid medium to obtain optimum amount of the expression vector. expression and purification of the fusion protein positive clones of e. coli bl21 were cultured at 37°c in ampicillin containing lb liquid medium. isopropy-d-thiogalactoside (iptg) was added to the culture at a final concentration of 1 mmol/l when the optical density at 600 nm (od600) reached a value of 0.6~0.8 to induce expression of fusion protein. after 5 hours incubation, the culture was centrifuged for 3 min at 10000g at 4°c to collect the cell. the precipitate was washed 3 times in 10 ml phosphate-buffered saline (pbs) for 10 min at 6000g and 4°c. cells was re-suspended in 15 ml pbs and sonicated with 400w at 6s and 15s interval for 90 cycles. cells were centrifuged again for 10 min at 6000g and 4°c. the supernatant and precipitate were collected separately. the precipitate was then diluted in pbs, and 2× loading buffer was added and boiled at 100°c for 5 min. sds-page electrophoresis was used to check the characteristics of protein expressions. the expressed recombinant protein band was cut from the gel and eluted in phosphate-buffered saline and kept at -80°c until used (yu et al., 2007). sociobiology 64(1): 85-91 (march, 2017) 87 production of polyclonal antibodies and western blotting validation fifty μg of purified recombinant protein was injected into balb/c type female mice (6 weeks old) with an equal volume of freund’s complete adjuvant. this was immunized for the second time after two weeks. negative serum was collected from the injected mice’s tail before immunization. the tail of experimental mice was cut to check the antibody through western blotting two weeks after the final immunization. blood was sampled from mice eyes and centrifuged at 5000 rpm and 4°c for 20 min. the serum was decanted and stored at -80°c for further use. polyclonal antibody specificity was then checked using western blotting. at this step, total protein from b. lantschouensis was extracted and separated on 12% sds-page, then the gel was transferred onto nitrocellulose membrane in a western blot system (biorad, usa) for 60 min. nitrocellulose (nc) membrane was blocked with a commercial blocking buffer for 1.5 hours (comwin, beijing). the polyclonal antibody was diluted at a ratio of 1:3000 and used for incubating with the transferred nc membrane. the membrane was then washed three times with 1*tbst containing 0.1% tween 20 and the europium chelate coated mice igg was used as secondary antibody to be incubated with nc membrane. finally, the treated nc membrane was scanned and images were detected on microplate spectrophotometer (molecular devices, usa). smpd expression characteristics in bumblebee in order to understand the smpd expression status, total proteins from different tissues and development stages of b. lantschouensis queens and workers were extracted and protein concentration was determined using a bca method. twenty μg of total protein was then electrophoresed by sdspage and immunoblotting was carried out as described above. protein expression levels in different tissues (antenna, head, muscle, leg, body wall, midgut, fat body, ovary, and poison gland) of three egg-laying queens were quantified. the different development stages including eggs, larvae (at day 5 and 10) and pupae (pw, pp, pb, pb1, pbdand pha) were also detected for the protein expression levels (duchateau & velthuis, 1989; dallacqua et al., 2007). results smpd cdna cloning and construction of its expression vector a part of smpd cdna was pcr amplified using a pair of specific primers designed from b.terrestris cdna sequence and pcr products were electrophoresed on 1% agarose gel. according to the result, a pcr product with noti and ecori restriction enzyme sites has produced a clear band at about 1200 bp (fig 1). furthermore, the dna sequence and homogenous analyses of this product has clearly elucidated that it is a segment of smpd gene. it has been also predicted that its relative molecular mass and isoelectric point were 50.8 kda and 6.17, respectively. the pcr product was subcloned into the pgex-6p-1 expression vector, and verified with double enzyme digestion and dna sequencing (fig 2). moreover, the recombinant vectorpgex-6p-1-smpd was successfully transformed into e. coli bl21 (de3) cells. fig 2. recombinant plasmid pgex-6p-1-smpd validated by restriction enzyme ecori and noti at agarose gel electrophoresis. m, molecular mass marker d2000; lane 1,pgex-6p-1 empty plasmid; lane 2, pgex-6p1-smpd recombinant plasmid; lane 3,pgex-6p-1-smpd recombinant plasmid product cut by enzyme ecoriand noti; lane 4: smpdpcr amplification product. fig 1. pcr amplification of smpd gene sequence from b. lantschouensis. m, molecular mass marker d2000; lane 1 and 2, amplified product (about 1200bp). recombinant protein expression and purification small-scale culture of positive pgex-6p-1-smpd clone was induced by iptg to express the recombinant protein. accordingly, polyacrylamide gel electrophoresis showed that the fusion protein has been successfully expressed at an expected molecular mass of about 70 kda. simultaneously, a similar protein band was not observed from the control treatment lanes (fig 3). large-scale expression of smpd fusion protein was separated by sds-page and the predicted l han et al. – bumblebee development affected by smpd88 smpd band was then collected. furthermore, electrophoresis has confirmed that the fusion protein was the major band on the sds-page indicating its purity (fig 4) which further indicated that we have successfully obtained the fusion gstsmpd protein from e. coli. fig 4. purified gst-smpd protein separated in sds-page electrophoresis. m, protein molecular weight standards; lane1, recombinant protein was purified by gel isolation. fig 3. sds-page profile of smpd expressed in pgex-6p-1/bl21 system. m, protein molecular weight standards; lane 1, protein expressed in bl21 cell with pgex-6p-1-smpd plasmid; lane 2, protein expressed in bl21 cell with un-induced pgex-6p-1-smpd plasmid; lanes 3 and 4, protein expressed in bl21 cell with induced and uninduced pgex-6p-1 plasmid respectively. polyclonal antibody preparation and western blot identification in this experiment, 500 μl of blood samples were collected from each of the experimental mouse at the end of two mice immunizations. furthermore, the antiserum was then divided into aliquot 200 μl tubes and stored at -80°c until use. accordingly, western blot analysis of the smpd polyclonal and his-tag monoclonal antibodies against hissmpd fusion protein demonstrated that both antibodies produced a similar band size at about 40 kda (fig 5). the smpd polyclonal antibody which was hybridized with the bumblebee protein produced a 70 kda protein band as was expected. simultaneously, the pre-immunized mice serum, the negative control, didn’t produce a detectable band signal (fig 6). based on the results from western blotting, it has been confirmed that we have successfully developed the smpd polyclonal antibody for the first time which could be used to characterize smpd protein in the future. fig 5. smpd polyclonalantibody (a) and his-tag monoclonal antibody (b) against the expressed his-smpd fusion protein. fig 6. polyclonal antibody and negative antiserum hybrid with bumblebee total protein using western blotting. sociobiology 64(1): 85-91 (march, 2017) 89 expression characteristics of smpd in bumblebee in order to understand the expression profile of smpd in bumblebee, the developed polyclonal antibody was used to detect smpd expression levels at different tissues and developmental stages of b. lantschouensis. accordingly, western blotting analysis showed significantly varied in smpd expression levels base on the same loading volume of total protein electrophoresis. among the nine majorly considered tissues, seven (antenna, head, muscle, leg, body wall, fat body and ovary) of them were detected with signals on western blotting. more specifically, smpd was highly expressed in the muscle, ovary and head. however, we didn’t detect smpd expression in the midgut and venom gland (fig 7(a)). furthermore, it has been confirmed that smpd expression decreased with increasing developmental stages. specifically, its expression levels were higher at egg and 5 days old larvae whereas smpd expression levels were lower at the pbd and pha developmental stages (fig 7(b)). according to our experiment, smpd gene recombinant plasmid was successfully constructed and expressed in a prokaryotic expression system which was a fast and inexpensive way to obtain plenty of interesting proteins. despite the high solubility of gst fusion protein, we didn’t get solubilized proteins during the expression conditions of our experiment. it was, thus, confirmed that there was no fusion protein found in the supernatant of cell disruption which may be associated with the high efficiency expression of pgex-6p-1 system (zhao et al., 2000; ding et al., 2006; liu et al., 2011). therefore, it needs to change the expression to acquire the soluble protein for studying the activity of smpd. as western blot results of this work suggested, fusion protein from prokaryotic expression had immunogenicity characteristics and successfully produced the smpd polyclonal antibody in mice. both the his-tag antigen and bumblebee total protein western blot demonstrated that the polyclonal antibody was effective and specific to smpd protein. generally, the specificity of this kind of polyclonal antibody has been found to be lower than the monoclonal antibody (chu & englund, 2013). however, our western blots suggested that the specificity of this kind was higher than expected which may require using a eu-labeled goat anti-mouse kit which also could improve sensitivity and reduce background to guaranty higher blotting quality (park et al., 2015; lynch et al., 2016). smpd protein has been described to play an important role in sphingolipid metabolism reactions. in line with this, consequently, we have found that smpd expression was higher at the early ages (egg and larval stages) of a bumblebee. this is because smpd might be produced at those stages in higher quantity to involve in sphingolipids metabolism. in this case, similar results were reported from drosophila embryos (renault et al., 2002). furthermore, higher expression of smpd observed at ovary and head could imply that these regions might be the functional sites of smpd in bumblebees. in conclusion, this study has displayed successful amplification of smpd gene cdna fragment from bumblebee for the first time. agst-smpd recombinant protein was successfully expressed in e. coli and a highly specific polyclonal antibody against smpd was produced. the antibody has also demonstrated that it can serve as a good tool in the analysis of smpd protein profiles in future studies. moreover, smpd expression has indicated that its role might be associated with egg and larvae development. finally, we believe that this work could provide an immense importance in further investigation of smpd functions in bumblebees. acknowledgements at this point, we would like to thank abebe jenberie and tolera kumsa for their help during manuscript revision. this work was financially supported by china agriculture research system (cars-45) and caas agricultural science and technology innovation program (caasastip-2015-iar). fig 7. expression levels of smpd proteinat different tissues(a) and different development stages(b) determinedusing prepared polyclonal antibody. the different tissues include: antenna (an), head (he), muscle (mu), leg (le), body wall (bw), midgut (mi), fat body (fb), ovary (ov) and poison gland (pg) of the queen. likewise, the differentstages include: egg, larvae atday 5, larvae atday 10, pw, pp, pb, pbl, pbd and pha stage. discussion recently, the development of next generation sequencing technologies made the predictions about biological features in the genome of non-model organisms easier. furthermore, such technologies also facilitated and supported the possible characterization of more and more novel genes and proteins. based on bombus terrestris vogt (hymenoptera: apidae) genome annotation and previously conducted experiment in our laboratory, smpd was identified from 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(2002). nutrient control of gene expression in drosophila : microarray analysis of starvation and sugardependent response. the embo journal, 21: 6162-6173. doi: 10.1093/emboj/cdf600 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5851sociobiology 68(1): e-5851 (march, 2021) introduction the managed honey bees are the most important pollinators for many crops and wild flowering species. many countries around the world, particularly in the northern hemisphere, rely on the western honey bee, apis mellifera, for commercial pollination of certain crops, but over the recent years some regions of the world have been suffering from an increase in losses in their managed honey bee colonies. the colony collapse disorder (ccd) has been reported for the first time in 2006 in the usa (neumann & carreck, 2010). abstract the western honey bee (apis mellifera l., hymenoptera: apidae) is a species of crucial economic, agricultural and environmental importance. in the last ten years, some regions of the world have suffered from a significant reduction of honey bee colonies. in fact, honey bee losses are not an unusual phenomenon, but in many countries worldwide there has been a notable decrease in honey bee families. the cases in the usa, in many european countries, and in the middle east have received considerable attention, mostly due to the absence of an easily identifiable cause. it has been difficult to determine the main factors leading to colony losses because of honey bees’ diverse social behavior. moreover, in their daily routine, they make contact with many agents of the environment and are exposed to a plethora of human activities and their consequences. nevertheless, a number of different factors are considered to be contributing to honey bee losses, and recent investigations have established some of the most important ones, in particular, pests and diseases, bee management, including bee keeping practices and breeding, the change in climatic conditions, agricultural practices, and the use of pesticides. the global picture highlights the ectoparasitic mite varroa destructor as a major factor in colony loss. last, but not least, microsporidian parasites, mainly nosema ceranae, also contribute to the problem. thus, it is obvious that many factors are involved in honey bee colony losses globally. increased monitoring and scientific research should throw new light on the factors involved in recent honey bee colony losses. this review focuses on the main factors which have been found to have an impact on the increase in honey bee colony losses. sociobiology an international journal on social insects peter hristov1, rositsa shumkova2, nadezhda palova3, boyko neov1 article history edited by evandro nascimento silva, uefs, brazil received 14 september 2020 initial acceptance 08 january 2021 final acceptance 21 january 2021 publication date 22 february 2021 keywords honey bee losses, colony collapse disorder, varroa destructor, viral diseases, nosematosis, negative pressures. corresponding author peter hristov department of animal diversity and resources institute of biodiversity and ecosystem research, bulgarian academy of sciences “acad. g. bonchev” str., bl. 25, sofia, 1113, bulgaria. e-mail: peter_hristoff@abv.bg although some bee losses have also been reported in japan and china, published data from various investigations have shown that honey bee colony numbers have been stable for the past ten years in these regions (taniguchi et al., 2012; liu et al., 2016). a significant rate of honey bee colony losses has been reported in south america as well. in a large-scale study in five countries, argentina, uruguay, chile, brazil, and venezuela, the main factors leading to the honey bee losses have been described (maggi et al., 2016). these include the ectoparasitic mite varroa destructor and the widespread use of acaricides for its treatment, agriculture intensification, 1 department of animal diversity and resources, institute of biodiversity and ecosystem research, bulgarian academy of sciences, sofia, bulgaria 2 research centre of stockbreeding and agriculture, smolyan agricultural academy, bulgaria 3 scientific center of agriculture, sredets, agricultural academy, bulgaria review honey bee colony losses: why are honey bees disappearing? p. hristov, r. shumkova, n. palova, b. neov – honey bee populations decline2 unbalanced bee nutrition leading to honey bee-associated viruses (sacbrood disease in brazil) and parasitic diseases (nosemosis in uruguay and argentina), etc. other studies have highlighted local honey bee losses in different countries in south america. for example, during the period 2013-2014 a survey among bee keepers in uruguay detected around 30% colony losses annually (antúnez et al., 2017). the main factors for this reduction include queen failure, diseases and parasites, and widespread use of pesticides. in a 5-year online survey in brazil (2013-2017) around 50% annual losses were observed, and pesticide exposures were suspected to be the main reason for the colony losses (castilhos et al., 2019). in argentina, coinfections with different pathogens (virus-fungi, mites-virus-fungi, and virus-mite) have been discussed as the main stressors for honey bees, but, according to some authors, there are much more complex reasons leading to honey bee population reduction (garcia et al., 2019). the global picture has shown that there are no significant honey bee colony losses reported in africa and australia. in the middle east, the high temperatures and droughts in the summer are the main factor leading to colony losses because many plants which are important sources for bee forage suffer from heat stress. another factor aggravating the problem is the lack of comprehensive laws and legislations concerning the importation of bee families (muli et al., 2014). indeed, bee colony losses are not a new phenomenon and historical records show that extensive losses were not unusual in the past. whilst recent problems may give the impression that there has been a massive decline, global research on honey bee colonies has shown that numbers actually increased between 1961 and 2007, mostly in asia (426%), africa (130%), south america (86%), and oceania (39%) (fao, 2009). in europe, on the other hand, an extensive study involving 18 european countries during the period 1965-2005 found out a decline of honey bee colonies of about 16.1% (potts et al., 2010). moreover, honey bee losses in europe have revealed a geographic pattern. for example, the number of bee colonies has been decreasing in northern and central european countries, and, likewise, in these countries a falling number of beekeepers has also been noted (potts et al., 2010). on the other hand, in the mediterranean and many south european countries the number of managed bee colonies is increasing (potts et al., 2010). it has been difficult to establish a common pattern for the colony losses, but different investigations confirm that it is a phenomenon characteristic of the western honey bee, while the asiatic honey bee, present in southern, southeastern, and eastern asia, appears to be more resistant to various pests and diseases. role of pests and diseases in honey bee colony losses to understand what is causing the current decrease in honey bee colonies worldwide, it is important to shed light on the key pests and diseases affecting bee health. honey bees are affected by a number of pests and diseases including mites, honey bee-associated viruses, microsporidia, bacterial infections and fungal diseases. due to the burden of infectious diseases and their agents, honey bee colonies may manifest significant weakness or even death. only recently have scientists come to better understand the importance of the development and interactions of these pests and diseases. ectoparasitic mites the hive of the honey bee is a suitable habitat for several mites (acari), including nonparasitic, omnivorous, pollenfeeding species, and parasites. out of different mite species associated with honey bees, varroa destructor, acarapis woodi, varroa jacobsoni and tropilae clareae are economic pests of honey bees and their infestation may lead to the destruction of the beekeeping industry in many cases (sammataro et al., 2000; dhooria, 2016). v. destructor is the most serious pest of honey bee colonies worldwide, and an obligate parasite which is able to attack different developmental stages and castes of a. mellifera (shen et al., 2005). it is interesting to note that varroa mites have been established in new zealand since 2000, but yet, australia remains varroa-free (iwasaki et al., 2015). for several decades in the 20th century v. jacobsoni was the sole cause of “varroosis” in apis mellifera (rosenkranz et al., 2010). for this reason, v. jacobsoni was mentioned in all literary sources in the last century as the main cause of “varroosis”, although in most cases v. destructor was involved. based on sequence analysis of mitochondrial dna (cox1 gene), it became possible to distinguish the new mite species v. destructor as morphologically and genetically different from v. jacobsoni (anderson & trueman, 2000). the european honey bee a. mellifera was introduced for the first time in asia in 1877 in order to improve the productive qualities of apis cerana (sakai & okada, 1973). nearly 80 years after the introduction of the western honey bee in asia, it was observed that v. destructor had switched hosts from a. cerana to a. mellifera by 1957 in japan (sakai & okada, 1973) and by 1963 in hong kong (delfinado, 1963). its range expanded quickly through global humanmediated honey bee trade – both legal and illegal – and most probably via shipping. currently, v. destructor can be found all over the world, except in australia, some extreme northern territories, and remote islands such as the seychelles and comoros archipelagoes (locke, 2016; roberts et al., 2017). in africa, african honey bees survive despite the presence of v. destructor, as do the africanized honey bees in south america. this increased resistance of the africanized honey bees against v. destructor may be explained with their more aggressive behaviour compared to the western honey bee (medina flores et al., 2014; oddie et al., 2018). v. destructor has been present for many years in most countries and is currently considered the biggest threat related to colony losses, not just as a vector in the transmition of honey bee-associated viruses but also due to its detrimental sociobiology 68(1): e-5851 (march, 2021) 3 effects on honey bee colonies (le conte et al., 2010). in the usa, it has been reported that during the winters of 19951996 and 2000-2001 honey bee colony deaths reached 50 to 100% in many apiaries (le conte et al., 2010; pettis & delaplane, 2010). no less were the reported losses due to the v. destructor infestation in europe (le conte et al., 2010; moritz et al., 2010). in central europe, a high number of colony losses was observed in the winter of 2002-2003 (hendrikx et al., 2009) and in southern europe – especially in the winter of 2007-2008 (mutinelli et al., 2010), with most beekeepers reporting v. destructor as the main causative agent of mortality. similar data for the role of v. destructor as a major threat to a. mellifera have been reported in south america (maggi et al., 2016). in contrast to the data mentioned above, it seems that the savannah honeybee a. m. scutellata in africa and a. cerana in asia have the ability to maintain mite populations at low levels, which reflects the low impact on thеsе honeybee populations (strauss et al., 2015; chantawannakul et al., 2016). typical control of v. destructor involves the use of fluvalinate, a pyrethroid, treated strips placed in the hive during times of no honey production. intensive use of these strips has selected for resistance in some parts of europe (floris et al., 2001), the united states (macedo & ellis, 2002), israel (mozes-koch et al., 2000), and mexico (rodríguez-dehaibes et al., 2005). the spread of pyrethroid resistance in europe roughly follows that of the initial spread of the mite according to bee movement, suggesting that resistance evolved once and spread thereafter (büchler et al., 2010). coumaphos, an organophosphate insecticide, was soon introduced for emergency use after control problems with fluvalinate, but resistance to coumaphos is now present in florida (elzen & westervelt, 2002) and northern italy (spreafico et al., 2001). resistance to both pyrethroids and amitraz, an amidine, has been reported in the united states (elzen et al., 2000) and in mexico (rodríguez-dehaibes et al., 2005). thus, the increased resistance of v. dectructor against various insecticides creates a precondition for additional difficulty in combating mites and seeking alternative approaches. viral infections about 24 honey bee-associated viruses have been identified in the western honey bee (apis mellifera) (gisder & genersch, 2015). some of them generally persist in the bee’s body, without causing a disease or manifestation of any clinical signs. in general, virus infestations were not considered to be a significant problem to honey bee health. on the other hand, some viruses are more virulent and infective, and thus may cause a significant loss in honey bee colonies as well as a decline in honey bees’ health and production. some viruses show pathogenicity only under certain favorable environmental conditions. the mite v. destructor is considered to be the main vector of many honey bee-associated viruses: the deformed wing virus (dwv); the acute bee paralysis virus (abpv), the kashmir bee virus (kbv), and the israeli acute paralysis virus (iapv) (locke, 2016; ramsey et al., 2019). furthermore, there are three viruses for which varroa seems to play no significant role in the transmission of, namely, the chronic bee paralysis virus (cbpv), sacbrood virus (sbv), and the black queen cell virus (bqcv) (tentcheva et al., 2004; nielsen et al., 2008). it is interesting to note that about 40 years ago there was no increase in colony losses despite the presence of varroa mites; such losses, however, have become more and more apparent over the last ten years. this fact allows us to think that varroa mites alone are not the cause of honey bee losses. the negative influence of v. destructor results from its role as a reservoir and important vector of some honey bee-associated viruses (shen et al., 2005); the mite promotes replication of honey bee viruses like the dwv (levin et al., 2016). due to its feeding behavior, the varroa mite directly injects viruses in the hemolymph, which has been associated with oral or sexual transmission of these viruses (francis et al., 2013). a large number of studies reflect the relationship between honey bee-associated viruses and colony losses (nielsen et al., 2008; soroker et al., 2011; cornman et al., 2012; granberg et al., 2013; li et al., 2014). research conducted in this direction has shown strong indications for iapv and abpv (both are members of the abpv/kbv/iapv clade) being involved in winter colony losses (cox-foster et al., 2007; genersch et al., 2010) and dwv being a key factor for overwintering colony losses in germany (genersch et al., 2010). the results from cox-foster et al. (2007) revealed that sbv, bqcv, dwv, and abpv viruses were found in both ccd and non-ccd colonies, while iapv and kbv were found only in the ccd colony. the obtained results allowed the authors to determine iapv as a significant marker for ccd. these observations were subsequently confirmed by the study of cornman et al. (2012), who established that viruses were significantly more abundant in ccd colonies, but in contrast to a previous study (cox-foster et al., 2007), they found no positive association between the presence or infection load of iapv and ccd. microsporidia microsporidia are fungal, obligate intracellular parasites infectious to honey bees. microsporidia are possibly the smallest single-cell organisms with a true nucleus. the genus nosema is a parasitic fungus that infects insects such as honey bees, bumble bees and silkworms. two described species of microsporidia, nosema ceranae and nosema apis, parasitize on adult honey bees (paris et al., 2018). it is well known that n. apis is specific for the western honey bee, apis mellifera l., whilst the asiatic bee, apis cerana, harbors n. ceranae (fries et al., 1996). for a long time, it was believed that n. ceranae and n. apis were species-specific. since the beginning of this millennium (mainly post 2003), many investigations have revealed that n. ceranae has switched hosts and has become p. hristov, r. shumkova, n. palova, b. neov – honey bee populations decline4 the dominant species in many countries (klee et al., 2007; paxton et al., 2007; chen et al., 2008; invernizzi et al., 2009; stevanovic et al., 2011). thus, it has been suggested that n. ceranae is possibly more virulent than n. apis. it has been well documented that microsporidia invade the midgut epithelial cells of worker bees, queens and drones (papini et al., 2017). nosema has adverse effects on the bee colony. the negative effect of nosemosis at the colony level relates to productivity and survival of honeybee colonies, including adult bee longevity, queen bees, brood rearing, bee biochemistry, pollen collection and other bee behaviors (botías et al., 2013). in contrast to n. apis, which rarely leads to the death of a diseased colony, since its emergence as a novel pathogen of the western honey bee a. mellifera, n. ceranae has been generally associated with heavily diseased honey bee colonies (vejsnaes et al., 2010). considering n. ceranae as a potential factor in ccd, we may summarize that almost any given disease organism has to persist over time (i.e., there has to be an increase in larval / adult incidence of infection) before causing colony mortality, generally, n. ceranae acts simultaneously with other pathogens. natural n. ceranae infestation can cause a sudden collapse of bee colonies and colony death in autumn or winter, and poor honey production and colony depopulation is six times higher in colonies infected with n. ceranae than in uninfected ones (higes et al., 2008). a metagenomic survey for detection of various pathogens showed a prevalence of some nosema spp. in ccd in contrast to non-ccd colonies (cox-foster et al., 2007). moreover, the presence of more than one of the four pathogens – kbv, iapv, n. ceranae, and n. apis – was observed in ccd colonies. the study on the connections between pathogens and ccd in collapsed colonies revealed an increase in the pathogen level in ccd colonies that was not observed in weak colonies (cornman et al., 2012). the authors also found that in ccd colonies, n. ceranae loads were significantly correlated with the levels of dwv and kbv, which supports the association between nosema spp. infestation and increased susceptibility to other pathogens. bicyclohexylammonium fumagillin, an antibiotic isolated from the fungus aspergillus fumigatus, has been the only widely used treatment for nosemosis, or “nosema disease”, in western honey bees, apis mellifera (higes et al., 2011). the practice of periodic fumagillin treatment results in decreasing but nearly constant exposure of multiple generations of bees and pathogens to the drug. although this practice appears to provide an environment conducive to selection of fumagillin-resistant nosema strains, n. apis has evidently not developed resistance to the drug; however, studies have shown that n. ceranae can reestablish to pretreatment prevalence 6 months after treatments are terminated (pajuelo et al., 2008). some studies have indicated that protein profiles of bees fed fumagillin confirmed our hypothesis that fumagillin affects bee physiology at concentrations that no longer suppress n. ceranae. thus, the use of fumagillin may increase the prevalence of n. ceranae and is potentially a factor in replacement of n. apis by n. ceranae in usa apiaries (huang et al., 2013). in addition to having negative effects on host physiology, fumagillin increases management costs, and residues may persist in the hive, posing risks to human health through honey consumption (van den heever et al., 2015). therefore, there is a need for alternatives to hard chemicals for nosema spp. management. synergistic effects of various diseases and parasites the interaction of nosema spp. and honey beeassociated viruses has been reported for bees co-infected with n. ceranae, cbpv or dwv. one study showed that co-infection of bees with n. ceranae and cbpv resulted in increased replication of cbpv but not mortality (toplak et al., 2013). costa et al. (2011) found a significant negative correlation between n. ceranae spore load and dwv titer in midgut tissues of workers bee. an interesting study has evaluated co-parasitism with varroa (v. destructor) and nosema (n. ceranae/n. apis) on honey bees (a. mellifera l.) with different defense levels (bahreini et al., 2015). the obtained results showed that highmite-mortality-rate (high-mmr) of bees in the nosema (-) group showed greater reductions in mean abundance of mites over time compared with low-mite-mortality-rate (low-mmr) bees, when inoculated with additional mites. however, high-mmr bees could not reduce mite load as well as in the nosema (-) group when fed with nosema spores. mean abundance of nosema spores in live bees and dead bees of both strains of bees was significantly greater in the nosema (+) group. molecular analyses confirmed the presence of both nosema species in inoculated bees but n. ceranae was more abundant than n. apis and unlike n. apis increased over the course of the experiment. collectively, this study showed differential mite mortality rates among different genotypes of bees, however, nosema infection restrained varroa removal success in high-mmr bees (bahreini et al., 2015). other authors found significant colony level variation in infection levels, and subtle differences between the microbiota of colonies with high infection levels versus those with low infection levels (rubanov et al., 2019). two exact sequence variants of gilliamella, a member of the honey bee “core gut microbiome” that has previously been associated with gut dysbiosis, were significantly more abundant in bees from colonies with high nosema loads versus those with low nosema loads. stress of long-distance transportation of honey bee colonies for crop pollination the stress of migratory beekeeping is also a risk factor for the health of bee colonies. for that reason, projects have been developed to test a year-round bee management scheme for large migratory and smaller non-migratory beekeeping sociobiology 68(1): e-5851 (march, 2021) 5 operations with an emphasis on the larger migratory operations that pollinate california almonds (almost half of all managed bees in the usa) (pettis & delaplane, 2010). for a more accurate analysis, observations need to be made in different regions because colony growth and disease epidemiology vary markedly in different parts of the country. the obtained data will provide an opportunity for evaluation of honey bee health between stationary versus migratory practices (pettis & delaplane, 2010). it is well known that in most countries large numbers of hives are transported to multiple locations throughout the country by truck to pollinate seasonal fields and orchards. during transportation, colonies are challenged by a variety of stressors. the condition of a hive prior to transportation is often locally acclimated to ecological conditions which often differ greatly from those of the destination. they are moved between locations at interstate highway speeds and deployed in fields and orchards prior to the bloom. changes in temperature, day length, and nutrient supplementation that bees experience after transportation can increase foraging activity and brood production earlier than would have occurred before relocation and in agricultural environments prior to floral bloom with low availability of resources (fewell & winston, 1992). transportation has been described as a likely contributor to colony loss but the focus has been on changing forage quality and consistency, not stress endured during transportation (oldroyd, 2007). transportation stress has received less attention because of the difficulty of collecting data during shipping. even though transportation lasts only a few days, colonies experience confinement, increased variation in temperature, air pressure, and vibration. during shipping, colonies experience a rapid progression of changing elevation and latitude. proper ventilation is a primary concern because poorly ventilated colonies often die from overheating. the consequences of low-temperature stress are less obvious. a colony may experience extended periods of sub-lethal chill stress and loss of thermoregulation (lt) that affects longterm colony survival without proximate mortality by inducing developmental defects in new brood (groh et al., 2004; jones et al., 2005). colonies have many possible locations on the trailer and may be oriented inward toward a center aisle or outward toward the road which may affect airflow, especially at interstate highway speeds. other factors influencing colony health and survival in addition to different diseases, there are some other factors that lead to colony losses. in certain cases, it is the interaction of these factors that leads to morbidity and mortality, and colony losses. climate conditions climate is a crucial factor affecting temperature and humidity. the humidity in the hives must be maintained as low as possible, while the temperature of the brood must be maintained at 34 °c, and in winter the core temperature of the hive must not fall below 13 °c (nürnberger et al., 2018). this is essential and honey bee colonies must have sufficient access to carbohydrates to maintain these temperatures and survive. prolonged periods of cold or wet weather or the food source becoming depleted can also have a negative influence on honey bee colony health. it can inhibit the flying activity and interrupt nectar and pollen supplies to the hive. in contrast to low temperature, if the brood temperature rises above 34.5 oc, the bees display behavioral differences combined with learning and memory difficulties (wang et al., 2016). the effect of weather on bee colonies as a key factor in ccd has been reported in a survey of honey bee colony losses in the usa (vanengelsdorp et al., 2010). ccd has been linked to changes in bee habitats and malnutrition, both of which are indirectly caused by climate change. in addition, climate change allows invasive species to take over bee hives, spoil stored food, and disrupt many processes within these hives, causing a further decline in bee populations (memmott et al., 2007; thomson, 2010). habitat loss and landscape changes the loss and fragmentation of natural habitats resulting from urbanization or intensification of agriculture lead to the reduction of sources of alternative foraging for the honey bee, and the nesting places of wild bees – hollows and tree hollows, bushes, holes, caves and others (goulson et al., 2008; brown & paxton, 2009). the process of reducing and degrading the terrain occupied by natural vegetation – grassy or tree, escalated in the 20th century and continues today. growing honey bees in an urban environment is considered to have its advantages – alternative food sources throughout the season (parks, alleys, etc.) and a lower risk of pesticide intoxication. however, if the environment is highly urbanized, flowering vegetation may not be sufficient. in addition, there is a risk of man-made pollution as well as collision of flying bees with moving vehicles (goulson et al., 2015). pollinators, managed or wild, cannot escape the various and massive impacts of industrial agriculture: they suffer simultaneously from the destruction of natural habitats caused by agriculture, and, because pollinators’ natural ranges inevitably overlap with industrial farming landscapes, the harmful effects of intensive agricultural practices (kovács-hostyánszki et al., 2016; belsky & joshi, 2019). fragmentation of natural and semi-natural habitats, expansion of monocultures and lack of diversity have a negative impact on honey bee health and survival (patrícioroberto et al., 2014; rollin et al., 2016). destructive practices that limit bee-nesting ability, and the spraying of herbicides and pesticides, industrial agriculture one of the major threats to pollinator communities globally. on the other hand, agriculture systems that work with biodiversity and without chemicals, such as ecological farming systems, can benefit p. hristov, r. shumkova, n. palova, b. neov – honey bee populations decline6 pollinator communities, both managed and wild (földesi et al., 2016; sponsler et al., 2019). by increasing habitat heterogeneity for bees, for example, ecological mixedcropping systems can provide additional flower resources for pollinators. this emphasises the potential beneficial roles of ecological/organic agriculture methods. studies on the arrival of the bees to pollinate almond flowers in california orchards – primarily in five counties between los angeles and the san francisco bay area – marks the start of a brief frenzy of activity. it is the world’s largest pollination event. depending on where your farm is located, some pollination strategies may be more appropriate than others. wild bees are more often found in orchards near natural habitat (potts et al., 2010; lee et al., 2019). in these areas, maintaining natural habitat will be important. growers with orchards far from habitat can diversify pollination strategies by using alternative managed bees, like the blue orchard bee, in addition to honey bees, and by adding flowering resources to support those managed bees and attract wild species. diet and nutrition feeding bees is often insufficient, due to overcrowding of the hives or irregular foraging, and in the conditions of prolonged cold and rainy weather, it is lacking (vanengelsdorp et al., 2009). feeding is deficient in areas with intensive agricultural production, where the so-called stress from a monotonous or “monocultural” diet is observed (goulson et al., 2015). this refers to the continuous foraging of bees on crops in mass flowering, grown in large areas, such as sunflower or rapeseed, as well as acacia, where the purpose is to produce honey or just pollination of plants. other factors related to insufficient nutrition include low-nutrient pollen and nectar, plant species including crops, flowers that contain natural but toxic to bee substances. this is the case for the amygdalin glycoside found in almond flowers (london-shafir et al., 2003; unep, 2010). it is well known that large quantities of food are required for the development and health status of honey bee colonies. undoubtedly, the most important characteristics of food sources are regularity, quality and quantity of the nectar and pollen (decourtye et al., 2011). within intensively farmed agricultural landscapes, nectarand pollen-producing crops may provide a narrow window with mass flowering followed by a shortage and even a complete lack of pollen and nectar resources. a typical example is observed with oilseed crops such as rape (brassica napus l.) and sunflower (helianthus annuus l.), where nectar and pollen resources are usually abundant during the blooming, but only for a short period. the subsequent temporal dearth of resources requires the creation of additional vegetation such as field margins (strips bordering crop fields), hedgerows (linear scrub along field boundaries), woodlands, ponds, ditches, and fallow farm fields (decourtye et al., 2010). the role of nectar and pollen for honey bee health and survival is indisputable, so beekeepers now provide supplements in the form of syrup or pollen in case of deficiency. these cannot fully replace natural compounds in terms of nutritive value. consequently, making sure that honey bees have access to pollen and nectar at the right time in their natural environment, remains the best way to guarantee colony survival (decourtye et al., 2011). the analysis of the nutritional composition of bee bread (mixture of pollen and nectar or honey) has shown a close relation with local land use and therefore available floral resources (donkersley et al., 2014). the finding that bee bread protein content correlates with land use suggests that landscape composition may have an impact on insect pollinators, as poor nutrition may contribute to the widespread and ongoing pollinator population decline by increasing the vulnerability to various stresses. the role of nutrition in the immune response to viral pathogens transmitted by v. destructor has also been analyzed (degrandi-hoffman & chen, 2015). according to this study, the role of the nutritional value of pollen and nectar and the relationship between diet and immunity are crucial in determining individual immune response. doubtlessly, improved nutrition can optimize colony growth and immune responses to honey bee-associated viruses and varroa infestation the influence of pollen nutrients on bee health has also been investigated in healthy and varroa-parasitized bees through digital gene-expression (dge) (alaux et al., 2011). the obtained results have shown that pollen-induced molecular mechanisms have a positive influence on some immune genes expression, thus affecting longevity and the production of some antimicrobial peptides, which helps to increase the immune defense of honey bees. however, the negative impacts of varroa on the bee metabolism and immune functions cannot be overcome by pollen feeding. this demonstrates that varroa infestation is extremely virulent and difficult to control, probably due to the influence of the multiple viruses vectored by the mite. a similar study investigated the influence of varroa infestation on immunological and nutritional status of the honey bee (aronstein et al., 2012). it was observed that protein content was depressed and free amino acid content elevated in varroa-infested pupae, suggesting that protein synthesis is impaired, which affects growth in honey bee. the relationship between the values of nutritional and immunerelated indices was more complex, and the effects on the colony showed the reduced weight of pupae in colonies with high varroa abundance. beekeeping practices specific peculiarities of beekeeping can be the direct cause or a supplement to the complex of stressors that can contribute to colony breakdown. these include artificial, unilateral feeding, antibiotics, acaricides and insecticides applied in the hive, exposure to adverse temperatures and temperature fluctuations, infections and parasites, overexploitation of bee products, unreliable sources of bees sociobiology 68(1): e-5851 (march, 2021) 7 and queens (schierow et al., 2012; capri & marchis, 2013). one-sided selection of the honey bee results in genetic erosion in the species population and a lack of resistance to infectious diseases, mites, beekeeping acaricides applied in hives, etc. (capri & marchis, 2013; johnson & corn, 2015). gmos soybean and cotton varieties, followed by corn, with genetically incorporated genes for insecticide synthesis and herbicide tolerance, were first introduced in the united states in 1996. in 2007, 113 million hectares in different parts of the world (eu is among the exceptions) were sown with genetically modified crops (usda-biotech crop data, 2009). with the expansion of the area planted with these crops, concerns have arisen about the safety of bees and other pollinators. researchers have conducted a number of studies, involving dozens of plant species carrying bt genes of bacillus thuringiensis for resistance to insect pests. the results have been summarized by johnson et al. (2010) and johnson (2015). no evidence of a negative effect of genetically modified plants on the bees feeding on them has been established. exposure to pesticides in the past decades, beekeepers have begun to use agrochemical pesticides not only for many crops, but in forests and other environments for the control of insect pests (moritz & erler, 2016; hladik et al., 2018). the exposure of bees to pesticides is through ingestion of residues found in the pollen and nectar of contaminated plants (crop plants or the weeds around the fields), which is why they pose the greatest danger to bees (pilling et al., 2013; al naggar et al. 2015). in recent years, the application of a new generation of pesticides – neonicotinoids – has been broadly discussed among the scientific and beekeeping communities. they are used worldwide and are widely applied for plant protection (crops, vegetables, and fruits), veterinary products, and biocides for invertebrate pest control in fish farming (simon-delso et al., 2015). a large number of studies reflect the role of neonicotinoids as a factor leading to colony losses (blacquière et al., 2012; goulson, 2013; godfray et al., 2015). in general, the effects of neonicotinoid insecticides exposure may be summarized as: (1) loss of reproduction (brood) which threatens the existence of the colony (decourtye & devillers, 2010), (2) as neurotoxic agents they affect the mobility of bees by inducing symptoms such as knockdown, trembling, uncoordinated movements, hyperactivity, and tremors (medrzycki et al., 2003; colin et al., 2004), (3) influence on olfactory learning and memory (decourtye et al., 2005), (4) increased overwintering bee colony lossеs (faucon et al., 2005; wood et al., 2020) etc. in addition to their individual negative impact, pesticides and various pathogens may interact to have stronger negative effects on honey bee colonies. for instance, the combination of the fungicides chlorothalonil and pyraclostrobin has been found to increase more than twice n. ceranae infection rates in bees that consumed greater quantities of them, suggesting that some fungicides have a stronger impact on bee health (pettis et al., 2013). the interaction between v. destructor infestation and neonicotinoids has resulted in significantly reduced survival of long-lived winter honeybees (straub et al., 2019). the exposure to the formamidine miticide amitraz increases mortality associated with viral infections (fine et al., 2017; o’neal et al., 2017), while synergistic interaction when bee larvae are exposed to clothianidin or the organophosphate dimethoate, in combination, negatively affects the survival and cellular responses in american foulbrood-infected honey bee larvae (lópez et al., 2017). concluding remarks the recent investigations have reported an increase in colony losses in some regions and have prompted investment in more co-ordinated monitoring of bees and research into how pests and diseases, bee diversity, beekeeping practices and bee foraging environment are affecting bee vitality. in addition, land management and environmental conditions affect the availability and quality of food sources as well as the conditions in the hive. effective management of bee colonies under changing situations is dependent on beekeeping practices and bee selection/breeding. all of these factors can impact on bee vitality and bees’ ability to deal with pests and diseases. funding this work was funded by the national scientific fund of the bulgarian ministry of education and science, [grant numbers 06/10 17.12.2016]. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. author’s contributions all authors have equally contributed to the idea of this review. p.h. and b.n. prepared the original draft with considerable contributions from r.s. all authors have substantially contributed to the writing of the final text. moreover, all authors have read and agreed to the published version of the manuscript. conflicts of interest statement the authors declare that they have no conflict of interest. references al naggar, y., codling, g., vogt, a., naiem, e., mona, m., seif, a. & giesy, j.p. 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(2016). low-temperature stress during capped brood stage increases pupal mortality, misorientation and adult mortality in honey bees. plos one, 11: e0154547. doi: 10.1371/journal.pone.0154547 wood, s.c., kozii, i.v., medici de mattos, i., de carvalho macedo silva, r., klein, c.d., dvylyuk, i., moshynskyy, i., epp, tasha. & simko, e. (2020). chronic high-dose neonicotinoid exposure decreases overwinter survival of apis mellifera l. insects, 11: 30. doi: 10.3390/insects11010030 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.6021sociobiology 68(1): e-6021 (march, 2021) the vast majority (>83%) of stingless bee species (hymenoptera: apidae: tribe meliponini) occur in the neotropical region, and they account for about half of the pollinators of native tropical plants, also providing pollination to important crops (camargo, 2013). stingless bees in brazil sum almost 300 species (pereira et al., 2017). however, they have been threatened by emergent pests and pathogens previously associated only with apis (teixeira et al., 2020), and also by anthropogenic activities such as deforestation, habitat loss, and illegal trade. several traits make stingless bees attractive to parasites of honey bee colonies (e.g., food storage, production of volatile substances from fermenting hive products, proteinrich larvae). possibly this is the case of the small hive beetle, abstract the small hive beetle (shb) aethina tumida, a honey bee colony scavenger/parasite native to sub-saharan africa, has been recorded in brazil since 2016 in africanized honey bees. now we detected the intruder parasitizing other susceptible social bees. the present study reports the first occurrence of shb in stingless bees in brazil, and is an alert to authorities and stingless beekeepers to prevent infestations. sociobiology an international journal on social insects sergio nogueira pereira1, luis henrique soares alves2, renata falcão r da costa1, fabio prezoto3, erica weinstein teixeira4 article history edited by evandro nascimento silva, uefs, brazil received 23 november 2020 initial acceptance 21 january 2021 final acceptance 26 january 2021 publication date 03 march 2021 keywords nitidulidae; meliponini; invasive pest; pollinator. corresponding author érica weinstein teixeira https://orcid.org/0000-0003-2141-0650 laboratório especializado de sanidade apícola (lasa) instituto biológico/apta/saa-sp pindamonhangaba, são paulo, brasil. e-mail: erica.teixeira@sp.gov.br aethina tumida murray, 1867 (coleoptera: nitidulidae), a honey bee parasite native to sub-saharan africa, where it was first identified, and now present on almost all continents (neumann et al., 2016). in latin america, this invasive pest was first observed in 2012 in mexico. it was detected in cuba that same year and continued to spread southward, reaching el salvador (2013), nicaragua (2014), costa rica (2015), and brazil in 2015 (antúnez et al., 2019). in africa, shb is a colony scavenger/parasite considered only a minor pest, and, in african subspecies, the beetle does not seem to have negative impacts (neumann & elzen 2004), what seems to be the same on african-derived honey bees present on those latin-american countries. european-derived bees, on the other hand, suffer considerable 1 secretaria de estado de agricultura, pecuária e abastecimento do rio de janeiro (seapa-rj). defesa agropecuária. coordenadoria de defesa sanitária animal. niteroi, rj, brasil. 2 centro universitário de valença (unifaa). valença-rj, brazil 3 universidade federal de juiz de fora (ufjf). departamento de zoologia, laboratório de ecologia comportamental e bioacústica (labec). juíz de fora-mg, brazil 4 laboratório especializado de sanidade apícola (lasa)/instituto biológico/apta/saa-sp, pindamonhangaba-sp, brazil short note occurrence of the small hive beetle (aethina tumida) in melipona rufiventris colonies in brazil id https://orcid.org/0000-0003-2141-0650 https://orcid.org/0000-0003-2141-0650 sn pereira, lhs alves, rfr costa, f prezoto, ew teixeira – aethina tumida in stingless bees colonies in brazil2 damage when parasitized by shb (neumann & elzen 2004), suggesting a potential threat to other susceptible social bees. in brazil, shb has been recorded in rio de janeiro apiaries since 2016 (pereira et al., 2017). local observations indicate that problems may arise when good management practices are not adopted and hives are kept in precarious condition, culminating in high levels of infestation and a large presence of shb in their immature, destructive stage (pereira s. n., unpubl. data). although almost three hundred native bee species are known in brazil, very few are properly managed. stingless bees of the genus melipona are important for honey production (pereira et al., 2017). melipona rufiventris (lepeletier, 1836) is endemic to the cerrado (savanna) biome, where it is designated as an endangered species (silveira et al., 2018). the present study reports the first occurrence of shb in stingless bees in brazil. in january (summer) and april (autumn) 2019, the rio de janeiro official veterinary service (ovs) received reports of shb invasion in two meliponaries (m1 and m2) in the metropolitan area of the capital (2–3 km from the urban center). there were no nearby apiaries. meliponary m1 kept colonies of four stingless bee species: tetragonisca angustula (latreille, 1811) (n = 4); melipona quadrifasciata lepeletier, 1836 (n = 2); m. rufiventris (n = 2); and melipona scutellaris latreille, 1811 (n = 1). the hives of m. rufiventris and m. scutellaris were on the balcony of a house. the other hives were in the orchard on individual stands with ant protection and individual roofs. the soil was sandy and well provided with organic matter (decomposing leaves) from fruit trees (mangifera indica) and garden shrubs. the stingless bee keeper reported finding a beetle inside a m. rufiventris hive three weeks after splitting it. upon inspection, ovs agents found that the colony was weak, no signs of population growth or queen. one shb was aspired from the middle of debris at the bottom of the hive and fixed in 70% alcohol, as well as the specimen captured by the bee keeper. no immature or other adult beetles were found. meliponary m2 contained hives of m. rufiventris (n = 17), m. quadrifasciata (n = 14), nannotrigona testaceicornis lepeletier, 1836 (n = 4), t. angustula (n = 3), and plebeia nigriceps (friese, 1901) (n = 1). the hives were in the backyard on individual stands, under fruit trees (m. indica and citrus spp.) with no fruits on the ground. the soil was moist, sandyloamy. the stingless bee keeper had found two beetles in one m. rufiventris hive. no larvae were present. the hive was weak and the box was precarious. at the time of the visit, ovs inspectors did not find any beetles in the hive that had been exchanged, which contained food reserves and healthy brood combs. beetles were confirmed to be a. tumida by the biological institute, brazil. these results corroborate previous findings indicating that non-apis bees can serve as alternative hosts for the opportunistic parasite shb, particularly when the colony is weak or the beehive is in a poor state. even though there was no damage to the colonies and the infestation rate was extremely small, the possible implications underscore the importance of adopting good beekeeping practices in order to maintain strong and stressfree colonies, especially during the multiplication period, whether natural or managed. some actions to prevent colony invaders are suggested: (i) keep beehive boxes in good condition, (ii) avoid opening the hive unnecessarily or for prolonged periods, (iii) strengthen colonies by providing non-apis-origin supplemental foods, and (iv), whenever necessary, transfer workers from other healthy, strong hives. another point worth noting is that the state of rio de janeiro is mainly covered by atlantic forest and is not endemic for m. rufiventris. thus, it can be assumed that the environment may also be a predisposing factor for shb infestation, generating stress, a fragilizing factor to health problems. there are few reports of shb infestation in colonies of stingless bees. in australia, the strong tetragonula carbonaria colonies quickly eliminated adult beetles, or mummified them (greco et al., 2010). although the lack of coevolutionary history might have prevented the development of defenses (gonthier et al., 2019), some species can have natural defensive traits to deal with invading predators. the loss of a weak tetragonula colony after splitting was reported in the philippines (cervancia et al., 2016). in north america, spiewok and neumann (2006) reported that bombus impatiens colonies were affected by the infestation of shb. in cuba, a. tumida was found in natural colonies of m. beecheii (peña et al., 2014). bobadoye (2019) found shb infestation in three melipona species in kenya (africa) and detected similar volatile chemicals attractive to shb. gonthier et al. (2019) demonstrated that shbs can complete an entire life cycle in association with nests of the solitary bee megachile rotundata. it is premature to say whether shb has the potential to wreak havoc on stingless bee colonies in brazil. nevertheless, this report is an alert to authorities and producers to take precautions to avoid infestations. the absence of shb in a meliponary located 40–50 m from infested honey bee hives, in são paulo, brazil (al toufailia et al., 2017), points to the importance of careful technical management, and good beekeeping practices to reduce vulnerability. acknowledgements we thank the agricultural defense superintendency and the department of animal health of the rio de janeiro state secretariat for livestock, agriculture, and supply for supporting this study. following the notification process, data was sent to the ministry of agriculture, livestock and food supply. authors’ contributions snp: conceptualization, investigation and writing; rfrc: conceptualization, investigation and writing; sociobiology 68(1): e-6021 (march, 2021) 3 fp: conceptualization and writing lhsa: investigation and writing ewt: writing data availability statement data sharing not applicable to this article as no data sets were generated or analysed during the current study. references al toufailia, h., alves, d.a., bená, d.c., bento, j.m.s., iwaniki, n. s. a., cline, a. r.,ellis, j. d. & ratnieks, f. l. w. 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(2020) european foulbrood in stingless bees (apidae: meliponini) in brazil: old disease, renewed threat. journal of invertebrate pathology, 172: 107357. doi: 10.1016/j.jip.2020.107357 https://link.springer.com/chapter/10.1007/978-1-4614-4960-7_2 https://link.springer.com/chapter/10.1007/978-1-4614-4960-7_2 http://ainfo.cnptia.embrapa.br/digital/bitstream/item/166288/1/criacaoabelhasemferrao.pdf http://ainfo.cnptia.embrapa.br/digital/bitstream/item/166288/1/criacaoabelhasemferrao.pdf about:blank about:blank about:blank about:blank open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.317-322sociobiology 60(3): 317-322 (2013) centris aenea (hymenoptera, apidae): a ground-nesting bee with high pollination efficiency in malpighia emarginata dc (malpighiaceae) ga oliveira1, cml aguiar1, m silva2, m gimenes1 introduction the production of west indian cherry or acerola (mal� pighia emarginata dc) is increasing in brazil and a significant part of the fruit production has been exported mainly to the united states, japan, and countries of the european community. the fruit is not only consumed fresh but is also used as raw material for the food and pharmaceutical industries (musser, 1995). brazilian agro-business has had a growing interest in commercializing acerola but pollinator deficit, which results in low fruit set, is one of the factors hindering increases in productivity (schlindwein et al., 2006; guedes et al., 2011). interactions between m. emarginata and its pollinators are based primarily on the availability and collection of floral oils and pollen (oliveira & schlindwein, 2009; vilhena et al., 2012; see freitas et al., 1999, and siqueira et al., 2011) for details on floral morphology). in m. emarginata, flower abstract this study aimed to assess the efficiency of centris aenea lepeletier as pollinator of malpighia emarginata dc. the pollination efficiency was determined according to three criteria: 1) the pollen deposition rate of m. emarginata and other plant species on the thorax ventral region of females, 2) the rate of m. emarginata fruit set after a single visit, and 3) the pollen deposition rate on the stigma after one single visit. we found that 59% of the pollen grains deposited on the ventral region of c. aenea came from flowers of m. emarginata. the fruit set after a single visit by c. aenea (21%) was higher than that reported previously for centris tarsata smith. we argue that pollination efficiency of c. aenea can be related to its body size, since that females of c. aenea can carry out larger amounts of pollen grains of m. emarginata than c. tarsata, as well females of c. aenea are able to touch a larger stigma area, resulting in higher fruit sets after a single visit. our study suggests that only one visit by c. aenea ensures fruit set in m. emarginata. sociobiology an international journal on social insects 1 universidade estadual de feira de santana uefs, feira de santana, bahia, brazil 2 faculdade de tecnologia e ciências, salvador, bahia, brazil article history edited by kleber del-claro, ufu, brazil received 20 july 2013 initial acceptance 09 august 2013 final acceptance 20 august 2013 key words pollination deficit, crop pollinator, bee-plant interaction, oil-collecting bee, west indian cherry corresponding author cândida maria lima aguiar univ. estadual de feira de santana av. transnordestina, novo horizonte 44036-900, feira de santana, bahia brazil e-mail: candida.aguiar@gmail.com pollination is performed only by specialist floral visitors and its pollinators are bees (apidae) belonging to tribe centridini (freitas et al., 1999; siqueira et al. 2011; guedes et al., 2011) (see freitas et al.,1999 for scheme and details on pollinator’s behavior). some cavity-nesting centris species have been suggested as promising candidates for pollination of m. emar� ginata (buchmann, 2004; oliveira & schlindwein, 2009). the main advantage of these species lies in the easiness to obtain and relocate its nests because they undergo nidification in movable trap-nests (jesus & garófalo, 2000; roubik & villanueva-gutiérez, 2009; aguiar & garófalo, 2004; pina & aguiar, 2011). on the other hand, other studies have found that ground-nesting centridini species such as centris aenea lepeletier and centris varia (erichson), reach high abundance in some regions and visit a large number of m. emarginata flowers (siqueira et al., 2011; vilhena et al., 2012), and are important for their pollination services to the crop. research article bees ga oliveira, cml aguiar, m silva, m gimenes pollination efficiency of centris aenea 318 comparative studies on the efficiency of several pollinators are needed to support the choice of the best species to be managed for increases in crop productivity. although several cavity-nesting bees actually have favorable characteristics for management as crop pollinators, the role of each species in crop pollination has been scarcely studied. studies on the nesting-cavity bees in crop areas have often focused on the bee high ability to occupy trap-nests (oliveira & schlindwein, 2009; pina & aguiar, 2011; magalhães & freitas, 2012) rather than on the abundance of bee species in crop flowers (e.g. vilhena et al., 2012) or the efficiency of pollinators (freitas et al., 1999; freitas & paxton, 1998; freitas et al., 2002). preliminary samples obtained in the study area indicated that c. aenea, a ground-nesting bee, was the most abundant visitor on m. emarginata flowers whereas the cavitynesting species centris analis (fabricius) and centris tarsata smith were much less frequent (unpublished data). despite its low frequency in flowers, these two cavity-nesting species use the orchard as their nesting site (pina & aguiar, 2011), and females of c. analis provision their brood cells mainly with pollen of m. emarginata (unpublished data). c. aenea has been registered in other areas as the most frequent potential pollinator of m. emarginata flowers (siqueira et al., 2011; guedes et al. 2011; vilhena et al., 2012) but its pollination efficiency has not yet been studied. for this reason, this study aimed to assess the efficiency of this species as pollinator of m. emarginata, and to support the choice of a potential species for the management to pollination of this crop. materials and methods study site the sample was collected at an acerola orchard (12°16’s, 38°58’w) located in the countryside of the municipality feira de santana, bahia, brazil. the climate is semiarid with average annual precipitation of 802 mm and average annual temperature of 24ºc (cei, 1994). the orchard contains approximately 300 plants of m. emarginata, grown under natural conditions with no irrigation or pesticides. flower visitors the visitors of m. emarginata flowers were sampled using an entomological net once a month, from july 2010 to june 2011, from 0700 to 1700 hours, for 30 minutes every hour. in each sample, two collectors walked throughout the orchard collecting bees in flowering plants. we measured twenty-five females of the most abundant species (c. aenea). their entire body length (between the median ocellus and the end of the abdomen) and intertegular width (the distance between wing bases) were measured with a digital paquimeter. the reproductive system reproductive biology tests (dafni et al., 2005) were performed in november 2010 and april 2011, when more than 50% of the plants were in bloom. two-hundred and forty flowers in pre-anthesis were previously bagged with voil bag and received the following treatments: 1hand self-pollination (autogamy); 2spontaneous self-pollination; 3hand cross-pollination (xenogamy); 4control 1, marked flowers kept under natural conditions and available to flower visitors (natural pollination). flowers were kept bagged for approximately 15 days until fruit set, when bags were removed and the fruit set for each treatment was quantified. pollination efficiency of centris aenea the pollination efficiency of c. aenea was determined according to three criteria. first, the pollen deposition rate of m. emarginata and other plant species on the thorax ventral region of female c. aenea collected while visiting acerola flowers. to reach this goal, all pollen grains from the females ventral region (n=12) were removed with glycerinated gelatin. next, the pollen grain-glycerinated gelatin mixture was dissolved in warm water and centrifuged at 2500 rotations per 10 min; the pollen grains were transferred to assay tubes containing acetic acid for at least 24 h for dehydration and then the material was processed using the acetolysis technique (erdtman, 1960). after this chemical treatment, the pollen grains were kept for at least one hour in a glycerin aqueous solution at 50% for rehydration. for the analysis of the pollen types deposited on females ventral region, four slides were prepared using pollen sample obtained in each female. slides were prepared with colorless gelatin, and one slide also for each sample was prepared with gelatin colored with safranin at 1%. the slides were examined under an optical microscope to identify and count the pollen of m. emarginata and other pollen types. at least 1,000 pollen grains per sample were counted. the second criterion determined the rate of m. emar� ginata fruit set after a single visit by c. aenea. fifty-two flowers in pre-anthesis were bagged for the experiment; twentyfive of them were emasculated. on the next day, flowers were exposed to a single visit by c. aenea and were rebagged to prevent further visitations until fruit set. two procedures were conducted in this experiment: in the first, 77 flowers were kept bagged, insect visits forbidden; in the second, 78 flowers were exposed to unrestricted bee visits (natural pollination, control 2). fruit set was determined 15 days later for both experiments. the third criterion analyzed pollen deposition rate on the stigma. the use of this technique aimed to complement the fruit set rate experiment after a single visit by c. aenea and count the pollen grains of m. emarginata that females of c. aenea were able to deposit on the stigma of flowers after sociobiology 60(3): 317-322 (2013) 319 one single visit. fifty flowers in pre-anthesis were bagged and on the next day, 20 flowers were exposed to a single visit by c. aenea (10 of which were emasculated). after the first visit, the stigmas were removed from flowers and fixed on slides with glycerinated gelatin colored with safranin. ten of the remaining 30 flowers were left bagged to prevent visits and 20 (10 of which were emasculated) were exposed to unrestricted bee visits from 0700 to 1700 h. after this period, the stigmas were also removed and fixed in slides with glycerinated gelatin colored with safranin. the deposited pollen grains were counted in the laboratory. the kruskal-wallis test was used to compare the number of pollen grains deposited on the stigma in each treatment. differences in fruit set rates among treatments of the reproductive system of m. emarginata and after the single visit on c. aenea were analyzed using the chi-square test (χ²). results pollination biology and flower visitors results of the reproductive biology experiments show the occurrence of fruit set in treatments with hand cross-pollination (14.5%) and natural pollination (control 1) (24%); fruit sets did not occur in spontaneous or hand selfpollination experiments (table i). no significant difference in fruit set rates between natural and hand cross-pollination (χ²=1.5905, p=0.207) were found. the number of fruits produced by natural pollination was different from the number of fruits obtained by hand self-pollination or spontaneous self-pollination (χ²=14.524, p<0.001). the number of fruits obtained by hand cross-pollination also differed from hand self-pollination and spontaneous self-pollination (χ²=8.3258, p=0.004). fruits produced by natural pollination (n=96 fruits, control 1) had 2.95 ± 0.2 seeds per fruit. pollination efficiency of centris aenea of the pollen grains (n=8,496) deposited on the ventral region of c. aenea, 59% came from flowers of m. emar� ginata. other pollen types, such as solanum paniculatum, poincianella microphylla, stigmaphyllon sp. and salvia sp. were also found in the samples and comprised approximately 40% of all grains. table 1. fruit set of west indian cherry (malpighia emarginata) in a semiarid area in brazil. table 2. number and proportion of fruits set per malpighia emar�malpighia emar� ginata flower after a single visit by centris aenea, in a west indian cherry crop area in a semiarid area in brazil. fruit set after a single visit by c. aenea was 21% (table ii). there was no difference in the number of fruits produced after a single visit by c. aenea on emasculated (16%) and non-emasculated flowers (26%) (χ²=0.76, p=0.38). similarly, results of these experiments did not differ from experiments obtained from fruit sets in flowers open to visitation (n = 78, 30.7%) (control 2) (χ²=1.46, p=0.22) (table ii). bagged flowers did not produce fruits. after the single visit by c. aenea, pollen grains were deposited on stigmas of all the 20 flowers studied. the number of grains deposited on stigmas of emasculated flowers varied from 6 to 51 (31.7±12.9), whereas it varied from 13 to 67 (33±17.8) on non-emasculated flowers, and no difference in deposition rates among treatments was observed (kruskal-wallis h=10.7, p=0.9296). the number of pollen grains deposited on the stigmas of flowers with unrestricted visits were between 2 and 371 in emasculated flowers and between 6 and 844 in non-emasculated flowers. no significant difference was observed between these two treatments (kruskal-wallis h=10.7, p=0.18). discussion results of the reproductive biology experiments show that the population of m. emarginata we studied did not produce fruits in self-pollination experiments, only in crosspollination experiments and is considered self-incompatible flowers of m. emarginata were visited only by bees belonging to tribe centridini: centris flavifrons (fabricius), c. aenea lepeletier, c. analis (fabricius), c. fuscata lepeletier, c. trigonoides lepeletier, c. tarsata smith, c. obsole� ta lepeletier, c. sponsa smith, epicharis flava (friese) and three other non-identified species of centris. among these, the most abundant species was c. aenea (66% of all bees), with females measuring 15.3±0.9 mm in length and 0.59± 0.23 mm in width. trea tment number of flowers number of fruits set flowers tha t set fruits (%) sponta neous self-pollina tion 53 0 0 ha nd self-pollina tion 53 0 0 ha nd cross-pollina tion 55 8 14.5 na tura l pollina tion (control 1) 54 13 24 trea tment number of flowers number of fruits set flower tha t set fruits (%) flowers non-ema scula ted (exposed to one visit) 27 7 26 flowers ema sculed (exposed to one visit) 25 4 16 flowers ba gged (without visits) 77 0 0 na tura l pollina tion (control 2) 78 24 30.7 ga oliveira, cml aguiar, m silva, m gimenes pollination efficiency of centris aenea 320 and dependent on biotic agents (centridini bees) for pollination. however, the fruit set rate by cross-pollination was low in unrestricted visits experiments (natural pollination), as also observed in other brazilian regions (freitas et al., 1999; guedes et al., 2011). the lack or low fruit set rates for the crop by self-pollination was also observed by other researchers (schlindwein et al., 2006; guedes et al., 2011, siqueira et al., 2011). self-pollination rates between 11% and 35% were observed for several varieties in irrigated m. emargina� ta orchards (siqueira et al., 2011). as such, low fruit set per inflorescence and dependence on entomophilus pollination seem to be traits of this plant species (freitas et al., 1999; siqueira et al., 2011). a plausible hypothesis to explain the low fruit set observed in m. emarginata is that this reproductive factor is directly related to the frequent ovule abortion in this plant species (miyashita et al., 1964; freitas et al., 1999), whereby few fruits are produced in spite of flower abundance. on the other hand, the low m. emarginata fruit set rate has also been explained by climatic conditions such as temperature, rainfall and luminosity, by genetic factors, and the low abundance of orchard pollinators (yamane & nakasone, 1961; schlindwein et al., 2006, carpentieri-pípolo et al., 2008; siqueira et al., 2011; guedes et al., 2011). guedes et al. (2011) recently registered pollination deficit in m. emargina� ta in a brazilian semiarid region during the dry season, when pollinator abundance was much lower than during the rainy season. those authors obtained a significantly higher fruit set after applying complementary hand cross-pollination, after natural pollination performed by bees during the dry season. in our study, pollination experiments were conducted whenever pollinator abundance was high, and therefore the low fruit set rate after natural pollination might not be related to the lack of pollinators. centris aenea was the most important of all species of centris visiting flowers of m. emarginata, not only due to its abundance but also for its high floral fidelity, noticeable primarily by the frequent presence (more than 50% of all pollen grains) on the thorax ventral region of m. emarginata. c. aenea was also the most abundant species on acerola flowers at our study site, particularly during blooming peaks (unpublished data). an even higher abundance (above 80%) of c. aenea on acerola flowers was reported in a study conducted in the semiarid region of bahia state (siqueira et al., 2011). these data show that c. aenea prefers foraging for pollen on m. emarginata flower in acerola orchards, thus a positive assessment of the bee efficiency as effective crop pollinator. the average number of pollen grains observed on the stigma after the single visit by c. aenea was enough to fertilize the ovules and produce fruits because only three pollen grains are required for fruit formation in m. emarginata. cruden (2000) argued that the number of pollen grains deposited on the stigma indicates pollinator efficiency and depends on several factors such as size of the stigma area and duration of stigmatic receptivity. stigmatic receptivity in m. emarginata lasts more than six hours (freitas et al., 1999; schlindwein et al., 2006), a trait that can increase the exposure time of flowers to pollinators as well as the chances for the effective pollination to occur because a larger number of co-specific pollen grains can be deposited on the stigmas, as already known (dafni et al., 2005). as a consequence, there would be fewer chances for stigma contamination with hetero-specific pollen, avoiding clogging and leading to higher fruit sets and more effective pollinator visitations (slaa & biesmeijer, 2005). we observed that m. emarginata fruit set after a single visit by c. aenea (21%) was much higher than that found for c. tarsata (7%) (freitas et al., 1999). they reported that more than one visit per flower by c. tarsata is necessary for a high fruit set to be achieved, whereas our study suggests that only one visit by c. aenea ensures fruit set in m. emarginata. the different efficiencies in pollination after a single visit between the two bee species might not be due to behavior because both species have the same behavior pattern during floral oil collection on these flowers (freitas et al., 1999; schlindwein et al., 2006; siqueira et al., 2011). however, pollination efficiency can be related to body size because c. aenea is approximately 1.5 times bigger than c. tarsata (n= 9, length = 10.4 ± 0.5 mm; width = 3.5 ± 0.25 mm; personal observation). body size can be related to the amount of pollen remaining on the pollinator body and by consequence, the amount of pollen to be deposited on the stigma, as also observed by cruden and miller-ward (1981) and by cruden (2000). due to its size, females of c. aenea can be more efficient in transporting larger amounts of pollen grains of m. emarginata than c. tarsata, as well females of c. aenea are able to touch a larger stigma area, resulting in higher fruit sets after a single visit. pollination of the m. emarginata population studied was conducted primarily by a ground-nesting bee, c. aenea. this bee was a highly efficient pollinator very abundant in flowers, whereas other species of centridini, including the cavity-nesting species were much less frequent. maintaining areas with adequate soil conditions for nesting of c. aenea, and also surrounding areas with vegetation that supplements the supply of nectar and even pollen when m. emarginata blooming is low are recommended strategies to keep the local populations of this pollinator. acknowledgments we thank to the national council for scientific and technological development, brazil (cnpq) (proc. #475715/2008-0; #562518/2010-0) and to the fundação de amparo à pesquisa do estado da bahia (fapesb) (to app0042/2009) for financial support for this project. c.m.l. aguiar received a research fellowship from cnpq (proc. 306403/2012-9), g.a. oliveira received a m.sc. scholarship sociobiology 60(3): 317-322 (2013) 321 from capes and m. silva received a postdoctoral fellowship from fapesb. we are grateful to the universidade estadual de feira de santana (uefs) for logistic support. references aguiar, c.m.l. & garófalo, c.a. 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(2012). acerola pollinators in the savanna of central brazil: temporal variations in oil-collecting bee richness and ga oliveira, cml aguiar, m silva, m gimenes pollination efficiency of centris aenea 322 a mutualistic network. apidologie, 4: 51–62. doi: 10.1007/ s13592-011-0081-1 yamane, g. & nakasone, h.h. (1961). pollination and fruit set studies of acerola malpighia glabra l. in hawai. proc. am. soc. hortic. sci., 78: 141-148. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i2.1615sociobiology 64(2): 159-165 (june, 2017) urn:lsid:zoobank.org:pub:b3e44fcc-b5f0-44fd-a054-0ab8405fc043 thaumatomyrmex fraxini sp. nov. (hymenoptera: formicidae), a new ant species from the brazilian atlantic forest introduction thaumatomyrmex (hymenoptera: formicidae) is a small neotropical ant genus originally proposed by mayr (1887) for the brazilian species thaumatomyrmex mutilatus in the subfamily ponerinae lepeletier de saint-fargeau, 1835. the genus remained monotypic during a long time until the second known species, thaumatomyrmex ferox, was described by mann (1922). currently the genus accounts 12 valid species (bolton et al., 2016). weber (1939) offered the first available species identification key and this was later updated by kempf (1975) who first revised the genus, also describing thaumatomyrmex contumax from brazil. due to its very peculiar morphology, emery (1901) first placed this genus in its own tribe thaumatomyrmecini [thaumatomyrmii] but, recently, based on the results of a detailed phylogenetic analysis using molecular and morphological data, schmidt abstract a new species of ponerinae, thaumatomyrmex fraxini d’esquivel and jahyny (hymenoptera: formicidae), is described from several localities in northeastern brazil, after the morphology of the worker. this species is easily distinguished from any other ones in the genus by a unique combination of characters that justify its allocation to the species-group ferox, sensu kempf (1975). the known distribution of this species reveals that it inhabits different ecosystems of the atlantic forest biome. sociobiology an international journal on social insects ms d’esquivel1, 2, bjb jahyny3, ml oliveira2, lsr lacau2, jhc delabie1, 4, s lacau1,2 article history edited by gilberto m. m. santos, uefs, brazil received 25 march 2017 initial acceptance 10 may 2017 final acceptance 01 july 2017 publication date 21 september 2017 keywords biodiversity, tabuleiro forest, taxonomy, ponerinae. corresponding author sébastien lacau universidade estadual do sudoeste da bahia laboratório de biossistemática animal itapetinga-ba, brasil. e-mail: slacau@uesb.edu.br and shattuck (2014) combined the genus thaumatomyrmex inside the tribe ponerini lepeletier de saint-fargeau, 1835. further additional information on the species taxonomy for this genus is offered in bolton et al. (2016). thaumatomyrmex species are distributed between mexico and northern argentina, with occurrence in caribbean islands (jahyny et al., 2015). they are small to median ants inhabiting a range of environments, such as tropical wet or dry forests, savannas and semi-arid regions with xerophytic vegetation, being collected up to 2000m altitude. their colonies, the smallest for population size in the formicidae family, with about 3-4 individuals for some species, live in natural cavities in the soil, the leaf-litter or the tree trunks (delabie et al., 2000; jahyny, unpub. data). they are specialist-predator feeding on polyxenid millipedes (diplopoda: penicillata) (brandão et al., 1991; jahyny et al., 2008; rabeling et al. 2012). 1 pós-graduação em zoologia, universidade estadual de santa cruz (uesc), ilhéus-ba, brazil 2 laboratório de biossistemática animal, universidade estadual do sudoeste da bahia (uesb), itapetinga-ba, brazil 3 colegiado de ciências biológicas, universidade federal do vale do são francisco (univasf), petrolina-pe, brazil 4 laboratório de mirmecologia, comissão executiva do plano de lavoura cacaueira (ceplac), ilhéus-ba, brazil research article ants ms d’esquivel et al. – a new ant species from the brazilian atlantic forest160 hereafter we describe a new species of thaumatomyrmex after the morphology of the worker. this was discovered in a remnant fragment of forest from southern bahia, brazil belonging to the “tabuleiro forest” ecological system, native vegetal formation known as “floresta ombrófila densa das terras baixas” (ibge, 2012) or “floresta pluvial dos tabuleiros” (thomas, 2003). in the central corridor of atlantic forest biome, the ant’s fauna inhabiting this ecosystem remains poorly known (oliveira et al., 2015), since very few studies on its diversity were published for this region. the only available studies on ant’s communities were published by leal et al. (1993) and delabie et al. (1997) who, respectively, recorded a list of 61 and 66 species of ants in a same remnant fragment of tabuleiro forest from northern espírito santo state. also, delabie et al. (2007) studied the ant fauna inhabiting the leaflitter of a cocoa plantation in the mucuri region (southern bahia state), farmed following the “cabruca” production system in a such remnant fragment of tabuleiro forest. however, some recent taxonomic studies suggest a strong endemism locally. for example, fernandez et al. (2009) and oliveira et al. (2015) respectively described a new myrmicine genus (diaphoromyrma) and a new pheidole species (pheidole protaxi) from a same remnant fragment of tabuleiro forest from southern bahia, all being endemic. also, fernandez et al. (2014) described a new monotypic genus (kempfidris) from which the type species has locus typicus in a remnant fragment of tabuleiro forest from southern bahia. for this reason, since the last remnant fragments of native forest are strongly threatened in the “biodiversity crisis” context (wilson, 1988), the discovery of a new thaumatomyrmex species here described significantly contributes to increase our knowledge on the ants’ diversity in this ecosystem. this paper is a partial result of an integrative research project named: “contribution to the study of the ants fauna in lowland ombrophilous forests from the atlantic forest biome”. material and methods the taxonomic definition of the new ant species here described result from the integrative establishment of its diagnosis and taxonomic affinities, based on a complex analytical process of a large amount of elementary morphological and taxonomic data relative to all valid species of thaumatomyrmex mayr, 1887. taxonomic nomenclature follows bolton et al. (2016). taxonomic and specimens’ data were managed by using the software mantis® version 2.0 (http://140.247.119.138/mantis/) (naskrecki, 2008). all samples bear a unique specimen-level identifier (id) affixed to the pin, being written in the text as: “[lbsa_sa_specimen codes]”, where lbsa refers to the acronym for collection of the laboratório de biossistemática animal, universidade estadual do sudoeste da bahia, itapetingaba, brazil. other depository collections for type material are referred to by the following acronyms: cpdc, centro de pesquisas do cacau, comissão executiva do plano de lavoura cacaueira (ceplac), itabuna-ba, brazil; mpeg, museu paraense emilio goeldi, belém, pará, brazil; mzsp, museu de zoologia da universidade de são paulo, brazil”. one time this new taxon will be published, in order to avoid the concentration of all types in only brazilian institutions, we will ask for official governmental authorizations allowing us to send some paratypes in several museum of europe and u.s.a. depending of the morphological structure considered, morphological concepts and terminology in this paper follow richards (1956), eady (1968), kempf (1975), harris (1979), gauld and bolton (1988), goulet and huber (1993), kugler (1994), bolton (1994) and keller (2011). direct morphological examination of specimens was completed at various magnifications using a light stereomicroscope olympus szx7. morphometric measures were made with a carl zeiss measuring microscope and recorded to the nearest 0.01 mm. all measurements are given in millimeters, using the following definitions and abbreviations: el (eye length): the maximum diameter of the eye. gl (gaster length): the length of the gaster in lateral view from the anteriormost point of first gastral segment (third abdominal segment) to the posterior most point. hl1 (head length 1): is the distance between two parallels drawn through the anteriormost point of projecting frontal lobes and the posteriormost point of vertex, in full-face view (this corresponds to the measure of hl in kempf (1975)). hl2 (head length 2): maximum distance from the mid-point of the anterior clypeal margin to the mid-point of the posterior margin of vertex, measured in full-face view. hw1 (head width 1): the maximum width of cephalic capsule proper measured anterior to the eyes, the head in dorsal view. hw2 (head width 2): the maximum width of cephalic capsule proper measured posterior to the eyes, the head in dorsal view. hfl (metafemur length): maximum length of metafemur, measured from the junction with the trochanter to the junction with the tibia. ifw (interfrontal width): the maximum distance measured between the outer borders of frontal lobes across the front. ml (mandible length): length of a mandible measured in ventral view from its basal articulation to its apex. pth (petiolar node height): maximum height of petiolar node measured in lateral view. ptl (petiole length): the maximum length of the petiole in lateral view. ptw (petiolar node width): maximum petiolar node width, measured in dorsal view. pw (pronotal width): maximum width of pronotum measured in dorsal view. sl (scape length): maximum scape length, excluding basal condyle and neck. tl (total length): tl= hl+wl+ptl+gl wl (weber’s length): diagonal length, measured in lateral view, from the anterior margin of the pronotum (excluding the collar) to the posterior extremity of the metapleural lobe. sociobiology 64(2): 159-165 (june, 2017) 161 indices have the following abbreviations and definitions: ci (cephalic index): hw1/hl1x100 hfi (hindfemur index): hfl/hw1x100 ifi (inter frontal index): ifw/hw1x100 mi1 (mandibular index 1): ml/hl1x100 mi2 (mandibular index 2): ml/hflx100 mi3 (mandibular index 3): ml/hw1x100 pi (pronotum index): hw1/pwx100 standard high-resolution microphotographs were produced through a multi-focused montage processing using las v4.4 software (https://www.leica-microsystems. com/applications/education/details/product/leica-las-ez/), from a series of source images taken by a leica ch-9435 heerburugg digital camera attached to a leica m165c microscope. each final microphotograph was improved using the tools of the photo editor module in the software adobe element photoshop (version 6. 0). when available, highresolution microphotographs of type and non-type specimens of other valid thaumatomyrmex species and their synonyms were studied, either being downloaded from internet (i.e.: iconographical banks of mcz type database @ (http://insects. oeb.harvard.edu/mcz/), smithsonian ant type specimen image database (http://ripley.si.edu/ent/nmnhtypedb/public/ browse.cfm) and antweb (www.antweb.org) or extracted from publications referring to its original descriptions or revisions. management of all morphological illustrations produced or collected (microphotographs and drawing) with its identification and indexation by key-words was carried out using the organizer module of the software adobe photoshop elements® version 6.0. comparative of all morphological and morphometric data originated from direct observations or from taxonomic literature was performed by using the software xper²® (http:// infosyslab.fr/?q=en/resources/software/xper2) (lis, 2016). results taxonomic treatment class insecta linnaeus, 1758 order hymenoptera linnaeus, 1758 family formicidae latreille, 1809 subfamily ponerinae lepeletier de saint-fargeau, 1835 tribe ponerini lepeletier de saint-fargeau, 1835 genus thaumatomyrmex mayr, 1887 thaumatomyrmex fraxini d’esquivel and jahyny, new species urn:lsid:zoobank.org:act:5bf88cb0-5a114207-9 013-cf79fdfa8f2d type material. holotype: one worker deposited in cpdc and labeled [see data]: [lbsa_sa_14015217], [brazil: bahia, belmonte, barrolândia, ceplac/egreb, 16°5’33.04”s, 39°12’17.64”w, elev. 109 m] and [col. s. lacau, l.b. godinho, m.r. da silva jr, m.l. oliveira, 05.09.2008]. paratypes (n=28): 2 workers with the same data as holotype, in cpdc ([lbsa_sa_14015192], [lbsa_sa_14015582]); 1 worker [brazil: bahia, ilhéus, cepec] and [col. j. maia, 07.06.1996], in cpdc ([lbsa_sa_14015569]); 1 worker [brazil: bahia, ilhéus, cepec] and [col. p. terra, 14.04.1987], in cpdc ([lbsa_sa_14015570]); 1 worker [brazil: bahia, ilhéus, cacaual] and [col. j.c.s. carmo, 05.1998], in cpdc ([lbsa_sa_14015572]); 1 worker [brazil: bahia, ilhéus, cepec] and [col. j.c.s. carmo, 04.1996], in cpdc ([lbsa_sa_14015573]); 1 worker [brazil: bahia, ilhéus, cepec, zoologia] and [10.07.1998], in cpdc ([lbsa_sa_14015580]); 2 workers [brazil: bahia, ilhéus, banco do pedro, 144051 s, 0391524 w] and [col. j.r.m. santos, j.c.s. do carmo, 12.01.1998], in cpdc ([lbsa_sa_14015571], [lbsa_sa_14015583]); 2 workers [brazil: bahia, ilhéus] and [col. j.c.s. carmo, 06.1997], in cpdc ([lbsa_sa_14015574], [lbsa_sa_14015575]); 3 workers [brazil: bahia, ilhéus, olivença] and [col. v.r.l. mello, 06-09.08.1996], deposited in cpdc ([lbsa_ sa_14015576], [lbsa_sa_14015577]), mzsp ([lbsa_ sa_14015578]) and mpeg ([lbsa_sa_14015579]); 2 workers [brazil: bahia, camacan] and [col. j.r.m. dos santos, 27.08.1999], in cpdc ([lbsa_sa_14015592], [lbsa_sa_14015593]); 2 workers [brazil: bahia, una] and [col. j.r.m. dos santos, 24.08.1998], in cpdc ([lbsa_ sa_14015612], [lbsa_sa_14015613]); 1 worker [brazil: bahia, mascote] and [col. j.r.m. dos santos, 18.06.1999], in cpdc ([lbsa_sa_14015562]); 2 workers [brazil: bahia, canavieiras, oiticica] and [col. j.r.s. carmo, 30.03.1998], in cpdc ([lbsa_sa_14015563], [lbsa_sa_14015564]); 3 workers [brazil: bahia, canavieiras, oiticica], [col. j.c.s.carmo, j.r.m. santos, 09.10.1998], in cpdc ([lbsa_ sa_14015565]), mzsp ([lbsa_sa_14015566]) and mpeg ([lbsa_sa_14015567]); 3 workers [brazil: bahia, itapebi] and [col. j.r.m santos, 16.07.1997], in cpdc ([lbsa_sa_14015609], [lbsa_sa_14015610], [lbsa_ sa_14015611]); 1 worker [brazil: bahia, santa cruz cabrália, 455321 s, 8203867 w] and [col. j.r.m. santos, j.c.s. carmo, 08.08.2006], in cpdc ([lbsa_sa_14015568]). other examined material: 5 workers [brazil: sergipe, crasto, santa luzia] and [col. j. jardim, 28.11.1993], in cpdc ([lbsa_sa_14015594], [lbsa_sa_14015595], [lbsa_sa_14015596], [lbsa_sa_14015597], [lbsa_ sa_14015598]); 2 workers [brazil: bahia, taboquinha] and [col. j.r.m. santos, 06-20.12.1996], in cpdc ([lbsa_sa_14015602], [lbsa_sa_14015603]); 1 worker [brazil: bahia, ilhéus, cepec, zoologia] and [10.07.1998], in cpdc ([lbsa_sa_14015581]); 1 worker, [brazil: bahia, ilhéus] and [col. j.r.m. santos, 22.09.1997], in cpdc ([lbsa_sa_14015587]); 1 worker [brazil: bahia, ilhéus] and [j.r.m. santos, j.c.s. carmo, 06.10.1997], in cpdc ([lbsa_sa_14015588]); 1 worker [brazil: bahia, ilhéus, olivença] and [col. j.c.s. carmo, j.r.m. santos, 09.11.1998], in cpdc ([lbsa_sa_14015584]); 1 worker ms d’esquivel et al. – a new ant species from the brazilian atlantic forest162 [brazil: bahia, ilhéus, olivença] and [col. j.r.m. dos santos, 16.11.1998], in cpdc ([lbsa_sa_14015591]); 1 worker [brazil: bahia, buerarema] and [col. j.r.m. santos, 22.11.1996], in cpdc ([lbsa_sa_14015586]); 1 worker [brazil: bahia, una] and [col. h.j. santos, 24.08.1996], in cpdc ([lbsa_sa_14015590]); 3 workers [brazil: bahia, una, cacaual] and [col. j.c.s. carmo, 09.05.1998], in cpdc ([lbsa_sa_14015599], [lbsa_sa_14015600], [lbsa_sa_14015601]); 2 workers [brazil: bahia, ibicaraí, km 41] and [col. j.r.m dos santos, 21.11.1998], in cpdc ([lbsa_sa_14015607], [lbsa_sa_14015608]); 1 worker [brazil: bahia, itororó, 14°58’28s, 40°03’01w] and [col. j.c. carmo, 11.08.2000], in cpdc ([lbsa_sa_14015589]); 6 workers [brazil: bahia, itapetinga, mata uesb] and [col. m. oliveira, 2008], in cpdc ([lbsa_sa_14015614] [lbsa_sa_14015615], [lbsa_sa_14015616], [lbsa_ sa_14015617], [lbsa_sa_14015618], [lbsa_sa_14015 619]); 3 workers [brazil: bahia, itambé] and [20.08.2006], in cpdc ([lbsa_sa_14015604], [lbsa_sa_14015605], [lbsa_sa_14015606]); 1 worker [brazil: bahia, vitória da conquista, pau brasil] and [col. j.r.m. dos santos, 16.07.2003], in cpdc ([lbsa_sa_14015585]). etymology: this species is named in honor to the professor dominique fresneau, a french ethologist who devoted his life studying the behavior of neotropical ponerines. the specific name “fraxini” is the genitive of fraxinus, the latin genus name of the ash tree (oleaceae) described by linnaeus, 1753. the family name fresneau is an old french name for these trees. diagnosis: the worker morphology of this new species exhibits all the diagnostic characters of the genus thaumatomyrmex. it differs from all other known species by the unique combination of the following characters: 1. head in dorsal view, with cephalic capsule subquadrate, slightly longer than wide (ci = 88.1-98.3). 2. maximal width of head less than 1,5 times the maximal width of pronotum (pi = 130-147.4). 3. maximal width of frons greater than that of pronotum. 4. mesosoma in lateral view, with outline of the dorsal face of mesonoto-propodeal complex drawing a single convexity (metanotal groove and suture absent), weak and symmetrical. 5. mesosoma in lateral view, with outline of the dorsal face of mesonoto-propodeal complex separated from that of the postero-lateral margin of propodeum by a nearly right angle. 6. propodeum with postero-lateral margins sharply marked and straight, nearly angulated but without defining any carinae. 7. segment abdominal 2 (petiole) in lateral view, with the node not differentiating a dorsal face, the outline of anterior face meeting that of posterior face in an acute angle. 8. mandible with a proximal tooth well differentiated (triangular shaped, as long as wide basally). 9. mandibles slightly shorter than the maximal head width in front of eyes (mi3 = 86.8-98.2). 10. mandibles slightly shorter or longer than the maximal length of femur (mi2 = 88.9-111.1). 11. median part of clypeus smooth and shining. 12. lateral parts of clypeus with longitudinal carinae. 13. frontal lobes with longitudinal carinae. 14. front mostly smooth and shining, except its more anterolateral parts at the posterior end of frontal carinae with dense longitudinal rugulae. 15. vertex smooth and shining. 16. genae smooth and shining, except the supra-ocular areas with some longitudinal microrugulae. 17. mesosoma smooth and shining. 18. metasoma smooth and shining. 19. clypeus with two pair of erect setae (long, slightly curved, forward directed, thin, only apically acute) situated at lateral margin of anterior half of clypeus, the most anterior being one third longer. 20. postero-lateral margins of propodeum with two erect setae (long, strongly curved, inward directed, thin, only apically acute). description: here we provide some complementary descriptive elements of the morphology of this new species through the presentation of high resolution microphotographs (see figures 1-12) and the following morphometric data: data for holotype given in [brackets]; means with standard deviations for paratypes (n=15) given in (parenthesis); maximum range for paratypes (n=15) given in {brace}. measurements: el [0.17] (0.16±0.01) {0.14 – 0.18), gl [1.11] (1.10±0.14) {0.95 – 1.47), hfl [0.50] (0.52±0.03) {0.45-0.57}, hl1 [0.60] (0.59±0.02) {0.55-0.62}, hl2 [0.58] (0.55±0.02) {0.52-0.58}, hw1 [0.57] (0.55±0.02) {0.52–0.59}, hw2 [0.57] (0.56±0.02) {0.54–0.60}, ifw [0.40] (0.39±0.02) {0.36-0.42}, ml [0.52] (0.51±0.03) {0.46-0.57}, pth [0.55] (0.46±0.05) {0.40-0.55}, ptl [0.32] (0.26±0.02) {0.23-0.32}, ptw [0.52] (0.48±0.03) {0.44-0.54}, pw [0.41] (0.40±0.01) {0.38-0.42}, sl [0.43] (0.41±0.02) {0.40-0.45}, tl [2.86] (2.77±0.17) {2.57-3.19}, wl [0.83] (0.81±0.03) {0.73-0.86}. indices: ci [95] {88.198.3}, ifi [70.2] {64.3-75}, mi1 [86.7] {79.3-95}, mi2 [104] {88.9-111.1}, mi3 [91.2] {86.8-98.2}, pi [139] {130-147.4}. geographic range: thaumatomyrmex fraxini sp. nov. is only known from the states of bahia and sergipe in brazil, with a geographic distribution limited to a latitudinal range between 11°21’s 16°15’s and a longitudinal range between 37°25’w 40°43’w. its altitudinal distribution reaches 1010m. it is encountered in several types of native forests in the central corridor of the atlantic forest biome where it may be locally sympatric either with t. mutilatus or t. contumax. also, t. fraxini may be found in agroecosystem like cocoa plantation, and in others anthropic environments. biology: the colonies inhabit small cavities in the leaflitter, with a preference for snail shells. this very small species is not a strict predator of penicillata (myriapoda, diplopoda), but also capture specimens of collembola (hexapoda, insecta), not so much small comparatively to its size. this species forms very small colonies without queen. sociobiology 64(2): 159-165 (june, 2017) 163 discussion regarding its morphological definition, the new species here described is easily distinguished from any other one in the genus. plate 1. thaumatomyrmex fraxini sp. nov., holotype worker [lbsa_sa _14015217]. fig. 1: habitus, left lateral view. fig. 2: head, dorsal view. plate 2. thaumatomyrmex fraxini sp. nov., holotype worker [lbsa_ sa_14015217]. fig. 3: mesosoma, dorsal view. fig. 4: petiole, dorsal view. plate 3. thaumatomyrmex fraxini sp. nov., worker [lbsa_sa_ 14015561]. images from antweb (www.antweb.org); photographer r. keller. fig. 5: head with detail of mouthparts, antero-ventral view. fig. 6: head, anterior view. fig. 7: head with details of eye and gena, left lateral view. fig. 8: head (partial) with detail of mandibles shape, dorsal view. plate 4. thaumatomyrmex fraxini sp. nov., worker [lbsa_sa_ 14015561]. images from antweb (www.antweb.org); photographer r. keller. fig. 9: mesosoma, left lateral view. fig. 10: petiole, left lateral view. fig. 11: gaster, left lateral view. fig. 12: gaster (partial) with detail of pygidium and hypopygium, left lateral view. ms d’esquivel et al. – a new ant species from the brazilian atlantic forest164 if using the taxonomic identification key of kempf (1975), thaumatomyrmex fraxini sp. nov. differs from t. contumax and t. mutilatus by its diagnostic characters 1, 11, 13, 14, 15, 16, 17, 18 and 19 (see dichotomy 1 and 2). moreover, t. fraxini also differs from these species by its diagnostic characters 5 and 6. also, t. fraxini differs from thaumatomyrmex cochlearis creighton, 1928 by its diagnostic characters 11, 14, 16, 17, 18 and 20 (see dichotomy 1 and 2). finally, t. fraxini may not be any one of the species keyed at dichotomy 4 (thaumatomyrmex atrox weber, 1939; t. ferox; thaumatomyrmex manni weber, 1939, thaumatomyrmex paludis weber, 1942 and thaumatomyrmex zeteki smith, 1944) because of its diagnostic character 5 and 6. moreover, t. fraxini differs from t. atrox, t. ferox and t. manni by its diagnostic characters 1 and 4. also, t. fraxini differs from t. zeteki by its diagnostic characters 4. considering the thaumatomyrmex species described posteriorly to the revision of kempf (1975), t. fraxini differs from thaumatomyrmex bariay fontenla rizo, 1995 by its diagnostic characters 1, 5, 7 and 14. also, t. fraxini differs from thaumatomyrmex mandibularis baroni urbani & de andrade, 2003 by its diagnostic characters 1, 5, 7 and 14. thaumatomyrmex fraxini differs from thaumatomyrmex nageli baroni urbani & de andrade, 2003 by its diagnostic characters 5 and 7. thaumatomyrmex fraxini differs from thaumatomyrmex soesilae makhan, 2007 by its diagnostic characters 1 and 2. we attempted to attribute thaumatomyrmex fraxini to one of the species groups proposed by kempf (1975). we concluded that this new species may not belong to the mutilatus or cochlearis groups, since it exhibits several incoherence’s with their respective definition. thus, t. fraxini differs from the members of the mutilatus group by the following morphological characteristics: different patterns for the sculpture (diagnostic characters 11 to 18) and chaetotaxy (diagnostic characters 19 and 20). also, t. fraxini differs from members of the cochlearis group by the following morphological characteristics: a different petiolar shape (diagnostic character 7); a different pattern for the sculpture (diagnostic characters 11, 14 and 15); a different pilosity on propodeum (character 20). moreover, the small denticle present at basis of apical tooth of mandible in t. fraxini lacks in all species of the cochlearis group. finally, we could attribute this new species to the ferox group since it morphology agrees with the definition of this (see diagnostic characters: 4, 7,11, 13-18, 19, 20). inside the ferox group, it should be noted that t. fraxini is morphologically more similar to t. paludis and t. zeteki (subgroup 1 sensu kempf (1975)) than to other members since it shares exclusively with these two species a subquadrate head shape (diagnostic character 1; ci [95] {88,1-98,3}), a similar interfrontal index (ifi [70,2] {64,3-75}), a more similar petiole in scalelike shape (diagnostic character 7) and some mandibles with apex not noticeably projecting laterad beyond genae when in closed position. after all, t. fraxini seems to be more related to thaumatomyrmex paludis since these two species share exclusively a mesosomal shape without a metanotal suture and transverse groove (see diagnostic character 4). finally, beside the morphological peculiarities of this new species above discussed, the comparison of its morphometrical pattern with all other valid species reveals that t. fraxini is the smaller species of the genus. acknowledgements we sincerely acknowledge josé antonio de souza (ceplac/cepec/egreb), leandro braga godinho, milton rodrigues da silva jr. and the staff of the research unit “laboratório de mirmecologia” of cepec-ceplac for its logistical support in the field work. we also thank to the staff of the research unit “laboratório de biossistemática animal” of uesb for its technical support in the laboratory study. we are also grateful to dr. keller for 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(ed) (1988). biodiversity, washington, dc: national academy of sciences/smithsonian institution. doi: 10.13102/sociobiology.v65i3.1932sociobiology 65(3): 358-369 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 canopy ant assemblage (hymenoptera: formicidae) in two vegetation formations in the northern brazilian pantanal introduction ants are social organisms that have a vast capacity for adaptation, exploration and colonization of a wide range of terrestrial environments (hölldobler & wilson, 1990; baccaro et al., 2015). ants have multiple feeding habits and spectra of interactions with other biological groups, which explains their high distribution and occurrence, especially in tropical regions (longino et al., 2002; wilson & hölldobler, 2005; lach et al., 2010; castaño-meneses, 2014; del-claro et al., 2018; philpott et al., 2018), including wetlands such as the brazilian pantanal (battirola et al., 2005; marques et al., 2011; soares et al., 2013; meurer et al., 2015; yamazaki et al., 2016). abstract the landscape of the northern pantanal region is a mosaic of fields and forests, distributed according to topography and hydrology of this floodplain, resulting in a particular pattern of vegetation distribution. among the forest formations, mixedspecies and monodominant landscape units can be found which are associated with floodable or non-floodable habitats. our study tested the hypothesis that forest formations with greater tree richness and which are non-floodable (cordilheiras) maintain distinct richness and composition in canopy ant assemblages in relation to the seasonally floodable monodominant forests (cambarazais). sampling was performed in 10 sample areas (five cambarazais and five cordilheiras) by means of canopy insecticide fogging during the dry and high water seasons of the pantanal’s hydrological cycle. the canopy ant assemblages revealed 105 species belonging to 30 genera and nine subfamilies. myrmicinae (41 spp.), formicinae (20 spp.) and pseudomyrmecinae (17 spp.) predominated. our results revealed that the composition of canopy ant assemblages varied between cambarazal and cordilheira forests, as well as between the dry and high water periods. nevertheless, the richness was homogeneous between these forests and in the dry and high water periods. these results show the specificity of each forest, as well as its structure, in maintaining distinct compositions in ant assemblages in canopies in the pantanal of mato grosso. sociobiology an international journal on social insects j dambros1, vf vindica1, jhc delabie2, mi marques³, ld battirola1 article history edited by ricardo solar, ufmg, brazil received 08 august 2017 initial acceptance 29 june 2018 final acceptance 01 august 2018 publication date 01 october 2018 keywords canopy; social insects; canopy fogging; vegetation types corresponding author juliane dambros universidade federal de mato grosso câmpus universitário de sinop sinop, mt, brazil e-mail: julianedambros84@gmail.com wetlands are ecosystems characterized by strong hydrological seasonality, influencing the distribution and availability of habitats and their use by fauna in an annual cycle (junk et al., 1989; wantzen at al., 2016). thus, the pantanal of mato grosso, the largest continuous floodplain on the planet, presents peculiar characteristics regarding its composition of habitats, influenced by the frequency, amplitude, duration and dynamics of the seasonal hydrological cycle (junk et al., 2013; nunes-da-cunha & junk, 2015). the pantanal of mato grosso is composed of a mosaic of vegetation formations including fields, savannas, pastures (native and exotic) and forest formations, incorporating into its floristic composition elements of adjacent phyto1universidade federal de mato grosso, câmpus universitário de sinop, sinop, mt, brazil 2universidade estadual de santa cruz, ilhéus, ba, brazil 3universidade federal de mato grosso, câmpus universitário de cuiabá, cuiabá, mt, brazil research article ants sociobiology 65(3): 358-369 (september, 2018) 359 geographic provinces such as the cerrado, amazônia and chaco (adámoli, 1982; silva et al., 2000; junk et al., 2006; nunes-da-cunha & junk, 2015). in the northern region of the pantanal, among the forest formations, mixed-species and monodominant landscape units can be found (alho et al., 2000). monodominant forests are defined as vegetational formations in which a single species dominates more than 50% of the community (connell & lowman, 1989). among the available habitats in forest formations, the canopy stands out as being responsible for mechanisms that regulate key ecosystem processes involving both nutrient cycling and species interaction (basset et al., 2002; dial et al., 2006; adis et al., 2010; fotis et al., 2018). the trees, isolated at the canopy or connected to each other by lianas and branches, constitute the main element of this stratum in tropical forests, harbouring important communities for the maintenance of forest diversity, resilience and functioning, being considered important habitat models in the ecology of communities and populations (nadkarni, 1994). despite the high density and proximity of tree crowns as canopies in forests, trees can be considered insular habitats for some taxa (adams et al., 2017), contributing to the complexity of this forest stratum. the structural complexity of the canopy resulting from the variety of tree species, as well as their architecture, tends to maintain high species richness associated in this forest stratum due to a greater diversity and distribution of microhabitats, food resources and niches available to fauna (farrel & erwin, 1988; pianka, 1994; erwin, 2013; yamazaki et al., 2016; yusah et al., 2018), which are important factors in the structure and composition of animal assemblages, including those of ants (e.g. battirola et al., 2005; ribeiro et al., 2013; meurer et al., 2015; yusah & foster, 2016). the structure of vegetation in monodominant and mixed-species habitats associated with seasonal flooding makes both, fauna and flora, present specific adaptations which allow for their survival in this type of habitat (adis et al., 2001; arieira & nunes-da-cunha, 2006; battirola et al., 2007, 2009, 2017a,b; rebellato et al., 2013; tissiani et al., 2015). in the northern pantanal region of mato grosso, specific strategies were recorded for different species of arthropods, including ants, involving vertical displacement to higher habitats such as tree trunks and canopies, horizontal displacement along the flood line (adis et al., 2001; wantzen et al. 2016), and temporary displacement between terrestrial habitats (meurer et al., 2015; yamazaki et al., 2015, 2016, 2017). considering the habitats specificity present within the flood plains of the northern pantanal region of mato grosso, we tested the hypothesis that the forests’ formations with greater tree richness and non-floodables (cordilheiras) maintain distinct richness and composition in canopy ant assemblages in relation to the seasonally floodable monodominant forests (cambarazais), influenced by the hydrological cycle of the pantanal as well as the structure of the vegetation. so, we compared the composition and richness of canopy ant assemblages between cambarazal and cordilheira during dry season and high water season, with the aim of revealing the habitats’ species richness, as well as the relationship between habitat structure and the maintenance of canopy ant assemblages in these habitats. material and methods study area this study was carried out in the floodplain of the northern pantanal region of mato grosso, poconé-mt, in areas between the bento gomes (16°18’s and 56°32’w) and cuiabá (16°30’s and 56°24’w) rivers. the region’s climate is described as tropical savanna, type aw under the köppen climate classification system, characterized by dry winters and rainy summers, with temperatures oscillating between 22ºc and 32ºc. annual rainfall ranges from between 1,000 to 1,500 mm (hasenack et al., 2003). the seasonality of the region is determined by four well-characterized periods (dry, rising water, high water and receding water seasons) that define the alternation between the terrestrial and aquatic phases of this region (heckman, 1998). sampling was performed in monodominant (cambarazais) and mixed-species (cordilheiras) forest formations. the monodominant areas correspond to seasonally flooded forests with predominance of vochysia divergens pohl. (vochysiaceae), regionally known as cambará, a tree species considered invasive to the pantanal’s natural fields (pott & pott, 1994; arieira & nunes-da-cunha, 2006; signor et al., 2010; uriu et al., 2017). this kind of forest is 5-19 m high and it is common to find other tree species such as calophyllum brasiliense cambess. (calophyllaceae), alchornea discolor poepp. (euphorbiaceae) and licania parvifolia huber (chrysobalanaceae) (arieira & nunes-dacunha, 2006). the areas of mixed-species formations, locally known as cordilheiras, are areas located in the upper parts of the floodplain and are therefore not subject to inundation during the seasonal high water and rising water periods. these areas are characterized by dense arboreal savanna vegetation composed of different species (3-17 m high), dominated by astronium fraxinifolium shott. (anacardiaceae), attalea phalerata mart. ex spreng. (arecaceae), qualea grandiflora mart. (vochysiaceae) and protium heptaphyllum (aubl.) marchand (burseraceae) (silva et al., 2000; nunes-dacunha et al., 2010; morais et al., 2013). formicidae assemblage sampling the sampling of ant assemblage was carried out in all 10 areas (five areas of cambarazal and five of cordilheira), throughout the dry season in september 2012 and 2013, j. dambros et al. – pantanal flood cycle and canopy ant assemblages in two vegetation formations 360 and in the high water periods in march 2013 and 2014. these 10 areas are located in the same region, but they are not connected (minimum distance of 3 km). in each of these 10 areas, three independent 5x5 m sample quadrats were delineated (minimum distance of 200m), totalling 15 quadrats per forest formation in each seasonal period. the phytosociological information (richness of tress, relative density and relative vegetation dominance data), were obtained for each of the sample quadrats, and was estimated based on plants with a height above 2m and dbh above 1cm. for the collection of ants, canopy fogging with insecticide was used, in accordance with procedures adapted from adis et al. (1998) and battirola et al. (2004). canopy fogging was carried out for five minutes, commencing at 6:00 a.m., when air circulation is less intense (e.g. adis et al., 1998). the swingfog sn50 fogging machine was used with 0.5% lambdacyhalothrin (icon ®) insecticide, a non-residual synthetic pyrethroid, diluted in diesel oil at a concentration of 1%, and combined with the synergist (0.1% ddvp). for each sample quadrat, arthropods were collected in 25 nylon collecting funnels (1m² area each), with 92% alcohol at the base of the collection flasks. samples were collected two hours after insecticide application. when removing collection flasks, the funnels walls were manually shaken and washed with a spray containing 92% alcohol. all collected material was packed in flasks containing 92% alcohol and transported to the acervo biológico da amazônia meridional (abam) of the universidade federal de mato grosso, sinop campus, where the testimonial material is deposited. ants were separated from the other arthropods and identification was conducted following the bolton (2015) and fernandez (2003) classification. data analysis the composition of canopy ant assemblages was evaluated based on data ordination through non-metric multidimensional scaling (nmds) and jaccard dissimilarity measurement, using a presence-absence species matrix (qualitative data). for the multivariate analysis of variance (manova), the scores of the two nmds dimensions were used to identify variations in ant assemblage composition in relation to the variables (i) forests (cambarazal and cordilheira) and (ii) seasonality (dry and high water periods). the significance level for each variable, as well as for the assumed models was α <0.05, through pillai trace test. normality of the data was evaluated by the shapiro-wilk test. in order to evaluate the effect of plant richness, relative density and relative vegetation dominance on the composition of canopy ant assemblages, multiple regressions were applied. the analysis of variance (anova) was used to evaluate variation in ant species richness between cambarazal and cordilheira and between dry and high water seasons. the jackknife 1 estimator was applied to analyse the sampling efficiency of canopy ant richness. all analyses were performed using the free software r, version 3.0.1 (r core team, 2013) in conjunction with the vegan (oksanen et al., 2013) and car (fox et al., 2013) packages. results assemblage composition canopy ant assemblages in areas of cambarazal and cordilheira were represented by 105 species belonging to 30 genera, 12 tribes and nine subfamilies. myrmicinae presented 41 species (39.1%), followed by formicinae (20 spp.; 19.1%), pseudomyrmecinae (17 spp.; 16.2%) and dolichoderinae (11 spp.; 10.5%) (table 1). ponerinae (8 spp.; 7.6%), ectatomminae (4 spp.; 3.8%), dorylinae (2 spp.; 1.9%), amblyoponinae (1 sp.; 0.9%) and paraponerinae (1 sp.; 0.9%) corresponded to subfamilies with lower species richness (table 1). the estimated species richness for the cordilheira according to the jackknife 1 estimator was 81 ant species for dry and 92 for high water season, however, 79% was sampled in the dry season and 77% during the flood season. in cambarazal jackknife 1 estimated 78 ant species for dry and 85 for high water season, with our sampling representing 81% and 82%, respectively. analysing each forest individually, a total of 85 ant species were collected in cordilheira areas (27 genera, 12 tribes and nine subfamilies), and 82 species were recorded in the cambarazal areas (25 genera, 11 tribes and eight subfamilies). in these samplings, 23 ant species occurred exclusively in cordilheira (21.9%) and 20 in cambarazal (19.1%), while 62 species occurred in both areas (59.0%) (table 1). of the 82 species recorded in cambarazal areas, 49 occurred in both the dry and high water periods (59.8%), with 16 restricted to the dry (19.5%) and 17 to the high water period (20.7%). in the cordilheira areas, 49 ant species were common to the dry and high water periods (57.6%), 19 occurred only in the dry (22.4%) and 17 were sampled only during the high water period (20.0%). the two nmds ordination axes captured 70% of the variation in ant species composition (stress = 0.22). the indirect ordination, with presence and absence data of the ant species, showed differences between cambarazal and cordilheira areas (manova: f = 20.18; df = 1; p < 0.001) (fig. 1a) and between dry and high water seasons (manova: f = 4.42; df = 1, p = 0.018) (fig. 1b). species richness ant species richness did not vary between cambarazal and cordilheira forests (anova, f = 3.73; df = 1; p = 0.06). also, no variation was observed between dry and high water periods, both in the cambarazal (anova, sociobiology 65(3): 358-369 (september, 2018) 361 f = 3.37; df = 1; p = 0.082) as well as in the cordilheira (anova, f = 3.68; df = 1; p = 0.07). multiple regression analysis including phytosociological variables and canopy ant species richness in cordilheira revealed an effect of vegetation dominance on ant assemblage (r2 = 0.41; df = 16; p = 0.006), while plant richness (p = 0.12) and vegetation density (p = 0.19) were not significant. the cambarazal areas did not reveal any effect of phytosociological variables on ant species richness. discussion the composition of canopy ant assemblages varies according to the monodominant (cambarazal) and mixedspecies (cordilheira) forests, as well as to the dry and high water periods. however, the richness of ant species was similar between these forests formations, and in the dry and high water periods. studies carried out in the same region of the pantanal showed similar results in relation to the species composition variation, but different for richness of ant assemblages throughout the seasonal periods (marques et al., 2011; yamazaki et al., 2016), as well as for other taxonomic groups of canopy arthropods (battirola et al., 2016, 2017a,b). the presence of greater tree richness in cordilheira areas was not preponderant in relation to the richness of ant species, showing that only variations in composition of the ant assemblage may be related to the habitat structure and seasonality in these forests formations. distinct from our results, studies evidenced that ant assemblages associated with canopies may respond positively to an increase in tree richness, which is related to the greater availability of food and nesting resources, consequently determining which species and how many will occupy these habitats (e.g. hölldobler & wilson, 1990; mausdsley, 2000; ribas et al., 2003; ribas & shoereder, 2007). klimes et al. (2012) showed in a comparative study between primary and secondary forests that reduction in plant taxonomic diversity in secondary forests is not the main driver for the reduction in canopy ant species richness, indicating that the majority of arboreal species losses in secondary tropical forests are attributable to simplification of the vegetation structure. due to the monodominance of v. divergens, the cambarazais demonstrate distinct conditions in relation to the cordilheira forests. the lower number of tree species characteristic of these forests leads to changes in the variety and distribution of resources available to fauna (e.g. tavares et al., 2001). due to the smaller number of tree species and a canopy stratum dominated by v. divergens, the distribution of food and nesting resources, for example, becomes further homogenized and less diversified, as well as more concentrated in regard to species adapted to these conditions (e.g. southwood, 1961; root, 1973). additionally, canopy ant assemblages were influenced most likely by the phenology and seasonal variations in these habitats (basset et al., 2003). variations in plant phenology are directly related to the availability of food resources for ants, considering, for example, the increase of these resources during fruiting and flowering events, as well as the reduction in leaf fall and renewal periods (basset et al., 2003; castaño-meneses, 2014). besides this the phenology of v. divergens depends on the pantanal’s hydrological regime, with flowering during the dry period, fruiting during the rising water and high water periods, and foliage renewal during the receding water and dry periods (nunes-da-cunha & junk, 2001; uriu et al., 2017). the occurrence of the flowering, fruiting and foliage exchange stages may directly or indirectly impact assemblages in canopies, as well as figure 1. evaluation of the composition of formicidae assemblage based on the comparison between scores generated by nmds from species presence/absence data between different vegetation formations (a) and between the dry and high water periods (b) in the northern pantanal region of mato grosso, brazil. j. dambros et al. – pantanal flood cycle and canopy ant assemblages in two vegetation formations 362 that observed for other arthropod groups in the pantanal (battirola et al., 2009; marques et al., 2006, 2014; meurer et al., 2015; yamazaki et al., 2016). in cambarazal areas, it is possible to suppose that the v. divergens phenology influences the distribution of associated arthropod assemblages, as well as the seasonal flooding causing the soil in these areas to remain waterlogged for up to four months, preventing its use by edaphic fauna (battirola et al., 2009, 2010), requiring the organisms to develop survival strategies for these conditions (adis et al., 2001; soares et al., 2013; marques et al., 2014; battirola et al., 2017a,b). it is common to observe the migration of soil-dwelling ant species to tree trunks and canopies in these floodplain formations (adis et al., 2001; battirola et al., 2005; meurer et al., 2015). this displacement, even if temporary, alters the structure of canopy assemblages (battirola et al., 2009), either by greater competition for resources or for niches available to ants (ribas et al., 2003; ribas & shoereder, 2007). among the species of ants captured exclusively during the high water period, some are commonly associated with the edaphic environment, such as neivamyrmex diana (forel, 1912), ectatomma brunneum smith, 1858, gnamptogenys moelleri (forel, 1912) and atta sexdens rubropilosa forel, 1908, indicating a possible temporary displacement from the soil to the canopy of this monodominant forest. unlike the cambarazais, the cordilheiras located on higher portions of land in the pantanal floodplain are not subject to inundation during the high water season, and can be used as areas of refuge for local fauna (guarimneto, 1992; silva et al., 2000; nunes-da-cunha et al., 2011; morais et al., 2013). in addition to permanent stability due to the absence of seasonal flooding, the higher diversity of tree species gives rise to a greater variety and distribution of resources in comparison to monodominant forests (e. g. mausdsley, 2000). this stability is associated with the fact that the different plant species have distinct phenological phases, providing fauna with various resources throughout different periods of the year. this succession in the supply of resources allows for a greater number of generalist species and opportunistic exploitation. the fact that these forests do not suffer direct impacts from seasonal floods further increases their stability and constancy over time, maintaining richer assemblages adapted to the local conditions (corrêa et al., 2006; ribas & shoereder, 2007). in these mixedvegetation areas, it was observed that among the analysed vegetation variables, only dominance had an effect on canopy ant richness, demonstrating the heterogeneity of the area and the distribution of available resources (e. g. lange et al., 2008). in general, the composition of canopy ant assemblages varied according to the evaluated vegetation formations (habitat heterogeneity) as well as the different seasonal periods of the mato grosso pantanal. however, in our study, no effects of the greater richness of arboreal species in cordilheira areas on the species richness of canopy ants was observed. in the same way, no variation in ant richness between the dry and full water / flood seasons was observed for both areas. our results demonstrate the importance of habitat variety and the complexity of their functioning in these flood plains, as well as the need for the maintenance and conservation of these habitats, for the conservation of ant assemblages associated with this ecosystem. acknowledgments to the programa de pós-graduação em ciências ambientais (ppgcam/ufmt/sinop) for the opportunity to carry out this study, and the fundação de amparo à pesquisa do estado de mato grosso, for the scholarship awarded to the first author. to the conselho nacional de desenvolvimento científico e tecnológico (cnpq), process no. 484679/2012-0 (universal announcement 14/2012), to the núcleo de estudos da biodiversidade da amazônia matogrossense (nebam/ufmt/sinop), and to the laboratório de ecologia e taxonomia de artrópodes (leta/ib/ufmt) for logistic support. jhcd acknowledges her research grant from cnpq. references adámoli, j.a. 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(continuation) cephalotes minutus (fabricius, 1804) 1 1 1 1 cephalotes pallidus de andrade, 1999 1 1 1 1 cephalotes pavonii (latreille, 1809) 1 cephalotes persimilis de andrade, 1999 1 1 1 1 cephalotes pusillus (klug, 1824) 1 1 1 cephalotes umbraculatus (fabricius, 1804) 1 1 cephalotes gr. laminatus sp. 2 1 1 cephalotes sp. 1 1 mycocepurus goeldii (forel, 1893) 1 pheidole astur wilson, 2003 1 1 1 pheidole (gr. fallax) sp. 1 1 1 1 1 pheidole (gr. flavens) sp. 5 1 1 pheidole (gr. flavens) sp. 6 1 1 1 pheidole sp. 2 1 pheidole sp. 3 1 procryptocerus hylaeus kempf, 1951 1 1 trachymyrmex sp. 1 wasmannia rochai forel, 1912 1 1 1 1 crematogastrini crematogaster brasiliensis mayr, 1878 1 1 1 crematogaster crinosa mayr, 1862 1 crematogaster curvispinosus mayr, 1862 1 1 1 1 crematogaster victma smith, 1858 1 1 1 1 crematogaster sp. 1 nesomyrmex tristani (emery, 1896) 1 1 1 1 nesomyrmex wilda (smith, 1943) 1 nesomyrmex sp.3 1 xenomyrmex sp. 1 1 1 1 1 xenomyrmex sp. 2 1 1 1 1 strumigenys trinidadensis wheeler, 1922 1 solenopsidini solenopsis sp. 1 1 1 1 1 solenopsis sp. 2 1 1 1 1 solenopsis sp. 3 1 1 solenopsis sp. 4 1 1 1 1 ponerinae ponerini hypoponera opaciceps (mayr, 1887) 1 hypoponera sp. 1 1 hypoponera sp. 2 1 neoponera curvinodis (forel, 1899) 1 1 1 neoponera foetida (linnaeus, 1758) 1 neoponera unidentata (mayr, 1862) 1 neoponera villosa (fabricius, 1804) 1 1 1 1 odontomachus bauri emery, 1891 1 paraponerinae paraponerini paraponera clavata (fabricius, 1775) 1 pseudomyrmecinae pseudomyrmecini pseudomyrmex atripes (smith, 1860) 1 1 1 pseudomyrmex curacaensis (forel, 1912) 1 1 1 pseudomyrmex dendroicus (forel, 1904) 1 pseudomyrmex gracilis (fabricius, 1804) 1 1 1 1 pseudomyrmex pupa (forel, 1911) 1 1 1 1 pseudomyrmex rochai (forel, 1912) 1 1 1 1 pseudomyrmex schuppi (forel, 1901) 1 1 1 1 pseudomyrmex tenuis (fabricius, 1804) 1 pseudomyrmex tenuissimus (emery, 1906) 1 1 1 1 pseudomyrmex termitarius (smith, 1855) 1 1 1 1 pseudomyrmex triplarinus (weddell, 1850) 1 sociobiology 65(3): 358-369 (september, 2018) 369 table 1. presence (1) and absence (-) of formicidae specimens sampled in the canopy of monodominant (cambarazal) and mixed-species (cordilheira) vegetation formations during dry and high water periods in the northern pantanal region of mato grosso, brazil. (continuation) pseudomyrmex gr. pallidus sp. 1 1 1 1 1 pseudomyrmex gr. pallidus sp. 2 1 1 pseudomyrmex gr. pallidus sp. 3 1 pseudomyrmex gr. pallidus sp. 4 1 1 1 1 pseudomyrmex gr. pallidus sp. 5 1 1 pseudomyrmex gr. pallidus sp. 6 1 1 1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1174sociobiology 64(1): 7-13 (march, 2017) the role of senescent stem-galls over arboreal ant communities structure in eremanthus erythropappus (dc.) macleish (asteraceae) trees introduction ants (hymenoptera: formicidae) are an ecologically important group of arthropods because of their considerable species richness and biomass, and the important roles they play in the functioning of ecosystems (hölldobler & wilson, 1990; lach et al., 2010). they are very conspicuous organisms in the arboreal environment due to the diversity of renewable food sources available to them, either produced by host plants, such as extrafloral nectaries, or by herbivorous insects abstract the extensive occupation of canopy trees by ants can be attributed to many factors, such as the presence of structures that provide food and shelter. structures induced by other insects in host plants, like senescent galls, can provide shelter and a nesting place for many species of ants. the main objectives of this work were: (1) to describe the ant communities found in canopies of candeia trees (eremanthus erythropappus), including the species which use galls as nesting sites; (2) verify the role of galls in determining the structure and composition of the ant communities and (3) to evaluate whether the size and shape of galls are important to the choice of nesting sites by ants. specifically, the following questions were investigated: 1 – are larger galls more frequently occupied by ants than smaller galls? 2 – does gall shape (globular and fusiform) influence occupation? 3 – which species of ants are present in the canopies of candeias and which are occupying galls? senescent galls were collected in locations in the southern portion of the espinhaço mountain range, state of minas gerais, southeastern brazil. in total, 3,195 galls were collected and 19 ant species were recorded. only 176 galls (5.5%) had been occupied by ants, and these were represented by 11 species. the most frequent species found occupying galls were myrmelachista nodigera, with 48 colonies; nesomyrmex spininodis, with 37 colonies; and crematogaster complex crinosa sp. 1, with 29 colonies. the ants occupied galls with greater volume and diameter. even considering the low occupation frequency, senescent galls in e. erythropappus are used by ants, either as outstations or satellite nests of polydomic colonies, and may be important in determining ant species composition in canopy trees. sociobiology an international journal on social insects lr santos1, rm feitosa2, maa carneiro1 article history edited by kleber del-claro, ufu, brazil received 25 july 2016 initial acceptance 27 september 2016 final acceptance 16 december 2016 publication date 29 may 2017 keywords cerrado, formicidae, galls, ant-plant interactions, nesting. corresponding author leonardo rodrigues dos santos universidade federal de ouro preto. laboratório de entomologia ecológica do instituto de ciências exatas e biológicas campus morro do cruzeiro cep 35400-000, ouro preto-mg, brazil e-mail: bio.leorodrigues@gmail.com associated with plants, such as hemipteran exudates (oliveira & freitas, 2004; del-claro, 2004; fagundes et al., 2012). other important resources provided by plants to ants are nesting sites and shelter, such as domatias or cavities produced by the activity of endophytic insects (oliveira & freitas, 2004; schoereder et al., 2010; nascimento et al., 2012; almeida et al., 2014). therefore, the supply of food resources and places for shelter and nesting are both determining factors for the establishment and survival of arboreal ants on trees (oliveira & freitas, 2004). 1 universidade federal de ouro preto, lab. de entomologia ecológica do instituto de ciências exatas e biológicas, ouro preto-mg, brazil 2 universidade federal do paraná, departamento de zoologia, curitiba-pr, brazil research article ants lr santos, rm feitosa, maa carneiro – the arboreal ant community in senescent stem-galls of eremanthus erythropappus trees8 gall inducing insects are among the most sophisticated and specialized organisms that exist (shorthouse et al., 2005). galls are vegetable tumors induced by insects or other organisms; they are free of chemical compounds, rich in nutrients and provide a location for the larvae of gallers to develop (mani 1964; price et al., 1987; dregerjauffret & shorthouse, 1992; raman et al., 2005; fernandes & santos 2014). stem-galls persist on the plant and, after being abandoned by the inductor (= senescent galls), can be colonized by different organisms such as spiders, coleoptera, lepidoptera and, mainly, ants (longino & wheeler, 1987, 1988; craig et al., 1991; almeida et al., 2014). due to the capacity of larvae to manipulate and modify their host plants, creating “new habitats” for other organisms, gall insects can be considered ecosystem engineers (jones et al., 1994, 1997; wright & jones 2006). these “new habitats” are ecologically important for increasing both the species richness and abundance of local communities (cornelissen et al., 2016). eremanthus erythropappus (dc.) macleisch (asteraceae), also known as “candeia”, is a pioneering tree species that forms dense, monodominant patches in areas in early stages of natural succession (pedralli et al., 2000). it is native to south america, and is found mainly in brazil, argentina and paraguay (pereira et al., 2014). in brazil, it is commonly found in the south and southeast, as well as in the states of bahia (northeast) and goiás (central brazil) (pedralli et al., 1997; lorenzi, 2009). on the espinhaço mountain range, which extends throughout the states of minas gerais and bahia, e. erythropappus is distributed across the entire altitudinal gradient, occupying high regions, rocky outcrops and riparian forests (carneiro et al., 2009b). due to its wide distribution in south america, and its pioneering character, candeia can be a key species in maintaining insect biodiversity. so far, six morphotypes of gall inducing insects have been described on candeia (carneiro et al., 2009b). recently, almeida et al. (2014) found eight ant species occupying senescent galls of candeia trees and suggested that the presence of senescent galls, an abundant resource, can increase the diversity of ant assemblages in the canopy of candeia trees. therefore, this work aims at expanding the knowledge about ants associated with candeia galls on a regional scale, by describing the ant communities found in the canopies of candeia trees, including the species that use galls as nesting sites. furthermore, this work also aims to verify the role of galls in determining the structure and composition of the ant communities and to evaluate whether the size and shape of galls are important to the choice of nesting sites by ants. specifically, the following questions were investigated: 1 – are larger galls more frequently occupied by ants than smaller galls? 2 – does gall shape (globe and fusiform) influence occupation? 3 – which species of ants are present in the canopy of candeias and which are occupying galls? material and methods the study was conducted in seven regions of the state of minas gerais (fig 1), which all possessed rupestrian field vegetation (vegetation in quartzite or ferruginous rocky outcrops) and a tropical montane climate. six of the regions are near the municipality of ouro preto in the southern portion of the espinhaço range: 1) itacolomi state park (peit), 2) environmental protection area of andorinhas waterfall (apa); 3) uaimií state forest (uaimií), 4) populations along mg 356 inconfidentes highway (rodovia); 5) serra do ribeiro (srib); and 6) private natural heritage reserve caraça sanctuary (caraça). peit (20°22’30” s, 43°32’30” w) encompasses approximately 7,500 ha, with its highest point being the itacolomi peak at 1,772 m. some mountain rainforest patches and araucária trees complement the landscape (araújo et al., 2003; ief 2015). apa (20º22’34’’ s, 43º29’27’’ w) has an area of approximately 18,700 ha with an altitude of 1,300 m, and is located on the hills of ouro preto encompassing the headwaters of rio das velhas, a very important river in the state of minas gerais. uaimií (20°14’43” s, 43°35’15.9” w) was established in 2003 in the area of apa, and has an area of 4,398 ha and elevations reaching to about 1,800 m (rezende et al., 2011). the location called rodovia (20º22’27” s, 43º32’22” w) is comprised of fire-damaged mesophilic vegetation, currently undergoing rehabilitation (pedralli et al., 1997, 2000). srib (20°27’55.4” s, 43°35’59” w) is a mountain formation located inside the itatiaia state natural monument, a conservation unit with an area of 3,200 ha and an altitude of 1,568 m (ief, 2015). caraça (20°05’53.4” s, 43°29’17.7” w) is a private nature reserve with 11,233 ha of area at 1,240 m of altitude. in addition, samples were also collected in ibitipoca state park (peib) located in the mantiqueira mountain range, in lima duarte, mg. peib (21°42’47.2” s, 43°54’07.7” w), with an area of 1,923 ha and elevations reaching 1,784 m. seasonal montane forest of semi-deciduous trees and rupestrian fields cover most of the area of peib (neto et al., 2007). ants and senescent galls were sampled during the period of january 2014 to march 2015. the study included seven regions throughout the host plant range to maximize sampling of ant species associated with e. erythropappus. each plant and the population of trees were sampled once during the study period. in each region, two sites, separated by at least 1 km, were selected, and 15 individuals of candeia were randomly sampled from each, for a total of 30 individuals for each region and 210 for the entire study. only plants no taller than three meters were sampled to facilitate canopy access. in addition to galls, foraging ants in the canopy were sampled with an entomological blow-type aspirator (gibb & oseto, 2006) during 15 minutes of inspection per plant. all senescent stem galls were collected and immediately deposited in a plastic bag specific for each plant. once in the laboratory, the galls were measured using a digital caliper sociobiology 64(1): 7-13 (march, 2017) 9 (error: ± 0.01mm) to determine largest diameter, smallest diameter and height, and to calculate the volume of globular (v = 4/3 πr³) and fusiform (v = r²hπ) galls according to the method described by almeida et al. (2014). in addition, all galls were opened to verify if ant colonies were present or not. ants were identified as specifically as possible (down to genus or species) by rmf, who consulted as references kempf (1959), longino (2003) and nakano et al. (2013). specimens were subsequently deposited in the entomological collection padre jesus santiago moure (dzup) at the universidade federal do paraná, curitiba, brazil. statistical analyses were performed using the statistical package r (r development core team, 2012). to answer if the occupation of galls by ants increases with gall size, an adjusted a logistic regression model was used in which the presence / absence of ants was the response variable (binary) and gall size the predictor variable. however, residual analyses did not support the assumptions of the logistic regression model. therefore, the non-parametric wilcoxon test was used to determine preferences regarding size and shape, where the explanatory variables were occupation categories (unoccupied and occupied), and the responses variables were gall diameter and volume (fernandes et al., 1988; sprent & smeeton, 2007). non-parametric alternative methods for the analysis of two samples were used because the residual analyses did not support the parametric model assumptions (sprent & smeeton, 2007). results in total, 3,195 senescent stem-galls were collected of two morphological types: 1,872 (58.6%) fusiform galls and 1,323 (41.4%) globular galls. only 176 (5.5%) of the senescent stem-galls had been occupied, of which 113 (64%) were fusiform and 63 (36%) globular. occupancy rates were low (occupied galls/total galls), not exceeding 5.5% of available galls and in each of the populations: {peit [1= 5/238 (2,1%) and 2=17/182 (9,3%)]; apa [1= 17/367 (4,6%) and 2= 14/339 (4,1%)]; caraça [1= 20/255 (7,8%) and 2= 7/192 (3,6%)]; peib [1= 4/110 (3,6%) and 2= 1/102 (0,98%)]; uaimií [1= 14/231 (6,1%) and 2= 7/289 (2,4%)]; rodovia [1= 17/197 (8,6%) and 2= 31/446 (6,9%)]; srib [1= 11/78 (14,1%) and 2= 11/169 (6,5%)]}. nineteen ant species belonging to eight genera and three subfamilies were found (fig 2). myrmicinae was the richest subfamily, with nine species, followed by formicinae, with seven species (table 1). we found 11 ant fig 1. map showing sampling regions in the state of minas gerais, brazil. fig 2. venn diagram showing number of ant species found in galls of the two morphotypes and in canopies of candeia trees. lr santos, rm feitosa, maa carneiro – the arboreal ant community in senescent stem-galls of eremanthus erythropappus trees10 species occupying senescent stem-galls in candeia trees. among these, two species were found only in fusiform galls, three only in globular galls and six in both morphological types (table 1). myrmicinae was the most common subfamily in galls (7), followed by formicinae (3) and pseudomyrmecinae (1) (table 1). the species with the highest number of colonies present in galls was myrmelachista nodigera mayr, 1887 (formicinae), with 43 colonies; nesomyrmex spininodis (mayr, 1887) (myrmicinae), with 37; and crematogaster complex crinosa sp. 1 (myrmicinae) with 29 colonies (table 1). in general, the occupied galls had greater volume (w = 342.370; p = 0.001; n = 3195) (fig 3a) and mean diameter (w = 336.890; p < 0.001; n = 3195) (fig 3b) than unoccupied galls. when both gall morphotypes were considered separately, the same pattern was found. occupied fusiform galls had greater volume (w = 124.240; p < 0.001; n =1872) and diameter (w = 119.160; p < 0.001; n =1872). likewise, the globular galls with higher volume (w = 57.604; p < 0.001; n = 1323) and higher diameter (w = 57.236; p = 0.001; n = 1323) were also more occupied by ants. discussion except for the genus solenopsis and some species of camponotus, all ant species found in the present study represent typically arboreal genera (brown, 2000). in fact, camponotus rufipes and c. crassus are commonly reported as foraging on plants (espírito santo et al., 2011; fagundes et al., 2013). fig 3. size (volume and diameter) of senescent stem galls on candeia trees eremanthus erythropappus (dc.) macleisch (asteraceae). the volume (w = 342.370; p = 0.001; a) and the diameter (w = 336.890, p < 0.001; b) of occupied galls were higher than those of galls not occupied by ants. taxa number of colonies in galls globular fusiform galls morphology canopy myrmicinae crematogaster complex crinosa sp. 1 29 x x x crematogaster complex crinosa sp. 2 2 x nesomyrmex spininodis (mayr, 1886) 37 x x x nesomyrmex aff. echinatinodis forel, 1886 15 x x x solenopsis sp. 4 x cephalotes pusillus (klug, 1824) x cephalotes eduarduli (forel, 1921) x cephalotes maculatus (smith, 1876) 1 x x procryptocerus goeldii forel, 1899 1 x x formicinae camponotus crassus mayr, 1862 x camponotus rufipes (fabricius, 1775) x camponotus blandus (smith, 1858) x camponotus novogranadensis mayr, 1870 x brachymyrmex sp. 9 x x x myrmelachista nodigera mayr, 1887 43 x x myrmelachista sp. 18 x x x pseudomyrmecinae pseudomyrmex gracillis (fabricius, 1804) x pseudomyrmex gr. pallidus sp. x pseudomyrmex sp. 2 1 x x table 1. ant species found in senescent stem-galls and canopy of e. erythropappus from the study sites. “x” means presence and “-“ means absence. sociobiology 64(1): 7-13 (march, 2017) 11 four ant genera among those occupying galls, represented by four morphospecies (brachymyrmex sp., cephalotes maculatus, pseudomyrmex gr. pallidus sp. and solenopsis sp.), were not found in candeia galls by almeida et al., (2014). so, the present work records, for the first time, these four morphospecies occupying galls on candeia trees, thus increasing the list presented in almeida et al. (2014), from eight to twelve species. this increase is likely due to more sampling areas and to the greater sampling effort of the present work; n= 3195 galls in 210 plants in this study, as compared to n= 227 galls in 100 candeia trees in almeida et al. (2014). however, crematogaster goeldii (forel, 1903), which was found by almeida et al. (2014), was not recorded in the present study, and seems to be uncommon in candeia canopies (almeida et al., 2014). despite the high number of species found in candeia galls in the present study, the occupancy rate was very low. in secondary vegetation, monodominant populations (as in the candeias studied here and in areas that have suffered some kind of environmental impact), ant diversity is usually low, as compared to primary and heterogeneous forests (ribas et al., 2003; klimes et al., 2012; floren et al., 2014). therefore, the low local richness of ants associated with candeia is likely a reflection of low structural complexity and environmental heterogeneity in the rupestrian fields of the study sites (klimes et al., 2012; stein et al., 2014). this work supports the previous results of almeida et al. (2014), which showed that larger galls have higher rates of occupation by ants. the preference for occupying larger galls has not only been related to ant body size and colony size, but also to the skill they have in modifying the habitat to accommodate the entire population of the colony as safely as possible (fernandes et al., 1988; araújo et al., 1995; almeida et al., 2014). moreover, the oldest and largest galls are naturally more lignified and, therefore, more resistant. they can offer greater protection to the colony from variation in the climate and other common stresses of the arboreal environment, such as strong winds and intense insolation, which can result in desiccation (yanoviak & kaspari, 2000). during the opening of galls, we noticed that some galls contained only adults, or adults and immatures, or just the founding queen. this indicates the possible formation of new nests or the expansion of colonies by polydomy, a very common phenomenon among ants (debout et al., 2007). polydomy can be a strategy adopted by ants in the face of low environmental heterogeneity (pfeiffer & linsenmair, 1998) and low availability of nesting sites (cereto et al., 2011). these characteristics are very common in monodominant candeia patches (almeida et al., 2014), which are often found in highly impacted localities and areas affected by fire (pedralli, 2000). another similar strategy – outstations consists of ants establishing nests on pre-existing structures in the environment (such as senescent galls or any physical structure) and using them as a rest place and/or shelter during territory patrols (anderson & mcshea, 2001). furthermore, these outstations enable ants to quickly respond to invasions, making them an important defense strategy to dominate their territories (anderson & mcshea, 2001; lanan et al., 2011). both polydomy and outstations are strategies that seem to be characteristic of the ant species found in this work, since we found multiple occurrences of the same species occupying different galls on the same plant, although no aggression tests were performed to determine the territoriality of the ants involved. even with a low frequency of gall occupation, not exceeding 5.5% of the total available, the galls sheltered more than half (11 of 19 species) of ant species collected in association with candeia. thus, in general manner, the role of senescent stem galls is relevant for ant occurrence and coexistence, and for greater species diversity of ants associated with candeia trees. acknowledgments we thank anonymous reviewers for their comments on a previous version of the manuscript. financial support was provided by the universidade federal de ouro preto (programa de pós-graduação em ecologia de biomas tropicais – ufop) and by the authors themselves. logistical support and licenses were provided by ufop, instituto estadual de florestas de minas gerais (ief) and instituto chico mendes de conservação da biodiversidade (icmbio). references almeida, m. f. b., santos, l. r. & carneiro, m. a. a. 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(2000). community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 259-266. doi: 10.1034/j.1600-0706. 2000.890206.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 65-68 (2013) the unusual neotenic system of the asian dry wood termite, neotermes koshunensis (isoptera: kalotermitidae) y miyaguni1, k sugio2, k tsuji1 introduction termites are highly eusocial insects that show reproductive division of labor among morphologically distinguishable castes (weesner, 1969). the reproductive castes of termites are classified broadly into primary reproductives, namely the queen and king, which are the dealates that found new colonies after a swarming, and the secondary reproductives, which are recruited within an established colony (roisin, 2000). depending on circumstances of the emergence of secondary reproductives, they are classified into either replacement reproductives, which emerge after the death (or disappearance) of the queen or the king, or both, and supplementary reproductives, which emerge even in the presence of the primary reproductives (roisin, 2000). on the basis of their ontogenetic origin, secondary reproductives are classified into neotenics, which are reproductives raised from immature individuals (they are more specifically called nymphoids, ergatoids, or pseudergatoids, depending on their origin from nymphs, workers or pseudergates), and adultoids, which are dealate imagoes and are therefore indisabstract in most lower termites, colonies are headed by neotenic reproductives of both sexes after the primary reproductives (i.e., the queen and king) are lost. the production of a neotenic sexual is inhibited by the presence of a primary reproductive of the same sex. we found an exception in the caste system of the dry wood termite neotermes koshunensis (kalotermitidae). the neotenic caste is exclusively male. moreover, production of male neotenics is completely inhibited not only by the presence of a king but also by the presence of a queen. therefore, it is likely to be difficult for n. koshunensis colonies to replace their reproductive pairs. sociobiology an international journal on social insects 1 kagoshima university, kagoshima, japan 2 university of the ryukyus, japan research article termites article history edited by: og desousa, ufv brazil received 12 october 2012 initial acceptance 09 december 2012 final acceptance 03 january 2013 keywords neotenic reproductives, orphaned colony, caste differentiation, neotermes koshunensis corresponding author yasushi miyaguni department of environmental science and conservation biology, united graduate school of agricultural sciences, kagoshima university, kagoshima 890-8580, japan e-mail: neotenic_of_termite@yahoo.com.jp tinguishable from the primary reproductives (roisin, 2000). the neotenic castes are widespread in various termite taxa, whereas adultoids are rare (myles, 1999). termite society is in principle bisexual, and males and females in most species perform the same social tasks (roisin, 2000). the neotenic castes in termites are also often bisexual, although a biased numerical sex ratio is reported in neotenics of some species (myles, 1999). the termite genus neotermes (kalotermitidae) shows an interesting diversity in the neotenic caste system. in neotermes connexus and neotermes papua (kalotermitidae) neotenics are found only in males (myles & chang, 1984; roisin & pasteels, 1991), whereas in neotermes tectonae and neotermes jouteli the common pattern of bisexual neotenic production is observed (kalshoven, 1930 cited by myles, 1999; nagin, 1972). to understand the biological reasons for this variation in the neotenic system more species should be studied in this genus. lüscher (1961) proposed the currently well-acknowledged model of sex-specific inhibition of neotenic production in termites, i.e., the emergence of male and female neoteny miyaguni, k sugio, k tsuji the unusual neotenic system of the asian dry wood termite66 ics is inhibited by the presence of the primary reproductive of the same sex. in kalotermes flavicollis (kalotermitidae), these inhibitory effects are lost when the transmission of anal excretions from royal reproductives to pseudergates is blocked, suggesting that there is a pheromone in the anal excretions (lüscher, 1961; miller, 1969). here, we report an unusual neotenic system in the asian dry wood termite, neotermes koshunensis (kalotermitidae); the production of male neotenics is totally inhibited by the presence of a queen. although partial inhibition of male neotenic production by the presence of a queen was reported in a related species, n. jouteli (nagin, 1972), the system found in n. koshunensis is exceptional and differs markedly from the model proposed by lüscher (1961). materials and methods termite n. koshunensis is distributed from taiwan to okinawa (ikehara, 1966). colonies nest in dead branches of living trees and in dead trees that serve as sources of both food and shelter (i.e., the termites are of the “one-piece” type, sensu abe, 1987); this enabled us to collect entire colonies. this species has a linear caste development pathway: all castes, including neotenics (pseudergatoids) differentiate from pseudergates (older larvae of the functional worker caste in termite species with a linear caste development pathway) after molting (roisin, 2000; katoh et al., 2007). effect of the presence of primary reproductives on the emergence of neotenics we collected 22 colonies of n. koshunensis on the main island of okinawa during 2007–2010. collected individuals were classified into the following castes according to katoh et al., (2007): small larvae, pseudergates, nymphs, pre-alate nymphs, alates, pre-soldiers, soldiers, the queen (we found a maximum of one individual in each colony) and the king (also a maximum of one individual in each colony). from each colony, we established subcolonies receiving two or three of the following four treatments (note that it was impossible to apply treatments i and ii, or i and iii, simultaneously in the same colony). (i) normal colony: 50 haphazardly sampled pseudergates + a queen and a king (n = 7), (ii) queen colony: 50 haphazardly sampled pseudergates + a queen (n = 9), (iii) king colony: 50 haphazardly sampled pseudergates + a king (n = 13), (iv) orphaned colony: 50 haphazardly sampled pseudergates (n = 22). each subcolony was put into a petri dish (90 mm diameter × 16 mm height) with a piece of damp filter paper, which served as a source of both food and moisture. the subcolonies were then kept in the laboratory (25 ± 1 °c) in the dark for 6 weeks. the caste composition of each subcolony was investigated every 7 days, and the filter paper was replaced at the same time. all statistical tests were performed using r version 2.12.2 (r development core team, 2011). results neotenics were produced only in the orphaned subcolonies: the presence either a queen or a king, or both, led to total inhibition of neotenic production (fig. 1). the proportion of subcolonies that produced neotenics differed significantly between the orphaned and any other treatments (all pairs p < 0.001, fisher’s exact test followed by bonferroni correction). this result holds true after controlling for the possible confounding effect of colonies: in the likelihood ratio test after applying the generalized linear model (glm) with binomial error structure (dependent variable = the proportion of subcolonies producing male neotenic(s), explanatory variable = treatment and colony, link function = logit), a significant effect of treatment was detected (χ2 = 58.958, p < 0.0001), but the effect of colony was non-significant (χ2 = 17.526, p = 0.6788). moreover, all of the neotenics that emerged in these orphaned subcolonies were males (fig. 1). at the end of the experiment, we carefully checked the contents of all petri dishes. we found eggs in the normal and queen subcolonies, figure 1. effects of the presence of either a king or a queen, or both, on the production of neotenics in neotermes koshunensis. normal colony: subcolonies with both a queen and a king; queen colony: subcolonies with a queen but no king; king colony: subcolonies with a king but no queen; orphaned colony: subcolonies without reproductives. numbers in parentheses denote sample sizes, i.e., the number of subcolonies of each type formed. sociobiology 60(1): 65-68 (2013) 67 whereas eggs were absent in the orphaned and king subcolonies. immediately after the experiment, we haphazardly chose 4 of the 19 orphaned subcolonies that had produced at least one neotenic and dissected all individuals to observe their gonads. in these colonies, the numerical sex ratio in females was 25% (10 of 40), 26% (10 of 39), 36% (16 of 45) and 40% (18 of 45), but no individuals except male neotenics had clearly developed gonads. in 16 of the 19 orphaned subcolonies, each subcolony produced a maximum of one male neotenic during the entire experimental period. in 3 subcolonies, a maximum of two male neotenics were found at the same time, however only one individual survived after 1-2 weeks. emergence of a neotenic was apparent by day 7 in 1 of the 19 orphaned subcolonies, by day 14 in 15 subcolonies, and by day 21 in 3 subcolonies. discussion the neotenic system of n. koshunensis is unusual for two reasons. first, the neotenic caste is male-specific. second, the inhibitory effect is not sex-specific, i.e., both the king and the queen, whether present individually or together, inhibit the production of male neotenics. the apparent absence of female neotenics is not explained by the presence of cryptic female neotenics that had no body color change and therefore went unrecognized, because no eggs were found in the orphaned and king subcolonies, which lacked a queen. furthermore, none of the females in the orphaned subcolonies that produced male neotenics had developed gonads. the experimental period seemed to be long enough at least for males, because the testes size (the width of a testis) of neotenics increased twice that of the testes of pseudergates during the experimental period of 6 weeks (miyaguni, y. unpublished data). there is a rather well-circulated description on the biology of this species by myles (1999) asserting the existence female neotenics in this species. there is a historical reason why this incorrect information was circulated in the scientific literature. myles (1999) referred to takuya abe’s personal communication in which abe wrote that in late autumn and winter female reproductives (neotenics) were produced in this species. it was also written that female neotenics were not as darkly colored as males but they have mature ovaries. however, abe’s descriptions were all based on the results of examination of gonad size of pseudergates by our coauthor koji sugio who was a student of abe in those days. what sugio’s data showed was only that female pseudergates in late autumn and winter had on average larger ovary size than in summer. this phenomenon is a simple reflection of higher fat storage of individuals in winter (sugio, k. unpublished data). unlike in caste differentiation, no specific individual showed more prominent ovarian development than the others. we conclude that abe simply misinterpreted sugio’s data, which has since been communicated to myles (personal communication). indeed, in addition to the current experimental data, we have much evidence in the field and laboratory strongly indicating the true absence of female neotenics in this species (miyaguni, y. and sugio, k. unpublished data). male-specific neotenic systems also occur in n. connexus and n. papua (myles & chang, 1984; roisin & pasteels, 1991). however, in these two species whether both the queen and the king inhibit male neotenic production remains to be determined. our finding that the presence of not only the king but also the queen inhibited the production of male neotenics is not explained by the model of lüscher (1961), which assumes the sex specificity of pheromonal inhibition (see also matsuura et al. 2010). however, there are a few known examples that the lüscher sex-specific inhibition does not fully account for. the possible inhibition of neotenic production by the presence of a reproductive caste of the opposite sex has also been suggested in mastotermes darwiniensis (mastotermitidae), which has both male and female neotenics (watson et al., 1975). in n. jouteli, the functional queen (female neotenic) and the functional king (male neotenic) inhibit independently the emergence of new neotenics of both sexes, although the inhibition is not as complete as in n. koshunensis (nagin, 1972). these previous data together with our current results suggest that the production of neotenics in some termites is controlled by a non-sex-specific factor. our results suggest that it is difficult for the reproductive pairs of n. koshunensis colonies to be recovered when colonies lose one or both primary reproductives. one possibility, which occurs in n. papua, is the recovery of reproductive castes through the production of both male neotenics and female adultoids in orphaned laboratory colonies (roisin & pasteels, 1991). at this stage, it is difficult to understand the adaptive significance of the production of male-only neotenics that are inhibited by the presence of a queen or king, or both. to answer this question we need more information, especially on the life history of this species and on the reproductive ability of its neotenics. acknowledgments we thank s. dobata, m. k. hojo and ed vargo who kindly gave us invaluable advice during the course of the study. two anonimous reviewrs provid valuable coments that improved this article. references abe, t. 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(eds.). biology of termites vol. 1 (pp. 19–47). academic press. new york. doi: 10.13102/sociobiology.v64i1.1190sociobiology 64(1): 50-56 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 evaluation of motor changes and toxicity of insecticides fipronil and imidacloprid in africanized honey bees (hymenoptera: apidae) introduction pollination by wild animals is a key ecosystem service that is linked to human well-being through the maintenance of ecosystem health and function, wild plant reproduction, crop production and food security (potts et al., 2016). bees, including western honey bees (apis mellifera), bumble bees and solitary bees, are the prominent and economically most important group of pollinators worldwide; 35% of the global food crop production depends on pollinators (klein et al., 2007). the worldwide economic value of the pollination service provided by insect pollinators was estimated in €153 billion annually for the main crops used directly for human food (gallai et al., 2009). abstract honey bees are important pollinators and are essential in beekeeping. honey bees get exposed to systemic pesticides while foraging in contaminated fields, and it is important to know the toxicity (ld 50 ) and evaluate the impacts of bees’ exposure to these molecules. fipronil and imidacloprid are systemic pesticides widely used in brazil and other countries. the objective of this study was to determine the ld 50 (24 hours) and evaluate motor changes in africanized honey bee foragers exposed to lethal and sublethal doses of fipronil and imidacloprid. to determine the ld 50 , foraging honey bees were exposed by ingestion and contact to five doses of fipronil (regent 800wg®) and imidacloprid (appalus 200sc®) insecticides. after 24 hours of exposure, the number of dead bees was counted, and the results were subjected to probit analysis. the motor activity of bees exposed by ingestion or contact to ld 50 and sublethal doses (1/500th of the ld 50 ) of both pesticides was assessed 4 hours after exposure using a behavioral observation box. the ingestion and contact with ld 50 of fipronil were 0.0528±0.0090 and 0.0054±0.0041 μg/bee, respectively; the ingestion and contact with ld 50 of imidacloprid were 0.0809±0.0135 and 0.0626±0.0080 μg/ bee, respectively. bees exposed to lethal and sublethal doses of both insecticides experienced significant motor alterations compared to the control, except for exposure to sublethal doses of fipronil by contact. fipronil and imidacloprid are highly toxic and promote motor changes in bees. thus, it is important to establish management methods to reduce pollinators’ exposure to these pesticides. sociobiology an international journal on social insects js lunardi, r zaluski, ro orsi article history edited by denise a. alves, esalq-usp, brazil received 06 september 2016 initial acceptance 09 january 2017 final acceptance 07 february 2017 publication date 29 may 2017 keywords toxicology, systemic pesticides, behavior, apis mellifera, beekeeping, pollinators. corresponding author ricardo de oliveira orsi faculdade de medicina veterinária e zootecnia, fmvz campus botucatu distrito de rubião junior s/n caixa postal 560, cep: 18618-970 botucatu, são paulo, brasil e-mail: orsi@fmvz.unesp.br among pollinators, bees are the most important group, visiting more than 90% of the leading 107 global crop types; and a. mellifera is the most commonly managed bee in the world for crop pollination (klein et al., 2007). furthermore, the beekeeping that is based in the nurturing of honey bees to explore honey, wax, pollen, propolis, royal jelly, and apitoxin (bee venom) has high importance in employment and income generation. despite their importance, intense reductions of managed and wild bees have been observed worldwide (oldroyd, 2007; stokstad, 2007; van engelsdorp & meixner, 2010; van der sluijis et al., 2013; goulson et al., 2015). many factors has been investigated as responsible for the overall reduction of pollinators, including habitat destruction, universidade estadual paulista (unesp), faculdade de medicina veterinária e zootecnia, botucatu, são paulo, brazil research article bees sociobiology 64(1): 50-56 (march, 2017) 51 reduction of floral resources, presence of pathogens and parasites, climate change, and indiscriminate use of pesticides for crop protection (for review, see potts et al., 2010, 2016; gonzález-varo et al., 2013; goulson et al., 2015). the use of pesticides in crops to control insect pests, nematodes, weeds, and diseases has harmed the survival of native pollinators and beekeeping practices (sanchez-bayo & goka, 2016). among pesticides widely used, fipronil (phenylpyrazole) and imidacloprid (neonicotinoid) are systemic insecticides used for seed treatment, foliar application, and soil treatment in many crops. because they are systemic, they contaminate all parts of the plants, including the nectar and pollen (bonmatin et al., 2015), which are resources collected by worker bees. besides the cultivated plants, water sources and shrubs and plants growing in nearby areas may be contaminated (krupke et al., 2012) due to the dispersion of residues by drift and lateral water flow (greatti et al., 2006; sanchez-bayo et al., 2007), increasing pollinators’ exposure to these molecules. worker bees are exposed to pesticides through direct contact during or immediately after pulverization or by ingestion of contaminated resources (nectar, pollen, water); and the exposure to high doses of insecticides in field may be sufficient to kill a bee immediately (sanchez-bayo & goka, 2014). residual levels of systemic insecticides can contaminate the resources collected by bees for long periods and can be stored in nests (chauzat et al., 2006; mullin et al., 2010; orantesbermejo et al., 2010). in the nest, the contaminated resources are used to feed larvae and adults, generating physiological and behavioral changes in bees and compromising the productivity and colony maintenance (orantes-bermejo et al., 2010; zaluski et al., 2015). in brazil, fipronil and imidacloprid insecticides have authorization for commercialization in various formulations and to control pests in sugarcane, cotton, rice, bean, corn, barley, soybean, wheat, and other crops (ministério da agricultura, pecuária e abastecimento, 2016; agência nacional de vigilância sanitária, 2016). currently, the authorization of the use of imidacloprid is under review process and the aerial application of fipronil and imidacloprid are forbidden in flowering crops (ministério da agricultura, pecuária e abastecimento, 2013). the release of pesticides on crops requires regulatory measures that include ld50 tests for pollinators, and are based on european protocols (sanchez-bayo & goka, 2016). the ld50value is determined based on laboratory tests corresponding to the dose capable of causing the death of 50% of the population over a period of 24 or 48 hours. currently, the tests to regularize the use of pesticides do not regard the damages that bee exposure to doses below the ld50 (sublethal doses) can cause to honey bees individually and to the entire colony (zaluski et al., 2015; sanchez-bayo & goka, 2016). in brazil are found africanized honey bees originated from crossing of imported african apis mellifera scutellata with local populations of european a. mellifera previously introduced. the poly-hybrid bees resulting from these crossings expressed high defensive behavior, have good resistance to diseases, and are excellent pollinators (pereira & chaudnetto, 2005). some studies performed with africanized bees demonstrate that fipronil is highly toxic to newly emerged bees (roat et al., 2013) and to foragers (zaluski et al., 2015); whereas studies performed by de almeida rossi et al. (2013) demonstrate that imidacloprid is highly toxic to newly emerged bees. considering the wide use of fipronil and imidacloprid in agricultural crops and the presence of africanized bees in americas that are widely used in beekeeping and crop pollination, is important to determine the ld50 and potential behavioral changes that worker bees can have if they are exposed to lethal or sublethal doses of these molecules. in this study, the acute ld50 of fipronil and imidacloprid insecticides for africanized a. mellifera foragers was evaluated by ingestion and contact testing. additionally, based in neurotoxic effects of fipronil and imidacloprid (brown et al., 2006; narahashi et al., 2010) that can trigger motor changes in bees, were conducted tests to evaluate the motor activity of bees exposed to ld50 and sublethal doses (1/500th of the ld50) of these insecticides. material and methods the experiment was developed in the beekeeping production area of lageado experimental farm, faculty of veterinary medicine and animal science, unesp, botucatu, são paulo state, brazil (22°50’30”s; 48°25’41”w), in a humid subtropical climate, and an average elevation of 623 m. the area where the experimental apiary is located and bees were collected to perform the experiments is free of application of pesticides, what reduces the risk of bees’ contamination during foraging. taking into account that foraging honey bees are most likely to be exposed to pesticides, all tests were performed with forager honey bees to better understand effects of exposition of these bees to fipronil and imidacloprid. the study used africanized a. mellifera (hymenoptera: apidae) workers aged 20 days collected in a single hive, in order to reduce genetic variations that can interfere inld50 tests (suchail et al., 2001; zaluski et al., 2015). a bee trap was installed in the entrance of the beehive and was closed during the collection of bees; thus, only aged bees that returned from the field (foragers) were collected. seven hundred and twenty bees were collected between 7:00 a.m. and 8:00 a.m. and anesthetized in a freezer at -10 ºc for 1–2 minutes to determine ingestion and contact ld50 (zaluski et al., 2015). in all tests, the active ingredients used were from commercial formulations that are used in the field: fipronil (regent 800wg®) and imidacloprid (appalus 200 sc®). a stock solution containing 1g l-1of these insecticides was prepared separately in distilled water, considering only the amount of fipronil and imidacloprid active ingredient present in commercial formulations. there were no problems with solubility. the doses used in the tests were prepared from js lunardi, r zaluski, ro orsi – systemic pesticide toxicity in honey bees52 these solutions that were stirred during preparation and before use to ensure that they were always at a proper concentration. the ingestion ld50 was determined followed oecd guidelines (1998a) according to the method described by miranda et al. (2003) with modifications proposed by zaluski et al. (2015). after collection, groups of 10 bees were placed in disposable wooden boxes (25 × 15 × 10 cm) with side screens, and they remained unfed for 3 hours. then, the bees received 1 ml of food (honey syrup 50%) in a plastic tube (50 × 10 × 10 mm). the syrup consumption of 50 μl per bee, the volume corresponding to the average consumption per bee (crane, 1990), has been associated with consumption of 0.00, 0.010, 0.020, 0.050, 0.100, 0.200, and 0.400 µg of fipronil and 0.000, 0.012, 0.025, 0.050, 0.100, 0.200, and 0.400 µg of imidacloprid. the contaminated food was provided to bees for 3 hours and then exchanged for uncontaminated food. the volume of contaminated syrup unconsumed by each group of bees was measured to confirm the approximate dose ingested by bees in each box. to determine the contact ld50, were followed oecd guidelines (1998b).collected honey bees were anesthetized and directly transferred to disposable petri dishes (25 × 15 × 10 cm) with a perforated lid to allow adequate ventilation. then, with an automatic micropipet, the bees received 2 µl of solutions containing different amounts of fipronil (0.0000, 0.0017, 0.0035, 0.0070, 0.0160, 0.0320, 0.0640 µg) or imidacloprid (0.0000, 0.0050, 0.0100, 0.0200, 0.0400, 0.0800, 0.1600 µg) in the thorax. during all contact tests, the bees received sugar syrup ad libitum. doses used to determine the ld50 were based on preliminary tests conducted using the same method described above. all tests were performed in triplicate, using 10 bees for each dose tested. bees that showed behavioral changes or lethargy before the tests were rejected and replaced with healthy bees. the bees were kept in a b.o.d. incubator at a temperature of 33 ± 1 ºc and humidity between 60 and 70%. the occurrence of behavioral alterations in bees 3 hours after exposure to insecticides was observed. twenty-four hours after starting the tests, the number of dead bees per treatment was recorded, and the results were used to determine ld50. to study motor function in bees exposed to fipronil and imidacloprid, 360 adult bees were collected and exposed by ingestion and contact to ld50 or sublethal doses of these insecticides. the sublethal dose supplied to bees corresponded to 1/500th of the ld50 of ingestion and contact determined in the present study. the collection and exposure of bees were performed as described for the ld50 measurements. the motor activity of bees was assessed 4 hours after exposure, according to the method described by zaluski et al. (2015). the tests were performed in the laboratory using a wooden behavioral box (60 × 35 × 04 cm) divided into five lanes (50 × 05 × 04 cm), containing a fluorescent lamp in the top and covered with glass through which the bees could be observed. the tests were performed in the dark, with the box tilted at 45° and the lamp turned on, stimulating locomotion of the bees by positive phototaxis (lambin et al., 2001). the bees were released into the box, one per lane, and the time that it took each bee to travel 50 cm was recorded. for each tested dose, 10 bees were exposed to the pesticides and 10 served as controls. all tests were performed in triplicate. the ingestion and contact ld50 were determined on the basis of the mortality of bees per dose using probit analysis with maximum likelihood. the results of the motor activity analyses were compared by non-parametric mann–whitney u test and presented as the median and interquartile intervals (q1−q3). a p value of less than 0.05 was considered significant. data analyses were performed using minitab statistical software (v. 16, state college, pa, usa). results the ingestion and contact fipronil ld50 (24h) were 0.0528 ± 0.0090 μg/bee and 0.0054 ± 0.0041 μg/bee (figures 1a and 1b), respectively. the ingestion and contact imidacloprid ld50 (24h) were 0.08092 ± 0.0135 and 0.0626 ± 0.0080 μg/ bee (figures 2a and 2b), respectively. from these values, were calculated the sublethal doses equivalent to 1/500th of the ld50 to be used in motor activity tests: fipronil sublethal doses of ingestion and contact were 0.0001056 and 0.0000108 μg/bee, respectively; and imidacloprid sublethal doses of ingestion and contact were 0.0001618 and 0.0001252 μg/bee, respectively. behavioral changes verified during the ld50 tests included agitation, seizures, tremors, and paralysis in bees. these behaviors occurred in higher frequency in bees exposed to doses between 0.100 and 0.400 μg in the ingestion tests for both insecticides, between 0.0160 and 0.0640 μg for contact tests with fipronil, and between 0.0400 and 0.1600 µg in contact tests with imidacloprid. fig 1. mortality of africanized apis mellifera (24 hours) after the intoxication with fipronil by ingestion (a), and by contact (b). sociobiology 64(1): 50-56 (march, 2017) 53 in tests that analyzed motor activity, it was found that bees exposed by ingestion or contact to the fipronil and imidacloprid ld50 took longer to pass through the 50-cm track compared to bees from the control group (p < 0.05; mann–whitney u test) (table 1). in the exposure to sublethal doses, there was a reduction of motor activity in bees exposed by ingestion and contact to imidacloprid and in bees exposed by ingestion to fipronil (p < 0.05; mann–whitney u test) (table 2). for exposure by contact to a fipronil sublethal dose, there was no significant motor impairment (p > 0.05; mann–whitney u test) (table 2). highly toxic to africanized honeybee foragers, causing motor and behavioral changes after exposure by ingestion or contact to lethal and sublethal doses of these systemic insecticides. in order to evaluate the ld50 of the insecticide fipronil to european newly emerged bees, the ingestion and contact ld50 were 0.0041 and 0.0059 µg/bee, respectively (agritox database, 2016). as for the imidacloprid insecticide, the ingestion and contact ld50 values were 0.0810 and 0.0037 µg/bee, respectively (agritox database, 2016). in studies performed with africanized newly emerged bees, the ingestion and contact ld50 of the insecticide fipronil were 0.00127 and 0.00106 µg/bee, respectively (roat et al., 2013); for exposure with imidacloprid by contact ld50 was 0.0809 µg/ bee (de almeida rossi et al., 2013). the values of ingestion ld50 for africanized honey bee foragers in this study were higher for both insecticides, whereas the contact ld50 value was similar, comparing to european and africanized newly emerged bees. higher values of ld50 ingestion may be related to metabolization of toxic compounds due to the presence of enzymes in bees’ digestive system, and malpighian tubules (miranda et al., 2003). ld50 values of fipronil in africanized honey bee foragers exposed by ingestion and contact ranged from 0.19 to 0.28 µg/bee and 0.006 to 0.012 µg/bee, respectively (carrillo et al., 2013; zaluski et al., 2015). for imidacloprid, ld50 values ranged from 0.04 to 0.10 µg/bee (suchail et al., 2001; carrillo et al., 2013). changes in ld50 values can occur due to the genetic variability of bees, origin of the population, methodology, and difference in detoxification ability of bees (suchail et al., 2001). the tests in this study showed that bees intoxicated by ingestion or contact with the pesticides ld50 had impaired motor activity. these changes can be explained by the neurotoxic action of fipronil and imidacloprid. fipronil and the metabolites resulting from its degradation have an antagonistic action on gamma amino butyric acid (gaba) neurotransmitters and glutamate-activated chloride channels (glucls) (narahashi et al., 2010). unlike fipronil, imidacloprid and its metabolites act as nicotinic acetylcholine receptor (nachr) agonists, which provide the majority of the excitatory neurotransmission in the table 1. median and interquartile intervals (q1−q3) of time (seconds) spent by africanized apis mellifera to travel 50 cm 4 hours after exposure by ingestion or contact to ld50 of fipronil and imidacloprid insecticides. treatment exposure (µg/bee) time (s) control ingestion 0.0000 6.00 (4.00 – 8.50) ld 50 ingestion fipronil 0.0528 35.50 (21.00 – 58.50)* ld 50 ingestion imidacloprid 0.0809 17.50 (13.00 – 29.25)* control contact 0.0000 7.00 (5.00 – 10.00) ld 50 contact fipronil 0.0054 12.00 (8.75 – 14.50)* ld 50 contact imidacloprid 0.0626 19.00 (12.00 – 31.00)* *p < 0.05 compared to the control group using the mann–whitney u test. discussion according to the toxicological classification of johansen and mayer (1990), pesticides whose ld50 is less than 2 μg/bee are highly toxic to a. mellifera. the results of this study demonstrate that fipronil and imidacloprid are fig 2. mortality of africanized apis mellifera (24 hours) after the intoxication with imidacloprid by ingestion (a), and by contact (b). table 2. median and interquartile intervals (q1−q3) of time (seconds) spent by africanized apis mellifera to travel 50 cm 4 hours after exposure by ingestion or contact to sublethal doses (sd) of fipronil and imidacloprid insecticides. treatment exposure (µg/bee) time(s) control ingestion 0.0000000 6.00 (4.00 – 8.50) sd ingestion fipronil 0.0001056 9.00 (6.00 – 12.00)* sd ingestion imidacloprid 0.0001618 10.00 (4.75 – 17.75)* control contact 0.0000000 7,00 (5.00 – 10.00) sd contact fipronil 0.0000108 7,00 (4.75 – 13.25) sd contact imidacloprid 0.0001252 10,00 (5.75 – 14.75)* *p < 0.05 compared to the control group using the mann–whitney u test. js lunardi, r zaluski, ro orsi – systemic pesticide toxicity in honey bees54 insects’ central nervous system (deglise et al., 2002; brown et al., 2006). besides motor changes, the effects of these insecticides in the bees’ nervous system trigger agitation, seizures, tremors, and paralysis, behaviors that were observed during ld50 tests. the results of this study also show that motor abnormalities occur in bees exposed to sublethal doses of fipronil and imidacloprid. studies by colin et al. (2004) found that sublethal doses of fipronil and imidacloprid reduce the foraging activity of a. mellifera. these results indicate that low doses of these pesticides can compromise vital functions for the maintenance of colonies, inducing effects on the nervous system that drive alterations in the orientation and consequently in the behavior. these alterations can compromise the orientation of bees for the location and collection of resources for the colony. motor and behavioral changes, as observed during this study, may occur after the pulverization of blooming crops, in which the bees collect resources. in addition, fipronil and imidacloprid residues in nectar and pollen can be found when crop seeds are treated with these molecules during planting; this is due to the high persistence and systemic nature of these insecticides (bonmatin et al., 2015). repetitive spraying of these insecticides on crops results in increased environmental contamination, and surrounding areas also can be contaminated, representing a risk to wild pollinators and to commercial beekeeping (greatti et al., 2006; sanchez-bayo et al., 2007). bees’ exposure to lethal and sublethal doses of systemic pesticides may increase motor and behavioral alterations due to continuous exposure of these pollinators to contaminated resources. the fipronil and imidacloprid detection in resources collected by bees and stored in hives (chauzat et al., 2006; pareja et al., 2011) confirmed the exposure of pollinators to sublethal doses of these insecticides, which can compromise the maintenance of whole colonies. studies also have demonstrated the high toxicity of these molecules to native stingless bees (tomé et al., 2012; rondeau et al., 2014; costa et al., 2015; soares et al., 2015). the use of the systemic insecticides fipronil and imidacloprid was banned in france, italy, germany, slovenia (ghisi et al., 2011), and uruguay (pareja et al., 2011) due to the high toxicity of these pesticides to pollinators. in brazil, the authorization of the use of imidacloprid is under review process, and the aerial application of neonicotinoids (imidacloprid, tiamethoxam, clotianidine) and fipronil are forbidden in flowering crops (ministério da agricultura, pecuária e abastecimento, 2013).the review of the use authorization of fipronil and imidacloprid in countries where these products are authorized, such as brazil, is important to reduce the risk that these substances represent to honey bees, whose loss affects beekeeping and compromises the survival and maintenance of native pollinators. measures to replace fipronil and imidacloprid and related insecticides with products with lower toxicity to pollinators must be taken in order to avoid honey bee losses and to preserve the native pollinators that are essential to the maintenance of ecosystems. the adoption of strategies to reduce the use of fipronil and imidaclopride on crops, including biological control and integrated pest management, can contribute to the conservation of bees and reduce the environmental contamination caused by the use of systemic pesticides. references agência nacional de vigilância sanitária (br). 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(2014). four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae. plos one, 9: e77547. doi: 10.1371/ journal.pone.0077547 doi: 10.13102/sociobiology.v62i1.10-17sociobiology 62(1): 10-17 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 field evaluations of broadcast, and individual mound treatments for red imported fire ant, solenopsis invicta buren, (hymenoptera: formicidae) control in virginia, usa introduction prior to 2009, all reported red imported fire ant (rifa), solenopsis invicta buren, infestations in virginia were documented and managed by the virginia department of agriculture and consumer services (vdacs). in spite of vdacs’ best efforts however, rifa infestations within the state continued to increase and spread. therefore, in 2009 the united states department of agriculture (usda) in conjunction with vdacs implemented the imported fire ant quarantine in the following areas of virginia: the counties of james city and york, and the cities of chesapeake, hampton, newport news, norfolk, poquoson, portsmouth, suffolk, virginia beach and williamsburg. consequently, vdacs is no longer responsible for treating rifa mounds in the quarantined areas. fire ant control in the quarantined counties/cities is now the responsibility of homeowners, nurseryman, and pest management abstract field evaluations were conducted to determine efficacy, residual activity, and knockdown potential for fire ant control products. broadcast granular products (advion, 0.045% indoxacarb; and top choice insecticide, 0.0143% fipronil) were individually evaluated, and compared with a combination of two products applied together, and with individual mound applications of maxforce fire ant killer bait (1.0% hydramethylnon). after application, the greatest percent reductions (90 days) were observed in the advion/ top choice combination plots (100.0%), followed by top choice alone (96.4%). advion and maxforce produced significantly lower foraging reductions at 90 days (61.2% and 27.5% respectively). at the conclusion of the test (day 360), significantly fewer ants were collected in the advion (777.7), top choice (972.8), and combination plots (596.2) than in the control plots (1257.8) (df 13, f = 8.3, p < 0.05). the mean number of ants collected from maxforce treatment plots was not significantly different from controls (p > 0.05). overall, the efficacy and residual studies suggested that the advion/ top choice combination produced both the most rapid reduction in ant foraging and the longest lasting control (90%) at 300 days. when evaluating time to knockdown of foraging populations, the advion/top choice combination also provided the most complete and rapid results by day 7, reducing foraging by 100%. while other products also performed well (75.6 95.9% reductions), both the maxforce and advion plots had significant increases in foraging at 30-90 days. overall, foraging knockdown was the most complete in the avion/top choice combination plots at 90 days. sociobiology an international journal on social insects hr allen, dm miller article history edited by kleber del-claro, ufu, brazil received 22 september 2014 initial acceptance 31 october 2014 final acceptance 25 november 2014 keywords red imported fire ant, solenopsis invicta buren, broadcast, control, bait. corresponding author dini m. miller department of entomology virginia tech university 216a price hall, blacksburg, va 24061, usa e-mail: dinim@vt.edu professionals. however, vdacs is still responsible for managing rifa infestations in cities outside of the designated quarantine areas. as of 2014, the quarantine has not been expanded and vdacs is still responsible for controlling infestations in the large majority of the state. the standard control method used by vdacs for treating rifa mounds in virginia in 2009 was to apply maxforce® fire ant bait (1.0% hydramethylnon) (bayer cropscience, kansas city, mo) around each active mound. bait applications were followed six weeks later by an acephate mound drench. although effective, these individual mound treatments (imt) required that all mounds be located prior to application and then treating them one at a time. while imts are labor intensive and time consuming, the direct chemical application to the mound does greatly enhance the amount of insecticide contact with colony members (barr & best, 1999). research article ants virginia tech university, blacksburg, va, united states sociobiology 62(1): 10-17 (march, 2015) 11 baits and liquid insecticides are the typical formulations used for imts, but aerosols, granules, and dusts are also frequently used. imts are the most useful when 20 or fewer mounds are present in an acre of land (barr & best, 2002). imts are also beneficial because they are only applied specifically to rifa mounds, thus preventing native ant mortality. however, because imts are only applied to visible mounds, fire ant recolonization can easily occur in treated areas where small mounds are overlooked and not treated. multiple applications are often necessary to control all the mounds in a particular area. in contrast to imts, broadcast insecticide treatments for rifa do not require individual mounds to be located. therefore, broadcast products greatly reduce the time and labor needed to treat a large area. broadcast fire ant control products are currently formulated as either granules or baits and are applied using either a hand or tractor mounted spreader (drees et al., 2006). broadcast products are typically applied in locations where mound densities exceed 30-40 per hectare. bait formulations are frequently applied as a broadcast rifa control method. rifa baits are usually formulated by combining a slower acting toxicant with soybean oil or some other food matrix that is attractive to foraging fire ants (williams et al., 2001). once the ants transport the bait back inside the colony, the ants transfer the active ingredient throughout the colony by trophallaxis. because the active ingredient must be spread throughout the colony via the worker ants feeding the queens and brood, it may take several weeks to months before significant colony reductions are observable in the field (drees et al., 2006). therefore, colony suppression may take significantly longer using broadcast baits when compared with imts that provide reductions in a single day. however, a study conducted by barr and best (1999) suggested that the benefits of large scale ant suppression that could be achieved with broadcast bait products far out-weighed the delay in short term (but eventual) results (barr & best, 1999). some broadcast fire ant products have been used to treat fire ants in virginia, but they have been used infrequently due to vdacs’ preference for individual mound treatments. however, now that vdacs is no longer responsible for treating infestations in quarantined counties and cities, residents in these locales have the burden of managing fire ants on their own. with the quarantine implementation, the need for broadcast rifa product evaluations and other control recommendations are vital, if not for stopping rifa, at least for slowing the spread of rifa in virginia. two of the leading broadcast fire ant control products are advion® fire ant bait (indoxacarb 0.045%; syngenta, research triangle park, nc) and top choice granular (fipronil 0.143%; bayer environmental sciences, cary, north carolina). advion is a fast acting bait (furman & gold, 2006) that contains the active ingredient, indoxacarb, which belongs to the oxadiazine chemical class. oxadiazines block sodium channels in the insect nerve axon. immediately after bait ingestion, ant feeding begins to decrease and target individuals usually succumb to death within 48 hours (barr, 2002a). top choice contains the active ingredient fipronil which belongs to the phenylpyrazole chemical class. fipronil is a nerve poison that blocks the passage of chloride ions through gaba receptor and glutamate-gated chloride channels causing nerve hyperexcitation in target insects (kolaczinski & curtis 2001). previous studies have shown that advion significantly reduced fire ant foraging 24 hours after treatment (barr, 2004) and eliminated > 95% of colonies after one week (hu & song, 2007) after application. top choice has a longer residual activity than advion but is much slower acting. barr and best (2004) reported that top choice® reduced the mean number of active fire ant mounds by 80% five weeks after treatment and greater than 90% control was observed 52 weeks later. the purpose of this study was to evaluate the performance of specific broadcast red imported fire ant treatment in virginia, and compare their efficacy with that of an imt. our goal was to determine which application method might have the longest residual activity, and therefore the greatest potential to prevent fire ant spread. in this study, field applications of the rifa control products: advion® fire ant bait (indoxacarb 0.045%; syngenta, research triangle park, nc); top choice granular (fipronil 0.143%; bayer environmental sciences, cary, north carolina); a combination application of advion and top choice; and an imt treatment using maxforce® fire ant bait (1.0% hydramethylnon), were monitored for efficacy for one year. the following year, the same field applications were reapplied to determine the rapidity of initial knock-down. materials and methods study area although our initial research plots were established on an infested vacant lot in hampton roads, virginia (2008; prior to the implementation of the federal fire ant quarantine (ffaq), the research site came to the attention of a neighboring school facilities manager who demanded that vdacs treat the location. to avoid further conflict within virginia we moved our research site 161 km due south to north carolina where the entire state was already under the ffaq. our new research plots were established within fun junktion park, a converted landfill located in elizabeth city, nc. the study was conducted from 5 august 2008 to 26 july 2009. elizabeth city is located on the northeast coast of north carolina in pasquotank county (36°17’44’n; 76°13’30’w). average monthly temperatures range from a low of 0° c during the winter months to a high of 31.8° c during the summer months. the city receives about 122 centimeters of rainfall annually. research plots fourteen 30 x 30 m (900 m2) research plots (fig 1a-b) were established within three different locations within the park. hr allen & dm miller field evaluation of red imported fire ant control products 12 eight plots were located on a driving range that was covered with grass and mowed weekly. four plots were established in a grass covered field located near an artificial lake. two plots were located in a weed covered field that was not mowed. an untreated buffer zone (7.6 m) separated each plot to reduce potential ant foraging between research plots. plots were randomly assigned to different treatments so that each of the four insecticide treatment had three replicates. the two remaining plots served as untreated controls. treatment products the broadcast products evaluated in the study were advion® fire ant bait (0.045% indoxacarb; syngenta, research triangle park, nc), top choice® insecticide (0.0143% fipronil; fig. 1a-b. placement of 1-year rifa treatment plots located at fun junktion park, elizabeth city, nc (google earth 2010)advion (adv), top choice (tc), maxforce (mf), advion /top choice combination (com) and untreated control (con). a b bayer environmental science, research triangle park, nc) and maxforce® fire ant killer granular bait (1.0% hydramethylnon; bayer crop science, kansas city, mo). advion and top choice were also used in a combination treatment where they were applied together in the same plot. all broadcast products were applied at the label rate (advion: 1.68 kg/hectare (1.5 lbs./acre), top choice: 209 kg/hectare (85 lbs./acre), maxforce: 14-28g/ mound (0.5-1.0 oz./mound), combination: advion/top choice) using scott’s handy green ii hand spreaders (scotts international b.v., scotts professional, geldermalsen, the netherlands). the maxforce bait is labelled for application as a broadcast or as an individual mound treatment. however, for the purposes of this study, maxforce was used as an imt and was applied directly to individual mounds from the product container. treatment applications were made on 12 july 2008 between 5:00 p.m. and 7:00 p.m. each broadcast treatment was applied to three plots. maxforce bait was applied to seven active fire ant mounds located in three experimental plots. sampling regimen to quantify foraging activity prior to treatment applications, slices of uncooked hot dog wieners were used as baits to quantify foraging activity in each of the plots. pre-treatment bait counts were taken on 11 july 2008 between 5 and 7:00 p.m. eight beef hot dog (gwaltney, smithfield va) slices (0.5 cm thick) were placed in each plot. the hot dog slices were arranged in two rows of four and each row was spaced 7 m apart. hot dog slices were left in place for one hour, after which photographic images were taken of each slice with a sony cybershot digital camera (sony electronics inc., san diego, ca). all images were downloaded onto a computer so that the species and number of ants in each hot dog photograph could be counted and recorded. post-treatment ant sampling with hot dog slices was conducted between 5:00 p.m. and 7:00 p.m. at 3, 7, 14, and 30 days after treatment and every month thereafter for one year. during the initial study, post-treatment data collected on 7, 14, 30, and 60 days were lost after the computer laptop holding that data was stolen. in addition, sampling was not conducted during the winter months (121 and 239 days after treatment) because of low temperatures that eliminated ant foraging. product reapplication to determine time to knockdown after the one year completion of the study described above, all plots were sampled again (as previously described) to determine ant foraging activity. after determining that the ant pressure had rebounded and was still very high, all products were reapplied. treatment applications were made on 21 july 2009 between 5:00 p.m. and 7:00 p.m. maxforce bait was applied to 5 mounds. control plots (2) were left untreated. post-treatment sampling was conducted between 5:00 p.m. and 7:00 p.m. sampling was conducted on days 3, 7, 14, 30, 60, and 90 after treatment to determine the time to knockdown for all treatment products and combinations. sociobiology 62(1): 10-17 (march, 2015) 13 statistical analysis the mean number of foraging fire ants collected per treatment on each sampling date was calculated by adding the total number of ants foraging on all 8 hot dogs in each treatment plot, and dividing that total by number of plots per treatment. to determine if the treatment applications had any effect on the mean number of foraging ants, data were transformed (√(x +⅜)) (zar 1984) and subjected to repeatedmeasures multivariate analysis of variance (manova), with the post treatment date as the repeated measure. repeatedmeasures manova was also used to determine if the residual activities of each treatment were significantly (p < 0.05) different from one another. each treatment plot was significantly lower (p < 0.05) than that collected in the controls. in addition, contrast comparison tests revealed that the mean number of ants collected in each of the treatment plots were all significantly different from each another. the ancova was conducted to compare the mean number of foraging ants in each treatment, on each sampling day. ls means calculated by the ancova were used to calculate the percent change in the mean number of active foragers on each post treatment sampling date (table 1). three days after treatment the mean number of foraging fire ants in the advion, maxforce, and advion/top choice combination plots was significantly lower than that in the untreated controls (p < 0.05). the greatest percent reduction in foraging three days after treatment was observed in advion/top choice combination plots (82.7%) followed by advion alone (79.5%), maxforce (68.4%), and top choice alone (6.6%). although sampling data was collected between for dat-7 through dat-60, these data were lost. when post-treatment sampling resumed on dat-90 there were significantly fewer ants collected from the advion (355.5), top choice (38.2), and advion/top choice (0.0) plots than in the maxforce (995.6) and control plots (1369.1). the greatest percent reduction in foraging at dat-90 was observed in the advion/top choice combination plots (100.0) followed by top choice (96.4), advion (61.2), and maxforce (27.5). for the remainder of the test (dat-90 – dat-360), fewer fire ants were collected in combination and top choice treatment plots than in all of the other experimental treatment plots. at the conclusion of the test on dat-360, there were significantly fewer ants collected in advion (777.7), top choice (972.8), and combination plots (596.2) than in the control plots (1257.8) (df 13, f = 8.3, p < 0.05). however, the mean number of ants collected from maxforce treatment plots was not significantly different from controls (p > 0.05). fig 2. mean number of foraging rifa in experimental plots before and after product applications (one-year study). trend lines followed by the same letter are not significantly different (α = 0.05). fig 3. mean number of foraging rifa in experimental plots before and after product re-applications (90-day study). trend lines followed by the same letter are not significantly different (α = 0.05). overall, the results suggest that the advion/top choice combination, and the advion treated plots had the greatest reductions in ant foraging by day 3, causing foraging reductions of 82.7 and 79.5 percent respectively. however, advion, top choice, and the advion/top choice combination treatment also provided the longest lasting control with significant reductions in foraging at 360 days. differences in the mean number of rifa collected in each treatment on each sampling date was determined using two by one way repeated-measures ancova, with the mean number of foraging ants collected on dat-0 as a covariate. significant differences among treatment means on each post treatment sampling date were separated by tukey’s hsd test (p < 0.05). ls means produced in the ancova were used to calculate percent change in the mean number of rifa foraging ants after treatment relative to the initial number of foragers on dat-0 (vickers 2001). separate repeated-measures manova and ancova analyses were conducted on both the initial application (year-long test), and re-treatment (knockdown) data. results product efficacy tests repeated-measures manova was used to determine whether product applications had any effect on the mean number of foraging ants collected in plots. results of the repeated measures manova indicated that there was a significant overall treatment effect on the mean number of foraging fire ants (f = 72.0; df = 9, p < 0.0001) (figure 2). contrast comparison tests between the mean number of foraging fire ants collected from treatment plots and control plots indicated that the mean number of foragers collected from hr allen & dm miller field evaluation of red imported fire ant control products 14 d ay a ft er t re at m en t ( d a t ) tr ea tm en t d a t0 d a t3 d a t90 d a t12 0 d a t24 0 d a t27 0 d a t30 0 d a t33 0 d a t36 0 to p c ho ic e l s m ea n (± s e ) pe rc en t c ha ng e 10 64 .3 99 4. 0a (± 5 1. 1) (6 .6 ) 38 .2 b (± 1 08 .7 ) (9 6. 4) -3 .3 b (± 5 6. 7) (1 00 .0 ) 28 .5 c (± 5 2. 6) (9 7. 3) 8. 7c (± 6 3. 8) (9 9. 2) 34 5. 0b c (± 1 01 .2 ) (6 7. 6) 75 2. 4b (± 7 0. 3) (2 9. 3) 97 2. 8b c ( ± 83 .9 ) (8 .6 ) a dv io n l s m ea n (± s e ) pe rc en t c ha ng e 91 6. 7 18 8. 1b c ( ± 52 .2 ) (7 9. 5) 35 5. 5b (± 1 11 .1 ) (6 1. 2) 26 0. 1a b (± 5 8. 0) (7 1. 6) 18 6. 3b c (± 5 3. 7) (7 9. 7) 57 7. 3b (± 6 5. 2) (3 7. 0) 51 0. 2a b (± 1 03 .5 ) (4 4. 3) 73 4. 3b c (± 7 1. 8) (1 9. 9) 77 7. 7c (± 8 5. 8) (1 5. 2) m ax f or ce l s m ea n (± s e ) pe rc en t c ha ng e 13 72 .3 43 3. 4b (± 5 3. 7) (6 8. 4) 99 5. 6a (± 1 14 .3 ) (2 7. 5) 23 9. 7a b (± 5 9. 6) (8 2. 5) 35 2. 7a b (± 5 5. 3) (7 4. 3) 66 8. 7b (± 6 7. 0) (5 1. 3) 72 3. 6a b (± 1 06 .4 ) (4 7. 3) 11 89 .4 a (± 7 3. 9) (1 3. 3) 13 18 .3 ab (± 8 8. 2) (3 .9 ) a dv io n/ to p c ho ic e c om bi na tio no l s m ea n (± s e ) pe rc en t c ha ng e 94 4. 0 63 .3 c (± 5 1. 9) (8 2. 7) -2 4. 4b (± 1 10 .4 ) (1 00 .0 ) -1 5. 2b (± 5 7. 6) (1 00 .0 ) 3. 9c (± 5 3. 4) (9 9. 6) -1 8. 2c (± 6 4. 8) (1 00 ) 78 .0 c (± 1 02 .8 ) (9 1. 7) 39 6. 5c (± 7 1. 4) (5 8. 0) 59 6. 2c (± 8 5. 2) (3 6. 8) u nt re at ed c on tr ol l s m ea n (± s e ) pe rc en t c ha ng e 12 40 .5 94 3. 7a (± 6 3. 1) (2 3. 9) 13 69 .1 a (± 1 34 .3 ) (1 0. 4) 51 9. 6a (± 7 0. 0) (5 8. 1) 50 8. 9a (± 6 5. 0) (5 9. 0) 11 55 .3 a (± 7 8. 8) (6 .9 ) 10 76 .7 a (± 1 25 .0 ) (1 3. 2) 10 29 .6 ab (± 8 6. 8) (1 7. 0) 12 57 .8 a (± 1 03 .7 ) (1 .4 ) f 58 .6 19 .1 9. 5 11 .7 38 .1 8. 5 12 .6 8. 3 df 13 13 13 13 13 13 13 13 p <0 .0 00 1 0. 00 03 0. 00 3 0. 00 1 <0 .0 00 1 0. 00 46 0. 00 13 0. 00 5 m ea ns w ith in a c ol um n fo llo w ed b y th e sa m e le tte r a re n ot s ig ni fic an tly d if fe re nt (t uk ey s h sd m ea n se pa ra tio n te st ; α = 0 .0 5) . t ab le 1 l ea st s qu ar e m ea ns (± s e ) a nd m ea n p er ce nt c ha ng e in n um be r o f f or ag in g r if a p ri or to a nd d ay s af te r a pp lic at io n. sociobiology 62(1): 10-17 (march, 2015) 15 product reapplication to determine time to knockdown repeated-measures manova results indicated that the insecticide products had a significant overall treatment effect on the mean number of foraging fire ants (f = 76.1; df = 9, p < 0.0001) (table 2). contrast comparison tests between the mean number of foraging fire ants collected from insecticide treated plots and control plots indicated that the mean number of foragers collected from insecticide treated plots were significantly lower (p < 0.05) than that of the controls. additionally, contrast comparisons also indicated that the greatest reductions in the number of active foragers occurred in the advion (82.9%), maxforce (79.6%), and advion/top choice (85.7%) combination plots. these reductions were far greater than reductions observed in top choice (17.5%) and control plots (0.9%). the ancova results indicated that throughout the test the mean number of ants collected in all chemical treatments was significantly lower (p < 0.05) than the mean number of ants collected in control plots on each sampling date. additionally, from dat-3 to dat-30 the mean numbers of ants collected in chemical treatment plots were significantly lower (p < 0.05) than the mean number collected from the control plots. however, the mean number of ants collected from dat-3 to dat-30 in each of the treatments plots were not different from each other. however on dat-60, the mean number of foraging ants increased in all plots except those treated with top choice. dat (days after treatment) treatment dat-0 dat-3 dat-7 dat-14 dat-30 dat-60 dat-90 top choice ls mean (± se) percent change 972.8 809.0b (± 64.4) (17.5) 239.0b (± 53.4) (75.6) 83.0b (± 46.0) (91.5) 75.0b (± 49.3) (92.4) 68.3c (± 46.7) (93.0) 107.6c (± 106.5) (89.0) advion ls mean (± se) percent change 777.7 149.1c (± 70.0) (82.9) 70.4b (± 58.0) (91.9) -21.5b (± 50.0) (100) 39.2b (± 53.5) (95.5) 349.7b (± 50.7) (59.9) 441.4bc (± 115.6) (49.4) max force ls mean (± se) percent change 1318.3 264.2c (± 97.7) (79.6) 53.3b (± 81.0) (95.9) 187.0b (± 69.7) (85.5) 120.3b (± 74.7) (90.7) 429.7b (± 70.7) (66.8) 989.6ab (± 161.4) (23.5) advion/top choice combination ls mean (± se) percent change 596.2 90.2c (± 106.7) (85.7) -18.5b (± 88.5) (100) -85.1b (± 76.1) (100) 44.0b (± 81.7) (93.0) 116.5bc (± 77.3) (81.6) 44.3bc (± 176.4) (93.0) untreated control ls mean (± se) percent change 1257.8 1227.9a (± 99.5) (0.9) 1489.1a (± 82.5) 20.1 1414.8a (± 71.0) 14.1 1005.8a (± 76.1) (18.9) 1039.0a (± 72.0) (16.2) 1460.7a (± 164.4) 17.8 f 36.7 79.5 91.3 45.5 59.0 20.3 df 13 13 13 13 13 13 p <0.0001 <0.0001 <0.0001 <0.001 <0.0001 0.0002 table 2. least square mean (± se) and mean percent change of foraging rifa prior to and after treatment reapplication. means within a column followed by the same letter are not significantly different (tukeys hsd mean separation test, α = 0.05). while advion and maxforce still had significant reductions in foraging at day 60, the reductions were significantly less than those of top choice and the advion/top choice combination at 60 days. at the conclusion of test on dat-90, percent reductions in foraging were greatest in the advion/top choice combination and top choice treated plots. at 90 days, the maxforce bait had the lowest reduction in foraging, but this reduction was not significantly different from that in advion or advion/top choice combination plots. overall, the knockdown of foragers was the most rapid and complete in the advion/top choice treated plots on day 7 (100%). however all insecticide treatments produced between 90-100% knockdown in 7-14 days. the advion/top choice combination and top choice treatments had the longest lasting effect, suppressing foraging by 89-93% for 90 days. discussion results obtained from the year-long field efficacy trial indicated that the advion/top. choice combination treatment provided the both the most rapid control of fire ants and the greatest residual activity. while both products were very effective at controlling fire ants in the field, the advion had most rapid activity although it did not suppress the populations as long as top choice. top choice did not produce the most rapid knockdown but did have the longest residual activity both alone and in the combination treatment (table 1). hr allen & dm miller field evaluation of red imported fire ant control products 16 therefore it was no surprise that the two products combined produced results that were superior to either of the broadcast products used alone, and to the imt using maxforce bait. in the 90 day knockdown evaluations, the advion/ top choice combination provided the most complete and rapid results by day 7, effectively reducing foraging by 100%. however, the other products also performed very well (75.6 95.9% reductions in foraging) and there were no significant differences between the treatments at 7 days (table 2). again at day 14 all of the treatments were equally effective with both advion and the advion/top choice reducing rifa foraging by 100%. interestingly, the trend we observed over 30 to 90 days after application was that there was significant increase in rifa foraging in both the maxforce and advion treatments, yet efficacy for the top choice alone and the advion/top choice combination remained relatively high. foraging suppression was significantly greater in the avion/top choice combination at 90 days than in all other treatments. overall, the advantage of the combined broadcast treatment application was that one formulation (advion) was very fast acting, while the slower acting top choice provided the persistent residual activity that was limited in the advion formulation. the data provided in these studies indicated that broadcast fire ant control products can provide longer residual control, and therefore that may slow the spread of rifa colonies into untreated areas more effectively than imts (williams et al. 2001, banks et al. 1988). in both the residual study, and 90 day knockdown tests, advion, top choice, and the advion/top choice combination provided faster, longer lasting results than the maxforce mound treatments. studies evaluating advion conducted by barr (2002a, 2002b) reported similar rapid knock-down results. in 2002, barr conducted two tests, one in the summer and one in the fall to evaluate the efficacy of indoxacarb to control rifa colonies. both tests were conducted at an airport located in yoakum, texas. in both tests, barr (2002a-b) compared the efficacies of different fire ant products: amdro® fire ant bait (0.73% hydramethylnon; ambrands, atlanta, ga), extinguish® fire ant bait (0.5% s-methoprene; wellmark international, schaumburg, il), talstar® 2g (0.2% bifenthrin; fmac professional solutions, philadelphia, pa) and three different concentrations of indoxacarb (0.025%, 0.05%, and 0.1%). results from tests conducted in the summer (barr 2002a) indicated that the three indoxacarb formulations provided faster control of rifa colonies than the other rifa products tested. one week after treatment, the mean number of active mounds in all of the indoxacarb treated plots ranged from (0.25 – 1.25) while the mean number of active mounds in plots treated with amdro was 4.0; extinguish 16.25, and talstar was 3.25. however, 6 weeks after treatment the number of active colonies in all the indoxacarb treated plots began to increase. the number of active mounds found in the other treatment plots also began to increase, however fewer active colonies were documented in extinguish treatment plots. mound density in plots treated with indoxacarb continued to increase for the remainder of the test. barr (2002b) replicated the airport test again in the fall, to determine if colony foraging or reproductive status influenced his summer results. barr (2002b) found that in the fall the product efficacy results were similar to those of the previous summer. overall, the three indoxacarb formulations provided more rapid foraging reductions than the other fire ant control products tested. similarly, studies evaluating top choice also found that the product provided longer residual fire ant control than other fire and products tested. barr and best (2004) conducted a test to evaluate the efficacy of two granular formulations of fipronil (0.0143%; 0.00015% fipronil) amdro ant bait (0.73% hydramethylnon,ambrands, atlanta, ga), and talstar 2g (0.2% bifenthrin, fmc, philadelphia, pa). the study results demonstrated that fipronil provided greater long-term control than the other fire ant control products. five weeks after treatment, granular applications of 0.0143% fipronil still provided an 83% 98% reduction in the number of active mounds. by week 52 the number of active mounds began to rebound in all treatment plots except those treated with the fipronil (granular 0.0143%; barr & best, 2004). because the combination treatment used in our tests consisted of advion (one of the fastest acting baits on the market) and top choice, (which provides long residual control) we expected the combination treatment to outperform the other products and provide long lasting control. our results indicated that our expectations were correct. however, it should be noted that these products are most efficiently used in large scale situations where many mounds are present, and fire ant spread is a concern. broadcast products are relatively expensive, costing $7-10 per hectare. product costs for individual mound treatments are ~25 cents per mound, not including the labor (drees et al., 2006). therefore, for small infestations in a residential yard or in some other more contained area, individual mound treatments would still be the most desirable and effective method of fire ant management. conclusions the overall results of this study determined that broadcast fire ant control products tested were faster acting and had a longer residual than the imt. presently, vdacs manages fire ant infestations outside of virginia’s fire ant quarantined areas while homeowners and pest control operators are responsible for treating infestations within quarantine borders. vdacs currently uses imts to treat all fire ant mounds outside of the quarantine area. given the evidence provided in this study, it is reasonable to assume that in confined locations where all active fire ant mounds are visible, the imts will provide adequate control. however, in large areas that contain many mounds, like a vacant lot, a broadcast application can provide better and longer lasting control. as proven by the implementation of the rifa quarantine, the imt method used alone was not enough to slow the spread of the red imported sociobiology 62(1): 10-17 (march, 2015) 17 fire ant in virginia. while broadcast rifa products are more expensive than imts they require very little labor to apply. thus government agencies like, vdacs could possibly save money on the application costs outside the quarantine area by adding broadcast application products to their rifa arsenal. acknowledgements the authors would like to thank dr. clay scherer of dupont professional products (now syngenta professional products) for providing the funding for this study. we would also like to thank the virginia department of agriculture office of plant and pest services for their initial oversight of this study prior to our having to move our field site to north carolina. references banks, w.a., williams, d. f. & c. s. lofgren. (1988). effectiveness of fenoxycarb for control of red imported fire ants (hymenoptera: formicidae) journal of economic entomology, 81:83-87. barr, c. l. (2002a). the active ingredient indoxacarb as a broadcast bait for the control of red imported fire ants. texas coop. ext., texas a&m univ. system. https://insects.tamu. edu/fireant/research/arr/year/99-03/res_dem_9903/pdf/4_ai_ indoxacarb.pdf. (accessed date: 12 march 2009) barr, c. l. (2002b). indoxacarb bait effects on mound activity and foraging of red imported fire ants. texas coop. ext., texas a&m univ. system. https://insects.tamu.edu/fireant/ research/arr/year/99-03/res_dem_9903/pdf/1_indoxacarb_bait_ effects.pdf. (accessed date: 12 march 2009) barr, c. l. (2004). how fast is fast?: indoxacarb broadcast bait. p. 46-50. in proceedings of the annual red imported fire ant conference, baton rouge, la. 201 p. barr, c. l., & best, r. l. (1999). comparison of amdro®, spectracide® fire ant bait and diazinon using broadcast and individual mound treatment applications. results and demonstration handbook. 1997-1999. texas agricultural extension service. bryan, tx. 70 p. barr, c.l. & best r. l. best. (2002). product evaluations, field research and new products resulting from applied research. southwestern entomologist, supplement, 25:47-52. barr, c. l. & best, r. l. (2004) comparison of different formulations of broadcast fipronil for the control of red imported fire ants. result demonstration handbook 1999– 2003. 2004. texas agric. ext. serv. bryan, tx. http://fireant. tamu.edu. (accessed date: 18 feb. 2011). drees, b. m., vinson, s. b., gold, r. e. merchant, m. e., brown, e., keck, m., nester, p. kostrom, d., flanders, k., graham, f., loftin, k. hopkins, j., vail, k., wright, r. smith, w., thompson, d. c., kabashima, j., layton, b., koehler, p. g., oi, d. h. & callcott, a. m. (2006). managing imported fire ants in urban areas. texas coop. ext., texas a&m univ. system. mp426. 23 p. furman, b.d. & r.e. gold (2006). determination of most effective chemical form and concentration of indoxacarb, as well as the most appropriate grit size, for use in advion. sociobiology. 48: 309-334. hu, x, & song, d. (2007). field evaluation of label-rate broadcast treatment with baits for controlling the red imported fire ant, solenopsis invicta (hymenoptera: formicidae). sociobiology. 50: 1107-116. kolaczninski, j. & curtis, c. (2001). laboratory evaluation of fipronil, a phenylpyrazole insecticide, against adult anopholes (diptera: culicidae) and investigation of its possible cross-resistance with dieldrin in anopheles stephensi. pest managemet science, 57: 41-45. vickers, a. j. (2001). the use of percentage change from baseline as an outcome in a controlled trial is statistically inefficient: a simulation study. bmc medical research methodology, 6: 1-4. williams, d. f, collins, h. l. & oi, d. h. (2001). the red imported fire ant (hymenoptera: formicidae): a historical perspective of treatment programs and the development of chemical baits for control. american entomologist, 47:146149. zar, j.h. (1984). biostatistical analysis. second edition. prentice-hall inc., engelwood cliffs, new jersey. 718 pp. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4. 483-489sociobiology 61(4): 483-489 (december, 2014) size variation in eggs laid by normal-sized and miniature queens of plebeia remota (holmberg) (hymenoptera: apidae: meliponini) introduction variation in queen body size has been registered for several species of stingless bees. dwarf or miniature queens have been described as small sized queens that may be eventually present in the nests. however, details related to proportion of occurrence and behavior when heading colonies, have been investigated practically only for schwarziana quadripunctata (lepeletier) and plebeia remota (holmberg) (ribeiro, 2002; ribeiro & alves, 2001; ribeiro et al, 2003; wenseleers et al., 2005; ribeiro et al, 2006a, b). these studies reported that miniature mated queens occur naturally at a low frequency in the population and, at least for p. remota, they can be as efficient in laying eggs as typical-sized queens. nevertheless, even when miniature queens lay the same amount of eggs as normal-sized queens, it is unknown whether the eggs of both queen morphotypes differ in size, and whether this variation is related to body size. in case the eggs produced by small queens are also small, this could result in smaller individuals, or when hatching out, the larvae would need larger amounts of food to develop into normal-sized adults. as suggested for honeybees (henderson, 1992), the amount of food available to the larvae is, in fact, abstract miniature stingless bee queens have been studied concerning frequency distribution, production and egg laying performance. this study aimed to investigate size variation in eggs laid by plebeia remota (holmberg) queens and whether it is due to differences in queen size or colony conditions. a sample of 10 queens (8 of typical size and 2 miniature) was measured morphometrically (head width, interorbital distance, and intertegular distance) as well the eggs they laid (length, width and volume). initially, eggs were analyzed when laid by queens in their own colonies. significant differences were found for length, width and volume of eggs considering the total group of queens or both queen morphotypes. however, no significant correlations were found between queen size and egg size. afterwards, two experiments were performed to evaluate the influence of colony conditions on egg size. firstly, we shifted the queens from their original colonies (i.e., a typical queen was placed into a miniature queen colony, and vice-versa). secondly, they were put into another colony (both types of queens, one each time, were placed on a third colony, a ‘host colony’). in all situations, both queen morphotypes laid eggs of similar or different sizes than before, often with significant differences. the results indicate that variation in egg size is due to conditions imposed to queens in the colony (e.g. queen feeding status, number of cells available to be oviposited), and not due to variation in queen body size. sociobiology an international journal on social insects mf ribeiro1,2, ps santos filho1 article history edited by vera l. imperatriz-fonseca, ibusp, brazil received 26 september 2014 initial acceptance 27 october 2014 final acceptance 25 november 2014 keywords queen body size, egg morphometry, stingless bees. corresponding author márcia de fátima ribeiro embrapa semiárido, br 428, km 152 zona rural, c.p. 23, 56302-970 petrolina pe, brazil e-mail: marcia.ribeiro@embrapa.br more important to determine adult size rather than egg size. however, to date, the question remains unclear and it is not possible to determine the implications of small and large eggs to the colony. actually, egg size in bees has been poorly studied. in honeybees, egg size varies due to several factors: between castes, i.e. queen and workers (woyke & wongsiri, 1992; woyke, 1994; gençer & woyke, 2006); according to seasons (henderson, 1992); and due to changes in the metabolic process (woyke, 1998). in stingless bees, eggs differ in size according to the species (velthuis & sommeijer, 1991), but there is little information on intra-specific variation. eggs can also differ in morphology due to their different functions: trophic eggs (laid by workers) or reproductive eggs (laid by queens) (koedam et al., 1996; 2001). variation in egg size produced by a single queen was studied only for scaptotrigona aff. depilis moure and s. quadripunctata (lacerda, unpublished data; lacerda & simões, 2006a, b; ribeiro et al., unpublished data). in this context, this study aimed (i) to investigate the size of eggs laid by queens of p. remota; (ii) to check for the relationship between the variation in egg size and queen body size, and (iii) the verify influence of the environment (colony) research article bees 1 universidade de são paulo, são paulo, sp, brazil 2 empresa brasileira de pesquisa agropecuária (embrapa), petrolina, pe, brazil m. f. ribeiro, p. s. santos filho plebeia remota queens: body size and egg size variation 484 on egg size variation. the hypothesis is that small queens do lay smaller eggs than normal-sized queens, similarly to that found for s. quadripunctata (ribeiro et al., unpublished data). material and methods queen measurements ten p. remota queens of different sizes were collected with an insect aspirator, directly from colonies kept at the bees laboratory, university of são paulo (usp), são paulo. their maximum width, average interorbital distance, and intertegular distance were measured under a stereomicroscope with a micrometer eyepiece (for details on the measuring method see ribeiro & alves, 2001). the queens were separated into two morphotypes: normal-sized (or typical) and miniature queens based on a previous study (ribeiro et al., 2006a). a queen was classified as miniature using the equation head < 2.76 – 0.378.iod – 0.416 iteg, where head: head width; iod: interorbital distance, and iteg: intertegular distance. although the group of queens used in this study does not represent the total range of sizes, they were the only ones available at the laboratory when the experiments were performed, and they were classified into both morphotypes using the formula described above without any restriction. egg measurement: non-experimental and experimental situation eggs were collected from the periphery of the upper combs, in which the cells have been oviposited recently. once the hatching of the larvae is virtually identified by the horizontal position of the egg (sommeijer et al., 1984), it was possible to collect eggs even without knowing exactly when the queen had laid them. in this way, we collected only eggs in the upright position, which therefore did not go through embryonic development. after opening the brood cell with a warmed entomological pin, the egg was collected using another pin, curved at the extremity. the egg was then immediately placed on aluminum foil, with a little amount of larval food, to prevent its dehydration. after that, the egg was measured under a stereomicroscope with a micrometer eyepiece, for length and width. egg volume was then calculated considering the egg as a prolate spheroid, and using the formula: v= 4/3.π.l/2.(l/2)2, where v= volume, l= length and w= width of the egg. to analyze the possible effects of colony conditions, some experiments were performed considering other situations for eggs collection. thus, in the first situation, nonexperimental, queens were in their own colonies (qown). in the second situation, we shifted the position of queens (qexch), i.e., a miniature queen was placed into the colony of a typical sized queen, and vice-versa. in the third situation, queens (miniature and typical sized), one at time, were placed into a third colony, a ‘host colony’ (qhost). the use of this third colony ensured that both queens (normal-sized and miniature) were subjected to the same colony conditions, which could be eventually different from their own colony, or the colony to which they were shifted to. the queen of the host colony was simultaneously placed in the colony from where the queen was removed to be tested. in order to provide time for adaptation of the queen time to a new colony, an interval of three days was allowed before a new egg collection. this method also assured that the sampled eggs were originated from the newly inserted queen and not from the former one; eggs were collected in the same way already described. in some cases, a new sample of eggs was obtained after the return of queens to their own colonies. in this way, in tables 3, 4 and 5, the numbers 1 and 2 after the code indicate the first and second times the queen was subjected to that situation. for example, qown 2 means that the queen returned to its own colony, after a shift; qexch 2 means the second time the queen was shifted, and so on. the table also mentions, in parentheses, in which colony the queen was at the moment of egg collection. statistical analysis statistical tests (zar, 1999) were applied (1) to check for normality of the data (kolmogorov-smirnov); (2) to test the differences between the eggs laid by different queens (kruskal-wallis); (3) to compare the eggs laid by both morphotypes of queens (mann-whitney u test); and (4) to check for correlation between the queen size and the egg size (spearman correlation). the software used to perform these tests was spss. results measurements of queens and eggs in own colony (qown) from the group of ten queens, eight were classified as normal-sized (numbered from one to eight) and two, as miniature (numbered from nine to ten). their body measurements are presented in table 1. considering all situations, a total of 642 eggs were collected, and for each individual queen, in each situation, up to 30 eggs were collected. because data showed no normal distribution, non-parametric tests were applied. table 1 lists the mean values (and sd) of egg measurements (length, width and volume) in qown 1. significant differences were found for all variables (kruskal-wallis, p= 0.000, n= 275 eggs, for length, width and volume). when comparing the two sets of queens, all variables analyzed for their eggs were also significant (mann-whitney, p= 0.000, n= 275 eggs, for length, width and volume). queen body size (head width, interorbital distance and intertegular distance) presented negative non-significant correlations with all variables of eggs (egg length, egg width and egg volume; table 2). sociobiology 61(4): 483-489 (december, 2014) 485 on the other hand, when we included qown2 in the analysis of all queens (i.e. qown1 + qown2), significant differences were also found for all variables (kruskal-wallis, p= 0.000, n= 275 eggs, for length, width and volume). this was not observed when the queens were compared between morphotypes, for egg length and volume (mann-whitney, p= 0.427, p= 0.517, respectively, n= 365 eggs). the differences for egg width were significant (mann-whitney, p= 0.050, n= 365 eggs). measurements of queens and eggs in another colony (qexch) the results found for egg measurements under qexch situations (1 and 2), including qhost and qown (situations 1 and 2), as well as the p values for the statistical tests are shown in tables 3, 4 and 5. when queens were interchanged between colonies (qexch 1 and qexch 2), eggs presented significant differences (kruskal-wallis, p= 0.000, for all variables, n= 277 eggs). when both morphotypes were compared, differences were significant only for egg volume (mann-whitney, p= 0.879, p= 0.134, and p= 0.000, respectively for length, width and volume, n= 277 eggs). it is remarkable that the lowest and highest mean values of egg length were presented by a miniature queen (number 10), respectively, 1.16 mm and 1.29 mm. in relation to egg width, the lowest mean value (0.43 mm) was once again presented by the queen 10, but the highest (0.52 mm) was exhibited by a normal-sized queen (number 5). with respect to egg volume, the lowest mean value was found for the queen 10 (1.04 mm3) and the highest by the queen 5 (1.35 mm3; tables 2, 3 and 4). fig. 1 illustrates the changes observed in egg volume for the queens subjected to more than two different experimental situations. two normal-sized queens (numbers 2 and 5) laid smaller eggs after the shift, while the others (one normal-sized: 1, and both miniatures: 9 and 10) laid larger eggs after the shift. when they returned to their own colonies, table 1. morphometric measures of body and eggs (averages + sd) fromnormal and miniature queens of plebeia remota in their own colonies, in the first analyzed situation (qown 1). (n = number of eggs). table 2. spearman rank correlations (rho) and p values for comparisons between queens’ size (head width, interorbital distance and intertegular distance) and average values for eggs’ measures (length, width and volume) of plebeia remota queens in their own colonies (qown1). (n = number of eggs). queens (morphotypes) head width (mm) interorbital distance (mm) intertegular distance (mm) egg length (mm) x ± sd egg width (mm) x ± sd egg volume (mm3) x ± sd 1 (normal) 1.93 1.48 1.78 1.20 ± 0.05 (n=28) 0.45 ± 0.04 (n= 28) 1.13 ± 0.12 (n= 28) 2 (normal) 1.93 1.48 1.78 1.24 ± 0.04 (n= 30) 0.46 ± 0.04 (n= 30) 1.20 ± 0.13 (n= 30) 3 (normal) 1.85 1.41 1.70 1.21 ± 0.05 (n= 30) 0.47 ± 0.04 (n= 30) 1.18 ± 0.14 (n= 30) 4 (normal) 1.85 1.41 1.70 1.20 ± 0.04 (n= 30) 0.46 ± 0.03 (n= 30) 1.16 ± 0.11 (n= 30) 5 (normal) 1.78 1.33 1.63 1.24 ± 0.05 (n= 16) 0.52 ± 0.04 (n= 16) 1.35 ± 0.13 (n= 16) 6 (normal) 1.78 1.41 1.63 1.25 ± 0.04 (n= 30) 0.47 ± 0.05 (n= 30) 1.24 ± 0.14 (n= 30) 7 (normal) 1.78 1.33 1.56 1.20 ± 0.04 (n= 30) 0.44 ± 0.04 (n= 30) 1.10 ± 0.12 (n= 30) 8 (normal) 1.70 1.33 1.48 1.22 ± 0.03 (n= 30) 0.45 ± 0.03 (n= 30) 1.14 ± 0.10 (n= 30) 9 (miniature) 1.63 1.26 1.41 1.17 ± 0.04 (n= 21) 0.45 ± 0.03 (n= 21) 1.09 ± 0.08 (n= 21) 10 (miniature) 1.48 1.11 1.18 1.16 ± 0.04 (n= 30) 0.43 ± 0.03 (n= 30) 1.04 ± 0.09 (n= 30) eggs measures head width (mm) interorbital distance (mm) intertegular distance (mm) average length (mm) rho= 0.127 p= 0.727 (n= 10) rho= 0.211 p= 0.559 (n= 10) rho= 0.166 p= 0.646 (n= 10) average width (mm) rho= 0.267 p= 0.456 (n= 10) rho= 0.295 p= 0.408 (n= 10) rho= 0.274 p= 0.444 (n= 10) average volume (mm3) rho= 0.268 p= 0.454 (n= 10) rho= 0.334 p= 0.346 (n= 10) rho= 0.296 p= 0.406 (n= 10) fig 1. average volume of eggs (mm3) laid by plebeia remota queens (number 1, 2 and 5: normal-sized; 9 and 10: miniature) in the different situations (own colony or another colony). legend: qown 1: queen in her own colony, and analyzed for the first time; qown 2: queen returned to her own colony, after being in another colony; qexch 1: queen in another colony, for the first time; qexch 2: queen in another colony, for the second time. arrows show the host colony. m. f. ribeiro, p. s. santos filho plebeia remota queens: body size and egg size variation 486 queens 1 (normal-sized) and 10 (miniature) laid smaller eggs again, but the queen 10 (the smallest queen) laid even larger eggs. after qexch2, all queens laid similar or smaller eggs than in the previous situation. regarding the third situation (qhost: colonies 2 and 5), queens of both morphotypes showed similar performance (table 5). thus, no significant differences were detected between the eggs from normal-sized (1 and 3) and miniature queens (9 and 10), when considered both morphotypes for egg length and width, but not for egg volume (mann-whitney, p= 0.147, p= 0.306, and p= 0,004, respectively, n= 113 eggs). when queens were considered in pairs, i.e., queens 1 and 9 in host colony 2, the results were similar (mann-whitney, p= 0.658, p= 0.366, p= 0.000, respectively for length and width, and volume, n= 60 eggs). however, when queens 3 and 10 in the host colony 5 were compared, no differences were significant for all analyzed variables (mann-whitney, p= 0.056 and p= 0.160, and p= 0.601, respectively for length, width, and volume n= 53 eggs). discussion the results for the situation in which the queens remained in their own colonies (qown1) could suggest that normalsized queens lay larger eggs than miniature queens (table 1). however, analyzing in detail the results of queens in other colonies (especially qhost, table 5; fig 1), it was verified that sometimes miniature queens are able to lay larger eggs than normal-sized queens. in fact, queens of different sizes laid, in all situations analyzed (i.e., in qown 1 and 2, qexch 1 and 2, or qhost), eggs of similar sizes, smaller or larger than before, and often these differences were significant (tables 3, 4 and 5; fig. 1). moreover, non-significant correlations between body size and egg size evidenced that the variation in egg size is not due to differences in the queen size. likewise, in different honeybee species, woyke et al. (2003) found no relationship between eggs’ size among queens of different species and sizes. table 3. comparisons of morphometric measures (averages + sd) obtained for eggs laid by the different normal-sizedqueens of plebeia remota in their colonies and other colonies. the number after qown or qexch refers to the situation, i.e., the first or second time the queen was in that situation. (legend: qown = queen in own colony; qexch = queen exchanged, in another colony; n = number of eggs; col. = colony). table 4. comparisons of morphometric measures (averages + sd) obtained for eggs laid by the different miniature queens of plebeia remota in their colonies and other colonies. the number after qown or qexch refers to the situation, i.e., the first or second time the queen was in that situation. (legend: qown = queen in own colony; qexch = queen exchanged, in another colony; n = number of eggs; col. = colony). queens comparison experimental condition egg length (mm) x ± sd mann-whitney test p egg width (mm) x ± sd mann-whitney test p egg volume (mm3) x ± sd mann-whitney test p 1 qown 1 (col. 1) vs. qexch 1 (col. 9) 1.20 ± 0.05 (n=28) 1.25 ± 0.04 (n=24) p= 0.001** 0.45 ± 0.04 (n= 28) 0.46 ± 0.05 (n= 24) p= 0.181 1.13 ± 0.12 (n= 28) 1.20 ± 0.14 (n= 24) p= 0.009** 1 qown 1 (col. 1) vs. qown 2 ( col. 1) 1.20 ± 0.05 (n=28) 1.22 ± 0.03 (n= 30) p= 0.014* 0.45 ± 0.04 (n= 28) 0.44 ± 0.01 (n= 30) p= 0.771 1.13 ± 0.12 (n= 28) 1.12 ± 0.04 (n= 30) p= 0.105 1 qexch 1 (col. 9) vs. qown 2 (col.1) 1.25 ± 0.04 (n=24) 1.22 ± 0.03 (n= 30) p= 0.024* 0.46 ± 0.05 (n= 24) 0.44 ± 0.01 (n= 30) p= 0.034* 1.20 ± 0.14 (n= 24) 1.12 ± 0.04 (n= 30) p= 0.040* 2 qown 1 (col. 2) vs. qexch 1 (col. 1) 1.24 ± 0.04 (n=30) 1.23 ± 0.04 (n=30) p= 0.196 0.46 ± 0.04 (n= 30) 0.44 ± 0.03 (n= 30) p= 0.002** 1.20 ± 0.13 (n= 30) 1.12 ± 0.10 (n= 30) p= 0.030* 2 qown 1 (col. 2) vs. qexch 2 (col. 9) 1.24 ± 0.04 (n=30) 1.24 ± 0.03 (n=30) p= 0.938 0.46 ± 0.04 (n= 30) 0.43 ± 0.02 (n= 30) p= 0.002** 1.20 ± 0.13 (n= 30) 1.12 ± 0.08 (n= 30) p= 0.049* 2 qexch 1 (col. 1)vs. qexch 2 (col. 9) 1.23 ± 0.04 (n=30) 1.24 ± 0.03 (n=30) p= 0.169 0.44 ± 0.03 (n= 30) 0.43 ± 0.02 (n= 30) p= 0.603 1.12 ± 0.10 (n= 30) 1.12 ± 0.08 (n= 30) p= 0.146 5 qown 1 (col. 5) vs. qexch 1 (col. 3) 1.24 ± 0.05 (n=16) 1.24 ± 0.05 (n=30) p= 0.626 0.52 ± 0.04 (n= 16) 0.51 ± 0.04 (n= 30) p= 0.399 1.35 ± 0.13 (n= 16) 1.32 ± 0.13 (n= 30) p= 0.388 5 qown 1 (col. 5) vs. qexch 2 (col. 10) 1.24 ± 0.05 (n=16) 1.24 ± 0.05 (n=30) p= 0.921 0.52 ± 0.04 (n= 16) 0.51 ± 0.04 (n= 30) p= 0.258 1.35 ± 0.13 (n= 16) 1.32 ± 0.12 (n= 30) p= 0.466 5 qexch 1 (col. 3) qexch 2 (col. 10) 1.24 ± 0.05 (n=30) 1.24 ± 0.05 (n=30) p= 0.597 0.51 ± 0.04 (n= 30) 0.51 ± 0.04 (n= 30) p= 0.811 1.32 ± 0.13 (n= 30) 1.32 ± 0.12 (n= 30) p= 0.852 queens comparison experimental condition egg length (mm) x ± sd mann-whitney test p egg width (mm) x ± sd mann-whitney test p egg volume (mm3) x ± sd mann-whitney test p 9 qown 1 (col. 9) vs. qexch 1 (col. 1) 1.18 ± 0.04 (n=21) 1.22 ± 0.05 (n=20) p= 0.001** 0.44 ± 0.03 (n= 21) 0.47 ± 0.04 (n= 20) p= 0.027* 1.09 ± 0.08 (n= 21) 1.20 ± 0.12 (n= 20) p= 0.001** 9 qown 1 (col. 9) vs. qown 2 (col. 9) 1.18 ± 0.04 (n=21) 1.28 ± 0.04 (n= 30) p= 0.000** 0.44 ± 0.03 (n= 21) 0.47 ± 0.04 (n= 30) p= 0.004* 1.09 ± 0.08 (n= 21) 1.25 ± 0.10 (n= 30) p= 0.000** 9 qexch 1 (col. 1) vs. qown 2 (col.9) 1.22 ± 0.05 (n=20) 1.28 ± 0.04 (n= 30) p= 0.000** 0.47 ± 0.04 (n= 20) 0.47 ± 0.04 (n= 30) p= 0.799 1.20 ± 0.12 (n= 20) 1.25 ± 0.10 (n= 30) p= 0.028* 10 qown 1 (col. 10) vs. qown 2 (col. 10) 1.16 ± 0.04 (n=30) 1.29 ± 0.07 (n=30) p= 0.000** 0.43 ± 0.03 (n= 30) 0.46 ± 0.05 (n= 30) p= 0.011* 1.04 ± 0.09 (n= 30) 1.24 ± 0.17 (n= 30) p= 0.000** sociobiology 61(4): 483-489 (december, 2014) 487 thus, our results indicate that it is not the size of the queens that influences the size of the eggs, but rather the conditions imposed to the queens. probably the condition of each colony was different and influenced the egg size, whether positively (larger eggs) or negatively (smaller eggs). on the other hand, when queens laid similar-sized eggs in both situations, all the colonies probably provided similar conditions. a possible explanation for the differences in egg size is the development of the embryos. thus, changes in egg size would imply in embryonic development. the exact moment of egg laying by the queen was not observed in this work. nevertheless, eggs were always collected in very new combs, i.e., with cells recently operculated and, therefore, the possibility of collecting eggs at an advanced stage of development is excluded. moreover, as already mentioned, when eggs hatch they did not stay in a vertical position, but lay down on the larval food and only eggs in vertical position were collected. egg size variation may be caused by nutritional conditions to which queens were exposed. therefore, in colonies with abundant food reserve, queens probably received more food, and could produce larger eggs. simultaneously, queens placed in colonies with fewer reserves, and supposedly lower food intake, would produce smaller eggs. opposite results, however, were found for s. aff. depilis (lacerda, unpublished data; lacerda & simões, 2006b). queens of this species in ‘weak’ colonies produced larger eggs than in ‘strong’ ones. however, the authors suggested that these eggs were haploid eggs, which are larger than diploid eggs. in p. remota, most haploid eggs are produced by the queen (tóth et al., 2002). nevertheless, the variation in egg size found herein was not due to male production, since adult males were not registered in the studied colonies after the experimental period. so, if the variation in egg size is associated to queen feeding status, this would be imposed by colony condition, and in some way, by the workers. queens get food through three different ways: by eating larval food from the open cell just before laying the egg, by eating trophic eggs laid by the workers or by trophallaxis with workers. this food ingestion could be important for the production and development of eggs inside the queen ovarioles. in this way, the workers, besides controlling the number of cells available for egg laying (ribeiro, 2002), indirectly are also responsible for the size of the eggs laid by queens. on the other hand, as suggested by j. woyke (personal communication, march 8, 2008) egg size may still be related to the cell production rate of the colony. analyzing the relationship between the number of cells available for oviposition in each colony with egg size, negative correlations were recorded for egg length and width (spearman, rho= -0.040, rho= -0.144, respectively, p= 0.01, n= 642 eggs). the longer the queen had to wait to lay eggs, the larger these eggs could become, probably by accumulation of vitellogenin. certainly, before the end of egg development, during vitellogenesis, a deposit of vitello can be established thus increasing egg volume up to the moment of ovulation (i.e. the expulsion of the egg through the oviducts) (cruz-landim, 2009). in this work, when the queen was transferred to a smaller colony, this would result in a delay in the oviposition and consequently could result in table 5. comparisons of morphometric measures (averages + sd) obtained for eggs laid by the different queens of plebeia remota in their colonies and other colonies. the number after qown or qexch refers to the situation, i.e., the first or second time the queen was in that situation. (legend: qown = queen in own colony; qexch = queen exchanged, in another colony; qhost= queen in host colony; n = number of eggs; col. = colony). queens (morphotypes) comparison experimental condition egg length (mm) x ± sd mann-whitney test p egg width (mm) x ± sd mann-whitney test p egg volume (mm3) x ± sd mann-whitney test p 1 (normal) qown 1 (col. 1) vs. qexch 2 (col. 2) = q host 1.20 ± 0.05 (n=28) 1.20 ± 0.04 (n= 30) p= 0.974 0.45 ± 0.04 (n= 28) 0.44 ± 0.03 (n= 30) p= 0.355 1.13 ± 0.12 (n= 28) 1.10 ± 0.10 (n= 30) p= 0.627 1 (normal) qexch 1 (col. 9) vs. qexch 2 (col. 2) = q host 1.25 ± 0.04 (n=24) 1.20 ± 0.04 (n= 30) p= 0.000** 0.46 ± 0.05 (n= 24) 0.44 ± 0.03 (n= 30) p= 0.026* 1.20 ± 0.14 (n= 24) 1.10 ± 0.10 (n= 30) p= 0.001** 1 (normal) qown 2 (col.1) vs. qexch 2 (col. 2) = q host 1.22 ± 0.03 (n= 30) 1.20 ± 0.04 (n= 30) p= 0.007* 0.44 ± 0.01 (n= 30) 0.44 ± 0.03 (n= 30) p= 0.307 1.12 ± 0.04 (n= 30) 1.10 ± 0.10 (n= 30) p= 0.015* 9 (miniature) qown 1 (col. 9) vs. qexch 2 (col. 2) = q host 1.18 ± 0.04 (n=21) 1.20 ± 0.05 (n= 30) p= 0.112 0.44 ± 0.03 (n= 21) 0.43 ± 0.02 (n= 30) p= 0.102 1.09 ± 0.08 (n= 21) 1.08 ± 0.09 (n= 30) p= 0.862 9 (miniature) qexch 1 (col. 1) vs. qexch 2 (col. 2) = q host 1.22 ± 0.05 (n=20) 1.20 ± 0.05 (n= 30) p= 0.029* 0.47 ± 0.04 (n= 20) 0.43 ± 0.02 (n= 30) p= 0.001** 1.20 ± 0.12 (n= 20) 1.08 ± 0.09 (n= 30) p= 0.001** 9 (miniature) qown 2 (col.9) vs. qexch 2 (col. 2) = q host 1.28 ± 0.04 (n= 30) 1.20 ± 0.05 (n= 30) p= 0.000** 0.47 ± 0.04 (n= 30) 0.43 ± 0.02 (n= 30) p= 0.000** 1.25 ± 0.10 (n= 30) 1.08 ± 0.09 (n= 30) p= 0.000** 3 (normal) qown 1 (col. 3) vs. qexch 1 (col. 5) = q host 1.21 ± 0.05 (n=30) 1.25 ± 0.06 (n=23) p= 0.012* 0.46 ± 0.05 (n= 30) 0.49 ± 0.04 (n= 23) p= 0.008** 1.18 ± 0.14 (n= 30) 1.29 ± 0.15 (n= 23) p= 0.001** 10 (miniature) qown 1 (col. 10) vs. qexch 1 (col. 5) = q host 1.16 ± 0.04 (n=30) 1.28 ± 0.05 (n=30) p= 0.000** 0.43 ± 0.03 (n= 30) 0.48 ± 0.05 (n= 30) p= 0.000** 1.04 ± 0.09 (n= 30) 1.27 ± 0.15 (n= 30) p= 0.000** 10 (miniature) qown 2 (col. 10) vs qexch 1 (col. 5) = q host 1.29 ± 0.07 (n=30) 1.28 ± 0.05 (n=30) p= 0.477 0.46 ± 0.05 (n= 30) 0.48 ± 0.05 (n= 30) p= 0.058 1.24 ± 0.17 (n= 30) 1.27 ± 0.15 (n= 30) p= 0.329 m. f. ribeiro, p. s. santos filho plebeia remota queens: body size and egg size variation 488 an increased size of the eggs ready to be laid. in an opposite situation, one could expect a contrary result. therefore, the results of the present study indicate that egg size is influenced by colony conditions (and consequently, food supply for the queens and/or rate of cell construction) imposed to the queen, rather than by body size. acknowledgments we are grateful to dr. j. woyke, dr. vera l. imperatrizfonseca, dr. dirk koedam, dr. beatriz a. j. paranhos and two anonymous referees for the critical reading of the manuscript; fundação de amparo à pesquisa do estado de são paulo (fapesp, proc. n. 98/01679-5), conselho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação cearense de apoio ao desenvolvimento científico e tecnológico (funcap, proc. n. 35.0781/2004-4) for financial support; and erica m. t. de alencar, for language advice. references cruz-landim, c. 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(1999). biostatistical analysis. 4th ed. new jersey: prentice hall, 663 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.1049sociobiology 63(2): 851-854 (june, 2016) antifungal effect of silver nanoparticles on rickia wasmannii cavara (ascomycota: laboulbeniales) infecting myrmica scabrinodis nylander (formicidae) ants laboulbeniales are obligate ectoparasites of arthropods with a peculiar biology (weir & beakes, 1995; haelewaters et al., 2015). insect-laboulbeniales relationships have several interesting but rather understudied aspects. recent results showed that ant parasitic laboulbeniales fungi can influence the physiology and behavior of their hosts (csata et al., 2014; báthori et al., 2015a; konrad et al., 2015), although penetration of the host cuticle was not observed (tragust et al., 2016). ants are optimal model organisms for studying the effects of insect parasitic microorganisms, as there is usually a high number of infected (often heavily infected) hosts available (and easily maintained) and methods for studying survival, overall health, behavioral changes, etc. have been established. furthermore, ant-associated laboulbeniales are also relatively easy to count using a binocular microscope (csata et al., 2014; báthori et al., 2015a; markó et al., 2016). the importance of uninfected controls in such survival/ behavior oriented studies is beyond question. this may constitute abstract rickia wasmannii cavara (ascomycota: laboulbeniales) is an ectoparasitic fungus infecting myrmica ants. ant-parasitic laboulbeniales and their interactions with the hosts have been in the focus of several studies. to assess the effects of these fungi, comparison of infected and uninfected or completely treated ants are needed. so far, treating laboulbeniales infection was only achieved with cockroaches, but not with ants. we present a simple, yet relatively long, agnp topical treatment that reduces or eliminates rickia infection from myrmica scabrinodis ants without affecting their lifespan. we discuss the possibilities of the proposed treatment in the light of the biology of rickia. sociobiology an international journal on social insects wp pfliegler1,2, l tálas1, f báthori3, a tartally3, i pócsi1, g szemán-nagy1 article history edited by evandro nascimento silva, uefs, brazil received 22 april 2016 initial acceptance 17 may 2016 final acceptance 17 may 2016 publication date 15 july 2016 keywords antimycotic, fungal parasite, ectoparasite, agnps, treatment. corresponding author andrás tartally department of evolutionary zoology and human biology university of debrecen egyetem tér 1., h4032 debrecen, hungary e-mail: tartally.andras@science.unideb.hu a problem as infected and uninfected specimens are not necessarily available from the same colony or even the same location (báthori et al., 2015a). thus, an alternative approach may be treating the hosts with an antifungal compound and thereby making comparisons between infected and uninfected insects possible. in the case of ants, it could give the possibility to have infected and uninfected individuals from the same colonies (e.g. sister or closely related workers). control of a laboulbenialean genus, herpomyces thaxt. on cockroaches was achieved using the fungicide benomyl {methyl n-[1-(butylcarbamoyl)benzimidazol-2-yl]carbamate} (gemeno et al., 2004). recently, this compound was found to be ineffective in treating myrmica rubra l. ant queens infected with r. wasmannii and the antifungal treatment also negatively affected the host (pech & heneberg, 2015). the main target of benomyl is the polymerization of tubulin, resulting in the inhibition of cell proliferation and division (wride et al., 2014). rickia thalli are attached firmly to the 1 department of biotechnology and microbiology, university of debrecen, hungary 2 postdoctoral fellowship programme of the hungarian academy of sciences (mta), budapest, hungary 3 department of evolutionary zoology and human biology, university of debrecen, hungary short note wp pfliegler – silver nanoparticles against rickia infection on myrmica852 host, presumably even if the cells of the thallus are dead, e.g. we observed rickia and other laboulbeniales thalli on decadesold museum specimens (báthori et al., 2014, 2015b) and the fungus does not produce hyphae at all. thus, antimycotics inhibiting cell proliferation may not be suitable to decrease the number of mature rickia thalli on infected hosts at all. additionally, the lack of structures penetrating the host in the case of ant-infecting laboulbeniales fungi (tragust et al., 2016) also probably accounts for the ineffectiveness of oral treatments administered to the host insect. on the other hand, antimicrobial compounds with cytotoxic effects may be more effective against rickia. silver nanoparticles (agnps) can interact with the cell surface causing ruptures in the cell membrane of eukaryotic cells, they are known to generate oxidative stress and disturb metabolic pathways and are also promising anti-biofilm agents (you et al., 2012; cavalieri et al., 2014). our aim was therefore to test whether agnps are able to exterminate r. wasmannii efficiently from its host ants. myrmica scabrinodis nylander ants infected with r. wasmannii were obtained from rakaca (48°27’n, 20°47’e, ne hungary) and maintained as a colony in laboratory. workers of age groups 3 and 4 (see: cammaerts-tricot, 1974) were selected to avoid variation in expected lifespan. treated (19 specimens) and water-treated control workers (18 specimens) were kept in individual plastic containers. conditions are described in báthori et al. (2015a). thalli of r. wasmannii were counted using a leica binocular microscope, using 20x to 100x magnification with hosts handled with a delicate pincer. only thalli on the dorsal part of the heads of specimens were counted (antennae and mouthparts excluded; the mean number of thalli counted on a single ant was 26.6, sd= 12.98, for 37 specimens). thalli were counted before the experiment and every 7 days during the four-week-long treatment. ants were monitored and thalli counted for 2 more weeks after treatment. thalli on dead specimens were not counted. treatment involved submerging ants into 20, 10 or 5 ppm agnp (pérez et al., 2008) solution (bay nano, miskolc) for 5 seconds using a pincer on a daily basis. specimens of the control group were treated the same way with distilled water. survival of agnp-treated and water-treated hosts was compared by kaplan-meier survival analysis and its log-rank test using the medcalc software. as rickia is not a mycelial fungus (weir & beakes, 1995), we could also assess the survival of the thalli (with a weekly resolution) using the same method (considering thalli on dead hosts as censored). student’s 2-sample t-test (or welch’s test depending on the equality of variance) was applied to test the significances of the differences in the proportional decrease of rickia thalli on the two groups of ants. during our initial trial experiments, we determined the optimal concentration of agnps to treat m. scabrinodis workers with r. wasmannii infection. 20 to 10 ppm agnps proved to be unsuitable for the treatment of ants as these concentrations caused argyria and death of the host. the 5 ppm concentration showed no such effects: compared to the control (water-treated) group, the mortality of the ants was not different (log-rank test for kaplan-meier survival analysis, p<0.6), with only 4-4 hosts surviving the whole 6-week-long period of observations. thus, we concluded that the agnp treatment did not affect the lifespan of workers during the period of experiments. the low survival rate in both groups is probably associated with the negative effects of social isolation (koto et al., 2015). it is noted that isolation was only necessary for tracking the changes on the number of thalli on each specimens. treatment to ants may be applied without isolation in further experiments. the effect of the 4-week-long daily agnp treatment resulted in the decrease in the number of thalli on the ants significantly more effectively than water treatment (number of thalli on ants in the group treated only with water also decreased in most cases). significance was p<0.05 for the first week of treatment and p<0.01 for the second to fourth week. after 4 weeks of agnp treatment, the mean decrease of thalli number was 82.99 % (sd=18.13) and all thalli disappeared from 4 of the surviving 10 agnp treated ants (40 %) (fig 1c). none of the control water-treated ants lost more than 75 % of thalli during this period and the mean decrease was 39.68 % (sd=26.62) for the surviving 9 control ants. post treatment observation after 2 weeks also confirmed the significant (p<0.05 and p<0.1) difference between the two groups’ loss of thalli, although the number of surviving ants was low. fig 1a summarizes the observations on the decrease in thalli number on the two ant groups. the results of the analysis and the comparison of the two survival curves using kaplanmeier survival analysis for the thalli of rickia confirms that the treatment was effective with high statistical support (p<0.0001), backed by a high number (n=983) of tracked thalli (fig 1b). our results offer a possible treatment of laboulbeniales infection of ants that could be exploited in the study their hostparasite interactions. infected and post-treatment uninfected ants from the same colony could be used for experiments, allowing better comparisons to assess the effect of rickia on behavior (especially social behavior between infected/uninfected sister ants), mortality, etc. as far as we know, our method is the first topical treatment of an insect for a fungal parasite and also the first effective treatment for laboulbeniales on ants. we also tried to apply the treatment to blatta lateralis l. cockroaches infected with herpomyces stylopygae speg., but the number of thalli quickly increased in both the treated and control groups (results not shown). this may be related to the fact that hesperomyces spp. penetrate the host’s cuticle (richards & smith, 1956), while r. wasmannii was recently shown to be only attached to the surface of the ant cuticle (tragust et al., 2016), but this question requires further study. studying other host-laboulbeniales pairs was not possible due to the rarity of these fungi and the usually small number of thalli on other insects (e.g. beetles). thus, topical treatment is so far only effective for ants. sociobiology 63(2): 851-854 (june, 2016) 853 acknowledgements at and fb was supported by the ‘antlab’ marie curie career integration grant (of at), part the 7th european community framework programme. at was supported by a ‘bolyai jános’ scholarship of the hungarian academy of sciences (mta). references báthori, f., pfliegler, w. p. & tartally, a. 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(2012). the progress of silver nanoparticles in the antibacterial mechanism, clinical application and cytotoxicity. molecular biology reports, 39: 9193-201. doi: 10.1007/s11033-012-1792-8. doi: 10.13102/sociobiology.v65i1.2097sociobiology 65(1): 88-100 (march, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 relative occurrence of the family kalotermitidae (isoptera) under different termite sampling methods introduction social insects are dominant inhabitants of tropical landscapes (wilson and hölldobler, 2005), and assessing their ecological significance requires astute taxonomic knowledge. species identifications rely on keys and descriptions of known and new taxa that are often obtained by deliberate collecting expeditions. field surveys to assess the known and unknown diversity and abundance of social hymenoptera and termites require different collecting methods. because of their open foraging behavior, most ants are collected with baits, trapping by pitfall or malaise devices, or passively sifting litter with abstract the termite family kalotermitidae constitutes a wood-nesting termite family that accounts for about 15% of all extant termite species. in recent decades, field studies have been carried out to assess termite diversity in various wooded habitats and geographic locations. three sampling methods have been favored expert, transect, and alate light-trap surveys. expert collecting is not spatially quantifiable but relies on field personnel to recognize and sample termite niches. the transect method aims to standardize and quantify termite abundance and diversity. light trapping is a passive method for sampling nocturnal alate flights. we compared our expert survey results and results of published sampling methods for their proportional yields of kalotermitid versus non-kalotermitid encounters. using an odds ratio statistic, we found that worldwide, there is about a 50.6-fold greater likelihood of encountering a kalotermitid sample versus a non-kalotermitid using the expert survey method and a 15.3-fold greater likelihood using alate trapping than using the transect method. there is about a 3.3 -fold greater likelihood of collecting a kalotermitid specimen versus a non-kalotermitid sample using the expert survey method than using the alate trap method. transect studies in which only termite species diversity was reported gave similar low kalotermitidae yields. we propose that multiple biases in sampling methodology include tools, time constraints, habitat type, geographical location, topographical conditions, and human traits account for the divergent outcomes in sampling the abundance and diversity of kalotermitidae compared to other termite families. sociobiology an international journal on social insects rh scheffrahn1, ja chase2, jr mangold3, hh hochmair1 article history edited by reginaldo constantino, unb, brazil received 27 september 2017 initial acceptance 13 december 2017 final acceptance 09 january 2018 publication date 30 march 2018 keywords expert survey, transect survey, alate trap survey. corresponding author r. h. scheffrahn university of florida, fort lauderdale research & education center, 3205 college avenue, davie, florida 33314 usa. e-mail: rhsc@ufl.edu winkler bags (agosti & alonso 2000, ellison et al., 2007, longino et al., 2002). similarly, social wasps are most commonly collected in baited traps and malaise traps (de souza & prezoto, 2006, noll & gomes, 2009, rezende diniz & kitayama, 1998, silveira, 2002). eusocial bees can be attracted with fragrances or caught in trap nests (feja, 2006). active collecting by hand or net is also used for ants, bees, and wasps. because of their cryptic nature, termites cannot be collected passively with traps or baits but must be acquired directly from within the substrata they inhabit. this requires digging, chopping, scraping, or probing into soil, wood, nest 1 university of florida, fort lauderdale research & education center, davie, florida, usa 2 marietta, georgia, usa 3 gulfport, florida, usa research article termites sociobiology 65(1): 88-100 (march, 2018) special issue 89 material, and foraging tubes to expose and collect termites. as discussed later, imagoes that disperse at night can be collected with light traps and, on occasion, open-foraging termites have been collected in pitfall traps (willis et al., 1992) or attracted to scattered baits such as instant oatmeal flakes (scheffrahn & rust, 1983). long-term cellulose baiting has been used to attract some termites as well (dawes-gromadzki, 2003, davies et al., 2013). in recent years, a series of termite sampling studies was published with the stated goal of defining species abundance and diversity of termites at a given locality. to accomplish this, the transect protocol of jones and eggleton (2000), or slightly modified versions thereof, were developed from the protocols first set forth in the 1990s to examine the contribution of soil fertility by soil-inhabiting termites (anderson & ingram, 1993). more recent publications continue to address termite sampling protocols, but again, from a “soil” perspective (bignell, 2009, moreira et al., 2008). therefore, the emphasis on sampling soil-inhabiting termites was a intended priority in transect surveys. for example, a compilation of 87 transects from 29 tropical locations (davies et al., 2003b) revealed that only 13 out of 1511 termite occurrences (0.86%) consisted of non-soil inhabiting termites (i.e., kalotermitidae). the termite family kalotermitidae is composed of nearly 500 extant species worldwide (krishna et al., 2013a). the kalotermitidae are broadly distributed and their diversity is the greatest near the equator. with one exception, all studied kalotermitids nest in galleries excavated within single pieces of sound or partially decayed wood. the dimensions of their host wood can range from mere twigs to massive boles with the majority of field-collected kalotermitids taken from dead tree limbs. unlike the kalotermitids, species of the other two major termite families (rhinotermitidae and termitidae, ca. 2,400 spp.) forage beyond their nest boundaries; either below the soil surface, under earthen or fecal tubing, or on open substrata. results of termite diversity surveys using alate traps have yielded very different taxonomic compositions compared to transect methods. this indicates that kalotermitids are underrepresented in transect surveys. of the five studies employing light traps (all neotropical: bourguignon et al., 2009, gomes da silva medeiros et al., 1999, martius, 2003, martius et al., 1996, robello & martius, 1994) 11.4% of the imagoes collected were kalotermitids. a tree canopy transect in panama (roisin et al., 2006) yielded an even higher proportion (34.9%) of kalotermitids. from 1990 to 2014, we conducted termite survey expeditions in the new world with the goal of collecting as much diversity (“species richness”) over the largest area and most varied habitats and microhabitats as time and cost permitted (e.g., scheffrahn et al., 2003, 2005, 2006). our “expert” surveys and those in taiwan by li, h. f, et al. (2011, 2015) also yielded higher proportions (2.6-61.5%) of kalotermitid colony occurrences (unique encounters) than those of transect studies from comparable habitats. in this paper, we review termite sampling methods and compare them for their proportional yields of kalotermitid versus non-kalotermitid encounters. we offer some hypotheses to explain the disparate ratios of encounters between these two distinct termite groups. materials and methods expert survey our expert collecting method was devised (scheffrahn et al., 1990) to maximize termite taxon diversity while minimizing collecting time at a given site. the primary goal of expert collecting was to build and enrich the holdings of the university of florida termite collection (uftc) in davie, florida. the flrec currently housed 43,426 unique colony samples. when planning termite surveys, site locations were not determined in advance of expeditions, although general travel routes were selected to include as many habitats and biomes that could be sampled in the time allotted. most site selections were dictated by daylight visibility, motor vehicle travel routes, vehicle parking (roads with shoulders or turnoffs), and human accessibility (steep slopes or flooded areas were avoided). habitats of little human disturbance were preferred but often not available. barbed wire fences, drainage ditches, and noxious roadside vegetation were often traversed to access collectable areas. sites above arm’s reach were seldom sampled. each site was surveyed from about five to 90 minutes by groups (one to nine team members) of experienced collectors. each collector was free to choose his or her own search strategy at each site. if a species at a given locality was found to be very common, it was likely that multiple samples of that species were collected, however, it was also communicated among the team members to stop collecting abundant species, e.g. nasutitermes corniger, and focus on other termites. it was also unlikely that multiple samples were collected from the same colony because disturbance or damage denoted that the spot had already been collected by a team member. if multiple samples were collected from a single colony, e.g. a large log containing kalotermitids, the vials are bound by rubber bands to denote a single sample. sites generally covered 0.1 to about 50 hectares and yielded from zero to over 60 colony samples. in most cases, travel time between sites was approximately equal to time spent collecting termites. the primary tool for each collector was a 32-cmlong, 0.9 kg hatchet. the hatchet was used to open sound to rotten branches, logs, stumps, fence posts, nest carton, epigeal mounds, etc. the hatchets were also used to dig into soil or to pry or lift objects lying on the soil surface such as logs, stones, dung pats, etc. a teflon®-lined cooking pan was used to hold extracted termites and associated debris. an aspirator was used to suction termites from debris for photography and transfer into 2-dram glass vials (fisher scientific no. cat. no. 03-339-26b) filled with 85% ethanol after each expedition, samples were cleaned by removing soil and other fine debris rh scheffrahn, ja chase, jr mangold, hh hochmair – kalotermitidae encounters by survey method90 and adding more 85% ethanol. a unique inventory code label was added to each vial for curation and long-term storage in the uftc. all collection sites were georeferenced either from paper maps (before 1997) or gps receiver coordinates. site elevations were confirmed using google™ earth. samples are stored in an air-conditioned room inside closed storage cabinets. using this curation method, many uftc samples > 20 years-old have yielded full 654 bp barcode (co1) dna sequences. in recent years, only two other taxon-quantified expert surveys have been published outside the new world. li et al. (2011) used axes and aspirators to sample termite diversity in a taiwanese pangolin habitat. li, h. f, et al. (2015) collected termites within 100 m diam plots in a tropical forest ecosystem in taiwan. collecting tools were not specified. expert collecting has occasionally been called “casual collecting” as in the case of gathorne-hardy & jones, 2000. transect surveys and alate trapping the transect protocol of jones & eggleton (2000) was developed as a standardized termite sampling protocol; “to measure termite species richness and functional diversity in tropical forests.” [of note: in response to roisin and leponce (2004), jones et al. (2006) gave a conflicting goal for their transect protocol: …..“measuring species density and relative abundance is for us more illuminating than estimating species richness.”]. regardless, the method of jones and eggleton (2000) specifies that transects be divided into 5x2 m sections and each section sampled for one hour “in the following microhabitats….samples of surface soil (each about 12x12 cm, to 10 cm depth); accumulations of litter and humus at the base of trees and between buttress roots; the inside of dead logs, tree stumps, branches and twigs; the soil within and beneath very rotten logs; all subterranean nests, mounds, carton sheeting and runways on vegetation, and arboreal nests up to a height of 2 m aboveground level” (italics emphasize typical kalotermitid microhabitats). all transect studies listed in table 1 cite either the method of jones and eggleton (2000) or specify a modification thereof, but all mention sampling from potential kalotermitid microhabitats. a single canopy transect, conducted by roisin et al. (2006), employed professional tree climbers who cut dead branches from trees and inspected standing stock greater than 10 m above ground. the cut limbs were then searched for termites. table 1. studies listing occurrences of kalotermitid and non-kalotermitid termites in wooded habitats by sampling method. region or biome sampling method no. kalo occurrences no. non-kalo occurrences total termite occurrences % kalo of total occurrences reference subtropical & tropical new world new world expert survey 10368 20208 30576 33.9 current study central america expert survey 795 4044 4839 16.4 current study ecuador, amazonia expert survey 28 1035 1063 2.6 current study nw mainland expert survey 3058 15623 18681 16.4 current study panama expert survey 138 1373 1511 9.1 current study peru, amazonia expert survey 31 952 983 3.2 current study south america expert survey 692 8108 8800 7.9 current study united states expert survey 1270 1066 2336 54.4 current study west indies expert survey 7310 4585 11895 61.5 current study argentina, chaco std. transect 3 29 32 9.4 godoy et al., 2012 argentina, chaco mod. transect 40 175 215 18.6 roisin & leponce, 2004 brazil, amazonia, manaus alate traps 1581 9991 11572 13.7 martius, 2003 brazil, amazonia, manaus alate traps 1919 23555 25474 7.5 martius et al., 1996 brazil, amazonia, manaus alate traps 2095 8625 10720 19.5 robello & martius, 1994 brazil, amazonia, manaus std. transect** 0 306 306 0.0 ackerman et al., 2009 brazil, amazonia, manaus std. transect 2 162 164 1.2 dambros et al., 2013 brazil, amazonia, manaus std. transect 0 692 692 0.0 dambros et al., 2016a brazil, atlantic forest alate traps 1140 10227 11367 10.0 gomes da silva et al., 1999 brazil, atlantic forest std. transect 26 701 727 3.6 cancello et al., 2014 brazil, atlantic forest std. transect 11 314 325 3.4 reis & cancello, 2007 brazil, atlantic forest std. transect 5 189 194 2.6 vasconcellos et al., 2010 brazil, atlantic forest std. transect 2 178 180 1.1 viana-junior et al., 2014 brazil, atlantic forest mod. transect 20 162 182 11.0 bandeira et al., 2003 brazil, atlantic forest mod. transect 11 406 417 2.6 couto et al., 2015 brazil, atlantic forest mod. transect 8 390 398 2.0 souza et al., 2012 brazil, atlantic forest mod. transect 5 160 165 3.0 vasconcellos et al., 2005 brazil, central amazonia mod. transect 12 4375 4387 0.3 dambros et al., 2016b brazil, northeast mod. transect 12 612 624 1.9 ernesto et al., 2014 brazil, northeast mod. transect 1 431 432 0.2 almeida et al., 2017*** brazil, pantanal std. transect 0 53 53 0.0 da cunha et al., 2015 brazil, plantation mod. transect 0 160 160 0.0 calderon & constantino, 2007 sociobiology 65(1): 88-100 (march, 2018) special issue 91 brazil, presidente figueiredo std. transect 3 268 271 1.1 dambros et al., 2012 brazil, southeast cerrado std. transect 0 219 219 0.0 oliveira et al., 2013 brazil, southeast cerrado mod. transect 0 64 64 0.0 carrijo et al., 2009 brazil, southeast cerrado std. transect 0 754 754 0.0 silva et al., 2016 brazil, southeast cerrado mod. transect 7 108 115 6.1 alves et al., 2011 colombia, amazonia mod. transect 0 278 278 0.0 florian et al., 2017 french guiana std. transect 6 3184 3190 0.2 davies et al., 2003a french guiana, amazonia mod. transect 5 851 856 0.6 bourguignon et al., 2011 panama canopy transect 22 41 63 34.9 roisin et al. 2006 panama alate traps 84 467 551 15.2 bourguignon et al. 2009 panama mod. transect 1 242 243 0.4 roisin et al., 2006 panama mod. transect 1 143 144 0.7 basset et al., 2017 peru, amazonia mod. transect 0 967 967 0.0 dahlsjo et al., 2015 peru, amazonia* std. transect 1 246 247 0.4 palin et al., 2011 tropical africa benin std. transect 0 443 443 0.0 hausberger & korb, 2016 burundi std. transect 0 1070 1070 0.0 nduwarugira et al., 2017 cameroon mod. transect 0 849 849 0.0 deblauwe et al., 2007 guinea std. transect 0 473 473 0.0 dosso et al., 2012 ivory coast std. transect 0 626 626 0.0 dosso et al., 2010 malawi std. transect 0 190 190 0.0 donovan et al., 2002 nigeria std. transect 0 120 120 0.0 kemabonta & balogun, 2015 south africa std. transect, baits 0 1015 1015 0.0 davies et al., 2013 asia asia, southeast std. transect 15 3509 3524 0.4 gathorne-hardy et al., 2002 borneo std. transect 1 195 196 0.5 jones et al., 2000 borneo std. transect 2 439 441 0.5 jones et al., 2010 borneo std. transect 2 378 380 0.5 jones & prasetyo, 2002 china, guangdong mod. tansect 0 179 179 0.0 li, z.q., et al., 2015 indonesia, krakatau std. transect 11 498 509 2.2 gathorne-hardy et al., 2000 java std. transect 0 88 88 0.0 pribadi et al., 2011 malaysia std. transect 0 265 265 0.0 hanis et al., 2014 malaysia mod. transect 10 1259 1269 0.8 eggleton et al., 1999 sarawak std. transect 0 25 25 0.0 jamil et al., 2017 sri lanka std. transect 1 160 161 0.6 hemachandra et al., 2010 sumatra std. transect 3 1178 1181 0.3 gathorne-hardy et al., 2001 sumatra std. transect 1 283 284 0.4 jones et al., 2003 taiwan expert survey 28 105 133 21.1 li, h. f., et al., 2011 taiwan expert survey 275 397 672 40.9 li, h. f., et al., 2015 thailand std. transect 1 198 199 0.5 inoue et al., 2006 vietnam mod. transect 0 154 154 0.0 duc et al., 2017 vietnam mod. transect 0 228 228 0.0 van quang et al., 2017 tropical australia e. australia mod. tansect 0 597 597 0.0 houston et al., 2015 n. australia mod. tansect 0 57 57 0.0 dawes-gromadzki, 2005 n. australia mod. tansect 0 80 80 0.0 dawes-gromadzki, 2008 worldwide review old & new world tropics std. transect 38 2105 2143 1.8 davies et al., 2003b *three peruvian habitats were surveyed. the amazonian rainforest yielded 72% of species; none were kalotermitids. **jones and eggleton (2000). ***kalotermitid misidentified as paraneotermes. table 1. studies listing occurrences of kalotermitid and non-kalotermitid termites in wooded habitats by sampling method. (continuation) region or biome sampling method no. kalo occurrences no. non-kalo occurrences total termite occurrences % kalo of total occurrences reference subtropical & tropical new world as with many other flying insects, light traps are effective in capturing termite alates but due to greater morphological ambiguity of this caste, most termite collectors seek soldiers and workers. earlier studies of regional termite diversity (e.g., new world: banks & snyder, 1920, emerson, 1925, mathews, 1977; old world: e.g. sjöstedt, 1925, emerson, 1928) did not quantify taxon occurrences but reported collections of alates around lights. the first taxon-quantified termite survey from alate trap catches was conducted by robello & martius (1994) near manaus, brazil. ultraviolet light traps were placed at 3.6 or 4 m height and sampled for 24 months. similar techniques were used in subsequent alate trapping studies. rh scheffrahn, ja chase, jr mangold, hh hochmair – kalotermitidae encounters by survey method92 we compiled our expert survey results and summarized literature results from transect, alate trap, and expert studies of wooded habitats with regard to kalotermitid and nonkalotermitid occurrences (table 1). we also compiled results in which species diversity of kalotermitid and non-kalotermitid termites was reported from transect surveys, but individual occurrences were not enumerated (table 2). statistical analysis an odds ratio (or) statistic was used to measure the effect of collection method on kalotermitid vs. nonkalotermitid encounters of the three main collection methods in table 1 (canopy transect excluded). three pairs of comparisons were considered: 1) transect vs. alate traps, 2) transect vs. expert survey, and 3) alate traps vs. expert survey. for each method pair, a 2x2 matrix comparison of percentages was used: 1) number of kalotermitids collected in method 1 (e.g. transect), 2) number of non-kalotermitids collected in method 1 (e.g. transect), number of kalotermitids collected in method 2 (e.g. alate traps), and number of non-kalotermitids collected in method 2 (e.g. alate traps). odds-ratios were computed for geographic regions where at least two survey methods were applied using two-sided p values for the test of independence with fisher’s exact test. region or biome sampling method no. kalo spp. no. non-kalo spp. % kalo spp. reference se brazil mod. transect 0 14 0.0 araújo et al., 2007 panama std. transect 1 16 6.3 basset et al., 2017 brazilian amazonia mod. transect 9 76 11.8 bandeira, 1989* brazilian amazonia mod. transect 0 78 0.0 constantino, 1992 ne brazil mod. transect 5 45 11.1 couto et al., 2015 thailand std. transect 0 16 0.0 davies, 1997 french guiana std. transect 4 96 4.2 davies, 2002 cameroon std. transect 0 88 0.0 eggleton et al., 1995 borneo std. transect 3 63 4.8 eggleton et al., 1997 tropical west africa std. transect 1 132 0.8 eggleton et al., 2002a congo std. transect 0 80 0.0 eggleton et al., 2002b benin std. transect 0 20 0.0 hausberger et al., 2011 cameroon std. transect 0 117 0.0 deblauwe et al., 2007 cameroon std. transect 0 47 0.0 jones & eggleton, 2000 sabah malaysia std. transect 0 33 0.0 jones & eggleton, 2000 peninsular malaysia std. transect 0 29 0.0 jones & eggleton, 2000 sumatra, indonesia std. transect 1 54 1.9 jones et al., 2003 vietnam std. transect 0 43 0.0 neoh et al., 2015 sumatra, indonesia std. transect 0 21 0.0 neoh et al., 2016 se brazil std. transect 1 49 2.0 nunes et al., 2017 thailand mod. transect 0 35 0.0 sornnuwat et al., 2003 * occurrence of incisitermes is dubious. table 2. studies in which occurrences of kalotermitid and non-kalotermitid termites was not enumerated. wood was sampled in each study. results table 3 gives the ors (p < 0.001) for each pairing of survey methods by geographic region (table 1). worldwide, there is about a 50.6-fold greater likelihood using the expert survey method and a 15.3-fold greater likelihood using alate trapping of encountering a kalotermitid sample versus a nonkalotermitid than using the transect method. there is about a 3.3 -fold greater likelihood of collecting a kalotermitid versus a non-kalotermitid using the expert survey method than with the alate trap method. for the new world, the results show about a 10-fold higher likelihood of collecting a kalotermitid versus a non-kalotermitid using the alate traps method than with the transect method, a 33.8 times higher chance of collecting a kalotermitid versus and non-kalotermitid using the expert survey than with the transect method, and a 3.3 times higher chance of collecting of collecting a kalotermitid versus a non-kalotermitid using the expert survey method than with the alate trap method. for asia, the results show about 115.7 times higher odds of collecting a kalotermitid (and not a non-kalotermitid) with the expert survey than with the transect method. of the 20 transect studies in which species were recorded but the number of individual occurrences were not enumerated, 13 failed to detect any kalotermitids, and seven reported the composition of kalotermitid species between 0.8 and 11.8% (table 2). sociobiology 65(1): 88-100 (march, 2018) special issue 93 discussion the first notable taxonomic treatises based on expert field surveys of termites were conducted around the start of the previous century (e.g., haviland, 1898, sjöstedt, 1900, silvestri, 1903, holmgren, 1906) all of which recorded many new kalotermitids. with the assistance of expert collectors, nathan banks described 12 new kalotermitid species between 1901 and 1920 (data from krishna et al., 2013a). the “emerson era” (krishna et al., 2013b) began with emerson’s (1925) expert survey of guyana where he collected 12 kalotermitid species, 11 of which were new. expert collections by emerson, light, krishna, snyder and their collaborators added an additional 100 new extant kalotermitid species between 1918 and 1962 (data from krishna et al., 2013a). since, 1962, approximately 170 new kalotermitid species have been described as a result of expert collections. using the expert survey method, we attempted to maximize termite taxon diversity while minimizing collecting time. in addition to finding many novel termitids, we have collected and described 22 new kalotermitid species and expanded the range of many more taxa. at least 20 more new kalotermitids are pending description from our expert surveys. as far as we know, only one new kalotermitid species, cryptotermes chacoensis roisin, has been collected and described from transect survey material (roisin & leponce, 2004). the stated goal of the transect protocol is to assess both termite species richness (diversity) and functional group (feeding niche) in tropical forests (jones & eggleton, 2000). our finding of kalotermitid underrepresentation in transect surveys when compared to other methods is likely rooted in multiple sampling biases. these might include collecting tools, time constraints, habitat type, geographical location, topographical conditions, and human traits (experience, search patterns, collecting skills, hiking ability, eyesight, etc.). alate traps may offer the most unbiased estimate of local termite composition, albeit for crepuscular or nocturnal fliers. because different species have different flight seasons, traps must be tended over long periods; time-costly and difficult to accomplish in remote sites. the five alate trap surveys given in table 1 yielded between 7.5 and 19.5% kalotermitid occurrences. the actual ratio of kalotermitidto-nonkalotermitid colonies is probably greater than absolute occurrence because kalotermitid colonies have lower populations than their nonkalotermitid counterparts. central to the performance of field surveys are the tools used and how selected tools affect collecting time and efficiency. for termites, two types of collecting tools are required; tools to access the termites and tools to handle and transfer the termites into ethanol. for access, a hoe is more efficient in excavating soil than an ax, but a hoe is not designed to split and open wood. curiously, the types of tools to use for standard transect surveys have never been specified although they are likely variants of a hoe as used by reginaldo constantino or tiago carrijo (scheffrahn pers. obs.). darlington (1992, 1997) used a cutlass (machete) to split wood. emerson (1938) mentions the use of a hatchet for opening nests during his collecting expedition in guyana (emerson, 1925). li et al. (2011) used axes. roisin et al. 2006 “cut down” tree branches using saws free from time constraints of ground-based transects. it is interesting to note that bandeira (1989), who collected the highest proportion of kalotermitid species (11.8%, table 2), used a chain saw to systematically sample dead wood. for handling and transfer, forceps can be used (e.g., ackerman et al., 2007) which excludes the transfer of termites from their niche detritus, but forceps require more field time than an aspirator. aspiration also collects large groups of termites before they can escape with little or no specimen damage. aspirated samples are rinsed of detritus in the laboratory allowing more field collecting time. kalotermitids require more time to collect than non-kalotermitid species. the transect protocol allocates 30 min sampling time by two trained people per 5x2 m plot (jones & eggleton, 2000). but even with an ax, splitting wood is strenuous and consumes time in the search for morphologically identifiable castes in the galleries of sound wood. whereas excavating rotten wood, nests, or the surface soil is less strenuous and may not require an ax. expert surveys accommodate sampling for kalotermitids because they employ the tools for splitting wood, are not confined to a defined search area (but within walking distance), and are not limited to a specific search time. comparisons of termite diversity yields between transect and expert collecting methods is problematic survey methods world total subtropical & tropical new world tropical asia expert survey vs. transect or 50.5 33.8 115.7 ci (44.6, 57.6) (29.4, 39.0) (83.7, 162.7) expert survey vs. alate traps or 3.3 3.2 n/a ci (3.2, 3.4) (3.2, 3.4) alate traps vs. transect or 15.5 10.4 n/a ci (13.7, 17.7) (9.0, 12.0) table 3. odds ratios (or) and their 95% confidence intervals (ci) between different survey methods for encountering a kalotermitid sample. all odds ratios in the table are significantly different from 1.0 (p<0.001). rh scheffrahn, ja chase, jr mangold, hh hochmair – kalotermitidae encounters by survey method94 because field sampling is biased by human traits (experience, collecting skills, hiking ability, physical condition, etc.) and the physical environment. jones & eggleton, 2000 stated that their field assistants were sufficiently trained to conduct transect sampling after one practice transect but suggested that their performance could be affected by motivation. other unquantifiable human qualities affecting collection efficiency include recognition of termite microhabitats, willingness to enter thick and/or spiny undergrowth or standing water, strength, and work performance in uncomfortable weather. motivation to discover novel taxa for our expert surveys is driven by a competitive spirit and peer pressure. but what is the difference in cost of an expert collecting expedition versus a transect survey? to get a rough idea, we compare our 2012 country-wide expert survey of paraguay with our 2011 on-foot expert survey of parque nacional yasuní, ecuador. the ecuador expedition is a reasonable proxy for a transect survey because it was conducted at a single locality. the cost of vehicle rental, fuel expense, and tolls for paraguay (2,075 km driven by two vehicles) amounted to about $3,500 usd, an expense not needed for yasuní. the collecting time lost to driving was substantial, however, travel allowed for sampling fauna in paraguay’s highly varied biomes. the non-monetary “cost” of traveling great distances on unfamiliar foreign roads is the inherent danger of a traffic accident. the paraguay expedition yielded 1,288 colony samples (54 kalotermitid samples) with a 60 person-day collecting effort. yasuní yielded 1,035 samples (28 kalotermitids) with a 45 person-day effort. for species richness, probably the most comparable of the two sampling methods is the jones & eggleton (2000) single transect in mbalmayo forest reserve, cameroon (+3.45°, +11.47°), and our first full day of expert collecting at yasuní (-0.674°, -76.398°). both are tropical rain forests, but probably with greater overall termite diversity at mbalmayo. jones and eggleton (2000) yielded 47 species (no kalotermitids) after four person-days of sampling. we collected 59 species (8 kalotermitid samples) after 7.5 person-days, suggesting rather similar diversity yields, excepting for the kalotermitidae. but does the transect method really reflect the local fauna, specifically the kalotermitidae? jones & eggleton (2000) concluded that “the taxonomic and functional group composition of the transect samples did not differ significantly from that of the known local fauna.” we offer limited evidence that in similar biomes, at least for kalotermitidae, that these may differ. table 4 compares the kalotermitid and nonkalotermitid samples collected in the peruvian amazon from palin et al. 2011, dahlsjö et al., 2015, and data from our unpublished 2014 survey. the former two transect studies yielded a single unidentified kalotermitid, collected at 1500 m, from a combined total of 1,214 nonkalotermitid samples collected from 190 m to 1500 m. our survey yielded 31 kalotermitids from six different genera from a total of 983 samples. geographical attributes, habitat types, and topographical conditions also bias kalotermitid encounters. our experience in the neotropics suggests that oceanic islands (e.g. west indies) offer the greatest abundance and relative diversity of kalotermitids followed by littoral mainland localities (e.g., panama) with inland forests showing the least in abundance and diversity (e.g., amazonian peru). for example, our expert survey results revealed that the island of jamaica has 10 endemic kalotermitid species compared to 4 non-kalotermitids, while panama yielded 23 kalotermitids compared to 53 nonkalotermitids, and the peruvian amazon produced only 12 kalotermitid species compared to 95 non-kalotermitids (scheffrahn et al. unpublished). because of their overwater dispersal abilities (yamane et al., 1992, scheffrahn & postle, 2013), islands become filters for kalotermitidae. littoral zones support tree species that are tolerant to tidal exposure and sandy soil. the low kalotermitid abundance in inland rain forests may be an artifact of the inability to assess high tree branches (roisin et al., 2006). it is likely that transect plots are set on relatively flat surfaces while expert sampling allows for collecting along steep trails and hillsides, roadsides, and narrow shorelines. while it is impossible to collect all termite species in a particular locality, all survey methods contribute to the discovery of new species and termite diversity in a given area. herein we show that the expert colleting method should be preferred for collecting greater diversity and abundance of the kalotermitidae. palin et al. 2011 dahlsjö et al. 2015 current study method std. transect mod. transect expert elevation (m) 190 925 1500 190 ≤190 204 1104 ≥1500 latitude (°) -12.83 -12.95 -13.05 -12.82 -8.60 to -8.37 -11.29 to -8.49 -10.72 to -10.71 longitude (°) -69.28 -71.53 -71.54 -69.27 -74.94 to -74.72 -76.04 to -74.67 -75.18 to -75.14 kalo./nonkalo. 0/189 0/55 1/3 0/967 5/153 25/810 1/20 kalo. genera unknown calcaritermes glyptotermes neotermes calcaritermes comatermes cryptotermes glyptotermes rugitermes rugitermes table 4. kalotermitid versus nonkalotermitid sampling yields from three peruvian amazon termite surveys. sociobiology 65(1): 88-100 (march, 2018) special issue 95 acknowledgements we thank terminix international and basf corp. for financial assistance. we were privileged to have had worked in the field with many expert collectors: paul ban (deceased), brian bahder, tiago carrijo, reginaldo constantino, robin giblin-davis, julian de la rosa guzman, robert hickman, solange issa, natsumi kanzaki, jan krecek, eko kuswanto, boudanath maharajh (deceased), aaron mullins, timothy myles, thomas nishimura, josé perozo, yves roisin, robert setter, and john warner. two anonymous reviews greatly improved this manuscript. references ackerman, i. l., constantino, r., gauch jr., h. g., lehmann, j., riha, s. j., & fernandes, e. 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(1992). recolonization of the krakataus by hymenoptera and isoptera (insecta). geojournal, 28: 213-218. doi: 10.13102/sociobiology.v60i3.242-251sociobiology 60(3): 242-251 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 protection mutualisms and the community: geographic variation in an ant-plant symbiosis and the consequences for herbivores eg pringle1, dm gordon2 introduction mutualistic interactions between species can strongly affect the structure of ecological communities (stachowicz, 2001; bruno et al. 2003; hay et al., 2004). for example, animal seed dispersal can determine the composition of tropical-tree communities (terborgh et al., 2008), and pollinator preferences can favor the spread of certain plant species (chittka and schürkens, 2001). protection mutualisms, in which a mutualist defends its partner against natural enemies, are frequently shown to affect the growth and fitness of individual organisms, but very little is known about how these interactions structure the communities in which they are embedded. the paucity of such studies may give the false impression that the community effects of protection mutualisms are rare abstract protection mutualisms mediate trophic interactions in many systems, but their effects on the surrounding community are rarely studied. ant-plant symbioses are classic examples of protection mutualisms: myrmecophytic plants provide nesting space and food for symbiotic ants in exchange for ant defense. ant defense should thus reduce the abundance of herbivores, but studies of ant-plant symbioses usually measure damage to the plant without quantifying the herbivores themselves. in this study, we investigated whether geographic variation in the quality of ant defense in a symbiotic mutualism between cordia alliodora trees and azteca ants was associated with the abundance and species richness of plant herbivore communities. in three tropical-dry-forest sites in middle america, we found that the density of azteca ants within trees was negatively associated with the levels of leaf herbivory. at sites where ants were effective tree defenders, tree herbivores were less abundant and herbivore assemblages on trees exhibited lower species richness than at a site where ants were poor defenders. in addition, in a site where ants reduced herbivory, herbivore communities were less abundant and diverse in the presence of ants than in their absence, where as in a site where ants did not reduce herbivory, there were no differences in herbivore abundance or richness between trees with or without ants. we conclude that geographic variation in the quality of ant defense drives variation in myrmecophytic-plant herbivore communities. moreover, ant-plant protection mutualisms should have important but rarely considered effects on herbivore population dynamics and food-plant specialization. sociobiology an international journal on social insects 1 university of michigan, ann arbor, michigan, usa. 2 stanford university, stanford, california, usa. research article ants article history edited by kleber del-claro, ufu, brazil received 02 july 2013 initial acceptance 31 july 2013 final acceptance 12 august 2013 key words ant-plant mutualism, azteca, context dependence, cordia alliodora, seasonally dry tropical forest, trophic cascades corresponding author elizabeth g. pringle university of michigan 830 n university st ann arbor, mi 48103, usa e-mail: epringle@umich.edu or unimportant, when in fact the studies so far suggest that protection tends to have strong effects on local diversity and abundance. interestingly, it has been suggested that the overall effect of protection is to reduce local species diversity (rudgers and clay, 2008; rudgers et al., 2010; but see jani et al., 2010), which contrasts with other wellknown mutualisms that clearly enhance local diversity (e.g., fig trees, corals) (terborgh, 1986; stachowicz, 2001; hay et al., 2004). such community consequences have been so little studied, however, that we do not know how general such local effects may be, or how these mutualisms affect systems on larger spatial and temporal scales. in terrestrial communities, ant protection frequently determines the strength of trophic cascades (sensu hunter and price, 1992). ants both decrease herbivory as mutualists sociobiology 60(3): 242-251 (2013) 243 of plants by predating and harassing herbivores (hölldobler and wilson, 1990) and increase herbivory as mutualists of honeydew-producing hemiptera by protecting them from other predators (way, 1963; del-claro and oliveira, 2000). in both mutualistic roles, ant protection can reduce the diversity and abundance of other arthropods in a system, but the strength of these effects varies among taxa and depends on environmental context (fowler and macgarvin, 1985; wimp and whitham, 2001; kaplan and eubanks, 2005; oliveira and del-claro, 2005; mooney, 2007; rudgers et al., 2010). indeed, although ant-plant interactions tend to have positive effects on individual plants (styrsky and eubanks, 2007; chamberlain and holland, 2009), which suggests that ants should reduce the abundance, if not also the diversity, of untended herbivores on those plants, these effects are often non-significant at both plantand experimental-plot-scales (kaplan and eubanks, 2005; mooney, 2007; rudgers et al., 2010). this is inconsistent with the evidence that ants reduce herbivory levels (chamberlain and holland, 2009; styrsky, 2007) unless most herbivores collectively consume very little and ants defend only against the few herbivores that inflict the most damage. whether or not such conditions are generally true, they raise interesting and mostly unanswered questions about the relative abundances of herbivore species in these systems, and about the relative specialization of those species on ant-defended food plants. the strength of any given ant-plant interaction is geographically variable. the identities and abundances of ants, herbivores, and even other predators vary over space and affect how much ants benefit plants (bronstein, 1994; rudgers and strauss, 2004; mooney, 2007; rosumek et al., 2009). geographic variation in the effectiveness of ant defense will also affect food availability for herbivores of a given plant, with potential cascading effects on herbivore population sizes and even food-plant specialization (i.e., in a geographic mosaic of coevolution, sensu thompson, 2005). in one of the few studies to examine the community effects of a geographically variable ant-plant interaction, rudgers et al., (2010) reported that, in three arizona populations of wild cotton, there was a positive relationship between how much ant presence within a site increased plant fitness and influenced arthropod community composition. geographic variation in the effects of ants in that system resulted from differences in total ant abundance and the relative abundance of a particularly aggressive ant species (rudgers and strauss, 2004). surprisingly, however, even at the site where ants produced the greatest benefits for plants, by decreasing the abundance of a specialist caterpillar (rudgers and strauss, 2004), ants did not reduce the per-plant abundances of total arthropods or generalist herbivores (rudgers et al., 2010). ant-plant symbioses are specialized mutualisms: myrmecophytic plants supply ants with nesting space and food, and ants defend plants against herbivores (davidson and mckey, 1993; heil and mckey, 2003). such highly specialized ant-plant interactions should consistently reduce the abundances of the herbivores that eat the plant. yet although ants' capacity to reduce herbivory and increase plant fitness in such systems is well documented (chamberlain and holland, 2009; rosumek et al., 2009), and some studies have contrasted the effects on plants of different herbivore species (e.g., frederickson 2005; palmer and brody, 2013), very few studies have investigated the community of herbivores associated with a given myrmecophyte (but see, e.g., janzen, 1967; gaume et al., 1997; itino and itioka, 2001). this lacuna probably results in part from the difficulty of identifying insect larval forms, which often inflict most of the leaf damage in the diverse tropical forests where myrmecophytes are common (davidson and mckey, 1993). surmounting this difficulty will be essential for understanding the community role played by these classic study systems. the myrmecophytic tree cordia alliodora (ruiz & pavón) oken (boraginaceae) hosts colonies of azteca ants (hymenoptera: formicidae: dolichoderinae) in neotropical forests. ants nest in hollow swellings, known as domatia, at stem nodes, and tend honeydew-producing scale insects (hemiptera: coccidae and pseudococcidae) that feed on plant phloem from inside the domatia. azteca ants can reduce folivory on c. alliodora (tillberg, 2004; trager and bruna, 2006; pringle et al., 2011), but the magnitude of these effects depends on the size of the ant colony, the size of the tree, and geographic location (pringle et al., 2011; pringle et al., 2012a). there have been few studies of c. alliodora herbivores, but many of the lepidopteran larvae that eat the tree's leaves in middle america can be identified by comparison with a database of caterpillars and their food plants from northwestern costa rica (janzen and hallwachs, 2009). in this study, we investigated geographic variation in c. alliodora herbivore communities.we investigated variation in the density of ants within and among c. alliodora trees and in the effectiveness of ant defense in three sites in middle america. by conducting surveys of the herbivore assemblages on individual trees, we asked whether the abundance and species richness of known c. alliodora herbivores were negatively associated with the density of ants and the effectiveness of ant defense at a site. we then asked whether trees occupied by azteca ants supported lower abundance and richness of known herbivores than unoccupied trees within each of two sites that differed in the typical effectiveness of ant defense. finally, we consider the implications of our results for herbivore population dynamics and food-plant specialism. material and methods study sites and system the study was conducted in three tropical dry forests located on the pacific coast of middle america: the chamela-cuixmala biosphere reserve, jalisco, mexico (19°30’ n, 105°02’ w), huatulco national park, oaxaca, mexico (15°42’ n, 96°10’ w), and the area de conservación guanaeg pringle, dm gordon variation in ant defense affects herbivore communities244 azteca density estimates to estimate the density of azteca ants in trees, a variable that is positively related to the effectiveness of ant defense against herbivores (pringle et al., 2011), we divided the number of ants in a tree by the number of domatia counted or estimated for that tree. we counted the number of ants and domatia for six entire trees (of heights 1.6-4.5 m) at each of the three sites by cutting trees down, collecting domatia in press-seal bags, and opening all domatia to count ants. we estimated the number of ants and domatia in 14 additional trees (of heights 2-8 m) in chamela and santa rosa. estimates were made from counts of the ants in three domatia and diameter measurements of the trees (for more details, see pringle et al., 2011).we tested whether ant density varied among sites with an anova that treated site as a fixed factor and post-hoc tukey-kramer hsd. survey of tree ant occupants we conducted a survey of tree ant occupants to determine the proportion of trees at each site occupied by azteca spp. in chamela and santa rosa, three 0.5-ha tree plots were established in july-august, 2007; all c. alliodora individuals were located within these plots, and the ant occupant was identified when possible (santa rosa n = 170 of 302 total trees, chamela n = 117 of 166 total trees). when trees were too tall to examine domatia directly, we shook the tree vigorously and examined the branches for ants with binoculars. trees in which no ants were visible were recorded as unknowns and were not included in the analysis. at all three sites, we also surveyed trees along the trail system, looking for c. alliodora individuals whose branches were low enough to examine domatia for the ant occupant(s) (chamela n = 108, caste, sector santa rosa, guanacaste, costa rica (10°50’ n, 85°36’ w). these three sites (henceforth chamela, huatulco, and santa rosa) are all characterized by strong rainfall seasonality: the annual 4-6 mo rainy season is followed by an intense dry season (bullock, et al., 1995). average annual precipitation between 1979 and 2009 was 778.5 mm in chamela, 1033.5 mm in huatulco, and 1686.4 mm in santa rosa, according to records from meteorological stations associated with the protected areas in chamela and santa rosa and with nearby towns in huatulco. cordia alliodora is common throughout the neotropics, from mexico to argentina, including the caribbean, and populations have recently been introduced to east africa and the south pacific (dawson et al., 2008). the most common ant symbiont throughout middle america is the c. alliodora specialist azteca pittieri forel, but other ant species do colonize the tree, especially at lower latitudes (longino, 1996; pringle et al., 2012b). notable among these other species are azteca beltii emery, which is a generalist live-stem nester and the second-most-common azteca species found in c. alliodora after a. pittieri, and cephalotes setulifer emery (hymenoptera: formicidae: myrmicinae), which is the secondmost-common c. alliodora specialist after a. pittieri, and whose known range extends from el salvador and honduras to panama (longino, 1996). cephalotes setulifer provides c. alliodora with little or no defense against herbivores (tillberg, 2004). because it is difficult to distinguish a. pittieri workers from those of other azteca species in the field (longino, 2007), we will refer to azteca ants throughout the rest of the manuscript by the genus name only. however, genetic data gathered from random samples of colonies in the field indicate that these ants are usually a. pittieri at all three sites (pringle et al., 2012b). figure 1. (a) number of worker ants per tree domatium (mean ± se) averaged for all domatia within trees at the three sites (chamela n = 19, huatulco n = 6, santa rosa n = 20), listed from north to south. different letters indicate significant differences by anova and tukeykramer hsd (p < 0.008). (b) percentage of c. alliodora trees occupied by azteca ants in surveys of trees at the three sites (chamela n = 225, huatulco n = 91, santa rosa n = 226). black portions of the bar indicate trees occupied by azteca spp.; gray portions indicate trees occupied by other ant species (c. setulifer, crematogaster spp., p. viduus); white portions indicate trees unoccupied by ants. sociobiology 60(3): 242-251 (2013) 245 huatulco n = 91, santa rosa n = 56). trees were recorded as unoccupied by ants only when an extensive examination of domatia revealed no ant entrance holes. herbivory estimates to compare the leaf herbivory experienced by trees in the presence of azteca spp. between sites, we measured standing levels of herbivory. levels of herbivory were estimated on n = 40 trees in chamela in august 2008, n = 27 trees in huatulco in july 2009, and n = 41 trees in santa rosa in june 2008. herbivory was estimated by e.g. pringle on ~100 leaves from ≥ 3 branches per tree of 2-8 m height using a standardized index (dirzo and domínguez, 1995). leaves were categorized by eye according to the following levels of leaf area eaten: 0 = 0%, 1 = 1-6%, 2 ≥ 6-12%, 3 ≥ 12-25%, 4 ≥ 25-50%, 5 ≥ 50-100%. the number of leaves in each category was multiplied by its category value, and the sum of these products was divided by the total number of leaves to generate the index. because herbivory is positively related to tree size in this system (pringle et al., 2012a), differences among sites were analyzed with an anova, in which site was treated as a fixed factor and tree basal diameter as a covariate, followed by a post-hoc tukey hsd test. herbivore surveys to investigate differences in the abundance and species richness of c. alliodora herbivore communities at the three sites, we conducted surveys in the early rainy season in june-august 2009 and 2010. because herbivory in tropical dry forests is highest early in the rainy season (janzen, 1988; dirzo and domínguez, 1995), herbivore surveys were conducted within the first third of the rainy season at each site in both years. in both years, we surveyed ~40 trees of 3 m average height (height ranges: chamela 1.3-7.5 m; huatulco 1.2-12.0 m; santa rosa 0.9-8.5 m) haphazardly chosen along the trail system at each site (chamela: 2009 n = 43, 2010 n = 40; huatulco: 2009 n = 53, 2010 n = 39; santa rosa: 2009 n = 38, 2010 n = 39). surveys were performed by visually scanning the tree, and particularly the undersides of the leaves, for 5 min. we searched the leaves from the ground, from a 2-m ladder, and with binoculars if necessary. tree size (basal diameter) was used as a covariate in all analyses. surveys were conducted simultaneously by three investigators, except in santa rosa in 2009 and chamela in 2010, when, for logistical reasons, they were conducted by two investigators. herbivores were identified to order and morphospecies were assigned to individuals on each tree. lepidopteran larvae were identified to genus, and to species when ~3rd instar or larger, based on the database of janzen and hallwachs (2009). in addition, larvae of coptocycla leprosa boheman (coleoptera: chrysomelidae) beetles are c. alliodora folivores (trager and bruna, 2006) and are common and easily identified at all sites. all arthropods seen during the survey were identified as non-herbivorous (predators, scavengers) or potentially herbivorous (i.e., omnivorous insects were classified as potential herbivores); potentially herbivorous morphospecies were then hierarchically classified as: (1) herbivorous arthropods and (2) known c. alliodora herbivores. the second category was based on food-plant records of janzen and hallwachs (2009), which come in part from the santa rosa site, and on personal observations by e.g. pringle. the ant occupant of each tree used for an herbivore survey was identified by examining domatia. in santa rosa, trees that were occupied by both azteca spp. and c. setulifer were treated in analyses as trees occupied by azteca. in chamela, all survey trees were occupied by azteca spp.; in huatulco and santa rosa, some survey trees were not occupied by azteca spp. (huatulco: 2009 n = 18, 2010 n = 9; santa rosa: 2010 n = 19). because we were interested in how geographic variation in azteca defense affects herbivore communities, only trees occupied by azteca spp. were included in the analysis of differences in herbivore communities between sites. analysis was conducted using a nested anova, in which site and year nested within site were treated as fixed factors and tree size was included as a covariate, followed by post-hoc tukey hsd. to test whether differences in herbivore communities between sites were caused in part by differences in ant defense between sites, and not just by other environmental variables, we also compared herbivore communities between azteca-occupied trees and unoccupied trees within huatulco and within santa rosa. stepwise regression of ant occupant and survey year with the huatulco data indicated that survey year was nonsignificant in all cases (herbivorous arthropod abundance p = 0.3, c. alliodora herbivore abundance p = 0.6, c. alliodora herbivore richness p = 0.08), so huatulco data from 2009 and 2010 were pooled for this analysis. the analysis of santa rosa data included only the 2010 survey, because we did not survey unoccupied trees in 2009. differences between azteca-occupied trees and unoccupied trees were assessed with an anova, in which ant occupant was the factor and tree size was included as a covariate. statistical software all analyses were conducted with jmp® pro 10.0 (sas institute inc. 2010). results the density of azteca ants within trees and of azteca colonies among trees varied significantly between sites. ant workers nested at higher densities within trees in chamela and huatulco than in santa rosa (fig 1a; anova f2,42 = 10.19, eg pringle, dm gordon variation in ant defense affects herbivore communities246 p < 0.0002; tukey-kramer hsd p < 0.008). a higher proportion of trees contained colonies of azteca spp. in chamela (97.3%) than in huatulco (44.0%) or santa rosa (34.5%) (fig 1b). chamela contained the fewest trees completely unoccupied by ants (1.3%), and there were fewer completely unoccupied trees in santa rosa (37.6%) than in huatulco (56.0%) (fig 1b). in chamela, 1.3% of trees were occupied by crematogaster crinosa mayr; in santa rosa, 25.7% of trees were occupied by c. setulifer alone (an additional 4.0% of trees contained both c. setulifer and azteca spp., although this is probably an underestimate because not all of the domatia were examined in any given tree (longino, 1996)), 1.3% of trees were occupied by crematogaster curvispinosa mayr, and 0.9% of trees were occupied by pseudomyrmex viduus smith. azteca-occupied trees experienced less herbivory in sites where ants occupied trees at higher densities. standing levels of herbivory in azteca-occupied trees were significantly lower in chamela and huatulco than in santa rosa (fig 2; anova: f3,104= 38.07, p < 0.0001, site: f = 41.53, p < 0.0001, tree basal diameter: f = 7.05, p < 0.01). the abundance and richness of herbivores found on c. alliodora trees was significantly higher in santa rosa, the site where ants occupied the tree at the lowest densities and appeared to be the least effective plant defenders. herbivores belonged to the orders coleoptera, hemiptera, lepidoptera, orthoptera, and phasmida. the composition of herbivorous arthropod orders varied between sites (table 1); the proportion of individuals from coleoptera and lepidoptera was highest in santa rosa. the abundance of herbivorous arthropods was greater in santa rosa than in the other two sites (fig 3a; anova f6,198= 9.25, p < 0.0001, site: f = 13.51, p < 0.0001, year[site]: f= 9.32, p < 0.0001, tree basal diameter: f = 2.73, p = 0.1). in addition, both the abundance and the species richness of known c. alliodora herbivores per tree was higher in santa rosa than figure 2. standing herbivory level based on an index of percent leaf area eaten (0-5; mean ± se) on trees occupied by azteca ants at the three sites (chamela n = 40, huatulco n = 27, santa rosa n = 41). different letters indicate significant differences by a mixed-effect anova and tukey hsd (p < 0.05). table 1. percentage of the total herbivorous arthropods found at each site on azteca-occupied c. alliodora trees belonging to each of five insect orders. in either chamela and huatulco (fig 3b-c; abundance: anova f6,198= 13.35, p < 0.0001, site f = 20.48, p< 0.0001, year[site] f = 14.17, p < 0.0001, tree basal diameter f= 3.00, p = 0.08; richness: anova f6,198 = 22.67, p < 0.0001, site f = 31.93, p < 0.0001, year[site] f = 27.27, p < 0.0001, tree basal diameter f = 3.89, p < 0.05). in all cases, the significant effect of survey year was driven by santa rosa, not by the other two sites. santa rosa figure 3. herbivore communities harbored by c. alliodora trees occupied by azteca ants at the three study sites (chamela, huatulco, and santa rosa). (a) abundance of all herbivorous arthropods. (b) abundance of herbivores known to eat c. alliodora per plant. (c) species richness of herbivores known to eat c. alliodora per plant. bars indicate mean ± se. different letters indicate significant differences by anova and tukey hsd (p < 0.05). orders of herbivorous arthropods site n coleoptera hemiptera lepidoptera orthoptera phasmida chamela 67 34% 21% 19% 24% 1% huatulco 146 21% 42% 29% 8% 0% santa rosa 239 42% 1% 54% 3% 0% sociobiology 60(3): 242-251 (2013) 247 surveys in 2010 showed higher abundances of herbivorous arthropods (anova f1,55 = 19.78, p < 0.001), abundances of c. alliodora herbivores (f1,55= 14.07, p < 0.0004), and species richness of c. alliodora herbivores (f1,55 = 33.86, p < 0.0001) than in 2009. trees occupied by azteca spp. harbored significantly fewer herbivores and lower herbivore species richness than unoccupied trees in huatulco, where ants nested at high densities and appeared to reduce leaf herbivory, but not in santa rosa, where they did not (fig 4). in huatulco, the abundance and richness of c. alliodora herbivores was significantly lower in the presence of an azteca colony (fig 4a-b; abundance: anova f2,89 = 8.89, p < 0.003, presence of azteca f = 16.83, p < 0.0001, tree basal diameter f = 0.26, p = 0.6; richness: anova f = 9.71, p < 0.0002, presence of azteca f = 16.78, p < 0.0001, tree basal diameter f = 0.01, p = 0.9); the abundance of all herbivorous arthropods was lower on azteca-occupied trees, but not significantly so (anova f2,89 = 2.24, p = 0.1). in santa rosa, azteca presence did not affect the abundance of herbivorous arthropods (f2,36= 1.42, p = 0.3), the abundance of c. alliodora herbivores (f2,36= 1.28, p = 0.3), or c. alliodora herbivore species richness (f2,36= 0.56, p = 0.6) (fig 4c-d). discussion despite the prominence of ant-plant mutualisms as study systems for evolutionary ecology (bronstein 1998; heil and mckey 2003), very few studies have quantified the effects of ant defense on herbivore communities. here we show that azteca symbiotic plant-ants can significantly reduce both the abundance and diversity of herbivores on their c. alliodora host plants, but that these reductions occur only at geographic locations where ants are effective plant defenders. janzen (1966) reported the first experimental evidence that symbiotic ants can reduce herbivory on myrmecophytic acacia plants, and he went on to identify the common acacia herbivores and characterize their interactions with defensive ants (janzen, 1967). since that time, much more attention has been paid to how ants affect plant damage than to how they affect the plant's herbivores (but see, e.g., gaume et al., 1997; itino and itioka, 2001). the few studies so far of the community-level effects of ant protection have focused on free-living (i.e. non-symbiotic) mutualisms, and have shown that the effects of ants on plant arthropod communities are context-dependent (fowler and macgarvin, 1985; wimp and whitham, 2001; kaplan and eubanks, 2005; mooney, 2007; rudgers et al., 2010). although herbivory reduction by ants tends to be greater in symbiotic mutualisms than in free-living ones (chamberlain and holland, 2009; rosumek et al., 2009), our results demonstrate that the effects of symbiotic ants on plant herbivore communities are also contextdependent, depending strongly on geographic variation in the effectiveness of ant defense. understanding the evolutionary ecology of species interactions requires studies at large spatial scales (thompson 2005), but interpreting such studies is challenging because there are many potential explanatory variables. in this study, the site with the least effective ant defense exhibited the highest per-plant abundance and diversity of herbivores, but ant defense is one of many factors that vary among the sites. for example, there is also considerable variation in annual rainfall among these sites, which can directly affect insect communities (janzen and schoener 1968). in support of an important role for ant defense among these variables, however, we also found that the presence of azteca ants was associated with lower abundance and richness of c. alliodora herbivores within a site where ants appeared to be effective plant defenders (huatulco), but not within a site where they did not (santa rosa). this latter result is consistent with the lack of effect of experimental ant exclusionon herbivory rates in santa rosa (pringle et al., 2011). overall, our results concord with those of rudgers et al. (2010), who reported stronger effects of freeliving ants on cotton-plant arthropod community composition at geographic locations where ants more strongly increased plant fitness. geographic comparisons of species interactions are necessary to decipher the selection pressures on species across their ranges, and such within-site controls render these comparisons more interpretable. we found that the effectiveness of ant defense was negatively associated with herbivore species richness across sites. species richness is an important metric for community comparisons, but its meaning can be confounded by differences in abundance (gotelli and colwell, 2001). this problem is particularly acute in diverse tropical forests where commufigure 4. abundance and richness of c. alliodora herbivores on trees occupied by azteca ants or not in huatulco (a,b) and santa rosa (c,d). (a,c) abundance of known c. alliodora herbivores per plant. (b,d) species richness of known c. alliodora herbivores per plant. bars indicate mean ± se. asterisks (*) indicate significant differences by anova (p < 0.0001). eg pringle, dm gordon variation in ant defense affects herbivore communities248 nities are usually undersampled, such that increased sample size usually leads to increased richness estimates. although there may be true differences in herbivore alpha diversity among the sites studied here, the negative association we found across sites between ant defense and herbivore richness results at least partly from the differences across sites in herbivore abundance (table 1). many of the lepidopteran herbivore species that we find regularly in santa rosa are also found in chamela, but less frequently (e.g. pringle, personal observation). when ant defense is effective, therefore, the primary effect seems to be to reduce overall herbivore abundance. the species that can reach the highest densities typically persist on azteca-defended trees, whereas other species persist only rarely or on unoccupied trees. interestingly, the rank order of species abundances for c. alliodora herbivores was similar in our three sites. for example, in our surveys, cropia connecta (lepidoptera: noctuidae) caterpillars comprised 13.3% of known c. alliodora herbivores in chamela, 12.8% in huatulco, and 13.0% in santa rosa. future work is necessary to identify species and evaluate variation among sites within non-lepidopteran orders. this and previous studies represent only the first step towards a comprehensive understanding of the effects of ants on herbivore communities. in particular, our results raise two important questions for future research about the community effects of protection mutualisms. first, are ants driving differences in the population sizes of herbivore species among sites? and second, what is the relationship between ant defense and herbivore food-plant specialization? ant defense of plants affects realized food availability for herbivores, which should affect herbivore population sizes. many ant-plant mutualisms conflate the traditional definition of trophic levels (sensu hairston et al., 1960), and the resulting distinction between "bottom-up" and "top-down" control of herbivore populations, because plants attract ants that evolved from predatory ancestors but that may not actually eat their herbivores (janzen, 1966). higher ant density in trees leads to a higher probability that ants will attack and chase away herbivores (pringle et al., 2011; palmer and brody, 2013), which should reduce the survivorship of vulnerable arthropod larvae (vencl and srygley, 2013). higher ant densities may also affect food availability by reducing female oviposition (sendoya et al., 2009). within a site, food availability will be affected not just by ant density within plants, but also by ant and tree population sizes, and the resulting proportion of trees that are occupied by defensive ants. we found a positive association between ant density within trees and colony density among trees at two of our sites (chamela and santa rosa), but not at our third site (huatulco), where individual ant colonies grew large within trees, but more than half of the surveyed trees did not contain ants (fig 1). this suggests that c. alliodora food availability is intermediate in huatulco, which may lead to intermediate herbivore population sizes at this site. ant defense is of course not the only potential driver of herbivore population sizes: site climate also has important direct and indirect effects. seasonality in tropical dry forests leads to particularly dramatic indirect effects on herbivores through changes in food availability. the near-synchronous flushing of new foliage in the early rainy season strongly affects the first generation of herbivores (janzen, 1988; dirzo and domínguez, 1995). variation in the timing and amount of rainfall early in the rainy season can also dramatically affect the survivorship and fitness of dry-forest herbivores (agosta, 2008), and these effects may have driven the significant temporal variation in santa rosa herbivore communities between our surveys in 2009 and 2010. abundance of herbivorous arthropods was much lower in our 2009 surveys than in 2010 in santa rosa (2.2 ± 0.5 versus 7.8 ± 1.5 herbivorous arthropods per plant, respectively), and these differences may have resulted from less overall rain in 2009 or from the "false start" to the 2009 rainy season, which began in april but did not continue until midmay. in addition to its effects on leaf flush, precipitation may also drive patterns of local adaptation in azteca (pringle et al., 2012b), and these effects may extend to variation in ant defensive behaviors. indirect effects of climate on herbivore population sizes are rarely documented (boggs and inouye, 2012), but such effects can be important, even for arthropods that are considered food-plant generalists (belovsky and slade, 1995). a next important step will be determining the relative importance of these different direct and indirect effects on herbivore population sizes over time. although any herbivore that eats c. alliodora could be affected by geographic variation in ant defense, the effects on specialist herbivores may be particularly important and complex. of the three most abundant lepidopteran larvae in our surveys, c. connecta is a specialist on boraginaceae (and also eats a few species in the malvaceae (sensu stevens, 2001)), paridnea holophaealis (lepidoptera: pyralidae) has only been found to eat cordia spp. and varronia spp. (boraginaceae), and stauropides persimilis (lepidoptera: noctuidae) has only been found to eat cordia spp. (janzen and hallwachs 2009). we consider food-plant specialism to be a continuum, and thus these three most abundant lepidopteran herbivores are specialists, considering the many thousands of plant species found in northwestern costa rica that they do not eat. moreover, ongoing dna barcoding of these lepidoptera continues to reveal cryptic species, and often further specialism, within the present taxonomic nomenclature (hajibabaei et al., 2006). it is unclear what the outcome of geographic variation in ant defense should be for population sizes of specialist herbivores. on the one hand, specialist herbivores of antdefended plants should have strategies that allow them to continue feeding in the presence of ants (heads and lawton, 1985), which could buffer the effects of geographic variation in ant defense on food availability. on the other hand, spesociobiology 60(3): 242-251 (2013) 249 cialist herbivores have fewer alternatives if c. alliodora is a primary food plant and ant density within and among trees is high. caterpillar defenses against ants in this system, which include thrashing, biting, regurgitating, dropping, and shelterbuilding, should also be effective against other predators and parasitoids (gentry and dyer, 2002), and it remains unclear whether any of these defenses have evolved in response to azteca specifically (see, e.g., vencl and srygley, 2013). nevertheless, the evolution of defenses against natural enemies may be one reason that tropical lepidoptera are generally more specialized than temperate lepidoptera (dyer et al., 2007). interestingly, gange (2002) suggested that plantmycorrhizal mutualisms may also increase herbivore foodplant specialism by enhancing plant chemical defenses, which suggests that although plant protection mutualisms seem to decrease diversity on a per-plant scale (rudgers et al., 2010), they may actually increase diversity at a community scale. nearly 50 years of research on ant protection has revealed that ants usually benefit plants by reducing herbivory (styrsky and eubanks, 2007; chamberlain and holland 2009; rosumek et al. 2009; trager et al., 2010), but the effects of ants on herbivore communities remain unexplored. detailed studies are necessary to determine how important protection mutualisms are to their communities, an important step towards the greater inclusion of mutualisms in ecological theory (stachowicz, 2001; bruno et al., 2003; hay et al., 2004). moreover, such research will address questions that have rarely been considered in the tropical environments where ant-plant interactions are most abundant. in particular, it seems likely that these interactions drive arthropod population dynamics, which are important to understand in the face of rapid environmental change. in addition, such research would shed light on the role of mutualisms in such key processes as food-plant specialization and plant-herbivore coevolution. acknowledgments we thank h. donaghe, g. griffin, a. loggins, i. medina, e. murphy, k. tanabe, a. sinha, g. verduzco-robles, and i. will for help in the field. egp thanks w. hallwachs and d. janzen for helpful discussion. this work was funded by a hubert shaw and sandra lui stanford graduate fellowship and a michigan society of fellows postdoctoral fellowship to egp, and by a national science foundation grant (deb0918848) to dmg. references agosta, s.j. 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(2001) biodiversity consequences of predation and host plant hybridization on an aphidant mutualism. ecology, 82: 440-452. doi: 10.1890/00129658(2001)082[0440:bcopah]2.0.co;2. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.259-265sociobiology 60(3): 259-265 (2013) coexistence of aphid predators in cacao plants: does ant-aphid mutualism play a role? en silva1 and i perfecto2 introduction arboreal foraging ants are the most abundant group of arthropods in the canopy of trees in forests and structurally complex managed ecosystems in the tropics (adis et al., 1984; perfecto et al., 1997). a well known characteristic of ant communities in natural forests and in tree-based cropping systems is the existence of a three-dimensional mosaic determining the spatial distribution of ant species (majer, 1972; leston, 1973; majer & camer-pesci, 1991; armbrecht et al., 2001). this mosaic is comprised of a group of dominant species which are territorial and tend to have mutually exclusive distribution patterns, a sub-dominant group with species that have high number of individuals but do not hold large territories and non-dominants which occur in between or are tolerated within the territories of dominant species (majer et al., 1994; dejean et al., 2003). abstract mutualism between ants and hemipterans that produce honeydew has important implications for biological control because hemipterans defended against predators can reach economic injury levels. we tested the hypothesis that ant-aphid mutualism can mediate competition and promote the coexistence of aphid natural enemies. a quadrate in the field measuring 30 x 30 meters (10 plants in 10 rows = 100 plants) was established in a cacao plantation and a whole quadrate survey was carried out in vegetative shoot flushings from the trunk. the number of ants and predators, the identity of ant and natural enemy species and colony occupancy by ants were recorded. spatial association indexes were used to evaluate the degree of overlap in ant-ant and ant-predator spatial distributions. the ant crematogaster victima f. smith was selected for a test on differences in its attack behavior against larvae of the syrphid ocyptamus antiphates (walker) and a species of ladybird beetle (coleoptera: coccinelidae). five species of ants were found tending aphids more frequently and their level of spatial association was slightly negative with remarkable mutual exclusion from aphid colonies. two of them, cr. victima and cr. erecta mayr, were potential defendants of aphids and were selected to study their spatial association with the distribution of natural enemies. it was found that spatial association between ants and aphid predators is slightly positive. the results suggest that the occurrence of attack behavior of cr. victima against syrphids, but not against coccinellids, can increase coexistence of predators by generating independent spatial distribution. sociobiology an international journal on social insects 1 universidade estadual de feira de santana, feira de santana, bahia, brazil. 2 university of michigan, ann arbor, michigan, usa. article info edited by kleber del-claro, brazil received 02 july 2013 initial acceptance 30 july 2013 final acceptance 18 aug 2013 key words arboreal ants, biological control, ant-natural enemies interactions, spatial association, toxoptera aurantii corresponding author evandro n. silva univ. estadual de feira de santana depto. de ciências biológicas av. transnordestina s/n feira de santana, ba 44036-900 brazil e-mail: evandro@uefs.br the spatial distribution of ants in a three-dimensional mosaic has important implications for biological control of insect pests in agroforestry systems (perfecto & castiñeiras, 1998; dejean et al., 2003; philpott & armbrecht, 2006). in africa, dominant arboreal ants have been effective against defoliator insects in cacao and coconut plantations (majer 1976a, b; bigger, 1993). in bahia, brazil, majer & delabie (1993) also found ants to be potential biological control agents of cacao pests. nevertheless, ant mosaics are essentially structured by the interaction of ants with hemipterans that produce honeydew as energy source (floren & lisenmeir, 2000; delabie, 2001; vandermeer et al., 2008) and hence along with effective biological control of other herbivores it is also desirable that hemipterans protected by ants do not cause economic loss. perfecto and castiñeiras (1998) highlight two situations in which this goal can be achieved in cacao plantations. first, when the hemipterans protected by ants are not significant research article ants en silva, i perfecto tending ants mediating coexistence of aphid predators in cacao plants?260 pests of the crop and, second, when hemipterans are pests but a much more noxious pest is effectively controlled by ants attracted by hemipteran honeydew. overall, interactions between ants, hemipterans, other herbivores, other predators and the crop are complex and have to be evaluated, at least qualitatively, in order to develop effective and sustainable pest management programs (perfecto & castiñeiras, 1998). one aspect of these interactions that requires attention both for theoretical comprehension of predator-prey dynamics and applied biological control is the ability of ants to defend tended hemipterans from the attack of predators. it can be hypothesized that ants that tend aphids are prone to defend them from predators and parasitoids and, by doing so, they can also determine spatial distribution of natural enemies and effectiveness of biological control. an empirical example in a natural ecosystem (brazilian savanna – the so called cerrado) showed that spiders, parasitoid wasps and syrphid larvae had a reduction in their density when colonies of the treehopper guayaquila xiphias (membracidae) were tended by ants that attacked these natural enemies (del-claro & oliveira, 2000). another example in support for this hypothesis was found by michaud and browning (1999) whom observed that the imported fire ant solenopsis invicta buren was ubiquitous and the most common species in attendance of the brown citrus aphid toxoptera citricida (kirkaldi) in citrus groves in puerto rico. in this study, the observed frequency of larvae of syrphid flies and coccinellid beetles in colonies of t. citricida was lower than what would be expected by chance, indicating an impact of s. invicta in the distribution of natural enemies of t. citricida. in traditional coffee farms in southern mexico, the mutualism between the ant azteca instabilis f. smith and the green scale coccus viridis (green), offers protection against the adult coccinellid azya orbigera mulsant, thus driving the distribution pattern of c. viridis (vandermeer & perfecto, 2006; vandermeer et al., 2010). toxoptera aurantii (boyer de fonscolombe) is an aphid that develops colonies in patchy and short-lived leaf flushes of cacao. species of predators belonging to the families syrphidae and coccinellidae and the order neuroptera have been recorded in different regions of the world (entwistle, 1972; kauffman 1973; firempong & kumar, 1975; cortez-madrigal, 1996). these records indicate that the guild of t. aurantii predators consists of four to seven species from local to regional scales, respectively. the predators are very effective in reducing aphid populations even in low densities because they attack aphid colonies when aphid density is still low (e. n. silva, unpublished data). furthermore, there is a good synchronization of predator and prey colonization, predators are able to reach optimum prey consumption at higher prey densities and there is a complementary action of the different predator species (firempong & kumar, 1975). this phenomenon of complementary effects was also observed by nahas et al. (2012) regarding herbivory reduction as an effect of multiple predators. up to date, there is no detailed study on the interaction between ants and predators of t. aurantii. our study aimed to investigate the degree of spatial association between ants and natural enemies of t. aurantii in a cacao plantation as well as the attack behavior of the ant cr. victima against two predaceous species. we tested the hypothesis that ant-aphid mutualism determines the spatial association and coexistence of predators of t. aurantii. material and methods spatial association on march of 2006 a survey was carried out in an experimental area of the cacao research center, in ilhéus, bahia, brazil (14º47’45”s 39º11’21”w). a whole quadrate survey was conducted to quantify populations of t. auranti, its predators and mutualistic ants. the quadrate consisted of 100 cacao plants, in an array of 10 rows with 10 consecutive plants (an area of 30 x 30 m). aphids and natural enemies were counted once only in vegetative shoot flushings arising from the tree trunk up to 1.5 m height. all colonies present in the chupons flushing from the trunks were included in the survey. the survey was carried out once. in each observed colony the presence or absence, species identity and number of ants, larvae of hoverflies, ladybird beetles and lacewings were recorded. attack behavior of ants against two predators during a survey of specimens of t. aurantii and its predators for species identification it was noticed that aphid colonies tended by the ants cr. erecta and cr. victima (e. n. silva personal observation) seemed to have less syrphid larvae than colonies not tended by the ants. these two ant species were seen attacking syrphidae larvae after encounters in the colonies. on the other hand, no attack of ants against larvae of coccinellids was observed. a behavioral experiment was set up in the field in order to test for differences in the frequencies of attack and removal of larvae of the syrphidae ocyptamus antiphates (walker) and a unidentified species of ladybird beetle (coccinellidae) by workers of cr. victima. this species was the one chosen for experimentation because it was numerically easier to find them tending aphids, making replication possible in our study. a day before the experiment 20 first instar larvae of o. antiphates were collected in the field and kept in the laboratory in petri dishes with abundant food (nymphs of t. aurantii). thus, under food satiation, they would simply set down and not search for prey when put in the presence of aphids in the behavioral trials. larvae of the coccinellidae species proved to be more sensitive to manipulation and thus another method was used to manipulate them. it consisted of locating sociobiology 60(3): 259-265 (2013) 261 individual larvae established in aphid colonies in trees where workers of cr. victima were foraging. the ants foraging in the branch to which the colony belonged to were assumed to have previous experience with the individual larvae and were brushed away. then, ants from neighbor branches (supposedly without previous experience with individual larvae) were allowed to forage in the colony where the coccinellid larvae were present. a five minute count was started whenever a minimum number of three cr. victima workers were foraging in the cacao leaf harboring aphids and one coccinellid larva. the following behaviors were quantified during the five minute counting: 1) events of attack and/or removal of the larva; 2) number of ants at the beginning of trial; and 3) mean time (in seconds) at which first encounter and attack occurred. after the trial, the coccinelid larva was removed with a needle. then, one larva of o. antiphates was placed in the same leaf and trial procedures were carried out as described before. species identity the identity of o. antiphates (walker) larvae was established by observation of color pattern (transparent body and white dorsal fat bodies) (fig. 1a). the brown lacewing nusalala tessellata gerstaecker has very conspicuous larvae and hence its identity was easily established in the field (fig. 1b). one morphospecies of coccinellidae is still to be identified, but its larva is very conspicuous and, hereafter, it is referred to as coccinellidae specie (fig. 1c). ants were identified in the field up to the generic level and then categorized in morphospecies for observation purposes, following sampling of specimens for identification in the cacao research center. ectatomma tuberculatum (olivier) was the only species directly identified in the field. vouchers of the ant biological material are deposited in the myrmecology laboratory collection in this research center. data analysis the spatial association between paired species (ant-ant and ant-predator) was determined by an index of spatial association (the four point correlation coefficient) according to the following equation (sevenster, 1996): φ = ad-bc/√[(a+b)(c+d)(a+c)(b+d)]; where a, b, c and d are the number of colonies in the four cells of a standard 2 x 2 absence-presence contingency table for the frequencies of two competing predator species; a and d refer to cells containing double absences and double presences, respectively. the index value ranges from -1 to +1, representing respectively high negative and positive spatial association between two species. a linear regression analysis was carried out to test for a significant relationship between the number of ants present in an aphid colony and the time of first attack. a chi-square test was performed to test whether aphid tending frequencies represented an association between the five most frequent tending ant species and t. aurantii. a fisher’s exact test was carried out to test for differences in ant attack behavior against two species of predators using the statistical software spss for windows 13.0. a 0.05 significance level was used for statistical inference in these tests. a b c d figure 1. a) larva of ocyptamus antiphates; b) larva of nusalala tessellata; c) larva of coccinellidae sp; d) larva of ocyptamus antiphates attacked by two workers of the ant c. victima. results ants surveyed there were nine species of ants present in 42 out of 211 colonies of aphids observed in 100 plants. the most frequent species were cr. erecta, cr. victima, e. tuberculatum, dolichoderus bidens l. and pseudomyrmex gracilis (fabricius). they were present in 9, 7, 10, 17 and 13 colonies respectively. a chi-square test (a 5 x 2 contingency table) allowed verifying that these frequencies do not support a hypothesis of association between ant species and t. aurantii different from what would occur by chance (χ2 = 5,73; g.l = 5; p = 0,37). other less frequent species included cephalotes atratus (l.), camponotus fastigatus roger, camponotus (myrmaphaenus) sp. and ectatomma brunneum smith (fig. 2). 0 5 10 15 20 25 30 o. antiphates coccinellidae sp. n. tessellata predator abundance colony occupation figure 2. total number of predators and colony occupation in 211 surveyed colonies of toxoptera aurantii. en silva, i perfecto tending ants mediating coexistence of aphid predators in cacao plants?262 spatial association all association indexes for pairs of ant species showed slightly negative to neutral values (range = -0.07 to 0), except for the pair cr. victima – p. gracilis which had a slightly positive index (table 1). this trend towards negative spatial association is remarkably determined by the fact that all ant species pairs, except cr. victima – p. gracilis, never had overlapped distribution even in a single colony. the spatial distribution of ants was very segregated at the colony level. the majority of the colonies of t. aurantii were not tended by ants. the number of colonies occupied by ants ranged from a maximum of 17 out of 211 for d. bidens to a minimum of 7 out of 211 for cr. erecta (fig. 3). the association indexes for the pairs of species o. antiphates cr. erecta and o. antiphates – cr. victima were slightly positive. coccinellidae sp. as well as n. tesellata had attack behavior of ants against two predators three ants were observed attacking larvae of o. antiphates during the plot survey: ce. atratus (one event), cr. erecta (two events) and cr. victima (two events). the ant ce. atratus was foraging on the cacao leaf infested with aphids when encountered and palpated the syrphid larva with the antennae, then grabbed the larva with the jaw and threw it out of the colony. the whole process from encounter to removal lasted about five seconds. the attack behavior of cr. erecta and cr. victima consists of two phases. they were generally tending aphids and when they reached a part of the colony with a larva they palpated the larva with antennae for some time (from a few to 40 seconds) and then inflicted jaw bites and stings on the larva. most frequently, the larvae were encountered by one ant, but after this ant engaged in the attack other ants came together and the removal effort started. it is appropriate to say that there was a handling stage (after the biting/stinging stage and before removal). the removal stage consisted either of the larva being dropped on the ground or carried out to the ant nest. all three species occurred in less than 10 colonies inside the survey plot but cr. victima was more abundant in other plants outside the survey plot. therefore, cr. victima was selected for a comparative study of attack behavior against syrphidae and coccinellidae larvae. this ant presented a distinct attack behavior when in contact with o. antiphates or coccinellidae sp. the fisher’s exact test showed that the frequency of attack against o. antiphates was higher (9 in 12 trials) than it would be expected by chance (4.5 in 12 trials; n=12; d.f. = 1; p<0.001) while there was never a single event of attack against coccinelidae sp. attacks to syrphid larvae consisted either of jaw bites or stings. regarding events of removal, only syrphid larvae were removed by the ants (3 in 12 trials). nonetheless, based on the fisher’s exact test the frequency of removal events was not significantly different (n=12; d.f. = 1; p=0.21) from what would be expected by chance (2 in 12 trials). the lack of significance occurred because some attacked larvae were successful in defending themselves against the attacks. some syrphid larvae regurgitated a substance over the ants engaged in the attack. after table 1 – indexes of spatial association between ants in 211 colonies of toxoptera aurantii surveyed in a field quadrate in ilhéus, ba, brazil. table 3 – indexes of spatial association between species of predators of toxoptera aurantii in 211 colonies surveyed a field quadrate in ilhéus, ba, brazil. a slightly negative association with cr. erecta and a slightly positive association with cr. victima (table 2). the spatial association index for the pair of species o. antiphates – coccinellidae sp. was -0.05. the other association indexes were slightly positive (table 3). very few aphid colonies had natural enemies present during the survey. the number of colonies with the respective presence of syrphid, ladybird beetle and lacewing larvae were 21, 4 and 7 out of 211. the abundance of predators was also very low (fig. 3). table 2 – indexes of spatial association between arboreal ants and species of predators of toxoptera aurantii in 211 colonies surveyed a field quadrate in ilhéus, ba, brazil. crematogaster erecta cr. victima ocyptamus antiphates 0.11 0.09 coccinellidae sp. -0.03 0.14 nusalala tessellata -0.02 0.09 figure 3. colony occupation by ants in 211 surveyed colonies of toxoptera aurantii. cr. erecta cr. victima e. tuberculatum d. bidens crematogaster erecta crematogaster victima -0.04 ectatomma tuberculatum -0.04 -0.05 dolichoderus bidens -0.05 -0.06 -0.07 pseudomyrmex gracilis -0.05 0.14 -0.06 0.0 o. antiphates coccinellida e sp. ocyptamus antiphates coccinellida e sp. -0.05 nusalala tessellata 0.10 0.18 sociobiology 60(3): 259-265 (2013) 263 the substance was in contact with the ants’ tegument they presented a shaking/trembling behavior for a few seconds and then disengaged and failed to proceed towards the removal phase of the attack. the mean number (±se) of ants present in the aphid colony during the 300 seconds trial was 6.1±1.70 (n=9). the mean time (±se) for first attack on a syrphid larva by ants was 121.9±28.02 seconds (n=9). a linear regression analysis did not show a significant relationship between the number of ants present in an aphid colony during the trial and the time of first attack (r2=0.05; f=0.36; p=0.56). the increase in the number of foraging ants does not increase the probability of a faster attack against syrphid larvae. discussion although ant-hemipteran mutualisms are prevalent in nature, we were able to find only one other study about ants protecting aphids against predators in cacao agroecosystems in bahia. silva (1944) mentioned that the ants azteca chartifex forel, d. bidens and solenopsis geminata medusa mann. tended and protected t. aurantii against natural enemies. in our study three new ant species that protect aphids against predators are recorded. we also provide details on the defensive behavior of cr. victima benefiting from aphids in cacao. it is very conspicuous that the attack of cr. victima is frequent against o. anthiphates larvae, but not at all against coccinellids. one possible mechanism causing this difference in the attack behavior of cr. victima against one predator but not against the other is chemical mimicry of aphids by their predators. lohman et al. (2006) studied profiles of cuticular hydrocarbons (chcs) from the north american woolly alder aphid, prociphilus tessellatus (hemiptera: aphididae), their tending ants, and three species of aphid predators: feniseca tarquinius (lepidoptera: lycaenidae), chrysopa slossonae (neuroptera: chrysopidae) and syrphus ribesii (diptera: syrphidae). the results showed that while the chc profile of each predatory species was distinct, each was chemically more similar to the aphids than to tending ant species. furthermore, the chcs of each predator species were a subset of the compounds found in the aphids’ profile. therefore, by producing or acquiring chc compounds similar to those of aphids the predator coccinelidae sp. could be mimicking aphids and circumventing ant recognition and aggressiveness. in order to support this hypothesis it is necessary to determine if this coccinelid is a myrmecophile species, since this degree of ant-aphid predator interaction is quite rare. according to slipinski (2007) cited by godeau et al. (2009) among the 6,000 species of ladybird beetles, seven are considered to be true myrmecophiles and four are suspected to at least live in association with ants. in a previous study on the ant mosaic in the canopy of cacao trees carried out in the same locality where this research was conducted, five species of ants were considered dominants: azteca instabilis, a. chartifex, e. tuberculatum, wasmannia auropunctata (roger) and cr. erecta (majer et al., 1994). one of the characteristics they found to determine the ant mosaic was that dominant ants exhibit at least one negative association with another dominant ant and that the 10 sub-dominant ants also exhibit negative association with at least one dominant ant. there was also a tendency to symmetrical exclusion based on ant size with small a. chartifex and cr. erecta excluding small ants from their territory and large e. tuberculatum excluding large ants. the results obtained in this study are in accordance with those cited above. the quadrate survey conducted in this study found that two of the species (cr. erecta and e. tuberculatum) considered dominants by majer et al. (1994) were present on aphid colonies. in general, nine out of 10 association indexes for pairs of the five ants considered more frequent were slightly negative. only one index was slightly positive, reflecting the only case in which double presence of ants was observed (cr. victima and p. gracilis) in the survey. p. gracilis uses to forage on aphid colonies as individuals and is sometimes attacked by cr. victima. however, p. gracilis is a large size ant (ca. 14 mm) while cr. victima is a small size ant (ca. 5 mm). perhaps this asymmetry in size is more favorable to p. gracilis and might be the cause of the common presence of both species in some colonies. therefore, negative association and body size might be better studied as possible components determining the distribution of ants in aphid colonies. whenever o. antiphates larvae were present, ant-predator association indexes were slightly positive. in the case of n. tesellata and coccinellidae sp. association indexes were negative against cr. erecta but positive against cr. victima. positive indexes for ant-predator spatial distribution should be expected, given the defense capability of syrphid larvae and the absence of attack from ants against coccinellidae sp. how different the indexes can be depends on how much syrphid larvae can escape ants’ attack and on the abundance of coccinellidae larvae. in this study, the spatial association with coccinellidae sp. was slightly positive and did not differ too much from indexes related to o. antiphates. the fact that some syrphid larvae are removed and coccinellid larvae are not, has certainly an effect in the distribution and coexistence of both species. however, the frequency of ants and predators in aphid colonies were too low to draw more emphatic conclusions in this study based on association indexes. nonetheless, conceptually, we can conclude that the ant-aphid mutualism can mediate the coexistence of these two predators (and others) because the removal of o. antiphates larvae by cr. victima might generate a habitat fraction exclusively occupied by coccinellids, a “competitor free-space”. in the future, carefully planned and well manipulated field and laboratory experiments would prove valuable in answering interesting questions based on this conceptual framework. the ants ce. atratus, cr. victima and cr. erecta vary en silva, i perfecto tending ants mediating coexistence of aphid predators in cacao plants?264 in size (large, medium, small, respectively) and so does o. antiphates along its ontogenetic development (first to third instar larvae are small, medium and large sized). it would be interesting to test whether the efficiency of ant species in the removal of syrphids and the successful self-defense of syrphids against them depend on the asymmetry of ant-predator size, ant behavior, aggressiveness and agility. these factors were found to be important in mediating ant-hemiptera interactions in a tropical savanna tree (del-claro & oliveira, 2000). another research approach that deserves attention is the chemical mimicry in the guild of predators of t. auranti and its relation with spatial overlap. finding answers to these questions will certainly shed light on syrphid-coccinellid coexistence in cacao plants mediated by ants. acknowledgements we thank dr. kazuyiuky nakayama, gilmar costa, gidevaldo gonçalves and josé raimundo maia dos santos in the section of entomology, cacao research center/ceplac, brazil, for logistical support with field and laboratory work. we thank dr. luciane marinoni and dr. sergio de freitas (in memoriam) for the identification of syrphidae and neuroptera species, respectively. dr. jacques h. c. delabie gently identified ant species and reviewed a draft of this manuscript. we also thank two anonymous referees for helpful remarks in an early version of this manuscript. this work was made possible by a doctoral scholarship granted to e. n. silva by capes foundation-ministry of education (bex 1609-00-9), brazil, and grants from the rackham school of graduate studies, the university of michigan, usa. references adis, j., lubin, y.d. & montgomery, g.g. 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(2010). ecological complexity and pest control in organic coffee production: uncovering an autonomous ecosystem service. bioscience, 60: 527-537. doi: 10.1525/bio.2010.60.7.8 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5783sociobiology 68(3): e5783 (september, 2021) introduction for the brazilian territory, 1.678 known species of bees belonging to 207 genera are documented (moure et al., 2007), and together of the different regions of the world, the number of species is estimated at 25 to 30 thousand, divided into 4.000 genera responsible for the pollination of, approximately, 60% of higher plants (michener, 2007). in the state of maranhão, brazil, there are about 230 species of bees recognized in biological collections (rêgo & albuquerque, 2012; ferreira et al., 2020) and many of these records occur on the coast. a massive number of works on the apifauna of the state of maranhão were carried out, exploring different environments such as restinga (silva et al., 2009; gostinski et al., 2016), brazilian savanna (albuquerque & mendonça, 1996; rebêlo & cabral, 1997; mendes et al., 2008), dunes (gottsberger & silberbauer-gottsberger, 1988; abstract surveys of the bee fauna on islands are scarce due to the difficult access to the study area. thus, the current study intended to establish the species of bees present in an island of the delta of the americas, called grande do paulino, tutóia, maranhão. together with the bees, the plants visited by these insects were recorded, in order to document the relationships between these organisms. between july 2017 and june 2018, once a month, 1,095 individuals, distributed in 16 tribes, 30 genera, and 48 species, were collected with active (entomological net) and passive (bowl traps) sampling methods. data from plants and their visiting bees are presented in an interaction network in the form of a bipartite graph, showing xylocopa cearensis as the most collected bee species, and chamaecrista ramosa as the most visited plant by bees. in addition to providing information about the bee fauna of the state of maranhão and, consequently, from the brazilian northeast, this study explores the apifauna of a place never before explored and, because it is an island, of difficult access, also providing information about the floristic interactions of these insects. sociobiology an international journal on social insects carlos l neves jr1, harryson c barros2, maira r diniz2, bruna ef correia2, luciano ac ferreira2, albeane g silva-almeida2, eduardo b almeida jr.2, márcia mc rêgo2 article history edited by antonio jc aguiar, unb, brazil received 24 august 2020 initial acceptance 25 january 2021 final acceptance 27 july 2021 publication date 13 august 2021 keywords restinga, floral visitors, floral resources, apifauna, species checklist. corresponding author carlos luis neves jr. federal university of viçosa (ufv) av. peter henry rolfs, s/nº cep: 36570-900 viçosa-mg, brasil. e-mail: carlos.njr@outlook.com albuquerque et al., 2007; serra et al., 2009; oliveira et al., 2010), amazon (silva & rebêlo, 1999; ferreira et al., 2019), as well as secondary vegetation regions (gonçalves et al., 1996; albuquerque et al., 2001; brito & rêgo, 2001) and islands (silva & rebêlo, 2002). although the bee fauna is well represented in the state, some places do not have information about the species. in the brazilian coast, few islands have data on bee fauna (zanella et al., 1998) due to their distance from the continent and/or the researchers’ difficult access. we can mention as an example grande do paulino island, the study area of this work, located on the coast of state of maranhão. one of the fundamental factors for the survival of bees is the plants, which provide resources for these insects and, consequently, are pollinated (michener, 2007). bees and plants have coevolved since the cretaceous to form a beneficial relationship that lasts to the present day (grimaldi, 1999). 1 federal university of viçosa, viçosa-mg, brazil 2 federal university of maranhão, são luis-ma, brazil research article bees bees from an island in the delta of the americas (maranhão state, brazil) and their floristic interactions mailto:carlos.njr@outlook.com carlos l neves jr et al. – bees from grande do paulino island (brazil, maranhão)2 parallel to these factors, we have the historical records of the disappearance and loss of several bee colonies, and the causes of this great episode are still discussed. some say they are motivated by parasitic interactions with mites (eliash & mikheyev, 2020) and climate changes (lima & marchioro, 2021; raven & wagner, 2021), but the most accepted reason today is the use of toxic substances in crops (almasri et al., 2020; faita et al., 2020). thus, we find ourselves in a situation of aggravating loss of the diversity of bees, and we do not even know many of the species in the national territory. although research on the diversity of brazilian bee fauna has occurred frequently in recent years (viana & lourenço, 2020; almeida et al., 2019), the vastness of the brazilian territory prevents all areas from being covered with this type of work, thus leaving some regions lacking such information, mainly restricted access regions, such as islands off the brazilian coast, for example. thus, the current study intended to establish the species of bees and their floristic interactions in an island of the delta of the americas, called grande do paulino, tutóia, maranhão, an area devoid of apifaunistic sampling. with the information obtained, we can contribute to the removal of wallacean (species distribution data), prestonian (abundance data) and eltonian (interactions data) biodiversity shortfalls (hortal et al., 2015). material and methods sampling area this study was conducted on grande do paulino island (42°11’23.4”s, 02°43’58.2”w,), which is 40.5 km2 and is located in the parnaíba delta, also known as the delta of the americas. this island belongs to the municipality of tutóia, state of maranhão, and is 2 km from the continental coast. the vegetation of the island is predominantly characterized by the presence of mangrove and restinga ecosystems, the latter characterized by the coastal location and the presence of undergrowth, shrub and tree vegetation (marques et al., 2015). according to the köppen-geiger classification, the climate of the locality is type aw, with two well-defined seasons: a rainy season from january to june, and a drought from july to december (alvares et al., 2013). the area used for the collection has about 4.940m2 (42°11’23.929”s, 2°44’0.877”w; 42°11’12.570”s, 2°43’57.112”w) and consists of an open restinga composed of a sandy field with creeping plants and shrubs, with the presence of flooded environments. temperature and humidity data were obtained on the collection days using a thermo-hygrometer and rainfall information was obtained by consulting the inmet digital platform database. sampling of the bees the collections were performed between july 2017 and june 2018, once a month, from 6h to 18h, by two collectors, totaling 288 hours of sampling. bees were collected using an entomological net to capture the bees while visiting the flowers, when at rest, or in flight (sakagami et al., 1967). the insects collected by entomological net were placed in paper bags with identification of the plant on which the bee was collected. simultaneously, plastic bowl traps (moerike traps, 15 cm wide, 5 cm high, 300 ml of water and drops of dishwashing detergent) were used (portman et al., 2020). bowls had different non-fluorescent colors (blue, green, red, white and yellow) and were deployed monthly for 24 h (total effort: 1.440 h). the bowls were placed on the ground (five groups with one bowl from each color) at 5 m intervals with interspersed colors. the collected insects were preserved and stored in 70% alcohol (krug & alves-dos-santos, 2008). all bees were mounted on pins and deposited in the bee collection of the laboratory of bee studies (leacol/ universidade federal do maranhão – ufma). they were identified to the morphospecies and species level by comparison with the reference collection, the assistance of taxonomists and the aid of taxonomic keys (silveira et al., 2002). michener’s classification scheme was used to identify bees (michener, 2007). sampling of melittophilous flora the plants that received visits from the inventoried bees were collected with the help of pruning shears and placed in a plastic bag for later herborization according to the usual techniques of peixoto and maia (2013). identification was performed with the aid of analytical keys and comparison with exsiccates from maranhão herbarium (mar), department of biology, federal university of maranhão, in which all the collected material was processed and deposited. descriptive community analysis the shannon-wiener index (h’) was applied to calculate diversity using past software version 3.13 (hammer et al., 2016). the abundance distribution for each species was analyzed using the whittaker diagram (rank/abundance). determination of the sample sufficiency was evaluated through the creation of the species accumulation curve with randomized samples. additionally, using estimates version 9.10 software (colwell, 2013), three non-parametric estimators (ace, jackknife, and bootstrap) were used to infer an approximation of the real richness of the community. the use of these estimators is justified by their complementarity, in which they relate the parameters: rarity, abundance and total wealth, respectively for the construction of the species accumulation curve, bees collected in both sampling methods were counted. occurrence frequency (of = number of samples with species i / number of samples x 100) and dominance (dm = abundance of species i / total abundance x 100) classes were calculated for bees collected with entomological net. if of ≥ 50%, then is considered a very frequent species (vf); of < sociobiology 68(3): e5783 (september, 2021) 3 50% and > 25% is considered frequent (f); and of ≤ 25% is considered infrequent (if). if dm > 5% = dominant species (d); dm ≤ 5% and > 2.5% = accessory species (a) and dm ≤ 2.5% = occasional species (oc). the of + dm combination divides the species into three groups: common species (vf or f + d), rare species (if + oc), and intermediate species (other combinations) (aguiar & gaglianone, 2012). interaction networks a bipartite weighted interaction network of bees and visited plants was created using r software (the r project for statistical computing), version 3.5.1, with the bipartite package (dormann et al., 2018). a bipartite network displays members of a trophic level connected to members from another trophic level (e.g., flowers and bees) (pigozzo & viana, 2010). among the available metrics for the description of a qualitative interaction network, the metrics were used to calculate the connectivity, the average degree for plants and animals. connectance (c), which measures the proportion of connections that are actually observed, is the ratio between the number of observed interactions (e) and the number of possible interactions, which in turn is given by the product of the number of plants (p) and animals (a) from the network: c = e/a.p (pigozzo & viana, 2010). for percentage values, the value of c was multiplied by 100. the average degree of plants and animals was obtained from the arithmetic mean of the degrees of all plant/animal species, the degree being the number of interactions in which each species was involved. (pigozzo & viana, 2010). the average degree of plants, corresponding to the number of interactions, was obtained from the arithmetic mean of all interactions performed by botanical species in relation to bees. the same principle was used to establish the average degree of animals. the distribution of the degree was performed graphically, in a vertical bar representation, in which the number of established interactions (degree) is represented on the x axis, while the y axis represents the number of species that presented a certain degree, either of plants or bees (pigozzo & viana, 2010). results species richness and diversity a total of 1,095 individuals were collected, distributed in 16 tribes, 30 genera, and 48 species, belonging to the andrenidae, apidae, colletidae, halictidae, and megachilidae families. specifically, with the entomological net, 1,089 individuals were collected (table 1), while in the bowl traps, only six bee specimens were collected (five bees from the blue bowls and one from the yellow bowls. the other bowls colors did not have the presence of bees) (table 2). table 1. families, tribes, and bee species collected in grande do paulino island, using entomological net. n = relative abundance; dm = dominance (d = dominant; a = accessory; oc = occasional); fo = frequency of occurrence (vf = very frequent; f = frequent; if = infrequent); cl = classification (c = common; i = intermediate; r = rare). family/tribe/species or morphospecies abbrev. specimen n (%) dm fo cl andrenidae protomeliturgini protomeliturga turnerae (ducke, 1907) pt 1 0,09% oc if r apidae apini apis mellifera (linnaeus, 1758) am 47 4,32% a f i centridini centris (xanthemisia) bicolor (lepeletier 1841) cb 1 0,09% oc if r centris (centris) byrsonimae (mahlmann & oliveira, 2012) cby 2 0,18% oc if r centris (centris) caxiensis (ducke, 1907) cc 194 17,81% d vf c centris (centris) decolorata (lepeletier, 1841) cd 55 5,05% d vf c centris (centris) flavifrons (fabricius, 1775) cf 3 0,28% oc if r centris (paracentris) hyptidis (ducke, 1908) ch 1 0,09% oc if r centris (melacentris) obsoleta (lepeletier, 1841) co 41 3,76% a f i centris (hemisiella) tarsata (smith, 1874) ct 3 0,28% oc if r centris (hemisiella) trigonoides (lepeletier, 1841) ct 1 0,09% oc if r epicharis (epicharis) bicolor (smith, 1854) eb 2 0,18% oc if r epicharis (epicharis) sp. ep 11 1,01% oc f i emphorini melitoma segmentaria (fabricius, 1804) ms 11 1,01% oc f i carlos l neves jr et al. – bees from grande do paulino island (brazil, maranhão)4 ericrocidini mesonychium asteria (smith, 1854) ma 31 2,85% a vf i eurytis funereus (smith, 1854) ef 6 0,55% oc if r eucerini florilegus sp. fl 13 1,19% oc if r euglossini eulaema (apeulaema) nigrita (lepeletier, 1841) em 1 0,09% oc if r meliponini frieseomelitta doederleini (friese, 1900) fd 12 1,10% oc f i melipona (melipona) subnitida (ducke, 1910) msu 63 5,79% d vf c tapinotaspidini paratetrapedia duckei (friese, 1910) pd 1 0,09% oc if r paratetrapedia sp. pa 1 0,09% oc if r xylocopini ceratina (crewella) pubescens (smith, 1879) cp 1 0,09% oc if r ceratina (crewella) rotundiceps (smith, 1879) cr 20 1,84% oc f i ceratina (crewella) maculifrons (smith, 1854) cm 13 1,19% oc f i ceratina (crewella) cf. asunciana (strand, 1910) ca 14 1,29% oc if r ceratina sp1 ce1 5 0,46% oc if r ceratina sp2 ce2 1 0,09% oc if r xylocopa (neoxylocopa) aurulenta (fabricius, 1804) xa 4 0,37% oc if r xylocopa (neoxylocopa) cearensis (ducke, 1910) xc 407 37,37% d vf c xylocopa (neoxylocopa) frontalis (olivier, 1789) xf 2 0,18% oc if r xylocopa (schonnherria) muscaria (fabricius, 1775) xm 1 0,09% oc if r colletidae hylaeini hylaeus sp. hy 1 0,09% oc if r diphaglossini ptiloglossa sp. pti 24 2,2% oc f i halictidae augochlorini augochloropsis aff. vivax (smith, 1879) av 10 0,92% oc if r augochlorini sp1 au1 1 0,09% oc if r augochlorini sp2 au2 1 0,09% oc if r augochlorini sp3 au3 2 0,18% oc if r halictini dialictus sp. di 1 0,09% oc if r megachilidae anthidiini dicranthidium arenarium (ducke 1907) da 47 4,32% a f i epanthidium tigrinum (schrottky, 1905) et 18 1,65% oc vf i megachilini coelioxys sp. coe 2 0,18% oc pf r hypanthidium maranhense (urban, 1998) hm 4 0,37% oc f i megachile sp1 me1 5 0,46% oc f i megachile sp2 me2 3 0,28% oc if r megachilini sp1 meg 1 0,09% oc if r total 1089 table 1. families, tribes, and bee species collected in grande do paulino island, using entomological net. (continuation) family/tribe/species or morphospecies abbrev. specimen n (%) dm fo cl sociobiology 68(3): e5783 (september, 2021) 5 the apidae family presented the highest abundance, with 88.58% of the collected specimens (970 individuals), of which xylocopa (neoxylocopa) cearensis (ducke, 1910) was the most abundant species, with 407 collected individuals, representing 37.17% of the abundance. the next most abundant family was megachilidae (7.4%, 81 individuals), followed by colletidae (2.28%, 25 individuals), halictidae (1.64%, 18 individuals), and andrenidae (0.09%, 1 individual). the highest abundance of apidae is mainly due to bees of the genera xylocopa (414 individuals) and centris (301 individuals), representing, respectively, 37.81% and 27.49% of the sampled specimens. xylocopini and centridini were the most abundant tribes, with 42.92% (470 individuals) and 28.68% (314 individuals). apidae was the family with the greatest richness, comprising 30 sampled species (62.5%), followed by halictidae (8 species, 16.67%), megachilidae (7 species, 14.58%), colletidae (2 species, 4.17%), and andrenidae (1 species, 2.08%). family/tribe/species or morphospecies bowl trap color specimen apidae xylocopini ceratina (crewella) maculifrons (smith, 1854) blue 1 ceratina (crewella) pubescens (smith, 1879) blue 1 halictidae augochlorini augochlorella tredecim (vachal, 1911) blue 1 halictini halictini sp1 blue 1 halictini sp2 blue 1 megachilidae anthidiini dicranthidium arenarium (ducke 1907) yellow 1 table 2. families, tribes, and bee species collected in grande do paulino island using bowl traps. fig 1. (a) temperature and number of individuals collected per hour in the study area and (b) seasonal distribution (specimens per month). activity pattern during all the months of collection, bees were captured with entomological net, while in the bowl traps, only in the months of july, november, december (2017) and april (2018): 551 specimens were collected in the dry period and 544 were collected in the rainy season. the month of february showed a peak of abundance (fig 1b), with 214 bees collected (19.54% of the entire sample). the morning period demonstrated higher activity of individuals (fig 1a), between 6:00 and 10:00. the occurrence of bees gradually decreased during the day, increasing again between 16:00 and 18:00 (fig 1a). carlos l neves jr et al. – bees from grande do paulino island (brazil, maranhão)6 regarding the frequency of occurrence, six species (12.24%) were considered very frequent (vf), 11 (22.45%) frequent (f), and 32 (65.31%) infrequent (if). centris (centris) caxiensis (ducke, 1907), c. (centris) decolorata (lepeletier, 1841), melipona (melipona) subnitida (ducke, 1910), and xylocopa (neoxylocopa) cearensis (ducke, 1910) were considered dominant (d) species (8.16%), four species (8.16%) accessory (a), and 41 (83.67%) occasional (oc). four species were considered common (8.16%), 13 intermediate (26.53%), and 32 rare (65.31%) (table 1). few very abundant species and many species considered not abundant were represented in the rank/ abundance graph (fig 2), demonstrating an unevenness of the bee community collected in grande do paulino island. the shannon-wiener diversity index (h’) presented a value of 2.4. the species accumulation curve together with the ace, jackknife, and bootstrap richness estimators are shown in figure 3. for ace, the samples correspond to 62,26% of the total richness of the location, while for jackknife and bootstrap, the values were, respectively, 70,41% and 84,45%. interactions network of the 1,089 bees sampled from the active search, 984 were collected during flower visits, while 105 were collected in flight and were not included in the construction and calculation of the interaction network metrics. bees were collected from 19 plant species, corresponding to 14 botanical families (tab 3). the most commonly visited plants were chamaecrista ramosa (vogel) h.s irwin & barneby (357 bees), byrsonima crassifolia (l.) kunth (144 bees), borreria verticillata (l.) g.mey. (141 bees), and euploca polyphylla (lehm.) j.i.m. melo & semir (133 bees). of the 14 families, only three were visited by one species, while the others interacted with four or more bee species. during the dry season, eight plant species were visited by bees, in contrast to 16 botanical species visited in the rainy season. the interaction network presented in the study area (fig 4) consisted of 44 bee species and 19 plant species, theoretically resulting in 836 possible interactions. however, only 118 interactions were observed between mellitophilous fauna and associated flora, equivalent to 14.11% of possible interactions. about 50% (59) interactions observed were concentrated in only eight bee species, which together made up 18.18% of all apifauna present in the network: xylocopa (neoxylocopa) cearensis (ducke, 1910) (12 interactions; 10.17%), centris (centris) caxiensis (ducke, 1907) (9; 7.63%), apis mellifera (linnaeus, 1758) (8; 6.78%), dicranthidium arenarium (ducke 1907) (7; 5.93%), epanthidium tigrinum schrottky, 1905 (7; 5.93%), centris (centris) decolorata (lepeletier, 1841) (6; 5.08%), centris (melacentris) obsoleta (lepeletier, 1841) (5; 4.24%), and melipona (melipona) subnitia (ducke, 1910) (5; 4.24%). fig 2. rank/abundance graph of bee species collected on grande do paulino island. fig 3. species accumulation curve with richness estimators. sociobiology 68(3): e5783 (september, 2021) 7 table 3. botanical families and species with their respective visiting bee species in the study area on grande do paulino island, tutóia, ma. n = nectar; p = pollen; o = oil. abbreviations for visiting bee species are presented in table 1. family/species abbrev. visiting bee species visits. (n) aizoaceae sesuvium portulacastrum (l.) l. sp am (2), av (9), fd (5), msu (1) 17 amarantaceae alternanthera brasiliana (l.) kuntze ab au3 (1), da (9), di (1), et (1), hm (2) 14 anacardiaceae anacardium occidentale l. ao cb (1), cc (12), cd (2), ct (1), co (1), ep (1), et (1), msu (2), xc (10) 31 boraginaceae euploca polyphylla (lehm.) j.i.m. melo & semir epo am (3), cc (11), cd (1), cm (1), cr (2), da (5), et (3), fl (7), msu (2), pt (1), xa (1), xc (96) 133 convolvulaceae ipomoea maurandioides meisn. im am (1), ce1 (5), cm (11), cp (1), en (1), ms (8), xc (7) 34 jacquemontia tamnifolia (l.) griseb. jt au1 (1), au2 (1), cc (3), fl (1), me2 (1), xc (1) 8 euphorbiaceae jatropha mollissima (pohl) baill. jm am (1) 1 fabaceae ancistrotropis peduncularis (fawc. & rendle) a. delgado ap xc (10) 10 andira surinamensis (bondt) splitg. ex pulle as xc (1) 1 canavalia sp. can xf (1) 1 chamaecrista ramosa (vogel) h.s irwin & barneby cra am (4), au3 (1), av (1), cby (1), cc (35), cd (23), ch (1), cm (1), co (26), ct (2), da (4), ef (3), et (3), fl (3), hm (1), ma (1), msu (36), pti (24), xc (187) 357 zornia reticulata sm. zr da (5), et (3), fl(1), hm (1), me1 (1) 11 loganiaceae spigelia anthelmia l. sa xa(1) 1 malpighiaceae byrsonima crassifolia (l.) kunth bc cc (91), cd (25), cf (3), co (9), eb (2), ep (8), fd (2), pa (1), pd (1), xc (2) 144 ochnaceae ouratea hexasperma (a.st.-hil.) baill oh cc (2), cd (2), msu (3), xc (7), xm (1) 15 rubiaceae borreria verticillata (l.) g. mey. bv am (2), cc(30), cd (2), co (2), cr (1), da (19), et (3), ep (1), hy (1), me1 (3), me2 (1), msu (8), xa (2), xc (66) 141 mitracarpus strigosus (thunb.) p.l.r. moraes, de smedt & hjertson mst am(3), cby(1), cc(1), da (1), ma(1) 7 sapotaceae manilkara triflora (allemão) monach. man xc(1) 1 turneraceae turnera melochioides cambess. tm am (1), ca (14), cc (4), ce2 (1), cr (17), co (2), da (2), ef (2), ep (1), et (2), ma (2), meg (1), xc(8) 57 carlos l neves jr et al. – bees from grande do paulino island (brazil, maranhão)8 about plant species, 68 interactions (57.63%) were concentrated in five species, which together corresponded to 26.32% of the entire mellitophilous flora of the network: chamaecrista ramosa (vogel) h.s irwin and barneby (19; 16.1%), borreria verticillata (l.) g.mey. (14; 11.86%), turnera melochioides cambess. (13; 11.02%), euploca polyphylla (lehm.) j.i.m. melo & semir (12; 10.17%), and byrsonima crassifolia (l.) kunth (10; 8.47%). the degree of bees ranged from 1 to 12 plant species (fig 5a), with the average bee community degree being 2.68. it is noteworthy that 15 bee species (34.09%) visited more plants than the average, and 22 species (50%) depended on or preferred only one plant species. for the plants, the degree ranged from 1 to 19, with a mean degree of 6.21 (fig 5b). seven plant species (36.84%) received above average visits, and six species (31.58%) received only one bee species as visitors. fig 4. bipartite interaction network: interactions between bees (left) and plants (right) of grande do paulino island. the vectors represent the interactions between these species. the thickness of the vectors is proportional to the relative abundance of visiting bees. abbreviations of species names are given in tables 1 and 2. fig 5. degrees of bee species (a) and plant species (b) involved in the interactions of grande do paulino island. discussion in this pioneering survey on grande do paulino island, 48 species of bees were collected with entomological net and trap dishes, three of which were new records for the state of maranhão. the data collected on grande do paulino island contribute to the removal of wallacean (species distribution data), prestonian (abundance data) and eltonian (interactions data) shortfalls (hortal et al., 2015) present in the state of maranhão and, consequently, in brazil. sociobiology 68(3): e5783 (september, 2021) 9 the apidae family presented the highest abundance in this study. this was expected, as commonly documented in apicultural surveys in different regions and/or ecosystems of the brazilian territory, such as dunes (oliveira et al., 2010; gostinski et al., 2016), restinga (madeira-da-silva & martins 2003; kamke et al., 2011), caatinga (lopes et al., 2010), the amazon (albuquerque et al., 2001), and atlantic forest (mouga et al., 2015, somavilla et al., 2018). the genera xylocopa and centris were represented in most of the months of collection, probably due to the availability of floral resources and nesting sites (schlindwein et al., 2003; aguiar & gaglianone, 2003). bees of the genus xylocopa have a cosmopolitan distribution, presenting greater diversity in the tropical and subtropical regions of the new and old world (gerling et al., 1989). these bees are described by viana and alves-dos-santos (2002) as abundant in open environments of dunes and beaches of northeastern brazil. viana and alves-dos-santos (2002) presented similar results to the present study regarding the abundance of xylocopa cearensis, in a survey carried out in a coastal dune area in abaeté, ba, where the authors obtained a total of 42.7% of the total collected individuals. in the same work, the species is cited as documented in the states of maranhão, paraíba, and bahia. it is noteworthy that x. cearensis, together with centris caxiensis, was a dominant and constant species in the restinga area of the lençóis maranhenses national park (gostinski et al., 2016), information also observed in other restinga areas of the brazilian northeast (viana & kleinert, 2006; oliveira et al., 2010), in agreement with our results. among the sources of floral resources for x. cearensis, the species chamaecrista ramosa, abundant in our study area, is also reported by viana et al. (2002) as the most commonly visited plant, with the same authors interpret the vibrating behavior of poricidal anthers as evidence of specialization between these organisms. this botanical species is recognized as an important pollen source for bee fauna in tropical environments (gottsberger & silberbauer-gottsberger, 1988). bees of the centridini tribe visit flowers to obtain oil, pollen, nectar, essential products for their maintenance and reproductive activity (aguiar et al., 2017; santos et al., 2020). studies point to the essential role of these bees as pollinating agents of various neotropical species (gottsberger & silberbauer-gottsberger, 1988), which include oil producing plants (rêgo & albuquerque, 1989), such as byrsonima crassifolia, which attracts a large number of bees to its inflorescences, the centridini tribe being the main pollinators (mendes et al., 2011). this species has a dependency link with its visitors, since its self-incompatibility was reported by rêgo and albuquerque (1989). to meet their energy needs and to collect material for nest building and provisioning, bees of the centridini tribe visit many flowers during their flight. apparently, in the collection area, b. crassifolia was the only source of floral lipids for these bees. the species of the centridini tribe represented in the current study, c. caxiensis, c. byrsonimae, c. flavifrons, c. decolorata, c. trigonoides, c. tarsata, c. obsoleta, c. bicolor, and epicharis bicolor, were also documented in the survey by gostinski et al. (2016) in a restinga area of the lençóis maranhenses national park. rêgo and albuquerque (2012) point to bees of the centridini tribe, along with the tetrapediini and tapinotaspidini tribes, as subsamples in the state of maranhão. the occurrence of c. hyptidis was documented for the first time at this study site (neves jr et al., 2020). ceratina pubescens and cer. rotundiceps are recorded for the first time in maranhão territory in the present study. cer. pubescens had been described only for the states of amazonas and pará; cer. rotundiceps was considered to occur only in the state of pará (silveira., et al., 2002). contrary to what previous studies point out (krug & alves-dos-santos, 2008, gostinski et al., 2016), few bees were collected in the bowl traps due here to use of non-uv colors. the low occurrence of bees in the trap dishes may be due also to the proximity of the flower dishes from the place, causing a loss of attractiveness to the bees, even though there is still no evidence to show this correlation (portman et al., 2020). this methodology tends to be selective for certain groups and should not be used in isolation in inventories (gostinski et al., 2016). although this method still has a low incidence of use in brazilian surveys, it is included in many of the traditional techniques of sampling hymenoptera fauna in north america and europe (pinheiro-machado & silveira 2006). for this method, usually the colors used are yellow, blue and white. here in this study we decided to include the colors green and red to test the possibility of attraction to bees. however, only the yellow and blue colors were attractive. still, three species were collected exclusively using this method. like the others caught in bowl traps, these species are small and hardly seen by the collector in active collection. bowl trap sampling can be useful when testing the sampling effort of collections, as no sampling bias by the collector is demonstrated and the results will not be influenced by the ease and/or difficulty of catching some species (gostinski et al., 2016). in this way, passive collection methods help standardize differences in catch rates between species. the bees demonstrated higher activity in the morning, between 6:00 and 10:00. it is possible to relate the greater activity observed during the morning period due to the supply of pollen and nectar at that time, limited resources that, once collected from a flower, are only offered again the other day (kuppler et al., 2021). the shannon-wiener diversity index found (h’= 2.4) was similar to those found in the dune and restinga areas of intermares beach, cabedelo, pb (h’ = 2.45; madeira-dasilva & martins, 2003) and lençóis maranhenses national park, barreirinhas, ma (h’= 2.41; gostinski et al., 2016) and it was higher than the indices obtained in dune areas of panaquatira beach, são josé de ribamar, ma (h ‘= 2.28; oliveira et al., 2010), são marcos beach, são luís, ma (h’ = 2.05; albuquerque et al., 2007), and abaeté beach, ba (h’= 1.99; viana & kleinert, 2006). carlos l neves jr et al. – bees from grande do paulino island (brazil, maranhão)10 the species accumulation curve together with the ace, jacknife, and bootstrap richness estimators demonstrate that there was a sample insufficiency, showing no stabilization after the twelve months of collection. this indicates that in the collection area there are probably still unregistered species. the ace, jacknife, and bootstrap richness estimators suggested that between 70% and 84% of the collection site bee fauna was effectively sampled, however, there was no stabilization of the curves of these estimators, indicating that this percentage may be lower. pigozzo and viana (2010) expect that, mathematically, the richness of species involved in interaction networks and the number of possible interactions themselves would be directly proportional. however, olesen et al. (2006) point to an inverse trend, where a small part of the possible interactions take place, a trend corroborated by the present study, in which, out of 779 possible interactions, only 115 were established (14.76%). the connectivity value found for the grande do paulino island interaction network (14.11%) was higher than that calculated for other networks between bees and flowers in open areas, such as restinga environments (viana & kleinert, 2006 – 13.9% of 1,044 possible interactions; pigozzo & viana, 2010 – 10.6% of 2,800 possible interactions) and caatinga (rodarte et al., 2008 – 13.90% of 1,722 possible interactions). the higher the value of the connectivity, the more abundant the actual interactions observed, compared to the possible total value of interactions. conclusion in tropical environments, as well as large plant diversity, it is expected to find a wide diversity of visiting floral and pollinating insects, mostly represented by bees. the present survey conducted on grande do paulino island reflected these expectations for tropical coastal environments, presenting a median diversity index when compared to other surveys in related areas. in addition, the results presented here contribute to the knowledge of the bee fauna of the state of maranhão. the species x. cearensis was the most abundant species in the study area, being commonly represented in coastal ecosystems. bees of the centridini tribe presented a high abundance value, corresponding to the supply of resources and nesting substrate available at the collection site. the occurrence of the species ceratina pubescens, and cer. rotundiceps had not been confirmed for the state of maranhão, until the present study. the plant chamaecrista ramosa was the most visited by the bee fauna. the visits to botanical species were also well represented by byrsonima crassifolia, which demonstrated an important relationship with bees of the centridini tribe. the majority of bee species presented interactions with only one plant species, a proportion not found in relation to plants, where a small number were visited by only one species. a low number of bee and plant species concentrating most of the interactions was observed, connected with the other species with few interactions. in addition to providing information about the bee fauna of the state of maranhão, this study was necessary to establish the various interactions between bees and plants from grande do paulino island. although it is a pioneer work for this site, it is important to emphasize that the nonstabilization of the collector curve indicates the need for future collections in order to increase the apifaunistic data of the region. acknowledgements fapema, josé eustáquio dos santos junior (ufmg), patrícia maia correia de albuquerque (rede bionorte), josé manuel macário rebêlo (ufma). references aguiar, c.m.l., caramés, j., frança, f. & melo, e. 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(2003). o gênero xylocopa latreille no rio grande do sul, brasil (hymenoptera, anthophoridae). revista brasileira de entomologia, 47: 107-118. doi: 10.1590/s0085-5626200 3000100016 serra, b.d.v., drummond, m.s., lacerda, l.m. & akatsu, i.p. (2009). abundância, distribuição espacial de ninhos de abelhas meliponina (hymenoptera, apidae, apini) e espécies vegetais utilizadas para nidificação em áreas de cerrado do maranhão. iheringia. série zoologia, 99: 12-17. doi: 10.1590/ s0073-47212009000100002 silva, f.s. & rebêlo, j.m.m. (1999). euglossine bees (hymenoptera: apidae) of buriticupu, amazonia of maranhão, brazil. acta amazonica, 29: 587-599. doi: 10.1590/1809-439 21999294599 silva, f.s. & rebêlo, j.m.m. (2002). population dynamics of euglossinae bees (hymenoptera, apidae) in an early secondgrowth forest of cajual island, in the state of maranhão, sociobiology 68(3): e5783 (september, 2021) 13 brazil. brazilian journal of biology, 62: 15-23. doi: 10.1590/ s1519-69842002000100003 silva, o., rêgo, m.m.c., albuquerque, p.m.c. & ramos, m.c. (2009). abelhas euglossina (hymenoptera: apidae) em área de restinga do nordeste do maranhão. neotropical entomology, 38: 186-196. doi: 10.1590/s1519-566x2009000200004. silveira, f.a., melo, g.a.r. & almeida, e.a.b. (2002). abelhas brasileiras: sistemática e identificação. belo horizonte, 253 p somavilla, a., schoeninger, k., nogueira, d.s. & kohler, a. (2018). diversidade de abelhas (hymenoptera: apoidea). e visitação floral em uma área de mata atlântica no sul do brasil. entomobrasilis, 11: 191-200. doi: 10.12741/ebrasilis. v11i3.800 viana, b.f. & alves-dos-santos, i. (2002). bee diversity of the coastal sand dunes of brazil. in: kevan, p.g. & imperatrizfonseca, v.l. (eds.), pollinating bees: the conservation link between agriculture and nature (pp 135-153). brasília: mma viana, b.f. & kleinert, a.m.p. (2006). structure of bee-flower system in the coastal sand dune of abaeté, northeastern brazil. revista brasileira de entomologia, 50: 53-63. doi: 10.1590/ s0085-56262006000100008 viana, b.f., kleinert, a.m.p. & silva, f.o. (2002). ecologia de xylocopa (neoxylocopa). cearensis (hymenoptera, anthophoridae) nas dunas litorâneas de abaeté, salvador, bahia. iheringia. série zoologia, 92: 47-57. doi: 10.1590/ s0073-47212002000400007 viana, t.a. & lourenço, a.p. (2020). surveys of the bee (hymenoptera: apiformes) community in a neotropical savanna using pan traps. papéis avulsos de zoologia, 60: 1-12. doi: 10.11606/1807-0205/2020.60.31 zanella, f.c.v., schwartz-filho, d.l. & laroca s. (1998). tropical bee island biogeography: diversity and abundance patterns. biogeografica, 74: 103-115 _hlk529374872 doi: 10.13102/sociobiology.v64i4.2087sociobiology 64(4): 492-494 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 social carrying and defensive behavior during colony emigration in the leaf-cutting ant atta sexdens social carrying behavior is one of the most common behaviors performed by ants; workers constantly transport eggs, brood, and adults to target areas inside the nest (moglich & holldobler, 1974; dahbi et al., 1997; pie, 2002). yet poorly investigated, social carrying seems to be primarily required in colony emigration, where it plays defensive roles as a response to the threats of the colony (fowler, 1981; nickele et al., 2012). colony emigration occurs more frequently than previously thought (hölldobler & wilson, 1990). in acromyrmex leafcutting ants, emigration process can take an entire week; they swiftly transport the symbiotic fungus, brood, males, gynes and queens to distances up to 100 meters (nickele et al., 2012). in atta species, emigration occurs subterraneously through underground galleries deeper than 1 meter that could reach distances up to 300 metres away from the original nest (autuori, 1941; fowler, 1981). according to fowler (1981), in leaf-cutting ants, emigrations are generally induced by pesticide intoxication or intraand interspecific competition. abstract in this work, we describe for the first time and under laboratory conditions, the behaviors related to social carrying and defensive strategies during colony emigration in the leaf-cutting ant atta sexdens. once colonies were laid on a tray under suboptimal conditions, groups of workers aggregated all over the body surface of the queen and brood, with mandibles half open and legs widely open in a ‘entangle’ formation. queens were the first caste to be reallocated, followed respectively by the transportation of brood, newly-emerged workers, and pieces of fungus garden to the new nesting site. contrary to what have been reported to the myrmicinae species, adult transport followed a stereotyped sequence of acts involving approach, seize and transportation of newly-emerged workers to new target areas. our results suggest that, in front of rapid unfavorable changes, leaf-cutting ants are capable of reorganize the nest in order to protect their members and resources. sociobiology an international journal on social insects l valadares, fs nascimento article history edited by yi-juan xu, south china agricultural university, china received 21 september 2017 initial acceptance 05 october 2017 final acceptance 06 october 2017 publication date 27 december 2017 keywords animal behavior, adult transport, atta sexdens. corresponding author lohan valadares universidade de são paulo (usp) laboratório de comportamento e ecologia de insetos sociais, departamento de biologia avenida bandeirantes, 3900 ribeirão preto-sp, brasil. e-mail: valadareslohan@usp.br in order to gain new insights into defensive strategies and social carrying behavior in leaf-cutting ants during colony emigration, in this work we adapted the methodology proposed by möglich and hölldobler (1974) where we stimulated emigration by keeping the population under suboptimal conditions and provided a new nesting site with optimal conditions. such conditions were promoted by removing a portion of fungus garden with queen, brood, and workers from plastic containers of the original nest to an open tray where individuals were exposed to low humidity (approximately 30%). we then offered a new nesting site made up of a 2 l acrylic chamber with 1 cm of plaster in the bottom to keep the humidity high, according to the preference of the species for rearing site and fungus culturing (araújo et al., 2011). the acrylic chamber was connected to the disturbed tray by a 45 cm plastic tube. the experiments were conducted with four mature colonies of a. sexdens collected in ribeirão preto, brazil, and for each colony we stimulated emigration only once. laboratório de comportamento e ecologia de insetos sociais, departamento de biologia, universidade de são paulo, ribeirão preto-sp, brazil short note mailto:valadareslohan@usp.br sociobiology 64(4): 492-494 (december, 2017) 493 the observations were made with the aid of a digital video camera and the behavior acts were registered and described. once laid on the perturbed tray, workers displayed alarm behavior; they moved rapidly with open mandibles, fig 1. behaviors associated to colony defense and social carrying during colony emigration, in a. sexdens. a, b: aggregation of median and major workers around the body surface of larvae (a), and queen (b). c, d: social carrying behavior of a newly-emerged worker during colony emigration. while few others stayed motionless with mandibles widely open to upward position. parallel to this immediate response, a group of workers, comprised mainly media workers (head width around 1.2 mm) and major workers (head width around 2.0 mm), aggregated all over the body surface of queen and brood, with mandibles half open and the pair of legs widely open in an ‘entangle’ formation (fig 1 a-b). after approximately 5 minutes of exposure to the perturbed tray, workers started to reorganize themselves. first, a group of dozens of workers entered and explored the new optimal chamber, moving and touching the wall of the chamber with their antennae and the tips of the gaster. in all observed emigrations, the first caste to be transported was the queen, which happened after a soldier have returned from the new chamber, and induced the queen to move out from the disturbed tray by an attempt to grab her with the mandibles by the head or gaster (fig 1b). we interpreted this behavior as a signalization to allow the queen to emigrate. such behavior, only performed by soldiers, induced the workers, which were entangled onto the queen’s body, to come down and rearrange themselves around the queen, sometimes pulling her by the legs, until the moment she arrived at the new nesting site. after the queen’s reallocation, workers transported respectively to the new site: brood, newly-emerged workers (new) and pieces of fungus. contrary to what have been reported to the myrmicinae species (moglich & holldobler, 1974), adult transport in a. sexdens followed a stereotyped sequence of acts separated into three categories: approach, seize and transportation. 1) approach, when the transporter worker approached the new by touching her with the antenna or legs, sometimes performing front leg boxing; 2) seize, when the transporter pulled the new by the legs, petiole or gaster; 3) after being seized, new assumed the ‘pupae’ position (fig 1c), by contracting the pair of legs close to the body, allowing the transporter to grab her by the petiole or gaster and reallocate them together with queen and brood items (fig 1d). our results suggest that, in front of adversities, leaf-cutting ants are capable of promptly reorganize the nest in order to protect their members and resources. acknowledgments this work was supported by the national council of scientific and technological development (cnpq), and fundação de amparo à pesquisa do estado de são paulo (fapesp, 2010/10027-5). references araújo, m.s., ribeiro, m.m.r., marinho, c.g.s., oliveira, m.a., della-lucia, t.m.c. (2011). fundação e estabelecimento de formigueiros. in della-lucia (ed.), formigas-cortadeiras, da biologia ao manejo (pp 173-189). viçosa: editora ufv autuori, m. (1941). contribuição para o conhecimento da saúva (atta spp. hymenoptera: formicidae) i – evolução do l valadares, fs nascimento – colony emigration of leaf-cutting ants494 sauveiro (atta sexdens rubropilosa forel, 1908). arquivos do instituto biológico, 12: 197–228 dahbi, a., cerdá, x., hefetz, a., lenoir, a. (1997). adult transport in the ant cataglyphis iberica: a means to maintain a uniform colonial odour in a species with multiple nests. physiologycal entomology, 22: 13-19. doi: 10.1111/ j.1365-3032.1997.tb01135.x fowler, h. g. (1981). on the emigration of leaf-cutting ants colonies. biotropica, 13: 316 hölldobler, b. & wilson, e.o. (1990). the ants. cambridge: harvard university press, 732 p möglich, m., hölldobler, b. (1974). social carrying behavior and division of labor during nest moving in ants. psyche, 81: 219–236. doi: 10.1155/1974/25763 nickele, m. a., pie, m. r. p., filho, w. r. (2012). emigration of a colony of the leaf-cutting ant acromyrmex heyeri forel (hymenoptera, formicidae). revista brasileira de entomologia, 56: 385-386. doi: 10.1590/s0085-562620 12005000045 pie, r. (2002). behavioral repertoire, age polyethism and adult transport in ectatomma opaciventre (formicidae: ponerinae). journal of insect behavior, 15: 25-35. doi: 10.1023/a:1014475927822 doi: 10.13102/sociobiology.v60i3.266-276sociobiology 60(3): 266-276 (2013) pollen collected and foraging activities of frieseomelitta varia (lepeletier) (hymenoptera: apidae) in an urban landscape kp aleixo1, lb de faria1,2, ca garófalo1, vl imperatriz fonseca3, ci da silva1,4 introduction according to dearborn and kark (2009) there are seven possible motivations for urban biodiversity conservation: preserving local biodiversity, creating stepping stones to nonurban habitat, understanding and facilitating responses to environment change, conducting environmental education, providing ecosystem services, fulfilling ethical responsibilities, and improving human well-being. although expansion of urban areas are among the human activities that result in the loss of native fauna and flora (mckinney, 2008), there is evidence that cities can provide potential resources for the animal species living within them or in their surroundings, including arthropods such as bees (nates-parra et al., 2006; silva et al., 2007; wojcik et al., 2008) and butterflies (bergerot et al., 2010). indeed, cities can also act as ecological corridors (owen, 1991). abstract cities provide resources for animal species that live within them or inhabit their surroundings. this has motivated an increase in ecological studies of urban areas, including the interactions between plants and pollinators. from march 2010 to february 2011, the flowering plants present in the study area, located at the universidade de são paulo, ribeirão preto, were sampled to evaluate how floral sources were distributed throughout the year. concurrently, worker bees with pollen loads were collected from four colonies of frieseomelitta varia (lepeletier, 1836) to identify the sources used by bees. despite an increase in plant species abundance in july, plants were in bloom year-round and consequently, the production and supply of floral resources were continuous. the workers collected resources from 77 plant species, but only three were extensively exploited. delonix regia (leguminosae), poincianella pluviosa (leguminosae) and ceiba speciosa (malvaceae) accounted for 42% of total pollen grains quantified during the year, showing that f. varia intensify pollen collection at few sources at spatiotemporal scale. this study emphasizes the importance of native urban flora to maintain f. varia and other bee species. the list of plants presented in this study can be used in the design and planning of urban areas. sociobiology an international journal on social insects 1 universidade de são paulo ribeirão preto, são paulo brazil. 2 universidade de são paulo são paulo, são paulo brazil. 3 universidade federal rural do semi-árido mossoró, rio grande do norte brazil. 4 universidade de santo amaro (unisa) são paulo, são paulo brazil. article history edited by candida m l aguiar, uefs, brazil received 20 may 2013 initial acceptance 26 june 2013 final acceptance 13 july 2013 key words food resource, pollen analysis, urban flora, social bees corresponding author: kátia paula aleixo faculdade de filosofia, ciências e letras de ribeirão preto. universidade de são paulo av. bandeirantes, 3900, 14040-901 ribeirão preto, são paulo, brasil. e-mail: katialeixo@yahoo.com.br in cities, communities of bees and wasps are highly diverse (frankie et al., 2005; zanette et al., 2005; matteson et al., 2008) and they are affected by variables that compose the urban landscape such as the availability of resources (cane et al., 2006; kearns & oliveras, 2009). floral generalist bee species are favored in urban areas (zanette et al., 2005; natesparra et al., 2006), while specialist bee species are severely affected by urbanization because the plants they usually visit to collect resources are rarely ornamental and therefore, are not often found in urban gardens (frankie et al., 2009). in brazil, studies concerning the use of floral resources by bees in urban areas are uncommon (knoll et al., 1994; taura & laroca, 2001; agostini & sazima, 2003; silva et al., 2007) and do not approach the topic from an urban landscape planning standpoint to promote the conservation of the diversity found within these areas. the social bees known as stingless bees comprise approximately 400 species distributed in the research article bees sociobiology 60(3): 266-276 (2013) 267 neotropical region (camargo & pedro, 2007). they live in perennial colonies and the worker bees forage year-round. although stingless bees are generalists, collecting pollen and nectar from an array of plant species (roubik, 1989; ramalho et al., 1990), the workers can intensify collection at certain sources for an amount of time (eltz et al., 2001; faria et al., 2012). while seeking food in flowers, the worker bees play an important ecological role as pollinators of many plant species, which makes them good candidates for commercial pollination, plus the fact that they can easily be kept in hives and are nonaggressive (slaa et al., 2006). frieseomelitta varia (lepeletier, 1836) is a neotropical medium-sized species of stingless bee with occurrence in several regions of brazil (camargo & pedro, 2007). colonies of this species are found not only in areas of natural vegetation (teixeira et al., 2007), but also in urban areas (marquessouza, 2010). given stingless bees’ ecological importance, the present study aimed (i) to understand the interactions between f. varia and urban flora by analyzing the pollen loads from foragers and (ii) to assess the influence of the availability of floral resources and climatic factor in the pollen-foraging workers in an urban landscape. materials and methods study area the present study was conducted in the campus of universidade de são paulo (21º10’30’’ s 47º48’38’’ w), located in the city of ribeirão preto, in the northeastern part of the state of são paulo, brazil, at altitudes ranging from 510 to 800 m.a.s.l.. the campus covers an area of 574.75 ha and the climate of the region is characterized by marked seasonality: cold, dry winters (april to september) and hot, wet summers (october to march). prior to the advent of coffee production in the region, the vegetation of the campus was predominantly seasonal semideciduous forest. the present day campus represents an urban area that consists of native and exotic plant species used in urban landscaping. there is a forest covering 75 ha of the campus comprised of plant species native of the vegetation of this area (pais & varanda, 2010). data collection from march 2010 to february 2011, plant species and individuals in bloom were sampled once a month and during five consecutive days in an area with 500 m radius from the meliponary of faculdade de filosofia, ciências e letras de ribeirão preto (ffclrp), totaling 78 hectares. the radius used to sample the plant species in bloom is according to flight range of the f. varia (lichtenberg et al., 2010). flower buds of each species were also collected, and the pollen grains were removed and mounted on slides, which were deposited in the reference pollen collection of ffclrp. the plant species were sampled throughout the vertical strata as suggested by silva et al. (2012), in which trees are considered woody individuals with circumference at breast height (cbh) ≥ 15 cm and more than 2 m high; shrubs are woody individuals with cbh < 15 cm and high between 1 and 2 m; herbaceous are not woody individuals with < 1 m high and lianas are woody individuals that grow laid on other plants without causing damages. the plant species collected in the study area were classified as ornamental and as native or exotic to brazil based on literature (lorenzi, 2008; souza & lorenzi, 2008). concomitantly to plant species sampling, the pollenforaging activity of worker bees in four colonies of f. varia was studied in relation to climatic factors and the availability of floral resources in the sampled area. these four colonies were kept in the meliponary of ffclrp and the maximum distance between them was 3 m. they were about the same age and the same size, considering the number of workers and number of storage pots. once a month, the number of workers with pollen loads returning to their colonies was quantified for five minutes at a time with the aid of a manual counter at intervals of 30 minutes between 5:30 am and 12:30 pm. from 1:30 pm, the workers were quantified every hour until 5:30 pm because the collection of resources by workers f. varia decreases during this time period. temperature, relative humidity, wind speed and rainfall data were collected at the meteorological station in the departamento de biologia, ffclrp, on the days in which pollen was sampled. after quantifying the foraging activity of the colonies, three intervals were determined: the beginning, the peak and the end of the foraging activity. on the following day, five workers returning to each colony with pollen in their corbiculae were sampled in each of the three intervals. thus, fifteen workers with corbiculae loaded with pollen were sampled from each colony (n = 4) per day, totaling 60 samples per month. bees were captured using an aspirator after closing the entries of the colonies, and the pollen loads were removed using a probe. the pollen loads were stored in 70% alcohol, following silva et al. (2010). after 24 hours, the alcohol was discarded, and the samples were placed in 4 ml of glacial acetic acid for at least 24 hours for subsequent acetolysis according to erdtman (1960). the pollen grains on the slides were identified by comparison with pollen deposited in the reference pollen collection of ffclrp. the plant material was deposited at spfr herbarium (são paulo filosofia ribeirão) of departamento de biologia, ffclrp. data analysis circular statistics was employed to assess seasonality in resource availability considering the number of plant species kp aleixo, lb de faria, ca garófalo, vl imperatriz-fonseca, ci da silva pollen collected and foraging activities of frieseomelitta varia268 and individuals in bloom from march 2010 to february 2011. to test for the occurrence of seasonality, the rayleigh´s test (z) was applied to determine the significance of the mean date of flowering event (in months; α=5%) (zar, 1999). the null hypothesis (h0) states that when the flowering event is distributed uniformly throughout the year, there is no seasonality. if h0 is rejected, the mean date is significant and there is a seasonal phenological pattern. the intensity of the concentration around the mean date, denoted by r, can be considered a measure of the degree of seasonality. the vector r has no units and may vary from 0 (when phenological activity is distributed uniformly throughout the year) to 1 (when phenological activity is concentrated around one single date or time of the year) (morellato et al., 2000). the circular statistics analysis was performed by the oriana software (kovach, 2011). the same test was applied to assess the frequency of workers of f. varia with pollen loads returning to the colonies throughout the year. pearson's correlation coefficient (r) (zar, 1999) was calculated to test the relationship between the number of species and individuals in bloom and the mean monthly temperature (oc) and rainfall (mm) from march 2010 to february 2011. pearson's correlation coefficient was also calculated to test the relationship between the number of pollen-foraging workers and the climatic factors of the day that they were counted [1. temperature (oc); 2. relative humidity (%); 3. wind speed (km/h)], and the diversity of pollen in the samples of the respective month. the relationship between the latter variable and the number of species that had bloomed throughout the year was also analyzed. the analyses were conducted using r version 2.13.1 (r development core team 2011). analysis of the pollen collected by the bees was performed using a binocular microscope with magnifications up to 2560x, which allowed images of pollen grains to be taken with an attached digital camera. the first 400 pollen grains were counted for each sample, as suggested by montero and tormo (1990). the percentages were then calculated according to the classification proposed by maurizio and louveaux (1965) using the following categories: dominant pollen (> 45% of the total grains on the slide), accessory pollen (from 15 to 45%), important isolated pollen (3 to 15%) and occasionally isolated pollen (< 3%). the plants with pollen classified as dominant and pollen that occurred over a period of at least six months were considered to be key plant species in the maintenance of f. varia. results during the study period, 235 plant species and 3285 individuals in bloom were sampled in the meliponary and the surrounding area. the mean flowering date of plant species was significant (z = 20.59, p < 0.01) and corresponded to the month of april, revealing a seasonal pattern despite the low concentration of species around this mean date (r=0.16) (fig. 1a). the mean flowering date of individuals in bloom was also significant (z = 167.48, p < 0.01) and corresponded to the month of may, revealing a seasonal pattern despite the low concentration of individuals around the mean date (r = 0.23) (fig. 1b). considering separately the native and exotic species and individuals in bloom the seasonal pattern was also observed [(nativespecies: z = 10.18, r = 0.13; exoticespecies: z = 12.69, r = 0.24) (nativeindividuals: z = 115.90, r = 0.22; exoticindividuals: z = 59.03, r = 0.27); p < 0.01] (fig. 1). the number of flowering species and individuals were not correlated with temperature and rainfall [(especies: rtemperature ( o c)= -0.24, rrainfall= -0.31) (individuals: rtemperature ( o c)= -0.54, rrainfall= -0.55); p > 0.05]. the monthly number of pollen foraging workers varied in the four studied colonies, and presented a seasonal distribution throughout the year [(zcolony1= 403.09, r = 0.67) (zcolony2 = 477.65, r = 0.61) (zcolony3 = 214.92, r = 0.50) (zcolony4 = figure. 1 distribution of total, native and exotic plant species (a) and individuals (b) that bloomed in the meliponary and its surrounding area from march 2010 to february 2011. sociobiology 60(3): 266-276 (2013) 269 733.75, r = 0.60); p < 0.01] (fig. 2). according to the pattern of this event observed in all colonies, the period of highest pollen collection activity by the bees (october) did not coincide with the period with highest availability of floral resources (july) (fig. 2). the pollen-foraging activity in f. varia was not influenced by climatic factors (rtemperature ( o c) = 0.37, rrelative humidity (%) = 0.40, rwind speed (km/h) = 0.40; p>0.05). seventy-seven pollen types distributed in 36 families and 60 genera were identified in the studied samples. of these, 63 were identified at species level and three remained as undetermined types (table 1). in general, the workers collected floral resources from a small fraction of the total number of plant species available, corresponding to 32.76% of the sampled plants that bloomed throughout the study period. more than half of the plant species used by f. varia were distributed in seven of the 36 botanical families. the families with the highest number of visited species were leguminosae and myrtaceae, which were represented by 11 and 7 species, respectively. however, most of the pollen came from species of leguminosae and malvaceae, which were represented by 44.30% and 9.50% of total pollen grains quantified in the samples. considering monthly pollen analysis, delonix regia (leguminosae), ceiba speciosa (malvaceae) and poincianella pluviosa (leguminosae) were intensively visited as they showed more than 45% of the pollen grains in the samples (table 1). throughout the year, the three species accounted for figure. 2 distribution of the monthly number of workers of frieseomelitta varia per colony and monthly number of individuals that bloomed in the meliponary and its surrounding area from march 2010 to february 2011. kp aleixo, lb de faria, ca garófalo, vl imperatriz-fonseca, ci da silva pollen collected and foraging activities of frieseomelitta varia270 42% of the total pollen grains. there is almost no overlap in the use of d. regia, c. speciosa and p. pluviosa by f. varia (table 1), which reflects the flowering period of these species in the study area. eucalyptus moluccana (myrtaceae), leucaena leucocephala (leguminosae), paspalum notatum (poaceae) and schinus terebinthifolius (anacardiaceae) were also important for f. varia because they provided food to the colonies for a period of at least six months (table 1). there was no correlation between mean number of pollen-foraging workers and pollen diversity (r=0.12; p>0.05), and no correlation between pollen diversity of samples and the number of species bloomed (r=0.00; p>0.05). although there was a low richness of plant species in bloom, the highest numbers of pollen types in the samples were found in october and november, with 33.33% and 45.45% of the available sources used, respectively. in contrast, the samples from march and april showed lower numbers of pollen types in relation to the numbers of plant species in bloom, with 16.49% and 13.27% of the available sources used, respectively. among the 63 identified species, 59 belong to plants used in urban landscape planning or plants that have a high potential to be used for this purpose. of these 59 ornamental species that were exploited as food sources, 64.50% species are native to brazil (table 1). discussion despite displaying a seasonal flowering pattern, species were in bloom year-round in the study area, and therefore the production and supply of floral resources was continuous. in a phenological study performed on the campus of the universidade de campinas sp, brazil (22º46’57’’ s 47º04’47’’ w), which is also an urban area, the species bloomed throughout the year but did not exhibit marked seasonality (agostini & sazima, 2003). those authors argue that species with different flowering periods are used to enhance ornamentation in urban settings, maintaining flowers available for most yearround. in our study, both native and exotic species contributed significantly to the seasonal pattern observed. besides the revegetated area, the campus also comprises trees species of the seasonal semideciduous forest for the purpose of shading. one of the main factors for the increased species richness of plants in urban habitats is human-aided dispersal of exotic species (mckinney, 2008). however, using native plants in the planning of green areas is important from the conservation point of view, since it favors the maintenance of a vast richness of plant-pollinator interactions. in the study area, the absence of correlation between the flowering phenology and climatic factors can be explained by the increased irrigation, which is one of the manipulator of floral blooming within the urban landscape. in urban areas, irrigation can influence the continuous production of flowers (frankie et al., 2005), mainly in shrub and herbaceous strata. in tropical areas, the flowering pattern, and consequently the resource availability, is associated with seasonal variations in rainfall (van schaik et al., 1993). our study shows that the period of highest food collection activity by f. varia is not associated with the period when the resources are more abundant in the field. this result is not in accordance with literature for other species of stingless bees. although roubik (1982) and biesmeijer et al. (1999) have discussed that pollen foraging activity in colonies of melipona species decreased due to reduced pollen availability in the environment, the authors did not evaluate the flowering phenology of plant species. although there is no relationship between climatic factors and the mean number of f. varia pollen-foraging workers, the flight activity of stingless bees is known to be influenced by such variables, and their pollen-foraging activity reflects the influence of meteorological parameters (pick & blochtein, 2002). for example, the flight activity of plebeia pugnax foragers, a small species of stingless bee, may be relatively constant over a wide range of temperatures (hilário et al., 2001), while the highest intensities of p. saiqui worker pollen-foraging activity coincided with the highest temperatures throughout the year (pick & blochtein, 2002). kajobe and echazarreta (2005) found that the number of worker bees for two medium-sized species of stingless bees decreased in the field when the relative humidity was higher than the optimal value of 78%. the worker bees of p. pugnax flew over a wide range of relative humidity, from 30% to 100%, decreasing slowly the number of bees flying after 50% (hilário et al., 2001). other factors may affect the flight activity of bees including the population size (hilário et al., 2000), the reproductive status of the colony (nunes-silva et al., 2010) and even the body size of different species, which affects the maximal flight distance and the onset of this activity (teixeira & campos, 2005). although f. varia workers collected resources from 77 plant species, only three species were extensively exploited, showing that the bees intensify pollen collection at few sources at spatiotemporal scale. these results are consistent with the work of marques-souza (2010) on f. varia in an urban area located in manaus am, brazil. that author identified pollen of 79 plant species distributed among 60 genera and 37 botanical families, but only four of these species provided 71% of the total pollen collected. this pattern was also found in natural environments by teixeira et al. (2007) when they analyzed brazilian studies on the use of floral resources by bees of the frieseomelitta genus in areas with different types of vegetation. those authors mention that workers visited a large spectrum of plant species but only focused on some of them. other studies have also shown that stingless bees obtain most of their food from a small number of plant species (imperatriz-fonseca et al., 1989; ramalho, 1990; pick & blochtein 2002). the species intensively visited d. regia, c. speciosa and p. pluviosa have individuals close to the colonies, which sociobiology 60(3): 266-276 (2013) 271 ta bl e 1. p la nt s pe ci es /p ol le n ty pe s us ed b y f ri es eo m el itt a va ri a an d th ei r r el at iv e ab un da nc e, b as ed o n an al ys es o f t he p ol le n lo ad s fr om w or ke rs , f ro m m ar ch 2 01 0 to f eb ru ar y 20 11 . d om in an t p ol le n (> 4 5% ), a cc es so ry p ol le n (1 5 to 4 5% ), im po rt an t i so la te d po lle n (3 to 1 5% ) a nd o cc as io na lly is ol at ed p ol le n (< 3 % ). o rn : o rn am en ta l s pe ci es . n : n at iv e sp ec ie s. e : e xo tic s pe ci es . fa m ily sp ec ie s/ po lle n ty pe s* o rn 20 10 20 11 m ar a pr m ay ju n ju l a ug se p o ct n ov d ec ja n fe b a ca nt ha ce ae th un be rg ia gr an di flo ra r ox b. e x 14 .2 9 0. 05 a m ar an th ac ea e c ha m is so a al tis si m a (j ac q. )k un th n x 0. 07 0. 03 sp .1 * 0. 04 a na ca rd ia ce ae a na ca rd iu m oc ci de nt al el . n x 0. 60 sc hi nu st er eb in th ifo lia r ad di n x 11 .6 9 0. 10 0. 89 18 .4 3 24 .4 0 0. 13 0. 06 15 .7 1 a po cy na ce ae sp .1 * 0. 02 a re ca ce ae a rc ho nt op ho en ix al ex an dr ae (f . m ue ll. ) h . w en dl . & d ru de e x 15 .3 5 0. 06 c oc os nu ci fe ra l . n x 0. 08 sa ba lm ar iti m a (k un th )b ur re t e x 10 .0 0 sp .1 * 3. 85 sp .2 * 4. 33 sy ag ru sr om an zo ffi an a (c ha m .) g la ss m an n x 23 .4 6 0. 60 0. 02 4. 60 a st er ac ea e b id en ss ul ph ur ea (c av .) sc h. b ip . n x 0. 04 sp .1 * 9. 32 sp .2 * 0. 11 9. 98 ti th on ia di ve rs ifo lia (h em sl .) a .g ra y e x 12 .6 4 1. 95 b ig no ni ac ea e h an dr oa nt hu si m pe tig in os us (m ar t. ex d c .) m at to s n x 4. 52 0. 02 h an dr oa nt hu sr os eo -a lb us (r id l.) m at to s n x 0. 15 0. 55 1. 48 0. 02 0. 03 b or ag in ac ea e c or di a tr ic ho to m a (v el l.) a rr áb .e x st eu d. n 0. 64 0. 05 c an na ba ce ae c el tis ig ua na ea (j ac q. )s ar g. n 7. 00 0. 13 0. 02 0. 05 0. 06 tr em a m ic ra nt ha (l .) b lu m e n 1. 48 10 .7 5 c ar ic ac ea e c ar ic a pa pa ya l . e x 5. 73 2. 13 3. 02 c om m el in ac ea e tr ad es ca nt ia pa lli da (r os e) d .r .h un t n x 3. 10 e up ho rb ia ce ae c ro to n ur uc ur an a b ai ll. n x 0. 04 3. 56 1. 28 0. 92 jo an ne si a pr in ce ps v el l. n x 0. 03 0. 06 r ic in us co m m un is l . e x 2. 65 3. 98 7. 70 0. 03 16 .1 7 l eg um in os ae a ly si ca rp us va gi na lis (l .) d c e x 0. 14 a na de na nt he ra m ac ro ca rp a (b en th .) b re na n n x 0. 10 3. 63 23 .4 4 2. 69 b au hi ni a lo ng ifo lia d .d ie tr . n x 0. 08 c ae sa lp in ia pu lc he rr im a (l .) sw . e x 0. 80 1. 63 0. 06 0. 54 d el on ix re gi a (b oj er ex h oo k. )r af . e x 0. 11 38 .8 6 53 .2 3 62 .7 8 50 .9 1 20 .3 5 in ga sp .* 0. 03 le uc ae na le uc oc ep ha la (l am .) de w it e x 20 .3 3 8. 72 21 .4 0 1. 11 1. 18 12 .5 8 0. 04 4. 60 12 .5 0 10 .5 6 m ac ha er iu m ac ul ea tu m r ad di n x 3. 85 m im os a sp .1 * 0. 83 0. 91 m im os a sp .2 * 9. 26 0. 44 0. 08 p oi nc ia ne lla pl uv io sa (d c .) l .p .q ue iro z. n x 0. 02 0. 02 0. 03 4. 23 67 .7 3 45 .3 3 15 .0 4 0. 05 9. 81 h el ic on ia ce ae h el ic on ia ps itt ac or um l .f . n x 0. 03 ir id ac ea e n eo m ar ic a ca er ul ea (k er g aw l.) sp ra gu e n x 2. 64 0. 02 kp aleixo, lb de faria, ca garófalo, vl imperatriz-fonseca, ci da silva pollen collected and foraging activities of frieseomelitta varia272 l am ia ce ae p le ct ra nt hu sc ili at us e .m ey .e x b en th am e x 0. 04 m al pi gh ia ce ae m al pi gh ia em ar gi na ta d c . e x 0. 13 0. 05 0. 02 0. 02 m al va ce ae c ei ba sp ec io sa (a .s t.h il. )r av en na n x 31 .4 2 56 .7 6 0. 20 1. 04 g ua zu m a ul m ifo lia l am . n x 0. 04 3. 90 si da sp in os a l . n x 0. 20 tr iu m fe tta rh om bo id ea ja cq . n x 10 .0 0 m el ia ce ae c ed re la od or at a l . n x 0. 03 m un tin gi ac ea e m un tin gi a ca la bu ra l . e x 0. 04 1. 68 0. 02 0. 02 m yr ta ce ae e uc al yp tu sc itr io do ra h oo k. e x 0. 04 0. 02 0. 38 e uc al yp tu sm ol uc ca na r ox b. e x 10 .4 8 0. 07 19 .8 6 12 .5 0 0. 02 1. 00 0. 03 0. 19 6. 13 e ug en ia br as ili en si sl am . n x 0. 02 e ug en ia in vo lu cr at a d c . n x 0. 09 0. 63 e ug en ia py ri fo rm is c am be ss . n x 0. 04 0. 12 0. 05 0. 40 e ug en ia un ifl or a l . n x 0. 10 sy zy gi um m al ac ce ns e (l .) m er r. & l .m .p er ry e x 1. 93 0. 13 0. 15 o le ac ea e li gu st ru m lu ci du m w .t .a ito n e x 0. 07 1. 88 27 .2 0 o na gr ac ea e lu dw ig ia el eg an s( c am be ss .) h .h ar a n x 1. 04 1. 23 ph yt ol ac ca ce ae g al le si a in te gr ifo lia (s pr en g. )h ar m s n x 20 .0 0 0. 08 0. 08 pi pe ra ce ae p ip er um be lla tu m l . n 2. 63 po ac ea e p as pa lu m no ta tu m a la in ex fl üg gé n x 0. 13 15 .6 5 11 .1 4 1. 50 8. 46 0. 94 po ly ga la ce ae sp .1 * 0. 02 po nt ed er ia ce ae p on te de ri a co rd at a l . n x 2. 25 pr ot ea ce ae g re vi lle a ro bu st a a .c un n. ex r .b r. n x 8. 25 r ub ia ce ae g en ip a am er ic an a l . e x 8. 51 ix or a ch in en si sl am . n x 3. 68 20 .2 3 sp .1 * 0. 72 r ut ac ea e c itr us lim on ia o sb ec k e x 11 .9 3 16 .6 1 0. 18 m ur ra ya pa ni cu la ta (l .) ja ck e x 8. 17 13 .7 5 12 .5 0 sa pi nd ac ea e p au lli ni a el eg an sc am be ss . n x 5. 79 5. 56 se rj an ia le th al is a .s t.h il. n x 6. 23 0. 05 0. 02 so la na ce ae a cn is tu sa rb or es ce ns (l .) sc hl td l. n x 1. 09 b ru nf el si a un ifl or a (p oh l) d .d on n x 0. 15 st re lit zi ac ea e r av en al a m ad ag as ca ri en si ss on n. e x 19 .8 0 u rt ic ac ea e c ec ro pi a pa ch ys ta ch ya t ré cu l n x 8. 06 0. 06 v er be na ce ae d ur an ta re pe ns l . n x 6. 90 u nd et er m in ed 1* 11 .0 5 u nd et er m in ed 2* 0. 14 u nd et er m in ed 3* 0. 04 3. 45 n um be ro fp ol le n sa m pl ed /m on th 16 13 11 25 20 20 9 20 20 14 7 16 ta bl e 1 (c on tin ue d) . sociobiology 60(3): 266-276 (2013) 273 facilitated the access to resources by workers once f. varia has a small estimated flight distance compared with estimates for other medium-sized stingless bee species (lichtenberg et al., 2010). besides that, those species bloomed in sequence which resulted in a temporal structuration with complementarity in the use of those sources. factors such as high protein content may also explain the high frequency with which the three plants were exploited (kleinert et al., 2009). the inflorescences of e. moluccana, l. leucocephala, p. notatum and s. terebinthifolius with large numbers of flowers and anthers, plus their long flowering period, provide a large amount of source to stingless bee populations. the eusocial bees, even showing a generalist habit accepted as standard (ramalho et al., 1990), may exploit the most lucrative sources, being temporarily selective in their food choice (eltz et al., 2001). the leguminosae and myrtaceae families, which were intensely visited by f. varia in the present study, have also been recorded in other surveys of plants used by stingless bees as demonstrated by ramalho et al. (1990). in a study by teixeira et al. (2007), bees of the frieseomelitta genus focused their visits on the caesalpinioideae (leguminosae), malpighiaceae, and anacardiaceae families. myrtaceae was the second most important family found in samples of f. varia by marques-souza (2010) and occurred in 11 months of the study period. of the plants visited by bees in the study by agostini and sazima (2003), leguminosae was one of the two best represented families, with 13 species. leguminosae is the family with the highest species richness in the area covering the meliponary and its surroundings, which was similar to the study conducted in campinas. when considering a monthly analysis, f. varia collects a greater diversity of pollen when there are more foragers in the field. because this species’ workers are solitary foragers (jarau et al., 2003), the interaction with other species of stingless bees would explain this tendency to diversify. the species of stingless bees whose workers forage in large groups through effective recruiting mechanisms dominate the sources of pollen and change the foraging patterns of species such as f. varia (lichtenberg et al., 2010). consequently, by foraging independently, f. varia workers would visit a higher number of different plant species in the same spatiotemporal scale than the group-foraging species, which is reflected in the diversity of resources brought to the colonies. lichtenberg et al. (2010) experimentally removed the group-foraging workers and found that the solitary foragers, including workers of f. varia, had greater access to sucrose solution feeders and collected more of it. this observation is most evident in the period with the fewest species and plants in bloom (august to january), which overlaps with the highest pollen diversity and number of pollen-foraging workers in the field. instructing more workers to collect pollen would be a strategy for f. varia colonies to ensure their supply of protein even when collecting from low-yield sources. indeed, when resources are in short supply, workers of group-foraging stingless bee species visit a wider range of sources. conversely, in periods of greater resource availability, the workers collect from fewer sources, focusing instead on those that offer more profitable resources (eltz et al., 2001; faria et al., 2012). another implication of the solitary foraging strategy is the lower importance of mass flowering plant species for the maintenance of colonies of f. varia in comparison with other stingless bees species. although e. moluccana was found during nine months of the study period, its relative abundance was low. furthermore, the other species of eucalyptus and species of eugenia, another mass flowering genus, were included in the occasionally isolated pollen category, with pollen of low visibility, among the species selected to maintain the colonies. the generation of a high number of flowers providing pollen and/or nectar in a short period is a valuable resource for species of stingless bees that recruit nest mates for foraging. it has been noted that the species of the eucalyptus genus are some of the most frequently used species by bees of the scaptotrigona genus (ramalho, 1990; faria et al., 2012), which includes the group-foraging species scaptotrigona aff. depilis (jarau et al., 2003). our study indicates that ornamental plants can maintain populations of f. varia, especially when native flowering species are used to enhance the green areas. this conclusion was also made in other studies about urban ecology (frankie et al., 2005; cane et al., 2006). in urban areas exotic flowers generally account for most of the ornamental species, especially in gardens (acar et al., 2007). therefore, exotic species are important nutritional resources not only for bees (agostini & sazima, 2003; zanette et al., 2005; nates-parra et al., 2006; frankie et al., 2009) but also for butterflies with generalist habits (bergerot et al., 2010). flowers of the exotic species tecoma stans (bignoniaceae), which is commonly used in landscaping, were visited extensively by bees for collection of nutritional resources; therefore, this species is an important source of resource for maintaining the bee populations in the three brazilian urban areas studied (silva et al., 2007). flowers of t. stans are similar to the flowers of handroanthus and tabebuia species, two genera of bignoniaceae native to brazil important as sources of nectar for bee populations. studies that consider the resource availability for bees usually sample only plants of the tree and shrub strata (agostini & sazima, 2003). nevertheless, some important sources of nectar can be found in herbaceous and liana strata as we could see in this study and in faria et al. (2012) that was developed in the same area. this result suggests that all the vertical strata are important for the bee´s foraging and therefore future studies about bee´s trophic niche should consider herbaceous and liana strata on floristic survey. urban areas, such as the campus, which nowdays has a beneficial and positive history of land management can act as a refuge for native bees. urban areas seem to act as a refuge for pollinators, as discussed by mcfrederick and lebuhn (2006) kp aleixo, lb de faria, ca garófalo, vl imperatriz-fonseca, ci da silva pollen collected and foraging activities of frieseomelitta varia274 and lópez-uribe et al. (2008), for bees of the bombini and euglossini groups, respectively. the present study has enabled the understanding of resource use by colonies of the eusocial bee species f. varia, which is one of the first steps in the management and conservation of this species’ populations. the data generated by the present study will be useful for designing and managing green areas in urban environments. plant species with different flowering periods are recommended, as also planting native species in green areas as gardens and parks. as previously discussed in the literature (silva et al., 2007; lópez-uribe et al., 2008; wojcik et al., 2008), the designs should aim to increase the number of plants that effectively attract and maintain pollinator populations. this present study emphasizes the importance of plant’s diversity to maintain frieseomelitta varia in urban area, as also showed to other species of pollinators (mckinney, 2008). acknowledgments we thank maurício m. n. castro and hipólito f. p. neto for helping us with the collection of plants, joão paulo castro for the collection and identification of plants, cristiano menezes for lending us the bee colonies, milton groppo júnior, curator of the spfr herbarium, and research center on biodiversity and computing (bio comp). we also thank klaus h. hartfelder and two anonymous referees for helpful remarks in an early version of this manuscript. this study was funded by fundação de amparo à pesquisa do estado de são paulo fapesp (processo nº 2010/10285-4) and programa nacional de pós doutorado pnpd of coordenação de aperfeiçoamento de pessoal de nível superior – capes (processo nº 02958/09-0). references acar, c., acar, h. & eroglu, e. 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(1999). biostatistical analysis (fourth edition). new jersey: prentice hall international editions. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1073sociobiology 64(1): 101-104 (march, 2017) nesting habits of social wasps (hymenoptera: vespidae) in forest fragments associated with anthropic areas in southeastern brazil introduction social wasps belong to the subfamily polistinae (hymenoptera: vespidae) and comprise more than 950 described species; they are most diverse in the neotropical region (pickett & carpenter, 2010). social wasps interact with the various fauna and flora. some species acting as floral visitors, constituting a significant portion of the foraging guild (heithaus, 1979; aguiar & santos, 2007; clemente et al., 2013); they are also predaceous, impacting population dynamics in various prey taxa (richter, 2000; souza & zanuncio, 2012). the survival of social wasps depends both on successful colony foundation and strong foraging ability (dejean et al., 1998; richter, 2000; oliveira et al., 2010). these insects can establish nests at various sites in urban or natural environments, and the nesting substrates used are extremely diverse, including the abaxial or adaxial surface of abstract social wasp communities and nesting habits were evaluated in forest environments associated with areas of human disturbance from march to september 2015. three hundred forty-seven (347) colonies belonging to eighteen (18) species distributed in six genera were recorded. the species with the highest number of colonies were mischocyttarus cassununga (r. von ilhering, 1903) (194 colonies), mischocyttarus cerberus richards, 1940 (50 colonies) and polybia paulista (h. von ihering, 1896) (23 colonies). other species colony numbers ranged from one to eighteen. sociobiology an international journal on social insects tct oliveira1, mm souza2, ep pires3,4 article history edited by gilberto m. m . santos, uefs, brazil received 19 may 2016 initial acceptance 07 october 2016 final acceptance 13 february 2017 publication date 29 may 2017 keywords hymenoptera, colony, nesting substrate, synanthropism. corresponding author epifânio porfiro pires departamento de entomologia universidade federal de lavras – ufla cep 37200-000, lavras, mg, brasil e-mail: epifaniopires@yahoo.com.br leaves, plant stems, abandoned termite mounds, rocks, and human-made structures (lima et al., 2000; alvarenga et al., 2010; lópez et al., 2012; souza et al., 2014). social wasps colonies can be classified as stelocyttarus, gymnodomous, astelocyttarus, or phragmocyttarus (richards & richards, 1951; richards, 1978; carpenter & marques, 2001). stelocyttarus nests are formed by one or more combs attached to the substrate by a peduncle, which may or may not contain a protective involucrum. gymnodomous nests have no involucrum (characteristic of mischocyttarus spp. saussure, 1853, polistes spp. latreille, 1802, agelaia spp lepeletier, 1836 and apoica spp. lepeletier 1836), while calyptodome nests have involucrum (pseudopolybia spp. saussure, 1863 and parachartergus spp. r. von ihering 1904). astelocyttarus nests present a single comb with protective involucrum and cells built directly on the substrate (synoeca spp. saussure, 1852 and metapolybia spp. ducke, 1905). in phragmocyttarus1departamento de biologia, universidade federal de lavras, minas gerais, brazil 2 instituto federal de educação, ciência e tecnologia de minas gerais, inconfidentes-mg, brazil 3 departamento de entomologia, universidade federal de lavras, minas gerais, brazil research article wasps tct oliveira, mm souza, ep pires – nesting habits of social wasps (hymenoptera: vespidae)102 type nests, the initial comb is broadly attached to the protective casing and the subsequent combs are constructed in contact with the sides of the previous combs (polybia spp. lepeletier, 1836 and brachygastra spp. perty, 1833). colony foundation can occur via swarming or independently (jeanne, 1991; wenzel, 1998; carpenter & marques, 2001). swarm founding consists of one or more queens and several workers leaving the original colony to seek a suitable place to build the new colony. for independentfounding species, one or more queens (reproductive females) build a nest, oviposit, and feed larvae (jeanne, 1991; wenzel, 1998; carpenter & marques, 2001). several studies have pointed out that some social wasp species prefer nesting in human-made structures. these sites provide protection from weather, and reduce the threat of predation and competition for nesting substrates (marques & carvalho, 1993; lima et al., 2000). in contrast, species that nest only in natural environments prefer specific features and environmental conditions (dejean et al., 1998; souza et al., 2010; souza et al., 2014). studies of social wasps nesting habits in urban sites surrounded by natural vegetation are still scarce. the claim that the social wasps prefer urban environments may be misleading, as association with urban areas could be due to invasion and subsequent reduction of natural areas. studies on the nesting behavior of these insects in the associated environments may provide important information on the ecological characteristics of organisms and their interactions with the environment (dejean et al., 1998; souza et al., 2010; souza et al., 2014). in this study we characterize the social wasp fauna and describe the various nesting substrates in forest fragments associated with anthropogenically impacted areas in southeastern brazil. material and methods study area the study was conducted in atlantic forest fragments with high human disturbance. the forest area has an elevation of 864 m and contains 215.62 hectares, and is located in ifsuldeminas (22°19’2’’ s, 46°19’42’’) in inconfidentes campus, minas gerais. regional vegetation landscape is characterized by fragments of semideciduous montane seasonal forest and riparian forest, which are undergoing constant human interventions in association with economic cycles for agriculture and livestock production. the climate is classified as cwb (mesothermal with well-defined seasons), with an annual average temperature ranging from 13.1 to 23.7oc and average annual rainfall of 1,800 mm (sarmento et al., 2013). methodology the survey was conducted from march to september of 2015, with a total sampling effort of 30 days. nest searching and specimen collection was carried out by means of active search (souza & prezoto, 2006). buildings on the campus and in forest areas were inspected. when colonies were discovered we recorded the species, the number of colonies, and types of substrates used. the types of substrates used by different species were grouped into the following categories: man-made structures (windows, metals, aluminum structures, concrete masonry units, poles, pipes, water tanks, wooden slats, and roofs), barranco, and plant substrate (all plants in which nests were found). specimens were collected species confirmation. species identities were determined using a taxonomic keys by richards (1978), carpenter and marques (2001) and carpenter (2004). species identities were confirmed by professor dr. orlando tobias silveira of the pará emilio goeldi museum in belém, pará. results and discussion a total of 347 social wasps colonies were found, belonging to 18 species distributed among six genera. of this total, 73% belonged to the genus mischocyttarus, 15% to polybia, 8% to polistes, 2% to synoeca and 1% each to parachartergus and brachygastra (table 1). the greatest proportion of colonies (87%) occurred in man-made structures (table 1), however diversity and species richness was higher in plant substrates (table 1). the species with the greatest numbers of colonies were mischocyttarus cassununga (r. von ihering, 1903) (194 colonies), mischocyttarus cerberus richards, 1940 (50 colonies) and polybia paulista (h. von ihering, 1896) (23 colonies). other species had between one and 18 colonies each (table 1). many studies conducted in urban environments have revealed high diversity in social wasps, with higher incidence of species in the genera mischocyttarus, polybia, polistes and protopolybia (lima et al., 2000; alvarenga et al., 2010; lópez et al., 2012). colonies of m. cassununga, polistes simillimus (zikán, 1951), polistes versicolor (olivier, 1791) and mischocyttarus drewseni (saussure, 1857) occurred only in man-made structures, suggesting synanthropism in these species (jeanne, 1972; raposo-filho & rodrigues, 1984; marques & carvalho, 1993; lima et al., 2000; torres et al., 2011; castro et al., 2014). mischocyttarus rotundicollis (cameron, 1912), polybia scutellaris (white, 1841), p. paulista, m. cerberus, and polybia fastidiosuscula (saussure, 1854) were recorded in both natural and man-made substrates. in contrast, polybia platycephala slyventris (richards, 1978), synoeca cyanea (fabricius, 1775), polybia jurinei (saussure, 1854), parachartegus fraternus (griboldo, 1892), polybia occidentalis (olivier, 19711), polybia sericea (olivier, 1971), polybia chrysothorax (lichtenstein, 1796), brachygastra lecheguana (latreille, 1824) and polybia minarum (ducke, 1906) were found only in plant substrates (table 1). based on the results, the availability of specific substrates for nesting seems to be a limiting factor for the species occurrence in certain environments. this is especially true sociobiology 64(1): 101-104 (march, 2017) 103 for species that construct astelocyttarus-type nests, such as synoeca and metapolybia which build a single comb with a protective involucrum with cells attached directly to the substrate (richards & richards, 1951); this partially explains the absence of species in these genera in several studies in urban areas (dejean et al., 1998; souza et al., 2010). the results from this study provide a better understanding of social wasp communities associated with human-modified environments. it is clear that although most species have generalist habits with respect to substrate choice for nest-building, others seem to prefer specific characteristics for colony construction. areas of vegetation in forest fragments associated with urban environments are vital for the maintenance of social wasp populations, and consequently play a role in maintaining environmental services provided by these insects such as biological control, pollination, and maintenance of trophic webs (alvarenga et al., 2010). further studies are needed in these environments that evaluate the potential use for social wasps as indicators of environmental quality, as well as to ensure species conservation, especially for those which require specific features for nesting. references aguiar, c.m.l. & santos, g.m.m. 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(2010). social wasps (hymenoptera: vespidae) as indicators of conservation degree of riparian forests in southeast brazil. sociobiology, 56: 387-396. souza, m.m. & zanuncio, j.c. (2012). marimbondos-vespas sociais (hymenoptera: vespidae). viçosa: editora ufv, 79p. souza, m.m., pires, e.p., elpino-campos, a. & louzada, j.n.c. (2014). nesting of social wasps (hymenoptera: vespidae) in a riparian forest of rio das mortes in southeastern brazil. acta scientiarum-biological sciences 36: 189-196. doi: 10.4025/ actascibiolsci.v36i2.21460 torres, v.o., montagna, t.s., fernandes, w.d. & antoniallijunior, w.f. (2011). colony cycle of the social wasp mischocyttarus consimilis zikán (hymenoptera, vespidae). revista brasileira de entomologia, 55: 247-252. wenzel, j.w. (1998). a generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae: vespinae, polistinae). american museum novitates, 3224: 1-39. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i3.1276sociobiology 64(3): 261-275 (september, 2017) ant assemblage structure in a secondary tropical dry forest: the role of ecological succession and seasonality introduction species diversity can be influenced by processes acting at different spatial and temporal scales (godfray & lawton, 2001). spatial scales have received increased attention in the analysis of the relationship between biodiversity and changes in landscape structure (ribas et al., 2003; kalacska et al., 2004), although recently few studies focused on the effects of time scales. this shift in the focus of the studies occurred because the variation in the number of species in a landscape depends on the ecological processes that operate on multiple time scales (lambi, 1996). ecological succession and seasonality are processes that occur in different time scales and can influence the species diversity of a forest in different ways (neves et al., 2014). abstract this study identified the main biological mechanisms governing the diversity of ants on different ecological time scales. ants were sampled in 15 plots distributed in early, intermediate and late stages of succession (five plots per stage) at the parque estadual da mata seca, brazil. at each sample point, unbaited pitfall traps were installed in hypogaeic, epigaeic and arboreal strata. we collected 95 ant species from 26 genera and nine subfamilies. our results indicated that there was an increase in species richness in advanced stages of succession. we also observed that ant assemblages were different among successional stages. for the arboreal and epigaeic strata, species richness did not change with succession progression, but species composition of these two strata differed among successional stages. unlike to arboreal and epigaeic ants, hypogaiec ant species richness was higher in the intermediate and late stages of succession and the composition of hypogaeic ants differed among successional stages. similarity between ant species foraging in arboreal and epigaeic strata decreases with succession progression and β-diversity was higher in advanced successional stages. additionally, species richness was higher in the dry season, whereas the composition of ant assemblages did not change between seasons. a considerable fraction of the ant assemblage was found only in advanced stages of succession, demonstrating the importance of secondary habitats in maintaining biodiversity in dry forests. sociobiology an international journal on social insects t marques1, mm espiríto-santo2, fs neves3, jh schoereder4 article history edited by gilberto m. m. santos, uefs, brazil received 14 december 2016 initial acceptance 13 february 2017 final acceptance 13 february 2017 publication date 17 october 2017 keywords formicidae; spatial scales; temporal scales; β-diversity; conservation strategies. corresponding author tatianne marques instituto federal de educação, ciência e tecnologia do norte de minas gerais fazenda varginha km 02 39560-000, salinas-mg, brasil. e-mail: tatianne.marques@ifnmg.edu.br two basic models of ecological succession have been described: assemblages controlled by dominance and assemblages controlled by foundation. in the first model, altered habitats will initially be colonized by pioneer species, which are later replaced by intermediate and climax species. this replacement occurs due to the niche requirements of the species and/or through competition, which is altered throughout the succession. in this model, there is a directional succession, and the species composition in the final stage is predictable (dauber & wolters, 2004; 2005). in the assemblages governed by the foundation model, some species may colonize the altered environment while inhibiting, facilitating, or tolerating the establishment of other species. if the early colonizers inhibit the establishment of other species, the assemblage 1 instituto federal de educação, ciência e tecnologia do norte de minas gerais campus salinas, salinas-mg, brazil 2 universidade estadual de montes claros, campus darcy ribeiro, montes claros-mg, brazil 3 universidade federal de minas gerais, belo horizonte-mg, brazil 4 universidade federal de viçosa, viçosa-mg, brazil research article ants t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages262 composition will remain the same throughout the succession process. this model is typically called “lottery model” (yu et al., 2001). conversely, if the initial species facilitate or tolerate the replacement of species, there will be change in the composition of species in the area. tropical dry forests (tdfs) have been subjected to intense and constant depletion because they are historically targeted for agriculture and human occupation (sanchezazofeifa et al., 2005). these factors can increase the area of tdfs undergoing ecological succession. in this scenario, vegetation and environment change mutually during secondary succession and in turn also affect biota, for example, by influencing resource availability and interactions among organisms (sousa-souto et al., 2016). according to ribas et al. (2002, 2003), competition is an important force that structures the ant assemblages which is regulated by habitat heterogeneity. however, some studies agree with a limited role of competition between ant species and pointed that environmental constraints may be more important for ant assemblages (baccaro et al., 2011; jacquemin et al., 2016). elucidating the pattern of variation in the ant assemblages along the succession and to identify environmental variables that explain this variation consists of subjects which have been poorly documented (sousa-souto et al., 2016). the seasonal changes that occur in the plant structure of tdfs influence different groups of animals and constitute another important temporal process. the main structural change of this ecosystem results from high foliar deciduousness, which is caused by a prolonged dry season that restricts the productivity of plants to the 3-5 months per year when rainfall is highly concentrated (sanchez-azofeifa et al., 2005). the seasonality of this forest affects the phenological patterns of plants, as well as the abundance, richness and composition of the associated fauna (cuevas-reyes et al., 2006). this explains why certain species may specialize in the use of resources under more severe conditions, resulting in a temporal variation of diversity and assemblages composition (silva et al., 2014). according to gove et al. (2005), the terrestrial ant richness increased in the dry season in mexican tdf. in studies conducted in a brazilian tdf, the arboreal ant species richness did not differ between seasons, but the abundance and richness of dung beetle species were higher in the wet season, and the composition of ant and dung beetles species changed between the seasons (neves et al., 2010a,b). among the various organisms that could be used in diversity studies, ants are considered an excellent model because they are highly abundant, diverse and have a wide geographical distribution (underwood & fisher, 2006). moreover, ants occupy multiple trophic levels and contribute substantially to ecosystem functioning (holldobler & wilson, 1990). in addition, the diversity of ants has been positively related to habitat complexity, as ant species richness increases with habitat heterogeneity and the availability of resources in tropical forests (ribas et al., 2003; ribas & schoereder, 2007; klimes et al., 2012). it is expected that land-use intensification of tdf may result in higher biotic homogenization (solar et al., 2015), which will in turn, decrease β-diversity among forests within each succession stage. in this study, we study the dynamics and structure of the ant assemblages at different time scales (ecological succession and seasonal variation). on the time scale for ecological succession, we tested the following hypotheses: (1) succession dynamics are controlled by dominance. thus, the ant species richness is higher in more advanced stages of succession, possibly due to the competitive exclusion of pioneer species by specialist species, leading to a shift in assemblage composition. (2) β-diversity is higher in advanced successional stages. with increasing environmental heterogeneity in succession process, there is a higher species turnover in these stages because ants occupy different spatial niches. we measured variables that could estimate the availability of resources and habitat conditions for the ant assemblages in order to identify factors that could explain the diversity patterns found within the succession models. on the time scale of seasonal variation, we tested the following hypothesis: (3) ant species richness is higher during the dry season and the composition of ant species changes between seasons. material and methods study area and sampling design the study was carried out at the parque estadual da mata seca (pems), a protected conservation area that was established by the expropriation of four farms in 2000. the park is currently managed by the instituto estadual de florestas. the pems has an area of 15,466.44 ha and is located in the middle são francisco valley in the municipality of manga, minas gerais (14°48’36” – 14°56’59”s and 43°55’12” – 44°04’12”w). the original vegetation of the park is tdf, which is dominated during the dry season by trees that are 90-95% deciduous (mayoctober) and grow on flat, nutrient-rich soil (pezzini et al., 2014). the climate is considered tropical semi-arid (köppen classification) and is characterized by a pronounced dry season during the winter. the region’s average temperature is 24.4ºc, and the mean annual precipitation is 818 ± 242 mm (pezzini et al., 2014). the main economic activities in the pems area before the expropriation of the farms were the cultivation of bean and maize, as well as livestock. approximately 1,525 ha (15%) of the pems comprise abandoned pastures in different stages of succession. in january 2006, 15 plots, each with an area of 20 x 50 m (0.1 ha each), were installed in forest patches inside the pems. these plots were undergoing early, intermediate or late successional stages (five plots for each stage; fig 1), as classified by the history of the land use (i.e., the time since the land was abandoned) and by the vertical (tree height, sociobiology 64(3): 261-275 (september, 2017) 263 number of forest strata) and horizontal (density of trees) structures in the vegetation areas (madeira et al., 2009). the early successional plots were used as pasture for at least 20 years and abandoned in 2000. they are characterized by herbaceous–shrubby vegetation with sparse trees forming a discontinuous canopy up to four meters in height. the intermediate successional plots were used as pasture for an unknown period and abandoned at the late 1980s. they have two vertical strata: the first is composed of 10–12 m tall deciduous trees and the second comprises a dense understory with many young trees and abundant lianas. the late successional plots have no records of clear-cutting for the last 50 years and are also have two strata. the first stratum is composed of deciduous trees that form a closed canopy 18–20 m high. the second stratum comprises a sparse understory of low liana density (see madeira et al., 2009; neves et al., 2014). plots from the same regeneration stage were located in a sequence and ranged 0.2-1.0 km from each other. plots were located along a 7 km transect encompassing all successional stages (fig 1). the plots had similar topographical, soil and microclimate characteristics, which reduced the variation in physical conditions that could affect the succession process and chronosequence study (vargas et al., 2008). ant sampling ants were sampling twice in each plot: in september 2008 (13.6 mm of rain), during the late dry season, and in february 2009 (247.5 mm of rain) at the onset of the wet season. in each of the five plots, five sampling points set 10 m apart were selected, representing a total of 25 points per stage. as different strata may have different ant assemblages (schmidt & solar, 2010), unbaited pitfall traps were installed at each sample point in hypogaeic (underground), epigaeic (ground level) and arboreal strata, totaling 75 points overall (5 plots x 5 sampling points x 3 pitfall traps = 75). arboreal pitfalls were designed based on the description in ribas et al. (2003), and epigaeic pitfall were similar to the arboreal pitfall but was buried so that the opening of the container was located at ground level. hypogaeic pitfalls were designed according to schmidt and solar (2010). to test the completeness of the sampling, we calculated the adequacy of the sampling effort with a species accumulation curve, both in dry and wet season. the traps in the epigaeic and arboreal strata were left in the field for 48 h. the impact caused by soil disturbance and the lack of attraction bait could hinder the performance of subterranean traps. so, we increased the sampling time of this type of trap to 72 h. this increased time would be also sufficient for the ants to reconstruct their tunnels (schmidt & solar, 2010). after trap removal, ants were sorted and taken to the laboratory, where they were mounted and identified. ants were sorted to genus and then to species or morphospecies and the similarity between epigaeic and arboreal ants (%) was calculated for each of the 75 points. ant nomenclature follows bolton (2016). voucher specimens are deposited at the reference collection at the laboratório de ecologia de comunidades at the universidade federal de viçosa (ufv), in viçosa, brazil. fig 1. map of the study region. in the detailed figure, square represent the landscape sampled at parque estadual da mata seca and sampling design of the 15 permanent plots. circles represent plots distributed in early stages of succession; squares represent intermediate and triangles represent late stages of succession. t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages264 explanatory variables we estimated variables that may represent the availability of resources and also the local conditions for the resident ants in each sampled stratum. to estimate conditions and resource availability to arboreal antes, we used two surrogates. basal circumferences of all trees found in a two-meter radius around each sample point were measured, as trees with higher basal circumferences occupy a larger area and could provide a higher number of nesting places (siemann et al., 1999) for arboreal ants. thus, these data yielded an estimate of the available resources for arboreal ants. also, we obtained a digital image next to the tree where the arboreal pitfall of each sample point was installed. images were taken using a nikon coolpix 4500 camera with a fish-eye lens attached, mounted on a leveled tripod at 1.5 m above the soil, with the fish eye focus set to infinity. the black and white images were taken in the late afternoon to avoid over-exposure from the midday sun. we used the software gap light analyzer to measure the percentage of canopy cover from the resulting photos (frazer et al., 1999). as photos were taken in the late dry season, at a time of canopy thinning, the canopy cover was estimated by the wood area that shades the forest floor. while acknowledging that this provided an underestimate of wet season canopy cover, this measure was used as an estimate of the climatic conditions of the stratum, because canopy cover can affect the availability of light and humidity in the understory. areas with greater canopy cover may maintain a microclimate that is more suitable for arboreal ant species (sousa-souto et al., 2016). we collected three 20 x 20 cm samples of leaf-litter one meter away from each o epigaeic trap. these samples were mixed, packed in plastic bags and transported to the community ecology laboratory (laboratório de ecologia de comunidades) at the ufv. leaf-litter was then transferred to paper bags and oven-dried at 60ºc until constant litter weight. the dry weight was used to estimate the availability of resources for epigaeic ants because the leaf-litter serves as a source of food and provides possible nesting sites for ants (muscardi et al., 2008). the percentage of canopy cover was also used as an estimate of abiotic conditions for epigaeic ants. the methodology was similar to the procedures conducted for the arboreal stratum, but the digital photographs were taken with the camera positioned at ground level close to the pitfall trap. to estimate conditions and resource availability to hypogaeic ants, a soil sample was removed with a metal ring of known volume (91.61 cm3) to determine the moisture content, as well as the density and porosity of the soil at each collection point. these two estimates were used as surrogates of conditions of this stratum for resident ants. schmidt et al. (2013) pointed soil density as determining factor for structure hypogaeic ant assemblages. the analyses were performed at the soil physics laboratory (laboratório de física do solo) at the ufv. we measured the tree species richness assisted by experts. we counted the number of tree species with a circumference at breast height (cbh) of more than 10 cm present in each plot of the successional stages in the tdf that was studied. tree richness was used as an estimate of the soil conditions for ants in all strata because the physical structure of soil is modified by the establishment of plant species in the area (sagar & verma, 2010). data analyses the data on the ants sampled in the dry season were used to test the first two hypotheses, concerning ecological succession. the sampling carried out during the wet season did not include the estimation of explanatory variables, it was used only to compare the ant assemblages in two different seasons. we constructed the species accumulation curves using the numbers of species and the number of pitfall traps sampled in different seasons, performing 10000 randomizations with replacement to generate confidence intervals using the vegan package in r 3.1.2 software (r development core team, 2014). due to variation in behavior of individual species and their interactions with the environment, any biodiversity sampling method must face the issue of underestimating true species richness. for this reason, estimation techniques are often used in diversity studies, particularly in samples taken over brief periods of time (gotelli et al., 2011). we used the sample-based species estimator first-order jackknife to estimate ant species number at each successional stage, stratum and season (appendix s1). these analyses were performed from presence/absence matrices. jackknife estimators provide the most accurate estimates of the true value of species richness observed in the site (gotelli & colwell, 2011). the jack1 estimator has performed consistently well in comparisons of various estimators of species richness, and is widely used for estimating species richness (gotelli & colwell, 2011). moreover, the jack1 estimator provides reliable estimates of local species richness even when habitats are heterogeneous within sampling units. calculations were performed using estimates software (colwell, 2006). β-diversity resulting from gamma diversity (total ant species in succession stages) minus alpha diversity (species richness per plots) and the similarity between epigaeic and arboreal ants (%) was calculated using the percentage of species sampled in two strata in each plot. we verified the response of estimated ant species richness, percentage of similarity between epigaeic and arboreal ants and β-diversity to successional stages and to seasons through general linear models (glms). all levels (stages) were compared in a contrast analysis for the aggregation levels and comparing the changes in deviations (crawley, 2013). analyses were followed by residual analyses to assess the adequacy of the models and error distribution (crawley, 2013). all analyses were performed in r platform (r development core team, 2014). the responses of estimated ant species richness to variables measured in the arboreal (richness of tree species, sociobiology 64(3): 261-275 (september, 2017) 265 basal circumference and canopy cover), epigaeic (richness of tree species, leaf-litter dry weight and canopy cover) and hypogaeic (richness of tree species, moisture, density and porosity of the soil) strata were verified using glms. succession stage was added to the models as a covariate. estimates of resources and conditions were used as explanatory variables, and the estimated ant species richness in each stratum was the response variable in each model. non-significant explanatory variables were withdrawn from the complete models, which were subjected to residual analyses to assess the adequacy of the model and the distribution of errors used (crawley, 2013). all tests were performed using r software (r development core team, 2014). to test the relationship between the environmental variables with successional stages we used glms, using normal errors when the response variable was an average and poisson errors when the response variable was count data. the analyses were performed using r (r development core team, 2014). using permutation multivariate analysis of variance (permanova, anderson, 2001) we tested the influence of successional stages, stratum and seasons and their interaction on ant assemblage composition, using the jaccard dissimilarity measure and 999 permutations. permanova is a permutation anova, which was developed to test the simultaneous response of one or more variables to one or more factors in analyses of variance (anova). a permutation multivariate analysis of dispersion (permdisp) was then run on this jaccard dissimilarity matrix to test the differences in the homogeneity of ant assemblages across successional stages, stratum and seasons (anderson, 2001; 2006). permdisp compares the distances from observations to their group centroid (analogous to a measure of variance) and thus allowed us to compare the heterogeneity of ant assemblage between the factors. the permanova used the ‘adonis’ procedure and the permdisp used the ‘betadisper’ procedure in the vegan package in r. results ant fauna we collected 95 ant species from 26 genera. the subfamily with the highest diversity was the myrmicinae (55 species), followed by formicinae (21 species), pseudomyrmecinae (six species), dolichoderinae (five species), dorylinae (three species), ponerinae (two species each). amblyoponinae, ectatomminae and heteroponerinae were each only represented by a single species. only four species of ants were found in all three successional stages and in both seasons: camponotus blandus, cyphomyrmex transversus, pheidole sp. 01 and pheidole sp. 07. the species camponotus substitutus was found in all three stages of succession but only in the wet season, and ectatomma edentatum, camponotus pr. lespesii, cephalotes grandinosus, solenopsis sp. 01, solenopsis sp. 11 and solenopsis saevissima were found in all three stages but only in the dry season (appendix s2). in the species accumulation curve we observed a tendency to an asymptote, indicating that the sampling effort was sufficient (appendix s3). these data suggest that the number of ant species sampled is close to the total number of species in dry and wet season. ant species richness the estimated ant species richness, accumulated in all strata was significantly higher in the intermediate and late stages of succession (f = 3.90; p = 0.049; fig 2a, appendix s1). nevertheless, arboreal and epigaeic ant richness did not change with the progress of ecological succession, but hypogaeic ant species richness was higher in the intermediate and late stages of succession (f = 6.31; p = 0.013; fig 2b). similarity between epigaeic and arboreal ants change along the succession. a higher similarity between the two strata was found in the initial stage and decreased significantly in the intermediate and late stages (f = 13.47; p = 0.0004; fig 2c). with respect to seasonal effects, ant species richness was higher in the dry season (25 ant species) than in the wet season (17,8 ant species) (f = 13.06; p = 0.001). β-diversity was significantly higher in the intermediate and late stages of succession (deviance = 23.661; p < 0.001; fig 2d), which may be a response to floristic differentiation along succession process (solar et al., 2015). arboreal and epigaeic ant species richness were not correlated to any of the variables used as surrogates of resources and conditions (p > 0.05). hypogaeic ant species richness increased significantly with soil porosity (f = 8.20; p = 0.013; fig 3a), soil moisture (f = 6.48; p = 0.024; fig 3b) and plant species richness (f = 6.71; p = 0.022; fig 3c). soil density was negatively correlated with porosity (r = 95.1%). successional stage affected only four surrogates of resources and conditions: soil average porosity (f = 20.54; p = 0.0001), leaf-litter average dry weight (f = 8.68; p = 0.004) and plant species richness (p < 0.0001) had an increasing trend along succession process while the soil average moisture (f = 5.48; p = 0.035) had a decreasing trend. changes in ant assemblage composition ant assemblage composition changes among successional stages (permanova, p < 0.001, table 1) and stratum (permanova, p < 0.001, table 1), that is, the position of centroids differed. moreover, there was not a significant interaction between factors (p > 0.05). when we consider the seasonal effect, ant composition differed significantly between seasons (permanova, p < 0.001, table 1). permdisp revealed significantly greater average distance from centroids in early (0.35) than intermediate and late successional stages (0.19 and 0.12) (f-value = 28.11, p < 0.0001, table 1). average distance in arboreal (0.43) is also greater than in epigaeic t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages266 fig 2. estimated total ant species richness (mean±se) (a), estimated hypogaeic ant species richness (mean±se) (b), similarity between epigaeic and arboreal species of ants (c), and beta diversity in a tropical dry forest from different the successional stages (d). different letters represent statistically significant differences between successional stages as determined by contrast analysis (p<0.05). factors permanova permdisp r2 f-value ant assemblages successional stages 0.08** 28.11*** stratum 0.42** 9.27** stages*stratum 0.50 seasons 0.11** 65.24*** significant differences are in bold. **<0.001, ***<0.0001. table 1. pair-wise permutation tests of differences in position and dispersion of ant species assemblages sampled in secondary tropical dry forest located in minas gerais, brazil. (0.32) and hypogaeic stratum (0.36) from centroid (f-value = 9.27, p < 0.001, table 1). moreover, ant composition in dry season is more distant from centroids (0.34) than wet season (0.12) (f-value = 65.24, p < 0.0001, table 1). e st im at ed a nt s pe ci es 0 5 10 15 20 25 30 35 a b b a e st im at ed a nt s pe ci es r ic hn es s 0 5 10 15 20 a b b b sp ec ie s si m ila ri ty ( % ) 0 10 20 30 40 a b b c successional stages b et a di ve rs ity 0 5 10 15 20 25 30 35 early intermediate late a b b d e st im at ed a nt s pe ci es 0 5 10 15 20 25 30 35 a b b a e st im at ed a nt s pe ci es r ic hn es s 0 5 10 15 20 a b b b sp ec ie s si m ila ri ty ( % ) 0 10 20 30 40 a b b c successional stages b et a di ve rs ity 0 5 10 15 20 25 30 35 early intermediate late a b b d e st im at ed a nt s pe ci es 0 5 10 15 20 25 30 35 a b b a e st im at ed a nt s pe ci es r ic hn es s 0 5 10 15 20 a b b b sp ec ie s si m ila ri ty ( % ) 0 10 20 30 40 a b b c successional stages b et a di ve rs ity 0 5 10 15 20 25 30 35 early intermediate late a b b d e st im at ed a nt s pe ci es 0 5 10 15 20 25 30 35 a b b a e st im at ed a nt s pe ci es r ic hn es s 0 5 10 15 20 a b b b sp ec ie s si m ila ri ty ( % ) 0 10 20 30 40 a b b c successional stages b et a di ve rs ity 0 5 10 15 20 25 30 35 early intermediate late a b b d discussion biological mechanisms driving succession the variation on species richness found in the ant assemblages in the studied tdf is consistent with the model of an assemblage controlled by dominance (see dauber & wolters, 2004; 2005). this model predicts that ant diversity will be lower in the early stages of succession and that the area will be colonized by generalist species that are common in open and modified environments, such as acromyrmex landolti, brachymyrmex patagonicus, cyphomyrmex gr. rimosus sp. 01, pheidole sp. 03 and solenopsis spp. the early stage revealed a differentiated species composition, and most species that were present can tolerate extreme conditions, such as low humidity and high temperatures caused by high solar irradiation from the discontinuous canopy of the forest (neves et al., 2013). throughout the succession process, other species colonized the plots, which caused an increase in the diversity of ants in the advanced stages of succession. the following mechanisms might explain the response of ant assemblages to ecological succession: (1) competition-colonization balance, in which competing species exclude species that are competitively inferior when both occupy the same area; and (2) the availability of adequate environmental conditions and resources to colonizing species. the first mechanism is centered on the processes of interaction among species, and assumes that interspecific competition is a limiting factor for the species richness of ant assemblages. although competitive interactions are usually considered important in the structure of ant assemblages (baccaro et al., 2011), some authors have reservations about the widespread importance of competition as a structuring force in the ant assemblages in the neotropical region (e.g. ribas & schoereder, 2002). the second mechanism is grounded in changes in physical structure of the environment that occur during the successional process. in tropical forests, plant succession sociobiology 64(3): 261-275 (september, 2017) 267 after a disturbance is often characterized by an increase in the structural complexity of the habitat, along with an increase in the quantity and diversity of resources (kalacska et al., 2004; madeira et al., 2009), both considered important factors for structuring ant assemblages (ribas et al., 2003; ribas & schoereder, 2007; klimes et al., 2012). with an increase of resources and changes of conditions along successional stages, a higher number of ant species can coexist in the same area because they occupy different spatial niches. solar et al. (2015) found higher β-diversity within all disturbance classes (logged, burnt, pasture, agriculture) in tropical forests, indicating less evidence for biotic homogenization within forests. accordingly, secondary forests maintained a high level of β-diversity among sites despite the initial disturbance (e.g. conversion to pasture or agriculture) removing the original biological assemblages, which reflects the importance of variation introduced by different successional pathways. then, the higher ant richness in the advanced successional stages may respond to an increase in environmental heterogeneity, which holds a higher β-diversity of ants in these stages. much of this variation in diversity occurred in the subterranean ant fauna, because in terms of richness only the ants of the hypogaeic stratum responded to ecological succession in the studied tdf. during natural regeneration, soil goes through physical changes due to the establishment of different plant species in the disturbed area. the areas in the early successional stage are characterized by bush vegetation and highly compacted soil because they were previously used as pasture until 2000. in the intermediate and late stages, there is a higher diversity of plants, and the soil is more porous. the hypogaeic stratum presents change in the ant fauna (schmidt & solar, 2010) that are closely linked to processes occurring in the vegetation, which could also contribute to the movement of water and soil as well as nutrient cycling (sousa-souto et al., 2007). thus, the changes in soil following disturbance exert pressure on the ant assemblage in this stratum, which results in a decrease in diversity. schmidt et al. (2013) found a similar relationship between hypogaeic species richness and soil density in atlantic rainforest fragments. in the studied tdf, the colonization of new ant species parallels the physical structure and condition changes of the soil, as well as the increase of plant diversity along the successional process. moreover, these environmental changes reflect not only in the richness, but also in the composition of the hypogaeic ant assemblage, which was sensitive to changes that occur in its stratum. the importance of mechanisms determining the richness of ants differs between strata. unlike hypogaeic ants, arboreal and epigaeic ant assemblages are consistent with the model of an assemblage controlled by foundation. this model predicts that diversity would not vary throughout successional process. inhibition, facilitation and tolerance are the three mechanisms involved in the diversity regulation in this assemblage model (connell & slatyer, 1977). the dynamics of each mechanism is based on different species interactions. these mechanisms fig 3. responses of the estimated hypogaeic ant species richness to soil porosity (a), soil moisture (b) and tree species richness (c) in a tropical dry forest from different successional stages in parque estadual da mata seca, minas gerais, brazil (p<0.05). e st im at ed h yp og ae ic a nt s pe ci es r ic hn es s soil porosity 0.46 0.48 0.50 0.52 0.54 0.56 0.58 0 5 10 15 a soil moisture (%) 0.08 0.09 0.10 0.11 0.12 0.13 0.14 0 5 10 15 b plant species richness 10 15 20 25 30 0 5 10 15 c t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages268 are more commonly observed in plant assemblages that are undergoing succession (connell & slatyer, 1977). for arboreal and epigaeic ant assemblages of our study, we hypothesize that habitat conditions influence more strongly ant species richness than the mechanisms that involve intraspecific interactions (inhibition, facilitation or tolerance). we found some evidence to our hypothesis that habitat condition is an important regulator of ant species richness in arboreal and epigaeic strata. our results point to a pronounced vertical stratification of the ant assemblages, which presents a decrease in similarity among epigaeic and arboreal strata along succession process. neves et al. (2010b) found the same results in the same study site. this vertical stratification occurs with the establishment of ant dominant species along succession. crematogaster obscurata is found in dry forests and nests in the dead portions of live trees (longino, 2003). species of this genus, and other members of the azteca and camponotus genera, are usually very aggressive and dominate the resources that are exploited by their assemblages. despite being dominant and found in dry habitats, c. obscurata was collected only in intermediate and late stages of succession, along with the azteca species and camponotus arboreus. therefore, the occurrence of these species in advanced successional habitats may be due to the presence of specific resources for structuring large nests, such as trees that have larger cbh, and not because some early ant species inhibit, tolerate or prepare the environment for the establishment of these ant species. seasonal variation in the tropics, some insect populations are influenced by seasonal changes in temperature and moisture (guedes et al., 2000), and ant assemblages are generally more diverse during the wet season (torchote et al., 2010). contrary to most studies in humid tropical forests, we found higher ant species richness during the dry season, demonstrating that such information is not adequate to the studies conducted on tdfs. this pattern of seasonal variation has been found in other tdfs. for example, assemblages of arboreal and terrestrial ants in a mexican tdf (gove et al., 2005) also have higher species richness in the dry season. however, neves et al. (2010b) using baited pitfall traps, found no seasonal differences in the arboreal ants species richness in the same tdf investigated in the present study. the presence of baits could induce distinct foraging patterns and alter the natural feeding pattern because high-protein and high-calory resources are being offered. furthermore, we believe that the presence of bait provided an even distribution of resources, which made the areas more equivalent in terms of resources. thus, the results of our study show a natural increase in the ant species number foraging in the dry season during different successional stages. the mechanism proposed by lassau and hochuli (2004) for the diversity of ants in less complex habitats might explain the higher ant species number collected in the tdf during the dry season. the foliar deciduousness of the trees could have simplified the physical environment by reducing the moisture and shade levels, which could have facilitated nest building and foraging by the ants, especially because most species are thermophilic (holldobler & wilson, 1990). moreover, as the availability of resources decreased in the dry season, the ants needed to increase their foraging area to find food, thereby increasing the chances that they would fall into the traps. during the wet season, the opposite process would occur, explaining the variation in species number and composition change between seasons. secondary tdfs conservation although the history of tdfs is marked by human exploitation, the future of this unique and diverse ecosystem depends on conservation, management and restoration. our results showed that environments with a simple physical structure (e.g., degraded forests) did not support species from the forest. a considerable fraction of species only appeared in the advanced stages of succession, reinforcing the importance of secondary habitats in maintaining the ecosystem biodiversity (gomes et al., 2014). we hypothesize that the restoration of the ant diversity in tdfs is faster than in rainforests. bihn et al. (2008) found that even after 35-50 years of regeneration in an atlantic forest, the ant assemblage did not show the species richness and composition expected for a mature forest. based on the pattern of succession in tdf, it is likely that 25 years of regeneration in this forest would be sufficient to restore the ant composition and richness, as also showed in the estimates made by neves et al. (2010b). tropical rain forests usually have a high level of alpha diversity (lopez & zambranatorrelio, 2006), and when interactions are disrupted by habitat disturbance, species recolonization and the establishment of inter and intraspecific interactions can take longer than the same processes in tdfs, which explains the difference in the resilience times for these different areas. acknowledgments the authors would like to thank c. f. sperber, o. desouza, r. c. solar and anonymous referees for reviewing the manuscript as well as a. queiroz and d. de paula for their help conducting fieldwork. we also thank r. s. m. feitosa and v. e. sandoval-gómez for their assistance with identifying the ants. we are grateful to ief for allowing this work to be performed at the pems and for the logistical support. this work received financial assistance from fapemig and the inter-american institute for global change research crn ii # 021, which is funded by the u.s. national science foundation (geo 0452325). finally, we would like to thank capes and cnpq for the authors’ grants. sociobiology 64(3): 261-275 (september, 2017) 269 references anderson, m.j. 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(2001). an empirical model of species coexistence in a spatially structured environment. ecology, 82: 1761-1771. doi: 10.2307/2679816 t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages272 sucessional stage arboreal epigaeic hypogaeic total observed estimated observed estimated observed estimated observed estimated early 6 9.2 12 20 2 2.8 18 27.4 early 3 3.8 9 13.8 5 8.2 12 16.8 early 5 7.4 9 13 5 7.4 15 22.2 early 4 6.4 5 7.4 2 2.8 7 8.6 early 9 14.6 6 9.2 5 7.4 14 25.6 intermediate 10 14.8 3 3.8 9 14.6 19 29.4 intermediate 8 11.2 11 15.8 8 11.2 22 30.0 intermediate 12 19.2 8 10.4 11 17.4 25 36.2 intermediate 8 11.2 10 13.2 6 8.4 18 25.4 intermediate 6 10 11 14.2 7 11 19 28.8 late 8 12 15 22.2 6 10 23 32.6 late 8 13.6 10 14 8 12 18 25.2 late 9 13 10 16.4 4 6.4 16 24.8 late 6 10.8 8 9.6 5 9 15 22.2 late 4 5.6 7 11 8 14.4 13 21.2 * all analyses were performed both with observed and estimated richness and results were similar. appendix s1. observed and estimated ant species richness* collected in a tropical dry forest fragment from different successional stages in parque estadual da mata seca, minas gerais, brazil. taxa stages / seasons initial intermediate late dry rainy dry rainy dry rainy amblyoponinae prionopelta punctulata mayr, 1866 h h h dorylinae acanthostichus serratulus (smith, 1858) h h cheliomyrmex morosus (smith, 1859) h labidus coecus (latreille, 1802) h h dolichoderinae azteca sp. 01 a azteca (gr. alfari) sp. 02 a dorymyrmex sp. 01 e forelius brasiliensis forel, 1908 e e e tapinoma melanocephalum (fabricius, 1793) a h ectatomminae ectatomma edentatum roger, 1863 e aeh e formicinae brachymyrmex sp. 01 h brachymyrmex sp. 02 e brachymyrmex coactus (mayr, 1887) eh h brachymyrmex pr. coactus h e brachymyrmex pr. longicornis h h brachymyrmex patagonicus mayr, 1868 aeh camponotus sp. 01 a camponotus sp. 02 ae ae appendix s2. ant species collected in a tropical dry forest fragment from different successional stages in parque estadual da mata seca during the dry and rainy seasons. a = arboreal stratum, e = epigaeic stratum and h =hypogaeic stratum. sociobiology 64(3): 261-275 (september, 2017) 273 appendix s2. ant species collected in a tropical dry forest fragment from different successional stages in parque estadual da mata seca during the dry and rainy seasons. a = arboreal stratum, e = epigaeic stratum and h =hypogaeic stratum. (cont.) taxa stages / seasons initial intermediate late dry rainy dry rainy dry rainy formicinae camponotus arboreus (smith, 1858) ae ae a ae camponotus atriceps (smith, 1858) ae ae camponotus blandus forel, 1901 ae ae ae ae e e camponotus cingulatus mayr, 1862 ae ae camponotus crassus mayr, 1862 a ae ae camponotus germaini emery, 1903 aeh e camponotus pr. lespesii ae ae aeh camponotus melanoticus emery, 1894 e camponotus renggeri emery, 1894 ae ae a camponotus substitutus emery, 1894 aeh ae ae camponotus pr. westermanni a camponotus vittatus forel, 1904 ae a ae a nylanderia pr. guatemalensis h heteroponerinae acanthoponera mucronata (roger, 1860) a myrmicinae acromyrmex landolti (forel, 1885) e e acromyrmex octospinosus (reich, 1793) e a acromyrmex rugosus (smith, 1858) eh e atta sexdens forel, 1908 e cephalotes atratus (linnaeus,1758) a a a cephalotes betoi de andrade, 1999 a cephalotes christopherseni (forel, 1912) a cephalotes grandinosus (smith, 1860) a ae aeh cephalotes minutus (fabricius, 1804) a cephalotes pavonii latreille, 1809 a e cephalotes pusillus (klug, 1824) a a ae a crematogaster abstinens forel, 1899 e crematogaster ampla forel, 1912 ae a a crematogaster pr. bruchi e e crematogaster evallans (forel, 1907) a crematogaster obscurata (emery, 1895c) a crematogaster torosa mayr, 1870 ae a crematogaster pr. torosa a a crematogaster victima smith, 1858 a cyphomyrmex gr. rimosus sp. 01 h cyphomyrmex transversus emery, 1884 e h h eh e e pheidole sp. 01 eh aeh ae aeh aeh aeh pheidole sp. 02 e e e eh pheidole sp. 03 e pheidole sp. 04 e eh e pheidole sp. 05 eh a h h pheidole sp. 06 aeh eh t marques, mm espiríto-santo, fs neves, jh schoereder – the role of succession and seasonality in ant assemblages274 appendix s2. ant species collected in a tropical dry forest fragment from different successional stages in parque estadual da mata seca during the dry and rainy seasons. a = arboreal stratum, e = epigaeic stratum and h =hypogaeic stratum. (cont.) taxa stages / seasons initial intermediate late dry rainy dry rainy dry rainy myrmicinae pheidole sp. 07 e e eh eh aeh aeh pheidole sp. 08 aeh e eh eh aeh pheidole sp. 09 h eh pheidole sp. 10 h pheidole sp. 11 h pheidole sp. 12 e rogeria blanda (smith,1858) h h solenopsis (gr. globularia) sp. 01 h h h solenopsis sp. 02 h e solenopsis sp. 03 h h solenopsis sp. 04 h solenopsis sp. 05 h solenopsis (gp. globularia) sp.06 h eh solenopsis sp. 07 eh h h h solenopsis geminata (fabricius, 1804) h e solenopsis sp. 09 eh eh solenopsis sp. 10 h h solenopsis sp. 11 e ah h solenopsis sp. 12 h h solenopsis sp. 13 eh solenopsis sp.14 h solenopsis sp.15 h h solenopsis sp.16 h solenopsis sp.17 e-h solenopsis saevissima (smith, 1855) e ah h strumigenys lilloana (brown, 1950) h wasmannia auropunctata (roger, 1863) e eh e wasmannia lutzi forel, 1908 a e ponerinae odontomachus bauri emery, 1892 e e h e e neoponera villosa (fabricius, 1804) e a pseudomyrmecinae pseudomyrmex flavidulus (smith, 1858) a pseudomyrmex gracilis (fabricius, 1804) a a pseudomyrmex (gr. pallidus) sp. 03 a pseudomyrmex (gr. pallidus) sp. 04 a pseudomyrmex schuppi (forel, 1901) a a a pseudomyrmex termitarius (smith, 1855) ae e e e sociobiology 64(3): 261-275 (september, 2017) 275 appendix s3. sample-based species accumulation curve of the total number of ant species collected with arboreal, epigaeic and hypogaeic pitfall traps placed in tropical dry forest fragments from different successional stages in parque estadual da mata seca, minas gerais, brazil. shaded area represents one standard deviation around mean values. 0 10 20 30 40 50 0 20 40 60 80 number of samples s pe ci es r ec or de d dry season wet season open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1210sociobiology 64(1): 69-77 (march, 2017) the similar usage of a common key resource does not determine similar responses by species in a community of oil-collecting bees introduction the comprehension of the factors that affect variations in abundance and species richness in natural systems is one of the most explored questions in ecology (morin, 1999; agrawal et al., 2007). in many situations, such variations seem to be determined by an interaction between both biotic (such as competition or predation) and abiotic (such as temperature and rainy fluctuations) factors (morin & lawler, 1995; ritchie, 2000; dyer & letourneau, 2003). however, there are groups of species that present similar specialized traits related to foraging behavior, predator avoidance strategies or even adaptations to specific conditions. in these cases, it may be that the existence of a similar adaptation makes the species bearing it more dependent on the variation of one or a abstract variations in abundance and species richness among communities are often determined by interactions between biotic and abiotic factors. however, for communities composed of species that share a common specialization (such as similar foraging adaptations) it may be a key ecological factor involved in the common specialization that affects community variations. to evaluate this possibility, we characterized the guild of oil-collecting bees of a neotropical savanna in brazil and tested whether differences in byrsonima abundance and availability of floral oil explain differences in species richness and abundance of oil-collecting bees of different tribes. both the number of species and total abundance of centridini species increased with the abundance of byrsonima. one plausible explanation for the stronger adjustment between the abundance of centridini and byrsonima is that the abundance of these plants affects not only the availability of floral oil, but also of pollen. these findings indicate that the existence of a common specialization among different species does not homogenize their response to variations in a common explored resource. sociobiology an international journal on social insects cml aguiar1, s lua2, m silva3, pec peixoto1, hm alvarez4, gmm santos1 article history edited by kleber del-claro, ufu, brazil received 10 october 2016 initial acceptance 16 december 2016 final acceptance 17 february 2017 publication date 29 may 2017 keywords centridini, oil quantification, oil-flowers, oil-bees, savanna. corresponding author cândida m. l. aguiar departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina, s/n, novo horizonte 44036-900, feira de santana-ba, brasil e-mail: candida.aguiar @gmail.com few key ecological factors. consequently, variations in such factors should trigger similar responses in the species that share similar adaptations. one species group that may respond to variations in a key ecological factor is oil-collecting bees that explore the resources produced by oil-flower species of different families. these plant-pollinator systems are highly specialized, with interactions based one resource of high energetic value (oil). few plant families provide oil on specialized flower structures (elaiophores), which are accessible only to flower visitors that are able to “scratch” epithelial elaiophores with specialized structures (combs) on their legs, or use specialized setae on their legs to obtain oil from trichomatic elaiophores (neff & simpson, 1981; vogel & machado, 1991). as consequence of these adaptations, oil-collecting bees and oil-producing 1 departamento de ciências biológicas, universidade estadual de feira de santana, bahia, brazil 2 ppg zoologia–uefs, universidade estadual de feira de santana, bahia, brazil 3 faculdade de tecnologia e ciências, salvador, bahia, brazil 4 departamento de ciências exatas, universidade estadual de feira de santana, bahia, brazil research article bees cml aguiar et al. – oil-collecting bees responses to oil/byrsonima abundance70 plants have interactions of high intimacy: floral oil is essential to feed the larvae and build nests (vinson & frankie, 2000; aguiar & garófalo, 2004; michener, 2007), while the pollination services provided by these bees are needed for the plant reproduction (vogel & machado, 1991; sigrist & sazima, 2004; costa et al., 2006; sazan et al., 2014). despite the high dependence between oil-collecting bees and oil-producing flowers, the effects of the abundance of floral resources on the communities of oil-collecting bees had been poorly studied. the few existing studies report that both the abundance and richness of centridini species (a tribe composed of oil-collecting bee species) may be determined by the abundance malpighiaceae species, mainly byrsonima sericea dc. this relationship between bee abundance or richness and malpighiaceae species seems to hold both in local and regional scales (ramalho & silva, 2002; rosa & ramalho, 2011). however, it is important to note that other bee tribes, such as tetrapediini and tapinotaspidini possess similar specializations and dependency to explore oil-flowers as centridini bees. consequently, if the foraging specialization observed in these bees is responsible for the strong relationship between centridini and malpighiaceae species, this pattern should also be detected in the other two bee tribes. in addition, since oil is the main food source for oil-collecting bees, it is possible that the abundance and richness of these insects are more dependent on oil availability than plant abundance. to evaluate the hypothesis that the specialization in foraging behavior is responsible for the tight relationship between the abundance and richness of oil-collecting bees and oil producing flowers, we made focal samplings of the flower visitors of two byrsonima species. we tested whether differences in the amount of floral oil available and in the local abundance of two byrsonima species explain differences in the richness and abundance of oil-collecting bees of different tribes at local scale. material and methods study area we carried out the study in the surroundings of the chapada diamantina national park (12°25’ s; 41°29’ w), palmeiras municipality, state of bahia, northeastern brazil. the local climate is hot, with humid summer, and four to five dry months. rains occur from december to april (jesus et al., 1983; nimer, 1989) and the annual rainfall varies from 600 to 1,000 mm (cei, 1994). the region of chapada diamantina has diverse vegetation, including campos rupestres (sandstone outcrop vegetation), campos gerais (grasslands), cerrados (savannas), caatingas (deciduous thorny woodlands), and montane forests, which form a vegetation mosaic (conceição et al., 2005). we collected samples in a cerrado area, with a predominance of herbaceous plants and shrubs, and sparse trees with up to 5 m. sampling we carried out field work during the flowering period of byrsonima sericea and byrsonima cydoniifolia a. juss (malpighiaceae), the most common oil-producing plants in this area. the total abundance of byrsonima was variable among the sampled sites (23 to 233 individuals per transect), but in general there were more than 55 individuals per transect. details on the morphology and floral biology of these plant species can be found in teixeira and machado (2000), and sazan et al. (2014). the flowers of b. sericea are attractive to bees for about a day and a half (teixeira & machado, 2000), and the flowers of b. cydoniifollia for approximately two days (sazan et al., 2014). in this region, b. sericea flowering season lasts from december to march, while b. cydoniifolia blooms in december and january (personal observation). we selected four sites, with distances varying from 1.1 to 3.8 km from one another. in each site, we established three transects, 1,000 m in length. in each month, we randomly selected four transects (one per site) for sampling on consecutive days. each whole transect was sampled from 0900 h to 1600 h, by two collectors. the collection of bees on b. sericea and b. cydoniifolia flowers lasted eight months, from january to march 2011, from october to december 2012, and from january to february 2013, a total of 175 h of sampling effort. the specimens were deposited in the johann becker entomological collection (zoology museum of the state university of feira de santana, mzfs) and in the entomological collection of the university of brasília. quantification of the number of flowers to estimate the number of flowers produced by b. sericea and b. cydoniifolia, we randomly picked ten individuals of each species, during the peak flowering period. for each plant individual, we randomly placed on the canopy a “square box” measuring 25 x 25 cm x the height of the “square box” (range 1.40 m – 1.70 m), which varied according to plant height. then, we counted all inflorescences that fitted inside the “square box”. after counting, we removed ten inflorescences of each individual plant to count the total number of open flowers. we also measured the radius and height of the tree canopy of each plant and used these measures to estimate the volume of flowers per canopy on each plant individual (10 individuals per each byrsonima species). hence, based on the volume of the “square box” and the volume of the canopy flowers, we estimated the amount of inflorescences per byrsonima individual (number of inflorescences per canopy on blossom= number of inflorescences x (volume of the canopy on blossom/ volume of the “square box”). to estimate the total number of flowers produced per individual of b. sericea and b.cydoniifolia, we multiplied the total number of flowers per inflorescence by the average number of flowers per inflorescence. finally, we counted all individuals of b. sericea and b. cydoniifolia observed in each transect, to estimate the abundance of byrsonima. sociobiology 64(1): 69-77 (march, 2017) 71 oil quantification in byrsonima flowers to estimate the amount of oil produced by b. sericea and b. cydoniifolia in each transect, we multiplied the average amount of oil produced by one flower by the estimated number of flowers per plant. we used the value obtained as an estimate of the amount of oil produced by an individual plant. then, to estimate the total amount of oil available in each of the 12 transects, we multiplied the amount of oil produced by an individual plant by the number of individual b. sericea and b. cydoniifolia with elaiophores recorded in each transect. to quantify oil production per flower, we collected ten inflorescences from ten individuals of b. sericea and b. cydoniifolia separately, with pre-anthesis flowers that had opened on the same day. we dried the flowers in an oven for 72 h at 60 ºc, and then removed the petals and reproductive structures of each flower, leaving only the floral receptacle and the elaiophores. then, for each of the two byrsonima species, we separated the dried material into eight samples of 1 g each. we extracted floral oil using the soxhlet system, with hexane solvent. we placed each sample (n = 8) on a cellulose disk (filter paper) and maintained it in the extractor system for 24 h. we subjected the resulting mixture (solvent plus floral oil) to a low pressure system in a rotaevaporator, to remove the excess of solvent and recover the oil. we weighted the oil obtained per sample in a high precision analytical balance. the oil content (oc) per sample was calculated as a percentage, using the equation: where: bu = biomass of flowers used (g); v% = humidity extracted of the flowers (%) to calculate the amount of oil produced per flower, we divided the amount of oil extracted from 1 g of flowers by the total number of flowers used for the extraction. data analysis to estimate the species richness of bees collected on byrsonima flowers, we used the chao-2 and jackknife1estimators. these estimators are based on the incidence of unicates and duplicates and on the number of singletons, respectively, using 150 randomizations (colwell, 2013). to oil-collecting bee species number of individuals on bs flowers number of individuals on bc flowers total of individuals centridini 259 115 374 centris (centris) aenea lepeletier 108 60 168 centris (centris) nitens lepeletier 2 4 6 centris (centris) spilopoda moure 24 9 33 centris (centris) sp.1 1 1 2 centris (hemisiella) tarsata smith 10 2 12 centris (ptilotopus) moerens (perty) 8 0 8 centris (ptilotopus) sponsa smith 3 0 3 centris (trachina) longimana fabricius 1 0 1 epicharis (epicharis) bicolor smith 32 10 42 epicharis (epicharis) sp.1 4 0 4 epicharis (epicharitides) cockerelli friese 0 2 2 epicharis (epicharoides) xanthogastra moure & seabra 2 0 2 epicharis (epicharoides) sp.1 35 17 52 epicharis (triepicharis) analis lepeletier 29 10 39 tapinotaspidini 137 17 154 lophopedia sp.1 15 0 15 monoeca mourei aguiar 1 0 1 paratetrapedia punctata aguiar & melo 1 1 2 tropidopedia nigrocarinata aguiar & melo 42 4 46 xanthopedia sp.1 21 5 26 urbanapsis diamantina aguiar & melo 57 7 64 tetrapediini 80 21 101 tetrapedia amplitarsis friese 72 21 93 tetrapedia diversipes klug 8 0 8 total 476 153 629 table 1. oil-collecting bees visiting byrsonima sericea (bs) and byrsonima cydoniifolia (bc) (malpighiaceae) flowers in a cerrado area in chapada diamantina, northeastern brazil. cml aguiar et al. – oil-collecting bees responses to oil/byrsonima abundance72 individuals b. cydoniifolia b. sericea no. flowers/ plant amount of oil/canopy (g) no. flowers/plant amount of oil/canopy (g) 1 3,803.77 0.82 341,004.00 181.88 2 13,972.95 3.01 51,606.53 27.52 3 25,162.70 5.42 220,912.31 117.83 4 226,324.37 48.77 145,068.00 77.37 5 47,481.72 10.23 5,411.45 2.89 6 20,216.98 4.36 162,225.76 86.52 7 112,486.56 24.2 204,863.65 109.27 8 79,943.49 17.23 98,626.34 52.60 9 13,975.71 3.01 60,345.88 32.19 10 2,426.59 0.52 123,855.67 66.06 table 2. number of flowers and amount of oil in ten individuals of byrsonima cydoniifolia and b. sericea (malpighiaceae) in a cerrado area in the chapada diamantina, northeastern brazil. assess whether the sampling effort was suitable, we built a rarefaction curve based on the species observed (mao tau). these analyses were made in the program estimates 8.20. we used generalized linear mixed-effects models to assess whether the number of bee species and individuals of different tribes varied with oil availability and plant abundance. we built models containing bee richness or abundance as the response variables, and tribe, oil abundance, and plant abundance as fixed factors. however, as oil availability and plant abundance were correlated (r = 0.53, p < 0.001), we built different models containing only oil availability or plant abundance as explanatory variables. to assess which model better explained the variations in abundance and richness of bees, we used the akaike information criterion corrected for small samples (aicc burnham and anderson 2002). the aicc represents a heuristic approach used to select the most parsimonious model that best approximates the true process under investigation among a set of candidate models. consequently, for each response variable, the model with the smaller aicc value will represent the best one. in all models, we fitted each sampled area nested per field trip as a random factor. whenever necessary, we performed planned contrasts to test for differences in response variables among centridini, tapinotaspidini, and tetrapediini tribes. we made all analyses using the packages lme4 (bates et al., 2014), bbmle (bolker & r development core team, 2012), and multcomp (hothorn et al., 2008) in r software (r development core team, 2014). results the guild of oil-collecting bees sampled on flowers of b. sericea and b. cydoniifolia was composed of 22 species (table 1). the species rarefaction curve showed signs of stabilization, with the sampled species richness corresponding to 89% and 97% of the estimates provided by jackknife-1 and chao-2, respectively. the centridini tribe showed higher richness (14 species; 64%) and abundance (n=374; 59% of all oil-collecting bees sampled) than the other tribes. centris aenea (lepeletier) and tetrapedia amplitarsis friese were the most abundant species (table 1). the total number of open flowers per crown in blossom (n=10 plants) varied from 5,411 to 204,863 flowers in b. sericea, and from 2,426 to 226,324 flowers in b. cydoniifolia (table 2). the oil content extracted from flowers (1 g samples) was higher in b. sericea (0.074%) than in b. cydoniifolia site/transect total oil estimated (g) number of individuals byrsonima centridini tapinotaspidini tetrapediini sampling effort (h) bs bc a r a r a r i t1 7,773.4 102 7 29 4 15 3 14 2 21 i t2 1,602.4 20 8 1 1 10 3 31 1 7 i t3 3,005.8 7 210 56 8 0 0 0 0 14 ii t4 3,025.6 19 135 40 5 6 2 0 0 21 ii t5 4,021.4 20 213 17 7 2 2 0 0 14 ii t6 1,320.4 10 48 10 5 1 1 0 0 7 iii t7 9,575.8 127 0 26 3 8 2 1 1 14 iii t8 4,321.4 57 2 1 1 45 4 23 1 7 iii t9 14,627.6 194 0 43 10 15 4 13 2 21 iv t10 16,397.2 217 3 146 10 22 4 1 1 28 iv t11 4,297.8 57 0 5 3 30 5 13 2 14 iv t12 1,734.2 23 0 2 1 0 0 5 2 7 table 3. abundance of the two species of byrsonima (bs: b. sericea, bc: b. cydoniifolia), the amount of floral oil estimated at each transect, abundance (a) and richness (r) of each tribe of oil-collecting bees per transect, sampling effort (hours) to capture bees in each transect in a cerrado area in the chapada diamantina, northeastern brazil. sociobiology 64(1): 69-77 (march, 2017) 73 (0.024%) (t = 13.5; df = 13; p<0.001). the average amount of oil produced per flower was 0.0005 g in b. sericea and 0.0002 in b. cydoniifolia. the amount of oil produced per plant varied from 2.89 to 181.88 g (µ = 75.41 ± 34.98 sd, n=10) in b. sericea, and from 0.52 to 48.77 g (µ = 11.75 ± 210.53 sd, n = 10) in b. cydoniifolia. the abundance of the two byrsonima species varied largely among the 12 transects (from 23 to 233 individuals/ transect), which resulted in differences in floral oil availability among transects. the estimates of floral oil produced per transect varied from 1,320.4 to 16,397.2 g (table 3). despite the high abundance of byrsonima in transects t3 and t5, they had relatively low oil availability (t3: 3,005.8 g and t5: 4,021.4 g). this low oil availability resulted from the predominance of b. cydoniifolia in these transects. richness (1-10 species) and abundance (1-146 individuals) of centridini varied largely among transects. centridini was more abundant in the transects t10, t9, and t3, which are three out of the four transects with the highest number of byrsonima plants (194-220 individuals) (table 3). the abundance of oil-bees was more strongly related to the abundance of byrsonima than to floral oil availability (table 4). the most parsimonious model indicated that the relationship between total oil-bee abundance and byrsonima abundance differed among tribes (χ2 = 334.18, df = 5, p < 0.001). in fact, the removal of the interaction term between tribe and plant abundance from the model indicated that this interaction was important to explain variations in total bee abundance (χ2 = 248.41, df = 2, p < 0.001). the total abundance of centridini increased with byrsonima abundance (fig 1a), fig 1. total number of individuals of centridini (a), tapinotaspidini (b) and tetrapediini (c) in relation to the number of byrsonima plants in a cerrado area in chapada diamantina, northeastern brazil. each dot represents one sampled transect per month (there are superimposed dots). cml aguiar et al. – oil-collecting bees responses to oil/byrsonima abundance74 whereas the effect was the opposite for tetrapediini species (fig 1b). the total abundance of tapinotaspidini species was unrelated to plant abundance (fig 1c, table 5). the number of oil-collecting bee species was also more strongly related to byrsonima abundance than to oil availability (table 4). the most parsimonious model indicated that the relationship between oil-collecting bee richness and byrsonima abundance differed among tribes (χ2 = 74.42, df = 5, p < 0.001). the removal of the interaction term between tribe and plant abundance from the model indicated that this interaction was important to explain variations in oil-collecting bee richness (χ2 = 19.04, df = 2, p < 0.001). the number of centridini species increased with byrsonima abundance (fig 2a), whereas the number of tetrapediini and tapinotaspidini species was unrelated to byrsonima abundance (fig 2b and 2c, table 5). response model aicc df δi wi abundance tribe + plant abundance + tribe*plant abundance 470.8 8 0.0 1 tribe + oil availability + tribe*oil availability 615.4 8 144.6 <0.001 tribe + oil availability 711.7 6 240.9 <0.001 tribe 712.9 5 242.0 <0.001 tribe + plant abundance 714.2 6 243.4 <0.001 oil availability 791.9 4 321.1 <0.001 plant abundance 794.1 4 323.3 <0.001 richness tribe + plant abundance + tribe*plant abundance 241.7 8 0.0 0.988 tribe + oil availability 251.3 6 9.6 0.008 tribe + oil availability + tribe*oil availability 255.0 8 13.3 0.001 tribe 255.1 5 13.4 0.001 tribe + plant abundance 255.8 6 14.0 <0.001 oil availability 300.3 4 58.5 <0.001 plant abundance 304.8 4 63.0 <0.001 table 4. summary of the models describing variation in total bee abundance and richness in relation to the tribe (centridini, tapinotaspidini, and tetrapediini), plant abundance, and oil availability, in two species of byrsonima (malpighiaceae) in a cerrado area in chapada diamantina, northeastern brazil. aicc represents the value of the akaike information criterion corrected for small samples; δi is the difference between the most parsimonious model i, wi is the akaike weight of model i. wi is the probability that the model is the best one among all candidate models. response contrast estimate se z p abundance centridini vs. tapinotaspidini 18.47 0.19 -9.60 <0.001 centridini vs. tetrapediini 36.07 0.35 -10.38 <0.001 tapinotaspidini vs. tetrapediini 17.60 0.33 -5.38 <0.001 richness centridini vs. tapinotaspidini 0.783 0.28 -2.78 0.027 centridini vs. tetrapediini 15.89 0.42 -3.80 <0.001 tapinotaspidini vs. tetrapediini 0.81 0.43 -1.85 0.241 table 5. planned contrasts for slope differences between tribes. each slope was calculated from the relationship between bee abundance or bee richness (response variables) and plant abundance (explanatory variable). estimated values indicate the difference in slope values between the first and the second tribe mentioned in each contrast. discussion the guild of oil-collecting bees of the studied savanna was diverse, with many rare and a few dominant species. the richness of oil-collecting bees of the studied savanna in chapada diamantina was in part a result from concentrating the sampling effort on byrsonima species, with which oilbees have a close relationship (ramalho & silva, 2002; rosa & ramalho, 2011). in others studies on bee communities in the same region (martins, 1994; silva-pereira & santos, 2006), sampling effort was equally distributed among all melittophilous flora. the savannas in chapada diamantina seem to harbor much richer bee faunas than sandstone outcrop vegetation, which are contiguous to the savannas in many sites of this region. centridini, in particular the genus centris, was the group with highest richness in the studied guild, similarly as recorded in other brazilian savannas (martins, 1994; silveira & campos, 1995; andena et al., 2005; 2012) and other neotropical biomes (araújo et al., 2006; santos et al., 2013; silva et al., 2015). mello et al. (2013), who used a network approach to assess the interactions between oil-flowers and oil-bees at the biome level, highlighted the importance of sociobiology 64(1): 69-77 (march, 2017) 75 centris and epicharis to the guild of oil-collecting bees. the authors reported that these two taxa occupy highly central roles in each network, because they showed a very large number of interactions (hubs) or connected different modules in the networks (connectors). although presenting similar foraging needs, distinct taxonomic groups that compose the local guild of oil-bees responded differently to the abundance of byrsonima. the abundance of centridini increased with the abundance of byrsonima, whereas the abundance of tetrapediini (tetrapedia spp) decreased and the abundance of tapinotaspidini showed no correlation. a similar trend was observed in species richness: the number of centridini species increased with the abundance of byrsonima, whereas the number of tetrapediini and tapinotaspidini species showed no correlation. these findings indicate that centris and epicharis species are more dependent on the resources provided by byrsonima than tetrapedia and tapinotaspidini species. perhaps species of tetrapedia and tapinotaspidini use other plants more intensively as food sources, as suggested by studies on the diet of the larvae of tetrapedia diversipes klug (menezes et al., 2012; neves et al., 2014) and tetrapedia curvitarsis friese (campos, unpubl. data). in addition, females of centris and epicharis species are often larger than females of tetrapedia and tapinotaspidini species. because oilcollecting bees typically “hug” the flower while foraging, it may be that the greater body size of centridini species prevent flower exploitation by other species. the abundance of centris and epicharis species associated with this possible exclusion behavior may result in exploitation dominance of byrsonima by centridini. the total abundance of oil-collecting bees was more strongly related to the abundance of byrsonima than to the amount of oil offered by these flowers. one possible explanation for the best adjustment between abundance of centridini and byrsonima plants (in detriment of oil availability) is that the abundance of these plants affects not only floral oil, but also pollen availability. pollen represents one of the main aspects of habitat quality for bee populations. many centridini species use byrsonima as a pollen source (aguiar & gaglianone, 2003; dórea et al., 2010; rabelo et al., 2012), and, therefore, we expect that fluctuations in byrsonima abundance directly influence bee reproduction. the local availability of resources to supply food and material to build nests (e.g., floral oil used to coat brood cells and nest plugs) should strongly influence the movement of females in the landscape. this movement in search of resources is expected to affect the local abundance of these bees in parts of the habitat. we can assume that centridini females that decide to nest in areas with a higher abundance of byrsonima, and, therefore, with high availability of oil and pollen, face lower costs to produce offspring. hence, this decision would contribute to an increase in fitness. as a result, we would expect an increase in the local abundance of these fig 2. number of bee species belonging to centridini (a),tapinotaspidini (b) and tetrapediini (c) in relation to the number of byrsonima plants in a cerrado area in chapada diamantina, northeastern brazil. cml aguiar et al. – oil-collecting bees responses to oil/byrsonima abundance76 bees in habitats with high availability of resources. the species richness and abundance of centridini increased with the abundance of byrsonima, which corroborates rosa and ramalho (2011). since we developed our study in a different vegetation type than previous studies, it is probable that the positive relationship between the abundance of centridini and byrsonima does not depend on phytophysiognomy. however, we showed that plant abundance per se was more important to determine the abundance and richness of centridini than the amount of oil available. this finding suggests that other resources, together with oil, are important for the development and maintenance of these bees. however, such relationship does not seem to be the result of the foraging specialization observed in oil-collecting bees, since the abundance and richness of species of tetrapediini and tapinotaspidini were unrelated to the abundance of byrsonima. consequently, it seems that even for species that share a common dependency and similar adaptations to exploit a specific resource (e.g. floral oil), other traits may be equally important in affecting community dynamics. acknowledgments we are grateful to prof. antonio j.c. aguiar (university of brasília, unb), prof. maria c. gaglianone (uenf), and prof. fernando c.v. zanella (unila), for identifying bee species. we also thank the national council for scientific and technological development, brazil (cnpq, no. 558228/2009-7) for financial support for this project. cnpq granted research fellowship to c.m.l. aguiar and g.m.m. santos (no. 306403/2012-9; 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(1991). pollination of four sympatric species of angelonia (scrophulariaceae) by oil collecting bees in ne brazil. plant systematics and evolution, 178: 153-178. doi: 10.1007/bf00937962 doi: 10.13102/sociobiology.v65i2.2048sociobiology 65(2): 112-129 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 review of ants (hymenoptera: formicidae) as bioindicators in the brazilian savanna introduction the majority of environmental problems are related to anthropic action, with habitat loss and fragmentation being a major threat to biodiversity (bascompte & sole, 1996; dirzo & raven, 2003; balmford et al., 2005; oliver et al., 2015). this affects the richness, species abundance, distribution and biodiversity in general. (lovejoy, 1986; kattan et al., 1996; kellman et al., 1996; turner, 1996; steffan-dewenter & tscharntke, 1997; kageyama & gandara, 1998; golden & crist, 1999). fragmentation is more threatening than habitat loss, depending on the existence of ecological effects that can be attributed to changes in the spatial pattern of habitat regardless of their quantity (fahrig, 2003). the effects of area loss and fragmentation are complex, since both spatial abstract the brazilian savanna is threatened mainly by the expansion of agriculture and livestock. regarding environmental problems, habitat loss in the biome and the need to verify studies on ants as bioindicators, the goal of this paper was to carry out a bibliographic review of the literature about ants as bioindicators in this biome from the last 30 years. we searched papers about ants as bioindicators in the brazilian savanna from the last 30 years, refining the search between the years 1986 to 2016 and we analyzed 16 papers about ants as bioindicators. monitoring studies focusing on ants as bioindicators in the brazilian savanna started in 1992 and increased since 2002. the results obtained in the studies presented changes in the species richness and composition in relation to potential bioindication. in general, 167 species are defined as bioindicators of the brazilian savanna and are related to specific habitats. we verified that most studies were done minas gerais state. we noted that the absence of rigorous analysis damaged the results of the studies, as well as the knowledge of ant fauna biology for correct attribution of indication characteristics of preserved or degraded areas. sociobiology an international journal on social insects m tibcherani1,2, vaf nacagava2,3, r aranda3, rl mello1 article history edited by kleber del-claro, ufu, brazil received 29 august 2017 initial acceptance 07 november 2017 final acceptance 13 november 2017 publication date 09 july 2018 keywords bioindicator; brazilian savanna; degradation; formicidae; hotspot; preservation. corresponding author mariáh tibcherani laboratório de sistemática de diptera instituto de biociências – inbio universidade federal de mato grosso do sul campo grande-ms, brasil. e-mail: mariah.biologia@gmail.com phenomena exhibit a strong correlation (mortelliti et al., 2010). however, as a result, we always have the change of the configuration, with the replacement of the original vegetation by another type of habitat and, consequently, connectivity becomes disrupted (smith et al., 2009). defining the factors and effects of these changes on the remaining environments and their implications for species richness and composition is part of the management practice of degraded areas, which include the effect of size, isolation and vegetation type within the spots to ensure biological viability in this area (fahrig, 2003). in this context, the brazilian savanna, with about two million km², is the second largest brazilian biome and is considered a hotspot for the conservation of biodiversity (myers et al., 2000; silva & bates, 2002; klink & machado, 2005; carvalho et al., 2009), which is primarily threatened 1 laboratório de sistemática de diptera, instituto de biociências – inbio, universidade federal de mato grosso do sul, campo grande-ms, brazil 2 programa de pós-graduação em biologia animal, instituto de biociências – inbio, universidade federal de mato grosso do sul, campo grande-ms, brazil 3 laboratório de ecologia de comunidade de insetos, universidade federal de mato grosso, departamento de ciências biológicas, rondonópolis-mt, brazil review mailto:mariah.biologia@gmail.com sociobiology 65(2): 112-129 (june, 2018) 113 by the expansion of agriculture and livestock (cunha et al., 2008; carvalho et al., 2009; strassburg et al., 2007). between the 1960s and 1980s, 67% of the brazilian savanna areas were modified by burning, deforestation, fertilizer and agrochemicals use due to the expansion of agriculture and livestock (ramos et al., 2003a). in addition, according to machado et al. (2004), deforestation rates of the biome ranged from 22,000 to 30,000 km² by the end of the 20th century. such expansion and degradation of the brazilian savanna severely threatens the biome and endemic species, making monitoring and policies for future biome prevention necessary (klink & machado, 2005; ferreira et al., 2013; carranza et al., 2015; strassburg et al., 2017). considering the increasing environmental problems due to habitat loss, methods for environmental monitoring have been developed using biological indicators (tolmasquim, 2001; hunter, 2002; henry et al., 2007), which consider the presence of certain species, or taxa, as an estimate of welldefined environmental conditions (wilson, 1994; hughes et al., 2010; gerlach et al., 2013; beiroz et al., 2014; siddig et al., 2016). the presence of certain organisms in a habitat provides information about the environmental situation from reactions when exposed to different types of degradation (allaby, 1992; mazzoni-viveiros & trufem, 2004). according to mcgeoch (1998) bioindicator species are divided into environmental, ecological, and diversity indicators, each one indicating some sensitive and changes in environment or in biodiversity. the most commonly used indicators are environmental quality and integrity (42%), pollution and contamination (18%), and management and restoration of ecosystems (18%). however, syntheses and revisions are infrequent (2%) (siddig et al., 2016). in general, invertebrates correspond to approximately 30% of the work on bioindication (siddig et al., 2016), among them ants, which can be classified as environmental, ecological and diversity bioindicators, since they have a sensitivity to environmental change, great abundance, are present in both intact habitats and disturbed areas, and are taxonomically well known (majer, 1983a). secondly, they have high species diversity, behavioral plasticity, besides being of ecological and functional importance in almost all trophic levels of terrestrial ecosystems (majer, 1983b; greenslade & greenslade, 1984; winston, 1995; alonso, 2000). among the studies on ants as bioindicators, ribas et al. (2012) carried out a bibliographic review that covered a period of 25 years (1987 to 2010) on ants as bioindicators in brazil, but did not separate studies by brazilian biomes. one aspect that has been poorly addressed in reviews, theoretical and applied paper in ants is the importance of conducting assessment of ants as biological indicators dealing particurlarly with a biome. since species or group of species that may be biological indicators of conservation in one biome, may not be in another or may indicate similar and different states (andersen, 1997; nordén & appelqvist, 2001). such separation is still unclear and the knowledge of ant fauna is important for this statment. thus, considering the growing environmental problems and habitat loss in the brazilian savanna, and the need to verify studies of ants as bioindicators in the biome, the target of this study was to fulfill a bibliographical review on ants as bioindicators in the brazilian savanna within the last 30 years, examining: (i) the development of the theme over the years, (ii) the most frequent types of bioindication, (iii) the parameters of the myrmecofauna analyzed, (iv) the collection methodologies used, (v) to find out which and if ants are good bioindicator species of preserved and degraded environments and (vi) the most frequent state of research in the area. materials and methods considering the work of ribas et al. (2012a), we analyzed the studies that were executed in the brazilian savanna and searched out for other papers about ants as bioindicators in that biome within the last 30 years, refining the search between 1986 and 2016. the search was done from september to november/2016, using the same key words used by ribas et al. (2012a): “ant”, “bioindicator”, “indicator”, “brazil”, adding the words “brazilian savanna” and “cerrado” to our research. this was made in four databases: scielo, google scholar, scopus, and periódicos capes. the same key words were used also in portuguese: “formiga”, “bioindicador”, “indicador” and “brasil”. for all the papers found, we recorded information about the general idea of the article, objectives (descriptive or hypothetical), perturbation (agriculture, human activity, conservation, fire, restauration or ecological succession), collection methodology (pitfall, attractive baits, winkler extractor, active search, attractive baits or observation), stratum (arboreal, epigeal, hypogea and litter ants), ant fauna parameters (richness and diversity or composition), observed environmental parameters (regeneration areas, baits, anthropic action or land use), results obtained (preservation or degradation) and conclusions reached in the study (table 1). however, the papers were analyzed by verifying the description of ants as bioindicators in the methodology or objectives, as well as presenting “bioindicators” or “indicators” as key words. in addition, ant collection methodology was analyzed to verify the most commonly used collection methods in the studies. we used the definitions of mcgeoch (1998) to analyze whether the papers examined presented ants as environmental, ecological, or diversity bioindicators. also, we created a list of bioindicator ant species when the papers displayed their occurrence in specific places or showed descriptions throughout the article. the following tests were used to analyze the data: cubic regression between number of papers and the years of publication, chi-square for the proportions of perturbation types, fauna parameters analyzed, and sampling methodologies used in the work, with an alpha of 0.05 for all analyzes. m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators114 information category description objectives descriptive only description of ants species. hypothetical hypothesis test comparing sampled areas. perturbation agriculture studies carried out only on agricultural habitats. human activity studies on natural habitats and agricultural habitats. conservation studies carried out only on natural habitats. fire studies that evaluated the presence of fire, whether natural or by anthropic actions. restoration studies that evaluated different techniques for the restoration of degraded habitats. ecological succession studies that evaluated the natural succession of the habitat. collection methodology pitfall falling trap for soil or vegetation ants sampling. attractive baits sardine bait and / or honey. winkler extractor sampling of litter. active search manual collection of ants. pitfall with attractive baits fall trap using attractive baits inside. observation observation of nests. stratum arboreal ants collected in trees or shrubs. epigeal ants collected above ground. hypogea ants collected in soil. litter ants collected in leaf-litter. parameters of ant fauna richness and diversity number of species and variety of species in a community. composition composition of ant species in the study areas. environmental parameters regeneration of areas regeneration of degraded areas by fire, mining. the age of regeneration is also considered. baits application of formicidal baits. anthropic action brazilian savanna subjected to anthropic impacts. land use agriculture and eucalyptus plantation. results obtained preservation presence of bioindicators of preserved areas (brazilian savanna). degradation presence of bioindicator ants from degraded areas. conclusions conclusions reached in each study. table 1. description of the information evaluated, categories of analysis and description of disturbances of the papers analyzed. results sixteen papers on ants as bioindicators in the brazilian savanna were analyzed, which covered a period of 30 years (from 1986 to 2016). of these, seven were obtained through the literature review of ribas et al. (2012) and nine from the new search. of all the papers analyzed, only two did not present descriptions about ants as bioindicators either in the introduction or in the objectives. instead, they presented results with potential for bioindication. the other papers mentioned an intention to use ants as bioindicators, by either utilizing the term “indicator” or using ants to indicate environmental parameters (table 2). monitoring studies about ants as bioindicators in the brazilian savanna began in 1992, increasing since 2002. this may be related to the publication of the book “ants: standard methods for measuring and monitoring biodiversity” in 2000, since prior to publication there were no established methodological criteria for ant sampling and, hence, with ants were intensified. there has been an increase in the studies in the last years until 2016 (r² = 0.39; p> 0.05, figure 1a). the disturbances of the study areas were also analyzed and we found that there were more studies related to perturbation (75%) than to preservation (25%) (χ2 = 16, d.f. = 1, p < 0.05). from the 16 papers, four were related to agriculture (25%), three to habitat restoration and to human activity (18.75% each theme), and two to fire effect, conservation, and succession (12.5% each theme). the studies differed between the parameters of ant fauna analyzed (χ2 = 33.33, df = 1, p < 0.05), so, we divided them between those which evaluated only richness (six papers, 37.5%) and those that assessed diversity and/or species composition (ten papers, 62.5%). in relation to studies that analyzed only the species richness of ants, four (25%) had a reduction in species richness in disturbed areas and another two studies (12.5%) showed no reduction in richness. sociobiology 65(2): 112-129 (june, 2018) 115 regarding the studies that analyzed the diversity and/or composition of species (ten papers, 62.5%), only one (6.25%) showed no change in the diversity and/or composition of ant species in degraded areas. in relation to the ant collection methodologies, pitfall was the most common one (six studies, 37.5%, χ² = 59.61, df = 6, p < 0.05), with attractive baits in the interior of pitfalls used in two other studies. we also verified the use of winkler extractors (five studies, 31.25%), in order to sample the ants present in the litter, attractive baits were used to sample ants in four studies (25%) and active search at the study sites in four studies (25%) to complement other collection methodologies. moreover, we checked the use of entomological umbrella in one study and observation of nests for another study (6.25% each) (figure 1b). we emphasize that four of the 16 analyzed papers used multiple (two or three) ant sampling methodologies. with the view to relate the answers obtained in the studies on ant composition and richness with the sampling methodologies used, we observed that the studies using winkler extractor, multiple sampling techniques, pitfall, and attractive baits showed a change in the composition and richness of the species. however, in three studies, which used pitfall and attractive baits methodologies, there was no change in ant composition and richness (figure 1c). among the analyzed studies, we verified that the ants were sampled in different stratum. thus, seven papers only collected epigeal ants, followed by litter in four studies. however, in five studies the sampling was carried out in two stratum, three of epigeal and hypogea ants and two of epigeal and arboreal ants. the ants were used as environmental indicators in 14 of the papers, as environmental and diversity indicators in one study, and as ecological indicators in another study. of the 16 papers, only six did not present lists of species and, therefore, it was possible to make a list of species of brazilian savanna bioindicating ants. the parameters used to define the species were presence/absence and frequency of occurrence, both found in five papers each. in general, 167 species were defined as brazilian savanna bioindicators and related to specific habitats according to results found in the articles. these could be brazilian savanna sensu stricto (preserved), disturbed brazilian savanna, disturbed brazilian savanna (post fire), eucalyptus plantation, agriculture, natural and intermediate regeneration of the brazilian savanna. the genera with the highest number of indicator species were camponotus (17), pachycondyla (8), acromyrmex (7), ectatomma (7), pseudomyrmex (7), cephalotes (5), dolichoderus (5), odontomachus (5) and anochetus (3) (table 3). following the description of the study areas of the papers that presented a survey of the species collected, we verified that linepithema humile, pachycondyla villosa, paratrechina longicornis, pseudomyrmex oculatus and wasmannia rochai were considered bioindicators of degradation, and were related to eucalyptus plantation and agriculture. the species fulakora armigera, camponotus latangulus, cephalotes atratus, cephalotes. borgmeieri, dolichoderus bispinosus, pachycondyla ferrugínea, pheidole fimbriata, strumigenys zeteki and strumigenys perparva were considered indicators of preserved areas (table 3). these criteria for species definition are presented by the authors, however assuming species as bioindicator should be considered some aspects, such as those presented in the discussion just below. fig 1. (a) papers analyzed using ants as bioindicators in the brazilian savanna in relation to publication years; (b) collection methodologies used in the studies analyzed.the papers presenting two or more methods were computed separately, adding up to 100%; (c) percentage of papers with or without change in richness and composition of species in relation to the sampling methodologies. m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators116 in addition to all the analyzes, we found that 75% of the analyzed papers were carried out in the state of minas gerais, followed by mato grosso (12.5%), mato grosso do sul (6.25%), and maranhão (6.25%). this information demonstrates the need for studies in the states such as bahia, goiás, paraná, piauí, rondônia, são paulo and tocantins (figure 2). discussion through a bibliographic review we were able to determine the history of ant research as bioindicators in the brazilian savanna in the last 30 years. between 1986 and 2016, we observed that most of the studies evidenced ants as indicators of environmental quality, diversity, and/or ecology in the introduction and/or objectives. when comparing the number of studies obtained between 1987 and 2010 (seven papers) by ribas et al. (2012a) with the studies found between the years 2011 and 2016 (nine papers), we verified that there was an increase in the studies on ants as bioindicators in the brazilian savanna by more than 128%. this may be related to the increasing environmental problems in the biome caused by habitat loss, and the need to monitor disturbed areas. thus, when analyzing the most frequent disturbances we verified that there was a greater amount of studies carried out in degraded areas than in preserved areas, related to agriculture, since there is such great expansion of the agricultural frontier in the brazilian savanna. in relation to the studies carried out in preserved areas, we checked that they were related to conservation and succession. this may be related to the need to preserve the areas, since the biome is threatened by agriculture and livestock. although most analyzed papers used the terms “bioindicators” and “indicators” in the introduction and/ or objectives, not all of them reported results on ants as bioindicators. that because they did not rigorously analyze their results through statistical analysis or because sampling was not fully effective. among some of the problems we highlight: (i) they did not present control study areas (preserved), (ii) they only sampled one area in different periods or (iii) only compared different plant strata in the same area. fig 2. states sampled and studies conducted between 1986 and 2016 in the brazilian savanna. some studies are in transitional areas such as mato grosso do sul (ms) and são paulo (sp). sociobiology 65(2): 112-129 (june, 2018) 117 we noted that most of the papers were related to ants as environmental indicators, since, according to mcgeoch (1998), they are species or group of species that respond to environmental disturbances and are easily observed and quantified. however, we also verified studies on ecological and diversity indicators, which are species that demonstrate the effects of environmental changes and reflect some measures of diversity of other species in a habitat, respectively. the richness and diversity and/or species composition were the most common parameters of the ant communities analyzed in the papers. we noted that the richness of species is influenced by disturbances, since most of the studies presented a reduction in the richness of ants in disturbed areas. we also verified this in relation to species diversity and/or composition, since most of the analyzed papers also showed a change in the composition of the community in disturbed areas. regarding the collection methodology, we verified that most of the studies used only one method. however, considering that ants are present in different stratifications, the use of combined techniques would allow a more complete sampling of the ant community. however, we emphasize that the choice of collection methods is related not only to the sampling of the ants, but also to the financial cost of the study and the time availability for the collection and processing of material. the fact that the most common collection method was the pitfall trap, which is a sampling technique to collect soil and vegetation insects, allows attractive baits to be used in the interior and selects what for the organisms that will be attracted. we emphasize that the use of such methodology can also be related to the low cost to develop it, the duration of the sampling, and the facility to allocate it in the study area. from the list of species generated and the description in the results of each study the definition of bioindicating species still presents gaps to be filled. due to the lack of specific analysis of results, these may not be precise. thus, a more rigorous analysis, such as the individual indicator value, would be required to ensure that species really indicate quality or degradation. this lack of rigor can be verified in the table, since there are various contradictory patterns of occurrence, and species occurring in degraded areas were also present in preserved areas, such as: atta laevigata, acromyrmex subterraneus brunneus, acromyrmex rugosus, among others generic and suprageneric classification (table 3). these results on bioindication were obtained from the studies analyzed. thus, it is important to consider the biology of the species, since the contradictory patterns may be related to possible errors in the delimitation of the study area and sample points. the compilation of species data and bioindications provided by them can be explored in the matter of directing strategies for defining a protocol to take these species as real bioindicators. we see the need to solve the question of the inclusion criteria of the species as a bioindicator. in this way, for example, the area of occurrence of the species and the frequency of records in a particular habitat type (eg the species must always be recorded in perserved environments) should be taken into account. assume that the timely recording of a given species in a given environment may be detrimental to the exact definition of bioindicator species. it is the suggestion for the creation of a robust database based on previous records of the species and type of habitat where collected. in this way it will be possible to determine the exact definition of the species as bioindicator. such an adjustment would solve the definition of bioindicators for the various biomes. the brazilian savanna is predominant in 11 brazilian states, present in bahia, goiás, maranhão, mato grosso, mato grosso do sul, minas gerais, paraná, piauí, rondônia, são paulo, and tocantins. although, we noted from the bibliographical review that there was higher sampling from the state of minas gerais in comparison to the other states and, consequently, a smaller amount or even absence of studies about ants as bioindicators in most of the brazilian states (fig 2). this may be related to the reduced number of researchers in each state, the difficulty of sampling areas, the difficulty of access to remaining areas in some states, and even the lack of funding for studies. conclusion thus, based on the literature review from the last 30 years, we verified that the absence of rigorous analyzes impairs the results of the studies, since the inclusion of statistical analyzes would allow a more precise analysis of the results. as well as knowledge of the ant fauna biology would attribute characteristics of indication of preserved or degraded areas. in addition, the use of a sampling protocol, in which standardization of the sampling method and analyzes to be carried out in future studies, should make the standardization of the work on bioindicators possible, avoiding possible patterns of contradictory occurrence in the inventories. when using collection protocol aimed at answering questions about bioindicators, it is necessary to create protocol of which parameters are used to define ants as bioindicators. as already shown, species with a double occurrence (+ and bioindication) should not be bioindicators, or they fit in both categories due to the imprecision of the authors in their categorization. in addition, the use of rare or low-frequency species may mask the patterns, using the most frequent species in each habitat type or desired indication profile, thus generating reliable or generic lists of species for effective monitoring purposes. in addition, a better description of the study areas would allow the inclusion of habitat data, allowing the development of a more robust list of ant species for the brazilian savanna. we suggest the creation of a database to solve the definition of bioindicators for the different biomes. furthermore, from this review we also verified the need for further studies on bioindicators in the brazilian savanna, mainly in the states of goiás and tocantins, which contain larger areas of brazilian savanna in comparison to the other states and have not presented studies in the past 30 years. m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators118 p er tu rb at io n o bj ec ti ve c ol le ct io n si te in di ca ti on k ey w or d e nv ir on m en ta l p ar am et er s e ff ec ts o n th e an t c om m un it y st ra tu m t yp e of in di ca to r r ef er en ce r es to ra tio n t o in ve st ig at e w hi ch a nt s ha ve re co lo ni ze d re cl ai m ed a re as a nd to e va lu at e th e ef fe ct o f d if fe re nt re ha bi lit at io nt ec hn iq ue s, co m pa ri ng th e re su lts w ith a us tr al ia . po ço s de c al da s m g y es n o a ge o f re ge ne ra tio n, s oi l pe ne tr at io n, n um be r of lo gs , m ea su re s of li tte r an d ve ge ta tio n. in cr ea se d sp ec ie s ri ch ne ss a nd al te re d co m po si tio n (p c oa ). e pi ge al e co lo gi ca l m aj er ( 19 92 ) h um an a ct iv ity to in ve nt or y th e fa un a of a nt s in e uc al yp tu s pl an ta tio ns w ith di ff er en t a ge s of u nd er st or y. b om d es pa ch o m g y es n o a ge o f eu ca ly pt us pl an ta tio n. h ig he r d en si ty o f s pe ci es in th e b ra zi lia n sa va nn a th an in th e eu ca ly pt us p la nt at io n an d si m ila r s pe ci es ri ch ne ss es tim at io n am on g th e ar ea s. l itt er e nv ir on m en ta l m ar in ho e t al . ( 20 02 ) a gr ic ul tu re (g ra nu la te d fo rm ic id al b ai ts ) t o ev al ua te th e ef fe ct o f t he lo ca liz ed u se a nd th e sy st em at ic us e of g ra nu la te d ba its fo r t he co nt ro l o f l ea fcu tti ng a nt s on th e no nta rg et a nt c om m un ity o f th e lit te r i n eu ca ly pt us . b om d es pa ch o m g y es n o fo rm s an d tim in g of ap pl ic at io n of f or m ic id al ba its . n o ef fe ct o f b ai t t yp e w as fo un d on th e ri ch ne ss o f a nt s pe ci es . r ed uc tio n in s pe ci es ri ch ne ss ob se rv ed o nl y in th e co nt ro l m et ho d, w ith s ys te m at ic ap pl ic at io n be in g m or e da m ag in g. e pi ge al e nv ir on m en ta l r am os e t a l. (2 00 3a ) c on se rv aç ão t o st ud y th e an t c om m un ity of a n ar ea o f p re se rv ed n at iv e b ra zi lia n sa va nn a an d to an th ro pi c im pa ct , e va lu at in g th e co ns er va tiv e qu al ity o f d iv er si ty in th e bi om e. b om d es pa ch o m g y es n o a nt hr op ic a ct io n. r ed uc tio n of d iv er si ty in im pa ct ed s ite s. l itt er e nv ir on m en ta l r am os e t a l. (2 00 3b ) c on se rv aç ão to te st th e hy po th es is th at d ir t ro ad s ar e si te s fa vo ra bl e to co lo ni za tio n an d es ta bl is hm en t of n es ts b y fo un di ng q ue en s of th e a tta la ev ig at a. u be rl ân di a m g po te nt ia lly n o a nt hr op ic a ct io n. t he n um be r o f a tte m pt s to co lo ni ze o n ro ad s w as 5 to 1 0 tim es g re at er th an in a dj ac en t ve ge ta tio n. e pi ge al e nv ir on m en ta l v as co nc el os et a l. (2 00 6) h um an a ct iv ity to e va lu at e th e ef fe ct o f d ay a nd ni gh t c ol le ct io n on th e fa un is tic co m po si tio n of e pi ge ic a nt s vi si tin g ba its in a re as p la nt ed b y eu ca ly pt us a nd n at iv e b ra zi lia n sa va nn a ve ge ta tio n. pa in ei ra s m g po te nt ia l n o l an d us e. e ff ec t o f c ol le ct io n tim e w as m or e im po rt an t f or a nt fa un a st ru ct ur e th an v eg et at io n ef fe ct . e pi ge al e nv ir on m en ta l ta va re s et a l. (2 00 8) r es to ra tio n to e va lu at e th e bi oi nd ic at io n of a nt s in im pa ct ed h ab ita ts . m or ro d o e sp in ha ço m g y es y es ti m e si nc e re st or at io n, di st an ce o f im pa ct a nd ph ys ic al p ro pe rt ie s of s oi l. g re at er s pe ci es ri ch ne ss in th e b ra zi lia n sa va nn a th an in th e re st or at io n ha bi ta ts a nd a ls o gr ea te r i n th e ec ot on ic a nd in te rm ed ia te z on es th an in th e be ac h an d ch an ge in ab un da nc e an d co m po si tio n. e pi ge al e nv ir on m en ta l c os ta e t a l. (2 01 0) t ab le 2 . p ap er s an al yz ed fo r a nt s as b io in di ca to rs in th e b ra zi lia n sa va nn a, in di ca tin g th e di st ur ba nc es in ve st ig at ed , o bj ec tiv es , c ol le ct io n si te , i nd ic at io n to b e us ed in b io in di ca to r s tu di es , e nv ir on m en ta l p ar am et er s, a nt c om m un ity re sp on se , s tr at um , t yp e of in di ca to r, an d re fe re nc e of p ap er s. sociobiology 65(2): 112-129 (june, 2018) 119 p er tu rb at io n o bj ec ti ve c ol le ct io n si te in di ca ti on k ey w or d e nv ir on m en ta l p ar am et er s e ff ec ts o n th e an t c om m un it y st ra tu m t yp e of in di ca to r r ef er en ce su cc es si on ( so il co nt am in at io n by a rs en ic ) to e va lu at e th e po te nt ia l u se o f an ts co m m un iti es a s bi oi nd ic at or s of th e en vi ro nm en ta l i m pa ct c au se d by ar se ni c re si du es in th e so il. n ov a l im am g y es n o r ec ov er y of a re as . a nt c om m un iti es w er e se ns iti ve to ch an ge s in th e ef fe ct s of a rs en ic th ro ug h ch an ge s in h ab ita t c on di tio n an d av ai la bi lit y of e nv ir on m en ta l r es ou rc es . e pi ge al a nd ar bo re al e nv ir on m en ta l an d bi od iv er si ty r ib as e t al . ( 20 11 ) a gr ic ul tu re (s oy be an ) to u nd er st an d th e re sp on se o f an ts ’ c om m un iti es o n th e bo rd er s be tw ee n b ra zi lia n sa va nn a ar ea s ex po se d to th e us e of s oy be an la nd . c ha pa da d as m an ga be ir as m a y es n o l an d us e (a gr ic ul tu re ). e vi de nc e of e dg e ef fe ct s on s pe ci es co m po si tio n in a nt c om m un iti es o f so il. b ra ch ym yr m ex p at ag on ic us , e . pa ci ve nt re , c . c ra ss us , l . c er ra de ns e an d w . a ur op un ct at a w er e cl as si fie d as sp ec ie s in di ca tin g ch ar ac te ri st ic s of th e in te ri or o f th e b ra zi lia n sa va nn a. e pi ge al e nv ir on m en ta l b ra nd ão e t al . ( 20 11 ) r es to ra tio n e va lu at e th e ef fe ct iv en es s of di ff er en t r eh ab ili ta tio n ef fo rt s af te r go ld m in in g ac tiv iti es u si ng a nt s as bi oi nd ic at or s. n ov a l im am g y es n o r ec ov er y of a re as . t he r es ul ts o f th e in dv al te st s ho w ed a sp ec ie s of h yp op on er a as a n in di ca to r of a re a re ha bi lit at io n af te r m in in g ac tiv iti es b ei ng d om in at ed b y gr as se s. e pi ge al a nd hy po ge a e nv ir on m en ta l r ib as e t al . ( 20 12 b) h um an a ct iv ity e xa m in e th e as se m bl ag e of s pe ci es of a nt s in d if fe re nt p la ce s of tr ai ls . sã o g on ça lo do a ba té , t rê s m ar ia s an d a nd ré qu ic ém g y es y es l an d us e (e uc al yp tu s pl an ta tio n) . a ss oc ia tio n of a nt s is c ha ra ct er iz ed b y th e ha bi ta t t yp e an d th e an t f au na o f th e b ra zi lia n sa va nn a is r es ili en t t o th e su rr ou nd in g im pa ct s in te rm s of s pe ci es ri ch ne ss b ut n ot s pe ci es c om po si tio n. e pi ge al a nd hy po ge a e nv ir on m en ta l c os ta m ila ne z et al . ( 20 14 ) a gr ic ul tu re a nd l iv es to ck to e va lu at e th e an ts c om m un ity of th e so il in a n in te gr at ed c ro pliv es to ck s ys te m . d ou ra do sm s y es n o l an d us e (a gr ic ul tu re an d eu ca ly pt us pl an ta tio n) . t he m or ph os pe ci es o f st ru m yg en ys s p. an d h yp op on er a sp . w er e fo un d on ly in a gr ic ul tu ra l a re as . l itt er e nv ir on m en ta l c re pa ld i et a l. (2 01 4) fi re to v er if y th e ef fe ct s of fi re in b ra zi lia n sa va nn a se ns u st ri ct o tr ai ls u si ng a nt s as to ol s to e va lu at e ch an ge s in b io di ve rs ity a nd en vi ro nm en ta l c on di tio ns . sã o g on ça lo d o a ba té a nd a nd ré qu ic ém g y es y es l an d us e (p os t fi re ). in cr ea se d sp ec ie s ri ch ne ss in th e b ra zi lia n sa va nn a af te r fir e. e pi ge al a nd ar bo re al e nv ir on m en ta l c os ta m ila ne z et al . ( 20 15 ) a gr ic ul tu re to v er if y th e im pa ct s ca us ed o n th e bi od iv er si ty o f th e lo ca l m yr m ec of au na , d ue to th e re pl ac em en t o f th e na tiv e ar ea of b ra zi lia n sa va nn a. l uc as d o r io v er de a nd v er am t y es y es l an d us e (a gr ic ul tu re an d eu ca ly pt us pl an ta tio n) . t he b io in di ca to r pa pe r of th e ge nu s p he id ol e w as v er ifi ed , b ei ng o nl y fo un d in th e en vi ro nm en t o f b ra zi lia n sa va nn a, w hi ch c or re sp on ds to it s ch ar ac te ri st ic o f co lo ni zi ng c on se rv ed en vi ro nm en ts . l itt er e nv ir on m en ta l c or as a et al . ( 20 15 ) su cc es si on to e va lu at e th e sp ec ie s of a nt s th at o cc ur a ss oc ia te d to d ia m on d m in in g, b es id es id en tif yi ng th e po ss ib le s pe ci es w ith p ot en tia l of e nv ir on m en ta l b io in di ca to rs fo r ar ea s re la te d to th is e co no m ic ac tiv ity . po xo ré u m t y es y es r eg en er at io n of a re as (m in in g) . t he s pe ci es f or el iu s br as ili en si s ca n be c on si de re d a bi oi nd ic at or o f en vi ro nm en ta l d eg ra da tio n in d ia m on d m in in g an d an th ro pi za tio n, w hi le c am po no tu s at ri ce ps , p ac hy co nd yl a cr as si no da a nd p ar ap on er a cl av at a ca n be c on si de re d bi oi nd ic at or s of pr es er ve d b ra zi lia n sa va nn a. e pi ge al e nv ir on m en ta l r oc ha e t al . ( 20 15 ) fi re to e va lu at e th e im pa ct o f th e en vi ro nm en ta l d is tu rb an ce c au se d by th e an th ro pi c fir e on th e co m m uni ty o f ep ig ei c an d hy po gi ca l a nt s l ag oa s an ta , pe dr o l eo po ld o an d m at oz in ho s m g y es y es r eg en er at io n of a re as (p os t fi re ). a nt c om m un iti es a re p ar tia lly a ff ec te d by fi re o cc ur re nc es , a nd e pi ge ne tic as se m bl ag es a re m os t a ff ec te d co m pa re d to h yp og ly ce m ic a nt s. e pi ge al a nd hy po ge a e nv ir on m en ta l c an ed ojú ni or e t al . ( 20 16 ) t ab le 2 . p ap er s an al yz ed fo r a nt s as b io in di ca to rs in th e b ra zi lia n sa va nn a, in di ca tin g th e di st ur ba nc es in ve st ig at ed , o bj ec tiv es , c ol le ct io n si te , i nd ic at io n to b e us ed in b io in di ca to r s tu di es , e nv ir on m en ta l p ar am et er s, a nt c om m un ity re sp on se , s tr at um , t yp e of in di ca to r, an d re fe re nc e of p ap er s. (c on tin ua tio n) m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators120 subfamily species parameter collection site habitat bioindicator references amblyoponinae fulakora armigera (mayr, 1887) presence/ absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) dolichoderinae azteca alfari (emery, 1893) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) dolichoderus bispinosus (olivier, 1792) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) dolichoderus germaini (emery, 1894) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna perturbado degradation costa-milanez et al. (2015) dolichoderus imitator (emery, 1894) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) dolichoderus lamellosus (mayr, 1870) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) dolichoderus lutosus (smith, 1858) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) dorymyrmex brunneus (forel, 1908) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) dorymyrmex pyramicus (roger, 1963) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation corasa et al. (2015) dorymyrmex spurius (santschi, 1929) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) dorymyrmex thoracicus (gallardo, 1916) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) forelius brasiliensis (forel, 1908) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) presence / absence poxoréu mt disturbed brazilian savanna degradation rocha et al. (2015) forelius maranhaoensis (cuezzo, 2000) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) gracilidris pombero (wild & cuezzo, 2006) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) linepithema anathema (wild, 2007) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) linepithema cerradense (wild, 2007) presence/ absence nova lima mg natural regeneration of the brazilian savanna preservation ribas et al. (2012b) linepithema humile (forel, 1908) presence/ absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) dorylinae labidus coecus (latreille, 1802) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) labidus praedator (smith, 1858) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna perturbado degradation ramos et al. (2003b) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. sociobiology 65(2): 112-129 (june, 2018) 121 subfamily species parameter collection site habitat bioindicator references dorylinae neivamyrmex orthonotus (borgmeier, 1933) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) ectatomminae ectatomma brunneum (smith, 1958) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) ectatomma edentatum (roger, 1863) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) presence / absence bom despacho mg natural regeneration of the brazilian savanna preservation ribas et al. (2011b) ectatomma lugens (emery, 1894) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) ectatomma muticum (mayr, 1870) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) ectatomma permagnum (forel, 1908) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) ectatomma planidens (borgmeier, 1939) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) ectatomma tuberculatum (olivier, 1792) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) gnamptogenys acuminata (emery, 1896) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) gnamptogenys striatula (mayr, 1884) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) formicinae camponotus atriceps (smith, 1858) presence / absence poxoréu mt brazilian savanna sensu stricto preservation rocha et al. (2015) camponotus bidens (mayr, 1870) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) camponotus blandus (smith, 1858) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) camponotus bonariensis (mayr, 1868) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) camponotus burtoni (mann, 1916) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) camponotus cameranoi (emery, 1894) frequency of occurrence são gonçalo do abaté, três marias and a ndrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) camponotus crassus (mayr, 1862) presence / absence bom despacho mg natural regeneration of the brazilian savanna preservation ribas et al. (2011b) camponotus fastigatus (roger, 1863) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) presence / absence bom despacho mg natural regeneration of the brazilian savanna preservation ribas et al. (2011) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators122 subfamily species parameter collection site habitat bioindicator references formicinae camponotus latangulus (roger, 1863) presence / absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) camponotus melanoticus (emery, 1894) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) camponotus novogranadensis (mayr, 1870) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) camponotus punctatus minutior (forel, 1912) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) camponotus renggeri (emery, 1894) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) camponotus rufipes (fabricius, 1775) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003) presence / absence lagoa santa, pedro leopoldo and matozinhos mg disturbed brazilian savanna degradation canedo-júnior et al. (2016) camponotus sericeiventris (guérin-méneville, 1838) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) camponotus sexgutattus (fabricius, 1793) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna perturbado (pós fogo) degradation costa-milanez et al. (2015) camponotus trapezoideus (mayr, 1870) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) gigantiops destructor (fabricius, 1804) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) nylanderia fulva (mayr, 1862) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) paratrechina longicornis (latreille, 1802) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequência de ocorrência chapada das mangabeiras ma agriculture degradation brandão et al. (2011) myrmicinae acromyrmex balzani (emery, 1890) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) acromyrmex coronatus (fabricius, 1804) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) acromyrmex landolti (forel, 1885) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) acromyrmex niger (smith, 1858) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) acromyrmex rugosus (smith, 1858) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) sociobiology 65(2): 112-129 (june, 2018) 123 subfamily species parameter collection site habitat bioindicator references myrmicinae acromyrmex subterraneus brunneus (forel, 1912) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) acromyrmex subterraneus subterraneus (forel, 1893) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) apterostigma pilosum (mayr, 1865) presence / absence dourados ms brazilian savanna sensu stricto preservation crepaldi et al. (2014) atta laevigata (smith, 1858) frequency of occurrence são gonçalo do abaté, três marias and a ndrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) atta sexdens rubropilosa (linnaeus, 1758) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) blepharidatta conops (kempf, 1967) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) cardiocondyla emeryi (forel, 1881) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) carebara urichi (wheeler, 1922) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) cephalotes atratus (linnaeus, 1758) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) cephalotes borgmeieri (kempf, 1951) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) cephalotes cristopherseni (forel, 1912) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) cephalotes pavonii (latreille, 1809) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) cephalotes pusillus (klug, 1824) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) crematogaster erecta (mayr, 1866) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) crematogaster tenuicula (forel, 1904) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) cyphomyrmex peltatus (kempf, 1966) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) cyphomyrmex transversus (emery, 1894) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) monomorium florícola (jerdon, 1851) presence / absence nova lima mg intermediate regeneration of the brazilian savanna preservation ribas et al. (2012b) mycetagroicus cerradensis (brandão & mayhé-nunes, 2001) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) mycocepurus goeldii (forel, 1893) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators124 table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) subfamily species parameter collection site habitat bioindicator references myrmicinae mycocepurus smithii (forel, 1893) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) myrmicocrypta foreli (mann, 1916) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) nesomyrmex asper (mayr, 1887) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) nesomyrmex brasiliensis (kempf, 1958) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) nesomyrmex spininodis (mayr, 1887) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) ochetomyrmex semipolitus (mayr, 1878) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) octostruma jheringhi (emery, 1887) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) pheidole fimbriata (roger, 1863) presence / absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) pheidole gertrudae (forel, 1886) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) pogonomyrmex abdominalis (santschi, 1929) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) rogeria scobinata (kugler, 1994) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) sericomyrmex parvulus (forel, 1912) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) solenopsis saevissima (smith, 1855) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) solenopsis substituta (santschi, 1925) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) strumigenys elongata (roger, 1863) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) strumigenys perparva (brown, 1958) presence / absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) strumigenys subdentata (mayr, 1887) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) strumigenys zeteki (brown, 1959) presence / absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) trachymyrmex bugnioni (forel, 1912) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) trachymyrmex dichrous (kempf, 1967) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) trachymyrmex fuscus (emery, 1834) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) wasmannia auropunctata (roger, 1863) presence / absence nova lima mg natural regeneration of the brazilian savanna preservation ribas et al. (2012b) sociobiology 65(2): 112-129 (june, 2018) 125 table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) subfamily species parameter collection site habitat bioindicator references myrmicinae wasmannia rochai (forel, 1912) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) frequency of occurrence são gonçalo do abaté and andrequicé mg disturbed brazilian savanna (post-fire) degradation costa-milanez et al. (2015) paraponerinae paraponera clavata (fabricius, 1775) presence / absence poxoréu mt brazilian savanna sensu stricto preservation rocha et al. (2015) ponerinae anochetus bispinosus (smith, 1858) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) anochetus diegensis (forel, 1912) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003a) anochetus inermis (andré, 1889) frequency of occurrence lucas do rio verde and vera mt eucalyptus plantation degradation corasa et al. (2015) dinoponera gigantea (perty, 1833) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) ectomomyrmex apicalis (smith, 1857) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) hypoponera foreli (mayr, 1887) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003a) odontomachus bauri (emery, 1892) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence chapada das mangabeiras ma brazilian savanna sensu stricto preservation brandão et al. (2011) presence / absence dourados ms agriculture degradation crepaldi et al. (2014) odontomachus bruneus (mayr, 1887) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003a) odontomachus chelifer (latreille, 1802) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003a) odontomachus haematodus (linnaeus, 1758) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) frequency of occurrence lucas do rio verde and vera mt eucalyptus plantation degradation corasa et al. (2015) odontomachus meinerti (forel, 1905) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003a) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2014) pachycondyla crassinoda (latreille, 1802) presence / absence poxoréu mt brazilian savanna sensu stricto preservation rocha et al. (2015) pachycondyla harpax (fabricius, 1804) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) pachycondyla striata (smith, 1858) presence / absence bom despacho mg brazilian savanna sensu stricto preservation marinho et al. (2002) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003b) presence / absence nova lima mg natural regeneration of the brazilian savanna preservation ribas et al. (2012b) pachycondyla villosa (fabricius, 1804) frequency of occurrence bom despacho mg disturbed brazilian savanna degradation ramos et al. (2003b) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) pseudoponera gilberti (kempf, 1960) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) m tibcherani, vaf nacagava, r aranda, rl mello – ants (hymenoptera: formicidae) as bioindicators126 table 3. species of ants defined as bioindicators for preservation or degradation in the reference studys, indicating the parameter used to relate to the habitat type, and the reference of the papers analyzed. (continuation) subfamily species parameter collection site habitat bioindicator references ponerinae pseudoponeraa stigma (fabricius, 1804) frequency of occurrence são gonçalo do abaté and andrequicé mg brazilian savanna sensu stricto preservation costa-milanez et al. (2015) rasopone ferrugínea (smith, 1858) presence / absence dourados ms brazilian savanna sensu stricto preservation crepaldi et al. (2014) thaumatomyrmex mutilatus (mayr, 1887) frequency of occurrence bom despacho mg brazilian savanna sensu stricto preservation ramos et al. (2003a) pseudomyrmecinae pseudomyrmex flavidulus (smith, 1858) frequency of occurrence chapada das mangabeiras ma agriculture degradation brandão et al. (2011) pseudomyrmex gracilis (fabricius, 1804) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) pseudomyrmex oculatus (smith, 1855) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) frequency of occurrence são gonçalo do abaté, três marias and andrequicé mg eucalyptus plantation degradation costa-milanez et al. (2014) pseudomyrmex pupa (forel, 1911) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) pseudomyrmex simplex (smith, 1877) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) pseudomyrmex tenuis (fabricius, 1804) frequency of occurrence lucas do rio verde and vera mt brazilian savanna sensu stricto preservation corasa et al. (2015) pseudomyrmex termitarius (smith, 1855) presence / absence bom despacho mg eucalyptus plantation degradation marinho et al. (2002) presence / absence nova lima mg intermediate regeneration of the brazilian savanna preservation ribas et al. (2012b) acknowledgment we are very grateful to the programa de pós graduação em biologia animal of universidade federal de mato grosso do sul (ppgba/ufms) and to coordenação de aperfeiçoamento de pessoal de nível superior (capes) by the fellowship to the first author and the anonymous reviewer for comments in improved earlier versions of the manuscript. we thank rony peterson s. almeida for reviewing the list of ant species. references allaby, m. (1992). the concise oxford dictionary of zoology. oxford university press, oxford, p. 442. alonso, l.e. 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(1995). co-occurrence of morphologically similar species of stream fishes. american naturalist, 145: 527-545. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5830sociobiology 68(3): e5830 (september, 2021) introduction despite the importance of apis bees, there has been a worldwide decline in the populations of these insects in recent years. such decline is attributed to climatic factors, use of agrochemicals, diseases, parasites, and habitat and food (e.g. nectar and pollen) reduction, all of which directly impair the maintenance of colonies (goulson et al., 2015; wood et al., 2020). among these factors, the use of agrochemicals in monocultures contributes to selecting pests that are resistant to the active ingredients used, culminating in the use of more and more toxic products, which are highly harmful to the environment and the pollinators (johnson et al., 2010). abstract inadequate quantity and quality of proteins in honey bee diet can cause weakening of their colonies and damage their resistance to agrochemical contamination, such as fipronil, which is highly toxic to bees. thus, we tested the hypothesis if protein supplementation would improve longevity and locomotion of honeybees exposed to fipronil pesticide. colonies of apis mellifera africanized were distributed into control group without protein supplementation and supplemented group with 25% crude protein provided as a paste form at 100 g per week. after four weeks, frames with sealed brood were removed and kept in an incubator until the emergence of worker bees, which were marked, returned to their hives and recaptured six days later to measure protein concentration in the hemolymph. the bee population development was measured by evaluating frames containing the queen’s oviposition from each colony. also, nursing bees were recaptured exposed by contact to fipronil ld 50% (0.009 ± 0.003 μg/bee), and the longevity and motor activity were measured. the results showed that the bee swarms protein supplementation promoted a significant increase in the sealed brood area. however, it did not promote changes in the protein content of the hemolymph. protein supplementation of bee swarms did not influence the survival of bees exposed to fipronil in the locomotion tests; however, fipronil was toxic to bees and promoted changes in the locomotion of bees. sociobiology an international journal on social insects iloran rc moreira, daniel cb barros, juliana s lunardi, ricardo o orsi article history edited by evandro n. silva, uefs, brazil received 01 september 2020 initial acceptance 03 may 2021 final acceptance 18 may 2021 publication date 13 august 2021 keywords beekeeping, neurotoxicity, nutrition, protein, supplementation. corresponding author ricardo de oliveira orsi faculdade de medicina veterinária e zootecnia – fmvz campus botucatu distrito de rubião junior s/nº caixa postal 560, cep 18618-97. botucatu, são paulo, brasil. e-mail: ricardo.orsi@unesp.br when bees collect resources (i.e., nectar and/or pollen) that contain high doses of agrochemicals, acute contamination of bees can occur, which causes their mortality over a short period. however, when applied in low doses, considered sublethal, it is often transported to the colony by the bees together with the collected resources, which may compromise the viability of breeding and the maintenance of colonies (villa et al., 2000; long & krupke, 2016). the contamination of colonies by agrochemicals reduces the longevity (colin et al., 2004; pettis et al., 2004; desneux et al., 2007) and affects the vitality of the colony (belien et al., 2009), amongst other behavioral and physiological changes that impair its survival (holdera et al., 2018). one of the agrochemicals harmful to honeybees is fipronil, which belongs to the phenylpirazole group and targets center of education, science and technology in rational beekeeping (nectar), college of veterinary medicine and animal sciences, unesp. botucatu, são paulo, brazil research article bees effect of protein supplementation in the bee apis mellifera l. exposed to the agrochemical fipronil id https://orcid.org/0000-0001-8099-8277 iloran rc moreira, daniel cb barros, juliana s lunardi, ricardo o orsi – honeybee nutrition and fipronil2 the receptor of γ-aminobutyric acid as an antagonist. this agrochemical is used worldwide as a pesticide in agricultural and veterinary practices (kairo et al., 2017). commonly used on seeds, this systemic insecticide is absorbed by the growing plant and distributed through its tissues, including the flowers (nauen & jeschke, 2011). thus, bees are exposed to residual levels of this insecticide when they collect nectar and pollen from the treated crops (chauzat et al., 2011). currently, neonicotinoids and fipronil represent one-third of the global insecticide market (simon-delso et al., 2015). in european countries such as italy, germany, and slovenia, their use has caused a reduction of colonies of a.mellifera bees, leading the european community to ban these in 2013 (eisenstein, 2015). depending on applied concentrations, fipronil may not cause the immediate mortality of bees. they can decrease the toxic effects of the pesticide through the action of enzymes such as glutathione-s-transferase, catalase, and cytochrome p450 (johnson et al., 2012). in general, the detoxication system converts fat-soluble substances to insoluble substances in an aqueous environment (berenbaum & johnson, 2015). the quality and quantity of nutrients can modulate this system, making it extremely important for bees to maintain an adequate diet. wahl and ulm (1983) verified increased agrochemical resistance in young bees after receiving a pollen diet of adequate quality and quantity. thus, the supply of a protein diet to bee swarms in areas at risk of contamination by agrochemicals can increase the hemolymph protein levels in nursing bees and improve the resistance of bees exposed to the agricultural pesticide fipronil. we tested the hypothesis if protein supplementation would help the honeybee’s longevity and locomotion exposed to fipronil pesticide materials and methods treatments the experiment was undertaken at geographic coordinates 22°49′ s; 48°24′w, with a cfa type climate (subtropical with summer of the higher temperatures) and an average altitude of 623 m. six colonies apis mellifera africanized (hymenoptera: apidae) were used, housed in standard langstroth hives, and distributed randomly into groups, with three colonies each: g1 – control group without protein supplementation and g2 – supplemented group with 25% crude protein for the formulation of the diet containing 25% crude protein, we used 47.6% of cornmeal, 47.0% of soybean meal, 5.0% of sugar, and 0.4% of oil, obtained from a single batch fmvz feed factory, unesp, botucatu. the calculated nutritional levels were 4,035 kcal of crude energy and 25% of crude protein (rostagno et al., 2011). a bromatological analysis was performed to verify the calculated levels of the diet, which obtained values of 26.9% of crude protein and 3,846 of crude energy (lime/g). from the bee bread collected from the experimental colonies, 20% of crude protein and 4,050 crude energy (lime/g) were obtained. the feed was provided in a paste form (a mixture of ingredients with a standardized amount of honey) and placed on the top bar of the central frame containing the bee swarms, at 100 g per week during december 2017. the leftovers from the rations were removed at the end of each week and weighed to measure consumption. all colonies had free access to nectar and pollen near the apiary. harvesting of bees for evaluation protein concentration in hemolymph, longevity, and locomotion of bees four weeks after the start of protein supplementation, two frames with sealed brood from the colonies (g1 and g2 groups) were removed, marked, wrapped in tissue (to keep the bees newly emerged in the frame), and kept in an incubator at 32°c and relative humidity of 80% until the emergence of the worker bees (roat et al., 2014). after the emergence, the newly emerged workers from each beehive were marked in the dorsal side of the thorax using a nontoxic pen (posca paint pens, mitsubishi pencils, japan) and returned to their respective hives. the marked bees were recaptured on their sixday-old, and the concentration of proteins in their hemolymph was measured. so, the honeybees were exposed to fipronil at a concentration of 0.009 ± 0.003 μg/bee (ld50%) (zaluski et al., 2015), and the longevity and motor activity measured in the laboratory. evaluation of the population development of a. mellifera for the evaluation of population development (g1 and g2 groups), two frames containing the queen’s oviposition were located in the center of the nest. this nest had its brood area open and sealed. it was evaluated from the first day of the experiment, for four weeks, according to the methodology adapted from al-tikrity et al. (1971). evaluation of the longevity of bees exposed to fipronil on the seventh day, the nursing bees from colonies (g1 and g2 groups, totaling 30 bees/treatment) were recaptured, anesthetized in a freezer, and housed in a petri dish (150 × 20 mm) with perforated lids to ensure ventilation. the bees of g2 received 2µl of a solution containing ld50% of fipronil in the dorsal side region using a micropipette. the g1 group received 2µl of distilled water. the petri dishes were kept in a dark incubator at 32 ± 1°c and relative humidity of 70 ± 10%. the bees received syrup and 50% sugar and water ad libitum during all trials. these experiments were performed in triplicate. the number of dead bees was recorded daily, and the dead bees were removed from the plates. the experiment was conducted until all the bees had died. sociobiology 68(3): e5830 (september, 2021) 3 evaluation of the motor activity of bees exposed to fipronil the evaluation of the motor activity of bees was performed according to the methodology described by zaluski et al. (2015). motricity was included in the study to evaluate the possible effects on the motor activity of bees after supplementation with a 25% crude protein diet and contamination with agrochemicals. a total of 10 nursing bees were collected from each experimental colony (g1 and g2 groups) and exposed by contact to ld50% of the agrochemical fipronil. we used a wooden box in the form of a drawer to perform the locomotion tests, with dimensions of 60 cm long, 40 cm wide, and 4 cm high. the box was capped with a glass plate to observe the bees. a fluorescent lamp was placed at the top of the box to attract the insects via positive phototaxis. the tests were performed in a dark room with the box tilted at 45°. the box presented five lanes with 50 cm for bee observations during the locomotion test. the bees were separated into two groups. one was subject to contact with fipronil at the ld50%, and the other not. bees belonging to the experimental groups were placed at the box and released simultaneously. the time spent by bees to travel a 50 cm distance was recorded at two different periods: the first was 1h after contamination, and the second was 4h after contamination. protein quantification in the hemolymph of bees a total of 10 nursing bees were recaptured from the colonies (t1 and t2 groups; totaling 30 bees/treatment) and anesthetized on ice for 10 min. hemolymph was collected using a micropipette through an incision made at the base of the bee’s wing (cremonez et al., 1998). the protein concentration from the hemolymph sample obtained from each hive was measured using the bradford (1976) method with a quick start™ bradford protein assay (cat. nº. #5000201; biorad). the readings were performed on a spectrophotometer (spectrophotometer evolution 60 thermo fisher scientific, usa) at a wavelength of 595 nm. bovine serum albumin was used to prepare the standard curve (cremonez et al., 1998). the reading of each sample was made in triplicate. statistical analysis the protein concentration in the hemolymph, motor activity, population development, and bee longevity were evaluated using a student’s t-test. in all tests, the results were considered statistically different when p<0.05. results during the experimental period, the average consumption of 17.6 ± 13.1% of the crude protein ration provided to the bee swarms was measured. supplementation with 25% crude protein significantly influenced the offspring in the sealed brood area compared to the control; however, it did not significantly affect the open brood area (table 1). protein supplementation did not affect bee survival compared to the control. however, bees of the control group and those supplemented with 25% crude protein and contaminated with fipronil showed 100% mortality 24 h after exposure (fig 1). population development brood area open brood area sealed g1 174.4 ± 85.8a 1548.0 ± 531.3a g2 191.2 ± 67.7a 2335.0 ± 892.0b lowercase letters in the column represent a significant difference between the means (p<0.05). g1: without proteic supplementation; g2: supplemented with 25% crude protein. table 1. open and sealed brood area (cm2) of honeybees apis mellifera africanized supplemented or not with 25% of crude protein. fig 1. survival curve of apis mellifera africanized supplemented (g2) or not (g1) with 25% of crude protein and challenged or not with ld 50% of fipronil. iloran rc moreira, daniel cb barros, juliana s lunardi, ricardo o orsi – honeybee nutrition and fipronil4 in the control group, which did not receive crude protein supplementation, the contamination of bees with ld50% fipronil affected their locomotion compared to those not contaminated with fipronil for 1 and 4 h. however, there was no difference in locomotion between the bees that received protein supplementation and were contaminated with fipronil compared to bees not supplemented and challenged with ld50% fipronil for 1 and 4 h (table 2). 2016), and protein supplementation showed a positive effect in the sealed breeding area of the swarms compared to the control, suggesting an improvement in the nutrition of bees because the balanced diet could help in the development and reproduction of these insects (behmer, 2009; lihoreau et al., 2015). protein supplementation of bee swarms did not interfere with the protein content of the hemolymph of the bees, although a direct relationship between these two parameters may occur (de jong et al., 2009). nicodemo et al. (2018) verified an increase in hemolymph protein content in bees at seven days of age who had received protein supplementation under laboratory conditions. in the present experiment with protein, supplementation was conducted in field conditions, whith bees performing their normal foraging activities and using the protein provided to balance their needs, which did not promote an increase in the protein content of the hemolymph. the tests performed under laboratory conditions to evaluate the toxicity of fipronil showed significant mortality of bees with or without supplementation with the protein diet, showing the high toxicity of this agrochemical (zaluski et al., 2015). diet supplementation with 25% crude protein did not influence the survival of bees. one way that bees protect themselves from the toxic effects of compounds released in nature is via their detoxification system, which is composed of enzymes such as p450 monooxygenase, glutathione transferase, and carboxylesterase (li et al., 2007; rand et al., 2015). wahl and ulm (1983) found that young bees showed high resistance after exposure to agrochemicals when they received a protein diet in adequate quality and quantity, suggesting an improvement in the bee detoxification system. however, in the present study, there was no effect of protein supplementation, probably because there was no increase in protein content in the hemolymph of the bee swarms. thus, the detoxification system was not influenced. fipronil significantly affected the locomotor activity of bees, with or without supplementation with 25% crude protein. this result suggests the action of the neurotransmitter gamma-aminobutyric acid (gaba), which is responsible for the reestablishment of the resting state of the central nervous system and muscles of insects (dowson, 1977; aajoud et al., 2003; narahashi et al. 2010). fipronil may have acted on gaba receptors, promoting neurological and muscular alterations, evidenced in the present study by the locomotor alterations of bees exposed to it. the result of the locomotion test is important because the normal motor activity of bees is essential for their foraging activities, and it was shown that fipronil directly affected this activity, which may compromise the swarm as a whole and promote behavioral, biochemical, and neurological alterations (sanchez-bayo & goka, 2014). conclusion the present study showed that protein supplementation promoted improvement in the development of bee swarms locomotion test 1 hour 4 hour g1 control 7.0 ± 3.1a 7.22 ± 2.5a g1 control + ld50% 14.31 ± 7.6b 18.37 ± 6.9b g2 + ld50% 13.13 ± 3.2b 21.13 ± 9.3b lowercase letters in the column represent a significant difference between the means (p<0.05). g1: without proteic supplementation; g2: supplemented with 25% crude protein. table 2. locomotion (seconds) of honeybees apis mellifera africanized supplemented or not with 25% of crude protein and challenged or not with ld 50% of fipronil. supplementation did not influence the protein content of the hemolymph of bees compared to the control (table 3). discussion in the present study, an average intake of 17.6% of the protein diet supplied to the bee swarms was found, corresponding to a 5% intake of the supplied protein. therefore, the bees supplemented with the protein feed in the field consumed what they needed for their protein needs, which was approximately 25% crude protein (manning, 2016) because the protein content found in the bee bread harvested from the experimental swarms was 20%. although bees can receive their protein needs by foraging floral resources, such as pollen, the protein concentration varies depending on the region and plant variability near the apiary (forcone et al., 2011). thus, protein supplementation is important in beekeeping, especially during the off-season period, to help the swarms balance their nutritional needs. bees can balance their diets to meet their needs (hendriksma & shafir, hemolymph g1 g2 18.20 ± 0.36a 18.52 ± 0,60a lowercase letters in the column represent a significant difference between the means (p<0.05). g1: without proteic supplementation; g2: supplemented with 25% crude protein. table 3. protein content in hemolymph of nursing honeybees apis mellifera africanized supplemented or not with 25% of crude protein. sociobiology 68(3): e5830 (september, 2021) 5 and is a management practice that beekeepers should adopt, especially during the off-season period when the natural bee forage is reduced. however, this supplementation did not influence the survival of bees when exposed to the agrochemical fipronil, which proved to be toxic for honey bees and promoted changes in their locomotion. acknowledgment this work was funded by the research support foundation of the state of são paulo – fapesp (process 2017/05695-8). authors’ contributions ircm: conceptualization, methodology and formal analysis, writing and editing roo: conceptualization, methodology and formal analysis, writing and editing dcbb: writing and editing jsl: writing and editing references aajoud, a., ravanel, p. & tissut, m. 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(2015). fipronil promotes motor and behavioral changes in honey bees (apis mellifera) and affects the development of colonies exposed to sublethal doses. environmental toxicology and chemistry, 34: 1062-1069. doi: 10.1002/etc.2889 https://www.ncbi.nlm.nih.gov/pubmed/?term=buhl j%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=buhl j%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=buhl j%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=charleston ma%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=charleston ma%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=charleston ma%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=charleston ma%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=charleston ma%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=sword ga%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=sword ga%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=sword ga%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=sword ga%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=sword ga%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=raubenheimer d%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=raubenheimer d%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=raubenheimer d%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=raubenheimer d%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=simpson sj%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=simpson sj%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=simpson sj%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=simpson sj%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=simpson sj%5bauthor%5d&cauthor=true&cauthor_uid=25586099 https://www.ncbi.nlm.nih.gov/pubmed/?term=zhao x%5bauthor%5d&cauthor=true&cauthor_uid=20563240 https://www.ncbi.nlm.nih.gov/pubmed/?term=ikeda t%5bauthor%5d&cauthor=true&cauthor_uid=20563240 https://www.ncbi.nlm.nih.gov/pubmed/?term=salgado vl%5bauthor%5d&cauthor=true&cauthor_uid=20563240 https://www.ncbi.nlm.nih.gov/pubmed/?term=yeh jz%5bauthor%5d&cauthor=true&cauthor_uid=20563240 https://doi.org/10.1371/journal.pone.0094482 https://doi.org/10.1371/journal.pone.0094482 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.295-305sociobiology 60(3): 295-305 (2013) comparing the structure and robustness of passifloraceae floral visitor and true pollinator networks in a lowland atlantic forest cr benevides1, dm evans2, mc gaglianone1 introduction according to the concept of pollination syndromes, the characteristics of flowers, such as colour, odour, shape, rewards, position of reproductive structures and flowering strategies are some of the attributes that can determine the group of pollinators visiting different plant species (gentry, 1974; faegri & pijl, 1979). on the other hand, the foraging behaviour of floral visitors, frequency of visits and the movements between flowers of the same species influence their potential to promote pollination (waddington, 1983) and, consequently, the reproductive success and genetic diversity of plants (vogel, 1983; richards, 1986). ecological networks describe the interactions between species, the underlying structure of communities and the function and stability of ecosystems (montoya et al., 2006). they are an important tool for understanding the complex interabstract we investigated the plant-pollinator interactions of passifloraceae occurring in fragments of lowland semi-deciduous atlantic forest. we described floral biology, pollination syndromes and the pollinators of passiflora alata, passiflora kermesina, passiflora suberosa, passiflora malacophylla and mitostemma glaziovii. we examined the robustness of the interaction networks to species loss, a plausible scenario resulting from forest fragmentation. the effects of pollination syndrome (flower size) on network robustness was also examined. passiflora alata, p.malacophylla and p.suberosa were pollinated by bees of different corporal sizes. p.kermesina and m.glaziovii presented the highest diversity of visitors and were pollinated mainly by hummingbirds and butterflies, respectively. through the analysis of the networks we differentiate the structures of the flower-visitor network with the ‘true’ plant-pollinator network. the robustness of the flower-visitor network to animal loss was generally high, but it declined when only true pollinators were included in the network. the sequential loss of plants from the flower-visitor network resulted in low robustness: the loss of key plants could have significant cascading effects on the animals feeding on them within the forest fragment. future studies should consider the interactions between all flowering plants and animals in this habitat in order to guide conservation and management plans for these forest fragments. sociobiology an international journal on social insects 1 universidade estadual do norte fluminense, campos dos goytacazes, rio de janeiro, brazil 2 university of hull, cottingham road, hull, united kingdom article history edited by kleber del-claro, ufu, brazil received 12 july 2013 initial acceptance 07 august 2013 final acceptance 15 august 2013 key words floral syndromes, pollination ecology, plant-pollinator network structure, crop pollinators corresponding author maria cristina gaglianone programa de pós-graduação em ecologia e recursos naturais univ. estadual do norte fluminense av. alberto lamego, 2000 campos dos goytacazes, rj, brazil e-mail: mcrisgag@gmail.com actions between communities such as plants and pollinators and have the potential to quantify the effects of environmental changes, such as habitat fragmentation (memmott et al., 2007; tylianakis et al., 2008). in analyses of multiple plant animal mutualistic networks, bascompte et al. (2003) and jordano et al. (2003) reported high levels of generalization and highly nested networks (although networks with high level of generalization not always will be nested (see almeida-neto et al. 2008). nested interaction networks are thought to be highly cohesive, because generalist plants and animals tend to interact with each other. as a result, the concept of pollination syndromes has been widely questioned, since plant-pollinator interactions have been shown to be more generalist than was previously thought (hingston & mcquillan, 2000; ollerton et al., 2009). however, a recent study of a plant-pollinator network showed that 69% of the total interactions resulted from the functional group of pollinators predicted by the plant research article bees cr benevides, dm evans, mc gaglianone floral visitorand pollinator-passifloraceae networks 296 syndrome (danieli-silva et al., 2012). of the numerous ecological network properties, network ‘robustness’ [a measure of the tolerance of the network to species extinctions (dunne et al., 2002; memmott et al., 2004)] has received particular attention, partly driven by advances in computational modelling (kaiser-bunbury et al., 2010; staniczenko et al., 2010), but mostly by the desire to understand the real threat of biodiversity loss to ecosystem services and functioning (see santos et al., 2012). recent work suggested that plant-pollinator networks are less robust to species extinction than other plantanimal interaction networks such as invertebrate-parasitoid and bird-seed feeder networks (pocock et al., 2012), which is pertinent given the current decline of pollinator populations in many parts of the world (e.g. biesmeijer et al., 2006). however, to our knowledge, no study has considered the importance of floral syndromes in robustness analysis of plant-pollinator networks. the flowers of passifloraceae are considered primarily nectar sources for pollinators, and these plants may depend on these agents for their reproduction, since many species are self-incompatible (sazima & sazima, 1978; koschnitzke & sazima, 1997). interspecific variation in floral morphology, relative to the perianth and corona orientation, size and colour of the sepals, petals and filaments, and volume and sugar concentration on nectar (varassin et al., 2001), can distinguish species that attract different pollinators. in this case, the identification of pollination syndromes appears to be a valuable tool for prediction of the main pollinators of passifloraceae species. however, the association between plants and pollinators may not be so evident, and analysis of the composition of visiting animals, their body size, behaviour and frequency of visits are essential to better understand pollination processes. passifloraceae are distributed in the tropics and subtropics, and among the 600 species, 150 occur in brazil (souza & lorenzi, 2005), represented mostly by vines and scandent shrubs. studies about their pollinators indicate different functional groups in several biomes. moths and bats are the main pollinators of species with nocturnal anthesis (sazima & sazima, 1978; buzato & franco, 1992; varassin et al., 2001), while bees (koschnitzke & sazima, 1997; varassin et al., 2001), hummingbirds (vitta, 1997; fischer & leal, 2006; varassin et al., 2001), wasps and butterflies (koschnitzke & sazima, 1997; varassin et al., 2001) pollinate species with diurnal flowers. for some passifloraceae however, plantpollinator interactions are poorly understood, which may be important given the ecosystem service provided by animal pollinators for cultivated species such as p. alata (gaglianone et al., 2010) and p. edulis (benevides et al., 2009; yamamoto et al., 2012). in this study, we examine the plant-pollinator interactions of passifloraceae occurring in fragments of atlantic forest. the lowland seasonal atlantic forests are biodiversity hotspots but have been severely devastated and fragmented over the last century. the remnants in the southern part of its distribution are small and isolated fragments subjected to a range of anthropogenic pressures, such as proximity to urban areas, intensive cultivation and pasture. investigating the robustness of these plant-animal interactions to species loss in these forest fragments is thus to plan possible management actions. by examining whether the body parts of animals contact the anthers and stigmas of the flowers, we use our data to construct and compare the structures of the ‘true’ plant-pollinator network with the general plant-flower visitor network, the latter widely (and wrongly) termed in the literature as ‘plant-pollinator’ networks despite lack of evidence of actual pollination. in this study our aims are: 1) to describe the floral biology and pollinators of sympatric species of passifloraceae in a remnant of lowland seasonal atlantic forest, including new pollination data for two species not previously studied; 2) to compare the structure of quantitative plant-pollinator and plant-flower visitor networks; 3) to examine the robustness of the networks to simulated plant and animal extinctions. our expectation is that the true plant-pollinator network is less robust than the more complex plant-flower visitor network; 4) to examine the importance of pollination syndrome on network robustness. material and methods study sites the study was conducted in two fragments of lowland seasonal semi-deciduous forest guaxindiba ecological station (21°24’s and 41°04’ w, circa 1200 ha) and funil forest (21°33’s and 41°02’ w, 130 ha), in rio de janeiro state, brazil. the average annual rainfall in the region was 1023 mm. this vegetation physiognomy also known as “tabuleiro” forest occupies a large tertiary plain area near the coast with plant species distributed along a coastal-inland climate gradient (rizzini, 1979). the sclerophylly is also a distinguishing feature of these forests, where in general, epiphytic species are rare (rizzini, 1979; silva & nascimento, 2001). the ecological station represents the largest remnant of this forest type in the state of rio de janeiro, which suffered high impact in the past due to deforestation for plantation crops, pasture, charcoal production, and logging of commercial timber species (villela et al., 2006). five species of passifloraceae with diurnal flowers occurred in the area: passiflora alata curtis, passiflora kermesina link & otto, passiflora malacophylla mast., passiflora suberosa l. and mitostemma glaziovii mast., and we studied aspects of their floral biology as well as animal visitors. the floral biology and pollinators of passiflora malacophylla and m. glaziovii have not been previously described. sampling and data collection the flowering period of the five species was monitored sociobiology 60(3): 295-305 (2013) 297 monthly and blooming plants were monitored weekly from may 2004 to october 2005. morphological features including colour, shape and size, and also odour were obtained from fresh material. the time and duration of anthesis was determined by monitoring marked flowers from pre-anthesis to the closing of the petals. to check the volume and solute concentration in the nectar throughout the day, we isolated buds (n=5) in preanthesis and monitored the same flowers during one day. in 1-hour intervals, the entire content of nectar was collected, using graduated microcapillary (± 5μl) or syringes (± 0.3 ml). the solute concentration in the nectar was measured using the brix scale with a manual refractometer (bs eclipse model). the nectar was collected until the closing of the petals or until the total absence of this resource. we tested the self-pollination through the bagging of flowers (n=4 to 19) in pre-anthesis phase, without manipulation (spontaneous self-pollination) and after they were manually pollinated with their own pollen (hand self-pollination). tested flowers were monitored until fruit maturation or flower senescence. the fruit set was calculated from the number of tested flowers (n=4 to 19) and formed fruits in each treatment. we attributed categories of pollination syndromes using the following floral traits: size, height of stigmas, colours, presence of odour, anthesis time, nectar volume and nectar concentration (faegri & pijl, 1979). the melittophily was considered according to the functional groups of bees in pollination by large (height of the thorax more than 6mm), medium sized (between 3 and 6mm) or small (less than 3mm) bees (scale adapted by the authors). we captured the floral visitors with entomological nets on the flowers during their visits for taxonomic identification. the vouchers of plant and pollinator species were deposited at the ‘universidade estadual do norte fluminense darcy ribeiro’, in campos dos goytacazes, rj, brazil, in the herbarium (huenf) and zoology collection of laboratory of environmental sciences, respectively. in order to analyse the frequency of visits we counted all visitors during timed observation sessions totaling at least 6 hours in one to four observation-days for each plant species. besides the frequency of visits, behaviour features such as landing site, intra-floral behaviour and time spent on the flower were recorded by focal observations throughout the day (dafni, 1992). visitors were considered legitimate pollinators when they contacted the reproductive parts of the plants during the visits for nectar collecting; visitors whose size did not permit contact with the reproductive parts were considered robbers, as well as floral visitors arriving on the flower illegitimately or damaging parts of the plant. analysis we tested the differences among the values of volume and concentration of nectar along the day through analysis of variance (anova) in r 3.0.1 (r core team, 2013). for each passifloraceae species, we calculated the diversity of visitors using the shannon index (magurran, 1988). for the network analysis, we pooled all plant-flower visitor interaction data into a single matrix incorporating plants, animals and the total number of interactions observed in the field. we created a separate plant-pollinator matrix by excluding the animals observed visiting the flowers, rather than pollinating them (e.g. some nectar feeders). we visualized the quantitative networks and examined the robustness (r) of the two networks to simulated species extinctions using package ‘bipartite’ in r 3.0.1 (r core team, 2013). first, we simulated the sequential loss of pollinating animals and recorded the proportion of plants still remaining, calculating robustness as the area under the curve (burgos et al., 2007). if r → 1, this is consistent with a very robust system in which, for instance, most of the plant species survive even if a large fraction of the animal species go extinct. conversely, if r → 0, this is consistent with a fragile system in which, for instance, even if a very small fraction of the animal species are eliminated, most of the plants loose all their interactions and go extinct.the order of extinction was based on the most-to-least connected animals. this is the most extreme case, where the most generalist species goes extinct first (see memmott et al., 2004). second, we simulated the sequential loss of flower-visitors and recorded the proportion of plants still remaining. this was to compare the robustness of the two networks. third, we simulated the sequential loss of plants (most-to-least connected) to examine the cascading effects on the interacting animals. in all cases robustness values were compared with null models (n = 100) using t-tests to determine whether robustness values were significantly different to random. finally, to examine the importance of pollination syndromes, we calculated robustness values for the networks based on the sequential loss of large-to-small flowers and large-to-small body-size animals. results flowering period and plant reproductive systems mitostemma glaziovii flowered only in the dry-season, for approximately six weeks, with simultaneous production of a large number (averaging 53) of open flowers per individual. passiflora alata, p. malacophylla and p. suberosa flowered exclusively in the rainy season (during one to three months). passiflora alata opened only a few flowers per day and presented low synchrony between individuals. in contrast, p. kermesina flowered throughout the year with few open flowers per plant (on average 2 flowers) and few flowering individuals simultaneously. the flowers opened up to 5am and lasted until one day. passiflora malacophylla had the shortest anthesis (six hours), while p. kermesina had the longest one (24 hours) (table 1). passiflora alata, p. kermesina and p. malacophylla cr benevides, dm evans, mc gaglianone floral visitorand pollinator-passifloraceae networks 298 produced no fruit in selfing experiments, whereas p. suberosa and m. glaziovii produced fruits in hand self-pollination experiments (table 2), in different rates. pollinators attraction passiflora kermesina and p. alata have the largest flowers considering the diameter and height of the stigmas (table 2, fig 1). the flowers have four (m. glaziovii) or five (the others) petals, the ovary is elevated on an androgynophore. the flowers of p. alata have petals and sepals purplishred and corona with long filaments striped violet and white. flowers of p. malacophylla have white petals, sepals and filaments. the petals and sepals of p. kermesina are dark pink and short filaments of corona are purplish-violet, densely arranged. passiflora suberosa has yellow-green sepals and filaments. the flowers of m. glaziovii have sepals and petals white and orange filaments of corona (table 2, fig 1). flowers of passiflora alata, p. kermesina and p. malacopylla are axillary and solitary; m. glaziovii presents axillary or terminal inflorescences, whereas p. suberosa has axillary flowers solitary or in pairs. the volume of nectar produced per flower during the day did not change significantly for p. alata (p> 0.05), although slightly higher values were observed at 11am, when it reached 42 µl in one hour. the average concentration of solute in the nectar for this species also did not vary during anthesis (p> 0.05) and reached 45% (fig 2). nectar production of p. kermesina occurred throughout the day, peaking between 10am and 12pm reaching 44 µl per hour at 11am. after this time the production decreased until 4pm, when only about 4 µl were produced in a flower during one hour. the total concentration of solutes present in the nectar was kept constant during anthesis (fig 2). for m. glaziovii, nectar production was higher at the beginning of anthesis, between 5 and 6.30am (fig2), 8µl per flower on average, decreasing continuously (p <0.05) up to 12pm when the average production was 0.2 µl per flower per hour. the concentration of solutes in the nectar of this species did not differ (p> 0.05) along the day. based on the analysed features, the passifloraceae species were closer to the patterns described for species melittophilous, psicophyllous, ornithophilous and pollination by small insects (table 2). floral visitors and pollinators mitostemma glaziovii was visited more frequently by hesperiidae butterflies (table 3, fig 1f). they land on the corona and insert the proboscis between the corona and androgynophore in search of nectar, and after that pollen grains were observed in the proboscis. large bees such as eulaema nigrita lepetelier, eulaema cingulata (fabricius), xylocopa ordinaria smith and xylocopa frontalis oliver were rarely observed visiting flowers of m. glaziovii, and in those cases always contacted the reproductive parts of the flower with the thoracic and metasomal sterna (fig 1g). medium-sized bees table 1. flowering of passifloraceae species studied in lowland seasonal semi-deciduous forest, rj, brazil. fp: flowering period; an: beginning of anthesis; da: duration of anthesis in hours; nf: numbers of flowers per plant; n = number of flowers analysed; ni= number of plants analysed (m= mean e sd = standard deviation). fp an (n) da (n) nfm ± sd (ni) passiflora kermesina jan to dec 5 to 5:30h (10) 24 (10) 2 ± 0.8 (6) passiflora malacophylla jan 5 to 6:00h (14) 6 (14) 12 ± 7 (3) passiflora alata feb to apr 5 to 6:00h (20) 10 (20) 2.5 ± 1.3 (11) passiflora suberosa mar to may 5 to 7:30h (19) 12 (19) 8 ± 4.4 (3) mitostemma glaziovii jul to aug 5 to 6:30h (27) 12 (27) 53 ± 8 (3) table 2. floral biology characteristics and pollination syndrome of passifloraceae species in lowland seasonal semi-deciduous forest (guaxindiba ecological station and funil forest) in rio de janeiro state, brazil. n= total number of tested flowers in each reproductive experiment, nm= not measured. floral diameter (mm) hight stigmas (cm) colour petals colour corona odour maximal nectar volume (µl) nectar concentration pollination syndrome % self pollination % hand self pollination passiflora kermesina 8.3 2.2 pink purple yes 44 µl (11am) 34 to 30% ornitophily 0 (n=4) 0 (n=4) passiflora alata 8.2 1.9 purplish purple/white yes 42 µl (6am) 45 to 36% melittophily/ large bees 0 (n=10) 0 (n=10) passiflora malacophylla 5.4 0.8 white white yes nm nm melittophily/ medium to large bees 0 (n=5) 0 (n=5) mitostemma glaziovii 4.1 0.8 white orange yes 8 µl (6am) 22 to 10% psycophily nm 10 (n=19) passiflora suberosa 1.7 0.4 greenish purple yes nm nm small insects 0 (n=9) 50 (n=8) sociobiology 60(3): 295-305 (2013) 299 apis mellifera l. was the most frequent visitor of p. malacophylla, collecting pollen and nectar from flowers in visits that lasted on average 23 seconds (table 3). these bees landed directly on the anthers to collect pollen and they could empty their content during one visit. sporadically xylocopa frontalis and xylocopa ordinaria visited flowers in search of nectar and always contacted the reproductive parts with the dorsal thorax. the most frequent visitors of p. suberosa were small bees, such as plebeia sp. (table 3) that visited flowers in search of pollen and nectar. these bees landed directly on the anthers and collected large amounts of pollen. when seeking nectar, they landed on the sepals and walked to the ring nectary. individuals of medium-sized bees hypanthidium foveolatum (alfken) visited the flowers of p. suberosa less frequently, contacting the reproductive parts on thoracic terga while feeding on the ring nectary (table 3, fig 1e). the highest richness of visitors was observed for mitostemma glaziovii, while the highest diversity was observed for p. kermesina and m. glaziovii (table 3). fig 2. volume and concentration of nectar (mean and standard deviation) in flowers of passiflora alata (n=5), passiflora kermesina (n=1), and mitostemma glaziovii (n=5), taken at intervals of one hour. fig 1. passifloraceae flowers and pollinators / robbers. a: epicharis flava visiting flower of passiflora alata; b: heliconius ethila narcaea on passiflora kermesina; c: flower of p. malacophylla; d and e: passiflora suberosa: flower and visit by hypanthidium foveolatum; f, g and h: mitostemma glaziovii visited by hesperiidae, augochlorini (halictidae) and eulaema cingulata, respectively (photographs by paulo augusto ferreira). such as augochloropsis patens (vachal) visited these flowers in search of nectar (fig1h). the flowers of p. alata were visited exclusively by bees, and epicharis flava (friese) was the most frequent visitor (table 3, fig1a); it searched for nectar, remaining on average 16 seconds in each flower. except for plebeia sp. that landed directly on the anthers in search of pollen, the other visitors (table 3) entered the flower between the corona and androgynophore to the nectary ring. we considered epicharis flava to be a pollinator as we observed contact between floral reproductive parts and thoracic terga, whereas no such contact by euglossa cordata was observed. butterflies (lepidoptera) and hummingbirds (trochilidae) were the most frequent visitors of p. kermesina (table 3, fig1b). the hummingbirds performed quick visits (about 7 seconds on each flower) and always contacted the reproductive parts of the flower with their head. heliconius ethilla narcaea remained on the flowers for 40 seconds on average and the contact, less frequent, could occur via both antennae and wings (table 3, fig 1b). cr benevides, dm evans, mc gaglianone floral visitorand pollinator-passifloraceae networks 300 fig 3. quantitative networks for (a) the plant-pollinators and (b) plant-flower visitors of a lowland atlantic forest fragment, brazil. black rectangles, left represent the passifloraceae species (a= m.glaziovii; b= p.alata; c= p.kermesina; d= p.suberosa; e= p.malacophylla) interacting with animal species (colour rectangles, right), with the gray triangles representing the frequency of interactions. for animal species rectangles, red represents lepidoptera, orange represents diptera, yellow represents hymenoptera and white represents aves. including species only observed contacting the plant reproductive parts resulted in a loss of 8 species from the network (a), including all diptera, leading to lower network complexity. work to plant extinctions was low (r = 0.38), suggesting that the network was particularly fragile (fig4). in all cases the robustness values were significantly different to the null models (p < 0.001, table 4). sequentially removing the plants based on flower size resulted in higher robustness values than removing plants based on their number of interactions (r = 0.60 and 0.64 for the flower-visitor and plant-pollinator networks respectively). when considering the loss of animals based on body size, the flower-visitor network was highly robust (r = 0.84). however, when examining the plant-pollinator network, robustness to animal loss was considerably lower (r = 0.67) as many insects such as apis mellifera and plebeia sp. although visiting the plants, did not pollinate them. discussion flowering period, floral biology and pollinators species of passifloraceae in the studied semi-deciduous forest fragments differ in their flowering strategies and morphological features, such as colour, size, orientation and position of the corona and perianth and consequently vary in the main visitor groups associated with them. the importance of these animals for the five species was confirmed through pollination experiments showing that these plants could not self-pollinate. different flowering strategies were observed among the plant species. the high intensity of flowering of mitostemma glaziovii over a period of several weeks was attractive to numerous groups of visitors, including opportunistic species, a phenomenon observed by others when multiple plants concurrently offer of flowers (gentry, 1974; ratchcke & lacey, 1985). this may explain the greater richness and diversity of visitors, including different groups of animals, to the flowers of m. glaziovii. although the floral characteristics of this species point to pollination by lepidoptera, the offer of abundant resources by intense flowering associated with a sweet odour and exposed nectary facilitates the exploitation of nectar by other insects too, as observed in our work. the flowering of m. glaziovii restricted to the dry season possibly also contributes to the high species richness of visitors, since this is the season of lower availability of flowers (morellato et al., 2000), indicating the high importance of m. glaziovii for numerous groups of insects. this is probably the case for large bees of the genus xylocopa and eulaema, whose adults are active throughout the year in the region (aguiar & gaglianone, 2008; bernardino & gaglianone, 2013). despite the low concentration of nectar, when compared to a typical melittophilous species such as p. alata, the flowers of m. glaziovii must be important for these bees by intense flowering in a period of reduced availability of floral resources in the environment more generally. mitostemma glaziovii should be considered in future studies because of this relevant ecological role in a seasonal forest and also because of their geographical network plants-pollinators the flower-visitor network (fig3b) consisted of 20 animals with 1.16 links per species (l/s) and connectance (l/s2) and interaction evenness values of 0.29 and 0.47 respectively. the plant-pollinator network (fig3a) had 8 less animals, leading to lower complexity and structure values (0.82 links per species and connectance and interaction values of 0.23and 0.32 respectively). the robustness of the flower-visitor network to animal loss was generally high (r = 0.77, table 4), although robustness declined when only true pollinators were included in the network (r = 0.68). the robustness of the flower-visitor netsociobiology 60(3): 295-305 (2013) 301 species (h’) visitors vt±sd fv % fr bc b passiflora alata hymenoptera (h´= 0.33) apidae epicharis flava (fr) 16±6 91.5 n thorax dorsal po euglossa cordata (l.) 9±3 6.9 n no contact ro plebeia sp. 20±5 1.6 p/n no contact ro passiflora kermesina lepidoptera (h´= 1.44) heliconiidae heliconius ethila narcaea gordat 43±14 41.3 n wings and antennae po hesperiidae hesperiidae sp. 53±8 17 n no contact ro pieriidae phoebis sennae l. 22 3.4 n no contact ro hymenoptera apidae euglossa cordata (l.) 10 3.4 n no contact ro plebeia sp. 42±7 6.9 p no contact ro aves trochilidae trochilidae sp. 7±1 28 n head po passiflora malacophylla hymenoptera (h´= 0.73) apidae apis mellifera l. 23±5 78 p/n no contact ro plebeia sp. 25 1.4 p/n no contact ro xylocopa frontalis ol. 5 1 n thorax dorsal po xylocopa ordinaria sm. 4 1.4 n thorax dorsal po diptera syrphidae syrphidae sp. 42±29 2.2 n no contact ro lepidoptera hesperiidae hesperiidae sp. 46±30 16 n no contact ro passiflora suberosa hymenoptera (h´= 0.49) apidae plebeia sp. 72±49 85.9 p/n no contact ro halictidae augochloropsis patens (vachal) 15 3.8 n thorax dorsal po megachilidae hypanthidium foveolatum (alfken) 18±7 10.3 n thorax dorsal po mitostemma glaziovii lepidoptera (h´= 1.32) arctiidae utheteisa ornatrix l. nm 0.5 n no contact ro hesperiidae hesperiidae spp 40±12 71.8 n proboscis po nymphalidae dione juno stoll 39±21 6.2 n proboscis po pieriidae pieriidae sp. 14 0.5 n proboscis po diptera syrphidae ordinia obesa fab. 10±6 1.9 n no contact ro hymenoptera apidae eulaema cingulata (fab) 4 3 n ventral side po eulaema nigrita lep. 4±1 0.7 n ventral side po xylocopa frontalis ol. 10±0.7 0.5 n ventral side po xylocopa ordinaria sm. 4 0.2 n ventral side po halictidae augochloropsis patens (vachal) 25±11 13 n no contact ro aves trochilidae trochilidae sp. 3.3±0.5 1.7 n no contact ro table 3. visitors/pollinators of passifloraceae flowers in lowland seasonal semi-deciduous forest in rio de janeiro state, brazil, and features of their behaviour. vt = average visit time; sd = standard deviation; fv = average relative frequency of visits; fr = floral resource collected; bc = body parts that contact anthers and stigmas; b = behaviour, n = nectar; p = pollen; po = pollinator; ro: robber. h’ = shannon diversity index. nm= not measured. cr benevides, dm evans, mc gaglianone floral visitorand pollinator-passifloraceae networks 302 visitors. these insects are very abundant, especially in the late morning, when the volume of nectar produced by the flowers of p. kermesina reached the highest values. this high frequency of visits, associated with the possible pollination behaviour through the movement of their wings or the touch of antennae in floral reproductive parts, suggests that heliconius butterflies may be important pollinators, especially in the low frequency of visits or absence of hummingbirds. this suggestion has already been made by benson et al. (1976) and our observations confirm these insects as potential pollinators, although less efficient than the hummingbirds. the flowering of passiflora alata differs from the previous species because of the opening of only a few flowers on each plant per day, but in higher numbers of flowering plants simultaneously. beyond flowering, floral traits are compatible with the description of the melittophily with pollination by large bees: large flowers with great volume (especially early in the morning) and high concentration of the nectar and sweet odour. epicharis flava was the main pollinator due to its large body. as noted in other studies with natural populations (varassin & silva, 1999) or even in cultivations of p. alata (gaglianone et al., 2010), large oil bees of the tribe centridini are their main pollinators. the use of passifloraceae flowers as nectar sources by these bees had been highlighted by gaglianone (2006). some features of p. malacophylla such as the size of flowers and short period of anthesis (only in the morning), suggested it as primarily melittophilous with pollination by medium or large sized bees. based only on body size, honey bees could be considered potential pollinators. however, the behaviour of these bees on the flowers indicated them as robbers. this behaviour is similar to that observed in flowers of passiflora edulis flavicarpa, the yellow passion fruit, cultivated in the study region (benevides et al., 2009). in flowers of both species, honey bees seek the pollen, which is taken directly from the anthers, without contact with the stigmas. the intense pollen removal directly from the anthers without promoting pollination was also observed by plebeia sp in flowers of passiflora suberosa. flowers of this plant, the smallest among the studied species, present pollination syndromes by small insects. however, only bees visited them in the study area and medium sized bees were the pollinators. unlike other species, p. suberosa showed high self-compatibility, which had already been described in other study (varassin & silva, 1999). our observations suggest that pollen removal made by plebeia can even prevent selfpollination, by pollen removal mainly in the period in which the stigmas were still curving themselves, before they were receptive. network plants-pollinators the construction and analysis of our relatively simple plant-animal interaction networks within atlantic forest fragments enabled us to consider how robust they are to simulated table 4. the robustness (r) of a) the pollinator network to animal extinctions (m1) and b) the flower-visitor network to animal and plant extinctions (m2 and m3 respectively). if r → 1, this is con-if r → 1, this is consistent with a very robust system in which, for instance, most of the plant species survive even if a large fraction of the animal species go extinct. conversely, if r → 0, this is consistent with a fragile system in which, for instance, even if a very small fraction of the animal species are eliminated, most of the plants loose all their interactions and go extinct. observed r null mean lower ci upper ci t p m1 0.679 0.562 0.549 0.574 -18.813 < 0.001 m2 0.765 0.788 0.778 0.797 4.648 < 0.001 m3 0.378 0.660 0.651 0.668 64.716 < 0.001 distribution restricted to the atlantic forest (bernacci et al., 2013). the flowering pattern of p. kermesina, in contrast to m. glaziovii, presents shorter flowering periods during the year and produces fewer flowers per day in plants sparsely distributed. this strategy is associated with trapline behaviour of pollinators such as hummingbirds and orchid bees (janzen, 1971), also observed in our study. the floral morphology, however, with petals and filaments pink and red, high volume of nectar, reduced corona and long distances between nectaries and reproductive organs indicate the ornithophilous pollination syndrome (faegri & pijl, 1979). hummingbirds were undoubtedly the most efficient pollinators, by their behaviour and body size, although lepidoptera were the most frequent fig 4. the robustness of the passifloraceae-animal interaction networks to simulated species extinction (based on the losing the mostto-least connected species). the robustness of the plant-pollinator (a) and plant-flower visitor network (b) to sequential animal loss is relatively high, whereas the sequential loss of passifloraceae species in the plant-flower visitor (c) leads to low robustness. sociobiology 60(3): 295-305 (2013) 303 species extinction. moreover, by examining which animals contacted the reproductive parts of the plants we were able to differentiate the structures of the flower-visitor network with the ‘true’ plant-pollinator network. we found differences in network structure and complexity. although the robustness of the flower-visitor network to animal loss was generally high, robustness declined when only true pollinators were included in the network. this has implications for studies of ecological networks that in the past have considered flower-visitor networks as pollination networks and have used the terms interchangeably (e.g. pocock et al., 2012). we found that the flower-visitor network had low robustness when plants were sequentially lost (based on the most-to-least connected) and was particularly fragile. although body size can predict degree in plant-animal mutualistic networks (e.g.chamberlain & holland, 2009), we found that the frequency of interaction of key plants was more important to network integrity than plant and animal size, although we concede that our network was too small and incomplete to test this conclusively. considering the possible effects of the forest fragmentation on the ecological interactions (e.g. hagen et al., 2012), our results suggest that the loss of passifloraceae could have considerable cascading effects on the animals feeding on them within the forest fragment. future studies should consider the wider interactions between all flowering plants and animals in this habitat. acknowledgments we are grateful to probio/mma (0115-00/04) and procad/capes (158/07) for funding this project, faperj/uenf for the scholarship to cr benevides (msc) and fellowship to dm evans (visiting professor), to cnpq for the fellowship to mcgaglianone (pq), to inea-rj for permission to study in guaxindiba ecological station, to dr. joão marcelo alvarenga braga (jardim botânico do rio de janeiro, rj) and dr. teonildes sacramento nunes (universidade estadual de feira de santana, ba) for plants identification, and paulo augusto ferreira for the photographs. we also thank two anonymous reviewers for helpful suggestions that improved this article. references aguiar, w.m. & gaglianone, m.c. 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(2012). the role of bee diversity in pollination and fruit set of yellow passion fruit (passiflora edulis forma flavicarpa, passifloraceae) crop in central brazil. apidologie, 43: 515-526, doi: 10.1007/s13592-012-0120-6 doi: 10.13102/sociobiology.v61i4.510-516sociobiology 61(4): 510-516 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 pollination services provided by melipona quadrifasciata lepeletier (hymenoptera: meliponini) in greenhouses with solanum lycopersicum l. (solanaceae) introduction ecosystem services are benefits provided by organisms that interact with each other for human welfare (daily, 1997). among them, pollination has proven to be an essential service, especially that performed by insects, accounting for approximately 35% of global agricultural production (klein et al., 2007). in this context, bees play a key role in various ecosystems (bawa, 1990). they have increasingly been spotlighted as the main agent responsible for pollinating many species of native and cultivated plants, since they ensure the maintenance of genetic variability in the former and the productivity and fruits quality in the latter (kremen, 2005). it is estimated that of a total of 40,000 pollinators, 20,000 are bees (michener, 2007) and 73% of cultivated plant species worldwide are pollinated by these insects (fao, 2004). in brazil, the efficiency of pollination performed by bees has been confirmed for various crops, either in greenhouses or open areas. among the crops grown in greenhouses, abstract stingless bees are considered particularly promising for use as commercial pollinators. however, studies testing the effectiveness of these bees in pollinating grape tomatoes in greenhouses are not yet available. this work aimed to analyze the floral biology of the grape tomato and evaluate whether the additional pollination performed by melipona quadrifasciata lepeletier generates an effective increase in production and improves the quality of this variety of tomato when grown in greenhouses. the research took place in two greenhouses located in araguari, minas gerais state, brazil. in one of them, we only used a mechanical method of pollination and, in the other, pollination by the mechanical method was associated with pollination performed by bees. the productivity was compared by recording the number of flowers and fruits formed on different branches, and tomato quality was assessed by analyzing their size, weight, number of seeds and total sugar content. tomatoes originating from flowers visited by m. quadrifasciata workers produced about 47% more seeds and their concentration of sugar was approximately 14% higher. these results suggest that using m. quadrifasciata for tomato pollination shows to be promising, since their use as pollinators entails positive effects on fruit quality. sociobiology an international journal on social insects bf bartelli, fh nogueira-ferreira article history edited by cândida maria l. aguiar, uefs, brazil received 23 september 2014 initial acceptance 31 october 2014 final acceptance 27 november 2014 keywords stingless bee, horticulture, fruit set, quality. corresponding author fernanda helena nogueira ferreira programa de pós-graduação em ecologia e conservação de recursos naturais, instituto de biologia, universidade federal de uberlândia, ceará st., umuarama, 38400-902, uberlândia, minas gerais, brazil e-mail: ferferre@inbio.ufu.br especially noteworthy are strawberry pollination, tested with tetragonisca angustula (latreille) (cruz & campos, 2009), scaptotrigona aff. depilis (moure) (roselino et al., 2009) and nannotrigona testaceicornis (lepeletier) (roselino et al., 2009); sweet pepper, with melipona subnitida ducke (cruz et al., 2005), melipona quadrifasciata anthidioides lepeletier (roselino et al., 2010) and melipona scutellaris latreille (roselino et al., 2010); and tomato, tested with nannotrigona perilampoides (cresson) (cauich et al., 2004; palma et al., 2008), m. quadrifasciata (del sarto et al., 2005; bispo dos santos et al., 2009) and apis mellifera l. (bispo dos santos et al., 2009). tomato, solanum lycopersicum l. (solanaceae), is one of the most widespread vegetable crops in the world. the fruit is widely consumed and is of great importance to the human body, mainly due to the presence of lycopene, an antioxidant that fights free radicals and slows aging. tomatoes are grown both in open areas and in greenhouses. their flowers are self-fertile, bisexual, do not produce nectar research article bees universidade federal de uberlândia (ufu), uberlândia, mg, brazil sociobiology 61(4): 510-516 (december 2014) 511 and present poricidal anthers. therefore, in order to release the pollen and, consequently, enable pollination to occur, vibration of the anthers with consequent opening thereof is necessary (buchmann, 1983). when grown in open areas, pollen release is performed by the wind (mcgregor, 1976; free, 1993) and natural pollinators, especially bee species, which are capable of a vibratory behavior or buzz-pollination. in greenhouse cultivation, pollination is normally accomplished by mechanical vibration of the flowers, as an alternative to the absence of wind and natural pollinators. however, the mechanical method increases the production costs as they require additional labor and can cause damage to flowers and thus to fruits (cribb et al., 1993; ilbi et al., 1994; dogterom et al., 1998). for this reason, the number of studies involving the management of pollinators in greenhouses has grown steadily in recent years. among tomato pollinators in greenhouses are amegilla chlorocyanea (cockerell) (hogendoorn et al., 2006), bees of the genus xylocopa (lestis) lepeletier & serville (hogendoorn et al., 2000) and bombus latreille (morandin et al., 2001), and the stingless bees n. perilampoides (cauich et al., 2004; palma et al., 2008) and m. quadrifasciata (del sarto et al., 2005; bispo dos santos et al., 2009; hikawa & miyanaga, 2009). stingless bees are particularly promising for use as commercial pollinators (cruz & campos, 2009), given that they do not present a functional sting, are easy to handle (usually low aggressive-nonstinging), have populous and perennial nests, present a marked worker recruitment behavior and stock a large amount of food (heard, 1999). m. quadrifasciata is a stingless bee that has a wide distribution throughout brazil and is present in argentina and paraguay (camargo & pedro, 2012). this species proved to be efficient in pollinating different tomato varieties. however, studies testing its pollination efficiency on grape tomato, grown mostly in greenhouses, are yet to be performed. accordingly, this study aimed to: (1) analyze the floral biology of grape tomato to allow a richer discussion about the effectiveness of pollinators; and (2) evaluate the pollination efficiency of m. quadrifasciata on grape tomato in greenhouses, to verify whether introducing nests of this species generates an effective increase in production and improves fruit quality. material and methods study area the study was conducted in two identical greenhouses located in “chácara paraíso”, in the municipality of araguari (18º39’3.55”s/48º11’7.51”w), minas gerais state, brazil. this municipality is configured as a hub of food production, with approximately 98,487 ha of arable area and an estimated production of 70,631 tons of tomatoes per year (ibge, 2012). the regional climate is marked by two distinct seasons, the rainy season that extends from october to march and the dry season, from april to september. annual rainfall varies between 1,160 and 1,460 mm/year and the average annual temperature is between 23 and 25ºc, being uniform throughout the year (alves & rosa, 2008). the greenhouses comprised approximately 1,344 m2 (48 m x 28 m), covered at the top with an extra long life (ell) diffuser antivirus plastic film with ultraviolet filter, and fully enclosed on the sides with anti-aphid screens. the greenhouses presented 24 planting rows, and each of these had an average of 112 tomato plants, totaling 2,688 plants. floral biology the floral biology analyzes were performed in one of the two greenhouses. the parameters evaluated included aspects of floral morphology, number of open flowers per plant, duration of the flower, pollen viability and stigma receptivity. the structures of a total of 20 flowers of different plants were analyzed. the floral diameter and length of anther, pistil and style were measured using a digital pachymeter. counting the number of open flowers per plant was performed on 40 different randomly selected individuals two months after the onset of flowering. fifty floral buds were marked and monitored to determine the time of flowers opening, duration of anthesis and flower senescence. pollen viability was tested indirectly by staining 20 pre-anthesis flower buds from different plants with 2% acetic carmine (kearns & inouye, 1993), and estimated through observation of the first 300 pollen grains from each bud using an optical microscope. stigma receptivity was tested in 10 buds three days before anthesis, 10 buds in pre-anthesis and a total of 55 newly opened flowers from 7 am to 6 pm (five flowers every hour) using 3% hydrogen peroxide (h2o2). receptivity was detected by the formation of air bubbles (kearns & inouye, 1993). pollination experiments with m. quadrifasciata for pollination experiments, the plants presented the same development time in both greenhouses. one greenhouse was used as control, where only mechanical method of pollination was performed (flowers were vibrated using a leaf blower). in the other greenhouse, six nests of m. quadrifasciata with similar population sizes were introduced in march 2012 at the onset of flowering. in this greenhouse, pollination occurred by the mechanical method associated with pollination by bees. given the difficulty in bee orientation inside the greenhouse (bartelli et al., 2014), before the introduction of bees, the old foragers were removed to avoid their loss in the enclosed space, following the methodology used by cauich et al. (2004). moreover, for the same reason, the nests were placed in the greenhouse after dark (cuypers, 1968). bf bartelli, fh nogueira-ferreira pollination services by m. quadrifasciata in s. lycopersicum512 in order to allow a homogeneous distribution of the bees on flowers, nests were arranged uniformly in the central region of the greenhouse (free, 1993) and supported by plastic boxes installed in eucalyptus logs located within the planting rows. containers with water, mud and cerumen (alternative source for plant resin) of t. angustula were placed on the plastic boxes. nests were fed biweekly with pollen collected by a. mellifera (5g) macerated with sugar and water until the onset of flower visitation, and fed weekly with syrup (a mixture of a. mellifera honey, sugar and water in the ratio 1:1) over the entire period of confinement. for pesticide application, a common management practice, the entrances of all nests were sealed with paper, the nests themselves were protected with plastic bags and the containers covered with cardboard boxes. these protections were removed only after the pesticide had dried. then, the entrances of the nests were unobstructed after dark. to evaluate the pollination efficiency of m. quadrifasciata after initiation of foraging activities by the bees on flowers, the productivity of the two greenhouses was compared by marking 20 branches of different plants (randomly selected) in each. the total number of flowers and fruits produced over time on each of the branches was recorded. fruits decrease in size from the base to the apex of the branches. thus, to determine whether the additional pollination performed by m. quadrifasciata improved the quality of the tomatoes, the position that each fruit occupied on the branch was taken into consideration. we collected 25 fruits originating from flowers effectively visited by bees (after observation of the visit and marking of the flower) in the experimental greenhouse and 25 corresponding fruits in the control greenhouse. all of them were collected at an intermediate stage of maturation (early in the reddish stage) and analyzed on the day of harvest. as tomatoes decrease in size and weight from the first crop through the subsequent flowerings, depending on the age of the culture (hill, 2001), the fruits of both greenhouses were collected during the same period. fruit quality was evaluated by the following parameters: size (longitudinal and equatorial diameters), using a digital pachymeter; weight, using a digital balance; number of seeds, counted directly; and concentration of total sugars (°brix), by means of an analogical portable refractometer, after removal of the seeds and maceration of the rest of the fruit. statistical analyzes to verify the extent to which tomato production was dependent on the introduction of nests, we conducted an analysis of covariance (ancova) for the number of fruits produced, with the number of flowers on the branches as a covariate (zar, 2010). to determine if the quality of the fruit (size, weight, number of seeds and sugar concentration) was affected by the additional pollination effected by bees, we performed a paired t-test (zar, 2010) for each of the analyzed parameters. results floral biology regarding floral morphology, the average floral diameter was 31.31 mm and the average length of anther, pistil and style were, respectively, 8.79, 10.56 and 8.01 mm (table 1). the average number of open flowers per plant was 16.07 (table 1) and the flowers lasted an average of five days, closing at night and opening again between 6:30 and 7 am, being totally open by 8 am. an average of 98% of the pollen grains present in anthers was viable. the stigma of newly opened flowers was receptive during the entire period of evaluation, from 7 am to 6 pm. even before opening, the grape tomato flowers were able to be fertilized, considering that the stigma of all buds in pre-anthesis as well as buds three days before anthesis were receptive. pollination experiments with m. quadrifasciata of the six nests introduced, only two remained over the entire 7-months cycle of flowering of the tomato plants in the greenhouse. two nests did not survive the conditions of confinement and two were removed from the greenhouse after being held in the greenhouse for three months to prevent their loss. the worker bees began the pollen collection from tomato plants after about six months of confinement. table 1. minimum, maximum and mean (± standard deviation) values of the floral diameter, length of anther, pistil and style and number of open flowers per tomato plant in a greenhouse of grape tomato, in araguari, minas gerais state, brazil. statistical data floral diameter (mm) anther length (mm) pistil length (mm) style length (mm) number of flowers/plant minimum 28.76 8.36 9.96 7.53 5 maximum 33.85 9.15 11.3 8.87 31 mean (± sd) 31.31 (± 1.33) 8.79 (± 0.22) 10.56 (± 0.36) 8.01 (± 0.39) 16.07 (± 6.39) the introduction of nests of m. quadrifasciata did not generate an effective increase in production of grape tomatoes, since the average number of fruits did not differ between treatments (f = 0.70; df = 1; p = 0.41; fig 1). however, fruits originating from flowers visited by bees presented about 47% more seeds (/t/ = 4.231; df = 24; p < 0.001) and had a sugar concentration approximately 14% higher (/t/ = 5.91; df = 24; p < 0.001) compared to those derived from flowers pollinated only by the mechanical method. regarding the other parameters associated with fruit quality, additional pollination by bees did not generate an increase in size, i.e., longitudinal diameter (/t/ = 0.811; df = 24; p = 0.425), equatorial diameter (/t/ = 0.053; df = 24; p = 0.958) or weight (/t/ = 0.169; df = 24; p = 0.867) of the tomatoes produced (table 2). sociobiology 61(4): 510-516 (december 2014) 513 discussion through genetic improvement, several varieties of tomatoes were and have been bred to meet the different tastes of consumers. among those, grape tomato has become increasingly popular, since the sweetness, small size and elongated shape (similar to grape) of the fruits make them attractive for culinary use, for example, as ingredients in salads (hill, 2001). hence the importance of knowing its productive potential, and efficiency in fruit formation. in relation to this, the studied tomato variety shows promising prospects. two important aspects to consider are the large percentage of viable pollen grains present in anthers and the extensive period of stigma receptivity. the stigma showed receptivity three days before anthesis and in newly opened flowers, over an extended period of the day. results similar to those obtained in the present study were found for the long-life tomato, variety rodas, regarding the time of flowers opening (del sarto et al., 2005). when compared to variety forty, grape tomato flowers are larger and the number of open flowers per plant is considerably higher (unpublished data). as expected, grape tomato flowers have the anthers longer than the styles, like other tomato varieties, favoring the occurrence of self-pollination. different results have been found in various studies that used stingless bees for tomato pollination in greenhouses (cauich et al., 2004; del sarto et al., 2005; palma et al., 2008; bispo dos santos et al., 2009; hikawa & miyanaga, 2009). in one such study, the inefficiency of m. quadrifasciata in increasing tomato production was explained by the overlap of only 30 minutes between the peak activity of the workers and the period of highest stigma receptivity of the flowers (del sarto et al., 2005). however, as previously mentioned, the stigma grape tomato flowers are receptive from 7 am to 6 pm, a time interval that covers almost the whole period of foraging by m. quadrifasciata workers (bartelli et al., 2014). biology and foraging behavior differ between species of stingless bees and, in addition, the pollination requirements of different cultivated plant species and different varieties of the same species are not identical. such factors must be taken into consideration when making comparisons. despite different tomato varieties being used, a more direct comparison can be made with a study developed by del sarto et al. (2005). according to these authors, two colonies of m. quadrifasciata would be sufficient to pollinate a greenhouse with 858-1,534 tomato plants (a proportion of one nest per 429–767 plants). these estimates were made considering that, in a strong nest of m. quadrifasciata, about 6% (jarau et al., 2000) of an approximate total of 890 bees (michener, 1974) are forager workers. moreover, for pollen collection, each worker visits 28–50 tomato flowers per trip (del sarto et al., 2005). in the present study, however, beyond the small number of active forager workers, the density was 0.001 nest/m2 and the proportion was one colony to 1,344 tomato plants, insufficient for an increase in production. thus, considering the size of the studied greenhouse, the inability of m. quadrifasciata to increase the production of tomatoes in this study is probably related to the low density of nests inside the greenhouse, inasmuch as only two of the six that were introduced remained at the end of the experiments. additionally, the number of active forager workers in each of the nests was very low compared to the number of flowers available for pollen collection. nevertheless, in general, an improvement could be observed in fruit quality. the highest number of seeds found in the tomatoes originated from flowers visited by m. quadrifasciata workers, denoting the efficiency of this species on tomato pollination. compared to the mechanical method, the buzz pollination behavior carried out by several species of bees, such as m. quadrifasciata, results in an increase in the number of pollen grains deposited on the stigma of the flowers, thereby increasing the quantity of seeds in the fruits (hogendoorn et al., 2000). although several studies had shown the existence of a positive correlation between the number of seeds and the size table 2. mean values (± standard deviation) of each of the parameters related to fruit quality in the two different treatments (mechanical method of pollination alone and pollination by mechanical method + pollination by bees), in araguari, minas gerais state, brazil. fig 1. mean number (± standard error) of fruit set per branch in the two greenhouses of grape tomato, one control (mechanical method of pollination alone) and the other where melipona quadrifasciata bees were introduced (pollination by mechanical method + pollination by bees), in araguari, minas gerais state, brazil. the same letters indicate no significant difference between treatments. fruit attribute pollination treatment mechanical mechanical + bees longitudinal diameter (mm) 29.14 (± 1.89)a 29.67 (± 2.81)a equatorial diameter (mm) 21.40 (± 1.82)a 21.38 (± 1.89)a weight (g) 7.261 (± 1.512)a 7.196 (± 1.733)a number of seeds 56.92 (± 23.18)a 83.72 (± 23.30)b concentration of sugars (ºbrix) 6.51 (± 0.51)a 7.40 (± 0.69)b bf bartelli, fh nogueira-ferreira pollination services by m. quadrifasciata in s. lycopersicum514 and weight of tomatoes (dempsey & boynton, 1965; imanishi & hiura, 1975; hogendoorn et al., 2010), there was no difference in these last two parameters between the treatments performed. among other factors, the size and weight of the fruit are related to the amount of water used in irrigation, determining a higher or lower concentration of soluble components. furthermore, high pluviometric indices also trigger the production of larger tomatoes, but with lower nutrient content and less accentuated flavour (casquet, 1998). however, the greenhouses used were positioned one beside the other and were managed the same way, eliminating such possibilities. our knowledge about the variety of tomato studied is still fairly rudimentary. however, the fruits apparently develop to an equal extent regardless of the degree of pollination. in other words, they grow independent of the number of pollen grains deposited on the stigma of the flowers and, consequently, of the number of seeds. this might explain the similarity in relation to the size and weight attributes found between fruits pollinated only by the mechanical method and those additionally pollinated by bees. nevertheless, tomatoes originating from flowers visited by bees had a considerably higher concentration of sugars. little is known about the mechanisms involved in accumulating sugars in the process of tomato development (damon et al., 1988). however, the higher concentration of carbohydrates in fruits resulting from the additional pollination carried by the bees may be related to the greater number of seeds therein. through the production of hormones, the number of seeds may modify and stimulate the processes of division and cell expansion, increasing the storage capacity for soluble solids in the cells (gillaspy et al., 1993; prudent et al., 2009). from the food science standpoint, quality comprises the features that distinguish the individual units of a product, being significant in determining the degree of acceptability by the purchaser. regarding tomatoes, quality is strongly associated with the soluble solids content, and sugars are the main components of this fraction (damon et al., 1988). thus, based on the results obtained in this study, one can conclude that the use of m. quadrifasciata for tomato pollination is promising, since the effects on the quality of the tomatoes originating from flowers visited by workers were inequivocally positive. in the last few years, the use of stingless bees in the brazilian agricultural systems under greenhouse conditions has become increasingly widespread and presented exceptional results (cruz & campos, 2009), contrasting with the alternative use of exotic pollinators and providing greater awareness of the importance of conservation of stingless bees. however, it is still necessary to improve techniques of nest multiplication and species management specific to different species, especially in protected environments. such actions would provide the availability and utilization of these insects in agriculture on a large scale. with improvements in these management techniques, the appropriate density of bees for pollination of different crops could be determined by maintaining greater numbers of nests inside greenhouses. even so, the use of m. quadrifasciata is already a good strategy for producers, since the additional pollination performed by bees improves tomato quality. acknowledgements the authors are grateful to the farmers edson, clóvis and joão, who allowed and gave full support to this study, to dr. marcela yamamoto for providing instructions on the floral biology experiments, to ms. camila nonato junqueira for the incentive and suggestions given to this study, to the biologist alexandre oliveira resende santos for the contributions in the field and to conselho nacional de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado de minas gerais (fapemig) and coordenação de aperfeiçoamento de pessoal de nível superior (capes) for their financial support. references alves, k. a. & rosa, r. 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(2010). biostatistical analysis fifth edition. new jersey: prentice-hall/pearson. doi: 10.13102/sociobiology.v61i1.100-106 sociobiology 61(1): 100-106 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 effect of the habitat alteration by human activity on colony productivity of the social wasp polistes versicolor (olivier) (hymenoptera: vespidae) rf torres1, vo torres1, yr súarez2, wf antonialli-junior2 introduction eusocial wasps are represented by 29 genera in the neotropical region, 22 of which are recorded in brazil (carpenter & marques, 2001). these wasps occupy many kinds of habitats, primarily associated with human constructions (lima et al., 2000; prezoto et al., 2007), i.e., show a high degree of synanthropy (fowler, 1983). among the numerous factors that contribute to the success of social wasps, the colony productivity stands out. productivity depends on ecological factors including changes in temperature, prey availability, and number of founders, among others (gamboa et al., 2005; inagawa et al., 2001). according to gamboa (1978), for example, colonies of polistes metricus (say) founded by association are more productive than those initiated by a single female. tibbetts & reeve (2003) found that polistes dominula (christ) colonies initiated by association better abstract currently, the main impacts on biodiversity are generated by human activities in natural environments. monitoring the number of species of social wasps nesting attached to buildings is important to evaluate the effect of this activity on colony productivity. this study evaluated the effect of habitat alteration, particularly by human activity on the productivity of colonies of the wasp polistes versicolor. we evaluated 20 abandoned nests and compared the productivity parameters: number of cells constructed, number of adults produced, nest dry mass, proportion of productive cells, number of generations, and diameter of the petiole. most of these parameters showed higher values in the colonies nesting in the habitat less altered by human activity. therefore, productivity was significantly higher in this habitat. in the nests, regardless of the site, the cells that were central and closer to the petiole were the most productive. colonies in the two habitats used different strategies: in the habitat more altered by human activity, the wasps invested more in reusing cells than in enlarging the nest. however, the species continues to nest in the urban area, probably because of decreased interspecific competition, predation, and interference from climate variations. sociobiology an international journal on social insects 1 universidade federal da grande dourados, dourados, mato grosso do sul, brazil. 2 universidade estadual de mato grosso do sul, dourados, mato grosso do sul, brazil. article history edited by fernando noll, unesp, brazil received 14 october 2013 initial acceptance 12 november 2013 final acceptance 28 november 2013 keywords nest, independent foundation, polistinae, reutilization of cells, synanthropy corresponding author romario ferreira torres programa de pós-graduação em entomologia e conservação da biodiversidade universidade federal da grande dourados fac. de ciências biológicas e ambientais, 241 dourados, mato grosso do sul, brazil 79804-970, e-mail:romario_torresmn@hotmail.com defend their colonies from predators and/or conspecific wasps, and this cooperation increases the colony productivity. environmental factors may also be related to colony success and productivity, for example, the nesting site, as evidenced by inagawa et al. (2001) and nadeau & stamp (2003) in polistes snelleni (saussure) and polistes fuscatus (fabricius), respectively. these studies showed that colonies founded in locations with higher mean temperatures had higher productivity. for colonies under the same environmental conditions, giannotti (1997) noted that the productivity can be influenced by intrinsic factors such as the number of cells, reuse of cells, duration of immature stages, and the mortality of the immatures in the different stages. as observed by montagna et al. (2010), the productivity can vary within the comb; the central cells of the combs of mischocyttarus consimilis (zikán) are more productive than the peripheral cells. research article wasps sociobiology 61(1): 100-106 (march, 2014) 101 santos & gobbi (1998), inagawa et al. (2001) and gamboa et al. (2005) evaluated the effect of habitat on the colony productivity of the social wasps, while others such as penna et al. (2007), montagna et al. (2010), oliveira et al. (2010) and sinzato et al. (2011) evaluated the productivity in urban environments. however, only michelutti et al. (2013) compared the productivity of colonies between environments with high and low degrees of human interference, in m. consimilis. according to samways (2005, 2007), disturbances caused by humans in natural environments are the main factors acting to reduce biodiversity in the tropics. the human presence alters habitat quality (raupp et al., 2010; schowalter, 2012). the expansion of ranching and other agricultural activities as well as urbanization are the main factors modifying the natural habitats (abensperg-traun & smith, 2000; new, 2005; raupp et al., 2010) and often have forced wasps to search for new nesting environments. it is common to find wasps nesting on or in human constructions (clapperton, 2000; mead & pratte, 2002). this can be explained by the abundance and quality of available sites, and the reduced interspecific competition and predation by vertebrates in these environments. among the polistinae, several species of polistes use human constructs as nesting substrates (fowler, 1983; butignol, 1992; giannotti & mansur, 1993). however, gould & jeanne (1984) and michelutti et al. (2013) suggested that the urban environment negatively affects the productivity and development of the colonies, since it offers fewer resources than do preserved environments. polistes versicolor (olivier, 1791) builds nests formed by a single, uncovered comb attached to the substratum by a single petiole (richards, 1978). this species is facultatively synanthropic, since it is associated with human constructions (oliveira et al., 2010) and also occurs in natural environments. studies of predation (butignol, 1992; prezoto et al., 2006) and productivity (ramos & diniz, 1993; oliveira et al., 2010) have been performed with this species, but none has evaluated the alteration of habitat by human activity on this aspect. therefore, this study evaluated the effect of the habitat alteration, particularly by human activity, on the productivity of p. versicolor. material and methods data collection and field procedures to evaluate the effects of human activity on colony development, we compared the final productivity of colonies of p. versicolor in two habitats in the municipality of mundo novo in the state of mato grosso do sul, brazil. we collected 10 abandoned nests in each habitat during april 2012 to august 2012. all these colonies were monitored weekly to determine the end of the colony cycle. we used only nests that reached the decline stage as defined by jeanne (1972), which showed widespread presence of empty cells in the comb and nest abandonment. following the parameters proposed by michelutti et al. (2013), productivity was measured from the number of cells constructed, number of adults produced, and dry mass of the nest. we also measured the diameter of the petiole, proportion of productive cells and number of generations in each cell for estimated productivity of the central and peripheral cells. by counting the number of layers of meconium in each cell of the comb was estimated the number of adults produced. this meconium layer is formed on the floor of the cells that produced adults, since just before pupation, when the last-instar larva eliminates feces (gobbi & zucchi, 1985; giannotti, 1999). each cell of the comb was sectioned with the aid of tweezers to extract the meconium layer and for analysis of the nest dry mass, the nest was weighed on a precision balance according michelutti et al. (2013). areas of study we compared the degree of alteration of the environment by human activity, according to the parameters suggested by michelutti et al. (2013). thus, the first area (23°93’77”s; 54°33’90”66w), categorized as habitat less altered by human activity, was located 8 km from the urban perimeter, and has little human activity. this area consists of rural properties with grassland, agricultural crops and forest fragments predominating, associated with rivers and streams. the second area (23°93’79”s; 54°29’47”w), categorized as habitat more altered by human activity, contains numerous buildings of wood or brick, most of them inhabited. there is intense movement of people, and mainly areas of pavement and grass lawn adjacent to the buildings. statistical analyses the t-test for independent samples was used to evaluate possible differences between the colony productivity parameters of the two populations. we applied a pearson correlation analysis to compare the number of cells constructed with the number of adults produced, to evaluate possible differences in strategies of comb use between the two populations. we also estimated the correlation between the diameter of the petiole and the nest dry mass. for all analyses was used the software systat 11 and the variable was considered when the resulting regression coefficient was significant at the 0.05 level. results in the habitat more altered by human activity, 60% of the nests occurred on human constructions and 40% on trees. in the habitat less altered by human activity, 100% of the nests were in trees, even if buildings were nearby. for the 10 nests in the habitat less altered by human activity, the mean values (±se, n=10) were: 411.30 ± 123.27 rf torres on colony productivity of the social wasp polistes versicolor102 cells constructed, 493.90 ± 213.03 adults produced, nest dry mass of 9.50 ± 4.28 g, 90.20 ± 10.87% of cells were productive, 24.76 ± 14.90% of cells were reused, and 1.16 ± 0.26 adults produced per cell (table 1). in this environment we found a significant positive correlation between the number of adults produced and the number of cells constructed (r = 0.86, p <0.01, n = 10) (fig. 1). the correlation between the diameter of the petiole and the nest dry mass was also significantly positive (r = 0.75, p = 0.01, n = 10) (fig. 2). for the 10 nests in the habitat more altered by human activity, the mean values (±se, n=10) were: 203.00 ± 44.79 cells constructed, 200.90 ± 65.69 adults produced, nest dry mass of 3.17 ± 0.99 g, 69.23 ± 17.18% of cells productive, 23.19 ± 15.95% of cells reused and 1.02 ± 0.34 adults produced per cell (table 1). in this environment there was no significant correlation between the number of adults produced and the number of cells constructed (r = 0.30, p = 0.40, n = 10) (fig. 1), or between the diameter of the petiole and the nest dry mass (r = 0.33, p = 0.36, n = 10) (fig. 2). the results of t-tests showed that the productivity in the habitat less altered was significantly higher than in the habitat more altered, with respect to the number of cells constructed (t = -5.02, df = 11.3, p <0.001), number of adults produced (t = -4.15; df = 10.7, p = 0.002), dry mass of nests (t = -5.98, df = 11.4, p <0.001) and proportion of productive cells (t = -3.29, df = 15.5, p = 0.005) (table 1). however, the proportion of reused cells (t = -1.11, df = 17.6, p = 0.28) and the number of adults produced per cell (t = -0.62, df = 18, p = 0.53) did not differ significantly between the two habitats (table 1). regarding the cell productivity in the habitat less altered, we observed that were older cells and therefore closest to the petiole produced more adults, up to 3 generations of adults (fig. 3a), and the cells that were farther from the petiole, generally the younger cells, produced fewer individuals. in the habitat more altered a similar pattern was observed with cells producing up to 4 generations (fig. 3b). discussion in the habitat less altered, the colonies nested only on plants; whereas in the habitat more altered, most nests were sited on human constructions. this is probably related to the availability of the types of substrate in each environment, although sometimes in the habitat less altered, a few buildings were located near the nests. sinzato et al. (2011) found significant differences in the nesting sites of p. versicolor, in which 99% of the nests were located on human constructions and only 1% on vegetation. these authors found that the nests were located high on the buildings, which afforded greater protection from human interference, weather, and direct sunlight. thus, there was a high degree of synanthropy. butignol (1992) and sinzato & prezoto (2000) found similar results, demonstrating that the choice of the nesting site is based on the influence of physical climate factors such as temperature and luminosity (butignol, 1992). the number of cells constructed and the number of adults produced were larger in the habitat less altered (table 1). oliveira et al. (2010) found a positive correlation between the number of cells constructed and the total number of adults produced in colonies of p. versicolor; however, all these colonies were evaluated in an urban environment. these authors reported that the colonies produced on average 244 ± 89.5 cells and 171.67 ± 109.94 adults, values close to those found in this study for the habitat more altered. the colony productivity was determined by the size of the nest in both habitats; larger nests are generally more productive. the results of t-tests (table 1) showed that most of these parameters differed significantly between the two habitats, and nests in the habitat less altered were more productive. in the study of michelutti et al. (2013) with m. consimilis, the results were similar, and nests in the habitat with less human interference are more productive; however, the proportion of productive cells showed no significant differences, as table 1. comparison of the colony productivity of the social wasp polistes versicolor nesting in two environments. 10 nests were used for each habitat. parameters habitat less altered by human activity habitat more altered by human activity mean se mean se t df p cells constructed 411.30 123.27 203.00 44.79 5.02 11.3 <0.001 adults produced 493.90 213.03 200.00 65.69 4.15 10.7 0.002 nests dry mass (g) 9.50 4.28 3.17 0.99 5.98 11.4 <0.001 productive cells (%) 90.20 10.87 69.23 17.18 3.29 15.5 0.005 reused cells (%) 24.76 14.90 23.19 15.95 1.11 17.6 0.28 adults produced/cell 1.16 0.26 1.02 0.34 0.62 18 0.53 sociobiology 61(1): 100-106 (march, 2014) 103 observed in this study. therefore, as previously suggested by michelutti et al. (2013), colony productivity of the social wasps can be affected by the habitat quality, and negatively impacted by human activity. the habitat more altered has a large concentration of buildings and predominantly grassy vegetation, which suggests a low availability of resources for the colonies. anjos et al. (1986) and zanuncio et al. (1991) emphasized that habitat quality contributes to lower productivity in degraded environments, which may have limited resources available during unfavorable periods, especially those used in feeding immatures, such as the larvae of other insects. mead & pratte (2002) demonstrated that populations of p. dominula in situations that differed in prey availability also differed in colony growth and in the rate of production of offspring. however, judd (1998) and mcglynn (2012) suggested that nesting on human buildings is advantageous to reduce interspecific competition and to protect against predation, especially by vertebrates, since these factors are less intense than in preserved natural environments. moreover, human structures can provide greater protection against variations of climatic factors (michelutti et al., 2013). the proportion of productive cells was lower in the habitat more altered, and the maximum number of generations was higher (figs. 1 and 2). ramos & diniz (1993) and oliveira et al. (2010) analyzed the productivity of p. versicolor colonies and found cells producing 4 and 6 generations, respectively, values close to those observed here. however, the difference between the 3 or 4 generations produced by the two populations is probably due to different strategies for reuse of the comb, since colonies in the habitat more altered invest more in reusing cells, while in the habitat less altered the colony productivity is ensured by construction of new cells, resulting in large nests with a higher proportion of productive cells (figs. 1 and 2). the existence of different strategies in colonies in the two habitats is reinforced by the significant positive correlation between the number of cells constructed and the number of adults produced, and also between the diameter of the petiole and the nest dry mass (figs. 2 and 3) in the habitat less altered, since in these colonies the population increases concomitantly with the size of the nest. downing & jeanne figure 1. linear correlation between the number of cells constructed and the number of adults produced in nests of polistes versicolor in two habitats. habitat a: less altered by human activity (r = 0.86, p <0.01, n = 10) and habitat b: more altered by human activity (r = 0.30, p = 0.40, n = 10). figure 2. linear correlation between the nest dry mass and the diameter of the petiole in nests of polistes versicolor in two environments. habitat a: less altered by human activity (r = 0.75, p <0.01, n = 10) and habitat b: more altered by human activity (r = 0.33, p = 0.36, n = 10). figure 3. productivity and number of adults produced per cell in nests of polistes versicolor in two habitats. a) habitat less altered by human activity. b) habitat more altered by human activity. note: the asterisk indicates an area of cells that were destroyed at the time of collection. rf torres on colony productivity of the social wasp polistes versicolor104 (1986) stated that in groups of independent foundation, the increase in the diameter of the petiole is associated with the construction of new cells, a relationship also observed by montagna et al. (2010) for m. consimilis. according to giannotti (1997), the petiole is reinforced with additional pulp and salivary secretions to support the weight of the nest. a nest with more cells can produce more adults. a likely explanation for these different strategies is that in environments with more human activity, a larger nest may attract more attention and is therefore more likely to be eliminated. the insects may have adapted to this situation, preferring to reuse cells instead of increasing the size of the nest. however, michelutti et al. (2013), comparing data for m. consimillis in environments with features similar to this study, did not observe differences in reuse of the comb cells, although the nests of this species in a forest environment were larger and more productive than those in an urban environment. on the other hand, fig. 3 shows a pattern of cell use in which the cells near the petiole are more productive, probably because these cells are older and therefore are used more often. oliveira et al. (2010) found that cells that are central and closer to the petiole are more productive because they are older and better protected from the pressure of predators, parasites and reproductive conflicts, as also noted by gobbi et al. (1993). therefore, both the position and the age of the cells are important in determining the productivity of a cell. according to montagna et al. (2010), the central cells of the combs of m. consimilis are more productive than the peripheral cells, because they provide better physical conditions for the development of immature individuals; this region has a higher concentration of adults and is less exposed to attack by predators. however, in m. consimilis the petiole is central (montagna et al. 2010), and therefore the more productive cells are also older. finally we conclude that colony productivity in p. versicolor is significantly higher in habitat less altered by human activity; and regardless of the habitat, the cells central and closer to the petiole are more productive. furthermore, colonies in habitat more altered by human activity prefer to invest more in reuse of cells than to enlarge the nest structure, perhaps because a smaller nest is less conspicuous. the species probably continues to nest in this habitat because of reduced interspecific competition and greater protection against variations in climatic factors, as suggested by michulleti et al. (2013). acknowledgments the authors thank orlando t. silveira (museu paraense emílio goeldi) for the identification of the species, and janet w. reid (jwr associates) for the revision of the english text. we are grateful to capes for a doctoral fellowship awarded to the second author. wfaj acknowledges his research grants from cnpq. references abensperg-traun, m. & smith, g.t. (2000). how small is too small for small animals? four terrestrial arthropod species in different-sized remnant woodlands in agricultural western australia. biodiversity and conservation, 8: 709-726. anjos, n., santos g.p. & zanuncio, j.c. (1986). pragas do eucalipto e seu controle. informe agropecuário, 12(141): 50-58. butignol, c.a. (1992). observações sobre a bionomia da vespa predadora polistes versicolor (oliver, 1791) (hymenoptera: vespidae) em florianópolis/sc. anais da sociedade entomológica do brasil, 21: 201-206. carpenter, j.m. & marques o.m. 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(2007). manejo de vespas e marimbondos em ambiente urbano, p. 123-126. in: a. s. pinto; m. n. rossi & e. salmeron (eds.) manejo de pragas urbanas. piracicaba: cp 2, 208p. ramos, f.a. & diniz, i.r. (1993). seasonal cycles, survivorship and growth of colonies of polistes versicolor (hymenoptera, vespidae) in the urban area of brasilia, brazil. entomologist, 112(3-4): 191-200. raupp, m.j., shrewsbury, p.m., herms, d. a. (2010). ecology of herbivorous arthropods in urban landscapes. annual review of entomology, 55: 19-38. doi: 10.1146/annurev-ento-112408-085351 richards, o.w. (1978). the social wasps of the americas, excluding the vespinae. london, british museum, natural history, 580p samways, m.j. (2005). insect diversity conservation. cambridge, uk: cambridge university press. 342p samways, m.j. (2007). insect conservation: a synthetic management approach. annual review of entomology, 52: 465487. doi:10.1146/annurev.ento.52.110405.091317. sinzato, d.m.s. & prezoto, f. (2000). aspectos comportamentais de fêmeas dominantes e subordinadas de polistes versicolor olivier,1791 (hymenoptera, vespidae) em colônias na fase de fundação. revista de etologia, 2(2): 121-127. sinzato, d.m.s., andrade, f.r., de souza, a.r.; del-claro, k. & prezoto, f. (2011). colony cycle, foundation strategy and nesting biology of a neotropical paper wasp. revista chilena de história natural, 84: 357-363. doi: 10.4067/ s0716-078x2011000300004 santos, g.m.m. & gobbi, n. (1998). nesting habits and colonial productivity of polistes canadensis canadensis (l.) (hymenoptera, vespidae) in a caatinga area, bahia state, brazil. journal of advanced zoology, 19: 63-69. schowalter, t.d. (2012). insect responses to major landscapelevel disturbance. annual review of entomology, 57: 1-20. doi:10.1146/annurev-ento-120710-100610. rf torres on colony productivity of the social wasp polistes versicolor106 tibbetts, e.a . & reeve, h.k. (2003). benefits of foundress associations in the paper wasp polistes dominulus: increased productivity and survival, but no assurance of fitness returns. behavioral ecology, 14: 510-514. doi: 10.1093/beheco/arg037 zanuncio, j.c., batista, l.g, zanuncio, t.v., vilela, e.f. & pereira, j.f. (1991). levantamento e flutuação de lepidópteros associados à eucaliptocultura: viii região de belo horizonte, minas gerais, junho de 1989 a maio de 1990. revista árvore, 15: 83-93. 1521 habitat associations of red imported fire ants (solenopsis invicta) and introduced populations of pseudacteon spp. phorid flies by robert t. puckett1*, marvin k. harris1, & roger e. gold1 abstract habitat selection is one of many aspects of red imported fire ants, solenopsis invicta buren, (rifa) that has been intensively studied since their initial introduction in the united states. concurrently, innumerable studies have attempted to identify effective chemical insecticides for rifa control. more recently, several species of pseudacteon spp. phorid flies (diptera: phoridae) have been intensively evaluated to determine their potential utility in biological control of rifa. these flies belong to a suite of natural enemies of rifa in their native south american range, and have been selected for release in the united states as biological control agents against rifa. it is hypothesized that phorid flies, through parasitism and suppression of rifa foraging, will provide measureable rifa population suppression as phorid ranges expand and fly densities increase. relatively little scientific attention has been paid to habitat selection and partitioning by these flies in their introduced range(s) in the us. we assessed rifa and phorid (pseudacteon curvatus and pseudacteon tricuspis) densities in four central texas habitats. the habitat types selected represent a continuum of habitat diversity, micro-habitat availability, and plant heterogeneity. within the habitat types studied, rifa mound densities were significantly different, but foraging intensity was not significantly different in three of four habitat types. population densities of p. curvatus were determined to be significantly higher in one habitat than all others. alternatively, p. tricuspis densities followed a trend within the surveyed habitats that correlated with rifa densities observed in this study. these data imply that the successful release, establishment, and range expansion of p. curvatus may require a greater degree of critical consideration regarding the ecolog y 1department of entomolog y, texas a&m university, 2143 tamu, college station, tx 778432143. 1*corresponding author e-mail: rpuck@tamu.edu 1522 sociobiolog y vol. 59, no. 4, 2012 of each system into which they are released, as opposed to p. tricuspis. these implications also suggest that the successful establishment of additional phorid species in the us should take into consideration the habitat matrix and habitat-based expansion corridors available to the flies. key words: solenopsis, pseudacteon curvatus, pseudacteon tricuspis, invasive species, biological control introduction the red imported fire ant, solenopsis invicta buren (hymenoptera: formicidae), hereafter referred to as rifa, is an invasive species that has become established in the united states and elsewhere (morrison et al. 2004). these ants are native to south america and were discovered in mobile, al in the 1930’s (vinson 1997). rifa populations have since undergone an explosive dispersal and range expansion across the gulf-coast and eastern seaboard states in the continental united states. their contiguous range now extends from western texas east to florida and north to north carolina. in addition, disjunct populations have become established in maryland, new mexico, arizona, and california. like many other invasive species of plants and animals, rifa were liberated from the population regulatory effects of natural enemies in their native range when they arrived in the us. as a result, rifa pose a significant ecological and economic threat to invaded areas (lofgren 1986, porter et al. 1992). chemical control measures for suppression of rifa densities have been the focus of intensive research efforts. additionally, the past decade has witnessed a large-scale effort to introduce and manipulate populations of natural enemies of rifa in the us, with the goal of suppressing rifa populations via biological control (porter 1998; gilbert et al. 2008; oi and valles 2011). one such suite of natural enemies of rifa, the pseudacteon spp. phorid flies (diptera: phoridae), are known to parasitize workers of the solenopsis saevissima complex of fire ants (including s. invicta) throughout their native south american range (folgarait et al. 2000; calceterra et al. 2005). many species of these flies have been released within the united states (porter 1998; graham et al. 2003; vogt and street 2003; porter et al. 2004; allen et al. 2010; and plowes et al. 2011). female phorids seek rifa hosts at 1523 puckett, r. t., et al. — fire ants and phorid flies mounds and along foraging trails, and oviposit into the thorax of ant workers (morrison et al. 1997). a significant amount of fundamental research has been conducted with the goal of understanding rifa/phorid interactions and phorid developmental biolog y (feener 1981; feener and brown 1992; folgarait and gilbert 1999; morrison 2000; orr et al. 2003; consoli et al. 2001; porter and pesquero 2001). development of a successful biological control program which seeks to introduce pseudacteon spp. phorids for suppression of rifa throughout their non-native range depends upon successful introduction and establishment, and then expansion of phorid range as well as population density increase. in order to successfully choose appropriate release sites, a critical evaluation of the biotic and abiotic factors necessary for phorid establishment is necessary. pseudacteon spp. phorid fly species assemblages are known to partition available niche space based upon selectivity of seasonal and diurnal activity patterns, as well as size of ant hosts (campiolo et al. 1994; fowler et al. 1995; pesquero et al. 1996; folgarait et al. 2003; callcott et al. 2011). relatively little is known regarding habitat utilization and niche partitioning among phorids in their introduced range in the united states. pseudacteon tricuspis was released and became established at 5-eagle ranch in burleson county, texas (30º 38’ 15” n; 96º 40’ 59” w) in 2002. pseudacteon curvatus was released in the spring of 2004 at the same site. pseudacteon curvatus is physically smaller than p. tricuspis and was selected for its ability to attack polyg yne rifa colonies, which predominate in most of texas. these colonies are characterized by greater mound densities and a larger proportion of small worker ants relative to monog yne rifa colonies (macom and porter 1996). the first recovery of adult p. curvatus occurred during the spring of 2005, and flies have been collected during all subsequent sampling periods. the presence of rifa, and establishment of these two phorid species at 5-eagle ranch afforded an opportunity to examine the interactions and spatial distributions of ant hosts and introduced parasitoids. materials and methods experimental field sites this field study was conducted at 5-eagle ranch, located in caldwell, tx (burleson co.). the interior area of the ranch is approximately 1,133 1524 sociobiolog y vol. 59, no. 4, 2012 ha (2,800-acre) and lies in the east central texas forest ecoregion of southcentral texas (olson et al. 2001). it is presumed that the ranch first became infested with s. invicta in early 1970 (vinson 1997). phorid flies, p. tricuspis and p. curvatus were released at the ranch as part of the usda-ars “areawide suppression of imported fire ants in pastures project” (pereira 2003), and are known to have become established by 2003 and 2005, respectively (vander meer et al 2007, gilbert et al 2008). the entire area encompassed by 5-eagle ranch was mapped using esri arcgis® v10.0 (esri 2011) software and the interior of the ranch was digitally divided into a grid of 100 m x 100 m cells. all cells were assigned a unique numerical identification code. esri arcgis® v10.0 (esri 2011) software was used to determine the center (centroid) of each selected cell, and the coordinates of those points were generated and stored in a trimble® geoxt datalogger . sampling sites for this study were then established by first classifying 5-eagle ranch based upon its constituent habitat types with esri arcgis® v10.0 (esri 2011) software. next, four common central texas habitat types were selected (hay pastures, cattle pastures, unmanaged habitat and full canopy forest; fig. 1a,b,c,d respectively). these habitat types represented a continuum of habitat diversity and plant species heterogeneity. the description of each habitat type is as follows: a) hay pastures these pastures consisted of coastal bermudagrass cynodon dactylon (l.) pers. (bogdan) monoculture, which received one herbicide treatment/year, and were fertilized twice yearly with nitrogen supplements. cattle were not permitted to graze in these pastures, and hay harvesting occurred 2-3 times annually between may and september. b) cattle pastures these pastures consisted of a mixture of coastal bermudagrass cynodon dactylon (l.) pers. (bogdan) and a variety of native grasses and shrubs, received one herbicide treatment/year, were fertilized once yearly with nitrogen supplements only, and required occasional (< once/year) shredding. cattle grazing occurred on a rotational basis. c) unmanaged habitat these pastures were created by removing trees ~25 yrs ago and existed in a transitional ecological state that, if continued to be unmanaged, would presumably revert to the original post oak savanna ecotype. the habitat consisted of grasses and shrubs including goldenrod 1525 puckett, r. t., et al. — fire ants and phorid flies solidago sp., croton croton sp., milkweed asclepias sp. and senna beans senna 0btusifolia (l.) irwin & barneby. cattle grazing occurred on a rotational basis. d) forest full canopy of oaks quercus spp. with dense yaupon ilex sp. understory. in addition to cattle, feral hogs, deer and other wildlife had access to much of this habitat, it was not considered part of the managed portion of 5-eagle ranch and was not manipulated in any way. sampling sites were established by selecting four grids of nine contiguous cells within each habitat type (9 cells per habitat types). where possible, grids were selected from within habitat types in a 3 x 3 block formation. the scale and distance between sampling points in the forest and unmanaged habitats required an alternative configuration of sampling points than hay and cattle pastures. the sampling points were a minimum of 100 m from each other and from alternative habitat types. this is considered a sufficient distance to fig. 1. a) typical hay pasture, b) cattle pasture, c) unmanaged habitat, and d) forest at 5-eagle ranch. these habitats, from a)-d) respectively, represent a continuum of habitat diversity and plant species heterogeneity. 1526 sociobiolog y vol. 59, no. 4, 2012 eliminate potential competition for attraction of phorids between traps and among habitat types (puckett et al. 2007). to determine rifa densities and their utilization of each habitat type, rifa were monitored by mound counts which involved a direct census of all active rifa mounds within circular 0.05 ha plots within each selected grid cell. the radius of each circular plot was 12.67 m. mound counts were accomplished by first locating the center of each cell. next, a 1.5 m length of rebar was placed at the cell center and a 12.67 m length of cord attached. the cord was pivoted around the center of the cell while four researchers were evenly spaced along its length. researchers identified, and assessed for activity, all rifa mounds within the plot until one full revolution was made around the cell center. only active rifa mounds were recorded. additionally, hot-dog lures were used to assess rifa density and foraging activity within all selected cells. for this assessment, one 0.5 cm slice of bar-s hot dog was positioned in the center of a 7.62 x 12.7 cm notecard and placed on the ground at the center of the cell. one hot dog slice was placed 5 m to the east and west of the cell center (totaling three hot dog baits per sampling site). after a period of 45 min elapsed, hot dog bait ‘hits’ (rifa recruitment to and domination of hot dog bait, to the exclusion of other ant species) were recorded. comparisons of active mounds and hot dog bait ‘hits’ were compared statistically via analysis of variance (anova) and means were separated using tukey’s honestly significant difference (hsd). the statistical package ibm spss v.20.0 (ibm spss inc. 2011) was used to perform these analyses (values significantly different when p < 0.05). to assess phorid density and habitat selection differentials, phorid sampling devices known as pts traps (puckett et al. 2007) were deployed at the center of each sampling plot and retrieved after a period of 24 hrs. traps were returned to the laboratory where phorids were identified to species and counted. beginning on sept. 12, 2008, sampling occurred once per week for a total of nine consecutive weeks. analysis of variance (anova) was used to analyze the mean number of each individual phorid species per habitat type, and means were separated using tukey’s honestly significant difference (hsd). students t-test was used to compare the mean number of p. curvatus and p. tricuspis within similar habitat type. again, the statistical 1527 puckett, r. t., et al. — fire ants and phorid flies package ibm spss v.20.0 (ibm spss inc. 2011) was used to perform these analyses (values significantly different when p < 0.05). results rifa activitymound countsrifa mounds were significantly less numerous in forest habitat than any other habitat type (f (3,35) = 20.22, p < 0.01; fig. 2 and table 1), and were significantly less abundant in unmanaged habitat than in cattle pastures (tukey’s hsd post hoc analysis, p < 0.01; fig. 2 and table 1). mean number of rifa mounds were not significantly different between hay and cattle pastures (tukey’s hsd post hoc analysis, p = 0.22; fig. 2 and table 1). hot dog baitshot dog bait hits demonstrated that rifa were significantly less abundant in forest than in any other habitat types (f (3,35) = 5.58, p < 0.01; fig. 3 and table 1). there were no significant differences in the mean number of foraging rifa on hot dog baits in the remaining habitat types (fig. 3 and table 1). phorid activity pseudacteon curvatusthese flies were significantly more abundant on pts traps which were deployed in unmanaged habitat than in any other table 1. mean number of active rifa mounds and hot dog bait ‘hits’ from within plots in each habitat type. rifa sampling methods habitat type mound counts hot dog baits hay pasture 12.33 (a,b) 1.55 (a) cattle pasture 16.78 (a) 1.44 (a) unmanaged habitat 7.33 (b) 1.67 (a) forest 0.01 (c) 0.11 (b) p value < 0.01 < 0.01 f stat 20.22 19.96 1528 sociobiolog y vol. 59, no. 4, 2012 habitat type (f (3,323) = 19.96, p < 0.01; fig. 4 and table 2). the mean abundance of p. curvatus was significantly lower in forest habitat as compared to all other habitat types. pseudacteon tricuspisthis phorid fly species was significantly more abundant in cattle pastures as compared to densities observed in unmanaged habitat and forest (f (3;323) = 17.44, p < 0.01; fig. 4 and table 2) and their abundance was not significantly different in hay and cattle pastures (fig. 4 and table 2). the mean abundance of p. tricuspis was significantly lower in forest habitat as compared to all other habitat types. pseudacteon curvatus vs p. tricuspisp. curvatus was significantly more abundant than p. tricuspis in unmanaged habitat (t (160) = 4.57, p < 0.01; fig. 4 and table 2). there was no significant difference in the relative abundance of p. curvatus and p. tricuspis in hay pastures (t (160) = 0.86, p = 0.39; fig. fig. 2. mean # of rifa mounds in all habitat types. mound densities were significantly higher in cattle pastures than in unmanaged habitat and forest, but were there was no significant mound density difference between cattle and hay pastures. mound densities were significantly lower in forest than any other habitat type. 1529 puckett, r. t., et al. — fire ants and phorid flies 4 and table 2), cattle pastures (t (160) = 0.35, p = 0.72; fig. 4 and table 2) or forest (t (160) = 1.69, p = 0.09; fig. 4 and table 2). discussion in this study, rifa population differentials were related to habitat type. that is, rifa colonies were significantly more abundant in cattle pastures, and least abundant in unmanaged habitat. with this in mind, a reasonable presumption would be that parasitoids of these ants, such as phorid flies, would likewise be spatially partitioned throughout the available habitat in a similar density composition. in fact, p. tricuspis densities followed an identical habitat-specific trend as that of the mean number of rifa mounds throughout the habitat types surveyed in this study. these flies were most abundant in cattle pastures, followed by hay pastures, unmanaged habitat, and finally forest, where they were almost non-existent. alternatively, p. fig. 3. mean # of rifa ‘hits’ on hot dog baits in all habitat types. there were no significant differences in rifa ‘hits’ on hot dogs in hay pastures, cattle pastures, or unmanaged habitats, and ‘hits’ in each of these habitat types was significantly greater than in forest plots. 1530 sociobiolog y vol. 59, no. 4, 2012 table 2. mean number of phorids collected in each habitat type. mean # of phorid fly species habitat type p. curvatus p. tricuspis p value t stat hay pasture 1.71 (b;1) 1.42 (a,b;1) 0.39 0.87 cattle pasture 1.99 (b;1) 2.15 (a;1) 0.72 0.36 unmanaged habitat 5.76 (a;1) 1.19 (b;2) < 0.01 4.57 forest 0.13 (b;1) 0.01 (c;1) 0.09 1.69 p value < 0.01 < 0.01 f stat 19.96 17.44 df 3,323 3,323 ***means followed by different numbers in the same row are significantly means followed by different letters in the same column are significantly different (tukey’s hsd, p = 0.05) different (student’s t-test, p = 0.05) fig. 4. relative abundance of pseudacteon curvatus and pseudacteon tricuspis across and within each of four habitat types. 1531 puckett, r. t., et al. — fire ants and phorid flies curvatus was significantly more prevalent in unmanaged habitat than the remaining habitat types. thus, the habitat-specific population density of p. tricuspis was more closely correlated to habitat-specific rifa population density than was that of p. curvatus. very few rifa or flies of either species were observed in forest habitat. there were no significant differences observed regarding foraging intensity of rifa in hay pastures, cattle pastures, and unmanaged habitats; however, foraging in all of these habitat types was significantly more intense than in forest habitat, where negligible rifa foraging on hot dog baits occurred. again, despite the similarities in foraging intensity among hay pastures, cattle pastures, and unmanaged habitats, phorid abundance was found to be partitioned based upon habitat. these aspects of rifa / phorid associations and habitat partitioning of each species suggest that phorid population densities are not solely dependent upon rifa population densities. rather, these data suggest that maintenance of successful phorid populations is possible once a host (rifa foragers) availability density threshold is met. while this study did not seek to determine this threshold, it appears that the foraging intensity in each habitat except forest exists above it. evidence of this is provided by the fact that both phorid species were ubiquitous among all but the forest habitat. further evidence of this is provided by the fact p. curvatus appears to be selectively partitioning their population among at least one habitat-specific niche (unmanaged habitat), despite the fact that mean rifa mound counts were significantly lower in this habitat type than in hay and cattle pastures. unquestionably, the difference in degree of habitat heterogeneity among the habitat types surveyed in these trials contributes to differences in a variety of abiotic micro-habitat metrics among these systems, such as relative humidity, degree of shade, and temperature. additionally, these habitats are likely to support very different communities of invertebrate and vertebrate predators of phorids. an attempt to fully understand the interactions of phorids and abiotic/biotic aspects of these habitats which affect the population dynamics of the flies was outside of the scope of this project. however, the fact that p. tricuspis and p.curvatus populations are both supported in all but forest habitat indicates that at the time of this study, phorids were capable of coexistence in this system. additionally, the significantly greater population 1532 sociobiolog y vol. 59, no. 4, 2012 density of p. curvatus observed in unmanaged habitat relative to that of p. tricuspis suggests that these habitats may be important for release, long-term success, and expansion of field-released populations of p. curvatus. further, the habitat-specific data related to population densities of p. tricuspis suggests that this phorid species may be more compatible with, and more successful in, a wider variety of release site habitats. further monitoring of this system will provide insight into these unanswered questions regarding long-term sympatric phorid competitive success, and will allow for more accurate strategic planning as it pertains to release and establishment of additional species of pseudacteon phorid parasitoids in the u.s. and elsewhere. acknowledgments monte eagleton and glenn rutherford allowed this work to be conducted at 5-eagle ranch. maggie toothaker, sarah skrivanek, and jessica honaker have our gratitude for their help in executing these field trials. we thank mrs. laura nelson for her helpful reviews and comments on earlier drafts of this document. references cited allen, h.r., s.m. valles, and d.m. miller. 2010. characterization of solenopsis invicta (hymenoptera: formicidae) populations in 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peng & christian, 2006; van mele, 2008). according to dejean (1991) a colony with 12 nests can capture approximately 45,000 prey items per year and may in this way suppress insect pest populations. o. smaragdina could increase economic benefit compared to commonly used conventional pesticides in australian mango and cashew (peng & christian, 2005a; peng et al., 2004) and asian citrus orchards (offenberg et al., 2013). furthermore, weaver ants are known to improve the quality abstract oecophylla ants are currently used for biological control in fruit plantations in australia, asia and africa and for protein production in asia. to further improve the technology and implement it on a large scale, effective and fast production of live colonies is desirable. early colony development may be artificially boosted via the use of multiple queens (pleometrosis) and/or by adoption of foreign pupae in developing colonies. in the present experiments, we tested if multiple queens and transplantation of pupae could boost growth in young oecophylla longinoda colonies. we found out that colonies with two queens artificially placed in the same nest, all perished due to queen fighting, suggesting that pleometrosis is not used by o. longinoda in benin. in contrast, pupae transplantation resulted in highly increased growth rates, as pupae were readily adopted by the queens and showed high survival rates (mean = 92%). within the 50-day experiment the total number of individuals in colonies with 50 and 100 pupae transplanted, increased with 169 and 387%, respectively, compared to colonies receiving no pupae. this increase was both due to the individuals added in the form of pupae but also due to an increased per capita brood production by the resident queen, triggered by the adopted pupae. thus pupae transplantation may be used to shorten the time it takes to produce weaver ant colonies in ant nurseries, and may in this way facilitate the implementation of weaver ant biocontrol in west africa. sociobiology an international journal on social insects 1 université d’abomey calavi, cotonou, république du bénin. 2 aarhus university denmark, denmark. 3 ecole de gestion et de production végétale et semencière, université d'agriculture de kétou, kétou, république du bénin 4cirad, upr hortsys, montpellier, france. iita, 08 bp 0932, cotonou, benin. research article ants article history edited by: wesley dáttilo, u. veracruzana, mexico received 11 july 2013 initial acceptance 09 august 2013 final acceptance 21 august 2013 key words biological control, entomophagy, nest mate recognition, weaver ant colony production, oecophylla longinoda corresponding author issa ouagoussounon université d’abomey calavi 03 bp 2819 cotonou jericho république du bénin e-mail: ouagous@yahoo.fr of crops such as cashew nuts (peng et al., 1995; peng et al., 2004), citrus (barzman et al., 1996) and mango (sinzogan et al., 2008; peng & christian, 2005b). therefore, oecophylla is increasingly being utilized as a substitute to synthetic chemical pesticides as they are often equally or even more efficient in controlling pests and at the same time cheaper to use. several recent studies on applied weaver ant research have been carried out in asia and australia on o. smaragdina, however, the life history and the behavior of the african o. longinoda is less well documented. emerging markets for organic and sustainably-managed african fruit (mango, citrus) and nut (cashew) products (van mele & vayssières, 2007) asks for more research and investment in o. longinoda in west africa, as also here the economic potential is high. for example, dwomoh et al. (2009) showed that o. longisociobiology 60(4): 374-379 (2013) 375 noda can be used to control pest hemipterans as effectively as insecticides in ghanaian cashew plantation and were able to increase yields four-fold compared to plots without any control measures. on top of the high potential as a biocontrol agent, oecophylla is also used as a commercial food product (sribandit et al., 2008) a tradition especially well developed in thailand and other southeast asian countries (van huis et al., 2013). the double utilization of the ants has led to an increasing interest in the development of oecophylla management. for the effective implementation of oecophylla ants in pest management and ant farming, several aspects need to be considered. it is difficult to collect a queenright colony since the queen nest is well hidden in less accessible places (peng et al., 1998). established “wild” ant colonies produce several winged queens (flying ants) each year, which individually leave their colony and start new colonies alone. however, under natural circumstances, the mortality of these queens is high (> 99%) and they are therefore difficult to obtain. furthermore, it takes, for the few survivors, approximately 2 years before their colony contains enough ants to be used for pest control (vanderplank, 1960; peng et al., 2004) or for ant larvae production (offenberg & wiwatwitaya, 2010). so far all implementation has been based on collection of wild colonies or natural establishment in orchards which also takes several years (peng et al., 2005a). therefore, to make the oecophylla technology accessible to non-specialists and to implement it on a large scale, cheap production of live colonies in ant nurseries is needed. artificial rearing of colonies from newly mated queens may lead to a stable and quick production of oecophylla colonies (krag et al., 2010) and improve the chances of a wide implementation. for an effective production of live weaver ant colonies in ant nurseries faster growth of young colonies is desired. two different ways may be used to boost early colony growth. firstly, oecophylla (peeters & andersen, 1989) and other ant species (bernasconi & strassmann, 1999) are known to found new colonies with multiple queens (pleometrosis) in order to increase the probability of survival during the initial phase of colony development via a faster production of more workers. secondly, the adoption of non-nestmate brood from other colonies may increase colony growth as several ant species are known to rob intraspecific brood from neighbor colonies and in this way accelerate colony growth by adding these robbed individuals to their worker force (bartz & hölldobler, 1982; rissing & pollock, 1987). it is not known if o. longinoda uses pleometrosis during colony founding nor is it known if they accept and adopt pupae transplanted from foreign colonies. in this study we tested if more than one queen could be merged in founding colonies of o. longinoda (pleometrosis) and we tested the effect of pupae transplantation on the growth of newly founded colonies. materials and methods in a mango plantation in the parakou area (09° 37’ 01”n/02° 67’ 08”e) of benin 54 o. longinoda queens were collected after their nuptial flight with the use of artificial nests during the wet season in 2012. artificial nests were made on 15 mango trees by rolling a single leaf together, fixing it with a plastic ring (1.3 cm in diameter) in the middle part and sealing the tip end with a paper clip. nests were colonized by founding queens right after their nuptial flight as they constitute safe nesting sites (j. offenberg, unpublished data). nests were inspected 2-3 times a week and all queens were collected 1-3 days after their mating flight. at this developmental stage all colonies were composed of a single queen and her eggs, as no pleometrotic founded colonies were found. after collection, the queens and eggs were put into open cylindrical transparent plastic containers (φ = 4.5 cm; height = 10.5 cm) with a mango leaf inside to increase humidity and sealed with mesh nylon materials at the open end. the brood transplantation experiment was divided into two sub-experiments; one where queens were kept individually with their brood and transplanted pupae, and a second where two queens were artificially merged into one colony to test for pleometrosis, as has been observed for o. smaragdina (peeters & andersen 1989; offenberg et al., 2012a; offenberg et al., 2012b). in the first experiment with single queen colonies, 30 fertilized queens were used, which were divided into three pupae transplantation treatments with 0 (control), 50 or 100 non-nestmate pupae being transplanted to each colony, resulting in 10 replicates per treatment. in the first six replicates pupae were transplanted to the queens 7-14 days after they were collected in the field whereas in the last four replicates, pupae transplantation took place the day after they were collected. every time a new queen was collected in the field, it was sequentially allocated to one of the three treatments (i.e. the first mated queen no transplantation, the next 50 pupae transplantation, the third 100 pupae transplantation etc.). in the second experiment with two queens per colony, colonies were divided into two pupae transplantation treatments with 0 (control) or 50 non nestmate pupae being transplanted, respectively, and with five replicates per treatment (= 20 queens in total). in both experiments transplanted nonnestmate pupae were obtained from a single mature o. longinoda colony. during transplantation, each colony was transferred to a cylindrical transparent plastic vial (φ = 8 cm and height = 5 cm) with a mango leaf and the relevant number of pupae placed inside the vial. all colonies were kept at ambient temperature ranging between 24.3 °c and 29.7 °c (mean 27.5 °c) on a table protected from intruding ants by placing each table leg in a tray with water. during the experiment, all colonies were provided a few drops of pure water every day to allow the queens to drink. after the emergence of the first imago workers drops of 20% sucrose water were provided to i ouagoussounon et al pupae transplantation to boost oecophylla longinoda colonies376 each colony every day. one week after emergence of imago workers, protein food in the form of canned cat food and fish was provided to all colonies ad libitum. the transparent plastic containers allowed daily inspection and counting of brood in their different developmental stages. the numbers of intrinsic eggs, larvae, pupae and imago workers (defined as the brood produced by the resident queens) and adopted workers, were counted 50 days after the pupae transplantation in all the colonies. at this point all adopted pupae and the oldest intrinsic brood had developed into imago workers. however, intrinsic imagines could be distinguished from adopted individuals due to the size difference between the pupae from the mature colony and the much smaller nanitic workers produced by the founding queens (porter & tschinkel, 1986; peng et al., 2004). based on the number of live adopted imago workers, the survival rate from transplanted pupae into imago workers was calculated [(no. of emerged workers / no. of transplanted pupae) x 100] and mean numbers of brood and pupae survival were compared with anovas using jmp 8.0.1 statistical software. due to the difference in the number of days from queens were collected until pupae were transplanted, the total number of days from queen collection until the experiment was terminated (50 days after pupae transplantation) was recorded for each colony and used as a covariate in the subsequent analyses. results in the single queen experiment the survival from transplanted pupae into imago workers ranged between 88 and 96% (mean % survival and sd = 92.05 and 2.52) and was not significantly affected by transplantation rate (mean % survival and sd, 50 pupae = 92.4 and 2.45, 100 pupae = 91.7 and 2.66; anova including development time as a co-factor, f (2, 17) = 1.1; p = 0.36) indicating that non-nestmate pupae were readily accepted by the queens and suggesting that pupae required no or only minimal amount of nursing. fifty days after the transplantation, intrinsic imago workers were present in all colonies, however, with significantly more individuals in colonies with more pupae transplanted (f (2,26) = 147.1, p < 0.0001). the mean (sd) number of intrinsic workers was 31.2 (4.37), 44.8 (3.04) and 74.5 (8.25) in the colonies that received 0, 50 and 100 pupae, respectively (table 1). pupae transplantation also led to increased production of the remaining developmental stages of intrinsic brood. this was true both for the number of eggs, larvae, pupae, workers and their sum (p < 0.0001 in all cases) at the end of the experiment (table 1). the average total intrinsic production in colonies without added pupae was 46.5 (6.33) individuals during the first 50 days of colony development. in comparison, 50 pupae transplantation led to a 70 % increase in the per capita queen production, and a 100 pupae transplantation led to 190% increase compared with no pupae transplantation (fig 1). thus, the transplanted pupae stimulated the fertilized queen’s egg production and increased her brood production with approximately 1.4 and 1.9% per adopted pupae, respectively. in addition, the total colony size (all intrinsic brood plus adopted workers) was 46.5 (6.33), 125.2 (11.01) and 226.7 (15.32), respectively, in the colonies that received 0, 50 and 100 pupae. in comparison to the treatment without pupae transplantation, the total number of individuals increased 169% with 50 transplanted pupae and 387% with 100 transplanted pupae (fig. 1). thus, adopted workers led to a considerably increase in total colony size. in the second experiment with two queens in each colony, one of the queens, in all cases, killed the other before the emergence of imago workers from the transplanted pupae, suggesting that pleometrosis induced in the laboratory is not possible. it should, however, be noticed that we found 5 claustral colonies with two queens out of a total of 87 collected in 2013 (all the others being singly founded)(i. ouagoussounon, table 1: mean (± sd) number of intrinsic brood (egg, larvae, pupae, imago workers and their total) produced by the resident queen in the colonies 50 days after the transplantation of pupae. transplantation (no. of pupae) eggs per colony larvae per colony pupae per colony workers per colony total intrinsic production per colony mean (sd) two-way anova mean (sd) two-way anova mean (sd) two-way anova mean (sd) two-way anova mean (sd) two-way anova 0 7.3 (4.06) f(2, 26) =58.3 p < 0.0001 4.3 (2.11) f(2, 26)= 25.1 p < 0.0001 3.7 (4.03) f(2, 26) = 23.3 p < 0.0001 31.2 (4.37) f(2,26)=147.1 p < 0.0001 46.5 (6.33) f(2, 26)=165.1 p < 0.0001 50 16.8 (4.71) 10.5 (5.62) 6.9 (5.26) 44.8 (3.04) 79.0 (11.74) 100 25.3 (3.59) 17.6 (4.81) 17.6 (4.67) 74.8 (8.25) 135.0 (14.83) total development time (days) f(1, 26) = 8.9 p < 0.0001 f(1, 26) = 5.8 p = 0.023 f(1, 26) = 0.3 p = 0.60 f(1, 26) = 0.06 p = 0.80 f(1, 26) = 4.0 p = 0.056 whole model f(3, 26) =43.6 p < 0.0001 f(3, 26) =19.6 p < 0.0001 f(3, 26) =15.8 p < 0.0001 f(3, 26) =98.2 p < 0.0001 f(3, 26) =113.2 p < 0.0001 sociobiology 60(4): 374-379 (2013) 377 unpublished data), suggesting that pleometrosis is sometimes used under natural conditions as also described by (dejean et al.,2007). because all queens were involved in fatal fights, no further analyses were conducted on this experiment. discussion the results showed no difference in survival between queens receiving 50 pupae and queens receiving 100. pupae in both treatments were readily accepted by the queen ants and more than 88% were reared to the imago stage. this means that potentially colonies may be boosted with even higher numbers of pupae and boosting can take place before the emergence of the first workers as a worker force is not needed for nursing. offenberg et al. (2012b) and peng et al. (2013) obtained similar results with a mean survival rate of 84%, when transplanting 30 and 60 non-nestmate pupae to o. smaragdina queens in darwin, australia. also in that case survival was unaffected by transplantation rate. the same pattern may not hold true if larvae were transplanted instead of pupae as they need to be fed and groomed by members of the receiver colony. larvae may well be accepted by receiver colonies, as described by krag et al. (2010) for o. smaragdina, however, it is questionable if they can be added in high numbers as with pupae, because of their need for food, which is available in only limited amounts, especially in very young colonies with only a single queen and no worker force. a further advantage to colonies adopting pupae from mature colonies derives from the fact that young ant colonies, in order to reserve resources, produce only smaller and slimmer workers (nanitics) with an associated narrow task repertoire compared to older and larger colonies that produce larger major workers (peng et al., 2004). after pupae transplantation the workers eclosing from the transplanted pupae are of a larger size as they originated from a mature colony. as a consequence these transplanted individuals may conduct wider tasks compared to the nanitic imago workers intrinsic to the young colonies. if larvae were transplanted these may turn into smaller imagines due to food shortage if they are transplanted prior to the determination point of their final size. this is not the case with pupae as they have already attained their final size. krag et al. (2010) showed that o. smaragdina nonnestmate larvae showed chemical insignificance (i.e. were without colony specific odor) as they were adopted by colonies containing mature workers (but no queens). subsequently it was found that also o. smaragdina pupae seems to be chemical insignificant and that the presence of queens in the colonies did not hinder adoption of foreign brood as transplanted pupae developed into imagines in queenright colonies (offenberg et al., 2012b). from the present study we conclude that the same holds true for o. longinoda suggesting that chemical significance does not develop until beyond the pupal stage as also suggested by lenoir et al. (2001), to be the case for other ant species. also the present study shows that the presence of the maternal queen does not preclude adoption of foreign brood. the addition of pupae to the colonies did not only increase colony size with the numbers added. in addition, the transplantation of pupae stimulated the fertilized queen´s own egg production and thereby increased the intrinsic brood production of the colonies. thus, the presence of brood at the pupal stage (or beyond), increased egg laying rates. this seems to be adaptive to queens as younger brood is associated with expenditures to the queens in terms of nursing time and food allocation, whereas pupae will soon eclose and develop into workers that can take over the nursing of brood and forage for food. this result follows the findings by gibson & scott (1990) and offenberg et al. (2012b), showing that pupae increased egg laying in camponotus spp. and o. smaragdina queens, respectively. on the other hand, other researchers have shown that only the number of late stage larvae is responsible for queen fertility in e.g. monomorium pharaonis and solenopsis invicta (tschinkel, 1988; børgesen & jensen, 1995; cassill & vinson, 2007). this suggests that different mechanisms may operate in different ant species. further, the triggering of an increased fecundity in the present study shows that egg laying rates in o. longinoda are plastic and can be manipulated via the presence of pupae and/or workers. in the present study, the total population size (all brood stages) in the colonies that received 0 pupae was 1.99 times higher (mean = 46.5 ± 3.6 se) compared to the results obtained by offenberg et al. (2012a) in the hapleometrotic colonies of o. smaragdina (mean = 23.3 ± 2.0 se) after 68 days. this lower production in o. smaragdina was likely affected by the fact that the colonies in that study were transported under cold conditions at the start of the experiment which may have delayed their development. on the other hand, the total population size (all brood stages) after 68 days was 1.6 times fig. 1: the mean (± sd) number of individuals (egg, larvae, pupae and imago workers) per colony, 50 days after the transplantation of pupae. i ouagoussounon et al pupae transplantation to boost oecophylla longinoda colonies378 higher in the pleometrotic o. smaragdina (offenberg et al., 2012a) colonies (mean = 74.9 ± 10.5 se) compared to the haplometrotic colonies in this study (mean = 46.5 ± 3.6 se). this highlights the strong effect of multiple queens in founder colonies. under natural circumstances, it takes approximately two years before a colony contains enough ants to be used for pest control (vanderplank, 1960; peng et al., 2004) as each colony is expected to occupy approximately 10 trees in the receiver plantation (peng et al., 2004). this minimum colony size may be achieved more quickly by boosting the growth via pupae transplantation. in the present study colony size increased up to almost 5-fold in only 50 days and with only a single pupae transplantation event. multiple transplantations with potentially even higher numbers of pupae being transplanted may lead to much higher boosting, considerably shortening the otherwise slow development to an acceptable colony size. secondly, it is evident that the biological control efficiency of weaver ants depends on the density of the worker ants (van mele et al., 2007; peng & christian, 2005b, 2007). thus, keeping high densities of o. longinoda is essential in biocontrol programs and may be accomplished by pupae transplantation during critical periods. this study suggests that queen right o. longinoda colonies accept foreign brood and that pupae transplantation facilitate colony growth. this knowledge may ease the implementation of the weaver ant technology in africa where the ants can be used to control pest species on various crops. future studies should test if the immediate presence of pupae will trigger higher egg-laying rate by the queen before the pupae emerge as workers. also it would be interesting to test if pupae transplantation and the following higher numbers of larger workers will lead to the production of a larger intrinsic worker caste in receiver colonies compared to colonies that develops naturally. acknowledgements we would like to thank, mm. zoumarou, for permission to do research in his plantation at korobororou. this study was supported by the project “increasing value of african mango and cashew production”. danida (dfc no. 10 – 025au). references bartz, s.h. & hölldobler, b. 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(1960). the bionomics and ecology of the red tree ant, oecophylla sp., and its relationship to the coconut bug pseudotheraptus wayi brown (coreidae). j. an. ecol., 29: 15–33. way, m.j. & khoo, k.c. (1992). role of ants in pest management. annu. rev. entomol., 37: 479-503. doi: 10.13102/sociobiology.v65i3.2726sociobiology 65(3): 370-374 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 leaf volatiles from two corydalis species lure a keystone seed-dispersing ant and enhance seed retrieval introduction myrmecochory (i.e., seed dispersal by ants) is a common ecological interaction in nature. ants benefit nutrition from elaiosomes attached to seeds and plants benefit from having their seeds dispersed (beattie, 1985). the plants that rely on ant dispersal can benefit from interaction in several ways, including directed dispersal, distance dispersal, fire avoidance and seed predator avoidance (beattie, 1985; giladi, 2006). myrmecochorous plants have evolved some features, which referred to dispersal syndromes, to increase the probability and effectiveness of seeds being transported by ants. to date, diverse features of plants such as elaiosome size and chemistry (e.g., hughes & westoby, 1992; gammans et al., 2006), seed morphology (e.g., nakanishi, 1994), predispersal mode (e.g., nakanishi, 1994; giladi, 2006), plant stature (e.g., leal et al., 2015), the timing of seed release (e.g., abstract it has been reported that a suit of plant traits can regulate the ant-seed interaction and subsequently affect the seed dispersal. however, the role of plant volatiles in attracting the ants for seed dispersal remains little examined. we used a y-tube olfactometer to test behavior response of a keystone seed-dispersing ant (pristomyrmex pungens mayr) to leaves and seeds of five co-occurring myrmecochorous corydalis species (c. wilfordii regel, c. racemosa (thunberg) persoon, c. sheareri s. moore, c. balansae prain and c. incisa (thunberg) persoon). of the five species, only c. wilfordii and c. racemosa leaves emits heavily volatiles. we also performed seed cafeteria experiments to assess the effect of leaf volatiles from c. racemosa on seed retrieval by presenting simultaneously the seeds near the fresh leaf and the leaf immersed by diethyl ether both in the field and lab. the experiment using y-tube showed that the ants were only significantly attracted by the fresh leaves of two species, c. wilfordii and c. racemosa. the cafeteria experiments showed that ants spent less time to detect the c. racemosa seeds which were near the fresh leaf, and transported these seeds more quickly. this indicated that the leaf volatiles can function as an attractant for the dispersing ants, and ant preference in turn enhance the seed retrieval. the findings reveal that leaf volatiles can play an important but underestimated role in shaping the ant-seed dispersing interactions. sociobiology an international journal on social insects y zhu, d wang article history edited by gilberto m. m. santos, uefs, brazil received 09 february 2018 initial acceptance 28 march 2018 final acceptance 10 april 2018 publication date 01 october 2018 keywords ants; corydalis; myrmecochory; plant volatiles; seed dispersal. corresponding author dong wang school of life sciences central china normal university key laboratory for geographical process analysis & simulation hubei province, wuhan, 430079, china. e-mail: 395351998@qq.com dw@mail.ccnu.edu.cn oberrath et al., 2002; boulay et al., 2007; arandarickert & fracchia, 2012), and spatial dispersion of seeds (e.g., gorb and gorb, 2000), have been reported to influence the association between seed-dispersing ants and myrmecochorous plants. many plants emit volatile organic compounds (vocs) from most parts of their anatomy (baldwin, 2010). the volatiles, functioning as either a deterrent to defend them against herbivores and pathogens or an attractant to lure pollinators and seed dispersers, are presumed to serve as cues that mediate various interactions with animals (inui et al., 2003; raguso, 2008; unsicker et al., 2009; baldwin, 2010). previous studies found that ants which are involved in interactions with plants, such as protective ant-plant interaction (e.g., brouat et al., 2000; edwards et al., 2007) and ant-flower interaction (e.g., wanjiku et al., 2014; vega et al., 2014), could respond to the vocs from host plants.however, it is stillless known about the role of volatiles in ant-seed school of life sciences, central china normal university, key laboratory for geographical process analysis & simulation, wuhan, p.r. china research article ants sociobiology 65(3): 370-374 (september, 2018) 371 dispersal interactions (sheridan et al., 1996; youngsteadt et al., 2008). the few existing results remain inconsistent (borges, 2015), with ants being attracted by seed odor of antgarden (ag) plant in amazonian rainforests (youngsteadt et al., 2008), but not for the myrmecochorous seeds asarum canadense (sheridan et al., 1996). apart from odor-induced seed retrieval by ants, volatiles from vegetative parts such as leaves can also mediate the interaction between ants and plants (brouat et al., 2000; pichersky & gershenzon, 2002). until now, whether volatiles from vegetative organs such as leaves can attract the dispersing ants and such the effect of attractiveness it may has on subsequent seed retrieval remains largely unclear. the corydalis (papaveraceae) is a north-temperate genus with about 465 species, about 357 of which are distributed in china (zhang et al., 2008). the species of corydalis have elaiosomes attached to seeds to attract ants (lengyel et al., 2010). of them, some species can readily emit volatiles from vegetative organs, whereas others do not (wu et al., 1999; zhang et al., 2008). we hypothesized that (1) leaf volatiles of corydalis can function as an attractant for keystone seed-dispersing ant, and (2) the resultant attractiveness would influence seed collection. methods study organisms in this study, five corydalis species including c. wilfordii, c. racemosa, c. sheareri, c. balansae and c. incisa were used. all the five species are ant-dispersed plants, and they co-occur in temperate forests at the margins of mufushan mountains (31°49′n, 113°55′e), hubei province, china. of them, only c. wilfordii and c. racemosa emit heavily volatiles, especially the leaves (wu et al., 1999; zhang et al., 2008). the leaves are still present on the fruiting plants of all five species. olfactometer assay to test the behavior response of ants to leaves and seeds from the five studied species, we conducted olfactometer assay by using y-tube olfactometer. our previous study showed that the pristomyrmex pungens mayr (formicidae: myrmicinae) was a keystone seed disperser of corydalis species in the study site (zhu & wang, 2014). the olfactometer was a y-shaped glass tube with a 2.6cm diameter and each of the three arms 10 cm in length. air flow of 750 ml/min was generated with an air pump (qc-4s, china) that directed ambient air through a charcoal filter, odor source bottles before entering a y-shaped teflon tube that split air flow evenly between the two arms of the olfactometer. odorant sources were placed into an odor source bottle (the other bottles was left blank), and 50–80 ant workers were induced to the basal arm. the initial choices of the first 30 different ant workers that walked into and proceeded at least 5 cm down the right or left arm of the olfactometer were recorded. thirteen replicates were conducted for each treatment. the y-tube was washed, and the two side arms were exchanged in turn for treatments and control after each replicate. the odor sources tested in the experiments were as follows: (1) leaf of corydalis spp. vs blank; (2) seed of corydalis spp. vs blank. the t-test was used to test the response of workers to the odor sources and blank. cafeteria experiments to assess the effect of volatiles from leaf on seed retrieval, we used c. racemosa seeds to conduct a cafeteria experiment both in the field and lab. in the field, we assembled 20 quadrats (1 × 1 m) 10 m apart in a 4 × 5 grid at a site which was at least 20 m away from the nearest c. racemosa individual. each quadrat included two seed depots 1 m apart. for each depot, five seeds were randomly placed near two types of leaves which mimic the presence or absence of volatiles in the field: (1) fresh leaf; (2) immersed leaf — leaf was immersed by diethyl ether for 8 h for removal of volatiles, and were then left overnight at room temperature to allow the diethyl ether to evaporate. the experiment was conducted during peak foraging periods (0800–1100 h). observations lasted 12 min, at which point the number of seeds removed from each depot was recorded.twenty trials were conducted. the workers of p. pungens could recruit workers to collect seeds of some corydalis species directly from their dehiscing capsules (fig 1). we collected four colonies of p. pungens in the field, and maintained them in the laboratory for bioassays. the colonies were reared in plaster nests (10 × 10 × 0.4 cm) and placed in an area (50 × 37 × 8 cm) with walls coated with fluon to prevent ants from escaping. each nest contained approximately 300 − 400 workers and 30 − 40 larvae. the laboratory temperature and relative humidity were maintained in a range from 21 to 26ºc and approximately 36%, respectively. we supplied ants with water and sucrose water (1m), and twice a week with mealworms. in the lab, we placed simultaneously five seeds near the two types of leaves (fresh leaf and immersed leaf) on the sites which left ca. 15 cm to the nest entrance in the laboratory (interleaf distance ca. 20 cm). observations lasted 12 min, at which point the number of removed seeds from each leaf was recorded. eight trials (4 colonies × 2 replicates) were conducted. data analysis spss v20 (ibm, inc.) was used for statistical analyses and the significant difference was set at p < 0.05. data were transformed prior to analysis when necessary. t-test was used to test the response of workers to the odor sources and blank. general linear model (glm) was used to test the effects of leaf type on the number of removed seeds (ln-transformed) in the lab and nest identity was included in this model as a random factor. t-test was also used to test the effects of leaf type on the number of seed removals in the field. y zhu, d wang – leaf volatiles help seed retrieval by ant372 fig 1. workers of pristomyrmex pungens directly collected corydalis racemosa seeds from the dehiscing carpels in the field (a); workers were attracted by capsules of c. racemosa in the lab (b) fig 2. responses of p. pungens (mean ± se) in a y-tube olfactometer when given a choice between plant and clean air. (a) leaf vs blank, (b) seed vs blank.the asterisk indicates significant difference within a choice test. results in olfactometer assay, workers of p. pungens significantly preferred the arm containing leaves of c. wilfordii (t = 3.477, df = 24, p = 0.002) and c. racemosa (t = 2.657, df = 24, p = 0.014) over the empty control arm, while workersshowed no obvious preference to leaves of the remaining three species (fig 2a). workers did not prefer the arm containing seeds than the empty control arm, regardless of plant identity (fig 2b). in cafeteria experiments, four species of ants were observed to retrieve seeds in the field, with p. pungens being responsible for the vast majority of seed removal (73.7%; table 1). ants located the seeds which were near the fresh leaf threefold and fourfold earlier than the seeds which were near the immersed leaf in the field and lab, respectively (fig 3). in the field, the number of seed removals near the fresh leaf was significantly greater than that near the immersed leaf (t = 3.365, df = 23.235, p = 0.003; fig 3). in the lab, the number of seed fig 3. number of mean seeds remaining (± se) of c. racemosa in the field (a) and lab (b). sociobiology 65(3): 370-374 (september, 2018) 373 this implied that the leaf volatiles of some corydalis species may be one important trait promoting the association between plants and ants. although the myrmecochorous seeds can be found and then transported by ants because of the attached elaiosome, the results of our study indicated that leaf volatiles of some myrmecochorous corydalis species can function as an attractant for ant dispersers, and such attractiveness in turn enhances the seed retrieval. it suggests that leaf volatiles of some corydalis species may be one of several factors shaping the ant-seed dispersing interactions. further research is needed to identify the chemical basis of volatiles that trigger ant foraging activity, and to assess to what extent the seed dispersal of corydalis species depend on leaf volatiles. acknowledgements this research was supported by the national natural science foundation of china (grant no 31170310, 31110103911), science and technology basic work (2013fy112100) and the specimen platform of china, teaching specimens sub-platform, web, http://mnh.scu.edu. cn/ (2005dka21403-jk). references arandarickert, a. & fracchia, s. 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(1990). neotropical ant gardens ii. bioassays of seed compounds. removals near the fresh leaf was also significantly greater than that near the immersed leaf (f = 4.572, df = 1, p = 0.017), while the nest identity had no effects on seed removals (f = 1.907, df = 3, p = 0.305). comparing seed-removal dynamics, ants removed nearly all seeds which were near the fresh leaf after 12 min (95% in both the field and lab), whereas they only removed 69.0% and 32.5% of seeds which were near immersed leaf in the field and lab, respectively (fig 3). ant species fresh leaf immersed leaf pristomyrmex pungens 74 36 pheidole nodus 12 7 paratrechina yerburyi 8 14 pachycondyla luteipes 1 0 table 1. number of removed seeds by each dispersing ant in the field. discussion this study showed that leaf volatiles from c. wilfordii and c. racemosa can lure the keystone dispersing ants pristomyrmex pungens, and subsequently promote the seed retrieval. previous studies have investigated the attractiveness of volatiles to ants in the ag plant and myrmecochorous plants, and some studies found the attractiveness of seed odor to ants (davidson et al., 1990; youngsteadt et al., 2008), and others did not (sheridan et al., 1996; gammans et al., 2006; youngsteadt et al., 2010). in the olfactometer assay, we did not observe the behavior response of p. pungens to seeds from the five corydalis species, implying that the seeds may not attract dispersing ants by olfactory cues to some extent. the possible reason is that the lower volatility of compounds in the diaspores insufficiently serves as gustatory cues (borges, 2015). indeed, turner et al. 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(in chinese with english abstract). doi: 10.5846/stxb201301010005 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1780sociobiology 65(1): 31-37 (march, 2018) special issue survivorship and walking behavior of inquilinitermes microcerus (termitidae: termitinae) in contact with host workers and walls from host nest introduction a range of species lives in close associations (i.e. symbiosis) (dimijian, 2000; redman et al., 2001; duarte et al., 2014). the maintenance of these associations should require characteristics that allow involved species to deal with each other. nests of social insects (e.g. wasps, bees, ants and termites) allow the maintenance of homeostasis (noirot & darlington, 2000) and show a space free of enemies, which provides greater longevity to their colonies. on the other hand, these same characteristics also favor the use of such structures abstract constrictotermes sp. nests are frequently inhabited by colonies of inquilinitermes microcerus. in this association, i. microcerus colonies usually establish their colonies spatially isolated from constrictotermes colonies. here, we investigated whether the apparent spatial isolation of i. microcerus colonies in constrictotermes nests should be related to their needs (e.g. feeding) in relation to the central part of the nest or to a possible stress provoked by the presence of the host. for this, survival and walking behavior bioassays were performed to test the hypothesis that the survivorship of inquilines is: (i) reduced in the presence of host, mainly of those from different nests, (ii) increased in contact with inner walls compared with external walls; and that the distance walked and walking velocity of inquiline is: (iii) increased in the presence of the host and (iv) reduced in contact with the internal walls compared with external walls of host nest. the mean time to death of inquiline workers is lower in contact with host (independently from the same or different nest) compared with control and the mean time to death of inquiline workers is lower in contact with external walls of host nest compared with control group and the inner walls. the distance walked and walking velocity of inquiline workers in contact with their hosts (from the same or different nest) did not differ from control, however, these parameters were reduced when workers were in contact with inner and external walls compared with control. in general, our results showed that i. microcerus adopt behavioral strategies to avoid perception by its host. sociobiology an international journal on social insects js cruz1, pf cristaldo1,2, jjm sacramento1, mlr cruz1,3, dv ferreira1,3, apa araújo1 article history edited by og desouza, ufv, brazil received 31 may 2017 initial acceptance 31 may 2017 final acceptance 09 july 2017 publication date 30 march 2018 keywords inquilines, isoptera, symbiosis, behavior. corresponding author ana paula a. araújo laboratório de interações ecológicas departamento de ecologia centro de ciências biológicas e da saúde universidade federal de sergipe (ufs) avenida marechal rondon, s/n rosa elze cep 49100-000, são cristóvão-se, brasil. e-mail: anatermes@gmail.com by symbionts species (see hughes et al., 2008). colonies of social insects, stand out in relation to the great diversity of symbionts that cohabit their nests, including parasites, mutualists and commensal species (hughes et al., 2008). termite nests, for example, can house a range of species, such as vertebrates (brightsmith, 2000; dechmann et al., 2009) and invertebrates (cunha & brandão, 2001; costa et al., 2009), called as termitophilous. among these cohabitants, other termite species than that building the nest are included, which are called inquilines (redford, 1984). some inquiline species are obligatory, since they do not have the ability to 1 laboratório de interações ecológicas, departamento de ecologia, centro de ciências biológicas e da saúde, universidade federal de sergipe, são cristóvão-se, brazil 2 programa de pós-graduação em agricultura e biodiversidade, universidade federal de sergipe, são cristóvão-se, brazil 3 programa de pós-graduação em ecologia e conservação, universidade federal de sergipe, são cristóvão-se, brazil research article termites js cruz et al. – survivorship and behavior of termite inquiline32 build their own nests and depend intrinsically of the host nest for its survivorship. these examples are found in species of the genus inquilinitermes mathews, 1977 (termitidae: termitinae) (e.g. i. fur, i. inquilinus and i. microcerus), which inhabit nests of constrictotermes sp. (mathews, 1977) and in serritermes serrifer (hagen & bates, 1858) (serritermitidae), which is obligatory inquiline of cornitermes cumulans (emerson & krishna, 1975) (termitidae: syntermitinae). differently from observed in hymenoptera, the high incidence of cohabitation in termite nests is intriguing (campbell et al., 2016; marins et al., 2016). like other social insects, in termites, colonies are able to recognize nestmate from non-nestmates, and also invest energy in the production of a specific caste to nest defense (šobotník et al., 2008). since obligatory inquilines have a closer relationship with their hosts, they are expected to exhibit strategies that allow the temporal stability of this interaction. although inquilines benefit from the nest of host colony, with no costs to constructing this structure (jirošová et al., 2016), a series of evidences suggest that the inquiline seems to avoid (physically and chemically) its host. studies have shown that the colonies of inquilinitermes sp. are small in size relative to their host and they are usually found in the central parts of the host nest where individuals of host colony are rarely observed (cunha et al., 2003). the aggregation of the inquilines in the central part of host nest may be related to the fact that they use this structure as a food resource (mathews, 1977; bourguignon et al., 2011; cristaldo et al., 2012; florencio et al., 2013; barbosa-silva et al., 2016). in addition, recent studies have shown evidence that individuals of i. microcerus can use chemical cues as a strategy to avoid conflicts, since they avoid chemical cues of the whole body, as well as the trail and alarm signals emitted by its host, c. cyphergaster (cristaldo et al., 2014; 2016). however, in spite of such advances in the understanding this interaction, the final balance for both partners still need to be unraveled. in the present study, we investigated whether the apparent spatial isolation of i. microcerus colonies in constrictotermes sp. nests should be related to their needs (e.g. feeding) in relation to the central part of the nest or to a possible stress caused by the presence of host colony. for this, survival and walking behavior bioassays were performed to test the hypothesis that the survivorship of inquilines is: (i) reduced in the presence of host, mainly of those from different nests, (ii) increased in contact with inner walls compared with external walls; and that the distance walked and walking speed of inquiline is: (iii) increased in the presence of the host, mainly of those from different nests and (iv) reduced when in contact with the internal walls of nest compared with external walls. material and methods study site and termite collection nests of constrictotermes sp. (n= 9) were collected in june 2016 at campus rural of federal university of sergipe, in são cristóvão (11º 01’s e 37º 12’o), northeast, brazil. the mean monthly precipitation and temperature in the region are 1.200 mm and 25 ºc, respectively. according to köppen classification, the clime is as’ type (tropical with rainy winter and dry summer) (pidwirny, 2011). collection consisted in the complete removal of nests from the field using spades and picks. the nests were taken to the laboratory and fragmented to verify the presence of inquiline colonies, which were found in three of the nests sampled. the identification of species was conducted in comparison with samples from the isoptera collection of university of brasília (unb), in which samples were deposited. the host was identified as constrictotermes sp. (#10745 and 10747) and the inquiline as inquilinitermes microcerus (#10746 and 10748). bioassays bioassays were conducted to check whether the survivorship and walking behavior of i. microcerus workers are affected by the presence of its host (from the same or different nest) and by the contact of inquilines with its host nest walls (inner and external walls). survival bioassays to check whether the presence of host affects the survivorship of inquiline workers, the following treatments were established: (i) 20 inquiline workers alone (control), (ii) 10 inquiline workers with 10 host workers from the same nest (sh) and (iii) 10 inquiline workers with 10 host from different nest (dh). bioassays were conducted in a petri dish (ø 9 cm x 1.5 cm [height]) covered with a filter paper. to check whether the contact with host nest wall affects the survivorship of inquiline workers, the following groups were established: (i) 10 inquiline workers alone (control), (ii) 10 inquiline workers with 7 g of substrate from inner wall of host nest (iw) and (iii) 10 inquiline workers with 7 g of substrate from external wall of host nest (ew). fragments from inner (easily recognized by its dark coloration) and external walls of host nest were ground with a mortar and pestle and sieved through a 6-mesh sieve. then, the material was weighed in a precision balance and distributed at the bottom of the petri dish on the filter paper. in control group, petri dishes were covered only with filter paper. for both survival bioassays, three replicates were performed per nest for each treatment, totalizing 27 replicates. the mean value of three replicates/nest were then used in the statistical analyses. petri dishes were kept in a biochemical oxygen demand (bod) incubator (25°c, without light) and the quantification of dead individuals was performed at onehour intervals in the first day and then two-hours intervals until the death of all individuals. walking behavior bioassays walking behavior bioassays were conducted in a petri dish (ø 9 cm x | 1.5 cm) covered with a black paper with the sociobiology 65(1): 31-37 (march, 2018) special issue 33 same treatments described above. however, in this bioassay, only one individual was placed in the control group and nest wall treatments and two individuals in the host treatments. three replicates were performed per nest and treatment, totalizing 27 replicates. the mean value of three replicates/ nest were then used in the statistical analyses. to allow the visualization and recorded of walking behavior of inquiline workers, individuals placed in the petri dish were previously marked with a mixture of white gouache and glue (2:1) following the procedure described in marins et al. (in press). the movement of individuals was video-recorded for 10 min using a camera (panasonic sd5 superdynamic model wv-cp504), equipped with spacecom lens (1/3 “3-8 mm) coupled to a computer. the distance walked and walking velocity were captured by ethovision xt® software (version 8.5, noldus integration system, sterling, va) and the data were analyzed using the studio 9 software (pinnacle systems, moutain view, ca). statistical analyses to check whether survivorship of i. microcerus workers (y-axis) is affected by the presence of their hosts (from the same or different nests) or by the contact of host nest wall (inner or external wall), data were submitted to survival analysis using weibull distribution. the mean time to death of all individuals in the petri dish was calculated in each treatment x nest and then, data were submitted to analysis of deviance (anodev). to check whether the distance walked (mm) and the walking velocity (mm/s) of inquiline workers (y-axis) is affected by the presence of their hosts (from the same or different nests) or by the contact of host nest wall (inner or external wall), data were submitted to analysis of deviance (anodev). all analyses were performed in the r statistical software (r development core team 2015) using generalized linear modelling (glm) followed by residual analyses to check the suitability of distribution choose. statistical simplification among treatments was performed via t test using multcomp package. results survival bioassays the survivorship of i. microcerus workers was significantly lower in the treatments with presence of host than in the control (f2,6 = 6.905, p = 0.027; fig 1a). however, no significant differences in the mean time to death of inquilines was observed in the treatments with host from same or different nests (p = 0.65). inquiline workers in contact with external wall of host nest (ew) showed a lower mean time to death compared with inquiline workers in contact with the inner wall of host nest (iw) and with control (inquiline workers alone) (f2,6 = 28.070; p< 0,001; fig 1b). control and inquiline workers in contact with the inner wall of host nest (iw) did not show significant differences between them (p = 0.83). walking behavior bioassays most representative trails of i. microcerus workers in all treatments are showed in fig 2. the presence of host (from same or different nest) did not affect significantly the distance walked (f2,6 = 1.13, p = 0.32) and the walking velocity (f2,6 = 0.19, p = 0.82) of i. microcerus workers. the distance walked (f2,6 = 7.31, p < 0.001) and the walking velocity (f2,6 = 0.654, p = 0.02) of inquiline workers were significantly reduced when in contact with host nest walls (inner or external) compared with the control group (fig 3a-b). however, there were no significant differences in the distance walked and the walking velocity of inquiline workers in contact with the inner or external walls of host nest (p = 0.10). control sh dh m ea n tim e to d ea th ( m in .) 0 500 1000 1500 2000 2500 control iw ew m ea n tim e to d ea th ( m in .) 0 500 1000 1500 2000 2500 a a b a b b a b fig 1. survival of inquilinitermes microcerus workers in different treatments. a) control (inquilines workers alone), sh (inquiline workers with host workers from the same nest), dh (inquiline workers with host workers from different nest; b) control (inquiline workers alone), iw (inquiline workers in contact with inner wall of host nest), ew (inquiline workers in contact with external wall of host nest). different letters in each graphic indicates significant difference among treatments. js cruz et al. – survivorship and behavior of termite inquiline34 discussion in the present study, we investigated how the presence of the host species (constrictotermes sp.) as well as the contact with host nest wall can modulate the survivorship and the walking behavior of its obligatory inquiline i. microcerus. in general, our results suggest that i. microcerus adopt strategies to avoid perception by its host, as already showed by previous studies. the mean time to death of i. microcerus workers was lower in the presence of host than in the absence of it (fig 1a). interestingly, the mean time to death of inquiline workers was not dependent of host origin (i.e. from same or different nest). such result can indicate that inquilines do not acquire the odor of the host colony, which could minimize the stress with its host colonies. in fact, c. cyphergaster and its obligatory inquiline i. microcerus do not share the same profile of cuticular hydrocarbons (cristaldo, unpublished data), a strategy usually used by inquilines of social insects. in hymenoptera, for example, the integration of social inquiline in the host nests is facilitated by the acquisition of host colony odor by inquilines [e.g. bees (dronnet et al., 2005), wasps (sledge et al., 2001), and ants (lenoir et al., 2001)]. thus, it is expected that the relationship of obligatory inquilinism observed here is mediated by other evolutionary stable strategies, allowing a faster establishment of i. microcerus in nests of constrictotermes sp. the fact that inquilines apparently did not acquire the odor of its host colony suggests that they should use strategies to avoid meeting the host. however, here we found that the distance walked and the walking velocity of inquiline workers have not been changed in contact with host workers. this lack of changes in the walking behavior of inquiline workers in the presence of host can be a strategy to not becoming apparent, which would reduce possible conflicts. thus, inquiline workers seem to minimize any reaction of perception and aggressiveness on the part of its host. ours results are in agreement with recent studies that showed that i. microcerus is able to recognize the trail pheromone of its host c. cyphergaster. by doing so, the inquiline can avoid galleries occupied by its host, facilitating the coexistence between them (cristaldo et al., 2014). inquilines are also able to recognize the alarm pheromone of its hosts, using it for their own benefit, either to avoid spaces inhabited by hosts or to escape from predators (cristaldo et al., 2016). recently, jirošová et al. (2016) showed that soldiers of i. inquilinus, an obligatory inquiline of c. cavifrons, produce chemical substances that are repellent to its host, suggesting that spatial separation of colonies is chemically mediated, which allow the coexistence by reduction of direct conflicts. to avoid the host, inquiline individuals could also contribute to reduce the chances of selection of strategies of counterattack by the host. in addition, reports in the literature suggest that inquiline colonies are usually found spatially isolated in the host nest and that they use galleries that do not overlap with those used by its host colonies (mathews, 1977; cunha et al., 2003; cristaldo et al., 2012). this apparently spatial segregation could occur to avoid direct contact with the host (see mathews, 1977; florencio et al., 2013). the inner wall of host nest is formed not only by excrement of host but also by excrement of inquilines. contrary to our hypothesis, inquiline workers in contact with inner wall of host nest did not survival more than those in the control group (fig 1b). this absence of significant variation can be related with two factors: i) the inability of inquiline workers to ingest the substrate of inner wall, as it was used in our bioassay and ii) the obstacle represented by the substrate. once the wall substrate has been macerated, it can be assumed that the inquiline workers were unable to ingest such particles, thereby reducing a possible effect of food on the increment in the survival of them. in addition, the presence fig 2. most representative trails of inquilinitermes microcerus workers in the following treatments: a) inquiline workers alone, b) inquiline workers with host workers from the same nest, c) inquiline workers with host workers from different nest, d) inquiline workers in contact with substrate from the inner wall of host nest and e) inquiline workers in contact with substrate from the external wall of host nest. sociobiology 65(1): 31-37 (march, 2018) special issue 35 of this substrate may have been an obstacle to walking, increasing the energy expenditure of these individuals, thus masking a possible positive effect of this treatment. this last explanation is supported by the fact that the walking behavior was reduced when in contact with the inner and external walls in the same way when compared to the control group (fig 2 and 3a-b). on the other hand, the contact with the external wall (ew) of host nest significantly reduced the survivorship of inquiline workers compared to iw and the control group (fig 1b). the external wall of host nest should present less signs that indicate to the inquiline workers the presence of a known environment and it should limit the contact among the individuals of its colonies, which would explain the greater mortality of this treatment when compared to the control. this suggests that the reduction of walking behavior may be more related to some obstacle to any behavioral change due to chemical signals in these structures. on the other hand, survival seems to have been more influenced by the presence of chemical signals of the colony. a series of hypothesis have been proposed to explain the cohabitation in termite nests: (i) inquilines become imperceptible in the nest (e.g. perceiving the chemical cues of its host or using different spaces in the host nest) (cristaldo et al., 2012; 2014; 2016), (ii) inquilines have ability to repel their hosts (jirošová et al., 2016) or (iii) inquilines do not overlap diet with their host (diet segregation; florencio et al., 2013). the present study supports the idea that avoidance strategies used by inquilines can be the primary factor of coexistence between the obligatory inquiline i. microcerus and its host constrictotermes sp. previous studies have already indicated that avoidance strategy seems to occur by perception of chemical cues from host, here we showed that i. microcerus also have behavioral mechanisms that can avoid possible conflict easing the cohabitation in its host nests. acknowledgements we are grateful to prof. reginaldo constantino (unb) for species identification. p.f.c. thanks cnpq/fapitec-se (#302246/2014-6) and pnpd-capes. jsc is supported by pibic/fapitec-se (#019.203.01192/2016-1). the other co-authors were supported by capes or cnpq grants. authors contribution apa araújo and pf cristaldo conceived and design the experiments. js cruz, pf cristaldo, jj marques, dv ferreira and apa araújo carried out the fieldwork. js cruz, pf cristaldo, jj marques, ml cruz, dv ferreira and apa araújo performed the bioassays. apa araújo and pf cristaldo performed the data analyses. js cruz, pf cristaldo and apa araújo wrote the manuscript. references ackerman, i.l., teixeira w.g., riha s.j., lehmann j.& fernandes e.c.m. 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(1981). behavior and ecology of foraging termites. in t. abe, d.e. bignell & m. higashi (eds.), termites: evolution, sociality, symbioses, ecology (pp. 141-168). dordrectht, netherlands: kluwer academic publishers. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i4.1178sociobiology 63(4): 1051-1057 (december, 2016) diversity of wasps (hymenoptera: vespidae) in conventional and organic guarana (paullinia cupana var. sorbilis) crops in the brazilian amazon introduction the replacement of natural areas by monocultures and pastures is resulting in widespread local and global biodiversity loss. besides habitat fragmentation, the use of pesticides and insecticides reduces the diversity of pollinators (durigan et al., 2007; lindenmayer et al., 2013; pimentel et al., 1992). pimentel et al. (1992) assert that it is important to conserve biological diversity in agricultural ecosystems, which, along with human settlements, cover approximately 95% of terrestrial environment. farmland conservation programs aim at improving the value of agricultural landscapes for biodiversity, and a key conservation strategy of these programs is the retention of remnant natural vegetation (kleijn et al., 2011; phalan et al., 2011). the maintenance of the edge vegetation is considered one of the most effective measures for sustaining insects abstract diversity of wasps (hymenoptera: vespidae) in conventional and organic guarana (paullinia cupana var. sorbilis) crops in the brazilian amazon. the present study aimed to determine the diversity of wasp species associated with the guarana crop and the difference in composition of species associated to organic and conventional crops, as well as among environments established in each management (adjacent forest, crop edge and guarana crop). we collected 977 individuals and 59 species, in 23 genera of vespidae, sixteen of polistinae (52 species) and seven eumeninae (seven species). polybia was the most abundant and rich genus with 553 specimens and 15 species, followed by agelaia (139, nine) and protopolybia (103, five). in organic management crop, 686 individuals allocated in 18 genera and 47 species were collected, whereas in conventional management crop 291 individuals allocated in 18 genera and 41 species were collected. according to the three sampling points, in both management types, the edge of the crop field shows the highest abundance of wasps with a total of 519 individuals allocated in 19 genera and 45 species. given the intense use of both environments (forest and crop) by the wasps, it is important to grow crops in regions near native forests, where the chances of social wasp colonies to be founded are increased. sociobiology an international journal on social insects a somavilla1, k schoeninger1, dgd de castro1, ml oliveira1, c krug2 article history edited by gilberto m. m. santos, uefs, brazil received 05 august 2016 initial acceptance 25 september 2016 final acceptance 07 october 2016 publication date 13 january 2017 keywords agroecosystem, crop edge, polybia, social wasps. corresponding author inpa instituto nacional de pesquisas da amazônia prog. de pós-graduação em entomologia av. andré araújo, 2936 aleixo cep 69080-971, caixa-postal 2223 manaus-am, brasil e-mail: alexandresomavilla@gmail.com diversity in crops (attwood et al., 2008; tscharntke et al., 2008). it allows native insects to persist in the transformed landscape by providing undisturbed refuge and supplementary resources within the agricultural mosaic (benton et al., 2003; duelli & obrist, 2003; gaigher et al., 2015). in the amazon region, guarana (paullinia cupana var. sorbilis (mart.) ducke) is one of the most cultivated plants; it is a native amazonian plant belonging to the sapindaceae family. according to ibge (2013), the planted area in brazil is 14.952 hectares and the amazonas state is one of the largest producers. currently, there is a great demand for organic products of guarana (tavares et al., 2003; tavares & garcia, 2009). however, the absence of basic studies (biology and ecology) and data about the composition of fauna biodiversity, especially of natural enemies as as predatory and parasitoids hymenoptera, make it impossible or delay the development of alternative techniques to biodiversity conservation and pest control. 1 instituto nacional de pesquisas da amazônia, manaus, amazonas, brazil 2 embrapa amazônia ocidental, manaus, amazonas, brazil research article wasps a somavilla et al. – wasps in guarana crop1052 the vespidae family occurs over a wide range of habits and presents varying levels of social complexity, serving as regulators of other insect populations (prezoto, 1999; carpenter & marques, 2001), as well as pollinators (sühs et al., 2009). the most part of protein acquired by wasps in their foraging comes from the capture of caterpillars, the main group of insects that feed on cultivated plants (prezoto et al., 2008). thus, wasps play a major role in agricultural systems (carvalho & souza, 2002). the most common subfamilies in the brazilian amazon are polistinae and eumeninae. the social polistinae comprises 26 genera and 958 described species widely distributed in the neotropical region (pickett & carpenter, 2010). the polistinae social wasps are important components of neotropical ecosystems due to their ubiquity and diversity, as well as their complex interactions with other organisms (silveira, 2002). the species composition of a determined area is an important factor on which to base comprehensive scientific studies of its ecological characteristics (humprhey et al., 1999). the highest diversity of polistinae is found in brazil (319 species recorded) and silveira (2002) pointed out that 200 species were recorded in the brazilian amazon. the eumeninae solitary wasps include 3.579 species, the subfamily with the highest number of species among vespid wasps and about 300 species have been recorded from brazil (carpenter & garcetebarrett, 2002; pickett & carpenter, 2010). in this context, the present study aimed to determine the diversity of wasp species associated with the guarana crop and the difference in composition of associated species to organic and conventional crop, as well as among different environments in each management (adjacent forest, crop edge and guarana crop). material and methods study area the study was conducted within the experimental fields of western amazon embrapa (brazilian enterprise of agriculture and cattle-raising), in manaus, amazonas, brazil, where there are two guarana crop fields, one under organic management (2°53’29.14”s / 59°58’45.80”w) and the other under conventional management system (2°53’42.18”s / 59°59’10.58”w). the conventional management crop was established in 1986 in an area of 1.6 ha with 710 plants cultivated in a spacing of 5 m x 5 m. in this crop field, the application of insecticide happened only once, on january 1st, 2013. the organic management crop was established in 2003 in an area of 3.9 ha with 1,595 plants cultivated in a spacing of 5 m x 5 m. sampling design in this study, we used two distinct and usual sampling methods, the malaise traps and the möericke traps. with malaise traps (townes model), the wasps were collected in a container located in the upper region of the trap, which had alcohol 70%. the möericke trap consists of a yellow container measuring 25 cm of length x 15 cm width x 5 cm of height, and water solution with 2 ml of neutral detergent. in each sampling occasion, which happened at biweekly intervals, the traps were set up for four days, and the water from the möericke trap was replaced every 24 hours in order to avoid loss and/or deterioration of biological material. a total of 12 samplings were conducted from september 2012 to february 2013. in each crop field (conventional and organic management), a diagonal sampling line was established along the three sampling points: 1) in the interior of the crop field; 2) at the edge of the crop field, and 3) outside the crop field, in the adjacent forest area. the distance between each sampling point was 60 m. at each point, one malaise trap was installed. the distance between conventional and organic management crops is approximately 5 km. data analysis the specimens of vespidae were sorted and identified at the hymenoptera laboratory of the national institute of amazonian research (inpa). the vouchers were deposited at inpa’s invertebrate collection. in this study, several measurements (samples) were made in the same experimental units (organic and conventional crop fields) over a period of time. such data are called ‘repeated measures’ (crowder & hand, 1990; davis, 2002; gotelli & ellison, 2004). in order to verify if the variables ‘type of crop’ (conventional and organic management), ‘sampling techniques’ (malaise and möerike), and ‘sampling points’ (adjacent forest, edge and interior of crop field) influenced wasps species richness, we performed an analysis of variance (anova) for repeated measures, since the samples were taken in the same crop fields across time. similarly, we performed the anova with repeated measures to test the influence of the variables ‘type of crop field’, ‘sampling techniques’, and ‘sampling points’ on the species composition of wasps. with both metrics (richness and composition) we tested whether there was an interaction between samples (over time) and other variables used in anova models. if required, anova analysis was followed by the post hoc tukey multiple-range test (yandell, 1997). the dimensionality of wasps abundance data was reduced using nonmetric multidimensional scaling (nmds, minchin, 1987) based on the bray-curtis dissimilarity index. additionally, we calculate the shannon diversity index (h’) and the pielou equitability index (j’). all analyses were conducted in the free software r, version 3.1.0 (r development core team, 2014), using the vegan package (oksanen et al., 2013). to calculate the frequency of the species, which is the proportion of individuals of a species in relation to all individuals in the sample, we used this formula: f = n/n x 100, where f = frequency (in percentage); n = number of individuals of each species and; n = total number of individuals. sociobiology 63(4): 1051-1057 (december, 2016) 1053 table 1. vespidae species collected in guarana organic and conventional management and sampling points, in the brazilian amazon. forest = adjacent forest, edge = edge of the crop and interior = interior of the crop. fr (%) = frequence. taxon conventional guarana crop organic guarana crop total fr (%) forest edge interior forest edge interior agelaia angulata fabricius, 1804 9 0 5 18 0 0 32 3.3 agelaia cajennensis (fabricius, 1798) 0 0 0 0 1 1 2 0.2 agelaia centralis cameron, 1907 0 2 0 0 0 0 2 0.2 agelaia constructor de saussure, 1854 3 0 1 2 0 1 7 0.7 agelaia flavipennis (ducke, 1905) 0 0 2 2 1 0 5 0.5 agelaia fulvofasciata degeer, 1773 2 2 3 2 10 5 24 2.5 agelaia myrmecophila ducke, 1905 1 1 1 1 1 1 6 0.6 agelaia pallipes olivier, 1792 0 1 1 8 12 38 60 6.2 agelaia testacea fabricius, 1804 0 0 0 1 0 0 1 0.1 angiopolybia obidensis ducke, 1904 0 0 0 4 0 0 4 0.4 angiopolybia pallens lepeletier, 1836 2 1 0 14 5 2 24 2.5 angiopolybia paraensis spinola, 1851 0 7 2 3 4 0 16 1.6 apoica pallida olivier, 1792 0 0 0 0 0 1 1 0.1 apoica strigata richards, 1978 0 0 0 1 0 0 1 0.1 brachygastra billineolata spinola, 1841 0 2 1 0 0 0 3 0.3 chartergellus amazonicus richards, 1978 0 0 1 0 2 0 3 0.3 chartergellus jeannei andena & soleman, 2015 0 2 0 0 1 0 3 0.3 chartergus chartarius olivier, 1791 0 0 0 0 1 0 1 0.1 charterginus xanthura (de saussure, 1854) 0 0 0 0 3 0 3 0.3 hypalastoroides sp.1 1 0 0 0 0 0 1 0.1 leipomeles dorsata fabricius, 1804 0 1 0 0 0 0 1 0.1 leipomeles spilogastra cameron, 1912 0 0 1 0 0 0 1 0.1 metapolybia cingulata fabricius, 1804 0 1 0 0 0 1 2 0.2 mischocyttarus labiatus fabricius, 1804 3 3 1 0 1 1 9 0.9 mischocyttarus rotundicollis cameron, 1912 0 1 0 0 0 1 2 0.2 mischocyttarus sp.1 0 0 0 0 0 3 3 0.3 montezumia sp.1 0 0 0 0 0 1 1 0.1 omicron sp.1 0 1 0 0 1 0 2 0.2 pachodynerus sp.1 0 4 0 1 2 1 8 0.8 pachymenes sp.1 3 6 3 2 10 5 29 3.0 parachartergus smithii de saussure, 1854 0 0 0 0 4 1 5 0.5 parachartergus griseus (fox, 1898) 0 0 1 0 0 0 1 0.1 parachartergus sp.1 0 0 1 0 0 0 1 0.1 polistes geminatus fox, 1898 0 1 1 0 0 0 2 0.2 polistes pacificus fabricius, 1804 1 10 16 2 0 0 29 3.0 polybia belemensis richards, 1970 0 2 1 0 14 5 22 2.3 polybia bistriata fabricius, 1804 0 0 0 0 3 5 8 0.8 polybia dimidiata olivier, 1791 0 1 0 0 0 0 1 0.1 polybia dubitata ducke, 1910 0 0 1 1 0 0 2 0.2 polybia emaciata lucas, 1879 1 1 1 0 4 2 9 0.9 polybia gorytoides fox, 1898 0 0 0 0 1 0 1 0.1 polybia ignobilis haliday, 1836 0 1 1 0 0 0 2 0.2 polybia jurinei de saussure, 1854 0 0 0 0 6 10 16 1.6 polybia liliacea fabricius, 1804 3 2 1 17 23 4 50 5.1 polybia occidentalis olivier, 1791 3 24 18 17 107 13 182 18.7 a somavilla et al. – wasps in guarana crop1054 results we collected 977 wasps specimens. the subfamily polistinae was the most abundant with 925 specimens, while eumeninae was represented by 52 individuals. polybia lepeletier was the most abundant genus, followed by agelaia lepeletier and protopolybia ducke with 553, 139 and 103 specimens, respectively. we determined 59 species of vespidae in 23 genera, 52 species in sixteen genera of polistinae and seven species in seven genera of eumeninae. again, polybia, agelaia and protopolybia were the most rich, with fifteen, nine, and five species, respectively (table 1). polybia rejecta (fabricius, 1798) was the most abundant and frequent (n = 247; 25.3%) species in guarana crop, followed by polybia occidentalis olivier, 1791 (n = 182; 18.7%). our collections include species that merit particular notice; we collected species that represent significant records because they have not been recorded by recent surveys in amazonas state: chartergellus amazonicus richards, 1978, chartergus chartarius olivier, 1791, charterginus xanthura (de saussure, 1854), leipomeles dorsata fabricius, 1804, leipomeles spilogastra cameron, 1912, protopolybia minutissima (spinosa, 1851), protopolybia picteti (cameron, 1907). in organic management crop, we collected 686 individuals allocated in 18 genera and 47 species. in conventional management crop, we collected 291 individuals allocated in 18 genera and 41 species. polybia was the most abundant and species-rich genus, in both crops. the shannon diversity index was similar for both managements after the species richness was approximated (h ‘= 2.71). regarding the pielou equitability index, an almost homogeneous distribution between the species both in the conventional management (j ‘= 0.72) and in organic (j’ = 0.70) was observed, except for p. occidentalis and p. rejecta, whose number of individuals were higher than the other species. in the three sampling points, we collected 519 individuals allocated in 19 genera and 45 species in the edge of the crop, 313 individuals allocated in 18 genera and 43 species in the interior of the crop and 145 individuals allocated in 10 genera and 25 species in the adjacent forest. in both management types, the edge of the crop field showed the highest number of individuals. differences in richness of vespidae were detected between the organic and conventional crop (anova(1,84) f=29.078; p<0.001*), and among sampling points adjacent forest, edge and interior of crop field (anova(1,84) f=27.931; p<0.001*). the other variables did not affect the richness of wasps. the variable sample (over time) showed no interaction effect with the others variables used in the model, that is, it did not affect the detection of the individual effect of tested variables (fig 1; table 2). table 1. vespidae species collected in guarana organic and conventional management and sampling points, in the brazilian amazon. forest = adjacent forest, edge = edge of the crop and interior = interior of the crop. fr (%) = frequence. (continuation) taxon conventional guarana crop organic guarana crop total fr (%) forest edge interior forest edge interior polybia parvulina richards, 1970 0 0 0 0 5 0 5 0.5 polybia rejecta (fabricius, 1798) 1 35 53 10 86 62 247 25.3 polybia sericea olivier, 1792 0 0 1 0 0 0 1 0.1 polybia velutina ducke, 1907 0 1 0 0 0 0 1 0.1 polybia (myrapetra) sp.1 0 3 0 1 1 1 6 0.6 protopolybia bituberculata silveira & carpenter, 1995 0 5 2 2 21 4 34 3.5 protopolybia chartergoides gribodo, 1891 0 2 0 0 2 3 7 0.7 protopolybia minutissima (spinosa, 1851) 0 7 1 1 35 12 56 5.8 protopolybia picteti (cameron, 1907) 0 2 0 0 2 1 5 0.5 protopolybia sp.1 0 0 0 0 1 0 1 0.1 pseudodynerus sp.1 0 1 0 0 3 0 4 0.4 pseudopolybia vespiceps de saussure, 1863 0 0 0 0 1 1 2 0.2 synoeca virginea fabricius, 1804 0 2 1 2 6 2 13 1.3 zethus sp.1 0 0 1 0 4 2 7 0.7 total of individuals 33 (3.4) 135 (13.8) 123 (12.6) 112 (11.4) 384 (39.3) 191 (19.5) 977 100 variables degrees of freedom f value p value type of crop field 1 29.078 <.0001* sampling point 2 27.931 <.0001* samples 11 0.781 0.655 interaction between type of crop field and samples 11 0.795 0.643 interaction between sampling point and samples 22 0.780 0.714 table 2. analysis of variance with repeated measure results for a model using type of crop field, sampling point, sampling techniques and samples for vespidae richness. significance level: p< 0.001*. sociobiology 63(4): 1051-1057 (december, 2016) 1055 on the other hand, differences in composition of vespidae species were not detected between the organic and conventional crop (anova(1,84) f=0.972; p<0.3286), or among sampling points (anova(1,84) f=1.065; p<0.352). therefore, the variable sample (over time) showed no interaction effect with the others variables used in the model, that is, it did not affect the detection of the individual effect of tested variables (tab. 3). however, comparing this study with protected areas or reserves in amazon rainforest, we noticed a lower species richness in guarana crop. for example, in an inventory at ducke reserve in amazonas state, 58 species of social wasps (polistinae) in 13 genera were collected (somavilla et al., 2014) and in caxiunã reserve, in para state, 65 species in 12 genera of polistinae were collected (silva & silveira, 2009), both with a higher species richness than this inventory in cultivated area, but with a lower number of genera. polybia rejecta and polybia occidentalis are two very abundant species in amazon region, most often recorded in open areas but rarely captured in close rainforest (somavilla et al., 2014). furthermore, these two species found colonies in open areas, such as the edge of guarana crop, enhancing the collection of these wasps in crops. the high abundance of agelaia and polybia registered in the current and other studies indicates that species of these genera find it easy to colonize several different types of microhabitats due to their protected nests, method of foundation and great number of individuals, which gives their colonies greater chances of success (hermes & köhler, 2004). in this study, their abundance was higher in adjacent forest and interior of the crop, showing a great capacity for dispersion, unlike the results of others studies where usually the uniform environments and interior of the crops lower their abundance, and suggests that these species encounter barriers to use resources outside the better-conserved environments (klein et al., 2015). the adjacent forest and the edge of cultures is very important for maintaining the biological diversity in agricultural ecosystems. the vespidae richness and composition in a crop depend on the diversity of vegetation within or in the edge, presence of only one or various cultures, the intensity of management, the distance of the natural vegetation and the presence of food (gaigher et al., 2015). the wasps use directly vegetation to building their nests and search for food, for this, vegetation improves the chances of social wasp colonies to be successfully founded because of their ability to migrate between these habitats. fig 1. the vespidae richness in guarana organic and conventional management (a) and sampling points (b), in the brazilian amazon. table 3. analysis of variance with repeated measure results for a model using type of crop field, sampling point, sampling techniques and samples for vespidae composition. significance level: p< 0.001*. variables degrees of freedom f value p value type of crop field 1 0.972 0.329 sampling point 2 1.065 0.352 samples 11 0.782 0.655 interaction between type of crop field and samples 11 0.795 0.643 interaction between sampling point and samples 22 0.780 0.714 discussion wasp surveys are lacking in agricultural ecosystems in brazil, especially in the amazon region, making this a pioneer study for the guarana crop. the vespidae richness and composition obtained in this study were higher than previous studies conducted in different crops. for example, in an area of silvipastoral culture of embrapa dairy cattle research center in minas gerais, a total of 205 social wasp specimens, distributed in 13 morphospecies and four genera were captured (auad et al., 2010). in forest fragments with different surrounding matrices of sugarcane and citrus crops in são paulo, a total of 1460 social wasp specimens, distributed in 29 morphospecies and 10 genera were captured (tanaka junior & noll, 2011). no studies including the eumeninae were conducted so far. a somavilla et al. – wasps in guarana crop1056 the sampling points within the forest and within the interior of the crop, for both conventional and organic crops, were similar in terms of composition of vespidae. this similarity may have reflected the environment conditions, which present preserved areas in the surroundings, composed of several different plants from the main crop. these areas provide shelter and food for the wasps (altieri & nicholls, 2004) and increase the concentration and distribution of wasps inside the crop. the abundance of vespidae was higher in organic management (70%) when compared to conventional. the greatest abundance can may be linked to the fact that no influence of chemical pesticides and fertilizers, which allows the entry of wasps inside of the crop. the wasps, in turn, enter the crop in order to establish a colony or to get resources for their survival, such as food or material needed to build their nests, which explains the presence of a lot of wasps social this type of management. in the conventional crop field, the edge and the interior crop had similar richness. surprisingly, the most similar environments in the organic crop field were the adjacent forest and the edge. these results have differed from the expected (adjacent forest and edge of crop field more similar to each other in the conventional crop system; and edge and interior of crop field in the organic crop system). perhaps, these results indicate that it is occurring the entry of wasp species into in interior crop for search food and nesting resources. concerning the two sampling methods, the use of several sampling methods increases the chances of sampling all potential niches (longino et al., 2002). however, it also increases the amount of time and money employed during the studies. thus, for the purpose of surveying vespidae fauna in guarana crops, it is ideally recommended the use of malaise trap, for the möericke traps do not have an efficiency for the collection of vespidae. this study highlights the importance of conserving natural fragments for maintaining biodiversity in agricultural landscapes, which supports existing set-aside programs. this is an important consideration in a region where high-value crops and biodiversity-rich natural land coincide (fairbanks et al., 2004) and where the potential for natural habitat conversion for agricultural expansion is high (underwood et al., 2009; viers et al., 2013; gaigher et al., 2015). therefore, it 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(1997). practical data analysis for designed experiments. chapman & hall/crc press, london/cleveland. 440p. http://www.r-project.org/ open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1663sociobiology 65(1): 15-23 (march, 2018) special issue phylogenetic community structure of southern african termites (isoptera) introduction the mechanisms that structure communities are still highly debated (chase & leibold, 2003; chave, 2004; zhou & zhang, 2008; holt, 2009). environmental factors such as temperature and rainfall can act as filters that limit, for instance, the distribution of species and define part of a species niche, especially over larger geographic scales. an understanding of the importance of local versus regional processes depends on the spatial scale, divided into spatial grain (the size of the sample unit) and the spatial extent (the total area of the study), at which species communities are defined and studied, and the scale at which actual processes operate (graham & fine, 2008; emerson & gillespie, 2008; weiher et al., 2011). classically, interspecific competition was thought to be a major driving force in structuring communities of ecologically similar species (members of the same guild, functional analoga) (diamond, 1978; schoener, 1982). the resulting concept of limiting similarity states that species abstract the processes that structure communities are still largely unknown. therefore, we tested whether southern african termite communities show signs of environmental filtering and/or competition along a rainfall gradient in namibia using phylogenetic information. our results revealed a regional species pool of 11 species and we found no evidence for phylogenetic overdispersion or clustering at the local scale. rather, our results suggest that the assembly of the studied termite communities has as strong random component on the local scale, but that species composition changes along the climatic gradient. sociobiology an international journal on social insects j schyra1, b hausberger1, j korb1, 2 article history edited by paulo fellipe cristaldo, ufs, brazil received 19 april 2017 initial acceptance 14 june 2017 final acceptance 05 july 2017 publication date 30 march 2018 keywords savanna, ß-diversity, namibia, neutral theory, filtering. corresponding author janine schyra behavioural biology university of osnabrueck barbarastr nº 11, 49076 osnabrueck. e-mail: janine.schyra@biologie.uniosnabrueck.de can only co-exist if they differ in their niche axes (abrams, 1983). there is supporting evidence, for example, when striking patterns of size or morphological differences were found for co-existing guild members (bowers & brown, 1982; leibold, 1998). yet, in many cases the concept was taken for granted and local communities were not tested against the null hypothesis of random assemblages from the regional species pools. in more recent years the development of the unified neutral theory of biodiversity and biogeography challenged the classical niche theory and considered niche differences less important (hubbell, 2001). according to this theory, trophically similar species are demographically equivalent and ecological communities are mainly structured by ‘ecological drift’ (i.e. stochastic factors such as birth, death, random migration and extinction) (hubbell, 2001; volkov et al., 2009). a ‘niche versus neutrality’ debate is largely unproductive and attempts exist to incorporate elements of both theories into more general explanations (holt, 2009; weiher et al., 2011). 1 behavioral biology, university of osnabrueck, germany 2 evolutionary biology and ecology, albert-ludwigs-university of freiburg, germany research article termites j schyra, b hausberger, j korb – community structure of termites16 the high species diversity of tropical ecosystems, where many species of seemingly identical niches coexist, is a challenge for classical niche theory. the fact that tropical insect communities have even less specialists than those of temperate regions (schleuning et al., 2012; ollertonet al., 2012) supports the theory that niches may not play such an important role in the tropics. yet, data that explicitly test structuring processes in tropical animal communities are scarce (vamosiet al., 2009). this applies especially for insects whose sheer species richness can hardly be explained by niche differences. many termite species occupy apparently very similar niches: they have similar abiotic requirements and are decomposers of organic matter. currently, four feeding groups are recognized by analysis of gut content (donovan et al., 2001), representing different stages of decomposition from sound wood to soil organic matter. this classification is also reflected in the four feeding types; wood-, leaf litter-, soiland true soilfeeders (eggleton & tayasu, 2001). recent stable isotope analyses of a termite assemblage from a forest implied that there is subtle niche differentiation within feeding groups (bourguignon et al.,2009). yet, they cannot explain how more than 20 species of leaf litter feeders can co-exist in african savannas that all forage from the same dead plant material (hausberger et al., 2011). we tested whether local termite communities in southern africa differ from random assemblages of the regional species pool with regard to phylogenetic composition, by looking at communities across a north/south rainfall gradient in namibia. the northern region is characterized by higher precipitation and therefore more diverse vegetation than the more arid southern region with less vegetation (jürgens et al., 2010; grohmann et al., 2010). we investigated whether ß-diversity between sites is related to distance along this gradient. to do this, we applied phylogenetic community analyses (webb et al., 2002, 2008; cavender-bares et al., 2009; kembel et al., 2010 ), which allowed us to explicitly test real communities against null models of random assemblages. by using phylogenetic information, we tested whether local communities are more or less closely related than assemblages drawn randomly from the regional species pool. as ecological traits are phylogenetically conserved for the species studied here (inward et al., 2007), related species share ecological traits, and therefore assemblages of less closely related species (i.e. phylogenetic overdispersion) indicate interspecific competition because these similar traits prevent species from coexistence. the reverse (i.e. phylogenetic clustering) suggests environmental filtering due to similar ecological preferences (temperature, rainfall, vegetation density, soil type) (webb et al., 2002, 2008). combined with analyses of species turnover between localities (ß-diversity; whittaker, 1972) and its phylogenetic signal (phylogenetic ß-diversity; bryant et al., 2008; kembel et al., 2010), and an analysis of the impact of environmental variables (helmus et al., 2007a, 2007b), we tested whether these southern african termite communities differed from random assemblages. materials and methods termite sampling termites were collected in january 2010 from 6 sampling sites (22°50’ to 26°11’n; 18°5’ to 16°8’e; namibia; fig 1) when they were most active, that is, at the beginning of the rainy season, using a standardized transect sampling protocol (hausberger et al., 2011). this protocol developed for termite diversity assessments consists of soil sampling and a thorough search of dead plant material on the ground, in trees, and in mounds (jones & eggleton, 2000). such transect sampling is recommended to assess termite diversity in regions of intermediate to moderately low rainfall (davies et al., 2013). a plot size of one ha was chosen because the foraging range of termite colonies is within 100m (korb & linsenmair, 2001) and hence one ha represents the local scale where interactions among colonies occur, i.e. it reflects the darwin-hutchinson-zone, which is most relevant to study the assembly of local communities (vamosiet al., 2009). three transects each measuring 2 m x 50 m, divided into ten 2 m x 5 m sections, were arbitrarily located within each plot. each transect section was searched thoroughly for termites for 15 minutes by a trained person; additionally, we sampled eight soil pits per transect section, each measuring 12 cm x 12 cm x 10 cm. all encountered samples were stored in pure ethanol for subsequent molecular analysis. due to limitations of the study, additional sampling along the climatic gradient was not possible. however, this transect method is designed to obtain the best possible ‘snapshot’ of conditions in a site and has been tested and used frequently in termite diversity studies (jones & eggleton, 2000; donovan et al., 2002; eggleton et al., 2002; inoue et al., 2006) and our study design is comparable to other termite studies (houston et al., 2015; dahlsjö et al., 2015). all samples were identified to species level. first, samples containing soldiers were identified to the genus level using the keys by webb (1961) and uys (2002). then these morphological identifications were confirmed by molecular genetic analyses (see below). for samples with workers only, morphological identification was difficult, they were genetically analysed (see below). the presence/absence of each species within a plot was documented as well as the encounter rate (i.e. the number of samples per species and plot), which is used as a surrogate of species abundance (davies, 2002). genetic analyses to allow unambiguous species identification, we isolated dna and sequenced fragments of three genes as described elsewhere (hausberger et al., 2011) (see also supplementary sociobiology 65(1): 15-23 (march, 2018) special issue 17 material, table s1): cytochrome oxidase subunit i (coi; total length 680bp), cytochrome oxidase subunit ii (coii; total length 740 bp), and 12s (total length 350 bp). these sequences were used to re-construct phylogenetic trees using three approaches (bayesian method, maximum-parsimony analysis, and maximum-likelihood analysis) to delimitate and identify species (for more details and genbank accession numbers see supplementary material, table s2). as in former termite studies (legendre et al., 2008; hausberger et al., 2011), coii was most useful for ‘barcoding’ (i.e., assigning species to samples) because it amplified well and gave appropriate resolution for species identification. all collected samples were sequenced for identification. phylogenetic community structure analyses we analysed the local community structure with phylocom 4.2 (webb et al., 2008). as input tree for the phylogenetic community structure analyses we used the coii gene tree, which was pruned prior to analysis so as to have only species of the regional species pool included and only one representative per species in the tree. we calculated the net relatedness index (nri) that measures whether locally co-occurring species are phylogenetically more/less closely related than expected by chance. it uses phylogenetic branch length to measure the distance between each sample to every other terminal sample in the phylogenetic tree, and hence the degree of overall clustering (webb et al., 2008). the nri is the difference between the mean phylogenetic distance (mpd) of the tested local community and the mpd of the total community (regional) divided by the standard deviation of the latter. high positive values indicate clustering; low negative values overdispersion (webb et al., 2002).we chose the nri and not the nearest taxon index (nti) because the nri quantifies overall clustering of taxa on a tree and takes deeplevel clustering into account. the nti quantifies terminal clustering, independent of deep-level clustering and gave qualitatively the same results as the nri. we tested whether our data significantly deviated from 999 random communities derived from null models using the independent swap algorithm on presence/absence data (gotelli & entsminger, 2003; hardy, 2008). the swap algorithm creates swapped versions of the sample/species matrix and constrains row (species) and column (species’ presence or absence) totals to match the original matrix. the regional species pool consisted of all species from all studied localities. as suggested by webb et al. (2008), we used two-tailed significance tests based on the ranks that describe how often the values for the observed community were lower or higher than the random communities. with 999 randomizations, ranks equal or higher than 975 or equal and lower than 25 are significant at p < 0.05 (bryant et al., 2008). comparison between study sites we quantified the compositional similarity (ß-diversity) between all localities using the sorensen and bray-curtis index, which were calculated in estimates version 8.2.0 (colwell, 2013). high values indicate high similarities between plots with regard to species composition and low values the reverse. a mantel test was used to analyse whether the similarity correlates with distance between plots using xlstat, version 2013.2.06. the phylosor index quantified the phylogenetic ß-diversity (bryant et al., 2008). it ranges from 0 (two communities only share a very small root, which means they consist of phylogenetically very different taxa) to 1 (both communities are composed of related taxa). equivalent to the phylocom analyses it was tested whether phylogenetic similarities between plots deviated from random by using ‘independent swap’ null models with 999 runs and applying rank tests. analyses were done with picante 1.2.0 (kembel et al., 2010) as implemented in r 3.0.3. influence of environmental variables we also tested if environmental factors influence the assemblage of termite communities using logistic regressions as suggested by helmus et al. (2007a, b). we retrieved monthly data for the bioclimatic variable mean annual precipitation for our study areas from the worldclim database (www. worldclim.org; hijmans et al., 2005), which is a set of global climate layers (climate grids) with a spatial resolution of 1km2. we only used one variable because of the small sample size and precipitation seems the most influential variable in arid regions. as termite colonies are perennial and sedentary this data seems more appropriate than recordings for a certain year. we correlated co-occurrence of species pairs with phylogenetic co-occurrence of species pairs, both before and after accounting for environmentally caused variability (mean annual precipitation), and calculated the change in occurrence correlations when including mean annual precipitation as implemented in the r-package “picante” (kembel et al., 2010). fig 1. map of the six sampling sites in namibia. one sampling site (plot) represents one community with one ha in size. j schyra, b hausberger, j korb – community structure of termites18 results diversity the phylogeny revealed a regional species pool of 11 species in nine genera (fig 2). nine species belonged to the higher termites (termitidae), with two macrotermitinae (allodontotermes sp., microtermes sp.), four termitinae (microcerotermes sp., angulitermes sp., promirotermes sp., amitermes sp.), and three nasutitermitinae (trinervitermes bettonianus, trinervitermes trinervoides and trinervitermes sp.). from the lower termites hodotermes mossambicus (hodotermitidae) was sampled as well as one representative of the genus psammotermes (rhinotermitidae). the phylogenetic analyses for the gene coii showed appropriate resolution, which was not the case for the genes coi and 12s. here sequencing of all samples was difficult and the phylogenetic trees did not give appropriate resolution for all species (fig 2, supp. fig s1 and s2). this similarly applied for the other phylogenetic methods used. phylogenetic community structure there were no significant signals of overdispersion or clustering within local communities. the nri indices ranged from 0.21 to 1.12. they never deviated from random communities generated with the ‘independent swap’ algorithm (always p>0.05). species richness per plot did not correlate with nri either (spearman-rank correlation: nri: r= 0.313, p= 0.545) (fig 3). comparison between study sites the compositional similarity between plots varied, with the sorensen index ranging from 0.40 to 1.00, the bray-curtis index ranging from 0.19 to 0.74 (supp. fig s3 and s4) and the phylosor index ranging from 0.53 to 1.00 (supp. fig s5). the sorensen and bray-curtis indices correlated significantly with the phylosor indices (spearmanrank correlation: sorensen: r= 0.742, p= 0.002; bray-curtis: fig 2. input bayesian phylogeny for phylocom based on the gene cytochrome oxidase ii using mr bayes v3.1.2. analysis was done with 107 generations, as well as a number of chains=4, sample frequency=1000 and a finalizing burn-in of 2500. fig 3. indices of the phylogenetic community structure using the null model independent swap with the presence/absence data showing the nri for all plots with the number of species per plot. the number of species did not correlate significantly with the nri. r=0.717, p=0.003). the phylosor and sorensen index showed that the community pairs located in the arid, southern region were more similar to each other compared to the remaining community pairs. tiras1 and oamseb 1, with a phylosor and sorensen index of 1, were significantly more similar with regard to phylogenetic composition than expected by chance and had identical species composition (fig s3 and s5; supp. table s3). community pairs tiras1 / oamseb2 and oamseb1 / oamseb2 had phylosor values of 0.89 and sorensen index value of 0.86. the remaining community pairs had index values ranging from 0.53 – 0.75 (phylosor index) and 0.40 – 0.67 (sorensen index). the geographical distance between plots did not significantly correlate with ß-diversity (manteltest: sorensen index: r= -0.144, p=0.611; bray-curtis index: r = -0.050, p= 0.861) or phylogenetic ß-diversity (mantel-test: r=-0.341, p = 0.205) (supp. fig s6). environmental variables the observed pairwise correlations between plots did not correlate significantly before and after including mean annual precipitation with the phylogenetic correlations (always p> 0.05) (fig 4a, b). further, the change in occurrence correlations, when including mean annual precipitation, did not significantly correlate with the pairwise phylogenetic correlations (p> 0.05) (fig 4c), indicating that it is not a major variable influencing species co-occurrences. discussion local community assembly this study aimed at uncovering processes that structure termite communities in namibia at a local scale, along a sociobiology 65(1): 15-23 (march, 2018) special issue 19 climatic gradient (supp. table s4). we did not find evidence that habitat filtering or interspecific interactions play major roles in structuring these communities locally. the highest termite diversity is located in african tropical forests (eggleton et al., 1994; davies et al., 2003). compared to forests, savannas harbour much lower species diversity. in west african savannas at least 20 species were identified, mostly fungus-growing macrotermitinae (hausberger et al., 2011). this is higher than the uncovered regional species pool of 11 species from nine genera in this study (supp. table s3). this might be due to the more arid conditions in namibia as previous studies revealed less species under drier conditions (de bello et al., 2006; traill et al., 2013). for namibia, a total of 17 termite genera have been identified (jürgenset al., 2010). this is considerably more than what we found, but our study covered a smaller area and did not include all vegetation zones (e.g. woodland savanna). we concentrated on analysing local community assembly, which supplements the work of jürgens et al. (2010), which monitored regional species occurrence and distribution. when comparing overlapping regions between both studies we had similar species occurrences (supp. table s2). this is the first study investigating the phylogenetic community structure of southern african termite communities and explicitly testing their composition against a null model of random assemblages. termites are mainly sessile organisms in that the colonies stay on one site over many years.therefore, they may be more prone to certain local environmental conditions and interspecific competition than highly mobile species that can (temporarily) escape unsuitable conditions, for example birds (canales-delgadillo et al., 2012; harmonthreatt et al., 2013). nest building in termites can be beneficial, in that it makes them less sensitive to abiotic environmental conditions. however, our results did not reveal significantly clustered or overdispersed communities according to the nri, indicating a random component to local species assembly. it has been shown that the opposing forces of habitat filtering and interspecific competition can counteract one another (lessard et al., 2009), resulting in seemingly neutral communities. in order to test for such effects the environmental regression analyses were done that tested for and ‘statistically’ removed environmental effects to reveal potentially hidden patterns of competition (kembel et al., 2010). assuming that rainfall is the major environmental factor in arid regions, including mean precipitation still could not reveal such a hidden pattern. for other tropical species, a variety of assembly patterns have been found, ranging from phylogenetic overdispersion to phylogenetic clustering along an environmental gradient (graham et al., 2009; webb, 2000; gòmez et al., 2010; cavender-bares et al., 2004). several reasons may explain these differences such as the studied taxon, its ecological requirements and sampling effort as well as the geographic history and conditions of the respective region.sampling effort could influence the results fig 4. influence of environmental factors on community structure. shown are correlations of pairwise species co-occurrence and pairwise phylogenetic correlations a) without environmental variables, b) including environmental variables,and the c) change in species co-occurrence once environmental variables are taken into account. there were no significant results a) without and b) with environmental variables included (p > 0.05). also c), the changes in correlation were not significant either (p > 0.05). j schyra, b hausberger, j korb – community structure of termites20 of community structure analyses as there might be a minimal diversity needed to detect a certain pattern. other studies had a termite species diversity of around 20 species and here community structure was detectable, even in local sites harbouring only 3-5 species (hausberger & korb, 2015, 2016). community structure along the climatic gradient the analysis of environmental variables showed that the effect of annual precipitation on community structure along the north / south gradient does not seem to be clear, despite the climatic gradient covered (annual precipitation in the north: 303 mm and south: 144 mm; supp. table s4). this may be due to a small sample size and the fact that other factors influencing community structure may compensate for the low rainfall in the arid region. this should be investigated in future studies. by contrast, other community studies have shown that broad-scale variation in climate, along latitudinal or altitudinal gradients, influences both the structure of species communities and the activity rates of certain species, which can modify species interactions and influence the degrees to which food resources are accessible (lessard et al., 2011; graham et al., 2009; gòmez et al., 2010; machac et al., 2011). as the ß-diversity and phylogenetic ß-diversity of the sampling sites revealed that communities are more similar in the southern part of the sampling area, some environmental or historical processes seem to influence species assembly, although we did not find an effect of the rainfall gradient. species diversity was higher in the northern area and next to a few other species, only trinervitermes sp. and the ‘desert’species psammotermes sp. were sampled frequently in the arid southern region. this is similar to what has been shown in previous studies (coaton & sheasby, 1972) and can be explained by the fact that trinervitermes sp. are grass-feeders and occur more frequently in open grass savannas with little other vegetation. although more research is clearly needed, our study contributes to the identification of african termite species with the goal to understand their distribution and species assembly processes. authors contribution j. korb obtained funding, conceived and designed the experiments. j. korb and b. hausberger carried out the fieldwork. j. schyra performed the laboratory work and data analyses. j. schyra, b. hausberger and j. korb wrote the manuscript. supplementary material h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfiles/1663/0 http://dx.doi.org/10.13102/sociobiology.v65i1.1663.s1935 acknowledgments we thank the landowners in namibia for allowing us to sample the termites on their land and abbo van neer for helping with field work, biota south and its team for their assistance on-site, and the ministry of environment and tourism of namibia for the collection permit (2010: 1456/2010) and export permit (2010: 78091). references abrams, p. 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(2011). advances, challenges and a developing synthesis of ecological community assembly theory. sociobiology 65(1): 15-23 (march, 2018) special issue 23 philosophical transactions of the royal society b-biological sciences, 366: 2403-2413. doi: 10.1098/rstb.2011.0056 whittaker, r.h. 1972. evolution and measurement of species diversity. taxon, 21: 213-251. zhou, s. & zhang, d. (2008). neutral theory in community ecology. frontiers in biology in china, 3: 1-8. doi: 10.1007/ s11515-008-0008-z doi: 10.13102/sociobiology.v65i2.2670sociobiology 65(2): 312-319 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 niche overlap and daily activity pattern of social wasps (vespidae: polistinae) in kale crops introduction insects from the vespidae family, order hymenoptera, popularly known by wasps, include species with solitary or eusocial habits. social wasps comprise three subfamilies (stenogastrinae, polistinae and vespinae) (carpenter & marques, 2001). wasps from the subfamily polistinae are the only social species that occur in brazil and belong to three tribes, polistini, mischocyttarini and epiponini, representing 21 genera and 343 species (hermes et al., 2017). social wasps forage to find water for nests and cooling, for plant fibers for nest materials, and for carbohydrates, such as pollen and nectar, for adult and larval nutrition (raveretrichter, 2000; lima & prezoto, 2003; clemente et al., 2017). in addition, they forage for animal protein, especially insects abstract kale (brassica oleraceae var. acephala) is of great importance in human nutrition and local agricultural economies, but its growth is impaired by the attack of several insect pests. social wasps prey on these pests, but few studies report the importance of this predation or the potential use of wasps as biological control for agricultural pests. this study aimed to survey the species of social wasps that forage in kale (b. oleraceae var. acephala), recording the influence of temperature and time of day on the foraging behavior of these wasps. the research was conducted at the federal institute of education, science and technology of minas gerais bambuí campus, from july to december 2015, when twelve collections of social wasps that foraged on a common area of kale cultivation were made, noting the temperature and time of collection for each wasp. polybia ignobilis, protonectarina sylveirae and protopolybia sedula were the most common wasp species foraging in fields of kale. interspecific interactions between wasp species did not affect their coexistence within kale fields, with peak foraging occurring between 1000 and 1100 hours. social wasps are important predators of herbivorous insects in the agricultural environment and the coexistence of a great diversity of these predators can help control pest insects that occur in the crop. moreover, knowing factors that influence foraging behaviors of common wasp species that occur in this crop is important for effective use of these insects in the biological control of pests. sociobiology an international journal on social insects gc jacques¹,², tg pikart³, vs santos³, lo vicente², lcp silveira¹ article history edited by gilberto m. m. santos, uefs, brazil received 23 november 2017 initial acceptance 19 december 2017 final acceptance 07 february 2018 publication date 09 july 2018 keywords biological control, foraging, polistinae. corresponding author gabriel de castro jacques departamento de entomologia universidade federal de lavras av. doutor sylvio menicucci, 1001, kennedy cep 37200-000, lavras-mg, brasil. e-mail: gabriel.jacques@ifmg.edu.br from the orders diptera, hemiptera, hymenoptera, and lepidoptera, which comprise about 90-95% of captured prey (prezoto et al., 2005; bichara-filho et al., 2009). these prey are torn, macerated and fed to larvae, being the main source of protein for social wasps in their early stages of development (rabb & lawson, 1957; jeanne et al., 1995; gomes et al., 2007). nutrients, carbohydrates and proteins, can be stored inside the cells for later consumption, constituting a reserve for unfavorable periods (barbosa et al., 2017; michelutti et al., 2017). foraging activity is one of the most important and complex behaviors exhibited by social wasps (lima & prezoto, 2003) and it depends on the insects’ ability to interact with the environment, as well as the availability of essential resources to support the colony (gomes et al., 2007). these wasps, like other generalists, forage predominantly on the most abundant 1 universidade federal de lavas, minas gerais, brazil 2 instituto federal de educação, ciência e tecnologia de minas gerais, campus bambuí-mg, brazil 3 universidade federal do acre, rio branco-ac, brazil research article wasps sociobiology 65(2): 312-319 (june, 2018) 313 resource, without preference or selective behavior (raveretritcher, 2000; santos et al., 2007). however, these wasps may return to hunt in locations of previous successful predation activity and feed several times on the same prey species, with individuals acting as facultative specialists (raveretrichter, 2000; bichara-filho et al., 2009). in general, higher temperatures, higher light intensity, lower humidity and lower wind speed are favor foraging conditions (lima & prezoto, 2003; ribeiro-junior et al., 2006). kale (brassica oleraceae var. acephala) belongs to the brassica family, which has the most oleaceous species, totaling 14 vegetables (filgueira, 2008), and is of great importance to human nutrition, as they exhibit good adaptation to a variety of climates (filgueira, 2008). several species of insect pests attack kale, such as the whitefly, bemisia tabaci (genn.) (hemiptera: aleyrodidae), and the aphids brevicoryne brassicae (l.) and myzus persicae sulzer (hemiptera: aphididae) (galo et al., 2002). these insects weaken plants by sucking sap and introducing toxins into their vascular system, causing leaf tissue deformations and gall formation, as well as contributing to the appearance of sooty mold on the foliage through honeydew excretion (galo et al., 2002; van emden, 2013). several lepidoptera also attack kale crops, such as the black cutworm agrotis ipsilon (hufnagel) (noctuidae), the cabbage looper trichoplusia ni (hübner) (noctuidae), the cabbage caterpillar ascia monuste orseis (godart) (pieridae) and the diamondback moth plutella xylostella(l.) (plutellidae) (gallo et al., 2002). a. monuste orseis and p. xylostella constitutes key-pests of this crop in the neotropical region, mainly in brazil (maranhão et al., 1998; barros & zucoloto, 1999). these species larvae feed on the leaves (gallo et al., 2002), resulting in losses of up to 100% of crop production (vendramim & martins, 1982; chen et al., 1996). the control of kale pests is mainly carried out by the application of insecticides (gallo et al., 2002; andrei, 2013). these synthetic chemicals can lead to a number of problems, such as residues in food, the death of natural enemies, poisoning of the applicators, and the emergence of resistant pest populations. therefore, the use of biological control agents could be an effective, cheap and safe alternative to the use of these toxic products. social wasps have been recorded preying on kale pests (picanço et al., 2010), however few studies report the importance of this predation or potential for the use of wasps in the biological control of agricultural pests (rabb & lawson, 1957; morimoto, 1961; prezoto & machado, 1999a,b; freitas et al., 2015). this research aimed to survey the species of social wasps that forage in kale (b. oleraceae var. acephala) crops, recording the influence of temperature and time of day on the foraging activity of these wasps and the temporal niche overlap of this community. with these data, we can identify which species have the greatest potential to be used in biological control programs for kale pests. material and methods this research was carried out at the federal institute of education, science and technology of minas gerais bambuí campus, from july to december 2015. the campus has a total area of 328 ha, being a human dominated but diverse landscape with a predominance of agricultural areas and buildings. a total of 175 ha are used for agricultural crops (corn, beans, sugarcane, orange, banana, coffee and vegetables) and pastures, and 34 ha are occupied by buildings, most of them are close to the cultivars. the campus has a high diversity of social wasps, with 29 species and 8 genera (jacques et al., 2015). twelve collections were carried out in a 5 x 10 m area of kale (b. oleracea var. acephala) (adapted from picanço et al., 2010) between the period from 09h00 to 15h00, in which the social wasps that were foraging on the crop were collected with an entomological net, placed in a bottle containing ethyl ether and preserved in 70% ethanol (adapted from souza et al., 2013). the time and temperature of the day at the time of collection of each wasp were recorded throughout the experiment to be correlated with the foraging activity. collection data were used to analyze the daily patterns of activity, diversity, dominance and temporal niche overlap of social wasp species. the collected individuals were identified with entomological keys (richards, 1978; carpenter, 2004), and the diversity of species calculated via shannon-wiener diversity (h’) and berger-parker dominance (dpb) indices, using the program past, v. 2.17c (hammer et al., 2005). temporal niche overlap for each possible pair of wasp species was determined using the schoener index (schoener, 1986). the kolmogorov-smirnov test for two samples was used to evaluate the interspecific differences between activity patterns for each pair of species (siegel, 1956). temporal niche overlap was calculated only for species of social wasps represented in the samples by more than eight specimens to minimize the effect of low abundance on the essay. the results of the regression analysis were performed for the foraging frequency of the wasps, taking into account the variables “air temperature” and “time of day” with p <0.05 (picanço et al., 2010). for all species, it was considered the linear regression model or the model with quadratic effects. the selection of the best model was performed based on the determination coefficient. the assumptions of the model were verified through graphical analysis and through the shapiro-wilk, durbin-watson and white tests to verify error normality, residual autocorrelation and variance heterogeneity, respectively. all analyzes were conducted in the 3.3.1 version of r software (r core team, 2017). for the durbin-watson test, the lmtest package was used (zeileis & hothorn, 2002). results and discussion three hundred and fifty-eight specimens belonging to six genera and 16 species of social wasps were collected gc jacques, tg pikart, vs santos, lo vicente, lcp silveira – social wasps (vespidae: polistinae) in kale crops314 in kale crops with a shannon-weiner diversity of h' = 1.84 (table 1). the species diversity was similar to visitation in cherry trees (eugenia uniflora linnaeus) (souza et al., 2013) and higher than other studies which analyzed only one species of plant (de souza et al., 2010; santos & presley, 2010; de souza et al., 2011; barbosa et al., 2014). social wasps flew over the crop and explored the kale plants, especially those with damaged leaves. this foraging behavior may be related to the presence of a. monuste orseis, b. brassicae and b. tabaci in kale plants. these pests belong to the orders of insects that are most captured by the social wasps (prezoto et al., 2005; bichara-filho et al., 2009; freitas et al., 2015). the presence of leaves damaged by herbivores in herbaceous plants attracted polybia occidentalis (olivier), polybia diguetana buysson and polybia fastidiosuscula saussure (saraiva et al., 2017). tobacco plants previously damaged by manduca sexta (l.) (lepidoptera: sphingidae) and trichoplusia ni (hübner) (lepidoptera: noctuidae) attracted more foraging individuals from mischocyttarus flavitarsis (de saussure) than non-damaged plants (cornelius, 1993). assumptions of error normality, autocorrelation and variance heterogeneity were met in all adjusted models, considering the graphical analysis and the appropriate usage of the tests (results not shown). the foraging behavior of the social wasps was related to the timeof day (p = 0.025) with the maximum number of wasps collected between 1000 and 1100 hours (fig 1). results similar to those found in cashew trees (anacardium occidentale l.) (anacardiaceae), with greater foraging between 0900 and 1200 hrs (santos & presley, 2010). the air temperature directly affects foraging behavior of social wasps (santos et al., 2009; de castro et al., 2011), with foraging occurring mainly during warmer times of day (picanço et al., 2010; barbosa et al., 2014), however air temperature was not an effective predictor of wasp activity (p = 0.2184). other factors not analyzed, such as the amount of light, wind speed and humidity, also affect foraging behavior (santos et al., 2009; de castro et al., 2011). there was high dominance (dbp = 0.41), with polybia ignobilis (haliday) (n= 147), protonectarina sylveirae (saussure) (n= 80) and protopolybia sedula (saussure) (n= 41) representing 75% of the total sampled individuals. the other most frequently collected species were polybia paulista (r. von ihering) (n= 22), p. occidentalis (n= 15), polistes satan (bequaert) (n= 12), brachygastra lecheguana (n= 11), polistes versicolor (olivier) (n= 9), mischocyttarus drewseni (saussure) (n= 8) and polybia sericea (olivier) (n= 4). mischocyttarus latior (fox), polistes simillimus (zikán) and polybia jurinei (saussure) (n= 2) and mischocyttarus labiatus (fabricius), polistes ferreri (saussure) and polybia fastidiosuscula (saussure) (n= 1) were uncommon and considered of accidental occurrences. species 09:00 10:00 11:00 12:00 13:00 14:00 15:00 total 1 brachygastra lecheguana (latreille, 1824) 3 4 1 2 1 11 2 mischocyttarus drewseni (saussure, 1857) 1 1 2 2 1 1 8 3 mischocyttarus labiatus (fabricius, 1804) 1 1 4 mischocyttarus latior (fox, 1898) 1 1 2 5 polistes ferreri (saussure, 1853) 1 1 6 polistes simillimus (zikán,1951) 1 1 2 7 polistes versicolor (olivier, 1971) 3 1 1 3 1 9 8 polistes satan (bequaert, 1940) 1 1 2 4 3 1 12 9 polybia fastidiosuscula (saussure, 1854) 1 1 10 polybia ignobilis (haliday, 1836) 28 29 22 23 21 19 5 147 11 polybia jurinei (saussure, 1854) 1 1 2 12 polybia occidentalis (olivier, 1971) 5 1 2 6 1 15 13 polybia paulista (r. von. ihering, 1896) 2 7 4 3 4 2 22 14 polybia sericea (olivier, 1971) 2 2 4 15 protonectarina sylveirae (saussure, 1854) 16 17 13 13 11 8 2 80 16 protopolybia sedula (saussure, 1854) 8 4 4 4 7 11 3 41 number of individuals 59 72 55 56 57 47 12 358 species richness (s’) 7 11 12 11 10 9 5 16 shannon-wiener index (h’) 1,35 1,78 1,85 1,81 1,88 1,58 1,59 1,84 berger-parker dominance (dpb) 0,48 0,40 0,40 0,41 0,36 0,41 0,38 0,41 table 1. number of individuals, species richness (s’), shannon-wiener diversity (h’) and berger-parker dominance (dpb) of the collected social wasps, per hour, in twelve collections made in kale (brassica oleracea var. acephala) at the federal institute of education, science and technology of minas gerais (ifmg), bambuí campus, minas gerais. sociobiology 65(2): 312-319 (june, 2018) 315 polybia ignobilis is the main predator of a. monuste orseis (picanço et al., 2010), and the predation of this wasp on the cabbage caterpillarswas recorded (fig 2). thus, the presence of this pest may have stimulated the greater presence of p. ignobilis in the crop. social wasp workers forage alone and opportunistically (jeanne et al., 1995; michelutti et al., 2017), being able to return to hunt in locations of previously successful hunts and feeding several times of the same species of prey (raveret -richter, 2000; bichara-filho et al., 2009). to optimize this form of foraging, signals can be exchanged among workers to facilitate the acquisition of resources for the colony (taylor et al., 2011). this behavior was reported in colonies of p. occidentalis (hrncir et al., 2007; schueller et al., 2010). species of the genus polybia (lepeletier) are also dominant in cashew, mango (mangifera indica l.) and cherry trees (santos & presley, 2010; souza et al., 2013; barbosa et al., 2014). wasps of this genus form large colonies, founded by a swarm composed of dozens of queens and hundreds of workers, which makes their local abundance greater than that of species whose colonies can be founded by one or a few wasps (barbosa et al., 2014). the highest number of p. ignobilis was found at 0930 h, using a quadratic model for time of day (fig 3a), different from that observed in the same crop in viçosa/mg, where the highest foraging occurred at 1330 h (picanço et al., 2010). through a linear model for the air temperature it was observed that the amount of p. ignobilis individuals tended to decrease with increasing temperature (fig 3b). in viçosa/ mg the result was similar, with the number of individuals increasing up to 29ºc, and decreasing after this temperature (picanço et al., 2010). time of day was a better predictor than temperature and accounted for 87% of the variation in mean p. ignobilis abundance (fig 3). higher temperatures occur after the period of greatest foraging for predation, 0930 h, thus we find a smaller number of individuals after this time of day. in the period of higher temperatures, the wasps are concentrated in the collection of water to cool the colony (akre, 1982; montefusco et al., 2017). fig 1. effect of time of day on average number of social wasps foraging in kale (brassica oleraceae var. acephala) from july to november 2016, in bambuí, mg. time of day w as ps fig 2. social wasp polybia ignobilis (hymenoptera: vespidae) preying on ascia monuste orseis (lepidoptera: pieridae) on a kale plant (brassica oleracea var. acephala). time of day air temperature (ºc) m ea n ab un da nc e of p ol yb ia ig no bi lis m ea n ab un da nc e of p ol yb ia ig no bi lis fig 3. average number of wasps of polybia ignobilis (hymenoptera: vespidae) foraging in kale plants (brassica oleracea var. acephala) as a function of: a -timeof day and; b air temperature, from july to november 2016 in bambuí, mg. the quadratic model for time of day explained 96% of the variation in mean p. sylveirae captures, with peak abundance observed early in the day (0900 hr) (fig 4). this time is different from the time found for all species, which was between 1000 and 1100hrs. temperature did not affect p. sylveirae abundance. the presence of a. monuste orseis gc jacques, tg pikart, vs santos, lo vicente, lcp silveira – social wasps (vespidae: polistinae) in kale crops316 may attract p. sylveirae, as this is a common prey species of p. sylveirae (bueno & souza, 1993). in addition, this species preys on hemiptera such as aleurothrixus floccous (maskell) (hemiptera: aleyrodidae) (souza & zanuncio, 2012), probably being attracted by the presence of b. brassicae and b. tabaci. on a. floccous (souza & zanuncio, 2012) and probably is also attracted by the presence of b. brassicae and b. tabaci. the temporal niche overlap between pairs of species of social wasps varied between 0.310.96 (schoener index) (table 2), being smaller between b. lecheguana and p. versicolor and higher between p. ignobilis and p. sylveirae. in general, the activity overlap was relatively high, being greater than 50% in 31 of the 36 pairs, and there was no significant difference based on kolmogorov-smirnov 2-sample tests. this high value of temporal niche overlap was also found in a. occidentale and e. uniflora (santos & presley, 2010; souza et al., 2013), suggesting a trend of coexistence among species of this group. the agricultural features of the campus, with the presence of many crops, and consequently many herbivores, may have led to a decrease in interspecific competition allowing greater coexistence of wasps in the crop. the division of resources by guild members and the resulting competitive structure can only be seen if they are maintained over time by competition for limiting resources (pianka, 1980). moreover, the generalist tendencies of social wasps allows them to have low dependence on particular food resources (santos et al., 2007). this dietary flexibility likely facilitates coexistence by reducing interspecific competition. m ea n ab un da nc e of p ro to ne ct ar in a sy lv ei ra e time of day fig 4. effect of time of day onaverage number of wasps of protonectarina sylveirae (hymenoptera: vespidae) species foraging in kale plants (brassica oleracea var. acephala) from july to november 2016 in bambuí, mg. none of the statistic models fit the data considering the time of day for p. sedula. in contrast, observations of colonies of this species indicate that peak foraging activity occurs between 1030 and 1430 (detoni et al., 2015). for the air temperature, the quadratic regression model was selected, in which the minimum number of wasps was obtained around 29º c (fig 5). the climatic variables possibly have a low effect over the foraging rhythm of this wasp (detoni et al., 2015). the presence of an envelope that protects p. sedula nests can generate a certain level of homogenization of the internal environment in terms of temperature and humidity, increasing the importance of internal colony stimuli when it comes to foraging outflows (detoni et al., 2015). moreover, this research recorded the foraging behavior only in kale, and this species may be foraging in another plant species. this species preys m ea n ab un da nc e of p ro to po by bi a se du la air temperature (ºc) fig 5. effect of air temperature on average number of wasps of protopobybia sedula (hymenoptera: vespidae) species foraging in kale plants (brassica oleraceae var. acephala) from july to november 2016 in bambuí, mg. br. le. mi. dr. po. ve. po. sa. po. ig. po. oc. po. pa. pr. sy. pr. se. brachygastra lecheguana ns ns ns ns ns ns ns ns mischocyttarus drewseni 0,61 ns ns ns ns ns ns ns polistes versicolor 0,31 0,46 ns ns ns ns ns ns polistes satan 0,62 0,75 0,47 ns ns ns ns ns polybia ignobilis 0,77 0,71 0,54 0,70 ns ns ns ns polybia occidentalis 0,72 0,58 0,36 0,60 0,61 ns ns ns polybia paulista 0,82 0,63 0,49 0,73 0,81 0,77 ns ns protonectarina sylveirae 0,79 0,68 0,51 0,71 0,96 0,62 0,83 ns protopolybia sedula 0,64 0,69 0,65 0,62 0,79 0,50 0,65 0,75 table 2. temporal niche overlap (schoener index) among pairs of species of social wasp (species with more than eight individuals) collected in kale (brassica oleracea var. acephala) crops at the federal institute of education, science and technology of minas gerais (ifmg), bambuí campus, minas gerais. significance (p≤0.05) for the kolmogorov-smirnov test for two samples used to evaluate differences between the patterns of temporal activity among pairs of species of social wasps is indicated above the diagonal. sociobiology 65(2): 312-319 (june, 2018) 317 we recorded 4 species, m. labiatus, p. ferreri, p. fastidiosuscula and p. sylveirae that had not been collected previously at the ifmg – bambuí campus according to a diversity survey of social wasps previously performed by jacques et al. 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(2002). diagnostic checking in regression relationships. r news 2(3), 7-10. retrived from: https://cran.r-project.org/doc/rnews/ open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.1620sociobiology 65(2): 225-231 (june, 2018) wind speed affects pollination success in blackberries introduction ecosystem services represent the goods and services derived from the functioning of ecosystems and utilized by humans (constanza et al., 1997). pollination is a clear example of an ecosystem service, with the majority of all animalmediated pollination provided by bees (roubik, 1995; klein et al., 2007). it is estimated that 87.5% of the angiosperm species in the world depend on animal-mediated pollination (ollerton et al., 2011). similarly, 87 of the leading global crops are dependent on animal pollination (klein et al., 2007), with pollinators contributing about 9.5% of the total value of the production of human food worldwide (gallai et al., 2009). pollination is therefore important for both biodiversity abstract pollination of wild plants and agricultural crops is a vitally important ecosystem service. many landscape and environmental factors influence the pollination success of crops, including distance from natural habitat, wind speed, and solar radiation. although there is a general consensus that increasing distance from forest decreases pollination success, few studies have examined the influence of specific environmental factors. in this study, we examined which environmental factors influence the pollination success of blackberries (rubus glaucus). we measured the number of fruitlets per berry, a proxy for pollination success, as well as the weight and sweetness of each berry. our results indicate that number of fruitlets is positively correlated with wind speed, but number of unripe red berries per bush is negatively correlated with wind speed. in addition, sweetness increased with increasing numbers of red berries per bush but was lower when flowers and berries were present, though this result should be considered with caution due to methodological limitations. our findings suggest that a little studied environmental factor, wind, has a large impact on the number of fruitlets in blackberries. although our findings should be confirmed in other locations to draw broader conclusions, they suggest that producers should consider the effect of wind on blackberry yield to optimize blackberry production. sociobiology an international journal on social insects am young1, pa gómez-ruiz2, ja peña3, h uno4,5, r jaffe6 article history edited by evandro nascimento silva, uefs, brazil received 29 march 2017 initial acceptance 02 february 2018 final acceptance 17 march 2018 publication date 09 july 2018 keywords fruit-set, landscape, agroecology, sweetness. corresponding author allison young department of integrative biology michigan state university 288 farm lane, natural science building east lansing, mi, usa. e-mail: younga46@msu.edu and human food security (hadley & betts, 2012). however, pollination is currently threatened by anthropogenic changes such as deforestation and agricultural intensification (kremen et al., 2002; chacoffet al., 2008; brown & paxton, 2009; hadley & betts, 2012). because of this threat, understanding the factors that influence the pollination success of crops, mediated by bees, is critical to protect global biodiversity and food security (klein et al., 2007; chacoff et al., 2008; hadley & betts, 2012). a number of recent studies have indicated that largescale landscape changes have profound impacts on pollinator communities and on their ability to successfully pollinate agricultural crops (hadley & betts, 2012; boreux et al., 2013; kennedy et al., 2013; garibaldi et al., 2016). because bees return to a fixed nest site after foraging, their foraging (and 1 department of integrative biology, michigan state university, mi, usa 2 conacyt-universidad autónoma del carmen (unacar), ciudad del carmen, campeche, méxico" 3 department of biology, university of puerto rico, san juan, pr 4 department of integrative biology, university of california berkeley, ca, usa 5 center for ecological research, kyoto university, kyoto, japan 6 instituto tecnológico vale, belém-pa, brazil research article bees am young, p gómez-ruiz, ja peña, h uno, r jaffe – blackberry pollination and the environment226 therefore pollinating) range is limited by their flight capacity (ricketts et al., 2006). factors that impact the flight ranges of bees therefore have large impacts on pollination success. distance from natural or semi-natural habitat is one of the most important factors (ricketts et al., 2006; ricketts et al., 2008), as both the abundance and diversity of bee pollinators decreases with increasing distance from forest (de marco & coelho, 2004; blanche et al., 2006; carvalheiro et al., 2010), influencing pollination success in crops such as coffee, mango, and macadamia (blanche et al., 2006; vergara & badano, 2009; carvalheiro et al., 2010). although many studies have explored the relationship between distance to forest edge and bee abundance and diversity, few have examined how other environmental factors influence pollination success and crop yields (but see vergara & badano, 2009; krishnan et al., 2012). wind speed, for example, could also have large impacts on the ability of bees to successfully pollinate crops. for instance, wind speed influences the speed at which honeybees (apis mellifera) fly (wenner, 1963). a study on stingless bees showed that flight activity is reduced or even completely halted in high wind (kleinert-giovannini & imperatriz-fonseca, 1986). thus, wind speed can potentially impact the amount of foraging and the provision of pollination services. ambient temperature and solar radiation have also been shown to impact the foraging decisions of honey bees (burrill & dietz, 1981). higher temperatures increased the number of foraging trips these bees made, while high levels of solar radiation strongly reduced the number of trips (burrill & dietz, 1981). it is therefore likely that the interaction of many environmental factors, and not only distance from natural habitat, influence pollination success. here we investigated how different environmental factors affect the pollination success of blackberries (rubus glaucus) in costa rica by examining fruit production. blackberries are a common crop in many latin american countries, including costa rica, where they are produced for both local markets and for exportation to countries such as the united states, holland, canada, the united kingdom, and nicaragua (castro & cerdas, 2005). the blackberry industry in costa rica has grown 55% since 1995 and is expected to continue to increase (strik et al., 2007). increased agriculture, including that of blackberries, will likely be one of the biggest threats to tropical forests, ecosystems, and biodiversity this century (laurance et al., 2014). this threat is exacerbated by the inefficiency of tropical agriculture, with most farmers producing significantly less than the full potential of their land for many crops (tilman et al., 2002; laurance et al., 2014). hence, understanding how landscape factors influence pollination of crops such as blackberries is necessary to improve crop yields and help design more sustainable agricultural practices, which maximize production while reducing the need to deforest new land (laurance et al., 2014). blackberries are an ideal model system in which to quantify pollination success because they produce a compound flower and fruit (fig 1), which contains many pistils that must be individually pollinated to produce a fruitlet (the small fruits that make up compound berries (cane, 2005)). because each fruitlet must be individually pollinated, the number of fruitlets on a berry can be used as a direct proxy for pollination success. although fruits such as blackberries are capable of limited self-pollination, the flower structure prevents complete selfpollination because only the outermost stigmata can be reached by the stamen, which leads to small, malformed berries made up of few fruitlets (cane, 2005). adequate bee pollination is therefore vital to blackberry production. we hypothesized that blackberry fruitlet set is strongly dependent on environmental and landscape factors that influence bee foraging ability, and predicted that distance from the forest edge and wind speed would have the strongest effects on pollination success compared to other factors such as slope, tree height, canopy cover, and flower production. specifically, we predicted that fruit-set would decrease with both wind speed and distance to forest edge. to our knowledge, this study is the first to examine the influence of environmental factors on blackberry pollination success. our results provide preliminary management recommendations to producers regarding how to optimize pollination of blackberry crops and maximize yields. materials and methods this study was conducted in a blackberry field (about 10 ha) owned by the cuericí biological station in the orosi district of cartago, costa rica from july 7 – 8, 2016. we selected 62 blackberry bushes that had more than 5 ripe (hereafter referred to as “black”) blackberries on the bush, and were at least 20 meters apart from each other (fig 2). ideally bushes with more than 5 black blackberries would have been used in this study, but black berries are harvested weekly at cuericí biological station. this weekly harvesting prevented us from measuring bushes with large numbers of intact ripe berries. distance was estimated using gps waypoints (gps map 60csx garmin). wind speed was measured once for each blackberry bush over a period of 10 minutes to account for fig 1. structure of blackberry flowers and fruits. each blackberry flower has multiple stigmata that are individually pollinated (see leftmost flower). the number of fruitlets in each berry depends on the number of stigmata successfully pollinated (photograph by pilar gómez). sociobiology 65(2): 225-231 (june, 2018) 227 gusts of wind. for each bush, we recorded several landscape predictor variables summarized in table 1. in addition, we recorded the number of fruitlets per berry and berry weight as proxies of pollination success of the blackberry bush for a minimum of 3 black blackberries per bush. weight of each black berry was first recorded, and then number of fruitlets per berry was counted. in addition, we measured sweetness of each sampled black blackberry through a taste test. a score for sweetness was assigned to each berry independently (see table 1). because black berries were picked weekly, we also recorded the number of unripe (hereafter referred to as “red”) berries per bush to compare fruit set across bushes (table 1). fig 2. distribution of sampled blackberry bushes surrounding cuericí biological station, costa rica (n = 63) and location of cuericí on a map of costa rica (inset). bushes were located more than 20 meters apart (~10 ha area). we analyzed the data using linear models and linear mixed models, using the "lme4" r (3.1.0) package. for each model, we performed model selection using the drop1 function. we modeled the number of fruitlets per berry, weight of each berry, number of red berries per bush, and sweetness of black berries as response variables in 4 separate models (table 2). for number of fruitlets, weight, and sweetness we included all environmental variables and bush characteristics as predictor variables in initial models, and bush identity as a random effect to account for the fact that some samples came from the same bush. for the number of red berries per bush we included only environmental (not landscape) variables. we used four tasters to assess the sweetness of the berries, and included taster id as a random factor in the sweetness model to account for differences in taste preference. we also fit poisson generalized linear mixed models (glmm) for the continuous response variables, but excluded these models because they failed to converge. results we found that the number of fruitlets per berry was best explained by wind speed (table 2), with the number of fruitlets positively correlated with wind speed (estimate = 4.3, variables data collection method predictor distance to forest edge rangefinder canopy cover densitometer slope inclinometer wind speed measured directly above top of bush using paper anemometer, categorized 1: no wind, 2: flagging tape moves, 3: anemometer moves slowly, 4: anemometer moves quickly presence of other flowering plants visual inspection of surrounding 1 meter, recorded presence or absence presence of ferns visual inspection of surrounding 1 meter, recorded presence or absence number of black blackberries visual inspection of bush presence of flowers on blackberry bush visual inspection of bush height of sampled berry measuring tape height of bush measuring tape distance to nearest blackberry bush measuring tape distance to nearest blackberry bush with fruit measuring tape response number of red blackberries counted total number of unripe (red) berries per bush number of fruitlets per berry individually counted fruitlets of 3-5 ripe (black) berries per bush berry mass weighed 3-5 ripe (black) berries per bush berry sweetness individually ranked 3-5 ripe (black) berries per bush from 1-5, where 5 is extremely sweet and 1 is extremely sour table 1. description of measured variables and their respective data collection methods. p= 0.0312, df = 59.78; fig 3a). in contrast, the number of red berries per bush was best explained by slope and wind speed (table 2), so that the number of red berries was negatively correlated with both wind speed (estimate = -12.9, p = 0.031, df= 59; fig 3b) and slope (estimate = -1.0, p= 0.036, df= 59). black berry sweetness was best explained by the number of red berries per bush and the presence of flowers on the bush (table 2), such that black berries were sweeter on bushes with more red berries (estimate = 0.0044, p = 0.043, df= 194.06; fig 4a) and in the absence of flowers (estimate = -0.64, p= 0.00016, df= 181.64; fig 4b). berry weight was best explained by a null model containing no predictor variables. however, berry weight was strongly correlated to number of fruitlets per berry (estimate = 0.021, p = < 2e-16, df = 196). in addition, wind speed was found to be correlated with slope (estimate = 0.013, p= 0.0251, df = 196) and distance to forest edge (estimate = 0.010, p= 1.04e-8, df = 196). am young, p gómez-ruiz, ja peña, h uno, r jaffe – blackberry pollination and the environment228 discussion our study aimed to determine how environmental factors influence fruit production and sweetness in blackberries (r. glaucus). we found that the pollination success of blackberries, as indicated by the number of fruitlets per berry, was positively related with wind speed. conversely, the number of red berries per bush was negatively associated with wind speed. also, blackberries were sweeter on bushes with more red berries and no flowers. we found no direct evidence linking blackberry weight to any of the assessed environmental variables, but did find a strong correlation between number of fruitlets per berry and berry weight. we predicted that wind speed would reduce fruitlet set, but actually found the opposite effect. a positive effect of wind speed on the number of fruitlets could be explained by the pollination syndrome of r. glaucus, because some species of rubus can self-pollinate with the assistance of wind (jennings, 1988; as reviewed by cane, 2005). a previous study found that increased visitation by pollinators can decrease fruitlet set in raspberries (saez et al., 2014), a crop with similar reproductive biology. if this also applies to blackberries, the positive effect of wind on fruitlet number could indicate that windy conditions are reducing flower visitation rates. windy conditions could also exclude the smallest pollinators, which usually have the shortest foraging ranges (greenleaf et al., 2007), and/or increase the amount of time pollinators spend on flowers (brown & mcneil, 2009). although we did not collect flower visitors, we observed that honeybees and bumblebees were the most common bees visiting blackberry flowers in our study site (smaller native bee species are also known to occur in the region (jarau & barth, 2008)). our results could suggest that windy conditions are favoring pollination by larger bees (i.e. honeybees and bumblebees), but future studies are needed to experimentally test the effect of wind on bee-mediated blackberry pollination. one major limitation of our study was that we only measured wind speed once for each blackberry bush, at the time of our measurements of berry production not at flower anthesis. wind speed is variable, so it is possible that wind speed might have been different during flowering than at the time of data collection. however, wind was significantly correlated with slope and distance to forest edge in this experiment, suggesting that the terrain plays a large part in determining wind speed in this environment regardless of time of day or season. wind speed can therefore be viewed as a measure of how sheltered blackberry bushes are. if some bushes are more sheltered than others, that would suggest there might be differences in pollinator visitation due to landscape differences, mediated through their effects on wind speed. that a significant relationship was found between wind and fruitlet set despite the confounding factors of terrain and time of year, highlights the importance of the wind effect found in this study. we found no significant effect of distance to forest on our response variables, similar to the findings of chacoff et al. (2008). these results contradict those of other studies, which found that fruit yields decreased with increasing distance from forest (de marco & coelho, 2004; blanche et al., 2006; carvalheiro et al., 2010). we posit that the lack of an effect is probably due to the limited distance gradient we measured in response variable number of observations predictor variable(s) chi-square value p-value number of fruitlets per berry 198 wind 4.8385 0.02783 berry weight 198 none n/a n/a berry sweetness 198 198 flowers number of red berries 14.0627 4.4381 0.0001768 0.0351444 number of red berries 198 slope wind speed -4.168 -3.998 4.61e-05 9.06e-05 table 2. likelihood ratio test results for final models chosen for each of the four tested variables: number of fruitlets per berry, berry weight, black berry sweetness, and number of red berries per bush. the null model was selected as the best model for berry weight based on the aic values. fig 4. effects of (a) the number of red berries and (b) the presence of flowers (where 'y' means present and 'n' means absent') on the sweetness of the berries at a blackberry farm in cuericí, costa rica. fig 3. effect of wind speed on (a) the number of fruitlets per berry and (b) the number of red berries per bush at a blackberry farm in cuericí, costa rica. sociobiology 65(2): 225-231 (june, 2018) 229 this study. we only sampled bushes scattered across a distance gradient of 2-120 m to the forest edge, which is a fraction of a bee’s flight range. honey bees, for example, usually forage between 1-2 km from their nest (steffan-dewenter & kuhn, 2003; greenleaf et al., 2007). even smaller bees, such as stingless bees, have flight ranges between 540 m and 2,000 m depending on their body size (araújo et al., 2004). all of the blackberry bushes we measured could therefore be easily reached by native bees. we found no effect of the environmental variables measured on blackberry weight, contradicting previous studies that found factors such as number of fruit per bush (link, 2000; whiting et al., 2005) and wind exposure (dry et al., 1988) influence fruit weight. however, a strong positive correlation between berry weight and number of fruitlets per berry was found, suggesting that environmental factors have an indirect effect on berry weight that is mediated through number of fruitlets per berry. a positive correlation between fruitlet number and berry weight has been found in other cultivars of blackberries (strik et al., 1996), supporting our finding. in our study, we found that blackberries were sweeter in bushes that had more red berries, likely due to high resource investment in fruits on a given bush. we also found that blackberries were less sweet on bushes with flowers. these results should be considered with caution, though, as sweetness is usually measured using ˚brix, which gives the sugar content of an aqueous solution (as seen in jayasena & cameron, 2008). measuring sweetness based on human subjects will likely be biased due to differences in taste receptors and preference. however, by taking into account taster id as a random factor in our model some of that bias was removed, providing support to our findings. our results, if true considering the limited methods, could be explained as the consequence of a trade-off between resources investment in the production of berries and flowers on the bush at a given point in time. investments in a particular type of structure can limit resources investment in other functions and structures in plants (bazzaz et al., 1987). alternatively, it is possible that bushes without flowers began producing fruits earlier in the year, and so had fruits with a higher sugar content; sugar is known to increase with ripeness in blackberries (tosen et al., 2008). however, sugar levels in a similar compound fruit, raspberries, are not significantly different between harvested fruit that is 50% mature and that which is 100% mature (wang et al., 2009). as all of the berries tested in this experiment were visibly mature and ripe, this issue likely did not play a large role in our results. our results suggest that wind speed has implications for the blackberry farming industry. with crops such as blackberries, which have long been a favorite wild fruit, common in several countries, and picked for commercial use (strik et al., 2007), knowledge about the environmental factors that affect yield could improve fruit production. as of 2005, the blackberry industry has grown 55% since 1995 (strik et al., 2007) and continues to grow, making it an important crop for many costa rican farmers. for local production in small costa rican farms, improvements to the field conditions related to wind speed could enhance blackberry production. because factors such as slope and wind speed, as likely indicators of how sheltered bushes are, impact the number of fruitlets per berry, the number of berries produced, and (indirectly) the weight of the berries, farmers could determine the best market for their blackberry yield based on the structure of their fields, or possibly even manipulate how sheltered bushes are to produce more berries. though this work is too preliminary to produce extensive recommendations, it can serve as a starting point for further research into how environmental factors impact blackberry production. supplementary information may be found online: http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1620/0 appendix i. raw data doi: 10.13102/sociobiology.v65i2.1620.s1523 appendix ii. r script used for analysis doi: 10.13102/sociobiology.v65i2.1620.s1524 author contributions all authors were involved in designing and performing the study. all authors except r. j. wrote an initial draft of the manuscript, which a. m. y. then revised and finalized. r. j. assisted with data analyses and writing of the manuscript. acknowledgements we would like to thank jenny stynoski, patricia salerno, and victor acosta chaves for their support in the field and constructive criticism that helped improve this manuscript. we thank don carlos and don alberto for kindly letting us study the blackberries on their property, and for providing accommodations at cuericí biological station, orosi district, costa rica. we also thank the anonymous reviewers who reviewed this manuscript. this study was part of a research project undertaken during the tropical biology: an ecological approach course from the organization for tropical studies. rodolfo jaffé thanks the organization of tropical studies for a travel grant. references araújo, e. d., costa, m., chaud-netto, j., & fowler, h. g. 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(2005). rootstock and training system affect sweet cherry growth, yield, and fruit quality. hortscience, 40: 582-586. https://doi.org/10.17660/actahortic.2008.777.31 http://dx.doi.org/10.1080/00218839.1963.11100053 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 125-127 (2013) karyotype description of cephalotrigona femorata smith (hymenoptera: apidae) and the c-banding pattern as a specific marker for cephalotrigona rv miranda1, a fernandes2, dm lopes1 cytogenetic analyses allow for morphological and quantitative characterization of the chromosomes of a determined species, which may contribute to the understanding of phylogeny and improvements in the taxonomy of some groups of hymenoptera (rocha et al., 2003). a very peculiar taxon within hymenoptera is the tribe meliponini, the stingless bees. these bees play an important role in economic and ecological processes of tropical regions because they are important pollinators of native plants (heard, 1999; michener, 2000). in brazil two species of the genus cephalotrigona schwarz, 1940 are found among the five species described taxonomically, cephalotrigona capitata and c. femorata (moure, 2011). cephalotrigona capitata is the only species of the genus for which the karyotype has already been described, and 2n = 34 chromosomes were observed (rocha et al., 2003). the present study evaluates the species c. femorata in order to characterize it cytogenetically and thus to obtain more knowledge on stingless bees. abstract cephalotrigona femorata (smith, 1854) was submitted to cytogenetic techniques to study and describe its karyotype. conventional staining allowed the counting (2n=34) and observation of chromosome morphology. the amount and distribution of heterochromatin in this species was different from cephalotrigona capitata (smith, 1854), another species of the genus already analyzed. our results indicate that heterochromatin is a potential marker for the genus, at least for the species found in brazil. this region was marked by dapi, revealing a high content of a:t. the cma 3 marked two pairs, and it seems to be polymorphic in one pair. sociobiology an international journal on social insects 1 universidade federal de viçosa, viçosa, minas gerais, brazil 2 universidade do estado de mato grosso, tangará da serra, mato grosso, brazil. article history edited by: cândida m. l. aguiar, uefs brazil received 31 october 2012 initial acceptance 07 december 2012 final acceptance 14 december 2012 keywords hymenoptera, stingless bees, cytogenetics, heterochromatin. corresponding author denilce meneses lopes universidade federal de viçosa departamento de biologia geral av. peter henry rolfs, s/n. campus ufv, 36570-000, viçosa, mg, brazil. e-mail: denilce.lopes@ufv.br post-defecant larvae of four colonies of c. femorata, were obtained in the municipality of urbano santos, maranhão, brazil (3°12’29”s; 43°24’18”w), as part of a project to rescue wildlife in the area of deforestation. larvae (n= 40) were collected and processed according to the method described by imai et al. (1988) to acquire the mitotic metaphase chromosomes. conventional staining was performed using diluted giemsa in sörensen buffer (0.006 m, ph 6.8) at the concentration of 4% for 20 minutes. for c-banding the bsg (barium hydroxide/saline/giemsa) method was used according to sumner (1972). fluorochrome staining (cma3/da/dapi) was performed according to the protocol proposed by schweizer (1980). ten slides from each hive were analyzed along with an average of 10 metaphases per slide. the material was observed under an olympus bx60 microscope and images were used for assembly of the karyotypes utilizing the image analysis program image-pro plustm (version 6.3, media cybernetics®, 2009). karyoshort note rv miranda, a fernandes, dm lopes karyotype description of cephalotrigona femorata126 types were organized as reported by imai (1991), taking into consideration heterochromatin distribution. the conventional staining technique showed that c. femorata presents a diploid number of 2n = 34 in females (fig. 1a and b). this data is similar to that of the other species of the genus, c. capitata. the c-band technique permitted observation of heterochromatic arms on all chromosomes (fig. 1b). often these portions represent regions larger than the euchromatic arms. two pairs also showed heterochromatin in the pericentromeric region (fig. 1b). the observed heterochromatin pattern differed signifi cantly from c. capitata where only 8 of the 17 pairs had a heterochromatin arm. according to the classifi cation proposed by imai (1991), c. femorata had a karyotypic formula of 2k=30am +4amc while for c. capitata this was 2k=18a+16 am, this variation can therefore be used to distinguish the two species since they are morphologically similar. the heterochromatic regions were positively stained by dapi revealing a high content of a:t in heterochromatin similar to other meliponini (fig. 1c). the fl uorochrome cma3 showed four markings. in one of the pairs this staining showed a large polymorphism of size (fig. 1c) in one homolog. these polymorphisms are common in bees and have been well characterized for other stingless bee species such as oxytrigona fl aveola (krinski et al., 2010). as previously observed in other stingless bees, these gc-rich regions may be related to the nucleolus organizer regions (brito et al., 1997, maffei et al., 2001; rocha, 2002; brito-ribon et al., 2005; lopes et al., 2011). thus, although the number of chromosomes in general is constant in the genus, other characteristics such as the amount and distribution of heterochromatin vary among species and may be used as an important tool that may aid in the taxonomy and conservation of this species. acknowledgments the authors thank the following research funding agencies: conselho nacional de desenvolvimento científi co e tecnológico (cnpq), fundações de amparo à pesquisa de minas gerais (fapemig) and mato grosso (fapemat), and fundação arthur bernardes (funarbe) for their fi nancial support to the research. references brito, r.m., costa, m.a. & pompolo, s.g. (1997). characterization and distribution of supernumerary chromosomes in 23 colonies of partamona helleri (hymenoptera, apidae, meliponinae). braz. j. genet. 20: 185-188. brito-ribon, r.m., pompolo, s.g., martins, m.f., magalhães, m.f.m., barros, e.g. & sakamoto-hojo, e.t. (2005). cytogenetic characterization of two partamona species (hymenoptera, apinae, meliponini) by fluorochrome staining and localization of 18s rdna clusters by fish. cytologia 70: 373-380. heard, t.a. (1999). the role of stingless bees in crop pollination. annu. rev. entomol. 44,183-206. imai, h.t. (1991). mutability of constitutive heterocromatin (c-band) during eukaryotic chromosomal evolution and their cytological meaning. jap. j. genet., 66: 635-661. imai, h.t., taylor, r.w., crosland, m.w.j. & crozier, r.h. (1988). modes of spontaneous evolution in ants with reference to the minimum interaction hypothesis. jap. j. genet. 63:159-185. krinski, d., fernandes, a., rocha, m.p. & pompolo, s.g. (2010). karyotypic description of the stingless bee oxytrigona cf. fl aveola (hymenoptera, apidae, meliponina) of a colony from tangará da serra, mato grosso state, brazil. genet. and mol. biol. 33: 494-498. http://dx.doi.org/10.1590/ s1415-47572010000300020 lopes, d.m., fernandes, a., praça-pontes, m.m., werneck, h.a., resende, h.r. & campos, l.a.o. (2011). cytogenetics of three melipona species (hymenoptera, apidae, meliponini). sociobiology 58: 185-194. maffei, e.m., pompolo, s.g., silva-junior, j.c. & caixeiro, a.p. (2001). silver staining of nucleolar organizer regions figure 1. karyotype of a cephalotrigona femorata female. a. conventional staining. b: c-banding. c: fluorochromes dapi and cma3. polymorphic pair indicated by arrow. scale bar: 5 µm. sociobiology 60(1): 125-127 (2013) 127 (nor) in some species of hymenoptera (bees and parasitic wasp) and coleoptera (lady-beetle). cytobios 104: 119-125. michener, c.d. (2000).the bees of the world. johns hopkins university press, baltimore, 913 pp. moure, j.s., urban, d. & melo g.a.r. (2011). catalogue of bees (hymenoptera, apoidea) in the neotropical region online version. available from http://www.moure.cria.org.br/ catalogue. rocha, m.p. (2002). análises citogenéticas em abelhas do gênero melipona (hymenoptera, meliponinae) ds. universidade estadual de campinas, campinas, são paulo, 86pp. rocha, m.p., pompolo, s.g. & campos, l.a.o. (2003). citogenética da tribo meliponini (hymenoptera, apidae), pp 311-320. in g.a.r. melo & i. alves dos santos (eds.) apoidea neotropica:homenagem aos 90 anos de jesus santiago moure., unesc, criciúma. schweizer, d. (1980). simultaneous fluorescent staining of r bands and specific heterochromatic regions (da-dapi bands) in human chromosomes. cytogen. cell genet. 27: 190-193. sumner, a.t. (1972). a simple technique for demonstrating centromeric heterochromatin. exp. cell res. 75: 304-306. doi: 10.13102/sociobiology.v61i1.68-77sociobiology 61(1): 68-77 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 does forest phisiognomy affect the structure of orchid bee (hymenoptera, apidae, euglossini) communities? a study in the atlantic forest of rio de janeiro state, brazil wm aguiar¹, gar melo², mc gaglianone³ introduction orchid bee communities have been widely sampled in different neotropical ecosystems in recent decades, including the atlantic forest (tonhasca et al., 2002a; sofia & suzuki, 2004; nemésio & silveira, 2006a; ramalho et al., 2009; mattozo et al., 2011; silveira et al., 2011; aguiar & gaglianone, 2012; ramalho et al., 2013), the amazonian forest (e.g., 1985; powell & powell, 1987; storck-tonon et al., 2009; abrahamczyk et al., 2011), central american forests (e.g., ackerman, 1983; brosi 2009), the savannas of central brazil (cerrado) (souza et al., 2005; alvarenga et al., 2007; faria & silveira, 2011), and the dry forest of northeastern brazil (caatinga) (souza et al., 2005; alvarenga et al., 2007; andrade-silva et al., 2012). these studies have shown structural differences in bee communities from distinct biogeographical regions, particularly in relation to composition, richness, patterns of dominant species and numbers of endemic species. the differences have usually been attributed mainly to historical factors, although regional differences in community structure at less encompassing spaabstract we describe and discuss the composition, abundance and diversity of euglossine in three vegetation types of the atlantic forest (lowland seasonal semideciduous, submontane seasonal and dense montane ombrophilous forest) in rio de janeiro state, brazil, compare them to previous studies in the region and investigate the importance of the vegetation types, climatic and geomorphological factors on the species composition. male euglossine bees attracted by fragrances were sampled monthly from august/2008 to july/2009 using entomological nets and traps. euglossine bee communities exhibited differences in their species composition and abundance along the year and in the vegetation types. the precipitation, altitude and vegetation types demonstrated a significant influence on the ordination of the euglossine communities. our study found differences in the composition of euglossine bee communities as well as in their patterns of abundance and dominance among different vegetation formations, stressing the importance of the conservation of landscape mosaics in the region. sociobiology an international journal on social insects 1 universidade estadual de feira de santana, departamento de ciências exatas, feira de santana, ba, brazil 2 universidade federal do paraná, departamento de zoologia, curitiba, pr, brazil 3 universidade estadual do norte fluminense , laboratório de ciências ambientais, campos dos goytacazes, rj, brazil article history edited by celso f martins, ufpb brazil received 14 august 2013 initial acceptance 23 october 2013 final acceptance 26 november 2013 keywords biodiversity, geographic transition conservation, neotropics, pollinators corresponding author willian moura de aguiar universidade estadual de feira de santana departamento de ciências exatas av. transnordestina s/n novo horizonte feira de santana, ba, brazil 44036-900. email: wmag26@yahoo.com.br tial scales can be analyzed based on current ecological characteristics related to climatic, geomorphological, and/or vegetational parameters (silveira et al., 2002; sydney et al., 2010). recently, nemésio and vasconcelos (2013) evaluated the beta diversity of euglossina in the atlantic forest and noted that climate variations explain twice as much variation in the species data than the spatial variation in species distribution. nevertheless, part of the observed latitudinal changes in community composition appears to be explained by a concomitant seasonal gradient of precipitation. similarly, low temperatures and a seasonal rainfall may help explain the relative specificity of the fauna of some of the most western atlantic forest. this tropical forest extends along almost the entire eastern coast of brazil and it is composed of a mosaic of rainforest, “restinga” (coastal vegetation on quaternary sandy soils) and mangrove swamp ecosystems (galindo-leal & câmara, 2003). the wide latitudinal extension and significant longitudinal width of this biome, associated with altitudinal variations throughout the region, constitute important factors that define different vegetation types (galindo-leal & câmara, research article bees sociobiology 61(1): 68-77 (march, 2014) 69 2003; ribeiro et al., 2009) and likewise favor the high diversity and endemism of animals found there (myers et al., 2000). comparative studies of orchid-bee communities between different vegetational formations have been carried out, and, in the atlantic forest, include those by nemésio and silveira (2007), sydney et al. (2010), mattozo et al. (2011) and nemésio and vasconcelos (2013). these authors reported differences between euglossine bee species composition in dense ombrophilous and semideciduous forests, along the northern and the southern coast of brazil. additionally, nemésio (2008) demonstrated the strong influence of altitude on spatial distributions and species composition of communities living in the same geographical region. dense ombrophilous forest areas within the atlantic forest biome generally occur from the scarps of the coastal mountains, which are directly influenced by masses of humid air moving in from the sea, to the coastline inland. forested regions in the northern part of rio de janeiro state, however, exhibit somewhat unusual features due to the occurrence of the “campos dos goytacazes gap”, which designates a geographic transition between the serra do mar forest domain (which extends from north of paraná to rio de janeiro) and the central atlantic forest domain (which covers the state of espírito santo, small areas of eastern minas gerais and southern bahia). the low humidity associated with this geographical transition (gap) between the two atlantic forest domains favors the appearance of seasonal forests that extend to the coast (oliveira-filho & fontes, 2000; oliveira-filho et al., 2005). this incursion of semideciduous forest to the coast in the northern region of the state of rio de janeiro makes the vegetation show limited number of species common to both areas of dense ombrophilous forest in the state of rio de janeiro (silva & nascimento 2001). oliveira-filho and fontes (2000) consider the weather and especially the temperature as the factor most strongly related to the floristic variation observed. this interposition of a seasonal forest between two large belts of ombrophilous forests might be expected to result in differences in the composition, richness and abundance of the orchid bee communities, as these bees are highly dependent on local floral resources for food, nesting, and reproductive resources (see roubik & hanson, 2004). the present study was therefore designed to: (1) describe and discuss the community structure of euglossine bees in three distinct vegetation types of the atlantic forest in the northern portion of rio de janeiro state; (2) comparatively analyze the structure of those bee communities with other communities previously studied in the same region (tonhasca et al., 2002a; aguiar & gaglianone, 2008, 2011, 2012; ramalho et al., 2009;); (3) investigate the importance of the different vegetation types and different climatic conditions (temperature, humidity, precipitation) and geomorphological factors (altitude) on the species composition of those bee communities. materials and methods study sites we selected three forest fragments, from the three different atlantic forest vegetation types that originally covered the northern portion of the rio de janeiro state (veloso et al., 1991), based on their large area, the advanced stage of regeneration, and their degree of conservation, considering that they have not been completely devastated in the past. 1“mata do carvão” (21º24’s 41º04’w), located in the conservation unity “estação ecológica estadual guaxindiba”, in the municipality of são francisco de itabapoana, is a fragment of lowland seasonal semideciduous forest (lssf) covering approximately 1,200ha, and situated at 40 m.a.s.l. (silva & nascimento, 2001). 2“mata da prosperidade” (21º24’s 42º02’w), located within a private farm in the municipality of são josé de ubá, is a fragment of semideciduous submontane seasonal forest (sssf) covering approximately 900 ha, and situated between 350 and 500 m.a.s.l. (dan et al., 2010). 3“mata da cabecinha” (22°05’s 42º05’w), located in the municipality of trajano de moraes, is a fragment of dense montane ombrophilous forest (dmof) covering approximately 900ha, at 750 to 1,000 m.a.s.l. (riorural/gef, 2007). the climate in the first two areas (lssf and sssf) is classified as aw (köppen & geiger, 1928), with an average total annual rainfall of approximately 1,100 mm (radambrasil, 1983) and a well-defined dry season from may to september (radam brasil, 1983). during the study year, the total precipitation was 1,600mm, with average temperatures of 25.3ºc and 24.8ºc in the two areas respectively (data: instituto nacional de meteorologia). in the dmof area, the climate is classified as cwa (köppen & geiger, 1928), with an average total annual rainfall of approximately 1,300 mm and a predominantly humid climate with no (or only small) water deficits during the year. during the study year, the precipitation was 1,600mm, with an average temperature of 21.4°c (data: instituto nacional de meteorologia). data collection male euglossine bees were sampled once a month from august, 2008 to july, 2009. at each collecting day, fragrance baits (methyl cinnamate, vanilla, eucalyptol, benzyl acetate, and methyl salicylate) were exposed from 09:00 to 15:00h, using two quantitative sampling methods: insect nets and traps, totaling 144 sampling hours (72 hours with an insect net and 72 with traps). the traps and sampling methodologies were the same described in aguiar and gaglianone (2011); baits were applied to cotton balls and inserted in the traps or hung on bushes for direct capture using the net. in the capture using an insect net, a wm de aguiar et al structure of orchid bees communities in the brazilian atlantic forest 70 single collector inspected the cotton balls throughout the sampling period and all bees that landed on cotton were collected. the attractors were placed 1.5m from the ground and 2m apart from each other. the minimum distance between collection stations was approximately 500m. two different capture methods were used to maximize the number of captured species. the data were used together to characterize the euglossine bee community. previous studies in the region (aguiar & gaglianone, 2011, 2012) suggested that the methodologies can be complementary, as traps collect more individuals of eulaema while more euglossa are captured with nets. sampling was consistently undertaken on sunny days, and never during periods of atypical low temperatures. all voucher specimens are deposited in the zoological collection of the laboratory of environmental sciences at the universidade estadual do norte fluminense. data analysis community descriptions three descriptive indices were estimated, shannonwiener diversity (h´), berger-parker dominance (d), and pielou’s evenness (j) (magurran, 2003), using the software past version 1.91 (hammer et al., 2001). in order to compare the diversity among the three euglossine communities in the study area, we generated a 95% confidence interval for the shannon-wiener diversity index using the jackknife method (zahl, 1977) and sites with nonoverlapping confidence intervals were considered significantly different. diversity estimates were generated using the software spade (chao & shen, 2005). species abundance distribution patterns were determined using the rank-abundance plot, with their relative abundance plotted in descending order (whittaker, 1965). all species whose relative abundance was larger than 10% were considered dominant for that area. rarefaction curves for species richness of each study area were obtained using 999 randomizations, following magurran (2003). this procedure was undertaken to evaluate the sampling effort based on the species richness in the study areas. the analyses were carried out with the software ecosim 7 (gotelli & entsminger, 2001) and the results plotted using statistica 8.0 (statsoft, 2007). the nonparametric richness estimators (chao1, jack1 and bootstrap) were also calculated using estimates 8.0 (colwell, 2006). comparative analyses with other atlantic forest communities the structure of the euglossine communities sampled were compared with those reported in studies previously undertaken in rio de janeiro state, based on the following criteria: (1) we considered surveys that used baited traps or direct capture with entomological net in baits, that undertook sampling at least on a monthly basis for at least one year, and used at least five fragrance baits (and at least one of which was cineol or eucalyptol); (2) in the case of surveys on a quarterly basis, we considered only those that lasted for at least two years. the study areas examined in the present work, as well as those selected for comparisons (according to the criteria delimited above), are presented in table s1 (available online as supplemenatry material). detrended correspondence analysis (dca) was used to evaluate the ordination of the communities being compared and a data matrix was built to that end containing abundance data for the individuals in each area (considering the methodological criteria described above for comparisons between areas). the first two axes of the dca were correlated with climatic (temperature, humidity, precipitation) and geomorphological (altitude) parameters using pearson’s linear correlations. the same axes were utilized to evaluate the influence of the forest vegetation types on the ordination of the euglossine bee communities using nonparametric variance analysis (kruskalwallis); the vegetation types were categorized according to table s1. these analyses provided representations of the patterns of gradual species substitutions along environmental gradients, as suggested by ter braak (1995), and were carried out in statistica 8 (statsoft, 2007). some of the species names used in the original publications were updated to correct identification, or in response to taxonomic changes. results description of the studied communities a total of 1,710 male euglossine bees were collected, belonging to four genera and 15 species. euglossa cordata (linnaeus), eulaema nigrita lepeletier and eulaema cingulata (fabricius) were the dominant species, representing more than 80% of the individuals collected (table 1). in the sssf fragment 978 individuals belonging to four genera and 11 species were collected; 444 individuals belonging to four genera and 10 species were collected in the lssf fragment and 288 individuals belonging to three genera and 12 species were collected in the dmof forest fragment (table 1). the estimated shannon diversity in sssf (h’ = 1.52, d.f = 0.024) significantly differed from the lssf and dmof sites (h’ = 1.23, d.f = 0.05 and h’ = 1.36, d.f = 0.08, respectively). similarly, the evenness in the sssf area showed a higher value than those in lssf or dmof (table 1). species composition differed in the three forest fragments: eulaema atleticana nemésio was sampled only in the lssf site, eufriesea violacea (blanchard) only in sssf, and euglossa annectans dressler, e. bembei nemésio, and e. truncata rebêlo & moure were restricted to the dmof site; euglossa clausi nemésio & engel was found only in sssf and dmof (table 1). euglossa cordata was dominant in lssf (52% of the total), while eulaema cingulata was dominant in both sssf and dmof (31% and 59%, respectively) (table 1). four species sociobiology 61(1): 68-77 (march, 2014) 71 were dominant in sssf, three species in lssf, and two species in dmof (fig 1). in spite of the differences in species dominance, the curves obtained for species importance did not demonstrate significant differences among the different vegetation types (kolmogorov-smirnov test, p > 0.05), suggesting essentially the same distribution patterns in all three studied sites (fig 1). the rarefaction curves based on species richness as a function of abundance revealed that the dmof site requires a higher sampling effort (fig 2). this result was also corroborated by the richness estimators (table 2). the rarefaction curves in the other two areas demonstrated a tendency toward stabilization beyond the abundance of 400 individuals, confirming the results of the richness estimators, which likewise indicated values very close to those actually observed (table 2). comparisons of the richness curves calculated for the study areas indicated that species richness was significantly greater in dmof than in the other two areas (fig 2). the studied euglossine communities exhibited differences in their species composition and abundance along the year and among the different vegetation types (fig 3). in lssf and sssf, the communities showed a peak of abundance during the dry season, as well as another smaller peak during the rainy season (although less prolonged in sssf). the highest abundance in the dmof area was observed between december and february, during the rainy season (fig 3b). temporal variation in abundance was largely determined by the dominant species. eulaema cingulata was more abundant during the dry season in sssf and lssf, but did not demonstrate a noticeable temporal pattern in the dmof site (fig 3d). the abundance peak of eulaema nigrita occurred during the rainy season (january to april) in lssf and dmof, while its abundance in sssf varied very little along the year (fig 3e). euglossa cordata showed a peak of abundance in the lssf site only during the dry period; this species showed two similar abundance peaks in the sssf site, one during the dry season and one in the rainy season, while a single peak was observed in dmof during the rainy season (fig 3f). euglossa securigera dressler demonstrated two peaks of abundance in sssf (fig 3c) that were similar to those of euglossa cordata; a few individuals were collected in the other two areas, making interpretations of its abundance more difficult. eufriesea violacea and e. surinamensis (linnaeus) were sampled only during the rainy period (between november and january). comparative analyses with other atlantic forest communities and correlation with abiotic factors the correspondence analysis (dca) of euglossine communities resulted in four major groupings (fig 4): (1) one group clustered all of the lowland seasonal semideciduous forest areas (lssf), between 0.0 and 0.4 on axis 1 and between -0.2 and -0.4 on axis 2, plus one of the seasonal semideciduous submontane forest sites (sssf-5); (2) the second group encompassed the areas of dense lowland ombrophilous forests (dlof) between -0.3 and -0.6 on axis 1 and between 0.0 and -0.4 on axis 2; (3) the third group, represented by the artable 1: composition, abundance (ab.), relative frequency (fr), richness, diversity, dominance, and evenness of the bee communities of the subtribe euglossina in different vegetation types of the atlantic forest in rio de janeiro state, brazil. lssf: lowland seasonal semideciduous forests; sssf: submontane seasonal semideciduous forest; dlof: dense lowland ombrophilous forest; dsof: dense submontane ombrophilous forest; and dmof: dense montane ombrophilous forest. species lssf sssf dmof total ab. fr ab. fr ab. fr ab. fr eufriesea surinamensis (linnaeus) 1 0.2 1 0.1 4 1.4 6 0.4 e. violacea (blanchard) 0 0 3 0.3 0 0 3 0.2 euglossa annectans dressler 0 0 0 0 9 3.2 9 0.5 e. bembei nemésio 0 0 0 0 1 0.3 1 0.1 e. cordata (linnaeus) 230 51.8 171 17.5 20 7 421 24.6 e. clausi nemésio & engel 0 0 6 0.6 1 0.3 7 0.4 e. despecta moure 5 1.1 2 0.2 1 0.3 8 0.5 e. fimbriata moure 2 0.5 22 2.3 4 1.4 28 1.6 e. pleosticta dressler 23 5.2 10 1.0 5 1.7 38 2.2 e. securigera dressler 12 2.7 213 21.8 9 3.1 234 13.7 e. truncata rebêlo & moure 0 0 0 0 1 0.3 1 0.1 eulaema atleticana nemésio 1 0.2 0 0 0 0 1 0.1 e. cingulata (fabricius) 48 10.8 306 31.3 170 59 524 30.5 e. nigrita lepeletier 120 27.0 242 24.7 63 22 425 24.9 exaerete smaragdina (guérin-méneville) 2 0.5 2 0.2 0 0 4 0.2 total 444 100 978 100 288 100 1710 100 richness 10 11 12 15 diversity (h’) 1.26 1.52 1.32 1.61 dominance (d) 0.518 0.313 0.590 0.306 evenness (j) 0.570 0.650 0.528 0.595 wm de aguiar et al structure of orchid bees communities in the brazilian atlantic forest 72 eas of sssf and dmof, was situated between 0.6 and 1.3 on axis 1 and between 0.0 and 0.4 on axis 2; (4) the fourth group, which encompassed the areas of dsof, was more scattered in the diagram, with clear indication of subgroups within it. the eigenvalues of axis 1 and axis 2 were 0.20 and 0.19, respectively, while the percentage of variance explained by axis 1 was 24.9%, and 23.7% by axis 2. the correlation between axis 1 of the dca with the abiotic variables demonstrated a significant negative influence of precipitation (person’s r = 0.56, n = 21, p = 0.001) and a positive influence of altitude (pearson’s r = 0.523, n = 21, p = 0.003) on the ordination of the euglossine communities. only altitude had a significant positive influence on the second axis of the dca (pearson’s r = 0.61, n = 21, p = 0.0004) (table 3). the vegetation type likewise demonstrated an influence on the ordination of the euglossine communities on both axes 1 and 2 (kruskal-wallis test: h= 7.85, p = 0.049 and h= 15.39, p= 0.0015 respectively). fig 1. abundance of orchid bee species in three forest communities in rio de janeiro state. lssf: lowland seasonal semideciduous forests; sssf: submontane seasonal semideciduous forest; and dmof: dense montane ombrophilous forest. for descriptions of the areas, refer to table s1. fig 2. rarefaction curves (1,000 simulations) for the species richness of orchid bees as a function of their abundance in areas with different vegetation types of forest in rio de janeiro state. dotted lines indicate the upper and lower limits (95%) of each curve. the abbreviations follow those of table s1. fig 3. climatic data (a) and temporal variations of euglossine communities (b) in three vegetation types of forest in northern and northwestern rio de janeiro state, between august/2008 and july/2009. 3c to 3f represent the temporal variations of the most abundant species. table 2 richness estimators for euglossine bee communities sampled between august/2008 and july/2009 in three different vegetation types of forest in rio de janeiro state, brazil. the abbreviations follow those of table s1. richness estimator lssf sssf dmof jack1 12.75 ± 1.43 13.75 ± 1.97 17.6 ± 2.85 chao1 10.33 ± 0.92 11 ± 0.16 23 ± 10.2 bootstrap 11.17 ± 0 12.2 ± 0 14.93 ± 0 fig 4: diagram of the detrended correspondence analyses (dca) based on the composition of the orchid bee fauna found in areas in northern and northwestern rio de janeiro state. lssf: lowland seasonal semideciduous forests; sssf: submontane seasonal semideciduous forest; dlof: dense lowland ombrophilous forest; dsof: dense submontane ombrophilous forest; and dmof: dense montane ombrophilous forest. the abbreviations follow those of table s1. table 3 pearson correlations between axes 1 and 2 of the detrended correspondence analysis (dca) and the variables of precipitation, temperature, humidity, and altitude in study areas in the northern and northwestern regions of rio de janeiro state, brazil, relative to the compositions of their orchid bee fauna. precipitation temperature humidity altitude axis 1 r = -0.56; p = 0.001 r = 0.014; p = 0.94 r = 0.24; p = 0.20 r = 0.52; p = 0.003 axis 2 r =0.009; p = 0.96 r = -0.20; p = 0.291 r = 0.08; p = 0.632 r = 0.61; p = 0.0004 a b c d e f sociobiology 61(1): 68-77 (march, 2014) 73 discussion the results of the present study, plus those obtained by tonhasca et al. (2002a), aguiar and gaglianone (2008, 2011, 2012), and ramalho et al. (2009) in other areas in the northern portion of the rio de janeiro state, revealed a total of 32 euglossine bee species for this region. this number corresponds to about 60% of the total orchid-bee fauna reported by nemésio (2009) for the atlantic forest biome. considering the result of the rarefaction curves and richness estimators, the orchid bee fauna in dense montane ombrophilous forest was underestimated and the real number of species is probably higher in the region. studies in other ombrophilous forests in rio de janeiro state (tonhasca et al., 2002a; ramalho et al., 2009) indicated higher number of species. in addition to insufficient sampling in the studied area, its regeneration time (approximately 60 years, according to local inhabitants), may not have been sufficient for more restrictive species to recolonize it. species such as euglossa marianae nemésio and euglossa iopoecila dressler are known from areas of well-preserved ombrophilous forest (tonhasca et al., 2002a; ramalho et al., 2009). these results point out to a great richness of orchid bee species in dense montane ombrophilous forest of the atlantic forest biome, even at the small spatial scales examined. this is possibly due to the great diversity of habitats, influenced by the wide geomorphological and climatic variation, besides the “campos dos goytacazes gap”, extending the lowland seasonal semideciduous forest to the coastline. this configuration forms a mosaic of landscapes probably favoring the occurrence of orchid bees in the region. the euglossine bee communities sampled in dense montane ombrophilous forest demonstrated the highest species richness among the studied physiognomies. the greatest average relative humidity values (up to 80%) seem to be the most important abiotic factor for this result. it was expected given the fact that other studies have indicated preference for humid forests by orchid bees (see roubik & hanson, 2004). eufriesea surinamensis and eulaema atleticana were not found in lowland seasonal semideciduous forest in a previous study (aguiar & gaglianone, 2008) using the same methodology. also four other species previously found there (eulaema niveofasciata (friese), euglossa gaianii dressler, euglossa leucotricha rebêlo & moure, and euglossa truncata) were not present in the current study. this reflects the dynamics of the bee communities in a short time scale, which might result in smaller chances of resampling rare species (roubik, 2001). euglossa annectans (treated as a junior synonym of euglossa stellfeldi moure in nemésio, 2009) had not been cited in previous surveys undertaken in rio de janeiro state although the species has been originally described from specimens collected at the tijuca forest, rj (dressler, 1982b). this species is endemic to the atlantic forest domain, being distributed from the states of bahia to santa catarina, and then into argentina (faria & melo, 2007; andrade-silva et al., 2012). in this study e. annectans along with e. bembei were found only in dense montane ombrophilous forest, which, together with data from other studies (tonhasca et al., 2002a; darrault et al., 2006; moure et al., 2007; ramalho et al., 2009; cortopassi-laurino et al., 2009), indicates their close association with dense ombrophilous forests in brazil. eulaema atleticana, also endemic to the atlantic forest, has been considered restricted to coastal areas in northeastern and southeastern brazil (nemésio, 2009). however, more recent surveys have indicated that this species is common at altitudes above 700m (a. nemesio, personal communication). the occurrence of eulaema atleticana in the lowland seasonal semideciduous forests in campos dos goytacazes gap may represent the southern limit of its distribution. eufriesea violacea was collected only in submontane seasonal semideciduous forest. this species has been previously found in other vegetation type of the atlantic forest domain (tonhasca et al., 2002a; nemésio & silveira, 2006a; giangarelli et al., 2009), but always in well-preserved areas. giangarelli et al. (2009) reported that it was sensitive to habitat fragmentation and that its abundance becomes significantly reduced in small forest fragments. this can explain its reduced abundance in the present study. effects of fragmentation in the orchid bee communities were detected by aguiar and gaglianone (2012), who found changes in the pattern of species dominance in small fragments of atlantic forest. originally the lowland seasonal semideciduous forests extended from southern rio grande do norte to northern of rio de janeiro state. this forest was severely fragmented and the studied fragment represents only a very small portion of its original cover in the region (silva & nascimento, 2001). the number of species recorded by bonilla-gómez (1999) for this vegetation type was much higher and some relatively common species in this physiognomy have become rare in small forest fragments. the dominance of euglossa cordata, eulaema nigrita and eulaema cingulata in the present study was likewise observed in other surveys in the atlantic forest (peruquetti et al.,1999; aguiar & gaglianone, 2008; farias et al., 2008; ramalho et al., 2009; nemésio & silveira, 2010). ramalho et al. (2009) and aguiar and gaglianone (2012) demonstrated that these species were abundant in areas at different stages of conservation and do not confirm that they are indicators of disturbed areas, as suggested previously (e.g. morato et al., 1992; peruquetti et al., 1999). however, their tolerance to disturbance areas is unquestionable. furthermore, the communal nesting or social behavior could favor their dominance in many of the communities studied (zucchi et al., 1969; garófalo, 1992; 1994; augusto & garófalo, 2004; roubik & hanson, 2004), as well as the great flight distance of eulaema nigrita and e. cingulata (dressler, 1982). despite exhibiting high relative abundances in all of the vegetation types studied, euglossa cordata and eulaema cingulata showed variations in their dominance patterns. euglossa cordata, for example, was not dominant in dense montane ombrophilous forest, although its abundance was greater than 50% in lowland seasonal semideciduous forest. eulaema cingulata was the most abundant species in dense montane ombrophilous forest but the third one in lowland seasonal semideciduous forests. euglossa securigera also showed great variation in their dominance patterns, represented 23% of the individuals collected in submontane seasonal semideciduous forest but only about 3% of the individuals sampled in lowland seasonal semideciduous forests and wm de aguiar et al structure of orchid bees communities in the brazilian atlantic forest 74 dense montane ombrophilous forest. vegetation types and differences in the availability of key resources can change population patterns of pollinators (lázaro & totland, 2010), including euglossine communities (ramirez et al., 2002; souza et al., 2005; nemésio & silveira, 2007); detailed analyses of resource availability in the forests would be important to clarify this issue. according to smith et al. (2012) the floral landscape in the neotropical forest is spatially and temporally heterogeneous for foraging bees, promoting considerable change in the abundance pattern and amount of brood in the nests for bees. the abundance distribution patterns observed in the present study were similar to those described by other authors in different atlantic forest areas: few dominant species; usually two to four species, each one with more than 10% of abundance and many rare species (rebêlo & garófalo, 1997; sofia et al., 2004; aguiar & gaglianone, 2008; nemésio, 2007; ramalho et al., 2009). however, this pattern differs from that found by aguiar and gaglianone (2012) for small fragments of atlantic forest, where most species had each more than 10% of abundance, suggesting the loss of rare species in small forest fragments. the euglossine communities in lowland seasonal semideciduous forests and submontane seasonal semideciduous forest demonstrated seasonal peaks of abundance, with one conspicuous peak during the dry period that was influenced by the abundances of euglossa cordata, e. securigera and eulaema cingulata, as well as another peak in the rainy season with a predominance of eulaema nigrita (thus similar to data presented by aguiar and gaglianone [2008, 2011]). the peak of abundance in dense montane ombrophilous forest occurred during the rainy season but the same was not demonstrated for the studied semideciduous forests. long-term studies are necessary to confirm the different seasonal pattern observed in these areas. short periods of adult activity were observed for eufriesea violacea and e. surinamensis. seasonality of eufriesea species was observed in different regions in brazil, for example, semideciduous forest in paraná (giangarelli et al., 2009), ombrophilous forest in the rio de janeiro (tonhasca et al., 2002a; ramalho et al., 2009) and sandbanks in maranhão state (silva et al. 2009). our data corroborate the seasonality of these bees in different physiognomies of atlantic forest. three of the four main groups detected in the detrended correspondence analysis (dca) (groups 1, 2 and 4 in the results) showed a close correspondence of the euglossine communities with the predominant vegetation types. a single group combined fragments from distinct vegetation types, seasonal semideciduous submontane forest and dense montane ombrophilous forest. this grouping may be partly explained by their uniquely sharing of euglossa clausi, as well as a higher relative abundance of eulaema cingulata. as stressed by linsley (1958) and abrahamczyk et al. (2011), different distribution patterns along natural gradients reflect different responses to changes in the biotic and abiotic factors acting along those gradients. in the case of the present study, the original atlantic forest was a continuum of forest, “restinga”, and mangrove swamp ecosystems stretching along the entire eastern coast of brazil (galindo-leal & câmara, 2003) and included diverse natural gradients within its latitudinal and longitudinal extensions, as well as altitudinal variation going from the coast to inland (galindo-leal & câmara, 2003). rainfall and altitude, and also vegetation types, were the main factors influencing the ordination of the euglossine communities in the study areas. according to abrahamczyk et al. (2011), these factors were found to have a significant effect on the ordination of euglossine communities in the western amazon region. altitudinal variation, can drastically reduce abundance patterns and alter the species compositions of euglossine communities. our data suggest that altitude is important and can modify species composition and abundance of euglossine bees, even at small scales of variation. this result has to be tested in future studies. temperature is influenced by altitudinal variations, and it is an important factor in the regulation of flowering time in the atlantic forest (talora & morellato, 2000; pereira et al., 2008), altering plant resource availability for bees. according to hegland and boeke (2006) and lázaro and totland (2010) the diversity and density of plants affect the foraging behavior and community of pollinators. indeed, the incursion of the lowland seasonal semideciduous forests, due to the campos of goytacazes gap, significantly modified the structure of vegetation in the region and can represent a great influence on the community structure of euglossine bees. precipitation has been found to be significantly related to the abundance and species richness of euglossine bees in the western amazon region (abrahamczyk et al., 2011). the lowland seasonal semideciduous forests area has relatively high average temperatures (25.3ºc) associated with lower relative humidity levels (70%), which influenced the ordination of euglossine communities. in addition to the climate and type of vegetation identified as strong influences on euglossine bee communities, other components, such as competition with similar species, historical occurrences, and habitat homogeneity (see armbruster, 1993; rosenzweig, 1995; roubik, 2001; tonhasca et al., 2002b; roubik & hanson, 2004) are also determinant for these bees. also cleptoparasitic euglossine bees, such as those of the genus exaerete, require additional biotic factors for their occurrence (such as the occurrence of host species in the genera eulaema and eufriesea) (wcislo & cane, 1996; nemésio & silveira, 2006b); however these factors have not been evaluated in this study, but should be considered in future studies. our data confirmed that a set of climatic, geomorphological and vegetational factors act to strongly influence euglossine species richness and composition (ramirez et al., 2002; souza et al., 2005; nemésio & silveira, 2007). euglossa annectans and e. bembei, for example, were found only in dense montane ombrophilous forest, the survey region with the highest altitude (850 m) and humidity and the lowest average temperature (21 ºc). altitude, however, does not appear to be a determinant factor in the geographical distribution of these species, as they have been also found in areas of dense ombrophilous forest at lower altitudes in southern brazil (with warmer temperatures) (cortopassi-laurino et al., 2009). our study reinforces the importance of the conservation of landscape mosaics that include various vegetation types, as the composition of euglossine bee communities as well as their patterns of abundance and species dominance have been found to differ among these distinct sites. furthermore, the sociobiology 61(1): 68-77 (march, 2014) 75 occurrence of rare species, such as eulaema atleticana in the lowland seasonal semideciduous forest, demonstrates the need to preserve these areas, as was similarly observed for euglossa annectans and e. bembei in dense montane ombrophilous forest and for eufriesea violacea in seasonal semideciduous submontane forest. the conservation of this important group of neotropical pollinators is essential for the maintenance of ecological services and the genetic diversity of their host plant populations, and attention should be given to studies focusing on these points. acknowledgements we thank instituto nacional de meteorologia (inmet) for climatic data; frederico machado teixeira, marcelita frança marques, mariana scaramussa deprá and giselle braga menezes for their help in the field; faperj for fellowships to the first author. we also thank the financial support provided by procad/capes (158/07), rio rural/seappa/gef, faperj, and cnpq for the pq scholarships to mcg and garm. supplementary material table s1 available at: http://periodicos.uefs.br/ojs/index.php/ sociobiology/rt/suppfilemetadata/231/0/418 doi: 10.13102/sociobiology/.v61i1.68-77.s418 references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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(1969). biological observations on a neotropical parasocial bee, eulaema nigrita, with a review on the biology of euglossinae. journal of the faculty of sciences of hokkaido univerdity, 17: 271-380. doi: 10.13102/sociobiology.v64i3.1257sociobiology 64(3): 284-291 (september, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 spectrum of pollen stored by melipona mandacaia (smith, 1863) (hymenoptera: apidae, meliponini) in an urban arid landscape introduction the melipona mandacaia (smith, 1863), is one of the mostknown stingless bee in the semiarid regions in northeastern brazil, thereby being an animal of permanent coexistence in the rural areas (alves et al., 2006), distributed in the caatinga (dry bushland) biome (batalha-filho, et al., 2011). this is a generalist bee, endemic, and little studied, possibly due to its region of occurrence (prado-silva et al., 2016; nunes, 2008). the bees of the caatinga biome play an important role in the pollination of native and cultivated plants, which contributes to the formation of more robust and better quality fruits for the producer market (siqueira et al., 2011). the flora abstract the objective of this study was to identify the species of pollen supplying plants that constitute the trophic niche of melipona mandacaia in an urban area in the caatinga domain (a tropical arid landscape). the collection of pollen in the colonies was carried out every 15 days, from october 2014 to september 2015. the pollen was removed directly from the storage pots in three distinct colonies. a total of 24 samples were analyzed and compared with the reference pollen collection, pollen catalogs and specialized literature. for the quantitative analysis, at least 1000 pollen grains per sample were identified. a total of 39 pollen types were identified, distributed in 17 botanical families, being one an indeterminate type. the most represented family was the fabaceae (n = 16). the most frequent types were leucaena leucocephala, mimosa pudica and melochia sp. there was a significant positive correlation between temperature and the number of pollen types throughout the study. relative humidity and rainfall were abiotic variables that did not present a significant correlation. the rarefaction curve showed that probably most of the pollen types collected by the bees studied were sampled, since the accumulation curve showed a progressive tendency to stabilization, indicating that there was sample adequacy of the pollen types. the analysis of similarity revealed a high sharing of pollen sources between colonies. sociobiology an international journal on social insects tfs carneiro-neto¹, po rebouças¹, je pereira¹, pm duarte², mhlc santos¹, gc silva¹, kmm siqueira¹ article history edited by evandro n. silva, uefs, brazil received 28 november 2016 initial acceptance 24 may 2017 final acceptance 05 july 2017 publication date 17 october 2017 keywords stingless bee, collection of pollen, trophic niche, caatinga. corresponding author thiago francisco de souza carneiro neto departamento de tecnologia e ciências sociais, universidade do estado da bahia rua edgar chastinet, s/n são geraldo cep 48905-680, juazeiro-ba, brasil. e-mail: thiagofs_10@hotmail.com of the caatinga basically provides the food resources to the bee’s population when the orchard plants are not flowery. however, knowledge of the bee fauna of this biome and the resources used are still very scarce, although rich in species and endemism. however, natural ecosystems have been affected for many years by the destruction of the original vegetation in order to use them for agriculture or livestock (ramírezarriaga et al., 2011). the simplification of these landscapes due to the intensive use of the soil has led to changes in the pollinator community structure. among the anthropogenic actions, the indiscriminate use of agrochemicals, fires and deforestation are considered the main threats to biodiversity (steffan-dewenter et al., 2006). the intense deforestation 1 state university of bahia, dtcs, juazeiro-ba, brazil 2 federal university of ceará, departament of agronomy, fortaleza-ce, brazil research article bees mailto:thiagofs_10@hotmail.com sociobiology 64(3): 284-291 (september, 2017) 285 of the caatinga has brought harmful consequences for m. mandacaia (batalha-filho et al., 2011). given their ecological importance as pollinators of many native plant species, knowledge of the sources of food used by stingless bees, as well as the spatio-temporal distribution of the resources that maintain their colonies, and the strategies adopted by them in the face of variations of environmental factors have great ecological and economic importance in this group of bees. (aleixo et al., 2013; imperatriz-fonseca et al., 2012). this way, the complementation of ecological data, obtained through the palynological analyses, is an important step in the rational exploration for the development of the preservation programs for these bees. the planning of urban green spaces using native species is important from a conservation perspective, since it favors the maintenance of several kinds of interactions between plants and pollinators (aleixo et al., 2014). this may help direct efforts to recover vegetation in affected areas using botanical species that guarantee food supply (luz et al., 2011; wiese, 1985). in this context, the objective of this study was to identify the species of plants supplying pollen, to verify the existence of correlation between biotic variables (pollen types) and abiotic variables, and the sharing of the trophic niche of melipona mandacaia in an urban area in the caatinga vegetation domain (a tropical arid landscape). material and methods the experiment was conducted in the meliponary of the department of technology and social sciences dtcs of campus iii uneb in juazeiro-bahia (09º25’43.6” s, 40º32’14” w, 384m) (fig 1). the climate of the region according to the classification of köppen is bswh’ hot dry, semi-arid with an annual average rainfall of 542 mm, with rainfall concentrated in the period from october to april (embrapa, 2015). the pollen was collected every 15 days during the period from october 2014 to september 2015. the pollen was collected directly from the storage pots in three colonies of m. mandacaia. they were found open, indicating their recent use. a total of 24 samples were analysed. the macroclimatic data, such as rainfall, temperature and relative humidity, were obtained through the uneb-campus iii meteorological station. preparation of slides and identification of pollen types the samples were placed in eppendorf type microtubes and taken to the entomology laboratory. for each sample, three slides were prepared in kisser’s glycerin gelatin, following the method of maurizio and louveaux (1965), without acetolysis. in the same sampling period, flowering plants were photographed for subsequent identification, being collected in transects up to 500 m from the meliponary. reference slides with pollen from anthers were made, using the same methodology already described. the identification of the pollen types was carried out under a zeiss primo star optical microscope with a 40x objective, comparing with the reference pollen collection, pollen catalogs and specialized literature. for the quantitative analysis, a minimum of 1000 pollen grains per sample was recorded. fig 1. geographical location of melipona mandacaia occurrence and of the study area, campus iii-uneb, bahia, northeast brazil. tfs carneiro-neto et al. – pollen stored in an urban landscape286 statistical analysis for the analysis of the pollen types the frequency was expressed in percentage and grouped in the following classes according to novais et al. (2009): very constant (vc), present in >75% of the samples; constant (c), >50%≤75%; low constancy (lc), >25%–≤50%; occasional (o), ≥5%–≤25%; and rare (r), <5%, considering only the simple presence or absence of a pollen type in any of the samples. the trophic niche amplitude was calculated by the shannon index (h ‘), using the algorithm h’ = σpk x ln pk, where pk is the ratio of the number of grains counted by each pollen type (k) and the total counted pollen. the uniformity of resource use was calculated by the pielou index (j ‘), using the formula j’ = h ‘/ h’max (ludwig & reynolds, 1988). for this the past software, version. 1.85 was used (hammer et al., 2001). the normality of the data was tested by the shapirowilk test (w), verifying that the data did not present normal distribution. for this reason, a simple regression analysis was applied through the spearman correlation coefficient (ρ) to verify the existence of correlation between biotic variables, pollen types and abiotic variables such as temperature, rainfall and relative humidity. significant correlations were considered when p <0.05, by the student t test. the analyses were done using past software version 1.85 (hammer et al., 2001). the collector curve was constructed by accumulating the number of pollen types collected by the workers and stored in the pollen pots of their nests during the period of this study. to estimate the total richness of the pollen types that occurred in the study area, colwell & coddington (1994) were used. in addition, the chao 2, jacknife 1 and bootstrap wealth estimators, 100 times randomized by the estimate s 9.1.0 program (colwell, 2013), were used to determine the sample sufficiency of the bees’ guild. the similarity coefficient proposed by sörensen (cs) (sörensen, 1948 apud magurran, 2011) was used to evaluate the degree of similarity in the composition of the pollen types of the boxes of m. mandacaia, varying from 0 to 1. considering high similarity when values of cs > 0.7. the coefficient of similarity of sörensen was calculated using the formula cs = 2a / 2a + b + c, where: a = number of common types occurring in the two colonies (box a and b); b = number of types occurring in colony a and not in b; c = number of types that occurs in colony b and not in colony a. results a total of 39 pollen types were identified, distributed in 17 botanical families, being one an indeterminate type (table 1). the most represented family was fabaceae with 16 pollen types distributed in three subfamilies: caesalpinioideae (n = 4), mimosoideae (n = 11) and papilinioideae (n = 1). the most frequent types were leucaena leucocephala (35.82 %), mimosa pudica (15.66%) and melochia sp. (13.32%). most of the pollen types found during the study period belonged to the mimosoideae (fabaceae). the largest pollen spectrum (21 pollen types) was found in sample i (oct/2014), however the highest contribution of pollen grains was concentrated in the l. leucocephala type (62.26%), which resulted in a reduced uniformity of the trophic resources (j ‘= 0.5) stored in the pollen pots of m. mandacaia. the samples xviii (jun/2015), xix (jul/2015) and xxiii (set/2014) presented the lowest spectrum, with six pollen types each. the amplitude of the trophic niche, based on the pollen stored in the pots of the studied colonies, ranged from 0.64 to 2.27, given that sample xxiii (set/2015) obtained the lowest diversity value (h ‘) and sample vi (dec/2014) presented the highest value. the uniformity of use of these resources in pollen samples ranged from 0.36 to 0.87, given the lowest value found for sample xxiii for september/2015, and the highest value for sample iv, which was collected in november, 2014. there was a significant positive correlation between temperature and the number of pollen types (ρ = 0.6455; p = 0.0234) throughout the study (fig 2). the relative humidity (ρ = -0.2035, p = 0.5258) and rainfall (ρ = 0.4205; p = 0.1735) were abiotic variables that did not present a significant correlation with the number of pollen types collected by bees. fig 2. relationship between the monthly average temperature and the number of pollen types during the period from october/2014 to september/2015in an urban arid landscape. comparing the analyses made with richness estimators the number of pollen types varied between 39 (chao 2) and 46 (jack 1) found along the 24 samples collected in the study period (fig 3). however, the estimator curves did not stabilize, indicating that the increase in sample effort could increase the number of estimated types. for the similarity analysis, a qualitative metric was used (sorensen index), which revealed a high share of the pollen sources among the colonies of m. mandacaia studied since the value of this index was high (cs> 0.7). the colony i + colony ii group presented the highest similarity among the three boxes studied (table 2). sociobiology 64(3): 284-291 (september, 2017) 287 po lle n ty pe s sa m pl es ( % ) t r f (% ) c c 20 14 20 15 o ct ob er n ov em be r d ec em be r ja nu ar y fe br ua ry m ar ch a pr il m ay ju ne ju ly a ug us t se pt em be r i ii ii i iv v v i v ii v ii i ix x x i x ii x ii i x iv x v x v i x v ii x v ii i x ix x x x x i x x ii x x ii i x x iv a na ca rd ia ce ae a na ca rd iu m o ci de nt al le 0. 97 4. 21 3. 74 1. 78 0. 84 1. 18 0. 51 o sc hi nu s ty pe 0. 13 0. 48 0. 03 o sp on di as tu be ro sa 2. 35 0. 29 0. 27 o a re ca ce ae a re ca ce ae ty pe 0. 94 0. 99 0. 07 o a st er ac ea e b id en s sp . 14 .6 0. 53 r b eg on ia ce ae b eg on ia s p. 0. 29 0. 09 0. 01 o b ig no ni ac ea e te co m a sp . 0. 13 0. 01 r c on vo lv ul ac ea e m er re m ia s p. 0. 26 0. 74 0. 35 0. 10 0. 09 0. 07 o c uc ur bi ta ce ae m om or di ca c ha ra nt ia 0. 20 0. 88 5. 01 0. 19 0. 27 o e up ho rb ia ce ae e up ho rb ia ce ae ty pe 1. 14 0. 45 0. 41 0. 08 o c ro to n sp . 0. 17 0. 44 0. 09 0. 59 0. 69 0. 09 o r ic in us c om un ni s 0. 33 0. 04 r f ab ac ea ec ae sa lp in io id ea e c as si a fis tu la 1. 99 1. 33 0. 27 1. 29 0. 33 o c ha m ae cr is ta s p. 0. 09 5. 79 1. 50 0. 46 3. 46 2. 94 2. 36 3. 00 3. 86 1. 28 0. 95 l c li bi di a fe rr ea 1. 77 0. 61 0. 28 0. 59 0. 79 0. 17 o se nn a ty pe 0. 19 7. 50 11 .5 1 0. 30 0. 75 o f ab ae ae m im os oi de ae a na de na nt he ra s p. 1. 06 0. 57 0. 09 0. 14 0. 09 3. 17 1. 21 0. 89 5. 90 0. 57 l c d es m an th us s p. 1. 43 0. 06 r le uc ae na le uc oc ep ha la 62 .2 6 67 .8 1 12 .5 9 5. 06 0. 69 5. 53 1. 85 9. 99 13 .1 5 10 .2 2 31 .4 0 33 .1 4 25 .8 3 32 .6 1 39 .8 0 52 .8 1 47 .1 1 52 .4 2 56 .3 3 74 .5 4 78 .0 3 66 .9 6 35 .8 2 v c m im os a ca es al pi ni ifo lia 1. 63 2. 29 8. 39 9. 62 5. 12 4. 12 1. 41 4. 63 0. 29 1. 33 0. 84 6. 10 1. 47 4. 40 25 .8 6 0. 75 14 .3 1 30 .5 3 8. 46 5. 32 9. 24 5. 73 v c m im os a op ht ha lm oc en tr a 2. 45 3. 05 2. 86 4. 96 1. 77 1. 96 4. 44 1. 03 l c t ab le 1 . d iv er si ty a nd u ni fo rm ity in di ce s pr es en t i n th e po lle n sa m pl es a nd to ta l r el at iv e fr eq ue nc y of th e po lle n ty pe s co lle ct ed b y m el ip on a m an da ca ia f ro m o ct ob er /2 01 4 to s ep te m be r/ 20 15 ,in an u rb an a ri d la nd sc ap e. tfs carneiro-neto et al. – pollen stored in an urban landscape288 t ab le 1 . d iv er si ty a nd u ni fo rm ity in di ce s pr es en t i n th e po lle n sa m pl es a nd to ta l r el at iv e fr eq ue nc y of th e po lle n ty pe s co lle ct ed b y m el ip on a m an da ca ia f ro m o ct ob er /2 01 4 to s ep te m be r/ 20 15 ,in an u rb an a ri d la nd sc ap e. (c on tin ua tio n) po lle n ty pe s sa m pl es ( % ) t r f (% ) c c 20 14 20 15 o ct ob er n ov em be r d ec em be r ja nu ar y fe br ua ry m ar ch a pr il m ay ju ne ju ly a ug us t se pt em be r i ii ii i iv v v i v ii v ii i ix x x i x ii x ii i x iv x v x v i x v ii x v ii i x ix x x x x i x x ii x x ii i x x iv p ip ta de ni a sp . 0. 33 0. 04 r m im os a m is er a 0. 63 0. 29 1. 88 5. 71 7. 09 4. 13 2. 41 0. 79 2. 47 5. 67 1. 40 0. 49 5. 69 4. 91 6. 94 9. 31 0. 10 0. 68 3. 25 2. 78 v c m im os a pu di ca 6. 57 1. 90 0. 20 0. 20 1. 26 0. 09 37 .0 2 11 .4 9 11 .1 5 40 .2 1 10 .0 7 10 .4 3 13 .5 7 15 .6 6 v c m im os a sp . 4. 94 0. 57 2. 72 5. 23 0. 49 3. 91 c m im os a te nu ifl or a 0. 53 2. 48 0. 84 3. 54 0. 39 0. 39 0. 28 0. 30 0. 79 3. 92 2. 17 0. 59 12 .0 9 18 .0 2 4. 15 4. 85 18 .6 6 0. 56 8. 06 8. 36 v c se ne ga lia b ah ie ns is 0. 86 0. 19 0. 10 o f ab ac ea ep ap ili ni oi de ae d es m od iu m u nc in at um 0. 23 0. 03 r m al va ce ae m el oc hi a ty pe 5. 56 17 .1 3 3. 38 13 .8 1 36 .3 6 68 .8 7 71 .2 8 28 .3 5 19 .2 0 27 .5 6 10 .8 8 11 .0 0 0. 29 0. 74 11 .3 3 13 .3 2 c si da s p. 0. 48 0. 02 r w al te ri a sp . 0. 07 o m yr ta ce ae m yr ci a ty pe 10 .5 5 8. 67 8. 75 4. 86 l c e uc al yp tu s sp . 2. 06 19 .5 5 7. 64 3. 54 1. 08 0. 26 0. 15 1. 58 l c p ol yg on ac ea e a nt ig on on s p. 0. 38 0. 09 2. 30 0. 79 0. 09 0. 68 c r ha m na ce ae zi zy ph us jo az ei ro 0. 27 0. 03 r sa pi nd ac ea e se rj an ia s p. 1. 77 2. 30 3. 52 1. 39 0. 39 o sa po ta ce ae m an ilk ar a za po ta 0. 30 2. 70 0. 16 o tu rn er ac ea e tu rn er a sp . 0. 53 0. 54 0. 09 o in de te rm in at e ty pe 8. 10 1. 39 2. 02 0. 46 2. 11 0. 10 0. 56 o t o ta l 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0. 00 to ta l o f po lle n ty pe s (s ) 21 18 14 12 14 17 16 18 11 11 13 13 10 10 11 9 10 6 6 8 7 8 6 7 h ’ i nd ex 1. 53 1. 49 2. 06 2. 23 1. 99 2. 27 2. 05 2. 02 1. 13 1. 19 1. 57 1. 60 1. 83 1. 78 1. 72 1. 58 1. 69 1. 26 1. 14 1. 35 1. 05 0. 86 0. 64 1. 13 j ’ i nd ex 0. 50 0. 50 0. 78 0. 87 0. 75 0. 80 0. 72 0. 68 0. 47 0. 48 0. 59 0. 62 0. 79 0. 77 0. 72 0. 72 0. 73 0. 70 0. 64 0. 65 0. 54 0. 42 0. 36 0. 58 v c = ve ry c on st an t, pr es en t i n >7 5% o f t he sa m pl es ; c = co ns ta nt , > 50 % –≤ 75 % ; l c = lo w c on st an cy , > 25 % –≤ 50 % ; o = oc ca si on al , ≥ 5% –≤ 25 % ; r = ra re , < 5% . h ’ i nd ex = sh an no nw ie ne r’ a nd j ’ i nd ex = un if or m ity p ie lo u; t r f= t ot al r el at iv e fr eq ue nc y; c c = c on st an cy c la ss es . sociobiology 64(3): 284-291 (september, 2017) 289 discussion in the present study, it was observed that many plants contributed to guarantee the maintenance of colonies of m. mandacaia. it is worth mentioning that the study area is a fragment of the riparian forest of the são francisco river, made up of caatinga species and exotic plants, also composed of irrigated crops, being located in the urban area. possibly, accidental pollen grains may have contaminated the mass transported during bees’ activities in the flowers, contributing to increase the number of pollen types with low representativity (carvalho et al., 1999). from this assumption, and considering that the total frequencies of pollen types less than 0.5% ,as proposed by eltz et al. (2001) in their study, which should be considered as contaminations, there would remain only 18 pollen types in our study representing the plants visited by the bees for pollen collection. this result shows a high selectivity and floral preference for plants that supply large quantities of pollen (maia-silva et al., 2014). a few species was collected in ribeirão preto (sp), 23 plant species of melipona quadrifasciata, and 18 species of melipona subnitida (maia-silva et al., 2014). conceição (2013) recorded 17 pollen types when identifying the botanical profile of the stored pollen (samburá) of melipona quadrifasciata anthidioides, in the semi-arid state of bahia-brazil. in native vegetation of the caatinga in the state of paraiba, 14 pollen types collected by melipona subnita were identified (maiasilva et al., 2015), whereas 52 pollen types for the same bee in the lençóis maranhenses national park-brazil (pinto et al., 2014). the intrinsic learning of each colony, genetic characteristics, flower characteristics, availability of trophic resources and different levels of competition, are factors that influence the bees’ collection behavior (modro et al., 2011; oliveira et al., 2009). the pollen type l. leucocephala is a widely cultivated forage, with irrigation for animal feed in the study area, in addition to what occurs in a sub spontaneous way. in a study carried out in a forest fragment in manaus, northern brazil, oliveira et al. (2009) of the eight species of fabaceae-mimosoideae identified in the pollen samples, l. leucocephala was the most important, being widely used by melipona seminigra merrillae and melipona fulva. according to the authors, l. leucocephala blooms the entire year and its inflorescence in cream-colored flower head provides large amount of pollen, which are collected by bees early in the morning. in a study conducted in petrolina-pe, located in the san francisco valley, northeast brazil, just as with m. mandacaia, the l. leucocephala species showed a total frequency between 16-45% in samples collected in an urban area (braga et al., 2012), corroborating the results reported here. inthe assu-rn national forest (biome caatinga), in studies with the species of stingless bee melipona subnitida and plebeia aff flavocincta, this species was also recorded, but with low frequency in the samples (azevedo-costa et al., 2013).several genus and species recorded in their study were also recorded in our study, such as the chamaecrista sp., anadenanthera colubrina (vell.) brenan, eucalyptus spp., mimosa tenuiflora (willd.) poir., senna spp., croton spp., anacardium sp. and turnera melochioides cambess. antagonistically to our study, the most constant pollen types were m. tenuiflora, senna sp., chamaecrista sp. and m. arenosa. the melochia species, flowers throughout the year and offers two floral resources (nectar and pollen) during the rainy season of the caatinga ecosystem, favoring the maintenance of bee populations and other floral visitors, being a key resource in the northeastern semi-arid of brazil (machado & sazima, 2008). when small flowers are arranged in dense inflorescences, they allow the visiting of medium and large bees. examples of species with small flowers that are, however, very attractive due to their organization in dense inflorescences are found in many legumes (fabaceae), such as a. colubrina, m. tenuiflora (machado & lopes, 2004), m. pudica, m. misera and m. caesalpiniifolia. this justifies the bees’ preference for the fabaceae family. in a floristic survey in southeastern brazil, the fabaceae family with the highest species richness was recorded in the study area (aleixo et al., 2014), being also observed by conceição (2013) in the baiano-northeastern brazilian semi-arid. a number of studies with native stingless bees highlight the importance of the fabaceae family as the main supplier of pollen (aleixo et al., 2013;azevedo-costa et al., 2013;maiasilva et al., 2014). studies performed by alves et al. (2006) to identify m. mandacaia nectar sources in the state of bahiafig 3. rarefaction curve and richness estimators (chao 2. jack 1. and bootstrap) of the pollen types collected in pollen pots of melipona mandacaia nests in an urban arid. colony ii colony iii colony i 0.93 0.79 colony ii 0.83 table 2. sörensen’s similarity coefficient in the composition of the pollen types of the three colonies of m. mandacaia, from october/2014 to september/2015, in an urban arid landscape. cs> 0.7. tfs carneiro-neto et al. – pollen stored in an urban landscape290 brazil, souza et al. (2015) of m. scutellaris on the north coast of bahia, matos and santos (2016) of m. scutellaris in an atlantic forest area in the state of bahia, and martins et al. (2011) of melipona fasciculata in the state of maranhãobrazil, indicated that the greatest diversity of types was also for the fabaceae family. therefore, this family, besides being the main supplier of pollen throughout the year, is one of the main sources of nectar for the stingless bees. in the southeast of brazil, in ribeirão preto (sp), the period with the highest number of plants supplying pollen was observed during the rainy season with positive correlation (aleixo et al., 2014). the highest number of pollen types collected by bees in our study also occurred in the period considered as rainy (october to april) in the region of the submediumvalley of san francisco. the period with the highest number of worker bees collecting pollen for frieseomelitta varia, another species of stingless bee, was recorded in october (aleixo et al., 2013) and the pollen activity was not influenced by the climatic factors. in the present study, it was recorded that the greatest diversity of pollen types was found in october and influenced by temperature. the rarefaction curve showed that probably most of the pollen types collected by the studied bees were sampled, since the accumulation curve presented a progressive tendency to stabilization, indicating that there was sufficient sample of the pollen types. the fact that the accumulation curve did not stabilize indicates that there may still be pollen types that were not recorded in the study area, suggesting that the diversity of pollen types in the study area is larger than that observed. based on the values obtained by the richness estimators, one may suggest that between 85% and 93% of the pollen types collected by bees population of m. mandacaia the study site has been effectively sampled, although the curves of these estimators have been completely stabilized.through pollen analysis, it was observed that the studied colonies share several pollen types. acknowledgments the authors are grateful to dra. gertrudes macario de oliveira for all the meteorological data, dra. lúcia h. p. kiill and tatiana ayako taura for the support in the making of the map, the anonymous reviewer for helpful comments on an earlier version of the manuscript and the state university of bahia for providing the necessary infrastructure for this experiment. references aleixo, k.p., faria, l.b., groppo, m., castro, m.m.n. & silva, c.i. 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(1985). novo manual de apicultura. porto alegre: agropecuária.493 p. doi: 10.13102/sociobiology.v65i3.2821sociobiology 65(3): 506-514 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 breeding patterns and population genetics of eastern subterranean termites reticulitermes flavipes in urban environment of nebraska, united states introduction termite infestation of urban structures and landscapes can create very serious economic and environmental problems (chapman & bourke, 2001). in social insects, successful expansion of new intrusive species is largely due to a breeding system (ross, 2001). the breeding system consists of important reproductive characteristics of social organisms, including the number of breeders in a social group, the genetic relationships of these breeders, and the extent of variation in parentage among same sex-breeders (wade & kalisz, 1990; ross et al., 1993). in addition, dispersal of intrusive species into a new territory affects the propensity to monopolize a new habitat. the most likely reason is the new environment and ecology lead to a variation in the breeding system (porter et al., 1997). abstract reticulitermes flavipes (kollar) has become the most destructive subterranean termite pest, on urban structures in nebraska. in this study, we used seven microsatellite loci to infer the colony breeding system and population genetic structure among 20 infested urban structures in nebraska. our data revealed that 17 structures were infested by simple family colonies of r. flavipes, while, the remaining three were infested with mixed family colonies. the measure of population differentiation, f ct value (0.459) indicated that all the 20 urban colonies (10 410 km apart) represented pronounced levels of genetic differentiation. the mantel test disclosed a weak and significantly-positive correlation between genetic and geographic distance (slope = 0.0009, p = 0.001). the urban populations of r. flavipes in nebraska possessed a breeding system characterized by monogamous pairs of outbred reproductives with excessive heterozygosity. sociobiology an international journal on social insects ah ab majid1, 2, st kamble2, h chen3 article history edited by qiuying huang huazhong, agricultural university, china received 30 december 2017 initial acceptance 29 july 2018 final acceptance 17 august 2018 publication date 02 october 2018 keywords ubterranean termites, reticulitermes flavipes, urban environment, colony breeding, genetic structure. corresponding author dr. abdul hafiz ab majid household & structural urban entomology laboratory, vector control research unit, school of biological sciences universiti sains malaysia miden, 11800 penang, malaysia e-mail: abdhafiz@usm.my therefore, a thorough investigation of the breeding system of intrusive social insects is fundamental to further comprehend the association between their social structure, dispersal and invasion success. coptotermes spp. and reticulitermes spp. are among well-known members of the subterranean termite species, causing numerous damages to structures and/or buildings (curl, 2008). in general, the colony of the subterranean termite consists of a single pair of primary (winged) reproductives, which results in a simple family structure. the winged reproductives can disperse long distances, leading to a gene flow across the spatial scales. the pair of reproductives usually drop off their wings and starts a new colony (perdereau et al., 2010). later in the life cycle, brachypterous nymphs or workers produce secondary reproductives (neotenics) that can 1 household & structural urban entomology laboratory, vector control research unit, school of biological sciences, universiti sains malaysia, penang, malaysia 2 urban entomology laboratory, department of entomology, university of nebraska, lincoln, nebraska, united states 3 volusia mosquito control district, new smyrna beach, florida, united states research article termites sociobiology 65(3): 506-514 (september, 2018) 507 replace the primary reproductives. the neotenics are unable to fly, hence, inbreeding within the colonies can occur. colonies can be formed by multiple neotenics, resulting in extended colonies and occasionally forming spatially diffuse networks of interconnected reproductive centers (perdereau et al., 2010; vargo & husseneder, 2009). for example, r. labralis and r. chinensis can produce alates that can disperse colonies in the surrounding areas for approximately 5 years and 9 years, respectively, once the colonies are established. meanwhile, colonies headed by neotenics begin to produce alates on an average of 2.29 years (xing et al., 2015; ma, 1989; lieu et al., 2002; goodisman & crozier 2002; crissman et al., 2010). a study in massachusetts using allozyme and mitochondrial dna (mtdna) markers revealed a wide range of differences in the breeding structure of r. flavipes between two sites located 0.5 km apart (bulmer et al., 2001). the site with rocky and poorly-drained soil consisted of a mixture of extended families (60%) and simple families (40%), where the foraging ranges were limited. meanwhile, the site with more porous soil had equal numbers of spatially expansive extended families and mixed family colonies. in other studies, r. flavipes from central north carolina identified with microsatellite markers provided consistent results concerning colony breeding structure in this region (vargo, 2003a, b; deheer & vargo, 2004). these authors reported that most colonies (75%) were simple families, while, the remaining colonies (24%) were neotenic-headed extended families, descendants of the simple families. there was also a single colony with genetically diverse individuals originating from the fusion of two distinct colonies. the populations of r. flavipes show a variation in the breeding structure at both small and large spatial scales, depending upon ecological conditions. in the united states, the colony breeding structures of r. flavipes are classified into two types, type 1 and type 2. populations of type 1 comprise a majority of simple families (>50%), with some extended families headed by a few neotenics. meanwhile, introduced populations and one population in the new orleans, putatively a population from french, are classified as type 2. two populations i.e. mf massachusetts and ln nebraska are intermediate where extended and/ or mixed families are predominant and headed by hundreds of neotenics, particularly the massachusetts population. both populations also have a substantial proportion of simple-family colonies (clement, 1981; jenkins et al., 1999; bulmer et al., 2001; matsuura & nishida, 2001; deheer & vargo, 2004, 2008; deheer & kamble, 2008; perdereau et al., 2010; perdereau et al., 2015; ab majid et al., 2013). while a large body of work on genetic structure of subterranean termite colonies in north america has been published, specific data on the population genetic of r. flavipes associated with seasonal temperature fluctuations in the midwest of the united states are, lacking (bulmer et al., 2001; bulmer & traniello, 2002; vargo, 2003a, b; deheer & vargo, 2004, 2006, 2008; vargo & carlson, 2006; vargo et al., 2006b; deheer & kamble 2008; parman & vargo, 2008). porter et al. (1997) and tsutsui et al. (2003) reported that a new environmental condition can lead to an alteration in the breeding system due to ecological conditions such as the absence of competitors or parasites, or genetics changes due to behavioral traits associated with dispersal behavior. furthermore, climatic variables (mean annual temperature and seasonality) are also strongly affecting the inbreeding in r. flavipes. non-climatic variables, including the availability of wood substrate and soil composition are among other inbreeding factors as well, however, are more evident in r. grassei. these results indicated that termite breeding structure was shaped by local environmental factors and that the species can vary in their responses to these factors (vargo et al., 2013). in china, reticulitermes chinensis snyder, an important pest of trees and buildings from the genus reticulitermes were also genotyped at eight microsatellite loci. analysis of genetic clusters showed that the two subpopulations in chongqing city were headed by outbred unrelated pairs (huang et al., 2013). their study also showed that dispersal by primary reproductives was relatively limited due to the short range mating flight or due to the frequent colony reproduction. in recent years, molecular markers have become unequivocal identification tools for large numbers of termite colonies, particularly in investigating colony foraging areas, population dynamics, as well as colony breeding structure (husseneder et al., 2003). in this study, we used seven microsatellite markers to determine the colony genetic and breeding structures of r. flavipes sampled from 20 infested buildings in the urban habitats of nebraska. the phylogeny of r. flavipes colonies in the nebraska ecosystem was also discussed in this study. materials and methods termites samples of r. flavipes were collected from 20 infested buildings in nebraska (fig 1, table 1) during 2010 2011. the addresses and geographic location of termite feeding sites were recorded using a hand-held magellan® gps unit (sportrak™ map, thales navigation, huntington beach, ca). several termite samples from the infested buildings were provided by the pest control operators (pcos). distances between the sites were in the range of 10 to 410 km as measured by the google earth 7 (google inc., 2013). all workers from each collection point were stored in 95% ethanol and at -20o c immediately after the collection prior to dna extraction procedures. all termites were morphologically identified as r. flavipes according to husen et al. (2006). dna extraction genomic dna was individually extracted from heads of ten workers per site using a qiagen dneasy kit (qiagen, gaithersburg, md, usa). the manufacturer’s protocols were ah ab majid, st kamble, h chen – breeding patterns and population genetics of r. flavipes508 followed except that treatments with proteinase k solution and rnas were omitted. dna was eluted in 80 μl of 1x te solution. the dna concentration was quantified using an nd 1000 spectrophotometer (nanodrop technologies, inc. wilmington, de, usa). microsatellite genotyping we genotyped 10 termite workers per site at seven microsatellite loci: rf 1 3, rf 5 – 10, rf 6 – 1, rf 11 – 1, rf 1 – 2, rf 15 – 2 and rf 21 – 1 (vargo 2000). the pcr reactions were setup in 96 well plates in 15 µl reaction mixtures containing 1.5 µl of 10x of pcr buffer, 1.2 µl mgcl2, 0.75 µl dntps mixture, 1.5 µl forward primer 1.5 µl reversed primer, 0.15 µl taq dna polymerase, 1 µl dna template and 7.4 µl ddh2o. all loci were amplified using pcr thermal cycler program with an initial denaturation step at 95˚c for 30 sec, followed by 35 cycles at 95˚c (30 sec), 54˚c (30 sec), and 72˚c (30 sec). the reaction was concluded with one cycle 72˚c (5 min) and then cooled to and kept at 4˚c until removed from the pcr thermal cycler. fragments were separated by capillary electrophoresis using a beckman ceq 8000 genetic analyzer (beckman coulter, fulleeton ca, usa). data were visualized and hand-scored using ceq 8000 fragment analysis software version 8.0. microsatellite data analysis summary of population statistics (allelic diversity, expected and observed heterozygosity) was calculated using the fstat 2.9.3.2 (goudet 2001). exact tests of genotypic differentiation were performed using the webbased genepop (goudet et al.1996, raymond and rousset, 1995 [http://wbiomed.curtin.edu.au/genepop/index.html]). when two independent samples of workers were drawn from the same colony, we are sampling from the distribution of fig 1. sampling sites for reticulitermes flavipes in various counties of nebraska. al = lincoln; ap = arapahoe; ba = lincoln; bh = omaha; bl = bellevue; bx = lincoln; cb = cambridge; dc = david city; er = lincoln; fs = lincoln; gt = gothenburg; je = lincoln; ke = kearney; ky = kearney; mc = mccook; me = mead; mn = minden; nf = norfolk ; ri = lincoln; wy = wymore population abbreviation city county collection date ap arapahoe furnas 12 june 2011 bl bellevue sarpy 12 june 2011 cb cambridge furnas 12 june 2011 dc david city butler 12 june 2011 gt gothenburg dawson 17 june 2011 ke kearney buffalo 20 july 2011 ky kearney buffalo 20 july 2011 al lincoln lancaster 30 june 2011 ba lincoln lancaster 30 june 2010 bx lincoln lancaster 30 june 2010 er lincoln lancaster 30 june 2010 fs lincoln lancaster 30 june 2010 je lincoln lancaster 30 june 2010 ri lincoln lancaster 30 june 2010 mc mccook red willow 13 june 2011 me mead saunders 21 july 2011 mn minden kearney 22 july 2011 nf norfolk madison 22 july 2011 bh omaha douglas 1 july 2011 wy wymore gage 22 july 2011 table 1. collection sites for subterranean termites, reticulitermes flavipes in nebraska. genotypes within that colony. conversely, when two samples of workers are drawn from two different colonies, we are sampling from two different distributions of genotypes. this is true, regardless of the specific breeding pattern of colonies involved. therefore, if we test the differences in genotype frequencies between two workers, we expect the test to be significant if they come from different colonies, and nonsignificant if they come from the same colony. sociobiology 65(3): 506-514 (september, 2018) 509 colony breeding pattern breeding pattern was classified using techniques of vargo (2003a) and deheer and vargo (2004). individual workers from the same colony were grouped together to determine the simplest breeding system that could be invoked to explain the genotype distribution within each colony. colonies were considered as simple-families when genotypes and the frequencies of genotypes did not significantly differ from the assumed mother and father (primary reproductives) under simple mendelian patterns of inheritance (vargo, 2003b). colonies with five or more alleles at least one locus were regarded as mixed colonies headed by more than one pair of primary reproductives. the breeding structure could not be resolved unambiguously in the case of colonies that did not fit the expected genotype frequencies for progeny of a simple family and that had four or few alleles at all loci.. this is due to the fact that an extended family colony that contains secondary reproductives and a mixed colony in which the kings and queens happen to share the same four or fewer alleles cannot be distinguished clearly. genetic structure and relatedness the breeding patterns and genetic differentiation among colonies were further examined with hierarchical f statistics classification using techniques of vargo (2003a), deheer and vargo (2004). the fstat 2.9.3.2 (goudet, 2001) were also used for all genetic structure analyses. f-statistics followed the notation of thorne et al. (1999), with the subscripts i, c and t representing the individual, colony, and total components of genetic variation, respectively. the 95% confidence intervals were obtained by bootstrapping over loci 10, 000 times, and the significance of the estimates (coefficient) was further tested by permuting alleles among individuals (thorne et al., 1999; bulmer et al., 2001; copren, 2007; vargo & carlson, 2006; vargo et al., 2006a, b; parman & vargo, 2008). fic is a colony-level inbreeding coefficient which is perhaps the most useful measure as it varies with the number of reproductives as well as their spatial distribution within colonies. the number of reproductives and their mating patterns within a social group is reflected by fic values (crozier & pamilo, 1996; thorne et al., 1999; deheer et al., 2005; deheer & vargo, 2008; pedereau et al., 2010). for simple families, fic is expected to be strongly negative.fic values should approach zero with increasing number of reproductives within colonies, and to become positive if there is assortive mating among multiple reproductives within colonies or there is mixing of individuals from different colonies (thorne et al., 1999; deheer et al., 2005). the fit is analogous to the inbreeding coefficient fis, and thus, measures deviation from random mating in the population (the t subscript is synonymous with s because there is no subpopulation structure in this analysis). fct is comparable to the fst measure of differentiation among colonies which is very similar to relatedness (r) (thorne et al., 1999; deheer et al., 2005; deheer & vargo, 2008; pedereau et al., 2010). genetic relatedness among workers was estimated for each colony and averaged over colonies of the same site using the computer program fstat v. 2.9.3.2 (goudet, 2001). the correlation between genetic and geographic distances was carried out using the mantel test command in genalex6.4 (peakall & smouse, 2005). the mantel test was executed for matrix correspondence at permutation of 999 so as to test the isolation-by-distance effect. the unweighted pair-group method with arithmetic average clustering (upgma) we used the program tfpga (miller, 1997) to visualize the cluster of genetic similarity among the 20 subterranean termite colonies. the genetic distances were calculated using allelic frequencies and the dendogram was constructed using nei’s unbiased minimum distance (nei, 1978) and 1,000 permutations of bootstrapping. results colony diversity allelic diversity ranged between 1 and 6 alleles per locus, with an average of 2.293. the mean percentage of polymorphic loci was 85%. the observed heterozygosity (ho) (0.690) was higher than the expected heterozygosity (he) (0.416) (table 2). colony breeding pattern there was a strong and highly significant differentiation among the r. flavipes and among the pairwise sample points (p < 0.0001). therefore, all 20 sample sites represented different colonies. based on the genotypes at the seven microsatellite loci and mendel’s laws, 17 samples were classified as simple family colonies, and the remaining three samples i.e. ap (arapahoe), cb (cambridge), and ba (lincoln) were identified as mixed family colonies (table 2). colony genetic structure the genetic differentiation between the simple and mixed family colonies was evaluated by the f statistics and relatedness (table 2). the inbreeding coefficient fis (0.099, p < 0.001) and the relatedness r (0.846) in the simple family colonies were higher than those of in the mixed family colonies (fis = 0.077, p = 0.089; r = 0.667), indicating that the individuals in the simple family colonies had a higher genetic similarity. the colony-level inbreeding coefficient fic value (-0.6840) in the simple family colonies was negatively higher than that of the mixed family colonies (-0.440), suggesting fewer reproductives involved in the simple family colonies. the genetic differentiation fct among the simple family colonies was 0.465 (p < 0.001), higher than that of ah ab majid, st kamble, h chen – breeding patterns and population genetics of r. flavipes510 colony code breeding pattern number of alleles per locus mean of allele/locus ho he rf 11-3 rf 5-10 rf 6-1 rf 11-1 rf 11-2 rf 15-2 rf 21-1 dc simple 1 1 2 2 3 2 2 1.857 0.714 0.388 bh simple 2 1 2 2 1 2 2 1.714 0.557 0.311 bl simple 3 2 4 2 2 2 2 2.429 0.900 0.524 fs simple 2 2 4 2 3 2 2 2.429 0.729 0.449 al simple 2 3 3 4 2 2 4 2.857 0.629 0.499 ap mixed 2 3 6 3 4 3 3 3.429 0.786 0.520 mc simple 2 2 2 1 2 2 2 1.857 0.786 0.416 cb mixed 2 2 3 6 2 2 1 2.571 0.529 0.377 ky simple 4 1 2 2 3 3 4 2.714 0.686 0.459 ke simple 2 3 4 3 3 2 2 2.714 0.871 0.515 nf simple 2 1 2 4 2 2 2 2.143 0.657 0.366 wy simple 2 2 4 2 1 1 1 1.857 0.300 0.205 er simple 3 2 2 2 2 1 1 1.857 0.586 0.349 gt simple 3 1 2 2 2 3 1 2.000 0.557 0.334 mn simple 1 2 2 2 3 2 3 2.143 0.857 0.446 ri simple 1 2 2 3 3 3 3 2.429 0.857 0.464 je simple 2 1 2 2 2 2 2 1.857 0.786 0.411 bx simple 2 1 2 3 1 2 2 1.857 0.443 0.288 ba mixed 5 3 3 2 2 2 2 2.714 0.743 0.491 me simple 2 3 2 2 3 1 4 2.429 0.826 0.459 average 2.3 1.9 2.8 2.4 2.3 2.1 2.3 2.293 0.690 0.416 table 2. statistics of microsatellite loci and inferred breeding patterns in the 20 colonies of reticulitermes flavipes collected from infested urban structure in nebraska. the mixed family colonies (0.359; p < 0.001), revealing more genetic separation among the simple family colonies. although the 95% confident intervals were overlapped (table 2), all estimations displayed the same trends in genetic differentiation between the simple and mixed family colonies, supporting our classification of the colony breeding patterns in this study. we found the total inbreeding coefficient fit (0.111, p < 0.001) and overall relatedness r (0.826) were significant and high. the fct value (0.459) was also significant (p < 0.01) (table 3), strongly suggesting that the genetic structure was shaped by non-random mating among the studied colonies. on the other hand, the mantel test gave a weak but positive correlation between genetic and geographic distances (y = 0.0009x + 10.782, p = 0.001), indicating a subordinate distance effect on the genetic differentiation among the studied colonies. aupgma dendrogram is presented in fig 2. several colonies from nearby sites (e.g., je and ri) or neighboring counties (e.g., ky and gt) were clustered together as well as a few colonies from far distances such as ap, me, mc, mn, je, and ri. these showed that the effect of geographic distance was sporadic and minor, consistent with the conclusion that the genetic structure among the termite colonies was mainly influenced by their breeding patterns as revealed by the relatedness, f-statistics and mantel test. colony location fit p-value fct p-value fic p-value r empirical values all colonies (n=20) 0.111 0.000 0.459 0.000 -0.642 0.000 0.826 (ci) (-0.014 ̶ 0.22) (0.401 ̶ 0.51) (-0.721 ̶ -0.545) (0.791 ̶ 0.863) simple family colonies (n=17) 0.099 0.000 0.465 0.000 -0.684 0.000 0.846 (ci) (-0.027 ̶ 0.201) (0.405 ̶ 0.516) (-0.751 ̶ -0.605) (0.816 ̶ 0.877) mixed family colonies (n=3) 0.077 0.089 0.359 0.000 -0.440 0.000 0.667 (ci) (-0.108 ̶ 0.252) (0.268 ̶ 0.444) (-0.627 ̶ -0.219) (0.535 ̶ 0.774) table 3. f-statisticsand relatedness coefficient: reticulitermes flavipes worker relatedness estimates (r value) from 20 infested urban structures. confidence intervals of 95% are shown in parentheses and the sample size n refers to the number of colonies studied in each population). p-values were estimated by permutations. sociobiology 65(3): 506-514 (september, 2018) 511 discussion overall, the subterranean termites from 17 sites belonged to the simple family colonies, whereas the other three sites/locations (ap, cb, and pa) were found to be of mixed family colonies. no extended family was found in this study. all samples had excess heterozygositybut only the simple family colonies showed significant inbreeding (fit = 0.099, p < 0.001). the negative values of fic were observed in all colonies regardless of the family types. it can be assumed that colonies with spatially-separated reproductive centers were most likely absent, as this configuration can yield fic values greater than zero under a large range of conditions (thorne et al., 1999; nobre et al., 2008). in contrast, our data yielded negative fic values, indicating no contribution of colonies being headed by multiple neotenic reproductives inbred for several generations to the reproduction of the population studied. the presence of at least four alleles in each colony was compatible with the hypothesis that all breeders from a colony were descended from a single pair of unrelated primary reproductives. the f statistics for the simple family colonies were consistent with monogamous reproductive pairings (table 3) and all colonies in this category should have a single feeding site (thorne et al., 1999; deheer et al., 2005; deheer & vargo, 2008; pedereau et al., 2010). the frequency of the simple families was 85% in this study. the frequency of the simple families in r. grassei populations from francewas 73% (clement, 1981; deheer et al., 2005). in coptotermes formosanus, 90% of the colonies infesting structures in nagasaki, japan consisted of simple families (vargo & hussender, 2009). based on the single polymorphic allozyme marker, clement (1981) reported a high proportion of simple families of r. santonensis population in the la coubre forest. in the central north carolina, ~75% of r. flavipes colonies were simple families, ~ 25% contained low numbers of neotenic reproductives descended from simple families, and 1-2% were mixed families (vargo, 2003a, b; deheer & vargo 2004). in massachusetts, most colonies possessed numerous neotenics, of which ~ 33% were simple families and ~ 10% were mixed families (bulmer et al., 2001). the negative values of fic in the three mixed family colonies indicated that colony fusion was uncommon if ever occurred. fic value could be positive as two or more unrelated colonies mixed (vargo & husseneder, 2011). in addition, the high relatedness (r > 0.5) in this study suggested that kin selection might occur in this species, especially in mixed family colonies (crozier et al., 1987). according to thorne (1997), the kin selection proposed for the diploid termites was the mechanism to generate similarly-skewed degrees of relatedness among sibs in comparison to the relatedness between parents and offspring. these would facilitate kin selection as the driving force toward eusociality in the termites and raised unrelated nymphs by indirect fitness and altruism in the colonies. furthermore, relatedness in these colonies remains high enough for sterile workers to achieve some indirect fitness benefits (goodisman & crozier, 2002). the correlation between genetic and geographic distances is an indication of genetic differentiation attributed to separation-by-distance even though the correlation is weak. this correlation reflects where populations at greater distances are more genetically distinct than those of populations that are geographically close. the weak correlation between genetic and geographic distances in this study could be due to inbreeding wahlund effect, founder effect, or technical issues including null alleles and errors (vargo & husseneder, 2011; pusadee et al., 2009). it occurs when colonies fuse either they are in the early stages of the fusion process or because there is no interbreeding between reproductives in the two (or more) original colonies (perdereau et al., 2015). the presence of null alleles could lead to an overestimation of both fct and genetic distance. errors during the allele scoring and data analyses could affect the correlation between geographic distance and genetic distance (chapuis & estoup, 2007). furthermore, according to vargo and husseneder (2011), ecological factors could shape the colony breeding structure, especially factors that select against inbreeding. a study in the region of southeastern nebraska with the dissected till plains and the great plains (fraser, 1998) demonstrated that the lands are flat, and lack of distinct geographic factors such as big mountains and lakes to impact the dispersal of r. flavipes. colony reproduction by budding, if common, should lead to a significant positive relationship between main parental colony and the satellite nest. moreover, if both parents and offspring colonies contain large numbers of replacement fig 2. the upgma dendrogram of nei’s unbiased minimum distance (nei,1978) over all 20 populations of reticulitermes flavipes that are denoted in abbreviation. the number at the nodes indicate the bootstrap percentages > 50%. al = lincoln; ap = arapahoe; ba = lincoln; bh = omaha; bl = bellevue; bx = lincoln; cb = cambridge; dc = david city; er = lincoln; fs = lincoln; gt = gothenburg; je = lincoln; ke = kearney; ky = kearney; mc = mccook; me = mead; mn = minden; nf = norfolk ; ri = lincoln; wy = wymore ah ab majid, st kamble, h chen – breeding patterns and population genetics of r. flavipes512 reproductives (neotenics), it may take several generations for genetic drift to allow one to detect significant genetic differentiation between these physically-separated colonies (thorne et al., 1999; vargo, 2003a, b). nonetheless, no genetic data are available to support the view of budding thus far. in addition, budding in reticulitermes spp. is not common. during mating flights, the reproductives disperse relatively far or they actively avoid relatives when forming tandem pairs (bulmer & traniello, 2002; deheer & vargo, 2004; vargo et al., 2006). in this study, colonies from two locations, namely mccook (mc) and mead (me), were clustered together in the upgma dendrogram and were inferred as simple families although both locations were > 300 km apart. (fig 2 and table 2). furthermore, these two samples shared the same ecological conditions and genetic similarities, thus determining which of these two factors is of more importance for shaping the breeding structure was hardly possible. other factors may involve such as founder effects; typically occur when a small number of individuals invade a new habitat and ultimately produce a new population. moreover, houses built on natural populations can lead to habitat fragmentation due to human disturbance (booth et al., 2012; vargo et al., 2006). vargo and husseneder (2011) suggested that genetic structure in termite populations with dispersal andcolony breeding regime should be studied in order to gain a better understanding of the roles of genetic drift and selection that may eventually lead to speciation in the termites. however, from the above-mentioned explanations, it is common to encounter distant populations that are genetically similar, and close populations that are genetically different due to the weak correlation between genetic and geographic distance. given the limited data on breeding and population genetic structure, there could be an ample opportunity to study the natural selection pressures affecting termite populations and colony breeding structure. long-term studies across nebraska either in urban or rural areas concerning r. flavipes breeding structure are needed to determine its overall patterns. in addition, further studies examining other urban population in big cities e.g. omaha and lincoln will facilitate us to better understand the breeding structure of r. flavipes in the nebraska ecosystem, mainly after the buildings have been treated for termite infestation. more studies are also required to investigate the effect of urbanization on overall breeding and genetic structures of the termites. our study documents the initial data on the breeding and population genetic structure of r. flavipes from the urban environment in nebraska. acknowledgments we greatly appreciate the support and guidance from urban entomology laboratory members, department of entomology, university of nebraska-lincoln, tim husen and ralph narain. references ab majid a.h., kamble s.t., miller n.j. 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(2015). the ‘floppy-wing’ morph of the subterranean termite reticulitermes labralis has a secondary reproductive function. insectes sociaux, 62: 183-191. doi: 10.13102/sociobiology.v61i2.234-236sociobiology 61(2): 218-220 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 assessing the utility of a pcr diagnostics marker for the identification of africanized honey bee, apis mellifera l., (hymenoptera: apidae) in the united states al szalanski, ad tripodi the africanized honey bee was first detected in texas in 1990 (sugden & williams, 1990), and usda reports it has been established in ten states: arizona, arkansas, california florida, louisiana, nevada, new mexico, oklahoma, texas and utah (anonymous, 2011) following a tragic death in 2010, africanized honey bees were confirmed in georgia (berry, 2011). hybrids between africanized and european honey bees are morphologically similar and difficult to distinguish from one another. two kinds of laboratory-based techniques, morphometric analysis (rinderer et al., 1993) and molecular diagnostics (sheppard & smith, 2000), are commonly employed to determine africanized status, but to our knowledge, these have not been compared. the typical morphometric approach to africanized honey bee detection is the fast africanized bee identification system (fabis) method (rinderer et al., 1987), although more precise methods such as automatic bee identification system (abis) (steinhage et al. 2001) are now available. morphometric diagnostic techniques require measurements from 10 or more freshly collected specimens (meixner et al., 2013) to assign a colony to one of four categories: 1) africanized (ahb), 2) ahb with european (ehb) traits, 3) ehb with ahb traits and 4) ehb (sylvester & rinderer abstract an assessment of a molecular diagnostic technique for distinguishing africanized honey bees from european honey bees in the united states was conducted. results from multiplex pcr diagnostics of a mitochondrial dna cyt-b marker corresponded with results based on coi-coii sequencing analysis, but differed from morphometric analysis results. we suggest utilizing both multiplex pcr and morphometric methods for africanized honey bee diagnostics in the united states, when possible. sociobiology an international journal on social insects university of arkansas, fayetteville, arkansas, usa. article history edited by gilberto m m santos, uefs brazil received 17 december 2013 initial acceptance 20 february 2014 final acceptance 05 march 2014 keywords apis mellifera, molecular diagnostics, africanized honey bee, mtdna, usa corresponding author allen szalanski dept. entomology, university of arkansas, 319 agriculture building fayetteville, ar 72701 e-mail: aszalan@uark.edu 1987). molecular diagnostics rely upon mitochondrial dna (mtdna), typically a region of the cytochrome b gene (pinto et al. 2003). identification of africanized bees with mtdna is relatively easy, because a single worker in any condition can represent a colony’s mtdna lineage (sheppard & smith, 2000). yet, since mtdna is maternally inherited, mtdna markers cannot determine if a european queen has mated with africanized drones, and such colonies will remain undetected with mtdna-based techniques. molecular identification of africanized bees is also conducted with an mtdna cytochrome b marker (cyt-b) as it has a low level of intraspecific variation in honey bees (crozier et al., 1991; szalanski & mckern, 2007). techniques typically used are either pcrrflp (pinto et al., 2003) or multiplex pcr using a primer specific for africanized honey bees (szalanski & mckern, 2007). both of these methods yield identical results. despite the utility of molecular markers, it is unknown how well they correlate with morphometric identification and how the cyt-b marker can distinguish the a (african) lineage of honey bees relative to the o (middle eastern), c (eastern europe) and m (western europe) lineages (ruttner, 1987), which are often determined through coi-coii sequencing. the objective of this study was to determine how a cyt-b multiplex technique short note sociobiology 61(2): 234-236 (june, 2014) 235 for distinguishing africanized from european bees compares with coi-coii dna sequence and fabis morphometric techniques. a total of 968 samples from swarms, feral colonies and managed honey bee colonies collected from arizona, arkansas, california, florida, georgia, hawaii, kansas, louisiana, missouri, mississippi, nebraska, new mexico, oklahoma, texas and utah during 1991-2013 were analyzed. samples were collected by various agencies and preserved in 70-100% ethanol. samples collected from texas from 1991 to 2008 were identified as africanized (ahb, n = 54), ahb with evidence of introgression of european genes (ahb.e, n = 23), ehb with evidence of africanized genes (ehb.a, n = 22) or ehb (ehb, n = 39) using fabis following rinderer et al. (1993). for the multiplex pcr diagnostics, dna was extracted from two workers from each sampled colony, and pcr of a portion of the cyt-b region was conducted per szalanski and mckern (2007). this multiplex results in a control amplicon of 485 bp for both africanized and european bees and a 385 bp amplicon unique to africanized bees. samples exhibiting a single electrophoretic band are diagnosed as european (multiplex ehb) and those with two are diagnosed as africanized (multiplex ahb). dna sequencing analysis of a coi-coii marker was conducted following szalanski and magnus (2010), and samples from that study were additionally subjected to our multiplex procedure for comparison (n = 360). dna sequences were identified to haplotype and assigned to lineage using genbank blast searches and our own database. africanization diagnoses from cyt-b multiplex diagnostics, coi-coii dna sequencing and fabis morphometric analysis were then compared with one another. of the 968 samples subjected to multiplex diagnostics, 318 samples were diagnosed as africanized and 650 were diagnosed as european (table s1). a total of 12 haplotypes from the a (african) lineage were observed, and four o (middle eastern), five m (western europe) and 11 c (eastern europe) haplotypes were compared with the multiplex results (table 2). all of the multiplex pcr identified africanized samples fell within the a lineage while the european samples were o, m or c lineages. the multiplex method reaches exactly the same diagnoses as the sequencing method but can be carried out in a single pcr step without subsequent dna sequencing. africanization diagnoses in the multiplex and fabis methods differed quite dramatically (table 3). of the 107 samples the exhibiting africanized signature in the pcr-multiplex, only 84 were diagnosed as of african descent (ahb, table 1. sampled states and results of multiplex pcr identification of samples as africanized or european honey bees. state n multiplex ahb multiplex ehb arizona 1 0 1 arkansas 143 1 142 california 3 3 0 florida 1 1 0 georgia 2 1 1 hawaii 124 1 123 kansas 18 0 18 louisiana 24 0 24 mississippi 20 0 20 missouri 25 0 25 nebraska 19 0 19 new mexico 62 45 17 oklahoma 178 58 120 texas 140 109 31 utah 208 99 109 total 968 318 650 table 2. correlation of mitochondrial dna coi-coii dna sequence lineages with cyt-b multiplex pcr identification. lineage (n haplotypes) n multiplex ahb multiplex ehb c (11) 173 0 173 m (5) 12 0 12 o (4) 19 0 19 a (12) 156 156 0 total 360 156 204 ahb.e and ehb.a) using fabis. this suggests that 21% of africanized bees in our sample were cryptically africanized and undetectable using morphology-based methods. similarly, of the 99 bees exhibiting morphometric characteristics of africanization (ahb, ahb.e and ehb.a), 15% were misdiagnosed as european by the multiplex. interestingly, our results suggest that only a small proportion (15%) of colonies that exhibit africanized morphology were founded by european queens inseminated by africanized drones. coi-coii lineage data includes haplotype determinations from szalanski and magnus (2010). this study verified the utility of using a cyt-b pcrmultiplex technique for identifying honey bees of the a lineage in the united states. identification of africanized matrilines through cytb-b diagnosis parallels classic coicoii matriline determination through sequencing, but in a single-step, lesser cost procedure. we also found evidence that using either fabis or mtdna determination alone may underestimate the occurrence of africanized populations. for greater certainty in diagnosing africanized populations of honey bees, we suggest utilizing both morphological and molecular methods when the number of samples makes this possible. al szalanski & ad tripodi africanized honey bee diagnostics236 acknowledgements we thank numerous beekeepers and the utah department of agriculture and food, along with ed levi, richard grantham, danielle downey, lisa bradley and clarence collison for providing samples. we thank lisa bradley for doing the morphometric analysis of the texas samples, and clinton trammel for assisting with the genetic analysis. this research was supported in part by the university of arkansas, arkansas agricultural experiment station. references anonymous (2011). map of the spread of africanized honey bee by year. http://www.ars.usda.gov/research/docs. htm?docid=11059&page=6. berry, j. 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(2010). mitochondrial dna characterization of africanized honey bee (apis mellifera l.) populations from the usa. journal of apicultural research, 49: 177-185. doi: dx.doi.org/10.3896/ibra.1.49.2.06 table 3 comparison of morphometric and multiplex pcr identification of africanized and european honey bees from texas. morphometric identification* n multiplex ahb (%) multiplex ehb (%) ahb 54 48 (89) 6 (11) ahb.e 23 19 (83) 4 (17) ehb.a 22 17 (77) 5 (23) ehb 39 23 (59) 16 (41) total 138 107 31 *ahb: africanized; ahb.e: africanized with evidence of introgression of european genes; ehb.a: european with evidence of introgression of africanized genes; ehb: european (rinderer et al., 1993). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.133-135sociobiology 61(2): 133-135 (june, 2014) activity patterns of the red harvester ant in a mexican tropical desert l. ríos-casanova1, g. castaño2, v. farías-gonzález1, p. dávila1, h. godínez-alvarez1 introduction red harvester ant (pogonomyrmex barbatus smith) is one of the most common and abundant species in deserts of usa and mexico (johnson, 2000). this ant harvests high proportions of seeds from annual and perennial plants, affecting their abundance and distribution. activity patterns determine periods in which ants forage seeds, therefore it is essential to document them and how they change along year in sites located at different latitudes. activity patterns of several pogonomyrmex species, including p. barbatus, have been studied in north american temperate deserts. diurnal ants are active in the morning and late afternoon with a period of decline around midday when temperatures reach maximum values, showing a bimodal pattern of activity most of the year (whitford, 1978; mackay & mackay, 1989; pol & lopez de casenave, 2004). ants diminish activity during winter changing to inactivity or to a unimodal pattern by being active during the hottest hours of the day (whitford & ettershank 1975; hölldobler & wilson, abstract red harvester ant (pogonomyrmex barbatus) inhabits deserts of usa and mexico. its activity patterns are well known in temperate deserts, but they have not been studied in tropical ones. we studied these patterns in the tehucan valley, a tropical desert in central mexico. it had bimodal activity patterns in spring, summer, and fall while unimodal patterns in winter. these patterns differ from those reported for this species in temperate deserts where activity stopped in winter. our results suggest that p. barbatus extends its activity periods and remains active all year round in the tehuacan valley. sociobiology an international journal on social insects 1 ubipro, fes-iztacala, universidad nacional autónoma de méxico, tlalnepantla, estado de méxico, méxico. 2 facultad de ciencias, campus juriquilla, universidad nacional autónoma de méxico, querétaro, méxico. article history edited by gilberto m m santos, uefs, brazil received 02 december 2013 initial acceptance 05 february 2014 final acceptance 19 may 2014 keywords pogonomyrmex barbatus, deserts, soil surface temperature, tehuacan valley corresponding author leticia ríos-casanova ubipro, fes-iztacala universidad nacional autónoma de méxico av. de los barrios 1, los reyes iztacala tlalnepantla, estado de méxico, méxico 54090 e-mail: leticiarc@campus.iztacala.unam.mx 1990; crist & macmahon, 1991). although these activity patterns are known in temperate deserts, they have not been studied in tropical ones. pogonomyrmex barbatus is distributed in north american temperate and tropical deserts; however, no reports exist about its activity in the tropics. we study activity patterns of this ant species in the tehuacan valley, a tropical desert in central mexico. this study represents the southernmost site where activity has been studied until now (figure 1). because seasonal and daily temperature variation is lower in tropical than in temperate regions (mackay & mackay, 1989), we expect p. barbatus will be active all year round at the tehucan valley compared to northern locations. this paper reports the number of ants returning to the nest and soil surface temperature for all seasons of the year. although activity patterns depend on environmental factors such as seed availability, daily and seasonal temperatures, we only measured soil surface temperature because it is considered the most important factor regulating ant activity in deserts (whitford, 1999). research article ants l ríos-casanova, et al activity patterns of the red harvester ant in a tropical desert134 material and methods this study was conducted at san rafael coxcatlán (18° 12’ 18° 14’ n, 97° 07’97° 09’ w; 1000m a. s. l.; fig 1) in the tehuacán valley, central mexico. the mean annual temperature is 25°c and the mean annual rainfall is 394.5 mm (valiente, 1991). the main vegetation type is tropical deciduous forest dominated by fouquieria formosa, bursera aptera, and ceiba parvifolia (ríos-casanova et al., 2006). we selected 8 nests separated by at least 10 m each, to count the number of ants returning to nest during 5-min periods. counts were conducted from 0700–1900 h during one day in fall (september 2010), winter (december 2010), spring (march 2011), and summer (july 2011) for a total of 384 observation periods. nocturnal activity was not recorded. soil surface temperature was recorded every hour (0700–1900 h) by placing one thermometer at 30 cm from nest entrance and burying the mercury containing bulb at 1 cm depth. results ants showed a bimodal activity pattern during fall, spring, and summer (fig. 2). foraging activity started when surface temperature rose to 21°c. the first activity peak (6071 ants/5 min) occurred between 11-13 h, when soil surface temperature was 36-42ºc. the second peak (10-40 ants/5 min) occurred in the afternoon (16-17 h), when temperature was 39-47ºc. foragers stopped activity between peaks in spring when maximum temperature was ca. 50ºc while they were always active between peaks in summer and fall. the maximum temperature in these seasons was 45-47ºc. ants only showed one activity peak in winter (20 ants/5 min), which occurred during midday when temperature was 40°c, which was the highest temperature recorded in the season (fig. 2). discussion our results showed that p. barbatus has bimodal activity patterns in spring, summer, and fall, and unimodal activity patterns in winter. activity peaks in bimodal patterns occurred when soil surface temperature was 36-47ºc. activity stopped when temperature rose to 50ºc. activity peak in unimodal patterns occurred when temperature was 40ºc. the activity pattern found in our study differs from that reported for p. barbatus in a temperate desert (garcíapérez et al., 1994), where bimodal activity patterns occurred in spring and summer, and unimodal patterns ocurred in fall. this ant species was not active in winter (garcia-perez et al., 1994). our findings therefore suggest that p. barbatus inhabiting a tropical desert extends its activity periods and remains active all year round, according to our predictions. despite differences in activity patterns, it seems that soil surface temperature is one of the main environmental facfig 1. localities where pogonomyrmex activity has been studied. 1 = p. owyheei, 2 = p. occidentalis (bernstein, 1979), 3 = p. californicus, p. rugosus, (bernstein, 1974), 4 = p. montanus, 5 = p. rugosus, 6 = p. subnitidus (mackay & mackay, 1989), 7 = p. barbatus (gordon, 1983), 8 = p. rugosus (whitford & ettershank, 1975, 9 = p. badius (golley & gentry, 1964), 10 = p. barbatus (garcía-pérez, et al., 1994), 11 = p. barbatus (this study). fig 2. mean soil surface temperature ± 1 standard error (solid line) and average number of active ants (p. barbatus) outside the nest ± 1 standard error (dashed line) hourly and four seasons of the year in san rafael coxcatlán, puebla, mexico. sociobiology 61(2): 133-135 (june, 2014) 135 tors regulating activity of this ant species in temperate and tropical deserts. johnson (2000) in analyzing physiological thermal tolerance of p. barbatus from the chihuahuan desert found that ant foragers stopped their activity at 47°c because they were unable to survive at higher temperatures. our data on soil surface temperature indicate that ant activity stopped at 50°c. other pogonomyrmex species such as p. montanus, p. subnitidus, p. apache, and p. rugosus from temperate north american deserts also reduce or stopped their activity when surface temperature was > 50°c (mackay & mackay, 1989; hölldobler & wilson, 1990; whitford, 1999). these findings suggest that activity patterns of pogonomyrmex species are regulated by soil surface temperature. this may be interpreted as an adaptation of these ants to high desert temperature, independently of latitude, which may be a phylogenetically conserved trait (macmahon et al., 2000). our results however should be interpreted cautiously because they assume that soil surface temperature is the only factor regulating ant activity. other factors such as seed availability, presence of other harvester ants, predation, and plant relationships which also regulate activity were not measured in this study (wilby & shachak, 2000; pol & lópez de casenave, 2004). these factors should be considered in future studies to determine their relative importance on ant foraging activity. in short, p. barbatus extends its activity period and remains active all year round in the tehuacan valley. evaluation of activity patterns throughout its distribution is essential to understand seed foraging impact on plant abundance and distribution. acknowledgments we thank authorities of san rafael coxcatlán, puebla for their permission to work in their lands. this work was supported by grants from facultad de estudios superiores iztacala, universidad nacional autónoma de méxico, proyecto papca no. 2010-2011 to l.r-c. and v.f-g. we thank two anonymous referees for their comments and criticisms on the manuscript. references bernstein, r. 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(2004). activity patterns of harvester ants pogonomyrmex pronotalis and pogonomyrmex rastratus in the central monte desert, argentina. journal of insect behavior, 17: 647-661. doi: 0892-7553/04/09000647/0 ríos-casanova, l., valiente-banuet, a. & rico-gray, v. (2006). ant diversity and its relationship with vegetation and soil factors in an alluvial fan of the tehuacán valley, mexico. acta oecologica, 29: 316-323. doi:10.1016/j.actao.2005.12.001 valiente, b.l. (1991). patrones de precipitación en el valle semiárido de tehuacán, puebla, méxico. tesis de licenciatura. facultad de ciencias, unam, méxico. 61 pp. whitford, w.g. (1978). structure and seasonal activity of chi-structure and seasonal activity of chihuahua desert ant communities. insectes sociaux, 25:79-88. whitford, w.g. (1999). seasonal and diurnal activity patterns in ant communities in a vegetation transition region of southern new mexico (hymenoptera: formicidae). sociobiology, 3: 477-492. whitford, w.g. & ettershank, g. (1975). factors affecting foraging activity in chihuahuan desert harvester ants. environmental entomology, 4: 689-696. wilby, a. & shachak, m. (2000). harvester ant response to spatial and temporal heterogeneity in seed availability: patterns in the process of granivory. oecologia, 125: 495-503. doi:10.1007/s004420000478 checklist of social.indd open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 101-106 (2013) checklist of social (polistinae) and solitary (eumeninae) wasps from a fragment of cerrado “campo sujo” in the state of mato grosso do sul yc grandinete1 fb noll2 introduction wasps are commonly known by their great variety of nest construction and, sometimes, their painful stings. with more than 5,000 species (pickett & carpenter, 2010), vespidae has six known subfamilies (carpenter, 1982; pickett & carpenter, 2010): euparagiinae, stenogastrinae, masarinae, eumeninae, polistinae and vespinae. in brazil, only masarinae, eumeninae and polistinae are recorded (carpenter & marques, 2001). polistinae wasps are the well-studied group, although it comprises only one fifth of the diversity of the family (pickett & carpenter, 2010). they are used as a model of evolution of social behavior but, besides that, they are considered as important “ecological engineers” (jones et al. 1994), because they change the physical environment and affect the availability of resources for other organisms (kears et al., 1998; alonso & agosti, 2000; kaspari, 2000). besides that, they contribute for the soil formation, biological control, abstract cerrado is one of the richest biomes in the world but it is still very threatened by human actions that affect not only the flora, but also the fauna, and certainly the largest known group, the insects. we present here a list of species of polistinae and eumeninae wasps, based on three different methodologies in a fragment of cerrado “campo sujo”, very affected by livestock raising, on paranaíba, mato grosso do sul state. we recorded 22 species of polistinae within 8 genera, with polybia and agelaia as the most representative. for eumeninae, 21 species within 10 genera were recorded, with montezumia and pachodynerus as the most representative. furthermore, this work shows the first records of eumeninae on mato grosso do sul state and some new of polistinae, compared to the literature. sociobiology an international journal on social insects 1 universidade de são paulo (ffclrp-usp), ribeirão preto, são paulo, brazil 2 universidade estadual paulista “julio de mesquita filho" (unesp), são josé do rio preto, são paulo, brazil research article wasps article history edited by: fabio nascimento, usp brazil received 24 october 2012 initial acceptance 11 december 2012 final acceptance 14 february 2013 keywords cerrado, polistinae, eumeninae, wasps. corresponding author yuri campanholo grandinete universidade de são paulo faculdade de filosofia, ciências e letras de ribeirão preto av. bandeirantes, 3900. ribeirão preto, sp, brazil, 14040-901 e-mail: grandineteyc@gmail.com pollination (van mele & cuc, 2000). according to pickett and carpenter (2010), this cosmopolitan subfamily has 958 described species in 26 genera, but the main diversity is in the neotropical region, mainly in brazil, with 21 genera and 304 recognized species (carpenter, 2004). commonly known as “potter wasps” due to the shape of the brood cells of some species, eumeninae is the most diverse in vespidae with more than 3,500 described species in 210 genera and brazil has approximately 277 species in 31 genera (pickett & carpenter, 2010). these wasps have solitary behavior, however there are some species with primitively social behavior, like zethus miniatus de saussure, which groups of females build communal nests made by plant material and resin (bohart & stange, 1965; westeberhard, 1987). according to mma (2010), the cerrado has 5% of all biodiversity from the planet. it is the richest biome in the world, although it is one of the most threatened. in brazil, it is the second in extension and, until 2008 the biome had yc grandinete, fb noll social and solitary wasps from cerrado102 already lost almost 50% of the original vegetation, because it has conducive characteristics to agriculture, livestock and the demand for charcoal for the steel industry. this dynamics of substitution includes deforestation and forest fire, causing environmental changes, habitat fragmentation, species extinction, invasion of exotic species, erosion, aquifer pollution, siltation of rivers and the imbalance of the carbon cycle. although it is obvious the importance of the cerrado and its biodiversity and preservation (oliveira & marquis, 2002), there are still a few studies focusing on insects, mainly wasps, which still need more research on their taxonomy, phylogenetic, behavior and ecology. for these reasons, this study was conducted to improve the knowledge or polistinae and eumeninae of cerrado, giving the first records of solitary wasps to mato grosso do sul state, and updating those from polistinae. material and methods the study was conducted on paranaíba municipality, state of mato grosso do sul, more specifically at “fazenda prata” (19º 48’45.83’’ s; 51º 06’ 25.22’’ o), in a “campo sujo” cerrado, characterized by a mixed flora, comprised of forests and grassland elements. besides that, the area has herbaceous vegetation, with predominance of poaceae, many shrubs and arboretums (coutinho, 1978, 2002). data sampling was carried out on a monthly basis, between october 2009 and september 2010, for five consecutive days, except in november due to constant rainfall. three methodologies were used to evaluate the diversity of polistinae and eumeninae: i) ten “attractive traps” were made by using pet bottles, where two openings were made with approximately 50 cm2 on center (adapted from jacques et al., 2012) and later they were strung and hung (using string) on vegetation between a 1.5 m and 2.0 m from the soil. inside the bottles we put industrial orange juice solution (about 200 ml juice per bottle) to attract the wasps. each month, we chose a random transect and set the “attractive traps” up along a 200 m, transect 20 m apart from each other. the traps were installed on the first and third days, the insects were collected and the orange juice was exchanged. on the fifth day, the same procedure was performed. the insects were kept on 70% alcohol for being pinned and identified later; ii) active collection, with entomological net. in each collection, 40 plants with flowers were chosen randomly and the insects were collected during five minutes per plant; iii) attractive solution (noll & gomes, 2009). this method uses a 10l dorsal spray bag, which contained the attractive solution made of a water based solution of crystal sugar (sucrose – 200g/l) and salt (sodium chloride – 25 g/l). the attractive solution was sprayed in 10 points equally separated within a 200 m transect. at each point, an average of 500 ml of solution was applied. the application was done following a zigzag pattern from left to right, generally applied on green vegetation, with solar incidence in an area of 3 m2. after the application of the attractive solution, each point was individually observed for five minutes and wasps that visited these points were collected with an entomological net. after collecting wasps at the 10 points, the solution was applied again at every point. four applications were made during the day, usually between the periods from 10:00 h to 16:00 h. results and discussion polistinae we collected 574 specimens of social wasps, belonging to 22 species within eight genera: agelaia, apoica, brachygastra, mischocyttarus, parachartergus, polistes, polybia and synoeca (table 1). the most representative genus was polybia (46.51%), followed by polistes and mischocyttarus, although this last had only one or two species collected. polybia lepeletier is the group within epiponini (vespidae: polistinae) with the great number of species (58, being 44 in brazil), and it is considered the most common genera of social wasps on south america (richards, 1978; carpenter & marques, 2001). the most collected species was agelaia pallipes (table 1), having almost a half of the abundance of social wasps (42.68%). agelaia is the third most representative genus on epiponini and has species that build huge nests with millions of wasps (zucchi et al., 1995; carpenter, 2004). the other species whose had the most representative were polybia sericea (20.55%), p. occidentalis (13.24%) and p. ignobilis (10.10%). there are only a few studies on the distribution, colony density and species seasonality from the cerrado: diniz and kitayama (1998) in mato grosso state, henriques et al. (1992) and raw (1998b) in distrito federal, mechi (1996) and mechi and moraes (2000) in são paulo, elpino-campos et al. (2007) and souza and prezoto (2006) in minas gerais and pereira & antonialli-junior (2011) and pereira-bomfim and antonialli-junior (2012) in mato grosso do sul (in a riparian forest). in other ecosystems, silva-pereira and santos (2006) studied wasp diversity in “campos rupestres”, silveira (2002) and silveira et al. (2008) in amazon rainforest, santos et al. (2007) in mangrove, atlantic forest and restinga vegetation and souza et al. (2010) in riparian forest. the predominance of agelaia pallipes was similar to the results of elpino-campos et al. (2007), who collected 29 species of social wasps distributed in 10 genera in four different areas, and mechi (1996) collected 32 species within nine genera in two different areas. diniz and kitayama (1998) sampled 36 species, the large part of them belongsociobiology 60(1): 101-106 (2013) 103 era (table 2). the most representative genera were montezumia and pachodynerus, followed by zethus and the most sampled species was montezumia nigriceps (15%), pachodynerus brachygaster (13.2%) and alphamenes campanulatus (11.3%). eumenines have solitary behavior and build their nests, commonly, with plant resin, mud, in pre-existing cavities and even on surfaces (evans, 1966; cowan, 1991; camillo et al., 1995, 1997). for these reasons, maybe it is more difficult to capture them than social wasps, whose build a variety kinds of nests, many times with characteristics that make possible to identify genera or even species (wenzel, 1998). one of the most representative work that sampled specimens of eumeninae from cerrado was mechi (2006), who collected 35 species within 16 genera, but there are other authors who sampled solitary wasps cerrado, but they used trap-nests methodology, which restrict the sampling to species that make nests on pre-existing cavities (camillo et al., 1995; camillo & assis, 1997). on other ecosystems, hermes and köhler (2004) collected 37 species in the green belt of “rio grande do sul” and morato (2001), on central amazon, colleted four species, although all of them were identified to genera. in the last years, due to anthropic actions (especially agriculture and pasture), the physiognomies found in mato grosso do sul have suffered intense degradation. these modifications cause environmental fragmentation table 1. species of social wasps sampled in “fazenda prata”, paranaíba, state of mato grosso do sul. species abundance proportion (%) agelaia pallipes (olivier) 245 42.6 apoica flavissima van der vetch 2 0.34 brachygastra augusti (de saussure) 1 0.17 brachygastra lecheguana (latreille) 5 0.87 brachygastra moebiana (de saussure) 1 0.17 mischocyttarus frontalis (fox) 1 0.17 mischocyttarus latior (fox) 1 0.17 mischocyttarus cerberus ducke 2 0.34 mischocyttarus mattogrossoensis zikán 1 0.17 parachartergus smithii (de saussure) 4 0.69 polistes canadensis (linnaeus) 7 1.21 polistes billardieri fabricius 12 2.09 polistes subsericeus de saussure 5 0.87 polistes versicolor (olivier) 16 2.78 polybia ignobilis (haliday) 58 10.10 polybia jurinei (de saussure) 8 1.39 polybia liliacea (fabricius) 5 0.87 polybia occidentalis (olivier) 76 13.24 polybia paulista (von ihering) 1 0.17 polybia ruficeps (richards) 1 0.17 polybia sericea (olivier) 118 20.55 synoeca surinama (linnaeus) 4 0.69 total 574 100 table 2. species of solitary wasps sampled in “fazenda prata”, paranaíba, state of mato grosso do sul. species abundance proportion (%) alphamenes campanulatus (fabricius) 6 11.3 alphamenes insignis (fox) 1 1.8 alphamenes sp1 1 1.8 alphamenes sp2 2 3.7 ancistroceroides venustus (brèthes) 4 7.5 cyphomenes anisitsii (brèthes) 3 5.6 hypalastoroides brasiliensis (de saussure) 2 3.7 hypalastoroides nitidus giordani soika 2 3.7 montezumia azurescens (spinola) 1 1.8 montezumia nigriceps (spinola) 8 15.0 montezumia petiolata saussure 1 1.8 pachodynerus brachygaster (saussure) 7 13.2 pachodynerus brevithorax (saussure) 1 1.8 pachodynerus guadulpensis (saussure) 1 1.8 pachodynerus reticulatus (cameron) 1 1.8 pachymenes laeviventris (fox) 2 3.7 parancistrocerus areatus (fox) 1 1.8 zeta argilaceum (linnaeus) 3 5.6 zethus hilarianus (saussure) 3 5.6 zethus miscogaster (saussure) 2 3.7 ing to the genera polybia, who was the most representative, polistes and mischocyttarus. this last, in this work, was very few sampled, with only five specimens (table 1). raw (1998b) and henriques et al. (1992) studied colonies of social wasps, while the first sampled 13 species within eight genera (polybia was the most representative), the second found only seven colonies of four species. until now, there were no studies of diversity of social wasps in mato grosso do sul state. a study on “riparian forest” (pereira & antonielli-junior, 2011) sampled 18 species belonging to six genera, with polybia as the most representative, but agelaia pallipes had almost half of the total abundance. in addition to the list of species from pereira and antonielli-junior (2011) and pereira-bomfim and antonielli-junior (2012), we present here new records of species from mato grosso do sul state: apoica pallens, brachygastra lecheguana, b. moebiana, mischocyttarus frontalis, m. latior, m. cerberus, m. mattogrossoensis and polistes canadensis. eumeninae this work presents the first records for eumeninae species in mato grosso do sul. we collected 53 specimens of solitary wasps belonging to 21 species within 10 genyc grandinete, fb noll social and solitary wasps from cerrado104 table 4. abundance and proportion of solitary wasps collected by using different sampling methods. species a.s. act. a.t. tot. alphamenes campanulatus (fabricius) 3 3 0 6 alphamenes insignis (fox) 1 0 0 1 alphamenes sp1 1 0 0 1 alphamenes sp2 2 0 0 2 ancistroceroides venustus (brèthes) 2 2 0 4 cyphomenes anisitsii (brèthes) 2 1 1 4 hypalastoroides brasiliensis (de saussure) 1 1 0 2 hypalastoroides nitidus giordani soika 1 1 0 2 montezumia azurescens (spinola) 1 0 0 1 montezumia nigriceps (spinola) 2 6 0 8 montezumia petiolata saussure 0 0 1 1 pachodynerus brachygaster (saussure) 1 6 0 7 pachodynerus brevithorax (saussure) 1 0 0 1 pachodynerus guadulpensis (saussure) 0 1 0 1 pachodynerus reticulatus (cameron) 0 1 0 1 pachymenes laeviventris (fox) 0 2 0 2 parancistrocerus areatus (fox) 0 1 0 1 zeta argilaceum (linnaeus) 1 2 0 3 zethus hilarianus (saussure) 3 0 0 3 zethus miscogaster (saussure) 1 1 0 2 zethus romandinus (saussure) 1 0 0 1 total 24 28 2 54 proportion 44.44% 51.86% 3.7% 100% and degradation, which promotes population reduction, local extinctions and consequently biodiversity loss (primack, 2002). besides the cerrado, mato grosso do sul has areas that consist of different physiognomies, like pantanal and chaco, with marsh, seasonal forests and paths, constituting a mosaic ecosystem (spichiger et al., 2004; morrone, 2006; uetanabaro et al., 2007). despite the biogeographic importance of the state, there are still few studies concerning the local fauna, mainly invertebrates diversity, ecology, behavior and phylogenetic relationships. comparison of methodologies according to table 3, the “attractive traps” sampled 76.31% of the total abundance collected (438 wasps), six genera and 13 species of polistinae wasps. active search had 9.23% (53), seven genera and 11 species, while attractive attractive solution had 14.46% (83), six genera and 15 species. even this last methodology did not collected the most abundance of wasps, it sampled the greatest richness, corroborating with gomes and noll (2009) and noll and gomes table 3. abundance and proportion of social wasps collected by using different sampling methods. species a.s. act. a.t. tot. agelaia pallipes (olivier) 28 3 214 245 apoica flavissima van der vetch 0 0 2 2 brachygastra augusti (de saussure) 1 0 0 1 brachygastra lecheguana (latreille) 4 0 1 5 brachygastra moebiana (de saussure) 0 1 0 1 mischocyttarus frontalis (fox) 1 0 0 1 mischocyttarus latior (fox) 1 0 0 1 mischocyttarus cerberus ducke 1 1 0 2 mischocyttarus mattogrossoensis zikán 1 0 0 1 parachartergus smithii (de saussure) 3 1 0 4 polistes canadensis (linnaeus) 0 0 7 7 polistes billardieri fabricius 2 9 1 12 polistes subsericeus de saussure 0 1 4 5 polistes versicolor (olivier) 0 1 15 16 polybia ignobilis (haliday) 4 5 49 58 polybia jurinei (de saussure) 2 0 6 8 polybia liliacea (fabricius) 0 0 5 5 polybia occidentalis (olivier) 13 3 60 76 polybia paulista (von ihering) 1 0 0 1 polybia ruficeps (richards) 1 0 0 1 polybia sericea (olivier) 20 27 71 118 synoeca surinama (linnaeus) 0 1 3 4 total 83 53 438 574 proportion 14.46% 9.23% 76.31% 100% (2009), which showed that this method is very effective on capture social wasps. the table 4 show the efficiency of the sampling methods on solitary wasps. the attractive solution and active search were the most effective methods, with 24 wasps (44.44%), 16 species within 9 genera and 28 wasps (51.86%), 13 species within 10 genera, respectively. the attractive traps sampled only two species of two different genera, with 3.7% of the total abundance. the first work that collected eumeninae using the attractive solution method was noll and gomes (2009), which collected 61 wasps, being the third group more representative (polistinae and ichneumonidae were first and second). as they, this work shows that this method is very effective not only for social wasps, but for solitary too, even on different ecosystems. acknowledgments we wish to thank marcel gustavo hermes for the identification of solitary wasps and getulio minoru tanaka junior, paulo vittor parecis silva and patricia pereira do nascimento for the assistance during collection. the work was funded by grants from “conselho nacional de desenvolvimento científico e tecnológico (cnpq)” (grant# 303249/2010-2). a.s. = attractive solution act. = active a.t. = attractive traps tot. = total a.s. = attractive solution act. = active a.t. = attractive traps tot. = total sociobiology 60(1): 101-106 (2013) 105 references alonso, l. e. & agosti, d. 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(1995). agelaia vicina, a swarm-founding polistine with the largest size among wasps and bees (hymenoptera: vespidae). j. n. y. entomol. soc., 103(2):129-137. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1050sociobiology 63(3): 919-924 (september, 2016) induced biotic response in amazonian ant-plants: the role of leaf damage intensity and plant-derived food rewards on ant recruitment introduction mutualistic interactions are characterized by a network of mutual benefits in which two species increase their fitness (survival, reproduction) when occurring together (bronstein, 1998). mutualistic interactions between plant and animals occur when plants provide resources to animal partners, and animals, in turn, provide services (e.g., transport, protection) (del-claro et al., 2016). a widespread mutualistic interaction in nature is the association of myrmecophyte plants and ants (fonseca & ganade, 1996). whereas myrmecophytes benefit ants offering shelter (domatia) and/or food resources (food bodies), ants defend myrmecophytes from natural enemies and could decrease herbivory (bronstein, 1998; bronstein et al., 2006). host plant defense is mediated by the recruitment of ant workers to plant regions with herbivore or herbivore abstract although plant-ants respond to cues indicating the presence of herbivores, it remains unclear how ant workers are stimulated by herbivory cues with varying intensity. i hypothesized that ants respond more quickly to severe foliar damage and that ants inhabiting myrmecophytes that provide food resource recruit workers to any intensity of foliar damage. i tested the induced response in three ant-plant systems: two that provide food resources and another one that does not provide. i simulated leaf damage using different concentrations of foliar extracts. in all systems, the plant-ant recruitment increased after damage simulations. in food providing systems, ants did not distinguish between different intensity damages, although one system has shown a mixed response to that intensity. this result indicates that ants defend more intensely their host plant to avoid injuries that diminish food provisioning. in the non-food providing system ants responded more quickly to the simulation of high foliar damage. this faster recruitment to leaves with high damage suggests that ants are searching for hot spots that potentially have more insects for feeding on. i suggested that future works could manipulate plant size and quality, and food provisioning to better understand why mutual ant-plant systems with very distinct characteristics are evolutionarily stabilized. sociobiology an international journal on social insects t gonçalves-souza article history edited by gilberto m. m. santos, uefs, brazil received 25 april 2016 initial acceptance 24 august 2016 final acceptance 18 september 2016 publication date 25 october 2016 keywords myrmecophytes, herbivory, protective mutualism, ant-plant interaction. corresponding author thiago gonçalves-souza laboratório de ecologia filogenética e funcional, departamento de biologia área de ecologia universidade federal rural de pernambuco (ufrpe) rua dom manoel de medeiros, s/nº cep 52171-900, recife-pe, brasil e-mail: tgoncalves.souza@gmail.com cues, such as leaf volatile compounds released after the damage (agrawal, 1998; agrawal & rutter, 1998; brunaet al., 2004). ant protection increases host plant leaf area, survival rates, and seed set (heil & mckey, 2003). the inducible defense, driven by plant tissue volatiles, is known as induced biotic response. in general, plants allocate resource to ants when the provisioning of this resource increases their fitness (karban & myers, 1989). theoretically, these complex changes in plant inducible response enhance the resistance of plants to further attack of herbivores (reviewed by karban & myers, 1989). experimental studies have been showing that herbivory simulations (e.g., leaf damage or chemical stimuli with leaf extracts) stimulate ant recruitment to those damaged regions (agrawal, 1998; romero & izzo, 2004). however, those studies have focused on how ants respond to different stimuli (visual, chemical), but few studies compared ant-plant universidade federal rural de pernambuco (ufrpe), recife-pe, brazil research article ants mailto:tgoncalves.souza@gmail.com t gonçalves-souza – induced biotic response in amazonian ant-plants920 systems that have different features, such as food rewards and ant partners. the intensity (velocity and number of workers recruited) of the response of ant partners to the damaged regions dependent on several factors. for instance, it has been suggested that plant age and structure and the provisioning of food resources are essential elements, determining the response of ant partners to herbivore cues and, thus, their efficiency in host plant protection (heil & mckey, 2003; izzo & vasconcelos, 2005; trager & bruna, 2006). for instance, calixto et al. (2015) showed that anti-herbivore defenses of qualea multiflora (vochysiaceae) vary along plant development, from higher sugar concentration in young leaves attracting more ants (a biotic protection), to higher foliar toughness (an abiotic defense), which both contribute to decreasing herbivore attacks (see also del-claro et al., 2016). previous studies showed that plant size is positively related to colony size which, in turn, affects the number of available ant workers for patrolling plant leaves (christianini & machado, 2004). consequently, plant species with varying sizes and leaves with different ages could have different responses to herbivore attacks because they have more (or less) ant workers (christianini & machado, 2004; calixto et al. 2015). accordingly, different cues could favor (or disfavor) the attraction of ants and the consequent defense of plants against their natural enemies (heil & mckey, 2003; ricogray & oliveira, 2007). whereas some myrmecophytes offer both food resources and shelter (e.g., maieta), other species offer only shelter (e.g., hirtella) (vasconcelos & davidson, 2000; izzo & vasconcelos, 2002; christianini & machado, 2004). the variety of ant-plant systems with and without food rewards can potentially attract ant partners that respond differentially to herbivore cues, from protective partners to commensals. moreover, even within the same plant species, the quality and amount of resources offered affect ant species (reviewed in heil & mckey, 2003). ants associated with myrmecophytes that do not provide food depend on arthropods (mainly herbivores) that forage on their host plants. thus, it is expected that only reliable cues of herbivory (high concentration of chemical volatiles) could elicit ant recruitment in an ant-plant system without food rewards. conversely, plant-ants associated with myrmecophytes that offer both food and shelter have the advantage of feeding directly on plants and, thus, should respond to any herbivory cues because they are interested in the vigor, growth and survival of their host plant, especially in those obligate inhabitants (heil & mckey, 2003). i compared the response of ants to herbivory cues (different concentrations of leaf extracts) in three ant-plant systems in central amazonia with and without provisioning of food rewards. i hypothesized that ant workers recruitment depends (i) on the intensity of leaf damage (low vs. high cues of herbivory) and (ii) that ant recruitment varies with the provisioning of food rewards. i predicted that the recruitment of ant workers associated with myrmecophytes that do not provide food reward (e.g., hirtella) is triggered only by strong herbivory cues, such as in treatments with a high concentration of leaf extract. on the other hand, in ant-plants that provide food (e.g., maieta) the ant recruitment occurs at any intensity (from low to high concentrations of leaf extract) of herbivory cues. material and methods study site and organisms the experiments were performed on august 2007 in a terra-firme amazonian rainforest (reserve 1501: 2°24’ s, 59°43’’w) administrated by biological dynamics of forest fragment project of the national institute for amazonian research (inpa). this forest is located 80 km north of manaus city, amazonia, brazil. the climate is classified as am in the köppen system with average rainfall from 1900 to 2500 mm/year, with a dry season between june and october and rainy season from november to may (lovejoy & bierregaard, 1990). i used three myrmecophytes, maieta guianensis and tococa bullifera (melastomataceae), and hirtella myrmecophila (chrysobalanaceae) commonly found in the understory of terra-firme forest. m. guianensisis a shrub with height varying from 0.5 to 1.5 meters that has the plant-ant pheidole minutula as the main mutualistic partner; their leaves have two-three domatia and plants produce food bodies used by p. minutula ants (vasconcelos, 1991). specimens of p. minutula are obligate plant-ants and the dominant species occurring on m. guianensis (vasconcelos,1991,1993). t. bullifera is a shrub with height varying from 0.5 to 1.5 meters. this ant-plant produces mullerian bodies that are used mainly by azteca spp. (vasconcelos & davidson, 2000).their domatia are heartshaped and inserted in the leaf base, where queens of azteca spp. establish a colony (vasconcelos & davidson, 2000) (fig 1a). i did not quantify the nutritional content of food bodies produced by these two myrmecophytes, but other studies have indicated the presence of glycogen, lipids, and carbohydrates (reviewed in rico-gray & oliveira, 2007; see also vasconcelos, 1991). h. myrmecophila (chrysobalanaceae) is a shrub with height varying from 0.5to 8 meters that houses the plant-ant allomerus octoarticulatus (fonseca, 1999). ant workers of a. octoarticulatus feed exclusively on insects that forage over h. myrmecophila leaves because these plants do not offer any food reward for ants. their leaves have a pair of domatia inserted in the base (romero & izzo, 2004). all organisms will be hereafter mentioned by their genus. effects of damage intensity on ant recruitment to test how damage intensity affects the velocity of ant workers recruitment i selected 30 plants of each species sociobiology 63(3): 919-924 (september, 2016) 921 varying from 0.5 to 2.5 meters in the understory of the terrafirme forest. i randomly picked one young leaf on the top of each plant to perform the experiment. i divided those 30 plants into three different treatments: 1) high damage concentration (n = 10 plants), in which the leaf received three drops of an extract with concentration of 20 g of leaf tissue macerated per 80 ml of water (0.25 g/ml); 2) low damage concentration (n = 10), in which the leaf received three drops of an extract with concentration of 1 g of young leaves per 80 ml of water (0.0125 g/ml); 3) water control (n = 10), in which each leaf received three drops of water. those drops were applied with syringes. i counted the number of ant workers immediately before the application (hereafter mentioned as moment 0), and 1, 3, 5, 8 and 10 minutes after the application of foliar extracts or water. i performed this procedure on all three systems, pheidole–maieta, azteca–tococa and allomerus– hirtella and selected plants occurring at the same site. the amount of leaf tissue per water that i categorized “high” and “low” concentration was defined after previous attempts that manipulate the same ant plant systems; lapola et al. (2003) with pheidole–maieta, bruna et al. (2004) with azteca– tococa, and romero and izzo (2004) with allomerus–hirtella. these authors compared water vs. foliar extract (without fig 1. basic description of studied ant-plant systems concerning (a) the provisioning of food rewards and (b) the time the number of ants recruited after the application of foliar extracts is doubled. ant recruitment (average number of ants ± 1se) to young leaves of (c) maieta guianensis, (d) tococa bullifera, and (e) hirtella myrmecophila after the application of foliar extracts with high (red colour) and low concentration (orange colour) and water control (blue colour). different letters comparing the average values mean significant difference (p < 0.05: tukey hsd). detailed information about treatment and time effects can be found in table 1. comparison of intensity) and arbitrarily defined an amount of leaf tissue per water (or methanol). for example, whereas romero and izzo (2004) used 5 g of fresh leaves per 80 ml of water, bruna et al. (2004) used 5 g of fresh leaves per 60 ml of methanol. thus, the treatments were categorized by arbitrarily multiplying the amount of 5 g of leaf tissue by four to define high concentration and dividing this amount by five to define low concentration. importantly, the studies i used to define my treatments found a strong effect of leaf extract on ant recruitment. in fact, the use of leaf tissue extracts is common in studies with myrmecophytes, and they are considered a chemical stimulus, indicating possible attacks of herbivores (lapola et al., 2003; romero & izzo, 2004). statistical analyses i used repeated measures analysis of variance (anova) to test if the concentration of leaf extracts affects the number of ant workers over time. i considered treatments as a fixed effect and time as the factor of repetition. to avoid sphericity, i corrected probabilities with the greenhouse-geisser (gg) method (quinn & keough, 2002). i used tukey hsd a posteriori test for comparing the number of ants between t gonçalves-souza – induced biotic response in amazonian ant-plants922 treatments at each time (quinn & keough, 2002). i utilized the software statistica13.0 to run these analyses. results i found on average 19.03 (±10.4 sd) pheidole ants per domatia of maieta, 50.4 (±36.6 sd) allomerus ants per domatia of hirtella, and 60.6 (±29.6 sd) azteca ants per domatia of tococa. the number of ant workers was higher on leaves with foliar extract (high and low concentration) than on leaves with water drops in all studied species. there was a significant effect of both treatment and time (table 1). whereas the number of pheidole ants increased 5.1 times after 1 minute of the chemical stimuliin maieta leaves (fig 1b, c), the number of allomerus and azteca increased 2.89 and 4.96 times in hirtella and tococa leaves, respectively (figs 1b, d, e). the number of pheidole ants did not differ between leaves with high and low stimuli (p>0.05 for all time comparisons, tukey hsd), although they both differed from water control (p<0.001; fig 1c). however, the number of azteca ants has a mixed response to the chemical stimuli through time. even though ant recruitment was higher in treatments compared with water control, ant numbers were similar between low and high stimuli only after 3 minutes of the application (fig 1d). conversely, the number of allomerus ants was greater in high concentration stimuli than on low concentration (p<0.05 for all time comparisons, tukey hsd) and water control (p<0.05 for all time comparisons, tukey hsd) after of the application of leaf extract (fig 1e). discussion in this study, i examined whether the intensity of leaf damage and the provisioning of food rewards affect ant workers recruitment. as predicted, ant workers associated with food providing systems did not distinguish between damage intensity, confirming the hypothesis that the provisioning of resource increases plant defense by ants. also, i confirmed the prediction that ant workers associated with plants that do not provide foods respond only to strong herbivory cues. previous studies have indicated that the intensity of ant response to herbivory varies depending on ant species and the host plant they are associated with (heil & mckey, 2003; bruna et al. 2004; heil 2015; del-claro et al., 2016). specifically, the provision of food rewards by myrmecophytes could elicit best ant partners (i.e., a more efficient defense against herbivores) when compared with plants that do not provide those rewards (risch & rickson, 1981; heilet al., 1997; izzo & vasconcelos, 2002). in fact, previous studies demonstrated that the provisioning of food increase ant aggressiveness (against plant herbivores) and survival rates (reviewed in heil, 2015). the results showed that the number of ant workers of pheidole, azteca, and allomerus increases rapidly after the application of leaf extracts, which are cues indicating herbivore attacks. as expected, the number of allomerus ants was three times higher in leaves with high chemical stimuli compared with low stimuli. indeed, only strong and reliable cues indicating the presence of herbivores stimulated the recruitment of ants that need to catch these insects for feeding. table 1. repeated measures anova showing the effects of treatments (high and low concentration extracts, and water control) and time (0, 1, 3, 5, 8, and 10 minutes after the application of the treatments) on ant recruitment. myrmecophytes / plant-ants effect df f p maieta guianensis / pheidole minutula treatment 2 35.25 < 0.001 error 27 time 5 75.26 < 0.001 time vs. treatment 10 17.98 < 0.001 error 135 hirtella myrmecophila / allomerus octoarticulatus treatment 2 6.339 0.006 error 27 time 5 27.84 < 0.001 time vs. treatment 10 3.45 < 0.001 error 135 tococa bullifera / azteca sp. treatment 2 12.72 < 0.001 error 23 time 5 12.41 < 0.001 time vs. treatment 10 4.86 < 0.001 error 115 sociobiology 63(3): 919-924 (september, 2016) 923 besides, it is possible that allomerus workers respond only to strong chemical stimuli because hirtella plants are big and they architecture is too complex (izzo & vasconcelos, 2005). therefore, low chemical stimuli concentration may be not strong enough to trigger a fast and localized response of ants to leaves with low damage because they are not detected by ants and/or because the higher plant size does not favor stimuli transmission. in fact, the number of ant workers in leaves with low concentration extract did not differ from leaves with water. the results i found reinforce previous studies showing that ants respond to leaf volatile compounds by increasing recruitment to damaged areas (agrawal, 1998; agrawal & rutter, 1998; bruna et al., 2004; christianini & machado, 2004; romero & izzo, 2004). notwithstanding, i did not found any difference between the concentration of foliar extracts in pheidole-maieta system; actually, pheidole ants responded to the minimum cue of herbivory. i suggest that the small size of maieta facilitates the transmission of chemical cues throughout the plant favoring ant recruitment. accordingly, by increasing the intensity of ant recruitment those workers could decrease leaf herbivory and benefit their mutualistic host plant. indeed, agrawal and rutter (1998) demonstrated that foliar damage decline plant vigor which, in turn, reduces resource quality to ants. therefore, in ant-plants with limited food supply and with a small number of leaves, the intensity of ant recruitment is essential to guarantee food provisioning (itino et al., 2001) and to stabilize these mutualistic systems. however, contrary to my expectations, there was a mixed, time-dependent response of azteca ants to chemical volatiles intensity. on one hand, these ants did not differentiate low concentration from water control after 1, 5, 8, and 10 minutes after the stimuli, which disagree with the expected response. on the other hand, ants responded similarly between high and low concentrations after 3 minutes, which was expected based on the rewards provided by tococa plants. these mixed results reinforce the importance of food rewards to ant recruitment, but also shows there are other factors dictating plant defense by ants (see, e.g., calixto et al. 2015). for example, ant patrolling can be associated with the number of leaves with domatia (christianini & machado, 2004) and, therefore, a fast response to chemical stimuli may depend on the real number of domatia present in the host plant (see also del-claro et al., 2016 for other important cues eliciting host plant defense by ants). the results showed that chemical cues associated with herbivory increase ant recruitment to potentially damaged leaves in three amazonian ant-plant systems. more importantly, these findings demonstrated that in ant-plant systems based on food rewards (such as maieta) ant workers defend their host plants in any chemical clue indicating the presence of potential herbivores, although the response of tococa ants was mixed. conversely, ant partners foraging on one plant species that do not provide food resource respond mainly to chemical cues indicating intense foliar damage. these results suggest the provisioning of food in ant-plant systems drive the intensity of ant recruitment to damaged areas. however, it is important to recognize that these finding should be tested in other systems (mainly from plants that do not provide rewards). for example, plant size could affect the way ant workers respond to that chemical stimulus (christianini & machado, 2004). future works could manipulate plant architecture (size, number of branches), quality (number of active domatia) and food provisioning to understand better why mutual ant-plant systems with very distinct characteristics are evolutionarily stabilized. acknowledgments i would like to thank the students of the ecology discussion group (ffun-club) that kindly revised early versions of this manuscript: t.n. bernabé, p.h.p. gusmão, l.s.o. melo, and p.h.a. sena. t. izzo, g. machado and g. q. romero helped with fundamental ideas before the experiment. the biological dynamics of forest fragments project (inpa/smithsonian) has partially supported this study. this is the publication 703 in the technical series of the biological dynamics of forest fragments project. references agrawal, a.a. 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(2000). relationship between plant size and ant associates in two amazonian antplants. biotropica, 32: 100-111. doi: 10.1111/j.1744-7429. 2000.tb00452.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i4.2107sociobiology 64(4): 423-429 (december, 2017) positive relation between abundance of pericarpial nectaries and ant richness in tocoyena formosa (rubiaceae) introduction there are several cases of mutualism regarding plants and ants (del-claro et al., 1996; heil & mckey, 2003; mondal et al., 2013), ranging from opportunistic to mandatory interactions (heil & mckey, 2003; duffy & hay, 2010; byk & del-claro, 2011; mayer et al., 2014). such interactions are very common among plants having extrafloral nectaries (efns), a nectar secretory structure that is not involved in pollination (koptur, 1994; fiala & linsenmair, 1995). ants are attracted to efns due to the presence of nectar, a highly energetic and nutritious substance, being composed mainly of sugar (byk & del-claro, 2011). in the cerrado, there is a high incidence of efns (fiala & linsenmair, 1995; ratter et al., 1997; cogni & freitas, 2002; del-claro & marquis, 2015), and previous studies have reported a mutualistic interaction mediated by efns, having the ant acting as a protector against herbivores while searching for nectar in plants (del-claro et al., 1996; oliveira, 1997). abstract ants can interact with plants in several ways, being one of the most common visitors of extrafloral nectaries (efns). however, pericarpial nectars (pns) may represent to the ant community a similar resource as efn would do. here, we investigate how an ant community interacts with pns by an individual-based network, using tocoyena formosa as a model. we hypothesized that plants with more pn’s would present a higher ant richness in comparison to plants with less pns and will occupy a central position in the network interaction. we observed 36 individuals of t. formosa and recorded both the ant species and abundances on each plant, as well as the number of active pns. to test this hypothesis, we performed a linear regression between pns and ant richness. a network analysis was performed to obtain both the specialization and centrality metrics of each plant, and we also conducted linear regressions between the number of pns and both the specialization and centrality. the hypothesis was confirmed, the ant community was more rich in individuals of t. formosa with more active pns, and these individuals were more central, being important for maintaining the interactions with ants. we believe that the coexistence between ants foraging is possible in t. formosa due to the seasonality and short time prevalence of the pns, whose dominant ants do not have able time to master the resource and exclude the others, allowing different species to use the same plant. sociobiology an international journal on social insects ds queiroga, rf moura article history edited by kleber del-claro, ufu, brazil received 05 october 2017 initial acceptance 10 october 2017 final acceptance 16 october 2017 publication date 27 december 2017 keywords individual-based network; efn; pn; interaction ecology; mutualism. corresponding author drielly da silveira queiroga universidade federal de uberlândia instituto de biologia curso de pós-graduação em ecologia e conservação de recursos naturais rua joão naves de ávila nº 2121 campus umuarama, bloco 2d sala 28 cep 38400-902, uberlândia-mg, brasil. e-mail: drielly.bio@gmail.com pericarpal nectaries (pn), a similar structure to efns, occur when the corolla falls and the nectariferous disc remains active throughout the fruit development (del-claro et al., 2013a). pns are often seen as a type of efn due to their morphological and functional similarities (del-claro et al., 2013a; paiva, 2009). despite their analogous importance in mutualistic ant-plant interactions, pns are often underexplored structures in studies that consider mutualistic effects on antplant interactions (del-claro et al., 2013a). mutualistic interactions present distinct characteristics in comparison to other types of interactions. for instance, previous studies already identified that mutualistic facultative networks have a particular organization, which is called a nested pattern (bascompte et al., 2003). it means that facultative networks have specialist species interacting mostly with generalist species, and these generalists are responsible for the network cohesion, acting as interconnectors among the network (bascompte et al., 2003; guimarães et al., 2006). universidade federal de uberlândia, curso de pós-graduação em ecologia e conservação de recursos naturais, uberlândia-mg, brazil research article ants mailto:drielly.bio@gmail.com ds queiroga, rf moura – abundance of pericarpial nectaries and ant richness424 according dattilo et al. (2014), it is possible to compare the individual-based networks of ant-plant interactions with the species-area model proposed by macarthur and wilson (1967), with each plant acting as an “island of resources”. in this model, it is expected an expansion of ant richness throughout the plant ontogeny due to an increase of resource availability (campos et al., 2006). high quality resources provided by plants are more susceptible to receive a greater ant visitation, which in turn is related to a better ant protection against herbivores (heil & mckey, 2003). efns, however, are not simply related to the number of ant visits, but also on how these visits are organized in time, meaning that plants with efns present differences between day and night visiting ant fauna, whereas plants without efns do not (anjos et al., 2017). several studies have focused in finding attributes of the ant-plant interaction that can predict the organization of such mutualistic associations (chamberlain & holland, 2009; dáttilo, 2012; gomez & perfectti, 2012; dáttilo et al., 2014, dátillo et al., 2017). commonly used metrics are species degree (k), specialization (d), centrality betweenness (cb) and centrality closeness (cc). species degree (k) and specialization (d) may be seen as a predictor of nestedness and are commonly used in order to investigate non-random patterns in networks (okuyama & holland, 2008, blüthgen et al., 2008, 2015). it is believed that species having a high cc have a potential to affect many other species, while species with high cb are important to the maintenance of the network cohesion (gonzález et al., 2010). both metrics (cc and cb) indicate the importance of a species to the network as a whole, in which species with high values of cb and cc will likely be key species for the maintenance of mutualistic interactions, such as the plants with in efns (gomez & perfectti, 2012). in the present study, we used as a model tocoyena formosa (rubiaceae) (cham. & schlecht.) k. schum 1889, a typically shrub from cerrado, which presents pns, to measure the importance of such resource in the mutualistic interactions among ants in a brazilian cerrado area. specifically, we tested the hypotheses that i) the number of pns available influences the ants search for this resource ii) and plants with more active pns are more visited by ants (richness and abundance). material and methods study area we carried out the study at serra de caldas novas state park (pescan, 17 ° 47’13 ‘’ s / 48 ° 40’12’’o), in the municipality of caldas novas, go, brazil. the region is at an average elevation of 1,000 m above ground level and we collected the data in a plateau area, a typical stricto sensu cerrado. the climate in the region is aw according to the classification proposed by köppen (dias cardoso et al., 2014). studied species tocoyena formosa is a shrub from the rubiaceae family, with 1.7 m of height on average (carlos-santos & del claro, 2001). it mostly occurs on savanna-like vegetation, dry forests, thorn-scrub vegetation and humid rain forests (silberbauer-gottsberger et al., 1992). the inflorescence has yellow flowers with long corolla tubes. the blooming occurs from october to december, but there are reports of blooming in january as well (silberbauer-gottsberger et al., 1992). after the pollination, the corolla falls, but the nectariferous disk located at the chalice remains active (carlos-santos & del claro, 2001). the persistence of the nectariferous disk gives rise to the pericarpial nectary (fig 1). study design we conducted the field work in november 2016, during the rainy season, coinciding with the flowering period of t. formosa. we used a track of 1km as transect, where individuals were screened for up to 5 m on each track side. we selected 36 individuals under the same conditions of luminosity, with measurements ranging from 1.50 to 1.70 high. we counted all active pns of each selected plant and considered an active pn when the corolla had already fallen, leaving the nectariferous disk exposed. plants that had any ant resources other than pns (e.g. hemipteran honeydew) or had no active pns or ants at all, were disregarded. these conditions were stipulated to evaluate only the effect of active efns, minimizing confounding factors (e.g. plant size, mutualistic interactions with other animals). observations were taken from 08:00 to 11:30 am, and we collected the visitors from the nectaries of each plant. in order to determine which ant species in fact were visiting the pns, we observed each branch inflorescence for about five minutes. we counted the effective visitors and two individuals of each species were collected for a more accurate identification after the field procedures. the identification was performed with the collaboration of a specialist and based on the published listings of the park’s myrmecofauna. network analysis and statistics we conducted all data analyses in the r software version 3.3.2. (r core team, 2016). we performed a rarefaction curve to investigate whether a satisfactory sample of ant richness visiting t. formosa was obtained. we performed a simple linear regression to investigate the relationship between the number of pns and ant richness. an individual-based network was performed using the package bipartite (dormann et al., 2008). network analysis shows associations (links) between species (nodes) in a community, and is a commonly used approach in order to study how interactions are organized (dáttilo et al., 2014; blüthgen et al., 2015; del-claro et al., 2016). several studies have been using this method for either community networks (lange et al., 2013; maruyama et al., 2014, 2015) or individual-based ones (baker-méio & marquis, 2012; gomez & perfectti, 2012; dáttilo et al., 2014). this analysis provides a great number of metrics and, for our aim, we selected six: complementary specialization (h2’), species specialization (d’), degree (k), sociobiology 64(4): 423-429 (december, 2017) 425 specialization and 1 depicts a perfect specialization (blüthgen et al., 2006; dormann et al, 2009). species specialization (d’) is characterized by the sum of links per species, which is the richness of ants that interact with an individual plant. degree (k) shows each species specialization based on a randomly selected partners discrimination. closeness centrality (cc) describes the centrality of a species in the network by counting the number of links to other nodes. it indicates that the higher the closeness, the biggest the effect to other nodes would be; and, at least, betweenness centrality (bc) that describes the centrality of the species in a network analysis. it also takes as a reference the position of a species in relation to other nodes, as well as the importance of this node as a connector to other parts of the network. (dormann, 2011). our matrix has each individual of t. formosa in the rows, while ant species are placed at the columns, with cells expressing the abundance of each ant species. we tested all these indices in a general linear model (glm), having the number of pns acting as a predictor variable to check if they are a good predictor to organizing ant-plant networks. we conducted a null model with 1000 randomizations using the function "oecosimu" from the packpage vegan (oksanen et al., 2007) in order to investigate if the pattern of the network and its metrics were not randomly obtained. we used the method r0 to maintain the t. formosa frequencies (row) and to fill presences anywhere on the row disregarding the ant species frequencies (column) (oksanen et al., 2007). results we found a total of 818 pns (min = 2; max = 86; mean ± sd = 22.72 ± 23.11) in the 36 individuals of t. formosa and 610 ants distributed in four sub-families, from 13 species (table 1). the rarefaction curve showed a degree of stabilization, which supports that most ant species that interact with t. formosa were sampled, indicating a strong robustness of the network (nielsen and bascompte, 2007) (fig 2). the simple linear regression between pns and the ant richness was statistically significant (r² = 0.11; f(1,34)=5.19; b = 10,857; p = 0.03), showing that the increased resource is accompanied by an increased number of links in the analyzed network (diversity). in the network analysis, we have a high specialization (h’2= 0.799), whitout compartment. with the glms, we found a negative relationship of pn abundance with the specialization (d’) (r² = 0.08; f1,34 = 4.14; b = -7.61; p = 0.05) and a positive relationship between the network’s betweenness (r² = 0.09; f1,34 = 4.41; b = 123.99; p = 0.04) and closeness (r² = 0.39; f1,34 = 23.45; b = 3473.48; p < 0.01) (fig 3). the relationship between species degree (k) and number of pns was not significant (r² = -0.03; f1,34 = 0.02; b = -2.39; p = 0.88). the randomizations showed that the model did not represent a random organization (p<0.01) which supports the non-random pattern of mutualistic networks found in other studies. fig 1. a) a view of tocoyena formosa. b) inflorescence with some active pericarpial nectaries (pns). c) camponotus sp. foraging on a recently available pn and d) camponotus sp. foranging in a pn with a growing fruit. closeness centrality (cc) and betweenness centrality (bc). h2’ is an index that measures network the specialization for quantitative matrices ranging from 0 to 1; 0 means no ds queiroga, rf moura – abundance of pericarpial nectaries and ant richness426 sub-family species abundance formicinae brachymyrmex sp1 63 camponotus blandus 113 camponotus senex 203 myrmicinae cephalotes atratus 3 cephalotes pusillus 4 cephalotes sp1 8 crematogaster sp1 27 ectatomminae ectatomma brunneum 120 ectatomma sp1 12 pseudomyrmecinae pseudomyrmex sp1 13 pseudomyrmex sp2 14 pseudomyrmex sp3 6 pseudomyrmex gracilis 24 table 1. list of ant species found in tocoyena formosa plants with the total number of observed individuals. fig 2. a rarefaction curve with each individual of t. formosa as a sample in the x axis and the number of ant species in the y axis. discussion the main hypothesis of our study was confirmed, indicating that pns are indeed capable of influencing an individual-based ant-plant mutualistic network. we found a positive relationship between pns abundance and ant richness. this result agrees with lange et al. (2017), where they find a positive relation between the amount of nectar produced and ant richness in many plants with efns in a cerrado area. lange et al. (2013) also found that a greater availability of efn may increase the co-ocurrence of ants in plants. this may occur as interactions of ant-plant with efns are mostly opportunistic and ants rarely monopolize the nectar produced by these plants (floren & linsenmair, 2000). for example, blüthgen et al. (2000), studied ant-plant interactions in amazonian forest, found a co-occurrence of different ant species in plants having efns, which was not the case for plants with aphids. they argue that efns are visited by both aggressive and non-aggressive ants, with more than one foraging species on the same plant. this occurs because the nectar is not continually produced throughout the year, thus, it may be difficult for ants to be exclusive users of a single plant species having efns, as plants are periodically generating and interrupting the nectar production. conversely, honeydew is a constant resource, allowing the “loyalty” of the ants. on the other hand, the pns are located in the fruits, the most distal parts of the plants (del-claro et al., 2013a; alves-silva et al., 2015). this might provide a sufficient spatial segregation among ants, which can minimize the encounters between ants and, consequently, the conflicts. nectar quality may be influencing the results if we consider a trade-off between protection and ant exclusion, happening only if the reward (efns), such as the energy provided by nectar, is high enough (blüthgen et al., 2000; davies et al., 2012). all these factors may be contributing to the pattern found in t. formosa about the ant richness correlated with pns. unlikely our findings, dáttilo et al. (2014) did not find a clear correlation between ant richness and efns, even when analyzing three plant-bearing efn species. they indicated two alternative not mutually exclusive hypotheses, though: 1) that efns has been monopolized by few dominant ant species that have competitively excluded other ant species or, 2) that the tree height in cerrado are relatively low, which eases the resource domination by few ant species. however, the second justifications presented by them is not extrapolated to our system, because t. formosa is a shrub, according to the arguments used by them, and yet we found this relation. our results are in accordance with other studies (blüthgen et al., 2000; lange et al., 2013, lange et al., 201), indicating that an increase in efns abundance is followed by an expansion of the ants richness, having an association with the resource type and maybe with ant aggressiveness (blüthgen et al., 2000). because of that, we consider that future works should take into account the ant aggressiveness factor. the absence of modulation in the network is commonly found in facultative mutualistic networks, which suggests a homogeneous and cohesive network (díaz-castelazo et al., 2013; dáttilo et al., 2014). in our system, the non-modularity may be explained by the short period that pns are active during a year, preventing the emergence of restrictive relations with some species of ants. it also occurs because ants do not usually show foraging fidelity in the same group of plant individuals (dáttilo et al., 2014), which is indicated by the low specialization that we found in t. formosa individuals with high numbers of pns. these two results (modularity and specialization) are complementary and reinforce the idea of seasonality acting on the organization of these interactions. pns are associated with betweenness and closeness (centrality metrics), as indicated in the results of the present study. centrality is a metric related to the plant fitness in pollinatorplant interactions and indirectly to ant-plant interaction, as shown by gomez and perfectti (2012), which found that centrality is positively related to the number and richness of visitant pollinators in individual-based networks. according to santos and del claro (2001), the main function of pns in t. formosa is to protect their fruits, meaning that when fruit herbivory is reduced, t. formosa produces larger fruits with more seeds. in this way, we may admit that for t. formosa, sociobiology 64(4): 423-429 (december, 2017) 427 centrality and pns may also be studied as a fitness measure, since individuals with higher values of centrality were correlated with the increase in pns. we also found a negative relationship between pns and individual specialization, indicating that the increasing pns may also expand the ant richness in this plant-model. this pattern is supported by the literature, where betweenness is associated with generalist species. martín gonzález et al. (2010) found in pollination networks that high generalist and central species can act as keystone in a network, interacting with most other species and connecting subgroups. on the present study, we state that mutualistic interactions between ant community and t. formosa plants having pericarpial nectaries (pns) can be strongly influenced by the abundance of resource. we believe that this pattern might be preserved at other plants of the rubiaceae family, which also have pns. acknowledgments we would like to thank lino a. zuanon and dr. renata pacheco for their assistance in identifying the collected ants. we also thank the graduation course of ecology and conservation of natural resources from the federal univeristy of uberlândia; the serra de caldas novas /go state park for the logistic structure and support; and capes (coordination for the improvement of higher education personnel) for the graduate scholarship provided to the authors. references alves-silva, e., bächtold, a., barônio, g. j., torezan-silingardi, h. m. & del-claro, k. 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(1992). hybrid speciation and radiation in the neotropical woody genus tocoyena (rubiaceae). plant systematics and evolution, 181: 143-169. doi: 10.1007/bf00937441 doi: 10.13102/sociobiology.v60i2.162-168sociobiology 60(2): 162-168 (2013) spatial distribution of acromyrmex balzani (emery) (hymenoptera: formicidae: attini) nests using two sampling methods l sousa-souto, ab viana-junior, es nascimento introduction the spatial distribution of organisms in an environment is a key parameter in studies of population ecology (krebs, 1989; begon et al., 1996). it allows the inference of ecological aspects of great importance such as the dispersal pattern of individuals, possible existence of competition or other agonistic interactions, the density of individuals in the study area and species dominance at the community level (brower et al., 1997). these inferences, in turn, enable the development of pest management programs (ferguson et al., 2003), to cope with invasive species (zhu et al., 2007) and to plan conservation strategies in the case of rare and endangered species (beissinger & westphal, 1998). although it is a good tool in population studies, spatial distribution is dependent of several variables that determine the suitability of sites for the establishment of individuals in the environment, such as sun exposure, soil or air humidity, altitude, availability of food and shelter, and sites for nesting and breeding (soares & schoereder, 2001; van gils & vanderwoude, 2012). abstract the spatial distribution (sd) of organisms is a key parameter in studies of population ecology. among the methods to describe the sd of sessile organisms, sampling by way of plots and transects are widely used. the measurement of the distance between individuals (“nearest neighbor”) is a simple method that has not been employed in population studies with ants. this study aimed to evaluate the sd of ant mounds of acromyrmex balzani (emery, 1890), using both plot sampling and nearest neighbor methods in order to evaluate which method is more appropriate for determining sd of this species. in january 2013 we established 359 plots of 10 m2 on a fragment of grassland in sergipe, brazil. in the same study area 25 colonies were randomly selected and the distance of the closest neighbor colony was determined. in total, 153 ant mounds were sampled (plots) and the density was estimated in 975 × colonies ha-1. colonies were clumped in the environment either by plot sampling (χ2 = 453.93; p < 0.05) as well as by the method of nearest neighbor (ax = 0.67, t = -1.72, p < 0.05). the aggregation of a. balzani colonies found in this study may be due to habitat heterogeneity or relate to the strategy of colony foundation. we conclude that the use of the nearest neighbor method was as accurate as the plot sampling method, providing the same results with much lower sampling effort. sociobiology an international journal on social insects 1 universidade federal de sergipe, são cristóvão, se, brasil research article ants article history edited by gilberto m m santos, uefs, brazil received 10 april 2013 initial acceptance 17 may 2013 final acceptance 22 may 2013 key words population ecology, leaf-cutting ants, nest density, nearest neighbor corresponding author: leandro sousa-souto universidade federal de sergipe programa de pós-grad. em ecologia e conservação, são cristóvão-se, brazil 49100-000 e-mail: leandroufv@gmail.com in general, the distribution (dispersal) of organisms in the environment can be differentiated in three spatial arrangements: (1) random, (2) aggregate or contagious, when the organisms tend to be distributed in groups, and (3) regular or uniform, when the individuals are uniformly distributed in a population (taylor, 1984; krebs, 1989; begon et al., 1996). in nature, organisms are rarely distributed in an arrangement as uniform as plants in agroecosystems; instead, they are usually aggregative (gao, 2013). for ants, clumped distributions may reflect the spatial heterogeneity of the habitat in relation to resource availability (belchior et al., 2012; van gils & vanderwoude, 2012), reflect the outcome of the reproductive strategy (rissing et al., 1986; nicholas & vilela, 1996; debout et al., 2007) or are related to the social behavior of some species (soares & schoereder, 2001; schatz & lachaud, 2008). a completely random distribution (in which the position of an organism is completely independent of any other position in the same population) may reflect an environment with homogeneous provision of resources, where intraspecific competition is negligible (soares & schoereder, 2001). on the other hand, uniform distribution may also indicate the sociobiology 60(2): 162-168 (2013) 163 existence of strong intraspecific competition between individuals in the population, such as when animals defend sites for mating or foraging (bernstein & gobbel, 1979; schatz & lachaud, 2008). two main methodological approaches are used to describe the spatial distribution of organisms (brower et al., 1997). one involves sampling through plots (frequently referred to as plot sampling or quadrat sampling) where the sampling is done by marking plots in different locations in the community and counting the number of organisms (ant nests) in each plot, followed by data comparisons using indices and probability models (i.e. poisson distribution and morisita’s index) (soares & schoereder, 2001; schatz & lachaud, 2008; silva junior et al., 2013). another method involves sampling the distance between individuals (usually plants and sessile organisms) or between the individual and a random point previously established, known as “nearest neighbor” method (brower et al., 1997; gao, 2013). distance methods can help us to determine whether ant mounds are growing or having foraging territories in discernible (and often ecologically important) patterns or are randomly dispersed. many intra-specific relationships among ant mounds are difficult to observe without using distance based sampling techniques. moreover, the effectiveness of plot sampling depends on plot size and shape and this method fails in giving the relative positions of individuals within plots (gao, 2013). however, due to the influence of several environmental factors involved, more than one index should be estimated before concluding about the spatial arrangement of a particular species (brower et al., 1997; mollet et al., 1984). leaf-cutting ants of the genera atta and acromyrmex are considered important modifiers in their environments, promoting the turnover and aeration of soil in areas adjacent to the mounds, incorporating organic matter to the system (moutinho et al., 2003; sousa-souto et al., 2008), acting in carbon and nutrient cycling (stenberg et al., 2009; sousasouto et al., 2012a) and directly affecting the plant community structure (garrettson et al., 1998; leal & oliveira, 1998; corrêia et al., 2010). however, species of these two genera are also considered major pests in brazilian agroecosystems by causing considerable damage due to intense and constant attacks on plants at all stages of development (della lucia 2011; nickele et al., 2013). for this reason, the majority of studies examining the spatial distribution of leaf-cutting ant mounds were made in reforestation areas, with the goal of developing pest management plans to control these ants (caldeira et al., 2005; cantareli et al., 2006; nickele et al., 2009). this study evaluates the spatial distribution of mounds of acromyrmex balzani using the methods of plot sampling and nearest neighbor distance. if different sampling methods produce similarly robust results, the simplest method would thus be more appropriate for determining the spatial distribution of this ant species in the future. material and methods study area the study was performed on a fragment of grassland, located in the municipality of são cristovão, sergipe, brazil (11 º 00’54 “s, 37 º 12’21” w). the dominant vegetation consists of grasses and herbs, mainly paspalum notatum flügge (poaceae), cynodon dactylon l. (poaceae) and richardia brasiliensis gomes (rubiaceae) (poderoso et al., 2009). the average annual temperature is 29 ºc with average annual rainfall of about 2000 mm. the soil type is spodozol, mainly sandy clay, deep, with low fertility, high porosity (draining rainfall), high acidity and salinity (sousa-souto et al., 2012b). the leaf-cutting ant a. balzani is commonly found and is clearly the most abundant leaf-cutting ant species in the study area. nests of a. balzani are small (0.2 to 1m2 of area) compared with other leaf-cutting ant species but with population densities that may reach beyond 100 nests per hectare (poderoso et al., 2009; sousa-souto et al., 2012b; silva junior et al., 2013). each nest has a single entrance (fig. 1 a-b) with a depth varying from 12 to 150 cm. this ant species relies mainly on leaves from monocotyledons (fowler et al., 1986) which are deposited within the nest chamber (poderoso et al., 2009). the waste material is delivered outside the colony, forming small piles of refuse (mendes et al., 1992; sousa-souto et al., 2012b). fig. 1 – acromyrmex balzani (emeri) in the study area. a – workers in the nest entrance. b – detail of a nest entrance formed by two tubes of straw and other vegetable waste. c general view of the area with nest mounds (blank spots). l sousa-souto, ab viana-júnior, es nascimento spatial distribution of acromyrmex balzani nests164 ant sampling in a fragment of approximately 0.5 ha, during january 2013 (dry season), we established 360 plots of 2 x 5 m (10 m2), with a total sampled area of 3,600 m2 (72% of the total area of fragment). in each plot, the number of colonies of a. balzani was obtained and the data were subjected to analyses of dispersion in order to determinate the type of spatial distribution. we used three index of dispersion: the poisson probability model of distribution, morisita’s index of dispersion and the nearest neighbor method (brower et al., 1997). for poisson model, the observed frequency is compared with the expected frequency, calculated by the formula: p(x) = (µx e-µ)/x!, where: p(x) = probability of finding x ant nests within plot; µ = average number of nests; a positive real number (1, 2, 3...) and e = is the base of the natural logarithm (= 2.71828...). the distribution pattern obtained can be compared by chi-square statistic (brower et al., 1997). in addition to the adjustment of the poisson distribution, data were submitted to the morisita’s index of dispersion: id = n (∑x2-n)/(n(n-1)) where n is the number of plots, ∑x2 is the squares of the number of ant mounds per plot, summed over all plots and n is the total number of mounds counted on all n plots (brower et al., 1997). for the nearest neighbor method, random points (ant mounds) were located in the 5,000 m2 stand and the distance from each sampling point to the nearest ant nest was measured (n = 25 mounds). only one measurement was made from each random nest, and all distances for all mounds were summed and divided to yield one average distance. we then determined whether mounds were distributed randomly, regularly, or were clumped using an aggregation index a1 = (d/μ)2 (clark & evans, 1954), where d is the mean distance between these pairs of ant mounds; that is the mean nearest neighbor distance. the statistical significance of a1 is determined by μ=1/(2√(n/a)), where se = 0.26136/√(n2/a) and the critical value of t is that for infinite (∞) degrees of freedom. polydomy for a. balzani has been described (caldato, 2010), meaning, two or more ant mounds in the same plot could be subnests associated with a single colony. however, for the purpose of this study, any ant mound was considered a sampling unit, considering that the physical presence of an ant mound can affect the plant community as well as the establishment of another colony, regardless of being a subnest or the main colony. results we sampled 351 mounds of a. balzani in the 360 plots, with values ranging from 0 to 6 mounds per plot (figs. 1c and 2). the nest density of 0.04 nests/m2 and an average number of 0.98 ant mounds/plot found in this study is high (975 × colonies ha-1), since the densities for this species can vary from 80-210 colonies × ha-1 in similar environments of fig. 2 – schematic representation of the 359 plots distributed in the study area (3,600 m2 of a total of 5,000 m2) and the frequency of a. balzani colonies per plot. table 1 – the observed data from figure 01 and the probabilities expected from the poisson distribution with a mean (μ) of 0.98 ant mounds per plot. number of ant mounds in plot (x) observed frequency ƒ(x) observed probability p(x) poisson probability p(x) 0 154 0.43 0.38 1 110 0.31 0.37 2 54 0.15 0.18 3 33 0.09 0.06 4 6 0.02 0.01 5 0 0.0 0.0 6 2 <0.01 0.0 sociobiology 60(2): 162-168 (2013) 165 the study region (poderoso et al., 2009; sousa-souto et al., 2012b). for another leaf-cutting ant species (ac. landolti) nest density can reach values of 2,000 colonies per hectare (fowler et al., 1986). the values obtained in the plot sampling method (χ2 = 37.22; p < 0.05 and iδ = 1.35) indicate that the colonies are clumped in the environment (table 1). the same results were obtained using the nearest neighbor method (t = -1.72; p < 0.01). the mean distance between the pairs of ant mounds was 4.05 m and most of them (70%) were in a range of 2 to 6 m (fig 3). discussion the present study evaluated the spatial distribution of a. balzani through sampling plots and nearest neighbor methods. both methods indicated the ant mounds were aggregately distributed, corroborating our hypothesis that different sampling methods give similar results for nest spatial distribution. these results contrast with previous studies of leaf-cutting ant species in eucalyptus spp. forests, where the distribution of nests was random (caldeira et al., 2005; nickele et al., 2009). however, the aggregate spatial arrangement of ant nests has also been observed in pasture (silva junior et al., 2013) as well as in environments with native vegetation (rissing et al., 1986; soares & schoereder 2001). other ant species also show an aggregate distribution pattern, such as solenopsis invicta (buren) (almeida et al., 2007), mycetophylax simplex (emery) (albuquerque et al., 2005) and several litter ant species (soares & schoereder, 2001). for social insects, aggregate distribution of nests may occur in response to physical (habitat heterogeneity) and biological factors (low rate of dispersion, nest budding or chance of survival increased when the organisms are grouped) (nicholas & vilela, 1996; elisei et al., 2012; leal et al., 2012). termites, for example, have aggregated distribution when favorable soil conditions to the nest establishment are distributed in patches (dias et al., 2012) or when foraging sites are sparsely distributed (filho et al., 2012). the uniform pattern of distribution is common in some ant species and occurs when there is high nest density (nicholas & vilela, 1996). such a distribution may be due to high intraspecific competition for resources, involving territory defense by colonies (bernstein & gobbel, 1979; soares & schoereder, 2001). the usual limiting resources for ants are food and nesting sites (fowler et al., 1983; leal et al., 2012). it is possible, however, that these resources are not limited for leafcutting ants in pasture and cultivated fields or eucalyptus forests, so that intra and interspecific competition might not be strong because these habitats have a constant abundance of food resources. the high density of aggregately distributed ant mounds found in this study indicate that a. balzani do not show aggressive defense behavior for their foraging sites. in fact, it is common to find workers of different colonies in the same foraging site without any incidents of aggressive behavior (personal obs.). besides, the occurrence of subnests around the main nest (polydomy) was found for a. balzani (caldato, 2010). one could say that polydomy can mask the real dispersion pattern of this ant species, because a proportion of ant mounds could be subnests from a same main colony, leading to a clumped pattern in the landscape. since the confirmation of polydomy is only possible through experiments of aggression (caldato, 2010), and considering that the impact of an ant mound on herbivory is similar, regardless of the origin of the mound (subnests or the main nest), we can assume that the aggregate pattern of ant colonies is prevalent, even with a high rate of polydomy. fig 3. – frequency of ant mounds of a. balzani according to the distance between pairs of mounds. the high density of ant mounds found in the present study can be explained by the preference of these ants for open and human perturbed areas. previous studies have found that other leaf-cutting ant species such as acromyrmex lobicornis (emery), ac. landolti forel and atta laevigata (f. smith) are abundant in areas with greater sun exposure, mostly open places (grasslands and pastures) or sites with trees or shrubs with reduced canopy cover (bucher & montenegro, 1974; clark & evans, 1954; nickele et al., 2009; silva junior et al., 2013). it is possible that interspecific competition also act in determining the aggregate pattern of colonies of leaf-cutting ants. colonies of species with large numbers of workers and aggressive behavior, such as atta sexdens or at. laevigata may be present in foraging areas coexisting with acromyrmex spp., confining the latter to restricted sites within the fragment or forcing these colonies to emigrate (fowler, 1977; l sousa-souto, ab viana-júnior, es nascimento spatial distribution of acromyrmex balzani nests166 1983). in his study about distribution of leaf-cutting ants in paraguay, fowler (1983) proposed that the most important factor determining the local abundance of leaf-cutting ant species was interspecific competition. in other words, in sites where acromyrmex species were locally abundant, atta species were rare or absent, and vice versa. indeed, in our study area we found two colonies of at. opaciceps with an average area of 5 m2 which were located on plots which also hadcolonies of ac. balzani. it is possible that competition is minimized by differentiation of the time of foraging (ac. balzani is diurnal whereas at. opaciceps is nocturnal) and the type of resources collected (leaves of grasses versus shrubs and trees). in a previous study, wetterer (1991) investigated the overlap of foraging areas between colonies of at. cephalotes and ac. octospinosus, noting that there was only a single case of simultaneous exploitation of of the same resource (flowers). several other differences in foraging strategies (e.g., type of substrate harvested, foraging time, size range of foragers) were also concluded to contribute to the sustained coexistence of these species (wetterer, 1991). future studies focusing on the interaction of these two genera will help clarify whether the dispersion of colonies is determined by the presence of interspecific competitors. the use of plots is a well-established method for studying the dispersion pattern of colonies in leaf-cutting ants. this method is appropriate in reforestation areas (pinus and eucalyptus spp.), where planting areas are divided into quadrats according to the age of trees. however, the use of the nearest neighbor method, as seen in this study, proved to be just as effective as plot sampling for determining distribution patterns of colonies in environments under native vegetation and fragments with irregular shape, making it a preferred method since we can provide the same results with lower sampling effort, compared with plot sampling method. acknowledgments the authors are grateful to hans kelstrup for the revision of the english and to bianca ambrogi for her comments in a previous version. we thank two anonymous reviewers for helpful comments on the manuscript. this study was supported by the coordenação de aperfeiçoamento de pessoal de nível superior (capes). references albuquerque, e. z., diehl-fleig, e. & diehl, e. 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(2007). predicting the spatial distribution of an invasive plant species (eupatorium adenophorum) in china. landsc. ecol., 22(8): 1143-1154. doi: 10.1007/s10980-007-9096-4 doi: 10.13102/sociobiology.v64i4.1934sociobiology 64(4): 404-416 (december, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ants (hymenoptera: formicidae) and spiders (araneae) co-occurring in the ground of vineyards from douro demarcated region introduction ants (hymenoptera, formicidae) and spiders (arachnida, aranaea) are two ubiquitous arthropods and keystone taxa in terrestrial communities, where they can interact directly or indirectly, resulting in strong effects on the abundance and distribution of each other (marín et al., 2015). ants are one of the most abundant organisms in terrestrial surface being found in most ecosystems (huang et al., 2011), where they impose a strong ecological footprint abstract this study, held in vineyards from douro demarcated region, aimed to: a) identify the communities and main functional groups of spiders and ants; b) check patterns of cooccurrence between the two communities; and c) evaluate the impact of ground cover and non-crop habitats adjacent to vineyards, on the two communities. samplings were done using pitfall trapping. twenty species of ants and 44 species of spiders were identified. the most abundant were: aphaenogaster gibbosa (latreille 1798), aphaenogaster iberica emery 1908, cataglyphis hispanica (emery 1906), cataglyphis iberica (emery 1906), messor barbarus (l. 1767) and tapinoma nigerrimum (nylander 1856) which totalized 71.21% of ants; and alopecosa albofasciata (brullé 1832), callilepis concolor simon 1914, eratigena feminea simon 1870, zodarion alacre (simon 1870) and zodarion styliferum (simon 1870) which accounted for 38% of spiders. three iberian endemic ants and nine iberian endemic spiders were also identified. abundance of both ant-mimicking and ant-eating spiders were positively correlated with abundance of formicinae, while only abundance of ant-eating spiders showed positive correlation with abundance of myrmicinae ants. all genera/ species of antassociated spider were associated with one or more genera/specie of ants. both spiders and ants have not benefited from the adjacent non-crop habitat. sheet web weaver spiders were found to be positively correlated with the percentage of ground cover. these results show that a) vineyard agroecosystem support a rich assemblage of ants and spiders evincing that wine production and species conservation is possible and b) non-random co-occurrence between ants and ant-associated spiders exist in the field. sociobiology an international journal on social insects f gonçalves1, v zina2, c carlos1,3, l crespo4, i oliveira1, l torres1 article history edited by kleber del-claro, ufu, brazil received 09 august 2017 initial acceptance 10 october 2017 final acceptance 13 november 2017 publication date 27 december 2017 keywords formicinae, myrmicinae, zodarion, co-occurrence, myrmecophagy, myrmecomorphy. corresponding author fátima gonçalves centre for the research and technology of agro-environmental and biological sciences, university of trás-os-montes and alto douro 5001-801 vila real, portugal e-mail: mariafg@utad.pt through their diverse ecological functions, mainly as biological regulators and ecosystem engineers (ward, 2006). most species are omnivorous and generalists (cerdá & dejean, 2011). others are important predators (karhu, 1998), herbivorous (albert et al., 2005; rodriguez et al., 2008) and scavengers (feeding on decaying organic matter) (perez & dupo, 2013). while others feed on honeydew (detrain et al., 2010), pollen (urbani & de andrade, 1997) and extrafloral nectar and glandular corpuscles (kost & heil, 2005; stefani et al., 2015; del-claro et al., 2016). ants are involved in mutualistic relationships with 1 centre for the research and technology of agro-environmental and biological sciences, university of trás-os-montes and alto douro, vila real, portugal 2 centro de estudos florestais, universidade de lisboa, lisboa, portugal 3 association for the development of viticulture in the douro region, vila real, portugal 4 department of evolutionary biology, ecology and environmental sciences (arthropods), biodiversity research institute (irbio), universitat de barcelona, spain research article ants mailto:mariafg@utad.pt sociobiology 64(4): 404-416 (december, 2017) 405 hemipterans, protecting them from their enemies, in exchange for honeydew (styrsky & eubanks, 2007). moreover, they are important for pollination (hickman, 1974), and dispersal of numerous plants (beattie & culver, 1981). in addition, they are ecosystem engineers, because of their effects on soil structure and processes, which directly and indirectly affect the flow of energy and material in ecosystems, as well as the habitats of other species (folgarait, 1998). through their activity, ants modify the physical, chemical and microbiological properties of soil (dauber & wolters, 2000; dostál et al., 2005; jouquet et al., 2006). through evolution, ants developed both structural (stings, spines, strong mandibles, chemical secretions) and behavioral (aggressiveness, deployment of polymorphic workers) defense mechanisms that allow them to escape from most predators (hunt, 1983). this evolutionary advantage makes them ideal models for mimics (mciver & stonedahl, 1993) and also available food for specialist predators. in fact, hunters that evolves strategies for overcoming ant’s defenses and aggression faces relatively little competition for a nearly unlimited food resource (cushing, 2012), since that they are rare and typically use special hunting tactics (mciver & stonedahl, 1993). three types of associations between ants and other species can be find: myrmecomorphy (species that mimic ants acquiring morphological, behavioural, chemical or textural similarity to the model, currently known as ant-mimics), myrmecophagy (species that feed on ants, currently known as ant-eaters), myrmecophily (species that live in symbiosis with ants in or near of their nests) (cushing, 1997; 2012; pekár et al., 2012a). spiders are ubiquitous predators in terrestrial ecosystems that feed primarily on insects but also on other arthropods including other spiders (wise, 1993). although it is assumed that in general, they are euryphagous or generalists, being able to subsist on a wide variety of prey types (cardoso et al., 2011), the fact is that some species specialize in hunting in a singular prey group. from this point of view, pekár et al. (2012a) report six categories of stenophagy among spiders, namely: araneophagy, lepidopterophagy, termitophagy, dipterophagy, crustaceophagy and myrmecophagy. indeed, although most of spiders are averse to ant predation most of the specialized species are myrmecophagous, perhaps as a consequence of ants being numerous in their habitats (pekár, 2004). examples of myrmecophagy are found in a variety of families, including gnaphosidae, oecobiidae, salticidae, theridiidae, thomisidae, and zodariidae (reviewed by pekár, 2009). moreover, some species of spiders also mimic ants; spiders disguise as ants to deceive primarily their predators (ceccarelli, 2013) being considered as a case of batesian mimicry (nelson & jackson, 2012), where a palatable mimic spider escapes from predators that have experienced unpalatable ants (hölldobler & wilson, 1990). the spiders corresponding mimicking mechanisms includes morphological, chemical and/or behavioral resemblance to ants (mciver & stonedahl, 1993). constrictions on the abdomen giving the illusion of three body regions (cushing, 2012), shiny opistossoma (ceccarelli, 2008), and long and thin legs (oliveira, 1988; durkee et al., 2011) are some of these mechanisms. also, their movement frequently becomes antlike, including wave their forelegs to mimic the antennal movement of ants (in a phenomenon referred as ‘‘antennal illusion’’) (ceccarelli, 2008; cushing, 2012). examples of myrmecomorphy spiders are mainly found in salticidae and clubionidae. however, theridiidae, araneidae, eresidae, thomisidae, gnaphosidae, zodariidae and aphantochilidae also have ant-like members (review by oliveira, 1988). in vineyards, ants are one of the most abundant arthropod group (addison et al., 2013; thomson et al., 2004), while spiders are one of the most abundant predator group (gaigher & samways, 2010; 2014; pérez-bote & romero, 2012). in douro demarcated region (ddr) vineyards, little is known about the composition of ant and spider communities, and how they interact each other. the aims of this study were to: a) assess the richness and functional diversity of ant and spider communities in vineyards of ddr; b) evaluate the cooccurrence pattern between ants and ant-associated spiders and c) evaluate the impact of ground cover and of non-crop habitats adjacent to vineyards on ant and spider communities. material and methods study area the study was carried out during the growing season of 2013, in five commercial vineyard farms (aciprestes (41°12’30”n, 7°25’58”w), arnozelo (41°8’1”n, 7°18’25”w), carvalhas (41°10’36”n, 7°32’26”w), cidrô (41°8’30”n, 7°22’51”w) and granja (41°15’4”n, 7°28’50”w)) from ddr. in each farm, one vineyard with ground cover in the inter-row and an adjacent non-crop habitat, was selected as study site. adjacent non-crop habitats were shelterbelts (mainly composed by cistus albidus l., cistus ladanifer subsp. ladanifer l., cistus salvifolius l., cytisus multiflorus (l’hér.) sweet, erica arborea l., erica umbellata loefl. ex l., genista anglica l., genista triacanthos brot., halimium lasianthum (lam.) spach, juniperus oxycedrus l., lavandula pedunculata (mill.) cav., pistacia terebinthus l., rubus ulmifolius schott, ulex minor roth, xolantha guttata (l.) rafin.), woodlands (mainly composed by arbutus unedo l., pinus pinaster aiton, quercus x coutinhoi samp., quercus faginea lam., quercus pyrenaica willd., quercus rotundifolia lam.) and groves (olea europaea l., prunus dulcis (mill.) d. a. webb). the ground cover of each vineyard consisted in a complex of resident vegetation (predominantly andryala integrifolia l., bromus madritensis l., coleostephus myconis (l.) rchb. f., cynodon dactylon (l.) pers., echium plantagineum l., ornithopus compressus l., silene gallica l., sonchus oleraceus l., trifolium sp.). in all farms, the vegetation in the inter-row was mowed in the beginning of spring, then allowed to regrow, mature and set seed, and again mowed. moreover, f gonçalves, v zina, c carlos, l crespo, i oliveira, l torres – ants and spiders co-occurring in vineyards406 while in two vineyards (arnozelo and carvalhas), the same procedure was performed to control the vegetation in the vine row, in the other three farms, herbicide was applied in the vine row (cidrô, aciprestes and granja). the mowed residues remained in the soil, acting as mulching. sampling data spiders and ants collection spiders and ants were sampled between april and september 2013, with a periodicity of 35 to 45 days, totalizing four sampling dates in each farm (two samplings per each of the season’s spring and summer). the first period was between april 20th and june 6th and the second was between july 1st and september 23rd. nine pitfall traps were placed in the interrow of each vineyard, at the rate of three traps per each of the following distances, 5, 50 and 100 m, from the adjacent noncrop habitat inward the vineyard. the pitfall traps consisted on plastic cups measuring 16 cm in height and 9 cm in diameter. the cups were filled to half its volume with a 3:1 mixture of water and polypropylene-glycol solution and were left in the field for 72 hours. ant specimen’s identification was based on collingwood and prince (1998) and gómez and espadaler (2007). spider specimen’s identification was based on world spider catalog (2017) and nentwig et al. (2016). individuals were identified to the species level whenever possible and in a minority of cases, fragmented specimen’s identification was considered reliable only to the genus level. ground cover vegetation for each plot, in each trap location, the richness (number of plant species) and the percentage of ground cover were assessed in 1 m2 area. observations were done between the end of may and the beginning of june. data analysis classification of ants and spiders using roig and espadaler (2010) classification, formicidae was separated in four functional groups: (1) generalists and/or opportunistic (g/o), (2) cold-climate specialists and/or shade habitats (ccs/sh), (3) hot climate specialists and/or open habitats (hcs/oh), (4) and cryptic (cr). following the same authors, these groups can be classified as to their relative importance in three global indicators: global indicator of disturbance (g/o), global indicator of stability (hcs/oh and ccs/sh) and a silent group of cryptic ants (c). moreover, trophic guild was also assessed by using the literature (see table s1 in supplementary materials). araneae were classified following cardoso et al. (2011), based on their foraging strategy (type of web and hunting method), prey range (stenophagous or euryphagous), vertical stratification (ground or foliage) and circadian activity (diurnal or nocturnal). eight guilds were assessed: (1) sensing web weavers (sew), (2) sheet web weavers (shw), (3) space web weavers (spw), (4) orb web weavers (orw), (5) specialists (sp), (6) ambush hunters (ah), (7) ground hunters (gh), and (8) other hunters (oh). ant associated spiders were divided into two groups: anteating “myrmecophages” and ant-mimicking “myrmecomorphs” (cushing, 1997, 2012; mciver & stonedahl, 1993; nentwig et al., 2016; pekár et al., 2012a; pekár & cárdenas, 2015; pekár & jarab, 2011). statistical analysis non-metric multidimensional scaling (nmds) was performed to visualize and to examine the similarities of functional groups of ant and spider communities among the studied sites and seasons. prior to analysis, observations with no individuals were removed. nmds were obtained fixing a 2-dimensional solution and using all the available dissimilarities indices in “vegdist” function, choosing the convergent solution with lowest stress value under 0.2. analysis were performed by using the vegan package facilities r (oksanen et al., 2016) and nmds plots were produced by using the r package ggplot2 (wickham, 2009). for the co-occurrence studies, spearman correlations were used to explore correlations between abundance of ant-associated spiders and ants. moreover, null models were performed to detect possible non-random of co-occurrence. the co-occurrence analyses allowed to test for non-random patterns of species co-occurrence in a presence-absence matrix. the null-model matrices are monte-carlo randomizations of the real matrix in order to create null expectations for the c-score. the c-score was used as an index which measure the degree to which pairs co-occur. a “fixed rows-equiprobable columns” model for 5,000 randomizations was performed. the model randomizes the occurrence of each species among the sites, assuming that sites are equiprobable. when used in fixed-equiprobable’ model, c-score has good statistical properties and are invulnerable to type i errors (false positives) (gotelli, 2000). for a better understanding of the results, standardized effect size (s.e.s) are presented in the results. s.e.s is calculated as (observed c-score – mean of simulated c-scores)/standard deviation of simulated c-scores; values of s.e.s greater than 1.96 and less than -1.96 indicate a negative and positive non-random co-occurrence pattern, respectively. these analyses included only species/genera with more than five individuals collected during the study. generalized linear mixed models (glmm) (with poisson error distribution or negative binomial distribution to account with over dispersion when necessary, and a log link function) were developed to fit both the abundance and richness of spiders and ants as a function of the season (spring vs summer), the distance to the adjacent non-crop habitat (5, 50, 100 m), and the richness and percentage of ground cover. the farm was included into the model as random factor, as it was intended to generalize the results of this experiment sociobiology 64(4): 404-416 (december, 2017) 407 to all field. when a random effect for farm is added, this characterizes idiosyncratic variation that is due to individual differences. the procedure was done for the most significant functional groups of spiders and ants. for each season, data from each trap were pooled resulting in a single sample per trap. co-occurrence analyses were carried out using ecosim program version 7.0 (acquired intelligence, inc., & pinyon publishing, 2016). glmm and spearman correlations were performed by using ibm spss v20 (spss inc. ibm company, 2010). significance was reported at the level of p < 0.05. results ant community a total of 6,322 ant individuals from three subfamilies and 12 genera were found, which corresponded to 20 species (table s1). the most abundant subfamily was myrmicinae (45.7%), followed by formicinae (42.3%) and dolichoderinae (12.0%). however, the one represented by higher number of taxa was formicinae (with six genera and 13 species), followed by myrmicinae (five genera and six species) and lastly by dolichoderinae (one genus and one species). the most abundant ant species, which together amounted 32.8% of the catches, were messor barbarus (myrmicinae), which was captured in all farms, and cataglyphis hispanica (formicinae), which was captured in four farms. other abundant species were aphaenogaster gibbosa, aphaenogaster iberica, cataglyphis iberica and tapinoma nigerrimum that totalized 38.5% of the catches. nine ant species were present in all farms (i.e. a. gibbosa, crematogaster auberti, camponotus cruentatus, iberoformica subrufa, m. barbarus, pheidole pallidula, cataglyphis sp., plagiolepis schmitzii and t. nigerrimum), while two species, (i.e. camponotus foreli and camponotus piceus) were collected in a single farm. three species, i.e. c. hispanica, c. iberica and a. iberica are endemic from iberian peninsula. according to the trophic group, 1.4% of the collected ants were classified as nectar feeders (c. foreli, c. piceus and camponotus pilicornis), 2.5% as honeydew feeders (camponotus sylvaticus, lasius grandis and p. schmitzii), 44.4% as omnivorus (a. gibbosa, a.iberica, c. auberti, c. cruentatus, formica fusca, i. subrufa, p. pallidula, p. pygmaea, solenopsis sp. and t. nigerrimum), 17.4% as herbivorous (m. barbarus) and 27.3% as scavengers (all cataglyphis), in spite of some of them could complement their diet with other diets (table s1). ant community was grouped in four functional groups: hot climate specialists and/or open habitats (hcs/oh) (46.2% of captures), generalists and/ or opportunistic (g/o) (32.4%), cryptic (cr) (11.2%) and cold climate specialists and/or shade habitats (ccs/sh) (2.1%) (fig 1, table s1). about 30.0% of the species were g/o, 45.0% hcs/oh, 15.0% ccs/sh and 10.0% cr (fig 2). the nmds analysis, showed a high degree of overlap between different sites for functional groups of ants without a formation of clear groups concerning season and farms (having in mind that farms have different soil management, namely the use of herbicide in the row in three of them) (fig 3). fig 1. percentage of ants per functional group. ac – aciprestes; ar – arnozelo; ca – carvalhas; c – cidrô; g – granja; t – all sites combined; g/o generalists and/ or opportunistic; hcs/oh hot climate specialists and/or open habitats; cr cryptics; ccs/sh cold climate specialists and/or shade habitats. fig 2. richness of ants per functional group. ac – aciprestes; ar – arnozelo; ca – carvalhas; c – cidrô; g – granja; t – all sites combined; g/o generalists and/ or opportunistic; hcs/oh hot climate specialists and/or open habitats; cr cryptics; ccs/sh cold climate specialists and/or shade habitats. spider community a total of 511 spiders were captured. about 10.4% of those individuals were damaged and could not be classified into families; the remaining were grouped in 19 families and 40 genera. only adults (about 67.3%) were identified, totalizing 44 species (table s2 in supplementary materials). the most abundant family was zodariidae (29.2%), followed by gnaphosidae (23.5%), lycosidae (13.5%), thomisidae (8.9%) and agelenidae (8.7%). nine other families accounted, each one, for less than 1% of the total catches. gnaphosidae had the highest number of species (13), followed by salticidae (10), lycosidae and thomisidae (both with 6), and agelenidae (5). f gonçalves, v zina, c carlos, l crespo, i oliveira, l torres – ants and spiders co-occurring in vineyards408 the most abundant species was zodarion styliferum (zodariidae) (20.3%), followed by alopecosa albofasciata (lycosidae) (5.9%) and eratigena feminea (agelenidae) (5.2%). z. styliferum and e. feminea were present in all farms. nine species (representing together about 12.2% of the catches) (i.e. eratigena bucculenta, e. feminea, eratigena montigena and tegenaria ramblae from agelenidae; zodarion alacre and zodarion duriense from zodariidae; castianeira badia from corinnidae; nemesia athiasi from nemesiidae and oecobius machadoi from oecobiidae) are endemic species from iberian peninsula (table s2). spiders were grouped into eight guilds: ground hunters (gh) (corinnidae, gnaphosidae, lycosidae and oonopidae) (37.5% of the identified individuals), specialist (sp) (zodariidae) (29.2%), sheet web weavers (shw) (agelenidae and agyneta fuscipalpa from linyphiidae) (9.8%), ambush hunters (ah) (sicariidae and thomisidae) (9.4%), other hunters (oh) (other linyphiidae, oxyopidae philodromidae, salticidae and scytodidae) (8.3%), space web weavers (spw) (dictynidae, theridiidae and titanoecidae) (4.6%), sensing web weavers (sew) (nemesiidae and oecobiidae) (0.9%) and orb web weavers (araneidae) (0.4%) (fig 4; table s2). the nmds analysis, evidenced a high degree of overlap between different sites; even though, a trend could be observed for the establishment of distinct groups (fig 5). arnozelo and carvalhas (both farms without herbicide in the row) showed a tendency to be separated from granja and aciprestes (both with herbicide in the row). while sew, oh and ah spiders were more associated with the first two farms, gh and sp spiders were more associated with the last two. fig 3. nmds ordination plots of functional groups of ants associated with the study vineyards. the ultimate 2-dimensional nmds solution was found with euclidean dissimilarity and had an associated stress of 0.108. ac – aciprestes; ar – arnozelo; ca – carvalhas; c – cidrô; g – granja; sp – spring; su – summer; g/o generalists and/ or opportunistic; hcs/oh hot climate specialists and/or open habitats; cr cryptics; ccs/sh cold climate specialists and/or shade habitats. in ac, c and g the row was treated with herbicide while in ar and ca no herbicide was applied. fig 4. percentage of spiders per functional group. ac – aciprestes; ar – arnozelo; ca – carvalhas; c – cidrô; g – granja; t – all sites combined; gh – ground hunters; sp – specialists; shw – sheet web weavers; ah – ambush hunters; oh – other hunters; spw space web weavers; sew sensing web weavers; orw orb web weavers. fig 5. nmds ordination plots of functional groups of spiders associated with the study vineyards. the ultimate 2-dimensional nmds solution was found with euclidean dissimilarity and had an associated stress of 0.194. ac – aciprestes; ar – arnozelo; ca – carvalhas; c – cidrô; g – granja; sp – spring; su – summer; gh – ground hunters; sp – specialists; shw – sheet web weavers; ah – ambush hunters; oh – other hunters; spw – space web weavers; sew – sensing web weavers; orw – orb web weavers. ac, c and g were treated with herbicide in the inter-row while ar and ca were not treated with herbicide. co-occurrence between ants and ant-associated spiders six genera of spiders were found to include ant-eater individuals, namely callilepsis, euryopis, nomisia, oecobius, oxyopes and zodarion. individuals of these genera accounted for 37% of the total catches. on the other hand, 2% of spiders belonged to three genera included ant-mimic spiders: castianeira and micaria (both known as ant-mimicking sac spiders) and myrmarachne (known as ant-mimicking jumping spiders). euryopis episinoides and zodarion spp. are also mimic ants; however, they were included in the ant-eaters group as their mimic behavior, viewed as inaccurate mimics, has as adjacent objective the predation of ants. sociobiology 64(4): 404-416 (december, 2017) 409 a strong and positive correlation was found between the abundance of ant-associated spiders and abundance of total ants; moreover, a strong and positive correlation was also found between the abundance of ant-associated spiders and that of both formicinae and myrmicinae, but no correlation could be found the abundance of dolichoderinae. on the other hand, the abundance of ant-eating spiders was significant and positively correlated with that of both formicinae and myrmicinae, while ant-mimics spider numbers only correlated positively with those of formicinae (table 1). the abundance of callilepis spiders was significant and positively correlated with that of cataglyphis ants, more precisely with the species c. pilicornis and c. auberti (table 2). null model tests of co-occurrence detected significant association between callilepis and both c. auberti and m. barbarus ants, while the association was near of significance between c. pilicornis and cataglyphis genus (table 3). the abundance of nomisia spiders correlated significant and positively with the abundance of ants from plagiolepis and aphaenogaster genera and a. iberica and a. gibbosa species (table 2). null model tests only found significant association with plagiolepis genus (table 3). ant-associated spiders ants total dolichoderinae formicinae myrmicinae total 0.494 *** 0.152 n.s 0.517 *** 0.331 ** ant-eaters 0.476 *** 0.010 n.s 0.495 *** 0.338 ** ant-mimics 0.205 n.s 0.136 n.s 0.257 * -0.131 n.s ***p<0.001; **p<0.01; * p<0.05; n.s p≥0.05 table 1. spearman correlation coefficients (rho) between abundance of ants (total and subfamily groups) and abundance of ant-associated spiders (total, ant-eaters and ant-mimics). the abundance of zodarion spiders correlated significant and positively with that of cataglyphis and aphaenogaster genera, as well as the species i. subrufa, a. iberica, a. gibbosa and m. barbarus (table 2); null model tests detected significant association between spiders from zodarion genus and ants from aphaenogaster genus, and i. subrufa, a. iberica and a. gibbosa species (table 3). z. styliferum abundance, the most abundant spider in this study, was significant and positively correlated with that of ants from camponotus, cataglyphis and aphaenogaster table 2. spearman correlation coefficients (rho) between abundance of ant-associated spiders and abundance of ants (genera and species). only genera/species with at least five occurrences were included in the analysis. ant subfamily/genus/species spiders ant-eaters ant-mimics callilepis spp. nomisia spp. zodarion spp. z. styliferum z. alacre micaria spp. dolichoderinae tapinoma nigerrimum 0.098 n.s -0.150 n.s 0.099 n.s 0.167 n.s -0.125 n.s -0.006 n.s formicinae camponotus spp. 0.096 n.s 0.163 n.s 0.116 n.s 0.228 * -0.114 n.s 0.332 ** c. cruentatus 0.104 n.s 0.127 n.s 0.115 n.s 0.210 * -0.083 n.s 0.341 ** c. pilicornis 0.212 * 0.161 n.s 0.189 n.s 0.289 * -0.125 n.s 0.476 *** cataglyphis spp. 0.286 ** -0.123 n.s 0.228 * 0.235 * -0.082 n.s 0.276 ** c. hispanica 0.104 n.s -0.193 n.s 0.141 n.s 0.131 n.s 0.004 n.s 0.083 n.s c. iberica 0.108 n.s -0.139 n.s 0.029 n.s 0.099 n.s -0.096 n.s -0.023 n.s iberoformica subrufa 0.084 n.s 0.140 n.s 0.408 *** 0.307 ** -0.107 n.s 0.467 *** plagiolepis spp. 0.186 n.s 0.252 * 0.067 n.s 0.085 n.s -0.094 n.s -0.017 n.s p. pygmaea 0.177 n.s 0.131 n.s 0.053 n.s 0.027 n.s -0.016 n.s 0.057 n.s p. schmitzii 0.177 n.s 0.169 n.s 0.056 n.s 0.051 n.s -0.125 n.s -0.094 n.s myrmicinae aphaenogaster spp. 0.046 n.s 0.225 * 0.401 *** 0.488 *** -0.046 n.s 0.195 n.s a. iberica -0.072 n.s 0.265 * 0.236 * 0.331 ** -0.230 * 0.288 ** a. gibbosa 0.073 n.s 0.219* 0.448*** 0.433*** 0.018 n.s 0.033 n.s crematogaster auberti 0.288 ** 0.090 n.s 0.176 n.s 0.254 * -0.159 n.s -0.046 n.s messor barbarus 0.190 n.s 0.117 n.s 0.274 ** 0.252 * -0.066 n.s -0.004 n.s pheidole pallidula -0.070 n.s 0.022 n.s 0.046 n.s 0.047 n.s -0.011 n.s -0.144 n.s *** p<0.001; **p<0.01; * p<0.05; n.sp≥0.05 f gonçalves, v zina, c carlos, l crespo, i oliveira, l torres – ants and spiders co-occurring in vineyards410 genera, and the species c. cruentatus, c. pilicornis, i. subrufa, a. iberica, a. gibbosa, c. auberti and m. barbarous (table 2). null model tests of co-occurrence detected a significant association between z. styliferum and the former taxa, except for cataglyphis genus and m. barbarous (table 3). the abundance of z. alacre spiders was correlated negatively with that of a. iberica ants and negative nonrandom co-occurrence (p (o>=e) = 0.018 and ses = 2.535) was also found between them. both spearman correlations and null model tests revealed significantly positive associations between micaria spiders and ants from the camponotus and cataglyphis genera, and c. cruentatus, c. pilicornis, i. subrufa and a. iberica species. (tables 2 and 3). impact of adjacent non-crop habitats and ground cover on the spider and ant communities the abundance and richness of generalists/ opportunists (g/o), specialist of warm climates and/ or open habitats (hcs/oh) as well as of cryptic (cr) ants was significantly higher in summer than in spring (table 4). moreover, the abundance of g/o and cr ants was higher in the distance of 100 m from adjacent non-crop habitat than in the distance of 5 m, and the abundance of hcs/oh ants was higher in 100 m than in both 5 m and 50 m. also, the abundance of hcs/oh ants was positively related to the richness of ground cover. concerning spiders, the abundance and richness of sp spiders and the richness of oh spiders was significantly higher in summer than in spring, while no differences between seasons occurred in the abundance and richness of gh, shw and ah spiders. both the abundance and richness of gh spiders, as well as the abundance of sp spiders, were higher in the distance of 100 m of the adjacent non-crop habitat than in the distances of 5 and 50 m (table 4). on the other hand, the richness of oh spiders was higher in the distance of 5 m from the adjacent non-crop habitat than in the distances of 50 or 100 m. both abundance and richness of gh spiders were positively related to the richness of ground cover (table 4), while the abundance and richness of shw spiders were positively related to the percentage of ground cover (table 4). the relationship between abundance of sp spiders and the percentage of ground cover was positive, although only near of significance. ant subfamily/genus/species ant-eaters ant-mimics callilepis spp. nomisia spp. zodarion spp. z. styliferum z. alacre micaria spp. dolichoderinae tapinoma nigerrimum -0.603 n.s 1.235 n.s -1.209 n.s -1.085 n.s 0.714 n.s 0.035 n.s formicinae camponotus spp. -0.273 n.s -1.894 n.s -1.391 n.s -2.286 ** 1.184 n.s -2.303 * c. cruentatus -0.598 n.s -1.413 n.s -1.547 n.s -2.192 * 0.872 n.s -2.518 * c. pilicornis -1.892 n.s -1.513 n.s -1.556 n.s -2.457 * 1.243 n.s -3.715 ** cataglyphis spp. -1.794 n.s 1.111 n.s -0.605 n.s -0.299 n.s 0.1295 n.s -1.926 * c. hispanica -0.094 n.s 1.964 n.s -0.076 n.s 0.185 n.s -0.097 n.s -0.892 n.s c. iberica -0.968 n.s 1.374 n.s 0.306 n.s -0.682 n.s 0.764 n.s 0.106 n.s iberoformica subrufa -0.7650 n.s -1.407 n.s -3.235 *** -2.490 ** 0.924 n.s -3.314 *** plagiolepis spp. -1.331 n.s -2.243 * -0.109 n.s -0.845 n.s 0.813 n.s -0.146 n.s p. pygmaea -1.508 n.s -1.452 n.s 0.450 n.s -0.764 n.s 0.039 n.s -1.682 n.s p. schmitzii -0.987 n.s -1.526 n.s 0.450 n.s -0.047 n.s 1.250 n.s -0.480 n.s myrmicinae aphaenogaster spp 0.097 n.s -0.322 n.s -2.648 ** -2.420 ** 0.079 n.s -1.228 n.s a. iberica 0.313 n.s -1.644 n.s -1.831 * -2.307 ** 2.535 n.s -2.178 * a. gibbosa -0.096 n.s -0.968 n.s -2.975 *** -2.579 ** -0.109 n.s -0.787 n.s crematogaster auberti -1.993 * -1.053 n.s -1.697 n.s -2.327 ** 1.307 n.s 0.254 n.s messor barbarus -3.834 *** 0.713 n.s -0.942 n.s -1.071 n.s 1.158 n.s -0.526 n.s pheidole pallidula 0.576 n.s -0.2533 n.s 0.325 n.s 0.369 n.s 0.591 n.s 1.176 n.s *** p<0.001; **p<0.01; * p<0.05; n.sp≥0.05 table 3. standard effect size (ses) for the co-occurrence of ant associated spiders and ants (genera and species) in null model tests. significant values are reported in bold. the significance level of p<0.05 is reported for the number of samples for which the observed value of the index was significantly less than the expected by chance (o<e). only genera/species with at least five occurrences were included in the analysis. sociobiology 64(4): 404-416 (december, 2017) 411 discussion ant community a total of 20 ant species were identified. more than half of the collected individuals belonged to five species (m. barbarus, c. hispanica, a. gibbosa, a. iberica and c. iberica). messor barbarus is widely distributed in the mediterranean region, being an herbivorous species, commonly known as “harvest ant”, which collect and feed on seeds and fruits from grassland and scrubland plants (azcárate et al., 2005). cataglyphis hispanica and a. iberica, two iberian endemic species, are scavengers that feed mainly on dead arthropods (lenoir et al., 2009; boulay et al., 2005); cataglyphis iberica is also scavenger (martínez et al., 2002) while a. gibbosa is an omnivorous species (lázaro-gonzález et al., 2013). season distance to non-crop habitat % ground cover ground cover richness f p f p f p f p hcs/oh n 387.524 <0.001 53.675 <0.001 2.479 0.119 23.992 <0.001 s 12.665 0.001 0.963 0.386 0.255 0.615 0.571 0.452 go n 488.977 <0.001 11.460 <0.001 0.377 0.541 0.375 0.542 s 14.559 <0.001 0.227 0.797 1.379 0.244 0.026 0.871 cr n 271.880 <0.001 8.783 <0.001 3.787 0.055 0.075 0.785 s 2.073 0.154 0.129 0.879 0.035 0.852 0.565 0.452 gh n 0.023 0.879 4.893 0.010 1.638 0.204 5.493 0.021 s 3.471 0.066 3.787 0.027 0.247 0.620 3.956 0.050 sp n 37.620 <0.001 5.452 0.006 3.763 0.056 0.369 0.545 s 7.458 0.008 1.126 0.329 1.525 0.220 0.546 0.462 shw n 0.062 0.804 2.140 0.124 13.760 <0.001 0.178 0.674 s 0.229 0.634 0.351 0.705 4.872 0.030 0.350 0.556 oh n 2.462 0.120 1.823 0.168 1.382 0.243 1.751 0.189 s 8.188 0.005 3.378 0.039 2.442 0.122 3.667 0.059 ah n 2.643 0.108 0.265 0.768 0.011 0.917 0.161 0.690 s 1.084 0.301 0.478 0.622 0.001 0.976 0.002 0.969 table 4. generalized linear mixed model (glmm) analysis for main functional groups of spiders and ants captured in pitfall traps evaluating the impact of season (spring and summer), distance from adjacent vegetation (5, 50 and 100 m), and richness and percentage of ground cover in their abundance (n) and richness (s). gh ground hunters; sp specialists; shw sheet web weavers; oh other hunters; ah ambush hunters; hcs/oh hot climate specialists and/or open habitats; g/o generalists/ opportunists; cr cryptic. in the present study, the number of ant species belonging to g/o, the group associated with disturbance was, in general, lower than the number of species belonging to hcs/oh and ccs/sh groups, that together constitute an indicator of stability (roig & espadaler, 2010). although the percentage of disturbance was expected to be higher in study sites with herbicide in the row, it was only slightly higher in these plots (between 35.3 and 37.5%) compared to the study sites without herbicide (33.3%). on the other hand, nmds analysis also did not show clear differences between farms, with respect to the functional groups of ants. spider community a total of 44 species of spiders were collected, from which nine are iberian endemic species, which represent about 11% of the endemic species known in the iberian peninsula (cardoso & morano, 2010). ground hunters and specialist spiders accounted, respectively, for 37.5% and 29.2% of the total individuals, and included the three most representative families (gnaphosidae and lycosidae, in the first case and zodariidae, in the second). ground hunters were reported as the most abundant spider guild in the ground of vineyards from italia (caprio et al., 2015), south africa (gaigher & samways, 2014), california (hogg & daane, 2010), north america (bolduc et al., 2005) and australia (d’alberto et al., 2012). from the most abundant species collected, two i.e. z. styliferum and e. feminea were present in all farms indicating that they are widely distributed and clearly adapted to ddr region. although the nmds have shown two distinct groups of farms (the first one consisting of two farms where no herbicide was applied, and a second one consisting of two farms where herbicide was applied on the row) we believe that this difference was mainly due to the percentage of ground cover of the inter-row, which was higher in the last two farms (data not shown). co-occurrence between ants and ant-associated spiders results suggest that the specificity of the association between ants and spiders was relatively extensive, with ant f gonçalves, v zina, c carlos, l crespo, i oliveira, l torres – ants and spiders co-occurring in vineyards412 associated spiders correlating with more than one ant genera or species. it was found that both ant-mimicking and anteating spiders were positively correlated with formicinae ants while only ant-eating spiders were positively correlated with myrmicinae. rákóczi and samu (2014) also only found positive association between ant-mimicking spiders and formicinae, indicating that this could be related to the numerical dominance of formicinae, which makes them a better model for batesian-mimicry. the correlation between ant-eating spiders and formicinae ants was higher than the correlation between ant-eating spiders and myrmicinae ants, and this was also reported by rákóczi and samu (2014), who explained that this difference could be related with the different types of defenses that these ant groups exhibit; thus, while myrmicinae have thick cuticle, cuticle structures, a distinct postpetiole and a functional sting, formicinae are deprived of postpetiole and functional stings and their defense is based on the use of their strong mandibles and toxin exudation (formic acid or anal gland secretions). zodarion, the most abundant ant-eating spider genus collected, is considered to be adapted to hunt ants (pekár, 2004; pekár et al., 2005; 2012a; pekár & toft, 2009), presenting nutritional limitations if non-ant prey is used as food (cushing, 2012 and references therein). moreover, it seems to be behaviorally and physiologically constrained to feed only on certain ant species (pekár, 2004; pekár & toft 2009). some zodarion species are batesian mimics, with various colour patterns and morphological resemblance to ants (korenko et al., 2013). in the present study, the abundance of zodarion was correlated with that of both formicinae and myrmicinae ants. despite pekár (2005) and pekár et al. (2005) stated that formicinae are paralyzed more quickly by the venom of zodarion than myrmicinae, some zodarion species have demonstrated the preference for myrmicinae (e.g. zodarion cyrenaicum which mimic and feed on messor spp. ants). however, the generalization to the subfamily level should be viewed with caution since it was found that some species (such as z. rubidum) are specialists at an even lower taxonomic rank (cárdenas et al., 2012). in this case, for instance, spiders are able to recognize intraspecific chemical signals produced by the ants. in fact, a study conducted by pekár et al. (2012b), suggests that the speciation of ant-eating zodarion spiders is induced by prey-shifting. moreover, pekár et al. (2012b) found that while individuals of z. styliferum from mainland portugal hunted predominantly messor ants (myrmicinae), those from madeira island hunted lasius ants (formicinae). on the other hand, pekár (2009), showed that in some spider species (e.g. zodarion asiaticum), the feeding habits change over the lifespan; thus, initially they are adapted to feed primarily on species of small dolichoderinae and myrmicinae ants, while the large juvenile and the adults are adapted to feed on large formicinae ants. spiders from micaria genus, that were found to be associated with camponotus (the genus as a whole, as well as the species c. cruentatus and c. pilicornis), cataglyphis spp., i. subrufa, and a. iberica, are thin-legged ant-mimic spiders whose bodies are often banded and peppered with iridescent scales, and their anterior lateral spinnerets are comparatively small (adams, 2014). other ant-associated spiders found in the present study in very low numbers, were not included in statistical analysis. this is the case of the ant-eaters from euryopis, oecobius and oxyopes genera, as well as of the ant-mimics from castianeira and myrmarachne genera. euryopis episinoides, is a tiny, 3-mm-long spider that catches ants, particularly from messor spp. (pekár & cárdenas, 2015) but also attacks crematogaster spp. (cushing, 2012). conveniently, females lay her egg sacks close to the nests of these ant genera and once hatched, the young spiders experience their first meal acquiring information about preferred prey and how successfully locate them (pekár & cárdenas, 2015). the spiders of the castianeira genus are mimics of large ants. though these spiders do not really resemble ants in shape and appearance, they behave like them. some species raise their first pair of legs and wave them like antennae, and walk on six legs (deeleman-reinhold, 2001). also, the members of myrmarachne are regarded as perfect ant mimics and are very similar to ants both morphologically and behaviorally (huang et al., 2011). the males of some species of myrmarachne have greatly enlarged chelicerae that extend anteriorly apparently to mimic encumbered ants (worker ants carrying items in their mandibles) (see cushing, 2012). species of this genera resemble ants, to avoid predation, through the first pair of legs movement to look like the ant antennal movement (ceccarelli, 2008). impact of adjacent non-crop habitats and ground cover on the spider and ant communities abundance of both g/o, hcs/oh and cr ants was high at the highest distance (100 m) from adjacent non-crop habitat, suggesting that their populations are established in the vineyards and apparently do not recolonize vineyards from these natural habitats. concerning to spiders, the impact of the season, the distance to the adjacent non-crop habitat, and the ground cover differed between guilds. while abundance and richness of sp spiders and richness of oh spiders were higher in summer than in spring, abundance and richness of gh, shw and ah spiders did not differ between seasons. results showed that, in general, spiders did not benefit from the proximity of adjacent non-crop habitats. abundance and richness of gh spiders was higher at the distances of 5 and 100 m from noncrop habitat than in 50 m. in a study conducted in californian vineyards, hogg and danne (2010) also found that dispersal activity of ground spiders did not change over the time; they also stated that ground spiders were unaffected by transect distance, from the adjacent non-crop habitat to the inside of sociobiology 64(4): 404-416 (december, 2017) 413 vineyard, speculating that or transects may not have been long enough to observe an effect on ground spiders or, ground spiders may be unaffected by seasonal changes in vineyards, and may not recolonize vineyards from natural habitats. other studies have suggested that spiders did not move from these non-crop habitats to adjacent crop fields (wu et al., 2009; d’alberto et al., 2012). the abundance of sp spiders was also higher in the distance of 100 m from adjacent non-crop habitat than in 5 or 50 m. being sp spiders specialist ant-eating spiders, it is admissible that their presence could have been related to the presence of ants, whose abundance was also higher at 100 m from the adjacent non-crop habitat than in the other studied distances. a strong influence of ants on the abundance and spatial distribution of the linyphiidae spiders, pocobletus sp. was also documented by marín et al. (2015). the positive effect of the percentage of ground cover on abundance and richness of shw spiders found in the present study, is in line with balfour and rypstra (1998) who observed high densities of shw spiders in areas with high densities of weeds. as reviewed by these authors web-building spiders are particularly sensitive to structural features of their environment because of the specific spatial requirements of web placement. no significant impact of ground cover on abundance and richness of the remaining spider guilds was found, although a positive effect of the percentage of ground cover in abundance of sp group was found to be near of significance. in olive groves, cárdenas et al. (2012), found that the cover crop removal affected positively the abundance of ground spider’s populations, namely z. styliferum (specialist) and other running spiders belonging to the families gnaphosidae and lycosidae (ground hunters), but not their diversity. still, according to the same authors, the preference of z. styliferum to uncovered soils could apparently be related with an easier facility to found shelter under stones; assuming this, the surface of soils in ddr vineyards is rich in stones even when ground cover is present. in short, results indicate that the ddr vineyard agroecosystem support a rich assemblage of ants and spiders, as evidenced by the occurrence in the studied vineyards, of 20 species of ants and 44 species of spiders, of which three and nine respectively are iberian endemics. the abundance of spiders seems to be mostly related with ground cover as well as the occurrence of prey. thus, the abundance of shw spiders seems to be particularly dependent on the ground cover, where they can build their webs, while that of sp spiders seems to be mostly related with the abundance of ants, since that it was higher in the areas where ant density was also higher. results still support the hypothesis that non-random co-occurrence between ants and ant-associated spiders exist in the field. notwithstanding further studies should be done in order to confirm the ability of these spiders to mimic and/or feed on ants. acknowledgments we thank the two wine companies involved on this study, real companhia velha (rcv) and sogevinus finewines s.a (sgv) and their technicians for their cooperation in the study. we also thanks mónica martins, for the identification of plants. this research was supported by: european investment funds by feder/compete/poci– operacional competitiveness and internacionalization programme, under project poci01-0145-feder-006958, by national funds by fct portuguese foundation for science and technology, under the project uid/agr/04033/2013 and by the interact project – “integrated research in environment, agro-chain and technology”, no. norte-01-0145-feder-000017, in its line of research entitled vitalitywine, co-financed by the european regional development fund (erdf) through norte 2020 (north regional operational program 2014/2020). vera zina was supported by fct portuguese foundation for science and technology, under the strategic project pest-oe/agr/ui0239/2014. supplementary material http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/1934/0 supplementary table s1 doi: 10.13102/sociobiology.v64i4.1934.s1646 suplementary table s2 doi: 10.13102/sociobiology.v64i4.1934.s1647 references adams, r.j. 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(2009). responses of ground-dwelling spiders to four hedgerow species on sloped agricultural fields in southwest china. progress in natural science, 19: 337-346. doi: 10.1016/j. pnsc.2008.05.032 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i3.1014sociobiology 63(3): 881-888 (september, 2016) mandibles of leaf-cutting ants: morphology related to food preference introduction the mandibular morphology reflects the species’ adaptations to particular foraging habits and social life (gronenberg et al., 1997). leaf-cutting ants (atta and acromyrmex), in particular, use different parts of plants for cultivation of their symbiotic fungus, with some species preferring dicotyledons and others grasses (monocotyledons) (fernandez et al., 2015). grass-cutting ants tend to have more massive and shorter mandibles, while the mandibles of leafcutting ants are longer and less massive (fowler et al., 1986). actually, comparing atta species with these different plant preferences has demonstrated they had similar mandible morphology, primarily differing in distal tooth length, while acromyrmex exhibited a higher variation in the mandible format as a whole (camargo et al., 2015). in general, the leaf blade of grasses contains abundant cells with lignified walls (scheffer-basso et al., 2002; pelegrine abstract feeding adaptation is crucial for the ecological success of animals, which explore specific or varied resources according to the suitability of the selected morphological feature. we hypothesized that the mandibles of leaf-cutting ants exhibit a specialization because of the specific food preference of some species. the objective of this study was to highlight possible morphological differences between the mandibles of leaf-cutting ant species of the genus atta related to food preference, i.e., to investigate the morphofunctionality. a detailed description of the mandibles of workers of the grass-cutting ant atta bisphaerica and of the leaf-cutting ant atta sexdens rubropilosa was provided. for morphometric analysis, 50 individuals of four size classes were dissected for removal of the right mandible and one sample of each caste was processed for scanning electron microscopy. it was observed differences between species and among castes, demonstrating the clear specialization of workers. although the results of mandibular morphometry accept the hypothesis of morphofunctionality, further investigation is needed taking into consideration the body size of foragers, robustness and factors that confer greater resistance to the mandibles such as zinc content, the force employed during cutting, and mandibular biomechanics. sociobiology an international journal on social insects lc silva1, rs camargo1, jfs lopes2, lcforti1 article history edited by evandro nascimento silva, uefs, brazil received 02 march 2016 initial acceptance 19 june 2016 final acceptance 11 september 2016 publication date 25 october 2016 keywords hymenoptera, atta, social insects, leafcutting ants. corresponding author roberto da silva camargo laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas/unesp rua dr. josé barbosa de barros, 1780 cep: 18610-307 botucatu-sp, brasil e-mail: camargobotucatu@yahoo.com.br et al., 2009), which renders them more resistant to cutting, as well as solid particles of hydrated silica (sio2nh2o) found in the epidermal cells (motomura et al., 2002). the accumulation of silicon has been associated with a variety of functions, including protection against fungal attacks, resistance to water loss, stimulation of photosynthesis, and structural support (lux et al., 2003; müller, 2003; hattori et al., 2005). moreover, silica plays an important role in the interaction between herbivores and plants. massey et al. (2006a, 2006b, 2007, 2008, 2009) showed that silica accumulation in grasses increases leaf abrasiveness, causing the wear and deterioration of mandibles and impairing insect feeding. in view of this previous knowledge, we hypothesized that the mandibular morphology of workers differs according to the plants preferentially cut by them. therefore, this study compared the morphology of worker mandibles between castes (soldier, forager, generalist and gardener according to wilson, 1980). it is expected that the mandible of grass1 faculdade de ciências agronômicas/unesp, botucatu-sp, brazil 2 universidade federal de juiz de fora, juiz de fora-mg, brazil research article ants lc silva et al. – mandibles of leaf-cutting ants882 cutting ants is more massive and shorter, while the mandible of leaf-cutting ants would be longer and less massive. material and methods two leaf-cutting ant species, a. s. rubropilosa and a. bisphaerica, were used in this study. field and laboratory colonies were used. for the collection of a. bisphaerica workers, a single fungus chamber of an adult nest was excavated on the santana farm located near the lageado experimental farm of unesp, botucatu, são paulo, brazil (geographic coordinates: 22o 50’ 46” s and 48o26’2” w). in this farm, the pasture was predominantly covered with paspalum sp. with some patches of brachiaria here and there. a. s. rubropilosa workers were obtained from an adult laboratory colony (10 years old, 50 liters of fungus garden). for morphometric analysis, 50 individuals of each caste were dissected for removal of the right mandible under a stereoscope: gardener (head width 0.8-1.2 mm; n=50), generalist worker (head width 1.3-1.6 mm; n=50), forager (head width 1.7-2.2 mm; n=50), and soldier (head width ≥ 3.3 mm; n=50). this caste determination was proposed by wilson (1980, 1983). the observations were made on the ventral side of right mandibles. the following measurements were made using a micrometer eyepiece coupled to a stereomicroscope: 1) length of the mandible; 2) width of the mandible; 3) length of the first tooth; 4) length of the second tooth; 5) number of teeth and 6) head width (mm). to detect morphological differences between mandibles of the different ant species, samples of the right mandible of workers of all size classes were fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer, ph 7.3, critical point dried, sputtered with gold (robards, 1978), and examined by scanning electron microscopy (sem) (fei quanta™ 200) at the center of electron microscopy, institute of biosciences (centro de microscopia eletrônica do instituto de biociências – cme-ibb), unesp, botucatu, sp. for determination of the occurrence of wear, the mandible of an a. s. rubropilosa forager was collected 1 day after emergence and submitted to the same procedures for sem analysis. we analyzed the data and verified that all variables weren’t normally distributed (shapiro-wilk normality test), being thus log-transformed. principal component analysis (pca) was used to examine variation in the morphometric data among species using the free software pc-ord 5.15 (mccune & mefford, 2011), with exception of the number of teeth. the number of principal components (pcs) was determined considering eigenvalues higher than those generated by the brokenstick method. the morphometric measurements that most contributed to the axes, were selected by pearson (r > 0.7). after that, we performed an anova comparing the pca scores with tukey hsd post-test (α=0.05), to point out the differences among the variables. results we found differences between species and among castes (anova, f 7;391=455.2, p <0.001). all castes differed among them, except between soldiers from a. s. rubropilosa and a. bisphaerica (p=0.31) and a. bisphaerica generalist workers with a. s. rubropilosa foragers (p=0.11) (table 2). pca analysis found that the first principal component explains 91.5% of the variation. also it is strongly correlated with the four morphometric variables. the first principal component increases with decreasing length mandible (r= -0.96), width mandible (r= -0.96), length of the first teeth (r= -0.98) and length of the second teeth (r=-0.93). this suggests that these four morphometric measures vary together. if one decreases, then the remaining also decreases. it would follow that species and castes would tend to be grouped due to their mandible features, in terms of the morphometric measurements (figure 1). differences were also detected within the different castes of each species. soldiers of the two species had larger and more robust mandibles compared to the other castes. mandibular morphology of foragers although similar to that of soldiers, were smaller mandibles and with teeth more worn (figure 6-7). the mean number of teeth was 7 or 8 for a. bisphaerica and 7 to 9 for a. s. rubropilosa (table 1). tooth fig 1. principal components analysis of mandible morphometric data of atta sexdensrubropilosa and atta bisphaerica in each worker caste for the variables width mandible (wm), length mandible (lm) length of the fisrt tooth (lft) and length of the second tooth (lst) first two principal components (pcs) are plotted with the proportion of variance explained by each component printed next to the axes labels. sociobiology 63(3): 881-888 (september, 2016) 883 ant species caste head width mandibular length mandibular width length of 1st tooth length of 2nd tooth number of teeth atta bisphaerica gardener 0.76±0.20 0.40±0.10 0.25±0.09 0.08±0.06 0.04±0.02 10.14±2.12 generalist 1.29±0.10 0.67±0.06 0.46±0.04 0.20±0.04 0.09±0.03 7.92±0.73 forager 2.13±0.19 0.99±0.08 0.70±0.06 0.26±0.05 0.13±0.04 7.78±0.79 soldier 4.79±0.43 1.58±0.08 1.11±0.07 0.49±0.07 0.14±0.03 7.78±0.68 gardener 0.89±0.13 0.51±0.10 0.30±0.06 0.10±0.03 0.05±0.02 9.46±1.15 atta sexdens generalist 1.32±0.08 0.65±0.06 0.40±0.05 0.13±0.03 0.07±0.02 8.84±1.08 rubropilosa forager 2.02±0.18 0.88±0.11 0.52±0.07 0.17±0.05 0.09±0.03 8.36±1.05 soldier 3.79±0.23 1.54±0.07 0.94±0.05 0.41±0.04 0.17±0.03 8.64±0.80 table 1. morphometric measures (mean and standard deviation) (mm) of the mandibles and teeth of atta bisphaerica and atta sexdens rubropilosa. wear was observed when adult foragers of undetermined age (figure 10) were compared with an adult on day 1 after hatching (figure 11). as can be clearly seen in figure 10, the teeth of probably older foragers were slightly rounder, probably due to wear out, when compared to the naive forager worker (1-day old) (figure 11), in which a larger number of teeth and more pointed teeth can be seen. generalist workers of a. bisphaerica had more elongated mandibles when compared to a. s. rubropilosa (figure 4-5) and presented no tooth wear. however, figure 4 shows wear out of the mandible of generalists a. bisphaerica workers. the elongated mandibles of a. bisphaerica were also observed in gardeners of this species (figure 2-3). no tooth wear was observed in this caste of the both species. gardeners had a larger number of teeth and more pointed teeth than the other castes analyzed (table 1). in all castes of the two species the teeth had a serrated appearance characterized by a progressive reduction in their size. the main difference between species was the larger size of the distal tooth in atta bisphaerica. these results demonstrate that the mandible morphology is different between species and within castes. however, a difference within castes seems to be found at the same features in both species. discussion leaf-cutting ants in general (atta and acromyrmex) use different parts of plants for cultivation of the symbiotic fungus. some species prefer dicotyledons, while others prefer grasses (monocotyledons) (fernandez et al., 2015), a fact raising the hypothesis of a greater degree of specialization in view of the specific food preferences of these species. the results of the present study accept the hypothesis of morphofunctionality of leaf-cutting ant (a. s. rubropilosa) and grass-cutting ant mandibles (a. bisphaerica) is related to food preference. this hypothesis was raised by fowler et al. (1986) based on the morphological differences observed in these groups. in this respect, in species that preferentially cut grasses the foragers tend to have shorter mandibles compared to foragers of leaves comparisons mean difference confidence intervals pvalue adjustedlower bound upper bound fs-js 0.606 0.491 0.720 < 0.0001 fs-sb -0.756 -0.870 -0.641 < 0.0001 fs-ss -0.670 -0.785 -0.555 < 0.0001 gs-gb 0.173 0.058 0.288 0.0001 jb-gb 0.742 0.627 0.857 < 0.0001 js-gb 0.502 0.386 0.617 < 0.0001 sb-gb -0.860 -0.975 -0.745 < 0.0001 ss-gb -0.774 -0.889 -0.659 < 0.0001 jb-gs 0.569 0.454 0.684 < 0.0001 js-gs 0.328 0.214 0.443 < 0.0001 sb-gs -1.033 -1.148 -0.918 < 0.0001 ss-gs -0.947 -1.062 -0.833 < 0.0001 js-jb -0.240 -0.355 -0.125 < 0.0001 sb-jb -1.603 -1.717 -1.488 < 0.0001 ss-jb -1.517 -1.632 -1.402 < 0.0001 sb-js -1.362 -1.477 -1.247 < 0.0001 ss-js -1.276 -1.391 -1.162 < 0.0001 ss-sb 0.085 -0.029 0.200 0.3097 fs-fb 0.253 0.139 0.368 < 0.0001 gb-fb 0.357 0.242 0.473 < 0.0001 gs-fb 0.531 0.416 0.645 < 0.0001 jb-fb 1.100 0.985 1.215 < 0.0001 js-fb 0.859 0.745 0.974 < 0.0001 sb-fb -0.502 -0.617 -0.387 < 0.0001 ss-fb -0.416 -0.531 -0.302 < 0.0001 gb-fs 0.104 -0.011 0.219 0.1096 gs-fs 0.277 0.162 0.392 < 0.0001 jb-fs 0.846 0.732 0.961 < 0.0001 *significant difference at 95% family-wise confidence level by tukey hsd test. table 2. statistical summary of multiple comparison by tukey hsd (α=0.05). ss: a. s. rubropilosa soldiers; fs: a. s. rubropilosa foragers; gs: a. s. rubropilosa generalist workers; gs: a. s. rubropilosa gardeners; sb: a. bisphaerica soldiers; fb: a. bisphaerica foragers; gb: atta bisphaerica generalist workers; gb: a. bisphaerica gardeners. lc silva et al. – mandibles of leaf-cutting ants884 which tend to have less massive mandibles. as a consequence, these foragers cut plants differently. for example, during foraging atta capiguara assumes a downward position while cutting the leaf blade of grasses, performing the cut and immediately returning to the nest. in contrast, in leaf-cutting ants the metathoracic legs serve as a pivot during cutting and their mandibles slide across the leaf blade. behavioral differences between grass-cutting and leaf-cutting ants are also observed during cultivation of the symbiotic fungus (lopes, 2004). leaf-cutting ants exhibit a higher degree of processing of the foraged material that resembles grinding before incorporation of the symbiotic fungus. in contrast, in the case of grass-cutting ants the degree of processing is lower, i.e., the leaf blades are just arranged on the fungus garden and the symbiotic fungus is subsequently incorporated (lopes, 2004). this behavior is probably related to the greater difficulty in processing the material due to the higher proportion of lignified tissues in grass leaves (lopes, 2004). in the present study, mandible morphometry was useful to discriminate between species and castes (figure 1). these results are possibly related to the recent evolutionary history of the two species. the genus atta is monophyletic (bacci et al., 2009), with relatively recent adaptive radiation about 8 to 13 million years ago (brady et al., 2006). however, the identification of adaptive radiation is based on the phylogenetic relationships of species and not on the dating of known fossils figs 2-3. ventral side of right mandibles of atta bisphaerica (2) and atta sexdens rubropilosa (3) gardeners. figs 4-5. ventral side of right mandibles of atta bisphaerica (4) and atta sexdens rubropilosa (5) generalist workers. sociobiology 63(3): 881-888 (september, 2016) 885 of atta, which are scarce for the genus. there is only one trace fossil of an atta nest from the miocene located in patagonia, argentina (laza, 1982). this intimate phylogenetic relationship between the species studied (a. s. rubropilosa and a. bisphaerica) and their recent evolutionary history explain the morphological differences of the mandibles, at least in terms of the features evaluated (table 1). these species are widely distributed in the neotropical region and differ in nesting site, food preference and behavior. the grass-cutting ant a. bisphaerica is only found in brazil (wilson, 1986), particularly in the states of são paulo, minas gerais, rio de janeiro and mato grosso do sul (forti & boaretto, 1997). in contrast, atta sexdens and its subspecies are distributed from costa rica to argentina and paraguay (wilson, 1986), with a. s. rubropilosa occurring in the midwestern and southeastern regions of brazil (forti & boaretto, 1997). the nesting site is a result of the biome. in this respect, a. bisphaerica nests are shallow and are generally found in open areas such as pastures characterized by a high incidence of insolation, while a. s. rubropilosa nests are relatively deep and are only found in shadow areas, such as native and eucalyptus forests (forti et al., 2011). morphological differentiation between castes was observed in both species studied (figures 2-3, 4-5, 6-7, 8-9, and 10-11) and was supported by the separation of species and grouping of the same castes by pca analysis, using the mandible morphometric data. gardeners are the smallest workers which are always found inside the nest and in larger quantities. the typical tasks attributed to this set of workers occur exclusively inside the nest, including brood care, final treatment of the substrate and hyphal inoculation into the fragments incorporated into the fungus garden (wilson, 1983). their mandibles are small in a. bisphaerica they are more elongated compared to the other castes since the tasks of gardeners are the most laborious and precise, with their teeth showing no wear (figure 2-3). generalist workers perform a relatively larger number of tasks that range from the elimination of residues from the colony and caring for the brood and queen to tasks related to cultivation of the symbiotic fungus (wilson, 1983). the mandibles of a. bisphaerica generalists are visibly more elongated than those of a. s. rubropilosa, probably to facilitate the grass blades manipulation (figure 4-5). gardeners and generalists are the predominant size classes in the population of a leaf-cutting ant colony and are responsible for a large number of tasks related to preparation of the plant material during the process of incorporation (peregrine & cherrett, 1974, 1976; littledyke & cherrett, 1976). the elongated mandibular morphology observed only in a. bisphaerica gardeners and generalists might be related to the way grasses are processed. the leaf blades of grasses are not chopped or processed before incorporation as observed for dicotyledon leaf fragments which have a pulp-like appearance after processing by workers. according to lopes et al. (2004), the latter processing facilitates the implantation of hyphal tufts. on the other hand, the function of foragers is to explore, to excavate, to recruit other ants, and mainly to cut new plant material at field (wilson, 1983), i.e., in both species studied these workers are not involved in the more laborious activities of plant processing, but rather in the exploration and cutting of plant material. as a consequence, the mandibles of these workers exhibited more marked tooth wear (figure 6-7) reflecting the repetitive cutting effort. this assumption is mandibles of different age workers (figure 10-11). finally, the task attributed to soldiers is the defense of the nest (wilson, 1983), an activity that requires larger and more robust mandibles (figure 8-9) and is not related figs 6-7. ventral side of right mandiblesof atta bisphaerica (6) and atta sexdensrubropilosa (7) foragers. lc silva et al. – mandibles of leaf-cutting ants886 figs 10-11. ventral side of right mandibles of atta sexdens rubropilosa foragers at different ages, showing natural wear due to foraging activity. adult of undetermined age (10) and adult one day after hatching (11). with plant manipulation, explaining why soldier mandibles were not different between species. like foragers, this group is not involved in the processing of plant material and no morphological differentiation between species is therefore observed. also, a. bisphaerica generalist mandibles did not differ from a. s. rubropilosa forager mandibles suggesting that task allocation between species must be different. actually, task allocation is different between species in function of which abilities the task require. the mandibles of ants have many functions, such as catching prey, fight, cutting leaves, brood care, and communication (paul, 2001). although an important tool for many tasks (hölldobler & wilson, 1990), there is little information on the morphology of ant mandibles and no quantitative data regarding mandibular differences are available. the specialization of mandibles does not only depend on their morphology, but is also related to the velocity of movement and the force generated by them. for example, catching prey obviously requires movement characteristics that differ from those necessary for cracking seeds (paul, 2001). thus, the veins patterns of grasses and dicotyledons and the greater abundance of lignified cells in grasses should also influence the figs 8-9. ventral side of right mandibles of atta bisphaerica (8) and atta sexdens rubropilosa (9) soldiers. sociobiology 63(3): 881-888 (september, 2016) 887 specialization of mandibles. although the results of mandibular morphometry accept the hypothesis of morphofunctionality, further investigation is needed taking into consideration the body size of foragers, robustness and factors that confer greater resistance to the mandibles such as zinc content, the force employed during cutting, and mandibular biomechanics. acknowledgements we are grateful to fundação de amparo à pesquisa do estado de são paulo (fapesp) for the financial support and stipends to the authors [grant numbers 2007/04010-0 and 2007/07091-0]. r.s. camargo thanks coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the postdoctoral fellowship. l.c. forti thanks conselho nacional de desenvolvimento cientifico e tecnológico for the research assistance (301917/2009-4). the authors are indebted to dr. rafael barbieri for help with statistical analyses and helpful comments. references andrade app, forti lc, moreira aa (2002). behavior of atta sexdens rubropilosa (hymenoptera: formicidae) workers during the preparation of the leaf substrate for symbiont fungus culture. sociobiology, 40: 293-306. bacci m, solomon se, mueller ug (2009). phylogeny of leafcutter ants in the genus atta fabricius (formicidae: attini) based on mitochondrial and nuclear dna sequences. molecular phylogenetics and evolution, 51: 427-437. doi: 10.1016/ 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(2002) tooth hardness increases with zinc-content in the mandibles of young adult leaf-cutter ants. naturwissenschaften, 89: 579-83. doi: 10.1007/s00114-002-0381-4 vicari m, bazely dr (1993). do grasses fight back the case for antiherbivoredefenses. trends in ecology and evolution, 8: 137-141. doi: 10.1016/0169-5347(93)90026-l. weber na (1956). treatment of substrate by fungus-growing ants. anatomic records, 125: 604-605. wilson eo (1980). caste and division of labor in leaf-cutter ants (hymenoptera, formicidae). i: the overall pattern in a. sexdens. behavioral ecology and sociobiology, 7: 143-156. doi: 10.1007/bf00299521 wilson eo (1983). caste and division of labor in leaf-cutter ants (hymenoptera, formicidae: atta). iv: colony ontogeny of a. cephalotes. behavioral ecology and sociobiology, 14: 5560. doi: 10.1007/bf00366656 http://dx.doi.org/10.1007/s00442-007-0703-5 http://dx.doi.org/10.1017/s0007485300047842 http://dx.doi.org/10.1017/s0007485300047842 http://dx.doi.org/10.1016/s1095-6433(01)00458-5 http://dx.doi.org/10.1016/s1095-6433(01)00458-5 h.30j0zll doi: 10.13102/sociobiology.v66i2.3390sociobiology 66(2): 274-278 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 herbicide application on genetically modified maize influence bee visitation introduction brazil is one of the world’s largest producers of maize (zea mays l.) with a total production of 60 million tons and average yield in the first harvest of 5.4 t ha-1 (conab, 2016). however, the average national productivity is considered low, since the industries that operate on a high technological level obtained values three times higher than the average productivity presented for the same harvest. several factors are responsible for the low productivity of the crops, though interference imposed by weeds and pests is highlighted (constantin et al., 2007). several technologies, including the use of herbicides and toxic proteins, help increase agricultural productivity. however, there are consequences linked to technologies use, such as the different effects on non-target organisms (rosa et al., 2010). abstract brazil is one of the world’s largest producers of maize (zea mays l.). cry proteins derived from the bacterium bacillus thuringiensis (bt) have been widely used in transgenic maize due to their toxicity and specificity against insects that damage crops. in addition, these plants have been stacked with different herbicide tolerance genes. non-target insects end up being exposed to bt proteins and herbicide applications. there is little information on the effects of bt transgenics and their cultural practices on the behavior of pollinators in genetically modified crops. the aim of this research was to verify the impact of genotypes of genetically modified maize, herculex®, powercore®, and the conventional isohybrid, pulverized or not with herbicides (atrazine, glufosinateammonium and nicosulfuron) in bee populations. in order to evaluate the presence of insects, a zig-zag tour was carried out throughout the experimental field, ascertained from visual analysis and direct counting of six plants per plot (the dimensions of the plots were 2.5 x 10 m with five maize lines spaced 0.50 m between rows and 0.36 m between plants) randomly, 18 days after spraying herbicides in the area. apis mellifera (l.) (hymenoptera: apidae), tetragonisca angustula (l.) (hymenoptera: apidae) and trigona spinipes (f.) (hymenoptera: apidae) were the pollinator species identified in the crop. it was observed that the incidence of pollinator insects varied according to cultivars and herbicides tested; however, the powercore® genotype experienced more visitation of pollinating bees independently of the herbicide treatments. sociobiology an international journal on social insects hc monteiro¹, mwr souza¹, lac reis¹, ea ferreira¹, vgm de sá2, ma soares¹ article history edited by solange augusto, ufu, brazil received 27 april 2018 initial acceptance 24 july 2018 final acceptance 31 january 2019 publication date 20 august 2019 keywords zea mays, transgenic, pollinating insects. corresponding author marcus alvarenga soares universidade federal dos vales do jequitinhonha e mucuri (ufvjm) campus jk, rodovia mgt 367 km 583, nº 5000, diamantina-mg, brasil e-mail: marcusasoares@yahoo.com.br bees play an important role in plant production, which are secondary targets of the effects of chemicals. thus, several researchers in the laboratory and in the field have attempted to evaluate and determine the effect of herbicides on bees (chambó et al., 2010). reductions and delays of plant flowering is caused by herbicide lesions that can disrupt pollinator communities (bohnenblust et al., 2016). herbicide lesions can also create a direct negative impact on pollinators. bees exploit floral sources of pollen and nectar influenced by the selection pressure of the environment, noting that environmental contaminants and the time that food sources remain available for the visitor are fundamental variables (brizola-bonacina et al., 2012). crystal proteins derived from the bacterium bacillus thuringiensis (bt) have been widely used in transgenic crops due to their toxicity against insects that damage crops. 1 departamento de agronomia, universidade federal dos vales do jequitinhonha e mucuri ufvjm, diamantina, minas gerais, brazil 2 dow agrosciences industrial ltda, zionsville rd, indianapolis, indiana state, eua research article bees sociobiology 66(2): 274-278 (june, 2019) 275 however, the distribution and metabolism of these toxins in the tissues and organs of non-target insect has remained obscure (zhao et al., 2016). the purpose of this innovative technology is to reduce insects that are considered crop pests. thus, the advancement of biotechnology in the field of transgenic development is very critical. in addition to the positive impact to principal crops of the global economic market, the technology can also give rise to biological, environmental and social problems. the effect of bt maize cultivation on non-target insects seems to depend on the geographic location and crop management of the agricultural ecosystem. other factors such as spraying chemicals in the maize field may thus exert a stronger influence in this process (resende et al., 2016). the impact of bt proteins on non-target arthropods is less understood than their effects on target organisms, where the mechanism of toxic action is known (yuan et al., 2014). some studies have been carried out investigating possible effects of transgenic plants on non-target organisms, but there is little information on the effects on the behavior of pollinator populations when visiting transgenic cultivars. the aim of this research was to verify the impact of genotypes of genetically modified maize, herculex®, powercore®, and the conventional isohybrid, pulverized or not with herbicides (atrazine, glufosinate-ammonium and nicosulfuron) in bee populations. material and methods the experiment was set up at the rio manso experimental farm of the federal university of the jequitinhonha and mucuri valleys (18° 4’ 25’’s; 43° 28’ 16’’o), located in the municipality of couto de magalhães de minas, minas gerais state, brazil. the experiment was performed in a field in dbc (randomized block design) in a 3 × 4 factorial scheme with 3 blocks where parcels were drawn within each block. as the a factor represented by the genotypes herculex® transgenic maize [bt cry1f protein (resistant to spodoptera frugiperda, je smith, lepidoptera: noctuidae)], powercore® stacked transgenic maize [bt cry1f, cry1a.105, cry2ab2 proteins (resistant to s. frugiperda) and cp4 epsps protein (glyphosate tolerance) and the conventional isohybrid maize sensitive to the s. frugiperda and herbicide. factor b was represented by the herbicides: atrazine, glufosinate-ammonium and nicosulfuron applications, plus a control without application. each plot was assembled in the dimensions 2.5 × 10 m, with five maize lines spaced 0.50 m between rows and 0.36 between plants, containing 3 blocks. the experiment was composed of a total of 36 plots with a total experimental area of 10 × 90 m. the soil was fertilized and corrected according to a soil analysis conducted one month prior to planting, which also indicated a medium texture. with the chemical analysis, the following results were obtained: ph (water) of 5.2; organic matter content of 5.1 daq kg-1; p and k of 3.3 and 65 mg dm-3, respectively; ca, mg, al, h + al and effective ctc of 1.9; 0.8; 0.1; 7.1 and 2.9 cmolc dm-3, respectively. 0.90 tonnes ha-1 of dolomitic limestone was applied. fertilizer with 500 kg ha-1 of the formulation 04:14:08 of n:p:k was applied and irrigation was monitored throughout the experiment. planting occurred on march 9, 2016. three seeds per linear meter were sowed. the plants emerged on march 13, 2016 and the herbicides (atrazine 6.0 l/ha-1, glufosinateammonium 2.0 l/ha-1 and nicosulfuron 1.5 l/ha-1) were applied on april 8, 2016; 26 days after the emergence of maize plants. maize is tolerant to the atrazine herbicide, which is recommended for the control of dicot plants in grass crops and its site of action is the protein d1 in photosystem ii. nicosulfuron is recommended for the control of monocot and dicot plants in maize crops whose mechanism of action is the inhibition of the acetolactate synthase (als) enzyme. glufosinate-ammonium is a herbicide that inhibits the glutamine synthetase (gs) enzyme in the nitrogen assimilation log and is classified as a non-selective herbicide (silva & silva, 2007). these herbicides are alternatives to glyphosate in brazil, where some weeds have resistance. to identify the natural presence of bees, six plants per plot were analyzed during the flowering period (18 days after spraying herbicides in the area) by zig-zag walking in the useful area of each plot, with direct counting of individuals in each plant. bee visitation was observed on the maize plants tassel (the tops of maize plants) and male part of the maize plants, where the pollen grains are produced. the observations at the different treatments were performed at the same time for each herbicide/transgenic factor. the data were submitted to analysis of variance and when significant, to the scott-knott grouping criterion at 5% probability of error. the analyses were developed using sisvar computer program (ferreira, 2014). results apis mellifera (l.), tetragonisca angustula (l.) and trigona spinipes (f.) (hymenoptera: apidae) were the pollinator species identified in the crop field. there was interaction among all the genotypes and herbicides in this research. apis mellifera was the most frequent visitor in maize plants in relation to the other species, t. angustula and t. spinipes (fig 1), in genetically modified and in the isohybrid genotypes, independent of herbicide treatments (table 1). the incidence of a. mellifera bees in the herculex® genotype was lower in the plots treated with atrazine and nicosulfuron (table 1). the plots treated with glufosinateammonium and the control were more visited by this species, showing difference in comparison to the other treatments. when evaluating the powercore® genotype, it was observed that the incidence of bees showed no difference for the hc monteiro et al. – herbicide application influence bee276 herbicide and control treatments (table 1). on the other hand, the isohybrid with glufosinate-ammonium showed less bee visitation than all the other genotypes, demonstrating that the isohybrid was severely affected by the application of this herbicide. the incidence of t. spinipes in the herculex® genotype was lower when these plants were treated with the herbicides. with the powercore® and isohybrid genotypes, no difference of visitation between herbicide and control treatments was observed (table 3). the herculex® genotype submitted to nicosulfuron application showed a lower incidence of this species when compared to the other genotypes. in the control, the lowest visitation of t. spinipes was observed on the isohybrid (table 3). the powercore® genotype was the most visited by all species of bees regardless of herbicide application (table 1, 2 and 3). fig 1. verification of bees on the genetically modified maize submitted to herbicide application. *averages followed by the same letter do not differ by scott-knott’s grouping criteria at 5% of probability. herculex® powercore® isohybrid atrazine 7.00 bb 23.33 aa 22.00 aa gluphosinato 13.33 ab 23.33 aa 6.67 bc nicosulfuron 8.00 bb 22.00 aa 20.00 aa control 14.33 ab 24.33 aa 22.33 aa coefficient of variation cv (%) 23.04 *average number of bees observed followed by the same lowercase letter in the column and the same uppercase letter in the row do not differ by scottknott’s grouping criterion. table 1. presence of a. mellifera on the genetically modified maize submitted to herbicide application. when evaluating the herbicides within each genotype, the herculex® genotype plots treated with atrazine, nicosulfuron and control had a lower incidence of a. mellifera bees. in the plots cultivated with the powercore® genotype, no difference was observed between the herbicide treatments and the control. in the isohybrid, a lower occurrence of bees was observed in plants treated with glufosinate-ammonium compared to the other treatments. the isohybrid was highly sensitive to the glufosinate-ammonium, with severe developmental delays, which may have reduced the demand for this cultivar by a. mellifera (table 1). the incidence of t. angustula in the herculex® and powercore® genotypes showed no difference between the herbicide treatments and the control (table 2). the isohybrid demonstrated higher visitation of t. angustula in the cultivated plants without the application of herbicides (control). in the plots treated with atrazine, glufosinate-ammonium and nicosulfuron, no difference was observed in the visitation of bees among the maize genotypes evaluated (table 2). regarding the control, there was a lower incidence of t. angustula on the herculex® and powercore® genotypes when compared to the isohybrid (table 2). herculex® powercore® isohybrid atrazine 1.67 aa 4.33 aa 2.67 ba gluphosinato 1.00 aa 1.67 aa 1.33 ba nicosulfuron 3.00 aa 4.00 aa 2.33 ba control 3.67 ab 3.00 ab 6.33 aa coefficient of variation cv (%) 63.79 *average number of bees observed followed by the same lowercase letter in the column and the same uppercase letter in the row do not differ by scottknott’s grouping criterion. table 2. presence of t. angustula on the genetically modified maize submitted to herbicide application. herculex® powercore® isohybrid atrazine 4.00 ba 5.00 aa 2.67 aa gluphosinato 3.33 ba 4.33 aa 2.67 aa nicosulfuron 1.33 bb 4.00 aa 3.67 aa control 7.00 aa 6.33 aa 3.67 ab coefficient of variation cv (%) 41.51 *average number of bees observed followed by the same lowercase letter in the column and the same uppercase letter in the row do not differ by scottknott’s grouping criterion. table 3. presence of t. spinipes on the genetically modified maize submitted to herbicide application. discussion the side effects of herbicides and toxic proteins on non-target insects such as pollinators need to be studied. indirectly, this class of insects ends up being exposed to genetically modified organisms and their cultural tracts. apis mellifera being the most frequent visitor in maize plants can be explained by pires et al. (2014) who studied genetically modified cotton and observed that the visitation of the species a. mellifera dominated in relation to other species of bees on the crop. this can be explained by the greater natural occurrence of this species. it is also a species of economic sociobiology 66(2): 274-278 (june, 2019) 277 interest and can thus be multiplied and commercialized in large scale. along with having an abundant presence in several environments a. mellifera is considered a dominant species. naturally, t. spinipes demonstrates highly aggressive behavior to other bee species (brizola-bonacina et al., 2012). considering this aggressive behavior, t. angustula avoids areas where t. spinipes are present (damascena et al., 2017). therefore, t. spinipes can negatively influence pollination, inhibiting grain production and reducing the attractiveness of these plants for other pollinators. thus, the species t. spinipes and t. angustula could be avoided themselves in the field and, consequently, controlling the population level of each other. considering that t. angustula is small in size and less aggressive than the others studied, it could be occupying a habitat of lesser interest to the others. in addition to the verification of more occurrence of a. mellifera in this experiment, it is also possible to conclude that the visitation of this species on the maize cultivars varied according to each genotype and the herbicide treatments. thus, herbicides may have had a strong influence on the visitation of pollinating species on maize genotypes. the isohybrid was highly sensitive to herbicides tested and the herculex® and powercore® genotypes demonstrated some resistance. physiological and anatomical variations may exist due to this sensitivity and resistance. the toxic proteins seem not to have influenced the visitation of bees at this experiment, and this is corroborated by other authors. grabowski and dabrowski (2012) tested the effect of genetically modified maize on the behavior of a. mellifera and found that there was no impact on the choice of bees between the flowers of the genetically modified cultivars and the isogenic line (without the cry1ab protein). it is possible that the exogenous protein made the transgenic cultivars less sensitive to the physiological and anatomical changes caused by herbicides application. the changes occurring in the isohybrid genotypes caused by the herbicides may have reduced bee visitation. such changes are confirmed by field observations, with the genotype powercore® was more visually attractive and with outstanding anatomical conditions. conclusions visitation of bees on the maize is affected by genotypes and herbicide application. the stacked powercore® was the only genotype that did not show alteration in bee visitations, regardless of the applied herbicide, with dominance of the species a. mellifera. this work constitutes an approach for evaluating the impact of genetically modified maize on pollinating insects. the method applied is sufficiently sensitive to detect differences between plants and herbicides applications; however, further work on the methodology, as well as the study of plants expressing other gene products and herbicides would be necessary before proposing a reliable test for verifying the safety of such plants. acknowledgments to the brazilian agencies “conselho nacional de desenvolvimento cientifico e tecnológico (cnpq)” and “fundação de amparo à pesquisa do estado de minas gerais (fapemig)” for scholarships and financial support. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. references bohnenblust, e.w., vaudo, a.d., egan, j. f., mortensen, d.a., tooker, j.f. 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(2016). distribution and metabolism of bt-cry1ac toxin in tissues and organs of the cotton bollworm, helicoverpa armigera. toxins, 8: 212. doi: 10.3390/toxins8070212 doi: 10.13102/sociobiology.v65i4.3293sociobiology 65(4): 760-765 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 fermentation of a pollen substitute diet with beebread microorganisms increases diet consumption and hemolymph protein levels of honey bees (hymenoptera: apidae) introduction protein from pollen is needed for brood rearing and for the satisfactory development of adult bees (crailsheim, 1990; roulston & cane, 2000; hoover et al., 2006). the nutritive value of bee bread for honey bees is higher than that of fresh bee collected, laboratory stored, or frozen pollen, with few exceptions (hagedorn & moeller, 1968; herbert & shimanuki, 1978; dietz & stevenson, 1980; cremonez et al., 1998; pernal & currie, 2002). however, there is recent evidence that processing of pollen by the bees results in pollen preservation and not nutrient conversion (anderson et al., 2014). protein levels in the diet affect the resulting protein levels in honey bee hemolymph (basualdo et al., 2013). honey bees also ferment pollen to preserve it from harmful microorganisms (herbert & shimanuki, 1978; vasquez & olofsson, 2009). during dearth periods, when insufficient pollen is available or when they need to build up colonies for pollination abstract pollen substitute diets have become increasingly important for maintaining strong and healthy honey bee colonies. palatability and nutritional value are key attributes of a good diet. since beebread, which is pollen fermented by the bees, is the main food of the worker nurse bees that feed and care for the bee larvae, pollen substitutes should have similar attributes. in an attempt to simulate this natural food source, an inoculum prepared from beebread was used to ferment a pollen-substitute diet. newly emerged bees were fed on the diets for seven days. they consumed significantly more fermented than unfermented diet. hemolymph protein levels were significantly higher in bees that had been fed a fermented versus an unfermented diet, though still significantly lower than in bees fed on beebread. vitellogenin (a key storage protein for honey bees) levels were also increased significantly in bees fed the fermented versus the nonfermented diet. survival rates were higher for bees fed the fermented versus the non-fermented diet, though the difference was not significant. we conclude that fermentation by beebread-derived microorganisms can improve the acceptance and utility of an artificial protein diet for honey bees. sociobiology an international journal on social insects jm almeida-dias1, mm morais2, tm francoy3, ra pereira1, ap turcatto1, d de jong1 article history edited by denise alves, esalq-usp, brazil received 28 march 2018 initial acceptance 27 april 2018 final acceptance 20 august 2018 publication date 11 october 2018 keywords apis mellifera, africanized honey bees, fermented, artificial diet. corresponding author joyce mayra volpini almeida dias departamento de genética faculdade de medicina de ribeirão preto universidade de são paulo (usp) cep 14040-901, ribeirão preto-sp, brasil. e-mail: joycemayra@usp.br contracts, beekeepers often invest in pollen substitutes to feed their bees (somerville, 2005). adequate diet formulation, deterioration during storage, attractiveness to bees and diet costs are major concerns (herbert et al., 1977). unfortunately, many nutritional supplements are poorly accepted by bees and have low nutritional value (schmidt & hanna, 2006). in order to develop artificial protein diets that are nutritious and attractive to bees, it would make sense to make them as similar as possible to their natural proteinaceous food in the hive, beebread. diet efficiency can be measured by various means, including brood and honey production (herbert et al., 1977; winston et al., 1983; paiva et al., 2016) and by measuring the protein levels in bee hemolymph (cremonez et al., 1998; cappelari et al., 2009; basualdo et al., 2013, 2014; barragan et al., 2016). generally, natural forage is better for bee health and production than artificial diets (degrandihoffman et al., 2010, 2016), but it is not always available in sufficient quantity (somerville, 2005). 1 departamento de genética, faculdade de medicina de ribeirão preto, universidade de são paulo (usp), ribeirão preto, são paulo, brazil 2 departamento de ecologia e biologia evolutiva, universidade federal de são paulo (unifesp), diadema, são paulo, brazil 3 escola de artes, ciências e humanidades, universidade de são paulo (usp), são paulo, brazil research article bees mailto:joycemayra@usp.br sociobiology 65(4): 760-765 (october, 2018) special issue 761 a good bee diet should be acceptable to the bees and provide nutrients essential for colony growth and development, bee health and colony production capacity (herbert & shimanuki, 1978; winston et al., 1983; rousseau & giovenazzo, 2016). although various diets have been developed for pollen replacement during dearth periods (abbas et al., 1995; de jong et al., 2009; ellis & hayes, 2009; morais et al., 2013a, b), common problems include a lack of attractiveness (robinson & nation, 1968; pernal & currie, 2002) and inefficient conversion into bee protein compared to beebread (cremonez et al., 1998). here, we tested whether fermentation with microorganisms from beebread would improve consumption and utility of a pollen substitute diet, whose components are defined below. material and methods the feeding trials were conducted in 2012–2013 with unselected africanized bees in our university apiary in ribeirão preto, sp, brazil (21°10’39’’ s, 47°48’24’’ w). inoculum preparation for fermentation inoculum was developed and prepared in our lab. all the glassware and mixing implements were sterilized with 70% ethanol prior to inoculum preparation. beebread was collected with a metal spatula from 12 brood combs, retrieved from four different colonies. after pooling and mixing, 10 g of freshly collected beebread was added to 300 ml of previously boiled sucrose syrup (50% w/v). this mixture was manually homogenized with a spatula, divided into two equal aliquots in 250 ml amber-colored glass bottles, and placed in an incubator at 35 °c and controlled relative humidity (70%) for 25 days. in order to release the co2 produced during fermentation, the bottles were briefly opened every 48 h. after the end of this fermentation period, the bottles were sealed and stored at 6–8 °c for up to 20 days. a new inoculum was prepared from freshly collected beebread every 20 days to help reduce contamination with opportunistic fungi and other microorganisms. diet preparation diets were formulated as follows: 1. beebread diet -100 g of beebread mixed with 80 ml of distilled water, forming a paste; 2. sucrose syrup –70% (w/v); 3. unfermented protein diet 20 g powdered sugar cane yeast, 16.7 g powdered soy meal., 43.3 g rice meal, 20 g sucrose, and sufficient 50% sucrose syrup to make a paste; 4. fermented protein diet – same ingredients as diet 3, with 40 ml fermented inoculum added and mixed and then stored in an incubator in a loosely covered plastic food-grade container at 35°c for 28 days. the artificial protein diet contained 25% crude protein in the dry components, before adding the sucrose syrup. considering the final weight of approximately 180 g (including the sucrose syrup and fermented inoculum, which is about 50% sucrose), the diet paste offered to the bees had approximately 14% protein. caged bees and feeding combs with emerging worker brood from four africanized honey bee colonies were placed in an incubator at 34 °c and 80% relative humidity. the workers that emerged within a period of 15–20 hours were collected, mixed as uniformly as possible and groups of 100 of these workers were placed in each plastic confinement cage (8 x 11 x 13 cm) (morais et al., 2013b). on days 0, 2, 4 and 6, 4 g of each diet was offered to the bees in plastic feeders (50 ml falcon centrifuge tubes partially cut lengthwise to fashion troughs) introduced into the cages through a hole drilled in the side. sucrose syrup (70% w/v) was provided ad libitum to all groups. each diet was tested in eight cages. the bees in the cages were maintained in an incubator in the dark at 34°c and 80% relative humidity. quantification of protein in the hemolymph after three and seven days of confinement and feeding, 10 workers were randomly collected from each cage and hemolymph was collected with a pipette from a small incision made with an entomological scissors at the base of the bees’ wings. the total protein in the hemolymph was determined by spectrophotometry (ultrospec 2100 pro pharmacia), at 595 nm, using the methodology proposed by bradford (1976). a standard curve was prepared using bovine serum albumen at 0, 4, 10, 16, 20, and 30 µg protein/µl. the standards and the hemolymph samples were pipetted into 96 well elisa plates and read in an elisa microplate reader (morais et al., 2013b). measurements of vitellogenin levels soluble hemolymph (proteins were separated by sdspage, according to the method of laemmli (1970) in a 7.5% polyacrylamide gel; 0.5 µl of hemolymph was obtained from a pool of 10 workers after seven days of confinement and feeding. the hemolymph was collected using the same technique as above, then mixed and centrifuged at 4000 rpm for 4 min at 4 ºc, added to sample buffer and subjected to a constant current of 15 ma at 7–10 °c. the buffer was made from 3.03 g of tris pm = 121.14) in 50 ml of distilled water; the ph was adjusted to 6.8 and the volume completed to 100 ml with distilled water; 1.25 ml of this solution was added to 0.5 ml 70% (w/w) sucrose, and 3 ml distilled water, 1.2 g bromophenol blue and 0.25 ml mercaptoethanol. after electrophoresis, the gels were stained with 1% coomassie brilliant blue dissolved in a solution of glacial acetic acid, ethanol and distilled water (1:5:5 v/v/v), which was also used for the gel discoloration. survival rate of caged bees fed natural, fermented and unfermented diets twelve confinement cages were prepared, each containing 100 newly emerged bees. bees in three of these cages were fed with the beebread diet (4 g), three were jm almeida-dias, mm morais, tm francoy, ra pereira, ap turcatto, d de jong – fermentation improves honey bee diets762 fed with sucrose syrup (ad libitum), three were fed with the unfermented protein diet (4 g) and three were fed with the fermented protein diet (4 g). the cages were kept in an incubator, under the same conditions of temperature and humidity mentioned above. we counted and removed dead bees from each cage daily. the food was renewed every 48 hours. survival rates were analyzed by kaplan-meier survival analysis using spss (version 17.0.2). determination of the consumption rate and preference for each diet ten confinement cages were prepared, each containing 100 newly emerged bees. each cage was provided with 4 g of unfermented protein diet and 4 g of fermented protein diet, made available at the same time, so that the bees could choose between diets. the cages were kept under the same incubator conditions mentioned above. the experiment lasted eight days and the diet was renewed every two days. remaining diet was weighed when the protein diet was replaced and on the last day to determine the consumption rate. statistical analysis data obtained from the protein quantification in the hemolymph and measurements of vitellogenin levels were compared using anova on ranks, and pair-wise comparisons were made using the student-newman-keuls test and the t-test. survival rates were analyzed using kaplan-meier survival analysis. the mean consumption rates were analyzed using the wilcoxon signed rank test, in sigmastat © 3.5. results total protein levels in the hemolymph of caged workers the mean levels of protein in the hemolymph of workers in cages fed with protein diets were significantly higher than in those fed sucrose syrup alone. additionally, bees fed with fermented protein diet had significantly higher levels of protein in their hemolymph than those fed with unfermented protein diet during seven days of confinement (table 1). after three days of feeding, the levels of protein in the hemolymph of bees fed unfermented and fermented protein diet were not significantly different from those of beebread fed bees (p = 0.809 and p = 0.437, respectively). all the groups fed protein diets had higher levels of protein in the hemolymph when compared to bees fed sucrose syrup (p < 0.01). after seven days of feeding, both the fermented and unfermented protein diet groups had significantly lower levels of protein in the hemolymph, when compared to workers fed beebread (p ≤ 0.001 and p = 0.007, respectively). vitellogenin levels the density of vitellogenin bands of hemolymph from bees fed the fermented diet was significantly greater (optical density of vitellogenin bands 165.85 ± 6.79 arbitrary optical density units (d.u.) compared to that from bees fed on unfermented diet (123.17 ± 7.71 d.u.; p = 0.026, student-neuman-keuls test). these levels were similar to and not significantly different when compared to beebread-fed bees (138.22 ± 5.60 d.u.). fig 1. survival of caged newly emerged bees fed different diets in confinement cages. diets were beebread, fermented protein diet, unfermented protein diet and sucrose syrup. age (days) diets (concentration of total protein μg/μl) sucrose syrup beebread diet unfermented protein diet fermented protein diet 3 14.38a ± 5.40 23.00b ± 4.35 23.65b ± 6.25 26.15b ± 5.97 7 12.14a ± 3.45 54.29d ± 7.13 32.24b ± 2.40 39.46c ± 6.64 table 1. mean and standard deviation of the concentration of total protein (μg/μl) in the hemolymph of honey bees confined in plastic cages at emergence, after feeding on beebread diet, sucrose syrup, unfermented protein diet or fermented protein diet for 3 7 days. pools of hemolymph from 10 bees were analyzed from each of eight cages for each diet. identical letters in the same row indicate the absence of significant statistical differences. different letters in the same row indicate significant differences (anova on ranks and pair-wise comparisons were made using the student-newman-keuls test and the t-test, p < 0.05). survival rates of adult workers confined to cages bees fed with the protein diets beebread, unfermented and fermented protein diet survived longer than those fed only with sucrose syrup (p ≤ 0.001, kaplan-meier survival analysis, fig 1). when compared with beebread, fermented and unfermented diet showed no significant difference (p = 0.188 and p = 0.05), indicating that feeding the artificial diet did not negatively affect adult survival rates. the comparison of the survival rate between bees fed fermented and unfermented diet also showed no significant difference (p = 0.178). sociobiology 65(4): 760-765 (october, 2018) special issue 763 consumption rates and preferences the mean consumption rates of unfermented and fermented protein diets were 3.5 ± 0.93 mg and 6.1 ± 1.25 mg per bee during eight days, respectively. fermentation of the diet significantly increased consumption (p = 0.002, wilcoxon signed rank test). in the preference test, when the two protein diets were offered together in the same cage, the bees preferred the fermented over the unfermented diet (2.63 ± 0.75 mg versus 1.09 ± 0.59 mg per bee, respectively; p < 0.001, wilcoxon signed rank test). discussion fermentation significantly increased diet consumption and the protein levels of bees fed on the pollen substitute diet (table 1). the highest protein levels were found in bees that consumed beebread, as also found by cremonez et al. (1998), van der steen (2007) and basualdo et al. (2014). a sucrose syrup diet (control) resulted in lower protein levels in the hemolymph, as also found by de jong et al. (2009) and morais et al. (2013b). in apis mellifera linnaeus the accumulation of storage proteins (vitellogenin) in the hemolymph of adult workers is significantly influenced by nutrition. consequently, a diet that is able to maintain proteins in the hemolymph both qualitatively and quantitatively helps guarantee the health of the bees (bitondi & simões, 1996). we found that workers fed with the fermented protein diet produced vitellogenin at similar levels when compared to beebread fed bees, but significantly higher than in bees fed unfermented diet. schmidt et al. (1987) and manning et al. (2007) observed that differences in the protein levels of pollen affect bee longevity. herbert and shimanuki (1978) found that pollen substitutes can efficiently substitute pollen; however, inducing the bees to consume artificial diets can be a major difficulty. they also stated that diets need to be both nutritious and palatable in order to be useful, as also concluded by mattila and otis (2006) and standifer et al. (1973). ellis and hayes (2009) found that bees consume more of a fermented diet than an unfermented diet, though they used a probiotic yogurt inoculum instead of beebread microorganisms. the main microorganisms present in beebread are: bacteria, including the genera lactobacillus, bacillus and agrobacterium, and fungi of the genera penicillium and aspergillus, as well as yeasts (gilliam, 1997). these are responsible for the fermentation process. alterations made by the microorganisms in beebread help preserve, increase palatability and facilitate consumption of bee-collected pollen (loper et al., 1980; gilliam et al., 1989; vasquez & olofsson, 2009). we used the same microflora to ferment a pollen substitute diet to determine if it would make the diet more attractive and useful for the bees. the bees preferred and consumed more of the fermented diet. on the other hand, carroll et al. (2017) found that bees preferentially consumed freshly stored pollen (one day old) over pollen that had been stored (and fermented) for a longer period, when choosing what was available in the hive. beebread from africanized hives could have a different microflora than that from european bees, as it has been found to be preferred by both types of bees (degrandihoffman et al., 2013). fermenting the diet with bee-derived microorganisms could help correct diet-related gut dysbiosis and protect the bees against pathogens (maes et al., 2016). in conclusion, fermenting a pollen substitute diet for bees can make it more useful as a substitute for natural pollen sources, resulting in greater protein levels in the hemolymph and consequently greater brood production in honey bee colonies. this fermentation process requires no specialized equipment and the inoculum can be made from beebread collected from local beehives. acknowledgments the authors thank cnpq (conselho nacional de desenvolvimento científico e tecnológico), faepa (fundação de apoio ao ensino, pesquisa e assistência) and fapesp (fundação de amparo à pesquisa do estado de são paulo) for financial assistance. authors’ contribution study conception and design: jmad; mmm; ddj. acquisition of data: jmad; apt; rap. analysis and interpretation of data: jmad; tmf; mmm. drafting of manuscript: jmad; tmf; mmm; ddj. critical revision: jmad; tmf; mmm; ddj. references abbas, t., abid, h. & ali, r. 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(2007). effect of a home-made pollen substitute on honey bee colony development. journal of apicultural research, 46: 114-119. doi: 10.1080/00218839.2007.11101377 vasquez, a. & olofsson, t. (2009). the lactic acid bacteria involved in the production of bee pollen and bee bread. journal of apicultural research, 48: 189-195. doi: 10.3896/ ibra.1.48.3.07 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3366sociobiology 66(1): 75-80 (march, 2019) an overview on honeybee colony losses in buenos aires province, argentina introduction losses of honey bees and other pollinators threat biodiversity as well as food and agricultural production (simondelso et al., 2014; watson & stallins, 2016). the phenomenon called colony collapse disorder (ccd) is expressed carrying out a complete absence of adult bees in a hive often plenty of both capped brood and food reserves (vanengelsdorp et al., 2009). this scenario has been observed around the world during the last two decades. some beekeepers in the united states have reported losses of up to 75% of their hives between 2006 and 2007 (oldroyd, 2007; vanengelsdorp et al., 2009; 2017; ellis et al., 2010; vanengelsdorp & meixner, 2010). in europe, similar phenomena have been reported (dainat et al., abstract honey bees (apis mellifera) are essential for the ecosystem, so their loss threatens biodiversity and agriculture. several factors have been proposed as possible causes of both massive losses and colony collapse disorder. in august 2017 episodes of colony losses were registered in general alvear, buenos aires province. the aim of the present study was to find possible causes of these events. the samples were screened for presence of several pathogens and the determination of maternal lineages was also performed. seven out of ten colonies were positive for pathogens, but there was no high prevalence of any of them. it will be necessary to carry out a standardization of studies, and delineate boundaries that allow comparing cases in order to discriminate different types of mortality of colonies that occur worldwide. sociobiology an international journal on social insects mlg garcía1,2,3, s plischuk4,5, cm bravi2,4, fj reynaldi3,4 article history edited by kleber del-claro, ufu, brazil received 23 april 2018 initial acceptance 14 may 2018 final acceptance 06 june 2018 publication date 25 april 2019 keywords colony collapse disorder, apis mellifera, pathogens, maternal lineages. corresponding author m. l. genchi garcia facultad de ciencias veterinarias laboratorio de virología universidad nacional de la plata calle 60 y 118 s/n (1900) la plata buenos aires, argentina. e-mail: ml.genchigarcia@gmail.com 2012; meana et al., 2017), but in those cases the symptoms would not have been the same as in the united states (stokstad, 2007). in recent years, some cases of ccd have also been reported in asia and south america (farooqui, 2013; antúnez et al., 2017) but there are not any documented cases in africa or oceania. in south america, particularly in argentina, even though there are no documented cases, several records of beekeepers suggest a 30% of losses in the last years (maggi et al., 2014). in 2017, 154 out of 170 commercial colonies nucleated in three apiaries from general alvear (35°55′ 23.67″s, 59°57′29.58″w), buenos aires province showed the sudden massive loss of their bees and died at the end of summer, according ccd symptoms. the aim of the present study was to analyze the possible causes of these episodes. 1 cic – comisión de investigaciones científicas de la provincia de buenos aires, buenos aires, argentina 2 imbice – instituto multidisciplinario de biología celular – cic-conicet-unlp, buenos aires, argentina 3 laboratorio de virología, facultad de ciencias veterinarias – unlp, buenos aires, argentina 4 conicet – consejo nacional de investigaciones científicas y técnicas, buenos aires, argentina 5 cepave – centro de estudios parasitológicos y de vectores – conicet-unlp, buenos aires, argentina research article bees mlg garcía, s plischuk, cm bravi, fj reynaldi – colony losses in argentina76 materials and methods ten samples from three different apiaries located in general alvear were collected in spring 2017 during episodes of colony losses. particularly, four samples were from he apiary, three from ca apiary and three from ho apiary. each hive was considered a sample unit, comprising 400 live honey bees that were frozen at -32 °c. afterwards, they were stored at -80 °c in the laboratory prior to the analysis. bees were screened for presence of seven virus, mites (varroa destructor), bacteria (melisococcus plutonius and paenibacillus larvae), fungi (nosema spp. and ascosphaera apis), and protists (malpighamoeba mellificae, apicystis bombi, nephridiophaga sp.) all of them already known to be present in the country (ringuelet, 1947; cantwell, 1970; rossi & carranza, 1980; alippi, 1992a; undeen & vávra, 1997; plischuk & lange, 2010; 2011; plischuk et al., 2011; reynaldi et al., 2010; 2011). bee maternal lineages were also analyzed. the virus detection was performed according to sguazza et al. (2013) with modifications. ten honeybees from each sample were homogenated in a stomacher with 2 ml of sterile pbs (free of nucleases), followed by extraction and purification of viral rna with trizol® reagent (thermo fisher scientific) according to the manufacturer protocol. subsequently, reverse transcription of the rna was made using reverse transcriptase (mml-v) and random primers, in order to synthesize the complementary dna. then, a multiple pcr reaction was performed using specific primers for seven bee virus abpv (acute bee paralysis virus), bqcv (black queen cell virus), cbpv (chronic bee paralysis virus), dwv (deformed wing virus), kbv (kashmir bee virus), sbv (sacbrood virus) and iapv (israeli acute paralysis virus) (table 1). the results were analyzed in a 2% agarose gel electrophoresis stained with ethidium bromide. both detection and quantification of v. destructor were performed according to the validated world organization primer virus nucleotide sequence (5’-3’) product length (bp) reference aivf iapv ggtgccctatttagggtgagga 158 sguazza et al. (2013)iapvr gggagtattgctttcttgttgtg dwvf dwv tggtcaattacaagctacttgg 269 sguazza et al. (2013)dwvr tagttggaccagtagcactcat sbvf sbv cgtaattgcggagtggaaagatt 342 sguazza et al. (2013)sbvr agattccttcgagggtacctcatc aivf abpv ggtgccctatttagggtgagga 460 sguazza et al. (2013)abpvr actacagaaggcaatgtccaaga bqcvf bqcv ctttatcgaggaggagttcgagt 536 sguazza et al. (2013)bqcvr gcaatagataaagtgagccctcc cbpvf cbpv aacctgcctcaacacaggcaac 774 sguazza et al. (2013)cbpvr acatctcttcttcggtgtcagcc primer bacteria nucleotide sequences (5’-3’) product length (bp) reference melifor m. plutonius gttaaaaggcgctttcgggt 281 garrido bailón et al. (2013)melirev gaggaaaacagttactctttccccta primer 1 p. larvae aagtcgagcggaccttgtgtttc 973 govan et al. (1999) primer 2 tctatctcaaaaccggtcagagg primer fungi nucleotide sequence (5’-3’) product length (bp) reference ascosfor a. apis tgtgtctgtgcggctaggtg 136 garrido bailón et al. (2013)ascosrev gctagccaggggggaactaa n. ceranae sense n. ceranae cggataaaagagtccgttacc 250 chen et al. (2009) n. ceranae antisense tgagcagggttctagggat n. apis sense n. apis ccattgccggataagagagt 269 chen et al. (2009) n. apis antisense ccaccaaaaactcccaagag primer maternal lineage nucleotide sequence (5’-3’) product length (bp) reference cytb f cytochrome b tatgtactaccatgaggacaaatatc 485 crozier et al. (1991)cytb r attacacctcctaatttattaggaat table 1. analyses of a. mellifera from general alvear, buenos aires, argentina: primers used for pathogens and maternal lineages detection. [abpv = acute bee paralysis virus; bqcv = black queen cell virus; cbpv = chronic bee paralysis virus; dwv = deformed wing virus; iapv = israeli acute paralysis virus; kbv = kashmir bee virus; m. plutonius = melissococcus plutonius; n. apis = nosema apis; n. ceranae = nosema ceranae; p. larvae = paenibacillus larvae; sbv = sacbrood virus]. sociobiology 66(1): 75-80 (march, 2019) 77 for animal health method (world organization for animal health [oie], 2008) with modifications. briefly, three hundred bees per hive were treated with surfactants to allow separation from mites. then a double sieve was used, the upper one retained the bees and the lower one the mites. mites were counted and the percentage of infection was estimated by multiplying by 100 the number of mites per bee (number of mites/number of bees) in each sample. the detection of m. plutonius, p. larvae and a. apis was carried out by the homogenization and subsequent dna extraction of ten individuals per sample with dnazol® reagent (thermo fisher scientific). subsequently, multiple pcr amplification was performed following garrido-bailón et al. (2013) (table 1). the presence of nosema spp. as well as protists was determined by individual homogenization in 1 ml of bidistilled water and observation of a drop in a phase contrast microscope (x400; x1000). for prevalence estimation, 100 individuals per sample were scrutinized. each positive homogenate was filtered and infective stages were quantified using an improved neubauer chamber (cantwell, 1970). a pcr analysis was performed according to chen et al. (2009) in order to determine the infective species of nosema spp. (table 1). the determination of maternal lineages was performed from total dna extraction. the dna was extracted from the thorax of one individual per sample using dnazol® reagent (thermo fisher scientific). pcr-rflp technique was carried out to discriminate introgression of african genes in the samples (pinto et al., 2003, with modifications) (table 1). the variation in the 485 base pair fragment restriction patterns was obtained and analyzed in 1% agarose gel electrophoresis stained with gelred™ (biotium). results the results of the detection of honey bee diseases and the genetic determination of maternal lineages are shown in table 2. samples have exhibited the presence of virus in three of the ten hives studied, five hives presented variable percentages of v. destructor, and in three hives (one of each apiary) bees with low spore loads (<10,000 spores/bee) of nosema spp. were detected. all of them were determined as nosema ceranae. none of the hives exhibited the presence of m. plutonius, p. larvae, a. apis, or any protist. all the individuals analyzed belonged to european maternal lineages. table 2. analyses of a. mellifera from general alvear, buenos aires, argentina: presence of pathogens and maternal lineage determination. results are expressed as (+) for those pathogens present in the sample and (-) for those that did not exhibit presence. maternal lineage is indicated with (x). [a. bombi = apicystis bombi; abpv = acute bee paralysis virus; bqcv = black queen cell virus; cbpv = chronic bee paralysis virus; dwv = deformed wing virus; iapv = israeli acute paralysis virus; kbv = kashmir bee virus; m. mellificae = malpighamoeba mellificae; m. plutonius = melissococcus plutonius; n. apis = nosema apis; n. ceranae = nosema ceranae; p. larvae = paenibacillus larvae; sbv = sacbrood virus; v. destructor = varroa destructor]. apiary hive virus mites bacteria fungi protists maternal lineage a b pv b q c v c b pv d w v k b v sb v ia pv v. d es tr uc to r. m . p lu to ni us p. la rv ae n os em a sp p. a . a pi s m . m el lifi ca e a . b om bi n ep hr id io ph ag a sp . e ur op ea n a fr ic an h e i + + x ii 1.5% x iii x iv x c a i x ii + 2.0% + x iii 1.0% x h o i + + 0.3% x ii + x iii 1.3% x mlg garcía, s plischuk, cm bravi, fj reynaldi – colony losses in argentina78 discussion interactions between multiple drivers or risk factors could be the most probable explanation for elevated mortality rates in honey bee colonies (potts et al., 2010; meana et al., 2017), but the reasons that trigger ccd are still in debate (stockstad, 2007; williams et al., 2010;stavely et al., 2014). several factors have been proposed as possible causes of massive losses related to ccd. some hypotheses suggested that pathogens like n. ceranae, v. destructor, bacteria, and several viruses could be responsible of these losses (coxfoster et al., 2007; mcmenamin & genersch, 2015; brutscher et al., 2016; meana et al., 2017), as well as pesticides (chauzat et al., 2006). unfavorable weather conditions and consequent lack of available food, large-scale transhumance practices, nutrition, genetic, or even a combination of several factors are considered some of other potential drivers (stokstad, 2007; vanengelsdorp et al., 2009; ellis et al., 2010; potts et al., 2010; ratnieks & carreck, 2010; huang, 2012; francis et al., 2014; watson & stallins, 2016; maggi et al., 2016; richardson, 2017). moreover since both honey bee host and pathogens (if involved) are genetically diverse, symptoms and causes of colony losses may well change in different regions (neumann & carreck, 2010). on the other hand, some authors proposed that extensive colony losses are not unusual and have occurred repeatedly over decades and regions (oldroyd, 2007; ratnieks & carreck, 2010). the loss of colonies is well documented in the northern hemisphere (oldroyd, 2007, vanengelsdorp et al., 2009; 2017; vanengelsdorp & meixner, 2010; neumann & carreck, 2010; ellis et al., 2010; dainat et al., 2012; meana et al., 2017) but case studies in the southern hemisphere are almost nonexistent. according to antúnez et al. (2017) 28% of annual losses were estimated in uruguay. in argentina, even though there are no documented cases, several records of beekeepers suggest a 30% of losses in the last years (maggi et al., 2014). particularly in buenos aires province, a survey over 200.000 colonies showed that 54% of producers had less than 10% of dead hives, 33% between 10-20%, and 13% more than 20% (reynaldi & guardia lópez, 2011). this scenario seems to alert about the need of document these cases and to find out the possible mortality causes as well as to compare regional cases with others worldwide. in this study, seven out of ten colonies harbored different pathogens. three of them presented coinfections between virus–fungi, mites–virus–fungi, and virus-mites. however, pathogens varied between hives and, at the same time, the coinfections did not occur among the same pathogens. even though other studies support the hypothesis of pathogens as the main cause of losses (cox-foster et al., 2007; mcmenamin & genersch, 2015; meana et al., 2017), our results suggest that their presence could not explain the losses by themselves. not only because there was not a high prevalence of any pathogen, but also because the identity and coinfections were not repeated between hives. regarding a possible effect of agrochemicals, the studied apiaries were sited 20-30 km away from general alvear city and no extensive crop cultivation exist in these area, making unlikely the situation of intoxication or weakening by agrochemicals. until now there is no single documented cause for ccd in argentina. instead, many causes arise from the hypothesis that this is a multifactorial complex syndrome. reframing discussions in a pluralistic way is needed, but reductionism should not be rejected outright (watson & stallins, 2016). a clearer separation that delineates the boundaries between the different cases of bee mortality is necessary to make estimations and comparisons between them and to be able to define ccd causes. aknowledgements conicet grant (pip) 0726 (2015-2017) and foncyt grant pict 2016-0289. author’s contribution ml genchi garcía, fj reynaldi and s plischuk conceived the study, analyzed the samples and wrote the manuscript; cm bravi contributed to genetic analysis and revised the final version of the manuscript. references alippi, a.m. (1992a). characterization of bacillus larvae white the causative agent of afb of honey bees. first record of its occurrence in argentina. revista argentina de microbiología, 24: 67-72. antúnez, k., invernizzi, c., mendoza, y., vanengelsdorp, d. & zunino, p. 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(2008). varroosis on honey bee (infestation of honey bees with varroa spp). chapter 2.2.7. in oie terrestrial manual. http://www.oie.int/fileadmin/ home/eng/health_standards/tahm/2.02.07_varroosis.pdf doi: 10.13102/sociobiology.v61i2.198-206sociobiology 61(2): 198-206 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 survey of subterranean termite (isoptera: rhinotermitidae) utilization of temperate forests ns little1, na blount2, ma caprio2, jj riggins2 introduction termites are ecologically and economically important insects, which can be found in nearly all forest ecosystems. subterranean termites (isoptera: rhinotermitidae) play important roles in cellulose decomposition, nutrient cycling, and soil mineralization across a multitude of environments (harris, 1966; wood & sands, 1978; black & okwakol, 1997). although they are known to cause significant economic damage to wooden structures in urban areas throughout the u.s., native subterranean termites (reticulitermes spp.) provide a valuable service to forest ecosystems as the predominant invertebrate decomposers of woody materials (la fage & nutting, 1978). the role of subterranean termites in forest ecosystem nutrient cycles is extremely important, and their presence can contribute up to 22% of total nitrogen input in tropical forest ecosystems (yamada et al., 2006). the non-native formosan subterranean termite (coptotermes formosanus shiraki), is reported to infest 17 species abstract both native and invasive subterranean termites (isoptera: rhinotermitidae), including the formosan subterranean termite, are well known pests of urban areas, but little is known about their distribution or impact in forest ecosystems of the southeastern united states. recently harvested timber stumps were mechanically inspected for the presence of subterranean termites in multiple locations across southern mississippi and eastern louisiana. a systematic line plot cruise with 100 x 200m spacing and 1/20th ha plots was implemented, and all stumps with a diameter greater than 7.6cm were inspected. in total, 7,413 stumps were inspected for the presence of subterranean termites, and 406 of those contained native subterranean termites (reticulitermes spp.). light traps were also placed at 8 sites to detect the presence of subterranean termite alates. while no invasive formosan subterranean termites were found during mechanical inspection of tree stumps, alates were captured in light traps at three sites. the proportion of stumps infested with subterranean termites was negatively correlated with the number of stumps in each plot. although 6.27% of pine stumps and 1.86% of hardwood stumps contained subterranean termites, no correlation was found between subterranean termite presence and type of stump (pine or hardwood) inspected. subterranean termite presence in stumps ranged from 0.94% to 14.97% depending on site. sociobiology an international journal on social insects 1 usda-ars, southern insect management research unit, mississippi, usa. 2 mississippi state university, mississippi, usa. article history edited by gilberto m m santos, uefs, brazil received 14 november 2013 initial acceptance 14 april 2014 final acceptance 23 may 2014 key words formosan subterranean termites, coptotermes formosanus, timber stumps, hardwood, softwood corresponding author nathan s. little usda-ars, southern insect managem. research unit 141 experiment station road stoneville, mississippi, usa 38776 e-mail: nathan.little@ars.usda.gov of living trees (chambers et al., 1988) and at least forty other species of plants (lai et al., 1983). formosan subterranean termites are known to cause significant economic damage to standing timber in many regions of the world (harris, 1966). greaves et al. (1967) reported that coptotermes spp. were responsible for up to 92% of tree losses in virgin eucalyptus forests and approximately 64% of losses in younger forests, which were predisposed to fire stress. while studies have investigated subterranean termite ecology in tropical forest ecosystems, little is known about their ecology in temperate forests. loblolly pine (pinus taeda l.) is a predominant species in the southern u.s., comprising 45% of commercial forestland and contributing $30 billion annually to the economy (schultz, 1999). considering the large industry for loblolly pine in the southeastern u.s. and the lack of research on formosan subterranean termites in forested areas, the impact of this invasive insect could be substantial. for instance, native subterranean termites, which are ubiquitously reported in the research article termites sociobiology 61(2): 198-206 (june, 2014) 199 literature to rarely infest living trees, have been observed readily utilizing blue-stained portions of bark beetle-attacked trees before any foliage chlorosis could be detected (little, 2013). while formosan termites commonly infest living trees in urban settings, loblolly pines are more frequently infested than other tree species (osbrink et al., 1999; guillot et al., 2010). additionally, morales-ramos & rojas (2001) observed that formosan subterranean termites consumed loblolly pine wood at a higher rate relative to other wood species in laboratory no-choice tests. biological differences between formosan and native subterranean termites create the possibility that formosan subterranean termites could impact forest ecosystems and individual trees differently than our native subterranean termites. for example, formosan subterranean termite colonies are considerably larger and they are more aggressive feeders than native species (su & scheffrahn, 1988). formosan subterranean termites also exhibited higher survival and wood consumption rates than native reticulitermes spp. in laboratory assays (smythe & carther, 1970). they can also nest above-ground with no connection to the soil, which allows them to utilize resources previously inaccessible to native species. la fage (1987) noted that in certain instances in new orleans, la, formosan termites completely replaced native termites as the predominant termite species. more recently, su (2003) confirmed that formosan subterranean termites were outcompeting native reticulitermes spp. in multiple urban locations. despite these concerns, little research has been done on the distribution or impact of formosan subterranean termites in non-urban forested environments of the u.s. the first indication that formosan subterranean termites were established in forested areas of the southeastern u.s. was when sun et al., (2007) reported that formosan subterranean termite alate catches were higher in forested settings than in urban areas. in addition, alate catches increased throughout the four year trapping study, which may have indicated that formosan subterranean termite populations were well established and colony expansion was occurring in mississippi (sun et al., 2007). native and formosan subterranean termite ecology in local forested settings of the southern u.s. has yet to be quantified. we hypothesized that elevated light trap captures of formosan subterranean termite alates in rural forested areas by sun et al. (2007) would be explained by infestations of living trees in surrounding forest stands. therefore, the objectives of this study were to determine the extent of subterranean termite utilization of living trees within localized forested settings, quantify subterranean termite presence in forested areas, determine if the proportion of stumps infested with subterranean termites was correlated with stump density or size, and quantify the effect of tree type (hardwood or softwood) on subterranean termite utilization of woody resources. material and methods stump inspections eleven sites encompassing 476.8ha throughout southern mississippi and louisiana were inspected during 2011 and 2012 (fig 1). there were nine sites surveyed in four mississippi counties (pearl river, harrison, jackson, and lamar) and two additional sites were surveyed in st. tammany parish, louisiana (table 1). according to sun et al. (2007), the four mississippi counties inspected during this study were among the top five counties in the state for formosan subterranean termite alate captures. additionally, formosan subterranean termites have been found in st. tammany parish, louisiana since the late 1980s (la fage, 1987; messenger et al., 2002). clear cut pine plantations were inspected for subterranean termite presence in residual stumps within three weeks of harvest because they offered the best opportunity to discover the presence of subterranean termites in living trees without damaging standing timber. a variety of methods have been used over the years to detect subterranean termites in living trees (e.g. acoustic devices, dogs, co2 detectors, coring, etc.); however, most have limitations, and are often less effective than physical inspections with hatchets and shovels (osbrink et al., 1999). site selection criteria (recent timber harvest and proximity to established formosan subterranean termite populations) led to sporadic site availability; therefore sites were inspected as they became available. to limit the possibility of post-harvest colonization of stumps, all inspections took place within three weeks of timber harvest; however, this only ocfig 1. map of inspecion sites. ns little et al utilization of woody resources by the formosan subterranean termite200 table 1. summary of sites inspected for this study site county/parish hectares % cruise gps coordinates soil conditions site designation a pearl river 83.0 5 n30° 46’ 58” w89° 29’ 56” dry upland b harrison 89.4 2.5 n30° 31’ 25” w89° 11’ 47” dry upland c harrison 117.4 2.5 n30° 31’ 43” w88° 53’ 22” wet lowland d jackson 25.9 2.5 n30° 36’ 43” w88° 47’ 17” dry upland e lamar 13.4 5 n31° 8’ 23” w89° 30’ 52” moderate upland f st. tammany 39.3 2.5 n30° 27’ 10” w89° 59’ 37” moderate lowland g st. tammany 19.8 5 n30° 25’ 1” w89° 48’ 45” moderate lowland h harrison 8.5 5 n30° 37’ 3” w89° 12’ 30” moderate lowland i lamar 19.4 5 n31° 15’ 13” w89° 37’ 49” moderate upland j lamar 39.3 2.5 n31° 15’ 11” w89° 38’ 20” moderate upland k pearl river 21.4 5 n30° 52’ 5” w89° 24’ 16” moderate upland curred for one site. the majority of sites were inspected while logging was still occurring, with the balance being inspected within a week of harvest. a 2.5% systematic line plot cruise (avery & burkhart, 2002) was implemented on post-harvest sites that were 20ha and larger to insure adequate sampling across each site and to limit sampling bias (with the exception of the first site inspected, a). sites smaller than 20ha received a 5% cruise. plots were 1/20th ha and circular, with 100m between plots and 200m between transects (fig 2). a global positioning system (gps) unit (gpsmap 60csx®, garmin ltd., olathe, ks) was used to record plot centers. all stumps greater than 7.6cm in diameter and located within plots were inspected with shovels and hatchets. stumps less than 7.6cm in diameter were not sampled based fig 2. example of systematic line plot cruise. on findings from prior studies, which indicated that native subterranean termites primarily occur in coarse woody debris with diameters above 7.6cm (wang & powell, 2001; wang et al., 2003). one-quarter of each stump was inspected for subterranean termite presence from the cut surface of the stump down to 15cm below the soil line. this method has been previously used to successfully detect infestations of formosan subterranean termites in living trees throughout urban areas, even when visual symptoms were not present (osbrink et al., 1999). if termites were present in stumps, they were immediately identified to genera, reticulitermes (native) or coptotermes (formosan), using the distinct morphological characteristic of head shape in the soldier caste (gleason & koehler, 1980). stump diameter was recorded for each occurrence of termites in stumps, which was subsequently labeled as hardwood or pine (softwood). each stump was then marked and labeled on a gps unit. alate survey light traps were used during spring of 2012 to confirm the presence of formosan subterranean termite alates near sites selected for physical inspections. methods similar to sun et al. (2007) were used to construct alate traps to ensure proper design. traps were constructed using a 1.52m (5-foot) t-post, white polyvinyl chloride (pvc) pipe, 20ga 2.54cm (1-inch) poultry netting (garden plus), a solar powered light emitting diode (led) light (model sps2-p1-bk-t24, lg sourcing inc., n. wilkesboro, nc), and a glue board (trapper® ltd, bell laboratories, inc., madison, wi) (fig 3). to avoid non-target catches of vertebrates attempting to prey on captured insects, poultry netting was wrapped around the glue board and t-post to achieve cage dimensions of 16.5 (9.5-inches) x 35.6cm (14-inches). a wire top was fashioned to give easy access for glue board replacement. the t-post was driven into the ground to insure firm placement of the trap. the led light came on approximately 20 min after sunset, and ran for six hours throughout the trapping season, sociobiology 61(2): 198-206 (june, 2014) 201 since peak formosan subterranean termite alate flight activity occurs at dusk (bess, 1970). formosan subterranean termite alates have been documented to swarm as early as mid-april in mississippi, with peak activity often occurring near the latter part of may (sun et al., 2007; lax & wiltz, 2010). traps were placed on sites at the end of the first week in april, early enough to detect initial swarms. glue boards were replaced once a week for a seven week period, with a final collection date of may 26. this sampling window allowed ample time to catch alates during their swarming periods determined by previous studies (sun et al., 2007; lax & wiltz, 2010). all glue boards were dated and labeled by site when removed, wrapped in plastic wrap, and placed in a freezer for preservation. alates were identified in the laboratory using morphological characteristics provided by gleason & koehler (1980). a total of twelve alate light traps were placed on eight sites. the lamar county sites (e, i, & j) were excluded from the alate trapping portion of this study due to travel constraints. additionally, lamar county had a low number of alate captures during a previous study (sun et al., 2007) relative to all other counties sampled in mississippi. the number of traps placed per site was determined by area, with five sites receiving one trap each (<40.5ha), two sites receiving two traps each (83 and 89.4ha respectively), and one site receiving three traps (117.4ha). traps were placed at the approximated center of each site. on sites with multiple traps, the location was partitioned according to area, with traps located in the approximate center of each partition. statistical analyses the proportion of stumps infested with termites was compared to stump density and site using the beinf family of the gamlss package (rigby & stasinopoulos, 2001; stasinopoulos & rigby, 2007; stasinopoulos et al. 2009), in the r statistical package (r core team, 2012). while an arcsine transformation is commonly used for proportional data, this transformation has a number of limitations and has been superseded by a variety of alternative techniques (warton & hui, 2011). a zero-inflated distribution was required because the data demonstrated a bimodal distribution with an excess of zeros (fig 4). the beinf family of gamlss models the data using a beta distribution and allows for inflation of both zeros and ones. inflation at both zero and one was chosen over zero-inflation alone because it was at least theoretically possible that all stumps in a plot could be infested with termites. this model assumes that there at least two processes that combined to produce the original data. the beta distribution models the range of proportion infestations between zero and one, while a binary process models an excess of plots with no infestations due to another process. the high number of plots with zero infestations and the potential for numerous infested trees within infested plots is potentially explained by the social/colonial nature of subterranean termites. subterranean termite workers only feed within a finite distance of the colony. this biological process likely explains the zeroinflated distribution of our data. pearson’s correlation coefficients (proc corr) were used to assess the relationship between infested pine and infested hardwood stumps in sas 9.2 (sas institute, cary, nc). significance for all analyses was determined at α≤0.05. figure 4. the distribution of the square root transformed proportion of stumps in a plot infested with termites compared to a kernel density distribution for the same variable. an excess of zeros is indicated by the large bar corresponding to zero infested stumps. fig 3. formosan subterranean termite alate light trap. ns little et al utilization of woody resources by the formosan subterranean termite202 results and discussion stump inspections a total of 7,413 stumps were inspected, consisting of 6,072 softwoods (hereafter pines) and 1,341 hardwoods. no formosan subterranean termites were found; however, 406 stumps containing reticulitermes spp. were recorded, resulting in 5.48% of all stumps containing subterranean termites. these included occurrences in 381 pine (6.27% of total pines) and 25 hardwood stumps (1.86% of total hardwoods) (table 2). site f had the highest overall occurrences of reticulitermes spp., with 14.97% of all stumps containing subterranean termites. sites c and g followed, with 10.81% and 7.42% of stumps containing subterranean termites, respectively. site i had the fewest subterranean termite occurrences among all sites with 0.94% of stumps containing termites, followed closely by sites d (1.33%) and k (1.58%). several sites had low occurrences of hardwood stumps. no hardwood stumps were encountered during inspections on site k, while site b had only 3 hardwood stumps in the plots. sites d and f also had low numbers of hardwood stumps, 16 and 33 respectively. five sites contained hardwood stumps that had no subterranean termites (b, d, h, i, and j). for further analysis and uniformity, stumps that contained reticulitermes species were converted to a per hectare basis using a conversion factor relative to the percent cruise conducted on each site. overall, the mean number of subterranean termites per hectare across all stumps and sites was 27.41 (se=0.28, n=7,413). mean number of pine stumps containing subterranean termites per hectare was 26.36 (se=0.30, n=6,072), with hardwood stumps at 1.05 (se=0.06, n=1,341). subterranean termite occurrences per hectare for all stumps on a per site basis ranged from 5.06 (site a) to 79.39 (site f). sites a, b, and i had fewer than 7.16 occurrences per hectare. site c had the second highest occurrence of subterranean termites per hectare, 65.08, followed by site g, 38.72. subterranean termite occurrence in hardwood stumps per hectare for site c was 7.16, which was the only site with more than 1.68 infested hardwood stumps per hectare. table 2. summary of stumps containing subterranean termite infestations by site. stumps inspected stumps infested % infestation site 1p 2h total 1p 2h total 1p 2h total a 530 360 890 20 1 21 3.77 0.28 2.36 b 411 3 414 16 0 16 3.89 0.00 3.86 c 1499 268 1767 170 21 191 11.34 7.84 10.81 d 585 16 601 8 0 8 1.37 0.00 1.33 e 181 166 347 15 1 16 8.29 0.60 4.61 f 488 33 521 77 1 78 15.78 3.03 14.97 g 258 52 310 22 1 23 8.53 1.92 7.42 h 223 204 427 11 0 11 4.93 0.00 2.58 i 304 121 425 4 0 4 1.32 0.00 0.94 j 387 118 505 19 0 19 4.91 0.00 3.76 k 1206 0 1206 19 0 19 1.58 0.00 1.58 ∑ 6072 1341 7413 381 25 406 6.27 1.86 5.48 1p = pine; 2h=hardwood fig 5. the distribution of the proportion of stumps in a plot infested with subterranean termites by site. sociobiology 61(2): 198-206 (june, 2014) 203 the distribution of the proportion of stumps infested varied with site (fig. 4). in the inflated beta distribution analysis, both stump density (χ2 = 8.49, df = 1, p = 0.0035) and sites (χ2 = 38.42, df =10, p = 3.21e-5) were significant. these data suggest that across the range of overall densities experienced at various sites, native termite species infested a greater proportion of stumps in plots with lower stump densities and presumably larger stumps (fig 6). in this agroforestry setting, lower stump densities indicate an older forest, with larger diameter trees. the beinf family of gamlss fits four parameters. two are the shape parameters for the beta distribution (μ = 0.1201, σ = 0.1942), while the other two are related to the probability of the additional processes leading to zero or one values (ν = 1.047, τ = 3.77e-09). these resulted in predicted probability densities of 0.511 at zero and 1.842e-9 at one. the frequency of subterranean termite infestations in hardwood stumps was significantly correlated with the frequency of infestations in pine stumps across all sites (pearson r = 0.68, p = 0.03). in other words, sites with higher termite abundance had more infestations of both pine and hardwood stumps. however, the frequency of infestation was always higher in pine stumps than in hardwood stumps. our results agree with previous studies (osbrink et al., 1999; guillot et al., 2010) that reported subterranean termite prefer loblolly pine over hardwoods. although no formosan subterranean termites were found in stumps of recently logged forested stands in mississippi and louisiana during this study, native reticulitermes spp. were found across all sites inspected. subterranean termite were more prevalent in pine stumps than hardwoods. additionally, our results confirmed findings from previous studies, which documented that the number of subterranean termite occurrences decreased with increasing wood hardness, with pines generally having higher occurrences of termites than hardwoods (lin, 1987; peralta et al., 2004). alate survey during the seven week trapping period a total of 14 formosan subterranean termite alates were caught among three sites (fig 5). two alates were detected on the may 11 trap check of site a in pearl river county. on may 12, one alate was present on the site d trap in jackson county. alates were not present again until the may 25 check, when 10 alates were caught on site a and one alate was present on site g within st. tammany parish, la. both traps on site a caught alates during this study. since reticulitermes spp. primarily swarm during daylight hours, no native termite alates were captured during this study. due to limited alate catches, no statistical analyses were performed. alates of formosan subterranean termites were captured at three of the sites sampled. site a had multiple alate captures among two traps, while only one alate was caught on fig 6. the predicted proportion of stumps infested in a plot based on site and stump density (the total number of stumps in the plot). each of the other two sites. formosan subterranean termite alates have the capability to disperse distances of nearly 900m in open areas (messenger & mullins, 2005); however, other studies have shown flight distances of 100m to be more common (higa & tamashiro, 1983). although it is unknown if formosan subterranean termite colonies were located on the inspection sites where alates were caught, it is likely that colonies were present within close proximity if common dispersal distances are taken into account. additionally, since two sites only had an individual alate captured, it is possible that these alates dispersed a greater distance than the site with multiple alate captures. multiple alates were captured on two different traps at site a, therefore it was likely that these traps were closer to parent colonies than the traps at other sites. it is also possible that site a contained a larger colony or greater number of colonies than the other sites. site d, which was located in a very rural location and completely surrounded by forest, only captured a single alate. the site was over 1.6km (1-mile) away from the nearest house or residential area. this increases the likelihood that the alate dispersed from an established colony in a forested area. formosan subterranean termite populations may not currently be high enough in forested settings for easy detection, but our capture of them at secluded sites indicates that colonies may already be established in localized forested areas. there are multiple plausible reasons as to why no formosan subterranean termites could be located during physical stump inspections in our study. formosan subterranean termites are known to commonly utilize living trees in urban areas (osbrink et al., 1999). urban environments, in general, contain far less downed woody debris than most forested areas. less woody debris would concentrate subterranean termites into fewer resources, making them easier to detect. soil moisture levels were also reduced during the hot summer months, which may have caused subterranean termites to travel deeper into the soil to prevent desiccation, thus avoiding detection. ns little et al utilization of woody resources by the formosan subterranean termite204 additionally, the trees on the sites inspected were mature prior to harvest, possibly limiting the import of any significant amount of infested woody debris for decades. given the propensity of formosan termites for attacking living trees in urban trees in the u.s. and plantation forestry settings in other parts of the world, additional research is needed to ensure that formosan subterranean termite populations do not pose a threat to native forests. more forested sites need to be inspected for the presence of formosan subterranean termites, utilizing stricter site criteria such as distance to railroads and major roadways, documentation of previous alate trap catches, and proximity to urban areas. imposing these additional requirements on inspection sites should increase the probability of locating formosan subterranean termites in forested areas. however, due to the difficulty of locating potential sites, it may take several years to adequately complete the investigation. continuation of alate monitoring within forested and rural areas is also very important as data collected can be useful in monitoring alate populations and range expansion. further confirmation of formosan subterranean termite establishment in localized forest settings would also raise the importance of determining feeding preferences and the likelihood of native subterranean termite displacement in forests, issues this study was not able to address. we found no formosan subterranean termites during physical stump inspections; however, only pine plantations were inspected during this study. hardwood bottoms and older forests with less active management and higher proportions of downed woody material (dwm) may harbor formosan subterranean termites. furthermore, living trees might only be infested once populations in dwm are high enough and competition for woody resources is strong, a possibility that should be explored in future research. it is still debatable why more alates were caught in rural areas than in forested settings by sun et al. (2007). formosan subterranean termites may utilize dwm in rural forested areas. another possibility is that light pollution may decrease alate trap efficacy in urban areas. as a consequence, traps placed in urban settings may underestimate alate numbers. at the conclusion of this study, native reticulitermes spp. were found in recently harvested timber stumps at the eleven sites inspected, but the introduced formosan subterranean termite was not found. however, formosan subterranean termites were located within the general area of 3 sites, as evidenced by the alate trap catches and proximity to homes. due to lack of findings on inspected sites, feeding preference data for formosan subterranean termites could not be collected, and native reticulitermes spp. displacement is not a current problem within the study areas. stump inspection data did reveal that reticulitermes spp. occurred in a higher percentage of pine stumps than in hardwoods, but stump type did not significantly affect the number of occurrences per site. the proportion of stumps infested by native subterranean termites was negatively correlated with the number of stumps on a given plot. it is important to note that formosan subterranean termites could be impacting forested stands in other locations, even though they were not located on the tracts inspected during this study. with rising populations, the invasive formosan subterranean termite could still pose a major ecological threat to forested settings and native termite species, issues that need to be addressed with continued research. acknowledgments the authors would like to thank jared seals, kevin chase, brady self, and zach senneff for their contributions to this research project. their help was instrumental in achieving the objectives of this study. this research was funded by the usda, forest service, forest health protection and southern research station, and the mississippi agricultural and forestry experiment station. the authors would like to thank the mississippi forestry commission and weyerhaeuser for providing vital assistance in locating and accessing research sites for this study. references avery, t. & burkhart, h. 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(2006). nitrogen fixation by termites in tropical forests, thailand. ecosystems, 9: 75-83. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2895sociobiology 65(3): 433-440 (september, 2018) ants (hymenoptera: formicidae) attracted to rabbit carcasses in three different habitats introduction the combined weight of all living ants has been estimated to constitute half the mass of all extant insects (sleigh, 2003) and individual ants outnumber most other terrestrial animals (taylor, 2007). ant species constitute the largest family in the order hymenoptera, which, in itself, is the third most numerous order of insects after diptera (true flies) and coleoptera (beetles) (taylor, 2007). ants are one of the most successful groups of insects, exploiting a variety of habitats around the world (sleigh, 2003) and are absent from only a few areas, even able to exist in extreme arid and hot desert environments. for example, species of the genus cataglyphis have developed very long legs so as to lift their abstract this study reports the ant species that were attracted to rabbit carcasses in three different habitats (agricultural, desert, urban) in the city of riyadh, saudi arabia from may to july 2014. rabbit carcasses were used as a model for decomposition. carcasses were categorized as exposed, clothed, shaded and burnt. a total of 726 ants belonging to three subfamilies and 14 species were collected during the decomposition process. trichomyrmex mayri (forel) was the only ant species attracted to the carcasses placed in the desert site. at the agricultural site, there was one ponerine species, five formicine species, and three myrmicine species were attracted, while at the urban site, five formicine species and one myrmicine species were recorded. the agricultural site attracted the highest number of ants. in contrast, the desert site attracted the lowest number of ants. tr. mayri was the most prevalent species occurring in both the agricultural and desert sites. the bloated stage of decomposition attracted the highest number of ants followed by the decay, fresh and dry stages, respectively. clothed carcasses attracted the highest number of ants followed by the exposed and burnt carcasses, respectively. the shaded carcasses attracted the fewest number of ants. this study found that ants are attracted to carcasses at all stages of decomposition and are common components of the necrofauna of central saudi arabia. sociobiology an international journal on social insects ashraf mashaly1, 3, mostafa sharaf2, medghom al-subeai1, fahd al-mekhlafi, abdulrahman aldawood2, gail anderson4 article history edited by evandro n. silva, uefs, brazil received 14 february 2018 initial acceptance 31 march 2018 final acceptance 02 june 2018 publication date 02 october 2018 keywords ants, decomposition, forensic entomology, riyadh, saudi arabia. corresponding author ashraf mashaly department of zoology, college of science p. o. box 2455, king saud university riyadh 11451, saudi arabia. e-mail: mmashely@ksu.edu.sa bodies up off the substrate, together with hairs which reflect light, allowing them to cope with air temperatures as high as 55 °c (keller & gordon, 2009; shi et al., 2015). ants can have an impact on overall carrion ecology. catts and haskell (1990) reported that ants may feed directly on carcasses or prey on other arthropods associated with carcasses. ants can also accelerate the decomposition process by producing abrasions or injuries facilitating the attraction of other species of necrophagous insects (gunn, 2006). sometimes, these ants cause lesions that can be misinterpreted as premortem mutilations, which can lead to errors in forensic analyses (moretti & ribeiro, 2006; campobasso et al., 2009; lindgren et al., 2011; moretti et al., 2011). for example, the red imported fire ant, solenopsis invicta (buren) can 1 department of zoology, college of science, king saud university, riyadh, saudi arabia 2 plant protection department, college of food and agriculture sciences, king saud university, riyadh, saudi arabia 3 department of zoology, faculty of science, minia university, el-minia, egypt 4 school of criminology, simon fraser university, university drive, burnaby, bc, canada research article ants ashraf mashaly et al. – ants attracted to rabbit carcasses in three different habitats434 cause damage to tissue that could be mistaken for pre-mortem burns (byrd & castner, 2001). in some instances, initial colonization of dipterans or other organisms on carcasses may be delayed by two to three days due to the high predation rate of fly eggs by ants (byrd & castner, 2001); these instances appear more common if the carcass is located close to an ant nest. for example, acrobat ants of the genus crematogaster feed on immature stages of many flies. the literature shows that there are a number of factors that may affect insect colonization of carcasses, including habitat, the presence or absence of clothing, sun exposure and whether the carcass has been burned. clothing may affect insect diversity, abundance, and feeding activities and consequently may increase the decomposition rate, by offering protection from ambient conditions and predators (mann et al., 1990; dillon, 1997; campobasso et al., 2001; card et al., 2015). it has been reported that carcasses exposed to the sun decompose faster than ones in the shade, as well as exhibiting different patterns of insect succession (joy et al., 2006; sharanowski et al., 2008; castro et al., 2011). heo et al. (2009) studied ant succession in malaysia and noted differences in species attracted to burned and unburned pig carcasses. in saudi arabia only one published study recorded ants attracted to the carcasses, where shaalan et al. (2017) recorded pheidole megacephala (f.) in al-ahsaa oasis, in the eastern region of the kingdom. hence, the importance of the present article is that it is the first to provide a qualitative assessment of the major ants taxa attracted to decomposing carcasses placed in an agricultural area, a typical desert site, and an urban site, in the city of riyadh using four carcass treatments, namely, exposed, clothed, shaded and burnt. material and methods study sites the study was conducted in three different habitats: an urban area (24°43’40.89” n, 46°36’54.92” e) located within 200 m of human dwellings; an agricultural area (24°44’36.54” n, 46°33’45.12” e,) with many palm trees (phoenix canariensis chabaud) and typical grasses (setaria verticillata l.), where the nearest human dwelling was approximately 1 km from the study site, and a desert site (24°44’36.54” n, 46°33’45.12” e) with a substrate made up primarily of hard packed dry rock, where the nearest human dwelling was approximate 4.5 km away. the experiment was repeated three times during may to july 2014. temperatures (fig 1) were recorded using a lascar el-usb-2 data logger. each data logger was attached to the top of a 1 m vertical post at the centre of each of the experimental sites. carcasses most carrion research utilizes pig (sus scrofa l.) carcasses, but pigs cannot be used in ksa. therefore, thirtythree (n = 33) live mature rabbits, oryctolagus cuniculus (l.), were obtained. each animal was euthanized using chloroform inhaled in a chamber. the treatment of the rabbits followed the regulations of the animal research and ethics committee (fu-care). several studies in ksa and other countries have used rabbit carcasses to study the process of decomposition and insect succession; for example, tantawi et al. (1996); abou zied (2014); silahuddin et al. (2015) and shaalan et al. (2016). each rabbit weighed 1.57 ± 0.44 kg. at each of the agricultural and urban sites, twelve rabbits were divided into three groups of four. at the desert site, nine rabbits were divided into three groups of three, since the experiment in shaded conditions was excluded from the desert habitat. at each study site, rabbit carcasses were arranged in a regular, symmetrical pattern of three parallel rows, with four carcasses in each row, with a 2 m space between rabbits. three rabbit carcasses (one from each row) were partially burned at the site using wood without accelerant to a level 2 on the crowglassman scale (glassman & crow, 1996). three carcasses were clothed in a white cotton short-sleeved clean t-shirt to simulate a clothed body. three rabbit carcasses were placed in a shaded area under a tree which was 2 °c cooler than in direct sun and three rabbits were exposed, with no additional treatment. each carcass was placed inside a steel cage (55cm x 40cm x 24cm) to exclude scavengers. the cages did not have a bottom, allowing the carcasses to be in direct contact with the ground. all carcasses were monitored from initial exposure to almost complete skeletonization. four stages of decomposition were recognized following payne (1965), gennard (2007) and abouzied (2014), namely fresh, bloated, active decay, and dry remains. sampling and identification each carcass was examined for 10 minutes hourly from 9 am to 4 pm, for the first three days, then daily, at 9 am from the fourth day after death, until the carcasses were almost completely skeletonized. ants were collected with forceps (for the running ants around the carcasses) from all parts of the rabbit carcasses including underneath fig 1. summary of the temperature data at the study sites over the duration of the experiment (may to july 2014). sociobiology 65(3): 433-440 (september, 2018) 435 the carcases. pitfall traps were also used to reduce carcass disturbance and to continue to monitor beyond the active collection times. collection was carried out without depleting the ants on the carcass so as to avoid any impact on the next collection. all ant specimens were preserved in 70% ethanol. the specimens were identified by the second author (m. r. sharaf) using collingwood (1985), hölldobler and wilson (1990), bolton (1994) and collingwood and agosti (1996). voucher specimens of each species are deposited at the king saud university museum of arthropods, king saud university, riyadh, saudi arabia. statistical analysis significant differences in diversity (as quantified by the number of ant species) among habitats, decomposition stages and carcass status was evaluated using the kruskalwallis test (minitab 2017). results in our study, the rabbit carcasses attracted different species of ants during the decomposition stages. the carcasses in the desert and urban sites progressed from the fresh to the dry remains stage in six days and in approximately 12 days at the agricultural site (fig 2). ants were observed feeding on immature insects on the rabbit carcasses. ants were attracted significantly to the carcasses at the agricultural site (454 ants) than the urban and desert sites, with 165 ants and 106 ants, respectively. the abundance of ants varied between treatments and sites. by far, the greatest number of ants were collected from the carcasses at the agricultural site and ants were present in all decompositional stages on the clothed and exposed carcasses and in the fresh, bloat and decay stages on the shaded and burnt carcasses, but were not present during the dry remains stage of these treatments. at the desert site, ants were much rarer in general. the urban site was the only one of the three in which ants were collected in all decay stages and on all treatments, although far fewer were collected than in the agricultural habitat. table 1 presents a summary of ant species collected at the three different sites from the rabbit carcasses. only one species, trichomyrmex mayri (forel) (myrmicinae) was collected from carcasses placed at the desert site. nine species of ants belonging to three subfamilies were found on carcasses placed at the agricultural site, brachyponera sennaarensis (mayr), camponotus aegyptiacus (emery), c. maculatus (fabricius), c. sericeus (fabricius), c. xerxes (forel), cataglyphis semitonsa (santschi), pheidole sp., tr. mayri, and tetramorium lanuginosum (mayr). at the urban site, six species were attracted, c. aegyptiacus, c. sericeus, ca. holgerseni (collingwood and agosti), ca. livida (andré), ca. savignyi (dufour) and monomorium venustum (smith). post-mortem interval (days) habitat 1413121110987654321 agricultural urban desert drydecaybloatedfreshkey: fig 2. the duration of the decomposition stages in the three different habitats. ant species were very habitat specific, with only tr. mayri observed in two different sites (agricultural and desert). at the agricultural and urban sites, ants were attracted to all decomposition stages, but at the desert site, ants were active only during the bloated and decay stages. also, at the agricultural and urban sites, ants were attracted to all types of carcasses although, at the urban site, ca. livida was the only ant species attracted to all types of carcasses and during all stages of decomposition. at the agricultural site, tr. mayri was attracted in high numbers followed by t. lanuginosum, c. sericeus and c. xerxes. cataglyphis holgerseni, ca. livida, ca. savignyi and m. venustum were not attracted to the carcasses at this site (fig 3). at the desert site, tr. mayri was the only ant species attracted in large numbers in comparison with other species at the other sites (fig 3). at the urban site, ca. savignyi was fig 3. abundance of ants according to the habitat in riyadh, saudi arabia from may to july (2014). ashraf mashaly et al. – ants attracted to rabbit carcasses in three different habitats436 fig 4. abundance of ants according to the decomposition stage of rabbit carcasses placed at the three habitats in riyadh, saudi arabia from may to july (2014). attracted in significantly higher numbers than the other ants, followed by ca. livida, ca. holgerseni and m. venustum. camponotus aegyptiacus was attracted significantly lower numbers that the other species (fig 3). the species richness of ant species showed significant difference among the three habitats (kruskal-wallis test, table 2). in the fresh stage, all ant taxa were represented except c. xerxes, ca. savignyi and m. venustum. tetramorium lanuginosum was the most abundant species attracted to the fresh stage. in the bloated stage, only ca. semitonsa was not attracted. in the bloat stage, trichomyrmex mayri was the most abundant species collected (fig 4). in the active decay stage, all of the ant taxa reported here were present, with the exception of ca. holgerseni. camponotus xerxes and c. aegyptiacus were attracted in a higher numbers in this stage. subfamily species occurrence of ants / agricultural habitat fresh bloated decay dry e c s b e c s b e c s b e c s b ponerinae brachyponera sennaarensis √ √ √ formicinae camponotus aegyptiacus √ √ √ √ camponotus maculatus √ √ √ camponotus sericeus √ √ √ √ √ camponotus xerxes √ √ √ cataglyphis semitonsa √ √ √ myrmicinae pheidole sp. √ √ √ √ √ √ √ trichomyrmex mayri √ √ √ √ √ √ √ tetramorium lanuginosum √ √ √ √ urban habitat formicinae cataglyphis livida √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ cataglyphis holgerseni √ √ √ √ √ √ √ √ cataglyphis savignyi √ √ √ √ √ √ camponotus sericeus √ √ √ √ √ camponotus aegyptiacus √ √ √ √ √ √ myrmicinae monomorium venustum √ √ √ desert habitat myrmicinae trichomyrmex mayri √ √ e: exposed, c: clothed, s: shaded, b: burnt √: presence of ant table 1. ant attracted to the rabbit carcasses in three different habitats. comparison kruskal-wallis test statistic d.f. p-value notes no. of ant species by habitat 9.57 2 0.008 * no. of ant species by decomposition stage 6.74 3 0.081 ns no. of ant species by carcass status 3.74 3 0.291 ns *; significant, ns; not significant (α = 0.05). table 2. kruskal-wallis test results. in the dry stage, only six ant species were still present, c. sericeus, ca. livida, ca. savignyi, ca. semitonsa, pheidole sp. and tr. mayri, the latter being the most abundant (fig 4). the kruskal-wallis test indicated that there were no significant differences in the number of ant species between the different stages of decomposition (table 2). the exposed and clothed rabbit carcasses attracted the greatest number of ants (237 and 289, respectively). in contrast, the burnt and shaded rabbit carcasses attracted relatively few ants (98 and 101, respectively). all species sociobiology 65(3): 433-440 (september, 2018) 437 of ants were attracted to the exposed carcasses (control and clothed carcasses), except c. maculatus, c. xerxes, ca. savignyi and m. venustum. brachyponera sennaarensis, c. maculatus, c. sericeus and ca. semitonsa were not apparently attracted to the clothed carcasses (fig 5). only c. maculatus, c. sericeus, ca. livida, ca. savignyi and m. venustum were attracted to the shaded carcasses. the burnt carcasses attracted c. aegyptiacus, c. sericeus, c. xerxes, ca. livida, ca. savignyi, pheidole sp. and tr. mayri. brachyponera sennaarensis and ca. semitonsa were present only on exposed carcasses and c. maculatus was only attracted to shaded carcasses (fig 5). also, no significant difference in the number of ant species was indicated among the different carcass status using the kruskal-wallis test, table 2). we did not observe ants feeding directly on the carcasses, but rather, feeding only on the immature stages of flies (lucilia sericata meigen, musca domestica l. and sarcophaga hirtipes wiedemann) during the succession process, as has also been reported in the studies of campobasso et al. (2004), heo et al. (2009), nazni et al. (2011) and chen et al. (2014). smith (1986) stated that, habitat and ambient temperatures are important factors that determine the composition and structure of carrion fauna. in our study, high air temperatures affected the rates of decomposition at all study sites. almost complete decomposition of the rabbit carcasses took six days in the desert and urban sites and about 12 days at the agricultural site. similar results were reported by mashaly (2016). anderson (2009) indicated that the length of time it takes for decomposition to complete is very variable, depending on a number of parameters, including geographical region. abou zied (2014) conducted a decomposition study in the southwestern mountains of ksa and found that the rate of decomposition of rabbit carcasses was affected by high temperature (about 30 °c). in kuwait, al-mesbah et al. (2012) illustrated that carcasses at an urban site were found to decompose significantly faster than in the agricultural, coastal or desert sites (as measured by percentage weight loss). throughout the course of the study, a total of 726 ants were identified as being attracted to the rabbit carcasses. the agricultural site attracted a significantly higher number of ants compared to the desert and urban sites. al-mesbah et al. (2012) did not collect ants from rabbit carcasses at an urban site in kuwait. we recorded only one species of ant from the carcasses placed at the desert site, but nine and six species of ants from the agricultural and urban sites, respectively. this may be due to the high temperature and relatively barren substrate of the desert site. trichomyrmex mayri is the most prevalent species compared with other species in all habitats and this is consistent with sharaf et al. (2016), who said that t. mayri is the most widely-distributed species in the arabian peninusla. the differences in diversity and numbers of ants attracted to the carcasses in the three different habitats are probably due to the differences in biogeoclimatic zones or ecozones as each habitat was very different (anderson, 2009). conditions in the desert habitat are very extreme and only species highly adapted to the high temperatures and arid conditions are able to survive there. the ant species richness is sensitive to plant cover and diversity (morrison, 1998), soil type (peck et al., 1998). in our study, no differences were found in the rate of decomposition between the four types of exposure (exposed, shaded, clothed, and burned) during the study time. heo et al. (2008), gruenthal et al. (2012). mashaly (2016) reported that burning not to have an effect on the rate of decomposition but to have an effect on insect succession. beckerdite et al. (2014) found that burning chicken carcasses accelerated the decomposition process and affected the succession of insects on the bodies. clothes can increase the rate of decomposition fig 5. abundance of ants according to the condition if the carcasses placed at the three habitats in riyadh, saudi arabia from may to july (2014). discussion in forensic entomological studies, ants attracted to corpses are categorized as omnivorous species (tabor et al., 2005). ants can feed on both the corpse and associated fauna. luederwaldt (1926) reported collecting several species of ants from vertebrate carrion. in addition, smith (1986) reported that, in the context of forensic entomological analyses, ants are considered to be predators. throughout our study period, ants were observed actively preying upon the eggs, larvae and newly emerged flies of calliphoridae, muscidae and sarcophagidae. campobasso et al. (2009) and chen et al. (2014) reported similar observations. mashaly et al. (2013) stated that b. sennaarensis preferred proteinaceous food over other types of food. carpenter ants of the genus camponotus are generally considered to be predators (sanders & pang 1992), and others feed on live and dead arthropods (dejean, 1988). although sharaf et al. (2016) recorded workers of tr. mayri in areas rich in decaying organic matter. in our study, we recorded ants on the carcasses from shortly after placement during the early post-mortem period and in all the subsequent stages of decomposition, apart from the dry stage. ashraf mashaly et al. – ants attracted to rabbit carcasses in three different habitats438 and insect activity (mann el al., 1990; campobasso et al., 2001), although anderson (2009) said that clothes slowed the decomposition and the insect succession, whereas kelly et al. (2009) found no difference in the succession of insects between unclothed and clothed carcasses. castro et al. (2011) found a significant effect of the interaction between the decomposition stage and insolation regime. carcasses exposed to the sun decompose faster than the ones in the shade (shean et al., 1993; joy et al., 2006) and show different patterns of insect succession (sharanowski et al., 2008). anderson (2009) reported that clothing cadavers affects relative humidity and provides protection for some insects, which may increase insect abundance and diversity. in contrast, matuszewski et al. (2015) reported that clothing has little importance when considering insect abundance and diversity. also, ant species differ according to the condition of the carcass. some ant species were restricted by the treatment types. for example, b. sennaarensis and ca. semitonsa were only attracted to the exposed carcasses and c. maculatus only to the shaded carcasses. heo et al. (2009) reported six species of ants associated with pig carcasses placed on the ground. oecophylla smaragdina (fabricius) was collected only from the burned carcass and was found throughout decomposition, tetramorium sp. was only collected from the burned carcass but only in the dry remains stage, whereas odontoponera sp., diacamma sp. and anoplolepis gracilipes f. smith were specific to the unburned carcass; a. gracilipes was found only in the fresh and bloated stages and odontoponera sp. and diacamma sp. only in the fresh and dry remains stage. pheidologeton sp. was the only species found on both burned and unburned carcasses, primarily in the dry remains stage (heo et al., 2009). as in our study, heo et al. (2009) reported that the ants predated on fly larvae. kolver (2009) stated that ants preyed on the fly eggs, fly larvae and adult beetles, but also fed on the burnt skin and soft tissue of the burnt carcasses, causing feeding damage in the form of a characteristic pitted appearance formed by the removal of minute portions of tissue. in conclusion, this is the first study to report the species of ants that are attracted to rabbit carcasses exposed in four different conditions (exposed, clothed, shaded and burnt) and in three different sites (agricultural, desert and urban) in riyadh, ksa. the study recorded a total of 726 ants belonging to three subfamilies and 14 species. the agricultural site attracted the highest number of ants. trichomyrmex mayri was the most abundant species in both the agricultural and desert sites. decomposition stage or the carcass status did not effect on the number of ants attracted. acknowledgements the authors extend their appreciation to the deanship of scientific research at king saud university for funding the work through the research group project no. rgp028. the authors are indebted to dr. boris kondratieff (colorado state university) for valuable comments. references abou zied, e.m. 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(2011). decomposition and insect succession of clothed and unclothed carcasses in western australia. forensic science international, 211: 67-7 5. doi: 10.13102/sociobiology.v66i1.3401sociobiology 66(1): 186-189 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a scientific note on a stingless bee hive model for ecological and behavioral studies and for environmental education introduction stingless bees are among the most important pollinators in the south american tropics (duarte et al., 2016). they occupy a wide variety of habitats and are remarkably diverse in hot tropical-subtropical regions. all known meliponini bee species are eusocial and live in sessile colonies. most species build their nests in tree hollows, though some build exposed nests or occupy hollows in the ground or within abandoned ant or termite nests (nogueira-neto, 1997; camargo, 2007). suitable nesting sites and building resources are crucial and limiting factors for stingless bees occurrence, especially in habitats that have been altered by man (sakagami, 1982; roubik, 1989). the most widely used hive models in brazil and mesoamerica can be categorized into horizontal and vertical models (leão et al., 2016). the horizontal model, the most traditional, is usually non-modular, without any internal divisions, although more elaborate options that have internal abstract stingless bees (apidae, meliponini) occupy a broad range of habitats in the tropical and subtropical regions of the world. they are eusocial and live in sessile colonies. most meliponini species build their nests in pre-existing cavities, such as tree hollows. current stingless bee hive models imitate the conditions of natural nests. however, they are not convenient for scientific studies, especially those focusing on ecological and behavioral characteristics. we developed and tested a hive model that ensures clear visibility of the interior of the hive, facilitating ecological and behavioral studies and environmental education. our new model was successfully used to house and maintain ten stingless bee species and one semi-social orchid bee species. sociobiology an international journal on social insects c barbieri1,2, gl pinheiro2, pm drago1, tm francoy1 article history edited by kleber del-claro, ufu, brazil received 01 may 2018 initial acceptance 28 june 2018 final acceptance 24 july 2018 publication date 25 april 2019 keywords meliponini, nesting biology, entomology, beekeeping, apidae, meliponiculture. corresponding author tiago mauricio francoy escola de artes, ciências e humanidades universidade de são paulo – usp rua arlindo béttio, 1000, bloco a1, sala t10l cep 03828-000, são paulo-sp, brasil. e-mail: tfrancoy@usp.br divisions to separate the food storage pots from the brood (nogueira-neto, 1997; sommeijer, 1999). this kind of model allows one to view the inner part of the hive. the vertical models follow the natural nest organization of most meliponini species. their popularity is growing among stingless bee beekeepers (venturieri, 2008). they usually have detachable modules, such as the fernando oliveira model (oliveira & kerr, 2000). this model type has many derivations created according to personal preferences; it is organized to separate the base chamber, which contains the brood cells, from the management modules above the nest, where the food storage pots usually are placed. most experienced meliponists build and test various hive models, and they adapt their management practices according to their observations (jaffé et al., 2015). unfortunately, the most widespread hive models are not convenient for scientific studies. here, we present a hive model that allows behavioral and ecological studies, developed by gerson luiz pinheiro, co-author of this paper and member of the ngo sos abelhas sem ferrão (stingless bees). 1 escola de artes, ciências e humanidades, universidade de são paulo, sp, brazil 2 sos abelhas sem ferrão, são paulo-sp, brazil short note sociobiology 66(1): 186-189 (march, 2019) 187 this hive model was developed in 2014 for environmental education purposes. the most relevant aspect of this model is its concept; the dimensions vary since each species needs different cavity sizes (table 1). table 1. bee species raised in the sos stingless bee hive model. bee species time in the hive (months) colonies (n) hive internal measurements (cm) friesella schrottkyi 39 4 10 x 10 x 10 frieseomelitta varia 36 3 15 x 15 x 15 leurotrigona muelleri 4 3 6 x 6 x 8 melipona marginata 36 2 15 x 15 x 15 melipona quadrifasciata 25 5 20 x 20 x 20 plebeia droryana 15 2 15 x 15 x 15 plebeia nigriceps 42 2 15 x 15 x 15 plebeia remota 36 6 13 x 13 x 13 scaptotrigona postica 14 2 20 x 20 x 30 tetragonisca angustula 24 5 15 x 15 x 15 euglossa sp. (a communal species) 7 1 15 x 15 x 15 material and methods the materials used to build this hive model are: 1 wood boards, at least 1 cm thick; 2 transparent acrylic or polycarbonate panels, 3 mm thick; 3 eva (ethyl vinyl acetate) layer, 3mm thick; 4 bolts; 5 a pressure closure; 6 wooden knob; 7 hinges 8 small wooden strips the basic structure of the hive model is made with the wood boards and then bolted. the pressure closure, hinges, feet and knob are also bolted. the eva protection layer is attached to the movable wall and roof cover using pva (polyvinyl acetate) glue due to its vulcanization properties. the observation window is attached to the beehive by embedding it in a low-relief cutout and keeping the hive closed for a few days. the bees will seal the observation window to the hive themselves, using propolis. the hive model is composed of 11 main parts: a) walls; b) feet; c) hive entrance; d) pressure closure; e) movable wall; f) protection layer; g) roof cover; h) knob; i) observation window; j) feeder support; k) hinges; (figure 1). fig 1. sos abelhas sem ferrão (stingless bees ) hive model (illustrations by paula drago). c barbieri, gl pinheiro, pm drago, tm francoy – new hive model for ecological and behavioral studies.188 the choice of materials was based on various trials and practical observations. explanations on the use of materials and the functions of the parts: a) walls (and the basic structure): we used pine wood. however other untreated wood types can be used. b) feet: we used two identical wooden strips below the hive to reduce the contact between the hive and the surface where it is installed. c) hive entrance: the central hole allows the bees to enter and exit the hive. the entrance size can be changed to accommodate species of various sizes. the low-relief halo is for attaching protection against ants and lizards, or an exit tunnel made with a plastic tube. the exit tunnel permits the bees to leave the hive to forage outside even when the hive is kept indoors. in order to transport the colonies from place to place, the entrance must be sealed in the previous night. d) pressure closure: the pressure closure seals the hive efficiently against light by pressing the movable wall against the observation window. we do not recommend other types of closure, since they may not block the external light entirely even with the protection layer. the pressure closure prevents light from entering the hive. consequently, the bees do not propolize the observation window, allowing clear visibility of the hive, when the movable wall is hinged open. e) movable wall: an articulated wall of the hive connected with hinges and a pressure closure. a protection layer is attached to the movable wall’s internal side. f) protection layer: the recommended material is eva, since it protects the observation windows from mechanical shocks; its opacity and flexibility blocks the light when the movable wall is appropriately closed. g) roof cover: protects the hive against weather conditions, such as rain, hail and sunlight. the cover’s protection layers also reduce mechanical shocks against the upper observation window. since the roof cover is not in contact with the bees, it is not coated with propolis and therefore not glued to the rest of the hive. h) knob: facilitates removal of the roof cover. i) observation window: the best materials are transparent acrylic or polycarbonate; they are thinner and more resistant than glass, and easier to clean. when the observation window is excessively covered with propolis, it can be replaced, and the “dirty” window can be cleaned with a prolonged soak in water or alcohol. the bees attach the observation window to the structure of the beehive by themselves, by sealing the sides with propolis. j) hive feeder support: made with wooden strips. k) hinges: allows the movable wall to open and close when necessary. measurements: we adopted various measurements for the hive model, according to bee species particularities (table 1). the most relevant measurements are the internal size and wood thickness. the internal size changes according the requirement of each species, such as brood chamber volume, the volume of stored food and others. the wood thickness varies according to species thermoregulation and humidity necessities. we recommend thicker wood in regions with larger temperature variation or lower average temperatures. we transferred all the meliponini colonies from trap-nests or other hive models. the only one that was spontaneously colonized was the hive with euglossa sp. fig 2. closed hive (photo by andré matos). results we successfully used this hive model for ten meliponini species and one communal euglossini species (table 1). all the colonies developed well and successfully occupied the hives without problems. all the colonies were used in environmental education activities about once a week, and no damage to colony health was noticed. this hive model allows excellent visibility of the interior of the hive (figures 2 and 3). fig 3. internal view (photo by andré matos). sociobiology 66(1): 186-189 (march, 2019) 189 this hive model has an alternative version with two movable walls and three observation windows. this variation allows other observation angles and use of light for photography and studies (figure 4). it is a good option for entomological studies, bee behavior and ecological research and environmental education. discussion our new hive model offers a good view of the interior of the nest and does not interfere with the natural development of the colonies. current horizontal models allow clear visualization of the interior of the nest; however, they result in unnatural colony component placement for most species, which may affect colony biology and behavior. some species, such as melipona marginata and melipona subnitida develop well in the horizontal models (celso barbieri & gerson luiz pinheiro, personal observations), but the concept of our model can easily be adapted for these particular cases. in addition to the advantages for scientific and educational use, our model facilitates colony management as it gives access to the inter area of the nest from two or more sides, which is not possible with traditional models. acknowledgements dr david de jong has kindly helped us to improve the english of our manuscript. we thank the ngo sos abelhas sem ferrão (www.sosabelhassemferrao.com.br) for their support in this project. rights of use commercialization of this hive model is reserved for the non-profit organization sos abelhas sem ferrão (sos stingless bees). it can be produced for private use, research and education, except if it involves profit interests. fig 4. two movable walls version (photo by celso barbieri). contribution of authors celsobarbieri jr: collected data, wrote the paper paula marques drago: made the illustrations, wrote the paper gerson luiz pinheiro: developed and built the hives tiago mauricio francoy: helped detail the concept and wrote the paper references camargo, j. m. f. (2007). trigona spinipes. in j. s. moure, d. urban & g.a.r. melo (orgs.), catalogue of bees (hymenoptera, apoidea) in the neotropical region. curitiba, sociedade brasileira de entomologia, 1058p. duarte, r.s., souza, j. soares, a.e.e. (2016). "nest architecture of tetragona clavipes (fabricius) (hymenoptera: apidae: meliponini)." sociobiology, 63: 813-318. doi: 10.13102/ sociobiology.v63i2.1019. jaffé, r., pope n., carvalho a.t., maia u.m., blochtein b., carvalho c.a.l., carvalho-zilze, g.a., freitas, b.m., menezes, c., ribeiro, m.f., venturieri, g.c. & imperatriz fonseca, v.l. (2015) bees for development: brazilian survey reveals how to optimize stingless beekeeping. plos one, 10: e0121157. doi: 10.1371/journal.pone.0121157 leão, k.l., queiroz, a.c.m., contrera, f.a.l.,veiga, j.c., venturieri, g.c. (2016). “colony development and management of the stingless bee scaptotrigona aff. postica (apidae: meliponini) using different hive models.” sociobiology, 63: 1038-1045. doi: 10.13102/sociobiology.v63i4.1041. nogueira neto, p. (1997). vida e criação de abelhas indígenas sem ferrão. são paulo: nogueirapis, 445p. http://eco.ib.usp. br/beelab/pdfs/livro_pnn.pdf oliveira, f. & kerr, w.e. (2000). divisão de uma colônia de japurá (melipona compressipes manaosensis) usando-se uma colmeia e o método de fernando oliveira. manaus: instituto nacional de pesquisas da amazônia, 10p roubik, d. w. (1989). ecology and natural history of tropical bees. cambridge university press, 514p sakagami, s.f. (1982). stingless bees. pp. 361-423 in: herman h r (ed.). social insects. academic press, new york. venturieri, g.c. (2008). caixa para a criação de uruçu amarela melipona flavolineata friese, 1900. comunicado técnico embrapa amazônia oriental, 212: 1-8. https://www.infoteca. cnptia.embrapa.br/bitstream/doc/409428/1/com.tec212.pdf doi: 10.13102/sociobiology.v65i2.2742sociobiology 65(2): 340-344 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ants that frequently colonize twigs in the leaf litter of different vegetation habitats in tropical forests, the leaf litter harbors a number of resources for ant nesting (hölldobler & wilson, 1990), including twigs derived from fallen tree branches. this resource is colonized by different ant species, supporting the presence of workers, reproductives and brood (souza et al., 2012; fernandes et al., 2012), albeit for a short period of time (byrne, 1994). ant species that colonized 10 or more twigs in 2,880 m2 of leaf litter in the amazon forest were considered “common inhabitants” of this type of resource by carvalho and vasconcelos (2002). however, there are no studies examining these communities in the atlantic forest domain. therefore, we analyzed the composition of “common inhabitants” and the diameter of colonized twigs in different vegetation habitats. specifically, we tested if any species showed preference for a certain twig diameter, regardless of habitat. samples were collected from urban parks (n = 9), eucalyptus plantations with a developed understory (n = 9) and native forest sites (atlantic forest; n = 9); all sites were situated in the state of são paulo, brazil (fig 1). at each site, six 16 m2 leaf litter plots were established 50 m apart along a linear transect. all twigs occupied by ant colonies were abstract ants often colonize twigs in the leaf litter, but some species use this resource more frequently than others. we analyzed the composition of the community and the diameter of colonized twigs to test if any species had a size preference. samples were collected in different vegetation habitats (urban parks, eucalyptus plantations and native forests). in each site, all twigs with an ant colony in six 16-m2 plots were collected and measured, and the ants occupying them were identified. for the analyses, we only included species recorded in 10 or more twigs; these species were considered “common inhabitants” of the twigs (approximately 19.7% of the fauna analyzed). our results indicate that the community is richer and uses a larger number of twigs of different diameters in the native forest. in addition, some species colonized twigs of similar sizes in different vegetation habitats, suggesting possible selection by ants. sociobiology an international journal on social insects tt fernandes¹, dr souza-campana¹, rr silva2, msc morini¹ article history edited by jacques delabie, uesc, brazil received 11 december 2017 initial acceptance 15 january 2018 final acceptance 26 march 2018 publication date 09 july 2018 keywords atlantic forest, urban fragment, diversity, nesting, understory. corresponding author maria santina castro morini laboratório de mirmecologia do alto tietê núcleo de ciências ambientais (nca) mogi das cruzes-sp, brasil. e-mail: morini@umc.br collected, and their size was described by three diameter measurements, one at each end of the twig and one in the middle. diameter has been identified as the variable that is most strongly associated with ant species richness in twigs (carvalho & vasconcelos, 2002; souza-campana et al., 2017) or with events of bamboo traps occupation by arboreal ants (cobb et al., 2006). ants were identified based on suguituru et al. (2015). ant vouchers of all the samples were deposited at laboratório de mirmecologia do alto tietê (myrmecology laboratory of alto tietê) of university of mogi das cruzes (são paulo, brazil). for the analyses, we only included ant species that were “common inhabitants” as defined by carvalho and vasconcelos (2002). first, we used a kruskal-wallis test, followed by dunn’s post hoc test, to analyze if the “common inhabitant” species occupied twigs of different diameters within each habitat (i.e., within-habitat comparisons of the local species pool). next, we used a mann-whitney test to examine if each species (i.e., a common inhabitant) was occupying twigs of different diameters in different vegetation habitats (betweenhabitat comparisons of each “common inhabitant” species). the significance level used for both tests was 5%. 1 laboratório de mirmecologia do alto tietê (lamat), núcleo de ciências ambientais (nca), mogi das cruzes-sp, brazil 2 museu paraense emílio goeldi, coordenação de ciências da terra e ecologia. belém-pa, brazil short note sociobiology 65(2): 340-344 (june, 2018) 341 we collected 496 twigs (0.19 twigs/m2) in 2,592 m2 of leaf litter, which were colonized by ants belonging to 60 species/morphospecies. among them, only 12 species (19.7%) were considered “common inhabitants” (table 1), being found in 274 twigs (0.10 twigs/m2 of leaf litter). although most of the “common inhabitants” were leaf-litter ants (suguituru et al., 2015), 25% were arboreal species myrmelachista catharinae mayr, 1887, myrmelachista ruzskyi forel, 1903 and pseudomyrmex phyllophilus (smith, 1858) that may use the twigs to expand their colonies, forming polydomous nests (davidson et al., 2006; debout et al., 2007). our results show that different vegetation habitats strongly influence ant richness and the number of “common inhabitant” nests in twigs. none species was recorded in urban parks in 864 m2 of leaf litter; in the eucalyptus plantations, four species (33.3%) were observed in 57 twigs (= 0.06 twigs/ m2 of leaf litter); and in the native forest, 11 species (91.7%) in 217 twigs (= 0.25 twigs/m2 of leaf litter). other studies on ants that colonize twigs in native forest and agroecosystems (murnen et al., 2013; souza-campana et al., 2017) have also shown that the less the anthropogenic influence, for the best the ant diversity. in addition to differences in species richness and number of nests, we found differences in species composition among vegetation habitats (table 1). ant community diversity is limited in areas with few nesting resources (fowler et al., 1991). however, resource quality is also important, because decayed and hollow twigs with higher moisture content (carvalho & vasconcelos, 2002) and larger diameters (souzacampana et al., 2017) are colonized more often. in eucalyptus plantations, twigs are very rigid and dense (pereira et al., 2007), making it more difficult for “common inhabitant” species to colonize them; as a result, these communities are poorer (table 1). even when the eucalyptus plantations have a developed understory, the structure of the twigs is homogeneous (souza-campana et al., 2017). therefore, “common inhabitants” colonize twigs of similar diameters (kw = 3.9113; df = 3; p = 0.2712), in contrast with the native forest, where “common inhabitants” exploit a greater diversity of twig sizes (kw = 27.9747; df = 10; p = 0.0018), especially gnamptogenys striatula mayr 1884, linepithema neotropicum wild, 2007, pheidole sarcina forel, 1912 and solenopsis sp.2 (table 1). regarding the native forest, our results suggest that, in the atlantic forest, the “common inhabitants” colonize more twigs/m2 of leaf litter compared to the amazon forest, because we found 0.25 twigs/m2 of leaf litter, while carvalho & vasconcelos (2002) found 0.16 twigs/m2 of leaf litter. in addition, the “common inhabitants” in twigs collected by us may be considered richer (0.01 species/m2) compared to the amazon forest (0.006 species/m2), according to data from carvalho and vasconcelos (2002). our results also show that, compared to the amazon forest, the pool of species classified as “common inhabitants” in the atlantic forest is more diverse and colonizes more twigs, despite the presence of a lower number of species by m2 in the leaf litter. for instance, in the amazon forest, there is approximately 0.8 species/m2 (vasconcelos et al., 2000), but only about half of that in the atlantic forest, ca. 0.45 species/m2 of leaf litter (suguituru et al., 2013). this suggests that, in the atlantic forest, ants colonize more twigs due to a lower availability of other nesting resources. of all species recorded in 10 or more twigs from the study sites, only l. neotropicum, p. sarcina and pheidole sospes fig 1. study sites in different vegetation habitats: urban parks (n = 9), eucalyptus plantation with a developed understory (n = 9) and native forests (n = 9). tt fernandes, dr souza-campana, rr silva, msc morini – twig colonization by ants342 species/morphospecies urban parks eucalyptus plantations native forest number of nests twig diameter (mm) number of nests twig diameter (mm) number of nests twig diameter (mm) acanthognathus rudis 1 27.82 acanthognatus ocellatus 1 9.2 brachymyrmex admotus 7 13.49 (7.27-19.12) 9 13.8 (5.66-32.41) 3 11.56 (7.53-21.36) brachymyrmex heeri 5 17.56 (11.15-21.13) camponotus crassus 4 13.75 (11.05-14.97) camponotus novogranadensis 3 15.35 (12.72-42.82) camponotus sp.2 4 14 (9.41-21.68) camponotus sp.5 4 16.46 (14.69-32.44) 4 19.74 (15.85-33.55) camponotus sp.9 2 10.77 (7.6-13.94) camponotus sp.10 19 14.31 (10.26–17.18) a cardiocondyla wroughtonii 1 17.94 cephalotes pusillus 2 10.49 (9.43-11.55) crematogaster curvispinosa 6 11.56 (6.14-20.82) crematogaster rochai 1 14.86 crematogaster sp.1 1 8.2 6 11.58 (8.82-23.96) crematogaster sp.17 2 10.29 (7.18-13.39) crematogaster sp.18 2 19.61 (19.54-19.69) 3 12.28 (10.13-32.77) crematogaster sp.20 2 12.96 (11.4-14.52) gnamptogenys striatula 5 22.6 (17.04-29.13) 5 14.34 (10.32-22.1) 17 20.97 (9.76–46.19) ab heteroponera dentinodis 3 15.59 (14.38-22.74) heteroponera dolo 1 37.49 heteroponera mayri 2 9.38 (7.54-11.23) 6 15.48 (5.67-21.23) hylomyrma reitteri 1 20.33 hypoponera sp.4 1 11.98 1 19.88 hypoponera sp.7 2 17.45 (13.06-21.85) 5 12.69 (10.9-20.69) hypoponera sp.8 1 12.71 hypoponera sp.10 1 33.1 2 39.61 (37.02-42.21) linepithema iniquum 2 12.56 (12.13-12.98) 6 13.79 (7.53-21.43) 2 16.37 (16.19-16.55) linepithema leucomelas 1 6.8 linepithema neotropicum 2 21.56 11 13.18 (8.12–24.19) 18 20.88 (10.58–42.93) ab megalomyrmex goeldii 1 26.66 megalomyrmex iheringi 1 14.77 mycetarotes parallelus 1 28.9 1 26.29 myrmelachista catharinae 1 12.76 1 16.02 19 16.83 (6.36–25.98) a myrmelachista nodigera 1 5.14 myrmelachista ruzskyi 3 8.94 (6.91-18.35) 15 13.26 (6.23–17.76) a neoponera crenata 3 15.85 (13.56-17.41) 1 12.13 nylanderia sp.1 3 15.52 (10.65-15.52) 3 17.43 (11.19-22.93) pheidole cf. dione 1 24.37 pheidole flavens 1 13.52 11 11.68 (4.18–26.83) a pheidole sarcina 2 14.13 (12.13-16.14) 11 11.41 (6.1–26.79) 47 19.51 (7.87–42.52) ab pheidole sigillata 8 13.49 (7.66-19.69) 15 11.91 (9.29–22.50) a pheidole sospes 1 13.45 16 12.67 (6.86–24.28) 14 13.2 (6.79–33.89) a pheidole sp.9 3 30.03 (20.66-38.44) pheidole sp.37 1 17.44 table 1. taxonomic diversity based on vegetation habitat, number of colonized twigs, and twig diameter (median with range in parentheses). in bold, common inahbitants species. sociobiology 65(2): 340-344 (june, 2018) 343 table 1. taxonomic diversity based on vegetation habitat, number of colonized twigs, and twig diameter (median with range in parentheses). in bold, common inahbitants species. (continuation) forel, 1908 were found both in the eucalyptus plantations and the native vegetation (table 1). of these, only l. neotropicum (mann-whitney = 1.7401; p = 0.0818) and p. sospes (mannwhitney = 0.0985; p = 0.9215) colonized similar-diameter twigs in different vegetation habitats (fig 2), although the leaf litter in the native forest shelters a higher twig diversity (murnen et al., 2013) compared to eucalyptus plantations (pereira et al., 2007). fig 2. diameter of colonized twigs in a colony of the same ant species in different vegetation habitats. the lines inside each box indicate the median, and same letters indicate non-significant differences between vegetation habitats (mann-whitney test). species/morphospecies urban parks eucalyptus plantations native forest number of nests twig diameter (mm) number of nests twig diameter (mm) number of nests twig diameter (mm) pheidole sp.39 1 26.14 pheidole sp.43 6 15.23 (10.01-19.44) 27 18.09 (5.65–23.36) a pheidole sp.44 5 15.98 (10.47-29.50) procryptocerus sp.1 3 13.84 (11.32-25.37) 7 12.91 (10.46-16.32) procryptocerus sp.2 2 18.52 (15.91-21.14) 2 13.84 (12.32-15.36) procryptocerus sp.4 5 18.35 (8.77-20.38) pseudomyrmex gracilis 2 16.22 (15.67-16.77) pseudomyrmex phyllophilus 3 15.83 (9.28-16.77) 19 11.54 (4.33–16.92) 3 11.33 (9.84-23.64) pseudomyrmex sp.8 2 9.20 (6.54-11.85) 6 11.12 (8.92-23.8) solenopsis sp.2 2 10.29 (8.62-11.96) 1 11.74 15 8.81 (3.32–18.79) ac solenopsis sp.3 2 17.74 (9.19-26.29) solenopsis sp.4 1 9.57 solenopsis sp.5 3 5.62 (5.58-11.26) strumigenys crassicornis 1 16.5 wasmannia auropunctata 7 19.88 (8.32-22.53) kruskal-wallis 3.9113; df = 3; p = 0.2712 27.9747; df = 10; p = 0.0018 *different letters: p<0.05 according to dunn’s post-hoc test. our study is the first to compare “common inhabitants” of twigs among different vegetation habitats in the brazilian atlantic domain, demonstrating that these communities are affected by habitat structure, as shown by souza-campana et al. 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(2000). responses of ants to selective logging of a central amazonian forest. journal of applied ecology, 37: 508-515. doi: 10. 1046/j.1365-2664.2000.00512.x. https://doi.org/10.1111/j.1095-8312.2007.00728.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v64i1.1192sociobiology 64(1): 57-68 (march, 2017) diversity of bees and their interaction networks with ludwigia sericea (cambessides) h. hara and ludwigia peruviana (l.) h. hara (onagraceae) flowers in a swamp area in the brazilian atlantic forest introduction the family onagraceae consists of 17 genera and approximately 650 species distributed in subtropical and temperate regions (hoch et al., 1993), and is predominant in the americas (cabrera, 1965). this family is divided into the subfamilies ludwigioideae and onagroideae. ludwigioideae, according to the former classification, presented a single tribe – jussieeae (wagner et al., 2007). this tribe has been excluded and ludwigia l. is the only genus (wagner et al., 2007). in fact ludwigia is considered one of the largest and most diverse genera in the family onagraceae, which has a generalized morphology and records of five species that abstract in southern brazil the great diversity of bees is due to the richness of ecosystems in the region and when studies attribute the processes for identification of the floral resources used by bees we can get important steps for the elaboration of management and conservation plans of the species involved. based on this setting and considering that the study area is a "várzea" (swamp) with a large concentration of flowers of ludwigia sericea and ludwigia peruviana, important sources of resources for bees, the aim of this study was to find out about the floral visitor bees and through palynological studies and interaction networks, understand the role of these plants as providers of pollen grains to bees and how the bees interact with them and other plants, also found in this habitat. ludwigia sericea, ludwigia peruviana and the other plant species are presented as generalist in the interactions with the bee species, and the same occurs for most of the bee species in relation to these plants. generalist bee species (bombus pauloensis, apis mellifera, augochlora amphitrite and melissoptila paraguayensis) tend to be more abundant and more resistant to disturbances than the specialist species. it was the first time that plebeia emerina, centris varia, mourella caerulea and augochlorella ephyra were recorded on the ludwigia flowers in brazil. based on the results presented in this study, these plants are important for maintaining the bee community in the region of the atlantic forest in southern brazil, because they provide resources (nectar and pollen) to bee species that only occur in this region. sociobiology an international journal on social insects l gonçalves, ml tunes buschini article history edited by wesley dátillo, instituto de ecología a.c., mexico received 18 september 2016 initial acceptance 25 october 2016 final acceptance 27 february 2017 publication date 29 may 2017 keywords apis mellifera, bombus, floral resources, food web, pollen grains. corresponding author lia gonçalves universidade estadual do centro-oeste rua saldanha marinho 1604, apto 11 centro, cep: 85010-290. guarapuava-pr, brasil e-mail: liagoncalves22@hotmail.com are self-incompatible (l. elegans (camb.) hara, l. irwinii ramamoorthy, l. nervosa (poiret) h. hara, l. pseudonarcissus (chodat & hassler) ramamoorthy and l. sericea) (ramamoorthy & zardini, 1987). out of 82 species of the genus ludwigia considered mesophytic (growing in humid areas), 45 occur in south america, mainly in brazil, argentina and paraguay (ramamoorthy & zardini, 1987). in brazil, species from the family onagraceae are concentrated in the south and southeast (falkenberg, 1988), where the genus ludwigia is known as “cruz-de-malta”. the place of origin of the family onagraceae has not been determined precisely, but the region of south america has been suggested as the most probable (raven & axelrod, programa de pós-graduação em biologia evolutiva, universidade estadual do centro-oeste, guarapuava-pr, brazil research article bees l gonçalves, ml tunes buschini – bees diversity and interaction networks with ludwigia sericea and ludwigia peruviana 58 1974; raven 1988). according to some authors, l. peruviana originated in the americas (ramamoorthy & zardini, 1987) and later introduced into wet areas in different regions, making it a dominant species in a short period of time and an important source of floral resources for pollinators, especially for bees (jacobs et al., 1994). studies about interactions between the ludwigia flowers and their visitors demonstrated the existence of important characteristics of both the flowers and the bees (the most important group of ludwigia pollinators). their flowers produce pollen grains in large tetrads (diameter > 100 µm), with the presence of viscin threads (hesse, 1984), and only specialist bees with long rigid hairs and few branched in their scopa, and with rapid body and leg movements can be the potencial pollinators of these plants (cruden, 1981; gimenes, 1997). schlindwein (2004), michener (1979, 2000), gimenes et al. (1993, 1996) and gimenes (2003) observed an association between plants of the family onagraceae, more specifically the genus ludwigia, and oligoletic bees in the south and southeast of brazil. the brazilian apifauna consists of five families (andrenidae, apidae, colletidae, halictidae and megachilidae) and 1.600 species of described bees, however, approximately 3.000 species are estimated to exist in brazil (silveira et al., 2002). in the south, the great diversity of bees is due to the richness of ecosystems in the region, which is influenced by andean elements from the temperate south and the dry west of the continent, as well as from subtropical components from the “cerrado” (brazilian savanna) and the tropical forest to the north (wittmann & hoffman, 1990; alves dos santos, 1999). even presenting such diversity, the rate of exploitation of brazilian ecosystems is alarming. high levels of richness and endemism, associated with past destruction, has definitively included the atlantic forest on a world scale as one of the 35 hotspots of biodiversity, as this biome is the fifth richest hotspot in endemism (mittermeier et al., 2004). its original area (in brazil) is estimated to have covered between 1.300.000 km2 to 1.500.000 km2 and current data indicate that only 8.5% remains (morellato & haddad, 2000; câmara, 2005). based on this setting and considering that the study area is a “várzea” (swamp) with a large concentration of ludwigia the aim of this study was to find out about the floral visitor bees of the ludwigia sericea and ludwigia peruviana in this habitat and with palynological studies mapping their interactions and also know the other plants that interact to form the network. materials and methods study area this study was carried out in the parque municipal das araucárias (araucárias municipal park), located in the municipality of guarapuava (paraná state) (25o21’06”s; 51o28’08”w) (third plateau of paraná) (altitude = 1.120 m.a.s.l.). the area of the park is approximately 104 ha, consisting of araucaria forest (43% of the area), gallery forest (10.09%), grasslands (6.8%), swamps (7.13%) and altered areas (33.23%) (niesing, 2003). the swamp areas are located in the loweraltitude regions of the park and are composed of mainly grasses and members of the family asteraceae (buschini & fajardo, 2010). according to the köppen classification, the climate in the region is humid mesothermal, with no dry season, and a moderate winter with frost. the average annual temperature is approximately 22oc. sample design the studied species of ludwigia were l. sericea and l. peruviana, both ones present in the swamp area of the park. observations were carried out in the area beginning in august/2011 to accompany the start of the flowering of the species. after the commencement of flowering, the captures of bees were carried out three times per week from december/2011 to april/2012. the collection time was established after observation of floral receptivity, which occurred from 09h to 16h, totaling 176 hours. the flowering bushes of the two species of ludwigia were used randomly in the collections, until the end of flowering or visitation by the bees. in 20min per-hour intervals, all of the bees present in the flowers (chosen randomly in each bush) were collected using small sequentially-numbered transparent flasks. posteriorly some bees separated in morphospecies for identification at universidade federal do paraná (ufpr), by gabriel a. r. melo. the specimens are deposited in the collection of bees and wasps of the biology and ecology laboratory, of universidade estadual do centro-oeste (unicentro). diversity and composition of bees species the diversity of bees visiting l. sericea and l. peruviana flowers was calculated using the indices of shannon-wiener diversity h’ (measures community diversity), margalef richness dmg (degree of species richness) and pielou evenness j’ (indicates if different species possess similar or divergent abundances). these indices were calculated using the program past© (version 1.98. paleontological statistics 1999-2010). the frequency of occurrence (fo) and the species dominance were calculated for each bee species obtained. frequency of occurrence is the percentage of the number of collections with a given species and was calculated as fo= (f/n)·100 (silveira neto et al., 1976), where “f” is the number of collections with the species and “n” is the total number of collections performed. the bee species were classified as primary (fo > 50%), secondary (fo = 25% -50%), or accidental (fo < 25%). the species dominance of bees (d) was calculated as d= (d/n)·100 (palma 1975), where “d” is the abundance of a specific species and “n” the total abundance. the species were classified as dominant (d > 5%), accessory (d = 2.5% 5%), sociobiology 64(1): 57-68 (march, 2017) 59 or accidental (d < 2.5%). according to palma (1975), the fo and d indices when used together group and determine the species as common, intermediary or rare. pollen collection pollen material was retrieved from floral visiting bee bodies washing them with 70% alcohol. from this material slides were elaborated for observation with a petrographic microscope based on the method proposed by erdtman (1960). bees of the same species collected on the same day and time were grouped. the simultaneous removal of pollen from their bodies was performed for the preparation of only one slide to represent the occurrence of that species in that day and time. plants in a flowering state, or presenting flower buds that were found in the proximity, were collected for the elaboration of slides, which aided in the identification of pollen types found on the bees bodies. after identifying each pollen type, the first 400 grains per slide were counted. the species and their respective authors were consulted on the botanical database site tropicos® of the missouri botanical garden. the slides were labeled and deposited in the palynotheca of the department of biology, universidade estadual do centro-oeste, guarapuava (paraná state), brazil. interaction networks the network of interaction was built between the bees found in l. sericea and in l. peruviana flowers and the pollen grains obtained from their bodies. the network size was calculated through the formula m =a·p (where m is the maximum number of possible interactions; a is the number of bees and p is the number of pollen types). connectance (c = e/(a·p)) measures the ratio between the number of observed interactions (e) and the number of possible interactions, given that p is the number of plants (pollen types), and a is the number of bees in the network. to transform these values into percentages, results were multiplied by 100 (jordano, 1987). the connectance is a qualitative measure of network specialization and also represents the density of interactions in a network. thus, a highly specialized community presents a low value of connectance (jordano, 1987; blüthgen, 2010). the specialization index (h’2) was calculated from the weighted matrix (quantitative) (blüthgen et al., 2006, 2008), and its result varies from 0 (maximum generalization) and 1 (maximum specialization) (blüthgen et al., 2006). the network specialization index evaluates niche overlap among species. a highly specialized community has a high value of network specialization index (blüthgen, 2010). on the qualitative interaction networks (binary matrix), the average degree (ҟ) was determined, which is the average number of observed connections for species of plants or bees (blüthgen et al., 2006, 2008). the degree is possibly the simplest measure of specialization. a species can be described as a specialist if it reveals a low degree, compared with degrees of the other species in the network or the number of potential partners. nevertheless, this interpretation is based on the premise that all interactions are equally possible (olesen & jordano, 2002). the dependency of species was ascertained from frequency of floral visits, with quantitative pollen analysis (bascompte & jordano, 2007). in this way, for each interaction two dependency values were obtained from plant species to bee species, and vice versa. from the adjacency matrix, built with data on the presence and absence of plant and bee species, eulerian graphs were prepared, using software r, version 3.0.1. the evaluation of the network degree of nestedness based on the adjacency matrix, was performed using the nodf metric (nestedness metric based on overlap and decreasing fill), for more consistent statistical properties. nestedness was estimated using the aninhado 3.0 software (guimarães & guimarães, 2006). nestedness is used in ecology to describe one of the possible distribution patterns of species among discrete environments (atmar & patterson,1993). in a nested network generalist species in general interact among themselves and with the specialists, but specialists rarely interact among themselves (bascompte et al., 2003). results the flowering period of l. sericea was different from that of l. peruviana, presenting a few months of overlap between them. ludwigia sericea started to flower in december/2011, lasting until april/2012, with the flowering peak in february; whereas l. peruviana flowered between february/2012 and may/2012, with the flowering peak in march. no bee was collected in l. peruviana in may. during the period in which the two species were flowering, the quantity of l. sericea specimens was 5 times larger than the quantity of l. peruviana specimens (20 and 4 flowering plants respectively). during the collections, 908 bees (837 in l. sericea and 71 in l. peruviana) were captured belonging to 24 species and five families (andrenidae, apidae, colletidae, halictidae and megachilidae). apis mellifera was the most abundant species in the two species of ludwigia (n = 317 in l. sericea and n = 28 in l. peruviana), followed by augochlora amphitrite (n = 105) and bombus pauloensis (n = 93) in l. sericea and melissoptila marinonni (n = 14) and tetraglossula amphitrite (n = 7) in l. peruviana ( 1). diversity and composition of bees species the values of the indices of richness (dmg) and diversity (h’) for l. sericea (dmg = 2.972; h’ = 2.043) were superior compared to those obtained for l. peruviana (dmg l gonçalves, ml tunes buschini – bees diversity and interaction networks with ludwigia sericea and ludwigia peruviana 60 = 2.815; h’ = 1.906). on the other hand, the index of pielou evenness was higher for l. peruviana (j’ = 0.743) than for l. sericea (j’ = 0.671). uniting the results of the indices of frequency of occurrence (fo) and dominance (d) in l. sericea, only a. mellifera (fo = 54%; d = 38%) was considered a common species, eight species (b. pauloensis, m. marinonii, m. paraguayensis, p. emerina, t. diversipes, t. spinipes, t. anthracina and a. amphitrite) were considered intermediate (fo = 36.3% to 48.5%; d = 2% to 12%) and the others rare. as regards l. peruviana, no species was classified as common, six (a. mellifera, b. pauloensis, m. marinonii, m. paraguayensis, t. anthracina and m. fiebrigi) were intermediate (fo = 9.09% to 30.3%; d = 4% to 39%) and the others rare ( 1). bee-plant interaction networks the interaction network established in the area of study consisted of 15 bee species (a) and 49 plant species (p), and 735 interactions (m) were theoretically possible (fig 1). however, among these interactions only 194 were observed (e) (c = 0.26; 26.3%). ludwigia sericea was visited by 15 bee species, while l. peruviana by 10 species. nine bee species were common to the two species of ludwigia. in this interaction network, some plant species were visited by almost all the bee species that visited the ludwigia species, for example, polygonum punctatum (12 bee species), cinnamomum amoenum (11 species) and styrax leprosum (10 species).out of the 49 plant species that made up the network, 30 maintained interaction with only 3 or less bee species. in the interaction network, the average degree of interactions with the plant species observed, was relatively low (k= 3.95), as a heterogeneous distribution, i.e., having few species with many interactions and many species with few interactions (fig 2a). the bees present the average degree of interaction (k= 12.93), higher than that observed for the plants. the level distribution, as well as for the plants, was also heterogeneous (fig 2b). fig 1. pollen types observed during pollen analysis of floral visitor bees of ludwigia sericea and ludwigia peruviana: 1-arecaceae sp; 2-asteraceae sp1; 3-asteraceae sp2; 4-asteraceae sp3; 5-asteraceae sp4; 6-asteraceae sp5; 7-asteraceae sp6; 8-baccharis anomala; 9-baccharis microdonta; 10-baccharis sp; 11-campovassouria cruciata; 12-chrysolaena platensis; 13-enechtites valerianifolius; 14-gochnatia polymorpha; 15-stevia tenus; 16-trixis sp; 17-vernonanthura westiniana; 18-begonia cucullata; 19-senna araucarietorum; 20-lobelia camporum; 21-lobelia sp; 22-lonicera japonica; 23-ipomoea grandifolia; 24-ipomoea purpurea, 25-erythroxylum deciduum; 26-mimosa flocosa; 27-lycopus sp; 28-tectona grandis; 29-cinnamomum amoenum; 30-heimia myrtifolia; 31-janusia guaranitica; 32-leandra sp; 33-tibouchina cerastifolia; 34-acacia recurva; 35-ludwigia peruviana; 36-ludwigia sericea, 37-phytolacca dioica; 38-polygala sp; 39-polygonum punctatum; 40-galianthe dichasia; 41-rubiaceae sp; 42-citrus sp; 43-serjania sp; 44-solanum americanum; 45-solanum variabile; 46-styrax leprosum; 47-vochysia ferruginea; 48-indet1; 49-indet. sociobiology 64(1): 57-68 (march, 2017) 61 lu dw ig ia s er ic ea lu dw ig ia p er uv ia na su bf am ily o f b ee s/ be e sp ec ie s n um be r of in di vi du al s f o d cl as si fic ati on n um be r of in di vi du al s fo d cl as si fic ati on a nd re ni da e rh op hi tu lu s fla vi ta rs is (s ch lin dw ei n & m ou re 1 99 8) 6 18 0. 7 ra re a pi da e a pi s m el lif er a (l in na eu s 17 58 ) 31 7 54 38 co m m on 28 30 .3 39 in te rm ed ia te bo m bu s (f er vi do bo m bu s) p au lo en si s (f ri es e 19 13 ) 93 48 11 in te rm ed ia te 5 18 .2 7 in te rm ed ia te ce nt ri s (c en tr is ) v ar ia (e ri ch so n 18 49 ) 2 6. 06 0. 2 ra re ex om al op si s an al is (s pi no la 1 85 3) 1 3. 03 0. 1 ra re m el is so pti la m ar in on ii (u rb an 1 99 8) 50 39 .3 6 in te rm ed ia te 14 18 .2 20 in te rm ed ia te m el is so pti la p ar ag ua ye ns is (b rè th es 1 90 9) 87 48 .5 10 in te rm ed ia te 6 18 .2 8 in te rm ed ia te m ou re lla c ae ru le a (f ri es e 19 00 ) 4 9. 09 0. 5 ra re pa ra te tr ap ed ia v ol ati lis (s m it h 18 79 ) 2 3. 03 0. 2 ra re pl eb ei a em er in a (f ri es e 19 00 ) 15 36 .3 2 in te rm ed ia te 2 6 3 ra re te tr ap ed ia d iv er si pe s kl ug 1 81 0 44 42 .4 5 in te rm ed ia te tr ig on a sp in ip es (f ab ri ci us 1 79 3) 51 42 .4 6 in te rm ed ia te 1 3. 03 1. 4 ra re co lle ti da e te tr ag lo ss ul a an th ra ci na (m ic he ne r 19 89 ) 40 36 .4 5 in te rm ed ia te 7 15 .1 10 in te rm ed ia te h al ic ti da e a ug oc hl or a (a ug oc hl or a) a m ph it ri te (s ch ro tt ky 1 90 9) 10 5 42 .4 12 in te rm ed ia te a ug oc hl or el la e ph yr a (s ch ro tt ky 1 91 0) 1 3. 03 0. 1 ra re d ia lic tu s m ic he ne ri (m ou re 1 95 6) 1 3. 03 0. 1 ra re ps eu do ga po st em on p ru in os us (m ou re & s ak ag am i 1 98 4) 6 9. 09 0. 7 ra re m eg ac hi lid ae co el io xy s cf r. ch ac oe ns is (h ol m be rg 1 90 3) 1 3. 03 1. 4 ra re co el io xy s to lte ca (c re ss on 1 87 8) 1 3. 03 1. 4 ra re m eg ac hi le a ff. b ra si lie ns is (d al la t or re 1 89 6) 1 3. 03 1. 4 ra re m eg ac hi le (a us tr om eg ac hi le ) f ac ia lis (v ac ha l, 19 09 ) 1 3. 03 0. 1 ra re 1 3. 03 1. 4 ra re m eg ac hi le (a us tr om eg ac hi le ) fi eb ri gi (s ch ro tt ky 1 90 8) 1 12 .1 0. 1 ra re 3 9. 09 4 in te rm ed ia te m eg ac hi le (m ou re ap is ) m ac ul at a (s m it h 18 53 ) 8 21 .2 1 ra re 1 3. 03 1. 4 ra re m eg ac hi le (m ou re ap is ) p le ur al is (v ac ha l 1 90 9) 2 6. 06 0. 2 ra re to ta l n um be r of in di vi du al s 83 7 71 to ta l n um be r of s pe ci es 21 13 t ab le 1 . s pe ci es o f be es c ol le ct ed in th e flo w er s of l . s er ic ea a nd l . p er uv ia na in a “ vá rz ea ” (fl oo dp la in ) ar ea in p ar qu e m un ic ip al d as a ra uc ár ia s (g ua ra pu av a st at e of p ar an á) a nd th ei r in di ce s: fr eq ue nc y of o cc ur re nc e (f o ), d om in an ce in de x (d ) a nd d om in an ce c la ss ifi ca tio n. l gonçalves, ml tunes buschini – bees diversity and interaction networks with ludwigia sericea and ludwigia peruviana 62 bombus (fervidobombus) pauloensis was the most generalist species, visiting 34 plant species, including the two species of ludwigia. the plants used by b. pauloensis, corresponded to 69.39% of the total number of plant species which comprise the network and 17.53% of all the network interactions. apis mellifera was the second most generalist bee species, visiting 33 plant species, 67% of the total number of plants and 17.01% of all the interactions, followed by augochlora (augochlora) amphitrite and trigona spinipes (20 plant species, 40.82% of the total number of plants; 10.31% of all the interactions) and megachile (moureapis) maculata (18 plant species; 36.73% of the total number of plants; 9.28% % of the total number of interactions). the interaction network ludwigia-visitors presented a nested pattern (nodf= 37.06; p< 0.001), with asymmetry shown in the split graphical representation. when the diversity of interaction is analyzed considering the weighted matrix, the value of the specialization index was low (h2’= 0.26), showing that most of the species maintain, in general, few interactions in relation to the proportion of possible interactions (connectance). concerning the dependence of the species of this network, it can be said that most of the bee species that visited the two species f ludwigia, collected flower resources preferentially in l. sericea, presenting between 78% (megachile maculata) to 100% (rhophitulus flavitarsis x megachile (moureapis) pleuralis) of dependence, according to the quantitative analyses of the pollen grains in the samples. from the bees that visited the two species of ludwigia, only plebeia emerina presented pollen grains dominant from another plant species, cinnamomum amoenum (52%) (fig 3a). fig 2. average degree distribution of the connections on plant species (a) and bee species (b). the line represents the expected decline if the distribution follows a power law. the horizontal axis represents the number of connections of each point in a network. the vertical axis represents the probability of a point in the network to have a number “k” or more of connections. when the dependence of the plants by bees was analyzed, 18 out of the 49 plant species presented dependence by some bee species, ranging between 72% for vernonanthura westiniana x megachile maculata to 99% for baccharis sp. x a. mellifera. the rest of the plant species (n=31) were more generalist (fig 3b). discussion a variation in the flowering period of species of the genus ludwigia has been verified, according to the region of brazil. in this study, the flowering periods observed were from december to april for l. sericea and from february to april for l. peruviana. in the são paulo state region, l. elegans (camb.) hara flowered practically the whole year (except in the cold months from august to october), with its flowering peak in february and march (gimenes, 2003; gimenes et al., 1993). ludwigia tomentosa also flowered almost the whole year (except in the very dry months from june to august) in the mato grosso do sul state region (pott & pott, 2000). in minas gerais state, ludwigia suffruticosa (l.) gomez had its flowering peak in june (martins & antonini, 1994). the fact that the region of guarapuava (paraná state) possesses well-defined seasons, with cool summers (average temperature of 22oc) and winters with frequent severe frosts, and an average temperature superior to 3oc and inferior to 18oc (maack, 1981), emphasizes the results presented here, where, during the hot months, the flowering period of the studied species occurred, as well as the foraging activity of the collected bees. according to bazilio (1997) and mendes and rêgo (2007), the seasonal pattern of the activities of various species of bees in subtropical climate regions is associated with the flowering of angiosperms. in the present study, the indices of diversity and richness of the apifauna were higher for l. sericea. a high value for these indices indicates, in most cases, a wellstructured community, with many rare species (costa et al., 1993). the fact that l. sericea had flowered before, for more time and with more specimens than l. peruviana, may explain the result of the indices of diversity and richness of the bees collected in these plants. both the diversity and the richness of the species of bees visiting the flowers of the genus ludwigia in the guarapuava region were higher than those recorded in the southeast and midwest of brazil (gimenes, 2003; steiner et al., 2010; ruim et al., 2011; carvalho et al., 2012). in addition, in these studies, the richness of solitary species was higher than social species. the fact that the diversity of solitary bees is greater in the south than in other regions of brazil (michener, 2000; silveira et al., 2002; santos, 2002; buschini, 2005) is possibly one of the factors that makes the diversity of bees in the flowers of ludwigia in guarapuava greater than the diversity in other studied regions of the country. this result deserves to be highlighted, because it shows the importance of solitary species in the pollination of the some plants in southern of brazil. sociobiology 64(1): 57-68 (march, 2017) 63 the nested standard presented in this study as in other studies (bascompte & jordano, 2007; hernández-yáñez et al., 2013) suggests that the specialized species were interacting with the generalists (and these among themselves) contributing to increase the strength of the generalists in the network. of all of the species of bees captured during this study, including solitary and social bees, apis mellifera was the most abundant in the two species of ludwigia. this species was also very frequent in flowers of l. elegans (gimenes, 1997, 2002, 2003) in são paulo state and in the flowers of ludwigia tomentosa (cambess.) (carvalho et al., 2012) in mato grosso do sul state. although it is a visiting generalist, a. mellifera is considered an efficient pollinator in flowers of ludwigia due to its size and behavior, which facilitate contact with the reproductive structures during the collection of nectar and pollen (gimenes, 1997). apis mellifera also has its foraging activity synchronized with the flowering peak of ludwigia (gimenes, 1997, 2003) and its abundance in these flowers is associated with its being a highly eusocial species, with a very efficient communication system between the individuals of the colony (seeley, 1985). fig 3. interaction network between pollen types (right) and floral visitor bees ludwigia sericea and ludwigia peruviana. the binary matrix (a) qualitatively demonstrates the interactions between species. the weighted matrix (b) represents quantitatively interactions. the width of the lines in the weighted matrix indicates total interactions frequency between one pair of species. abbreviations: see table 2. l gonçalves, ml tunes buschini – bees diversity and interaction networks with ludwigia sericea and ludwigia peruviana 64 botanical family pollen type abbreviation subfamily of bees bee species abbreviation arecaceae arecaceae sp aresp andrenidae rhophitulus flavitarsis rhofla asteraceae asteraceae sp1 astsp1 apidae apis melífera apimel asteraceae sp2 astsp2 bombus (fervidobombus) pauloensis bompau asteraceae sp3 astsp3 centris (centris) varia cenvar asteraceae sp4 astsp4 exomalopsis analis exoana asteraceae sp5 astsp5 melissoptila marinonii melmar asteraceae sp6 astsp6 melissoptila paraguayensis melpar baccharis anomala dc. bacano mourella caerulea moucae baccharis microdonta dc. bacmic paratetrapedia volatilis parvol baccharis sp l. bacsp plebeia emerina pleeme campovassouria cruciata (vell.) r.m.king & h.rob. camcru tetrapedia diversipes tetdiv chrysolaena platensis h. rob. chrpla trigona spinipes trispi erechtites valerianifolius (wolf.) dc. eneval colletidae tetraglossula anthracina tetant gochnatia polymorpha (less.) cabrera gocpol halictidae augochlora (augochlora) amphitrite augamp stevia tenus steten augochlorella ephyra augeph trixis sp trisp dialictus micheneri diamic vernonanthura westiniana (less) h. rob. verwes pseudagapostemon pruinosus psepru begoniaceae begonia cucullata willd. begcuc megachilidae coelioxys cfr. chacoensis coecha caesalpinaceae senna araucarietorum h.s. irwin & barneby senara coelioxys tolteca coetol campanulaceae lobelia camporum pohl. lobcam megachile aff. brasiliensis megbra lobelia sp pohl. lobsp megachile (austromegachile) facialis megfac caprifoliaceae lonicera japônica thunb. lonjap megachile (austromegachile) fiebrigi megfie convolvulaceae ipomoea grandifolia l. ipogra megachile (moureapis) maculata megmac ipomoea purpurea roth. ipopur megachile (moureapis) pleuralis megple erythroxilaceae erythroxylum deciduum a.st.-hil erydec fabaceae mimosa flocosa l. mimflo lamiaceae lycopus sp l. lycsp tectona grandis l.f. tecgra lauraceae cinnamomum amoenum (nees) kosterm. cinamo lythraceae heimia myrtifolia cham. & schltdl. heimyr malpighiaceae janusia guaranítica (a.st. hil.) a. juss. jangua melastomataceae leandra sp leasp tibouchina cerastifolia cogn. tibcer mimosaceae acacia recurva dc. acarec onagraceae ludwigia peruviana (l.) h. hara ludper ludwigia sericea (cambessides) h. hara ludser phytolaccaceae phytolacca dioica l. phydio table 2. pollen types (plant species) and bee species belonging the interaction networks of floral visitors of ludwigia sericea and ludwigia peruviana, with their respective abbreviations. sociobiology 64(1): 57-68 (march, 2017) 65 the second most frequent and abundant species in this study was augochlora amphitrite, which was also recorded by steiner et al. (2010) in flowers of l. peruviana in santa catarina state and by gimenes (2002) in flowers of l. elegans in the region of state of são paulo as a frequent species, but without an important role in pollination. paratetrapedia volatilis (rare species in the present study) and bombus spp. were also collected by these researchers in the flowers of ludwigia. the genus bombus, whose behavior is generalist (alves dos santos, 1999; steiner et al., 2010), is considered an important pollinator in elevated altitudes of brazil (freitas & sazima, 2006). pollen analysis showed that bombus pauloensis was the most generalist bee species, having interactions with 34 pollen types. bombus was also recorded in other species of ludwigia, being the most frequent visitor and abundant in flowers (gimenes, 2002; silveira et al., 1993) and the generalist behavior (alves dos santos, 1999; steiner et al., 2010), probably because it is a social species that during nesting seasons increase its foraging activities and its morphological and behavioral aspects, which facilitate the transportation of a large quantity and variety of pollens. generalist bee species tend to be more abundant and more resistant to disturbances than the specialist species (vazquez, 2005; vazquez & aizen, 2004). bees of the genus tetrapedia were considered effective pollinators of l. sericea in são paulo state based on records of pollen collection behavior, frequency and time spent in the flowers (sazima & santos, 1982). in the present study, tetrapedia diversipes was classified as an intermediate species and possible pollinator of flowers of ludwigia, due to the presence of a structure (scopa) that facilitates the transport of pollen. silveira et al. (1993), alves dos santos (1999), steiner et al. (2010), ruim et al. (2011) and menezes et al. (2012) verified the interaction of tetrapedia diversipes with flowers of ludwigia in mata atlântica areas in the states of minas gerais, rio grande do sul, santa catarina, paraná and rio de janeiro, respectively. in this study, pseudagapostemon puinosus, rhophitulus fiavitarsis and the species of megachile can be considered accidental pollinators in the flowers of ludwigia. according to gimenes (1997, 2002), the first two species cited also presented this type of behavior in flowers of ludwigia during the collection of resources. on the other hand, the genera exomalopsis, dialictus and coelioxys did not behave like pollinators when they visited the flowers. although l. sericea, l. peruviana and the other plants species sampled in this study are presented as generalist in the interactions with the bee species, and the same occurs for most of the bee species in relation to these plants, the network showed that megachilidae species largely depend on the ludwigia pollen grains. megachile aff. brasiliensis, collected only from the l. peruviana, megachile (moureapis) pleuralis and rhophitulus flavitarsis collected only from the l. sericea, had oligoletic habits. these habits are generally associated to plants which occur in open areas (schlindwein 2000). many plant species that occur in these environments have large pollen grains such as those of the ludwigia, which requires from the bees of the megachilidae family a scopa (ventral) with long and unbranched hairs enabling them to adequately handle the pollen (schlindwein, 2004). these results confirmed what buschini et al. (2009) had observed when analyzing the pollen grains found in megachile (moureapis) sp. nests. according to these authors, l. peruviana and l. sericea are the main pollen sources for the offspring of these bees, considered specialists at collecting ludwigia pollen. tetraglossula anthracina and pseudagapostemon pruinosus also largely depend on the l. sericea pollen. these species were recorded as the main visitors to the ludwigia flowers in other regions in brazil (gimenes, 1997; gimenes et al., 1993, 1996; schlindwein, 2004; steiner et al., 2010) and are specialists at collecting pollen and nectar from the flowers of this genus. moreover, t. anthracina is still considered an efficient pollinator due to the morphological and behavioral botanical family pollen type abbreviation subfamily of bees bee species abbreviation polygalaceae polygala sp l. polsp polygonaceae polygonum punctatum michx. polpun rubiaceae galianthe dichasia (sucre & c.g. costa) galdic rubiaceae sp rubsp rutaceae citrus sp l. citsp sapindaceae serjania sp radlk. sersp solanaceae solanum americanum mill. solame solanum variabile mart. solvar styracaceae styrax leprosum hook and am. stylep vochysiaceae vochysia ferrugínea pohl. vocfer unidentified indet1 indet1 indet2 indet2 table 2. pollen types (plant species) and bee species belonging the interaction networks of floral visitors of ludwigia sericea and ludwigia peruviana, with their respective abbreviations (continuation). l gonçalves, ml tunes buschini – bees diversity and interaction networks with ludwigia sericea and ludwigia peruviana 66 adaptations (size and quick abdominal movements while collecting), which favored collecting the resources (gimenes, 1997, 2003; gimenes et al., 1993, 1996). the presence of plebeia emerina, centris varia, mourella caerulea and augochlorella ephyra on the l. sericea and l. peruviana flowers is an unprecedented result of this study, i.e. it was the first time that these species were recorded on the ludwigia flowers in brazil. this can be attributed to the distribution of these species, which, apart from of c. varia, occur preferentially in southern brazil (moure et al., 2007). based on the results presented here, it can be concluded that the diversity of bees is high on the ludwigia flowers in this region of brazil, especially the solitary species. these plants are important for maintaining rare species like those from the family megachilidae. furthermore, l. sericea and l. peruviana are part of a more complex system interacting directly with 24 species of bees and, indirectly, with 49 species of plants. thus, they connect various solitary bee species to other plants that are part of this important brazilian biome, which is severely under threat. acknowledgments we thank the graduate program in evolutionary biology (unicentro) and capes for financial support. we also thank prof. dr. gabriel augusto r. melo – ufpr (curitiba, paraná state) for identification of the bees, prof. dr. juliano cordeiro – ufpr (palotina, paraná state) for identification of the plants and cláudia i. silva – ufc (fortaleza, ceará state), for the help with palynological study and all support. references alves dos santos, i. 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(1990). bees of rio grande do sul, southern brazil (insecta, hymenoptera, apoidea). iheringia, série zoologia, 70: 17-43. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.1146sociobiology 65(3): 497-505 (september, 2018) population diversity of odontotermes formosanus (shiraki) (termitidae, macrotermitinae) from different geographic locations in anhui province, china introduction odontotermes formosanus (termitidae, macrotermitinae), a soil-dwelling termite, is mainly distributed in the southern provinces along the yellow and yangtze rivers in china (mühlemann et al., 1995). although o. formosanus promotes balance in the agroecosystem and biomass degradation in forest systems (black & okwakol, 1997), this species is an important pest which can damage many garden trees, plantations and dikes with high economic losses (huang et al., 2000; huang et al., 2006; ge et al., 2008; appel et al., 2012). as such a most destructive pest, o. formosanus are usually controlled and treated in urban areas by chemical pesticide in time (huang et al., 2006). abstract genetic differentiation, genetic exchange, and influence of natural geographic barrier on the genetic structure of 20 geo-populations of odontotermes formosanus sampled from different regions in anhui province, china were detected using issr. seventy-nine polymorphic loci were detected with nine issr primers, and the percentage of polymorphic bands was 87.78%. the average number of alleles per locus was 1.8778 ± 0.3294, and the effective number of alleles was 1.4741 ± 0.3438. the nei′s gene diversity and shannon information index were 0.2832 ± 0.1696 and 0.4307 ± 0.2274, respectively. all the populations were divided into two groups through upgma clustering analysis based on nei’s genetic distance. one group comprised geo-populations a, c, and j, and the other group consisted of the remaining clusters. mantel test results revealed no significant correlation between genetic similarity and geographical distance, as well as between elevation. high levels of genetic diversity, genetic mutation, and genetic differentiation were also detected among the geo-populations of o. formosanus. this study revealed the gene flow and possible migration paths of o. formosanus, which are necessary for continuous monitoring and prevention of this species. sociobiology an international journal on social insects yq yang1*, lj pu2, 3*, q wang4, z wang5, zp pang6, yh long2 article history edited by qiuying huang, huazhong agricultural university, china received 24 june 2016 initial acceptance 24 february 2017 final acceptance 12 june 2018 publication date 02 october 2018 keywords genetic diversity, genetic variation, intersimple sequence repeat, odontotermes formosanus. corresponding author yanhua long department of biotechonology, school of life science anhui agricultural university 130 changjiang road, hefei, china. e-mail: longyanhua@ahau.edu.cn an abundant geographic division and significantly different climatic conditions, topography, and vegetation exist in anhui province as a result of the diversity in terrain and geomorphology, as well as the fact that the yangtze river and the huai river both traverse anhui province from west to east (https://en.wikipedia.org/wiki/anhui#geography). because of the influence of natural and anthropogenic factors, o. formosanus is capable of long-distance migration and shortrange dispersal (hu et al., 2007). geographical location, environmental situation, and host pressure may lead to genetic differentiation and changes in the genetic structure of insects in different geographical populations (yang et al., 2015). the relationship between genetic similarity and the distances of geographic locations has been pointed out when focused on 1 state key laboratory of tea plant biology and utilization, anhui agricultural university, hefei, china 2 college of life science, anhui agricultural university, hefei, china 3 peking-tsinghua joint center for life sciences, peking university, beijing, china 4 institute pasteur of shanghai, chinese academy of sciences, shanghai, china 5 institute of termites control of bengbu, bengbu, china 6 institute of termites control of wujin, changzhou, china * authors contributed equally to this work. research article termites yq yang, lj pu, q wang, z wang, zp pang, yh long – population diversity of o. formosanus 498 individual intraand interspecific populations in east china (long et al., 2009). obviousely, study on the genetic diversity or similarity through methods of molecular biology and genetics to determine whether there is gene exchange between different populations of one species seems very important. also, through those studies, the possible insect-source site and migration path could be well pointed out, which will be great helpful for controlling of this pest. molecular genetic markers are useful tools for distinguishing colony distinctness, inferring colony breeding structure, tracking colonies after insecticide treatment and determining whether termites were exterminated entirelly (vargo, 2003a). many researchers obtained results about microsatellite markers on some termites, such as macrotermes michaelseni (kaib et al., 2000), cubitermes subarquatus (harry et al., 2001), mastotermes darwiniensis (goodisman et al., 2001), coptotermes lacteus (thompson et al., 2000), coptotermes formosanus (vargo & henderson 2000; vargo et al., 2003), reticulitermes flavipes (vargo, 2000, 2003b), reticulitermes chinensis (huang et al., 2013) and reticulitermes speratus (hayashi et al., 2002). such studies shed light on the genetic studies on termites. while as a kind of representative fungus-growing termites, most current literatures about odontotermes contain the studies of the evolution and transmission of its symbiotic fungal (aanen et al., 2002 and 2009; osiemo et al., 2010; yashiro et al., 2011), the cellulases of symbiotic bacterium (su et al., 2016; duan et al., 2017), the biology and ecology of this insect (huang et al., 2008; appel et al., 2012). husseneder (2013) surveyed the population genetic of o. formosanus sampled from some provinces of south china through microsatellites techonology which showed that populations tested were genetically differentiated and the gene flow was appreciable with long geographical distance. but considering the effect of the special geographic condition, such as some mountains and rivers, as mentioned in last paragraph, more genetic studies on o. formosanus in anhui province need to be done. inter-simple sequence repeat (issr) is more reliable and easily used for studies on genetic diversity than other molecular markers based on pcr technology, such as random amplified polymorphic dna (rapd), amplification fragment length polymorphism (aflp) and restriction fragment length polymorphism (rflp) (nybom, 2004; long et al., 2009). in this system, pcr is performed on a short dna sequence between two simple sequence repeat (ssr) sequences in genomic dna with a single oligomerized nucleotide primer containing anchored 1-4 bases in the 3′ or 5′ end of the sequence, which are widely available and exhibits fast evolutionary rate (esselman et al., 1999; tanya et al., 2011; hu et al., 2015). in addition, the features of long sequence and high annealing temperature ensure that issr-pcr would exhibit high stability and reproducibility (nagaoka & ogihara, 1997; tsumura et al., 1996; qian et al., 2001; naik et al., 2017). although issr is widely used in studies on various plants (qian et al., 2001; cao et al., 2006; tanya et al., 2011), fungi (menzies et al., 2003) and some insects (li et al., 2009; dutta et al., 2012; barbosa et al., 2014), but it was less used on the study of termites (long et al., 2009). so in this study, the relationship between genetic and geographical distance, as well as genetic similarity and elevation, were investigated among geographical populations of o. formosanus sampled from different regions in anhui province of china. materials and methods sample collection and identification thirty natural populations were sampled from different sites with typical geographical features in anhui province, china from june 2011 to october 2011. twenty populations were identified as o. formosanus. all of the samples were stored in anhydrous ethanol and stored at −20 °c. collected data are shown in table 1 and fig 1. samples were first identified using morphological features and then through molecular identification by amplifying the mitochondrial dna coii gene (sperling et al., 1994; wallman & donnellan, 2001; beckenbach et al., 1993). the total dna was extracted from the head of a soldier termite to avoid contamination from termite intestinal symbiotic microbes. genomic dna was extracted following previously described methods (long et al., 2009). dna precipitate was dissolved in 20 μl of 1×te buffer (10 mm tris-hcl and 1 mm edta, ph 8.0). dna quality and quantity were determined using a nanodrop 2000 uv–vis spectrophotometer (thermo fisher scientific, waltham, ma, usa), and the concentration was regulated at 20 ng μl-1. the dna samples were then stored at −20 ºc prior to issr–pcr analysis.the primers c2f2 (lo et al., 2004) and btlys (liu & beckenbach, 1992) were used to amplify a partial sequence of the coii gene through pcr on a c1000 touch™ thermal cycler (bio-rad hercules, ca, usa). fig 1. locations where o. formosanus populations were sampled in china. the explanation of a-t can be found in table 1. the green shadow represented the yangtze river. sociobiology 65(3): 497-505 (september, 2018) 499 geo-population sampling site geographical coordinates elevation (m) collection date a qianshan 116°33′02.63″ e 30°39′39.97″ n 50 2010.09 b qimen 117°42′55.44″ e 29°51′05.89″ n 117 2010.09 c lu’an 116°32′48.36″ e 31°45′32.70″ n 66 2010.07 d lu’an 116°30′14.28″ e 31°45′42.96″ n 42 2010.07 e lu’an 116°29′38.70″ e 31°45′24.54″ n 67 2010.07 f bengbu 117°11′08.49″ e 32°44′54.61″ n 103 2010.08 g shucheng 117°01′21.83″ e 31°19′25.71″ n 92 2010.07 h qianshan 116°33′04.89″ e 30°39′39.97″ n 53 2010.09 i hefei 117°10′27.86″ e 31°51′04.76″ n 45 2010.07 j huangshan 118°08′43.20″ e 30°05′05.58″ n 576 2010.09 k hefei 117°09′41.04″ e 31°50′41.90″ n 66 2010.07 l hefei 117°15′14.82″ e 31°51′42.31″ n 32 2010.07 m hefei 117°15′57.75″ e 31°51′27.83″ n 33 2010.07 n qianshan 116°33′06.72″ e 30°39′40.88″ n 54 2010.09 o tongling 117°47′59.10″ e 30°56′50.73″ n 9 2010.08 p huangshan 118°08′48.72″ e 30°10′58.68″ n 522 2010.09 q hefei 117°09′40.04″ e 31°50′39.90″ n 67 2010.07 r lu’an 116°32′46.80″ e 31°45′37.26″ n 67.2 2010.07 s hefei 117°10′11.35″ e 31°51′03.94″ n 64 2010.07 t lu’an 116°29′40.08″ e 31°45′21.48″ n 69.1 2010.07 table 1. collection data of geo-populations of o. formosanus. pcr was carried out in a 20 μl reaction mixture containing the following components: 13.8 μl of sterile deionized water, 2.0 μl of 10× pcr buffer, 1.2 μl of 25 mm mg2+, 0.4 μl of 10 mm dntps, 0.3 μl of 10 μm primer1, 0.3 μl of 10 μm primer2, 0.5 μl of 5 u μl-1 rtaq dna polymerase, and 1.5 μl of 20 ng μl-1 templates. the following thermal profile was used for pcr amplification: initial denaturation for 3 min at 94 ºc; 29 cycles of denaturation for 30 s at 94 ºc, annealing for 30 s at 55 ºc, and extension for 1 min at 72 ºc; and final extension for 10 min at 72 ºc. pcr products were sent to shenggong inc. (shanghai, china) for purification and sequencing. issr amplification and product detection according to 96 issr primers released by columbia university in canada (ubc set 9) and former research results (long et al., 2009), nine primers, which can generate clear, bright, and multifarious fragments, were used to provide polymorphic markers for detection of genetic diversity (table 3). pcr was carried out in a 20 μl reaction mixture containing the following components: 13.2 μl of sterile deionized water, 2.0 μl of 10× pcr buffer, 2.4 μl of 25 mm mg2+, 0.3 μl of 10 mm dntps, 0.6 μl of 10 μm primers, 0.5 μl of 5 u μl-1 rtaq dna polymerase, and 1.0 μl of 20 ng μl-1 template. amplification table 3. inter-simple sequence repeat (issr) primers and corresponding information. primer name core sequence (5’–3’) attached bases annealing temperature (ºc) number of amplified bands number of polymorphic bands ppb † (%) h ‡ i § is01 (ac)8 t 55 10 9 90 0.3009±0.2041 0.4461±0.2687 is07 (atg)6 53 10 9 90 0.3333±0.1683 0.4917±0.2341 is09 (tg)6 cc 50 10 8 80 0.2804±0.2069 0.4153±0.2839 is13 (ca)6 ag 50 11 9 81.82 0.3050±0.2039 0.4482±0.2751 is14 -(ga)5 caa46 10 9 90 0.2941±0.1956 0.4418±0.2525 is16 (cac)4 rc 50 8 7 87.50 0.3105±0.1809 0.4611±0.2534 is18 (ga)8 yt 48 11 9 81.82 0.1953±0.1695 0.3149±0.2344 is23 (gag)4 rc 50 10 10 100 0.3267±0.2007 0.4805±0.2582 is24 (gaa)6 50 10 9 90 0.3207±0.1904 0.4735±0.2525 total 90 79 87.78 † percentage of polymorphism bands ‡ nei’s (1973) gene diversity § shannon’s information index [lewontin (1972)] yq yang, lj pu, q wang, z wang, zp pang, yh long – population diversity of o. formosanus 500 was performed on the pcr apparatus (c1000 thermal cycle, bio-rad) under the following cycle profiles: initial denaturation for 5 min at 94 °c; 39 cycles of denaturation for 45 s at 94 °c, annealing for 45 s at 46.0 °c to 55.0 °c (depending on primers used, table 3), and extension for 1.5 min at 72 °c; and final extension for 10 min at 72 °c. the amplification products were resolved electrophoretically on 1.0% agarose gels at 180 v in 1× tae buffer (2 m tris, 1 m acetic acid, and 100 mm edta, ph 8.0) for 1 h. the products were then stained with sybr® green and photographed under the geldoc-it® imaging system (uvp, llc, usa). data analysis the nucleic acid sequences of the coii gene of all samples were sorted, edited, and subjected to blast search using bioedit 7.0.5.3 (hall, 1999) and submitted to genbank to select geo-populations of o. formosanus. all the issr amplification bands were read using the gel-pro analyzer software (media cybernetics, silver spring, md, usa). the bands, no matter strong or weak, were scored as present (1) or absent (0) for each dna sample to form a two-dimensional matrix. population genetic parameters were statistically analyzed using two assumptions: 1) the populations a/h/n b c/r d e/t f g i/k/l m/q/s j/p o a/h/n 0 b 139357 0 c/r 124204 240274 0 d 125760 241508 1603 0 e/t 135127 269331 60110 60596 0 f 265635 342305 154643 153155 197820 0 g 83845 213251 46609 48187 56865 200527 0 i/k/l/m/q/s 146078 223962 68809 68333 128914 122830 99615 0 j/p 197413 61886 286005 287061 321862 367616 265044 257440 0 o 122652 121722 152763 153440 201658 223071 148062 112271 145957 0 table 2. geographical distance among all populations. (unit: m) populations were in hardy–weinberg equilibrium and 2) the bands with the same electrophoretic mobility were considered as the amplification products of identical dna fragments in the genome (lu et al., 2009). the number of polymorphic loci, percentage of polymorphic bands (ppb), nei’s gene diversity (h), shannon’s information index (i), genetic distance, and genetic similarity were calculated using popgen32 v 1.31 (yeh et al., 1997; zhao et al., 2009). to represenet the relationship among populations, a upgma dendrogram was generated using nei’s unbiased genetic distance with the ntsyspc v 2.1 software package (rohlf, 2000). a clustering map was generated through a tree plot module. the geographical distance of 20 o. formosanus populations were obtained using distance calculator (www. daftlogic.com/projects-google-maps-distance-calculator. htm) to analyze the existence of isolation by distance among populations from different locations (table 2), and elevation difference was also calculated using the altitude of collecting sites. the correlation of the symmetric matrix constituted by genetic and geographical distance, as well as genetic similarity and elevations, was further compared and analyzed using mxcomp programs and mantal test in ntsyspc-2.1. results issr profile nine issr primers generated 90 bands, which correspond to an average of 10 bands per primer. of these bands, 87.78% were polymorphic among the 20 populations. each band in the same size presented an unique issr genotype, which indicated an extensive genetic variation among the collected populations. the electrophoregram of amplification products using the primer is16 was shown in fig 2, and information on all issr primers and the corresponding amplification are shown in table 3. all primers generated multiple distinct bands. is16 and is09 generated least amounts of polymorphic bands, namely, 7 and 8 bands, fig 2. amplification products of the primer is16 for analyzed populations (a to t). m in the lane 1 represent dl2000 marker which produce 5 different sized bands (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp). sociobiology 65(3): 497-505 (september, 2018) 501 p op ul at io n a b c d e f g h i j k l m n o p q r s t a ** ** 0. 65 56 0. 83 33 0. 67 78 0. 57 78 0. 63 33 0. 55 56 0. 64 44 0. 61 11 0. 80 00 0. 60 00 0. 58 89 0. 60 00 0. 57 78 0. 61 11 0. 56 67 0. 68 89 0. 66 67 0. 60 00 0. 56 67 b 0. 42 23 ** ** 0. 55 56 0. 64 44 0. 74 44 0. 82 22 0. 72 22 0. 74 44 0. 71 11 0. 56 67 0. 74 44 0. 66 67 0. 67 78 0. 70 00 0. 68 89 0. 77 78 0. 67 78 0. 70 00 0. 76 67 0. 80 00 c 0. 18 23 0. 58 78 ** ** 0. 62 22 0. 52 22 0. 53 33 0. 43 33 0. 56 67 0. 53 33 0. 92 22 0. 50 00 0. 48 89 0. 50 00 0. 52 22 0. 51 11 0. 46 67 0. 65 56 0. 61 11 0. 54 44 0. 48 89 d 0. 38 89 0. 43 94 0. 47 45 ** ** 0. 70 00 0. 71 11 0. 70 00 0. 63 33 0. 73 33 0. 63 33 0. 65 56 0. 68 89 0. 63 33 0. 70 00 0. 68 89 0. 64 44 0. 74 44 0. 67 78 0. 72 22 0. 68 89 e 0. 54 86 0. 29 51 0. 64 97 0. 35 67 ** ** 0. 76 67 0. 84 44 0. 75 56 0. 78 89 0. 53 33 0. 80 00 0. 81 11 0. 82 22 0. 80 00 0. 78 89 0. 83 33 0. 64 44 0. 73 33 0. 86 67 0. 85 56 f 0. 45 68 0. 19 57 0. 62 86 0. 34 09 0. 26 57 ** ** 0. 72 22 0. 70 00 0. 77 78 0. 54 44 0. 74 44 0. 73 33 0. 72 22 0. 70 00 0. 71 11 0. 77 78 0. 76 67 0. 76 67 0. 76 67 0. 82 22 g 0. 58 78 0. 32 54 0. 83 62 0. 35 67 0. 16 91 0. 32 54 ** ** 0. 73 33 0. 76 67 0. 44 44 0. 77 78 0. 83 33 0. 75 56 0. 77 78 0. 74 44 0. 78 89 0. 64 44 0. 62 22 0. 80 00 0. 83 33 h 0. 43 94 0. 29 51 0. 56 80 0. 45 68 0. 28 03 0. 35 67 0. 31 02 ** ** 0. 81 11 0. 55 56 0. 84 44 0. 78 89 0. 75 56 0. 73 33 0. 74 44 0. 74 44 0. 64 44 0. 71 11 0. 77 78 0. 72 22 i 0. 49 25 0. 34 09 0. 62 86 0. 31 02 0. 23 71 0. 25 13 0. 26 57 0. 20 94 ** ** 0. 54 44 0. 83 33 0. 82 22 0. 76 67 0. 74 44 0. 80 00 0. 77 78 0. 72 22 0. 72 22 0. 81 11 0. 77 78 j 0. 22 31 0. 56 80 0. 08 10 0. 45 68 0. 62 86 0. 60 80 0. 81 09 0. 58 78 0. 60 80 ** ** 0. 51 11 0. 50 00 0. 51 11 0. 53 33 0. 50 00 0. 45 56 0. 64 44 0. 60 00 0. 53 33 0. 50 00 k 0. 51 08 0. 29 51 0. 69 31 0. 42 23 0. 22 31 0. 29 51 0. 25 13 0. 16 91 0. 18 23 0. 67 12 ** ** 0. 92 22 0. 77 78 0. 77 78 0. 76 67 0. 78 89 0. 64 44 0. 66 67 0. 82 22 0. 78 89 l 0. 52 95 0. 40 55 0. 71 56 0. 37 27 0. 20 94 0. 31 02 0. 18 23 0. 23 71 0. 19 57 0. 69 31 0. 08 10 ** ** 0. 76 67 0. 76 67 0. 77 78 0. 80 00 0. 70 00 0. 67 78 0. 81 11 0. 82 22 m 0. 51 08 0. 38 89 0. 69 31 0. 45 68 0. 19 57 0. 32 54 0. 28 03 0. 28 03 0. 26 57 0. 67 12 0. 25 13 0. 26 57 ** ** 0. 77 78 0. 81 11 0. 78 89 0. 64 44 0. 77 78 0. 80 00 0. 78 89 n 0. 54 86 0. 35 67 0. 64 97 0. 35 67 0. 22 31 0. 35 67 0. 25 13 0. 31 02 0. 29 51 0. 62 86 0. 25 13 0. 26 57 0. 25 13 ** ** 0. 72 22 0. 81 11 0. 66 67 0. 64 44 0. 77 78 0. 81 11 o 0. 49 25 0. 37 27 0. 67 12 0. 37 27 0. 23 71 0. 34 09 0. 29 51 0. 29 51 0. 22 31 0. 69 31 0. 26 57 0. 25 13 0. 20 94 0. 32 54 ** ** 0. 82 22 0. 67 78 0. 78 89 0. 85 56 0. 80 00 p 0. 56 80 0. 25 13 0. 76 21 0. 43 94 0. 18 23 0. 25 13 0. 23 71 0. 29 51 0. 25 13 0. 78 62 0. 23 71 0. 22 31 0. 23 71 0. 20 94 0. 19 57 ** ** 0. 63 33 0. 72 22 0. 81 11 0. 86 67 q 0. 37 27 0. 38 89 0. 42 23 0. 29 51 0. 43 94 0. 26 57 0. 43 94 0. 43 94 0. 32 54 0. 43 94 0. 43 94 0. 35 67 0. 43 94 0. 40 55 0. 38 89 0. 45 68 ** ** 0. 73 33 0. 68 89 0. 70 00 r 0. 40 55 0. 35 67 0. 49 25 0. 38 89 0. 31 02 0. 26 57 0. 47 45 0. 34 09 0. 32 54 0. 51 08 0. 40 55 0. 38 89 0. 25 13 0. 43 94 0. 23 71 0. 32 54 0. 31 02 ** ** 0. 77 78 0. 74 44 s 0. 51 08 0. 26 57 0. 60 80 0. 32 54 0. 14 31 0. 26 57 0. 22 31 0. 25 13 0. 20 94 0. 62 86 0. 19 57 0. 20 94 0. 22 31 0. 25 13 0. 15 60 0. 20 94 0. 37 27 0. 25 13 ** ** 0. 83 33 t 0. 56 80 0. 22 31 0. 71 56 0. 37 27 0. 15 60 0. 19 57 0. 18 23 0. 32 54 0. 25 13 0. 69 31 0. 23 71 0. 19 57 0. 23 71 0. 20 94 0. 22 31 0. 14 31 0. 35 67 0. 29 51 0. 18 23 ** ** t ab le 4 . g en et ic s im ila ri ty c oe ffi ci en t ( ab ov e di ag on al ) a nd g en et ic d is ta nc e (b el ow d ia go na l) a m on g ov er al l p op ul at io ns b as ed o n is sr m ar ke rs . respectively, whereas is23 generated the highest amount of polymorphic bands, namely, 10 bands. overall, 90 bands were generated with nine primers and the ppb was 87.78%. analysis of genetic diversity according to the genetic similarity and genetic distance of o. formosanus geo-populations, the average number of alleles (na) per locus was 1.8778 ± 0.3294 and the effective number of alleles (ne) was 1.4741 ± 0.3438. the nei’s gene diversity (h) and shannon information index (i) were 0.2832 ± 0.1696 and 0.4307 ± 0.2274, respectively, and the total gene diversity was 0.2832 ± 0.0288. these data revealed high genetic diversity at the population level among all samples and the abundant intraspecific variation in o. formosanus. by analyzing genetic similarity among the populations (table 4), we obtained 231 correlation coefficients ranging from 0.4333 to 0.9222 with an average of 0.6828, which indicated a high range of genetic variation among the populations studied in this research. the highest level of genetic similarity (0.9222) was observed between geo-populations k and l, as well as geo-populations c and j, which indicated that these populations were genetically related and shared the same genetic components. relationship between population genetic differentiation and geographical location the relationships of two sets of data (between genetic distance and geographic distance, between genetic identity and difference in elevation) are shown in figs. 3 and 4. data points were scattered without a discernible pattern. mantel test results revealed no significant correlation between genetic and geographical distance (r = 0.10517, p = 0.8062), as well as between genetic similarity and elevation (r = −0.12512, p = 0.0707). this finding indicated no significant isolation by distance. thus, proximity of sample collection did not affect genetic similarity. fig 3. correlation between genetic and geographical distance. the data points were generated by mxcomp programs and shown as a scattered pattern. no significant correlation were identified between genetic and sample geographical distance by mantel test (r = 0.10517, p = 0.8062). yq yang, lj pu, q wang, z wang, zp pang, yh long – population diversity of o. formosanus 502 upgma cluster analysis based on nei’s genetic distance a 0/1 matrix was constructed for all issr amplification bands by using the gel-pro analyzer software, and a dendrogram was generated with the ntsyspc-2.1 software (fig 5). twenty geo-populations sampled from anhui province were separated into two groups if 0.68 (genetic similarity coefficient) was set as a boundary. group i included geopopulations c, j and a. geo-populations a and c were distributed near and were both located in the northwest of anhui province, whereas geo-population j clustered into this group despite being sampled from huangshan and separated from geo-populations a and c by a natural geographic barrier (i.e., the yangtze river). within a single clade, which is divided into at least two groups, twelve geo-populations (e, s, p, t, o, m, g, n, k, l, i and h) from the central part of anhui province formed one sub-group in group ii. similar to geopopulations c and j, geo-populations b and f gathered into another sub-group despite being separated by the yangtze fig 5. dendrogram after unweighted pair-group method with arithmetic mean of o. formosanus based on nei’s genetic distance. population codes are explained in table 1. fig 4. correlation between genetic identity and difference in elevation. the data points were generated by mxcomp programs and shown as a scattered pattern. no significant correlation were identified between genetic similarity and sample elevation by mantel test (r = −0.12512, p = 0.0707). river (geo-population b was sampled from qimen and f from bengbu). these two clusters gathered with geo-population r and then combined with another cluster, which was generated by geo-populations d and q. discussion as botstein et al. (1980) proposed the range of polymorphic loci by ppb, all the nine issr primers presented high polymorphism and are potentially effective markers for the analysis of genetic diversity and phylogenetic relationship of geo-populations. in this study, only primer is23 can get as high as 100% ppb. but all the same primers except for is16 generated 100% ppb in another study on termites (long et al., 2009). the difference should be related to the species of termites. the objects of this study were all o. formosanus, while inter-species termites from odontotermes, reticultitermes and coptotermes were analyzed with high polymorphic loci. twenty populations investigated here are distributed in those natural areas, including jianghuai hilly area, dabie mountain area, plains along the yangtze river, and mountainous areas of south anhui, which generally cover the whole anhui province. the relationship between genetic variation and the geographical environment is important in research on genetic diversity and genetic distance, which represent the extent of genetic differentiation between two populations. the pattern of dendrogram was consistent with the result calculated by popgen32, but not the geographical structure entirely. for example, the result that the nearest sociobiology 65(3): 497-505 (september, 2018) 503 differentiation distance between geo-population c and j or between k and l were consistent with the result of genetic similarity (0.9222). in fact, geo-population c (from lu’an) and j (from huangshan) are separated by the yangtze river and the yellow mountain with 286km long distance, while geo-population k and l are sampled from the same city. the genetic structures of geo-populations b, f, p, and t were the same condition (table 2, fig 1). the lowest level of genetic similarity was observed between geo-populations c and g, which indicated that their genetic components varied greatly despite that the distance between them was only about 46km. these results showed no significant correlation between genetic similarity and geographical distance. and the genetic diversity of populations also depends on the breeding structure and the origin of the nest (husseneder et al., 2012). this phenomenon was also similar with the study done by husseneder (2013). he pointed out that populations from different provinces (guangdong, jiangxi, and hubei) were genetically differentiated while the genetic distance between populations was surprisingly small. huang (2013) found that the huanggang population was separated from other populations indicated by the largest genetic distance, while the populations of changsha, chongqing-1 and chongqing-2 shared smaller genetic distance although they were far enough in the actual geography. the long-range dispersal by alates and/or transport by human perhaps were the reason. so here, we also considered that this may be due to artificial factors that gave rise to one population as the offspring of the other population, or similar gene selective pressures, such as host, terrain features, geomorphology, and climatic factors. whether it was due to the similar gene selective pressure among populations or other artificial factors, further experiment need to carry out, such as the confirmation of the presence of gene exchange and gene flow among the populations of o. formosanus in the central part of anhui province. frequent communicating activities or inborn ability of short-range dispersal of the species might be the possible reason. long-distance migration of o. formosanus should also be noted for prevention and control. however, this study only analyzed o. formosanus in anhui province; thus, research on population genetic relationships, genetic structure, and migration pattern in a wider range must be performed. acknowledgement this study was supported by the national natural science foundation of china (grant 31300426, 31870635), the natural science foundation of universities of anhui province (kj2013a121), the anhui provincial key r & d projects 1804e03020320, and skltof201801109.” references aanen, d.k., eggleton, p., rouland–lefevre, c., guldberg– froslev, t., rosendahl, s. & boomsma, j.j. 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to become noisier upon exposure to organic pesticides, while silent upon exposure to neonicotinoid pesticides (seccomb, 2015). the use of beehive sound recordings has been developed for the detection of chemical-laced airborne threats under sbir agreement from the united states army center for abstract as the study of honey bee health has gained attention in the biology community, researchers have looked for new, non-invasive methods to monitor the health status of the colony. since the beehive sound alters when the colony is exposed to stressors, analysis of the acoustic response of the colony has been used as a method to identify the type of stressor, whether it is chemical, pest, or disease. so far, two feature sets have been successfully used for this kind of analysis, being these low-level signal features and mel frequency cepstral coefficients (mfcc). here we propose using soundscape indices, developed initially to delineate acoustic diversity in ecosystems, as an alternative to now used features. in our study, we examine the beehive acoustic response to trichloromethane laced-air and blank air and compare the performance of all three feature sets to discern the colony’s sound between the hive being exposed to the chemical and not. our results show that sound indices overperform the alternative features sets on this task. based on these findings, we consider sound indices to be a valid set of features for beehive sound analysis and present our results to call the attention of the community on this fact. sociobiology an international journal on social insects mz sharif1, 2, f wario3, n di1,2, r xue1,2, f liu1,2 article history edited by denise alves, usp, brazil received 22 september 2020 initial acceptance 20 november 2020 final acceptance 07 december 2020 publication date 28 december 2020 keywords apis mellifera, bioacoustics, low-level signal features, machine learning, trichloromethane, soundscape index corresponding author fanglin liu https://orcid.org/0000-0002-8371-6316 institute of technical biology and agriculture engineering hefei institutes of physical sciences chinese academy of sciences hefei, anhui, p.r. china. e-mail: flliu@ipp.ac.cn environmental health research. the most dynamic thing about the results obtained from the investigates is that the bee colony sound not only alters in response of exposure to variant stressor, but in some cases also alters to delineate the particular type of chemical, pest, and disease (bromenshenk et al., 2009, 2004). a beehive is a small ecosystem, consisted of 30,000-50,000 individual bees. the soundscape indices, used to describe the sound scene, may be suitable to delineate the beehive sounds emitted by different individuals. but their efficacy in classifying beehive audio samples has not been explored so far. audio-based classification models have been explored to detect the statuses of a beehive, such as queenless (howard et al., 2013), swarming (ferrari et al., 2008), and diseases or pests (robles-guerrero et al., 2017) etc. however, the automatic classification of beehive audio samples remains a challenge yet. 1 institute of technical biology & agriculture engineering, hefei institutes of physical science, chinese academy of sciences, hefei, 230031, p.r. china 2 university of science and technology of china, hefei, 230026, p.r. china 3 university of guadalajara av. juárez n° 976, col. centro, 44100, guadalajara, jalisco, méxico research article bees soundscape indices: new features for classifying beehive audio samples sociobiology 67(4): 566-571 (december, 2020) 567 classification of audio samples involves two stages, the first in which certain features are extracted from waveform files, and the second in which a classification model is built on the extracted features. several classification schemes have been proposed, including statistic models, such as linear discrimination analysis (qandour et al., 2014) and self-organizing maps (howard et al., 2013), and machine learning algorithms (nolasco & benetos, 2018), such as support vector machine (qandour et al., 2014), random forest (robles-guerrero et al., 2017) and neural network (rybin et al., 2017). it has been shown that the schemes are not different in the classification accuracy (qandour et al., 2014). given the current state of audio classifiers, further advances could be made by developing powerful features, rather than building new classification schemes. so far, two feature sets are used for the classification of beehive audios. they are the low-level signal feature set, including zero-crossing rate, bandwidth, spectral centroid, and signal energy (qandour et al., 2014), and the mel spectra set, which are dozens of mel frequency cepstral coefficients (mfcc) (robles-guerrero et al., 2017) (nolasco et al., 2019). recently, ecologists have been developing soundscape indices to assess biodiversity at landscape scale (mammides et al., 2017). in the present study, we examine the beehive sound response to an organic chemical compound (trichloromethane [chcl3]), as the beehive sonograms are altered when bees are exposed to different chemicals (bromenshenk et al., 2015). we also compare the performance of the soundscape indices with that of the low-level signal properties in discriminating beehive sound response to this chemical. the application of soundscape indices in classifying beehive audio data is also discussed. material and methods experimental setup the experiment was conducted in the spring and the summer, 2019, respectively, at the hefei institutes of physical sciences, chinese academy of sciences. a beehive with a. mellifera was kept in a lab room. a pre-cut circular hole in the glass window of the room fits tightly around a transfer entrance/exit tube. the bees were allowed to come and go through the transfer tube freely. an outdoor-facing landing platform was fixed outside right below the entrance/exit end of the transfer tube, giving our bees a convenient perch for takeoff and landing. the beehive was set for one week. a vacuum pump was used to pump the air at a ~ 5 meters distance. the air was first pumped to a glass jar via a flexible plastic pipe in which some filters with or without trichloromethane were placed. later, the air was pumped to the beehive via beehive entrance. the content of trichloromethane in the glass jar was 250 mg/ m3, which is slightly higher than the maximum allowable concentration in the open air (240 mg/m3). an airflow rate of 0.4 m/s was controlled by an air-flow meter. data collection audio data were collected at night time in good weather days with a microphone, positioned inside the hive to avoid propolization, at a sampling rate of 22.05 khz (16 bits, one channel). to ensure the data are correctly labeled with ventilating trichloromethane-laced air, only the continuous recordings within 3-23 min after the air was released to the hive were used. the audio files were saved in a waveform (wav). the hive air was expelled out by a fan at the hive entrance for two days before blank air was pumped. the procedure alternated ventilating trichloromethane-laced air, and ventilating blank air was repeated five times (3 times for the spring and two times for the summer). feature extraction each 20-min audio file, either labeling with blank or trichloromethane-laced air, was divided into 20-s samples without overlap. the common low-level signal features, such as zero crossing rate (zcr) and spectral centroid (sc), and 12 mel-frequency cepstral coefficients, were extracted from each 20-s sample with a 20-ms frame with 50% overlap. mel-frequency cepstral is a standard extraction approach for similar classification tasks (robles-guerrero et al., 2017). in the r programming language (teamrcore, 2015), such features were extracted according to previously described methodology (li et al., 2001). the common soundscape indices, acoustic complexity index (aci), acoustic diversity index (adi), acoustic evenness index (aei), and bioacoustic index (bi), were calculated with the “soundecology” package (villanueva-rivera and pijanowski 2016). aci was analyzed with a cluster size of 10 seconds and limited to 6000 hz. the maximum frequency of adi and aei is set to 6000 hz. the size of the frequency bands is set to 1000 hz. other indices were calculated with the default settings. feature performance feature performance was estimated using random forests. briefly, 75% of the 20audio samples were randomly chosen as a training group, the others as a test group. for the training group, the features of an audio sample were grouped into either blank ventilating air or trichloromethane-laced air. a binary classification model was built based on the features of the audio samples by the randomforest (rf) package “v4.6-12” (liaw & wiener, 2002). details about this classification method are described in a publication (breiman, 2001). the trained rf model classified each audio sample in the test group to find which class it most probably belongs to. the overall misclassification rate, estimated from the confusion matrix, was used as an essential performance measure. random forest possesses a couple of methods to evaluate the importance of every predictor variable in the model; the methods included are mean decrease in accuracy (mda) and mean decrease in gini (mdg) (cutler et al., 2007; calle & urrea, 2011). but, we had chosen mda in our current investigation to predict the importance of indices (fig 1a, d & g) as computed by random forest. mz sharif, f wario, n di, r xue – monitoring honeybee colony: the role of acoustic features568 fig 1. the importance order of three feature sets and their changes with time. black circles denotes ventilating blank air, red circles denotes trichloromethane-laced air. each point represents the datum extracted from a 20-s audio sample. czr: zerocrossing rate, cs: spectral centroid, c[1] to c[12]: mfcc coefficients, sp.ent: spectral entropy, meanfreq: mean frequency, meanamp: mean amplitude, energy: energy of singal, rms: root-mean-square level, aci: acoustic complexity index, adi: acoustic diversity index, aei: acoustic evenness index, bi: bioacoustic index results concerning time, each treatment (i.e., blank air and trichloromethane laced-air) yielded significant differences among all the indices. regarding the upper panels, they showed the importance order of the low-level signal features and their changes with time (fig 1b and c). zcr and sc were the most critical features (fig 1). they significantly fluctuated with time (fig 1b and c); either blank air or trichloromethane-laced air was ventilated into the beehive. visual examination designates that the values of zcr and sc for trichloromethane laced-air fluctuate and decrease with time and remain below their values for blank air. based on the two features, the beehive sound responses to the two air types could not easily be discriminated against via a classification model, such as linear discrimination analysis. the middle panels showed the importance order of mfccs and their changes with time. the mfcc coefficients (c[1] and c[3]) were the most fundamental features (fig 1d). the graphic representation exhibits that the values of c[1] and c[3] features for chemical laced-air fluctuated, but the former upsurge and later declined with time. inversely, for blank air, the c[1] feature oscillated below the chemical laced-air, while the values of c[3] oscillated above the chemical laced-air. the changes detected in c[1] coefficient for trichloromethane and blank air were clear to observe the acoustic variations, but the changes seen in c[3] were mixed at some points (fig 1e and f). according to the classification accuracy, mfcc showed good performance than the low-level features, but the low performance was noted compared to the soundscape indices (table 1). anyhow, the mean decrease in accuracy values for c1 and c3 were higher than other coefficients, which represent sociobiology 67(4): 566-571 (december, 2020) 569 them as a critical feature for acoustic discrimination (fig 1d). based on mfcc features, especially c[1], the beehive sound responses to the two air types can be discriminated. regardless of upper and middle panels, the lower panels showed more importance in soundscape indices and their change time. aci and adi were also the most significant indices (fig 1h and i); based on these indices, beehive sound responses to blank air remained stable. however, bees response to trichloromethane-laced air dramatically fluctuated, either below (fig 1i for aci) or above (fig 1h for adi), the response of bees to blank air. by visual examination, the soundscape indices were more discriminative power than the low-level signal features and mfccs for binary classification. the classification accuracy calculated using rf model is summarized in table 1. in terms of classification accuracy, the soundscape indices have 9.99% to 11.66% better performance than other feature sets including mfcc, and low-level signal features, respectively. based on the four crucial low-level signal features (zcr, sc, mean free, and sp.ent) extracted from each 20-s audio sample, the classification accuracies were 75% for trichloromethane-laced air and 81.67% for blank air, respectively. based on the four important mfccs, the classification accuracies were 76.90 % for trichloromethanelaced air and 80.00% for blank air, respectively. according to the soundscape indices (aci, adi, ae, bi), the accuracies were 88.46 % for trichloromethane-laced air and 91.67 % for blank air, respectively. furthermore, the mean decrease in accuracy values for aci and adi is higher than other feature sets like bi, aei, sc, and others (fig 1a, d & g). that is why both the soundscape indices (aci and adi) were designated as the most central indices, followed by other indices and feature sets. discussion audio-based beehive monitoring becomes a partial solution to precision apiculture (ferrari et al., 2008). several acoustic features have been explored to classify beehive audio samples (cecchi et al., 2019; qandour et al., 2014). in this study, we show that the soundscape indices are more potent than the low-level signal features and mfccs in discriminating bee response to chemicals. anyhow, mfcc features also achieved good classification accuracy than the low-level features, although less than the soundscape indices (fig 1e and 1f). similar results for classification accuracy of mfcc features were presented in a research by zgank (2019). this research describes that with the medium and high complexity acoustic models, the mfcc features achieved 75.43% and 82.27% classification accuracy without cepstral mean normalization, and 74.81% and 81.96% with it, respectively. but, the soundscape indices may be more effective and can be used as new acoustical features to classify the beehive audio data. why are soundscape indices suitable for the classification of beehive sounds? temporal variations of the basic features are essential for classification (breebaart & mckinney, 2004). the greater the variability of the basic features, the more likely it achieves a high classification accuracy. unlike lowlevel signal features, which are extracted from the stationary periods (robles-guerrero et al., 2017), the soundscape indices are designed to assess changes in acoustic energy in time or frequency domain (or both) as an analog for the number of vocalizing animals in an area (parsons et al., 2016). the extraction process for the soundscape indices includes signal variations as much as possible. therefore, the soundscape indices are suitable representations in the complex context of a beehive sound scene. most studies on avian soundscapes use 1-min audio file to extract soundscape indices. in our research, we found that the soundscape had a time scale. for the spring samples, as the frames increased from 20 to 30 s, the classification accuracy for trichloromethane air remained 88.46% (table 1). this is why we chose 20 s as the frame size for the calculation of the soundscape index. whether it is the optimum sample size for calculation of the soundscape indices should be further examined. nonetheless, from a perceptual point of view, this is a remarkably short section of audio files on which the soundscape indices are calculated. improvements in classification performance could indeed be made if classification were based on more than a one-time scale. table 1. comparison of the discriminative power between three feature sets based on random forest. low-level signal features: spectral centroid (sc), zero-crossing rate (zcr), mean frequency (meanfreq), and spectral entropy (sp.ent); mel frequency cepstral coefficient (mfccs): c[1], c[3], c[7] and c[5]; soundscape indices: acoustic complexity index (aci), acoustic diversity index (adi), acoustic evenness index (aei), and bioacoustic index (bi). feature set sample size frame size classification accuracy trichloromethane-laced air blank air low-level signal features 20 s 20 ms 75.00% 81.67% mfcc 20 s 20 ms 76.90% 80.00% soundscape indices 20 s 88.46% 91.66% soundscape index 30 s 88.46% 86.67% mz sharif, f wario, n di, r xue – monitoring honeybee colony: the role of acoustic features570 in short, we show that the performance of the soundscape index is superior to that of the standard low-level signal features in classifying the beehive audio samples. the soundscape indices, which include signal variations as much as possible, may be necessary for their suitability to represent the beehive-inside complex sound scene. several chemical pesticides (chlorpyrifos, cypermethrin, carbofuran, bifenthrin, clothianidin, and others) have previously been checked for harmful effects on the lives of honey bees (yao et al., 2018; yang et al., 2020; dai et al., 2010). therefore, we recommend that researchers should use soundscape features as a valuable tool for evaluating the health of colonies in future studies. moreover, the automatic classification of beehive audio samples can be optimized using the soundscape features. acknowledgments this work was supported by the key research program of the chinese academy of sciences (grant no. kgfzd-135-17-010). the authors would like to thank zhuqing lv, sabah mushtaq puswal, jianjun liao, and fenmei wang for their assistance in the trails. authors' contribution fl and mzs conceived and designed the analysis, and did the data collection. fl and mzs wrote the manuscript. mzs refined the manuscript with grammatical corrections and improved the description of the result section of the manuscript. fl did the data analysis. fw helped in manuscript preparation, write up and suggesting improvements in data analysis. nd and rx revised the manuscript and contributed to significant improvements. all the authors coordinated to revise and approve the final version of the manuscript. references 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(2020). acute and chronic toxicity of acetamiprid, carbaryl, cypermethrin and deltamethrin to apis mellifera larvae reared in vitro. pest management science, 76: 978-985. doi: 10.1002/ps.5606. zgank, a., 2020. bee swarm activity acoustic classification for an iot-based farm service. sensors. 20: 21. doi: 10.33 90/ s20010021 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3431sociobiology 66(2): 287-292 (june, 2019) post-embryonic development of the seminal vesicle in the stingless bee melipona quadrifasciata lepeletier, 1836 (apidae: meliponini) introduction the male reproductive tract of insects has a pair of testes connected to an ejaculatory duct via two vasa deferentia (snodgrass, 1935). in many insects, including those of the order hymenoptera, there are regions termed seminal vesicles along the vasa deferentia, modified for spermatozoa storage. accessory glands connect to the vasa deferentia or near the ejaculatory duct. the number of accessory glands may differ among species (baer et al., 2000; chapman, 2013). the seminal vesicle is mesodermal in origin and consists of dilated portions of vasa deferentia (chapman, 2013). in eusocial honeybees, the seminal vesicle is an elongated tube (cruz-landim & cruz-höfling, 1969; sawarkar & tembhare, 2014), whereas in stingless bees (meliponini), it is spherical (dallacqua & cruz-landim, 2003; cruz-landim, abstract the male accessory glands of stingless bees (apidae: meliponini) are absent and the morphology of their seminal vesicle indicate probable secretory function by this organ. this study investigated the post-embryonic development of the seminal vesicles in males of the stingless bee melipona quadrifasciata by histology and histochemistry. white-eyed pupae, pink-eyed pupae, brown-eyed pupae, black-eyed pupae, newly emerged and sexually mature males were studied. seminal vesicle has a wall with a single layered epithelium onto a thin basement membrane, followed by a well-developed muscle layer. the epithelium is polarized in the pupal stage with basal cell region strongly positive for glucoconjugates and carbohydrates. the seminal vesicle has an enlarged lumen from the young pupal stages with luminal content increasing gradually with glucoconjugates along the pupal development. in the newly emerged and mature males, the histochemical tests to carbohydrates were negative. in the sexually mature males, spermatozoa clusters are embedded by the glucoconjugates content of the seminal vesicle lumen. in conclusion, the seminal vesicle of m. quadrifasciata has a secretory function during the pupal stage and in newly emerged males, whereas in adult males this organ stores the spermatozoa. sociobiology an international journal on social insects rp ferreira, ha werneck, j malta, ad teixeira, lao campos, je serrão article history edited by eduardo almeida, usp, brazil received 09 may 2018 initial acceptance 01 july 2018 final acceptance 14 december 2018 publication date 20 august 2019 keywords hymenoptera, reproductive system, male accessory glands. corresponding author ríudo de paiva ferreira universidade federal de viçosa departamento de biologia geral avenida ph rolfs s/nº, cep: 36570000 viçosa, minas gerais, brasil. e-mail: riudopaiva@gmail.com 2009). in meliponini, newly emerged males have empty seminal vesicles with epithelial cells characterized by apical microvilli and collapsed lumen, whereas in sexually mature males, the seminal vesicle is filled with spermatozoa with a cubic epithelium (lima et al., 2006). the male accessory glands in bees are structures of mesodermic origin and open into the ejaculatory duct, releasing compounds that are transferred into the female together with spermatozoa during mating (chen, 1984; gillot, 1988, 2003). the accessory glands are already formed in newly emerged adult insects and their activity increases during the first days of emergence in the adult insect (chapman 2013, xu et al., 2015). in bees, accessory gland secretions are proteins, but can also include carbohydrates and lipids (blum et al., 1962; blum et al., 1967; chen, 1984; baer et al., 2000; boomsma et al., 2005; colonello & hartfelder, 2005; gorshkov et al., 2015). departamento de biologia geral, universidade federal de viçosa, viçosa-mg, brazil research article bees rp ferreira et al. – development of the seminal vesicle of a stingless bee288 this secretion affects female reproductive biology after mating, including protection, storage and activation of sperm, sperm competition, responsiveness inhibition, and fecundity enhancement (fausto et al., 2000; baer & boomsma, 2004). stingless bees are particularly interesting, because male accessory glands are absent (ferreira et al., 2004), suggesting that the epithelial cells of seminal vesicles, vasa deferentia or the ejaculatory duct play a role in the secretion of compounds to form sperm (dallacqua & cruz-landim, 2003; araújo et al., 2005). it has been shown (dallacqua & cruz-landim, 2003; brito et al., 2010) that the epithelial cells of the seminal vesicle melipona bicolor lepeletier, 1938 have secretory activity. this study describes the post embryonic development of the seminal vesicle in melipona quadrifasciata lepeletier, 1836, providing further evidence that seminal vesicles, in substitution for male accessory glands, produce compounds in stingless bees. materials and methods male pupae and adults of m. quadrifasciata were obtained from three colonies kept at apiário central (“central apiary”), universidade federal de viçosa (viçosa, mg, brazil). brood combs were transferred to laboratory at 28 oc and five individuals of each white-eyed pupae (wep), pink-eyed pupae (pep), brown-eyed pupae (bwp) and black-eyed pupae (blp), were collected from brood cells with forceps. the newly emerged males (n=5) were collected immediately after emerging from the brood cells. sexually mature males (n=5) were collected 15 days after emerging from the brood cells. vouchers specimens were deposited in the entomological collection of apiário central-universidade federal de viçosa (ufv). males were dissected in presence of 0.1 m sodium phospate buffer ph 7.6 (pbs) and the reproductive tracts transferred to zamboni’s fixative solution (stefanini et al., 1967) for 4 h. after washing in pbs, samples were dehydrated in a graded ethanol series (70, 80, 90, 95o) and embedded in jb-4 historesin. tissue seccions of 2 μm thin were obtained from the leica rm2255 rotary microtome and were stained with hematoxylin and eosin. to characterize the biochemical composition of the luminal contents of the vesicles, some tissue sections were submitted to the following histochemical tests: pas for glycogen and neutral carbohydrates (junqueira & junqueira, 1983); alcian blue ph 2.5 for glucoproteins; nile blue for lipids and mercury bromophenol blue for total proteins (bancroft & gamble, 2008). all sections were examined in zeiss primo star light microscopy. results the seminal vesicle of m. quadrifasciata is a paired spherical structure found in all pupal stages. it has an enlarged lumen lined by a single layer of columnar cells in all pupal stages and in newly emerged males (fig 1a-g). in the adult males the epithelium becomes cubic (fig 2). all stages are presents onto a thin basal membrane followed by thin muscle layer (figs 1,2). fig 1. histology of the seminal vesicles of pupae and adult males of melipona quadrifasciata. (a-d) white-eyed pupae (wep). (a) wep with wide lumen. (b) clear areas in the basal cell region (dashed rectangle). (c-d) positive pas reaction in the basal region of the epithelium (asterisks) and lumen (arrow) of the vesicle of the wep. (e-g) pink-eyed pupae as seen for the wep seminal vesicles. abbreviations: (ep) seminal vesicle epithelium, (l) seminal vesicle lumen, (n) nuclei, (m) muscular sheath, (pov) post vesicular deferent duct. scale bar = 20µm. sociobiology 66(2): 287-292 (june, 2019) 289 hematoxylin-eosin staining (fig 1a) revealed that the epithelial cells of seminal vesicles have nuclei in the median cell portion, whereas the basal cell portion presents clear areas (fig 1b), which shows a positive pas reaction (fig 1c,d). the clear areas in the basal cell portion increased in size with advancement of pupal development (fig 1d, 2a). however, in both newly emerged and sexually mature adult males, the epithelial cells of the seminal vesicles were uniformly stained (fig 2d,h), although in mature males the cells were cubical (fig 2h). in newly emerged males, the seminal vesicles showed an amorphous luminal content (fig 2d-g). the clear basal cell region of the seminal vesicle epithelium was strongly positive for pas test showing the presence of neutral carbohydrates in all pupal stages (figs 1c,d,f,g, 2b,c,g). the luminal content of the seminal vesicle was pas-positive in white-eyed pupae (fig 1c, d). in pink-eyed pupae, the apical surface of the seminal vesicle epithelium had pas-positive granules (fig 1e-g). in brown-eyed pupae, carbohydrates were stored throughout the cell cytoplasm of the seminal vesicle (fig 2b-c,g). in the black-eyed pupae, the amount of carbohydrates stored was higher in the basal cell region (fig 2 c) and lumen. small lipid droplets (fig 2 e) were fig 2. histology of the seminal vesicles of pupae and adult males of melipona quadrifasciata. (a) detail of the columnar epithelium of the browneyed pupae (bwp). (b) seminal vesicle of the bwp. (c) black-eyed pupae (blp). the asterisks indicate the carbohydrates in cytoplasm of the cells in the basal region of the epithelium. (d) vesicle of the newly emerged male with secretion in the lumen. (e) lipid droplets (arrow) of uniform size in the apical portion of the epithelium. (f-g) sexually mature male. (f) seminal vesicle showing the luminal region (l) filled with sperm and, the cubic epithelium (ep). (g) the pas-positive granules involve the spermatozoa bundles (arrow) in seminal vesicle luminal region. scale bar = 20µm. rp ferreira et al. – development of the seminal vesicle of a stingless bee290 found in the apical cell region of the seminal vesicle epithelium of brown and black-eyed pupae. protein content was found in all cells of the seminal vesicle, but not stored in secretory granules. in newly emerged males, the cells of the seminal vesicle were pas-positive (fig 2d, e), whereas in sexually mature ones, when the seminal vesicle was filled with spermatozoa, the epithelial cells did not possess neutral carbohydrate storage (fig 2f), despite the presence of pas-positive granules in the lumen of the seminal vesicle (fig 2g). these granules were found to be associated with the luminal content of the vesicle along with the spermatozoa bundles (fig 2g). the main changes in the epithelium of seminal vesicles of m. quadrifasciata during the pupal development and after adult emergence are summarized in the table 1. discussion the histology of the seminal vesicle was similar along the entire pupal stage of m. quadrifasciata. it suggests that the differentiation of this region of the male reproductive tract occurs in early white-eyed pupae. in fact, spermatogenesis in stingless bees begins during the pupal stage (cruzlandim & dallacqua, 2002) and the spermatozoa storage organ should be functional during this developmental stage. the seminal vesicle of m. quadrifasciata pupae is enlarged and filled with amorphous content, like that in melipona mondury smith, 1863 (lima et al., 2006), contrasting with narrow and empty lumen of seminal vesicles of newly emerged males of scaptotrigona xanthotricha moure, 1950 (araujo et al., 2005) and friesella schrottkyi (friese, 1900) (brito et al., 2010). our histochemical tests show the presence of glucoconjugates in the epithelial cells of the seminal vesicles of pupae and newly emerged males, but its absence in mature ones. in the latter, glucoconjugates occur associated with spermatozoa bundles in the seminal vesicle lumen, suggesting that these compounds are produced during the pupae stage and released to the seminal vesicle. in mature males of other meliponini species, it has been reported that glycoprotein coating spermatozoa bundles into the seminal vesicle (moreira et al., 2004; cruz-landim, 2009). the presence of lipid droplets in the apical region of the seminal vesicle epithelium in late black-eyed pupae suggests that this compound may be used for spermatozoa maintenance during storage in adult. fatty acids are found in male accessory glands in bumblebees (baer et al., 2000). because secretions of male accessory glands are released during insect mating and transferred together with spermatozoa to the female (colonello & hartfelder, 2005; den boer et al., 2009, 2015), compounds similar to those produced in these glands may be synthesized in the seminal vesicle of stingless bees. tozetto et al. (2007) pointed out an increase in the protein content in the seminal vesicle of late pupae of apis mellifera linnaeus, 1758, which may be due to organ growth. furthermore, sawarkar & tembhare (2014) showed that the seminal vesicle in apis cerana indica fabricius, 1798 has secretory activity with high protein content, followed by lipids and carbohydrates, which may aid in the storage and survival of spermatozoa in the seminal vesicle. stage of development histochemical tests characteristics of seminal vesicle pas nile blue mercuric bromophenol blue white-eyed pupae + + lumen enlarged, filled with amorphous content, epithelium high, nucleus in the middle portion of the cell, basal region with weakly stained clear areas. carbohydrate storage in the basal region of the epithelium. pink-eyed pupae + + lumen enlarged, filled with amorphous content, epithelium high, nucleus in the middle portion of the cell, basal region with weakly stained clear areas. basal region of the epithelium with pas-positive granules. brown-eyed pupae + + + lumen enlarged, filled with amorphous content, epithelium high, nucleus in the middle portion of the cell, large clear areas in the basal region of the epithelium. high carbohydrate storage in the basal portion, pas-positive granules dispersed in the cell cytoplasm. lipid droplets of uniform size in the apical portion of the epithelium. black-eyed pupae + + + lumen enlarged, filled with amorphous content, epithelium high, nucleus in the middle portion of the cell, basal region with weakly stained clear areas. basal portion of the epithelium strongly pas-positive. lipid droplets present. luminal content with carbohydrate. newly emerged male + + lumen enlarged, epithelium high and uniformly stained. low carbohydrate storage in the basal portion of the epithelium. luminal content with carbohydrate. sexually mature male + lumen enlarged, epithelium cubic uniformly stained, without lipids and carbohydrates storage. luminal content with pas-positive granules associated with spermatozoa bundles. table 1. main characteristics of the seminal vesicle during post-embryonic development of males of melipona quadrifasciata, by histochemical tests. test positive (+) and negative (-). sociobiology 66(2): 287-292 (june, 2019) 291 the histochemical composition of the luminal contents of the seminal vesicle of m. quadrifasciata during the pupal stages shows a large amount of carbohydrates, low lipid and protein contents, which may act in reducing oxidative stress during the storage of spermatozoa in the organ. although experimental manipulations suggest multiple mating in m. quadrifasciata (lopes et al., 2003) and microsatellite analyses confirm polyandry in m. mondury (viana et al., 2015), the ejaculated material during sperm transfer associated with the physical barrier of the chitin-lined organs of the reproductive tract in m. quadrifasciata may favor the monarchical behavior of this bee, preventing multiple mating. the epithelium of the seminal vesicle of m. quadrifasciata is polarized during the pupal stage with the basal cell region storing neutral carbohydrates and glucoconjugates. in addition, the seminal vesicle lumen is enlarged in early white-eyed pupae being gradually filled by pas-positive content, whereas in adult males, storage of organic compounds in the lumen is reduced. these findings, together with similar results among other meliponini (dallacqua & cruz-landim, 2003; araújop., 2005; lima et al., 2006), suggest that the seminal vesicle plays a dual role in stingless bees: in pupae and newly emerged males, it is a secretory organ, whereas in sexually mature males, the seminal vesicle functions as a spermatozoa storage organ, overlaying the absence of male accessory glands in meliponini. acknowledgments this research was supported by the brazilian research agencies fapemig, cnpq and capes. the authors are also grateful to the two anonymous reviewers for their suggestions for improving the quality of the manuscript. references araújo v.a., zama u., neves c.a., dolder h. & linoneto j. 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(2015). nutrition regulation of male accessory gland growth and maturation in tribolium castaneum. scientific reports, v. 5, p. 10567. doi: 10.1038/ srep10567. doi: 10.13102/sociobiology.v65i2.2345sociobiology 65(2): 280-284 (june, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 estimating colonies of plebeia droryana (friese, 1900) (hymenoptera: apidae: meliponini): adults, brood and nest structure introduction stingless bees are found in tropical and subtropical regions all around the world (sakagami, 1982) and they have populated tropical regions for over 65 million years – longer than apis, the honeybees (camargo & pedro, 1992; michener, 2000). however, stingless bees have 50 times more species and, as emphasized here, differ from apis in many biologically significant ways (roubik, 2006). the colonies have individuals active every day that perform their activities and, therefore, have supported climate impact of the environment (roubik, 1989; hansell, 1993). the colonies of stingless bees present structural, behavioral and physiological adaptations, which protect bees from the external environment (roubik & peralta, 1983). their nests are perennial, and they are active throughout the year (michener, 1974; sakagami, 1982). abstract estimate of stingless bee colonies including nest structures and quantitative brood and adult individuals are scarce. here, we describe a new approach to estimate colonial parameters from nest structure, adults and brood. we used five colonies of plebeia droryana (friese, 1900) to evaluate colony size and weight of adult and brood. nest architecture in p. droryana is similar to the species of the same genus but differ to the other stingless bees. in this species, we counted a total of 9 to 12 brood combs and a total of 19 to 25 food pots in the nests. the number of individuals in the colonies is considered small and our estimate was based on individual and group weight. our study approach may contribute to further detailed studies of the species nest and considering the stingless bees to the pollination of agricultural crops and native flora of tropical regions, it is important to add information about the biology of p. droryana. sociobiology an international journal on social insects ys roldão-sbordoni, fs nascimento, s mateus article history edited by denise alves, usp, brazil received 19 october 2017 initial acceptance 04 december 2017 final acceptance 13 march 2018 publication date 09 july 2018 keywords stingless bees; nest estimative; population size. corresponding author yara sbrolin roldão-sbordoni faculdade de filosofia, ciências e letras de ribeirão preto são paulo (usp) departamento de biologia, laboratório de comportamento e ecologia de insetos sociais avenida bandeirantes, 3900 cep 14040-901 ribeirão preto-sp, brasil. e-mail: yara.roldao@hotmail.com stingless bees usu ally build their nests in holes in trees, cavity in the soil, and even in cavities in human constructions, but some also construct aerial nests (nogueira-neto, 1997), and some species that regularly establish their nests within nests of nasutitermes or ants (michener, 2000). in contrast to the apini, new stingless bee nests are started by workers going back and forward from an existing old colony. they transfer building materials and food to the new site. ultimately, a young queen flies to the new site, workers stay there, and independence from the old colony is gradually attained over a period of weeks or months (michener, 1946; inoue et al., 1984; van-veen & sommeijer, 2000). nests are made of wax secreted from the metasomal terga mixed with resins and gums collected by the foragers. some species add mud, feces, or other materials in several parts of the nest. in all species, the composition and texture differ in different parts of the nest (michener, 2000). in some species, faculdade de filosofia, ciências e letras de ribeirão preto são paulo usp, departamento de biologia, laboratório de comportamento e ecologia de insetos sociais, ribeirão preto-sp, brazil research article bees sociobiology 65(2): 280-284 (june, 2018) 281 the brood combs are covered by layers of wax involucrum and the storage pots are made with cerumen. several bee species build a strong structure with cerumen, mud, and resin called scutellum or batumen (zucchi & sakagami, 1972). some of the supports and batumen plates are of tough material, and the external sheet of batumen around some exposed nests is harder and brittle (michener, 2000). nest characteristics such as the nesting site, architectural complexity, and building materials may be taxon-specific and provide an excellent opportunity to assess information about underlying behavioral evolution of the respective taxa and higher groups (rasmussen & camargo, 2008). the nest architecture of stingless bees consists of an entrance, access tunnel, food pots (nectar and pollen storage) and brood area, with horizontal combs or in cluster (zucchi & sakagami, 1972; wille & michener, 1973; michener, 1974). michener (1974) described the colony size of various social bees, especially the genus trigona. in addition, lindauer and kerr (1960) presented an estimate of adult workers in colonies of stingless bee colonies. they evaluated 10 species and among them plebeia droryana (formerly known as trigona droryana) had 2,000 to 3,000 adults. wille and michener (1973) showed another estimate in which measured nest structures as food pots and brood cells diameter and length. using lindauer and kerr (1960) estimative references to p. droryana and other bees, duarte et al. (2016) studied nest architecture of tetragona clavipes and presented structures and individuals estimative. the genus plebeia schwarz, 1938 consists a very diverse group and is distributed in the neotropical region. plebeia is morphologically the most primitive taxa within stingless bees and is mainly found in the southeast region in brazil (wittmann, 1989; michener, 1990; witter et al., 2007). the stingless bee p. droryana has a wide geographic distribution, which includes argentina, bolivia, paraguay and some brazilian states as bahia, espírito santo, minas gerais, paraná, pernambuco, rio grande do sul, rio de janeiro, santa catarina and são paulo (moure et al., 2007; camargo & pedro, 2013). these bees are small (4 mm) and not aggressive, build royal cells for queen production (nogueira-neto, 1970). although some estimative recordings for p. droryana nests and other stingless bee species have been made, studies on basic biology aspects are still scarce. the stingless bees are important for pollination of agricultural crops and native flora of tropical regions and income source as the meliponiculture. therefore, new studies that provide information about the biology of neotropical stingless bees and can collaborate with the conservation of the species by measuring the nest structures and to allow better handling conditions are necessary (cortopassi-laurino et al., 2016; jaffé et al., 2015). here, we present a description of the nest structure and an evaluation of adults and brood of p. droryana. moreover, we provide a new method to estimate stingless bee colonies. material and methods study site the study was carried out in the campus of the university of são paulo at ribeirão preto, brazil (21°10’39’’s, 47°48’24’’w). five colonies of the stingless bee p. droryana were kept in hives made with wood (14.0 ́ 14.0 ́ 12 cm; walls: 2 cm; volume of 2.352 cm3) in the laboratório de comportamento e ecologia de insetos sociais. the hives were connected to the outside via plastic tubes through which the bees could freely exit and enter in the nest. the bees collected pollen and nectar freely because they no received artificial feeding. colony size we evaluated colony size by weighting p. droryana colonies (hereafter pd1, pd2, pd3, pd4 and pd5) between 17th september and 27th september 2013. we used carbon dioxide to anesthetize individuals and to preserve the structure of the nest. we weighted the combs, the involucrum wax and the adult individuals. we estimated the weight of food pots before removing the combs. workers and males were weighted together, but males were found only in one nest. we counted the number of the food pots (honey and pollen), brood combs with egg, larvae (small and medium) and pupae, resin piles and garbage dump. we also estimated the population size of adult and immature individuals. to estimate adult population, we weighted separately 42 workers in order to know the individual weight, and divided by the total weight of nest individuals, representing the total estimated number of the nest. we used a formula to calculate the total weighted of adult bee population and nest material: totalcol size = (in w + c + boxs + q + w) + totalap where, the total colony size (totalcol size) was equal to the sum of the involucrum wax (inw), combs (c), box with structures (boxs), queen (q), waste (w) and the total weight of the adult individuals (totalap estimative). the waste was the variation found in the colonies weighted because the manipulation of the colonies and lost of some materials (wax, honey and others). to estimate immature individuals, we weighted one comb and counted the number of cells. we measured the comb (length x width) and related the number of cells with the number of combs for each nest (fig 2). the data were analyzed with microsoft excel. results and discussion nest and colony estimative nests had from 9 to 12 brood combs and 19 to 25 food pots. also, there were one to four resin piles and one to three garbage dumps (table 1). in the total combs in every nest, we found a proportion of three to four combs with eggs and larvae stage, and six to eight combs with pupae stage (table 1). we counted one pollen pot only in the pd4 nest. in the pd1 nest, we observed one royal cell and one giant male individual (fig 1; table 1). ys roldão-sbordoni, fs nascimento, s mateus – estimating plebeia droryana colonies282 the characteristics of nest architecture in p. droryana were similar to those described by zucchi and sakagami (1972) and wille and michener (1973) in other species. we found the occurrence of an involucrum and the resin pile, typical of this species. in the brood area, the involucrum layers keep the temperature by retaining the heat production from brood metabolism (wille, 1983; engels et al., 1995; barbosa et al., 2013). the presence of garbage dump deposit was observed in this study such as previously reported by nogueira-neto (1997) in other stingless bee species. weight estimative although some authors described previously the nest structures, the weight of them was not reported as in our study. the weight of the five colonies ranged from 987.7 to 1109.9g (table 2). the brood combs weighted 31g in the pd1 colony, 44.1g in the pd2 colony, 38.3g in the pd3 colony, a b c d fig 1. nest architecture of the stingless bee plebeia droryana. (a) food pots (honey and pollen). (b) resin pile. (c) the nest with all structures. (d) the amount of the combs (brood area). scale bar 3 mm. pd1 pd2 pd3 pd4 pd5 mean ± sd total brood combs 11 10 10 9 12 10.40 ± 1.14 combs (eggs and small larvae) 3 4 3 3 4 3.40 ± 0.55 combs (larvae medium and pupae) 8 6 7 6 8 7.00 ± 1.00 total adult individuals estimative 2507 1548 2414 1917 1069 1891.00 ± 601.45 total immature estimative 4158 5915 5137 4212 4727 4829.80 ± 727.25 food pots 25 22 19 19 22 21.40 ± 2.51 resin piles 1 4 2 2 1 2.00 ± 1.22 garbage dump 1 3 3 3 2 2.40 ± 0.89 table 1. number of the nest structures, brood combs, adult individuals estimative and immature estimative and the mean ±sd, respectively. fig 2. brood comb of the plebeia droryana used to estimate immatures. scale bar = 3 cm. sociobiology 65(2): 280-284 (june, 2018) 283 31.4g in the pd4 and pd5 colonies (mean of 35.2 ± 5.82g). the weight of the involucrum wax varied between 3.6 to 8.9g (mean±sd = 6.62 ± 2.0g) (table 2). queens weighted 0.045g (pd1 colony), 0.058g (pd2 colony), 0.053g (pd3 colony), 0.048g (pd4 colony) and 0.049g (pd5 colony). the average of the weight to the total adult individuals was of 13.6g to pd1 colony, 8.4g to pd2 colony, 13.1g to pd3 colony, 10.4 g to pd4 colony and 5.8g to pd5 colony (mean: 10.26 ± 3.26) (table 2). finally, the weight of foragers and comb workers were obtained (fig 1) and we used the total weight (42 individuals) for the total individual nest estimative (fig 3). the number estimated of adult individuals in the colonies was 2,507 (pd1), 1,548 (pd2), 2,414 (pd3), 1,917 (pd4) and 1,069 (pd5), mean of the 1,891 ± 601.45 (table 1). the estimated number of immature individuals was 4,158 (pd1), 5,915 (pd2), 5,137 (pd3), 4,212 (pd4) and 4,727 (pd5), mean of the 4,829.8 ± 727.25 (table 1). the weight of individuals has an important role for the estimative of the population. duarte et al. (2016) have estimated nest architecture by measuring (in centimeters), and they found an approximate population of 50,927 individuals in t. clavipes by using a formula (ihering, 1930). our study pd1 pd2 pd3 pd4 pd5 mean ± sd total nest hive 1075.3 987.7 1094.7 1042.6 1109.9 1062.04 ± 48.57 involucrum wax 3.6 8.9 6.1 6.7 7.8 6.62 ± 2.00 combs 31 44.1 38.3 31.4 31.4 35.24 ± 5.82 empty wooden boxes 701.5 701.5 701.5 701.5 701.5 box with structures 878.1 916 702.3 985.1 1096.6 915.62 ± 145.34 structures without the box 176.6 214.5 0.8 283.6 395.1 214.12 ± 145.32 queen 0.045 0.0582 0.0536 0.0484 0.0499 0.051 ± 0.005 total adult individuals 13.6 8.4 13.1 10.4 5.8 10.26 ± 3.26 table 2. weight of the structures and individuals in the five different nests (in grams) and the mean ± sd to each item. the weight of the empty wooden boxes was the average between all colonies studied. the “box with structures” refers to the weight of the wooden box with food pots, garbage dump and resin piles; and “structures without the box” refers to the weight without the mean of the empty wooden box, to calculate approximately the weight of the structures that remained inside the box. individuals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 w ei gh t ( g) 0,000 0,002 0,004 0,006 0,008 foragers comb workers showed an effective estimative by using the weight. nests of p. droryana presented 1,069 to 2,507 individuals, observed also by lindauer and kerr (1960) that estimated to p. droryana between 2,000 and 3,000 individuals. cortopassilaurino (1979) estimated the adults’ population of the p. droryana using another parameter. the colonies were considered “strong” when they had approximately 1,500 to 2,000 individuals and this result agreed with our estimated adult population (1,069 to 2,507). the immature estimate was of 4,727 individuals. conclusion we provided a new method to estimate plebeia droryana. the present study contributes to provide more details about the nests of this species and the estimate can be useful to further studies as well to the management and conservation of stingless bees. acknowledgments the authors acknowledge to financial support by capes (coordenação de aperfeiçoamento de pessoal de nível superior) and fapesp (proc. 2015/25301-9). sam morris helped with language editing. references barbosa, f.m., alves, r.m.o., souza, b.a. & carvalho, c.a.l. 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(1972). capacidade termoreguladora em trigona spinipes e em algumas espécies de abelhas sem ferrão (hymenoptera: apidae: meliponinae). in: homenagem a warwick e. kerr (pp. 301-309). rio claro. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 107-113 (2013) temporal polyethism and life expectancy of workers in the eusocial wasp polistes canadensis canadensis linnaeus (hymenoptera: vespidae) vo torres1, e giannotti2, wf antonialli-junior3 introduction according to wilson (1990), the non-reproductive division of labor among nestmates is the evolutionary advantage that most promoted the ecological success of social insects. this division of labor among workers that continues throughout their lifetimes is known as temporal polyethism; or if according to the morphology of the individual, morphological polyethism (naug & gadagkar, 1998a). this phenomenon is most common in termites (noroit, 1989) and ants (hölldobler & wilson, 1990), and was recently discovered in a species of bee, tetragonisca angustula (latreille) (grüter et al., 2011). temporal polyethism is evident in honey-bees (seeley, 1995) and stingless bees (sommeijer, 1984). both temabstract the division of tasks among nestmates is one of the most important traits of the social hymenoptera, and is responsible for their evolutionary success. this division of labor, which occurs among workers throughout their lives, is called temporal polyethism. this study investigated how temporal polyethism occurs, and its effects on the life expectancy of the eusocial wasp polistes canadensis canadensis linnaeus. to evaluate the different activities and determine longevity, newly emerged females were individually marked with colored dots on the mesosome. during their lives, workers perform 22 behavioral acts, and the nonmetric multidimensional scaling analysis revealed a variation in the pattern of behaviors performed until the fifth week of life, i.e., close to their mean longevity. the mean life expectancy was 37.06 ± 29.07 days, with a mortality rate of 24.14% in the first week, coinciding with the onset of foraging activity. in other, lessderived species this began early in the life of the workers. the high entropy value (h = 0.750) reflects the high mortality rate in the first weeks of life. sociobiology an international journal on social insects 1 universidade federal da grande dourados, dourados, mato grosso do sul, brazil 2 universidade estadual paulista “julio de mesquita filho”, rio claro, são paulo, brazil 3 universidade estadual de mato grosso do sul, dourados, mato grosso do sul, brazil research article wasps article history edited by: gilberto m. m. santos – uefs, brazil received 27 november 2012 initial acceptance 31 january 2013 final acceptance 14 february 2013 keywords division of labor, worker age, longevity, foraging activity corresponding author viviana de oliveira torres programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, faculdade de ciências biológicas e ambientais, 241, 79804-970, dourados, mato grosso do sul, brazil. e-mail: vivianabio@yahoo.com.br poral and morphological polyethism are less evident in halictinae bees (michener, 1974), bombus (cameron, 1989) and polistinae wasps (jeanne, 1991). according to shorter and tibbetts (2008), social insects can also be divided into two categories, the leastderived or basal, and the most-derived. in most derived social insects there is a clear temporal polyethism, in which young workers care for the offspring, more often performing intra-nest tasks, while older workers forage and defend the colony, as described for apis mellifera (linnaeus) (huang & robinson, 1996), polybia occidentalis (olivier) (o’donnell, 2001), pheidole dentata (mayr) (wilson, 1976), trachymyrmex septentrionalis (wheeler) (beshers & traniello, 1996) and agelaia pallipes (olivier) (nascimento et al., 2005), all species that are considered more derived within vo torres, e giannotti, wf antonialli-junior temporal polyethism in polistes canadensis108 their respective groups. less-derived social insects have a less-clear division of labor: they are able to perform different tasks according to the needs of the colony (beshers & fewell, 2001). among the species that have been studied are bombus griseocollis (de geer) (cameron, 1989), bombus bifarius nearcticus (handl) (o’donnell et al., 2000), polistes lanio lanio (fabricius) (giannotti & machado, 1994) and belonogaster petiolata (de geer) (jeanne, 1991), in which no clear temporal polyethism was observed. however, other species such as polistes canadensis (linnaeus) (giray et al., 2005), ropalidia marginata (lepeletier) (naug & gadagkar, 1998b), polistes metricus (say) (dew & michener, 1981), polistes jadwigae (dalla torre) (tsuchida, 1991) and polistes dominula (christ) (pardi, 1950) have a clearer division of labor. the degree of temporal polyethism also seems to be related to the size of the colony (wilson, 1971; thomas & elgar, 2003), since the division of labor among workers in general is directly proportional to colony size (jeanne, 1986). in colonies of ants, bees, wasps and termites with thousands to millions of individuals, the division of tasks is clear and workers are highly specialized (jeanne, 1986; hölldobler & wilson, 1990). however, in small colonies of fewer than a hundred individuals, the workers tend to perform different daily tasks, showing “behavioral plasticity,” for example, species of the genera polistes (post et al., 1988), bombus (cameron & robinson, 1990) and belonogaster petiolata (jeanne, 1991). the different tasks performed by workers determine their life expectancy. antonialli-junior et al. (2007) found that the life expectancy of workers of ectatomma planidens (borgmeier) gradually decreases after the onset of foraging activity. other studies conducted with wasps, by miyanno (1980) with polistes chinensis antennalis (pérez) and jeanne (1972) with mischocyttarus drewseni (saussure) found that the mortality rate increases after the wasps initiate extra-nest activities. the first tasks performed by workers are usually intra-nest, which are safer and less costly. as the workers become older and more experienced, they perform outsidenest tasks (wilson, 1971). jeanne (1986) and tofilski (2002) suggested that these sequences of tasks must maximize the workers’ longevity. this study investigated how temporal polyethism occurs and its effects on life expectancy and entropy in the eusocial wasp polistes canadensis canadensis (linnaeus), because of the importance of the division of labor in understanding the level of organization in social-insect colonies. material and methods the observations were made on colonies of p. c. canadensis constructed in urban areas of the central-western brazil (23º56’17” s and 54º16’15” w). the study was conducted between march 2005 and june 2006, totaling 200 hours of observation, in which 145 workers were monitored in 15 colonies. workers were marked as they emerged with dots of nontoxic paint on the mesosome, similarly to method used by nakata (1996), allowing monitoring of their behaviors. marking allowed us to collect data on life expectancy and to calculate the entropy for the workers. temporal polyethism marked workers were observed for one hour, three times a week, for qualification and quantification of behavioral acts performed throughout their lives, to characterize the temporal polyethism. we used the method of all occurrences (“ad libitum” sensu altmann, 1974), which involves observing the animal’s behavior including its entire performance, i.e., its movements and/or immobility. a description of how each action was executed was given by torres et al. (2009). the behavioral repertoire of the workers was grouped by week and analyzed by nonmetric multidimensional scaling (nmds). the bray-curtis similarity matrix was used for the nmds ordination, based on the workers’ behavioral repertoire according to the weeks of life. behavioral data for the first six weeks (days 1 to 49) were used for the nmds analysis, because by that time more than 50% of the workers had died. determination of life expectancy and entropy the workers’ life expectancy was calculated according to the methodology proposed by carey (1993) and for the life-table construction and life-expectancy calculation, data were used for 145 workers, which were observed from emergence until death, by using the equation: where: tx= total number of days at age x of survivors until the last possible day of life; x= age interval of 1 day; w= maximum age in days reached by the last surviving individual; lx= mean probability of surviving between successive ages, calculated as lx= lx-(dx)/2; lx= proportion of survivors among newly emerged workers in relation to age interval; dx= proportion of individuals that died between age x and x +1, calculated as dx= lx-lx +1. the remaining life expectancy (ex) of individuals at the beginning of each interval was calculated as ex= tx/ lx. entropy (h), a measurement of the heterogeneity in the survival pattern, defined as the ratio between the number sociobiology 60(1): 107-113 (2013) 109 of days lost by the population due to deaths and the number of days that the workers lived, was calculated by the equation: where: ex= life expectancy at age x; dx= proportion of individuals that died between ages x and x +1; e0= life expectancy at age 0; and ew= maximum age reached by the last surviving individual. according to carey (1993), the lower the value of entropy, the later individuals tend to die; and the higher the entropy value, the higher is the mortality in the initial intervals. therefore, in populations in which individuals reach their maximum longevity, the entropy value is closer to zero; while in populations where the mortality rate is very high in the initial intervals, this value is closer to 1. results temporal polyethism the workers of p. c. canadensis performed 22 different types of tasks during their lives, and the frequency of each act varied according to age (fig 1). the behaviors of vibrating wings, cleaning cells, larva-adult trophallaxis, suffering physical dominance, checking cells with antennae, walking on the nest or on the substrate, moving the gaster, self-grooming, adult-adult trophallaxis, checking cells and immobile on the nest were performed more often by younger workers. on the other hand, the behaviors of removing larvae from the nest, destroying cells, rubbing the gaster on the nest and oophagy were performed more often by older workers (fig. 1). with respect to foraging activity, the workers of p. c. canadensis started this behavior since the beginning of the first week of life, and intensified with the aging of the worker; however, the older females in the colonies did not forage (fig 1). behaviors such as recognizing a newly emerged female, larviphagy, and applying oral secretion on the nest or on the substrate were performed with similar frequencies in both younger and older workers. the nonmetric multidimensional scaling analysis (nmds: r2 = 0.78, f = 14.5, p <0.02, gl = 4) showed significant variation in the behavior of workers until the fifth week of life (fig 2). determination of life expectancy and entropy the mean longevity of the workers was 37.06 ± 29.07 days (1-126 days). the estimated life expectancy was ex= 36.56 days just after emergence, increasing to ex= 40.00 days on the eighth day of life. from that time until the worker reached the maximum longevity, life expectancy decreased more gradually (fig 3). the highest mortality rate occurred during the first week of life; 24.14% of the workers had died by the seventh day. half of the workers did not reach 45 days, and only 9.65% exceeded 84 days (fig 3). the entropy found in p. c. canadensis was h = 0.750 (fig 4), indicating a high mortality rate during the first intervals. the survival curve remained between 0.5 and 1.0 until the last life interval of workers (fig 4). because of the high number of deaths during the first days of life, the survival curve for this species has a convex shape. this curve is more pronounced until the 10th day of life and then increases more gradually until the last day of life. figure 1. frequency with which workers of polistes canadensis canadensis performed the 22 behavioral acts throughout their lives. vo torres, e giannotti, wf antonialli-junior temporal polyethism in polistes canadensis110 discussion the results found here demonstrate that the behavioral repertoire of the workers varies according to the age (figs 1 and 2), although the nmds indicated that this variation occurs only until the fifth week of life, close to the worker’s mean life expectancy. the lack of a clear pattern of task execution after the fifth week shows that despite the existence of temporal polyethism, the workers have a strong “behavioral plasticity” after this age. similar patterns were observed by jeanne (1991) and karsai and wenzel (1998) in colonies with fewer than 100 individuals. 2001), a. pallipes (nascimento et al., 2005) and vespula germanica (fabricius) (hurd et al., 2007). the degree of temporal polyethism also seems to be related to the size of the colony (wilson, 1971; thomas & elgar, 2003), since the division of labor among workers increases as the colony grows (jeanne, 1986). there is a clear division of tasks and workers in colonies of ants, bees, wasps and termites with thousands to millions of individuals, and they are highly specialized (hölldobler & wilson, 1990; pie, 2002). however, in small colonies of fewer than 100 individuals, the workers tend to perform different daily tasks, as in species of the genera polistes (post et al., 1988; reeve, 1991), bombus (cameron & robinson, 1990) and belonogaster (jeanne 1991). polyethism in m. mastigophorus is also affected by dominance interactions between queen and workers and between workers (o’donnell, 1998a). according to o’donnell (1998b), the pattern of independent foundation in these wasps suggests that the state of dominance in queens and workers affects the performance and the division of labor among individuals in the colony. in p. c. canadensis, the queens become more aggressive with workers as they age. the increased aggressiveness of the queen with older workers is known as gerontocracy (strassmann & meyer, 1983), in which the behaviors of physical dominance increase with the aging of the worker. this phenomenon was also observed by pardi (1948) in polistes gallicus (linnaeus); dew and michener (1981) in p. metricus; and by strassmann and meyer (1983) in polistes exclamans viereck. however, west-eberhard (1969) and jeanne (1972), investigating colonies of polistes erythrocephalus (latreille) and m. drewseni, respectively, observed that younger females are in positions just below the queen. figure 2. ordination by nonmetric multidimensional scaling (nmds), indicating the behavioral variation according to age in workers of polistes canadensis canadensis. although p. c. canadensis is considered a less-derived species, it displays temporal polyethism, which is considered “weak”. studies such as those of jeanne (1991) and giray et al. (2005) found that species of independent-foundation show little or no correlation between age and the tasks performed by workers. as seen in other studies, younger workers of p. c. canadensis perform mainly intra-nest tasks, and as they age they begin to perform extra-nest tasks. however, after the fifth week they perform a wider variety of tasks, both intraand extra-nest. studies by o’donnell (1998a), naug and gadagkar (1998b), shorter and tibbetts (2008) and togni and giannotti (2010) with mischocyttarus mastigophorus (richards), r. marginata, p. dominula and mischocyttarus cerberus (richards), respectively, have shown that even less-derived eusocial species have some degree of temporal polyethism. however, temporal polyethism is more evident and defined in the more-derived eusocial species, with large colonies and morphological differences between the castes (naug & gadagkar 1998a), as observed in p. occidentalis (o’donnell, figure 3. life expectancy of workers of polistes canadensis canadensis under field conditions. another factor responsible for behavioral changes with age is the changes in hormones, especially juvenile hormone (jh) (giray et al., 2005). shorter and tibbetts (2008) have shown, for example, that foraging activity is initiated earlier by workers of p. dominula when newly emerged workers are treated with jh. however, in r. marginata, alsociobiology 60(1): 107-113 (2013) 111 though jh accelerates ovarian development, it does not affect the temporal polyethism (agrahari & gadagkar, 2003). the estimated mean longevity of the workers was close to that reported for p. c. antennalis (miyano, 1980). the survival curve was relatively convex, similar to that described by jeanne (1972) in m. drewseni, whose workers live on average 31 days. giannotti (1999) described a mean life expectancy of only 14.1 days in mischocyttarus cerberus styx (richards), shorter than the species described above. these authors argue that the onset of foraging activity is directly linked to the longevity of the workers. life tables and survival curves of polistes (epicnemius) cinerascens (saussure) (giannotti, 1997) and polistes lanio (fabricius) (giannotti & machado, 1999) also show high rates of mortality in the first age interval, which coincides with the beginning of foraging activity. this relationship was also observed by simões and zucchi (1980) in protopolybia exigua exigua (saussure) and by post et al. (1988) in polistes fuscatus variatus (cresson). according to o’donnell and jeanne (1995), foraging activity implies a greater risk of predation, energy cost and physiological stress, thus reducing the life expectancy and longevity of the worker, and resulting in an entropy value closer to 1. the high mortality rate in p. c. canadenis also coincides with the beginning of foraging activity, as 65% of workers began foraging activity in their first week of life. the entropy value of h = 0.750 found here was close to that observed by giannotti and von zuben (2000) for p. l. lanio (h = 0.869) and m. cerberus (h = 0.767), also lessderived species with small colonies in which workers begin foraging activity early. although p. c. canadensis has relatively small colonies, in which workers exhibit certain behavioral plasticity, they perform most of their repertoire in the first weeks of life; however, it is possible to perceive a clear division of tasks by the fifth week. thus, longevity and entropy are directly influenced by foraging activity early in the first week of life. acknowledgments to the mato grosso do sul state university for the grants of pibic/uems, process no. 364/uems in march 2005. the “national counsel of technological and scientific development” (cnpq) for the grants to antonialli-junior, w.f. the authors thank also janet w. reid (jwr associates) for the revision of the english text. two anonymous reviewers provided valuable comments to the submitted manuscript. references agrahari, m. & gadagkar, r. 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(1990). success and dominance in ecosystems: the case of the social insects. oldendorf/lube: ecology institute. doi: 10.13102/sociobiology.v61i2.178-183sociobiology 61(2): 178-183 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 seasonality of epigaeic ant communities in a brazilian atlantic rainforest psm montine1, nf viana1, fs almeida2, w dáttilo3, as santanna1, l martins†, ab vargas1 introduction ants are extremely abundant and diverse in tropical rainforests (hölldobler & wilson, 1990; ward, 2000; lach et al., 2010). previous studies showed that ant diversity is influenced by several biotic factors, such as plant richness and density (gomes et al., 2010a; tews et al., 2004), as well as by abiotic factors, such as leaf litter depth (nakamura et al., 2003; vargas et al., 2007), temperature (almeida et al., 2007), rainfall (kaspari, 2000; speight et al., 2008), and physical and chemical properties of the soil (gomes et al., 2010b). moreover, due to the high spatial abundance of ants in tropical environments, they play several ecological roles (hölldobler & wilson, 1990). in the soil-litter interface, ants also are related with essential ecological processes, such as decomposition of organic matter, soil aeration, seed dispersal, and population control of other arthropods (hölldobler & wilson, 1990; folgarait, abstract in this study we assessed the leaf-litter ant community in the cicuta forest, a semideciduous forest located in the state of rio de janeiro, southeastern brazil. specifically, we tested the following hypotheses: (1) ant richness and diversity are higher in the rainy season, due to higher resource availability and better temperature and humidity conditions; and that (2) the structure of the ant community is influenced by climate seasonality. we collected 83 ant species of 35 genera and eight subfamilies. in total, 64 species were collected in the dry season and 73 species in the rainy season. based on rarefaction curves with confidence intervals, we observed that species richness in the dry and rainy seasons did not differ significantly from each other. shannon diversity did not differ significantly (t = -1.20; p = 0.23) between the dry (3.43) and rainy seasons (3.52). we did not observe a significant effect of climate seasonality neither on ant species composition, richness, and diversity, nor on community structure. these results may be explained by the degree of isolation and degradation of this forest remnant. in short, our study contributes to knowledge on how seasonal variations affects ant communities. sociobiology an international journal on social insects 1 centro universitário de volta redonda (unifoa), volta redonda, rio de janeiro, brazil. 2 universidade federal rural do rio de janeiro, três rios, rio de janeiro, brazil. 3 universidad veracruzana, xalapa, veracruz, mexico. † in memorian. article history edited by gilberto m m santos, uefs, brazil received 31 january 2014 initial acceptance 02 april 2014 final acceptance 25 april 2014 keywords myrmecology, richness, diversity community structure corresponding author andré barbosa vargas centro universitário de volta redonda av. paulo erlei alves abrantes, 1325 volta redonda, rio de janeiro, brazil 27240-560 e-mail: andrebvargas@yahoo.com.br 1998; passos & oliveira, 2004; dattilo et al., 2009). the structure of ant assemblages varies in time and space, following environmental heterogeneity (lassau & hochuli, 2004; jankowski et al., 2009). it is well known that variations in rainfall intensity affect temperature and humidity, and so influence ecosystem productivity (kaspari, 2001). therefore, rainfall seasonality is expected to lead to variations in the activity, abundance, and species richness and composition of ants (coelho & ribeiro, 2006; almeida et al., 2007; castro et al., 2012). indeed, rainfall may affect ant assemblages in two ways, by altering microclimate at a local scale or ecosystem productivity at a regional scale (castilho et al., 2011; castro et al., 2012). on the other hand, seasonal variations may not affect ant richness and composition due to environmental simplification and changes in habitat structure, either at a local or at a regional scale, which makes these variations less pronounced (vasconcelos & laurance, 2005; silva et al., 2011). research article ants sociobiology 61(2): 178-183 (june, 2014) 179 for example, castro et al., (2012) did not observe variations in the structure of ant assemblages in an impacted area, but only between different areas. in this study, we assessed temporal variations in ant assemblages that live in the soil-litter interface in the cicuta forest. this area is a rainforest remnant located in an urban matrix in southeastern brazil. specifically, we hypothesized that, due to higher resource availability and better temperature and humidity conditions during the warm and rainy season, ant richness and composition should differ between dry and rainy seasons. material and methods study area the cicuta forest has 125.14 ha and is located within the municipalities of volta redonda and barra mansa, in the southern region of rio de janeiro state, southeastern brazil. in the 18th and 19th centuries this region was economically very important due to coffee production (dean, 1996). classified as an area of relevant ecological interest, according to the brazilian environmental law, the reserve was created by the conama resolution #05 of june 5th, 1984 and by the decree #90,792 of january 9th, 1985. the local vegetation is classified as submontane semi-deciduous seasonal forest (ibge, 1992). the surrounding landscape is classified as an urban matrix, with small forest fragments at different successional stages, eucalyptus plantations, and mainly pastures. the altitude ranges between 300 and 500 m a.s.l. (monsores et al., 1982) and the climate is cwa (dry winter and warm and rainy summer) in koeppen classification. the average annual rainfall is 1,300mm; february is the warmest month (24ºc) and july is the coldest (17ºc). the local geomorphology is characterized by flattened hilly terraces, isolated structural hills, and dissected tablet-shaped terraces. the red-yellow podzolic soil, whose constitution may facilitate the occurrence of erosion, predominates in the region (brasil, 1983; dias et al., 2001). the cicuta forest, which is considered an important atlantic forest remnant, harbors large tree species such as jequitibá (cariniana estrellensis), chichá (sterculia chicha) and figueira branca (ficus guaranítica). sampling for sampling ants we used pitfall traps at three equidistant sites, 500 m apart from one another. at each site, we placed 20 pitfalls at every 10 m in four parallel transects. pitfall traps were made of 500-ml plastic cups (8 cm diameter) containing approximately 150 ml of ethanol 70% as preservative fluid, which remained in the field for seven days. during the exposure period, the traps were constantly checked so that they always contained the preservative fluid. we repeated sampling four times: two times in the rainy season and two times in the dry season (june 2001 dry season, march rainy season and june dry season in 2002, and march 2003rainy season). data analysis we used the mao tau moment-based rarefaction (gotelli & colwell, 2001) to build rarefaction curves of ant species for the dry and rainy seasons. in spite of our sampling effort, this technique eliminates the need for replicates, as it allows a direct comparison of richness between seasons of the year (colwell et al., 2004). in addition, we used the first order jackknife non-parametric species richness estimator for extrapolating species richness in the study area. both analyses (rarefaction and richness estimation) were made in estimates 7.5.2 (colwell, 2005). we calculated the shannon diversity index (h’) for each season (winter and summer) and compared them with a t test in past (hammer et al., 2001). we also tested for a turnover in the composition of dominant ant species between the dry and rainy seasons. we considered dominant the species that were present in more than 25% of the samples. to summarize the structure of the ant community in dry and rainy seasons, we ordered the samples with non-metric multidimensional scaling (nmds). this type of ordination is one of the most robust, as it summarizes more information in fewer axes compared with other techniques (legendre & legendre, 1998). the nmds was performed based on a distance matrix calculated with the bray-curtis dissimilarity index. next, we tested for differences in ant species composition between samples collected in the dry and rainy seasons, using a permutation test (10,000 permutations) based on an analysis of similarities (anosim) (clarke, 1993). both the ordination and similarity analysis were performed in r 2.13.1 (r development core team). in order to not overestimate the ant species with more efficient systems for recruiting and / or those whose colonies are closer to the bait (gotelli et al. 2011), all analyses used in this study were calculated based on the frequency of species occurrence in the pitfall traps and not based on the number of workers. results we collected 83 ant species of 35 genera and eight subfamilies (table 1). the subfamily myrmicinae had the largest number of species (42 51%), followed by formicinae (17 20%), ponerinae (12 14%), and ecitoninae (5 6%). in total, 64 species were collected in the dry season and 73 in the rainy season. although the rarefaction curves evidenced a fast increase in the number of species in both seasons (dry and rainy), no curve reached an asymptote. this suggests that more species could have been added with a larger sampling effort or with the addition of other sampling methods (figure 1). according to the jackknife 1 richness estimator, the sampling efficiency was 71.2 % in the dry season (observed richness: 64 species; estimated richness: 89.7 species) and 73.9 % in the rainy season (observed richness: 73 species; estimated richness: 98.78 montine et al seasonality of ant communities 180 table 1 list of species by subfamilies sampled in two different periods (dry and wet) in cicuta forest, volta redonda, rio de janeiro. subfamilies/species sampling period functional groups dry wet dolichoderinae 14 2 linepithema sp. 1 1 omn linepithema sp. 2 14 1 omn ecitoninae 32 12 eciton cf. vagans olivier 3 arm labidus praedator (fr. smith) 28 8 arm neivamyrmex sp. 1 2 1 arm neivamyrmex sp. 2 1 arm neivamyrmex sp. 3 1 arm ectatomminae 11 2 ectatomma edentatum roger 7 1 lit-dom gnamptogenys minuta (emery) 1 1 gen-pred gnamptogenys sp. 3 3 gen-pred formicinae 43 43 acropyga sp. 1 1 1 sub brachymyrmex sp. 1 1 11 omn brachymyrmex sp. 2 5 2 omn brachymyrmex sp. 3 5 2 omn brachymyrmex sp. 4 4 1 omn brachymyrmex sp. 5 1 omn camponotus crassus mayr 1 omn camponotus sericeiventris guérin 1 1 omn camponotus sp. 1 5 5 omn camponotus sp. 2 1 3 omn camponotus sp. 3 1 omn camponotus sp. 4 16 11 omn camponotus sp. 5 1 omn camponotus sp. 6 1 1 omn camponotus sp. 7 1 omn camponotus sp. 8 1 omn nylanderia sp. 1 1 1 omn heteroponerinae 1 heteroponera sp. 1 1 gen-pred myrmicinae 263 291 acanthognathus brevicornis smith 6 2 lit-pred acromyrmex sp. 1 5 11 fung apterostigma gr. pilosum 5 2 fung atta sp. 1 1 fung carebara urichi (wheeler) 3 1 omn cephalotes pallens (klug) 1 omn crematogaster sp. 1 2 omn crematogaster sp. 2 1 2 omn cyphomyrmex sp. 2 1 7 fung cyphomyrmex sp. 3 1 fung cyphomyrmex sp. 4 1 fung hylomyrma balzani (emery) 4 4 lit-pred leptothorax sp. 1 1 2 lit-pred megalomyrmex sp. 1 6 6 lit-pred mycetarotes carinatus mahyé-nunes 3 fung octostruma sp. 1 3 7 omn pheidole gertrudae forel 1 omn pheidole sp. 1 1 16 omn pheidole sp. 10 1 3 omn pheidole sp. 11 2 3 omn pheidole sp. 2 23 21 omn pheidole sp. 3 14 13 omn pheidole sp. 4 24 40 omn pheidole sp. 5 15 10 omn pheidole sp. 6 28 11 omn pheidole sp. 7 21 16 omn pheidole sp. 8 17 7 omn pheidole sp. 9 1 omn procryptocerus sp. 1 1 omn solenopsis sp. 1 1 4 lit-omn solenopsis sp. 2 24 40 lit-omn solenopsis sp. 3 39 19 lit-omn solenopsis sp. 4 4 10 lit-omn solenopsis sp. 5 2 lit-omn solenopsis sp. 6 2 lit-omn solenopsis sp. 7 1 7 lit-omn strumigenys apretiata 3 5 lit-pred strumigenys sp. 1 2 3 lit-pred strumigenys sp. 2 4 3 lit-pred strumigenys sp. 3 1 lit-pred strumigenys sp. 4 1 lit-pred trachymyrmex sp. 1 2 fung ponerinae 116 110 hypoponera sp. 1 3 8 gen-pred hypoponera sp. 2 1 gen-pred hypoponera sp. 3 6 2 gen-pred hypoponera sp. 4 8 1 gen-pred leptogenys sp. 1 1 1 lit-pred leptogenys sp. 2 1 lit-pred odontomachus chelifer (latreille) 7 8 gen-pred odontomachus meinerti forel 22 20 gen-pred pachycondyla harpax (fabricius) 26 15 gen-pred pachycondyla sp. 1 1 gen-pred pachycondyla sp. 2 3 1 gen-pred pachycondyla striata fr. smith 39 52 gen-pred pseudomyrmecinae 1 2 pseudomyrmex sp. 1 1 2 omn total richness 64 73 species). diversity (h’) was 3.43 in the dry season and 3.52 in the rainy season, which did not differ significantly from each other (t = -1.20; p = 0.23). although some species occurred only in one season, the structure of the ant assemblage did not differ between the dry and rainy seasons (figure 2) (nmds followed by anosim: r = 0.048; p < 0.001). in addition, we observed that 10 species (15.6 %) were particularly dominant in the dry season and only five species (6.8 %) in the rainy season. we recorded 27 (33%) rare species (table 1). sociobiology 61(2): 178-183 (june, 2014) 181 discussion several studies have shown that abiotic factors, such as rainfall and temperature, are directly related to the availability of food and nesting sites for insects (speight et al. 2008). these factors may also influence the foraging activity of ants (levings, 1983; almeida et al., 2007). some previous studies carried out in semi-deciduous areas of the brazilian atlantic forest pointed to an effect of climate seasonality on ant assemblages (vargas et al., 2007; castilho et al., 2011). in this study, despite the highest ant richness found in the rainy period, we did not observe an effect of climate seasonality on species richness, diversity, or assemblage structure. however, the expressive number of exclusive species and their frequency in each season suggest that some species may be influenced by seasonality. our results corroborate castro et al., (2012) who also did not found a relationship between ant species richness and seasonality in a degraded area. the lack of correlation observed in the present study may be explained by environmental degradation, which makes the environments simpler (vasconcelos et al., 2006; sobrinho & schoereder, 2007), and increases the competition and abundance of generalistic species (schoereder et al., 2004). we observed the same pattern in the hypogaeic fauna, as also observed by figueiredo et al., (2013). the cicuta forest has faced different impacts throughout the years, such as fire, hunting, cattle farming, and logging, mainly in its surroundings, which influence vegetation structure. these impacts certainly restrict the occurrence of more demanding species and contribute to the dominance of a smaller number of generalist species. currently access to the forest is prohibited, except for scientific research, in order to minimize human impacts and help its conservation. this forest remnant is extremely important to biodiversity maintenance in the region, mainly because it is located within an urban matrix. one example of its importance is the occurrence of a population of alouatta guariba clamitans cabrera, 1940 (red howler monkey), which may be considered one of the last populations in the paraíba valley (alves & zaú, 2005). the ant richness found in the cicuta forest is high compared to other forest remnants (veiga-ferreira et al., 2005; castro et al., 2012; vargas et al., 2013). the assemblage composition of ants is highly generalist, but also we can found a high occurrence of rare species (33%). in addition, it is worth to mention the record of a well-documented pattern for tropical forest fragments with myrmicinae, formicinae, and ponerinae as the subfamilies with highest diversity. the same is true for the genera pheidole, camponotus, and solenopsis, which stood out as the most diverse (ward, 2000 ward, 2010; castilho et al., 2011; miranda et al., 2013; dattilo et al., 2011). however, subfamilies as amblyoponinae, cerapachyinae, and proceratiinae, which are usually recorded in forests, were not represented. this has possibly happened due to the sampling technique used here (see vargas et al., 2009), as most of their species present cryptic behavior, and reduced size and they rarely forage in the leaf litter (hölldobler & wilson, 1990; see longino et al., 2002; figueiredo et al., 2013). hence, biodiversity conservation in the atlantic forest is related to the maintenance of its forest remnants, even if they are relatively small. therefore, the cicuta forest is important for the conservation of biodiversity in semi-deciduous seasonal forests, especially if we consider that variations in rainfall and temperature are important to regulate the ant community. in short, our study contributes to knowledge of how seasonal variations affects ant communities. acknowledgements to the university center of volta redonda (unifoa), faperj, and cnpq. we thank the brazilian institute for the environment and natural resources (ibama) for permission to collect ants in the cicuta forest. we also thank two anonymous reviewers for valuable comments that improved this article. fig 1. rarefaction curves of ant species richness (mao tao) for dry and wet seasons based on the number of individuals collected. the thinner lines represent the confidence interval of 95%. fig 2. non-metric multidimensional scaling (nmds) composition of ants collected in dry (triangles) and rainy seasons (squares) between june 2001 and march 2003 in the hemlock forest, state of rio de janeiro, brazil. this ordination analysis was calculated from bray-curtis dissimilarity index’s (stress = 0.615; 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(2010). taxonomy, phylogenetics and evolution. in: lori lach, catherine l. parr, and kirsti l. abbott (ed.). ant ecology, 429p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2685sociobiology 65(2): 285-290 (june, 2018) epidemiological survey of ascosphaera apis in small-scale migratory apis mellifera iberiensis colonies introduction the benefits of safeguarding pollinators, especially the honey bee apis mellifera as the most economically important pollinator insect, have been recently articulated (potts et al., 2016). in this sense, sustainable hive management is among the strategies to avoid pollinator decline, as it has been shown that techniques such as migratory beekeeping increase oxidative stress levels in honey bees (simone-finstrom et al., 2016), and also favor the spread of pathogens (cavigli et al., 2016). recently, attention has been paid to the impact of transporting beehives on spreading diseases (ahn et al., 2012; zhu et al., 2014; simone-finstrom et al., 2016; traynor et al., 2016; guimarães-cestaro et al., 2017). while in north america honey bee hives are moved yearly for pollination of monocultures, in spain beehives are mainly moved to abstract honey bee hives are moved yearly mainly for pollination, but also to take advantage of consecutive flowering events to get as many harvests of honey as possible and/or to find favorable sites for food sources and summer temperatures. such movements may lead to pathogen spill-over with consequences on the honey bee health and finally on population decline. ascosphaera apis is the causative agent of the chalkbrood disease, a pathology affecting honey bee larvae that significantly harms population growth and colony productivity. in this study, we detected the presence of a. apis in adult worker honey bees by pcr-amplification of the intergenic transcribed spacer (its1) of the ribosomal gene (rdna). we first optimized the dna extraction by testing different protocols in individual and pooled (colony level) adult honey bee samples. subsequently, the presence of the fungus a. apis was assessed in both stationary and migratory colonies (subjected to small scale regional level movements) to determine the effect of migratory practices on the dispersal of this pathogen. results confirmed a higher prevalence of a. apis in migratory apiaries when compared to stationary ones, indicating that migratory colonies are more likely to develop chalkbrood disease. given these results, we suggest that beekeepers should be aware of the risks of pathogens spreading while moving beehives, even within a reduced geographic range. sociobiology an international journal on social insects l jara, d martínez-lópez, i muñoz, p de la rúa article history edited by cândida aguiar, uefs, brazil received 29 november 2017 initial acceptance 22 january 2018 final acceptance 28 february 2018 publication date 09 july 2018 publication date keywords beekeeping, migratory management, honey bee health, chalkbrood disease, spain. corresponding author pilar de la rúa área de biología animal, departamento de zoología y antropología física facultad de veterinaria universidad de murcia 30100 murcia, spain. e-mail: pdelarua@um.es take advantage of consecutive flowering events (to get as many harvests of honey as possible) and to find favorable sites for food sources and summer temperatures. migratory beekeeping is an extended practice in this country. there are 2.7 million beehives (data from 2015; rega 2016), of which 80% are moved by professional beekeepers (a beekeeper is considered to be professional when he/she manages more than 150 beehives per year; mean is 406 beehives per beekeeper). although this practice usually leads to increased honey production, it can imply higher costs due to transportation and colony losses that may counteract the actual economic benefits. pathogen dissemination has been cited as one of the main causes of the severe decline of honey bee populations (neumann & carrek, 2010). among pathogens, fungi might play an important role since spores can resist up to 15 years (gilliam, 1986). ascosphaera apis, the causative agent of área de biología animal, facultad de veterinaria, universidad de murcia, spain research article bees l jara, d martínez-lópez, i muñoz, p de la rúa – chalkbrood in stationary and migratory honey bee colonies286 chalkbrood disease in honey bee larvae, can survive one year in honey and two in pollen (flores et al., 2005a, b). this disease is found worldwide, is typically common during spring (aronstein & murray, 2010) and causes significant harm to population growth and colony productivity in weak colonies through a loss of honey bee workforce (evison, 2015); however, those colonies that grow stronger over the summer can fully recover (evison & jensen, 2018) and often show no symptoms of the disease. in these cases, diagnostic techniques such as molecular assays are needed to detect the pathogen. a. apis has been shown to be difficult to identify due to the lack of distinctive morphological features and the requirement of a special medium and growth conditions (jensen et al., 2013 but see chorbinski & rypula, 2003). the first biochemical method for identifying this fungus was based on isozyme analysis (gilliam & lorenz, 1993; chorbinski, 2003). later, internal spacers (its1, its2) of ribosomal rdna have been shown to be particularly useful in elucidating the relationships between closely related species of ascosphaera (anderson et al., 1998; chorbinski, 2004; borum & ulgen, 2008). these analyses facilitated the design of primers specific for the its1-5.8s-its2 rdna region for each ascosphaera species (james & skinner, 2005; murray et al., 2005; yoshiyama & kimura 2011). more recent diagnostic methods rely upon information from the ascosphaera genome (qin et al., 2006) depicting polymorphic loci that can be used to differentiate haplotypes in a. apis (jensen et al., 2012). as aronstein and murray (2010) noted, ‘the migratory nature of commercial beekeeping in north america and australia is probably the most important factor contributing to the rapid spread of chalkbrood disease within these two continents’. it can be then hypothesized that migratory hives have a higher incidence of a. apis due to the close contact among hives while transporting them, and the intrinsic management of this beekeeping practice. to test this hypothesis, we first implemented the molecular characterization of the causal agent of chalkbrood in adult worker honey bees. then we searched for a. apis in symptomatic and asymptomatic colonies of both stationary and migratory apiaries from murcia, a southeastern mediterranean spanish province. murcia has 93,954 beehives grouped in 485 apiaries of which 420 usually practice migratory beekeeping, and transportation occurs at different scales, nationally (around 700 km) to regionally (80 km). this study investigates a possible correlation between the prevalence of a. apis in honey bee colonies and the practice of small-scale migratory beekeeping. material and methods samples (20 to 25 honey bee workers) were taken from the inner frames of 46 beehives of a. m. iberiensis during spring (2015), when the incidence of a. apis is presumably higher (flores et al., 1996; borum & ulgen, 2008). from these 46 beehives, 21 were stationary (8 belonging to an apiary at the locality of miravetes and 13 from our research apiary at the, university of murcia, espinardo campus) i. e. those colonies are not moved from the apiary in any moment of the year; the remaining 25 beehives were moved yearly around 60 km from their original locations (10 from loma ancha, 6 from el llano and 9 from pantano puentes migratory apiaries) (figure 1 and table 1). fig 1. location of the apiaries sampled in murcia (se spain). stationary hives were located in 1 (miravetes) and 2 (espinardo campus), while migratory hives were located in 3 (loma ancha), 4 (el llano) and 5 (pantano puentes). different protocols were tested to optimize dna extraction and pcr amplification of the fungus a. apis. dna was first extracted at the individual level using two legs of each worker honey bee following either the ivanova et al. (2006) or chelex® methods (evans et al., 2013). at the colony level, two tests were performed: one consisted on a macerate of five workers per colony, from which dna was extracted by following a modified protocol from martínhernández et al. (2007), and the second consisted of a pool of ten legs (two from each of the five workers), from which dna was extracted with a modified chelex® method by adding 200 µl of 5% chelex® and 10 µl of proteinase k (10 mg/ml). dna extraction yields obtained from each protocol were assessed by using nanodrop 1000 spectrophotometer (nanodrop technologies). the rdna region of a. apis was amplified using the primers 3f1f: 5’-tgt ctg tgc ggc tag gtg-3’ y ascoaii2r: 5’-gaw cac gac gcc gtc act-3’ (james & skinner, 2005) in a thermocycler ptc 100 (mj research), by adding 2 µl of dna of each sample to a final reaction volume of 15 µl. the program cycle used was as follows: denaturation for 3 min at 94 ºc; 36 cycles of 30 s at 96 ºc, 30 s annealing at 55 ºc and extension for 30 s at 68 ºc; then a final elongation step of 20 min at 65 ºc (modified from yoshiyama & kimura, 2011). positive (dna extracted directly from the fungus isolated from a mummy honey bee worker) and negative controls were included in each reaction. amplicon size was confirmed after electrophoretic separation on 1.5% agarose gels. sociobiology 65(2): 285-290 (june, 2018) 287 correlation between the variables “stationary” and “migratory” and “presence of a. apis” was analyzed using the chi-squared test, the fisher exact test, and cramer’s v for contingency tables (past v3.17 program, hammer et al., 2001). results dna concentration obtained from the different tested extraction protocols was appropriate for pcr amplification in all cases, yielding from 9 to 115 ng/µl of dna. the highest extraction yields were obtained with the chelex® protocol at both individual and colony levels. therefore, we adopted the protocol adapted to pools of legs of honey bee workers for processing all subsequent samples at the colony level. to set up the amplification reaction of the its region of a. apis we extracted dna from honey workers selected from a colony with chalkbrood symptoms (figure 2) and directly from the fungus as a positive control. when using 55 ºc of annealing temperature, amplicons of the expected size (442 base pairs) were obtained. stationary migratory miravetes (1) campus of espinardo (2) loma ancha (3) el llano (4) pantano puentes (5) gps data latitude/ longitude 38º5’54”/1º53’45” 40°1’34.71”/3°10’33.82” 38º5’50”/2º0’058.8” 38º6’11.8”/1º51’58.4” 37º43’54.3”/1º47’41.5” no. of hives 8 13 10 6 9 % of infected colonies 50 76.92 100 100 100 table 1. geographical information about the apiaries included in the study and percentage of colonies in which a. apis was found in stationary and migratory apiaries. number in brackets refer to the apiaries in locations from fig 1. the next step was to detect a. apis in the migratory and stationary apiaries at the colony level. the fungus a. apis was detected in all 25 colonies belonging to the three studied migratory apiaries. in the case of stationary apiaries, 50% of the colonies from miravetes and 77% of the colonies from espinardo campus were positive for the presence of a. apis (table 1). in total, 100% of migratory colonies and 67% of stationary colonies were positive for the fungus. a significant difference (χ2 = 23.95, df = 1 and p < 0.00; fischer exact test < 0.001) between the presence of a. apis and the beekeeping management of the colonies was observed. additionally, cramer’s v (0.722) showed that beekeeping management was strongly associated with the presence of a. apis, suggesting that these factors (stationary or migratory colonies) affected the probability of occurrence of chalkbrood disease. fig 2. chalkbrood at the entrance of the beehive, indicating the development of the disease. discussion one of the drivers of pollinator decline at the global level is the dispersion of pathogens and parasites; therefore, the study of the prevalence of these organisms is of increasing interest. in this study, we detected the fungus a. apis by extracting dna with the chelex method directly from adult worker honey bees sampled from brood combs. this extraction method is widely used in population genetic studies of the honey bee (evans et al., 2013), and it has also proven to be useful for detecting the fungus both at the individual and colony levels. this is particularly relevant in the case of social insects, as population studies include large number of samples, hence the need for cheap, fast, and effective molecular tools to monitor individual or colony health and exposures. in our study we have set up the detection of a. apis spores in adult honey bees to further increase the knowledge of the impact of beekeeping techniques as migration of hives, on the spread of this fungus and its associated disease. in this sense, although some condition (temperature, habitat) differences exist in the stationary apiaries, the percentage of colonies positive for a. apis in the stationary apiary at the espinardo campus was higher (77%) than that found in the apiary of a private beekeeper (50%). colonies at the university are subjected to continuous management and experimentation procedures what may be considered as stress factors. it is possible that the higher proportion of infected colonies is the result of fungus transmission through the use of contaminated beekeeper material, because spores can accumulate in all parts of the beehives and in all beehive products (e.g. foundation wax, stored pollen and honey), and remain viable for at least 15 years (gilliam, 1986). it is therefore recommended to carry out management procedures with clean instruments, to minimize the dispersion of the fungus spores and the possible l jara, d martínez-lópez, i muñoz, p de la rúa – chalkbrood in stationary and migratory honey bee colonies288 development of the disease. likewise, combs should be replaced yearly to avoid transfer of combs between potentially infected beehives (flores et al., 2005a, b). the results of the statistical analysis confirm the initial hypothesis about the effects of migratory beekeeping on the dispersion of the fungus a. apis. the percentage of detection of the pathogen was significantly lower in stationary than in migratory colonies (50-77% stationary, 100% migratory). the intensive management of migratory colonies makes them prone not only to a higher prevalence of the fungus, but also to a rapid dispersal of spores across distant areas; both transport stress and spore contamination may explain the high incidence of chalkbrood disease in moved colonies mentioned by aronstein and murray (2010). during migratory events, apiaries dedicated to economic exploitation need constant supervision and management, including handling with the same equipment, which increases the possibility of contagion from one colony to another. in addition, because of continuous management to produce more honey, colonies may suffer a nutritional deficiency that increases the likelihood of disease development, since shortage of essential amino acids will impair the correct functioning of the immune system (di pasquale et al., 2013). other undesirable factors that affect migratory colonies in relation to a higher incidence of a. apis fungus could be stress upon honey bees resulting from truck noise and vibrations, changing temperatures among the visited sites (simone-finstrom et al., 2016) and a higher drift of workers during migratory operations (fries & camazine, 2001). similar results to those obtained here have been observed in studies in turkey including other pathogens as nosema spp (tokzar et al., 2015) although this microsporidium has a different biology. these authors noted a higher prevalence of nosema ceranae in migratory colonies than in stationary colonies. however, any significant relationship between hive management and pathogen dispersal was found in other studies in which the prevalence of pathogens and parasites related to the decline of honey bee colonies (for instance paenibacillus larvae, varroa destructor, n. apis and n. ceranae) in stationary and migratory apiaries in brazil has been evaluated (guimarães-cestaro et al., 2017), suggesting that there may be also other factors that influence the spread of pathogens such as environment stressors as climate (van engelsdorp et al., 2013), and even the honey bee subspecies or evolutionary lineage that could show different resistance traits (jara et al., 2012). a. apis spores geminate in the anaerobic environment of the closed hindgut of two–four day old larvae after their activation by co2 (bamford & heath, 1989), therefore adult honey bees are not susceptible to chalkbrood disease, but they can transmit the pathogen within and between beehives because fungal spores carried by foraging honey bees are passed to nursing honey bees that then feed larvae with contaminated food (aronstein & murray, 2010). this possibility should be taken in consideration by beekeepers, as one of the drivers of pollinator decline at the global level is the dispersion of pathogens and parasites. in conclusion, beekeepers should find a balance between economic benefits of migrating colonies (even at a small-scale) for a continuous honey harvesting and the impact of spreading pathogens and stressing the colonies that increases the incidence of diseases. acknowledgments we very much appreciate comments from prof. josé serrano and two anonymous reviewers on an earlier version of the manuscript. this study was supported by inia-feder (grant number rta2013-00042-c10-05) and fundación séneca (plan of science and technology of the region of murcia, grant of regional excellence 19908/germ/2015). lj and pdlr are presently members and receive support from cost action fa1307 (sustainable pollination in europe: joint research on bees and other pollinators (super-b). irene muñoz is supported by fundación séneca (murcia, spain) through the post-doctoral fellowship “saavedra fajardo” (20036/sf/16). references ahn, k., xie, x., riddle, j., pettis, j. & huang, z. y. 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(2014). transportation and pollination service increase abundance and prevalence of nosema ceranae in honey bees (apis mellifera). journal of apicultural research, 53: 469-471, doi: 10.3896/ibra.1.53.4.06. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.1783sociobiology 65(2): 149-154 (june, 2018) a new protocol using artificial seeds to evaluate dietary preferences of harvester ants in semi-arid environments introduction seed predation is widespread among ants, mainly in arid environments where harvester ants are ecologically dominant (wilson & hölldobler 1990; taber 1998; rico-gray & oliveira 2007; dáttilo & rico-gray 2018). in the american continent, harvester ants (pogonomyrmex spp. mayr, 1868) are recognized as one of the main seed predators, since they influence the ecological dynamics of natural communities by exerting effects on the spatial distribution and demography of plants (macmahon et al. 2000; m. herrera & pellmyr 2002). however, although the ecology of pogonomyrmex species is well studied in northern and southern environments of the new world (macmahon et al. 2000; pirk & de casenave abstract the preferences of seed intake by harvester ants (pogonomyrmex spp.) have been debated for a long time, mainly due to the lack of repeatable methods to draw clear conclusions. however, several characteristics of the food resource are well recognized as the drivers of such selective predation. for instance, resource quality (i.e., availability of carbohydrates, lipids, and proteins) is one factor that could explain the observed foraging patterns of pogonomyrmex species. in this sense, experimental approaches using artificial resources (e.g., synthetic seeds/diaspores) have provided a useful and alternative tool to study ant’s food foraging behavior. therefore, it is expected that the use of artificial seeds also could offer a versatile way to assess the influence of resource quality on the resource selection by harvester ants. on the other hand, empirical experiments involving harvester ants and artificial seeds are still rare in the literature and it is not known if such methodology is efficient with different pogonomyrmex species. in this study carried out in a neotropical arid environment of central mexico, we tested a simple but fundamental question: do harvester ants (pogonomyrmex barbatus) predate artificial seeds with manipulated nutrient content (lipids and proteins) in the field? we found that the proportion of native seeds removed was lower than the proportion of artificial seeds removed. however, we found no difference between the removal of artificial seeds containing only lipids and the seeds containing lipids + proteins. these findings indicate that the artificial seeds synthesized by us could be an effective method to test different ecological hypothesis involving harvester ants. moreover, our empirical experiment offers a benchmark to study the influence of resource quality on the food foraging behavior of harvester ants in neotropical arid environments. sociobiology an international journal on social insects p luna, w dáttilo article history edited by jean santos, ufu, brazil received 05 june 2017 initial acceptance 27 september 2017 final acceptance 01 january 2018 publication date 09 july 2018 keywords experimental methods, granivorous ants, myrmecochory, pogonomyrmex, seed predation. corresponding author wesley dáttilo red de ecoetología instituto de ecología a.c. carretera antigua a coatepec nº 351 el haya, cp 91070, xalapa veracruz, mexico. e-mail: wdattilo@hotmail.com wesley.dattilo@inecol.mx 2006; pirk & de casenave 2011), little is known about the ecology of harvester ants in neotropical arid environments (but see garcía-chávez et al. 2010; belchior et al. 2012; guzmán-mendoza et al. 2012). as central place foragers, the foraging activity of pogonomyrmex species is limited to small distances (between 5 to 25 m) around their nests (holder & polis 1987; macmahon et al., 2000; pirk et al., 2009; belchior et al., 2012). to avoid the costs generated by having such limited foraging areas, pogonomyrmex harvester ants predate seeds from a great variety of plant species (rico-gray & oliveira 2007), and in some cases, a unique ant colony can predate seeds up to 30 different plant species (pirk et al., 2009; belchior et al., 2012). however, most of pogonomyrmex species exhibit particular red de ecoetología, instituto de ecología a.c., xalapa, veracruz, mexico research article ants mailto:wdattilo@hotmail.com mailto:wesley.dattilo@inecol.mx p luna, w dáttilo – artificial seeds fora harvester ant150 preferences in their seed intake, predating some plant species more than others (i.e., differential seed predation) (taber, 1998; macmahon et al., 2000; rico-gray & oliveira, 2007). on the other hand, in the wide geographical distribution of pogonomyrmex species, there is not a consensus about their seed preferences and which are the main factors influencing the seed selection by harvester ants (macmahon et al., 2000; pirk & de casenave, 2006; belchior et al., 2012; schmasow & robertson, 2016). in addition, in arid environments, where harvester ants are found, the landscape is highly heterogeneous (in terms of vegetation, climatic conditions, topography and geology) in both spatial and temporal scales (bestelmeyert & wiens 2001; rietkerk et al., 2002; whitford, 2002), which has led to the implementation of particular methodologies conceived to be used in a given arid environment (since they cannot operate in the same way and need to be readjusted for other localities (e.g. huenneke et al., 2001) although there is no agreement about which factors shape the differential predation of seeds, studies have showed that some factors can directly influence the seed selection by pogonomyrmex spp., such as: spatial abundance and richness of plants (macmahon et al., 2000; pirk & lopez de casenave, 2006), nutritional quality of seeds (whitford & steinberger, 2009; schmasow & robertson, 2016), seed size (macmahon et al., 2000; pirk & de casenave, 2010; pirk & de casenave, 2011), seed density (flanagan et al., 2012), and the seed distance from the nests (hölldobler, 1976; holder & polis, 1987; schmasow & robertson, 2016). however, due to the difficulty to test ecological hypothesis in the field that consider the effect of seed traits (e.g., nutrient content, morphology and physiochemical defenses) on the differential predation by pogonomyrmex species, these questions have been rarely explored. on the other hand, the use of artificial methods in experimental protocols has proved to be an excellent benchmark to test ecological hypothesis in the field, mainly because they can be implemented and repeated in different environments and under different conditions (e.g. dáttilo et al., 2016; roslin et al., 2017). in this sense, some studies have implemented the use of artificial seeds to evaluate dietary preferences by ants (raimundo et al., 2004; bieber et al., 2014; rabello et al., 2014). specifically for harvester ants, whitford and steinberger (2009) used artificial seeds with manipulated nutrient content and found that the harvester ant pogonomyrmex occidentalis (cresson, 1865) exhibits preferences for seeds with different protein content in north america. in fact, the use of artificial seeds could be an alternative tool to study the seed intake by pogonomyrmex harvester ants, since this subject is supported by little empirical evidence in the literature. however, it is not known if the approach of artificial seeds is efficient for all the pogonomyrmex species, particularly in the neotropical region. therefore, in this study we synthesized two types of artificial seeds containing different proportions of lipids and proteins to test if one harvester ant distributed in neotropical regions [pogonomyrmex barbatus (smith, 1858)] could discriminate among native and artificial seeds with manipulated contents of lipids and proteins in a mexican semi-arid environment. methods study area the study was conducted during april 2017 within the neotropical semi-arid region of zapotitlán, state of puebla, mexico, located within the biosfere reserve of tehuacáncuicatlán. the mean altitude is 1,400 ma.sl., annual mean precipitation is 400mm and the mean temperature is 22° c (zavala-hurtado et al., 1996 and references therein). the vegetation of this region can be categorized as a xeric shrub land (rzedowski, 2006), and the plant association where we performed the study corresponds to “tetechera”, in which the dominant plant species are the columnar cactus neobuxbaumia tetetzo (j.m. coult.) backeb. (cactaceae), and the thorny shrubs prosopis laevigata (willd.) m. johnston (fabaceae), mimosa luisana brandegee (fabaceae), parkinsonia praecox (ruiz &pav. ex hook.) hawkins (fabaceae) and vachellia constricta (benth.) seigler and ebinger (fabaceae) (zavalahurtado, 1982, pavón et al., 2000, dáttilo et al., 2015). the study area exhibits strong seasonality, with a long dry season from october to may and a short rainy season from june to september. artificial seeds we synthesized two types of seed, one with lipids (9% of the content) and other with lipids and proteins (9% and 6% of the content respectively). artificial seeds were prepared by mixing 150 g of wheat flour, the desired amount of casein protein, 100 ml of water and 25 ml of vegetable oil. the addition to the vegetable oil served to mask the small differences in caloric value of the wheat flour and casein (see whitford & steinberger, 2009). we used these nutrients since ants can forage selectively for protein, lipids and carbohydrates to redress specific colony requirements (e.g. ontogeny) (mayntz et al., 2005; whitford & jackson, 2007; dussutour & simpson, 2008). moreover, it is worth to mention that the content of our mixture represents highly nutritive resources, thus, they can influence the foraging behavior of harvester ants (crist & macmahon, 1992; whitford & steinberger, 2009; schmasow & robertson, 2016). the mixture was poured in a thin layer and allowed to air dry for 24h before the experiments. we then cut the dried mixture into smaller pieces of the same size (4 × 4 mm) (fig 1). sampling design in order to test if p. barbatus predates artificial resources containing lipids or both lipids and protein, we selected 14 active colonies of the harvester ant pogonomyrmex barbatus (smith, 1858) (myrmicinae). in order to control the effect of colony size (i.e. number of ants), we used only colonies that presented mounds of approximately one meter in diameter. sociobiology 65(2): 149-154 (june, 2018) 151 we used three types of seeds: a non-artificial native seed from opuntia pilifera f.a.c. weber (cactaceae) (mean size ± se: length = 3.85 ± 0.07 mm, width = 3.78 ± 0.09 mm, n=20), and the two artificial seeds synthesized by us, with lipids (9% of the content) and with lipids and proteins (9% and 6% of the content, respectively). we selected o. pilifera since it is frequently used by the ants and are widely distributed in the study area (luna 2016; luna et al., 2018). moreover, it is well documented the predation of opuntia spp. mill. seeds by harvester ants (gonzález-espinosa & quintana-ascencio, 1986; montiel & montaña, 2003; gárcia-chavez et al., 2010). in order to establish the experimental units, first, we selected a random orientation in respect to the entrance of the p. barbatus colonies. then at a distance of four meters, in the selected orientation, we eliminated all branches and small rocks and leaves on the soil surface, leaving three 20 x 20 cm quadrants of bare soil with a distance of 10 cm between them. approximately 24 hours later, we placed 20 seeds of each category (i.e., native, only lipids or lipids + proteins, respectively) within each quadrant. the seeds were placed at 06:00h (before starting the activity of the ant colonies) and were checked 12 hours later to count the number of seeds removed. we visited the colonies 24 hours after the experiments to verify if the ants left the seeds out of the colony, which did not happen with any seed type in any colony. we took care that only ants removed the seeds. in fact, in a previous study in the region, garcía-chavez and collaborators (2010) showed that ants are the dominant seed predators, displacing birds and rodents. data analysis since our experimental design corresponds to a mixed effect model, we applied a generalized linear mixed model with a binomial error distribution with logit link (crawley 2012) to test if the proportion of seeds removed at the end of the experiment differs between the seed types (native, lipids and lipids + proteins). for our data analysis, the fixed effect was seed type (native, lipids, and lipids + proteins) while the random factor was the ant colonies. in addition, we applied a post-hoc analysis to look for differences between the levels of the factor “seed type” through wald χ2 tests. all analysis where performed in r (version 3.4.0) (r core team 2017) with the packages lme4 (bates et al. 2015) and phia (helios 2015). results we found that all of the studied colonies removed the offered artificial seeds. further, we observed that the proportion of removed artificial seeds containing only lipids reached 100% in five colonies (35.7%), while the proportion of seeds removed containing lipids and proteins reached 100% in seven colonies (50%). moreover, we recorded no removal of native seeds in seven colonies (50%). note that, we observed ants from the genus pheidole westwood, 1839 (myrmicinae) in contact with the artificial seeds in two experimental units, but we did not record any removal by this ant genus. we observed that, the seed type predicted the proportion of seeds removed (χ2 = 120.31, df = 2, p < 0.001). specifically, the proportion of native seeds removed was lower than the proportion of artificial seeds removed (post-hoc: native v.s. lipids: χ2 = 103.63, df = 1, p < 0.001; native v.s. lipids + proteins: χ2 = 115.51, df = 1, p < 0.001) (fig 2). however, we found no difference in the number of seeds removed between the artificial seeds containing only lipids and both lipids and proteins (posthoc lipids v.s. lipids + proteins: χ2 = 1.968, df = 1, p = 0.16) (fig 2). fig 1. workers of pogonomyrmex barbatus (myrmicinae) manipulating seeds: (a) of opuntia pilifera (cactaceae) and (b) artificial seeds with lipids (9% of the content). photo credits: (a) j. h. garcía-chavez and (b) p. luna. p luna, w dáttilo – artificial seeds fora harvester ant152 the foraging strategies of p. barbatus in the dry and warm season, we found that p. barbatus can switch its foraging activity into the harvest of novel resources (artificial seeds in this case), leaving behind a commonly predated resource (seeds of o. pilifera) (garcía-chávez et al., 2010). additionally, the complete removal of artificial seeds in many ant colonies, suggest that workers of p. barbatus can rapidly focus its foraging efforts on the collection of highly nutritious resources. the higher predation rates on resources with manipulated contents of lipids and proteins suggest that the food quality is quickly communicated among foragers, leading to a greater number of recruited workers to the seed patch (whitford & steinberger, 2009; flanagan et al., 2012). moreover, the nonrejection of our artificial seeds shows that our they could be stored for further consumption (macmahon et al., 2000). in conclusion, we found that our manipulation of lipids and proteins in the artificial seeds generated an increase in the seed predation rates by p. barbatus, which indicates that the manipulation of nutritional components could be useful on future experiments. therefore, the experiment conducted by us using artificial seeds offers an excellent benchmark to study the influence of resource quality on the food foraging behavior of harvester ants in neotropical arid environments. however, note that the results of our experiments could be restricted to the red harvester ant p. barbatus since we do not know how other pogonomyrmex species could react to our artificial seeds. morevoer, it is important to mention that to draw clear conclusions about the differential seed predation by harvester ants, and to answer particular ecological hypothesis, the use of artificial seeds require an adequate experimental design. in short, our study is, therefore, an important advance to implement repeatable methods in the study of harvester ants in order to better understand its influence as seed predators on ecological communities. acknowledgments p. luna thank consejo nacional de ciencia y tecnología (conacyt) for the postgraduate scholarship (no. 771366), the comisariado de bienes comunales de zapotitlan salinas, the pacheco-gonzález family for their help during fieldwork and to juan h. garcía-chávez and vinicio sosa for their suggestions on the experimental approach. d. tokman provided insightful comments and helped the authors with insect nutrition topics. references bates, d., mächler, m., bolker, b. & walker, s. 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(1996).vegetation environment relationships based on a life-forms classification in a semiarid region of tropical mexico. revista de biologia tropical, 44: 581-590. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.281-285sociobiology 61(3): 281-285 (september 2014) evidences of batesian mimicry and parabiosis in ants of the brazilian savanna mc gallego-ropero1, rm feitosa2 introduction mimicry is considered a conspicuous demonstration of darwinian selection (fisher 1930) and can incorporate a wide range of sensory modalities, including visual, auditory, vibrational and chemical (pasteur 1982). batesian mimicry has been described as the mechanism by which a palatable species look similar to an unpalatable one to avoid predation. mimetic systems have been recorded in a vast range of invertebrates and despite the fact that the majority of quantitative studies of batesian mimicry have examined defensive visual mimicry among lepidopterans (e.g. joron & mallet 1998), social insects are one of the most common “models” of batesian mimics (hölldobler &wilson 1990; mciver & stonedahl 1993; cushing 1997; ceccarelli & crozier 2006). it occurs because social insects may have strong defense mechanisms that are effective against predators. nevertheless, records of social insects mimicking themselves are relatively rare. batesian mimicry has been suggested in few ant species (ward 1984; hölldobler & wilson 1992; gobin abstract despite the numerous records of ant-mimicking arthropods, reports of ant species that are mimics among themselves are still rare. in the savanna of central brazil we found two ant species that are remarkably similar in color pattern and body size, pseudomyrmex termitarius and camponotus blandus. both species are widely distributed in the neotropical region, but the cases of mimicry between them are apparently restricted to populations inhabiting nests of the termite cornitermes cumulans in the brazilian cerrado. field observations and excavation of the termitaries revealed that camponotus blandus shares nest chambers and foraging trails with p. termitarius, and workers of both species are mutually tolerant. our observations suggest that the morphological and behavioral similarities between these species represent a batesian mimicry relationship in which the relatively palatable camponotus blandus mimics the unpalatable p. termitarius for predator avoidance. the pacific association between the termitophilous colonies of these species may also suggest some level of parabiotic interaction. sociobiology an international journal on social insects 1 university of cauca, department of biology, popayán, colombia. 2 departamento de zoologia, universidade federal do paraná, curitiba, brazil. article history edited by gilberto m m santos uefs, brazil received 11 february 2014 initial acceptance 28 may 2014 final acceptance 15 june 2014 keywords camponotus blandus, pseudomyrmex termitarius, termites, mimicry evolution, parabiosis, brazilian cerrado. corresponding author maría cristina gallego ropero university of cauca department of biology calle 5 no. 4 – 70, popayán, colombia. e-mail: macrisgaro@yahoo.es et al. 1998; merrill & elgar 2000; ito et al. 2004), despite the vast diversity of arthropods that mimic ants (e.g. pie & del-claro 2002; taniguchi et al. 2005; nelson & jackson 2009). here we provide a novel account of a remarkable similarity between two species of unrelated ants. we found workers of the formicine ant camponotus blandus (fr. smith) reproducing the body coloration and sharing nest chambers and foraging trails of the pseudomyrmecine pseudomyrmex termitarius (fr. smith). the two species are exclusively neotropical, occurring from central america to northern argentina. however, the records of batesian mimicry in these species are apparently restricted to the populations inhabiting termite nests in the savanna of central brazil. like most members of pseudomyrmecinae, p. termitarius is a very aggressive ant with precise mandibles and a painful, venomous sting which serves as a solid deterrent to many potential predators. as its name suggests, p. termitarius has a preference for establishing its colonies inside termite nests, although subterranean nests can also be found (mill 1981; jaffé et al.1986; pulgarín 2004). this species is research article ants mc gallego-ropero & rm feitosa batesian mimicry and parabiosis in ants282 an opportunistic predator and, when inhabiting termitaries, it can prey upon termite brood or inquiline arthropods that also occupy the termite nests. populations of p. termitarius are widespread in the brazilian cerrado, where they can they can most commonly be found inside nests of the termite cornitermes cumulans (kollar) (redford 1984), although colonies can also be found in open soil and in association with other termite species (kempf 1960). in contrast, the defense mechanisms of formicine ants are limited to a formic acid spray, since these ants do not possess a functional sting. this is the case with camponotus blandus. the formic acid of these ants renders some of them unpalatable to some predators. however, lacking more substantial defenses, many formicines may be relatively more vulnerable to predation (lamon & topoff 1981; montgomery 1985; merrill & elgar 2000). for example, ants of the genus camponotus are among the most commonly preyed upon by australian birds (barker & vestjens 1990). like many species of this genus, c. blandus is a polymorphic, generalist ant (fernández 2003). the nests are found in the soil, in preexisting cavities under rocks, decaying logs or termite mounts. the foraging is largely arboreal but the workers can be efficient ground predators, preying on terrestrial arthropods and even raiding termite galleries (fowler & crestana 1987; mendonça & resende 1996). morphologically, camponotus blandus can be highly variable regarding the color patterns along its wide geographical distribution, but as far as we know, only in the brazilian cerrado this species assumes the reddish and black pattern typical of p. termitarius. both species can share nests of cornitermes cumulans in central brazil. our morphological, geographical and behavioral lines of evidence indicate that one species is a visual model of the other. we argue that c. blandus, a member of a normally highly predated genus, mimics an aggressive and venomous ant, p. termitarius, in order to reduce the risk of predation in the open areas of central brazil. this is the first formal report of mimicry of an ant by another ant in the neotropical region and may also represent a new case of parabiosis between ant species. materials and methods collecting and observations on interactions between camponotus blandus and pseudomyrmex termitarius were carried out in two field stations. the first one, estação experimental embrapa cerrados, is located in the planaltina municipality, near brasília, in brazil’s federal district (15,36’35,5”s; 47,44’09,5”w). it is a 3,500 hectares site with permanent ecological reserves and 10 different cerrado phytophysiognomies (embrapa 2010). the second station, reserva acangaú, is situated in the paracatu municipality, state of minas gerais (17,12’08,2”s; 47,4’19,6”w). it is classified as a private natural heritage reserve of 3,000 hectares, where the cerrado sensu strictu is the prevalent physiognomy (inmet 2011). we made qualitative behavioral observations of workers from five colonies of camponotus blandus and p. termitarius cohabiting in termitaries of cornitermes cumulans (fig. 1a). termitaries containing colonies of both species were excavated to determine the position of the ant nests and internal organization of the colonies. then, eight and three additional colonies were collected for p. termitarius and camponotus blandus, respectively, from termitaries of both study sites. these additional colonies were found to be isolated, without the presence of the second species. our observations were made throughout the day, primarily to establish the periods when the ants were most active and abundant above the ground. when possible, all the individuals present in the colonies were collected, including sexuals and brood, and fixed in 70% ethanol. voucher specimens are deposited at the insect collection of the instituto de biologia, universidade de brasília (unb), df, brazil. fig. 1 a typical termitary of cornitermes cumulans in the brazilian cerrado (a), worker of pseudomyrmex termitarius foraging solitarily on a termitary (b), mixed sample from a nest chamber containing individuals of camponotus blandus (cb) and pseudomyrmex termitarius (pt) (c). results the foraging behavior of pseudomyrmex termitarius is predominantly solitary (fig. 1b); however, workers may follow disperse trails on the surface of termitaries and in the adjacent areas. we found workers of the mimic camponotus blandus sharing the trails of p. termitarius in colonies studied from both study sites, though workers of camponotus blandus could be seen foraging independently. termitaries excavation revealed that p. termitarius forms polydomous colonies, occupying several chambers inside the termite nests. these chambers were normally found on the hypogaeic portion of the termitaries, from 40 cm high to 15 cm below the soil surface and, in many cases, were shared with colonies of camponotus blandus (fig. 1c). it was not possible to observe if brood of both species was kept together, but no agonistic interaction was observed between workers of the two species. the presence of the termitary builder sociobiology 61(3): 281-285 (september 2014) 283 (cornitermes cumulans) is not a requirement for the presence of the ants, since ant nests were found in both abandoned and occupied termite nests. in general, colonies of p. termitarius were larger than those of camponotus blandus, with an average number of individuals of 45 and 23, respectively, including brood and sexuals. morphologically, the most conspicuous similarity between these two species is their color pattern. both share the reddish antennae, mesosoma, waist, anterior segments of gaster, and distal portion of legs, and the black head, coxae and apical segments of gaster. additionally, the total body length is also very similar in both species, about 7 mm (fig. 2). extensive collecting was performed in other areas where the species co-occur and the study of specimens of both species deposited in myrmecological collections indicates that the morphological similarity only occurs in populations of termitaries in central brazil. most of the camponotus blandus specimens found without the association with p. termitarius are predominantly blackish in color. still, regarding all the species phylogenetically related (subgenus myrmaphaenus) to camponotus blandus, the termitophilous populations of this species are the only ones to exhibit the color pattern observed here. discussion the color combination exhibited by p. termitarius and camponotus blandus is most likely aposematic. the black and reddish coloring can frequently be seen as a warning pattern in unpalatable plants and animals of different groups and regions (lythgoe 1979). many ichneumonoid wasps and butterflies of the “tiger-complex” combine these colors, apparently to advertise mechanical or chemical defenses to potential predators (quicke et al. 1992; beccaloni 1997). similar reddish and black color patterns can be found within a variety of species in the australian ant genus myrmecia. species of these ants are among the most aggressive ants in the world (haskins & haskins 1950; hölldobler & wilson 1990). populations of camponotus blandus occupying termite nests in the brazilian cerrado differ in worker caste from the majority of the conspecific populations with arboreal habits and also from those of different biomes and geographic regions. while most species of camponotus have a wide range of size classes, termitophilous camponotus blandus exhibits relatively little variation in the size of the foragers. this conservative caste system helps to create a more consistent congruence in size between camponotus blandus and p. termitarius foragers, and thus increases the efficacy of mimicry, given that p. termitarius is a monomorphic species. mimicry will be most effective when the ranges of the mimic and its model overlap broadly, such that predators encountering the mimic are likely to have had some experience with the model (pough 1994). the range of camponotus blandus lies entirely within that of p. termitarius, although the mimicry cases between both species are apparently restricted to the termitaries in central brazilian cerrado. the geographic sympatry of these species is even tighter at the ecological level. both camponotus blandus and p. termitarius share the same nesting and foraging strategies in the open areas of the cerrado. more significantly, however, is the fact that workers of both species can share nest chambers inside the termitaries, showing an almost complete ecological overlapping. theory also predicts that mimicry will be most effective when the model is relatively abundant (lindström et al. 1997), and in all our observations p. termitarius is relatively more common than camponotus blandus within their range, with more individuals per colony. museum collections corroborate this finding; p. termitarius is well represented in the ant collection of the museu de zoologia da usp (mzsp), with more specimens than for most of its congeners (rmf, pers. obs.). the striking similarities between p. termitarius and camponotus blandus are obviously unlikely to be derived from a common phylogenetic ancestry given that the formicines and pseudomyrmecines are very divergent phylogenetic lineages (brady et al. 2006). instead, our observations suggest that these similarities represent a batesian mimicry relationship fig. 2 lateral view of workers: pseudomyrmex termitarius (a) and camponotus blandus (b). mc gallego-ropero & rm feitosa batesian mimicry and parabiosis in ants284 in which the relatively palatable camponotus blandus mimics the unpalatable p. termitarius in order to escape predation. batesian mimicry in ants has been reported in pheidole nasutoides, camponotus bendigensis, polyrhachis rufipes and camponotus sp. the major workers of pheidole nasutoides mimic soldiers of nasutitermitinae termites, which have formidable chemical defenses against predation in the costa rican tropical forest (hölldobler & wilson 1992). camponotus bendigensis shares body size and color patterns with myrmecia fulvipes, a very aggressive australian ant with a painful venomous sting (merrill & elgar 2000). in the oriental tropics, the species polyrhachis rufipes is often found on trails of gnamptogenys menadensis. workers of polyrhachis rufipes can follow the trails of g. menadensis and thus reach sugar sources (gobin et al. 1998). finally, workers of an undescribed species of an arboreal camponotus were exclusively observed on foraging trails of the myrmicine ant crematogaster inflata in western malaysia. the bright yellow and black color pattern, as well as the walking behavior, are very similar in both species (ito et al. 2004). the most remarkable result of our study is the finding of camponotus blandus sharing nest chambers and foraging trails with pseudomyrmex termitarius, and the mutual tolerance between workers of both species during foraging and intranidal activities. it must be confirmed in the future whether camponotus workers can recognize signals from pseudomyrmex termitarius and vice versa, but our observations may suggest some level of parabiotic interaction between these species. parabiosis is defined as a particular form of facultative or obligatory symbiosis in which two or more species utilize the same nest structure and sometimes even the same odor trails, but normally keep their broods separate (hölldobler & wilson, 1990). it is important to emphasize the facultative nature of this particular association, since neither of the participating species is dependent on the other. parabiotic associations have been described for ant species from all over the globe, but most records report facultative relations concentrated in the neotropics, where several cases, generally involving members of different subfamilies, have been recorded (e.g. adams 1990; ipinza-regla et al. 2005; sanhudo et al. 2008). it remains to be explained why this tight association between the wide distributed camponotus blandus and pseudomyrmex termitarius involves only the termitophilous populations of these species in the brazilian savanna. the variables favoring this mimetic and probably parabiotic system may include the predation of camponotus blandus by birds and terrestrial reptiles, both are extremely common in the savanna of central brazil. it is likely that these predators have sufficient spectral sensitivity to detect the color pattern exhibited by these ants, especially in the open lands of the cerrado, where foragers are more exposed than in the forests and woodlands where camponotus blandus also occurs. the efficient defensive mechanisms of pseudomyrmex termitarius and the solid protection of the termitaries of cornitermes cumulans have apparently resulted in the evolution of the mimicry and parabiotic syndromes in camponotus blandus. acknowledgements we thank the staff of the termitology laboratory of universidade de brasília for their assistance in the field and the colleagues of embrapa and reserva acangaú for the collecting permits. thanks to the pec-pg program for the grant offered and to universidad del cauca, colombia. this study was funded by capes, pronex-fapdf-cnpq project (proc.193.000563-2009). we acknowledge the research grant received from fundacão de amparo à pesquisa do estado de são paulo to r.m. feitosa (no. 11/24160–1). references adams, es. 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(1984) a revision of the ant genus rhytidoponera (hymenoptera: formicidae) in new caledonia. australian journal of zoology, 32: 131-175. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.413-420sociobiology 60(4): 413-420 (2013) differential gene transcription in honeybee (apis cerana) larvae challenged by chinese sacbrood virus (csbv) y zhang1, x huang2,1, zf xu2, rc han1, jh chen1 introduction insects have diverse mechanisms to combat infection by pathogens (evans et al., 2006), including cellular and humoral immune defenses. the innate immune response of insect actions include the secretion of antimicrobial peptides, phagocytosis, melanization and the enzymatic degradation of pathogens (hoffmann, 2003; hultmark, 2003), which result from triggering the four non-autonomous pathways, toll (transmembrane signal transducing pathway serves in both immunity and development), imd (immune deficiency), jak/stat (the janus kinase/signal transducers and activators of transcription) and jnk (intracelular signalling pathways) (boutros et al., 2002). viral double-stranded rna is also recognized as a pathogen-associated molecular pattern and processed into small interfering rnas (sirnas) by the host ribonuclease dicer. after amplification by host rna-dependent rna polymerases in some cases, these virus-derived sirnas guide specific antiviral immunity through rna interference and related rna silencing effector mechanisms (ding, 2010). abstract honey bees are economically important social insects. they are suffering from all kinds of pathogens, especially the viruses. in response to pathogens, different immune pathways such as toll, imd, jak-stat and rnai are involved. in the present study, the transcription analysis of 32 immune-related genes from apis cerana challenged by chinese sacbrood virus (csbv), the most widely distributed virus in a. cerana, was carried out by qrt-pcr to provide cues for the antiviral mechanism and the effective control of bee viruses. the expression level of 22 genes were statistically changed, including 11 up-regulated genes (cactus-2, lys-2, vir, s3a, mta1, faa, vhdl, co-1-iv, ago-1, ago-3, aub) in which 3 (ago-1, ago-3, aub) were related to rnai pathway, and 11 down-regulated genes (kenny, pgrp-lc, pgrp-s2, abaecin, lys-1, lys-3, domeless, tepa, mlc, dscam, rpl8) related to toll, imd, and jak-stat pathways. the results indicated that csbv infection in a. cerana may activate the whole immunity systems, including the rna-based antiviral immunity system. this work constituted the first report, under laboratory conditions, about induction of immune related genes in response to csbv. sociobiology an international journal on social insects 1 guangdong entomological institute, guangzhou, guangdong, china. 2 south china agricultural university, guangzhou, guangdong, china. research article bees article history edited by: evandro n silva, uefs, brazil received 19 july 2013 initial acceptance 11 september 2013 final acceptance 02 october 2013 keywords apis cerana, qrt – pcr, rnai, antiviral immunity corresponding author richou han guangdong entomological institute 105 xingang road west, guangzhou guangdong, 510260, china e-mail: richou-han@163.net apis mellifera and apis cerana are the major honey bee species in the global beekeeping industry (gallai et al., 2009; garibaldi et al., 2011). they are heavily infected by different vital viruses (chen et al., 2004, 2006; ai et al., 2012). the a. mellifera genome (honeybee genome sequencing consortium, 2006) revealed that honey bees possess homologues of members of the four pathways implicated in humoral immune responses (evans et al., 2006). chinese sacbrood virus (csbv) is the most stricken pathogen of a. cerana, which results in severe and deadly infections of the colony and eventually losses of the entire colony (yan et al., 2008; liu et al., 2010; han et al., 2013). this virus was first observed in guangdong province, china in 1972 and spread to the whole china and the counties of southeast asia (liu et al., 2010). some efforts have been made to study this viruse, such as diagnostic methods (electron microscopy, enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (rt-pcr)) (yan et al., 2009), and the control of this disease by rna interference (liu et al., 2010). it is interesting that a. mellifera is not sensitive to csbv in general beekeeping practice y zhang, x huang, zf xu, rc han, jh chen gene transcription in honeybee challenged by csbv 414 (zhang et al., 2001). furthermore, a. mellifera may develop cellular and humoral immune responses to various pathogens such as bacteria (chan et al., 2009; lourenço et al., 2013), viruses (azzami et al., 2012), microsporidian (antúnez et al., 2009; chaimanee et al., 2012) and varroa mites (gregorc et al., 2012). however, no information on the expression of immune-related genes from a. cerana after infection of csbv is reported. in the present study, the transcription analysis of the immune-related genes from a. cerana challenged by csbv was conducted to provide cues for the antiviral mechanism of the honey bees and the effective control of the bee viruses. materials and methods honey bees second instar larvae of a. cerana were obtained from a single mated honeybee queen in a healthy apiary at conghua, guangdong province (113°17’ e, 23°8’’ n), china. to obtain age controlled larvae, the queen was caged on a comb and left to lay eggs for six hours. twenty hours after larval eclosion, or ninety-two hours after oviposition, the comb containing second instar larvae was retrieved from the colony, and placed in the laboratory for treatments at 32°c 34°c. all the larvae used in this study were detected by rt-pcr method for the absence of the following viruses, black queen cell virus (bqcv), chronic bee paralysis virus (cbpv), acute bee paralysis virus (abpv), deformed wing virus (dwv), kashmir bee virus (kbv), csbv and israeli acute paralysis virus (iapv), with the primers from ai et al. (2012). rna was extracted using trizol reagent (invitrogen) and cdna synthesis was performed using primescripttm 1st strand cdna synthesis kit with dna eraser (takara, japan). amplification profile of pcr consisted of an initial 2-min denaturation at 94 °c, followed by 35 cycles of 30 s at 95 °c, 30 s at 55 °c, 1 min at 72 °c and finally 7-min cycle at 72 °c. no signs of clinical american foulbrood or viral disease were observed in these larvae. virus csbv-infected larvae with typical symptom were collected from an apiary at xinhua village from hunan province, and kept at -80 °c for less than 10 days before use. the presence of csbv virus was confirmed by observing the morphological symptom under electronic microscopy and rt-pcr according to chen et al. (2004, 2006) and liu et al. (2010). to obtain csbv, the larvae (approximately 0.2 g) infected by csbv were ground in 6 ml sterile phosphate-buffered solution (pbs) (1x: 137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 1.8 mm kh2po4, ph 7.4) with a sterile grinder. the resulting solution was centrifuged at 12,000 rpm at 4 °c for 10 min, and the supernatant was further passed through a 0.45 µm cell filter first, then through a 0.22 µm cell filter (ghosh et al., 1999). csbv concentration was quantitated by absolute quantification assay (liu et al., 2010). csbv polyprotein gene (sbv1) was amplified to detect copy number of csbv according to liu et al. (2010). pcr reactions were carried out in triplicate in an mx3000ptm real-time pcr system (stratagene, california, usa), using sybr_green (brilliant ii sybr-green qpcr master mix; stratagene, california, usa). csbv concentration for the experiments was 7.20 ×104 copies/ml. viral challenge and sample collection second instar larvae were collected from a comb with a special grafting tool and transferred to a 96-well tissue culture plate filled with 30 µl of basic diet prepared according to patel et al. (2007). the larvae were maintained in an incubator (sanyo, tokyo, japan) at 33°c 34°c and 75% relative humidity. twenty-four hours later, each larva was fed with 30 µl csbv suspension or pbs for 24 h. then, twenty larvae were picked from each replicates, sterilized with 75% ethanol (prepared with 0.2% diethylpyrocarbonate (depc) -water) for 5 min, washed with depc-water for three times, immediately frozen in liquid nitrogen, and kept at -80°c until total rna isolation (liu et al., 2010; zhang et al., 2010). three replicates with 50 larvae in each were used for both treatments. gene selection and primers a total of 32 genes from a. cerana were selected for quantitative analysis according to references on the innate immune pathways in insects (toll, imd, jak/stat and rnai pathways) (table 1) (christophides et al., 2004; clem et al., 2005; dostert et al., 2005; royet et al., 2005; yang & cox-foster, 2005; evans et al., 2006; ding et al., 2010). primers and genebank accession numbers of the corresponding genes was summarized in table 1. quantitative real-time pcr (qrt-pcr) quantitative real-time pcr was carried out according to our previously described method with modifications (zhang et al., 2010). briefly, total rna and cdna were prepared as described above. expression of actin gene (gi: 406122) was used as an internal control. the efficiency of each primer set was first validated by constructing a standard curve through five serial dilutions. pcr reactions were carried out in triplicate in an mx3000ptm real-time pcr system (stratagene, usa), using sybrgreen (sybr® premix ex taq™ ii, takara, japan). a control without template was included in all batches. the pcr program began with a single cycle at 95 °c for 10 min, 40 cycles at 95 °c for 15 s, 55 °c for 30 s and 72 °c for 45 s. afterwards, the pcr products were heated to 95 °c for 30 s, cooled to 55 °c for 30 s and heated to 95 °c for 30 s, in order to measure the dissociation curves sociobiology 60(4): 413-420 (2013) 415 and to determine a unique pcr product for each gene. mrna levels were calculated relative to actin expression using the mx3000ptm software (version 4.1) (agilent, usa). the fold change was calculated using the 2−δδct method. each sample was analyzed independently and processed in triplicate. pcr products were diagnosed by 1% agarose electrophoresis. positive samples were cloned to pmd-19t (takara, japan), then sent to invitrogen company china for sequencing with corresponding specific primers. data analysis data were analyzed using spss statistical software (version 16.0) and the significance between treatments in each experiment was evaluated by one-way anova test followed by tukey’s test for pair comparisons at p < 0.05. the values are expressed as means ± s.d. (p < 0.05) was defined as statistically significant. results bee colonies did not present any visible clinical symptoms of any disease (i.e., american foulbrood or chalkbrood). nosema spp. spores were not detected from the bee samples. no csbv, bqcv, cbpv, dwv, kbv, sbv, or iapv viruses were detected in all the larvae used in this study. csbv was only present in the larvae fed with csbv. rna expression levels of 32 genes were determined using qrt-pcr 24 h after csbv challenge. a set of 32 genes showed differential expression in the larvae challenged by csbv. gene-specific amplification was confirmed for 32 primer pairs by sequencing, by a single peak in melt-curve analysis and a single band with the expected size in agarose gel electrophoresis. no primer-dimer formation was detected and the standard curves derived from five-fold serial dilutions of pooled cdna from the larvae (csbv-challenged/ control) gave correlation coefficients greater than 0.995 and efficiencies between 80% and 103%. among 32 differential expression genes, 11 genes (cactus-2,p = 0.0269; lys-2, p = 0.0173; vir, p = 0.0451; s3a, p = 0.0399; mta1, p = 0.0219; faa, p = 0.0233;vhdl, p = 0.0400;co-1-iv, p = 0.0141;ago-1, p = 0.0018; ago-3, p = 0.0186; aub, p = 0.0142) were up-regulated, 11 genes (kenny, p = 0.0057; pgrp-lc, p = 0.0418; pgrp-s2, p = 0.0352; abaecin, p = 0.0247;lys-1, p = 0.0048;lys-3, p = 0.0276; domeless, p = 0.0434; tepa, p = 0.0336; mlc, p = 0.0389; dscam, p = 0.0012;rpl8, p = 0.012;) down-regulated, and 10 gene (galectin, p = 0.2860; cactus-1, p = 0.1460; imd, p = 0.0991; basket, p = 0.1010; def2, p = 0.1070; tpi, p = 0.1200; ago-2, p = 0.0765; dicer-1, p = 0.0629; dicer, p = 0.2570; piwi, p = 1.9400;) showed no significant difference in expression (anova, tukey’s test , p < 0.05) (fig 1). of the up-regulated expression genes, cactus-2, lys-2 were genes related to toll pathway; ago-1, ago-3, and aub were members of rnai pathways; vhdl and co-1-iv were refig 1 relative transcription analysis of selected genes in csbv infected apis cerana larvae by qrt-pcr. csbv the larvae fed with csbv for 24 h. pbs the larvae fed with phosphate-buffered solution as control. results are reported as an average of the triplicates plus the standard deviation. bars with different letters indicated significant differences by tukey’s test at p < 0.05(*). lated to cellular component and development; mta1, faa, and s3a were involved in cellular metabolic and regulated progress. among the down-regulated genes, kenny, pgrp-lc were the important members in imd pathways; imd is specific for antimicrobial defense and normal development; pgrp-s2 coding peptidoglycan recognition protein critically was involved in sensing bacterial infection and activation of the toll signaling pathway (evans et al., 2006); abaecin, lys-1, lys-2 and lys-3 were antimicrobial effectors of toll pathway; domeless, vir and tepa were the members of jak pathway; mlc was related to cellular component and development; dscam was related to humoral immunity (evans et al., 2006); rpl8 was involved in cellular metabolism and regulation. no selected genes specific to rnai pathway displayed down-regulated expression, although ago-2, dicer-1, dicer, and piwi showed no significant difference in expression. y zhang, x huang, zf xu, rc han, jh chen gene transcription in honeybee challenged by csbv 416 g en e n am e g en e id g en e an no ta tio n pr im er s r eg ul at io n1 r ef er en ce s2 r ef er en ce s ho st pa th og en s r eg ula tio n ga le ct in g b 10 02 6 ce llu la r r eg ul at io n f: a c c a c c t t c a g g t c c t g g t t r : c c c a g a g a g c c a a g g t t t c c d co -1 -i v g b 14 56 4 co lla ge n f: g g t ta c g t t c g t t c c c g g t t r : t a c c t t g c t c g c c c c t g ta a u tp i gi |1 48 22 42 76 g ly co ly tic e nz ym e f: c a t g g g ta a t t c t t g g t c r : c a t g g g ta a t t c t t g g t c e fa a g b 13 98 2 hy dr ol as e f: a g g g a ta t t t g g t t t g a t t g g r : g t g g a g a a g a t g c t c t g g g a t u z ha ng e t a l., 20 10 a .c er an a v ar ro a de st ru vt or d pg rp -l c g b 17 18 8 im d pa th w ay f: t c c g t c a g c c g ta g t t t t t c r : c g t t t g t g c a a a t c g a a c a t d g eo rg e et a l., 20 02 a no ph el es e . c ol i u ke nn y g b 17 10 6 im d pa th w ay f: g c t g a a c c a g a a a g c c a c t t r : t g c a a g t g a t g a t t g t t g g a d e va ns e t a l.e t a l., 20 06 a .m el li fe ra e . c ol i p . l ar va e d im d g b 18 60 6 im d pa th w ay f: t g t ta a c g a c c g a t g c a a a a r : c a t c g c t c t t t t c g g a t g t t e e va ns e t a l.e t a l., 20 06 a .m el li fe ra e . c ol i p . l ar va e d ba sk et g b 16 40 1 im d pa th w ay f: a g g a g a a c g t g g a c a t t t g g r : a a t c c g a t g g a a a c a g a a c g e e va ns e t a l.e t a l., 20 06 a .m el li fe ra e . c ol i p . l ar va e d ds ca m g b 11 87 1 im m un og lo bu lin f: t t c a g t t c a c a g c c g a g a t g r : a t c a g t g t c c c g c ta a c c t g d e va ns e t a l.e t a l., 20 06 a .m el li fe ra e . c ol i p . l ar va e d te pa g b 18 78 9 ja k pa th w ay f: c a a g a a g a a a c g t g c g t g a a r : a t c g g g c a g ta a g g a c a t t g d e va ns e t a l., 20 06 a .m el li fe ra e . c ol i p . l ar va e d do m el es s g b 16 42 2 ja k pa th w ay f: t t g t g c t c c t g a a a a t g c t g r : a a c c t c c a a a t c g c t c t g t g d x i e t a l., 2 00 8 a ed es a eg yp ti (d en gu e vi ru s se ro ty pe 2 ) d e n v -2 u vi r 10 08 72 98 4 ja k pa th w ay f: g g g ta g t g ta g g c a g a g g g g r : c t t g t g t c c a a g g g c g a c t t u d os te rt e t a l., 20 05 d ro so ph il a d ro so ph ila c v iru s (d c v ) u vh dl g b 72 61 82 l ip op ro te in f: g c a t c a c c t t c t g a c c a a c c r : a c c t c g t c c a a c a t c c t t c t u z ha ng e t a l., 20 10 a . c er an a va rr oa u m lc g b 40 98 81 m yo si n re gu la to ry f: a a t c t c t t c g c a t c t c g c r : c g c a t c g t t g a c t t c c t t d sc ha rla ke n et a l., 20 08 a . m el li fe ra e .c ol i d ta bl e 1 l oc us n am es , g en e an no ta tio n, p ri m er s an d ge ne ba nk a cc es si on n um be rs fo r t he s el ec te d ge ne s 1 r eg ul at io n in th is s tu dy ; 2 r eg ul at io n, c ha lle ng ed h os ts a nd p at ho ge ns fr om th e re fe re nc es ; u : u pre gu la te d ge ne s; d : d ow nre gu la te d ge ne s; e : n o di ff er en tia l e xp re ss io n ge ne s. sociobiology 60(4): 413-420 (2013) 417 m ta 1 gi |1 10 75 68 03 n u r d f: c a t c t c t g t g c t t c t c c t c r : a c t c g a t c t g g t t g t t t t c u s3 a gi |6 65 47 34 0 r ib os om al p ro te in f: g t g c g g g ta g t a a g a g c g a a g a a g r : g t g c g g g ta g ta a g a g c g a a g a a g u rp l8 g b 17 62 9 r ib os om al p ro te in f: t g g a t g t t c a a c a g g g t t c a t a r : c t g g t g g t g g a c g t a t t g a t a a d ag o1 g b 12 26 54 r n a i p at hw ay f: t g g c c c a g a t c a a g ta g a g c r : a a t t t g a ta g c g t t t g t g g t g a t u d in g, 2 01 0 ag o2 g b 15 46 4 r n a i p at hw ay f: g a g c ta t t g c g c g c t ta g a g a r : t a t c a a c a a t g g t g c c c g c c e d in g, 2 01 0 d ro so ph il a fl oc k ho us e vi ru s (f h x ) m ut an t ag o3 g b 19 38 9 r n a i p at hw ay f: c c c a a t g c c c g a t g t g a t g t r : t c c a t t c t g c a g g c t t t g c c u d in g, 2 01 0; k ee ne e t a l., 2 00 4 a no ph el es ga m bi ae o ’n yo ng -n yo ng vi ru s m ut an t au b g b 10 29 3 r n a i p at hw ay f: t g g t t g g g a t g t t t t g c c a c a r : t c g c a ta g c a c c t c t t c c c a u d in g, 2 01 0 di ce r g b 15 17 0 r n a i p at hw ay f: a g c a g ta g c t g a t t g t g t g g a r : t t c a g a a g c g c a a g g c a t g t u d in g, 2 01 0 di ce r1 g b 11 96 6 r n a i p at hw ay f: a a g a g c t c c a g a t g c c c t g t r : t g c a t c a c c t c c a t c a a g t g g e d in g, 2 01 0 pi w i 41 24 27 r n a i p at hw ay f: t g c a a a a g a a a c a g t g c t g g a r : t c g c a ta g c a c c t c t t c c c a e d in g, 2 01 0 ab ae ci n g b 18 32 3 t ol l p at hw ay f: c a g c a t t c g c a ta c g ta c c a r : g a c c a g g a a a c g t t g g a a a c d e va ns e t a l., 2 00 6 a . m el li fe ra e . c ol i p . l ar va e u ca ct us -1 g b 10 65 5 t ol l p at hw ay f: c a c a a g a t c t g g a g c a a c g a r : g c a t t c t t g a a g g a g g a a c g e e va ns e t a l., 2 00 6 a .m el li fe ra e . c ol i p . l ar va e u ca ct us -2 g b 13 52 0 t ol l p at hw ay f: t ta g c a g g a c a a a c g g c t c t r : c a g a a a g t g g t t c c g g t g t t u x i e t a l., 2 00 8 a ed es ae gy pt i (d en gu e vi ru s se ro ty pe 2 ) d e n v -2 d de f2 g b 10 03 6 t ol l p at hw ay f: g c a a c ta c c g c c t t ta c g t c r : g g g ta a c g t g c g a c g t t t t a e e va ns e t a l., 2 00 6; a .m el li fe ra e . c ol i; p . l ar va e u ly s1 g b 10 23 1 t ol l p at hw ay f: g a a c a c a c g g t t g g t c a c t g r : a t t t c c a a c c a t c g t t t t c g d e va ns e t a l., 2 00 6 a .m el li fe ra e . c ol i p . l ar va e u ly s2 g b 15 10 6 t ol l p at hw ay f: c c a a a t ta a c a g c g c c a a g t r : g c a a t t c t t c a c c c a a c c a t u e va ns e t a l., 2 00 6 a .m el li fe ra e . c ol i p . l ar va e d ly s3 g b 19 98 8 t ol l p at hw ay f: t c g a t g a a t g c g a a g a a a a t c r : t c g a t g a a t g c g a a g a a a a t c d e va ns e t a l., 2 00 6 a .m el li fe ra e . c ol i p . l ar va e u pg rp -s 2 g b 19 30 1 t ol l p at hw ay f: t a a t t c a t c a t t c g g c g a c a r : t g t t t g t c c c a t c c t c t t c c d e va ns e t a l., 2 00 6 a .m el li fe ra e . c ol i p . l ar va e u ac ti n g b 40 61 22 r ef er en ce g en e f: a t g c c a a c a c t g t c c t t t c t g g r : g a c c c a c c a a t c c a ta c g g a (c on tin ua tio n) y zhang, x huang, zf xu, rc han, jh chen gene transcription in honeybee challenged by csbv 418 discussion as social animals, honey bees are at considerable risk from parasites and pathogens (gregory et al., 2005; yang & coxfoster, 2005; evans, 2006; azzami et al., 2012). specifically, increased genetic relatedness and the high population densities of honey bee societies can strongly favour pathogen spread and epizootic outbreak. however, compared to the sequenced drosophila and anopheles genomes, honey bees are relatively immunologically deficient (i.e., express fewer immune response proteins) (evans et al., 2006). usually, in response to bacterial pathogens, fungal pathogens, parasite or acaricides, the expression level of antimicrobial peptides were up-regulated in the infected bees (gregory et al., 2005; evans, 2006; scharlaken et al, 2008; aronstein et al., 2010; dussaubat et al, 2012; gregorc et al, 2012; hamiduzzaman et al, 2012; garrido et al., 2013). the up-regulated genes were detected from a. mellifera after response to bacteria (erp60, obp17, propo, hsps; lys, hymenoptaecin, transferrin-1abaecin, defensin, hymenoptaecin, pgrp-s3, b-glc-2, cactus-2, dorsal-1b, relish) (scharlaken et al., 2008; chan et al., 2009; lourenço et al., 2013), microsporidian (abaecin, defensin, hymenoptaecin ) (antúnez et al., 2009; chaimanee et al., 2012) and varroa mites (pgrp-sc, abaecin, defensin1, hymenoptaecin ) (gregorc et al., 2012). however, infection of a. mellifera with abpv produced neither elevated levels of specific antimicrobial peptides (amps), such as hymenoptaecin and defensin, nor any general antimicrobial activity, such as nodulation (azzami et al., 2012). these data suggest that bees use a distinct mechanism to counter different viral infections. the present results showed that a. cerana produced expression changes of immune-related genes when responding to csbv infection. rnai based antiviral pathway might be very important for a. cerana. in the present study, after challenged by csbv, a. cerana larvae produced 3 down-regulated (pgrp-s2, abaecin, lys-1) and 2 up-regulated (cactus-2, lys-2) genes for toll pathway. however, in response to paenibacillus larvae, the genes (pgrp-s2, abaecin, lys-1 and lys-3) in a. mellifera were strongly up-regulated, while cactus-2 and lys-2 were downregulated (evans et al., 2006). the differences in gene expression patterns of toll pathway may be related with the bee species and responding pathogens. moreover, from the gene regulation pattern, to a certain extent, it seemed that the toll pathway was suppressed by csbv infection. of 4 genes (imd, basket, kenny, pgrp-lc) related to imd pathway, 2 genes (kenny and pgrp-lc) were down-regulated and others (imd and basket) showed no significant difference in expression. pgrp-lc is the activator of the imd signaling process, which elicited by peptidoglycan, fungi and varroa mites (werner et al., 2003; stenbak et al., 2004; gregorc et al., 2012). it was apparent that csbv infection suppressed the expression of pgrp-lc. among 3 genes related to jak/stat pathway, 2 genes (domeless, tepa) were up-regulated, but vir (virus-induced rna) was down-regulated. vir was not induced by pathogenic bacteria or fungi in drosophila (dostert et al., 2005). but jak kinase hopscotch was involved in the control of the viral load in infected flies (dostert et al., 2005). previous studies also indicated that rna interference as an intrinsic defense against viral infection play a major role in antiviral immunity in insects, e.g. d. melanogaster, anopheles gambiae, bombyx mori, caenorhabditis elegans (keene et al., 2004; galiana-arnoux et al., 2006, 2007; van rij. et al., 2006; kemp & imler, 2009; azzami et al., 2012; xu et al., 2012). central to the rnai mechanism are the slicing enzymes of the argonaute (ago) family (five members in drosophila), which mediate highly specific cleavage of target rna molecules. the specificity of ago enzymes is achieved by their association with small rnas, which guide them to complementary sequences (ding, 2010). three rnai pathways, involving different members of the ago family (ago-1, ago-2, ago-3), have been defined in drosophila (kemp & imler, 2009) in rna silencing, ago proteins are the effector molecules of specific gene silencing, the specificity of which is determined by the ago-bound sirna (kemp & imler, 2009; ding, 2010). in our results, ago-1, aub, and ago-3 in a. cerana larvae were significantly up-regulated after csbv infection, although ago-2, dicer-1, dicer and piwi showed no significant expression difference, indicating that in honey bees there may be a rna-based antiviral immunity system, though it can active the whole immunity systems. future studies are of importance to reveal the specific role of rnai in the antiviral response of honey bees. acknowledgments this work was supported by natural scientific foundation (10251026001000001) from the national natural science foundation of guangdong, china, and guangzhou agricultural bureau ([2011]584), guangdong department of science and technology (2011b01050002) and guangdong academy of sciences (sf201208). we thank two anonymous reviewers for comments that contributed to improve this article. reference ai, h.x., yan, x., & han, r.c. 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(2010). differential gene expression of the honey bees apis mellifera and a. cerana induced by varroa destructor infection. j. insect physiol., 56: 1207 1218. doi: org/10.1016/j. jinsphys.2010.03.019 * y zhang, x huang and rc han contributed equally to this work. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i1.1840sociobiology 65(1): 79-87 (march, 2018) special issue effects of usnic, barbatic and fumarprotocetraric acids on survival of nasutitermes corniger (isoptera: termitidae: nasutitermitinae) introduction termites are eusocial insects that live in large colonies, in which the work is well divided, and all tasks are performed by distinct groups of individuals organized into castes of wingless or winged termites. nasutitermes corniger (motschulsky), a member of the termite family termitidae, is one of the most dominant and broadly distributed species of the genus nasutitermes. it is a neotropical species found in roughly 42 caribbean islands and other countries spanning a longitudinal distance of 6000 km to include parts of central and south america, in brazil it is found in the semi-arid region (scheffrahn et al., 2005). n. corniger is highly adaptable abstract lichens (algal-fungal association) synthesize unique chemical substances with different biological activities. three pure lichen compounds were assayed to evaluate their potential insecticidal activity against the termite nasutitermes corniger on petri dishes. usnic, fumarprotocetraric and barbatic acids were isolated and purified from the lichens cladonia substellata, c. verticillaris and cladia aggregata, respectively, using thin-layer and high-performance liquid chromatography for attesting their purity. nuclear proton magnetic resonance and infrared spectrophotometry was used for their chemical characterization. after exposure, mortality of termites (workers and soldiers) was determined during 11 days period. the termiticidal effect was influenced by the exposure time and the type of member colony. the results showed that lichen substances, tested at 5, 7 and 10 mg ml-1, have a termiticidal activity (~100%) on worker termites after eight days of treatment, in comparison with controls. however, no significant effect on soldiers was found. these findings indicate that usnic, fumarprotocetraric and barbatic acids are potential compounds for use in the control of this urban pest. abbreviations: usnic acid (usn), barbatic acid (bar) and fumarprotocetraric acid (fum). sociobiology an international journal on social insects mcb martins1, rs lopes2, ps barbosa1, r santiago1, brm rodrigues1, ac albuquerque3, eps falcão4, vlm lima2, nh silva1, ec pereira5 article history edited by og desouza, ufv, brazil received 01 july 2017 initial acceptance 06 october 2017 final acceptance 21 october 2017 publication date 30 march 2018 keywords cladoniaceae, lichen substances, arboreal termite, termiticidal activity. corresponding author eugênia c. pereira universidade federal de pernambuco departamento de ciências geográficas av. prof. moraes rego, 1235 cep 50.740-530, recife-pe, brasil. e-mail: verticillaris@gmail.com.br to colonization of contrasting habitats in urban, agricultural, and natural environments. usually termites are evolutionarily and ecologically very successful insects, which preferentially build their nests in trees, soil, roofs and even furniture, feeding on cellulose or lignocellulose digested by symbiotic intestinal microbiota (breznak et al., 1982). living in complex societies and being able to digest wood with the aid of a diverse symbiotic gut fauna, seems to be the basis for this success story. these arthropods have considerable ecological importance as primary consumers, assisting in the decomposition process and recycling of nutrients from plants, and also as nitrogenfixing organisms. they are responsible for the distribution of important nutrients in the soil, and can be used as a bioindicator 1 universidade federal de pernambuco, laboratório de produtos naturais, recife-pe, brazil 2 universidade federal de pernambuco, laboratório de lipídios, recife-pe, brazil 3 universidade federal rural de pernambuco, departamento de biologia, recife-pe, brazil 4 universidade federal de pernambuco, centro acadêmico de vitória de santo antão, pernambuco, brazil 5 universidade federal de pernambuco, departamento de ciências geográficas, recife-pe, brazil research article termites mailto:verticillaris@gmail.com.br mcb martins et al. – usnic, barbatic and fumarprotocetraric acids on nasutitermes corniger80 of soil quality and fertility. however, in urban areas some species are considered pests due to the destruction caused to building materials and the deterioration of paintings, books, old documents and monuments of historical importance (verma et al., 2009). one estimate from 2005 put the annual damage caused by termites at about us$ 50 billion worldwide, with the us alone investing more than us$ 11 billion in termite control in 1994 (korb, 2007). in brazil, termite damage affected 42.7% of constructions in 2002, with losses of about 3.5 billion dollars in the past 20 years in the state of são paulo (milano & fontes, 2002). the usual method for controlling termites involves the use of synthetic compounds. however, insecticides, such as organophosphates and pyrethroids, are characterized by their extremely high toxicity against ecosystems and their biota. thus, research has been carried out on natural products from plants (flavonoids, alkaloids, terpenoids and tannins) (santana et al., 2010) and lichens (lectins, depsides and depsidones) (cetin et al., 2008), due to their biological activities, including anti-herbivory and insecticidal properties. lichens are the result of a symbiotic relationship between fungi and algae and/or cyanobacteria in a process denominated lichenization. the characteristics that make lichens different from other fungi are the formation of an air-exposed thallus, slow growth and the longevity of the fruiting bodies (sipman & aproot, 2001). lichens are complex biological structures with unique characteristics, such as the production of distinctive substances grouped in four well differentiated chemically classes (depsides, depsidones, dibenzofurans and depsones), named as lichen acids (howell et al., 2003; eisenreich et al., 2011). those compounds are phenolic acid derivatives with free hydroxyl groups, which can be toxic to most other living beings. the interest in the properties of lichen substances goes as far back as the 17th century. according to solhaug et al. (2009), the deterrent action against herbivory is the most likely ecological function of such substances. several studies have reported herbicidal, anti-herbivory and insecticidal activity of different lichen metabolites (lawrey, 1980; reutimann & scheidegger, 1987; giez et al., 1994; rundel, 1978; nimis & skert, 2006; asplund et al., 2009; silva et al., 2009; pöykkö et al., 2010). however, the effectiveness of most lichen acids against higher termites (termitidae) has not been evaluated. the aim of the present study was to evaluate the insecticidal potential of phenolic compounds: usnic acid (usn), barbatic acid (bar) and fumarprotocetraric acid (fum), extracted and purified from the lichens: cladonia substellata, c. verticillaris and cladia aggregata, respectively, collected in northeastern brazil against n. corniger, a termite of ecological importance that is well adapted to urban areas. materials and methods harvesting and storage of lichens c. substellata vainio was collected in the municipality of mamanguape (paraíba, brazil), c. verticillaris (raddi) fr. was collected in the municipality of alhandra (paraíba, brazil) and c. agreggata (sw.) nyl. was collected in the municipality of bonito (pernambuco, brazil). about 150 g of each species were harvested in the selected areas. thalli were dried in air flow and stored in the dark at room temperature (28 3 °c). a sample of each species was deposited in the herbárioufp-geraldo mariz, universidade federal de pernambuco (brazil), whose register numbers are 34402 (c. substellata), 36431 (c. aggregata) and 361638 (c. verticillaris). acquisition of organic extracts the thallus sample from c. substellata (100 g) was washed, ground and submitted to extraction with diethyl ether (darmstadt, germany) in a soxhlet extractor at 40 ºc for 16 h. the extract was filtered through whatman filter paper (no. 1) and evaporated in a rotary evaporator (buchler instruments, fort lee, nj, usa) in a water bath at 40 ºc until dry. the same procedure was performed for the thallus samples of c. aggregata (100 g). the ether extracts of c. substellata and c. aggregata were used for the isolation and purification of usn and bar, respectively. the thallus sample from c. verticillaris (100 g) was washed, ground and submitted to acetone extraction (merk®, darmstadt, germany), following the same procedures used for c. substellata and c. aggregata. the acetone extract of c. verticillaris was used for the purification of fum (asahina & shibata, 1954). lichen acids: isolation and purification usnic acid (usn) usn was obtained from the ether extract of c. substellata. isolation and purification were performed using a classic chromatographic procedure in a silica gel column (porosity 70 to 230 mesh). the mobile phase was chloroform: n-hexane (80:20 v/v) (odabasoglu et al., 2006). barbatic acid (bar) the ether extract of c. aggregata was successively washed with chloroform in a g4 funnel under pressure to obtain pure crystals of bar. additionally details related to the extraction and purification of bar are provided by martins et al. (2010). fum the acetone extract of c. verticillaris was successively treated with cold methanol to obtain fum crystals as described gudjnsdottir and ingolfsdottir (1997). identification of extracted phenolic compounds, purified substances, confirmation of molecular structure identification and degree of purity (> 95%) were evaluated using thin-layer chromatography (tlc) and highperformance liquid chromatography (hplc). confirmation of the molecular structure was performed with proton nuclear magnetic resonance (1h nmr) and infrared (ir) spectroscopy. the usn standard was obtained from merk® sociobiology 65(1): 79-87 (march, 2018) special issue 81 darmstadt, germany, and bar and fum were obtained from the natural products laboratory of the universidade federal de pernambuco, brazil. thin-layer chromatography (tlc) samples of 0.1 mg of the purified compounds, organic extracts and phenolic standards were dissolved in acetone (0.5 ml). next, 1 µl of the solution was applied to a silica gel plate (gel 60 f254+366merk® darmstadt, germany) measuring 20 x 20 cm. tlc assays were performed under ascending conditions using the solvent systems a (toluene/dioxane/ acetic acid, 36:9:1, v/v/v) for usn and bar and b (n-hexane/ diethyl ether/formic acid, 6.5:4:1 v/v/v) for fum. bands were observed under short (256 nm) and long (366 nm) ultraviolet (uv) light and visualized using a sulfuric acid solution (10%) and heating to 50 °c for 20 minutes to promote the color reaction. the phenolic composition was evaluated by determining rf values and comparisons with those obtained for standard usn, bar and fum (culberson, 1972). high performance liquid chromatography (hplc) the same material analyzed by tlc was submitted to hplc assays as described legaz and vicente (1983), in a hitachi chromatograph (655 a-11, tokyo, japan) coupled to a cg437-b uv detector set at 254 nm. for the separation, a c-18 reverse phase column micropack mch-18 de 300 mm × 4 mm, berlin, germany (merck® kgaa, darmstadt, germany) was used. the samples were injected at a concentration of 0.1 mg ml and were developed in an isocratic mode using as mobile phase methanol/deionized water/acetic acid, 80:19.5:0.5 (v/v). other analytical parameters were: volume of injection 20 μl, attenuation 0.16, pressure 87 atm, flow rate1.0 ml min-1 at room temperature (28°c ± 3°c). the results were analyzed by comparing the retention time (rt) of substance in the column, and peak area, taking the standards as reference. proton nuclear magnetic resonance (1h nmr) and infrared (ir) spectroscopy the chemical structures of the isolated compounds (usn, bar and fum) were confirmed using mnr1h and ir spectroscopy. the mnr1h data were obtained from a varian unity plus 300 mhz spectrometer using dmso-d6 as solvent in 5 mm tubes at room temperature. ir analyses were performed in a bruker fourier spectrometer (model ifs 66) with kbr disks. termiticidal assay a colony of n. corniger termites was collected from the campus of the universidade federal de pernambuco (brazil) and the termites were identified by dra. auristela c. albuquerque (ufrpe, brazil). termite colony was maintained in the vegetation house at the department of plant biology (ufrpe). termiticidal activity was performed using a method based on kang et al. (1990). filter paper disks (4 cm in diameter) were soaked with solutions (200 µl) of the usn, bar and fum at concentrations of 5, 7 and 10 mg ml-1 solubilized with acetone. each experiment unit consisted of a petri plate with the deep plate covered by filter paper disk. the disks were dried at 28 °c and placed in petri dishes (90 x 15 mm). a total of 20 termites (workers and soldiers, in the proportion of 4:1 respectively) were carefully transferred to each plate and kept in the dark at 27 ± 1 °c with 80% relative humidity. evaluation of percentage insect survival was made daily until 11-day period. bioassay was achieved in quintuplicate and survival rates were obtained for each treatment and expressed as mean ± sd. statistical analysis the statistical analyses were performed with the proc lifetest of the sas program (sas institute, 2001), using probit analysis with 95% confidence intervals. significant differences were determined using the student’s t-test (p < 0.05). results chemical characterization and identification of lichen acids diagnostic secondary metabolites for the three lichens species are well known with rf values and spot characteristics. in the thin layer chromatogram (tlc) of c. verticillaris acetone extract spot with rf 0.18 was shown, tlc data demonstrate the presence of fum as the major phenolic compound in this extract. traces of two other compounds were also found, with rfs of 0.08 and 0.19. the same result was observed for ether extracts of c. substellata and c. aggregata. spot with rf 0.50 demonstrates the presence of usn the major phenolic in c. substellata ether extract and bar in c. aggregata ether extract (rf 0.31). other unidentified substances were also found, with rfs of 0.22 and 0.30 from the former and rf of 0.31 from the later. data were in agreement with the purified and standard acids (figure 1). the tlc data were confirmed in the hplc analyses. the degree of purity of the isolated compounds was 100% for fum (rt 5.147 min), 95% for usn (rt 14.53 min) and 98% for bar (rt 22.99 min) (figures 2a, b and c, respectively). the chemical structures of the isolated compounds from c. verticillaris, c. substellata and c. aggregata (figures 3a, b and c) were confirmed by the determination of the melt point and through h1-mnr and ir spectroscopy. the data are described as follows: fum: white solid, mp. 252260oc (decomposition), 1h nmr (300 mhz, dmso-d6) δh (h; mult.; int.): 2.38 (3h; s; ch3-9), 2.41 (3h; s; ch3-9’). 5.26 (2h; s; ch2-8’), 6.60 (2h; s; ch-2’; ch-3’), 6.80 (1h; s; ch-5), 10.53 (1h; s; ch-8), 11.93 (1h; s; c-4-oh or c-2’oh). ir. (kbr): 3490, 3095, 2950, 2851, 1745, 1701, 1654, 1329, 1259, 1229, 1209, 1156, 1105 cm-1; usn: yellow solid crystal, mp, 201-203°c, [α]d a 25 °c + 493° (chcl3 c, 1.00 1h nmr (300 mhz, dmso-d6) δh (h; mult.; int.): 1.73 (3h; s, ch3-13), 2.08 (3h; s, ch3-16), 2,64 (3h; s, ch3-15), 2.65 (3h; s, ch3-18), 5.92 (1h; s, c-4-h), 11.04 (1h; s, c-10-oh), mcb martins et al. – usnic, barbatic and fumarprotocetraric acids on nasutitermes corniger82 13.29 (1h; s, c-8-oh), 18.89 (1h; s, c-3-oh). ir. (kbr): 3090, 3007, 2930, 1692, 1632, 1560, 1454, 1370, 1357, 1340, 1330, 1289, 1230, 1190, 1143, 1118, 1070, 1039, 959, 940, 840, 818, 700 cm-1; bar: gray solid crystal, mp, 188-189°c, 1h nmr (300 mhz, dmso-d6): 1.99 (3h; s; ch-8’), 2.00 (3h; s; ch3-8), 2.47 (3h; s; ch-9’), 2.56 (3h; s; ch3-9), fig 1. thin-layer chromatogram of 1: fumarprotocetraric acid (fum) standard (rf 0.18); 2: purified fum (rf 0.18); 3: acetone extract of cladonia verticillaris (rf 0.12); 4: usnic acid (usn) standard (rf 0.50); 5: purified usn (rf 0.50); 6: ether extract of cladonia substellata (rf 0.50); 7: barbatic acid (bar) standard (rf 0.31); 8: purified bar (rf 0.31); 9: ether extract of cladia aggregata (rf 0.31). fig 2. high-performance liquid chromatogram acquired at 254 nm of a: purified fumarprotocetraric acid (fum) (rt 5.14 min); b: purified usnic acid (usn) (rt 14.93 min); c: purified barbatic acid (bar) (rt 22.99 min). fig 3. chemical structure of purified substances according to spectroscopic analysis. a: fumarprotocetraric acid (fum); b: usnic acid (usn); c: barbatic acid (bar). numbers refer to carbon of the molecule. 3.86 (3h; s; ch3-o-4), 6,60 (1h; s; ch-5), 6.69 (1h; s; ch5’), 10.73 (1h; s; c-2-oh). ir. (kbr): 3092, 2992, 2984, 2858, 1659, 1631, 1399, 1290, 1272, 1233, 1154, 1080 cm-1. the chemical analysis revealed a total absence of other lichen acids and purity of lichen acids, employed to termiticidal assay, was confirmed. termiticidal assay the insecticidal potential of the purified usn, fum and bar was evaluated on n. corniger. three concentrations were assayed (5, 7 and 10 mg ml-1) and the percentage of survival was evaluated over an 11-day period. survival of n. corniger incubated in the absence (control treatment) or in the presence of each phenolic are shown in tables 1, 2 and sociobiology 65(1): 79-87 (march, 2018) special issue 83 3. contact with three purified lichen acids in 7 and 10 mg ml-1 concentrations induced mortality of workers after 9 days, while 5 mg ml-1 concentration promoted 100% mortality after 10 days. results described in these tables shown significant differences found in the mortality of workers after 7 days for fum and bar treatment in all concentrations, and 10 mg ml-1 for usn. at a concentration of 5 mg ml-1 (table 1), survival on 7 day had dropped to 0,2±0.9% for workers treated with fum, 0.1±0.9% for those treated with bar and 15±0.8% for those treated with usn. on day 8 and 9, worker survival was less than 1% for those treated with usn acid, while fum and bar promoted 100% mortality for workers. no statistically significant differences were found between treatments. contact with lichen acids at a concentration of 7 mg ml-1 induced mortality of workers after day 9 (table 2). treatment with all lichen acids in this concentration induced mortality higher than 50% after day 5. for fum and bar on day 7 percentage survival of workers was 0.5±0.9% and 0.2±0.9%, respectively. on day 8, worker survival was less than 1% for those treated usn and 0% for those treated with fum and bar. treatment with 10 mg ml-1 concentration showed the following data (table 3). survival workers was lower than 50% for all compounds (usn: 38±0.6%; bar: 37±0.6%; fum: 37±0.6%) after day 5 of treatment. while contact with all lichen acids in this concentration induced death of workers higher than 99% on day 7, and 100% after day 9. statistical analysis using student´s t-test (p˂0.05) showed that the effects of three lichen acids on workers was not influenced by the concentrations of the purifies lichen compound, while effect of exposure time was demonstrated. all treatments tested pointed equal mortality against termites after day 8, with significant differences in the percentage of survival in comparison to the control group. the mortality rates after day 7 of the treatment with the maximum concentration (10 mg ml-1) of three lichen acids was determined as ˃99%. mortality rates after day 7 of treatment with the 5 and 7 mg ml-1 concentrations were found with ˃99% only for bar and fum compounds. the analysis in tables tested the hypothesis that the assayed lichen compounds (fum, bar and usn) influenced on n. corniger (workers) survival. significant differences did not occur in the effect of lichen acids on survival of soldiers in comparison with the control group. additionally, similar effect of three phenolic was detected in all treatments and for this reason, results obtained for soldiers are not shown. 5 mg ml-1 survival (%) day 0 1 2 3 4 5 6 7 8 9 10 11 control 100±0 90±0 89±0.1 82±0.1 75±0.2 60±0.4 43±0.5 35±0.7 33±0.6 33±0.6 33±0.6 33±0.6 usn 100±0 93±0.7 89±0.1 86±0.1 79±0.2 64±0.3 33±0.6 15±0.8 0.2±0.9* 0.1±0.9* -* -* fum 100±0 95±0 93±0 90±0.1 68±0.3 43±0.5 17±0.8 0.2±0.9* -* -* -* -* bar 100±0 96±0 88 ±0.1 84±0.1 79±0.2 51±0.4 20±0.8 0.1±0.9* -* -* -* -* 7 mg ml-1 survival (%) day 0 1 2 3 4 5 6 7 8 9 10 11 control 100±0 90±0 89±0.1 82±0.1 75±0.2 60±0.4 43±0.5 35±0.7 33±0.6 33±0.6 33±0.6 33±0.6 usn 100±0 88±0.1 84±0.1 80±0.2 63±0.3 38±0.6 25±0.7 15±0.8 0.3±0.9* -* -* -* fum 100±0 97±0 96±0 93±0 75±0.2 42±0.5 20±0.8 0.5±0.9* -* -* -* -* bar 100±0 96±0 93 ±0 86±0.1 62±0.3 32±0.6 15±0.8 0.2±0.9* -* -* -* -* table 1. percentage of termite worker survival following treatment with usnic (usn), fumarprotocetraric (fum) and barbatic (bar) acids at concentration of 5 mg ml-1. symbol (-) indicates 0% survival. treatments were compared with the control and among treatments. asterisk (*) indicates a significance difference at p< 0.05. 10 mg ml-1 survival (%) day 0 1 2 3 4 5 6 7 8 9 10 11 control 100±0 90±0 89±0.1 82±0.1 75±0.2 60±0.4 43±0.5 35±0.7 33±0.6 33±0.6 33±0.6 33±0.6 usn 100±0 90±0 77±0.1 64±0.3 59±0.4 38±0.6 16±0.8 0.7±0.9* -* -* -* -* fum 100±0 98±0 95±0 91±0 63±0.3 37±0.6 16±0.8 0.2±0.9* -* -* -* -* bar 100±0 95±0 88±0.1 83±0.1 60±0.4 37±0.6 20±0.8 0.8±0.9* 0.1±0.9* -* -* -* table 2. percentage of termite worker survival following treatment with usnic (usn), fumarprotocetraric (fum) and barbatic (bar) acids at concentration of 7 mg ml-1. symbol (-) indicates 0% survival. treatments were compared with the control and among treatments. asterisk (*) indicates a significance difference at p< 0.05. table 3. percentage of termite worker survival following treatment with usnic (usn), fumarprotocetraric (fum) and barbatic (bar) acids at concentration of 10 mg ml-1. symbol (-) indicates 0% survival. treatments were compared with the control and among treatments. asterisk (*) indicates a significance difference at p< 0.05. mcb martins et al. – usnic, barbatic and fumarprotocetraric acids on nasutitermes corniger84 discussion describing the phenolic composition of c. verticillaris occurring in northeastern brazil, ahti et al. (1993) identified the presence of the fum, protocetraric acid, orcinol, methylorcinol carboxylate and atranorin. for c. substellata and c. agregatta, the same authors describe the presence of the compounds identified in this study, namely, usn in c. substellata and bar in c aggregata, which are dominant chemotype. however, the authors also found stictic, constictic, cryptostictic, norstictic and connortistic acids in the c. substellata from ne of brazil. for c. aggregata the chemotype with the stictic acid group is also known. this variety of chemotypes was confirmed in the extracts tlc analyses, where traces of other compounds were also found (figure 1). in the present study, we tested dominant chemotypes to improve their amount of purification, but the other compounds as stictic and norstictic acids has also been described as biocative compounds (de paz et al., 2010; pejin et al., 2013). studies on the termiticidal activity of lichen substances are scarce. however, previous investigations have demonstrated that arthropods, such as moths, mites and beetles, avoid feeding on species of lichens that produce fum, stictic and pulvinic acids. these substances are described as toxic compounds with a bitter taste (lawrey, 1987; reutimann & scheidegger, 1987). according to nimis and skert (2006), there is a close relationship between the presence of the lichen metabolites and grazing phenomena. the observations of the cited authors indicate that species that contain atranorin, calcium oxalate, gyrophoric acid, lecanoric acid, usn and zeorin have an important ecological function by either repelling or attracting insects, depending on the compounds found in the thallus. based on this, some studies demonstrated that the toxicity of extracts isolated from lichen samples against pests was related to their components (emmerich et al., 1993; balaji et al., 2007; cetin et al., 2008). extracts isolated from different lichen species might have different toxicity levels, which can be attributed to their different chemical composition (sahip et al. 2008; yildirim et al. 2012; emsen et al. 2015). therefore, lichens can be potential sources of natural compounds for pest management. natural products are now being considered as alternatives to the arsenal of synthetic compounds. the insecticidal activity of natural products is of considerable interest to researchers, as these compounds may be the basis for strong new insecticides, offering an alternative to extremely toxic synthetic insecticides. termites consume many types of food such as cellulosic materials, for example paper and wood. the evaluation of lichens acids effect on n. corniger was performed using filter paper soaked with the purified lichen compounds. the results showed that the survival of termite workers treated with lichen substances was significantly reduced in relation to the control group within a short period of time. moreover, the insecticidal efficiency of these substances may be extended to other types of insects. for example, assays involving spodoptera larvae submitted to a diet with oxyphysodic (11.2 µmol g-1wt) and fum (30.5 µmol g-1 wt) acids or calicin (37.6 µmol g-1 wt) obtained from lichens demonstrated a strong increase in the larval period and a high incidence of malformations, indicating a prolonged effect of the lichen substances tested (giez et al., 1994). depsides, such as bar, lecanoric acid, rosolic acid and orcein, and depsidones, such as fum, protocetraric and stictic acids derived from orcinol, are found in lichens, particularly those of the genera cladonia and cladia. the insecticidal activity of these substances has also been described. for example, the depside lecanoric acid and the depsidone physodic acid are lethal to larvae of the caterpillars helicoverpa armigera and cleorodes lichenaria, respectively (balaji et al., 2007; pöykkö et al. (2010). in this study, it has been demonstrated that bar and fum are lethal to worker termites at 5, 7 and 10 mg ml-1 concentrations after 7 days of treatment (tables 1, 2 and 3). dibenzofuran usn, which is a major compound in c. substellata, caused 100% mortality of termite workers at a concentration of 10 mg ml-1 after 7 days of treatment (table 3). this compound has demonstrated considerable efficacy against several genera of insects of interest to public health. cetin et al. (2008) describe the action of usn against culex pipiens under laboratory conditions, demonstrating 100% lethality in day 3 and 4 instar larvae at concentrations of 5 and 10 ppm, with lc50 at 0.8 ppm. the data indicate that usn may be used as a new bioinsecticide and its chemical structure can be used as a prototype for stronger insecticidal compounds. besides the phenolic derivatives (depsides and depsidones) from the secondary metabolism, the lichen thallus is rich in substances from its primary metabolism, such as complex carbohydrates and lectins. lectins have an important function in the symbiotic association with fungi and algae (díaz et al., 2016) and have proven to be effective against n. corniger. lectin (clavell) extracted and purified from c. verticillaris exhibits termiticidal activity, inducing the death of both workers (lc50: 0.196 mg ml -1) and soldiers (lc50: 0.5 mg ml -1) after 10 days of treatment (silva et al., 2009). the results obtained in the present study showed that fum purified from c. verticillaris reduced the survival of termite workers at all concentrations tested these findings, supported by silva et al. (2009), justify c. verticillaris lectin to be a potential bioinsecticide. termites have the ability to digest cellulose due to the presence of endoglucanases, exoglucanases and β-glucosidases with endogenous and/or symbiotic origin in their gut (breznak & brune, 1994). according to yamaoka (1996), cellulose digestion in the intestine of termites occurs with the assistance of symbiont protozoa or more types of bacteria present therein. zhou et al. (2008) tested efficacy of three prototype termite cellulase inhibitors as novel termite control agents. lichen substances have many biological activities described sociobiology 65(1): 79-87 (march, 2018) special issue 85 by different authors. for example, bactericidal effects have been found for bar on staphylococcus aureus, fum on klebisiela peneumonie and usn on mycobacterium tuberculosis (martins et al., 2010, neeraj & behera, 2011; lucarini et al., 2012). it is quite possible that the effect of such chemical substances is associated with their antibacterial activity or their acidic nature and bitter taste, which reduces the palatability of the thallus (ivanova & ivanov, 2009). moreover, lichens produce complex carbohydrates, such as evernin, inulin, lichenin and isolichenin, which cause irritation of the intestinal tract and hinder the digestion of many substances (lawrey, 1987). the precise action mechanism of lichen compounds as toxic molecules is still unknown. termites are ecologically beneficial, however biodeterioration of cellulose-based materials by termites is a serious problem and are considered as plague-insects. the control of these insects is currently based on the use of chemical insecticides, which can contaminate the environment, are toxic to humans and animals and can lead to the emergence of resistant insects. thus, new effective methods for termite control have been searched. fum, bar and usn exhibited termiticidal effect for worker caste of n. corniger, indicating their potential as bioinsecticides. this way, our findings offers an opportunity to develop alternative strategies to control this pest. further studies are needed for discovering new potential termiticidal compounds produced by lichens and characterization of their action mechanisms against pests. acknowledgments the authors are very grateful to the brazilian fostering agencies capes and cnpq and the universidade federal de pernambuco for sponsoring this study. references ahti, t., stenroos, s., filho, l.x. 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(2008). inhibition of termite cellulases by carbohydrate-based cellulase inhibitors: evidence from in vitro biochemistry and in vivo feeding studies. pesticide biochemistry and physiology, 90: 31–41. doi: 10.1016/j.pestbp.2007.07.011 doi: 10.13102/sociobiology.v62i3.400sociobiology 62(3): 446-449 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 post-embryonic development of mischocyttarus latior (fox) (hymenoptera: vespidae) introduction mischocyttarus latior (fox) occurs in bolivia and has been registered in the brazilian states of mato grosso, minas gerais and são paulo (richards, 1978). this species has also been recorded in cerrado areas (a type of savanna) in the southeast region of brazil (elpino-campos et al., 2007; lima et al., 2010). there are few documented descriptions of this species and at first glance, m. latior adults are very similar to polybia ignobilis (haliday). richards (1978) provides one of the few descriptions of m. latior adults, describing the species as a black wasp, with “ferruginous” fore tibia. the same author reports a narrow margin of metanotum, and a single dorsal streak on the mid coxa and two on the hind coxa. richards (1978) also observed yellow dots on the thorax, at the articulation points of the mid and hind legs, but also highlights “striking” variation in the morphology of the adults he studied. in order to increase our knowledge of m. latior biology the objective of the present study was to describe m. latior immatures, determine the period of development of the immature stages and determine which factors affect its duration (seasons, position of cells and stage of development). abstract mischocyttarus latior (fox) occurs in bolivia and brazil and has been recorded in cerrado areas from southeast brazil. the objectives of the present study were to describe the immature stages of m. latior, determine the development time of the immature stages and determine which factors affect its duration. daily mappings were performed from march 1994 to august 1995 in 22 nests. two nests were also collected and dissected to characterize immatures. larvae presented five instars and unlike most mischocyttarus, m. latior has only a single lobe on the abdominal sternite, which is vestigial in third instar, emergent in fourth instar and fully developed in the fifth instar larvae. the mean duration of the immature stages (from egg laying to adult emergence), was 67.38 ± 9.41 days, a longer period than the total duration of the immature stages of other previously studied species. the development time of the immatures was variable, depending on the colony development stage, with development times being quicker in pre-emergence colonies. sociobiology an international journal on social insects dss cecílio1, aa da rocha2, e giannotti1 article history edited by gilberto m. m. santos, uefs, brazil received 21 april 2014 initial acceptance 24 may 2014 final acceptance 10 november 2014 keywords larva, lobes, mischocyttarini, polistinae. corresponding author: agda alves da rocha universidade federal da bahia campus anísio teixeira rua rio de contas, quadra 17, lote 58, bairro candeias, cep 45055-090, vitória da conquista, bahia, brazil e-mail: rocha.agda@gmail.com materials and methods daily mappings were performed from march 1994 to august 1995 in 22 m. latior nests, located in the campus of the são paulo state university (“universidade estadual paulista júlio de mesquita filho”), and in the district of ferraz, in the municipality of rio claro, são paulo state, brazil (22º24’36’’ s; 47º33’36’’ w). at each visit the number of cells was recorded and their content classified into four groups: empty, egg, larva, or pupa. the position of the egg in the cell (giannotti & fieri, 1991) and the position of cells occupied by immatures were also recorded. in addition the nest size was classified (small: up to 30 cells and large: greater than 30 cells) and colony development stage (preand postemergence) were also recorded. to describe the immatures two nests were collected. all the eggs, larvae and pupae were removed from the two nests and morphometric measurements taken with the aid of a stereomicroscope coupled to a micrometer viewer. the morphometric measurements were: head capsule of the larvae and largest diameter and length of the eggs. the kruskal-wallis test was used to check for significant differences in the mean duration of immature stages between research article wasps 1 universidade estadual paulista (unesp), rio claro, sp, brazil 2 universidade federal da bahia, vitória da conquista, ba, brazil sociobiology 62(3): 446-449 (september, 2015) 447 annual seasons. the mann-whitney u test was used to test for differences in the duration of the immature stages and position in the cells (center or periphery) and colony development stage (preand post-emergence) (siegel & castellan, 2006). results and discussion description of immature stages the appearance of m. latior eggs was typical of members of the vespidae family. the eggs were white, elongated, narrower at the base and slightly curved and dilated at the tip. m. latior eggs were attached to one of the angles of the nest cell walls by an adhesive secretion. the m. latior eggs were on average 1.63 ± 0.15 mm long and 0.55 ± 0.06 mm wide (n = 66, measured at the widest diameter). m. latior eggs were larger than those of m. cassununga (ihering), which measured 1.36 mm long and 0.48 mm wide and m. cerberus styx richards, which were 1.13 mm long and 0.43 wide (giannotti & fieri, 1991; giannotti & silva, 1993; giannotti, 2006a). the larval stage showed five instars (fig. 1), as found in m. cassununga (giannotti & fieri, 1991), m. drewseni saussure (giannotti & trevisoli, 1993) and m. cerberus styx (giannotti, 2006a). the average width of the head capsule of the different instars was as follows: larva i was 0.53 ± 0.09 mm, larva ii, 0.88 ± 0.10 mm, larva iii, 1.22 ± 0.10 mm, larva iv, 1.51 ± 0.02 mm and larva v, 1.78 ± 0.09 mm. the average growth rate was 1.37, in agreement with dyar´s rule (wigglesworth, 1965). raposo-filho (1981) and silva (1984) reported the occurrence of only four larval instars in m. extinctus zikán and m. atramentarius zikán respectively. the species has only one lobe on the first abdominal segment, unlike m. drewseni (giannotti & trevisoli, 1993). m. latior presented a vestigial lobe in third instar larvae, emergent in fourth instar, and fully developed in fifth instar larvae (fig 2). development of immature stages the mean total duration of the immature stages (from egg laying to adult emergence), was 67.38 ± 9.41 days. this duration was longer than the total duration of the immature stages of other studied species, which showed values from 46.1 to 65.9 days (table 1). previous studies showed that the average duration of eggs in eight other mischocyttarus s. species ranged from 11.1 to 16.1 days (table 1). these values were similar to the values we found for m. latior (table 1). the average duration of the larvae of the other eight species ranged from 16.1 to 34.7 days (table 1), while the average larval stage in m. latior was longer at 36.88 days, a value similar to those found in m. drewseni and m. cassununga. the average duration of m. latior pupae was closer to those found in m. atramentarius z., m. extinctus z., m. labiatus (f.), m. cassununga and m. mexicanus saussure (litte, 1977; 1981; raposo-filho, 1981; silva, 1984; giannotti & fieri, 1991). the average duration fig 1. dorsal view of larval instars from mischocyttarus latior (fox) (hymenoptera: vespidae): l1 – l5 1º to 5º instar larvae. species location (state and country) average duration (in days) of development stages references egg larva pupa total mischocyttarus atramentarius z. rj and mg, brazil 12.70 25.10 16.80 54.60 silva (1984) mischocyttarus cassununga i. sp, brazil 13.20 32.60 15.60 61.20 giannotti and fieri (1991) mischocyttarus cerberus styx (r.) sp, brazil 11.27 31.90 19.80 61.70 giannotti, (2006b) mischocyttarus drewseni s. pa, brazil 11.10 20.20 14.80 46.10 jeanne (1972) pr, brazil 11.30 34.70 19.90 65.90 dantas-de-araújo (1980) sp, brazil 15.10 26.50 18.90 58.00 giannotti and trevisoli (1993) mischocyttarus extinctus z. rj and mg, brazil 11.40 20.60 16.80 48.80 raposo-filho (1981) mischocyttarus flavitarsis b. arizona, usa 14.10 23.10 19.70 56.90 litte (1979) mischocyttarus labiatus (f.) cali, colombia 16.10 16.10 16.30 48.50 litte (1981) mischocyttarus mexicanus (s.) florida, usa 13.90 24.8 16.3 55.0 litte (1977) mischocyttarus latior (f.) sp, brazil 14.08 36.88 16.42 67.38 present study table 1. comparative data on the duration of the developmental stages of immature individuals of nine species of the genus mischocyttarus so far studied. dss cecílio et al. post-embryonic development of mischocyttarus latior448 of the pupal stage of the other eight mischocyttarus species ranged from 14.8 to 19.9 days (table 1). at this stage a smaller variation in duration was observed between species. the only significant seasonal difference in m. latior immature duration was found in larvae (h = 113.615, p = 0.000, d.f. = 3) (table 2). these results suggest that warmer temperatures (spring and summer) cause m. latior individuals to mature faster on average especially during the larval stage (table 2). litte (1979) conducted a study in the american southeast (temperate climate) and found that the average total duration of the immature stages of m. flavitarsis also varied according to the months of the year, as well as in the spring and summer seasons, when these colonies develop. in brazil, giannotti and machado (1994) also found that there is seasonal variation in the duration of the immature stages of polistes lanio lanio. there is a tendency for the duration of the immature stages to shorten when there is an increase in temperature, as observed for m. drewseni (jeanne, 1972; dantas-de-araújo, 1980; giannotti & trevisoli, 1993). temperature seems to also have a strong influence on the maturation time of eggs of these insects. the eggs of m. cassununga showed a longer development time in the cold season (giannotti & fieri, 1991). the data on duration of immature stages were also analyzed with respect to the nest position occupied by immatures (table 3). in the center (seven oldest cells) of the nest, the mean duration of eggs was 13.24 ± 5.24 days (6-29), larvae, 28.54 ± 16.82 (12-92) and pupae, 15.41 ± 3.51 (7-26), (total 57.19 ± 8.52 days ). in the other remaining (i.e. newer or “peripheral”) cells the mean duration of eggs was 14.28 ± 5.55 days (6-29), larvae, 38.86 ± 18.25 (11-92) and pupae, 16 .53 ± 4.54 ( 9-26), (total 69.67 ± 11.90 days). tests indicated that there was no significant difference between the maturation times of eggs (u = 13431.5, p = 0.170, d.f. = 1) or in the duration of the pupae (u = 3137.0, p = 0.349, d.f. = 1). however, a significant difference was found in the mean duration of larvae (u = 2109.0, p = 0.018, d.f. = 1). our findings suggest that the larvae localized in cells which occupy a central position in the nest usually receive a larger amount of food from the adults. similar results were also reported by west-eberhard (1969) in polistes canadensis (l.). it is possible to suggest two (non-mutually exclusive) explanations for the observed differences between the duration of development at the center and periphery of nests. the center of the nest is usually the first place where foragers arrive after collection. therefore, the returning foragers usually begin by feeding the central larvae and often there is little or no food for the larvae located to the periphery. another possibility is that due to their position in the comb the temperature of these first seven cells is more stable than that of the cells on the periphery. this would explain the shorter development time of the eggs and pupae, which do not depend on the food provided by forgers for their development. the fig 3 shows the data of the average duration of immature stages of m. latior, analyzed in relation to the stages of colony development (preand post-emergence). results of the mann-whitney u test indicated that there were significant differences in the duration of the immature stages, with quicker stages in the pre-emergence colonies: egg (u = 5168.0, p = 0.000, d.f. = 1), larva (u = 1273.5, p = 0.000, d.f. = 1) and fig 2. a – lateral view of 1º to 5º instar larvae of mischocyttarus latior (fox) (hymenoptera, vespidae); b detail of the lobe in the first abdominal segment of 5º instar larva. season average duration (in days) of immature stages climatic factors (mean values) egg larva pupa temperature (ºc) rainfall (mm) relative humidity (%) spring (september – november) 14.18 ± 5.54 22.58 ± 6.82 15.98 ± 2.49 24.01 107.55 64.40 summer (december – february) 14.52 ± 5.92 19.63 ± 4.40 15.41 ± 3.62 25.00 347.06 75.71 autumn (march may) 13.16 ± 5.15 38.84 ± 13.93 16.88 ± 4.99 21.80 95.58 75.04 winter (june – august) 14.83 ± 5.63 44.78 ± 15.98 17.40 ± 4.94 18.67 10.41 67.53 mean total duration 14.08 ± 5.50 36.88 ± 17.98 16.42 ± 4.77 22.37 140.14 70.67 table 2. average duration (in days) of the developmental stages of immature individuals of mischocyttarus latior (fox) (hymenoptera: vespidae), in relation to seasons and climatic factors. sociobiology 62(3): 446-449 (september, 2015) 449 pupa (u = 2181.0, p = 0.020, d.f. = 1). this fact is important for colony survival as it shortens the period when the founder remains alone in the nest, allowing the swift emergence of the colony workers. jeanne (1972) also found that maturation of larval stages in m. drewseni colonies was quicker in the preemergence stage. according to a study conducted by silva (1984), the duration of eggs, larvae and pupae, also varied according to the stage of colony development (pre and post-emergence). giannotti, e. (2006b). fatores que afetam o tempo de desenvolvimento dos estágios imaturos da vespa social mischocyttarus cerberus (hymenoptera: vespidae). in: resumos do xxi congresso brasileiro de entomologia. recife, pe. p. 354. giannotti, e. & fieiri, s.r. (1991). on the brood of mischocyttarus (monocyttarus) cassununga (ihering, 1903) (hymenoptera, vespidae). revista brasileira de entomologia, 35: 263-267. giannotti, e., machado, v.l.l. (1994). the seazonal variation of brood stages duration of polistes lanio (fabricius, 1775) (hymenoptera, vespidae). naturalia, 19: 97-102. giannotti, e. & silva, c.v. (1993). mischocyttarus cassununga (hymenoptera, vespidae): external morphology of the brood during the post-embrionic development. revista brasileira de entomologia, 37: 309-312. giannotti, e. & trevisoli, c. (1993). desenvolvimento pósembrionário de mischocyttarus drewseni saussure, 1857 (hymenoptera, vespidae). insecta, 2: 41-52. jeanne, r.l. (1972). social biology of the neotropical wasp mischocyttarus drewseni. bulletin of the museum of comparative zoology, 144: 63-150. lima, a.c.o., castilho-noll, m.s.m., gomes, b. & noll, f. b. (2010). social wasp diversity (vespidae, polistinae) in a forest fragment in the northeast of são paulo state sampled with different methodologies. sociobiology, 55: 613-626. litte, m. (1977). behavioral ecology of the social wasp mischocyttarus mexicanus. behavioral ecology and sociobiology, 2: 229-246. doi: 10.1007/bf00299737 litte, m. (1979).mischocyttarus flavitarsis in arizona: social and nesting biology of a polistine wasp. z. tier psychol., 50: 282-312. doi: 10.1111/j.1439-0310.1979.tb01033.x raposo-filho, j.r. (1981). biologia de mischocyttarus (mischocyttarus) extinctus zikán, 1935 (polistinae-vespinae). rio claro: ibunesp, 110p. richards, o.w. (1978). the social wasps of the americas excluding the vespinae. london: british museum (natural history), 580p. siegel, s. & castellan, n.j. (2006). estatística não paramétrica para as ciências do comportamento. são paulo: artmed-bookman, 448p. silva, m.n. (1984). aspectos do desenvolvimento e do comportamento de mischocyttarus (kappa) atramentarius zikan, 1949 (hymenoptera, vespidae). rio claro: ibunesp, 151p. strassmann, j.e. &orgren, m.c.f. (1983). nest architectures and brood development times in the paper wasp, polistes exclamans (hymenoptera, vespidae). psyche, 90: 237-248. doi: 10.1155/1983/32347 west-eberhard, m.j. (1969). the social biology of polistine wasps. miscellaneous publications, 140: 1-101. wigglesworth, v.b. (1965). the principles of insect physiology. london: methuen, 741p. fig 3. average duration (in days) of the developmental stages of immature individuals of mischocyttarus latior (fox) (hymenoptera, vespidae), in pre-emergent (dark grey bars) and post-emergent (light grey bars) colonies. stage length of mischocyttarus latior immatures first seven nest cells other nest cells mean s.d. range mean s.d. range egg 13.24 5.24 (6 29) 14.28 5.55 (6 29) larva 28.54 16.82 (12 92) 38.86 18.25 (11 92) pupa 15.41 3.51 (7 26) 16.53 4.54 (9 26) total 57.19 8.52 69.67 11.90 table 3. average duration (in days) of the developmental stages of immature individuals of mischocyttarus latior (fox) (hymenoptera: vespidae), raised in the first seven cells constructed and in the other nest cells. references dantas-de-araújo, c. z. (1980). bionomia e comportamento social comparado de mischocyttarus drewseni drewseni de saussure, 1987, nas regiões subtropical (curitiba, pr) e tropical (belém, pa) do brasil (hymenoptera, vespidae). curitiba: ufpr, 110p. elpino-campos, a., del-claro, k. & prezoto, f. (2007). diversity of social wasps (hymenoptera, vespidae) in cerrados fragments of uberlândia, minas gerais state, brazil. neotropical entomology, 36: 685-692. doi: 10.1590/s1519566x2007000500008 giannotti, e. (2006a). caracterização morfológica dos estágios imaturos e número de ínstares larvais da vespa social mischocyttarus cerberus (hymenoptera: vespidae). in: resumos do xxi congresso brasileiro de entomologia. recife, pe. p. 354. doi: 10.13102/sociobiology.v61i1.88-94sociobiology 61(1): 88-94 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 co-existence of ants and termites in cecropia pachystachya trécul (urticaceae) ac neves1, ct bernardo2, fm santos3 introduction the neotropical genus cecropia (urticaceae) includes 60-70 species, 80% of them are trees inhabited by obligatory simbiotic ants of at least four subfamilies: dolichoderinae, formicinae, myrmicinae e ponerinae (davidson & fisher 1991, davidson & mckey 1993, folgarait et al. 1994). the relationship between cecropia spp. and azteca spp. (hymenoptera: formicidae) – one of the most conspicuous and well studied mirmecofilic interactions – is considered a symbiotic relationship because ants live only in the large hollow internodes of some species, feeding on müllerian bodies produced in modified petiole bases (trichilia) (wheeler 1942, janzen 1969, dejean et al. 2009). these food bodies are rich in carbohydrates, lipids, proteins and primarily glycogen, which remarkably is the principal storage carbohydrate found in animals and is abstract individuals of cecropia pachystachya trécul (urticaceae) host azteca (hymenoptera: formicidae) colonies in their hollow internodes and feed them with glycogen bodies produced in modified petiole bases (trichilia). in turn, ants keep trees free from herbivores and lianas. here, we report for the first time the association of nests of nasutitermes ephratae rambur (isoptera: termitidae) with these trees, in south-pantanal (brazil). we aimed to describe the cecropia-ant-termite relationship and to investigate how their coexistence is made possible. we hypothesize that: 1) the frequency of termite nests in c. pachystachya is lower than in neighbor trees; 2) termite nests occur in trees with lower density of foraging ants; 3) the time that ants take to find and remove live termite baits in c. pachystachya trees is lower in leaves (close to trichilia) than in trunks; 4) termite nests are fixed preferentially in the smallest and less branched trees; and 5) termite nests are fixed preferentially distant from the canopies. unexpectedly, termitaria occurred in c. pachystachya at the same frequency as in other tree species; there was no relationship between ant patrol activity and the occurrence of termite nests in c. pachystachya; and they occurred mainly in the tallest and more branched trees. however, termite nests generally were fixed in the trunk, fork or basal branches, where there is better physical support and ant patrol is more modest. the segregation of termite and ant life-areas may represent a escape strategy of termites in relation to ants inhabiting c. pachystachya, specially during nest establishment. the isolation of termites in fibrous nests and galleries may complete their defense strategy. sociobiology an international journal on social insects 1 universidade federal de minas gerais, belo horizonte, mg, brazil. 2 universidade de brasília, brasília, df, brazil. 3 universidade estadual de campinas, campinas, sp, brazil. article history edited by kleber del-claro, ufu, brazil received 06 january 2014 initial acceptance 27 february 2014 final acceptance 18 march 2014 keywords myrmecophytism, azteca, pantanal, nasutitermes ephratae corresponding author ana carolina de oliveira neves departamento de biologia geral instituto de ciências biológicas universidade federal de minas gerais caixa postal 486 31270-901, belo horizonte, mg, brazil e-mail: ananeves@gmail.com extremely rare in plants (rickson 1971, rico-gray & oliveira 2007). azteca ants supplement their diet with invasive insects, which they aggressively attack (dejean et al. 2009). there is strong evidence that ants provide nutrients to cecropia individuals (putz & holbrook 1988, sagers et al. 2000) and keep them free from herbivores and vines, thus acting as allelopathic agents and increasing tree competitive ability (janzen 1966, downhower 1975, schupp 1986, vasconcelos & casimiro 1997). ants involved in obligate mutualisms reduce their foraging area to plant’s surface and develop specialized behaviors to protect food resources, becoming extremely aggressive (carroll & janzen 1973). they are able to detect physical disturbances and chemical signals, such as volatile substances from leaves, responding with rapid recruitment of numerous soldiers (agrawal et al. 1998, 1999, dejean et al. 2009). some studies show that research article termites sociobiology 61(1): 88-94 (march, 2014) 89 herbivores prefer cecropia trees unocuppied by azteca individuals, including trees smaller than 2 m tall, which are usually not inhabited by ants or that present a smaller ant patrol activity (downhower 1975, schupp 1986, vasconcelos & casimiro 1997). however, some insects escape from predation even in trees occuppied by ants. ant genus such as cephalotes, crematogaster and pseudomyrmex, were found foraging on cecropia pachystachya trécul (urticaceae) branches (vieira et al. 2010) and camponotus, solenopsis and procryptocerus were found in cecropia insignis (liebm.) inhabited by azteca ants (longino 1991). herbivore larvae such as ophtalmoborus (coleoptera: curculionidae) are usually present in the spikes of cecropia pistillate inflorescences (berg et al. 2005). schupp (1986) showed that the leaf damage made by chewer beetles was lower in ant-occupied cecropia obtusifolia bertol. than in unoccupied individuals. on the other hand, according to the same author, gall flies (diptera) and phloem-feeding homoptera (hemiptera) activity was not affected by presence of ants in these plants. he attributed ant ineffectiveness to gall flies small size and rapid oviposition, and to the motionless feeding pattern of homoptera that must be contacted by patrolling ants, while beetles are detectable from a distance due to leaf vibration caused by chewing and body movements. also, when contacted, homoptera ‘explode’ off the leaf and alight undetected elsewere on the plant. ants are the main predators of termites (hölldobler & wilson 1990). there are at least six ant genera specialized in feeding on such insects, and arboreal termitaria are negatively influenced by predator ants (wilson 1971, gonçalves et al. 2005). termites are often attacked during mating flights or after the accidental breakage of nests and galleries (weber 1964, shepp 1970, carroll & janzen 1973). some ant colonies, such as solenopsis, carebara, centromyrmex and hypoponera, invade termite nests and attack their eggs, nymphs and adults (lemaire et al. 1986, delabie 1995, dejean & feneron 1999). however, early in the 20th century about 200 ant species were described living in pacific association with termites (wheeler 1936), including the mutualism between amitermes laurensis mjoeberg (hymenoptera: termitidae) and two ant species of the genus camponotus, which inhabit termite mounds and protect them from attack by the “meat ant” iridomyrmex sanguineus forel (hymenoptera: formicidae) (higashi & ito 1989). little is known about the opposite situation, i. e., the occupation of ant colonies by termites (quinet et al. 2005). nasutitermes ephratae rambur (hymenoptera: termitidae) and other arboreal termite species are often found in southern-pantanal. the presence of this termite species in cecropia pachystachya is remarkable because such trees host entire colonies of aggressive ants, mainly of the genus azteca, which do not establish peaceful association with termites. thus, the objective of this study was to describe the cecropia-ant-termite relationship and to investigate how their coexistence is possible. we hypothesize that: 1) the frequency of termite nests in c. pachystachya is lower than in neighbor trees; 2) termite nests occur in trees with lower density of foraging ants; 3) the time that ants take to find and remove live termite baits in c. pachystachya trees is lower in leaves than in trunks; 4) termite nests are fixed preferentially in the smallest and more branched trees; and 5) termite nests are fixed preferentially distant from the canopies (where trichilia are concentrated). material and methods study area the pantanal is a seasonal floodplain in tropical south america, located in the upper paraguay river basin. it occupies an area of 147.572 km2, between 80 and 150 m above sea level (alho & gonçalves, 2005). there is a rainy season from november to march corresponding to 72% of the total annual rainfall (1,182.5 mm) and a dry season from april to october. the average annual temperature is 25.5 ° c, but the absolute maximum exceeds 40° c and the minimum is close to 0° c. the average relative humidity is 82% (soriano et al. 1997). annual floods occur during summer (february to may), although there are also multi-annual floods, which produce long periods of pronounced dry and wet seasons. flooding results mainly from drainage difficulties caused by the low declivity of the terrain, which varies from 3 to 5 cm/km (east-west), and from 1 to 30 cm/km (north-south) (alho & gonçalves, 2005). sampling was done along the highway ms-184 (park road), between the coordinates 19°38’56.4’’s, 057°01’37.6’’w and 19°22’20.28’’s, 57°02’32.40’’w, in the municipality of corumbá, mato grosso do sul state, brazil. the tree comunity along the road was dominated by c. pachystachya, vitex cymosa (verbenaceae), copernicia alba (arecaceae), enterolobium contortisiliquum (fabaceae) and tabebuia spp (bignoniaceae). study species cecropia pachystachya occurs throughout the pantanal, where it is common in riparian flooded areas and non flooded forested patches (“capões” and “cordilheiras”) (pott & pott 1994). their large-hollow internodes provide nesting space for ants, and the thin spots in their upper wall (prostomata) allow ants to circulate from inside to outside the tree (berg et al. 2005). they also present trichilia formed by patches of dense indumentum abaxially at the base of the petiole of adult leaves, which produce food corpuscles called müllerian bodies (berg et al. 2005). in the study area, c. pachystachya are inhabited by azteca ovaticeps forel, a.isthmica wheeler and a. alfari emery (vieira et al. 2010). azteca are territorial ants that feed on müllerian bodies and form dense colonies in cecropia. during foraging activity, workers move randomly through the ac neves et al. co-existence of ants and termites in cecropia pachystachya90 tree until they find any invader. they recruit soldiers by alarm pheromones and aggressively attack the invader (even when it is dead), killing and throwing it from the tree, and eventually feeding on it (carroll & janzen 1973, quinet et al. 2005, dejean et al. 2009). nasutitermes ephratae is a neotropical termite that feeds on plant-debris and inhabits lowland areas (thorne 1980, vasconcellos & moura 2009). it builds arboreal, spherical or ellipsoidal, carton nests in trunks or branches, with internal or external galleries spreading from nests toward food sources (thorne 1980, thorne & haverty 2000). there are records of aggressive encounters between nasutitermes individuals and azteca ants (braekman et al. 1983; noirot & darlington 2000, quinet et al. 2005). although these termites feed mainly in litter wood, they are usually found, attacked and removed by ants that inhabit cecropia trees when used as baits (e. g. oliveira et al. 1987, dejean et al. 2009). nasutitermes soldiers defend themselves throwing a viscous secretion produced by frontal glands (see eisner et al. 1976). sampling methods to compare the frequency of termitaria between c. pachystachya and its neighbor trees, we covered a path of ~ 30 km of a dirt road counting trees from several species, with and without nests. we sampled only trees whose trunks and crowns were fully visible (not hidden by other trees or vines), a total of 140 individuals of c. pachystachya and 78 from other tree species. to verify whether ant patrol activity differed between cecropia trees with or without termitaria, we counted the number of ants that crossed an area of 5 x 2 cm2, defined by a carton frame disposed in the tip of a branch and in a leaf (near petiole insertion). the count was done after a soft tap in the branch. the procedure lasted for two minutes and was done between 8 am and 11 am, in 17 trees without termite nests and in eight trees with termite nests. to test how long ants took to find and remove live termite baits in different parts of the trees, we used white school glue to adhere n. ephratae soldiers on leaves (in the abbaptial surface), petioles (next to trichilia) and trunks. we repeated this procedure in 20 c. pachystachya individuals and observed for up to seven minutes. to characterize the architecture of c. pachystachya individuals with and without termitaria, we estimated the height of trees (h), we counted the number of branches per tree (n) and calculated the ratio branches: tree height (n/h) as an indicator of canopy density, in 90 trees, 22 of which had termitaria. histograms of h and n/h of trees were made, highlighting the ocurrence of termite nests in the population of c. pachystachya. in order to have an indication of termitarium distance from the canopy, we estimated the height (h) of 27 nests and then calculated the ratio nest height: tree height (h/h) and separated the results into four categories. thus, the closer the ratio h/h was to zero, the nearest to ground the nest was. we also determined the tree structure in which 18 nests were fixed (trunk, fork, lower or upper branches). data analysis we compared the frequency of termite nests between c. pachystachya and its neighbour tree species with the chisquare test. ant patrol activity in leaves and branches were compared between c. pachystachya individuals with and without termitaria using the student’s t test. results and discussion in the study area, termitaria were composed by a carton nest with rigid galleries made of dirty and fibrous material, spreading toward branches (fig 1). while ants were present in all the cecropia pachystachya trees with which we had direct contact, termite nests were present in 42.9% of the c. pachystachya individuals and in 36.8% of the other tree species, with no statistical difference between them (χ2 = 0.105, p = 0.74, n=218). ants crossed the defined 10 cm2 area in c. pachystachya individuals with and without termite nests, respectivefig 1: cecropia pachystachya trécul (urticaceae) in south-pantanal, brazil, with a nest of nasutitermes ephratae rambur (termitideae). note the galleries spreading in trunk and branches. sociobiology 61(1): 88-94 (march, 2014) 91 ly, 70.75±27.12 and 56.47±18.16 times in branches, and 3.63±2.56 and 10.94±3.80 in leaves (average ± standard error of mean). ant patrol activity was similar between trees with and without termite nests in branches (t = -0.44, p=0.60, df =23, n=25) and in leaves (t =1.25, p =0.22, df =23, n=25). thirty five per cent of termites used as baits were found and attacked by ants. in some cases, especially in trunks, they were found but were not attacked. attacks occurred in 65% of 20 trials in petioles, 25% in leaves and 15% in trunks. the time elapsed for attacks were 112.54±32.53 seconds (n=13) in petioles, 151.40±56.94 s (n=5) in leaves and 67.77±28.26 s (n=3) in trunks. it was not possible to compare statistically the time of ant atack due to the small size of samples. we also observed that the galleries spreading from nests were not examined or attacked by ants. in the sampled population (n=90), c. pachystachya individuals varied from 3-12 m tall, but termitaria (n=22) ocurred only in trees to 7-11 m tall, with a modal class of frequency in 8 m that coincided with the population mode (fig 2). the number of branches per height varied from 0.20 – 6.86. termitaria ocurred in trees with one to 6.86 branches per meter. again, the modal classes of trees with termitarium coincided with the modal class of branching in the population, i. e. 1.51 – 2.50 branches per meter (fig 3). almost fifteen percent (14.8%) of termite nests were fixed in the lower one-quarter of trees, 63% in the second, 18,5% in the third and only 2.7% in the fourth one-quarter (from bottom to up). most of them were located at the base of the lower branches (60%), followed by the fork and upper branches (15% each), and finally the trunk (10%). opposite to our hypothesis’ predictions, nasutitermes ephrateae termitaria occur in cecropia pachystachya individuals in the same frequency than in non-mirmecophyte trees, as well as in c. pachystachya individuals with intense or modest ant patrol activity. although it is an unexpected result, considering the mirmecophilic life story of cecropia, we sugest that physical mechanisms mediate the relationship between n. ephrateae and ants of the genus azteca, namely the occupation of distinct parts of the tree by termites and ants, and the physical isolation of termitaria (nests and galeries) by fibrous structures. although the distribution of c. pachystachya individuals with termitarium have the same modal class than the population as a whole (regarding tree height and number of branches per height), termite nests did not occur randomly. termite nests were found only in a restricted range of tree height within the population studied. by their turn, ants are expected to occur in c. pachystachya trees taller than 2 m after downhower (1975) and schupp (1986). one might expect that termites fixed their nests preferentially on small trees, which are not inhabited by ants or fig 2. abundance of individuals of cecropia pachystachya trécul (urticaceae) in pantanal sul, brazil, regarding their height. black bars: trees with termitarium of nasutitermes ephratae rambur (termitideae); gray bars: trees without termitarium. fig 3: abundance of individuals of cecropia pachystachya trécul (urticaceae) in pantanal sul, brazil, regarding their ratio branches: height of the trees. black bars: trees with termitarium of nasutitermes ephratae rambur (termitideae); gray bars: trees without termitarium. present a smaller ant patrol activity, being prefered by herbivores (downhower 1975, schupp 1986). however, besides the scape of predation, physical support must be a determinant of termitaria establishment, as it is suggested by the absence of termite nests in c. pachystachya individuals smaller than 7 m and its scarcity in trees with less than 1.51 branches per meter of height. in fact, cunha (2000) found that constrictotermes cyphergaster silvestri (isoptera: termitidae) usually fixed their nests in trees with intermediate trunk circumference. although termite nests predominated in trees with 1.51 to 2.5 branches per height (the modal class of branch density in the population), they were disproportionally underrepresented in the smaller classes. termitaria predominance in more branched trees once more indicates that its occurrence is not random. densely branched individuals may provide more opportunity for nesting and better support conditions, and although they have more dense canopies, it does not necessarily imply in larger density of trichilia and therefore greater ac neves et al. co-existence of ants and termites in cecropia pachystachya92 patrol by ants, since the lower branches often had no leaves or ants. in other species of mirmecophyte cecropia it was observed that ant patrol differed between leaves in different portions of the canopy, as well as herbivory. in cecropia peltata linnaeus, 53.9 % of ants were located in leaves in the upper canopy and only 11.2 % in the leaves of basal branches, resulting in proportional rates of herbivory (downhower 1975). in c. pachystachya termite nests were more frequent in the lower half of the trees and in lower branches, i. e. distant from young-active trichilia in the top canopy, where ant patrol may be more intense. this suggests that there is a displacement of the location of termites and ants, avoiding overlap. this finding is corroborated by the fact that termites are rarely attacked by ants in trunks, but they are attacked and removed when experimentally placed in leaves or petioles, which are close to trichilia. one hypothesis to explain the coexistence of azteca sp. and n. efrateae in c. pachystachya is that they do not engage in agonistic encounters. however termites were readily attacked and removed from leaves and petioles, and sometimes from trunks when experimentally introduced. ant’s attacks to termite would probably be more frequent, especially in branches, if they did not construct galleries with fibrous and dust material. we suggest that, after a period of large vulnerability during the establishment of termitaria, nest and galleries built with rigid material contribute to the coexistence of these groups of insects, allowing termites to move protected inside them, even in the canopy. this strategy is consistent with the one adopted by other termite species that coexist with ants without predation. in cases that ants colonize termitaria, these insects often dwell distinct portions. for example, crematogaster rochai forel (hymenoptera: formicidae) inhabit termite nests of n. ephrateae and nasutitermes corniger motschulsky (hymenoptera: termitidae) and maintains a physical separation with its hosts by plugging the cells that they inhabit with fibrous material (quinet et al. 2005). the observed trend of tree occupation is also in agreement with the distribution of vines in cecropia, that, as well as herbivores, are cut and removed by ants (janzen 1969, downhower 1975, vasconcelos & casimir 1997). vines are often found on the stem and the lower parts of the lower branches, but not higher up (berg et al. 2005). this was attributed to the pattern of branches’ growth and loss – lower branches depart from the trunk in angles of 45 degrees and lower leaves are continuously lost –, thus affecting the growth of vines (berg et al. 2005). we suppose that in mirmecophyte cecropia it may also be attributed to the pattern of tree occupation by ants and their vine cutting activity. in conclusion, this study shows that n. ephrateae nests occur at the same frequency in cecropia individuals than in non mirmecophyte trees, as well in cecropia individuals with itense or modest ant foraging activity. the determinants of nest occurrence are probably the mechanical support offered by trees and nest distance from trichillia, i. e. from the area more intensively patrolled by ants, that may influence specially the colonization time. after that, isolation of termites inside fibrous nests and galleries may contribute to their coexistence with ants. so, our results indicate that the coexistence between termites and ants in c. pachystachya may be possible due to spatial segregation of their colonies, avoiding agonistic interactions. to the best of our knowledge, this is the first report of the occurrence of termites on cecropia trees, although this is a common association in the study area. further research on the interaction between these insects would help to understand how their coexistence is made possible, as well as the influence of the flood pulse in the pantanal in the evolution of this ecological relationship. acknowledgments we thank two anonymous referees for valuable comments in the manuscript; r. constantino and i. leal for termite and ant identification; g. a. f. medina for language editing and comments on the manuscript; and students of the under-comments on the manuscript; and students of the under-students of the undergraduate course of biological sciences in the of universidade federal de mato grosso do sul (ufms) for helping with sampling. this study was conducted during the 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(1971) the insect societies. cambridge: belknap press of harvard university press, 560 p. doi: 10.13102/sociobiology.v65i4.3393sociobiology 65(4): 722-726 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 floral traits and foraging behavior of bee species visiting martynia annua l. (martyniaceae) in a coastal habitat introduction martynia annua l. (martyniaceae) is an herbaceous perennial plant native of america. it ranges from 350 to 950 m of elevation from southern arizona, through the neotropical area of mexico, central america and the antilles (calderón, 1998). it was introduced in the old world around the 18th century and now occurs in all of its continents where is considered an invasive species (calderón, 1998; khuroo et al., 2012; cabi, 2018). m. annua reaches 1–3 m height and is covered with dense glandular sticky hairs, almost glabrous when adult. stems are branched and could be lignified at the abstract floral visitors are often overlooked in those plants considered invasive and widespread weed species. martynia annua l. is an example of an introduced species to the old world being native from tropical america, however, information of its endemic pollinators in the neotropical region is missing. in this study, the floral visitors of m. annua were evaluated in chamela field station (chamela-cuixmala biosphere reserve) in jalisco, mexico. our aim was to provide information of the breeding system of m. annua and then indicate the potential pollinators. we included morphological and sexual features to estimate the outcrossing index (oci). the frequency, behavior, and pollen loads were considered to find the potential pollinator. despite the evidence of protandry and oci indicating a xenogamous breeding system, the lack of herkogamy suggests m. annua is a facultative xenogamous species. the highest frequency of visits corresponded to the maximum diameter of corolla. euglossa viridissima friese was the most recurrent visitor. however, this species often carried a high proportion of heterospecific pollen and did not touch any sexual structure of the flower. in contrast, centris agilis smith performed as the most likely pollinator. flowers of m. annua offer valuable rewards to its visitors and may be a good source of energy to those foragers capable of reaching the nectaries, though small bees are apparently unable to penetrate the flowers. we consider that there are evidence that suggests m. annua is a specialized melittophilic plant, pollinated by moderate to large-sized hairy bees throughout its distribution. sociobiology an international journal on social insects d cárdenas-ramos1, a falcón-brindis2, r badillo-montaño3, i hinojosa-díaz4, r ayala5 article history edited by kleber del-claro, ufu, brazil received 29 april 2018 initial acceptance 16 june 2018 final acceptance 22 july 2018 publication date 11 october 2018 keywords floral biology, pollen loads, bee-plant interactions, outcrossing index. corresponding author a falcón-brindis av. instituto politécnico nacional, 195 playa palo de santa rita sur, la paz c. p. 23096, baja california sur, méxico. e-mail: armandofalcon123@gmail.com base in old individuals. leaves are simple, blades reniform to broadly ovate, margins entire to deeply lobed ending in an acute apex. petioles are 3–25 cm long. the inflorescence is a short raceme with 10–20 purplish white flowers, which are campanulate, zigomorph, sympetalae, pedicels are 1–2 cm long; flower tubes slightly to strongly ventricose. calyx is greenish, 1.5 cm long with five free sepals. the corolla is five-lobed, with pink to reddish spots; the basal lobe widened to form a landing surface and presenting a yellow nectar guide. two fertile stamens, two staminodes, and sometimes one rudimentary (calderón, 1998). these morphological features are commonly associated to melittophily (bee 1 instituto de ecología, universidad nacional autónoma de méxico, ciudad de méxico, méxico 2 academia de ecología de zonas áridas, centro de investigaciones biológicas del noroeste, s.c. la paz, méxico 3 red de estudios moleculares avanzados, instituto de ecología a.c., xalapa, ver, méxico 4 instituto de biología, universidad nacional autónoma de méxico, ciudad de méxico, méxico 5 estación de biología chamela (sede colima), instituto de biología, universidad nacional autónoma de méxico, jalisco, méxico research article bees mailto:armandofalcon123@gmail.com sociobiology 65(4): 722-726 (october, 2018) special issue 723 pollination) and are shared with most genera of martyniaceae: ibicella, martynia, and proboscidea (phillippi & tyrl, 1979). nonetheless, the breeding system of m. annua has never been studied and data on floral biology is poor. assuming the type of breeding system based on floral traits (i.e. pollination syndrome hypothesis) should be addressed with caution (fenster et al., 2004). since the spatiotemporal plantpollinator assemblage can play a role in the outcrossing process (herrera, 1988; lázaro et al., 2008), it becomes imperative to evaluate the assemblage of pollinators in correspondence to multiple floral traits (johnson & steiner, 2000). moreover, studies on the pollinator systems of invasive species may incorporate useful information (e.g. ecological predictor data) for risk modelling of invasive species (jiménez-valverde et al., 2011), thus effective eradication programs (gardener et al., 2010). only carpenter bees, digger bees and hawkmoths have been reported visiting flowers of m. annua in india, where this plant is considered invasive (raju & reddi, 2000). in mexico, trigona fulviventris guérin-méneville is the only known bee that visits m. annua (ayala, 2004). therefore, literature about floral visitors of m. annua along its natural rage is scarce. our work focused on the bee community that visited m. annua in a biosphere reserve located within the natural distributional range of the plant. because of its external morphology, we hypothesized that (a) m. annua attracts only visitors of the bee community (melittophilous) and that (b) larger and hairy bees may perform a better pollination fitness. material and methods study area. the study was conducted in september 2017 in the surrounding facilities of chamela biological station, unam, part of the “reserva de la biósfera chamela-cuixmala (rbcc)”. it is placed in jalisco state, mexico, located 1.7 km from the nearest point to the pacific coast (19.49891 n, 105.04441 w). it belongs to a tropical subhumid region with contrasting dry and rainy season. annual mean temperature is 25.2°c, averaging 745.1 mm/year of rainfall (trejo, 1999). the predominant vegetation is deciduous tropical dry forest (miranda & hernández, 1963). data collection. during the last week of september 2017, we monitored the floral visitors of m. annua from 06:00 to 00:00 h within a span of three consecutive days. every two hours, three observers recorded information during 10 min (i.e. 900 min of field observations). in situ data considered information on the foraging conduct, beginning and end of activity, visit, recognition fly, and time spent inside the flower. since individuals of m. annua usually contained less than four opened flowers, each observer often recorded information on groups of 2-4 different flowers distributed in small plots containing 3-4 flowering plants (n=10/plants/ day). each monitoring round, observers switched places to minimize individual perception bias. individual flowers pre-anthesis were selected from 10 different plants to collect biological (i.e. anthesis period and floral lifespan) and morphological data. the latter included 12 morphological attributes of reproductive and nonreproductive floral traits. the total nectar production and sugar concentration per individual flower were evaluated in flowers covered with soft tulle mesh to prevent the entrance of any visitor (fig 1a). the bees that visited the flowers were caught and killed in separated tubes. then we used fuchsin jelly to remove all pollen grains from the vertex and scutum of the bees. we made preparations of each individual bee to observe and count the pollen grains under 40 – 100x. previously, we collected pollen from virgin flowers of m. annua as a reference for the species and identify the different pollen morphotypes by using palacios-chavez et al. (1989) and quiroz-garcía et al. (1994). data analysis. morphometric and sexual data were used to estimate the outcrossing index of m. annua (oci), to give an approximation of the plant´s breeding system (cruden, 1977). the oic ranges from 0 to 4; where 0 = cleistogamy, 1 = obligate autogamy, 2 = facultative autogamy, 3 = facultative xenogamy and 4 = xenogamy. considering the spatial separation between stigma and anthers, we used a paired t test to proof herkogamy in flowers. changes in the frequency of visitation rate through time were evaluated with a chi-square test of goodness of fit. statistical tests were done with r v3.4.4 (r core team, 2018). fig 1. exclusion and foraging behavior during floral anthesis of martynia annua. a. floral exclusion with soft tulle mesh. b. trigona fulviventris; c. euglossa viridissima; d. representation of foraging behavior of e. virissima on m. annua: a) recognition flight, b) landing, and c) obtaining nectar. diagram by falcón-brindis, a. d cárdenas-ramos; a falcón-brindis; r badillo-montaño; i hinojosa-díaz; r ayala – floral traits and visitors of m. annua724 results floral traits and floral resources flowers of m. annua opened between 05:00 and 08:00 h, reaching the maximum diameter of the corolla at midday (3.46 ± 0.77 cm, hereafter standard error (se), n=10). anthesis varied from 1–2 days. flowers kept partially opened at night (i.e. lobes of corolla gently retracted inward), even when plants exhibited nyctinasty (upward folding position of leaves) when sunlight started vanishing at 17:00 –18:00 h. leaves returned to their position until morning. each plant displayed 2–3 opened flowers simultaneously during blooming (n=12). nectar production averaged 0.021 ± 0.086 ml with a concentration of 36.16 ± 2.28 °bx/flower (n= 6). the corolla diameter of m. annua was greater than 6 mm (table 1) and did not exhibit herkogamy, since anthers and stigma were not spatially separated (t = 0.00064, df = 6, p = 0.49). dehiscence of anthers occurred since flower opening and before stigma receptivity, which denoted protandry. the outcrossing index (oci), m. annua displays a xenogamous reproduction system (supplementary material). the floral measurements of m. annua are described in table 1. the most abundant visitor was e. viridissima (70.6 % of total records), the vast majority were males (93.3% n=15) occurring within 6:00 and 18:00 h. the other recurrent visitor was t. fulviventris, occurring between 10:00 and 16:00 h. individuals of this species, along with f. nigra frequently slipped at the entrance of the corolla while trying to make their way inside the flowers (fig 1b). thus, they failed the attempts to reach the nectar in >90% of observations (n = 13). noctuid moths were observed reaching the nectar with their proboscis while standing on the landing lobe of the corolla. none of the floral visitors were seen removing pollen from the anthers. only c. agilis was observed touching the reproductive organs of m. annua. in general, the visitation period had short duration (< 5.5 s), though e. viridissima spent 2-12 s in recognition flights around the flower, and then stayed 2-15 s inside the flower during the nectar consumption (fig 1c-d). pollen loads in total, 12 different pollen morphotypes were found on the scutum surface among the floral visitors of m. annua (supplementary material). moreover, the composition and amount of pollen loads were variable (table 2). in general, the amount of pollen extracted from the scutum of c. agilis was overwhelming if compared with the rest of individuals collected of other species. e. viridissima transported 50 to 80% of heterospecific pollen. table 1. floral attributes of martynia annua in the studied location. se = standard error. floral attributes mean (cm) se non-reproductive flower lenght 5.76 0.66 corolla diameter 2.96 0.86 inner diamater of corolla 1.81 0.82 pedicel length 1.48 0.79 pedicel diameter 0.21 0.20 reproductive gynoecium length 2.44 0.73 stigma lenght 1.95 0.42 ovary (polar) 0.48 0.20 upper length of ovary 0.26 0.17 ovary (equatorial) 0.26 0.22 stamen length 2.53 0.84 anther length 1.86 0.33 floral visitors and foraging behavior six floral visitors were identified, including five species of bees: centris (melanocentris) agilis smith, euglossa viridissima friese, frieseomelitta nigra (cresson), t. fulviventris, and caenaugochlora sp., and one species of moth (lepidoptera: noctuidae). most of the visits occurred between 10:00-12:00 h. the frequency of floral visitors differed along the day (χ2 = 81.91, d.f. = 8, p< 0.001), presenting the highest rates of visitation at noon with 8.66 ± 2.60/individuals, which agrees with the maximum diameter of corolla (fig 2). fig 2. assemblage of floral visitors and their frequency per hour in martynia annua. discussion this is the first study describing the reproductive system of m. annua in the area of its native distribution and summarizing aspects on floral biology, visitors, and their behavior. our results indicate that m. annua has hermaphroditic flowers with absent herkogamy but with temporary separation between the release of pollen and the receptivity of the stigma (i.e. protandry). according to the oci, m. annua has a xenogamous reproductive system (cruden, 1977), which is usually present among plants who require outcrossing sociobiology 65(4): 722-726 (october, 2018) special issue 725 pollen transfer either by biotic or abiotic vectors (fenster et al., 2004). despite protandry being a method to avoid selfpollination (richards, 1986), the absence of herkogamy could eventually allow autogamy in m. annua in the absence of the appropriate vectors that promote outcrossing. this alternative is also present in proboscidea louisianica (mill.) (the closest relative of m. annua), which is a facultative xenogamous species (phillippi & tyrl, 1979). floral traits as predictors of the expected type of visitors (i.e. pollination syndromes), could depend on the plant-pollinator assemblage co-existing in a particular space and time (herrera, 1988; lázaro et al., 2008). however, syndrome concepts have been broadly tasted, supporting that adaptations in the most effective pollinator group can drive the convergent floral evolution (wolfe & krstolic, 1999; lázaro et al., 2008; gong & huang, 2009; rosas-guerrero et al., 2014). therefore, some important traits in m. annua, such as reward, color, fragrance, and zygomorphism may have such convergence ruled by specialized visitors. plants with zygomorphic flowers may restrict the approaching angle of desired pollinators, supporting the idea of the ecological coherence between the flower and the suitable floral visitors (fenster et al., 2004; sargent, 2004). in all visitation records, we noticed that small bees slipped on when intended to alight and climb up to take the reward. thus, they were unable to reach the nectar. probably both the thin waxy layer in the internal surface of the corolla and the flower bearing angle (>70°) cloud restrict the access to small bees. among the most common and generalist bee species occurring at the rbcc, e. viridissima has been reported interacting with 24 plant species (ayala, 2004). that would explain why e. viridissima transported a great diverse pollen grains. however, heterospecific pollen transfer can reduce the fitness of plants by loss of pollen and physical blocking (raine et al., 2007). moreover, this euglossine species was not necessarily transporting conspecific pollen nor touching reproductive organs of m. annua. despite the usual lower frequency of large bees, the higher rate of pollen deposition (>50 times that of small bees) makes them important pollinators of wild plants (liu & huang, 2013). in our results, the amount of pollen grains carried by an individual of c. agilis was stunning. its size and abundant pilosity allowed for transportation of large pollen loads on the scutum. similarly, in p. louisianica, the bee species melissodes communis cresson and bombus pensylvanicus (de geer) were found to be the main pollinators (phillippi & tyrl, 1979). both bee species are moderate to large sized and densely clothed with long hairs. our results suggest that c. agilis would have the potential to be among the few specialized effective pollinators of m. annua. in terms of our two proposed hypotheses we conclude that: (a) the reproductive biology of m. annua suggests a strong relationship with some specialized bees to perform outcrossing; (b) the effective outcrossing must be driven by large hairy bees that touch reproductive organs during visits hand have high rates of pollen deposition. in addition, some bees such as e. viridissima may use m. annua as an energetic supply rather than a protein source. considering the wide range of distribution of m. annua, it is likely that bee species of the genera centris, bombus, xylocopa or the tribe eucerini, may be the associated pollinators in other regions of america. finally, since m. annua is tolerant to human disturbance (calderón, 1998), it is conceivable to think that the pollinator community vary along gradients of conservation and fragmentation, but also to have reproductive adaptations that allow their prevalence. acknowledgments we thank posgrado de ciencias biológicas of unam, the sponsor of the course in which we developed this work. we thank workers of the estación biológica chamela for their kind support during the usage of the facilities. d cárdenasramos, a falcón-brindis, and r badillo-montaño received a scholarship from conacyt, méxico. supplementary material doi: 10.13102/sociobiology.v65i4.3393.s2221 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3393/0 authors’ contribution d cárdenas-ramos contributed with the floral biology and botanical analysis of the studied plant. a falcón brindis is responsible of the analysis, structure, and integration of this family species pg hpg sa (cm2) time apidae euglossa viridissima 10 ± 9 2 ± 10 0.25 ± 0.01 6.4 ± 5.3 centris agilis 798 3 0.49 3.4 frieseomelitta nigra* 1 0 0.04 4.1 trigona fulviventris* 12 ± 3 2.2 ± 1.3 0.04 3.8 ± 0.88 halictidae caenaugochlora sp* 1 3 0.09 3.7 * never entered to the flower. table 2. pollen loads on floral visitors and average time spent within the flowers of martynia annua. pg = pollen grains of m. annua (mean ± se); 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(1999). symmetry and its influence on variance in flower size. the american naturalist, 154: 484-488. http://www.cabi.org/isc http://www.cabi.org/isc doi: 10.13102/sociobiology.v62i1.46-51sociobiology 62(1): 46-51 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the insecticidal and repellent activity of soil containing cinnamon leaf debris against red imported fire ant workers introduction the red imported fire ant (solenopsis invicta), from parana river basin of south america, is a voracious consumer of numerous other arthropod species, and often is the most abundant predaceous arthropod in crop fields throughout united states, new zealand, and australia (nattrass & vanderwoude, 2001; ascunce et al., 2011). they were introduced to mainland china in 2005 (zhang et al., 2007) and widely distributed in south china, causing severe damage to humans, animals, and the environment. traditional methods for managing the red imported fire ant are through insecticides or baits might lead to groundwater contamination, nontarget species, and other environmental considerations, more consumers are turning to organic solutions for their pest problems. (vogt et al., 2002). using chemicals from natural products in insect pest management is generally considered as a safer alternative than using synthetic contact insecticides (chen, 2009). while, abstract in the study, the amount of cinnamaldehyde and eugenol in soil containing cinnamon leaf debris were determined at different depths by high performance liquid chromatography (hplc). the insecticidal activity and repellence of the soil was tested separately. results showed that higher contents of cinnamic aldehyde and eugenol were found in soil at depths of 5 10 cm. in the insecticidal toxicity bioassay, the corrected mortality of major workers treated with cinnamon soil at depths of 5 10 cm, which was higher than the other soil depths, increased from 13.3% to 80.0% with contact time from 1 5 d. likewise, the corrected mortality of minor workers also increased from 6.7% to 100.0%. in the repellent activity bioassay, the repellency (96.3%) of major and minor workers treated with cinnamon soil at depths of 5 10 cm for 24 h were significantly higher than the other treatments. this result revealed ecological value of cinnamon. soil underneath cinnamon contained cinnamaldehyde and eugenol from fallen leaves, and these components showed insecticidal activity and repellence against red imported fire ants. perhaps we could control the red imported fire ants by planting cinnamon in some possible regions or by incorporating cinnamon leaves into soil where cinnamon will not grow. sociobiology an international journal on social insects cl huang1, jt fu 1, yk liu 1, dm cheng 1, 3, zx zhang 1, 2 article history edited by evandro n. silva, uefs, brazil received 27 march 2014 initial acceptance 19 january 2015 final acceptance 22 february 2015 keywords cinnamaldehyde, eugenol, solenopsis invicta, repellency, insecticidal toxicity corresponding author zhi-xiang zhang key lab. of nat. pesticide and chemical biology, ministry of education south china agricultural university guangzhou, china, 510642 e-mail: zdsys@scau.edu.cn many natural products are extremely toxic, so we perhaps need new strategies in red imported fire ant control. studies on the toxicity of botanical essential oils on red imported fire ant have shown that various essential oils are repellent and/or toxic to the ant (tang et al., 2013). mint oil granules were proven to be toxic and repellent to red imported fire ants (appel et al., 2004). the essential oils of camphor, artemisia annua, eucalyptus, mugwort, turpentine wintergreen, chrysanthemum, and forsythia showed effective fumigation toxicity against the red imported fire ant (tang et al., 2013). in a previous study on the anti-termitic activity of essential oils and their chemical constituents, 5 mg/g each of benzaldehyde, r-terpineol, neral, geraniol, eugenol, cinnamyl alcohol, and cinnamaldehyde exhibit 100% mortality after 1 d post-treatment (chang & cheng, 2002). cinnamomum aromaticum nees, lauraceae trees, which is native to south china and widely planted in guangdong, hong kong, guangxi, hainan, and yunnan, as well as in other subtropical areas. cinnamon oil is commonly used in food research article ants 1 south china agricultural university, guangzhou, china 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 zhongkai university of agriculture and engineering, guangzhou, china sociobiology 62(1): 46-51 (march, 2015) 47 and chemical industries because of its special aroma. studies have shown that the essential oil of indigenous cinnamon (c. osmophloeum) leaf contains cinnamaldehyde and eugenol. the essential oil and trans-cinnamaldehyde of indigenous cinnamon leaf also elicit an excellent inhibitory effect that controls red imported fire ant. eugenol, one of the six major components in essential balm, is an insect-repellent substance that negatively affects the workers of red imported fire ant at varied concentrations (chen, 2009). clove powder applied at 3 and 12 mg/cm2 provided 100% ant mortality within 6 h, and repelled 99% within 3 h and eugenol was the fastest acting compound against red imported fire ant compared with eugenol acetate, beta-caryophyllene, and clove oil (kafle & shih 2013). however, studies have been focused attention on cinnamon essential oil, few studies have reported that cinnamon could affect fire ants directly, maybe we could control fire ant by a more environment-friendly method, which is planting cinnamon in some possible place or just incorporating cinnamon leaves into soil. materials and methods standards cinnamal standard (purity is approximately 98.4%) and eugenol standard (purity is approximately 99.7%) were purchased from accustandard, inc. insects four s. invicta colonies were collected from the suburb of guangzhou. workers and nest material were placed into plastic boxes (40 cm × 52 cm × 12 cm) coated with teflon emulsion on the top, in which a test tube (25 mm × 200 mm) used as a water source was partially filled with water and plugged with cotton. the ants were fed with ham sausage and maintained in the laboratory at 25±2 °c. the major workers were 5 6 mm in length and the minors were 3-4 mm in length.(cheng et al., 2008) soil and cinnamon leaves we established six points under the c. aromaticum nees, which has been planted for about 30 years in the insecticidal botanical garden at south china agricultural university, and the stem diameter of the tree is about 30 cm. before sampling, fallen leaves, grasses and other small plants under the tree have been artificially removed, for excluding the interference of them. the distances of these points from the trunk were 0, 20, 40, 60, 80, and 100 cm. we collected soil samples at depths of 0 5 cm, 5 10 cm, 10 15 cm, 15 20 cm, 20 25 cm, 25 30 cm, 30 35 cm, and 35 40 cm by using a puncher with a diameter of 5 cm at each point. we also collected younger green leaves, older green leaves, yellow green leaves, yellow leaves, and tan leaves from the tree. fallen leaves were classified into yellow green, tan, and brown. insecticidal toxicity bioassay approximately 20 g soil was placed in a 250 ml beaker [71 mm (diameter) × 97 mm] with a vertical wall coated with fluon emulsion and allowed to dry for 24 h to prevent the ants from escaping. ten minor workers and ten major workers were placed at the bottom of the beaker. the soils in the treatment groups were obtained from the depths of 0 5 cm, 5 10 cm, 10 15 cm, 15 20 cm and close to the cinnamon tree. the soil in the control group was obtained outside the insecticidal botanical garden at south china agricultural university. mortality was assessed after 1, 2, 3, 4, and 5 d after the treatments with different soils. all treatments were replicated thrice. the workers were maintained at 25 ± 1 °c and relative humidity of 80%. we used the following equations: m(%) = nd / nt × 100, (1) mc(%)= (mt – mc ) /(1–mc )×100 , (2) where m is the mortality, nd is the number of dead ants, nt is the number of total ants, mc is the corrected mortality, mt is the mortality of treatment group, mc is the mortality of control group. repellent activities bioassay approximately 20 g soil sample was placed at the bottom of a 500 ml beaker (90 mm (diameter) × 122 mm) with a vertical wall coated with fluon emulsion. the upper part of the beaker was then covered with 20 g of control soil. we selected and placed 10 minor workers and 10 major workers in the breaker. in the experiment, four treatment groups and one control group were set up. all of these treatments were replicated thrice. we found that ants preferred to live in the soil without the repellent compared with the soil containing a repellent. the following equation was used to determine the repellency: rr(%)= (nc– nt ) / nc × 100 (3) where rr is the repellency, nc is the number of ants in control group, nt is the number of ants in treatment group. cinnamic aldehyde and eugenol contents in cinnamon soil determined by high performance liquid chromatography (hplc) dissolved cinnamicaldehyde and eugenol standard with fisher chemalert guide respectively, diluted to different concentrations, the concentrations of cinnamicaldehyde standard solution was 0.01, 0.05, 0.1, 0.5, 1 and 5 mg/l, and the concentrations of eugenol standard solution was 0.05, 0.1, 0.5, 1, and 5 mg/l. approximately 4 g soil from each soil sample was mixed with 3 ml fisher chemalert guide in a 10 ml centrifuge tube and then subjected to ultrasonic extraction for 30 min. the leaves were treated with the same method as in soil except 0.1 g leaves was mixed with 4 ml fisher chemalert guide. we then detected the cinnamic aldehyde and eugenol contents in the soil and leaves by hplc. the hplc system with c18 column (250 mm × 4.6 mm, 5 μm) was used. the following conditions were used: the cl huang, et al. insecticidal effect of cinnamon against s. invicta workers48 mobile phase, fisher chemalert: chromatographic methanol: guide-distilled water (65:35); flow rate, 1.0 ml/min; column temperature, 25 °c; detection wavelength, 290 nm; and sample size, 10 µl. statistical analysis statistical analyses were carried out and figures were produced using microsoft office excel 2003. the differences between the data were assessed by using the duncan-test with p< 0.05 regarded as statistically significant. results cinnamic aldehyde and eugenol contents in cinnamon leaves and soil cinnamicaldehyd and eugenol standard was separately quantified with an external standard curve between 0.01 and 5 mg/l (y = 114922x – 3444.7, r2= 0.9998), 0.05 and 5 mg/l (y=6277.5x-421.9, r2= 0.9999). fig 1 showed the chromatograms of cinnamaldehyde and eugenol standard. cm, 10 15 cm, 15 20 cm, and 20 25 cm. by comparison, contents of cinnamic aldehyde and eugenol at these depths were significantly higher than those at depths of 25 30 cm, 30 35 cm, and 35 40 cm (fig 3). fig 1. the chromatograms of cinnamaldehyde(a) 0.5 µg/ml and eugenol (b) 0.5 µg/ml standard. cinnamic aldehyde and eugenol contents in fresh leaves increased initially from 0.2% and 1.3% (younger green leaves) to 0.5% and 4.3% (fallen yellow green leaves) and then decreased to 0.2% and 1.7% (fallen brown leaves) (fig 2). contents of cinnamic aldehyde (0.0608 mg/kg) and eugenol (1.4161 mg/kg) in soil at depths of 5 10 cm were significantly higher than at other depths. however, no significant difference in contents was observed among depths of 0 5 fig 2. the content of eugenol and cinnamal in cinnamon leaves. (1=younger green leaves, 2=older green leaves, 3=yellow green leaves, 4=fallen yellow green leaves, 5=yellow leaves, 6=fallen yellow leaves, 7=tan leaves, 8=fallen tan leaves, 9=fallen brown leaves.) insecticidal toxicity the insecticidal toxicity tests showed effective insecticidal activity against red imported fire ant workers. in the test, the corrected mortality of workers varied significantly according to soil depth and treatment time. in all of the treatments, the corrected mortality of both minor and major workers increased with exposure time ranging from 1 d to 5 d. for the major workers, the corrected mortality increased from 3.3% to 64.0% (0 5 cm), from 13.3% to 88.0% (5 10 cm), from 13.3% to 80.0% (10 15 cm), and 6.7% to 72.0% (15 20 cm). for the minor workers, the corrected mortality increased from 0.0% to 75.0% (0 5 cm), from 6.7% to 100.0% (5 10 cm), from 3.3% to 91.7% (10 15 cm), and fig 3. the content of cinnamal and eugenol of cinnamon soil in different depth. sociobiology 62(1): 46-51 (march, 2015) 49 from 0.0% to 87.5% (15 20 cm). the corrected mortality of major workers showed no significant difference among different depths of soil at 1, 2, and 3 d after treatment. at 4 and 5 d after treatment, the corrected mortality at depths of 5 10 cm, 10 15 cm, and 15 20 cm was higher than that at 0 5 cm treatment, but no significant difference was observed among these results. at 3 d, the corrected mortalities at depths of treated soil 5 10 cm and 10 15 cm were significantly higher than those at 0 5 cm and 15 20 cm (figs 4 and 5). repellent activity we can calculate the repellency according to eq. (3). figure 5 shows that the repellency of major and minor workers reached 96.3% at the treated soil depth of 5 10 cm. this rate was significantly higher than the others. at 24 h of treatment, the repellency against minor workers were 49.2% (0 5 cm), 73.7% (10 15 cm), and 33.3% (15 20 cm). at 24 h of treatment, the repellency against the major workers were 52.4%, 79.6%, and 57.1% (fig 6). fig 4. corrected mortality of major workers caused by the cinnamon soil in different depth. fig 5. corrected mortality of minor workers caused by the cinnamon soil in different depth. fig 6. repellency of minor and major workers caused by the cinnamon soil in different depth. discussion the study of cinnamon soil indicated effective insecticidal and repellent activities against red imported fire ant. this may be caused by some components like cinnamaldehyde and eugenol in fallen leaves. this result is consistent with that in a previous report, in which eugenol, menthol, and methyl salicylate as the components of essential balm significantly suppress the digging abilities at 10 mg/kg (chen et al., 2009). in another study, toxicity tests indicated that the indigenous cinnamon leaf essential oil and trans-cinnamaldehyde exhibit an excellent inhibitory effect that controls red imported fire ant (cheng et al., 2008). in this study, the soil at a depth of 5 10 cm contained higher eugenol and cinnamic aldehyde than the soil at other depths. at depths of 10 15 cm, 15 20 cm, 20 25 cm, and 25 30 cm, higher contents of eugenol and cinnamaldehyde were found than at deeper soil layers. how could the chemicals were introduced into soil so deep? firstly, there were lots of fallen leaves covering on the ground every year, and sometimes the thickness of the litters could reach to 50 mm. fallen leaves decay as part of natural processes. as these leaves decay, the compositions inside which may be either decomposed or retained in soil. both cinnamaldehyde and eugenol are slightly soluble, solubility of the former is 0.01-0.1 g/100 g h2o(cao et al, 2010), while guangzhou is located in a subtropical area, where the average annual rainfall was nearly 2000 mm. as such, some compositions in the soil even slightly soluble cl huang, et al. insecticidal effect of cinnamon against s. invicta workers50 might be leached into the subsoil or deeper as rainfall was enough. however, cinnamic aldehyde in the topsoil can be easily oxidized and degraded upon exposure to the sun, rain, and wind, thereby producing cinnamic acid. cinnamaldehyde, epoxy cinnamyl alcohol, and cinnamic acid are considered as oxidation products of cinnamyl alcohol and cinnamaldehyde with the largest amounts (niklasson et al., 2013). while the substances in subsoil can be relatively stable. the red imported fire ants prefer open sunny disturbed areas like farmland, wasteland, green belts, roadside and so on. but ants also be found in the building, school, lawn, etc, where they are more likely to harm humans. the significance of this study is to provide a new way to drive away or kill red imported fire ants in places where people often appear by planting trees like cinnamon or incorporating leaf detritus into soil around the house, at school or on the lawn. compared with traditional chemical control methods, the new way is more safe and environment-friendly. further more, constantly falling leaves make the toxic and repellent effects continuous. in addition, in this way, we can also create a more comfortable and greener environment. the study have focused on two chemicals, cinnamicaldehyde and eugenol, two main insecticidal ingredients in cinnamon leaves. nonetheless, more research is needed to see if there are other chemicals in the leaves that might contribute to the toxicity or repellency against fire ants. acknowledgments this study was supported by the talent development program of joint training of postgraduates demonstration base in guangdong province, china (no.4200-214093). references appel, a. g., m. j. gehret. & m. j. tanley. (2004). repellency and toxicity of mint oil granules to red imported fire ants (hymenoptera: formicidae). journal of economic entomology, 97: 575-580. doi: 10.1603/0022-0493-97.2.575 ascunce, m. s., c. c. yang, j. oakey, l. calcaterra, w. j. wu, c. j. shih, j. goudet, k.g. ross & d. shoemaker. (2011). global invasion history of the fire ant solenopsis invicta. science, 331: 1066-1068. doi: 10.1126/science.1198734 batish, d. r., h. p. singh, n. setia, s. kaur. & r. k. kohli. (2006). chemical composition and inhibitory activity of essential oil from decaying leaves of eucalyptus citriodora. zeitschrift fur naturforschung c, 61: 52-56. cao, y. l. (2010). study on the process monitor of coupling alkaline hydrolysis-water vapor distillation of cinnamaldehyde. http://www.doc88.com/p-206874485419.html chang, s. t.& s. s. cheng. (2002). antitermitic activity of leaf essential oils and components from cinnamomum osmophleum. journal of agricultural and food chemistry, 50: 1389-1392. doi: 10.1021/jf010944n chen, j. (2009). repellency of an over-the-counter essential oil product in china against workers of red imported fire ants. journal of agricultural and food chemistry, 57: 618-622. doi: 10.1021/jf8028072 chen, j., c. l. cantrell, h. w. shang. & m. g. rojas. (2009). piperideine alkaloids from the poison gland of the red imported fire ant (hymenoptera: formicidae). journal of agricultural and food chemistry, 57: 3128-3133. doi: 10.1021/jf803561y cheng, s. s., j. y. lin, c. y. lin, y. r. hsui, m. c. lu, w. j. wu. & s. t. chang. (2008). terminating red imported fire ants using cinnamomum osmophloeum leaf essential oil. bioresouce technology, 99: 889-893. doi: 10.1016/j. biortech.2007.01.039 kafle, l. & c. j. shih. 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(1999). influence of large termitaria on soil characteristics, soil water regime, and tree leaf shedding pattern in a west african savanna. plant and soil, 206: 47-60. doi: 10.1023/a:1004321023536 tang, l., y. y. sun. & q. p. zhang. (2013). fumigant activity of eight plant essential oils against workers of red imported fire ant, solenopsis invicta. sociobiology, 60: 3540. doi: 10.13102/sociobiology.v60i1.35-40 vogt, j. t., t. g. shelton, m. e. merchant, s. a. russell, m. j. tanley. & a. g. appel. (2002). efficacy of three citrus oil formulations against solenopsis invicta buren (hymenoptera: formicidae), the red imported fire ant. journal of agricultural and urban entomology, 19: 159-171. yeh, h. f., c. y. lin, c. y. luo, s. s. cheng, y. r. hsu. & s. t. chang. (2013). methods for thermal stability enhancement of leaf essential oils and their main constituents from indigenous cinnamon (cinnamomum osmophloeum). journal of agricultural and food chemistry, 61: 6293-6298. doi: 10.1021/jf401536y sociobiology 62(1): 46-51 (march, 2015) 51 zhang, r., y. li, n. liu. & s. d. porter. (2007). an overview of the red imported fire ant (hymenoptera: formicidae) in mainland china. florida entomologist, 90: 723-731. doi: 10.1653 /0015-4040(2007)90[723:aootri]2.0.co;2 zhang, z. x., y. zhou. & d. m. cheng. (2013). effects of hematoporphyrin monomethyl ether (hmme) on worker behavior of red imported fire ant, solenopsis invcita. sociobiology, 60: 169-173. doi: 10.13102/sociobiology.v60i2.169-173. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.39-45sociobiology 62(1): 39-45 (march, 2015) impact of african weaver ant nests [oecophylla longinoda latreille (hymenoptera: formicidae)] on mango [mangifera indica l. (sapindales: anacardiaceae)] leaves introduction among tropical fruits, mango (mangifera indica l.) has the highest potential for food security (vayssières et al., 2008) and incomes (tinkeu et al., 2010) in west africa. costeffective, locally available and sustainable pest management options for this fruit are considered necessary to enhance its production, consumption and export. in africa, oecophylla longinoda latreille has been detected as an effective plant protector (way & khoo, 1992; dwomoh et al., 2009; olotu et al., 2013; anato et al. unpublished data) and can increase fruit yields and/or fruit quality compared to conventional synthetic insecticides (van mele, 2008; dwomoh et al., 2009; anato et al., unpublished data). abstract oecophylla ants are appreciated for their control of pests in plantation crops. however, the ants´ nest building may have negative impacts on trees. in this study we tested the effect of ant densities and nest building on the leaf performance of mango trees. trees were divided into three groups: trees without ants, trees with low and trees with high ant densities. subsequently, the total number of leaves, the proportion of leaves used for nest construction, and tree growth was compared between these groups. the percentage of leaves used for nests was between 0.42-1.2 % (mean = 0.7%±0.02) and the total number of leaves and tree growth was not significantly different between trees with and without ants. further, leaf performance was compared between shoots with and without ant nests and between leaves in or outside ant nests. the number of leaves and lost leaves per shoot, leaf size, leaf condition (withered), leaf longevity and hemipteran infection was compared between groups. in the dry season nest-shoots held more leaves than shoots without nests despite nest-shoots showed more lost leaves. leaves in nests were smaller than other leaves, more likely to wither and more often infested with scales. however, smaller nest-leaf size was probably due to the ants´ preference for young leaves and the higher incidence of withering resulting as leaves in nests cannot fall to the ground. in conclusion, the costs associated to ant nests were low and did not affect the overall number of leaves per tree nor tree growth. sociobiology an international journal on social insects fm anato1, a sinzogan1, a adandonon2, x hounlidji1, j offenberg3, dk kossou1, j-f vayssières4 article history edited by gilberto m. m. santos, uefs, brazil received 20 june 2014 initial acceptance 21 august 2014 final acceptance 04 september 2014 keywords weaver ants, leaf performance, ant nests, biocontrol, plant costs, benin. corresponding author florence mahouton anato université d’abomey calavi, 07 bp 1130, cotonou, république du bénin e-mail: anatoflorence@yahoo.fr the relationship, however, between the ants and their host plants involve negative as well as positive aspects, from a plant point of view. ants are engaged in mutualistic trophobiotic interactions with plant sap sucking hemipterans and may in this way indirectly damage plants (buckley, 1987; van mele et al., 2007). on the other hand, they also feed on various herbivorous arthropods and can in this way benefit host plants (way, 1954; hölldobler, 1983; van mele & cuc, 2003; lim et al., 2008). further, on the negative side, weaver ants weave leaves together (crozier et al., 2010) and deposit territorial marking on leaves (offenberg, 2007) which may reduce the efficiency of photosynthesis on their host plant. also, when ants interlace leaves and turn them out of their original position, they can increase tension on the leaf petiole research article ants 1 université d’abomey calavi, cotonou, république du bénin 2 université d’agriculture de kétou, cotonou, république du bénin 3 aarhus university, denmark 4 biocontrol unit for africa, cotonou, république du bénin fm anato, et al. african weaver ants impact on mango leaves40 and potentially cause a premature detachment of the leaf from the shoot (dillwith et al., 1991; riedell & blackmer, 1999; offenberg et al., 2006). thus, oecophylla nest building may result in indirect cost to the leaves used for their nest construction. in benin weaver ants are able to reduce fruit fly infestations in mango so efficiently that farmers´ negative perceptions of the ants are changing toward a more positive attitude (van mele et al., 2007; sinzogan et al., 2008; van mele et al., 2009). the ants are often integrated as a biocontrol component in ipmprograms. however, when promoting weaver ants, the scientific community is often facing questions from end users (farmers, extension officers) relating to the possible effect of the ants´ nest building on the trees leaf performance. some end users suspect that the presence of weaver ant nests (i) decreases the number of leaves on the trees and may have a negative impact on (ii) the size of leaves, (iii) their condition (more withering leaves), (iv) longevity and (v) on hemipteran infections. the effect of weaver ants (oecophylla spp.) on such plant responses has not yet been tested on crop plants. the objective of this study was therefore to evaluate the effect of the density of o. longinoda on the host tree responses listed above. materials and methods the study was conducted from september 2012 to september 2013 in n’dali in the borgou area located between longitude 02°04’ 03°33’e and latitude 09°24’ 12°08’n which is the major mango production area in benin (vayssières et al., 2008). the mango orchard selected for the experiments had on average 100 trees ha-1 at 10m × 10m spacing and were dominated by the variety “gouverneur”. all the trees were 15 years old and had similar size and appearance. the orchard was colonized by weaver ants 10 years ago and thus had trees with varying ant densities. the effect of ants on leaf numbers and tree growth mango trees in the plantation were inspected and divided in two groups – trees with and without weaver ants. after that, ant densities on ant-trees were assessed using two different methods – a trail density estimate and a nest density estimate. in the first approach ant densities were estimated with the forager density index (fdi) (offenberg and wiwatwitaya, 2010) which is based on the number of main trunks on a particular tree that is holding an ant trail (for more details see offenberg & wiwatwitaya, 2010). with this approach the fdi was collected fortnightly (12 times) from september 2012 to february 2013. based on the first survey, trees were divided into three groups: 1) trees without ants (fdi = 0); 2) trees with medium ant densities (fdi between 50 – 80%) and 3) trees with high ant densities (fdi >80%). twenty trees were then haphazardly selected from each group. in the second method the number of active ant nests in each tree was counted fortnightly (12 times) from april 2013 to september 2013. a nest was considered active if ants were defending it when shaken it with a pole. based on the initial number of nests (found in the first survey) in each trees, trees were divided into three groups: 1) trees without ant nests, 2) trees with a medium nest density (from 20 to 40 nests), and 3) trees with a high nest density (more than 60 nests). in this design seven weaver ant colonies were chosen and two trees per nest density were selected haphazardly in each colony equaling 14 trees per group. in both methods, trees that were originally without ants were kept ant-free by preventing ant access using a sticky band (oecotak 5; oecos ltd, u. k.) around the base of the tree trunks. as ant densities in ant trees may change over time the average density per tree was calculated for the whole period and based on this average trees were redistributed into the three different density groups before analyzing the effect of density on response variables. in addition to registering the fdi and number of nests (nn) in each tree, the trees total number of leaves (nl) was also estimated fortnightly, and their diameter at breast height (dbh) measured. diameter at breast height (dbh) was also taken on the main trunk at 1.30m above the ground with ribbon ‘‘pi’’ (specific ribbon used by foresters to measure tree dbh) of 1-cm precision. to estimate a trees´ total number of leaves a modified version of korine et al. (2000) method was used. originally, korine et al. (2000) categorized tree branches into main branches, secondary branches and small branches with leaves. the total number of main branches was counted for each tree and six branches were haphazardly selected. on these six main branches, all secondary branches were counted and one secondary branch haphazardly selected where all small branches were counted. the number of leaves was then assessed on 30 small branches, haphazardly selected from one secondary branch. based on this, the average number of leaves per small branch was calculated. the average number of leaves on a tree was then calculated as: nl = nal*nsmb*nsb*nmb, where nl is the number of leaves on the tree, nal is the average number of leaves per small branch, nsmb the number of small branches, nsb the number of secondary branches and nmb the number of main branches. we modified this method to adapt for the branching morphology of mango trees. as mango generally have less than 6 main branches but has secondary and tertiary branches, we changed the equation by introducing tertiary branches, to: nl = nal*nsmb*nsb*ntb*nmb, where ntb is the number of tertiary branches. the total number of leaves on a tree was then compared to the number of leaves used for ant nest construction – the latter found by multiplying the number of nests on the tree with the average number of leaves used per nest (see below). the proportion of nest leaves to the total number of leaves was then calculated for each tree. ant nests´ effect on mango leaves and shoots ten fortnightly surveys (five in the harmattan season [november to december 2012] and five in the rainy season sociobiology 62(1): 39-45 (march, 2015) 41 [may-june 2013]), were done to compare the performance of leaves inside and outside ant nests. fifteen trees (3 per survey) with ant nests were selected randomly in the orchard per season. on these trees all shoots with ant nests (nest shoots) were collected, and serving as control shoots, the nearest shoot to each nest shoot was also collected. collected shoots were placed individually in plastic bags and frozen before examination. leaves on nest shoots were categorized as either nest leaves if they were used for nest construction, or control leaves if they were not incorporated into an ant nest. on control shoots, all leaves were control leaves. on each shoot the following variables were registered / calculated: total number of leaves, percentage of leaves used for nest construction, the size of all leaves, each leafs´ condition (green vs. withered yellow), percentage of lost leaves, and the percentage of leaves colonized by trophobiotic hemipterans. leaf sizes were estimated using the method of pandey and singh (2011). each leaf was placed on paper and its outline cut out and weighed on an electronic balance. the mass of the paper outline was then divided by the mass of a 1 cm2 piece of paper to provide an estimate of the leaf area. the number of lost leaves was registered by counting leaf scars left by lost leaves on the petioles. data analysis the number of leaves and number of nests per tree, the number of leaves used for nest construction, the number of leaves per shoot and leaf areas were transformed with log10 [x+1] to stabilize the variance and achieve normality of the data before analysis. a multivariate repeated measures analysis of variance was performed to test if time and ant density affected the total number of leaves on the trees and one way anovas was used to test for the effect of ant density on the number of nests per tree, the number of leaves used for nest construction and the dbh. one way anovas was also used to compare the total number of leaves per shoot and the leaf area of leaves between nest shoots and control shoots. as it was not possible to transform the percentage of leaves used for nest construction, lost leaves, withered leaves and leaves with scale insects, to normal distributions, these responses were tested with non-parametric wilcoxon tests. all analyses were done with jmp 10.0.0. a log10 (x + 1) transformation was used on the number of nests and number of leaves used in nests constructions to stabilize variance and normalize the data (dagnelie, 2003). as the percentage of leaves used for nest construction could not be transformed to a normal distribution, a non-parametric wilcolxon test was performed. results the effect of ant density in the trees showed the same trend on the response variables if initial densities or the average densities were used to categorize trees into the three different density categories. thus, only the categorization based on the average densities was used in the presented results. the effect of ants on leaf numbers and tree growth table 1 shows the mean number of nests per ant-tree in the different density groups and from the sampling of nests from shoots it was found that weaver ants used an average of 12.68 ± 0.26se leaves to construct their nests. based on these measures it was found that the proportion of tree leaves used to construct nests was significantly different between trees with low and high trail densities (f1,478=45.81; p<0.0001), as well as between trees with low and high nest densities (f1,334=473.80; p<0.0001) (table 1). yet, the weaver ants used only on average 0.72 ± 0.02se % (0.42-1.20%) of the total number of mango leaves to construct their nests on the trees (table 1). this proportion was too low to affect an overall effect on the amount of leaves on the trees as there was no significant difference between density groups on table 1. statistics on the effect of ant density (oecophylla longinoda) on the number of ant nests and leaves on mango trees. numbers are mean values ±se per tree. number of nests number of leaves used for nest construction percentage of leaves for nest construction % trail density approach low density 22.77 ± 0.68 288.77 ± 8.61 0.49 ± 0.02 high density 35.31 ± 1.77 447.76 ± 22.44 0.77 ± 0.05 statistics* f(1,478)=45.84; p<0.0001 f(1,478)=45.84; p<0.0001 χ2= 27.65; df = 1; p<0.0001 nest density approach low nest 28.05 ± 0.73 355.67 ± 9.25 0.42 ± 0.01 high nest 62.82 ± 1.47 796.61 ± 18.70 1.20 ± 0.04 statistics* f(1,334)=473.79; p<0.0001 f(1,334)=473.79; p<0.0001 χ2= 186.52; df = 1; p<0.0001 * anova/wilcoxon statistics fm anato, et al. african weaver ants impact on mango leaves42 green, while a mean of 13.58 ± 1.31se % (rainy season) and 21.76 ± 1.25se % (harmattan season) of the leaves on nest shoots were withered. within nest shoots the majority of withered leaves were found among those leaves that were used for nests (rainy season: χ2 =181.59; df=1; p<0.0001 and harmattan season: χ2 = 122.02; df=1; p<0.0001). only 0.62 ± 0.39se and 6.12 ± 1.34se % of the control leaves on nest shoots were yellow versus 23.64 ± 2.03se and 33.78 ± 1.65se % of nest leaves on nest shoots in the rainy and in the harmattan seasons, respectively. consequently, withering was exclusively associated with nest shoots, and on these shoots, leaves used for nests constructions were more affected. scale insects were found on nest leaves in high numbers, whereas they were almost absent outside ant nests. on nest shoots 7.81 ± 1.76se and 16.69 ± 1.37se % of nest leaves were infected opposed to 3.52 ± 1.55se and 0.0% of the control leaves in rainy (χ2=150.9; df=1; p<0.0001) and harmattan seasons (χ2= 10.2; df=1; p=0.0014), respectively. during both seasons, all the leaves on control shoots were without scale insects (rainy season: χ2=311.2; df=2; p<0.0001 and harmattan season: χ2=37.6; df=2; p<0.0001). scale insects were not identified to the species level but were found to belong to coccidae. leaf loss was also affected by nest building. more nest shoots (79 of 289 in rainy season and 26 of 196 in harmattan season) had lost at least one leaf, compared to the control shoots (25 of 289 in rainy season and 11 of 196 in harmattan season). moreover, the average percentage of lost leaves per shoot was significantly different between the two types of shoot during the rainy (χ2=33.14, df=1, p<0.0001) and the harmattan (χ2=6.76, df=1, p=0.0093) season. a mean of 0.97 ± 0.21se % and 0.72 ± 0.23se % of the leaves on control shoots were lost, while a mean of 3.01 ± 0.37se % and 1.71 ± 0.35se % of the leaves on nest shoots were lost, respectively, in rainy and harmattan season. the total number of leaves per tree even after excluding those leaves that were used in ant nests (f2,57=1.38, p=0.26 in the trail density approach; f2,39=2.82, p=0.08 in the nest density approach) (table 2 and fig 1). the dbh of the trees was also not affected by ant densities. mean dbh was 80.04 ± 5.54se, 72.05 ± 3.38se and 73.94 ± 3.93se cm for control trees without ants, trees with low trail densities and trees with high trail densities, respectively (f2,57=0.92; p=0.41). a similar trend was seen when trees were categorized according to nest densities. in this case mean dbh was 95.52 ± 7.32se, 105.81 ± 4.65se, and 94.79 ± 5.05se, respectively, for the three density groups (f2,39=1.13; p=0.33). ant nests´ effect on mango leaves and shoots during the harmattan season, the total number of leaves per shoot was higher (f1,390=35.41, p<0.0001) on nest shoots (10.16 ± 0.28se leaves/shoot) than on control shoots (8.05 ± 0.22se leaves/shoot). on the contrary, during the rainy season, there was no significant difference between control shoots (8.22 ± 0.14se leaves/shoot) and nest shoots (8.55 ± 0.16se) (f1,576=1.61, p=0.21). in both seasons nest leaves were found to be smaller than other leaves. during the rainy seasons control leaves on nest shoots (39.47 ± 0.56se cm²) and control shoots (39.83 ± 0.22se cm²) were significantly (f2,4849 = 238.78; p<0.0001) larger than the leaves used for nest construction (33.63 ± 0.24se cm²). the same trend was observed in the harmattan season (f2,3558 = 120.66; p<0.0001) with mean leaf sizes of 40.24 ± 0.76se cm² on control leaves on nest shoots, 41.20 ± 0.41se cm² on control shoots and 33.15 ± 0.35se cm² on nest leaves. withered leaves were more abundant on nest shoots compared to control shoots in both seasons (rainy season: χ2=341.9; df=1; p<0.0001 and harmattan season: χ2= 164.2; df=1; p<0.0001) as all the leaves on control shoots were table 2. repeated measures statistics on the effect of time and oecophylla ant density on the total number of leaves per tree. seasonal mean values (per tree) are presented in fig 1. factors total number of leaves per tree including leaves used for nest construction total number of leaves per tree excluding leaves used for nest construction f df p f df p trail density approach treatment 1.26 2,57 0.29 1.38 2,57 0.26 time 28.73 11,47 <0.0001 28.81 11,47 <0.0001 weeks*treatment 1.26 22,94 0.22 1.26 22,94 0.22 nest density approach treatment 2.82 2,39 0.07 2.82 2,39 0.08 time 16.15 11,29 <0.0001 16.15 11,29 <0.0001 weeks*treatment 0.98 22,58 0.50 0.98 22,58 0.50 sociobiology 62(1): 39-45 (march, 2015) 43 discussion trees with weaver ants did not show significantly different number of leaves compared to control trees. this held true even if the leaves used in ant nests were considered “dead” to the tree and were subtracted from the total number of leaves. we can thus deduce from the current study that the foliar development and thus photosynthetic activities were unaffected by weaver ants. several reasons could explain this. firstly, the number of leaves used to form nests was low, 0.7% in average. similarly, offenberg et al. (2006) estimated that, o. smaragdina used as little as 0.25% of the leaves of the mangrove tree r. mucronata to form their nest. thus, weaver ants in general use only few of the available leaves on trees to build their nests. therefore, this potentially negative impact is limited and likely to be outweighed by the positive impacts of weaver ants. secondly, the agro-physiological factor, bud initiation, can be brought up. in most cases, when the terminal bud was used for nest construction, it led to the formation of secondary buds on the shoot and thus the formation of new flushes (anato, unpublished data). this observation may explain the higher average number of leaves per shoot on nest shoots compared to control shoots in the harmattan season. in the rainy season, however, rains led to extensive leaf flush which may have reduced the effect of bud initiation as there was no significant difference in the number of leaves on shoots between the two groups during this part of the year. in harmattan, the extra leaves on nest shoots may reduce the negative impact of nest building by increasing the leaf surface vacant for photosynthesis which may again affect fruit production positively. this is an important issue to be studied together with the direct effect of weaver ants in the future. the smaller size of leaves used in nests probably derived from the ants´ nesting behavior as they prefer to build their nests at the apical end of shoots where the young and still not fully grown leaves are situated. similarly, offenberg et al (2006) observed that o. smaragdina showed a strong preference for young leaves on r. mucronata in a thai mangrove. on the other hand, there was no size difference between the control leaves on nest and control shoots, showing that the nests did not affect the size of neighboring leaves. weaver ants engage in trophobiotic interactions with honeydew producing hemipterans, including scale insects (way, 1963; hölldobler & wilson, 1990; blüthgen & fiedler, 2002).this was also evident on mango in the present study as the presence of scale insects was strongly correlated with ant nests. thus, mango leaves indirectly support the ants via the scale insects and in this way leaves loose nutrients. however, ant-hemipteran trophobioses have most often only low costs to the plants unless the involved hemipterans are vectors of plants diseases. the scale species attended by the ants in this study has not been identified by a specialist taxonomist, however, based on morphology and behavior we believe it to be udinia catori (green), which is also the main hemipteran species protected by weaver ants on mango trees in benin (germain et al., 2010; vayssières, 2012). this species is a minor pest in benin and is not known as a vector of viruses (germain et al., 2010; vayssières, 2012). furthermore, a meta-analysis showed that plants in general benefit from anthemiptera interactions as the ants positive effects on plants (by deterring herbivores that are more detrimental than the hemipterans) more than outweigh the negative impact of a higher standing crop of sap-sucking hemipterans (styrsky & eubanks 2007). thus, counter intuitively, the presence of ant attended hemipterans may actually benefit host plant fitness. in the present study the increased presence of scale insects on ant trees did not seem to lead to any detrimental effects on leaf performance except, maybe, the increased incidence of withering and lost leaves on nest shoots. an increased incidence of withered leaves in the nests would be expected, though, as old leaves were weaved together with young leaves. this prevents the old leaves to fall to the ground when they wither. however, it cannot be ruled out that leaves also showed reduced longevity and thus earlier withering due to the physical stress of being bended out of position when used in an ant nests and by being associated with attended scale fig 1. mean number of mango leaves (excluding leaves used in ant nests) per tree by season on trees with different ant densities. a: ant trail density approach (september 2012 to february 2013); b: ant nest density approach (april to september 2013). a b fm anato, et al. african weaver ants impact on mango leaves44 insects. the physical stress on leaves may also have been the reason for the observed increase in lost leaves on ant nest shoots. our belief, though, is that these negative effects are small, as they did not affect the total number of leaves on trees, neither their dbh. regarding the dbh, though, it should be noted that these measurements were taken only once during the study and thus represent the growth of trees during their entire lifetime. as the plantation was colonized by weaver ants 10 years ago it is possible, e.g., that trees with abundant weaver ants during the study period may previously have had less or no weaver ants, as the ants may move between trees. therefore, the lack of an ant density effect on the dbh recorded in the present study should be interpreted with caution. further studies tracking tree growth under known ant densities are needed to elucidate this effect further. on the other hand, it has been shown by several authors that there is a strong correlation between stem volume and foliar biomass (bartelink, 1997; swenson & enquist, 2008), supporting that stem growth will be unaffected by ant density as long as leaf area is unaffected. in the context of weaver ants as biological control agents it has been claimed that ant trail density scores above 50% are desirable in plantations. above this level ant densities are sufficient to afford effective protection against pest insects (peng & christian, 2004; 2005). in the present study, trail densities were considerably higher (> 80%) in the high density group, yet, without showing any sign of a detrimental effect on leaf performance on the trees. thus, based on the present study, the high ant densities required for efficient biocontrol does not seem to conflict with leaf performance. in conclusion, the seemingly detrimental effects of nest building by weaver ants are either not real costs to the plants (smaller leaves and withered leaves) or are of limited impact (lost leaves and scale infections). as a consequence they do not translate into reduced leaf performance on ant trees. in other words, farmers using weaver ants for biocontrol should not worry about the effect of nest building on their crop trees. acknowledgements we would like to thank mr gaba lawal for allowing us to do this research in his field. we are grateful to pascal ayelo and other local people for technical help and assistance in the field. the danish international development assistance (danida) financed and supported this research (project: ‘increasing value of african mango and cashew production’ no.: 10 – 025au). references bartelink, h.h. 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(2012). tri-trophic relations between different food web structures about fruit flies in tropical fruit agroecosystems. habilitation à diriger des recherches. cirad-hortsys. université paris est, paris, france, p. 158. vayssières, j.-f., korie, s., coulibaly, t., temple, l. & boueyi, s. (2008). the mango tree in central and northern benin: cultivar inventory, yield assessment, early infested stages of mangos and economic loss due to the fruit fly (diptera: tephritidae). fruits, 63: 335-348. doi:10.1051/fruits:2008035 way, m.j. & khoo, k.c. (1992). role of ants in pest management. annual review of entomology, 37: 479-503. way, m.j. (1954). studies on the association of the ant oecophylla longinoda (latreille) (formicidae) with the scale insect saissetia zanzibarensis williams (coccidae). bulletin of entomological research, 45: 113-134. way, m.j. (1963). mutualism between ants and honeydew producing homoptera. annual review of entomology, 8: 307-344. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3464sociobiology 65(4): 645-653 (october, 2018) special issue two colors, one species: the case of melissodes nigroaenea (apidae: eucerini), an important pollinator of cotton fields in brazil introduction accurate species identification is crucial for biodiversity studies that aim to characterize patterns of species richness and abundance, and to identify recent changes in these patterns as a result of anthropogenic activities (austen et al., 2016; packer et al., 2018). a critical step for biodiversity studies is the reliable identification of collected individuals to the species level. in some cases, independent evolutionary units with very similar morphological characters might be classified as one species leading to an underestimation abstract accurate taxonomic delimitation in ecological research is absolutely critical as studies that seek to evaluate levels of biodiversity and quantify human effects on the environment are rapidly undertaken. coloration is a widely used morphological character for species identification through dichotomous keys. however, taxonomic identification based upon coloration is often unreliable because this character can exhibit high degree of intraspecific variation. an uncertain interpretation of mesosoma color-morphs (yellow or black) occurred when we used this character, in association with the bristle bands in the tii, for the identification of eucerine bee melissodes nigroaenea (smith), an important pollinator of cotton fields in south america. thus, we used a dna barcoding approach to investigate if different color morphs within this species reflected two distinct evolutionary lineages. our bayesian phylogenetic reconstruction revealed that both yellow and black individuals clustered in a highly supported monophyletic group. additionally, pairwise genetic distances between m. nigroaenea color-morphotypes were lower than 3%. these results indicate that both mesosoma color-morphs correspond to intraspecific variability within the same evolutionary unit. although the mesosoma coloration is not the unique character considered for taxonomic differentiation among melissodes species, our results indicate that this character is not reliable for melissodes species differentiation. the incorporation of dna barcoding approaches to taxonomic classification can help resolve some of the problems that originate while relying on purely morphological taxonomy. sociobiology an international journal on social insects c grando1,2, nd amon2,6, sj clough3, n guo3, w wei3, p azevedo1,4, mm lópez-uribe2, mi zucchi5 article history edited by eduardo almeida, usp, brazil received 10 may 2018 initial acceptance 02 july 2018 final acceptance 26 august 2018 publication date 11 october 2018 keywords cytochrome oxidase i, bayesian phylogeny, bees, barcoding gap. corresponding author maria i. zucchi, agência paulista de tecnologia dos agronegócios pólo regional centro sul rodovia sp 127, km 30 cep 13400-970, piracicaba-sp, brasil. e-mail: mizucchi@apta.sp.gov.br of biological diversity (vodă et al., 2015). evolutionary lineages which have a recent divergence time but cannot be differentiated morphologically are the result of a process called cryptic speciation (bickford et al., 2007). on the other hand, species may exhibit polymorphism, a phenomenon whereby conspecific individuals display marked variation in morphological characters. if these polymorphic characters are used for species identification, the same evolutionary unit may be assigned to different species, leading to an overestimation of biodiversity (sigovini et al., 2016). when cryptic or polymorphic species are present in a community, 1 institute of biology, university of campinas, campinas, são paulo, brazil 2 department of entomology, center for pollinator research, pennsylvania state university, university park, usa 3 department of crop science, university of illinois at urbana-champaign, urbana, usa 4 centro de estudos em insetos sociais, universidade estadual paulista “julio de mesquita filho”, rio claro, são paulo, brazil 5 agência paulista de tecnologia dos agronegócios, pólo regional centro-sul, piracicaba, são paulo, brazil 6 department of entomology, university of wisconsin-madison, madison, usa research article bees c grando et al. – color polymorphism in the eucerine bee melissodes nigroaenea646 species misidentification may be frequent, especially if morphological taxonomic keys are solely used to differentiate species (padial et al., 2010). even though morphological characters are widely used for species identification, some of the phenotypic traits that are easiest to be recognized visually often fail to delimit species accurately (arribas et al., 2012; lecocq et al., 2015). for example, several studies on insects, including flies (grella et al., 2015), butterflies (garzón-orduña et al., 2018), and bees (carolan et al., 2012; ferrari & melo, 2014; huang et al., 2015) have shown that traits like color can be hard to interpret as diagnostic characters for species identification. color traits may exhibit high rates of polymorphism due to environmental influences or genetic differences at a single single or multiple loci (miyanaga et al., 1999; uy et al., 2009; white & kemp, 2016; hines et al., 2017). still, color is commonly used as a trait in taxonomic keys for many organisms, including bees, birds, and dragonflies among many others (michener, 2007; mckay et al., 2014; rodrílguez et al., 2015). the advent of molecular biology has led to the development of diagnostic techniques that complement morphological species identification and may ameliorate some of the challenges with morphological taxonomic delimitation. dna barcoding is a common molecular technique for species determination and involves the amplification of a highly variable region of the genome through pcr (polymerase chain reaction) (hajibabaei et al., 2007). in animals, the mitochondrial gene cytochrome c oxidase subunit i (coi) is the marker of choice for dna barcoding because it can be easily amplified with universal primers across a large number of taxa (hebert et al., 2003), and is likely involved in the process of speciation (hill, 2016). coi fragments are aligned and genetic distances between individuals are calculated and used as diagnostic boundaries for species. low genetic distance among individuals indicates low divergence suggesting the presence of genetic diversity within one evolutionary lineage. on the other hand, large genetic distances among individuals may indicate the presence of independent evolutionary lineages; usually identified through a barcoding gap (hebert et al., 2004; čandek & kuntner, 2015). despite its known intraspecific variability, color has been a widely used character among bee taxonomists for species descriptions and dichotomous keys (michener, 2007; colla et al., 2011; ascher & pickering, 2018). some authors argue that because coloration has a well-characterized genetic basis, integument or hair color can be safely used as a morphological character for species identification (nemésio, 2009). however, color polymorphism is well characterized in several bee groups including bumble bees (hines & williams, 2012; lecocq et al., 2015; duennes et al., 2017), orchid bees (ferrari & melo, 2014) and sweat bees (miyanaga et al., 1999). a challenging taxonomic identification involving color characters occurs within the eucerine bees of melissodes genus. a taxonomic description of urban (1973) organized into a dichotomous key the important morphological characters for species differentiation of south american melissodes bees, besides had discussed the many taxonomic classifications proposed by previous authors. according to the author, there are two sympatric melissodes species in brazil, melissodes nigroaenea (smith) and melissodes sexcincta (lepeletier), whose females can be differentiated mainly by the fullness of the bristle band on the second tergum (tii), but also by the color of mesosoma hair, and the presence of maxillary subapical yellow spots. in a recent survey of bee pollinators in cotton (gossypium hirsutum) farmlands from three regions in brazil, we collected a large number of female bees that were identified as m. nigroaenea based in the dichotomous key of urban (1973). our specimens showed two distinct colormorphs of mesosoma (yellow and black) in all populations, and few individuals showed variation in the bristle band in tii that was not expected for m. nigroaenea. however, the proper identification of the individuals with these unexpected patterns of bristle bands was not supported by the use of the mesosoma color character, what brought us some concern about dealing or not with more than one evolutionary unit in our bee sampling. using the dna barcoding approach and the phylogenetic species concept, in which we consider a species as the smallest aggregation of sexual population or assexual lineages that presents a unique combination of character states (wheeler & platnick, 2000), we tested alternative hypotheses about the interpretation of color variation in m. nigroaenea for species identification. specifically, we investigate whether different color morphs we sampled correspond to one or more evolutionary units by quantifying the genetic distance of these individuals using the coi barcoding region. our hypothesis is that, if significant differences in coi sequences underpin variation in m. nigroaenea coloration, individuals identified as m. nigroaenea actually comprise two evolutionarily distinct lineages of bees. if instead we observe nominal genetic variation between individuals with distinct colormorphotypes, our alternative hypothesis is to attribute this variation to color polymorphism. material and methods species description m. nigroaenea (fig 1) is a eucerine bee (apidae: eucerini) distributed along the east coast of brazil, uruguay and argentina, and interiorly in paraguay and brazil (fig 2). in brazil, m. nigroaenea is the most abundant native pollinator of cotton (cardoso et al., 2007), a fiber crop which generated $ 74.1 billion usd to the brazilian economy (abrapa, 2017). this native bee is a highly efficient pollinator of cotton plants, transferring a larger number of pollen grains between flowers and generating more seeds per boll when compared with honey bees (apis mellifera l.) (cardoso et al., 2007). sociobiology 65(4): 645-653 (october, 2018) special issue 647 according to urban (1973), females of m. nigroaenea might be identified for having a predominant black mesosoma with some variations of yellow, presenting the second bristle brands in the tii incomplete, and the presence of maxillary subapical yellow spots. urban (1973) also describes m. sexcincta as a sympatric species, whose females have a predominant yellow fig 1. map of the distribution of melissodes nigroaenea (black dots), and sampling sites of this study (pink squares). data source: urban (1973), discover life database (ascher & pickering, 2018). fig 2. female specimens of melissodes nigroaenea collected in mato grosso, brazil, representing both mesosoma color morphs (a-b), and showing a continuum in the completeness of tii medial bristle bands (c-f). mesosoma, showing two complete bristle bands in the tii. we used these characters to identify 32 female specimens as m. nigroaenea, although we observed few individuals with variation in the bristle bands of tii (almost absent, almost complete and complete) that did not correspond with the description of this species (incomplete) collection sites we collected bees in cotton fields between february and may 2016 in three different localities in brazil (fig 2, pink squares). collections took place at one site within the campinas municipality (são paulo), one site in the sorriso municipality (mato grosso), and five sites at campo novo do parecis municipality (mato grosso) (table 1). in 50 x 50 meter plots within cotton fields, we sampled for bees using two methods: a) netting bees visiting cotton flowers while walking along four transects in each plot, and b) setting up four blue vane traps filled with propylene glycol. we stored specimens in tubes full of 70% ethanol at 10 °c after collection. c grando et al. – color polymorphism in the eucerine bee melissodes nigroaenea648 table 1. collection sites, coordinates, color of mesosoma and genbank accession number of the specimens used in this study. * individuals with variation in the second bristle band of tii not expected for m. nigroaenea (almost absent, few or complete) collection site coordinate specimen color of mesosoma genbank accession campo novo do parecis fazenda chapada -13.7873667; -57.5666667 cncha_1590 yellow mh667931 fazenda chapada -13.7873667; -57.5666667 cncha_bv575 black mh667928 fazenda gaúcha -13.7526781; -57.6417054 cngau_1351 black mh667923 fazenda gaúcha -13.7526781; -57.6417054 cngau_1362 black mh667934 fazenda gaúcha -13.7526781; -57.6417054 cngau_1363 black mh667924 fazenda gaúcha -13.7526781; -57.6417054 cngau_1380 black mh667925 fazenda gaúcha -13.7526781; -57.6417054 cngau_1387 yellow mh667926 fazenda gaúcha -13.7526781; -57.6417054 cngau_1389 black mh667927 fazenda graciosa -13.4755948; -57.9402887 cngra_1011 black mh667906 fazenda graciosa -13.4755948; -57.9402887 cngra_1013 black mh667909 fazenda graciosa -13.4755948; -57.9402887 cngra_1018 black mh667903 fazenda graciosa -13.4755948; -57.9402887 cngra_1049 black mh667912 fazenda graciosa -13.4755948; -57.9402887 cngra_1061 yellow mh667904 fazenda graciosa -13.4755948; -57.9402887 cngra_1066 yellow mh667907 fazenda graciosa -13.4755948; -57.9402887 cngra_1072 yellow mh667910 fazenda ramada -13.6845448; -57.8804554 cnram_1106 yellow mh667913 fazenda ramada -13.6845448; -57.8804554 cnram_1111 yellow mh667914 fazenda ramada -13.6845448; -57.8804554 cnram_1112 black mh667915 fazenda ramada -13.6845448; -57.8804554 cnram_1122* black mh667916 fazenda ramada -13.6845448; -57.8804554 cnram_1140 yellow mh667917 fazenda ramada -13.6845448; -57.8804554 cnram_1152 black mh667918 fazenda ramada -13.6845448; -57.8804554 cnram_1169 black mh667919 fazenda ramada -13.6845448; -57.8804554 cnram_1184* black mh667920 fazenda ramada -13.6845448; -57.8804554 cnram_1191* yellow mh667921 fazenda ramada -13.6845448; -57.8804554 cnram_1211 yellow mh667922 fazenda santa teresinha -13.5521948; -57.8435054 cnste_925 black mh667905 fazenda santa teresinha -13.5521948; -57.8435054 cnste_994 black mh667908 fazenda santa teresinha -13.5521948; -57.8435054 cnste_997 yellow mh667911 sorriso fazenda primavera -12.8651948; -55.8820887 sopp1_bv182* yellow mh667929 fazenda primavera -12.8651948; -55.8820887 sopp1_bv183 black mh667932 campinas fazenda santa elisa -22.8662783; -47.0770554 cpsel_1626 yellow mh667933 fazenda santa elisa -22.8662783; -47.0770554 cpsel_bv588 black mh667930 taxonomic identification bees were identified by danielle c. de luna between may and september 2016 in the department of biology at faculty of philosophy, sciences and literature of ribeirão preto at university of são paulo (ffclrp/usp). identifications were based on the taxonomic key to the genus melissodes developed by urban (1973), and the reference specimens from the entomological collection “prof. j. m. f. camargo” (rpsp), university of são paulo. dna extraction and quantification we followed a protocol developed by (boyce et al., 1989) in order to extract genomic dna from specimens. in a 1.5 ml tube containing 4 legs of a specimen and 4 metallic beads, we added a solution containing 700 ml ctab 2% buffer [2% (w/v) ctab; 1.4 m nacl, 20 mm edta ph 8.00; 100 mm tris-hcl ph 8.00; 1% (w/v) 40,000 pvp; ph 8.3], 2 µl of beta-mercaptoethanol and 10 µl of proteinase k at 65 °c. samples were then macerated and incubated at 65 °c for sociobiology 65(4): 645-653 (october, 2018) special issue 649 60 min. we then added 600 µl chloroform: isoamyl alcohol (24:1) to the tubes and centrifuged at 14,000 rpm for 10 min to separate the aqueous and organic phases, and the upper aqueous phase transferred to a new 1.5 ml tube. after adding 400 µl of cold isopropanol, the samples were incubated at -20 °c for 60 min to precipitate the dna, followed by a centrifugation at 14,000 rpm for 10 min to collect the dna pellet, which was subsequently washed with 70% and 95% etoh. the pellet was then air dried for approximately 2 hours. finally, 44 µl ultrapure water and 1 µl of pure rnase (20 mg/ µl) were added, and the samples incubated for 3 hours at 37 °c. to approximate the quantify of dna in the samples, aloquots were run on a 1% (w/v) agarose gel and band intensities compared to known amounts in bands of a 100 bp dna ladder. dna barcoding we performed test pcr amplifications with two combinations of coi primers, jerry-pat (simon et al., 1994) and hco-lco (folmer et al., 1994). we made a 25 μl reaction mix containing around 20 ng of genomic dna, 2 μl of forward primer (5 pmol/μl), 2 μl of reverse primer (5 pmol/μl), 0.5 μl of dntp mix (10 mm), 2.5 μl of 10× standard taq reaction buffer, 2 μl of mgcl2 (25 mm), and 1 u of taq dna polymerase (fermentas, thermo fisher scientific, pittsburgh, pennsylvania, usa). pcr programs for both primer pairs consisted of an initial denaturation step of three minutes at 94 ºc, followed by 40 cycles of 30 seconds at 94 ºc, 30 seconds for annealing at the specific temperature for each primer (tested with 48, 50, 51 and 52 ºc) and 1 min at 72 ºc, followed by a final extension step of 10 min at 72 ºc. we used a 1% (w/v) agarose gel to corroborate the amplification products. we used a qiagen pcr purification kit to clean the pcr products and used bidirectional sanger sequencing at the university of illinois at urbana-champaign core sequencing facility (usa). alignment of sequencing data raw sequences were edited and aligned in the software geneious (kearse et al., 2012). in addition to the 32 coi sequences from our study, we downloaded six coi sequences of other melissodes species from genbank and added them to the alignment (table 2). to select the optimal model of nucleotide evolution for phylogenetic reconstruction, we used the python program partitionfinder2 (lanfear et al., 2017). we ran mrbayes (ronquist & huelsenbeck, 2003) using the best nucleotide model to estimate the phylogenetic relationships among individuals using a bayesian inference. we adopted a sample size of 107 generations and a burn-in of 25% of the trees, sampling every 1,000 generations with markov chain monte carlo (mcmc) methods, which generated a consensus tree with high branch support. our alignment file was also read into mega7 (kumar et al., 2016) to estimate the pairwise genetic p-distance between our samples using 10,000 bootstraps. results we identified 13 yellow and 19 black individuals among the 32 samples we collected. even though urban (1973) described the mesosoma color variation as a continuum, we found two discrete coloration (yellow and black, fig 2a-b) among the individuals we collected in all populations from the 3 geographic regions. we also found variation in four individuals in the completeness of the second bristle band in the tii as described by urban (1973). two individuals presented almost no bristal bands (cnram_1122, fig 2c, and cnram_1184), one presented few bristle bands in the lateral (sopp1_bv182. fig 2d), and one individual presented complete bristle bands (cnram_1191, fig 2f). most individuals presented incomplete bristle bands, like cngau_1387 (fig 2e). pcr reactions with the primer pair lco 1490/hco 2198 were optimized for our specimens at an annealing temperature of 50° c, producing a single distinct band at 710 bp. the primer pair jerry-pat failed to amplify from our specimens. we identified gtr+g as the best nucleotide substitution model for our data. the consensus tree obtained from mrbayes shows that the coi fragments group both color morphs into one highly supported clade (posterior probability = 1) sister to melissodes ecuadorius (bertoni and schrottky), a species distributed in ecuador, peru, and chile. we found that the average pairwise genetic p-distance within yellow individuals was 0.56 % (± 0.20 %), between black individuals was 0.86 % (± 0.26 %) while between yellow and black individuals was 0.73% (± 0.23%) (table 3). this level of genetic differentiation in mitochondrial dna does not support the presence of two evolutionarily independent units between the different mesosoma colors. the average pairwise genetic distances among m. nigroaenea and other species were between 8% and 11.5% (table 3). discussion both pairwise genetic distances and the bayesian phylogenetic reconstruction supported that m. nigroaenea individuals with yellow and black coloration in the mesosoma belong to the same evolutionary unit (fig 3a-b; table 3). pairwise genetic distances within and between the two color-morphs were below 3%, considered a threshold for taxa reference genbank accession number melissodes agilis dorchin et al. (2018) mg251042.1 melissodes desponsa dorchin et al. (2018) mg251044.1 melissodes druriella hebert et al. (2016) kr801296.1 melissodes ecuadorius packer and ruz (2017) kx820894.1 melissodes illata sheffield et al. (2009) fj582332 melissodes paroselae dorchin et al. (2018) mg251041.1 table 2. taxa, reference paper and genbank accession number of coi sequences of additional melissodes species found in genbank. c grando et al. – color polymorphism in the eucerine bee melissodes nigroaenea650 differentiating species in other insect orders (hebert et al., 2003). these results indicate that the yellow and black color morphs are a polymorphism present in sympatric populations of m. nigroaenea. additionally, we also observed significant variation in the bristle band fullness of tii in four individuals, but the variation in this character was more continue than the observed variation in the color of the mesosoma (fig 3c-f). taxonomic delimitations in the tribe eucerini can be challenging primarily due to the morphological intergradation — meaning presence of intermediate characters (terrell, 1963) — among taxa (michener, 2007; dorchin et al., 2018). in the melissodes species description proposed by urban (1973), the color of the mesosoma is an important character to differentiate female m. nigroaenea (black) from m. sexcincta (yellow). however, this same author recognizes the presence of variation in the mesosoma coloration in m. nigroaenea, suggesting possible intergradation in this diagnostic character. another taxonomic character mentioned by urban (1973) to differentiate females of both species was the bristle bands in the tii, in which we observed a continuous variation not expected for m. nigroaenea, from the almost absence to the fullness of the second bristle band in tii. we identified four variants for this character, of which three are from fazenda ramada (campo novo do parecis region), and one from fazenda primavera (sorriso region), that do not constitute a separated evolutionary unit based in our analysis. although we might suggest the morphological intergradation for this character, further studies with more individuals from different regions will support if the bristle bands in tii could also be considered a polymorphic character for m. nigroaenea. fig 3. consensus phylogenetic tree based on bayesian inference generated from coi sequences for the 32 yellow and black morphs studied, plus six additional coi sequences for other melissodes genus found in genbank. the numbers over the branches correspond to posterior probability values. the scale in the bottom (0.03) means the number of substitutions per site. sociobiology 65(4): 645-653 (october, 2018) special issue 651 urban (1973) also made a description about the dentiform male genitalia for melissodes species identification. according to the author, males of m. nigroaenea presents the genitalia with a cut near the valve, while m. sexcincta does not present this cut. however, despite of the reliance on male genital characters to confirm the taxonomy in south american melissodes bees, our samples were composed mostly by females, while the few males we found were not presented in all populations. thus, this morphological character was not considered for analyses, but may merit further study, especially in combination with dna barcoding. additional studies using molecular techniques to resolve taxonomic issues within bees have also concluded that color should not be used as a primary character for taxonomic classification. ferrari and melo (2014) found that the integument color of three species of euglossa orchid bees present geographic variation in brazil, with both blue and green colors observed. however, the authors did not find phylogenetic support for the independence of the three euglossa species, nor did they observe a vast enough genetic distance to justify separating both color morphs into different species. another group of bees known for high variability in coloration pattern are the bumble bees. huang et al. (2015) found nine distinct color patterns for female bumble bees in different regions of china, but all color morphs clustered together as a monophyletic group in the bayesian analysis, belonging to bombus koreanus (skorikov). carolan et al. (2012) tested the hypothesis that color patterns could reliably distinguish three bombus in the lucorum complex from one another when compared to coi sequence data, and found that morphological data often resulted in incorrect classification of individual specimens, due to the cryptic nature of the lucorum complex. however, taxonomic characters can be informative to identify species, especially when they are interpreted in conjunction with another type of dataset like dna barcoding (freitas et al., 2018). here, we confirmed a mesosoma color polymorphism in m. nigroaenea, an important pollinator of cotton plantations in brazil. our results suggest that this species exhibits yellow and black individuals in multiple areas across its distribution. sampling a higher number of individuals from more populations and male individuals in future studies will contribute to a better understanding of the ubiquity of this polymorphism in m. nigroaenea, and also to confirm if the bristle bands in the tii might be considered a polymorphic character. in addition, more molecular markers will be necessary to determine whether these two color-morphs are the result of recent incipient speciation. species delimitation studies using museum materials identified as m. sexcincta may assist in clarifying whether this species and m. nigroaenea and are two evolutionary independent entities or should be synonymized. acknowledgements we would like to thank to são paulo research foundation fapesp for funding this research (grant number 2014/50738-9), and the coordination for the improvement of higher education personnel capes (programa biologia computacional, brazil grant number 1572813, and us grant number 060/2017-08) for the post-doctoral scholarship of c. grando at university of campinas and penn state university, respectively. we thank to danielle c. de luna for the precious help with bee collections and the identifications, as well as prof. eduardo a. b. de almeida for the use of his lab and the access to the entomological collection “prof. j. m. f. camargo” (rpsp), university of são paulo, during the identification. we are also grateful to instituto matogrossense do algodão (imamt) for making possible our bee collections in cotton farms of the state of mato grosso (brazil). last, but extremely important, we would like to express our admiration and gratitude for all the contributions of prof. danuncia urban for the taxonomy of bees in brazil. authors’ contributions cg, mmlu and miz conceived and designed the study; cg conducted the field sampling; cg, sc, ng and ww generated the molecular data; pa photographed bee individuals; cg and nda conducted the data analyses; cg, nda, miz, and mmlu drafted the manuscript. all authors gave final approval of the manuscript for publication. references arribas, p., abellán, p., velasco, j., bilton, d.t., millán, a. & sánchez-fernández, d. (2012). evaluating drivers of vulnerability to climate change: a guide for insect conservation strategies. global change biology, 18: 2135–2146. doi: 10.11 11/j.1365-2486.2012.02691.x melissodes nigroaenea black melissodes nigroaenea yellow melissodes ecuadorius melissodes illata melissodes desponsa melissodes druriella melissodes paroselae melissodes agilis melissodes nigroaenea black 0.86 % (± 0.26 %)* 0.73 % (± 0.23 %) 8.03 % (± 1.18 %) 9.74 % (± 1.31 %) 11.00 % (± 1.34 %) 11.00 % (± 1.39 %) 11.30 % (± 1.43 %) 11.50 % (± 1.42 %) melissodes nigroaenea yellow 0.73 % (± 0.23 %) 0.56 % (± 0.20 %)* 8.01 % (± 1.19 %) 9.59 % (± 1.31 %) 10.87 % (± 1.35 %) 10.90 % (± 1.39 %) 11.29 % (± 1.44 %) 11.50 % (± 1.43 %) table 3. average pairwise genetic p-distance between each color morph and other melissodes species, with their respective average standard error estimates. 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(2000). the phylogenetic species concept (sensu wheeler and platnick). in q.d. wheeler & r. meier (eds.), species concepts and phylogenetic theory: a debate. new york: columbia university press. _hlk517793547 doi: 10.13102/sociobiology.v60i2.198-203sociobiology 60(2): 198-203 (2013) antipredator behavior produced by heterosexual and homosexual tandem running in the termite reticulitermes chinensis (isoptera: rhinotermitidae) g li, x zou, c lei, q huang introduction many termite species generally reproduce by annual dispersal of alates that leave the parent colony and found new colonies from bisexual pairs (bordereau et al., 2002; peppuy et al., 2004). after the mating flight, individuals may exhibit calling behavior and tandem running (hanus et al., 2009; hartke & baer, 2011). finally, pairs of dealates look for a suitable nesting site (hartke & baer, 2011). the other castes and the nest itself can not protect the imagoes while encountering predators during the period from swarming to colony foundation, which is when they are the most vulnerable (de-when they are the most vulnerable (dethey are the most vulnerable (deligne et al., 1981). however, few studies focus on the role of tandem running in reducing predation risk during mating flights of termites. owing to the cryptic nesting habits and short swarming times in subterranean termites, it is very difficult to conduct extensive studies of their pairing behavior and antipreda-pairing behavior and antipreda-ing behavior and antipredaabstract heterosexual and homosexual tandem running can be observed together in the alate pairings in some species of termites. this study examined the effect of heterosexual and homosexual tandem running in the termite reticulitermes chinensis on the predation risk by a predatory ant, leptogenys kitteli. results showed that both heterosexual and homosexual tandem running reduced the predation risk of participants. when a male-male tandem encountered a female, the back male had a significant advantage over the front male in win-over the front male in winthe front male in winning a female. moreover, the back males were significantly heavier than the front males. these results indicated that the predation risk of dealates could be decreased by tandem running through the dilution effect. furthermore, these data suggest that male-male tandem running could induce selection pressure in favor of vigorous males and may play an essential role in indirect sexual selection. sociobiology an international journal on social insects 1 huazhong agricultural university, wuhan, china research article termites article history edited by: evandro n silva, uefs brazil received 10 march 2013 initial acceptance 13 may 2013 final acceptance 24 may 2013 keywords predation risk, tandem running, reticulitermes chinensis, dilution effect, sexual selection corresponding author qiuying huang hubei insect resources utilization and sustainable pest management key laboratory huazhong agricultural university wuhan, china 430070 e-mail: qyhuang2006@mail.hzau.edu.cn tor behavior in the field. instead, researchers mainly focus on laboratory simulations and mathematical models to investi-s and mathematical models to investiand mathematical models to investi-s to investito investigate social behavior in termites (hayashi et al., 2003; huang et al., 2008; kenne et al., 2000; lee et al., 2006; matsuura & kobayashi, 2007; jeon & lee, 2011). matsuura et al. (2002) demonstrated that homosexual tandem running was an antipredator behavior in the japanese subterranean termite, reticulitermes speratus. however, how widespread this antipredator behavior is within the isoptera is still unknown. thus, it is necessary to further study the effect of tandem running on predation risk in other species of termites. the termite r. chinensis is widely distributed in china, including beijing, tianjin, shanxi and the yangtze river drainage basin (wei et al., 2007). this termite species builds nests in the soil and wooden structures, and is an important pest of forest trees and urban buildings (li et al., 2010). however, knowledge about pairing behavior and antipredator behavior in r. chinensis is very limited currently. in this study, we examined the effect of heterosexual and homosexsociobiology 60(2): 198-203 (2013) 199 ual tandem running in r. chinensis on the risk of predation by a sympatric predatory ant, leptogenys kittel, to determine whether dealates of this species might also exhibit antipredator behavior. methods and materials insects the ant leptogenys kitteli was chosen for this study because it is common in the habitat of r. chinensis and has been observed preying on it. on march 30, 2011, we collected a colony of l. kitteli from the decayed stump of a pine, pinus massoniana, in wuhan city, china. the l. kitteli colony was maintained in a plastic box (75×75×60 mm3) which was connected by a plastic tube to a clear plastic case (75×75×60 mm3) used as a foraging arena where the ants were fed on live r. chinensis workers every 3 days. on april 20, 2011, alates of r. chinensis were collected together with nest wood in wuhan city just before the swarming season. they were housed in a plastic nest box (670×480×410 mm3) covered with nylon mesh and were held at 16 °c in a darkroom for 7 days to con-a darkroom for 7 days to condarkroom for 7 days to control the time of flight. just before starting the experiments, the plastic nest box was transferred to a room with artificial light at 30 °c so that alates emerged from the nest wood and began to fly (matsuura & nishida, 2001; matsuura et al., 2002). the alates or dealates were anesthetized with co2 and separated by sex using confi guration of the caudal sternites under a ste� sex using configuration of the caudal sternites under a ste�sternites under a ste-under a stereoscope (roonwal, 1975). then, the same-sex imagoes were put together in petri dishes containing moist filter paper until they shed their wings. each dealate was used only once, i.e. no dealate was re-used either within or between experiments. effect of unit type on post-encounter risk in this experiment, there were five treatments: single male, single female, male-male, female-female and malefemale. each unit type was selected randomly and placed in the foraging arena. after a single dealate was put in the forag-a single dealate was put in the forag-single dealate was put in the foraging arena, the entrance to the foraging arena was opened and then shut after an ant entered. once the ant encountered the single dealate, the capturing situations were recorded. when each pair of dealates began tandem running, the entrance was opened and then shut after one ant entered the foraging arena. because an ant could only capture one dealate at a time, enant could only capture one dealate at a time, en-ant could only capture one dealate at a time, en-a time, entime, encounters will result in one of the following three situations: (1) the front dealate is captured, (2) the back dealate is captured, or (3) both dealates escape (matsuura et al., 2002). each trial was run until the end of the ant's first attempt to capture a termite. each treatment was replicated 50 times. individual capture rates per predatory attack between single and tandem dealates, and between front and back dealates in tandems were compared. statistical significance was analyzed using fisher's exact probability test (spss inc., 1989–2002). effect of unit type on encounter risk as tandem running increased the size of the prey unit, it is easier for ants to fi nd prey. �he actual change in the en�easier for ants to fi nd prey. �he actual change in the en� for ants to fi nd prey. �he actual change in the en�find prey. �he actual change in the en� prey. the actual change in the encounter risk could not be evaluated in a laboratory experiment, because dealates tend to run along the perimeter of a container as previously described in r. speratus (matsuura et al., 2002). therefore, a mathematical model was needed to estimate the encounter risk of tandem dealates relative to a single dealate. the mathematical model of matsuura et al. (2002) was used to estimate the frequency of encounters (r). the parameters w and l show the body width (the biggest diameter of abdomen) and length of dealates for the r. chinensis, respectively. the parameter s represents the sensory range of an ant (the range between both antennae). effect of volatiles and vision on predator behavior if ants search for prey using visual or volatile cues, it is easier to detect tandem dealates than single dealate. thus, a choice test was tested in a modified t-shaped box. the test is as follows: the apparatus (200×20×20 mm) was connected to the ant nest by a plastic tube. we used glass or a 60-mesh stainless-steel screen to separate a compartment (20×20×20 mm) at both ends of the apparatus. two dealates were put at one end of the apparatus, but there were no dealates at the other end, as a control (fig. 1). the entrance was shut after an ant entered. the ant was allowed to search for prey for 60 s. then, the time spent on the dealates and control sides was recorded. each treatment was replicated 20 times. new white paper was laid in the junction each time to remove the influence of ant trail pheromones. if visual or volatile cues from delalates can attract ants, the ants should spend more time searching on the dealate side than the control side. in addition, we put dealates wrapped in nylon mesh in the foraging arena and observed the reaction of ants, so that we could detect whether ants considered dealates as prey without direct antennal contact. fig. 1. the experimental apparatus used to detect whether ants were attracted to dealates by visual or volatile cues. a: ant nest; b: search area; c: dealate area; d: control area; a: entrance switch; b: glass g li, x zou, c lei, q huang antipredator behavior in the termite reticuliterme chinensis200 plate or a 60-mesh stainless-steel screen. effect of tandem position on pairing opportunity an interesting phenomenon was found in our experiment as described previously in r. speratus (matsuura et al., 2002). when two males met, they would turn around in cirmet, they would turn around in cir-met, they would turn around in circles to compete for the back position, while such phenomenon did not occur in females. an important question was raised, whether the back position has dominance in subsequent pair-dominance in subsequent pair-ominance in subsequent pairing competition. therefore, the following test was performed to examine this possibility. two dealates of the same sex were chosen randomly and were placed in a 90 mm culture dish. a dealate of the opposite sex was chosen randomly and was put into the culture dish after the two dealates of the same sex beafter the two dealates of the same sex be-the two dealates of the same sex be-of the same sex be-began tandem running. there will be three results: (1) the front dealate successfully pairs, (2) the back dealate successfully pairs, or (3) triple tandem. then, the front and back dealates were anesthetized with co2 and weighed. each sex was replicated 20 times. results effect of unit type on post-encounter risk the post-encounter risk of the back dealate in tandem running was significantly lower than that of a single individual (in male-male tandems: p = 0.005; in female-female tandems: p = 0.003; in female-male tandems: p < 0.001, fisher’s exact probability test). furthermore, the post-encounter risk of the front dealate in tandem running was also significantly lower than that of a single individual, except in male-male tandems (in male-male tandems: p = 0.070; in female-female tandems: p = 0.027; in female-male tandems: p = 0.016, fisher’s exact probability test) (table 1). although the front individual is always captured at a higher rate than the one in the back, regardless of sex or pair type, there were no significant differences in the post-encounter risk between the back dealate and the front dealate (in male-male tandems: p = 0.412; in female-female tandems: p = 0.532; in female-male tandems: p = 0.284, fisher’s exact probability test). table 1. comparison of the post-encounter risk in different unit types. data in parentheses is the post-encounter risk relative to single dealates. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. unit type capture rate escape rate single male 0.64 0.36 single female 0.64 0.36 front captured back captured both escape male-male tandem 0.44 (0.69)ns 0.34 (0.53)** 0.22 female-female tandem 0.40 (0.63)* 0.32 (0.50)** 0.28 female-male tandem 0.38 (0.59)* 0.26 (0.41)*** 0.36 effect of unit type on encounter risk the predation risk of tandem dealates relative to single dealates is yielded by multiplying the relative encounter risk and the relative post-encounter risk. the relative encounter risk is r2/r1 = 1.341 (l = 4.92, w = 1.26, s = 5.27 mm) (table 2). the relative predation risk of each position was as follows: male-male tandems, front males: 0.93, back males: 0.71; female-female tandems, front females: 0.84, back females: 0.67; female-male tandems, front females: 0.79, back males: 0.55. because a value of 1 represents equal predation risk between tandem dealates and single dealates (matsuura et al., 2002), these results indicate that the total predation risk was reduced by tandem running relative to single dealates. table 2. body size of dealates and running speed of dealates and ants. † data were the average from 20 dealates and 20 ants. ‡ sensitive width was the interval between the tips of both antennae. § running speed was determined according to the time required to run 20 cm on a white paper at 25 °c. termite alate† predatory ant† male female body width w (mm) 1.220 ± 0.009 1.290 ± 0.005 — body length l (mm) 4.827 ± 0.030 5.014 ± 0.033 — sensitive width s (mm) ‡ — — 5.271 ± 0.072 running speed (mm/s) § 29.048 ± 0.974 32.882 ± 0.641 50.956 ± 3.902 effect of volatiles and vision on predator behavior �he differences were not significant in the residence times (visual: df = 19, t = -0.471, p = 0.643, volatile: df = 19, t = 1.344, p = 0.195, paired t-test) (fig. 2), suggesting that the ants were not attracted to the dealates separated by glass or a steel screen. the supplementary experiment showed that the ants were not interested in dealates wrapped in nylon mesh when they appeared in the foraging arena. these results dem-appeared in the foraging arena. these results demthe foraging arena. these results demonstrate that tactile hunting is primary in l. kitteli. fig. 2. differences in the residence times of ants between arms of the apparatus having two dealates and lacking dealates. error bars represent the standard error. ns, not significant; paired t�test: (■) two dealates, (□) no dealates. effect of tandem position on pairing opportunity when a male-male tandem encountered a female, the back male had a significant advantage in winning the female over the front male (p < 0.001, two-tailed binomial test) (fig. 3). the back male won the female 12 times, while the front male only won 2 times. the “triple-tandems” occurred 6 times. these results clearly showed that back males had the superiority over front males in the pairing competition. this advantage was supported further by the weight results. the front males were significantly lighter than the back males in male-male tandems (df = 19, t = -3.133, p = 0.005, paired t-test) (fig. 4). however, when a female-female tandem encountered a male, there was no significant difference between the front female and the back female in winning the male (p = 0.155, two-tailed binomial test) (fig. 3). the front female paired with the male 11 times, and the back female paired 6 times. the “triple-tandems” occurred 3 times. in addition, there was no significant difference in the weights between the front females and the back females in female-female tandems (df = 19, t = -1.371, p = 0.186, paired t-test) (fig. 4). discussion sociobiology 60(2): 198-203 (2013) 201 running speed (mm/s) § 29.048 ± 0.974 32.882 ± 0.641 50.956 ± 3.902 effect of volatiles and vision on predator behavior �he differences were not significant in the residence times (visual: df = 19, t = -0.471, p = 0.643, volatile: df = 19, t = 1.344, p = 0.195, paired t-test) (fig. 2), suggesting that the ants were not attracted to the dealates separated by glass or a steel screen. the supplementary experiment showed that the ants were not interested in dealates wrapped in nylon mesh when they appeared in the foraging arena. these results dem-appeared in the foraging arena. these results demthe foraging arena. these results demonstrate that tactile hunting is primary in l. kitteli. fig. 2. differences in the residence times of ants between arms of the apparatus having two dealates and lacking dealates. error bars represent the standard error. ns, not significant; paired t�test: (■) two dealates, (□) no dealates. effect of tandem position on pairing opportunity when a male-male tandem encountered a female, the back male had a significant advantage in winning the female over the front male (p < 0.001, two-tailed binomial test) (fig. 3). the back male won the female 12 times, while the front male only won 2 times. the “triple-tandems” occurred 6 times. these results clearly showed that back males had the superiority over front males in the pairing competition. this advantage was supported further by the weight results. the front males were significantly lighter than the back males in male-male tandems (df = 19, t = -3.133, p = 0.005, paired t-test) (fig. 4). however, when a female-female tandem encountered a male, there was no significant difference between the front female and the back female in winning the male (p = 0.155, two-tailed binomial test) (fig. 3). the front female paired with the male 11 times, and the back female paired 6 times. the “triple-tandems” occurred 3 times. in addition, there was no significant difference in the weights between the front females and the back females in female-female tandems (df = 19, t = -1.371, p = 0.186, paired t-test) (fig. 4). discussion the dilution effect could reduce an individual’s risk of predation in group-forming animals (hamilton, 1971; hall et al., 2009; marcoux, 2011; rodgers et al., 2011). our reour re-our results suggested that colonies of r. chinensis experienced the same dilution effect as described previously in r. speratus (matsuura et al., 2002). the alates of r. chinensis need to land and shed their wings in order to search for a mate, so it is easy for them to encounter potential predators during the period from swarming to colony foundation (bordereau & pasteels, 2011). our results suggest that the probability of tandem dealates as the victim of a predator was reduced com-the victim of a predator was reduced com-victim of a predator was reduced com-a predator was reduced com-predator was reduced comcom-compared to single dealates. this can be explained by the fact that one ant cannot capture two dealates at the same time. in other words, the escape probability of an individual to each predation attack should be increased through tandem running, because one ant can only capture one dealate in each type of tandem (matsuura et al., 2002). in summary, the phenomenon fig. 3. pairing success between front and back dealates in male-male tandems and female�female tandems. �wo�tailed binomial test: (□) front, (■) back, (□) triple�tandems. fig. 4. differences in body weight between front and back dealates in male-male and female-female tandems. error bars represent the standard error. ns, not significant; **, p < 0.01; paired t�test: (□) front, (■) back. g li, x zou, c lei, q huang antipredator behavior in the termite reticuliterme chinensis202 of homosexual tandem before dealates encounter the opposite sex is an adaptive strategy to minimize predation risk. more-strategy to minimize predation risk. moreto minimize predation risk. moreover, heterosexual tandem running had also reduced the predation risk of two participants. this result was different from r. speratus in which the predation risk of the following male was larger than that of a single male in female-male tandems (matsuura et al., 2002). the ant l. kitteli did not utilize visual or volatile cues to prey on the dealates in this study, indicating the encounter risk could not be increased by either the enlarged visual image or volatiles. therefore, we could use the mathematic model of matsuura et al. (2002) to estimate the encounter risk of r. chinensis by l. kittel. this model estimates that tandem running could reduce the total predation risk to an individual. that is to say, a dealate in a tandem run is safer than a single dealate. reduced predation risk cannot be explained by reduced encounter rates or reduced post-encounter success of the ant against tandems. average speed is the same between tandems and single dealates, the ant is apparently not utilizing termite pheromones as localization cues, and the escape rate is the same for both prey types (singles vs. tandems). rather, ants can only handle one item of prey at a time, with the result that post-encounter predation risk is spread over more individuals. this suggests that the longer “trains” of dealates observed in the field are an extension of this adaptive strategy to reduce individual predation risk, although the limits to this tactic have not been explored. when two males met, they turned round and round to compete for the back position. the likely interpretation of this phenomenon is that the post-encounter risk of the back dealate in male�male tandem running was significantly lower than that of a single individual. in fact, our results suggested that the ants less often captured back males than front males. in addition, the back males were significantly heavier than the front males in male-male tandems, and the back males have the superiority over front males in the pairing competi-pairing competicompetition. these results suggested that male-male tandem running would induce selection pressure in favor of heavy vigorous males. tandem running may therefor play a role in indirect sexual selection, if such "dominant" males contribute more to reproductive investment by both direct nutrient transfer and labor in the colony foundation stage (shellman-reeve 1990). in female-female tandem, we found that there were no differfemale-female tandem, we found that there were no differences in the number of pairing success and vulnerability between the positions in r. chinensis, consistent with the results in r. speratus (matsuura et al., 2002). in r. speratus, the cooperative colony foundation by female pairs was considered as one of the reasons that females do not compete for males as aggressively as males compete for females (matsuura et al., 2004; matsuura et al., 2002; matsuura & kobayashi, 2007). however, we need to further investigate whether there also is the cooperative colony foundation by female pairs in r. chinensis. antipredator effects of heterosexual and homosexual tandem running in termites has been tested only in r. speratus and r. chinensis until now, although homosexual tandem runs have been seen in many termite species. austin et al. (2004) found that r. speratus and r. chinensis were close relatives within the genus reticulitermes. thus, whether the antipredator effect of tandem running exists in only these two species, throughout reticulitermes or possibly beyond requires extensive studies in reticulitermes and in closely related genera such as coptotermes and heterotermes. our work extends the previous discovery of antipredator behavior in termites, however, the extent to which this is actually antipredator behavior needs to be measured under more realistic conditions. moreover, the evolutionary significance of termite homosexual tandem runs remains further investigations. also interesting is that the ants in this experiment were relatively smaller compared to the termites than in matsuura's experiment, judging from the relative values of l, w, and s, suggesting position dependent predation risk may be generalizable to many termite-ant pairings regardless of the relative sizes of the interactants, although it still remains to be tested with ants that are much smaller than the termites they are preying on. acknowledgments we thank dr. s.j. tan for identifying the ant species. we also thank drs. x.p. wang and w.w. zheng for revising the manuscript. we also thank the anonymous reviewers for providing valuable comments on earlier drafts of this manuscript. this work was supported by the national natural science foundation of china (31000978) and the international foundation for science (d/4768-1). references austin, j.w., szalanski, a.l. & cabrera, b.j. 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(2004). speciesspecific sex pheromones secreted from new sexual glands in two sympatric fungus-growing termites from northern vietnam, macrotermes annandalei and m. barneyi. insectes soc., 51: 91-98. doi: 10.1007/s00040-003-0718-9 rodgers, g.m., ward, j.r., askwith, b. & morrell, l.j. (2011). balancing the dilution and oddity effects: decisions depend on body size. plos one, 6: e14819. doi: 10.1371/ journal.pone.0014819 roonwal, m.l. (1975). sex ratio and sexual dimorphism in termites. j. sci. ind. res. india., 34: 402-416. shellman-reeve, j.s. (1990). dynamics of biparental care in the dampwood termite, zootermopsis nevadensis (hagen): response to nitrogen availability. behav. ecol. sociobiol., 26: 389-397. doi: 10.1007/bf00170895 wei, j.q., mo, j.c., wang, x.j. & mao, w.g. (2007). biology and ecology of reticulitermes chinensis (isoptera: rhinotermitidae) in china. sociobiology, 50: 553-555. * �he first two authors contributed equally to this work. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.3662sociobiology 67(1): 65-73 (march, 2020) introduction the atlantic forest biome is distributed along a vast extension of the brazilian coast. its territory covers tropical and subtropical regions and is considered one of the 35 hotspots (myers et al., 2000; mittermeier et al, 2004). nowadays, this biome only counts on approximately 10% of its original distribution area (silva & castelleti, 2003, ribeiro et al., 2009, sloan et al., 2014). deforestation has been causing environmental fragmentation that increases the number of habitat remnants and the distance between abstract endemic species of atlantic forest central corridor may have evolved under adverse climate conditions, and their response to modern climate change is unclear. the aim of this study is to evaluate the response of the endemic and endangered ant species dinoponera lucida to biotics and abiotics factors based on three scales: ecological factors inside forest fragment, physical attributes of landscape and climatic variables of the assessed region. data collection took place in a representative selection of forest fragments in the region where the species is distributed in. pitfalls were used to collect samples and to assure the presence and absence of the species in the site. we also checked the abundance of food resources and applied a hemispherical photography technique to measure shading inside the fragment, in loco. landscape attributes data and climatic predictors were collected through geoprocessing techniques. all predictors were associated with binary “presence” and “absence” data based applied in logistic models. there was no significant response to environmental aspects within the fragment or to landscape, but there was strong and peculiar response to climatic variables such as temperature and rainfall. accordingly, d. lucida presents a restricted realized niche, a feature shared among many endangered species that can disappear due to displacement and to habitat loss caused by climate and environmental changes. this species presents all the criteria necessary to be considered as rare, which is a controversial subject with political implications for espírito santo state, and makes d. lucida the ideal target for urgent conservation strategies. sociobiology an international journal on social insects ss simon1, jh schoereder1, mc teixeira2 article history edited by jacques delabie, uesc, brazil received 02 august 2018 initial acceptance 23 may 2019 final acceptance 12 february 2020 publication date 18 april 2020 keywords endemism; climate change; niche breadth; extinction risk; rare species. corresponding author sabrina simon faculdade de saúde pública da usp av. doutor arnaldo, 715, cerqueira cesar cep: 01246-904 são paulo-sp, brasil. e-mail: ssimon@usp.br them, given the decrease in habitat-remnant sizes. therefore, species adapted to conditions inside the forest are exposed to intense population decline (bender et al., 1998; ewers & didham, 2007; ewers & banks-leite, 2013). one of the most threatened portions of this tropical forest is the atlantic forest central corridor (afcc), a region under preservation priority that covers the south of bahia state and the whole espírito santo state. it is responsible for the north-south connection of the atlantic forest and has a remarkable biological representation for the biome (brasil, 2015). 1 federal university of viçosa, viçosa, brazil 2 federal university of espírito santo, são mateus, brazil research article ants environmental response of dinoponera lucida emery 1901 (hymenoptera: formicidae), an endemic threatened species of the atlantic forest central corridor ss simon, jh schoereder, mc teixeira – environmental response of dinoponera lucida66 among endemic species in the afcc, one finds the ant dinoponera lucida emery (1901), which belongs to the in-danger category in the red book of the brazilian fauna threatened with extinction (icmbio, 2018). this species presents low dispersion capacity and aggregated distribution pattern within fragments, nests with few individuals and distribution limited to southern bahia, eastern minas gerais and espírito santo states (campiolo et al, 2015; icmbio, 2018, see fig 1). this region faces intense deforestation pressure and landscape fragmentation, but d. lucida response to such environmental pressures remains poorly understood. assumingly, d. lucida is quite sensitive to the internal conditions of the fragment, to landscape structure and to regional climate, mainly to temperature and rainfall, given its biological features and degree of threat in their habitat. therefore, the aim of this study was to evaluate d. lucida response to environmental components, to landscape attributes and to climatic variables at local and regional scales. our hypotheses were that the presence and abundance of d. lucida would be negatively influenced by the shortage of biomass as food supply and low shading within the forest fragment, small area of the fragment, irregular shape and high levels of isolation between fragments in the landscape, and extreme climatic conditions in their distribution region. material and methods the spatial-ecological analysis was applied to assess d. lucida response to the environment at (a) microsite, (b) localities and (c) mesoscale levels, according to leibold et al. (2004) recommendations. its response to factors inside the fragment corresponds to the local level (= microsite), and it was assessed in fragments in espírito santo state (fig 1). the response to the landscape as a set of fragments corresponds to the landscape level (= patches or localities), which, together with the regional response analysis (= mesoscale), covered the southern bahia and espírito santo states (fig 1), where the response to climatic variables was also assessed. aligned pitfalls in 450 meters long linear transects emerged as an appropriate method to collect d. lucida representatives in the fragment, since it is expected that the ant does not have a homogeneous distribution, but presents aggregate patterns (peixoto et al., 2010). each transect had 10 pitfalls, which were separated 50 meters from each other. the number of transects used in each fragment was proportional to the logarithm of the fragment’s area, which ranged from one to four transects. in total, there were 300 pitfalls distributed in 30 transects, on 10 forest fragments and conservation unities listed in appendix 1-a. the transects were plotted between december and march, 2013. fig 1. data sampling points of this study and current geographic distribution of d. lucida, according to icmbio (2018). sociobiology 67(1): 65-73 (march, 2020) 67 sampling was conducted under the permissions icmbio nº 35895-1 and nº 35947-1. the collected specimens were identified by the authors based on lenhart et al. (2013), and were added to the collection at the laboratory of communities ecology of federal university of viçosa. microsites: species representatives, animal biomass (various invertebrates collected within the pitfalls, surrogate of food resources measure) and shading were recorded in 286 points distributed throughout the 10 fragments mentioned above (appendix 1-a), since, from the 300 placed pitfalls, 14 were removed or lost. pitfalls stayed on the ground for 48 hours to collect animal biomass. the contents found in the pitfalls were screened, dried in oven at 45 ºc for 24 hours and weighed in gehaka analytical scale (model ag200). for shading measurement, hemispherical pictures of the canopy were taken with fisheye 0.45 mm x 28 mm lens attached to a nikon coolpix 4500 4mpx digital camera vertically positioned on the ground. they were analyzed in the gap light analyzer software version 2.0 in order to calculate canopy opening, which was used as an indicator of environmental conditions in the forest. data about the presence and absence of the species were analyzed based on logistic regression models using quasibinomial distribution in r environment. data were logtransformed in the model, because biomass data were too close to zero. this calculation was conducted in order to conform the data to the linearity of the independent variable and to the log odds (saupe et al., 2015). landscape: d. lucida response to the physical attributes of the landscape (area, fragment shape and connectivity) was evaluated in 30 forest remnants in bahia and espírito santo states (list in appendix 1-b), where the presence or absence of the species were previously verified. those physical attributes resulted from data provided by the sos mata atlântica foundation, at ratio 1:50,000 in the shapefile format, made available by the laboratory of image processing and geoprocessing (lapig, 2015). data analysis was performed in the free version (2.18) of the qgis software in the sirgas projection system 2000 utm for zone 24. shape calculations of each fragment were based on the pantton index adapted to metric units: if = p/[200.(π.at)0.5], wherein p is the perimeter in meters and at is the total area of the fragment in hectares. this index measures shape as a circularity deviation, so that the if value is close to 1.0 when the polygon is almost round-shaped, which increases depending on the complexity of its boundaries (laurance & yensen, 1991). the connectivity analysis was performed through the visual classification of all remnants in a 10 km radius from the edge of each of the 30 assessed fragments. the area occupied by forest remnants around the fragment was calculated as the fraction of the total surrounding area within the 10 km radius. the connectivity index (ic) expresses the proportion of forested area around the central remnant, which is used as connectivity indicator: zero (0) indicates complete isolation and one (1) indicates total connectivity (simon et al., 2018, in print). the final model was based on logistic regression; the binary data of d. lucida presence in the fragments were related to landscape attributes by taking into consideration the quasibinomial distribution. the variable area was logtransformed, given its high variability. regional scale: data from 19 climatic variables concerning temperature and rainfall were collected to analyze population response to climate (listed in appendix 2). they were expressed in the raster format at 30-second resolution, released and made available at worldclim (version 2.0) (fick & hijmans, 2017). climatic predictors of each point were remotely collected in the qgis software (version 2.18) by using the sirgas 2000 projection system. a previous selection of climatic variables was made through principal components analysis (pca) associated with the biological plausibility criteria and exclusion of variables in the correlation matrix, since climatic variables usually present strong collinearity. pca was conducted in the factominer and factoextra packages for r. the selected predictors were applied to the multivariate logistic regression model with quasibinomial distribution. after the significant predictors were selected, the student’s t-test was used to compare the means of each predictor in the presence and absence responses. in all the analysis levels, presence records were assigned as 1 and absence records were assigned as 0. climatic predictor p-value presence (1) (n = 154) absence (0) (n = 96) test t student temperature annual range 5.76e-07 :14.47 σx̅: 0.466 s2: 33.4539 : 14.91 σx̅: 0.782 s2: 58.6843 1 < 0 p-value = 2.855e-06 annual mean temperature 0.000135 23.32 σx̅: 1.103 s2: 187.232 : 22.59 σx̅: 1.846 s2: 327.0748 1 > 0 p-value = 6.672e-04 precipitation of driest month 0.004738 : 41.48 σx̅: 0.423 s2: 27.5192 : 41.54 σx̅: 0.629 s2: 37.9489 1 = 0 p-value = 0.4697 fig 1. data sampling points of this study and current geographic distribution of d. lucida, according to icmbio (2018). ss simon, jh schoereder, mc teixeira – environmental response of dinoponera lucida68 fig 2. a-c: significant climatic predictors according to presence and absence data. fig 2c results d. lucida representatives at the three levels were found to be: 48% of pitfalls inside the assessed fragments (local scale), 83% of the 30 assessed landscape fragments (landscape scale) and in 65% of the 250 analyzed points, at regional level. the presence of the species was verified in different vegetation formations of the atlantic forest, including the dense ombrophilous forest, and its transition to restinga, as well as in the semidecidual seasonal forest. ant populations did not show significant response to environmental components inside the fragment (biomass (p-value = 0.1959) and shading (p-value = 0.1069)) or to landscape attributes (area (p-value = 0.336), shape (p-value = 0.655) and fragment connectivity (p-value = 0.827)). however, based on the regional scale, d. lucida recorded significant responses to three climatic predictors. the assessed populations showed remarkable sensitivity to temperature and to minimum rainfall conditions. they mainly settled in regions presenting higher annual temperatures and lower thermal seasonality. the variance of the response stands out, since presence records flow close to the mean (low variance), whereas absence data show broader dispersed behavior (high variance) (table 1; fig 2). discussion local and landscape scales understanding the responses and behaviors of species at different scales is essential to model and predict their future (leibold et al., 2004). endemic species are mainly threatened by environmental changes, therefore, knowing their distribution and responses to microclimatic factors within the limits of their habitat is essential to the development of biologically valuable conservation interventions (hannah et al., 2014). d. lucida responses to ecological factors inside the fragment or to landscape structure were not verified in this study. results suggested greater tolerance than the expected for food supply variations, which is probably due to its omnivorous and generalist eating behavior (peixoto et al., 2010). as the atlantic forest is a biome with a substantial variety of vegetal formations, d. lucida did not show response to the shading in the fragment. for ant communities diversity, shading inside the fragment is an overall important predictor, whereas, on the other hand, lack of response from these organisms to landscape structure in the atlantic forest (santos et al., 2006), caatinga (gomes & leal, 2010) and in the amazon (carvalho & vasconcelos, 1999) seems to be common. diversity patterns are observed in studies involving a whole set of ant species (gascon et al., 1999; schoereder et al., 2004; bieber et al., 2006; gomes et al., 2010), but such patterns cannot be seen when a single species is assessed. assumingly, the response of these organisms to landscape is modulated by other, yet unknown, biological factors. fig 2b fig 2a sociobiology 67(1): 65-73 (march, 2020) 69 except for the recent anthropogenic pressure on natural environments, the current landscape configuration results from past events closely related to climate. resende et al. (2010) suggested that d. lucida is adapted to small forest remnants and, therefore, it might not respond to area fragmentation. this outcome may be an evolutionary inheritance of selective fragmentation pressures during the pleistocene, which generated the high genetic and karyotypic diversity of the species (mariano et al., 2004; mariano et al., 2008; santos et al., 2012; simon et al., 2016). the lack of d. lucida direct response to landscape found in this study may indicate that it is not the spatial configuration itself, but the factors derived from it. it should be noted that it may also derive from the study limitations, since the research was conducted in the region where the species is distributed in. even though sampling within fragments provided a balanced proportion of presence and absence data, species representatives were found in most of the fragments and there were only a few records of species absence in the database. it takes substantial effort to assure species absence reliability. presumably, increased absence records would allow detecting responses at regional levels, although it was not verified in this study. in addition, it is important to highlight that, although data sampling has covered a considerable portion of the species current distribution, it may be possible to verify a different response to landscape configurations in the states of bahia or minas gerais, where atlantic forest deforestation rates are still higher when compared to the state of espírito santo (sos mata atlântica, 2018; sos mata atlântica & inpe, 2019). regional scale: climate, niche and habitat ströher et al. (2019) emphasize the importance of climatic response patterns are becoming clearer on the atlantic forest history, even though the underlying mechanisms such as how the niche of each species affects the way they are influenced by the climate and vegetation changes needs to be further investigated. the relation between the species and its environment throughout evolutionary time generates the ecological niche of the species itself. this niche corresponds to the environmental space available to the species, which is limited by environmental factors that determine species distribution in space (whittaker et al., 1973). beyond the biotic components, the niche of d. lucida depends on the combination of at least three climatic predictors, whereas the niche projection in physical space results in a limited region range which presents satisfactory environmental suitability. different from the expected, the annual rainfall volume does not influence d. lucida settlement. rainfall was only significant in the driest month; however, the mean rainfall between sites presenting, or not, the species was the same. the mean itself can mask important biological effects in biological studies, so variance becomes informative of the ecological responses of the species. based on variance, d. lucida gets close to the average to the values of rainfall in the driest month. the species does not tolerate locations drier or wetter than the average during the dry season. species tolerance to minimum rainfall values seems to restrict the species presence to ombrophilous and semidecidual forests, as water stress in the dry season gets more intense as the perennial forest turns deciduous (saiter et al., 2015). in addition, given the species low tolerance to annual temperature variation, its populations prefer to settle in places presenting temperature variation close to the regional average. climatic seasonality seems to be a determining factor for the species settlement in its own region, a fact that contributes to shape the geographic distribution of the species. range of geographic boundaries and suitable habitat availability are important dimensions of the hypervolumetric environmental space of the species, which is more important to face extinction threats than the tolerance of the species to limiting factors and its adaptation skills. the ability of the species to occupy its geographical space by filling its corresponding environmental space may be one of the most important aspects of its resistance to extinction (saupe et al., 2015). this issue is a concern since potential distribution analysis predicts that the occurrence of the species tends to expand to its southern limits in a climate change scenario (campiolo et al, 2015), where espírito santo becomes even more important and responsible for the preservation of this and other species with similar urgencies. rarity, threat and implications the region where d. lucida is currently distributed in, can be further reduced due to habitat loss caused by deforestation processes and also due to climatic and environmental changes (campiolo et al, 2015). the dense ombrophilous and semideciduous seasonal forests are seriously threatened. estimates point towards their intense geographic reduction due to climatic changes, which pose even more risks on highly vulnerable ecosystems (follador, 2016). recently, d. lucida was inadvertently removed from the state of espírito santo list of watched out species for political and economic motivations, while it has evolved from vulnerable (vu) to endangered (en) status in the brazilian national list (icmbio, 2018). however, the aforementioned information shows that it has the profile that matches one of the seven definitions of rare species, since it is locally abundant in a specific and geographically limited habitat (rabinowitz, 1981), presenting a high endogamy rate and consequently low genetic diversity within populations (resende et al, 2010; simon et al, 2016). these conditions are more threatening for social insects because of their reduced population effective size, even though a high number of individuals, suggests great abundance in ecological terms (wilson, 1963; chapman and bourke, 2001). the concept of rarity presented here takes into account small geographic range, narrow habitat specificity and small local ss simon, jh schoereder, mc teixeira – environmental response of dinoponera lucida70 population size (or effective size, for social insects) presented by rabinowitz (1981), and can be applied to all endemic species in the afcc, which have very similar evolutionary backgrounds (carnaval et al., 2009; alvarez-presas et al., 2013), and regions with high levels of endemicity should receive full and unquestionable consideration (fattorini, 2010). the present results provide the basis for future predictions about the impacts of environmental changes on representatives of this species distributed in a very relevant biological portion of the atlantic forest. therefore, the collected data are extremely important for those who plan biodiversity conservation strategies, since preservation actions developed for one specific endemic species can be extrapolated to several species that have evolved under similar environmental conditions. acknowledgements the authors thank benicio simon, filipe pola vargas, jhennifer queiroz curty, jardel pesca, sara soares simon, diego ernani, donias messias soares, nazareth, mamute and many others who, at some level, participated in this study, including “anonymous referees” for their great contribution. they thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for financial support. references alvarez-presas, m., sánchez-gracia, a., carbayo, f., rozas, j. riutort, m. 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(1963). social modifications related to rareness in ant species. evolution, 17: 249-253 sociobiology 67(1): 65-73 (march, 2020) 73 b. 30 forest fragments at (b) landscape and (c) mesoscale level. studied locations latitude longitude private property in são rafael -19.378583 -40.437583 fazenda sayonara private reserve of natural heritage -18.494583 -39.955267 pau a pique private reserve of natural heritage -20.127964 -40.552563 private property in regência -19.633744 -39.881015 duas bocas biological reserve -20.272617 -40.4797 córrego do veado biological reserve -18.3703 -40.141567 linda lais private reserve of natural heritage -19.952467 -40.752517 sooretama biological reserve -19.0446 -40.005583 goytacazes national forest -19.435983 -40.0733 augusto ruschi biological reserve -19.851683 -40.5633 private property in guaratinga -16.598277 -39.919411 private property in itamaraju -16.87 -39.65 private property in prado -17.3 -39.38 private property in serra dos aimorés -17.831675 -40.279922 private property in ibirapuã -17.698916 -40.231192 private property in são antônio do canaã -19.844816 -40.646719 private property in são joão de petrópolis -19.804809 -40.672172 private property in cariacica -20.253568 -40.462747 private property in viana -20.406439 -40.461068 private property in mathilde -20.557557 -40.815533 private property in marechal floriano -20.4 -40.66 private property in domingos martins -20.35 -40.65 goiapaba-açu municipal park -19.913928 -40.473891 david victor farina natural municipal park -19.930836 -40.12966 morro do aricanga natural municipal park -19.821565 -40.331407 private property in mata fria, santa maria de jetibá -19.990993 -40.797359 vale natural reserve -19.153167 -40.019667 santa lúcia biological station -19.97325 -40.528667 são lourenço municipal park -19.928262 -40.608416 embrapa marilândia -19.405004 -40.554187 appendix 2: list of all 19 climate variables evaluated in this study. code variable name bio1 annual mean temperature bio2 mean diurnal range (mean of monthly (max temp min temp)) bio3 isothermality (bio2/bio7) (* 100) bio4 temperature seasonality (standard deviation *100) bio5 max temperature of warmest month bio6 min temperature of coldest month bio7 temperature annual range (bio5-bio6) bio8 mean temperature of wettest quarter bio9 mean temperature of driest quarter bio10 mean temperature of warmest quarter bio11 mean temperature of coldest quarter bio12 annual precipitation bio13 precipitation of wettest month bio14 precipitation of driest month bio15 precipitation seasonality (coefficient of variation) bio16 precipitation of wettest quarter bio17 precipitation of driest quarter bio18 precipitation of warmest quarter bio19 precipitation of coldest quarter appendix 1: list of the sampling locations. a. 10 forest fragments at (a) microsite level. studied locations latitude longitude private property in são rafael -19.378583 -40.437583 fazenda sayonara private reserve of natural heritage -18.494583 -39.955267 pau a pique private reserve of natural heritage -20.127964 -40.552563 private property in regência -19.633744 -39.881015 duas bocas biological reserve -20.272617 -40.4797 córrego do veado biological reserve -18.3703 -40.141567 linda lais private reserve of natural heritage -19.952467 -40.752517 sooretama biological reserve -19.0446 -40.005583 goytacazes national forest -19.435983 -40.0733 augusto ruschi biological reserve -19.851683 -40.5633 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.6071sociobiology 68(2): e6071 (june, 2021) introduction human dependence on natural resources is continuously increasing and aims to meet our immediate needs for food, fiber, water, and shelter. since the industrial revolution, there has been an intensification in land use change and habitat conversion (which we define here as the conversion of natural habitats into anthropogenic land uses, abstract conversion of natural to anthropogenic environments affects biodiversity, and the understanding of these impacts may be improved by assessing how different functional groups respond to such land conversion. we studied land conversion impacts on ant functional groups, as ants are ecologically important and respond well to various environmental changes. we hypothesized that conversion of natural to anthropogenic environments modifies the composition of functional groups, fostering generalist and opportunistic groups over specialist ones, with more responses of this type in tropical than in temperate regions. we recovered 412 papers from isi web of science, of which we selected 17 studies, published between 1993 and 2018, that addressed our study’s question. we assessed whether each functional group responded positively or negatively to conversion of natural habitat into anthropogenic land uses and used monte carlo tests to assess significance. ants were affected by natural habitat conversion into monoculture and polyculture and by the conversion of savannas and of tropical and subtropical forests. land conversion affected six of the 13 functional groups assessed here. in the temperate zone, cryptic species, predators, subordinate camponotini, cold-climate specialists and tropicalclimate specialists were impaired, whereas hot-climate specialists were favored. in the tropics, land conversion negatively impacted fungus-growers and predators. in both climatic zones, several functional groups, mainly those with broad ecological niches, did not respond to land conversion. our results corroborate that land conversion effects vary among ant functional groups and indicate that the ant fauna of temperate ecosystems may be more susceptible than that of tropical regions. sociobiology an international journal on social insects roberta j santos1,4, pavel dodonov2, jacques hubert c delabie3,4 article history edited by eduardo calixto, university of florida, usa received 01 december 2020 initial acceptance 26 january 2021 final acceptance 27 april 2021 publication date 28 june 2021 keywords insects; land use; land cover; disturbance; habitat simplification. corresponding author roberta de jesus santos laboratório de mirmecologia centro de pesquisa do cacau, cepec/ ceplac caixa postal 7, cep: 45600-970 itabuna, bahia, brasil. e-mail: beta.biologia@gmail.com mainly agricultural ones), which has resulted in the global degradation of environmental conditions (foley et al., 2005; millennium ecosystem assessment, 2005; ellis et al., 2013). the effects of such land use change range from altering the structure and functioning of ecosystems to modifying the dynamics of interactions between ecosystems and atmosphere, water bodies and the surrounding lands (vitousek et al., 1997; foley et al., 2005). in addition, land use changes, 1 universidade estadual de santa cruz, programa de pós-graduação em ecologia e conservação da biodiversidade, campus soane nazaré de andrade, ilhéus-ba, brazil 2 universidade federal da bahia, laboratório de ecologia espacial, instituto de biologia, campus de ondina, salvador-ba, brazil 3 universidade estadual de santa cruz, departamento de ciências agrárias e ambientais, campus soane nazaré de andrade, ilhéus-ba, brazil 4 laboratório de mirmecologia, centro de pesquisa do cacau (cepec/ceplac), ilhéus-ba, brazil review effects of habitat conversion on ant functional groups: a global review mailto:beta.biologia@gmail.com roberta j santos, pavel dodonov, jacques hubert c delabie – habitat conversion effects on ant functional groups2 especially the conversion of natural and complex landscapes into anthropized environments, have led to the simplification, loss, and fragmentation of native habitats, contributing to the disappearance of biodiversity and consequently its functions and ecosystem services (pimm & raven, 2000; hansen et al., 2004; haines-young, 2009; almeida et al., 2016). negative impacts caused by habitat conversion and intensification have already been documented for several taxonomic groups, such as birds (donald et al., 2001, rittenhouse et al., 2012), mammals (sauvajot et al., 1998; sotherton, 1998; riffell et al., 2011; seki et al., 2017), insects (vasconcelos, 1999; philpott et al., 2008; winfree et al., 2011), and plants (philpott et al., 2008; meers et al., 2010). thus, assessing the effects of habitat conversion on biological communities helps to understand how environmental changes affect biodiversity, ecosystem functions and ecosystem services (haines-young, 2009; luck et al., 2009; geijzendorffer & roche, 2013). for several taxa, a commonly used functional approach is the classification of species in a community into functional groups (fgs), with functionally similar species being included in the same group (cummins, 1974; cianciaruso et al., 2009; laureto et al., 2015). species classification into fgs can be either a priori, according to a classification based on species characteristics or similarities, or a posteriori, with multivariate analysis techniques (petchey, 2004; calaca & grelle, 2016). the classification into fgs has been widely used in recent studies of biotic communities, including ants (hymenoptera: formicidae) (andersen, 1995; king et al., 1998; delabie et al., 2000; ottonetti et al., 2006; underwood & fisher 2006; crist, 2009, leal et al., 2012; lawes et al., 2017; assis et al., 2018). such groupings are based on taxonomic affinities, morphological patterns, and niche dimensions such as diet, nest location, foraging behavior, and habitat preference or environmental tolerance (andersen, 1990, 1995, 1997; hoffmann & andersen, 2003; silvestre et al., 2003; andersen & majer, 2004; weiser & kaspari, 2006; silva & brandão, 2010; brandão et al., 2012; koch et al., 2019). the classification of ants into fgs is useful in studies of habitat conversion, as such classifications permits to infer how community structure and function are affected by disturbances and environmental changes (andersen & majer, 2004; underwood & fisher, 2006). considering the importance of the ant fauna for the provision of important ecosystem supporting and regulating services, such as nutrient cycling, seed dispersal, population control of other arthropods, and formation and structuring of soil superficial layers (hölldobler & wilson, 1990; folgarait, 1998; del toro et al., 2012), the understanding of habitat conversion effects on different ant fgs is essential to better understand the impacts of environmental changes on biodiversity. since the first sketches of ant classification into fgs, with the greenslade’s (1978) pioneer study in australia, the fgs scheme has been modified and adapted to classify ant communities from other biogeographic regions (majer et al., 2004). one of the best known functional schemes developed from ant communities of australian savannas classifies the groups according to the ants’ relationship with climate, soil, vegetation, and disturbances, and the main fgs are: (i) dominant dolichoderinae; (ii) subordinate camponotini; (iii) generalized myrmicinae; (iv) opportunists; (v) climate specialists; (vi) cryptic species and (vii) specialist predators (see table 1) (andersen, 1995, 1997, 2000; king et al., 1998; hoffmann & andersen, 2003; andersen & majer, 2004; andersen et al., 2007). another important functional classification, based on the nutrition and bioecological aspects of the ants, was developed for the neotropical region, and resulted from information in previous studies (delabie et al., 2000; silvestre et al., 2003; brandão et al., 2009, 2012; silva & brandão, 2010). this classification includes ant guilds or groups organized around ant nutritional requirements and/or feeding habits: (i) generalist predators; (ii) specialists; (iii) arboreal predator ants; (iv) generalists; (v) fungus growers; (vi) legionary ants; (vii) dominant arboreal ants associated with carbohydrate-rich resources or domatia; (viii) pollen-feeding arboreal ants, and; (ix) subterranean ants (see table 1 and brandão et al., 2009, 2012). in general, habitat disturbance and conversion affect ant communities directly, by reducing the availability of resources and removing colonies, and indirectly, through changes in habitat structure, nesting site availability, temperature, and humidity (andersen, 2000; philpott et al., 2010). in addition, habitat changes promoted by agriculture, logging, grazing, mining, etc., may lead to the exclusion of sensitive groups and the advent of groups tolerant to disturbances, which can replace or compete with those present before the disturbance (andersen, 1995, 2000; hoffmann & andersen, 2003; andersen & majer, 2004; ottonetti et al., 2006; crist, 2009; leal et al., 2012; parui et al., 2015; assis et al., 2018; amaral et al., 2019). highly specialized fgs, such as cryptic species, tend to be sensitive to changes in habitat due to their preference for forested environments and nesting and foraging in soil and litter (hoffmann & andersen, 2003). on the other hand, other groups of ants, often called opportunists, are ruderal with low competitiveness and wider environmental tolerance and may be predominant in disturbed environments (andersen, 1997; king et al., 1998; andersen & majer, 2004). in addition, the effects of habitat conversion may affect ant fgs in different ways throughout the globe, as aspects associated with species richness, such as habitat heterogeneity and physical environment, differ between tropical and temperate zones. habitat heterogeneity is related to the variety of resources in the environment, with a greater availability of resources permitting a better partitioning of niche space and greater specialization (macathur & macathur, 1961, pianka, 1966; brown & lomolino, 1998). therefore, it is possible that conversion of native habitats to anthropogenic land use sociobiology 68(2): e6071 (june, 2021) 3 promotes greater loss of the original habitat heterogeneity in more structurally diverse and complex areas, impacting specialized fgs associated with native habitats, and may therefore be more significant in the tropics than in temperate regions. in order to assess the effects of habitat conversion on different ant fgs in tropical and temperate zones of the globe, we conducted a literature review to: i) identify how habitat conversion (conversion of native habitats to anthropogenic functional groups of australian ants group subdivision characteristics dominant dolichoderinae species of the dolichoderinae subfamily, with dominant behavior and preference for hot and open habitats. subordinate camponotini ants tend to be behaviourally submissive to dominant dolichoderines, and have large body size, nocturnal foraging, and/or arboreal habits. generalized myrmicinae group with subdominant behavior to dominant dolichoderinae from a global perspective, and with high tolerance to disturbance. opportunists group that is dominant in disturbed environments in which other groups lose their relative importance. climate specialists hot-climate specialists climate specialists that have specific habitat tolerance, with preferences related to temperature and humidity. cold-climate specialists tropical-climate specialists cryptic species species that can hide in their habitat and occur preferentially in forests and forage within soil and litter. specialist predators ants specialized in diet and sensitive to disturbance. functional groups of neotropical region group subdivision characteristics generalist predators epigaeic generalists predators ants with large and medium-sized predators that forage on the soil surface or above the litter. hypogaeic generalist predators medium and small-sized ants that forage within the leaf litter. specialists (with specialized morphology and biology) predation in mass and/or nomadism ants with hunting strategy in groups of workers in columns/ mass in predation or nomadic behavior in predation of certain preys. dacetini predators possess specialized jaws with morphology and mechanism different from other mymicinae. arboreal predator ants species which forage in vegetation and prey on a range of arthropods. generalists generalized myrmicines ants with a wide ecological niche.generalized formicines, dolichoderines and some myrmicines small-sized hypogaeic generalist foragers fungus growers leaf cutters use live or dead plant substrate for rearing their symbiotic fungus. litter-nesting fungus growers cryptobiotic attina ants living in the leaf litter and using a variety of substrates (e.g. leaves, flowers, fruits, seeds, feces, lichen, and carcasses of arthropods) to rear the symbiotic fungus or yeast. legionary ants (army ants) ants with behavioral and reproductive syndrome, with nomadism, dicthadiiform reproductive females (wingless queens) and mandatory collective foraging. dominant arboreal ants associated with carbohydrate-rich resources or domatia ants picking up liquid food resources (as nectar produced by floral or extrafloral nectaries, carbohydrate-rich exudates sucking hemipterans, and exudates of some lepidoptera larvae) or species living in association with myrmecophytes that present specialized structures aiming ant nesting (such as domatia) and provide food (nectaries and mullerian or food bodies). pollen-feeding arboreal ants components of the neotropical cephalotini tribe that remove anemophilous pollen deposited on the vegetation surface, an important item in the diet of these ants. subterranean ants ants that live in the deeper layers of soil. *andersen, 1995, 1997, 2000; king et al., 1998; hoffmann & andersen, 2003; andersen & majer, 2004; andersen et al., 2007; **brandão et al., 2009, 2012. table 1. details of functional groups scheme of australian ants based on their relationships to the climate, soil, vegetation, and disturbance*, and functional classification for ants of neotropical region based on their nutritional and bioecological aspects**. roberta j santos, pavel dodonov, jacques hubert c delabie – habitat conversion effects on ant functional groups4 land uses, especially agricultural areas) affects ant fgs; ii) assess whether these effects can be observed in different types of native habitats and anthropogenic land uses; and iii) verify how the different ant fgs respond to habitat conversion in the tropical and temperate zones. we hypothesized that: a) fgs in general are negatively affected by the conversion of natural to anthropogenic environments; b) opportunistic and generalist ants and ants with a preference for disturbed sites respond positively to habitat conversion; c) specialist ants respond negatively to such changes; and d) in tropical zones, more fgs of ants would have negative responses as compared to groups in temperate climatic zones. methods literature search this study addresses the effects of converting natural to anthropic environments on ant fgs based on a literature review and statistical significance tests (fig 1). the studies were identified through a comprehensive search (last updated in july 2018) in the isi web of science database, using the following terms: “(ants or ant or formicidae) and (land cover or land use) and (functional)’ in topic, ‘doctype=all document types, language=all languages’, from all databases”. the research resulted in 412 published studies. 412 records identified through database searching 412 records screened 93 full-text articles assessed for eligibility 17 studies included in review title and abstract revealed not appropriate 319 records excluded articles outside the inclusion criteria 76 full-text articles excluded screening elegibility included identification fig 1. the flow diagram showing an overview of the study selection process according to the prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines. adapted from moher et al. (2009). selection criteria after the first period dedicated to literature search, we selected studies that evaluated directly or indirectly, explicitly or implicitly, the effects of habitat conversion (natural to anthropogenic) on ant fgs and that sampled ant fauna in areas with native vegetation (control) and in converted areas. we understand conversion as the integral transformation of a natural environment into an anthropogenic environment (coppin et al., 2004), excluding uses such as selective logging, which only partially alter the natural environment, but including agroforestry. titles and abstracts were carefully examined to determine whether the paper met the criteria for inclusion in the review. then, the following criteria were used to select the studies for full reading: a) we included only studies on native vegetation that was completely converted to human activities for economic purposes and maintained for such purposes throughout the study; b) we excluded studies on fire ecology (burned vs. unburned areas), studies comparing nongrazed vs. grazed areas or studies comparing different logging managements in native areas; c) we excluded converted areas that were in natural regeneration, rehabilitation or succession; and d) we only included studies that included functional classification of species and figures or tables that permitted to compare the response of ant fgs between native vegetation (control) and converted areas. a study carried out in a deactivated mining region was excluded because it was the only study with this type of land use among the selected studies. thus, in our final dataset we had comparisons of natural areas with areas converted to monoculture, polyculture, pasture, and agroforestry. we did not perform a meta-analysis because many published studies did not provide information such as mean and standard deviation or correlation or regression coefficients, which are necessary to calculate effect sizes (borenstein et al., 2009). thus, we devised a new simulationbased approach, based on the general recommendations for monte carlo statistical tests (manly, 2007). as most of the studies reported responses in the absence of formal statistical significance tests, we first defined the type of response through sociobiology 68(2): e6071 (june, 2021) 5 visual analysis of figures and tables. our assessments of the responses of different fgs in any particular study are therefore necessarily qualitative. for each study, we assessed whether the response of fgs to habitat conversion was positive, negative or neutral by comparing the occurrence of different fg in native vegetation (control) and in converted areas. when the mean values reported for each fg in the two environments did not differ or differed slightly (e.g. difference in relative presence was less than 4%; difference in mean less than 0.25; or overlapping points in the scatter plot for fgs in native and anthropogenic habitat), we considered the effect as neutral. otherwise, we considered habitat conversion to have positive effects when the values were greater in the converted environment and negative effects when they were greater in the natural habitat. this approach is similar to that used by hoffmann & andersen (2003). due to the small number of studies that we included in the analysis according to the inclusion criteria above, it was not possible to characterize richness, frequency and abundance separately; we therefore combined these measures into a single response variable (table 3). thus, a positive effect may indicate a positive outcome on abundance, frequency and/or richness of a functional group, without differentiating between these measures. study characteristics our final selection included 17 papers published between 1993 and 2018, corresponding to studies carried out in several continental regions present in the tropical climatic zone [africa (2), asia (2), oceania (2), and south america (3)] and temperate zone [europe (1), north america (1), and oceania (6)]. the selected studies employed 11 methods of ant sampling: pitfall traps (15), winkler for leaf litter samples (3), baits (2), hand collection (2), baited arboreal pitfall traps (1), baited pitfall traps (1), baited subterranean traps (baited eppendorf’s tubes) (1), soil monolith method (1), quadrat sampling (insect vacuum and mouth aspirator) (1), sweep netting and foliage shaking (1), and tullgren funnels with leaf litter and surface soil samples (1) (table 3) (for a general review of sampling ants methods, see delabie et al., 2021). these methods were used for ant sampling in different strata (arboreal, leaf litter, soil, and subterranean). the anthropic land uses included in the studies were agroforestry (e.g. cocoa plantations with native woody plant or mixture of different crop plants), monoculture (e.g. coffee, sugarcane, eucalyptus plantation), pasture (e.g. alien grasses, grazing or managed areas), and polyculture [including crop rotation, mixed-crop fields, cultivated fields (cereals), and farmland]. group variations in nomenclature of some functional groups that occur in the studies arboreal arboreal ants, arboreal nesting dominant, territorially dominant arboreal species, non-dominant arboreal species army ants cryptic species dominant dolichoderinae fungus-growers attina, fungus-growing, fungivore/surface generalists omnivore, omnivore/surface generalized myrmicinae opportunists predators other predators, predator/surface, predator/litter, specialized predators, specialist predators, generalist predators, large, solitary predators subordinate camponotini cold-climate specialists hot-climate specialists tropical-climate specialists table 2. functional groups and variations in nomenclature of some functional groups present in the studies which met the selection criteria of this review. the classification of ants into fgs of these papers was according to functional schemes such as those of andersen (1990, 1995, 1997), delabie et al. (2000), silvestre et al. (2003), and weiser & kaspari (2006) (table 3 and see appendix 1). the classifications used in the selected articles are based on a particular functional grouping as previously mentioned and, in some cases, the authors of these articles made modifications and/or used literature with relevant information to group ants into ecological groups. we recognize the variety and specificity of the functional classifications of the set of studies selected in this review and, therefore, we made a broader and more general classification allowing us to analyze the extracted data. most papers (17) classified ants into fgs and only two classified ants into functional guilds based on feeding and foraging guilds (appendix 1). 13 fgs were used for the whole study while some fgs with differences in nomenclature were combined into a single group to facilitate data interpretation (table 2). roberta j santos, pavel dodonov, jacques hubert c delabie – habitat conversion effects on ant functional groups6 r ef er en ce n um be r of in de pe nd en t ca se s tu di es (i .e ., ob se rv at io n) c on ti ne nt h ab ita t t yp e an d la nd u se sa m pl in g m et ho d sa m pl ed st ra tu m m et ri cs u se d r ef er en ce s us ed fo r fu nc tio na l c la ss ifi ca tio n a ss is e t a l., 2 00 8 3 so ut h a m er ic a tr op ic al a nd s ub tro pi ca l ra in fo re st v s. m on oc ul tu re tr op ic al a nd s ub tro pi ca l ra in fo re st v s. p as tu re pi tf al l t ra ps so il r el at iv e pr es en ce (% ) d el ab ie e t a l., 2 00 0 l aw es e t a l., 2 01 7 1 o ce an ia tr op ic al a nd s ub tro pi ca l ra in fo re st v s. p as tu re pi tfa ll tra ps / b ai te d su bt er ra ne an tra ps (b ai te d e pp en do rf t ub es )/ b ai te d a rb or ea l p itf al l t ra ps / l ea f lit te r s am pl es w in kl er so il, a rb or ea l, su bt er ra ne an an d le af li tte r fr eq ue nc y of o cc ur re nc e a nd er se n, 1 99 5 pa ch ec o et a l., 2 01 7 2 so ut h a m er ic a sa va nn a vs . p ol yc ul tu re b ai te d pi tf al l t ra ps a nd b ai te d su bt er ra ne an tr ap s (c on ve nt io na l pi tf al l t ra ps ) so il an d su bt er ra ne an lo gtra ns fo rm ed m ea n nu m be rs o f a nt s pe r t ra p si lv es tr e et a l., 2 00 3 sa ad e t a l., 2 01 7 2 so ut h a m er ic a tr op ic al a nd s ub tro pi ca l ra in fo re st v s. m on oc ul tu re pi tfa ll tra ps , b er le se (s oi l s am pl es ) an d tu llg re n fu nn el s (s oi l s am pl es ) so il an d su bt er ra ne an to ta l s pe ci es ri ch ne ss w ei se r & k as pa ri, 2 00 6 l u et a l., 2 01 6 1 a si a tr op ic al a nd s ub tro pi ca l ra in fo re st v s. a gr of or es try pi tfa ll tra ps / s w ee p ne tti ng a nd fo lia ge s ha ki ng so il an d ar bo re al m ea n fu nc tio na l g ro up re la tiv e co nt rib ut io n (% ) a nd er se n, 1 99 0, 1 99 5 pa ru i e t a l., 2 01 5 2 a si a tr op ic al d ry fo re st v s. m on oc ul tu re pi tf al l t ra ps so il r el at iv e ab un da nc e (% ) a nd er se n, 1 99 5 k ua te e t a l., 2 01 5 1 a fr ic a tr op ic al a nd s ub tro pi ca l ra in fo re st v s. p ol yc ul tu re pi tf al l t ra ps / q ua dr at s am pl in g (i ns ec t v ac uu m a nd m ou th as pi ra to r) / b ai t so il an d le af li tte r r el at iv e oc cu rr en ce (% ) d el ab ie e t a l., 2 00 0; a nd er se n, 2 01 0 k on e et a l., 2 01 2 4 a fr ic a tr op ic al a nd s ub tr op ic al ra in fo re st v s. a gr of or es tr y l ea f l itt er s am pl es w in kl er / pi tfa ll tra ps / m on ol ith m et ho d/ h an d co lle ct io n l ea f l itt er , s oi l an d su bt er ra ne an r el at iv e ab un da nc e lé vi eu x, 1 98 3; m aj er , 1 98 3; a nd er se n, 1 99 5, 19 97 ; k in g et a l., 1 99 8; h of fm an n et a l., 2 00 0; r ea d & a nd er se n, 2 00 0; a nd er se n & m aj er , 20 04 ; d ej ea n et a l., 2 00 7; b ol to n & f is he r, 20 08 fr an kl in , 2 01 2 12 n or th a m er ic a sc ru b, h ea th a nd sh ru bl an ds v s. p as tu re pi tf al l t ra ps so il r el at iv e ab un da nc e a nd er se n, 1 99 5, 1 99 7 h ou se e t a l., 2 01 2 12 o ce an ia g ra ss la nd v s. p as tu re g ra ss la nd v s. p ol yc ul tu re w oo dl an d vs . p as tu re w oo dl an d vs . p ol yc ul tu re pi tf al l t ra ps so il pe rc en ta ge o cc ur re nc e (% ) g re en sl ad e, 1 97 8; a nd er se n, 1 99 7; b ro w n, 2 00 0 g ol la n et a l., 2 01 1 2 o ce an ia w oo dl an d vs . p as tu re pi tf al l t ra ps so il r el at iv e ab un da nc e a nd er se n, 2 00 1 y at es & a nd re w , 2 01 1 2 o ce an ia g ra ss la nd v s. m on oc ul tu re w oo dl an d vs . m on oc ul tu re pi tf al l t ra ps so il n um be r o f a nt s pe ci es a nd er se n, 1 99 5 n ak am ur a et a l., 2 00 7 2 o ce an ia t ro pi ca l a nd s ub tr op ic al ra in fo re st v s. p as tu re pi tfa ll tra ps , t ul lg re n fu nn el s (l ea f lit te r a nd s ur fa ce s oi l s am pl es ) so il an d le af lit te r m ea n fr eq ue nc y a nd er se n, 2 00 0 sc hn el l e t a l., 2 00 3 2 o ce an ia w oo dl an d vs . m on oc ul tu re w oo dl an d vs . p as tu re pi tf al l t ra ps so il r el at iv e ab un da nc e (% ) a nd er se n, 1 99 0 g óm ez e t a l., 2 00 3 4 e ur op e sc ru b, h ea th a nd sh ru bl an ds vs . p as tu re s cr ub , h ea th a nd sh ru bl an ds v s. po ly cu ltu re w oo dl an d vs . p as tu re w oo dl an d vs . p ol yc ul tu re pi tf al l t ra ps so il m ea n ab un da nc e a nd er se n, 1 99 5, 1 99 7a (b es te lm ey er & w ie ns , 1 99 6; a nd er se n, 1 99 7b , 2 00 0; r ea d & a nd er se n, 2 00 0; b ro w n, 2 00 0) k in g et a l., 1 99 8 2 o ce an ia tr op ic al a nd s ub tro pi ca l ra in fo re st v s. p as tu re pi tfa ll tra ps / l ea f l itt er s am pl es w in kl er / b ai ts so il, le af li tte r an d ar bo re al a bu nd an ce a nd er se n, 1 99 5 l ob ry d e b ru yn , 1 99 3 2 o ce an ia sc ru b, h ea th a nd sh ru bl an ds v s. p ol yc ul tu re w oo dl an d vs . p ol yc ul tu re pi tf al l t ra ps / h an d co lle ct io n so il pe rc en ta ge a bu nd an ce (% ) g re en sl ad e & g re en sl ad e, 1 98 4; a nd er se n, 19 86 , 1 98 7 t ab le 3 . d et ai ls o f t he a rt ic le s se le ct ed fo r t hi s re vi ew . sociobiology 68(2): e6071 (june, 2021) 7 most studies had more than a single land use or sampled in contrasting landscapes (i.e., different locations) or different years or sampled in different strata and/or with different traps, as well as having more than a single type of native vegetation. thus, when a study showed more than a single combination of native vegetation type versus converted environment, each combination was considered as a separate data set, i.e., independent case studies (hereafter observations), so that some papers corresponded to more than a single case study. studies with paired comparisons in different climatic zones were excluded. therefore, our review summed a total of 56 observations (table 3) for the analyses. statistical analysis due to the nature of our data, assuming values of -1, 0, and 1 (for negative, neutral, and positive responses, respectively – see below), we chose to use a monte carlo approach to calculate significance by comparing our observed results to simulations of the null hypothesis. monte carlo method works by comparing a test statistic with a large number of random samples generated under a given model, which often represents the null hypothesis (manly, 2007). one advantage of this test is its flexibility in defining the statistics and the model used for the simulation, permitting it to be adapted for specific study questions (manly, 2007). thus, we developed monte carlo test specifically to address our study questions and to test our null hypotheses. as we classified each response as negative, neutral, or positive, our statistical null hypothesis was that these values (-1, 0, +1) were randomly assigned to each response. we thus developed a simulation (monte carlo) model specifically to test this null hypothesis. we used this new monte carlo test to test whether the responses of ant fgs are significantly positive or negative (i.e. deviate significantly from what would be expected under our null hypothesis) and how these responses vary among the types of native vegetation, types of conversion, fgs, and climatic zone. due to sampling size limitation, we considered observations from the same study as independent. we coded each response (observation) as -1 (negative response, i.e., decreased fg richness or abundance in converted environment), 0 (neutral, i.e., no difference between natural and converted environments), or +1 (positive, i.e., increased fg richness or abundance in the converted environment). we then performed analyses separately for each 1) type of converted environment, 2) type of native vegetation, 3) climatic zone, 4) fg, and 5) fg separated by climatic zone (temperate and tropical); for analyses 1 to 3 we grouped all fgs due to insufficient sampling size for separate analyses. in each analysis, we calculated the average response for each level of the explanatory variable by summing the responses (coded as -1, 0, and 1) and dividing this sum by the number of responses assessed. thus, this index varies from -1 to 1. a value near -1 means that group most responses were negative, whereas a value close to 0 can mean that most response were neutral or that positive and negative responses were equally common and a value close to 1 means that most responses were positive. we then simulated the distribution of this index under the null model to assess whether the average responses differ significantly from zero. we are unaware of previous studies using exactly this type of simulation but, considering the flexibility of monte carlo test to adapt to specific research questions and nonstandard situations (manly, 2007), we believe that this approach is valid. the null model used for these simulations was that positive, negative, and neutral responses were all equally probable, i.e., that a study could have observed a negative (-1), neutral (0), or positive (1) response for a give fg with the same probability. thus, in each simulation, we randomly assigned a value of -1, 0, or 1 to each response (observation), simulating the null hypothesis that the responses were random, and calculated the average response with the randomized data. we repeated this procedure 9,999 times, thus obtaining a distribution of average responses under the null model, and calculated significance (p-value) as the proportion of times that the simulated absolute value was greater than or equal to the real absolute value; the real data were included as one of the possible results of the simulation (manly, 2007). following a traditional line of null hypothesis significance testing, we rejected the null hypothesis for p ≤0.05. all analyses were performed in r (r core team, 2018) and the scripts used are available as supplementary material 2 and at https://github.com/pdodonov/publications. results when the fgs were combined, the responses were either positive or neutral. thus, monoculture and polyculture had negative effects (p<0.007), whereas agroforestry and pasture did not have statistically significant effects (p ≥0.07) (table 4). the only habitats in which habitat conversion effects were consistently negative (p<0.007) were savanna and tropical and subtropical rainforests, and habitat conversion did not have statistically significant effects for other types of native habitats (p≥0.09). when combining the vegetation types, negative effects were observed both in the temperate and tropical zones (p<0.01). concerning the fgs, when combining the climate zones, six groups (cryptic species, fungus-growers, predators, subordinate camponotini, coldand tropical-climate specialists), responded negatively (p<0.02) to habitat conversion, whereas the other groups did not respond (p≥0.08) (table 5). the coldand tropical-climate specialists, as well as predators, cryptic species, and subordinate camponotini, responded negatively (p<0.02) in the temperate zone too. in the tropical zone, however, only the fungus-growers and predators were negatively affected (p<0.008) by habitat conversion, with the roberta j santos, pavel dodonov, jacques hubert c delabie – habitat conversion effects on ant functional groups8 other groups having neutral responses (p≥0.07). the only significant positive effect (p=0.04) of habitat conversion was observed for hot-climate specialists in the temperate zone. finally, arboreal ants, army ants, dominant dolichoderinae, generalists, generalized myrmicinae, and opportunists were not affected (p ≥0.07) by habitat conversion. disturbed habitat number of observation number of studies number of observations considering the fgs response significance agroforestry 5 2 30 -0.2 0.4324 monoculture 9 5 49 -0.3111 0.0057 pasture 29 9 149 -0.119 0.0702 polyculture 13 5 75 -0.2667 0.006 native habitat number of observation number of studies number of observations considering the fgs response significance grassland 7 2 35 -0.2 0.1809 savanna 2 1 12 -0.6667 0.007 scrub, heath and shrublands 15 3 68 -0.0441 0.7154 tropical and subtropical rainforest 16 8 89 -0.2969 0.0025 tropical dry forest 2 1 16 -0.2857 0.1653 woodland 14 6 83 -0.1471 0.0925 table 4. average response values of ant functional groups and significance for disturbed habitat and native habitat. significant results (p < 0.05) are in bold. discussion negative effects habitat conversion to monocultures and polycultures were observed, as well as a lack of effect of conversion to agroforestry and pastures. these changes were observed in both the temperate and the tropical zones, with more fgs affected in temperate ecosystems. in general, the more specialized fgs (e.g. predators and cryptic species) responded negatively whereas more generalist groups did not respond to the habitat conversion. these results are partially consistent with our hypotheses and highlight the sensitivity of more specialized fgs to land use changes, especially in the temperate zone. conversion to monoculture and polyculture can thus affect both the taxonomic and functional ant diversity (philpott et al., 2008; fayle et al., 2010; liu et al., 2016; groc et al., 2017; saad et al., 2017; rivera-pedroza et al., 2019). this may be due to the intensive management of some of these land uses, in which, in addition to removing vegetation cover and soil preparation, the use of fertilizers and pesticides impacts the ant fauna (lobry de bruyn, 1999; matlock & de la cruz, 2003; steinbauer & peveling, 2011; queiroz et al., 2012; nickele et al., 2013). additionally, the loss of diversity and simplification of vegetation structure that results of these processes modifies important habitat characteristics for ants, such as microhabitat structure, supply of nesting sites, temperature, availability/access to resources, as well as competitive interactions (andersen, 1995, 2000; hoffmann & andersen, 2003; philpott & foster, 2005; armbrecht et al., 2006; pacheco et al., 2009; amaral et al., 2019). in turn, agroforestry had a neutral effect, possibly due to land uses such as shade cocoa and coffee plantations that maintain a fraction of the native vegetation and have microclimates similar or close to those of natural vegetation, increasing the availability of food, nesting sites, and hiding places to the ants and other arthropods (perfecto et al., 1997; philpott & armbrecht, 2006; delabie et al., 2007; groc et al., 2017; amaral et al., 2019). although pastures did not have significant effects, this result should be treated with caution, as pastures are open areas with sun exposure and cattle trampling, with limited food resources and nesting sites that directly impact on the ant fauna (neves et al., 2012; cantarelli et al., 2015). still, it is possible that the lower intensity of management practices (e.g. the low rate of application of agricultural inputs) compared to monocultures, as well as the growth of shrubs and the occurrence of scattered trees, may result in smaller differences in ant fgs between pastures and native habitats (dias et al., 2008; neves et al., 2012; frizzo & vasconcelos, 2013; queiroz et al., 2017). the negative effects were prominent in the conversion of tropical and subtropical rainforest and savanna. these environments are structurally and compositionally complex, and the reduction of this complexity likely affects the availability of resources and conditions necessary for ants, impacting the abundance and composition of several fgs in these habitats (andersen, 1995, 2000; hoffmann & andersen, 2003). on the contrary, the conversion of tropical dry forest, scrub, heath and shrublands, grassland, and woodland to anthropogenic land uses, show non-significant effects on ant fgs. although this may be due to idiosyncrasies of the evaluated studies, it is possible that environments such as sociobiology 68(2): e6071 (june, 2021) 9 table 5. average response values of ant functional groups for climatic zone, functional group and functional group by climatic zone. significant results (p<0.05) are in bold. functional groups number of observations number of studies response significance arboreal 14 4 -0.3 0.3359 army ants 6 3 -0.5 0.215 cryptic species 17 9 -0.5625 0.0072 dominant dolichoderinae 44 12 -0.1053 0.3879 fungus-growers 7 3 -0.8333 0.0064 generalists 11 4 -0.1429 0.8198 generalized myrmicinae 45 13 0.0588 0.6491 opportunists 49 14 0.2174 0.0871 predators 28 11 -0.6818 0.0001 subordinate camponotini 29 10 -0.6316 0.0001 cold-climate specialists 9 4 -0.6667 0.0202 hot-climate specialists 37 8 0.1923 0.1288 tropical-climate specialists 7 4 -1 0.0013 functional group in temperate zone number of observations number of studies response significance cryptic species 8 3 -0.875 0.0032 dominant dolichoderinae 38 8 -0.1351 0.2829 generalized myrmecinae 38 8 0.0714 0.6256 opportunists 38 8 0.1579 0.2817 predators 6 3 -0.8 0.019 subordinate camponotini 24 6 -0.8571 0.0001 cold-climate specialists 8 3 -0.75 0.0147 hot-climate specialists 34 6 0.2917 0.0451 tropical-climate specialists 4 2 -1 0.0257 functional group in tropical zone number of observations number of studies response significance arboreal 14 4 -0.3 0.3411 army ants 6 3 -0.5 0.2147 cryptic species 9 6 -0.25 0.518 dominant dolichoderinae 6 4 1 0.0739 fungus-growers 7 3 -0.8333 0.0089 generalists 11 4 -0.1429 0.8193 generalized myrmecinae 7 5 0 1 opportunists 11 6 0.5 0.1286 predators 22 8 -0.6471 0.0008 subordinate camponotini 5 4 0 1 cold-climate specialists 1 1 0 1 hot-climate specialists 3 2 -1 0.0753 tropical-climate specialists 3 2 -1 0.0748 climatic zone number of observations number of studies number of observations considering fgs response significance temperate 38 8 198 -0.1386 0.0177 tropical 18 9 105 -0.3125 0.0002 roberta j santos, pavel dodonov, jacques hubert c delabie – habitat conversion effects on ant functional groups10 scrublands, heathlands, and grasslands select predominantly generalist species adapted to more stressful environmental conditions (such as the species of ants of dominant dolichoderinae, generalized myrmicinae, opportunists in relation to the habitat and/or diet generalists), thus being less affected by habitat conversion. on the other hand, a lack of effect could also have been observed if some groups (probably more specialized) were harmed while others (generalists) were favored, generating a substitution of fgs (andersen, 1997, 2000; hoffmann & andersen, 2003; underwood & fisher, 2006). as expected, the conversion of habitat as a whole (without separating between natural and anthropogenic land uses) negatively impacted several fgs of ants, with this effect being more pronounced in temperate habitats. although disturbance impacts tend to be greater in habitats that are structurally complex (arcoverde et al., 2018), it is possible that ant fgs of temperate native habitats are more vulnerable to conversion, as more groups have been affected in this zone (cryptic species, predators, subordinate camponotini, coldand tropical-climate specialists) than in the tropics (fungus-growers and predators). this suggests that the loss of structural heterogeneity and complexity, as well as the typical habitat conditions and resources in anthropogenic land uses, has more significant consequences for ant fgs in temperate native (andersen, 1995, 1997; gómez et al., 2003; underwood & fisher, 2006; gollan et al., 2011; castilloguevara et al., 2019). finally, it is possible that, as tropical areas are generally more species-rich, this high species richness results in greater resistance or resilience to habitat conversion (tilman et al., 2014). the most affected fgs were cryptic species, fungusgrowers, predators, subordinate camponotini, coldand tropical-climate specialists. such effects were also observed when evaluating temperate ecosystems separately, with the exception of hot-climate specialists, which were favored, and fungus-growers, which had significant losses recorded only in the tropics, similarly to the predators. in general, these groups are more specialized than those that were not affected. the response of predators and cryptic species to conversion may be due to their specificities to their own habitat conditions and high specialization of requirements, being especially sensitive to disturbances (andersen & majer, 2004; underwood & fisher, 2006; kone et al., 2012). this could also be the case of subordinate camponotini, which also tend to occur in complex, heterogeneous and shaded habitats with abundant leaf litter for nesting and foraging (andersen, 1997; hoffmann & andersen, 2003; hill et al., 2008; parui et al., 2015; assis et al., 2018). the fgs coldand tropical-climate specialists are composed of ants that have important restrictions in habitat tolerance, such as variation in temperature and humidity (andersen, 1995, 1997; castillo-guevara et al., 2019). in temperate regions, the occurrence of cold-climate specialists in native habitats, such as oak forests, is favored by the microclimate conditions of low temperature and sun exposure (cuautle et al., 2016; castillo-guevara et al., 2019); in addition, ant species richness and overall abundance of this fg may decrease as land use intensifies (gómez et al., 2003). it is possible that the same explanation applied to tropical-climate specialists. the fgs hot-climate specialists, in turn, were favored by habitat conversion in temperate regions. this may be due to increased temperatures in converted areas (see gómez et al., 2003; schnell et al., 2003; gollan et al., 2011). still, some studies observed this group only or mostly in native vegetation (see gómez et al., 2003; yates & andrew, 2011; house et al., 2012). although studies suggest that some fungus-growers (e.g. leaf-cutting ants) are favored by agricultural land uses, deforested areas, edges and disturbed environments (jonkman, 1979; vasconcelos & cherrett, 1995; wirth et al., 2007; siqueira et al., 2017), we found a negative effect on this group in the tropics. fungus-growers ants form a peculiar fg, with habits associated with fungal gardening, for which they use residues from arthropods and carcasses and/or live or dead plant material to grow their symbiotic fungus or yeast (delabie et al., 2000; mehdiabadi & schultz, 2010). forest habitat loss has been related to decreased abundance of this group in argentina (gonzález et al., 2018). the impacts of habitat conversion in fungus-growers fgs probably occurred because some land uses, with intensive management, in addition to homogenizing the habitat, affect strongly the ant fauna through the use of pesticides and soil preparation with effects on the structure of the nests, as well as the availability of the organic substrate for cultivation of the fungus (lobry de bruyn, 1999; queiroz et al., 2012; nickele et al., 2013). our results show that, although some species may be favored by habitat conversion, fungus-growers ants in general are negatively impacted by it. some fgs were not affected by the conversion of native habitats, including dominant dolichoderinae in temperate and tropical ecosystems, which can be explained by their environmental tolerance, being favored mainly by warmer conditions and open habitats (andersen, 1995, 1997; hoffmann & andersen, 2003). similarly, the lack of response of generalized myrmicinae is probably due to their broad environmental tolerance allowing them to predominate in environments with moderate levels of disturbance and shaded habitats (king et al., 1998; hoffmann & andersen, 2003; andersen & majer, 2004). opportunists also had a neutral effect on conversion, as it is a group with broad habitat tolerance that can occur in a range of environments, generally favored in disturbed sites and with low productivity (andersen, 1990, 1995, 1997). arboreal ants, army ants, and cryptic species also had neutral responses (in general or in one of the climatic zones), probably due to the existence of negative and positive responses of these fgs in different observations. the neutral response of arboreal ants is possible because some land uses https://www.linguee.com/english-portuguese/translation/together+with.html sociobiology 68(2): e6071 (june, 2021) 11 still maintain trees that provide nesting sites, a limiting factor for this fg (majer & delabie, 1999; schonberg et al., 2004; kone et al., 2012). army ants, in turn, are harmed in highly disturbed habitats (matsumoto et al., 2009) by the lack of adequate bivouac sites, unfavorable microclimate and few organisms living in leaf litter and fallen logs (roberts et al., 2000; peters et al., 2011). however, the active search for preys associated with the nomadic behavior (gotwald, 1995), the environmental tolerance and broader diet of some army ant species, may allow the occurrence of this fg in anthropic environments (perfecto, 1992; roberts et al., 2000; delabie et al., 2007; o’donnell et al., 2007; matsumoto et al., 2009; schleuning et al., 2011; assis et al., 2018). cryptic species are more diverse and abundant in forest habitats, associated with tree cover (majer et al., 2004; dalle laste et al., 2019), and in our study they were negatively affected by the conversion of habitat except in the tropical zone. it is then possible that in tropical environments some components of this group have plasticity that allows their occurrence in land uses beyond the forest. the neutral response of cold-climate specialists to conversion in the tropical region may not reliable as there were only two observations from a single study, for this group. generalist ants did not respond to habitat conversion, probably because of this group’s ability to use different sites for nesting and food sources (i.e., broad ecological niche), permitting their occurrence in native habitats (kone et al., 2012; pacheco et al., 2017; saad et al., 2017) as well as in anthropogenic land uses (kone et al., 2012; assis et al., 2018), with survival and even dominance in homogeneous and simplified environments (garcía-martínez et al., 2015; assis et al., 2018). in the tropical region, habitat conversion did not affect subordinate camponotini even though this group is usually associated with complex and shaded habitats (parui et al., 2015), suggesting a broader flexibility to their occurrence in this region. our results highlight that habitat conversion plays an important role in the loss of biodiversity. although we have not detected the effect of habitat conversion for all fgs of ants considered in the review, we have evidenced, in general, a negative impact on part of the groups, where polyculture and monoculture have stronger impacts than pastures and especially agroforestry. these effects are especially evident on the more specialized groups. in addition, although some groups were not affected by habitat conversion, positive effects were seldom observed. in addition, we found more observations in the temperate than in the tropical regions, indicating that further studies in tropical regions are needed to better understand these impacts. in general, our study points out the importance of remnants of native habitats for the shelter and protection of specialized organisms, while also ensuring the ecosystem services mediated by them – especially compared to more managed systems, like monocultures and polycultures. acknowledgments we thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes, finance code 001) for the scholarship granted to rjs and the post-doctoral pnpd scholarship granted to pd; jhcd acknowledges his research grant from conselho nacional de desenvolvimento científico e tecnológico. authors’ contributions all authors conceived this study. rjs collected and organized the data. pd developed the r scripts and rjs and pd performed the statistical analyses. all authors contributed to the writing, discussed the results and commented on the manuscript. this manuscript is part of rjs’s phd research, supervised by jhcd and cosupervised by pd. references almeida, d.s. 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(2011). comparison of ant community composition across different land-use types: assessing morphological traits with more common methods. australian journal of entomology, 50: 118-124. doi: 10.11 11/j.1440-6055.2010.00795.x https://doi.org/10.1046/j.1442-9993.2003.01312.x https://doi.org/10.1046/j.1442-9993.2003.01312.x http://dx.doi.org/10.1016/s0006-3207(97)00082-7 http://dx.doi.org/10.1016/s0006-3207(97)00082-7 https://doi.org/10.1016/j.cropro.2011.02.001 https://doi.org/10.1016/j.biocon.2006.03.022 https://doi.org/10.1023/a:1008891710230 https://doi.org/10.1023/a:1008891710230 https://doi.org/10.1080/01650529509360947 https://doi.org/10.1111/j.0307-6946.2006.00759.x https://doi.org/10.1111/j.1440-6055.2010.00795.x https://doi.org/10.1111/j.1440-6055.2010.00795.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i4.3743sociobiology 66(4): 527-535 (december, 2019) invasive ants affect spatial distribution pattern and diversity of arboreal ant communities in fruit plantations, in tarakan island, borneo introduction ants are an important ecological group both in natural and modified habitats (hölldobler & wilson, 1990; lachet al., 2010). in tropical areas, their biomass and species diversity are much higher, leading to the formation of complexant community structures. ant assemblages on forest canopies are suitable for exploring the factors influencing local community structures, species composition and richness, and spatial distribution (yanoviak & schnitzer, 2013). the canopies of individual trees are frequently isolated, functioning as a habitat island (southwood & kenedy, 1983; harris, 1984; adams et al., 2017). this is likely to limit the movement, resource abstract human activities influence ant community structure. in tropical areas, the habitat characteristics of crop plantations frequently shape the structure of arboreal ant communities. the present study investigated the spatial distribution of arboreal ants dwelling in durian durio zibethinus and citrus citrus amblycarpa plantations in the tarakan island, north kalimantan. specifically, it was investigated whether ant communities are dominated by native or invasive species; and if ant arboreal mosaics occur. this study included two sites (a and c) comprising durian and citrus plantations and one site b with only citrus plantations. ant workers dwelling on crop trees were collected by branch beating, and subsequently identified and counted. across all sites, a total of 64,360 workers, from 22 ant species, were collected from 59 durian and 63 citrus trees. in site a, the invasive species tapinoma melanocephalum and the native species oecophylla smaragdina were numerically dominant. a null model analysis of species co-occurrence revealed that species segregation existed in this site. conversely, in sites b and c the invasive species t. melanocephalum and technomyrmex albipes were dominant, and native arboreal ants almost co-occurred with the two species. moreover, the number of t. melanocephalum and t. albipes workers was negatively correlated with the species diversity index of arboreal ants. however, the number of o. smaragdina workers showed no significant correlation. the results suggest that the invasion and domination of non-native species dissasemble spatial structures and reduce the species diversity in arboreal ant communities. the community structures of arboreal ants in fruit plantations were varied, depending on the fruit species and the properties of dominant ants. sociobiology an international journal on social insects a rahim1, 2, k ohkawara1 article history edited by gilberto m. m. santos, uefs, brazil received 22 september 2018 initial acceptance 11 november 2018 final acceptance 10 november 2019 publication date 30 december 2019 keywords ants, plantation, invasive species, ant mosaic, species segregation corresponding author abdul rahim laboratory of ecology division of biological sciences graduate school of natural science and technology, kanazawa university kanazawa, 920-1192, japan. e-mail: arahim.ubt@gmail.com use, and habitat preference of ant assemblages. consequently, the habitat characteristics, tree species, tree size, and crown connectivity shape the structure of arboreal ant communities (tschinkel & hess, 1999; ribas et al. 2003; powell et al., 2011). moreover, arboreal ants account for up to 90% of the arboreal insects’ biomass, interacting with the other taxa and mediating a range of ecosystem processes (davidson et al., 2003). therefore, the community structures of arboreal ants strongly depend on the properties of the populations and communities of the taxa they interact with. the community structure of arboreal ants is highly influenced by human activities (morris, 2010). simple forestry systems composed of a single or a few crop trees are 1 laboratory of ecology, division of biological sciences, graduate school of natural science and technology, kanazawa university, kanazawa, japan 2 departement of agrotechnology, agriculture faculty, borneo university, tarakan, north kalimantan, indonesia research article ants a rahim, k ohkawara – structure of ant communities in fruit plantations528 often invaded by non-native species, which tend to be more dominant than native species; this results in an increase in negative interactions (sanders et al., 2003; fayleet al., 2013). additionally, in agricultural lands and disturbed secondary forests, the species composition and spatial distribution of arboreal ants frequently result in the formation of patterns that are referred to as ant mosaics. these are patchworks of territories dominated by different species that mutually exclude each other, and display nonrandom patterns of species cooccurrences (majer et al., 1994; jackson, 1984; blüthgen & stork, 2007; rizali et al., 2008). the development of ant mosaic is catalyzed by two significant factors, namely interspecific competition, including resource use patterns, and dominant species territoriality (room, 1975; ribas & schoereder, 2002). the existence of mosaic structures has been well-documented in plantations managed by farming activities, such as coffee, cacao, and cocoa farms, and palm oil plantations (majer, 1976; 1992; majeret al., 1994, dejeanet al., 1997; philpott, 2006; fayle et al., 2013; perfecto & vandermeer, 2013). pfeiffer et al. (2008) investigated palm oil plantations in the borneo and malay peninsulas and found that ant mosaics were dominated by anoplolepis gracilipes, technomyrmex albipes, and oecophylla smaragdina. in african cocoa plantations, o. longinoda, crematogaster spp., and tetramorium maculeatum are usually found and are usually found to be the most populous species among ant mosaics (tadu et al., 2014). the environmental condition in the plantations shape the peculiar structures of arboreal ant communities. however, the habitat characteristics of plantations differ among the planted crops, which differ in tree height, canopy area, crown connectivity, and other qualitative traits. furthermore, the species composition of the herbivorous insects and arthropods using each crop as host plants is different. this leads to the difference in the ant communities interacting with them. the durian durio zibethinus and citrus citrus amblycarpa fruits are traditionally grown in the agroforestry systems of indonesian kalimantan (siregar, 2006). to date, little is known about the communities of arboreal ants dwelling in the plantations of two these fruits. moreover, in any fruit plantation in kalimantan, it has been reported that ants monopolize the major part of the biomass in arboreal arthropods (pfeiffer et al., 2008; fayle et al., 2013; diamé et al., 2017). however, asfiya et al. (2015) suggested that intensive agroforestry practices promote the establishment of non-native ant species in the cocoa plantations of southeast sulawesi. in the region of and sulawesi and indonesian kalimantan, information on community structures of arboreal ants dwelling in plantations is currently lacking yet. the current study investigates the species composition and spatial distribution of arboreal ants in the plantations of durian and citrus fruits of the north kalimantan area of borneo. the following two topics were specifically investigated: (1) whether arboreal ant communities dwelling in the plantations of durian and citrus fruits are dominated by non-native species; and (2) whether ant mosaic structures occur in those communities. in addition, the effects of non-native species on the community structures were also investigated. materials and methods study sites field research was conducted in the tarakan island of borneo, indonesia (fig 1). here, the monthly mean rainfall ranges from199 – 2008 mm3. the mean annual temperature and humidity are 27.7 and 84%, respectively. three plantation sites were selected (fig 1): site a at mamburungan (3°18’15’’n, 117°37’12’’e); site b at east mamburungan (3°17’14’’n, 117°38’1’’e); and site c at kampung enam village (3°18’41’’n, 117°38’1’’e). the plantations at site a and c were established in open land where the densities of trees were relatively low. the plantation in site b was near to secondary forests. in the three plantations, we set up the study area (the area: 2.0 ha each) where many durian and citrus fruits were intensively planted. in site a and c, we selected 66 (durian: 44 and citrus: 22) and 30 (durian:15 and citrus: 15) trees as sampling trees, respectively (table 1). only 26 citrus trees were selected in site b. however, horticulture crops including included durians, citrus fruits, banana, mangoes, maize, cabbage, and other crops were planted and grown sporadically in all three sites. the horizontal positions of all selected trees were plotted on maps of each site by measuring the distribution within the study area. while the research was carried out farmers did not use the pesticide in the study area; however, weed killing and pest control were infrequently conducted with herbicides and insecticides. collection of arboreal ants on crop trees in studied sites the collections of ants were conducted from march to september in 2016 and during march in 2017. on trees in the studied sites, 10 branches (length: 50-80 cm, diameter: 5-10 cm) fig 1. location of the studied sites in the tarakan island of north kalimantan. sociobiology 66(4): 527-535 (december, 2019) 529 were selected and ants that were present on the branches were collected using the beating method. in addition, the number of ants walking on another 20 branches were also counted. the ant collection and counting were conducted 1-7 times in each site. all collected samples were stored in 99% ethanol and sorted in the laboratory. the species were identified using identification manuals and online resources i.e. bolton (1997) and antweb. org (accessed on 2017). they were classified as belonging to one of the following three categories using information from databases: native species (n); invasive or tramp species (i); and unknown (u) (antweb, 2017; pacific invasive ant group, 2017; antmaps, 2017). species collected in each site were classified as dominant species, if they met at least two the following criteria: (1) the collected number of workers was more than 5000 individuals in each site; (2) the frequency of collected workers was more than 25% of all collected workers in the site; and (3) there were polydomous nests in the sites (> 2 nests per tree were common finding). in site a, the territory ranges of colonies in dominant species were estimated by the observation of aggressiveness among workers. from each tree, 10 workers of each dominant species were collected. workers from different trees were put into a transparent plastic container (the diameter: 40 mm, the depth: 25 mm) and the response among them was checked for 2-5 minutes. if they were mutually attacked by aggressive behavior, biting or pulling, they were regarded as members of different colonies. in one observation for a pair of trees, 10 replicates were conducted by using different 10 workers. statistical analyses to analyze species composition and collection frequency in the sites, the average number of ant workers collected in one sample from one branch in each tree was calculated. the comparison of species diversity was evaluated using the shannon-wiener diversity index (h′) (krebs, 1989). the degree of overlap among species in a tree was evaluated using the pianka and czekanowski niche overlap index (pianka, 1973; albrecht & gotelli, 2001). then, principal component analysis (pca) was used to evaluate whether the ant communities were different among the sites or fruit trees. to confirm the existence of ant mosaics, we used c-scores as the metric to assess community-wide species co-occurrence (gotelli, 2000; pfeiffer et al., 2008; fayle et al., 2013). in our study, c-score was the number of pairs of species and pairs of trees where each species occurs only once and two species occur at different trees. the higher the c-score, the greater the number of non-overlapping species distribution. randomization of the original matrix was used to create the distribution of c-score expected under the null model which assumes random species co-occurrence. c-scores were simulated 1000 times randomly for the null model. standardized effect sizes (ses) were calculated to evaluate the difference between the observed and expected c-scores. positive and negative ses values indicate segregation between species and aggregation, respectively. the analyses were conducted using the ecosim r function in the r package (gotelli & elison, 2013). table 1. collection data and characteristics of ant communities in three studied sites. as the index of diversity, the average pairwise niche overlap among species was shown. site a b c area of studied site (ha) 2.0 2.0 2.0 number of observed trees 66 26 30 number of each crop species durio zibethinus 44 0 15 citrus amblycarpa 22 26 15 collection data species number of collected ants 22 21 15 total number of collected ants 53461 7203 3696 average of species number (/branch/tree) 11.3 ± 1.6 10.5 ± 2.1 3.9 ± 1.7 range of species number 8-15 7-16 1-8 average of collected number (/branch/tree) 810 ± 400.1 277 ± 89.2 123.2 ±83.8 range of collected number 202-2202 120-561 14-309 species diversity index shannon-wiener index (h′) 1.75 1.67 1.02 overlap index pianka index (α) 0.16 0.26 0.13 czekanowski index 0.15 0.19 0.1 a rahim, k ohkawara – structure of ant communities in fruit plantations530 results from 2016 to 2017, 64,360 workers were collected from 59 durian and 63 citrus trees in the three sites (table 1). they comprised of 22 species from 16 genera and five subfamilies (table 2). the species composition was significantly different between the three sites (χ2=22413.2, p<0.01, g-test). the average number of workers and species (in a branch per tree) was significantly different between the sites (collected number: f2,119=17.5, p<0.001; species number: f2,119=183.2, p<0.001, one-way anova) and they were larger in site a than at other two sites. the species diversity in site a was also higher (table 1). workers of t. melanocephalum, o. smaragdina and t. albipes were the majority, with more than 60% of all catches in each site (table 2). they usually monopolized the trees by nesting in branches and trees and building weaver nests on tree, therefore they were regarded as dominant species. five species, t. melanocephalum, t. albipes, anoplolepis gracilipes, iridomyrmex anceps, and trichomyrmex destructor were identified as invasive (table 2). it is noteworthy that the workers of non-native species were collected in all sampled trees in all sites. furthermore, more than 60% of all workers collected in each site were occupied by those of non-native species (site a: 74.5%, site b: 80.7%, site c: 66.1%). in particular, t. melanocephalum workers occupied more than 50% of trees in site a and b. the pca of the data collected from the 122 trees identified two main groups of ant communities with one group in site a and b and another group in site c, though the groups were not separated on the basis of the type of fruit trees from which the ants were collected. the first and second principal component explained only 11.6% and 8.3% of the variance of the communities respectively (fig 2). the ant communities in site a and b were comprised of tapinoma melanocephalum, oecophylla smaragdina, and other subdominant ants, whereas the community in site c primarily comprised technomyrmex albipes. the pattern identified by the pca suggest that the habitats of t. melanocephalum and t. albipes tended to be separated, although o. smaragdina were coexisting with them. subfamily species group site a site b site c n (%) n (%) n (%) ponerinae ponera sp. 1 n 4 0.01 1 0.01 0 0 dolichoderinae dolichoderus sp. 1 u 2 0.001 2 0.03 8 0.2 iridomyrmex anceps i 3771 7.1 371 5.2 64 0.2 philidris sp. 1 n 786 1.5 71 1.0 1 0.03 tapinoma melanocephalum i 26808 50.5 4020 55.8 45 1.2 tapinoma sp. 1 u 288 0.5 45 0.6 30 0.8 technomyrmex albipes i 3945 7.4 383 5.3 2363 63.9 formicinae anoplolepis gracilipes i 2325 4.4 697 9.7 11 0.3 oecophyllas smaragdina n 8131 14.6 294 4.1 1033 27.9 camponotus sp. 1 n 57 0.1 7 0.1 1 0.03 polyrhachis sp. 1 n 29 0.05 47 0.7 8 0.21 pseudomyrmicinae tetraponera sp. 1 n 325 0.6 66 0.9 8 0.21 tetraponera sp. 2 n 49 0.1 1 0.01 0 0 myrmiciane crematogaster sewardi n 2121 4.0 592 8.2 18 0.5 crematogaster sp. 1 n 806 1.5 65 0.9 48 1.3 crematogaster sp. 2 n 145 0.3 14 0.2 0 0 trichomyrmex destructor i 2712 5.1 341 4.7 20 0.5 monomorium sp. 1 u 402 0.8 0 0 0 0 monomorium sp. 2 u 140 0.3 37 0.5 0 0 tetramorium sp. 1 u 586 1.1 119 1.7 38 1.0 tetramorium sp. 2 u 32 0.1 26 0.4 0 0 pheidole sp. 1 u 16 0.03 4 0.06 0 0 table 2. species composition of ants collected in three studied sites. by the information of life history, they were classified with three groups: native species (n), invasive or tramp species (i), and unknown (u). sociobiology 66(4): 527-535 (december, 2019) 531 figure 3 shows the observed c-score index and frequency distribution of c-scores expected using null models in three studied sites. for site a, the observed c-score was significantly different from the mean value under null model (p<0.001, table 3), i.e. species segregation was found in arboreal ant communities. in this site, two dominant species had multiple colonies (t. melanocephalum: six colonies, o. smaragdina: five colonies). figure 4 shows the spatial distribution of territory ranges of the colonies. within and among species, the distribution tended to be spatially segregated, though that certain large territories overlapped. additionally, the average number (/branch/tree) of t. melanocephalum workers was negatively correlated with that of o. smaragdina workers (r2=0.08, p<0.05). probably, the two dominant species mutually avoid the overlap of territories. on the other hand, in site b and c, the observed c-scores were close to the mean values (fig 3) and the differences were not significant (table 3). ant species in these two sites therefore co-occurred. especially, the overlap index among species was highest in site b (table 1). fig 2. biplot for the data of frequency and species composition of ants collected in 122 trees of three sites. (a) first and second principal components of ordinations of the trees. the circle, rhombus and triangle symbols represent site a, b, and c respectively. black and white symbols mean durian and citrus trees. (b) the ordinations of the ant species. the names were shown in dominant and subdominant species of which the collection frequency was more than 1.0%. fig 3. the observed c-score index (broken lines) and the frequency distributions of c-scores expected using null models in which there are no interactions between ant species for a tree in three studied sites. in site 1, the scores were significantly different (p<0.001, onetailed t-test). study site c-score obs. mean null ses p a 70.5 69.1 2.82 <0.001 b 10.4 10.2 1.23 0.11 c 15.8 15.7 0.24 0.37 table 3. the observed c-scores(obs.), mean metric values under null models (mean null), standardized effect sizes (ses) and p-values (one-tailed t-test) for arboreal ant communities in three studied sites. large c-score ses values indicate a greater degree of species segregation than would be expected at random. a rahim, k ohkawara – structure of ant communities in fruit plantations532 in the three sites, many trees were occupied by workers of a few dominant species. the average number (/branch/tree) of t. melanocephalum and t. albipes workers, both considered to be invasive and tramp species, was negatively correlated with the species diversity of arboreal ants at trees in all sites (fig 5). however, the average number of o. smaragdina workers had no significant relationships, though it tends to be negatively correlated in site c. this suggests that the invasion and domination of non-native species reduces the number of ant species in trees and is associated with a reduction in species diversity of the arboreal ant community in fruit plantations. fig 4. spatial distribution of trees occupied by two dominant species, t. melanocephalum and o. smaragdina in site a. the symbol circle indicates fruit trees where ants were collected. dotted line shows the territory ranges of colonies in each species. discussion the present study revealed the structure of arboreal ant communities in durian and citrus fruit plantations in borneo. ant communities were dominated three species, t. melanocephalum, o. smaragdina and t. albipes, which are also widespread in nearby natural forest and urban areas of fig 5. relationships between average number of workers (/branch/ tree) in dominant species and the species diversity of arboreal ants in tree. species diversity is described as shannon’s diversity index. java, celebes, and kalimantan (rizali et al., 2008; rizali et al., 2011; asfiya et al., 2015) as well as in other forests of southeast asia (pfeiffer et al., 2008; elwood et al., 2016). the spatial distribution pattern of ants was different among three sites. the community structures of arboreal ants are varied, depending on the plantation conditions, micro environmenral factors, crop species and farming activities. similar observations in other studies of plantations have also been made. ribas and schoereder (2002) tested whether 14 ant comunities in various crop plantations fit to the prediction of the ant mosaic model and showed the model to be valid in only about half of these cases. in site a plantation, where t. melanocephalum and o. smaragdina were dominant, non-random spatial segregation was clear, suggesting that an ant mosaic may be present. in this site, the distribution of territory ranges in t. melanocephalum and o. smaragdina colonies were unlikely spatially overlapping. probably, the species segregation may be due to the distribution pattern. spatial distribution of arboreal ant assemblages is affected by several factors, including interspecific interactions and territoriality of dominant species. in general, invasive species including t. melanocephalum sociobiology 66(4): 527-535 (december, 2019) 533 heavily impacts their enviroments as competitors on other ants (holway et al., 2002; dejean et al., 2010; falcão et al., 2017). also, o. smaragdina is aggressive towards other ant species, defining its territory over multiple trees (hölldobler & wilson, 1990; van mele, 2008; devarajan, 2016; diamé et al., 2017). the interspecific interations with territoriality may be one of the factors giving rise to the species segregation. however, the effects on other ant taxa was different between the two species. the increase of o. smaragdina workers did not reduce the species diversity of ants on trees. as a reason, native ants that act as subdominant species could defend overlapping territories in the same way as dominant species (leston, 1973). the species segregation among dominant species and the interactions of o. smaragdina with other ants appear to lead to high diversity of arboreal ants in site a. it indicates that native ants acting as dominant species facilitate arboreal ant communities with high species diversity. in contrast, species aggregation was observed in site b and c where t. melanocephalum and t. albipes were generally and numerically dominant. the aggregation of species could be due to several factors. first, the subordinate ant community could have disassembled by t. melanocephalum and t. albipes. in many of the trees in these sites, >90% of ants collected were either t. melanocephalum or t. albipes. these two species are dominnant and frequently exclude other species, particularly in disturbed habitats (holway et al., 2002; pfeiffer et al., 2008; klimes et al., 2011). moreover, it has been suggested that the presence of dominant competitors increases the randomness of co-occurance in the subordinate ant communities (gotelli & arnett 2000; sanders et al., 2003; 2007). such behaviour leads to weaker separation of ant species. second, severe disturbance to ant habitats increases the degree of species segregation (floren et al., 2001; souza da conceição et al., 2015). particularly, the plantation in site b was established near the secondary forests and trees other than durian and citrus were present within and around the plantation. therefore, it is possible that the native ant communities move to the canopies of other native trees, resulting in the random distribution of native ants in durian and citrus fruit tress. to confirm this, the spatial distribution of ant species on the canopies of the native trees should be investigated in this site. third, the number of observed trees in site b and c may be insufficient for robust statistical analyses. the number of observed tress in site a, where spatial segregation was clearly observed, was more than twice that in theses sites. ant populations from additional trees in site b and c should be further evaluated to increase statistical robustness. however, since the increase of t. melanocephalum and t. albipes workers had negative effects on species diversity of arboreal ant communities, this suggests that the invasion and domination of non-native species can disassemble the spatial structures and reduce species diversity of arboreal ant communities in these fruit plantation. the structures of arboreal ant communities are different among the fruit plantations. the invasion of non-native species appear to have any negetive effects on the structures. it is known that arboreal ants have an important role on predation of herbivorous insects and other arthropods in the plantations. for example, o. smaragdina is a predator that negatively impacts other insect groups, including polllinators, herbivores and parasites (tsuji et al., 2004; tanga et al., 2016; appiah et al., 2014; migani et al., 2017). it is also reported that a few species of the genus crematogaster are predators of herbivorous insects in plantations (tanaka et al., 2012; castracani et al., 2017). our results indicate the interactions of arboreal ants with other insects are also affected by the invasion of non-native ant species and the change of the community structures. information about the factors affecting ant community structures will be useful for the efficient management of agroforestry system. acknowledgments this study was founded by bppln-ristekdikti scholarship of indonesian ministry of research, technology, and higher education. we are grateful to muttaqien, wisnu ageng, philipus, and other staffs of the agriculture faculty in borneo tarakan university and laboratory of ecology in kanazawa university. cordial thanks to the owners of the plantations as studied sites. we also thank to the editor and two referees for their reviews and comments on our manuscript. references adams, b.j., schnitzer, s.a., & yanoviak, s.p. 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(eds.), treetops at risk: challenges of global forest canopies. springer, pp. 209-214. doi: 10.13102/sociobiology.v65i3.2876sociobiology 65(3): 482-490 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 genetic variation in iranian honey bees, apis mellifera meda skorikow, 1829, (hymenoptera: apidae) inferred from pcr-rflp analysis of two mtdna gene segments (coi and 16s rdna) introduction intraspecific taxonomy of the honey bee apis mellifera l. has been based mainly on morphology. at present, 29 subspecies of a. mellifera are recognized on the basis of morphometric characters (ruttner, 1988, 1992; sheppard et al., 1997; sheppard & meixner, 2003; arias & sheppard, 2005; meixner et al., 2011 rahimi et al., 2017). the western honey bee originated in asia and entered africa and europe in three distinct evoluationary branches: branch (a), which included the subspecies from africa (a. m. lamarckii, a. m. abstract in this study, the genetic structure of iranian honey bee (apis mellifera meda) populations, mainly obtained from all of regions, were investigated at two different mitochondrial regions. a total of 300 worker bees were collected from 20 different populations in 20 different locations. portions of the mitochondrial 16s ribosomal rna (16s rdna) and cytochrome c oxidase i (coi) genes were amplified by pcr and then subjected to rflp pattern analysis using 8 restriction enzymes. nucleotide polymorphisms were revealed using restriction enzyme sau3a i, ssp i and taq i in coi and bsp143i, ssp i and dra i in the 16s rdna gene segment. in this study, 3 novel composite genotypes (haplotypes) were found in iranian honey bee populations. the average haplotype diversity (h) within populations was 0.0405. heterozygosity values, shannon index and the number of alleles of iranian honey bee populations were low that could be caused by low definite geographic structure of iranian honey bee populations. genetic distance (d) values were found to be low (0.0–0.0011) within iranian honey bee populations. cluster analysis based on upgma method revealed that all populations and samples groups be in one cluster. also, the phylogenetic tree based on neighbor-joining method divided 29 subspecies of honey bee to 5 distinct clusters. the iranian subspecies honey bee composed of a shared clade with subspecies of eastern mediterranean, near east and eastern parts of middle east (o branch). this result is very useful for the control of conservation of local honey bees, as the movement of colonies across the border line of these neighboring countries, may affect the genetic structure of honey bee populations. sociobiology an international journal on social insects a rahimi1,2, a mirmoayedi1, d kahrizi3, l zarei3, s jamali1 article history edited by marco costa, uesc, brazil received 04 february 2018 initial acceptance 03 june 2018 final acceptance 05 september 2018 publication date 02 october 2018 keywords apis mellifera; genetic structure, mtdna, iran. corresponding author ataollah rahimi department of plant protection faculty of agriculture, campus of agriculture and natural resources razi university, kermanshah, iran. e-mail: rahimi.ata.1@gmail.com yemenitica, a. m. scutellata, a. m. litorea, a. m. adansonii, a. m. capensis), branch (m) which included the subspecies of north african and west european (a. m. mellifera, a. m. iberica, a. m. intermissa and branch (c) which included the subspecies from eastern europe, northern mediterranean and middle east. afterwards, subspecies in branch c were divided into two groups, branch c included a. m. carnica, a. m. ligustica, a. m. macedonica, a. m. cecropia and a. m. sicula, branch o included the near and middle eastern subspecies (a. m. caucasica, a. m. armeniaca, a. m. meda, a. m. anatoliaca, a. m. syriaca, a. m.cypria, a. m. adami) (ruttner, 1988). the 1department of plant protection, faculty of agriculture, campus of agriculture and natural resources, razi university, kermanshah, iran 2department of animal science, college of agriculture kifri, garmian university, kalar, as sulaymaniyah, krg of iraq 3department of plant breeding and biotechnology, faculty of agriculture, campus of agriculture and natural resources, razi university, kermanshah, iran research article bees sociobiology 65(3): 482-490 (september, 2018) 483 phylogenetic relationships based on molecular data (cornuet & garnery, 1991; garnery et al., 1992; arias & sheppard, 1996) agree in general with those obtained with confirmed using mitochondrial and microsatellite variability (franck et al., 2000; palmer et al., 2000; kandemir et al., 2006; rahimi, 2014a,b). southwest asia included iran is a region of high morphological diversification and evolution for honey bees. one clearly distinct race has been evolved in this region, which include a diversity of habitats. honey bee race in this region include the subspecies a. m. meda (ruttner, 1988). honey bee subspecies from iran was studied extensively using morphometric and izoenzymic analysis (moradi & kandemir, 2004; rahimi & asadi, 2010; rahimi & mirmoaydi, 2013; rahimi et al., 2014a,b; rahimi et al., 2015a; rahimi et al., 2017); molecular markers, mitochondrial dna (mtdna) analysis (rahimi et al., 2014a,b; rahimi et al., 2015b; rahimi et al., 2016); microsatellite and rapd analysis (royan et al., 2007; kamrani et al., 2012; rahimi et al., 2014a,b). more recently, genetic systems such as allozymes (nunamaker & wilson, 1982; badino et al., 1988), nuclear dna (hall, 1990; tarès et al., 1993), mitochondrial dna (mtdna) (moritz et al., 1986; smith et al., 1989, 1991; hunt & page, 1992; garnery et al., 1993; oldroyd et al., 1995; arias and sheppard, 1996; pedersen, 1996; de la rùa et al., 2000) and microsatellites (estoup et al., 1993; garnery et al., 1998) have been used to study honey bee diversification. such analysis of population genetic differentiation at the molecular level contributes to better understanding of honey bee population structure by allowing the comparison of morphological, behavioural, geographical and molecular variation. the mtdna is a favourite tool in systematic and population biology. it is generally maternally inherited without recombination. only maternal inheritance of mtdna has been demonstrated for honey bees (smith, 1991; meusel & moritz, 1993; arias & sheppard, 1996; francisco et al., 2001; pinto et al., 2003). iran is a vast country comprising a land area about 1648195 km2. from the geographical point of view, iran has a very diverse climatic condition with diverse patterns of plant communities forming four climatic zones simultaneously; therefore it has a high potential for beekeeping and honey production. therefore, genetic structure maintenance and preservation of iranian honey bees is the first step to explore of this potential. the main aim of the present research was to determine the level of genetic differentiation among iranian honey bee populations as discriminated using pcr-rflp pattern analysis of the coi and 16s rdna gene regions of mtdna, and also the results of this study were compared with the results of other earlier mitochondrial studies of honey bees, such comparison thereby allowing much more complete estimation of the genetic structure of iranian honey bee populations than until now previously possible using morphometrics alone. fig 1. geographical locations of the 20 sampled honey bee populations in iran. n is the number of sampled bees per population. a rahimi, a mirmoayedi, d kahrizi, l zarei, s jamali – genetic variation in iranian honey bee484 materials and methods sampling a total of 300 young adult worker bees from 150 apis mellifera meda colonies were sampled in 100 different localities from 20 iranian provinces during june to october 2014 (fig 1, table 1). samples were taken from honey bee colonies in most active apiaries from five cities in each province, one apiary in each city and one to three hive per apiary. young adult worker bees directly were collected from the broad areas on the combs. the samples were immediately killed by immersion in absolute ethanol and kept at –20 ºc until dna extraction. molecular analysis dna extraction total genomic dna was extracted from the head and thorax sections of each honey bee worker, using the salting out method described by aljianabi and martinez, (1997) with slight modifications and then stored at −20 ºc. rflp analysis the mtdna variation was analyzed by rflp’s, performed on pcr amplified products. mitochondrial regions were amplified according to bouga et al. (2005). two sets of primers were used for amplifying 16s rdna and coi gene regions. these were 5’ caacatcgaggtcgcaaacatc-3’ and 5’-gtacctttt gtatcagggttga-3’ for 16s rdna and 5’-gatta cttcctccctcatta-3’ and 5’-aatctggatagtctg aataa-3’for coi segment. pcr was run in a total volume 25 µl of the following reaction mixture: 2.5 µl of 10 x reaction buffer with kcl as provided by the manufacturer (fermentas life sciences, vilnius, lithuania), 1 mm mgcl2, 0.5 mm of dntp mix, 1 µm of each primer, 2 u of taq polymerase and 20 ng of total purified honey bee dna. for each primer pair, the following reaction profile was used: initial denaturation 94 °c for 4 min, 35 cycles of 94 °c for 1 min, annealing at 55 °c for 1 min, and extension at 72 °c for 2 min, followed by a final extension step at 72 °c for 10 min. the amplified products obtained were next electrophoresed on 1% agarose gel to verify the size of the fragment. amplified mtdna regions from one individual of each bee were digested with 8 restriction enzymes to check for the presence of recognition sites. the informative restriction enzymes were then analyzed using one individual from each colony. the informative restriction enzymes used for the 16s rdna gene fragment were: sau3a i, ssp i, dra i, and ecor i, and for the coi gene fragment sau3a i, ssp i, taq i and ncoi. the digested fragments were separated electrophoretically on 2% or 3% agarose gels in 1× tbe buffer, stained with table 1. sampling localities, geographical positions and the number of honey bees used for pcr-rflp analysis in iranian honey bee. province/ locations abbreviation of the province/ locations the number of apiary the number of colonies the number of bees geographical position latitude longitude altitude kermanshah ker 5 7 14 34° 29′ 15.62 n 047° 05′ 57.65 e 1664 hamadan ham 5 8 16 34° 43′ 24.64 n 048° 31′ 01.27 e 2464 ilam ila 5 5 10 33° 34′ 31.97 n 046° 27′ 26.44 e 1548 lorestan lor 5 6 12 33° 32′ 01.81 n 048° 14′ 12.03 e 1802 esfahan esf 5 10 20 32° 36′ 56.09 n 051° 34′ 04.20 e 1618 chaharmahal and bakhtiari cha 5 7 14 32° 28′ 19.40 n 050° 06′ 30.53 e 2582 fars far 5 10 20 28° 59′ 19.33 n 053° 39′ 56.47 e 1563 sistan and baluchestan sis 5 5 10 29° 30′ 53.89 n 060° 50′ 17.17 e 1407 kerman krm 5 6 12 29° 18′ 19.25 n 057° 08′ 29.72 e 2838 kurdistan kur 5 7 14 34° 42′ 48.06 n 046° 51′ 34.16 e 1775 west azerbaijan wea 5 10 20 37° 34′ 04.14 n 044° 44′ 17.19 e 2138 east azerbaijan eaa 5 10 20 38° 06′ 13.12 n 046° 11′ 10.99 e 1345 ardabil ard 5 9 18 38° 13′ 58.28 n 048° 10′ 05.22 e 1455 zanjan zan 5 6 12 36° 42′ 37.00 n 048° 22′ 25.99 e 1546 tehran teh 5 6 12 35° 43′ 44.11 n 052° 07′ 00.70 e 2233 razavi khorasan rek 5 7 14 36° 25′ 56.06 n 059° 31′ 26.22 e 922 north khorasan nok 5 6 12 37° 29′ 16.88 n 057° 09′ 56.89 e 1454 golestan gol 5 5 10 36° 50′ 47.46 n 054° 40′ 12.88 e 181 mazandaran maz 5 10 20 36° 25′ 22.19 n 052° 14′ 39.71 e 135 gilan gil 5 10 20 36° 50′ 23.28 n 049° 25′ 57.83 e 329 total 150 300 sociobiology 65(3): 482-490 (september, 2018) 485 ethidium bromide, visualized under uv light and photographed using a vilber lourmat gel imaging system. the sizes of dna fragments were compared to the pcr marker (promega g316a, promega corp.) run on the same gel and were calculated using dna frag 3.03 (nash, 1991) program. data analysis composite genotypes for each individual were then defined from all the restriction patterns of the two mtdna segments. the restriction fragment data were converted to restriction data (gain or loss of restriction site). the popgene software version 1.31 was used for estimating population genetic structure. the genetic distance from restriction site data (nei & tajima, 1981; nei & miller, 1990) was estimated using the reap computer package (mcelroy et al., 1991). phylogenetic tree was constructed by the neighbor-joining method, based on results of genetic distance from restriction site data, using the past package version 2.02. results from analogous study on honey bees from different areas for other subspecies of honey bee were included in the above mentioned statistical process. results the sizes of the pcr-amplified mtdna regions for all populations studied were found to average 964 bp for 16s rdna and 1028 bp for coi mtdna gene regions. eight restriction enzymes were found to have at least one restriction site in the amplified 16s rdna and coi regions, respectively. observed fragment patterns generated by each restriction enzyme for the two mtdna regions are summarized in table 2. sau3a i, ssp i and taq i restrictions in coi and sau3a i, ssp i and dra i restriction in the 16s rdna gene each generated two different restriction profiles in iranian honey bee populations (table 2). diagnostic and novel patterns were revealed in the east azerbaijan (eaa), west azerbaijan (wea) and sistan and baluchestan (sis) populations after the digestion of 16s rdna and coi segment with the restriction enzymes bsp143i, ssp i and dra i and sau3a i, ssp i and taq i, respectively (pattern type a). the three different and novel haplotypes (composite genotypes) which were detected in the twenty populations studied and the haplotype frequencies and haplotype diversity values are presented in table 3. the three novel haplotypes were found in east azerbaijan (eaa), west azerbaijan (wea) and sistan and baluchestan (sis) samples. the average haplotye diversity (h) within populations was 0.0405 (table 3). the number of alleles (na) and the effective number of alleles (ne), the observed and expected heterozygosities (ho and he) and shannon index (i) per coi and 16s rdna coi 16s rdna restriction enzyme patterns observed (bp) restriction enzyme patterns observed (bp) sau3a i a: 338, 315, 168, 75, 72, 60 b: 398, 315, 168, 75, 72 sau3a i a: 964 b: 545, 419 ssp i a: 520, 210, 170, 80, 48 b: 520, 258, 170, 80 ssp i a: 515, 335, 114 b: 630, 334 taq i a: 350, 262, 244, 172 b: 432, 352, 244 dra i a: 314, 212, 118, 115, 90, 70, 45 b: 359, 212, 118, 115, 90, 70 ncoi 1028 ecor i 494, 470 table 2. restriction fragment patterns generated from analysis of coi and 16s rdna gene segments of iranian honey bee. fig 2. cluster analysis of 300 honey bee individuals from twenty provinces of iran based on upgma method. a rahimi, a mirmoayedi, d kahrizi, l zarei, s jamali – genetic variation in iranian honey bee486 mtdna gene and per restriction enzyme in iranian honey bee populations are shown in table 4. the genetic distance (d) values were found to be low (0.0 – 0.0011) within iranian honey bee populatioins (table 5). phylogenetic trees are generally plotted to find the genetic distances between the collected samples of honey bees. cluster analysis based on upgma method between all samples haplotype composite genotype haplotype diversity (h) ncoi 16s rdna sau3a i ssp i taq i ncoi sau3a i ssp i dra i ecor i kermanshah a a a a a a a a 0.00 14 hamadan a a a a a a a a 0.00 16 ilam a a a a a a a a 0.00 10 lorestan a a a a a a a a 0.00 12 esfahan a a a a a a a a 0.00 20 chaharmahal and bakhtiari a a a a a a a a 0.00 14 fars a a a a a a a a 0.00 20 sistan and baluchestan b b b a b b b a 0.18 10 kerman a a a a a a a a 0.00 12 kurdistan a a a a a a a a 0.35 14 west azerbaijan b b b a b b b a 0.28 20 east azerbaijan b b b a b b b a 0.00 20 ardabil a a a a a a a a 0.00 18 zanjan a a a a a a a a 0.00 12 tehran a a a a a a a a 0.00 12 razavi khorasan a a a a a a a a 0.00 14 north khorasan a a a a a a a a 0.00 12 golestan a a a a a a a a 0.00 10 mazandaran a a a a a a a a 0.00 20 gilan a a a a a a a a 0.00 20 haplotype diversity (h) 0.0405 table 3. composite genotypes (haplotypes), haplotype diversity and sample size of all the populations studied. mtdna gene restriction enzymes na ne ho he i coi ncoi 1 1 0.001 0.001 0.001 taqi 3 1 0.001 0.001 0.001 sspi 5.1 1.001 0.002 0.002 0.0009 sau3ai 6.1 1.002 0.004 0.004 0.0033 16s rdna ecori 2.2 1.003 0.006 0.006 0.0016 drai 6.1 1.003 0.003 0.003 0.0033 psti 2.1 1.003 0.005 0.005 0.0033 taqi 2.1 1.003 0.005 0.005 0.0033 table 4. genetic parameters: the number of alleles (na) and the effective number of alleles (ne), the observed and expected heterozygosities (ho and he) and shannon index (i) per coi and 16s rdna mtdna gene and per restriction enzymes in iranian honey bee populations. in this study shown in fig 2. the results of this cluster showed that all populations and samples groups be in one cluster. phylogenetic tree based on rflp analysis was drawn using neighbor-joining method to compare phylogenetic relationships iranian subspecies honey bee with other honey bee subspecies. phylogenetic tree showed that 29 honey bee subspecies could be divided into 5 distinct groups (fig 3). discussion in a recent study of iranian honey bee samples, we earlier showed that all honey bee samples then tested belonged to the east mediterranean (c) lineage as found using several restriction enzymes and morphological characters (rahimi, 2015b; rahimi et al., 2017). in the present study, we have used different two mtdna regions with different samples of the mitochondrial genome to verify the nature and distribution of genetic variation within and between iranian honey bee populations. the results of fig 3 showed that iranian honey bee belong to evolutionary lineages (c) and confirmed the results of previous studies. it is remarkable that several technical improvements introduced in beekeeping management may have interfered with the natural distribution of populations. the importation of foreign sociobiology 65(3): 482-490 (september, 2018) 487 w e a e a a a r d k u r z a n g il m a z g o l n o k r e k k e r h a m il a t e h l o r e sf c h a fa r k r m si s w e a ** * e a a 0. 00 00 ** ** a r d 0. 00 03 0. 00 03 ** ** k u r 0. 00 00 0. 00 00 0. 00 03 ** ** z a n 0. 00 04 0. 00 04 0. 00 08 0. 00 03 ** ** g il 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 ** ** m a z 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 ** ** g o l 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 ** ** n o k 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 ** ** r e k 0. 00 00 0. 00 00 0. 00 04 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** k e r 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** h a m 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** il a 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** t e h 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** l o r 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** e sf 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** c h a 0. 00 00 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 00 00 ** ** fa r 0. 00 03 0. 00 00 0. 00 10 0. 00 06 0. 00 11 0. 00 03 0. 00 00 0. 00 03 0. 00 00 0. 00 04 0. 00 03 0. 00 00 0. 00 04 0. 00 03 0. 00 00 0. 00 04 0. 00 01 ** ** k r m 0. 00 01 0. 00 01 0. 00 03 0. 00 01 0. 00 05 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 01 0. 00 07 ** ** si s 0. 00 06 0. 00 06 0. 00 10 0. 00 06 0. 00 11 0. 00 06 0. 00 06 0. 00 06 0. 00 06 0. 00 07 0. 00 06 0. 00 06 0. 00 06 0. 00 06 0. 00 06 0. 00 06 0. 00 01 0. 00 06 0. 00 01 ** ** t ab le 5 . g en et ic d is ta nc e (d ) v al ue s be tw ee n pa ir s of th e ir an ia n ho ne y be e po pu la tio ns . fig 3. phylogenetic tree between 29 honeybee subspecies based on neighbor-joining method. queens and the practice of moving colonies several times per year are factors that can affect the genetic structure of a local honey bee population through genetic introgression (garnery et al., 1998). in comparison our results with bouga et al. (2005), kekeçoglu et al. (2009) and ozdil et al. (2012), we find similar restriction profiles with generally small base differences in the fragments, except for sau3a i, ssp i and taq i and bsp143i, ssp i and dra i digestions in coi and 16s rdna gene regions, respectively (table 2). in these regions, different restriction profiles were detected in our samples or populations. sau3a i, ssp i and taq i restriction in coi revealed six or five, five or four and four or three restriction sites in this study, respectively, whereas in others subspecies such as a. m. anatoliaca, a. m. caucasica and a. m. meda (in turkey), restriction analysis were previously reported to have revealed four or six, three or two and two restriction sites for these enzymes, respectively (bouga et al., 2005, kekeçoglu et al., 2009, ozdil et al., 2012). correspondingly, the restriction of the 16s rdna gene region with sau3a i, ssp i and dra i revealed two or one, three or two and seven or six sites (table 2), compared with the three or two, two and two or seven as reported in turkish honey bees (bouga et al., a rahimi, a mirmoayedi, d kahrizi, l zarei, s jamali – genetic variation in iranian honey bee488 2005, kekeçoglu et al., 2009, ozdil et al., 2012). comparing our findings with these of ozdil et al. (2012), honey bees in iran and turkey (specially a. m. meda) were found to be well discriminated; it is noted that for the first time are reported diagnostic patterns that distinguish the honey bee populations of these neighboring countries. honey bees from iran are also discriminating from other subspecies of honey bees in turkey, iraq and syria. this study indicated that iranian honey bee populations were characterized by a low variability in the number of alleles (na) and the effective number of alleles (ne), the observed and expected heterozygosities (ho and he) and shannon index (i). according to results of cluster analysis (fig 2), it should be concluded that the honey bees of these twenty populations have formed one population, and there is no special divisions among them. also, the cluster analysis showed that honey bees from different cities and provinces are scattered across the branches of the phylogenetic tree and apparently belong to the same population. this information showed that genetic diversity between iranian honey bee populations is low, so we need a comprehensive breeding program for breeding of iranian honey bee. however, the above mentioned results are very useful for the conservation of iranian honey bee, but further follow up studies are needed to characterize mitochondrial dna variation in the honey bees of these regions along with morphometric analyses in order to accurately characterize the particular regional honey bee populations. acknowledgements the authors appreciate the kindness of those beekeepers who allowed us to collect honey bee samples from their apiaries and equally thank the authorities of plant protection and biotechnology departments of razi university who let us a free access to laboratory facilities which led to fulfill of the present study. references aljianabi, s.m. & martinez, i. 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(1993). characterization of an unusually conserved alu i highly reiterated dna sequence family from the honeybee apis mellifera. genetics, 134: 1195-1204. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3403sociobiology 65(4): 679-685 (october, 2018) special issue wing morphometrics reveals the migration patterns of africanized honey bees in northeast brazil introduction since its introduction to brazil, in 1956 (gonçalves, 1974), africanized bees have occupied almost the entire american continent, from northern argentina to the usa (rinderer et al., 1993). they can be easily identified based on morphometric features, like the pattern of wing venation (francoy et al., 2008) and are well adapted to the local environment, especially to northeast brazil, a region with very similar features to the original african environment where these bees were originally sampled. northeast brazilian covers an area of about 1.5 million square kilometers. approximately 60 percent of its total area presents semi-arid conditions (silva, 2004) corresponding to 11 percent of the abstract climatic differences can directly affect the population structure of organisms. the northeastern brazilian covers an area of about 1.5 million square kilometers, in which the semi-arid part corresponds to approximately 60%. it is probably the most vulnerable region to climatic variations in brazil. here, we investigated the variability of africanized honey bees in different localities from northeast brazil during the dry season and the influence of drought periods in morphological variation among populations. analyses were carried out with data collected by traditional and geometric morphometrics of bees sampled during the dry season and showed a subtle morphological variation in agreement to the climatic pattern. furthermore, once we added samples collected during the rainy season, we observed a change in its pattern, with a very different result from the same population sampled during drought periods. the geometric morphometrics results emphasized that samples collected during the rainy season in semi-arid areas would be more similar to bees from humid coastal areas. these results probably reflect the probable dispersion pattern of these bees between humid coastal and semi-arid areas. sociobiology an international journal on social insects cj moretti1, cp costa2, tm francoy3 article history edited by kleber del-claro, ufu, brazil received 01 may 2018 initial acceptance 04 july 2018 final acceptance 19 july 2018 publication date 11 october 2018 keywords apis mellifera; drought; geometric morphometric; semi-arid climate; traditional morphometric; migration patterns. corresponding author tiago maurício francoy escola de artes, ciências e humanidades universidade de são paulo av. arlindo bettio, 1000, vila guaraciaba cep 03828-000 são paulo-sp, brasil. e-mail: tfrancoy@usp.br area of brazil (marengo et al., 2017). this extensive area is the most vulnerable region in brazil regarding climatic variations where the periodicity and duration of the droughts usually are high (marengo et al., 2017; silva, 2004). previous studies have shown that the presence of africanized honey bee colonies in semi-arid is high during the rainy season, but these bees become scarce in the dry season (freitas et al., 2007). it suggests that africanized honey bees could migrate from semi-arid areas to regions where environmental conditions are more favorable during drought periods (freitas et al., 2007). in areas constantly subjected to disturbance, bees must continually adapt to these changes, otherwise absconding can occur towards more favorable areas, a fact that is even more exacerbated in africanized honey bees (freitas et al., 2007). 1 faculdade de filosofia, ciências e letras de ribeirão preto; universidade de são paulo, ribeirão preto, são paulo, brazil 2 faculdade de medicina ribeirão preto, universidade de são paulo, ribeirão preto, são paulo, brazil 3 escola de artes, ciências e humanidades, universidade de são paulo, são paulo, brazil research article bees cj moretti, cp costa, tm francoy – honey bee morphometrics680 these climatic differences between nearby localities can directly affect the population structure of organisms established in these regions. diniz-filho et al. (2000) observed that the variations among local populations of african subspecies of apis mellifera l. were correlated with their distribution in different climates. diniz-filho and malaspina (1995) observed spatial variation in populations of africanized honey bees distributed throughout brazil. hence, here we evaluate the variability of africanized honey bee populations within northeast brazilian region, including semi-arid, during the rainy season and dry season comparing traditional and geometric morphometrics of wings. our main expectation is that the populations’ variability to be related to the differences between the climatic regions and also to the migration flow. materials and methods sampling we sampled workers from 161 colonies of africanized honey bees, in 19 localities from five states of northeast brazil (table 1), representing different climatic regions in these states. bees were collected from september to december 2012 during the dry season, except for 37 colonies from mossoró rn, which were collected in march 2013 during the rainy season. all collected individuals were stored in 96% ethyl alcohol to preserve the samples for future molecular analysis. morphometric analyses the right forewings of approximately five workers per colony were mounted between a microscope slide and coverslip and photographed with a digital camera connected to a stereomicroscope. the traditional morphometric analyses were based on a dataset of measures from wings as described by ruttner et al. (1978). all measurements were performed with the aid of the software tsview version 7. to analyze the patterns of wing venation, a tps file was made from the images using the software tpsutil version 1.70 (rohlf, 2015). nineteen landmarks were plotted on the wing vein intersections using tpsdig2 version 2.26 (rohlf, 2015) according to francoy et al. (2008). traditional morphometric analyses were carried out using statistica7 software (statsoft, 2004). geometric morphometric analyses were conducted using the software morphoj version 1.03 (klingenberg, 2011). we first produced a procrustes fit to eliminate variation caused by differences in size, position, and orientation of the wings. the residuals of this regression were used as “size free” variables in the following statistical analyses. both morphometric data were used as input in canonical variant analyses (cva) and discriminant function analyses (dfa). bees were grouped according to their original sampling location and climate. a leave-one-out cross-validation test was performed to assess the accuracy of the data. we also calculated the morphological distances between the centroids of the groups’ distribution and state location latitude longitude climate mean precipitation n alagoas rio largo 09° 28’ 49’’ s 35° 51’ 29’’ w humid coastal 1,634 mm 8 alagoas barra de santo antônio 09° 24′ 58″ s 35° 30′ 33″ w humid coastal 1,634 mm 5 alagoas murici 09° 18’ 18’’ s 35° 56’ 30’’ w humid coastal 1,309 mm 2 alagoas maceió 09° 39′ 59″ s 35° 44′ 06″ w humid coastal 1,570 mm 1 alagoas joaquim gomes 09° 06’ 60’’ s 35° 44’ 15’’ w humid coastal 1,634 mm 3 paraíba joão pessoa 07° 06′ 55″ s 34° 51′ 40″ w humid coastal 1,874 mm 1 paraíba baraúna 05° 04′ 14″ s 37° 37′ 02″ w semi-arid 750mm 2 paraíba cuité 06° 28′ 54″ s 36° 08′ 59″ w semi-arid 735 mm 2 paraíba taperoá 13° 32′ 18″ s 39° 06′ 01″ w semi-arid 503 mm 5 paraíba maturéia 07° 15′ 59″ s 37° 20′ 57″ w semi-arid 726 mm 1 piauí são raimundo nonato 09° 00’ 54’’ s 42° 41’ 50’’ w semi-arid 697 mm 14 piauí bela vista 07° 58′ 58″ s 41° 52′ 40″ w semi-arid 847 mm 16 piauí isaias coelho 07° 44’ 16’’ s 41° 40’ 45’’ w semi-arid 847 mm 4 piauí parnaíba 09° 06’ 41’’ s 45° 55’ 50’’ w tropical 1,064 mm 13 rio grande do norte macaíba 05° 51′ 36″ s 35° 20′ 59″ w humid coastal 1,442 mm 2 rio grande do norte são paulo do potengi 05° 53’ 44’’ s 35° 45’ 29’’ w humid coastal 1,750 mm 5 rio grande do norte mossoró 05° 11’ 17’’ s 37° 20’ 39’’ w semi-arid 765 mm 57* sergipe brejo grande 10° 25’ 38’’ s 36° 28’ 12’’ w humid coastal 1,650 mm 7 sergipe são cristóvão 11° 00’ 49’’ s 37° 13’ 21’’ w humid coastal 1,372 mm 13 table 1. localities (state and city) and number of africanized honey bee colonies sampled in northeast brazil, during the dry season (in 2012). n = number of colonies sampled. *37 colonies from mossoró were collected in march 2013 during the rainy season. sociobiology 65(4): 679-685 (october, 2018) special issue 681 used it to construct a dendrogram of morphological proximity based on the neighbor-joining algorithm using mega 5.2 (tamura et al., 2011). we ran a mantel test using in tfpga software (miller, 1997) on the morphological distances between the centroids of the groups and the geographic distances among the sampling locations (measured by google earth 6.1.0.5001/2011). results analyses were carried out with data collected by traditional and geometric morphometrics of collected bees during the dry season. the workers from different climates presented a subtle separation for both traditional and geometric morphometrics (fig 1a-b). furthermore, once we added workers collected in the rainy season, we observed a change in the distribution pattern, with the homogenization of samples from the semi-arid region and humid coastal (fig 1c-d). therefore, we classified the individuals according to the climate, assigning mossoró two classifications (rainy and dry seasons), and we made further statistical analyses for both morphometric methods. traditional morphometrics using the data sets from traditional morphometric measurements, we found a subtle separation of bees according to the climates, with workers from mossoró entirely differentiated according to the sampling period, grouping oppositely in the scatter plot (fig 2a). the bees from mossoró collected during dry season were placed within the semi-arid group, while bees collected during rainy season grouped separately from the others. the two first canonical variate functions were more influenced by length wings, which affected most of the first axis (cva1), and angle 5, which influenced most of the second axis (cva2). we also observed that the differences in size were significantly different according to climatic groups (table s1, supplementary material). the bees from fig 1. africanized honey bee distribution to according climatic variation in northeast brazilian. canonical variant analyses on the data of all morphometric traits produced by traditional and geometric morphometrics. (a) cva on samples from northeast collected during dry season by traditional morphometric; (b) cva on samples from northeast collected during dry season by geometric morphometric; (c) cva on samples from northeast collected during dry and rainy seasons by traditional morphometric; (d) cva on samples from northeast collected during dry and rainy seasons by geometric morphometric. cj moretti, cp costa, tm francoy – honey bee morphometrics682 mossoró collected during the rainy season were the largest in both wings length and width (table s2, supplementary material). the mahalanobis distances among the centroids in the cva of the climatic groups were significant (table 2), and through these morphological distances, we confirmed that workers from mossoró diverged utterly according to the sampling period, positioning oppositely in the dendrogram (fig 2b). besides, bees sampled during the rainy season were more differentiated among the groups (table 2). assignment of the specimens to groups correctly classified approximately 53.2% of the total analyzed individuals (table 3). the crossvalidation test also showed the highest accuracy in classifying bees from mossoró during the rainy season, around 96.3%. on the other hand, the bees collected during the dry season in mossoró exhibited the lowest accuracy (11.5%) and a high error rate (35.7%) when we compared to the specimens from semi-arid regions. geometric morphometrics using 19 cartesian landmarks generated from the right wings, we also found a subtle divergence between semi-arid and humid coastal in which the tropical climate overlaps with semi-arid (fig 2c). the two first canonical variate functions were significant for the discrimination of the climate (p < 0.001) and explained 84.73% of the total data variability (cva1 accounted for 69.22%; cva2 accounted for 15.51%, fig 2c). fig 2. the dispersion patterns of africanized honey bee along the seasons in northeast brazilian. analyses on data collected by traditional and geometric morphometrics. (a) cva on samples from northeast collected during dry and rainy seasons by traditional morphometric; (b) dendrogram of clustering of samples from northeast collected during dry and rainy seasons by traditional morphometric; (c) cva on samples from northeast collected during dry and rainy seasons by geometric morphometric; (d) dendrogram of clustering of samples from northeast collected during dry and rainy seasons by geometric morphometric. mossoró – rainy, bees collected during the rainy season; mossoró – dry, bees collected during the dry season. sociobiology 65(4): 679-685 (october, 2018) special issue 683 the mahalanobis squared distances among the centroids of the climates were also significantly different from each other (table 4). workers from mossoró diverged completely according to the sampling period, placing oppositely in the scatter plot (fig 2c) being more differentiated among the groups (table 4), similar to results from traditional morphometric. although bees from mossoró collected during the rainy season were the most differentiated among the groups, these bees were close to the bees from humid coastal climate, while the bees from mossoró collected during dry season were close to the semi-arid group (fig 2d). assignment of the specimens to groups correctly classified approximately 52.2% of the total analyzed bees (table 5). the cross-validation test showed high accuracy about the bees from mossoró during the rainy season, around 76%. like traditional morphometric, the bees collected during the dry season in mossoró exhibited lower accuracy than bees from the same place but sampled in during another period, and a high error rate (34.4%) when compared to the specimens from semi-arid regions. discussion both traditional and geometric morphometrics indicated similar results regarding a subtle morphological variation in agreement to the climatic patterns. it suggests that groups may be relatively adapted to these environmental conditions, although there is probably a gene flow between them. tropical humid coastal semi-arid mossoró – rainy mossoró – dry tropical <.0001 <.0001 <.0001 <.0001 humid coastal 0.83 <.0001 <.0001 <.0001 semi-arid 2.77 1.58 <.0001 <.0001 mossoró – rainy 15.17 10.17 7.47 <.0001 mossoró – dry 1.04 2.21 2.90 18.54 table 2. mahalanobis square distances (below diagonal) and statistical significance (above diagonal) among the populations obtained from canonical variant analysis of traditional morphometric. mossoró – rainy, bees collected during the rainy season; mossoró – dry, bees collected during the dry season. (α = 0.05). table 3. percentage of correct classifications of individuals in the cross-validation test from traditional morphometric. mossoró – rainy, bees collected during the rainy season; mossoró – dry, bees collected during the dry season. values marked with * are assignments of the specimens to groups correctly classified. humid coastal tropical semi-arid mossoró – dry mossoró – rainy total humid coastal 45.4* 24.8 10.6 15.6 3.6 100 tropical 21 48.4* 17.7 12.9 0 100 semi-arid 11.9 16.7 59.5* 11.3 0.6 100 mossoró – dry 11.1 35.4 35.7 11.5* 6.3 100 mossoró – rainy 1.3 0 1.2 1.2 96.3* 100 humid coastal tropical semi-arid mossoró – dry mossoró – rainy total humid coastal 54.0* 4.8 24.6 6.3 10.3 100 tropical 28.2 17.2* 35.9 10.9 7.8 100 semi-arid 16.0 3.8 52.0* 11.3 16.9 100 mossoró – dry 28.1 8.3 34.4 27.1* 2.1 100 mossoró – rainy 14.0 0.6 9.4 0 76.0* 100 tropical humid coastal semi-arid mossoró – rainy mossoró – dry tropical <.0001 <.0001 <.0001 <.0001 humid coastal 1.5074 <.0001 <.0001 <.0001 semi-arid 1.4851 1.1687 <.0001 <.0001 mossoró – rainy 2.9128 2.3535 2.0903 <.0001 mossoró – dry 1.9457 1.5432 1.4874 3.6632 table 5. percentage of correct classifications of individuals in the cross-validation test from geometric morphometric. mossoró – rainy, bees collected during the rainy season; mossoró – dry, bees collected during the dry season. values marked with * are assignments of the specimens to groups correctly classified. table 4. mahalanobis square distances (below diagonal) and statistical significance (above diagonal) among the populations obtained from discriminant function analysis of geometric morphometric. mossoró – rainy, bees collected during the rainy season; mossoró – dry, bees collected during the dry season. (α = 0.05). cj moretti, cp costa, tm francoy – honey bee morphometrics684 previous studies indicated morphological variation gradients regarding geographical distribution. souza et al. (2009) showed two different morphometric groups of a. mellifera in northeast brazil. nunes (2012) found morphologically differentiated groups of honey bees regarding geographical distribution in five brazilian macro-regions (north, northeast, south, southeastern, and midwest). through traditional morphometric, we found significant size differences among groups, with bees from humid coastal climate being larger than others, while samples collected during the dry season in semi-arid areas are the smallest. besides, it is possible to observe that bees from semi-arid areas present a greater form variation among the individuals, whereas samples collected in humid coastal areas present the smallest variation. the areas characterized by a semi-arid climate presented more adverse environmental conditions than humid coastal areas (freitas et al., 2007). hence, bees collected in semi-arid areas are under greater environmental stress than samples from humid coastal areas. environmental stressors directly affect the development of organisms, what may imply changes in the wing shape (debat et al., 2003). these morphometric differences might also be related to the phenotypic plasticity linked to the environmental differences found in the different climatic regions. for instance, when wings of heliconius erato phyllis (fabricius) were analyzed using geometric morphometric, results showed a variation of wing size and shape among individuals fed with different plants, suggesting an environmental effect on the development of these individuals (jorge et al., 2011). environmental factors such as temperature, relative air humidity, food supply, and density have been related to phenotypic plasticity (batista et al., 2013). the morphometric differences in triatoma brasiliensis neiva may be related to the phenotypic plasticity, once the variations in the wing size and shape would be associated to different ecotypes resulted of the conditions in each microhabitat (batista et al., 2013). the morphometric data indicated that the two populations of mossoró (dry and rainy) are different from each other, with an important similarity observed between the sampling during the dry season and samples from semiarid areas. it is noteworthy that the city of mossoró is located in the semi-arid region. however, geometric morphometric emphasizes that samples collected in mossoró during the rainy season would be more similar to bees from humid coastal areas. freitas et al. (2007) monitored the flow of arrival and exit of swarms of two localities in northeastern brazil, one in the interior and another on the coast of ceará, in northeast brazil. they suggest that colonies migration of africanized honey bees is closely related to the seasons. honey bee colonies would nest in the semi-arid region only during the rainy season, absconding these areas during the dry season. only a few colonies would remain in this area during the entire year due to the scarcity of natural resources during the dry season (freitas et al., 2007). on the other hand, excessive precipitation would probably drive africanized honey bee colonies back to the semi-arid during the rainy season (freitas et al., 2007). our results suggest samples collected during the dry season (november) in mossoró were those that remained in their colonies even under adverse conditions and did not abscond like most of the other nest. the samples from the same place, but collected during the rainy season (march), were nests that settled in bait hives after the rainy period started when the conditions were once again favorable to the bees. the morphological proximity of these two groups (rainy and dry) respectively to the populations from humid coastal areas and semi-arid areas probably reflect the dispersion patterns of these bees along the seasons. the morphological differences found between bees that resisted drought and did not swarm and bees that migrated also reflect behavioral differences and could be used in future artificial selection studies to develop lineages with a low tendency to swarm. indeed, more studies are needed before this step of selection, but these data bring an exciting possibility for future works. acknowledgments the authors would like to thank dr. lionel segui gonçalves and many beekeepers for kindly providing part of the material used in this study. financial support was provided to tmf by grant process 2011/07857-9, fundação de amparo à pesquisa do estado de são paulo (fapesp, são paulo research foundation). fapesp (process 2013/021580) provided fellowships to cpc, and cnpq (national counsel of technological and scientific development – process 1343 47/2012-9) provided scholarships to cjm. supplementary material doi: 10.13102/sociobiology.v65i4.3403.s2225 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3403/0 authors’ contribution cj moretti: designed the experiment, performed experiments, and analysis, wrote the paper. cp costa: performed the analysis, wrote the paper. tm francoy: designed the experiment, performed the analysis and wrote the paper. references batista, v.s.p., fernandes, f.a., cordeiro-estrela, p., sarquis, o. & lima, m.m. 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(2011). mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution, 28: 2731-2739. doi: 10.1093/molbev/msr121 doi: 10.13102/sociobiology.v66i2.3598sociobiology 66(2): 316-326 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 occupation and emergence of solitary bees in different types of trap nests introduction the ecological (tscharntke et al., 1998; willcox et al., 2017; hung et al., 2018) and economic (potts et al., 2010; freitas & nunes-silva, 2012; ipbes, 2016) importance of bees make evident the need of studies identifying key species to natural and cultivated ecosystems, as well as their potential use in conservation and/or crop pollination programmes. in this sense, knowledge of the nesting habits of solitary species, such as the substrates used and nest attributes, may be important to guide new management practices. although most species of bees are solitary and play key roles within ecosystems, most studies are targeted at social species (schüepp et al., 2011; garófalo et al., 2012). abstract the study investigated the occupation and emergence of bees that nest in trapnests and assessed aspects of the structure of such nests, sex ratio, parasitism and mortality of bees in four areas of baturité massif, state of ceará. samples were taken using three types of trap-nests: dried bamboo internodes, cardboard tubes and rational boxes. in the four studied sites, a total of 185 artificial nests were offered monthly and 34 of them were occupied by bees. six species of bees, distributed in five genera (centris, mesocheira, euglossa, megachile and coelioxys) occupied the 34 trap-nests, but of this total nests, 24 presented emergence of individuals. in the rest of the nests there was mortality of the occupants. considering the total of nests with emergence, it was obtained 139 individuals: 131 bees (28 kleptoparasite bees) and 8 coleopterans. in 34 bee nests obtained, there were constructed 162 brood cells, the number of cells per trap-nest varied from 1 to 13 brood cells and the length of these nests varied from 2.4 to 14cm. thirteen nests were parasitized by hymenopterans (apidae and megachilidae) and coleopterans (meloidae), resulting in a parasitism rate of 38.2% of the total of nests founded. in addition, mortality occurred from unknown causes in 29.4% (n=10) of individuals before reaching adult stage. this work identified the bee species that use preexisting cavities in the baturite massif, determined their nesting requirements and constrains for their reproduction. this information may contribute to conservation efforts of these bee species as well as their potential use for pollination services. sociobiology an international journal on social insects mo guimarães-brasil1, df brasil2, ajs pacheco-filho3, ci silva4, bm freitas3 article history edited by gilberto m. m. santos, uefs, brazil received 20 july 2018 initial acceptance 21 december 2018 final acceptance 15 may 2019 publication date 20 august 2019 keywords nesting parasitism, nesting baits, structure of nesting, sex ratio, brood cells. corresponding author michelle o. guimarães-brasil instituto federal de educação, ciência e tecnologia do rio grande do norte campus pau dos ferros br 405, km 154, bairro chico cajá cep 59900-000 pau dos ferros-rn, brasil. e-mail: michelleguima@gmail.com the lack of bionomic knowledge of social or parasocial bees stems from the complexity of finding these bees on flowers and, mainly, from the difficulty in locating and accessing the nests of these insects with the habit to nest in places such as soil, logs, stumps or branches of trees and sometimes prefer to build their nests in preexisting cavities, which makes it difficult to locate them, to manage the nests and to collect pollen material (krombein, 1967; silva et al., 2012). the behavior of nesting in preexisting cavities causes females of these species to be attracted to artificial man-made cavities, called trap-nests (krombein, 1967; garófalo et al., 2012). through the use of these artificial cavities, nests can be observed and studied in the field and in the laboratory. 1 instituto federal de educação, ciência e tecnologia do rio grande do norte (ifrn), campus pau dos ferros, pau dos ferros-rn, brazil 2 universidade federal rural do semi-árido (ufersa), programa de pós graduação em ciência animal, mossoró-rn, brazil 3 universidade federal do ceará (ufc), setor de abelhas do departamento de zootecnia, campus universitário do pici, fortaleza-ce, brazil 4 departamento de ecologia, instituto de biociências, universidade de são paulo (usp), são paulo-sp, brazil research article bees sociobiology 66(2): 316-326 (june, 2019) 317 this makes possible to obtain information on the diversity and abundance of nesting solitary species, mortality of the immature, sex ratio of emerging bees, nesting behavior, architecture and materials used to construct nests, resources provided to larvae, and occurrence of parasites, predators and pathogens (dórea et al., 2010; seidelmann et al., 2016; araujo et al., 2018). studies have shown that some of the species of solitary bees that occupy trap-nests may be reared, rationally managed and potentially used in pollination programs (oliveira filho & freitas, 2003; magalhães & freitas, 2013; junqueira & augusto, 2017). however, much research is still needed to supoort the use of these bees at a commercial scale. thus, the possibility of obtaining artificial nests of these species and studying the aspects of their biology is the first step for rearing these insects on a large scale (garófalo et al., 2012). thus, this study aimed to investgate the occupation and emergence of bees nesting in trap-nests, as well as to evaluate aspects of nest structure, sex ratio, mortality and the presence of natural enemies of these insects in an area of atlantic forest located in the semi-arid region. material and methods study areas the study was conducted in the environmental preservation area (apa) of the baturité massif, state of ceará, brazil, also known as the baturité mountain range (fig 1). this mountain range stands out from the rest of the state of ceará because it is one of the highest, humid and biologically richest, being considered important for the maintenance of plant and animal diversity, thus representing a true genetic bank of biodiversity (pinheiro & sousa-silva, 2017). the local climate is hot and humid, with the greatest abundance of rainfall in the months of march to april, while in the months from september to november there is less rainfall (inmet, 2015). the vegetation that occurs in the studied areas is classified as ombrophylous montane forest (remnants of atlantic forest), located on the windward slope, at altitudes above 600 m. these areas are located in an isolated massif forming true islands of moisture in the middle of the semi-arid depressions of the caatinga domain (veloso et al., 1991 fernandes, 1998). the study was carried out from september 2012 to november 2013 in four areas of this massif, three located in the municipality of guaramiranga, state of ceará, namely: a1 in the alto da serra tourist complex (4º15’28.1” s and 38º55’39.8” w and 919 m altitude); a2 at café brasil inn (4º15’20.7” s and 38º 57’57.9” w and 888 m altitude) and a3 at remanso hotel da serra (4º14’33.5” s and 38º55’41.9” w and 830 m altitude). the a4 is an area in the chalet nosso sítio (4º13’24.6” s and 38º55’41.5” w and 760 m altitude) that is located in the municipality of pacoti, state of ceará. sampling for bee sampling, three types of trap-nests were produced: (1) dry bamboo internodes opened at one end and closed at the other by the node, approximately 20 cm in length fig 1. location of the two study areas regarding the nesting biology of solitary bees in the region of the baturité massif, state of ceará. mo guimarães-brasil et al. – nesting biology of solitary bees in trap nests318 and diameter ranging from 0.5 cm to 3.0 cm. the bamboo internodes were placed horizontally on the shelf of each station, inside building bricks, with 18 units available; (2) tubes made of black cardboard, measuring 20 cm in length, closed at one end with the same material and inserted into blocks of wood, with 6 holes of 1 cm and 6 holes of 2 cm, totaling 12 holes per block in each station; (3) rational boxes made of wood being, four with external dimensions of 12 cm high x 12 cm wide x 10 cm long; a box with external dimensions of 15 cm high x 20 cm wide x 22 cm long; a box with external dimensions of 18 cm high x 14 cm wide x 22 cm long and a box with external dimensions of 14 cm high x 9 cm wide x 19 cm long. boxes were 1.0 cm thick and 1.0 cm and 2.0 cm hole diameters. all the nests were arranged in collection stations, made with wooden rafters, covered by plastic tarpaulin and placed on wooden shelves fixed to the support rafters of the station at a distance of 1.3 m from the ground. all four study areas contained one collection station and the same number of trap-nests (fig 2). the inspections to verify the occupation of the substrates were performed fortnightly. when occupied and completed, trap-nests founded on bamboo internodes and cardboard tubes were collected, identified and taken to the laboratory. nests founded in rational boxes, when finalized, had their entry connected to a test tube, until the individuals began to emerge. when the bees emerged, the box was transported to the laboratory. after removed from the field, all trap-nests were replaced by new ones, of similar diameter and size, in order to keep the same number of nests at the station. the nests transferred to the laboratory had their entries individually coupled to the entrance of transparent pet plastic bottles and sealed with adhesive tape. they were kept at room temperature and inspected daily until individuals began to emerge. when all the individuals of the nest founded in rational box were emerged, the same box was taken to the collection station again. all specimens collected were sent to taxonomists at the university of são paulo. after the emergence of the individuals, trap-nests were opened, photographed and described in relation to the number of brood cells, length of the nest built, diameter of the trapnest, materials used in the construction of nests (sand, plant leaves or resin), number of cells and mortality register. nests without emergence were also opened and analyzed for the aforementioned aspects. the data of attack by natural enemies were obtained through the emergence of parasites of the nests. climatic data on temperature and rainfall collected during the study period were obtained through the national institute of meteorology (inmet, 2015). statistical analysis chi-square test (χ²) (zar, 1996) was applied to test whether sex ratio was significantly different from one male for each female (1: 1). the parasitism rate was calculated by the ratio: (total nests founded by bees/total parasitized nests) x 100. in order to evaluate if the population parameters (occupation of trap-nests, emergence, emergence time, nest size, mortality and parasitism) differ between rainy and dry periods, the mann-whitney test (u) (zar, 1996) was used. the statistical analyses were run using the past software version 2.17c (hammer et al., 2001). results occupation of trap-nests the study areas six species of nesting bees were sampled, distributed in five genera (centris, mesocheira, euglossa, megachile and coelioxys) and two families (apidae and megachilidae). of the species considered founders, centris (hemisiella) tarsata was the most frequently observed, occupying 45.0% of the trap-nests used in the studied areas, followed by centris (heterocentris) sp. (30.0%), megachile (austromegachile) aff. susurrans (15.0%) and euglossa pleosticta, which occupied 10.0% of the available nests. in addition to the founder bees, two kleptoparasite species occupied the nests: coelioxys (cyrtocoelioxys) sp. and mesocheira bicolor. a species of coleoptera (tetraonyx sp.) was also registered, whose individuals occupied four nests (fig 3). fig 2. a) front part of a collection station, showing dry bamboo internodes inside the holes of bricks and different types of rational boxes used as trap-nests. b) back of a collection station showing the black cardboard tubes inside wooden blocks and rational wooden boxes of different sizes. baturité massif, state of ceará, brazil. sociobiology 66(2): 316-326 (june, 2019) 319 perhaps because it was a small sampling, no significant association was detected between the occupation of trap-nests by the species and the mean monthly values of temperature and humidity (p > 0.05 for all analyses). there was also no difference in the occupation rate of trap-nests between dry and rainy periods, when all species were considered together (mann-whitney = 18.5, p = 0.272) or each species alone in each study area (p > 0.05 for all analyses). emergence of bees of the 24 nests registered with emergence, 139 individuals were obtained, being 131 bees (28 kleptoparasite bees) and eight coleopterans. bee emergence started in the second month after the installation of the traps and were more intense between october 2012 and january 2013 (fig 2). there was no significant association between bee emergence and mean monthly values of temperature and humidity for any of the species analyzed (p > 0.05). of the nests built by bees, centris (hemisiella) tarsata and centris (heterocentris) sp. were the most abundant founder species with 54 (52%) and 17 (16.5%) individuals respectively, emerging both in the rainy and in the dry period. euglossa pleosticta and megachile (austromegachile) aff. susurrans were less abundant in the areas, presenting together 31% of the total emergence, with individuals emerging only in the dry period, in july, september and october (fig 4). among the kleptoparasite species, coelioxys (cyrtocoelioxys) sp. had 93% (n=26) of emerged individuals and mesocheira bicolor 7% (n=2) (table 1). euglossa pleosticta and megachile (austromegachile) aff. susurrans were the least abundant founder species. they did not present a common pattern and built their nests in the two areas that presented the greatest richness of bee species. the mean time elapsed from the collection of nests to the emergence of individuals varied between species and also within the species (fig 5). fig 3. number of trap-nests occupied in the four study areas, between september 2012 and november 2013, in the baturité massif, state of ceará, brazil. fig 4. individuals emerged from the nests, from september 2012 to november 2013, from the study areas, located in the baturité massif, state of ceará, brazil. mo guimarães-brasil et al. – nesting biology of solitary bees in trap nests320 in centris (hemisiella) tarsata, the emergence time of individuals ranged from 33 to 38 days, with a mean of 35.5 ± 1.6 days. for centris (heterocentris) sp., the time of emergence ranged from 29 to 45 days with a mean of 35.6 ± 5.1 days. megachile (austromegachile) aff. susurrans presented an emergence time of 12 to 26 days, with a mean of 18.1 ± 5.4 days. for euglossa pleosticta, the time of emergence of the bees varied between 72 and 90 days, with a mean of 82.4 ± 6.0 days. the kleptoparasite bee coelioxys (cyrtocoelioxys) sp. ranged from 30 to 38 days with a mean of 31.8 ± 2.0 days and mesocheira bicolor, which had only two individuals emerged, ranged from 15 to 30 days with a mean of 22.5 ± 10.6 days (table 2). there was no significant difference in the mean time of emergence of the species studied between dry and rainy periods (p > 0.05 for all species). there was no evidence of diapause in the species studied. nest structure and sex ratio in the 34 bee nests obtained, 162 brood cells were constructed. the number of cells per trap-nest ranged from 1 to 13 brood cells and the length of these nests ranged from 2.4 to 14 cm. bees of the genus centris built 15 nests in preexisting cavities. the material used to construct the cells consisted basically of a mixture of sand grains and an oily binder substance, whose composition was not determined. the cells were oval in shape, with the concave back, thick wall and colors ranging from light yellow to dark brown. externally they presented a rough and irregular texture, whereas internally they had smooth, homogeneous, shiny and rigid aspect. some nests present vestibular cell, which is characterized by an empty space located between the last cell of the nest with brood and the closing wall of the nest, the cell closest to the entrance of the nest. these cells were made of sand, but were not lined with oily substances and were filled with uncompacted sand. cells of the nests of centris (hemisiella) tarsata were arranged horizontally, whereas cells of centris (heterocentris) sp. were arranged horizontally in some nests and in other nests, arranged obliquely in relation to the horizontal plane. all cells of both species were individualized in a linear series. fig 5. emergence time of individuals emerged from occupied nests, in the period between september 2012 and november 2013, in the study areas located in the baturité massif, state of ceará, brazil. table 1. number of emerged individuals (ei) in artificial nests and emergence time (days) (et), observed in the period from september 2012 to november 2013, in areas a1, a2, a3 and a4 in the baturité massif, state of ceará, brazil. family/species a1 a2 a3 a4 grand total e i *e t e i *e t e i *e t e i *e t e i *e t apidae centris (hemisiella) tarsata smith, 1874 32 36 ± 1.5 22 34.8 ± 1.7 54 35.5 ± 1.6 centris (heterocentris) sp. 3 31.3 ± 0.6 7 35 ± 7 7 38 ± 1.9 17 35.6 ± 5.1 mesocheira bicolor fabricius, 1804 1 30 ± 0 1 15 ± 0 2 22.5 ± 10.6 euglossa pleosticta dressler, 1982 16 82.4 ± 6.0 16 82.4 ± 6.0 megachilidae megachile (austromegachile) aff. susurrans haliday, 1836 5 12 ± 0 11 20.9 ± 4 16 18.1 ± 5.4 coelioxys (cyrtocoelioxys) sp. 6 31 ± 0 2 34.5 ± 4.9 5 30.8 ± 0.8 13 32.2 ± 2.0 26 31.8 ± 2.0 total 14 62 13 42 131 * elapsed time (in days) between nest collection in the field and adult emergence in the laboratory. sociobiology 66(2): 316-326 (june, 2019) 321 centris (hemisiella) tarsata used both bamboo internodes and cardboard tubes to build their nests, founding nine nests with 69 cells. the number of cells per nest ranged from 2 to 13 and the nest size ranged from 5.5 to 14 cm with a mean of (9.5 ± 3.9) cm. centris (heterocentris) sp. constructed six nests and 37 cells in bamboo internodes. the size of the nests ranged from 2.4 to 14 cm, with a mean of (8.3 ± 3.8) cm, with maximum and minimum number of brood cells observed inside nests ranging from 2 to 11. the three nests of megachile (austromegachile) aff. susurrans were constructed on bamboo internodes, in a linear series of cells, arranged horizontally in the cavity and separated by walls. the material used for the construction consisted basically of leaves with cuts in elliptical format. all megachile susurrans cells had a similar architecture, being elongated, cylindrical in appearance, slightly rounded at the bottom, with smooth inner surface of the cells. at the entrance of the nests, there were observed several layers of leaves cut in a rounded shape and loose, arranged in order to prevent the entry of natural enemies. for nests of this species, the number of brood cells ranged from 5 to 7. the nests had a length ranging from 9 to 13 cm, with a mean of (11 ± 2.0 cm) (table 3). there was no significant association between mean nest length and mean monthly temperature and humidity (p > 0.05 for all species). there was also no significant difference in mean nest length between dry and rainy periods (p > 0.05 for all species). the two nests of euglossa pleosticta were built in rational wooden boxes. females built their nests on the wall and the bottom of the box. each nest was formed by a set of nine cells, constructed with resin, of elliptic shape that after closed presented a structure at the apex similar to a nipple. one nest was constructed with yellow resin, whereas the other one was produced with dark brown resin. except for the species centris (hemisiella) tarsata, in which the proportion of females was higher than that of males, in all other species the proportion of males and females was similar. this difference in c. tarsata was significantly different (χ2 = 16.67, p <0.0001). for the other species, the sex ratio among the emerged bees did not differ statistically from 1: 1, being: centris (heterocentris) sp. (χ2 = 0.53; p = 0.467); megachile (austromegachile) aff. susurrans (χ2 = 0; p = 1) and euglossa pleosticta (χ2 = 0.62; p = 0.803) (table 2). table 2. number of nests founded, nest size and number of males and females of emergent species in artificial nests in areas a1, a2, a3 and a4, between september 2012 and november 2013, in the baturité massif, state of ceará, brazil. family/species number of nests founded number of individuals emerged number of males (♂) number of females (♀) sex ratio (♂/♀) nest size cm (±sd) apidae centris (hemisiella) tarsata 9 54 12 42 1:3.5 9.5 ± 3.9 centris (heterocentris) sp. 6 17 10 7 1:0.7 8.3 ± 3.8 euglossa pleosticta 2 16 7 9 1:1.28 * megachilidae megachile (austromegachile) aff. susurrans 3 16 8 8 1:1 11 ± 2.0 total 20 103 37 66 * nests founded on rational wooden boxes. because the cells were not in linear arrangement, nest size was not calculated. order family parasite species emerged individuals sex ratio host species nests parasitized by the species site of occurrence hymenoptera apidae mesocheira bicolor 1 1:1 centris (hemisiella) tarsata 2 a2 a3 1 * megachilidae coelioxys (cyrtocoelioxys) sp. 12 1:1 centris (heterocentris) sp. 7 a1 a2 a3 a4 14 * coleoptera meloidae tetraonyx sp. 8 centris (hemisiella) tarsata 4 a2 a4 total 36 13 * host unknown, only the species kleptoparasite emerged. table 3. parasite species and their respective host species in trap-nests founded in the four studied areas, between september 2012 and november 2013, in the baturité massif, state of ceará, brazil. mo guimarães-brasil et al. – nesting biology of solitary bees in trap nests322 parasitism and mortality of bees of the 34 bee nests occupied in the four studied areas, 13 were parasitized by hymenopterans (apidae and megachilidae) and coleopterans (meloidae), resulting in a parasitism rate of 38.2% of the total nests founded. the number of emerged parasites represented 25.9% (n = 36) of the total. in all, three parasite species were recorded parasitizing only nests of bees of the genus centris, being: coelioxys (cyrtocoelioxys) sp., mesocheira bicolor and tetraonyx sp. (table 3). euglossa pleosticta and megachile (austromegachile) aff. susurrans did not have natural enemies associated with their nests during the study period. coelioxys (cyrtocoelioxys) sp. was the most frequent kleptoparasite species attacking exclusively nests of centris (heterocentris) sp. in three of the studied areas: a1, a3 and a4. fourteen individuals of coelioxys (cyrtocoelioxys) sp. emerged from three distinct nests, from which the hosts were not born. of the seven nests parasitized by this species, 14 females and 12 males emerged and the sex ratio among them did not differ from 1: 1 (χ2 = 0.15; p = 0.695). nests of centris (hemisiella) tarsata were attacked by mesocheira bicolor and tetraonyx sp., the latter being the most frequent species parasitizing 44.4% of the nests. the sex ratio of individuals emerged from mesocheira bicolor was 1: 1, with 1 female and 1 male emerged (χ2 = 0; p = 1). an individual of mesocheira bicolor emerged from a nest in which the host was not known. among the three parasite species of the genus centris, coelioxys (cyrtocoelioxys) sp. was the most abundant species, presenting an important negative effect on the population of centris (heterocentris) sp., parasitizing 67% of the nests of this species. of the total number of bee nests founded, mortality from unknown causes occurred in 29.4% (n = 10) of the total number of individuals before reaching adult stage. in four nests (11.8%), only kleptoparasite species emerged, of which it was not possible to know the founder species. however, there is strong evidence that such nests belonged to the genera centris and megachile because of the material used in the construction, size and shape of the nest cells. in the nests with emergence of founder bees, the mortality rate of the individuals was considered low, when compared to the number of bees emerged, representing 1.42% (n = 2) of the total of constructed cells. the mortality of the individuals was recorded only in two nests of the species centris (heterocentris) sp. where it was verified the death of one individual in the adult phase and another one in the larval phase, both caused by unknown causes. discussion the pattern of occupation of trap-nests founded by bees in this study was characterized by the occurrence of few nesting species and few nests built. nevertheless, even with variables that may impede the nesting of bees in the artificial cavities, some species, especially those of the genus centris, were dominant in the occupation of the nests offered, mainly centris (hemisiella) tarsata, which also occur with higher density in some sites in the brazilian northeast (aguiar et al., 2005; melo & zanela, 2012; aguiar et al., 2013; vivallo & zanella, 2012). some authors suggest that this species has a preference in nesting in warm and sunny areas (aguiar & garófalo, 2004), in open spaces of secondary vegetation (pérez-maluf, 1993), in a dune environment (viana et al., 2001), caatinga (vivallo & zanella, 2012), as well as in forest fragments in northeast brazil (aguiar & garófalo, 2004). the baturité massif, being an area that has fragments of secondary atlantic forest, receives solar incidence mainly in the dry period favoring the nesting of this species, which had more intense emergence between october and january. these can be important and favorable characteristics for the management of this species in the programs of pollination of agricultural crops (aguiar et al., 2013). during the dry period, it was observed the exclusiveness in the emergence of euglossa pleosticta and megachile (austromegachile) aff. susurrans, suggesting bee preferences for the dry period. this is probably due to the fact that solitary bees are more sensitive to environmental conditions and therefore have the time development easily affected by climate (klein, 2017). the developmental period of the founder species is consistent with the patterns evaluated in other studies for the species of the genus centris (mendes & rêgo, 2007), euglossa (aguiar & garófalo, 2004) and megachile (teixeira et al., 2011). however, the average time of emergence of the species megachile (austromegachile) aff. susurrans was relatively short when compared to studies on species of the same genus (sabino & antonini, 2017). this result may be associated with relatively constant temperatures in the months of development and emergence of these individuals in the study areas. kemp and bosch (2000) experimentally proved that temperature has a considerable influence on the development of immature bees of the genus megachile and that, in general, development rates increase with increasing temperature, with mean temperatures between 22 °c and 29 °c being the most efficient for the rapid development, the low mortality rate and the rapid emergence of these individuals, being able to provide larger populations. these factors are important when intending to use insects of this genus in pollination programs. the structure of the nests founded by centris bees reveals a great similarity to the nests described in the literature for species of this genus, mainly, as to the type of material used, arrangement and number of cells (aguiar & garófalo, 2004; mendes & rêgo, 2007; mesquita et al., 2009). the presence of an oily binder substance for the construction of cells and the presence of a vestibular cell in the nests is discussed by some authors as a protection strategy against parasitism (jesus & garófalo, 2000; couto & camillo, 2014). sociobiology 66(2): 316-326 (june, 2019) 323 according to mesquita et al. (2009) as well as magalhães and freitas (2013), the fact that these species accept nesting in artificial nests and use oil to build their nests may be a good strategy to use these species in pollination programs applied in orchards of plants secreting oil, such as acerola tree (malpighia emarginata dc), proving effective both for the multiplication of populations of the genus centris and to provide considerable gains in the productivity of acerola (sazan et al., 2014). megachile susurrans nests were constructed with only cut leaves in a linear series of cells, with a similar structure to the nests described by sabino and antonini (2017) for megachile (moureapis) anthidioides and cardoso and silveira (2012) for megachile (moureapis) benign and megachile (moureapis) maculata. euglossa pleosticta, the only species that nested in a rational wooden box, built its nests on the wall and bottom of the box. garófalo et al. (1998) reported a similar result on the structure of euglossa annectans nests when testing small rational boxes of wood and bamboo internodes of different diameters and lengths as trap-nests for this bee species. the authors reported that the cells were constructed on the floor of the box with structure and arrangement similar to those described in this study. the similarity in the architecture of the nests founded by the species of bees in this study with those of other researches indicates that, regardless of the type of environment, the characteristics of the nesting biology of these species are preserved. according to some authors, the variation in the sex ratio is associated with the abundance of resources available in the environment for females. the similar proportion of males and females may be related to a stable availability of trophic resources preferred by these species in nature. however, in times of greater availability of resources, there is a greater production of females that require a greater amount of food for their development (pérez-maluf, 1993; mendes; rêgo, 2007). these factors may be related to the sex ratio of centris tarsata, which in this study had a significantly different proportion of males and females, with a deviant sex ratio for females of (1♂: 3.5♀), which may be related to high availability of preferred resources by this species during the study period. in addition, the size of the trap-nests (such as the length of the bamboo internodes and cardboard tubes used in this study) may also influence the sex ratio of bees, as observed by gruber et al. (2011) for osmia bicornis and alonso et al. (2012) for centris (heterocentris) analis. increased numbers of females in trap-nests can contribute positively to the use of these species in pollinating services, since females have a greater pollinator effect than males by collecting resources such as pollen and nectar for their brood (bosch & blas, 1994; cane et al., 2011). the association of the parasites coelioxys (cyrtocoelioxys) sp., mesocheira bicolor and tetraonyx sp. with bees of the genus centris has been documented in several studies carried out in brazil as the most frequent parasites of this genus (aguiar & garófalo, 2004; aguiar et al., 2006; drummont et al., 2008; gazola & garófalo, 2009). the high value in the parasitism rate of coelioxys (cyrtocoelioxys) sp. attacking bees of the genus centris may be related to the number of cells observed in the parasitized nests. according to aguiar and gaglianone (2003), nests with a higher number of cells may be more susceptible to parasite attack. in this study, coelioxys (cyrtocoelioxys) sp. attacked only the nests with the largest number of cells. moreover, the proximity and density of trap-nests may influence the attractiveness of these parasites (wcislo & cane, 1996). the mortality rates attributed to unknown causes (couto & camillo, 2007) and fungal proliferation (camarottide-lima & martins, 2005) have been diagnosed as the main causes of mortality of immature individuals of various bee species nesting in trap-nests. conclusions our study concludes that the species centris (heterocentris) sp. and c. (hemisiella) tarsata have important and potential characteristics to be reared and multiplied through the use of trap-nests. on the other hand, species such as euglossa pleosticta and megachile (austromegachile) aff. susurrans present limitations for mass rearing throughout the year, since they have a preference for nesting during the hottest periods of the year. the high rate of kleptoparasitism may also be a problem for population growth in trap-nests and should be controlled. these peculiarities may be relevant for the use of these species in crop pollination programs. references aguiar, c.m.l. & gaglianone, m.c. (2003). nesting biology of centris (centris) aenea lep., 1841 (hymenoptera, apidae, centridini). revista brasileira de zoologia, curitiba, 20: 601606. doi: 10.1590/s0101-81752003000400006 aguiar, c.m.l. & garófalo, c.a. 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(1996). biostatistical analysis. 3th ed. mcelroy, w.d; c.p swanson (eds.). new jersey, usa, prentice-hall inc, englewood cliffs, 662p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3367sociobiology 65(4): 583-590 (october, 2018) special issue bee pollination highly improves oil quality in sunflower introduction recently, the global population surpassed 7 billion people, and according to the united nations projections for 2050, it will reach 9.8 billion (un, 2017). this fast population growth has imposed significant challenges to meet considerably high demand for food production (godfray et al., 2010), including the expansion of agricultural lands, which contributes to the loss of biodiversity and its valuable ecosystem services (steffan-dewenter et al., 2005). among all the ecosystem services directly connected to agriculture, animal-mediated pollination is one of them, increasing the production of 75% of important crops worldwide for balanced human diet (klein et al., 2007). up to 90% of these leading global crops rely to some degree on pollination provided by bees, improving seed and fruit yield and quality (klein et al., 2007; potts et al., 2016). many crops have a positive relation between fruit set and pollinator richness and abundance (garibaldi et al., 2013) and abstract sunflower is a pollinator-dependent crop and one of the most cultivated oilseeds in the world, supporting important sectors of the agricultural industry, such as the food supply, because it is an important source of vitamin e and unsaturated fatty acids for human health. although it is well established that bee pollination improves sunflower seed set, it is still unknown if pollinators influence the nutritional composition. considering the economic importance of sunflowers for several brazilian agricultural sectors, the aim of this study was to evaluate the effect of the bee community for (1) achene quality (weight and nutritional composition) and (2) market value. exclusion experiments were performed with hybrid sunflowers and showed that bee pollination enhanced the achene weight by 91%, the levels of vitamin e by 45% and unsaturated fatty acids by 0.3%. also, it was estimated that due to the pollination services provided by bees, the grower of the sunflower cultivar used in this study nearly duplicates the sale value of the achenes per hectare of cultivated area. thus, the current study highlights the importance of bees as providers of crossand self-pollination to nutritional quality of sunflower achenes and provides useful baseline figures to further evaluations of the effects of pollinators on human diets and health. sociobiology an international journal on social insects cas silva, wac godoy, cro jacob, g thomas, gms câmara, da alves article history edited by solange augusto, ufu, brazil received 25 april 2018 initial acceptance 11 june 2018 final acceptance 18 august 2018 publication date 11 october 2018 keywords native bees, crop pollination, nutritional value, vitamin e, helianthus annuus, brazil. corresponding author denise araujo alves department of entomology and acarology luiz de queiroz college of agriculture university of são paulo avenida pádua dias nº 11 cep 13418-900, piracicaba-sp, brasil. e-mail: daalves@usp.br bee pollination leads to heavier fruits, with less malformations and higher commercial grades (i.e., increased fruit color, firmness and shelf life; klatt et al., 2014). also pollinatordependent crops are important sources of macroand micronutrients, which are essential for human health (eilers et al., 2011). beyond human food supply, bee-pollinated crops contribute to green manure, medicines, fibers and biofuels (potts et al., 2016). this ecosystem service, which is still disregarded in national and international agricultural policies, contributes to 35% of global crop production (klein et al., 2007) and its economic value is estimated at us$ 235-577 billion annually (potts et al., 2016). bees as crop pollinators are thus inextricably intertwined to food and nutritional safety, diversity and stability in the sale prices of agricultural products (steffan-dewenter et al., 2005). among over 20,000 described bee species (ascher & pickering, 2018), apis mellifera l. is the most commonly managed bee in the world to enhance agricultural production. however, since a. mellifera is not an effective pollinator of luiz de queiroz college of agriculture (esalq), university of são paulo, piracicaba, são paulo, brazil research article bees cas silva, wac godoy, cro jacob, g thomas, gms câmara, da alves – bees affect sunflower nutritional value584 many plants, its integrated management with wild bee species substantially increases the crop yields as well as farmers’ profits, and at the same time, it preserves biodiversity and the provision of multiple ecosystem services (garibaldi et al., 2013; isaacs et al., 2017). even though a. mellifera is the most abundant sunflower visitor (free, 1993; parker, 1981; carvalheiro et al., 2011), wild native bees species contribute significantly to seed set (greenleaf & kremen, 2006; carvalheiro et al., 2011). moreover, bee pollination also enhances the sunflower seed quality, rendering heavier seeds with higher oil contents (parker, 1981). even modern sunflower hybrids with high levels of self-compatibility benefit from pollination provided bees (degrandi-hoffman & chambers, 2006), since their visits are important to transfer pollen from male-phase to female-phase florets of the same flower head or of different plants (mcgregor, 1976; free, 1993). sunflower (helianthus annuus l.) is one of the most cultivated oilseed crops in the world since it supports important sectors of the agricultural industry, such as biofuel, rotation crops, cattle fodder and human food supply. the oil extracted from the sunflower seeds has high nutritional value since it is rich in fatty acids, such as oleic (omega-9) and linoleic (omega-6) acids, vitamin e (including alpha, betaand gamma-tocopherol) and phytosterols (fao/who, 2015). although brazil does not stand out as the main sunflower producers in the world, the national seed yield increased 155% between 2005 and 2015 (ibge, 2017). since sunflowers are highly pollinator-dependent crops, almost of 65% of the annual agricultural income of this crop (us$ 41 million) corresponds to the pollination mediated mostly by bees in brazil (giannini et al., 2015). combined with the brazilian climatic conditions, the expansion in sunflower productivity is benefited by the high number of bee species, being that brazil is the second country with greater bee richness worldwide (ascher & pickering, 2018). however, projections for the year 2050 show that climate change will reduce the probability of pollinator occurrence, causing a negative impact on 100% of brazilian municipalities where sunflowers are grown, and consequently affecting the brazilian gross domestic product and food security (giannini et al., 2017). considering the economic importance of sunflowers for several brazilian agricultural sectors and bees maximize seed set of several commercial cultivars, our aim was to evaluate the effect of the bee community for (1) achene quality (weight and nutritional composition) and (2) market value. material and methods experimental design the study was carried out at luiz de queiroz college of agriculture, university of são paulo (piracicaba, são paulo state; 22º42’26.70” s, 47º37’58.54” w) from october 2015 to february 2016 (hereafter 2015/16), and from october 2016 to february 2017 (2016/17). in october of each year, sunflower seeds (hybrid brs 323; embrapa, 2013) were sown in five rows, regularly spaced in 0.80 m, in an area of 25 x 4.8 m. four random blocks were made up of eight plots of 2.0 x 6.25 m, with 50 sunflowers per plot. sunflower planting experiments were homogeneously designed and set up aiming to avoid any random variation that could prevent analytical comparisons between the data sets. procedures essentially involved usual soil management (soil mixture, fertilization, irrigation), manual removal of weeds and insect pests and identical experimental timing between sampling time periods. in each of the four plots, before any floret had opened, white nylon mesh bags (1 mm mesh width) were placed over seven sunflower heads to exclude insect visitors, allowing only self-pollination. for each one of the other four plots, seven sunflower heads were tagged and left open to be accessible to visitors. at the end of the flowering period, the open sunflower heads were also bagged, in order to avoid seed predators, and all sunflower heads (n = 56 heads per period) were left that way up to seed maturation and the achene set was assessed. bee visitors of sunflowers each parcel was randomly walked for 5 min, every hour, and all bees found on the sunflower heads were collected with an entomological net. the sampling took place when over 90% of the sunflowers were blooming, on a sunny day, with temperature higher than 20 °c, between 7:00 a.m. and 4:00 p.m. bee richness and difference in the species composition were compared between 2015/16 and 2016/17 (kindt & coe, 2005). bee diversity was also estimated, using as reference simpson’s index, due to the evidence of dominant species, and consequently, for capturing distribution variance of species abundance. the difference in the species composition was initially estimated using ecological distance matrices, by the bray-curtis method, due to the need to consider the influence of the greater abundance differences of species, and also for high discrepancy between the number of individuals of some species, mainly plebeia droryana (friese), tetragonisca angustula (latreille), and a. mellifera. the ecological distance was also estimated by hellinger distance estimation, which takes into account the difference in the proportion of species, most importantly for considering species with low abundance. for this reason, the decision was to show the results only for this analysis. the distance matrix varies from 0 (complete similarity in the species composition between the two periods) to 1 (complete dissimilarity). due to the variation in the magnitude of abundance values for some species, the ecological distance matrices were analyzed taking into account the following scenarios: (1) all bee species; (2) solitary bees; (3) highly eusocial bees (a. mellifera and stingless bees); (4) highly eusocial bees, excluding the most abundant species, p. droryana, or t. angustula or a. mellifera. the analyses were performed using r package biodiversityr (kindt, 2016). sociobiology 65(4): 583-590 (october, 2018) special issue 585 achene quality and market value in january of 2015 and 2016, 56 sunflower heads were cut (28 bagged and 28 unbagged sunflower heads), dried at room temperature, and threshed by hand after which the achenes were obtained. for each plot, 1,000 achenes were randomly weighed. in order to evaluate nutritional quality, 500 g achenes for each group of sunflower heads were sent to food technology institute (instituto de tecnologia de alimentos – ital; campinas, são paulo state), where they were submitted to a cold pressing process for oil extraction and identification of tocopherols and fatty acids, using methods of the american oil chemists’ society (aocs). to distinguish methyl esters from fatty acids (saturated, monoand polyunsaturated), the samples were submitted to preparation according to hartman and lago (1973), with adaptations by firestone (2014). gas chromatography analyses (agilent technologies, model 7890a) were performed with a flame ionization detector. after saponification and esterification, the samples were diluted in hexane and agitated for phase separation, supernatant was collected, transferred to vial (2 ml) and injected into the chromatograph. the compounds were separated in fused silica capillary column cp-sil 88 (100 m x 0.25 mm x 0.20 mm). the column temperature programming started at 130 °c for 2 min., increased at a rate of 10 °c.min-1 up to 230 °c, and was kept in isotherm for 20 min. the temperatures used in the injector and in the detector were 230 and 260 °c, respectively. a volume of 1 µl of each sample was injected, adopting a splitter ratio of 1:75. the velocity of the hydrogen carrier gas was 30 ml.min-1. the fatty acids were identified through a comparison in retention time of pure standards of methyl esters of fatty acids with the compounds separated from the samples. the quantification was performed by area normalization (%) and the results were shown in g/100 g of samples. the detection and quantification of tocopherols (alpha, beta and gamma) were performed in equipment for liquid chromatography (prominence lc-20a) attached to a fluorescence detector rf10axl (shimadzu) using the excitation wavelength at 292 nm, and emission at 336 nm. 0.50 g of oil diluted in n-hexane was weighed. the analytes were separated in a lichrospher si 60 normal phase column (12.5 cm in length x 4 mm in d.i., and particles of 5 µm, merck), having as mobile phase a mixture with 97.6% of n-hexane, 1.8% de ethyl acetate, and 0.6% acetic acid, using an isocratic system. the quantification was performed by means of an external standard, using tocopherol set standard (article 613424, calbiochem). for the calculation of market value of achenes, it was assumed that (1) each sunflower head contained 1,000 fertile florets (pisanty et al., 2014), (2) 40,000 sunflowers were evenly spaced out, in a 1 ha area, as in this study, (3) weights of the 1,000 achenes from bagged and unbagged sunflower heads, and at (4) a sale price in the unites states (us$ 379.00/t of achenes (established in march 2017 by national supply company; conab, 2017). results bee community in total, 653 individuals belonging to 20 species of 18 bee genera were sampled (table 1). apidae was the most representative family, with 574 bees of 13 species (table 1), which corresponded to 87.9% of the total number of bees. the second sampled family was halictidae, with 79 bees from seven species (table 1). augochloropsis cupreola (cockerell), bombus morio (swederus), diadasina sp., friesella schrottkyi (friese), neocorynura codion (vachal), pseudaugochlora graminea (fabricius), tetragona clavipes (fabricius) were sampled exclusively in 2015/16, and represented 50% of the 18 species (table 1). the highly eusocial species, a. mellifera e t. clavipes, exhibited the highest frequency of individuals (36.47% and 27.65%, respectively). in 2016/17, geotrigona subterranea (friese) and melipona quadrifasciata lepeletier were collected exclusively during this period and corresponded to 18.18% of the 11 captured species, out of which, the stingless bees p. droryana were the most abundant, with 49.07% of the sampled individuals (table 1). species richness and the simpson index showed values slightly lower in second period: 18 and 0.77 in 2015/16, and 11 and 0.71 in 2016/17. however, general abundance increased substantially, resulting from 170 individuals in 2015/16 to 483 in 2016/17. the significant increase in overall abundance of bees may be explained by the dominance of some species, especially in 2016/17, when 237 individuals of p. droryana, 69 of t. angustula, 57 of a. mellifera visited sunflower heads. on the whole, observing hellinger ecological distance, there was a general tendency for low matrix value (0.42), showing increase in similarity in diversity patterns between 2015/16 and 2016/2017. when the analyzed scenarios in a comparative way were evaluated, highly eusocial bees showed matrix value (0.46) very close to the value obtained for the bees in total (0.42), which suggests that similarity patterns between the periods were determined by the eusocial species, probably due to the high abundance of some of them, as mentioned previously. when the investigated scenario was composed only by solitary bees, there was a decrease in the matrix value (0.33) when compared to the value found for eusocial bees (0.46). this may indicate that, when the proportion of species with low abundance is considered, the solitary species leaned towards similarity more than the eusocial bees. when the three most abundant eusocial bees were considered, the removal of p. droryana resulted in the lowest matrix value (0.28) of all scenarios (without t. angustula: 0.51; without a. mellifera: 0.52), suggesting that its removal indicated higher tendency to similarity between 2015/16 and 2016/17. therefore, the scenarios that indicated the highest similarity in the species composition between the two periods were the one with the removal of p. droryana from the eusocial species, and the one with solitary bees. cas silva, wac godoy, cro jacob, g thomas, gms câmara, da alves – bees affect sunflower nutritional value586 table 1. bee species and respective number of individuals sampled with entomological net in sunflower areas in 2015/16 and 2016/17. family tribe species number of bees 2015/16 2016/17 apidae apini apis mellifera linnaeus, 1758 62 57 bombini bombus (fervidobombus) morio (swederus, 1787) 1 0 ceratinini ceratina sp. 1 0 emphorini diadasina sp. 3 0 exomalopsini exomalopsis (exomalopsis) auropilosa spinola, 1853 2 24 meliponini friesella schrottkyi (friese, 1900) 1 0 geotrigona subterranea (friese, 1901) 0 6 melipona quadrifasciata lepeletier, 1836 0 1 nannotrigona testaceicornis (lepeletier, 1836) 3 25 plebeia droryana (friese, 1900) 5 237 tetragona clavipes (fabricius, 1804) 47 0 tetragonisca angustula (latreille, 1811) 10 69 trigona spinipes fabricius, 1793 7 13 halictidae augochlorini augochlora (augochlora) esox (vachal, 1911) 18 5 augochlora (oxystoglossella) morrae strand, 1910 3 14 augochlora sp. 2 32 augochloropsis cupreola (cockerell, 1900) 2 0 neocorynura codion (vachal, 1904) 1 0 pseudaugochlora graminea (fabricius, 1804) 1 0 halictini dialictus creusa (schrottky, 1910) 1 0 total 170 483 achene quality and market value the mean weight of achenes of unbagged sunflowers (mean ± s.d.: 74.27 ± 7.35 g) was substantially higher than those of bagged sunflowers (38.82 ± 10.27 g) (t = –7.9388; p = 0.000003). then, the flower visitors contributed with the increase in the mean weight of the achenes by 91.3%. when the periods were considered separately, the achenes of unbagged sunflowers in 2015/16 were 107.35% heavier than the bagged ones (t = –8.4938; p = 0.002762), while this weight difference was 79.72% for 2016/17 (t = –7.7933; p = 0.002395; fig 1). fig 1. mean weight (± s.d.) of 1,000 achenes of unbagged (grey) and bagged (black) sunflower heads in 2015/16 and 2016/17. * t test, p < 0.005. for each cultivated hectare, the estimated sale price of the achenes of bagged sunflowers was us$ 588.51, while unbagged sunflowers resulted in the estimate of us$ 1,125.93. then, the pollinators increased the sale price of sunflower achenes by 91.3%. considering the nutritional value of sunflower oil, the levels of unsaturated fatty acids were higher than the saturated ones, predominating the oleic acid (omega-9) among the monounsaturated, and the linoleic acid (omega-6) among the polyunsaturates (table 2). the alpha-tocopherol was highly predominant among the other tocopherols (table 2). in 2015/16, since most of the achenes of the bagged sunflowers were empty (when pollination fails, the floret results in an empty achene), it was not possible to extract oil for chemical analysis. with respect to unbagged sunflowers in the same period, 23% of the 500 g of achenes corresponded to lipids. among the tocopherols, the alpha-tocopherol corresponded to 96.34%, followed by the betaand gammatocopherol, 2.22% and 1.44%, respectively. concerning the fatty acids, omega-9 corresponded to 99.46% of the monounsaturates, omega-6 to 100% of the polyunsaturates, and the palmitic and stearic acids, at 51.69% and 32.77% of the saturates, respectively (table 3). in 2016/17, the oil of unbagged sunflowers had higher levels of gamma(160.71%), beta(54.90%) and alpha-tocopherol (40.06%), and linolenic (omega-3; 13.33%), behenic (9.88%), lignoceric (7.14%), arachidic (4.17%), stearic (4.05%) and linoleic (3.83%) acids, sociobiology 65(4): 583-590 (october, 2018) special issue 587 t. angustula: 5,000 workers (tóth et al., 2004)) that need constant nutritional supply to ensure their survival, especially during periods of floral resource scarcity (maia-silva et al., 2016). thus, there are a diversity of foraging strategies and signals used by bees to guide their nestmates to rich food sources (jarau & hrncir, 2009), as they need a constant supply of nectar and pollen that are obtained from a wide range of plant species (kleinert et al., 2009). sunflower is a highly attractive source of nectar and pollen to bees (parker, 1981), specially to stingless bees. due to their biological and behavioral characteristics previously mentioned, they are ecologically dominant in comparison to the other native flower visitors in the neotropical region (roubik, 1989). during 2015/16, t. clavipes was the most dominant native species, whereas p. droryana showed greater abundance among the flower visitors in the second period. when p. droryana was removed from the group of highly eusocial bees with respect to hellinger distance analysis, the species composition of this group was more similar taking into account the two periods. this result, as the one obtained for analysis with solitary bees, showed that high abundance of few species may influence negatively the general diversity of bee species, since both species richness and diversity had slight declines between the two periods. however, the expressive increase in the mean weight of the achenes, with 67.64 g in 2015/16 and 80.90 g in 2016/17, may be in part attributed to the increase in the overall bee abundance. even if the community of flower visitors was composed of several taxa, the pollinating efficiency of bees table 2. nutrients that were quantified in oil from achenes of unbagged and bagged sunflower heads in 2015/16 and 2016/17. components 2015/16 2016/17 unbagged bagged unbagged bagged tocopherols (mg/100g) alpha-tocopherol 57.68 40.66 29.03 beta-tocopherol 1.33 0.79 0.51 gamma-tocopherol 0.86 2.92 1.12 fatty acids (g/100g) saturated 8.30 8.77 8.70 monounsaturated 47.89 51.44 52.53 polyunsaturated 39.42 35.39 34.07 composition of fatty acid (g/100g) palmitic 4.29 4.25 4.34 palmitoleic (omega-7) 0.10 0.10 0.11 stearic 2.72 3.08 2.96 oleic (omega-9) 47.63 51.35 52.27 linoleic (omega-6) 39.42 35.22 33.92 arachidic 0.22 0.25 0.24 linolenic (omega-3) 0.00 0.17 0.15 behenic 0.78 0.89 0.81 lignoceric 0.29 0.30 0.28 compared with oil of bagged sunflowers in the same period. however, there was a decrease in the levels of palmitic (2.07%), palmitoleic (omega-7; 9.09%) and oleic (1.76%) acids (table 2). discussion this study shows that pollinators play an essential role for improving achene quality and thus market value in hybrid sunflowers. even though there are efforts for the development of hybrid sunflower cultivars that display high levels of selfcompatibility and are less dependent on cross-pollination, but surprisingly enough, pollinators contributed with an increase of 91% in the achene weight and in the increase of the levels of some fatty acids, and specially, of tocopherols in the oil. even in a small area of sunflower cultivation (100 m2), a high number of bee species was recorded, corroborating previous studies carried out in brazil (morgado et al., 2002), united states (parker, 1981; greenleaf & kremen, 2006), spain (hevia et al., 2016) and south africa (carvalheiro et al., 2011). taking into account the 20 species sampled in sunflower heads, 50% were highly eusocial species. these species were dominant flower visitors, and the native stingless bees (p. droryana, t. clavipes e t. angustula) and the exotic a. mellifera corresponded to 56.4% and 18.3% of the individuals, respectively. the dominance of highly eusocial bees was expected since their perennial colonies consist of one egg-laying queen and a large worker population (e.g., a. mellifera: tens of thousands of workers (michener, 1974); p. droryana: 3,000 workers; t. clavipes: 7,000 workers; cas silva, wac godoy, cro jacob, g thomas, gms câmara, da alves – bees affect sunflower nutritional value588 on a given crop depends on their behavior on the flowers and on the probability of transferring pollen from their bodies to the flower stigmas of conspecific plants (woodcock et al., 2013). even though a. mellifera is the most abundant flower visitor of several animal-pollinated crops, including sunflowers (carvalheiro et al., 2011; morgado et al., 2002; greenleaf & kremen, 2006), crop productivity increases substantially with pollination services provided by wild native insects (garibaldi et al., 2013). the results obtained in this study are consistent with previous studies, suggesting that native flower visitors interact effectively with crops and contribute significantly to pollination and yield of most crops regardless of a. mellifera abundance (garibaldi et al., 2013), reaffirming that the abundance of wild native bees is relevant for crop stability (garibaldi et al., 2011). higher flower-visitor density could increase crop yields by a median of 24% in fields with less than 2 hectares, which are very common in developing countries such as brazil, enhancing therefore small farmer livelihoods (garibaldi et al., 2016). taking into account that, due to the pollination services provided by bees, the growers of the sunflower cultivar used in this study nearly duplicate the sale value of the achenes per hectare of cultivated area, the restoration and conservation of habitats for wild pollinators, including floral resources and nesting sites, within agricultural landscape enhance biodiversity and the ecosystem services it provides (garibaldi et al., 2011; wratten et al., 2012). pollinators enhanced, besides achene weight, sunflower oil composition. the oil from achenes of unbagged sunflowers presented an increase of 44.7% of total tocopherols when compared to the oil of flowers in which pollinators had been excluded. tocopherols and tocotrienols compose the vitamin e family, which is considered the most important antioxidant in human diet (mathur et al., 2015), and a large portion is present in pollinator-mediated crops (alpha-: 36% of crops; beta-: 99%; gamma-tocopherol: 67%, eilers et al., 2011). among the tocopherols found in sunflower oil, alphatocopherol constitutes the largest portion, and increased 40.1% in unbagged sunflowers. however, gamma-tocopherol showed a substantial increase of 160.7%. even though alphatocopherol is the most known analog in vitamin e, more recent studies suggest that gamma-tocopherol show more potent antioxidant, cardioprotective and anti-inflammatory effects than alpha-tocopherol (reviewed in mathur et al., 2015). in relation to the fatty acids present in sunflower oil, slight difference between unbagged and bagged sunflowers was detected, contrary to the findings concerning tocopherols. among the polyunsaturated fatty acids, omega-6 level was 3.8% higher in achenes of unbagged sunflowers. although omega-3 level is extremely lower than omega-6 level, the pollinators affected the former by 13.3%. concerning the saturated acids, they composed less than 10% of fatty acids, the oil of unbagged sunflowers showed slightly lower levels of palmitic acid and a little higher level of stearic acid than bagged sunflowers. since sunflower oils with high contents of omega-9 (monounsaturated acid) and omega-6 (polyunsaturated acid) and low levels saturated acids, strategies have been implemented for the development of sunflower cultivars with these characteristics (raß et al., 2008), such is the case of hybrid brs 323 used in this study. oils with higher omega-9 than omega-6 contents showed increased level of oxidative stability, which is desirable for frying, refining and storage purposes, and also, better dietetic properties since it reduces cholesterol in blood (flagella et al., 2002). however, the same sunflower cultivar may show difference in fatty acid levels depending on environmental conditions, such as temperature, light and water availability, and on sowing period (flagella et al., 2002). similar to this study, pollinators improved the nutritional composition of fruits and seeds of several globally important crops. for example, insect pollinators, particularly bees, influenced the chlorophyll content in oilseed rape (brassica napus, bommarco et al., 2012), the proportions of oleic to linoleic acid in almond (prunus dulcis, brittain et al., 2014) and the sugar-acid-ratio in strawberry (fragaria x ananassa, klatt et al., 2014). thus, the current study highlights the importance of bees as providers of crossand self-pollination to nutritional quality and market value of sunflower achenes and provides useful baseline figures to further evaluations of the effects of pollinators on human diets and health (eilers et al., 2011; potts et al., 2016). acknowledgements we are especially thankful to eduardo a. b. almeida (ffclrp-usp) for bee identification, roberto gaioski jr, marcela m. barbosa and samanta l. nanzer for field assistance and josé b. malaquias for assisting in experimental design. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001and associação brasileira de estudo das abelhas (a.b.e.l.h.a.). authors’ contribution conceived and designed the experiment: cass, gmsc. performed the experiment: cass, croj. analyzed the data: wacg, gt. interpreted the data: daa, wacg, cass, croj. wrote the paper: daa, wacg, cass. all authors read and approved the final manuscript. references ascher, j.s. & pickering, j. 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(2012). pollinator habitat enhancement: benefits to other ecosystem services. agriculture, ecosystems & environment, 159: 112-122. doi: 10.1016/j.agee.2012.06.020 _hlk488845616 _hlk488847926 doi: 10.13102/sociobiology.v67i1.2897sociobiology 67(1): 48-58 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction algerian steppe is considered ecologically a buffer zone between coastal and saharan algeria (nedjraoui & bedrani, 2008). it is limited to the north by the tellian atlas and to the south by the saharan atlas and extends over a length of about 1,000 km from the tunisian border to the moroccan border. it covers an area of 20 million hectares out of a total of 42 million hectares of steppe for the entire maghreb region. this wide area makes the algerian steppe an ecosystem characterized by a diversity of landscapes submitted to a great variability of ecological factors (bencherif, 2011). despite this diversity, there is a lack of information on the fauna living in this ecosystem, particularly arthropods, including ants. with more than 12,500 species, ants are considered to be the most diverse and abundant group of abstract this paper describes the structure and composition of ant communities in the pre-saharan steppe of algeria. the study focused on three stations located in two regions with different climates: semi-arid (aflou) and arid (laghouat). ants were collected between march 2013 and february 2014, using pitfall trap sampling over a four-season period and quadrat counting techniques. a total of 20 ant species have been identified, which belong to 8 genera in three subfamilies: dolichoderinae, myrmicinae and formicinae. moreover, it was noticed that the study areas, which can be characterized by their floristic nature, physiognomic and even edaphic aspects, directly influence the ant community ecology and distribution. we classified them in both eurytopic and stenotopic species. it was also observed, using a correspondence factorial analysis (cfa), that the ants’ activity is seasonal and often correlated with temperature fluctuation and trophic availability. sociobiology an international journal on social insects y amara1, h tliba1, f bounaceur2, s daoudi1 article history edited by jacques delabie, uesc, brazil received 16 february 2018 initial acceptance 28 april 2018 final acceptance 16 december 2019 publication date 18 april 2020 keywords steppe, myrmecofauna, diversity, trophic availability, seasonal variation, algeria. corresponding author yacin amara department of forest and agricultural zoology national high school of agricultural sciences avenue hassan badi, el harrach algiers, es1603, algeria. e-mail: yacin.amara@hotmail.com social insects (dieng et al., 2016; passera & aron, 2005). they can be found everywhere, in forests as well as in open areas, near water or in dry places (cagniant, 1973). ants are widely applied in several biodiversity assessment programs (agosti et al., 2000), as indicators of ecological change and environmental control (andersen & majer, 2004), which is undoubtedly related to their importance in terms of biomass (hölldobler & wilson, 1990). in algeria, ants have been the subject of previous studies, particularly in the northern regions with a humid and sub-humid bioclimatic stage. these include studies by cagniant (1966, 1968, 1969, 1970 and 1973) and djioua et al. (2014) on ant communities in algerian forests. belkadi (1990) and oudjiane et al. (2007) studied the ants in kabylia region, while barech (2014) and dehina et al. (2007) investigated the ants in the algiers sahel. 1 department of forest and agricultural zoology, national high school of agricultural sciences, el harrach algiers, algeria 2 department of natural and life sciences, faculty of sciences and technology tissemessilt university, tissemessilt, algeria research article ants diversity, richness and composition of ant communities (hymenoptera: formicidae) in the pre-saharan steppe of algeria sociobiology 67(1): 48-58 (march, 2020) 49 however, the myrmecofauna of the steppe and southern algeria was unknown until bernard’s (1958) comparative study of ant communities of france and northern africa, including some of the algerian steppes, as well as the recent study of souttou et al. (2011) and barech et al. (2016). therefore, this study aims to provide an updated inventory of ants in algeria, to assess the diversity and distribution of ant species represented in the northern algerian sahara (steppe zone) and to determine their temporal succession in relation to seasonal variations. materials and methods study area and sample sites this study was carried out in the algerian steppe. it is characterized by an arid to semi-arid mediterranean climate, with low irregular rainfall (100 to 450 mm/year), high temperature ranges (over 40°c in summer and below 0°c in winter) and an altitude of 700 to 1,500 m a.s.l. (bencherif, 2011). the investigation was conducted in two distinct regions: (1) aflou: (34°06’n 02°06’e) which is located on the mountains of the saharan atlas, notably in the massive heart of djebel amour, at an altitude between 1,000 and1,500 m a.s.l. with slopes of 12,5 to 25%; and (2) laghouat: (33°47’n 02°51’ e) which is located in the southern foothills of the saharan atlas, at an altitude between 700 and 1,000 m a.s.l. and slopes from 0 to 3% (fig 1). the research was carried out in three types of habitats, the distance between them being up to 100 km: (i) pine forest (34°07’n 02°05’e): located on the high plain of the aflou region, at an altitude of 1,450m a.s.l. this habitat is a planted area aiming to reduce the creep of desertification and its spread to the fertile northern part of the country; it includes different types of plants, including aleppo pine (pinus halepensis mill.) associated with a formation of halfah grass (stipa tenacissima l.); (ii) dry river (33°08’n 02°53’e): situated in m’zi, laghouat region. the study site was a wadi (dry river), at an altitude of 760m a.s.l., characterized by herbaceous plants and represented mainly by the aristide grass (aristida pungens desf.) dispersed on sandy soil; (iii) dhaya (33°9’n 03°20’e): located in tilghimt, 100 km south of laghouat, at an altitude of 700m a.s.l. this station is a closed depression where non saline water accumulates and then evaporates or infiltrates very slowly into fine soils (pouget, 1992). the dhaya are mainly marked by the presence of the atlas pistachio (pistacia atlantica desf.) and the wild jujube (ziziphus lotus (l.) lam.) (fig 2). ant sampling ant sampling was carried out using two collecting techniques. the first one was the quadrat method, which consists of manually counting and capturing ant species over an area of 100 m2 (10 m x 10 m) (cagniant, 1973; suguituru et al., 2011), noting that the quadrat is randomly selected with three repetitions per station. the second method used was pitfall traps. this process consists in burying metal pots (7.5cm in diameter and 10.5cm deep) up to ground level; they are placed on a transect plane of 10 traps, with a space of 5m. two-thirds of the buried pots are filled with an attractive mixture (water with detergent) and left in place for 2 days (48 hours) (berville et al., 2016; el keroumi et al., 2012). fig 1. geographic situation of study area (steppe of algeria) and distribution of sampling sites. y amara, h tliba, f bounaceur, s daoudi – diversity, richness and composition of ant communities50 it should be noted that ants were sampled once a month for one year, from 2013 to 2014. ant species were counted by the quadrat method from early sunrise to noon, while the pitfall trapping was done from noon to noon on the second day. in each station, the total number of samples is therefore 3 x 12 quadrats and 10 x 12 pitfalls. as there are three stations, our whole study corresponds to 460 samples of ants. all ants were placed in vials, stored in 70% ethanol and labeled according to the type of collecting, trap number and any ecological information. ants were identified using the keys of cagniant (1996, 1997, 2009), cagniant and espadaler (1997) and the internet site of b. taylor (“ants of africa”). part of the ants was sent to professor henri cagniant (paul sabatier university, toulouse, france) for identification. voucher specimens of the ants were deposited in the collections of h. cagniant and zoology department of the national school of agricultural sciences (algeria). data analysis despite the great sampling effort, when we analyzed our data, our samples showed a fairly low independence and we preferred to consider the abundance rather than the frequency of occurrence of the collected ants. to analyze the ant diversity, the data of the pitfall traps of the various prospected habitats are evaluated by the following indices: total species richness index (s), relative abundance (ra%) is calculated as (ra = ni / n*100) where ni is number of individuals of taxon i and n is the total number of individuals of all species, dominance (d) it was calculated as d=σ(ni/n)2 and occurrence frequency (o %) was calculated according to dajoz (1985): (o = pi / p*100) where pi is the total number of samples containing the species taken into consideration and p is the total number of samples. from the ant occurrence frequency (o %), we distinguished: constant species (o ≥ 50%), accessory species (25% < o < 50%), accidental species (o ≤ 25%). in addition, the diversity of the three habitats was estimated using the shannon’s index (h) it was estimated as h = -σ(pi log pi), where pi is the proportion of individuals of the i species (pi = ni / n;), hmax is the maximum possible value of h, and is equivalent to (log2s) and pielou’s evenness index (j), it is calculated as j = h / hmax (the value of j varied between 0 (a single species dominates) and 1 (all species are equally abundant)). finally, the data were processed via correspondence analysis, in order to determine the activity of myrmecofauna during one year of study using past software version 2.17 (hammer et al., 2001) results myrmecofauna we found a total of 20 species of ants in the three studied stations (table 1), belonging to three sub-families: dolichoderinae represented by a single species: tapinoma nigerrimum (nylander, 1788), myrmicinae represented by messor arenarius (fabricius, 1787), messor aegyptiacus (emery, 1878), messor sanctus emery, 1921, messor medioruber santschi, 1910, messor capitatus (latreille, 1798), tetramorium biskrensis forel, 1904, tetramorium sericeiventre emery, 1877, monomorium salomonis (linnaeus, 1758), monomorium subopacum (smith, 1858), pheidole pallidula (nylander, 1848) and cardiocondyla batesii forel, 1894. fig 2. photographs showing the physiognomy of the three prospected stations (a) pine forest, (b) dry river and (c) dhaya. sociobiology 67(1): 48-58 (march, 2020) 51 the formicinae sub-family is represented by two genera only: i) camponotus with camponotus thoracicus (fabricius, 1804), camponotus foreli emery, 1881, camponotus alii forel, 1890 and camponotus barbaricus emery, 1905; ii) cataglyphis, represented by four species, namely cataglyphis bicolor (fabricius, 1793), cataglyphis bombycina (roger, 1859), cataglyphis albicans (roger, 1859) and cataglyphis rubra (forel, 1903). the abundance of each ant species was different according the sampling techniques used, since certain species were occurred only in the quadrat method, as it is the case of m. aegyptiacus, t. sericiventre, m. subopacum, c. thoracicus. the difference between the number of ant species taken by the two sampling methods was yet not statistically significant (wilcoxon signed-rank test; pitfall traps: p > 0.05, quadrat traps: p = 0.65) fig 3. occurrence frequency (o%) of ant species in the three habitats. total specimens (pin forest: 2264; dry river: 2511; dhaya: 1772) number of pitfalls, 10. a greater richness was recorded with the use of pitfall traps for pine forest, with nine species: m. capitatus, m. salomonis, t. biskrensis, c. foreli, c. barbaricus, c. alii, c. bicolor, c. albicans and c. rubra. in the dhaya of tilghimt, eight species of ants were identified, including m. sanctus, m. medioruber, t. biskrensis, m. salomonis, p. pallidula, c. batesii, c. thoracicus and c. bicolor. in the dry river of m’zi, only five species were detected: t. nigerrimum, m. arenarius, m. salomonis, c. bombycina and c. bicolor. sometimes abundance reflects better the ant activity in the locations we studied according the ant sampling mode. in the 3 habitat types (table 2), the pitfall traps revealed that m. capitatus is especially abundant exclusively in forest, while the highest relative abundance was noted for t. nigerrimum (39.3% of the whole individuals in the traps used for sampling) in the dry river station, followed by c. bombycina (36.6%). in the dhaya of tilghimt, c. bicolor is dominant (41.5% of the whole individuals in the traps used for sampling), followed by p. pallidula (20.4%) (table 2). for the frequency of occurrence (o %) of ants species recorded by pitfall, in the pin forest, on the nine species of ants reported, c. bicolor is considered as the only constant species, the rest of the ants are presented in the category of accessory species in the dry river t. nigerrimum with c. bombycina are constant ants while c. bicolor is classified in the category of accessory species; however m. arenarius and m. salomonis have appeared less in dry river. the rates of occurrence of ants in the daya are as follows: c. bicolor and p. pallidula have appeared consistently, the species of messor and c. batesii are classified as accessory species and the last species represented by c. thoracicus and m. salomonis appeared accidentally. accidental species accessories species constant species y amara, h tliba, f bounaceur, s daoudi – diversity, richness and composition of ant communities52 species captured by pitfall traps. however, there are only two species common in the three types of vegetation, namely c. bicolor and m. salomonis. the other ants are considered as characteristic to each region (table 2). subfamily species pitfall quadrat dolichoderinae tapinoma nigerrimum (nylander, 1788) 986 1,095 myrmicinae messor capitatus (latreille, 1798) 763 241 messor arenarius (fabricius, 1787) 13 298 messor aegyptiacus (emery, 1878) 0 192 messor medioruber santschi,1910 288 678 messor sanctus emery, 1921 221 552 tetramorium biskrensis forel, 1904 184 95 tetramorium sericeiventre emery, 1877 0 17 monomorium salomonis (linnaeus, 1758) 430 359 monomorium subopacum (smith, 1858) 0 23 pheidole pallidula (nylander, 1849) 362 468 cardiocondyla batesii forel, 1894 149 133 formicinae camponotus barbaricus emery, 1905 296 5 camponotus foreli (roger, 1859) 126 132 camponotus alii forel, 1890 116 282 camponotus thoracicus (fabricius, 1804) 1 12 cataglyphis rubra (forel, 1903) 224 67 cataglyphis albicans (roger, 1859) 76 43 cataglyphis bicolor (fabricius, 1793) 1,421 1,322 cataglyphis bombycina (roger, 1859) 918 823 total richness 17 20 table 1. abundance of ant species collected at the steppe of algeria by two sampling methods the study areas are very different from the points of view of their floristic nature and their physiognomic and even edaphic aspects, and seem to directly influence the ant community ecology and distribution, particularly the 17 table 2. ant species collected by pitfall trapping in the three habitats. subfamily species pine forest dry river dhaya ni ra% ni ra% ni ra% dolichoderinae tapinoma nigerrimum (nylander, 1788) 986 39.3 myrmicinae messor capitatus (latreille, 1798) 763 34 messor arenarius (fabricius, 1787) 13 0.5 messor medioruber santschi,1910 288 16.3 messor sanctus emery, 1921 221 12.5 tetramorium biskrensis forel, 1904 38 6.5 38 2.1 monomorium salomonis (linnaeus, 1758) 298 12.8 92 3.7 49 2.8 pheidole pallidula (nylander, 1849) 362 20.4 cardiocondyla batesii forel, 1894 149 8.4 formicinae camponotus barbaricus emery, 1905 269 11.9 camponotus foreli (roger, 1859) 126 5.6 camponotus alii forel, 1890 116 5.1 camponotus thoracicus (fabricius, 1804) 1 0.1 cataglyphis rubra (forel, 1903) 224 9.9 cataglyphis albicans (roger, 1859) 76 3.4 cataglyphis bicolor (fabricius, 1793) 255 11.3 255 20 664 37.5 cataglyphis bombycina (roger, 1859) 918 36.6 ni: number of individuals, ra%: relative abundance sociobiology 67(1): 48-58 (march, 2020) 53 species diversity the high total specific richness of ants in forest environment (9 species) also matched with the results obtained for the shannon index of diversity. the results showed that this environment is the most diversified (h=1.96), followed by dhaya (h=1.64) and the dry river (h=1.20). on the other hand, the dominance index in the three habitats studied is slightly high in the dry river of m’zi (d=0.3) compared to that of the dhaya (d=0.2) and the pine forest (d=0.1). the pielou’s evenness index varies between j= 0.74 to j=0.89 at the three locations. the values tend towards 1, which means that the number of species remains in equal proportion and tends to be stable (table 3). pine forest dry river dhaya taxa (s) 9 5 8 individuals 2264 2511 1772 dominance (d) 0.17 0.32 0.23 shannon index (h’) 1.96 1.20 1.64 simpson (1-d) 0.82 0.67 0.76 evenness index (j) 0.89 0.74 0.78 table 3. diversity indexes and evenness for the three habitats. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% iii iv v vi vii viii ix x xi xii i ii o cc up an cy r at e % month pine forest camponotus alii camponotus foreli camponotus barbaricus cataglyphis rubra cataglyphis albicans cataglyphis bicolor monomorium salomonis tetramorium biskrensis messor capitatus 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% iii iv v vi vii viii ix x xi xii i ii o cc up an cy r at e % month dhaya camponotus thoracicus monomorium salomonis tetramorium biskrensis cataglyphis bicolor cardiocondyla batesii pheidole pallidula messor sanctus messor medioruber 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% iii iv v vi vii viii ix x xi xii i ii o cc up an cy r at e % month dry river monomorium salomonis messor arenarius cataglyphis bombycina cataglyphis bicolor tapinoma nigerrimum fig 4. seasonal variation in ant community trapped by pitfall in the three habitats prospected. seasonality effect on ants’ activity most species show a variation in their seasonal activity according to their habitats (fig 4). the overall average abundance y amara, h tliba, f bounaceur, s daoudi – diversity, richness and composition of ant communities54 of ant populations recorded by the pitfall traps was calculated for each season in all studied stations to better understand the activities of the ants living in algerian steppe. in figure 1, we can observe the maximum activity peak of each ant species in each habitat. in the pin forest, the peak activity of ants is recorded mainly for the spring and summer months. this is not the case in the dry river and the dhaya, there are some species which have the peak in spring, other in summer and also in autumn. in the pine forest, there is less seasonality than in the other two habitats. the activity of myrmecofauna during one year is illustrated by a factorial analysis of correspondence (fig 5). the analysis is satisfactory since the sum of the variables’ projections on the plane f1 and f2 is greater than 50% of the total variance. the correspondence analysis of ant showed that the seasons are represented in three quadrants. each quadrant reflects a different spatial and temporal distribution of ant species. the first quadrant corresponds to the ant populations that are active in the spring season, represented by t. nigerrimum, c. albicans, c. alii, c. foreli and c. batesii. the second quadrant concerns the most abundant ants during the summer, namely c. bicolor, c. bombycina, c. rubra,c. barbaricus, c. thoracicus, m. capitatus, t. biskrensis and m. salomonis. finally, the third quadrant is characterized by ants observed during autumn period. it includes the following species: messor (m. sanctus, m. medioruber, m. arenarius) and p. pallidula (fig 5). spring summer automn w inter t. nigerrimum c. bicolor c. bombycina m. arenarius m. salomonis m. capitatus t. biskrensis c. albicans c. rubra c. barbaricus c. f oreli c. alili m. medioruber m. sanctus p. pallidula c. batesii c. thoracicus -0,9 -0,6 -0,3 0,3 0,6 0,9 1,2 1,5 1,8 -0,60 -0,48 -0,36 -0,24 -0,12 0,12 0,24 0,36 0,48 ax is 2 (1 8. 43 % ) axis 1 (70.99%) group 03 group 01 group 02 c. alii fig 5.correspondence analysis of ant activity according to season. discussion diversity and distribution of ants based on the steppe inventory, a total of 20 ant species were recorded using the quadrat method and 17 species using the pitfall trapping. the difference between the richness values of the two sampling techniques was evident, particularly for species of the genus messor, c. barbaricus, c. thoracicus, m. subopacum and t. sericeiventre. this can be explained by some behavioral traits of these species. messor spp., for example, are more abundant in quadrats than in pitfalls probably due to the use of trails for foraging, and it is less likely than the workers fall into traps except when a trail ends on a pitfall. in this case, they fall in large number and it seems to be the case of m. capitatus (x. cerdá, personal communication). as many camponotus, c. barbaricus and c. thoracicus are mostly nocturnal, and this is certainly the reason why they are less detected in quadrats (cagniant, 2009). bernard (1958) identified 16 species of ants living in the algerian-tunisian steppes, close from the location of the present study, souttou et al. (2011) reported 15 species of ants in the steppe of the djelfa region, while in another part of djelfa, bouzekri et al. (2015) recorded nine species of ants only. until this moment, the single study on an algerian steppe ant community showing a higher richness (s = 24 species) is that of barech et al. (2016). furthermore, between12 and 14 species were found in the tunisian steppe (heatwole & muir, 1989) while in iran, from 7 to15 species were sampled in several steppe locations (paknia & pfeiffer, 2011). sociobiology 67(1): 48-58 (march, 2020) 55 the richness of the forest environment (pine forest) is consistent with djioua et al. (2014) with s= 15, including 13 species nesting in forest environment. furthermore bougherara (2009) studied an oak grove at chréa (algeria) (1,450 meters a.s.l.) where he reported the occurrence of 9 ant species. the study on the heterogeneity of the distribution of ant fauna between study stations is consistent with cagniant (1973). the author noted that this heterogeneity can be explained by the influence of climate and composition of the vegetation cover. similarly, gaspar (1972) showed that climatic and soil factors directly influence the ant distribution. at salt lake chott el hodna (algeria), barech et al. (2016) also noted that the distribution of ants is dependent not only to soil texture but also to its composition (salinity). our study suggests that the distribution of formicidae can be classified into two groups. the first one is represented by eurytopic species, which have a wide distribution and occupy various biotopes. this is the case of c. bicolor, common in the three stations surveyed. this observation corroborates cagniant (1969, 1970) who indicated that this cataglyphis is common throughout algeria as well as in the sahara oases with color variability; the darkest forms are those of the saharan atlas nesting in open areas. m. salomonis is a second species that can be attributed to this group. according to bernard (1958) this omnivorous ant is dominant especially in the arid steppe and desert environments. the second group includes stenotopic species, frequently localized in a specific area; this is the case of camponotus spp., which use to climb trees and are widespread in all forests (cagniant, 1996). on the other hand, bernard (1945, 1958) reported that t. nigerrimum are found near wadis. this statement is in agreement with our own observations since this species has been recorded only in the dry river at m’zi station. according to bernard (1968), t. nigerrimum is sensitive to soil nature and its nests are dug in moist, clayey soils or highly permeable sand. the genus of harvester ants messor, represented by five species in our study, includes m. capitatus which is frequent in forests and mountains at an altitude between 1,000 and 1,700 m (cagniant & espadaler, 1997). cagniant (1997) corroborates our observation about m. arenarius since he noted that this ant is a sabulicolous species found in the saharan ergs, wadi sands, high plains and sahara atlas. the distribution of m. aegyptiacus is observed mainly in the northern edges of the sahara (cagniant, 1997), which corresponds to one of the locations of this study. two species of the genus cataglyphis were common in the pine forest: c. albicans and c. rubra. this result agrees with bouzekri et al. (2015) and bernard (1958), who reported the dominance of c. albicans in the algerian-tunisian steppes where it lives in dry, hard and steppe-like habitats (wehner, 1983). cagniant (2009) confirmed the occurrence of c. rubra in the entire pre-saharan region of morocco in the steppes and other stony areas between 800 and 1,400 m a.s.l., as well as in the algerian high lands. sheltered by tufts of aristida pungens, the nests of c. bombycina are dug in pure sand and can occupy a very large area. this “silver ant” is common on dunes and other sandy areas (cagniant, 2009). the ants’ diversity is reported to be more variable in forest areas (sommer & cagniant, 1988). indeed, this diversity uses to be high when it is not subjected to any external disturbance (faurie et al., 2006). this is consistent with the results of this study (table 3) and with barech et al. (2016), with a diversity of h= 1.4 and h = 1.35, respectively. on the other hand, a low diversity (h between 0.02 and 0.7) was observed in algerian cultivated zones as a citrus orchard by fekkoun et al. (2011) and in cultivated areas by bouzekri et al. (2015) and dehina et al. (2007). seasonal activity of ants according to the correspondence analysis, the temporal dynamics of the ants in the different habitats sampled here was categorized into three groups. as a result, temperature is the most important factor which affects the spatial and temporal variation of ants which can be then considered as thermophilic (hölldobler & wilson, 1990). pfeiffer et al. (2003) also noted that the distribution of the ants in the mongolian steppes depends on temperature amplitude. furthermore, seed-harvesting ants, such as m. sanctus, m. medioruber, m. arenarius and p. pallidula, seem to be more abundant and active in autumn. these species are important seed consumers in several desert ecosystems (cagniant, 1973; pirk et al., 2009). this explains their dominance during this period coinciding with seed maturation when they found an amount of this interesting trophic resource. similarly, cros et al. (1997), in a study on the activity of mediterranean ants, reported a maximum foraging activity of ants of the genus messor and p. pallidula during autumn (september and october), except for m. capitatus which is more active during summer. in addition, cros et al. (1997) observed that this species is mainly active at dusk and at night during the summer months, as well as c. thoracicus and c. barbaricus, also observed in this study. in the case of the xerophilic genus cataglyphis, sunlight and temperature rise seem to be the main factors determining worker foraging (tohmé et al., 1985; cerdá et al., 1994; lenoir et al., 2009). this is the case of c. bicolor, c. bombycina, c. rubra and c. albicans. a strictly springtime activity were observed in t. nigerrimum, c. batesii and two species of camponotus, represented by c. foreli and c. alii. our observations of the ant seasonal activity corroborates a study on the impact of the seasons on the abundance, diversity and species composition of an ant community in a semi-arid zone of australia (andersen, 1983). similarly, basu (2008) observed significant seasonal fluctuations in ants’ activity in a tropical forest in the western ghats (india).consequently, ants’ activity in algerian steppe strongly seems to be influenced by y amara, h tliba, f bounaceur, s daoudi – diversity, richness and composition of ant communities56 seasonal variations, which are often correlated with temperature variations (retana & cerdá, 2000; bollazzi & roces, 2002) and trophic availability (steinberger et al., 1992). acknowledgments we would particularly like to acknowledge the reviewers and express our gratitude to professor xim cerda for his valuable advice. special thanks to jacques delabie and elmo koch for their revisions on earlier versions of the manuscript. we also extend our gratitude to professor henri cagniant for the confirmation of ant identifications. references agosti, d.; majer, j. d.; alonso, l. e., schultz, t. r. 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(1985). contribution à l’étude systématique et bioécologique de cataglyphis frigida (hymenoptera, formicidae). revue française d'entomologie, 7: 83-88. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.145-154sociobiology 61(2): 145-154 (june, 2014) richness of termites and ants in the state of rio grande do sul, southern brazil e diehl1, e diehl-fleig1, ez de albuquerque2, lk junqueira3 introduction studies on biodiversity are urging in face of diversity loss (wilson, 1997; mcgeoch & chown, 1998). termites (mill, 1982; constantino, 1992; eggleton et al., 1995; black & okwakol, 1997; lavelle et al., 1997; jones, 2000; gathorne-hardy et al., 2001; jones et al., 2003) and ants (majer, 1983; hölldobler & wilson, 1990; andersen, 1997; silva & brandão, 1999; ward, 2000; ribas et al., 2012) have been considered biodiversity indicators due to their biological and ecological characteristics. these groups have also been considered as surrogate groups in evaluations of conservation status, degradation, or recovery of terrestrial ecosystems (new, 1996; majer, 1996; andersen, 1997; majer & nichols, 1998; lobry de bruyn, 1999; jones & eggleton, 2000; bandeira & abstract previous studies on the effects of environmental factors, such as altitude, latitude, temperature, deforestation, forest fragmentation, fire, and flood on the community structure of termites and ants were conducted in various regions of brazil; few of them were carried out in the southernmost brazilian state of rio grande do sul. here we describe termites and ants diversity at different sites along the four geomorphologic units of this state. we recorded 16 taxa of termites, of which three are new state records, increasing to 19 the number of termite species known to occur in the state. accordingly, we also found 73 species and 115 morphospecies of ants, of which only one was a new record, raising to 265 taxa the number of ant species known to occur in the state. as expected, we found a higher species richness of ants than termites. the low richness of both groups relative to other brazilian regions could be a consequence of the subtropical to temperate climate in the state, since most portions of the state are below 30o latitude, the study areas be above 500 m altitude, and other environmental characteristics of each site. we suggest a positive relationship between species richness of termites and altitude, while ant richness indicated an inverse relationship. however, our data are not conclusive, due to the low number of replications in each altitude, particularly for termites. this study is unique in presenting an updated checklist of termites and ants in the state of rio grande do sul. sociobiology an international journal on social insects 1 universidade federal do rio grande do norte, natal, rn, brazil. 2 museu de zoologia, universidade de são paulo, são paulo, sp, brazil. 3 pontifícia universidade católica de campinas, campinas, sp, brazil. article history edited by gilberto m m santos, uefs, brazil received 18 december 2013 initial acceptance 28 january 2014 final acceptance 06 april 2014 keywords social insects, distribution, altitude. corresponding author eduardo diehl-fleig instituto do cérebro, universidade federal do rio grande do norte av. nascimento de castro 2155 natal, rn, brazil 59056-450 e-mail: edf.formiga@gmail.com vasconcello, 2002; diehl et al., 2004). like ants, termites are entirely eusocial and have profound ecological significance in the tropics (engel et al., 2009). studies have shown that ants and termites play a crucial role to create soil structure, influence aeration, water infiltration and nutrient cycling acting as ecosystem engineers (lobry de bruyn & conacher, 1990; lavelle et al., 1997; mora et al., 2005; jouqueta et al., 2011; del toro et al., 2012). in some cases, termites and ants are also responsible for increased crop yield under dry conditions through soil water infiltration due to their tunnels and improved soil nitrogen (evans et al., 2011). environmental disturbances, such as deforestation, forest fragmentation, fire, and floods may affect the community structure of termites (de souza & brown, 1994; eggleton et research article ants diehl et al termites and ants from rio grande do sul, brazil146 al., 1994, 1995; davies, 2002; bandeira et al., 2003; inoue et al., 2006) and ants (hölldobler & wilson, 1990; majer & nichols, 1998; diehl et al., 2004; schmidt & diehl, 2008). previous studies have shown that local species richness of ants and termites is influenced by environmental factors, such as temperature, rainfall, and vegetation (benson & harada, 1988; silva & brandão, 1999; gathorne-hardy et al., 2001; oliveira & del-claro, 2005; del-claro, 2008; jones & eggleton, 2011). there has also been some evidence that increasing altitude and decrease of temperature decrease species richness (kusnezov, 1957; collins, 1980; ward, 2000; gathorne-hardy et al., 2001) and that the phylogenetic structure might be involved in this process, specially in the temperate forests (machac et al., 2011). the neotropics rank in the third position in termite richness among the biogeographic regions, with the oriental and ethiopian ones presenting the highest number of species recorded so far (krishna et al., 2013). about 72% of the neotropical species occur in south america (constantino, 2014) and the sites with good termite surveys are between the parallels 0° s and 20° s, which include the biomes amazon and atlantic forests, cerrado and caatinga (constantino & acioli, 2006). a review of the termite fauna in across brazil can be found in constantino & acioli (2006). recent studies recorded 33 species for five fragments of semideciduous atlantic forest in northern brazil, varying locally from 11 to 27 species (souza et al., 2012). with different levels of perturbation, 26 species were recorded in a region of semiarid caatinga (vasconcellos et al., 2010). in southern brazil, termite fauna is poorly known. studies mention only 16 termite species to rio grande do sul (araújo, 1977; constantino, 1998; fontes, 1998; castro & diehl, 2003; diehl et al., 2005b; florencio & diehl, 2006). this low number can be due to the climate (eggleton et al., 1994; constantino & acioli, 2006), or lack of taxonomists and local studies (diehl-fleig et al., 1995; constantino & acioli, 2006). despite its importance, little is known about the edaphic insects, specially ants and termites, in southern brazil. thus, here we present the richness and a checklist of termites and ants in the four geomorphic units of rio grande do sul, reporting new records for state. the checklist also includes species from other inventories conducted in the state of rio grande do sul by researchers apart from our group leaded by e. diehl. most aimed at collecting species of acromyrmex and atta or urban ant fauna and few have focused on ant species inventories in forests and other relevant ecosystems (please see supplementary material for further details at): http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/313/408 doi: 10.13102/sociobiology/.v61i2.145-154.s451 material and methods study areas geomorphological units of rio grande do sul the southernmost brazilian state of rio grande do sul has four geomorphologic units: southern plateau, central depression, sul-riograndense shield, and coastal plain (fig 1). two main vegetation types originally occurred in the state: forests and grasslands occupying 34% and 46% of the state area, respectively. the remaining was occupied by coastal vegetation, wetlands, and other vegetation types. the vegetation reflects soil type and origin and forests usually occur where the soil is deep and fertile. grasslands develop where the soil is siliceous or shallow. while the southern region of the state is dominated by grasslands, the northern grasslands are interspersed with araucaria forest (rambo, 1994). the weather in southern brazil is subtropical humid, according to the köppen classification. while the average annual temperature in the coastal plain is around 18°c, temperature approaches 20°c in the central depression, which also has mild winters. conversely, the annual average temperature in the southern plateau does not exceed 15°c, due to its high altitude, winters are harsh, with often negative temperatures, severe frosts, and even deep snow. the campanha, in the sul-riograndense shield, is an open landscape and receives all continental winds in the winter, but due to their large grassland areas it receives much sunlight resulting in summers with high temperatures (rambo, 1994). sampling sites (fig 1) are described below and are mostly under different levels of disturbance: 1) remnants of seasonal semideciduous forest and suburban areas around são leopoldo (29° 45’ 29° 47’ s, 51° 05’ 51° 10’ w; 15 m a.s.l.) in the central depression (mayhénunes & diehl-fleig, 1994; diehl-fleig & fleig, 1997; haubert et al., 1998; flores et al., 2002; diehl et al. 2006; florencio & diehl, 2006; marchioretto & diehl 2006); 2) periurban and urban areas in canela (29° 21’ s, 50° 49’ w; 819 m a.s.l.) and gramado (29° 22’ s, 50° 52’ w; 825 m a.s.l.) in the southern plateau (diehl-fleig, 1997); 3) dunes and restingas in torres (29° 20’ 29° 23’ s, 49° 43’ 49° 46’ w; 0 16 m a.s.l.) in the northern coastal plain (diehl-fleig et al., 2000; hameister et al., 2003; albuquerque et al. 2005; diehl, unpub. ms.); 4) grasslands in the caçapava do sul (30° 47’ s, 52° 24’ w; 220 m a.s.l.) in soils with different copper levels, including a copper mine and a native savanna area in the camaquã river basin in the sul-riograndense shield (diehl et al., 2004); 5) restingas in morro da grota (30° 21’ s, 50° 01’ w; 263 m a.s.l.) near lagoa dos patos and a typical forest of granite sociobiology 61(2): 145-154 (june, 2014) 147 soil, sandy and rocky areas in pedreira beach (30° 21’ s, 50° 02’ w; at the sea level), on the banks of the guaíba lake, both at the itapuã state park in viamão, which is within both sul-riograndense shield and coastal plain (sacchett & diehl, 2004; diehl et al., 2005a; diehl, unpub. ms.); 6) eucalyptus plantation in restingas in capivari do sul (30° 11’ s, 50° 21’ w; 12 m a.s.l.) and tramandaí (29° 59’ s, 50° 13’ w; 8 m a.s.l.) in the northern coastal plain (fonseca & diehl, 2004); 7) wetland used as pastures for cattle or irrigated rice in santo antônio da patrulha (29° 54’ s, 50° 33’ w; 25 m a.s.l.) in the coastal plain (diehl et al., 2005b; moraes & diehl, 2009); 8) an area of mixed ombrophilous forest in the são francisco de paula national forest (29° 23’ 29° 27’ s, 50° 23’ 50° 25’ w; 930 m a.s.l.) and in the pró-mata research and nature conservation center (29° 28' s, 50° 13' w; 900 a.s.l.) in the southern plateau (diehl et al., 2005c; pinheiro et al., 2010); 9) vineyards (29° 56’ s, 51° 33’ w; 645 m a.s.l.) in bento gonçalves, within the northeastern central depression (sacchett et al., 2009); 10) yerba mate plantations in mato leitão (29° 31’ s, 52° 07’ w; 81 m a.s.l.), ilópolis (28° 55’ s, 52° 07’ w; 683 m a.s.l.), and putinga (29° 00’ s, 52° 09’ w; 435 m a.s..l) in the northeastern central depression (junqueira et al., 2001; steffens, 2006); 11) primary forest and reforestation area in alto ferrabraz, sapiranga (29° 34’ s, 50° 56’ w; 500 m a.s.l.). this is a transition zone between the southern plateau and the northeastern central depression (haubert, 2006); 12) secondary forest, with acacia plantation and an area with reforestation in rolante (29º 36’ 32.2” s, 50º 31’ 39.1” w; 370 m a.s.l.) in the central depression (schmidt & diehl, 2008); 13) grasslands in campos de cima da serra (29° 10’ s, 50° 10’ w; 1,200 m a.s.l) in the southern plateau (albuquerque & diehl, 2009); at each termite sampling site, we established a transect (100 m x 3 m), further divided into 20 sections of 15 m2. we then sampled ten non-contiguous sections on the right and left sides of the transect alternately, with a sampling effort of 60 minutes per section. in each section, we search for termites in leaf litter and humus at the base of trees and roots, under both rocks and fallen trees, inside logs, hollow twigs, decomposing fallen branches, epigean and arboreal nests. we also set up one termitrap® bait per section (almeida & alves, 1995) at 15 cm deep, 60 days before samplings. furthermore, we also searched for termites in ten blocks (340 cm3) of soil horizon a per section. termites were stored in individual amber glass with 80% ethanol. termites were identified to the genus level (fontes, 1995; constantino, 1999; 2002) and eventually to the species level or morphospecies. we also consulted specialist taxonomists to confirm the identification of termites. we used five techniques to sample ants: 1) manual capture of all ants on the ground, under stones, in hollow trees, and branches; 2) baits with sardines in vegetable oil on a piece of filter paper (6 cm2); 3) leaf litter samples, firstly sieved in the field and further using winkler extractors, in which they remained for 72 hours; 4) pitfall traps with and without attractive, which consisted of 200-300 ml plastic cups buried up to the top containing 70% ethanol, remaining on the ground for 24 to 48 hours. attractive traps were composed by sardines in vegetable oil pierced in a galvanized wire rod; 5) underground trap with attractive, which was a small plastic pot (3.3 cm wide by 5 cm high) with small holes on the sides. within each trap a piece of 1 cm3 sardines in vegetable oil was added. traps were buried 20 cm deep by 48 hours. in grasslands we used direct sampling, sardine baits and pitfall traps. in dunes, we only used direct sampling and sardine baits. at each site, we established two to three 100 m transects, along which we sampled every 10 m. sampling effort was at least 2 hours in each site. we classified ants into subfamilies following bolton (2014) and by genus following bolton (1994) and fernández (2003). the subfamilies ponerinae and ecitoninae were treated according to the most recent reviews see supplementary material for details. we used several identification keys and also consulted specialists to identify species. we assigned specimens to morphospecies when specific identification fig 1. distribution of combined termite and ant sampling (crossed circles) and other ant inventoried sites (open circles) in the four geomorphologic units of rio grande do sul, southern brazil by diehl group (geomorphologic map adapted from seplag, 2008). diehl et al termites and ants from rio grande do sul, brazil148 was not possible. at least three specimens of each species/ morphospecies were mounted with entomological pins and the remaining preserved in 70% ethanol. specimens of termites and ants are housed at the collection of social insects (isoptera and formicidae) of the universidade do vale do rio dos sinos (unisinos), são leopoldo, rio grande do sul, brazil. the checklist presented results of ca. 30 years of collections leaded by e. diehl added to most ant inventories conducted by few other researchers in the state of rio grande do sul. we reviewed papers published from 1972 to 2014 included in the databases isi web of science, scielo and google scholar that contained the keyword combination: ant or formicidae and rio grande do sul. only papers referring nominal species and site location (municipality or more specific location) were considered, except those with doubtful identification (approximate identification, to be confirmed, compared to, of group, subgenus or species complex). three studies on atta and acromyrmex distribution were excluded because site location was not provided. species previously registered to the state was provided by the catalog of kempf (1972) with additions of brandão (1991). results we recorded 16 isopterans from two families (table 1). we only found one species of the kalotermitidae genus rugitermes. we also recorded a new genus of termitidae, besides seven morphospecies and seven species from four subfamilies (apicotermitinae, nasutitermitinae, syntermitinae, and termitinae). termite species richness was very low. we recorded 10 species in the southern plateau (181-1,200 m a.s.l.) and 15 in the central depression (20-30 m), whereas in the sul-riograndense shield (23-29 m) and northern coastal plain (5-15 m) we found the lowest richness: four and six species, respectively. we found 26 ant genera from seven subfamilies (dolichoderinae, dorylinae, ectatomminae, formicinae, heteroponerinae, myrmicinae, and ponerinae) that were stored in 70% ethanol. furthermore, we recorded 51 genera from nine subfamilies (amblyoponinae, dolichoderinae, dorylinae, ectatomminae, formicinae, heteroponerinae, myrmicinae, ponerinae, and pseudomyrmecinae), which were stored in entomological drawers. in total, we recorded 73 species and 115 morphospecies in rio grande do sul. compiling only nominal ant species registered by our group and other researchers, 127 species distributed among 55 genera from 10 subfamilies occur in the state (an extended list is available as supplementary material). a single species, monomorium floricola, is reported as new record for the state of rio grande do sul. thus, ant species richness in rio grande do sul raises to 265, considering published data on nominal species from the catalogs (kempf, 1972; brandão, 1991), from our research group and the other groups. this number would likely increase, if we family/ subfamily taxon kalotermitidae rugitermes sp. (1, 3, 4)* termitidae apicotermitinae apicotermitinae 1 (1, 2, 3, 4) apicotermitinae 2 (1, 2, 3, 4) grigiotermes bequaerti (snyder & emerson, 1949) (3) grigiotermes sp. (3) ruptitermes sp. (3) tetimatermes sp. (3) new genus 1 (3) nasutitermitinae araujotermes caissara fontes, 1982 (1) cortaritermes fulviceps (silvestri, 1901) (1, 2, 3, 4) nasutitermes aquilinus (holmgren, 1910) (3, 4) nasutitermes jaraguae (holmgren, 1910) (3, 4) syntermitinae cornitermes cumulans (kollar, 1832) (2, 3, 4) termitinae dihoplotermes inusitatus araújo, 1961 (3, 4) neocapritermes sp. (1, 3, 4) termes sp. (3, 4) *geomorphologic units: 1, coastal plain; 2, sul-riograndense shield; 3, central depression; 4, southern plateau table 1. termites recorded in the state of rio grande do sul, brazil. consider the morphospecies not yet identified to the species level and other published studies with nominal species and site location indicated (see studies available as supplementary material). ant richness and composition varied along the geomorphologic units. we found 88 species/morphospecies in the southern plateau (900-1,200 m a.s.l.), 40 in the transition between the southern plateau and the central depression (500 m), 108 in the central depression (10-683 m), 51 in the sulriograndense shield (220 m), 81 in the transition between the sul-riograndense shield and the coastal plain (16-263 m), and 97 in the coastal plain (0-80 m). of the 188 species/morphospecies, most (40.43%) were restricted to a single geomorphologic unit, while only ca. 2.5% were common to all units (pheidole sp.3, pheidole sp.6, pheidole sp.15, wasmannia sp. and wasmannia sp.1). excluding three sites where ant fauna was inventoried exclusively in agricultural or urban areas, there is some indication that species richness and composition could vary in response to an altitudinal gradient. the highest ant richness, 56, 81, and 73 species were found in sites at the sea level, 10 m, and 26 m, respectively. conversely, we found 51, 35, 39, 55, and 33 species in sites at 220 m, 370 m, 500 m, 930 m, and 1,200 m, respectively. of the 158 ant species/morphospecies in these eight areas, none were present in all sites, only ca. 3% were common to seven areas. the great majority (35.44 %) of ants were restricted to a single site. sociobiology 61(2): 145-154 (june, 2014) 149 discussion according to krishna et al. (2013) there are 2,937 termite species worldwide, of which 569 are found in the neotropics. the on-line termite database maintained by constantino (2014) refers 2,882 termite species in the world, of which 562 species occur in the neotropics (database was last updated in september 2012). estimates of termite richness for brazil are not yet accurate, ranging from 250 to 364 species (cancello & schlemmermeyer, 1999; constantino, 1999; 2014; fontes & araújo, 1999). however, the estimated species richness should increase with termite sampling in other areas of the country. our results provide records of a new genus likely belonging to apicotermitinae, another unidentified species of this subfamily (apicotermitinae 1), and dihoplotermes inusitatus. thus, the number of known termite species in rio grande do sul increases to 19. drywood termites of the family kalotermitidae have major economic importance and a wide geographic distribution. these termites are poorly known because they inhabit places difficult to access (jones & eggleton, 2011). the largest and most diverse isopteran family is termitidae (eggleton & tayasu, 2001). we found species from four out of the eight termite subfamilies previously known to occur in rio grande do sul (apicotermitinae, nasutitermitinae, syntermitinae, and termitinae). a negative correlation between altitude and species richness has been reported for various organisms (rahbek, 2005; mccain, 2009; mccain & grytnes, 2010). this correlation seems to be dependent on altitude in tropical and subtropical areas. altitude is positively correlated with richness in latitudes below 30° and below 500 m, but negative in latitudes above 30° and at altitudes above 500 m (kusnezov, 1957; ward, 2000). we found indication that termite species richness relates positively to altitude. previous studies (inoue et al., 2006) found the species richness of nasutitermitinae increased with altitude, while species richness and abundance of macrotermitinae decreased with altitude. the other groups were apparently not influenced by altitude. recently, palin et al. (2010) evaluated the influence of the variation along an amazon–andes altitudinal gradient in peru on richness, abundance, and diversity of functional groups of termites. they registered 49 species and verified that, in general, the diversity declined with increased elevation. the functional groups responded differently to the upper distribution limit: for the soil-feeding it was between 925 and 1,500 m a.s.l., while the wood-feeding termites was between 1,550 and 1,850 m a.s.l. and this differential response led the authors to suggest that the energy requirements for each group are a key factor in shaping their occurrence associated with the altitude and temperature. termite species richness seems to be influenced by local environmental conditions, such as altitude, temperature, rainfall, and vegetation (collins, 1980; gathorne-hardy et al., 2001; davies, 2002; inoue et al., 2006; jones & eggleton, 2011). the species richness of termites we found is as low as reported in subtropical and temperate areas. for example, previous studies found two to eight morphospecies in the atlantic forest of southeastern brazil, whereas an average of 30 morphospecies were found in tropical forests near bahia, northeastern brazil (cancello et al., 2002). the far southernmost part of south america has often been considered as a separate biogeographic sub-region. it is depauperate in termite species due to its high latitude (eggleton, 2000), and countries like chile and uruguay have even fewer species registered: six and nine, respectively, while 37 are reported for paraguay (constantino, 2014). argentina, on the other hand, has 57 species reported (constantino, 2014), even though this number could increase to 95 (torales et al., 2008). argentina local termite species varies from 12 in the chaco province (godoy et al., 2012) to 26 species in northeast argentina (laffont et al., 2004). we present the first two records of the rare, monotypic genus dihoplotermes to rio grande do sul. a colony of d. inusitatus was found inhabiting a nest cornitermes bequaerti in gallery forests in mato grosso (mathews, 1977), whereas other studies (constantino, 1999) found d. inusitatus in the cerrado and disturbed habitats in the southeast. we found the first record of d. inusitatus within a nest of c. cumulans in grasslands surrounding the são francisco de paula national forest. however, we could not find this species in further surveys. this is a rare species that could have went locally extinct after a pinus plantation covered the area. the other record of d. inusitatus was also in a nest of c. cumulans in the suburb of são leopoldo, more than 500 km away from the first site. with the increase of urbanization in the region, this rare species is subject to rapid local extinction. the family formicidae comprises more than 15,700 species and subspecies (antweb, 2013); recent estimates suggest that this number should exceed 23,000 species (antweb, 2013). however, previous studies mention 208 (kempf, 1972) or 224 species brandão (1991) to rio grande do sul. since its last catalog update (brandão, 1991), 23 years ago, our study is the first to present an updated checklist of the ant species in rio grande do sul. if we take into account the determination of species level of the all morphospecies in our collection at unisinos and in other studies, the species list would likely increase. only few list are available for other states, such as santa catarina (ulysséa et al., 2011), where ant richness (366 species and 17 subspecies) is greater than the 265 species now updated to rio grande do sul. apart from different collection efforts, historical aspects linked to the myrmecology development are part of this numerical difference reported. ant inventories in rio grande do sul until the early 2000s focused primarily in the genera atta and acromyrmex (e.g., diehl-fleig, 1997). this attines have long been known for their pest status and major impact on agriculture, fairly diehl et al termites and ants from rio grande do sul, brazil150 common and economically relevant throughout the state. it is clear that ant fauna inventories in rs are still very scarce and the geomorphic units are undersampled, particularly the pampa biome. this grassland ecosystem is unique in fauna and vegetation among brazilian biomes and less than 42% remains preserved (roesch et al., 2009). so far, ant fauna has been surveyed mostly in the atlantic forest biome in the state (diehl et al., 2005c; albuquerque & diehl, 2009; pinheiro et al., 2010) while a single study included the pampa (marques & schoereder, 2014). the diversity of habitats, vegetation, altitude, latitude, and climate between sampling sites should be the main factors responsible for the differences found in termite and ant species richness along the study areas and also other tropical areas in brazil (oliveira & del-claro, 2005). currently, 106 ant genera are known to occur in brazil (antwiki, 2013), while the termite diversity is much lower, only 74 genera (constantino, 2014). as expected, we found a higher richness of ant genera than termite along the studied areas. species diversity decreases with increasing distance from the equator (kusnezov, 1957; eggleton et al., 1994; ward, 2000). therefore, the lowest species richness of termites and ants found by us was expected. additionally, the low richness may be due to the subtropical climate, the low latitude (< 30º), and high altitudes (> 500 m), as might be case for our findings that ant richness was lower at higher altitudes. moreover, sampled sites varied in the degree of habitat complexity, from single crop to native forest, which also play a key role in shaping species richness and composition with different outcomes (e.g., lassau & hochuli, 2004; silva et al., 2007; pacheco & vasconcelos, 2012). this study is unique in compiling the richness of both termites and ants so far known in the state of rio grande do sul and we hope it provides a landmark in exposing the necessity for future studies and establishing efforts in unraveling our eusocial insect diversity. acknowledgements we would like to express our gratitude to three anonymous reviewers, to dr. jacques h. delabie, dr. antônio j. mayhé-nunes, dr. rodrigo dos s.m. feitosa, dr. carlos e. sanhudo and thiago ranzani who helped us to confirm either/or identify ant species, in addition to providing several dichotomous keys. we also thank dr. reginaldo constantino and dr. luiz r. fontes for identification of termites. daniela f. florencio, laura v.a. menzel and carlos e. sanhudo helped in field and laboratory work. fapesp provided financial support to e.z. de albuquerque (proc. n. 2010/02560-5). e. diehl was supported by cnpq. formigas do brasil research group provided much of the ant bibliographic review. we thank unisinos for the laboratory facilities.. references albuquerque, e.z.; 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(1997). a situação atual da diversidade biológica. in e.o. wilson & f.m. peter (eds.), biodiversidade (pp. 3-24). rio de janeiro: nova fronteira. doi: 10.13102/sociobiology.v62i1.116-119sociobiology 62(1): 116-119 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 clonal composition of colonies of a eusocial aphid, ceratovacuna japonica introduction altruistic behavior, in which sterile individuals benefit their kin individuals, has evolved in eusocial insects of some taxonomic groups (trivers & hare, 1976; hölldobler & wilson, 1990; pike & foster, 2008). eusocial aphids produce sterile individuals (so-called soldiers) that protect their colony mates from predators (stern & foster, 1996; pike & foster, 2008). soldiers have morphological, behavioral and physiological traits that have specialized functions against predators (kutsukake et al., 2004; hattori & itino, 2008; hattori et al., 2013a, b). for example, soldiers of the eusocial aphid ceratovacuna japonica (homoptera, hormaphidinae) have longer horns and forelegs than non-soldiers (hattori & itino, 2008). when they encounter a predator, they grasp it with their forelegs and then stick their frontal horns into the body of the predator to kill it or to delay its predation (hattori et al., 2013a). kin selection theory predicts that such a reproductive division of labor is maintained by high genetic relatedness within a colony (hamilton, 1964). in accordance with this abstract high degrees of relatedness among colony mates and kin recognition ability are important factors maintaining eusociality because kin-selection favors eusociality when donor and recipient of altruistic acts are related to each other. however, in eusocial aphids, clone mixing between different colonies occurs frequently, suggesting a lack of kin recognition. studies investigating the clonal composition of eusocial aphid colonies have focused on the aphid generation on the primary host plant (gall generation) and showed that clone mixing occurs frequently among galls (= nest). to test whether clone mixing also occurs in open colonies of eusocial aphids on their secondary host plants (opencolony generation), we carried out an amplified fragment length polymorphism analysis to investigate clonal composition within colonies of the eusocial aphid ceratovacuna japonica. the results showed that clone mixing occurred frequently in open-colony generation. they suggest that not only relatedness but also other factors (e.g., ecological background) are important for maintaining eusociality in eusocial aphids. sociobiology an international journal on social insects shinshu university, matsumoto, nagano, japan article history edited by kleber del-claro, unb, brazil, brazil received 26 may 2014 initial acceptance 30 june 2014 final acceptance 05 august 2014 keywords amplified fragment length polymorphism, clone mixing, kin recognition, sociality. corresponding author mitsuru hattori department of biology, faculty of science, shinshu university, 3-1-1 asahi, matsumoto, nagano 390-8621, japan e-mail: mtr.hattori@gmail.com hypothesis, some hymenopteran insects can recognize nonkin individuals and prevent them from invading the colony (hölldobler & wilson, 1990). such kin recognition may generally help maintain eusociality (hamilton, 1964 but see abbot, 2009), because an ability of kin recognition can keep high relatedness between donor and recipient in altruistic interactions (gadagkar 1985). however, some eusocial aphids (e.g., c. japonica, ceratoglyphina bambusae, and pseudoregma bambucicola) cannot recognize kin individuals (aoki et al., 1991 carlin et al., 1994; shibao, 1999; for a review see pike & foster, 2008). several eusocial aphids show a cyclic parthenogenesis which is composed of a single sexual generation and multiple parthenogenetic generations, and host alternation (i.e., seasonally migrating from the primary host to the secondary host vice versa) (stern & foster, 1996). in the primary host generation, aphid often construct gall (= nest) (aoki & kurosu, 2010). several studies have shown that even within a eusocial aphid gall on the primary host (= nest), the degree of relatedness among colony mates is low (abbot et al., 2001; johnson et al., 2002; wang et al., 2008). this low relatedness indicates that research article social artrhopods m hattori, t yamamoto, t itino sociobiology 62(1): 116-119 (march, 2015) 117 clone mixing occurs due to the absence of kin discrimination and exclusion of non-kin individuals at the nest entrance. therefore, we can hypothesize that clone mixing frequently occurs not only on primary hosts but also (even more frequently) on secondary hosts where free-living generation of aphids inhabits without a gall. in this study, to determine whether clone mixing occurs in colonies on secondary host plants, we surveyed the clonal composition of colonies of the eusocial aphid, c. japonica, by using amplified fragment length polymorphism (aflp) analyses. material and methods the eusocial aphid ceratovacuna japonica takahashi is a eusocial aphid that parthenogenetically produces sterile “pseudoscorpionlike” first-instar soldiers, which defend their colony on secondary host plants. this species has one primary host plant, styrax japonica sieb. et zucc. (ebenales, styracaceae), and several secondary host plants (poaceae species; sasa senanensis rehd. in central japan) (aoki & kurosu, 1991, 2010). adult individuals on secondary hosts produce alate individuals in the fall, which disperse to the primary host plants on which they overwinter. however, in central japan where this study was conducted, c. japonica aphids are rarely observed on primary hosts. this suggests an overwinter on the secondary host s. senanensis. in fact, we observed apparently overwintering individuals (alone or with a few mates on a leaf) in the fall and winter season on s. senanensis. in this paper, we define an aphid colony as an aggregation of aphid individuals on a single leaf of a secondary host plant. field sampling field sampling was conducted in an aphid population parasitizing a s. senanensis patch (about 10 m × 5 m in size) at the edge of a deciduous coniferous forest (dominated by larix kaempferi [pinales, pinaceae]) at the foot of mt. norikura, nagano, central japan (1600 m above sea level; 36°8’16.64n, 137°36’24.5e). in august 2012, we randomly selected five aphid colonies, each consisting of 200–1000 aphid individuals, and randomly collected 10 soldiers and 10 adults from each colony. we preserved the collected specimens in 100% ethanol before aflp analyses. genetic analyses we extracted dna from the ethanol-preserved aphids by using a “salting-out” protocol (sunnucks & hales, 1996). we performed aflp analysis according to the method of vos et al. (1995) with some modifications. genomic dna was digested with the restriction enzymes ecorι and msei at 37°c for 1.5 h. double-stranded adaptors were then ligated to the ends of the digested dna fragments at 20°c, overnight. we performed pre-selective amplification performed for 20 cycles, using a primer pair with one additional nucleotide on each restriction enzyme (msei-c/ecori-a) with the following cycle profile: a 30-s dna denaturing step at 94°c, a 1-min annealing step at 56°c, and a 1-min extension step at 72°c. we performed selective amplification performed for 30 cycles with the following cycle profile: a 30-s denaturing step at 94°c, a 20-s annealing step, and a 2-min extension step at 72°c. the annealing temperature of the first cycle was 66°c, then for each of the next 10 cycles, it was reduced by 1°c each cycle, and finally it was maintained at 56°c for the remaining 19 cycles. selective polymerase chain reaction (pcr) amplifications were then conducted with a fluorescence-labeled primer combination: ecori-aca (fam)/msei-ccg. a dice tp600 pcr thermal cycler (takara bio, shiga, japan) was used with the aflp amplification core mix (applied biosystems, foster city, ca, usa) for both the pre-selective and selective amplifications. aflp fragments were detected with an abi prism 3130 automated sequencer (applied biosystems) and gene mapper software v.4.0 (applied biosystems). in this analysis, we selected 8 aflp bands and obtained fragment data from 57 of the 100 collected aphid individuals. we did not include the fragment data from the remaining 43 individuals in the following analyses because the quality of the data from these specimens was low. to observe the clonal composition of the aphid colonies, we analyzed the fragment data of each aphid individual by conducting a principle coordinate analysis (pcoa) (gower, 1966) with r version 2.13.0 software (r development core team, 2011). in this analysis, we considered specimens with identical pcoa scores to be clones. results we found 15 clones (designated by the letters a–o) among 57 individuals from five colonies of c. japonica (table 1, fig 1). the number of clones per colony ranged from four to seven, and many clones occurred in several different colonies. for example, the ‘a’ clone was found in only colony 1, and the ‘o’ clone was found in all five colonies (table 1). each clone may consist of (1) only soldiers (first-instar larvae) (‘i’ and ‘m’ table 1. clonal composition of the five c. japonica colonies. lowercase letters indicate identical clones. s, soldier; n, non-soldier adult. colony no. clone (number of individuals by caste*) 1 a (1s, 2n), c (2s), f (1n), g (1n), j (1n), m (1s), n (2n), o (1s) 2 b (1n), c (2n), d (1n), i (1s), j (1s), n (2s, 1n), o (3s, 4n)) 3 b (1s), c (1s), l (1n), n (n1), o (3n) 4 c (1s, 1n), e (1n), f (1n), g (1n), k (1n), n (1s), o (2s, 2n) 5 d (2s), h (1n), j (5n), o (2n) *s: soldiers, n: non-soldier adults m hattori, t yamamoto, t itino clonal composition of ceratovacuna japonica colonies118 clones), (2) only non-soldier adults (‘f’, ‘g’ and ‘h’ clones), or (3) both soldiers and non-soldier adults (‘a’, ‘b’, ‘d’, ‘e’, ‘j’, ‘l’, ‘n’ and ‘o’ clones) (table 1). furthermore, pcoa showed that frequency of clone mixing did not relate to genetic similarity among clones (table 1, fig 1). for example, in colony 1, distant clones (e.g., ‘a’ and ‘j’) coexisted. fig 1. two-dimensional principal coordinate analysis based on the result of aflp. pcoa axis 1 and pcoa axis 2 explained 26% and 21% of the total variance, respectively. lower cases indicate clones (c.f., table 1). distance between lower cases shows a difference of fragment pattern in the result of aflp. to fill a hole (pike & foster, 2004). such behavior may be selected against predators and conspecific cheating intruders. on the other hand, surprisingly, c. japonica in the secondary host, cannot discriminate kin. without kin recognition in the free-living, secondary host generation, the aphids may suffer intensive invasion by non-kin cheaters. kin selection theory predicts that a high degree of relatedness among individuals within a colony is a mechanism for the maintenance of sociality (hamilton, 1964). in fact, in the gall-forming social aphid pemphigus obesinymphae (homoptera, pemphigidae), which does not have kin-recognition ability, intruders to non-kin colonies behave and develop selfishly; they rarely attack predators and are more than three times as likely to be reproductive as individuals that remain in their natal galls (abbot et al., 2001). theoretically, acceptance of intrusions by unrelated selfish individuals should gradually increase dominance of non-kin offspring, which may eventually break the sociality of this aphid species. however the eusociality of c. japonica is maintained despite the occurrence of clonal mixing as shown in this study. this fact implies the maintenance of eusociality in this species is not explained by relatedness only. for example, ecological background such as predation pressure may be important for the maintenance of eusociality in aphids because predation may select for colonies composed of only altruistic clones even if relatedness between clones is low. to understand how some individuals come to migrate to and to invade into other colonies and the precise mechanism that allows the maintenance of eusociality in c. japonica despite clonal mixing, future studies should observe the migration and competition of clone-identified individuals. acknowledgments we thank y. nagano for advice on the aflp analysis. this work was supported in part by the nagano society for the promotion of science (no. 24-1-9) to m. h., and by a grantin-aid for scientific research (c-22570015) from the japan society for the promotion of science and research to t. i., and by education funding for japanese alps inter-universities cooperative project, ministry of education, culture, sports, science and technology, japan, to t. i. references abbot, p. 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(2008). social parasitism and behavioral interactions between two gall-forming social aphids. insectes sociaux, 55: 147152. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2744sociobiology 65(3): 375-382 (september, 2018) new records and potential distribution of the ant gracilidris pombero wild & cuezzo (hymenoptera: formicidae) introduction gracilidris pombero wild & cuezzo, 2006 is the only extant species of the dolichoderinae ant genus gracilidris wild & cuezzo, 2006. the natural history of this species is poorly known but previous studies indicate that colonies are relatively small (wild & cuezzo, 2006). this ant builds its nest in the ground, and foraging is predominantly or strictly nocturnal, which may explain why g. pombero is poorly represented in entomological collections (wild & cuezzo, 2006). the species was described based on workers collected in a few localities in paraguay, argentina and the central and northeastern regions of brazil (wild & cuezzo, 2006; guerrero & sanabria, 2011). more recently, g. pombero abstract gracilidris pombero wild & cuezzo, 2006 is an ant that remains poorly studied. endemic from south america, its geographical distribution is known from few and scattered collection points. in this study, we present new occurrence records of g. pombero obtained through extensive collections along the cerrado biome and the atlantic forest of northeastern brazil. based on the new and existing occurrence records we produced a model of the geographic distribution of g. pombero. modelling method was chosen based on maximization of model performance after evaluating a series of modelling approaches, including different parametrizations of the maxent algorithm and distinct runs of the garp algorithm. we found a total of 43 new records of g. pombero in brazil, including the first records of this species in the states of goiás, mato grosso do sul, piauí, sergipe and tocantins. based on our model, the areas of highest suitability of occurrence of g. pombero are located in two main zones in south america: one ranging from midwestern brazil to southeastern bolivia and paraguay; and the other spanning the south of brazil and uruguay. sociobiology an international journal on social insects eba koch1,2, jpso correia3, rst menezes4,5, ra silvestrini6, jhc delabie2, hl vasconcelos6 article history edited by gilberto m. m. santos, uefs brazil received 12 december 2017 initial acceptance 30 may 2018 final acceptance 19 july 2018 publication date 01 october 2018 keywords biogeography, dolichoderinae, garp, maxent, neotropical region. corresponding author elmo borges de azevedo koch universidade federal da bahia instituto de biologia programa de pós-graduação em ecologia e biomonitoramento rua barão de geremoabo, 147, ondina cep 40170-290, salvador-ba, brasil. e-mail: elmoborges@gmail.com was found in the extreme south of brazil, extending its previous known latitudinal range by about 450 km to the south (feitosa et al., 2015). gracilidris pombero seems to be associated with relatively dry and open habitats, such as grasslands and savannas (wild & cuezzo, 2006; feitosa et al., 2015). however, populations of this species have also been found in the colombian amazon and in the atlantic forest of northeastern brazil. nevertheless, in both cases, the ants were not found in forest areas, but rather in human-managed habitats (such as cattle pastures or cocoa plantations) (wild & cuezzo, 2006; guerrero & sanabria, 2011). based on the known distribution of the living species (g. pombero), and a fossil from the dominican amber [gracilidris humilioides (wilson, 1985)], guerrero & 1 universidade federal da bahia, programa de pós-graduação em ecologia e biomonitoramento, salvador-ba, brazil 2 laboratório de mirmecologia, centro de pesquisas do cacau comissão executiva plano da lavoura cacaueira, ilhéus-ba, brazil 3 fundação oswaldo cruz, instituto oswaldo cruz, programa de pós-graduação em biodiversidade e saúde, rio de janeiro-rj, brazil 4 department of entomology, national museum of natural history, smithsonian institution, washington-dc, usa 5 departamento de biologia, faculdade de filosofia, ciências e letras universidade de são paulo (ffclrp/usp), ribeirão preto-sp, brazil 6 instituto de biologia, universidade federal de uberlândia, uberlândia-mg, brazil research article ants eba koch et al. – new records and niche modelling of gracilidris pombero376 sanabria (2011) proposed that during drier periods of the pleistocene gracilidirs occurred from paraguay to puerto rico (including the amazon), but as the climate became wet again during the holocene, its distribution retracted, resulting in the isolation of populations in the colombian amazon. the same hypothesis may also explain the occurrence of g. pombero in the brazilian atlantic forest biome. assuming that g. pombero was once much more widely distributed, it is more likely that the atlantic forest populations represent relictual populations rather than populations that recently expanded into that region as the result of land cover and land use changes (guerrero & sanabria, 2011). in this study, we present new occurrence records of g. pombero obtained through extensive collections of grounddwelling ants along the cerrado biome (south american savanna) of central brazil and along the atlantic forest of northeastern brazil. based on the new and published information we produced a model of the potential geographic distribution of g. pombero in order to identify areas where new discoveries are more likely, and to determine the main climatic variables that help to explain the current distribution of the species. materials and methods occurrence records part of the new records of g. pombero presented here come from standardized sampling of the ant fauna conducted in 29 well-preserved savanna sites using pitfall traps (vasconcelos et al., 2018). these sites were distributed across the entire extension of the brazilian cerrado biome, in a region spanning ca. 20° of latitude and 18°of longitude (vasconcelos et al., 2018). records recently published (santos et al., 2017) obtained from ant surveys performed in several landscapes in the atlantic forest biome of the state of bahia, brazil, were also included in our analysis. in addition, we examined material deposited in the collection of the laboratório de mirmecologia in the centro de pesquisa do cacau – cepec/ ceplac (cpdc) (ilhéus, bahia, brazil), and which include specimens collected in the cerrado and pampas biomes, cocoa plantations, or urban areas. finally, we compiled all published records of occurrence of g. pombero through a detailed survey of the literature (wild & cuezzo, 2006; guerro & sanabria, 2011; brandão et al., 2011; neves et al., 2013; costa-milanez et al., 2014; camacho & vasconcelos, 2015; feitosa et al., 2015; meurer et al., 2015; solar et al., 2016). ecological niche modeling (enm) the distribution probability map of g. pombero was obtained by applying a filtered data set of the occurrence points on several bioclimatic variable layers from the worldclim database (hijmans et al., 2005). the modelling method was chosen based on maximization of model performance. this was done after evaluating a series of modelling approaches, including different parametrizations of the maxent algorithm (using maxent software version 3.4.1) and distinct runs of the genetic algorithm for rule set production (garp) (available in the openmodeller software v1.1.0; muñoz et al., 2009). we chose to work with the maxent and garp algorithms due to their superior ability and better performance even with a small data set in comparison with other modelling methods (wisz et al., 2008). these algorithms have shown success to modeling distributions of several species using occurrence records and environmental data (peterson et al., 1999; anderson et al., 2003; raimundo et al., 2007; zhang et al., 2015). additionally, we used the auc (average area under curve) and sensitivity values to test the performance of our models. both auc and sensitivity values ranges from 0 to 1, where a score higher than 0.9 can be considered good predictors of favorable conditions for the occurrence of a species (phillips et al., 2006). data filtering of the 57 available occurrences (see table 1) was necessary to avoid biased probability estimations (kramer-schadt et al., 2013; boria et al., 2014), since it was observed a spatial aggregation pattern along the map, as a result of intense sampling efforts within some specific regions. filtering criteria was based on distance between points: for a given group of neighboring occurrences whose distance between one another was less than 2 km, only the central point was maintained. hence, the filtered data set consisted of 33 occurrence points, which were divided into calibration (27) and validation (6) in order to allow an independent and robust validation. the calibration data were used as the only occurrences available to perform the modeling process, whereas the validation data were considered for validation purposes only. in order to obtain a consistent model, we evaluated the degree of correlation between the bioclimatic variables based on principal component analysis (pca), using the software past v3.02 (hammer et al., 2001) as performed by menezes et al. (2017). thus, we extracted values of each occurrence point associated to each of the 19 bioclimatic layers using quantum-gis v2.8 (open source geospatial foundation project, beaverton, or, usa) and we checked autocorrelated variables by pca. simultaneously, we verified the percent of contribution of each bioclimatic layer and we excluded some non-contributing variables. finally, from the 19 bioclimatic variables available, only 16 were passed into model algorithms either because of high correlation or due to absence of contribution for our species distribution modelling. the 27 occurrence calibration points and the 16 bioclimatic variables were passed to several maxent and garp algorithms, with different parameters. the resultant simulated distributions consisted of two maxent distributions surfaces and three probability distributions from garp. the best model was chosen based on the maximization of calibration and validation sensitivity values. validation was performed by calculating the percentage of validation points located in high probability areas. the final model consisted sociobiology 65(3): 375-382 (september, 2018) 377 of maxent algorithm run with the following parameters: quadratic, product, threshold and hinge, 500 iterations, regularization multiplier equal 1, thirty percent of random test and ten replicates subsampled. all modelling procedures were developed for the neotropical region at a spatial resolution of 2.5°. results new occurrence records we found 43 new records of g. pombero for brazil, including the first records of this species for the states of goiás, mato grosso do sul, piauí, sergipe and tocantins (table 1). nevertheless, most of the new records were largely coincident or complementary to the previous known range of g. pombero in south america. among the new records, most (70%) were obtained in typical cerrado vegetation (savanna-like vegetation), 18.5% in agricultural systems (cocoa plantations and small-scale agriculture) or pastures, 7% in the atlantic forest biome (fragments with low forest cover and in its surrounding matrix in some cases), 2.3% in urban areas (e.g., university campus), and 2.3% in an abandoned mining area in the south of brazil (see table 1). enm using maxent the performance analysis of the geographic distribution for g. pombero, obtained through the best-fit maxent model, showed high accuracy values, as demonstrated by both auc and sensitivity metrics. auc values ranged from 0.92 for calibration dataset and 0.88 for validation data, while sensitivity varied from 0.85 for the calibration and 0.83 considering validation. the small variation observed between calibration and validation performance metrics shows model’s ability to predict with high accuracy even in areas in which occurrence points were not available for model fitting, indicating a high degree of confidence on the predicted distribution. the areas of highest suitability for the occurrence of g. pombero were located in two main zones in south america: one ranging from midwestern brazil to southeastern bolivia and paraguay; and the other spanning the south of brazil and uruguay (fig 1). among these places, for uruguay and bolivia have not been registered any occurrence of the species yet. other suitable regions include the coast of northeastern brazil. moreover, the model predicted high suitability for the occurrence of g. pombero also in central america, notably in the south of mexico, cuba, honduras, and the dominican republic, indicating that the entire region has climatic conditions suitable for the species. fig 1. potential geographical distribution of gracilidris pombero in the neotropical region. the intensity of color gradient from blue to red (the redder color means the higher probability of occurrence) is proportional to probability of occurrence. fig 2. modelled probability of occurrence of gracilidris pombero in relation to annual rainfall. the line represents a scatterplot smoothing of the data. annual rainfall (mm) m od el le d pr ob ab ili ty o f o cc ur re nc e suitable areas were mainly guided by three variables in the model, which alone explained 83% of model prediction: annual precipitation (42.6%), mean temperature of driest quarter (21.4%), and temperature seasonality (18.8%). furthermore, the model indicates that sites receiving 1,000 to 1,500 mm of rain per year have the highest probability of occurrence of g. pombero (see fig 2). eba koch et al. – new records and niche modelling of gracilidris pombero378 discussion our study represents a four-fold increase in the number of distributional records of g. pombero, from 14 to 57 records. we recorded g. pombero in eight brazilian states, of which five (goiás, mato grosso do sul, piauí, sergipe and tocantins) have the ant recorded for the first time. over half of the new records (26 out of 43) were obtained during collections along the brazilian cerrado biome and more specifically in cerrado vegetation in biological reserves (vasconcelos et al., 2018). most of the remaining records were in eastern bahia, within the atlantic forest domain, were it was found in atlantic forest fragments of low forest cover and in human-managed habitats, including cattle pastures, cocoa plantations and other small-scale agriculture systems. one record corresponds to an urban area (university campus) in sergipe. similarly, the only new record in southern brazil (rio grande do sul) was in a human-disturbed area (an abandoned mine). these data reinforce the idea that g. pombero is a species typical of savannas, shrublands and grasslands, which nevertheless can also be found in disturbed habitats, such as forest edges, pasture, crops and urban areas. despite the limited information about the ecology and other specific aspects of the biology of g. pombero, we suggest that the large number of new records presented in this study is related to the sampling method used, since most of these records came from pitfall traps (bestelmeyer et al., 2000), which collect ants with both diurnal and nocturnal habits. g. pombero is thought to exhibit nocturnal habits (wild & cuezzo, 2006), and this may help to explain the difficulty of collecting it when using other sampling methods, such as visual searches, baits and winkler traps. it is difficult to find the species even when performing more elaborate soil searches (depths up to 10 cm) if they are carried out during daytime (guerrero & sanabria, 2012). our distribution modelling suggests that although g. pombero has not been collected in bolivia, it is very likely to be present there. similarly, there is a strong chance of occurrence of g. pombero in northern uruguay. the areas with highest probability of occurrence of g. pombero correspond largely with the distribution of the cerrado, chaco, and pampa biomes of south america. although g. pombero has been recorded several times in the atlantic forest, it was always found in relatively open forest sites (three cases; table 1) or in non-forest habitats, particularly in cocoa plantations and urban areas, reinforcing the view that this species can tolerate some degree of anthropic disturbance (guerrero & sanabria, 2011; feitosa et al., 2015). the expansion of g. pombero to areas of this biome may be related to the historic impact of land use change by deforestation, with the replacement of native forests by agriculture or urban centers, intensified principally during the nineteenth and twentieth centuries (young, 2003) and which continues – at an estimated rate of 29,075 ha deforested per year (sos mata atlântica and inpe, 2017) – to date. the brazilian atlantic forest currently accounts for about 11.6% of its original vegetative cover (ribeiro et al., 2009), in a highly fragmented state with more than 80% of these fragments having an area of less than 50 ha (ribeiro et al., 2011) surrounded by a matrix that can be pasture, agriculture, eucalypt plantations, or urban areas (joly et al., 2014). interestingly, our model indicates a low probability of occurrence of g. pombero in the caatinga, even though this biome is part of the “diagonal of dry biomes”, which also includes the cerrado and chaco. in fact, several studies have failed to record this species in the caatinga (leal et al., 2003; ulyssea & brandrão, 2013; silva & delabie, 2014). this biome is drier than the cerrado and chaco and, as our model suggests, the probability of occurrence of g. pombero declines sharply at relatively dry regions with an annual rainfall of less than 1,000 mm, such as in caatinga (ab´saber, 1993). finally, the climatic conditions in central america seem highly suitable for the occurrence of g. pombero although the ant seems totally absent from this region. this finding plus the gracilidris dominican fossil suggests that the genus gracilidris, in the past, could have occurred both in central and south america as shown by the known records of the genus (guerrero & sanabria, 2011). a similar pattern is suggested for other dolichoderine genera such as technomyrmex and bothriomyrmex with a putative wide geographic distribution in the past but currently restricted to a few isolated forests (fernandez & guerrero, 2008). however, further studies exploring the biogeographic history and the divergence time of gracilidris will be necessary to elucidate the geographic origin of the genus. acknowledgments records of g. pombero presented here come from surveys conducted with financial support from the projects “rede de pesquisa biota do cerrado (rpb cerrado 6) isoptera e hymenoptera”; “efeito da redução da cobertura vegetal e do histórico biogeográfico sobre limiares de extinção: um estudo multi-táxon na mata atlântica da bahia”; and “projeto integrando níveis de organização em modelos ecológicos preditivos” (inomep/pronexfapesb-cnpq). ebak is thankful to bahia research foundation (fapesb) by grant 0483/2015. jpsoc is thankful to conselho nacional de desenvolvimento científico e tecnológico (cnpq). rstm is grateful to são paulo research foundation (fapesp) by grants 2015/02432-0 and 2016/21098-7. jhcd acknowledges his research grant from cnpq. we thank fernando fernández and rodrigo feitosa for assisting in the identification of the individuals of g. pombero deposited in the entomological collections at the museu de biodiversidade do cerrado (mbc). sociobiology 65(3): 375-382 (september, 2018) 379 table 1. records of gracilidris pombero in the south america. lt = literature data; sc = data of scientific collection (cpdc*); pd = project data**. city/locality state/province country coordinates record type habitat type reference longitude latitude santiago del estero santiago del estero argentina -61,9113 -25.8547 lt chaco wild & cuezzo (2006) barreiras bahia brazil -44,9281 -12,1466 sc cerrado (savanna) this study/ vasconcelos et al., 2018 barreiras bahia brazil -45,1496 -12,1482 pd cerrado (savanna) this study/ vasconcelos et al., 2018 barreiras bahia brazil -45,1382 -12,1446 pd cerrado (savanna) this study/ vasconcelos et al., 2018 cruz das almas bahia brazil -38,3494 -11,7858 sc pasture (cattle pastures) this study/cpdc ilhéus bahia brazil -39,1000 -14,7361 lt cocoa plantation wild & cuezzo (2006) ilhéus bahia brazil -39,0336 -14,8016 pd atlantic forest (5% coverage); cocoa plantation this study/santos et al., 2018 inhambupe bahia brazil -39,1063 -12,6775 sc cocoa plantation this study/cpdc jaguaripe bahia brazil -39,0219 -13,1727 pd pasture; small scale agriculture this study/santos et al., 2017 nilo peçanha bahia brazil -39,1961 -13,6488 pd pasture; small scale agriculture this study/santos et al., 2017 pres. tancredo neves bahia brazil -39,3158 -13,3900 pd atlantic forest (less than 15% of canopy coverage); cocoa plantation this study/santos et al., 2017 serra grande bahia brazil -39,1000 -14,4833 pd cocoa plantation this study/cpdc ubaíra bahia brazil -39,6702 -13,1216 pd pasture (cattle pastures) this study/santos et al., 2017 una bahia brazil -39,0616 -15,1897 pd cocoa plantation this study/cpdc valença bahia brazil -39,1908 -13,3308 pd small scale agriculture this study/santos et al., 2017 wenceslau guimarães bahia brazil -39,7019 -13,5538 pd atlantic forest (less than 40% of canopy coverage); pasture this study/santos et al., 2017 mineiros goiás brazil -53,0085 -17,9088 pd cerrado (savanna) this study/ vasconcelos et al., 2018 mineiros goiás brazil -52,9897 -17,9130 pd cerrado (savanna) this study/ vasconcelos et al., 2018 mineiros goiás brazil -52,9687 -17,9191 pd cerrado (savanna) this study/ vasconcelos et al., 2018 bacabal maranhão brazil -44,7800 -04,2250 sc cerrado (savanna) this study/ vasconcelos et al., 2018 balsas maranhão brazil -46,7178 -08,5721 lt cerrado (savanna) brandão et al., (2011) barra do garça mato grosso brazil -52,2674 -15,8505 pd cerrado (savanna) this study/ vasconcelos et al., 2018 barra do garça mato grosso brazil -52,2540 -15,8573 pd cerrado (savanna) this study/ vasconcelos et al., 2018 cacéres mato grosso brazil -57,8143 -16,0825 lt cerrado (savanna) wild & cuezzo (2006) chapada dos guimarães mato grosso brazil -55,9678 -15,3542 pd cerrado (savanna) this study/ vasconcelos et al., 2018 eba koch et al. – new records and niche modelling of gracilidris pombero380 cuiabá mato grosso brazil -56,1053 -15,5808 pd cerrado (savanna) this study/ vasconcelos et al., 20188 nova xavantina mato grosso brazil -52,3571 -14,7174 pd cerrado (savanna) this study/ vasconcelos et al., 2018 nova xavantina mato grosso brazil -52,3116 -14,6969 pd cerrado (savanna) this study/ vasconcelos et al., 2018 novo são joaquim mato grosso brazil -52,7876 -15,0785 pd cerrado (savanna) this study/ vasconcelos et al., 2018 novo são joaquim mato grosso brazil -52,7798 -15,0695 pd cerrado (savanna) this study/ vasconcelos et al., 2018 novo são joaquim mato grosso brazil -52,7773 -15,0592 pd cerrado (savanna) this study/ vasconcelos et al., 2018 ricardo franco mato grosso brazil -60,0646 -14,9076 pd cerrado (savanna) this study/ vasconcelos et al., 2018 vila bela mato grosso brazil -59,7793 -15,0540 pd cerrado (savanna) this study/ vasconcelos et al., 2018 vila bela mato grosso brazil -59,7845 -15,0623 pd cerrado (savanna) this study/ vasconcelos et al., 2018 vila bela mato grosso brazil -59,7908 -15,0511 pd cerrado (savanna) this study/ vasconcelos et al., 2018 campo grande mato grosso do sul brazil -54,6857 -20,4228 pd cerrado (savanna) this study/ vasconcelos et al., 2018 andréquicé minas gerais brazil -44,8475 -18,4524 lt wetland areas in cerrado (“veredas”) costa-milanez et al., (2014) pandeiros minas gerais brazil -44,7558 -15,5072 lt cerrado (savanna) neves et al., (2013) pandeiros minas gerais brazil -44,6828 -15,4938 pd cerrado (savanna) this study/ vasconcelos et al., 2018 pandeiros minas gerais brazil -44,7266 -15,5004 pd cerrado (savanna) this study/ vasconcelos et al., 2018 paracatu minas gerais brazil -47,0661 -17,1906 pd cerrado (savanna) this study/ vasconcelos et al., 2018 paracatu minas gerais brazil -47,0583 -17,1790 pd cerrado (savanna) this study/ vasconcelos et al., 2018 paracatu minas gerais brazil -47,0440 -17,1812 pd cerrado (savanna) this study/ vasconcelos et al., 2018 são gonçalo do abaeté minas gerais brazil -49,7661 -18,3377 lt wetland areas in cerrado (“veredas”) costa-milanez et al., (2014) uberlândia minas gerais brazil -48,4000 -19,1666 lt cerrado (savanna) camacho & vasconcelos (2015) paragominas pará brazil -47,3639 -02,9793 lt pasture and agricultural areas solar et al., (2016) sete cidades piauí brazil -41,7095 -4,9910 pd cerrado (savanna) this study/ vasconcelos et al., 2018 aceguá rio grande do sul brazil -54,1572 -31,6486 lt pampas feitosa et al. (2015) candiota rio grande do sul brazil -53,6737 -31,4914 sc abandoned mining area this study lavras do sul rio grande do sul brazil -53,9813 -30,7005 lt pampas feitosa et al. (2015) agudos são paulo brazil -48,9399 -22,4971 lt cerrado (savanna) feitosa et al. (2015) table 1. records of gracilidris pombero in the south america. lt = literature data; sc = data of scientific collection (cpdc*); pd = project data**. (continuation) city/locality state/province country coordinates record type habitat type reference longitude latitude sociobiology 65(3): 375-382 (september, 2018) 381 references ab’saber, a.n. (eds.). 2003. os domínios da natureza no brasil: potencialidades paisagísticas. são paulo, ateliê editorial, 160 p. anderson, r.p., lew, d. & peterson, a.t. (2003). evaluating predictive models of species’ distributions: criteria for selecting optimal models. ecological modelling, 162: 211-232. brandão, c.r.f., silva, r.r. & feitosa, r.m. 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(2008). effects of sample size on the performance of species distribution models. diversity and distributions, 14: 763-773. doi: 10.1111/j.1472-4642.2008.00482.x young c.e.f (2003) socioeconomic causes of deforestation in the atlantic forest of brazil. in: galindo-leal, c. & câmara, i.g (eds.) the atlantic forest of south america: biodiversity status, threat and outlook. island press, conservation international, washington, p. 103-117. sociobiology 65(1): march (2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 editorial special issue on termites due to their astonishing abundance and typical cellulose-digesting abilities, termites (blattodea: isoptera) impact ecosystem functioning, from biogeochemical cycles to species interactions. their interindividual interactivity provides firm ground for the study of self-organized collective behaviour while their diplo-diploidy foments rich discussion on the evolution of sociality. all this, coupled with termites’ economic impact, implies that scientific interest in this prominent group of organisms is burgeoning and will continue to do so. in this special issue, thirteen articles provide a cross-diciplinary sample of termite studies which, directly or indirectly, shed light on the above subjects. pequeno et al (page 1) tackle the life-history allocation strategies of termites inspecting, in the field, how growing colonies should invest in growth, reproduction an defense. as for species coexistence, we start from the global question of “how many termite species are there?” (constantino, page 10). we then proceed with schyra et al (page 15) analysing at the intra-continental scale, whether southern african termite communities are niche or neutrally assembled and if environmental filtering and or competition influence community structure. at the local scale, latifian et al (page 24) present a survey of coexisting termite species in iranian date palm orchads. finally, drivers of species coexistence are investigated at the intra-termitarium scale, from a behavioural perspective. in doing so, cruz et al (page 31) and rosa et al (page 38) inspect, respectively, the strategies employed by inquilines and termitophiles to cohabit termitaria along with its builders. in another group of articles in this issue, we are offered a thorough analysis of the environmental drivers of termite infestation on live trees (fernandes et al, page 48), dead trees (roh et al, page 59), and also in the urban scenario (botch& houseman, page 67). in addition to understanding drivers of infestation, we sometimes need to interfere directely to control it. here, martins et al (page 79) demonstrated the termiticidal activity of three pure lichen compounds which could, potentially, help efforts in termite control a special volume on termites would be not complete without a methodological section. here, scheffrahn et al (page 88) provides a analysis of the sampling biases for the divergent outcomes in sampling the abundance and diversity of kalotermitidae compared to other families. as for laboratory methodology, nunes et al (page 101) propose the use of tympanic arenas for vibroacoustic assays on termites and carvalho et al (page 108) provides the best light regimes that could be innocuously used for video recording termite bioassays. in summary, the studies composing this special issue provide important bases for the advancement of termitology at both, theoretical and empirical fronts. this special issue was promoted as part of the iv symposium of termitology (iv symtermes, https://www.zenodo.org/communities/symtermes/), which was held in viçosa-mg, brazil, from 7 to 10 november 2017. some of the above papers were also presented at this symposium. we thank all authors and reviewers for their rich and important contribution for this special issue. dr. paulo f. cristaldo (universidade federal de sergipe, brazil) dr. og desouza (universidade federal de viçosa, brazil) editors for this special issue sociobiology an international journal on social insects open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.155-163sociobiology 61(2): 155-163 (june, 2014) does ant community richness and composition respond to phytophysiognomical complexity and seasonality in xeric environments? em silva1; am medina1; ic nascimento2; pp lopes1; ks carvalho2; gmm santos1 introduction it is common that communities suffer changes in species composition and richness in seasonal environments, such as tropical dry forests (murphy & lugo, 1986). a remarkable feature of these vegetation types is the loss of leaves by trees during the dry season (veloso et al., 1991) , which interspersed with wetter periods and higher productivity, determine changes in the amount and quality of resources and, consequently, the structure of local communities (sánchez-azofeifa et al., 2005). this explains why certain species may specialize in the use of resources under more severe conditions, resulting in a temporal partition of the same, reflecting a temporal variation of community composition (pianka, 1980). apart from climate change, another factor that can influence the animal community is the structural complexity of vegetation. the ecological prediction states that the occurrence of more speabstract this study aimed to analyze how the vegetation structure (physiognomy) and seasonal changes between seasons (wet and dry) influence richness, diversity and composition of ant species of arboreal and shrubby savanah (caatinga) environments. the vegetation structure was significantly different among the three strata for all parameters (mean diameter of vegetation, level of herbaceous cover, degree of coverage and depth of litter and percentage of canopy cover). we collected 127 ant species. the mean number of species was approximately two times higher in the rainy season than in the dry season. there was no difference in species richness between the arboreal and shrubby caatinga physiognomies nor interaction between season and physiognomy. despite the similarity in richness, species composition differed between physiognomies, however we found no difference in composition between seasons. the seasonal differentiation may be mainly related to the variation in the overall numbers of individuals circulating in the environment, since the enhancement of resource availability during rainy season allows the colony to grow or expand foraging activities, which increases local diversity. water restriction explains the limited diversity in both environments, while the occurrence of species with greater resource specificity may determine differences in ant composition. differences in composition of each of caatinga’s physiognomy enhance beta diversity, therefore, raising the overall diversity in the caatinga domain. sociobiology an international journal on social insects 1 universidade estadual de feira de santana, feira de santana, bahia, brazil 2 universidade estadual do sudoeste da bahia, campus jequié, bahia, brazil. article history edited by kleber del-claro, ufu, brazil received 16 may 2014 initial acceptance 07 june 2014 final acceptance 18 june 2014 keywords environmental heterogeneity, species composition, formicidae, seasonality corresponding author ivan c. nascimento univ. estadual do sudoeste da bahia departamento de ciências biológicas av. jose moreira sobrinho jequiezinho jequié, ba, brazil 45200-000 e-mail: icardoso@hotmail.com cies in a community is seen as a response to the greater complexity of vegetation structure (pacheco et al., 2009; corrêa et al., 2006), which provides a greater amount of realizable niches by species of animals and therefore a greater number of species in a given community (tews et al., 2004). to test this hypothesis, several studies involving the comparison of areas with distinct physiognomies have been done throughout the world, using ant communities, such as armbrecht and ulloa-chacón (1999) in colombia; fisher and robertson (2002) in madagascar; wilkie et al. (2009) in peruvian amazonic forest; lindsey and shinner (2001) in south africa. in brazil we highlight the studies of fowler et al. (2000) comparing forests from bahia and pará states; corrêa et al. (2006) in forest patches from mato grosso do sul and delabie et al. (2007) comparing shaded cocoa agroecosystem developed under atlantic forest vegetation or other native vegetation in bahia state. the caatinga, a native dryland registered in the northresearch article ants silva et al., effect of environmental heterogeneity in ant community156 eastern brazil, shows a marked variation in its vegetation structure (andrade-lima, 1981), particularly with regard to the density and size of the plants. these differences can be perceived at the local level, where even within a few dozen meters we recognize differences, usually related to a clearly identifiable environmental change as rock extrusions (‘lajedos’ formations), that determine shallow soils and lower water availability (amorim et al., 2005). however, studies with animal communities are still incipient, particularly in relation to communities of ants (neves et al., 2006; leal, 2003). in this context, the present study attempted to answer the following question: does the type of vegetation (physiognomy) that makes up the caatinga and seasonal variations between the dry and rainy seasons in this environment, influence the richness, diversity and species composition of ants? to answer this question, the following hypotheses were formulated: the richness, diversity and composition of ant species respond positively to the increased structural complexity of the environment and negatively to conditions of the dry season due to the scarcity of food and nesting resources. material and methods study area in this study, we compared two physiognomies of caatinga, an arboreal and a shrubby caatinga, both located in the city of milagres, bahia, brazil (12º52’36s 39º51’22w). the arboreal caatinga is characterized by having tall trees reaching up to 20 meters, straight stems and understory consisting of smaller trees and ephemeral subshrubs (ferreira, 1997). the shrubby caatinga, in turn, is marked by more sparse trees and greater representation of cactaceae and euphorbiaceae with a formation that resembles the vegetation of fields (ross, 2001). the region has a semi-arid tropical climate with an average temperature of 24.3ºc and average rainfall of 551 mm/year, although large variations between years may occur (142 to 1206 mm/year). the rainy season generally extends from december to february, although there are annual variations, with at least five dry months during the year (bahia, 1994). in order to characterize each vegetation type in each sampled area we evaluated vegetation variables which were compared using two-sample independent tests (t test or mann-whitnney): cbh (circumference at breast height), herbaceous cover, litter cover, litter depth and percent cover of vegetation canopy, measured at each sample point of fauna. cbh of trees was measured at 1.30m above ground, within a 5m radius circular plot (78,5m²) marked from the sampling points; in plants smaller than 1.50m in height, cbh was replaced by the circumference of the trunk below the first branch (soares, 1999). the herbaceous cover is given by counting the herbaceous plants in a radius of 1.50m from the sample point. coverage of litter was measured according to the scale of fornier (1974): 1 (0-25%=small); 2 (2650%=medium 1); 3 (51-75%=medium 2); 4 (76-100%=large). the depth of the litter was classified according to pacheco et al. (2009) comprising four classes of arbitrary amplitude: very shallow (0-2cm), shallow (2-4cm), deep (4-6cm) and very deep (>6cm). the percentage cover of the vegetation canopy was evaluated through a modification of the methodology for indirect estimation of canopy proposed by monte et al (2007). we used a sony cybershot camera (7.2 mp) to capture the canopy image, and through the photoshop 7.0 software we created a binary image (black-white) in order to quantify the amount of black pixels, estimating canopy coverage. ant sampling between may 2009 and january 2010 we carried out four field incursions, two during the dry season and two during the rainy season. in each incursion we sampled the ant fauna associated with three areas of arboreal caatinga and three areas of shrubby caatinga. samples were taken on a transect of 350 meters in each area, each transect containing 15 pitfall traps 25m distant from one another. the traps were kept active for 48 hours in the field. additionally, we installed 15 traps with attractive bait sardines in vegetable oil (1cm³), at each transect, exposed for a period of 30min. to avoid interference, the baits were installed only after the removal of the pitfalls. this collection protocol had three replicates at each physiognomy and repeated in all four incursions in the field totaling 12 transects in arboreal caatinga and 12 transects in the shrubby caatinga. collected ants were identified following the classification proposed by bolton et al. (2006) and witness individuals were deposited in the prof. johann becker entomological collection from zoology museum of the universidade estadual de feira de santana (mzfs) and in the entomological collection from the myrmecology laboratory from the comissão executiva de pesquisa da lavoura cacaueira (cepec/ceplac), in itabuna, bahia. data analysis we tested our hypothesis with analyzes based on the components of community structure. the first analysis was based on species richness and used the sample points as local units. for this, we used a generalized linear mixed model with poisson distribution and log link function to assess the influence of vegetation type and season (explanatory variables) on species richness (response variable). furthermore, we use the sampling points, and collection areas incursions as random factors to control the temporal pseudoreplication. we conducted this analysis in r software (r development core team, 2013) using the lme4 package (bates et al., 2013). sociobiology 61(2): 155-163 (june, 2014) 157 the second analysis was based on species composition and areas used as sampling points. for this purpose, we build a similarity matrix using the jaccard index and from this we performed a non-metric multidimensional scaling (nmds). this technique is an ordering method more robust to non-linear situations and often summarizes more information in fewer axes than other indirect ordination techniques (legendre & legendre, 1998). we tested changes in species composition between seasons (dry and wet) and between vegetation types (arboreal and shrubby caatinga) using a two level similarity analysis (two-way anosim). we conducted this analysis using primer® 6.0 software (clarke & gorley, 2006). results vegetation structure the vegetation structure of arboreal and shrubby caatinga were different in all traits, with the arboreal physiognomy presenting a cbh 1.4 times greater than in shrubby caatinga (arboreal = 30.0±21.4mm; shrubby = 42.2±22.03mm; t = 2.652; n = 45; p ≤0.001), herbaceous cover twice as large (arboreal = 1.9±0.9, shrubby = 0.7±0.4; t = 7.0; n = 45; p <0.001), larger leaf litter coverage (u = 122.5; n = 90; p <0.001), larger leaf litter depth (u = 701; n = 90; p <0.001), and canopy coverage was two times larger (arboreal = 70.9±9.2%, shrubby = 33.6±22.2%, t = 14.74; d.f. = 118.57; p < 0.001). mirmecofauna we collected 127 ant species (apendix 1), with the most frequent species being dinoponera quadriceps (68%), camponotus sp6 (56%) and ectatomma muticum (41%). a total of 32 ant species was recorded exclusively on arboreal caatinga and 29 species exclusively in the area of shrubby caatinga. between stations, 18 species of ants were collected only in the dry season and 20 species of ants were collected exclusively in the wet season. the mean number of species by point sampled was approximately two times higher in the wet season than in the dry season (χ² = 5.45; d.f. = 1; p <0.05). moreover, there was no difference in species richness among the caatinga physiognomies (χ² = 0.4796; d.f.=1; p = 0.49) nor interaction between physiognomy and season (χ² = 6.95; d.f. = 3; p = 0.07) (fig. 1). despite the similarity in richness, composition of ant species differed between the physiognomies (r = 0.849; p <0.01), however we found no difference in species composition of ants between the dry and wet seasons (r = 0.118; p = 0.10) (fig. 2). discussion the fact that there were differences in species composition between the physiognomies, despite the similarity of species richness, indicates that there are two distinct communities in the caatinga a specialist in arboreal caatinga and another in shrubby caatinga. increasing environmental complexity can change the types of resources and their availability. once the resources are different, the environment may become less advantageous to the dominant species and completely change the structure and composition of the community (perfecto & vandermeer, 1996). an example of how the heterogeneity of vegetation may determine the occurrence of specialist species is the presence of gnamptogenys concinna in the area of arboreal caatinga. it was believed that this ant species was restricted to wet forest environments, and recently recorded for the state of bahia in cacao shaded figure 1. mean of ant species richness for point sampled in two caatinga phytophysiognomies (arboreal and shrubby) (χ²=0.4796; d.f.=1; p = 0.49) in dry (gray columns) and rainy (black columns) seasons. the mean number of species for point sampled was approximately two times higher in the wet season than in the dry season (χ²=5.45; d.f.=1; p < 0.05). bars represent standard errors. fig 2. non-metric dimensional scaling (nmds) based on jaccard similarity index, comparing ant species composition in milagres, bahia, brazil. circles represent arboreal caatinga and squares represent shrubby caatinga. dry season in gray and rainy season in black. silva et al., effect of environmental heterogeneity in ant community158 by large trees (delabie et al., 2010). g. concinna is the only one belonging to the arboreal specialist genus (lattke, 1990; longino, 1998), with strong links with canopies with high numbers of epiphytes of the families bromeliaceae and orchidaceae (delabie et al., 2010). a link to these epiphytes may explain its occurrence in the area of arboreal caatinga, considering that the area where it was found has a canopy with lots of large epiphytic bromeliaceae. studies focusing on the influence of grazing on the ant community in semi-arid regions also found effects restricted to species composition (bestelmeyer & wiens, 2001). however, in the present study species richness was similar because once the two areas are subject to stress caused by lack of rain, the food resources must be scarce in both environments. we found no effect of the interaction between environmental complexity and seasonality on the ant diversity, but we found a difference in the richness of ant species between seasons, but without a difference in species composition. due to the unpredictability in the acquisition of resources caused by the shortage of rainfall, most species in caatinga should be able to pull through in xeric conditions (like ants in american desertlands e.g: whitford et al., 1999), and the species should only reduce the number of active individuals in the colony during the dry season and invest in foraging activities when the environment has more resources (bernstein, 1979). if there are less active individuals, the probability of finding more species will be smaller. it is worthwhile to point out that, although fluctuations in abundance may occur between seasons, we work only with occurrences, missing evidence to support this hypothesis. a study addressing ants that use floral resources in a tropical dry forest demonstrated that dietary overlap is higher in the dry season (brito et al., 2012) indicating a decrease in food resources. since there are large differences in species composition in the two physiognomies, this leads to an increase in beta diversity of caatinga on a regional scale, similar to what happened with other groups such as bees (martins, 2002). given that there is more than one type of physiognomy in the caatinga, the importance of environmental heterogeneity for increasing beta diversity in the region shown in the present study may be underestimated. two factors influence the effect of the plant structure on ants and are characteristics that distinguish arboreal and shrubby caatinga. the first is the increase in shading in the environment caused by the presence of trees (reyes-lópez et al., 2003). the second is the deposition of litter which is a factor that positively influence the activity of ant species (perfecto & vandermeer, 1996). similar to that reported in this study, other studies have also found no influence of environmental complexity in species richness of ants (corrêa et al., 2006; neves & braga, 2010). however, the use of only one feature as a surrogate of environmental complexity may fail to find an effect even if it exists (see discussion on the review by tews et al., 2004), so an indirect analysis can more easily detect the environmental heterogeneity even without setting a key structure, provided focusing on the species composition. our study shows the following pattern: while the variation in rainfall is responsible for the increase in the number of species that may be present in the environment on a local scale, the difference of environmental complexity on both physiognomies is detectable in species composition and is responsible for increasing the beta diversity through differences in species composition. acknowledgments many colleagues contributed to this study in different ways, especially j.j. resende who helped during sampling. we thank two anonymous reviewers for valuable comments. the brazilian national council for scientific and technological development (cnpq) supported this study. g.m.m. santos received productivity fellowship from cnpq. references amorim, i.l., sampaio, e.v.s.b. & araújo, e.l. 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(2009). diversity of ground-dwelling ants (hymenoptera: formicidae) in primary and secondary forests in amazonian ecuador. myrmecological news, 12: 139-147. sociobiology 61(2): 155-163 (june, 2014) 161 appendix 1. list of ant species collected in areas of arboreal caatinga and shrubby caatinga, during dry and rainy seasons in milagres, bahia, brazil. species phytophisiognomy arboreal caatinga shrubby caatinga dry season rainy season dry season rainy season acanthoponera sp01 x x acanthostichus sp01 x acromyrmex sp01 x x anochetus sp02 x apterostigma sp01 x atta sexdens rubropilosa x x x x azteca sp01 x azteca sp02 x azteca sp03 x brachymyrmex sp01 x x brachymyrmex sp02 x brachymyrmex sp03 x x brachymyrmex sp04 x x camponotus sp01 x x x x camponotus sp02 x x x x camponotus sp03 x x x x camponotus sp04 x x x x camponotus sp05 x x x camponotus sp06 x x x x camponotus sp07 x x x camponotus sp08 x x x x cephalotes prox. goeldi x x cephalotes atratus x x cephalotes clypeatus x x x cephalotes depressus x x x cephalotes grandinosus x x cephalotes minutus x x x x cephalotes pussilus x x x x cephalotes sp01 x cephalotes ustus x crematogaster sp01 x x x crematogaster sp02 x x x crematogaster sp03 x x crematogaster sp04 x crematogaster sp05 x x crematogaster sp06 x x x x crematogaster sp07 x x x cyphomyrmex sp01 x x cyphomyrmex sp02 x x dinoponera quadriceps x x x x dorymyrmex sp01 x x dorymyrmex thoracicus x x silva et al., effect of environmental heterogeneity in ant community162 ectatomma edentatum x x x x ectatomma muticum x x x x ectatomma sp01 x x ectatomma sp02 x x ectatomma suzanae x x x x forelius brasiliensis x x gnamptogenys concinna x gnamptogenys sp01 x x gnamptogenys sp02 x hylomyrma balzani x x x hylomyrma sp01 x labidus coecus x x labidus mars x labidus praedator x x linepithema humile x linepithema sp01 x x x linepithema sp02 x x x linepithema sp03 x linepithema sp04 x mycetophylax sp01 x neivamyrmex sp01 x x nylanderia sp01 x x ochetomyrmex sp01 x octostruma sp03 x odontomachus chelifer x x odontomachus haematodus x x x x oxyepoecus sp02 x pachycondyla bucki x x pachycondyla prox. magnifica x pachycondyla prox. venusta x x x pachycondyla striata x pheidole sp01 x x x x pheidole sp02 x x x x pheidole sp03 x x x x pheidole sp04 x x x pheidole sp05 x x x pheidole sp06 x pheidole sp07 x x x x pheidole sp08 x x x pheidole sp09 x x x pheidole sp10 x x x x pheidole sp11 x x pheidole sp12 x x x x pheidole sp13 x x x x pheidole sp14 x x x x pheidole sp15 x x x pheidole sp16 x x x sociobiology 61(2): 155-163 (june, 2014) 163 pheidole sp17 x x pheidole sp18 x pheidole sp19 x x x x pheidole sp20 x x pheidole sp21 x x x x pheidole sp22 x x x x pheidole sp23 x x x x pheidole sp24 x x x x pheidole sp25 x x pogonomyrmex (e.) naogeli x x x procryptocerus sp01 x pseudomyrmex elongatus x x pseudomyrmex gracilis x pseudomyrmex sp01 x x x x pseudomyrmex sp02 x x pseudomyrmex sp03 x pseudomyrmex sp04 x x pseudomyrmex sp05 x pseudomyrmex tenuis x x x pseudomyrmex termitarius x x rogeria sp01 x solenopsis sp02 x x x x solenopsis sp03 x x x x solenopsis sp04 x x x x solenopsis sp05 x x x solenopsis sp06 x x x solenopsis sp07 x x x x solenopsis sp08 x solenopsis sp09 x x strumigenys sp02 x tapinoma sp01 x tapinoma sp02 x x tapinoma sp03 x x trachymyrmex sp01 x x x x trachymyrmex sp02 x x wasmannia sp01 x x x wasmannia sp02 x x x wasmannia sp03 x x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.239-242sociobiology 61(2): 239-242 (june, 2014) temporal activity patterns and foraging behavior by social wasps (hymenoptera, polistinae) on fruits of mangifera indica l. (anacardiaceae) bc barbosa, mf paschoalini, f prezoto foraging of social wasps comprises a collection of the following resources: carbohydrates (used mainly for adult diet), animal protein (used for immature diet), plant fiber (used for nest building), and water (used for cooling and building the nest) (hunt, 2007, prezoto et al. 2008, elisei et al. 2010, clemente et al. 2012). while foraging for these resources, social wasps show a generalistic and opportunistic behavior, and evidence of foraging optimization. this behavior has been documented in wasps foraging on different fruit species, such as grapes (hickel & schuck 1995), cacti (santos et al 2007b), jabuticaba trees (de souza et al. 2010), cashew trees (santos & presley 2010), guava trees (brugger et al. 2011), pitanga trees (souza et al 2013), and spanish prune (prezoto & braga, 2013). in these studies, the authors reported that the wasps might prey on crop pests that damage fruits to collect carbohydrates. despite the growing number of studies on social wasps in the past decade, information on the role of these insects in orchards is still scarce, despite these insects having their highest diversity in the neotropics (rafael et al. 2012). hence, there is a need for studies that answer questions such as: which species of social wasps forage on fruits? what types of behavior do they display? do wasps offer risk of accidents to fruit farmers? abstract this study had as objective to determine which species of social wasps visit mango fruits, to record the behaviors displayed by them while foraging and to verify which the species of wasps visitors offer risk of accidents to farmers. the studied area was monitored during february 2012, from 8:00 to 17:00, in a 144 hour effort, and the data collected included the time of activity, wasps diversity, aggressiveness and the general behavior of social wasps around the fruits. there were registered a total of 175 individuals of 12 different species. social wasps damaged the healthy fruits, and we registered the abundance and richness peaks during the hot period of the day. this study indicated the need for special care during the harvest, as aggressive wasps are indeed present and abundant, resulting in a possible increase of accident risk for the workers. sociobiology an international journal on social insects laboratório de ecologia comportamental e bioacústica (labec), universidade federal de juiz de fora, juiz de fora, mg, brazil. article history edited by gilberto m m santos, uefs, brazil received 05 september 2013 initial acceptance 15 october 2013 final acceptance 14 november 2013 keywords fruit trees, harvest, hymenoptera, vespidae corresponding autor bruno corrêa barbosa lab. de ecologia comportamental e bioacústica (labec), universidade federal de juiz de fora 36036-900, juiz de fora, mg, brazil. e-mail: brunobarbosabiologo@hotmail.com this study aims to increase the knowledge about the occurrence of social wasps on mango tree plantations and to describe the richness and abundance of social wasps that forage on fruits throughout the day, as well as to describe the behaviors displayed by them while foraging. the study was conducted in a farm in the municipality of juiz de fora, zona da mata mineira (21°43’55”s, 43°22’16”w, 800 m a.s.l.). observations were made in february 2012, during the fruiting of mango trees, from 8:00 to 17:00, in a total of 144 h of observation. for each observation event, we established 4-m² quadrants on the base of trees to record visiting wasp and their behavior (ad libitum sensu altmann 1974) during foraging on fallen fruits. all were record by direct observation. to record behavioral information we defined four types of arrival behavior of wasps on fruits: (i) direct landing on fruit, (ii) hovering before landing on fruit, (iii) hovering over other fruits before landing, and (iv) landing elsewhere and moving to the fruit. after observation, wasps were collected with an insect net for identification, using for genera and species keys proposed by richards (1978) and carpenter & marques (2001), vouchers were deposited in the laboratory of behavioral ecology and bioacustics at the federal university of juiz de fora (labec). short note bc barbosa et al. foraging behavior of social wasps on fruits of mangifera indica240 we also classified behavioral displays in the presence of other insects as: no aggressive behavior (the wasp remained on the fruit even after being touched by other insects); aggressive behavior (attack or threat to other insects landed on the fruit). for the correlation test between richness and abundance of social wasps we used the pearson coefficient test (r). we also determined the berger-parker index of dominance in r 3.0 (freeware). we recorded 175 individual wasps of four genera and 12 species foraging on mango fruits (table 1). most species (87.8%; n = 9) were swarm-founding wasps (whose nests are founded by a swarm composed of tens of queens and hundreds of workers), which have large biomass, and, therefore, need a large amount of food. these species form large colonies, which makes their local abundance higher than that of species of independent foundation (whose colonies may be founded by one or a few wasps) and may determine resource consumption. these studies corroborate studies carried out in areas of eucalyptus plantations (ribeiro junior 2008), silvopastoral systems (auad et al. 2010), rainforests (souza & prezoto 2006), and arid (santos et al 2009) and island environments (santos et al 2007a) in which swarming species were more abundant than species of independent foundation. the abundance peak occurred from 10:00 to 14:00h (fig. 1). there was a positive correlation between abundance and the warmest times of the day (r = 0.7635; p = 0.0062). these results corroborate studies on social wasp foraging, in which the activity peak of wasps was observed in the warmest times of the day (rezende et al. 2001; elisei et al. 2010; bichara filho et al. 2010; castro et al., 2011). there was no wasp visit from 14:30 to 17:00h. we believe that this may have occurred, because after 14:00h there was shading on fallen fruits, causing a decrease in the temperature in this environment and probably interfering in the foraging of wasps. the species polybia table 1: frequency of social wasps, arrival behavior, aggressiveness, and dominance while foraging on fruits of mangifera indica l. (ab aggressive behavior, na did not exhibited aggressiveness). species abundance arrival behavior of wasps on fruits dominance agressiveness i ii iii iv agelaia vicina 3 x 0.017 na polybia bifasciata 8 x 0.045 na polybia ignobilis 28 x 0.160 ab polybia jurinei 18 x x 0.102 na polybia sp 8 x 0.045 ab polybia platycephala 62 x x 0.354 na polybia scutellaris 1 x 0.005 na polybia fastidiosuscula 15 x 0.085 na synoeca cyanea 3 x 0.017 na mischocyttarus araujo 3 x 0.017 na mischocyttarus cassununga 22 x 0.125 na polistes versicolor 4 x 0.022 na total 175 fig 1. relationship between abundance and richness (a), and variations in abundance and richness (b) throughout the day. a b sociobiology 61(2): 239-242 (june, 2014) 241 platycephala richards, 1951, polybia ignobilis (haliday, 1836), and mischocyttarus cassununga (von ihering, 1903) presented the highest dominance indices (d = 0.354; d = 0.160, and d = 0.120, respectively) and polybia scutellaris (write, 1841) was recorded only once (table 1). all species exploited fruits with pre-existent orifices (mainly caused by other insects such as atta ants and the bee trigona spinipes fabricius, 1973. the only exception was the species synoeca cyanea (fabricius, 1775), which was always observed breaking the skin of fruits. this behavior suggests that this species may become a pest in some environments due to its potential to damage fruits. the same behavior was observed by de souza et al. (2010) in jabuticaba trees and by brugger et al. (2011) in guava trees. however, prezoto & braga (2013) recorded that this behavior of s. cyanea in spanish prune results from wasp predation on larvae of the fruit fly zaprionus indianus gupta, 1970, which qualifies this wasp as a natural enemy of this pest. most wasp species landed directly on the fruit (i). only the species polybia jurinei saussure, 1854 and p. platycephala displayed more than one arrival behavior (table i). the wasps p. ignobilis and polybia sp. were the only species that displayed aggressive behavior (table i). all the other species were recorded using the same fruit without displaying aggressive behavior (table i). although the species p. ignobilis and polybia sp. represent only 20% (n = 36) of the wasps recorded in the study, the aggressive potential of these species should be taken into account to avoid accidents, since they are swarming species, whose colony population easily surpass hundreds of individuals. we also emphasize that p. ignobilis was also described by hermes & kohler (2004) as an aggressive species. based on our results, we suggest that in the period from 10:00 to 14:00, characterized as the activity peak of wasps on fruits, the collectors have an extra care during their activities, as for example the use of personal protective equipment, or even the interruption of the collection activity to reduce the risk of accidents by stings. references altmann, j. (1974). observation study of behavior: sampling methods. behaviour, 49: 223-265. auad, a.m.; carvalho c.a.; clemente m.a. & prezoto, f. (2010). diversity of social wasps in a silvipastoral system. sociobiology, 55: 627-636. bichara filho, c. c., santos, g. m. m., santos filho, a.b., santana-reis, v.p., cruz, j. d. da, & gobbi, n. (2010). foraging behavior of the swarm-founding wasp polybia (trichothorax) sericea (hymenoptera, vespidae): daily resource collection activity and flight capacity. sociobiology, 55: 899-907. brugger, b.p., souza, l.s.a., souza, a.r. & prezoto, f. (2011). social wasps (synoeca cyanea) damaging psidium sp. (myrtaceae) fruits in minas gerais state, brazil. sociobiology, 57: 533-535 carpenter, j. m. & marques, o. m. (2001). contribuição ao estudo dos vespídeos do brasil (insecta, hymenoptera, vespoidea), cruz das almas. universidade federal da bahia. serie publicações digitais, 2: 147. castro, m. m., guimaraes, d. l., prezoto, f. (2011). influence of environmental factors on the foraging activity of mischocyttarus cassununga (hymenoptera, vespidae). sociobiology, 58: 133-141. clemente, m.a., lange, d., del-claro k., prezoto f., campos, n.r. & barbosa, b. c. (2012). flower-visiting social wasps and plants interaction: network pattern and environmental complexity. psyche: a journal of entomology. doi: 10.1155/2012/478431 de souza, a.r., venancio d. & prezoto, f. (2010). social wasps (hymenoptera: vespidae: polistinae) damaging fruits of myrciaria sp. (myrtaceae). sociobiology, 55: 297-299. elisei, t.; nunes, j.v.e.; ribeiro junior, c.; fernandes junior, a.j. & prezoto, f. (2010). uso da vespa social polistes versicolor no controle de desfolhadores de eucalipto. pesquisa agropecuária brasileira, 45: 958-964. hermes, m.g. & köhler, a. (2004). chave ilustrada para as espécies de vespidae (insecta, hymenoptera) ocorrentes no cinturão verde de santa cruz do sul, rs, brasil. edunisc. caderno de pesquisa-série biologia, 16(2): 65-115. hickel, e.r. & schuck, e. (1995). vespas e abelhas atacando a uva no alto vale do rio do peixe. agropecuária catarinense, 8: 38-40. hunt, j. h. (2007). the evolution of social wasps. oxford university press, new york, 259 p. prezoto, f. & braga, n. (2013). predation of zaprinus indianus (diptera: drosophilidae) by the social wasp synoeca cyanea (hymenoptera: vespidae). florida entomologist, 96: 670-672. doi: 10.1653/024.096.0243 prezoto, f., cortes, s.a.o. & melo, a.c. (2008). vespas: de vilãs a parceiras. ciência hoje, 48: 70-73. rafael, j.a.; melo, g.a.r.; de carvalho, c.j.b.; casari, s.a. & constantino r. (2012). insetos do brasil: diversidade e taxonomia. ribeirão preto. holos editora, 810 p. resende, j.j., santos, g. m. m., bichara filho c.c. & gimenes m. (2001). atividade de busca de recursos pela vespa social polybia occidentalis occidentalis (olivier, 1791) (hymenoptera, vespidae). revista brasileira de zoociências, 3: 105-115 ribeiro junior c. 2008. levantamento de vespas sociais (hymenoptera: vespidae) em uma cultura de eucalipto. dissertação de mestrado. universidade federal de juiz de fora, juiz de fora, mg, brasil. 68p. bc barbosa et al. foraging behavior of social wasps on fruits of mangifera indica242 richards, o. w. (1978). the social wasps of the americas excluding the vespinae. london, british museum (natural history), 580 p. santos, g. m. m. & presley, s. j. (2010). niche overlap and temporal activity patterns of social wasps (hymenoptera: vespidae) in a brazilian cashew orchard. sociobiology, 56: 121-131. santos, g. m. m., cruz, j. d. da, marques, o. m. & gobbi, n. (2009). diversidade de vespas sociais (hymenoptera: vespidae) em áreas de cerrado na bahia. neotropical entomology, 38: 317-320. doi: 10.1590/s1519-566x2009000300003 santos, g. m. m., bichara filho, c. c., resende, j. j., cruz, j. d. da, & marques, o. m. (2007)a. diversity and community structure of social wasps (hymenoptera: vespidae) in three ecosystems in itaparica island, bahia state, brazil. neotropical entomology, 36: 180-185. doi: 10.1590/s1519-566x2007000200002 santos, g. m. m., cruz, j.d. da, bichara filho, c.c., marques, o.m. & aguiar, c.m.l. (2007)b. utilização de frutos de cactos (cactaceae) como recurso alimentar por vespas sociais (hymenoptera, vespidae, polistinae) em uma área de caatinga (ipirá, bahia, brasil). revista brasileira de zoologia, 24: 1052–1056. doi: 10.1590/s0101-81752007000400023 souza, m.m. & prezoto, f. (2006). diversity of social wasps (hymenoptera: vespidae) in semideciduous forest and cerrado (savanna) regions in brazil. sociobiology, 47: 135-147. souza, g. k.; pikart, t.g.; jacques, g. c.; castro, a. a.; souza, m. m.; serrao, j. e. & zanuncio, j. c. 2013. social wasps on eugenia uniflora linnaeus (myrtaceae) plants in an urban area. sociobiology, 60: 204-209. doi: 10.13102/sociobiology. v60i2.204-209 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.402sociobiology 63(2): 841-844 (june, 2016) internal armature of the hindgut of pericapritermes nitobei (shiraki) many subterranean termite species have a cuticular armature on the internal surface of the digestive tract; this is assumed to serve as a valvular apparatus, which is generally termed an “enteric valve” (noirot & noirot-timothée, 1969). valve morphology differs among species, and is sometimes used as an identification and/or taxonomic character (e.g., grassé & noirot, 1954). notably, the structure is extensively divergent in soil-feeding species, and is an important taxonomic character for soldierless termites (e.g., sands, 1972; fontes, 1992; sands, 1998; noirot, 2001; donovan, 2002). two species of soil-feeding termites, pericapritermes nitobei (shiraki) and sinocapritermes mushae (oshima and maki), have been reported from japan (ikehara, 1957; takematsu, 1994). however, the detailed characteristics of their enteric valves have not yet been examined. in the present study, we examined the enteric valve and the other hindgut armature of p. nitobei. two colonies of p. nitobei were obtained from soil of iriomote island, okinawa prefecture, japan (location: 24º36′33′′ n, 123.79′61′′ e, 27.3 m a.s.l.) on june 5 2013. the abstract the internal armature of the hindgut of pericapritermes nitobei was examined under a light microscope. termites were obtained from iriomote island, okinawa prefecture, japan, identified based on the typological characters of soldiers and 12s (mitochondrial) ribosomal rna sequencing, and dissected to examine hindgut morphology. the hindgut of p. nitobei could be separated into five parts, p1-p5, and p1-p3 had cuticular spines on the inner surface. p1 bore small triangular spines. p2 formed an enteric valve, composed of six finger-shaped enteric pads with triangular spines, and six marginal regions with small dot-like spines. p3 bore three different types of spines: relatively large star-shaped spines; thorn-like small spines, and long curved spines with brush-like tips. sociobiology an international journal on social insects n. kanzaki, w. ohmura article history edited by rudolf scheffrahn, ufl, usa received 21 april 2014 initial acceptance 28 january 2016 final acceptance 16 february 2016 publication date 15 july 2016 keywords enteric valve, diagnosis, morphology, soil-feeding termite. corresponding author natsumi kanzaki forestry and forest products research institute 1 matsunosato, tsukuba, ibaraki 305-8687 japan e-mail: nkanzaki@affrc.go.jp colonies were fixed on-site in absolute ethanol and brought back to the laboratory. the species was identified based upon the external morphology of soldiers and the sequence of the mitochondrial 12s ribosomal rna of workers. soldiers were transferred to 70% ethanol, and observed under a dissection microscope (leica s8 apo: leica japan, tokyo, japan). we initially focused on head and mandible morphology, and compared our data to those of a previous study (yasuda et al., 2000). several workers were next dissected to observe the hindgut structure. the workers were transferred to 70% ethanol, and entire digestive system was extracted with tweezers and insect pins, and the hindgut elements were dissected separately using sharpened insect pins. the dissected elements were mounted using 70% ethanol, and photomicrographs were taken using a digital camera (dxm1200: nikon, tokyo, japan) fitted to a light microscope facilitated with a dic optics (nikon eclipse 80i: nikon, tokyo, japan). the head capsule was transferred to prepmantm ultra dna sample preparation reagent (applied biosystems, foster city, ca), digested following the manufacturer’s instructions, and used forestry and forest products research institute, tsukuba, ibaraki, japan short note n. kanzaki, w. ohmura – hindgut armature of pericapritermes nitobei842 as a pcr template. pcr amplification and sequencing were performed as described previously (inward et al., 2007). the termites were identified as p. nitobei, the only pericapritermes species reported from japan, because the soldiers had a strongly curved left mandible and a long and round-to-square head. the sequence of the 12s mitochondrial rna of dissected workers was identical to the p. nitobei sequence stored in genbank (accession numbers: ab006584 and dq441790). the hindgut of p. nitobei was clearly separable into five parts, p1 (= ileum), p2 (= enteric valve), p3 (paunch), p4 (= colon) and p5 (= rectum). p1 was relatively tube-like, followed by a narrow constriction (p2). p2 formed an enteric valve, and the hind gut of ethanol-fixed material was often broken at the posterior end of p2 (= the p2/p3 junction). p3 was expanded to form a large sac, narrowing at the posterior end, and smoothly connected to a tube-like colon (= p4) which is smoothly connected to a little expanded short rectum (= p5). of these five sections, p4 and p5 had no obvious internal armature, but the other three parts bore various forms of cuticular structures. the locations of the various types of hindgut spines in p1-p3 are shown schematically in figure 1. the internal armature of p3 was of interest. three different types of spine were evident. these were large, dark, amber-colored star-shaped spines formed by two palm-like components adherent at their bases (fig. 4a, b); small, light amber-colored thornor arrowhead-like spines (fig. 4c); and long, dark, amber-colored curved spines with brush-like split tips (fig. 4d). the positions of these spines within p3 were distinct. the thorn-like and star-shaped spines were located ~20% of the total p3 length from the anterior end. although the detailed arrangements could not be clearly observed, these two types of spine occurred together and formed a narrow spiny band. the long spines covered the remainder of p3, but were most dense in the anterior and middle regions, becoming fig 1. schematic drawing of the hindgut of pericapritermes nitobei. the sections are indicated as p1, p2 (ev), p3, and p4. the locations and densities of spines are coded using a grayscale and are indicated by arrows. p4 and p5 are not drawn because these parts do not have cuticular spines. the internal surface of p1 bore amber-colored triangular spines, which were relatively simple and small (fig 2). the spinal arrangement seemed to be irregular, and the spines occupied almost all of p1. p2 formed a moderately developed enteric valve, separable into six enteric pads and six marginal regions. the enteric pads were finger-shaped (i.e., with rounded posterior ends) and bore light amber-colored triangular spines (fig 3). the tips of the spines were relatively blunt, forming somewhat flattened triangles (fig 3). the marginal parts were membrane-like, with small dot-like cuticular spines (fig 3). compared to some other pericapritermes species, the enteric valve of p. nitobei was somewhat similar to that of p. papuanus bourguignon, leponce & roisin 2008. thus, p. papuanus bears triangular spines on enteric pads, and dot-like cuticles on the marginal regions, although the shape of the triangular spines and the density of dots differ from those of the species under discussion in the present report (bourguignon et al., 2008). fig 2. internal armature of the p1 section of pericapritermes nitobei. fig 3. the enteric valve (p2 section) of pericapritermes nitobei. a: an entire enteric valve was expanded and mounted on a slide; b, c: an enteric pad (center) and marginal regions (right and left sides) in different focal planes. sociobiology 63(2): 841-844 (june, 2016) 843 gradually sparser posteriorly. brown glue-like materials, assumed to be organic in nature, were adherent to the bases of the long spines and were evident in all p3 regions bearing such spines. the organic material could be removed by sonication, but not by tweezers or insect pins. thus, the spines, especially the long spines, may trap organic nutrient materials. the structures and arrangement of spines were consistent among all individual workers examined. we did not examine soldier structure because only one soldier was obtained from each colony. more soldier materials are required for detailed observation. the enteric valve structure of higher termites has become an important taxonomic character (donovan, 2002; bourguignon et al., 2008; scheffrahn, 2013). scheffrahn (2013) reported small spiny flap-like cuticular structures on the posterior end of the enteric valve seat of compositermes vindai. also, bignell et al. (1980, 1983) described long spines in the p3 regions of procubitermes aburiensis and cubitermes severus. further, noirot (2001) photographed a row of saw blade-like spines baring of p3 of p. magnificus which is clearly different from any of p3 spines of p. nitbei. homology among those cuticular structures and the structures found in the present study are necessary to be examined in the future with developmental and/or molecular works, e.g., analysis of tissue-specific gene expression pattern. however, in most instances, the p1 and p3 sections of termite guts have been inadequately studied. nevertheless, such characters may be useful in terms of functional morphology and termite taxonomy, and warrant close examination. acknowledgement the authors thank prof. dr. rudolf h. scheffrahn, university of florida for his advises on the general morphology of enteric valve of pericapritermes. references bourguignon, t., leponce, m. & roisin, y. (2008). revision of the termitinae with snapping soldiers (isoptera: termitidae). zootaxa, 1769: 1-34. bignell, d.e., oskarsson, h. & anderson, j.m. (1980). specialization of the hindgut wall for the attachment of symbiotic microorganisms in a termite procubitermes aburiensis (isoptera, termitidae, termitinae). zoomorphologie, 96: 103-112. bignell, d.e., oskarsson, h., anderson, j.m., ineson, p. & wood, t.g. (1983). structure, microbial associations and functions of the so-called “mixed segment” of the gut in tow soil feeding termites procubitermes aburiensis sjöstedt and cubitrmes severus silvestri (termitidae, termitinas). journal of zoology, 201: 445-480. donovan, s.e. (2002). a morphological study of the enteric valves of the afrotropical apicotermitinae (isoptera: termitidae). journal of natural history, 36: 1823-1840. fontes, l.r. (1992). key to the genera of new world apicotermitinae (isoptera: termitidae). in d. quintero & a. aiello (eds.), insects of panama and mesoamerica. selected studies (pp. 242-248). oxford: oxford university press. grassé, p.p. & noirot, c. (1954). apicotermes arquieri n. sp.: ses constructions et sa biologie. considérations sur la sousfamille des apicotermitinae. annales des sciences naturelles, zoologie, 16: 345-388. ikehara, s. (1957). the termite fauna of the ryukyu islands and its economic significance (i). bulletin of arts and science division, university of the ryukyus mathematics & natural sciences, 1: 44-61. inward, d.j., vogler, a.p. & eggleton, p. (2007). a comprehensive phylogenetic analysis of termites (isoptera) illuminates fig 4. internal armature of the p3 section of pericapritermes nitobei. a, b: star-shaped spines in different focal planes; c: thorn-like spines; d: long curved spines. n. kanzaki, w. ohmura – hindgut armature of pericapritermes nitobei844 key aspects of their evolutionary biology. molecular phylogenetics and evolution, 44: 953-967. doi: 10.1016/j.ympev.2007.05.014 noirot, c. (2001). the gut of termites (isoptera). comparative anatomy, systematics, phylogeny. ii. higher termites (termitidae). annales de la societé entomologique de france, 37: 431-471. noirot, c. & noirot-timothée, c. (1969). the digestive system. chapter 3. in k. krishna & f. m. weesner (eds.), biology of termites, vol. 1 (pp. 49-88). new york: academic press. sands, w.a. (1972). the soldierless termites of africa (isoptera: termitidae. bulletin of the british museum (natural history) entomology, 18: 1-244. sands, w.a. (1998). the identification of worker castes of termite genera from soils of africa and the middle east. wallingford: cab international, 512 pp. scheffrahn, r.h. (2013). compositermes vindai (isoptera: termitidae: apicotermitinae), a new genus and species of soldierless termite from the neotropics. zootaxa, 3652: 381391. doi: 10.11646/zootaxa.3652.3.6 takematsu, y. (1994). first record of sinocapritermes mushae (oshima and maki) (isoptera, termitidae, termitinae) from japan, with redescription of soldier and worker castes. japanese journal of entomology, 62: 709-712. yasuda, i., nakasone, y., kinjo, k. and yaga, s. (2000). morphology and distribution of termites in ryukyu islands and north and south daito islands. japanese journal of entomology, 3: 139-156. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2069sociobiology 65(2): 345-347 (june, 2018) discovery of the termite specialist ant genus centromyrmex, mayr 1866 (hymenoptera: formicidae: ponerinae) for the guiana shield the ant genus centromyrmex mayr, 1866 (formicidae: ponerinae) is a relatively small genus with 15 species worldwide. morphologically, the genus is easily recognized by the lack of eyes, smooth cuticle, presence of spiniform setae on the mesotibiae and laterally-opening metapleural gland orifices, situated just below the propodeal spiracle. of these characters, only the last is truly autapomorphic for centromyrmex (schmidt & shattuck, 2014). very little is known about the behavior of centromyrmex, and the few observations on the species biology suggest that all species in this genus are obligatory predators of termites (schmidt & shattuck, 2014). the habits are clearly subterranean and the species of centromyrmex are well adapted to a hypogeic and fossorial lifestyle. this is evidenced by the presence of morphological features commonly found in other hypogeic ants such as their relatively smooth cuticles, lack of eyes in the workers, and the short, powerful, spiny legs that facilitate their movement abstract the ponerine ant genus centromyrmex is recorded for the first time in french guiana. the specimens reported here were collected by the seag (société entomologique antilles-guyane) team in the commune of saül, french guiana. the ants were collected in a region of amazon forest with flight interception traps. two out of the three species currently recognized for the genus in south america were here recorded in french guiana, centromyrmex alfaroi and centromyrmex gigas. in total, 24 specimens were collected, all represented by alate queens. this record expands the knowledge about the distribution of this rarely collected genus and reinforces the importance of alternative sampling techniques for collecting cryptobiotic ants and unknown alate forms. sociobiology an international journal on social insects w franco, rm feitosa article history edited by gilberto m. m. santos, uefs, brazil received 06 september 2017 initial acceptance 02 november 2017 final acceptance 12 november 2017 publication date 09 july 2018 keywords new records, distribution, guiana shield, amazon forest, poneroid. corresponding author weslly franco laboratório de sistemática e biologia de formigas – lsbf departamento de zoologia universidade federal do paranáufpr av. cel. francisco h. dos santos, 210 cep 81531-970 curitiba-pr, brasil. e-mail: weslly.franco@gmail.com through the substrates they inhabit (kempf, 1967; dejean & fénéron, 1999; bolton & fisher, 2008). nesting sites are usually near or inside termitaries. despite rarely collected, workers are usually found inside their prey’s nests, upper soil layers, beneath leaf litter, or in rotten logs (delabie, 1995; bolton & fisher, 2008). centromyrmex is a cosmopolitan genus with a mostly tropical distribution. most of species in the genus are found in the afrotropics, where 11 species are registered. three species are currently known from the neotropics: centromyrmex alfaroi emery, 1890; centromyrmex brachycola (roger, 1861) and centromyrmex gigas forel, 1911. according to kempf (1967), these three neotropical species represent the brachycola group of the genus. in the neotropics, centromyrmex is relatively widely distributed. the species of the brachycola group are known for argentina, brazil, bolivia, colombia, costa rica, ecuador, panama, paraguay, trinidad and tobago, and venezuela. 1 departamento de zoologia, universidade federal do paraná (ufpr), curitiba-pr, brazil short note w franco, rm feitosa et al. – first record of centromyrmex for the guianas shield from flight interception tramps346 the northernmost occurrence locality for the genus is costa rica and the southernmost limit is the province of misiones, argentina. despite this, the distribution of centromyrmex present several localities, principally in the south america, where there is no record for the genus. in this work, we report the first records of two species of centromyrmex for french guiana, extending the distribution limits for the genus. the specimens reported here were identified among the ants collected by the seag (société entomologique antillesguyane) team during the year 2011 in the commune of saül, french guiana (3º37’22”n and 53º12’57”w), an overseas department of france in south america. the locality where the genus was recorded belongs to the parc amazonien de guyane, a national park created to protect part of the amazon forest in the country. ant specimens were collected with slam traps (megaview science, taichung city, taiwan) a variant of the malaise trap (sand, land and air malaises). malaise traps are among the most widely used trapping devices for insect collection (matthews & matthews, 1971). the operation of the trap is based on the interception of the flight trajectory of the insects by a fine mesh fabric which acts as a vertical barrier. the slam trap is a self-supporting trap mounted on a flexible “igloo tent” structure (dodds et al., 2015; mccravy, 2017). the ants collected by the seag team were donated to the laboratório de sistemática e biologia de formigas da universidade federal do paraná, curitiba, paraná, brazil, where the samples were processed. the species of centromyrmex were identified using the key available in kempf (1967). vouchers were deposited in the padre jesus santiago moure entomological collection at the universidade federal do paraná (dzup). high resolution images were obtained under a leica m125 stereomicroscope attached to a leica dfc 295 video camera at the dzup. the images of the layers were aligned and combined in program zerene stacker v. 1.04, and posteriorly treated in adobe photoshop cs6 for brightness and contrast corrections. the distribution map was generated by the software qgis 2.18.11 with coordinates imported from google earth after consulting the records for centromyrmex in the literature. two species of centromyrmex were identified among the ants collected by seag, namely centromyrmex gigas and centromyrmex alfaroi, with 19 and five individuals recovered from the samples, respectively. all the 24 specimens captured are alate queens, which could be securely identified to species since they match most of the diagnostic characters of the species (fig 1). this represents the first record of the genus from french guiana. both species recorded here appear to be termitophagous, as commonly reported for the genus (baccaro et al., 2015). specimens of centromyrmex gigas are relatively large, with distinctly dentate mandibles, head distinctly broader than long, clypeus without a median tumulus, and the petiole with a long subpetiolar process. the integument is reddish brown to amber-colored (kempf, 1967). this species is frequently found in nests of syntermes spp. termites (delabie, 1995). centromyrmex alfaroi is smaller than centromyrmex gigas, with a broader head, clypeus with an elevated median tumulus subpetiolar process rather short. the color is dark ferruginous. the biology is unknown, but the few specimens observed alive are presumed entirely subterranean (kempf, 1967). until now, centromyrmex gigas was known only from argentina and brazil. the species was recorded in the lower amazon valley (states of pará, amapá, roraima, and amazonas), and in the central and southeastern states of brazil (goiás, bahia, são paulo, and rio de janeiro). the only record of c. gigas outside brazil so far corresponds to the province of missiones, argentina, representing the southernmost locality for the occurrence of this genus in the neotropical region (fig 2). fig 1. centromyrmex species of french guiana. centromyrmex gigas (a, b); centromyrmex alfaroi (c, d). a, c: full-face view; b, d: body in lateral view. fig 2. distribution map of centromyrmex gigas in the neotropics based on literature records and the present study. sociobiology 65(2): 345-347 (june, 2018) 347 centromyrmex alfaroi is a relatively widely distributed species, with records from brazil (states of roraima, bahia, goiás, minas gerais, and são paulo); bolivia; ecuador; colombia; costa rica and panamá (fig 3). the records presented here extend the distribution of c. gigas and c. alfaroi in the amazon region in more than 900 km to the west from the previously westernmost record in roraima, brazil. the cryptic habits, small colonies and the low local abundance make it rare for ant inventories to detect species of centromyrmex by the conventional sampling techniques applied for ants. for this reason, the genus distribution presents major gaps in the neotropical region. the centromyrmex specimens recorded here are alate (sexual) forms captured by flight interception traps. information on the sexual forms are of primary interest in ant taxonomy, since most of the specimens deposited in myrmecological collections are wingless (workers). this is especially true for cryptobiotic ants. also, information about the phenology of ant nuptial flights is largely unavailable, and limited to a few taxa (feitosa et al., 2016). our results reinforce the idea that applying different sampling methods is essential to improve our ability of recording cryptobiotic groups (bestelmeyer et al., 2000), and that the use of flight interception traps has the potential to greatly improve our knowledge on ant biology and distribution. bestelmeyer, b.t.; agosti, d.; alonso, l.e.; brandão, c.r.f; brown, jr w.l.; delabie, j.h.c.; silvestre, r. (2000). field techniques for the study of ground-dwelling ants: an overview, description, and evaluation. in: agosti, d.d.; majer, j.d.; alonso, l.e.; schultz, t.r. (eds.), ants: standard methods for measuring and monitoring biodiversity (pp.222–144). washington: smithsonian institution press. bolton, b. & fisher b.l. (2008). afrotropical ants of the ponerine genera centromyrmex mayr, promyopias santschi gen. rev. and feroponera gen. n., with a revised key to genera of african ponerinae (hymenoptera: formicidae). zootaxa, 1929: 1-37. dejean, a. & fénéron, r. (1999). predatory behavior in the ponerinae ant, centromyrmex bequaerti: a case of termitolesty. behavioural processes, 7: 125-133. delabie, j.h.c. (1995). inquilinismo simultâneo de duas espécies de centromyrmex (hym., formicidae, ponerinae) em cupinzeiros de syntermes sp. (isoptera, termitidae, nasutermitinae). revista brasileira de entomologia, 39: 605-609. dodds, k.j., allison, j.d., miller, d.r., hanavan, r.p., sweeney, j. (2015), considering species richness and rarity when selecting optimal survey traps: comparisons of semiochemical baited flight intercept traps for cerambycidae in eastern north america. agricultural forest entomology, 17: 36-47. doi: 10.1111/ afe.12078 feitosa, r.m.; silva, r.r.; aguiar, a.p. (2016). diurnal flight periodicity of a neotropical ant assemblage (hymenoptera, formicidae) in the atlantic forest. revista brasileira de entomologia, 60: 241-247. doi: 10.1016/j.rbe.2016.05.006 kempf, w.w. (1967). a synopsis of the neotropical ants of the genus centromyrmex mayr (hymenoptera: formicidae). studia entomologica, 9: 401-410. matthews, r.w. & matthews, j.r. (1971). the malaise trap: its utility and potential for sampling insect populations. michigan entomologist, 4: 117-122. mccravy, k.w.; geroff, r.k; gibbs, j. (2017). malaise trap sampling efficiency for bees (hymenoptera: apoidea) in a restored tallgrass prairie. florida entomologist, 99: 321323. doi: 10.1653/024.099.0230 schmidt, c.a. & shattuck, s.o. (2014). the higher classification of the ant subfamily ponerinae (hymenoptera: formicidae), with a review of ponerine ecology and behavior. zootaxa, 3817: 1-242. doi: 10.11646/zootaxa.3817.1.1. author contributions wf, rmf– identified specimens, wrote the manuscript. wf – compiled the data and prepared the distribution map. fig 3. distribution map of centromyrmex alfaroi in the neotropics based on literature records and the present study. acknowledgements we thank to the brazilian council of research and scientific development (cnpq) for supporter. wf (grant 130642/2016-9). rmf (grant 302462/2016-3). reference baccaro, f.b.; feitosa, r.m.; fernandez, f.; fernandes, i.o.; izzo, t.j.; souza, j.p. de; solar, r. (2015). guia para os gêneros de formigas do brasil. manaus: editora impa, 388 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2822sociobiology 65(3): 383-396 (september, 2018) zoogeography of the ants (hymenoptera: formicidae) of the segura river basin introduction the mediterranean basin, including the iberian peninsula, is one of the most important biodiversity hotspots in the world, with a high level of endemism (médail & quézel, 1997; myers et al., 2000; gómez & lunt, 2007; hewitt, 2011). médail and quézel (1997), based on floral endemism, identified ten mediterranean basin hotspots, and the baetic-rifan complex (including the baetic ranges), one of them being. our study area, the segura river basin, is located in the central sector of southeastern iberian peninsula (fig 1) and is representative of the environmental diversity of the area. it is framed in the western border by the baetic ranges, abstract the work submitted in this paper presents the first checklist of the ant species of the segura river basin based on a review of specific literature and biological material collected during field work conducted from 2012 to 2016. our findings recorded 110 species that belong to 30 genera of ants and twenty-two of these species have been recorded for the first time in this area. the zoogeographical composition is dominated by the species of the mediterranean zone (75.2%), followed by the mixed and deciduous forest zone (19.1%). the most important zoogeographic elements are: iberian (20%), holomediterranean (17.1%) and westmediterranean (13.3%). there are only six cosmopolitan species (5.71%). there is a greater proportion of species from the mixed and deciduous forest zone in the high-mid altitudes in the segura river basin, where the climate is cooler, and more humid. the euro-caucasian and euro-west siberian elements tend to be more associated to forest with a higher precipitation, whilst the south palearctic elements seem to be more associated to ecosystems more similar to the foreststeppe zone with intermediate precipitation. the existence of these different zoogeographic origins in this area is probably linked with: the position between africa and europe; the complex geotectonic, paleogeographic, and paleoclimatic history during the last 7 my; the complex geomorphology; and the high climate and habitat diversity. based on ant studies and other taxa, possible explanations of the zoogeographic origin of these ant chorotypes are proposed. sociobiology an international journal on social insects c catarineu1, gg barberá2, jl reyes-lópez3 article history edited by gilberto m. m. santos uefs, brazil received 30 december 2017 initial acceptance 15 may 2018 final acceptance 09 july 2018 publication date 02 october 2018 keywords biodiversity, biogeography, checklist, endemism, faunistics, new records. corresponding author chema catarineu asociación de naturalistas del sureste plaza del pintor josé maría párraga nº 11 30002, murcia, spain. e-mail: chema@asociacionanse.org a result of the convergence between the african and iberian plates. the baetic ranges intercept atlantic cyclones in such a way that at some points of the mountainous area rainfall reaches 900-1,200 mm per year, whilst a rain shadow lies immediately to the east and dominates the region. the segura river basin (19,025 km2) has large orographic variety, with mountains that reach up to 2,000 m.a.s.l., as well as contrasting coastal areas and extensive plains and valleys (confederación hidrográfica del segura, 2017). mountain lithology consists of limestone, dolomite and shale, and other metamorphic siliceous materials, whilst the plains and valleys between mountains are usually filled with marls, alluvial, and colluvial quaternary deposits and some volcanic outcrops (mellado et al., 2002). the territory is very 1 asociación de naturalistas del sureste (anse), murcia, spain 2 department of soil and water conservation, csic-cebas, murcia, spain 3 área de ecología, universidad de córdoba, córdoba, spain research article ants c catarineu, gg barberá, jl reyes-lópez – ants of southeastern iberian peninsula384 singular, both for the iberian peninsula and europe as a whole, and is dominated by a mediterranean semiarid climate, yet, also presents strong rainfall and temperature gradients (vidalabarca et al., 1987). the vegetation, predominantly xerophytic, is a product of climate aridification during mid-to-late holocene and human induced degradation (carrión et al., 2010). the contrasts in altitude, lithology, precipitation, temperature and also in relation to human occupation, generate a great diversity of environments that allows a high biodiversity. the complex paleogeographic and paleoclimatic history of the area (carrión et al., 2010) has also contributed to the existence of a rich biodiversity. the iberian ant fauna is progressively more widely known, although this knowledge is still very fragmentary and limited to studies of specific areas. there are only a few sub regional checklists of the myrmecofauna in this area (tinaut, 1981; ortiz & tinaut, 1988; espadaler, 1997a; espadaler, 1997b; catarineu & tinaut, 2012; del campo et al., 2014). there are also very few publications focusing on the zoogeography of european ants: northern europe (baroniurbani & collingwood, 1977), greek islands (collingwood, 1993), poland (czechowski et al., 2002; czechowski et al., 2012), ukraine (radchenko, 2011), and montenegro (karaman, 2011). dynamic lists, such as antmaps (antmaps.org, 2016), also provide species distribution information. the southeastern iberian peninsula is included in only four publications mentioning ant zoogeography, in the sierra nevada (tinaut, 1981), cabo de gata (tinaut et al., 2009), se-spain (piñero et al., 2011), and región de murcia (catarineu & tinaut, 2012). the main objectives of this paper are: 1) to compile a checklist of ant species in the segura river basin based on a review of the literature and material collected during sampling conducted from 2012 to 2016; 2) to classify the ant species of the segura river basin into zoogeographic chorotypes; 3) to analyze if zoogeographic chorotypes are related to climatic gradients in the segura river basin. material and methods ant data sources the checklist presented is based on three different sources: (i) a literature review (23 papers that cite southeastern iberian ants, see table 1); (ii) systematic sampling of 60 locations spread over ten 10×10 km utm squares representative of the climatic, geological and geomorphological variation of the region [in each location, a 100-m transect was set up, consisting of 10 pitfall traps separated by 10 metres and open for one week (in july 2014 and july 2015); pitfall traps were polystyrene tubes (2 cm diameter, 10.5 cm long) filled with 5 ml of 50:50 propylene glycol and water solution with a few drops of liquid soap added to reduce surface tension]; (iii) non-systematic sampling by pitfall trapping and hand collecting over the whole region, adding up to 103 different localities during the period 20102016 (see fig 1 for the 163 locations, and table s1 for the whole information about locations and dates). all ant samples were sorted and identified to species level using a stereo microscope and iberian ant taxonomic keys (hormigas.org, 2016), taxonomic revisions of some ant genera (seifert, 1988, 1992, 2002), and species descriptions and highresolution pictures provided by antweb (2017). it was not possible to identify species of leptanilla emery, proformica ruzsky, and solenopsis westwood due to a lack of modern revisions and complete taxonomic keys in these genera. in relation to tapinoma cf nigerrimum and tetramorium cf caespitum, recently seifert et al. (2017) and wagner et al. (2017) confirmed the existence of different cryptic species within these species complexes found in the iberian peninsula. unfortunately, there is not a simple nor precise method for phenotypical species delimitation in these species complex as we have no access to the specific optical equipment required for this task. identified ants were transferred to vials with ethanol 70º, or were mounted. all specimens are deposited fig 1. map of spain with the segura river basin outlined (on the left) and a map of the segura river basin with the 163 localities sampled indicated with grey squares (on the right). sociobiology 65(3): 383-396 (september, 2018) 385 in the premises of asociación de naturalistas del sureste (murcia, spain) and some duplicates samples in the university of córdoba (área de ecología, córdoba, spain). zoogeographic categories a review of the present geographic distribution of the segura river basin ants was undertaken, based mainly on the following sources: czechowski et al. (2002); glaser (2006); csösz et al. (2007); karaman (2011); czechowski et al. (2012); seifert (2012a, 2012b); antarea (2015); antmaps (2016); hormigas.org (2016); janicki et al. (2016); antweb (2017); and lebas et al. (2017). considering their current geographical distribution in the palaearctic and saharo-arabian zoogeographic realms (sensu holt et al., 2013), the native ant species were classified into two main zoogeographical classes corresponding to the two main palaearctic vegetation zones, in accordance with czechowski et al. (2002) and czechowski et al. (2012): (i) mixed and deciduous forest zone; and (ii) the mediterranean zone sensu lato. within each zone, species ranges were classified into the following scheme: elements represented in the segura river basin ants (modified from czechowski et al., 2012): i. mixed and deciduous forest zone. 1) the central and southern european element (cse)species distributed mainly in southern and central europe. 2) the central and southern european/maghrebian element (cse/m)species distributed mainly in southern and central europe and also in the maghreb. 3) the eurocaucasian element (ec)species distributed mainly in the zone of deciduous forest, partly in mixed forest areas within europe and the caucasus; possibly reaching asia minor and the near east. 4) the eurocaucasian/maghrebian element (ec/m) eurocaucasian species also present in the maghreb. 5) euro-west-siberian element (ews)species widely distributed in europe (usually also in the caucasus) and west siberia, reaching the east altai mts. 6) euro-west-siberian/maghrebian element (ews/m)eurowest-siberian species also present in the maghreb. 7) the south-palaeartic element (sp)generally transpalaeartic forms, often distributed from the atlantic to pacific oceans, whose ranges cover mainly the southern part of the forest zone and the forest-steppe zone; ecologically associated with dry forest or dry grasslands. ii. mediterranean zone sensu lato. 8) the tethyan element (t)species with a wide area that includes wholly or at least mostly the mediterranean region, south of central and eastern europe, the caucasus, asia minor, the middle east, iran, kazakhstan and middle asia. 9) the holomediterranean element (hm)species distributed in the european mediterranean region, south of central and eastern europe, north africa and, in asia minor (or in a significant part of this area). they can also occasionally reach near and middle east, central europe, and the southern part of east europe. 10) the north-mediterranean element (nm)species distributed in the european mediterranean region but absent in north africa. 11) the west-mediterranean element (wm)species distributed in the west-mediterranean region: iberian peninsula, france, the apennine peninsula and the maghreb. in some cases, they are also present in balearic islands, corsica, sardinia or sicily. 12) the north-west mediterranean element (nwm) – species distributed in the iberian peninsula and southern france. they can also reach the northern apennine peninsula. 13) the iberian elements (i)species distributed only in the iberian peninsula. we include some iberian sensu lato species, that can also reach the french pyrenees or the balearic islands. 14) the iberian/maghrebian elements (i/m)species distributed in the iberian peninsula and also in the maghreb. relation of zoogeographic chorotypes to environmental variability data sources are heterogeneous and the sampling effort was heavily unbalanced across the region. therefore, we adopted a strategy designed to deal with this problem. firstly, we resampled data at 10×10 km resolution on the utm grid in order to obtain a higher number of species by sampling unit and a more homogeneous definition of the locality concept. in total there are data from fifty-seven 10×10 km utm squares. then, we studied the relationship between the proportion of species belonging to the class of mixed and deciduous forests and the precipitation (computed as the mean of the localities sampled within a 10×10 km utm square) by logistic regression, being the number of success the number of species belonging to the class of mixed and deciduous forests, and the number of trials the total number of species. the species richness of a particular chorotype is clearly influenced by sampling intensity, however one can expect that the proportion of species in a chorotype is independent of the sampling intensity although values on less sampled areas are not as reliable. moreover, the fisher scoring algorithm for logistic regression weighs observations proportional to the number of species, allocating low weights to sampling units with low sampling intensity and high weights to adequately sampled units. therefore, estimates of parameters is much more influenced by best sampled localities, while marginal information of other areas is not absolutely neglected. mean annual precipitation and temperature data for the whole basin were obtained from worldclim 2 (fick & hijmans, 2017), and altitude from aster satellite imagery. there was a very high correlation (>0.95) between the three variables as a result of the natural correlation of precipitation (positive) and temperature (negative) with altitude in the area, but it was probably exacerbated by the interpolation methodology in worldclim 2 as altitude is incorporated in the interpolation algorithm. because of redundancy between the three variables, we chose precipitation as the target climatic predictor variable. c catarineu, gg barberá, jl reyes-lópez – ants of southeastern iberian peninsula386 localities with ant data are a biased sample of the basin. highest altitudes (>1200 m) are overrepresented in the sample (fig s1.a) while low-mid altitude (200-1100 m) are in general underrepresented, but lowest altitudes (<200 m) are again overrepresented. since the correlation between altitude and precipitation (positive) and temperature (negative) is high in the area, this means the coldest and rainiest areas are overrepresented in the sample, while intermediate temperature and precipitation are underrepresented except in the warmest areas (fig s1b-c). the sampling bias is a consequence of much higher proportion of natural habitats on the mountain ranges than in the inter-range plains and valleys, mostly occupied by agricultural lands (lópezbermúdez et al., 1986) which were rarely sampled. a second approach to better understanding the relationship between chorotypes and environmental variability was carried out using elements instead of zones as zoogeographic units. at this higher zoogeographic resolution, the number of species per chorotype becomes rather reduced, therefore we used only data obtained from the systematic sampling of ten 10×10 km utm squares (item ii; ant data sources). these squares were selected as representing the diversity of climates, lithologies and geomorphology of the basin, after clustering data with those variables using k-means method (data not shown). sampling intensity was exactly the same across the 10 squares and therefore data are strictly comparable. in this analysis, we grouped cse and cse/m; ec and ec/m; and csw and csw/m (cse, ec/m and ews/m had only 1, 1 and 2 species respectively). then, we calculated the richness per chorotype element (permitting the grouping indicated) and the richness matrix by element by 10×10 km utm square was submitted to a non-metric multidimensional analysis (nmds; bray-curtis distance; dimensions = 2; stress = 0.13). coordinates in the nmds space are represented on the geographic space of the basin and sampling unit coordinates on nmds axis 1 and 2 were individually regressed on precipitation. results the checklist of the segura river basin ants includes 110 ant species from 30 genera of five subfamilies (dolichoderinae, formicinae, leptanillinae, myrmicinae, and ponerinae). two subfamilies were dominant: myrmicinae with 53 species and formicinae with 46 species (table 2). the most represented genera are temnothorax mayr (19 species), camponotus mayr (14 species), lasius fabricius (8 species), formica linnaeus, and plagiolepis mayr (6 of each). twenty two species were recorded for the first time in this area (table 1). one species previously reported from the area is excluded: lasius niger l. there are only two records of l. niger for the segura river basin (collingwood & yarrow, 1969; martínez et al., 2002), and these records possibly correspond to lasius grandis forel, because the first record was before seifert (1992) raised l. grandis to species, and the second was based on one queen only. seifert (1992) argues that in most mesophilic areas of spain l. niger is completely displaced by l. grandis and our findings appear to support him. one new species was discovered as a result of our field work, temnothorax ansei, recently described by catarineu et al. (2017). there are three undescribed species in the checklist: camponotus sp.1 (a parasite of camponotus pilicornis roger); plagiolepis sp.1 (a parasite of plagiolepis schmitzii forel); and temnothorax sp.1. camponotus sp.1 and plagiolepis sp.1 are being described by x. espadaler (x. espadaler, personal communication); temnothorax sp.1 is being described by a. tinaut (a. tinaut, personal communication). subfamily no. of genera (%) no. of species (%) dolichoderinae 3 (10.0) 6 (5.5) formicinae 10 (33.3) 46 (41.8) leptanillinae 2 (6.7) 2 (1.8) myrmicinae 13 (43.3) 53 (48.2) ponerinae 2 (6.7) 3 (2.7) total 30 110 table 2. the composition of the ant fauna of the segura river basin, with species and genus numbers/percentages given for each subfamily. zoogeographical composition table 3 shows the zoogeographical composition of the ant fauna in the segura basin. for this classification, we consider only 105 species, discarding those not identified as species (leptanilla sp., proformica sp., solenopsis sp., tapinoma cf nigerrimum and tetramorium cf caespitum). three quarters of the species (79 spp., 75.2%) are associated with the mediterranean zone, while nearly a fifth (20 spp., 19.05%) are associated with the mixed and deciduous forest zone. the other species are cosmopolitan (6 spp., 5.7%), tramp species introduced in this region with larger distribution globally, which on the other hand indicates that 94.3% of the species collected can be considered as native from the segura river basin region. the four principal zoogeographical elements are clearly: iberian (21 spp., 20 %), holomediterranean (18 spp., 17.1%), west-mediterranean (14 spp., 13.3%) and north-west mediterranean (11 spp., 10.5%). over two-fifths of the segura basin ants are also encountered in the maghreb region (43 spp., 41%). chorotypes and environmental variability there is a clear relationship between the proportion of ant species in the mixed and deciduous zone and precipitation (fig 2). it is rare to find species of this zone in areas with less than 350 mm of annual precipitation. with rainfall over 450 sociobiology 65(3): 383-396 (september, 2018) 387 mm these species were only absent in 10×10 km utm squares with very low intensity sampling (very low total observed ant richness consequently). with rainfall between 300-400 mm (the most frequent precipitation range in the region, fig s1.c) there is a considerable spread of the proportion of species in the class. it may be related to the large environmental differences between south and north-face of the mountain ranges, the latter faces being considerably mesic as compared to areas on flat or south-faced slopes, and therefore having denser vegetation, less insolation and higher humidity. focusing on elements only for homogeneously sampled 10×10 km utm squares, the pattern that appears clearly separates most of the mixed and deciduous forest zone elements from the mediterranean elements (fig 3). the first axis (nmds1) separates euro-caucasian (ec + ec/m) and euro-west siberian (ews + ews/m) elements from the rest. the second axis (nmds2) clearly separates south paleartic (sp), ec and ews elements from the rest, but ec and ews and sp are on opposite sides of the axis (fig 3). nmds1 separates presumably forest associated elements ec and ews from the rest, while nmds2 separates clearly sp element more associated to steppe eurosiberian ecosystems. this is clearer on the geographical representation of the nmds coordinates of the 10×10 km utm squares (fig 4.a-b). the euro-caucasian and euro-west siberian elements seem to be more associated with subhumid and humid forests on the nw segura river basin mountains. the south paleartic elements seem to be more associated to the nsegura river basin, an area of transition to the central spain plateau, characterized by mid-height mountains surrounded by relatively high valleys and plains more similar to the forest-steppe zone with more open forests and grasslands (fig 4a and 4b). fig 2. relationship between proportion of species on the mixed and deciduous forest class and mean annual precipitation. solid line, logistic regression model expectation. dots, empirical data grouped on 10×10 km utm squares. size of dots is proportional to the total number of ant species recorded on the square. two squares with one and two species, respectively, are not represented on the figure as they are out of range (proportion of mixed and deciduous forest class 0.5 and 1). intercept: -4.4427 ± 0.5479, z= -8.108, p=<0.001; precipitation: 0.0054 ± 0.0013, z= 4.271, p=<0.001. fig 3. non metric multidimensional scaling (nmds) of zoogeographic elements on 10×10 km utm squares representing the geological, geomorphological and climatic variation of the segura river basin. cse = central and southern european, ec = euro-caucasian, ews = euro-west siberian, hm = holomediterranean, i = iberian, i/m = iberian/ maghrebian, nm= north-mediterranean, nwm = northwest mediterranean, sp = south-paleartic, t = tethyan, wm = west-mediterranean. fig 4. geographical representation of coordinates of 10×10 km utm squares of the systematic sampling (see methods) on nmds space of figure 3. (a) coordinates on axis nmds1. (b) coordinates on axis nmds2. a) both axes have a clear relation with precipitation (fig 5). thus, nmds1 coordinate is linearly negatively correlated with precipitation (p = 0.04; linear regression; r2 adj = 0.61), while nmds2 shows a peak at intermediate precipitations which is better fitted by a linear regression with linear and quadratic terms of precipitation as predictors (p = 0.02; r2 adj = 0.79). b) c catarineu, gg barberá, jl reyes-lópez – ants of southeastern iberian peninsula388 discussion at this point 110 ant species belonging to 30 genera and five subfamilies were recorded in the segura river basin but the actual species number is undoubtedly higher. a greater sampling effort, the study of hypogaeic species such as leptanilla spp., the delimitation in the tapinoma cf nigerrimum and tetramorium cf caespitum species complexes, and the revision of genera such as proformica or solenopsis, will increase the inventory of species. ant diversity in the segura river basin seems similar to other iberian peninsula regions like the comunitat valenciana (108 sps, 23,255 km2, mediterranean coast; del campo et al., 2014) or burgos (99 sps, 14,022 km2, northern plateau in the transition between mediterranean and cantabrian ranges with oceanic climate; garcía & cuesta, 2017). the segura river basin ant fauna is formed by species with different evolutionary and zoogeographic origins (two zoogeographical zones and 14 zoogeographical elements; table 3). the existence of these different zoogeographic origins in this area is probably linked with: the position between africa and europe; the complex geotectonic, paleogeographic, and paleoclimatic history during the last 7 my; the complex geomorphology; and the high climate and habitat diversity. ant species from the mixed and deciduous forest zone the ant species from the mixed and deciduous forest zone in the segura river basin accounted for 19.1% of the species inventoried. these species possibly suffered contraction in their ranges during the ice ages and survived in several southern refuges, such as the baetic ranges. the climatic oscillations during the pleistocene ice ages, and particularly over the last 2 my, are known to have had an important influence on the zoogeographic history of europe driving the repeated contraction/expansion of the fig 5. coordinates of 10×10 km utm squares of the systematic sampling (see methods) on nmds space of figure 3 against precipitation. dots, empirical data; lines fitted regression lines between nmds coordinates in each axis and precipitation (see methods). zoogeographical zones zoogeographical elements acronym n % zone of the mixed and deciduous forest central and southern european cse 1 0.95 central and southern european/ maghrebian cse/m 3 2.86 euro-caucasian ec 5 4.76 euro-caucasian/maghrebian ec/m 1 0.95 euro-west-siberian ews 3 2.86 euro-west-siberian/maghrebian ews/m 2 1.90 south palearctic sp 5 4.76 20 19.05 mediterranean zone holomediterranean hm 18 17.14 iberian i 21 20.00 iberian/maghrebian i/m 5 4.76 north-mediterranean nm 6 5.71 north-west mediterranean nwm 11 10.48 tethyan t 4 3.81 west-mediterranean wm 14 13.33 79 75.24 cosmopolitan cosmopolitan c 6 5.71 total number of species 105 100.00 table 3. zoogeographical composition of the ant fauna of the segura river basin (south-west spain). it includes only the 105 species in which it has been possible to reach the species level. sociobiology 65(3): 383-396 (september, 2018) 389 zoogeographical ranges (hewitt, 2011). the iberian peninsula was one of the most important refuges in europe during the pleistocene ice ages (hewitt, 2000, 2001, 2011; gómez & lunt, 2007). after the last glacial maximum period (20-14 ky bp) some species have expanded their range northward from their iberian refugia, or tracked suitable habitats higher up along elevation gradients within mountain ranges (gómez & lunt, 2007; hewitt, 2001). the baetic ranges, where the segura river basin is located, were one of the iberian refugia (carrión et al., 2003; gómez & lunt, 2007, hewitt, 2011). consistent with this, in the nmds analysis, both nmds1 and nmds2 axis are mainly determined by zoogeographic elements of mixed and deciduous forest class (fig 3). there is a greater proportion of species from the mixed and deciduous forest zone in the high-mid altitudes in the segura river basin, where the climate is cooler, the precipitation regime is subhumid or humid, and vegetation is dominated by pinus l. and quercus l. forests (fig 2, 4 and 5). this happens especially with euro-west siberian (ews + ews/m) and eurocaucasian (ec + ec/m) elements. south paleartic (sp) species have a similar pattern but peaks at intermediated precipitation/elevation and, especially at the transition zone between the baetic ranges and valleys typical of the segura basin and the central spain plateau. cse elements, on the contrary, appear clearly grouped with typically mediterranean elements. therefore, the general pattern of the class shows a clear gradient of elements where species are mainly associated to eurosiberian forests (ec, ews) than those on the transitional areas forest –steppe (sp) and then transitional to mediterranean and cse elements. some euro-caucasian and euro-west-siberian species were recorded only at high altitudes, with more humid climate: temnothorax unifasciatus latreille (ec/m, 1161 m.a.s.l.); polyergus rufescens latreille (ews, 8581282 m); strongylognathus testaceus schenck (ews, 2000 m); camponotus fallax nylander (ews/m, 1280-1286 m). other species classified under the same zoogeographical elements, were regularly collected at high altitudes but also at lower altitudes in humid habitats such as gallery forests or gardens: myrmica specioides bondroit (ec, 640-1275m); temnothorax affinis mayr (ec, 6401100 m); formica rufibarbis fabricius (ews, 40-1359m); and camponotus vagus scopoli (ews/m, 640-1356 m). some sp species were also collected only at high altitudes: formica pratensis retzius (1208-1280 m); formica sanguinea latreille (1280 m); and lasius carniolicus mayr (1563 m). global climate change is probably inducing altitudinal shifts of some ant species that will track suitable habitats along elevation, but the ant species currently limited to the mountaintops have no further elevation range to track habitats with suitable climatic conditions. talavera et al. (2014), studying a recently described and endemic ant from the balearic islands (lasius balearicus talavera et al., 2014) state that this species, elevationally constrained to island summits, is endangered by climate change and is potentially facing extinction. this could also be the case of species that inhabit only at high altitudes in the segura river basin which could be in danger of local extinction, as f. pratensis (tinaut et al., 2011). ant species from the mediterranean zone the species from the mediterranean zone accounted for three quarters of the species collected (79 spp., 75.2%), with the iberian species, the most commonly represented zoogeographical element (21 spp., 20%). the iberian peninsula is known to present high endemism of plants (25-30%; castroviejo et al., 1986), iberian carabid (43.1 %; serrano et al., 2003), and other taxa as reptiles, amphibians, mammals, fishes or butterflies (abellán & svenning, 2014). many of these endemic species possibly evolved during the pleistocene ice ages from tertiary species, when conditions in the different iberian refugia enabled long periods of allopatry, with speciation occurring as a result of adaptive selection and genetic drift processes (huseman et al., 2013). gómez and lunt (2007) stated that many species and species complex show strong genetic subdivisions in the iberian peninsula which provides evidence of their isolation. a pleistocene origin is also proposed for other endemic taxa as dytiscidae water beetles: most iberian endemics have an apparent recent origin, differing less than 2% in mdna from their sister species, which implies an origin from middle to late pleistocene (ribera, 2003). a possible example of the pleistocene speciation may be found in cataglyphis foerster ants: villalta et al. (2017) investigated the phylogeography of the cataglyphis albicans group and suggested the existence of at least three clades in the iberian peninsula and five in the maghreb. the three iberian clades are monophyletic and parapatric, and estimation of divergence times suggests a speciation process initiated after the messinian salinity crisis and the last reopening of the gibraltar straits (≈5,33my). other iberian ant endemics such as aphaenogaster iberica emery and a. dulcineae emery could also be derived from a north-african ancestor, bearing in mind that nowadays there are 40 aphaenogaster mayr native species and morphospecies in morocco (antmaps, 2016). medail and quézel (1997) proposed the baetic rifan complex, which includes the baetic ranges, as the most important areas of plant biodiversity inside the mediterranean basin hotspot. gómez-campo et al. (1984) state that the baetic ranges display the highest plant biodiversity in continental europe with great iberian endemic species richness, many of which are baetic ranges endemic species. this high endemism is the result of the long-term survival, genetic divergence and speciation in these refugia (gómez & lunt, 2007; hewitt, 2011). the mountainous geomorphology is particularly important in these processes of isolation, divergence and speciation during the past 3my (hewitt, 2000). the number of endemic plants in the baetic ranges is c catarineu, gg barberá, jl reyes-lópez – ants of southeastern iberian peninsula390 outstanding: 418 taxa (pérez-garcía et al., 2012) and these endemics concentrate on highland disjunct areas (mota et al., 2002). there are also a lot of species of terrestrial animals that are local endemics, as the amphibian alytes dickhilleni arntzen and garcía-parís or the spanish lizard algyroides marchi valverde (gómez & lunt, 2007). piñero et al. (2011) studying the arid areas of south-east iberian peninsula found a 8.4% insect and a 9.6% spiders species endemic to this area. in the case of ants, piñero et al. (2011) report 9.5% of endemic species but, in the segura river basin, we found only four endemic ants of the south-east iberian peninsula, accounting for 3.6% of the total number of species: camponotus haroi espadaler, goniomma collingwoodi espadaler, temnothorax ansei and temnothorax cristinae espadaler. holomediterranean elements account for 17.1% (18 species). these species are possibly tertiary species that found suitable habitats and survived the pleistocene ice ages in the iberian peninsula and other refuges around the mediterranean basin. the paleoecological similarities of the maghreb and southern europe have led to a circum-mediterranean distribution of many taxa (huseman et al., 2013). there are 43 species of the segura basin ants also present in the maghreb (cse/m, ec/m, ews/m, hm, i/m, t and wm) accounting for 41% of the recorded species. the presence of ibero-north african species has been reported in other taxonomic groups, for example, the baetic ranges have the highest values of ibero-north african plants in the iberian peninsula (gómez-campo et al., 1984) and this occurrence is also found in spiders and in various insect orders (piñero et al., 2011). the maghreb was also an important differentiation and speciation center during the pliocene and pleistocene, where many taxa may have evolved or survived and later expanded to the iberian peninsula and europe (huseman et al., 2013). two paleogeographical events allowed the exchange of terrestrial organisms between africa and the iberian peninsula. the first event was the separation of parts of the baetic region from the iberian mainland and its connection by the south to africa during the baetic crisis 16-14 mya (veith et al., 2004). the second event was the closure of the mediterranean-atlantic connections due to tectonic processes during the messinian salinity crisis 5.96-5.33 mya (veith et al., 2004; agustí et al., 2006; hewitt, 2011; gibert et al., 2013). the strait of gibraltar has been an effective barrier to genetic exchange since this event, although there is evidence that some terrestrial reptiles or amphibians crossed during the pleistocene, possibly with lower sea levels during the glacial maxima (veith et al., 2004; hewitt, 2011; huseman et al., 2013). the i/m and wm ants in the segura basin might represent tertiary species that colonized the iberian peninsula, and in some cases southern france, from north africa during the messinian salinity crisis. there are no ant species in the segura river basin also present in the afrotropical realm. serrano et al. (2003), studying iberian carabidae (coleoptera), state that the iberian peninsula is poor in afrotropical elements, probably because of the strong isolation derived from the sahara desert. the proportion of species from the mediterranean zone is greater in the lowland areas, and decreases with the altitude in the segura river basin (fig 2), possibly because they are more thermophilic species. in the nmds analysis, all the mediterranean elements are strongly aggregated together and with the central and southern european element, only iberian/ maghrebian elements are lightly separating from the rest (fig 3). cosmopolitan ant species the cosmopolitan ant species are all introduced tramp species, and account only for 5.7% (6 species). none of them seem to have much ability for invasion in the segura river basin. the most widespread, linepithema humile mayr, as well as the scarcer pheidole indica mayr and cardiocondyla mauritanica forel are present only in degraded coastal habitats and in anthropogenic environments such as gardens. paratrechina longicornis latreille was collected at only two sites in urban habitats, while strumigenys membranifera emery was collected at a single site in a semiarid and altered area. it is likely the harsh conditions of the semi-arid areas of the segura river basin are not suitable for the invasion of the exotic species. future directions unfortunately, zoogeographical studies are so scarce that we are not able to compare the zoogeographical ant composition of the segura basin to other mediterranean regions. further comparative phylogeographical analyses are needed to elucidate the range changes and the evolutionary history of the myrmecofauna in the iberian peninsula during the cenozoic. there is also further research required on the zoogeographic consequences of the changes we currently face living in the present epoch, the anthropocene. the segura river basin has suffered during thousands of years the effects of deforestation, fire, and pastoralism. during the last decades, the area has suffered dramatic ecosystem changes caused by uncontrolled urban development, expansion of intensive agriculture, abandonment of extensive livestock farming, inadequate reforestations, soil degradation, habitat fragmentation, and a deficiency in the conservation management (piñero et al., 2011). climate change is already occurring and, as it progress, it will also cause ecosystem changes with important effects on biodiversity. some species will suffer a rapid loss of suitable habitat and face extinction (thomas et al., 2004; wilson et al., 2005). other species are less vulnerable or may even be favoured by these changes. the evolutionary origin of species and their elevational distribution seem to be important in determining vulnerability to climate change. management for biodiversity conservation requires monitoring ecosystem changes and their effects on the biocenosis. for this task, ants could be crucial bioindicators in the coming years. sociobiology 65(3): 383-396 (september, 2018) 391 subfamily scientific valid name z.c. z. e. elevation range references dolichoderinae bothriomyrmex meridionalis (roger, 1863) 2 wm 275–1,223 r, y linepithema humile (mayr, 1868) 3 c 1–1,148 c, n, o, r, t, y tapinoma erraticum (latreille, 1798) 1 sp 2 r tapinoma madeirense forel, 1895 2 nwm 248–1,429 r, v, y tapinoma cf nigerrimum (nylander, 1856) 2 ? 2–1,507 b, c, r, v, y tapinoma nigerrimum (nylander, 1856) 2 nwm 339–392 y formicinae camponotus aethiops (latreille, 1798) 2 t 640–1,282 c, r, v, y camponotus cruentatus (latreille, 1802) 2 wm 858–1,442 r, y camponotus fallax (nylander, 1856) 1 ews/m 1,280–1,282 r, v, y camponotus figaro collingwood & yarrow, 1969 2 i 858–1,359 y camponotus foreli emery, 1881 2 i/m 1–1,280 b, m, r, v, x, y camponotus haroi espadaler, 1997 2 i 484 y camponotus lateralis (olivier, 1792) 2 hm 51–1,359 r, v, y camponotus micans (nylander, 1856) 2 wm 286–1,113 b, r, u, y camponotus piceus (leach, 1825) 2 nm 248–1,507 b, c, r, v, y camponotus pilicornis (roger, 1859) 2 nm 590–1,442 c, p, r, y camponotus ruber emery, 1925 2 i/m 15–238 r, t camponotus sp.1 espadaler in prep. 2 i 1148–1,155 p, r camponotus sylvaticus (olivier, 1792) 2 wm 1–1,406 b, c, d, n, r, t, v, x, y camponotus vagus (scopoli, 1763) 1 ews/m 640–1,356 y cataglyphis iberica (emery,1906) 2 i 1–1,282 e, j, k, l, m, n, r, t, v, x, y cataglyphis piliscapa (forel, 1901) 2 nwm ? c cataglyphis rosenhaueri santschi, 1925 2 i 905–1,304 y cataglyphis velox santschi, 1929 2 i 640–1,406 r, v, y colobopsis truncata (spinola, 1808) 2 hm 1,280–1,282 r, v, y formica cunicularia latreille, 1798 1 ec 42–1,406 c, r, v, y formica decipiens bondroit, 1918 2 i 242–1,282 r formica frontalis santschi, 1919 2 i 1,356 y formica pratensis retzius, 1783 1 sp 1,077–1,282 q, r, v, y formica rufibarbis fabricius 1793 1 ews 42–1,359 r, v, y formica sanguinea latreille, 1798 1 sp 1,280 v iberoformica gerardi (bondroit, 1917) 2 i 1,356 y iberoformica subrufa (roger, 1859) 2 i 15–1,359 b, r, v, x, y lasius alienus (foerster, 1850) 1 sp ? c lasius brunneus (latreille, 1798) 1 ec 1,282 r lasius carniolicus mayr, 1861 1 sp 1,563 y lasius cinereus seifert, 1992 2 nwm 640–2,000 i, r, v, y lasius emarginatus (olivier, 1792) 1 ec 495 r lasius grandis forel, 1909 2 wm 2–1,307 c, n, r, t, v, y lasius lasioides (emery, 1869) 2 hm 248 y table 1. checklist of the ant species of the segura river basin. the list is arranged alphabetically by subfamily, genus and species. species names in bold characters refer to species recorded for the first time in this area. zoogeographical classes (zc) and zoogeographical elements (ze) abbreviations: 1 = mixed and deciduous forest zone, 2 = mediterranean zone, 3 = cosmopolitan, cse = central and southern european, cse/m = central and southern european/maghrebian, c = cosmopolitan, ec = euro-caucasian, ec/m = euro-caucasian/maghrebian, ews = euro-west siberian, ews/m = euro-west-siberian/maghrebian, hm = holomediterranean, i = iberian, i/m = iberian/ maghrebian, nm = north-mediterranean, nwm = north-west mediterranean, sp = south-palearctic, t = tethyan, u = unknown, wm = west-mediterranean, ? = zoogeographical element questionable. elevation range units: masl. bibliographic references are as follows: a = emery 1924, b = santschi 1932, c= collingwood & yarrow 1969, d = martínez & espadaler 1986, e = cerdá 1988, f = seifert 1988, g = lópez et al. 1990, h = lópez, 1991, i = seifert, 1992, j = haro et al. 1995, k = dahbi et al. 1996, l = espadaler 1997c, m = martínez et al. 1997, n = martínez et al. 2002, o = morcillo et al. 2006, p = espadaler et al. 2007, q = tinaut et al. 2011, r = catarineu & tinaut 2012, s = martínez et al. 2012, t = arcos et al. 2013, u = del campo et al. 2014, v = asociación ibérica de mirmecología 2016, x = catarineu et al. 2017, y = present paper. for locations, see table s1. c catarineu, gg barberá, jl reyes-lópez – ants of southeastern iberian peninsula392 formicinae lasius myops forel, 1894 1 cse/m 793–1,661 r, y paratrechina longicornis (latreille, 1802) 3 c 73 r, y plagiolepis grassei le masne, 1956 2 nwm 400 s plagiolepis pygmaea (latreille, 1798) 1 cse/m 87–1,429 c, n, r, t, v, y plagiolepis schmitzii forel, 1895 2 hm 2–1,341 n, r, t, v, x, y plagiolepis sp.1 espadaler in prep. 2 i/m 2–392 y plagiolepis taurica santschi, 1920 1 sp 437–1,046 y plagiolepis xene stärcke, 1936 1 cse 107–1,280 n, r, t, y polyergus rufescens (latreille, 1798) 1 ews 858–1,282 r, y proformica ferreri (bondroit, 1918) 2 i 472 y proformica nasuta (nylander, 1856) 2 nwm ? c proformica sp. ? ? 640–923 y leptanillinae leptanilla sp. ? ? 242–1,258 x, y leptanilla theryi forel, 1903 2 i/m ? r myrmicinae aphaenogaster dulciniae emery, 1924 2 nwm 329–1,155 c, r, y aphaenogaster gibbosa (latreille, 1798) 2 hm 277–1,492 r, v, y aphaenogaster iberica emery, 1908 2 i 1–2,000 b, c, l, m, n, r, t, v, x, y aphaenogaster senilis mayr, 1853 2 wm 15 r cardiocondyla batesii forel, 1894 2 hm 2–714 c, r, x, y cardiocondyla mauritanica forel, 1890 3 c 2–10 r crematogaster auberti emery, 1869 2 wm 1–1,359 a, c, r, t, v, x, y crematogaster laestrygon emery, 1869 2 hm 2–465 r crematogaster scutellaris (olivier, 1792) 2 hm 10–1,359 b, r, t, v, y crematogaster sordidula (nylander, 1849) 2 hm 192–1,359 a, c, r, t, y goniomma blanci (andré, 1881) 2 nwm 392–1,280 r, v, y goniomma collingwoodi espadaler, 1997 2 i 472 y goniomma hispanicum (andré, 1883) 2 wm 244–640 r, v, x, y goniomma kugleri espadaler, 1986 2 i 244–465 x, y messor barbarus (linnaeus, 1767) 2 wm 1–1,359 b, l, m, n, r, v, x, y messor bouvieri bondroit, 1918 2 nwm 1–1,313 b, c, r, v, x, y messor capitatus (latreille, 1798) 2 hm 248–1,442 r, v, y messor structor (latreille, 1798) 1 t 1,200 c, y monomorium andrei saunders, 1890 2 i/m 2–192 r, t, y monomorium subopacum (smith, 1858) 2 hm 1–275 r, y myrmica aloba forel, 1909 2 nwm 905–1,307 c, f, r, y myrmica specioides bondroit, 1918 1 ec 248–1,275 v, y oxyopomyrmex saulcyi emery, 1889 2 wm 198–881 x, y pheidole indica mayr, 1879 3 c 10–16 r pheidole pallidula (nylander, 1849) 2 t 1–1,442 a, c, n, o, r, t, v, x, y solenopsis latro forel, 1894 2 hm 192–793 c, r, t solenopsis sp. 4 u 6–1,359 n, r, v, y strongylognathus caeciliae forel, 1897 2 i 238 r, x strongylognathus testaceus (schenck, 1852) 1 ews 2,000 y strumigenys membranifera emery, 1869 3 c 154 n temnothorax affinis (mayr, 1855) 1 ec 640–1,077 v, y temnothorax angustulus (nylander, 1856) 2 hm 640 v, y table 1. checklist of the ant species of the segura river basin. the list is arranged alphabetically by subfamily, genus and species. species names in bold characters refer to species recorded for the first time in this area. (continuation) subfamily scientific valid name z.c. z. e. elevation range references sociobiology 65(3): 383-396 (september, 2018) 393 myrmicinae temnothorax ansei catarineu, barberá & reyes-lópez, 2017 2 i 244–591 x,y temnothorax blascoi (espadaler, 1997) 2 i 854–1,339 y temnothorax cristinae (espadaler, 1997) 2 i 107–787 l, r, t, x, y temnothorax curtulus (santschi, 1929) 2 i/m 1307–1,356 y temnothorax formosus (santschi, 1909) 2 wm 465–1,359 r, v, x, y temnothorax gredosi (espadaler & collingwood, 1982) 2 i 248 r temnothorax kraussei (emery, 1916) 2 hm 1,307 y temnothorax kutteri (cagniant, 1973) 2 nwm 120 y temnothorax luteus (forel, 1874) 2 nm 79–1,406 c, x, y temnothorax niger (forel, 1894) 2 nm 2–1,077 r, x, y temnothorax pardoi (tinaut, 1987) 2 wm 248–1,406 r, v, y temnothorax racovitzai (bondroit, 1918) 2 nm 79–1,359 r, t, x, y temnothorax recedens (nylander, 1856) 2 hm 107–1,291 r, t, v, y temnothorax sp.1 tinaut in prep. 2 i 1,231 y temnothorax specularis (emery, 1916) 2 nm 44–1,359 v, x, y temnothorax unifasciatus (latreille, 1798) 1 ec/m 1,162 y temnothorax universitatis (espadaler, 1997) 2 i 465 x tetramorium biskrense forel, 1904 2 hm 88–1,406 v, y tetramorium cf caespitum (linnaeus, 1758) 4 u 1–2,000 g, h, l, m, r, v, y tetramorium forte forel, 1904 2 wm 392–1,507 r, y tetramorium semilaeve andré, 1883 2 hm 2–1,359 c, g, h, l, m, r, x, y ponerinae hypoponera eduardi (forel, 1894) 3 c? ? c ponera coarctata (latreille, 1802) 2 hm 500 c, r ponera testacea emery, 1895 1 cse/m 1155–1,280 y table 1. checklist of the ant species of the segura river basin. the list is arranged alphabetically by subfamily, genus and species. species names in bold characters refer to species recorded for the first time in this area. (continuation) subfamily scientific valid name z.c. z. e. elevation range references acknowledgments many thanks to xavier espadaler and alberto tinaut for their invaluable help; to alexander radchenko for his helpful suggestions; to bernhard seifert for identifying a tapinoma nigerrimum nylander sample; to fernando ochotorena for finding goniomma collingwoodi and for providing useful information; to nuria álvarez, sofía catarineu, gonzalo catarineu, pepe illueca, eduardo illueca and pablo delis for helping in the field work; and to karen peel for the english version revision. finally, we thank the two anonymous reviewers for their helpful suggestions. references abellán, p. & svenning, j.c. (2014). refugia within refugia-patterns in endemism and genetic divergence are linked to late quaternary climate stability in the iberian peninsula. biological journal of the linnean society, 113: 13–28. doi: 10.1111/bij.12309 agustí, j., garcés, m. & krijgsman, w. 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(2005). changes to the elevational limits and extent of species ranges associated with climate change. ecology letters, 8(11): 1138–1146. doi: 10.1111/ j.1461-0248. 2005.00824.x doi: 10.13102/sociobiology.v63i3.1031sociobiology 63(3): 858-880 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 studies of social wasp diversity in brazil: over 30 years of research, advancements and priorities introduction in the last decade, neotropical social wasps have stood out as role models in studies on ecology, biology and animal behavior (prezoto et al., 2011; prezoto & souza, 2015). this growing interest in the group is due to the acknowledgement of the wasps’ role in the trophic balance of ecosystems, since they can contribute both as pollinators (during the collection of nectar and pollen) and as predators (during their search for the animal protein used in the nourishment of their larvae); thus, wasps show potential as possible agriculture pest control agents (hunt, 2007; prezoto et al., 2008; elisei et al., 2010; clemente et al., 2013; clemente et al., 2012, barbosa et al., 2014). furthermore, some species abstract the first records of social wasps in brazil were made during expeditions focused on the taxonomy and distribution of the species throughout the country. from the 1970s the essence of publications on the diversity of social wasps has been changing, with studies focusing on specific areas and incorporating the use of sampling methodologies and analysis of results through ecological indexes. since then, the neotropical social wasps have gained more prominence due to the acknowledgement of their decisive role in the trophic balance of ecosystems, which has been increasing the interest in studying these insects. therefore, we aimed to make a detailed analysis of the social wasp diversity studies published in brazil over the past 33 years, looking to build knowledge on the research history of the group. for the literature review, selected publications must have attended to the following criteria: including keywords addressing the matter and being indexed in databases within the defined period. we found 78 publications, most of them (70.52%) published in scientific journals. diversity studies featured in publications in a regular basis from the year 2005 on, and the years 2010, 2012 and 2014 were the most productive; there was also a concentration of studies in the ba, mg and sp states. there were 11 different collection methods used, from which the active search and attractive trap methods stood out as most common; however, we found no pattern regarding study duration or collection methodology. the contribution of this analysis is to extend the current status of knowledge of social wasps research, as well as to guide and encourage future studies in unexplored areas. sociobiology an international journal on social insects bc barbosa, m detoni, tt maciel, f prezoto article history edited by kleber del-claro, ufu, brazil received 24 march 2016 initial acceptance 11 august 2016 final acceptance 26 september 2016 publication date 25 october 2016 keywords diversity, vespidae, polistine, metadata, scientific production, scientometric analysis. corresponding author bruno corrêa barbosa laboratório de ecologia comportamental e bioacústica labec universidade federal de juiz de fora campus universitário, martelos, juiz de fora, minas gerais, cep 36036-900, brasil e-mail: barbosa.bc@outlook.com are sensitive to environmental changes, being acknowledged as effective indicator organisms (urbini et al., 2006; souza et al., 2010). the first records on social wasps in brazil were made during expeditions focused on taxonomy and species distribution by von ihering (1904), ducke (1904, 1905, 1907, 1918), zikán (1949, 1951) and araújo (1944, 1946, 1960). in 1978, richards publishes the book “the social wasps of the americas, excluding the vespinae”, which comprehends an extensive review on the neotropical species, with details on their distribution, morphology and biology; this publication instantly became a milestone for posterior studies on the group, and references to it can still be found in recent publications (e.g. melo et al., 2015; jacques et al., 2015). universidade federal de juiz de fora, juiz de fora-mg, brazil review sociobiology 63(3): 858-880 (september, 2016) 859 after the publishing of professor vilma maule rodrigues’s paper in 1982 on the wasps in the horto florestal navarro de andrade garden in the city of rio claro/sp, brazil, there was a major change in the essence of publications regarding social wasps; researchers began to focus their sampling effort on a chosen locality, and, as years went by, applied sampling methodologies and data analysis through ecological indexes. it is estimated that we know less than 10% of the brazilian insect species (lewinsohn & prado, 2005) and although diversity studies are essential to the conservation of species, particularly for social wasps, these efforts must be carried out as to enhance the existing knowledge, thus allowing the comparison between studies and providing information to guide future investigations on the matter. therefore, we aimed to analyze in detail the publications on social wasps diversity in brazil for the last 30 years, aiming to increase the knowledge on these studies through a discussion on the advancements and research priorities regarding the methods applied and the attained results. methods method and data search criteria on this study we followed the protocol suggested by the prisma method for systematic studies and metaanalysis (moher et al., 2009) adapted by moher et al. (2015). the methodological approach included the development of the selection criteria, the definition of search strategies, the evaluation of the studies’ quality and the extraction of relevant data. the criteria for selection and inclusion of publication were: publications approaching the matter; publications indexed on the google scholar, scientific electronic library online (scielo), scopus and web of science databases; papers published in journals within the period limited between january 1982 and october 2015. the key words used to search publications were ‘social wasps’ and ‘diversity’. publications such as monographies, theses and books were added through cross-referencing. we recorded the following data from each publication: study area, focus, duration, sampling methods and identified social wasp species. based on this information, we generated: (1) a map of the distribution of the publications by state and (2) a table of social wasp species and the methods used to sample them, also sorted by state. data analysis in order to assess social wasp species richness, we generated species rarefaction curves (sensu gotelli & colwell, 2001) in the software estimates 9 (colwell, 2013) with 5000 randomizations. this software generates 5000-species accumulation curves by randomizing the order of samples; this way, each point along the curve represents the mean of the accumulated richness for the 5000 curves and is associated to a standard deviation value. each publication was considered a sample, therefore resulting in 76 samples; two studies were not considered since their authors did not identify organisms to species-level. we calculated the constancy index suggested by bodenheimer (1955) in order to assess social wasp species constancy recorded in studies in the brazilian territory. to perform the calculations, once again, each publication was considered a sample. species present in more than 50% of the samples were considered constant; the ones present in 25% to 50% of the samples were considered accessory; species present in less than 25% of the samples were considered accidental. results and discussion we selected 78 publications, from which eight (10.25%) were books, 15 (19.23%) were unpublished studies (monographies and theses) and 55 (70.52%) were papers publishedin scientific journals (table 1). regarding the papers, 25 distinct journals were used, most of them being brazilian (n= 16). the most used journals were: socibiology (n= 13), revista brasileira de entomologia (n= 5), neotropical entomology, mgbiota and entomobrasilis, these last four featuring a single publication each (table 1); altogether, they make up 54.54% (n= 30) of the published papers (n= 55). almost one quarter of the papers (23.63%) were published on the sociobiology journal. the first publication on social wasp diversity in brazil dates from 1982 (rodrigues and machado, 1982); from 1985 to 2002, 12 more studies werepublished on an uneven frequency. from 2005 on, however, some publication regularity started to appear, the years of 2010, 2012 and 2014 being the most productive (with eight, eight and 12 publications respectively) (fig 1, table 1). in this chronological sense, it stands out that most papers (n= 45; 57.69%) were published in the last six years (from 2010 to 2016) (fig 1). the distribution of publications throughout the brazilian states showed a concentration in the states of minas gerais (n= 24), são paulo (n= 13) and bahia (n= 10), which together make up more than half of all studies (fig 2), while nine states still haven’t had any published studies on their social wasp fauna. fig 1. number of publications per year, in national and international journals, on brazilian social wasp diversity, in the period between 1982 to 2015. bc barbosa et al. – social wasps of brazil: a review860 paper no author/year journal study duration (months) sampling methods i ii iii iv v 1 rodrigues and machado, 1982 naturalla 144 as sp 33 10 20 13 2 lorenzato, 1985 agron. sulriograndense 16 bt sc 12 5 9 3 3 marques, 1989 m.sc dissertation 16 bt ba 13 8 11 2 4 marques et al., 1993 insecta 63 as, fs ba 20 10 14 6 5 marques and carvalho, 1993 insecta 52 as ba 17 10 12 5 6 diniz and kitayama, 1994 j. hymenopt research 5 q mt 30 15 22 8 7 santos, 1996 agrárias 13 bt go 9 5 9 0 70 diniz and kitayama, 1998 rev. de biologia tropical 4 as mt 36 12 26 10 8 raw, 1998a rev. brasileira de zoologia 47 days* as df 13 8 9 4 74 raw, 1998b book as rr 36 13 26 10 9 lima et al., 2000 rev. bras. zoociências 13 as mg x 5 x x 10 silveira, 2002 papéis avulsos de zoologia 7 as, mt pr 79 18 51 28 62 mechi, 2005 book fs sp 28 8 17 11 11 melo et al., 2005 book 8 as, fs ba 23 10 14 9 12 silveira et al., 2005 entomological science 8 bt pr 6 2 6 0 53 hermes and kohler, 2006 rev. bras. entomologia 32 fs rs 25 7 13 12 75 mechi and moraes, 2006 book 25 fs sp 26 8 17 9 13 santos et al., 2006 sociobiology 13 fs ba 13 5 7 6 14 silva-pereira and santos, 2006 neotropical entomology 8 fs ba 11 6 8 3 15 souza and prezoto, 2006 sociobiology 15 as, bt, fs, q mg 38 10 20 18 16 elpino-campos et al., 2007 neotropical entomology 12 as, bt mg 29 10 16 13 18 santos et al., 2007 neotropical entomology 37 as ba 21 11 14 7 19 lima, 2008 m.sc dissertation 27 as, fs, lb sp 31 11 21 10 20 morato et al., 2008 acta amazonica 24 days* mt ac 20 7 16 4 21 ribeiro-junior, 2008 m.sc dissertation 12 as, bt mg 12 6 6 6 22 silveira et al., 2008 acta amazonica as, bt am/ap 46 15 38 8 23 souza et al., 2008 mg.biota 24 as, bt, fs, q mg 42 12 23 19 61 carbonari, 2009 ph.d thesis 23 as, mt, tt ms 19 8 11 8 24 clemente, 2009 m.sc dissertation 12 as, bt mg 21 8 12 9 25 gomes and noll, 2009 rev. bras. entomologia 6 ba, lb sp 14 7 11 3 26 santos el al., 2009a neotropical entomology 8 as ba 19 13 15 4 68 santos et al., 2009b environmental entomology 6 as ba 17 10 11 6 27 silva and silveira, 2009 iheringia série zoologia 6 as, mt pr 63 12 40 23 29 alvarenga et al., 2010 sociobiology 2 as mg x 5 x x 63 arab et al., 2010 sociobiology 37 st sp 10 4 7 3 30 auad et al., 2010 sociobiology 24 mt mg 13 4 11 3 31 coró, 2010 m.sc dissertation 12 mt sp 20 9 16 4 32 lima et al., 2010 sociobiology 15 as, fs, lb sp 30 10 20 10 33 prezoto and clemente, 2010 mg.biota 12 as, bt mg 23 10 14 9 45 souza et al., 2010 mg.biota 12 as, bt, bf mg 32 9 15 17 50 ribeiro, 2010 monography 6 as pr 13 5 7 6 66 de souza et al., 2011 j. economic entomology 12 bt, as mg 17 9 13 4 table 1. papers, mean of publication, study duration, sampling methods, study area (states) and number of genera and species recorded in brazilian social wasp diversity studies from 1982 to 2015: as – active search; bt – bait traps; f – fogging technique; fs – flower search; lb liquid bait; lt – light traps; mo – möericke; mt – malaise trap; q – quadrant; st –shuey trap; tt – tray traps; i – study state; ii – nº of species found; iii – nº of genera found; iv – nº of swarming species and v – nº of independent species. sociobiology 63(3): 858-880 (september, 2016) 861 paper no author/year journal study duration (months) sampling methods i ii iii iv v 35 henrique-simões et al., 2011 checklist 12 as, bt, mt mg 34 10 16 18 36 pereira and antonialli-jr, 2011 sociobiology 27 days* as, bt, lb ms 18 6 10 8 37 silva el al., 2011 rev. bras. entomologia 13 as ma 31 13 25 6 69 silva, 2011 m.sc dissertation 6 as, bt mg 13 7 9 4 34 tanaka and noll, 2011 psyche 25 as, lb sp 29 10 21 8 60 bomfim et al., 2012 sociobiology 6 as, bt ms 18 6 10 8 38 henrique-simões et al., 2012 iheringia série zoologia 12 as, bt mg 32 10 15 17 39 jacques et al., 2012 sociobiology 3 as, bt mg 25 10 16 9 72 noll et al., 2012 book 9 as, mt, lb sp 32 11 25 7 41 silva, 2012 m.sc dissertation 12 as, bt mg 20 7 11 9 42 silveira et al., 2012 rev. bras. entomologia 44 days* mt, as pa 30 6 21 9 43 somavilla, 2012 m.sc dissertation 5 as, bt, f, lt, mt am 86 17 70 16 44 souza et al., 2012 mg.biota 12 as, bt mg 38 10 21 18 76 auko et al., 2013 book 11 days* as, mt ms 8 6 7 1 55 auko and silvestre, 2013 biota neotropica 11 as, mt, mo ms 18 9 10 8 71 gomes, 2013 ph.d thesis 8 bt, lb ro 76 15 67 9 47 grandinete and noll, 2013 sociobiology 12 as, bt, lb ms 22 8 14 8 46 silva et al., 2013 entomobrasilis 4 as, bt mg 10 4 7 3 48 almeida et al., 2014 sociobiology 10 as mt 14 8 13 1 51 andena and carpenter, 2014 book mt x 74 17 45 29 59 brugger, 2014 m.sc dissertation 12 as, bt mg 23 8 17 6 40 locher et al., 2014 sociobiology 13 as, bt, lb sp 31 8 18 13 54 rocha and silveira, 2014 entomobrasilis as pi 12 6 10 2 28 togni et al., 2014 checklist 13 as, bt sp 21 8 14 7 67 klein, 2014 monography 13 as, bt, lb rs 16 7 10 6 77 silvestre et al., 2014 book 17 as, bt, mt, tt, mo ms 31 8 13 18 56 somavilla et al., 2014a rev. bras. entomologia 16 days* as am 58 13 46 12 57 somavilla et al., 2014b entomobrasilis 17 bt, mt, lt ma 38 12 36 2 49 souza et al., 2014a bioscience journal 12 as mg 38 10 22 16 52 souza et al., 2014b acta sciemtiarum 24 as mg 29 10 16 13 64 barbosa, 2015 m.sc dissertation 32 as, bt mg 36 10 21 15 65 clemente, 2015 ph.d thesis 6 as, bt sp 31 8 17 14 73 freitas et al., 2015 revista agrogeoambiental 4 bt mg 19 8 14 5 78 jacques et al., 2015 sociobiology 26 as, bt mg 29 8 15 14 17 melo et al., 2015 checklist 12 fs ba 8 5 5 3 58 somavilla et al., 2015 entomobrasilis 7 days* as, lt, mt am 49 14 42 7 table 1. papers, mean of publication, study duration, sampling methods, study area (states) and number of genera and species recorded in brazilian social wasp diversity studies from 1982 to 2015: as – active search; bt – bait traps; f – fogging technique; fs – flower search; lb liquid bait; lt – light traps; mo – möericke; mt – malaise trap; q – quadrant; st –shuey trap; tt – tray traps; i – study state; ii – nº of species found; iii – nº of genera found; iv – nº of swarming species and v – nº of independent species. (continuation) this asymmetry regarding publications may be explained by the fact that minas gerais, são paulo and bahia host some of the core social wasp research groups in brazil, present in universities, research institutes and technology centers; these groups perform important roles not only by carrying out studies on the group, but also by developing human resources which would organize new research groups dispersed in other brazilian states. bc barbosa et al. – social wasps of brazil: a review862 however, the increased amount of studies in the states of minas gerais e são paulo does not grant the southeastern region the status of most studied in the country, since the states of espírito santo and rio de janeiro still have, on their territories, 10.5% and 18.6% (respectively) of the original fragments of the atlantic rainforest, which is considered one of the most endangered of the brazilian biomes (sos mata atlântica, 2013); therefore, it is surprising that there aren’t any publications on their social wasp fauna. data on the duration of studies was present on 73 (93.58%) publications and ranged from a few days to 144 months, being 12 months the most usual duration (n= 15). in 28 publications the duration was superior to 12 months (from 13 to 144 months), while in other studies (n= 30) the duration was less than 12 months (from 7 consecutive days to 11 months) (table 1). it is evident that there is no uniformity in the duration of the sampling period, and there is a necessity to create a pattern for study duration in order to enable data comparison between studies. one of the consequences of the variable duration of studies can be observed when a species accumulation curve is generated (fig 3). taking the 24 months-long study performed by barbosa (2015) as role model, we can relate the curve’s behavior to the potential of species to be sampled through time. the species accumulation curve is asymptotic and grows in a decreasing rate, since for each sampling event the potential for finding new species decreases. on the first six months of study this rate is very high, which shows a possibility of sampling a greater number of species in the area. this rate is noticeably lower between six and 12 months, and is minimum after this period (between 12 and 24 months). this curve pattern shows that short-term studies tend to underestimate the number of species in an area, thus reassuring the precision of long-term studies. for the social wasps, it is evident that studies with 12 or more months of samplings have a better estimation of the species diversity when compared to the expected value. regarding the sampling, we recorded 11 different methods used to capture of social wasps: active search (n= 60), bait trap (n= 35), malaise trap (n= 14), flower search (n =13), liquid bait (n= 9), quadrant (n= 3), light trap (n =3), tray trap (n= 2), möericke trap (n= 2), shuey trap (n= 1) fig 2. geographical representation of the number of publications on social wasp diversity per brazilian state from 1982 to 2015. sociobiology 63(3): 858-880 (september, 2016) 863 and fogging technique (n= 1) (table 1). furthermore, most publications (55.12%, n= 43) used more than one sampling method, which became a trend after 2006; before that, the use of a single sampling method prevailed on most studies. this trend agrees with many studies (silveira, 2002; souza & prezoto, 2006; togni et al., 2014) that highlight the importance of conciliating methods in order to better record the fauna of wasps in an area. sampling through bait traps was the second most used method (present in 35 studies), right after active search, and was also the method that varied most on its way of application. there were different kinds of baits used, usually made of various fruit juices or sardine-based protein broths; the amount, disposition, duration and confection of traps also varied a lot (e.g. santos, 1996; souza & prezoto, 2006; clemente, 2009; locher et al., 2014). this methodological diversity observed for bait traps is mainly due to the lack of a study that tests the best layout for this method; such possibility would generate data to optimize the distance between traps, the setting height for them, the kind of bait used (natural or industrialized juice), the setting duration on field, the container size, and so on. this standardization would bring direct benefits for a more fitting comparison of sampling efforts in future studies, and also to optimize time and money costs to set the traps on the field. almost half of the sampling methods used (45.45%, n= 5) recorded exclusive species (recorded by means of a single method). among those, the methods that recorded the most exclusive species were: active search (n= 23) and malaise trap (n= 15) (table 2). curiously, sampling through light traps, recorded for only three studies and characterized as effective for capturing species with night habits, stood out for sampling eight species in the apoica genus, which is known for its night activity, but also for other 63 species belonging to 13 genera (agelaia, angiopolybia, asteloeca, brachygastra, clypearia, leipomeles, mischocyttarus, parachartaegus, polistes, polybia, protonectarina, pseudopolybia and synoeca), which are all active during the day (table 2). a possible explanation for the capture of that many day species may be due to the light traps being controlled by photosensors, which make the traps trigger by the end of the afternoon, a time in which many foragers of wasp species active during the day are still returning to their nests. social wasps, in the same way as bees, have positive phototropism, which makes them attracted to the luminosity in the trap and therefore captured by it. only six studies focused on the difference of the setting height for the sampling methods (silveira, 2002; de souza et al., 2011; somavilla 2012; somavilla et al., 2014b; clemente, 2015; barbosa, 2015), usually adopting two different heights, fig 3. species accumulation curve model based on barbosa (2015), with 24 months of sampling of social wasps. the vertical lines separate the sampling intervals in six, 12 and 18 months. bc barbosa et al. – social wasps of brazil: a review864 the canopy (close to 5 meters high) and the understory (chestheight, approximately 1,5 meters high). two of these studies (de souza et al., 2011; barbosa, 2015) recorded greater species richness for the traps set in canopy height and also exclusive species for each setting height. therefore, these studies show the importance of sampling the different levels of the vegetal mosaic in the environment. regarding the use of diversity indexes, we observed that mechi (2005) was the first study to apply a diversity index; on this particular case, the author used the shannonwiener index (h’) while studying the social wasp fauna in estação ecológica jataí, são paulo state; the second study to use a diversity index was published by souza and prezoto (2006). most publications (77.27%, n= 34) applied at least one diversity index, which shows the emergence of a trend to use this kind of test in order to discuss the results found since 2006. based on the studies that properly identified the social wasp species, 235 species were recorded, belonging to 19 different genera; of these, the most representative ones were the mischocyttarus (n= 68), polybia (n= 44) and polistes (n= 25) genera (table 2). the calculated constancy index showed that among the 233 species identified in the publications, most (88.1%, n= 207) were accidental, followed by accessory (8.5%, n= 20) and constant (3.4%, n= 8). the latter, present in most of the studies, were: polybia sericea (n= 61), polybia ignobilis (n= 58), polistes versicolor (n= 56), polybia occidentalis (n= 54), brachygastra lecheguana (n= 49), polybia paulista (n= 46), e protonectarina sylveirae (n= 42), apoica pallens (n= 39). the presence of few constant and accessory species may mean that they are more widespread throughout the brazilian territory; however, we cannot ignore the polarization of social wasp studies on the southeastern region, which would make endemic species seem constant when this data is extrapolated to the whole country. regarding the occurrence of species per state (table 2), we noted that five species were present in 14 or more sampled states: brachygastra lecheguana (n= 15), polybia ignobilis (n= 15), synoeca surinama (linnaeus, 1767) (n= 15), polybia sericea (n= 15) e polybia occidentals (n= 14). however, 36.17% (n= 85) of the identified species were recorded only for a single state. amongst these, the amazonas state stands out with the most species recorded (n =125), while the goiás state has the least species recorded (n= 9) (table 2). this impressive number of species recorded for amazonas surely does not yet represent the region’s mega diversity, since there were only four studies carried out on this state; further investigations should lead to a significant increase of recorded species. on the other hand, the small amount of species recorded on the single study carried out in goiás (santos, 1996) shows the particular characteristics of its methodology, since the study which took place at an orchard and not at the state’s typical biome environments. while representatives of the polistinae are found throughout the whole world, its greatest diversity is achieved in tropical regions (specially the neotropical region); its worldwide fauna is made of 26 genera and more than 1000 species (carpenter and andena, 2013). some authors (e.g. fox, 1889; richard, 1978; carpenter, 1991; carpenter and marques, 2001; carpenter and andena, 2013) estimate that brazil holds 22 genera and 346 species of social wasps. therefore, based on the properly identified species in the 76 publications hereby listed (table 1), we observe that the 233 species recorded correspond to 77.74% and 68.62% of the total estimated species. by generating a species accumulation curve based on the studies and recorded species (fig 4), the estimators jack 1 and jack 2 estimated, respectively, 301.08 and 341.38 species for brazil, a lower amount than the described in the literature; however, we believe this percentage to be a little higher when we add, to the diversity studies, publications on natural history, biology and ecology of social wasps. considering the potential of social wasps as role models for studies in biology, behavior and ecology due to their importance as ecological service providers in the ecosystem, we must highlight the value of studies that investigate the ecology of these species in detail, aiming to further understand this group of organisms. the small amount of studies on social wasp ecology may be a consequence of the sometimes exaggerated behavior of human societies when concerning wasps (by associating wasps to the risk of accidents provoked by their stings) or even of disregard (by believing that these species have no value). therefore, the analysis presented here may guide and subside future research on social wasp diversity and its ecological relations on the different brazilian biomes. finally, our study’s contribution is to widen the possibilities on social wasp research scenario and to give directions for future researchers on their work through the material listed on this paper. fig 4. rarefaction curves for species richness estimators and species accumulation curves generated through social wasp diversity studies in brazil from 1982 to 2015, made from 5.000 randomizations on the sample order (see details in data analysis). sociobiology 63(3): 858-880 (september, 2016) 865 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy a ge la ia a cr ea na s ilv ei ra & c ar pe nt er , 1 99 5 a m a m m t 43 1. 28 a ge la ia a ng ul at a (f ab ri ci us , 1 80 4) a m , a t, u e a m , m a , m g , p a , r o , r r , s p b t, m t, s t, a s, l b 10 , 1 2, 2 0, 2 7, 2 8, 4 3, 4 4, 56 , 5 7, 5 8, 63 , 7 1, 7 4 16 .6 7 a ge la ia a ng ul ic ol lis (s pi no sa , 1 85 1) a m a m , p a b t, m t, a s 10 , 2 7, 4 2 3. 85 a ge la ia b re vi st ig m a (r ic ha rd s, 1 97 8) a m a m , a p m t 22 1. 28 a ge la ia c aj en ne ns is (f ab ri ci us , 1 79 8) a m , c a , s a a m , a p, b a , p a , r o b t, l t, m t, a s, l b 10 , 1 2, 2 7, 4 2, 4 3, 5 1, 5 6, 7 1 10 .2 6 a ge la ia c en tr al is (c am er on , 1 90 7) a g , a m , a t, c a a m , a p, m a , m g , p a , r o b t, l t, m t, a s, l b 10 , 2 2, 2 7, 4 2, 4 3, 4 4, 5 1, 56 , 5 7, 5 8, 7 1 15 .3 8 a ge la ia c on st ru ct or (d e sa us su re , 1 85 4) a m a m b t, a s 43 , 5 6 2. 56 a ge la ia fl av ip en ni s (d uc ke , 1 90 5) a m , s a a m , m t m t, a s 58 , 7 0 2. 56 a ge la ia fu lv of as ci at a (d eg ee r, 17 73 ) a m a m , a p, m a , p a , r o , r r b t, l t, m t, a s, l b 10 , 1 2, 2 0, 2 2, 2 7, 4 2, 4 3, 5 6, 5 7, 5 8, 7 1, 7 4 15 .3 8 a ge la ia h am ilt on i ( r ic ha rd s, 1 97 8) a m r o b t, l b 71 1. 28 a ge la ia lo bi pl eu ra (r ic ha rd s, 1 97 8) a m , s a a m , m t, r o m t, l b , q 6, 2 0, 7 0, 7 1 5. 13 a ge la ia m el an op yg a c oo pe r, 20 00 a m r o b t, l b 71 1. 28 a ge la ia m ul tip ic ta (h al id ay , 1 83 6) a g , a m , a t, e u , r f, r g , s a , u e m g , m s, m t, r s, r r , sc , s p b t, m t, s t, a s, t t, fs , l b , m o , q 1, 2 , 1 5, 1 9, 2 1, 2 3, 2 4, 2 8, 3 0, 3 2, 3 4, 3 9, 40 , 4 1, 4 5, 4 9, 5 2, 5 3, 6 1, 6 3, 6 4, 6 5, 6 6, 6 7, 70 , 7 2, 7 3, 7 4, 7 5, 7 6, 7 7, 7 8 41 .0 3 a ge la ia m yr m ec op hi la (d uc ke , 1 90 5) a m , s a a m , a p, m a , p a , r o b t, l t, m t, a s, f , l b 10 , 2 0, 2 2, 2 7, 4 2, 4 3, 5 6, 5 7, 7 0, 7 1 12 .8 2 a ge la ia o rn at a (d uc ke , 1 90 5) a m a m , r o a s, l b 43 , 5 6, 5 8, 7 1, 7 4 6. 41 a ge la ia p al lid iv en tr is (r ic ha rd s, 1 97 8) a m a m , r o a s, l b 43 , 7 1 2. 56 a ge la ia p al lip es (o liv ie r, 17 91 ) a g , a m , a t, c a , e u , r f, a s, u e a m , b a , c e , g o , m a , m g , m t, m s, p a , p i, r o , r s, s p b t, l t, m t, a s, f s, l b 1, 7 , 1 0, 1 2, 1 6, 1 9, 2 5, 2 7, 3 1, 32 , 3 4, 3 6, 37 , 4 0, 4 2, 4 3, 4 7, 5 0, 51 , 5 4, 5 6, 5 7, 5 8, 6 0, 62 , 6 5, 6 7, 7 0, 7 1, 7 2, 7 3, 7 7 41 .0 3 a ge la ia te st ac ea (f ab ri ci us , 1 80 4) a m a m , a p, m a , p a , r o , r r , b t, l t, m t, a s, l b 10 , 2 7, 4 2, 4 3, 5 6, 5 7, 5 8, 7 1, 7 4 11 .5 4 a ge la ia ti m id a c oo pe r, 20 00 a m r o b t, l b 71 1. 28 a ge la ia v ic in a (s au ss ur e, 1 85 4) a g , a t, c a , e u , r f, r g , s a ,u e a l , b a , c e , m g , s c , sp , r s b t, l t, m t, a s, f s, l b , q 2, 1 5, 2 1, 2 3, 2 4, 2 5, 2 6, 2 8, 3 0, 31 ,3 2, 3 3, 34 , 3 9, 4 0, 4 1, 4 6, 4 9, 5 1, 5 3, 5 9, 6 2, 6 4, 65 , 6 6, 6 9, 7 2, 7 3, 7 5 37 .1 8 a ng io po ly bi a ob id en si s (d uc ke , 1 90 4) a m a m b t, a s 43 , 5 6 2. 56 a ng io po ly bi a pa lle ns (l ep el et ie r, 18 36 ) a m , a t, c a , m g , r e , u e a m , a p, b a , m a , p a , pe , r r , s p b t, l t, m t, a s, f s 10 , 1 2, 1 8, 2 0, 2 2, 2 7, 2 8, 4 2, 4 3, 5 1, 5 6, 57 , 5 8, 7 4 17 .9 5 a ng io po ly bi a pa ra en si s (s pi no sa , 1 85 1) a m , c a a m , m a , b a , p a , r o b t, l t, m t, a s, l b 10 , 1 2, 2 0, 2 7, 4 2, 4 3, 5 1, 5 6, 5 8, 7 1 12 .8 2 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . bc barbosa et al. – social wasps of brazil: a review866 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy a ng io po ly bi a zi sc hk ai r ic ha rd s, 1 97 8 a m r o b t, l b 71 1. 28 a po ic a al bi m ac ul a (f ab ri ci us , 1 80 4) a m a m lt , a s 43 1. 28 a po ic a ar bo re a sa us su re , 1 85 4 a m a m , m a , p a b t, l t, m t, a s 10 , 4 3, 5 6, 5 7, 5 8 6. 41 a po ic a fla vi ss im a v ec ht , 1 97 2 a m , a t, u e ,c a , e u , a s a m , a p, m s, p b , r r , s p b t, l t, m t, a s, t t, l b , m o 1, 1 9, 2 2, 3 7, 4 7, 5 1, 5 5, 6 1, 7 4, 7 7 12 .8 2 a po ic a ge lid a v ec th , 1 97 2 a g , a m , c a , r f, s a a m , m g , p e , r o , s p b t, l t, m t, l b 23 , 3 5, 3 8, 4 3, 5 1, 5 2, 6 5, 7 1, 7 3 12 .8 2 a po ic a pa lle ns (f ab ri ci us 1 80 4) a g , a m , a t, c a , r g , e u , m g , p a , r e , r f, s a , u e a m , a p, g o , b a , m a , m g , m t, m s, p a , s p b t, l t, m t, s t, a s, f s, l b , q 1, 3 , 4 , 5 , 6 , 7 , 1 0, 1 1, 1 5 1 6, 1 8, 21 ,2 2, 2 3, 2 4, 2 6, 2 7, 2 8, 3 3, 3 6, 3 9, 40 , 4 2, 4 3, 4 4, 4 5, 4 8, 5 0, 5 1, 5 6, 5 7, 58 , 6 0, 6 3, 6 4, 6 5, 6 8, 7 2, 7 5, 7 7 51 .2 8 a po ic a pa lli da (o liv ie r, 17 91 ) a m , c a a m , a p, b a , c e , m a , pa , r r b t, l t, m t, a s 10 , 2 2, 4 3, 5 1, 5 7, 5 8, 7 4 8. 97 a po ic a st ri ga ta r ic ha rd s, 1 97 8 a m a m , m a , p a b t, l t 27 , 4 3, 5 7 3. 85 a po ic a th or ac ic a b uy ss on , 1 90 6 a m a m , a p, r r b t, l t, m t, a s 22 , 4 3, 5 8, 7 4 5. 13 a st el oe ca tr ai li (c am er on , 1 90 6) a m m a , p a b t, l t 10 , 5 7 2. 56 b ra ch yg as tr a al bu la r ic ha rd s, 1 97 8 a m a m , r o m t, l b 20 , 7 1 2. 56 b ra ch yg as tr a au gu st i ( sa us su re , 1 85 4) a m , a t, e u , p a , r f, s a , u e a m , a p, d f, m a , m g , m s, m t, r o , s p b t, m t, a s, f s, l b 1, 8 , 1 5, 1 9, 2 2, 2 3, 3 2, 3 4, 3 6, 4 0, 4 4, 47 , 4 8, 5 2, 5 7, 5 8, 5 9, 6 0, 6 2, 6 4, 7 0, 71 , 7 2, 7 5 30 .7 7 b ra ch yg as tr a bi lin eo la ta (s pi no sa , 1 84 1) a m , s a a m , a p, m t, p a , r o b t, m t, a s, l b , q 6, 1 0, 2 2, 3 7, 5 8, 7 0, 7 1 8. 97 b ra ch yg as tr a co op er i ( r ic ha rd s, 1 97 8) a m r o b t, l b 71 1. 28 b ra ch yg as tr a le ch eg ua na (l at re ill e, 1 82 4) a g , a m , a t, c a , e u , m g , pa , r e , r f, r g , s a , u e a m , a p, b a , g o , m g , m s, m t, p a , p b , p e , p i, r o , r s, s c , s p b t, l t, m t, a s, t t, fs , l b , m o 1, 2 , 3 , 4 , 5 , 7 , 1 0, 1 1, 1 3, 1 4, 1 6, 17 ,1 8, 1 9, 2 0, 2 2, 2 4, 2 6, 3 2, 3 3, 3 4, 35 , 3 8, 3 9, 4 0, 4 1, 4 4, 4 7, 4 8, 4 9, 5 1, 53 , 5 4, 5 5, 5 8, 5 9, 6 1, 6 2, 6 4, 6 5, 6 6, 67 , 6 8, 7 0, 7 1, 7 2, 7 5, 7 7, 7 8 62 .8 2 b ra ch yg as tr a m oe bi an a (s au ss ur e, 1 86 7) a m , a t, s a , u e a m , m s, s p m t, a s, l b 20 , 3 4, 4 7, 7 2, 7 5 6. 41 b ra ch yg as tr a m ou le ae r ic ha rd s, 1 97 8 a t, s a m s, s p b t, m t, a s, t t, f s, l b , m o 34 , 5 5, 6 1, 6 2, 7 2, 7 7 7. 69 b ra ch yg as tr a sc ut el la ri s (f ab ri ci us , 1 80 4) a m , c a a m , p e , p i, r o m t, l b 43 , 5 1, 5 8, 7 1 5. 13 b ra ch yg as tr a sm ith ii de s au ss ur e, 1 85 3 a m r r a s 74 1. 28 c ha rt er ge llu s am az on ic us r ic ha rd s, 1 97 8 a m a m , r o m t, a s, l b 43 , 5 6, 7 1 3. 85 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) sociobiology 63(3): 858-880 (september, 2016) 867 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy c ha rt er ge llu s co m m un is r ic ha rd s, 1 97 8 a m , c a , s a b a , m g , m t, p a , r o b t, m t, a s, l b , q 6, 1 6, 2 6, 2 7, 3 7 51 , 7 0, 7 1 10 .2 6 c ha rt er ge llu s ni ge rr im us r ic ha rd s, 1 97 8 a m a m , a p m t 22 1. 28 c ha rt er ge llu s pu nc ta tio r r ic ha rd s, 1 97 8 a m a m , a p m t 22 1. 28 c ha rt er ge llu s zo na tu s (s pi no la , 1 85 1) a m r o b t, l b 71 1. 28 c ha rt er gi nu s fu lv us f ox , 1 90 4 a m a m , p a , r o b t, m t, a s, l b 10 , 4 3, 5 6, 7 1 5. 13 c ha rt er gu s ch ar ta ri us (o liv ie r, 19 71 ) a m , s a a m , a p, m t, r r m t, a s, q 6, 2 2, 5 8, 7 0, 7 4 6. 41 c ha rt er gu s gl ob iv en tr is s au ss ur e, 1 85 4 a m , c a , p a , s a , u e b a , m t, p a , r o b t, m t, a s, l b 10 , 2 6, 3 7, 4 8, 5 1, 7 1 7. 69 c ha rt er gu s m et an ot al is r ic ha rd s, 1 97 8 a m pa b t, m t 10 1. 28 c ly pe ar ia a ng us tio r d uc ke , 1 90 6 a t, c a , s a b a , m g b t, l t, m t, a s, f s 11 , 2 3, 4 4, 5 1, 5 9, 6 8 7. 69 c ly pe ar ia a pi ci pe nn is (s pi no sa , 1 85 1) a m a m a s 43 , 5 6 2. 56 c ly pe ar ia d uc ke i r ic ha rd s, 1 97 8 a m a m , a p m t 22 , 4 3 2. 56 c ly pe ar ia s ul ca ta (d e sa us su re , 1 85 3) a m a m , a p, p a b t, m t, a s 10 , 2 2, 4 3, 5 6, 5 8 6. 41 c ly pe ar ia w ey ra uc hi r ic ha rd s, 1 97 8 a m a m , a p m t 22 1. 28 e pi po na ta tu a (c uv ie r, 17 97 ) a m , s a a m , d f, m a , m g , m t, p a a s, b t, l t, m t, q 6, 8 , 1 0, 4 3, 5 6, 5 7, 7 0 10 .2 6 le ip om el es d or sa ta (f ab ri cu s, 1 80 4) a m , c a a m , b a , c e , p a , r o b t, l t, m t, a s, l b 10 , 2 7, 4 2, 4 3, 5 1, 5 6, 5 8, 7 1 10 .2 6 le ip om el es p us si la (d uc ke , 1 90 4) a m a m a s 43 1. 28 le ip om el es s pi lo ga st ra c am er on , 1 91 2 a m a m m t, f 43 1. 28 m et ap ol yb ia a lfk en ii (d uc ke 1 90 4) a m a m m t 22 1. 28 m et ap ol yb ia c in gu la ta (f ab ri ci us 1 80 4) a m , a t, c a , m g , r e , s a , u e a m , a p, b a , m g , m t, p a , pe , s p a s, b t, m t, l b , q 6, 1 0, 1 8, 2 5, 2 6, 3 1, 3 7, 4 4, 5 1, 7 2 12 .8 2 m et ap ol yb ia d ec or at a (g ri bo do , 1 89 6) a m a m , a p m t 22 1. 28 m et ap ol yb ia d oc ili s r ic ha rd s, 1 97 8 sa sp m t, a s, l b 34 , 7 2 2. 56 m et ap ol yb ia n ig ra r ic ha rd s, 1 97 8 a m a m a s 43 , 5 8 2. 56 m et ap ol yb ia r uf at a r ic ha rd s, 1 97 8 a m a m , a p m t 22 , 4 3 2. 56 m et ap ol yb ia s uf fu sa (f ox , 1 89 9) sa sp a s 37 1. 28 m et ap ol yb ia u ni lin ea ta (i he ri ng , 1 90 4) a m , s a a m , r r , s p m t, a s 37 , 4 3, 7 4 3. 85 m is ch oc yt ta ru s ad ol ph i z ik án , 1 94 9 a m a m , p a b t, m t, a s 10 , 2 7, 4 2 3. 85 m is ch oc yt ta ru s al bo ni ge r r ic ha rd s, 1 97 8 a m r r a s 74 1. 28 m is ch oc yt ta ru s ar ac at ub ae ns is z ik an , 1 94 9 sa sp fs 62 1. 28 m is ch oc yt ta ru s ar au jo i z ik án 1 94 9 a t, e u , r f m g , s p b t, a s 1, 1 5, 2 3, 4 4, 4 9, 6 4 7. 69 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) bc barbosa et al. – social wasps of brazil: a review868 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy m is ch oc yt ta ru s ar tif ex (d uc ke , 1 91 4) a m , r f, s a a m , a p b t, m t, a s 22 , 2 3, 5 2 3. 85 m is ch oc yt ta ru s ba hi ae r ic ha rd s, 1 94 5 a g , c a , u e b a , m g lt , l t, m t, a s, f s 4, 5 , 5 1, 7 8 5. 13 m is ch oc yt ta ru s ba hi ae ns is z ik án , 1 94 9 c a b a , m g a s, b t, l t, m t 44 , 5 1 2. 56 m is ch oc yt ta ru s be rt on ii d uc ke , 1 91 8 u e pa a s 50 1. 28 m is ch oc yt ta ru s ca rb on ar iu s (s au ss ur e, 18 54 ) a m a m , p a , r r b t, m t, a s 10 , 2 7, 4 2, 7 4 5. 13 m is ch oc yt ta ru s ca ri nu la tu s z ik án , 1 94 9 c a b a lt , m t 51 1. 28 m is ch oc yt ta ru s ca ss un un ga (i he ri ng , 1 90 3) a g , a t, c a , e u , r f, r g sa , u e b a , d f, m g , p a , p e , s p b t, l t, m t, a s, f s, l b , q 1, 8 , 1 5, 1 6, 1 9, 2 1, 2 3, 2 4, 2 6, 2 8, 30 , 3 2, 3 5, 3 8, 3 9, 4 0, 4 1, 4 4, 4 5, 49 , 5 0, 5 1, 5 2, 5 9, 6 2, 6 4, 6 8, 7 5, 7 8 37 .1 8 m is ch oc yt ta ru s ce ar en si s r ic ha rd s, 1 94 5 c a b a lt , m t, f s 17 , 5 1 2. 56 m is ch oc yt ta ru s ce rb er us d uc ke , 1 91 8 a g , a t, c a , e u , s a , u e b a , d f, m t, m g , m s, s p b t, l t, m t, a s, f s, l b , q 1, 6 , 8 , 1 3, 1 6, 1 9, 2 5, 2 6, 3 1, 3 2, 34 , 3 7, 4 7, 5 1, 6 2, 7 0, 7 2, 7 5 24 .3 6 m is ch oc yt ta ru s co lla re llu s r ic ha rd s, 1 94 0 a m a m , p a b t, m t, a s 10 , 2 7, 4 2 3. 85 m is ch oc yt ta ru s co lla ri s (d uc ke , 1 90 4) a m a m a s 43 , 5 6 2. 56 m is ch oc yt ta ru s co nf us us z ik an , 1 93 5 a t, r f, r g , s a m g b t, a s, f s 15 , 2 3, 2 4, 3 3, 3 5, 3 8, 4 4, 4 5, 5 2 11 .5 4 m is ch oc yt ta ru s co ns im ili s z ik án , 1 94 9 a t, s a sp m t, a s, l b 34 , 7 2 2. 56 m is ch oc yt ta ru s dr ew se ni s au ss ur e, 1 95 4 a g , a m , a t, c a , e u , r f, r g , s a , u e a m , b a , m g , m t, m s, r s, s p b t, l t, m t, a s, f s, l b , q 1, 4 , 6 , 1 1, 1 4, 1 5, 1 6, 1 9, 2 3, 2 4, 26 , 3 0, 3 2, 3 3, 3 5, 3 6, 3 8, 3 9, 4 0, 41 , 4 4, 4 5, 4 9, 5 1, 5 2, 5 3, 5 8, 5 9, 60 , 6 2, 6 4, 6 5, 6 6, 6 8, 6 9, 7 0, 7 3, 7 8 48 .7 2 m is ch oc yt ta ru s du ck ei b uy ss on , 1 90 8 a m a m , p a b t, m t, a s 10 , 2 0, 2 7, 4 2 5. 13 m is ch oc yt ta ru s fla vi ca ns (f ab ri ci us , 1 80 4) a m a m , p a , r o b t, a s, l b 24 , 4 3, 5 6, 7 1 5. 13 m is ch oc yt ta ru s fla vi co rn is z ik án , 1 93 5 c a b a lt , m t 51 1. 28 m is ch oc yt ta ru s fla vo sc ul le ta tu s z ik án , 19 35 a t m g b t, a s 44 , 6 4 2. 56 m is ch oc yt ta ru s flu m in en si s z ik án , 1 94 9 a t m g b t, a s 44 1. 28 m is ch oc yt ta ru s fo ve at us r ic ha rd s, 1 94 0 a m a m , a p, p a b t, m t 10 , 2 2 2. 56 m is ch oc yt ta ru s fr on ta lis (f ox , 1 89 8) a s, u e m g , m s a s, b t, l b 44 , 4 7 2. 56 m is ch oc yt ta ru s fu ne ru lu s z ik án , 1 94 9 a t m g b t, a s 15 , 2 3, 4 4, 4 9 5. 13 m is ch oc yt ta ru s go m es i s ilv ei ra , 2 01 3 a m r o b t, l b 71 1. 28 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) sociobiology 63(3): 858-880 (september, 2016) 869 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy m is ch oc yt ta ru s go ya nu s z ik án , 1 94 9 sa d f a s 8 1. 28 m is ch oc yt ta ru s gr an ad ae ns is z ik an , 1 94 9 a m r r a s 74 1. 28 m is ch oc yt ta ru s ig no tu s z ik án , 1 94 9 a g , a t, s a m g , s p m t, a s, l b 34 , 7 2, 7 8 3. 85 m is ch oc yt ta ru s ih er in gi z ik án , 1 93 5 u e m g a s 64 1. 28 m is ch oc yt ta ru s im ita to r z ik án , 1 93 5 a m a m , a p, p a b t, m t 10 , 2 2, 4 3 3. 85 m is ch oc yt ta ru s in ju cu nd us (s au ss ur e, 1 85 4) a m , c a , s a a m , a p, b a , p a , r r , s p b t, l t, m t, a s 10 , 2 2, 3 7, 5 1, 7 4 6. 41 m is ch oc yt ta ru s in te rr up tu s r ic ha rd s, 1 97 8 a m r o b t, l b 71 1. 28 m is ch oc yt ta ru s ju ra nu s r ic ha rd s, 1 97 8 a m pa b t 10 1. 28 m is ch oc yt ta ru s la bi at us (f ab ri ci us , 1 80 4) a m , a t, c a , e u , s a a m , b a , m t, p a , r o , r r , s p b t, m t, a s, l b , q 1, 6 , 1 0, 2 0, 4 3, 5 1, 5 6, 5 8, 7 1, 7 4 12 .8 2 m is ch oc yt ta ru s la ne i z ik án , 1 94 9 a g , c a , u e b a b t, l t, m t, a s, f s 4, 5 , 1 3, 5 1 5. 13 m is ch oc yt ta ru s la tio r (f ox , 1 89 8) a g , a t, e u , r g , s a , u e m g , m s, s p b t, a s, f s, l b 1, 1 6, 1 9, 3 2, 3 5, 3 8, 4 5, 4 7, 7 7, 7 8 12 .8 2 m is ch oc yt ta ru s le co in te i ( d uc ke , 1 90 4) a m a m , a p, p a , r o b t, m t, a s, l b 10 , 2 2, 2 7, 4 2 ,4 3, 5 6, 7 1 8. 97 m is ch oc yt ta ru s m al ar is r ic ha rd s, 1 97 8 a m a m m t 42 1. 28 m is ch oc yt ta ru s m ar ac ae ns is r aw , 1 99 2 a m r r a s 74 1. 28 m is ch oc yt ta ru s m ar gi na tu s (f ox , 1 89 8) a t c a , r g , s a b a , m g , p a , s p b t, l t, m t, f s, l b 19 , 3 2, 3 5, 3 8, 4 5, 5 1 7. 69 m is ch oc yt ta ru s m at to gr os so en si s z ik án , 1 93 5 a g , c a , r f, s a , u e b a , m g , m t, m s, s p a s, b t, f s, l b , q 4, 5 , 6 , 4 0, 4 7, 6 5, 7 0, 7 8 10 .2 6 m is ch oc yt ta ru s m el an op s c oo pe r, 19 96 a m pa b t 10 1. 28 m is ch oc yt ta ru s m et at ho ra ci cu s (s au ss ur e, 1 85 4) a m , s a a m , m t, p a b t, m t, a s, q 6, 1 0, 2 7, 4 3, 5 8 6. 41 m is ch oc yt ta ru s m ir ifi cu s z ik án , 1 93 5 r g m g b t, a s 45 1. 28 m is ch oc yt ta ru s m on te i z ik án , 1 94 9 c a b a lt , m t 51 1. 28 m is ch oc yt ta ru s no m ur ae r ic ha rd s, 1 97 8 a g , c a b a , m g lt , m t, a s 51 , 7 8 2. 56 m is ch oc yt ta ru s oe co th ri x r ic ha rd s, 1 94 0 a m a m , p a b t, m t, a s 10 , 2 7, 4 2 3. 85 m is ch oc yt ta ru s pa ra gu ay en si s z ik án , 1 93 5 a g m g a s 78 1. 28 m is ch oc yt ta ru s pa ra lle llo gr am m us z ik án , 1 93 5 a t, u e m g , s p b t, a s 28 , 3 9 2. 56 m is ch oc yt ta ru s pa ul is ta nu s z ik án , 1 93 5 a t, s a sp m t, a s, l b 34 , 7 2 2. 56 m is ch oc yt ta ru s pr om in ul us r ic ha rd s, 1 94 1 a m r r a s 74 1. 28 m is ch oc yt ta ru s pu nc ta tu s (d uc ke , 1 90 4) a m m g , p a a s, b t 27 , 4 4 2. 56 m is ch oc yt ta ru s ri og ra nd en si s r ic ha rd s, 1 97 8 a t r s fs 53 1. 28 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) bc barbosa et al. – social wasps of brazil: a review870 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy m is ch oc yt ta ru s ro tu nd ic ol lis (c am er on , 1 91 2) a g , a m , a t, c a , e u , r g , r f, s a , u e a m , b a , m g , m t, r s, s p b t, m t, a s, f s, l b 1, 1 5, 1 9, 2 1, 2 3, 2 5, 2 8, 3 1, 3 2, 3 4, 35 , 3 8, 4 0, 4 1, 4 3 44 , 4 5, 4 9, 5 1, 5 3, 56 , 5 9, 6 2, 6 4, 6 5, 6 7, 7 0, 7 2, 7 5, 7 8 38 .4 6 m is ch oc yt ta ru s sa tu ra tu s z ik án , 1 94 9 a m a m , p a b t, m t 10 , 4 3 2. 56 m is ch oc yt ta ru s so ci al is (s au ss ur e, 1 85 4) a m , a t, r f, r g , s a , u e a m , s p, m g b t, m t, a s, f s, l b 15 , 1 6, 1 9, 2 3, 2 8, 3 2, 3 3, 3 5, 3 8, 3 9, 43 , 4 5, 5 2, 5 6, 5 8, 6 4 20 .5 1 m is ch oc yt ta ru s su ri na m en si s (s au ss ur e, 1 85 4) a m , c a a m , b a , c e , p a , r r b t, m t, a s 10 , 4 3, 5 1, 5 6, 5 8, , 7 4 7. 69 m is ch oc yt ta ru s sy lv es tr is r ic ha rd s, 1 94 5 a m a m , p a b t, m t, a s 10 , 2 7, 4 2 3. 85 m is ch oc yt ta ru s sy no ec us r ic ha rd s, 1 94 0 a m a m , a p, p a b t, m t, a s, f 10 , 2 2, 2 7, 4 2, 4 3 6. 41 m is ch oc yt ta ru s te ct us c oo pe r, 19 96 a m pa b t, m t 10 1. 28 m is ch oc yt ta ru s tim bi ra s ilv ei ra , 2 00 6 sa sp a s 37 1. 28 m is ch oc yt ta ru s to m en to su s z ik án , 1 93 5 a m pa , r o b t, m t, l b 10 , 7 1 2. 56 m is ch oc yt ta ru s tr ic ol or r ic ha rd s, 1 94 5 a t, s a m g b t, a s 15 , 2 3, 3 5, 3 8, 4 4, 4 9, 7 5 8. 97 m is ch oc yt ta ru s w ag ne ri (b uy ss on , 1 90 8) a t, r f, r g m g b t, a s 15 , 2 3, 4 4, 4 5, 4 9, 5 2, 6 4 8. 97 m is ch oc yt ta ru s yp ir ag ue ns is f on se ca , 1 92 6 r g m g b t, a s 45 1. 28 p ar ac ha rt er gu s fa sc iip en ni s d uc ke , 1 90 5 a m a m m t 43 1. 28 p ar ac ha rt er gu s fla vo fa sc ia tu s (c am er on , 1 90 6) a m r o b t, l b 71 1. 28 p ar ac ha rt er gu s fr at er nu s (g ri bo do , 1 89 1) a m , a t, p a , r g , r f, s a , u e a m , a p, d f, m a , m g , m t, p a , r o , s p b t, m t, a s, f s, l b , q 6, 8 , 1 0, 1 9, 2 2, 2 3, 2 7, 3 2, 3 3, 3 7, 38 , 3 9, 4 3, 4 4, 4 5, 4 8, 5 2, 5 7, 5 8, 64 , 6 9, 7 0, 7 1 29 .4 9 p ar ac ha rt er gu s fu lg id ip en ni s (s au ss ur e, 1 85 4) a m a m , a p, p a b t, m t 22 , 2 7 2. 56 p ar ac ha rt er gu s gr is eu s (f ox , 1 89 8) a m a m m t 43 1. 28 p ar ac ha rt er gu s le nk oi r ic ha rd s, 1 97 8 a m r o b t, l b 71 1. 28 p ar ac ha rt er gu s ps eu do ap ic al is w ill in k, 1 95 9 a g , a m , a t, c a , e u , m g , r e , a s, u e b a , m g , p e , r o , s p b t, l t, m t, a s, f s, l b 1, 4 , 5 , 1 6, 1 8, 2 6, 3 5, 5 1, 6 8, 7 1 12 .8 2 p ar ac ha rt er gu s ri ch ar ds i w ill in k, 1 95 9 a m a m , p a m t 10 , 4 3 2. 56 p ar ac ha rt er gu s sm ith ii (s au ss ur e, 1 85 4) a m , a t, s a , u e a m , a p, m s, m t, r o , s p m t, a s, f s, l b 22 , 3 4, 4 7, 6 2, 7 0, 7 1, 7 2 8. 97 p ol is te s ac ta eo n h al id ay , 1 83 6 a g , a t, e u , r f, a s, u e m g , r s, s p b t, a s, f s, l b 15 , 2 1, 2 3, 3 5, 3 8, 3 9, 4 0, 4 4, 4 9, 5 2, 53 , 5 9, 6 4, 6 6, 6 7, 7 8 20 .5 1 p ol is te s bi gu tta tu s h al id ay , 1 83 6 a t r s fs 53 1. 28 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) sociobiology 63(3): 858-880 (september, 2016) 871 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy p ol is te s bi lla rd ie ri f ab ri ci us , 1 80 4 a g , a t, c a , e u , m g , r e , r f, r g , s a , u e b a , m g , m s, m t, p b , p e , r s, s p b t, l t, m t, a s, f s, l b 1, 4 , 1 3, 1 5, 1 6, 1 8, 2 4, 3 3, 3 5, 3 6, 3 8, 4 0, 4 1, 4 3, 4 4, 4 5, 4 6, 4 7, 4 9, 51 , 5 2, 5 3, 6 0, 6 5, 6 8, 7 0 33 .3 3 p ol is te s br ev ifi ss us r ic ha rd s, 1 97 8 a m , a t, c a , r f b a , m s, p b , r r b t, l t, m t, a s, l b , a s 36 , 5 1, 6 0, 7 4 5. 13 p ol is te s ca na de ns is (l in na eu s, 1 75 8) a g , a m , a t, c a , e u , m g , r e , r g , s a , u e a m , a p, b a , c e , m g , m t, m s, p b , p e , p i, r o , s p a s, b t, l t, m t, t t, f s, l b , m o , q 1, 3 , 4 , 5 , 6 , 1 1, 1 3, 1 4, 1 7, 1 8, 1 9, 2 2, 32 , 3 7, 4 3, 4 4, 4 7, 5 1, 5 4, 5 5, 5 6, 5 8, 6 1, 68 , 7 0, 7 1, 7 6, 7 7, 7 8 37 .1 8 p ol is te s ca rn ife x (f ab ri ci us , 1 77 5) a m , a t, c a , m g , r e , u e a m , b a , m g , p e , s p a s, b t, l t, m t 18 , 2 8, 4 4, 5 1, 5 8 6. 41 p ol is te s ca va py ta s au ss ur e, 1 85 3 a t r s fs 53 1. 28 p ol is te s ca va py tif or m is r ic ha rd s, 1 97 8 a t r s fs 53 1. 28 p ol is te s ci ne ra sc en s sa us su re , 1 85 4 a g , a t, c a , m g , r e , r f, r g , s a a l , b a , c e , m g , m s, r s, s p b t, l t, m t, a s, f s, l b , q 1, 1 1, 1 3, 1 5, 1 6, 1 8, 2 3, 2 4, 3 3, 3 5, 3 6, 38 , 4 0, 4 1, 4 4, 4 5, 4 9, 5 1, 5 2, 5 3, 6 0, 6 2, 64 , 6 5, 6 7, 7 3, 7 5 34 .6 2 p ol is te s co ns ob ri nu s sa us su re , 1 85 3 a g , a t, e u r s, s c , s p b t, a s, f s 1, 2 , 5 3 3. 85 p ol is te s da vi lla e r ic ha rd s, 1 97 8 r g m g b t, a s 45 1. 28 p ol is te s fe rr er i s au ss ur e, 1 85 3 a g , a t, c a , r g , r f, s a , u e b a , m g , m s, p a , s c , s p b t, l t, m t, a s, t t, m o , q 2, 1 5, 1 6, 2 3, 2 4, 3 3, 3 5, 3 8, 4 0, 44 , 4 5, 4 9, 5 0, 5 1, 5 5, 6 1, 7 7 21 .7 9 p ol is te s ge m in at us f ox , 1 89 8 a t, c a , s a , r f b a , m g , m s, m t, s p b t, l t, m t, a s, f s, l b 34 , 3 5, 3 6, 3 8, 4 0, 5 1, 6 0, 6 2, 7 0, 7 2 12 .8 2 p ol is te s go el di i d uc ke , 1 90 4 a m a m , m g , p a b t, m t, a s 10 , 3 5, 4 3, 5 6 5. 13 p ol is te s la ni o (f ab ri ci us , 1 77 5) a g , a t, c a , e u , a s, u e b a , m g , s p, p a b t, l t, m t, s t, a s, f s 21 , 3 5, 3 8, 4 0, 5 0, 5 1, 6 2, 6 3, 6 5, 7 3, 7 5 14 .1 0 p ol is te s oc ci pi ta lis d uc ke , 1 90 4 a m m g , p a , r o a s, b t, m t, l b 10 , 2 7, 4 4, 7 1 5. 13 p ol is te s pa ci fic us f ab ri ci us , 1 80 4 a m , a t, c a , r f, s a a m , b a , m g , m t, p a , r s b t, l t, m t, a s, f s, q 6, 1 0, 1 5, 2 3, 4 3, 4 4, 4 9, 5 1, 5 2, 5 3, 56 , 6 4, 7 5 16 .6 7 p ol is te s ru fiv en tr is d uc ke , 1 90 4 a m pa b t, m t 27 1. 28 p ol is te s sa ta n b eq ua er t, 19 40 a g , s a d f, m g b t, a s 8, 7 8 2. 56 p ol is te s si m ili m us z ik án , 1 95 1 a g , a t, c a , e u , r g , r f, sa , u e b a , m g , m s, r s, s p b t, l t, m t, s t, a s, t t, fs , l b , m o 1, 1 4, 1 5, 1 7, 2 1, 2 3, 2 4, 3 1, 3 4, 3 6, 3 9, 40 , 4 1, 4 4, 4 5, 4 9, 5 1, 5 2, 5 5, 6 0, 6 1, 6 2, 63 , 6 4, 6 5, 6 6, 6 7, 7 2, 7 3, 7 5, 7 7, 7 8 41 .0 3 p ol is te s su bs er ic iu s sa us su re , 1 85 4 a g , a t, c a , r f, s a , u e b a , m g , m t, m s, s p b t, l t, m t, a s, f s, l b , q 15 , 1 6, 1 9, 2 3, 3 2, 3 5, 3 6, 3 8, 4 0, 4 1, 4 4, 45 , 4 7, 4 9, 5 0, 6 1, 6 2, 6 5, 7 0 24 .3 6 p ol is te s te st ac ei co lo r b eq ua er t, 19 37 a m a m , m a m t, a s 43 , 5 6, 5 7 3. 85 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) bc barbosa et al. – social wasps of brazil: a review872 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy p ol is te s th or ac ic us f ox , 1 89 8 sa m t a s 70 1. 28 p ol is te s ve rs ic ol or (o liv ie r, 17 91 ) a g , a m , a t, c a , e u , m g , pa , r e , r f, r g , s a , u e a m , a p, b a , m a , m g , m s, m t, p a , r r , r s, s c , s p b t, l t, m t, s t, a s, t t, f s, l b , m o 1, 2 , 3 , 4 , 5 , 1 0, 1 1, 1 3, 1 5, 1 6, 1 8, 1 9, 2 1, 22 , 2 3, 2 5, 2 6, 2 8, 3 1, 3 2, 3 3, 3 4, 3 5, 3 6, 38 , 3 9, 4 0, 4 1, 4 4, 4 5, 4 6, 4 7, 4 8, 4 9, 5 0, 51 , 5 2, 5 3, 5 5, 5 7, 5 9, 6 0, 6 1, 6 2, 6 3, 6 4, 65 , 6 6, 6 7, 6 8, 6 9, 7 2, 7 3, 7 4, 7 5, 7 7, 7 8 73 .0 8 p ol yb ia a ffi ni s b uy ss on , 1 90 8 a m a m , p a b t, m t 10 , 2 7, 5 8 3. 85 p ol yb ia b el em en si s r ic ha rd s, 1 97 0 a m a m , a p, p a b t, m t, a s 22 , 2 7, 4 3, 5 6, 5 8 6. 41 p ol yb ia b ic yt ta re lla r ic ha rd s, 1 95 1 a m , s a sp , a m , a p, p a b t, m t, a s 10 , 2 2, 3 7, 4 3, 5 6, 5 8 7. 69 p ol yb ia b ifa sc ia ta s au ss ur e, 1 85 4 a g , a m , a t, r f, s a ,u e a m , m g , r o , s p b t, a s, f s, l b 15 , 1 9, 2 3, 2 8, 3 2, 3 5, 3 8, 3 9, 4 0, 43 , 4 4, 5 2, 5 9, 6 4, 7 0, 7 1, 7 8 21 .7 9 p ol yb ia b is tr ia ta (f ab ri ci us , 1 80 4) a m a m , b a , m a , p a , r o b t, m t, l t, a s, l b 10 , 2 7, 4 3, 5 1, 5 7, 5 6, 5 8, 7 1 10 .2 6 p ol yb ia b ru nn ea (c ur tis , 1 84 4) a m pa b t 27 1. 28 p ol yb ia c at til lif ex m oe bi us , 1 85 6 a m , c a , u e a m , s p, p a , r o b t, l t, m t, a s, l b 20 , 2 7, 2 8, 4 2, 5 1, 7 1 7. 69 p ol yb ia c hr ys ot ho ra x (l ic ht en st ei n, 1 79 6) a g , a m , a t, c a , e u , p a , r f, r g , s a , u e b a , c e , g o , m a , m g , m s, m t, s p b t, l t, m t, a s, t t, f s, l b , m o 1, 3 , 4 , 5 , 7 , 1 1, 1 4, 1 5, 2 3, 3 1, 3 4, 35 ,3 6, 37 , 3 8, 4 0, 4 4, 4 5, 4 8, 4 9, 51 , 5 2, 5 5, 5 7, 59 , 6 0, 6 1, 6 4, 6 5, 7 0, 7 2, 7 6, 7 8 42 .3 1 p ol yb ia d ep re ss a (d uc ke , 1 90 5) a m , c a a m , b a , m a , p i, r o m t, a s, l b 43 , 5 1, 5 6, 5 7, 5 8, 7 0, 7 1 8. 97 p ol yb ia d ig ue ta na b uy ss on , 1 90 5 a m r o b t, l b 71 1. 28 p ol yb ia d im id ia ta (o liv ie r, 17 91 ) a g , a m , a t, c a , e u , r f, s a a m , a p, b a , m g , m t, p a , r o , r r , s p b t, m t, a s, l b 1, 1 0, 2 2, 2 7, 34 , 4 0, 4 2, 4 3, 4 4, 5 1, 5 6, 7 0, 71 , 7 2, 7 4, 7 5 20 .5 1 p ol yb ia d im or ph a r ic ha rd s, 1 97 8 a m , s a a m , p a , r r b t, m t, a s 10 , 2 0, 2 7, 3 7, 4 3, 5 6, 5 8, 7 4 10 .2 6 p ol yb ia d ub ita ta d uc ke , 1 91 0 a m a m a s 56 1. 28 p ol yb ia e be rh ar da e c oo pe r, 19 93 a m r o b t, l b 71 1. 28 p ol yb ia e m ac ia ta l uc as , 1 87 9 a m a m , p a , r o b t, m t, a s, l b 20 , 2 7, 4 2, 7 1 5. 13 p ol yb ia e ry th ro th or ax la r ic ha rd s, 1 97 8 a g , s a m g , m t a s, q 6, 7 0, 7 8 3. 85 p ol yb ia fa st id io su sc ul a sa us su re , 1 85 4 a g , a m , a t, c a , e u , r g , r f, s a , u e a m , b a , d f, m a , m g , p a , r s, s c , s p b t, m t, s t, a s, fs , l b 1, 2 , 8 , 1 5, 1 9, 2 3, 2 4, 2 8, 3 1, 3 2, 33 ,3 4, 3 5, 38 , 3 9, 4 0, 4 3, 4 4, 4 5, 4 9, 5 0, 5 1, 5 3, 5 6, 57 , 5 9, 6 2, 6 3, 6 4, 6 5, 7 2, 7 3, 7 5 42 .3 1 p ol yb ia fl av ifr on s sm ith , 1 85 7 c a , s a , u e b a , m t, s p lt , a s, f s 26 , 5 1, 6 2, 7 0, 7 5 6. 41 p ol yb ia fl av iti nc ta f ox , 1 89 8 a t, m g , r e b a a s 18 1. 28 p ol yb ia g or yt oi de s fo x, 1 89 8 a m a m , p a , r o b t, m t, a s, l b 10 , 2 7, 4 2, 7 1 5. 13 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) sociobiology 63(3): 858-880 (september, 2016) 873 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy p ol yb ia ig no bi lis (h al id ay , 1 83 6) a g , a m , a t, c a , e u , m g , pa , r e , r f, r g , s a , u e b a , c e , d f, g o , m a , m g , m s, m t, p a , p b , p i, r r , r s, s c , s p b t, l t, m t, a s, t t, fs , l b , m o , q 1, 2 , 3 , 4 , 5 , 7 , 8 , 1 1, 1 3, 1 4, 1 5, 1 6, 17 , 1 8, 1 9, 24 , 2 5, 2 6, 2 8, 3 0, 3 1, 3 2, 3 3, 3 4, 3 5, 3 6, 3 8, 3 9, 40 , 4 1, 4 4, 4 5, 4 6, 4 7, 4 8, 4 9, 5 0, 5 1, 5 3, 5 4, 5 5, 57 , 5 9, 6 0, 6 1, 6 2, 6 4, 6 5, 6 6, 6 7, 6 8, 7 0, 7 2, 7 3, 74 , 7 5, 7 6, 7 7, 7 8 75 .6 4 p ol yb ia ju ri ne i s au ss ur e, 1 85 4 a g , a m , a t, c a , e u , r f, r g , p a , s a , u e a m , b a , c e , m a , m g , m t, m s, p a , r o , s p b t, m t, a s, f s, l b , q 1, 6 , 1 0, 1 5, 1 6, 1 9, 2 1, 2 5, 2 7, 2 8, 32 , 3 4, 3 6, 3 7, 38 , 3 9, 4 0, 4 1, 4 3, 4 4, 4 5, 4 7, 4 8, 4 9, 5 1, 5 6, 5 7, 58 , 5 9, 6 0, 62 , 6 4, 6 5, 7 0, 7 1, 7 2, 7 3, 7 5, 7 8 50 .0 0 p ol yb ia li lia ce a (f ab ri ci us , 1 80 4) a m , s a , u e a m , m a , m s, m t, p a , r o , r r b t, m t, a s, l b 10 , 2 7, 4 2, 4 3, 4 7, 5 7, 5 6, 5 8, 7 0, 71 , 7 4 14 .1 0 p ol yb ia lu gu br is d uc ke , 1 90 5 u e m g b t 64 1. 28 p ol yb ia m ic an s d uc ke , 1 90 4 a m , c a b a , m a , p a , r o b t, l t, m t, l b 10 , 2 7, 5 1, 5 7, 7 1 6. 41 p ol yb ia m in ar un d uc ke , 1 90 6 a g , a t, c a , r f, s a , u e b a , m g , p a , r s, s p b t, l t, m t, a s, fs , l b 4, 1 5, 1 9, 3 2, 3 5, 3 8, 4 4, 4 9, 5 0, 5 1, 5 3, 6 5, 7 3 16 .6 7 p ol yb ia o cc id en ta lis (o liv ie r, 17 91 ) a g , a m , a t, c a , e u , m g , pa , r e , r f, r g , s a , u e a m , b a , d f, g o , m a , m g , m t, m s, p b , p e , p i, r r , s c , s p b t, m t, l t, a s, t t, fs , l b , q 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 1 1, 1 3, 1 4, 1 5, 16 , 1 7, 1 8, 1 9, 23 , 2 5, 2 6, 2 8, 3 1, 3 2, 3 3, 3 4, 3 5, 3 6, 3 8, 4 1, 4 3, 44 , 4 5, 4 7, 48 , 4 9, 5 1, 5 4, 5 6, 5 7, 5 8, 5 9, 6 0, 6 1, 62 , 6 4, 6 5, 6 6, 6 8, 7 0, 7 2, 7 3, 7 4, 7 5, 7 7, 7 8 69 .2 3 p ol yb ia p ar vu lin a r ic ha rd s, 1 97 0 a m a m , p a , r o b t, m t, a s, l b 10 , 2 7, 4 3, 5 6, 7 0, 7 1 7. 69 p ol yb ia p au lis ta (i he ri ng , 1 89 6) a g , a t, c a , e u , m g , r f, r g , r e , s a , u e b a , d f, m g , m t, m s, p i, sp b t, l t, m t, a s, f s, l b , q 1, 3 , 4 , 5 , 6 , 8 , 1 1, 1 3, 1 4, 1 5, 1 6, 1 8, 1 9, 2 3, 2 4, 25 , 2 6, 3 1, 3 2, 3 3, 3 4, 3 5, 3 6, 3 8, 3 9, 4 0, 4 1, 4 5, 47 , 5 1, 5 2, 5 4, 6 0, 6 2, 6 4, 6 5, 6 8, 7 2, 7 3, 7 5, 7 8 58 .9 7 p ol yb ia p la ty ce ph al a r ic ha rd s, 1 95 1 a g , a m , a t, c a , e u , r g , r f, s a , u e a m , b a , m g , p a , r o , r s, s p b t, m t, s t, a s, fs , l b 1, 1 0, 1 5, 1 6, 2 3, 2 7, 3 7, 3 9, 4 1, 4 3, 44 , 4 5, 4 6, 49 , 5 1, 5 2, 5 3, 5 6, 5 9, 6 3, 6 4, 6 6, 7 1 29 .4 9 p ol yb ia p ro ce llo sa z av at ta ri , 1 90 6 a m a m , r o a s, l b 43 , 7 0, 7 1 3. 85 p ol yb ia p un ct at a b uy ss on , 1 90 8 a t, c a b a , s p lt , m t, s t 51 , 6 3 2. 56 p ol yb ia q ua dr ic in ct a sa us su re , 1 85 4 a m , s a a m , a p, m t, p a , r o b t, m t, a s, l b , q 6, 1 0, 2 2, 2 7, 4 3, 5 6, 5 8, 7 0, 7 1 11 .5 4 p ol yb ia r ej ec ta (f ab ri ci us , 1 79 8) a g , c a , s a , a m , a t, m g , r e a m , a p, b a , g o , m a , m g , p a , pe , r o , r r b t, l t, m t, a s, l b 7, 1 0, 1 8, 2 0, 2 2, 2 7, 3 7, 4 3, 4 4, 5 1, 5 6, 5 7, 58 , 7 1, 7 4, 7 8 20 .5 1 p ol yb ia r or ai m ae r aw , 1 99 8 a m r r a s 74 1. 28 p ol yb ia r ufi ce ps s ch ro ttk y, 1 90 2 a t, c a , p a , r f, s a , u e b a , c e , m g , m s, m t, p i, sp b t, l t, m t, a s, f s, l b , q 6, 1 6, 1 7, 1 9, 2 5, 3 2, 3 6, 4 7, 4 8, 5 1, 5 4, 60 , 7 0, 7 2 17 .9 5 p ol yb ia r ufi ta rs is d uc ke , 1 90 4 a m a m , a p, p a , r o b t, m t, a s, l b 10 , 2 2, 2 7, 4 3, 5 6, 7 1 7. 69 p ol yb ia s cr ob al is r ic ha rd s, 1 97 0 a m a m , m a , p a , r o b t, m t, l t, a s, l b 10 , 2 7, 4 3, 5 7, 5 8, 5 6, 7 1 8. 97 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) bc barbosa et al. – social wasps of brazil: a review874 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy p ol yb ia s cu te lla ri s (w hi te , 1 84 1) a g , a t, c a , e u , r f, r g , sa ,u e b a , m g , p a , r s, s c b t, l t, m t, a s, f s, l b 2, 1 5, 1 6, 2 3, 3 3, 4 4, 4 5, 4 6, 4 9, 5 0, 5 1, 52 , 5 3, 6 6, 6 7 19 .2 3 p ol yb ia s er ic ea (o liv ie r, 17 92 ) a g , a m , a t, c a , e u , m g , pa , r e , r f, r g , s a ,u e a m , b a , g o ,m a , m g , m s, m t, p a , p b , p i, r o , r r , r s, s c , s p b t, m t, s t, a s, f s, l b , q 1, 2 , 3 , 4 , 5 , 6 , 7 , 1 1, 1 3, 1 4, 1 5, 1 6, 18 , 19 , 2 1, 2 4, 2 3, 2 5, 2 6, 3 0, 3 1, 3 2, 3 3, 3 4, 35 , 3 6, 3 8, 3 9, 3 7, 4 0, 4 1, 4 3, 4 4, 4 5, 4 6, 47 , 4 8, 4 9, 5 0, 5 1, 5 2, 5 3, 5 4, 5 6, 5 7, 5 9, 60 , 6 2, 6 3, 6 4, 6 5, 6 6, 6 8, 6 9, 7 0, 7 1, 7 2, 73 , 7 4, 7 5, 7 7, 7 8 79 .4 9 p ol yb ia s in gu la ri s d uc ke , 1 90 9 a m , s a a m , a p, m a , m t, p a , r o b t, m t, a s, l b , q 6, 1 0, 2 2, 2 7, 4 2, 4 3, 5 6, 5 7, 5 8, 7 1 12 .8 2 p ol yb ia s tr ia ta (f ab ri ci us , 1 78 7) a m , s a , u e a m , a p, m g , m a , p a , r o b t, m t, l t, a s, l b 10 , 1 6, 2 2, 2 7, 3 0, 4 2, 43 , 5 7, 5 8, 59 , 6 4, 71 15 .3 8 p ol yb ia ti nc tip en ni s fo x, 1 89 8 a m a m , r o m t, l b 20 , 4 3, 7 1 3. 85 p ol yb ia v el ut in a d uc ke , 1 90 7 a m a m a s 43 , 5 6 2. 56 p ro to ne ct ar in a sy lv ei ra e (s au ss ur e, 1 85 4) a g , a t, c a , e u , m g , r e , r f, r g , s a , u e b a , c e , m g , m s, p i, r s, sc , s p b t, l t, m t, a s, t t, f s, l b , m o 1, 2 , 3 , 4 , 5 , 1 1, 1 3, 1 5, 1 7, 1 8, 1 9, 23 , 2 4, 25 , 2 6, 3 1, 3 2, 3 3, 3 4, 3 5, 3 8, 3 9, 4 1, 4 4, 45 , 4 6, 5 1, 5 2, 5 3, 5 4, 5 5, 5 9, 6 1, 6 2, 6 4, 65 , 6 6, 6 7, 6 8, 7 2, 7 3, 7 5, 7 7 55 .1 3 p ro to po ly bi a ac ut is cu tis c am er on , 1 90 7 a m r o b t, l b 71 1. 28 p ro to po ly bi a bi tu be rc ul at a si lv ei ra & c ar pe nt er , 1 99 5 a m a m , m a , m t, a s 43 , 5 6, 5 7, 5 8 5. 13 p ro to po ly bi a ch ar te rg oi de s (g ri bo do , 1 89 1) a m , s a a m , a p, m a , m t, p a , r o b t, m t, a s, l b , q 6, 1 0, 2 0, 2 2, 4 3, 5 6, 5 7, 5 8, 7 1 11 .5 4 p ro to po ly bi a du ck ei (b uy ss on , 1 90 5) c a b a lt , m t 51 1. 28 p ro to po ly bi a du ck ei an a r ic ha rd s, 1 97 8 a m a m ? 43 1. 28 p ro to po ly bi a em or tu al is (d e sa us su re , 1 85 5) a m a m , p a b t, m t 10 , 4 3 2. 56 p ro to po ly bi a ex ig ua (s au ss ur e, 1 85 4) a g , a m , a t, c a , e u , m g , r e , r g , s a ,u e , b a , c e , m a ,m t, m g , m s, p a , p e , p i, r r , s p b t, l t, m t, a s, f s, t t, l b , m o , q 1, 3 , 4 , 5 , 6 , 1 1 13 , 1 4, 1 8, 1 9, 26 , 2 7, 2 8, 32 , 3 3, 3 4, 3 7, 3 9, 5 1, 5 4, 5 5, 5 7, 6 1, 6 2, 64 , 6 6, 6 8, 7 0, 7 2, 7 4, 7 5, 7 7 41 .0 3 p ro to po ly bi a ho lo xa nt ha (d uc ke , 1 90 4) a m a m a s 43 1. 28 p ro to po ly bi a ni tid a (d uc ke , 1 90 4) a m a m m t 58 1. 28 p ro to po ly bi a ro tu nd at a d uc ke , 1 91 0 a m r o b t, l b 71 1. 28 p ro to po ly bi a ru gu lo sa d uc ke , 1 90 7 a m a m m t 43 1. 28 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . ( c on tin ua tio n) sociobiology 63(3): 858-880 (september, 2016) 875 sp ec ie s st ud ie d e nv ir on m en t st ud ie d st at e sa m pl in g m et ho ds r ef er en ce s c on st an cy p ro to po ly bi a se du la (s au ss ur e, 1 85 4) a g , a m , a t, c a , e u , r g , r f, s a a m , b a , c e , m g , s p b t, l t, a s, q 1, 1 5, 3 3, 3 5, 3 7, 3 8, 4 5, 5 1, 52 , 5 8, 5 9, 64 , 7 3, 7 8 17 .9 5 p se ud op ol yb ia c om pr es sa (s au ss ur e, 1 85 4) a m , c a , s a a m , a p, b a , m t, r o a s, l b , q 6, 2 2, 4 3, 5 1, 7 0, 7 1 7. 69 p se ud op ol yb ia d iffi ci lis (d uc ke , 1 90 5) a m pa b t, m t 10 , 2 7 2. 56 p se ud op ol yb ia la ng i b eq ua er t, 19 44 a m a m ? 43 1. 28 p se ud op ol yb ia v es pi ce ps (s au ss ur e, 1 86 4) a m , a t, c a , r f, s a b a , d f, m a , m g , m t, p a , p e , r o , r r , s p b t, l t, m t, a s, f s, l b , q 6, 8 , 1 5, 1 6, 1 9, 2 3, 2 7, 3 2, 3 5, 3 7, 3 8, 4 4, 51 , 5 2, 5 7, 7 1, 7 4 21 .7 9 sy no ec a ch al ib ea d e sa us su re , 1 85 2 a m r o b t, l b 71 1. 28 sy no ec a cy an ea (f ab ri ci us , 1 77 5) a g , a m , a t, c a , e u , m g , r e , r g , s a , e u b a , m g , p a , p e , r s, s p b t, l t, m t, a s, f s, l b 1, 3 , 4 , 5 , 1 0, 1 1, 1 4, 1 8, 1 9, 2 1, 2 4, 25 , 2 6, 28 , 3 0, 3 1, 3 2, 3 3, 3 5, 3 8, 3 9, 4 0, 4 4, 4 5, 5 1, 52 , 5 3, 6 4, 6 5, 6 7, 6 8, 6 9, 7 2, 7 3, 7 5, 7 8 46 .1 5 sy no ec a su ri na m a (l in na eu s, 1 76 7) a g , a m , a t, c a , p a , sa , u e a m , a p, b a , d f, g o , m a , m g , m t, m s, p a , pb , p e , r o , r r , s p b t, l t, m t, a s, l b , q 7, 6 , 8 , 1 0, 1 6, 2 2, 2 7, 3 1, 3 4, 3 7, 43 , 4 7, 4 8, 51 , 5 6, 5 7, 5 8, 7 0, 7 1, 7 2, 7 4 26 .9 2 sy no ec a vi rg in ea (f ab ri ci us , 1 80 4) a m , c a a m , a p, m a , p a , p i, r o , r r b t, l t, m t, a s, l b 20 , 2 2, 2 7, 4 3, 5 1, 5 6, 5 7, 5 8, 7 1, 7 4 12 .8 2 t ab le 2 . s oc ia l w as p sp ec ie s, st ud ie d en vi ro nm en t, sa m pl in g m et ho ds a nd p ub lic at io n re fe re nc es sa m pl in g sp ec ie s d iv er si ty in b ra zi l f ro m 1 98 2 to 2 01 5: s tu di ed e nv ir on m en t: a g – a gr oe co sy st em ; a m – a m az on ra in fo re st ; a t – a tla nt ic ra in fo re st ; c a – c aa tin ga ; e u – e uc al yp tu s; m a – m an gr ov e; p a – p an ta na l; r e – r es tin ga ; r f – r ip ar ia n fo re st ; r g – r oc ky g ra ss la nd ; s a – s av an na ; u e – u rb an e nv ir on m en t. st ud ie d st at e: a c – a cr e; a p – a m ap á; a m – a m az on as ; b a – b ah ia ; d f – d is tr ito f ed er al ; g o – g oi ás ; m a – m ar an hã o; m t – m at o g ro ss o; m s – m at o g ro ss o do s ul ; m g – m in as g er ai s; p a – p ar á; p r – p ar an á; p i – pi au í; r s – r io g ra nd e do s ul ; r o – r on dô ni a; r r – r or ai m a; s c – s an ta c at ar in a; s p – sã o pa ul o. s am pl in g m et ho ds : a s – a ct iv e se ar ch ; b t – b ai t t ra ps ; f – f og gi ng te ch ni qu e; f s – fl ow er s ea rc h; l b l iq ui d b ai t; l t – l ig ht t ra ps ; m o – m öe ri ck e; m t – m al ai se t ra p; q – q ua dr an t; st – sh ue y t ra p; t t – t ra y t ra ps . r ef er en ce s: s ee t ab le 1 . 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togni oc, locher ga, giannotti e, silveira ot (2014). the social wasp community (hymenoptera, vespidae) in an area of atlantic forest, ubatuba, brazil. check list, 10(1): 10–17. urbini a, sparvoli e, turillazzi s (2010). social paper wasps as bioindicators: a preliminar research with polistes dominulus (hymenoptera: vespidae) as a trace metal accumulator. chemosphere, 64: 697-703. von ihering r (1904). as vespas sociais do brasil. revista do museu paulista, 6:9-309. zikán jf (1949). o gênero mischocyttarus saussure (hym, vespidae), com a descrição de 82 espécies novas. parque nacional do itatiaia. boletim do parque nacional do itatiaia, 1:125. zikán jf (1951). beitrage zur kenntnis der arten der gattung polistes latreille, 1802, nebest beschreibung. arten dusenia, 2: 225-236. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.4308sociobiology 66(3): 515-522 (september, 2019) evaluation of inter and intraspecific differences in the venom chemical compositions of polybia paulista wasps and ectatomma brunneum ants using ftir-pas introduction the hymenoptera, one of the most significant orders of insects, is composed of wasps, bees, ants, and sawflies. within this order, some individuals are eusocial, which have as essential characteristics the division of reproductive labor, cooperative parental care, and overlapping generations (wilson, 1971). an essential characteristic of hymenoptera, specifically the aculeata, is the presence of a sting apparatus that is used to inject venom into prey and enemies. this sting apparatus, which contains a venom reservoir, is a modification of the ovipositor device from ancestral groups. it consists of abstract wasp and ant venoms represent complex mixtures of compounds such as proteins, peptides, lipids, vasoactive amines, enzymes, besides of amino acids and compounds with low molecular weight. these venoms have as function capture prey and assist in defense of their colonies. parameters such as geographic location, genetics, environment, sex, age, season of the year, and diet determine the composition of the venom. however, studies on the compositional variability of venom are still limited due to the difficulty in obtaining samples and the complexity of these substances. this work describes the use of the fourier transform infrared photoacoustic spectroscopy (ftir-pas) to investigate inter and intraspecific variability in the venom chemical composition of the social wasp polybia paulista (von ihering 1896) and the ant ectatomma brunneum (smith 1858). the results reveal significant differences in venom chemical composition between the ant and wasp, even for samples obtained from the same environment. the genetic component, therefore, seemed to be the predominant factor determining the compounds present. the findings also showed that exogenous factors, such as diet, could also be responsible for intraspecific differences, especially in wasps. the ftir-pas technique proved to be a reliable way of assessing intraand interspecific differences in social hymenoptera venom chemical composition. sociobiology an international journal on social insects a mendonça1,3, rc bernardi2,3, elb firmino2,3, lhc andrade2, sm lima2, wd fernandes1, wf antonialli-junior1,2,3 article history edited by fernando noll, unesp, brazil received 09 december 2018 initial acceptance 28 january 2019 final acceptance 24 june 2019 publication date 14 november 2019 keywords eusocial insects; exogenous component; genetic component; vespidae, formicidae. corresponding author angelica mendonça programa de pós-graduação em entomologia e conservação da biodiversidade universidade federal da grande dourados rodovia dourados-itahum, km 12 cidade universitária, c.p. 533 cep 79804-970, dourados-ms, brasil. e-mail: angel_bio1@yahoo.com.br two different regions: a glandular portion, where the venom and other substances are produced, and a motor portion composed of chitinous and muscle structures that act together in the injection of venom and sting protrusion and extrusion (manzoli-palma & gobbi, 1997). its primary function is to capture prey, but it has become effective in the intimidation of vertebrates. also, it has acquired a significant defensive role, notably in social species (macalintal & starr, 1996). venom is defined as a secretion produced by specialized glands, which is capable of altering or disrupting the normal biological or physiological processes of the target organism (casewell et al., 2013). in addition to the offensive and defensive functions, volatile compounds present in glandular 1programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil 2 centro de estudo em recursos naturais, programa de pós-graduação em recursos naturais, universidade estadual de mato grosso do sul, dourados-ms, brazil 3-laboratório de ecologia comportamental labeco, universidade estadual de mato grosso do sul, dourados-ms, brazil research article wasps a mendonça et al. – inter and intraspecific differences between venoms of the wasp and ant using ftir-pas516 venom can act as an alarm pheromone (schmidt, 1982; orivel et al., 2001). mateus (2011) observed that the parachartergus fraternus social wasp uses its venom to mark a new site for colony foundation. thus, venom (or its components) also plays an essential role as chemical communication signal in wasp societies. hymenopteran venom consists of complex organic molecules of proteins, peptides, lipids, vasoactive amines (octopamine, dopamine, and histamine), and enzymes such as phospholipases, hyaluronidases, and phosphatases (edstrom, 1992; lima & brochetto-braga, 2003). besides, it is composed predominantly of amino acids and complex mixture of compounds with low molecular weight (palma, 2006). the venom can have antiseptic activities against bacteria, fungi, protozoa, and viruses (orivel et al., 2001; baracchi & tragust, 2017). the antiseptic function of venom is part of the innate immunity of hymenoptera and its initial evolutionary value is associated with the need to minimize potential contamination by prey (orivel et al., 2001; baracchi & tragust, 2017). it has been found that variation in venom chemical composition among organisms is related to their geographic position, genetic, environmental conditions, sex, age, diet and also depends on seasonal factors (daltry et al., 1996; badhe, 2006; abdel-rahman et al., 2009, 2011). polybia paulista (von ihering, 1896) social wasp is widely distributed in south america (richards, 1978). this genus builds phragmocyttarus nests without any pedicel, so the first comb is established directly in the substrate, with a protective jacket built around it (carpenter & marques, 2001). the ectatomma brunneum ant (smith, 1858) is widely distributed in the neotropical region, occurring from panama to argentina (kempf, 1992). it is a generalist predator species, with welldeveloped stingers. the nests are constructed on the ground, with more significant abundance in open fields or degraded areas including pastures, crops, grassland, unpaved roads, and clearings (gomes et al., 2009). although there is a significant number of studies with the venom of p. paulista (souza et al., 2009; santo et al., 2011; pinto et al., 2012; hoshina et al., 2013; jacomini et al., 2013; gomes et al., 2014; dias et al., 2014, 2015; leite et al., 2015; vinhote et al., 2017; perez-riverol et al., 2018) and some with the venom of e. brunneum (pluzhnikov et al., 2014; touchard et al., 2015; aili et al., 2016) only a few of them investigated intraspecific differences in venom compositions, for example, p. paulista (mendonça et al., 2017) and e.brunneum bernardi et al. (2017). as the occurrence of these two species is ample, they end up being good study models to evaluate intraspecific differences of their venoms. in the last few years, fourier transform infrared photoacoustic spectroscopy (ftir-pas) has proven to be a reliable technique for using in comparative studies of the venoms of social hymenoptera (bernardi et al., 2017; mendonça et al., 2017), as well as their cuticular hydrocarbons (antonialli-junior et al., 2007, 2008; neves et al., 2012, 2013; bernardi et al., 2014; soares et al., 2014; torres et al., 2014). due to the versatility of photoacoustic detection, which enables analysis of the optical absorption of materials that are opaque in the infrared region, it can be applied to different systems and various sizes of the sample. the mid-infrared absorption band is sensitive to the vibration of molecular chemical groups, enabling the identification and distinction of molecular radicals and the types of chemical bonds present in a sample. the present study aimed to evaluate interand intraspecific variability in the venom chemical composition of the p. paulista social wasp and the e. brunneum ant using ftir-pas. materials and methods hymenoptera sample collection five colonies of p. paulista wasps (fig 1a) were collected in dourados city, brazil. three of these colonies were located at the universidade estadual de mato grosso do sul (at 22º11’50.1”s, 54º55’48.5”w), denoted as “area a”, and two colonies were located in the urban area of dourados (at 22º14’38.8”s, 54º49’36.6”w and 22º15’23.5”s, 54º47’59.1”w), denoted as “area b”. the colonies were collected by wrapping plastic bags around the nests and then detaching the nests from the substrates to which they were fixed. analyses were performed using between 25 and 40 wasps from each colony. the wasps were killed and preserved by freezing, and the samples were stored in eppendorf vials for subsequent analysis. the e. brunneum ants (fig 1b) were collected in the same areas (a and b) where the wasps were sampled. active individuals were captured randomly at different sites to obtain an adequate representation of the populations, considering possible genetic variability. for each area, 100 forager ants were collected and the contents of their glands were analyzed. the ants were killed and preserved by freezing, in the same methodology described for the wasps. extraction and fourier transform infrared photoacoustic spectroscopy (ftir-pas) the ftir-pas analyses were performed using a thermo nicolet nexus 670 spectrophotometer, with photoacoustic detection at 4000-400 cm-1. during the experiment, the spectrometer was purged with dry air to remove water vapor. the photoacoustic cell was purged with helium gas before each reading. for standardization, a piece of black carbon was used to collect reference infrared spectra, with a new background measurement being performed every 100 minutes. spectra were obtained as the average of 128 scans at a resolution at 16 cm-1. in order to enable comparative analysis, the area of each spectrum was determined, and one of them was used to normalize all the spectra. sociobiology 66(3): 515-522 (september, 2019) 517 the wasps and ants were prepared for the infrared analyses by dissection with tweezers under a stereomicroscope. the glands and their storage reservoirs were extracted by pulling the stinger, and they were immediately placed into a photoacoustic cell crucible and analyzed, to avoid any risk of protein degradation. for each spectrum, five venom reservoirs were inserted into the photoacoustic cell, and 15 spectra were recorded for each colony. statistical analysis the interand intraspecific differences in venom chemical composition of the two species were evaluated by discriminant function analysis (dfa) (quinn & keough, 2002). all absorptions peaks were identified based on literature data (lin-vien et al., 1991; smith, 1999). the main absorption intensities in the infrared spectrum were selected, with the wavenumbers of the absorption peaks as variables and the peak intensities as experimental values. all peaks were integrated and normalized by the sum of the area. the statistical procedure identified the linear combination of variables that provided the best explanation of the differences among the analyzed groups. the wilks’ lambda statistic parameter was used as a measure of the differences among the groups: values close to zero indicate there is no overlap among the groups, and values close to 1 indicate considerable overlap (manly, 2008). the squared mahalanobis distance was used to detect the differences between the species and the collection areas. results the average photoacoustic infrared spectra obtained for the venoms of p. paulista wasps and of e. brunneum ants sampled in different areas are shown in fig 2. by the spectra, it is possible to identify three regions (highlighted in the figure) that can be used as biomarkers of wasps and ants venoms. the highlighted regions represent the most important differences observed for intraand interspecific interpretation, which were more precisely confirmed by data from fig 3. fig 1. wasp polybia paulista (a) and ant ectatomma brunneum (b). (mendonça, a.) fig 2. average infrared absorption spectra of venom glands and reservoirs from polybia paulista wasps and ectatomma brunneum ants. the highlighted regions represent the most important differences observed for intraand interspecific interpretation, which were more precisely confirmed by data from figure 3. the first range of the fig 2, indicated as (1) around 970-1190 cm-1, corresponds to the absorption of the functional group c-h with vibrational mode bending in the plane. it is possible to observe that in this region, the wasp venom has absorption intensity higher than the ant venom. this effect is an indication that the venom chemical composition of p. paulista wasp has in its composition aromatic rings in higher concentration than the venom of e. brunneum ant. following, observing (2) in 1360-1490 cm-1 range, which corresponds to the absorption of the dc-n functional group from amide i and dcnh from amide ii (approximately at 1396 and 1457 cm-1, respectively with vibrational modes defined as stretching and stretch-bend in cis configuration), it is possible to note that the ratio among the intensities of dc-n and dcnh (dc-n/dcnh ratio) in the wasps venom spectra is different, if they are compared with the same ratio observed in the ant spectra. although the amide i and ii concentrations a mendonça et al. – inter and intraspecific differences between venoms of the wasp and ant using ftir-pas518 are different among the studied species, it is impossible to note any difference among the studied area comparing within the species. finally, the indication (3) in 28003000 cm-1 range shows that ch composition is strongly different among ants and wasps. by the e. brunneum ant venom spectra is noted that the absorption peak at 2877 cm-1, due to nsch3 (symmetric stretching), is less intense if compared to the absorption peaks at 2931 and 2962 cm-1, which correspond to asymmetric stretching of nasch2 and nasch3, respectively. in opposite, the venom of p. paulista wasp exhibits the peak at 2931 cm-1 with higher absorption intensity than the e. brunneum ant venom. it is interesting to note that there is difference in the relationship between the intensities i1 and i2 of the peaks 2931 and 2962 cm-1, respectively: for p. paulista species, the i1/ i2 relationship is > 1 and for area a is higher than area b. in opposite way, for e. brunneum the relation i1/i2 is < 1 (fig 2). this was observed for both a and b areas, and suggests that these vibration modes can be used as a reference for distinguishing venom for wasp and ants. to interpret the infrared data statistically, the mentioned absorption intensities together with other peaks of amide, like 1542 cm-1 due to stretch-bend (trans) of dcnh, 1650 cm-1 corresponding to stretching of c=n, and 3293 cm-1 due to stretching of nsn-h, were used as experimental data, using the wavenumbers as the variables, it was possible to construct a matrix to discriminate the four investigated groups. fig 3 shows the obtained scatter plot by the statistical analysis. it reveals that the differences among venom chemical composition of ants and wasps are significant (wilks’ lambda = 0.005; f = 14.347; p < 0.001). the first canonical root explains 98.8% of the differences, being the most important root to discriminate among the venom of wasps and ants. among the investigated infrared absorption peaks, those at 1041, 1079 and 1157 cm-1 were significant to separate the groups. these peaks are located in the highlighted region (1) in fig 2. the second canonical root differentiates the area a from b for p. paulista wasps, while the venom chemical composition of e. brunneum ants is similar among the areas a and b. the squared mahalanobis distances obtained with the infrared experimental data are listed in table 1, confirming that the p. paulista wasps and e. brunneum ants venom chemical composition are significantly different (p < 0.001). fig 3. scatter plot showing the differences in venom chemical composition among polybia paulista wasps and ectatomma brunneum ants collected in two different areas (a and b) of dourados-ms. p. paulista area a p. paulista area b e. brunneum area a e. brunneum area b p. paulista area a 0.000 18 164 135 p. paulista area b 18 0.000 170 142 e. brunneum area a 164 170 0.000 5 e. brunneum area b 135 142 5 0.000 table 1. squared mahalanobis distances between groups of polybia paulista wasps and ectatomma brunneum ants, comparing the venom chemical composition among samples from two different areas of dourados-ms. discussion the results indicate that the chemical composition of venom of wasps and ants are significantly different, independent if they were sampled in areas a or b. this can be seen as well the absorption spectra in the medium infrared (fig. 2) as by the statistical interpretation of the data (fig. 3). comparing the spectra of p. paulista wasps collected in areas a and b it is possible to notice a high degree of similarity. this can be confirmed by fig. 3, where the ellipses of wasps and ants are practically aligned with respect to the first canonical root, which accounts for 98.8% of the data. this observation can be explained by genetic factors since the insects belong to different families. this result has been observed previously in works concerning the influence of the environmental component on venom chemical composition (tsai et al., 2004; badhe et al., 2006; abdelrahman et al., 2011; cologna et al., 2013). studies with social hymenoptera have also shown that genetic factors and age can be responsible for the venom chemical composition and some compounds are characteristic of particular species (ferreira-junior et al., 2010; cologna et al., 2013). similar variation in venom chemical composition among species has also been reported for snakes (daltry et al., 1996). in this case, it was observed that snakes born in captivity had venom chemical composition similar to those of wild snakes collected in the same region of origin of those kept in captivity. the authors suggest that the association between the venom chemical composition and the prey is an inherited characteristic and is not induced by the environment. sociobiology 66(3): 515-522 (september, 2019) 519 however, venom is a complex mixture of substances and the variation in its chemical composition cannot be attributed to any single factor. studies have found that in addition to genetic factors, environmental variables, such as geographic location and climate, can also affect the distribution of food and therefore influence the diets of the organisms (tsai et al., 2004; badhe et al., 2006; abdel-rahman et al., 2009, 2011; cologna et al., 2013). hence, environmental factors also exert an influence on venom chemical composition. the studies of mendonça et al. (2017) analyzing the venom of p. paulista and bernardi et al. (2017) with e. brunneum, both by ftir-pas, highlight the environmental effect on the chemical profiles of venom. in addition, mendonça et al. (2017) discuss that both environmental and genetic factors can affect the composition of p. paulista venom, since venom of wasps from nesting colonies in closer areas are more similar than those of more distant ones, because the former have the closest kinship level and share the same type of environments. variations in venom chemical composition according to the location was also observed in maurus palmatus scorpions (abdel-rahman et al., 2009). studies analyzing only the protein portion of the p. paulista venom also show variation in the chemical composition between different colonies (dias et al., 2014), as well as studies with ants that found variation in the venom composition of colonies of the same population (aili et al., 2017), in which the authors suggested genetic polymorphism or small environmental variations between areas. the results presented here corroborate the findings of badhe et al. (2006), who evaluated the role of genetic factors in different populations of red scorpions (mesobuthus tamulus) and suggested the existence of genetic variation among the populations. nonetheless, it concludes that environmental conditions also significantly affected the lethality of the venom. it can be seen from fig 3 that there is no overlap among the ellipses for the venom chemical composition of wasp from different colonies (areas a and b), opposing to the observed for the ants venom chemical composition. this effect can be explained by the difference in food resources utilized by each species. wasps often prey on the larvae of lepidoptera (giannotti et al., 1995; prezoto et al., 2006; bichara-filho et al., 2009; fernandes et al., 2010), which should result in differences between the two sampled areas regarding venom chemical composition. on the other hand, e. brunneum ant as well as other ants of this genus, prey on a greater diversity of organisms (overal, 1986; giannotti & machado, 1992; marques et al., 1995), leading to a potential dilution of the differences between the two sampled areas. an additional and essential point to be considered is that wasps fly further from the nest to forage. therefore, the diversity of plants visited by wasps is likely to be greater, which might influence the venom chemical composition. the influence of diet on the composition of e. brunneum venom was also reported by bernarid et al. (2017). in a study with dinoponera quadriceps ants sampled from four different regions, cologna et al. (2013) noted that the venoms of individuals collected in the closest areas showed greater similarity when compared to the venom of specimens collected in far regions. among the 335 compounds detected in the venom, only 48 were shared by all four colonies, indicating that the environment highly influenced the venom chemical composition. in fact, studies have shown that some animals can synthesize the venom from precursors or sequester the chemical elements of the environment through diet to compose their venom (savitzky et al., 2012). therefore, environmental as well as genetic factors significantly influence the venom composition of different species of animals. thus, it is clear that the two species synthesize significantly different elements to compose their venoms. these differences have a genetic basis, however, as reported in the literature, exogenous factors may also be determinant to compose the differences between the venom of different populations of a species, especially in this case that of social wasps. conclusions we can conclude that there is a significant difference between the venom chemical composition of ants and wasps and the genetic component seems to be predominant in determining the compounds of venom. this proves that the venom chemical composition varies among families in the hymenoptera. on the other hand, exogenous factors such as the diet in different environments can be responsible for intraspecific differences, particularly in social wasps. finally, statistical analysis applied to the infrared absorption data obtained by ftir-pas proved to be a reliable and efficient methodology for evaluating differences in the venom chemical composition of social hymenoptera. therefore, the methodology can be used as an auxiliary taxonomic tool. acknowledgements the authors thank dr. orlando t. silveira (museu paraense emílio goeldi, mpeg) for the identification of the species, fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect), coordenação de aperfeiçoamento de pessoal de nível superior (capes), and universidade estadual de mato grosso do sul (uems). wfaj, lhca, and sml acknowledge research grants from conselho nacional de desenvolvimento científico e tecnológico (cnpq). authors’ contribution the original idea for this research was conceived by a mendonça and wf antonialli junior. a mendonça, a mendonça et al. – inter and intraspecific differences between venoms of the wasp and ant using ftir-pas520 rc bernardi, elb firmino, lhc andrade and sm lima performed the experiments and analyzed the data. a mendonça drafted the manuscript. all the authors reviewed and approved the final manuscript. references abdel-rahman, m.a., omran, m.a.a., abdel-nabi, i.m., ueda, h. & mcvean, a. 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(1971). the insect societies. cambridge, belknap press, 548p. doi: 10.13102/sociobiology.v66i1.3434sociobiology 66(1): 88-96 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 pollen analysis of the post-emergence residue of euglossa bees (apidae: euglossini) nesting in an urban fragment introduction the euglossini tribe (hymenoptera: apidae) is distributed only in the neotropical region and it is divided into five genera: euglossa latreille, eulaema lepeletier, and eufriesea cockerell consist of pollen-gathering species, whereas aglae lepeletier and serville as well as exaerete hoffmannsegg are cleptoparasites (michener, 2007). the euglossa genus has the largest number of species, with 129 species already described (nemésio & rasmussen, 2011), which are distributed from mexico to northern argentina and in the caribbean islands (cameron, 2004; michener, 2007). in the corbiculate apidae, euglossini bees are the only species that are not, strictly speaking, social. certain euglossa species are solitary or can form community nests with various females born in the nest (garófalo et al., 1998; michener, 2007; augusto & garófalo, 2011). initially, when a euglossa abstract euglossini bees are considered pollinators of a wide variety of plants in the neotropical region, but little is known about their floral preferences. in this study, we identified the botanical species used as pollen and nectar sources by three euglossa species, euglossa cordata (linnaeus), e. townsendi cockerell, and e. securigera dressler using pollen residue found in brood cells from trap nests installed in an urban fragment in são luís, maranhão, brazil. in 14 analyzed e. cordata nests, 23 pollen types were observed, in seven e. townsendi nests, ten pollen types were observed, and in one nest of e. securigera, six morphotypes were identified. solanum (solanaceae), zanthoxylum (rutaceae), mimosa pudica l. (fabaceae), and chamaecrista (fabaceae) pollen types were common to all three bee species. a principal component analysis showed 83.04% variability on the first two axes, demonstrating substantial similarity among the samples. solanum, mimosa pudica, and zanthoxylum were the principal components in the ranking. larger diversity values (mean = 0.80) in some samples indicated that the species gathered resources in a heterogeneous manner; this is consistent with the findings of other studies of euglossa. in general, the bees exhibited overlapping niches with regard to the most abundant pollen grains in the nests, but the females showed individual plasticity when gathering floral resources. sociobiology an international journal on social insects rs pinto, ag silva, mmc rêgo, pmc albuquerque article history edited by denise alves, esalq-usp, brazil received 06 may 2018 initial acceptance 22 june 2018 final acceptance 12 september 2018 publication date 25 april 2019 keywords floral preferences, nectar, trap nests, orchid bees. corresponding author patrícia maia correia de albuquerque universidade federal do maranhão departamento de biologia laboratório de estudos sobre abelhas av. dos portugueses nº 1966 cep: 65080-805, são luís-ma, brasil. e-mail: patemaia@gmail.com female finds an appropriate place to build her nest, she forms the first cell made with resin, where she stores the larval food. next, she lays her egg and closes the cell, then begins to build new brood cells (garófalo et al., 1998; ramírez et al., 2002). nests of euglossa can be aerial or constructed in different types of cavities found in natural substrates; additionally, they can also be hosted by artificial man-made structures (augusto & garófalo, 2004; cameron, 2004). the use of trap nests has enabled researchers to become acquainted with the different species of bees nesting in such cavities in different localities, as well as providing information on species biology (garófalo et al., 1993). this includes the food resources utilized by adult bees and larvae (arriaga & hernández, 1998; silva et al., 2016). euglossini bees are pollinators of a wide variety of plants and can fly long distances in tropical forests (janzen, 1971). to date, the resources that these bees use for food are not well known; most previous studies are based on universidade federal do maranhão, são luís, maranhão, brazil research article bees sociobiology 66(1): 88-96 (march, 2019) 89 direct observations of flowers (ramírez, 2002; rocha-filho et al., 2012). some studies have identified the floral resources through pollen analysis of larval food; e.g. euglossa atroveneta dressler (arriaga & hernández, 1998), e. annectans dressler (cortopassi-laurino et al., 2009), e. viridissima friese, e. dilemma bembé & eltz (villanueva-gutierrez et al., 2013), e. nigropilosa moure (otero et al., 2014), and e. townsendi cockerell (silva et al., 2016). in spite of this, there is a need for more research into the different biomes in which these bees are found. euglossa cordata (linnaeus), e. securigera dressler, 1982, and e. townsendi are widely distributed in the neotropics. in brazil, they occur in the humid amazon forest and in vast areas in the northeast and the central-south (rêbelo et al., 2003). in the state of maranhão, these species are widely distributed throughout the various phytogeographic regions (rebêlo & silva, 1999). maranhão is undergoing rapid deforestation, and is located in a region of transition between the amazon forest, the cerrado of the central plains, and the semi-arid caatinga in the northeast (rêbelo & silva, 1999). therefore, it is necessary to conduct further research to understand the biodiversity and ecological relationships of these flora and fauna. to this end, the aim of the present study was to determine the floral resources utilized by e. cordata, e. townsendi, and e.securigera via the analysis of post-emergence pollen residue collected from trap nests in an urban fragment. material and methods study area the study was conducted at the 120.95-ha integral protection conservation unit in the sítio do rangedor state park, located in the municipality of são luís, maranhão, brazil (decree no. 23303/2007). the park serves as a climate control and water capture unit for the municipality (sema, 2017). the vegetation in rangedor state park is a remnant fragment of the amazon forest. estimates indicate that there are more than 120 botanical species in the region, distributed in 52 plant families, with syagrus oleracea (mart.) becc. (arecaceae), attalea speciosa mart ex. spreng. (arecaceae), pithecellobium sp. (fabaceae), and himatanthus drasticus (mart.) plumel (apocynaceae) constituting approximately 45% of the tree population (sema, 2017). the city of são luís is located on the northern coast of maranhão. it has a humid tropical climate, with average temperatures ranging from 26 to 28 ºc. annual rainfall reaches 2000 mm3, with the period of most intense rainfall from january to june, and the dry season from july to december (silva et al., 2008). nest sampling trap nests were installed in the study area for one year (april 2010 to april 2011). the nest diameters were 8, 10, 12, 14, and 16 mm. they consisted of two pieces of wood joined together with adhesive tape (3 cm high x 3 cm wide x 16 cm long), with a 10 cm deep longitudinal slit. in all, 500 nests were used, with 100 of each diameter. they were organized into 100 clusters protected by pet bottles, with one nest of each diameter in the cluster. the blocks were placed in two sampling units, selected according to the physiognomic characteristics of the vegetation. area i (2°9’54”s, 44°15’51”w) was selected for its open vegetation, mainly composed by shrubs and herbaceous plants, and greater exposure to the sun. area ii (2°29’48”s, 44°16’07”w) was chosen for its naturally regenerating vegetation and shaded environment; this area corresponds to dry land forest, which represents 50% of the plant cover of the park (sema, 2017). in each area, we installed 50 blocks on the branches of trees, hanging at a height of 1.5 m from the ground. every two weeks, the trap nests were inspected. those in which bees had established were taken to the laboratory until the individuals emerged. they were then sacrificed in chambers with ethyl acetate, pinned, and labeled. the euglossa genus, identified by a specialist, was common in the trap nests in both area i and area ii. the species were found to belong to the subgenus euglossa (euglossa) and to be highly similar morphologically. identification was made possible through the males that were born in the nests, since they were the only ones with identification keys. pollen samples each nest occupied by bees was considered a sample (e. cordata: ec.01–ec.14, e. townsendi: et. 01–et.07, and e. securigera: es.01). after all the individuals had emerged, the residual pollen grains and feces found in the brood cells were obtained so that the floral resources utilized by the bees. the pollen material was prepared using the acetolysis method described by erdtman (1960). under the optical microscope, pollen types were identified and 500 pollen grains were counted per sample. in this manner, we were able to determine the frequency of pollen type occurrence. when possible, we identified pollen grains at the species level, and we also used the pollen type criterion for genus or family, in which pollen grains that present a set of very similar morphological characteristics comprise a group of plants that share the same characteristics (barth, 1989). pollen slides from the palynotheca of the federal university of maranhão and pollen catalogues were used to identify the botanical affinity of the pollen types (e.g. roubik & moreno, 1991; silva et al., 2014; lorente et al., 2017). data analysis to verify the similarity of pollen types among samples, multivariate analysis was conducted using principal components analysis (pca). first, a matrix with the absolute values for each pollen type present in the samples was transformed into natural logarithms. next, ranking was conducted using a covariance matrix and employing the past program. variability among samples was expressed using the first two axes of the pca. rs pinto, ag silva, mmc rêgo, pmc albuquerque – pollen analysis of the post-emergence residue of euglossa90 the amplitude of the trophic niche, assessed by the species richness (s), the shannon diversity index (h’), and the equitability index (j’), were calculated for all samples. these indexes were utilized to determine the diversity and degree of uniformity of pollen types found in the bee nests (odum, 1988). results three species of euglossini nested in trap nests in the sítio do rangedor state park. fourteen nests were occupied by e. cordata, which nested in cavities with three diameters (12 mm: three nests, 14 mm: five nests, 16 mm: six nests). in total, eight of these nests were in area ii, while six were in area i. e. townsendi occupied nests of four different diameters (10 mm: one nest, 12 mm: two nests, 14 mm: three nests, 16 mm: one nest) and only in area ii. the only nest of e. securigera was found in a 16mm cavity in area i. during the study period, nesting occurred in all months except from october to december 2010 (tables 1 and 2). twenty-eight pollen types were found in the 22 analyzed nests, belonging to 14 botanical families and 24 genera; only three pollen types were not identified. the botanical family with the largest number of types was fabaceae (10), followed by arecaceae and myrtaceae (2 each); all others included only one pollen type. euglossa cordata samples ec.01 ec.02 ec.03 ec.04 ec.05 ec.06 ec.07 ec.08 ec.09 ec.10 ec.11 ec.12 ec.13 ec.14 mean date may/10 jun/10 jun/10 jul/10 jul/10 jul/10 jul/10 jul/10 aug/10 aug/10 sep/10 jan/11 apr/11 apr/11 diam. (mm)/site 14 / i 16 / ii 12 / ii 12 / ii 14 / ii 16 / i 12 / ii 14 / ii 16 /i 16 / ii 14 / i 14 / i 16 / i 16 / ii number of cells 4 7 4 3 3 4 4 5 3 5 5 6 6 9 pollen types frequencies of pollen types (%) arecaceae copernicia prunifera 0.4 0.2 0.04 mauritia 0.6 0.04 asteraceae wedeliapilosa 0.4 0.2 0.04 bignoniaceae bignonia 0.6 0.8 4.4 1.4 0.51 euphorbiaceae dalechampia 0.8 0.6 1.2 10.4 1.2 3.8 1.29 fabaceae chamaecrista 0.2 0.4 28.2 7.6 7.8 2.2 1.4 3.41 cassia 9.0 0.64 centrosema brasilianum 6.4 0.46 copaifera 0.2 0.01 delonix 0.4 0.2 25.8 1.89 dioclea 0.4 0.03 mimosa pudica 41.4 13.4 0.8 28.6 68.4 60.8 3.8 37.8 33.4 4.4 20.91 mimosa type 2 0.8 0.06 schrankia leptocarpa 0.6 1.0 0.4 0.14 swartzia 0.4 0.03 myrtaceae eugenia 50.2 3.59 myrcia fallax 2.4 0.17 rubiaceae manettia 0.4 0.03 rutaceae zanthoxylum 11.4 15.0 34.8 8.8 38.4 31.2 11.4 18.4 7.0 12.2 7.6 23.4 15.69 solanaceae solanum 88.0 43.4 1.0 89.6 2.6 0.4 26.4 97.8 64.4 41.6 65.6 63.4 57.6 67.0 50.63 verbenaceae lantana 2.2 1.8 0.29 not identified ni1 0.2 0.01 ni2 1.2 0.09 table 1. frequency of occurrence of pollen types in euglossa cordata nests. sociobiology 66(1): 88-96 (march, 2019) 91 twenty-three pollen types occurred in the nests of e. cordata, belonging to 10 families and 20 genera; two were not identified (table 1). for e. townsendi, 10 pollen types were identified, distributed among seven families and nine genera; one was not identified. e. securigera, with just one nest evaluated, exhibited six pollen types belonging to five families and six genera (table 2). the most frequent types of pollen grains for e. cordata were solanum (50.63%) (fig 1a), mimosa pudica l. (20.91%) (fig 1b), zanthoxylum (15.69%) (fig 1c), eugenia (3.59%) (fig 1d), chamaecrista (3.41%), delonix (1.89%), and dalechampia (1.29%) (fig 1e); the remaining 16 pollen types constituted less than 1% of grains counted (table 1). for e. townsendi, solanum (55.06%), mimosa pudica (31.49%), zanthoxylum (11.97%), and copernicia prunifera (mill.) h.e. moore (1.06%) were the most frequent pollen types; the remaining six pollen types represented less than 1%. for e. securigera, the most important pollen types were solanum (56%), zanthoxylum (23.80%), byrsonima (11.40%), mimosa pudica (4.40%), and micinia (3.80%) (table 2). the pollen grains in common for all three bee species were chamaecrista, m. pudica, zanthoxylum, and solanum. sixteen pollen types were exclusive to e. cordata, three to e. townsendi, and only one to e. securigera (table 3). euglossa townsendi e. securigera samples et.01 et.02 et.03 et.04 et.05 et.06 et.07 mean es.01 date jun/10 jul/10 aug/10 mar/11 mar/11 apr/11 apr/11 feb/11 diam. (mm) / site 14 / ii 14 / ii 16 / ii 14 / ii 10 / ii 12 / ii 12 / ii 16 / i number of cells 4 8 6 8 9 9 9 3 pollen types frequency of pollen types (%) arecaceae copernicia prunifera 7.4 1.06 fabaceae chamaecrista 1.0 0.14 0.60 mimosa pudica 56.4 30.8 1.8 89.0 2.2 40.2 31.49 4.40 schrankia leptocarpa 0.8 malpighiaceae byrsonima 0.4 0.06 11.40 malvaceae hibiscus 0.2 0.03 melastomataceae micinia 3.80 rutaceae zanthoxylum 38.6 4.0 13.4 3.2 13.2 5.6 5.8 11.97 23.80 sapindaceae cupania diphylla 0.4 0.06 solanaceae solanum 4.2 65.2 84.8 83.4 54.2 93.6 55.06 56.00 not identified ni3 0.2 0.03 table 2. frequency of occurrence of pollen types in euglossa townsendi and e. securigera nests. table 3. exclusive pollen types (*) shared (+) by euglossa cordata, e. townsendi and e. securigera nesting in trap nests in the sítio do rangedor state park, maranhão, brazil. e. cordata e. townsendi e. securigera e. cordata * mauritia. wedelia pilosa. bignonia. dalechampia. cassia. centrosema brasilianum. copaifera. delonix. dioclea. mimosa tipo 2. eugenia. myrcia fallax. manettia. lantana. not identified (ni 1. ni 2) + copernicia prunifera. chamaecrista. mimosa pudica. schankia leptocarpa. zanthoxylum. solanum + chamaecrista. mimosa pudica. zanthoxylum. solanum e. townsendi * hibiscus. cupania diphylla. not identified (ni 3) + chamaecrista. mimosa pudica. byrsonima. zanthoxylum. solanum e. securigera * micinia rs pinto, ag silva, mmc rêgo, pmc albuquerque – pollen analysis of the post-emergence residue of euglossa92 fig 1. photomicrographs of certain pollen types observed in the nests of euglossa females. (a) solanum, (b) mimosa pudica, (c) zanthoxylum, (d) eugenia, (e) dalechampia and (f) centrosema brasilianum (scale bars = 10 μm). the pollen type samples were ordered using pca according to the similarity of their occurrence. figure 2 shows that the variability covered 83.04% on the first two pca axes, which demonstrates high similarity among the samples. solanum, m. pudica, and zanthoxylum were the principal components used for ranking the samples. the aggregate of types, on the left side of the pca, indicates species with few grains or those encountered in just a few samples. the ec.10 sample had the highest richness of pollen types (10). in contrast, samples ec.01, et.02, ec.06, et.03, and et.06 had the lowest number of pollen types (3 each). on average, there were 4.95 types per nest [standard deviation (sd): ± 1.78]. fig 2. ranking of principal components analysis (pca) for nests of euglossa cordata (ec.01 to ec.14), euglossa townsendi (et.01 to et.07) and euglossa securigera (es.01). (● = pollen type). sociobiology 66(1): 88-96 (march, 2019) 93 as for the shannon diversity index, sample ec.10 presented the highest value (h’ = 1.371) and ec.08 had the lowest (h’= 0.135) (0.80 ± 0.34) (fig 3). the mean equitability index was 0.52 (± 0.18); sample et.06 had the highest value (j’ = 0.782) and ec.08 had the lowest value (j’ = 0.084) (fig 3). discussion the predominance of nests obtained from e. cordata was due to the fact that the species is one of the most frequent species in the ecosystems of the northern region of the state of maranhão (rêbelo et al., 2003) and in several other regions, while e. townsendi and e. securigera are less frequent in surveys (e.g. neves & viana, 1997; mendes et al., 2008; ramalho et al., 2009). in spite of the difference in the number of nests obtained, the floral resources identified in this analysis contribute to the knowledge of the plants used by the bee species studied, particularly for e. securigera, as the food resource usage of which has not been previously assessed. the euglossa species are considered generalists when foraging for food resources. nevertheless, they show preferences for certain plant families. overall, regardless of the area of the park in which they nested, e. cordata, e. townsendi, and e. securigera exhibited important source sharing. this result was similar to those of a previous study on e. viridissima and e. dilemma, which demonstrated overlapping niches (villanueva-gutierrez et al., 2013). in this study, 23, 10, and 6 pollen types were observed in the nests of e. cordata, e. townsendi, and e. securigera, respectively, which was similar to that observed by boff and alves-dos-santos (2018) in nests of e. cordata (20 pollen types), but less than that observed in nests of e. atroveneta (74) (arriaga & hernández, 1998), e. annectans (74) (cortopassilaurino et al., 2009), e. viridissima, and e. dilemma (45) (villanueva-gutierrez et al., 2013). however, a smaller number of nests was assessed in the sítio do rangedor state park, especially for e. securigera¸ for which only a single nest was obtained for the analysis. furthermore, the pollen analyzed in our study was the residue present in the brood cells. even so, the floral preferences were quite similar to those found inprevious studies. even for e. townsendi, which had 10 pollen types identified in seven nests, we observed the lowest degree of exploited species richness compared to a previous study of the same bee species (silva et al., 2016), in which 21 pollen types were found in six nests. because the sítio do rangedor state park consists of a forest fragment with reduced plant diversity (sema, 2017), the number of botanical species exploited by e. townsendi in the locality may have been limited. however, both studies showed that this bee species has botanical preferences and, in the state of são paulo, the greatest preference was for miconia chamissois naudin (melastomataceae), followed to a lesser extent by solanum palinacanthum dunal (solanaceae) (silva et al., 2016). plant species of solanaceae (solanum), fabaceae (chamaecrista and cassia), and melastomaceae (micinia) have flowers with poricidal anthers; in order to obtain pollen from these flowers, bees must powerfully vibrate their wings fig 3. richness (s), diversity index (h’) and equitability (j’) of pollen grains found in euglossa cordata (ec.01 – ec.14) and euglossa townsendi (et.01 to et.07) nests. rs pinto, ag silva, mmc rêgo, pmc albuquerque – pollen analysis of the post-emergence residue of euglossa94 in a behavior known as “buzz pollination” (buchmann, 1985; nunes-silva et al., 2010). furthermore, other polleniferous species without poricidal anthers are important for euglossa (arriaga & hernandez, 1998), such as eugenia (myrtaceae) and m. pudica (fabaceae), which exhibited high frequencies in some samples. as for the latter, m. pudica is a ruderal species with flowering throughout the year but greater abundance in months with higher rainfall, and is considered crucial for the survival of several bee species (marques et al., 2011). if euglossini females do have preferences regarding pollen gathering, their search for nectar seems to be less specific (lópez-uribe et al., 2008). plants with nectar are frequently cited as being visited by euglossini females and males (janzen, 1971; ramírez et al., 2002; rocha-filho et al., 2012), and some foraging by females was recorded in this study: e.g. c. prunifera (arecaceae), wedelia pilosa baker (asteraceae), bignonia (bignoniaceae), centrosema brasilianum (l.) benth., copaifera, delonix and dioclea (fabaceae), hibiscus (malvaceae), manettia (rubiacae), zanthoxylum (rutaceae), cupania diphylla vahl (sapindaceae), and lantana (verbenaceae). the dalechampia (euphorbiaceae) pollen type, which occurred in six samples of e. cordata, may be singled out as the main source of floral resin for nest building (armbruster, 1993; opedal et al., 2016). plants of this botanical genus are frequently pollinated by euglossini bees, which has been well documented in the literature (armbruster & webster, 1981; sazima et al., 1985; opedal et al., 2016). the pollen of byrsonima, which was shared between e. townsendi and e. securigera (with a relative frequency of 11.40% for the latter), is not commonly gathered by these bees. the malpighiaceae family is frequently visited by ‘oilgathering bees’ because of the use of floral oil to feed the larvae (rêgo & albuquerque, 2006); however, they are also visited for pollen gathering, as recorded among eulaema (ramírez et al., 2002). thus, nothing prevents euglossa females from also visiting these plants in search of this resource. the only nest of e. securigera occurred in area i, and based on our observations, the locality contains many byrsonima shrubs.on the other hand, e. townsendi also collected pollen from this plant, which is significant because it only nested in area ii, where plants of this genus were unusual; this indicates that the bees were not limited to the immediate vicinity of their nesting area for foraging. according to janzen (1971), plants foraged by euglossini bees provide high quality rewards as they flower over long periods of time. thus, these bees habitually and repeatedly visit the same species when it is flowering. an example of a species foraged over a long period of time is zanthoxylum (rutaceae), which was very important for bee species in this study, possibly as a source of both pollen and nectar. additionally, the pollen of rutaceae (citrus) was observed over nine months in e. atroveneta samples, although with low pollen frequency (arriaga & hernández, 1998). on the other hand, shorter flowering species can also be visited by these bees, as shown by the rare occurrence of these pollen types in the samples. notably, the pollen types of solanum, m. pudica, and zanthoxylum were observed in most months during which the nests were established. they were of greater or lesser importance depending on the sample employed as the major pollen in the pca ranking. even samples of nests obtained in the same month, i.e. both e. cordata and e. townsendi, varied in terms of importance of the principal pollen type. according to arriaga and hernández (1998), an interesting behavior of these bees is that they can forage the same plants at the moment of nidification, but the way the resource is exploited can change; furthermore, individual preferences may differ among females, indicating a plasticity at the individual level. this is consistent with the findings of cortopassi-laurino et al. (2009), where the contents of brood cells were not homogeneous, and the polylectic behavior of euglossa was observed over a matter of days. the higher diversity values in some samples show that the bees gathered resources in a heterogeneous manner, with up to ten pollen types in nest ec.10. the mean diversity value of 0.80 was similar to that found for e. atroveneta (arriaga & hernández, 1998), which was 0.83. as for the uniformity of the samples, et.06 had the greatest index, which shows that the female of that nest foraged resources in a more homogenous manner (solanum 54.2%, m. pudica 40.2%, and zanthoxylum 5.6%). on the other hand, sample ec.08, with the least uniformity (0.084), indicates that the female gathered her resources in a heterogeneous manner, while having the greatest preference for solanum (97.8%). this variation in the uniformity and diversity of pollen grains encountered in the nests can also be explained by the individual-level plasticity of female foraging. the euglossini bees have considerable ability to fly several distances in search of food resources (janzen, 1971). thus, we consider that, although the bees founded their nests in different areas (open vegetation vs. shaded areas), this does not preclude species from foraging for their resources throughout the region of the park and surrounding area and sharing their preferred resources. the euglossa bees that nest and forage in the rangedor state park contribute to the pollination of the plant species present in the area, and by their capacity for displacement, can mediate gene flow with individuals located in other urban fragments, which is of prime importance for the conservation of vegetation. acknowledgements the authors wish to thank professor angela corrêa from botanical institute of são paulo for her collaboration in the identification of pollen types, as well as denilson martins for identifying the specimens of euglossa. this work was supported by the maranhão foundation for support for research and scientific development (fundação de amparo à pesquisa e ao desenvolvimento científico e tecnológico do maranhão – fapema) through grants to the rsp and pmca. sociobiology 66(1): 88-96 (march, 2019) 95 references armbruster, w.s. 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(2016). high prevalence of miconia chamissois (melastomataceae) pollen in brood cell provisions of the orchid bee euglossa townsendi in são paulo state, brazil. apidologie, 47: 855-866. doi: 10.1007/s13592-016-0441-y silva, c.i., imperatriz-fonseca, v.l., groppo, m., bauermann, s.g., saraiva, a.m., queiroz, e.p., evaldt, a.c.p., aleixo, k.p., castro, j.p., castro, m.m.n., faria, l.b., ferreira-caliman, m.j., wolff, j.l., paulino-neto, h.f. & garófalo, c.a. (2014). catálogo polínico das plantas usadas por abelhas no campus da usp de ribeirão preto. ribeirão preto: holos, 153p. villanueva-gutierrez, r., quezada-euan, j. & eltz, t. (2013). pollen diets of two sibling orchid bee species, euglossa, in yucatán, southern mexico. apidologie, 44: 440-446. doi: 10.1007/s.13592-013-0194-9 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.2878sociobiology 65(4): 777-779 (october, 2018) special issue biological data of stingless bees with potential application in pesticide risk assessments brazil has the greatest global richness of stingless bees (michener, 2013) and is also the world’s largest consumer of pesticides (mapa, 2017). therefore, it is essential that ecotoxicological information on native fauna be made available to agencies responsible for environmental assessments of pesticides, represented in the case of brazil by the instituto brasileiro do meio ambiente e dos recursos naturais renováveis (ibama). according to ibama (2017), among the main limitations for pesticide risk assessments for native bees is the lack of basic data on the biology of these bees, such as food consumption at different life stages and the mass of the individuals. in addition, the safety of the current use of the exotic species apis mellifera l. as a substitute for the other native species of brazil in these assessments is questioned. abstract due to the current practice of intensive pesticide use in brazil on crops with flowers that are attractive to bees, biological information about brazilian native bees is required in order for public authorities that are responsible for environmental safety to use them for calculations of risk assessments. thus, the present study aimed to obtain biological data on stingless bees: melipona scutellaris latreille, scaptotrigona postica latreille and tetragonisca angustula latreille. the food consumed by larvae and by adults and the mass of forager workers were obtained. the results provide essential inputs for the risk assessment of stingless bee exposure to pesticides, which combined with information about the concentrations of these substances in crops with flowers that are attractive to bees, may be used in risk calculations. sociobiology an international journal on social insects as dorigo1, as rosa-fontana1, if camargo2, rcf nocelli2, o malaspina1 article history edited by cândida aguiar, uefs, brazil received 04 february 2018 initial acceptance 02 may 2018 final acceptance 14 august 2018 publication date 11 october 2018 keywords model organism, pesticides, pollinators, risk assessment, sensitivity to pesticides, stingless bees’ larval food. corresponding author annelise rosa-fontana universidade estadual paulista júlio de mesquita filho unesp centro de estudos de insetos sociais av. 24-a, 1515 bela vista código postal 199 cep: 13506900 rio claro-sp, brasil. e-mail: annesouzar@gmail.com melipona scutellaris latreille, scaptotrigona postica latreille and tetragonisca angustula latreille are interesting species to be used as model organisms for risk assessment. the first two are considered as priorities for pesticide risk assessments, according to the recent list of species selection, which consider several criteria such as geographic distribution, association with crops, and importance as pollinators, among others (pires & torezani, 2018). a species of the scaptotrigona genus (s. postica) was chosen by both its broad distribution in brazil and its intermediate size among the other species. in addition to composing the native brazilian fauna, there is a need for biological data on stingless bee species, which may be useful for carrying out risk assessments for native species (ibama, 2017). based on the data gaps mentioned, the present study aimed to provide 1 universidade estadual paulista júlio de mesquita filho (unesp), rio claro, são paulo, brazil 2 universidade federal de são carlos (ufscar), araras, são paulo, brazil short note as dorigo, as rosa-fontana, if camargo, rcf nocelli, o malaspina – stingless bees and pesticide risk assessments778 data to fill the gaps in knowledge about aspects related to nutrition and body mass of stingless bees. these results will provide essential inputs for the risk assessments of exposure of stingless bees to pesticides. to quantify the larval food consumed by brood cell, as well as the daily syrup consumption and the individual mass of worker foragers, three non-parental colonies of each species, maintained at universidade estadual paulista, rio claro, brazil, were used. to measure the amount of larval food consumed per brood cell, the food of 20 newly oviposited cells per colony was collected by random selection. before food collection, the eggs were removed from cells. with an automatic micropipette, the food from each colony was homogenized in eppendorf microtubes with volumetric graduation. they were weighed in an analytical balance before and after the food collection. the total volume and mass were divided by the number of brood cells to estimate the amount of individual food per cell. to estimate the food consumption and individual mass of worker foragers, individuals from the three species (total number/species = 30 for each test) were collected at the entrances of the nests when they left to forage. the mass of the individuals was verified using an analytical balance. to check the food consumption, 10 individuals from the same colony were placed on plastic perforated pots for air circulation. eppendorf microtubes syrup feeders (sugar and water 1:1,v:v), simulating nectar, were placed on the pots, and the bees fed ad libitum. for 4 days, every 24 hours, the feeders were weighed using an analytical balance in order to verify daily consumption. in addition to the establishment of the data for the stingless bee species, the same data for honey bees were taken from literature, in order to compare them. the mean values of the larval food consumed by brood cell, the daily syrup consumption by forager workers, the individual mass of the bees and the data taken from literature are shown in the table 1. the data on the amount of food consumed by larvae and syrup by forager workers, combined with information on the concentrations of these substances in agricultural crops with flowers that are attractive to bees, may be used for risk assessments. understanding this relationship is extremely relevant since brazil currently bases its protocols on standards of the oecd (organisation for economic co-operation and development), which uses a. mellifera as a model organism. these protocols were adopted by the united states epa (environmental protection agency) for the schematization of risk assessment, which was used as a reference for the current brazilian risk assessment scheme. nevertheless, among the main uncertainties noted by ibama (2017), the use of a. mellifera stands out, since stingless bees are part of the native pollinator fauna. one way to remedy this issue would be to carry out a comparison of a. mellifera with native species regarding the toxicity of and exposure to pesticides, and our work provides important information that contributes to this evaluation. the differences between the groups are evident in biological parameters, such as their larval feeding systems. while a. mellifera nurse workers progressively deposit the food to the brood, in stingless bees, they do it all at once, depositing all the food that will be consumed (velthuis, 1998). in addition, the volume of food consumed by the brood also differs between groups: in a. mellifera, each larva consumes a total of 160 µl (aupinel, 2005), and in the stingless bee species, m. scutellaris, s. postica and t. angustula, each larva ingests 130.58, 25 and 5.6 µl, respectively. the nectar consumption by the forager workers and the individual mass of the bees also give rise to the uncertainty about using a. mellifera as a study model in brazil. according to cresswell et al. (2012), one forager worker of a. mellifera consumes a daily average of 50 mg of nectar and weighs on average of 79.1 mg, showing greater food consumption and body mass comparing to stingless bees (table 1). in addition to that, the mass of individuals presented herein may be a relevant factor in the sensitivity of the different species to a particular type of pesticide, for example. comparing the sensitivity of bee species to exposure to pesticides, a meta-analysis study conducted by arena and sgolastra (2014) indicated the sensitivity of stingless bees was in general higher than bumblebees. in addition, devillers et al. (2003) suggested the sensitivity of different bee species is generally inversely proportional to their mean body mass. volume of larval food by brood cell (µl) mass of larval food by brood cell (mg) daily syrup consumption by worker (mg) individual mass of forager worker (mg) melipona scutellaris 130.58 ± 0.52 158.04 ± 5.18 35.06 ± 9.15 76.65 ± 2.9 scaptotrigona postica 25 ± 1.14 32.5 ± 0.8 9.45 ± 2.41 17.02 ± 0.35 tetragonisca angustula 5.6 ± 0.77 7.96 ± 1.03 7.23 ± 1.74 4.1 ± 0.37 apis mellifera 160† 50† 79.1±4.9† †the data of volume of larval food by brood cell, daily syrup consumption by worker and individual mass of forager worker were obtained, respectively, from aupinel (2005), cresswell et al. (2012) and blatt and roces (2001). table 1. amount of larval food consumption by cell, daily syrup consumption and individual weight of forager workers.± indicates standard deviations values. “–” indicates absence of data. sociobiology 65(4): 777-779 (october, 2018) special issue 779 nevertheless, it is interesting to observe that, in the workers of the species of smaller body size of this study, t. angustula, the amount of food consumed corresponds to twice the body mass, compared to the other species of stingless bees and also to a. mellifera. this is an interesting point to be considered for future investigations regarding species-sensitivity. in this work, some information required for the characterization of pesticide exposure risks to stingless bees was presented. although there is a high richness of stingless bee species in brazil, little is known about their basic biological data (ibama, 2017). for the inclusion of native species in risk assessments of pesticides in brazil, it is necessary that this approach continues in future studies. acknowledgements we thank capes and fundação de amparo à pesquisa do estado de são paulo (fapesp process nº 2016/00328-4) for financial support. authors’ contribution all of the authors conceived research. authors 1, 2 and 3 conducted experiments. authors 1 and 2 analysed data and conducted statistical analyses. all of the authors wrote, read and approved the manuscript. references arena, m. & sgolastra, f. (2014). a meta-analysis comparing the sensitivity of bees to pesticides. ecotoxicology, 23:324334. doi: 10.1007/s10646-014-1190-1 aupinel, p., fortini, d., dufour, h., tasei, j., michaud, b., odoux, j. & pham-delegue, m. (2005). improvement of artificial feeding in a standard in vitro method for rearing apis mellifera larvae. bulletin of insectology, 58:107. blatt, j. & roces, f. (2001). haemolymph sugar levels in foraging honeybees (apis mellifera carnica): dependence on metabolic rate and in vivo measurement of maximal rates of trehalose synthesis. journal of experimental biology, 204: 2709-2716. cresswell, j.e., page, c.j., uygun, m.b., holmbergh, m., li, y., wheeler, j.g. & tyler, c.r. (2012). differential sensitivity of honey bees and bumble bees to a diet ary insecticide (imidacloprid). zoology, 115: 365-371. doi:10.1016/j.zool.20 12.05.003 devillers, j., decourtye, a., budzinski, h., pham-delegue, m.h., cluzeau, s. & maurin, g. (2003). comparative toxicity and hazards of pesticides to apis and non-apis bees. a chemometrical study. sar and qsar in environmental research, 14:389-403. doi: 10.1080/10629360310001623980 ibama (2017). avaliação de risco de agrotóxicos para insetos polinizadores e lacunas de conhecimento. http:// www.ibama.gov.br/phocadownload/noticias/noticias2017/ nota_tecnica_avaliacao_de_risco_de_agrotoxicos.pdf. (accessed date: 30 june, 2017) mapa. (2017). agrotóxicos. ministério da agricultura, pecuária e abastecimento. http://www.mma.gov.br/segurancaquimica/agrotoxicos. acessed date:10 july 2017. michener, c.d. (2013). pot-honey: a legacy of stingless bees. new york: springer. 654 p. pires, c.s.s. & torezani, k.r.s (2018). seleção de espécies de abelhas nativas para avaliação de risco de agrotóxicos, brasília: ibama. 84 p. velthuis, b.j., & velthuis, h.h.w. (1998). columbus surpassed: biophysical aspects of how stingless bees place an egg uprighton their liquid food. naturwissenschaften, 85: 330-333. _hlk515368606 _hlk515368663 _goback doi: 10.13102/sociobiology.v62i1.28-33sociobiology 62(1): 28-33 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 recognition and aggression of conspecific and heterospecific worker in acromyrmex subterraneus subterraneus (forel) (hymenoptera: formicidae) introduction an important feature of social species is the existence of elaborate recognition systems that facilitate cooperation among members of a group, because they help to maintain the integrity of the society, reducing the negative impact caused by predators, competitors and social parasites (crosland, 1990; fishwild & gamboa, 1992; crowley et al., 1996; wiley, 2013). ants maintain coherence within the colony through chemical recognition of nestmates, allowing workers to differentiate other individuals between friend and foe through specific chemical signatures of each colony (lenoir et al., 2001; akino, 2008; guerrieri et al., 2009). leaf-cutting ants (hymenoptera: formicidae: myrmicinae: attini) are dominant herbivores in the neotropics (hölldobler & wilson, 1990). acromyrmex mayr, with 62 species and nine subspecies (forti et al., 2006; souza et al., 2007; brandão et al., 2011), causes economic losses in agriculture and is one of the most important pests of forest plantations (boulogne et al., 2012). abstract aggressive behavior is important for social insects because it makes possible for the colony to defend itself and the offspring from the action of invasive species. we studied the recognition and aggressiveness of the leaf-cutting ant acromyrmex subterraneus subterraneus (forel) to co-specific workers from other nest and heterospecific workers of acromyrmex subterraneus molestans santschi, acromyrmex subterraneus brunneus (forel) and acromyrmex niger (smith); and queens of their social parasite acromyrmex ameliae de souza, soares and della lucia. workers of other species were placed in contact with those of a. subterraneus subterraneus for three minutes and during this period the behavioral interactions were quantified. the aggressiveness index (ai) for each agonistic encounter was obtained. acromyrmex subterraneus subterraneus workers exhibited greater aggressiveness against heterospecific than against conspecific competitors. aggressiveness is connected to differences in the chemical profiles, which are larger in heterospecifics colonies. sociobiology an international journal on social insects 1 universidade federal de viçosa, viçosa, mg, brazil 2 universidade do estado de santa catarina, lages, sc, brazil article history edited by gilberto m. m. santos, uefs, brazil received 12 june 2014 initial acceptance 10 july 2014 final acceptance 29 november 2014 keywords behavior, competition, defense, etogram, heterospecifics, leaf-cutting ant. corresponding author terezinha maria castro della lucia departamento de biologia animal universidade federal de viçosa 36570-000 viçosa, minas gerais, brazil e-mail: tdlucia@ufv.br these ants are models for behavioral studies because of their elaborate social organization and interactions between individuals of the colony and with other organisms (camargo et al., 2006). workers of different sizes exhibit different behaviors and division of labor (polyethism), which maximizes foraging (hart et al., 2002). however, there are activities of the colony that are still poorly studied, such as some interactions between the workers of a nest. social insects exhibit aggressive behavior towards individuals from other nests and invading organisms (hölldobler & michener, 1980). this behavior allows the nests to store resources (hamilton, 1972) and protect their workers and offspring from external threats (pollock & rissing, 1989; sakagami, 1993; mori et al., 2000; allon et al., 2012). the recognition of nestmates occurs through a specific “label” called “colony odor” (crozier & dix, 1979). this label is made of cuticular hydrocarbons in social insects (lenoir et al., 2001a) and allows ants to recognize nestmates and distinguish them from intruders (hölldober & michener, research article ants tg pikart1,2, pg lemes1, wc de c morais1, jc zanuncio1, tmc della lucia1 sociobiology 62(1): 28-33 (march, 2015) 29 1980). soil characteristics, food source and other nest materials participate in the formation of this label (heinze et al., 1996). diet is more important than the influence of the queen and their genetic origin in the formation of odor in nests of acromyrmex subterraneus subterraneus (forel) (hymenoptera: formicidae) and, consequently, in the recognition of the ants (richard et al., 2004). the fungi recognition behavior (viana et al., 2001) and trophallaxis (moreira et al., 2006) were studied in this subspecies. however, several types of behavior of a. subterraneus subterraneus workers such as aggressiveness have not yet been evaluated. we hypothesized that workers of a. subterraneus subterraneus exhibit greater aggressiveness against heterospecifics than against conspecific ones. the objective of this study was to describe and compare the behavior of recognition and aggression of a. subterraneus subterraneus workers in relation to workers of: (1) a. subterraneus subterraneus from different colonies; (2) acromyrmex subterraneus molestans santschi; (3) acromyrmex subterraneus brunneus (forel); (4) acromyrmex niger (smith); and their social parasite queens (5) acromyrmex ameliae de souza, soares & della lucia. material and methods study site and species the study was conducted at the laboratório de formigas cortadeiras (lfc) of the universidade federal de viçosa (ufv) in viçosa, minas gerais state, brazil in november 2011. acromyrmex subterraneus subterraneus, a. subterraneus molestans, and a. niger naturally co-occur in the region of viçosa. the social parasite a. ameliae occurs only in nests of a. subterraneus subterraneus and a. subterraneus brunneus in another region of minas gerais state, at about 300 km from viçosa. all individuals used in the experiment were obtained from a total of ten colonies maintained in the lfc at 24 ± 2ºc and ± 75% rh; and fed with ligustrum japonicum thunb. (oleaceae) and acalipha wilkesiana müll. arg. (euphorbiaceae) leaves. one of the colonies of a. subterraneus subterraneus was collected on the ufv campus and kept under the same conditions as the others, in a separate room. recognition and agressiveness evaluation the aggressiveness and recognition among workers were assessed in a neutral arena, a plastic container (250 ml) with the inner upper half covered with talc. five workers of a. subterraneus subterraneus were randomly selected and introduced into the arena and, after 10 minutes, another individual was introduced as the following treatments: nestmate worker (control), co-specific worker of nest collected in the field (ass); worker of a. subterraneus molestans (asm); worker of a. subterraneus brunneus (asb); winged queens of a. ameliae (aa); and worker of a. niger (an). we avoided the use of a. subterraneus subterraneus workers that were weak, sick or injured in the experiment, because the use of non-healthy individuals may produce inconsistent results (roulston et al., 2003). agonistic encounters were observed for 3 minutes and the evaluated behavioral interactions were divided into six different levels of aggression (modified from suarez et al., 1999 and velasquez et al., 2006): 0 ignore; 1 quick or repeated antennation on another individual; 2 retreat towards contrary direction after contact; 3 intimidation by opening the mandibles; 4 biting and gaster flexing; and 5 hold/dominate the other individual and try to remove it from the arena. the latency, the time between the release of the stimulus and the first reaction of the workers of a. subterraneus subterraneus, was also recorded. each arena was washed with distilled water and neutral detergent and then wiped with 70% alcohol after each evaluation to remove any substance that could influence the ants’ behavior. ten replicates were conducted for each treatment. the frequency of each behavior was determined for each treatment and an aggressiveness index (ai) was calculated (velasquez et al., 2006): where obi is the observed behavior i, fi is the frequency of each behavior during three minutes of observation and n is the total number of interactions observed during the period. statistical analysis the difference in total frequencies in each agression level per treatment was determined using the total number of behaviors per treatment. the average proportion of each independent aggression level and the aggressiveness index were compared among treatments using analysis of variance (anova) and the fisher test (lsd) with the statistica 7.0 software (statsoft inc., tulsa, usa). fig 1. aggressiveness index (ai) resulting from observed behaviors during interactions among workers of a. subterraneus subterraneus and conspecific nestmate workers (control), conspecifics collected from a colony in the field (ass), workers of a. subterraneus molestans (asm), a. subterraneus brunneus (asb), queens of a. ameliae (aa) or workers of a. niger (an) with their standard errors. different letters indicate significant differences by post hoc fisher test (lsd). tg pikart et al. recognition and aggression in a. subterraneus subterraneus30 results the aggressiveness index (ai) was higher in encounters between workers of a. subterraneus subterraneus and those of a. subterraneus molestans, a. subterraneus brunneus and a. niger (fig 1) compared to the other treatments. acromyrmex subterraneus subterraneus workers showed only antennation and ignored their nestmates (table 1). the workers in control treatment showed agression levels between 0 and 1, while all other treatments showed all aggression levels (fig 2). level 0 was more common in the control, followed by treatment with the parasite queen a. ameliae (fig 2). aggression levels 1, 2 and 5 did not differ among treatments (fig 2). levels 3 and 4 were more frequent in treatments with workers of a. subterraneus brunneus and a. niger, respectively. table 1. observed behaviors during the interactions among workers of a. subterraneus subterraneus and conspecific nestmates (control), conspecifics of colony collected in the field (ass), workers of a. subterraneus molestans (asm), a. subterraneus brunneus (asb), queens of a. ameliae (aa) or workers of a. niger (an). behavior control ass asm asb aa an ignore 74 14 6 2 49 2 antennation 79 103 120 130 107 123 retreat 0 6 19 16 6 10 intimidation 0 37 80 79 41 56 bite 0 28 49 24 13 47 gaster flexing 0 20 23 20 2 35 grab/dominate 0 7 10 8 1 7 remove 0 0 1 2 5 2 total 153 215 308 281 224 282 fig 2. average ratio of six aggression levels (0-5) in six treatments: conspecific nestmate workers (control); conspecific from colony collected in the field (ass), workers of a. subterraneus molestans (asm), a. subterraneus brunneus (asb), queens of a. ameliae (aa) or workers of a. niger (an) with their standard errors. different letters indicate significant differences by post hoc fisher test (lsd). sociobiology 62(1): 28-33 (march, 2015) 31 discussion the presence of workers of a. subterraneus molestans, a. subterraneus brunneus and a. niger induced more aggression than the other intruder workers. workers of a. subterraneus subterraneus made distinction between conspecific nestmates and non-nestmates, being more aggressive when in contact with non-nestmates, unlike the observed for a. subterraneus molestans (souza et al., 2006). this is probably related to the ants diet. colonies of a. subterraneus subterraneus maintained in the laboratory and the field had different food sources; this was the condition reported by souza et al. (2006). although the diet of colonies of a. subterraneus subterraneus, a. subterraneus molestans, a. subterraneus brunneus and a. niger kept in the laboratory was the same, the high aggressiveness of workers on these heteroepecifics is a sign that other factors, such as genetic influence, interfere with the composition of the “colony odor” (vanzweden et al., 2010; krasnec & breed, 2013). the low aggressiveness of workers of a. subterraneus subterraneus when in contact with winged queens of a. ameliae differs from that observed for workers of the genus temnothorax (hymenoptera: formicidae), which responded more aggressively to the presence of workers of the social parasite protomognathus americanus (emery) (hymenoptera: formicidae) than to conspecifics or heterospecifics (pamminger et al., 2011; scharf et al., 2011). the tolerance observed in this experiment may be related to the phenomenon called “chemical insignificance”. just after emergence, immature ants are devoid of cuticular chemicals, acquiring the odor of the colony and integrating to other workers later (lenoir et al., 1999). the weak signal of young workers allows them to be more easily accepted in other colonies than older workers (lenoir et al., 2001a), similar to what can have occurred with the queens of a. ameliae. adaptations in the morphology of these insects and the production of similar chemicals are possibly linked to nonaggression towards these queens (martin et al., 2010; bauer et al., 2009). chemical camouflage, which occurs when the social parasite acquires the odor by direct contact with the host when entering the host colony, may also be occurring (lenoir et al., 2001b). males of bombus vestalis vestalis (fourcroy) (hymenoptera: apidae) lack the morphological and chemical adaptations that females have to infiltrate the host colony, but they produce a repellent odor that prevents them from being attacked by workers of the host species (lhomme et al., 2012). it should be investigated if a similar phenomenon occurs with winged queens of a. ameliae. leaf-cutting ants defend their territories against conspecific and heterospecific intruders, protecting food resources and their offspring. the species a. subterraneus subterraneus, a. subterraneus molestans, a. subterraneus brunneus and a. niger co-exist in their natural range, besides having habits that make them potential competitors for feeding resources. on the other hand, the possible similarity of the cuticular chemical profile of different colonies of a. subterraneus subterraneus tested and the strategy of the parasite a. ameliae to infiltrate in the host colony may explain the lower intensity of aggression suffered by the latter. workers of a. subterraneus subterraneus are more aggressive when in contact with heterospecifics competitors compared to the co-especifics and the social parasite a. ameliae. the mechanisms involved in the reduced aggression towards these treatments have to be evaluated to have a better understanding of the ecological process between competitors and social parasites in these ants. acknowledgments we are greateful to the laboratory technician manoel ferreira for his help in performing the experiments. we also thank "conselho nacional de desenvolvimento científico e tecnológico (cnpq)", "coordenação de aperfeiçoamento de pessoal de nível superior (capes)" and "fundação de amparo à pesquisa do estado de minas gerais (fapemig)" for financial support. references akino, t. 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(hymenoptera: formicidae): a new social parasite of leaf-cutting ants brazil. insect science, 14: 251-257. suarez, a.v., tsutsui, n.d., holway, d.a. & case, t.j. (1999). behavioral and genetic differentiation between native and introduced populations of the argentine ant. biological invasions, 1: 1-11. vanzweden, j.s., brask, j.b., christensen, j.h., boomsma, j.j., linksvayer, t. & d’ettorre, p. (2010). blending of heritable recognition cues among ant nestmates creates distinct colony gestalt odours but prevents within-colony nepotism. journal of evolutionary biology, 23, 1498-1508. velásquez, n., gómez, m., gonzález, j. & vásquez, r.a. (2006). nest-mate recognition and the effect of distance from the nest on the aggressive behaviour of camponotus chilensis (hymenoptera: formicidae). behaviour, 143: 811-824. sociobiology 62(1): 28-33 (march, 2015) 33 viana, a.m.m., frézard, a., malosse, c., della lucia, t.m.c., errard, c. & lenoir, a. 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(2013). specificity and multiplicity in the recognition of individuals: implications for the evolution of social behavior. biological reviews of the cambridge philosophical society 88: 179-195. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i4.3408sociobiology 66(4): 597-601 (december, 2019) genetic characterization of melipona subnitida stingless bee in brazilian northeast melipona subnitida is highly adapted to extreme environmental conditions and can survive under long drought periods, since during water and resource scarcity, the bees reduce the colony size to a minimum number of brood and workers. thus, the species maintains only essential tasks for the nest, being an important mechanism to save the resources stored during the bloom of rainy periods (maia-silva et al., 2014; 2015). assuming the climate change predictions for northeastern brazil (marengo et al., 2011) in the semi-arid region, where average reductions in precipitation rates of up to 40–50% are expected (pbmc, 2013), the study bees resistant to these conditions, such as m. subnitida, is noteworthy. considering the importance of this species regarding resistance to extreme climatic conditions, distributional modeling abstract the study of m. subnitida, a stingless bee well adapted to extreme environmental conditions, is noteworthy once the northeastern brazil faces climate changing predictions in which the precipitation rates are expected to decrease, and the average of temperatures to increase. the well-studied populations are limited to the caatinga biome, where the species was considered endemic. however, the occurrence of this species has been reported in contrasting environments from arid region, such as mangrove and sandbanks in maranhão state. our primary goal was to characterize samples from these different environments and compare them with previously studied populations. we identified a unique mitochondrial haplotype per region. the haplotype found in lençóis maranhenses national park was exclusive from this location and differed regarding the amino acid sequence when compared to the literature presented haplotypes from caatinga, which might be related with different evolutionary processes in the distinct environments, though further studies are needed to confirm. sociobiology an international journal on social insects mm barbosa1, v bonatti2, js galaschi-teixeira3, mmc rêgo1, tm francoy4 article history edited by solange augusto, ufu, brazil received 02 may 2018 initial acceptance 11 june 2018 final acceptance 23 september 2019 publication date 30 december 2019 keywords conservation, habitat, haplotypes, pollinators, mitochondrial dna. corresponding author marcela de matos barbosa universidade federal do maranhão departamento de biologia laboratório de estudos sobre abelhas av. dos portugueses nº 1966 cep: 65080-805. são luís-ma, brasil. e-mail: marmbarbosa5@gmail.com studies have suggested a shift in the potential occurrence area of m. subnitida and its main resource plant for the near future (maia et al., 2014; 2015; giannini et al., 2017). even though the impact of climate change on geographic distribution of this native bee is predictable, studies dedicated to understand how its genetic variability is distributed are still scant. m. subnitida is usually considered endemic from the caatinga (zanella & martins, 2002), but it has been observed in biomes with distinct landscapes from semi-arid region, such as in the coast of maranhão, in lençois maranhenses national park (pnlm) (rêgo & albuquerque, 2006) and in ilha grande dos paulinos (rêgo et al., 2017) (fig 1). these areas, unlike caatinga biome, are composed of sand dunes and lakes, annual rainfall ranging from 1600 to 1800 mm (ibama, 2002) and surrounded by mangrove. 1 universidade federal do maranhão, são luís, brazil 2 faculdade de medicina de ribeirão preto universidade de são paulo, ribeirão preto, brazil 3 faculdade de filosofia, ciências e letras de ribeirão preto universidade de são paulo, ribeirão preto, brazil 4 escola de artes, ciências e humanidades universidade de são paulo, são paulo, brazil short note mm barbosa et al. – low genetic diversity of melipona subnitidain maranhão state598 the populations of m. subnitida present in such areas differed regarding the tree species used as nesting sites often found in the caatinga region (câmara et al., 2004; bruening 2006; rêgo et al., 2017). in order to evaluate if differences among bees are restricted to behavioral characteristic or if they are reflected in the genetic signatures, we have genetically compared populations from maranhão with mitochondrial haplotype data available in the literature. we searched for natural nests and sampled 28 workers of m. subnitida in pnlm (20) and in ilha grande dos paulinos (8) and the nests were approximately 1km apart from each other. the bees were stored at -20ºc and dna was extracted from antennae of one bee per colony using chelex method (walsh et al., 1991). for analysis of the cytochrome c oxidase i (coi) gene partial sequence were used primers mtd6 and mtd9 (simon et al., 1994). for pcr it was used 5μl of dna, 2.5 μl of each primer, 25 μl of 10x pcr buffer and 15 μl of nuclease-free water. genetic material was amplified, sequenced and analyzed as described in bonatti et al. (2014). we amplified a 436pb of coi gene fragments from 28 individuals. these sequences were compared to 11 haplotypes from bonatti et al. (2014), which sampled individuals from maranhão, piauí, ceará and rio grande do norte states of northeastearn brazil. haplotypes network was built using network software v.4.6 (polzin & daneshmand, 2003) and mega software v.7.0.14 (tamura et al., 2011) to calculate the distances among haplotypes using tamura 3-parameter with gamma distribution (t92+g). our results found one haplotype per sampled region. nonetheless, the haplotype h4 (in ilha grande dos paulinos) was shared with mossoró-rn, fortaleza-ce, jandaíra-rn and parnaíba-pi populations (table 1) previously analyzed by bonatti et al. (2014) and identified likely as ancestral haplotype. on the other hand, the pnlm had its unique haplotype, h12, not shared with any other areas (fig 2). this haplotype diverges from h7 (found in barreirinhas-ma and parnaíba-pi) and h10 (found in fortaleza-ce) by a single mutational step (fig 2a). according to the genetic proximity between h7 and h12 haplotypes, showed by dendrogram (fig 2b) and geographic distance between barreirinhas and pnlm, we believe the h7 may have originated h12. bonatti et al. (2014) also observed one exclusive mitochondrial haplotype per locality as occurred in pnlm. this pattern may be associated with the process of colonization of new areas in meliponini by one or few original colonies (miranda et al., 2016). furthermore, the low dispersal ability of daughter colonies during swarming process, the dependence upon mother colony, as well as the queen philopatry may help to explain these patterns (nogueira-neto, 1954; engels & imperatriz-fonseca, 1990). other researches using population genetic approaches with different stingless bee species have fig 1. map of brazil highlighting the collecting sites of m. subnitida in brazilian northeast at different biomes. the greendots represent the locations of sampling in maranhão (pnlm and ilha grande dos paulinos) and the black dots represent the samples used from genbank. haplotypes localities h1 mossoró; parnaíba h2 jandaíra h3 jandaíra h4* mossoró; jandaíra; parnaíba; fortaleza; ilha grande dos paulinos h5 areia branca; jandaíra; parnaíba h6 jandaíra h7 barreirinhas; parnaíba h8 fortaleza h9 barreirinhas h10 fortaleza h11 areia branca h12* pnlm *haplotypes found in this present work table 1. localities where each haplotype are originally found. sociobiology 66(4): 597-601 (december, 2019) 599 also found similar results (tavares et al., 2007; francisco et al., 2008; batalha-filho et al., 2010; brito et al., 2013; bonatti et al., 2014, miranda et al. 2016; galaschi-teixeira et al., 2018). when we analyzed and compared the dna sequence of the two haplotypes identified here (h4 and h12), we observed that they differed from each other in two mutational steps. the replacement of a guanine by an adenine at 128 position translates into changes in one amino acid (valine-isoleucine). this non-synonymous change at the amino acid level was not observed in bonatti et al. (2014) where all mutations found in the 11 haplotypes were neutral, but has been observed in brito et al. (2013) for partomana mulata. it is noteworthy that, albeit it has been observed that m. subnitida has a wide flight range (silva et al., 2014), the dunes might act as geographic barriers, hindering the gene flow among these populations. these dunes are surrounded by lakes, and altogether they form a large complex of landscape without any vegetation or any other substrate, which could allow bee dispersion to long distances. the change in the amino acid sequence of coi protein from pnlm may reflect a random factor or it may be the result of selective pressures driven by the environment where these bees live. these areas differ in the type of trees where they build their nests, climatic conditions, and vegetation structures. notwithstanding, the natural process of desertification may play a role limiting gene flow among populations, since the movement of dunes ends up covering green areas, limiting the few chances of nesting sites. genomic studies with a more thorough sampling will be necessary to better understand the evolutionary history of bees in this region. acknowledgments we thank irene aguiar, mr. emídio and mrs. maria for letting us develop field work inside their land and their assistance guiding us through the areas to find bee nests. the authors are also grateful to the anonymous referees for comments and suggestions. to cnpq for the first author’s masters scholarship and for research support. authors contributions the authors provided performance data and genetic analysis, including laboratories procedures and availability of laboratory from university of são paulo, ribeirão preto. references batalha-filho, h., waldschmidt, a.m., campos, l.a.o., tavares, m.g. & fernandes-salomão, t.m. 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(2002). abelhas da caatinga: biogeografia, ecologia e conservação. in: i.r. leal, m. tabarell & j.m.c. silva (eds.), ecologia e conservação da caatinga (cdd 5745265). recife: editora universitária, ufpe. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3383sociobiology 66(1): 107-112 (march, 2019) stingless bees fed on fermented soybean-extract-based diet had reduced lifespan than pollen-fed workers introduction stingless bees (apidae, meliponini) constitute a group of eusocial insects that form perennial colonies, and comprise over 500 species. these species occur in tropical and subtropical regions, where rational breeding for honey production and, more recently, for agricultural pollination is common (cortopassi-laurino et al., 2006; slaa et al., 2006; michener, 2013). the management of stingless bees for honey production is the most traditional, but the potential for agricultural pollination has greatly increased the demand for new colonies (cortopassi-laurino et al., 2006; jaffé et al., 2015). however, several obstacles must be surpassed to enable abstract nectar and pollen are the basic food resources of stingless bees. the current advance of meliponiculture led to the search for supplementary feeding. despite little is known about native bee supplementation, several alternative foods have been tested as protein substitutes, with soy being one of the most commonly used. in this study, we compared the effect of a semiartificial soy-based diet versus a natural diet on the longevity of adult worker of melipona flavolineata friese and scaptotrigona aff. postica (latreille). a total of 200 workers of each species (40 from each colony) were used, of which 100 comprised the control group (consumed honey and pollen) and 100 the experimental group (honey and a semiartificial food based on soybean extract). the workers were divided into groups of 20 individuals confined in mdf boxes not completely enclosed, without a queen, and kept in bod incubators. dead bees were counted and removed daily. kaplan-meier survival curves were plotted for each species. we found greater longevity in workers who consumed only natural pollen (71 days for m. flavolineata, 78 days for s. aff. postica, in average) compared to those consuming the soy-based diet (62 days for m. flavolineata, 61 days for s. aff. postica, in average). workers of m. flavolineata that consumed pollen lived nine days more (21.8%) than those fed on the soybased diet, while s. aff. postica workers lived seven days more (12.7%). as longevity was only slightly reduced, we can recommend a soy-based diet for stingless bees during dearth periods or for supplemental feeding of newly formed colonies. sociobiology an international journal on social insects acm queiroz1, kl leão1,2, jcs teixeira1, fal contrera2, c menezes1 article history edited by denise alves, esalq-usp, brazil received 27 april 2018 initial acceptance 07 june 2018 final acceptance 25 september 2018 publication date 25 april 2019 keywords protein feed, pollen, soybean, meliponini. corresponding author ana carolina martins de queiroz laboratório de botânica embrapa amazônia oriental travessa enéas pinheiro, s/nº cp: 18 cep: 66095-100, belém-pa, brasil. e-mail: carolinamqueiroz@gmail.com the large-scale production of meliponine colonies, including the development of an efficient artificial diet (venturieri et al., 2012; menezes et al., 2013). the bees obtain the nutrients necessary for their development and activities from nectar and pollen collected from the flowers (roubik, 1989; brodschneider & crailsheim, 2010). the nectar is composed basically of sugars and, therefore, is the energy source of the adult workers. pollen is much more nutritionally complex, being the main source of proteins, lipids, vitamins and minerals. pollen is used to feed the larvae, as well as newly emerged workers, for the development of glands (mandibular and hypopharyngeal) needed to produce larval food (cruz-landim & akahira, 1966; nogueira-neto, 1997). 1 laboratório de botânica, embrapa amazônia oriental, belém-pa, brazil 2 laboratório de biologia e ecologia de abelhas, universidade federal do pará, belém-pa, brazil research article bees acm queiroz, kl leão, jcs teixeira, fal contrera, c menezes – stingless bees fed on fermented soybean-extract-based diet108 the stingless bees store the pollen in cerumen (‘wax’) pots (nogueira-neto, 1997). within the colonies, this pollen is subjected to the action of microorganisms that change considerably its flavor, odor, color and texture transforming it into what we know as “saburá” (camargo et al., 1992; souza et al., 2004; menezes et al., 2018). the quality of pollen can affect bee longevity (schmidt et al., 1987), ovarian activation (hoover et al., 2006; human et al., 2007; pirk et al., 2010) and physiological mechanisms (alaux et al., 2011; di pasquale et al., 2013). the lack of pollen, therefore, directly affects colony development by reducing larval food production, nutrient intake of egg-laying queens, and the number of offspring, consequently reducing colony health (cruz-landim, 2009). nectar has a simpler composition, and therefore has been easily replaced by beekeepers by sugar-rich artificial feed made from sugar cane (nogueira-neto, 1997). however, pollen is not so easily replaced because of its nutritional complexity, and fully replacing it is still a challenge (venturieri et al., 2012). the pollen collected and processed by stingless bees is indeed very complex but very little is known about it yet. the knowledge is still limited to their general composition such as protein, carbohydrates and lipids content; nothing is known about micronutrients. this is an important gap to find better ingredients to complement their nutritional needs (silva et al., 2006; rebelo et al., 2016; hartfelder & engels, 1989; de oliveira alves & carvalho, 2018; de oliveira alves et al., 2018). several alternative foods have been tested as protein substitutes. penedo et al. (1976) observed a regular hypopharyngeal glands development of scaptotrigona postica (latreille) workers fed with a mixture of yeast (25%) and pollen (75%). zucoloto (1976), also studying s. postica workers, chose a mixture of yeast (18%) and sucrose (82%) as a good alternative for pollen among eight substitute compositions tested. still for this species, a mixture of 75% pollen from s. postica and 25% pollen from a. mellifera was a satisfactory pollen substitute (testa et al., 1980). a fermented mixture of yeast, pollen and sucrose solution (50%) showed equivalent results in hypopharyngeal glands development to scaptotrigona depilis (moure) workers, compared to the natural diet (fernandes-da-silva & zucoloto, 1990). soybean is one of the most commonly ingredient used, because of the low cost and wide availability in the market. in the genus melipona, studies showed an efficient semi-artificial composition based on soybean extract, sucrose solution and pollen. costa and venturieri (2009) pointed out no differences in the acini and oocytes sizes of melipona flavolineata friese workers, compared to natural diet (pollen). pires et al. (2009) recommended the use of a similar composition (soybean extract, 30% sucrose solution and pollen) to the melipona fasciculata smith workers, in scarcity periods. the main parameters evaluated so far were the development of the ovaries and of the hypopharyngeal glands of the workers. however, other parameters should also be used to efficiently evaluate the nutritional value of protein diets for stingless bees, especially at the colony level (e.g., the effect on worker longevity, colony brood cell production rate and larval development), despite the importance of the aforementioned factors in evaluating an artificial protein diet (menezes et al., 2012). therefore, the objective of this study was to compare the effects of a semi-artificial soybean extract diet on the longevity of workers of two stingless bee species, m. flavolineata and s. aff. postica. these species are widely kept by local beekeepers in the brazilian state of pará. melipona flavolineata is a species of restricted distribution in brazil, occurring in the states of pará, maranhão and tocantins, while scaptotrigona aff. postica has a much broader distribution (bahia, ceará, goiás, maranhão, mato grosso, mato grosso do sul, minas gerais, pará, pernambuco, piauí, são paulo, tocantins), being commonly called “canudo” (camargo & pedro, 2013). material and methods experimental design the experiments were conducted at the meliponiculture laboratory, eastern amazon embrapa, belém, pará, brazil (1°26’11.52’’s, 48°26’35.50’’w), from june to october 2013. newly emerged workers obtained from 10 nests of the scientific bee nursery (herein ‘meliponary’) of the eastern amazon embrapa were used to evaluated the effect of the consumption of a semiartificial soy-based diet on the survival of workers of the species m. flavolineata and s. aff. postica (five nests per species). a total of 200 bees were used for each species (40 per colony), of which 100 were used in the control group (natural pollen) and 100 for each experimental group (soy). the workers were collected as soon as they emerged (first day of age) to form groups of 20 individuals from different colonies, because few workers emerge from a single comb in a single day, especially in m. flavolineata combs. they were placed in mdf (medium density fiberboard) boxes (8.2 x 8.2x 3.5 cm), without a queen. boxes were kept in bod incubators (model dl-sedt 02) at 28 ± 1 °c and relative humidity between 70% and 80%, from emergence to death of all the bees. the number of live bees was monitored and dead bees were removed daily. in addition, the trash was removed and replaced, and water added to maintain moisture (adapted from costa & venturieri, 2009). the workers were submitted to two diets, soy-based and pollen-based, according to the treatment used. bees on the soy-based diet consumed its own honey and semiartificial food based on soybean extract (recipe below) stored in a refrigerator at 4 °c for the experimental period, while those on the pollen-based diet consumed honey and pollen from the colonies of the species itself, stored in a freezer at – 6 °c throughout the experimental period. bees received approximately 0.1 ml of honey and 0.15 g of soy-based food or pollen per bee per day, depending on the treatment. food stocks were renewed daily. sociobiology 66(1): 107-112 (march, 2019) 109 semi-artificial food used in experiments the production of the semi-artificial food based on soybean extract followed costa and venturieri (2009), and pires et al. (2009), with some modifications. the food consists of 500 g soybean extract, 500 ml sucrose syrup diluted to 50% and 50 g fermented pollen. pollen was collected from the colony pots of the study species and used immediately. the 500 g of soybean extract was homogenized in 500 ml of diluted syrup at about 60 °c. 50 g fermented pollen was added when the temperature of the mixture was reduced to 28 °c. the food was kept in an oven at 28 °c for 15 days and homogenized daily to enable oxygen penetration and facilitate the fermentation process. once ready, the food was refrigerated at 4 °c during all the experiment period. data analysis kaplan-meier survival curves were plotted for each treatment, per species. the log-rank test was applied using the software statistica® 8.0, to compare the survival curves of the treatments, for each species. a 5% significance level was considered for all analyses. results the bees used the protein food (pollen or soy) for consumption throughout the experimental period. despite consumption was not measured, we could observe a higher consumption in the first days of bee life. in some boxes food pots were built by bees, in these cases, they were filled of honey. the effect of the semi-artificial diet on the longevity of the bee species m. flavolineata and s. aff. postica was observed, and a higher mean longevity was recorded for the control groups (pollen treatment) than for the experimental groups (soy treatment) (table 1). the s. aff. postica individuals that consumed pollen exhibited a greater longevity, surviving on average 17 days more than individuals on the soy treatments. similarly, individuals of the species m. flavolineata survived on average 9 days more than individuals on the soy treatment (table 1). the maximum longevity of s. aff. postica was recorded in an individual submitted to pollen treatment (control) (121 days), while the maximum in the soy treatment was 83 days. bee mortality occurred from the 9th day for the soy treatment, and from the 19th day for the control. comparison of survival curves showed that a highly significant difference in results (log-rank test, p < 0.01; fig 1). species/treatment n pollen (days) soybean (days) p scaptotrigona aff. postica 200 78 ± 22.50 61 ± 11.90 <0.001 melipona flavolineata 200 71 ± 43.56 62 ± 30.07 <0.001 table 1. longevity in days (mean ± standard error) for bees of the species melipona flavolineata and scaptotrigona aff. postica confined in group of 20 individuals and submitted to dietary treatments. pollen (control): bees fed honey and its own pollen, soybean: bees fed honey and a semi-artificial food based on soybean extract. it was applied a log rank test using 5% significance level. fig 1. survival curves for scaptotrigona aff. postica confined in group of 20 individuals and submitted to dietary treatments. pollen (control): bees fed honey and its own pollen, soybean: bees fed honey and a semi-artificial food based on soybean extract. the value t refers to log rank test using 5% significance level (p). the longest that m. flavolineata lived in the control treatment was 141 days compared to 138 days in the soy treatment, bee mortality began on the 5th day in the pollen (control) treatment, and on the 6th day in the soy treatment. the difference among survival curves of each treatment was highly significant (log-rank test, p < 0.01; fig 2). fig 2. survival curves for melipona flavolineata confined in group of 20 individuals and submitted to dietary treatments. pollen (control): bees fed honey and its own pollen, soybean: bees fed honey and a semi-artificial food based on soybean extract. the value t refers to log rank test using 5% significance level acm queiroz, kl leão, jcs teixeira, fal contrera, c menezes – stingless bees fed on fermented soybean-extract-based diet110 discussion in this study, the longevity of workers that consumed exclusively natural pollen was greater than of those that consumed the soy-based diet. the m. flavolineata workers fed on pollen lived 22% more than those that consumed soybased diet, while the s. aff. postica workers lived 13% more. on the one hand, it is a positive result as it shows that the soy-based diet did not drastically affect the longevity of adult bees (camargo, 1982; gomes et al., 2015), and therefore it is a good basis for the elaboration of an artificial diet. however, it is also evidence that the diet still requires enhancements to fully meet the nutritional needs of stingless bees. improvement of artificial diets is essential in the breeding activity of these bees, to assist the growth of newly formed colonies and to strengthen them during periods of food shortage. pollen availability is essential for the development of the colony and to increase population size. pollen availability also plays a key role in bee’s morphology and physiology and may affect longevity if it is not suitable (ramalho et al., 1998). as in other social insects, an altered longevity of the workers will affect the growth rate and final size of the colony, which can be increased both by a higher birth rate and/or by a decreased mortality rate of workers (carey, 2001). bees require basic nutrients such as carbohydrates and lipids (non-essential nutrients), amino acids, vitamins and minerals throughout their life stages. pollen is the main protein source (souza et al., 2004; menezes et al., 2018), and acts directly on offspring production, larval development and longevity of adult individuals (sagili & pankiw, 2007; brodschneider & crailsheim, 2010; alaux, 2011; frias et al., 2016). in this study, differences in the nutrient concentration in the food offered to the bees could justify the increased longevity found for bees fed on natural food (pollen). the higher protein concentration in natural food may be one of the causes of the differences in longevity found between treatments. little is known about chemical composition of pollen collected by stingless bees (rebelo et al., 2016), included these species studied. bee pollen usually has a high protein content. three amazonian species of melipona showed protein content between 15.7 and 23.8 (mean 19.5%) (souza et al., 2004). rebelo et al. (2016) obtained the protein percentage mean values of 24 (for m. interrupta) and 37.63 (m. seminigra). costa and venturieri (2009) verified different results due to protein content in diets. using a semi-artificial food soy-based (25 g of soybean extract, 25 g of sucrose, 50 ml of water, 2.4 g of processed pollen from m. flavolineata) similar to the one used in this study, they reported a protein content of 12%, lower than protein bees requirement of 20% (souza et al., 2004; somerville, 2005). m. flavolineata workers fed with this diet had smaller acini than workers fed with natural pollen, however no differences were reported in oocytes development (costa & venturieri, 2009). on the other hand, m. flavolineata workers fed with a different composition soybased diet (43 g soybean extract, 14 g of sucrose, 43 ml of water, 2.4 g of pollen) that reach a protein content of 18%, presented greater development of the hypopharyngeal glands and oocytes, being even superior to the control treatment (pollen). probably it is due to the higher energetic value of soybean compared to pollen. the higher protein concentration in natural food may be one of the causes of the differences in longevity found between treatments. somerville (2000) observed that higher protein levels in the body of a. mellifera workers resulted in greater longevity. in another important species for beekeeping in the states of pará and maranhão, m. fasciculata, a higher longevity has been reported in workers submitted to a semiartificial diet (a mixture of soybean extract, sucrose syrup diluted to 30% and pollen) compared to bees that consumed natural pollen (pires et al., 2009). these authors present a formula that can be offered during periods of flower shortage with no apparent damage to colonies. semi-artificial food based on soybean extract has proven to be a viable option for critical periods of scarcity of natural resources, or even to be used in large-scale colony production despite the reduction in longevity. teixeira (personal communication, april 02, 2018) compared the size of individuals submitted to the aforementioned treatments and reached the conclusion that individuals fed on soy were larger than those that consumed natural food (pollen) and reported no differences in the survival or size of immature m. flavolineata individuals when compared to those that consumed natural food (pollen). similar longevity-reduction results were obtained for s. aff. postica individuals that consumed soy-based food compared to natural food consumption (pollen), although precise data for the species are lacking. the physical and chemical compositions of the food tested possibly resemble those found for m. flavolineata (costa & venturieri, 2009). besides the concern of ensuring a minimum percentage of components (e.g., proteins), an excess of elements must also be considered. the minerals sodium, sodium chloride and calcium, for example, may be toxic to honey bees (a. mellifera), affecting the production of offspring (somerville, 2005). therefore, the addition of new ingredients to the artificial diet needs to be done with caution. future studies are important in trying to match the components to the nutritional needs of bees. until this optimal composition is reached, the results obtained in this study contribute by reinforcing the recommendation to use semi-artificial food based on soybean extract for stingless bees in periods of scarcity of natural resources or for newly formed colonies. acknowledgements we would like to thank janete teixeira gomes, lorival juracy lucas for their valuable contributions. we also thank alistair campbell for linguistic advice and two sociobiology 66(1): 107-112 (march, 2019) 111 anonymous referees for insightful suggestions. we thank capes/embrapa (15/2014) for providing grants to kll. this research was funded by cnpq (479710/2011-2) through the project universal 14/2011. we dedicate this work to the memory of prof. warwick e. kerr (1922 2018), a pioneer on studies of stingless bees. author’s contribution acmq, kll, jcst and cm conceived the experimental design; acmq, kll and jcst collected and analyzed data; falc assisted with data analyses; acmq and kll wrote initial draft of the manuscript and all authors contributed to subsequent revisions and gave final approval for publication. references alaux, c., dantec, c., parrinelo, h. & le conte, y. 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(1987). survival of honeybees apis mellifera (hymenoptera: apidae) fed with various pollen sources. annals of the entomological society of america, 80: 176-183. doi: 10.1093/aesa/80.2.176 slaa, e. j., sánchez-chaves, l. a., malagodi-braga, k. s. & hofstede, f. e. (2006). stingless bees in applied pollination: practice and perspectives. apidologie, 37: 293-315. doi: 10.1051/apido:2006022 somerville, d. (2000). honey bee nutrition and supplementary feeding, agnote dai/178, nsw. agriculture. somerville, d. (2005). fat bees skinny bees a manual on honey bee nutrition for beekeepers. australian government. rural industries research and development corporation. 150 p. souza, r.c.s., yuyama, l.k.o., aguiar, j.p.l. & oliveira, f.p.m. (2004). valor nutricional do mel e pólen de abelhas sem ferrão da região amazônica. acta amazonica, 34: 333336. doi: 10.1590/s0044-59672004000200021 silva, t.m.s., camara, c.a., lins, a.c. da s., barbosa-filho, j.m., da silva, e.m.s., freitas, b. m. & santos, f. de a. r. dos (2006). chemical composition and free radical scavenging activity of pollen loads from stingless bee melipona subnitida ducke. journal of food composition and analysis, 19: 507511. doi: 10.1016/j.jfca.2005.12.011 testa, p.r., silva, a.n. & zucoloto, f.s. (1980). nutritional value of different pollen mixtures for nannotrigona (scaptotrigona) postica. journal of apicultural research, 20: 94-96. doi: 10.1080/00218839.1981.11100479 venturieri, g.c., alves, d.a., villas-boas, j.k., carvalho, c.a.l., menezes, c., vollet-neto, a., contrera, f.a.l., cortopassilaurino, m., nogueira-neto, p., & imperatrizfonseca, v.l. (2012). meliponicultura no brasil: situação atual e perspectivas futuras para uso na polinização agrícola. in: imperatrizfonseca, v.l., canhos, d.a.l., alves, d.a. & saraiva, a.m. (eds.), contribuição e perspectivas para a biodiversidade, uso sustentável, conservação e serviços ambientais (pp. 213-236). são paulo: edusp. zucoloto, f.s. (1976). valor nutritivo de alguns carboidratos para nannotrigona (scaptotrigona) postica (hymenoptera, apoidea). ciência e cultura, 28: 193-194. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2902sociobiology 65(3): 531-533 (september, 2018) “the road to reproduction”: foraging trails of constrictotermes cyphergaster (termitidae: nasutitermitinae) as maternities for staphylinidae beetles termite nests present sophisticated micro-environments compared to the external environment. the association of other insect orders, such as beetles and other termite species (kistner, 1969; grassé, 1986; jacobson & pasteels, 1992) can be linked to the abiotic conditions inside the nest (temperature, humidity, luminosity) (kistner, 1979). the association with termite is important to beetles, as they provide protection and potential food resources (noirot & darlington, 2000), and have apparently evolved over millions of years (cai et al., 2017). according to the particular types of insect-insect interactions (kistner, 1969), the guests are referred to as inquilines (termite species that live inside other termite colonies), termitophilous (non-termite taxa that live in association with abstract corotoca (coleoptera: staphylinidae) beetles are known for their close integration in the nests of the termite constrictotermes cyphergaster (termitidae: nasutitermitinae). although this relationship is regarded as ancient, many details are still obscure, such as their reproduction and the processes that lead to the dispersal of new beetles. we observed the use of termite foraging trails by staphylinidae females to deposit and disperse their larvae. we recorded the deposition of larvae of c. melantho, c. fontesi, and c. sp. n. on the dorsal surfaces of termite host workers. the workers continue to follow the foraging trail until the newborn larvae freed themselves and fell into the leaf litter, subsequently burrowing into the ground. information regarding the life stages of those staphylinidae larvae outside the termite nest is important to understanding their full lifecycle as those taxa have strong relationships with the nest environment but also require dispersal strategies. sociobiology an international journal on social insects mh oliveira1, rv silva-vieira2, ie moreira3, cm pires-silva3, hvg lima3, mrl andrade3, ma bezerra-gusmão1,3 article history edited by paulo cristaldo, ufs, brazil received 18 february 2018 initial acceptance 26 march 2018 final acceptance 04 june 2018 publication date 02 october 2018 keywords termitophily, corotoca, larvae, dispersal. corresponding author mário herculano de oliveira laboratório de ecologia de térmitas departamento de biologia universidade estadual da paraíba rua das baraúnas nº 351 campus i, bairro universitário campina grande-pb, brasil. e-mail: mariohecules@hotmail.com termites colonies), or termitariophilous (other invertebrates/ vertebrates that live in associations with the nest). staphylinidae is one of the largest beetle families, comprising approximately 61,300 described species (newton, 2015). although showing wide morphological variability, the group can be distinguished from other beetles by their small elytra (newton et al., 2001). additionally, numerous species within this family are physogastric, including those of the genus corotoca. species of corotoca are only found in the nests of constrictotermes spp. (asenjo et al., 2013). previous studies have shown that individuals of c. melantho show morphological congruency with the workers of its host species (cunha et al., 2015), as well as chemical mimicry 1 laboratório de ecologia de térmitas, programa de pós-graduação em ecologia e conservação, universidade estadual da paraíba, campina grande-pb, brazil 2 laboratório de herpetologia (integrado ao laboratório de etnoecologia), departamento de biologia, universidade estadual da paraíba, campina grande-pb, brazil 3 laboratório de ecologia de térmitas, departamento de biologia, universidade estadual da paraíba, campina grande-pb, brazil short note mh oliveira et al. – foraging trails of constrictotermes cyphergaster as maternities for staphylinidae beetles532 through cuticular hydrocarbon compounds acquired from the host (rosa et al., 2018). those strategies allow beetles to live in close association with their hosts, although relatively little is known about the roles of each partner in that relationship, nor why, contrary to nearly all other groups of insects, those coleopterans are so successful in their invasions of social insect colonies (see yamamoto et al., 2016). staphylinidae beetles have been observed walking among their ant and termite hosts on open foraging trails (quinet, 1995), but it was not known why they left the nest to follow their hosts’ trails. the present study therefore investigated the behavior of those beetles on the foraging trails of costrictotermes cyphergaster silvestri, 1901. we monitored the foraging trails of 14 c. cyphergaster colonies from 18:00 to 06:00. individuals of corotoca spp. were observed and collected at the start of colony foraging events. observations were carried out in october/2017 (three days; at four nests) at fazenda almas (rppn fazenda almas), located in the municipality of são josé dos cordeiros (36º52’w; 7º28’s); and in november/2017 (three days; at five nests) and janeiro/2018 (21 days; at five nests) at the são joão do cariri experimental station, located in the municipality of são joão do cariri (36º31’w; 7º22’s), both sites in paraíba state, brazil. we registered the occurrence of individuals of c. melantho (n = 13), c. fontesi (n = 9), and c. sp. n. (n = 7) in all colonies sampled. additionally, 25 beetles were collected from different nests, dissected and observed the presence of larvae inside their abdomen. staphylinidae identifications were made by consulting fontes (1977) and zilberman (2018). collected specimens were deposited as vouchers in the termite ecology laboratory of the state university of paraiba, brazil. the beetles only left the termitarium after the establishment of the foraging trails (fig 1) and kept in the middle of the trails (among the workers); they were never observed at the trail edges where the soldiers stand. we recorded the presence of up to eight individuals of corotoca from the same colony during a single foraging event. during termite foraging events, the female beetles deposited a well-developed larva on the pronotal and heads of c. cyphergaster workers (supplementary material movie sm01). this was accomplished after traveling a short distance from the nest along the foraging trail (up to 50 cm), after which the females of corotoca started to release their larval offspring (which remained attached to the end of the mother’s abdomen). the female beetle was observed actively moving its antennae and moving towards the workers. a worker passing close to the beetle stop and beetle female deposited the larva; then the worker continuing along the foraging trail with the larva on its dorsal surface. further along the trail, when the larva apparently detected the presence of leaf litter, it squirmed over the worker’s back and detach itself – falling fig 1. female of corotoca sp. releasing its larva during the foraging of its host constrictotermes cyphergaster. sociobiology 65(3): 531-533 (september, 2018) 533 to the ground and quickly burying itself into the soil. after depositing the larva on the worker termite, the adult beetle returned to the nest (still pregnant). we could not determine whether the same corotoca individual would leave the nest again during the same foraging event, on the same night, to deposit the second larva. the comprehension of this termitophile’s life cycle is essential to understanding the benefits of their termiteguest relationship. the caatinga domain presents extreme environmental conditions (drought and high temperatures), and the survival of corotoca beetles depends on the support of their hosts’ nests. according to costa and vanin (2010), some species of beetles closely associated with termites can confuse their hosts using mimicry cues, and beetle larvae are recognized as “termites”. we hypothesize that this strategy is essential to larval dispersal during foraging events. furthermore, the existence of a lifecycle stage outside of the nest could require mimetic compounds to be acquired or produced by the larvae, as that mimicry would be required for the re-colonization of host nests (see rosa et al., 2018). the interactions between termites and aleocharinae beetles have been studied by several authors, highlighting seevers (1957) and grassé (1986), but the full life cycle of corotoca individuals is not yet clearly understood in regard to its reproduction and nest colonization. the present work provides important information concerning the interactions of corotoca individuals and c. cyphergaster that will aid future studies with termitophilous species. references asenjo, a., irmler, u., klimaszewski, j., herman, l.h. & chandler, d.s. (2013). a complete checklist with new records and geographical distribution of the rove beetles (coleoptera, staphylinidae) of brazil. insecta mundi, 0277, 1-419. cai, c., huang, d., newton, a.f., eldredge, k.t., michael, s. e. (2017). early evolution of specialized termitophily in cretaceous rove beetle. current biology, 27: 1-7. doi: 10.1016/j.cub.2017.03.009. costa, c. & vanin, s.a. (2010). coleoptera larval fauna associated with termite nests (isoptera) with emphasis on the “bioluminescent termite nests” from central brazil. psyche: a journal of entomology, 2010: 1-12. doi: 10.1155/ 2010/723947. cunha, h.f., lima, j.s., de souza, l.f., dos santos, l.g.a. & nabout, j.c. (2015). no morphometric distinction between the host constrictotermes cyphergaster (silvestri) (isoptera: termitidae, nasutitermitinae) and its obligatory termitophile corotoca melantho schiødte (coleoptera: staphylinidae). sociobiology, 62: 65-69. doi: 10.13102/sociobiology.v62i1.65-69. fontes, l. r. (1977). notes on the termitophilous genus corotoca, with a new species from brazil (coleoptera: staphylinidae). revista brasileira de entomologia, 21: 69-74. grassé, p.p. (1986). termitologia: comportement, socialité, ecologie, evolution, systématique. masson, paris: foundation singer-polignac,715p. jacobson, h.r. & pasteels, j.m. (1992). staphylins termitophiles de nouvelle-guinée: une nouvelle espèce de termitoptocinus et nasutiptochus nn.nov. (coleoptera: staphylinidae, corotocini). sociobiology, 20: 49-55. kistner, d.h. (1969). the biology of termitophiles. in k. krishna & f.m. weesner (eds), biology of termites (pp. 525–557). new york: academic press. kistner, d.h. (1979). social and evolutionary significance of social insect symbionts. in: h. herman (eds). social insects (pp. 339–413). new york: academic press. kistner, d.h. (1982). the social insects bestiary. in: h. hermann (eds). social insects (pp. 1-244). new york academic press. newton, a.f. (2015). beetles (coleoptera) of peru: a survey of the families. part i. overview. journal of the kansas entomological society, 88: 135-139. doi: 10.2317/00228567-88.2.135. newton, a.f., thayer, m.k., ashe, j.s., & chandler, d.s. 2001. staphylinidae latreille, 1802. in r.h. arnett & m.c. thomas (eds). american beetles (pp. 272–418). boca raton: crc press. noirot, c., & darlington, j.p.e.c. (2000). termite nest: architecture, regulation and defence. in t. abe, d.e. bignell & m. higashi (eds). termites: evolution, sociality, symbioses, ecology (pp.121–139). dordrecht: kluwer academic publishers. rosa, c.s., cristaldo, p.f., martins, a., florencio, d.f., lima, e.r., & desouza, o. (2018). on the chemical disguise of a physiogastric termitophilous rove beetle. sociobiology, 65: 38–47. doi: 10.13102/sociobiology.v65i1.1942 quinet, y. (1995). trail following and stowaway behaviour of the myrmecophilous staphylinid beetle, homoeusa acuminata, during foraging trips of its host lasius fuliginosus (hymenoptera: formicidae). insects sociaux, 42: 31-44. seevers, c.h. (1957). a monograph on the termitophilous staphylinidae (coleoptera). fieldiana: zoology, 40: 1-334. doi: 10.5962/bhl.title.3797. yamamoto, s., maruyama, m., and parker, j. (2016). evidence for social parasitism of early insect societies by cretaceous rove beetles. nature communications, 7: 13658. doi: 10.1038/ncomms13658. zilberman, b. (2018). new species and synonymy in the genus corotoca schiødte, 1853 (coleoptera, aleocharinae, corotocini). zootaxa, in press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.547-553 sociobiology 61(4): 547-553 (december 2014) pattern of the daily flight activity of nannotrigona testaceicornis (lepeletier) (hymenoptera: apidae) in the brazilian semiarid region introduction the daily activity patterns of eusocial stingless bees are mainly associated with activities as collection of pollen, nectar, resin, and clay. these activities occur during the day, but in higher intensity at specific moments, influenced by climatic factors (hilário et al., 2001; souza et al., 2006; rodrigues et al., 2007; ferreira-junior et al., 2010). according to corbet et al. (1993) there would be an ideal temperature range in which the eusocial bees are active, able to carry out activities that are external to the nest. the light intensity is another climatic factor that influences the flight activity of the bees (kleinert-giovannini, 1982; corbet et al., 1993), acting as an indicator for the onset of the flight activities (lutz, 1931). heard and hendrikz (1993) studying tetragonula carbonaria (smith) verified a diurnal pattern of activity with the influence of temperature and radiation. for these authors, there was no good correlation between activity and temperature, but it is not conclusive. factors such as rainfall, for melipona asilvai moure (nascimento & nascimento, 2012), and relative air humidity, for plebeia remota (holmberg) (hilário et al., 2007), can also influence the daily activities of the stingless bees. abstract the flight activities of meliponini (stingless bees) are associated with a series of particular behaviors as collection of pollen, nectar, resin, and clay during the day, which can be influenced by extrinsic (e.g. abiotic factors) or intrinsic factors (e.g. internal conditions of the colony, morphology, physiology of the individuals, and others). this study aims to analyze the pattern of the daily flight activity of nannotrigona testaceicornis (lepeletier) and the influence of climatic factors (temperature, relative humidity, and light intensity) on these activities in chapada diamantina, bahia, brazil. the study was conducted every two months between october 2010 and october 2011 during three consecutive days in two colonies (one managed and the other unmanaged). the managed colony was more active than the unmanaged one in all the months. nevertheless, both colonies showed regularity of times for their first activities and for the preferential time for the most part of the activities analyzed, which occurred generally in the morning, until noon. this daily pattern of activity of both colonies was mainly influenced by light intensity. in this sense, these activities began with the sunrise and become more intense with the increase of the light intensity in the environment. sociobiology an international journal on social insects wp silva, m gimenes article history edited by denise araujo alves, esalq-usp, brazil received 31 march 2014 initial acceptance 12 may 2014 final acceptance 03 december 2014 keywords stingless bees, daily flight activity, meliponini, biological rhythm. corresponding author miriam gimenes departamento de ciências biológicas, universidade estadual de feira de santana av. transnordestina, s/nº, novo horizonte, 44036-900, feira de santana, ba, brazil e-mail: mgimenes@uefs.br the time to supply with floral resources also exerts great influence on the foraging of eusocial bees. since most plants show another dehiscence in the morning, the presence of a large quantity of bees in the flowers is common during this period for pollen collection (roubik, 1989). many factors, such as those mentioned above are important for interpreting the patterns of daily activity, mainly related to the behavior in the collection of resources. however, the patterns of daily activity can also be manifestations of the circadian timing system, which can be related to many of the aspects of rhythmic daily activities of meliponini (bloch, 2008). endogenous rhythmicity in the activity of meliponini has already been observed by bellusci and marques (2001). according to moore (2001), eusocial bees present a daily rhythm in the collection of floral resources such as nectar and pollen and these rhythms are generally synchronized with environmental (light/dark) or climatic (temperature) cycles. in this sense, there are very few studies in literature that analyze the daily activities based on the biological rhythm. moreover, the pattern of daily activity can occur in a different way in the different months of the year, depending on the climatic research article bees universidade estadual de feira de santana (uefs), feira de santana, ba, brazil wp silva, m gimenes daily activity of nannotrigona testaceicornis 548 characteristics of every month, as noted by nunes-silva et al. (2010) when they observed the flight activity of p. remota. in order to verify the pattern of daily activity of meliponini and the influence of environmental and climatic factors, an analysis was performed on two nests of nannotrigona testaceicornis (lepeletier), a neotropical bee that occurs in several regions of brazil (moure et al., 2012). material and methods study area the work was developed in the valley of capão, municipality of palmeiras, chapada diamantina (bahia state, brazil) (12º 31’s, 41º 33’w, at 1000 m of altitude). this area has low, fairly homogenous, and dense vegetation. the canopy is high, reaching 10 m height with trees emerging over 15 m mainly in the lower altitudes, which can be characterized as dry forest (semideciduous) (queiroz et al., 2005). the climate of palmeiras varies between sub-humid and dry with a yearly average temperature of 24.3 oc (köppen-geiger: bsh). the rainy period occurs between october and july and the annual rainfall index is 1,361.7 mm (sei, 2011). the climatic data were collected at intervals of one hour, from 5:00 to 18:00 during the observations. the data of temperature and relative air humidity were collected with a digital thermo hygrometer fixed above the soil (ca. 1.5 m) in a shadowy area and the data of light intensity (illuminance) were measured with a digital luximeter (lutron lx-107) at a distance of approximately 1 m from the soil. sunrise and sunset times were obtained from the almanac of the national observatory (http://euler.on.br/ephemeris/index.php). flight activity for this study were used two colonies of n. testaceicornis, one managed and one unmanaged. the managed colony of n. testaceicornis was captured in the valley of capão (chapada diamantina), and had been maintained in a wooden box, under a roof, for eight years. the unmanaged colony was located in the trunk of a tree which was transferred from caatinga environment (semiarid region, located around palmeiras, bahia, brazil) to valley of capão, approximately four years ago and had been kept in an open area. individuals of n. testaceicornis were collected and deposited in the entomological collection “johann becker” at the museum of zoology of the state university of feira de santana. the observations of the managed colony were made every two months from october 2010 to october 2011 while the unmanaged colony was observed in february, june, and october 2011. the observations in each colony were conducted between 5:00 and 19:00 (intervals of 15 min/hour) over three consecutive days. quantification of the number of bees was made by counting the bees that entered and left the nest (observation of the individuals at the entrance of the colonies). the bees were separated in categories: a) did not carry apparent material; b) carrying pollen, and c) carrying resin on the corbiculae. statistical analyses of data the analyses of the daily activities of n. testaceicornis colonies were performed using the rayleigh test of the circular statistics method (batschelet, 1980; zar, 2010). preferential times (or acrophases) of activities were considered for values with the significant vector (r) above of 0.7, which can range from 0 to 1, according to dispersion of data (p<0.05). the analyses were only applied when the number of each activity observed reached 10 or more in the month. the daily activities of both colonies were compared using the watsonwilliams test (p<0.05) (batschelet, 1980; zar, 2010). pearson’s correlation coefficient (r) was applied for verifying the correlation between the abiotic variables (temperature, relative humidity, and light intensity in open area and light intensity in hive entrance) and biotic variables (entrance, entrance with pollen, entrance with resin and exit). correlations were considered statistically significant when p<0.01, by student’s t test. correlations were made using the program past version 1.85 (hammer et al., 2001). they were considered moderate correlations when the values of r (pearson’s correlation coefficient) were between 0.39 and 0.69 and strong correlations when the r values were above 0.7 (dancey & reidy, 2005 in figueiredo-filho et al., 2009). results during the observations of the daily flight activities of n. testaceicornis in the study area, the temperature varied from 17 oc to 28 oc with an average temperature variation of 25 oc (october 2010 and february 2011) to 21oc (june 2011) (table 1). the lowest values of temperature and light luminosity were registered close to sunrise and sunset and the highest values were registered in the afternoon between 12:00 and 17:00, generally higher than 25 oc. the relative humidity varied from 41% to 90% with the average varying from 55% (august) to 83% (december) (table 1). sunrise times oscillated between 5:10 (october) and 6:00 (june) and sunset occurred between 17:27 (june) and 18:19 (february). table 1. time of the first activities of the unmanaged and managed colonies of nannotrigona testaceicornis and the meteorological factors in these times (light intensity at the entrance of the colony, li, sunrise, temperature, temp. at the onset of the activity and monthly average, relative humidity, rh at the onset and monthly average) from october 2010 to october 2011 in valley of capão (chapada diamantina, bahia, brazil). months hour li (lux) sunrise temp. (°c) temp. average rh (%) rh average managed colony oct/10 7:00 504 – 1110 5:15 19 25 82 59 dec/10 6:00 28 – 126 5:11 20 24 90 83 feb/11 6:00 1 – 95 5:39 20 25 77 61 abr/11 6:00 78 – 85 5:50 20 23 76 70 jun/11 8:00 410 – 786 6:03 18 21 80 69 aug/11 8:00 12500 – 19400 6:00 19 23 70 55 out/11 6:00 125 – 157 5:14 20 24 78 59 unmanaged colony feb/11 6:00 3 – 230 5:39 20 25 77 61 jun/11 8:00 2100 – 3850 6:03 18 21 80 69 oct/11 6:00 798 – 1333 5:14 20 24 78 59 sociobiology 61(4): 547-553 (december 2014) 549 monthly activities the managed colony of n. testaceicornis showed daily activity in all the seven months of observation. the numbers of total daily flight activities (entrance plus exit), entrance with pollen, and entrance with resin were always higher for the managed colony than for the unmanaged colony in all the months when both colonies were observed (figs 1 and 2). in october 2011 (temperature average: 24oc), a higher number for total activity and entrance with pollen in both colonies could be observed and also in august 2011 (temperature average: 23oc) for the managed colony (fig 1). daily activities the opening and closing of the cerumen tube at the nest entrance by the bees were observed daily and these movements marked respectively the starting and the ending of the activities for both colonies. the first activities of both colonies of n. testaceicornis occurred at the same time of the day. however, these times varied in different months of the year (table 1; fig 1). the first total activities and the first entrance activities with pollen occurred earlier (interval between 6:00 and 7:00) in december 2010, february 2011, april 2011, and october 2011 for the managed colony and in february 2011 and october 2011 for the unmanaged one. the first activities of the bees occurred later (between 8:00 and 9:00) in june 2011 (managed and unmanaged colonies) and august 2011 (managed colonies). during fig 1. total activity (entrance plus exit, without materials apparent) of nannotrigona testaceicornis and temperature values in colony unmanaged (in february, june and october/11) and managed (from october 2010 to october 2011) in the valley of capão (chapada diamantina), bahia, brazil. tm: temperature. fig 2. entrance activity with pollen of nannotrigona testaceicornis and temperature values in colony unmanaged and managed, in february, june and october/11 in the valley of capão (chapada diamantina) bahia, brazil. tm: temperature these activities, the temperature usually ranged from 18oc (june 2011) to 20oc (in the other months), the relative humidity ranged from 76% to 90% and the values of light intensity were always higher than 1 lux. the last total activities and collection of pollen by the bees of the two colonies occurred between 17:00 and 18:00, generally a little before sunset while the light intensity still above 1 lux, with the exception of february 2011 in which the activities ended later (between 18:00 and 19:00), month that values of temperature were higher (media 24.5oc) (fig 1) and sunset occurred later (18:19). total activities (fig 1) and the entrance with pollen (fig 2) in the managed and unmanaged colonies occurred at a higher intensity in the morning until 12:00, declining after this time. through the analysis of circular statistics was observed the presence of preferential times (or acrophases) for most of the flight activities of the bees for both colonies (table 2). although no difference was found between the times of the first and last activities of the managed and unmanaged colonies, there was a difference in the acrophases of the colonies. in the unmanaged colony, located in an open area, the acrophases of all activities occurred generally earlier than in the managed colony (table 2). the acrophases values for the unmanaged colony varied from 8:57 (october 2011) for the activity of entrance with pollen to 12:00 (june 2011) for the activity of exit. in the managed colony, the times of the acrophases varied from 9:31 (october 2011) for the entrance with pollen to 12:46 (june 2011) for the entrance into the nest (table 2). furthermore, the watson-williams test showed significant difference between the acrophases of the two colonies in all wp silva, m gimenes daily activity of nannotrigona testaceicornis 550 activities that were compared (entrance, exit, and entrance with pollen) in february 2011, june 2011, and october 2011 (table 3). all bees’ acrophases were later in october 2010 than in october 2011. although the value of mean temperature was the same in october 2010 and october 2011 (24oc), the light intensity was different. the mean of light intensity in open area was 17,160 lux in 2010 and 39,314 lux in 2011 and in the entrance of the managed colony, a light intensity reached 807 lux in 2010 and 2,446 lux in 2011. the acrophases for the entrance with resin were detected only for the managed colony; this was generally later than for the other activities, varying from 10:04 (october 2010) to 13:26 (june 2011). there were significant positive correlations (moderate) between the light intensity and entrance activities (r = 0.48, p<0.01) and exit (r = 0.50, p<0.01) in the managed colony at the open area. also, there were significant positive correlations (moderate) between the light intensity at the entrance of the colony and the activities of entrance (r = 0.40, p<0.01) and exit (r = 0.44; p<0.01) in unmanaged colony. there were no significant correlations between the two colonies with temperature and also with the relative humidity (table 4). table 2. acrophases times of exit and entrance activity; entrance with pollen and entrance with resin for nannotrigona testaceicornis from october 2010 to october 2011 in the valley of capão (chapada diamantina, bahia, brazil). table 3. comparison of the daily flight activities of the managed and unmanaged colonies of nannotrigona testaceicornis in february, june and october 2011 in the valley of capão (chapada diamantina, bahia, brazil). significance determined by watson-williams test (f) (p < 0.05). month/activity f critical value of f statistical significance february/11 entrance 316.93 0.05(1).1.7582 = 3.86 s exit 218.51 0.05(1).1.4968 = 3.86 s pollen 83.91 0.05(1).1.3719 = 3.86 s june/11 s entrance 62.56 0.05(1).1.5517 = 3.86 s exit 41.71 0.05(1).1.4268 = 3.86 s pollen 78.06 0.05(1).1.2148 = 3.86 s october/11 entrance 61.57 0.05(1).1.17794 = 3.86 s exit 90.95 0.05(1).1.13387 = 3.86 s pollen 45.45 0.05(1).1.5725 = 3.86 s managed colon y temperature li – ec li – ao rh entrance 0.15 0.32 0.48* -0.18 exit 0.09 0.31 0.50* -0.15 polen -0.02 0.26 0.39 -0.03 resi n 0.05 0.14 0.06 0.17 unmanaged colony entrance 0.08 0.40* 0.31 -0.05 exit -0.01 0.44* 0.32 0.05 polen -0.03 0.27 0.26 0.09 resi n -0.01 0.19 0.05 -0.02 * moderat e significance bee/activity oct/10 dec/10 feb/11 apr/11 jun/11 aug/11 oct/11 unmanaged colony entrance 11:01 12:21 * exit 10:49 12:00 10:23 pollen 10:27 11:42 08:57 resin * managed colony entrance 11:25 11:03 12:18 11:12 12:46 11:43 09:45 exit 11:20 10:48 12:02 11:06 12:22 11:32 09:51 pollen 09:51 10:35 11:34 10:56 12:38 11:49 09:31 resin 10:04 11:06 11:41 * 13:26 11:11 * value non-significant (r < 0.7) table 4. correlation analysis between abiotic variables (temperature, relative humidity, rh and light intensity in open area, li–ao, and in hive entrance, li-ec) and biotic variables (bees activities), of the managed (from october 2010 to october 2011) and the unmanaged colonies (in february, june and october 2011) in the valley of capão (chapada diamantina, bahia). discussion the highest values of the activities in the managed colony of n. testaceicornis observed in all months of the study may be an indication that this colony is stronger than the unmanaged one, since the number of flights of the colony characterized it as weak, medium, or strong (hilário et al., 2000). in general, the two colonies were more active in october 2011 and the managed colony also in august 2011. the onset of the daily flight activities of n. testaceicornis showed regularity in the months investigated for both colonies. the beginning of activities occurred generally in the early morning and the end of the activities in the late afternoon for both colonies. this regularity was also observed by the occurrence of a preferential time for most part of the flight activities of the bees, which was statistically significant and occurred generally in the morning. based on the first and last activities and on the acrophases observed in n. testaceicornis, it can be suggested that the two colonies showed a daily biological rhythm for most of the activities that were observed in the study. in literature, there is a great number of studies showing that daily flight activities occur at specific times through the day (iwama, 1977; kleinert-giovannini, 1982; guibu & imperatriz-fonseca, 1984; kleinert-giovannini & imperatriz-fonseca, 1986; souza et al., 2006). however, few studies relate these activities with rhythmic aspects of the foraging behavior of the bees. heard and hendrikz (1993), working with t. carbonaria in australia, noted the presence of activity peaks of the workers, indicating the presence of a daily periodicity independently from the meteorological variables considered (temperature, radiation, and relative humidity). some studies in brazil consider the pattern of the daily activity of meliponini as rhythmic manifestations that may have an endogenous origin. hilário et al. (2003) studying melipona bicolor lepeletier in the southeast, and gouw and gimenes (2013) studying melipona scutellaris, latreille and frieseomelitta doederleini (friese) in the northeast of brazil observed the occurrence of the daily activity rhythm in these species. *value non-significant (r < 0.7) *moderate significance sociobiology 61(4): 547-553 (december 2014) 551 the daily activities rhythm observed for the two colonies of n. testaceicornis may be synchronized by environmental meteorological factors or even by the time that the floral resources are available to the flower visitors. the influence of light/dark and photoperiodic cycles as synchronizers of the activities of n. testaceicornis can be observed in the first and last flight activities of both colonies since these activities occur very close to the time of sunrise and sunset, respectively. moreover, the acrophases occur earlier in october and december (end of spring and beginning of summer, when the sun rises earlier) compared to june (winter, when the sun rises later) in the area of the study, mainly for the managed colony. besides this, in february 2011, the activities finished later than in the other months in both colonies, and in this month the sunset occurred later. the entrainment of the daily activities by the light/dark cycle was observed by bellusci and marques (2001) for the workers of scaptotrigona aff. depilis (moure), suggesting an endogenous character of this synchronization. the entrainment of the daily rhythm of insect activities by the daily light/dark cycles and the annual photoperiodic cycle has already been discussed in the chronobiological literature (saunders, 2002; dunlap et al., 2004). this entrainment would lead to the adjustment of the time activities of the insects (such as bees) with the most favorable moments of the day, when the abiotic factors are favorable for the flight, allowing the organisms to anticipate the favorable or unfavorable cyclical environmental conditions (dunlap et al., 2004). the synchronization of the bees with the flowers may also be related to the time of the presentation of resource, as pollen or nectar. in this study, the workers from both colonies of n. testaceicornis presented their acrophases for the entrance with pollen in the morning, perhaps related to the greater availability of pollen by the plants at this time of the day, as also considered by roubik (1989). moreover, the light/dark and photoperiodic cycles can act in the synchronization of daily rhythm of foraging bees with the flower opening, the pollen exposure, and nectar production as were observed by gimenes et al. (1993; 1996) in the interaction between flowers of ludwigia elegans (cambess.) h.hara (onagraceae) and flower-visiting bees. the daily flight activities of both colonies of n. testaceicornis were probably influenced by the light intensity because it showed positive correlations between the activities of entrance and exit in both colonies. moreover, the bees were never active when the light intensity was under 1 lux. the effects of light intensity can be better observed when the activities of both colonies of n. testaceicornis are compared. the fact that the acrophases of the unmanaged colony had occurred earlier may be related to the positive influence of light intensity. this influence is due to the higher values of light intensity that acts on the entrance of the unmanaged colony whose nest was located in an open area favoring the earlier exit of the bees. another point to be considered is the fact that in october 2011 all acrophases of the managed colony occurred earlier than in the same month in 2010. in both months, the temperature average did not vary, but the intensity light was lowest in 2010. also, the light intensity seems influencing the end of the flight activity of n. testaceicornis in both colonies. according to corbet et al. (1993) the flight activities of social bees can be more related to radiation than to temperature, especially at the onset of daily activity, next to sunrise. lutz (1931) also observed the influence of light intensity at the beginning of flight activity of one species of meliponini, trigona mosquito (smith). such effect of light intensity could be modulator of the daily flight activities of bees. in this regard, bellusci and marques (2001) observed the endogenous circadian rhythm having light intensity as a modulator factor, in what clearer days influenced positively the increase of the external activities of s. aff. depilis. statistical analysis showed no significant effect of temperature on the activities of n. testaceicornis. however, the onset of the activities always occurred when the temperature was over 18°c. in other studies in the literature, it was observed a correlation between temperature and flight activities of the meliponini, mainly for melipona spp. (souza et al., 2006; ferreira-junior et al., 2010), plebeia pugnax moure (in litt.) (hilário et al., 2001), and tetragona clavipes (fabricius) (rodrigues et al., 2007). the effect of the temperature on the activities of the bees may be related to the body size; species considered small (such as n. testaceicornis) start their activity at a higher temperature than those that are of a larger size. according to teixeira and campos (2005) the onset of the external activity of bees of a small body size such as plebeia droryana (friese), frieseomelitta varia (lepeletier), friesella schrottkyi (friese), and n. testaceicornis occurred at temperature values ranging from 18°c to 21°c, while larger meliponini such as melipona quadrifasciata anthidioides lepeletier and m. bicolor started their activities at lower temperatures (ca. 12°c). the fact of no significant correlation between the daily flight activities of n. testaceicornis and the temperature may be related to the fact that bees living in tropical regions, where the average temperature ranges from 20°c to 30°c, so, there are not great thermal stresses (heinrich, 1993). however, heard and hendrikz (1993) correlated daily activity with both climatic factors, temperature and light intensity in flight activities of t. carbonaria. the results from this study showed the influence of light/dark cycle in different months of the year and also the light intensity in the daily flight activities of n. testaceicornis in an area of the brazilian semiarid, where temperatures are relatively favorable to the flight of bees. therefore, the synchronization of the flight activities with the times of day, and favorable climatic factors can ensure bee survival, especially for meliponini of small size, as n. testaceicornis. acknowledgments the authors would like to thank dr. favízia f. de oliveira (universidade federal da bahia ufba), for identifying the bee species; mr. lars erich rellstab for their permission to work in the area with their bees; and capes for the masters study grant. wp silva, m gimenes daily activity of nannotrigona testaceicornis 552 references batschelet, e. 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(2010).biostatistical analysis. 5th ed. new jersey: pearson prentice-hall, upper sandller river, 944 p. doi: 10.13102/sociobiology.v66i3.4354sociobiology 66(3): 408-413 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a new species of cryptopone emery (hymenoptera: formicidae: ponerinae) from brazil with observations on the genus and a key for new word species introduction the monophyly of ponerinae is strongly supported by the detailed molecular phylogenetic study of schmidt (2013). within the subfamily, substantial changes were made to the taxonomy of the genus pachycondyla smith, 1858, found to be paraphyletic and recently fragmented into 19 genera (schmidt & shattuck, 2014), one of which is the genus cryptopone emery, 1893. the genus was also found as polyphyletic by borowiec et al. (2019), which used two species belonging to different lineages in their study, c. gilva (roger, 1863) and c. hartwigi arnold, 1948, the former is a part of the ponera genus-group and the latter is sister to fisheropone schmidt and shattuck, 2014. cryptopone is a moderately large genus (25 described species and subspecies) with its probable radiation center in east and southeast asia (brown, 1963; bolton et al., abstract cryptopone is a moderately large genus with 25 described species and subspecies. in the new world, only four species are known, c. gilva, c. guianensis, c. holmgreni and c. mirabilis. recently four workers of an unidentified cryptopone species were collected in the state of rondônia, brazil, in soil samples from the floresta nacional do jamari (flona jamari). these workers are described as cryptopone pauli sp. nov. this is the first record of this genus for that state. we also present new records of cryptopone for the neotropical region with some comments on its biology, systematics, and an updated key to the workers of cryptopone in the new world. sociobiology an international journal on social insects io fernandes1, jhc delabie2,3 article history edited by john lattke, ufpr, brazil received 02 february 2019 initial acceptance 01 april 2019 final acceptance 08 may 2019 publication date 14 november 2019 keywords amazon, morphological convergence, grounddwelling ants, genus record, hypogeic ant. corresponding author itana oliveira fernandes instituto nacional de pesquisas da amazônia coordenação em biodiversidade coleção de invertebrados av. andré araújo, 2936 petrópolis. cep: 69067-375, manaus-am, brasil. e-mail: itanna.fernandes@gmail.com 2006; schmidt & shattuck, 2014). in the new world, we consider three species as currently valid: c. gilva (roger, 1863) (united states to panama), c. guianensis (weber, 1939) (mexico to southeastern brazil), and c. holmgreni (wheeler, 1925) (caribbean islands to southeastern brazil). c. mirabilis (mackay & mackay, 2010) was described from specimens collected in bolivia and mato grosso state, brazil, but longino (2015) examined the c. mirabilis holotype from the natural history museum of los angeles county and concluded that it is in fact centromyrmex brachycola (roger, 1861). longino also comments that the type series of c. mirabilis (collected by mann from bolivia, rosario on lake rocagua) was obtained from a series already studied by kempf (1966) and considered as centromyrmex brachycola in his revision of the neotropical members of the genus. as such, we exclude further consideration of c. mirabilis in the present study. 1 instituto nacional de pesquisas da amazônia – inpa, coordenação em biodiversidade, coleção de invertebrados, manaus-am, brazil 2 laboratório de mirmecologia, centro de pesquisas do cacau – cepec/ceplac, itabuna-ba, brazil 3 departamento de ciências agrárias e ambientais, universidade estadual santa cruz-uesc, ilhéus-ba, brazil research article ants urn:lsid:zoobank.org:pub:5ac25387-8f20-4a00-b447-5be17642862c sociobiology 66(3): 408-413 (september, 2019) 409 cryptopone workers are particularly well-adapted to a hypogaeic lifestyle, with small body size, reduced or absent eyes, flattened scapes, and stout setae on the mesotibiae (schmidt & shattuck, 2014). almost nothing is known about the social organization of cryptopone, but colonies are typically small (c. butteli forel, 1913: wilson, 1958; c. guianensis: weber, 1939). creighton and tulloch (1930) observed a single colony of c. gilva with five dealate queens and smith (1934) observed polygynous colonies of c. gilva, noting they could have as many as several hundred workers. after the revision by mackay and mackay (2010), no other cryptopone species have been described for the new world. recently an unidentified cryptopone species was collected in soil samples from the state of rondônia, brazil, and it is hereafter described as a new species. material and methods the samples came from the floresta nacional do jamari (flona do jamari), a reserve spread out amongst the municipalities of cujubim, porto velho, ariquemes and itapuã do oeste, in the state of rondônia, brazil. the ants were extracted from a soil piece of 25x25cm taken at the depth of 10 cm (anderson & ingram, 1993). the soil samples were then put in winkler bags (bestelmeyer et al., 2000) for 48 hours for extraction of the ants. museum collections are referred by the following acronyms: cpdc: centro de pesquisa do cacau, laboratório de mirmecologia, ilhéus, bahia, brazil. inpa: instituto nacional de pesquisas da amazônia, manaus, brazil. mpeg: museu paraense emílio goeldi, belém, pará, brazil. we follow morphological terminology of bolton (1994) and schmidt and shattuck (2014). specimens were identified and measured using a leica m250a stereomicroscope, with magnifications 20x, 40x and 60x under a white light. digital images were made using the same equipment. all measurements are given in millimeters and the abbreviations used are: hw: head width. in full-face view, maximum width of head. hl: head length. in full-face view, from the anterior edge of the clypeus to the medial posterior margin of the head. sl: scape length. in frontal view, measured from apex of first antennal segment to base, excluding the basal condyle. wl: weber’s length. in lateral view, measured from the anterior surface of the pronotum (excluding the collar) to the posterior margin of the propodeal lobes. pw: pronotal width. the maximum width of pronotum in dorsal view. ph: petiole height. in lateral view, the distance from the ventrum of the petiolar sternite to the apex of the petiolar tergite, taken as a vertical measurement perpendicular to the longitudinal axis of the petiole. pl: petiole length. in lateral view, maximum longitudinal distance between the anterior and posterior extensions of the petiolar node, excluding the anterior and posterior condyles. ptl(a3): posttergite length (a3). in lateral view, diagonal measurement, starting from the upper point of the limit between the preand posttergite, and going until the posterior most point of posttergite. ptl(a4): posttergite length (a4). in lateral view, straight measurement, starting from the upper point of the limit between the preand posttergite, and going until the posteriormost point of posttergite. gl: gaster length. in lateral view, maximum length of the gaster. tl: total length. the total outstretched length of the ant from the mandibular apex to the gastral apex. results cryptopone pauli fernandes & delabie, new species (fig. 1ªb; 2ª-d) urn:lsid:zoobank.org:act:8ef3d318-14bd-4a70-9179-52ae1a521036 type material: holotype. brazil, rondônia, floresta nacional do jamari (flona jamari), 63°01'24"w 9°09'40"s, serra da onça, 29.v.2018, in soil sample, d.c. castro col., inpa (1 w.). paratypes: 3 workers with same data as holotype, deposited in inpa (1 w.), mpeg (1 w.) and cpdc (1 w.). etymology: the species honors the first author’s husband, paulo vilela cruz, who has dedicated his life to studying mayflies, not ants (nobody is perfect!). the name is the genitive form of paulus, his latin name. diagnosis: mandibles elongate-triangular, masticatory margin bearing 7 teeth with apical tooth large and acute; clypeus unarmed; petiole rectangular, with anterior margin almost straight in lateral view, its posterior margin straight in dorsal view; body large tl: >7.00 mm. measurements: paratypes (holotype). sl: 0.98-1.02 (1.00), hw: 1.39-1.41 (1.41), hl: 1.39-1.40 (1.39), wl: 1.83-1.85 (1.85), pw: 0.90-0.93 (0.92), pl: 0.47-0.49 (0.49), ph: 0.59-0.67 (0.67), ptl (a3): 0.86-0.95 (0.89); ptl (a4): 0.64-0.68 (0.65); gl: 2.60-2.64 (2.63), tl: 7.05-7.11 (7.10). worker description head and mesosoma punctate, mesopleuron and propodeum punctate-reticulate in lateral view. mandible smooth and shining in dorsal surface (frontal view) with scattered punctures. antennae and legs overall punctate. propodeum punctate and shining in dorsal view; declivious (posterior) face of propodeum, petiolar dorsum and posterior face shining. petiole punctate and shining, gaster shining and sparsely foveolate in dorsal view. body covered with dense pubescence which does not hide sculpturing, pubescence sparse on propodeal dorsum; body brown. long suberect golden hairs on the occipital io fernandes, jhc delabie – new cryptopone from brazil410 margin and ventral face of head. four long suberect, golden hairs present on anteromedian clypeal margin. mandible (dorsal surface) covered with sparse short golden hairs and ventral face with sparse long suberect hairs. antennae and legs with dense pubescence. inner surface of protibia with spiniform setae mixed with finer long pilosity. surfaces of mesotibia with spiniform setae mixed with finer long pilosity. protarsus, mesotarsus, and metatarsus with spiniform setae mixed with finer long pilosity (fig. 2b-d). head subrectangular head in dorsal view, slightly longer than broad, broadened posteriorly, sides convex; frontovertexal margin shallowly concave. mandible elongatetriangular; bearing 7 teeth along masticatory margin; diastema between apical tooth (at) and tooth ii (t2) (fig 2a); basal portion of mandible with distinct oval fovea dorsolaterally. scape barely reaching posterior margin of head; frontal lobes small and closely approximated. eye rudimentary, with 6–7 facets, placed at level of antennal torulus. anterior margin of clypeus convex in frontal view. pronotum in dorsal view with dorsal and lateral faces meeting at blunt angle. mesonotum approximately half as big as pronotum; promesonotal suture distinct; mesometanotal suture feeble; mesopleuron separated from mesonotum by notopleural suture; anapleural sulcus weakly impressed between katepisternum and anepisternum. propodeum with strong constriction between mesonotum, distinctly narrower than mesonotum; propodeum in dorsal view much narrower than mesonotum, its lateral margins slightly diverge posteriorly; propodeum in lateral view slightly depressed below level of mesonotum, forming rounded angle with oblique, evenly convex declivity; metanotal spiracle covered by distinct mesepimeral lobe; propodeal spiracle round, situated close to metapleural gland. protibial apex with one pectinate spur and one simple spur; mesotibial apex with one simple spur and one barbulate spur; metatibial with two spurs, one barbulate and one simple. petiole in profile robustly subrectangular, narrowed toward apex, anterior margin slightly straight, posterior margin slightly straight in dorsal and lateral views; petiolar node in dorsal view trapezoid, node summit rounded in anterior view; subpetiolar process forming reduced keel with inconspicuous teeth. gaster mostly cylindrical, segments gently curving ventrally, abdominal tergite 3 in lateral view with anterior margin weakly convex raising almost vertically; preand postsclerites of a4 separated by distinct constriction; prora absent; sting long, sharp, and upcurved. queen and male: unknown distribution. brazil (rondônia). other material observed for this study (all in cpdc): cryptopone guianensis (weber, 1939): costa rica: heredia, estación biológica la selva, 10°26’n 84°01’w, 50150m, 2-12.vii.2004, ant course 2004 (1 q., 1 w.) [b. jahyny, leg.]; french guiana: macoupa, 1999, p. lavelle col. (1 w) [l. cellini leg.]. fig 1. cryptopone pauli sp. nov. (a) lateral view and (b) dorsal view. fig 2. cryptopone pauli sp. nov. (a), mandible with seven teeth along masticatory margin and diastema between the apical tooth (at) and tooth ii (t2) (fig 2a) (front-lateral view); (b) legs in front-lateral view, note spiniform setae on mesotibia; (c) mesotibia and metatibia in front-lateral view; (d) metatibia and mesotibia in posterior view. sociobiology 66(3): 408-413 (september, 2019) 411 cryptopone holmgreni (wheeler, 1925): brazil: amazonas, manaus, rs 3209, #4832, 19.i.1994, a.b. casimiro col. (1w.); bahia, boa nova, mata úmida t4, pitfall 03, 14°24’08.9"s 40°07’40.8"w, 12.ix.2014, a.s. souza col. (1 w.); bahia, ilhéus, cepec quadra g, #4483g, 17.xii.1991, a.l.b. souza col. (3 w.); idem, i. nascimento col. (4 w.); french guiana: macoupa, 1999, p. lavelle col. (4 w.) [l. cellini, leg.]; petit-saut, xi.2001, s. lacau leg. (1 w.); sainteugène, 19.xi.2001, s. lacau leg. (1 w.). observations about neotropical species of cryptopone: most of the ants observed here were taken in soil samples, except a single specimen of c. holmgreni caught with a pitfall trap. all the neotropical species of cryptopone seem almost strictly cryptobiotic, but in rare cases, as already observed for c. gilva (haskins, 1931 in schmidt & shattuck, 2014) and here for c. holmgreni, workers can forage for short periods on the ground. the macoupa (french guiana) specimens were found in soil samples (tsbf traps) during earthworm diversity studies. although cryptopone species are always rare and we have no natural history details about this series of specimens, the macoupa material suggests that two species (c. guianensis and c. holmgreni) can live in sympatry. due to its cryptobiotic habits, cryptopone presents an important morphological convergence (mesotibial spiniform setae, figure 2b-d) with several other ponerinae genera (feroponera bolton & fisher, 2008, promyopias santschi, 1914, buniapone schmidt & shattuck, 2014 and, especially, centromyrmex mayr, 1866). these setae seem well adapted to allow rapid circulation in narrow tunnels built by earthworms and other underground organisms, as any movement of the legs in contact with the oblique walls would facilitate ant locomotion. furthermore, the morphological convergence with centromyrmex suggests that some species of cryptopone, such as c. holmgreni, could be specialist predators of termites living in the ground (delabie et al., 2000). key to the workers of cryptopone species from the new word (modified from mackay & mackay, 2010). 1. mandibles nearly elongate-triangular, oblique masticatory margin nearly continuous with basal border, the latter having 4 5 teeth; tooth at mid-length of mandible noticeably longer than others; petiolar node as seen from side subtriangular, tapered markedly to a narrowly rounded apex; mexico (veracruz), central america to southeastern brazil (são paulo) ............................................................. c. guianensis (weber) 1’. mandibles triangular, basal and masticatory margins meeting at distinct angle, with at least 6 teeth, approximately equal in length; petiole as seen from side nearly subquadrate, feebly tapered dorsally .........…..........….......…………........ 2 2. dorsal face of petiole narrowly convex, shorter than posterior margin in lateral view; anterior half of subpetiolar process translucent and conspicuously larger; united states (missouri south to florida), mexico, central america at least as far south as panama ……………............... c. gilva (roger) 2’. dorsal face of petiole broadly convex, nearly equal in length to posterior margin in lateral view; anterior half of subpetiolar process not translucent; peru east to the guianas, south to bolivia ..................................................................... 3 3. small species tl: >5.00 mm; mandible with 7 teeth; clypeus is slightly concave medially with a small raised medial tooth along the anterior margin; caribbean islands, colombia to brazil (são paulo) ................................. c. holmgreni (wheeler) 3’. large species tl: >7.00 mm; mandibles with at least 7 teeth; clypeus medially convex and unarmed (rondônia) ...... ....................................................................... c. pauli sp. nov discussion cryptopone pauli n. sp. is easily distinguished from others in the genus by the combination of large size (tl: >7.00 mm) and its clypeus lacking a concave median indentation. the worker of c. holmgreni can be separated from c. pauli sp. nov. by size, since the total length of c. holmgreni does not reach 5.00 mm and the triangular mandible is not elongatetriangular such as in c. pauli sp. nov. also, c. holmgreni is easily recognized as it is the only one in the genus with medial clypeal tooth. cryptopone guianensis can be separated from c. pauli sp. nov. by the elongate mandibles with an unusual tooth at mid-length, noticeably longer than the others. finally, c. gilva can be easily separated from c. pauli sp. nov. by its subpetiolar process, which is translucent and also by the presence of a prora, while in c. pauli sp. nov., it is dull and absent, respectively. schmidt (2013) recovered c. gilva and c. sauteri (wheeler, 1906) in the ponera genus group, closely related with species from eumeryopone forel, 1912, mesoponera emery, 1901 and pseudoponera emery, 1900, but morphological synapomorphies have not yet been discovered (schmidt & shattuck, 2014). although cryptopone most closely resembles pseudoponera, it differs in the presence of the mesotibial spiniform setae. mackay and mackay (2010) synonymized cryptopone with pachycondyla and also noted that wadeura guianensis, the type species of wadeura weber, 1939, is basically a cryptopone with unusual mandibles (schmidt & shattuck, 2014). the genus wadeura differs from typical cryptopone chiefly in mandibular shape (narrower and with longer teeth in wadeura), in the shape of the mesonotum (bulging in wadeura, with the consequent appearance of a depressed propodeum), body size (wadeura is somewhat larger than most cryptopone with tl 1.7–6.1 mm), and metatibial spur count (one in cryptopone, two in wadeura) (schmidt & shattuck, 2014). our new species, c. pauli sp. nov. differs from the typical cryptopone by its large size (tl: >7.00), with spur formulae 1:2:2. c. pauli sp. nov. also differs from the typical wadeura by its mandibular shape, elongate-triangular (narrower and longer in wadeura) and the size tl: >7.00 (tl: 4.10-4.60 in wadeura) (weber, 1969). io fernandes, jhc delabie – new cryptopone from brazil412 wadeura could actually be unrelated to cryptopone and represent a remarkable case of convergence and considering the morphological features mentioned above. c. pauli sp. nov. could shed light on this relationship. the study by borowiec et al. 2019 implies the non-monophyly of cryptopone, with cryptopone hartwigi, a southern african species, more closely related to fisheropone, while cryptopone gilva, a new world species, is closer to ectomomyrmex mayr, 1867. as noted by schmidt and shattuck (2014) and borowiec et al. 2019, the resolution of cryptopone taxonomy would require a more thorough revision and sampling of all species attributed to the genus. regarding its distribution, c. pauli sp. nov. is only known from a single location in the state of rondônia, brazil. this is the first record of the genus for rondônia. with exception of c. gilva, all other new world species of cryptopone are found in south america. brazilian records of the genus are very scarce, probably due to its hypogeic habits. winkler extraction or other common sampling techniques (see bestelmeyer et al., 2000) may be inadequate for sampling subterranean species (fernandes et al., 2015). additionally, nothing is known about the life histories, population structures, or reproductive biology of most rare ant species (brandão et al., 2008). the discovery of c. pauli sp. nov. emphasizes the need for increased exploration of hypogeic ant diversity sometimes called “myrmecology’s last frontier” (ryder wilkie et al., 2007; delabie et al., 2015; wong & guénard, 2017). acknowledgments thanks are due to inpa for providing the facilities for the sorting and identification of the species, and maria aurea pereira silveira for providing the ants from flona do jamari (brascan recuperação ambiental). the authors are grateful to john lattke, john longino, and michael branstetter for their constructive suggestions. i.o.f. is also thankful to the invertebrate collection of inpa for allowing the use of the photographic equipment for this study, and to paulo v. cruz for the help with the figures. doctoral e posdoctoral (pnpd/inpa) scholarship. j.h.c.d. acknowledges his research grant by cnpq. references anderson, j.m. & ingram, j.s.i. 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(2017). subterranean ants: summary and perspectives on field sampling methods, with notes on diversity and ecology (hymenoptera: formicidae). myrmecological news, 25: 1-16. doi: 10.25849/myrmecol. news_025:001 doi: 10.13102/sociobiology.v65i4.3480sociobiology 65(4): 696-705 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 comparative molecular cytogenetics of melipona illiger species (hymenoptera: apidae) introduction melipona is a neotropical genus of stingless bees, that occurs from mexico to argentina (michener, 2007) and with greater diversity in the amazon basin (silveira et al., 2002). moure divided it into four subgenera, eomelipona, melikerria, melipona and michmelia (moure et al., 2007). michener (2007) did not recognize the subgenera for considering them very similar. silveira et al. (2002) also recognize subgenera, but suggest some modifications in moure’s proposal. molecular analyzes of mitochondrial and nuclear genes support this classification (fernandes-salomão et al., 2002; ramirez et al., 2010; rasmussen & cameron, 2010). melipona shows unique characteristics among the meliponini, such as the geneticalimentary process of caste determination and absence of morphologically distinct queen cells (michener, 2007). abstract cytogenetic studies of melipona are scarce with only 24 species analyzed cytogenetically. of these, six species had the rdna sites physically mapped and characterized by fluorescent in situ hybridization (fish). the aim of this study was to perform karyotype analyzes on melipona species from different regions of brazil, with a greater sampling representative of the amazonian fauna and using conventional, fluorochrome staining and fish with heterologous rdna probes. the predominant chromosome number was 2n = 18, however, the subspecies melipona seminigra abunensis cockerell and melipona seminigra pernigra moure & kerr showed 2n = 22 chromosomes. the karyotypes were symmetrical, however melipona bicolor smith, melipona quadrifasciata guérin, melipona flavolineata friese, melipona fuscopilosa moure & kerr, melipona nebulosa camargo presented the first pair heteromorphic in length. cma 3 + blocks also exhibited heteromorphism of size and in almost all cases coincided with rdna sites, except for melipona crinita moure & kerr and m. nebulosa, which presented additional non-coincident cma 3 + blocks. the cma 3 /rdna sites were terminal and interstitial in species with high heterochromatic content, and pericentromeric in the species with low heterochromatic content. in addition to describing cytogenetic features of cytotaxonomic importance, the reorganization of the genome in melipona is also discussed. sociobiology an international journal on social insects v andrade-souza1,4, omp duarte2, ccc martins1, is santos3, mgc costa1, ma costa1 article history edited by cândida aguiar, uefs, brazil received 10 may 2018 initial acceptance 12 june 2018 final acceptance 22 august 2018 publication date 11 october 2018 keywords fish, ribosomal genes, fluorochromes, heteromorphism, amazonian bee fauna. corresponding author marco antonio costa universidade estadual de santa cruz departamento de ciências biológicas rodovia jorge amado, km 16, salobrinho cep 45662-900 ilhéus-ba, brasil. e-mail: costama@uesc.br although there are several studies involving melipona biology, there is a paucity of molecular cytogenetic studies. so far, 23 out of 74 described melipona species (camargo and pedro, 2013; pedro, 2014), were included in cytogenetic studies (tavares et al., 2017), however, with concentrated samplings in the south and southeast brazil. the chromosome numbers n = 9 and 2n = 18, are conserved in this genus and were found in almost all species (rocha et al., 2007; tavares et al., 2017) except for melipona seminigra merrillae cockerell (2n = 22) (francini et al., 2011). in melipona quinquefasciata lepeletier (2n = 18 to 2n = 22) (rocha et al., 2007) and melipona rufiventris lepeletier (2n = 18 to 2n = 19) (lopes et al., 2008) the differentiated chromosome number varied due to the presence of supernumerary chromosomes. in the first species 2n = 18 is the chromosome number of the regular complement, and there is a variable number of b chromosomes. 1 universidade estadual de santa cruz, ilhéus, bahia, brazil 2 universidade federal do sul da bahia, porto seguro, bahia, brazil 3 instituto federal bahiano, santa inês, bahia, brazil 4 universidade federal de roraima, boa vista, roraima, brazil research article bees mailto:costama@uesc.br sociobiology 65(4): 696-705 (october, 2018) special issue 697 in the second species only one supernumerary chromosome was recorded. most of the previous cytogenetic analyzes basically included conventional staining with giemsa, staining with the cma3/dapi fluorochromes and c-banding. only melipona compressipes (fabricius) (rocha et al., 2002), m. quinquefasciata, melipona capixaba moure & camargo, melipona quadrifasciata lepeletier, melipona scutellaris latreille and melipona bicolor smith (rocha et al., 2007) were analyzed using fluorescence in situ hybridization (fish) with probes for localization of rdna sites. characteristics such as conserved dna sequences and large variation in the number of copies make ribosomal genes good cytological markers in the characterization of the chromosome set by in situ hybridization, making possible to make inferences about genetic variability, intra and interspecific divergence (rafael et al., 2003; sochorová et al., 2018). this technique has been used in studies of different insect orders, such as lepidoptera (nguyen et al., 2010), diptera (roy et al., 2005), hymenoptera (carabajal paladino et al., 2013) and hemiptera (salanitro et al., 2017) using homologous probes. in the coleoptera (vitturi et al., 1999), orthoptera (cabrero & camacho, 2008; loreto et al., 2008), lepidoptera (vershinina et al., 2015) and hymenoptera (hirai et al., 1996) heterologous probes were also used. among the bees, stingless bees are still poorly studied in this respect. in this study a comparative karyotype analysis was performed using fish with a 45s rdna heterologous probe, conventional staining and fluorochrome staining, among melipona species from different regions of brazil, with the inclusion of scarcely studied species of the amazonian fauna. material and methods the analyzed samples were collected directly from bees’ nests in their natural habitat or in colonies maintained by research institutions, such as ceplac (research center for cacao crops ilhéus/ba), universidade federal de viçosa (mg), embrapa (pa) as listed in table 1. a minimum number of five individuals per species and 10 metaphases per slide were analyzed. specimens of vouchers identified by dr. gabriel a. r. melo were deposited in the entomological collection pe. j. s. moure of the universidade federal do paraná (dzup), curitiba, brazil. mitotic metaphases were obtained from cerebral ganglia of last-instar larvae, treated with colchicine (0.005%) for 20 min following imai et al. (1988). the chromosomes were stained with giemsa 3% in phosphate buffer, ph 6.8. the selected metaphases were photographed under an olympus cx-41 microscope, equipped with a digital camera. the karyotypes were organized using the adobe photoshop cs4 program. the nomenclatura used was of levan et al. (1964). after air drying, the slides were stained with cma3 (chromomycin a3) (0.5 mg / ml) and counterstained with dapi (4 ‘, 6-diamidino-2-phenylindole) (2 μg / ml) according to guerra and souza (2002). the images were analyzed using the leica dmra2 epifluorescence microscope, captured with the im50 software and overlaid using adobe photoshop cs4. the slides were decolorized and stored at -20°c for further in situ hybridization with 45s rdna probe. the rdna sites were localized using heterologous 45s rdna probe r2 arabidopsis thaliana (brassicaceae), a fragment of 6.5 kb containing copies of the 18s-rdna unit species locality geographic coordinates m. (eomelipona) bicolor smith viçosa-minas gerais 42 w 52’ 54”, 20 s 45’ 15” m. (melikerria) fasciculata smith belém-pará 48 w 30’ 15”, 1 s 27’ 21” m. (melikerria) grandis guérin xapuri-acre 68 w 30’ 15”, 10 s 39’ 06” m. (melipona) quadrifasciata guérin viçosa-minas gerais 42 w 52’ 54”, 20 s 45’ 15” m. (michmelia) fuscopilosa moure & kerr xapuri-acre 68 w 30’ 15”, 10 s 39’ 06” m. (michmelia) seminigra pernigra moure & kerr santarém-pará 54 w 42’ 29”, 2 s 26’ 36” m. (michmelia) seminigra abunensis cockerell rio branco-acre 67 w 48’ 35”, 9 s 58’ 29” m. (michmelia) crinita moure & kerr xapuri-acre 68 w 30’ 15”, 10 s 39’ 06” m. (michmelia) flavolineata friese belém-pará 48 w 30’ 15”, 1 s 27’ 21” m. (michmelia) mondury smith viçosa-minais gerais 42 w 52’ 54”, 20 s 45’ 15” m. (michmelia) nebulosa camargo xapuri-acre 68 w 30’ 15”, 10 s 39’ 06” m. (michmelia) scutellaris latreille ilhéus-bahia 39 w 02’ 57”, 14 s 47’ 21” m. (michmelia) aff. flavolineata capixaba-acre 67 w 40’ 31”, 10 s 34’ 2” m. (michmelia) aff. flavolineata brasiléia-acre 68 w 44’ 52”, 11 s 00’ 9” m. (michmelia) aff. flavolineata xapuri-acre 1 68 w 30’ 15”, 10 s 39’ 06” m. (michmelia) aff. flavolineata xapuri-acre 2 68 w 30’ 15”, 10 s 39’ 06” table 1 melipona species analyzed and collection sites. v andrade-souza, omp duarte, ccc martins, is santos, mgc costa, ma costa – comparative molecular cytogenetics of melipona species698 5.8-25s (wanzenböck et al., 1997). the fish procedures were done according to the protocol of moscone et al. (1996) with small modifications, at 72% of stringency. the probes were labeled with cyanine 3 (cy3-dutp) by nick translation (invitrogen). the hybridization mixture contained 50% v/v formamide, 5% w/v dextran sulfate and 2xssc, 2-5 ng/ μl of the probe. the preparations were hybridized in situ overnight followed by stringency washing. the preparations were counterstained with dapi (2 μg/ml) and mounted on vectashield mount medium. the images were obtained using the leica drma2 microscope. results the chromosome number found for most species analyzed was 2n = 18, except for melipona seminigra abunensis cockerel and melipona seminigra pernigra moure & kerr, which presented 2n = 22 (fig 1). the species melipona grandis guérin, melipona nebulosa camargo and m. seminigra abunensis and m. seminigra pernigra had the karyotypes determined for the first time. the species had symmetrical karyotypes, with slight gradual decrease of size in the chromosomal pairs. fig 1. melipona karyograms based on giemsa staining. species with 2n = 18: melipona bicolor (a), melipona quadrifasciata (b), melipona flavolineata (c), melipona fuscopilosa (d), melipona nebulosa (e), melipona scutellaris (f), melipona grandis (g), melipona mondury (h), melipona fasciculata (i), melipona aff. flavolineata (capixaba-ac) (j), m. aff. flavolineata (brasiléia-ac) (k), m. aff. flavolineata (xapuri-ac) (l), m. aff. flavolineata (xapuri-ac) (m), melipona crinita (n), and with 2n = 22 melipona seminigra pernigra (o) and melipona seminigra abunensis (p). the chromosomes were arranged in descending order of size. bar = 10 µm. sociobiology 65(4): 696-705 (october, 2018) special issue 699 nevertheless they differed substantially in other features. the species m. bicolor, m. quadrifasciata, melipona flavolineata friese, melipona fuscopilosa moure & kerr, m. nebulosa have the first heteromorphic chromosome pair, with one of the chromosomes of the pair with a distinctly longer size compared to the other chromosomes (fig 1a-e). in m. scutellaris, m. seminigra abunensis and m. seminigra pernigra the first pair is found in the homomorphic or heteromorphic condition (fig 1f, o, p). the karyotypes of m. flavolineata, m. fuscopilosa, m. nebulosa, m. scutellaris, m. grandis, melipona mondury smith, melipona fasciculata smith, m. aff. flavolineata, melipona crinita moure and kerr, m. seminigra pernigra, m. seminigra abunensis presented chromosomes with the euchromatin distribution restricted to the terminal regions and a high heterochromatic interstitial content, making it difficult to locate the centromere and determine the morphology of the chromosomes (fig 1c-p). m. bicolor presented submetacentric, acrocentric chromosomes, and the first pair metacentric (fig 1a). m. quadrifasciata showed submetacentric and acrocentric chromosomes, and the first heteromorphic pair composed of a longer metacentric and shorter submetacentric (fig 1b), as previously described. the cma3/dapi fluorochrome staining revealed heteromorphisms in relation to the length of the cma3 + blocks, differences in the chromosomal locations and in the number of karyotypic markings. m. fasciculata and m. grandis (fig 2a, b) had interstitial markings, m. quadrifasciata and m. bicolor (fig 3a, f), pericentromeric markings and in the other species (fig 2c-h, 3b -e, h) markings occurred in the terminal regions. in most species a single chromosomal pair with cma3 + regions has been observed. m. nebulosa and m. crinita (fig 3g, h), however revealed a distinct pattern, showing four fig 2. cma3/dapi staining in metaphases of: melipona fasciculata (a), melipona grandis (b), melipona flavolineata (c), melipona scutellaris (d), melipona fuscopilosa (e), melipona mondury (f), melipona seminigra pernigra (g) and melipona seminigra abunensis (h). cma3 + (green bands). bar = 10 μm. v andrade-souza, omp duarte, ccc martins, is santos, mgc costa, ma costa – comparative molecular cytogenetics of melipona species700 cma3 + blocks, located in the first and second chromosome pairs. in the first species the blocks were homomorphic and in the second species there was heteromorphism related to the location of the cma3 + block between the two pairs. the dapi evenly stained almost the entire chromosome, except the terminal regions that were weakly stained. the signals of hybridizations with the rdna probe coincided in number, size and chromosomal pair with the cma3 + bands (fig 4 and 5), except for m. crinita and m. nebulosa (fig 5g, h) in which only two of the markings cma3 + sites coincided with rdna sites. most of the samples had the rdna band in the first pair except m. flavolineata, m. scutellaris, m. crinita, m. seminigra abunensis and m. seminigra pernigra, which was located in the second chromosomal pair and m. fuscopilosa, with marking located in the 6th chromosomal pair. discussion the chromosomal number 2n = 18 obtained in most of the species analyzed in this study is consistent with the predominant chromosome number in the genus melipona. except for the m. seminigra species in which the chromosome number 2n = 22 (fig 1o, p) was similar to that found for the subspecies m. seminigra merrillae (francini et al., 2011). rocha and pompolo (1998) reported the different chromosome number, 2n = 18, for m. seminigra fuscopilosa species. however, camargo and pedro (2013) propose the specific status for this form as m. fuscopilosa. our results support this specific status by recording the differentiated chromosome number in the two subspecies maintained in melipona seminigra friese. fig 3. cma3/dapi staining in metaphases of melipona quadrifasciata (a), melipona. aff. flavolineata (xapuri-ac) (b), m. aff. flavolineata (brasiléia-ac) (c), m. aff. flavolineata (xapuri-ac) (d), m. aff. flavolineata (capixaba-ac) (e), melipona bicolor (f), melipona crinita (g) and melipona nebulosa (h). cma3 + (green bands). bar = 10 μm. sociobiology 65(4): 696-705 (october, 2018) special issue 701 the different chromosome number obtained for the subspecies of m. seminigra suggests that the increased chromosome number is a derived character within the michmelia subgenus, and may have originated by chromosome fission (francini et al., 2011; tavares et al., 2017) with subsequent addition of heterochromatin, resulting in a similar size to the other chromosomes of the karyotype. analysis of other species of this group, however, may reveal whether the chromosome number 2n = 22 is maintained or if there is variation between closely related species. the heteromorphism in the first chromosome pair was observed in eight of the 13 species analyzed. this record had already been made in m. bicolor and m. quadrifasciata (rocha & pompolo, 1998), and m. mondury (lopes et al., 2008), however, for m. flavolineata in the analysis of lopes et al. (2011) this pair was homomorphic. this heteromorphic difference, according to lopes et al. (2008) and rocha and pompolo (1998) are due to an additional amount of heterochromatin in the larger chromosome, indicating c-band heteromorphism. the great similarity as to the relative size fig 4. fish with 45s rdna in metaphases of: melipona fasciculata (a), melipona grandis (b), melipona flavolineata (c), melipona scutellaris (d), melipona fuscopilosa (e), melipona mondury (f), m. seminigra pernigra (g) and m. seminigra abunensis (h). bar = 10 μm. v andrade-souza, omp duarte, ccc martins, is santos, mgc costa, ma costa – comparative molecular cytogenetics of melipona species702 and morphology of this pair among the species suggests the homeology and consequently stability of this chromosomal pair in the karyotype evolution of melipona. cytogenetic confirmation of this hypothesis would require additional analyzes such as microdissection and chromosome painting by heterologous hybridization. according to rocha et al. (2002) species of group ii, those with high interstitial heterochromatic content (michmelia and melikerria subgenera), form a natural group and these authors suggest that the higher content of heterochromatin is a derived character. however, due to the polyphyletic character of the subgenus eomelipona, whose diagnostic morphological characters are symplesiomorphies (silveira et al., 2002) and the fact that the representatives of the subgenus are separated in the molecular phylogeny of ramirez et al. (2010), the consideration of group ii as natural should be discussed and investigated with the cytogenetics of a larger group of species. interestingly, although there is a large interspecific difference in relation to heterochromatic content, the chromosome number is conserved in melipona. these results suggest that there was conversion of euchromatin into heterochromatin or vice versa besides the heterochromatin addition. this transformation was proposed to explain this karyotype difference between mytilus species (mollusca) (martínez-expósito et al., 1997). in melipona, the presence of the low eucromatic content in the species of the supposed group i, compared to those of the group ii (sensu rocha & pompolo, 1998) reinforces this hypothesis of alteration in the content of euchromatin and heterochromatin. fig 5. fish with 45s rdna in metaphases of m. quadrifasciata (a), melipona aff. flavolineata (xapuriac1) (b), m. aff. flavolineata (brasiléia-ac) (c), m. aff. flavolineata (xapuri-ac2) (d), m. aff. flavolineata (capixaba-ac) (e), melipona bicolor (f), melipona crinita (g) and melipona nebulosa (h). bar = 10 μm. sociobiology 65(4): 696-705 (october, 2018) special issue 703 the location of the cma3 + blocks was also different among the species with low and high heterochromatic content analyzed. among the former are m. quadrifasciata and m. bicolor analyzed in the present study (fig 3a, f) and melipona asilvae moure, melipona marginata lepeletier, melipona subnitida ducke (rocha et al., 2002) and melipona mandacaia smith (rocha et al., 2003), which had markers on the pericentromeric regions coincident with the respective c-band regions (rocha et al., 2002). the second group comprises the species that had the cma3 + markings in the terminal regions or interstitial euchromatic regions, which stained weakly with dapi. these characteristics were also observed in other species of this genus by rocha et al. (2002) and lopes et al. (2008). the interstitial marking in m. fasciculata was previously observed by lopes et al. (2011). m. mondury and m. scutellaris, exhibited moderate gc content, whose fluorescence was less intense, in the terminal regions of all chromosomes. lopes et al. (2008) also observed this characteristic in m. mondury and m. rufiventris, however in that study it was more evident possibly due to the method of staining applied with cma3 and distamycin (cma3da), which confers higher contrast in the regions rich in gc. the registration of four cma3 + markings in m. nebulosa and m. crinita is unprecedented in the genus and differs from rocha et al. (2002), who recorded only one chromosomal pair with cma3 + band in m. crinita from the same locality. in the phylogeny of ramirez et al. (2010) these species are in separate groups, and in m. flavolineata that is in the same group of m. crinita only two cma3 + sites were observed, suggesting that the presence of the two additional cma3 + sites may have arisen independently in the respective groups. the correlation between cma3 + regions and rdna sites observed here was also found in other stingless bee species, such as partamona peckolti (friese) and partamona cupira (smith) (brito et al., 2003; marthe et al.,2010) and melipona (rocha et al., 2002). fish analysis in the present study revealed that the additional cma3 + bands in m. crinita and m. nebulosa do not represent additional rdna sites (fig 3g, h, and 5g, h). these bands may correspond to a satellite dna region rich in gc base pairs resulting from translocation or chromosomal inversion, since they are located in the interstitial region. however, in the review on insect satellite dna, at content in hymenoptera ranged from 46-72%, indicating that there is no predominance of gc content (palomeque & lorite, 2008). confirmation of this information would reveal a different type of satellite dna in these species. for clarification, fish with specific satellite dna probe would be recommended. a characteristic present in all species analyzed was heteromorphism of size of the cma3 + sites and rdna sites. different hypotheses attempt to explain this variation, including the terminal localization of rdna sites that influence variation in the number of copies of the genes (hanson et al., 1996), the occurrence of unequal crossing over and the differential amplification of intergenic sequences in nor (nucleolar organizer regions) (fernandes & martinssantos, 2006). the heteromorphism in these sites has been well documented in megoura viciae (aphididae) buckton (mandrioli et al., 1999), drosophila (roy et al., 2005), maxillaria (orchidaceae) (cabral et al., 2006) and salvelinus (salmonidae) (śiliwińska-jewsiewicka et al., 2015). within the michmelia subgenus we found differences regarding the location of clusters of ribosomal genes and their position on the chromosome, including closely related groups such as the species m. flavolineata from the state of pará and m. aff. flavolineata from the acre region. the chromosomal number 2n = 22 for m. seminigra species suggests a derived state in the subgenus. these characteristics together with the presence of additional cma3 + sites in m. crinita and m. nebulosa suggest that the genome of this group undergoes a more accelerated restructuring process. other analyzes including a larger number of species, especially the subgenus eomelipona, melipona and melikerria, should be carried out in order to expand information on karyotype evolution in the entire genus melipona and to correlate them with available phylogenetic proposals. acknowledgments authors thank dr. marcelo guerra for having kindly provided the rdna r2 probe. this work was supported by the fundação de amparo a pesquisa do estado da bahia (fapesb) and by the coordenação de aperfeiçoamento de pessoal de nível superior (capes), which granted a phd. scholarship to v.a.s. authors’ contribution v andrade-souza, mgc costa and ma costa conceived the study; v andrade-souza wrote the manuscript and analyzed the data; omp duarte, ccc martins and is santos helped in the preparation of the slides. references brito, r.m., caixeiro, a.p.a., pompolo, s.g. & azevedo, g.g. 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(1997). ribosomal transcription units integrated via t-dna transformation associate with the nucleolus and do not require upstream repeat sequences for activity in arabidopsis thaliana. plant journal, 11: 1007-1016. doi: 10.1046/j.1365313x.1997.11051007.x http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6wnh-4yygh08-1&_user=10&_coverdate=08%2f31%2f2010&_alid=1370872262&_rdoc=3&_fmt=high&_orig=search&_cdi=6963&_sort=r&_st=13&_docanchor=&_ct=167&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=183165c47dd91d32224f15ceb324b8d4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6wnh-4yygh08-1&_user=10&_coverdate=08%2f31%2f2010&_alid=1370872262&_rdoc=3&_fmt=high&_orig=search&_cdi=6963&_sort=r&_st=13&_docanchor=&_ct=167&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=183165c47dd91d32224f15ceb324b8d4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6wnh-4yygh08-1&_user=10&_coverdate=08%2f31%2f2010&_alid=1370872262&_rdoc=3&_fmt=high&_orig=search&_cdi=6963&_sort=r&_st=13&_docanchor=&_ct=167&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=183165c47dd91d32224f15ceb324b8d4 _hlk521227562 _hlk521336030 _hlk521336995 _hlk521228171 _hlk521227987 _hlk521229323 _hlk521253275 _hlk521336102 _hlk521335841 _hlk521336538 doi: 10.13102/sociobiology.v66i1.3665sociobiology 66(1): 154-160 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 richness of lichens consumed by constrictotermes cyphergaster in the semi-arid region of brazil introduction termites are eusocial insects and the most important decomposers in tropical forest, savanna, and desert ecosystems (lo & eggleton, 2011). in a remarkable display of adaptive radiation of feeding habits, termite diets include, among different species, necromasses in different stages of decomposition, herbaceous plants, litter, fungi, termite nests or mound materials, animal excrement, carcasses, soil organic material (humus), and lichens (lee & wood, 1971; wood, 1978; edwards & mill, 1986; bignell & eggleton, 2000; donovan et al., 2001). lichens have been reported being used as food resources by hospitalitermes, grallatotermes, longipeditermes, and lacessitermes termites (roisin & pasteels, 1996; miura & abstract the consumption of lichens by constrictotermes cyphergaster termites is suggested in the literature, but not yet demonstrated with concrete evidence. we examined the use and richness of lichens consumed by c. cyphergaster during both the dry and rainy seasons in a semiarid environment in northeastern brazil by monitoring the foraging of five termite colonies for ten consecutive days during each period. twenty-nine species of corticolous lichens were consumed by c. cyphergaster, with seasonal variations in the richness of their ingestion. chrysothrix xanthine, pertusaria flavens, and dirinaria confluens were the lichen species most consumed. tlc analyzes of termite gut contents revealed twelve secondary lichen compounds ingested in both seasons, while staining showed fragments of fungal hyphae, green algae, and typical lichen spores. this study represents the first systematic survey of the abundances of lichens that compose the diet of c. cyphergaster and indicates the seasonal selectivity of that resource related to the chemical compositions of the lichen stalks. sociobiology an international journal on social insects am barbosa-silva¹, ac silva¹, ecg pereira2, mll buril2, nh silva2, mes cáceres3, a aptroot4, ma bezerra-gusmão5 article history edited by evandro n. silva, uefs, brazil received 07 august 2018 initial acceptance 07 september 2018 final acceptance 21 october 2018 publication date 25 april 2019 keywords food resources; nutritional ecology; foraging; seasonality; termites. corresponding author ana márcia barbosa-silva. universidade federal da paraíba departamento de sistemática e ecologia laboratório de termitologia cidade universitária cep 58059-900 joão pessoa-pb, brasil. e-mail: anamarcia1983@hotmail.com matsumoto, 1997, 1998; martius et al., 2000), and one of the six described species of the genus constrictotermes (bourguignon et al., 2011). according miura and matsumoto (1997), lichens can provide 10 to 60 times more nitrogen to those insects than wood (their main food source). except for the energy roles of lichens in termite diets, little is known about interactions between those organisms, their foraging strategies, or the species richness of lichens that make up their diet. lichens are stable, self-sustaining unions between fungi (the mycobiont) and photoautotrophic algae or cyanobacteria (the photobiont), and represent a significant portion of the earth’s biological diversity (honda, 2012). among other ecosystem services provided by lichens is the production of a variety of antiherbivory compounds that can be tolerated by some animals (gerson & seaward, 1977; 1 universidade federal da paraíba, joão pessoa, brazil 2 universidade federal de pernambuco, recife, brazil 3 universidade federal de sergipe, itabaiana, brazil 4 abl herbarium, nl-3762 xk soest, the netherlands 5 universidade estadual da paraíba, campina grande, brazil research article termites sociobiology 66(1): 154-160 (march, 2019) 155 richardson & young, 1977) and used for their own benefit (hesbacher et al., 1995). the termite constrictotermes cyphergaster (termitidae), found in the caatinga and cerrado biomes in brazil, has been described as a potential lichen consumer (bordereau & pasteels, 2011; mathews, 1977), but there is no firm evidence to date of that behavior. moura and vasconcellos (2006) observed that the termite c. cyphergaster maintained contact with lichens during foraging, but those authors were unable to conclude if the lichens were actually being consumed. furthermore, bezerra-gusmão (pers. comm.) observed termite individuals foraging extensively on lichens and assumed they were used as food resources. in order to demonstrate the consumption of lichens by c. cyphergaster, we surveyed the diversity of lichen species ingested by that termite in an area of caatinga vegetation in the semiarid region of northeastern brazil and tested the hypothesis that this consumption would be influenced by seasonal factors. materials and methods study area field observations were conducted at the são joão do cariri experimental station (eesjc) (7o20’34”s, 36o31’50”w) belonging to federal university of paraíba (ufpb) in northeastern brazil. the station area covers 381 ha, at elevations ranging from 400 to 700 m a.s.l. the region has an average annual precipitation rate of 386 mm, 50% humidity, and mean monthly temperatures ranging from 28 to 35 ºc (paraíba state government, 1985). the regional climate is hot and dry, with irregular distributions of short periods of rainfall, with a prolonged dry season. the regional vegetation is open, tree-shrub caatinga, dominated by the plants cenostigma pyramidale tul., croton blanchetianus müll. arg., combretum leprosum mart., jatropha mollissima (pohl) baill, aspidosperma pyrifolium mart., and tacinga palmadora (britton and rose) n.p. taylor and stuppy (barbosa et al., 2007). the soils are classified as chromic/verticluvisols (alfisols usda) in lowland areas, with litholic neosols associated with rock outcrops in an undulating landscape at higher elevations (chaves et al., 2000). sampling procedures five nests of c. cyphergaster spaced at least 50 m one from another were monitored during foraging activities in both the dry (november of 2012) and rainy (march of 2013) seasons. observations were made over ten consecutive days for each climatic period (focusing always on the same nests), from 18:00h to 06:00h. all established foraging columns were monitored from the time the termites first left their nests to the farthest points of their foraging routes (about 18 m). all of the lichen thalli explored were marked to determine termite visitation frequencies (= no. of times). the study did not take into account trails in the vegetation canopy, limiting its focus to the lower portions of the vegetation (up to ca. 2 m). at the end of the ten days of monitoring, the lichens visited were collected for identification and chemical analyses. lichen identifications were made using thalli characteristics such as shape, ascoma type, and ascospore color, size, and septation. transversal sections of the thalli and ascoma were treated with iodine (i) and/or potassium hydroxide (koh). the species were identified by consulting cáceres (2007), lücking and rivas-plata (2008), and marbach (2000). lichen intake in order to evaluate lichen intake by termites, 20 workers from each nest population were captured after leaving a lichen thallus and placed in distilled water. a portion of the gut (crop) of each worker was dissected and its contents extracted.that material was stained with neutral red, phenol red, or 10% toluidine blue to detect the presence of lichen structures. lichen chemical analysis the presence of lichenic compounds in c. cyphergaster guts was evaluated in 100 workers from each nest that were collected directly in the field while foraging on lichens. the workers were dissected, their digestive guts removed, and extracts were prepared using cold acetone. the lichen patches visited by c. cyphergaster were scraped with a steel blade and 10 mg of the biomass of each species was also extracted with acetone (1ml) to obtain their secondary metabolic compounds. the extracts were subjected to thin layer chromatography (tlc) on f254+366 merck silica gel plates that were run in solvent system a (toluene/dioxane/acetic acid 180: 45: 5, v/v/v) (culberson, 1972). after solvent evaporation, the plates were developed under long and short uv light, sprayed with 20% sulfuric acid, and subsequently heated on a hot plate at 50 °c until the bands became visible. the compounds were identified based on their rf and band colors, which were compared to standards of atranorin, divaricatic, usnic, norstictic, parietin, estictic, tamnolic, and didimic acids, compounds frequently encountered in lichens. literature reviews on lichen biochemistry were consulted, and the descriptions of rambold et al. (2001) were used to identify bands not comparable to the standards used. data analysis the relative frequencies of termite visits to the lichens were determined by the numbers of times each species was visited during the survey. differences in termite visitation frequencies to lichens between the dry and rainy seasons were evaluated using the kruskal-wallis test. lichen community am barbosa-silva et al. – consumption of lichens bytermites156 similarities between the two seasons were evaluated using nonmetric multidimensional scaling (nmds), and the structures of lichen clusters were compared through similarity analysis (anosim), at a significance level of 5%. the analyzes were performed using primer 6.1.1 software. results twenty-nine crustose lichen species distributed among 17 genera were found to be associated with the diet of c. cyphergaster (table 1). the richness of lichen species consumed by termites was dissimilar between seasons (fig 1). the dissimilarity observed using nmds was confirmed by anosim (global test: r = 0.192; p = 0.04). eleven lichen species were ingested only in the rainy season, ten only in the dry season, while eight were consumed during both climatic periods (table 1). table 1. lichens consumed by constrictotermes cyphergaster, relative frequency (%) of visits to the lichen thalli and secondary compounds observed in a semiarid region, ne brazil. legend: ds – dry season; rs – rain season. lichens consumed relative frequency (%) secondary compounds dry season ds rs anisomeridium subprostans (nyl.) r.c. harris 4.2 0 unidentified substance anisomeridium tamarindi (fée) r.c. harris 4.2 0 no substance detected graphis submarginata lücking 2 0 norstictic acid graphis sp. adans 2 0 norstictic acid lecanora achroa nyl. 8.3 0 norstictic acid; 2’-o-methylperlatolic acid lecanora helva stizenb. 4.2 0 2’-o-methylperlatolic acid opegrapha cf. arengae vain 2.1 0 2’-o-methylperlatolic acid pertusaria quassiae (fée) nyl. 2.1 0 stictic acid pertusaria sp. dc. 2.1 0 unidentified substance polymeridium proponens (nyl.) r.c. harris 2.1 0 no substance detected rainy season anisomeridium albisedum (nyl.) rc harris 0 1.2 no substance detected arthonia sp. ach. 0 7.4 unidentified substance coniocarpon cinnabarinum dc. 0 1.2 no substance detected canoparmelia sp. elix & hale 0 1.2 stictic acid clandestinotrema sp. rivas plata, lücking & lumbsch 0 2.5 no substance detected graphis cincta (pers.) aptroot 0 4.9 tamnolic acid mycoporum eschweileri (müll. arg.) r.c. harris 0 1.2 no substance detected lecanora leprosa fée 0 8.6 stictic acid polymeridium albocinereum (kremp.) r.c. harris 0 1.2 no substance detected polymeridium amyloideum r.c. harris 0 2.5 lichexanthone polymeridium subcinereum (nyl.) r.c. harris 0 1.2 no substance detected lichens consumed in both periods rainy dry chrysothrix xanthina (vainio) kalb 18.7 6.2 vulpinic acid vulpinic acid arthopyrenia cinchonae (ach.) müll. arg. 8.3 6.2 no substance detected no substance detected dirinaria confluens (fr.) d. d. awasthi 12.5 10.2 divaricatic acid; terpenes divaricatic acid; triterpenes dirinaria leopoldii (stein) d.d. awasthi 4.2 1.2 divaricatic acid triterpenes glyphis scyphulifera (ach.) staiger 4.2 11.1 no substance detected no substance detected leucodecton occultum (eschw.) frisch 4.2 7.4 stictic acid norstictic acid; stictic acid pertusaria flaven nyl. 10.4 23.4 didymic acid unidentified substance pyrenula anomala (ach.) vain. 4.2 1.2 unidentified substance no substance detected fig 1. non-metric multi-dimensional scaling ordination of similarity of lichen diversity ingested by constrictotermes cyphergaster in the dry (2012) and rainy (2013) seasons in the semiarid region of northeastern brazil. sociobiology 66(1): 154-160 (march, 2019) 157 termite visitation frequencies to the lichens did not differ significantly between the two seasons (p = 0.69; df = 1; h = 0.15). the lichens chrysothrix xanthina, dirinaria confluens and pertusaria flavens and lecanora achroa were the most visited ones. fig 2. thin layer chromatography of lichens organic extracts ingested by constrictotermes cyphergaster and crop food content of workers during foraging in dry season (2012) in a semiarid region of northeastern brazil. chromatography points: 1-18: (lichens): 1anisomeridium subprostans; 2a. tamarindi; 3arthopyrenia cinchonae; 4chrysotrix xanthina; 5dirinaria confluens; 6d. leopoldii; 7glyphis scyphulifera; 8graphis submarginata; 9graphis sp.; 10lecanora achroa; 11l. helva; 12leucodecton occultum; 13opegrapha cf. arengae; 14 pertusaria flavens; 15p. quassiae; 16pertusaria sp.; 17polymeridium proponens; 18pyrenula anomala. 19 to 23: (craw food content of workers who ate lichens): 19nest population 1; 20 nest population 2; 21nest population 3; 22nest population 4; 23nest population 5. 24-27: (standard substances): 24atranorin; 25divaricatic acid; 26usnic acid; 27nortistic acid. fig 3. thin layer chromatography of lichens organic extracts ingested by constrictotermes cyphergaster and crop food content of workers during foraging in wet season (2013) in a semiarid region of northeastern brazil. chromatography points: 1-19: (lichens): 1 anisomeridium albisedum; 2arthonia sp; 3arthopyrenia cinchonae; 4canoparmelia sp.; 5chrysothrix xanthina; 6clandestinotrema sp.; 7coniocarponcin nabarinum; 8dirinaria confluens; 9d. leopoldii; 10glyphis scyphulifera; 11graphis cincta; 12mycoporumes chweiler; 13lecanora leprosa; 14leucodecton occultum; 15pertusaria flavens; 16polymeridium albocinereum; 17p. amyloideum; 18 p. subcinereum; 19pyrenula anomala. 20 to 24: (food contents of the workers who ate lichens): 20nest population 1; 21 nest population 2; 22nest population 3; 23nest population 4; 24nest population 5. 25-30: (standards substances): 25divaricatic acid; 26tamnolic acid and didymic; 27stictic acid; 28usnic acid; 29atranorin; 30parietin. tlc analyses revealed twelve secondary lichen compounds ingested in both seasons (table 1; figs 2 and 3). in the dry season, six tlc bands were observed in the acetone extracts of termite crop samples. bands with rf = 0.82 and 0.42 were identified as atranorin and divaricatic acid respectively (fig 2). § § 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 lichen species ingested food content of workers standard substances 0.60 0.53 0.82 0.64 0.06 triterpenes 0.50 0.69 0.42 0.51 0.56 0.62 0.07 0.07 0.06 0.30 stictic vulpinic 2’-o–methylperlatolic acid atranorin usnic divaricatic norstictic 0.79 usnic 0.62 0.62 0.620.51 0.51 0.40 0.6 0.13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 lichen species ingested food contente of workers standard substances vulpinic 0.890.91 stictic divaricatic 0.64 0.79 terpenes 0.84 0.79 0.84 0.84 0.85 didymic 0.84 divaricatic lichexanthone stictic tamnolic am barbosa-silva et al. – consumption of lichens bytermites158 the rf = 0.64 band was apparently equivalent to usnic acid, but the staining reaction did not correspond well to that standard (possibly due to its low concentration in the gut contents of the termites). other compounds with rf = 0.60, 0.53, and 0.42 did not coincide with any of the standards used and could not be identified (fig 2). four bands were observed in the gut contents of termites in the rainy season, and three of them coincided with the standards for didimic (rf = 0.84), usnic (rf = 0.79), and tamnolic acid (rf 0.62). an additional band was observed at rf = 0.89 that did not correspond to any of the chemical standards utilized in this analysis (fig 3). color analyses of the solid material collected from c. cyphergaster worker crops revealed algal cells, different spore types, and fungal hyphae (fig 4). sp. in their digestive tracts (collins, 1979). the consumption of pertusaria spp. and lecanora spp. by snails and lepidopterans was previously observed (plitt, 1934; bailey, 1970; boch et al., 2015). those lichens were among the most visited by termites in the present study, and their thalli were the most frequent along the foraging trails of c. cyphergaster. boch et al. (2015) reported that snails appear to prefer the lichens that are most frequent in their habitat, and they avoid less common species through their recognition of secondary compounds – supporting the idea that consumers adapt more readily to the most abundant foods. some lichenivorous invertebrates are considered potential dispersers of photobionts and mycobionts through their feces (nash, 2008; fröberg et al., 2001; boch et al., 2011), and it is possible that c. cyphergaster makes up part of that group. many of the green algae and spores found in that termite’s digestive tract were apparently viable, and could form new lichens. tests of the viability of those structures are currently being undertaken to clarify that hypothesis (barbosa-silva et al., in preparation). according to lawrey (1980), lichen-derived compounds can show direct toxicity to the intestinal microflora of invertebrates that consume them. bezerra-gusmão et al. (2015) were able to inhibit the growth of eight bacterial symbiont morphotypes in the guts of c. cyphergaster with usnic acid. other compounds, such as norsictic and divarictic acids, and atranorin and parietin present in the lichens consumed by c. cyphergaster, also have antifungal and/or antibiotic effects (bustinza, 1950; ribeiro et al., 2002; tay et al., 2004; manojlovic et al., 2005). however, the roles of those compounds in termite diets still need to be investigated. the seasonal differences observed in the species of lichens exploited by c. cyphergaster may reflect physicochemical variations in lichen steles (dixon & paiva, 1995; giordani & incert, 2007) as seasonality can promote changes in the activities of antioxidant enzymes, flavonoids, anthocyanins, and phenolic compounds found in lichens (ghorbanli et al., 2012) that presumably alter the palatability of lichen stems (gauslaa et al., 2013). thus, knowing that the caatinga domain experiences extreme seasonal abiotic conditions (sampaio, 1995; silva et al., 2009), we accept the hypothesis that those variations can promote a seasonal selectivity of lichens by the termites. the overall diversity of lichens ingested by c. cyphergaster is almost certainly greater than that documented in the present study, as it was impossible to investigate their foraging trails in the canopy – limiting our observations to the lower vegetation levels (up to 2 m). the list of lichen species that constitute the diet of c. cyphergaster will almost certainly be extended by future studies in other areas where it occurs. in conclusion, the lichen species recorded in the present study are common in caatinga environments (cáceres, 2007; cáceres et al., 2008; menezeset al., 2011), and c. cyphergaster consumed 29 lichen species (with seasonal variations). this fig 4. lichen structures observed in the solid substrate of the crops of the workers of constrictotermes cyphergaster after consuming lichens in the semiarid region of northeastern brazil. algae cells (a and b); fungal cells (c and d); fungal hyphae (e and f). discussion the presence of lichen structures and compounds in the digestive tract of c. cyphergaster confirms their consumption by that termite. information about cost/benefit ratios between those two organisms are relatively scarce, and there are many more questions than conclusive answers available concerning their interactions. crustose lichens are common in the diets of hospitalitermes termites, as confirmed by the presence of spores of phaeotrema sp., phaeographis sp., and melaspilea sociobiology 66(1): 154-160 (march, 2019) 159 report represents the first systematic examination of lichen intake by those termites and raises the following questions: (1) what is the cost/benefit balance of interactions between termites and lichens? (2) what evolutionary advantage(s) does feeding on lichens offer? and, (3) which lichen compounds play important roles in termite diets? acknowledgements the authors wish to thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for financial support. references bailey, r.h. (1970). field and study notes: animals and the dispersal of soredia from lecanora conizaeoides nyl. ex. cromb. lichenologist, 4: 256. doi: 10.1017/s002428 2970000294 barbosa, m.r.v., lima, i.b., cunha, j.p., agra, m.f. & thomas, w.w. (2007). vegetação e flora no cariri paraibano. oecologia brasiliensis, 11: 313-322. bezerra-gusmão, m.a., soares, r.k.l., costa, h.a., oliveira, m.h., barbosa-silva, a.m., silva, a.k.o., abad, a.c.a., mota, r.a. & pereira, e.c. 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(2009). dinâmica espaço temporal da vegetação no semiárido de pernambuco. revista caatinga, 22: 195-205. tay, t., türk, a.o., yilmaz, m., türk, h. &kivanç, m. (2004). evaluation of the antimicrobial activity of the acetone extract of the lichen ramalina farinacea and its (+)-usnic acid, norstictic acid, and protocetraric acid constituents. verlag der zeitschriftfür naturforschung, 59: 384-388. wood, t.g. (1978). food and feeding habits of termites. in m.v. brian (ed), production ecology of ants and termites (pp. 55-80). cambridge university. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.347-354sociobiology 60(4): 347-354 (2013) “marimbondos”: a review on the neotropical swarm-founding polistines fb noll introduction hymenoptera is the most well-known group of social insects (wilson 1971). ants and bees, probably because of their economical importance, are very well studied groups, while wasps are relegated to a marginal condition. also, ants and bees are specific monophyletic groups (single evolutionary origin), while wasps are termed as everything other than ants and bees. vespidae is a family which comprises highly eusocial wasps, including vespinae and polistinae (carpenter 1991). yellow jackets and hornets (vespinae) are commonly known for their solitary nest initiation, annual colony life cycle and strikingly divergent morphological variation between castes, attributed to a necessary condition of winter survival (westeberhard 1969). polistinae, on the other hand, is much more diverse in terms of social organization. polistes is the group model for several sociobiological theories developed for wasps, but it does not reflect the real diversity and complexity of this subfamily which remains poorly known regarding some genera (noll & wenzel 2008). polistinae can be classified into two behavioral groups: the plesiomorphic (ancient) condition, shared with vespinae, abstract neotropical swarm-founding polistines, the epiponini, compose a highly social tribe of vespids which displays several complex social characteristics, such as: intricate nest building, polygyny, swarm reproduction, and the absence of morphology caste discontinuities, a trait that would not be expected for a highly eusocial group. the biology of epiponini will be reviewed, evidencing the scarcity of information for this group and also that not all social insects might fit in traditional theories for the evolution of social behavior. sociobiology an international journal on social insects universidade estadual paulista, ibilce, são josé do rio preto, são paulo, brazil review article history edited by: marcel hermes, ufpr, brazil received 20 august 2013 initial acceptance 17 october 2013 final acceptance 24 october 2013 keywords social wasps, swarming process, polygyny, colony cycle corresponding author fernando b. noll depto. de zoologia e botânica ibilce-unesp, rua cristovão colombo, 2265 15054-000 são josé do rio preto, sp, brazil e-mail: noll@ibilce.unesp.br which is the independent foundation, of which polistes, mischocyttarus and ropalidiini (partially) are representatives. on the other hand, ropalidiini in part (polybioides and some ropalidia) and epiponini present colony foundation by organized swarms in which female filling different roles (castes) are highly dependent upon each other (jeanne 1991). in contrast to well-studied social insects, such as corbiculate bees, and temperate zone wasps and ants; neotropical swarm-founding wasps present a chimera of characteristics that make them singular and hard to accommodate in current theories on the evolution of social insects (noll &wenzel 2008). the main characteristics are: the complex and diverse nesting habits and construction, polygyny, swarm reproduction, castes lacking a strong morphology variation, and caste flexibility which creates a window for worker reproduction or a relaxation of ovarian control (west-eberhard 1978). it is hard to sketch an evolutionary scenario for the origin of such complex traits consistent with classical theory based on polistes. it is believed that the origin of such wasps is tropical (evans & west-eberhard 1970; carpenter 1993). in contrast to temperate zone areas, seasonal variation is modest and may not constrain colony development, and as a consequence these wasps face a completely different situation in which colonies are perennial and asynchronous (jeanne 1991). as a result, fb noll neotropical swarm-founding polistines348 colonies are multivoltine and not univoltine (when counting production of reproductive individuals) as observed in temperate zone wasps. another important factor that may influence strongly the evolution of these wasps is the extensive predation of their colonies by ants (jeanne 1979), which forces relocation of colonies unpredictably. rapid nest abandonment and swarming, nest architecture, polygyny, and castes lacking clear-cut morphological discontinuities suggest evolutionary adaptations against ant predation as well as the optimization of work (jeanne 1975; wenzel 1998). in this review, some important aspects of the biology of neotropical swarm-founding wasps will be addressed, specifically systematics, swarming process, polygyny, castes and colony cycle of these insects that depart from standard theory. systematics and studied groups because theories of evolution are necessary exercises in reconstructing ancestral states, it is important to have an understanding of the state of the systematics and phylogenetic relationships among taxa in question. the polistinae includes four tribes: polistini (cosmopolitan, except in new zealand), mischocyttarini (argentina through the southeastern united states and the western mountains to british columbia), ropalidiini (australia, sub-sahara africa, the arabian peninsula, eastern tropics, china, korea, japan, iran, equatorial africa, and india) and epiponini (argentina through the southwestern united states (polybia and brachygastra occur in texas and arizona)). the polistinae exhibits greater diversity in tropical regions, especially in the neotropics. there are 26 genera worldwide, with approximately 900 species. epiponini is diverse, especially in the neotropical region, with 19 genera and approximately 234 species described. the first phylogenetic hypothesis for polistinae was proposed by carpenter (1991) (fig. 1a) using primarily morphological data, which presented several polytomies. a much more well-resolved tree was proposed by wenzel and carpenter (1994) (fig. 1c) with the combination of morphology of adults and larvae and nest architecture (wenzel 1993) (fig. 1b). pickett and carpenter (2010) proposed a phylogeny of vespidae based on morphological and molecular data with a different topology for some clades of polistinae determined previously by wenzel and carpenter (1994) (fig. 1d). in general, the clade formed by synoeca, clypearia, metapolybia and asteloeca is placed quite differently, sometimes appearing as sister to the modular, enclosed, stacked brood comes nest building genera, such as polybia, protonectarina, epipona, and sometimes as a component nested within this clade. in any case, irrespective of internal differences in the clades, it is clear that the epiponini composes a monophyletic group. the systematics for each genera of epiponini is a very recent subject. andena et al. (2007b) published the first phylogeny of the genus angiopolybia using morphological data. the other genera that have been subject to cladistic analyses were: pseudopolybia (andena et al. 2007a), angiopolybia (andena et al. 2007b), apoica (pickett & wenzel 2007), synoeca (andena et al. 2009a), epipona (andena et al. 2009b), charterginus (andena et al. 2009c), and brachygastra (andena & capenter 2012). larger genera still need review, such as agelaia and polybia, even though it is already in progress (carpenter jm pers. com.). studies on polistines do not cover the diversity of genera and species well. a previous survey on the number of citations to all social wasps from the subfamily polistinae showed the vast majority of the papers was written on polistes and only on few species (wenzel & noll 2006). for the epiponini, this scenario is not different, with a single genus (polybia) representing the vast majority of studies (wenzel & noll 2006). such restriction is problematic because using a few species as explanatory models may compromise the application of proposed theories for the whole group (see the section caste for more details). in this regard, generalizations should be viewed with caution when they are based on a very small subset of the diversity of species. swarming highly eusocial insects that initiate their colonies by swarms migrate as a cohort of queen(s) and workers from the old nesting site to a new one. brood predation by ants (bouwma et al. 2007), parasites in the brood (west-eberhard 1982), vertebrate attacks on the nest and storm damage (richards 1978) are causes for absconding swarms and may happen at any time. in contrast, reproductive swarms (forsyth 1978; west-eberhard 1982) occur when the colonies are producing gynes (young queens and males) (ezenwa et al 1998). the coordination of a swarm to a new place requires some sort of communication, which is apparently mediated by pheromones (jeanne 1980). in the epiponini, it was observed that pheromones are released in the air to coordinate the swarm (hunt et al. 1995; howard et al. 2002; mateus 2011), in other cases the workers rub their abdomen in leaves or substrates located between the old and new nest sites, as well as in the new nest site (mateus 2011). other colony members land on these marked points, apparently looking for new points along the route to the new nest site (mateus 2011). jeanne (1981) experimentally demonstrated that the secretion produced by richards’ gland, located at the fifth sternal metasomal segment, is used to mark the trail to the new nest in polybia sericea. in parachartergus fraternus, mateus (2011) suggests that venom is used to mark the new nest site and the trail markings. in any case, it is still unknown how workers that mark the trail inform the rest of the colony about the new nest location (sonnentag & jeanne 2009). scout wasps have to induce the rest of the colony to relocate to the new spot and guide them (sonnentag & jeanne 2009; mateus 2011). it was proposed that mechanical signal, such as “bumping” besociobiology 60(4): 347-354 (2013) 349 haviors in which a female bumps her head against idle wasps are responsible for activation and migration to the new spot (sonnentag & jeanne 2009; mateus 2011). a flight around the nest has been suggested by west-eberhard (1982) as a stimulus to idle wasps to relocate as a swarm. a clear signal that a swarm is starting is the buzz running behavior in epiponines and it is triggered by older workers (naumann 1970; forsyth 1978; west-eberhard 1982; mateus 2011). scout wasps are responsible for choosing the new nest site that will be suitable for the colony survival, depending on the type of substrate, height, solar incidence and, probably, food and water availability (mateus 2011). polygyny it is not hard to imagine that the origin of polygyny (presence of several egg-layers coexisting in a given nest) was molded by the same factors as swarming and nest design, for example, ant predation (jeanne 1979). considering that the loss of a single queen would be fatal, the origin of several queens would be advantageous for the colony in terms of survival. among polistines, polygyny appeared three times: polybioides, ropalidia and epiponini (carpenter 1991). epiponines are characterized as permanently polygynous or primarily polygynous with variation (decrease) in queen figure 1 phylogenetic trees for the epiponini based on: a carpenter (1991), bwenzel & carpenter (1994), cwenzel (1993) and dpickett & carpenter (2010). fb noll neotropical swarm-founding polistines350 number during the colony cycle, sometimes resulting in monogyny (west-eberhard 1978; richards 1978; jeanne, 1991). the presence of polygyny in the basal clades of this tribe indicates that the common ancestor already presented long-term polygyny (carpenter 1991). the presence of several females laying eggs could represent a problem in the interpretation of the conflict of interests that usually occurs in social insect colonies. even though relatedness is low in independent founding polistes (pickett et al., 2006), an additional problem arises in the epiponini: because reproduction would be divided among several females, kinship rates would be even lower than what it is found in polistes, and would not be high enough to justify the sterility of workers. however, relatedness studies in epiponini colonies indicate that kinship is relatively high (queller et al. 1988, 1993; hughes et al. 1993). these findings are well understood by west-eberhard (1978, 1981), which showed that after a swarm, colonies of metapolybia aztecoides start with several queens and workers, and as the colony develops some queens disappear or adopt a worker role. as the colony develops further, the number of queens reduces until one or a few queens remain at last. daughter queens and swarms occur predominantly after the establishment of monogyny or oligogyny, restricting the production of gynes to only a few individuals. such a pattern is observed in several other species, such as in polybia occidentalis (west-eberhard 1978; queller et al. 1993), parachartergus colobopterus (strassmann et al. 1991), polybia emaciata (strassmann et al. 1992), protopolybia exigua (gastreich et al. 1993), brachygastra mellifica (hastings et al. 1998), and agelaia multipicta (west-eberhard 1990). in this regard, the cyclical oligogyny was coined to define the decrease in the number of queens, with the production of gynes only during the period with fewer queens, increasing the kinship among daughter-queens, as predicted by the kinselection theory (hamilton 1964 a, b; 1972). another important point for the maintenance of high rates of relationship is that queens in epiponines are singly mated (goodnight et al. 1996). also, the possibility that epiponines may simply be inflexible in terms of losing sociality should not be discarded. castes size differences between castes appeared independently in various taxa and a phylogenetic interpretation shows several distinctly different syndromes, representing a picture more complex than formerly thought (reviewed in noll et al. 2004; noll & wenzel 2008). castes in this tribe challenge standard definitions. as expected for highly eusocial insects, pre-imaginal caste determination has been reported in at least five genera (noll et al. 2004). however, in at least six genera (noll et al. 2004), reproductive females resemble non-reproductives in terms of morphology, and castes also lack physiological discontinuities. in these cases, castes are flexible and determined by disputes among adults rather than by larval manipulation (west-eberhard 1981; mateus et al. 2004; noll & wenzel 2008). also, in several colonies the absence of workers´ sterility (termed “intermediates” by richards & richards 1951) has been identified. their role is debatable, because they are considered as trophic egg or male producers (richards 1971) or young queens (forsyth 1978; west-eberhard 1978; gastreich et al. 1993). the level of ovarian development is inversely related to the number of queens, i.e. in the presence of a few females intermediates present more developed ovaries and vice versa (richards 1971; west-eberhard 1978). also, they are absent in several other taxa (reviewed in noll et al. 2004). anyway, intermediates are part of a more complex scenario related to the evolution of castes and will be more detailed below. the complex scenario of low morphological differentiation and non-sterility of workers is caused by the fact that pre-imaginal determination (wheeler 1991) of castes, highly widespread in social insects, is not a plesiomorphic condition for all epiponini. instead, caste flexibility (west-eberhard 1981) is probably plesiomorphic for these wasps. the classical theory for the origin of sociality in wasps is the polygynous family hypothesis (west-eberhard 1978) in which polistes would be the model. however, as pointed out by noll and wenzel (2008) this reasoning is not so straightforward because in primitive social vespids females compete for reproduction, restraining these societies to solitary or short-term monogynic conditions. that is the opposite of what is seen in the epiponini, in which queens are more tolerant (simões 1977; naumann 1970; herman et al. 2000). in this case, queens of epiponines seem similar to subordinates of polistes that are tolerant of other’s reproduction than the dominant, intolerant, monogynic queens of polistes (fig. 2). if queens in epiponines are loose in terms of controlling reproduction, workers are not. workers test and remove queens from the colony (west-eberhard 1978, 1981; herman et al. 2000, platt et al. 2004) which seems to be important in keeping levels of genetic relatedness high enough to achieve “workers interests” (strassmann 1997, 1998). thus, the main difference between short-term monogyny and polygyny is that in the former, queen-policing is more prevalent and in the latter, worker-policing is (platt et al. 2004). figure 2 shows how eliminating the intolerant monogynic egg-layer and replacing her with her several tolerant subordinates could achieve the first step toward polygyny in the epiponini. the origin of highly tolerant egglayers can have some consequences. the first consequence is that many females would aspire for some chance of reproduction that would lead to caste totipotency (strassmann et al. 2002). thus, it would be expected to find ovary development widespread in members of polygynous societies, and, in fact, several epiponines have laying workers (noll et al. 2004). several basal genera of epiponini fit in the scenario proposed for polygynous groups (west-eberhard 1978, 1981) that is, caste flexibility due to the absence of morphological differences between castes and the sociobiology 60(4): 347-354 (2013) 351 extensive ovarian activation in all females, which is primarily found in the genera angiopolybia, pseudopolybia, parachartergus, chartergellus and leipomelles (noll et al., 2004). thus, the distribution of this characteristic is consistent with the presence of laying workers being, in fact, a primitive state rather than derived. in contrast to the intuitive pattern of complex societies is that obligatorily two morphs, the queen and the worker, epiponines lack such a morphological skew, and so may have been thought to be less complex than species with morphological castes. however, epiponines show a unique set of advanced characteristics, such as that they do not maintain their reproductive position by aggression, present marked refined communication, show age polyethism, and display intricate nest construction (jeanne 1991), all indicative of complexity. in fact, jeanne (2003) suggested another definition that supports self-organization instead of morphological skew patterns. if self-organization is prevalent, there is no need to have an evolution of two morphs in some complex societies, as in the epiponini. in fact, the egglayer’s role is a plesiomorphic condition, once all solitary hymenoptera also lay eggs. it is possible that, at least in some societies, the evolution of complexity would drive selection favoring totipotent individuals that may develop complex tasks, related to worker castes, but keeping their reproductive potential. in fact, as suggested by west-eberhard (2003), the origin of morphologically distinct queens is much more a condition of colony stability and the level of defense rather than an indicative of social complexity. from this perspective, the complex societies of epiponini may have passed through a phase of monomorphic, totipotent females (where egg laying has no relationship to dominance of polistes queens) to various systems of morphologically distinct queens in different lineages. noll et al. (2004) show that such a theory plots perfectly onto a cladogram of genera of epiponini. colony cycle the colony cycle in the swarm-founding polistine wasps is poorly known. as defined by jeanne (1991) colony cycle “is the period of development lasting from the end of one reproductive episode to the end of the next” and, apparently, the tropical environment in which the epiponini thrive has rendered colony cycles much more plastic (asynchronic) than those under the temperate climates (synchronic) (jeanne 1991). the onset of nest foundation, for instance, which is limited to the start of the favorable season in the temperate zone species, shows a wide distributional pattern scattered along several months in the neotropics. in the rainy season, the population increases and a reproductive swarm figure 2. a model for the evolution of epiponine polygyny. a. ancient (plesiomorphic) society, as seen in short-term, monogynic polistinae, in which the dominant female is intolerant of reproduction by other females. the egglayer performs policing tasks, whereas workers are tolerant of reproduction by others. occasionally, workers do reproduce, perhaps as replacement reproductives. b. epiponine polygyny, with no intolerant primary egglayer. rather, reproduction is performed by several tolerant females. the role of policing is adopted by workers, who suppress each other, and select among reproductives as well. this system is not what would result from merely adding several short-term queens together. fb noll neotropical swarm-founding polistines352 may occur (jeanne 1991). an exception is in agelaia vicina (oliveira et al. 2010) which shows an increase in population during the dry season. interestingly, in new colonies or swarms, producing queens may not necessarily be linked to the production of swarms. in fact, in parachartergus colobopterus (strassmann et al. 1998), the queens present in swarms were rarely mothers of the workers of those swarms. it is suggested that the queens who join the swarms were losers in the race for dominance in original colonies. because the production of queens and new colonies are not connected, the interests of workers in the production of males and queens prevail. as discussed above, castes develop in different ways for epiponines and the colony cycle certainly influences some of its aspects. in some species studied so far, morphological discontinuities might increase through the progression of the colony cycle (noll & zucchi 2000, 2002). such a phenomenon has also been reported in some ropalidiini (fukuda et al. 2003 a, b). combining morphological differences between castes, physiological potential of the females to develop ovaries and colony cycle, five different syndromes have been proposed (noll & zucchi 2000, 2002). in two syndromes, morphological differences between castes are absent and workers present a wide range of ovarian development in one case and in the other workers are physiologically distinct (sterile). in other two syndromes there are morphological and physiological discontinuities between castes, which are subjected to variation according to colony cycle, with queens larger than workers. initially, queens have a variable size, but at the end of the cycle they are among the larger individuals (noll & zucchi 2000, 2002). these two syndromes differ from each other by the presence of non-inseminated laying females that appear always (in one case) or in some phases of the colony cycle (other syndrome). in the final syndrome, it is more similar to be basic pattern of social insects, in which castes are highly distinct based on both morphology and physiology. concluding remarks this review demonstrates that the diversity and complexity that may be found in epiponini cannot be easily explained by conventional theory extending a polistes-like system, but seem instead to require a new perspective that separates egglaying from dominance, and reproduction from regulation of work. from the other side, the scarcity of studies (and scholars) on this subject delays considerably the advance on the knowledge of this group. hopefully, this review may serve as trigger to inspire young students to choose this group as their own. acknowledgements i thank ronaldo zucchi (usp) for his encouragement for writing this review and guidance throughout my scientific career, and financial support from fapesp and cnpq. references andena, s.r., noll, f.b., carpenter, j.m. & zucchi, r. 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(1971).. the insect societies. belknap press. harvard. university. press. cambridge, pp 548 doi: 10.13102/sociobiology.v66i2.3554sociobiology 66(2): 218-226 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 dear enemy phenomenon in the ant ectatomma brunneum (formicidae: ectatomminae): chemical signals mediate intraspecific aggressive interactions introduction as in other insects’ societies, ants are characterized by a high degree of cooperation between different individuals in the colony. colony members display social behaviors that complement each other, resulting in an overall development of the colony (zinck et al., 2008). workers, for example, actively work on building the nests, protecting the colony against predators, foraging and nursing (ratnieks et al., 2006). therefore, the integrity of the colony depends exclusively on the social relationships between its individuals and thus their abstract the integrity of ant colonies depends exclusively on social relationships between their individuals, especially the ability of communication between group members, which is mainly mediated through chemical signals. another important feature of ant behavior is territory defense, since they need to gather large amounts of food to feed their larvae, males and breeding females. thereby, ants might display behavioral strategies to defend their territories from intruders. here we investigated whether ectatomma brunneum displays the dear enemy phenomenon, what is the relationship between cuticular hydrocarbon composition and levels of aggression during their intraspecific interactions and which compounds and/or classes of compounds might be the most important to modulate the level of aggression. to test our hypothesis, we evaluated the levels of aggression through behavioral observations during interactions between 23 pairs of colonies nested in two distinct sites at varied distances. then, we analyzed the cuticular chemical profile of the individuals involved in the interactions, and compared these results with the levels of aggression displayed between colonies tested. the results allow us to confirm our hypothesis that the dep occurs in e. brunneum. the higher tolerance between closer colonies can be explained due to their kinship level in addition to sharing the same microhabitats. the results also showed there are significant differences in chcs profiles, especially between colonies nested at relatively greater distances, and it is likely that differences in content of some branched alkanes are the most important to establish these differences and, therefore, the levels of aggression during the interactions. sociobiology an international journal on social insects mc pereira1,2, elb firmino2, rc bernardi2,3, ld lima2, ic guimarães2,3, cal cardoso3, wf antonialli-junior1,2,3 article history edited by jean santos, ufu, brazil received 27 june 2018 initial acceptance 14 february 2019 final acceptance 22 february 2019 publication date 20 august 2019 keywords aggressiveness; behavior; cuticular hydrocarbons; gas chromatography corresponding author ingrid c. guimarães universidade estadual de mato grosso do sul cidade universitária rodovia dourados itahum, km 12 caixa postal 351, cep: 79804-970 dourados, mato grosso do sul, brasil. e-mail: guimaraes_ingrid@yahoo.com.br ability of communication between group members (crozier & pamilo, 1996). social insects use mainly chemical signals for communication, especially the cuticular hydrocarbons (chcs) which might vary between colonies or even between castes (e.g. blomquist et al., 1998; lenoir et al., 1999; antonialli-junior et al., 2007). chcs have profiles both genetically and environmentally determined (sorvari et al., 2008), which are used by workers as “signatures” for intra and interspecific recognition (arnold et al., 2000). the existence of these recognition mechanisms is of great value, especially in intraspecific interactions. considering that social insects are 1 programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados, brazil 2 laboratório de ecologia comportamental, centro de estudos em recursos naturais, universidade estadual de mato grosso do sul, dourados, brazil 3 programa de pós-graduação em recursos naturais, universidade estadual de mato grosso do sul, dourados, brazil research article ants sociobiology 66(2): 218-226 (june, 2019) 219 not really clones, but groups of individuals with high degree of kinship that live in colonies and might have different interests (cassill & tschinkel, 1999; ratnieks et al., 2006), conflicts between colonies are expected to be frequent. ants generally control and defend their territories, from where they extract resources to feed the large number of larvae, males and breeding females of their colonies (newey et al., 2010). thus, in most species, encounters between foragers from different colonies might trigger conflicts that can turn into clashes with physical contacts resulting in death of individuals (matthews & matthews, 2010). these conflicts can happen between individuals of several colonies or species for access to the food source, territory, or reproductive partners. such disputes, indeed, generally involve aggressive behaviors (huntingford & turner, 1987). on the other hand, some species respond less aggressively to the entrance of neighbors in their territories than the entrance of a non-neighbor (stranger). this difference in intensity of aggressive response is known as “dear enemy phenomenon” (dep) and has been described in several species of vertebrates and invertebrates (e.g. ydenberg et al., 1988; temeles, 1994; dimarco et al., 2010). in ants, the higher tolerance between neighbors has been described in species such as acromyrmex octospinosus reich (formicidae: myrmicinae) (jutsum et al., 1979), temnothorax nylanderi foerster (formicidae: myrmicinae) (heinze et al., 1996), pheidole gilvescens creighton and gregg (formicidae: myrmicinae) and pheidole xerophila wheeler (formicidae: myrmicinae) (langen et al., 2000), formica pratensis retzius (formicidae: formicinae) (pirk et al., 2001), cataglyphis fortis forel (formicidae: formicinae) (knaden & wehner, 2003), acromyrmex lobicornis emery (formicidae: myrmicinae) (dimarco et al., 2010) and ants of the neoponera apicalis complex latreille (formicidae: ponerinae) (yagound et al., 2017). one explanation for this phenomenon is based on familiarity between neighbors, and once the territory boundaries are established, these neighbors show little or no threat, while an individual from a distant site corresponds to a stranger who may be looking for a new territory (temeles, 1994). another hypothesis is based on the reduction of energy costs of an aggressive interaction and preventing fights with neighbors, since there are frequent encounters during foraging (ydenberg et al., 1988). investigations in several species such as camponotus fellah dalla torre (formicidae: formicinae) (boulay et al., 2004), cataglyphis niger andré (formicidae: formicinae) (soroker et al., 1995), aphaenogaster senilis mayr (formicidae: myrmicinae) (lenoir et al., 2001) and neoponera apicalis latreille (formicidae: ponerinae) (soroker et al., 1998), have shown that chcs undergo a homogenization between colony members through social interaction. thus, many authors have evaluated the levels of aggression during intraspecific interactions in order to investigate the origin of individual recognition cues in different species of ants, since members of the same colony generally ignore each other during encounters, but show different levels of aggression towards individuals from different colonies or species (stuart & herbers, 2000; menzel et al., 2009). ectatomma brunneum smith (formicidae: ectatomminae) is an ant with widespread distribution in latin america, occurring from panama to argentina and can be found throughout the brazilian territory, usually in areas with open vegetation such as forest borders or clearings, but also in crops, pasture and secondary vegetation (brown, 1958). it is a generalist predator of terrestrial arthropods, alive or recently dead (giannotti & machado, 1992; maques et al., 1995), and the food is generally collected on the ground, rarely on vegetation (del-claro et al., 1992). despite being a species of widespread occurrence, little is known about many aspects of its biology. here we investigated whether e. brunneum displays the dep, what is the relationship between the chc and levels of aggression during their intraspecific interactions and which compounds and/or classes of compounds might be the most important to modulate the level of aggression. we tested whether worker ants display different levels of aggression towards neighbors and non-neighbors. our hypothesis was that if the dep occurs in this species, ants should be more aggressive towards non-neighbors, since these ants could represent a threat to their already established territorial organization. secondly, we analyzed the cuticular chemical composition, establishing the relationship between the chc and levels of aggression during conspecific interactions and identifying which compounds and/or classes of compounds might be the most involved in modulating the level of aggression between these ants. material and methods behavioral study we collected nine colonies of e. brunneum (table 1), five of them nested in the surroundings of the universidade estadual de mato grosso do sul campus in dourados, mato grosso do sul, brazil (22°11′53.32′′s, 54°55′50.90′′w), in an area of pasture, with predominance of grass and shrub species (area 01). other four colonies were nested in a forest board of fazenda coqueiro (22°12′45.78′′s, 54°54′43.15′′w) (area 02), classified as a semideciduous forest, according to the instituto brasileiro de geografia e estatística ibge classification system (veloso et al., 1991). the two areas were around 3 km apart from each other. colonies were ranked according to collection site. we collected colonies nested at different distances and environments because the cuticular chemical compounds may be affected by both genetic and environmental factors (sorvari et al., 2008), and this type of variation could influence on our results. colonies were collected according to the method described by lapola et al. (2003). in order to standardize the time during which the colonies would be under controlled conditions and to control habituation, the period between collections was never greater than twenty days. then, all mc pereira et al. – dear enemy phenomenon in the ant ectatomma brunneum220 individuals were placed in artificial nests in laboratório de ecologia comportamental of universidade estadual de mato grosso do sul. all adults of each colony were marked with non-toxic paint in the pronotum region for identification. the mean colonial population in area 01 was of 124.5 ± 4.9 individuals and 142.5 ± 19 in area 02, all colonies had immature (larvae and pupae) and, except for colony 6, all of the others had a queen. we only used colonies for the experiment after two days of acclimatization in the artificial nests. the experimental design consisted of two colonies at a time connected to a single foraging arena, consisting of a wooden box of 51cm x 42.5cm x 6 cm connected to both nests, where it was offered water in a petri dish, larvae of tenebrio molitor linnaeus (coleoptera: tenebrionidae), and molasses. in this arena, we could observe the interactions between foragers of the two colonies during their activities outside the nest. for every new experiment, we used a new and neutral arena. we kept the connection between colonies and performed observations for three days maximum, in order to avoid familiarity between them. before connecting different colonies, we induced encounters in the foraging arena between workers from the same colony as control tests. when the first worker left the colony, we captured it and waited until the next worker entered the foraging arena. then, we placed the workers close to each other in order to induce the encounter, and recorded their behaviors. we observed each pair of colonies every day, 5 hours per day, totaling 15 hours of observation for every pair tested. five induced encounters were performed between workers from the same colonies as a control test. we evaluated the interactions between 23 pairs of colonies: nine encounters between colonies nested in the pasture (pp); six encounters between colonies nested in the forest (ff); and eight between colonies nested in forest and pasture (fp). we observed the colonies in pairs because it would be difficult to perform the experiment with all colonies at the same time, since the maintenance period under laboratory conditions, which implies standardization of diet, can lead to similarity in patterns of chcs, interfere in habituation and generate different behavioral responses. during the interactions, we observed and recorded all behaviors through the ad libitum method (altman, 1974). we considered only the encounters one by one. the behaviors observed were: ignore, touch, escape, attempted seizure, seizure, antennal boxing, body lift, display of abdomen and fight. in order to evaluate the intensity of aggression between foragers, the behaviors exhibited received a rating scale, modified from suarez et al. (1999), from 0 to 2, as follows: 0 for touch, escape and ignore; 1 for attempted seizure, seizure, antennal boxing, body lift and display of abdomen; and 2 for fight. at the end of 15 hours of observation, we compiled an arithmetic mean between the intensities of aggression displayed during each encounter between foragers of each pair of colonies. chemical analyses in order to assess whether the mean level of aggression displayed during interactions correlates with the similarity or dissimilarity of each colony’s chcs composition, the cuticular chemical profile of the body of at least nine foragers from each colony that interacted in the arena were assessed by gas chromatography coupled to mass spectrometry (gc-ms). for gc-ms analyses, the cuticular compounds were extracted with 5 ml of hexane (hplc-vetec) in ultrasonic bath for 3 min. after filtration, the solvent was dried under fume hood, and the dry extract was solubilized in 100 µl of hexane prior to analysis. analyses were performed employing a gas chromatograph (gc2010 shimadzu, kyoto, japan) coupled to a mass spectrometer detector (qp 2010), using fused silica capillary column db -5 (j & w, folsom, ca, usa) 5% phenyldimethylpolysiloxane (30 m length × 0.25 mm diameter × 0.25 mm of film thickness). the analysis conditions were: helium as carrier gas (99.999%) at flow rate of 1.0 ml1, injection volume 1 µl in splitless mode. the temperature ramp was programmed as follows: initial temperature of 50 °c, reaching 85 °c at a rate of 5 °c min-1, followed by a ramp of 8 °c min-1 up to 280 °c, and a second ramp of 10 °c min-1 up to 300 °c remaining at the final temperature for 35 minutes. temperature of injector, detector and transfer line was kept at 280 °c. the parameters of mass spectrometer included scanning ms voltage electron impact ionization of 70 ev, ranging from 45 to 600 m/z, with 0.5 s of scan interval. data processing was performed using a signal to noise ratio of three. the criteria for accepting a detected compound was a minimum of 900 of similarity match and manual inspection of the quality of the mass spectrum of each compound. identification of compounds was performed employing the calculated retention index (van den dool & kratz, 1963), using a standard mixture of linear alkanes (c7c33), and comparing the calculated value with the retention index of literature associated with interpretation of mass spectra obtained from the samples and compared with the data bases nist21 and wiley229. the relative area for each chromatographic peak was employed as abundance approach to evaluate the contribution of each compound area to the total table 1. distance (m) between the nine colonies of ectatomma brunneum. p means colony nested in pasture and f nested in forest. #1 f #2 f #3 f #4 f #5 p #6 p #7 p #8 p #9 p #1 f 0 #2 f 173 0 #3 f 711 720 0 #4 f 707 712 235 0 #5 p 2087 2086 2480 2482 0 #6 p 2083 2073 2475 2501 4 0 #7 p 2006 2205 2386 2387 192 151 0 #8 p 2100 2015 2397 2390 203 168 135 0 #9 p 2077 2078 2485 3342 172 160 187 196 0 sociobiology 66(2): 218-226 (june, 2019) 221 area and for comparison between the samples. the sum of all peak areas was considered 100% of the sample and to each peak was assigned a percentage corresponding to its area. statistical analyses in order to assess whether differences in levels of aggression between colonies of the two areas is significant, the normality of data was checked and then a t-test was performed with the mean levels of aggression between colonies from the same area and between different areas using the r software. in order to evaluate the relationship between the level of aggression and the distances between colonies we used the pearson’s correlation coefficient with the data of aggressiveness and distances between colonies nested in the two different areas. these analyses were performed in the r (r development core team 2010) software. the gc-ms data were analyzed by stepwise discriminant function analysis, using the systat 10.2 software, which can reveal the set of variables that best explains the difference between the groups, indicated by wilk’s lambda (quinn & keough, 2002). with these results, which show the compounds that were most significant for groups’ separation, we performed a cluster analysis, in order to assess the relationship between colonies based on their cuticular chemical profiles. this analysis was also performed using the r software. finally, in order to assess the relationship between differences in chcs composition, distance and aggressiveness between colonies, we performed the mantel test in the ntsys-pc 2.1 software. results we did not observe any aggressive behavior in control tests. we observed an average of 236 ± 144 encounters between foragers from distinct colonies. the t-test performed with mean values of levels of aggression between colonies nested in the same area, regardless of distance, did not indicate significant differences (ff x pp = p > 0.05). however, comparing the values obtained from encounters between colonies nested in the same area and in different areas, there were significant differences (ff + pp x fp p < 0.05). the correlation analyses showed that there is a certain level of intolerance between colonies regardless of nesting site and distance between them (fig 1). the highest level of aggression between colonies was fig 1. scatter plot depicting the pearson’s correlation tests results between the level of aggression and distances of colonies of ectatomma brunneum. markers (●) correspond to mean levels of aggression obtained from the interactions between colonies. (a) = interactions between colonies nested in the pasture area; (b) = interactions between colonies nested in the forest area; (c) = interactions between colonies nested in the pasture and colonies nested in the forest area; (d) = general mean levels of aggression regardless of nesting site. mc pereira et al. – dear enemy phenomenon in the ant ectatomma brunneum222 table 2. relative area (%) of chcs compounds identified in workers from colonies of ectatomma brunneum, obtained by gc-ms. 0.95 and they were 3.3 km apart from each other (fig 1c); and the lowest was 0.008 and colonies were 3 m apart from each other (fig 1a). the analyses showed that there was significant correlation between the level of aggression and distance in encounters between colonies nested in the forest area (fig 1b), and when it was considered all encounters (fig 1d). however, the correlation between the level of aggression and distance in encounters between colonies nested in the pasture area (pp) and between colonies nested in different areas (fp) was not significant (fig 1a and c). forty-seven (47) chcs, ranging from 20 to 31 carbon atoms. linear alkanes represented 25% of compounds, branched alkanes 21%, alkenes 7% and non-identified compounds 47% (table 2). linear alkanes, branched alkanes and alkenes represented more than 75% of the total area of compounds. the results of the discriminant function analysis revealed that the chcs profile of the nine colonies are significantly different (wilk’s lambda = 0.000, f = 987.369, p < 0.05). five compounds were the most significant for groups’ separation (table 2), four branched alkanes and one linear alkane. the cluster analysis displays the dissimilarities between the colonies based on their chcs profiles (fig 2; cophenetic correlation coefficient = 0.818). the mantel test (t = 4.13, p < 0.05) showed that colonies nested closer to each other, especially those from the same area, are the ones with the most similar cuticular chemical profiles and the lowest levels of aggression. compounds relative area (%) per colony #1 #2 #3 #4 #5 #6 #7 #8 #9 c20 n-eicosane 0.516 0.759 0.754 0.782 1.526 1.604 1.666 1.731 1.764 c21 n-heneicosane 0.493 0.492 0.522 0.456 0.514 0.538 0.528 0.504 0.487 c22 n-docosane 0.21 0.211 0.27 0.256 0.306 0.29 0.293 0.29 0.308 c23 n-tricosane 7.697 7.839 7.953 7.944 7.066 7.078 6.993 6.928 7.06 c24 n-tetracosane 0.197 0.203 0.29 0.269 0.298 0.298 0.29 0.294 0.298 c25 n-pentacosane 13.74 13.82 12.96 12.93 11.81 11.73 12.02 12.01 11.06 c26 n-hexacosane 2.013 1.991 1.713 1.665 2.49 2.596 2.666 2.731 2.523 c27 n-heptacosane 12.70 12.86 12.77 12.93 15.12 15.38 15.17 15.19 15.18 c28 n-octacosane 0.206 0.209 0.27 0.254 0.294 0.28 0.293 0.29 0.306 c29 n-nonacosane 0.206 0.216 0.271 0.252 0.284 0.292 0.293 0.29 0.302 c30 n-triacontane 0.209 0.213 0.27 0.257 0.292 0.29 0.293 0.29 0.307 c31 n-hentriacontane 2.263 2.254 1.815 1.807 1.898 1.738 1.764 1.813 2.255 13-mec25 13-methylpentacosane 2.347 2.219 2.327 1.95 2.008 2.008 1.887 2.41 2.32 3-mec25 3-methylpentacosane 2.27 2.446 2.487 3.013 2.4 2.402 2.666 2.087 2.526 13-mec27 13-methylheptacosane 3.013 2.92 2.576 2.974 3.028 3.024 2.866 3.014 2.903 5-mec27 5-methylheptacosane 1.419 1.286 1.514 1.119 1.538 1.538 1.431 1.143 1.313 3-mec27 3-methylheptacosane 3.975 3.871 4.028 4.014 4.02 4.018 4.017 3.867 3.871 15-mec29 15-methylnonacosane 2.664 2.727 2.986 2.559 3.244 3.252 2.776 2.665 2.727 14-mec28 14-methyloctacosane 1.12 0.985 1.225 0.918 1.304 1.302 1.035 0.886 0.986 15-mec29 15-methylnonacosane 3.777 3.839 3.517 4.018 3.666 3.668 3.987 4.038 3.808 13-mec31 13-methylhentriacontane 1.976 1.929 2.088 1.948 2.112 2.108 1.853 1.78 1.987 13-mec33 13-methyltritriacontane 1.558 1.488 1.521 1.508 1.246 1.248 1.519 1.386 1.488 c27:1 n-heptacosene 1.288 1.166 1.33 1.018 1.24 1.234 1.086 1.115 1.168 c29:1 n-nonacosene:1 1.128 1.178 1.02 1.089 0.888 0.886 1.107 0.987 1.178 c29:2 n-nonacosene:2 0.487 0.546 0.6 0.519 0.558 0.558 0.726 0.849 0.548 c31:1 n-hentriacontene 0.756 0.815 0.51 0.836 0.556 0.558 0.675 0.907 0.802 most important chcs for groups’ separation. sociobiology 66(2): 218-226 (june, 2019) 223 discussion our results demonstrate that among the groups of encounters between colonies nested in the same area (ff and pp), the levels of aggression were not significantly different, but these changes when compared to the level of aggression observed in encounters between colonies from different areas (fp). considering the correlation analyses performed with encounters between colonies from the same area (fig 1a and b), the significant correlation observed in ff encounters is probably related to the kinship level between colonies, which tends to be lower between more distant colonies. meanwhile, colonies nested in the pasture area were, in general, nested closer therefore the level of kinship and consequently the level of aggression should be lower than the ones of colonies from the forest, which explains the correlation result (fig 1a). this fact becomes clearer when we evaluate the level of aggression of encounters between colonies from different areas, whose correlation was not significant (fig 1c). in this case, colonies were at least 2 km apart from each other, so the level of kinship and probability of encounter during foraging activity under natural conditions should be so low that ants displayed high levels of aggression in all encounters regardless of the distance. finally, considering the levels of aggression from all encounters, the correlation is highly significant confirming that indeed the greater the distance the higher the level of intolerance between colonies (fig 1d). these results agree with other studies with the ant species acromyrmex octospinosus reich (formicidae: myrmicinae) (jutsum et al., 1979), temnothorax nylanderi foerster (formicidae: myrmicinae) (heinze et al., 1996), pheidole gilvescens creighton and gregg (formicidae: myrmicinae) and pheidole xerophila wheeler (formicidae: myrmicinae) (langen et al., 2000), formica pratensis retzius (formicidae: formicinae) (pirk et al., 2001), cataglyphis fortis forel (formicidae: formicinae) (knaden & wehner, 2003), which also present the dep, since ants are more tolerant towards conspecifics from neighboring colonies. temeles (1994) argues that this feature can be explained due to familiarity between neighbors, because once the territory boundaries are established, these neighbors show little or no threat, while an individual from a distant site corresponds to a stranger who may be looking for a new territory. in addition, there are frequent encounters between neighbors during foraging, so preventing fights would be an effective way of reducing energy costs due to aggressive interactions (ydenberg et al., 1988). the discriminant function analysis showed that four of the five most important compounds for groups’ separation, were branched alkanes and one linear alkane. these results corroborates the discussions of leconte and hefetz (2008), blomquist and bagnères (2010), richard and hunt (2013) and lorenzi et al. (2014) regarding the role of branched alkanes as important signals for intraspecific recognition. however, some studies also discuss the role of linear alkanes for this function (lorenzi et al., 2004; tannure-nascimento et al., 2007; ferreira et al., 2012). colonies nested at closer sites have more similarity in chcs composition, and can use this information to mediate interactions during encounters (fig 2). groups’ separation observed in the similarity dendrogram shows, therefore, that there is a direct relationship between chcs composition and colonies’ nesting site (fig 2). although each colony had its own chemical signature as suggested by studies on chcs in other ant species, such as a. senilis (lenoir et al., 2001) and ectatomma vizottoi almeida (formicidae: ectatomminae) (antonialli-junior et al., 2007), there is greater similarity between profiles of colonies nested in nearby sites. one possible explanation is the higher level of kinship between these colonies due to a restricted dispersal strategy and/or by the phenomenon of colony fission already documented for e. tuberculatum olivier (formicidae: ectatomminae) (zinck et al., 2008). on the other hand, it is well known that chcs composition is determined by two main components: genetic and environmental (sorvari et al., 2008), thus this similarity in chcs profile, as well as the low level of aggression, might be partially explained by environmental similarity, especially microhabitats shared by neighboring colonies. in the particular case of the interaction between colonies #5 and #6, collected 3m apart from each other, in the same area, the level of aggression was the lowest (fig 1a). the analysis based on these colonies’ chcs profiles showed that they are indeed relatively similar (fig 2). ten hours after being connected by the single foraging arena, they merged to form a single colony with adults and immature accepted by other colony members. this suggests that these two colonies could be one that had been divided into distinct nesting sites, thus consisting of a polidomic nest, supporting the hypothesis suggested by lapola et al. (2003) and vieira and antoniallijunior (2006) for the same species. on the other hand, the low level of aggression may be due to microhabitat similarity, which could lead to similarity in chcs profiles because of both the higher level of kinship and the same types of food resource (pirk et al., 2001). in this context, some authors have shown that colonies kept under the same diet during a large period can have their chcs profile modified leading to similarity in composition (e.g. zweden et al., 2009; bernardi et al., 2014; valadares et al., 2015). the importance of the environmental component especially microhabitats on chcs profiles is enhanced by the greater similarity between colonies #8 and #9 presented in the cluster analyses (fig 2). although these colonies were nested in pasture area, there were small trees and shrubs in their nesting sites, unlike the others, which were surrounded by grasses. this is likely the reason why these colonies separated from the others nested in the same area (fig 2). therefore, these results demonstrate that the environmental component is also important to determine the mc pereira et al. – dear enemy phenomenon in the ant ectatomma brunneum224 composition of chcs. indeed, heinze et al. (1996) describe that when colonies share the same microhabitat there might occur similarity in odor produced by them, and as a result, workers reduce the aggressive response towards neighbors. some authors suggest, for example, that diet is one of the factors that influence odor formation, and colonies that use similar resources should present similar colonial odor, therefore, have higher levels of tolerance when in situations of interaction (jutsum et al., 1979; liang & silverman, 2000). conclusion the results confirmed the hypothesis that e. brunneum presents the dep, since ants were indeed more aggressive towards non-neighbors than towards neighbors. the lower level of aggression displayed between workers from closer colonies is likely explained due to their higher kinship levels, in addition to environmental factors such as sharing the same microhabitats. furthermore, the results showed that there are significant differences in chcs profiles, especially between colonies nested at relatively greater distances, and according to the analyses it is likely that the differences in content of some branched alkanes are the most important to establish these differences and, therefore, the levels of aggression during the interactions. acknowledgments the authors thank universidade federal da grande dourados and universidade estadual de mato grosso do sul for technical support, pronex/fundação de amparo à pesquisa do estado da bahia, coordenação de aperfeiçoamento de pessoal de nível superior (capes), fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect) for financial suppport, and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for: calc, grant number 311599/20125 and wfaj scholarship, grant number 307998/2014-2. authors’ contribution mc pereira and wf antonialli-junior conceived and design the experiments. mc pereira and elb firmino performed the behavioral study. rc bernardi and cal cardoso performed the chemical analyses. mc pereira perfomed the data analyses. mc pereira, ld lima, ic guimarães, cal cardoso and wf antonialli-junior wrote and reviewed the manuscript. references altman, j. 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(2009). disentangling environmental and heritable nestmate recognition cues in a carpenter ant. journal of insect physiology, 55: 158-163. doi: 10.1016/j.jinsphys.2008.11.001 doi: 10.13102/sociobiology.v65i4.3447sociobiology 65(4): 534-557 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 economic and cultural values of stingless bees (hymenoptera: meliponini) among ethnic groups of tropical america introduction a persistent interaction with the environment has shaped human experience and resultant knowledge across diverse groups and societies. therefore, intricate links between ecosystem use, management and human cultural values have produced numerous indigenous, local knowledge systems (gómez-baggethun et al., 2013; jax et al., 2013). the millennium ecosystem assessment (ma) defined cultural ecosystem services as “the nonmaterial benefits people obtain from ecosystems through spiritual enrichment, cognitive abstract stingless honey bees — commonly known as stingless bees — have long provided food and materials to the inhabitants of tropical america. we conducted a literature search to codify available information, including non-peer reviewed ‘grey literature’, on the purported value of stingless bees to indigenous people. among > 400 species of neotropical stingless bees several are widely used in beekeeping. varied cultural and economic values are associated with their use, and in some cases husbandry, as a consequence of ongoing contact between people and these social insects. adapting new species to husbandry is being attempted in many countries. the bees remain culturally important, and beliefs associated with them are significant for different groups, beyond utilization as commodities. we find values in food, craft, religion and medicine, with cultural values ranging from utilitarian to mythological. values transmitted across generations allow cohesion and communal identity of native organisms associated with any indigenous society. such cultural values seem in danger of extinction, primarily due to external factors. we provide examples of successful regional strategies in averting cultural and economic loss in natural human heritage, in this case bees that provide honey and other benefits. preserving stingless bees and the cultural heritage around them provides a good example of sustainable use of native species in human communities. bees are important agents for conservation of the environment. sociobiology an international journal on social insects jjg quezada-euán1, g nates-parra2, mm maués3, vl imperatriz-fonseca4, dw roubik5 article history edited by cândida aguiar, uefs, brazil denise alves, esalq-usp, brazil received 10 may 2018 initial acceptance 25 june 2018 final acceptance 19 july 2018 publication date 11 october 2018 keywords indigenous local knowledge, stingless honey bee, meliponiculture, honey, cerumen, cultural value. corresponding author josé javier g. quezada-euán departamento de apicultura tropical campus ciencias biológicas y agropecuarias universidad autónoma de yucatán mérida cp 97100, méxico. e-mail: javier.quezada@correo.uady.mx development, reflection, recreation, and aesthetic experience, including, e.g., knowledge systems, social relations, and aesthetic values” (ma, 2005). from a biological perspective, the importance of traditional knowledge often relies on its practical nature, particularly in agriculture, fisheries, health, horticulture and forestry, including the development and preservation of locally bred plants and animals. recently, the importance of indigenous cultural values and traditional knowledge as essential components of biodiversity models and policy has been recognized (hill et al., 2016: ipbes (2016)). nonetheless, for centuries if not 1 universidad autónoma de yucatán, mérida, méxico 2 universidad nacional de colombia, bogotá dc, colombia 3 embrapa amazônia oriental, belém, brazil 4 instituto tecnológico vale, belém, brazil 5 smithsonian tropical research institute, balboa, panama review mailto:javier.quezada@correo.uady.mx sociobiology 65(4): 534-557 (october, 2018) special issue 535 millennia, traditional culture has been neglected and now has great difficulty resisting the onslaught of outside influences. cultural values are in danger of being lost at an increasingly rapid rate. stingless honey bees are a tropical phenomenon whose time has apparently come, evidenced by burgeoning volumes documenting their biology and use, including in beekeeping (e.g. roubik, 2006; vit et al., 2013; cortopassi-laurino & nogueira-neto, 2016; heard, 2016; jalil & roubik, 2017; quezada-euán, 2018). in addition, the americas provide one of the best examples of a link involving cultural values and traditional knowledge. it has developed over millennia, but now is disappearing from indigenous communities. before the european era, stingless bees played an important role in the economies and traditions of american indigenous cultures (de landa, 1566; cobo, 1653, cited in roubik, 2000; schwarz, 1948; quezada-euán et al., 2001; koedam, 2018). because it was both a sweetener and a rich source of energy stingless bee honey was frequently used in trade and in traditional medicine and ceremonies (schwarz, 1948; weaver & weaver, 1981). honey and other nutritional or useful substances were removed from wild nests. but very significantly, in a few, scattered cultures, particularly the maya, bee husbandry evolved, and bees were protected in artificial domiciles or ‘hives’ (de landa, 1566; schwarz, 1948). notwithstanding the untimely disappearance of organized cities after the european conquest, in particular the ‘corn-culture’ centers which supported permanent settlement and human populations in much of pre-hispanic mesoamerica (coe & koontz, 2013), we find evidence of a steady decline in stingless beekeeping since the 16th century. a constant reduction of natural forest due to agriculture and settlement expansion has diminished nesting, foraging and reproduction by many species of bees and other animals used by the human population, particularly in forested or rural areas. we propose that the decline in stingless beekeeping is a multifactorial problem that involves ecological, social and economic factors (quezada-euán et al., 2001; villanueva-gutiérrez et al., 2013). moreover, native indigenous peoples are now progressively more in contact with and influenced by western customs and media, and thereby abandon some traditions as a consequence (stearman et al., 2008). there is strong evidence that bees are in decline in certain manmade settings (biesmeijer et al., 2006; fitzpatrick et al., 2007) with a number of factors thought to play major causal roles (kremen et al., 2002; woodcock et al., 2017; tsvetkov et al., 2017). native bees are considered the most important pollinators of crops world-wide; their contribution seems difficult to replace by commercially managed pollination (garibaldi et al., 2013; roubik, 2018a). in the neotropics, stingless bees may represent the main pollinators in abundance and species (roubik, 1989, 2018b). given the projections for human population growth to ∼9 billion by 2050 (united nations, 2004), particularly pronounced in tropical and subtropical latitudes, and the corresponding increasing need for food, the importance of native bees to human survival and ecosystem resilience can only increase in the coming years. traditional practices and knowledge associated with the use of native species like the stingless bees almost certainly will diminish, if gone unacclaimed, at precisely the time when they are most relevant and important. here we summarize and evaluate the literature, both published in peer-reviewed journals and among our judicious selection of opinions posted on the worldwide web, regarding the values and traditional practices associated with stingless honey bees and their products in tropical america. our interest is in revealing and summarizing as much as possible, in areas where traditional knowledge, without becoming part of anthropological field research, does not attain the level of academic publication. thus, we include a sampling of the so-called electronic ‘grey literature’, or that subject to rapid database searches. our aim is not only to identify cultural values associated with these insects and to raise public awareness of the significance of preserving them as part of biocultural human heritage, but also to acknowledge their current role in agricultural and ecosystem resilience, in the face of global change. materials and methods we searched published peer reviewed literature on traditional knowledge and the use and cultural values of stingless bees. databases included organizational web searches, web search engines and bibliographic checking. many documents were in spanish and portuguese. the main key words were queried in three languages, spanish, portuguese and english, and web searches were based upon “native bee, cerumen, wax, cultivation, cultural value, exploitation, honey, meliponiculture, husbandry, myth, melipona, stingless bee, trigona, indigenous and traditional knowledge”. we selected publications referring to the americas. for each category, we recorded the type of web search, date, search details, search terms, hits, and output, after replicate removal. our review also examined book information that may be obtained from governments, dissertations, web sites of individual professionals, business and industry — in print and electronic format. these are not controlled by commercial publishers (grey literature report; accessed 21/03/18 http://www.greylit.org/home). we also contacted academic researchers, usually with advanced degrees in the subject. when few studies were available, we focused on the methodology as the main criterion for scientific quality and inclusion in our survey. our findings were grouped according to the main subject of the manuscript. we further categorized economic and cultural values (as described by ma, 2005), given to stingless bees by local people, by taking the number of citations for each as a measure of relative importance. we http://www.greylit.org/home jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees536 also considered it important to distinguish between reports of species used in rational beekeeping (generally termed that performed using hives of some sort, usually those in which colonies can be propagated or fed) and those related to other cultural/medicinal use that does not involve bee management in some form. the main features and cultural values associated with stingless honey bees in mesoamerica and south america were considered separately (kent, 1984; crane, 1992). we also constructed a map including species used in beekeeping on a larger scale, and a table with all species of stingless bees for which evidence of traditional use was found across the americas, including the indigenous groups that use them, and the major economic and cultural values given to the bees and/ or their products. finally, we compiled some of the beliefs and traditions associated with stingless bees, among various indigenous peoples. results although we conducted an extensive search of the literature, no published information could be found for many areas. in particular, when we refer to mesoamerica it should be noted that most available literature comes from mexico and guatemala, some from honduras, el salvador, nicaragua, costa rica and panama, but no information could be obtained from belize. in south america, information was found from brazil, colombia, venezuela, ecuador, peru, paraguay, bolivia and argentina. one general finding was that current stingless beekeeping, most often with a local, indigenous origin, is based on only a few species. in this regard, we are not considering species of stingless bees that may be occasionally exploited, or with limited relevance in current beekeeping. indeed, there are many reports of several species recently promoted for honey production (yurrita-obiols & vásquez, 2013; ruano-iraheta et al., 2015; arnold et al., 2018). in brazil, for instance, the use of 61 species in different biomes, with their popular names, was recognized (venturieri et al., 2012). the most important species, geographically, in beekeeping is tetragonisca angustula (latreille) (fig 1). however, melipona beecheii bennett, is probably the most important culturally (de landa, 1566). apart from these species only five or six more are used extensively in beekeeping in the americas. species of the genus melipona seem to be particularly appreciated because of the amount and quality of their honey (fig 1). we note that some of these species are possibly species complexes, as indicated by molecular analyses (quezada-euán et al., 2007; mayitzá et al., 2012; francisco et al., 2014; hurtado-burillo et al., 2016, 2017). thus, a few more distinct bee species may be involved in indigenous beekeeping. at least 100 more species have cultural and medicinal importance. further, the use of common names reveals that, in an analysis of 76 ethnic names for ecuador (vit et al., 2017), and over 50 names used by two indigenous peoples in amazonas state, brazil (oliveira et al., 2013), the species with multiple names are likely most important. in mesoamerica m. beecheii has at least 20 different local names (arnold et al., 2018; quezadaeuán, 2018), while t. angustula had seven common names in ecuador and melipona grandis guérin, in central brazil, had eight (table 1). finally, in brazil, only about 10% of the stingless honey bees have common names, while an estimate based on 12 countries, worldwide, indicates 25% of local species are utilized and have such names (roubik et al., 2018). if common names indicate utility, or exploitation, then there is an indication here for both underexploited and perhaps potentially overexploited species (table 1). fig 1. geographic distribution of the main species of stingless bees used in beekeeping in tropical america. data from catalogue of bees (hymenoptera, apoidea) in the neotropical region. available on line at http://www.moure.cria.org.br/catalogue. discussion we identified many economic and cultural values associated with stingless bees and their products. we also found several major categories within each value type. food, tool and craft, medicinal, economic or mythological categories predominated. the results are presented in table 1. the most important overall value given to stingless bees is economic, as a source of food and additional income. honey is the main sociobiology 65(4): 534-557 (october, 2018) special issue 537 commodity in this respect, used either for local consumption or trade (fig 2). honey is also used to treat various illnesses and is sometimes mixed with other natural products. other bee colony products are also used for consumption but are minor, such as the pollen, resin and brood. resin (propolis) and cerumen are used primarily in tools and handicraft. a second economic use concerns traditional medicine practiced by shamans and midwives, which in the process become figures of authority and respect in their communities (fig 2). the number of ways in which stingless bees are valued contrasts between mesoamerica and south america. although all the surveyed native groups make extensive use of meliponini as a source of food, the medicinal and religious values associated with stingless bees are, evidently, a more currently relevant and also an ancient feature in mesoamerica. in contrast, the mythological significance of these insects is a common feature to indigenous peoples throughout tropical america. although the stingless honey bees have been extensively used and appreciated among native groups, major geographic differences are found today in the management of different stingless bees. as discussed further below, the northern hemisphere cultures, particularly those in large, permanent settlements, practiced husbandry, with the possibility of reproducing their colonies and maintaining them, while the southern hemisphere peoples relied on bees as one of the renewable resources extracted directly from the forest. to date, there is no evidence of extensive stingless bee husbandry in south america, where use has always concentrated on hunting nests mostly for honey (brown, 2006; rosso & imperatriz fonseca, 2017). people usually open and remove meliponine nest contents, leaving the damaged colonies in the wildlands (kerr et al., 2001). there was no systematic reproduction of such colonies, and most succumb to phorid fly depredation (roubik, 1989). the brazilian kayapó are perhaps an exception, practicing what is considered semi-domestication, collecting the nests and bringing them to the vicinity of their homes to remove bee products from time to time (camargo & posey, 1990). south american societies had no religious relationship like that in mesoamerica, but for some indigenous peoples (andoque, uwa, tupinambá, kayapó), stingless bees were important in cosmogony and mythology (jara, 1996; de jong, 1999; falcheti & nates-parra, 2002; lima & moreira, 2005; posey & camargo, 1985). in the following sections we present summaries of the most important cultural manifestations related to stingless bees and their cultivation by different ethnic groups. mesoamerica the main feature in mesoamerican stingless bee exploitation was the development of husbandry with an annual propagation of colonies, or colony “splitting”. such a rational form of stingless beekeeping likely originated in the seasonally dry lowland forests within the maya region of the yucatan peninsula, northern guatemala and belize, more than 1400 years ago (kent, 1984; crane, 1992; yurritaobiols & vásquez, 2013). unmentioned is perhaps the equally important fact that ‘corn culture’ and the potential for permanent settlements developed in the same region. as the forest was cleared for corn cultivation, and local stingless bees were overexploited, the indigenous people may have devised means for sustainably maintaining their colonies of m. beecheii, and perhaps other species. it is believed that due to the maya, stingless beekeeping spread to other civilizations across mesoamerica (schwarz, 1948; kent, 1984). a key aspect of husbandry in mesoamerica was the development of hives of standard measures to accommodate colonies. interestingly, two types of hive exist today in mesoamerica, one, the hollow log (locally known as ‘hobon’) was extensively used in the maya area. another type consists of clay pots (‘mancuernas’ or ‘ollitas’) used as hives in the mexican highlands of puebla, veracruz and oaxaca (fig 3; weaver & weaver, 1981; gonzálezacereto, 2008; arnold et al., 2018; quezada-euán, 2018). for the maya, stingless beekeeping became part of cosmogony and mythology, being of similar importance to the cultivation of maize, a staple food for mesoamerican civilizations (de jong, 1999). no other culture conferred such spiritual significance upon one of the bees they coveted, the “xunan-kab” (the lady bee, or lady of the honey), known to science as m. beecheii. evidence of the economic and religious importance of xunan-kab is found in maya art and manuscripts (crane, 1992; zralka et al., 2014; sotelo-santos & asomoza, 2018; quezada-euán, 2018). the most important is the codex of madrid, one of the three surviving maya codices, in which stylized images of the xunan kab and guardian gods are represented in various scenes, probably associated with honey harvest and colony multiplication (zralka et al., 2014). the scenes depicted in the codex and other maya creations reveal that many deities looked after the bees and the shelter “na hil kab”, where their nests in hollow logs “hobones” were maintained. some of these deities are ah-mucen-kab (the descending honey god), noh yum kab, hobnil, balam-kab and moc-chí (schwarz, 1948; de jong, 1999). all are represented with a mixture of anthropomorphic and bee-like features, sometimes involving characteristics of other sacred animals like the jaguar (as seen on the face of balam-kab) (lópez-maldonado, unpublished data, see supplementary material 1; quezada-euán, 2018). like their mayan ancestors, peasant farmers in the yucatan peninsula fig 2. a summary of literature reports of the main uses given to stingless bee products by different indigenous groups of tropical america. jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees538 and guatemala still use hollow logs, often of the “chicle” tree, manilkara, to keep colonies of m. beecheii (yurrita-obiols & vázquez 2013; bianco et al., 2017). large numbers of managed colonies were kept in precolumbian yucatan, which produced large amounts of honey supporting intense trade within and outside the region (schwarz 1948; calkins, unpublished data; batún-alpuche unpublished data, see supplementary material 1). the production of m. beecheii honey in the yucatán peninsula seems to have occurred as a large-scale cottage industry and was organized at household and neighborhood levels (batún-alpuche, unpublished data, see supplementary material 1). thus, the accumulation of wealth in maya society may have been a consequence of honey trade (quezada-euán et al., 2001). in maya mythology, it is believed that the xunan-kab was directly given to humans by the major god “kun’ ku” or “yumbil dios”, therefore, the beekeepers are seen as guardians and caregivers of this creature, which in essence does not belong to them (de jong, unpublished data, see supplementary material 1). as a tribute to the real owner of the bees, during annual rituals the beekeepers are obliged by tradition to give back part of the honey harvested from the colonies in a ceremony called the “u hanlil-kab”, or the feast of the bees (weaver & weaver, 1981). only the honey from the xunan kab is accepted by the gods. this has to do with the concept of the earth as a living entity composed of spirit, blood and flesh. honey from xunan-kab is considered “hot” (schwarz, 1948) and is seen as a living and essential fluid from the land where the bees dwell, which men have extracted from it. it is a concept parallel to that of blood as part of the human body (de jong, 1999). xunan-kab honey is also associated with fertility and is applied by midwives to the abdomen during child birth, and it also purportedly increases the production of milk consumed in beverages (schwarz, 1948; gonzálezacereto et al., unpublished data, see supplementary material 1). honey of this bee is used to treat disease, which in this traditional cosmology is thought to derive from an imbalance in body temperature (schwarz, 1948). interestingly, the pollen is considered as feces or filth from the bees and is not used for human consumption. this is probably an idea derived from the massive infestation by phorid flies that may occur in pollen pots. during the harvest, pollen pots are discarded and buried underground (gonzálezacereto, 2008). stingless bees other than xunan-kab are considered masculine and their honey, including that of the western hive honeybee, apis mellifera l., is regarded as “cold” (de jong, 1999), although it is consumed and used in traditional medicine (gonzález-acereto et al. unpublished data, see supplementary material 1). being considered such a sacred creature, it is not surprising that the traditional cultivation of xunan-kab is strongly associated with various symbols related to their divine origin and the creation of the world in maya cosmogony (schwarz, 1948; weaver & weaver, 1981; de jong, 1999; gonzález unpublished data, see supplementary material 1). in contrast with the wide use of honey, cerumen seems to have been mainly used in “lost wax” gold foundry outside the mayan area, but not in candle-making, which started only after contact with europeans (schwarz, 1948). in mesoamerica, outside the yucatan peninsula and northern guatemala, pre-columbian stingless beekeeping seems only to have reached a moderate level of development, but was probably widely practiced (calkins, unpublished data; kent, 1984: aguilar et al., 2013a; arnold et al., 2018). to the north, remnants of this activity are still found in the mexican states of puebla and veracruz on the east coast, and on the pacific coast in the state of guerrero. indigenous nahua groups practiced stingless beekeeping in those regions but not with xunan-kab. because m. beecheii is found from the tropical regions of mexico to costa rica (quezada-euán et al., 2007; roubik & camargo, 2012), but colonies were kept and multiplied only in the yucatan peninsula (foster, 1942), we may ponder the reasons why this particular species was more prolific, or managed with greater success in some regions and not in others. along the highlands of eastern mexico, the totonaco and nahua peoples still keep and propagate colonies of scaptotrigona mexicana (guérin)“p isil-nek-mej” or “taxcat” in clay pots (schwarz 1948; garcía-flores et al., unpublished data, see supplementary material 1). similarly, in the highlands of oaxaca on the pacific coast, the mixteco, zapoteco, chontal and chinanteco cultivate plebeia fulvopilosa ayala, in clay pots (arnold et al., 2018). in contrast, the zapoteco and mixteco on the pacific and the zoque and popoluca of the gulf coast rarely cultivate m. beecheii, melipona fasciata latreille, melipona solani cockerell, scaptotrigona pectoralis (dalla torre), s. mexicana, t. angustula and frieseomelitta nigra (cresson), in hives. these indigenous people keep nests of those species either in their original trunks close to their homes, or mostly hunt them in the wild (vásquez-dávila unpublished data, see supplementary material 1; arnold et al., 2018). on the pacific coast the tlapaneco keep m. fasciata in log hives (gonzálezacereto, 2008); this is probably the bee mentioned in the account of stingless beekeeping in western mexico provided by dixon (1987). there is some evidence of nannotrigona perilampoides (cresson) being used along the mexican pacific coast (bennett, 1964). there are very few records concerning indigenous stingless bee keeping in southern mesoamerica, and it possibly did not play an important role in the religion of native people (aguilar et al., 2013a). south america unlike mesoamerica, where most traditional knowledge is related to a few bee species, south american native people used a wide range of species, but in contrast to mesoamerica and mexico, we found no reference to bee husbandry in precolumbian times. most indigenous groups exploited the bees in their natural environment and/or in their natural nests kept near their homes. sociobiology 65(4): 534-557 (october, 2018) special issue 539 before europeans, indigenous peoples in south america knew stingless honey bees (espinosa, 1529, cited in samper pizano, 2003; gumilla, 1741; castellanos, 1852; acuña, 1986; crane, 1999; medrano & rosso, 2010). one south american locale in which a primitive form of meliponiculture was practiced was on the slopes of the sierra nevada de santa marta, in northeastern colombia (patiño, 1965-1966, cited in patiño, 1990) — perhaps under the influence of the maya (oviedo & valdés, 1959, cited in crane, 1999). a secondary center of stingless beekeeping likely existed in northern venezuela (sanoja & vargas 1974, cited in patiño, 1990). according to rivero oramas (1972) the oldest references on stingless bees in venezuela, from 1612-1613, are those of fray pedro simon. although references to ancient forms of stingless beekeeping were found for these regions, it is not clear if it was practiced by native people before the spanish conquest. in colombia the bee species used by indigenous peoples are at least partly known. the nukak, traditional nomadic peoples of the northwestern amazon, harvested products of around 43 native bee species (cabrera & nates-parra, unpublished data, see supplementary material 1). within the mythology of the uwa (sierra nevada del cocuy, colombia), the bees are considered important as beings that made life evolve in the universe, and honey is associated with fertility and procreation (falcheti & nates-parra, 2002). according to jara (1996), for the andoke tribe, honey is considered a food crop (and female in gender, like the cassava) while the stingless bees themselves represent the male gender. the andoke evidently failed to recognize male versus female bees. in the vaupés state of colombia, rosso and parra (2008), estrada (2012) and rosso-londoño (unpublished data, see supplementary material 1), documented indigenous traditional knowledge related to stingless bees, especially within the ethnic groups tatuyo, syriano and bara. among these societies there is detailed knowledge of at least 25 ‘ethnospecies’ of stingless bees, used in traditional medicine, food, tool making and musical instruments and as bait for fishing, among other things. for these societies, as for the maya, the pollen was not used because it was considered dirty (bee excrement) (rosso-londoño, unpublished data, see supplementary material 1). nates-parra and rosso-londoño (2013) recorded nearly 50 common names used for the stingless bees, as did posey and camargo (1983) among the kayapó of brazil, with wide variation between regions and informants. one of the oldest references to stingless bees in south america is that written by hans staden in 1577, the “warhaftig historia” (engels, 2009). the report describes three species and nests, probably melipona quadrifasciata lepeletier, scaptotrigona postica (latreille) and t. angustula used by the tupinambá, who lived in the coastal region of santos, state of são paulo, brazil. another study from lima and moreira (2005) reports that the tupinambá associate the stingless bees with their cosmology. they have named constellations with bee names such as the “seichu-jurá” or “jirau-de-abelha”, “seichu” or “eichu”, meaning master bee or queen bee. noteworthy among south american indigenous groups are the kayapó from the eastern amazon forest, who developed a solid tradition on knowledge of stingless bees (posey, 1983; camargo & posey, 1990; melatti, 2007). the stingless bees compose part of their cosmology and social organization models, while maintaining importance in food and medicine. when hunting colonies, the kayapó recommend leaving part of the colony and nest intact after harvesting honey, as an offering to “bep-kororoti”, a powerful shaman. although fig 3. indigenous stingless bee husbandry in mesoamerica involves the use of specially made hives: hollow logs or ‘hobones’ in the mayan area, and clay pots or ‘mancuernas’, in the highlands of puebla, veracruz and oaxaca (photos courtesy of humberto moo and margarita medina). jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees540 it was perhaps unintentional, by following that practice, a colony could recover, and again provide honey for the kayapó (posey, 1986). the kayapó group can identify 56 species of “stingless bees” (note that posey, 1986, included bees of taxa other than meliponini), in strong correlation with scientific classification, and they recognize the nest architecture of each in detail, as well as the different stages of bee development (posey, 1983). in addition, the kayapó developed an ability to locate bee nests by listening for the noise made by bees performing nest ventilation, which they recognized for each bee species. aggressive bees like oxytrigona and the exotic colonist a. mellifera were managed with smoke of a liana (tanaecium nocturnum (barb. rodr.) bureau & schumann, bignoniaceae), which narcotized the bees, so that honey could be taken (camargo & posey, 1990).the kayapó people have a semi-domesticated system of beekeeping. some wild nests are taken to the village, while others are harvested in their natural environment, and small forest clearings are made to encourage nesting there by certain stingless bee species. no other amazonian tribe is reported to maintain stingless bees in the manner of the kayapó. the kayapó associate honey, wax, and bees with the heavens and rains because of the fondness that “bepkororoti” has for honey. they burn the cerumen to produce a smoke that is believed to attract storm clouds and rain, as well as to repel evil spirits, purge houses from lingering ancestral spirits, and protect children from witchcraft. the cerumen has multiple uses, such as (when melted) a glue to attach feathers to an arrow, and to strengthen and lubricate bow strings (posey, 1983; stearman et al., 2008). a most impressive and symbolic use of cerumen is for the “me-kutom”, a hat worn by young men about to receive ceremonial names upon reaching adulthood. cerumen for the hat is inherited and kept in the family for generations (posey, 1983). other groups like the enawenê-nawê from the southwestern amazon, mato grosso state, also have an excellent classification system and recognize 48 species of stingless bees (santos & antonini, 2008). the pankararé from the northeastern “caatinga” savannah (steppe), bahia state, name 23 folk species (costa-neto, 1998) and the kaiabi, also from mato grosso state and guarani m’byá from the atlantic forest, southwestern and southern brazil, recognize 27 and 13 species, respectively (ballester unpublished data, see supplementary material 1; rodrigues unpublished data, see supplementary material 1). the kawaieté people that live in xingu river basin in brazil have names and knowledge for 37 stingless bee species, identify 28 forest trees they use for nesting and 19 plants for feeding (villas-bôas, 2012). bees are also important in the cosmology and diet (honey, pollen and larvae) of the guarani from the atlantic forest. they call the bees “mother of the gods” (nogueira-neto 1997; rodrigues unpublished data, see supplementary material 1). to the pankararé from the arid zones of northeastern brazil, all “bees” (bees and wasps are placed in a single category) are believed to be enchanted organisms, protected from human exploitation by guardian spirits of plants and animals called “encantados” (costa-neto, 1998). eleven species provide 13 raw materials used for remedies to treat or prevent 16 illnesses (costa-neto, 1998). the pankararé classify the “abeias” (bees) according to their behavior as either “angry” or “gentle”. they also divide the “abeias” into three ethnofamilies, depending on the presence of a sting (costa-neto, 1998; castro et al., 2017). the knowledge on bees held by brazilian indigenous tribes is passed on orally from one generation to another and has likely influenced many involved in stingless beekeeping. indeed, widely used stingless bee names, such as “jataí, uruçu, tiúba, mombuca, irapuá, tataíra, jandaíra, guarupu, manduri” have indigenous linguistic origins (lenko & papavero, 1979; nogueira-neto, 1997; villas-bôas, 2012). the kaingang tribes from southern brazil believe that stingless bee honey has two strongly contrasting values. honey and raw vegetables are “cold” foods. they are only allowed to widows or widowers who, if they eat meat or any cooked food, are at risk of suffering from an internal heating, followed by death (lévi-strauss, 2004b). nevertheless, other groups of kaingang prepare a fermented manioc beverage in two different ways, with or without stingless bee honey, and the type sweetened with honey is considered more intoxicating, and if imbibed while fasting, may induce vomiting. it is thus called a “hot” food. lévi-strauss (2004b) reports that among certain northern tupi indians, honey occupies a central place in ceremonial life and religious thought. like their relatives from the tembé tribe, the guajajara from maranhão state, northeastern brazil, celebrate honey in their principal festivals. it is also reported that the umutina believe man was created from wild fruits and honey (lévi-strauss, 2004 a, b). the cerumen of native bees also played a central role in amerindian cultures that practiced metallurgy. golden ornaments like jewelry used the cerumen of stingless bee nests to fashion their model, and a mold was made into which molten gold was poured, after the cerumen or “lost wax” had been melted away (falchetti, 1999). another product widely used was “propolis”, or pure collected plant resin. in colombia the propolis of “brea bees”, ptilotrigona occidentalis (schulz), called “canturron”, served for the elaboration of “embil”, a name given the torches used by chocoan people (patiño, 2005). the ‘canturron’ is also used by other local communities (valle de cauca and antioquia, western colombia) for waterproofing boats and for the healing of minor wounds (galvis, 1987; nates-parra, 2005). in argentina, especially in misiones and yungas, there is evidence of stingless honey bee product use in traditional medicine, along with many plants (zamudio & hilgert, 2011, 2012; flores et al., 2015). the stingless bees were well known by different cultures in northern argentina before the arrival of europeans; sociobiology 65(4): 534-557 (october, 2018) special issue 541 the traditional knowledge of stingless bees in argentina is reflected in the many and accurate names by which different species have been known by diverse ethnic groups, such as the guaraní, guaycurú, toba, wichí, and quichua people, as well as the criollos. the more exploited species of stingless bees in northern argentina are tetragonisca fiebrigi (schwarz) (‘yatei’, ‘rubiecito’), scaptotrigona jujuyensis (schrottky) (‘negrito’, ‘tapezua’, ‘yana’) and t. aff. angustula (‘rubiecito’, ‘mestizo’) and some plebeia (schwarz) species (roig-alsina et al., 2013). in bolivia, t. angustula are called “señorita bees”, and there are indigenous communities (yuqi, ayoreo, sirionó) that use this bee for food and medicine. the ayoreo possess an extraordinary knowledge of bees and honey. they report 16 species of bees such as “ajidabia” (t. angustula) and “cuteri” (scaptotrigona polysticta moure), and classify honey by flavor and texture, acknowledging different properties. for example, acid honey causes diarrhea, so they don’t eat it. the acid and sour taste were due to the origin, that is to say, certain flowers — a biologically correct observation (szabó & stierlin, 2005). currently, the sirionó keep various species of stingless bees in hives as a state-supported production project. honey is used as food, especially to give strength to children and to make drinks like “mead” and “hichi”. the cerumen is used as glue in the making of arrows (lehm & lehm, 2004). in the upper amazon of ecuador, the achuar people collect bee nests to consume their larvae and obtain honey “mishik” from t. angustula “wapaa yumir”. pot-honey is used to treat throat inflammation (descola, 1996; guerrini et al., 2009). the kichwa people are recognized for their knowledge of stingless honey bees. they are able to describe and to differentiate honey types without previous sensory training. this suggests ecuadorian native kichwa maintain a sensory legacy by teaching new generations how to identify different honeys by taste (vit et al., 2017). although we cannot claim there is clear documentation of ancient cultures in the hot, seasonal savanna-forest of northeastern brazil, there are many cave paintings, dated over 20 millennia in age (lahaye et al., 2013; see hoffman et al., 2018). one of them is probably related to harvest of bee brood or other material from an aerial nest of trigona (fig 4). this fact indicates that further study is required to fill many gaps and clarify our knowledge of the archeology of bee use and management in the americas. status and perspective of indigenous knowledge related to stingless bees in tropical america stingless bees have been widely used across tropical america. many were documented here, but probably many more remain undocumented. native groups have developed and still maintain strong traditions and myths based upon their perception of these insects. in spite of the varied evidence of a rich heritage associated with stingless bees, it could be at risk of disappearing. in yucatan, a steady decline of stingless beekeeping is occurring, probably as a consequence of deforestation, changes in the economic system and the introduction of honeybees (quezada-euán et al., 2001; villanueva-gutiérrez et al., 2005a, b; gonzález-acereto et al., 2006; villanueva-gutiérrez et al., 2013). in south america, a similar situation has occurred; there was a rich background of knowledge of various native tribes that is disappearing in the younger generation across the region (stearman et al., 2008). it seems in many countries traditional knowledge associated with these species is disappearing. one of the important factors for the decline of traditional knowledge is the decline of the indigenous peoples themselves. geographic displacement, encroachment and the erosion of their culture occur as outside elements have caused the breakdown of their customs and traditions (lyver et al., 2014). nonetheless, different educators and groups of producers across the americas have been aiming at rescuing stingless bees through their inclusion in beekeeping. a mainstay of this activity concerns dissemination of methods to efficiently maintain and reproduce colonies, which reduces the impact of hunting wild populations (gonzález-acereto et al., 2006; quintal & roubik, 2013; hill et al., 2016). attempts are also made to improve the quality and marketability of products for better economic rewards and also by increasing the value of colonies from additional services such as commercial pollination (quezada-euán et al., 2001; vit et al., 2013; roubik et al., 2018). stingless beekeeping is showing signs of recovery for various indigenous groups. people outside these communities are also getting involved in stingless beekeeping and commercialization of bee products (gonzález-acereto et al., 2006). across the americas, there are numerous examples of projects to promote stingless beekeeping and for the recovery of this ‘threatened’ activity (ferrufino & aguilera, 2006; gonzález-acereto et al., 2006; meriggi et al., unpublished data, see supplementary material 1; pérez & salas, 2008; fig 4. an ancient cave image from the serra da capivara, northeast brazil, probably related to hunting stingless bee honey. courtesy of acervo da fundação museu do homem americano. jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees542 rosso & parra, 2008; venturieri, 2008a, 2008b; infante et al., unpublished data, see supplementary material 1; estrada 2012; gómez unpublished data, see supplementary material 1; aguilar et al., 2013b; herrera-gonzález et al. unpublished data, see supplementary material 1; villanueva-gutiérrez et al., 2013; arnold et al., 2018). countries like brazil are developing a vigorous following of meliponiculture, especially in the northeast and north regions (villas-bôas, 2012; jaffé et al., 2015). melipona scutellaris latreille, m. quadrifasciata, melipona rufiventris lepeletier, melipona subnitida ducke, melipona compressipes (fabricius), t. angustula and scaptotrigona spp. are the most common bees maintained in brazilian meliponaries (rosso unpublished data, see supplementary material 1; jaffé et al., 2015). in rio grande do sul, an extensive work was done to improve meliponiculture techniques (witter et al., unpublished data, see supplementary material 1) and melipona bicolor schencki gribodo, plebeia spp., and scaptotrigona spp. are important species for honey production and pollination. in el gran chaco, the honey extracted from stingless bee colonies often caused destruction of the colony and the tree sheltering it (arenas, 2003; meriggi et al., unpublished data, see supplementary material 1, medrano & rosso, 2010), but meliponiculture is now encouraged as a tool to preserve nature in the argentinian part of this region. this was followed by the implementation of modern techniques to raise and manage stingless bees (meriggi et al., unpublished data, see supplementary material 1). the government and nongovernmental organizations have promoted meliponiculture projects in different provinces (cedit, 2005; baquero & stamatti, 2007; gennari unpublished data, see supplementary material 1). key elements for the recovery of stingless bees have been the development of a market for their products and “extension” courses and instruction. many young people of surviving ethnic groups have lost the connection to their ancestors or even have no knowledge of the bees. training offered to indigenous peoples can also make honey hunting sustainable by preventing colony destruction (gonzálezacereto, 2008; perichon, 2013). most importantly, a respect for the local habits and traditions is essential to preserve ancestral knowledge and management techniques that may help in preserve both stingless bees and beekeeping (lyver et al., 2014; jaffé et al., 2015; quezada-euán, 2018). there are many examples in which outsiders try to change traditional systems and take advantage of indigenous communities depriving them from their land and resources. there should be a way to regulate such interactions, protecting the latter (rosso & imperatriz-fonseca, 2017). the lesson to be learned is that the traditional knowledge and heritage can survive as long as they are linked to the economics and well-being of local people (gonzálezacereto et al., 2006; villanueva-gutiérrez et al., 2013; hill et al., 2016), as they are the ones in direct interdependence with them. we suggest that it is essential, for the future preservation of stingless bees and the cultural heritage that surrounds them, to consider the different aspects of indigenous local knowledge in the development of policies for conservation of the bees. they are needed in the global effort to support programs for the development of indigenous peoples. traditional knowledge can strengthen the capacity of human societies to address disturbances and to maintain ecosystem services under conditions of uncertainty and change (berkes & turner, 2006; gómez-baggethun et al., 2013). there is hope that stingless bees can recover from an apparent imminent extinction and go on to become important agents for the conservation of the environment and human heritage. acknowledgments this manuscript was inspired by the chapter “biocultural diversity, pollinators and socio-cultural values, from the ipbes assessment on pollinators, pollination and food production” and highlights additional contents on latin american ethnic groups concerning stingless bees. we thank ro hill, peter kwapong and simon potts (ipbes assessment co-chair) for support and discussion. we thank two anonymous reviewers for their comments and suggestions. we are deeply thankful to eduardo almeida and anne costa for kindly providing images of stingless bees deposited in coleção entomológica “prof. j.m.f. camargo”, ffclrp/usp used to illustrate some species listed in table 1. images are also provided by gnp, rdw, jjgqe, margarita medina, humberto moo-valle and fundação museu do homem americano. jjgqe thanks project conacyt 103341 “the conservation of the stingless bees of mexico”, gnp thanks universidad nacional de colombia , and vlif thanks cnpq 311857/2013-2. supplementary material doi: 10.13102/sociobiology.v65i4.3447.s2224 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3447/0 references acuña, c de. 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(2014). excavations in nakum structure 99: new data on protoclassic rituals and precolumbian maya beekeeping. estudios de cultura maya, 44: 85-117. doi: 10.19 130/iifl.ecm.2014.44.790 https://ethnobiomed.biomedcentral.com/ jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees548 table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. scientific name local name country indigenous groups product used value (e = economic, c = cultural) refs. cephalotrigona capitata (smith) ei tapexua brazil guarani m’byá honey, cerumen c: food c: craft: cerumen used for art crafts. rodrigues (unpublished data, supplementary material 1) cephalotrigona zexmeniae (cockerell) ta ´ah cab ehool mexico maya honey, cerumen e: food e: craft making of figurines to magically protect crops. gonzález-acereto (2008) friesella schrottkyi (friese) ei mirî ‘i brazil guarani m’byá honey, larvae, pollen, e: food – honey, pollen, larvae c: religious & medicinal honey is used in new-born children mouth as bactericide. rodrigues (unpublished data, supplementary material 1) frieseomelitta sp. mykrwãt brazil kayapó honey, pollen, brood e: food – honey, larvae, pupae and pollen posey (1983) frieseomelitta nigra (cresson) sak xic mexico maya honey e: medicinal diluted to treat pterygium. mixed with resin of the same bee to treat wounds. gonzález-acereto (2008) frieseomelitta silvestrii (friese) abeia-brancado-fundinhobranco brazil pankararé larvae, pollen (food) cerumen, e: food – larvae and pollen c: medicinal cerumen used to treat influenza. costa-neto (1998) frieseomelitta varia (lepeletier) mehnodjanh brazil kayapó honey, cerumen, e: food – honey c: medicinal cerumen has medicinal use smoke from cerumen used for curing/ healing. posey (1983) sociobiology 65(4): 534-557 (october, 2018) special issue 549 geotrigona leucogastra (cockerell) abeja de la tierra ecuador kichwa community honey e: food – honey vit et al. (2017) melipona sp. bísara, bichara, bíjara colombia uwa honey e: food falchetti and nates parra (2002) ei ruyu or akã-moto brazil guarani m’byá honey, cerumen e: food – honey; c: craft: cerumen used for art crafts. rodrigues (unpublished data, supplementary material 1) melipona beecheii bennett xunan kab kolel kab pool kab paisil kab mexico (yucatan peninsula) maya honey e: medicinal: treat conjunctivitis mixed with herbs and deer flesh for women fertility. mixed in a drink with leaves of the tree guazuma ulmifolia and applied on the abdomen of women during labor c: religious: beverage balché. in ceremonies for good crops and rain (u hanlil kab) c: mythology: vital fluid of the land. gonzález-acereto et al., (unpublished data, see supplementary material 1) weaver and weaver (1981) gonzález (unpublished data, see supplementary material 1) de jong (1999) cerumen e: craft: candle making in honor of the death. gonzález-acereto (2008) the bees c: mythological: gods: ah mucen cab the great divine bee. bees related to the creation of the world (chilam balam) quezada-euán et al. (2001) mediz-bolio (1952) yum chab mexico (tabasco) chontal honey e: food vásquez-dávila and hipólito-hernández (unpublished data, see supplementary material 1) ajachab mexico (chiapas) tzeltal and zoque honey e: food vásquez-dávila (unpublished data, see supplementary material1) oy mëj tsin mëj pa’ak tsin pa’ak mexico (oaxaca) mixe honey e: food and medicinal arnold et al. (2018) busdoo mexico (oaxaca) zapoteco honey e: food and medicinal arnold et al. (2018) criolla colmena grande guatemala honey e: food and medicinal: wounds, gastritis, post-partum. enriquez et al. (unpublished data, see supplementary material 1) jicote gato costa rica honey e: food herrera-gonzález et al. (unpublished data, see supplementary material 1) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees550 melipona bicolor lepeletier guaryuka brazil guarani m’byá honey, propolis (batumen) e: food – honey c: medicinal: propolis (extracted from the batumen) used as infusion as contraceptive for men and woman. rodrigues (unpublished data, supplementary material 1) melipona compressipes (fabricius) ngày-re tiubá brazil kayapó honey, cerumen e: food – honey; e: craft & medicinal cerumen has utilitarian and medicinal use c: religious: cerumen has ceremonial use posey (1983) kerr et al. (2001) melipona fasciata latreille colmena real mexico tlapaneco honey e: medicinal gonzález-acereto (2008) tinzuca guatemala honey e: food enriquez et al. (unpublished data, see supplementary material 1) melipona fulva lepeletier to beroa colombia tatuyo honey, larvae cerumen e: food: honey, larvae and for fishing bait e: craft: cerumen estrada (2012) rosso-londoño (unpublished data, see supplementary material 1) melipona fuscopilosa moure & kerr isabitto venezuela paria grande (estado amazonas) uothuja (piaroa) honey, pollen cerumen, e: food: honey e: medicinal : honey : treatment of cataracts pollen: treatment of spots on the skin, facial cleaning e: craft: cerumen has utilitarian use c: cerumen has ceremonial use vit et al. (2011) infante et al. (unpublished data, supplementary material 1) melipona grandis guérin bunga negra abeja de la tierra ecuador kichwa honey e: food vit et al (2017) melipona mandacaia smith mandassaia kagnàrà-krã-tik kagnàrà-udja-ti kagnàrà-ti brazil pankararé kayapó honey honey, larvae, pollen, cerumen c. religious & medicinal: honey has medicinal use for snake bites. e: food – honey, larvae and pollen; e: craft: cerumen has utilitarian use; c: religious & medicinal: cerumen has ceremonial and medicinal use. costa-neto (1998) posey (1983) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) sociobiology 65(4): 534-557 (october, 2018) special issue 551 melipona marginata lepeletier mandori brazil guarani m’byá honey, cerumen e: food – honey. c: craft: cerumen used for art crafts. rodrigues (unpublished data, supplementary material 1) melipona nebulosa camargo niti dobea colombia bará honey, larvae cerumen e: food: honey, larvae and for fishing bait e: craft: cerumen. estrada (2012) rosso-londoño (unpublished data, see supplementary material 1) melipona quadrifasciata lepeletier ei raviju brazil guarani m’byá honey, cerumen e: food – honey c: religious; cerumen has ceremonial and religious use c: craft: cerumen used for art crafts. rodrigues (unpublished data, supplementary material 1) melipona cf. rufescens friese melipona eburnea friese to dobea colombia bará honey, larvae cerumen e: food:honey, larvae and for fishing bait e: craft: cerumen estrada (2012) rosso-londoño (unpublished data, see supplementary material 1) melipona rufiventris lepeletier ngày-kumrenx brazil kayapó honey, cerumen e: food – honey; e: craft & medicinal: cerumen has utilitarian and medicinal use c: religious: cerumen used for “me-kutom” (ceremonial use). posey (1983) quigüeté bolivia yuquí, brood cerumen (irití) e: food: immature e: craft: cerumen has utilitarian use: elaboration of arrows stearman et al. (2008) ere reu bolivia tacana community honey e: food: honey gomez et al. (2003) m. r. flavolineata friese quigüeguá bolivia yuqui brood cerumen (irití) e: food: immature e: craft: cerumen has utilitarian use: elaboration of arrows stearman et al. (2008) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) https://www.researchgate.net/ profile/robert_wallace7/ publication/269398245_ investigacion_colaborativa _un_pilar_para_la_conservacion _de_la_biodiversidad_a_nivel_del _paisaje_basada_en_comunidades _locales/links/54b680b80cf24 eb34f6d267f/investigacioncolaborativa-un-pilar-para-laconservacion-de-la-biodiversidada-nivel-del-paisaje-basada-encomunidadesjjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees552 melipona scutellaris latreille uruçu brazil pankararé honey c: medicinal:snake bites, rabid dog bites, impotence. costa-neto (1998) melipona seminigra friese ngài-ny-tyk-ti brazil kayapó honey, cerumen e: food – honey; e: craft & medicinal: cerumen has utilitarian and medicinal use c: ceremonial: bee parts used for hunting magic; c: ceremonial: cerumen has ceremonial use. posey (1983) nannotrigona perilampoides (cresson) trompetilla bees beú mexico nahua zapoteco honey e: food padilla-vargas et al. (unpublished data, see supplementary material 1) arnold et al. (2018) oxytrigona tataira (smith) kagnàrà·krãkamrek brazil kayapó honey, brood pollen, cerumen, e: food – honey, larvae and pollen; e: craft: cerumen has utilitarian use c: religious & medicinal: cerumen has ceremonial use and medicinal use; c: cultural: cut entire tree to take kamrek honey. posey (1983) paratrigona sp. yvy ei brazil guarani m’byá honey, brood e: food – honey and larvae rodrigues (unpublished data, supplementary material 1) partamona sp. ngày-càk-ñy kukoire·ka brazil kayapó honey cerumen e: food – honey; c: cultural: cerumen in magic to make enemy weak. nests preserved in termite mounds. posey (1983) partamona bilineata (say) cho´ch kuitanektsin mexico maya nahua honey e: medicinal – mixed with the blood of the bird crotophaga sulcirostris is used to treat diphtheria e: food quezada-euán et al. (2001) padilla-vargas et al. (unpublished data, see supplementary material 1) partamona cupira (smith) cupira brazil pankararé honey e: medicinal: honey has medicinal used, for throat inflammation treatment. costa-neto (1998) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) sociobiology 65(4): 534-557 (october, 2018) special issue 553 plebeia sp. xalnektsin besinbea mexico nahua zapoteco honey e: food padilla-vargas et al. (unpublished data, see supplementary material 1) arnold et al. (2018) ei mir~i brazil guarani m’byá honey e: food – honey; e: medicinal: honey is used in new-born children mouth as bactericide rodrigues (unpublished data, supplementary material 1) plebeia mosquito (smith) mosquito-preto brazil pankararé honey e: medicinal: honey has medicinal use, eaten and massaged for throat ache and oral mycosis. costa-neto (1998) ptilotrigona occidentalis (schulz) canturrona brea bees colombia afro descendant communities western colombia resin called ‘canturrón’ e: medicinal: minor wounds. c: cultural: elaboration of the embil, name of the torches that the chocoan people used for lighting. waterproofing boats galvis (1987) nates-parra (2005) patiño (2005) scaptotrigona hellwegeri (friese) abeja bermeja mexico pacific coast of mexico honey e: food gonzález-acereto (2008) scaptotrigona mexicana (guérin) pisil nek mej taxkat congo mexico nahua, totonaco honey propolis e: food e: medicinal respiratory illnesses. gonzález-acereto (2008) padilla-vargas et al. (unpublished data, see supplementary material 1) congo guatemala honey e: food enriquez et al. (unpublished data, supplementary material 1) scaptotrigona pectoralis (dalla torre) kan ts´ak alazán mexico guatemala maya honey e: food gonzález-acereto (2008) enriquez et al. (unpublished data, see supplementary material 1) scaptotrigona polysticta moure suro, cuteri bolivia ayoreo honey pollen (gebora) larvae cerumen e: food: honey, pollen, larvae e: craft: cerumen has utilitarian use. stearman and szabó (2005) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) http://amazoniabolivia. com/amazonia_bo.php?id_ contenido=304&opcion= detalle_des jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees554 scaura longula (lepeletier) mehnorã-tyk brazil kayapó cerumen, resin e: medicinal: cerumen has medicinal use; c: cultural: resin used for jaguar hunting magic posey (1983) tetragona sp. kedari colombia nukak honey, larvae e: food cabrera and nates-parra (1999) o-i ton-my ri brazil kayapó cerumen, resin e: craft: cerumen has utilitarian use e: medicinal: cerumen has medicinal use; c: cultural: bee thought to be “stupid” and weak posey (1983) tetragona clavipes (fabricius) ajavitte venezuela paria grande (estado amazonas) uothuja (piaroa) honey e: food: honey vit et al. (2011) wáno venezuela colombia yukpa-yuko people honey, larvae cerumen e: food: honey and larvae e: craft: cerumen has utilitarian use: waxing thread used to bind arrows e: economic: bait by fishermen. ruddle (1973) ei pytã brazil guarani m’byá honey, cerumen, larvae (brood) e: food – honey e: medicinal: larvae mixed with plant root used as fertility stimulant; honey mixed with cerumen passed on children mouth (bactericide). rodrigues (unpublished data, supplementary material 1) tetragona goettei (friese) mehn-xi·we’i brazil kayapó honey, cerumen e: food e: craft: cerumen has utilitarian use c: religious & medicinal: cerumen has ceremonial and medicinal use. posey (1983) tetragona quadrangula (lepeletier) menire-udja menire-udga brazil kayapó honey, cerumen e: food – honey e: craft: cerumen has utilitarian use e: medicinal: cerumen c: religious cerumen has ceremonial and medicinal use. posey (1983) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) sociobiology 65(4): 534-557 (october, 2018) special issue 555 tetragonisca angustula (latreille) uux pa’ak mexico (oaxaca, chiapas) mixe, zapoteco, tzeltal, honey e: food arnold et al. (2018) chumelo guatemala honey e: food e: medicinal: cataracts enriquez et al. (unpublished data, see supplementary material 1) angelita wapasa yumiri colombia ecuador, alto amazonas achuar honey honey called “mishik” larvae e: medicinal respiratory and ocular illnesses (conjunctivitis, pterygium) e: food: honey and larvae treat throat inflammation with pot-honey nates-parra and rosso-londoño (2013) descola, (1996) guerrini et al. (2009) señorita bolivia yuracaré tacana community honey e: food: honey e: medicinal: honey: treatment of cataracts, gastritis, anemia. camacho, 2012. http://www.opinion. com.bo/opinion/ articulos/2012/0413/ noticias.php?id=51805 gomez et al. 2003 señorita, ajidabia bolivia ayoreo honey pollen (gebora) larvae cerumen e: food: honey, pollen, larvae e: craft: cerumen has utilitarian use. stearman and szabó (2005) jatei brazil guarani m’byá honey, brood cerumen, resin, pollen e: food – honey, brood e: medicinal: honey, resin, pollen and cerumen have medicinal use (flue, stomach diseases, rheumatism, parasitosis e: craft: cerumen used to make candles and art crafts c: religious: honey mixed with corn to prepare “tambojape”, a special bread for the baptism ceremony rodrigues (unpublished data, supplementary material 1) trigona sp. mumia colombia bará honey, larvae cerumen e: food: honey, larvae and for fishing bait craft: cerumen. estrada (2012) trigona sp. ngoi·tenk brazil resin e: medicinal: resin is mixed with a leaf to treat rheumatism rodrigues (unpublished data, supplementary material 1) http://amazoniabolivia. com/amazonia_bo.php?id_ contenido=304&opcion= detalle_des table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) http://www.opinion.com.bo/opinion/articulos/2012/0413/noticias.php?id=51805 http://www.opinion.com.bo/opinion/articulos/2012/0413/noticias.php?id=51805 http://www.opinion.com.bo/opinion/articulos/2012/0413/noticias.php?id=51805 http://www.opinion.com.bo/opinion/articulos/2012/0413/noticias.php?id=51805 jjg quezada-euán, g nates-parra, mm maués, vl imperatriz-fonseca, dw roubik – economic and cultural values of stingless bees556 ngoi·tenk brazil kayapó honey, cerumen e: food – honey e: craft: cerumen utilitarian use c: religious & medicinal: cerumen has ceremonial and medicinal use. posey (1983) trigona amalthea (olivier) udjy brazil kayapó resin; bees e: craft: resin used in artifacts c: cultural: bee parts mixed with urucu (bixa orellana) for hunting magic. posey (1983) trigona branneri cockerell mehñiy-tyk brazil kayapó honey, cerumen e: food – honey e: craft: cerumen has utilitarian use e: medicinal: cerumen has medicinal use; posey (1983) trigona chanchamayoensis schwarz imre-ti-re brazil kayapó honey, pollen, brood e: food – honey, brood and pollen posey (1983) trigona cilipes (fabricius) mehnorãkamrek brazil kayapó cerumen, resin e: medicinal: cerumen has medicinal use; c: cultural: skinny eyes like jaguar posey (1983) trigona corvina cockerell wuti be´ colombia nukak honey , larvae, cerumen e: food: honey and larvae for food. e: craft: the cerumen is used in the manufacture of musical instruments, hammocks and blowpipe cabrera and nates-parra (1999) trigona dallatorreana friese tu colombia nukak honey e: food cabrera and nates-parra (1999) kukraire brazil kayapó pollen e: food – pollen posey (1983) trigona ferricauda cockerell wuti butu colombia nukak honey, larvae e: food cabrera and nates-parra (1999) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) sociobiology 65(4): 534-557 (october, 2018) special issue 557 trigona fulviventris guérin pyka-kam brazil kayapó honey, cerumen, resin e: food – honey e: craft: cerumen has utilitarian use c: religious & medicinal: cerumen in ceremonial and medicinal use. posey (1983) trigona fuscipennis friese djô brazil kayapó honey, cerumen e: food – honey e: craft: cerumen has utilitarian use c: religious: cerumen ceremonial use. posey (1983) trigona pallens (fabricius) myre brazil kayapó honey, cerumen e: food – honey e: craft: cerumen has utilitarian use c: religious & medicinal: cerumen has ceremonial and medicinal use. posey (1983) trigona silvestriana (vachal) chiichiiyá bolivia yuqui cerumen e: craft: cerumen has utilitarian use: elaboration of arrows stearman et al. (2008) trigona spinipes (fabricius) mehñykamrek arapuá ei irapua brazil kayapó pankararé guarani m’byá honey, brood, pollen cerumen honey honey, pollen, e: food – honey, brood and pollen e: craft: cerumen has utilitarian use c: religious & medicinal: cerumen has ceremonial and medicinal use; c: cultural: cerumen burned; smoke causes dizziness c: religious & medicinal: honey has medicinal use, eaten for diabetes treatment e: food – honey and pollen are eaten posey (1983) costa-neto (1998) rodrigues (unpublished data, supplementary material 1) trigona trinidadensis vachal venezuela colombia yukpa-yuko people honey, larvae cerumen e: food: honey and larvae e: craft: cerumen has utilitarian use: waxing thread used to bind arrows e: economic: bait by fishermen. ruddle (1973) trigona williana friese kajawo dawa colombia nukak honey, larvae e: food cabrera and nates-parra (1999) table 1. economic and cultural values associated with stingless bees by different ethnic groups of tropical america. (continuation) _hlk515455205 _hlk515274688 _hlk513463858 _hlk513450723 _hlk515365273 _hlk517862760 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3467sociobiology 65(4): 751-759 (october, 2018) special issue influence of wild bee diversity on canola crop yields introduction pollinators play an important functional role within ecosystems by providing environmental services essential to the reproduction and survival of plants (potts et al., 2016). among insects, bees stand out as the major pollinators of angiosperms in the world (ollerton et al., 2012). a full 33% of plants grown for human consumption is known to depend on beemediated cross-pollination (klein et al., 2007). this beneficial relationship between bees and plants can be threatened in several ways. studies on different crops have shown that many species and interconnected processes may collapse due to changes in adjacent habitats to crops, leading to decreased productivity and limiting yields (vicens & bosch, 2000). pollinator populations are subject to various effects of forest fragmentation, including agriculture, animal husbandry, abstract the foraging range of bees determines the spatial scale over which each species can provide pollination services. in agricultural ecosystems, productivity is related not only to the taxonomic diversity of bees per se, but also to the location of their nesting sites, which has impact on their flight range. within this context, the present study sought to assess how wild bee assemblages affect the yield of brassica napus at three different distances (25 m, 175 m and 325 m) from forest remnants in southern brazil. pan traps were employed to sample bees, and data analysis was carried out using the shannon diversity index and generalized linear models. we identified 11 species of native bees, both solitary and social, and high prevalence of the exotic species apis mellifera l. our findings show that canola crop yield was positively influenced by the diversity of bee species, demonstrating that native bees, not only a. mellifera, can greatly contribute to the productivity of canola crops. in addition, it was found that bee body size is significantly associated with flight distance travelled within the canola fields, and indicated a relationship with nesting sites. thus, we hypothesize that higher canola yields are associated with increased presence of wild bee species, both social and solitary, and that maintenance of these pollinators is directly related to practices adopted in rural areas, whether within crop fields or in forest remnants used as nesting sites by wild bees. sociobiology an international journal on social insects r halinski, cf dos santos, tg kaehler, b blochtein article history edited by solange augusto, ufu, brazil received 10 may 2018 initial acceptance 21 august 2018 final acceptance 12 september 2018 publication date 11 october 2018 keywords brassica napus, pollination, stingless bees, apis mellifera, agricultural ecosystems. corresponding author rosana halinski department of biodiversity and ecology school of sciences, laboratory of entomology pontifical catholic university of rio grande do sul (pucrs) av. ipiranga, 6681 partenon prédio 12a partenon, cep 90619-900 porto alegre rio grande do sul, brasil. e-mail: ro.halinski@gmail.com and other anthropogenic pressures (cresswell & osborne, 2004). this gradual reduction of natural landscapes significantly reduces the flow of animals, pollen, and seeds (samways, 1995). consequently, habitat fragmentation may have negative impacts on richness and diversity of several functional groups, including pollinator insects (kremen et al., 1993). assessment of insect diversity involves a variety of sampling methods designed to analyze the effects of the landscape on insect populations. investigations on anthophilous insects have sought to ascertain their diversity in different crops and to identify potential pollinators (westphal et al., 2008). historically, such studies have used collection nets for sampling. however, since the last decade, the use of pan traps has increased due to their efficiency in capturing a wide range of floral visitors and the absence of collector bias (westphal et al., 2008; vrdoljak & samways, 2012). pontifícia universidade católica do rio grande do sul, porto alegre, rs, brazil research article bees r halinski, cf dos santos, tg kaehler, b blochtein – wild bees and canola yield752 canola (brassica napus l., var. oleifera), or the oilseed rape, is the third most widely grown oleaginous plant in the world. the oil extracted from its seeds is used for human consumption and as biodiesel (marjanović-jeromela et al., 2008). in brazil, approximately 47,500 hectares of land, predominantly located in the south, are dedicated to the cultivation of canola (conab, 2017). the massive flowering of canola is highly attractive to bees, as it provides abundant floral resources (nectar and pollen) (holzschuh et al., 2016). studies have shown that insect visitation improves the yield of grain crops, of which apis mellifera l. is considered the main pollinator (abrol, 2007; rosa et al., 2010; bommarco et al., 2012; jauker et al., 2012; halinski et al., 2015; witter et al., 2015). in canada, where canola originates, sabbahi et al. (2005) introduced three a. mellifera hives per hectare in a canola field and found a 46% increase in seed yield. studies have shown that the presence of bees in this crop can increase grain yield by up to 47% (becker et al., 1992; bommarco et al., 2012; blochtein et al., 2014; witter et al., 2015). however, despite the well-established importance of a. mellifera to canola yields, wild bees may also contribute significantly to a more efficient pollination of this crop (garibaldi et al., 2013; koh et al., 2016; potts et al., 2016). the presence and diversity of these insects increases pollination and, consequently, the yield and market value of the crops (sabbahi et al., 2005; bommarco et al., 2012). considering that pollinators predominantly inhabit forest fragments adjacent to crop fields, and that bee body size is a predictor of their foraging range, they are expected to act on pollination at different scales (greenleaf et al., 2007; bommarco et al., 2012; bailey et al., 2014; wright et al., 2015). therefore, investigations on potential pollinators of commercially important crops and their nesting habits are necessary not only for the development of strategies to support conservation and management of these species, but also to enable their more efficient use in crop pollination. the present study was designed to evaluate whether the diversity and body size of bee species have influence on the productivity of canola fields located at varying distances from forest remnants in southern brazil. therefore, we sought to answer the following questions: (1) do the bee species present in flowering canola fields nest in nearby forest remnants? (2) is bee body size related to distances between crop fields and adjacent forest remnants? (3) does bee diversity increase canola crop yields? material and methods study area the study was conducted in four areas near and within b. napus fields (hyola 420 cultivar), in the municipality of esmeralda, southern brazil. the region is characterized by pasture land, forest fragments, and fields of annual crops (canola, soybean, maize, and wheat). the four investigated canola fields were 20 ha (field 1, fig 1), 80 ha (2), 100 ha (3), and 80 ha (4) in size, located 2.5 to 23.5 km from each other. these fields are in the upper plateau of serra do nordeste region, with average annual temperatures of 14.416.8 °c, relative humidity of 76-83%, annual precipitation of 1,412-2,162 mm, and altitude of 944 m (veloso et al., 1992). according to the köppen classification, the region is considered to have cfa – humid subtropical climate (alvares et al., 2013), and its original vegetation is composed of mixed ombrophilous forest and grassland. there are neither apiaries nor meliponaries in the municipality. bee sampling bees were sampled with blue, yellow, and white pan traps, which remained exposed for 24 h per sampling (adapted from westphal et al., 2008). these traps were laid out in plots, and consisted of five groups of three pots, keeping a distance of 15 m between groups and 3 m between pots, forming an equilateral triangle (adapted from fao, 2010; fig 1). the traps were placed within canola fields at three distances from the forest remnants: 25 m, 175 m, and 325 m, during flowering season (august-october), for a total of four samplings in 2010 (field 1 and 2) and seven in 2011 (fields 1 to 4). fig 1. schematic representation of pan traps’arrangement within the canola fields for bee sampling. the bees were identified and deposited at the bee collection of the museum of science and technology, at pontifícia universidade católica do rio grande do sul. after identifying the different species, we conducted a literature review to obtain relevant data on the characteristic nesting substrates and foraging range of each species (see supplementary material 1). bee size to test for correlation between distance flown from the forest remnants and bee body size, we measured the intertegular distance, or span, of the collected specimens. sociobiology 65(4): 751-759 (october, 2018) special issue 753 this morphological measurement is a robust estimator of bee body mass, size (bullock, 1999), and flight range (greenleaf et al., 2007; wright et al., 2015). measurements of intertegular distance were obtained using digital calliper only in female specimens due their role in brood care, therefore, return to their nests with provisions. productivity of canola crops to assess the productivity of canola crops, once siliques had matured, 20 plants were harvested and spaced 1.5 m apart, forming 225 m2 plots in each field, totaling three harvest plots per field. these plants were used to analyze seed production under free visitation of insects at three different distances from forest remnants: 25, 175, and 325 m. the number of seeds per plant was counted manually with the aid of a vacuum seed counter (ericksen de leo), and seed weight measured on an analytical balance (auy200). the mean seed weight per plant was obtained from the sum of the individual weight of each plant divided by the total number of plants. statistical analysis to assess species abundance and richness, we used the shannon diversity index, which responds more sensitively to changes in the importance of rarer species (peet, 1974). the diversity function of the “vegan” package in r was used for this purpose (oksanen et al., 2016). we then constructed a gaussian family (link = log) generalized linear model (glm) to assess whether mean canola grain weight (yield) could be explained by bee diversity. glm fit was assessed by the pseudo-r2 statistic, using the rsquared function in r package “piecewisesem” (lefcheck, 2015). the influence of bee size (intertegular distance) on the distance from the forest remnants (25, 175, 325 m) was assessed using a generalized linear mixed model (glmm) with negative binomial family estimated by maximum likelihood (laplace approximation). this error distribution family was selected after checking for overdispersion with a poisson-distributed glmm. in addition to the distance variable, nesting sites (supplementary material 1) and interaction between species and nests were used as predictor variables (fixed effects) for the glmm. fields and pan trap colors were regarded as random effects. analysis was conducted using function glmer.nb from package “lme4” (bates et al., 2015). finally, bee species collected in each of the four canola fields, and stratified by social vs. solitary, were plotted as a function of distance from the corresponding forest remnants. all analyses were carried out in r (r core team, 2018). we computed the accumulation curve of species and its respective interpolation/extrapolation of hill number based on sample size and abundance of individuals (bees per distance) which values of diversity (q=0) were estimated from chao1 (chao et al., 2014) after 2,000 replications. for this, we used the individual-based abundance data using the function inext from package of same name (hsieh et al., 2016). we also calculated the extrapolated species richness in our data matrix in order to estimate the number of unobserved species. we used the function specpool from “vegan”. the extrapolated richness was calculated with chao equation, that weights the number of individuals observed in a single sampling. we also performed a diversity profile according to diversity order from hill number (hill, 1973). such analysis was developed to widely evaluate bee diversity on canola fields. these values determine the sensibility of relative abundance of species by sites (chao et al., 2014), here assumed as distances from forest fragments. thus, we employed the bee community data matrix to generate the diversity profile using the function renyi (hill=true) from package “vegan”(oksanen et al., 2016). the similarity percentages, i.e. the pairwise comparisons of groups of sampling units and the average contributions of each bee species to the average overall bray-curtis dissimilarity, was estimated after 2,000 replications using the function simper from “vegan”. this function displays the most important species for each pair of groups (distances from forest fragments). as a result, these species must contribute at least to 70% of differences between groups. we also used bee community data to carry out a nonmetric multidimensional scaling (nmds). this ordination was employed to observe the dissimilarity on bee composition related to distance from forest fragments (25 m, 175 m, and 375 m). however, before performing this analysis, we standardized our community data using the function decostand (method = “total”). consequently, we applied the dissimilarity index of bray–curtis (proper to abundance data) using the function vegdist to implement nmds. thus, we used two functions from “vegan” (metamds and stressplot) to fit nmds and to find goodness of fit measure for points in nmds. stress values lesser than 0.2 are considered adequate. finally, we used our dissimilarity object provided by the function vegdist to fit a permanova type ii in function adonis.ii from package “rvaidememoire” (hervé, 2015). subsequently, we performed pairwise comparisons between group levels (distance from fragments) using the function pairwise.perm.manova from the same package with 2,000 permutations and statistic test by pillai method and p-value adjustment after “fdr”. results bee diversity within canola fields a total of 314 bees were collected from the four sampling areas, representing 11 native and one exotic species (a. mellifera).there are no managed colonies of any species of bee in the city, thus all collected bees were feral. the exotic species a. mellifera was the most abundant, accounting for 58% of all collected specimens (supplementary material 1). r halinski, cf dos santos, tg kaehler, b blochtein – wild bees and canola yield754 despite the apparent predominant role of a. mellifera bees in the canola fields, native bee species also warrant attention, whether because some of them were also present in all distances or because their behavior makes them as efficient as a. mellifera for pollination of this crop. in this regard, one native species stood out with a result very similar to a. mellifera: trigona spinipes (fabricius), which was collected in all distances except for 325 m in field 1 and 25 m in field 3 (fig 2). this is an interesting finding because, t. spinipes is smaller than a. mellifera (intertegular distance 1.79 ± 0.11 mm vs. 3.15 ± 0.03 mm). we have found at least 12 bee species visiting canola fields in southern brazil. however, as chao estimator suggests, if more bees are sampled the probability of finding more bee fig 2. location of bees collected within four canola fields at three distances from forest remnants (25 m, 175 m, 325 m). red circles represent social species, while green circles represent solitary species. fig 3. accumulation curve of species: number of species found versus number of expected species. bee diversity on canola fields in southern brazil. interpolation (solid lines) and extrapolation (dashed lines) of hill number with order q. note: shadow means confidence intervals (95%). species is higher (chao = 21 ± 9) (fig 3). as data indicate, it is more likely to find more bee species nearer (25 m) than far (175 m, 375 m) from forest fragments. it demonstrates the positive impact of having such ecosystem, as they provide cavities suitable for nesting and floral resources for potential pollinators. diversity profile of hill number indicates that diversity of bee species (richness, q = 0) near forest fragments (25 m) is higher than at larger distances (175 m, 375 m). as such, our results suggest that canola fields may largely benefit from bee species provided by natural vegetation around crops. on the other hand, if we observe the shannon-weiver index (q = 1) it is possible that both distances (175 m, 375 m) are similar, while at 25 m this diversity index remains higher than at longer distances from forest fragments (fig 4). the similarity percentages suggest that species of social bees (plebeia emerina (friese), a. mellifera, t. spinipes) greatly contribute for discriminating between different set of distances from forest fragments (table 1). thus, even though the smallest social bee (p. emerina) contributes to discriminate the nearest distance (25 meters), other larger social bees are relevant at longer distances, as a. mellifera and t. spinipes. on the other hand, solitary bees are also important to canola crops. for example, pseudagapostemon tesselatus cure appears as a relevant species aiding to discriminate all sets of distances (table 1). these results strongly suggest that canola crops may benefit from bees provided by wild vegetation and bee species inhabiting the soil within fields. sociobiology 65(4): 751-759 (october, 2018) special issue 755 bee body size and foraging range (distance from forest remnants) regarding body size, the largest forager sampled in this study was bombus pauloensis friese (intertegular distance: 4.87 mm – fig 7). however, only one individual was collected 25 m from a forest fragment, possibly because the canola crop is not so attractive to this species. the stingless bees t. spinipes, schwarziana quadripunctata lepeletier, and p. emerina, all of which have smaller intertegular spans, were found in nearly all sampled distances, except s. quadripunctata, which was found only at 325 m from forest remnants. it is worth noting that p. emerina is 2.6 times smaller than a. mellifera (intertegular distance: 1.19 mm and 3.15 mm, respectively), and that t. spinipes (1.79 mm) is 1.76 times smaller than a. mellifera. thygater mourei urban was collected at all distances in field 4, 175 m in field 2, and 325 m in field 3. caenohalictus essellates (moure) was the only species found solely at 25 m from the sampled forest fragment (fig 2). permanova type ii tests response: bee community data sum sq mean sq d.f. f p distances 0.92 0.92 1 2.57 0.04 residuals 0.46 0.03 13 0.55 14 pairwise comparisons between distances from forest fragments (p-values) 25 175 175 0.02 375 0.04 0.59 fig 4. diversity profile of hill number: bee diversity based on three distances from forest fragments on canola (brassica napus) crops in southern brazil. for moving x-axis to left the rare species become more important, while to opposite side (right) there is more weight to equability of bee species. some diversity indexes can be extracted from x-axis as: a) 0 = species richness; b) shannon index; c) simpson index; inf = berger-parker index. 25 175 meters 25 375 meters 175 375 meters plebeia emerina apis mellifera apis mellifera 0.23 0.26 0.34 pseudagapostemon tesselatus plebeia emerina pseudagapostemon tesselatus 0.45 0.48 0.65 apis mellifera pseudagapostemon tesselatus trigona spinipes 0.65 0.69 0.79 trigona spinipes trigona spinipes 0.80 0.80 table 1. cumulative contributions of most influential species to the differentiate set of distances from forest fragments around canola (brassica napus) fields in southern brazil. fig 5. non-metric multidimensional scaling: bee composition visiting canola crops in southern brazil. note: numbers inside polygons indicate the set of distances from wild vegetation; points means sample units; white lines are spider bodies calculated as centroids (averages) of polygons. the nmds ordination (stress = 0.13) indicates that bee composition nearer to forest fragments is significantly different than at larger distances (fig 5, table 2). we suggest that vegetation provides substrates as tree cavities to support social bees like p. emerina and a. mellifera, and aerial nesting areas for t. spinipes. further, data show that wider distances from wild vegetation statistically modifies the diversity of bees, suggesting that either larger bees with higher foraging range may visit the canola crops or solitary bees that nest on soil are also visiting canola flowers in southern brazil. table 2. cumulative contributions of bee community to different sets of distances from forest fragments around canola (brassica napus) fields in southern brazil. canola yield regarding bee diversity and canola grain yield, we found a shannon diversity index ranging from 0.78 in field 1 to 1.61 in field 4 (fig 6). our results demonstrate that bee diversity measured by the shannon index significantly affected canola yield (gaussian glm, f = 615, p < 0.001, pseudo-r2 = 0.99) (fig 6). r halinski, cf dos santos, tg kaehler, b blochtein – wild bees and canola yield756 we demonstrated a significant difference in body size among bees sampled at each of the three distances from the forest edge (negative binomial glmm, χ2 = 20.07, p < 0.001, fig 7). furthermore, we found significant differences between the nesting sites of the collected species (negative binomial glmm, χ2 = 2.25, p < 0.001), and in the interaction of individual size and nesting substrates of the sampled species (negative binomial glmm, χ2 = 6.25, p = 0.012). 2000). furthermore, food scarcity is common in the region during winter, making massive canola blooms attractive for bee species. in addition to this predominance, a. mellifera was the only species found in all sampled distances. this finding was expected, as similar studies suggested that this species can fly distances greater than 10 km (beekman & ratnieks, 2000), which far exceeds the distance assessed in our experiment (< 500 m). although the estimated flight range of t. spinipes (800 m; araújo et al., 2004) is much shorter than that expected for a. mellifera (10 km; beekman & ratnieks, 2000), the high population density of their nests (180,000 individuals; jaffé et al., 2014) greater than that of a. mellifera and their efficient recruitment habits accomplished through chemical signaling, i.e., odor-trail recruitment communication (nieh et al., 2004), may have contributed to the presence of this species in practically all distances. therefore, the native bee species t. spinipes stands out as a promising alternative to a. mellifera to improve cross-pollination in canola crops, particularly because their individuals remain longer on canola flowers when compared to a. mellifera (d’ávila & marchini, 2005). the yield results found highlight the importance of native bee species in addition to a. mellifera in canola fields in southern brazil. furthermore, pollination by bees in agricultural areas increases not only the quantity but also the quality of grain (ricketts et al., 2008) and, consequently, the biofuel productivity (durán et al., 2010). therefore, taxonomic identification of species and better understanding of their nesting habits provide inputs for preservation of the diversity of bee populations in canola-growing areas and to the strengthening of sustainable farming models. another species of native social bee that stood out in this study was p. emerina. this species was collected in all fields, except at 325 m in field 1 and 175 m and 325 m in field 4. witter et al. (2015) showed that this species is also an efficient pollinator of canola, contributing to grain yield with a 75% rate of fruit set; there was no difference in pollination efficiency between visits of p. emerina and a. mellifera. accordingly, other authors have pointed out the efficiency of native bees as pollinators of this crop (garibaldi et al., 2013; potts et al., 2016). on the other hand, regarding the occurrence of solitary bee species at different sampled distances, a notable finding was the genus pseudagapostemon, with species p. tesselatus and pseudagapostemon pruinosus moure and sakagami. solitary bees, especially halictidae, were found mainly at the 175 m and 325 m distances. it bears noting that these bees nest in soil (supplementary material 1), including within areas in which annual crops are grown (eickwort & sakagami, 1979; dalmazzo & roig-alsina, 2012; r. h., personal observation), as observed in the canola fields by the authors during this study. given this, we consider that these bees may contribute to the pollination of this crop, irrespective of distance from forest remnants. fig 6. relationship between canola grain yield (seed weight) and bee diversity (shannon index, h’). note: points denote observed values; line denotes the predicted model (gaussian family glm); pink-shaded area denotes the 95% confidence interval. model fit: pseudo-r2 = 0.99. fig 7. intertegular distance of bees in relation to distance from the sampled forest remnant (25 m, 175 m, 325 m). points denote the mean; error bars denote the 95% confidence interval. all measures of dispersion estimated after 999 replications (bootstrap). discussion bee diversity the predominance of a. mellifera may be related to several factors, including the presence of wild nests of this species in the region, the large population of individuals in nests, the effective recruitment behavior and the species plasticity regarding nesting substrates (beekman & ratnieks, sociobiology 65(4): 751-759 (october, 2018) special issue 757 supplemental contributions to pollination and importance of canola for bees studies have shown a 12 to 47% increase in canola crop yield secondary to cross-pollination with a. mellifera and other native bee species (rosa et al., 2010; bommarco et al., 2012; garibaldi et al., 2013; blochtein et al., 2014; witter et al., 2015; koh et al., 2016; potts et al., 2016). forest fragments and other semi-natural areas play a relevant role in wild bees nesting by facilitating their permanence in these sites. no-till farming, widely adopted in the investigated area for annual crops (including canola), may facilitate nesting of the solitary bee species recorded in this study (morandin et al., 2007). therefore, solitary and social bees play complementary roles in canola fields in southern brazil, influencing pollination processes and increasing grain yield. another important factor that contributed to bee species abundance and richness in canola fields was that this crop blossoms in the winter, a period of food scarcity, and its massive flowering provides copious and readily available pollen and nectar. considering that bees need to visit a large number of flowers to meet their individual needs, those of the brood and those of the colony (corbett et al., 1991), their significant presence in this crop is a good predictor of higher canola yields. foraging range of bees the intertegular distance proved to be an efficient estimator of foraging range based on mathematical equations, providing additional knowledge on their position regarding this scale and corroborating the findings of araújo et al. (2004). analysis showed that individual bees with larger intertegular span reached longer foraging distances than smaller bees (except b. pauloensis). these individuals tend to forage from plants closer to their nests, especially when there is an abundance of floral resources as in canola fields (morandin et al., 2007). in stingless bees, body size may act as a limiting factor for maximum flight range, making many species feed near their nests (araújo et al., 2004). however, although p. emerina has a small intertegular span, its specimens were found at all distances, probably as they are able to fly farther than 325 m (araújo et al., 2004). data on the foraging range of bees provided important information to help us understand the scale at which bee populations respond to the landscape (greenleaf et al., 2007), as well as to determine the spatial scale in which each bee can provide pollination services (kremen, 2005). changes such as specialization of foraging behavior to search for specific floral resources, spatial orientation methods, location and abundance of food resources as well as the availability of nesting sites, may influence the spacial scale occupied by bees (araújo et al., 2004). in some regions of southern brazil, beekeepers take honeybee hives into canola fields, exploiting its abundant pollen resources, and stimulating the growth of their bee colonies during periods of food scarcity (winter) by improving the number of individuals and keeping a “strong colony” for spring. this period is fundamental for multiplication of hives and for introducing pollination in different crops (blochtein et al., 2014). since a few years, these benefits for beekeepers allied to the increased quality and quantity of crop production led farmers and beekeepers to start a promising partnership in brazil. furthermore, in the future, beekeepers of native bees (meliponicultors) can contribute substantially to the improvement of canola yields (garibaldi et al., 2013). we have demonstrated that, in southern brazil, the diversity of bee assemblages has a positive influence on canola productivity. we also obtained evidence of the importance of forest remnants and canola fields as nesting sites for bees. in both landscapes, the practices adopted by farmers are fundamental to ensure the protection of bees’ nests, whether underground or arboreal. therefore, the preservation of wild bees – social and solitary – can greatly contribute to the productivity of canola fields depending directly on the practices adopted in rural areas. such preservation will reflect on the yields of this crop and protect the biodiversity of pollinators in the vicinity of canola fields. acknowledgments we thank prof. gabriel melo and prof. danuncia urban for bee identification and daniel guidi for his assistance with laboratory procedures. funding provided by capes (rh: 13190066-4; tgk: 13190065; cfs: pnpd), cnpq (call for pollinator research proposals 556635/2009-4), global environmental facility (gef), the united nations environment programme (unep), the food and agriculture organization (fao), the brazilian biodiversity fund (funbio), and the brazilian ministry of the environment (mma). supplementary material doi: 10.13102/sociobiology.v65i4.3467.s2218 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3467/0 authors’ contribution r halinski conceived the study, wrote the manuscript and analyzed the data; tg kaehler performed analyses of morphological measurements; cf dos santos conducted data analysis; and b blochtein reviewed the presented research process and findings. references abrol, d.p. 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(2015). evidence of forage distance limitations for small bees (hymenoptera: apidae). european journal of entomology, 112: 303–310. doi: 10.14411/eje.2015.028. _hlk513748802 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.3042sociobiology 65(3): 441-448 (september, 2018) how does landscape anthropization affect the myrmecofauna of urban forest fragments? introduction currently there are few ecosystems that do not experience anthropic pressure (barlow et al., 2012). these pressures are derived from the increasing human population and the consequent increase in urbanization, which leads to the reduction of green areas and biodiversity loss (uno et al., 2010). green areas in urban cities are generally restricted to street islands, tree lined streets, squares, home gardens, parks, and riparian forests (loboda & de angeles, 2005). since they are denominated as permanent preservation areas (ppa) by the brazilian forest code (law 12.651/2012), riparian forests are usually the largest, or the only remaining, green areas in cities. from the social point of view, the presence of green areas improves quality of life because they are related to leisure, abstract we evaluate whether landscape variables surrounding urban remnant forest fragments influence ant diversity and its components in urban areas. the study was conducted in six riparian forest fragments in midwestern minas gerais state, brazil, by sampling epigaeic and arboreal ants. arboreal ants respond to fragment isolation with changes in alpha, beta and gamma diversities. isolation likely hinders dispersion and re-colonization such that the more isolated a fragment is, the less likely that new species arrive there. on other hand, epigaeic diversity did not show any response to variables of the surroundings or fragments, probably because natural periodic floods constitute a more severe disturbance for these ants. in addition, throughout the process of urbanization, anthropogenic improvements, such as paving, that prevent the natural percolation of water, increase the flooding of riparian soil. arboreal ant species composition responds to percentage of urban area, fragment area and distance from the urban center, while epigaeic ants respond only to fragment area and percentage of urban area. we believe that even with the loss of species diversity and anthropogenic influences on fragments within urban centers, these areas are still important for species conservation. we also suggest the development of environmental protection projects for riparian areas within urban centers, including investments in ecological corridors connecting fragments and public policies seeking to preserve these areas. sociobiology an international journal on social insects gs santiago1, rbf campos1 & cr ribas2 article history edited by evandro n. silva, uefs, brazil received 07 march 2018 initial acceptance 30 april 2018 final acceptance 07 june 2018 publication date 02 october 2018 keywords urbanization, ants, planning, green areas, riparian forest, conservation. corresponding author graziele santiago programa de pós-graduação em ecologia setor de ecologia e conservação departamento de biologia universidade federal de lavras lavras, minas gerais, brasil. e-mail: grazielesantiago@hotmail.com landscaping, and environmental preservation (loboda & de angeles, 2005), as well as human health (thompson et al., 2012; campos & castro, 2017). in addition to social aspects, such areas have the potential to play a role in the conservation of biodiversity (doody et al., 2009). regarding birds and insects, urban green areas are important for provisioning shelter and different food resources, mostly for generalist species but also benefitting some specialist species that inhabit forest areas surrounding cities (goddard et al., 2009), thus ensuring a varied species composition (pacheco & vasconcelos, 2012). among insects, ants represent a group of abundant, diversified organisms that can serve as bioindicators (underwood et al., 2006; philpott, 2010; ribas et al., 2012), and which inhabit several strata including soil, litter, and trees. ants of different strata have distinct responses to environmental 1 instituto superior de educação de divinópolis, divinópolis, minas gerais, brazil. 2 laboratório de ecologia de formigas, departamento de biologia, universidade federal de lavras, lavras-mg, brazil research article ants gs santiago, rbf campos & cr ribas – myrmecofauna in urban forest fragments442 changes (vargas et al., 2007; schimdt & solar, 2010; neves et al., 2014). moreover, ants are involved in several ecosystem functions, such as defense against herbivores (lourenço et al., 2015), seed dispersal (dominguezhaydar & ambrecht et al., 2011), and nutrient cycling (souza-souto et al., 2007). in this sense, the conservational status of an area may determine the number and identity of species inhabiting it. for example, pacheco and vasconcelos (2007) found that large public squares close to natural areas have higher ant species richness. therefore, the area of a fragment, as well as its distance from the urban center, and the existence of natural vegetation in the surroundings, could be considered good predictors of the diversity of arthropods in urban areas (egerer et al., 2017). urbanization and landscape metrics have been shown to influence arthropod communities (mckinney, 2008). ortega and meneses (2015) found that ant diversity is related to the level of impact, while fattorini (2013) documented a rapid increase in the loss of tenebrionid beetles in an urban area. jost (2010) points to the need for understanding distribution patterns in the geographic space that butterfly species inhabit because it can contribute to decision-making by environmental managers regarding land use and occupation in urban cities. egerer et al. (2017) showed that an increase in percentage of urban area, a landscape metric, promoted an increase of invasive ant species, while soga et al. (2012) reinforced the importance of fragment metrics by arguing that circularizing the shape of forest patches maximizes the core areas to preserve biodiversity in urban areas where small forest remnants dominate. in this sense, understanding the spatial patterns of species richness is very important for the development of conservation strategies (marques & schoereder, 2013). on the other hand, the use of different parameters may lead to different results. in fact, in their review paper ribas et al. (2012) noted that papers using ant species richness as an indicator parameter for disturbance concluded that the number of species can increase, decrease or remain unchanged. thus, the authors concluded, richness is not a good bioindicator parameter and suggested that species composition is the most suitable parameter for evaluating the effect of disturbance on ant communities. in a multi-taxa study, kessler et al. (2009) also concluded that changes in species composition (referred to by them as beta diversity) are more consistent than changes in species richness (alpha diversity). therefore, we aimed to evaluate whether landscape metrics influence ant diversity (richness and composition) of forest fragments in urban areas. we also sought to understand whether this influence differs among different spatial scales (alpha, beta and gamma diversities). we investigated the hypothesis that fragments that are larger, more distant from the urban center, less isolated and with a smaller percentage of urban area and more forest cover in the surrounding area will have a greater number of ant species and dissimilar ant species composition. material and methods study area we conducted the study during the rainy season, from february to april, in six riparian forest fragments in the urban area of divinópolis, midwestern minas gerais state, brazil (20º 8’21” s and 44º 53’17” w). the municipality has an area of 716 km2 and the urban area consists of 192 km2 with approximately 228 thousand inhabitants (ibge, 2014). the original vegetation is predominantly cerrado (brazilian savanna) and the climate is temperate humid with a dry winter and hot summer, according to köppen’s index. the rainiest months are from december to march whereas the driest are from april to november. the municipality is crossed by the itapecerica and pará rivers; the first is the major source of water for the population and passes through the city for part of its 18 km length. four of the six fragments sampled in this study, are on the banks of the itapecerica river, while the other two are on the banks of the pará river (table 1). fragment arboreal ant richness epigaeic ant richness river riverbank coordinates extension f1 12 03 itapecerica left 20°08’30.2” s 44°53’00.6” w 600m length 180 m width f2 10 12 itapecerica right 20°08’05.8” s 44°52’51.4” w 190 m length 130 m width f3 12 08 itapecerica right 20°07’49.7” s 44°52’52.4” w 180 m length 60 m width f4 08 05 itapecerica right 20º11’32.5” s 44º53’36.9” w 1000 m length 620 m width f5 05 15 pará left 20°06’36.34” s 44°50’01.80” w 300 m length 410 m width f6 12 08 pará right 20°07’51.20” s 44°52’53.98” w 600 m length 800 m width table 1. description of studied urban fragments of riparian forest in the municipality of divinópolis, minas gerais, brazil. the fragment f1 is the closest to the urban center whereas f6 is the farthest. sociobiology 65(3): 441-448 (september, 2018) 443 sampling design since some of the studied remnants were very small, we used a 50-m transect, inserted perpendicular to the riverbank and at least 50 m from the fragment edge, to sample ants. for each transect, we established five sampling points 10 m apart from each other, with the first being 10 m from the riverbank. at every sampling point we collected ants in two strata (arboreal and epigaeic) by using pitfall traps (bestelmeyer et al., 2000; ribas et al., 2003), since this method is very common in bioindicator studies (ribas et al., 2012). pitfalls were made with plastic containers (10 cm high and 20 cm in diameter), containing sardine and honey as bait. the traps were kept in the field for 48 hours, after which the material was collected, sorted, mounted, identified to the level of genus using the key provided by bolton (1994) up dated by the key of baccaro et al. (2015), and separated to morphospecies by comparison with the reference collection of laboratório de ecologia de comunidades de formigas of the universidade federal de viçosa. in order to calculate landscape metrics, we used a land use map based on cartographic data from the terra class cerrado project, under the responsibility of the instituto nacional de pesquisas espaciais (inpe). for each fragment we calculated dits area and then arbitrarily defined the urban center as the intersection of the two main streets of the business center (avenida primeiro de junho and rua goiás, 20°08’50.30’’s; 44°53’17.43’’w), to measure the distance from the edge of the analyzed fragments to the urban center. distance from the nearest fragment, which we considered as an isolation metric, was calculated from the euclidian distance from the edge of each analyzed fragment to the edge of the next nearest fragment. the percentage of urban area and the percentage of forest cover in the surrounding area we calculated for a 500-m buffer from the fragment centroid. these metrics were calculated in arcgis 10.2 with softwares v-late and patch grid. statistical analyses we carried out analyses of diversity and composition separately for each stratum (arboreal and epigaeic). species richness was estimated by the jackknife technique of the vegan package (oksanen et al., 2007) in r-project software ver. 3.3.2. we determined gamma diversity as the total number of species collected per fragment, alpha diversity as the mean number of species collected by pitfall traps within each fragment, and the beta diversity as the difference between alpha and gamma diversities (magurran, 2004). in order to determine if landscape variables were correlated we used pearson correlation for normally distributed variables (distance of urban center, percentage of forest cover and percentage of urban area) and spearman correlation for non-normally distributed variables (fragment area and isolation). for correlated variables (>70%), the variable with the greater biological significance to the aim of the study was retained while the others were excluded from the analyses. we tested for correlations between explanatory variables related to fragments (area and isolation) and among those related to the surroundings (distance of urban center, percentage of forest cover and percentage of urban area) separately. we tested the hypothesis that landscape metrics influence ant diversity by constructing generalized linear models (glms) using landscape metrics as explanatory variables. since we did not have enough degrees of freedom to test all variables in the same model, we constructed two models, separating explanatory variables that related to fragments (area and isolation) from those related to the surroundings (distance of urban center, percentage of forest cover and percentage of urban area). because different groups of ants may exhibit distinct responses to different environmental factors, the analyses were carried out separately for epigaeic and arboreal ants. thus, alpha, beta, and gamma diversities of each stratum were considered separately as response variables. we tested for normality and corrected distributions when necessary. these analyses were performed with r software (r development core team, 2014). to investigate whether there were differences in myrmecofauna composition in relation to the landscape metrics, again using fragment variables and surrounding variables separately, we conducted a multivariate analysis based on the distance based linear models (distlm). we used the composition of each fragment as the response variable. tests were performed using the jaccard similarity index with 999 permutations, adjusted to the matrices of presence and absence. this analysis was done in the software primer v6 (clark & gorley, 2006). n um be r of s pe ci es samples n um be r of s pe ci es a b fig 1. accumulation of ant species collected in urban fragments of riparian forest: a) arboreal ants; b) epigaeic ants. gs santiago, rbf campos & cr ribas – myrmecofauna in urban forest fragments444 species f1 f2 f3 f4 f5 f6 atta sexdens (linnaeus, 1758) a a brachymyrmex sp. 1 e camponotus agra (smith, 1858) a camponotus atriceps (smith, f., 1858) a a a a camponotus crassus (mayr, 1862) a a camponotus melanoticus (emery, 1894) a camponotus rufipes (fabricius, 1775) a camponotus sericeiventris (guérin-méneville, 1838) a a camponotus (tanaemyrmex) sp. 1 a camponotus sp. e camponotus sp. 1 a camponotus sp. 2 a a a camponotus sp. 6 a a carebara sp. e cephalotes pusillus (klug, 1824) a a a a cephalotes sp. 1 e cephalotes sp. 3 a a crematogaster acuta (fabricius, 1804) a a, e a e crematogaster sp. 2 a a crematogaster sp. 4 a a a crematogaster sp. 7 e e e crematogaster sp. 8 e dolichoderus validus (kempf, 1959) a a a ectatomma edentatum (roger, 1863) e hypoponera sp. 1 a hypoponera sp. 2 e hypoponera sp. 9 e e e e labidus coecus (latreille, 1802) e leptogenys sp. 1 e linepithema sp. e linepithema sp. 1 a megalomyrmex modestus (emery, 1896) e mycocepurus sp. e neivamyrmex planidorsus (emery, 1906) e nesomyrmex sp. 1 a nylanderia sp. 1 a, e a, e e a octostruma balzani (emery, 1894) e e odontomachus bauri (emery, 1892) e odontomachus meinerti (forel, 1905) e e pachycondyla vilosa (fabricius, 1804) a pheidole gertrudae (forel, 1886) e pheidole radoszkowiskii (mayr, 1884) e e pheidole sp. 1 a a pheidole sp. 8 e e pheidole sp. 16 e e e e procryptocerus sp. 1 a a pseudomyrmex sp. 12 a solenopsis sp. 2 a, e e a, e a, e e strumygenys sp. e strumygenys sp. 1 e strumygenys sp. 2 e wasmannia auropunctata (roger, 1863) a, e a, e e a, e e wasmannia sp. 1 a wasmannia sp. 2 a wasmannia sp. 3 a a total arboreal species 11 11 12 8 5 12 total epigaeic species 3 12 9 5 15 8 table 2. ant species sampled in each of six urban fragments of riparian forest. the letter “a” refers to ants sampled in the arboreal stratum whereas the letter “e” refers to ants collected in the epigaeic stratum. sociobiology 65(3): 441-448 (september, 2018) 445 results we collected 55 species belonging to six subfamilies. twenty-six species were collected only in the arboreal stratum, 25 species were collected exclusively in the epigaeic stratum, and four species were common to both strata (table 2). our samples represented 72.3% and 65.4% of the total number of species estimated by the jackknife technique for the arboreal and epigaeic ant faunas, respectively (figure 1). percentage of urban area and percentage of forest cover were correlated (table 3); therefore, we opted to retain only percentage of urban area since we were interested in fig 2. relationship between isolation (calculated from euclidian distance from the edge of each analyzed fragment to the nearest fragment edge) and diversity of arboreal ants. alpha diversity: f(1,3)= 6.339; p = 0.004. beta diversity: f(1,3)= 6.340; p = 0.004. gamma diversity: f(1,3)= 6.339; p = 0.004. landscape variables p value r value fragment area x isolation 0.9493 -0.033 % urban area x % forest cover 0.0156 -0.896 % urban area x distance of urban center 0,0817 -0.756 % forest cover x distance of urban center 0.1866 0.622 table 3. correlation between landscape metrics. bold values indicate significant correlations. arboreal stratum epigaeic stratum alpha beta gamma alpha beta gamma fragment area 2.949 2.949 2.949 2.1990 1.1729 1.4681 isolation 66.339* 66.340* 66.340* 3.0088 3.6639 4.1293 % of urban area 0.4836 0.4836 0.4836 0.0964 0.0023 0.0063 distance of urban center 0.5558 0.5558 0.5558 0.6833 1.7221 1.4176 table 4. influence of landscape metrics (f-values) on alpha, beta and gamma ant diversity. values with * are significant p > 0.05. the impacts generated by anthropization. none of the other variables were correlated (table 3). with respect to the variables related to fragments (fragment area and isolation), the diversities of the arboreal stratum (alpha, beta and gamma) were not influenced by fragment area (table 4) but were negatively influenced by isolation (figure 2). the variables related to the surroundings (percentage of urban area and distance of urban center) did not have an influence on arboreal ant diversity (alpha, beta and gamma) (table 4). for the epigaeic stratum, none of the explanatory variables, either related to fragments or to surroundings, influenced alpha, beta and gamma diversities (table 4). of the variables linked to fragments, the composition of arboreal ants was influenced by fragment area (p = 0.011), which explained 7% of the variation. of the variables related to the surroundings, two influenced the composition of arboreal ants, the percentage of urban area (p = 0.015) and the distance of urban center (p = 0.045), with each explaining 6% of the variation. epigaeic ant species composition was influenced by fragment area (p = 0.044; 6%) and percentage of urban area (p = 0.017; 7%). discussion fragment isolation (distance from nearest fragment) was found to influence ant richness (diversity alpha, beta and gamma) of forest fragments in the studied urban area, and this influence is similar regard less of the spatial scale analyzed, but dependent on the stratum. arboreal ants were found to be responsive to the isolation of fragments while epigaeic ant diversity was not influenced by any variable. composition of arboreal ants was affected by fragment area, percentage of urban area and distance of urban center, while composition of epigaeic ants was responsive only to fragment area and percentage of urban area. gs santiago, rbf campos & cr ribas – myrmecofauna in urban forest fragments446 our findings support that arboreal ants are more responsive to landscape metrics than epigaiec ants, indicating that they are more affected by this anthropic impact, probably because vegetation suppression is one of the first actions of the process of urbanization. yasuda and koike (2009) observed that host tree species richness was an important factor in determining the abundance of ants and other arthropods. our results confirm the importance of this stratum, which can serve as shelter and a source of food, and contribute to the maintenance of favorable environments for ants (estrada et al., 2014). however, the only landscape metric that affected arboreal ant richness in the present study was fragment isolation, which was calculated as the distance from nearest fragment. our observation that arboreal ant diversity decreased with increased fragment isolation was also observed by badano et al. (2005). likewise, pacheco and vasconcelos (2005) observed that natural areas with native vegetation in the proximity of urban parks can be important for the species diversity therein. a hypothesis that may explain the reduced species richness of the arboreal stratum in more isolated fragments is the difficulty of dispersion and re-colonization, since new species are less likely to arrive to more isolated fragments (lucey et al., 2013; macarthur & wilson, 1967). we also observed that the effect of isolation was independent of spatial scale. epperson (2010) reported that such spatial scales are correlated, that is, the effect caused on a smaller scale may be reflected on a larger scale, which we believe to have been the case in our study. over the long term, urbanization can affect, and contribute more and more to, this scenario of isolated fragments having reduced diversity. it is noteworthy that the diversity of epigaeic ants was not influenced by any landscape metric. ives et al. (2013), and egerer et al. (2017) suggest that epigaeic ants respond more to local conditions and factors, such as interactions, rather than to landscape metrics. gomes et al. (2010) and forgs et al. (2015) also did not find a relationship between ant richness and abundance and a highly urbanized area, or percentage of the surrounding vegetation. likewise, gomes et al. (2010) did not find a relationship between leaf litter ant richness and fragment area, which they attributed to the very small corporal size of ants and, thus, the lack of a need for a large area to nest and to obtain alimentary resources. nevertheless, a possible explanation is that, for riparian soil ants, another factor may be more important, such as flooding. natural floods of riparian forest areas are in fact a sever source of disturbance and can be worsened by urbanization and improvements for the human population, especially paving. pavement prevents the natural percolation of water into the soil, thus forcing the water to reach rivers more rapidly and, consequently, increasing the frequency and intensity of flooding (soares et al., 2013). flooded and humid soils make it difficult for ants to establish colonies, which may be masking the effects of the variables tested in the riparian areas of the present study. this strong disturbance can also explain the reduced richness of ant species if we compare to epigaeic with arboreal strata. in this context, campos et al. (2008) found more species on the soil than in trees, even when using fewer traps on the soil than in arboreal stratum, unlike our study where we did not find higher richness on the soil in comparison to the trees when using a similar trapping effort in both strata. the compositions of arboreal and epigaeic ants were little affected by landscape metrics, only fragment area and percentage of urban area, plus distance from the urban center for arboreal ants. urban area surrounding fragments indicates the loss of natural habitat and is probably related to the homogenization of the environment, which previously supported a composition of specialized and demanding species. with the alteration and degradation of the environment only the most tolerant species remain, such as opportunistic and less demanding generalists, or even the replacement of native with exotic species (egerer et al., 2017). since fragments with a higher percentage of urban area in the surroundings are more subject to anthropic impacts, such as pollution, a large influx of people frequenting the interior of these fragments trampling the soil and/or the discarding of solid residues in the areas, they may experience unfavorable impacts on species that depend on a more preserved environment. in addition, larger areas may possess greater environmental complexity (i.e. more heterogeneous environments), as well as distinct tree species that support a greater diversity of ant species that utilize and exploit their resources (estrada et al., 2014). in contrast, more homogeneous environments will have less diversity of resources and, consequently, fewer species that exploit them. once the landscape changes, the natural environment is recharacterized and the loss and/or substitution of species are inevitable results. this is also true for the direct interference of human actions in the living areas of these species. beyond the different responses of ants to the environmental parameters tested in the present study, we note that only four species occur in both epigaeic and arboreal strata, evidencing the importance of sampling more than one stratum in ecological studies using ant assemblages as models in riparian areas since structuring can be influenced in different ways. species that forage and inhabit different stratum have different habits and behaviors and can respond in different ways to environmental changes. for example, canedo et al. (2016), found the dynamics of a hypogaeic ant assemblage to respond differently to fire disturbance. we believe that even with the loss of species diversity and anthropogenic disturbances of fragments within urban centers, these areas are still important areas for species conservation. this is particularly true because they connect forest remnants outside (downstream and upstream) of urban centers, and isolation, as evidenced by our data, is an important parameter for the richness of arboreal ants. with regard to the management of urban forest areas, we found that the most important variable was fragment isolation. in order for fragments to obtain greater ant richness, greater flow of species and increased colonization of areas, it is necessary to invest in ecological corridors and sociobiology 65(3): 441-448 (september, 2018) 447 the reforestation and recovery of green urban areas, because the greater the isolation of an area, the lower the richness of arboreal ant species. in addition, we suggest that urban centers develop environmental protection projects for riparian forests, such as investing in connecting fragments and instituting public policies that seek to conserve these areas. acknowledgements we thank rafael gonçalves cuissi and elisângela aparecida silva for their critical reading of the manuscript, and, andré tavares, cézar fonseca, chaim lasmar, ernesto canedo and guilherme demétrio for helping with the statistical analyses and discussions. daysy mara de andrade and ramsés martins ferreira kindly helped with sampling biological data. this paper has was partially produced during the course pec 533 – publicação científica em ecologia, at the ecologia aplicada post-graduation program at the universidade federal de lavras. references baccaro et al., (2015). guia para os gêneros de formigas do brasil. manaus: editora inpa, p.388 badano e.i.; regidor, h.a.; núñez, h.a.; acosta, r.; gianoli, e. 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(2007). efeitos de fatores ambientais sobre a mirmecofauna em comunidade de restinga no rio de janeiro, rj. neotropical entomology, 36: 28-37. yasuda m. & koike (2009). the contribution of the bark of isolated trees as habitat for ants in an urban landscape. landscape and urban planning, 92: 276-281. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2824sociobiology 65(3): 527-530 (september, 2018) floral resources partitioning by two co-occurring eusocial bees in an afromontane landscape eusocial bees with large colonies are crucial actors in many ecosystems. in addition to pollinating numerous plants, they affect other flower visitors’ behaviour and food choice by consuming a large part of floral resources due to their high numbers and biomass (e.g. whitfield et al., 2006). individual species of eusocial bees influence the food niche of each other as well. in the tropics, such interspecific interactions in competition for food sources have been studied mainly among various bee species with an introduced honeybee, apis mellifera linnaeus (e.g. sommeijer et al., 1983; ramalho et al., 2007; franco et al., 2009; liu et al., 2013; hilgert-moreira et al., 2014). on the other hand, we have surprisingly limited knowledge on resource partitioning among eusocial bees in the afrotropics, where a. mellifera is native (whitfield et al., 2006). to abstract floral preferences of generalist foragers such as eusocial bees influence the success of pollination of many flowering plants, as well as competition with many other bee species in tropical communities. eusocial bees are important for the pollination success of many flowering plants, as well as for food resources availability for many other species. however, their foraging preferences are still unknown in many tropical areas, especially in the afrotropics. we studied the foraging activity of two syntopic eusocial bees with large colonies, the honeybee apis mellifera linnaeus and the stingless bee meliplebeia ogouensis (vachal), on seven plant species in the bamenda highlands, cameroon, in two consecutive years. simultaneously, we quantified intraand inter-annual changes in the food resources. we observed resource partitioning among the two bee species. although both species are considered as generalists, their short-term food niches overlap was very low. their preferences to the most often visited plants differed even more strongly interannually. our results bring the first evidence on such relatively strong resource partitioning among two dominant eusocial bee species from west/central africa. sociobiology an international journal on social insects r tropek1,2*, e padyšáková1,2, š janeček1,3 article history edited by cândida aguiar, uefs, brazil received 02 january 2018 initial acceptance 02 may 2018 final acceptance 03 may 2018 publication date 02 october 2018 keywords apinae, apis mellifera; honeybee; stingless bees; interspecific interactions; pollination. corresponding author robert tropek department of ecology, faculty of science, charles university, viničná 7, cz-12844 prague, czechia email: robert.tropek@gmail.com our knowledge, the only study from this region focused on the pollen foraging of a. mellifera and two stingless bee species of meliponula in uganda (kajobe, 2007). nevertheless, such data are crucial to understanding the dynamics in flower visitor insect communities, as well as to the role of eusocial bees on the evolution of plant pollination systems. to fill this gap, we bring the first data on food partitioning among two dominant eusocial bees, a. mellifera and the stingless bee meliplebeia ogouensis (vachal), from the afromontane landscape of mendong buo, nw cameroon (6°5’21’’ n, 10°18’11’’ e). it is a mosaic-like landscape of montane forest remnants and open habitats (tropek & konvicka, 2010). from late november till early january (i.e. the first half of the dry season) of 2007/2008 and 2008/2009 we recorded 1department of ecology, faculty of science, charles university, prague, czechia 2biology centre, czech academy of sciences, institute of entomology, české budějovice, czechia 3institute of botany, czech academy of sciences, třeboň, czechia short note r. tropek et al. – resources partitioning by eusocial bees528 the two bee species visiting flowers of seven plant species whose flowers dominated the studied sites in stream gallery vegetation (acanthaceae: brillantaisia lamium, hypoestes aristata; campanulaceae: lobelia columnaris; hypericacea: hypericum revolutum, h. roeparianum; lamiaceae: pycnostachys eminii; rubiaceae: virectaria major; see bartoš et al. (2012, 2015) for the plants characterisation, pictures and functional specialisation). all of them produce nectar, except h. roeparianum (bartoš et al., 2012). only b. lamium, h. aristata and p. eminii have flowers of mellitophilous pollination syndrome. in our previous studies (padyšáková et al., 2013; bartoš et al., 2015), h. aristata and h. roeparianum were shown to be flowers more commonly visited by a. mellifera, while h. revolutum was visited mainly by m. ogouensis. the bee visits were recorded along sixteen 15 m transects of gallery vegetation, where 5-min observations of each plant species per sampling session were equally distributed within the day (between 9 to 16 h) and study periods (altogether 29 and 33 sampling days in the two sampling periods, respectively). during the sessions, only visitors of a single observed plant species were recorded; one visiting individual was counted just once independently on the number of visited flowers. each plant species was observed for about 10 hours in total (bartoš et al., 2012). the possible partitioning of resources by the bees was analysed by a chi-square test of a contingency table (7 plants × 2 bees) with frequencies of visits in individual cells, performed in r v. 2.15.0 (r development core team, 2012; fig. 1). we also calculated the schoener index of niche overlap combining the proportions of visits of both bee species on flowers of the particular studied plants (schoener, 1968; aguiar et al., 2017). the numbers of flowers along the transects were counted seven and four times in the two successive sampling periods, respectively (janeček et al., 2012), and they remained mostly constant during both sampled dry season beginnings (fig. 2). as the exceptions, flower numbers increased for l. columnaris and decreased for p. eminii during both sampled periods, whilst the flower numbers of v. major and b. lamium decreased during the second sampling period only. altogether 253 a. mellifera and 373 m. ogouensis bee-plant interactions were observed in both sampling periods: 121 and 238 visits in 2007/2008, and 132 and 135 visits in 2008/2009 (fig 1; online resource 1). during the sampling, a. mellifera visited mainly h. aristata (n = 60), followed by l. columnaris (n = 20), v. major (n = 17) and h. revolutum (n = 17) in 2007/2008, whereas it visited h. revolutum (n = 58), p. eminii (n = 41) and h. aristata (n = 25) in 2008/2009. m. ogouensis visited mainly h. revolutum (n = 182) and h. roeparianum (n = 34) in 2007/2008, and h. revolutum (n = 82) and l. columnaris (n = 51) in 2008/2009. although both bees are generalists, each species preferred only part of available floral sources (χ2 = 215.9089; df = 6; p ˂ 0.01; fig 1), revealing thus a food source partitioning between them and low overlap of their niches. this was confirmed also by low schoener index: 0.23 and 0.48 for the two periods, respectively, while the niches are considered to be overlapped for values over 0.6 (schoener, 1968; aguiar et al., 2017). such niche partitioning among a. mellifera and various stingless bees was already described from various regions, usually interpreted as a result of differences in foraging behaviour, although an aggressive interspecific interference driven by larger honey bees is also sometimes hypothesised (e.g. sommeijer et al., 1983; ramalho et al., 2007; kajobe, 2007). the visitation rates of individual plants by both bees, however, differed during and between sampling periods (fig 1 and 2). the strongest shift in resource partitioning among the two bee species involved the flowers of l. columnaris, between years, and the flowers of h. revolutum, intra-annually. during the first year, preference of a. mellifera toward l. columnaris followed its blooming during the second half of the period, while it visited h. aristata more or less during fig 1. number of visits to each plant species by the two bee species, in the afromontane landscape of mendong buo, nw cameroon, in two sampled dry seasons. lobcol: lobelia columnaris, hyprev: hypericum revolutum, hypari: hypoestes aristata, pycemi: pycnostachys eminii, hyproe: hypericum roeparianum, virmaj: virectaria major, brilam: brillantaisia lamium. sociobiology 65(3): 527-530 (september, 2018) 529 the whole period. a. mellifera strongly dominated on p. eminii in the beginning, switching to h. revolutum later after the first species overflowered, mainly in the second year. meliplebeia strongly preferred h. revolutum in the beginning of both sampled periods, but almost entirely switched to l. columnaris later in the second year. such temporal specialisation of honey bees and stingless bees for floral choice has already been described as a result of both optimisation of resource usage and competition avoidance (leonhardt et al., 2014); without other data we cannot exclude other possible factors. nevertheless, our study has confirmed an interesting phenomenon of food niche partitioning among two eusocial bee species in the overlooked landscape of afrotropical mountains. acknowledgements we would like to thank l. spitzer and m. janda for their help with the data collection, j. straka for meliplebeia identification, and m. sweney for english corrections. our research was funded by the czech science foundation (14-36098g, 16-11164y) and by the charles university (primus/17/sci/8 and unce 204069). references aguiar, c.m.l., caramés, j., frança, f. & melo, e. 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(2010). forest eternal? endemic butterflies of the bamenda highlands, cameroon, avoid close-canopy forest. african journal of ecology, 48: 428-437. doi: 10.1111/j.1365-2028.2009.01129.x whitfield, c.w., behura, s.k., berlocher, s.h., clark, a.g., johnston, j.s., sheppard, w.s., smith, d.r., suarez, a.v., weaver, d. & tsutsui, n.d. (2006). thrice out of africa: ancient and recent expansions of the honey bee, apis mellifera. science, 314: 642-645. doi: 10.1126/ science.1132772 doi: 10.13102/sociobiology.v62i3.359sociobiology 62(3): 382-388 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 influence of the vegetation mosaic on ant (formicidae: hymenoptera) distributions in the northern brazilian pantanal introduction floodplains, especially the brazilian pantanal, are characterized by the controlling influence of flood pulses and vegetation, which have a major impact on the population dynamics of species, thereby influencing community structure (junk et al., 1989; junk, 1997). another defining factor in the pantanal of the state of mato grosso, brazil is its landscape heterogeneity, which comprises a mosaic of forest, cerrado, grasslands and monodominant clusters of trees (por, 1995; silva et al., 2000). together, in association with flood pulses, influence animal diversity, especially invertebrates (brown, 1970). plant formation and landscape diversity in this ecosystem result from many climatic changes that occurred throughout geological formation (jimenez-rueda et al., 1998), which was also influenced by seasonal flooding (junk et al., 1989; junk & nunesabstract we examined how vegetation mosaic influences distribution of the edaphic ant (formicidae) community in the northern part of the pantanal in cáceres, state of mato grosso, brazil. plant formations (hereafter habitats) that characterize this area include several savanna types, such as: cerrado sensu stricto, cerradão, semi-deciduous forest, termite savanna, open fields and cerrado field/carandazal. pitfall traps were placed in ten 250 m transects each one separated by 1 km, within an area of 2 x 5 km (following rapeld methodology). five traps at intervals of 50 m were placed along each transect, in september and december 2008. forty-four ant species were collected. leaf litter predicted ant presence and influenced species occurrence in the different habitats. pantanal habitats are very different structurally from one to another, which has have resulted in areas with very specific ant assemblages. the understanding of the ant community structure in these areas is fundamental to floodplain management. sociobiology an international journal on social insects e meurer¹, ld battirola3, jhc delabie2, mi marques1 article history edited by edilberto giannotti, unesp, brazil received 17 september 2014 initial acceptance 12 february 2015 final acceptance 11 march 2015 keywords cerrado, semi-deciduous forest, wetlands. corresponding author: eliandra meurer programa de pós-graduação em ecologia e conservação da biodiversidade universidade federal de mato grosso av. fernando corrêa da costa nº 2.367 boa esperança, 78060-900, cuiabá-mt, brazil e-mail: eliandrameurer@gmail.com da-cunha, 2005) with input from adjacent phytogeographic provinces (adámoli, 1982; silva et al., 2000; alho & gonçalves, 2005; junk et al., 2006). this landscape diversity also helps the pantanal resist stressful conditions such as drought, floods and large fires (nunes-da-cunha et al., 2011). ant (formicidae) population dynamics are likely to be influenced by a variety of factors, including food resource availability, nest sites, competition and climate (kaspari, 2000). behavioral diversity also structures these communities, and organization is influenced by the distribution of unexploited resources and strategies used to obtain these resources (fowler et al., 1991). these behavioral differences are a consequence of the variety of available niches, which increase with vegetation diversity and complexity (hölldobler & wilson, 1990; tews et al., 2004). ants are an important component of terrestrial invertebrate communities in the pantanal because they research article ants 1 universidade federal de mato grosso, cuiabá, mt, brazil 2 convênio uesc/ceplac, itabuna-ba, brazil 3 universidade federal de mato grosso. sinop, mt, brazil sociobiology 62(3): 382-388 (september, 2015) 383 participate in most ecological processes and have complex social behaviors and a variety of strategies for survival (adis et al., 2001; battirola et al., 2005, castilho et al., 2005; santos et al., 2008; silva et al., 2013; soares et al., 2013). in an attempt to associate habitat complexity with the role played by ants in tropical ecosystems of the pantanal, we tested the interaction between the vegetation mosaic and the distribution and richness of the edaphic ant community in the pantanal in brazil. materials and methods study area ants were studied on the ranch baía de pedra, in cáceres, state of mato grosso, brazil (16º28’49”s 58º08’26”w). this area is in the pantanal sub-region of cáceres (silva et al., 2000), and comprises six plant formations (herafter habitats) belonging to the cerrado biome (brazilian savanna): cerrado sensu stricto (ss), cerradão (c), semi-deciduous forest (sdf), termite savanna (ts), open field (of) and cerrado field/ carandazal (cc) (e. g. nunes-da-cunha et al., 2011). methods samples were taken twice during the dry period, once in september and once in december 2008. for this, 10 transects were established, each 250 m in length, and with at least 1 km minimum distance between them. transects were established in a 2x5 km grid following rapeld protocol (magnusson et al., 2005). transects followed the topography to maintain a constant elevation (flooding regime) but independent of other natural habitat variation. this design provides a method to independent sample species distribution and abundance in each site. each sampling point was characterized by a 250 m transect with five pitfall traps placed 50 m apart. traps were on the field for five days when all samples were collected, for a total of 50 traps each, as well in september as in december. we also sampled leaf litter in ten 25 x 25 cm quadrants next to the pitfall traps. leaf litter was placed in paper bags and dried until a constant weight was reached. temperature and relative humidity were measured with a digital thermohygrometer at ground level next to all traps. individual ants were identified according (bolton, 2003) and nomenclature following bolton’s catalog (antweb, accessed 2014). reference collections were deposited in the laboratory of ecology and arthropod taxonomy (leta) of the federal university of mato grosso bioscience institute and the mirmecology laboratory collection of the cacau research center (cpdc), cepec-ceplac, ilhéus, bahia, under the reference number #5574. data analysis community distribution patterns were determined in each habitat type, by non-metric multidimensional scaling (nmds). we used presence/absence in the data matrix, and similarity was estimated using the jaccard index. we associated the ant community (dependent variable) with leaf litter, temperature and humidity (independent variables) using multivariate multiple regressions. significance level was established at 0.05, and we used the programs past (hammer et al., 2001) and systat 11 (wilkinson, 2004) to carry out the analyses. results the ant community comprised 44 species (fig 1, table 1), with the greatest species richness in the cerrado sensu stricto and open field (28 spp.), followed by semi-deciduous forest (26 spp.). while species richness was similar, individual species varied in each habitat. six species were exclusive to the open field and five exclusive to the cerrado sensu stricto. fig 1. ant presence and leaf litter mass of each phytophysiognomy in the pantanal of cáceres, mato grosso, brazil. cc cerrado field/ carandazal; ts – termite savanna; of open field; c cerradão; sdf semi-deciduous forest; ss cerrado stricto sensu. in the ant fauna, we noticed that gracilidris pombero wild and cuezzo was restricted to cerrado field/carandazal and open field while dinoponera mutica (emery) was restricted to semi-deciduous forest. nmds found two axes that explained 84% of the variation in the data (stress = 0.147). sampling locations formed three groups defined by the quantity of leaf litter in each sampling unit. the first group had dense vegetation (semi-deciduous forest), which thus have a greater amount of leaf litter. the second group was cerradão, cerrado stricto sensu, and open field, and the third was formed by cerrado field/carandazal, termite savanna and open field (fig 2). the amount of leaf litter (g) was the best predictor of ant occurrence in this vegetation mosaic (multivariate regression, f2.5 = 10.847, p = 0.003), and ant presence was independent of both humidity (f 2.5 = 3.884, p = 0.098) and temperature (f 2.5 = 3.224, p = 0.120). e meurer et al. formicidae distribution in the cáceres pantanal384 taxa phytophysiognomy *cc tm cf cf c sdf sdf ss ss ss sum myrmicinae acromyrmex rugosus (smith, f. 1858) 1 1 1 3 atta sexdens (linnaeus, 1758) 1 1 1 1 1 5 cardiocondyla obscurior wheeler, 1929 1 1 cephalotes persimplex de andrade, 1999 1 1 cephalotes pusillus (klug, 1824) 1 1 1 3 crematogaster brasiliensis mayr, 1878 1 1 1 1 1 1 1 7 crematogaster victima smith, f. 1858 1 1 2 cyphomyrmex major forel, 1901 1 1 cyphomyrmex transversus emery, 1894 1 1 1 1 1 5 mycocepurus goeldii (forel, 1893) 1 1 1 1 4 ochetomyrmex neopolitus fernández, 2003 1 1 1 1 1 1 1 1 8 pheidole prox. cursor 1 1 1 1 1 5 pheidole sp.1 grupo fallax 1 1 1 1 1 1 1 1 8 pheidole subarmata mayr, 1884 1 1 1 1 1 1 1 7 pogonomyrmex naegelii forel, 1886 1 1 1 3 sericomyrmex sp.1 1 1 1 1 1 5 solenopsis sp.2 1 1 1 1 1 1 1 1 1 1 10 solenopsis sp. complexo tridens sensu trager, 1991 1 1 1 1 1 1 1 1 1 9 solenopsis invicta buren, 1972 1 1 1 3 solenopsis globularia (smith, f. 1858) 1 1 1 1 1 1 1 1 8 strumigenys denticulata mayr, 1887 1 1 1 3 wasmannia auropunctata (roger, 1863) 1 1 1 1 1 1 1 1 1 9 xenomyrmex sp.1 1 1 dolichoderinae dorymyrmex brunneus forel, 1908 1 1 1 1 1 1 1 7 forelius pusillus santschi, 1922 1 1 1 1 1 1 1 7 gracilidris pombero wild & cuezzo, 2006 1 1 1 3 tapinoma melanocephalum (fabricius, 1793) 1 1 1 1 1 5 formicinae brachymyrmex sp.1 1 1 1 1 1 5 brachymyrmex heeri forel, 1874 1 1 1 1 1 1 6 brachymyrmex patagonicus mayr, 1868 1 1 1 1 1 5 camponotus melanoticus emery, 1894 1 1 1 1 1 1 1 1 8 camponotus novogranadensis mayr,1870 1 1 1 3 nylanderia sp.1 1 1 1 1 1 1 1 1 1 1 10 nylanderia sp.2 1 1 nylanderia fulva (mayr, 1862) 1 1 1 3 ectatomminae ectatomma brunneum smith, f. 1858 1 1 1 1 1 1 1 7 ectatomma permagnum forel, 1908 1 1 1 1 1 1 6 gnamptogenys acuminata (emery, 1896) 1 1 2 21 table 1. ant presence and richness (s) from pitfall traps in the pantanal of cáceres, mato grosso, brazil. *cc cerrado field/carandazal; ts – termite savanna; of open field; c cerradão; sdf semi-deciduous forest; ss cerrado stricto sensu. sociobiology 62(3): 382-388 (september, 2015) 385 taxa phytophysiognomy *cc tm cf cf c sdf sdf ss ss ss sum ponerinae dinoponera mutica emery, 1901 1 1 2 odontomachus bauri emery, 1892 1 1 1 1 1 1 6 odontomachus haematodus (linnaeus,1758) 1 1 pachycondyla apicalis (latreille, 1802) 1 1 1 1 1 5 pseudomyrmecinae pseudomyrmex termitarius smith, f. 1855 1 1 1 1 1 1 1 1 8 ecitoninae labidus praedator (smith, f. 1858) 1 1 1 3 total number of occurrence 15 15 28 12 25 26 19 24 28 22 144 leaf litter (g) 31 36 68 30 93 207 135 60 115 20 temperature (°c) 33 28 35 34 33 31 31 21 23 30 relative humidity (%) 40 50 54 40 50 46 42 41 40 51 table 1. ant presence and richness (s) from pitfall traps in the pantanal of cáceres, mato grosso, brazil. *cc cerrado field/carandazal; ts – termite savanna; of open field; c cerradão; sdf semi-deciduous forest; ss cerrado stricto sensu. (continuation) discussion species richness was clearly a consequence of habitat complexity, as observed too in other studies, such as in different successional stages (leal & lopes, 1992), vegetation types (leal, 2002), vegetation complexity (hölldobler & wilson, 1990; corrêa et al., 2006) and plant density and richness (leal, 2003). ant communities respond to changes in plant composition between different habitats (morrisson, 1998; gotelli & ellison, 2002). variation in ant diversity is influenced by environmental characteristics, and greater complexity leads to greater species diversity (matos et al., 1994; oliveira et al., 1995). soares et al. (2003) found a greater number of ant species in native forest than in montane savanna (campo rupestre), which reinforces fig 2. ordination (nmds) of the ant community (presence and absence) obtained by pitfall traps in the pantanal of cáceres, mato grosso, brazil. cc cerrado field/carandazal; ts – termite savanna; of open field; c cerradão; sdf semi-deciduous forest; ss cerrado stricto sensu. the idea that ant diversity is affected by vegetation structure. this relationship is direct (andow, 1991) since more complex vegetation leads to a more diverse ant community due to the greater support capacity. habitat types may determine species composition of a region (vasconcelos & vilhena, 2006). studies show that leaf litter can predict species richness in different plant formations and habitats (matos et al., 1994; carvalho & vasconcelos, 2002; leal, 2003). differences between composition and structure of the ant community are probably a consequence of resource and niche distribution and foraging strategies in each (fowler et al., 1991; kaspari, 2000). leaf litter thickness might be important because less litter implies reduced food abundance as well as less shelter and material for nest construction (hölldobler & wilson, 1990). variation in the occurrence of ant communities among the vegetation types is explained by the positive relationship between species richness and heterogeneous environmental conditions (cerdá et al., 1997; santos et al., 2008). locations with greater variety and availability of resources support a greater number of species than poorer habitats due to greater resource sharing (lassau & hochulli, 2004; cramer & willig, 2005). ant species richness in this study is similar to other studies in the pantanal (ribas & schoereder, 2007; marques et al., 2010; marques et al., 2011; silva et al., 2013; soares et al., 2013). wilson (1987) was the first to show that periodically flooded peruvian amazon forests had more species than dry forests. in the amazon, species that are associated with soil and leaf litter are more affected by natural and periodic disturbances than those that use other forest strata (majer & delabie, 1994). similar situation was observed in cocoa plantations inserted in places formerly occupied by gallery forest and irregularly flooded in the brazilian state of bahia (delabie et al., 2007). e meurer et al. formicidae distribution in the cáceres pantanal386 ant species richness in the pantanal is also less than that in other ecosystems (for example: delabie et al., 2000; silva & silvestre, 2004, santos et al., 2006; leal et al., 2012). low richness in the pantanal of mato grosso may be due to periodic flooding and the resulting effect of the flood pulse on pantanal biota (junk et al., 1989; 2006). habitat disturbances significantly affect species diversity and abundance (vasconcelos, 1998) since strong climatic variation in the pantanal limits local diversity (lange et al., 2008). gracilidris pombero is found in a variety of open environments (wild & cuezzo, 2006) including cerrado pastures with copernica alba morong ex. morong & britton (arecaceae). on the other hand, d. mutica is usually found in forests. biological and ecological aspects of this genus have received increasing attention during the last 20 years due to its very large size, its notable reproductive system and its wide occurrence in the atlantic forest, caatinga, cerrado and amazon (for example: monnin & peeters, 1998; peixoto et al., 2010; lenhard et al., 2013). high structural variation of pantanal habitats and the effects on ant communities have resulted in areas with specific characteristics, and learning more about these environments is fundamental to floodplain management and conservation. leaf litter was most influential for the distribution pattern of the edaphic ant community in this vegetation mosaic, and it was thus considered a predictor of this community among the different plant formations studied. the different occurrence in these plant formations and low diversity could be related to forage characteristics and habitat preference of ant species in this ecosystem. acknowledgments we thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes / mec) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for a ph.d. grant. further financial and logistic support came from the programa de pós-graduação em ecologia e conservação da biodiversidade at the instituto de biociências of the universidade federal de mato grosso (ufmt), the centro de pesquisas do pantanal (cpp), the programa de apoio a núcleos de excelência pronex/fapemat/cnpq, and the instituto nacional de ciência e tecnologia em áreas úmidas (inau). references adámoli, j.a. 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(1987). the arboreal ant fauna of peruvian amazon forests: a first assessment. biotropica, 19: 245-251. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.107-114sociobiology 61(1): 107-114 (2014) diversity of the nests of social wasps (hymenoptera: vespidae: polistinae) in the northern pantanal, brazil sm almeida1,3, sr andena², ej anjos-silva³ introduction the most known social insects are the bees and the ants (grimaldi & engel, 2005; melo et al., 2012), which belong to apidae and formicidae, respectively. the term “wasp” is applied to all other groups within the taxon (melo et al., 2012). most people, usually, have the perception that species of wasps live in a nest, sharing it with the other members of the colony, however only a small portion of the taxon is eusocial, in the sense that overlapping of generations, cooperative care over offspring and reproductive division of labor occurs. actually, in wasps the eusociality is restricted only to polistinae and vespinae, both within vespidae. polistinae, which comprises the tribes polistini, mischocyttarini and epiponini, is widely distributed in neotropics with 25 genera and around 900 species recorded (richards, 1978; carpenter & marques, 2001). abstract some species of wasps demonstrate plasticity with diverse nesting habits according to the environmental conditions and substrates used for building the nests, while others are restricted to habitats with specific conditions and may exhibit some degree of fidelity. the aim of this study was to estimate species richness and abundance of nests of polistini and epiponini wasps in four landscape units in the pantanal of poconé, retiro novo farm, southwestern mato grosso state, brazil. the nests of social wasps were sampled in four plant physiognomies locally known as cambarazal, landizal, pombeiral and campo limpo from august 25, 2011 to april 11, 2012, being recorded 308 nests of eight genera and 14 species of social wasps. the highest number of nests belongs to polybia ruficeps xanthops (32.69%), poly. sericea (24.27%) and synoeca surinama (15.21%). the highest species richness was recorded in cambarazal and the highest abundance of nests in pombeiral, while campo limpo showed the lowest richness and abundance of nests. the nests of s. surinama were associated with cambarazal and landizal (indval = 93.3, p = 0.001), while the nests of poly. ruficeps xanthops and poly. chrysothorax were associated with cambarazal, landizal and pombeiral (indval = 97, p = 0.001). there was lower abundance and lower species richness of wasps in campo limpo. these results demonstrate that the maintenance of forest environments in the pantanal is essential for the establishment and maintenance of social wasp nests. sociobiology an international journal on social insects 1 universidade do estado de mato grosso, nova xavantina, mato grosso, brazil. 2 universidade estadual de feira de santana, feira de santana, bahia, brazil. 3 universidade do estado de mato grosso, cáceres, mato grosso, brazil. article history edited by gilberto m m santos, uefs, brazil received 11 october 2013 initial acceptance 10 november 2013 final acceptance 22 december 2013 keywords indval, cambarazal, pombeiral, campo limpo, wetlands corresponding author evandson josé dos anjos-silva lab. de abelhas e vespas neotropicais universidade do estado de mato grosso departamento de biologia av. tancredo neves s/n, cavalhada cáceres, mt, brazil 78200-000 e-mail: beevandson@uol.com.br the social wasps play a vital ecological role as pollinators (see prezoto & machado 1999; vitali-veiga & machado 2001; silva-pereira & santos, 2006) and predators and act as natural agents of biological control (clapperton, 1999; carpenter & marques, 2001; hunt, 2007). also they have frequently been used to test evolutionary models for the origin of social behavior because of their different levels of sociality from solitary to eusocial (west-eberhard, 1978, 1996; itô, 1986; spradbery, 1991 as cited in noll et al., 2004). the nests can be the size of a thimble or more than a meter long, as durable as hard felt or more fragile than egg shells, more regular and uniform than the much-celebrated honeybee comb or wildly chaotic with an intricate mazelike interior (wenzel, 1998). except for four neotropical species that build nests of mud, the nests are all made of vegetable fiber without wax or plant resins (wenzel, 1998; hunt, 2007). research article wasps sm almeida et al. diversity of the nests of social wasps in the northern pantanal108 soil or glandular secretion may be used to reinforce or repair nests, but they rarely constitute the primary building material for mature colonies (wenzel, 1998). in a general sense, according to the shape, nests can be classified as: a) phragmocyttarous, where the initial comb is fixed on substratum and covered by an envelope. a second comb is built by adding new cells at the bottom of the first envelope, and also is covered by an envelope. each envelope has an entrance to the respective combs, b) astelocyttarous nest, where a single comb is built directly on substratum and covered by an envelope with one entrance; and c) stelocyttarous nest, where a comb or combs are suspended by stalks. they can or cannot have envelope and are called of calyptodomous and gymmodomous, respectively (saussure, 1853; richards and richards, 1951, richards, 1978, carpenter & marques, 2001). the preference for nesting substrates is different, depending on the physical and biological characteristics of the environment (dejean et al., 1998; cruz et al., 2006). nests can be built on natural substrate, such as plants rocks, cavities, besides termites and human constructions (carpenter & marques, 2001). some wasp species have great ecological plasticity and varied nesting habits according to environmental conditions and substrates for nest building (santos & gobbi 1998). however, other species have lower ecological plasticity and are restricted to habitats with specific conditions, and may have some allegiance to such environments (heithaus, 1979; dejean et al., 1998; cruz et al., 2006; silva pereira & santos, 2006; santos et al., 2009a; souza et al., 2010). such characteristics may elect wasps as bioindicators of environmental quality, as pointed out by souza et. al (2010). the choice of sites for nesting is more characteristic and less diverse than those for foraging (richards, 1978), and the diversity of these insects is associated more to the nesting habitat and not necessarily to foraging habitat (simões et al., 2012). the vegetation structure can favor the nesting of social wasps either by increasing the availability of physical support for nests or the number and diversity of available food resources, by imposing lower variability in microclimate characteristics (lawton, 1983). studies on social wasps have been conducted in brazilian areas of cerrado (richards 1978; henriques et al., 1992; diniz & kitayama, 1994; diniz & kitayama, 1998; silva-pereira & santos, 2006; santos et al., 2009a; santos et al., 2009b; silva et al., 2011), amazon forest (silveira, 2002; silveira et al., 2005, 2012; silva & silveira 2009; somavilla, 2012), atlantic forest (santos et al., 2007) and caatinga (santos et al., 2009b). in areas of cerrado 130 species were recorded (richards, 1978; diniz & kitayama, 1994), while in the amazon 20 genera and over 200 species were recorded (silveira, 2002; silva & silveira, 2009). in the cerrado of mato grosso, there are records of 88 species in nova xavantina (richards, 1978) and 36 species in chapada dos guimarães (diniz & kitayama 1998). despite the importance of social wasps for the maintenance of ecosystems, this group still has little information in the literature (prezoto et al., 2008) and for pantanal of mato grosso there is shortage or no data on the social wasps. given the importance of social wasps to ecological systems, the present study aimed at determining the species richness and abundance of polistinae wasp nests in four different landscape units in the north pantanal, poconé, mato grosso. material and methods study area the pantanal, considered the largest wetland in the world, is located in the middle of south america and covers parts of brazil, bolivia, paraguay and argentina. the pattern of flood inside the pantanal is strongly influenced by rainfall (± 1.250 mm per year). the climate in this floodplain is hot with average annual temperature around 25° c, a pronounced dry climate period from may to september, and a rainy season from october to april (junk et al., 2006; fantin-cruz et al., 2010; fernandes et al., 2010). the pantanal of poconé is a subregion belonging to the northern pantanal, located in the state of mato grosso (fernandes et al., 2010), and it was the region chosen to develop this study conducted in the retiro novo farm (16°15’12” s; 56°22’12” w), municipality of poconé (fig 1). in this area of the pantanal, the cerrado dominates the landscape in the form of natural grasslands, but has also feafig 1. study area located at pirizal district, retiro novo farm, municipality of poconé, southwestern mato grosso state, brazil. sociobiology 61(1): 107-114 (2014) 109 tured forest formations, locally known as landizal, cambarazal and pombeiral (nunes da cunha et al., 2010) but can occur as forests mosaic areas as show in the fig 1. the cambarazal (fig 2a) is a dense forest formation, semi-evergreen, in which vochysia divergens pohl (cambará), sometimes occurring in monospecific stands, dominates. vochysia divergens is a characteristic species of the pantanal. dense stands develop in low-lying humid areas because this species is flood-tolerant and in multi-year wet periods spreads into surrounding savannas, creating serious problems for farmers. the expansion of cambarazal is counteracted by the wild fires of extremely dry years. elongated and sinuous stretches of semi-evergreen forest in high-lying plains covered by cerrado vegetation point to the presence of landizal (fig 2b). this can be explained by the fact the, in periodically drying drainage systems, floods last longer and water availability during lowwater periods is better because of a high groundwater level (nunes da cunha et al., 2007). landi refers to the regional name of calophyllum brasiliense cambess. (calophyllaceae) trees, that forms the landizal plant life vegetation. the following species, which are also common in other seasonally flooded forests, are characteristic of this sub-type of forest: licania parvifolia huber (chrysobalanaceae), erythroxylum anguifugum mart. (erythroxylaceae), alchornea discolor poepp. (euphorbiaceae), c. brasiliense, mouriri guianensis aubl. (melastomataceae), ficus pertusa l.f. (moraceae), sorocea sprucei (baill.) macbr. (moraceae), eugenia florida dc. (myrtaceae), coccoloba ochereolata weed. (polygonaceae), and triplaris gardneriana wedd. (polygonaceae). in pombeiral (fig 2c), it is evident the abundance of combretum lanceolatum pohl ex eichler. scrubland with c. lanceolatum pohl ex eichler, which reaches a height of about four meters, is often monospecific. it is widespread in the pantanal and occurs near permanent water bodies in areas subject to several months of inundation. the campo limpo (fig 2d), is characterized by low density of trees and shrubs, dominated by grasses and are open areas subject to periodic flooding, being the vegetation dominated by hyptis brevipes (lamiaceae), richardia grandiflora (rubiaceae) e axonopus purpusii (poaceae) (nunes da cunha et al., 2007; nunes da cunha et al., 2010). methodology the search for nests of social wasps was performed from 25 august 2011 to 11 april 2012 in the landscape units known as landizal, pombeiral, cambarazal and campo limpo. in 68 days of field work (1024 hours of sampling), each of the four landscape units (17 days/locality) were visit using linear transects, about 3 km, from 7:00h to 17:00h, with a different landscape unit being sampled every day. sampling was carried out by two people who walked along the transect, extending about five meters on either sides of vegetation searching for nests. the nests and colony were taken off from the substrate. in nests that were located in substrates difficult to reach, a sample of 10 to 60 specimens were collected with telescopic entomological net. the nests recorded in this inventory were georeferenced using gps (global position system), being listed and individually marked with colored plastic tape. the height of nests above the ground was measured with the use of tape, recording the substrate used for nesting, such as termites, trunks, branches, thorns etc. the specimens were identified following the keys of richards (1978), carpenter and marques (2001), andena and carpenter (2012). voucher specimens were deposited in the collection of the laboratory for neotropical bees and wasps (licence number # 18147), department of biology, universidade do estado mato grosso (unemat), campus cáceres, in mato grosso; museum of zoology, universidade estadual de feira de santana (mzfs), feira de santana, bahia; and universidade estadual paulista júlio de mesquita filho (unesp), campus são josé do rio preto, são paulo. data analysis the number of active colonies of wasps in the four plant physiognomies was used as a measure of abundance, each sampling day being considered a sample. we did a hierarchical ordering cluster analysis and classification from binary matrices (cluster analysis). therefore, we used the jaccard dissimilarity measure relating them to the plant physiognomies with wasps’ species, through the connection method upgma (legendre & legendre, 2012). to verify the occurrence of wasps’ species that nest in specific physiognomies, the indval method via indicspecies program package (r development core team, 2011) was employed. such a method combines the degree of specificity for a particular species to an ecological status such as, for examfig 2. the four plant physiognomies found in the study area, retiro novo farm, pantanal de poconé, mato grosso, brazil. a – cambarazal; b – landizal; c – pombeiral; d – campo limpo, during flooded season. sm almeida et al. diversity of the nests of social wasps in the northern pantanal110 ple, the habitat type, and its fidelity within status, measured by the percentage of occurrence. this analysis gives a value of 0 to 100%, where 0% is equivalent to no indication of an indicator species for a particular environment, and 100% indicates that the occurrence of particular species is characteristic to the environment (dufrene & legendre, 1997). the analysis significance was performed by monte carlo test with 10,000 randomizations by accepting p <0.05 as significant. results a total of 308 nests of social wasps belonging to 14 species, distributed in eight genera were recorded in the four landscape units studied, with polybia accounting for 69.57% of the nests sampled and 42.85% of the species. except polybia and brachygastra, the other six genera, agelaia, apoica, chartergus, parachartergus, polistes and synoeca were represented by only one species each (table 1). the species with the highest number of nests cataloged were poly. ruficeps xanthops (32.69%), poly. sericea (24.27%) and s. surinama (15.21%). the highest number of nests recorded in cambarazal and pombeiral was of poly. ruficeps xanthops 40.48% and 42.7% of nests, respectively. in landizal was recorded the highest number of nests of s. surinama (n = 35, 39.78%) while in campo limpo was recorded the highest number of poly. sericea (n = 21; 44.68%) (table 1). the highest wasp species richness was recorded in cambarazal (s = 13) and highest abundance of nests in pombeiral (n = 89), landizal (n = 88) and cambarazal (n = 84), while campo limpo had the lowest richness (s = 7) and abundance (n = 47) (table 1; fig 3). fourteen species recorded nesting in the four landscape units sampled; nests of three species were associated to three landscape units. the nests of s. surinama were associated with cambarazal and landizal (indval = 93.3, p = 0.001), whereas nests of poly. ruficeps xanthops were associated with cambarazal, landizal and pombeiral (indval = 97, p = 0.001), as well as nests of poly. chrysothorax (indval = 77, p = 0.014). the cluster analysis shows that cambarazal, pombeiral and landizal are more related than campo limpo, which is a distinct branch. the cophenetic correlation coefficient (ccc) was 0.80, and this value demonstrates that the distance matrix is well represented (fig 3). the nests of wasps cataloged in this study were located, in average, at 2.0m (sd = 5.70) above the ground, those of agelaia sp. 1 (n = 2) were at lower height, constructed in soil cavities; and those of greater height, c. globiventris nests (n = 12; mean = 5.44; min = 1.96m, max = 7.71m, sd = 1.96), exposed in the vegetation. among the plants used as support by the wasps, 10.23% of the nests were associated with bactris glauscescens drude (arecaceae). regarding the substrate used for building the nests, table 1. absolute frequency (n), relative frequency (%), average height of the wasps nests from de soil (hs) and standard deviation (sd) at pirizal district. retiro novo farm, pantanal de poconé, mato grosso, brazil. tribe / species cambarazal landizal pombeiral campo limpo total hs (m) n (%) n (%) n (%) n (%) (mean ± sd) epiponini agelaia sp. 1 1 (1.19) 1 (1.12) 2 (0.64) 0 apoica pallens (fabricius. 1804) 2 (2.38) 1 (1.13) 3 (0.97) 2.0 ± 0.69 brachygastra augusti (de saussure, 1854) 2 (2.38) 2 (0.64) 0.6 ± 1.09 brachygastra lecheguana (latreille, 1824) 5 (5.95) 2 (2.27) 6 (12.76) 13 (4.21) 1.4 ± 1.25 chartergus globiventris de saussure, 1854 3 (3.57) 1 (1.12) 7 (14.9) 11 (3.56) 5.9 ± 1.96 parachartergus fraternus (gribodo, 1892) 7 (8.33) 2 (2.27) 2 (2.25) 4 (8.51) 15 (4.85) 3.2 ± 1.57 polybia chrysothorax (lichtenstein, 1796) 4 (4.77) 8 (9.1) 10 (11.24) 22 (7.12) 1.4 ± 0.41 polybia ignobilis (haliday, 1836) 1 (1.19) 2 (4.25) 3 (0.98) 0.8 ± 0.25 polybia jurinei de saussure, 1854 1 (1.19) 2 (2.25) 3 (0.98) 1.4 ± 0.96 polybia gr. occidentalis 1 (1.19) 1 (1.13) 5 (5.62) 4 (8.51) 11 (3.56) 0.7 ± 0.31 polybia ruficeps xanthops richards, 1978 34 (40.48) 26 (29.54) 38 (42.7) 3 (6.38) 101 (32.69) 1.5 ± 0.57 polybia sericea (olivier, 1791) 12 (14.28) 13 (14.78) 28 (31.46) 21 (44.68) 74 (24.27) 2.5 ± 5.86 synoeca surinama (l., 1767) 11 (131) 35 (39.78) 1 (1.12) 47 (15.21) 1.7 ± 0.66 polistini polistes versicolor (olivier, 1791) 1 (1.12) 1 (0.32) 1.1 total of nests 84 88 89 47 308 richness 13 8 10 7 14 sociobiology 61(1): 107-114 (2014) 111 beiral and landizal are physiognomies with more structured vegetation and retain most of the vegetation cover in the dry season (nunes da cunha et al., 2007). such environments may harbor more species of nesting social wasps in comparison to more open environments, such as grasslands, once the forest areas can provide greater protection, materials to build nests and substrates for attachment of wasp nests (santos et al., 2009a; santos et al., 2009b). studies on other animal groups, such as birds and beetles, in this region of the pantanal, shows higher species richness in cambarazal (pinho & marini, 2012, marques et al., 2010). in this study, conducted with birds in forestry environments in the area of the present survey, pinho & marini (2012) shows that the highest species richness and a greater number of bird nests were observed in cambarazal, suggesting some relationship with vegetation structure, food supply, protection from predators and suitable microclimate; being the cambarazal an important nesting habitat for several species that prefer forest habitats in this region of the pantanal. the landizal in turn, despite maintaining much of the vegetation cover during the dry season is characterized by few species with low density in the understory (pinho & marini, 2012). to edaphic beetles (coleoptera), the highest abundance and species richness were also recorded in cambarazal, while the campo limpo had the lowest values of abundance and richness in this region (marques et al., 2010). in this case, the type of vegetation determined the ecological condition such as microclimate, light, shelter, food supply, making it important to define the composition, richness and abundance of species of beetles (marques et al., 2010). other studies that investigated the social wasps nesting in different landscape units in brazil, also found higher species diversity in forest environments. for example, a study conducted in three landscape units of cerrado (agricultural systems, campo sujo and cerradão (arboreal plants), there were 19 nests of social wasps species, distributed in 13 genera, with the highest richness having been observed in cerradão, and lower richness in agricultural systems (santos et al., 2009a). a total of 319 nests belonging to 17 species of social wasps were recorded in three caatinga vegetation types, the highest richness found in arboreal caatinga, while agricultural systems and shrubby caatinga showed similar species richness (santos et al., 2009b). in the present study, the highest number of wasp nests and the second highest number of wasp species have been recorded in pombeiral, invader vegetation of natural and artificial pastures, and that reduces the foraging ability of cattle, which led some farmers from pantanal to try to eradicate it (nunes da cunha et al., 2007). according to the indval analysis, the nests of s. surinama were associated with landizal and cambarazal. nests of p. ruficeps xanthops and p. chrysothorax in turn, were associated with landizal, cambarazal and pombeiral. however, no wasp species had nests associated with campo limpo in the those of agelaia sp. 1 and poly. ignobilis were found in termite nests and fallen logs, while the nests of s. surinama were built directly on tree trunks. the nests of c. globiventris, par. fraternus and poly. jurinei were attached in branches of plants, while the nests of polistes versicolor were among thorns of b. glauscescens, as recorded for poly. chrysothorax, poly. gr. occidentalis, and b. lecheguana and b. augusti also built their nests on the branches. the nests of poly. ruficeps xanthops and poly. sericea were found in pre-existing cavities, branches of plants and between spines of b. glauscescens. discussion in this work 14 species of wasps, that represents 15.90% of the total of species recorded for the cerrado of nova xavantina, 88 species (richards, 1978) and 38.88% when compared with the species collected in chapada dos guimarães, 35 species (diniz & kitayama,1998). also in chapada dos guimarães diniz & kitayama (1994) found 100 nests, belonging to 30 species and 15 genera. richards (1978) recorded 199 colonies comprising 51 species in 14 genera nesting in six habits in nova xavantina and serra do cachimbo. the species with the largest number of colonies was poly. ruficeps (14%), followed by poly. occidentalis (9%), poly. jurinei (6%), poly. erythrotorax (5%), poly. ignobilis (5%), pa. fraternus (4%) and epipona tatua (4%). the genus polybia represents 60% of the colonies reported from the two regions of mato grosso state, ranging from 67% in nova xavantina and serra do cachimbo to 45% in rio manso (richards, 1978; diniz & kitayama, 1994). the highest species richness of wasps in cambarazal may be related to vegetation structure and foliage density, once the cambarazal is a semi-deciduous forest and maintains much of the vegetation cover during the dry season (nascimento & nunes da cunha, 1989). the campo limpo had the lowest species richness, while pombeiral and landizal showed intermediate values. environments such as cambarazal, pomfig 3. dendrogram of hierarchical clustering analysis classification according to vegetation types based on presence or absence of social wasp species (hymenoptera: vespidae) using upgma and cophenetic correlation coefficient (ccc = 0.80), retiro novo farm, pantanal de poconé, mato grosso state, from august, 2011 to april, 2012. sm almeida et al. diversity of the nests of social wasps in the northern pantanal112 study area, despite poly. sericea has presented a greater number of nests (44.58%) in this plant physiognomy. some wasp species are found nesting in environments with specific conditions, with some fidelity to these sites (heithaus, 1979; santos et al., 2009a). in the riparian forest of rio das mortes, minas gerais, the indval analysis showed that pseudopolybia vespiceps and polybia fastidiosuscula were associated with more conserved sites, while mischocyttarus drewseni was associated with disturbed areas and can be used as environmental indicators (souza et al., 2010). nests of mischocyttarus (mischocyttarini) were not found in the present study. according to silva et al. (2011) smaller colonies like mischocyttarus, or those camouflaged or built inside cavities (e.g. agelaia spp.) may easily go unnoticed. in the present study, nests of s. surinama were not record in campo limpo, just in forestry environments. this result can be attributed to the fact of synoeca presenting arboreal and sessile nests, which are built directly on the trunk of trees and occupy a large area of the substrate (wenzel, 1998). open areas with few trees and shrubs, such as campo limpo, may not provide the necessary conditions for establishing synoeca nests. in bahia, nests of s. cyanea were not observed in agricultural systems, although many individuals have been observed collecting nectar and water in such environments (santos et al., 2009a). in the cerrado lato sensu in mato grosso, despite s. surinama and p. sericea having been sampled foraging in different habitats, such as gallery forest, cerrado sensu stricto, campo sujo and campo úmido, both nested only in gallery forests and cerrado sensu stricto, respectively (diniz & kitayama, 1994). in this study, the wasp species with the highest number of nests recorded in campo limpo was poly. sericea, species widely distributed throughout south america, especially in open areas like fields, and various types of cerrado and dry forests (richards, 1978). it is known that b. lecheguana, poly. sericea, poly. ignobilis, poly. occidentalis have broad ecological tolerance and are generally dominant in open ecosystems and under adverse environmental conditions, being very important in simpler community structure and those subject to strict ecological conditions (santos, 2000). the nests of most social wasp species (85.71%) cataloged in this study were located at medium height compared to soils less than three meters, and only the nests of c. globiventris and pa. fraternus were recorded at heights above. the behavior of nesting near the soil can benefit social wasps because this type of environment offers greater availability of substrates for nesting, low temperatures and high humidity (raw, 1998). regarding the use of thorny plants as support for nests of social wasps, such as b. glauscescens, such behavior may be one of the strategies of social wasps to reduce nest predation, as noted in the study on the nest site selection in palm plants b. simplifrons and astrocaryum sciophilum (arecaceae) (dejean et al., 1998). the natural grasslands showed lower abundance and lower species richness in the study area, and none wasp species with nests associated with this vegetation type. these results show that maintaining forest environments in the pantanal of mato grosso is essential for the establishment of social wasp nests and therefore, it is important for maintenance of wasp species in this region. we hope that this work helps studies related to ecology, biology, distribution and abundance of social wasps in northern pantanal and stimulate additional surveys of social wasps in the pantanal of mato grosso. acknowledgements financial support was provided by fapemat (737955/2008; 285060/20010). we thank l.a. castro for providing the map used in figure 1. references andena, s.r. & carpenter, j.m. 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(1996). wasp societies as microcosms for the study of development and evolution. in: s. turillazzi & m.j. west-eberhard (eds.), natural history and evolution of paper-wasps (pp. 290-317). oxford: oxford university press. wilson, e.o. (1985). the sociogenesis of insect colonies. science, 228 (4704): 1479-1485. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.307-312sociobiology 61(3): 307-312 (september 2014) honey bee health in apiaries in the vale do paraíba, são paulo state, southeastern brazil lg santos1; mltmf alves2; d message3; fa pinto1; mvgb silva4; ew teixeira2 introduction although honey bees are the most frequently studied social insects due to their ecological and economic importance (winston, 1987; martin, 2001), a set of factors still not well understood has been affecting these arthropods in an increasing number of countries. various organisms can parasitize honey bees in the immature or adult phase, possibly leading to collapse of the colony depending on the level of virulence (bailey & ball 1991; ellis & munn, 2005). there have been many reports of colony collapse in the northern hemisphere in recent years, prompting strong concern in the scientific community, particularly due to the importance abstract bee health is a growing global concern due to phenomena with as yet undefined causes, such as the sudden population decline of colonies that has been observed in apiaries in many countries, recently including brazil. the main objective of this study was to assess the presence and/or prevalence of pathogens that afflict africanized apis mellifera bees in the vale do paraíba region of são paulo state, brazil. three sampling periods were established: period 1 – august and september 2009 (winter/early spring); period 2 – december 2009 and january 2010 (summer); and period 3 – april and may 2010 (autumn). samples were collected of honeycomb from the brood area, combs containing capped brood, adult bees that cover the brood and foraging bees, to evaluate the presence and prevalence of paenibacillus larvae, varroa destructor and nosema sp. the results indicated that the intensity of infection by nosema ceranae and infestation rates of v. destructor in the hives were low (mean 637x103 spores of nosema ceranae, 5.41% infestation of varroa in adult bees and 4.17% infestation of varroa in brood), with no detection of p. larvae spores in the samples. the prevalence of n. ceranae and v. destructor was high, at respective values of 85.2 and 95.7%. all told, 1,668 samples were collected from 438 hives, in 59 apiaries. these results demonstrate that although mites and microsporidia are widespread in the region’s colonies, the africanized bees are apparently tolerant to pathogens and parasites. however, the mechanisms related to defense against pathogens are not completely clear, and monitoring and prophylactic measures are essential to maintain the health of bee colonies. sociobiology an international journal on social insects 1 universidade federal de viçosa (ufv), viçosa, minas gerais, brazil. 2 agência paulista de tecnologia dos agronegócios, pindamonhangaba, são paulo. brazil. 3 universidade federal rural do semiárido. (pvns/capes), mossoró/rn, brazil. 4 embrapa gado de leite, juiz de fora, mg, brazil. article history edited by astrid kleinert, usp, brazil received 20 december 2013 initial acceptance 31 january 2014 final acceptance 30 march 2014 key words apis mellifera, varroa destructor, nosema sp., paenibacillus larvae corresponding author lubiane guimarães dos santos graduate program in entomology federal university of viçosa(ufv) avenida peter henry rolfs s/n. cep: 36.571-000. viçosa, mg, brazil e-mail: lubi.guimaraes@gmail.com of bees as pollinizers (neumann & carreck, 2010). this phenomenon, called colony collapse disorder (ccd), was first identified in the united states in the winter of 2006, when 23% of apiaries were affected, with average colony losses of 45% (van engelsdorp et al., 2007). besides the risks posed by ccd, another factor that must be considered is the potential problems caused by applying drugs to control diseases, because of the possibility of generating resistant populations and of contamination of bee products, with consequent risks to human health (lodesani et al., 2008). although honey bees are afflicted by numerous parasites and pathogens, including viruses, bacteria, protozoa and mites (bailey & ball, 1991), in brazil there are few research article bees lg santos et al. honey bee health in southeastern brazil 308 reports of diseases causing large-scale mortality. this pattern could be related to a series of biotic and abiotic factors typical of each region, among them the breed of bees used in local apiculture (moretto, 1997). africanized honey bees (ahb) have traits that favor resistance to parasites such as the mite varroa destructor, among other pathogens (de jong, 1996; rosenkranz, 1999; rosenkranz et al., 2010). nevertheless, in recent years honey production in some brazilian regions has been declining, accompanied by observations of apparent weakening of colonies, with adult bees and brood in many cases presenting anomalous symptoms (message et al., 2012). the explanation for this fact can be related both to increased virulence of parasites already present in the country and the introduction of new pathogenic agents, such as viruses (teixeira et al., 2008). preventive monitoring of the levels of infestation and prevalence of harmful agents is important to maintain the health of apiculture in the country. in this study, we investigated the prevalence and incidence of v. destructor, nosema sp. and paenibacillus larvae in apiaries of southeastern brazil. these agents have been indicated as responsible for causing large losses to apiculture worldwide. material and methods samples were collected in 438 colonies of 59 apiaries, located in 13 municipalities in the region of vale do paraíba, são paulo state. in this region, the climate is considered subtropical (koeppen climate classification), with temperatures varying from 18ºc to 29ºc during the year. samples included pieces of honeycomb from the brood area, comb containing capped brood, adult bees covering the breeding area from brood comb, and forager bees collected at the entrance of the hive, to assess the presence and prevalence of paenibacillus larvae, varroa destructor and nosema sp., respectively. we established three collection periods, to obtain samples from different moments of colony development as a function of the natural conditions and availability of food resources in the field (nectar and pollen flow): period 1 – august and september 2009 (winter/early spring); period 2 – december 2009 and january 2010 (summer); and period 3 – april and may 2010 (autumn). all the analyses were carried out in pindamonhangaba, são paulo, in the honey bee health laboratory of the são paulo state agribusiness technology agency (lasa/apta). the collection of the samples for analysis of varroa destructor, nosema sp. and paenibacillus larvae was based on teixeira & message (2010). evaluations of varroa destructor infestations were based on de jong et al. (1982). the protocol for microbiological analyses developed by schuch et al. (2001) and later considered as the brazilian method (brasil, 2003) was used to analyze samples for p. larvae, while the method of cantwell (1970) was used to count the spores for nosema. in order to identify the nosema species, samples from all apiaries were submitted to duplex pcr assay (martín-hernández et al., 2007), using the primers 321apis-for/rev, for identification of n. apis and 218mitoc-for/rev for n. ceranae. positive and negative controls were used. analysis of variance was carried out considering a statistical model whose representation is given by: ijkjiijk ecly +++= µ , where: yijk= dependent variables; μ = overall mean; li = effect of the i th site, cj = effect of the jth sample and eijk is the random effect of the error. the degrees of freedom for the sources of variation studied were decomposed into contrasts and evaluated through the f-test at a significance level of 1%. all analyses were performed using the glm procedure of the sas statistical package (2001). results during the three sampling periods, 438 samples of foraging bees were collected at the hive entrances, and for other types of samples it was possible to collect 432 samples of adult bees present in the brood area, 368 comb pieces containing honey from the brood area and 430 samples of comb containing at least 100 older pupae. table 1 shows the intensity of infection by the microsporidium nosema ceranae, represented by the average number of spores per bee, as well as the average infestation rate (%) by the mite v. destructor in adult bees and in brood in apiaries located in 13 locations in the vale do paraíba: cunha, bananal, lagoinha, lorena, monteiro lobato, natividade da serra, paraibuna, pindamonhangaba, redenção da serra, são josé dos campos, são luís do paraitinga, santo antônio do pinhal, taubaté e tremembé. only the species n. ceranae was detected by molecular analysis (fig. 1), and no p. larvae spores were detected in the honey samples. fig. 1. 2% agarose gel showing duplex nosema apis and nosema ceranae pcr products. m: 100 bp marker. column 13: samples showing n. ceranae amplification. column c+: positive control – nosema ceranae (218 bp) and nosema apis (321 bp). column c-: negative control. sociobiology 61(3): 307-312 (september 2014) 309 table 1. mean number of spores of nosema ceranae per bee, mean infestation rate (%) by the mite v. destructor in brood worker bees and mean infestation rate (%) by v. destructor in brood in different municipalities*. municipality no. of nosema sp. spores (x10³) v. destructor in adult bees (%) v. destructor in brood (%) bananal 623 ± 188a 5.49 ± 0.97a 6.63 ± 1.34a cunha 485 ± 128a 3.53 ± 0.69a 2.93 ± 0.78a lagoinha 258 ±106a 5.87 ± 0.56a 5.65 ± 0.66a monteiro lobato 623 ± 145a 5.92 ± 0.76a 5.70 ± 0.88a paraibuna 581 ± 99a 4.94 ± 0.51a 3.61 ± 0.61a pindamonhangaba (apta) 1.655 ± 114b 4.74 ± 0.59a 3.81 ± 0.69a pindamonhangaba 424 ± 110a 6.75 ± 0.59a 3.30 ± 0.68a redenção da serra 636 ± 153a 4.01 ± 0.79a 2.12 ±0.91a são jose dos campos 459 ± 145a 4.26 ± 0.75a 2.52 ± 0.88a santo antonio do pinhal 607 ± 275a 4.17 ± 1.42a 1.14 ± 1.66a são luis do paraitinga 369 ± 386a 5.66 ± 1.99a 2.90 ± 2.33a taubaté 1.055 ± 153b 4.90 ± 0.79a 3.55 ± 0.93a tremembé 658 ± 99a 5.82 ± 0.51a 4.65 ± 0.60a * in collection period 2, it was not possible to obtain samples in five localities. †means accompanied by different letters differ significantly (p<0.01). table 2. mean number of spores of nosema ceranae per bee, mean infestation rate (%) by the mite v. destructor in brood worker bees and mean infestation rate (%) by v. destructor in brood in different collection periods (1: winter 2009, 2: summer 2010, 3: autumn 2010). collection periods no. of nosema sp. spores (x10³) v. destructor in adult bees (%) v. destructor in brood (%) 1 379 ± 62a 6.03 ± 0.32a 4.58 ± 0.37a 2 689 ± 87b 3.57 ± 0.45b 1.95 ± 0.53b 3 879 ± 74b 5.65 ± 0.39a 4.66 ± 0.46a mean 637 ± 36 5.41 ± 0.20 4.17 ± 0.23 † means accompanied by different letters differ significantly (p<0.01). discussion this is the first comprehensive study (1,668 samples analyzed, collection periods during all seasons) conducted to identify the prevalence of three typical honey bee pathogens (nosema sp., varroa destructor and paenibacillus larvae) in brazil. although nosema apis was a highly problematic pathogen in the 1970s in the southern region of brazil (teixeira et al., 2013), it was not detected in the present study. in the first collection period (winter and early spring 2009), the number of spores detected per bee was very low, differing significantly (p<0.001) in relation to the other two periods (summer and autumn 2010). traver et al. (2011) also observed low levels of this pathogen in the winter, but in the autumn their results were opposite to ours, even though in the region of the united states studied by them the autumn temperatures are near those in the winter in são paulo. in turn, higes et al. (2008) observed higher intensities of infection by n. ceranae in the coldest months and lower intensities at the beginning of spring, while in germany, gisder et al. (2010) found that the intensity of infection by nosema spp. was greater in spring than in autumn. although comparisons of infection intensity between different regions are highly contradictory, even considering data from temperate regions where the seasons are well defined, the climatic peculiarities of the regions under analysis must be considered, because they can affect the intensity of infection, even if only indirectly (le conte & navajas, 2008). in brazil, no pattern in the infection intensity of nosema the apiaries in pindamonhangaba and taubaté presented the highest infection intensities, differing significantly (p<0.001) from the other municipalities studied. table 2 reports the intensities of infection by the microsporidium in the three sampling periods. the first period (winter and early spring 2009) presented the least intense infection by nosema ceranae, differing (p<0.001) from the intensities of periods 2 (summer 2010) and 3 (autumn 2010). the results for the number of adult v. destructor mites as well as descendants in the 13 locations and three collection periods can be observed in tables 1 and 2, respectively. the overall average infestation of v. destructor in adult bees was 5.41 ± 0.20 (table 1). the infestation rates observed in period 2 were lower in relation to the other two periods (1 and 3), both in adult bees and brood. therefore, there was a difference (p<0.01) of the infestation rates observed in these periods; but in all cases the infestation levels were low. lg santos et al. honey bee health in southeastern brazil 310 ceranae has been observed during the year (teixeira et al., 2013), considering the weather or region. in the two places (pindamonhangaba – sp and taubaté – sp) where infection by nosema ceranae was higher than at the other sites, the experimental apiaries are frequently managed for teaching/research purposes rather than production, so the frequent management practices (change of frames, common use of material between colonies, etc.) might have facilitated dispersion and/or made the bees more susceptible due to stress from frequent human intervention. the low numbers of spores in our samples and the absence of collapse of any of the colony analyzed, in contrast to the observations of higes et al. (2008), can possibly be credited to a higher tolerance of africanized a. mellifera honey bees. in fact, many questions can be posed in relation to nosema ceranae, mainly regarding the absence of clinical symptoms in infected colonies and the considerable variation of infection intensity within very short periods (d. message, unpublished data). in the state of são paulo, considering the samples analyzed so far, the presence of the species nosema ceranae has been confirmed in 100% of the cases where nosema has been observed (fig 1). according to teixeira et al. (2013), even with the proof of the high prevalence of n. ceranae in the country’s apiaries, the recommendation by researchers and technicians who work in the area of honey bee health is not to use chemical products, due to the inconsistency of the real effect of the presence of this pathogen in the colonies. the first report of the species nosema ceranae in brazil was by klee et al. (2007); these authors reported this species of microsporidium on four continents. according to various authors, n. ceranae jumped from apis cerana to apis mellifera probably in the 1990s, since then dispersing to most regions of the world (fries et al., 1996; higes et al., 2006; klee et al., 2007; paxton et al., 2007; fries, 2010). nevertheless, in brazil, teixeira et al. (2013) detected its presence in samples that had been collected in the southern state of rio grande do sul more than three decades ago. in asia, where it supposedly originated, recent articles have reported the presence of n. ceranae in vietnam (klee et al., 2007) and iran (nabian et al., 2011). in europe, studies have confirmed its presence since 1998 (fries et al., 2006; paxton et al., 2007), in several countries: spain (fries et al., 2006; higes et al., 2006; klee et al, 2007; martínhernandez et al., 2007), france (chauzat et al., 2007), germany (klee et al., 2007), sweden (klee et al., 2007), finland (klee et al., 2007; paxton et al., 2007), denmark (klee et al., 2007), greece (klee et al., 2007), hungary (tapaszti et al., 2009), holland (klee et al., 2007), united kingdom (klee et al., 2007), italy (klee et al., 2007), serbia (klee et al, 2007), poland (topolska &kasprzak, 2007), bosnia (santrac et al., 2009) and turkey (whitaker et al., 2010). in africa, higes et al., (2009) reported n. ceranae in algeria in 2008. in north america, this species has been present since 1995, affecting colonies mainly in the united states (klee et al., 2007; chen et al., 2008; williams et al., 2008), as well as canada (williams et al., 2008) and mexico (guzmán-novoa et al., 2011). in central america, n. ceranae was found in costa rica by calderón et al. (2010), and in south america it is present in brazil and argentina (klee et al., 2007; medici et al., 2012), uruguay (invernizzi et al, 2009) and chile (martínez et al., 2012). in oceania, recent studies reported this species in new zealand (klee et al., 2007) and australia (giersch et al., 2009). with respect to the mite varroa destructor, the infestation rates were low, as also reported by pinto et al. (2011) in africanized bees in a region with similar environmental conditions to those in the present study, although with a smaller number of samples. on the other hand, in evaluating the infestation levels in the forest zone of the state of minas gerais, a region with a tropical climate with temperatures varying from 14 to 26ºc during the year (koeppen climate classification), bacha junior et al. (2009) found an average value of 7.8% during the summer. apparently, there is great variation in the infestation rates of this mite according to the region of the country and the respective climate (moretto, 1997). showing the same tendency as the levels of infestation in adult bees, the rates of infestation by the mite in brood combs were low in comparison with those found in colonies without treatment in bulgaria, england and the entire united kingdom (18 to 49%, 15 to 40% and 6 to 42%, respectively) (martin, 1994; 2001). usually, when the temperature drops, especially during the autumn and the winter, the brood area shrinks, as a consequence of the reduced availability of food resources in the environment and the number of phoretic mites increases. this can explain the low number of the mites in the capped brood. the high infertility rate of this mite on africanized bees in brazil (message & gonçalves, 1995; rosenkranz, 1999; calderón et al., 2010; rosenkranz et al., 2010) could also explain the low infestation rate observed. however, garrido et al. 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(1987). the biology of the honey bee. harvard university press. cambridge. london. uk. doi: 10.13102/sociobiology.v67i2.3760sociobiology 67(2): 144-152 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 seasonal changes in sugar and amino acid preference in red wood ants of the formica rufa group introduction red wood ants (formica rufa group) include a number of species with high ecological importance in temperate forests. their presence has effects both above and below ground, playing an important role in nutrient cycling, seed dispersion and predator-prey dynamics, ultimately affecting plant and tree growth and providing benefits to a wide variety of myrmecophile species (jurgensen et al., 2008; stockan & robinson, 2016). wood ants’ diet is largely based on honeydew, which they use for metabolic maintenance as well as growth (hölldobler & wilson, 1990; domisch et al., 2009). therefore, as many other ant species, red wood ants establish mutualistic relationships with honeydew-producing insects, predominantly aphids. in exchange for honeydew, ants protect aphid colonies against (i) predators and parasitoids (buckley, abstract red wood ants of the formica rufa group are important ecosystem engineers throughout the northern hemisphere with potential to be commercially produced and used as predatory agents in biological control programs. however, in order to do that, their mutualistic relationship with aphids needs to be disrupted. this may be achieved by developing artificial sugar-based solutions with a composition that makes them more attractive than aphid honeydew. the present field study investigated formica rufa’s preference for several sugar and amino acid sources, as well as potential seasonal changes in these preferences. red wood ants consistently preferred sucrose to monosaccharides and were most attracted to solutions containing an amino acid source, albeit seasonal differences were observed with regard to which amino acid sources were most preferred. recruitment to offered sugar solutions was highest during july, when colony requirements were high, and during october, when alternative food sources were scarce. since ant preference for sugar solution constituents seems to be species-specific and show seasonal dynamics, artificial food aimed at disrupting ant-aphid mutualisms should be tailored to individual species and seasons. sociobiology an international journal on social insects nel madsen; j offenberg article history edited by gilberto m. m. santos, uefs, brazil received 01 october 2018 initial acceptance 15 may 2019 final acceptance 11 november 2019 publication date 30 june 2020 keywords ant nutrition, food selection, formicidae, biocontrol, carbohydrates, protein. corresponding author natalia e. l. madsen department of bioscience aarhus university vejlsøvej 25, 8600 silkeborg, denmark. e-mail: nel@bios.au.dk 1987; beltrà et al., 2015), (ii) diseases (nielsen et al., 2010) and (iii) weather (way, 1963). due to this mutualism, a great number of ant species can interfere with biological control of honeydew-producing pests by natural enemies (jiggins et al., 1993; stechmann et al., 1996; styrsky & eubanks 2007) and lead to increases in such pest populations (flatt & weisser, 2000; stewart-jones et al., 2008). to obtain the protein necessary to support brood rearing, red wood ants prey opportunistically on different arthropod species, depending on their availability (stockan & robinson, 2016). a medium-sized nest of formica polyctena has been estimated to kill eight million insects per year (way & khoo, 1992), and there is evidence that formica ants have the potential to suppress populations of unwanted arthropods in commercial crops (godzińska, 1986; paulson & akre, 1992). furthermore, wood ant nests are easily transplanted and established in commercial orchards (nielsen et al., 2018). department of bioscience, aarhus university, silkeborg, denmark research article ants sociobiology 67(2): 144-152 (june, 2020) 145 thus, these ants could potentially become successful biocontrol agents in horticulture when not tending harmful aphid species. in european forests, formica spp. predate on several species of defoliating pests (adlung, 1966; skinner & whittaker, 1981), potentially contributing to a reduction of herbivore damage on trees (warrington & whittaker, 1985). however, they also allow ant-tended hemipteran populations to reach damaging densities in some trees, e.g. fagus (way & khoo, 1992). similar patterns have been found in plantations. in an apple orchard the presence of introduced f. polyctena colonies led to reductions of the amount of winter moth larvae (an important pest in organic apple orchards) on ant visited trees; however, the ants also had a negative impact by increasing the number of aphids on trees (offenberg et al., 2019). for this reason, we need strategies to disrupt ant-aphid mutualisms. this would eliminate the adverse effects derived from aphid tending and open the door to commercial production of f. rufa as a biological control agent, allowing us to harness the benefits derived from ant presence. such a strategy would have an impact beyond economic benefits, as increasing the interest in wood ant production and transplantation may help improve their conservation status in nature. artificially provided carbohydrate solutions have successfully been used to decrease aphid tending by lasius niger, resulting in significant increases in natural enemy pressure on both green apple aphids (aphis pomi) and rosy apple aphids (dysaphis plantaginea) (nagy et al., 2013, 2015). similar results were found for lasius grandis in citrus orchards, further supporting the provision of artificial sugars as a viable strategy for ant management (wäckers et al., 2017). in addition, there is evidence that sugar-fed ants may start preying on aphids that were formerly tended (way, 1954; offenberg, 2001). aside from direct effects on ant-aphid dynamics, artificial sugar solutions may provide collateral benefits. in forests, the use of sugar baits on trees decreased pine weevil feeding activity on conifer seedlings by 30% (maňák et al., 2013), possibly due to wood ants aggressively protecting their sugar sources (maňák et al., 2015). due to the potential benefits of finding a sugar solution that can out-compete aphid honeydew, the field experiments in this study tested the preference of red wood ants towards different carbohydrate and amino acid sources, as previously done for lasius niger (madsen et al., 2017). additionally, preference tests were carried out at different times during the season in order to investigate potential seasonal changes in wood ants’ preferences. methodology the methodology and statistical analysis in the present study are largely based on those used by madsen et al. (2017). the sugar experiment tested formica’s preference for six combinations of monodiand tri-saccharides (glucose, fructose, sucrose, raffinose and melezitose). glucose, fructose and sucrose, all commonly found in aphid honeydew (fischer & shingleton, 2001), were used as main constituents and raffinose and melezitose were added to sucrose in small amounts to investigate their potential as attractants. the protein experiment tested formica’s preference for mixtures of pure sucrose with five different amino-acid-containing substances, i.e. three mixtures of pure amino acids (aa), casein hydrolysate (sigma-aldrich, product 22090) and egg powder (sigma-aldrich, product 55871). sucrose was selected as a carbohydrate constituent because it has been widely used in other studies and is readily accepted by many ant genera, including formica (sudd, 1985; tinti & nofre, 2001; blüthgen & fiedler, 2004). the composition of each solution is given in tables 1 and 2. aa mixtures were created by grouping aas according to their ability to stimulate chemoreceptor cells, following the categorization given by shiraishi and kuwabara (1970). mix a contained aas that do not stimulate chemoreceptor cells in flies while mix b contained those that stimulate sugar receptor cells. mix c combined all the aas in mix a and b at half the concentration. casein hydrolysate and whole egg powder provided a varied source of different peptides and aas (kay, 2004; arganda et al., 2014) and, in the case of the egg powder, also fats (dussutour & simpson, 2008). amino acid-only solutions, i.e. without sugar, were not included in the experiment, as they do not elicit strong ant recruitment (blüthgen et al., 2004; personal observations). ten independent f. rufa mounds were located in forest areas in silkeborg and gammel ry (denmark). the distance between mounds in the same area varied from 25 to 250 meters, with most colonies being at least 50 meters apart (additional information on the location and size of the nests can solutions: sugar preference experiment g per 100 ml of water glucose fructose glucose + fructose sucrose sucrose + raffinose sucrose + melezitosesubstance: glucose 20 – 10 – – – fructose – 20 10 – – – sucrose – – – 20 16 16 raffinose – – – – 4 – melezitose – – – – – 4 table 1. composition of solutions in the sugar preference experiment. nel madsen; j offenberg – sugar and amino acid preference in red wood ants146 be found in supplementary material 1). it should be noted that high intraspecific variability and hybridization make accurate identification to species level a great challenge (stockan & robinson, 2016), and therefore there is a chance that our colonies could be of f. polyctena. experimental solutions were offered simultaneously in two separate cafeteria experiments; i.e. one for sugar preference and one for amino acid preference, carried out on the same day. water was offered together with the baits in order to control that ants were not attracted to the different solutions due to thirst. each set of experiments was carried out in june, july, august and october 2017. five milliliters of each solution were offered in small petri dishes (35mm ø) mounted on a larger petri dish (135mm ø), which served as an arena. the location of each of the solutions on the dish was randomized to control for positional bias. the feeding arena was placed in the shadow of vegetation close to a nest entrance, in areas with moderate to high ant activity. all choices within each experiment were offered simultaneously to avoid differences in discovery time. we assumed that the short distance between adjacent food sources ensured that when a forager encountered one of the baits, the ant would readily discover the others and still make a choice among all the solutions available before deciding where to drink. during the experiment, ants were observed to repeatedly visit several baits before starting to drink from one. after offering the solutions, ants were allowed to forage freely for at least 10 minutes before the assessment of individual ant visits began. visiting ants were counted three times, at eight to ten minutes intervals. mean drinking time recorded for formica rufa was 121 seconds (± 14,2 seconds) with a maximum drinking time of 268 seconds (joachim offenberg, personal communication).thus, wood ants observed at each counting were considered different workers or workers revisiting the sugar baits after being in the nest. since all food sources were presented in liquid form, we assume that the differences in the number of ants at each bait is similar to the differences in the amount of food collected (kay, 2004). the total number of ants visiting each solution was summed for each colony and used as a response to analyze preferences. solutions: amino acid preference experiment g per 100 ml of water substance: sucrose sucrose + mix a sucrose + mix b sucrose + mix c sucrose + casein sucrose + egg powder sucrose 20 20 20 20 20 20 serine – 0,25 – 0,125 – – alanine – 0,25 – 0,125 – – threonine – 0,25 – 0,125 – – cysteine – 0,25 – 0,125 – – phenylalanine – – 0,25 0,125 – – valine – – 0,25 0,125 – – leucine – – 0,25 0,125 – – methionine – – 0,25 0,125 – – casein hydrolysate – – – – 1 – whole egg powder – – – – – 1 table 2. composition of solutions in the amino acid preference experiment. all amino acids are in l – form. statistical model initially, data within each of the experiments; i.e. sugar and amino acid, was pooled for the whole season and analyzed. subsequently, data collected in different months was analyzed separately in order to reveal potential seasonal changes in food preference. colonies where we recorded zero or one visit during a whole experiment were excluded from the analysis. because different food choices were offered simultaneously, our individual counts for each solution are not independent and cannot be treated with the most common statistical methods. therefore, data was analyzed using the bayesian model described in madsen et al. (2017), which estimates the distribution of possible pi values for each food source, allowing us to estimate the probability for an ant to visit a specific source. using the estimated distributions of probabilities, a resampling process permitted us to calculate the probability that a specific solution will be chosen over each of the alternatives. to estimate the most likely probability for an ant to choose a specific solution we use the dirichlet distribution (using k = 1), and the following formula: , where v is the total number of visits for the specific experiment, n is the number of available choices, vi is the total number of visits to source i and pi is the probability for a visit to source i. sociobiology 67(2): 144-152 (june, 2020) 147 to calculate the probability of solution a to be chosen over solution b, sets of pi values (one for each of the food sources) were randomly drawn from the probability distributions previously estimated by the dirichlet model. after 106 resamplings, and counting how many times solution a was preferred over b, we obtained the probability that a will be chosen over b. with this information, we assigned superscript letters to the previously estimated probabilities, in order to specify the hierarchies between each of the solutions. an r code for the resampling process is provided in supplementary material 2. an application of the model to pollinating networks can be found in sørensen et al. (2011) and mathematical proofs and details are given by frigyik et al. (2010). results ant recruitment to the sugar and protein experiments varied during the season (table 3). however, the number of ant visits was consistently higher in the protein experiments compared to sugar experiments. in all experiments, water was always among the least preferred options. when data from the sugar experiment was pooled for the whole season, the solutions with a higher chance of being visited were sucrose and the combination of sucrose and melezitose (table 4). results from the resampling process (table 5), revealed a clear preference for the disaccharide sucrose over monosaccharides. combinations of sucrose with a trisaccharide were either less preferred than sucrose alone (raffinose) or equally preferred (melezitose). the mixture of glucose and fructose was preferred over either of the pure monosaccharide solutions. the estimated probability for an ant to choose water becomes negligible, i.e. rounded to zero, because water collection represents only a very small fraction of the total number of visits for the whole season. analysis of the pooled data from the protein experiment showed that all solutions containing a protein component were significantly preferred over sucrose alone. among all the protein sources, casein was the most attractive to ants, followed by amino acid mixes b and a (table 6). table 3. total number of ant visits in each experiment by season and type of experiment. values in brackets represent the number of colonies that contributed to the visits. sugar experiment protein experiment june 35 (3) 116 (6) july 167 (8) 464 (9) august 39 (6) 79 (4) october 194 (10) 438 (10) table 4. estimated mean value for the probability of a foraging ant to choose each of the presented solutions. solutions with different superscript letters are significantly different at a significance level of 0.05 (see table 5). solutions with the same superscript letter are equally preferred. table 5. probabilities for pairwise preferences based on 106 simulations. values show the probability that the solution in the row is preferred over the solution in the column at a significance level of 0.05. i.e. if values > 0.95 the row is significantly preferred over the column and if values < 0.05 the column is significantly preferred over the row. solution sucrose 0.25a sucrose + melezitose 0.23a,b sucrose + raffinose 0.2b glucose + fructose 0.14c fructose 0.1d glucose 0.08d water 0.00e glucose fructose glucose + fructose sucrose sucrose + raffinose sucrose + melezitose fructose 0.750 glucose + fructose 0.991 0.954 sucrose 1.000 1.000 1.000 sucrose + raffinose 1.000 1.000 0.985 0.036 sucrose + melezitose 1.000 1.000 1.000 0.292 0.893 water 0.000 0.000 0.000 0.000 0.000 0.000 the preference matrix resulting from the resampling process for this analysis can be found in supplementary material 3. results for the individual analysis of each experiment are compiled in table 7, which presents the seasonal dynamics of the probabilities for an ant to choose a specific solution. the accompanying matrices showing the outcome of each resampling simulation (equivalent to table 5) can be found in annex 3. nel madsen; j offenberg – sugar and amino acid preference in red wood ants148 seasonal changes in sugar preference in june, all sugar solutions except for fructose were significantly preferred over the water control. sucrose had the highest probability of being selected, but this was not significantly different from that of the mixtures sucrose + trisaccharide or glucose + fructose. this lack of significance is likely due to the reduced sample size for this experiment. during july, all solutions with sucrose as a basic component were significantly preferred over those with monosaccharides, and the mixture of glucose and fructose was preferred over baits with only one of the two components, as seen in the analysis of the pooled data. the addition of a trisaccharide to sucrose did not make those solutions more attractive, as sucrose alone was significantly preferred over all other experimental solutions. this changed in august, where the mixture of sucrose and melezitose was significantly preferred over sucrose alone (and over monosaccharide solutions). as in july, in august there was a trend to prefer disaccharides over monosaccharides, although preference for sucrose and fructose was not significantly different (p = 0.055). in october, ants significantly preferred the mixture sucrose + melezitose over monosaccharides (but not over sucrose and sucrose + raffinose) and sucrose-based solutions over glucose. overall, seasonal results were similar to those obtained with the pooled data, as there was a tendency for ants to prefer sucrose-based solutions over monosaccharides, with trisaccharides sometimes increasing the attractiveness of sucrose. seasonal changes in protein preference in the protein experiment in june, casein was significantly preferred over all other experimental solutions and no other significant differences were found. in july,the combination of sucrose and casein was significantly preferred over the solution containing mix c, but no significant difference was found between the former and mixes a and b. no significant difference was found in ant preference when comparing the three mixtures of single amino acids. sucrose alone and in combination with whole egg powder were significantly disfavored, as they were the least preferred of all experimental solutions, only selected over the water control. solution pi sucrose + casein 0.27a sucrose + mix b 0.18b sucrose + mix a 0.17b,c sucrose + egg powder 0.15c sucrose + mix c 0.14c sucrose 0.09d water 0.00e table 6. estimated mean value for the probability of a foraging ant to choose each of the presented solutions. solutions with different superscript letters are significantly different at a significance level of 0.05 (see table 5). solutions with the same superscript letter are equally preferred. table 7. ranking of solutions from most to least preferred for each of the months in the sugar and protein experiment. in brackets, estimated mean value for the probability of a foraging ant to choose each of the presented solutions. solutions with different superscript letters are significantly different at a significance level of 0.05. solutions with the same superscript letter are equally preferred. suc = sucrose, glu = glucose, fruc = fructose, mel = melezitose, raf = raffinose, wat = water, cas = casein hydrolysate, ep = whole egg powder. june solution july solution august solution october solution sugar experiment suc (0.28) a suc (0.30) a suc + mel (0.39) a suc + mel (0.22) a suc + mel (0.17) ab suc + mel (0.21) b suc + raf (0.26) a,b suc(0.20) a,b suc + raf (0.17) ab suc + raf (0.20) b,c suc (0.17) b,c suc + raf (0.17) a,b glu + fruc (0.17) a,b glu + fruc (0.14) c fruc (0.07) c,d glu + fruc(0.15) b,c glu (0.12) b fruc (0.07) d glu (0.04) d fruc(0.14) b,c fruc (0.07) b,c glu (0.06) d glu + fruc (0.04) d glu (0.11) c wat (0.02) c wat (0.01) e wat (0.02) d wat (0.00) d protein experiment suc + cas (0.38) a suc + cas (0.22) a suc + cas (0.34) a suc + cas (0.27) a suc + mix a (0.15) b suc + mix a (0.21) a,b suc + mix a (0.22) a,b suc + ep (0.24) a suc + mix b (0.13) b suc + mix b (0.21) a,b suc + mix c (0.19) b,c suc + mix b (0.18) b suc + mix c (0.12) b suc + mix c (0.17) b suc + mix b (0.10) c,d suc + mix a (0.12) c suc + ep (0.11) b suc + ep (0.09) c suc + ep (0.07) d suc + mix c (0.11) c,d suc(0.09) b suc(0.09) c suc(0.07) d suc(0.08) d wat (0.02) b wat (0.01) d wat (0.01) e wat (0.00) e sociobiology 67(2): 144-152 (june, 2020) 149 in august, casein was preferred over all other experimental solutions except for mix a. mix a and mix c were similarly visited and significantly preferred over sucrose alone and sucrose mixed with whole egg powder. while mix a was preferred over mix b, no significant difference was found between mixes b and c. the least preferred solutions were sucrose alone, mix b and whole egg powder, with no significant differences among them. in october, solutions containing casein and egg powder were preferred over all other solutions, with no significant difference between them. amino acid mixes a and b were significantly preferred over pure sucrose. mix b was significantly more visited than mixes a and c. despite the variability observed during the season, f. rufa workers consistently preferred solutions containing a protein source over sucrose alone and our water control had always the lowest probability of being visited. discussion we have shown that wood ants prefer sucrose to monosaccharides and that adding an amino acid source increased the attractiveness of sugar solutions. furthermore, we argue that ant preferences for specific attractants is species-specific as different preferences have been found in other ant species (see below) and that preferences for specific attractants may change over the season. these findings have implications concerning the development of sugar formulations that may be used to interrupt the mutualisms between ants and attended honeydew-producing trophobionts. with regard to sugars, wood ants preferred sucrose to glucose and fructose. similarly, a preference for sucrose over monosaccharides has been previously observed in myrmica rubra (boevé & wäckers, 2003), lasius niger (völkl et al., 1999; tinti & nofre, 2001; madsen et al., 2017) and ten species of tropical ants (blüthgen & fiedler, 2004). to our knowledge, no studies to date have described the opposite pattern for any ant species. in light of these results, the sugar component of artificial solutions aiming at breaking ant-aphid mutualisms should be based on disaccharides, primarily sucrose. overall, wood ants recruited more foragers in the protein experiments than the sugar ones (poisson, chisq = 295,7, p = 2.2e-16). in addition, the sucrose-only control consistently ranked among the least preferred solutions when presented along with sugar solutions that contained a protein component. similarly, when protein components were added to sugar solutions, the vast majority of the twenty-three tropical ant species tested by blütgen and fiedler (2004) preferred mixtures of sucrose and amino acids over sucrose alone, and the rest were non-selective. for both l. niger and s. invicta, more workers were counted at sugar solutions containing a protein component than at sugar-only solutions (lanza, 1991; madsen et al., 2017). thus, despite evidence that two tropical ant species that discriminate against specific amino acids, in favor of sugar-only artificial nectars (lanza & krauss, 1984), it seems that amino acid sources increase the attractiveness of sugar solutions to most ant species. this supports the idea that protein can be added to sugar to make carbohydrate solutions more attractive to ants. results showed that both recruitment and preferences changed during the study season. in the early summer, ants were observed exploring the pure sugar solutions and leaving them without drinking while trees in the surrounding area showed heavy ant traffic up and down tree trunks. this suggests that, at that time, ants fulfilled their sugar needs with aphid honeydew. similarly, sudd (1985) observed that workers of formica lugubris accepted lower concentrations of sugar in the spring than they did in the summer, when aphid honeydew became available. thus, fluctuations in availability of alternative food in their territory could explain changes in wood ant’s seasonal foraging patterns. this is further supported by kay (2004), whose field experiments found that ant species that collect extrafloral nectar or honeydew, i.e. have easy access to carbohydrates, selected a higher protein:carbohydrate ratio than species who did not. another reason explaining the observed seasonal variability could be changes in the demographics of the ant colony. this response has been described in fire ants solenopsis invicta, where foragers adjusted food selection depending on which of the subcastes in the ant colony, e.g. reserve workers, nurses or larvae, had been starved (sorensen et al., 1985). similarly, colonies of lasius niger with and without brood showed significantly different foraging patterns regarding sugar and protein collection (portha, 2002). in our experiments, the highest number of visits recorded were in late july, when the needs of the colony (especially for protein) are at its peak due to high activity and brood production, and mid-october, when alternative food sources decline (stockan & robinson, 2016). in autumn, food availability decreases but colony demand is still high, as carbohydrates are collected and stored by young workers to use at the beginning of next season, when food is scarce (schmidt, 1974; as quoted in sudd. 1985). in early spring, when ant activity is resumed, workers use glands to convert stored lipids and proteins into food to feed (i) the queen and (ii) the sexual larvae resulting from the queen’s “winter eggs” (stockan & robinson, 2016). anticipation of this future need for nutrients in a time where alternative food is rare could explain the high recruitment observed in our experiments in october (table 3). lastly, laboratory experiments by cook et al., (2010) indicated that there might be additional, season-specific cues that affect the foraging behavior of fire ants, as they found that nutrient regulation strategies were seasonally dynamic independently of colony demographics, environmental conditions or availability of food. whether these cues are universal for all ant species remains to be investigated. seasonal variability was also observed regarding formica’s acceptance of trisaccharides. in our experiments, raffinose and melezitose decreased the attractiveness of sucrose in july, but increased it in august. in lasius niger, nel madsen; j offenberg – sugar and amino acid preference in red wood ants150 trisaccharides have been found to generally increase the attractiveness of sugar solutions, but while some studies found a preference for melezitose over raffinose (völkl et al., 1999; tinti & nofre, 2001; detrain et al., 2010), madsen et al. did not (2017). studies on several species of tropical ants did not find a preference for raffinose or melezitose over sucrose alone (cornelius et al., 1996; blüthgen & fiedler, 2004). since evidence indicates that preference for melezitose is speciesspecific, further studies on wood ants with larger samples sizes are needed in order to better interpret these conflicting results. in a similar way, results on amino acid preference are also inconclusive and need further investigation. casein was the protein source with the highest probability of being visited, both when results were pooled for the whole season and for each of the seasonal experiments. however, different solutions rank at the same preference level as casein in july, august and october (table 7). in particular, wood ant’s acceptance of egg powder should be explored, since it was clearly disfavored during summer but heavily collected (and as preferred as casein) in october (table 7). as previously discussed, changes in food availability and colony demographics may at least partially explain this shift. our results indicate that a solution aiming at disrupting ant-aphid mutualism should contain carbohydrates in form of sucrose and an adequate protein source. of all the components tested, ants showed a consistently high preference for casein, but further research is needed to gain insight on the ideal carbohydrate/protein ratio that would get transplanted red wood ants to abandon aphid tending while continuing to capture prey. despite trisaccharides seemingly being a good choice for increasing sugar attractiveness in other ant species, e.g. l. niger, our results cannot support their use for f. rufa, and thus additional components aiming at further increasing the solution’s attractiveness remain to be examined. furthermore, and in order to corroborate the existence of a true seasonal pattern, as opposed to changes brought by chance or fluctuations in local conditions, it would be necessary to replicate this study for at least another season, preferably more. ideally, artificial solutions should be tailored to each ant species and to the relevant season. for example, during the late spring and summer, when aphid populations are abundant, a more nutrient-rich solution is needed in order to attract ant attention. even after finding the ideal composition, wood ant workers might need some time to shift from foraging aphid honeydew to artificial solutions, as they exhibit high route fidelity (stockan & robinson, 2016). however, this strong site allegiance implies that once they start foraging on suitable artificial food, they will remain to do so for as long as it is available. thus, the best course of action would be to start offering artificial food early in the season, when aphid populations are still absent or low in numbers. in this way, we may ensure that ants forage on artificial solutions and build up site allegiance to artificial feeders. in conclusion, offering wood ants an artificial sugar-based solution that could nutritionally outcompete honeydew, would open the door to using red wood ants as biological control agents, e.g. in fruit orchards. besides, increased interest in optimizing commercial production of formica rufa colonies could also have a positive impact in the efforts towards conservation of this key species group. acknowledgements special thanks go to lise lauridsen for her intensive help with field experiments and to peter b. sørensen for his invaluable statistical advice and support. we also thank the anonymous reviewers who took the time to read and comment on this manuscript. this study was funded by the ministry of environment and food of denmark through their gudp program (project number 34009-130599) and developed in collaboration with borregaard bioplant aps and musca tec biosystems aps. authors’ contributions madsen, nel.: corresponding author, experimental design, data analysis and interpretation, drafting, editing and finalization of manuscript. offenberg, j.: conception of the study, substantial contributions to data interpretation, critical revision of manuscript at different stages, final approval of the version to be published. both authors agree to be accountable for all aspects of the work. supplementary materials h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfiles/3760/0 references adlung, kg. 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(1992). role of ants in pest management. annual review of entomology, 37: 479–503 doi: 10.13102/sociobiology.v66i3.4384sociobiology 66(3): 480-490 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 patterns of diversity and distribution of arboreal social bees’ beehives within chimpanzees’ home range in a forest-savanna mosaic (comoé national park, côte d’ivoire) introduction invertebrates are included in the diet of many primates (redford, 1987; pruetz, 2006). within the primates, chimpanzees abstract the goal of this study was to explain the patterns of diversity and distribution of arboreal social bees nesting in forests of the comoé national park, within the homeranges of wild chimpanzees, consumers of their honey. investigations were done using a total of sixteen plots of one hectare each, established in three habitat types (mature forest island, secondary forest island and gallery forest). the diversity and distribution of arboreal social bees was assessed using visual searches. the exploitation of the beehives of these bees by chimpanzees was also evaluated using honey dipping tools as indicators. five bee species belonging to two tribes, namely the meliponini (meliponula ferruginea, meliponula togoensis, meliponula bocandei, hypotrigona gribodoi) and the apini (apis mellifera) were collected. furthermore, frequent exploitation of the honey of stingless bees by chimpanzees was observed, excepted for h. gribodoi. beehives of meliponula ferruginea were identified as the most exploited ones by chimpanzees. a total of 114 beehives were found in the established plots leading to an estimated density of 2.4 beehives/ha within the study area. among the surveyed habitats, mature forest island was found to harbor the highest beehives’ density (4.2 beehives/ha), followed respectively by secondary forest island (1.9 beehives/ha) and gallery forest (1.1 beehives/ha). finally, all bee species were found nesting in cavities of trees with a dbh ranging from 15 to 87.3 cm, with a special preference for dialium guinneense. however, the dbh of the nesting trees and the beehives’ height measured from the ground level, were found not significantly influencing the honey exploitation by chimpanzees. in sum bee species diversity and distribution might be the most important variables in the survival of chimpanzees within forest-savanna mosaic landscape. sociobiology an international journal on social insects na soro1, j lapuente2, na koné3, k yéo4, s konaté5 article history edited by kleber del-claro, ufu, brazil received 27 february 2019 initial acceptance 18 june 2019 final acceptance 02 july 2019 publication date 14 november 2019 keywords bee species, honey, chimpanzee, nesting tree, habitat type, comoé national park. corresponding author nicodénin angèle soro nangui abrogoua university 02 bp 801, abidjan 01, côte d’ivoire. e-mail: nicodenin@gmail.com consume mainly social insects such as termites (macrotermes spp., cubitermes spp., thoracotermes spp.), ants (dorylus spp., oecophylla longinoda, camponotus spp.) and bees/honey (apis mellifera and the meliponini) (o’malley & power, 2012). 1 nangui abrogoua university, ufr des sciences de la nature (ufr sn), research station in ecology of lamto scientific reserve, research station in ecology of the comoé national park, abidjan, côte d’ivoire 2 research station in ecology of the comoé national park, abidjan, côte d’ivoire; animal ecology and tropical biology, biozentrum, universität würzburg tierökologie und tropenbiologie (zoologie iii), würzburg, germany 3 nangui abrogoua university, ufr des sciences de la nature (ufr sn), research station in ecology of the comoé national park, abidjan, côte d’ivoire 4 nangui abrogoua university, ufr des sciences de la nature (ufr sn), research station in ecology of lamto scientific reserve, abidjan, côte d’ivoire 5 nangui abrogoua university, ufr des sciences de la nature (ufr sn), abidjan, côte d’ivoire research article bees sociobiology 66(3): 480-490 (september, 2019) 481 these invertebrates offer a large, clumped biomass and/or a high nutritional pay-off (mcgrew, 2001; deblauwe et al., 2003). in african savannas, where chimpanzees have been studied extensively, it has been demonstrated that the consumption of insects by chimpanzees occurs seasonally. in tanzania and senegal, respectively located in eastern and western africa, termites were for example found mainly included in their diet during rainy seasons (mcbeath & mcgrew, 1982; goodall, 1986), while bees and honey were eaten during the late dry season (pruetz, 2006). the consumption of honey by chimpanzees has been reported in many sites across africa, suggesting the importance of bees as a significant food resource for these primates (mcgrew, 1992; sanz & morgan, 2007; 2009). in fact, some populations of the chimpanzee subspecies have been found to prey upon brood and stored honey of apini and meliponini (sommer et al., 2012). many of these studies revealed the use of tools by the chimpanzees to collect the honey (crickette & david, 2008; boesch et al., 2009; mclennan, 2011; sommer et al., 2012). indeed, apart from humans, only orangutans (van schaik, 2004) and chimpanzees (sanz & morgan, 2009) gain access to beehives using tools. honey gathering using tools typically involves inserting probes in beehives. these probes can be modified to obtain frayed ends for honey dipping (sommer et al., 2012). indeed, such tools with brush-tips allow collecting up to six times more honey than those with unmodified tips (tutin et al., 1995). like other great ape taxa, the western chimpanzee has come under enormous human pressures. for example, about 90% of the ivorian chimpanzee population was lost mainly due to anthropogenic disturbances (campbell et al., 2008). in this country, the chimpanzee populations are highly endangered since only those found in the taï and comoé national parks are considered as viable (hoppe-dominik, 1991; marchesi et al., 1995). recently, studies combining the use of camera trap videos and the indirect signs’ observations along transects, revealed the importance of honey consumption by the chimpanzees of the comoé national park (lapuente et al., 2016). however, a lack of information was identified on (i) the diversity of bee species exploited by these chimpanzee populations, (ii) the density of beehives within the chimpanzees’ habitats and (iii) the plant species on which these beehives are established. bees are recognized to exhibit a diverse array of nesting strategies closely dependent to the part of the habitat type, the nature of the used substrate to nest and the material required for the construction of the beehive (roubik, 2006). the most important role of these beehives is to protect colonies against environmental perturbations by maintaining a specific microclimate for brood development (roubik, 2006; siqueira et al., 2012; pavithra et al., 2013). social bees from the apidae family, including honeybees, bumblebees, and stingless bees, often use pre-existing cavities of trees, ground and termite mounds for building their beehives (potts et al., 2005; roubik, 2006; eardley et al., 2010). we started this research with the following questions in mind: (1) which social bees’ species produce honey consumed by chimpanzees of the comoé national park? in addition, (2) is there a selection of tree species by bees, for nidification within habitats? finally, (3) does the habitat type has an effect on the availability of tree species with suitable nesting sizes for bees? the overall goal of this study was to determine the patterns of diversity and distribution of arboreal social bee species’ beehives within habitats of a sudano-guinean savanna zone in order to assess their exploitation by the dwelling chimpanzee species. specifically, it aimed at (i) assessing the diversity of arboreal social bees nesting in the homeranges of chimpanzees within the comoé national park, (ii) determining the distribution and density of the beehives of these bee species within these habitats, (iii) identifying the preferred plant/tree species and the height of beehive’ positioning in the identified bee species. materials and methods description of study area this study was conducted in the comoé national park (cnp). this park covers around 11,500 km². it is a unesco world heritage site and a biosphere reserve, located in the north-eastern part of côte d’ivoire in west africa, between the 8°30’ 9°40’ n and the 3°10’ 4°20’ w. the cnp is covered by 91% of savanna habitats while gallery forests and forest islands cover around 8.4% of this park (mühlenberg et al., 1990). these forests are the main chimpanzee habitats within this park. the climate of the cnp is warm and dry, with a mean temperature of 27°c and an average annual precipitation oscillating around 1090 mm (hennenberg, 2005). from november 2017 to july 2018, this study was concentrated on a study area of 900 km² that the comoé chimpanzee conservation project established since 2014 in the south-western sector of the park. during this study, three forest types, which respective characteristics are described below, were surveyed: mature forest island (mfi): this habitat is an old, never exploited semi-deciduous forest island submitted to a natural evolution which is now close to the final stages (aging, mortality) of the silvigenetic cycle. it is an open pool of trees, up to 35 m in height, with a recovery of at least 40% (kouassi et al., 2014). this habitat is dominated by several species belonging to numerous plant families: malvaceae (ceiba pentandra, cola cordifolia), combretaceae (anogeissus leiocarpus), moraceae (antiaris toxicaria, milicia excelsa), oleaceae (schrebera arborea), zygophyllaceae (balanites wilsoniana), ulmaceae (celtis zenkeri, c. integrifolia), fabaceae (dialium guineense), ebenaceae (diospyros mespiliformis, d. abyssinica), sapotaceae (pouteria alnifolia, manilkara obovata, m. multinervis), dichapetalaceae (tapura fischeri) (lauginie, 2007). na soro, j lapuente, na koné, k yéo, s konaté – bee assemblages in the home range of savanna chimpanzees482 the herbaceous layer is dominated by poaceae family (oplismenus hirtellus, cyrtococcum chaetophoron, olyra latifolia and centotheca lappacea). this layer is sparse and has a height between 0 and 2 m (kouassi et al., 2014). secondary forest island (sfi): which is also a semideciduous forest island that has undergone different forms of anthropogenic disturbances at a given period; but regenerated over years (aubréville, 1949). this habitat is also dominated by most of the same plant species of the mature forest island; but in a lower density. however, other species were exclusively encountered in this habitat (i.e. lannea welwitschii, tetrapleura tetraptera, zanthoxylum zanthoxiloides, afraegle paniculata) and woody liana belonging to two families; namely the malpighiaceae (flabellaria paniculata) and the apocynaceae (alafia scandens, landolphia hirsuta, secamone afzelii, baissea zygodioides, cryptolepis sanguinolenta) (lauginie, 2007). the invasive plant species chromolaena odorata (asteraceae) is also abundant in this habitat. gallery forest (gf): this habitat is located along the comoé river. it presents a relatively closed pool of trees with a higher closed canopy of more than 30 m high. the recovery is between 40 and 70% for the upper stratum (kouassi et al., 2014). the dominant tree species is cynometra megalophylla (caesalpiniaceae). however, other plant species are found in this habitat. they mainly belong to the families euphorbiacece (drypetes floribunda, d. gilgiana, dichapetalum madagascariensis,), rubiaceae (oxyanthus recemosus), fabaceae (dialium guinnense), loganiaceae (strychnos sp.), linaceae (hugonia planchonii) and annonaceae (xylopia parvifolia). the herbaceous layer of this habitat dominated by three plant families namely acanthaceae (elytraria marginata), poaceae (acroceras zizanoides) and cyperaceae (hypolytrum heteromorphum). its characteristics are identical to those of the forest island. sampling design bee beehives survey sixteen plots of one hectare each (100 m x 100 m) were established within each of the surveyed habitats. visual searches of arboreal social bees’beehives were conducted in each plot, using a pair of binoculars when needed. trees with diameter at breast height (dbh) ≥ 15 cm, were considered as potential nesting ones (darchen, 1972; tornyie & kwapong, 2015). for every encountered beehive, the tree dbh and height at which the entrance of bees is located above the ground level, were recorded using a measuring tape. for trees with large buttresses, the dbh measurements were taken just above these buttresses. the height of the bees’ nests above the ground level was measured when located less than 6 m and just estimated when superior to this distance. the density of potential nesting trees (with a dbh ≥ 15 cm) was calculated per habitat and per species surveyed. the density of beehives per species was also determined in these established plots. some beehives were certainly missed, especially those located high in the canopy. the field surveys were done during the sunny days in order to make easier the observation of foraging bees’ flying in and out of their respective beehives. bees’ traffic and beehives entrances tubes were then searched in bases, on trunks and branches of all dead and live trees. the encountered beehives were all photographed and geo-referenced with a handheld gps. for each beehive found, samples of worker individuals were collected using an insect aerial net and stored in eppendorf tubes containing 70 % ethanol for future identification. the species identification of colonies nesting high in inaccessible tree trunks was done in situ using a pair of binoculars. when a beehive was found in a tree cavity, this tree was identified at the species or morphospecies level. the identification of plant species was confirmed by the national floristic centre located of the félix houphouët-boigny university (abidjan, côte d’ivoire). identification of social bees which honey is exploited by chimpanzees a recent study by lapuente et al. (2016) showed the use of tools for the consumption of honey by the chimpanzee communities of the cnp. based on these results, the chimpanzee honey dipping tools were used as indicators of beehives exploited by these primates. comoé chimpanzees have been found to leave honey collection tools inserted in the beehive entrance or at the base of the trees where the beehive has been exploited. therefore, we determined which beehives had been exploited by chimpanzees by the presence of used tools and traces of the exploitation such as honey dripping from the beehive entrance or remains of the honeycombs with chimpanzee teeth marks. we identified the used chimpanzee tools by the following characteristics: a portion of a branch cut to a determined length, stripped from leaves and lateral branches, often with a brush tip made by chimpanzees using their teeth and clear signs of wear from being used, such as frayed or blunt ends, remains of honey or wax, dirt left by the hand grip. identification of bees bees were first mounted, labeled and then identified using the determination keys of eardley (2004) and (eardley et al., 2010) under a low-power stereo binocular microscope. the reference collection of bee species of central côte d’ivoire collected in the lamto scientific reserve was also used during this work. voucher specimens of all the identified species are available at the lamto research station in ecology. sociobiology 66(3): 480-490 (september, 2019) 483 statistical analysis the sampling efficiency of bees was tested by constructing sample-based species accumulation curves and recording the mean similarity between plots of the same habitat type. indeed, accumulation curves were produced to show the evolution of the species richness in relation to the sampling effort. using the program estimates 8.0.0 (robert k. colwell, department of ecology & evolutionary biology, university of connecticut, storrs, ct 06869-3043, usa; website: http://purl.oclc.org/estimates), the observed and estimated species accumulation curves, respectively sobs and chao2, were constructed after randomizing 500 times the sample in order to ensure the statistical representation of the target assemblage. the diversity of bees’ assemblages and its evenness are measured by simpson’s index. the bray-curtis index was calculated for the measurement of the β-diversity on one hand and the determination of the similarity of species composition between plots of each habitat type on the other hand. based on the species composition of the surveyed habitats, a hierarchical ascending classification (hac) was performed in order to test the similarity them. these indexes were run on paleontological statistics (past) version 3.09 (hammer et al., 2001) at a significance level of 0.05. levene’s test for homogeneity of variance was used to test the distribution of our data before comparison between habitats. due to the non-normal distribution of data and the heterogeneity of variances, non-parametric multivariate analysis of variance kruskal-wallis and post hoc test (pairwise mann-whitney) was used for comparison. results overall taxonomic structure of the observed bee species a total of five bee species, belonging to the family apidae, were found being regularly exploited by comoé chimpanzees. four of these species belong to three genera of meliponini, the stingless bees (hypotrigona gribodoi magretti 1884, meliponula ferruginea lepeletier 1836, meliponula bocandei spinola 1853, meliponula togoensis stadelman 1895) and one to the apini, the honeybees (apis mellifera latreille 1804). sampling efficiency during this study the accumulation curves of the estimated and observed species richness were similar in all the surveyed habitats (fig 1), suggesting a good estimate of the expected species richness of these habitats. indeed, 100 % of the expected species of each of the surveyed habitat were sampled (table 1). species richness and species diversity of the recorded bees five bee species (h. gribodoi, m. ferruginea, a. mellifera, m. bocandei and m. togoensis) were all collected in each of the surveyed habitats. however, different values of simpson and evenness indexes were obtained for these habitats. the highest values of these indexes were found for the mature forest island, followed by the gallery forest and the secondary forest island. furthermore, the bray-curtis index based on the species composition of habitats, showed an important similarity between the mature forest island and the secondary forest island (fig 2). abundance and distribution of the beehives of arboreal social bee species exploited by chimpanzees a total of 114 beehives were recorded within all the surveyed habitats; 95 belonging to the stingless bees and 19 to the honeybees. the highest mean beehive density was observed in stingless bees (2 beehives/ha); while only 0.4 beehives/ha was registered in the honeybees. in terms of beehives’density and abundance at the habitat level, statistical analyses revealed significant differences (kruskal-wallis test: χ²=6.79; df=2; p=0.03), with a decrease ranking going from the mature forest island to the gallery forest through the secondary forest island (table 2). fig 1. accumulation curves of the observed (a) and estimated species richness (b) in the visited habitats. abbreviations: fi = mature forest island; sfi = secondary forest island, gf = gallery forest. habitat types species richness sampled coverage simpson’s index evenness’s index abundance mfi 5 100 0.76 0.91 67 sfi 5 100 0.56 0.62 30 gf 5 100 0.73 0.84 17 table 1. metrics of arboreal social bees’ diversity in the visited habitats. na soro, j lapuente, na koné, k yéo, s konaté – bee assemblages in the home range of savanna chimpanzees484 a total of 36 beehives (roughly 32 % of the total recorded ones) were found with chimpanzees’ honey exploitation tools. most of the beehives exploited by chimpanzees were found in stingless bees (i.e. 89.19 %, n=32). in contrast, only 11.11 % (n=4) of beehives exploited by chimpanzees were in honeybees. at the habitat level, 22 exploited beehives were observed in the mature forest island while 8 were found in the gallery forest and 6 in the secondary forest island. the most exploited bee species by chimpanzees was m. ferruginea respectively followed by m. bocandei, m. togoensis, h. gribodoi and a. mellifera. in contrast, the highest number of non-exploited beehives were observed in h. gribodoi, respectively followed by a. mellifera, m. togoensis, m. ferruginea and m. bocandei (fig 3). availability of the resource provided by bees to chimpanzees and the potential nesting trees we observed six aggregations of beehives for h. gribodoi with a maximum of seven beehives observed in the same dead tree. on the other hand, only one case of aggregation was observed for m. ferruginea, with two beehives found on dialium guinneense. concerning secondary forest island, we found the highest abundance of beehives for h. gribodoi within the mature forest island and the secondary forest island. on the other hand, in the gallery forest, the beehives of m. ferruginea were the most abundant (table 3). moreover, in the mature forest island, m. bocandei had the highest number of exploited beehives (n=7), while m. ferruginea had the highest number of exploited beehives in the gallery forest (n=6). fig 2. similarity of species composition of the visited habitats based on bray-curtis index. abbreviations: fi = mature forest island; sfi = secondary forest island, gf = gallery forest. fig 3. bee species-specific relative abundance of nests exploited and non-exploited by chimpanzees. habitats densities total observed beehives beehives/ha potential nesting trees/ha mature forest island 67a 4.2a 98.8a secondary forest island 30b 1.9b 58b gallery forest 17c 1.1b 94.6a table 2. densities of bees’ beehives and potential nesting trees in the three habitats sampled. the highest density of bee-nesting potential trees was found in the mature forest island (98.8 trees/ha) followed by the gallery forest (94.6 trees/ha) and the secondary forest island (58 trees/ha). these habitat-specific densities were found significantly different from a forest type to another (kruskalwallis test: χ²=16.72; df=2; p<0.05). mann-whitney pairwise test indicated a significant difference between the density of bee-nesting potential trees of the visited habitats (table 2). bee species habitats mfi sfi gf total and ra apis mellifera 10 (3) 5 (1) 4 (0) 19 (4) hypotrigonagribodoi 26 (3) 19 (1) 1 (1) 46 (5) meliponula bocandei 10 (7) 2 (1) 3 (1) 15 (9) meliponula togoensis 9 (4) 2 (1) 2 (0) 13 (5) meliponula ferruginea 12 (5) 2 (2) 7 (6) 21 (13) total 67 (22) 30 (6) 17 (8) 114 (36) table 3. values in brackets represent the number of beehives exploited by chimpanzees for each bees species in the habitats. beehives’ availability on tree species and their potential exploitation by chimpanzees beehives of arboreal bee species were encountered on 17 tree species during this study (dead trees were not taken into account). these species belong to 9 plant families sociobiology 66(3): 480-490 (september, 2019) 485 and 15 genera. most of the beehives were found on trees of the leguminoseae family (i.e. dialium guinneense, albizia adianthifolia, a. zygia, tamarindus indica and tetrapleura tetraptera, cynometra megalophylla). on the 114 observed beehives, 91 were found on living trees and 23 on dead ones. of the 91 beehives found on living trees, 33 were exploited. on the other hand, among the 23 beehives found in cavities of the dead woods, only 3 beehives were exploited by chimpanzees. the highest abundance of beehives was found in the cavities of dialium guinneense and manilkara multinervis. these main plant species were respectively followed by celtis integrifolia, albizia adianthifolia, cynometra megalophylla, anogeissus leiocarpus and vitex sp.. the lowest number of bee beehives were found in cavities of albizia zygia, tetrapleura tetraptera, liana, vitellaria paradoxa, antiaris toxicaria, ficus ingens, ficus sp., diospyros mespiliformis and cola gigantean (table s1). the number of tree species on which beehives were encountered was higher in mature forest island (13 species, 12 genera, 7 families) than the secondary forest island (6 species, 6 genera, 5 families) and the gallery forest (6 species 6 genera, 4 families). dialium guinneense was identified as the main nesting tree species of the mature forest island. in the secondary forest island, manilkara multinervis is the main plant species preferentially chosen by bee species for the establishment of their beehive (table s1). cynometra megalophylla is the most important plant species supporting the highest number of beehives within the gallery forest. moreover, a high significant difference was found between the total number of observed trees and the number of trees on which bee nest exploited by chimpanzees were found in the different habitats (mann-whitney test: mature forest island: χ²=35, df=1, p<0.05; secondary forest island: χ²=45.5, df=1, p<0.05; gallery forest: χ²=48.5; df=1; p<0.05) (table s1). beehives of the sampled bee species were respectively found established on a plant species diversity ranging from 4 to 8 species. meliponula ferruginea was identified as the bee species establishing beehives on the highest diversity of plant species (i.e. 8 species), followed by m. togoensis and m. bocandei with 7 species each one. beehives of h. gribodoi were encountered on 6 plant species while those of a. mellifera were generally found established on trees of 4 plant species. however, most of the beehives of a. mellifera, m. togoensis and m. bocandei, were recorded in the cavities of dialium guinneense’s trees. in contrast beehives of h. gribodoi, were registered in high quantities on trees of manilkara multinervis. no beehive of this bee species was observed on dialium guinneense while the highest number of beehives of m. ferruginea was found on trees of cynometra megalophylla. relationship between the diameter at breast height of trees, the nesting choice of bees and the honey exploitation by chimpanzees bee species recorded in this study were found nesting in cavities of trees with a dbh ranging from 15 to 87.3 cm. meliponula bocandei was found nesting in trees with a high dbh (mean dbh: 52.5 cm ± 3.6, n=15) while h. gribodoi nested in trees with the lowest dbh (mean dbh: 39.6 cm ± 3.7, n=46). however, no significant difference was found between the mean dbh of nesting trees of all bee species (kruskal-wallis test: χ²=6.41, df=4, p>0.05). nevertheless, mann-whitney pairwise test showed a significantly difference in the dbh of nesting trees between a. mellifera and m. bocandei (u=37, p<0.05), between m. bocandei and h. gribodoi (u=54, p<0.05) and between m. togoensis and m. bocandei (u=90.5, p<0.05) (table s2). finally, no significant difference was found in the dbh of nesting trees with beehives identified as exploited or not by chimpanzees (mann-whitney test: χ²=894.5, df=1, p>0.05) (table s3). the highest value of beehive’s height was observed in a. mellifera (6.3 m ± 1; n=19) followed by m. bocandei (5.9 m ± 0.7; n=15), m. ferruginea (5.3 m ± 0.7; n=21), m. togoensis (4.9 m ± 0.8; n=13) and h. gribodoi (3.8 m ± 0.3; n=46) (table s2). results also showed that chimpanzees have an exploitation preference of a. mellifera low height’s beehives. indeed, it is only in this species that a significant difference was found between the height of exploited beehives and non-exploited beehives (mann-whitney test: χ²=2, df=1, p<0.05) (table s4). status of nesting trees (dead or living) and nesting place on trees all the beehives of a. mellifera and m. ferruginea were found in cavities of living trees trunks, while only those of h. gribodoi beehives were observed in all the considered parts of trees. meliponula bocandei and m. togoensis were both found in only two parts of trees (i.e. trunks of living trees and dead trees) (table 4). table 4. relative abundance of bee species’ beehives found on living trees and dead trees. bee species relative abundance of beehives on parts of trees in brackets (%) trunks of living trees dead parts of living trees trunks dead branches of living trees dead trees apis mellifera 19 (100) 0 0 0 hypotrigona gribodoi 4 (8.89) 9 (20) 14 (30.43) 19 (42.22) meliponula bocandei 14 (93.33) 0 0 1 (6.67) meliponula ferruginea 21 (100) 0 0 0 meliponula togoensis 11 (84.62) 0 0 2 (15.38) na soro, j lapuente, na koné, k yéo, s konaté – bee assemblages in the home range of savanna chimpanzees486 discussion in this study, we found that chimpanzees of the comoé national park exploited all the five species of arboreal social bees which were recorded in three forest types surveyed during the present study. however, there was some evidence the stingless bees (h. gribodoi, m. ferruginea, m. bocandei, m. togoensis) are more exploited than the honeybees (a. mellifera). this observation could probably be explained the by the aggressive defense strategy adopted by the honeybees to secure their beehives and honey. indeed, african honeybees are notorious for defending their beehive through aggressive stinging – the stings are too painful for many animals that they can’t withstand for long time (hodgson et al., 2010). in contrast, stingless bees are recognized to mainly build passive defenses for their beehives such as thick walls of mud and wax. the main strategy they use to make their beehives inaccessible is to build them up to one meter underground (tornyie & kwapong, 2015). however, none of these passive defense systems were found in the stingless bees of the cnp. indeed, they only build thin wax wall or used thin cracks as entrance to their arboreal beehives on one hand or buildt heir beehives on high height branches (personal observation). in the stingless bees of the cnp, only h. gribodoi was seen with an aggressive behavior against the disturbance of their beehives. this species was not found stinging but workers invade the attacking by targeting all his orifices such as ears, eyes, mouth, nostrils (case of a disturbance caused by human) in order to destabilize him (personal observation). a mean density of 2.4 beehives/ha was found within the surveyed habitats. this density was found higher than those reported before in brazil (siqueira et al., 2012) and ghana (tornyie & kwapong, 2015). however, we can assume that the observed range of densities (i.e 1.4 2.4 beehives/ ha) was underestimated, due to human error in detecting beehives. indeed, the detection of bee beehives in natural ecosystems, especially in forests is difficult and may affect the assessment of their abundance as suggested by dorazio and connor (2014). moreover, the bee beehives’ density within habitat seems to depend on the habitat type, the availability and diversity of potential nesting trees. in fact, high beehives densities were found in forests with a high plant specific richness (e.g. high floral resource for bees) and large dbh trees (i.e. the forest island). in contrast, a relatively low density of beehives was observed in the gallery forest which was found mainly dominated by one plant species (cynometra megalophylla). these results are in line with those of eltz et al. (2002) and roubik (2006), who showed that the uneven distribution of bee species across habitats may be explained by various factors including food resources for bee foraging and the availability of suitable nesting places. in the present study, this is well demonstrated by the highest beehive density and the important number of chimpanzees’ honey exploitation tools found in the mature forest island in comparison to the other habitats. finally, one of the important reasons of this observed density can be the adaptability of bee species to habitat types. indeed, hypotrigona gribodoi and m. ferruginea had the highest number beehives in the surveyed habitats, with m. ferruginea as the most exploited bee species by the cnp’s chimpanzees. these observations can be explained by the fact that this species build beehive types adapted to various type of habitats including disturbed ones (hamisi, 2016). in addition, the high number of tools found under the beehives of m. ferruginea can also be explained by wide distribution of this species (e.g. high abundance within habitats). moreover, h. gribodoi was identified as the species with the highest monospecific nesting aggregation on the same tree, suggesting (i) the absence of an intra-specific nesting competition in this species (nkoba et al., 2012), (ii) the poor ability of this species in locating new nesting places by its scout bees (eltz et al., 2003) or (iii) its short flight ability and dispersion across fragmented habitats (araújo et al., 2004). as reported by cortopassi-laurino et al. (2009), the present study revealed that the most used trees used by the bees to beehive in cnp belong to the leguminosae’s family. in contrast, nkoba et al. (2012) identified the family euphorbiaceae as the mostly nested plant species by stingless bee species in kakamega forest. at the plant species level, dialium guinneense, manilkara multinervis and cynometra megalophylla were found with the highest number of beehives in their cavities. the highest beehive number was found on dialium guinneense probably due to the structure of this tree. the suitability of this species as nesting substrate may be related to relatively medium size of their trunks which very often have cavities. similar tendency was reported by kajobe (2007) where m. ferruginea seemed to have some selectivity preferences for parinari excelsa. in fact, as suggested by hubbell and johnson (1977), bee species are opportunistic in selecting nesting place and generally use tree species presenting cavities with correct dimensions and purpose. however, our result contrasts with nkoba et al. (2012), who observed a little selectivity within four meliponini species in the kakamega forest in kenya for a nesting trees preference. arboreal bee species, in the present study, were found nesting in cavities of trees with a dbh range from 15 to 87.3 cm, in agreement with the results of several authors (darchen, 1972; eltz et al., 2003; venturieri, 2009; tornyie & kwapong, 2015). bee species were found choosing nesting trees based on the presence of cavities in the trunk and the size of the dbh of the plant species. furthermore, the size of these cavities and their respective location on trees were found determinant in the establishment of beehives. however, the exploitation of beehives by chimpanzees was not plant species dbh size dependent. some bee species (a. mellifera and m. bocandei) were found nesting very high on trees. on the other hand, h. gribodoi had the lowest mean height of beehives and also a lowest number of honey exploitation tools found under its beehives. this is probably due to the small size of this species on one hand and the difficulty in locating the small cavities of its beehives. sociobiology 66(3): 480-490 (september, 2019) 487 most of the encountered bee species (m. bocandei, m. ferruginea, m. togoensis and a. mellifera) where found nesting preferentially in trunks of living trees; whiles the beehives of h. gribodoi were mostly found in dead parts of trees. however, beehives located in the cavities trees trunks were found more exploited by chimpanzees than those found on branches, probably due to the important quantities of honey generally found in trunks (michener, 2000). the choice of living or dead trees to beehive is may be a defense strategy of the colony. indeed, according to roubik (1989) and martins et al. (2004), a strategy of defense against predators and parasites, guarantee of a longevity of colonies, in some meliponini is the establishment of beehives on living trees. in conclusion, beehives density within habitats was found depending on the habitat type, the availability and diversity of the nesting trees. arboreal bee species were found choosing their nesting trees based on the presence of cavities in the trunk and the size of the dbh of the plant species. frequent exploitation of stingless bee’s honey by chimpanzees was observed, except for h. gribodoi. meliponula ferruginea was the most exploited species by chimpanzees in the comoé national park. these primates don’t have a particular choice for beehives when we consider the dbh of trees nesting but they tend to exploit a. mellifera nests with low height. complementary researches are needed to assess the influence of the quantity and quality of honey on one hand and the beehive’s structure on the other hand, in their exploitation choice by chimpanzees. data should also be collected on the involvement of the five bee species in the pollination of trees (e.g. production of fruits consumed by chimpanzees), through the melissopalynology. acknowledgements this work was partly supported by a grant of the rufford foundation (id 24051-1) and the comoé chimpanzee conservation project. we also would like to thank the agency in charge of ivorian parks and reserve (the so called oipr) for the authorization access and the research permit in the comoé national park. we are grateful the anonymous reviewers for the comments and edits on a first version of this manuscript. thank you to our field assistants (sylvain, ibrahim, moussa and abou) and to all the staff of comoé national park research station in ecology (koffi, lakado, richard, david and inza) for their support during our stay. authors’ contributions nicodénin angèle soro: investigation, data collection, data curation and writing the original draft. juan lapuente: investigation, data collection, methodology, conceptualization, writing, review and editing. n’golo abdoulaye kone, kolo yeo and souleymane konate: conceptualization, methodology, writing, review and editing references araújo, e.d., costa, m., chaud-netto, j. & fowler, h.g. 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(2009). the impact of forest exploitation on amazonian stingless bees (apidae, meliponini). genetics and molecular research, 8(2): 684-689. family tree species mfi sfi gf total relative abundance (%) leguminoseae dialium guinneense willd., 1796 69/15/10 21/7/3 41/2/1 131/24/14 26.37 albizia adianthifolia (schumach.) w. wight, 1909 75/7/1 4/0/0 11/0/0 90/7/1 7.69 albizia zygia j.f. macbr., 1919 19/1/0 1/0/0 5/0/0 25/1/0 1.1 tamarindusindica l., 1753 7/3/3 1/1/1 0 8/4/4 4.4 tetrapleura tetraptera (schumach. & thonn.) taub., 1891 11/0/0 22/0/0 4/1/1 37/1/1 1.1 cynometra megalophylla l., 1753 5/1/1 0 973/5/5 978/6/6 6.59 ulmaceae celtisintegrifolia lam., 1983 119/10/0 28/0/0 36/2/1 183/12/1 13.19 liana 0 1 0 1 1.1 sapotaceae manilkara multinervis (baker) dubard, 1915 19/8/3 20/11/2 4/2/0 43/21/5 23.08 vitellaria paradoxa c.f. gaertn., 1807 1/1/0 0 0 1/1/0 1.1 combretaceae anogeissus leiocarpa (dc.) guill. & perr., 1832 105/4/0 40/0/0 9/0/0 154/4/0 4.4 moraceae antiaris toxicaria lesch., 1810 10/1/0 3/0/0 3/0/0 16/1/0 1.1 ficus ingens (miq.) miq., 1867 6/0/0 6/1/0 0 12/1/0 1.1 ficus sp. 17/1/0 9/0/0 0 26/1/0 1.1 verbenaceae vitex sp. 5/2/1 0 4/2/0 9/4/1 4.4 ebenaceae diospyros mespiliformis hochst. ex a.dc., 1844 42/1/0 61/0/0 14/0/0 117/1/0 1.1 sterculiaceae cola gigantean a. chev., 1908 7/0/0 36/1/0 9/0/0 52/1/0 1.1 total 516/55/19 252/22/6 1113/14/8 1881/91/33 100 p values 0.0005 0.001 0.002 abbreviations: mfi = mature forest island; sfi = secondary forest island, gf = gallery forest data in cells are reported as follows: total individual species-specific observed trees/ number trees on which bee nests were found/ number of trees on which bee nest exploited by chimpanzees were found. ps: the lianas were not taken into account in the total of individual species-specific observed trees and in the number of trees on which bee nest exploited by chimpanzees were found. the p values compare the total number of observed trees and the number of trees on which bee nest exploited by chimpanzees were found. table s1: relative abundance of plant species on which arboreal social bees’ beehives were observed. supplementary material na soro, j lapuente, na koné, k yéo, s konaté – bee assemblages in the home range of savanna chimpanzees490 table s2: characteristics of beehives and the nesting trees of the encountered bee species. bees species range of beehives heights (m) beehives meanheights (m) exploited beehives non-exploited beehives exploited beehives non-exploited beehives apis mellifera 1 5 2.5 15 2.1 ± 1 7.7 ± 0.9 meliponula togoensis 2.5 11 2 7 5 ± 1.7 4.8 ± 6.1 meliponula bocandei 3.5 7 1.2 15 6.8 ± 0.9 4.4 ± 1 meliponula ferruginea 2.7 14 2.3 9 5.9 ± 0.9 4 ± 0.9 hypotrigona gribodoi 2.5 5 0.6 7 4.8 ± 1.3 3.8 ± 0.3 bees species dbh range (cm) mean dbh (cm) range of beehives’ height (m) mean height of beehives (m) apis mellifera 31.2 51.6 39.9 ± 1.9 1 15 6.3 ± 1 meliponula togoensis 28.7 67.8 47.1 ± 5.4 2 11 4.9 ±0.8 meliponula bocandei 32.2 87.3 52.5 ± 3.6 1.2 15 5.9 ±0.7 meliponula ferruginea 15 75.5 45.9 ± 4.1 2.3 9 5.3 ± 0.7 hypotrigona gribodoi 19.7 60.5 40.1 ± 2 0.6 7 3.8± 0.3 table s3: range and mean of dbh of the exploited and non-exploited beehives in the recorded bee species. table s4: height of exploited beehives or not by chimpanzees. bees species dbh range (cm) mean dbh (cm) exploited beehives’ trees non-exploited beehives’ trees exploited beehives’ trees non-exploited beehives’ trees apis mellifera 39.5 51.6 31.2 49 45.6 ± 3.5 38.3 ± 2.2 meliponula togoensis 22.3 54.1 28.7 67.8 39.4 ± 0.8 51.95 ± 7.7 meliponula bocandei 32.2 87.3 41.4 65.3 52.2 ± 5.5 53.1 ± 4.1 meliponula ferruginea 28.7 68.8 15 75.5 47.2 ± 4.7 43.3 ± 8.6 hypotrigona gribodoi 19.7 47.8 21.7 60.5 33.8 ± 8.1 40.7 ± 2.1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3394sociobiology 65(4): 727-736 (october, 2018) special issue honey from stingless bee as indicator of contamination with metals introduction bees can carry contaminants from the environment to the beehive and consequently to honey, due to their foraging activity. when foraging areas for bees are polluted, several undesirable chemicals may be introduced into honey through nectar, pollen or sugary exudates from plants growing on contaminated soil and/or absorbing contaminated water. additionally, elements in the atmosphere also are important source of contaminants that may mix with the resources collected by bees (porrini et al., 2003; stecka et al., 2014; di et al., 2016). honey can be contaminated with inorganic chemical elements during raw material collection by bees or in the honey extraction process. moreover, different weather conditions, seasons and honey botanical origin are variables that affect of metal contents in bee products. as bee products are the final stage of a bioaccumulation process, chemical study on honey abstract melipona scutellaris latreille (apidae, meliponini) is one of the main species of stingless bees used in beekeeping in the northeast of brazil. we examined the honey of m. scutellaris as an indicator to evaluate the levels of metals at sampling sites subject to a broad spectrum of environmental pollutants. the collections were carried out in the urban-industrial area of salvador, bahia and the metropolitan region. samples (n = 58) were submitted to the nitroperchloric digestion procedure. we used the inductively coupled plasma optical emission spectrometry technique (icp oes) to determine the concentration of metals (cd, co, cr, cu, fe, mn, mo, ni, pb and zn) in the samples. the studied metals were detected among the samples, which presented tolerable levels according to current brazilian legislation and recommendations from the world health organization (who), except for cr, which presented mean values higher than the threshold for all sampling sites. the detection of the analyzed metals indicates that the honey of m. scutellaris is a useful tool to evaluate the presence of environmental contaminants; therefore, it can be considered a good indicator of environmental contamination for monitoring a particular region and preventing issues due to the release of metals into the environment. sociobiology an international journal on social insects as nascimento1, ed chambó2, dj oliveira1, br andrade1, js bonsucesso1, cal carvalho1 article history edited by cândida aguiar, uefs, brazil received 30 april 2018 initial acceptance 13 june 2018 final acceptance 17 august 2018 publication date 11 october 2018 keywords meliponini, melipona scutellaris, bioindicators, environmental pollution, icp oes. corresponding author andreia santos do nascimento universidade federal do recôncavo da bahia centro de ciências agrárias, ambientais e biológicas rua rui barbosa nº 710, centro cep 44380-000, cruz das almas-ba, brasil. e-mail: asndea@gmail.com can provide useful information on the environmental quality of the area where bees feed (pisani et al., 2008; stecka et al., 2014; silici et al., 2016). plants can accumulate metals in their tissues due to their ability to adapt to various chemical conditions in the environment. therefore, some species are considered accumulators of metals found in the soil, water and air (malavolta, 1994). the geographic and botanical origin, as well as anthropogenic factors near colonies are determinant for the presence of high concentrations of metals in bee products (bogdanov et al., 2007; silici et al., 2016). in this sense, the pollen analysis of honey in bee products is very important because it helps a better understanding of the levels of metals in samples from different sites. the presence of toxic metals in honey can threaten human health (ru et al., 2013). additionally, it can serve as an indicator of environmental pollution. thus, many studies 1 universidade federal do recôncavo da bahia, cruz das almas, brazil 2 universidade federal do amazonas, benjamin constant, brazil research article bees as nascimento et al. – stingless bee honey as indicator of contamination728 have investigated metal concentration in the body of bees and in their products (porrini et al., 2003; batista et al., 2012; pohl et al., 2012; bastías et al., 2013; aghamirlou et al., 2015; nascimento et al., 2015; martin et al., 2016; steen et al., 2016; zarić et al., 2016; bonsucesso et al., 2018). however, our study is the first carried out in an industrial urban area using honey of melipona scutellaris latreille, a stingless bee, as an indicator of environmental quality. bees react to changes in the environment they inhabit, especially in relation to amounts of toxic metals in the soil, air and plants. this characterizes bees as reliable indicators of environmental quality, allowing their use in biomonitoring (zhelyazkova, 2012). stingless bees (meliponini) have potential for use as indicators of environmental contamination with toxic metals (nascimento et al., 2015). these bees have an atrophied sting, which facilitates management (camargo et al., 2017), and are commonly reared in an urban environment, where there are many anthropogenic activities near the beehives, thus the bees are more exposed to loads of pollutants, especially in large urban centers. m. scutellaris (apidae, meliponini) is one of the main species used in the beekeeping activity in northeastern brazil (villas-bôas, 2012). it was the most frequent species in the region evaluated in this study and presented a satisfactory honey production, allowing its use for this research. this study used honey from m. scutellaris as an indicator to evaluate the levels of metals at sampling sites subject to a broad spectrum of environmental pollutants. material and methods study site the collections were carried out in urban-industrial area of salvador and metropolitan region, bahia, brazil (fig 1). the selected meliponaries were installed in urban (sites a-d), semi-rural (site e) and rural environments (site f), near the petrochemical complex of camaçari (site e-f), sanitary landfill of salvador and highways cia-aeroporto (site a-d), base naval road and ba 093 (site a) (table 1). the colonies were exposed to a very high range of pollutants that may be present in the atmosphere, soil and water. one month before sample collection, we prepared the colonies used in the experiment, leaving these colonies with empty honey pots, which allowed collection of honey stored by bees during the sampling period. fig 1. geographical location of sampling sites in salvador and the metropolitan region, state of bahia, brazil. sociobiology 65(4): 727-736 (october, 2018) special issue 729 a meliponary installed in a non-urban area was used to collect control samples, located in baía do iguape, cachoeira, bahia. the marine extractive reserve of the baía do iguape (currently known as iguape) is a federal conservation unit in brazil categorized as an extractive reserve (resex) in the state of bahia. sites municipality environment anthropogenic influence meliponary a salvador 12º51’32.4’’ s 038º27’9.90’’ w urban area near (~ 600 m distance) highway base naval, with intense traffic of vehicles meliponary b salvador 12º49’58.7’’ s 038º22’27.4’’ w urban area region of the industrial center of aratu (cia), highway cia-aeroporto, with intense traffic of vehicles meliponary c salvador 12º51’28.3’’ s 038º21’54.3’’ w urban area region of the industrial center of aratu (cia), highway cia-aeroporto, with intense traffic of vehicles, near a landfill and unpaved road meliponary d lauro de freitas 12º50’38.1’’ s 038º21’12.1’’ w urban area metropolitan region of salvador, bahia. unpaved road, near (~ 800 m distance) an indian reserve meliponary e simões filho 12º43’55.5’’ s 038º23’51.6’’ w semi-rural metropolitan region of salvador, bahia. farm located approximately 300 m from highway ba 093, with intense traffic of vehicles, near the petrochemical complex of camaçari, bahia meliponary f dias d’ávila 12º32’28.0’’ s 038º21’42.3’’ w rural metropolitan region of salvador, bahia. farm located approximately 8 km from highway ba 093 with intense traffic of vehicles meliponary g cachoeira 12º38’25.3’’ s 38º51’42.0’’ w non-urban area federal conservation unit of brazil. marine extractive reserve of baía do iguape (control) table 1. description of sampling sites in salvador and the metropolitan region, bahia, brazil. thermo scientific icap 6000 series, model 6300 duo. table 2 presents the analysis conditions of icp oes. accuracy of the analytical method was evaluated in terms of repeatability of experimental results of real samples (triplicate) and expressed as standard deviation of the mean. accuracy was verified by calibration (using standard solutions). pollen analysis to determine the botanical origin of honey, samples were prepared according to the methodology described by jones and bryant jr. (2004) and later submitted to the acetolysis process of erdtman (1960). the resulting pellet was mounted on slides for microscopy, followed by the identification and counting of the pollen grains that compose the pollen spectrum of the sample. the pollen types were identified using specialized literature, such as barth (1989), roubik and moreno (1991), punt et al. (2007) and consulting database and images of the palinoteca of the universidade federal do recôncavo da bahia, brazil. frequency class of each pollen type was determined according to louveaux et al. (1978), classified as: predominant pollen ( pp> 45% of total grains), secondary pollen (sp-16 to 45%), important minor pollen (imp – 3 to 15%) and minor pollen (mp < 3%). sampling the samples (n = 58), composed each of approximately 250 g of honey, were collected directly from the colonies of m. scutellaris with disposable syringes, placed in properly identified sterile plastic containers. the sampling period was one year, between august 2014 and july 2015. preparation of samples we used the method of nitro-perchloric digestion proposed by malavolta et al. (1989) in the preparation of samples to identify the metals. we used 2 g of each honey sample, the evaluations of each sample being performed in triplicate. all the glassware used was placed in a 10% nitric acid solution (hno3) for 24 h for decontamination, after which all the material was washed with ultrapure water (18.2 mω.cm). we used a standard solution (blank solution) containing only acids, which was submitted to the same procedures for the digestion of honey samples. for the analyses, reagents of certified analytical grade were used. determination of metal concentration metals cadmium (cd), copper (cu), lead (pb), cobalt (co), chromium (cr), iron (fe), manganese (mn), molybdenum (mo), nickel (ni) and zinc (zn) were selected for this study because of their importance as environmental contaminants. to determine the concentration of metals in the samples, the inductively coupled plasma optical emission spectrometry (icp oes) technique was used in the icp (spectrometer) as nascimento et al. – stingless bee honey as indicator of contamination730 m et al sa m pl in g si te s b ra zi lia n l eg is la tio n* w h o * (m g/ kg /d ay ) a (n =9 ) b (n =8 ) c (n =1 0) d (n =1 2) e (n =9 ) f (n =1 0) g (n =1 0) c on tr ol m ea n± sd m ea n± sd m ea n± sd m ea n± sd m ea n± sd m ea n± sd m ea n± sd c da 0. 00 10 ±0 .0 03 0 <l d <l d <l d <l d <l d <l d 0. 50 00 0. 00 70 c oa 0. 00 20 ±0 .0 03 0 0. 00 69 ±0 .0 07 8 0. 00 60 ±0 .0 01 4 0. 00 38 ±0 .0 03 5 0. 00 10 ±0 .0 01 5 0. 00 38 ±0 .0 03 6 <l d c ra 0. 28 06 ±0 .3 62 1 0. 34 26 ±0 .2 99 7 0. 48 49 ±0 34 77 0. 51 68 ±0 .4 75 0 0. 55 13 ±0 .5 03 3 0. 30 97 ±0 .1 15 2 <l d 0. 10 00 0. 00 00 3 0 .0 00 13 c ub 0. 29 36 ±0 .1 58 0 0. 33 35 ±0 .1 65 9 0. 45 63 ±0 .1 28 1 0. 36 67 ±0 .1 20 3 0. 31 96 ±0 .1 44 7 0. 66 88 ±0 .3 86 9 <l d 10 .0 00 0. 05 0 0 .5 00 0 f ea 1. 21 08 ±0 .8 48 8 2. 48 29 ±1 .0 37 7 1. 88 02 ±0 .4 81 3 1. 53 11 ±0 .5 98 3 2. 95 85 ±1 .9 32 8 1. 48 01 ±1 .0 83 9 3. 60 00 ±0 .8 00 0 m nb 0. 26 29 ±0 .0 91 6 0. 42 74 ±0 .0 86 9 0. 60 60 ±0 .4 75 5 0. 24 05 ±0 .0 78 5 0. 77 82 ±0 .3 32 3 1. 20 35 ±0 .4 94 4 <l d m ob 0. 02 26 ±0 .0 37 1 0. 00 63 ±0 .0 11 7 0. 20 71 ±0 .1 18 0 0. 16 98 ±0 .1 44 6 0. 28 18 ±0 .1 75 6 0. 13 95 ±0 .0 55 3 <l d n ia 0. 01 52 ±0 .0 08 7 0. 01 37 ±0 .0 08 1 0. 01 45 ±0 .0 10 4 0. 02 41 ±0 .0 19 8 0. 01 76 ±0 .0 06 0 0. 01 79 ±0 .0 06 1 <l d 5. 00 00 0. 50 00 p ba 0. 00 07 ±0 .0 02 0 <l d <l d <l d <l d <l d <l d 0. 50 00 0. 05 00 z na 0. 40 00 ±0 .1 99 4 0. 91 66 ±0 .5 04 2 0. 70 44 ±0 .7 66 3 0. 84 28 ±0 .5 87 2 0. 90 49 ±1 .1 14 3 0. 92 78 ±0 .5 06 3 <l d 50 .0 00 0. 30 0 1 .0 00 0 a n o si gn ifi ca nt d if fe re nc es a m on g th e se ve n si te s by k ru sk al -w al lis te st . b si gn ifi ca nt o ve ra ll di ff er en ce a m on g si te s by k ru sk al -w al lis te st . l d = li m it of d et ec tio n, w he re : c d, c u an d m n (l d = < 0. 00 5 m g/ kg ); c o, c r, m o, n i a nd z n (l d = < 0. 00 2 m g/ kg ); f e an d pb (l d = < 0. 00 1 m g/ kg ) *b ra si l ( 19 65 ; 1 99 8; 2 00 9) a nd r ec om m en de d in ge st io n by w h o (w or ld h ea lth o rg an iz at io n) (w h o , 1 98 2; 1 99 1; 19 93 ; 1 99 6) . t ab le 3 . m ea ns (m g/ kg ) a nd s ta nd ar d de vi at io ns o f m et al c on ce nt ra tio ns d et ec te d in h on ey s am pl es o f m el ip on a sc ut el la ri s fr om d if fe re nt s ite s. parameters icp oes conditions of analysis power rf 1150 w nebulization flow 0.70 l/min auxiliary gas flow 0.50 l/min internal standard ítrio (y) integration time and reading 15 s purity of gas (argon) 99.999% metal wavelength (nm) cd 226.5 axial co 228.6 axial cr 267.7 radial cu 324.7 radial fe 259.9 radial mn 257.6 radial mo 202.0 axial ni 231.6 axial pb 220.3 axial zn 213.8 axial table 2. conditions of analysis in icp oes for quantification of metals in honey. data analysis first, the mean and standard deviation for all metals investigated and detected at each site were calculated. subsequently, the data were submitted to the shapiro-wilk and levene tests to verify the residue normality and homogeneity of the variances, respectively. the data did not present the assumptions for one-way anova. thus, we chose to analyze the data by the kruskal-wallis nonpanametnic rank analysis of variance (h statistic) to establish an overall difference between the seven sites, and then by post hoc bonferronidunn test with adjustment of p-values for the pairwise multiple comparison of mean ranks. statistical significance was set at p < 0.05. all analyses were carried out in triplicate. the “r” statistical and programming environment version 3.0.2 (r core team, 2015) was used. results the metals studied were detected in the samples and presented tolerable levels according to current brazilian legislation and recommendations of the world health organization (who) (brasil, 1965, 2009; who, 1982; 1983; mercosur, 2011), except for cr that presented mean values higher than that established for all sampling sites (table 3). honey samples analyzed in this study presented cd and pb values below the detection threshold in 98% of the samples (table 3), both detected only in one sample, cd in meliponary a and pb in meliponary b. sociobiology 65(4): 727-736 (october, 2018) special issue 731 the highest mean concentrations among the samples of the studied areas were recorded for fe, zn, mn, cr and cu. as expected among control samples, concentrations of the metals evaluated were below the detection limit, except for fe that presented a mean of m = 3.6000, sd = 0.8000 mg/kg. the kruskal-wallis test statistics was significant for cu (χ2(5) = 13.84; p = 0.02), mn (χ2(5) = 43.13; p = 0.0001), mo (χ2(5) = 34.59; p = 0.0001). there were no differences in the cd (χ2(5) = 2.33; p = 0.80), pb (χ2(5) = 6.25; p = 0.28), zn (χ2(5) = 9.65; p = 0.09), ni (χ2(5) = 4.85; p = 0.44), cr (χ2(5) = 5.28; p = 0.38) between sites (table 3). although the kruskalwallis test showed some differences for fe (χ2(5) = 14.37; p = 0.01) and co (χ2(5) = 12.30; p = 0.03) between sites, these possible differences were not found when adjusting the probability values for pairs of multiple comparisons. the mean rank of cu (mg/kg) was significantly greater in site f compared with site a (p = 0.03). the mean rank of mn (mg/kg) was significantly different between sites a and e (p = 0.003) and a and f (p = 0.0001). the mean rank of mn (mg/kg) was also significantly greater in site c compared with site d (p = 0.02) and different from sites e (p = 0.0001) and f (p = 0.0001). there was no difference in mo between sites a and b (p = 1.00) (table 4). additionally, in the determination of the botanical origin of samples, we identified 85 pollen types, distributed in 27 families. in all sampling sites, the fabaceae family showed the highest number of pollen types, representing 33% of the total, followed by the families anacardiaceae (9%), arecaceae (9%) and euphorbiaceae (6%). the honeys analyzed were classified as multifloral with expressive representation of pollen types of the family fabaceae, and the mimosa caesalpiniifolia type occurred among samples from all the studied sites. sample, the concentration determined for cd was 0.0050 mg/ kg. current standards for honey (brasil, 2009) establish a threshold of 0.5000 mg/kg for this metal. the world health organization (who) suggests as tolerable limit of daily intake of cd 0.0070 mg/kg for adults with 70 kg of body weight (who, 1993). in the sample where this metal (cd) was detected, the pollen analysis showed that it is multifloral honey. however, the pollen type mimosa caesalpiniifolia (fabaceae) occurred as secondary pollen (sp -16 to 45%) and mimosa quadrivalvis and mimosa tenuiflora (fabaceae) as minor pollen (mp <3%). note that types m. quadrivalvis and m. tenuiflora were identified only in the august sample of meliponary b. this may have contributed to cd detection in this sample, since species of fabaceae may be cd accumulators, according to barbosa et al. (2017). in addition, climatic conditions may have contributed to the detection of cd levels in the sample evaluated. cd is released into the environment through various industrial processes and enters the food chain from absorption by contaminated soil plant or water. thus, cd concentration in distinct areas depends on many variables, leading to a variation in the concentration in samples of different honey origin (naccari et al., 2014; aghamirlou et al., 2015; silici et al., 2016). this variation in concentration also occurred in our study for the evaluated metals and can be observed in the different sampling sites (table 3). perugini et al. (2011) found that cd levels were significantly higher in urban areas than in non-residential areas. aghamirlou et al. (2015) and silici et al. (2016) also reported a similar result. the samples evaluated in our study were obtained in industrial urban area and rural did not present high concentrations of the cd in the sample in which they was detected, indicating that the concentration of metals is variable and depends on factors such as botanical and geographical origin of the sample and climatic conditions. the lead metal (pb) in our study was also detected only in a sample of the meliponary b also collected in august 2014, in this sample concentration determined for pb was 0.0075 mg/kg. current standards for honey (brasil, 2009) establish a threshold of 0.5000 mg/kg for this metal. the who suggests as tolerable limit of daily intake of pb 0.0500 mg/kg for adults with 70 kg of body weight (who, 1993). lead was the metal that presented the lowest concentration in a study by di et al. (2016). morgano et al. (2010) reported different results by comparing pollen stored by bees, another hive product, from semi-rural environments and high-emission pollutants sites of automotive vehicles in brazil, where they observed very high pb concentrations (0.44 mg/kg). in our study, pb was detected in only one sample from site a, this meliponary is located near a highway where there is intense traffic of motor vehicles. considering that pb has limited accumulation in plants and that even in contaminated soils this metal is not readily bioavailable to plants (hladun et al., 2015, davies et al., 2003), sites mean ranks () cu mn mo a 19.7a 13.9ab 11.5a b 24.6ab 29.6abc 8.4a c 36.1ab 33.9ac 39.6b d 28.3ab 10.6b 33.5b e 22.3ab 43.6c 43.5b f 43.5b 49.1c 35.1b * different letters in the same column indicate significant differences (p <0.05) according to the bonferroni-dunn test. table 4. mean ranks ( jr ) of metals in honey samples from seven different sites. discussion although many metals can be sequestered and transferred to the body of bees and their products, those usually detected are cd and pb (satta et al., 2012; hladun et al., 2015). in our study the cd was only detected in a sample of the meliponary a collected in august 2014. in this as nascimento et al. – stingless bee honey as indicator of contamination732 its low concentration is justified in floral nectar, raw material for honey production. in addition, nectar is less exposed to environmental contamination from other sources such as air (silveira et al., 2013). this may have a correlation with pb concentration below the detection limit in 98% of the samples analyzed in our study. chromium (cr) presented a mean concentration ranging from 0.2806 (sd = 0.3621) to 0.5513 (sd = 0.5013) mg/kg. the presence of this element in the samples at concentrations above the limit stipulated by the brazilian legislation (brasil,1965) can be related to anthropogenic factors in the sites of origin of the samples, which are mostly located in an urban-industrial environment (table 1 and fig 1). cr is a natural element in the earth’s crust and is released into the environment by natural and anthropogenic sources. companies most contribute to cr release from metal processing, tanning facilities, chromate production, stainless steel welding and electroplate iron production. the population is exposed to cr by inhalation, food intake and drinking water contaminated with this metal (atsdr, 2012a). contamination by cr in honey probably comes from the load of pollutants in the atmosphere that can contaminate honey directly through, nectar or honeydew or the bees add them involuntarily. metals in the air can be deposited on bee body when during foraging activities; thus, they can be carried to the hive (porrini et al., 2003; bogdanov et al., 2007; orioan et al., 2016). the mean values of cr found by bogdanov et al. (2007) were higher in samples collected in the city (0.010 mg/kg) than in rural areas (0.004 mg/kg). according to the authors, the cr content in honey depends on climatic conditions and differences in the cr content in the different sample areas are attributed to environmental or geographic factors. zn concentration in the honey samples analyzed presented mean values ranging from 0.4000, (sd = 0.1994) to 0.9278 (sd = 0.5063) mg/kg (table 3). the maximum permissible concentration is 50.00 mg/kg for zn according to decree no. 55871/65 (brasil, 1965). however, in brazil, there are no updated guidelines on acceptable zn levels, specific for honey. the who establishes 0.30 to 1.00 mg/kg as the tolerable limit for daily intake (who, 1982). aghamirlou et al. (2015) reported that zn was the most abundant metal in all honey samples, with a mean of 148 μg/kg (ranging from 122 to 6638 μg/kg) and cu, cr, cd, pb and ni were also detected and presented lower means in comparison to zn. iron (fe) was the chemical species with the highest concentrations; however, in compliance with the limits required by the who (table 3). fe is considered a contaminant mainly in the oxide and hydroxide forms. fe is an essential metal and required by all life forms. in the human body, fe has important functions in metabolism, such as protein synthesis and oxidative metabolism. the mean daily fe intake for men is estimated at 17.00 mg and for women at 9-12.00 mg (who, 1983). acute toxicity of fe ingested from distinct dietary sources is not reported, and idiopathic hemochromatosis is a disease characterized by long and slow accumulation of fe in tissues without evidence of excessive dietary intake (who, 1983). copper (cu) was detected in samples from all sites (except in meliponary g control) with a mean concentration lower than 10.00 mg/kg, which is the maximum concentration established by ordinance no. 685/98 (brasil, 1998), presenting mean values that ranged from 0.2936 (sd = 0.1580) to 0.6688 (sd = 0.3869) mg/kg between samples (table 3). the mean rate of cu was significantly higher in site f compared to site a (table 4). meliponary a is located in an urban setting while site f is in a rural area. lower concentrations of cu in meliponary f was expected because it is far from the urban perimeter. however, this can be explained by the probable high pollutant load emitted by motor vehicle traffic from highway ba 093 that is approximately 8 km from this sampling site (table 1 and fig 1). in addition, as it is a rural area with cultivation of cocus nucifera l. (arecaceae), manihot esculenta crantz. (euphorbiaceae), musa spp. (musaceae), passiflora edulis sims. (passifloraceae) and zea mays l. (poaceae), farmers can adopt measures of pest control with agrochemicals. cu may be present in honey due to the use of agrochemicals (fungicides) in agriculture and concentration this metal varies according to the botanical origin, geographical and anthropogenic activities (orioan et al., 2016). in the honey samples of meliponary f, the pollen type cocus nucifera had a relative frequency of 50.00% and the euphorbiaceae type occurred as predominant pollen (pp = 45.20%), indicating that bees may have collected material contaminated with cu, which reveals the importance of considering the botanical origin of samples. families arecaceae and euphorbiaceae are the most representative of 27 botanical families identified in the pollen spectrum of the samples evaluated. detection of cu in the samples of all meliponaries studied is possibly because they are located in an environment with a certain anthropization degree, such as proximity to roads, a petrochemical center and industrial areas. urban areas are overloaded with pollution from various sources. vehicle traffic is one of the main pollution sources in urban centers (silva et al., 2014). the presence of cu in urban areas is generally explained by anthropogenic and industrial activities, and brake pads from motor vehicles are considered a significant cu source (zarić et al., 2016). cu is a vital element for the health of living beings. however, excessive intake of cu can cause adverse effects to human health. therefore, it is necessary to consider the daily intake of cu from different food sources, and daily intake for cu of 0.05 to 0.5 mg/kg of body weight is tolerable (who, 1982; aghamirlou et al., 2015). cobalt (co) was detected in honey samples with concentrations ranging from 0.0006 (sd = 0.0014) to 0.0069 (sd = 0.0078) mg/kg. co is a natural element found in rocks, sociobiology 65(4): 727-736 (october, 2018) special issue 733 soil, water and plants. this metal is used to produce alloys used in manufacture of aircraft engines, cutting and roughing tools. co compounds are also used in the industry to color glass, ceramics and paints (atsdr, 2004). therefore, co in the samples evaluated may come from natural sources of the environment and from anthropogenic activities. the tolerable limit for co in water is 0.050 mg/l established by resolution no. 357 of the national council of the environment – conama, published in march 2005. brasil (2009) does not establish a limit for this metal in honey. among the metals evaluated and detected in the samples, trace elements such as cu, fe, mn and zn are important for human metabolism and are required in small quantities (who, 1996; atsdr, 2012a; reynaud, 2014). thus, detection of these metals at a tolerable level (except cu) indicates that these samples do not present toxic levels that make their consumption impossible, considering only these metals. to ensure honey quality for human consumption, other complementary analyses are necessary, such as determination of physicochemical and microbiological parameters. manganese (mn) was detected in the samples (except meliponary g control) with mean concentrations ranging from 0.2405 (sd = 0.0785) to 1.2035 (sd = 0.4944) mg/kg. mn occurs naturally in many types of rocks and soils. mn is mainly used in steel production and processing, along with cast iron and superalloys. mn can also be used as a fuel additive for automotive vehicles, such as gasoline. mn in additives rapidly degrades the environment when exposed to sunlight, releasing this metal. in this way, its presence in the environment and in the products of the hive can come from natural sources or anthropogenic activities. emphasizing that manganese is a trace element and is necessary for good health (atsdr, 2012b). meliponaries e and f are respectively of semi-rural and rural environments and presented the highest mean concentrations of mn. as the soil is a natural mn source and many urban soils are no longer similar to original natural soils of a given site and may present lower availability of macro and micronutrients (burghardt et al., 2015), higher mn concentrations from meliponaries e and f are related to the greater mn availability in soils of rural and semi-rural areas. the tolerable limit for mn in water is 0.100 mg/l established by resolution no. 396 of the national council of the environment – conama, published in april 2008. brasil (2009) does not establish a limit for this metal in honey. molybdenum (mo) was also detected in samples of all meliponaries (except in meliponary g control), with the mean concentrations ranging from 0.0063 (sd = 0.0117) to 0.2818 (sd = 0.1756) mg/kg. bonsucesso et al. (2018) conducted a study in the same sampling site of our research and reported that mo was also detected in samples of m. scutellaris geopropolis of all meliponaries. the tolerable limit for mo in water is 0.700 mg/l established by resolution no. 396 of the national council of the environment – conama, published in april 2008. atsdr (2017) recommends ingestion of 0.045 mg/kg. data on toxicity of mo in humans are limited. the association between mo effects and thyroid diseases is indicated (atsdr, 2017; yorita & christensen, 2013). the variation (0.0137 (sd = 0.0081) to 0.0179 (sd= 0.0061) mg/kg) in the mean concentration of nickel (ni) in honey samples (table 3) may be related to several factors, such as nickel source and distance from the contamination source. ni is present in air, water and soil and is usually evenly distributed throughout the soil profile (aghamirlou et al., 2015). all samples presented ni levels within the threshold (5.00 mg/kg) established by the brazilian legislation (1965). batista et al. (2012) compared the concentration of metals in brazilian honeys to that in other countries and observed that honeys in brazil present higher average concentrations for al, mg and ni and lower average concentrations of cd, cu and pb. additionally, the authors found that the mean values for p, zn, mn and fe were very similar to those found for honey samples from other countries. our results were similar to those found by batista et al. (2012). metal concentration in honey and other hive products is associated to floral origin, agricultural practices and soil characteristics, as well as environmental factors, such as pollution, which vary depending on the geographical location (porrini et al., 2003; bogdanov et al., 2007; silici et al. 2016). in a study on soil samples and geopropolis (bee stingless propolis) of m. scutellaris, bonsucesso et al. (2018) found that most contents of the metals investigated in geopropolis had the soil as source. in addition, other external sources can contribute to the increase of metal concentration in geopropolis. similar to our study, the authors verified that both soil and geopropolis had low contamination with metals, although the samples were obtained mainly from an urbanindustrial region. as metal concentration in honey can be related to its floral origin and the analyzed honey samples presented in its pollen spectrum a greater representation of the family fabaceae (33% of total pollen types), this botanical family has some species that are considered hyperaccumulators of toxic metals. possibly, there is some metal accumulator species among the identified pollen types of fabaceae, as these metals can be absorbed by the plants and accumulated mainly in the roots and shoots (vasconcelos et al., 2012). barbosa et al. (2017) and souza et al. (2013) cited some examples. therefore, the appropriate choice for the site to install the meliponary with the bee colonies needs to be considered to avoid areas very close to contamination sources due to anthropogenic activities. malavolta (1994) highlighted that the useful life of toxic metals in the soil varies from 70-510 years for zn, 13-1100 years for cd, 300-1500 years for cu and 740-5900 years for pb. therefore, if present in the soil, these metals can be absorbed by plants that constitute the bee flora. the detection of the metals analyzed indicates that honey of m. scutellaris is a useful tool to evaluate the presence of as nascimento et al. – stingless bee honey as indicator of contamination734 environmental contaminants and can therefore be considered a good indicator of environmental contamination and be used to monitor a certain site to prevent problems caused by the release of metals into the environment. the samples in general presented tolerable levels of metals according to the current brazilian legislation and recommendations of the world health organization. acknowledgements this study was financed in part by the “coordenação de aperfeiçoamento de pessoal de nível superior brasil” (capes) finance code 001 and by the “fundação de amparo à pesquisa do estado da bahia” (fapesb) finance code pam0004/2014. we thank the “conselho nacional de desenvolvimento científico e tecnológico” (cnpq) by fellowship for calc (number 305885/2017-0). as nascimento wishes to thank capes for the postdoctoral fellowship pnpd20130760. authors’ contribution as nascimento, dj oliveira and js bonsucesso designed the study and interpreted the data. authors ed chambó performed statistical analysis. br andrade elaboration of figure 1 (map). authors as nascimento, cal carvalho, ed chambó, dj oliveira br andrade, and js bonsucesso participated in study conduction and data interpretation. author as nascimento drafted the manuscript. all authors read and approved the final manuscript. references aghamirlou, h.m., khadem, m., rahmani, a., sadeghian, m., mahvi, a.h., akbarzadeh, a. & nazmara, s. 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(2012). honeybees – bioindicators for environmental quality. bulgarian journal of agricultural science, 18: 435-442 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3370sociobiology 65(4): 603-611 (october, 2018) special issue diversity of flower visiting insects in dry grasslands and vineyards close to the city of vienna with special focus on wild bees introduction interactions between flowering plants and flowerpollinating insects belong to the most significant in the terrestrial synecology (richards, 1978; proctor et al., 1996). diversity and local distribution resp. commonness or rarity of both plants and insects are strongly dependent on the development and integrity of these relationships (e.g. fontaine et al., 2006). in the surrounding of the city of vienna open semidry grasslands are one of the most important hot spots of biodiversity for both plants and insects (adler & mrkvicka, 2003). but their part in the landscape is comparatively small, and patches of grassland are in most cases strongly isolated from each other by the surrounding forests, settlement abstract interactions between flower visiting insects and nectar resp. pollen producing plants belong to the most relevant in terrestrial ecosystems. their diversity and dominance relationship are important indicators for the stability and functionality of ecosystems and belong to the high ranking ecosystem services. potential pollinators should be strongly concerned especially regarding anthropogenic impacts on habitats. we studied the diversity and quantities of flower visiting insects with special focus on wild bees (apiformes) in two locations near the city of vienna (austria). insect sampling occurred from may until july 2015 every two weeks parallel to the vegetation surveys incl. records of the cover of flowering plants. in each location patches of semi-natural grassland as well as flowering strips within vineyards were investigated. we found a significant correlation between the number of insects or insect taxa (especially for hymenoptera) and the current flower cover. in some cases flowering strips in vineyards harbor higher numbers of insects and higher diversity of bee species than the semi-natural grassland due to temporarily higher values of flower cover. however, grassland patches provide a much more constant supply with nectar producing plants replacing each other in their flowering phase during the season. in contrast, flowering strips are often dominated by one or a few short-lived sown plants, which is of advantage for some oligolectic bees specialized on brassicaceae or fabaceae. flowering strips within organically farmed vineyards are more similar to semi-natural grassland regarding the diversity of flower visiting insects than to conventionally farmed vineyards. sociobiology an international journal on social insects l rasran, a diener, b pachinger, k-g bernhardt article history edited by kleber del claro, ufu, brazil cândida aguiar, uefs, brazil received 25 april 2018 initial acceptance 28 june 2018 final acceptance 13 july 2018 publication date 11 october 2018 keywords organic farming, flower cover, flower strips. corresponding author leonid rasran institute of botany university of natural resources and life sciences vienna gregor-mendel-str 33, a-1180 vienna, austria. e-mail: leonid.rasran@boku.ac.at infrastructure and agricultural landscapes including arable fields and vineyards. fragmentation and isolation of biotopes lead to the reduction of their functionality as habitat and dispersal corridors for insects (tscharntke et al., 2002; garibaldi et al., 2011). moreover, intensive agriculture presumes the application of pesticides, which either leads to the reduction of insect diversity and quantity. one of the expected consequences could be pollination limitation (reduction of fruit setting rate due to insufficient pollination, calvo & horvitz, 1990; jennersten & nilsson, 1993; larson & barrett, 2000). lack of pollinators is relevant not only for plant species diversity and population stability of rare and endangered semi-dry and dry grassland herbs, but also for the entire agricultural production and has a clear economical value (klein et al., 2007; neumayer, 2011). university of natural resources and life sciences vienna, austria research article bees mailto:leonid.rasran@boku.ac.at l rasran; a diener; b pachinger; k-g bernhardt – pollinator diversity in dry grasslands and vineyards604 bee species (apiformes) are especially sensitive to the decline of insect-pollinated plants in the landscape (biesmeijer et al., 2006) and thus are good indicators for the stability and integrity of plant/pollinator interactions and ecosystem services (burkle et al., 2013). the cultural landscape of low mountains around the city of vienna (parts of vienna forest, so called wiener hausberge leopoldsberg, nussberg, bisamberg) was significantly changed in the last decades. in some parts agricultural use (mainly in form of vineyards) was strongly intensified, while at the same time formerly moderately used grassland areas became abandoned and are now covered by shrubs or forest. in both cases the attractiveness of areas for pollinators has been reduced. some authors (see e.g. haaland et al., 2011) express the expectation, that flower strips within or besides the vineyards could serve as important step-stone biotopes and feeding grounds for bees and other plant pollinating insects. in these functions and ecosystem services flower strips would complete or even replace the species-rich grasslands formerly present in the area in high percentage. however, flowering strips are artificial sites with significant differences to (semi-) natural grasslands. following critical points can be mentioned in this context: (i) seed mixtures for flower strips usually include only a few dominant, but short-lived species which offer nectar and pollen for a rather narrow, restricted time span (van elsen et al., 2007; rundlöf et al., 2014); (ii) the seed mixtures for flower strips often include alien species or genetic varieties, which are either less adapted to local conditions or are potential invaders with a negative impact for the resident biota (keller et al., 2000); (iii) flower strips are involved in agricultural production and are affected by the application of pesticides and mineral fertilizers together with the wine rows. mowing of the flower strips is also linked to the demands of crop production, which means that mowing occurs earlier and more frequently than in the grassland and shorten significantly the flowering period of plant species and their offer of nectar and pollen (bruggisser et al., 2010). regarding the mowing time, significant differences could be expected between traditional and ecological viticulture, both present in the study area. the present study focuses on the comparison between semi-natural grassland patches and flower strips in vineyards. we recorded the floristic diversity, vegetation structure and flower cover (as a cue for pollen respectively nectar availability). parallel, we analyze the quantities and species composition of flower-visiting insects with special focus on wild bees. following research questions can be worded: i. how far does the flower cover correlate with diversity and quantity of flower visiting insects? ii. are there significant differences in composition of insect communities in general and bee communities in particular between semi-natural grasslands and flower strips? iii. are there significant differences between flower strips within traditional and ecological viticulture regarding the pollinator communities? material and methods study areas the present study was carried out at two low mountains on the outskirts of the city of vienna (austria): bisamberg north of the danube (n 48° 18` e 16° 22`, 358 m a. s. l.) and leopoldsberg/nussberg south (n 48° 16` e 16 21`, 425 m a. s. l.). these mountains belong to the most ne-part of vienna forest and build together the so called “vienna gate” of the danube valley. the south-exposed slopes of both mountains are covered by semi-dry deciduous forests (downy oak forests), different types of semi-dry calcareous grasslands (mesobromion) and vineyards. we selected ten plots per area (bisamberg – five grasslands and five vineyard flower strips; leopoldsberg/nussberg – three grasslands and seven vineyard flower strips). on bisamberg all vineyard flower strips and three of five grassland plots were situated within the research farm “götzhof” (federal department of viti and pomiculture), where ecological agricultural methods are tested. the vineyards and associated flower strips on nussberg are managed in a traditional way. the study areas are about 5 km away from each other and within the area all study plots were situated within the circle of about 1200 m diameter. the study plots were of different shape dependent on the landscape from 10 x 10 m squares in grassland to 1.5 x 66.6 m strips between vine rows, but of the same size of 100 m². the current management and the composition of seed mixtures for flower strips are listed in table 1. vegetation and insect surveys each study plot was monitored six times from may to july 2015 in a two week interval. the monitoring included an observation of flower-visiting insects performed by one person with a sampling time of 15 minutes by patrolling the plot (linear or zigzag dependent on plot configuration) and capturing the insects with help of an entomological hand-net. all captured insects were immediately counted. individuals, which could not be easily identified in the field, were killed with ethyl acetate and preserved for further identification. the focus insect group of bees (apiformes) and some easily recognizable coleoptera and macrolepidoptera were determined down to species level, while at some groups (e.g. diptera) we had to deal with morphospecies. bee species were categorized according to their food and nesting preferences following scheuchl and willner (2016). nomenclature of bee species follows gusenleitner et al. (2012). insect monitoring took place in the daytime and only in good weather (temperature above 15°c, no precipitation). subsequent to the insect record a vegetation relevée (scale based on cover percentages of single species) after londo (londo, 1976) was performed for the study plot, while besides cover of plant species their contribution to flower cover was estimated. entomophilous plants with currently open blossoms were seen as relevant sociobiology 65(4): 603-611 (october, 2018) special issue 605 table 1. description of the study sites at bisamberg (b1-b10) and at leopoldsberg/nussberg (l1-l10), habitat type (flower strip/grassland), plot shape (length x width; m), vegetation/land use, coordinates and exposition. all plots were of the same size of 100 m². habitat type plot size (m) vegetation/land use coordinates slope angle (°), exposition bisamberg b1 flower strip 5 x 20 spontaneous re-vegetation, lepidium drabadominant cover n48°18´47.7´´ e16°22´18.6´´ 20 s b2 flower strip 1.5 x 66.6 3-component-seed mixture* n48°18´45.7´´ e16°22´24.6´´ 10 s b3 grassland 10 x 10 mown in summer n48°18´45.2´´ e16°22´27.8´´ 8 s b4 flower strip 1.5 x 66.6 3-component-seed mixture* n48°18´45.2´´ e16°22´29.6´´ 8 s b5 flower strip 1.5 x 66.6 seed mixture “rebenfit”** n48°18´39.0´´ e16°22´28.1´´ 8 15 s b6 grassland 10 x 10 mown in summer n48°18´39.5´´ e16°22´26.5´´ 5 s b7 grassland 10 x 10 mown in summer n48°18´39.9´´ e16°22´20.7´´ 6 s b8 flower strip 1.5 x 66.6 seed mixture “rebenfit”** n48°18´39.7´´ e16°22´18.4´´ 6 s b9 grassland 10 x 10 mown in autumn/abandoned n48°18´45.8´´ e16°21´46.1´´ 2 3 s b10 grassland 10 x 10 mown in autumn/abandoned n48°18´46.9´´ e16°21´41.2´´ 15 18 s leopoldsberg/nussberg l1 grassland 10 x 10 mown in autumn/abandoned n48°16´29.4´´ e16°20´34.2´´ 12 e l2 grassland 10 x 10 mown in autumn/abandoned n48°16´29.6´´ e16°20´31.4´´ 8 e l3 grassland 10 x 10 mown in autumn/abandoned, orlaya grandiflora-dominant cover n48°16´35.1´´ e16°20´43.4´´ 40 s l4 flower strip 1 x 100 sown trifolium incarnatum, phacelia tanacetifolia, fagopyrum esculentum, sinapis alba, raphanus sativus and vicia sativa n48°16´07.9´´ e16°20´40.9´´ 4 10 ne l5 flower strip 10 x 10 sown trifolium incarnatum, camelina sativa, centaurea cyanus, trifolium repens, medicago sativa, medicago lupulina and plantago lanceolata n48°16´01.9´´ e16°20´54.6´´ 1 2 ne l6 flower strip 1.5 x 66.6 sown sinapis alba, raphanus sativus subsp. oleiferus, phacelia tanacetifolia, melilotus officinalis, vicia sativa, vicia pannonica, calendula officinalis and malva sylvestris n48°16´00.6´´ e16°20´57.5´´ 0 2 s l7 flower strip 1 x 100 sown melilotus officinalis, sinapis alba, raphanus sativus subsp. oleiferus, trifolium incarnatum, vicia pannonica, malva sylvestris, phacelia tanacetifolia, trifolium pratense, trifolium alexandrinum and cichorium intybus. n48°15´58.8´´ e16°21´03.7´´ 4 10 s l8 flower strip 10 x 10 sown medicago sativa n48°15´56.4´´ e16°21´10.9´´ 8 10 s l9 flower strip 1.5 x 66.6 sown trifolium repens n48°15´47.5´´ e16°21´22.0´´ 2 12 s l10 flower strip 1.5 x 66.6 sown medicago sativa n48°15´48.4´´ e16°21´26.6´´ 2 12 s *3-component-seed mixture* includes phacelia tanacetifolia, fagopyrum esculentum and trifolium incarnatum. ** seed mixture "rebenfit", includes camelina sativa, trifolium incarnatum, trifolium repens, medicago lupulina, plantago lanceolata and centaurea cyanus. l rasran; a diener; b pachinger; k-g bernhardt – pollinator diversity in dry grasslands and vineyards606 for flower-visiting insects. some species, normally seen as wind-pollinated (e.g. plantago lanceolata (l.)) were counted to the flower cover as well due to the potential importance of their pollen as food for insects (e.g. sharma et al., 1993). determination and nomenclature of plant species was according to fischer et al. (2008). we studied the relationship between species richness respectively quantities of insects and characteristic of study plots (incl. flower cover of different plant groups) applying linear regression and analysis of variances (anova). a repeated measurement anova was calculated to study the effect of observation time. additionally, factor “time” was included in a three-way anova together with the factors “area” and “habitat” (grassland vs. flower strips) to study the interactions between them . a canonical correspondence analysis (cca) was applied to put the entire species composition of bee communities in relation to land use and flower aspect. the statistical analyses were performed using the software package r 2.15.2 (r development core team, 2015). multivariate analyses were performed using canoco 5 (smilauer & leps, 2014). results vegetation and flower cover semi-natural grasslands on both study areas included 28 to 48 species of flowering plants per 100 m² (without poaceae). the plant species diversity on the flower strips was significantly lower, not above 20 species, in most cases about 10. the estimated flower cover, however, reached similar values for both grassland and flower strips and in some cases was even higher on the last mentioned (table 2, fig 1). most significant for the flower cover of strips was the contribution of sown species such as phacelia tanacetifolia benth., trifolium incarnatum l. or sinapis alba l., but some spontaneously established ruderal species such as lepidium draba l. could locally reach a high flower density. the flower cover of grassland plots was mainly polydominant, but strong contribution of one single species in a particular point of time (e.g. orlaya grandiflora (l.) hoffm. on one of the plots on leopoldsberg) was also observed. the temporal variation of the flower cover was especially high at flower strips (table 2, fig 1). generally, the flower cover tended to reduce during the recording period from may to july. flower strips between vine rows were mown usually once (ecologically managed flower strips on bisamberg) or twice (traditionally used flower strips on nussberg) during the observation period. in some cases vegetation could rapidly recover from these effects and developed high flower cover for the second time, but there was a significant time span, where the flower offer for pollinators was lowered. grassland patches were usually mown in late summer (after the observation period was finished) or remain unmown. dependent area time treatment (grassland vs. flower strips area x time area x treatment time x treatment area x time x treatment no. of plant species 3.6 ns 1.78 ns 41.71*** 0.1 ns 23.81*** 0.07 ns 0.15 ns sum flower cover 0.01 ns 6.07 *** 2.63 ns 2.77* 6.61* 1.61 ns 4.18** flower cover brassicaceae 0.01 ns 5.78*** 7.3** 0.84 ns 0.12 ns 2.14 ns 1.29 ns flower cover asteraceae 1.16 ns 0.96 ns 21.54*** 0.69 ns 11.66*** 1.02 ns 2.44* flower cover apiaceae 2.23 ns 0.42 ns 8.28 ** 1.32 ns 3.69 ns 0.82 ns 1.98 ns flower cover fabaceae 7.9** 1.19 ns 1.29 ns 0.87 ns 4.18* 0.22 ns 0.45 ns no. of insect species 1.64 ns 1.3 ns 6.52* 1.27 ns 28.84*** 1.31 ns 2.15 ns no. of insect ind. 8.17** 2.62* 4.07* 3.39** 12.04*** 2.49* 3.66** hymenoptera species 0.91 ns 0.24 ns 0.12 ns 1.41 ns 6.65* 1.37 ns 1.46 ns hymenoptera ind. 6.74* 1.86 ns 5.68* 3.05* 4.78* 2.37* 2.18 ns coleoptera species 4.33* 1.44 ns 4.98* 1.61 ns 20.44*** 1.27 ns 1.49 ns coleoptera ind. 4.09* 1.59 ns 0.05 ns 1.27 ns 12.76*** 0.86 ns 2.29 ns lepidoptera species 0.36 ns 2.07 ns 35.08*** 2.38* 20.27*** 2.72* 1.17 ns lepidoptera ind. 2.4 ns 1.81 ns 31.38*** 1.74 ns 20.11** 2.59* 2.14 ns diptera species 2.89 ns 3.89** 0.12 ns 2.38* 22.65*** 2.95* 3.08* diptera ind. 0.01 ns 2.56* 0.44 ns 3.02* 23.08*** 3.65** 5.92*** table 2. differences of plant species diversity, flower cover, flower visiting insects and insect groups between the investigated locations (bisamberg, leopoldsberg/nussberg) and habitats (flower strip and grassland) as well as their interactions. three-way full factorial anova. values of fisher-distribution (f-value) are shown. significant effects are highlighted and marked as follows: * p<0.05, ** p<0.01 and *** p< 0.001. ns – not significant. sociobiology 65(4): 603-611 (october, 2018) special issue 607 flower visiting insects in general during the study entirely 201 (morpho-) species of flower-visiting insects were recorded. the most species-rich insect group was hymenoptera with 62 species (incl. 41 species of apiformes), followed by diptera (48), lepidoptera (45), coleoptera (31 species), hemiptera (14) and neuroptera (1). the number of insect species per record was similar between different sites. the only slight significant differences could be seen between land use forms (table 2). semi-natural grassland possessed higher insect diversity, than flower strips. individual numbers (quantities) of insects recorded on study plots differ both between areas and land use forms and fluctuate strongly in time (repeated measurements anova, f = 2.97 p < 0.05), while these fluctuations have different rhythms in different treatments (table 2). the quantities of insects were significantly correlated with the flower cover (ad. r² = 0.325, f = 56.92, p < 0.001), but not with the diversity of plant species (ad. r² = -0.005, f = 0.35, n.s.). bee communities we identified entirely 41 bee species (apiformes) during the study, 14 of them occurring only in grassland, 19 only in flower strips within vineyards and 8 in both habitat types (table 3). number of bee species was significantly correlated to the plant species diversity per plot (ad. r² = 0.134, f = 19.42, p < 0.001), while number of individuals was clearly dependent on the entire flower cover (ad. r² = 0.261, f = 43.00, p < 0.001). most numerous were apis mellifera (l.), bombus lucorum (l.) / bombus terrestris (l.) and bombus lapidarius (l.). honey bees and large earth bumblebees reached the highest individual numbers on phacelia-dominated flower strips during its flowering time. regarding their pollen specification, the majority of detected bee species can be considered as polylectic, but there are also some specialists / oligolectic species. as specialists on flower strips species using brassicaceae (andrena agilissima (scop.) and andrena floricola (ev.)), apiaceae (andrena nitidiuscula fig 1. temporal dynamic of total flower cover in percent in relation to the total number of flower visiting insects, separated by location (bisamberg vs. leopoldsberg/nussberg) and habitat type (flower strip/ grassland). year of recording: 2015. mean and mean +/standard error is shown. (schenck)), asteraceae (colletes similis (schenck)), fabaceae (eucera nigrescens (pérez) and melitta leporina (panz.)) as well as convolvulus-species (systropha curvicornis (scop.)) could be detected. at grassland patches there were mainly specialists feeding on asteracea (heriades crenulatus (nyl.), hylaeus nigritus (fab.), megachile pilicrus (mor.) and osmia spinulosa (kir.)) and on ranunculaceae (chelostoma florisomne (l.)). there were also some differences in the bee species composition between habitats regarding their nesting behavior. the most common category were terricolous (ground nesting) species, wide spread on all sites. in contrast, cavity nesting could be mainly found on grassland sites (9 species) and only two of them on flower strips. all three species nesting in snail shells were only found in semi-natural grassland, among them the rare and in austria endangered species anthidium septemdentatum (latr.). l rasran; a diener; b pachinger; k-g bernhardt – pollinator diversity in dry grasslands and vineyards608 flower strips grassland pollen source nesting behavior andrena agilissima (scopoli, 1770) x brassicaceae t andrena chrysosceles (kirby, 1802) x polylectic t andrena floricola eversmann, 1852 x brassicaceae t andrena haemorrhoa (fabricius, 1781) x polylectic t andrena minutula (kirby, 1802) x polylectic t andrena minutuloides perkins, 1914 x polylectic t andrena nitidiuscula schenck, 1853 x apiaceae t anthidium septemdentatum latreille, 1809 x polylectic hs apis mellifera linnaeus, 1758 x x polylectic bombus hortorum (linnaeus, 1761) x polylectic t/h bombus hypnonum (linnaeus, 1758) x polylectic t/h bombus lapidarius (linnaeus, 1758) x x polylectic t/h bombus lucorum/bombus terrestris x polylectic t/h bombus pascuorum (scopoli, 1763) x polylectic t/h chelostoma florisomne (linnaeus, 1758) x ranunculus h colletes similis schenck, 1853 x asteraceae t eucera nigrescens pérez, 1879 x x fabaceae t halictus quadricinctus (fabricius, 1776) x x polylectic t halictus rubicundus (christ, 1791) x polylectic t halictus sexcinctus (fabricius, 1775) x polylectic t halictus simplex blüthgen, 1923 x x polylectic t halictus tumulorum (linnaeus, 1758) x polylectic t heriades crenulatus nylander, 1856 x asteraceae h hylaeus communis nylander, 1852 x polylectic h hylaeus gredleri förster, 1871 x polylectic h hylaeus nigritus (fabricius, 1798) x asteraceae h lasioglossum malachurum (kirby, 1802) x polylectic t lasioglossum marginatum (brullé, 1832) x x polylectic t lasioglossum nigripes (lepeletier, 1841) x polylectic t lasioglossum pauxillum (schenck, 1853) x x polylectic t lasioglossum politum (schenck, 1853) x polylectic t lasioglossum leucozonium (schrank, 1781) x polylectic t megachile pilicrus morawitz, 1877 x asteraceae h megachile rotundata (fabricius, 1787) x polylectic h melitta leporina (panzer, 1799) x fabaceae t nomada succincta panzer, 1798 x p p osmia bicolor (schrank, 1781) x polylectic hs osmia spinulosa (kirby, 1802) x asteraceae hs sphecodes gibbus (linnaeus, 1758) x x p p systropha curvicornis (scopoli, 1770) x convolvulus spp. t xylocopa violacea (linnaeus, 1758) x polylectic h table 3. distribution of bee species (apoidea) between the habitats (flower strips in vineyards vs. grassland), pollen source and nesting behavior after scheuchl & willner, 2016 (p cleptoparasite, h cavity breeder, t ground-nesting, t/h ground-nesting and cavity breeder, hs breeding in snail shells). sociobiology 65(4): 603-611 (october, 2018) special issue 609 the species composition and quantity of bee communities have a good explanatory value between the studied habitats (cca, aj. expl. variation was 39.26 %, axis 1 – 18.09 %, axis 2 – 16.68 %, pseudo-f on all axes =1.8, p = 0.013). in the ordination diagram bee communities clustered mainly in three groups corresponding to semi-natural grasslands (upper left part), flower strips in traditionally managed vineyards (right-hand part) and ecologically managed vineyards together with regularly mown grasslands (downer left part; fig 2). indeed, our study showed that semi-natural pannonian grasslands remain a higher diversity and higher quality habitat for insects, especially bee species, in comparison to farmland. even under pollinator-friendly conditions flower strips include a narrow selection of nectarand pollen-offering plant species, not including those relevant for foraging specialists (see wood et al., 2015). in our case flower strip sown mixtures contained no ranunculaceae and only a little amount of asteraceae-species, while these plant families were strongly represented in grassland patches. also wild growing apiaceae (o. grandiflora) and brassicaceae (lepidium draba) dominated locally some grassland patches and were important attractors for various insect species. seed mixtures showed higher cover of fabaceae (especially t. incarnatum, medicago sativa (l.)) and p. tanacetifolia, a plant not present in grassland. these species together with brassicaceae (s. alba) led flower strips within vineyards to provide very high values of flower cover. the entire number of insects, mainly generalists foraging on these species, was comparable or even higher, than on grasslands, especially mown (traditionally used) ones (see also aviron et al., 2011). generally, due to high quantities of flower visiting insects flower strips should be able to provide ecosystem (pollination) services for the surrounding landscape (korpela et al., 2013). further differences between grasslands and flower strips for bee species were manifested regarding the structure of microhabitats. this include the effects of nesting behavior (availability of cavity including snail shells) or in general the flower-richness of the surrounding and landscape heterogeneity (rundlöf et al., 2008; spiesman, 2017). cultivation of vine rows and associated flower strips destroy small cavities, necessary as nesting habitats for bees. semi-natural grasslands for their part are more suitable habitats for small snails, than flower strips. bee species dependent on these structures occur mainly or solely at undisturbed dry grasslands. differences between flower strips in traditional and organic vineyards are manifested in significant effects of area x treatment interaction, as vineyards of bisamberg were organic ones and of nussberg/leopoldsberg the traditional ones. flower cover of the strips differed only slightly between the two agricultural forms. but temporal dynamics (due to 2x cut of intensively used strips vs. 1x cut in ecological farming) differed more clearly. similar to holzschuh et al. (2007) we also found that the differences in insect (and especially bee) diversities and quantities could be to a significant part explained by the differences in flower cover. remarkable are the similarities between moderately used grasslands and flower strips in organic managed vineyards (compare holzschuh et al., 2010). besides the similarities in management (1x mowing in mid-summer) avoidance of pesticide use could be a good explanation for the high species diversity there compared to traditional farming (e.g. thompson, 2001, 2003). attraction of wild bee species and further pollinators (e.g. hoverflies) is an important improvement of pollination services of flower strips for the neighbored crops (campbell et al., 2017). fig 2. constrained ordination (cca) of studied plots (without consideration of time dynamics). vegetation cover (veg_cov), total flower cover (flo_cov) as well as flower cover of single plant groups: ranunculaceae (ran), lamiaceae (lam), asteraceae (ast), apiaceae (api), fabaceae (fab), convolvulus (conv) & phacelia (pha) were plotted as passive variables. b location bisamberg, l – location leopoldsberg/nussberg, seminat – semi-natural grassland, mown – intensively used grassland, ecol – flower strips in organic vineyards, int flower strips in traditional vineyards. bee species with the highest explanatory values are shown (see table 3). following species names are shortened: and_mino andrena minutuloides, bom_hyp bombus hypnonum, che_flo chelostoma florisomne, hali_tum halictus tumulorum, las_pol lasioglossum politum. discussion as expected, the flower cover was the most significant predictor for quantities of insects in general and for the most common / generalist flower visitors in particular (potts et al., 2003; holland et al., 2015). fluctuations of flower cover caused immediately changes at insect numbers. l rasran; a diener; b pachinger; k-g bernhardt – pollinator diversity in dry grasslands and vineyards610 conclusions flower strips in vineyards are able to provide food for high quantities of flower-visiting insects, comparable to seminatural grasslands of the surrounding. especially strips within ecologically managed vineyards are comparable to moderately used (mown) grassland patches. the diversity of insect and explicitly of bee species was however higher at grassland sites due to food and nesting specialists, dependent on them. the development of ecologically managed flower strips can thus provide the important ecosystem service of pollination in the landscape, but protection of grassland patches is either crucial for the preservation of biodiversity. acknowledgments we would like to thank dr. reinhard eder and stefan lassl from the research farm “götzhof” for their help and important information. dr. kati vogt and two anonymous reviewers helped us with their remarks to improve the manuscript. the research was supported by the university foundation of the city of vienna (hochschuljubiläumsstiftung der stadt wien). references adler, w. & mrkvicka, a.ch. 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(2015). pollinatorfriendly management does not increase the diversity of farmland bees and wasps. biological conservation, 187: 120126. doi: 10.1016/j.biocon.2015.04.022 doi: 10.13102/sociobiology.v62i3.422sociobiology 62(3): 340-346 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 population dynamics of acromyrmex crassispinus (forel) (hymenoptera: formicidae) and attacks on pinus taeda linnaeus (pinaceae) plantations introduction ants belonging to attini tribe cultivate a symbiotic fungus inside their nests. foragers of atta and acromyrmex genera cut fresh fragments of leaves and other plant parts and carry them to their nests to be used as substrate for the fungus cultivation (de fine licht & boomsma, 2010). leaf-cutting ants (atta and acromyrmex) are among the most polymorphic of all social insects and show an extensive division of labor among size castes. the division of labor among different ant worker castes improves efficiency and benefits the colony as a whole (hölldobler & wilson, 1990). leaf-cutting ants have a particularly complex foraging process involving individual scouts and mass recruitment (jaffe & howse, 1979), mass traffic flows involving tens of thousands of outbound and returning workers (fowler et al., 1986), and processing of leaf fragments by workers in the nest before they take them to the fungal gardens. foraging is conducted by medium to large size abstract this work aimed to study the population dynamics of acromyrmex crassispinus (forel) in pinus taeda l. plantations, evaluating the density and spatial distribution of nests over time, inferring about the period of the first nuptial flight of a. crassispinus colonies, and evaluating the levels of attack of this leaf-cutting ant on p. taeda plants. assessments were performed monthly in the first year after planting, every three months until the third year and every six months until the plantation was six years old. the presence of nests was observed only after 15 months after planting. the nest density gradually increased until the planting completed 30 months, and decreased when the forest canopy began to close (after 54 months). spatial distribution of a. crassispinus nests was random. probably, the first nuptial flight of an a. crassispinus colony occurred after the third year of the colony foundation. pinus taeda plants were not attacked by a. crassispinus throughout the evaluation period. then, when dealing with a replanting area of pinus plantation, where the previous forest has not been subject to pruning nor thinning, the problem with a. crassispinus is almost null if the clearcutting and the new planting occur during the winter. in this case, leaf-cutting ants control can be alleviated and it is not necessary to carry out systematic control of ants where a. crassispinus is the predominant leaf cutting ant species. acromyrmex crassispinus control should be done only if nests are located or if attacked plants by ants are detected. sociobiology an international journal on social insects ma nickele1, w reis filho2 article history edited by kleber del-claro, ufu, brazil received 21 may 2014 initial acceptance 21 september 2014 final acceptance 25 september 2014 keywords control, leaf-cutting ants, nest density, nuptial flight, spatial distribution. corresponding author mariane aparecida nickele universidade federal do paraná departamento de zoologia, caixa postal 19020, cep 81531-980 curitiba, pr, brazil e-mail: manickele@ufpr.br workers that cut and collect small vegetation fragments, which they transport along trails to the nest. in the nest, this material is further processed by smaller workers and incorporated into the fungus garden (weber, 1972, hölldobler & wilson, 1990). leaf-cutting ants are possibly the most important herbivores of terrestrial environments throughout the neotropics, leading to considerable economic losses to a variety of crops. these ants are considered the most important pest of forest plantations in brazil due to their intense and constant attacks on plants at all stages of development. however, the age of plants can influence the vulnerability of the forest plantation to the damage caused by these ants. seedlings planted in infested areas by leaf-cutting ants have no chance of survival, as they can be easily destroyed by the ants (cherrett, 1986; vasconcelos & cherrett, 1997). leaf-cutting ants are usually controlled by applying insecticides from different chemical groups and different formulations (granulated baits, fogging, and dust) (boareto & forti, 1997). however, these products cause serious environmental research article ants 1 universidade federal do paraná, curitiba, pr, brazil 2 epagri/embrapa florestas, colombo, pr, brazil sociobiology 62(3): 340-346 (september, 2015) 341 problems because they are toxic to non-target animals and persist in the environment (ying & kookana, 2006). the information about insect densities in cultivated areas is essential, because it allows inferences about the potential damage of the species (pereira et al., 1999). the knowledge of the density and spatial distribution of pests is also essential to perform control strategies, to improve sample technics, or to estimate economic losses of those insects (croft & hoyt, 1983, pereira et al., 1999). this study aimed to study the population dynamics of acromyrmex crassispinus (forel), the most common leaf-cutting ant species in southern brazil, in pinus taeda l. plantations. evaluating the density and spatial distribution of nests over time, inferring about the period of the first nuptial flight of a. crassispinus colonies, as well as, evaluating the levels of attack of this leaf-cutting ant on p. taeda plants. material and methods experiments were carried out in são mateus do sul city (25°58’56,33”s, 50°23’49,26”w, alt. 766 m) state of parana, brazil. recently-planted p. taeda plantations were selected (replanted areas, in other words, pinus was previously planted in the area). in the previous planting there were approximately 1.300 plants per hectare, and it has not been subject to pruning nor thinning. the clear cutting was done in june-july, 2007 and the new planting was done in august, 2007 (1.600 plants ha-1). three plots of one hectare without control against ants were demarcated (100x100 m). the number (density) and the size of acromyrmex nests in the total area of each plot were recorded. all nests were measured and the major axis length was taken as the approximated diameter of the nest. nests were categorized into three size classes: class i, up to 30 cm in diameter; class ii, between 31 and 60 cm, and class iii, more than 61 cm (nickele et al., 2009). the evaluations of density and size of nests were performed monthly in the first year after planting, every three months until the third year and every six months until the plantation was six years old (from september 2007 until august 2013). the location of acromyrmex nests in the total area of each plot was recorded. maps with the location of acromyrmex nests in each plot were made to study the spatial distributions. the evaluations of spatial distributions of nests were performed in the same period than the evaluations of density and size of nests (from september 2007 until august 2013). the total sampling area (30,000 m2) was subdivided into 192 sampling units of 156.25 m2, based in nickele et al., 2010. the data of the location of a. crassispinus nests in each evaluation were tested to see if they fitted poisson’s distribution (that represents the random spatial distribution), positive binomial distribution (that represents the regular spatial distribution) or negative binomial distribution (that represents the aggregated spatial distribution), following the expressions, as bliss and fisher (1953), davis (1993) and krebs (1989). the model fits the original data adequately when the observed and expected frequencies are in close proximity. such proximity was tested by the chi-square test ( 2χ ), given by: ,where: foi = observed frequency in class i; fei = expected frequency in class i; nc = number of classes in the frequency distribution. the number of degrees of freedom in the 2χ test is given by: d.f. = nc np – 1, where: np = number of parameters estimated in the sample. the test criterion adopted was that the fitting of the distribution under study should be rejected at the 5% probability level if: 2χ ≥ 2χ (nc np 1; α). to study the period of occurrence of the first nuptial flight of a. crassispinus, 10 nests were evaluated monthly during spring of 2009 and 10 nests were evaluated monthly during spring of 2010. we selected the largest nests present in the area. during the spring of 2009, sticky traps were installed around 10 nests to collect winged male or female ants. in the spring of 2010, we decided to open 10 nests looking for reproductive ants. all p. taeda plants in each plot (census) were evaluated, recording the number of plants that were attacked by leafcutting ants. the evaluations were performed monthly in the first year after planting, every three months until the third year and every six months until the plantation was six years old (from september 2007 until august 2013). results density and size of the a. crassispinus nests the presence of a. crassispinus nests was observed only from 15 months after planting (spring/2008), where there was one nest per hectare, in average (figure 1). nest density gradually increased in subsequent months, and 30 months after planting (summer/2010) there was, in average, 26 nests per hectare. however, a substantial decrease in nest density happened over time. at 54 months after planting, the forest canopy closed and at 72 months after planting, there was only 0.33 nests per hectare, in average. the few nests observed after 54 months after planting were located near tree gaps in the middle of planting. fig 1. density (mean ± standard deviation) of acromyrmex crassispinus nests in a pinus taeda plantation. são mateus do sul, pr, brazil, 2007-2013. ma nickele, w reis filho – population dynamics of acromyrmex on pinus plantations342 fig 2. frequency of acromyrmex crassispinus nests in a pinus taeda planting, by size class: class i, up to 30 cm in diameter, class ii, between 31 and 60 cm; and class iii, greater than 61 cm. são mateus do sul, pr, brazil, 2008-2013. until 18 months after planting, all nests were class i size (smaller than 30 cm in diameter) (figure 2). nests with class iii size (over 61 cm) were observed 27 months after planting. when nest density reached a peak (30 months after planting), 11, 53 and 36% of nests were classes i, ii and iii, respectively. at 72 months after planting, the only one nest found was class i size. spatial distribution of a. crassispinus nests until 24 months after planting, the spatial distribution of a. crassispinus nests fitted with the poisson distribution model, because the chi-square values were not significant. it represents random spatial distribution. the other models could not be tested due to insufficient frequency classes for the chi-square test, which was attributed to the low occurrence of nests in the sampled areas (table 1). months after planting period number of nests poisson positive binomial negativa binomial 2χ d.f. 1 2χ d.f. 1 2χ d.f. 1 1 sep/07 0 2 oct/07 0 3 nov/07 0 4 dec/07 0 5 jan/08 0 6 feb/08 0 7 mar/08 0 8 apr/08 0 9 may/08 0 10 jun/08 0 11 jul/08 0 12 aug/08 0 15 spring/2008 3 0,00006 ns 1 i i i i 18 summer/2009 2 0,00006 ns 1 i i i i 21 fall/2009 7 0,009 ns 2 i i i i 24 winter/2009 31 1,18 ns 2 i i 1,81 ns 1 27 spring/2009 47 0,41 ns 2 1,33 ns 1 1,49 ns 1 30 summer/2010 78 6,51 ns 3 8,39* 1 44,21* 2 33 fall /2010 50 3,83 ns 3 4,16* 1 0,72 ns 1 36 winter/2010 58 5,61 ns 3 6,05* 1 2,09 ns 1 42 summer/2011 15 1,74 ns 2 i i i i 48 winter/2011 14 0,07 ns 2 i i i i 54 summer/2012 11 0,04 ns 2 i i i i 60 winter/2012 3 0,00006 ns 1 i i i i 66 summer/2013 0 72 winter/2013 1 0,00005 ns 1 i i i i df: number of degrees of freedom of the chi-square test; * significant at 5% probability; ns: not significant at 5% probability; i: number of class insufficient for testing. table 1. chi-square test of adherence of observed frequencies to the expected poisson distributions, positive and negative binomial distributions for acromyrmex crassispinus nests in a pinus taeda plantation. sociobiology 62(3): 340-346 (september, 2015) 343 from 27 to 30 months after planting, there was adjustment with the poisson model. however, at 27 months there were also adjustments with the positive and negative binomial distribution. the best adjustment is represented by the frequency distribution with the lower value of the chi-square calculated. in this case, the best fit was with the poisson distribution, indicating random spatial distribution. (table 1). from 33 to 36 months after planting, there was adjustment to both, poisson distribution and negative binomial distribution. in this case, the best adjustment was to the negative binomial distribution, because it showed the lowest value of the chisquare calculated (table 1). it represents aggregated spatial distribution in this period. in these evaluations, there was a higher concentration of nests in an edge of the area. from 42 to 72 months after planting, there was adjustment with the poisson model again, indicating that the spatial distribution of a. crassispinus nests is random in most samplings. nuptial flights of a. crassispinus in the spring of 2009, winged male or female ants were not observed in the nests sampled. in the spring of 2010, winged ants were observed in 50, 20, 20 and 10% of the nests sampled in september, october, november and december, respectively (table 2). males emerge earlier than females. in several nests, while males were already adult, females were still in the pupal stage. the average diameter of nests monitored during the spring of 2010 was of 92.1 ± 18.51 cm (mean ± sd). the colonies with nest diameter below the average size contained no winged ants. in contrast, colonies with nest diameter greater than average size contained winged ants (table 2). after the nuptial flight, nests were with one aspect that they were languishing or dying. in the evaluation during the next station (summer/2011), all of these nests were empty, in other words, the colony probably emigrated or died (table 2). attacks of a. crassispinus on p. taeda plants during the first 15 months after planting there were no p. taeda plants attacked by ants in the plots evaluated. after this period, wherein a. crassispinus nests started to be located in the area, ants attacked some native plants or p. taeda plants that were growing between the planting lines. however, plants with commercial interest suffered no attacks of leaf-cutting ants throughout the observation period. discussion acromyrmex crassispinus is the most common leafcutting ant species in southern brazil (rando & forti, 2005). however, nest density and spatial distribution of this ant species have never been studied in the same area for a period longer than one year. in this study, a. crassispinus nests were located only when planting reached 15 months of age. in the previous rotation of the planting had not been performed any activity of pruning or thinning plants, which contributed to the lack of a. crassispinus nests, since the canopy was completely closed and without the direct sunlight within the planting, and absence of undergrowth, with a layer of dried needles covering the soil. these ants have preference for nesting in open areas (nickele et al., 2009). the clearcutting of the previous planting occurred during the winter of 2007, that is, before the period of nuptial flight of the ants, which occurs during the spring, to found new colonies (diehlfleig, 1993; reis filho & oliveira, 2002). when the area was replanted there were no ant nests and it is suspected that the a. crassispinus nests settled in the area from the spring of 2007, but as young colonies of acromyrmex are not easily located, they were observed only at 15 months after planting (spring 2008). this fact leads us to infer that when a. crassispinus nests started to be located, the colonies were more than one year old. young colonies of acromyrmex are not easily located, due to the size of its population. incipient nests of acromyrmex octospinosus (reich) have only 3-7 workers (fernandezmarin et al., 2003). in contrast, initial nests of atta spp. may contain approximately 100 workers when they were 3 months old and they can be easily located (autuori, 1942). the density of nests belonging to class i size (smaller than 30 cm) remained constant throughout the evaluation period, because of the nuptial flights that occurred in subsequent years. the establishment of a new nest starts with the nuptial flights, wherein the winged virgin females run away of their original nest and they are inseminated by one or more males. after mating, queens seek the most appropriate place to start building colony diameter of the nest (cm) sep/10 oct/10 nov/10 dec/10 summer/11 1 75 0 0 0 0 alive 2 80 0 0 0 0 dead/ emigrated 3 98 1 0 0 0 dead/ emigrated 4 82 0 0 0 0 alive 5 98 1 1 1 0 dead/ emigrated 6 62 0 0 0 0 alive 7 98 0 0 1 1 dead/ emigrated 8 98 1 1 0 0 dead/ emigrated 9 100 1 0 0 0 dead/ emigrated 10 130 1 0 0 0 dead/ emigrated table 2: occurrence of reproductive ants (winged male or female ants) in acromyrmex crassispinus nests. (0) absent; (1) present. ma nickele, w reis filho – population dynamics of acromyrmex on pinus plantations344 their nest (hölldobler & wilson, 1990). initial plantations are suitable areas for installation and establishment of queens shortly after the nuptial flight. queens seek sites destituted of vegetation to start building their nest (vasconcelos, 1990), because incipient colonies require some degree of insolation (jaffe & vilela, 1989) and because the first workers are smaller than the smallest workers produced by a mature colony and those workers forage efficiently in herbaceous plants (wetterer, 1994). the density of a. crassispinus nests gradually increased when planting completed 30-36 months of age, and decreased when the forest canopy began to close (after 54 months). these data are similar to results found by nickele et al. (2009) in rio negrinho and três barras, sc, brazil, where the density of a. crassispinus nests was lower in early development of p. taeda planting, almost doubling in three years old plantations and suffering a reduction when the forest was six years old. zanetti et al. (2000) found a similar situation in the nest density of atta spp. in eucalyptus plantations in joão pinheiro, mg, brazil. the reason for the occurrence of this situation is that in newly planted areas, new nests settle from the time at which the nuptial fligth occur. in the course of time, there is an increase in nest density in all size classes, because colonies grown and nuptial fligth occur every year. the fact that the nest density decrease when plants grown may be related to the closure of the forest canopy, which may hinder the installation of new colonies, or could be related to the low diversity of understory, in other words, the lack of plant resources for ant foraging (zanetti et al., 2000; nickele et al., 2009). at 60 months after planting, the nests were found near clearings and were class i and ii size. acromyrmex crassispinus showed the same behavior in a 6 years old plantation of p. taeda in três barras, sc city (nickele et al., 2009). the leaf cutting ants found in primary native forests also are concentrated in areas near clearings, where the sun and pioneer plant species are more available to the colonies (wetterer, 1994, farjibrener, 2001, peñaloza & farji-brener, 2003). the spatial distribution of a. crassispinus nests was random in most samples, similar to that described in nickele et al. (2010). the random distribution of leaf cutting ants also had been documented by zanuncio et al. (2002), caldeira et al. (2005) and cantarelli et al. (2006). the random distribution occurs when site conditions are similar, that is, in areas where there is homogeneity in temperature, soil type, cultural practices, and so forth. queens fall randomly in the areas and establish their nests anywhere in the forest (caldeira et al., 2005). in contrast, acromyrmex balzani (emery) and acromyrmex landolti forel have aggregated spatial distribution, in fragments of grassland. this type of distributions may reflect the habitat heterogeneity or can be relate to the strategy of colony foundation (sousasouto et al., 2013; silva junior et al., 2013). reproductive ants (winged males and females ants) only appear in the colonies at certain periods of the year to conduct the nuptial flight and founding new colonies (hölldobler & wilson, 1990). the first nuptial flight of atta occurs only after the colonies are 3 years old (autuori, 1941), being repeated every year after this period during the spring. the nuptial flight of acromyrmex species also occurs during the spring, however, it is not known how old the colony is when first nuptial flight occur. probably, the a. crassispinus colonies found in the experimental area had similar age to the age of the plantation. the clearcutting of the earlier planting occurred before the nuptial flight of the ants, suggesting that nests only settled in the area from the spring of 2007 (along with the new planting), since this leaf cutting ant species prefers nidificate in areas opened. the presence of reproductive ants in sampled colonies was observed only from the spring of 2010, that is, three years after planting. this suggests that the first nuptial flight of an a. crassispinus colony also occurs after the third year of the colony foundation, as occurs in atta species. colonies that performed the nuptial flight had nests size greater than 92 cm in diameter, showing that nest size is related to the age of the colony. in the first evaluations, all nests were less than 30 cm in diameter. however, over time nests were recorded with different size classes. all nests emigrated or died after the occurrence of the nuptial flight. the emigration of a. crassispinus colonies is very common, regardless of the colony size (nickele et al., 2009). a leaf cutting ant colony can emigrate after suffering some sort of disturbance, such as intoxication with formicide baits, floods, lack of resources for foraging or intra and interspecific competitive interactions (nickele et al., 2012a). probably, in this study there was the emigration of the a. crassispinus colonies after the nuptial flight, because it is assumed that it is more advantageous to this ant species build a new nest than to reform the old one, since a. crassispinus builds superficial nests with a single camera located in a shallow excavation. colony death is unlikely, since acromyrmex queens can live for up to 10 years, at least under laboratory conditions (weber, 1972). the fact is that the colonies disappeared after the nuptial flight, but it is unclear what happened to the colony. this subject deserves further investigation to clarify aspects that are still unclear. there were no p. taeda plants attacked by ants in the plots evaluated. when the a. crassispinus nests began to be observed in the area and began foraging with greater intensity, p. taeda plants were already more than one year old, that is, they were no more so vulnerable to acromyrmex attacks. in addition, there were several options of native plants between the pine rows after one year of planting. so, ants preferred foraging in plants derived from natural regeneration and they did not attack the plants of commercial interest, which is exotic. nickele et al. (2012b) found that higher percentages of acromyrmex attack in p. taeda plants occurs in the first months after planting, with larger impact in the first 30 days, in reform areas where earlier planting suffered pruning and thinning. in this case, a. crassispinus nests have been observed since the beginning of planting and the first months after planting, ants had no options of native plant resources for foraging due to soil preparation for planting (nickele et al., 2009). sociobiology 62(3): 340-346 (september, 2015) 345 another factor that contributed to ants do not attack plants after 15 months, is that older plants have a higher amount of chemical defenses against herbivores, being less susceptible to ant attacks than younger plants (farji -brener, 2001; mundim et al., 2009). when dealing with a replanting area of pinus plantation, where the previous forest has not been subject to pruning nor thinning, the problem with a. crassispinus is almost null if the clear cutting and the new planting occur during the winter. this occurs because the number of nests in the previous planting is very low or nonexistent, because this ant species prefers nidificate in open areas. if the new planting is done before the nuptial flight period, plants will not be as vulnerable to a. crassispinus attack when new nests settle and begin to forage with greater intensity. in this case, leaf-cutting ants control can be alleviated and it is not necessary to carry out systematic control of ants when a. crassispinus is the predominant species. acromyrmex crassispinus control should be done only if nests are located or if attacked plants by ants are detected. acknowledgements we thank nádia caldato, priscila strapasson and mila ferraz de oliveira martins for their assistance during field work. we also thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for granting fellowship to m.a.n, and mwv rigesa for granting the study area. references autuori, m. 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(2000). spatial distribuition of nests of the leaf-cutting ant atta sexdens rubropilosa (hymenoptera: formicidae) in plantationsof eucalyptus urophylla in brasil. sociobiology, 39: 231-242. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.3315sociobiology 65(3): 463-470 (september, 2018) differentiating iberoformica and formica (serviformica) with description of the sexual castes of formica (serviformica) gerardi bondroit, 1917 stat. rev. introduction iberoformica subrufa (roger, 1859) constitutes the monotypic genus iberoformica (tinaut, 1990) endemic to the iberian peninsula and the mediterranean coast of southern france (bernard, 1967). roger (1859) included this species into the genus formica together with formica cinerea mayr, 1853 and formica fusca linnaeus, 1758, thus, into the serviformica subgenus/fusca species group. bernard (1967) synonymized formica gerardi bondroit, 1917 and formica pyrenaea bondroit, 1918 with i. subrufa (= formica subrufa roger, 1859) despite its pilosity and the fact of being the most xerophilous species in the formica genus. he reasoned that types of these species were small, immature and seemed to be hybrids between i. subrufa and formica cunicularia latreille, 1798. subsequent references consider i. subrufa as a distinct valid species of formica (e. g. collingwood & yarrow, 1969). abstract a list of morphological characters to separate iberoformica and formica (serviformica) (f. fusca species group) is provided. sexual forms of formica gerardi bondroit are described based on iberian material and reinstated into the subgenus serviformica based on genetic data and morphological characters. the status of †f. horrida wheeler, 1915 is assessed. sociobiology an international journal on social insects k gómez1, p lorite2, f garcía3, a tinaut4, x. espadaler5, t palomeque2, o sanllorente2, j trager6 article history edited by jacques delabie, uesc, brazil received 04 april 2018 initial acceptance 22 may 2018 final acceptance 13 june 2018 publication date 02 october 2018 keywords phylogeny, formica fusca, formica horrida. corresponding author kiko gómez avda 303 nº 117, castelldefels, barcelona, spain e-mail: netodejulilla@gmail.com according to tinaut (1990), i. subrufa should be considered under a subgenus or species group different from those defined for formica based on the finding of the smaller males and with other differential characters presented in this article. this new subgenus was called iberoformica. the article also stated the defining characters for iberoformica workers and gynes. agosti (1994), synonymized iberoformica and the rest of formica subgenera within formica. since 2002 studies endorsed the status of iberoformica as a clade different from the rest of formica species groups first (lorite et al., 2002; lorite et al., 2004; lorite et al., 2012) and finally, as a genus closely related but separated from formica (muñoz-lópez et al., 2012). formica gerardi was described from banyuls, france and was considered close to f. cinerea and formica glebaria nylander, 1846 (now a synonym of f. fusca) (bondroit, 1917). although considered a synonym of i. subrufa by bernard (1967), it regained its status as species (collingwood 1 independent researcher, barcelona, spain 2 departamento de biología experimental, universidad de jaén, jaén, spain 3 barcelona, spain 4 departamento de zoología. facultad de ciencias. universidad de granada granada, spain 5 creaf, universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain 6 shaw nature reserve, gray summit, missouri, usa research article ants k gómez et al. – differentiating iberoformica and formica (serviformica)464 & yarrow, 1969; collingwood, 1978). it was moved to the subgenus iberoformica (czechowski et al., 2012: 39) without explanation. subsequent authors like borowiec (2014: 435) treat the species as belonging to the subgenus serviformica inside formica but without giving it an official status. bolton’s online catalog (accessed 12/2017) treats the species as belonging in the genus iberoformica. we propose in this paper that, with the data available, iberoformica is properly a monotypic genus with a single species, i. subrufa, and we consequently propose to exclude f. gerardi from iberoformica and reinstate it into the genus formica, subgenus serviformica. we also describe the sexual forms of f. gerardi, which add even more evidence to our proposal, also supported by genetic data. materials and methods type material for i. subrufa and f. gerardi were seen at bondroit collection (brussels) and forel collections (geneva) respectively. i. subrufa samples analyzed cover the whole distribution area, from southern spain to south france. f. gerardi samples were collected throughout its distribution range (cádiz, barcelona, burgos, granada and zaragoza). sexuals were collected jointly with workers at the nest credits of all images belong to www.antweb.org. code on image references belong to the unique identifier in www. antweb.org database and can be accessed via https://www. antweb.org/specimenimages.do?code=codenumber. genitalia were digested with naoh diluted in distilled water for 24 hours. resulting parts were mounted in hoyer fluid. collection references: atpc: alberto tinaut collection fgpc: federico garcía collection kgac: kiko gómez collection xegc: xavier espadaler collection numbers and/or letters after collection codes design collection reference numbers that individually identify the specimen. measurements and indexes. all measurements in mm. hl: head length in frontal view measured from the basal clypeal to the apical occipital lines hw: head width, maximum head width in frontal view sl: scape length, excluding the basal constriction and the condylar bulb el: eye length, length in mm. of the eye’s longest axis wl: weber length, in lateral view from the pronotal declivity to the farmost line of the propodeal lobe pw: pronotum width in dorsal view, measured just before tegulae pld: in dorsal view, distance between propodeal declivity and mesoscutellum, excluding propodeum. oi: ocular index. el/hw x 100 ci: cepahlic index. hw/hl x 100 si: scape index. sl/hw x 100 mi: mesosomal index. pw/hw x 100 mdi: pw/pld x 100 molecular analysis several ant species were used for molecular studies (table 1). pools of 3 to 5 workers were used for dna extraction. dna purification was carried out using the nucleospin® tissue kit from macherey-nagel. dna was finally eluted in 100 µl. three nuclear gene fragments were used for phylogenetic analysis; wingless (wnt-1), abdominal-a (abda) and long-wavelength rhodopsin (lwrh) as well as a fragment of the mitochondrial coi gene. several sequences of these genes were directly retrieved from the genbank (table 1). the remaining sequences were obtained by pcr. the nuclear gene fragments were amplified using the primers and pcr conditions described in palomeque & al. (2015). primers and pcr conditions described in sanllorente et al. (2012) were used for coi gene amplifications. pcr products were directly sequenced on both strands by the dideoxy sequencing method. the obtained sequences were deposited in the embl database (table 1). species location wnt-1 abda lwrh coi proformica longiseta sierra nevada, granada (spain) kx219899 lt883161 (ps) kx219939 hm126584 iberoformica subrufa canena, jaén (spain) lt623167 (ps) lt623178 (ps) lt632330 (ps) kj499818 polyergus rufescens tours (france) kx219902 lt883159 (ps) kx219942 kj499816 polyergus samurai natural history museum and institute, chiba (japan) kx219903 lt883160 (ps) kx219943 ab010930 formica cunicularia campus universidad jaén (spain) lt623165 (ps) lt623177 (ps) lt632328 (ps) ab010926 formica frontalis sierra nevada, granada (spain) kx219882 lt883157 (ps) kx219922 kx219955 formica sanguinea tuscany (italy) kx219883 lt883158 (ps) kx219923 kx219956 formica gerardi sierra de huetor, granada (spain) mf276903 (ps) lt883156 (ps) mf276901 (ps) mf276902 (ps) table 1. sequences used for molecular analyses. for each species it is indicated the location of the sample and the genbank accession numbers for the used genes. some of the sequences were obtained for the present study (ps) and the remaining sequences were retrieved from the genbank. sociobiology 65(3): 463-470 (september, 2018) 465 multiple-sequence alignments were performed using clustalw. this dataset was used for phylogenetic analyses using the maximum-likelihood method (saitou & nei, 1987) implemented in the program mega version 6 (tamura et al., 2013). the best-fit nucleotide substitution model with the lowest bic (bayesian information criterion) value was chosen for each molecular marker and for the concatenated nucleotide sequences of the four gene fragments (gtr+g+i). node support was assessed with 1000 bootstrap replicates. bayesian analyses were carried out for each locus separately and for concatenated nucleotide sequences using mrbayes version 3.2 (ronquist et al., 2011). two independent runs were performed with four mcmc chains and run for 1,000,000 generations. the reported value of the effective sample size (ess) was above 1000 and the potential scale reduction factor (psrf) was 1.0 for all parameters, so we considered the number of runs sufficient. trees were sampled each 100th generations and a burn-in was set to 25% of samples. finally, a 50% majority rule consensus tree was calculated from the obtained trees and the posterior probabilities were calculated using the command “sumt” in mrbayes. characterization of f. gerardi workers the following combination of morphological characters has been used to isolate f. gerardi from other close species (formica lemani bondroit, 1917, f. fusca, formica decipiens bondroit, 1918, and european formica of the rufibarbis group). ecological requirements and habitat types used by those species are also indicative – f. gerardi is the most xerothermic species within european formica– and are currently in use during field work. • colour light to dark brown, sometimes with reddish brown areas at the sides of pronotum and pro-mesonotum lateral juncture. • body surface appearing, at low magnification (<20x), as absolutely mat. • internal face of tibiae iii with diluted pubescence. pubescent setae could be described as flat, wide and very appressed over tibial surface. instead, in f. lemani, f. fusca, f. decipiens, and european f. rufibarbis group, the internal face of tibia iii pubescence density is much higher (see figure 2). formica picea nylander 1846, strangely enough, approaches f. gerardi condition in tibial pubescence density but has otherwise clear differences in surface shininess, pilosity and colouration. • pronotum and mesonotum with short erect setae • propodeum lower than in european f. rufibarbis group species. sexual castes description for f. gerardi queen revised material: spain: barcelona, el muntanyá (seva) 710m 18/07/2000, espadaler, x. leg (1q) [xe00205-2]; burgos, alto de solanillas (castrovido) 990m 29/04/2016 (f. garcía) (3w, 1q) fgpc; burgos, alto de solanillas (castrovido) 29/04/2016 (f. garcía) (3w, 1q) fgpc; cádiz, montes de propios (jerez de la frontera) 26/07/2008 (f. garcía) (3w, 1q) fgpc; granada, sierra alfaguara (1w, 2q) b. pascual leg (atpc 6363); same data (2w, 1q) (atpc 6364); same data, (1q each) (atpc 6366, atpc 6367) huesca, torre ventosa (sierra de alcubierre) 05/2009 (f. garcía) (3w, 1q) fgpc; palencia, carrión de los condes (2w, 1q) a. tinaut leg (atpc 6362); zaragoza, sierra de alcubierre (1q) fgpc hl=1.7 [1.36-1.91] , hw=1.71 [1.53-1.89] , sl=1.6 [1.31.81] , el=0.63 [0.55-0.74] , wl=3.13 [2.86-3.57] , pw=1.67 [1.3-1.9] , pld=2.61 [2.33-2.97] , oi (el/hw)=37 [33-48] , ci (hw/hl)=100 [95-112] , si (sl/hw)=93 [84-100] , mi (pw/ hw)=98 [78-133] , mdi (pw/pld)=64 [49-77] (n=9) head subquadrate, smaller basally. mandibles striated, strong and with 6-7 acute teeth in the masticatory margin. clypeus convex, with a medial carina. frontal ridges short. fig 1. queens of formica fusca (a, b) [casent0173171], formica gerardi (c, d) q [xe00205-2] and iberoformica subrufa (e, f) [kg030006-3]. images from www.antweb.org k gómez et al. – differentiating iberoformica and formica (serviformica)466 scapes long, surpassing the occipital border, funiculus with four first segments elongated, the remainder subquadrate. pronotum dorsally visible and relatively low in lateral view in the smaller specimens. scutum rounded in profile view, scutellum flat in profile view and subquadrate in dorsal view. propodeum a straight line to the insertion with petiole. scutoscutellum suture demarcated but not deep. petiole squamiform, high, narrowly ovate in profile view. subrectangular in frontal view, convex with a medial notch. gaster as wide as mesosoma in dorsal view. whole body covered with a short, whitish pubescence. long, erect to semierect white setae present following the frontal ridges to occipital margin of the head (5 to 10), abundant on clypeus (10-15), numerous (>10) and sudecumbent on pronotal dorsum, semierect on dorsum of mesosoma (>20) and abdomen. rest of the head, propodeum and petiole with appressed pubescence only. body and head entirely shagreened and dull due to heavy sculpturation except frontal suture, central-anterior line of scutellum and two symmetrical lateral lines in its posterior half. colour entirely dark brown to black, with the appendages lighter. a reddish band present in the borders of the pronotum and mesopleurae. head below eyes and mandibles with a more or less developed reddish tinge. fig 2. detail of microsculpture in f. gerardi (a), coarse and deep creating a matt appearance, and f. fusca (b), superficial. detail of pubescence in tibiae iii, f. gerardi (c) and f. lemani (d); scale bar = 100 micrometers fig 3. males of formica fusca (a, b) [casent0178770], formica gerardi (c, d) [xe00205-3] and iberoformica subrufa (e, f) [kg030006-4]. images from www.antweb.org diagnosis: the long scapes and dull frontal triangle places it in the serviformica subgenus, and its dull cuticle (figure 2) differentiates it from the rest of iberian serviformica queens in which scutellum is smooth and shining. male revised material: spain: granada, sierra alfaguara, b. pascual leg (1m each) [atpc 6365, atpc 6368]; barcelona, el muntanyá (seva) 710m 18/07/2000, espadaler, x. leg (2m) [xe00205-3, xe00206] xegc; cádiz, montes de propios (jerez de la frontera) 25/07/2008. (f. garcía) (1w, 1m) [fgpc0724] fgpchl=1.30 [1.27-1.36] , hw=1.53 [1.481.58] , sl=1.27 [1.23-1.33] , el=0.73 [0.71-0.76] , wl=2.73 [1.18-3.28] , pw=1.92 [1.66-2.14] , pld=2.70 [2.47-2.89] , oi (el/hw)=47 [46-49] , ci (hw/hl)=117 [113-123] , si (sl/hw)=83 [81-84] , mi (pw/hw)=146 [130-162] , mdi (pw/pld)=71 [67-80] (n=5). head triangular, widest at apex, wider than long (ci~117), vertex convex, lateral a straight line; clypeus convex, medial carina weak but present; mandibles sublinear, slightly rugulose; apical tooth present, followed by a edentate border with 0-1 denticles; eyes large (oi~47); three conspicuous ocelli present and elevated over the rest of the head, the center one oriented forward, the other two laterally; frontal ridges absent, vestigial laterally with exposed antennal sockets. sociobiology 65(3): 463-470 (september, 2018) 467 scapes long (si~83) surpassing the occipital border when laid back, the length between occipital border and apex clearly longer than distal to occipital border; funiculus filiform with all funicular segments longer than wide, similar in size. mesosoma clearly wider than head (mi~146). pronotum almost not visible in dorsal view, slightly depressed in the medial line. scutum rounded, notauli absent, parapsidal lines clearly demarcated. scutelllum rounded dorsally and laterally, elevated over the scutum. posteropropodeum clearly longer than dorsopropodeum, rounded in lateral view. petiole low; in profile view rounded, subovoid and almost symmetrical, with an acute apex; in frontal view trapezoidal, with straight dorsal line and vertical lateral sides connected by two 45 degree almost straight lines. gaster long and cylindrical. genitalia typical of genus formica (figures 4, 5). sagitta with apex recurved and rounded, the border of its ventral half dentated. digitus recurved. fig 4. dorsal view of formica gerardi (a) [xe00205-3] and iberoformica subrufa (b) [kg030006-4]. detail of genitalia of formica fusca (c) [casent0178770], formica gerardi (d, f) [xe00205-3] and iberoformica subrufa (e, g) [kg030006-4]. images from www.antweb.org fig 5. dissected genitalia of iberoformica subrufa (a, b, c) and formica gerardi (d, e, f). imaged by federico garcía. propodeum. petiole with scattered erect short setae, as long as the pubescence. whole body covered with yellowish, dense long pubescence, its length clearly longer than distance between pubescence lines, overlapping. diagnosis: as in the worker and queen castes, the dull, matt appearance differentiates the f. gerardi male from all the other serviformica males present in the iberian peninsula, which have shiny mesopleurae. discussion genetic evidence studies of chromosome numbers and molecular phylogenies provide support for our recognition of distinct generic placement of i. subrufa and f. gerardi in an earlier study the karyotype of f. gerardi had been studied with a haploid chromosome number of n=27, (lorite et al., 1998). later, lorite et al. (2002) showed that i. subrufa has a chromosome number of n=26. the authors already suggested in this work that cytogenetic data and morphologic differences (tinaut, 1990) supported the separation of i. subrufa from the subgenus serviformica and the maintenance of the subgenus iberoformica for this species. previously agosti (1994) had synonymized the subgenus iberoformica with formica. the chromosome number in the genus formica show low variation, with n=26 or n=27 (lorite & palomeque 2008). however, in spite of the low variation in chromosome numbers in the genus formica, the chromosome numbers showed a heterogenous distribution among the different formica subgenera. in the subgenus serviformica n=27 was to be the usual chromosome number, as had already been reported for f. gerardi. dark brown to black, with legs and funiculus light brown to brown. head surface matt except for the frontal line from clypeus to central ocellus. rest of body sculptured with the same pattern than worker, matt (figure 3). 1-2 pairs of semierect to erect setae on clypeus medially on clypeus, one on vertex and one setae more below each lateral ocelli. mesosoma dorsally with scattered yellow, short semierect to erect setae, slightly longer than pubescence, absent on k gómez et al. – differentiating iberoformica and formica (serviformica)468 iberoformica f. fusca species group figs. worker nodiform (not squamiform) petiole, becoming almost cylindrical petiole squamiform 7b, 7d, 7f concave mesonotum that follows smoothly into the dorsal propodeal line, not meeting at an angle mesonotum concave, followed by a horizontal propodeum, both meeting at an angle 7b, 7d, 7f whole body covered with short, thick, truncated white setae whole body bare, except for some scattered setae present in propodeum and mesonotum, these hairs thinner than those of subrufa 7 queen meso and metathorax less developed: metanotal species (details in tinaut & ruano, 1992) meso and metathorax very developed, similar to the rest of formica queens (macronotal species) 1b, 1d, 1f gaster with first segment almost rectangular, longer than wide, giving the gaster an elongated appearance gaster with first segment wider than long, the gaster not elongated 1b, 1d, 1f males small size, similar to workers size bigger than workers and only slightly smaller than queens head almost as wide as mesosoma head clearly less wide than mesosoma 4a, 4b frontal ridges present, short frontal ridges absent 3a, 3c, 3e petiole high, squamiform and only slightly concave on the apex petiole low, triangular and clearly concave on the apex 3b, 3d, 3f gaster long and narrow, narrower than mesosoma width gaster about the same width than mesosoma 4a, 4b sagitta pointy sagitta blunt, subrectangular 4c-4g, 5 volsella very slender, finger shaped volsella semicircular 4c-4g, 5 stipes longer than volsella and lacinia stipes approximately same long than than volsella and lacinia 4c-4g, 5 table 2. comparison of morphological characters between the genus iberoformica and subgenus serviformica. lorite & al. (2004) characterized the satellite dna in seven species of the genus formica: f. cunicularia, f. fusca, f. gerardi, f. rufibarbis, and formica selysi bondroit, 1918 (fusca group), formica frontalis santschi, 1919 (rufa group), formica sanguinea latreille, 1798 (sanguinea group) and in i. subrufa. the study showed that satellite dna sequences from i. subrufa were clearly different from those found in formica species, resulting in a phylogenetic tree separated in two well supported clades. in the formica clade the sequences of all species appeared intermixed, including the sequences of f. gerardi. these results support the differentiation of i subrufa in relation to the other formica species as well as the similarity of the f. gerardi sequences with other species of the fusca group. in a later study previous results were confirmed, and iberoformica was raised to a genus status only composed by i. subrufa. in the present study a phylogenetic study was carried out on several species of the genus formica and of its outgroup genera, polyergus and proformica, using sequences of nuclear satellite dna and the mitochondrial 16s rrna as molecular markers (muñoz-lópez & al. 2012). unfortunately f. gerardi was not included in this last study. all recent molecular phylogenies have shown that the genera formica, iberoformica and polyergus form a monophyletic clade (blaimer & al. 2015, sanllorente & al. 2017). in this paper we perform a new phylogenetic study including f. gerardi, and other species from the genera iberoformica, polyergus and proformica and using four different fig 6. phylogenetic tree using concatenated sequences of the abdominal a, wingless, long-wavelength rhodopsin nuclear genes and the mitochondrial cytochrome oxidase i gene. first number at nodes indicates the bootstrap values obtained in the maximum likelihood analysis (only when higher than 70%) and the second one the posterior probability values in the bayesian inference analysis (only when higher than 0.7). genetic markers, three nuclear genes (wnt-1, abda, lwrh) and a mitochondrial gene (coi). the phylogenetic approaches were carried out using one genetic marker alone or different combinations of them. the best bootstrap values are obtained when several molecular markers are considered together (figure 6). all molecular markers, or combination of them, cluster together all formica species in a well-supported clade and clearly separated from the iberoformica, its sister genus. morphological characters we summarize in table 2, the main characters which differentiate the genus iberoformica and the f. fusca species group. sociobiology 65(3): 463-470 (september, 2018) 469 behavioral and ecological differences iberoformica subrufa and f. gerardi share the fact of being thermophilic ants, living in the mediterranean forests clearings. although both species can share the same habitat occasionally, i. subrufa may be present in the same localities as other highly thermophilic species like camponotus foreli emery, 1881 or cataglyphis velox santschi, 1929, while f. gerardi is more dependent on shaded areas. being less thermophilic, f. gerardi extends its distribution to the northern iberian meseta, where i. subrufa is present only in isolated areas, and the opposite in the south iberian peninsula, where i. subrufa is common and f. gerardi needs forest-shaded areas to live. one main behavioral difference between both species is that i. subrufa has not known dulotic relation with polyergus species, while various ants belonging in the fusca group are commonly enslaved by them. in its revision of the genus polyergus, trager writes about polyergus rufescens (latreille, 1798): “i have series from the pyrenees with f. gerardi, where this is the most abundant potential host” (trager, 2013: 511). one of us (f. garcía) has also found these two species in dulotic relation in ayora (valencia province, 39°06’59”n 1°12’37”w 850m , 18/02/2017), in a pinus halepensis pinewood where f. gerardi was one of the most frequent species. i. subrufa was also present and frequent but was never found in dulotic relation with p. rufescens. the case of formica horrida the abundant pilosity formed by short and stout setae is the most visible character that defines iberoformica. the only formica species known to us that shares this character is the fossil formica horrida wheeler, 1915. this species was described from the baltic amber and might be a good candidate to be included into the genus iberoformica. we must refrain to include this species into iberoformica until more material is available. the material type seems to be lost and the only existing image does permit appreciation of the typical sinuous iberoformica mesonotal profile. conclusion we have presented evidence that formica gerardi presents genetic, morphologic and behavioral characteristics that place it in the ant genus formica, more concisely into the fusca-group, so we propose its reinstatement into the genus formica. acknowledgements to dr. bernhard seifert, for his comments and suggestions to this manuscript. to xim cerdá, director of the estación biológica de doñana (csic), for the suggestions and bibliography. to michele exposito (california academy of sciences), for imaging the specimens and dr. brian fisher (california academy of sciences) for allowing the use of the images in the reference site www.antweb.org. references agosti, d. 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(1915, “1914”). the ants of the baltic amber. schiriften der physikalisch-ökonomischen gesellschaft zu königsberg in pr.schr. phys.-ökon. ges. königsb, 55: 1-142 doi: 10.13102/sociobiology.v66i4.4271sociobiology 66(4): 536-544 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction leaf-cutting ants of the genera atta and acromyrmex (hymenoptera: formicidae) cultivate a symbiotic fungus, leucocoprinus gongylophorus (heim) (leucocoprini: agaricales), that forms the basis of their diet. for this purpose, the ants cut the leaves of different cultured plants and are therefore considered pests in agricultural and forest systems (mueller et al., 2018). ant baits are currently the most effective control method indicated for leaf-cutting ants (britto et al., 2016). however, there has been a growing search for alternative control, especially that involving natural botanical extracts. plant extracts with insecticidal and/or fungicidal activity against leaf-cutting ants are widely investigated. the most studied toxic plants are: ricinus communis (hebling et al., 1996; abstract chemical control of leaf-cutting ants is widely used, but alternative control with toxic plant extracts is promising. substances with insecticidal potential extracted from plants have numerous ecological advantages. this study evaluated the insecticidal and/or fungicidal potential of the plants asclepias curassavica (tropical milkweed), rosmarinus officinalis l. (lamiaceae) (rosemary) and equisetum spp. (horsetail) for control of the leaf-cutting ant atta sexdens rubropilosa forel, 1908 (hymenoptera: formicidae). forty laboratory-reared colonies of atta sexdens rubropilosa were used. the plants were collected, dried out in a circulating air oven for 48 hours, ground, and macerated in 96o ethanol until exhaustion. after filtration, the products were evaporated under reduced pressure to obtain the ethanolic extracts. acceptance of the reagent, topical application of the extracts, and application of baits containing 4% of the plant extracts were tested. the results showed that all plant extracts tested negatively influenced the development of the fungus garden. baits produced with asclepias curassavica caused the highest mortality of the colonies within 7 days. in conclusion, the ethanolic extracts of asclepias curassavica, rosmarinus officinalis and equisetum spp. exhibit insecticidal (contact and ingestion) and fungicidal activity in colonies of the leafcutting ant atta sexdens rubropilosa. sociobiology an international journal on social insects vm ramos, rg ferreira leite, vt almeida, rs camargo, jv souza cruz, rm leão, mv prado, mc sousa pereira article history edited by evandro n. silva, uefs, brazil received 26 november 2018 initial acceptance 26 november 2019 final acceptance 06 december 2019 publication date 30 december 2019 keywords atta spp., plant extracts, botanical insecticides, alternative control. corresponding author vania maria ramos laboratory of agricultural entomology agronomy department college of agricultural sciences university of western são paulo [universidade do oeste paulista – unoeste] presidente prudente-sp, brasil. e-mail: vaniaramos@unoeste.br bigi et al., 1998; kitamura et al., 1999; bigi et al., 2004; caffarini et al., 2008; alonso & santos, 2013), sesamum (hebling et al., 1991; bueno et al., 1995; ribeiro et al., 1998; peres filho et al., 2003; morini et al., 2005; bueno et al., 2004), canavalia ensiformis (hebling et al., 2000; rodriguez et al., 2008; valderrama-eslava et al., 2009; aubad-lopez, 2011;varon et al. 2007), tithonia diversifolia (giraldoecheverri, 2005; castano, 2009) and azidarachta indica (bigi et al., 2004; gruber & valdix, 2003; herrera, 2009). despite the large number of plants studied for the control of leaf-cutting ants, there are three plant species with a promising and unprecedented potential: asclepias curassavica, equisetum sp. and rosmarinus officinalis. the genus asclepias, which comprises about 490 species, is distributed in the paleotropical, holarctic and neotropical regions (pereira et al., 2004). some species of this genus laboratory of agricultural entomology, agronomy department, college of agricultural sciences, university of western são paulo [universidade do oeste paulista – unoeste], presidente prudente-sp, brazil research article ants bioactivity of asclepias curassavica, equisetum spp. and rosmarinus officinalis extracts against leaf-cutting ants sociobiology 66(4): 536-544 (december, 2019) 537 have medicinal properties. for example, a. curassavica (apocynaceae) acts on the central nervous system and is used for the treatment of rheumatism, tumors, inflammation and ophthalmological infections in mammals (li et al., 2009). high effectiveness of a. curassavica extract diluted in ethanol in the control of nomophila sp. caterpillars (lepidoptera: noctuidae) has been reported (costa et al., 2014). the genus equisetum belongs to the family equisetaceae and comprises about 30 species. popularly known as “horsetail”, these plants can reach approximately 1 meter in height and are used for therapeutic purposes. plants of this genus contain high levels of minerals, mainly silicon, and secondary metabolites such as saponins, flavonoids, tannins and alkaloids, which exert beneficial effects on metabolism maintenance and can treat different diseases (mello & budel, 2013). some equisetum species have been used as an alternative pest control system. the extract of this plant confers increased structural stiffness to plant tissues because of the high amount of silicon. this prevents the penetration of fungal hyphae and increases resistance to some phytophagous insects, in addition to influencing the accumulation of phenolic compounds (bertalot et al., 2010; mello & budel, 2013). finally, r. officinalis l., better known as rosemary, belongs to the family lamiaceae and is a spice recognized since antiquity for its medicinal effects (hentz, 2007). the medicinal purposes described are the use of the dry leaves as tea for the treatment of dyspepsia and inflammation (marsarojúnior, 2007). additionally, rosemary has been used for the control of thyrinteina arnobia leaf-stripping caterpillars (soares et al., 2011), storage pests (melo et al., 2011), aphis craccivora aphids (santos et al., 2011) and bacteria (ribeiro, 2011), and is also employed because of its antimicrobial activity (hentz, 2007). given the above and considering the harmful effects in insects, we postulate that a. curassavica, equisetum sp. and r. officinalis have a promising potential for the alternative control of leaf-cutting ants. therefore, the objective of this study was to evaluate the bioactivity of ethanolic extracts of a. curassavica, equisetum sp. and r. officinalis on the leaf-cutting ant atta sexdens rubropilosa under laboratory conditions. material and methods target species the experiment was conducted at the laboratory of agricultural entomology, university of western são paulo (unoeste), presidente prudente, são paulo, brazil, in a fully climatized room at a temperature of 23.0 ± 1.0°c and humidity of 60% ± 10%. the a. sexdens rubropilosa colonies were collected in the field and stored in plastic containers (1 liter) covered with a plaster layer for moisture balance. the colonies were daily supplied with acalypha leaves (acalypha wilkesiana). when the fungus reached a minimum volume of 250 ml, two new containers (500 ml) were interconnected at the ends of each colony, one reserved for food and the other used as waste disposal. plant extract for preparation of the extracts, a. curassavica, r. officinalis and equisetum spp. were collected in the region of presidente prudente sp. after cleaning and selection of plant parts, leaves and stems were stored in kraft paper bags and dried out in a circulating air oven at 60°c for 48hours (table 1). the dried plants were then ground in a willye knife mill to a particle size of 0.45 mm to obtain a fine powder. the powder was stored in hermetically sealed glass containers at 24°c in the dark until manipulation of the extracts. the powder of each plant was macerated in 96o ethanol solution and filtered once a day, through a conventional glass funnel, using germination paper as filter paper. after filtration, the flask was filled with 96o ethanol until it covered 4 cm of the powder volume. this procedure was performed until exhaustion in order to obtain the ethanolic extract (santana et al., 2013). when a color difference was observed during the filtration process, the whole added solvent was evaporated under reduced pressure in a rotary evaporator, yielding the crude ethanolic extracts of a. curassavica, r. officinalis and equisetum spp. fabrication of the baits the baits were fabricated by mixing ground citrus pulp (300 g), carboxymethylcellulose (0.8 g), vegetable oil (2.0 g), distilled water (300 ml), 96º ethanol (240 ml), and the plant extract at a proportion of 4% (w/w) (ramos et al., 2006). after grinding the pulp, carboxymethylcellulose, oil and water were added and the mixture was homogenized. the crude extract was dissolved in ethanol and added to the mixture in an autoclaved glass container, forming a paste that could be molded. the control treatment was prepared without the plant extract. the pasty mixture was inserted and compacted in a 3-ml syringe with the tip cut off and arranged on a tray covered with aluminum paper, forming strands of approximately 5 cm that were cut into smaller pieces (pellets). finally, the pellets were dried in an oven at approximately 50ºc for 24 hours. scientific name common name family parts used origin asclepias curassavica tropical milkweed asclepiadaceae leaves and stems presidente prudente sp rosmarinus officinalis rosemary lamiaceae leaves and stems paraguaçú paulista sp equisetum spp. horsetail equisetaceae leaves and stems presidente prudente sp table 1. denomination and origin of the plants used for preparation of plant extracts. vm ramos et al. – plant extracts against leaf-cutting ants538 bioassay for testing the repellency or rejection of plant extracts by worker ants this assay aimed to evaluate if the solvent used for preparation of the baits influences bait loading, i.e., if it promotes repellency or rejection. two treatments were compared to determine acceptance of the material. in the first treatment, 5-mm filter paper discs were immersed in 96º ethanol and immediately rolled in ground citrus pulp to increase their attractiveness. in the control treatment, the filter paper discs were immersed in distilled water before rolling in citrus pulp powder. the experimental unit was set up as the foraging chamber of a colony. we used a total of 30 experimental units, divided into fifteen colonies used as replicates per treatment. for each replicate (colony), 20 dry filter paper discs were simultaneously supplied, totaling 300 discs per treatment. loading, incorporation and return of the discs in the waste chamber were evaluated after 24 hours according to ramos (2005). bioassay for testing topical action of the plant extracts on workers the aim of this assay was to evaluate ant mortality resulting from contact application of the plant extracts. since ants exhibit a self-grooming and grooming behavior, they were used individually, ensuring that the effect of the extracts was not due to ingestion. the experimental unit was set up as a plastic container (75 ml) with plaster covering the bottom in order to maintain humidity, and one worker added to it. worker ants of medium size were removed randomly from healthy colonies and placed inside containers. a piece of fungus (0.5 cm³) was placed inside each container, which served as food for the ants. the fungus was always changed when it had lost its nutritional value as demonstrated by a decrease in its volume and color change. the experiment consisted of 5 treatments: ethanolic extract of a. curassavica (4%), ethanolic extract of r. officinalis (4%), ethanolic extract of equisetum spp. (4%), ethanol, and distilled water. for each treatment, 30 plastic containers with one ant were used as replicates, totaling 150 replicates. each worker received an application of 0.5 µl of the respective treatment on its pronotum. the evaluations of worker mortality were conducted at 2, 8, 12, 24, 48, 72, 168 and 384 hours after extract application. bioassay for testing the effect of the baits containing the plant extracts on colonies this assay aimed to evaluate the effect of baits containing the plant extracts on a. sexdens rubropilosa colonies. the experimental unit was comprised of a colony containing both foraging and waste chambers, and all colonies were the same age (approximately 1 year) and same size (500 ml fungus garden). forty colonies were randomly assigned into four treatments, which led to 10 replicates per treatment. the following treatments were applied: citrus pulp baits containing 4% ethanolic extract of a. curassavica, r. officinalis and equisetum spp. as well as ethanol as control. at the beginning of the experiment, all colonies had their residues discarded and no leaves were supplied for 48 hours. in addition, the initial volume of the fungus garden of each replicate was determined as the proportion of fungal volume in relation to the total volume of the container. each replicate received 1 g of bait (pellets) in their foraging chamber for 7 consecutive days. twenty-four hours after each supply, the number of pellets carried by the workers, pellets incorporated into the fungus garden, and pellets returned to the waste chamber was counted. additionally, the presence of fungus discarded in the waste and the number of dead ants were daily evaluated. in order to avoid interference with the effect of the treatments on the colonies, a. wilkesiana leaves was also daily supplied as foraging substrate. the volume of the fungus was evaluated on days three and seven after application of the treatments, determining whether the volume of the fungus garden remained the same or decreased as a result of application of the treatments. ant mortality was scored as 0, 25, 50 and 100. score 0 represents the natural mortality of ants, corrected for the control and daily observed with minimum numbers as a function of the normal life cycle of the ants. score 25 corresponds to the mortality of approximately 25% of ants present in the colony, score 50 to 50%, and score 100 to complete mortality of workers. statistical analysis first, the data were submitted to the shapiro-wilk normality test. since the data were not normally distributed, the behavior results were submitted to nonparametric analysis. the survival curves of workers were compared by the logrank test. bait loading, bait incorporation, fungal volume and worker mortality in the colonies were analyzed by the kruskalwallis test. if significance was observed, each variable was compared by the student-newman-keuls post-test, adopting a level of significance of 5%. separate analyses were performed at 24 hours, 72 hours, and 7 days. results bioassay of repellency and rejection of the plant extracts the filter paper discs of the two treatments were equally carried out by workers and incorporated into the fungus garden (100% loading and incorporation). these results suggest that citrus pulp has a superior odor to ethanol or that ethanol evaporates completely and leaves no residues. this finding confirms that ethanol does not cause repellency or rejection by ants and can be used in the formulation of toxic baits containing plant extracts without interfering with the result of the final product. bioassay of the topical action of the plant extracts on workers the results showed a strong effect of the plant extracts on worker mortality, particularly until 168 hours (7 days) after topical application (figure 1). the a. curassavica extract was the most promising, with a cumulative mortality of 70 to sociobiology 66(4): 536-544 (december, 2019) 539 90% between 48 and 72 hours after application of the extract. comparison of worker survival curves showed a significant difference between ethanol (control) and the equisetum spp. (log-rank test, x2=4.56, d.f.=1, p<0.05), a. curassavica (logrank test, x2=61.01, d.f.=1, p<0.001) and r. officinalis (logrank test, x2=9.91, d.f.=1, p<0.05) extracts. a significant difference was also detected between the survival curves of a. curassavica and equisetum spp. (log-rank test, x2=12.42, d.f.=1, p<0.001) and a. curassavica and r. officinalis (logrank test, x2=39.18, d.f.=1, p<0.001). there was no significant difference between the survival curves of workers receiving water and ethanol (log-rank test, x2=0.0023, d.f.=1, p>0.05). bioassay on the effects of the baits containing the plant extracts on colonies the results showed a deleterious effect of baits containing plant extracts on colonies of leaf-cutting ants (figure 2). regarding bait loading, a significant difference was observed between treatments at 24 hours (kruskalwallis test, h=35, d.f.=3, p<0.001), 72 hours (kruskal-wallis test, h=39, d.f.=3, p<0.001), and 7 days (kruskal-wallis test, h=38, d.f.=3, p<0.001). as can be seen in figure 2-a, loading declined after 24 and 72 hours. the control baits (ethanol) and baits containing the r. officinalis extract did not differ significantly from one another, but differed from those containing the a. curassavica and equisetum spp. extracts. on day 7, lower loading by workers was observed for baits containing a. curassavica extract, which differed from the other treatments (figure 2-a). incorporation of baits into the fungus garden also differed between treatments, with the observation of a significant difference at 24 hours (kruskal-wallis test, h=34, d.f.=3, p<0.001), 72 hours (kruskal-wallis test, h=37.5, d.f.=3, p<0.001), and 7 days (kruskal-wallis test, h=36, d.f.=3, p<0.001). a reduction in the rate of bait incorporation was observed at 24 and 72 hours (figure 2-b). the control baits (ethanol) and baits containing the r. officinalis extract did not differ significantly from one another, but differed from those containing the a. curassavica and equisetum spp. extracts. on day 7, baits containing the a. curassavica and equisetum spp. extracts were less incorporated by workers, differing from the other treatments (figure 2-b). figure 1. survival curves of leaf-cutting ants over a period of 384 hours. eq (equisetum spp. extract), as (asclepias curassavica extract), ro (rosmarinus officinalis extract), water, ethanol. vm ramos et al. – plant extracts against leaf-cutting ants540 a reduction in fungus garden volume was observed for each treatment (figure 2-c), with a significant difference at 72 hours (kruskal wallis test, h=34, d.f.=3, p<0.001) and 7 days (kruskal-wallis test, h=38, d.f.=3, p<0.001). the baits containing the a. curassavica and equisetum spp. extracts promoted the greatest reduction in the volume of the fungus garden (figure 2-c). the baits containing plant extracts also caused significant mortality of workers, with significant differences between treatments at 24 hours (kruskal-wallis test, h=22,29, d.f.=3, p<0.001), 72 hours (kruskal-wallis test, h=38,29, d.f.=3, p<0.001), and 7 days (kruskal-wallis test, h=38,36, d.f.=3, p<0.001). at 24 hours, high mortality was observed for colonies treated with baits containing the r. officinalis extract (figure 2-d). on day 7, high worker mortality was found in colonies treated with the a. curassavica extract (figure 2-d). figure 2. percentage of loading and incorporation of baits (a and b), fungus garden volume (c), and worker mortality (d) in atta sexdens rubropilosa colonies under laboratory conditions. different letters above the columns indicate a significant difference by the student-newmankeuls post-test (p<0.05). the same letters indicate the absence of a difference. discussion the present study results show significant insecticidal and fungicidal activity of the ethanolic extracts of a. curassavica, equisetum sp. and r. officinalis against a. sexdens rubropilosa colonies (figures 1 and 2). the topical application of the extracts to workers significantly reduced survival (figure 1), especially the a. curassavica extract. regarding the insecticidal activity of toxic plants, the hexane and dichloromethane fractions of leaf extracts of r. communis, applied topically, also exhibited insecticidal activity (bigi et al., 1998). fatty acids and ricin were found to be toxic to a. sexdens rubropilosa workers at concentrations of 0.2 and 0.4mg.ml-1 (bigi et al., 2004). contact activity has also been reported for extracts of amazon plants (banara guianensis, clavija weber baueri, mayna parvifolia, ryania speciosa, spilanthes oleraceae and siparuna amazônica) against a. sexdens, a. laevigata and acromyrmex subterraneus molestans at a concentration of 5mg.ml-1 (gouvea et al., 2010). in addition, compounds such as terpenoids, caryophyllene epoxide, nerolidol and kolaenol isolated from hymenaea courbaril, melampodium divaricatum and vismia bacciferae exhibited insecticidal activity when workers were supplied with an artificial diet containing these compounds (howard et al., 1988). feeding artificial diets, boulogne et al. (2018) observed insecticidal activity of the seed extract of mammea americana against ac. octospinosus workers whose lethal concentration was close to that of fipronil (0.03g.kg-1). the authors attributed this effect to the significant presence of alkaloids, phenolic compounds, and terpenes in this plant. the equisetum spp., a. curassavica and r. officinalis extracts clearly exhibited insecticidal activity by direct contact and ingestion, as demonstrated by worker mortality after topical application and the presence of dead workers in colonies that received the baits with the extracts (figure 1, figure 2-d). many compounds of botanical extracts exhibit this feature. for example, the crude seed extract of a. indica caused significant ingestion and contact toxicity in a. sexdens workers (santos oliveira et al., 2006). in a. cephatoles, the sociobiology 66(4): 536-544 (december, 2019) 541 extract of tithonia diversifolia, at a concentration of 1.5 ml.l-1, was efficient as topical application and when ingested (castano, 2009). substances purified from helietta puberula, including anthranilicacid, kokusaginine and dictamnine, provided in artificial diets were toxic to a. sexdens workers (almeida et al., 2006). the alkaloid, 5-methoxy-canthin6-one, isolated from the plant simarouba versicolor was also toxic to a. sexdens workers (penaflor et al., 2009). furthermore, boulogne et al. (2011) found that topically applied m. americana and nicotiana tabacum extracts were toxic to workers, and nerium oleander extract exhibited a delayed action, although many fractions were repellent to ants. another interesting topic to be addressed refers to the chemical compounds present in the extracts that are toxic to leaf-cutting ant workers. the seed oils of r. communis a nd jatropha curcas were toxic by ingestion at a concentration of 10 and 30mg.ml-1, respectively, and by direct contact at a concentration of 0.1 and 0.2mg.ml-1 (alonso & santos, 2013). the authors suggested that ricinoleic, oleic and linoleic acids are the toxic agents of ricinus oil. another compound is coumarin, identified in extracts of rue (ruta graveolens), jimsonweed (datura estramonium), cordia verbenaceae, peppermint (mentha pitanperita), goat weed (ageratum conyzoides) and tropical apricot (m. americana), which is a potential insecticide for leaf-cutting ants (araujo et al., 2008; boulogne et al., 2018). equisetum sp. plants are known for their high content of minerals, especially silicon, and secondary metabolites such as saponins, flavonoids, tannins and alkaloids (mello & budel, 2013). one of the main active compounds of a. curassavica is the glycoside asclepiadin (costa et al., 2014; carvalho et al., 2009), which causes toxicity in mammals (costa et al., 2014). r. officinalis contains different active compounds found in the essential oil prepared from the leaves and flowers (hentz, 2007). these compounds are probably toxic to leaf-cutting ants since they caused the death of workers. the mode of action of most of these compounds is unknown, probably because of the difficulty in performing physiological studies. few researchers have studied the mode of action of compounds from toxic plants. for example, bullatacin is a compound isolated from plants of the family annonaceae, which has a great insecticidal potential. this compound strongly inhibits cellular respiration, exerting an antagonistic effect on the electron transport in mitochondria, with a specific action on complex i (ahammadsahib et al., 1993). however, we do not know about the continuation of that research. other compounds with a neurophysiological focus are being studied; for example, silphinenes, compounds extracted from the aerial part of senecio palmensis. this tricyclic sesquiterpene acts as an antagonist of the ɤ-aminobutyricacid (gaba) system, more specifically on chloride receptor channels (bloomquist et al., 2008). another compound with neurotoxic activity is the monoterpenoid pulegone-1, 2-epoxide, isolated from lippiasteochadifolia (grundy & still, 1985). the authors demonstrated that this compound acts like carbamates, irreversibly inhibiting the enzyme acetylcholinesterase. little information is available on extracts of a. curassavica, equisetum sp. and r. officinalis and their active compounds, especially the mode of action of these compounds. expressive fungicidal activity was observed in the experiments (figure 2-c), demonstrated by the drastic reduction in the volume of the fungus garden of the colonies. synthetic fungicides such as cycloheximide have the same effect (sousa et al., 2017). previous studies have shown that natural botanical extracts promote a decrease in the fungus garden of leaf-cutting ants. after 6 weeks of treatment, m. americana seed extract caused total decline of ac. octospinosus colonies and the symbiotic fungus garden was completely infested with competitors (boulogne et al., 2018). pagnocca et al. (1990) found that sesamum indicum extract caused a decrease in the growth of leucocoprinus gongylophorus, but the authors did not identify the compounds of this extract. in a subsequent study, these authors obtained the same result for lignans of virola sebifera and otoba parvifolia (pagnocca et al., 1996). lignans are common components of plants. more than 500 compounds that have antitumor, antimitotic and antiviral activity have been described (macrae & towers, 1984). pagnocca et al. (1996) concluded that lignans are the most potent compounds against the symbiotic fungus and ants therefore do not cut v. sebifera leaves. sesamin exerted a fungicidal effect at a concentration of 70µg.ml-1 (bueno et al., 2004). this result disagrees with the findings of ribeiro et al. (1998) who demonstrated complete inhibition of the fungus at a low concentration (2.5µg.ml-1). bigi et al. (2004), studying the activity of r. communis extract, reported that fatty acids are important compounds against the fungus of leaf-cutting ants. however, it is not known which fatty acid was responsible for inhibition of the fungus. in a chromatographic analysis of s. indicum extracts, ribeiro et al. (1998) purified substances with fungicidal activity, including tetradecadonoic, hexadecanoic, 9, 12, 15-octadecatrienoic, octadecanoic, icosanoic and docosanoic acids, but an attempt to isolate the substances was unsuccessful. a similar result was found for the fatty acids and crude extract of canavalia ensiformis (monteiro et al., 1998). based on this knowledge, castro faria and sousa (2000) added s. indicum seeds to the culture medium of the symbiotic fungus of acromyrmex leafcutting ants and concluded that the seeds affect fungal growth. the dichloromethane and hexane fractions of cipadessa fruticosa and cedrela fissilis extracts exerted an inhibitory effect on the symbiotic fungus of leaf-cutting ants (bueno et al., 2005; leite et al., 2005). almeida et al. (2006) isolated 6 substances from helietta puberula, including anthranilicacid, kokusaginine and dictamnine, which exhibited a strong inhibitory effect on the symbiotic fungus of a. sexdens. these alkaloids are a promising source of new substances for the control of leaf-cutting ants. similar results were obtained for another alkaloid, 4, 5-dimethoxy-canthin-6-one, isolated vm ramos et al. – plant extracts against leaf-cutting ants542 from s. versicolor extract (penaflor et al., 2009). caffeine (1, 3, 7-trimethylxanthine) was also used to evaluate the in vitro growth of the symbiotic fungus of a. sexdens rubropilosa and concentrations of 0.1 and 0.5% caused fungal death in the laboratory (miyashira et al., 2011). taken together, these results show that the synergistic action of substances can affect the development of the fungus garden of leaf-cutting ants, similar to the results of the present study using a. curassavica, equisetum sp. and r. officinalis. in conclusion, the ethanolic extracts of a. curassavica (tropical milkweed) r. officinalis (rosemary) and equisetum spp. (horsetail) exhibit insecticidal (by contact and ingestion) and fungicidal activity in colonies of the leaf-cutting ant a. sexdens rubropilosa. however, further assays using different doses and bait application methods are necessary to obtain definitive results that can be recommended for the field. references ahammadsahib, k.i., hollingworth, r.m., mcgovren, j.p., hui, y.h. & mclaughlin, j.l. 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(2009). enforced herbivory on canavalia ensiformis and tithonia diversifolia and its effects on leaf-cutting ants atta cephalotes. journal of applied entomology, 133: 689-694. varon, e.h., eigenbrode, s.d., bosque-perez, n.a. & hilje, l. (2007). effect of farm diversity on harvesting of coffee leaves by the leaf cutting ant atta cephalotes. agricultural and forest entomology, 9: 47-55. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.345-347sociobiology 61(3): 345-347 (september 2014) brood hiding test: a new bioassay for behavioral and neuroethological ant research a szczuka, b symonowicz, j korczyńska, a wnuk, ej godzińska introduction adult workers of social hymenoptera usually engage first in intranidal tasks and then switch to extranidal ones (wilson, 1971; hölldobler & wilson, 2009). this transition is often called the transition nurse-forager (e.g., heylen et al., 2008). however, older ant workers and/or foragers may retain the ability to engage in intranidal brood care (lenoir, 1979; sorensen et al., 1984; seid & traniello, 2006; muscedere et al., 2009). moreover, several studies revealed that ant foragers are more attracted to brood found outside the nest than nurses and show higher readiness to retrieve it to the nest (weir, 1958; walsh & tschinkel, 1974; lenoir, 1977; 1981). on the basis of these findings lenoir (1977; 1981) proposed a bioassay acting as a reliable technique of identification of ant foragers: the brood-retrieving test. at the start of the test a simple artificial ant nest (a test tube equipped with a water reservoir and partly covered by a black paper tube to assure darkness in its humid zone) is inclined so that the brood falls on the dry cotton plug closing the other end of the tube. workers inhabiting the nest may then retrieve brood back to the dark zone close abstract we describe a new bioassay for behavioral and neuroethological ant research, the brood hiding test. a group of adult ants is taken out of the nest, confined together with brood and exposed to strong light. ants may interact with brood, and, in particular, transport it to the provided shadowed area. the brood hiding test may be accompanied by administration of neuroactive compounds and/or by measurements of their levels in the brain and/or in specific brain structures. during pilot tests with workers of formica polyctena the values of the score quantifying ant behavior were positively correlated with the group size. sociobiology an international journal on social insects nencki institute of experimental biology, warsaw, poland. article history edited by evandro n silva uefs, brazil received 19 november 2013 initial acceptance 30 march 2014 final acceptance 08 july 2014 keywords bioassay, brood, light, group size, formica polyctena corresponding author ewa joanna godzińska laboratory of ethology department of neurophysiology nencki institute of experimental biology pas, pasteur st. 3, pl 02-093 warsaw, poland e-mail: e.godzinska@nencki.gov.pl to the water reservoir. this test was successfully applied to identify about 80% of foragers in small colonies of lasius niger l. however, it is less suitable for experiments carried out to evaluate the effects of various experimental treatments, as the ants are tested in their home nests, and neither the number of workers, nor the quantity and quality of brood can be easily controlled. moreover, it is difficult to disentangle ant responses to humidity and illumination. therefore, we developeda new bioassay for behavioral and neuroethological ant research, the brood hiding test. during the test a group of adult ants is taken out of the nest and confined together with brood in a container exposed to strong light. the ants may interact with brood and, in particular, transport it to the provided shadowed area. experimental settings recommended for the application of that test to study the behavior of workers of the wood ant formica polyctena först (table 1) were chosen on the basis of the results of 20 pilot tests during which workers taken from a laboratory colony fragment (1-20) were confined together with 5 homocolonial pupae in various types of containers exposed to strong white light (fig. 1). single workers did not short note a szczuka et al. brood hiding test 346 show brood hiding behavior and their interactions with brood were limited to antennal contacts. more advanced interactions with brood appeared as a function of increasing group size. the values of the score quantifying the outcome of each test (1-9) were highly significantly positively correlated with the number of workers tested together (spearman rank correlation test: r = 0.8536, p < 0.000001). (fig. 1). this preliminary result was confirmed by another experiment in which brood hiding test was applied to compare the behavior of various subclasses of workers of f. polyctena (szczuka et al. unpublished results). in that experiment foragers collected from the trails performed less well than workers from other experimental groups. as reported by lenoir (1977; 1981), during the brood-retrieving tests brood is retrieved mostly by foragers. responses to brood displayed by ants during the brood hiding test and the brood-retrieving test are thus mediated by at least partly different proximate mechanisms. the brood hiding test may be used to investigate such questions as behavioral polymorphism within ant colonies, ontogeny of ant behavior, nestmate and species recognition, and behavioral differences between ants from various fig 1. the results of 20 min pilot tests (n = 20) investigating the responses of workers of the red wood ant formica polyctena to homocolonial brood (5 pupae) exposed to strong white light (5000 lx) in various types of containers containing a shadowed area. ant behavior was quantified as a score (1-9) denoting the most advanced form of interactions with brood observed during the test: 1: antennal contacts; 2: seizing; 3: attempt at transport (a short episode of transport no longer than 23 s); 4. transport; 5. hiding of 1 pupa in the hadowed area; 6. hiding of 2 pupae; 7. hiding of 3 pupae; 8. hiding of 4 pupae; 9. hiding of 5 pupae. two classes of nestmate workers were tested: nurses (taken out of artificial nest chambers from among workers employed in intranidal brood care), and foragers (taken from the foraging area of the same artificial nest). element of experimental procedure description experimental arena an open cylindrical glass container (5 cm high, inner diameter 10 cm) with the walls coated with fluon® shadowed area a small (25 mm x 30 mm x 5 mm) rectangular shelter made of aluminum foil with one of its longer side walls (facing the center of the arena) left open to allow the ants to enter the shadowed zone source of illumination strong white light (5000 lx) produced by the lamp “fotovita fv10” (ultra-viol sp. j.). chemical cues left by nestmates present (20 homocolonial ants are allowed to walk inside the container during 1 h preceding the first test carried out on a particular day) brood 5 homocolonial worker pupae time during which the ants are allowed to settle after the introduction into the test container and before the introduction of brood 30 min duration of the test 15 or 20 min recording of ant behavior digital video camcorder analysis of behavioral recordings software for the analysis of video recordings of behavior [for instance, “the observer video-pro” (noldus information technology)] table 1. experimental settings recommended for the application of the brood hiding test to study worker-brood interactions in the red wood ant formica polyctena först. these findings provide a new example of the crucial role of group size in the mediation of the expression of specific behavior patterns in social hymenoptera. the phenomenon of critical (threshold) group size necessary for the expression of a particular behavior trait was first described by chauvin (1954) and then reported in many other studies [reviewed in wilson (1971) and szczuka & godzińska (2004a)]. particularly clear-cut effects of group size on the expression of a specific behavior pattern were documented in a series of experiments during which workers of f. polyctena were confronted with dead adult houseflies (musca domestica l.) offered to them in the foraging areas of their nests (szczuka & godzińska 1997; 2000; 2004a; 2004b). the values of the score quantifying the responses of ants to prey increased as a function of increasing group size, and prey retrieval was observed only in groups counting at least 30-40 workers. several studies also reported similar relationships between worker group size and the degree of escalation of ant aggressive behavior (roulston et al., 2003; tanner, 2006; 2008; buczkowski & bennett, 2008). during our pilot tests brood hiding scores tended to be lower in the case of foragers than in the case of nurses sociobiology 61(3): 345-347 (september 2014) 347 systematic groups related both to phylogenetic distance and differences in ecology. groups of individuals tested together may be homogenous, but may also consist of individuals belonging to different castes and/or worker subclasses. not only homocolonial, but also allocolonial and/or allospecific brood may be used, and the tested ants may be subjected to the situation of choice between various categories of brood. behavior of the tested ants may be quantified by assigning a score to the outcome of each test, or by video recording the tests and analyzing the recordings by means of an appropriate software. the brood hiding test may also be accompanied by the administration of neuroactive compounds delivered by various techniques including acute and chronic oral administration, injections, and topical (transcuticular) application. the variables quantifying ant behavior may also be analyzed as a function of levels of specific neuroactive compounds in the brain or specific brain structures. such more complex versions of the brood hiding test may be applied to study neurobiological processes underlying such phenomena as task-related differences in responses to brood, effects of experience on expression of worker behavior, worker cooperation and communication, and inter-individual variability of behavior. acknowledgements this study was supported by the project n n303 3068 33 (2007-2011) of the ministry of science and higher education (poland). references buczkowski, g. & bennett, g. w. (2008) aggressive interactions between the introduced argentine ant, linepithema humile and the native odorous house ant, tapinoma sessile. biological invasions, 10: 1001-1013. doi: 10.1007/s10530-007-9179-9. chauvin, r. 1954. aspects sociaux des grandes fonctions chez l’abeille. la theorie du superorganisme. insectes sociaux, 1: 123131. heylen, k., gobin, b., billen, j., hu, t. – t., arckens, l. & huybrechts, r. s. (2008) amfor expression in the honeybee brain: a trigger mechanism for nurse–forager transition. journal of insect physiology, 54: 1400-1403. doi: 10.1016/j. jinsphys.2008.07015. hölldobler, b. & wilson, e. o. (2009) the superorganism. new york: w. w. norton. lenoir, a. (1977) sur un nouveau test éthologique permettant d’étudier la division du travail chez la fourmi lasius niger l. comptes rendus de l’acadèmie de sciences, paris, , série d, 284: 2557-2559. lenoir, a. (1979) le comportement alimentaire et la division du travail chez la fourmi lasius niger. bulletin biologique de la france et de la belgique, 113: 79-314. lenoir, a. (1981) brood retrieving in the ant, lasius niger l. sociobiology, 6: 153-178. muscedere, m. l., willey, t. a. & traniello, j. f. a. (2009) age and task efficiency in the ant pheidole dentata: young minor workers are not specialist nurses. animal behaviour, 77: 911-918. doi: 10.1016/j.anbehav.2008.12.018. roulston, t. h., buczkowski, g. & silverman, j. (2003) nestmate discrimination in ants: effect of bioassay on aggressive behavior. insectes sociaux, 50: 151-159. seid, m. a. & traniello, j. f. a. (2006) age-related repertoire expansion and division of labor in pheidole dentata (hymenoptera: formicidae): a new perspective on temporal polyethism and behavioral plasticity in ants. behavioral ecology and sociobiology, 60: 631-644. sorensen, a. a., busch, t. m. & vinson, s. b. (1984). behavioral flexibility of temporal subcastes in the fire ant, solenopsis invicta, in response to food. psyche, 91: 319-331. szczuka, a. & godzińska, e. j. (1997). the effect of past and present group size on responses to prey in the ant formica polyctena först. acta neurobiologiae experimentalis, 57: 135-150. szczuka, a. & godzińska, e. j. (2000) group size: an important factor controlling the expression of predatory behaviour in workers of the wood ant formica polyctena först. biological bulletin of poznan, 37: 139-152. szczuka, a. & godzińska, e. j. (2004a). the role of group size in the control of expression of predatory behavior in workers of the red wood ant formica polyctena (hymenoptera: formicidae). sociobiology, 43: 295-325. szczuka, a. & godzińska, e. j. (2004b). the effect of gradual increase of group size on the expression of predatory behavior in workers of the red wood ant formica polyctena (hymenoptera: formicidae). sociobiology, 43: 327-349. tanner, c. j. (2006). numerical assessment affects aggression and competitive ability: a team-fighting strategy for the ant formica xerophila. proceedings of the royal society of london b., 273: 2737-2742. doi: 10.1098/rspb.2006.3626. tanner, c. j. (2008). aggressive group behavior in the ant formica xerophila is coordinated by direct nestmate contact. animal behaviour, 76: 1335-1347. walsh, j. p. & tschinkel, w. r. (1974) brood recognition by contact pheromone in the imported fire ant, solenopsis invicta. animal behaviour, 22: 695-704. weir, j. s. (1958) polyethism in workers of the ant myrmica (part ii). insectes sociaux, 5: 315-339. wilson, e. o. (1971) the insect societies. cambridge, ma: belknap/harvard university press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3484sociobiology 65(4): 773-776 (october, 2018) special issue primitively eusocial behavior observed in colonies of augochlora amphitrite (hymenoptera: halictidae) reared in laboratory augochlora smith is a new world genus of halictid bees with a wide distribution, ranging from southern canada to the pampean region in argentina, where five species occur (dalmazzo & roig-alsina, 2011). two subgenera are recognized, oxystoglossella eickwort, which includes soil-dwelling, social species, and augochlora s.str. species, which nest in decaying wood (michener, 2007). species of the latter were considered solitary, based on limited data obtained from nests in the field (eickwort & eickwort, 1973; zillikens et al., 2001) and from laboratory study of the solitary species augochlora pura (say) (stockhammer, 1966). the existence of eusocial behavior in this subgenus was surmised from studies of nests of augochlora isthmii schwarz and augochlora amphitrite (schrottky) in the field (wcislo et al., 2003; dalmazzo & roig-alsina, 2012) and recently confirmed, based on the study of artificial nests of augochlora phoemonoe (schrottky) (dalmazzo & roig-alsina, abstract the present study provides evidence of primitively eusocial behavior in augochlora amphitrite (schrottky). bees were reared in laboratory nests and observed throughout their nesting cycle. introduced foundresses constructed nests solitarily, but upon the emergence of the first daughter their activities changed drastically, marking the onset of a social phase. the colonies presented two well defined female castes according to their physiology, size and behavior. foundresses monopolized oviposition, displayed low rates of nest construction, guarding, and pollen collection, they were the individuals that initiated social interactions, and were statistically larger. daughter bees were smaller, with undeveloped ovaries, performed most tasks at the nest and were the subordinate individuals in social interactions. sociobiology an international journal on social insects 1 museo argentino de ciencias naturales “bernardino rivadavia” conicet, ciudad autónoma de buenos aires, argentina 2 universidad nacional del litoral, conicet, santa fe, argentina article history edited by cândida aguiar, uefs, brazil eduardo almeida, usp, brazil received 15 may 2018 initial acceptance 16 july 2018 final acceptance 26 august 2018 publication date 11 october 2018 keywords artificial nests, social interactions, wild bees, augochlorini. corresponding author milagros dalmazzo cátedra de entomología departamento de ciencias naturales facultad de humanidades y ciencias universidad nacional del litoral conicet ciudad universitaria paraje el pozo cp: 3000, provincia de santa fe, argentina. e-mail: milidalmazzo@yahoo.com 2015). these findings suggest that eusocial behavior could be more frequent in the subgenus, and that solitary forms could be derived from eusocial ancestral behavior. more data on different species are needed to test this hypothesis in a phylogenetic framework. this study analyzes the social behavior observed in three colonies of a. amphitrite reared in artificial nests maintained in laboratory conditions. twenty-nine adult females foraging on flowers were collected in the surroundings of buenos aires during spring (october november) of 2008 and 2009, marked on the scutum with different colors of nail polish, and introduced into a flight room. they were placed into small holes in the substrate of the artificial nest (fig 1; for detailed description of artificial nests and flight-room construction, see dalmazzo, 2018). five females succeeded establishing active nests and produced at least one brood. two females produced only males. daughter females were marked with a two-color code on the scutum: m dalmazzo1,2, a roig-alsina1 short note mailto:milidalmazzo@yahoo.com m dalmazzo, a roig-alsina – eusocial behavior in colonies of augochlora amphitrite774 one color indicated to which nest a female belonged (same color as her mother), and the other one allowed to discriminate between daughters of the same nest. nests were observed daily from the introduction of foundresses until dissection of the nests at the end of the season (march). each observation session took place between 8:00 and 20:00, totaling an average of 45 hours per nest. during each observation session, each nest was watched by the same observer continuously for 20 min. the order in which the nests were observed was randomly assigned each day to avoid observation biases. focal sampling was used to trace the behavior of individuals; the number of times (frequency counts) that each individual performed each activity or interaction from the behavioral catalogue was recorded on nests that contained daughter females. the behavioral catalogue is the same as the catalog detailed for a. phoemonoe (dalmazzo & roig-alsina, 2015). the life cycle of a. amphitrite in the laboratory nests was characterized by a solitary phase in which the five introduced foundresses constructed a main tunnel and 1 to 3 clustered cells. these females foraged in the flight room, provisioned and laid eggs in the cells, and also guarded the nest entrances. this phase lasted an average of 32 (± 2.05) days. the developmental time of emerged females varied between 30 and 35 days (n = 7, median = 32) and that of emerged males varied between 28 and 31 days (n = 9, median = 30). the activities of the foundresses changed drastically upon the emergence of the first daughter, marking the onset of the social phase (table 1). two nests had three emerged daughters and one had a single daughter, the remaining two nests produced only males and their foundresses had unfertilized spermathecae. one daughter female emerged from one of these nests with 3 daughters, dispersed, and founded her own nest producing only males. nest productivity ranged from 2 to 6 individuals (adults and pupa stages), with an average of 4.33 ± 1.7 (n = 3). the cells produced 2 ± 0.82 (mean ± sd) of males, which emerged in february march. emerged males stayed within the nest for 12 – 20 hours and did not participate in any activity. at the time of dissection of the nests by the end of march, one individual (male) was still at the stage of pupa. we found statistical differences (mann–whitney u test) between frequencies of activities performed by foundresses (n = 3) and daughters (n = 6) during the social phase (table 1). daughter females had higher frequencies of construction and pollen collecting. guarding was mostly performed by daughters during the social phase (p value = 0.07, close to 0.05). on the other hand, interactions, such as passing, antennation-tarsation and following, were mostly initiated by foundresses, and oviposition was exclusively performed by them (table 1 indicates statistical differences). the bees were dissected at the end of the nest cycle and classified according to their ovarian development (groups a to c, from ovaries well developed to not developed) and wear of wings and mandibles (in a scale from 0 to 3, intact to much worn) (dalmazzo & roig-alsina, 2012). foundresses were characterized by well developed ovaries (groups a and b; n = 3) and worn wings (classes 2 and 3; n = 3) and mandibles (class 3 n = 3), while daughter bees had slender ovaries (group c; n = 6) and less worn wings (classes 1 and 2; n = 6) and mandibles (classes 1 and 2; n = 6). the mean values of length of body (lb) and length of forewing (lf) were significantly larger in foundresses (lb = 11.33 ± 0.94; lf = 5.48 ± 0.14 mm) than in daughter bees (lb = 8.46 ± 1.2; lf = 4.99 ± 0.34 mm wilcoxon test, p = 0.03). the only species of augochlora s. str. for which primitively eusocial behavior is well documented is a. phoemonoe (dalmazzo & roig-alsina, 2015). as in this species, we found in a. amphitrite two well defined castes according to their physiology, size and behavior. foundresses monopolize oviposition, and display low rates of nest construction, guarding, and pollen collection during the social phase (table 1); they are the individuals that initiate fig 1. aartificial nest where the colonies of augochlora amphitrite were reared; bdaughter female and sawdust from cell construction between the glass panes of the artificial nest. sociobiology 65(4): 773-776 (october, 2018) special issue 775 social interactions. daughter bees have undeveloped ovaries, perform most tasks at the nest and are the subordinate individuals in social interactions. castes are morphologically alike, except that mean size of foundresses was higher than mean size of daughter bees. sub-castes, such as guards or foragers (michener, 1990) were not identified. most daughter females stay in the nest helping in the production of a new generation, but one of them was able to initiate her own nest. disperser females are known in social species of the tribe halictini (michener, 1990), as well as in two species of augochlorini: augochlorella striata (provancher) (mueller, 1996) and augochloropsis iris (schrottky) (coelho, 2002), which are primitively eusocial. the present study provides evidence of primitively eusocial behavior (michener, 1990) in a. amphitrite, involving cooperative brood care among motherdaughter assemblages, wherein the foundress monopolizes oviposition and the daughters perform most of the tasks at the nest, and castes are differentiated by size only. foundresses (n=3) daughters (n=6) u p solitary phase social phase locomotion mean±sd 21±7.74 44.33±3.05 50.86±21.45 4.5 0.17 feeding mean±sd 7.33±4.93 12.67±1.53 19±11.40 8 0.57 construction mean±sd 18±8.72 3±2.64 20.57±15.46 2 0.05 pollen collecting mean±sd 6.67±1.15 0 8.14±6.47 1.5 0.03 guarding mean±sd 7±2.64 3.33±2.08 22±15.90 2.5 0.07 oviposition mean±sd 1.67±1.15 2±1 0 <0.001 0.003 sudden retreat mean±sd 0 20.33±7.50 0.86±1.07 <0.001 0.01 antennation-tarsation mean±sd 0 31±8.54 2.28±1.60 <0.001 0.02 passing mean±sd 0 18.33±1.53 1±0.58 <0.001 0.01 following mean±sd 0 12.33±6.03 0.57±1.13 <0.001 0.01 table 1. activities and interactions performed by foundresses and daughters observed in three nests of augochlora amphitrite. median frequencies and standard deviation (sd) are presented for each type of activity and interaction. bold values indicate statistical significant differences between foundresses and daughters during the social phase (u: mann–whitney u test, p≤ 0.05). acknowledgements we thank eduardo almeida and two anonymous reviewers for their comments on the manuscript. this study was supported by grants anpcyt, argentina, 2007-1238, and conicet, argentina, pip 2011-0288. references coelho, b.w.t. (2002). the biology of the primitively eusocial augochloropsis iris (schrottky, 1902). insectes sociaux, 49: 181-190. dalmazzo, m. (2018). biología de nidificación de la abeja eusocial primitiva augochlora phoemonoe (halictidae). revista del museo argentino de ciencias naturales, nueva serie, 20: 1-10. dalmazzo, m. & roig-alsina, a. (2011). revision of the species of the new world genus augochlora (hymenoptera, halictidae) occurring in the southern temperate areas of its range. zootaxa, 2750: 15–32. dalmazzo, m. & roig-alsina, a. (2012). nest structure and notes on the social behavior of augochlora amphitrite (schrottky) (hymenoptera, halictidae). journal of hymenoptera research, 26: 17–29. doi: 10.3897/jhr.26.2440 dalmazzo, m. & roig alsina a. (2015). social biology of augochlora (augochlora) phoemonoe (hymenoptera, halictidae) reared in laboratory nests. insectes sociaux, 62: 315–323. doi: 10.1007/s00040-015-0412-8 eickwort, g.c. & eickwort, k.r. (1973). notes on the nests of three wood-dwelling species of augochlora from costa https://doi.org/10.1007/s00040-015-0412-8 m dalmazzo, a roig-alsina – eusocial behavior in colonies of augochlora amphitrite776 rica (hymenoptera: halictidae). journal of the kansas entomological society, 46: 17–22. michener, c.d. (1990). reproduction and castes in social halictine bees. in w. engels (ed.), social insects: an evolutionary approach to castes and reproduction (pp. 77121). berlin heiderbelrg-new york: springer. michener, c.d. (2007) the bees of the world. 2nd ed. baltimore: johns hopkins university press. mueller, u.g. (1996). life history and social evolution of the primitively eusocial bee augochlorella striata. journal of the kansas entomological society, 69: 116-138. stockhammer, k.a. (1966). nesting habit and life cycle of a sweet bee, augochlora pura. journal of the kansas entomological society, 39: 157–192. wcislo, w.t., gonzalez, v.h. & engel, m.s. (2003). nesting and social behavior of a wood-dwelling neotropical bee, augochlora isthmii (schwarz), and notes on a new species, a. alexanderi engel (hymenoptera: halictidae). journal of the kansas entomological society, 76: 588–602. zillikens, a., steiner, j. & mihako, z. (2001). nest of augochlora (a.) esox in bromeliads, a previously unknown site for sweat bees (hymenoptera: halictidae). studies on neotropical fauna and environment, 36: 137–142. doi: 10.1076/ snfe.36.2.137.2133 doi: 10.13102/sociobiology.v61i3.250-257sociobiology 61(3): 250-257 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant assemblages (hymenoptera: formicidae) in three different stages of forest regeneration in a fragment of atlantic forest in sergipe, brazil. ecf gomes, gt ribeiro, tms souza, l sousa-souto introduction remaining forests are important for the maintenance of favorable environmental conditions for the establishment and persistence of native fauna (gibson et al., 2011; ulyshen, 2011). several studies have shown that part of the worldwide decline in biodiversity, threatening the functioning of ecosystems, is related to anthropogenic modification of the landscape (dirzo & raven, 2003; colombo & joly, 2010; tabarelli et al., 2010), including the atlantic forest, one of the main hotspots in the world (myers et al., 2000). deforestation of atlantic forest is considered a constant threat to biological diversity (melo et al., 2009; oliveira et al., 2004), including ant assemblages (leal et al., 2012) and the monitoring of areas in process of plant recovery can be an important tool in the diagnosis of these threats (conceição et al., 2006; delabie et al., 2006). due to its abundance in most terrestrial ecosystems ants are considered ecologically dominant and play complex ecological roles such as ecosystem engineers, predators, herbivores and seed dispersal agents (hölldobler & wilson, abstract in this study we compared the epigeic ant assemblages in forest fragments with three different status of plant recovery (an area reforested in 2007, another reforested in 2005 and another one of secondary forest, with over 35 years of plant regeneration), located in the municipality of laranjeiras, sergipe, brazil. the ants were sampled in february (dry season) and june (rainy season) of 2012. we tested the following hypotheses: (1) the species richness of ants increases with time after the process of forest restoration; and (2) there are significant changes in species composition of ants among the three stages of forest regeneration. twenty-five pitfall traps were installed in each area. a total of 82 morphospecies of ants were sampled, distributed in 31 genera and seven subfamilies. the richness of ants was similar among the three sites (f = 1.71, p = 0.19). the composition of ant species, however, was different in the area of late regeneration (35 years) compared to other areas of early reforestation (p <0.05). thus, epigeic ants were partially sensitive to changes in the habitat studied in response to reforestation, presenting changes in species composition but no differences in ant species richness among areas. we conclude that seven years after reforestation are not enough to restore the same ant diversity in disturbed environments. sociobiology an international journal on social insects universidade federal de sergipe (ufs), são cristóvão, sergipe, brazil article history edited by kleber del-claro, ufu, brazil received 27 may 2014 initial acceptance 10 june 2014 final acceptance 30 june 2014 keywords bioindicators, degraded areas, environmental monitoring, species composition, forest recovery. corresponding author genésio tâmara ribeiro universidade federal de sergipe cidade univ. prof. josé aloisio de campos av. mal. rondon, s/n, jardim rosa elze são cristovão-se, brazil 49100-000 e-mail: genesiotr@hotmail.com 1990; folgarait, 1998). in tropical ecosystems the importance of ants is more evident because they can represent up to 60% of all arthropod biomass, and approximately 90% of their abundance (hölldobler & wilson, 1990; floren & linsenmair, 1997). species richness and structure of ant assemblages can be used as response variables in environmental monitoring, as these insects are sensitive to anthropogenic activities, including agricultural practices (hernández-ruiz & castaño-meneses, 2006) and reforestation (pais & varanda de 2010; schmidt et al., 2013). therefore, the study of these insects is useful to assess the success of forest restoration practices (sobrinho et al., 2003; silva & silvestre, 2004; holway & suarez, 2006; wetterer, 2012). although recovery of degraded areas is commonly used to reduce the negative environmental impacts on forest remnants (metzger, 2009; calmon et al., 2011) and, in spite of several studies on the role of the replanting of native species in accelerating the recovery of degraded environments, there are still many questions about the time required for the recovering of ant fauna along a gradient of forest regeneration research article ants sociobiology 61(3): 250-257 (september 2014) 251 of fragments dominated previously by an agricultural matrix (neves et al., 2010; teodoro et al., 2010; leal et al., 2012). processes that influence the structure and species diversity of epigeic ants in agroecosystems are still poorly known (neves et al., 2010; teodoro et al., 2010), despite the increasing conversion of forest fragments in less diverse and structurally simple habitats (primack & corlett, 2005; barona et al., 2010). in this study, we investigated the response of epigaeic ant assemblages in forest fragments with three different status of plant recovery, aiming to test the following hypotheses: (1) species richness of ants increases with time after the process of forest restoration (following an increase in habitat complexity) and (2) the composition of ant species undergoes changes along a gradient of regeneration of reforested area, with reduction of generalist species. material and methods the study was conducted in three sites: two sites were previously plantations of sugar-cane that were reforested, one with 32ha in 2005 (rf1) and another with 30.7ha and reforested with native species in 2007 (rf2). the third area is a secondary atlantic forest fragment with 55ha (ff) used as “area of permanent preservation” (app). all sites are located at fazenda boa sorte, a large sugar-cane company, located in the municipality of laranjeiras (10° 48’ 44”s, 37° 10’ 16” w), state of sergipe, brazil. the studied region is dominated by agricultural land with altitude ranging from 30 to 68 m a.s.l. the mean annual temperature is 25.5 °c and annual average rainfall of 1,200 mm. the rainy season usually lasts from may to october. the original vegetation was dominated by atlantic forest and all remnants are embedded in a 20 year-old, homogeneous matrix of sugar-cane fields (cuenca & mandarino, 2007). the soil type is spodozol, mainly sandy clay, deep, with low fertility and high porosity (draining rainfall). the area reforested in 2005 (rf1) with 32 ha, is at an intermediate stage of development, with seven years of planting and composed of trees with canopy of approximately 4-6 meters (10°49’15,8”s; 37°09’41”w). the other area, reforested in 2007 (rf2), with 30.7 ha is in early stage of development, with five years of planting and composed by sparse patches of woody vegetation, shrubs, herbs and grasses with a single layer of treetops with up to 4 m tall (10°49’01,6”s; 37°09’40,7”w). fourteen species of trees native to the atlantic forest were used in reforestation: tapirira guianensis, caesalpinia echinata, genipa amerciana, spondias lutea, schinus terebinthifolius, erythrina velutina, enterolobium contorsiliquum, cleome tapia, caesalpinia leiostachya, inga marginata, cassia grandis, lonchocarpus sericeus, anadenanthera macrocarpa and hymenaea courbaril the fragment (ff) of atlantic forest is an area of secondary forest, protected from logging for over 35 years and consists of trees with 7-20 meters in height that forms a closed canopy (10°49’17”s; 37°11’13”w). epigeic ants were sampled in 15 transects of 50 m, being five transects per area. we established a minimum distance of 150 m between each transect. ant sampling was conducted using pitfall traps on the ground surface. in each transect, five pitfalls were installed at a distance of 10 m, totaling 25 pitfalls/site. pitfalls consisted of 1,000 cm3 plastic pots containing approximately 120 cm3 water with detergent and were kept for 48 h in the field (schmidt & solar, 2010). sampling was conducted in two periods, one during the dry season (february 2012) and another during the rainy season (june 2012). all ants collected were sorted to species level when possible or morphospecies, using identification keys from bolton (1994) and fernandez (2003) and later the identification was confirmed through comparison with specimens from the collection of the laboratório de ecologia de comunidades (ant collection), of the universidade federal de viçosa and laboratório de mirmecologia of the cepec/ ceplac, ilhéus, bahia, brazil. voucher specimens of all species are deposited in laboratório de pragas florestais of the universidade federal de sergipe. to verify the effect of habitat type (rf1, rf2, and ff) and sampling period (wet or dry season) (response variables) on the species richness of ants (explanatory variable) the linear mixed effect (lme) was used, followed by residuals analysis to verify the adequacy of the error distribution and the fit of the model. fixed factors were sampling sites (rf1, rf2, and ff), while samples (nested within sites) were treated as random factors. a minimum adequate model (mam) was obtained by extracting non-significant terms (p < 0.05) from the full model arranged by all variables and their interaction (crawley, 2007), using the software r (r development core team, 2009). a non-metric multidimensional scaling (nmds) analysis was carried out to verify differences in the composition of ant fauna among the forest regeneration types (neves et al., 2010). the ordination was conducted using the jaccard index. additionally, similarity analysis (anosim; clarke, 1993) were conducted to compare the difference between two or more groups of sampling units among sites. differences between r-values were used to determine similarity patterns among ant assemblages in the three sites. the analysis were conducted using the software past (hammer et al., 2001). results we collected 82 ant morphospecies, distributed in 31 genera (table 1). the subfamilies myrmicinae and formicinae presented 66% of all ant species sampled, with 42 and 12 morphospecies, respectively. the genera pheidole and camponotus presented the higher richness with 11 (13.5%) and 10 (12%) morphospecies, respectively. twelve species were restricted to rf1 site, six species were found exclusively in rf2 site while 34 species were restricted to ff site (table 1). ecf gomes et al ant assemblages in a fragment of atlantic forest252 table 1. relative frequency of epigeic ant species collected in pitfalls, during the wet and dry season of 2012 in three sites of different forest regeneration stages: a fragment of secondary forest (ff) one area of reforestation with five years (rf2) and other with seven years of reforestation (rf1). ant subfamilies occurrence in each season ff rf1 rf2 dry wet dry wet dry wet dolichoderinae dolichoderus lutosus 0.2 dolichoderus diversus 0.2 dolichoderus attelaboides 0.2 dorymyrmex biconis 0.2 azteca sp. 1 0.8 0.4 azteca sp. 2 0.2 azteca sp. 3 0.2 ecitoninae labidus praedator 0.4 labidus coecus 0.4 0.4 nomamyrmex esenbeckii 0.2 neivamyrmex diana 0.2 formicinae brachymyrmex pr. patagonicus 0.2 camponotus trapezoideus 0.2 camponotus renggeri 1 0.4 camponotus bispinosus 0.2 camponotus novogranadensis 0.6 1 camponotus fastigatus 1 camponotus arboreus 0.2 0.2 camponotus cingulatus 0.8 camponotus vittatus 0.4 0.8 1 0.6 0.8 camponotus (myrmaphaenus) sp.9 1 0.8 0.8 camponotus rufipes 0.4 0.2 1 nylanderia pr. fulva 0.4 0.2 myrmicinae piramica pr. perpava 0.4 0.4 piramica sp. 2 0.2 cephalotes atratus 0.6 cephalotes umbraculatus 0.2 cephalotes minutus 0.2 cephalotes maculatus 0.2 cephalotes pusillus 0.2 cephalotes depressus 0.4 acromyrmex balsani 0.6 0.4 0.2 acromyrmex rugosos rugosos 0.6 0.4 0.2 0.2 atta sexdens rubropilosa 0.2 0.2 cyphomyrmex minutus 0.2 cyphomyrmex transversus 0.6 0.2 0.8 0.4 mycetosoritis sp. 1 * 0.2 mycocepurus obsoletus 0.4 sericomyrmex sp. 1 0.2 0.2 sericomyrmex sp. 2 0.4 table 1. (continued) ant subfamilies ff rf1 rf2 dry wet dry wet dry wet trachymyrmex sp. 1 0.2 monomorium floricula 0.2 0.8 solenopsis tridens 0.2 0.2 solenopsis sp. 2 0.2 0.6 solenopsis sp. 3 0.6 solenopsis saevissima 0.2 0.6 0.6 0.8 0.4 solenopsis globularia 0.2 1 0.8 hylomyrma balzani 0.2 0.2 pheidole radoszkowskii 0.4 0.4 1 1 0.6 1 pheidole fimbriata 0.2 0.4 pheidole (gr. diligens) sp. 3 0.4 0.8 pheidole sp. 4 0.6 1 pheidole (gr. flavens) sp. 5 0.2 0.4 0.4 pheidole (gr. tristis) sp. 6 0.8 0.4 0.2 pheidole (gr. fallax) sp. 7 1 0.4 0.8 1 pheidole (gr. diligens) sp. 8 0.2 1 0.2 pheidole sp. 9 0.4 pheidole (gr. fallax) sp. 10 0.4 0.6 0.2 pheidole (gr. fallax) sp. 11 0.6 0.8 0.8 0.4 0.8 0.4 crematogaster abstinens 1 0.4 0.4 1 crematogaster sp. 2 0.2 crematogaster pr. distans 0.6 crematogaster sp. 4 0.4 crematogaster sp. 5 0.2 cardiocondyla emeryi 0.2 0.2 ponerinae odontomachus haematodos 1 1 1 0.6 0.8 1 leptogenys unistimulosa 1 0.6 0.6 0.8 1 0.4 hypoponera sp. 1 0.2 pachycondyla venerae 0.4 0.4 pachycondyla harpax 0.4 1 ectatomminae gnamptogenys acuminata 0.2 gnamptogenys sulcata 0.4 0.4 ectatoma bruneunn 0.4 0.6 0.6 ectatoma tuberculatum 0.2 0.6 0.2 ectatoma edentatum 0.4 0.8 0.2 0.6 pseudomyrmecinae pseudomyrmex tenuis 0.8 0.6 pseudomyrmex termitarius 0.2 0.2 pseudomyrmex sp. (gr. pallidus) 0.2 pseudomyrmex sp. 4 0.4 pseudomyrmex gracilis 0.2 pseudomyrmex sp. 6 0.2 pseudomyrmex sp. 7 0.2 ff34 ve/ rf1.12 vd/ rf2.6 p/ comuns30; * new genus sp.1, r. feitosa (personal communication, 19 september 2012) sociobiology 61(3): 250-257 (september 2014) 253 there was no significant difference in species richness of ants among the three sites of forest regeneration (f2,22 = 2.26, p = 0.12). however, the species richness of ants was lower in the wet season in the rf1 and rf2 sites, compared to ff area (f1,22 = 11.19, p = 0.002) (fig.1). the nmds analysis indicates the formation of two distinct groups (stress = 0.15) with one group represented by ff and another group formed by rf1 and rf2 (fig.2). besides, the analysis of similarity (anosim) indicated significant difference in the structure of ant assemblages between ff versus rf1 (p = 0.003) as well as ff versus rf2 (p = 0.001) (table 2). the simper analysis indicated that the morphospecies that contributed most to the differentiation among sites were pheidole (group fallax) sp.7, camponotus (myrmaphaenus) sp 9, crematogaster abstinens, camponotus vittatus, solenopsis saevissima, pheidole sp. 4, cyphomyrmex transversus, solenopsis globularia, ectatoma edentatum, camponotus renggeri and pseudomyrmex tenuis. these morphospecies together contributed to 31.5% of cumulative dissimilarity among stages of plant recovery (table 3). table 2. analysis of similarity (anosim) among three sites of different forest regeneration stages: a fragment of secondary forest (ff) one area of reforestation with five years (rf2) and other with seven years of reforestation (rf1). fmn rf1 rf2 fmn 0.0003** 0.0001** rf1 0.0003** 0.0732 rf2 0.0001** 0.0732 ** significant difference, p< 0,01. discussion in our study the species richness did not differ with time of restoration and, on the one hand, it suggests that five years are enough for the recovery of ant species richness. this time can be considered short compared to that from other studies conducted by vasconcelos (1999) and roth et al. (1994) (10 and 25 years, respectively). on the other hand, however, the differences found in species composition among ff and rf1 or rf2 indicate that other parameters, rather than species richness, are important to make decisions about the use of ants as bioindicators. an increase in species richness in ff might be correlated with a more complex environment, leading to an increase in availability of resources (matos et al., 1994, oliveira et al., 1995). the restoration of rf1 and rf2 sites with native species of trees might have created favorable conditions for colonization of ants and have led to comparable values of species richness in ff area. the colonization of ants, however, seems to have been carried out by ant species from adjacent agroecosystems and not from nearby forested areas, since the composition of species between ff and the other two areas differ greatly. difference in ant species composition between more complex areas and regenerating ones have been found by other authors (vasconcelos, 1999; schmidt et al., 2013). in general, generalist species have higher colonization rate of disturbed fragments than do specialist ants (schoereder et al., 2004). although there were no differences in species richness among sites, there were differences between season of sampling, with higher values in the dry season of rf1 and rf2 (seasonality was not tested as a hypothesis in this study, and thus we used this variable only as a source of variation in the statistical model). table 3. simper analysis among three sites of different forest regeneration stages: a fragment of secondary forest (ff) one area of reforestation with five years (rf2) and other with seven years of reforestation (rf1). species cumulative percent of dissimilarity (%) pheidole (group fallax) sp.7 3.407 camponotus (myrmaphaenus) sp.9 6.709 crematogaster abstinens 9.905 camponotus vittatus 12.89 solenopsis saevissima 15.77 pheidole sp. 4 18.58 cyphomyrmex transversus 21.25 solenopsis globularia 23.92 ectatoma edentatum 26.59 camponotus renggeri 29.05 pseudomyrmex tenuis 31.51 figure 1. species richness of ants in the dry season (white bars) and rainy season (hatched bars) (mean ± se) sampled in three areas with different stages of forest recovery. rf1 = fragment with 7 years of reforestation; rf2 = fragment of 5 years of reforestation and ff = forest fragment with 35 years of plant recovery (d and w indicate sampling in dry and wet seasons, respectively). different letters on bars indicate significant difference within the same site (p <0.05). ecf gomes et al ant assemblages in a fragment of atlantic forest254 changes in the frequency of foraging ants have been observed with environmental seasonality (wolda, 1988; kaspari, 2000; castro et al., 2011; cook et al., 2011). the availability of resources is reduced during the dry months of the year, and the increased mobility of ants in this period might explain the rise in species richness in the dry season (andow, 1991; dantas et al., 2011). these results, however, should be viewed with caution since the data were collected considering just a year and a longer period of collection is needed to establish more secure inferences about effects of seasonality on ant species. considering that thirty-four species were restricted to ff site (42% of all species found) our results make it clear that environmental differences among the areas were crucial for determining the composition of species. the most common species in the ff were camponotus renggeri, pheidole sp.4 and pseudomyrmex tenuis, indicating a preference of these species for less-disturbed environments. notably, several studies also demonstrate the occurrence of c. renggeri in seasonal forest formations such as cerrado (del-claro & oliveira, 1999; christianini et al., 2007; neves et al., 2012) and forests in the semi-arid region of brazil (hites et al., 2005). conceição et al. (2006) also have reported the presence of p. tenuis in environments with low disturbance. besides, predatory species of the genus pyramica (masuko, 2009) were also found only in the ff, suggesting that this fragment has a more suitable resource availability than the other areas. the genera pheidole (group fallax) sp.7, camponotus (myrmaphaenus) sp.9, crematogaster abstinens, solenopsis globularia and cyphomyrmex transversus, were found in rf1 and rf2, indicating that these species could have preference for colonization in degraded areas. other studies in the atlantic forest recorded s. globularia in a disturbed mangrove area (delabie et al., 2006) and c. transversus in grasslands (braga et al., 2010). in fact, the areas rf1 and rf2 have low density of tree species, allowing the establishment of herbaceous species. individuals of solenopsis saevissima exhibit aggressive behavior and are also usually associated with disturbed environments (silvestre et al., 2003). the presence of this species is favored in sites colonized by pioneer plant species, typically found in early sucessional areas (vasconcelos, 2008; schmidt et al., 2013). although camponotus vittattus was sampled in all three areas, this species had similar occurrence to s. saevissima being more frequent in samples from rf1 and rf2, thus suggesting its preference for opened sites. in contrast, we also reported the presence of some ant species in sites with late regeneration time (rf2 and ff), such as ectatoma edentatum. previous studies have associated the occurrence of this species with advanced stages of plant recovery (ramos et al. 2003; vasconcelos 2008). the rf1 site had a similar species richness of epigeic ants compared with rf2 or ff sites. however, the species composition differs considerably among environments with similar historical disturbances versus a forest fragment with late regeneration. our study shows that ant assemblages can vary greatly along a gradient of plant recovery and the conservation of forest fragments with different stages of reforestation is important to sustain more diverse ant fauna. therefore, reforestation programs that prioritize the conservation and the adoption of native plant species in these areas are a good alternative (gillespie et al. 2000), enabling the development of scientific studies and especially the maintenance of local biodiversity. acknowledgments the authors are grateful to júlio cezar mário chaul, laboratory of community ecology ufv and jacques h. c. delabie, laboratory of myrmecology cepec / ceplac for their help in species identification. this study was supported by the coordenação de aperfeiçoamento de pessoal de nível superior (capes). references andow, d.a. 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(1988). insect seasonality: why? annual review of ecology and systematics, 19: 1-18. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.4311sociobiology 66(2): 389-393 (june, 2019) occurrence of hymenoptera on pig carcasses in a tropical rainforest in central amazonia, brazil frequently many hymenoptera visit carcasses in natural environments, and being potential indicators of postmortem interval (pmi), seasonality, and body movement of carcasses (moretti et al., 2007). they can act as predators when feeding on immature and adult arthropods, such as scavengers, when consuming decaying tissues and parasites of larvae and pupae of dipterans and coleopterans that colonize the carcasses (oliveira-costa, 2011). succession studies of insect associated with decaying domestic pig carcasses in medellín, colombia, detected the presence of apidae epicharis klug, 1807, eulaema lepeletier, 1841, partamona and apis linnaeus, 1758 (wollf et al., 2001; pérez et al., 2005). two species of formicidae and one of apidae were reported in the succession of cadaveric entomofauna, using pig carcasses, in buenos aires, argentina (centeno et al., 2002). abstract hymenotpera species may act as necrophagous, consuming decomposing tissues, as predators, when they feed on other immature and adult insects, and parasites of larvae and pupae of dipterans and coleopterans that colonize the carcasses. in this way, the fauna of four hymenoptera families (vespidae, apidae, icheneumonidae and formicidae) associated to different decomposition stages of pig carcasses partially submerged in water stream (igarapé) of the terra-firme amazonian forest are presented. formicidae were the most abundant insects with 957 individuals collected all directly in the carcass, followed by vespidae (143), apidae (88) and ichneumonidae with nine individuals collected in the suspended trap. due to the aspect of the injuries caused by some hymenoptera to the carcasses, they may be mistaken as skin ulcers, burns or abrasions, which may mislead a forensic investigation. sociobiology an international journal on social insects a somavilla1, jlp souza1,2, ao da silva3, rlf keppler1 article history edited by evandro n. silva, uefs, brazil received 11 december 2018 initial acceptance 27 january 2019 final acceptance 01 may 2019 publication date 20 august 2019 keywords apidae, forensic entomology, formicidae, vespidae. corresponding author alexandre somavilla instituto nacional de pesquisas da amazônia – inpa coordenação de biodiversidade av. andré araújo, 2936, petrópolis cep: 69.067-375, manaus-am, brasil. e-mail: alexandresomavilla@gmail.com in brazilian amazonian terra-firme rainforests, bees like apis mellifera, trigona, partamona, cephalotrigona and melipona were collected in stages of domestic pig decomposition (vianna, 2010). in the same region, agelaia and angiopolybia wasps were collected in carrion traps within (silveira et al., 2005). and a. mellifera was also collected associated with pig carcass in the first stages of decomposition, in são paulo state (gomes et al., 2007) and partamona, trigonisca, plebeia and apis in dry forest in paraíba state (santos et al., 2014). for ants, an anoplolepsis longipes colony was used to estimate the pmi of human remains found in a metal box (goff & win, 1997) in hawaii. recently, studies have pointed out the use of social wasps and ants in forensic analyses (gomes et al., 2007; moretti et al., 2008, 2011; somavilla et al., 2019). however, 1 instituto nacional de pesquisas da amazônia – inpa, coordenação de biodiversidade, manaus-am, brazil 2 instituto nacional da mata atlântica-inma, santa teresa-es, brazil 3 instituto de proteção ambiental do amazonas – ipaam, manaus-am, brazil short note a somavilla et al. – hymenoptera on pig carcasses in central amazonia390 there are no published reports on postmortem injuries caused by these wasps. the real importance of necrophagy for brood nutrition in social wasps and ants is difficult to assess, but the skills at rapidly discovering pieces of carcasses (cornaby, 1974) may suggest carrion is an important source of protein or other nutrients. in this study, we evaluated the fauna of four hymenoptera families (apidae, vespidae, icheneumonidae and formicidae) associated to different stages of decom-position on pig carcass, sus scrofa, partially submerged in a water stream in the central amazonia. the sampling was carried out on ombrophilous terrafirme forest area at ducke reserve, near manaus, amazonas, brazil. the climate in the area is humid tropical, with a mean annual relative humidity around 80%, mean annual precipitation of 1,750-2,500 mm and mean annual temperature of 26 ºc. the vegetation is lowland tropical rainforest, with a closed canopy and shady understory, characterized by the abundance of palm trees (baccaro et al., 2008). samplings were made at barro branco water stream (02°55′49.4″ s, 59°58′28.1″ w), classified as a second order water stream (delgado, 1996). the collection occurred in two seasonal periods: dry season in july-august of 2009 and rainy season in november-december of 2009. in each experiment, we used three ~45 kg pig carcasses partially submerged. the animal was taken alive to the study site and was killed with a shot of a 38 mm caliber pistol in the frontal head. the carcass was placed in an iron cage (1.00 x 0.63 x 0.74 m; 3 x 3 cm mesh opening) to protect the carcass from large vertebrate scavengers. the choice of bait occurred because it is an animal similar to the human, in relation to diet, intestinal fauna, internal anatomy and decomposition patterns (amendt et al., 2004; marques, 2008). part of hymenoptera was obtained by manual collection with tweezers, and suspended traps (rafael & gorayeb, 1982), installed in the upper part of the iron cage approximately 30 cm (figure 1a). the specimens were identified and compared with the inpa invertebrate collection material. the voucher hymenoptera specimens were deposited in the same collection. the experiments were authorized and recorded under authorization number cep-ufam no 029/10, by the ethics committee on animal use, based on the brazilian control committee for animal experimentation (concea). we collected a total of 1,197 specimens in four hymenoptera families: vespidae, apidae, icheneumonidae (collected in the suspended trap and on the pig carcasses) and formicidae (collected directly on the pig carcasses). in the rainy season occurred greater abundance of vespidae, apidae, icheneumonidae and in the dry period a greater abundance of formicidae. independent of the seasonal period, formicidae represented the majority of individuals collected (957), followed by vespidae (143), apidae (88) and icheneumonidae with nine individuals (table 1). there was no variation of hymenoptera as a seasonal period and according the decomposition stages (paired t-test, p=0.917). formicidae and vespidae consumed the pig fresh and, occasionally, predated the immature calliphoridae (diptera), and apidae fed on body fluids, mostly. ichneumonidae did not participate in the decomposition process. table 1. absolute (n) and relative (%) abundances of the hymenoptera taxa collected in july-august (dry season) and november-december (rainy season) of 2009 in three stages (fresh, bloated and active decaying) on pig carcasses in the ducke reserve, manaus, am, brazil. taxa dry season rainy season total fresh stage bloated stage active decaying stage fresh stage bloated stage active decaying stage vespidae 22 10 66 45 143 agelaia angulata 02 05 07 agelaia constructor 03 02 05 agelaia fulvofasciata 05 02 12 07 26 agelaia pallipes 12 03 22 07 44 agelaia testacea 01 01 11 04 12 angiopolybia obidensis 01 02 09 17 angiopolybia pallens 04 03 14 11 32 apidae 20 09 38 21 88 trigona crassipes 15 06 29 15 65 trigona hypogea 05 03 09 06 23 ichneumonidae 01 02 06 09 ichneumonidae sp. 01 02 06 09 formicidae 291 237 172 257 957 camponotus femoratus 59 52 60 26 197 crematogaster carinata 169 142 43 126 480 nylanderia sp. 63 43 69 105 280 sociobiology 66(2): 389-393 (june, 2019) 391 vespidae: agelaia estacea (07 specimens), agelaia constructor (05), agelaia fulvofasciata (26), agelaia pallipes (44), agelaia estacea (17), angiopolybia obidensis (12) (figure 1b) and angiopolybia pallens (32) used the decaying pig as a source for feed, because of this habit of removing small pieces of decaying flesh, these species are of forensic entomological concern, since they remove tissue mainly from around the cavities of the carcasses, such as the nose, mouth, ear and anus (gomes et al., 2007, somavilla et al., 2019). in addition, some species were observed preying on calliphoridae (diptera) that were also colonizing the carcass, other wasps cutting pieces of meat in the natural carcass cavities, such as ears, nose, mouth and anus. wasps were collected in the initial stages of decomposition, fresh stage (1-24 hours) and bloated stage (24-48 hours). decomposing meat can be an important source of food for social wasps and for bees in the neotropical region (o’donnell, 1995). the wasps lacerated the skin and the musculature of the carcasses in the fresh and bloated stages, made by the jaws supported in the hind legs. the areas most affected were close to the jugular, the mucosa of the nose, eyes, mouth and genitalia, and less frequently in regions with lower hair density, such as the foot and abdomen. wasps feed on the superficial tissues of the carcasses, as well as the eggs and larvae of the first stage of diptera (gomes et al., 2007). this behavior has a potential to decrease the larval mass of diptera on the carcasses and, consequently, reduce the number of older fly larvae, and these older larvae are important to estimate the pmi. in addition, this behavior can increase the diameter of fig 1. acage model and suspended trap, with pig carcass; bwasp angiopolybia obidensis; cbee trigona crassipes; dant crematogaster carinata, on pig carcass in ducke reserve, manaus, am, brazil. a somavilla et al. – hymenoptera on pig carcasses in central amazonia392 the natural carcass cavities as well as the odor, speeding up the arrival of insects of great importance forensic (flies and beetles), as well as causing lesions that occasionally confuse the expert in the cadaveric analysis process, resembling perimortem lesions by puncture-blunt or puncture-sharp objects. therefore, it may have a relevance in determining the pmi, if such aspects are not considered (gomes et al., 2007). apidae: trigona crassipes (65 specimens) (figure 1c) and trigona hypogea (23) were collected in the bloated stage (24-48 hours) and active decaying stage (48-96 hours) on body fluids, probably as an alternative source of food, and santos et al. (2014) suggest some bees use animal carcasses as a possible source of water, and salts during inhospitable periods. probably these species of bees cut fresh pieces of meat in the carcass and when they arrive at their nests, regurgitate for the immature ones. regarding social bees, a number of stingless bee species have been observed collecting meat or juices from carcasses, and species of the trigona hypogea silvestri group are obligate necrophages, like trigona hypogea, t. crassipes, found in the amazon, and t. necrophaga, endemic to panama. (roubik, 1982; camargo & roubik, 1991). in t. hypogea and related species, pieces of flesh are not directly transported as such to the nest, being chewed and ingested at the feeding site, and contents from the crop are subsequently regurgitated to other workers in the nest. current evidence indicates that flesh is either metabolized into proteinaceous glandular secretions (camargo & roubik, 1991) or stored in protein storage pots mixed with honey (noll, 1997; serrão et al., 1997) to be used for nutrition of the larvae. ichneumonidae: these are immature parasitoid wasps of diptera and coleoptera influencing the decomposition of the corpse, delaying the degradation process, but not actively participating in it (catts & goff, 1992). in our study, they were collected in advanced decaying stage and remains stage, in the last four days. members of this family were also collected during active deterioration and pig remains in puerto rico wet forest (oliveira-costa, 2011). formicidae: crematogaster carinata (myrmicinae) (figure 1d), camponotus femoratus, and nylanderia sp. (both formicinae) (table 1). cr. carinata was the most abundant, with 480 individuals, followed by nylanderia sp. with 280, and ca. femoratus with 197 specimens, registered in both seasonal periods, bloated stage (24-48 hours) and active decaying stage (48-96 hours). in an urban area of são paulo, 77 specimens of crematogaster, camponotus and dolichoderus were collected (moretti et al., 2007). although ants be an abundant and constant group on carcasses, they have been the subject of few studies on its importance in criminal investigations. in the bloated stage, the ants feed on body fluids and then pig tissue. necrophagous ants can cause mutilation and other injuries when feeding in areas where they remove fragments of skin, small spots or scratches appear (campobasso et al., 2009). the influence of ants on the necrophagic community may be significant and will depend on their role as a necrophagous or as decomposer (moretti et al., 2007). formicidae occupies diverse ecological associations, being able to act as omnivores and predators, in the latter case they can reduce the decomposition process, already observed in rat carcass by the predation of eggs and larvae of colonizing flies (moretti & ribeiro, 2006; ramón & donoso, 2015). moreover, in the bloated stage and active decaying stage, individuals of cr. carinata preying on eggs and larvae of diptera in the two seasonal periods. in a fragment of the atlantic forest of pernambuco, ca. femoratus preyed eggs and larvae of calliphoridae in the first five days after death in pig carcass (jiron & cartin, 1981). knowledge on the behavior and injuries caused by hymenoptera is important for forensic examiners so as not to mistake the injuries as pre-mortem. this is relevant to crimes involving human beings, since the injuries that hymenoptera cause on the corpses during the different stages of decay, and the hymenopteran’ occurrence per se, can affect the conclusions of forensic investigations. the knowledge on the possible effects of the hymenoptera as consumers of fresh corpse is still incipient. further studies with these insects on carrion are required to analyze them as displacement species, consumers of carcasses’ resources, and to survey the rates of flesh and fly egg removal, thus assessing their importance to forensic entomology in the amazon region. acknowledgments the authors are grateful to tohnson sales and anastácio ribeiro (in memoriam) for support and help in the fieldwork. thanks the programa de apoio aos núcleos de excelência (pronex) cnpq-fapeam and the inpa/mcti/prj 12.24 sistemática integrada de insetos aquáticos, com ênfase em simuliidae (diptera: nematocera) na america do sul for financial support. we sincerely thank fundação de amparo à pesquisa do estado do amazonas for the postdoctoral scholarship (fapeam fixam) to a. somavilla. references amendt, j., krettek, r. & zehner, r. 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(2001). preliminary study of forensic entomology in medellín, colombia. forensic science international, 120: 53-59. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.4355sociobiology 66(3): 475-479 (september, 2019) drone production by the giant honey bee apis dorsata f. (hymenoptera: apidae) introduction the production and maintenance of male honey bees (drones) requires a major investment by colonies of eusocial honey bees (page 1981; page & metcalf, 1984; seeley, 1985; seeley, 2002). from the viewpoint of commercial honey bee management for the western honey bee species apis mellifera l., the investment in males has been long considered as a drain on overall honey production; to this point langstroth (1866) referenced drones as “useless consumers.” while this pragmatic view is understandable for a. mellifera production beekeeping, it falls outside the veil of reproductive ecology especially when considering asian apis species. for temperate a. mellifera biotypes males are produced on a seasonal basis with drone production commencing in the early spring, peaking in late spring to early summer and then declining in midto late summer (allen, 1963; page, 1981; lee & winston, 1987). for the cavity-nesting honey bee species a. mellifera and a. cerana f. and the two dwarf honey bee species a. florea f. and a. andreniformis smith abstract this study investigates male (drone) production by the giant honey bee (apis dorsata f.). the entire brood population from 10 colonies were counted to determine the immature population of drones’ relative to workers. after the condition of each cell was determined, the cell’s position and content were noted using the microsoft excel platform. the contents of the brood comb, including eggs, larvae, prepupae, capped worker pupae, capped drone pupae, pollen storage cells and finally empty brood cells, were recorded. results reveal the percent of pupal drones averaged 5.9 ± 6.8% with a range of 0.1 to 17.3%, of the total capped brood population. the size of the drone pupal population relative to the worker pupae was highly variable and displayed no correlation (r = 0.29). drone pupae distribution was scattered throughout the brood comb in a random manner when drone pupal populations were low; in instances of higher drone production, the drone pupae appeared in banded patterns concomitant with the worker pupal distribution. sociobiology an international journal on social insects b chuttong1, n buawangpong2, m burgett3 article history edited by celso martins, ufpb, brazil received 05 february 2019 initial acceptance 01 june 2019 final acceptance 27 july 2019 publication date 14 november 2019 keywords apis dorsata, giant honey bee, male investment, drone. corresponding author bajaree chuttong science and technology research institute chiang mai university chiang mai 50200, thailand. e-mail: bajaree@yahoo.com drones are reared in larger cells than those used to produce worker bees and a portion of the brood comb matrix is devoted to the larger drone cells. the amount of drone comb built by an a. mellifera colony is dependent on the size of the worker bee population and the time of year (free and williams, 1975). in a study of natural nests of a. mellifera seeley and morse (1976) found that an average of 17% of the total comb area is given over to drone comb. for the dwarf honey bee species a. florea burgett and titayavan (2004) reported 4.5% of the comb area of mature colonies is used for drone production. in a study of the natural nest of the eastern honey bee a. cerana somana et al. (2011) reported that 16.7% of the comb is available for drone production in the dry winter season and 5.7% in the wet summer season. the giant honey bee species a. dorsata f. is unique in that both drone and worker immatures are reared in the same sized brood cell (ramachandran 1937; lindauer 1957; thaker & tonapi 1961). tan (2007) and buawangpong et al. (2014) provide brood cell size ranges from 5.2 – 6.1 mm. tan (2007) reports that drone production is confined to brood cells with 1 science and technology research institute, chiang mai university, chiang mai, thailand 2 department of entomology and plant pathology, faculty of agriculture, chiang mai university, chiang mai, thailand 3 department of horticulture, oregon state university, corvallis, oregon, usa research article bees b chuttong, n buawangpong, m burgett – drone production by the giant honey bee 476 a diameter ≥ 5.5 mm; however, his data suggest that workers are reared in all cell diameters (5.2 – 6.1 mm). this poses a question that if for all other apis species, drone production is potentially limited by the presence of the differentially sized drone cell, what are the controlling factors for drone production with a. dorsata where there is no apparent cell size difference? an approach to partially address this question is to investigate male (drone) production by a. dorsata which is what we have done with this report. materials and methods for this study the single nest combs from 14 colonies of a. dorsata were obtained within a 15 kilometer radius of metropolitan chiang mai (18.71° n, 98.98° e) in northern thailand in 2016, 2017 and 2018. to determine a colony’s drone population requires the examination of the entire adult bee population as well as the immature stadia. the difficulty of capturing the entire adult bee cadre can be appreciated by any researcher who is at all familiar with giant honey bee colony defense. as described by wallace (1869) and morse and laigo (1969) the fierce defensive nature of a. dorsata is a considerable barrier. for our purpose it was decided to approach the question of drone investment by examining the ratio of worker to drone pupa. all colonies examined had resided on anthropogenic structures and were from 10 to 30 m above ground. to facilitate the removal of the brood comb, the adult bees were smoked off of the comb surface and by the use of a modified rice sickle, the brood combs were cut from their substrates and allowed to fall into an enlarged and strengthened sweep net outfitted with a plastic bag to contain the comb. combs were immediately returned to the chiang mai university campus where they were placed in a -20° c freezer. to determine the population of drone immatures relative to worker immatures (capped pupal stadia) we initiated a census of all brood cells. this was done by examining each contiguous brood cell commencing at the bottom of the comb and working our way to the top of the comb, row by row. as the condition of each cell was determined the cell’s position and content were noted using the microsoft excel platform. this allowed us to develop a spacial record of the contents of the brood comb, including eggs, larvae, prepupae, capped worker pupae, capped drone pupae, pollen storage cells and finally empty brood cells. the census data allow for a number of determinations such as area of brood comb, proportion of brood comb actively in use, ratios of immature stadia, and ultimately the proportion of male pupae to worker pupae. we tallied the complete brood population for 10 colonies. an additional 4 colonies had the brood counted on one comb side only. colonies ranged in age from ca. 3 to 6 months. precise colony ages could not be determined as all our colonies originated on structures where the occupants could provide only an estimate of when the bees first arrived. results & discussion drone and worker pupal populations table 1 provides the data contrasting drone and worker pupal counts. the percent of pupal drones averaged 5.9 ± 6.8% of the total pupal cohort with a range of 0.1 to 17.3% between the individual colonies. the size of the drone pupal population relative to the worker pupae was highly variable with an r = 0.29 indicating essentially no correlation. there is a weak correlation between the number of drone pupae and the total number of all brood stadia (r = 0.39). older colonies (>3 mo.) possessed higher numbers of drone pupae but this is an expected observation and because we do not possess more exact colony ages, we cannot attempt a statistical proof. comparative data for drone production for other apis species, with the exception of the western honey bee a. mellifera, is relatively sparse. allen (1963) in comparing worker and drone production for a. mellifera showed that 1.4% of seasonal brood production was given over to drones in colonies with limited drone comb. for colonies with excess drone comb she showed that 9.0% of the seasonal brood cohorts were drones. her data included all immature brood stadia, but our data for a. dorsata are narrowed to the pupal stage. this is because by using the same sized brood cell for producing workers or drones it is impossible to discriminate brood sex at the egg, larval or pre-pupal stages. our observation shows 5.9% of the total capped brood is limited to drones. page and metcalf (1984) show an average a. mellifera colony during the active brood rearing season, produced a drone population that was 7.9% of the total brood production. in western canada lee and winston (1987) examined a. mellifera drone production relative to swarming and from their data, 5.4% of the brood were devoted to drone production under the conditions of pre-swarming colonies. from the same study, drone brood from colonies established from primary swarms, was 6.2% of brood production. seeley and morse (1976) present data that show drones represent 4.8% of the adult bee population in feral a. mellifera colonies. colony # pupal sex worker drone % male c1 2,528 399 13.6 c2 5,354 5 0.1 c3 3,112 326 9.5 c4 3,040 2 0.1 c5 4,379 205 4.5 c6 12,157 81 0.7 c7 5,513 1,155 17.3 c8 9,803 1,467 13.0 c9 5,664 15 0.3 c10 4,760 4 0.1 average 5,631 366 6 sd ±3,075 ±523 ±7 table 1. worker drone pupal cohort and percent male. sociobiology 66(3): 475-479 (september, 2019) 477 drone investment data for the eastern honey bee (a. cerana) come from somana et al. (2011) who showed that 14.2% of total brood production was utilized for drones. when examining just the pupal worker and pupal drone populations they specify a figure of 14.7%. their data concerning the adult bee cohort revealed that drones comprised 4.3% of the adult bee population during the more active dry summer season of northern thailand. a study by burgett and titayavan (2004) examining a. florea colony biometrics, found that adult drones constituted 2.9% of the adult bee population in the wet summer season, but only 0.7% in the dry winter period. this can be compared to the findings of seeley and morse (1976) which showed the adult drone population for feral a. mellifera colonies to be 4.8%. in on-going research chuttong and burgett (unpublished) show that the adult drone population for a. florea colonies in the winter season to be 1.2% of the adult bee population and that the comb area dedicated to drone production is 11.2% of the total nest area. drone distribution figures 1-3 display the patterns and spatial distributions of drone pupae relative to worker pupae for three of the examined a. dorsata colonies. all three colonies (numbers 6, 7, 8) had well filled brood nests with brood occupancy rates of 95.3%, 89.2% and 90.2% respectively. colony 6 has a high worker/drone pupae ratio of 150 worker pupae for each drone pupa. colonies 7 and 8 possessed relatively robust drone populations with pupal worker/drone ratios of 4.8 and 6.8 workers per drone pupa respectively. colony 6 (fig 1) c6 side a c6 side b fig 1. colony 6: drone pupal distribution (red) relative to worker pupae (black) distribution. open areas house eggs, larvae and prepupae in this brood nest that had a 95.3% brood occupancy rate (brood cells utilized relative to brood cells available). this colony had a drone pupal population that was 0.7% of the total capped pupae. the drone brood appears in a random, scattered distribution which is typical of developing colonies that are commencing drone production. c7 side a c7 side b fig 2. colony 7: drone pupal distribution (red) relative to worker pupae (black) distribution. open areas housed eggs, larvae and prepupae in this brood nest that had an 89.2% brood occupancy rate (brood cells utilized relative to brood cells available). this colony had a drone pupae population that was 17.5% of the total capped pupae. colony 7 had the highest number of drone pupae of all the colonies in the study. the drone brood appears in bands which are concomitant with the banding pattern of the worker pupae. c8 side a c8 side b fig 3. colony 8: drone pupal placement (red) relative to worker pupae (black). open areas housed eggs, larvae and pre-pupae in this brood nest that had a 90.2% brood occupancy rate (brood cells utilized relative to brood cells available). this colony has a drone pupae population that was 13.9% of the total capped pupae. the drone brood appears as bands which are concomitant with the banding pattern of the worker pupae and is similar to the pattern of colony 7 (fig 2). exemplifies what we term a shotgun drone distribution pattern, where pupal drones appear randomly distributed throughout the capped brood area. colonies 7 and 8 (figs 2 and 3) show banding patterns as alluded to by tan (2004). no given quadrate of the comb surface is shown to preferentially host drone pupae, which is particularly well illustrated by the distribution of drone pupae in colonies 7 and 8. as concerns drone pupal placement relative to brood comb side, the data show that drone pupae are equally divided between the two comb sides (r = 0.89). regarding the equality of all broods when comparing the division between comb sides, the r = 0.89. b chuttong, n buawangpong, m burgett – drone production by the giant honey bee 478 summary our metric for a. dorsata drone investment of 5.9% of the pupal brood being given over to drones aligns reasonably well with published figures for a. mellifera; 9.0% (allen, 1963), 7.9% (page & metcalf, 1984) and 5.4 – 6.2% (lee & winston, 1987). it is lower than the 14.2% immature drone production for a. cerana (somana, 2011). drone production was highly variable between individual colonies in our study with a low of 0.1% to a high of 17.3%, which is in all likelihood connected to the maturity of colonies, with colonies commencing drone production at some unknown point as they mature. because of the migratory nature of giant honey bees (dyer & seeley, 1994; woyke et al., 2012) the period of development from colony initiation by a swarm, to the production of drones and queens is seasonal although there are no published data on average colony residence times but from our observations in northern thailand, following establishment via reproductive or absconding swarms, colonies require 4-6 months to reach sexual maturity, i.e., the production of drones and virgin queens. following virgin queen production colonies undergo swarming events and finally the remaining new queen and workers abscond. from our data on brood comb area along with data regarding immature bee development times, we can compute theoretical adult worker and drone populations. considering the immature development time of a. dorsata (egg to eclosion of adult) is 18.3 – 19.7 d for workers and 22 – 23.7 d for drones (qayyum & ahmad, 1967; tan, 2007) the number of brood cycles for a colony at 6 mo of age is ca. 9. from our data on an average brood comb area (4,054 ± 1,594 cm2) and hypothesized brood cycles, a colony at 6 months of age would have produced a population of some 97,000 adult bees (#brood cycles (9) x average brood area occupancy rate (71%) x average #brood cells available (15,243) which is calculated from the metric of 3.76 brood cells/cm2 (buawangpong et al., 2014). with putative drone production beginning with the 6th brood cycle (ca. 98 days’ post colony initiation) and continuing for another 4 cycles, the result would be a hypothetical adult drone production of ca. 2,500 males or approximately 2.6% of the adult population. this compares to published data for adult drone populations of 4.8% for a. mellifera (seeley & morse, 1976), 4.3% for a. cerana (somana et al., 2011) and 2.9% for a. florea (burgett & titayavan, 2004). as a result of the complete census of brood combs the following peripheral observations can be made: queen ovipositional rates ranged from 210 to 1,063 eggs per diem. eggs were rather equally distributed on the 2 comb sides of each colony. the correlation coefficient comparing the number of eggs per side resulted in an r of 0.66. this comb side brood equality suggests the queen is temporally efficient in her patrolling of both comb sides. of the colonies brood population, eggs averaged 12.1%, larvae 10.3%, pre-pupae 9.2% and capped pupae 68.4%. the brood occupancy rate (percent of cells with brood vs. total brood cells available) averaged 71.0 ± 24.8% with a range of 31.4 to 95.3%. the average brood comb area was 4,053 ± 1,594 cm2 with a range of 2,262 to 6,661 cm2. acknowledgements we sincerely thank dr. natdanai likhitrakarn who facilitated the collection of biological material on the mae jo university campus. we thank dr. yaowaluk chanbang of the postharvest technology research center, chiang mai university for generously providing the laboratory facilities. references allen md (1963). drone production in honey-bee colonies (apis mellifera l.). nature, 199: 789-790. buawangpong n, saraithong p, khongphinitbunjong k, chantawannakul p, burgett m (2014). the comb structure of apis dorsata f. (hymenoptera: apidae): 3-dimensional architecture and resource partitioning. chiang mai journal of science, 41(5.1): 1077-1083. burgett dm, titayavan, m (2004). apis florea f. – colony biometrics in northern thailand. proceeding 8th ibra conference on tropical beekeeping & vi encontro sobre abelhas, ribeirao preto, brazil. pp. 46-54. dyer fc, seeley td (1994). colony migration in the tropical honey bee apis dorsata f. insectes sociaux, 41(2): 129-140. free jb, williams ih (1975). factors determining the rearing and rejection of drones by the honeybee colony. animal behavior, 23:650-675. lee pc, winston ml (1987). effects of reproductive timing and colony size on the survival, offspring colony size and drone production in the honey bee (apis mellifera). ecological entomology, 12(2): 187-195. langstroth ll (1866). a practical treatise on the hive and the honey bee. 3rd ed. lippincott and co., philadelphia. lindauer m (1957). communication among the honeybees and stingless bees of india. bee world, 38: 3-14. morse ra, laigo fm (1969). apis dorsata in the philippines. monograph philippine association of entomologists no. 1, 97 pp. page re (1981). protandrous reproduction in honey bees. environmental entomology, 10(3): 359-362. page re, metcalf ra (1984). a population investment sex ratio for the honey bee (apis mellifera l.). the american naturalist, 124(5): 680-702. qayyum ha, ahmad n (1967). biology of apis dorsata f. pakistan journal of science, 19: 109-113. sociobiology 66(3): 475-479 (september, 2019) 479 ramachandran s (1937). bee-keeping in south india. bulletin department of agriculture madras no. 57 2nd ed. seeley td (1985). honeybee ecology. princeton university press. 202 pages. seeley td (2002). the effect of drone comb on a honey bee colony’s production of honey. apidologie, 33(1): 75-86. seeley td, morse ra (1976). the nest of the honey bee (apis mellifera l.). insectes sociaux, 23(4): 495-512. somana w, chanbang y, kulsarin j, burgett m (2011). biometrics of the natural nest of the eastern honey bee (apis cerana f.) as observed in northern thailand. [in thai] chiang mai university journal of agriculture, 27(3): 219-228. tan nq (2007). biology of apis dorsata in vietnam. apidologie, 38(3): 221-229. thaker cv, tonapi kv (1961). nesting behavior of indian honeybees i. differentiation of worker, queen and drone cells on the combs of apis dorsata. bee world, 42(3): 61-62. wallace ar (1869). the malay archipelago. macmillan and co., london. woyke j, wilde j, wilde m (2012). swarming and migration of apis dorsata and apis laboriosa honey bees in india, nepal and bhutan. journal of apicultural science, 56(1): 81-91. doi: 10.13102/sociobiology.v66i4.3436sociobiology 66(4): 568-574 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 species distribution of euglossini bees (hymenoptera: apidae) at an altitudinal gradient in northern santa catarina introduction the tribe euglossini (hymenoptera: apidae) is comprised of bees distributed in the neotropical region and more numerous in rain forests near the equator line (dressler, 1982). toward southern brazil, there is a decrease in richness and abundance probably due to either climatic and vegetation transitions (wittmann et al., 1988). besides the effects of latitude, the altitude variation of the landscape implies fast abiotic changes of temperature, humidity, solar radiation and wind velocity, causing variation in species composition (uehara-prado & garófalo, 2006; aguiar & gaglianone, 2012). however, it is difficult to identify which factors are responsible for the variation on species composition (hagen et al., 2008). the territory of santa catarina state, in the southern brazil, is ideal to explore the altitudinal variation on species composition, abstract euglossini bees are found from southern usa to central argentina and southern brazil. variations in latitude and altitude can influence the distribution of these bees. this study focused in recognizing the euglossini species in northern santa catarina state, evaluating the distribution over the gradient between sea level and 800 meters altitude. the bees were collected in six locations, between spring and summer from 2013 to 2015, using cotton balls containing the following odor baits: benzyl benzoate, 1,8 cineole, eugenol, menthol and vanillin. a total of 794 bees were sampled, belonging to 10 different species, including a possible new species. in terms of abundance eufriesea cockerell, 1908 (4 species) accounted for 552 individuals, followed by euglossa latreille, 1802 (5 species) with 218 and eulaema lepeletier, 1841 (2 species) 24 individuals. five species were not found above altitude of 400 meters. eufriesea violacea blanchard, 1840, euglossa annectans dressler, 1982, and eulaema nigrita lepeletier, 1841 were the only species found in every location along the altitudinal gradient, but their abundance declines toward higher altitudes. the results surpassed the known orchid bee species for santa catarina state from 9 to 14 and confirmed the expected tendency of richness and abundance reduction toward the highlands. sociobiology an international journal on social insects e dec¹, i alves-dos-santos² article history edited by solange augusto, ufu, brazil received 09 may 2018 initial acceptance 24 june 2018 final acceptance 08 august 2019 publication date 30 december 2019 keywords altitude, 26°s latitude, orchid bees, serra do mar, atlantic rainforest, subtropical climate corresponding author isabela alves dos santos universidade de são paulo rua do matão, travessa 14, n º 321 cidade universitária, são paulo-sp, brasil. e-mail: isabelha@usp.br because in short stretch (within the same latitude) it is possible to go from the sea level to the plateau of the mountains. efforts to recognize euglossini fauna of the state of santa catarina are presented by steiner et al. (2006; 2010), mouga (2009), dec and mouga (2014), and the compilations by nemésio (2009) e moure et al. (2012). the focus of this study is to survey the euglossini species occurring in the northern region of santa catarina, as well as estimating the richness and abundance in the altitudinal gradient that varies between sea level and 800 meters a.s.l. material and methods study area the study was performed in six locations in the northern region of state of santa catarina (all within the 26°s latitude): 1 laboratório de hymenoptera, departamento de entomologia, museu nacional – ufrj, rio de janeiro, brazil 2 departamento de ecologia., universidade de são paulo – ibusp, são paulo-sp, brazil research article bees sociobiology 66(4): 568-574 (december, 2019) 569 1. vila da gloria (vg) (são francisco do sul municipality) at the sea level, with lowland dense ombrophilous forest; 2. morro do finder (mf), 3. morro do boa vista (mbv) and 4. mutucas (mut), all at 200 meters a.s.l., with sub-mountain dense ombrophilous forest; 5. rio seco (rs) (400m); and 6. castelo dos bugres (cb) (800m), with sub-mountain and mountain dense ombrophilous forest, respectively. the locations 2 to 6 belong to the joinville municipality (fig 1). the locations of morro do finder and morro do boa vista are protected forest fragments with 525 and 390 hectares, respectively, inserted in an urban area, while the other locations constitute continuous areas of serra do mar (environmental protection area (apa) serra dona francisca). according to the köppen classification, the weather is humid subtropical climate (cfa), without dry season. the data collecting occurred between september/2013 to april/2014 and september/2014 to april/2015, which correspond to spring and summer in southern brazil. monthly, the bees were sampled in each site, by only one person, between 9 am to 4 pm. the bees were attracted with scents dripped to small cotton balls hanging on tree branches, 1.5 meters from the ground and approximately 10 meters of distance from one another. each cotton ball had one of the following baiting odors: benzyl benzoate, 1,8-cineol, eugenol, menthol and vanillin. when necessary, the refill was made to compensate for evaporation of the substance. the bees were captured with insect net and then they were sacrificed. in the laboratory they were pinned into entomological pins and identified through the key identification proposed by faria jr. and melo (2007) and nemésio (2009). the species identification was reviewed by specialists and then, deposited into the entomological collection cepann (ibusp). the pollinaria of orchidaceae found on the bees were identified by specialists. climatic data presented were obtained from the defesa civil of joinville. data analysis the statistical analyses were made with the software paleontological statistics (past 3.04). diversity, uniformity and dominance of the species were measured by shannonwiener index (h’), pielou index (j’) and simpson index (1-d), respectively. the similarity between study sites was calculated by the sorensen and renkonen indexes. this second is suggested to small samples (wolda, 1981). the change in species composition through the altitude gradient was verified by the whittaker index. results a total of 794 bees of the genus eufriesea cockerell, 1908 (ef.); euglossa latreille, 1802 (eg.) and eulaema lepeletier, 1841 (el.), were sampled and distributed through 10 species and potentially one new species, cryptic to eufriesea auriceps (friese, 1899), treated as eufriesea aff. auriceps (table 1). of the total, 531 bees (66.9%) belonged to eufriesea violacea blanchard, which were registered at all study sites. although its abundance this species was recorded only between november and january, mainly in the locations at 200 meters a.s.l. the second most abundant species was fig 1. specific location of the collecting areas for euglossini in the state of santa catarina. superior left: dark gray highlight for the south region of brazil. inferior left: south of brasil with dark gray highlight for the state of santa catarina. right: joinville and são francisco do sul counties, where the collecting occurred: 1. vila da gloria, 2. morro do finder, 3. morro do boa vista, 4. mutucas, 5. rio seco, 6. castelo dos bugres. e dec, i alves-dos-santos – euglossini at an altitudinal gradient in serra do mar/sc570 euglossa annectans dressler, with 143 individuals (17.9%), also recorded in the six locations throughout the year (except for september/2013 and april/2014). on the other hand, euglossa iopoecila dressler was sampled only in late summer (march and april/2014 and 2015). the shannon index of diversity (h’) varied between 0.8 in the morro do finder and morro do boa vista locations and 1.54 in the vila da gloria (table 1). the abundance of the species was more uniform at vila da gloria (j’= 0.8) compared to morro do finder and morro do boa vista locations (j’= 0.34 and 0.36, respectively), where a strong dominance of the species ef. violacea was evident. the sorensen’s similarity and renkonen index (table 2) were higher between the localities morro do finder and morro do boa vista, which share nine species. the location mutucas also presented a strong cluster with these two fragments of forest as well with vila da glória. the whittaker’s index has showed a low variation of species composition through the altitudinal gradient (βw=0.57). species number of bees total vg 20m mf 200m mbv 200m mut 200m rs 400m cb 800m eufriesea aff. auriceps 1 1 eufriesea dentilabris (mocsáry, 1897) 6 2 7 3 18 eufriesea mussitans fabricius, 1787 1 1 2 eufriesea violacea (blanchard, 1840) 8 218 209 64 23 9 531 euglossa cordata (linnaeus, 1758) 2 1 1 4 euglossa townsendi cockerell, 1904 1 2 3 euglossa annectans dressler, 1982 33 31 12 40 16 11 143 euglossa iopoecila dressler, 1982 13 1 14 euglossa stellfeldi moure, 1947 18 10 13 10 3 54 eulaema cingulata (fabricius, 1804) 1 3 4 2 1 11 eulaema nigrita lepeletier, 1841 2 5 3 1 1 1 13 richness abundance 7 81 10 274 9 252 7 121 6 45 3 21 11 794 shannon wiener (h’) 1.54 0.8 0.8 1.22 1.14 0.85 pielou (j’) 0.8 0.34 0.36 0.59 0.64 0.77 simpson (1-d) 0.74 0.33 0.34 0.61 0.61 0.54 table 1. captured species of euglossini during the period of september/2013 to april/2014, and september/2014 to april/2015. locations’ acronyms: vg = vila da gloria; mf = morro do finder; mbv = morro do boa vista; rs = rio seco; cb = castelo dos bugres. among the odor baits, 1,8-cineol, vanillin and benzyl benzoate attracted, each one, six species of euglossini, while eugenol and menthol attracted four species each (table 3). males of ef. violacea were collected mainly with cineol (275) and vanillin (149). they were not attracted by eugenol that, moreover, attracted no species of eufriesea. vanillin was the only substance to attract two rare species in the region: ef. aff. auriceps and ef. mussitans, however it did not attract any species of euglossa. the majority of males of euglossa were attracted by eugenol and benzyl benzoate. menthol was used for the capturing of 75 males of the genus eufriesea and eulaema. table 2. indexes applied to all possible combinatory pairs for the studied location. upper diagonal, sorensen’s index (in bold, the most similar locations); lower diagonal, renkonen’s index (in bold, the most similar locations). subtitles: vg = vila da gloria; mf = morro do finder; mbv = morro do boa vista; rs = rio seco; cb = castelo dos bugres. local vg mf mbv mut rs cb vg 0.83 0.75 0.93 0.77 0.60 mf 0.70 0.94 0.88 0.62 0.46 mbv 0.60 0.90 0.82 0.71 0.50 mut 0.75 0.70 0.77 0.71 0.54 rs 0.62 0.50 0.50 0.62 0.66 cb 0.42 0.30 0.30 0.42 0.50 species / baits bb c e m v eufriesea aff. auriceps 1 eufriesea dentilabris 4 7 7 eufriesea mussitans 2 eufriesea violacea 45 275 62 149 euglossa annectans 10 7 125 euglossa cordata 1 3 euglossa iopoecila 1 14 euglossa stellfeldi 6 48 euglossa townsendi 2 1 eulaema cingulata 3 5 3 eulaema nigrita 9 1 3 total number of individuals 65 299 190 75 165 total number of species 6 6 4 4 6 table 3. number of males for each species, baited by different odor baits in the periods between september/2013 and april/2015, in the northern region of santa catarina. subtitles: bbbenzyl benzoate; ccineole; eeugenol; mmenthol; vvanillin. sociobiology 66(4): 568-574 (december, 2019) 571 temperatures varied between 14º and 37ºc during the period of study, however, the bees appeared only above 21ºc. there were some picks of precipitation along the year, but no typical dry season. the means of monthly temperature and rainfall in the joinville and são francisco do sul municipalities are shown in figure 2, however there was difference among the localities due to the altitudinal variation. the sampled males were carrying 123 pollinaria of eight species of orchidaceae. by the total, 102 pollinaria were adhering to males of ef. violacea, being 91 of the orchid species gongora bufonia lindl (table 4). some males showed two, three or even four pollinaria, always adhering to the end of the scutellum. table 4. number of orchid pollinaria recorded on euglossini males captured in the six locations studied in the northern regions of santa catarina. orchid species eufriesea aff. auriceps eufriesea dentilabris eufriesea violacea euglossa annectans euglossa stellfeldi eulaema nigrita bifrenaria sp. 6 catasetum sp. 1 4 catasetum sp. 2 1 cirrhaea sp. 1 gongora bufonia lindl. 1 91 notylia sp. 15 huntleya meleagris lindl. 1 rodriguezia venusta rchb. f. 4 discussion with the records of this study the number of species of euglossini in the state of santa catarina was raised from 9 to 14: ef. auriceps, ef. dentilabris, ef. mussitans, eufriesea smaragdina (perty); ef. violacea, eg. annectans, eg. townsendi; eg. cordata, eg. iopoecila, euglossa mandibularis friese, euglossa stellfeldi moure, el. nigrita, eulaema cingulata fabricius and exaerete dentata linnaeus (steiner et al., 2006, 2010; nemésio, 2009; moure et al., 2012; dec & mouga, 2014). additionally, a potentially new species was registered, cited as ef. aff. auriceps. the species eg.cordata was considered by nemésio (2009) as occurring in the state (treated as euglossa carolina nemésio), and now confirmed in the present study. in an inventory made in antonina, paraná, 100 km north from joinville, mattozo et al. (2011) registered the occurrence of euglossa roderici nemésio and euglossa viridis perty, which were not captured in santa catarina nor in rio grande do sul (wittmann & hoffmann, 1990). thus, even though the occurrence in the dense ombrophilous forest, these species were never registered to latitudes farther than 26° south. moreover the region of northern santa catarina might be the meridional limit for the distribution of the species eg. iopoecila and eg. stellfeldi, since steiner et al. (2006; 2010) did not record these species in the island of florianópolis. future sampling on these two regions may refine data about the distribution and limits or geographical barriers for these species. fig 2. mean monthly temperature (grey bars) and mean monthly rainfall (red line) between september/2013 and april/2015 in the joinville and são francisco do sul municipalities. e dec, i alves-dos-santos – euglossini at an altitudinal gradient in serra do mar/sc572 through the altitudinal gradient, it was verified that no species were exclusive to a determined altitude. in the locations between 400 and 800 meters a.s.l., the richness and abundance tend to decrease: 6 species (45 individuals) and 3 species (2 individuals), respectively. however, the shannon’s diversity of the community of cb (800 m) found a similar value to the communities mf and mbv (200 m) due to dominance of ef. violacea in these two locations. the two most abundant species in this study, ef. violacea and eg. annectans, were found in the six studied localities. in other regions of the country ef. violacea was abundant in environments higher than 600 meters a.s.l., including inland areas in the states of paraná and minas gerais (sofia & suzuki, 2004; uehara-prado & garófalo 2006; nemésio & silveira, 2007a; knoll & penatti, 2012; cordeiro et al., 2013). the distribution of this species extends to the states of mato grosso do sul (ferreira et al., 2011), mato grosso, and parts of argentina (kimsey, 1982). in the present study ef. violacea was observed during 70 days, between november and january, with peaking activity in december. peruquetti and campos (1997) sampled this species for approximately 150 days, while wittmann et al. (1987), recorded individuals for 90 days in rio grande do sul. similarly eg. annectans, that was more frequent in low land sites usually is associated with higher altitudes in southeastern of brazil (knoll & penatti, 2012; garófalo et al., 1998). to some authors el. nigrita is considered to be a bioindicator of impacted environments (rebêlo & cabral, 1997; peruquetti et al., 1999; tonhasca et al., 2002; aguiar & gaglianone, 2008). in the studied region, the low number of el. nigrita may be related to the natural absent of great populations, since according to mattozo et al. (2011), rocha filho and garófalo (2013) and giangarelli et al. (2015) in low altitudes located on areas of dense ombrophilous forest in são paulo and paraná, the species already shows a small population number. another species, el.cingulata, is usually numerous in inventories (rebêlo & cabral, 1997; peruquetti et al., 1999; tonhasca et al., 2002; nemésio & silveira, 2006; rocha-filho e garófalo, 2013). aguiar and gaglianone (2012) considered this species fairly tolerant to anthropic perturbations. in the same way, tonhasca et al. (2002) found many individuals in fragment of the mata atlântica, in rio de janeiro, in areas considered to be disturbed. nemésio and silveira (2006) collected more el. cingulata in border vegetation than in the interior of the forest and nascimento et al. (2015) recorded this species in abundance in areas of eucalyptus in mato grosso state. on the other hand, mattozo et al. (2011) found only one specimen of el. cingulata in the municipality of sete barras (sp) and none in antonina (pr). in the present study the samples of el. cingulata were found in fragments as many as in continuous areas, with similar proportions to the el. nigrita. euglossa cordata is one of the most abundant species in the southeastern and northeastern cost of the country (viana et al., 2002; souza et al., 2005; aguiar & gaglianone, 2008, 2012; rocha-filho & garófalo, 2013), however in the present study, only four individuals were captured. mattozo et al. (2011) show that this species already have small population in the latitude 25°s. for the interactions recorded between orchids and bees it stands out the presence of g. bufonia lindl. in the five locations between sea level to submontane. there is a strict relation between ef. violacea and g. bufonia, since 91 pollinaria were on the males of this species, corroborating with singer and sazima (2004), that reported ef. violacea as pollinators of g. bufonia. this orchid presents a complex mechanism of pollination, therefore as other genus of stanhopeinae, being dependent exclusively to bees euglossini for pollination (dressler, 1993). according to williams (1982), the gongora produces an odor that has higher intensity in warmer hours of the day, coincidentally with the periods that most ef. violacea males were attracted to the odor baits, between 11:30 am and 12:30 pm. morro do finder and morro do boa vista were important scenario for this interaction, suggesting a well conserved state of these areas, since ef. violacea seems to be a species sensitive to degradation of habitat (giangarelli et al., 2009). therefore we sustain the importance of these ecological fragments in the north of santa catarina. acknowledgments the authors are grateful to capes for providing a scholarship to development this study; dr. gabriel a. r. melo for identifying the bees; dr. emerson r. pansarin and dra. ludmila m. pansarin for the identification of pollinaria. finally, we are grateful to the anonymous referees for their careful revision. references aguiar, w.m. & gaglianone, m.c. 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(1981). similarity indices, sample sizes and diversity. oecologia, 50: 296-302. doi: 10.13102/sociobiology.v67i2.4061sociobiology 67(2): 312-321 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the biodiversity of insects in brazil is one of the highest of the world, representing around 10% of all known insects (marinoni et al., 2006; hermes et al., 2015), distributed in six main brazilian biomes (amazon rainforest, atlantic forest, caatinga, cerrado, pantanal, and pampa). the amazon rainforest is the biggest biome within brazilian territory, covering an area of 4,196,943 km² (ministério do meio ambiente, 2018). the amazon biodiversity is considered as rich as unknown and one of the great challenges to its conservation is the establishment of a solid database on the distribution and abundance of organisms (borges et al., 2004). knowledge about biodiversity remains inadequate because most species living on earth were still not formally described (the linnean shortfall) and because geographical distributions of most species are poorly understood and usually contain many gaps (the wallacean shortfall) (bini et al., 2006). even today, mainly the geographic distribution of abstract the thematic network ‘biodiversity of insects in the amazon’ is the first network among researchers of the brazilian amazon in terms of the increase of knowledge and provision of subsidies for the conservation of amazonian biodiversity, focusing on insects, aiming as well to disseminate this knowledge to different sectors of society. in this way, expeditions to six localities in the amazonas state were carried out and we present here the results for social wasps (vespidae: polistinae). we used three modified malaise traps traps from july 2016 to june 2017. a total of 140 species and 20 genera were collected: 92 species and 18 genera in zf-2manaus area, which presented the greatest diversity, followed by tefé (73 species, 16 genera), careiro-castanho (72 species, 17 genera), novo airão (71 species, 16 genera), presidente figueiredo (62 species, 16 genera), and ipixuna (58 species, 17 genera). metapolybia rufata richards, 1978 and polybia diguetana du buysson, 1905 were new records for brazil, and other six species were first records for amazonas state. the results indicate that further investigations should significantly increase the knowledge of wasp species diversity in the amazon region and add more information on polistinae diversity. sociobiology an international journal on social insects a somavilla, rnm de moraes junior, ml oliveira, ja rafael article history edited by marcel hermes, ufla, brazil received 09 november 2018 initial acceptance 04 january 2019 final acceptance 10 january 2019 publication date 30 june 2020 keywords agelaia, amazon biome, entomologists network, hymenoptera, polybia. corresponding author alexandre somavilla instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo, 2936, cep: 69067-375 manaus, amazonas, brasil. e-mail: alexandresomavilla@gmail.com most organisms in the amazon is still poorly known, even among those most studied groups such as birds, primates and plants (borges et al., 2004; lomolino, 2004). regarding insects, the paucity of geographic distribution knowledge is much more dramatic, exemplifying very well both linnean and wallacean shortfalls (lomolino, 2004, bini et al., 2006). for vespidae, the knowledge on the geographic distribution of most species is incomplete, being most times inadequate at all scales. for this reason, a thematic network ‘biodiversity of insects in the amazon’ was created by a group of researchers and students interconnected by processing modules through a communication system about insects in the amazon. the network has as main objective to create and promote the exchange of the first entomologists network researchers of the brazilian amazon in terms of the increase of knowledge and provision of subsidies for the conservation of amazonian biodiversity, focusing on insects, aiming as well to disseminate this knowledge to different sectors of society. instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus, amazonas, brazil research article wasps biodiversity of insects in the amazon: survey of social wasps (vespidae: polistinae) in amazon rainforest areas in amazonas state, brazil sociobiology 67(2): 312-321 (june, 2020) 313 the brazilian amazon rainforest has one of the greatest biodiversities in the world, including the greatest diversity of social wasps (silveira, 2002; somavilla et al., 2014a; barbosa et al., 2016). polistinae is the most diverse group among the social wasps, with more than 950 species described (pickett & carpenter, 2010). the subfamily is divided in the tribes ropalidiini, polistini, mischocyttarini and epiponini, but the first not occurring in brazil (carpenter & marques 2001; carpenter, 2004). polistes latreille, mischocyttarus de saussure, and the 19 genera of epiponini compose the brazilian fauna of wasps, totalizing about 350 species, of which 104 are endemic (carpenter & marques 2001; carpenter, 2004; hermes et al., 2017). the knowledge of social wasps comes from some few studies carried out in forest fragments. ducke (1904, 1907) conducted one of the first surveys of wasp fauna in the eastern region of the brazilian amazon, mainly in pará state. recently, similar works have been carried out in the brazilian amazon, as follow in acre state (morato et al., 2008; gomes et al., 2018), amapá state (silveira et al., 2008), maranhão state (somavilla et al., 2014b), pará state (silveira, 2002; silva & silveira, 2009) and roraima state (raw, 1998; barroso et al., 2017). in the state of amazonas, six surveys have been carried out: mamirauá and alvarães reserves, with 46 and 42 species, respectively (silveira et al., 2008), jaú national park with 49 species (somavilla et al., 2015), madeira–purus rivers with 38 species (oliveira et al., 2015), embrapa-manaus with 52 species (somavilla et al., 2016), and ducke reserve with 103 species (somavilla & oliveira, 2017). despite the contributions of these works, somavilla et al., (2014a) stated “there are many sample gaps in the amazon region and distribution and occurrence studies are necessary for improving this prior knowledge”. in this way, several expeditions were carried out within the network ‘biodiversity of insects in the amazon’ and we present here the results of six different areas sampled in the amazonas state. amazonas is the largest brazilian state, and it has the largest tropical forest in the world. the amazon forest, also is part of bolivia, ecuador, colombia, guyana, french guiana, peru, suriname, and venezuela in south america. amazonas state has most of its land occupied by forest reserves and water, and the access to the region is mainly made by waterway or by planes. it is located in the north of brazil, bordering the states of mato grosso, rondônia, and acre to the south; pará and roraima, in the north east beyond the republics of peru, colombia, and venezuela to the southwest and northwest respectively (ministério do meio ambiente, 2018). most of the state is in the tropical rainforest climate zone (barbosa, 2015). the equatorial climate is denoted af in the köppen (1948) climate classification. the average temperature varies very little by season, between 26 and 28 °c, and relative humidity is around 80% (barbosa, 2015). regardless of the relief type, the region is formed basically by two geomorphologic types: upland and lowland or floodplains, locally known as “terrafirme” and “várzeas”, respectively (telles et al., 2003). material and methods amazonas state areas the social wasps were collected in six areas in amazonas state: zf-2, in manaus (2°35’21”s, 60o06’55”w), and presidente figueiredo (1°41’50.1”s, 59o36’43.5”w) to the north of the amazonas river; careiro-castanho (4°12’48”s, 60°49’04”w), ipixuna (7°21’46”s, 71°52’07”w), and tefé (3°25’19”s, 64°37’05”w) to the south of the amazonas river. finally, novo airão (2°48’58”s, 60°55’18”w) in the interfluvial area solimões and negro rivers (figure 1). figure 1. location of zf-2 manaus, careiro-castanho, tefé, novo airão, presidente figueiredo and ipixuna areas in amazonas state, brazil. a somavilla, rnm de moraes junior, ml oliveira, ja rafael – social wasps in amazon rainforest areas in amazonas state314 wasp collection the wasps were collected in the forests using three modified malaise traps model: 1. townes (1972) model 2-meter long; 2. gressit and gressit (1972) model 6-meter long with two collector vials in understory, and 3. suspended traps (rafael and gorayeb 1982) model in the canopy. both traps were active for fifteen consecutive days each month, for a period of one year between july 2016 to june 2017. the polistinae specimens were sorted and identified at the hymenoptera laboratory of the national institute of amazonian research (inpa). the vouchers were deposited into the inpa’s invertebrate collection. specimens were identified using the keys proposed by richards (1978), carpenter and marques (2001), and carpenter (2004) and were compared to previously identified species from the inpa invertebrate collection. data analysis we used the euclidean distance analysis to verify the similarity between the species composition in the six amazonas areas, according to the presence or absence of each species. this analysis was conducted in r version 3.3.3. (r core team, 2017) using vegan package 2.4-0 (oksanen et al., 2016). results a total of 140 species and 20 genera were collected (table 1). zf-2 manaus area presented the greatest diversity, with 92 species and 18 genera, on the other hand, ipixuna fragment presented the lowest diversity, with 58 and 17, respectively (figure 2). protonectarina ducke was the only genus not collected in these areas, and there are no records of its occurrence for the amazon biome. polybia lepeletier, 1836 was the richest genus for all studied areas with 33 species, followed by mischocyttarus de saussure (24), agelaia lepeletier (16), protopolybia ducke (11), and polistes latreille (10 species) (table 1). agelaia was the most abundant genus followed by polybia. regarding species composition, only 33 species were sampled in all six areas, 31 from epiponini and just one for mischocyttarus and polistes. of this number, ten of the 16 species of agelaia, nine of 33 polybia species, four of the seven apoica lepeletier species, two of the three angiopolybia araujo species, two of the three synoeca saussure species, and one species of brachygastra perty, chatergellus bequaert, clypearia saussure, and leipomeles möbius were collected in the all six sampled areas. still to species composition, 43 species were exclusive of only one fragment area, 12 mischocyttarus, seven protopolybia and six polybia. according to the euclidean distance analysis between the social wasps’ composition from six different areas in the amazonas, the relationship between the species of careiro-castanho and tefé fragments were the closest, sharing 59 species, followed by presidente figueiredo and ipixuna fragment with 45 shared species. the zf-2 manaus area presented the most diverse and the largest number of exclusive species, with 19 species, and in the analysis was the area with the longest distance between all sampled areas (figure 3). discussion silva and silveira (2009) and somavilla at al. (2014a) showed that fast inventories were efficient for sampling the most abundant species, recording three genera: polybia, mischocyttarus and agelaia. herein, we found the same most specious genera, which constituted more than 50% of the species collected. specimens of polybia has a very active foraging behavior, which facilitates the collection of the specimens in trap, and the genus with largest number figure 2. graph with the species and genera numbers, respectively, collected in the six different amazonas areas. sociobiology 67(2): 312-321 (june, 2020) 315 of species within epiponini (carpenter & marques, 2001). mischocyttarus is the genus with more species in social wasps (around 240), of which 117 occur in brazil, that can support the high diversity in this study (silveira, 2002). agelaia species usually form large colonies with millions of individuals (zucchi et al., 1995), and, consequently, they are more likely to be captured (silva & silveira, 2009), an expected result as they are present in all inventories of social wasps in amazonian rainforest (silveira, 2002; somavilla et al., 2014a). the amazon region has the highest diversity of polistinae species of the world (richards, 1978; carpenter & marques, 2001; silveira 2002; barbosa et al., 2016). in the brazilian amazon, 20 genera and more than 220 species have been recorded, which represents about two thirds of the brazilian diversity of social wasps (silveira, 2002; somavilla & oliveira, 2017). currently 161 species have been reported for the state of amazonas, despite six studies on social wasps having been carried out in the state to date (silveira et al., 2008; somavilla et al., 2014a; oliveira et al., 2015; somavilla et al., 2016; somavilla & oliveira, 2017) and reported by richards (1978) in your social neotropical wasps’ revision, which clearly indicates that the diversity of wasps in the region is still vastly underestimated. the records of chartergellus jeannei andena & soleman, 2015, necterinella manauara silveira & santos jr., 2016 and protopolybia rotundata ducke, 1910 represented the second record for each species, both of which were recorded only for the type locality so far, ducke reserve (amazonas) the first two and base camp (mato grosso), respectively. also, we produced the first records of metapolybia rufata richards, 1978 and polybia diguetana du buysson, 1905 for brazil, previously only registered for colombia and ecuador, and mexico to bolivia, respectively. we made the first records of agelaia lobipleura (richards, 1978), polybia minarum ducke, 1906, protopolybia nitida (ducke, 1904), protopolybia rotundata, protopolybia sedula (de saussure, 1854), and mischocyttarus punctatus (ducke, 1904) for amazonas state in brazil. the higher richness found at this six areas when compared to other biomes can be explained by the high effort of collection as well as by the higher structural complexity of these amazonian environments. they are composed by dense forest, clearings, bottomlands, streams, plateaus and canopies, that allow the establishment and survival of more species of social wasps, providing microhabitats for the organisms, greater protection from predators, and increased availability and diversity of food resources and nesting substrates (lawton, 1983; santos et al., 2007). vegetation exerts direct influence on social wasp communities, providing support for nesting and food resources, indirectly affecting those communities by variations in temperature, humidity, and amount of shade in the environment (wenzel, 1998; diniz & kitayama, 1994; dejean et al., 1998). the similarities of social wasp species composition between tefé and careiro-castanho areas was not surprising since they are both reserves located in comb of two distinctive areas lowland and terra-firme areas, in the south of solimões river. in compensation, the high diversity of social wasps in zf-2 manaus follows the results found in the ducke reserve (somavilla & oliveira, 2017), both in north of manaus, geographically close (100 km apart) and both areas have the same phytophysiognomy characteristics (ombrophilous dense and humid forest) (telles et al., 2003). but the proximity of presidente figueiredo and ipixuna species composition is something that needs to be better investigated, probably the wide geographical distribution of most species explains this, since the limiting is the lack of sampling in parts of amazon rainforest. despite the difficulties of collecting in amazonian environments due the difficult access to some isolate areas, and in the canopy height, the permanent collection is a good strategy. when comparing other areas already sampled in the brazilian amazon, such as mamirauá and alvarães reserves figure 3. cluster dendrogram for euclidean distance analysis to verify the similarity between the species composition in the six different amazonas areas. zf-2 manaus novo airão careiro-castanho tefé presidente figueiredo ipixuna a somavilla, rnm de moraes junior, ml oliveira, ja rafael – social wasps in amazon rainforest areas in amazonas state316 in amazonas (silveira et al., 2008), caxiuanã, pará (silveira, 2002), lagos region, amapá (silveira et al., 2008), serra do divisor, acre (morato et al., 2006), and gurupi park, maranhão (somavilla et al., 2014b), the diversity is lower than these six fragments sampled. in those studies, using a greater number of malaise traps per site, but the collections were made quickly at most seven days. regarding the methods used for samples, there are different methods to sample social wasps; however, few studies have attempted to propose a standardization of these methods or to establish comparable and adequate protocols to survey the fauna of a given site. an important factor to consider in the implementation of novel social wasp sampling protocols is the distribution pattern of these organisms (silveira, 2002; silva & silveira, 2009) or, when using traps, finding the most efficient ones to collect the target group and dispose them in a standardized manner (noll & gomes, 2009). somavilla et al (2014a) proposed the best would be to use active search + malaise trap, and for exploring other forest strata, such as the canopy (suspended malaise trap) or attraction traps. in this study, it was not possible to make active search for wasps, due to the long distances between the sampled areas, for that reason, we standardized the use combined use of malaise traps and suspended traps in each collected area, for 15 direct days. conclusion here was recorded 140 species of social wasps in six forest areas in the amazonas state, in the central brazilian amazon. this represents a little more than 85% of the species of social wasps known to the amazonas state, and based on this percentage is possible to conclude that the continuous collection effort overlaps on short collection efforts usually applied here in the amazon biome. metapolybia rufata and polybia diguetana were new records for brazil, and other six species were first records for amazonas state, these new records significantly increased the range for some species and filled distribution gaps for others. the results obtained in this study indicate that further investigations should significantly increase the species diversity of wasps in the amazon region and add more information to the knowledge of polistinae diversity. acknowledgments specimens were collected during a project coordinated by ja rafael and financed by the conselho nacional de desenvolvimento científico e tecnológico (cnpq, process: 407623/2013-2) through the program “rede bia – biodiversidade de insetos na amazônia”. we sincerely thank fundação de amparo à pesquisa do estado do amazonas for the postdoctoral scholarship (fapeam fixam) of a. somavilla and cnpq research productivity fellowship of ja rafael and ml oliveira. references barbosa, p.h.d., costa, a.c.l. da, cunha, a.c. da & silva junior, j.a. 2015. variabilidade de elementos meteorológicos e de conforto térmico em diferentes ambientes na amazônia brasileira. revista brasileira de climatologia, 17: 98-118. barbosa, b.c., detoni, m., maciel, t.t. & prezoto, f. 2016. studies of social wasp diversity in brazil: over 30 years of research, advancements and priorities. sociobiology, 63: 858880. doi: 10.13102/sociobiology.v63i3.1031 barroso, p.c.s., somavilla, a. & boldrini, r. 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(1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of new york entomological society, 103: 129-137. appendix table 1. species of social wasps (vespidae: polistinae) collected at the zf-2 manaus, careiro-castanho, tefé, novo airão, presidente figueiredo, and ipixuna areas over the network “biodiversity of insects in the amazon”. taxa zf-2 manaus careiro castanho tefé novo airão presidente figueiredo ipixuna agelaia angulata (fabricius, 1804) x x x x x x agelaia brevistigma (richards, 1978) x x agelaia cajennensis (fabricius, 1798) x x x x x x agelaia centralis (cameron, 1907) x x x x x x agelaia constructor (de saussure, 1854) x x x x x x agelaia flavipennis (ducke 1905) x x x x x x agelaia fulvofasciata (degeer, 1773) x x x x x x agelaia hamiltoni (richards, 1978) x x x x x x agelaia lobipleura (richards, 1978) x agelaia myrmecophila (ducke, 1905) x x x x x agelaia ornata (ducke, 1905) x x x x x x agelaia pallidiventris (richards, 1978) x agelaia pallipes (olivier, 1792) x x x x x x agelaia pleuralis cooper, 2002 x agelaia testacea (fabricius, 1804) x x x x x x agelaia vicina (de saussure, 1854) x angiopolybia obidens (ducke, 1904) x angiopolybia pallens (lepeletier, 1836) x x x x x x angiopolybia paraensis (spinola, 1851) x x x x x x apoica albimacula (fabricius, 1804) x x x apoica arborea de saussure, 1854 x x x x x x apoica gelida (van der vecht 1972) x x x x x apoica pallens (fabricius, 1804) x x x x x x apoica pallida (olivier, 1791) x x x x x x apoica strigata richards, 1978 x x apoica thoracica du buysson, 1906 x x x x x x asteloeca traili (cameron, 1906) x brachygastra augusti (de saussure, 1854) x x x x x brachygastra bilineolata spinola, 1841 x x x x x brachygastra lecheguana (latreille, 1824) x x x x x x brachygastra scutellaris (fabricius, 1804) x x x sociobiology 67(2): 312-321 (june, 2020) 319 chartergellus amazonicus richards, 1978 x x x x x x chartergellus jeannei andena & soleman, 2015 x chartergellus nigerrimus richards, 1978 x charterginus fulvus fox, 1898 x x x x chartergus artifex (christ, 1791) x chartergus globiventris de saussure, 1854 x x clypearia apicipennis (spinola, 1851) x clypearia duckei richards, 1978 x x clypearia sulcata (de saussure, 1854) x x x x x x epipona tatua (cuvier, 1797) x x x leipomeles dorsata (fabricius, 1804) x x x x x x leipomeles spilogastra (cameron, 1912) x x x x x metapolybia decorata (gribodo, 1896) x metapolybia docilis richards, 1978 x metapolybia nigra richards, 1978 x x metapolybia rufata richards, 1978 x metapolybia unilineata (r. von ihering, 1904) x x x mischocyttarus adolphi zikán, 1949 x mischocyttarus bertonii ducke, 1918 x x mischocyttarus carbonarius (de saussure, 1854) x mischocyttarus cerberus ducke, 1918 x mischocyttarus collaris (ducke, 1904) x x mischocyttarus drewseni de saussure, 1857 x x mischocyttarus flavicans (fabricius, 1804) x x x x mischocyttarus flavicornis zikán, 1935 x x mischocyttarus foveatus richards, 1941 x mischocyttarus imitator (ducke, 1904) x x mischocyttarus injucundus (de saussure, 1854) x mischocyttarus labiatus (fabricius, 1804) x x x x x x mischocyttarus lecointei ducke, 1904 x x x mischocyttarus metathoracicus (de saussure, 1854) x x mischocyttarus omicron richards, 1978 x mischocyttarus prominulus richards, 1941 x x mischocyttarus punctatus (ducke, 1904) x mischocyttarus rotundicollis (cameron, 1912) x mischocyttarus smithii de saussure, 1853 x mischocyttarus socialis (de saussure, 1854) x mischocyttarus surinamensis de saussure, 1854 x x x table 1. species of social wasps (vespidae: polistinae) collected at the zf-2 manaus, careiro-castanho, tefé, novo airão, presidente figueiredo, and ipixuna areas over the network “biodiversity of insects in the amazon”. (continuation) taxa zf-2 manaus careiro castanho tefé novo airão presidente figueiredo ipixuna a somavilla, rnm de moraes junior, ml oliveira, ja rafael – social wasps in amazon rainforest areas in amazonas state320 mischocyttarus synoecus richards, 1940 x mischocyttarus tomentosus zikán, 1935 x mischocyttarus xanvante silveira, 2010 x x nectarinella manauara silveira & nazareno jr, 2016 x nectarinella xavantinensis mateus & noll, 1998 x parachartegus amazonensis ducke, 1905 x x parachartergus fasciipennis ducke, 1905 x x parachartergus flavofasciatus (cameron, 1906) x parachartergus fraternus (gribodo, 1892) x x x x parachartergus richardsi willink, 1951 x x polistes canadensis (linnaeus, 1758) x x x x x polistes carnifex (fabricius, 1775) x x x polistes claripennis ducke, 1904 x polistes goeldi ducke, 1904 x x polistes lanio (fabricius, 1775) x x x polistes niger brèthes, 1930 x polistes occipitalis ducke, 1904 x x polistes pacificus fabricius, 1804 x x polistes testaceicolor bequaert, 1937 x polistes versicolor (olivier, 1792) x x x x x x polybia affinis du buysson, 1908 x polybia belemensis richards, 1970 x x x x polybia bifaciata de saussure, 1854 x x x polybia bistriata (fabricius, 1804) x x x x x x polybia catillifex möbius, 1856 x polybia chrysothorax (lichtenstein, 1796) x x polybia depressa (ducke, 1905) x x polybia diguetana du buysson, 1905 x x polybia dimidiata (olivier, 1792) x x x x x x polybia dimorpha richards, 1978 x x x x x x polybia emaciata lucas,1879 x x x x x x polybia gorytoides ducke, 1904 x x x x polybia ignobilis (haliday, 1836) x x x x x x polybia incerta ducke, 1907 x x polybia jurinei de saussure, 1854 x x x x x x polybia juruana r. von ihering, 1904 x polybia liliacea (fabricius, 1804) x x x x x x polybia micans ducke, 1904 x x table 1. species of social wasps (vespidae: polistinae) collected at the zf-2 manaus, careiro-castanho, tefé, novo airão, presidente figueiredo, and ipixuna areas over the network “biodiversity of insects in the amazon”. (continuation) taxa zf-2 manaus careiro castanho tefé novo airão presidente figueiredo ipixuna sociobiology 67(2): 312-321 (june, 2020) 321 polybia minarum ducke, 1906 x x polybia occidentalis (olivier, 1792) x x x x x x polybia parvulina richards, 1970 x x x polybia platycephala richards, 1951 x x polybia procelosa zavattari, 1906 x x x polybia quadricincta saussure, 1854 x x x polybia rejecta (fabricius, 1798) x x x x x x polybia rufitarsis ducke, 1904 x polybia scrobalis richards, 1970 x x x x x polybia sericea (olivier, 1792) x x x x polybia signata ducke, 1905 x x x polybia singularis ducke, 1905 x x x x polybia striata (fabricius, 1787) x x polybia tinctipennis fox, 1898 x x x polybia velutina ducke, 1907 x x x x protopolybia acutiscutis (cameron, 1906) x x protopolybia bituberculata silveira & carpenter, 1995 x x x protopolybia chartergoides gribodo, 1892 x x x x x protopolybia emortualis de saussure, 1855 x protopolybia exigua (de saussure, 1854) x protopolybia holoxantha (ducke, 194) x x x x x x protopolybia minutissima spinola, 1851 x x protopolybia nitida (ducke, 1904) x protopolybia rotundata ducke, 1910 x protopolybia rugulosa ducke, 1907 x protopolybia sedula (de saussure, 1854) x pseudopolybia compressa (de saussure, 1854) x x pseudopolybia difficillis (ducke, 1905) x pseudopolybia langi bequaert, 1944 x x pseudopolybia vespiceps (de saussure, 1863) x x x synoeca chalibea de saussure, 1852 x synoeca surinama (linnaeus, 1767) x x x x x x synoeca virginea (fabricius, 1804) x x x x x x table 1. species of social wasps (vespidae: polistinae) collected at the zf-2 manaus, careiro-castanho, tefé, novo airão, presidente figueiredo, and ipixuna areas over the network “biodiversity of insects in the amazon”. (continuation) taxa zf-2 manaus careiro castanho tefé novo airão presidente figueiredo ipixuna open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4504sociobiology 67(2): 163-172 (june, 2020) introduction ants are one of the most diverse and abundant arthropods in tropical forests (hölldobler & wilson, 1990), where up to 50% of them may be associated with the leaf litter (brandão et al., 2012). in this layer, ants are highly taxonomically diverse (ward, 2000). nine trophic (delabie et al., 2000) and morphological guilds (silva & brandão, 2010) are found in the atlantic forest. in the leaf litter, ants use various microhabitats, such as those derived from vertebrates (hölldobler & wilson, 1990) or invertebrates (leponce et al., 1999, jahyny et al., 2007) and live branches (hölldobler & wilson, 1990) or dead branches and twigs (souza et al., 2012, castro et al., 2017) that are used for colony expansion (carvalho & vasconcelos, 2002) and nesting (fernandes et al., 2012). the scarcity or absence of these resources is a limiting factor for ant diversity (fowler et al., 1991). abstract twigs in the litter derived from the fragmentation of tree branches form one microhabitat, where entire colonies of ants, both leaf litter and arboreal species, can be found. the objective was to survey ant species that are present in both the leaf litter and twigs simultaneously. we describe the nest type, the social structure of the colonies and the trophic guild membership of these species. samples were collected from 10 preserved fragments of brazilian atlantic forest. we used berlese funnels to collect leaf litter ants and manual collection for twig ants. we recorded 80 ant species; 60 species were in leaf litter samples and 35 species were in twigs. of the total species, only 15 (20%) occurred simultaneously in the leaf litter and in twigs. of these species, gnamptogenys striatula, pheidole sarcina, p. sospes and solenopsis sp. 2 were the most frequent among leaf litter dwellers, and myrmelachista catharinae was the most common arboreal species. most of these belonged to generalist and predator guilds, with “polydomous nests” and colonies monogynous. sociobiology an international journal on social insects tt fernandes1, rr silva2, dr souza-campana2, ns silva1, ogm silva2, msc morini1 article history edited by evandro nascimento silva, uefs, brazil received 27 may 2019 initial acceptance 17 december 2019 final acceptance 13 april 2020 publication date 30 june 2020 keywords dead wood, social structure, twig-dwelling ants. corresponding author maria santina de castro morini universidade de mogi das cruzes laboratório de mirmecologia do alto tietê mogi das cruzes, são paulo, brasil. e-mail: mscmorini@gmail.com in general, wood structures are frequently colonized by ants (hashimoto et al., 2006; king et al., 2013), but twigs are the most sought-after resource in the leaf litter (gomes et al., 2013). twigs are derived from the fragmentation of tree branches (fernández, 2003; silva et al., 2009). inside twig cavities there may be colonies comprised of hundreds of individuals (nakano et al., 2012; king et al., 2018), with different feeding habits (byrne, 1994). however, even if competition for nesting resources is high in the leaf litter (delabie et al., 2007), due to the high species diversity (silva & brandão, 2010), not all of the available twigs are colonized (sagata et al., 2010). twigs may be satellite structures, or temporary shelters, when only workers are present (carvalho & vasconcelos, 2002; debout et al., 2007); or part of a polydomous colony, when it includes both workers and immatures (kaspari et al., 1996). there are no descriptions of this kind of species found in both the leaf litter and twigs in the brazilian atlantic forest. 1 universidade de mogi das cruzes, laboratório de mirmecologia do alto tietê, mogi das cruzes, são paulo, brazil 2 museu paraense emílio goeldi, coordenação de ciências da terra e ecologia, belém, pará, brazil research article ants occurrence of ants (hymenoptera: formicidae) in both leaf litter and twigs in atlantic forest tt fernandes, rr silva, dr souza-campana, ns silva, ogm silva, msc morini – ants in both leaf litter and twig164 thus, in this work we analyse these ant communities, based on the hypothesis that twigs represent a satellite structure, because they are considered an ephemeral resource (byrne, 1994). in addition, we describe the nest type, the social structure and the trophic guild membership. in this case, we assumed that twigs were colonized more often by generalist leaf litter species, because they belong to the most abundant guild in this layer (silva & brandão, 2010). materials and methods collection sites in 2010, we conducted field expeditions in ten fragments of native atlantic forest located in the state of são paulo, brazil (figure 1). fragment sizes ranged from 20 to 350 ha and were located at elevations between 600 and 850 m. according to the köppen classification, the climate of this region is mesothermic with a dry winter (cwb), and annual precipitation is 1,500 mm. samples were collected in the months of september, october, november and december, which are between the end of the dry season and the middle of the rainy season according to cptec-inpe (centre for weather prediction and climate studies/national institute for space research, classification 2018). collection, colony characterization and ant identification at each site a linear transect was established 100-200 m from the forest edge, depending on the size of the fragment. six 16 m2 plots 50 m apart were established in each fragment. at the centre of each 16 m² plot, 50 cm2 was marked and all the leaf litter within it was scraped up. this material was placed in berlese funnels for seven days. in the 16 m² plots, all twigs on the surface of the leaf litter were collected and placed in individual plastic bags, including those from the spot where the leaf litter was removed. in total, we processed 30 m2 of leaf litter to collect ants and collected twigs from an area of 960 m2 of leaf litter on the forest floor. the twigs were opened and all colony components were removed, recorded and stored in 90% alcohol. colony presence in twigs was defined by (i) the presence of ten or more workers, with or without a queen(s) or (ii) if less than ten workers were present, the presence of immatures or alates (fernandes et al., 2012). fig 1. brazilian atlantic forest sites (1 to 10) where collections were conducted. nests were classified as a “satellite nest”, when only workers were found in the twigs (carvalho & vasconcelos, 2002), or as a “polydomous nest”, when they contained more than workers only and were likely part of a polydomous colony. in this case, there were two types of structure: workers and immatures (debout et al., 2007), or workers plus alates, or workers plus immatures plus alates. the social structure of the colony was classified as monogynous or polygynous (carvalho & vasconcelos, 2002). the guilds were classified according to brandão et al. (2012). ants were identified based on suguituru et al. (2015) and all morphospecies were numbered according to this literature. all vouchers were deposited at laboratório de mirmecologia do alto tietê (myrmecology laboratory of alto tietê) of university of mogi das cruzes, in the state of são paulo, brazil. results we recorded 31 genera and 80 species/morphospecies of ants. of these, 60 were in the leaf litter and 35 were inside twigs (table 1). no twigs contained more than a single ant species. in the leaf litter there were 0.49 species per 50 cm2. camponotus sp. 10, gnamptogenys striatula mayr, 1884, myrmelachista catharinae mayr, 1887 pheidole sospes forel, 1908, solenopsis sp. 2, and solenopsis sp. 3 were the most frequent species. sociobiology 67(2): 163-172 (june, 2020) 165 we found 0.03 species/m² in 335 twigs; linepithema neotropicum (wild, 2007), pheidole sarcina forel, 1912, pheidole sp. 43, and solenopsis sp. 2 were the most frequent species. among leaf litter dwellers, g. striatula and solenopsis sp. 2 were the species that most often colonized both the leaf litter and the twigs at the same time. among arboreal species, the most frequent colonizer was myrmelachista catharinae mayr, 1887 (table 1). out of all species, only 15 (20%) were simultaneously recorded in the leaf litter and in twigs, and most (63%) belonged to the generalist and predator guilds (table 1, figure 2). despite the low richness, these species colonized most twigs (211 = 60%) in the remaining twigs (40%), five of the recorded species were arboreal (myrmelachista ruzskyi forel, 1903; two procryptocerus species; and two pseudomyrmex species), and six species were possibly arboreal (five camponotus species and one crematogaster species). pheidole flavens roger, 1863 was the most frequent species in twigs, but it was not recorded in the leaf litter. fig 2. total number of ant species colonizing both leaf litter and twigs. we found 18 satellite nests (9 species, 57%) and 111 polydomous nests (14 species, 88%). the latter group included 96 nests with workers + immatures (14 species), 4 nests with workers + alates (3 species) and 11 nests with workers + immatures + alates (5 species). in addition, five species – brachymyrmex admotus mayr, 1887, camponotus sp. 9, p. sarcina, p. sospes, and pheidole sp. 43, formed polydomous and polygynous nests. most colonies were monogynous (figure 3). in total, we recorded 10 species with one queen and 5 species with more than one queen; four species had two queens (b. admotus, pheidole sp. 43, p. sarcina, p. sospes) and one species had three (pheidole sp. 43). fig 3. proportion of nests of species found in both leaf litter and twigs with different colony component patterns. the number of queens is shown in parentheses. tt fernandes, rr silva, dr souza-campana, ns silva, ogm silva, msc morini – ants in both leaf litter and twig166 table 1. list of taxa recorded from leaf litter and twigs, with social structure of the colony, nest type, and guild membership. the total number of nests is shown in parentheses. species in bold font were recorded both in the leaf litter and in twigs. taxon leaf litter twig social structure of the colony nest type trophic guild membership* acanthognathus ocellatus mayr, 1887 3 2 (0) monogyny (1) satellite structure dacetini predators with kinetic mandibles(0) polygyny (1) polydomy acromyrmex diasi gonçalves, 1893 1 leaf cutters acromyrmex disciger mayr, 1887 1 apterostigma sp. 1 2 litter-nesting fungus growers octostruma rugifera (mayr, 1887) 15 medium-sized epigeic generalist predators dacetini predators octostruma stenognatha brown & kempf, 1960 13 brachymyrmex admotus mayr, 1887 4 3 (0) monogyny (1) satellite structure generalist species dominant arboreal ants associated with carbohydraterich resources or domatia (1) polygyny (1) polydomy brachymyrmex cordemoyi forel, 1895 2 brachymyrmex heeri (forel, 1874) 15 camponotus sp. 2 4 (0) monogyny (0) satellite structure dominant arboreal ants associated with carbohydraterich resources or domatia (0) polygyny (4) polydomy camponotus sp. 5 2 (0) monogyny (0) satellite structure (0) polygyny (2) polydomy camponotus alboannulatus mayr, 1887 20 (3) monogyny (0) satellite structure (0) polygyny (17) polydomy camponotus sp. 8 2 (0) monogyny (0) satellite structure (0) polygyny (2) polydomy camponotus sp. 9 2 (0) monogyny (0) satellite structure (1) polygyny (1) polydomy carebara sp. 1 4 small-sized hypogeic generalist foragers neocerapachys splendens (borgmeier, 1957) 3 specialists predators living in the superficial layers of the soil crematogaster corticicola mayr, 1887 1 dominant arboreal ants associated with carbohydraterich resources or domatia crematogaster sp. 1 4 6 (1) monogyny (2) satellite structure (0) polygyny (3) polydomy crematogaster sp. 7 1 crematogaster sp. 18 3 (0) monogyny (2) satellite structure (1) polygyny (0) polydomy cyphomyrmex rimosus (spinola, 1851) 11 litter-nesting fungus-growers cyphomyrmex transversus (emery, 1894) 6 dyscothyrea sexarticulata (borgmeier, 1954) 1 specialist predators living in the superficial layers of the soil ectatomma edentatum roger, 1863 2 large epigeic generalist predators arboreal predators gnamptogenys continua (mayr, 1887) 2 medium-sized epigeic generalist predators medium-sized hypogeic generalist predators gnamptogenys striatula (mayr, 1884) 25 17 (0) monogyny (5) satellite structure (0) polygyny (12) polydomy sociobiology 67(2): 163-172 (june, 2020) 167 heteroponera dentinodis (mayr, 1887) 3 (0) monogyny (0) satellite structure medium-sized epigeic generalist predators (0) polygyny (3) polydomy heteroponera dolo (roger, 1860) 1 (0) monogyny (0) satellite structure (0) polygyny (1) polydomy heteroponera mayri kempf, 1962 8 7 (0) monogyny (0) satellite structure (0) polygyny (7) polydomy hylomyrma balzani (emery, 1894) 5 medium-sized epigeic generalist predatorshylomyrma reitteri (mayr, 1887) 3 hypoponera sp. 1 10 medium-sized hypogeic generalist predators hypoponera sp. 4 10 3 (1) monogyny (1) satellite structure (0) polygyny (1) polydomy hypoponera sp. 5 2 hypoponera sp. 7 8 4 (1) monogyny (0) satellite structure (0) polygyny (3) polydomy hypoponera sp. 10 2 (0) monogyny (0) satellite structure (0) polygyny (2) polydomy hypoponera sp. 11 3 linepithema iniquum (mayr, 1870) 2 (0) monogyny (0) satellite structure generalist species (0) polygyny (2) polydomy linepithema neotropicum (wild, 2007) 7 18 (3) monogyny (1) satellite structure (0) polygyny (14) polydomy megalomyrmex goeldii forel, 1912 1 medium-sized epigeic generalist predators mycetarotes parallelus (emery, 1906) 1 (1) monogyny (0) satellite structure (0) polygyny (0) polydomy mycetarotes senticosus kempf, 1960 2 myrmelachista catharinae mayr, 1887 1 18 (0) monogyny (2) satellite structure (0) polygyny (16) polydomy myrmelachista ruzskyi forel, 1903 14 (0) monogyny (1) satellite structure (0) polygyny (13) polydomy neoponera crenata (roger, 1861) 1 (0) monogyny (0) satellite structure (0) polygyny (1) polydomy nylanderia sp. 1 11 odontomachus affinis guérinméneville, 1844 1 large epigeic generalist predators odontomachus meinerti forel, 1905 3 oxyepoecus myops alburquerque & brandão, 2009 4 medium-sized epigeic generalist predators generalist species pachycondyla harpax fabricius, 1804 2 large epigeic generalist predators medium-sized hypogeic generalist predators arboreal predator pachycondyla striata smith, 1858 4 pheidole flavens roger, 1863 13 (8) monogyny (0) satellite structure medium-sized epigeic generalist predators(0) polygyny (5) polydomy table 1. list of taxa recorded from leaf litter and twigs, with social structure of the colony, nest type, and guild membership. the total number of nests is shown in parentheses. species in bold font were recorded both in the leaf litter and in twigs. (continuation) taxon leaf litter twig social structure of the colony nest type trophic guild membership* tt fernandes, rr silva, dr souza-campana, ns silva, ogm silva, msc morini – ants in both leaf litter and twig168 pheidole gertrudae (forel, 1886) 1 generalist species pheidole sarcina forel, 1912 20 56 (28) monogyny (2) satellite structure medium-sized epigeic generalist predators generalist species (1) polygyny (25) polydomy pheidole sigillata wilson, 2003 3 16 (10) monogyny (0) satellite structure (0) polygyny (6) polydomy pheidole sospes forel, 1908 40 13 (6) monogyny (3) satellite structure (1) polygyny (3) polydomy pheidole subarmata mayr, 1884 5 pheidole sp. 9 3 (1) monogyny (0) satellite structure (0) polygyny (2) polydomy pheidole sp. 12 8 pheidole sp. 15 1 pheidole sp. 16 4 pheidole sp. 18 5 pheidole sp. 20 1 pheidole sp. 28 2 pheidole sp. 43 7 26 (16) monogyny (2) satellite structure (2) polygyny (6) polydomy procryptocerus adlerzi (mayr, 1887) 6 (0) monogyny (0) satellite structure pollen-feeding arboreal ants (0) polygyny (6) polydomy procryptocerus sp. 2 2 (0) monogyny (0) satellite structure (0) polygyny (2) polydomy pseudomyrmex phyllophilus (smith, 1858) 3 (0) monogyny (0) satellite structure arboreal predators (0) polygyny (3) polydomy pseudomyrmex gr. pallidus 6 (1) monogyny (0) satellite structure (0) polygyny (5) polydomy solenopsis sp. 2 53 20 (9) monogyny (0) satellite structure small-sized hypogeic foragers small-sized hypogeic foragers (0) polygyny (11) polydomy solenopsis sp. 3 25 2 (2) monogyny (0) satellite structure (0) polygyny (0) polydomy solenopsis sp. 4 3 solenopsis sp. 5 4 (0) monogyny (0) satellite structure small-sized hypogeic foragers small-sized hypogeic foragers(0) polygyny (4) polydomy strumigenys appretiata (borgmeier, 1954) 2 dacetini predators with static pressure mandibles strumigenys cosmostela kempf, 1975 2 strumigenys crassicorns mayr, 1887 9 strumigenys denticulata mayr, 1887 17 strumigenys sanctipauli kempf, 1958 1 strumigenys schmalzi emery, 1906 2 wasmannia affinis santschi, 1929 20 generalist species richness 61 36 * according to brandão et al. (2012) table 1. list of taxa recorded from leaf litter and twigs, with social structure of the colony, nest type, and guild membership. the total number of nests is shown in parentheses. species in bold font were recorded both in the leaf litter and in twigs. (continuation) taxon leaf litter twig social structure of the colony nest type trophic guild membership* sociobiology 67(2): 163-172 (june, 2020) 169 discussion the richness of ants that exploit both the leaf litter and twigs is small relative to the diversity of species that inhabit the leaf litter (20% of total species). in this work, we observed that not all twigs had ant colonies, but 43% were colonized by species that were also present in the leaf litter. according to sagata et al. (2010), the fact that most twigs are not colonized, despite the diversity of leaf litter ants (silva & brandão, 2010), is probably not related to the availability of this nesting resource. souza-campana et al. (2017) showed that diameter explained the richness of ant communities in twigs. some species even colonize same-diameter twigs, regardless of habitat (fernandes et al., 2018). we observed that most ant species present in both the leaf litter and in twigs were inhabitants of the atlantic forest leaf litter (brandão et al., 2012). however, species that forage sporadically in the leaf litter (delabie et al., 2000), such as exclusively arboreal species (for instance, m. catharinae), were also recorded by us both in the leaf litter and in twigs. arboreal species exploit a habitat that is constrained in terms of nesting and feeding resources (wilson & hölldobler, 2005) and provides a drier environment relative to the leaf litter (davidson & patrell-kim, 1996; yanoviak & kaspari, 2000). thus, we suggest that the leaf litter and the twigs may help maintain the colony, especially during the dispersal of reproductive forms, a period of great energy expenditure (frank, 1987). for m. catharinae, along with alates, we found workers and immatures, but never any queens. in contrast, nakano et al. (2013) found colonies of m. catharinae, m. nodigera mayr, 1887 and m. ruszkyi roger, 1863 that included queens, demonstrating that twigs can contain all colony components and that this nesting site must be part of the life cycle of these arboreal species. in contrast to myrmelachista species, p. flavens is a leaf litter dweller (mcglynn & owen, 2002) that is rarely found in this layer in atlantic forest areas (pacheco et al., 2009; suguituru et al., 2011; 2013), but is very frequent in twigs (fernandes et al., 2012). here we only detected this species in twigs. given these reports, we suggest that this species is arboreal and forage sporadically in the leaf litter, in the same way as m. catharinae (nakano et al., 2013), azteca and crematogaster (delabie et al., 2000). conflicting with our hypothesis, most nests appeared to be from polydomous colonies and had workers and immatures, but no queen. the polydomous colony is a functional and cooperative unit (ellis et al., 2017), because it increases the probability of colony survival (silvestre et al., 2003; santos & del-claro, 2009) by expanding the foraging area (robinson, 2014) and the space for colony development (byrne, 1994) in the competitive environment of the leaf litter (yanoviak & kaspari, 2000). in addition, it protects the colony against predators (debout et al., 2007) by facilitating information sharing, such as mass recruitment (hamidi et al., 2017) and transfer of immatures and the queen (stroeymeyt et al., 2017). a polydomous colony structure is a strategy to reduce mortality, because this risk increases when there is only one nest (hölldobler & wilson, 1990; debout et al., 2007). the presence of immatures also increases the level of defence deployed by the colony (debout et al., 2007). species that colonize ephemeral habitats, such as twigs (byrne, 1994), tend to be polygynous (hölldobler & wilson, 1990), but our results showed that monogyny was more frequent. normally, a monogynous colony disperses farther away (hamidi et al., 2017) because they have larger energy supplies compared to polygynous colonies (deheer et al., 1999). given that the soil and the leaf litter are rich in ants (delabie et al., 2007; silva et al., 2017) and other invertebrates (decaëns, 2010; morais et al., 2010), the colony dispersal to more distant habitats may be a strategy to avoid competition; the twigs would act as a protective environment during this stage, when a higher accumulation of energy is needed. here we observed concurrent polydomy and polygyny in 6% of the species, which may indicate an influence of the environment over the strategy of colonization and colony dispersal of certain species. martins segundo et al. (2017) reported that colonies of crematogaster pygmaea forel, 1904 in environments subject to human disturbance and adverse and unstable climatic conditions had a higher rate of polydomous and polygynous colonies than crematogaster abstinens forel, 1899 recorded in a preserved environment. twigs host many ant genera that have different foraging habits, ranging from generalists to predators, which correspond to two of the nine most common guilds in the atlantic forest leaf litter (silva & brandão, 2010). this has also been observed in the amazon forest (carvalho & vasconcelos, 2002) and in different habitats within the atlantic forest region (souza-campana et al., 2017). predatory ants may feed on various arthropods that also colonize twigs, such as springtails (castaño-meneses et al., 2015), other ant species (brandão et al., 2012), isopods, beetles (baccaro et al., 2015) and larvae (ogogol et al., 2017), which makes this an attractive resource due to the presence of potential prey (lanan et al., 2014). our study presents biological information that contributes to the knowledge about ants that occupy the leaf litter and colonize twigs in the brazilian atlantic forest, since we show that most species are polydomous and monogynous. species survival strategies in a competitive habitat may be linked to the type of nest and social structure of the colony. the monogynous status suggests that a new colony may be founded in a site that is more distant than the origin site, a strategy that was unknown for twig-colonizing species until now. acknowledgements we would like to thank the são paulo research foundation (fapesp; protocol no. 10/50973-7; no. 10/50294-2; tt fernandes, rr silva, dr souza-campana, ns silva, ogm silva, msc morini – ants in both leaf litter and twig170 no. 2013/16861-5), the foundation for the support of teaching and research/university of mogi das cruzes (faep/umc), and the national council for technological and scientific development (cnpq; protocol no. 302363/2012-2) for their financial support, the biodiversity authorization and information system (sisbio; protocol no. 45492), and l. menino for preparing the map. financial support this work was supported by the fundação de amparo à pesquisa do estado de são paulo (fapesp), under grant number: 10/50973-7; no. 10/50294-2, and number: 2013/ 16861-5; national council for technological and scientific development (cnpq), under grant number 302363/2012-2) and foundation for the support of teaching and research/ university of mogi das cruzes (faep/umc). authors’ contributions tae tanaami fernandes: contribution to data collection, contribution to data analysis and interpretation, contribution to manuscript preparation. rogério rosa silva: contribution to data analysis and interpretation; contribution to manuscript preparation; contribution to critical revision, adding intelectual content. débora rodrigues de sousa-campana: substantial contribution in the concept and design of the study, contribution to data analysis and interpretation; contribution to manuscript preparation. nathalia sampaio da silva: contribution to data collection, contribution to manuscript preparation, contribution to critical revision. otávio guilherme morais da silva: contribution to data collection, contribution to data analysis and interpretation, contribution to manuscript preparation. maria santina de castro morini: substantial contribution in the concept and design of the study; contribution to manuscript preparation; contribution to critical revision, adding intelectual content. references baccaro, f.b., feitosa, r.m., fernandez, f. & fernandes, i.o. 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(2000). community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 259-266. doi: 10.1034/j.1600-0706. 2000.890206.x doi: 10.13102/sociobiology.v62i4.445sociobiology 62(4): 598-603 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 community of social wasps (hymenoptera: vespidae) in areas of semideciduous seasonal montane forest introduction social wasps (hymenoptera: vespidae), known as marimbondos and/or cabas (souza & zanuncio, 2012), possesses cosmopolitan distribution, with the greatest diversity of species in the neotropical region (carpenter, 1981; carpenter & marques, 2001). in brazil, the representatives of that group belong to the subfamily polistinae, with around 319 species reported in 26 genera (prezoto et al., 2007). they are found in natural environments (elpino-campos et al., 2007; souza et al., 2012), both cultivated (auad et al., 2010; de souza et al., 2012) and man-modified areas (jacques et al., 2012; souza et al., 2013). social wasps play an important role in the communities, whether in the natural whether agricultural ecosystems by the predation pressure exerted on the populations of other organisms (carpenter & marques, 2001; souza & zanuncio, 2012). another function which has been ascribed to those insects is the one of acting as important components of the guild of floral visitors in the neotropics (heithaus, 1979; aguiar & santos, 2007; clemente et al., 2013). abstract the composition of the fauna of social wasps in two areas of semideciduous seasonal montane forest in southeastern brazil was investigated. the collections were conducted between january 2011 and february 2013 with the use of two methodologies: bait traps and active collecting. thirtyfour species were recorded, distributed in 11 genera, namely, polybia lepeletier represented by 11 species (31%), followed by polistes latreille and mischocyttarus de saussure, both with seven species (20%); the other genera were represented only by one or two species. three new records were done for minas gerais: mischocyttarus consimilis zikán, mischocyttarus ignotus zikán and mischocyttarus paraguayensis zikán. the similarity of the fauna of social wasps of the different studies conducted in minas gerais presented negative and significant correlation with the distance among the areas (r2=0.1488, p= 0.02). sociobiology an international journal on social insects mm souza¹, ep pires², , r silva-filho³, te ladeira3 article history edited by gilberto m. m. santos, uefs, brazil received 15 june 2014 initial acceptance 17 july 2014 final acceptance 08 octuber 2015 keywords polistinae, survey, polybia. corresponding author epifânio porfiro pires departamento de entomologia universidade federal de lavras ufla 37200-000, lavras-mg, brazil e-mail: epifaniopires@yahoo.com.br in spite of the ecological importance of social wasps, a few species are potentially endangered of extinction, particularly in consequence of the human action by the destruction of the colonies, indiscriminate use of insecticides (prezoto, 1999; souza et al., 2012), in addition to the fragmentation and replacement of continuous forest areas, which has brought about the decrease of the nesting sites and food sources (souza et al., 2010; souza et al., 2014). in that way, the knowledge of the fauna of those insects becomes urgent with the purpose of supporting conservation and management actions and programs which aim at the maintenance of those species and environmental services (elpino-campos et al., 2007). in spite of several works of diversity of social wasps having been carried out in minas gerais in the latest years, are few the sampled areas of natural vegetation (souza & zanuncio, 2012). in this context, the achievement of that study proposes to obtain information of the fauna of social wasps in two areas of semideciduous seasonal montane forest in southeastern brazil. research article wasps 1 instituto federal de educação, ciência e tecnologia do sul de minas , inconfidentes-mg, brazil 2 universidade federal de lavras (ufla), lavras-mg, brazil 3 universidade federal de viçosa (ufv), viçosa-mg, brazil sociobiology 62(4): 598-603 (december, 2015) 599 material and methods study area the data of the present study were obtained in the “parque estadual serra do brigadeiro” (pesb) and in the municipality of “são gonçalo do sapucaí”. the pesb is situated at 20º 43’ s 42º 29’ w, with altitudes reaching 1,900 m. the region’s climate is of the type middle mesothermal (cwb), with annual average rainfall of 1,300 mm and annual average temperature of 18 ºc (engevix, 1995). the vegetation of the pesb is made up of seasonal semideciduous forest, of upper montane formation, with campos de altitude (altitude fields) occupying the isolated plateaus and the cliffs in some areas above the elevation height of 1,600 m (veloso et al., 1991). the municipality of “são gonçalo do sapucaí” (sgs) is situated at 21° 52′ s, 45° 35′ w, with altitude around 1,000 m. the climate of the region is of the type humid subtropical according to the köppen climate classification. the vegetation of sgs is of the type semideciduous seasonal montane forest, riparian forest and campo cerrado. method the species of social wasps were collected in “parque estadual serra do brigadeiro” in the period of january to december of 2011, with 20 days collection and in “são gonçalo do sapucaí”, in the period of april of 2012 to february of 2013, with 20 days of sampling. the collections in both the areas were conducted by means of the active collecting and bait traps (silveira, 2000; souza & prezoto, 2006). the active collecting was performed in the interval between 7 hours and 17 hours in existing trails, on plants with flowers, rocky outcrops, areas close to the streams existing in the area, holes in tree trunks, broad-leafed vegetation, canopy (with the aid of binoculars), abandoned buildings, farms nearby farms and termite hills. it amounted to a sampling effort of 200 hours’ collection per area. the bait traps for collection of social wasp species were manufactured with translucent “pet” type plastic bottles of two litters with two triangular side openings (2 x 2 x 2 cm) (souza & prezoto, 2006). passion fruit (passiflora edulis f. flavicarpa deg.; passifloraceae), mango (mangifera indica l.; anarcadiaceae) and sardine (sardinella brasiliensis steindachner 1789) were used as baits,. for preparation of the baits we have used, 1 kg of fruit pulp, 200 grams of crystal sugar and two litters of tap water. for the sardine, 250 kg for each two litters of tap water were used. the attractants were prepared with the aid of a blender in order to obtain a homogenous blend. eight sets of traps per area were set up, each set being made up of the three attractive substances (mango, sardine and passion fruit), suspended at 1.50m away from ground and 100 meters equidistant. for standardization purposes, in each trap 300 ml of the bait were placed. the traps were collected after a week in order to avoid the insects deterioration. the identifications of the specimens were done on the basis of keys proposed by richards (1978), carpenter and marques (2001), carpenter (2004) and by comparison with the specimens of the collection of social wasps of ifsuldeminas and those of the collection of the “museu paraense emílio goeldi”, belém, pará. the vouchers were incorporated to the heritage of the if-suldeminas collection, campus inconfidentes, minas gerais (http://vespas. ifs.ifsuldeminas.edu.br) and in the entomology collection of the museu paraense emilio goeldi, belém, pará. literature data for the similarity analysis among the faunas of social wasps collected in the state of minas gerais, data of areas of cerrado of the works of elpino-campos et al. (2007) in “uberlândia” (15º57’s, 48º12’w and 19º09’s, 48º23’w) and simões et al. (2012) in the “reserva biológica unilavrasboqueirão” (21º20’s e 44º59’w); “campo rupestre” of the works of prezoto and clemente (2010) in “parque estadual do ibitipoca” (21º40’s e 43º52’w) and souza et al. (2010) in the “apa são josé” (21°05’s and 44°10’w); area of evergreen forest, atlantic forest of the work of souza et al. (2012) in “parque estadual do rio doce” (19º38’s e 42º 31’w); transition area cerrado semideciduous forest of the works of souza and prezoto (2006) e souza et al. (2008) in “região da mata do baú” (21°12’s e 43º55’w) and man-modified area of the work of jacques et al. (2012) on the “campus da ufv” (20º46’s e 42º52’w) were utilized. data analysis the comparison between the faunas of social wasps collected in the state of minas gerais was done by the cluster analysis (upgma) by means of the jaccard similarity coefficient (krebs, 1998), which takes into consideration the occurrence of the two species in each area. in the similarity analysis were utilized only the species with identifications till the species level. species listed only as “sp.” in souza et al. (2012), elpino-campos et al. (2007) and jacques et al. (2012) were not included in the survey. the identifications at the subspecies level of the works of elpinocampos et al. (2007), souza et al. (2008), souza et al. (2010), souza et al. (2012) were not taken into account in the survey. the pearson analysis (r) (zar, 1999) was utilized to establish likely similarity relation among the faunas of social wasps of nine areas studied in minas gerais with their respective distances utilizing the software statistica for windows 5.0. in the analysis undertaken, the level of significance (α) of 0.05 was considered. the data of the distances (km) among the areas were obtained by the tool “rule” of the google earth pro. mm souza et al. – social wasps in semideciduous seasonal montane forest600 results and discussion during the period of study were recorded 34 social wasp species distributed into 11 genera, with representatives of the tribes polistini (seven species), mischocyttarini (seven species) and epiponini (nine genera and 20 species) (table 1). the richness of species recorded in this study for tribe epiponini when compared with mischocyttarini and polistini (table 1) can be related to the fact of the species of the first tribe presenting founding of its colonies by swarming, which can build large nests containing a great deal of individuals (jeanne, 1991; carpenter & marques, 2001), making this way the occurrence of a great deal species of that tribe frequent in the places where the nests are built (locher et al., 2014). the genus polybia lepeletier, 1836 was the most representative with 11 species (31%), followed by polistes latreille (1802) and mischocyttarus de saussure, 1853, both with seven species (20%), the other genera were represented by only one to two species (fig 1). the greatest richness of species recorded in this study for the genus polybia corroborates with other surveys done in minas gerais (elpino-campos et al., 2007; souza et al., 2014) and other regions in brazil (diniz & kitayama, 1994; somavilla et al., 2014). nevertheless, those results differ from those recorded by silveira (2002) and silva and silveira (2009) who inventoried the stretch of amazon rainforest in caxiuanã, pa and souza et al. (2012) in an area of campos rupestres in tiradentes, mg, in which the most representative genus was mischocyttarus. species pesb sgs epiponini agelaia multipicta (haliday, 1836) + agelaia vicina (de saussure, 1854) + + apoica gelida van der vecht, 1973 + brachygastra lecheguana (latreille, 1824) + + parachartergus fraternus (griboldo, 1892) + polybia chrysothorax (lechtenstein, 1796) + + polybia fastidiosuscula de saussure 1854 + + polybia ignobilis (haliday, 1836) + + polybia jurinei saussure, 1854 + + polybia minarum ducke, 1906 + + polybia occidentalis (olivier, 1791) + + polybia paulista von ihering 1896 + + polybia platycephala richards, 1978 + polybia punctata buysson, 1908 + polybia scutellaris (write, 1841) + + polybia sericea (olivier, 1791) + + protonectarina sylveirae (saussure, 1854) + + protopolybia sedula (saussure, 1854) + + pseudopolybia vespiceps (saussure, 1864) + synoeca cyanea (fabricius, 1775) + + polistini polistes actaeon haliday, 1836 + polistes billardieri ruficornis saussure, 1854 + polistes cinerascens saussure, 1853 + + polistes ferreri saussure, 1853 + + polistes lanio lanio (fabricius, 1775) + polistes simillimus zikán, 1951 + + polistes versicolor (olivier, 1971) + mischocyttarini mischocyttarus consimilis, zikán 1949 + mischocyttarus ignotus, zikán, 1949 + mischocyttarus atramentarius zikan 1949 + mischocyttarus cassununga (von.ihering, 1903) + + mischocyttarus drewseni saussure, 1857 + + mischocyttarus paraguaensis, zikán, 1935 + + mischocyttarus rotundicollis (cameron,1912) + + total 22 33 + (present in the area) and (absent in the area). table 1. list of social wasp species recorded in the “parque estadual serra do brigadeiro” in the period from january to december 2011, and the municipality of “são gonçalo do sapucaí” in the period from april 2012 to february 2013, minas gerais, brazil. fig 1. number of species per genus of social wasps recorded in the “parque estadual serra do brigadeiro” in the period from january to december 2011, and the municipality of “são gonçalo do sapucaí” in the period from april 2012 to february 2013, minas gerais, brazil. twenty-one species of social wasps were common to both areas. twelve species were unique to “são gonçalo do sapucaí” and only one to “parque estadual serra do brigadeiro” (table 1). the occurrence of particular species in the habitats can be related to the presence of substrates with conditions specific to nesting, in addition to the favorable conditions of temperature and humidity required to the development of the immature ones (dejean et al., 1998; kumar et al., 2009; souza et al., 2010; souza & zanuncio, 2012). three new records were done for minas gerais, mischocyttarus consimilis, zikán 1949; mischocyttarus ignotus, zikán, 1949 in “são gonçalo do sapucaí” and mischocyttarus paraguayensis, zikán, 1935 in “parque sociobiology 62(4): 598-603 (december, 2015) 601 estadual serra do brigadeiro” (souza et al., 2015). the record of new species of the genus mischocyttarus is due to the sum of factors, such as the tiny size of their nests with few individuals, in addition to the fact forming the largest group of social wasps, with 245 species of nine subgenera, which added to the regions little investigated as areas of forest on the mountain tops in minas gerais, increases the chance of unprecedented records (cooper, 1998; silveira, 2008; souza et al., 2012; souza et al., 2015). on the basis of the jaccard similarity coefficient among faunas of social wasps, some surveys done in the state of minas gerais, it was possible to observe the formation of basically two groups. one formed by the “parque estadual do rio doce” region, area of evergreen forest, atlantic forest (souza et al., 2012) and the other by the other areas (elpinocampos et al., 2007; souza et al., 2008; prezoto & clemente, 2010; souza et al., 2010; jacques et al., 2012; simões et al., 2012 and present study) (fig 2). greater similarity value between the composiiton of the species of “parque estadual serra do brigadeiro” and “são gonçalo do sapucaí” was found, both present in the study, with around 65% of similarity, followed by “mata do baú” with “apa tiradentes” (59%); “mata do baú” and “reserva biológica unilavras-boqueirão” (57%); “apa tiradentes” and “reserva biológica unilavras-boqueirão” (54%); “parque estadual serra do brigadeiro” and “mata do baú” (54%); “parque estadual do ibitipoca” and “parque estadual serra do brigadeiro” (51%). the other areas obtained similarity below 50%. “parque estadual do rio doce” presented smaller similarity index with the all the other areas, which ranged from 16 to 32% (fig 2). the similarity of the faunas of social wasps of the different studies conducted in minas gerais presented negative and significant correlation with the distance among the areas showing that the greater the distance, smaller the similarity (r2=0.1488, p= 0.02) (fig 3). fig 2. dendrogram similarity jaccard among the fauna of social wasps nine areas studied in minas gerais. data retrieved from the regions of “mata do baú” (souza & prezoto, 2006; souza et al., 2008), “uberlândia” (elpino-campos et al., 2007), “parque estadual do ibitipoca” (peib) (prezoto & clemente, 2010), “apa são josé” (souza et al., 2010), “reserve biológica unilavras-boqueirão” (rbub) (simões et al., 2012), “parque estadual do rio doce” (perd) (souza et al., 2012), “campus da ufv” (jacques et al., 2012) , “são gonçalo do sapucaí” (sgs) and “parque estadual serra do brigadeiro” (pesb) (this study). fig 3. correlation pearson between the similarity (jaccard) between the faunas of social wasps of nine areas studied in minas gerais with their respective distances. the greatest similarity values between pesb and sgs, in the present study, can be explained by the vegetation structure of the two areas (semideciduous seasonal montane forest) as well as historical and geographical factors. however, for the low similarity of the “parque estadual do rio doce” region as well as the other areas, can be ascribed to the fact that the region of the park relates to ecosystems with unique vegetation and climate features, which are important factors in determining the composition of social wasp species which occur in a given area (elpino-campos et al., 2007; santos et al., 2007; souza et al., 2012). the studies in semideciduous forest in the latter decade revealed eight new species of social wasps for the state of minas gerais and endemic to that ecosystem (souza & prezoto, 2006; prezoto et al., 2009; souza et al., 2015), which shows the importance of that site, even fragmented. nevertheless, with advance of deforestation ad the few areas inserted into conservation units can take to the loss of those species, mainly those which need particular conditions for nesting (souza et al., 2010; souza et al., 2014). the results point to a diversity of social wasps for minas gerais, showing that the region plays an important role in the distribution of this fauna for the southeast region of brazil. acknowledgments we thank orlando tobias silveira for species identification and the ifet-mg-campus inconfidentes; employees of the state of brigadier park; the sao goncalo do sapucai prefecture, in mm souza et al. – social wasps in semideciduous seasonal montane forest602 the person of marli education department oraboni, transport; 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(1991). classificação da vegetação brasileira, adaptada a um sistema universal. ibge, departamento de recursos naturais e estudos ambientais, rio de janeiro, 123p. doi: 10.13102/sociobiology.v61i3.332-337sociobiology 61(3): 332-337 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 repellent effects of annona crude seed extract on the asian subterranean termite coptotermes gestroi wasmann (isoptera:rhinotermitidae) mn acda introduction plant extracts have been studied in the past as potential sources of botanical insecticides to control a variety of arthropod pests. their use for pest management has been regarded as an alternative to synthetic chemical insecticides (logan et al., 1990; isman, 2006). a large pool of plant based materials belonging to the families meliaceae, rutaceae, asceraceace, labiateae, piperaceae and annonaceae, among others, have been investigated for their insecticidal properties (jacobson, 1975; schoonhoven, 1982; grainge et al., 1984; arnason et al., 1989; van beek & breteler, 1993). plant parts used for screening and evaluation include leaves, flowers, roots, stem, fruit peeling, seeds and bulbs. studies have shown that plant extracts containing limonoids and terpenoids are effective against various pests such as the colorado potato beetle (alford et al., 1987), the brine shrimp larva (culex quinquefascintus) (magadula et al., 2009), the larvae of the mosquitoes aedes aegypti (hoe at al., 1998) and anopheles stephensi (saxena et al., 2004), the diamondback moth, plutella xylostella (leatemia and isman 2004a; 2005), the cabbage looper trichoplusia abstract crude seed extract of three tropical fruits belonging to the family annonaceae, viz., sweetsop (annona squamosa l.), soursop (a. muricata l.) and biriba (rollinia mucosa baill.) were investigated for their repellent effects on the asian subterranean termite coptotermes gestroi wasmann (isoptera: rhinotermitidae). results of laboratory feeding bioassay (choice and no-choice) indicated that crude extract of a. squamosa, a. muricata and r. mucosa had feeding deterrent effects on c. gestroi. termites showed significant avoidance behavior to filter paper treated with extracts of the three annona species investigated. soil barrier test revealed that annona extracts were able to limit penetration of c. gestroi in laboratory tunneling test. the results suggest that annona seed extracts may offer an alternative source of natural insecticide against subterranean termites. sociobiology an international journal on social insects university of the philippines los banos, philippines. article history edited by og de souza ufv, brazil received 11 february 2014 initial acceptance 28 may 2014 final acceptance 15 june 2014 keywords annonaceae, repellent, acetogenins, coptotermes gestroi, feeding deterrence corresponding author menandro n acda department of forest products and paper science, college of forestry and natural resources university of the philippines los banos e-mail: mnacda@yahoo.com ni (leatemia & isman, 2004b), the lepidopteran spodoptera frugiperda (alvarez colom et al., 2007), the german cockroach (blattella germanica) (alali et al. 2000), the adult and egg masses of the snail biomphalaria glabrata (dos santos & santa ana, 2010). one potential application of botanical insecticides is the control of subterranean termites. termites are serious structural pests of homes and wood structures in tropical and sub-tropical regions of the world (lee, 1971). total worldwide damage and repair costs due to termite activity had been estimated to be about usd 40 billion annually (rust & su, 2012). recent studies showed that plant derivatives such as pyrethrins, terpenoids, azadirachtin and flavanoids have excellent termiticidal activity (sharma et al., 1994; cornelius et al., 1997; chen et al., 2001; zhu et al., 2001a, b). one of the best sources of botanical insecticides is tropical annona species, i.e., members of the custard apple family (annonaceae). annona species are important sources of fruit juices, frozen pulp, jelly and ice cream in southeast asia and thousands of tons of seeds from these fruits are generated annually from commercial processing plants (isman, 2006, scuc 2006). research article termites sociobiology 61(3): 332-337 (september 2014) 333 seeds of annona species yield upon extraction a mixture of long fatty acid derivatives known as acetogenins (chang et al., 1998; polo et al,. 1998; pettit et al., 2008). annonaceous acetogenins are widely reported for its high insecticidal, molluscicidal and nematicidal properties (rupprecht et al., 1990; mclaughlin et al., 1997; isman, 2006). however, limited studies have been conducted on the efficacy of annonaceous acetogenins against subterranean termites. the present paper reports on the repellent effects of the crude seed extracts of anonna squamosa l., a. muricata l. and rollinia mucosa baill. on the asian subterranean termite coptotermes gestroi wasmann (isopetra: rhinotermitidae). materials and methods plant extracts ripe fruits of sweetsop (a. squamosa), soursop (a. muricata) and biriba (r. mucosa) were purchased from several market locations in laguna province, philippines. the fruits were harvested from small farms in the area cultivated by local farmers free of applied insecticide. extraction of seeds to separate bioactive crude components was based on the method described by leatemia and isman (2004a). briefly, seeds from various locations were pooled, washed with water, air dried and ground in a wiley mill (1.0 mm). one hundred gram of each ground samples were extracted in 200 ml of 95% ethanol (5x) over 5 days by soaking. the suspension was stirred at intervals to facilitate uniform extraction. the supernatants were filtered (whatman #1) and concentrated in a rotary evaporator under vacuum (35ºc). the concentrated extracts were re-suspended in 95% ethanol then transferred to pre-weigh vials. the ethanol was dried in a fume hood and yield determined by re-weighing the vials to determine extract weight. termites secondary nests of three active field colonies of the asian subterranean termite c. gestroi were collected from infested buildings in the university of the philippines los banos campus. c. gestroi (formerly known as c. vastator in the philippines) is a major structural pest of wood structures in southeast asia (acda, 2004; lee et al., 2007). the nests were placed in black garbage bags, transported to the laboratory and placed inside 100 liter plastic containers with lids kept in a room at 25° c for three days. distilled water was sprayed on the sides of the container to keep the relative humidity above 80%. mature worker (pseudergates beyond the third instar as determined by size) and soldier termites were separated from nest debris by breaking apart and sharply tapping materials into plastic trays containing moist paper towels. termites were then sorted using a soft bird feather and used for bioassay within one hour of extraction and segregation. feeding test a no-choice and choice feeding bioassay was performed to determine toxicity and repellency of crude annona seed extracts on the asian subterranean termite c. gestroi. dry crude extracts were used to prepare stock solutions in 95% ethanol. the no-choice feeding test was conducted using petri dishes (5.5 cm in diameter) containing sterilized moist sand (1 mm) and a 2.54 x 2.54 cm filter paper (whatman #1) impregnated with 80 µl of extract solution (12.4 µl/cm2). extract concentrations tested were 0, 1, 5, 10, 20% (w/v). concentrations used were based on preliminary screening. filter paper wetted with distilled water served as control. fifty worker termites plus 5 soldiers of c. gestroi from three active field colonies were placed in each dish. experimental units containing the termites were placed in an incubator maintained at 28°c and 85% relative humidity and force-fed on treated paper for 14 days. after the prescribed exposure period, percent mortality was determined by examining the experimental units for dead or moribund termites. workers were considered moribund when they no longer walk or stand when probed with forceps. dead or moribund workers were recorded and removed from each experimental unit daily. termite mortalities were corrected by abbott’s formula (1925). the test was replicated three times for each colony with a total of nine replicates for each concentration. the amount of filter paper consumed by the termites was determined by estimating the loss in surface area (mm2) of treated paper. paper consumption reported was the average of the evaluation of three individuals. data from the feeding test were fitted in a completely randomized design and evaluated by analysis of variance (anova) using statgraphics centurion 16.1 software (2010). treatment means were separated by tukey’s honest significance difference (hsd) test (α = 0.05). choice feeding test was performed similar to the no-choice feeding test described above except that two filter papers, one treated with crude extract and the other untreated, were placed in the center 1.0 cm apart. papers were impregnated with 80 µl of extract solution (0, 1, 5, 10, 20% (w/v). fifty workers plus 5 soldiers of c. gestroi were introduced to each experimental unit and placed in an unlit incubator as described above for 14 days. two untreated papers wetted with water were used as control. percent termite mortality was monitored daily. the test was replicated three times for each colony with a total of nine replicates for each concentration. consumption of treated paper by termites was determined as described above. soil barrier test tunneling and penetration of c. gestroi through soil treated with crude annona seed extracts were evaluated using method similar to that described by su and scheffrahn (1990) with some modifications. the tunneling units consisted of a 12 cm glass tube (1.5 cm diameter) containing a 5 cm segment m. n. acda repellent effects of annona crude seed on termites334 of treated soil sandwiched between 2 cm layer of 10% nonnutrient agar. sterilized loamy soil was thoroughly mixed with the crude seed extract corresponding to 0, 1, 5, 10 and 20% [weight (crude extract)/weight (soil)] concentration. treated soil was left to stand in a fume hood for 24 hours to evaporate the ethanol solvent. soil wetted with distilled water served as control. fifty workers plus 5 soldier termites were introduced into the void space and a piece of moistened filter paper was included to serve as food source. both ends of the glass tube were sealed with aluminum foil and then placed vertically in an unlit incubator maintained at 28°c and 85% relative humidity. termites were allowed to tunnel freely for 7 days and cumulative tunneling distance was monitored and recorded daily. the assembly was disassembled after 7 days, number of surviving insects was counted and termite mortality calculated. the test was replicated three times for each colony with a total of nine replicates for each concentration. results and discussion feeding tests no choice forced feeding of c. gestroi on filter paper treated with crude seed extract of a. squamosa, a. muricata and r. mucosa resulted in significant increase (p < 0.001) in termite mortality with increasing extract concentration (fig. 1). termites feed on paper treated with 1 and 5% crude seed extract of all three annona species investigated resulting in 23-68% mortalities after 14 days of exposure. treated filter papers showed termite nibbling along the edges causing about 5.5–12 mm2 reduction of surface area. however, paper treated with 1 and 5% extract was not sufficient to produce high mortality in c. gestroi. in addition, final mortalities at 5% concentration were observed to rise only after 6-7 days after exposure. mortalities on the first 5 days for the three annona species were about 8-15%. the reason for this observation is not clear but suggests delayed toxicity of annona seed extracts on c. gestroi. acetogenins, the bioactive component of the plant family annonaceae, was reported to act as a slow acting stomach poison against chewing insects (guadano et al., 2000; leatemia & isman, 2004a, b). further study on the delayed toxicity aspect, however, needs be investigated. termite mortalities at 10-20% crude extract of a. squamosa and a. muricata were relatively higher at 87-100% after 14 days of exposure (fig.1). papers treated with r. mucosa showed lower mortalities (78-85%) at the same extract concentration. however, examination of filter papers treated with 10-20% extract for all three samples revealed that the material was undamaged and not consumed by the termites. in addition, termites partially or completely covered the treated filter papers with sand. apparently, the insects died of starvation and burying papers in sand could be an attempt to avoid contact with treated material. the results suggest that atleast 10% crude annona seed extracts had repellent or feeding deterrent effects on c. gestroi. toxicity by extract ingestion of treated paper could not be judged in these experiments. choice feeding test showed that c. gestroi consumed both untreated and treated papers up to 5% extract concentration (table 1). termite mortality with papers treated with 1 to 5% extract of a. squamosa, a. muricata and r. mucosa was relatively low at about 3-14% after 14 days of exposure. paper consumption with untreated and those treated up to 5% extract was about 8-12 mm2 and 6-15 mm2, respectively, indicating healthy feeding on both materials. this would agree with the results of the no choice feeding above that low concentration of annona seed extracts was not toxic by ingestion to cause high mortality in c. gestroi nor repellent to prevent feeding of treated materials. however, termites showed significant avoidance behavior on filter papers treated with 10% extract as indicated by non-consumption of treated papers (table 1). termite mortality at 10-20% extract was relatively low at about 11-18% for all annona species investigated with untreated paper consumption of about 15– 36 mm2. termites also buried or covered treated papers with sand similar to the no-choice test above to prevent contact with the extract. repellent or anti-feeding effect of plant extracts on c. formosanus shiraki, reticulitermes santonensis de feytaud, r. virginicus banks such as limonoids (serit et al., 1992), flavanoids (ohmura et al., 2000), nootkatone and vetiver oil (zhu et al., 2001a;b), isoborneol and cedar oil (blaske & hertel, 2001; blaske et al., 2003), matrine and oxymatrine (alkaloids) (mao and henderson, 2007), essential oils (sakasegawa et al., 2003; simaron et al., 2009), extracts from the heartwood of various hardwoods (chang et al., 1997; watanabe et al., 2005; roszaini et al., 2013) had been reported in the literature. although the repellency levels reported in this study was relatively high in comparison with synthetic insecticides, the availability of significant amount of cheap raw materials (waste seeds) may offset extraction cost to be commercially feasible. in addition, purified components of fig. 1. mortality of c. gestroi in no choice feeding with crude annona seed extracts after 14 days of exposure. sociobiology 61(3): 332-337 (september 2014) 335 annona extracts (i.e. acetogenins) may be more potent against termites and should be further investigated. soil barrier untreated and soil treated with 1% crude extract of a. squamosa, a. muricata and r. mucosa were fully breached by c. gestroi penetrating the full 5 cm barrier after 7 days in tunneling and penetration tubes (table 2). however, soil treated with 5% crude extract of a. squamosa and a. muricata were able to limit termite penetration to about 0.62-1.24 cm during the 7 day exposure period. extract of r. mucosa at 5% showed longer penetration of about 3.3 cm but prevented c. gestroi from breaching the barrier. penetration of soil treated with at atleast 10% extract prevented termite tunneling and penetration of treated soil. no dead termites were found in treated soil. termite mortality in all concentration tested was relatively low at about 7-16%. the result indicated that annona crude seed extracts could potentially be used as soil barrier to prevent tunneling and penetration of c. gestroi. apparently, the mechanism involved in preventing penetration of c. gestroi appeared to be repellency or avoidance of treated soil. reduction in penetration and tunneling of subterranean termites were also reported in laboratory trials in sand or soil treated with plant extracts such as isoborneol (cornelius et al., 1997; blaske & hertel, 2001; blaske et al., 2003), nootkatone and vetiver oil (maistrello et al., 2001), essential oils (peterson & wilson, 2003) and other plant oils (yoshida et al., 2003; acda, 2009). conclusions in general, the study showed that ethanolic crude seed extracts of sweetsop (a. squamosa), soursop (a. muricata) and biriba (r. mucosa) had repellent or feeding deterrent effects on the asian subterranean termite c. gestroi. nochoice and choice feeding tests indicated that crude extract of a. squamosa, a. muricata and r. mucosa prevented termite feeding on treated filter paper. termites showed significant avoidance behavior to filter paper treated with atleast 10% annona crude seed extract. soil treated with 5-10% crude extract of the three annona species investigated prevented tunneling and penetration of c. gestroi. termite behavior in both feeding and soil penetration tests indicated that extracts of a. squamosa, a. muricata and r. mucosa seed extracts had anti-feeding or repellent effect on c. gestroi indicating table 1. average mortality and paper consumption of c. gestroi in choice feeding test with filter paper treated with various concentration of annona seed extract. % extract concentration a. squamosa a. muricata r. mucosa % mortality consumption (mm2) % mortality consumption (mm2) % mortality consumption (mm2) 1 8.45 ± 2.13a 10.23 ± 2.65 3.63 ± 1.12a 5.78 ± 1.22 12.03 ± 2.64a 9.34 ± 2.44 control 12.45 ± 1.83 12.57 ± 3.76 10.25 ± 1.13 5 11.38 ± 1.47a 12.43 ± 0.75 7.48 ± 0.56b 8.26 ± 1.58 14..42 ± 1.47ab 15.12 ± 2.66 control 8.48 ± 1.27 7.97 ± 2.68 8.573 ± 0.46 10 15.34 ± 1.68b 0 12.34 ± 1.32c 0 11.34 ± 3.17a 0 control 36.34 ± 3.45 22.53 ± 3.52 30.34 ± 3.55 20 18.56 ± 3.18c 0 15.75 ± 2.75c 0 13.24 ± 3.68a 0 control 25.45 ± 2.78 15.48 ± 4.35 19.62 ± 2.43 aeach value is the mean of 9 replicates each; numbers within a column followed by the same letter are not significantly different (tukey’s hsd test, α = 0.05). table 2. average mortality and penetration distance of c. gestroi after 7 days in tunneling tubes containing soil treated with various concentration of annona seed extract. % extract concentration a. squamosa a. muricata r. mucosa % mortality penetration (cm) % mortality penetration (cm) % mortality penetration (cm) 0 5.23 ± 0.75a 5.0a 8.45 ± 1.26a 5.0a 8.56 ± 1.13a 5.0a 1 8.34 ± 1.45a 5.0a 9.75 ± 2.52a 5.0a 10.17 ± 1.55ab 5.0a 5 12.43± 1.78b 0.62 ± 0.11b 10.17 ± 1.35ab 1.24 ± 0.68b 14.53 ± 1.43b 3.30 ± 0.74b 10 15.17 ± 2.42c 0b 12.5 ± 2.11b 0.53 ± 0.14c 13.23 ± 2.24b 0.65 ± 0.12c 20 16.26 ± 3.67c 0b 14.01 ± 1.34c 0c 15.18 ± 1.24b 0c aeach value is the mean of 9 replicates each; numbers within a column followed by the same letter are not significantly different (tukey’s hsd test, α = 0.05). m. n. acda repellent effects of annona crude seed on termites336 that these compounds may be potentially useful in the development of an alternative source of natural insecticide or in combination with other control methods against subterranean termites. purification, identification and testing of bioactive components of seed extracts of annonaceae species used in this study against termites are now underway. acknowledgments the author wishes to thank the national 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(2001b). evaluation of vetiver oil and seven insect active essential oil against the formosan subterranean termite. journal of chemical ecology, 27: 1617-1625. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.323-328sociobiology 60(3): 323-328 (2013) research article bees implications of the floral herbivory on malpighiacea plant fitness: visual aspect of the flower affects the attractiveness to pollinators ca ferreira, hm torezan-silingardi introduction interactions among organisms have occurred since the origin of life on earth, and all species are involved in this process (thompson, 2010). among the relationships that occur between plants and animals, pollination is essential because it increases the adaptive value of plants in relation to the environment, provides resources to animals, and occurs in 87.5% of angiosperms (ollerton et al., 2011). insects, especially winged social hymenoptera (bees and wasps), are among the main pollinators in nature (gullan & cranston, 2006). conversely, herbivory is one of the most important antagonistic relationships because it harms angiosperms and reduces their fitness (strauss et al., 1996, 1997; price et al., 2011). herbivores can consume photosynthetic tissues, storage organs, and reproductive structures (romero & vasconcellos-neto, 2007; del-claro et al., 2013), thereby comabstract the malpighiaceae family is species-rich and is abundant in brazil. malpighiaceae flowers provide oil and pollen to pollinating bees and serve as food for herbivorous insects, which damage the floral structures. although common in the cerrado, florivory is still poorly studied. in the present study, we evaluated the effect of florivory in one of the most common genera of malpighiaceae in the cerrado (banisteriopsis) and the impact of florivory on fruiting. the florivory rate was quantified in flowers of b. malifolia belonging to two morphotypes and in flowers of b. variabilis. additionally, a petal-removal experiment was performed, which simulated the presence of damage in the flowers. the manipulation involved a control group with intact flowers, a group without the standard petal and a group of flowers without common petals. the florivory in the petals (floral area lost) differed between the species, and b. malifolia was the most damaged. the experimental manipulation revealed that intact flowers had a higher fruiting rate compared with the remaining flowers. these results reinforce the concept that florivory renders flowers less attractive to pollinating bees, which negatively affects the fruiting rate and the reproductive success of plants. we suggest that basic studies (such as the present investigation) be extended to further elucidate the effect of interactions between pollinators, plants, and herbivores on the general structure of communities. sociobiology an international journal on social insects universidade federal de uberlândia, minas gerais, brazil. article history edited by gilberto m m santos, uefs, brazil received 15 july 2013 initial acceptance 05 aug 2013 final acceptance 22 aug 2013 keywords experimental manipulation, florivory, fruiting corresponding author helena maura torezan-silingardi lab. de ecologia comportamental e de interações universidade federal de uberlândia uberlândia, minas gerais, brazil 38400-902 e-mail: torezan@inbio.ufu.br promising the growth, reproduction, and survival of plants (torezan-silingardi, 2011). florivory, or the consumption of floral structures, negatively affects pollination (krupnick et al., 1999; mccall & irwin, 2006), either by destroying the internal structures of the buds and/or by reducing the attractiveness of flowers to the pollinators. for example, flower damage recorded for sanicula arctopoides causes a reduction in the number and weight of the seeds produced (lowenberg, 1994), illustrating reproductive losses for the species. therefore, florivory may affect the maintenance of plants in natural environments (torezan-silingardi, 2007, 2011). the signaling of the production and provision of resources, by coloration, size, scent, shape, and texture (gumbert, 2000; miller et al., 2011), serves as a cue for floral visitors. several studies have demonstrated that bees respond to these signals by visiting mainly the flowers that offer cues of more profitable rewards such as a greater amount of pollen, ca ferreira, hm torezan-silingardi effects of florivory in banisteriopsis fitness324 at the moment of anthesis and in the intensity of the scent: one type has flowers with an intense pink coloration and a strong scent (b. mali ip morphotype 1), and the other type produces light pink flowers and a mild scent (b. mali lp morphotype 2). b. variabilis produces only shrubs with white flowers and no scent. nectar, scent, or inflorescences that are larger, more conspicuous, or more numerous (waser & price, 1981; krupnick et al., 1999; irwin & strauss, 2005). similarly, these signals are attractive for other types of visitors and directly affect the reproduction of plants (torezan-silingardi, 2007; alvessilva et al., 2012). the family malpighiaceae is very common in the brazilian cerrado (ratter et al., 1997; giulietti et al., 2005), and the genus banisteriopsis, one of the largest genera of the family, comprises approximately one half of the species that occur in this biome (mabberley, 1997). banisteriopsis flowers supply oil and pollen for pollinating bees (anderson, 1979, 1990; vogel, 1974, 1990; sigrist & sazima, 2004; gaglianone, 2005). however, its flowers, fruit, and leaves are also sought by animals from various taxa (torezan-silingardi, 2011; oki, 2005; diniz et al., 2000; alves-silva et al., 2013). therefore, the main objective of the present study was to investigate the hypothesis that florivory in banisteriopsis significantly reduces the reproductive potential (i.e., fruit formation). the specific objectives were (1) to determine the proportion of damage by floral herbivory in the species b. malifolia and b. variabilis and (2) to describe the effect of florivory on the attractiveness of flowers and on the reproductive success of b. malifolia. materials and methods study site the study was performed in an area of cerrado strictu senso (a savanna-like vegetation that is the main physiognomy of the cerrado biome) at the legal reserve area of the clube de caça e pesca itororó de uberlândia (ccpiu), located at 18° 59’ s and 48° 18’ w, with an altitude of 863 m and a total area of 640 ha. according to the koppen classification, the climate of the region is type aw, with two well-defined seasons: the dry season from may to september and the rainy season from october to april, with a mean annual temperature of 22°c and a mean rainfall of 1,500 mm (bachtold et al., 2012). the vegetation in the reserve includes the following physiognomies: grassland (campo limpo), shrubland (campo sujo), cerrado strictu senso, woodland (cerradão), palm swamps (veredas), and small patches of mesophyllous forest (see réu & del-claro, 2005, for details on the area). species b. malifolia variety malifolia (ness & martius) g. gates and b. variabilis b. gates are extremely similar species (fig. 1). their flowers are complete and conspicuous, with a slightly zygomorphic symmetry and five fimbriated petals, the standard petal has a size and insertion angle that is different from the remaining four petals. b. malifolia exhibits two types of shrubs that vary in the coloration of the flowers fig. 1. flowers of banisteriopsis malifolia morphotypes intense pink (a) and light pink (b) and of banisteriopsis variabilis (c) present at the reserve area of the clube de caça e pesca itororó de uberlândia, state of minas gerais, brazil. data collection b. malifolia and b. variabilis shrubs of similar size and phenological state were monitored throughout the flowering period, particularly between april and july. for quantifying the floral herbivory, the flowers of 15 individuals of b. malifolia morphotype 1 (b. mali ip), b. malifolia morphotype 2 (b. mali lp), and b. variabilis were collected, photographed, and measured. in total, 45 flowers were analyzed with the photoshop cs6 software, by measuring the total floral area and the floral area lost. for the experiment of simulated florivory, the inflorescences of 30 shrubs of b. malifolia (morphotype 2: light pink), divided in three groups of 10 individuals, were isolated at the pre-anthesis period and were subsequently manipulated using gloves and fine-tip tweezers. the manipulations were always performed before 07:00 to avoid the timepoint at the beginning of pollinator activity. in total, 150 flowers were manipulated (five per shrub), and each individual was submitted to one of the following treatments: (1) control, the flowers remained intact without the simulation of herbivory; (2) herbivory was simulated by removing only the standard petal, with the common petals intact; (3) herbivory was simulated by removing four common petals, with the standard petal intact. statistical analysis after determining the normality of the data, a one-way analysis of variance (anova) was used to evaluate whether sociobiology 60(3): 323-328 (2013) 325 the florivory differed between the b. malifolia and b. variabilis species and morphotypes. we also assessed whether there was variation in the fruiting rate among the different treatments of the damage-simulation experiment. the analyses were performed with the systat 10.1 software. results the flowering period occurred between the months of april and july 2012 (fig. 2). banisteriopsis malifolia was the first species to exhibit flowers (at the beginning of april), followed by b. variabilis, which had its first flowers recorded at the end of the same month and in the beginning of may. the flowering overlap encompassed approximately 20 days and resulted in a high number of available flowers, which attracted different groups of floral visitors. light-pink morphotype (df 2,42=14.307; p<0.001) (fig. 3). of the b. malifolia morphotypes, the light-pink one exhibited a higher number of damaged flowers; however, intensely pink flowers suffered a higher loss of floral tissue area. the florivory percentage differed significantly between the species (0.03±0.13; n= 45). b. malifolia suffered a greater loss of floral area than did b. variabilis (f2,42=4.86; p=0.013) (fig. 4), and there was no significant difference in these values between the b. malifolia morphotypes. regarding the site of the damage, both species had damaged flowers with signs of herbivory in the central area of the petals, the margin of the petals and in both parts. the results of the simulated florivory revealed that the visual aspect of the flower affects the attractiveness to pollinators because there was a significant difference among the fruiting rates in the different treatments (2,93±1,38; n= 30). the group of intact flowers (control) had the highest fruiting rate of all three treatments (f 2,27= 35,413; p<0.001) (fig. 5). the flowers began the process of anthesis between 06:15 and 10:00 in the morning, with a small delay on cloudy and rainy days. during this period, pollinators and other groups such as herbivores, predators, and parasitoids were recorded exploring the resources offered by both species. the signs of floral herbivory were easily recognized, and herbivores were found mainly at the onset of flowering, between the months of april and may. herbivores belonging to the orders coleoptera, lepidoptera, hemiptera, orthoptera, hymenoptera, and thysanoptera were recorded in flowers of both species studied the most common ones being the curculionidae beetles (present mainly at the bud stage) and lycaenidae butterflies and small thysanoptera (in the buds and flowers). the results demonstrated variations in the floral area, number of damaged flowers, and florivory rate. the number of damaged flowers was higher in b. malifolia than in b. variabilis (table 1 and 2). the floral area was higher in b. malifolia (2,95±0.76; n= 45), mainly in flowers of the fig. 2. flowering phenology of the species of banisteriopsis variabilis and banisteriopsis malifolia from the reserve area of the clube de caça e pesca itororó de uberlândia, state of minas gerais, brazil. species /damage number of flowers total area of the petal (cm²) number of damaged flowers (%) damaged area in cm² (%) b. malifolia ip 80 42.75 32 (40%) 2.80 (6.5%) b. malifolia lp 70 53.90 36 (51%) 2.08 (3.7%) b. variabilis 15 36.42 03 (20%) 0.24 (0.6%) discussion the results of the floral-manipulation experiments and quantification of the natural florivory and the fruiting rate revealed that florivory negatively affects the reproduction of plants by the genus banisteriopsis in the cerrado. table 1. estimate of the floral damage and its percentage recorded for the study species and the different morphotypes of banisteriopsis malifolia. fig. 3. floral area of banisteriopsis variabilis and of banisteriopsis malifolia and its morphotypes from the reserve area of the clube de caça e pesca itororó de uberlândia, state of minas gerais, brazil. ip and lp refer to the two morphotypes of banisteriopsis malifolia: intense pink and light pink, respectively. ca ferreira, hm torezan-silingardi effects of florivory in banisteriopsis fitness326 these findings thus corroborate the central hypothesis of the present study. plants have developed several strategies to attract the service of pollinators, and this investment favors the maintenance of the species in the ecosystem (stout, 2000). however, florivory may directly reduce the quality and quantity of the available resources (karban & strauss, 1993) by alterations in the floral traits, such as shape and size of the petals (fraze & marquis, 1994), and by reductions of the visual and olfactory cues (krupnick & weis, 1999; krupnick et al., 1999). the damage recorded in the flowers of both species of banisteriopsis is directly related to the use of buds for egg-laying by endophytic florivores (torezan-silingardi, 2011) or to the use of petals for feeding by exophytic herbivores (alves-silva et al., 2013), both in the juvenile and adult stages (torezan-silingardi, 2007). the use of petals and of the remaining internal bud structures has been associated with a reduction in the overall attractiveness of flowers to their effective pollinators and to the consequent decrease in fruiting success (leavitt & robertson, 2006). the flowers of b. malifolia displayed a more extensive floral area, a higher number of flowers attacked, and a greater proportion of damage. the herbivores responsible for the reported damage might be attracted by the same cues identified by pollinators to find the flowers, such as the coloration, intensity of scent, size of the flowers and density of flowers per area, which are more evident in b. malifolia than in b. variabilis. therefore, the coloration and size of the flower appear to be important characteristics for explaining the preference of floral herbivores. irwin and strauss (2005), frey (2004), and salomão et al., (2006) discuss similar results in which the attraction cues (such as size, color, shape, and scent) that are recognized by the pollinators and can also be identified by floral herbivores. table 2. contingency data based on the presence and absence of floral damage recorded for both morphotypes of banisteriopsis malifolia and for both species of banisteriopsis. fig. 4. percentage of damage by florivory in the flowers of banisteriopsis variabilis and of banisteriopsis malifolia and its morphotypes from the reserve area of the clube de caça e pesca itororó de uberlândia, state of minas gerais, brazil. ip and lp refer to the two morphotypes of b. malifolia: intense pink and light pink, respectively. fig. 5. mean number of fruits produced during the simulated-florivory experiment in banisteriopsis malifolia (morphotype 2: light pink) at the reserve area of the clube de caça e pesca itororó de uberlândia, state of minas gerais, brazil. the results obtained in the experiment of simulated florivory reinforce the importance of floral attractiveness in the selection of resources by the pollinator because a reduced number of petals negatively affected the production of fruits. flowers with damaged structures or eaten petals may indicate a deficit or absence of resources, causing the bees to refuse them. furthermore, the olfactory cues that act together on the floral attraction of pollinators may influence the process of selection. salomão et al. (2006) studied the effects of the floral-area size and of the floral herbivory in trichogoniopsis adenantha (asteraceae) and reported that large bushes and flowers undamaged by herbivory produce a higher proportion of seeds. the authors also noted that, just as pollinators prefer to visit larger bushes, herbivores may also respond in the same manner and cause greater damage in larger flowers, thereby reducing their attractiveness to pollinators. sober et al. (2010) studied the floral herbivory in verbascum nigrum and reported results similar to the present findings, in which the visitation rate was negatively correlated to florivory. the presence of herbivorous insects on the banisteriopsis flowers studied was responsible for the change in the floral traits, and such damage negatively affected the flowerpollinator interactions, causing a significant reduction in the fruit and seed production. this lower fruiting rate may be presence absence total b. malifolia ip 36 44 80 b. malifolia lp 32 38 70 total 68 82 150 presence absence total b. malifolia 68 82 150 b. variabilis 3 12 15 total 71 9 165 sociobiology 60(3): 323-328 (2013) 327 the first sign of a reduction in fitness, considering that the majority of interactions with herbivores cause similar losses. hendrix and trapp (1989) tested the hypothesis that the responses to floral herbivory in pastinaca sativa l. (apiaceae) may compensate or even increase the fitness of the individuals attacked. the authors found that, despite the compensatory responses generated, the reduced fitness is a result of damage by herbivory, and the species cannot compensate for this reduction. therefore, future investigations (e.g., byk & del-claro, 2010) will be important for describing the responses of plant species to floral damage over time. studies of the interactions that occur between organisms are critical for the survival and reproduction of the species involved. furthermore, the different forms of interactions generate ecological diversity and enable the formation of rich ecosystems, which are composed of species that have coevolved (thompson, 2012). we can conclude that the species b. malifolia and b. variabilis are damaged by floral herbivores in different proportions and that this phenomenon directly affects the floral attractiveness, resulting in lower fruit production; furthermore, the higher the damage that is caused, the lower are the visitation rate and the reproductive success. the study of these effects in the interactions between floral herbivores, flowers, and pollinators elucidates how these species relate to the environment and may support future investigations regarding the selection of areas for conservation of the local biodiversity. acknowledgments we thank dr. maria cândida mamede for identifying the species of banisteriopsis and dr. kleber del-claro for the suggestions regarding the manuscript. caf was granted a scholarship by the brazilian office for the advancement of higher education (coordenação de aperfeiçoamento de pessoal de nível superior capes). references alves-silva, e. barônio, g.j., torezan-silingardi h.m. & del-claro, k. 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(1981). pollinator choice and stabilizing selection for flower color in delphinium nelsonii. evolution, 35:376–390. doi: 10.13102/sociobiology.v61i3.318-323sociobiology 61(3): 318-323 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 termite infestation in historical buildings and residences in the semiarid region of brazil ap mello, bg costa, ac silva, amb silva, ma bezerra-gusmão introduction termites are recognized as insects that interact with the urban ecosystem in the most complex way (fontes, 1995). with approximately 3,105 described species in the world (krishna et al., 2013), of the registered 534 species in brazil, 18 are considered urban pests (constantino, 2005). meanwhile, the disorganized growth of urban areas has created an impact on natural ecosystems, changing the population dynamics and resulting in the adaptation of the new species to urban conditions, which has turned them into pest species (milano & fontes, 2002). termite infestations have become increasingly more frequent inside the urban perimeter. in tropical countries and coastal regions of continents, harris (1971) reports infestations caused mainly by reticulitermes spp. (europe and united states), coptotermes spp. (australia, asia and part of africa) and cryptotermes spp. (colombia, and the vast majority of south american countries). in south america the infestations are caused mainly by coptotermes gestroi abstract this study evaluated termite infestations in historical buildings (hb) and residences (rb) in five cities in the semiarid region of brazil (a1: fagundes; a2: pocinhos; a3: alagoa grande; a4: areia; a5: bananeiras). eighty-nine percent of infestation of historical buildings and 62% of residential buildings were caused by nine species of termites, belonging to six genera and three families (kalotermitidae, rhinotermitidae and termitidae). we observed greater richness in a3, a4 and a5 (amitermes amifer, heterotermes tenuis, h. sulcatus, h. longiceps, neotermes sp. and nasutitermes corniger), relating to a1 and a2 (nasutitermes sp.1, nasutitermes sp.2, n. corniger and microcerotermes strunckii). n. corniger and dry wood termite was responsible for 66.9% and 33.1% for infestations in hb and 29.7% and 72.3% in the rb, respectively. the economic impact for these infestations was estimated at 609,956.03 usd. no correlation between the quantity of infestations and the age of the buildings was found; nor was there a correlation between the humidity and the number of infestations. however, the infestations of the rb correlated with humidity. possibly the absence of preventative mechanisms of infestation control, along with the loss of natural habitat of these insects caused by urban expansion, explain the high indicators of registered infestations. sociobiology an international journal on social insects universidade estadual da paraíba, campina grande, paraiba, brazil. article history edited by alexandre vasconcellos, ufpb, brazil received 07 march 2014 initial acceptance 15 may 2014 final acceptance 22 july 2014 keywords infestation, isoptera, urban environment, damage, cultural patrimony. corresponding author antonio paulino de mello laboratório de ecologia de térmitas departamento de biologia universidade estadual da paraíba campina grande, pb, brazil 58109-753 e-mail: antonio.pmello@hotmail.com (wasmann) (rhinotermitidae), heterotermes tenuis (hagen), h. longiceps (snyder) (rhinotermitidae), nasutitermes corniger (motschulsky) (termitidae) and cryptotermes brevis (walker) (kalotermitidae), the last of which is considered the main species pest termite in urban regions (fontes, 1995; torales et al., 1997; bandeira et al., 1998; eleotério & bertifilho, 2000; constantino & dianese, 2001; vasconcellos et al., 2002; oliveira et al., 2006). the occurrence of termites in urban areas is seen as a complex that extrapolates the limit of economic damage to human populations. these insects can reach high levels of infestation in a short period of time, causing damage to the artistic, historic and cultural patrimony (bandeira et al., 1989; fontes, 1995). currently, the annual cost in building repairs estimated in urban areas worldwide for treatment and replacements is in the order of the 40 billion usd (rust & su, 2012). in brazil, research has generated only limited discussion about the pest potential and distribution of these species in specific regions of the country, mainly the southeast and research article termites sociobiology 61(3): 318-323 (september 2014) 319 center-west (lelis, 1995; fontes, 1999; eleotério & bertifilho, 2000; constantino & dianese, 2001; ferraz & cancello, 2001; fontes & milano, 2002 and costa et al., 2009). these studies revealed the potential of n. corniger and c. brevis as pest species. however, little is known about the urban fauna and termite control methods infestations in the northeastern region of brazil (bandeira et al., 1989; bandeira et al., 1998; matias et al., 2006; albuquerque et al., 2012), especially in the state of paraíba, with focus on the studies of bandeira et al. (1998) and vasconcellos et al. (2002). the objective of this study was to survey the termite occurrences in historical and residential buildings in the semiarid region of the northeastern region, assessing the richness of species, examining the determining factors for infestation, as well as estimating the economic damage caused by infestations. materials and methods the study was developed in five cities in the state of paraíba, in brazil’s northeast semiarid region. the collecting occurred between 2010 and 2012 in the city of fagundes 11,405 inhabitants (a1) (7o20’45,56”s/35o47’51,13”w); pocinhos 17,032 inhabitants (a2) (7o04’36,26”s/36o03’40,22”w); alagoa grande 28,479 inhabitants (a3) (07°05’20”s/35°38’36”w); areia 23,829 inhabitants (a4) (06º 57’46”s/35º41’31”w); and bananeiras 21,851 inhabitants (a5) (06º 45’ 00”s/35º 37’ 58”w) (ibge, 2010). the cities of fagundes and pocinhos present arid climatic characteristics, with an average temperature between 28ºc and 30ºc and an irregular precipitation pattern (inmet, 2012), while the cities of alagoa grande, areia and bananeiras are located in the region of “brejo de altitude”, with milder climatic conditions (10ºc to 28ºc), the yearly precipitation between 900 and 1300 mm (inmet, 2012). the municipalities contribute to the history of the state of paraiba and of brazil, and their houses are listed in the cultural patrimony of the cities, especially the city of areia, which is listed in the national historical patrimony. the general characteristics of the historical buildings present in the municipalities were analyzed, beyond 50 residences. these were chosen randomly through a raffle, placed within the 10 streets that border the central area, totaling a sample of 46 historical buildings and 250 residential buildings. the ages of the historical buildings vary from 40 to 150 years, and the residential buildings from four to 102 years. in each building, the existence of key factors indicate interaction with the termites were analyzed, such as the presence of wooden furniture, the structural conditions of the building, coverage, beyond the existence of leaks and internal indicators the unit obtained from the thermo-hygrometer in each compartment of the buildings. the number of infestations per building was considered based on quantifying the number of pieces of wood that contained termites. the termites were collected and conditioned in 80% alcohol, identified through specialized literature (constantino, 1999) and confirmed by researchers dr. luiz roberto fontes and dr. alexandre vasconcellos. in some cases, dry wood termites have not been identified in this study because they colonize the interior furniture and wooden structures which were not authorized to be broken down into pieces that made up the interior of the historic and residential buildings, making it difficult to collect soldiers. in these cases, we considered the presence of insect infestations (soldiers and workers) and the characteristic signs of their presence, as the existence of holes in the wooden parts for which they are expelled to be as fecal matter characteristic of dry wood termites. the economic damage caused by infestation was calculated from an estimated market price for replacement and repair of parts and damaged structures. the samples found were deposited in the entomological collection of termitology laboratory in the biology department of state university of paraíba. pearson correlation tests were performed using an array with information about the number of infestations by age and values of internal moisture in buildings. the frequency of infestations in wooden structures was associated with the presence of termites. the frequency of occurrence of the species in historical and residential buildings was counted according to the number of times a species was recorded. the chi-square test (χ2) was used to check for differences in the frequencies of infestation observed and expected by age class and type of building. results infestations were diagnosed in 89% of the historical buildings and 62% of the residential buildings, caused by nine species of termites, belonging to six genera and three families (kalotermitidae, rhinotermitidae and termitidae) (table. 1). fagundes and pocinhos present the least generic richness when compared with the municipalities’ termite fauna in alagoa grande, areia and bananeiras (table. 1). n. corniger and dry wood termites were present in real estate in the five inspected cities, with evidence such as damaged cabinets, wooden benches, tables, paintings, doors, windows and roofing (table. 1). the greater frequency of infestation by termites was registered in the roofing, followed by doors, windows and internal furniture in historical buildings (cabinets, secretaries, tables and painting frames) and in the residential (cabinets, tables and chairs) (table. 2). the economic damage caused by infestations was estimated in 609,956.02 usd, for repairs and/or replacements in damaged structures, being 555,149.59 usd for historical buildings and 54,806.43 usd for residential buildings. ap mello et al. termite infestation in historical buildings and residences320 p = 0.26) and residential buildings (r = 0.12, p = 0.06). no significant difference between the observed frequency and the expected frequency of infestation was observed, concentrated by age class in both types of buildings studied (historical: χ2 = 0.13, p = 0.99 and residential: χ2 = 3.72 p = 0.81). however, the historic buildings up to 40 years of age represent only about 15.22% of total observed and present the greatest amount of infestation (36.5%), while in residential buildings, with ages between 21 and 40 construction years, corresponding to 33.2% of total observed, present the largest quantities of infestation (46.2%). discussion the richness of termite species found in the studied areas was considered low (nine), when compared to studies with similar objectives to this study that totaled the registration of 22 species (vasconcellos et al., 2002; eleotério & bertifilho, 2000; constantino & dianese, 2001). nevertheless, there is a similarity in richness of the urban fauna of termites with the observations of albuquerque et al. (2012) in the city of recife in the northeast of brazil. the absence of ventilation mechanisms and inside temperature control (be it artificial or natural) through adequate constructive techniques, combined with precarious preservation conditions, with countless infiltrations in the walls and the roof, all favor the increase in internal humidity of real estate. these conditions combine to create a favorable environment for the nesting of new colonies and can explain the elevated infestation rate (lelis, 1999; hedges, 1998). despite the impossibility of specific identification of dry wood termites, we believe that the high number of attacks by dry wood termites verified may be related to the fact that some species of the genus cryptotermes submit acclimation capacity and high level of responses to orphans, especially table 1 frequency the termite’s species in historical and residential buildings in the municipalities of fagundes (a1), pocinhos (a2), alagoa grande (a3), areia (a4) and bananeiras (a5), located in the semiarid region of brazil. family species municipalities frequency (%)a1 a2 a3 a4 a5 termitidae amitermes sp.1 x 5.64 nasutiternes sp.1 x 6.65 nasutitermes sp.2 x 1.4 nasutiternes corniger (motschulsy) x x x x x 39.4 rhinotermidiae microcerotermes strunckii (sörensen) x 1.4 heterotermes longiceps (snyder) x 1.4 heterotermes tenuis (hagen) x 1.4 heterotermes sulcatus (mathews) x x 1.4 kalotermitidae neotermes sp. x 1.4 dry wood termite * x x x x x 40 total 09 04 04 04 04 02 100 * was not treated as a species. table 2 – wooden parts and/or structures with termite’s infestations in historical (hb) and residential (rb) buildings in the municipalities of de fagundes (a1), pocinhos (a2), alagoa grande (a3), areia (a4) and bananeiras (a5), located in the semiarid region of brazil. (hb – historical buildings; rb – residential buildings). structures and/or parts % infestation hb rb cabinet 6.1 6.43 chest 0.39 0.25 wooden benches 6.84 secretaries 3.33 2.23 bed 0.59 0.74 chair 1.18 9.65 shelf 4.73 5.45 stairs 0.59 6.68 window 6.08 4.7 table 5.1 15.1 door 10.39 4.21 roof lining 0.98 1.24 framing 25.1 roofing 28.6 43.32 total 100 100 problems related to humidity, which varied from 45% to 98%, and infiltrations in the walls and roofs were observed in 80% of the buildings and in 64.8% of the residences. meanwhile, there was no correlation between the humidity and the number of infestations in historic buildings (r = 0.23, p = 0.12), while a correlation was found between these parameters for residences (r = 0.16, p< 0.05). no correlation was found between the number of infestations and the age of the historic buildings (r = -0.17, sociobiology 61(3): 318-323 (september 2014) 321 cryptotermes brevis, considered a major pest of urban brazil (mcmahan, 1962; steward, 1983; edwards & mill, 1986; bacchus, 1987; fontes, 1995). moreover, the low durability and natural resistance of woods used in furniture may also be contributing to this frequency in the domestic environment (spear, 1970; silva et al., 2004). the elevated frequency of n. corniger in the study corroborates the importance of the species as one of main urban pests in brazil (eleotério & berti-filho, 2000; vasconcellos et al., 2002; costa et al., 2009; albuquerque et al., 2012). some researchers theorize that this species’ success in colonizing in urban space is due to its great biological plasticity, mainly related to its reproduction mechanisms (thorne & noirot, 1982), its feeding habits (bustamante & martius, 1998; vasconcellos & bandeira, 2000) and aspects related to its nesting (levings & adams, 1984; vasconcellos, et al., 2002). irregular occurrences of amitermes sp., heterotermes longiceps, h. sulcatus, h. tenuis, microcerotermes strunckii and neotermes sp. were also observed by constantino and dianese (2001); milano and fontes (2002); vasconcellos et al. (2002); costa et al. (2009); albuquerque et al. (2012). we believe that the low frequency of these species in the urban environment is a result of lack of planning and disordered growth of urbanization that stimulate adjustment factors and the occurrence of species that previously occurred only in natural areas, as well as the product from the competition with well-established species in this environment. in 1989, bandeira et al. highlighted the importance of heterotermes as a potential pest in brazil. these termites have been reported for being responsible for attacks on books, journals, painting frames and wooden shelves in an urban area (pizano & fontes, 1986; mill, 1991; lelis, 1995). the infestation indicators observed in the buildings’ wooden roofing and the reported attacks on doors and windows are possibly related to their state of conservation and exposure of these parts during the termites’ swarm. in general, the buildings present precarious structural conditions, with roof infiltrations, and in the case of historical buildings, without performing repairs and preventative restorations over time. according to fontes (1998), wooden sections facing the external side of buildings that are not directly exposed to weatherproofing offer good conditions for termite establishment. the presence of infiltrations in the roof promotes the elevation of environmental humidity, facilitating the proliferation of biologic agents such as fungi, organisms that facilitate the installment of termite colonies (bandeira, 1989; torales, 1997). however, even though the buildings present different levels of infestation, it was observed that the existence of reforms in some of the historic buildings and homes older than 50 years showed a trend toward a decrease in the number the infestations, and thus influenced the lack of correlation between age of the property and the number of reported infestations. importantly, even with problems of infiltration and considerable internal moisture content are responsible in the buildings do not have protocols or specific control techniques to combat and prevent infestations, they used from the indiscriminate use of insecticides of different classes resulting in failed attempts to control. no reports were found in the literature on techniques for termite control for this type of construction in paraíba. however, for some historic buildings, an attempt to control the termites may have interfered in our findings, even in the short term, resulting in the absence of correlation between the humidity and the number of infestations. even though resistance and natural durability tests were not performed on the woods used for the manufacturing of the observed structures that were infested with termites, it is possible that the termite attacks on the furniture in buildings is due to the weak durability of the wood used in the furniture, the precarious structural conditions of the real estate, and the absence of repair activities and prevention by the responsible parties. these factors associated to infiltration and humidity problems are determinant in the increase in likelihood of infestations (bandeira, 1989; torales, 1997; silva et al., 2004; milano & fontes, 2002). attacks on historical and residential buildings in the arid zone of paraíba, northeastern brazil, were caused by nine species of termites, causing severe economic consequences and damaging the historical and cultural patrimony of the inspected cities. we found that the problems of infiltration and indoor humidity coupled with few or no repairs to the structure of the buildings as well as the absence of diagnosis and prevention of termite infestation accounted for a part of the high percentage of the attacks on the buildings. however, based on the findings of the frequency of occurrence and the types of damage caused, it was discovered that only dry wood termites and n. corniger were present as pest species in the cities we analyzed. acknowledgements we would like to thank kátia cristina ferreira da silva for her support with field work. we also thank dr. luiz roberto fontes and dr. alexandre vasconcellos for the confirmation of the species. to the national council of scientific and technological development, brazil, and the state university of paraíba, brazil we express our appreciation for their financial support. references albuquerque, a.c., matias, g.r.r.s., couto, a.a.v.o., oliveira, m.a.p., vasconcellos, a. 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(2002). termites (isoptera) pests in buildings in joão pessoa, brazil. sociobiology, 40: 1–6. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3384sociobiology 65(4): 671-678 (october, 2018) special issue relationship between hydrocarbon composition on the cuticle of melipona quadrifasciata (hymenoptera: apidae) workers and the secretion of the cephalic salivary glands introduction insects produce chemicals of several natures that act in communicative interactions among individuals of different species, named allelochemicals, and among individuals of same species, known as pheromones (morgan, 2010). in social insects these compounds are found in the secretion of several exocrine glands and also in the epicuticle’s surface mediating social interactions among the nestmates (jarau et al., 2004, 2006; howard & blomquist, 2005; cruz-landim, 2009). the epicuticle, the outermost layer of the insect cuticle, is constituted of 90% of hydrocarbons. along evolution, epicuticle acquired informational roles about physiological state, age, sex, species and caste of individuals as well as mating and discrimination of nestmates (howard, 1993; abdalla et al., 2003; poiani et al., 2014; nunes et al., 2017; vollet-neto et al., 2017). abstract since chemical communication is pivotal for social insect success, the present paper aimed to quantify and qualify the chemical compounds that might have pheromonal role in both cephalic salivary gland and epicuticle of workers of melipona quadrifasciata lepeletier using gas chromatographymass spectrometry (gc/ms). the results indicated that the hydrocarbons were the main compounds in both cephalic salivary gland and epicuticle, followed by esters. positive mantel correspondence analysis suggests that the glands could contribute to replenishment of surface compounds as an auxiliary source. discriminant analysis also pointed out that gland and epicuticle chemical profiles were phase-related. sociobiology an international journal on social insects sb poiani1, ed morgan2, fp drijfhout2, c cruz-landim1 article history edited by denise alves, esalq-usp, brazil received 27 april 2018 initial acceptance 12 june 2018 final acceptance 16 august 2018 publication date 11 october 2018 keywords chc, labial gland, mantel, pheromone, stingless bee. corresponding author silvana b. poiani universidade estadual paulista unesp departamento de biologia instituto de biociências avenida 24a, 1515, bela vista cep 13506-900, rio claro-sp, brasil. e-mail: silbeani@gmail.com cuticular compounds are lost in a few weeks, suggesting that cuticular compounds must be constantly renewed (d’ettorre et al., 2006). oenocytes are of ectodermal origin and they are pointed out as the main endogenous source of surface compounds (kramer & wigglesworth, 1950; gu et al., 1995). epidermal cells are also of ectodermal origin, however, in the adult, they are inactive except in certain glandular regions. thus, in addition to the class iii exocrine glands, all glands of ectodermal origin such as tegumental class i gland and exocrine glands structured as organs (cruzlandim, 1994) have the potential to contribute to secretion and formation of surface compounds. in fact, cephalic salivary glands (gsc) and dufour’s have been pointed out as source of these hydrocarbons (kullenberget al., 1973; bergman & bergström, 1997; poiani & cruz-landim, 2017). in general, bee colonies are formed by adult and immature individuals. adult workers and queens are females 1 departamento de biologia, universidade estadual paulista, instituto de biociências, rio claro, são paulo, brazil 2 chemical ecology group, lennard jones laboratories, keele university, united kingdom research article bees sb poiani, ed morgan, fp drijfhout, c cruz-landim – gland and epicuticle chemical profiles of m. quadrifasciata672 and comprise different castes. the division of labor among workers presents age-correlated patterns of task performance (temporal polyethism) (wilson, 1985). worker bees generally pass through following phases/stages: callow/newly emerged (incubation and repairs of the brood chambre), nurse bee (construction and provisioning combs, cleaning the nest, and feeding larvae, young adults and the queen), housekeeper (further cleaning the nest, reconstruction of the involucrum, reception and ripening of nectar, and guard duty at the entrance of the nest) and forager (foraging for pollen, nectar, propolis and other materials) (wille, 1983). to perform most of the tasks, the worker bee also present developmental state and secretion content of glands changing according to phase or task (katzav-gozansky et al., 2001; poiani & cruz-landim, 2009, 2010a, b, c). the chemical profile of csg has been investigated among bees. hydrocarbons are present in newly emerged, nurse and forager workers of apis mellifera linnaeus and some males of bombus species (kullenberg et al., 1973; katzavgozansky et al., 2001; poiani & cruz-landim, 2017). esters are most likely found in newly emerged, nurse and forager workers of meliponini bees (jarau et al., 2006; schorkopf et al., 2007; stangler et al., 2009; poiani et al., 2015) and a mixture of oxygenated compounds and hydrocarbons were observed in virgin and old queens and workers of bombus terrestris (linnaeus) (amsalem et al., 2014). the function of csg varies among bee species. in meliponini, csg produces a trail pheromone to communicate food source to nestmates (jarau et al., 2006; schorkopf et al., 2007; stangler et al., 2009). also, it aids to handle resins collected in the field (santos et al., 2009). in a. mellifera and some bombus species, the hydrocarbons produced by csg are also present in the cuticle of workers (kullenberg et al., 1973; arnold et al., 1996; poiani & cruz-landim, 2017; martin et al., 2018). in a. mellifera, csg may take part as an auxiliary source of cuticular hydrocarbons (poiani & cruz-landim, 2017). males of some bombus species use csg content for territorial marking and female attraction (terzo et al., 2007; amsalem et al., 2014). melipona quadrifasciata lepeletier is a brazilian eusocial stingless bee. the body size of worker measures 10 to 11 mm. the population of this species ranges from 300-400 bees within the colony (lindauer & kerr, 1960). the present research aimed to analyze and to identify the chemical profile of csg and epicuticle of newly emerged, nurse and forager workers of m. quadrifasciata. the ultimate purpose is to verify if csg is a candidate for auxiliary source of epicuticle hydrocarbon by comparing their profiles. material and methods samples preparation workers of m. quadrifasciata were collected in different phases of life. newly emerged (ne) at the time of emergence from their brood cells; workers provisioning brood combs (ca); and forager workers returning to the nest with pollen (fo). all workers were taken from a single colony. the colony is located at the meliponary of sao paulo state university unesp, institute of biosciences, rio claro, brazil. twenty pairs of wings and 20 glands from ne, ca and fo were used. wings were chosen because they have the same chemical composition found on the body surface. also, they are less subject to contamination from tegumentary exocrine gland discharge (cruz-landim et al., 1965). chemical analysis chemical analyses were conducted on an agilent 6890 gas chromatograph (agilent, santa clara, ca, usa) equipped with hp-5ms column (30 m long, 0.25 mm diam; agilent). the gas chromatograph is coupled to an agilent 5973 mass detector with electron impact ionization at 70 ev. solid sample injection (morgan, 1990) was used. the glands and pair of wings from each individual were dissected and inserted into a thin glass capillary. glands and wings were analyzed separately. the capillary measures approximately 2–3 cm and was sealed at one end. the capillary was inserted into the solid sampler in the heated gc inlet, and crushed to release the compounds. samples were analyzed in splitless mode. the oven temperature program was held at 40 °c for 1 min then increased to 320 °c at 15 °c/min., and then held for 10 min. helium was used as carrier gas at a constant flow of 1.0 ml/min. relative quantification was based on the peak areas in the total ion chromatograms obtained (singer & espelie, 1992). the compounds were identified according to their mass spectra, comparing them with a database (ms-database), and consulting the wiley library. statistical analysis mantel test statistic was performed to verify correspondence between gland and cuticle surface compounds in each worker group. the relative amounts of each compound in csg and cuticle of each individual worker was used. the amount is related to the area of each peak in the chromatogram. the test statistic is the pearson productmoment correlation coefficient (r). the coefficient r falls in the range of –1 to +1. the r value close to –1 indicates strong negative correlation. the r value close to +1 indicates strong positive correlation. mantel tests performed here were conducted using the r package ade4 by running the function mantel.rtest (gla.dist, cut.dist, nrepeat = 9999) and at α = 0.05, where gla.dist is the distribution of distances between the relative quantity of chemical compounds in the gland for a given group – either ne, ca, fo); and cut.dist is the distribution of distances between the relative quantity of chemical compounds in the cuticle for the equivalent group yet, the distribution of distances was calculated with the command dist() and stored in the variables gla.dist and cut.dist. sociobiology 65(4): 671-678 (october, 2018) special issue 673 discriminant analysis (da) and fisher distance were used to verify whether the phases were correctly classified within the pre-established groups (ne, ca and fo) according to their chemical compounds in csg and cuticle surface. statistical significance for da was established at p < 0.0001. after running da, the confusion matrix generated from a cross-validation (method jackknife) was checked in order to assess the overall accuracy of original data and estimation of error rates for every group. for all groups we found correct classification rate of 100%. moreover, hierarchical cluster analysis (hca) and rho distance similarity was performed to identify which group of workers is more similar/closer to each other for both gland content and cuticle chemical profile. both hca and da were performed using average percentage original data and the software past. results chemical profile of cephalic salivary glands a total of 33 different compounds were found in the glands being 10 esters and 23 hydrocarbons. the hydrocarbons include alkanes, methyl-branched alkanes, alkenes and alkadienes. the hydrocarbons chain ranges from n-c21 to n-c31 (table 1). the quality and relative quantity of each compound varied according to worker phase (ne, ca and fo). however, the alkenes (z)-pentacosene and (z)-heptacosene were the major compounds in the ne, ca and fo glands. ne workers presented 20 compounds in their glands: three esters and 17 hydrocarbons. hexadecyl dodecanoate, (zz)nonacosadiene, 11 and 13-methylnonacosane and 11 and 13-methylhentriacontane are exclusive in ne glands. the glands of ca workers presented 29 compounds being nine esters and 20 hydrocarbons. the exclusive compounds found in ca workers were alkanes: 3-methyltetracosane and 11-methylhexacosane. the glands of fo workers presented 25 compounds being nine esters, and 16 hydrocarbons (table 1). chemical profile of cuticle surface a total of 28 different compounds were found in the cuticle surface being one ester, two acids, and 25 hydrocarbons. the hydrocarbons include alkanes, methyl-branched alkanes, and alkenes ranging from n-c21 to n-c31. the quality and relative quantity of each compound varied according to worker phase. the alkenes (z)-pentacosene and (z)-heptacosene were the major compounds in the ne, ca and fo cuticle surface. ne workers presented 24 compounds in their cuticle: one ester, two acids, and 21 hydrocarbons. the cuticle of ne workers contains exclusive compounds that were all alkanes: n-heneicosane, 5-methylheptacosane, 11 and 13-methylnonacosane, 5-methylnonacosane and 11 and 13-methylhentriacontane. the cuticle of ca workers contains 22 compounds being one ester, two acids and 19 hydrocarbons. the exclusive compound found in ca workers was 3-methylhentriacontane. the cuticle of fo workers presented 21 compounds being one ester, two acids, and 18 hydrocarbons (table 1). rt compounds cephalic salivary gland cuticle ne (n = 20) mean ± sd ca (n = 20) mean ± sd fo (n = 20) mean ± sd ne (n = 20) mean ± sd ca (n = 20) mean ± sd fo (n = 20) mean ± sd acids 13.60 hexadecanoic acid 0.47 ± 1.02 0.95 ± 1.02 0.44 ± 1.21 14.94 (z,z)-9,12-octadecadienoic acid 0.08 ± 0.01 0.09 ± 0.05 0.06 ± 0.13 esters 14.79 ethyl-9,12-octadecadienoate 3.82 ± 7.11 1.75 ± 1.53 0.21 ± 0.57 15.29 hexadecan-1-yl acetate 0.30 ± 0.41 0.16 ± 0.20 15.82 methyl octadecanoate 0.38 ± 0.53 0.13 ± 0.11 0.37 ± 0.23 16.17 ethyl oleate 0.32 ± 0.94 0.16 ± 0.25 16.27 (z)-13octadecen-1-yl acetate 0.18 ± 0.25 0.19 ± 0.18 16.40 octadecyl acetate 0.23 ± 0.32 0.33 ± 0.31 20.13 octadecyl dodecanoate 1.81 ± 2.00 0.21 ± 0.25 0.47 ± 0.48 19.89 hexadecyl dodecanoate 2.48 ± 2.22 20.84 eicosyl decanoate * 0.51 ± 0.71 22.10 docosyl decanoate 0.07 ± 0.11 0.66 ± 1.28 22.60 tetracosyl decanoate 0.15 ± 0.15 1.37 ± 3.06 hydrocarbons 15.53 (z)-heneicosene 0.16 ± 0.20 0.12 ± 0.10 15.67 heneicosane (c21) 1.02 ± 1.14 table 1. mean and standard deviation (sd) of relative concentration (%) of the compounds from cephalic salivary gland secretion and cuticle of workers of melipona quadrifasciata. sb poiani, ed morgan, fp drijfhout, c cruz-landim – gland and epicuticle chemical profiles of m. quadrifasciata674 16.65 (z)-x-tricosene 0.08 ± 0.15 2.32 ± 1.37 2.62 ± 1.53 0.41 ± 0.38 0.19 ± 0.14 * 16.76 tricosane (c23) 2.46 ± 1.15 1.29 ± 0.74 0.94 ± 0.50 9.28 ± 2.92 1.21 ± 0.62 0.60 ± 0.64 16.94 9-methyltricosane 0.12 ± 0.09 0.22 ± 0.06 17.30 3-methyltricosane 3.03 ± 1.46 3.20 ± 1.67 0.38 ± 0.32 0.52 ± 0.22 0.31 ± 0.29 17.32 tetracosane (c24) 0.48 ± 0.36 0.51 ± 0.28 0.25 ± 0.21 17.59 11-methyltetracosane 0.46 ± 0.54 0.27 ± 0.41 17.61 3-methyltetracosane 0.49 ± 0.53 18.06 (z)-pentacosene 19.33 ± 7.99 40.73 ± 2.99 38.77 ± 7.48 21.27 ± 4.60 19.85 ± 2.62 18.53 ± 5.20 18.37 pentacosane (c25) 4.99 ± 1.80 4.72 ± 3.43 4.22 ± 1.51 12.58 ± 4.20 16.34 ± 2.29 15.59 ± 5.38 18.41 9-methylpentacosane 1.45 ± 0.59 1.23 ± 0.43 1.24 ± 1.04 1.80 ± 0.42 0.44 ± 0.22 0.29 ± 0.23 17.58 5-methylpentacosane 2.41 ± 0.92 1.16 ± 0.57 1.89 ± 1.05 0.53 ± 0.26 0.82 ± 0.30 18.56 3-methylpentacosane 2.07 ± 0.56 5.29 ± 1.05 5.53 ± 1.53 2.26 ± 0.88 1.89 ± 0.46 1.82 ± 0.54 hexacosane (c26) 0.16 ± 0.26 1.08 ± 0.38 0.81 ± 0.31 17.79 11-methylhexacosane 0.54 ± 0.50 18.17 (z)-heptacosene 30.73 ± 5.95 27.85 ± 4.28 26.95 ± 5.56 18.84 ± 3.59 21.17 ± 3.43 24.78 ± 6.14 18.22 heptacosane (c27) 3.04 ± 1.15 1.07 ± 2.30 4.02 ± 1.21 14.01 ± 1.52 17.68 ± 4.66 18.44 11 and 13-methylheptacosane 2.86 ± 0.38 1.32 ± 0.57 2.07 ± 1.08 1.74 ± 0.65 17.72 7 and 9-methylheptacosane 0.92 ± 0.26 1.23 ± 0.44 17.78 5-methylheptacosane 2.90 ± 1.27 18.00 3-methylheptacosane 3.59 ± 1.29 1.25 ± 0.62 0.92 ± 0.36 18.53 (z)-octacosene 1.12 ± 0.18 1.23 ± 0.29 18.72 (z,z)-nonacosadiene 0.47 ± 0.63 19.72 (z)-nonacosene 14.51 ± 7.47 4.85 ± 2.25 7.37 ± 3.84 10.92 ± 3.59 6.92 ± 1.40 8.45 ± 2.45 19.78 nonacosane (c29) 1.45 ± 1.42 4.73 ± 1.00 5.74 ± 3.10 19.91 11 and 13-methylnonacosane 2.39 ± 1.21 1.25 ± 0.56 19.93 5-methylnonacosane 0.48 ± 0.83 0.16 ± 0.17 0.28 ± 0.16 0.39 ± 0.41 20.45 (z)-hentriacontene 0.23 ± 0.50 0.12 ± 0.15 0.41 ± 0.52 0.79 ± 0.55 0.39 ± 0.25 0.39 ± 0.30 20.49 hentriacontane (c31) 1.18 ± 0.97 0.60 ± 0.60 20.59 11 and 13-methylhentriacontane 0.34 ± 0.57 0.54 ± 0.49 21.16 3-methylhentriacontane 0.44 ± 0.27 ne = newly emerged; ca = workers from comb area; fo = foragers; rt = retention time *the compounds with relative concentration less than 0.05% were considered as traces. table 1. mean and standard deviation (sd) of relative concentration (%) of the compounds from cephalic salivary gland secretion and cuticle of workers of melipona quadrifasciata. (continuation) correlation among chemical profiles the correspondence analysis mantel r showed that there is positive correlation between csg and cuticle in ne (r = 0.61), ca (r = 0.47) and fo (r = 0.48). for all groups p < 0.0001 indicates that the results are statistically significant at α = 0.05. da analysis of chemical content of both glands (fig 1a) and cuticle surface (fig 1b) has shown that the three groups ne, ca and fo were completely segregated. the fisher distances among all groups were p < 0.001. hca dendrogram of csg content (fig 2a) separated the workers into three well defined groups, as expected, and showed that nurses and foragers are closest. hca dendrogram of cuticle surface compounds (fig 2b) also showed that nurses and foragers are closest. it was possible to observe nurse and forager individuals out of their predicted groups. discussion chemical compounds on body surface and exocrine glands are important chemical cues used for social interactions and community homeostasis in social insects (jarau et al., 2004, 2006; howard and blomquist 2005; cruz-landim, 2009). the present study provided information about quality and quantity of chemical profile in csg and epicuticle of workers of m. quadrifasciata in different phases of life comparing the results in order to seek for correspondence between both compartments. rt compounds cephalic salivary gland cuticle ne (n = 20) mean ± sd ca (n = 20) mean ± sd fo (n = 20) mean ± sd ne (n = 20) mean ± sd ca (n = 20) mean ± sd fo (n = 20) mean ± sd sociobiology 65(4): 671-678 (october, 2018) special issue 675 in stingless bees, csg has been pointed out as a producer of esters that act as scent trails to guide their nestmates to a food source (jarau et al., 2004, 2006; schorkopf et al., 2007; stangler et al., 2009; poiani et al., 2015). however, workers of m. quandrifasciata use sound pulses to recruit nestmates to food source (hrncir et al., 2000) suggesting that csg in m. quadrifasciata might has function other than scent trail. fig 2. dendrograms obtained from a hierarchical cluster analysis (hca). the groups were formed according to similarities in the cephalic salivary gland secretion (a) and cuticle surface (b) chemical profiles of melipona quadrifasciata. both dendrograms showed that nurses (ca) and foragers (fo) are closest. ne = newly emerged workers. fig 1. discriminant analysis graphs of chemical contents of cephalic salivary glands (a) and cuticle surface (b) of workers of melipona quadrifasciata. all groups differed from each other. the individuals were 100% allocated correctly (f1+f2=100%). ne = newly emerged; ca = working in comb area; fo = forager. it is known that oenocytes are the main responsible for cuticle hydrocarbon production. however, some epidermal cells that function as tegumental glands are also involved in cuticle hydrocarbon synthesis (kramer & wigglesworth, 1950; abdalla et al., 2003) and such compounds can be fully or partially stored in glands after being released as secretion (bagnères & blomquist, 2010). the present research showed that hydrocarbons were the major compounds in csg indicating that the studied gland may be an auxiliary source of cuticle hydrocarbons as observed in a. mellifera (poiani & cruz-landim, 2017; martin et al., 2018). in ants, the postpharyngeal gland (ppg) contains similar hydrocarbons found on cuticle surface (bagnères & morgan, 1991; kaib et al., 2000). the content of ppg can be transferred to cuticle by grooming (meskali et al., 1995; morgan, 2010). in honeybees, self-grooming enables bees to remove ectoparasites, dust, and pollen from their own bodies and helps disperse pheromones (boecking & spivak, 1999). it is described as involving biting and licking with the mouthparts, as well as movement of the proand/or mesothoracic legs (danka & villa, 2005). the grooming behavior displayed by m. quadrifasciata workers could contribute to spread the content of csg on the body surface. the final duct opening of csg on the glossa allows the content of these gland reach the mouthparts and be spread on the body surface using the proand/or mesothoracic legs. the present study has shown that the chemical profile of the glands of ne, ca and fo contained mainly hydrocarbons but esters were also present in small amount. in cuticle, esters were practically absent. the hydrocarbons composition was similar in both compartments. the positive mantel correspondence analysis for all groups indicated that there were correspondences between gland and cuticle compounds. these results indicate that csg of m. quadrifasciata may play a role as an auxiliary source of hydrocarbons replenishment in the epicuticle. similar results were found for a. mellifera (arnold et al., 1996; poiani & cruz-landim, 2017) and sb poiani, ed morgan, fp drijfhout, c cruz-landim – gland and epicuticle chemical profiles of m. quadrifasciata676 some bombus species (kullenberg et al., 1973; bergman & bergström, 1997; terzo et al., 2007). csg starts to produce lipid-based secretion (simpson, 1960; poiani & cruz-landim, 2016) soon as the worker emerge from brood comb (poiani & cruz-landim, 2009). then, additional use of secretion cannot be completely ruled out. the presence of small amounts of esters mixed with hydrocarbons may be used also to perform internal task in parallel of an auxiliary source of cuticle compounds. santos et al. (2009) suggested that the csg secretions of plebeia emerina (friese) nurses aid in the handling resins collected in the field. csg of ne, ca and fo contained most of the same compounds. however, the quantity of them was enough to distinguish the groups, as demonstrate by discriminant analysis. despite hydrocarbons were majority, the presence of esters increases in ca and fo and suggest that ca and fo are more related with each other than with ne. also, hca dendrogram supported that ca and fo are closest groups. such result is expected since the csg of ne in bees are starting to produce secretion while in ca these glands present alveoli almost full of secretion and in csg of fo were observed the peak of the secretion accumulation (poiani & cruz-landim, 2009; 2010a,b,c). in a. mellifera, the content of csg also showed more similarities between nurses and foragers (poiani & cruz-landim, 2017) while in scaptotrigona postica (latreille) nurses are subdivided and there are individuals close to newly emerged and others that are similar to foragers suggesting progressive change in csg content in this species (poiani et al., 2015). the most common cuticle lipids in insects are the hydrocarbons and they vary from c17 to c49, including alkanes, methyl-branched alkanes, and alkenes (morgan, 2010). in this sense, m. quadrifasciata does not differ of the other insect species and presented hydrocarbons ranging from c21 to c31. the proportion of alkanes, alkenes and methyl-branched alkanes vary within insect species (blomquist & bagnères, 2010). both alkanes and alkenes are related to chemical communication such as nestmate recognition, individual’s fertility, sex, gender, caste, sex pheromone (blomquist & bagnères, 2010). moreover, alkanes form an impermeable layer at cuticle that protects the insect against desiccation (gibbs, 2002). the most compounds in epicuticle of m. quadrifasciata workers were alkenes. the epicuticle chemical profile of workers of m. quadrifasciata provides information about the individual status as observed in castes or sex of other bee’ species (nunes et al., 2017; vollet-neto et al., 2017). since almost all compounds were present in ne, ca and fo workers, the quantity variation of the compounds was pivotal to separate the workers groups according to their phase of life using discriminant analysis. the exclusive compounds in ne and ca also act as markers that indicate to which group the workers belong to. hca dendrogram showed that nurse and forager workers share more similarities in their cuticle surface compounds in comparison with newly emerged. the acquisition of compounds increases according to age, contact with wax comb (breed et al., 1995) and possibly to csg development that may contribute to surface chemical profile. despite hydrocarbons were major compounds in cuticle, there were little amounts of oxygenated compounds. the presence of oxygenated compounds has been described for several species of insects (buckner, 1993) and for some species of bees (abdalla et al., 2003; nunes et al., 2010). in bees, it was related to wax contact in the nest and playing a role in nestmate recognition (breed et al., 1995). then, it is possible that some contact with the nest material was responsible for the acquisition of oxygenated compounds in m. quadrifasciata. in conclusion, csg secretion and the cuticle chemical profile undergo significant chemical changes that correspond to a worker’s phase of life suggesting a pheromonal role. the statistical test indicated positive correlation between csg and cuticle surface compounds in ne, ca and fo, suggesting that there is a possibility for the csg to be an auxiliary source of cuticular hydrocarbons. acknowledgments the authors would like to thank the são paulo research foundation (fapesp) for the support via a fellowship (grant no. 07/56682-1) for the reviewers for their valuable suggestions which helped to improve the article, and v.t. santana for help with graph and the correspondence analysis. authors’ contribution all authors designed the experiments; s.b.p. collected bees, dissected the glands and wings, and performed the chemical experiments; s.b.p., e.d.m. and f.p.d. analysed the chemical data; s.b.p. carried out statistical analyses; s.b.p. and c.c.-l. analysed the statistical results; s.b.p. and c.c.-l. wrote the paper. references abdalla, f.c., jones, g.r., morgan, e.d. & cruz-landim, c. 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(1985). the sociogenesis of insect colonies. science, 228: 1489-1495. doi: 10.1126/science.228.4707.1489 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.391sociobiology 62(4): 571-577 (december, 2015) diversity and temporal variation in the orchid bee community (hymenoptera: apidae) of a remnant of a neotropical seasonal semi-deciduous forest introduction the tribe euglossini consists of approximately 230 described species (nemésio & rasmussen, 2011). in the last years, the use of odorous baits to attract males has allowed detailed investigations of the ecological characteristics of these bees, the effects of habitat fragmentation on the diversity of their communities and aspects of their geographic distribution (tonhasca et al., 2002; brosi, 2009). euglossine bees have been studied in a variety of ecosystems, including the amazonian forest (e.g., powell & powell, 1987; abrahamczyk et al., 2011), central american forests (brosi, 2009), the cerrado (brazilian savannas) in central brazil (faria & silveira, 2011; silveira et al., 2015), and caatinga (dry scrub forests) in northeast brazil (andradesilva et al., 2012) . studies have also been conducted in the abstract multiple biotic and abiotic variables control the composition, diversity, and temporal fluctuations of insect communities. in particular, the assembly of bee communities is strongly influenced by climatic factors as well as variation in food resource availability, which in turn also fluctuates in response to the same factors. the goal of the present study was to investigate the species composition, the species richness and the abundance of the euglossine bees in a remnant of the seasonal semi-deciduous forest of northeastern são paulo state, and evaluate the temporal variation in those parameters over time. from january to december 1990, males of euglossine bees were sampled using three aromatic baits. we collected 643 males belonging to 12 species and three genera. euglossa imperialis cockerell was the most abundant species, followed by eulaema nigrita lepeletier and euglossa melanotricha moure. the composition and species richness showed temporal variation throughout the year, with the highest values being observed during the rainiest months. such variations occurred by the presence of males from six species which were sampled only during the rainiest months. of the five most abundant species, eg. imperialis and eg. melanotricha were the only species sampled throughout the year. only el. nigrita and eg. melanotricha, had the monthly abundance of males correlated with precipitation. based on the assumption that male euglossine capture rates reflect actual abundance at the sampled habitats, we believe that eg. imperialis, usually represented by few male in other studies, has a population very well established in the habitat here studied. sociobiology an international journal on social insects s mateus, acr andrade-silva, ca garófalo article history edited by cândida m. l. aguiar, uefs, brazil received 02 april 2014 initial acceptance 15 may 2014 final acceptance 10 november 2015 keywords euglossini, neotropics species richness, abundance, scent baits. corresponding author sidnei mateus departamento de biologia faculdade de filosofia, ciências e letras de ribeirão preto-usp av. bandeirantes, 3900 – monte alegre 14040-901,ribeirão preto-sp, brazil e-mail: sidneim@ffclrp.usp.br atlantic forest (aguiar & gaglianone, 2012; rocha-filho & garófalo, 2013), both in areas covered by rain forests and in areas covered by semi-deciduous forests, the two major vegetation types that comprise such biome (morellato & haddad, 2000). the atlantic rain forest covers mostly low to medium elevations (<= 1000 m.a.s.l.) of the eastern slopes of the mountain chain that runs along the coastline from southern to northeastern brazil, while the atlantic semideciduous forest extends across the plateau (usually > 600 m.a.s.l.) in the center and southeastern interior of the country (morellato & haddad, 2000). despite being a key area for conservation, the atlantic forest biome remains severely threatened due to its proximity to urban centers and areas of agricultural monoculture such as coffee, orange, sugar cane, and eucalyptus plantations (morellato & haddad, 2000). the seasonal semi-deciduous research article bees universidade de são paulo, ffclrp-usp, ribeirão preto-sp, brazil s mateus, acr andrade-silva, ca garófalo – orchid bee in a neotropical seasonal semi-deciduous forests572 forests of this biome are one of its most vulnerable and impacted ecosystems, especially in regions where the terrain facilitates the establishment of mechanized agriculture. these forests include distinct enclaves of habitat associated with specific environmental conditions, such as those found in the northeastern extreme of são paulo state (durigan et al., 2002). this region is characterized by a relatively high proportion of plant species that are either rare or have a restricted distribution, and has the highest priority for conservation due to its ecological and sociological characteristics, and the degree of habitat fragmentation, being one of the state’s most devastated areas over the past 30 years (kronka et al., 1998). given the importance of the conservation of the biodiversity of isolated fragments of habitat, the investigation of a specific group, such as euglossine bees, may contribute to the diagnosis of environmental quality, providing important insights for conservation management, as well as contributing to the investigation of distribution patterns. in this context, the present study describes the diversity and composition of the euglossine bee community in a remnant of the seasonal semideciduous forest of northeastern são paulo state, and evaluates the temporal variation in these parameters over time. material and methods study area the study was conducted in the furnas do bom jesus state park (20°14’55” s, 47º28’48” w), which is located in the municipality of pedregulho, são paulo, brazil. the park has an area of 2,069.06 ha with altitudes ranging from 750 to 1063 m above sea level. the territory of the park is mostly covered with shrubs and small trees (“capoeiras”) and seasonal semi-deciduous forests that occupy the scarp of caves in the valley of the córrego do pedregulho. in the highest regions, there are areas with different savanna physiognomies: “campo sujo”, “campo cerrado” and “cerrado” sensu stricto. the original forest cover has not significantly changed, and represents the typical semideciduous vegetation found in the region, now distributed in discontinuous patches (branco et al., 1991). the local climate is mesothermal, with warm summers (temperatures of 18–32ºc) and relatively dry winters (3– 13°c), the driest months being june, july, and august, with monthly precipitation of less than 100 mm. male sampling cineole (c), eugenol (e) and vanillin (v) were used as male attractants because these chemicals are considered to be the most effective for attracting males of most euglossine species (dressler, 1982). these chemicals were applied to small balls of filter paper, which were tied to tree branches 1.5 meters above the ground and 10 m apart, and approximately 50 meters from the edge of the forest. from january to december 1990, once a month, between 10 a.m. and 1 p.m., on sunny days, male euglossine bees were captured with an entomological net as they arrived at the chemical baits. the baits were replenished once an hour due to the volatility of the scents. local temperature was measured every 30 minutes during the sampling period. bees were identified in the laboratory and deposited in the entomological collection of the chemical ecology and animal behavior laboratory, biology department (ffclrp-usp), ribeirão preto, são paulo, brazil. data on precipitation were obtained from chapadão farm, located 3 km from the study area (20º15’75” s, 47º27’58” w) (fig 1). fig 1. mean temperature (line) of the periods of sampling of males and total precipitation (columns) recorded each month from january to december 1990, in the region of furnas do bom jesus state park, pedregulho, são paulo, brazil. data analyses the adequacy of the samples for the evaluation of species richness was assessed by plotting cumulative species curves, and analyzing the data using the richness estimators (chao 1, jackknife 1, bootstrap, and ice) available in the estimates 8.2 program (colwell, 2006). diversity was estimated by the shannon-wiener index (krebs, 1999). while this index may be the most appropriate for comparing diversity within and among species assemblages (magnussen & boyle, 1995), magurran (2004) has identified some limitations, such as the difficulty of comparing values and the assumption that all species are represented in the sample. to mediate these problems, the exponential values– exp (h’) – were used, allowing us to estimate effective number of species (hill, 1973). equitability and dominance were quantified following pielou index and berger-parker index, respectively (magurran, 2004). the potential effect of abiotic variable precipitation on the number of males collected monthly and number of species sampled monthly was evaluated using pearson correlation coefficient (r), based on the total precipitation recorded each month. differences in the number of individuals attracted by different types of baits were analyzed using the kruskalsociobiology 62(4): 571-577 (december, 2015) 573 wallis test followed by multiple mann-whitney comparisons with the bonferroni correction (sokal & rohlf, 1995). the statistical significance of differences in the attractiveness of each bait was calculated using a g test for independent samples (zar, 1996). the analyses were performed by using the statistical package sigmastat 3.1 for windows (point richmond, ca, usa, 2004). results a total of 643 males belonging to 12 species representing three genera were collected: (1) eufriesea (= ef.) with two species ef. auriceps and ef. violacea; (2) eulaema (= el.) with one species el. nigrita; and (3) euglossa (= eg.), represented by nine species eg. imperialis, eg. melanotricha, eg. fimbriata, eg. leucotricha, eg. pleosticta, eg. truncata, eg. securigera, eg. annectans and eg. cordata. species accumulation curve reached the asymptote well before the end of the study (fig 2) suggesting that an increase in sampling effort would probably not result in the addition of any meaningful number of new species, as confirmed by the species richness estimators. the most abundant species were eg. imperialis (44.5% of the males collected), el. nigrita (20.0%), eg. melanotricha (12.1%), eg. fimbriata (7.0%), and eg. pleosticta (7.0%). the remaining species (n = 7) accounted for 9.4% of the total males collected (table 1). the diversity and equitability indices of the fig 2. cumulative species curve and richness estimators for euglossine bees captured in the furnas do bom jesus state park, pedregulho, são paulo, brazil, from january to december 1990. the continuous lines represent the mean value, and the dotted lines the lower and upper limits of the standard deviation. species number of males captured jan feb mar apr may jun jul aug sep oct nov dec total (%) ef. auriceps friese 3 2 2 1 8 (1.0) ef. violacea blanchard 6 1 4 7 18 (3.0) eg. annectans dressler 1 1 3 5 (0.8) eg. carolina nemésio 3 1 1 5 (0.8) eg. leucotricha rebêlo & moure 7 7 (1.1) eg. fimbriata moure 4 9 14 10 2 2 2 1 4 48 (7.0) eg. imperialis moure 19 25 64 49 36 9 5 22 21 13 7 16 286(44.5) eg. melanotricha moure 11 6 12 5 2 3 1 5 10 9 6 8 78 (12.1) eg. pleosticta dressler 11 3 10 9 2 1 1 4 2 43 (7.0) eg. securigera dressler 2 3 1 1 7 (1.1) eg. truncata rebêlo & moure 2 2 1 3 2 10 (1.6) el. nigrita lepeletier 17 19 36 13 5 1 2 5 10 8 12 128 (20.0) total of males 85 65 144 86 45 15 7 33 45 39 27 52 643 number of species 10 7 9 5 4 4 3 6 6 8 6 8 12 table 1. number of male euglossine bees attracted to chemical baits in the furnas do bom jesus state park, pedregulho, são paulo, brazil, from january to december 1990. ef. (=eufriesea), el. (=eulaema), eg. (=euglossa). sampled community were h’ = 1.70 and j’= 0.88, respectively. euglossa imperialis, the most abundant species, reached a 0.44 dominance according to its berger-parker index (d). both the monthly species richness and abundance of males were significantly correlated with monthly precipitation values (r = 0.86 and r = 0.68; p < 0.05 for both, respectively). in addition, species richness and abundance of males were also correlated with each other (r = 0.65; p < 0.05). s mateus, acr andrade-silva, ca garófalo – orchid bee in a neotropical seasonal semi-deciduous forests574 of the five most abundant species, eg. imperialis and eg. melanotricha were the only species sampled throughout the year, with peaks of abundance in march. males of el. nigrita did not occur in july and males of eg. fimbriata and eg. pleosticta did not visit the baits in may, june, november and june, november and december, respectively. of these species, males of el. nigrita and eg. fimbriata were more abundant in march and the males of eg. pleosticta, in january. the remaining seven species had their males sampling scattered by the months from january to march and from september to december. except for ef. violacea and eg. annectans, that had the greatest number of males sampled in october, november and december, males of ef. auriceps, eg. cordata, eg. leucotricha, eg. securigera and eg. truncata were recorded in greater numbers in the first three months of the year (table 1). only two species, el. nigrita and eg. melanotricha, had the monthly abundance of males significantly correlated with precipitation (r = 0.81 and r = 0.76, p < 0.05 for both, respectively). significant differences in attractiveness between the odor baits were observed (kruskal-wallis: h = 23.49; p <0.05). furthermore, the attraction pattern between the species was different (g = 351.60; df = 22; p <0.05). the number of aromatic compounds that the individual species were attracted ranged from one (eg. imperialis, eg. fimbriata, eg. cordata, eg. leucotricha and ef. auriceps) to three (eg. pleosticta). cineole was the most attractive compound, having attracted males from 11 out of 12 sampled species and around of 85% of the collected males. vanillin and eugenol were more effective than cineole only on attracting males of ef. auriceps and ef. violaceae and males of eg. truncata, respectively (fig 3). in the present study, the stabilization of the cumulative species curve, reinforced by the richness estimators, indicates that the sampling effort (12 months) was adequate for the full inventory of the euglossine species present in the study area (12 species), considering the sampling procedure used. in other studies made in areas also covered by semi-deciduous forests the richness ranged from seven to 14 species (giangarelli et al., 2015). this variation must be interpreted cautiously because, in addition to the aspects mentioned above, the results may also be affected by other factors such as patch characteristics, isolation from other fragments and landscape context. the finding of a few species represented by a large number of males – they are the dominants in the community – whereas most of other species are relatively rare, an attribute of the communities, as observed in this study, has also been reported by other authors working with other communities (aguiar & gaglianone, 2008; rocha-filho & garófalo, 2013). the species composition of the community here studied is similar to those reported by other authors working in areas also covered by seasonal semi-deciduous forests (e.g., rebêlo & garófalo, 1991 ; silveira et al. 2011; knoll & penatti, 2012; giangarelli et al., 2015). nonetheless, even in areas with the same type of vegetation and great similarity in the structure of the communities some differences can be observed. these differences occur mainly due to the presence or absence of species usually represented by few males and by changes in the relative frequencies of each species in each community, as emphasized by knoll and penatti (2012). an example is the data reported by viotti et al. (2013) showing eg. leucotricha, usually represented by few males, as dominant species in a community of high-altitude rocky fields. a similar and surprising example is the occurrence of eg. imperialis as dominant species in the community here studied, a result unobserved in other studies in environments with the same phytophysionomy. this species has a geographic distribution from central america to brazil reaching são paulo state (rebêlo & moure 1995). analyzing a set of relevant morphological features, rebêlo and moure (1995) compared males sampled in this study with males from populations of the amazon basin and atlantic forest. according to the authors, no populations differed from each other, i.e., no significant difference between males can be found. although without any evidence, the occurrence of eg. imperialis in the regions west of minas gerais and north of são paulo led rebêlo and moure (1995) to suggest the connection of these populations with the population of the amazon basin through the central brazil. in this context, it is interesting to note the observations by moura and schlindwein (2009) reporting that species of the atlantic rainforest like eg. imperialis, eg. truncata and el. cingulata occur in the gallery forest of the são francisco river under the semi-arid climate of the caatinga region. in a study on the diversity of the euglossine bees in forest and woody savanna remnants within the brazilian savanna domain, silveira et al. (2015) reported the occurrence fig 3. frequency of euglossine males captured at each scent bait (cineole, eugenol and vanillin) in the furnas do bom jesus state park, pedregulho, são paulo, brazil, from january to december 1990. discussion different phytophysiognomies, forest fragment size (brosi, 2009), differences in sampling effort (silveira et al., 2011), number and aromatic compound used (rocha-filho & garófalo, 2013), and efficiency of sampling method (nemésio & morato, 2004) are some aspects that can affect the results on species richness and abundance in euglossine bee communities. sociobiology 62(4): 571-577 (december, 2015) 575 of eg. imperialis in seasonal semideciduous forests, woody savanna and in a gallery forest where the highest abundance of this species happened. according to those authors, the presence of typical species of the amazon and atlantic forest in the sampled remnants reinforces the hypothesis that both gallery and seasonal semideciduous forest acted as bio-corridors, especially for those species that depend on higher humidity, as emphasized by moura and schlindwein (2009). the importance of gallery forests as mesic corridors, that open the way to the colonization of the savanna landscape by forest-dependent organisms with ranges centered in the neighboring amazon and atlantic forest was suggested by sick (1966) and willis (1992). moreover, the occurrence of aglae caerulea, a cleptoparasite of eulaema nests, in the gallery forest of the vale do véu de noiva in the parque nacional da chapada dos guimaraes was interpreted by anjos-silva et al. (2006) as the “gateway” of that species to mato grosso state. recently, the role of gallery forests as important dispersal alternatives for several species, including a. caerulea, dwelling the amazon and the atlantic forest was highlighted by silva et al. (2013). regardless of the abundance, some recent records show the presence of eg. imperialis in savanna areas (nemésio & faria, 2004, alvarenga et al. 2007, silva & de marco, 2014; silveira et al., 2015). however, the close association of this species to forest habitats (rebêlo & moure, 1995) can be confirmed by the increased abundance of it in communities occurring in areas with that phytophysiognomy, as reported by silveira et al. (2015) and also observed in this study. multiple biotic and abiotic variables control the diversity, composition, and temporal fluctuations of insect communities. in particular, the assembly of bee pollinator communities is strongly influenced by climatic factors as well as variation in food resource availability, which in turn also fluctuates in response to climatic variables (ramirez et al., 2015). as reported by some authors (rebêlo & garófalo, 1991; silveira et al., 2011; andrade-silva et al., 2012; viotti et al., 2013; rocha-filho & garófalo, 2013) and observed in the present study, species richness showed temporal variation throughout the year, with the highest values being observed during the rainiest months. the variation in species composition and species richness here reported occurred by the presence of males from six species which were sampled only during the rainiest months. individual analysis of the five most abundant species showed the highest frequencies of male collection of eg. melanotricha and el. nigrita occurring during the rainy months, following the pattern most commonly reported to the orchid bees (e.g., silveira et al., 2011; andrade-silva et al., 2012; rocha-filho & garófalo, 2013), while this tendency was not observed for the males of eg. imperialis, eg. fimbriata and eg. pleosticta. in addition to these patterns of temporal variation, males of ef. violaceae, ef. auriceps, eg. annectans, eg. carolina, eg. leucotricha and eg. securigera were active only in the rainiest months. of these six species, only ef. auriceps and ef. violacea have one generation per year, with the immature undergoing a prepupal diapause during the coldest and driest months, and adults with activities for a relatively short period during the hottest and rainiest months (roubik & ackerman 1987; rocha-filho & garófalo, 2013). although the abundances of males of eg. imperialis, eg. pleosticta and eg. fimbriata have not been correlated with the values of precipitation, the abundances of eg. melanotricha and el. nigrita and the total abundance of sampled individuals were positively related to that climatic parameter. as the seasonal fluctuations in the abundance of adult bees are primarily associated with the food supply (rêbelo & garófalo, 1991), the tendency for a positive correlation between precipitation and abundance of males can best be explained with an increase in flowering of hosts plants and consequently in food resource availability, as suggested by thiele (2005). and, with nectar and pollen sources available, the bees could take advantage of the high level of food for the nesting activities or to coincide adult emergences for that period (rebêlo & garófalo, 1991). the efficiency of cineole, eugenol and vanillin in attractiveness of euglossine males, as observed in this study, have been shown in dozens of inventories carried out in several neotropical regions (rebêlo & garófalo, 1991 silveira et al., 2011; rocha-filho & garófalo 2013). in a study to investigate phenological patterns and seasonal and geographic variations in the preference for fragrances of euglossine males, in two coastal areas of atlantic forest in ubatuba, sp, rocha-filho and garófalo (2013) utilized 14 aromatic baits, three of them, cineole, eugenol and vanillin, in the first year and the other 11 in the second year. during the first year 18 species were sampled and 987 males were collected while in the second year 488 males belonging to ten species were attracted. these results confirm the efficiency of those three compounds in the attraction of euglossine males. although the species composition of the community here studied was similar to those occurring in areas also covered by seasonal semi-deciduous forests, the presence of eg. imperialis as the most abundant species was a main factor differentiating the communities. thus, based on the assumption that male euglossine capture rates reflect actual abundance at the sampled habitats (otero & sandino, 2003), we believe that eg. imperialis has a population very well established in the habitat here studied. acknowledgements the authors are grateful to josé manuel macário rebêlo and jesus santiago moure (in memoriam) for species identification, and henrique paulo barbosa de souza for providing the rainfall data for the period between 1989 and 1990. we thank túlio marcos nunes for the help with manuscript corrections and text improvements and silvia helena sofia for helpful comments. finally, the authors are grateful to the administration of furnas do bom jesus state park, pedregulho, são paulo, for permission to conduct fieldwork in the park. s mateus, acr andrade-silva, ca garófalo – orchid bee in a neotropical seasonal semi-deciduous forests576 references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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(1996). biostatistical analisys. 4th ed. new jersey: prentice hall 663 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.207-217sociobiology 61(2): 207-217 (june, 2014) trap-nesting solitary wasps (hymenoptera: aculeata) in an insular landscape: mortality rates for immature wasps, parasitism, and sex ratios al oliveira nascimento, ca garófalo introduction there are more than 34,000 described species of aculeate wasps in the world, of which about 14,700 species belong to the families pompilidae, crabronidae, sphecidae, and vespidae (subfamily eumeninae) (morato et al., 2008). for the neotropical region, 1,858 species of crabronidae and sphecidae have been recorded (amarante, 2002; 2005), of which 645 occur in brazil (morato et al., 2008). ninety per cent of the species known have solitary behaviour and diversified nesting habits (o’neill, 2001). the female captures several insects or spiders to provision the cells, exerting a very significant predation pressure on ecosystems (krombein, 1967). around 5% of all species of solitary wasps have the habit of nesting in pre-existing cavities (krombein, 1967). this characteristic has facilitated the study of these solitary species, because females are attracted to nest in human-made trap-nests. in studies made with solitary wasp species that nest abstract the aim of this study was to examine the species composition and the abundance of solitary wasps that nest in preexisting cavities in the ilha anchieta state park, brazil. sampling was made during two years utilizing trap-nests. of the 254 nests obtained, 142 nests were built by 14 species belonging to four genera and four families. in the remaining 112 nests all immatures were dead by unknown causes or had been parasitized by natural enemies. the occupation of trap-nests occurred almost throughout the study period and the wasps nested more frequently during the super-humid season. trypoxylon lactitarse, pachodynerus nasidens, trypoxylon sp.2 aff. nitidum and podium denticulatum were the most abundant species. the sex ratios of t. lactitarse and trypoxylon sp.2 aff. nitidum were significantly male-biased, whereas those of trypoxylon sp.5 aff. nitidum and p. nasidens were significantly female-biased. sex ratios of p. denticulatum and p. brevithorax were not significantly different from 1:1. natural enemies emerging from the nests were identified as belonging to the families chrysididae, ichneumonidae and chalcididae (hymenoptera), the genus melittobia (hymenoptera, eulophidae), and the species amobia floridensis (townsend, 1892) (diptera: sarcophagidae). the number of cells with dead immatures from unknown factors was significantly higher than the number of cells parasitized by insects. sociobiology an international journal on social insects universidade de são paulo (ffclrp-usp), ribeirão preto, sp – brazil article history edited by gilberto m m santos, uefs, brazil received 13 september 2013 initial acceptance 08 october 2013 final acceptance 17 october 2013 keywords atlantic forest, colonization, natural enemies, phenology, wasp abundance corresponding author ana l. oliveira nascimento faculdade de filosofia, ciências e letras de ribeirão preto universidade de são paulo av. bandeirantes, 3900 ribeirão preto, são paulo, brazil. 14040-901 e-mail: analuizanascimento@uol.com.br in pre-existing cavities, the use of trap-nests has provided information not only on the occurrence of species in a given habitat, but also on the wasp community composition and their natural enemies, on the nesting biology of the species, on food sources used to rear the immatures (e.g., krombein, 1967; gathmann et al., 1994; camillo et al., 1995; camillo & brescovit, 1999; zanette et al., 2004; tylianakis et al., 2006; buschini et al., 2006; asís et al., 2007; buschini, 2007; santoni & del lama, 2007; buschini & woiski, 2008; ribeiro & garófalo, 2010; musicante & salvo, 2010; loyola & martins, 2011; polidori et al., 2011). habitat quality, the effects of habitat fragmentation and of landscape complexity on community composition and predatory-prey interactions (tscharntke et al., 1998; morato, 2001; steffan-dewenter, 2002; kruess & tscharntke, 2002; tylianakis et al., 2007; loyola & martins, 2008; gonzález et al.,2009; holzschuh et al., 2009; schüepp et al., 2011), and how urban environments can support such insects (zanette et al., 2005) have also been assessed with the use of trap nests. research article wasps al oliveira nascimento, ca garófalo solitary wasps colonizing trap-nests in an insular landscape208 given the important role of solitary wasps in terrestrial ecosystems (lasalle & gauld, 1993), any effort to understand them and unsure their survival is fully justified. in this context, biological inventories are the basic tools for the initial survey of biological diversity, as well as for monitoring changes in different components of biodiversity in response to the impacts of both natural processes and human activities (lewinsohn et al., 2001). the present study reports data on the occupation of trap-nests by solitary wasps from a survey carried out in the ilha anchieta state park, a reserve within the atlantic forest domain. among tropical biomes, the atlantic forest is the one that has suffered the most fragmentation and degradation by human intervention, which threatens the high species diversity and the high degree of endemism of this biome (myers et al., 2000). material and methods study area the study was conducted within the ilha anchieta state park (45º 02’ – 45º 05’ w and 23º 31’ – 23º 34’ s). the park occupies the entire island (828 ha) and has only one perennial stream, which is located in an area of coastal forest (restinga). the topography is rugged and mountainous, with slopes typically greater than 24º. low-lying level areas (with slopes under 6º) are found at two beaches (‘grande’ and ‘presídio’). areas of intermediate slope are located in the valley bottoms and on the flat hilltops of the island (rocha-filho & garófalo, 2013). the vegetation found on ilha anchieta has been described by guillaumon et al. (1989), following rizzini (1977), and includes anthropogenic fields, rocky coast, atlantic forest, gleichenial, mangrove, and restinga. the climate of ilha anchieta is tropical rainy (koppen, 1948). it has two distinct seasons a year: one super humid, from october to april, with monthly rainfall above 100 mm, and the other less humid, from may to september, when the monthly rainfall is below 100 mm. during the study, from september 2007 to august 2009, the average monthly temperatures ranged from 22.5 °c to 25.8 °c in the super-humid periods and from 18.0 °c to 21.4 °c in the less humid periods. the monthly rainfall totals ranged from 190.3 mm to 488.3 mm in the super-humid periods and from 0 to 220.9 mm in the less humid periods. temperature and rainfall data of the ubatuba region were obtained from the centro integrado de informações agrometereológicas (http://www.ciiagro.sp.gov.br). the super-humid and less-humid terminology follows talora and morellato (2000). methods the design of the trap-nests used in the present study followed camilo et al. (1995): they consisted of tubes made of black cardboard, with one end closed with the same material. these tubes, with dimensions of 5.8 cm long × 0.6 cm diameter (small tube, st) and 8.5 cm long × 0.7 cm diameter (large tube, lt) were inserted into holes drilled into wooden plates (length: 30 cm, height: 12 cm, thickness: 5.0 cm). each wooden plate had a total of 55 holes available, 2.0 cm apart from each other and distributed in five rows. we also used another type of trap-nest made of bamboo canes (bc) which were cut so that a nodal septum closed one end of the cane. the bamboo canes had variable length and their internal diameter ranged from 0.4 to 2.9 cm although all sizes were not equally represented. in order to protect the bamboo canes from the sun and the rain, they were inserted, in bundles of 10-15 units, into a pvc tube, 40 cm long × 12 cm diameter. the plates containing the cardboard tubes and the pvc tubes were arranged on iron stands and placed at the sampling sites. three sampling sites (site 1: 45° 04’ 06.5” w and 23° 32’ 22.0” s; site 2: 45° 03’ 55.7” w and 23° 32’ 24.3” s, and site 3: 45° 03’ 52.1” w and 23° 32’ 23.6” s) were established on the ‘presídio’ beach. distances between sites ranged from 100 m, between sites 2 and 3, to 308 m, between sites 1 to 3. sites 1 and 2 were separated by a distance of 220 m. the iron stands were installed behind or next to existing buildings in the area. the geographical coordinates of all three sampling points were recorded with a gps receiver. the iron stands installed at each sampling site carried 55 large tubes, 55 small tubes, and 90 bamboo canes distributed in three pvc tubes. the trap-nests were inspected once a month from september 2007 to august 2009. each inspection was made using a penlight. when traps contained completed nests, they were collected and replaced with empty ones. these nests were taken to the laboratory in ribeirão preto, sp, and each trap-nest was placed in a transparent glass or plastic tube, 4.0-5.0 cm longer than the trap and with an internal diameter of 0.9 cm. the nests were kept at room temperature and observed daily until the adults emerged. as the adults emerged into the glass or plastic tube, the trap was removed and the individuals were collected. a few days after the last emergence, the nest was opened and its contents analysed. cells and nests from which no adult emerged were also opened, and the cause and stage of mortality were recorded. the analysis of the food present in brood cells allowed identification of the genus (or subfamily in the case of eumeninae) of the breeding species. voucher specimens of wasps were deposited in the collection of bees and solitary wasps (coleção de abelhas e vespas solitárias caves) of the department of biology of the faculty of philosophy, science and letters at ribeirão preto –usp. statistical analysis statistical tests followed zar (1999) and were performed using the statistical package sigmastat for windows, version 3.10 (2004 – systat software, inc.). kruskal-wallis (h) test was used to verify differences among the diameters of bamboo canes occupied by different species. to isolate the sociobiology 61(2): 207-217 (june, 2014) 209 species that differs from the others it was used a multiple comparison procedure (dunn’s method). chi-square tests were used to compare mortality rates for immature wasps, parasitism rates, and sex ratios. pearson correlation analyses were performed to test for the strength of associations between nesting frequency and climatic conditions (average monthly temperature and rainfall). throughout the text, all means (x) are reported with their associated standard error (se) as x ± se. results occupation of trap-nests by solitary wasps we collected 150 nests in the first year (september 2007 august 2008) and 104 nests in the second year (september 2008 august 2009). of the nests established in the first year, 78 nests were built by 12 species and of those established in the second year, 64 nests were made by six species. these species belong to four genera (trypoxylon, pachodynerus, podium, and auplopus) of four families (crabonidae, vespidae, pompilidae, and sphecidae). crabronidae was the family with the highest number of species (nine), followed by vespidae and pompilidae, each with two species. sphecidae was represented by only one species (table 1). in the 112 remaining nests (72 nests of the first year and 40 of the second one) all immatures were dead from unknown causes or had been parasitized by insects, so that only the genus of the nesting species could be identified. most species (n = 10) nested exclusively in the bamboo cane traps. pa. nasidens, pa. brevithorax, and trypoxytable 1. species of solitary wasps that nested in the trap-nests in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009, number of nests built and type of trap-nest (st = small tube; lt = large tube; bc = bamboo cane) occupied. species number of nests frequency of utilization of each type of trap-nest 1st year 2nd year st lt bc trypoxylon lactitarse (saussure, 1867) 13 21 34 trypoxylon aurifrons (shuckard, 1837) 4 4 trypoxylon punctivertex (richards, 1934) 2 2 trypoxylon albitarse (fabricius, 1804) 1 1 trypoxylon sp.1 aff. nitidum 3 3 trypoxylon sp.2 aff. nitidum 17 11 5 1 22 trypoxylon sp.5 aff. nitidum 5 5 trypoxylon (trypargilum) sp.1 3 3 trypoxylon (trypargilum) sp.2 1 1 pachodynerus nasidens (latreille, 1812) 7 24 13 11 7 pachodynerus brevithorax (saussure, 1852) 5 3 1 1 podium denticulatum (smith, 1856) 20 2 18 auplopus pratens (dreisbach, 1963) 2 2 auplopus sp. 1 2 1 3 total 78 64 21 15 106 lon sp.2 aff. nitidum were the only species that used all three types of trap-nest. pachodynerus nasidens nested more frequently in the small and large tubes whereas nests of trypoxylon (n = 75) were found mainly in bamboo canes (n = 69). podium denticulatum occupied only the large tubes and bamboo canes, but most nests were built in bamboo canes (table 1). the analysis of the diameters of bamboo canes occupied by t. lactitarse, trypoxylon sp.2 aff. nitidum, pa. nasidens and po. denticulatum revealed significant differences among them (kruskal-wallis h = 56.7; df = 3; p < 0.001). the cavities used by t. lactitarse were larger than those occupied by trypoxylon sp.2 aff. nitidum and pa. nasidens, and those used by po. denticulatum were larger than those used by trypoxylon sp.2 aff. nitidum. on the other hand, no significant difference was found among the diameters of bamboo canes used by po. denticulatum, t. lactitarse and pa. nasidens as well as between the canes occupied by pa. nasidens and trypoxylon sp.2 aff. nitidum (table 2). phenology of nesting trypoxylon lactitarse, trypoxylon sp.2 aff. nitidum, pa. nasidens, and auplopus sp.1 nested in both years, pa. brevithorax and auplopus pratens nested only in the second year, and the remaining eight species occupied the traps only in the first year (table 1). the occupation of trap-nests occurred throughout the study period, with the exception of december 2007, when no nests were built. in both years, the wasps nested more frequently during the super-humid season (109 nests in the first year and 76 in the second year) than al oliveira nascimento, ca garófalo solitary wasps colonizing trap-nests in an insular landscape210 during the less-humid season (31 nests in the first year and 28 in the second year). in both years, the highest frequency of nesting during the hot/super-humid season (average temperature = 23.9 ± 1.1°c) occurred in march. during the cool/ less-humid season (average temperature = 19.9 ± 1.06°c), july of the first year and august of the second year were the months with the highest numbers of nests built (fig 1). trypoxylon lactitarse, pa. nasidens, trypoxylon sp.2 aff. nitidum and po. denticulatum were the most abundant species in terms of number of nests. trypoxylon lactitarse showed an overall higher frequency of nesting in the second year (table 1). more traps were occupied in the cool/lesshumid season in the first year (n= 9 nests), and more traps in the hot/super-humid season in the second year (n = 13 nests). in both years, the females were active for only five months (fig 2). pachodynerus nasidens was the second most abundant species. this species built 7 nests in the first year and 24 in the second one (table 1). except for two nests constructed in july of the first year, the nests were constructed in the hot/super-humid season (fig 2). trypoxylon sp.2 aff. nitidum occupied more traps in the first year (17 nests) than in the second year (11 nests) (table 1). the nesting peak occurred in november of the first year, whereas january and april were the months with the highest number of nests in the second year. regardless of the total number of nests built the females of trypoxylon sp.2 aff. nitidum were active for a longer period than the females of other species (fig 2). females of po. denticulatum, the fourth most abundant species, occupied trap-nests only in february, march, and april of the first year. analysis of the temporal distribution of nesting of the four most abundant species in relation to average monthly temperature and rainfall only revealed a significant correlation between the number of nests and temperature for pa. nasidens (r = 0.45; p = 0.02; df = 24). period of development and sex ratio the total duration from oviposition to adult emergence was not determined. however, the maximum interval between nest collection and adult emergence may provide an estimate. excepting two males of t. punctivertex and one female and six males of po. denticulatum, which emerged from 165 to 192 days after the nests had been removed from the field, other individuals had the maximum interval ranged from 59 to 66 days (table 3). these figures indicate an egg-to-emergence table 2. diameter of bamboo canes occupied by solitary wasps in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009. species trap-nest diameter (cm) range x ± se (n) trypoxylon lactitarse 0.8-1.1 0.96 ± 0.02 (34) trypoxylon aurifrons 0.5-0.7 0.6 ± 0.05 (4) trypoxylon punctivertex 0.5-0.6 0.55 ±0.07 (2) trypoxylon albitarse 1.1 1.1 (1) trypoxylon sp.1 aff. nitidum 0.6 0.6 (3) trypoxylon sp. 2 aff. nitidum 0.4 – 0.7 0.5± 0.02 (22) trypoxylon sp. 5 aff. nitidum 0.6-0.7 0.6 ±0.02 (5) trypoxylon (trypargilum) sp.1 0.6 0.6 (3) trypoxylon (trypargilum) sp.2 0.9 0.9 (1) pachodynerus nasidens 0.6-0.9 0.67± 0.04 (7) pachodynerus brevithorax 1.0 1.0 (1) podium denticulatum 0.8-1.1 0.93 ±0.02 (18) auplopus pratens 1.2-1.5 1.35 ±0.14 (2) auplopus sp.1 1.1 -1.5 1.33 ±0.12 (3) fig 1. climatic conditions (average temperature and rainfall) and number of nests established in trap-nests by solitary wasps in the ilha anchieta, state of são paulo, from september 2007 to august 2009. fig 2. number of nests built by trypoxylon lactitarse, pachodynerus nasidens and trypoxylon sp.2 aff. nitidum in trap-nests in the ilha anchieta, ubatuba, state of são paulo, from september 2007 to august 2009. sociobiology 61(2): 207-217 (june, 2014) 211 table 3. period (in days) between the collection of the nests of solitary wasps that occupied the trap-nests in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009, and emergence of individuals produced. species period (in days) between collection date and emergence males females range x ± se(n) range x ± se(n) trypoxylon lactitarse 9-61 39.1±1.30(65) 20-56 37±1.35(30) trypoxylon aurifrons 17-20 17.4±0.39(8) 17-20 18±0.98(3) trypoxylon punctivertex 177 177(2) 09-12 10.5±1.49(2) trypoxylon albitarse 66 66(1) trypoxylon sp. 1 aff. nitidum 15-30 24.3±3.6(4) 22 22(1) trypoxylon sp. 2 aff nitidum 9-35 23.6±0.97(50) 9-37 22.9±1.36(22) trypoxylon sp. 5 aff. nitidum 26 26(1) 11-26 16.1±1.52(14) trypoxylon (trypargilum) sp. 1 28-42 32.2±2.59(5) 30 30(1) trypoxylon (trypargilum) sp. 2 24 24(1) pachodynerus nasidens 11-58 22±3.19(28) 5-59 21.5± 1.77(50) pachodynerus brevithorax 11-19 17±1.22(6) 11-21 17.3±2.15(4) podium denticulatum 11-177 91.1±20.0(14) 10-192 68.7±31.89(7) auplopus pratens 17-18 17.5±0.50(2) 21-22 21.5±0.50(2) auplopus sp.1 2/20 6.8±3.33(5) 21 21(1) table 4. number of males and females emerged and sex ratio for each species that nested in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009. (values of χ2 in bold indicate a sex ratio significantly different at p < 0.05 and df = 1, from 1:1). species number of males number of females sex ratio (no. ♂ : no. ♀) trypoxylon lactitarse 65 30 2.2♂: 1 ♀ (χ2= 12.9) trypoxylon aurifrons 8 3 2.6♂: 1 ♀ trypoxylon punctivertex 2 2 1♂: 1 ♀ trypoxylon albitarse 1 trypoxylon sp.1 aff. nitidum 4 1 4♂: 1 ♀ trypoxylon sp.2 aff. nitidum 50 22 2.3♂: 1♀ (χ2= 10.9) trypoxylon sp.5 aff. nitidum 1 14 0.1♂: 1 ♀ (χ2= 11.3) trypoxylon (trypargilum) sp.1 5 1 5♂: 1 ♀ trypoxylon (trypargilum) sp.2 1 pachodynerus nasidens 28 50 (0.56♂:1♀) (χ2= 6.2) pachodynerus brevithorax 6 4 1.5♂: 1 ♀ (χ2= 0.4) podium denticulatum 14 7 (2♂:1♀) (χ2= 2.33) auplopus pratens 2 2 1♂: 1 ♀ auplopus sp.1 5 1 5♂: 1 ♀ period about of two months. on the other hand, the results obtained for t. punctivertex and p. denticulatum indicate the occurrence of diapause in nests established in february (po. denticulatum), march (t. punctivertex and po. denticulatum), and april (po. denticulatum). the sex ratios of t. lactitarse and trypoxylon sp.2 aff. nitidum were significantly male-biased, whereas those of trypoxylon sp.5 aff. nitidum and pa. nasidens were significantly femalebiased. sex ratios of po. denticulatum and pa. brevithorax were not significantly different from 1:1 (table 4). females and males of the remaining species were produced in similar numbers from the nests of a. pratens and t. punctivertex, whereas from the nests of t. albitarse and trypoxylon sp.2 only males emerged (table 4). mortality of immatures of 768 brood cells constructed in 254 nests, 240 (31%) contained dead immatures from unknown causes, and 199 (26%) had been parasitized by insects. the number of cells with dead al oliveira nascimento, ca garófalo solitary wasps colonizing trap-nests in an insular landscape212 immatures was significantly higher than the number of cells parasitized (χ2= 4.10; p < 0.05; df =1) only in nests of trypoxylon whose species could not be determined (table 5). for trypoxylon (trypargilum) sp.2 and a. pratens, the offspring had a survival rate of 100%. in the case of t. albitarse, the only cause of nonemergence was death of the immature from unknown causes, whereas for trypoxylon (trypargilum) sp1 the one cell from which a wasp did not emerge had been parasitized. although in relatively small numbers all others species had immature mortality due to unknown causes and parasite attack (table 5). natural enemies and hosts the natural enemies were identified as belonging to the families chrysididae (hymenoptera), ichneumonidae (hymenoptera), and chalcididae (hymenoptera), the genus melittobia (hymenoptera: eulophidae), and the species amobia floridendis (townsend, 1892) (diptera: sarcophagidae). trypoxylon sp.2 aff. nitidum was the species with the highest number of nests attacked (15) and cells parasitized (21), followed by t. lactitarse, pa. nasidens, and pa. brevithorax with 17, 5 and 4 cells parasitized, respectively (table 6). among the nests that could not be identified to the level of species due to total mortality of the brood cells from unknown causes or parasitism, the largest number (61 nests) table 5. numbers of brood cells, individuals produced, cells with dead immatures and parasitized cells in nests of solitary wasps that occupied trap-nests in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009. (* nests of unidentified species) **(value in bold indicates a statistically significant, at p <0.05 and df = 1, difference between the number of immature wasps mortalities due to unknown causes compared to parasite attack). species no. of brood cells no. individuals produced no. of cells with dead immatures no. of parasitized cells χ2** trypoxylon lactitarse 133 95 21 17 χ2 = 0.42 trypoxylon aurifrons 16 11 2 3 trypoxylon punctivertex 9 4 2 3 trypoxylon albitarse 3 1 2 trypoxylon sp. 1 aff. nitidum 10 5 2 3 trypoxylon sp. 2 aff. nitidum 109 72 16 21 χ2 = 0.67 trypoxylon sp. 5 aff. nitidum 21 15 5 1 trypoxylon (trypargilum) sp. 1 7 6 1 trypoxylon (trypargilum) sp. 2 1 1 pachodynerus nasidens 91 78 8 5 pachodynerus brevithorax 17 10 3 4 podium denticulatum 27 21 4 2 auplopus pratens 4 4 auplopus sp.1 9 6 2 1 subtotal 457 329 67 61 trypoxylon* 265 0 149 116 χ2 = 4.10 podium* 29 0 16 13 χ2 = 0.31 eumeninae* 11 0 5 6 auplopus* 6 0 3 3 subtotal 311 0 173 138 total 768(100%) 329 (43%) 240 (31%) 199 (26%) belonged to the genus trypoxylon, followed by podium (10 nests), eumeninae (3 nests), and auplopus (2 nests)(table 6). no natural enemy was found in the nests of a. pratens, t. albitarse, or trypoxylon (trypargilum) sp.2. among the natural enemies associated with the nests, amobia floridensis (diptera: sarcophagidae) was the main enemy, attacking the nests of six species and responsible for 58.3% of all parasitized cells. melittobia sp. (hymenoptera: eulophidae) caused 23.6% of the combined mortalities of t. punctivertex, trypoxylon sp.2 aff. nitidum, pa. nasidens, and po. denticulatum. individuals of the family chrysididae were reared exclusively in the nests of trypoxylon, parasitizing 22 cells (11.1%) in 17 nests. individuals of ichneumonidae attacked more often nests of t. lactitarse, parasitizing 6 cells in 6 nests. in addition to this host, nests (n = 2) of two other species of trypoxylon also had cells parasitized (one in each nest), and a cell of auplopus sp. was also attacked by this enemy. individuals of chalcididae attacked exclusively nests of auplopus, and parasitized two cells in two nests (table 6). discussion although there are tendencies to make a comparative analysis between the number of species occupying trap-nests and/or their abundances obtained in inventories carried out sociobiology 61(2): 207-217 (june, 2014) 213 at different geographical locations, the results of these analyses should be interpreted with caution. differences in sampling methods, the type and arrangement of the trap-nests in the study area, the arrangement of natural cavities, and the sampling periods may hinder the formulation of reliable comparisons (aguiar et al., 2005). among the 14 species collected from the trap-nests set up on ilha anchieta, seven: t. lactitarse, t. albitarse (coville, 1981), t. aurifrons (amarante, 2002), t. punctivertex, po. denticulatum (bohart & menke, 1976), pa. brevithorax and pa. nasidens (willink & roig-alsina, 1998), have in common a broad geographic distribution. within brazil these species have been reported for three farmland habitats (santa carlota farm, cajuru, sp, camillo et al., 1995), a riparian forest area surrounded by crops and pastures (ituiutaba, mg, assis & camillo, 1997), an urban forest (belo horizonte, mg, loyola & martins, 2006), three habitats in a municipal park (araucárias municipal park, gurarapuava, pr, buschini & woiski, 2008), and tropical savanna and riparian forest areas (ingai, mg, pires et al., 2012). they have also been observed in a region of temperate deciduous forest in (ontario, canada, taki et al., 2008) and forest fragments covered by chaco serrano vegetation (córdoba, argentina, musicante & salvo, 2010). of the seven remaining species, a. pratens has been recorded only in brazil (fernandez, 2000), trypoxylon sp.1 aff. nitidum and trypoxylon sp.2 aff. nitidum had occurrences reported by assis and camillo (1997), and for the other four species, there is no information available. trypoxylon lactitarse, the species that occupied the highest number of trap-nests on ilha anchieta, was also the dominant species in the studies by camillo et al. (1995), assis and camillo (1997), buschini and woiski (2008), and musicante and salvo (2010), which evidences a great plasticity to adapt to different environments. when compared to these studies, t. lactitarse on ilha anchieta showed significantly elevated reproductive activity, occupying a large number of trap-nests. the only exception was the study by musicante and salvo (2010), carried out in forest fragments dispersed in an agricultural matrix strongly dominated by wheat in winter and soya beans or corn in summer, in which the number of t. lactitarse nests was smaller than observed on ilha anchieta. trypoxylon sp.2 aff. nitidum built a larger number of nests on ilha anchieta than reported for the habitats studied by assis and camillo (1997). likewise, po. denticulatum nested more frequently on ilha anchieta than in the areas studied by camillo et al. (1996) and assis and camillo (1997), who used sampling periods similar to those used in the present study. although most nests on ilha anchieta were built during the hottest and rainiest period of the year, individual analyses for the four most abundant species showed an association betable 6. host species, number of nests and brood cells attacked by natural enemies of trap-nesting solitary wasps in the ilha anchieta state park, ubatuba, state of são paulo, from september 2007 to august 2009. (* nests of unidentified species). host species natural enemies number of nests attacked and (number of brood cells attacked = c) amobia floridensis melittobia sp. chrysididae ichneumonidae chalcididae trypoxylon lactitarse 7(11c) 6(6 c) trypoxylon aurifrons 1(1c) 1(2c) trypoxylon punctivertex 1(2c) 1(1c) trypoxylon sp. 1 aff. nitidum 2(3c) trypoxylon sp. 2 aff. nitidum 3(5c) 4(7c) 7(8c) 1(1c) trypoxylon sp. 5 aff. nitidum 1(1c) trypoxylon (trypargylum) sp. 1 1(1c) pachodynerus nasidens 3(3c) 1(2c) pachodynerus brevithorax 2(4c) podium denticulatum 1(1c) 1(1c) auplopus sp.1 1(1c) subtotal 17(25c) 7(12c) 12 (15 c) 8(8c) 1(1c) trypoxylon* 42(79c) 11(27c) 5 (7c) 3(3 c) podium * 9(12c) 1(1c) eumeninae* 2(6c) auplopus* 1(1c) 1(1c) 1(1c) subtotal 51(91c) 15(35c) 5(7c) 4(4c) 1(1c) total 68(116c) 22(47c) 17(22c) 12(12c) 2(2c) al oliveira nascimento, ca garófalo solitary wasps colonizing trap-nests in an insular landscape214 tween temperature and nesting activity only for pa. nasidens. the relatively small number of occupied trap-nests hinders a more robust analysis of the action of the climatic factors on the phenology of these species. krombein (1967) and fricke (1991) have suggested that the diameter of the cavity used by a given species is not only related to the size of the female, but also to its prey. these parameters may explain the lack of overlap in the diameters of the bamboo canes used by t. lactitarse and other species of the same genus. the exceptions were t. albitarse and t. (tripargilum) sp.2, which presented marginal overlap with t. lactitarse, due to the larger size of the individuals of the latter species compared to the other species (assis & camillo, 1997) and/or the larger size of the spiders used to provision the cells. auplopus species occupied the bamboo canes with the largest diameters. this is certainly related to the fact that cells are built with material (clay) taken into the cavity, inside which the female needs space to work on the construction. it’s interesting to observe that among the most abundant species t. lactitarse, po. denticulatum and pa. nasidens nested in cavities with similar diameters. this could suggest the occurrence of competition among those species depending up of the availability of cavities with those diameters. considering all the variables that may contribute to the selection of a cavity for nesting, any project aimed at the preservation of species with the characteristic of nesting in pre-existing cavities should provide a large number of options in terms of diameters of the cavities offered. the method used in the present study does not directly yield data about the duration of the development period of immature wasps (from oviposition to emergence). however, by combining the time intervals between nest collection and the emergence of individuals with information on the distribution of nesting activities throughout the year, it is possible to affirm that species such as t. lactitarse, t. sp.2 aff. nitidum, pa. nasidens, and po. denticulatum, have more than one generation a year. for species such as t. lactitarse and pa. nasidens, the maximum periods observed between nest collection and emergence reflect mainly the influence of a lower temperature resulting in a longer development period. the exceedingly long periods observed for po. denticulatum indicate the occurrence of diapause in some immature wasps of this species, as also reported by ribeiro and garófalo (2010), based on studies carried out on the campus of the university of são paulo at ribeirão preto, state of são paulo. only two nests of t. punctivertex were collected, both in march 2008; from one nest a female rapidly emerged followed much later by two males that had evidently undergone diapause, from the other nest a female rapidly emerged. considering that diapause acts as a protection mechanism against unfavourable seasons, thereby reducing the extinction risk (martins et al., 2001), the prepupal diapause observed in po. denticulatum and t. punctivertex nests could be a survival strategy against adverse conditions. the occurrence of nests from which some wasps emerge with and others without diapause, as observed with t. punctivertex, shows that some adults should pass through an adverse season by sheltering somewhere. trap-nest diameter (buschini, 2007) and food availability (polidori et al., 2011) are two factors that may affect the sex ratio of the offspring of species that nest in pre-existing cavities. considering the four most abundant species sampled on ilha anchieta, t. lactitarse and t. sp.2 aff. nitidum showed a male-biased sex ratio, pa. nasidens a femalebiased sex ratio, whereas the sex ratio of po. denticulatum was not significantly different from 1:1. analogous results have been reported by borges and blochtein (2001) for t. lactitarse, whereas buschini (2007) reported similar proportions for the two sexes for the two generations of t. lactitarse collected during a study in the state of paraná. ribeiro and garófalo (2010) found a sex ratio of 1:1 for po. denticulatum, as observed in the present study. it is important to highlight that there are multiple factors that may affect the sex ratio of a population. a first step towards evaluating these factors will be the acquisition of more extensive data sets, based on larger sample of nests and to assess the food resources availability in the study area. several studies carried out with solitary wasps occupying trap-nests have shown wide variations in the mortality rates of immatures. the loss rate for immatures reported by some authors is typically close to 50% (krombein, 1967; freeman & jayasingh, 1975; jayasingh & freeman, 1980; camillo et al., 1996; buschini & wolf, 2006), but some species have reported much lower and others much higher mortality rates (danks, 1971; camillo et al., 1993; gathmann et al., 1994; assis & camillo, 1997; tormos et al., 2005). in the present study, the brood mortality rate based on the total set of collected nests was around 57%, but analysing separately each of the seven species that produced the largest number of brood cells (t. aurifrons, 16 brood cells to t. lactitarse, 133 brood cells), the loss rate for immatures ranged from 14.3% (pa. nasidens) to 41.2% (p. brevithorax). curiously, the two extremes were represented by eumeninae species. except for pa. brevithorax, the other six species that utilized more frequently the trap-nests showed loss rates for immatures that are much lower than the values reported by other authors. unknown factors and natural enemies caused brood mortalities in similar proportions in nests built by identified species. among the natural enemies that attacked the trapnests, parasitization by individuals of the family chrysididae was restricted to species of trypoxylon as the host, whereas individuals of the family chalcididae only attacked the nests of auplopus. considering the other identified enemies associated with the nests collected on ilha anchieta, amobia floridensis was the agent that caused the highest brood mortality, attacking the nests of trypoxylon, pachodynerus, and podium. with a distribution from the united states to brazil (pape et al., 2004), the associations between species of eusociobiology 61(2): 207-217 (june, 2014) 215 meninae and trypoxylon are well known through reports by krombein (1967), freeman and jayasingh (1975), and but the association between a. floridensis and podium is described here for the first time. according to krombein (1967), the amobia female follows the host female back to its nest, lands, and remains close to the nest entrance. when the host female leaves its nest to forage in the field, the fly enters the nest and larviposits. amobia floridensis larvae compete with the host species larvae for caterpillars and spiders placed in the cells of eumeninae and trypoxylon, respectively, and according to the results obtained in the present study, they also compete for the cockroaches stored in the cells of podium. the dispersal ability of flying arthropods is usually determined by body size, as larger species have a better flight capacity (gathmann et al., 1994; steffan-dewenter & tscharntke, 1999). there is no information available on the flight capacity of the wasp species sampled on ilha anchieta, but it is reasonable to propose that the colonization of the island may have occurred through the transportation of adult individuals on the vessels that daily cross the 600 m channel that separates the island from the mainland. another means by which cavity-nesting wasp species may cross water barriers is within pieces of floating wood, a process known to occur for solitary bees (michener, 2000). regardless of the colonization process for ilha anchieta, the number of nests collected in the present study may be considered relatively small, given the area of the island and the number of wasp species sampled. however, the number of trap-nests occupied by females may be related to the availability of natural cavities within the study area. one of the tourist attractions on ilha anchieta are the ruins of a prison with several buildings constructed in masonry, whose current dilapidated state affords many nesting opportunities for wasps. this fact together with the decline of 30.9% in the number of trapnests occupied and 50% of species nesting in the second year compared to the first suggests that the populations of solitary wasps on ilha anchieta may be dependent on the mainland populations for the permanent occupation of the island. acknowledgments we are grateful to j.c. serrano for wasp identifications, c.a. mello-patiu for amobia floridensis identification, e.s.r. silva for technical help, the instituto florestal de são paulo for permission to work in the ilha anchieta state park, the staff at ilha achieta for their support, two anonymous reviewers for providing valuable comments on the manuscript, and research center on biodiversity and computing (biocomp). a.l.o. nascimento received grants from fapesp (08/06023-4) and capes. research supported by fapesp (04/15801-0) and cnpq (305274/2007-0). references aguiar, c.m.l., garófalo, c.a. & almeida, g.f. 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(1999). biostatistical analysis. new jersey: prentice hall, 663p. doi: 10.13102/sociobiology.v67i4.5785sociobiology 67(4): 572-583 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction studies on nesting biology in wasp and bee species using preexisting cavities have enhanced our understanding of behavior, life cycle, trophic niche and sex ratio issues, among others, contributing to the enlightenment of the life histories, ecology and evolution of these insects (michener, 2007; costa & gonçalves, 2019). additionally, knowing the nesting biology of solitary species reveals the ecological relevance of these species and contributes to their conservation. the evolution of behaviors associated with different levels of parental investment (differences in nesting and provision) and sociality have been successfully investigated using species of the sphecidae family as model organisms (matthews, 1991; melo, 2000). abstract podium denticulatum occurs from mexico to southern brazil, including northeastern argentina. females use pre-existing cavities to build nests, consisting of cells separated by walls of mud and resin and massively provisioned with paralyzed cockroaches. trap-nests were disposed in three localities in the state of são paulo, brazil (araras, são carlos, rifaina), resulting in the sampling of 201 nests from december/2003 to june/2007. the founding nests were brought to the laboratory, opened and the pupae transferred to identified vials until the emergence of the adults, when they were then weighed, sexed and stored at -20 °c. the nesting activity was highly seasonal, since almost all nests were collected in the warm and rainy period of the year. the number of constructed cells ranged from one to nine per nest. the emergence rate of adults in the 716 brood cells was 74%, with mortality homogeneously distributed by egg, larva and pupa stages. this mortality was partly due to parasitism observed in 39% of nests, predominantly by melittobia sp. a 1:1 sex ratio was observed among the newly emerged adults of each area analyzed. strong sexual dimorphism was characterized by linear measurements of wings and body mass, with females and males showing a mass between 27-116 mg and 14-70 mg, respectively. the geometric morphometry confirmed this dimorphism and revealed significant variation of wing size and shape among individuals of the analyzed populations, a result that deserves subsequent studies to point out the factors that account for this differentiation. sociobiology an international journal on social insects l shibata, mm santoni, v oliveira e silva, ma del lama article history edited by marcel hermes, ufla, brazil received 14 august 2020 initial acceptance 20 october 2020 final acceptance 07 december 2020 publication date 28 december 2020 keywords nest architecture, sex ratio, geometric morphometrics, solitary wasps, trapnests. corresponding author marco antonio del lama https://orcid.org/0000-0002-3329-8953 departamento de genética universidade federal de são carlos rodovia washington luis km 235, monjolinho, cep 13565-905 são carlos-sp, brasil. e-mail: dmd@ufscar.br the species of the podium fabricius genus of the sphecidae family are grouped into the rufipes (four species), agile (four species) and fumigatum (twelve species) groups. foraging is preferably done with cockroach nymphs and adults and is one of the few known sphecids that uses resin in nest building (bohart & menke, 1976). podium denticulatum f. smith, 1856, a species of the p. (fumigatum group), occurs in a large area of the neotropical region, from mexico to southern brazil and northeastern argentina, whose females show the nesting behavior in preexisting cavities (bohart & menke, 1976; krombein, 1958; genaro, 1994; morato & campos, 2000; morato, 2001; gazola, 2003). information on the nesting biology of p. denticulatum has already been reported (camillo et al., 1996; assis & camillo, 1997; camillo, 2001; gazola, 2003), as well departamento de genética, universidade federal de são carlos, são carlos-sp, brazil research article wasps nesting biology, sexual dimorphism, and populational morphometric variation in podium denticulatum f. smith, 1856 (hymenoptera: sphecidae) sociobiology 67(4): 572-583 (december, 2020) 573 as aspects of its behavior (ribeiro & garófalo, 2010) and description of its immature stages (buys et al., 2004). data available in the literature also refer to the materials used for nest building, number and size of cells per nest, number of preys in each cell and their identification, cocoon size, adult size, natural enemies and nesting times in nests of podium rufipes fabricius, p. luctuosum f. smith, p. fulvipes cresson and p. angustifrons kohl (rau 1937, bohart & menke, 1976; camillo et al., 1996; assis & camillo, 1997; camilo, 2001; buschini & buss, 2014), as well as immature stages of p. fumigatum perty and p. aureosericeum kohl (buys et al., 2004). in this study, aspects of the nesting biology of p. denticulatum were investigated, seeking to emphasize traits not yet highly explored in previous studies, such as population sex ratio, size and shape sexual dimorphism and morphometric differences between individuals from different populations. for such, analyzes with a large number of nests and individuals from three populations coming from sampling in successive years were performed using a methodological approach that has not yet been explored in this species, the geometric morphometry, to estimate its intra and interpopulation variation. material and methods trap-nests the trap-nests consisted of bamboo canes of varying length and diameter, cut in half horizontally and sealed with masking tape; the closing of one end of the trap-nest was through the very knot of the bamboo. study sites and sampling method the study was conducted in three areas of são paulo state with favorable nesting conditions, such as areas of typical savanna (cerrado) or atlantic forest vegetation and water bodies. the geographic coordinates, altitude, pluviometric index, köpen climate, annual medium temperature and sampling period in the three sampling areas are: i) ufscar campus in araras (22º21’s, 47º23’w; 614 m; 1312 mm; cwa; 20,3ºc), from december/2003 to june/2007; ii) rio branco farm at rifaina (20º04’s, 47º25’w; 575 m; 1531,6 mm; aw; 23 ºc), from july/2004 to june/2007; iii) ufscar campus at são carlos (22º01’s, 47º53’w; 856 m; 1440 mm; cfa; 19,7ºc), from november/2004 to november/2006. the são carlos and araras locations are 64.6 km apart and their distance from rifaina is 220.1 and 253.1 km, respectively. in each sampling area, packages with 8-12 bamboo canes of similar length and diameter were disposed at selected sites 100-300 meters apart each other. in araras, rifaina and são carlos, 90, 60 and 24 packages were made available monthly at nine, six and three sites inside these sampling areas, respectively. samples were taken once a month at each site. the found nests were identified and replaced by new ones and taken to the laboratory (hymenopteran evolutionary genetics laboratory), then opened and some of their characteristics noted, such as: absence or presence of closing wall, vestibular cell, back wall, bottom cell, presence of parasitoids and the position of each cocoon inside the nest. after the annotations were made, the cocoons (spindleshaped, smooth, brittle, bright brown with one end rounded and the other tapering to a dark mass, probably feces) were placed individually in vials closed with cotton and remained there for room temperature until the brood emerged, when the date, sex and body mass of each emerged individual were recorded. then the adults were stored at –20ºc. if the individual was still in the larva phase when the nest was opened, the nest was closed again and only reopened when the nest was in the pupal phase. some p. denticulatum individuals were mounted on entomological pins and sent for identification by specialists (dr. sérvio túlio pires amarante/mzusp and josé carlos serrano/ffclrp-usp) in order to be used for the identification of new individuals. specimens were deposited at the museu de zoologia da universidade de são paulo and at the lgeh. wing linear measurements wings of 190 adults of p. denticulatum (97 females and 93 males) were mounted between microscopic blades. with the aid of a camera attached to a leica stereomicroscope, images were captured with the win tv 2000 system. then, measurements were taken with the leica las version 4.1 software for each of the following traits: right anterior (rawl) and posterior (rpwl) wings length; right anterior (raww) and posterior (rpww) wing width, number of hammuli of the right (hr) and left wings (hl). additionally, their body mass was also taken. each character was measured five times and the average of them was used as a measurement value to reduce errors. wing geometric morphometry images of the right anterior wings of 181 adults (90 females and 90 males, see table 1) were taken for geometric morphometric analysis by a leica s6d digital camera coupled to the leica dfc450 stereomicroscope. for the analysis of partial deformations, the images were converted to format.tps with the aid of the software tpsutil64 version 1.79 (rohlf, 2009). eighteen homologous landmarks (fig 1) were manually plotted twice independently at the junctions of the wing venation using the tpsdig2 software, version 2.16 (rohlf, 2010) (figure 1). this number of landmarks is similar to that of others studies in insects (benítez et al., 2013; perrard & loope, 2015). in the morphoj software version 1.6 (klingenberg, 2011), the images underwent procrustes adjustment, allowing the performance of discriminant function analysis (dfa), principal component analysis (pca) and canonical variables analysis (cva) from the dataset generated. an anova was performed, based on centroid shape and size data, to assess the variation in size and shape in samples from different sexes and populations. all software used here is freely accessible and is available at http://life.bio.sunysb.edu/morph and http:// www.flywings.org.uk/morphoj. l shibata, mm santoni, v oliveira e silva, ma del lama – nesting biology in podium denticulatum574 statistical analysis to perform the statistical analysis, the bioestat 5.0 software (ayres el al., 2007) was used. verification of a 1:1 sex ratio was performed by chi-square test. to evaluate the differences in mass between males and females and the asymmetry between individuals who entered or not in diapause, the kolmogorov-smirnov test and student’s t-test (α = 0.05) were used. wing measurements and masses were associated by pearson linear correlation. statistical analyzes were made in agreement with zar (1999). results number of nests and seasonal abundance in the three areas, 201 nests of p. denticulatum were obtained from december 2003 to june 2007. in araras, 153 nests were sampled at seven of the nine sites, and 79% of the nests were founded in two closer sites. in são carlos, 26 nests were obtained in two of the three sites. in rifaina, 22 nests were sampled at four of the six available sites, and 75% of the nests were founded in two sites only. in this study, we observed an effect of seasonality in the nest productivity (fig 2). nesting activity was concentrated in the hot and rainy period (september april), with very low activity in the cold and dry period (may august), since only one nest with four cells was collected in august 2004 in araras. nest architecture p. denticulatum nests presented linearly constructed cells separated by mud walls, with the inner surface rough and convex and the outer surface smooth and concave. the number of cells observed was from one to nine cells per nest, with an average of 3.7 ± 1.8 cells per nest (araras: 3.7 ±1.8; rifaina: 3.6 ± 1.5; são carlos: 3.7 ± 2.2 cells per nest). the presence of closure plug and deep plug that were observed in some nests is illustrated in fig 3. table 2 shows the number and percentages of these and others architectural elements of nests from each sampling site. the length of the trap-nests used by p. denticulatum ranged from 7.5 cm to 42.3 cm, with an average of 20.4 ± 5.6 cm (19.6 ± 4.9 cm, 26.7 ± 5.7 cm, and 19.3 ± 5.5 cm in the nests of araras, rifaina and são carlos, respectively) (see fig 4a). otherwise, the diameter of the trap-nests used ranged from 4.1 to 17.4 mm, with an average of 10.0 ± 2.7 mm (10.1 ± 2.7mm, 9.3 ± 2.2mm and 9.8 ± 2.4mm in the nests founded in araras, rifaina and são carlos, respectively) (fig 4b). study site city (state) study site code females males araras sp arr 73 65 rifaina sp rfa 10 10 são carlos sp sca 7 19 total 90 91 table 1. number of males and females of podium denticulatum from three study sites analyzed for wing size and shape by geometric morphometry. fig 1. right forewing of a female of podium denticulatum with 18 landmarks plotted at the vein intersections. fig 2. number of nests of podium denticulatum sampled monthly in three locations at são paulo state during 2004 – 2007. study site nn dp dc cp vc arr 153 (76.1%) 43 (28.1%) 19 (12.4%) 57 (37.2%) 68 (44.4%) rfa 22 (10.9%) 4 (18.2%) 3 (13.6%) 11 (50%) 7 (31.8%) sca 26 (12.9%) 1 (3.8%) 10 (38.5%) 6 (23.1%) total 201 47 (23.4%) 23 (11.4%) 78 (38.8%) 81 (40.3%) table 2. number of p. denticulatum nests (nn) from araras (arr), rifaina (rfa) and são carlos (sca), and number (%) of nests with deep plug (dp), deep cell (dc), closure plug (cp) and vestibular cell (vc). sociobiology 67(4): 572-583 (december, 2020) 575 in nine of the p. denticulatum trap-nest samples, a second species also built in the same cavity. (trypoxylon aurifrons shuckard, trypoxylon lactitarse saussure, trypoxylon rogenhoferi kohl, or centris sp.). in five nests, the first species to nest was p. denticulatum, followed by nesting by trypoxylon sp., whereas in the other four nests, the first species to nest was trypoxylon sp. (n = 3) or centris sp. (n = 1), followed by nesting by p. denticulatum (fig 5). mortality in the 201 nests sampled, 716 brood cells were observed, from which 531 adults (74%) emerged. the mortality of the offspring in the other cells was evenly distributed among the egg, larval or pupal stages (table 3). parasitoids were found in 39.8% of nests (n = 80), and in 97.5% (n = 78) of these nests was found the parasitoid melittobia sp. (eulophidae). in 2.5% (n = 2) of the nests, pupae of tachinidae (diptera) were found in the last nest cell. interval between collection date and emergency date the time elapsed between the date of nest collection and the emergence of individuals ranged from 1-41 days (mean = 17 ± 8.2), with slight differences among nests of different locations (araras: 18.0 ± 8.1 days; são carlos: 15.1 ± 7.7 days; rifaina: 13.0 ± 7.5 days). most of the individuals who emerged between 46 and 366 days after the date of collection (mean = 129.0 ± 73.5) entered diapause. in araras, 21.8% of those collected in 1 2 fig 3. p. denticulatum nest. closure plug (1) and deep plug (2) are shown. fig 4. lenght (a) and diameter (b) of the trap-nests used by the females of p. denticulatum in the three sampled areas. fig 5. nest foundation by centris sp., followed by nesting of p. denticulatum in a trap-nest. l shibata, mm santoni, v oliveira e silva, ma del lama – nesting biology in podium denticulatum576 january and 93.7% of them collected in april emerged from 60 to 366 days after the date of collection (mean = 151.0 ± 69.8). in são carlos, 68.4% of individuals collected in january and all collected in april entered diapause, emerging 46 to 129 days after collection (mean = 58.0 ± 22.3 days). in the nests where diapause was observed, all individuals of the nests entered diapause, except in a nest collected in araras in april 2005. in this nest, four cells had been built in the first two, the individuals emerged 7 days after collection; in the next two cells, brood entered diapause and emerged 166 days after collection. the emergence of individuals which entered diapause occurred throughout the year, even in the cold and dry season. sex ratio and nest position in the three studied localities, a 1:1 sex ratio was verified (araras – 155 females and 153 males; χ2 = 0.013, p>0.05; são carlos – 23 females and 22 males; χ2 = 0.022, p> 0.05; rifaina – 14 females and 26 males; χ2 = 3.6, p>0.05). the position of the sexes in p. denticulatum nests was not random, with the first cells (cells further away the nest entrance) presenting more females than males (fig 6); so, males usually emerged before females. body mass a large variation in body mass of females (x ± sd = 80.1 ± 20.1; amplitude: 27-116 mg; n = 157) and males (x ± sd = 35.46 ± 11.6; amplitude: 14-69 mg; n = 137) was observed (fig 7). when compared, these values were highly significant (student´s t = 19.63, p = 0.0001). females that entered (n = 30) and not entered in diapause (n = 129) presented body mass between 32 116mg (x ± sd = 67.6 ± 23.2) and 32 115mg (x ± sd = 74, 4 ± 19.9), respectively. males that entered (n = 28) and not entered in diapause (n = 112) had body mass between 17 69mg (x ± sd = 36.1 ± 12.7) and 9 70mg (x ± sd = 32, 9 ± 14.5), respectively. the results observed in females and males of the two groups are not significantly different (t = 1.617, p = 0.1084; t = 1.0683, p = 0.287, respectively). table 3. number of nests (nn) and cells (cn), number and percentage of emerged adults before collection (bc) or at the laboratory (lab). number of died individuals in egg, larva or pupa stage of p. denticulatum nests sampled in three study sites (arr: araras-sp, rfa: rifaina-sp, sca: são carlos-sp). sites nn cn emerged adults egg larva pupa bc lab arr 153 560 45 366 45 51 53 (8.0%) (65.4%) (8.0%) (9.1%) (9.5%) rfa 22 75 11 51 5 0 8 (14.7%) (68.0%) (6.7%)   (10.7%) sca 26 81 5 53 8 1 14 (6.2%) (65.4%) (9.9%) (1.2%) (17.3%) total 201 716 61 470 58 52 75 (8.5%) (65.6%) (8.1%) (7.3%) (10.5%) fig 6. emergence (%) of females and males in relation to the position of the cell. in some of the analyzed nests, one or more individuals died before emergency. wing linear measurements wings of 190 individuals (97 females, 93 males) were measured for the following traits: right anterior (rawl) and posterior (rpwl) wings length; right anterior (raww) and posterior (rpww) wing width, number of hammuli of the right (hr) and left wings (hl), and body mass. the range of variation, mean and standard deviation, and coefficient of variation for each of these characters in males and females are shown in table 4. it is interesting to note that the coefficient of variation for rawl, rpwl, and raww were higher in males than in females. fig 7. violin and box-plots were used to visualize the body mass (mg) distribution of newly emerged females (orange) and males (black) of p. denticulatum. sociobiology 67(4): 572-583 (december, 2020) 577 a discriminant analysis was performed on basis of 140 individuals (67 males, 73 females) from araras, for which we had the values for all the variables measured. the analysis showed a clear differentiation between males and females of p. denticulatum (fig 8) based on the traits analyzed, largely due to the association of body mass values to the wing linear measurements. the asymmetry estimated by the difference between the width of the right and left hind wings was not statistically different in diapausing and not diapausing individuals (student´s t = 0.397, p = 0.6922) (table 5). lwm (mm) females males (n = 97) (n = 93) range x ± sd cv (%) range x ± sd cv(%) rawl 7.98-13.20 11.6 ±0.9 7.7 7.24-12.20 9.89 ±1.00 10.0 rpwl 5.9-9.9 8.8 ±0.7 7.9 5.3-9.2 7.5 ±0.7 10.0 raww 2.15-3.40 3.01 ±0.24 7.9 1.7-3.1 2.5 ±0.3 11.0 rpww 1.3-2.8 2.25 ±0.27 16 1.2-2.4 1.9 ±0.3 13 hr 21-35 28.2 ±2.7 9 17-29 23.1 ±2.5 10 hl 21-35 28.3±3.1 14 16-34 23.5±2.9 12 table 4. wing linear measurements (lwm) in adult females and males of p. denticulatum. range, average (x), standard deviation (sd) and coefficient of variation (cv) for seven wing features: right anterior wing length (rawl), right posterior wing lenght (rpwl), right anterior wing width (raww), right posterior wing width (rpww), number of hammuli from rigth (hr) and left wings (hl). table 5. wing comparison (difference in mm between the right and left wing width) from females and males adults of p. denticulatum that entered (e) or not entered (n) diapause during the development. development females males range x ± dp range x ± dp e 0.000-0.094 0.0214±0.0198 0.000-0.056 0.0228±0.0175 d 0.002-0.074 0.0215±0.0202 0.000-0.075 0.0197±0.0204 body mass (mg) was highly correlated with rawl (r = 0.86), rpwl (r = 0.88), raww (0.87), and rpww (r = 0.73), indicating that mass is a good size estimator; it also contributed significantly to highlight the sexual dimorphism showed by the wing linear measurements (fig 8). a significant correlation was found between number of hammuli and rpwl in males (r = 0, 32; p = 0.013), but these two variables were much more correlated in females (r = 0.85; p = 0.0001). geometric morphometry (gm) of wing size and shape the males and females (n = 181) described in table 1 were also analyzed by geometric morphometry to detect possible differences in the size and shape of the right anterior wing of males and females. the pca generated 32 components responsible for the total variation of the data, and the first 10 components explained 86.16% of the variation (pc1 27.02%; fig 8. discriminant analysis based on four wing linear measurements (see table 4) and body mass of males (grey) and females (orange) of podium denticulatum. fig 9. canonical variate analysis (cva) showing morphometric dimorphism between males (grey) and females (orange) of p. denticulatum. l shibata, mm santoni, v oliveira e silva, ma del lama – nesting biology in podium denticulatum578 pc2 18.80%). the dfa showed that 95.18% of individuals were correctly assigned to their sites of origin, while the percentage in the cross-validation test was 83.51%. the accuracy in the correct attribution of the sexes was 97.78%, with an average of 93.37% in the cross-validation. the gm analysis showed a marked dimorphism between females and males (fig 9), and wing shape differences between males and females of the three areas were detected by cva (fig 10). differential interpopulational differentiation was detected through the mahalanobis distances of wing size among females, males and females+males of p. denticulatum from the three study sites (araras, rifaina, and são carlos) (table 6). the procrustes anova values for centroid size (cs) and shape (sh) in p. denticulatum females of the three study sites are presented in table 7, corroborating the morphometric variation among the three populations of this species. fig 10. canonical variate analysis (cva) for morphometric variation of the wing shape based on the joint analysis of males and females (a), only females (b) and only males (c). the samples of são carlos, rifaina, and araras are indicated by the colors orange, grey and black, respectively.     arr rfa females rfa 4.9545 sca 4.1055 5.6414 males rfa 6.7093 sca 2.9807 8.6982 females and males rfa 3.5097 sca 2.5613 4.2208 table 6. mahalanobis distances of wing size between females and males of p. denticulatum from the three study sites (arr: araras-sp, rfa: rifaina-sp, sca: são carlos-sp). tabela 7. procrustes anova for wing centroid size (cs) and shape (sh) in p. denticulatum females of the three study sites. sum of squares (ss) and mean squares (ms) are in dimensionless units of procrustes distances.  effect  ss ms df f p cs individual 6018364.469201 3009182.234601 2 7.10 0.0014 residual 36898857.494009 424124.798782 87 sh individual 0.00248785 0.0000388726 64 3.18 <0001 residual 0.03399638 0.0000122113 2784 discussion nesting activity by p. denticulatum in trap-nests has been previously reported by camillo et al. (1996), assis and camillo (1997) as well as ribeiro and garófalo (2010). camillo (2001) observed p. denticulatum reusing inactive nests of brachymenes dyscherus (saussure). it is noteworthy that the trap-nests we offered in the selected areas were also used by species of wasps of the family crabronidae (trypoxylon aurifrons, t. nitidum f. smith, t. lactitarse and t. rogenhoferi) and solitary bees of the genus centris sp. a seasonal nesting activity in p. denticulatum was found in those previous studies (camillo et al., 1996; ribeiro & garófalo, 2010). this seasonal pattern has been reported in other wasps, such as trypoxylon aurifrons (santoni & del lama, 2007), but not in bees as tetrapedia curvitarsis friese (campos et al., 2018). low nesting activity in the cold and dry period has also been previously reported by camillo et al. (1996) and camillo (2001). the greater nesting activity in araras suggests the existence of a well-structured population in this area, although the shorter sampling time in são carlos and rifaina may have contributed to the lower foundation rate in these locations. according to ribeiro and garófalo (2010), the nesting activities of a female of p. denticulatum are initiated by the search for a suitable nest building site, a determining factor for the success of nest foundation in p. rufipes (krombein, 1970). variation in nesting rates at different sites in the collection areas was found, a fact usually seen in many similar reports. santoni and del lama (2007) observed variation in trypoxylon aurifrons nesting rates in the same collection areas of our study, what was explained by the authors by factors as philopatric behavior, preference and/or competition for nesting sites, different periods of sampling or simply the availability of trap-nests. sociobiology 67(4): 572-583 (december, 2020) 579 the observed architecture of p. denticulatum nests, with linearly constructed cells separated by mud walls, was very similar to that found by camillo et al. (1996), assis and camillo (1997) as well as ribeiro and garófalo (2010). otherwise, krombein (1967, 1970) and gazola (2003) described that nests of p. rufipes are made up of a single cell that occupies almost the entire trap-nest cavity. the average number of cells per nest was higher in our study than in previous reports (camillo et al., 1996; assis & camillo, 1997; ribeiro & garófalo, 2010). nests with more than 6 cells were not observed in those previous studies, which may be explained by the trap-nests with longer length we offered. according to camillo et al. (1996), the closure and compartmentalization processes of p. denticulatum nests are very similar to p. rufipes (krombein, 1970) and penepodium goryanum (lepeletier) (garcia & adis, 1993) nests. these similarities include the use of mud and resin in partitions, the presence or absence of double walls and the insertion of pieces of leaves, lichens and wood fragments into the nest camouflage. these walls with physical insulation and sometimes camouflage functions are externally coated with resin. the habit of building a deep plug, this “initial mud deposit” or “preliminary wall” at the beginning of nesting was also observed in trypoxylon species (santoni, 2008). based on studies in t. rogenhoferi, garcia and adis (1995) suggest that this behavior is linked and restricted to species whose larvae use mud to make puparia. as p. denticulatum does not have this behavior, the frequency of construction of these walls is low. relative to the diameters and lengths of trap-nests used by the females, it is possible to verify their preference to trapnests with medium diameters and lengths, a result already reported by camillo et al. (1996), assis and camillo (1997) as well as ribeiro and garófalo (2010). however, nesting by p. denticulatum females in trap-nests over 25 cm in length is being described for the first time. the diameter of nesting cavities is selected by wasps as a function of species size (krombein, 1967), prey size (fricke, 1991; garcia & adis, 1995) and the effectiveness of internal wall thickness against parasitoids (coville & coville, 1980). trap-nests containing brood cells constructed by p. denticulatum and a female of a second species have already been described. camillo et al. (1996) reported this type of nesting involving females of centris sp., megachile sp. and trypoxylon sp.. however, this is the first description of nests founded by p. denticulatum that was also used by a wasp or bee female. the mortality rate estimated here is very similar to that observed in nests of the species analyzed by assis and camillo (1996). parasitoids such as melittobia sp., antrax sp., perilampidae, phoridae and tachinidae were observed by camillo et al. (1996). assis and camillo (1997) described the parasitoids melittobia sp. and antrax sp. in p. denticulatum nests, while ribeiro and garófalo (2010) observed the parasitoids melittobia sp., tachinidae and chrysididae in nests of this species. pre-pupae entered diapause were also observed by camillo et al. (1996) also ribeiro and garófalo (2010). diapause occurrence in the pre-pupal stage is a strategy used by many wasp and bee species in response to adverse weather conditions, which, in turn, also affect the availability of food resources used by these organisms, especially by decreasing their supply, a fact that can be explained by the transition from the hot and rainy period (september to april) to a cold and dry period (may to august). the time between nest collection and adult emergence was similar to that observed by camillo et al. (1996), except for individuals who entered diapause, whose emergence occurred over a longer period of time. it is possible that temperature variation inside the laboratory may have interfered with the brood developmental time and occurrence of diapause. camillo et al. (1996) proposed the occurrence of two generations per year in p. denticulatum, while ribeiro and garófalo (2010) suggested the occurrence of five/six generations per year in the species. data from our work, such as i) nests where individuals emerged prior to collection, ii) a nest collected in the cold and dry season, and iii) individuals that emerged during the cold and dry season indicate that more than six generations may occur, with one generation overlapping the other and generations where some individuals go through prolonged diapause. observing some nesting in the cold and dry period, when some nests went through the diapause phase, camillo et al. (1996) concluded that most of the population of p. denticulatum survives the adverse period of the year as adults. however, the low amount of nesting observed during this period indicates that most of the population survives the adverse period of the year in pre-pupal diapause, a condition that penepodium goryanum pupae use to cope with periods of adverse conditions (garcia & adis, 1993). a 1:1 sex ratio was verified in our three samples studied, confirming previous data in p. denticulatum (ribeiro & garófalo, 2010) and podium rufipes (krombein, 1967). otherwise, camillo et al. (1996) observed in p. denticulatum the ratio of 1.7 males: 1 female in one generation, but they warn that there are numerous factors related to sex ratio that have not been analyzed. in most panmitic populations, equal investment in both sexes is expected if fitness return is the same with a production of a daughter or a son (fisher, 1930). however, in many hymenopterans there are deviations of this proportion, often attributed to ecological, physiological and behavioral factors (trivers & hare, 1976). although in most nests male and female emerged, from a few nests only male or female emerged. the emergence of males only can be explained for different reasons, including: i) females are not fertilized; ii) option for the least expensive sex; iii) because they are of advanced age (tepedino & torchio, 1982; frohlich & tepedino, 1986). l shibata, mm santoni, v oliveira e silva, ma del lama – nesting biology in podium denticulatum580 the observed non-random pattern of sex emergence in trap-nests of p. denticulatum was already reported by camillo et al. (1996) also ribeiro and garófalo (2010) and in other solitary wasp species. in t. aurifrons, for example, from the first cells emerged more males than females (santoni & del lama, 2007), a pattern opposite to that observed here. the size sexual dimorphism showed here in p. denticulatum, with females larger than males, has been previously reported (camillo et al., 1996; ribeiro & garófalo, 2010) based on head width measurements, a trait especially useful for revealing sexual dimorphism in sphecidae (bohart & menke, 1976). the developmental interruption promoted by diapause did not result in significant changes in wing size (width) and body mass of males and females, an unexpected result if we consider diapause as due to some disturbance in the environment experienced by individuals. body size is one of the key features of organisms. ecological traits such as survival, resource acquisition rate, and reproductive capability usually are size-dependent (takahashi & blanckenhorn, 2015). differences in body size between males and females, so called sexual size dimorphism (ssd), are common in many taxa (fairbairn, 2005; stillwell et al., 2010). in insects, females are larger than males in >70% of the taxa in all major insect orders, except odonata (stillwell et al., 2010). ssd has been attributed to growth rate and developmental time in arthropods (blanckenhorn et al., 2007); however, our understanding of the causal proximate and ultimate factors of body size variation is still poor due to the low number of studies about the genetic and developmental mechanisms underlying ssd (takahashi & blanckenhorn, 2015). we found higher coefficient of variation for three wing linear measurements (rawl, rpwl, and raww) in males than in females. it is expected that the levels of genetic variance underlying body size traits would be larger in males if these traits are under lower intensity of sexual selection over them. evidences of a stronger sexual selection for female than for male size have been shown in many species due to a positive correlation between female body size and fecundity (molumby, 1997; strohm & linsenmair, 2000; peruquetti & del lama, 2003). the marked sexual dimorphism revealed by traditional (fig 8) and geometric morphometry (fig 9) demonstrates the high resolution of these methodologies to detect subtle differences in male and female morphologies, although gm can show higher resolution when compared to traditional morphometry (quezada-euán et al., 2015). on the other hand, when the population analysis was performed with the males and females (fig 10a), a better differentiation between populations is reached when males and females are analyzed separately, as shown in fig 10b and 10c, respectively. as wing morphology analysis of males and females clearly show that there are definite small-scale differences, these subtle shape differences between sexes plays a role when using wing morphology to distinguish between families (colonies), populations or species. so, a separate analysis of each gender rather just one analysis may be critical at lower taxonomic levels; at the higher levels, differences between sexes may not greatly affect results when males and females are considered together (see pretorius, 2005). based on wing shape and size analysis, the three populations of p. denticulatum showed morphometric variability. mahalanobis distances (table 6) and cva analyses (fig 10a, 10b and 10c) revealed that the rifaina population is differentiated from the populations of são carlos and araras, an expected result when we consider that rifaina is geographically more distant from araras and são carlos, and presents phytophysiognomy and other abiotic factors distinct from those verified in the two other localities. wing size accessed by the centroid size showed significant higher variation among populations than wing shape (table 7). this is an expected finding since size variation in insects are related to quantity and quality of the resources provided to the brood in the larval stage (peruquetti, 2003; campos et al., 2018). so, the higher wing size variation may reflect the differential offer of food resources at each environment, as the supply dynamics certainly vary temporally in different phytophysiognomies (campos et al., 2018). alternatively, this variation may be associated rather to differential responses of these populations to the features of their local environments (grassi-sella et al., 2018), or to local variations in landscape elements (ribeiro et al., 2019), and specially, to higher developmental constraints to shape than to size variation. the morphometric variation found may also be at least partially the result of genetic interpopulational variation. further studies are needed to investigate the significance of these findings, as well as to estimate the contribution of genetic and/or environmental factors to this variation, a theme little explored in bees and, in particular, wasps. concluding remarks the data obtained in this work generally confirm previous data on nesting biology in p. denticulatum. however, new information was obtained: i) nesting by females in longer trap-nests, which may have resulted in the larger number of cells per nest observed here; ii) longer periods of diapause; iii) the first description of nests founded by a p. denticulatum female that was also used by a second species (bee or wasp); iv) the possible occurrence of more than six generations per year. the sex ratio 1:1 in the three populations analyzed constitutes data that corroborates previous information obtained in populations from other areas. the demonstration of a marked sexual dimorphism by traditional and geometric morphometry (gm) confirms the effectiveness of these methodologies for detecting wing morphology variation. in addition, gm proved to be effective in demonstrating wing size and shape differences between individuals from geographically close populations, especially when such analysis is done separately from males and females from sociobiology 67(4): 572-583 (december, 2020) 581 different populations. this result deserves further studies to point out the factors that account for the observed variation, contributing to evaluate its possible functional significance, the central task of evolutionary biology. acknowledgments thanks are due to adhemar rodrigues alves, owner of rio branco farm at rifaina/ sp, and to dr. sérvio túlio pires amarante and josé carlos serrano by the species identification. l shibata thanks a fellowship from pibicufscar/cnpq, mm santoni thanks fellowships from fapesp (2004/02389-3; 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(1999). biostatistical analysis, 4th. ed. london: prentice hall, 663 p open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.2879sociobiology 66(2): 263-273 (june, 2019) temporal variation in production and nutritional value of pollen used in the diet of apis mellifera l. in a seasonal semideciduous forest introduction the quantity and quality of pollen collected by bees are closely related to the type of vegetation and availability of floral resources. bee pollen is the agglutination of flower pollen that receives small amounts of nectar and other salivary substances from apis mellifera (villanueva et al., 2002). this process facilitates the attachment of pollen to the corbicula of foraging bees and transportation to the colony. bee pollen contains more than 200 substances (komosinska-vassev et al., 2015), consisting mainly of proteins, amino acids, lipids, fibers, enzymes, minerals, sugars and vitamins (arruda et al., 2013; avni et al., 2014; bogdanov, 2015; sattle et al., 2015). this composition makes pollen essential for feeding brood as well as for maintenance of the colony of a. mellifera (marchini et al., 2006). abstract the flora of mountain formations in the caatinga biome is composed predominantly by semi-deciduous species with representatives of both atlantic and amazon forest. information on the potential for bee pollen production of these species is limited. in this study we evaluated the potential of production, the temporal variation, the botanical origin and the nutritional value of bee pollen produced in a seasonal semideciduous forest in northeastern brazil. we identified a total of 252 flowering plant species throughout the year. the diet of apis mellifera consisted of 74 pollen types distributed in 58 genera and 27 families. we identified two production peaks of bee pollen, the highest occurring in the rainy season. nutritional value considering crude protein, carbohydrates, lipids and mineral matter changed over the study period, with influence of rainfall on the dry matter level. some taxonomic groups of plants showed a strong relationship with nutrients, suggesting that although the diet of a. mellifera is broadly diversified, this species devoted most of its pollen foraging effort on the genus mimosa and the palm tree species of attalea speciosa. the results show that the seasonal semideciduous forest of the mountain range in the northeast brazil presents plant species: mimosa caesalpiniifolia, baccharis trinervis, mimosa tenuiflora myracrodruom urundeuva, cecropia pachystachya, attalea speciosa, with high nutritional level and potential for the pollen production. sociobiology an international journal on social insects jem nascimento¹, bm freitas¹, a js pacheco filho¹, es pereira¹, hm meneses¹, je alves², ci silva¹,³ article history edited by evandro n. silva, uefs, brazil received 05 february 2018 initial acceptance 13 february 2018 final acceptance 25 december 2018 publication date 20 august 2019 keywords bee flora, bee pollen, bee nutrition, beekeeping, pollen production. corresponding author josé elton de melo nascimento universidade federal do ceará departamento de zootecnia cca setor de abelhas, bloco 814 campus universitário do pici cep: 60.356-000, fortaleza-ce, brasil. e-mail: eltonzootec@gmail.com bee pollen is also an important source of income for beekeepers in different countries. worldwide, pollen production is approximately 1,500 tons/year, with spain and china as the two major producers (estevinho et al., 2012; yang et al., 2013). however, global production of bee pollen is concentrated on temperate countries and regions and little is known about the potential production in tropical and subtropical areas of the world (estevinho et al., 2012). in these warmer regions, beekeeping has faced consistent growth in recent years, but focusing on honey production. in brazil, for instance, much of the beekeeping production is based on semi-arid parts of the country (barreto et al., 2006), where the physiognomy varies from sparsely vegetated desert to areas with dry forests covered by dense tree layers (araujo-filho, 2013). nevertheless, pollen production 1 departamento de zootecnia, universidade federal do ceará (ufc), fortaleza-ce, brazil 2 centro de ciências agrarias e biológicas, universidade estadual vale do acaraú, sobral-ce, brazil 3 departamento de ecologia, universidade de são paulo (usp), são paulo-sp, brazil research article bees jem nascimento et al. – temporal variation of pollen used in the diet of apis mellifera l.264 in the region is limited especially by the lack of knowledge about the identity of polliniferous plants with potential to sustain beekeeping activity throughout the year (milfont et al., 2011). the present study investigates the potential of bee pollen production in a seasonal semideciduous forest of the semi-arid area of northeastern brazil and evaluates the temporal variation in the production of pollen used in the diet of a. mellifera and its nutritional value. material and methods study area samplings were carried out from november 2012 to october 2013 in a seasonal semideciduous forest, located in an environmental protection area in the municipality of meruoca (3°35’40.63” s and 40°24’11.91” w), state of ceará, brazil (fig 1a). the climate of the region is aw’, characterized as hot, humid and rainy in the summer (köppen, 1948). the average annual rainfall is 1,530.3 mm (fig 1b). the rainy season is concentrated between january and april, extending to june and with annual average rainfall of 1,194.3 mm (carvalho, 2013). in this type of geomorphological formation, a predominance occurs of deep soils, moderately drained with good fertility. the rainwater accumulated in the soil (eutrophic red-yellow argisols) favors the establishment of arboreal species. the original prevailing vegetation is pluvial-nebular subperennifolia (humid forests, serranas), but nowadays, besides the natural areas, there are also subsistence crops and a poorly developed beekeeping (funceme, 2015). floristic composition and bee plants in order to survey the floristic composition in the study area we set up a radius with approximately 1,000 meters (krebs, 1999), having the apiary as the central point. then, we examined the study area on a monthly basis and identified the flowering plant species, considering the entire vertical stratification (except poaceae species) (silva et al., 2012). we took three samples of each flowering plant species for the preparation of vouchers and subsequent identification of the species by experts. vouchers are deposited in the herbarium professor francisco josé de abreu matos-huva, state university vale do acaraú (uva), ceará brazil. preparation of reference pollen collection we collected the anthers of all species of flowering plants and maintained in 70% ethanol for 24 hours (silva et al., 2014). the material was then ground, subjected to the acetolysis procedure (erdtman, 1960) and maintained in 50% glycerin. for each plant species, we mounted three slides using kisser gelatin and sealed with paraffin (silva et al., 2014). slides were properly identified according to silva et al. (2010) and deposited in the pollen collection of the bee laboratory, department of animal sciences, federal university of ceará (ufc). pollen grains were photographed with a digital camera attached to a trinocular microscope. based on the images, we made measurements of pollen grains for morphological descriptions. subsequently, we organized a pollen catalogue with 252 species that was used to identify, by comparison, the pollen collected by a. mellifera foragers. pollen collection from apis mellifera colonies pollen samples were obtained installing pollen collectors in the entrances of ten langstroth hives, standardized to the number of bees (28 thousand ± 2 thousand), number of combs (n=10) and queen age (one year). these pollen collectors require bees to enter the hive through small holes that scrape the pollen from the bee corbiculae. we sampled pollen every other day, always at 5:30 pm, totaling 15 monthly collections for each colony. fresh pollen samples were cleaned by removing the debris attached to the pollen (dead bees, bee larvae, propolis) and weighed to the nearest 0.1 g and then cooled in a freezer at less than 20 °c. pollen analysis pollen of the 15 samples taken every month from each hive was pooled, forming a single monthly composite sample per hive. each composite sample was divided into two parts, one for chemical analysis and another for botanical origin fig 1. a) the study area (white circle 3 ° 35'40.63 "s, 40 ° 24'11.91" w) is located within the environmental protection area of the serra da meruoca (bounded by the white line). (b) rainfall in the municipality of meruoca during the study period (funceme cearense foundation of meteorology and water resources 2012-2013) sociobiology 66(2): 263-273 (june, 2019) 265 identification. thus, for the same composite monthly sample, we obtained information about the visited plants and the nutritional value of the diet of a. mellifera. chemical analysis chemical analyses were carried out in the laboratory of animal nutrition (lana-dzo-ufc) in department of animal sciences at the federal university of ceará. the bee pollen collected was kept in falcon tubes of 15 ml and frozen until the time of chemical analysis. pollen samples were thawed, dried in a forced air oven at 55°c for 72 hours, and ground to pass through a 1 mm screen (wiley mill, arthur h. thomas, philadelphia, pa, usa). all samples were analyzed for dry matter (dm; aoac 1990; method number 930.15), mineral matter (mm; aoac 1990; method number 924.05), crude protein (cp; aoac 1990; method number 984.13), lipids (fat; aoac 1990; method number 920.39). the total carbohydrate content (tc) was calculated by the formula tc (%) = 100 %cp %fat % mm), according to sniffen et al. (1992). identification of the botanical origin of pollen pollen samples, about two grams, were kept in 4 ml of 70% alcohol, in falcon tubes with capacity for 15 ml, for 24 hours, then they were centrifugated and the supernatant was discarded. after the alcohol was discarded, the material was kept for 24 hours in 4 ml glacial acetic acid (silva et al., 2014) and acetolyzed, for each sample, 5ml of the acetolysis mixture was used, nine parts of acetic anhydride for onepart sulfuric acid (9:1) following the method described by erdtman (1960). after acetolysis, the material was kept in 50% glycerin. we mounted three slides for each sample, using kisser gelatin and transparent lacquer for reading fast. in the qualitative analysis, the pollen types found on the slides were identified by comparison with the pollen types of the reference slides of the plants that flourished in the area during the study period. we also used specific literature for the identification of pollen collected by bees (silva et al., 2010; bauermann et al., 2013; silva et al., 2014). in the quantitative analysis, we identified and counted the first 400 pollen grains found on each slide (montero & tormo, 1990). then, we calculated the relative frequency (percent) of each pollen type and classified the material in accordance to occurrence, as proposed by barth (1970) and louveaux et al. (1970,1978): predominant pollen (dp, > 45% of the total pollen grains present on the slide), accessory pollen (ap, from 15 to 45%), important isolated pollen (iip, 3 to 15%) and occasional isolated pollen (oip, < 3%). we also measured the total volume of pollen grains of each plant species in each sample used by a. mellifera based on the mean length of the longitudinal axis measured on 25 pollen grains. based on these values, the mean volume pollen grain for each plant species was calculated according to the method proposed by villanueva-g and roubik (2004). total pollen volume expresses the pollen dominance in the diet of a. mellifera. data analysis generalized linear models (glm) were used to evaluate how the independent variables ‘number of pollen types’ and ‘precipitation (mm)’ influence the following response variables: (a) pollen production (g/month), (b) crude protein, (c) total carbohydrates, (d) lipids, (e) mineral matter and (f) dry matter. a model for each response variable was elaborated as follows: response variable = ‘number of pollen types’ + ‘precipitation (mm)’. as the response variables are continuous and normal, we used a glm with gaussian distribution and identity binding function. we performed a posteriori diagnostic tests to verify if the models were adequate (such as the analysis of the normality of residuals and verification of the influence of atypical values). a spearman correlation (rs) was performed to check if the dominance of some botanical groups (families or genera) is related to the chemical variables (crude protein, total carbohydrates, lipids, mineral matter and dry matter). dominance was estimated using the total number and volume of pollen grains of each plant species used by a. mellifera. a cluster analysis was run to classify pollen samples in accordance with colonies of a. mellifera, by using the bray-curtis similarity index and the paired group algorithm. the consistency of grouping pattern was tested by means of cophenetic correlation, in which values close to unity indicate good representativeness. the analyses were run using r 2.13.1 (r development core team, 2014). for the grouping and similarity analyses, the ‘vegan’ package was used (oksanen, 2013). for the other analyses the native functions of the r software were used. results during the study period the diet of a. mellifera consisted of 74 pollen types distributed in 58 genera and 27 families. families with the highest number of species were leguminosae (n = 16), asteraceae (11) and rubiaceae (6) (table 1). the genus with the highest number of species (n = 7) in bloom during the study period was mimosa (leguminosae). regarding the proportion of pollen types in the samples during the rainy season (table 1), the best represented species in january were mimosa tenuiflora (34.88% = ap) and psidium cattleianum (22.23% = ap); in february, march and april, mimosa caesalpiniifolia was dominant (47.38%, 74.75% and 72.88% = dp); wedelia calycina was relevant only in march (16.25% = ap); in may, pollen of leucaena leucocephala and mimosa niomarlei was accessory; in june, pollen of baccharis trinervis was accessory, but very close to dominant (44.93 = ap). in july, which is the transition period between the rainy and dry seasons, foraging bees collected pollen mostly on mimosa tenuiflora (78.90 = dp). in the dry season, the number of pollen types was lower compared to the rainy season (table 1). in august, attalea speciosa (46.38% = dp), borreira spinosa (23.88% = ap) and m. tenuiflora (17.60 = ap) had the highest proportions in samples. jem nascimento et al. – temporal variation of pollen used in the diet of apis mellifera l.266 table 1. temporal variation in the diet of apis mellifera in a seasonal semideciduous forest in the state of ceará, in the period from november 2012 to october 2013. months highlighted in gray correspond to the rainy season and months highlighted in white represent the dry season. the values in each month correspond to the percentage of each plant species of the whole pollen collected during each month. family species/pollinic types nov dec jan feb mar apr may jun jul aug sep oct acanthaceae ruellia asperula lindau. 0.05 0.10 amaranthaceae alternanthera brasiliana (l.) o. kuntze 0.08 alternanthera tenella colla 0.15 0.20 1.45 0.05 0.73 anacardiaceae anacardium occidentale l. 9.13 2.70 0.08 0.05 0.18 0.95 myracrodrum urundeuva allemão 0.20 1.35 95.33 13.00 arecaceae attalea speciosa (mart.) barb. rodr. 53.80 64.85 7.78 4.18 0.85 0.45 0.08 6.23 2.83 46.38 2.88 20.03 asteraceae acanthospermum hispidum dc. 0.03 baccharis trinervis pers. 2.88 5.05 0.38 6.90 44.93 0.70 0.18 0.40 bidens subalternans dc. 0.08 emilia sonchifolia (l.) dc. ex wight 0.23 0.20 0.03 melanthera latifolia (gardner) cabrera 0.65 4.65 0.15 pithecoseris pacourinoides mart. 5.30 0.55 0.10 stilpnopappus tomentosus mart. ex dc. 0.03 0.03 trichogonia salviifolia gardner 0.10 0.05 tridax procumbens l. 0.10 2.00 4.60 0.13 2.60 vernonanthura brasiliana (l.) h. rob. 0.08 0.08 1.03 0.28 0.05 wedelia calycina rich. 4.15 9.35 8.60 16.25 11.40 3.60 4.68 0.05 boraginaceae cordia trichotoma (vell.) arrab 0.70 0.93 0.15 0.68 1.28 1.15 commelinaceae aneilema brasiliense c.b. clarke 0.05 0.53 0.05 commelina benghalensis l. 0.15 3.50 0.80 3.55 0.65 commelina diffusa burmf. 0.15 0.05 0.33 0.03 convolvulaceae ipomoea piurensis o´donell 0.03 merremia macrocalyx o’donell 0.03 0.45 convolvulaceae sp1 0.03 0.03 turbina cordata choisy 0.03 euphorbiaceae croton floribundus spreng 0.25 0.03 croton jacobinensis baill 0.30 0.08 croton microcalyx mull. arg. 0.10 0.08 lamiaceae hyptis pectinata (l.) poit. 0.03 1.78 hyptis suaveolens (l.) poit 0.30 6.70 4.05 ocimum gratissimum l. 0.03 0.10 0.18 0.40 0.38 0.48 0.10 leguminosae anadenanthera columbrina (vell) brenan 0.15 2.83 0.50 0.33 0.25 bauhinia cheilantha (bong) steud. 0.03 0.05 0.35 0.05 bauhinia ungulata l. 0.05 delonix regia (bojer ex hook.) raf. 0.53 0.55 0.30 0.10 0.25 dioclea grandiflora mart ex benth 0.08 sociobiology 66(2): 263-273 (june, 2019) 267 indigofera suffruticosa mill. 0.30 leguminosae leucaena leucocephala wit. 37.78 2.78 0.05 mimosa caesalpiniifolia benth 1.55 47.38 74.75 72.88 3.05 0.08 mimosa candolei r. grether 0.33 0.45 mimosa invisa mart 0.18 2.35 2.68 mimosa niomarlei afr. fer. 0.40 28.00 1.85 mimosa sensitiva l. 0.08 0.55 0.95 mimosa setosa benth 0.23 1.50 0.28 0.58 mimosa tenuiflora (willd) poir. 4.18 5.30 34.88 0.28 0.80 4.65 2.20 78.90 17.60 6.50 piptadenia stipulacea (benth) ducke 0.13 senna splendida (vogel) h.s.i 0.38 loranthaceae struthanthus syringifolius (mart.) mart. 5.33 malvaceae guazuma ulmifolia lam 6.80 0.50 0.05 0.03 sida spinosa l. 0.03 0.08 0.03 triumfetta rhomboidea jacq 0.03 0.45 melastomataceae sp1 0.10 meliaceae azadirachta indica a. juss. 5.30 0.05 cedrela odorata l. 4.13 myrtaceae eucalyptus citriodora hook 0.08 0.18 1.08 0.33 0.18 eugenia uniflora l. 0.13 psidium cattleianum sabine 22.23 28.08 0.78 0.10 psidium guajava var. pomifera l. 7.00 6.35 0.03 0.33 0.25 1.28 nyctaginaceae boerhavia difusa l. 0.08 0.25 0.25 onagraceae ludwigia octovalvis (jacq.) p.m 0.08 passifloraceae passiflora cincinnata mast. 0.15 0.03 0.10 0.10 0.03 0.03 0.28 poaceae zeamays l. 0.03 0.65 1.33 0.03 rubiaceae borreria latifolia (aubl.) k. schum 2.10 borreira spinosa (l.) cham 3.93 3.08 13.08 23.88 diodella apiculata delpret. 0.03 0.05 0.05 manettia cordifolia mart. 0.05 0.20 0.05 0.03 spermacoce sp. 0.05 spermacoce verticillata l 1.10 rutaceae citrus limonia osbeck 1.28 0.68 0.13 sapindaceae cardiospermum corindum l. 0.08 0.03 0.10 0.10 0.18 solanaceae brugmansia suaveolens (humb) 0.03 turneraceae turnera sp. 0.05 0.28 0.03 0.03 0.03 urticaceae cecropia pachystachya trécul 32.68 11.00 0.13 0.83 57.53 verbenaceae lantana camara l. 0.33 taxa (s) 6 14 14 18 20 27 30 38 26 31 9 12 shannon index (h’) 1.065 1.343 1.909 1.480 0.885 1.169 1.866 2.280 0.838 1.591 0.246 1.230 equitability (j’) 0.595 0.509 0.724 0.512 0.295 0.355 0.549 0.627 0.257 0.463 0.112 0.495 berger-parker (d) 0.538 0.649 0.349 0.474 0.748 0.729 0.378 0.449 0.789 0.464 0.953 0.575 table 1. temporal variation in the diet of apis mellifera in a seasonal semideciduous forest in the state of ceará, in the period from november 2012 to october 2013. months highlighted in gray correspond to the rainy season and months highlighted in white represent the dry season. the values in each month correspond to the percentage of each plant species of the whole pollen collected during each month. (continuation) family species/pollinic types nov dec jan feb mar apr may jun jul aug sep oct jem nascimento et al. – temporal variation of pollen used in the diet of apis mellifera l.268 in september, the diet was monofloral, composed almost exclusively by pollen of myracrodruom urundeuva (95% = dp). in october and november, cecropia pachystachya (57.53% = dp; 32.68 = ap, respectively) and attalea speciosa (20.03% = ap; 53.80 = dp) stood out over the other species used by a. mellifera. in december, attalea speciosa predominated in the diet of honeybees (64.85%). the cluster analysis reliably showed (cophenetic correlation = 0.8545) that colonies have high similarity as to bee pollen, with a minimum similarity of approximately 60% (fig 2). pollen production was greater in the rainy season (344.76 ± 43.33 g/col./month) than in the dry season (149.20 ± 20.45 g/col./month) (fig 3a). in march and april, we found the highest productions of bee pollen (517.61 g/col. and 454.09 g/col., respectively). pollen production was positively related to rainfall (β = 1.23, t = 8.95, p <0.001, fig 4a), but the richness of pollen types was not associated with production (β = -2.40; t = -1.61, p = 0.142). the nutritional value of pollen sampled also varied throughout the study period (fig 3b-3f). the protein content showed higher levels in the dry-rainy-dry seasons transitions fig 2. similarity dendrogram (bray-curtis index and paired group algorithm) in the composition of pollen sampled in the colonies of apis mellifera in a seasonal semideciduous forest in the state of ceará, in the period from november 2012 to october 2013. fig 3. temporal variation in production of bee pollen used in the diet of apis mellifera and its nutritional value. (a) annual production of bee pollen. (b) total protein. (c)total carbohydrate. (d) lipids. (e) mineral matter. (f) dry matter. the symbol + represents the mean of each month. months highlighted in gray correspond to the rainy season and months highlighted in white represent the dry season. sociobiology 66(2): 263-273 (june, 2019) 269 table 2. results of generalized linear models (glm) of factors that influence chemical variables of bee pollen produced by apis mellifera in a seasonal semideciduous forest in the state of ceará, in the period from november 2012 to october 2013. dependent variables independent variables estimate (β) standard error t p crude protein number of pollen types 0.023 0.097 0.236 0.819 precipitation 0.003 0.009 0.349 0.735 total carbohydrates number of pollen types 0.028 0.153 0.182 0.859 precipitation 0.009 0.014 0.671 0.519 lipids number of pollen types 0.037 0.016 2.321 0.049 precipitation -0.001 0.001 -0.843 0.424 mineral matter number of pollen types -0.023 0.010 -2.385 0.041 precipitation -0.002 0.001 -2.003 0.076 dry matter number of pollen types -0.124 0.063 -1.969 0.085 precipitation -0.041 0.007 -5.684 0.000 (fig 3b). total carbohydrates were inversely proportional to the protein content (fig 3b and 3c). lipids concentration showed little variation, except for the january sample in which discrepant values when compared to the other months (5.46%) occurred (fig 3d). the average annual mineral matter of bee pollen was 2.15%, with highest level in november (2.76%) and lowest in july (1.33%) (fig 3e). regarding dry matter, in the rainy season we obtained lower levels (68%) when compared to the months that there was no precipitation. analyzing the relationship between nutrients (cp, tc, fat, mm, dm), number of pollen types and rainfall, we observed that the lipids was positively related to the number of pollen types (table 2, fig 4b). the mineral matter was negatively correlated with the number of pollen types (fig 4c), while the dry matter was negatively correlated with rainfall (table 2; fig 3f and 4d). no relationship was detected between protein (or carbohydrate) with rainfall and the number of pollen types (table 2). we also found a positive relationship between precipitation and pollen production (fig 4a). in addition, we found significant correlations between dominance of some taxonomic groups with some nutrients. fig 4. spearman correlation between dependent variables. (a) annual production of bee pollen and rainfall. (b) lipids and number of pollen types (c) mineral matter and pollen types (d) dry matter and rainfall. there was a positive correlation between the protein content and dominance of pollen of the genus mimosa (leguminosae) and of the family malvaceae (table 3). total carbohydrates were negatively correlated with the amount of malvaceae pollen and positively with the amount of poaceae pollen. the lipids was positively correlated with the presence of the family malvaceae species, while the mineral matter was negatively correlated with asteraceae, commelinaceae, lamiaceae and positively with arecaceae and urticaceae (table 3). these results were found in the correlation analysis based on the number of pollen grains of all plant species as well as in the analysis using the total pollen volume of each plant species in the bee diet. jem nascimento et al. – temporal variation of pollen used in the diet of apis mellifera l.270 discussion bee flora of the seasonal semideciduous forest studied proved to be well diversified, and the foraging workers of a. mellifera interacted with many plant species, as observed in other biomes (pacheco filho et al., 2015). however, the workers of the colonies tended to constantly search for floral resources in plants of specific taxonomic groups, possibly because such plants provide further floral resources (nectar or pollen) at the time of collection or because of the quality of such resources (hill et al., 1997). the family leguminosae was the most frequent in the study area (santos et al., 2014) and species such as mimosa caesapiniifolia and m. tenuiflora were constantly present in the diet of a. mellifera. these two plant species are responsible for the highest availability of nectar and pollen in the caatinga (maia-silva et al., 2012). the importance of the genus mimosa in maintaining the diet of stingless bee species was reported for trigona spinipes, partamona rustica, in the state of ceará (blochtein et al., 2010), melipona subnitida and melipona scutellaris state of rio grande do norte (maiasilva et al., 2015). pollen of myracrodrum urundeuva was collected in high proportions during the dry season and in august it was monofloral. its flowering in periods of low water availability highlights the importance of this species for the maintenance of a. mellifera colonies, as well as of native bees (maia-silva et al., 2012; araujo-filho, 2013). table 3. correlation between taxonomic groups and chemical variables of bee pollen produced by apis mellifera in a seasonal semideciduous forest in the state of ceará, in the period from november 2012 to october 2013. the rs values are presented for dominance in the diet (% of pollen grains) and pollen volume of each plant group. taxonomic groups crude protein total carbohydrates lipids mineral matter rs p rs p rs p rs p d om in an ce (% ) mimosa 0.594 0.042 -0.147 0.649 0.210 0.513 -0.329 0.297 asteraceae -0.105 0.746 0.406 0.191 0.413 0.183 -0.727 0.007 commelinaceae -0.011 0.972 0.377 0.227 0.168 0.602 -0.664 0.018 lamiaceae 0.015 0.964 0.370 0.237 0.348 0.268 -0.653 0.021 malvaceae 0.723 0.008 -0.668 0.018 0.679 0.015 -0.163 0.612 arecaceae -0.329 0.297 -0.196 0.542 -0.035 0.914 0.636 0.026 poaceae -0.184 0.568 0.684 0.014 -0.421 0.173 -0.388 0.213 urticaceae -0.460 0.132 -0.008 0.981 -0.460 0.132 0.663 0.019 v ol um e po lle n (c m ³) mimosa 0,678 0,015 -0,413 0,183 0,476 0,118 -0,252 0,430 asteraceae 0,028 0,931 0,217 0,499 0,490 0,106 -0,748 0,005 commelinaceae 0,116 0,720 0,310 0,327 0,146 0,652 -0,575 0,051 lamiaceae 0,015 0,964 0,370 0,237 0,348 0,268 -0,653 0,021 malvaceae 0,631 0,028 -0,638 0,026 0,653 0,021 -0,254 0,426 arecaceae -0,329 0,297 -0,196 0,542 -0,035 0,914 0,636 0,026 poaceae -0,184 0,568 0,684 0,014 -0,421 0,173 -0,388 0,213 urticaceae -0,460 0,132 -0,008 0,981 -0,460 0,132 0,663 0,019 attalea speciosa was present in the diet of bees throughout the year, becoming dominant in november, december and august. studies on pollen production in other regions of the northeastern brazil reported that species of arecaceae (e.g., cocos nucifera) are always frequent throughout the year in the diet of honeybees, emphasizing the importance of this family for a. mellifera (almeida-muradian et al., 2005; arruda et al., 2013; alves & santos, 2014). pollen production varied throughout the year with two peaks, one in the dry season with lower productivity and in the rainy season, with higher values attributed mainly to pollen provided by mimosa caesapiniifolia. an important plant for the commercial production of bee pollen, as was certified in samples from rio grande do norte, bahia, sergipe and são paulo states (melo et al. 2018). fluctuations in pollen production are common, due to the influence of many productivity factors (negrão et al., 2014), including the flowering of one or more species of abundant plants in the site, weather conditions, colony size, of brood area size and queen age (dimou & thrasyvoulou, 2007; rebolledo et al., 2011; avni et al., 2014). carbohydrates were the most abundant group of nutrients, followed by proteins. both, the protein and carbohydrate contents varied throughout the year, probably due to the variation in the botanical source, environmental conditions, and factors related to the handling and storage (villanueva et al., 2002; yang et al., 2013; sattle et al., 2015). protein increment in the bee diet is attributed mainly to the presence of mimosa because the volume of pollen sociobiology 66(2): 263-273 (june, 2019) 271 grains of this genus was ten times greater than the volume of any other plant species found in the diet. therefore, despite the positive correlation between total protein and malvaceae pollen, the total pollen grain volume of this group is small suggesting that malvaceae are less important protein sources when compared to mimosa. similarly, carbohydrate levels showed a negative correlation with malvaceae and positive correlation with poaceae, but their variability can be related mainly to the presence of poaceae pollen due to its greater volume, as observed by yang et al. (2013). in turn, lipids remained constant throughout the year, and the lipids content was attributed to polliniferous sources. bees select pollen with high levels of unsaturated fatty acids, which are better suited to bee metabolism (estevinho et al., 2012; avni et al., 2014). a fatty compound that contributes to levels of fatty acid esters is found in pollenkitt covering the whole grain surface, which is evident in many plant species (pacini & casadoro 1981; dobson, 1988). in addition, the positive correlation of pollen types richness with lipids suggests that the larger the number of pollen types harvested by bees, the larger the chances of increasing the average lipids content in the pollen taken to the colony. bees cannot synthesize minerals, and other nutrients, and it is through pollen that they get the mineral quantities required for the structural maintenance of the individuals and the colony (brodschneider & crailsheim, 2010). the content of mineral matter in this study proved to be quite stable, with no discrepancy in values. high levels of mineral matter may be due to incorrect handling of the product by beekeepers, or inefficient cleaning process (sattle et al., 2015). nevertheless, a negative correlation between the mineral matter and the number of pollen types implies that the plant species that were used as resources in the bee diet during the dry season (when there are less pollen types and lower plant species richness) contributed with higher levels of minerals. the level of dry matter is one of the main items to be monitored in pollen production. herein, the water content was slightly increased in the months with higher rainfall and higher relative humidity. high levels of humidity can compromise the quality and potentially promote microbial growth because the bee pollen is a highly hygroscopic product (barreto et al., 2005; melo et al., 2015). floristic diversity, nutritional quality, reduced exposure to pesticides coupled with adequate management are factors that can contribute to colony productivity and performance (colwell et al., 2017). even so, the percentages based on the dry matter of cp, fat, ct and mm are within the values found in the national and international literature (yang et al., 2013; negrão et al., 2014; sattle et al., 2015). conclusion it is known that a. mellifera has an extremely plastic behavior, and that its foraging on floral sources is mostly related to the abundance of resources as well to the floral density than to the species specific features (stang, 2007). here, we find that these honeybees invest most of its pollen foraging efforts in the genus mimosa (leguminosae) myracrodrum (anacardiaceae), cecropia (urticaceae) and the species attalea speciosa. in this way, it is very important to keep these plants in the vicinity of the apiaries. although the study area is under semiarid climate regime and subjected to long periods of water deficit, the studied seasonal semideciduous forest allows production of bee pollen throughout the year without the use of artificial feeding. in addition, the bee pollen produced has a nutritional value similar to that observed in other countries, indicating that this product is within national and international standards. acknowledgements we thank the national council for scientific and technological development (cnpq) for grant awarded during master’s degree process: 131596/2014-4. beecare bayer and technological development of engineering for support: process 001505. the online pollen cataloge network (rcpol) for the support in the pollen identification. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. references almeida-muradian l. b., pamplona l. c., coimbra s. & barth o. m. 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(2013) characterization of chemical composition of bee pollen in china. journal of agricultural and food chemistry, 61: 708718. doi: 10.1021/jf304056b open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(3): september (2013) editorial special issue on arthropod-plant interactions sociobiology is a journal dedicated to the study of social insects. thus, social behavior, taxonomic aspects, life history studies and behavioral ecology of ants, bees and wasps prevail in published papers. however, these animals present in their biology a long history of evolved and coevolved interactions with plants. this evolution timeline began firstly with social insects as herbivores, chewing on leaves, roots, buds and petals. then, later evolutionary radiation of some groups allowed the rise of those species that could feed on pollen or suck exudates, secretions and soft plnt tissues. this evolutionary history continued later on when social insects established several harmonic relationships with plants, such as pollination, seed dispersion and plant protection. these relationships are indeed very common in all natural systems. although the implications of these insect-plant interactions to the comprehension of the structure and function of natural communities are enormous, articles with such an approach are very few in sociobiology. for this main reason and to stimulate the publication of this type of studies in sociobiology the editors decided to support this special issue and we are very happy with the results. people of four continents contributed with research conducted in totally different ecosystems, involving bees, wasps, ants and associated fauna and flora. we hope that several of the papers published here will have a significant impact on our comprehension of how social insects impact plant-animal interactions in natural communities. we are very thankful to every colleague that believed in this idea. kleber del-claro helena maura torenzan-silingardi gilberto m. m. santos evandro n. silva cândida maria lima aguiar editors for this special issue sociobiology an international journal on social insects doi: 10.13102/sociobiology.v62i1.124-127sociobiology 62(1): 124-127 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 new species of scuttle flies (diptera: phoridae) associated with a ponerine ant (hymenoptera: formicidae) in brazil introduction the ant dinoponera lucida emery (ponerinae) is endemic of the atlantic rain forest in brazil (peixoto et al., 2008) and it was included in the list of brazilian threatened species in 2003 (campiolo & delabie, 2008). during 2012 marcos teixeira collected diptera attacking or hovering over this ant in the vicinity of six colonies at aracruz, espírito santo state, in david farina park (19o55`54” s 40o07`41” w). the scuttle flies (phoridae) were preserved in 70% ethanol and sent to rhld for slide mounting and identification. the fly specimens are deposited in the collection of the museu de zoologia, universidade de são paulo, brazil (mzsp) and the university of cambridge museum of cambridge museum of zoology (ucmz). genus apocephalus coquillett the larvae of this large genus are dominantly parasitoids of ants. we add a further example below. abstract among scuttle flies caught at colonies of the ant dinoponera lucida emery (ponerinae) were apocephalus exlucida disney new species and females of two species of megaselia rondani that, which in our present state of knowledge, cannot be named until associated with their males. sociobiology an international journal on social insects rhl disney1 , mal bragança2, mc teixeira3 article history edited by evandro do nascimento silva, uefs, brazil received 24 may 2014 initial acceptance 11 august 2014 final acceptance 12 august 2014 keywords ants, parasite. corresponding author r henry l disney department of zoology, university of cambridge, downing street, cb2 3ej, u. k. e-mail: rhld2@hermes.cam.ac.uk apocephalus exlucida disney new species in the key of borgmeier (1971) this species runs to couplet 60 but fits neither species nor subsequently described species running to the same couplet. among the latter are those covered by brown’s (2000) a. miracauda-group, which includes some with the most similar ovipositor sheaths, and species associated with leaf cutter ants in argentina (brown et al., 2010) but all with very different ovipositor sheaths. female. frons as fig 1, the microtrichia being well spaced. antennae pale (fig 2) and postpedicels lacking subcutaneous pit sensilla. palps as fig 3. proboscis with pale labrum and labella, the latter being narrow and lacking fields of spinules on their lower faces. thorax yellow apart from brown patches on pteropleuron. notopleoron with two bristles and no cleft in front of these. mesopleuron bare. scutellum with an anterior pair of small hairs and a posterior pair of bristles. abdominal tergites light brown, tending to yellowish at sides with small hairs apart from some longer more robust hairs at sides of t2 short note 1 -university of cambridge, cambridge, u. k. 2 universidade federal do tocantins, porto nacional, to, brazil 3 universidade federal do espírito santo, são mateus, es, brazil sociobiology 62(1): 124-127 (march, 2015) 125 and with some slightly longer fine hairs at rear of t6. venter pale apart from broad grey bands on flanks below the sides of the tergites, and with hairs, longer than those on tegites, below segments 3-6. terminal opvipositor sheath complex as fig 5, with its anterior ventral sclerite comprising an anterior long dark sclerotised rod and a broader greyish brown posterior diamond shape ending in a tapered posterior projection. legs pale straw yellow apart from brown tips to hind femora and patches on mid coxae. fore tarsi slender with a posterodorsal hair palisade on all five segments and 5 slightly longer than 4. dorsal hair palisade of mid tibia extends about 0.8 times its length. hairs below basal half of hind femur longer than those of the anteroventral row of outer half. hind tibia with about a dozen differentiated posterodorsal hairs, the last being longer and more robust, and spinules of apical comb all simple. wings as fig 6. knobs of halteres mainly brown. teixeira, to-115) (5-164, mzsp). paratypes: 9 females, 8 males, as holotype except some vii.2012 (to-114-122) (5160-165, mzsp & ucmz). comment. previous records of apocephalus attacking species of dinoponera are apocephalus miricauda borgmeier and apocephalus sp. at d. gigantea perry (silveira-costa & moutinho, 1996); it being shown that injured ants attracted more phorids than uninjured ants, but it was not the haemolymph that was the lure, a. miricauda at d. longipes emery, there being 4-9 eggs per host and oviposition being mainly between the propodeum and petiole but sometimes the antennal suture (brown, 2000). genus megaselia rondani in this huge genus, most females cannot be named until linked to their males in our present state of knowledge. megaselia species a in the keys of borgmeier (1962) this species belongs to abteilung vii, running down to couplet 8 but fails to fit either lead. female. frons brown, clearly broader than long, with about 80 hairs and devoid of very fine microtrichia. supraantennal bristles (sas) unequal, the lower pair being clearly shorter and finer. the upper sas slightly wider apart than the pre-ocellars. the antials very slightly lower on frons than anterolaterals, closer to als than to upper sas. pre-ocellars closer together than than either is from a mediolateral bristle, which is a little higher on frons. cheek without bristles and jowl with two. the subglobose postpedicels light brown, 0.09 mm in diameter, with 2-3 subcutaneous pit sensilla (sps) vesicles of which the diameter of the largest is subequal to that of the socket of a lower sa bristle. palps pale yellow, at most 0.038-0.039 mm at broadest, with 7-8 bristles (the longest clearly longer than palp breadth) and 4-5 hairs. labrum pale brownish yellow and about 0.95-0.10 mm wide. labella paler than palps and lacking small spinules below. thorax brown. three notopleural bristles and no cleft in front figs 1-6. apocephalus exlucida female. fig 1. frontal view of head; fig 2. antenna; fig 3. palp; fig 4. dorsal view of abdominal segments 3 to tip; fig 5; terminal (ovipositor sheath) segments of abdomen; fig 6. right wing. figs 7-10. apocephalus exlucida male. fig 7. frontal view of head; fig 8. left face of hypopygium; fig 9. right face of hypopygium; fig 10. left face of hypopygium. male. head as fig 7. thorax as female. abdomen with tergites with hairing similar to female but with stronger hairs at rear of t6 (fig 8) but while largely brown they are variably partly yellow, especially t5. venter entirely pale yellow and with hairs below segments 3-6 but those at rear of segment 6 longer than those at rear of t6 (fig 8). hypopygium as figs 8-10. legs, wings and halteres similar to female. material examined. holotype female, brazil, espírito santo, aracruz, vi.2012, at dinoponera lucida emery, (marcos disney, r.h.l. et al. scuttle flies associated with a ponerine ant in brazil126 female. frons as fig 14 and with fairly dense but very fine microtrichia. cheek with 2-3 small bristles and jowl with two that are longer and more robust. the subglobose postpedicels pale yellow, without subcutaneous pit sensilla (sps) vesicles, and 0.10-0.11 mm at widest. palps yellow, 0.05 mm greatest breadth with, 6 bristles (the longest being 0.08 mm long) and as many hairs. labrum yellow lightly tinged brown and 0.160.17 mm wide. labella coloured as palps and lacking short spinules below. thorax yellow, apart from brown patches on pteropleuron. two notopleural bristles and no cleft in front of these. mesopleuron bare. scutellum with an anterior pair of hairs and a posterior pair of bristles, the former being about 0.59 times as long as the latter. abdominal tergites 1-5 part brown and part whitish yellow. t6 whitish yellow apart from very narrow brown lateral margins and about twice as long as broad. tergite 7 similar but smaller. epiproct very pale. venter mainly pale apart from broad gray bands on the flanks extending from the sides of the tergites, and with hairs below segments 3-6. sternite 7 represented by hairs only. posterolateral lobes at rear of sternum 8 pale with 2 hairs at rear margin. cerci very pale and about twice as long as broad. furca not evident. dufour’s crop mechanism very pale, about twice as long as wide and slightly pointed at rear end. apart from brown patch on mid coxa and tip of hind femur legs yellow. fore tarsus with posterodorsal hair palisade on segments 1-5 and 5 slightly longer than 4. dorsal hair palisade of mid tibia extends about 0.8-0.9 times its length. hairs below basal half of hind femur longer than those of anteroventral row of outer half (fig 15). hind tibia with about 16 differentiated posterodorsal hairs, the last being the longest and strongest but the two preceding it clearly weaker and shorter than those above them; and spinules of apical combs simple (fig 15). wings (fig 16) 1.2-1.3 mm long. costal index 0.59. costal ratios 3.7 : 3.4 : 1. costal cilia (of section 3) 0.05 mm long. no hair at base of vein 3 . with 6 axillary bristles, the outermost being 0.10-0.11 mm long. sc reaching r1. haltere knob pale grayish brown. of these. mesopleuron bare. scutellum with an anterior pair of hairs (about as long as hairs of scutum adjacent to notopleuron) and a posterior pair of bristles. abdominal tergites brown. t5t7 as fig 11. venter brown, and with fine hairs below segments 3-6. sternite 7 a narrow bar, about 0.08 mm at widest and tapering a little anteriorly. epiproct pale contrasting with its sclerotised anterior apodemes. posterolateral lobes at rear of sternum 8 pale and with 3 longish hairs at hind margin (the longest about 0.050.06 mm). furca not evident. dufour’s crop mechanism pale, and at least twice as long as broad and rounded behind. apart from increasingly brown outer half of hind femur and dark brown patch on mid coxa, legs yellow. fore tarsus with posterodorsal hair palisade on segments 1-4 and 5 about as long as 4. dorsal hair palisade of mid tibia extends about 0.75 times its length. hairs below basal half of hind femur longer than those of anteroventral row of outer half (fig 12). hind tibia with 10 differentiated posterodorsal hairs, the last being stronger and longer than the rest, and spinules of apical combs simple. wings (fig 13) 1.2-1.3 mm long. costal index 0.56-0.57. costal ratios 3.6 : 3.3 : 1. costal cilia (of section 3) 0.04-0.05 mm long. without hair at base of vein 3. with two unequal axillary bristles, the outer being 0.06-0.07 mm long. sc not reaching r1. haltere with pale stem and brown knob. material examined. 1 female, espírito santo, aracruz, vii.2012, at dinoponera lucida emery, (marcos teixeira, to115, mzsp). figs 11-13. megaselia species a female. fig 11. abdominal tergites 5-7; fig 12. hind femur; fig 13. right wing. megaselia species b with the anterior scutellar strong hairs being about 0.59 times as long as the posterior bristles, in the keys of borgmeier (1962) this species belongs either to abteilung vi or vii, depending on whether it is considered to possess 2 or 4 scutellar bristles. in abteilung vi it runs to couplet 50, but is immediately excluded by its yellow thorax. in abteilung vii, it runs down to couplets 79 and 82-84, which cover males only but in all cases non sexually dimorphic details exclude this species. figs 14-16. megaselia species b female. fig 14. frontal view of head; fig 15. hind femur and tibia; fig right wing. sociobiology 62(1): 124-127 (march, 2015) 127 material examined. 1 female, espírito santo, aracruz, vii.2012, at dinoponera lucida emery, (marcos teixeira, to118, mzsp). acknowledgements rhld’s studies of phoridae are currently supported by grants from the balfour-browne trust fund (university of cambridge). malb thanks to cnpq for the financial support in brazil. filipe pola vargas for support in the field work. references borgmeier, t. (1962). versuch einer uebersicht ueber die neotropischen megaselia-arten, sowie neue oder wenig bekannte phoriden verschiedener gattungen (dipt. phoridae). studia entomologica, 5: 289-488. borgmeier, t. (1971). further studies on phorid flies, mainly of the neotropical region (diptera, phoridae). studia entomologica, 14: 1-172. brown, b. v. (2000). revision of the “apocephalus miricauda-group” of ant-parasitizing flies (diptera: phoridae). contributions in science. natural history museum of los angeles county, 482: 1-62. brown, b.v., disney, r. h. l., l. elizalde, l. & folgarait, p. j., (2010). new species and new records of apocephalus coquillett (diptera: phoridae) that parasitize ants (hymenoptera: formicidae) in america. sociobiology, 55: 165-190. campiolo, s., & delabie, j. h. c. (2008). dinoponera lucida emery. in: machado, a. b. m., drummond, g. m. & paglia, a. p. (eds.) livro vermelho da fauna brasileira ameaçada de extinção. 1st ed., brasília, df: ministério do meio ambiente; belo horizonte, mg: fundação biodiversitas. p. 388-389. peixoto, a. v., campiolo, s., lemes t. n., delabie, j. h. c. & hora, r. r. (2008). comportamento e estrutura reprodutiva da formiga dinoponera lucida emery (hymenoptera, formicidae). revista brasileira de entomologia, 52: 88-94. doi: 10.13102/sociobiology.v65i4.3470sociobiology 65(4): 784-788 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 pollen resources stored in nests of wild bees xylocopa ciliata burmeister and megachile pusilla pérez (hymenoptera: anthophila) in a temperate grassland-forest matrix pollen analysis of nest provisions is a useful tool to identify their botanical origin and to understand the pollen specialization in bees (cane & sipes, 2006; müller & kuhlmann, 2008). even extreme pollen specialists can take nectar from many floral species but forage pollen on a reduced number of host-plants (robertson, 1925; 1926; cane & sipes, 2006; minckley & roulston, 2006; müller & kuhlmann, 2008; vossler, 2013; 2014). threshold values of 10 and 5% total pollen counts have commonly been used to distinguish minor or potential contaminants from abundant pollen types (ramalho et al., 1985; kleinert-giovannini & imperatriz-fonseca, 1987; cane & sipes, 2006; müller & kuhlmann, 2008) which is necessary to correctly identify pollen specialization even in polylectic bees (vossler, 2018). the aim of the present study was to identify the botanical origin of nest pollen provisions of two wild bees (the exotic megachile pusilla pérez and the neotropical xylocopa ciliata burmeister) and their pollen specialization at a same site during the period of nesting activity. it is hypothesized that both species are polylectic, similar to most species of megachile and xylocopa. abstract pollen analysis was employed to study the diet composition of two wild bees in a patch of temperate grassland invaded by exotic plants. thirty pollen types from 14 families and two unidentified types were present in the seven samples analyzed. the three samples from megachile pusilla pérez were composed of 100% lotus glaber and the four samples from xylocopa ciliata burmeister of abundant pollen (˃5%) of lotus glaber, galega officinalis, adesmia bicolor and type senna-chamaecrista (all fabaceae). the latter resource likely foraged out of the park, which could be an evidence to support its pollen preference for fabaceae. however, a larger number of samples is necessary to identify the specialization status of x. ciliata. the preference for lotus in this site was due to temporal specialization as m. pusilla was identified as polylectic in its origin area, and this could help to explain its effective naturalization in the new world and other areas of the old world. sociobiology an international journal on social insects fg vossler article history edited by solange augusto, ufu, brazil received 11 may 2018 initial acceptance 12 august 2018 final acceptance 23 august 2018 publication date 11 october 2018 keywords adesmia; entomopalynology; galega; lotus; pollen feces; pollen provisions. corresponding author favio gerardo vossler laboratorio de actuopalinología cicyttp (conicet/uader/prov. entre ríos) entre ríos, materi y españa, e3105bwa diamante, entre ríos, argentina. e-mail: favossler@yahoo.com.ar m. pusilla was found nesting in small soil cracks of a bank and x. ciliata in internodes of only large stems of the invasive dipsacus fullonum l. (fig 1). their nests were hard to find in the site sampled and no studies on nest pollen provisions were performed for m. pusilla in areas where it was naturalized neither for x. ciliata. nests were provisioned during summer and sampled during summer and fall (three nests of m. pusilla sampled on 12th february 2011 and two samples from nest 1 (nest 1a and b) of x. ciliata on 27th december 2008 and one sample (nest 1c) from 1st january 2009 and one sample from nest 2 in june 2012) (table 1; fig 1). the study was carried out at the parque ecológico municipal de la plata (34° 51’-52’ s; 58° 03’-05’ w), a 200 ha patch of temperate grassland composed of many melittophilous species native to the oriental district of the pampean region sensu cabrera (1971) but heavily colonized by exotic plants (vossler et al., unpublished data), at villa elisa city, buenos aires province, argentina. the whole pollen provisions from nest cells of m. pusilla and dry feces of post-defecating larvae from nest centro de investigaciones científicas y transferencia de tecnología a la producción (conicet/uader/prov. entre ríos), entre ríos, argentina short note sociobiology 65(4): 784-788 (october, 2018) special issue 785 entrances of x. ciliata were sampled and stored in plastic tubes at 5 °c. in the laboratory, they were dissolved in distilled water at 80-90 °c for 10-15 minutes, pressed when necessary using a glass rod, stirred by hand for a few minutes, and filtered. finally, to obtain pollen sediment, samples were centrifuged at 472 x g for 5 minutes, and acetolized (erdtman, 1960). pollen types were identified at 400 and 1,000 x magnification and 500 grains per slide were counted (except in sample 1a as only 204 grains were found) using a light microscope leitz laborlux . pollen grain identification was carried out comparing nest pollen grains with reference pollen. the reference pollen collection was made from flowers of plant species mainly collected in parque ecológico municipal de la plata (villa elisa city) and la plata city, in the northeastern of buenos aires province, argentina. these plant specimens were pressed, dried, identified by the author and deposited in the herbarium lorentz (dte) of diamante, entre ríos, argentina. the vegetation and flowering of the site was quantitatively recorded from august 2008 to august 2009 (vossler et al., unpublished data). to identify pollen specialization categories, the lexica of cane and sipes (2006) and müller and kuhlmann (2008) were applied. fig 1. pollen host blooming: a_ lotus glaber; b_ adesmia bicolor; c_ galega officinalis. nesting sites of xylocopa ciliata (d) and megachile pusilla (e, f): d_ nest 1 of xylocopa ciliata in a thick stem of dipsacus fullonum surrounded by galega officinalis and lotus glaber during blooming; e_ leaf fragments in a completed nest cell (left) and transported by a female for nest building (right); f_ scopal pollen of megachile pusilla during nest provisioning. fg vossler – pollen resources stored in nests of wild bees786 a total of 30 pollen types from 14 families and two unidentified types were present in the seven samples analyzed (table 1). from them, most were minor pollen (≤5 %) and only four types from two subfamilies of fabaceae were abundant: lotus glaber, galega officinalis, adesmia bicolor and type senna-chamaecrista (table 1; fig 2). the three samples of m. pusilla were composed of 100% l. glaber. the four samples of x. ciliata were composed of abundant pollen of the four fabaceae in different representation and many minor pollen from many families (table 1; fig 2). the fact that only l. glaber was identified composing the diet of m. pusilla would indicate strong specialization on this species. however, as all the samples were from the same day, this result could be related to resources availability. moreover, pollen analysis of 24 loads from 18 localities reported lotus as abundant pollen and many families as hosts of m. pusilla in its area of origin, which is around the mediterranean sea in the old world (soltani et al., 2017), suggesting polylecty. therefore, the preference for lotus in this site was interpreted as temporal specialization. its polylectism could help to explain its effective naturalization in the new world and other areas of the old world. an intensive usage of lotus and other three genera in disparate tribes of fabaceae was observed in x. ciliata. plant family pollen type xylocopa ciliata megachile pusilla nest 1 a nest 1 b nest 1 c nest 2 nest 1 nest 2 nest 3 amaryllidaceae zephyrantes minima + apiaceae eryngium 0.39 apiaceae type ammi + arecaceae arecaceae + asteraceae, astereae grindelia pulchella + 2.39 asteraceae, astereae type baccharis + 1.19 asteraceae, cardueae carduus acanthoides 1.99 asteraceae, cardueae cirsium vulgare + 4.58 asteraceae, eupatorieae eupatorium buniifolium 0.39 asteraceae, heliantheae ambrosia tenuifolia + asteraceae, lactuceae picris echioides + + asteraceae, lactuceae type hypochaeris + 1.86 asteraceae, vernonieae vernonia + 1.39 brassicaceae type nasturtium + caprifoliaceae lonicera japonica + + casuarinaceae casuarina + celtidaceae celtis + + dipsacaceae dipsacus fullonum + + fabaceae, caesalpinioideae gleditsia triacanthos 0.15 + + fabaceae, caesalpinioideae type senna-chamaecrista 22.91 fabaceae, papilionoideae adesmia bicolor 32.94 0.79 + fabaceae, papilionoideae galega officinalis 28.43 49.84 56.19 22.51 + fabaceae, papilionoideae lotus glaber 69.12 49.84 7.81 40.04 100 100 100 fabaceae, papilionoideae vicia sativa 1.47 + + myrtaceae eucalyptus + poaceae poaceae type 1 + 0.79 + + poaceae poaceae type 2 0.98 + solanaceae salpichroa origanifolia 0.20 solanaceae solanum 1.18 verbenaceae verbena 0.15 unidentified unidentified 1 (4-colporate psilate) + unidentified unidentified 2 (monosulcate) 0.39 table 1. abundance (%) of the pollen types found in xylocopa ciliata and megachile pusilla nest pollen samples, in alphabetic order of their families. the abundant resources (> 5%) are shown in bold. ‘+’ includes pollen types present in the slides but not recorded during the counting. sociobiology 65(4): 784-788 (october, 2018) special issue 787 the high abundance of fabaceae in only four samples could be considered as a strong evidence of pollen specialization on the whole family: oligolecty (sensu cane & sipes, 2006) or broad oligolecty (sensu müller & kuhlmann, 2008). furthermore, as vegetation was recorded in this site where nests were sampled, the pollen types foraged could be ascribed to the plant species found within this area. moreover, this fact allowed for the identification of the abundantly foraged type senna-chamaecrista as not belonging to this natural plant community and likely belonging to an ornamental species cultivated in the urbanized landscape surrounding this park and therefore foraged out of the park. this fact could be a further evidence to support its pollen preference for fabaceae. palynological studies have not yet been carried out in this xylocopa species. some of the records on floral resources seem to suggest polylecty (sakagami et al., 1967; sakagami & laroca, 1971; hurd, 1978; schlindwein, 1998; schlindwein et al., 2003; gonçalves & melo, 2005; dalmazzo, 2010). however, the differentiation between males and females as well as nectar and pollen intake, necessary to identify the pollen specialization status (cane & sipes, 2006; müller & kuhlmann, 2008; vossler, 2013; 2014; 2018), was not done in most of these studies, but the fact that solanum has pollenonly flowers (vogel, 1978; buchmann, 1983) suggests that it is a legitimate pollen host of x. ciliata. the study of a larger number of nest pollen samples is necessary to identify the specialization status of x. ciliata and would likely include solanum as an abundant pollen host. acknowledgement in memory of my dear friend silvana durante, specialist in megachilidae, who encouraged me to start the study of the life history of bees. i also would like to thank luciana hiriart and ivana tapia for their help in the sampling of the vegetation of the parque ecológico municipal de la plata from august 2008 to august 2009 and the searching of bee nests. references buchmann, s.l. (1983). buzz pollination in angiosperms. in c.e. jones & r.j. little (eds.), handbook of experimental pollination biology (pp. 73–114). new york: scientific and academic editions. cabrera, a.l. (1971). fitogeografía de la república argentina. boletín de la sociedad argentina de botánica 14: 1–42. fig 2. abundant (a, b, c, and h) and minor (d, e, f, g, i) pollen types identified from nests of xylocopa ciliata, seen under light microscope. a_ lotus glaber; b_ galega officinalis; c_ adesmia bicolor; d_ solanum; e_ type hypochaeris; f_ picris echioides; g_ poaceae type 1; h and h’_ type senna-chamaecrista (h’: colapsed grain); i_ vernonia. fg vossler – pollen resources stored in nests of wild bees788 cane, j.h. & sipes, s. (2006). characterizing floral specialization by bees: analytical methods and a revised lexicon for oligolecty. in n.m. waser & j. ollerton (eds.), plant-pollinator interactions. from specialization to generalization (pp. 99– 122). chicago and london: the university of chicago press. dalmazzo, m. (2010). diversidad y aspectos biológicos de abejas silvestres de un ambiente urbano y otro natural de la región central de santa fe, argentina. revista de la sociedad entomológica argentina 69: 33–44. erdtman, g. (1960). the acetolysis method, a revised description. svensk botanisk tidskrift 54: 561–564. gonçalves, r.b. & melo, g.a.r. (2005). a comunidade de abelhas (hymenoptera, apidae s.l.) em uma área restrita de campo natural no parque estadual de vila velha, paraná: diversidade, fenologia e fontes florais de alimento. revista brasileira de entomologia 49: 557–571. doi: 10.1590/s008556262005000400017 hurd, p.d. (1978). an annotated catalog of the carpenter bees (genus xylocopa latreille) of the western hemisphere (hymenoptera: anthophoridae). washington: smithsonian institution press, 106 p. kleinert-giovannini, a. & imperatriz-fonseca, v.l. (1987). aspects of the trophic niche of melipona marginata marginata lepeletier (apidae, meliponinae). apidologie, 18: 69–100. minckley, r.l. & roulston, t.h. (2006). incidental mutualisms and pollen specialization among bees. in n.m. waser & j. ollerton (eds.), plant-pollinator interactions. from specialization to generalization (pp. 69–98). chicago: the university of chicago press. müller, a. & kuhlmann, m. (2008). pollen hosts of western palaearctic bees of the genus colletes (hymenoptera: colletidae): the asteraceae paradox. biological journal of the linnean society 95: 719–733. doi: 10.1111/j.10958312.2008.01113.x ramalho, m., imperatriz-fonseca, v.l., kleinert-giovannini, a. & cortopassi-laurino, m. (1985). exploitation of floral resources by plebeia remota holmberg (apidae, meliponinae). apidologie, 16: 307–330. robertson, c.h. (1925). heterotropic bees. ecology 6: 412–436. robertson, c.h. (1926). revised list of oligolectic bees. ecology 7: 378–380. sakagami, s.f., laroca, s. & moure, j.s. (1967). wild bee biocoenotics in são josé dos pinhais (pr), south brazil. journal of the faculty of science, hokkaido university. series 6, zoology, 16: 253–291. sakagami, s.f. & laroca, s. (1971). observations on the bionomics of some neotropical xylocopine bees, with comparative biofaunistic notes (hymenoptera: anthophoridae). journal of the faculty of science, hokkaido university. series 6, zoology, 18: 57–127. schlindwein, c. (1998). frequent oligolecty characterizing a diverse bee-plant community in a xerophytic buschland of subtropical brazil. studies on neotropical fauna and environment, 33: 46–59. schlindwein, c., schlumpberger, b., wittmann, d. & moure, j.s. (2003). o gênero xylocopa latreille no rio grande do sul, brasil (hymenoptera, anthophoridae). revista brasileira de entomologia, 47: 107–118. doi: 10.1590/s008556262003000100016 soltani, g.g., bénon, d., alvarez, n. & praz, c.j. (2017). when different contact zones tell different stories: putative ring species in the megachile concinna species complex (hymenoptera: megachilidae) biological journal of the linnean society, 20: 1–18. doi: 10.1093/biolinnean/blx023 vogel, s. (1978). evolutionary shifts from reward to deception in pollen flowers. in a.j. richards (ed.), the pollination of flowers by insects (pp. 89–96). london: academic press. vossler, f.g. (2013). the oligolecty status of a specialist bee of south american prosopis (fabaceae) supported by pollen analysis and floral visitation methods. organisms diversity and evolution, 13: 513–519. doi: 10.1007/s13127-013-0134-6 vossler, f.g. (2014). a tight relationship between the solitary bee calliopsis (ceroliopoeum) laeta (andrenidae, panurginae) and prosopis pollen hosts (fabaceae, mimosoideae) in xeric south american woodlands. journal of pollination ecology, 14: 270–277. vossler, f.g. (2018). are stingless bees a broadly polylectic group? an empirical study of the adjustments required for an improved assessment of pollen diet in bees. in p. vit, s.r.m. pedro & d.w. roubik (eds.), pot-pollen in stingless bee melittology (pp. 17–28). cham: springer. doi: 10.1007/9783-319-61839-5 _goback open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.409sociobiology 62(3): 457-459 (september, 2015) heat shock proteins expression during thermal risk exposure in the xerothermic ant formica cinerea sandy and arid landscapes are one of the most demanding environments for living organisms, leading to the evolution of many interesting adaptations in animal dwellers. in a longer perspective, food and water are elements limiting animal survival rate, but in the shorter – daily – perspective, temperature is an ultimate factor. for ectotherms (unable to actively control body temperature), inhabiting sandy habitats such as deserts or dunes, ground temperature is a factor limiting activity and survival rate (lenoir et al., 2009). the temperature of a sand surface at the sun’s zenith differs at every geographical range. for example, this temperature in the sahara desert is even 74ºc, while in a sandy forest clearing of a temperate climate, can reach 54ºc, but in both cases, it is beyond the thermal optimum of most living organisms. every animal exposed for a longer period of time to these conditions faces the same threat of experiencing heat stroke. despite individual strategies of avoiding heat (digging burrows, raising the body, etc.), at a cellular level, all organisms induce heat shock proteins (hsps) as molecular chaperones to protect their own proteins against denaturation (feder & abstract the abiotic conditions of the desert habitat fluctuate in a circadian rhythm of hot days and cold nights. species living in desert habitats evolved many adaptations to increase their chances of survival. however, abiotic conditions in xerothermic habitats of a temperate climate are much different. diurnal fluctuations are not as strong, but animals have to cope with seasonal changes and hibernate during the winter, which may potentially influence their adaptations to critical temperature conditions. we attempted to assess heat resistance adaptations using the example of a widely distributed xerothermic ant formica cinerea. using real-time pcr, we measured the expression of three heat shock protein genes (hsp60, hsp75, hsp90) and assessed the adaptations of f. cinerea to enable foraging in risk prone conditions. the analysis of gene expression using the generalized linear model surprisingly indicated that there was no significant effect of temperature when comparing workers from the control (23ºc) with workers foraging on the surface of hot sand (47-54ºc). as a next step we tried to estimate the threshold of a thermal resistance with the use of thermal chambers. expression of all hsps genes increase compare to the control group, expression of hsp60 and hsp90 continued up to 45ºc. sociobiology an international journal on social insects p ślipiński, jj pomorski, k kowalewska article history edited by gilberto m. m. santos, uefs, brazil received 05 may 2014 initial acceptance 19 january 2015 final acceptance 01 may 2015 keywords formicidae, temperature, limits, stress, resistance, physiology. corresponding author museum and institute of zoology polish academy of sciences wilcza 64, 00-679 warsaw, poland e-mail: piotrs@miiz.waw.pl hofmann, 1999). hsps recognize and bind to other proteins in non-native conformations during denaturing stress, serving as molecular chaperones. in proposed study hsps are used to asses a thermal risk during foraging in formica cinerea. f. cinerea is a typical thermophilic and xerophilic oligotope of dry open habitats. field observations of f. cinerea workers foraging on hot soil during the summer led to developing the main working hypothesis that foraging in such demanding conditions in the case of f. cinerea is most probably correlated with a high level of cellular stress and thus the expression of hsp. before obtaining the results, the only question seemed to be: how risky is foraging in these particular conditions? the aim of the field work part, was to measure the hsp expression level during worker foraging on the surface of hot sand, whereas the goal of the laboratory part, was to estimate the threshold of thermal resistance under laboratory conditions by exposing ants to high temperatures over a long period of time. in field experiment individuals of f. cinerea were collected directly from the nest entrance in the morning when short note museum and institute of zoology, polish academy of sciences, warsaw, poland p ślipiński, jj pomorski, k kowalewska – heat shock proteins expression in formica cinerea458 the nest temperature was low and the soil temperature around the nest was 23.5ºc (control group). another group of workers were collected from the hot soil in the middle of the sunny day (between 12:30 to 14:40 p.m.), when the soil surface temperature reached from 47ºc to 54ºc (see more detailed description of the material and methods in supplementary material). the generalized linear model (in spss v. 20) with a linear scale response and identity link function was applied to compare both groups. data before the glm were log transformed. glm model indicate that that there is no significant effect (p = 0.122) of group (control/workers form hot sand), and therefore no difference in hsps expression between ants collected in the morning when the external temperature was low (23ºc) and ants taken from hot sand during the middle of the day when the surface temperature was very high (up to 47 – 54ºc). no effect of group also means, in this case, no effect of temperature. the hsp family factor (hsp60, hsp75, hsp90) had a strong effect (p < 0.001) on expression. the graph presents (fig 1) an increase of hsp75 and hsp90 expression indicating some physiological response to risk-prone conditions, whereas hsp60 remained at the same level. the interaction between group and hsp family was not significant (p = 0.587). no effect of temperature rejects the main working hypothesis that foraging on hot soil is correlated with a high level of thermal stress and thus the significant increase of hsp expression. at the same time, this result generates at least two additional questions: (1) what really is the threshold of thermal resistance in this species and (2) if f. cinerea workers do not suffer from thermal stress during foraging on a hot soil surface, are they physiologically adapted to avoid this kind of stress? the laboratory part of this experiment can help in understanding the physiological barrier of thermal resistance and answer – at least partially – the issues raised. in laboratory experiment workers of f. cinerea were collected in the morning and they originated from the same colonies as the individuals used in the field experiment. immediately after transport, the ants were placed in thermal chambers for two hours at 40ºc or 45ºc and put in rnalater after thermal stress. similarly like in field experiment the generalized linear model with a linear scale response and identity link function was applied. glm model indicates significant effect of the temperature and gene family on the hsp expression level. interaction was close to significant (p = 0.055) indicating that different hsp genes respond differently to applied temperatures. the expression of hsp60 and hsp90 increased from 23ºc to 45ºc, however the expression of hsp75 reached maximum already in 40ºc, demonstrating that a temperature of 40ºc is already a strong enough factor for the maximum expression of this gene (fig 2). comparing this result with the field part of the experiment – where hsp75 expression was the highest in a group from hot soil – it is most probable that hsp75 is most “sensitive” to heat stress in temperatures up to 40ºc. similar results were fig 1. field experiment – expression of hsp60, hsp90 and hsp75 in f. cinerea workers during foraging in field conditions. mean log. value of gene expression and 95% confidence interval (cl) error bars. fig 2. laboratory experiment – expression of hsp60, hsp75 and hsp90 in f. cinerea workers in 40ºc and 45ºc thermal chambers comparing to control (23ºc). mean log. value of gene expression and 95% confidence interval (cl) error bars. obtained by gehring & wehner (1995) on hsp70 during the study of cataglyphis. the authors interpret this protein synthesis and accumulation in low temperatures as a preadaptation of the studied species to foraging in rapidly changed conditions when workers emerge from a relatively cold nest (where hsp accumulation already starts) to lethal outdoor temperatures. the authors also emphasize that hsp70 synthesis in f. polyctena stops above the temperature of 39ºc, whereas it continues up to 45ºc in cataglyphis, making this species more heat shock resistant. following this argument, in the case of our study with f. cinerea, the synthesis of hsp75 also reach maximum at a temperature of 40ºc, but the expression of the other two genes sociobiology 62(3): 457-459 (september, 2015) 459 (hsp60 and hsp90) increases up to 45ºc (as in cataglyphis), also demonstrating some physiological adaptations to high temperatures. this result partially answers the question about physiological adaptations of f. cinerea to thermal stress in field conditions. the limits of thermal resistance was measured by the number of ant demises resulting from temperature exposure. four individuals (out of 20) of f. cinerea did not survive to the end of the thermal exposure in 40ºc and ultimately died as a result of heat stroke (or/and desiccation). the results of the experiment suggests that expression of hsps in workers of f. cinerea foraging in conditions of thermal risk is similar to control group. is it an effect of short exposition on lethal temperatures – because ants simply shorten they foraging time or just avoiding warm patches during foraging – is totally not clear. observation during the field work suggest that many workers stay in the nest during most hazardous part of the day and only some part is foraging, but what proportion and what individuals (maybe only older and more experienced are foraging) is also a mystery. outcome of the experiment indicate that workers of f. cinerea are to some point physiologically adapted to cope with high temperatures but the level of such an adaptation cannot be specified by one experiment and require further effort. most present and past experiment was conducted on typical desert animals but the knowledge about the thermal response in a species from temperate climate is somehow neglected. references feder, m. e. & hofmann, g. e. (1999). heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. annual review of physiology, 61: 243-282. gehring, w. j. & wehner, r. (1995). heat-shock proteinsynthesis and thermotolerance in cataglyphis, an ant from the sahara desert. proceedings of the national academy of sciences u.s.a., 92: 2994-2998. lenoir, a., aron, s., cerdá, x. & hefetz, a. (2009). cataglyphis desert ants: a good model for evolutionary biology in darwin’s anniversary year a review. israel journal of entomology, 39: 1-32. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3558sociobiology 66(1): 61-65 (march, 2019) nest architecture and life cycle of small carpenter bee, ceratina binghami cockerell (xylocopinae: apidae: hymenoptera) introduction small carpenter bees of the genus ceratina latreille, are a widespread bee fauna belonging to the subfamily xylocopinae. they are bright metallic blue to green bees actively foraging on wide range of crops plants (michener, 2007). ceratina binghami cockerell (1908) is polylectic and were reported to be an efficient pollinator of wide range of crops temperate fruits and oilseed crops such as niger, safflower, mustard, linseed (navatha & sreedevi, 2015), apple, raspberry, cranberry, cosmos, sunflower, red gram, tomato, winged bean, mustard, alfalfa (mattu & kumar, 2016). native bee communities serve as a needed alternative in the event of honey bee declines and may contribute substantially to crop pollination (parker et al., 1987). ceratina bees construct their nests in dead woods and pruned pithy stems by making linear burrows (raju & rao, 2006). adult female bee chew opens the central pith of the abstract the small carpenter bee, ceratina binghami (xylocopinae: apidae) is an important pollinator of many agricultural and horticultural crops. the nests constructed by the bee in the pruned pithy stems of caesalpinia pulcherrima were collected to study its biology under laboratory conditions. the bee constructs its nest in the pithy stems of different plants by chewing out the pith. the bee is polylectic and provision with pollen balls to its brood cells with an interesting nest guarding behavior. the nests consisted of egg, larvae, prepupae, pupae and adult stages. life cycle of the bee was completed in 41.67 ± 3.12 days. pupal stage consisted of different colors of eye pigmentation. foraging activity of the bee started during morning hours approximately between 6.45 to 7.15 am and ended during the late evening hours of 4.50 to 5.15 pm. detailed nest architecture of the bee was studied. the nesting behavior and short life cycle of c. binghami in pithy stems of c. pulcherrima helps in the in-situ conservation and utilization of these bees in the pollination of agricultural crops. sociobiology an international journal on social insects a udayakumar, tm shivalingaswamy article history edited by celso martins, ufpb, brazil received 03 july 2018 initial acceptance 13 september 2018 final acceptance 12 october 2018 publication date 25 april 2019 keywords caesalpinia; ceratina; foraging; nesting: life cycle; nest architecture. corresponding author amala udayakumar division of germplasm conservation and utilization icar-national bureau of agricultural insect resources h a farm post, pb no 2491 bengaluru 560024, karnataka india. e-mail: amala.uday@gmail.com pruned stem and construct cells. then the female flies out, forage for the pollen, mould them into pollen balls and place them inside the cells (mcintosh, 1996). on this pollen ball provision, she lays the egg. after provisioning, few species of bees cap the nest entrance with a membranous layer using the chewed pith and leave the nest. but in few other species, females were found to remain in their nests, guarding their broods till they complete development (sakagami & maeta, 1977). the nesting biology of several species of ceratina viz., c. smaragdula (kapil & kumar, 1969; batra, 1976b) and c. calcarata (rehan et al., 2010) was well documented and could be easily conserved in-situ using pithy stems of different plants in different habitats. destruction of natural nesting habitats of these native bees will have a direct negative impact on the valuable pollination provided by them. the decreasing abundance and species richness of native bees in agricultural landscapes due to habitat loss and lack of foraging resources was well division of germplasm conservation and utilization, icar-national bureau of agricultural insect resources, bengaluru, karnataka, india research article bees a udayakumar, tm shivalingaswamy – nest architecture and biology of ceratina binghami62 reported (kevan, 1999; ricketts, 2004). understanding of nesting behavior and life cycle will enable us to conserve the bees and utilize them for efficient pollination in different crops. the present investigation on c. binghami, an efficient crop pollinator was undertaken to study its life cycle and nest architecture. materials and methods study location the study was carried out in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bangalore-yelahanka campus (13.096792n, 77.5659 76e). the stems of five-year-old peacock flower, caesalpinia pulcherrima (fabaceae) were pruned as a standard practice to enhance the branching and flowering of the plant during the first fortnight of september 2016. the cut ends of the stems were the site of attraction for the bees for nest building (fig 1) and were regularly monitored for the presence of nests constructed by the ceratina bees. nest architecture and biology of ceratina binghami the nest construction by the bees were identified by the presence of holes at the cut ends of the pruned stems in few cases and by the sighting the bees entering the holes in some places. twenty-five nests were destructively sampled from the c. pulcherrima plants located adjacent to the two parcels of farmland. the nests were collected during the evening hours to ensure the presence of adult bees in the nest after foraging during the day. the entrance hole of the nests was closed with a piece of cotton and tied with a rubber band to prevent the escape of the adult bees present inside the nest. the nests were cut opened carefully starting from the entrance hole giving a gentle split lengthwise using a sharp knife. once after making the linear split of the nest, the nest architecture including the nest length, brood cell length and brood cell breadth were measured and recorded. the number of immature stages, presence of pollen provisions, length and breadth of pollen provision were recorded. after examination, the two split stems were joined together with a rubber band to provide suitable micro-climate for the broods to develop and mature. the immature stages in the nest were reared till adult emergence in the laboratory (28 ± 1 ºc; 70% rh). the stems were kept inside the rearing boxes covered with a cotton cloth to provide aeration. the split stems were opened on regular basis to examine any change in the life stage of the bee. the duration of the different life stages of the bee viz., egg, larvae, pupae, adult and adult longevity was also recorded. classification of nests based on brood components the nests were classified into five categories viz., hibernacula nests, founding nests, active brood nests, full brood nests and mature brood nests as per the descriptions by daly (1966) based on the life stages of the bees present in the cells. hibernacula nests include the nests that were built during the previous season with their interior content filled with fecal remains, pollen and molted skins of previous broods. founding nests have only few cells with usually harboring adult bees actively involved in construing cells with partitions. these cells are devoid of pollen masses and immature stages of the bees. active brood nests contain pollen masses in all the formed cells with few immature stages of the bees. full brood nests inhabit the life stages of the bee at different stages of development with pollen masses of different proportions due to active feeding of the larvae. mature brood nests were found not to inhabit either pollen masses or immature stages of the bees. they contain only the fecal pellets, pupal exuviae and chewed up walls of the cells indicating the emergence and exit of the adult bees. foraging behavior of ceratina binghami the period of activity of the ceratina bees were observed and recorded in the study site. five prior marked nests were observed for a period of 10 days. the nests were inspected during early morning hours for the presence of adult bees. the time when the adult bee leaves the nest for foraging was noted down. the time spent by the bee in the nest back from foraging activity was also recorded. under field conditions, flowers visited by the bees, the reward collected and the average time spent by the bees in individual flower was also recorded. fig 1. pruned cut ends of caesalpinia pulcherrima with ceratina binghami showing nest building behavior. sociobiology 66(1): 61-65 (march, 2019) 63 results lifecycle of ceratina binghami a total of twenty five nests were collected comprising of following categories. six of them were active brood nests with a solitary mother were found along with the developing brood. eight were founding nests with clear interior walls found with signs of progressive cell construction by the adult female bee. five nests were of mature brood nests (fig 2) in nature with no pollen mass and inhabit only with late larval instars, pre-pupae, pupae and adults (in different stages of pigmentation) in the cells. four nests were of full brood nests with the pollen masses of different sizes with different stages of the larvae inside the cells. life stage life stage description duration (days) (n=25) egg 4.83±0.75 larvae one fifth of size of pollen ball 3.00±0.89 twice the size of pollen ball 3.67±0.52 small bit of pollen 3.33±0.52 fully grown larvae 3.17±0.98 total larval period 13.67±1.63 prepupae 4.50±0.54 pupae white eyed 2.67±0.81 pale brown eyed 1.67±0.52 dark brown eyed 2.17±0.40 black eyed 3.67±0.81 total pupal period 10.50±2.07 adult 1/2 body pigmented 1.17±0.41 1/4 body pigmented 1.50±0.55 3/4 body pigmented 1.33±0.52 full body pigmented 3.50±1.51 total adult period 7.67±1.50 egg to adult 41.67± 3.12 table 1. life cycle of ceratina binghami. fig 2. full brood nest and pollen provision made by ceratina binghami in pithy stems of caesalpinia pulcherrima. fig 3. pupae of ceratina binghami at different stages of eye colouration. the larvae required 13.67 ± 1.63 days (n = 25) to complete its development. pre-pupae metamorphose into white eyed pupae, later the eye color changed into pale brown, dark brown and then black in color (fig 3). the duration of the pupal stage lasted for 10.5 ± 2.07 days (n = 25) with changing eye pigmentation from lighter to dark at different levels. after reaching the black-eyed stage of the pupae, the body the bee gradually attains pigmentation at different stages. the body color of the adult changed from gradual pigmentation to complete pigmentation (fig 4), in 3.5 ± 1.51 days. after full pigmentation, the pupae molted into winged adults. the adult bees were found to chew upon the partition layer of the cells to ensure easier emergence from the nest. the total life cycle of c. binghami was completed in 41.67 ± 3.12 days. the life cycle of c. binghami consisted of egg, larvae, pupae and adult stages (table 1). the adult females laid single egg over the pollen provision in individual cells. the pollen provisions were yellowish brown in colour, slightly viscous, soft and ranged between 0.8 to 0.9 cm in length (n = 25). the egg appeared to be spindle shaped, translucent white in color, placed dorsally over the top of the pollen ball to ensure the immediate availability of food for the hatching larvae (fig 2). the apodous larvae were found to feed actively over the pollen mass in each cell. the pollen mass was found to be bigger in size in the cells containing the early instar larvae and vice versa in the case of mature larvae. this may be due to depletion of the nutritious pollen fed by the growing larvae. a udayakumar, tm shivalingaswamy – nest architecture and biology of ceratina binghami64 nest architecture ceratina binghami was found to construct linear nests in inside the cut ends of the pithy stems of caesalpinia pulcherrima (l.) (fabaceae) plants. when the nests were dissected, adult females were found to stay in a defensive position with their head or abdomen in some nests facing upwards guarding the brood cells. all the completed nests were found to harbor adult female bees guarding the nest entrance (fig 1). in few of the nests, the adult females were observed near the septa of the second or third cells. they were observed to repair the broken septa during the brood development. the nests were unbranched with average length of 8.04 ± 1.93 cm (n = 25). the pith of the stem was used by the bees to form the cell partition, the depth of which was recorded to be 0.57 ± 0.06 cm respectively. out of the twenty-five nests sampled, all the nests were found to harbor different life stages of the bees. none of the nests were either found to be orphaned (without adults) or attacked by any parasitoids. the diameter of the nest entrance hole was found to be 1.26 ± 0.11 cm wide. each nest was found to contain 6-7 cells with an average with a length and breadth of 1.0cm and 1.0cm respectively. foraging behavior ceratina bees were found to visit many species of flowering plant species and hence are polylectic in habit. among cultivated crops, the bees were found foraging on flowers of winged bean, tomato, pigeon pea, corn and sunnhemp. on an average, the bees spent 21.4 seconds in individual flower. the bees were found to collect pollen reward back to the nest. the pollen laden bee entered the nest through the entrance hole and spent approximately 14.8 seconds inside the nest and then started the next foraging bout. foraging started during the morning hours between a time period of 6.45 to 7.15 am and ended during the late evening hours of 4.50 to 5.15 pm. fig 4. adult of ceratina binghami at different stages of body pigmentation. discussion in the present study, c. binghami preferred the pithy stems of caesalpinia pulcherrima but we have also noticed its nesting activity in other plants like adhathoda zeylanica and adenanthera pavonina. the lifecycle, nest site selection and nest architecture vary between the species, climate, availability of plants and various other factors. ali et al. (2016) reported that ceratina (pithitis) smaragdula made its nests in wooden stalks of ravenna grass (saccharum ravennae l., poaceae), with their life cycle completed between 28 to 32 days under laboratory conditions in pakistan. adult females were found to guard the nest entrance with their head facing upwards in few nests and with their abdomen in other few nests. the adult bees were found crawling over the brood cells in few nests during the dissection. this might be due to the examination of the provisioned pollen provided by the females in the individual cells for the brood development. the sub social behavior of female bees crawling through the cell partitions in the nest to examine the offspring was reported by maeta et al., 1997. the nest guarding behavior in a completely provisioned nest by an adult female is advantage for the offspring as it can increase the offspring survival by protecting from the natural enemies. the dissected nests showed 100% emergence of adult bees without any signs of parasitization which might be due to the parental care provided by the adult bee as exhibited by their guarding behavior. increased offspring survival as a result of guarding strategy by adult females of c. cucurbitina was reported by mikat et al., 2016. ceratina calcarata was observed to be a sub-social species whose females build their nests solitarily but remain in the nest and continue to care for their offspring till they reach adulthood (durant et al., 2015). rehan and richards (2010) reported the similar trend in the nesting biology of c. calcarata with duration of 46 days to complete single generation. all the inspected nests were found to inhabit different life stages of the bees. in some nests, empty cells were found between the cells with life stages of the bees. few broods might have completed their development and emerged out of the nests leaving their cells empty amidst the other cells. those empty cells were found to possess eaten up cell partitions with molted exuviae in the cells as evident of their emergence. the pupae were found near the entrance in most of the nests. the results were in line with the observations made by yogi (2014) who reported the orientation of the pupae of c. simillima towards the entrance of the nest. in his study, c. sociobiology 66(1): 61-65 (march, 2019) 65 simillima was found to utilize caesalpinia stems as nesting site. hongjamrassilp and warrit (2014) reported the similar nest architecture of a candidate bee xylocopa nasalis belonging to the subfamily xylocopinae with linear unbranched nest with pithy partitions made by the founding female. conclusion in conclusion, the nest architecture and life cycle details provided in this study will be of immense importance to melittologists involved in trapping and conservation of ceratina bees. pithy stems of the caesalpinia can be used to conserve these bees by serving as the artificial nesting substrates for the bees for their valuable ecosystem service in terms of crop pollination. farmers can be encouraged to provide artificial nesting substrates using the pithy stems of c. pulcherrima to conserve these bees or by growing these plants as hedges in the farms and periodical pruning not only helps in conservation of the bees and enhanced farm scaping. author’s contribution the first author carried out the field visits and studied the nest architecture and nesting biology of the bee. the second author conceived of the study and participated in design and coordination. both the authors read and approved the final manuscript. references ali, h., alqarni, a.s., shebl, m. & engel m.s. (2016). notes on the nesting biology of the small carpenter bee ceratina smaragdula (hymenoptera: apidae) in northwestern pakistan. florida entomologist, 99: 89-93. bohart, g.e. (1967). pollination of alfalfa and red clover. annual review of entomology, 2: 355-80. durrant, d.r. berens, a.j., toth, a.l. & rehan s. (2015). transcriptional profiling of overwintering gene expression in the small carpenter bee, ceratina calcarata. apidologie, 47: 572-582. doi: 10.1007/s13592-015-0402-x. hongjamrassilp, w. & warrit, n. (2014). nesting biology of an oriental carpenter bee, xylocopa (biluna) nasalis westwood in thailand (hymenoptera, apidae, xylocopinae). journal of hymenoptera research, 41: 75-94. kapil, r.p. & kumar, s. (1969). biology of ceratina binghami cockerell (ceratinini: hymenoptera). journal of research, 6: 359-371. kevan, p.g. (1999): pollinators as bioindicators of the state of the environment: species, activity and diversity. agriculture ecosystems and environment, 74: 373-393. kidokoro, m. k. & higashi, s. (2010). flower constancy in the generalist pollinator ceratina flavipes (hymenoptera: apidae): an evaluation by pollen analysis. psyche: 1-8. maeta, y., sierra, e. a. & sakagami, s. f. (1997). comparative studies on the in-nest behaviors of small carpenter bees, the genus ceratina (hymenoptera: xylocopinae): i. ceratina cucurbitina. journal of entomology, 65: 471-481. mattu, v.k. & kumar, a. (2016). diversity and relative abundance of solitary bees on jatropha curcas crop in sirmour and solan hills of himachal pradesh, india. international journal of science and research, 5: 1815-1818. mcintosh, m. 1996. nest-substrate preferences of the twignesters ceratina acantha, ceratina nanula (apidae) and pemphredon lethifer (sphecidae). journal of the kansas entomological society, 69: 216-231. mikat, m., cerna, k. & straka, j. (2016). major benefits of guarding behavior in subsocial bees: implications for social evolution. ecology and evolution, 6: 6784-6797. michener, c.d. (2007). the bees of the world (2nd edition). johns hopkins university press, baltimore, maryland, 953p. navatha, l. & sreedevi, k. (2015). pollinator diversity of solitary bees in oilseed crops. current biotica, 8(4): 375-381. popov, v. b. (1967a). wild bees (hymenoptera, apoidea) of central asia and their relationships to flowering plants [in russian]. proceedings of the zoological institute of the russian academy of sciences, 38: 11-329. raju, a.j.s. & rao, s.p. (2006). nesting habits, floral resources and foraging ecology of large carpenter bees (xylocopa latipes and xylocopa pubescens) in india. current science, 90: 1210-1217. rehan, s. m. & richards, m. h. (2010). nesting biology and subsociality in ceratina calcarata (hymenoptera: apidae). canadian entomologist, 142: 65-74. ricketts, t.h. (2004). tropical forest fragments enhance pollinator activity in nearby coffee crops. conservation biology, 18: 1262-1271. sakagami, s.f. & maeta, y. (1977). some presumably presocial habits of japanese ceratina bees, with notes on various social types in hymenoptera. insectes sociaux, 24: 319-343. sundararaju, d. (2011). diversity of bee pollinators and flora in cashew. journal of horticultural sciences, 6: 52-55. yogi, m.k. & khan, m.s. (2014). nesting biology of the small carpenter bees ceratina propinqua and ceratina simillima (hymenoptera: apidae). animal biology, 64: 207-216. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2837sociobiology 65(3): 491-496 (september, 2018) nesting biology, seasonality and host range of sweat bee, hoplonomia westwoodi (gribodo) (hymenoptera: halictidae: nomiinae) introduction native wild bees provide a valuable pollination service in a wide range of crops in natural and managed ecosystems. with the recent decline in honeybee colonies due to various biotic and abiotic factors, there is an urgent and planned need to conserve the native solitary bees for their valuable ecosystem service. the pollen in some flowers might be concealed or trapped in the anthers and it needs natural or artificial vibratory movement to release the pollen for effecting pollination. the bee with an inherent ability to sonicate the flowers and collect the pollen from the poricidal anthers is an efficient pollinator. they make buzzing movement using their thoracic wing muscles as a result of which the pollen gets released from the terminal pores and gets coated over the bee’s abdomen thereby it reaches the nest (buchmann, abstract nesting biology and seasonal dynamics of halictid bee, hoplonomia westwoodi (nomiinae: halictidae) was studied at icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus (13.096792n, 77.565976e) india from july 2016 to may 2017. the bee built subterranean nests on a leveled soil surface with turrets and the main shaft running to a depth of 70.1 cm. in total, nineteen cells were observed in clusters at different depths. different life stages of the bee were observed in the cells. the life cycle of the bee was completed in 41.80 days. the bees were found actively foraging on different flora belonging to the different families like acanthaceae, asteraceae, convolvulaceae, fabaceae, lamiaceae, malpighiaceae, polygonaceae, rubiaceae and solanceae throughout the year with the peak population during the months of june to november. marked preference and behavior of buzz pollination was observed on the flowers of solanaceous crops like tomato and eggplant. sociobiology an international journal on social insects a udayakumar 1, tm shivalingaswamy2 article history edited by gilberto m. m. santos, uefs, brazil received 05 january 2018 initial acceptance 20 march 2018 final acceptance 28 march 2018 publication date 02 october 2018 keywords hoplonomia westwoodi, nomiinae, seasonality, solanaceae. corresponding author amala udayakumar division of germplasm conservation and utilization, icar national bureau of agricultural insect resources (nbair) bengaluru, 560024, india. e-mail: amala.uday@gmail.com 2000). major buzz pollinating bees belongs to four families viz., andrenidae, halictidae, and colletidae. few species of bees like blue banded bee, amegilla sp (apidae) are known to buzz pollinate the flowers with poricidal anthers (harter et al., 2002; hogendoorn et al., 2006; santos et al., 2014). native bee hoplonomia westwoodi (nomiinae: halictidae) is one such efficient buzz pollinators of solanum violaceum in sri lanka (wanigasekara & karunaratne, 2012), tomato solanum lycopersicum (amala & shivalingaswamy, 2017), cleome viscosa, lablab purpureus, vigna sp, ocimum basilicum (karunaratne et al., 2005). seasonal composition of available floral resources as a major factor in the attraction of foraging activity of the native bees (tomassi et al., 2004; cane et al., 2006). as the tomato flowers does not provide nectar reward and the pollen being accessible only for buzz pollinators and honey bees do visit the tomato flowers but 1 scientist (entomology), division of germplasm conservation and utilization, icar – national bureau of agricultural insect resources (nbair), bengaluru, india 2 principal scientist (entomology), division of germplasm conservation and utilization, icar – national bureau of agricultural insect resources (nbair), bengaluru, india research article bees a udayakumar , tm shivalingaswamy – nesting biology, seasonality and host range of sweat bee492 seldom pollinates them (buchmann, 1983; free, 1993). amala and shivalingaswamy (2017) reported the flower visitation and pollination in tomato by h. westwoodi resulting in a significant increase in fruit weight (46.96 g) and seed set per fruit (140.50 seeds) compared to the self-pollinated flowers. these buzz pollinator bees have been reported to improve self-pollination of the crops having poricidal anthers whose pollen grains released are deposited on the stigma of the same flower (king & buchmann, 2003; greenleaf & kremen, 2006). one of the major reasons for the poor fruit formation in some crops was the lack of temporal coincidence between the anthesis and occurrence of pollinators. a decline in pollinator populations due to the habitat loss and fragmentation of farm lands may be the reason for this non-synchrony of anthesis and presence of pollinators (klein et al., 2007, winfree et al., 2007). in view of this situation, there is an immense need to understand the nesting behavior of the native buzz pollinating bee, h. westwoodi so as to conserve this pollinator in agro-ecosystems and enhance their role in pollination of different crops. though it is a well reported buzz pollinator, information on nesting biology of h. westwoodi is scanty. the present study aims at studying the nesting biology of sweat bee, h. westwoodi, its seasonality and spectrum of foraging flora in a farm landscape. materials and methods study site the present study was carried out in yelahanka campus experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, india (13.096792n, 77.565976e) from july 2016 to may 2017. the study area of 22 acres comprising of cultivated crop lands with various annual crops like cereals and pulses, orchard blocks of mango, sapota, cherimoya. also, two patches of pollinator gardens of about 1.5 acres with over 100 plant species of diverse plant families were also part of this study location which is right in the heart of a rapidly growing hightech-city and capital of south indian state of karnataka. nest architecture and biology of h. westwoodi based on our experience on the natural nesting sites of h. westwoodi, we used two artificial nesting structures. first one was a polypropylene bag (30x30 cm) and the other one was an earthen flower pot (60x30cm) filled with soil which was similar to the natural nesting area. the soil surface was levelled properly in these structures. care was taken that these nesting structures were kept in close proximity to flowering plants so as to attract foraging h. westwoodi for nesting. twenty each of these simulated nesting structures were placed in different areas adjoining the study site to invite the bees for nesting. weekly observations of the nest building activity in the soil filled flower pots and polypropylene bags installed in experimental farm of icar-nbair yelahanka campus, bengaluru were made during july 2016 to may 2017. active nests were identified by the presence of turrets and tumulus with entrance holes in both the artificial structures provided for nesting. the adult bee activity of entering and exiting from the entrance holes of the nest was observed. presence of fresh turret and sighting of the pollen laden adult female bee entering the turret was a clear indication of the newly formed nest with broods. nest excavation was carried out by digging carefully the soil around the nest to study the nest architecture. the number of pollen collection trips made by the adult female was observed between 7.30 am to 5.30 pm and recorded. tunnel length and width, number of cells built, number of cells with active brood, number of empty cells, number of adult bees were observed and recorded from nineteen brood cells in the laboratory. the distance between each nest, nest dimensions such as length of turret, diameter of entrance, burrow diameters, nest depths were recorded. presence of foundress in the nest if any was also recorded. the brood clusters were cut opened carefully to examine the inner contents. the larvae observed in the brood cells were reared in the laboratory till pupation and observed for emergence of adults. egg, larvae and pupae (n=25 each) were taken in meshed boxes (9x4 cm) were maintained in the laboratory and the rate of survival of each stage was recorded. the duration of egg, larva and pupal stages of the bee was observed and recorded. mortality of life stages of the bee and causes for the mortality if any was also observed. seasonal incidence of hoplonomia westwoodi the seasonal incidence of h. westwoodi was studied in the pollinator garden of icar-nbair yelahanka campus between july 2016 and december 2017. bees foraging over the flora were sampled using sweep nets at weekly intervals. sweeping was done between 08.00 hrs to 14.00 hrs during the day of observation. the collected bees were killed using ethyl acetate and brought to the laboratory. the bee specimens were pinned, taxonomically identified and preserved. the total number of adult bees collected were observed and recorded. host plant preference of h. westwoodi a wide array of diverse flowering plants in the experimental farm was observed for the visitation by the bee between july 2016 and may 2017 at fortnightly intervals. the different families of the flowering plants preferred by the bee for foraging was observed and recorded. the number of visits made by h. westwoodi per five minutes time period per flower was recorded at different time points, 8.00-9.00 am, 10.0011.00 am, 12.00-1.00 pm, 2.00-3.00 pm and 4.00 to 5.00 pm. the reward in terms of pollen and nectar collected by h. westwoodi during each visit was also recorded. results and discussion sociobiology 65(3): 491-496 (september, 2018) 493 nesting site the nests of h. westwoodi were observed in the artificial nesting structuresearthen flower pots and soil filled polypropylene bags placed in the experimental farm of icar-nbair yelahanka campus bengaluru. the bees were observed to construct their nests in these structures placed in close proximity to the flowering plants. more number of nests were found in the earthen flower pots filled with dried leaf litters compared to bags. some of the nest entrances were exposed but a few was found to be hidden under the dried leaf litter. upon careful observation of adult bee activity of entering inside the entry hole, presence of few nests was confirmed. on a bright morning after a previous day rainfall, the surviving foundress was observed repairing its collapsed nest turrets. the bees were found to actively construct their nests during june to november as evident from the soil trap nests in the present study. active nesting of h. westwoodi during this period could be due to the copious availability of blooming flora providing rich pollen for its broods. nest architecture of h. westwoodi the nest entrance was circular with a diameter of 0.5 cm with a turret measuring 1.2 cm high sometimes appeared hard in few places. the female bee was observed to use more than one nest entrance to gain entry into the tunnel as the nests were branched and found connected with each other in soil. the main tunnel had branches wherein the branches ended in individual or group of cells (fig 1). the main shaft of the nest tunnel ran to a depth of 70.1 cm with a diameter of 0.5 cm (n=10). horizontal laterals originated from the main shaft at a depth of 41.7 cm. cells were observed in clusters at depth ranging from 62.8 to 70.00 cm from the soil surface. this might be to ensure protection of the broods by the foundress as flooding during heavy rains was very less likely to reach brood cells. similar observations on cell construction at greater depths in the genera lasioglossum, agapostemon and most of halictus species of bees has been reported by packer et al. (1988). each cell was typically oval in shape with the walls coated with a water proof secretion. the average length and width of each cell (n=20) ranged between 10.03 to 15.0 mm and 5.25 to 5.5 mm respectively. each cell was closed with a thin layer of soil. the pollen mass provision appeared to be spherical and measured 2.5 mm high and 5.0 mm diameter placed at the side end of each cell over the top. the translucent egg was laid on the pollen mass by the adult bee. the pollen mass was slightly wet might be due to the secretions of the adult bee. the cells contained different life stages viz., egg, pre-defecating and post defecating larvae (fig 2). abundance of h. westwoodi our observations indicated that population of h. westwoodi occur throughout the year with a peak occurrence during the month of june 2016 to may 2017. the active nests with foraging female bees were also found during the period of june to october. the reason could be that foundress bee could involve in digging activity in wet and loose soil due to the rains between june and october. the study area with abundance of blooming flora coinciding with the rainy season supplying pollen for the foraging females could also be the influencing factor for higher abundance of the bee during this period. also another factor could be the availability of standing crop of field bean, lablab purpureus l. planted in the experimental farm provided constant supply of pollen would have supported the active population of h. westwoodi. number of pollen and nectar rewarding plants in the farm was found to be constant throughout the study period but richness of the flora was comparatively lesser during the later period (mid november to early december) which was marked by decreased activity of h. westwoodi (fig.3). the fig 1. nest architecture of hoplonomia westwoodi. a udayakumar , tm shivalingaswamy – nesting biology, seasonality and host range of sweat bee494 seasonal incidence and activity of the bee is influenced by the phenology of flowering plants and their diversity, land use and nesting suitability (shebl et al., 2016). availability of an appropriate seasonal floral base that provides nectar and pollen to the foraging bees is vital for the survival and reproduction of the bees. the frequency of a particular flower visitor in the target crop is an important factor that determines its efficiency in pollination of that crop. biology of h. westwoodi the duration of egg, larva and pupal stages lasted for 5.6±1.14, 23.40±3.58 and 12.80±2.17 days, respectively. fig 2. a. nest turret; b. female bee visiting the nest; c. multiple entrance of the nest; d. egg; e. different larval stages; f. late larva and prepupa; g. pupa. fig 3. abundance pattern (number of visitors per flower) of the sweet bee, hoplonomia westwoodi (2016-2017). sociobiology 65(3): 491-496 (september, 2018) 495 overall egg to adult stage was completed in 41.8 days. the predefecating larvae appeared curved, whitish in color covered in the pollen provision. on the contrary, the post defecating larvae appeared fully grown, curved, yellowish white with fecal pellets scattered in the cells and devoid of pollen provision. a marked difference in the color of the eyes during pupal stage was observed. initially the pupae appeared white eyed, later the color changed to pale brown and finally dark brown in color. none of the pupae was found to be in diapause stage. there was no evidence of brood parasitism in the excavated nests as there was 100 percent rate of emergence of adult bees from the pupal brood. there were no signs of parasitism or predation in the excavated brood cells containing the larvae. during the process of excavation more than four females were found in the shaft, which could be the probable reason for nonincidence of parasitism of the broods inside the nest. the female bee was observed to conduct 8-10 pollen collection trips during in each nest per day. two females were observed entering through the same nest entrance might be due to the communal nesting behavior of h. westwoodi. wcislo, (1993) reported the communal nesting behavior in pearly-banded bee, nomia tetrazonata with multiple number of female bees occuring in the same nest. host plant preference of h. westwoodi h. westwoodi was found to be broadly polylectic in nature. our observations showed a marked preference of this bee to solanaceous crops like tomato and eggplant. the descending order of visitation range of h. westwoodi in the flora belonging to different families was found to be in the order of solanaceae >lamiaceae >fabaceae > asteraceae > malpighiaceae > acanthaceae > rubiaceae > convolvulaceae. this could be due to the need of buzzing activity of the native h. westwoodi which is essential for the pollination of tomato flowers. apart from the solanaceous plants, the bees were found to forage on a variety of the flowers of acanthaceae, asteraceae, convolvulaceae, fabaceae, lamiaceae, malpighiaceae, polygonaceae, rubiaceae in the study site (table 1). the bees were observed to collect the nectar reward from the flowers of the foraging plants. whereas in the solanaceous flowers viz., tomato and eggplant, the bees were found to buzz pollinate and collect the pollen over the hairs present in the legs and abdomen. batra (1966) reported many species of nomia viz., n. oxybeloides, n. capitata as polylectic in habit exhibiting social and semi-social behavior. with the growing decline in insect pollinators worldwide, there is an immense need to conserve the native, efficient buzz pollinating bees like h. westwoodi. studies on seasonal abundance and nesting habit of this native bees helps in the understanding their ecology, plant preferences by which they can be conserved in-situ in their natural breeding habitats. acknowledgments the authors are thankful to dr. chandish r. ballal, director, icar-nbair for providing facilities and constant encouragement for conducting the study. references amala, u., & shivalingaswamy, t. m. (2017). role of native buzz pollinator bees in enhancing fruit and seed set in tomatoes under open field conditions. journal of entomology and zoology studies, 5: 1742-1744 batra, s.w.t. (1966). social behaviour and nests of some nomiinae bees in india (hymenoptera: halictidae). insectes sociaux, 13: 145-154 buchmann, s.l. (2000). buzz pollination in angiosperms. handbook on pollination biology. 73-113. cane, j. h., minckley, r. l., kervin, l., & roulston, t. h. (2006). complex responses within a desert bee guild (hymenoptera: apiformes) to urban habitat fragmentation. ecological applications, 16: 632-644. greenleaf, s.s., & kremen, c. (2006). wild bee species increase tomato production and respond differently to surrounding land use in northern california. biological conservation, 13: 81-87. doi: 10.1016/2006.05.025. harter, b., leistikow, c., wilm, s, w., truylio, b., & engels, w. (2002). bees collecting pollen from flowers with poricidal anthers in a south brazilian araucaria forest: a community study. journal of apicultural research, 40: 9-16. doi: 10. 1080/00218839.2002.11101063. hogendoorn k, gross cl, sedgley m, keller ma. (2006). table 1. host plants and rewards collected by h. westwoodi. family plant species reward collected pollen nectar acanthaceae asystasia gangetica (l.) t. anderson ++ asteraceae gaillardia pulchella foug. ++ convolvulaceae jacquemontia sp. ++ argyreia cuneata ker gawl. ++ fabaceae crotolaria retusa l. ++ lamiaceae ocimum basilicum l. -++ ocimum gratissimum l. -++ strobilanthus barbatus nees -++ malpighiaceae tristellateia australasiae a. rich ++ polygonaceae antigonon leptopus hook. & arn. ++ rubiaceae hamelia patens jacq. ++ solanaceae solanum lycopersicon l. ++ -solanum melongena l. ++ -a udayakumar , tm shivalingaswamy – nesting biology, seasonality and host range of sweat bee496 increased tomato yield through pollination by native australian amegilla chlorocyanea (hymenoptera: anthophorinae). journal of economic entomology, 99: 828-833. king, j., & buchmann, s.l. (2003). floral sonication by bees: mesosomal vibration by bombus and xylocopa, but not apis (hymenoptera: apidae), ejects pollen from poricidal anthers. journal of the kansas entomological society, 76: 295-305. klein, a.m., vaissiere, b., cane, j.h., steffan-dewenter, i., cunningham, s.a., kremen, c., & tscharntke, t. (2007). importance of crop pollinators in changing landscapes for world crops. proceedings. biological sciences, 274: 303-313. doi:10.1098/rspb.2006.3721. packer, l., sampson, b., lockerbie, c., & jessom, v. (1988). nest architecture and brood mortality in four species of sweat bee (hymenoptera; halictidae) from cape breton island. canadian journal of zoology, 67: 2864-2870 santos aor, bartelli bf, nogueira-ferreira fh. (2014) .potential pollinators of tomato, lycopersicon esculentum (solanaceae), in open crops and the effect of a solitary bee in fruit set and quality. journal of economic entomology, 107: 987-994. shebl, m.a., al aser, r.m., & ibrahim, a. (2016). nesting biology and seasonality of long-horned bee eucera nigrilabris lepeletier (hym., apidae). sociobiology, 63: 1031-1037. tommasi, d., miro, a., higo, h.a., & winston, m.l. (2004). bee diversity and abundance in an urban setting. canadian entomologist, 136: 851-869. wanigasekara, r. w. m. u. m., & karunaratne, w. a. i. p. (2012). efficiency of buzzing bees in fruit set and seed set of solanum violaceum in sri lanka. psyche, id 231638. 1-7. doi:10.1155/2012/231638 wcislo, w.t. 1993. communal nesting in a north american pearly-banded bee, nomia tetrazonata, with notes on nesting behavior of dieunomia heteropoda (hymenoptera: halictidae: nomiinae). annals of the entomological society of america 86: 814-821 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 30-34 (2013) influence of camponotus blandus (formicinae) and flower buds on the occurrence of parrhasius polibetes (lepidoptera: lycaenidae) in banisteriopsis malifolia (malpighiaceae) e alves-silva ¹, a bächtold ², gj barônio ¹, k del-claro ¹ introduction lycaenids are abundant and widely distributed in the neotropics, accounting for 1200 species distributed in several biomes and vegetation types (brown jr., 1993; robbins & lamas, 2004). in brazil, lycaenid records are based mostly on adult individuals, thus little is known about the larval host range (emery et al., 2006; francini et al., 2011). lycaenid larvae generally feed on plant reproductive parts, such as flowers and flower buds (robbins & aiello, 1982). however, despite the vast diversity of flora in brazilian biomes, only recently the interactions among lycaenid larvae, their host plants, and mutualistic ants have been considered in ecological studies (kaminski & freitas, 2010; kaminski et al., 2010a; silva et al., 2011; bächtold & alves-silva, 2012). abstract in the brazilian savanna, myrmecophilous lycaenids are often found in many shrubs feeding on plant reproductive structures while are tended by ants, but only recently the role of both ants and food on the occurrence of lycaenids have received attention. in this study, we investigated the influence of camponotus blandus (formicinae) and flower bud abundance on the occurrence of parrhasius polibetes, a florivorous lycaenid species that occurs in banisteriopsis malifolia (malpighiaceae). we also examined to what extent larval florivory was deleterious to plant reproductive outputs. ant-exclusion experiments revealed that most p. polibetes individuals were found on branches with free c. blandus access. nonetheless, the occurrence of larvae was not related to the abundance of ants and flower buds, indicating that the presence, rather than the abundance of mutualistic ants and food, influenced the occurrence of p. polibetes. larvae were attended by c. blandus, which antennated frequently the dorsal nectary organ of larvae. larval florivory was not deleterious to the plant. banisteriopsis malifolia produces thousands of buds simultaneously and larvae feed only on a small portion of flower buds. the occurrence of p. polibetes in b. malifolia is advantageous for the larvae, since this plant supports mutualistic ants and plenty of food resources. sociobiology an international journal on social insects 1 universidade federal de uberlândia, uberlândia, minas gerais, brazil 2 universidade de são paulo, ribeirão preto, são paulo, brazil research article ants article history edited by: gilberto m m santos, uefs brazil received 07 january 2013 initial acceptance 02 february 2013 final acceptance 16 february 2013 keywords brazilian savanna; florivory; larval food; myrmecophily corresponding author estevão alves-silva instituto de biologia, universidade federal de uberlândia, rua ceará, s/n. bloco 2d campus umuarama uberlândia, mg, brazil, 38400-902 e-mail: estevaokienzan@yahoo.com.br lycaenidae has one of the most remarkable interactions within the lepidoptera – mutualistic associations with ants (pierce et al., 2002). larvae have dorsal nectary organs (dnos) and perforated cupolas organs (pcos) (fiedler, 1991). the former releases a sugared substance while the latter pacifies the aggressive behavior of tending ants (malicky, 1970). in this context, studies have shown that lycaenid female oviposition choices may be ant-mediated (seufert & fiedler, 1996; wynhoff et al., 2008). this trend was observed in parrhasius polibetes (stoll), a facultative myrmecophilous species that is frequent in the brazilian cerrado savanna (silva et al., 2011). kaminski et al. (2010a) showed that p. polibetes female oviposition was mediated by the presence of tending ants. ants may increase larval performance and survivorship by protecting them from natural enemies (weeks, 2003). in these cases, females will seek more fasociobiology 60(1): 30-34 (2013) 31 vorable plants, taking into account not only the presence of ants, but in the case of p. polibetes, females will also seek for high quality food (rodrigues et al., 2010). the availability of food items also influences the occurrence of lycaenids (wagner & kurina, 1997) and by feeding on plant reproductive structures, larvae are supposed to exert negative effects on plant fitness (oliveira & del-claro, 2005). for instance, badenes-pérez et al. (2010) discussed the use of lycaenids as biological control agents against the invasive species miconia calvescens dc. (melastomataceae) in costa rica, as larvae were observed to damage up to 30% of the reproductive structures of the plant (see also jordano et al., 1990). in the brazilian savanna (cerrado biome), the extrafloral (efn) nectaried shrub banisteriopsis malifolia (nees & martius) b. gates (malpighiaceae) is patrolled by a wide range of efn feeding ants (alves-silva, 2011). camponotus blandus (smith), formicinae, is one of the most abundant ant species in b. malifolia, being very aggressive towards other arthropods (alves-silva et al., 2012). nonetheless, c. blandus has mutualistic relationships with membracids (oliveira & del-claro, 2005). facultative myrmecophilous lycaenids are found in b. malifolia feeding on flower buds and are susceptible to ant contact, but whether larvae are attended by c. blandus and the role of ant presence on larval occurrence have not previously been studied. in this study, we investigated the influence of i) ants (presence and abundance) and ii) flower buds (presence and abundance) on the occurrence of lycaenids in b. malifolia. we also examined the quantity of flowers buds consumed by larvae to investigate whether larvae negatively influenced plant fitness. to conclude, we conducted observations of the behavior of c. blandus towards p. polibetes larvae and towards other herbivores. an appreciation of the factors involved in the occurrence of lycaenid larvae in plants of common occurrence can be a tool for the understanding of ant-lycaenid mutualisms in the neotropics, especially in the brazilian savanna (cerrado). material and methods study area the study was conducted in a strictu sensu cerrado area (18º59’ s – 48º18’ w) in uberlândia city, brazil, from march to may 2012, which corresponds to the reproductive season of b. malifolia. the cerrado covers about 230 hectares and is dominated by shrubs and trees ranging between 2 4m tall. the climate is markedly seasonal with a dry winter (may to september) and a rainy summer (october to april). plant species banisteriopsis malifolia is a small shrub (< 2 meters high). leaves have small trichomes on both sides and bear a pair of efns at the base, near the petiole. flower bud production starts in march and peaks in mid-april. buds are on average 7-10 mm in diameter, pinkish and bear eight oil glands in its circumference. flower buds grow on inflorescences located at the apex of branches. ant-exclusion experiment the role of ant presence on the occurrence of lycaenids was examined in 30 individuals of b. malifolia, distributed evenly in approximately 10 ha within the study area. all shrubs were patrolled by c. blandus. a control and a treatment branch containing flower buds and young leaves with functional efns were tagged in each plant individual in late march. at the base of treatment branches (n = 30), a layer of atoxic wax (tanglefoot® grand rapids, mi, usa) was applied to prevent the access of ants to the plant structures (nahas et al., 2012). the control branches (n = 30) were left unaltered, allowing the free access of ants to the plant parts. at this occasion, all b. malifolia shrubs were carefully examined and no immature lycaenids (egg or larva) were seen. sampling lycaenid sampling was performed once a week after wax application, from the first week of april until the end of may. on each occasion, buds, flowers, shoots, and both sides of leaves of the treatment and control branches were examined. parrhasius polibetes larvae found in the field were then collected, individualized in plastic containers (250 ml), and reared in the laboratory until pupation. other lycaenid larvae that were observed on the plant were also collected and reared in the laboratory. the comparison between the number of p. polibetes larvae found in the treatment and control branches was made with a chi-squared test with yates correction. the abundance of c. blandus and flower buds was also estimated at the beginning of the study. flower bud counting was made in a randomly selected inflorescence within each plant. the number of ants foraging on each b. malifolia individual was counted once. the difference in the abundance of ants and flower buds in plants with and without p. polibetes was made with student’s t tests (original data was log10 transformed to fit normal distribution). ant-lycaenid interactions in the field, we performed 30 hours of observation (ad libitum) on the behavior of c. blandus towards p. polibetes larvae. whenever c. blandus encountered a larva, we carefully observed whether ants attended the larvae or not. ant attendance was characterized by quickly and alternate antennation on the dorsal nectary organ, coupled with walking back and forth over or near the larva (ballmer & pratt, 1991). e alves-silva, a bächtold, gj baronio, k del-claro influence of ants on the occurrence of lycaenids32 ant hostility towards invaders camponotus blandus hostility towards invaders was examined by placing one live termite worker (nasutitermes sp. termitidae) on inflorescences of b. malifolia (n = 21 individuals). each plant received one termite. with this method, we intended to simulate possible wingless lycaenid natural enemies such as spiders, or other insects that might molest lycaenids or interfere with their feeding activity. termite baits are usually used to investigate the behavior of patrolling ants towards plant invaders (oliveira, 1997). the termites were followed for 10 minutes and interactions with ants were recorded. larval florivory the estimation of the daily bud consumption by p. polibetes larvae was made under laboratory conditions. five larvae were fed ad libitum with flower buds every 24h. florivory estimation was conducted in fourth instar larvae only, as in this stage larvae can feed on several flower buds. florivory rates were compared with the abundance of flower buds in the plants. all quantitative data is presented as mean ± standard error. results we found eleven (0.37 ± 0.11; n = 30 plants examined) p. polibetes in b. malifolia and only one larva was found in an ant-excluded branch, while all the other larvae (n = 10) were found in branches with free ant-access (χ2 = 5.82; df = 1; p < 0.05). besides p. polibetes, other three lycaenid species were found in b. malifolia, but in low abundance: rekoa sp. (0.1 ± 0.07; n = 3), r. marius (lucas) (0.03 ± 0.03; n = 1), and allosmaitia strophius (godart) (0.03 ± 0.03; n = 1). each b. malifolia had on average 16.42 ± 1.91 c. blandus individuals. this plant also presented many flower buds per inflorescence (340.07 ± 35.77). parrhasius polibetes larvae were found in nine b. malifolia and these plants had on average 10% more ants and 13% more flower buds. however, none of these variables was related to larvae occurrence (ants: t28 = 0.5523; p > 0.05; flower buds: t28 = 1.0968; p > 0.05; fig. 1). camponotus blandus attacked all termite baits placed on b. malifolia inflorescences. the ants bit the termites several times, often throwing them away from the plant. nevertheless, no lycaenid larva was preyed upon, attacked or molested by c. blandus. in fact ants attended p. polibetes larvae. the ants, alone or in groups of two to three individuals, walked over the larva and antennated the whole extension of the p. polibetes body, but the antennation was concentrated on the dorsal nectary organ (fig. 2). in the meantime, the larva continued to feed on flower buds. the other lycaenids sampled in b. malifolia were not observed being attended by ants. parrhasius polibetes larvae fed on the whole content of buds, often leaving only an empty shell comprising the external surface of buds. larvae consumed on average 17.0 ± 5.32 (range 5 – 35; n = 5 p. polibetes individuals) flower buds per day. this value refers to the feeding activity of the 4th instar larvae until pupation, which lasted on average seven days. thus each 4th instar larva can damage on average 119 flower buds, which corresponds to approximately 35% of flower buds per inflorescence (flower bud abundance per inflorescence = 340.07 ± 35.77). figure 1. relationship between the occurrence of parrhasius polibetes according to: a the abundance of camponotus blandus; and b the abundance of banisteriopsis malifolia flower buds per inflorescence. bars show the mean ± standard error. discussion according to price et al. (1995), cerrado vegetation supports a high diversity, but low abundance, of lepidopterans per species. concerning lycaenids, silva et al. (2011) showed that it is necessary large sampling effort to obtain a considerable abundance of larvae in different host plants; and despite considerable field sampling the authors found a low frequency of immature lycaenids in plants of common occurrence. this trend was also observed in our study, as we found a relative high community of lycaenids associated with b. malifolia, but the abundance of each species was low. parrhasius polibetes was the most abundant species (68.75% of individuals) while the other lycaenids were found in low numbers. the occurrence of p. polibetes in b. malifolia was observed to be mediated by the presence, but not the abundance, of c. blandus. furthermore, p. polibetes presence was not related to the abundance of flower buds, but no larvae were found in the end of b. malifolia flowering season (may). parrhasius polibetes was the only lycaenid species attended by c. blandus. ant-mediated occurrence seems to be common in p. polibetes and kaminski and rodrigues (2011) showed that p. polibetes tended by camponotus experienced increased performance and survivorship, as ants usually reduced the abundance of lycaenid natural sociobiology 60(1): 30-34 (2013) 33 enemies (see also kaminski et al., 2010a; rodrigues et al., 2010). in our study, c. blandus was very aggressive towards invaders, as demonstrated by the termite bait exposure experiment, suggesting that ants can molest or displace wingless lycaenid natural enemies. banisteriopsis malifolia supports a diverse community of arthropods, including herbivores and predators (alves-silva, 2011; alves-silva et al., 2012), but in the field no larvae was observed to be attacked or injured by natural enemies, such as spiders or parasitoids, or molested by any other insect. in our study, despite the limitation of larvae reared in the laboratory, p. polibetes fed on approximately 35% of flower buds per inflorescence, but its effect on b. malifolia fitness was negligible. banisteriopsis malifolia bears tens of inflorescences, which together may contain more than 5000 flower buds (pers. obs.). therefore, the reduction of up to 35 flower buds per day (4th instar larvae feeding estimation) will account for only a small portion of flower buds produced by the plant. in this context, the occurrence of p. polibetes in b. malifolia is unlikely to affect plant fitness to any great extent. malpighiaceae are considered as important hosts for misphere. in the neotropics, only recently the basic aspects of lycaenid life histories such as their distribution, host range, and ant-associations have received attention (kaminski & freitas, 2010; rodrigues et al., 2010; silva et al., 2011). given the risk of extinction of some species (see brown jr., 1993; kaminski et al., 2010b), lycaenid studies in the neotropics are imperative. further studies will aim at unraveling the relationships between lycaenids and malpighiaceae, as this seems to be an important host for lycaenid larval development (robbins, 1991; monteiro, 2000; kaminski & freitas, 2010; bächtold et al., 2013). acknowledgements we would like to thank fapesp (fundação de amparo à pesquisa do estado de são paulo); capes (coordenação de aperfeiçoamento de pessoal de nível superior) and cnpq (conselho nacional de desenvolvimento científico e tecnológico) for funding. references alves-silva, e. (2011). post fire resprouting of banisteriopsis malifolia (malpighiaceae) and the role of extrafloral nectaries on the associated ant fauna in a brazilian savanna. sociobiology, 58(2): 327-340. alves-silva, e., barônio, g. j., torezan-silingardi, h. m. & del-claro, k. (2012). foraging behavior of brachygastra lecheguana (hymenoptera: vespidae) on banisteriopsis malifolia (malpighiaceae): extrafloral nectar consumption and herbivore predation in a tending ant system. entomol. sci. 16(2):162-169. doi: 10.1111/ens.12004. bächtold, a. & alves-silva, e. (2012). behavioral strategy of a lycaenid (lepidoptera) caterpillar against aggressive ants in a brazilian savanna. acta ethol, in press, doi: 10.1007/ s10211-012-0140-2. bächtold, a., alves-silva, e., del-claro, k. (2013). notes on lycaenidae (lepidoptera) in inflorescences of peixotoa parviflora (malpighiaceae): a new host plant in a brazilian atlantic forest. j. lepidop. soc. 67(1): 65-67. badenes-pérez, f. r., alfaro-alpízar, m. a. & johnson, m. t. (2010). diversity, ecology and herbivory of hairstreak butterflies (theclinae) associated with the velvet tree, miconia calvescens in costa rica. j. insect sci. 10(209): 1-9. ballmer, r. g. & pratt, g. f. (1991). quantification of ant attendance (myrmecopily) of lycaenid larvae. j. res. lepidop., 30(1-2): 95-112. brown jr, k. s. (1993). neotropical lycaenidae: an overview. in t. r. new (ed.), conservation biology of lycaenidae (butterflies) (pp. 45-61). gland, switzerland: international union for conservation of nature and natural resources. figure. 2. camponotus blandus tending a parrhasius polibetes larva in banisteriopsis malifolia. the ant is drumming the last body segments of larva. ant size 5 mm. rekoa and a. strophius in the neotropics (robbins, 1991; monteiro, 2000; kaminski & freitas, 2010), but in our study the abundance of these lycaenids was low. both rekoa and a. strophius are polychromatic and this characteristic was observed in b. malifolia, where larvae turned pink. no rekoa and a. strophius larvae were observed in contact with ants in b. malifolia, but both species have ant-organs and are facultative myrmecophilous (robbins, 1991; monteiro, 2000; kaminski & freitas, 2010). the current knowledge about lycaenid-ant-host systems is based mostly on studies performed in the northern hee alves-silva, a bächtold, gj baronio, k del-claro influence of ants on the occurrence of lycaenids34 emery, e. o., jr, k. s. b. & pinheiro, c. e. g. 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(2008). looking for the ants: selection of oviposition sites by two myrmecophilous butterfly species. an. biol., 58(4): 371-388. doi: 10.13102/sociobiology.v65i4.3395sociobiology 65(4): 576-582 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 does seasonality affect the nest productivity, body size, and food niche of tetrapedia curvitarsis friese (apidae, tetrapediini)? introduction tetrapedia curvitarsis friese is a widely distributed species, frequently found in surveys in the states of minas gerais and são paulo (camillo, 2005; alves-dos-santos et al., 2007; pinto et al., 2015). like other species of the genus tetrapedia, this species has the behavior of nesting in preexisting cavities (garófalo et al., 2004; camillo, 2005; martins et al., 2012), allowing more detailed study of its nesting behavior. the species of tetrapedia with known nests, tetrapedia diversipes klug, t. curvitarsis, tetrapedia rugulosa friese, and tetrapedia garofaloi moure present more than one generation per year, higher activity of individuals in the wet season, and the occurrence of pre-pupal diapause (camilo, 2005). additionally, it was observed a biased female sexual ratio for t. diversipes, t. rugulosa, and t. garofaloi, but not abstract tetrapedia curvitarsis friese is a widely distributed species, frequently attracted by trap-nests. previous studies have revealed a higher frequency of nesting in the wet season and dimorphism between the sexes, with females exhibiting larger body size than males. we evaluated the effects of seasonality on the production of nests, food niche, and body size of t. curvitarsis. the study was conducted from april 2009 to march 2010 and from april 2012 to march 2013 at the água limpa experimental station, located in the triângulo mineiro, minas gerais state. the number of cells was positively correlated with length and diameter of trap-nests. however, the number of nests and the number of cells produced did not differed between the seasons. the females demonstrated a larger head width than males and both presented greater body size in the hot/wet season. a higher food niche breadth was observed in the hot/wet season and low similarity in the use of pollen sources between seasons (ps=39.05%). thus, it is concluded that the season has no effect on the production of nests or cells, but rather on the body size of males and females and food niche breadth. there was no interaction between sex and season, i.e., both factors influenced the individuals’ size independently. the production of smaller individuals in the dry season could be related not only to the quantity but also the quality of food offered to immature bees. sociobiology an international journal on social insects es campos1, tn araújo1, ls rabelo1, ema bastos2, sc augusto1 article history edited by cândida aguiar, uefs, brazil received 10 april 2018 initial acceptance 18 june 2018 final acceptance 18 august 2018 publication date 11 october 2018 keywords pollen sources, bee, trap-nest, brazilian savanna. corresponding author solange cristina augusto universidade federal de uberlândia instituto de biologia rua ceará s/nº, bloco 2d, umuarama cep 38400-902 uberlândia-mg, brasil. e-mail: solange.augusto@ufu.br for t. curvitarsis. all the species studied present females larger than males, except t. diversipes (camilo, 2005). availability of food resources is one of the factors that can affect body size of bees. studies have shown that the size of solitary bees is strongly influenced by larvae feeding (bosch & vicens, 2002; radmacher & strohm, 2010), with females generally receiving more food than males (stark, 1992; strohm, 2000; peruquetti & del lama, 2003). tetrapedia species, in addition to pollen and nectar, also use floral oil for the nourishment of bees’ immature. malpighiaceae species are important oil sources for tetrapedia (camillo, 2005; alves-dos-santos et al., 2007). studies on pollen analyses of larval food from nests of t. diversipes also revealed the presence of pollen grains of malpighiaceae species (menezes et al., 2012; neves et al., 2014). however, species of the genus dalechampia (euphorbiaceae) and ludwigia (onagraceae) were identified as the main sources of pollen for this species. 1 universidade federal de uberlândia, uberlândia, minas gerais, brazil 2 fundação ezequiel dias, belo horizonte, minas gerais, brazil research article bees sociobiology 65(4): 576-582 (october, 2018) special issue 577 in the brazilin savanna, the most important sources of pollen and oil sources for tetrapedia bloom mainly in the hot/wet season (carpentieri-pípolo et al., 2008; coelho & spiller, 2008; mendes et al., 2011), such as most woody and herbaceous species (batalha & mantovani, 2000). thus, considering the available information on nesting behavior of t. curvitarsis, we verified the possible effect of seasonality on nest productivity, food niche breadth, and body size of individuals in a brazilian savanna area. . material and methods study areas and trap-nests this study was conducted at the água limpa experimental station (ales) (19º05’48” s and 48º21’05” w) belonging to the federal university of uberlândia and located in the city of uberlândia, minas gerais state. the area covers a mixed system with 104 ha of natural areas of the brazilian savanna and 194.72 ha of pastures, crops, and orchards (neto, 2008). the area also contains 17 ha of predominantly fruit crops, including mango, mangifera indica l., west indian cherry, malpighia emarginata dc., passion fruit, passiflora edulis sims, pineapple, ananas comosus (l.) merr., guava, psidium guajava l., and tamarindo, tamarindus indica l. the area presents a seasonal tropical climate. it is characterized by cold/dry season, from april to september, and a hot/wet season from october to march (klink & machado, 2005). in the study area, two bee shelters, 2.5 m high and 1.5 m long, were built from wood rafters and plastic canvas covers, and they were situated 200m from each other. these bee shelters were supplied with trap-nests, consisting of bamboo sticks, commercially used for fishing. these bamboo canes were closed at one end by the node, with a length ranging from 8.00 cm to 20.10 cm, and diameters between 0.45 cm and 1.00 cm. new trap-nests were frequently added to maintain the availability of these substrates. the nests were organized inside building bricks, placed on shelves in the shelters. number of nests and cells the nests of t. curvitarsis were collected in two periods, from april 2009 to march 2010 (period 1 = p1) and from april 2012 to march 2013 (period 2 = p2). during p1, all operculated nests were collected at monthly visits. in p2, biweekly visits were made and only half of the total operculated nests were collected. the collected nests were taken to the laboratory of ecology and behavior of bees, federal university of uberlândia (leca-inbio/ufu). only the individuals from p2 were used in the analysis regarding the number of cells, according to the season. the length and diameter of each trap-nest collected in p2 were measured with the aid of a pachymeter. after emergence, all individuals were sacrificed, mounted with an entomological pin, and deposited in the collection of lecainbio/ufu. subsequently, the trap-nests were opened and their contents analyzed. body size of males and females all individuals of t. curvitarsis that emerged from nests collected during p2 were measured. the size of males and females was determined from the maximum head width (in millimeters) (fig 1) using the imagej 1.44p program (rasband, 1997-2011). for this, all individuals were photographed using a digital camera, positioned parallel to the measuring structure. the program was calibrated using the 10 mm scale in each photo. food niche during the p2, samples of larval food were removed from 22 active nests of t. curvitarsis, 11 from the cold/dry season and 11 from the hot/wet season. the samples were obtained by introducing pollen collectors made of wooden sticks and pins at the nest’s entrance. the larval food samples were stored into 15 ml centrifuge tubes, with 2 ml 70% alcohol, and processed using the acetolysis method (erdtman, 1960). three slides were prepared from each larval food sample (n = 66 slides). the slides were deposited in the pollen slide collection of the laboratory of plant morphology, microscopy and image of the federal university of uberlândia (lamovi-inbio/ufu). the characteristics of the pollen grains outlined by the acetolysis were used to compare the grain morphology of collected samples with the literature (salgado-labouriau, 1973; roubik & moreno, 1991), a database of pollen grain images (bastos et al., 2008), and a reference slide collection from lamovi-inbio/ufu. fig 1. measure of the head maximum width of tetrapedia curvitarsis. es campos, tn araújo, ls rabelo, ema bastos, sc augusto – nest productivity, body size, and niche of tetrapedia curvitarsis578 quantitative analysis of the larval food sample was performed by scanning the entire microscope slide using 800 × of magnification. for this, the cover slip was divided into four quadrants and approximately 400 pollen grains were counted, 100 grains in each quadrant and all grains present in quadrants that had less than 100 grains (vilhena et al., 2012). approximately 1,200 grains were counted per nest. pollen types occurring in abundances lower than 3% in each sample were excluded from the analysis, as they were considered as contaminants. data analysis to verify possible differences in the number of nests between the hot/wet and cold/dry seasons, a t-test was performed for the data collected in p1 and the mann-whitney test for the data collected in p2. a pearson correlation test was performed to verify if there was an association between the length and diameter of trap-nests and the number of brood cells produced, considering all nests, independent of the season. a t-test was also performed to evaluate differences between the number of cells produced and the seasons of the year. to evaluate if there was a difference in the size of the individuals between the sexes, according to the season, and whether there was an interaction between these factors, a two-factor anova was performed using the maximum head width measurement. all analyzes were performed in the r studio program (version 3.4.0) (r core team, 2017) and the normality was tested using kolmogorov–smirnov (lilliefors; p>0.05). the food niche breadth in each season was calculated from the shannon-wiener diversity index (camillo & garófalo, 1989; aguiar et al., 2013), and later compared by the hutcheson t-test. in order to establish the degree of uniformity of pollen collection in the species of plants visited by bees, the pielou index (j’) was used. these analyzes were performed in the past program 2.13 (hammer et al., 2001; zar, 2013). to verify the similarity in the use of pollen sources between the seasons of the year, the percentage of similarity index ps = σ of the lowest percentage of each pollen type was calculated (brower et al., 1997). results number of nests and cells a total of 51 nests of t. curvitarsis were collected during p1 and 45 nests in p2. nests were collected throughout the year, and no significant difference was observed in the number of nests between the hot/wet and cold/dry seasons (p1: t = 0.33, df = 10, p> 0.05; p2: u = 12.50, n1=6, n2= 6, p>0.05) (fig 2). the number of cells per nest ranged from 1 to 9. nests containing 5 to 7 cells were the most frequent, representing 66.67% of nests collected. the number of cells was positively correlated with bamboo length (r = 0.52, df = 43, p <0.05) (fig 3a) and diameter (r = 0.58, df = 43, p <0.05) of trapnests (fig 3b). however, as the number of nests produced, the number of cells did not differ significantly between seasons (t = 0.49; df = 43; p>0.05). fig 2. abundance of tetrapedia curvitarsis nests obtained in period 1 (april 2009 to march 2010) and in period 2 (april 2012 to march 2013), at the água limpa experimental station, uberlândia-mg. fig 3. number of brood cells produced in tetrapedia curvitarsis nests, according to length (a) and diameter (b) of trap-nests. sociobiology 65(4): 576-582 (october, 2018) special issue 579 body size of males and females a total of 112 individuals were measured, 44 males and 68 females, 46 individuals from the cold/dry season and 66 from the hot/wet season. the females presented larger head widths than the males (f1.108 = 9.36; p <0.05) (fig 4). both males and females presented a higher maximum head width in the hot/wet season (f1.108 = 28.46; p <0.05) (fig 4). however, there was no interaction between sex and season (f1.108 = 0.56; p> 0.05), i.e., both factors influenced the individuals’ size independently. food niche t. curvitarsis used 22 pollen types as sources of pollen for larval food, belonging to eight botanical families: asteraceae, clusiaceae, euphorbiaceae, fabaceae, lamiaceae, malpighiaceae, myrtaceae, and sapindaceae. according to abundance and frequency data, kielmeyera type was abundantly collected and presented in all nests in the cold/dry season and 72.72% of the nests in the hot/wet season. in the hot/wet season, 15 pollen types were identified. the most abundant pollen types were kielmeyera (37.24%), maprounea (27.55%), type 1 (12.93%), and type 2 (9.35%) (fig 5). twelve pollen types were identified in nests collected in the cold/dry season. the most abundant pollen types in this season were kielmeyera (73.89%), vernonia (9.04%), and baccharis (6.19%) (fig 5). t. curvitarsis presented a higher food niche breadth in the hot/wet season (h’= 1.83) compared to the cold/dry season (h’ = 1.04) (t = -43,947, df = 17.516, p <0.05). higher uniformity in the collection of pollen was also observed in the hot/wet season (j’ = 0.66) than cold/dry season (j’ = 0.42). in addition, similarity in the use of pollen sources between the seasons was low (ps=39.05%). fig 4 maximum head width in millimeters (averages ± standard error) of tetrapedia curvitarsis collected between the seasons (cold and dry season and hot and wet season) in the period from april 2012 to march 2013, at the água limpa experimental station, uberlândia-mg. fig 5. relative abundance of the pollen types sampled in the larval food of tetrapedia curvitarsis in cold/dry (samples 1 to 11) and hot/wet seasons (samples 12 to 22) in the period from april 2012 to march 2013, at the água limpa experimental station, uberlândia-mg. *the category “other” represents the other pollen types which presented relative abundance less than 5%. es campos, tn araújo, ls rabelo, ema bastos, sc augusto – nest productivity, body size, and niche of tetrapedia curvitarsis580 discussion in the present study, we did not observe an effect of seasonality in the nest productivity. camillo (2005) suggests that during the cold/dry season, mainly between june and september, immature of t. curvitarsis probably present a prepupal diapause, thus reducing the activity of the bees during this period. in our study, although the number of nests and cells found in the cold/dry season was numerically smaller, there was no significant difference between the seasons, as registered in other studies (camillo, 2005; mesquita & augusto, 2011). the highest number of cells was verified in nests with longer bamboo length and larger diameter, indicating that the females can optimize the use of the substrates for nesting. the number of brood cells produced by solitary bee species on each nesting substrate tends to increase with the greater space availability (pereira et al., 1999; aguiar & garófalo, 2004; ramos et al., 2010). it is probable that this mechanism is related to the high energy cost of searching for new places for the construction of nests. the number of cells produced in a nest is also dependent on availability of food (ramos et al., 2010). in addition, the choice of cavities by foundress females can affect the body size of the offspring. roulston and cane (2002) verified that species of cavity-nesting bees presented significantly greater variation in body size compared to ground-nesting species. thus, these authors suggest that the choice of cavity utilized may be a more important predictor of offspring body size than parental body size. females of t. curvitaris were larger than males, pattern observed for other species of solitary bees (pereira et al., 1999; camillo, 2005; bosch & vicens, 2006). the amount of food deposited in the brood cells strongly affects the size of the individuals produced (roulston & cane, 2002; bosch & vicens, 2002; radmacher & strohm, 2010). female bees and wasps are usually larger than males, due to the greater amount of food supplied by the nesting female for their development (stark, 1992; strohm, 2000; peruquetti & del lama, 2003). especially for females, the body size is a selective force that influences surviving and reproduction. a study realized with a solitary bee species, osmia cornuta, recorded that small females have larger chances of dying during their development, disperse more frequently from their natal nesting site, present lower provision rates and produce more males (bosch & vicens, 2006). in the present study, we also observed a seasonal effect in the size of both sexes males and females being significantly larger in the hot/wet season. greater amount of food stored in the brood cell could explain the occurrence of larger individuals, as observed in other studies (roulston & cane, 2002; bosch & vicens, 2002; radmacher & strohm, 2010). however, another possible explanation could be differences in composition and abundance of the pollen types used in the two seasons, as verified in the present study. while in the hot/wet season kielmeyera type and maprounea type together represented, approximately, 65% of the pollen grains sampled, in the cold/dry season kielmeyera type alone constituted 74% of the sample. in addition, in general, the similarity in pollen type diversity between seasons was low. the flexible pollen foraging performed by polylectic bees such as t. curvitarsis during the two different seasons can result in larval food consisting of pollen types that differ in protein content. it is known that the pollen quality of different plant families can vary, affecting bee growth and mortality (roulston & cane, 2002). experimental study conducted with a solitary bee species lasioglossum zephyrum (halictidae) recorded increase of body sizes of the individuals submitted to diet contained rich-protein pollens (roulston & cane, 2002). then, we suggest that changes in the pollen quality of larval food between seasons could also affect the body size of individuals of t. curvitarsis. in conclusion, although we did not observe an effect of season on the number of nests or cells produced we recorded an effect on the body size of both sexes and on food niche breadth. the results suggest the occurrence of a possible compensatory effect, in which the productivity of the nests is maintained, but there is a change in the quantity and/or quality of available food and, consequently, the production of smaller individuals. the influence of quantity and quality of floral resource, especially pollen, in the determination of body size is an issue that we intend to investigate in future studies. acknowledgments this study was supported by grants from fundação de amparo à pesquisa do estado de minas gerais (fapemig) and conselho nacional de desenvolvimento científico e tecnológico (cnpq). this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nivel superior brasil (capes) finance code 001. es campos received a fellowship from capes and s.c. augusto received research fellowship from cnpq (306298/2015-5). authors’ contribution es campos did field work and laboratory; 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(2012). acerola pollinators in the savanna of central brazil: temporal variations in oil-collecting bee richness and a mutualistic network. apidologie, 43: 51-62. doi: 10.1007/s13592-011-0081-1 zar, j.h. (2013). biostatistical analysis: pearson new international edition. pearson higher ed. _hlk512006104 _hlk512261357 doi: 10.13102/sociobiology.v66i1.3679sociobiology 66(1): 194-197 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 are orchid bees (apidae: euglossini) good indicators of the state of conservation of neotropical forests? as proposed by reyes-novelo et al., 2009, seven criteria have to be met by wild bees to be considered as bioindicators and in this work we highlight issues for each criteria when working specifically with orchid bees as bioindicators in the neotropics. ‘the taxonomy of the group must be well known and stable so that the species can be identified reliably’: although there are five well-defined genera (kimsey, 1987; roubik & hanson, 2004), a major issue is the lack of taxonomic identification of females for most species. ‘biology and lifestyle must be well known’: studies such as dressler (1982) or roubik and hanson (2004) provide general aspects of their biology. however, other basic knowledge remains unknown for most species. for instance, the impossibility to collect and identify females prevents the abstract this work discusses the criteria proposed to consider wild bees as bioindicators, and specifically applied to orchid bees in neotropical forests. some of the issues are: 1) the deficiencies of the sampling methods, which makes it difficult to accurately assess species inventories. 2) missing knowledge about the biology of many species. 3) spatial or temporal distribution of most species remains unknown, which may misslead the results of short-term studies. 4) it is not clear whether orchid bees are affected by climate change as seen in other bees, which weakens their predictive potential. 5) a measure of the economic benefits provided by orchid bees is needed to better appraise them and their conservation. finally, future studies should develop predictive models for conservation, accounting for evolutionary aspects like phylogeny or distributions; together with studies of the effect of disturbance on the physiology of the bees. sociobiology an international journal on social insects yj añino1,2, a parra3, d gálvez2,4,5 article history edited by gilberto m. m. santos, uefs, brazil received 08 august 2018 initial acceptance 02 october 2018 final acceptance 14 october 2018 publication date 25 april 2019 keywords bioindicators, conservation, euglossina, hymenoptera, neotropics. corresponding author dumas gálvez indicasat aip building 219, city of knowledge, clayton, panama. rep. of panama, pobox 0843-01103, panama 5. e-mail: dumas.galvez@mail.com estimation of sex-ratios in a population, which could be used for conservation purposes (murray et al., 2009). seasonal cycles, dynamics with natural enemies and other ecological factors could mislead studies that are based on indexes of diversity. in addition, the heterogeneity across sampled ecosystems, sharing the same species, makes the inference of the above aspects difficult. ‘the group should be composed of well-defined and rich trophic guild that should be important in the structure and functioning of ecosystems’: although the role of orchid bees to preserve plant diversity through pollination is undeniable (ackerman, 1986), information on the network of host plants remain unknown for most species. moreover, the fact that orchid bees can thrive in the absence of its orchid mutualists (pemberton & wheeler, 2006) is an example of the difficulty 1 museo de invertebrados g. b. fairchild, universidad de panamá, balboa, panamá 2 programa centroamericano de maestría en entomología, universidad de panamá, panamá 3 laboratorio de investigaciones en abejas, labun, universidad nacional de colombia, bogotá d.c., colombia 4 indicasat aip, panamá 5 sistema nacional de investigación, senacyt, panamá short note sociobiology 66(1): 194-197 (march, 2019) 195 to make predictions about forest conservation based on the presence of certain species. ‘organisms should be easily captured, manipulated and observed; the study of the group should not jeopardize its conservation’: orchid bees males collect fragrances in nature, which facilitates their collection by means of chemical fragrances placed as baits (parra-h et al., 2016) and collections are done manually with nets, which is prone to bias caused by the skills of the collector. fixed traps are also prone to bias since some species are never caught by these traps (prado et al., 2017) and their efficiency can be influenced by their design (sydney & gonçalves, 2015). another bias is the innate variation across species in the preference for certain fragrances, while some are not attracted by any fragrance and others are attracted by specific fragrances (dodson & dressler, 1969; nemésio & silveira, 2004). protocols that perform samplings twice a month often do not account for the uneven spatial and temporal distribution throughout the year, an issue in areas with strong seasonality (nemésio, 2012). overall, it has been difficult to establish systematic sampling methods (prado et al., 2017). ‘the geographical distribution of the group should be broad, including different habitats, allowing the use of a variety of experimental designs and comparisons’ orchid bees inhabit lowlands from sea level to more than 2000 meters above sea level, from mexico to northern argentina (kimsey, 1987; roubik & hanson, 2004). parra-h et al. (2016) suggest that their distribution is much more complex than currently documented or that specific events in the ecosystems generate population structuring, making it more dynamic in terms of displacement and occupation. again, many species are not attracted to chemical baits and some species are able to fly long distances to reach the baits, moving across different habitats, making it impossible to determine accurately their distribution ranges. ‘species should tend to specialize in a particular habitat, so that they are sensitive to habitat degradation and regeneration’ in a 20-year monitoring in panama, roubik (2001) observed that there was no aggregate trend in abundance, and richness was overall stable for most species. this contrasts with some theoretical predictive models for orchid bees (faleiro et al., 2018) and bee declines in other parts of the world as a result of climate change and other factors (goulson et al., 2015; rykken et al., 2014). many of the reported disappearances in short-term studies are in fact due to demographically rare species and not caused by local extinctions; thus, short-term studies may not provide reliable numbers (rasmussen, 2009; reyes-novelo et al., 2009; roubik, 2001). other studies showed that abundance and richness are not affected by habitat fragmentation and disturbance (botsch et al., 2017) or they respond positively to disturbances (brosi, 2009; otero & sandino, 2003). in agroecosystems, using orchid bees abundances to show differences between different types of monocultures may be influenced by plantation size, spatial distribution (hedström et al., 2006a) and seasonality (hedstöm et al., 2006b). moreover, species composition may not be a good indicator when comparing monocultures under different conditions (hedström et al., 2006a). an explanation is that orchid bees can fly up to 2 – 50km,exploiting a wide spectrum of resources across different habitats (elizondo, 2015; pokorny et al., 2015). then, individuals sampled in a disturbed environment may be flying in from another place, making them poor indicators of forest conservation. then again, orchid bees can exploit habitats that lack their orchid mutualists which points at our lack of knowledge about habitat specialization (pemberton & wheeler, 2006). ‘the group must have species with potential economic importance’ orchid bees are important pollinators of plant species in the forest (rocha-filho et al., 2012) and few of those are of economic importance (dressler, 1982). still, an economic measure of how much agroecosystems and forests benefit from them is missing, a credit often given to honeybees and other native bees (kremen et al., 2002; losey & vaughan, 2006). neither there is data on how honeybees interact with orchid bees, which generally is a negative relationship in which honeybees outcompete native bees (cane & tepedino, 2017). in the light of our considerations, we conclude that the use of orchid bees as bioindicators should be reevaluated. their use seem to be based on precarious concepts due to the lack of research. importantly, we are not aware of any study that investigated the effects of pollution on orchid bees (e.g. mortality, residues accumulation); another element to evaluate the state of conservation of habitats using orchid bees (celli & maccagnani, 2003). future studies should implement orchid bees phylogeny (ramírez et al., 2010) together with geographical distributions and the iucn conservation status to make predictions on the evolutionary diversity of the group, as an approach to conservation efforts (forest et al., 2015). references ackerman, j. 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(2015). the capture success of orchid bees (hymenoptera, apoidea) influenced by different baited trap designs? a case study from southern brazil. revista brasileira de entomologia, 59: 32-36. doi: 10.1016/j. rbe.2014.11.003 doi: 10.13102/sociobiology.v60i2.154-161sociobiology 60(2): 154-161 (2013) post-embryonic development of intramandibular glands in pachycondyla verenae (forel) (hymenoptera: formicidae) workers lcb martins1, jhc delabie2,3, jc zanuncio1, je serrão1 introduction hymenoptera have two mandibular gland types, the ectomandibular or mandibular glands, and the mesomandibular or intramandibular glands (cruz-landim & abdalla, 2002). unlike the mandibular glands that are the most studied, the current knowledge regarding intramandibular glands is restricted to occurrence and descriptive morphology in adult bees (nedel, 1960; toledo 1967; costa-leonardo, 1978; cruz-landim & abdalla, 2002; santos et al. 2009a, cruzlandim et al., 2011) and ants (schoeters & billen, 1994, billen & espadaler, 2002, grasso et al., 2004, roux et al., 2010, martins & serrão, 2011). the intramandibular glands in ants can be divided in three classes (martins & serrão, 2011). first, gland characterized by cubical or columnar epidermal cells, which are described as class i gland cells by noitot & quennedey (1974). second, isolated glands with spherical cells in the inner cavity of the mandible characterized by the presence of canaliculi that open in pores on the mandible surface, this abstract the current knowledge of intramandibular glands in hymenoptera is focused on occurrence and morphology in adult insects. this is the first report regarding the postembryonic development of intramandibular glands in a “primitive” ant, pachycondyla verenae. in this study, we analyzed mandibles of prepupae, white-eyed, pink-eyed and black-eyed pupae, pupa of pigmented body pupae, and adults. adult workers of p. verenae have intramandibular glands with epidermal secretory cells of class i and isolated glands of class iii, and both glands have onset differentiation in pink-eyed pupae. some histological sections were submitted to histochemical test for total proteins and neutral polysaccharides. histochemical tests showed occurrence of polysaccharides and proteins in epidermal secretory cells of class i from the white-eyed pupae, polysaccharides and proteins in pink-eyed pupae to black-eyed pupae in both glands classes i and iii and presence of polysaccharides in adult ants also in both gland classes i and iii. intramandibular glands of classes i and iii in p. verenae workers differentiate during pupation, with onset occurring in pink-eyed pupae, and completion occurring in black-eyed pupae. sociobiology an international journal on social insects 1 universidade federal de viçosa, viçosa, mg , brazil 2 ceplac/cepec, itabuna, ba, brazil 3 universidade estadual de santa cruz. ilhéus, ba, brazil research article ants article history edited by: gilberto m. m. santos, uefs brazil received 11 march 2013 initial acceptance 22 april 2013 final acceptance 21 may 2013 keywords histology; histochemistry; ponerinae; secretory cell; exocrine gland. corresponding author josé eduardo serrão departamento de biologia geral universidade federal de viçosa 36570-000 viçosa – mg, brazil e-mail: jeserrao@ufv.br gland class has a secretory and a conducting cell (billen 2009) and is classified as class iii gland cells by noitot & quennedey (1974). the third type occurs in attini, with hypertrophy of epidermal cells containing a reservoir (amaral & caetano, 2006; billen, 2009; martins & serrão, 2011). in ants, studies of salivary system are restricted to morphology of post-pharyngeal and hypopharyngeal (gama, 1978; amaral & caetano, 2005), mandibular (gama, 1978; pavon & camargo-mathias, 2005; ), labial or salivary glands (gama, 1978; meyer et al., 1993; lommelen, et al., 2002; 2003; amaral & machado-santelli, 2008) and intramandibular glands (martins & serrão, 2011) but the intramandibular glands development remains unclear. ponerini ants are an interesting model to study gland development since their representatives have features similar to ants ancestor (kusnezov, 1955; peeters & crewe, 1984, hölldobler & wilson, 1990). in this work we describe the histology and histochemistry of intramandibular glands from prepupae to adult workers of the neotropical ant pachycondyla verenae. sociobiology 60(2): 154-161 (2013) 155 material and methods five colonies of p. verenae were collected in the experimental field of the department of fruit science, federal university of viçosa, viçosa, state of minas gerais, brazil. worker ants at the following developmental stages were evaluated: prepupae, white-eyed, pink-eyed and black-eyed pupae, body pigmented pupae, and adults (fig. 1a, 1b). features that allowed for the identification of the successive post-embryonic developmental stages were the focus of these evaluations (soares et al., 2004). five individuals in each developmental stage were -anesthetized by thermal shock at 0 oc for 3 min and the mandibles were removed and transferred to zamboni fixative solution (stefanini et al., 1967). mandibles were dehydrated in a graded ethanol series, embedded in jb-4 historesin, and 3 μm slices were stained with hematoxylin and eosin. fig. 1: different post-embryonic developmental stages in pachycondyla verenae workers. a prepupae (pr). b white-eyed (wp), pink eyed (pp) and black-eyed pupae (bp) and body pigmented pupae (bb). arrows: mandibles. some mandible sections were subjected to histochemical analysis of mercury-bromophenol for protein detection and periodic acid-schiff (pas) for neutral polysaccharide detection according to protocols from pearse (1985) and bancroft & gamble (2007). negative control for pas test was performed by omitting periodic acid oxidation. results the morphology of the intramandibular glands was characterized in adult workers, followed by monitoring of their development from the prepupae stage. adult mandibles of p. verenae adult workers had two classes of intramandibular glands, class i epidermal secretory cells and class iii isolated glands. the class i secretory cells were characterized as columnar epidermal cells with well developed spherical nucleus and a homogeneously acidophilic cytoplasm (fig. 2a). class iii isolated glands were scattered within the mandible cavity and showed well developed spherical nuclei and strongly basophilic cytoplasm with many vacuoles (fig. 2b). fig. 2. histological sections of the mandibles in adult workers of pachycondyla verenae. a class i epidermal secretory cells (g1) characterized by cubic cells. trachea (tr). b class iii unicellular glands of (g3) with vacuoles (va) in the cytoplasm containing and spherical nucleus (nu) with predominance of uncondensed chromatin. cu: cuticle. he: hemocoel. hematoxyline and eosin stained. histochemical analyses of both intramandibular gland types were positive for the presence of neutral polysaccharides and proteins. prepupae in the mandibles of p. verenae prepupae, free cells were not found within the intramandibular cavity, which was filled with flocculent material. the epidermis was formed by flattened cells (fig. 3), without features of glandular secretory cells. fig. 3. histological section of mandible of pachycondyla verenae prepupae. a flattened epidermis (arrow) and mandibular cavity, hemocoel (he) filled with flocculent material. cu: cuticle. hematoxyline and eosin stained. white-eyed pupae in white-eyed pupae of p. verenae, the mandible cavity was characterized by intense cell reorganization, with cells displaying irregular distribution and filamentous projections. lcb martins, jhc delabie, jc zanuncio, je serrão post-embryonic development of intramandibular glands in ants156 cell identification was based on the presence of small nucleus (fig. 4a) as well as aggregation of protein rich rectangular cells (fig. 4b). the single layered epidermal cells were cubic (fig. 4a, 4c), although in some mandible regions they remained flattened. in addition to cells, the mandible cavity displayed an accumulation of flocculent material positive for proteins (fig. 4c) and neutral polysaccharides (fig. 4d). pink-eyed pupae in pink-eyed pupae of p. verenae, the mandible cuticle was thicker than in previous pupae and mandibular teeth were present (fig. 5a inset). the mandibular epidermal cells were similar to class i secretory cells found in adult workers, but with variations in size and shape. specifically, some cells were spherical while others were columnar (fig. 5a). the epidermis within the mandible was composed of columnar epithelium on one side and a pseudostratified-like epithelium on the opposed side (fig. 5a). in addition, we found sensitive cell precursors (neuroblasts) of mandibular sensilla closely associated with the epidermis (fig. 5b). in the mandible cavity, precursors of class iii gland cells were dispersed and had clear cytoplasm, making it difficult to identify the cell boundaries (fig. 5c, 5d). the nuclei of these cells had uncondensed chromatin (fig. 5b, 5c). in addition to cells dispersed in the mandible cavity, there were some chromatic bodies, which were small and strongly basophilic (fig. 5a, 5b). the basal and lateral regions of the class i epidermal cells were positive for neutral polysaccharides (fig. 5e) and proteins (fig. 5f). black-eyed pupae in black-eyed pupae of p. verenae, the mandible was almost entirely lined by cubic class i epidermal cells (fig. 6a). the class iii gland cells had well developed nuclei and cytoplasm that was homogeneous, without vacuoles (fig. 6b). chromatic bodies were rare within the mandible cavity. histochemical analyses were positive for neutral polysaccharides (fig. 6c) and proteins (fig. 6d) in both class i and iii gland cells. body pigmented pupae pupae of p. verenae with a pigmented body had a mandible epidermis with class i cubic gland cells and class iii gland cells with vacuolated cytoplasm (fig. 7). the histochemical tests results in different intramandibular glands of different developmental stages of p. verenae are summarized in table i. discussion the morphology and histochemistry of intramandibular glands in p. verenae adult workers described here are similar to those of other ponerini ants reported by martins & serrão (2011). the occurrence of intramandibular glands in p. verenae with class i epidermal secretory cells and class iii isolated glands was similar to that of adult ants (schoeters & billen, 1994, billen & espadaler, 2002; grasso et al., 2004; roux et al., 2010; martins & serrão, 2011). both classes i and iii of intramandibular glands in p. verenae workers began to differentiate during the pink-eyed pupae stage. in bees, intramandibular glands also differentiate in the pupal stage (cruz-landim & abdalla 2002). in addition, mandibular glands appear during the pupal development of the ant p. obscuricornis (lommelen et al., 2003). intramandibular gland differentiation in p. verenae was not observed in prepupa. during that larval stage, ants, likely other holometabolous insects, accumulates food reserves and increases in size (wheeler & martinez, 1995), followed by metamorphosis, which consists of the complete structural reorganization of larval organs to adult organs (wheeler & buck, 1992; rosell & wheeler, 1995; roma et al., 2006; 2009). the white-eyed pupae of p. veranae also had no intramandibular glands, however, cell reorganization at this stage did occur and was likely due to proliferation and differentiation of epidermal cells. the white-eyed pupae of hymenoptera is the earliest period of internal organs modifications, because it has yet larval organs and absence of those present in adults (soares et al., 2004, azevedo et al., 2008, santos et al., 2009b; cruz-landim et al., 2011). histochemical test developmental stage pp wep pep bep bpp adult g1 g3 g1 g3 g1 g3 g1 g3 g1 g3 g1 g3 protein ga ga ga ga ga ga + + + + + + neutral polysaccharide ga ga ga ga + + + + + + + + ga gland absent, + positive reaction table i histochemical tests in the intramandibular glands types i (g1) and iii (g3) of pachycondyla verenae (formicidae: ponerini) during different developmental stage of prepupae (pp), white-eyed pupae (wep), pink-eyed pupae (pep), black-eyed pupae (bep), body pigmented pupae (bpp) and adult. sociobiology 60(2): 154-161 (2013) 157 intramandibular glands characterized as class i epidermal secretory cells or class iii isolated glands can be definitively identified during the pink-eyed pupae stage of p. verenae in a manner similar to likely the labial, post-pharyngeal, hypopharyngeal, and mandibular glands that also differentiate in camponotus rufipes pupae (gama 1978). in body pigmented pupae of p. verenae, both classes i and iii of intramandibular glands are almost completely differentiated, similar to that reported from the salivary system of c. rufipes, which reaches maximum differentiation during the body pigmented pupae stage, despite the lack of secretory activity within the glands (gama, 1978). the same may be occurring in the intramandibular glands of p. verenae, as the amount of gland cells in body pigmented pupae seems low compared to the adult ants. the cellular processes that result in tissue and organ changes during metamorphosis include addition of new cells by cell division, cell loss due to programmed cell death, and cell migration (cruz-landim, 2009). in the present study, we are unable to detect some of these processes, but after the black-eyed pupae stage of p. verenae, intramandibular glands were similar to those found in adult workers, as is the case for other internal organs of hymenoptera (neves et al., 2002, 2003, soares, 2004, azevedo et al., 2008; santos et al., 2009b). the presence of proteins and neutral polysaccharides in intramandibular glands from white-eyed pupae of p. verenae suggests that during this developmental stage, there is a high level of cellular activity derived from the metabolic cell machinery. in the leaf-cutter ant, atta sexdens rubropilosa, class iii secretory cells contain carbohydrate and protein releasing glucoconjugate compounds (amaral & caetano, 2006). in conclusion, class i epidermal secretory cells and class iii isolated cells of the intramandibular glands of p. verenae workers differentiate during pupation, with onset occurring in pink-eyed pupae, and completion occurring in blackeyed pupae. fig. 4. histological sections of mandible of pachycondyla verenae white-eyed pupae. aepidermis (ep) with flattened cells and irregular cells in the mandible cavity with small nucleus (arrowheads) and flocculent material (*). hematoxyline and eosin stained. b cell aggregate (ca) and flocculent material (*) into mandible cavity positive (arrows) for protein. mercury-bromophenol blue stained. c epidermis (ep) with cubic cells and flocculent material (*), both positive (arrows) for proteins. mercury-bromophenol blue stained. d flocculent material (*) and epidermis (ep) positive for polysaccharides (arrows). periodic acidschiff stained. he: hemocoel. cu: cuticle. lcb martins, jhc delabie, jc zanuncio, je serrão post-embryonic development of intramandibular glands in ants158 fig. 5. histological sections of the mandible of pachycondyla verenae pinkeyed pupae. a columnar (ec) and pseudostratifiedlike (ee) epidermis note chromatic bodies (arrow) into mandible cavity insert general view of the mandible showing teeth (arrowhead). hematoxyline and eosin stained. b neuroblast (arrowhead) in the epidermis and chromatic bodies (arrow) into mandible cavity (he). hematoxyline and eosin stained. c class i epidermal gland showing columnar cells (gi). hematoxyline and eosin stained. d class iii precursor cells (arrows) with light cytoplasm into mandible cavity. hematoxyline and eosin stained. e positive reaction for polysaccharides (arrows) in the class i secretory cells. periodic acidschiff stained. f positive reaction for proteins (arrows) in class i secretory cells. mercury-bromophenol blue stained. cu: cuticle. ep: epidermis. he: hemocoel. sociobiology 60(2): 154-161 (2013) 159 fig. 6. histological sections of the mandibles of pachycondyla verenae black-eyed pupae. a class iii unicellular glands of (giii) showing well developed nucleus (nu) and homogeneous acidophilic cytoplasm. hematoxyline and eosin stained. b class i epidermal secretory cells (gi) showing nuclei with predominance of uncondensed chromatin. hematoxyline and eosin stained. c positive reaction for polysaccharides (arrows) in the class i epidermal secretory cells (g1) and class iii glands (g3). periodic acidschiff stained. d positive reaction for proteins in the class i epidermal secretory cells (g1) and class iii glands (g3). mercury-bromophenol blue stained. ep: epithelium. he: hemocoel. cu: cuticle. fig. 7. histological section of the mandible of body pigmented pupa of pachycondyla verenae. a well developed class i epidermal cells (g1) and class iii unicellular glands of (g3) with cytoplasm vacuoles (va). ep: epithelium. hematoxyline and eosin stained. he: hemocoel. cu: cuticle. nu: nuclei. lcb martins, jhc delabie, jc zanuncio, je serrão post-embryonic development of intramandibular glands in ants160 acknowledgements this research was supported by brazilian research agencies fapemig, cnpq and secti/fapesb-cnpqpnx0011/2009. authors are grateful to dr. ronara s. ferreira of the federal university of lavras (ufla), brazil, for help in ant collection. references amaral, j.b. & caetano, f.h. 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(1995). storage proteins in ants (hymenoptera: formicidae). comp. biochem. phsyiol. b, 112, 15-19. doi: 10.13102/sociobiology.v60i2.190-197sociobiology 60(2): 190-197 (2013) species composition of termites (isoptera) in different cerrado vegetation physiognomies de oliveira¹, tf carrijo², d brandão³ introduction termites (isoptera) are one of the most abundant soil invertebrates in tropical ecosystems (wilson, 1971; wood & sands, 1978; eggleton et al., 1996) and the most important soil ecosystem engineers of these environments (bignell, 2006; jouquet et al., 2011). in some arid and semi-arid tropical savannas, during the dry season, termites are the only active group of invertebrates able to decompose organic matter (jouquet et al., 2011) and provide ecosystem services such as soil formation and aeration (lavelle et al., 2006). most species of termites are tropical, and among more than 2800 described abstract little is known about the termite fauna of the different vegetation physiognomies in the cerrado biome. it is suggested that the species compositions in grassland and savanna areas are closely related to each other, and quite distinct from those of forests. this study compared the species composition from five different physiognomies of cerrado, and tested the hypothesis that the termite faunas of savannas and grasslands form a distinct group from that of forests. the study was conducted in the parque estadual da serra de jaraguá, state of goiás, brazil. termites were sampled from two physiognomies of savanna, one natural grassland, one pasture, and one gallery forest. a transect with 10 parcels of 5x2 m was established in each physiognomy. the relative abundance was inferred by the number of encounters, termites were classified in feeding guilds, and the dissimilarity in the species composition between the physiognomies was calculated. a total of 219 encounters, of 42 species of two families were recorded. the most abundant feeding guilds were the humivores (98) and xylophages (55). the physiognomies with the largest number of species were rupestrian cerrado (23 species) and cerrado sensu stricto (21). the physiognomies had a similar species composition (less than 55% dissimilarity), mainly the natural open areas. the hypothesis of a distinct fauna of termites in forest vegetation was refuted. the termite fauna of gallery forest is very different from that of pasture, but most species also occur in natural open areas. the impact of pasture on the diversity and composition of termites seems to be significant, but the impact is even greater on the proportion of the feeding guilds, reducing the proportion of xylophages and intermediates. sociobiology an international journal on social insects 1 universidade de brasília, brasília – df, brazil 2 universidade de são paulo, ribeirão preto – sp, brazil 3 universidade federal de goiás, goiânia – go, brazil research article termites article history edited by: alexandre vasconcellos, ufpb brazil received 12 december 2012 initial acceptance 24 january 2013 final acceptance 25 march 2013 keywords termite assemblage, biodiversity, forest, savanna, feeding guilds corresponding author danilo elias de oliveira laboratório de termitologia departamento de zoologia instituto de biologia universidade de brasília brasília, df, brazil 70910-900 e-mail: daniloelo@gmail.com species, approximately 500 occur in the neotropical region (kambhampati & eggleton, 2000; constantino, 2012). with about 150 species, 50% of them endemic (constantino, 2005), the cerrado biome in central brazil probably harbors the most diverse and highly endemic savanna termite fauna (domingos et al., 1986). the cerrado is the largest and richest tropical savanna in the world, and is also probably the most threatened one (silva & bates, 2002). ranked among the 25 most important biodiversity hotspots (myers et al., 2000), the cerrado is the second most extensive biome in brazil (klink & machado, 2005), and the most strongly affected by human disturbance. sociobiology 60(2): 190-197 (2013) 191 during the past 50-60 years, with the evolution of agricultural techniques, this savanna also has become the largest agricultural frontier in brazil (sano et al., 2010). the latest analysis of the conservation status of the cerrado showed that only 20% of the original area remained relatively undisturbed, and only 6.2% was preserved in protected areas (myers et al., 2000). the cerrado biome is a mosaic of 10 different kinds of vegetation formations (physiognomies) (ribeiro & walter, 1998). the development of these physiognomies depends on the soil, groundwater, and other environmental variables. the physiognomies can be distinguished from one another mainly by the species composition, richness pattern, and relative abundance of plant species (eiten, 1983). the termite faunas of most of these habitats are still little known, and for some of them no published information is available. the majority of termite surveys in the cerrado biome have been conducted in the cerrado sensu stricto physiognomy (mathews, 1977; coles, 1980; brandão & souza, 1998; constantino & schlemmermeyer, 2000; constantino, 2005; cunha et al., 2006; carrijo et al., 2009). some surveys have also described local faunas in the cerradão (mathews, 1977; constantino & schlemmermeyer, 2000; cunha et al., 2006), gallery forest (mathews, 1977; constantino & schlemmermeyer, 2000; cunha et al., 2006), and interfluve mesophytic forest (brandão & souza, 1998; cunha et al., 2006). the diversity of animals is often linked to the type of vegetation where they live or breed, and often differs between forests and grassy (grasslands and savannas) environments (bond & parr, 2010); termites can be expected to follow these patterns. constantino (2005) remarked that in the cerrado biome there are two termite faunas, one closely related to those of grasslands and savannas, and another one, quite distinct, related to forests. constantino (2005) also suggested that the species composition of termites from cerrado forests resembles faunas of the amazon and atlantic forest biomes. also, mathews (1977) observed a gradient in the species composition of termites that build epigeal (above-ground) nests, from grasslands and savannas to forests. the present study aimed to 1) compare the species composition, richness, and abundance of termites in five cerrado physiognomies including three types of savanna (ranging from shortgrass savanna to woodland), one forest, and one pasture, all situated in the parque estadual da serra de jaraguá, goiás, brazil; and 2) test if there are two different groups of termite species related to the vegetation in the cerrado biome: one in open areas (grasslands and savannas), and another in forests. material and methods this study was carried out at the parque estadual da serra de jaraguá, municipality of jaraguá, state of goiás, brazil (15.75º to 15.85º s and 49.27º to 49.37º w). the park covers an area of about 2,860 ha, ranging in altitude from 640 to 1,140 m. the predominant soil type is litolic neosol, and the köppen-geiger climate type is “aw” (agência ambiental, 2004). the park is surrounded by cattle grazing land and is mostly covered by cerrado sensu stricto (a vegetation type composed of tropical xeromorphic semideciduous broadleaf trees and scrub woodland of cerrado), which is more or less dense depending on the location. at higher altitudes (above 1,000 m) some rupestrian cerrado is present (tropical rupestrian semideciduous broadleaf trees and scrub woodland on rocky soil). also, gallery forests (tropical mesophytic semideciduous broadleaf gallery forest) extend along the permanent water courses. near the border of the park are some open shortgrass savannas (tropical xeromorphic, semideciduous broadleaf scrub with seasonal shortgrass). termites were sampled in these four physiognomies, and in a cattle pasture that was described by carrijo et al. (2009) as “a 7-year-old pasture cultivated with brachiaria brizantha stapf. (poaceae), originally covered by cerrado sensu stricto and cleared and turned into pasture approximately 50 years ago”. we used the same methods as carrijo et al. (2009), that is: in each physiognomy we established one linear transect with 10 plots of 5 x 2 m and 2 m height. each plot was 30 m distant from the next one, and the transect was at least 50 m distant from the border of the particular physiognomy. the plots were carefully searched for termites in all possible places where these insects can be found: epigean nests, litter, plant stalks, fallen branches, soil surface, and to a depth of 20 cm below ground. each plot was sampled by two collectors during 30 min. a total of 50 plots were sampled, comprising an area of 500 m². because of the difficulty of estimating the number of colonies (relative abundance) with this protocol, all individuals of the same species that were collected in the same plot were considered as one encounter. thus, the maximum abundance of each species in each plot is 1 (one), and for all plots combined, 50 encounters (following bignell and eggleton, 2000). samples from all colonies were identified using appropriate literature and/or comparing with the termitological collection of the universidade federal de goiás (ufg), where the vouchers were deposited. species were classified in four functional groups (feeding guilds) according to field observations and literature information (mathews, 1977; gontijo & domingos, 1991; desouza & brown, 1994): xylophages, humivores, grass/litter feeders and intermediates. the statistical analyses were performed with the software r (r development core team, 2010), using the packages ‘vegan’ (oksanen et al., 2011) and ‘vcd’ (meyer et al., 2006, 2011). the sampling effort for each physiognomy was evaluated using a species accumulation curve, constructed using the function ‘specaccum’. the number of de oliveira, tf carrijo, d brandão termite composition in different physiognomies of cerrado192 species of termites was estimated by the estimators jackknife 1 and bootstrap, which are two of the best-known richness estimators (colwell & coddington, 1994; magurran, 2004); for this, we used the function ‘poolaccum’. a pearson chisquare test was performed between the number of encounters observed for each feeding guild, and that estimated based on a poisson distribution; the results of this test were presented in a cohen-friendly association plot, generated by the function ‘assoc’ (meyer et al., 2006). the dissimilarity in species composition between the five physiognomies was assessed using the chao-jaccard index (chao et al., 2005), with the function ‘vegdist’, 1,000 permutations and method ‘chao’. the cluster was performed with the function ‘hclust’ and method ‘average’. results in the five physiognomies, a total of 42 species and 27 genera of termites from the families termitidae and rhinotermitidae were collected (table 1). rhinotermitidae was represented by two species, coptotermes sp. and heterotermes tenuis. of the four subfamilies of termitidae, apicotermitinae was the richest, with 15 species, followed by nasutitermitinae, with nine species. a slight tendency toward stabilization can be observed in the species accumulation curve (fig 1). the number of observed species was closer to the bootstrap estimate (48 species) than to the jackknife 1 estimate (58). the sampling completeness was 87.5% if we use the first estimator, and 72.4% for the latter (fig 1). the savannas (cerrado sensu stricto and rupestrian cerrado) were the physiognomies with higher numbers of species, followed by the grasslands (shortgrass savanna and pasture), and the gallery forest, respectively (fig 2). none of the accumulation curves from the five physiognomies showed a stabilization tendency, which means that the numbers of species are certainly higher (fig 2). the relative abundance of termites in all physiognomies was 219 encounters, with 5.214 ± 5.542 (average ± standard deviation) encounters per species (table 1). however, a strong dominance of some species was found. the most common species, amitermes sp., anoplotermes sp. 3 and nasutitermes sp. 1, had 23, 21 and 17 encounters, respectively, while 16 species were found only once, comprising 38% of the total pool of species surveyed. the subfamily apicotermitinae was the most abundant both in absolute number (90 encounters) and in number of encounters per species (six encounters per species). rhinotermitidae was the least abundant by both measurements: six in absolute number, and three encounters per species. the cerrado sensu stricto showed the highest abundance of termites, both absolute (61), and per species (2.9); while the gallery forest had the lowest absolute abundance (25), and the shortgrass savanna had the lowest abundance per species (1.8). the most abundant species in the savannas (cerrado sensu stricto and rupestrian cerrado) was armitermes sp., while anoplotermes sp. 3 was the most abundant in the pasture and gallery forest, and nasutitermes sp. 1 in the shortgrass savanna (table 1). considering all physiognomies together, the most abundant and diverse guild was the humivores (98 encounters and 20 species) followed, in number of encounters, by the xylophages (55 encounters and six species) and, in number of species, by the intermediates (25 encounters and nine species). the grass/litter feeders had 41 encounters and seven species. humivorous and xylophagous species were the most abundant in all the physiognomies considered separately; except in the pasture, where the grass/litter feeders had 18 encounters, against three of xylophagous species. the proportion of feeding guilds in cerrado sensu stricto and rupestrian cerrado was similar, with more humivores and xylophages, but also with a high proportion of intermediates, compared to the other physiognomies (fig 3). the pearson chi-square test showed that the shortgrass savanna had a lower proportion of grass/litter feeders than that expected by chance (fig 3). the gallery forest showed a higher proportion of humivores, while the proportions of the other guilds were lower than those expected by chance. the pasture was the most singular physiognomy; the proportions of humivores and, mainly of grass/litter feeders were much higher, while those of intermediates and xylophages were much lower than those expected by chance (fig 3). regarding the species composition, all physiognomies were very similar to each other (fig 4). all the open areas had a dissimilarity index less than 0.5. the gallery forest and pasture were the most dissimilar areas (chao-jaccard index = 0.8426). in fact, only three species were shared between gallery forest and pasture, and two of them (anoplotermes figure 1 accumulation curves with the observed termite richness and the estimates from bootstrap and jackknife 1 of five physiognomies pooled together from the parque estadual da serra de jaraguá. vertical lines are the standard deviations. sociobiology 60(2): 190-197 (2013) 193 fig 2 accumulation curves with the observed termite richness for each physiognomy present in the parque estadual da serra de jaraguá. vertical lines are the standard deviations. sp. 3 and nasutitermes sp. 1) were the only species that were present in all physiognomies. discussion the species richness at the parque estadual da serra de jaraguá was as expected, as the local richness of termites in the cerrado biome is about 40 to 60 species (constantino, 2005), ranging from 15 (cunha et al., 2006) to 114 (mathews, 1977). in previous surveys in the cerrado, the subfamily nasutitermitinae was frequently the richest and most common group, but in these studies the species of apicotermitinae were frequently pooled together due to limitations of identification (constantino, 2005; cunha et al., 2006). the true richness and abundance of apicotermitinae is expected to be very high (eggleton, 1999), which was the case in the present study. however, most previous studies in the cerrado cannot be properly compared, since the protocols used are not standardized. the gallery forest had the second smallest proportion of xylophages (6 species of a total of 25). this was an unexpected result, since the amount of wood is expected to be higher in forests than in the other physiognomies, and, consequently, more xylophagous species were expected in this environment. the reason is probably that, in the savannas, most of the wood needed by the xylophages is in shrubs, below two meters (the maximum height collectable by the protocol used), while in the forest the xylophages are situated much higher, in the canopy, and out of reach of our collections. in forests, a vertical gradient of termite species occurs and the xylophagous species found on the ground are only a subsample of those on the canopy (roisin et al., 2006). the grass/litter feeders were the most abundant termites in the pasture. this was expected, since grass is the most abundant food resource for termites in this environment. moreover, litter-feeding species occur in higher proportions in fig 3 cohen-friendly association plot for the alimentary guilds of termites for each physiognomy of the parque estadual da serra de jaraguá. the bar length is proportional to the contribution to the pearson chi-squared; and the bar area is proportional to the difference between the observed and expected frequencies. the independence values for each physiognomy are the vertical dashed lines. the black bars, to the right, are the values higher than expected by chance, and the white bars, to the left, are the values lower than the expected. grassland studied, which suggests that this is linked to human activity, turning cerrado sensu stricto into pasture, and not only the coverage of grass. the effects of pasture formation on termite communities are well known and include a reduction of richness and abundance of humivores and mostly xylophages, leading to high rates of local extinctions and an increase in the abundance of grass-feeders, which transforms some species into aesthetic pests (brandão & souza, 1998; constantino, 2002; cunha & orlando, 2011). the termite fauna studied here had a very similar composition between all physiognomies. although previous studies have concluded that each physiognomy has a different termite species composition (e.g., constantino & acioli, 2008), the natural open areas (rupestrian cerrado + cerrado sensu stricto + shortgrass savanna) investigated here have about the same termite species pool. mathews (1977) also found a gradient of termite species from open areas to forests, with a high number of shared species of two or more physiognomies. eiten (1983) observed a continuum of plant species between cerrado sensu stricto and rupestrian cerrado. in an inventory of plant species of the parque estadual da serra de jaraguá (unpublished data), the present authors found a continuum of plant species between all the natural open areas, with some exclusive species in each physiognomy. the gallery forest and the pasture each had a distinct cerrado than in forest biomes (constantino & acioli, 2008). however, the high proportion of grass/litter feeders led to a skewed pattern in the feeding-guild proportions. compared to the other physiognomies, the pasture had fewer xylophages and intermediates, and many more grass/litter feeders. this pattern was not as evident for the shortgrass savanna, the other de oliveira, tf carrijo, d brandão termite composition in different physiognomies of cerrado194 table 1 list of the number of termite encounters in different physiognomies of cerrado in parque estadual da serra de jaraguá, state of goiás, brazil. (feeding group x – xylophagous; h – humivorous; g – grass/ litter feeder; i – intermediate). family/subfamily/species feeding group pasture shortgrass savanna cerrado sensu stricto rupestrian cerrado gallery forest rhinotermitidae coptotermes sp. x 0 0 1 0 0 heterotermes tenuis x 0 2 1 0 2 termitidae: apicotermitinae anoplotermes sp.1 h 0 3 5 5 3 anoplotermes sp.2 h 1 1 2 1 0 anoplotermes sp.3 h 7 4 4 2 4 anoplotermes sp.4 h 0 3 3 2 4 anoplotermes sp.5 h 0 1 2 1 1 anoplotermes sp.6 h 6 1 0 2 0 anoplotermes sp.7 h 3 2 0 0 0 anoplotermes sp.8 h 1 0 0 0 0 anoplotermes sp.9 h 0 1 0 0 0 aparatermes sp. h 1 0 1 0 1 grigiotermes sp.1 h 1 0 0 0 0 grigiotermes sp.2 h 1 0 0 0 0 grigiotermes sp.3 h 0 0 0 1 0 ruptitermes sp. g 6 0 1 0 0 tetimatermes sp. h 0 0 1 0 0 termitidae: nasutitermitinae agnathotermes sp. h 1 0 0 0 0 anhangatermes sp. h 0 0 0 1 0 atlantitermes stercophilus i 0 1 0 0 0 nasutitermes sp.1 x 1 5 4 6 1 nasutitermes sp.2 x 0 0 5 1 2 nasutitermes sp.3 x 0 0 0 0 1 parvitermes bacchanalis g 0 0 1 0 0 subulitermes sp. h 0 0 0 0 1 velocitermes heteropterus g 0 1 5 1 1 termitidae: syntermitinae silvestriermes euamignathus i 1 0 2 3 0 cornitermes silvestrii g 2 0 3 3 0 cornitermes villosus g 0 0 0 0 3 curvitermes minor i 0 1 0 1 0 cyrilliotermes cupim h 0 1 0 0 0 embiratermes festivellus i 0 0 3 2 0 labiotermes emersoni h 1 0 2 6 0 procornitermes araujoi g 4 0 0 2 0 syntermes nanus g 6 1 0 1 0 termitidae: termitinae amitermes sp. x 2 3 10 8 0 dihoplotermes cf. inusitatus i 0 0 0 1 0 neocapritermes araguaia i 0 1 2 1 0 neocapritermes opacus i 0 1 0 0 0 neocapritermes talpa i 0 0 0 0 1 spinitermes trispinosus h 0 0 0 2 0 termes sp. i 0 0 3 1 0 encounters (total = 219) 45 33 61 54 26 species richness (total = 42) 17 18 21 23 13 sociobiology 60(2): 190-197 (2013) 195 subsample of termite species from the natural open areas, plus some exclusive species. for example, of all the termite species found in the gallery forest, four were exclusive to this physiognomy and the other nine species were also found in cerrado sensu stricto. in spite of their high overall similarity, the pasture and gallery forest had very different species compositions, with only three shared species. this is an expected result, since the vegetation structure in gallery forest is very different from pasture. in gallery forest there is an abundant amount of wood and a comparatively small stock of grass, and the opposite in pasture. in fact, two species of xylophages were found for each species of grass/litter feeders termites in the gallery forest. in pasture the opposite occurred, with two species of grass/litter feeders found for each species of xylophagous termites. although the gallery forest had a different species composition pool, there is no evidence in the present study to support the hypothesis that this physiognomy hosts species from the amazon and atlantic forest biomes. as mentioned above, the species pool of the gallery forest is a subsample of that in cerrado sensu stricto plus some exclusive species (nasutitermes sp. 1, subulitermes sp. 1, cornitermes villosus and neocapritermes talpa). of these species, cornitermes villosus only occurs in forests of the cerrado biome; nasutitermes and subulitermes have species occurring in open areas of cerrado; and only neocapritermes talpa occurs in both the amazon and cerrado forests. in the last decade, different physiognomies have undergone different rates of deforestation, and those with denser vegetation (i.e., forests and savannas) have been most affected (rocha et al., 2011). it is alarming that gallery forests, which have a relatively depauperate but little-known termite fauna, are among the physiognomies that are most affected by deforestation. this study showed that termites have a high number of species and abundance in cerrado areas, mainly in the savanna physiognomies. the physiognomies have a similar species composition, mainly the natural open areas, and there is no support in the present study for the hypothesis of a distinct fauna of termites in forest. as expected, the termite fauna of gallery forest is very different from pasture, but most of the species also occur in natural open areas. the implantation of pasture seems to impact the termite diversity and, mainly, the species composition and proportions of the feeding guilds. however, this statement should be reinforced by future studies, with a greater sampling effort. acknowledgements we thank our friends diogo a. costa and thiago santos for their help in discussing the project and also for their essential help in the fieldwork. this study was supported by the conselho nacional de desenvolvimento científico e tecnológico, brazil, and the coordenação de aperfeiçoamento de pessoal de nível superior, capes, brazil. the secretaria do meio ambiente e dos recursos hídricos, semarh, brazil, for granting a collecting permit. we also thank two anonymous reviewers for valuable comments that improved this article. references agência ambiental, g. 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(1978). the role of termites in ecosystems. in m.v. brian (ed.), production ecology of ants and termites (pp. 245–292). cambridge: cambridge university press. doi: 10.13102/sociobiology.v66i4.4477sociobiology 66(4): 592-596 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 first record of myrcia magna d.legrand (myrtaceae) as a myrmecophyte host for myrcidris epicharis ward, 1990 (formicidae: pseudomyrmecinae) in the neotropics, mutualistic relationships between plants and ants frequently provide protection against predators to the hosts and housing to the insects (goitía & jaffé, 2009). several independent plant-based myrmecophilous interactions have been registered in nature (heil & mckey, 2003). this category of mutualism is particularly notable in amazonian communities, where a large number of species of the formicidae are found inhabiting domatia of plants (dáttilo et al., 2013). the subfamily pseudomyrmecinae (formicidae) comprises the genera myrcidris ward, pseudomyrmex lund, and tetraponera f.smith. many species of these genera are arboreal, and some have association with plants that bear domatia (ward, 1990; ward & downie, 2005; baccaro et al., 2015). to date, myrcidris epicharis ward, 1990 is the only species described for this genus of ants (ward & downie, 2005) that establishes its colonies in domatia present at the nodes of branches of amazonian plants of the genus myrcia abstract the association of the ant myrcidris epicharis with the plant myrcia magna is reported for the first time. this association was registered in two localities along the negro river basin, in the region of manaus, amazonas state, brazil. the ants inhabit swollen shoots in apical and subjacent nodes of the branches. this record represents the second plant species of myrtaceae to be associated with myrcidris epicharis. sociobiology an international journal on social insects ph gaem1, f farroñay2, tf santos3, nb cabello1, ff mazine1, a vicentini2 article history edited by jean c. santos, ufs, brazil received 17 april 2019 initial acceptance 04 july 2019 final acceptance 23 august 2019 publication date 30 december 2019 keywords amazon rainforest, ant-plant association, hymenoptera, myrtales. corresponding author paulo henrique gaem universidade federal de são carlos campus sorocaba rodovia joão leme dos santos (sp-264) km 110, cep 18052-780, itinga sorocaba-sp, brasil. e-mail: phgaem@gmail.com dc. (myrtaceae) (passmore et al., 2012; vicente et al., 2012). this association has been registered only between myrcidris epicharis and myrcia madida mcvaugh [for a detailed description of this specialized relationship see ferreira and vasconcelos (2010) and vicente et al. (2012)]. here we report an interaction between myrcidris epicharis and another species of myrtaceae, myrcia magna d.legrand. the samples of myrcidris epicharis and myrcia magna were collected during field expeditions to the rio negro sustainable development reserve (rds rio negro, 03°02’56’’s, 60°06’11’’w) and to the alto cuieras river base of the national institute for amazonian research (cuieras, 02°34’07’’s, 60°19’15’’w) in january 2018 and july 2018 respectively (fig 1). the plants were identified based on specimens deposited at inpa herbarium (from national institute for amazonian research, manaus, amazonas, brazil) and on the information of the literature about the systematics 1 universidade federal de são carlos, sorocaba-sp, brazil 2 instituto nacional de pesquisas da amazônia, manaus-am, brazil 3 universidade federal do amazonas, manaus-am, brazil short note sociobiology 66(4): 592-596 (december, 2019) 593 of myrcia (lucas et al., 2016; 2018). the ants were identified following baccaro et al. (2015). pictures in the field were taken with a nikon d3300 camera and laboratory pictures were taken with a leica m205c stereomicroscope. voucher material of the plants was deposited at soro herbarium (from federal university são carlos, sorocaba, são paulo, brazil) and the ants were deposited at zoological collection paulo bührnheim (from federal university of amazonas, manaus, amazonas, brazil). additional material of myrcia magna was selected by examining the specimens deposited in herbaria in order to hypothesize the actual range of occurrence of this interaction. the specimens of myrcia magna deposited at inpa were analyzed directly, and the presence of domatia in materials from other plant collections was checked from online high-resolution images. the geographical coordinates were extracted from the specimens’ labels when available and estimated when missing. a map of plant species distribution was made using qgis version 2.18.20 (qgis development fig 1. distribution of myrcia magna: our records of plants associated with myrcidris epicharis (yellow stars), and records of plant material deposited in herbaria that bear domatia (blue circles) and without domatia (red circles). team, 2019). ants inhabiting domatia of myrcia magna were seen and collected from two plants, one in the cueiras and another in the rds rio negro reserve. the domatia in myrcia magna are presented as hollow cavities in the branches just below each leaf node and also at the base of the petioles, and they are accessed by the ants through circular holes (fig 2). workers and queens were observed and collected in both plant individuals (fig 3). however, differently from myrcia madida in which only one queen of myrcidris epicharis was reported per host plant (ferreira & vasconcelos, 2010), in myrcia magna we recorded two queens per plant in rds rio negro and one queen in the cuieras reserve. the mutualistic relationship between myrcidris epicharis and myrcia madida was reported to the central (ferreira & vasconcelos, 2010; passmore et al., 2012; dátillo et al., 2013) and southern amazon (vicente et al., 2012). other authors also observed a similar phenomenon between myrcidris epicharis and an unidentified species of myrcia in the region of manaus (ward, 1990). myrcidris epicharis was also registered in colombian amazon, but with no information about possible interactions with myrmecophytes (guerrero, 2009). several studies considered the association between myrcidris epicharis and myrcia madida as highly specialized and mandatory (e.g. bruna et al., 2005; dáttilo et al., 2013), but our report involving another species of plant is a first step into the discussion of the specificity of this interaction. not all herbarium collections of myrcia magna presented domatia (fig 1), but perhaps they only appear in recently grown branches, unlike those with flowers and fruits frequently preserved. myrcia magna is distinguished from myrcia madida by its inflorescences with opposite to alternate branching, flowers with glabrous floral disc and rounded fruits (vs. inflorescence branching exclusively opposite, floral disc distinctively pilose and ellipsoid fruits in myrcia madida). according to a recently published infrageneric molecular-based classification of the genus myrcia, myrcia magna belongs to myrcia sect. aulomyrcia (o.berg) griseb. whilst myrcia madida is ph gaem, f farroñay, tf santos, nb cabello, ff mazine, a vicentini – myrcia magnaas host of myrcidris epicharis594 placed within myrcia sect. myrcia (lucas et al., 2016; 2018). therefore, these two interactions appear to have originated independently within the genus. moreover, the domatia present in these two species of myrcia are very similar. it is unclear whether the ants trigger the development of domatia or escave them, and further investigation is required to better understand these associations. zoological material examined: brazil. amazonas: iranduba, rds do rio negro, galho de myrcia sp., 03°02’56”s, 60°06’11”w, 27 january 2018, coleta manual, col.: f. farroñay (czpb-hy000006–7); manaus, base alto rio cuieras, 02°34’07”s, 60°19’15”w, 09 july 2018, coleta manual, col.: f. santana. (czpb-hy000003–5). fig 2. ant-plant association: fertile branch of myrcia magna (gaem et al. 148)(a), fruit of myrcia magna (b), longitudinal section of a domatium (c), a domatium just below a node (d), an individual of myrcidris epicharis (f. farroñay, czpb) getting out of a domatium (e), and detail of an ant (f). sociobiology 66(4): 592-596 (december, 2019) 595 plant material examined: brazil. amazonas: iranduba, rds do rio negro, mata de terra firme, 03°02’56”s, 60°06’11”w, 27 january 2018 (fr.), p.h. gaem et al. 148 (soro 6575); manaus, proximidades da base alto cuieras do inpa, 02°34’07”s, 60°19’15”w, 09 july 2018, n.b. cabello 102 (soro 6574). additional plant material considered. inpa: d.f. coêlho s.n. (inpa 2247); d.f. coêlho et al. 59 (inpa 98378); a.h. gentry 12863 (inpa 128044); w.a. rodrigues 4004 (inpa 10569); w.a. rodrigues 4361 (inpa 10927). ny: d.f. coêlho et al. 59 (barcode: 01461064); c.a. cid ferreira et al. 6770 (barcode: 01461065) m.j.r. pereira et al. 3304.4967 (barcode: 01551639); g.t. prance et al. 3758 (barcode: 01461068); g.t. prance et al. 3714 (barcode: 01461067); w.a. rodrigues and j. lima 4119 (barcode: 01461066); m.g. da silva 919 (barcode: 01461063). spf: s.g. egler 1101 (barcode: 00131778). acknowledgements we thank the anonymous reviewers for the precious observations. we are also indebted to the myrtaceae specialist marcos sobral, who kindly helped us to identify myrcia magna. moreover, we thank the cenbam-ppbio group: armando dos santos, lúcia pinto, william magnusson, albertina lima, iderland viana, emilio higashigawa, ramiro melsinki, andresa viana, and the field assistants from the ramal uga-uga village. the collection at the rds rio negro reserve was made during inventory conducted by ff as part of his studies at the master program in botany at national institute of amazonian research (inpa) and as part of the cnpq research group ecology and evolution of amazonian plants of inpa. the collection at the cuieras reserve was obtained in july 2018 during the “i natural history course at the cuieras reserve” organized by the museu na floresta project (inpa-jica). we thank national council for scientific and technological development (cnpq) for research funding, grant 302309/2018-7. this study was financed in part by coordination for the improvement of higher education personnel (capes) finance code 001. finally, this paper is part of the results of a project supported by são paulo research foundation (fapesp), grant 2018/13985-9. authors’ contributions phg, ff, nbc & av collected the plants and ants during the field expeditions cited. phg, ff & tfs investigated the taxonomy of the organisms involved in the interaction. phg & ff made the literature review and verified the novelty of the interaction.all authors wrote the manuscript. phg, ff & tfs prepared the figures. finally, ffm & av advised this work as a whole. fig 3. myrcidris epicharis (f. santana, czpb). a worker in lateral view (a) and full-face view (b). queen in lateral view (c) and full-face view (d). ph gaem, f farroñay, tf santos, nb cabello, ff mazine, a vicentini – myrcia magnaas host of myrcidris epicharis596 references baccaro, f.b., feitosa, r.m., fernandez, f.; fernandes, i.o., izzo, t.j., souza, j.l.p. & solar, r. (2015). guia para os gêneros de formigas do brasil. editora inpa, manaus, am. 388pp. bruna, e.m., vasconcelos, h.l. & heredia, s. (2005). the effect of habitat fragmentation on communities of mutualists: amazonian ants and their host plants. biological conservation, 124: 209-216. doi: 10.1016/j.biocon.2005.01.026 dáttilo, w., izzo, t.j., vasconcelos, h.l. & rico-gray, v. (2013). strength of the modular pattern in amazonian symbiotic ant–plant networks. arthropod-plant interactions, 7: 455-461. doi: 10.1007/s11829-013-9256-1 ferreira, l.v. & vasconcelos, h.l. (2010). on a poorly known amazonian ant-plant association: myrcia madida mcvaugh (myrtaceae) and myrcidris epicharis ward (hymenoptera: formicidae: pseudomyrmecinae). boletim do museu paraense emílio goeldi. ciências naturais, 5: 363-367. goitía, w. & jaffé, k. (2009). ant-plant associations in different forests in venezuela. neotropical entomology, 38: 7-31. doi: 10.1590/s1519-566x2009000100002 guerrero, r.j. (2009). first record of the ant genus myrcidris (formicidae: pseudomyrmecinae) from colombia. revista colombiana de entomologia, 35: 103-104. heil, m. & mckey, d. (2003). protective ant-plant interactions as model systems in ecological and evolutionary research. annual review of ecology, evolution and systematics, 34: 425-53. doi: 10.1146/annurev.ecolsys.34.011802.132410 lucas, e.j., amorim, b.s., lima, d.f., lima-lourenço, a.r., nic lughadha, e.m., proença, c.e.b., rosa, p.o., rosário, a.s., santos, l.l., santos, m.f., souza, m.c., staggemeier, v.g., vasconcelos, t.n.c. & sobral, m. (2018). a new infrageneric classification of the species-rich neotropical genus myrcia s.l. kew bulletin, 73: 1-12. doi: 10.1007/s12225-0179730-5 lucas, e., wilson, c.e., lima, d.f., sobral, m. & matsumoto, k. (2016). a conspectus of myrcia sect. aulomyrcia (myrtaceae). annals of the missouri botanical garden, 101: 648-698. doi: 10.3417/2014015 passmore, h.a., bruna, e.m., heredia, s.m. & vasconcelos, h.l. (2012). resilient networks of ant-plant mutualists in amazonian forest fragments. plos one, 7: 1-8. doi: 10.1371/journal.pone.0040803 qgis development team. (2019). qgis geographic information system. open source geospatial foundation project. retrieved from: http://qgis.osgeo.org vicente, r.e., dátillo, w. & izzo, t.j. (2012). new record of a very specialized interaction: myrcidris epicharis ward 1990 (pseudomyrmecinae) and its myrmecophyte host myrcia madida mcvaugh (myrtaceae) in brazilian meridional amazon. acta amazonica, 42: 567-570. doi: 10.1590/s004459672012000400016 ward, p.s. (1990). the ant subfamily pseudomyrmecinae (hymenoptera: formicidae): generic revision and relationships to other formicids. systematic entomology, 15: 449-489. doi: 10.1111/j.1365-3113.1990.tb00077.x ward, p.s. & downie, d.a. (2005). the ant subfamily pseudomyrmecinae (hymenoptera: formicidae): phylogeny and evolution of big-eyed arboreal ants. systematic entomology, 30: 310-335. doi: 10.1111/j.1365-3113.2004.00281.x doi: 10.13102/sociobiology.v66i1.3178sociobiology 66(1): 44-51 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 fire effects on the ant community in areas of native and exotic vegetation introduction invertebrates have been used for years as indicators of the ecological conditions of an ecosystem. that is why they are highly diverse, functionally important, sensitive to variations in the environment and easy to sample (greenslade & greenslade, 1984; rosenberg et al., 1986; andersen, 1999; brown, 1997; mcgeoch, 1998). ants have all these characteristics and many authors consider them as better bioindicators than other groups of arthropods (andersen, 1991; peck et al., 1998; alonso, 2000; kaspari & majer, 2000). however, their use in monitoring programs in argentina is scarce. ants have been used as a tool to monitor sites under restoration or impacted by the functional groups scheme (andersen, 1995, 1997). this model has been widely used to abstract acacia melanoxylon is an invasive species of the mountain grassland of the southeastern part of the buenos aires province, argentina. fires are a natural disturbance, characteristic of the area, and favor the germination of this invasive plant. however, they are used as the first step in management systems for the acacia species. moreover, the use of ants in monitoring programs is very scarce for argentina. the objectives of this work are: 1) to analyze the response and resilience capacity of native and invaded sites by a. melanoxylon after a fire for controlling this invasive species; 2) to detect groups of ants considered to be indicators of the recovery phase subsequent to the burning; and 3) to apply the concept of groups of disturbances, proposed by roig and espadaler, as an effective tool for monitoring. the sampling design consisted of three replicates of 10 pitfall traps for each environment, native and invaded. ant species were grouped into functional groups, trophic guilds, and disturbance groups. the fire did not generate significant changes in the richness and abundance of ants in the mountain grassland. however, it generated a positive effect on the sites invaded by acacia during the first year after the fire. the groups of minimum specialists of vegetation and the dominant dolichoderinae are considered good bioindicators. finally, the disturbance indicators can be considered reliable management tools if the biology of the species that compose it is known beforehand. sociobiology an international journal on social insects jm arcusa article history edited by kleber del-claro, ufu, brazil received 16 march 2018 initial acceptance 23 april 2018 final acceptance 21 november 2018 publication date 25 april 2019 keywords invasive species, restoration , grassland, fire. corresponding author juan manuel arcusa universidad nacional de mar del plata instituto de investigaciones en biodiversidad y biotecnología (inbiotec) comisión de investigaciones científicas de la provincia de buenos aires mar del plata, argentina. e-mail: juan.arcusa@gmail.com analyze biogeographic patterns in community composition and changes caused by disturbances (king et al., 1998). the success of this methodology is based on the large number of papers that describe the response of the ant community to the disturbances. they have been able to identify groups of species whose responses are consistent, decreasing or increasing, in relation to the disturbance (andersen & majer, 2004). buenos aires province has important areas for conservation. however, several factors, influence the dynamics of the environment in those areas, such as floods caused by strong storms (rossel & garcía martínez, 2008), strong winds causing soil blasting (gaspari, 2007), fires (anthropic or natural) and the presence of invasive plants (arcusa, 2016). fire affects the soil fauna directly and indirectly, changing the microclimatic conditions of the ground (guazzelli, 1999). on the other hand, biological invasions usually accompany universidad nacional de mar del plata, instituto de investigaciones en biodiversidad y biotecnología (inbiotec), comisión de investigaciones científicas de la provincia de buenos aires, mar del plata, argentina research article ants sociobiology 66(1): 44-51 (march, 2019) 45 the processes of environmental deterioration resulting in the elimination of the invaded ecosystems resilience (bilenca & miñarro, 2004). for this reason, it is important to monitor areas under conservation to preserve them. the aim of this work was to evaluate the effects of fire and invasive plants on a natural environment using ants as bioindicators. we monitored a burned area for a 3-year period. the fire is expected to produce positives effects in the invaded sectors, increasing the richness and abundance of ant species. moreover, it is expected to produce changes in the species composition and functional groups of ants. finally, we used for the first time the concept of disturbance groups proposed by roig and espadaler (2010) in a long monitoring program. materials and methods study area the paititi private nature reserve (fig 1) is located in the mar del plata-balcarce mountain range. it is part of one of the sierra de difuntos outcrops, which in turn is part of la peregrina of the sierras de mar del plata (guazzelli, 1999). its access point is at kilometer 22.5 of route 226. the vegetation is characteristic of the pampas grassland where paspalum quadrifarium (lambert) and p. exaltatum (presl) (poaceae) are dominant. shrubs conformed by baccharis articulata (persoon), b. cordifolia (candolle), b. dracunculifolia (dc) (asteraceae), buddleja thyrsoides (lamarck) (scrophulariaceae), dodonaea viscosa (boissier) (sapindaceae), and colletia paradoxa (sprengel) (rhamnaceae) predominate in the mountains. however, large areas of the reserve are invaded by the species acacia melanoxylon (r.br) (fabaceae). this forms a layer of leaf litter that results in a highly homogeneous environment. in march 2015, a fire was unleashed and burned 1000 hectares of the mountain environment. as a result, practically the entire sierra de difuntos was affected by fire, completely losing its vegetation (fig 1). sampling the sampling design consisted of three replicates, separated by 200 m., of 10 pitfall traps for each environment, native and invaded (60). data from the summer prior to the fire were obtained from a sector that was already being studied. after the fire, the sample was continued until 2016, so that the data for the next two summers could be obtained. the traps used are the classic pitfalls, consisting of plastic pots measuring 850 cm³ for determining the densityactivity of different edaphic arthropods (agosti et al., 2000). a 30% alcohol solution and detergent were used as a preservative. pitfalls were placed at a minimum distance of 10 m from each other. the data corresponded to the summer months (january to march) of 2014, 2015, and 2016. the traps were active for one week per month. fig 1. location and condition of the study area for the sampling years. the orange rectangles indicate the sites with native vegetation, the sky blue ones the sectors invaded by a.melanoxylon. jm arcusa – fire effects on the ant communities46 we studied the difference in richness, abundance, composition, functional groups, trophic guilds, and disturbance groups between native and invaded sites before and after a fire (1 and 2 years after the fire). ant species were grouped into functional groups. these were conformed according to the criterion proposed by andersen (1997) based on differences in habitat use, foraging mode, activity, and dominance. then, the species were grouped into trophic guilds, using those proposed by silvestre et al. (2003). finally, the ants were grouped into three groups taken from roig and espadaler (2010) according to three criteria: indicators of disturbance and indicators of maturity and cryptic species. to assemble the ants into groups, biological and ecological data of each species were taken from bibliography. it was determined possible differences between the three years for richness and abundance, functional groups, trophic guilds and disturbance groups using the two-way anova test and man-whitney test. in order to determine the difference in the structure composition, the anosim analyses was applied. these analyses were carried out by the programs past 3 and statcato 1.0.2. species abundances in individual traps were transformed to a five-point scale (1= 1 ant, 2= 2± 5 ants, 3= 6± 20 ants, 4= 21± 50 ants, 5= <50 ants) to avoid misleadingly high abundances due to sampling near foraging paths or nest entrances (king et al., 1998). results in the paititi natural reserve, 26 species were identified as belonging to 15 genera and 8 subfamilies. these were grouped into 7 functional groups and 13 trophic guilds (table 1). after the fire, the richness and abundance of both habitats were significantly incremented (fig 2). nevertheless, in 2016, both variables had decreased their value regarding 2015. on the one hand, for the native environment, these variables reached the initial values. on the other hand, for the invaded environment, both variables decreased their values in relation to 2015. however, this amount kept on being significantly higher than the initial state. finally, there were no differences in 2016 comparing both sites. table 1. classification of the ants in the different groups proposed. species functional group trophic guilt disturbance group acromyrmex lundi attini leaf-cutting ants disturbance acromyrmex ambiguus attini leaf-cutting ants disturbance apterostigma bruchi attini cryptic-cutting ants cryptic mycetophylax sp. attini cryptic-cutting ants cryptic crematogaster quadriformis generalized myrmicinae omnivorous dominant of ground maturity pheidole sp.1 generalized myrmicinae omnivorous dominant of ground disturbance pheidole sp. 2 generalized myrmicinae omnivorous dominant of ground disturbance pheidole sp.3 generalized myrmicinae minimum specialist of ground cryptic solenopsis richteri climate specialist omnivorous dominant of ground disturbance solenopsis minutissima climate specialist minimum specialist of vegetation maturity oxyepoecus sp. cryptic species predator cryptic myrmicinae cryptic linepithema humile dominant dolichoderinae omnivorous dominant of ground disturbance linepithema micans dominant dolichoderinae arboreal of massive recruitment cryptic camponotus puntctulatus subordinate camponotini generalist camponotini maturity camponotus bonariensis subordinate camponotini generalist camponotini maturity camponotus rufipes subordinate camponotini generalist camponotini maturity camponotus sp.1 subordinate camponotini generalist camponotini maturity camponotus sp.2 subordinate camponotini generalist camponotini maturity nylanderia silvestrii opportunist omnivorous dominant of ground maturity brachymyrmex brevicornis cryptic species vegetation and soil omnivorous maturity ponera opaciceps cryptic species cryptic ponerinae cryptic discothyrea neotropica cryptic species minimum specialist of ground cryptic pseudomyrmex phyllophillus climate specialist agile pseudomyrmecinae maturity neivamyrmex sp.1 climate specialist nomadic species maturity neivamyrmex sp.2 climate specialist nomadic species maturity gnamptogenys striatula opportunist large epigean predator maturity sociobiology 66(1): 44-51 (march, 2019) 47 fig 3. functional groups of ants. only groups with a representative number of individuals were plotted. fig 2. n= native site; a= invaded site. numbers correspond to the sample year: 1 for 2014, 2 for 2015 and 3 for 2016. same symbols indicates differences between sites. from the seven functional groups, only the subordinate camponotini (cs) was not present in the sectors invaded by acacia. furthermore, the opportunists (o) and climate specialists showed differences only in 2014 regarding their abundance between native and invaded environments, with the former being more abundant before the fire than the latter. finally, the dominant dolichoderinae group (dd), which was completely absent before the fire, increased in number significantly in 2016 (fig 3). jm arcusa – fire effects on the ant communities48 fig 4. trophic guilt of ants. only groups with a representative number of individuals were plotted. in relation to the trophic guilds, only three of them show a difference between sectors or years. the group of omnivorous dominants of ground showed a difference between the native and invaded environment for the postfire years. minimum specialists of vegetation, a group composed only of solenopsis minutissima, presented differences between environments and was more abundant before the fire. in subsequent years, it was not present, or its abundance turned out to be very low. finally, the guild of the generalist camponotini repeated the obtained results for the functional group of the subordinate camponotini, because these groups are identical (fig 4). when analyzing the disturbance groups, a considerable increase of the species of disturbance indicators was observed in 2015. for the following year, they diminished in abundance, reaching a situation similar to the one before the fire episode. the maturity indicator species varied significantly during the 3 years, increasing after the fire and subsequently decreasing in abundance (fig 5). changes in functional groups and trophic guilds are reflected in differences in structure assemblage. the native environment in 2014 was different regarding the same in 2015 (p = 0,001; r = 0,12) but it returned to the basal state in 2016 (p = 0,1; r = 0,1). the same occurred in the invaded site. there was a difference between 2014 and 2015 (p = 0,04; r = 0,3) but not considering 2016 (p = 0,8; r = -0,07). however, the r values were relatively low in all comparisons. sociobiology 66(1): 44-51 (march, 2019) 49 discussion differences between both environments in their initial sate were mainly attributed to changes in the resource availability by a. melanoxylon (le maitre et al., 2011; arcusa, 2016). meanwhile, the slight variation among the variables belonging to the native sites was mainly due to the geological history of the place. certain species that inhabit areas where fire is a part of the system are expected to be resistant and resilient to conflagration such as those that happen in the brazilian savanna and rupestrian grassland (anjos et al., 2017). in addition, areas with low vegetation, such as grasslands, are dominated mainly by ground ants. the soil temperature does not change significantly below 5cm. underground in the savannas (anjos et al., 2017), which would explain why the ants are protected from the fire. moreover, the fire completely eliminated the leaf litter produced by the acacia. this produced a significant increase in available resources such as seeds, tender shoots, and nesting sites. this increase attracted species from the surrounding areas by increasing the richness. finally, as a result of the rapid recovery rate of acacia (gibson et al., 2011), by 2016, all the invaded systems had recovered their initial state, leading to a negative impact on the species richness. regarding the functional group of the climate specialists, it was equivalent to the minimum specialists of vegetation guild as the former consisted mainly of solenopsis minutissima and the latter exclusively of them. both groups were considerably affected by the fire, decreasing their density-activity. this occurred because this species is frequently found nesting inside the floral poles of the plants belonging to the genus eryngium, the dry stems of carduus and cortadeira (pers. obs.). after the fire, the rods and dry stems were reduced to ashes as they are very flammable. the detection of this ant after two summers is possible due to the recolonization of the new floral poles. for species that nest on branches, leaves, and trunks, nesting sites are a limiting factor after a fire (fagundes et al., 2015). likewise, s. minutissima was not found in invaded sites where only a. melanoxylon predominated and, therefore, the conditions for its nesting were not satisfied. the guild of the minimum specialist of vegetation was previously considered an indicator of recovery environments (silvestre et al., 2003), for this reason, i consider it an important group for the monitoring of affected systems. the increase of dominant dolichoderinae after the fire reflects what is already known about this group. it usually increases its abundance after a disturbance and remains low or absent at undisturbed sites (king et al., 1998, andersen et al., 2003; andersen & majer, 2004; villalba et al., 2014). this group has an omnivorous diet and forages preferentially in open sites with bare soil (silvestre et al., 2003), which is why it is not found in the invaded areas (arcusa, 2016). this functional group is included within the guild of the omnivorous dominant of ground. solenopsis richteri is included among the species that define this guild, a disturbance indicator species (folgarait, 1998), that increased its abundance after the fire in the native environment. being a heterogeneous group consisting of five species, it is affected by the responses of each one of them, making its interpretation difficult. however, as a whole, it proved to be a beneficial and valuable group for monitoring because it was made up of two species of known roles as indicators of disturbance. in this regard, this work reinforces the observations made by silvestre et al. (2003) for this group. ants of the genus camponotus are characterized by being opportunistic and generalist in terms of diet and nesting (silvestre et al., 2003). both reasons may explain why this group was less affected by the fire. other studies indicate that these species can become more abundant after a fire episode (fagundes et al., 2015). camponotus punctulatus, the most abundant species, does not feed on leaves or seeds (gonzalezpolo et al., 2004), the main resources within the acacia forest. camponotus bonariensis is a species associated with stems of plants such as eryngium spp., cicuta sp., cortadeira sp., and cardurs sp. (kusnezov, 1951). these plants were not detected in the invaded sectors. the opportunist group, wich consist of nylanderia silvestrii and gnaptogenys striatula, were reduced in abundance after the fire. this could be probably related to the increase of dominant dolichoderinae, the occurrence of s. richteri, and the fact that this group is noncompetitive (fuster, 2014). the disturbance indicator group could be considered a good management tool if the biology of the species that composes it was widely known. in this work, the group was made by grouping species or genera whose response to disturbances is documented (folgarait, 1998; tizón et al., 2010; fuster, 2014; villalba et al., 2014; arcusa, 2016). in this way, it was possible to monitor the increase of these species and estimate the time of resilience. this is not the case for indicative of maturity species. this group turned out to be very heterogeneous in its composition. with a high number of species and scarce information of a biological and ecological nature, it was difficult to analyze the changes in abundance. the first application of these groups as a monitoring tool was fig 5. disturbance indicators. jm arcusa – fire effects on the ant communities50 for olive groves in portugal (patanita et al., 2012), and, as in this work, the importance of disturbance indicators as a management and monitoring tool was emphasized. changes produced in the different groups (functional groups, trophic guilds, and disturbance groups) reflect changes in the assemblages of ants. this is in accordance with other works where it is indicated that the fire produces changes in the structure of the communities (neves et al., 2016; anjos et al., 2017; maravalhas & vasconcelos, 2014; costa et al., 2010). conclusion the fire did not generate significant changes in the richness and abundance of ants in the mountain grassland of paititi’s nature reserve. however, it generated a positive effect on the sites invaded by acacia during the first year after the fire. it increased the values of richness compared to those found in the native environment. these values were reduced again after two yars. the minimum specialist of vegetation, mainly solenopsis minutissima, and the dominant dolichoderinae stands out at as bioindicator groups. the former are indicators of recovery of nesting sites and, therefore, an improvement in the conditions of the system. the latter, as has already been shown in previous works, are indicators of disturbance. within the disturbance groups, the disturbance indicators can be considered reliable management tools if the biology of the species that compose them is known beforehand. references agosti, d.; majer, j. d.; alonso, l. e. & schultz, t. r. 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(2014). grupos funcionales dominantes de hormigas (hymenóptera: formicidae) es pastizales naturales con y sin pastoreo del noreste de buenos aires, argentina. revista de la facultad de agronomía, 113: 107-113. doi: 10.13102/sociobiology.v65i4.3490sociobiology 65(4): 744-750 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 plasticity of stingless bee melipona fuliginosa lepeletier to obtain food resources in amazonia introduction fights between social insect colonies are common in nature (sakagami et al., 1993; gloag et al., 2008; breed et al., 2012; cunningham et al., 2014; grüter et al., 2016), and tend to be motivated by two main reasons: robbing of food from inside the nests and robbing of the entire stock of food, including the nesting site (cunningham et al., 2014; grüter et al., 2016). such fights between colonies vary considerably in terms of mortality; they can generate from few to thousands of victims, and the conflicts can last hours, days or even weeks (sakagami et al., 1993; grüter et al., 2016). among the approximately 500 species of stingless bees (meliponini) (michener, 2013), several have developed robbing or usurpation behavior (roubik, 1989; michener, 2007). there are species of exclusive robbers, such as those abstract the stingless bee melipona fuliginosa lepeletier is described as being aggressive robber, but there is little information about its raids. here, we describe two different raids of m. fuliginosa on other melipona species: melipona paraensis ducke and melipona fasciculata smith. the robbing behavior was observed in the volta grande do xingu region (pará) and carajás national forest (pará), and the attacks by m. fuliginosa occurred at the end of the dry season, shortly before the start of the rainy season, a time of flower scarcity. the raid on m. paraensis hive lasted five days and involved no deaths of worker bees of both species; the robbers collected honey and wax. during the pillaging, m. fuliginosa workers dedicated themselves exclusively to this task; their flight activity peaked between 8:00 a.m. and 12:00 p.m. but lasted until 6:00 p.m, which is atypical for the species. the raid on m. fasciculata differed from the other event because it led to the extermination of all forager workers of five colonies, however, the brood combs as well as the callow workers were preserved; the robbers collected honey and wax. m. fuliginosa attack defensive and non-defensive colonies, the events can cause severe damage and may lead to death of the victim colony in natural conditions. flight activity varies from foraging on flowers during dawn to all day long robbing, showing considerable plasticity to obtain food resources. robbing behavior could be associated to flower scarcity and artificial feeding. sociobiology an international journal on social insects l costa1, js galaschi-teixeira1, um maia1,2, vl imperatriz-fonseca1 article history edited by denise alves, esalq-usp, brazil received 10 may 2018 initial acceptance 05 june 2018 final acceptance 20 august 2018 publication date 11 october 2018 keywords hymenoptera, apidae, meliponini, bee attack, robbing behavior, cleptobiosis. corresponding author luciano costa instituto tecnológico vale rua boaventura da silva, 955 nazaré, 66055-090, belém-pa, brasil. e-mail: luciano.costa@itv.org in the genera lestrimelitta and cleptotrigona, and this behavior is known as cleptobiosis (nogueira-neto, 1997; michener, 2007; breed et al., 2012). these species do not collect nectar and pollen from flowers but take these resources from the stock inside the nest of other species. there are also facultative robber species (facultative cleptobiotics) that normally forage on flowers but occasionally invade colonies of other species to steal their food storage. this is the case for species such as melipona fuliginosa lepeletier (nogueiraneto, 1997; camargo & pedro, 2008), trigona spinipes (fabricius), trigona hyalinata (lepeletier) and oxytrigona spp. (nogueira-neto, 1997). finally, there are species whose main objective during attacks is nest usurpation, and these take over all the stock as well as the nest, as in the case of tetragonula hockingsi (cockerell) (cunningham et al., 2014) and tetragonisca angustula (latreille) (sakagami et al., 1993). 1 instituto tecnológico vale, belém, pará, brazil 2 universidade federal do pará, belém, pará, brazil research article bees sociobiology 65(4): 744-750 (october, 2018) special issue 745 although fights are common and have serious consequences for bee colonies, little is known about the robbing strategies employed to acquire resources in meliponini, especially in the case of facultative robber species (grüter et al., 2016) such as m. fuliginosa. m. fuliginosa bees are widely distributed through the neotropics, from the amazonian region to the atlantic rain forest in brazil, reaching northern argentina; they are robust eusocial bees, the largest of the genus melipona, with approximately 15 mm in length (camargo & pedro, 2008). they are predominantly black in color and nest inside the trunks or branches of live trees (roubik, 2006; camargo & pedro, 2008). in the literature, m. fuliginosa has been described as an aggressive robber bee (nates-parra, 1995; nogueira-neto, 1997; roubik, 2006; camargo & pedro, 2008). the formation of mixed colonies with melipona fasciata latreille has been reported (roubik, 1981), however, such studies do not provide details of how such events occur. here we describe raids by m. fuliginosa on two other congeners: melipona paraensis ducke and melipona fasciculata smith. m. fasciculata is a relatively large and defensive bee, measuring approximately 12 mm. m. paraensis is a less defensive species of medium size, approximately 10 mm long. both species have overlapping distribution with m. fuliginosa in the amazon region (camargo & pedro, 2013). similar to m. fuliginosa, m. paraensis and m. fasciculata nest in preexisting cavities in the trunks and branches of live trees. in contrast with m. fuliginosa, which is not adapted for rearing in rational bee hives (roubik, 1981; nogueira-neto, 1997), m. paraensis and m. fasciculata are commonly reared for honey production in the amazon (cortopassi-laurino et al., 2006; venturieri et al., 2012). materials and methods study period and site we observed m. fuliginosa raids on m. paraensis and m. fasciculata colonies in two distinct sites, as follows. volta grande do xingu region, pará the first observation was done in a meliponary located at the margin of the xingu river, pará brazil (3°22’24.3” s, 63°56’25.8” w). this meliponary received wildlife rescue colonies following deforestation for the installation of the belo monte hydroelectric plant. the predominant vegetation at the site was a mosaic of “igapó” (blackwater-flooded) forest, dryland forest, “capoeira” (secondary forest) and abandoned pasture (salomão et al., 2007). according to the köppen classification, the local climate is equatorial am and aw type, with an average temperature of 26 °c and annual rainfall of approximately 1,680 mm, which is concentrated between december and may (alvares et al., 2013). serra dos carajás, pará the second observation of robbery was done in the meliponary of the instituto tecnológico vale, desenvolvimento sustentável – itvds, carajás national forest, pará, brazil (6°2’57.87” s, 50°4’51.46” w), located in a matrix of primary open ombrophilous forest (zappi, 2017). according to the köppen classification, the climate of the carajás national forest region fits within type awi (alvares et al., 2013). temperatures are always above 18 °c with averages between 23 °c and 25 °c. the rains are concentrated between december and april. robbery record number 1 m. fuliginosa vs m. paraensis this raid was identified at 2:00 p.m. on november 19, 2015, during the daily inspection of the meliponary, at which point we began to record data on the workers who entered and left the m. paraensis colony as well as the m. fuliginosa colony. we counted the total number of workers going in and out for five minutes every hour from 6:00 a.m. to 7:00 p.m., that is, from sunrise to sunset. we also observed whether there was aggressive behavior (dead bees inside and in front of the hive, or bees biting and grabbing one another). the observations were carried out for five days until the phenomenon stopped. for each day of observation, we compared the number of m. fuliginosa workers entering and leaving the robbed colony as well as entering and leaving their own colony. because our data did not follow normal distribution (shapiro-wilk, p < 0.05), we used kruskal-wallis test. we used r studio software (rstudio team, 2016) for statistical analysis. colonies observed m. paraensis: a colony found in a deforested area in a “cajá” (spondias mombin, anacardiaceae) tree trunk. the trunk containing the colony was transported to the meliponary and was transferred to a beehive one month prior to the attack. at the time of the raid, the colony exhibited three combs of approximately 8 cm in diameter (the pupae had already emerged from the transferred combs). there were approximately 40 food pots, most of them containing honey. there were enough workers to perform the tasks of the new colony, but they had not yet delimited the entrance to the colony, which is typically the width of a worker’s thorax. thus, the entrance to the beehive was maintained with an opening of approximately 10 mm, although the workers built a six-centimeter-long internal entry tube. m. fuliginosa: a colony in its original substrate (“acapu” trunk, vouacapoua americana, fabaceae) that had been rescued from a deforested area four weeks earlier. through the opening at the top of the hollow trunk, one could see no sign of phoridae flies (pseudohypocera sp.) and that there were l costa, js galaschi-teixeira, um maia, vl imperatriz-fonseca – plastic behavior of melipona fuliginosa 746 pollen (most of the pots) and honey pots (three in the upper part). due to the nest being in a good state and the fact that the species does not adapt well to beehives (nogueira-neto, 1997), we chose to keep the colony in the original trunk. this colony was approximately 100 m from the m. paraensis colony. both colonies (m. paraensis and m. fuliginosa) as well as the other colonies in the meliponary were given supplemental food (100 ml of 50% sugar syrup) two to three times per week following rescue. the two colonies were new to the place, which already contained other colonies of different species of melipona and other meliponini genera. robbery record number 2 m. fuliginosa vs m. fasciculata we identified this raid a few days after its occurrence during routine inspection of the meliponary on november 4, 2017 (visits were performed every month). in this case, we were able to evaluate the damage caused by m. fuliginosa in five colonies of m. fasciculata. the attack was inferred based on the observation of dead m. fasciculata and m. fuliginosa workers on the ground in front of the hives, as well as by comparing the status of the colonies with the previous month’s observations from photographic records taken periodically. observed colonies m. fasciculata: five colonies in an intermediate state for the species, all containing five to six brood combs measuring 8–12 cm in diameter and approximately 20 honey and pollen pots in the upper part of the nests (beehive model; venturieri, 2004) as well as workers foraging, cleaning and guarding as usual. the colonies received approximately 200 ml of 50 % sucrose syrup every two weeks. m. fuliginosa: wild colony established in a tree in the surrounding forest, so we could not determine the status of the robber colony. results m. fuliginosa vs m. paraensis: description of robbing behavior from the first day of observation, the number of m. fuliginosa workers entering the m. paraensis colony increased, reaching a peak on the third day, and then decreased, reaching values close to zero on the fifth day and fully ceasing on the sixth day (fig 1). on all observation days the robbing occurred from 7:00 a.m. until around 5:00–6:00 p.m., when it ceased altogether. except for the first and fifth days, the activity peaked between 8:00 a.m. and 12:00 p.m. and decreased after that. the number of workers of m. fuliginosa entering and leaving the colony of m. paraensis, as well as the number of workers leaving and entering the m. fuliginosa colony was statistically equal during all observation days (h(3) = 3.63–0.50, p > 0.30) . therefore, we conclude that the flow was constant; that is, for each worker entering the colony, another left carrying material from the colony. likewise, the number of workers leaving and entering the m. fuliginosa colony was statistically equal to that of workers entering and leaving the m. paraensis colony, indicating that during the days of robbing, all the foragers of the m. fuliginosa colony were dedicated to that task. fig 1. flight activity of melipona fuliginosa during the robbing of an melipona paraensis colony. (a) m. fuliginosa workers entering the m. paraensis colony. (b) m. fuliginosa workers leaving the m. paraensis colony. (c) workers leaving the m. fuliginosa colony. (d) workers returning to the m. fuliginosa colony. there were no statistical differences among a, b, c and d (h(3) = 3.63–0.50, p > 0.30). sociobiology 65(4): 744-750 (october, 2018) special issue 747 from the moment when we realized that the raid was taking place and throughout the observation period, we noticed that the behavior of the m. paraensis colony changed. the workers of this species were no longer guarding the entrance, leaving it free for the m. fuliginosa workers to access without any kind of reaction. likewise, there was no internal fighting or reaction on the part of m. paraensis, and there were no dead bees or any bees grabbing each other in front of the nest, as is typical in attack situations. during the days of the robbery, most m. paraensis workers were concentrated on the walls or at the bottom of the beehive or on the brood combs; the mother queen was not killed. there were also no foraging or flights to remove garbage from the colony, and egg-laying process was also interrupted. it is important to note that although this colony was not very populous (we had found other colonies of the same species that were very populous), the bees foraged and cleaned the nest normally before the robbing, considering what is commonly observed for the species. this led us to hypothesize that the attack by m. fuliginosa somehow changed the behavior of the m. paraensis workers. the robbing behavior consisted of stealing honey and wax from the food pots, and on the sixth day, after the robbing had ended, the m. paraensis workers resumed garbage removal as well as foraging flights and guarding the nest entrance. feeding and general care enabled the colony to recover. fig 2. upper left: melipona fuliginosa and melipona fasciculata workers dead, grabbing one another. center left: decapitated m. fasciculata worker. lower left: decapitated m. fuliginosa worker. upper right: m. fasciculata colony one month before the robbing. lower right: the same colony after robbing showing empty pots. l costa, js galaschi-teixeira, um maia, vl imperatriz-fonseca – plastic behavior of melipona fuliginosa 748 m. fuliginosa vs m. fasciculata: robbery description on the ground in front of the five m. fasciculata colonies, there was evidence of a fight between this species and m. fuliginosa. many headless sets of thoraxes and abdomens of m. fasciculata workers were found in front of the hives along with whole and parts of bodies of m. fuliginosa in a ratio of 10.5:1. workers of the two species were also found grabbing each other (fig 2). after the attack, the m. fasciculata colonies predominantly had workers that still could not fly and few forager workers (compared to the previous month). this, together with the bodies of dead bees in front of the hives, indicated that the entire population of adult bees of all colonies had been killed during the attack by m. fuliginosa. internally, all the honey and part of the wax of the pots had been robbed from the m. fasciculata colonies, and only the non-flying workers, the brood combs and a portion of the empty pots remained in the colony. only one colony was infested by phoridae larvae after the attack. control of phoridae with vinegar traps (nogueira-neto, 1997), replacement of the bottom of the beehive, and supplementary feeding allowed the colonies to recover. discussion apparently, the type of attack by m. fuliginosa depends on the reaction of the target bee colonies. in the case of m. paraensis, the workers did not present defensive behavior, which represented an advantage for the colony because there was no worker mortality, while the defensive behavior of m. fasciculata workers resulted in a violent attack, causing the death of the entire population of guards and forager workers of five colonies. this observation demonstrates that the diversity of robbing behaviors varies not only among species (grüter et al., 2016) but also within the same species when attacking different species, as previously observed on lestrimelitta raids (sakagami et al., 1993). in the case of the obligate cleptobiotic bee lestrimelitta, species phylogenetically closer to it are more frequently victims of raids (sakagami et al., 1993; quezada-euán et al., 2013). moreover, phylogenetically closer species had less aggressive response to the presence of cleptobionts, due to the similarity of cuticular hydrocarbons (quezada-euán et al., 2013). that could be also a plausible explanation for the pacific occupation of the m. paraensis colony, because m. fuliginosa and m. paraensis (both in michmelia subgenus) are phylogenetically closer to each other than to m. fasciculata (melikerria subgenus) (ramírez et al., 2010; camargo & pedro, 2013). the non-defensive response of m. paraensis could be related to the similarity of cuticular hydrocarbon profile of m. fuliginosa or to the fact that the attacked colony was already in weakened condition. pheromones could also be involved, although we did not recognize any smell, such as citral or related substances, known to occur in cleptobiotic lestrimelitta (sakagami et al., 1993). however, we saw workers of m. fuliginosa raising the abdomen and fanning the wings inside the m. paraensis nest as well as at the entrance, outside the nest, as described for lestrimelitta raids (sakagami et al., 1993). in both observed raids, m. fuliginosa bees robbed honey and wax from the pots, differently than lestrimelitta, which takes also stored pollen and larval food (sakagami et al., 1993). the attacks discussed here occurred in the same period, in the late dry season shortly before the onset of the rainy season, when there is a shortage of flowers. the rainy season is known to be a period of resource scarcity for the stingless bees of the amazon region (marques-souza et al., 1996, 2007). thus, in both situations, food scarcity could be a possible trigger for the robbing behavior in m. fuliginosa. in addition, the artificial feeding and the management of the colonies could have helped to attract the raids. flower scarcity has also been associated to violent robberies practiced by m. fuliginosa on apis mellifera l. hives (nates-parra, 1995). beekeepers in the region of santander (serra andina) and the llanos (amazon lowlands) have reported the decapitation of individuals and robbing of whole colonies of africanized bees by m. fuliginosa (nates-parra, 1995). in the present study, we observed the simultaneous attack of five m. fasciculata colonies, which certainly required the mobilization of a large number of m. fuliginosa workers. as we observed in the attack on the m. paraensis colony, all the forager workers of the m. fuliginosa colony were involved in the robbing, and this was the only activity carried out by the colony during the pillaging period. in addition, the flight activity of the m. fuliginosa colony changed considerably during the robbery period. normally, the workers of this species forage in the early hours of the morning, interrupting or considerably reducing their flight activity after 7:00 a.m. (cortopassi-laurino et al., 2007). however, as observed here, the flight activity of m. fuliginosa extended throughout the day during robbing and was concentrated between 8:00 a.m. and 12:00 p.m., which was different from its normal rhythm. this change in flight activity due to robbing could explain the sudden change in the flight activity of m. fuliginosa observed by cortopassi-laurino et al. (2007). they found the peak flight activity of workers to be at 6:00 a.m., but the activity peak changed to 12:00 p.m. during one of the observation days. this change occurred in the last month of the dry season (which is in october in xapuri, acre, brazil), the same environmental conditions recorded for the two raids described hereby. the raids presented here also help to clarify what roubik (1981) considered to be a natural mixed colony of m. fuliginosa and m. fasciata. he wrote that the brood chamber was composed of m. fasciata pupae and brood combs containing larvae and eggs laid by a physogastric queen of m. fuliginosa. the m. fasciata physogastric queen was not found sociobiology 65(4): 744-750 (october, 2018) special issue 749 in the colony. workers of m. fasciata coexisted pacifically with m. fuliginosa and kept working normally for the colony. this mixed colony was probably a m. fasciata colony invaded by m. fuliginosa, not for robbing food and wax but for nest usurpation. it seems clear that a former m. fasciata colony was gradually becoming a m. fuliginosa colony. however, the author considered it was the opposite. as well as m. paraensis, m. fasciata belongs to the michmelia subgenus, indicating again correlation between phylogenetic closeness to m. fuliginosa and less defensiveness against its raids. this observation is in accordance with the less defensive behavior of phylogenetically related species to the raids of lestrimelitta (quezada-euán et al., 2013). comparing the results discussed here as well as the literature reports and the review by grüter et al. (2016), we can conclude the following: attacks by m. fuliginosa are highly organized because the whole colony can be involved in the attack. the species can attack a single sparsely populated colony as well as five sparsely populated colonies simultaneously, the latter case being certainly equivalent to attacking a very populous colony. the attacked colonies did not have high-quality stocks because most of the stocked honey was from sugar syrup. m. fuliginosa attacks both nondefensive and defensive colonies and may steal resources or usurp nests. its attacks cause severe damage to the target colonies and could lead to death in natural conditions. flight behavior varies from foraging on flowers during dawn to all day long robbing, showing considerable plasticity to obtain food resources. flower scarcity could be a possible trigger of robbing behavior in m. fuliginosa. acknowledgements the authors thank alexandre castilho, sérgio souza júnior, jenaldo carvalho, luiz batista, leandro maioli, renan coelho, olegário reis and everyone else who helped us in the carajás national forest, laerte viola e patricia bertola for support at belo monte, and anonymous reviewers that helped to improve this manuscript. authors’ contribution lc, jsgt and umm were responsibly for fieldwork and data sampling as well as manuscript writing. lc performed statistical analysis. vlif revised and improved the manuscript with suggestions. references alvares, c.a., stape, j.l., sentelhas, p.c., de moraes gonçalves, j.l. & sparovek, g. 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(2017). paisagens e plantas de carajás, 1° ed. belém: instituto tecnológico vale (itv). https://doi.org/10.1016/j.anbehav.2013.01.028 doi: 10.13102/sociobiology.v61i3.258-264sociobiology 61(3): 258-264 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 competitive interactions in ant assemblage in a rocky field environment: is being fast and attacking the best strategy? ta sales, in hastenreiter, lf ribeiro, jfs lopes introduction the structure of an ant assemblage can be influenced by various factors, the main ones being mutualism, competition, parasitism and predation. competition plays an important role in the structure of the assemblage, since competitive interactions could control the access of different species to resources (food and nesting sites), thus determining the coexistence among species within an assemblage. evidence which supports the role of competition as a structuring factor of ant assemblages comes from observation of physical and chemical aggression among species to protect resources and territorial limits (parr & gibb, 2010). the high competitive potential of each ant species arises from the fact that most of them are omnivorous. this increases the competition effect abstract the ant assemblage structure can be molded by mechanisms such as competition and discovery-dominance trade-off. in harsh circumstances it is likely that ant species that control the food resource are the first to arrive at the food source, the most aggressive (behavioral dominance), abundant (numerical dominance) and, thus, ecologically dominant. by these characteristics combination, the discovery-dominance should not be a trade-off, but a positive relationship. here, we examined the interactions among nine ant species in a rocky field area, in the ibitipoca state park, minas gerais, brazil. by offering attractive baits at field, we determined the discovery-dominance ability and the frequency of attack, avoidance and coexistence behaviors of each species. we showed that crematogaster sericea, pheidole obscurithorax and pheidole radoszkowskii abundance has a positive and significant correlation with their discovery ability. they were the first to arrive at the baits (best discoverers) and were numerically dominant, being thus considered ecologically dominant. despite p. radoszkowskii being part of this relationship, this interpretation should be taken cautiously. its dominance was assured by their high discovery ability and abundance, but the behavioral strategy exhibited was avoidance, not attacking as c. sericea and p. obscurithorax. the discovery-dominance trade-off could be broken by the linked characteristics that define the ecological dominant status of the ant species studied. also, p. radoszkowskii demonstrates that other strategies could surpass the combination of being fast and attacking, and thus this is not the best strategy for all. in harsh circumstances each species has its own best strategy. sociobiology an international journal on social insects ppgcb: comportamento e biologia animal, universidade federal de juiz de fora, juiz de fora, minas gerais, brazil article history edited by gilberto m m santos uefs, brazil received 14 january 2014 initial acceptance 29 april 2014 final acceptance 06 june 2014 keywords behavior, competition, discovery ability, ecologically dominant corresponding author tatiane archanjo de sales ppgcb, comportamento e biologia animal universidade federal de juiz de fora instituto de ciências biológicas campus universitário s/n juiz de fora, minas gerais, brazil 36036-300 e-mail: tatiane.archanjo@gmail.com and its importance in structuring ant assemblages (benson & harada,1988), which leads ants to exhibit various strategies to obtain food resources (carroll & janzen, 1973; detrain & deneubourg, 1997). the competitive strategy of some species to control the food source is through numerical dominance, with the predominance of particular species in numbers, biomass and/or frequency of occurrence (davidson, 1998). others exhibit aggressive behaviors which forces their competitors to avoid them (behavioural dominance) (bestelmeyer, 2000; davidson, 1998; fellers, 1987). however, this dominant status is not immutable. a study conducted by markó and kiss (2002) indicates that myrmica ruginodis changes its behavior from aggressive to submissive in the presence of a stronger competitor (manica rubida). espírito-santo et research article ants sociobiology 61(3): 258-264 (september 2014) 259 al. (2012) showed that the presence of a strong competitor (camponotus sericeiventris) leads c. rufipes to show different degrees of aggressiveness. when conspecific from different origins meet, the behavior of camponotus sericeiventris workers also changes from the simple inspection to foreignerchasing (yamamoto & del-claro, 2008). another mechanism widely investigated related to competition and coexistence in ant assemblages is the discovery-dominance trade-off. various authors have cited that there is a balance between discovery ability and resource dominance (vepsäläinen & pisarski, 1982; fellers, 1987; lebrun & feener, 2007; pearce-duvet et al., 2011), wherein dominat species which are slower to find food have greater capacity to defend it. on the other hand, species that are good at finding food can be classified as subordinate and their strategy for success is to find food quickly so as to exploit it partially before being dislodged by a dominant species (vepsäläinen & pisarski, 1982; fellers, 1987; lebrun & feener, 2007). however, this trade-off can be broken by ecological dominant ant species (davidson, 1998), invasive ant species (holway, 1999) and the presence of parasitoids (lebrun & feener, 2007), in structurally complex habitats (gibb & parr, 2010) or at high temperatures (bestelmeyer, 2000). in highly diverse ant assemblages high variation of the competitive interactions is likely to exist (andersen, 2008). the rocky field area, located at state park of ibitipoca, minas gerais, brazil, besides having an impressive richness of ant species (lopes et al., 2012), is an environment considered extreme and hostile, where food resources become scarce in certain periods (fowler et al., 1991). matching up all these characteristics is likely to find at rocky field-type habitats a more enhanced competition among ant species, wherein species that will control the food resource will be very aggressive (behavioral dominance), abundant (numerical dominance) and, wherefore, ecologically dominants (davidson, 1998). if this is true, the discovery-dominance will be a positive relationship and not a trade-off. here, we examine the interactions among nine ant species in a rock field, considered a harsh environment due to extreme daily variation of temperature and humidity; patches with different levels of sun incidence daily; and swallow soils (rodela & tarifa, 2002). by offering baits, we aimed to verify which kind of relationship between dominance and food discovery is found for ants in such environment. also we evaluated if the aggressiveness level is related to the dominance status of ant species. material and methods study site the study was conducted in a rocky field area, at state park of ibitipoca (parque estadual do ibitipoca-peib), minas gerais, brazil (s 21º42.493’, w 043º53.738’). the region has a humid mesothermal climate, with a mild summer and dry winter seasons and at 1500m average elevation (rodela, 1998). mean annual precipitation is 1532 mm (being high between months december and january). the average summer-maximum and winter-minimum temperatures are 36ºc and -4ºc, respectively, with extremes daily fluctuations of temperatures (rodela & tarifa, 2002). the studied rocky field is characterized by grassland vegetation consisting of grass, herbs and shrubs on outcrops of quartzitic rocks associated with shallow soils and high solar incidence (rodela, 1998). vegetations composition includes velloziaceae, compositae, melastomataceae, orchidaceae, gramineae, asclepiadaceae, eriocaulaceae, bromeliaceae and cyperaceae (rodela, 1998). experimental design we carried out the observations between june 2010 and february 2011. the experimental system consisted of six contiguous plots measuring 8 x 8 m, each one included 25 points set out 2m from each other in a grid pattern. of these 25 points, the 16 edge points were not utilized, and the 9 internal points constituted the bait stations (delsinne et al., 2007 adapted) (fig. 1). the plots order for baits offer was randomly chosen, so that each one of the six plots was sampled 10 times. at each of the nine bait stations, we placed 3g of sardine with honey (1:1; g:g) over a square pvc plate (10x10 cm). the nine baits were monitored until the appearance of the first forager ant at one of the baits, which lasted at maximum 15 minutes. then we removed the other eight baits not visited, in order to reduce the excess of resources available, thus avoiding the distribution of potential competitors over the baits spread through the experimental system. in the first discovered bait, we started recording by filming for 40 minutes. the temperature and relative humidity were measured at the beginning of each recording session. this procedure was repeated until we obtained 60 recording sessions, totalizing 40 hours of records. the experiment was always carried out between 8:00 am and 3:00 pm. voucher specimens were collected for later identification in the laboratory. at the end of the observations we removed the bait with a plastic bag. fig 1. diagram of the plots with the bait stations. empty circles represent sites where the baits were offered. ta sales, et al. ant interactions and behavioral strategies260 the video allowed us to identify the species that was the first to find the food source, the number of species that visited each bait and its respective abundance. interactions at baits were also observed and registered by frequency of occurrence into three categories, following fellers (1987): attack, avoidance and coexistence. an attack constituted of when one ant bit, turned its gaster toward another ant or there was an outright fight. we considered it to be avoidance when ants fleed or avoided direct confrontation with an individual of another species. lastly, coexistence was considered when two or more workers of different species fed at the same bait without any interaction. using this classification, we registered the frequency of each behavioral category for ant species. data analysis we restricted our analyses to species which occurred in at least 10% of the baits in order to obtain sufficient numbers of behavioral interactions to reliably assess their dominance. using this method, we sorted the nine most common species, coincidentally the same number of species used in other studies (fellers, 1987; lebrun, 2005; delsinne et al., 2007). for data analysis, we divided the 40 minutes of observation into 5-minute intervals, in which we registered the abundance of each species, which enables the calculation of specific average abundance. the discovery ability of each species (da) was the number of baits at which the species was the first to arrive (nf) divided by the total number of baits in which that species was observed (no): (da = nf / no). values near 1 indicate higher discovery ability (pearce-duvet et al., 2011). we analyzed the relationship between the specific average abundance (log10x+1 transformed) and their respective discovery ability by fitting a linear regression model. to verify whether there was a dependence between the frequency of occurrence of each behavior category (attack, avoidance and coexistence) and the species, we subjected the data to the independence pearson’s chi-squared (χ2) test for contingency tables, in order to access if a species exhibits more often one of the behavioral categories. when attacks occurred more than expected, the species was considered aggressive. we used the r program for all the analyses, at 5% significance in all cases (r development core team, 2013). results we sampled a total of 20 ant species, which belong to 11 genera, distributed into 6 subfamilies: ectatomminae, ponerinae, formicinae, dolichoderinae, pseudomyrmicinae and myrmicinae. nine of these twenty species were observed at more than 10% of the baits (table 1). the majority of the baits were visited by more than one species, while only 7% attracted only one species (fig. 2). c. crassus and c. renggeri visited more baits than the other species, while pheidole sp.1 exploited the smallest number (fig. 2). the specific average abundance calculated for each species showed a positive and significant correlation with their discovery ability (df=7, p<0.001, r²=0.91), showing that the best discoverer species were also numerically dominant. conversely, for the species that were not the first ones to arrive at the baits, we registered lower values of abundance (fig. 3). during the experimental period, the average temperature was 29.2 ± 4.0 ºc (39.9 – 19.5 ºc) and the relative humidity was 50.5 ± 10.5% (75.4 – 28.6%). through the results of the regression analyses it was not possible to show a significant effect of these abiotic variables on the abundance for any of the nine species. subfamily species baits (%) formicinae camponotus crassus mayr, 1862* 65.0 formicinae camponotus renggeri emery, 1894* 48.0 ectatominae ectatomma edentatum roger, 1863* 38.3 formicinae camponotus genatus santschi, 1922* 35.0 myrmicinae pheidole obscurithorax naves, 1985* 35.0 ponerinae pachycondyla striata smith, 1858* 23.3 myrmicinae pheidole radoszkowskii mayr, 1884* 21.7 myrmicinae pheidole sp1* 18.3 myrmicinae crematogaster sericea forel, 1912* 16.7 dolichoderinae linepithema cerradense wild, 2007 8.3 myrmicinae pheidole sp3 8.3 myrmicinae pheidole sp2 6.7 myrmicinae cephalotes pavonii (latreille, 1809) 5.0 myrmicinae cephalotes pusillus (klug, 1824) 5.0 ectatomminae ectatomma sp1 5.0 ponerinae odontomachus sp1 3.3 myrmicinae solenopsis sp1 3.3 formicinae myrmelachista sp1 1.7 myrmicinae pheidole sp4 1.7 pseudomyrmicinae pseudomyrmex sp1 1.7 table 1. frequency of occurrence of the ant species sampled at 60 baits in ibitipoca state park, minas gerais, brazil. * indicate the species used in the analyses (observed at more than 10% of the baits). when two or more species were recorded foraging at the same bait, approximately 53% of the behaviors exhibited were avoidance. the chi-square test indicates dependence between the behavior category and species (pearson’s chisquared test: df = 16, χ2 = 356.84, p < 0.001), supplying further evidence that the behavioral strategy shown by a sociobiology 61(3): 258-264 (september 2014) 261 species varies according to the species with which it interacts. c. sericea, p. obscurithorax and c. renggeri attacked more than expected. however, such aggressiveness only assured dominance of the bait for the first two species, not for c. renggeri. in the present study, the aggression exhibited by c. renggeri did not assure dominance at the baits, probably due to its low abundance per bait (nmax = 2). in turn, c. sericea stood out for the absence of avoidance behaviors (table 2), which along with its abundance and discovery ability characterizes it as an aggressive species that can potentially exert a limiting effect of resource use by subordinate species. p. radoszkowskii also presented a high abundance, but the workers exhibited avoidance behaviors more often than expected, suggesting is the avoidance strategy that assures its ability to remain at the resource (table 2). fig 2. bait occupancy by the nine species. the species are separated only for illustrative purposes. table 2. relative frequency for the attack, avoidance and coexistence behaviors and standardized residuals in contingency tables (pearson’s χ2 test for standardized residuals in contingency tables). in boldface behaviors that occurred above the expected (>+1.96) and behaviors that occurred below the expected (<-1.96). species relative frequency of behaviors (%) *standardized residuals in contingency tables (z) attack avoidance coexistence attack avoidance coexistence camponotus crassus 27.43 51.43 21.14 -2.09 3.21 -1.79 camponotus genatus 16.85 47.19 35.96 -2.37 0.95 1.60 camponotus renggeri 73.63 2.75 23.63 7.72 -6.52 -0.61 crematogaster sericea 69.64 0.00 30.36 3.68 -3.72 0.54 ectatomma edentatum 29.00 31.00 40.00 -0.79 -1.07 2.41 pachycondyla striata 3.85 46.15 50.00 -2.17 0.44 2.06 pheidole obscurithorax 41.42 29.75 28.83 2.33 -2.93 1.11 pheidole radoszkowskii 10.13 73.00 16.88 -5.63 7.01 -2.61 pheidole sp1 22.06 50.00 27.94 -1.45 1.12 0.25 fig 3. relationship between the average abundance of the species at the baits and respective discovery ability. cc: camponotus crassus, cg: camponotus genatus, cr: camponotus renggeri, cs: crematogaster sericea, ee: ectatomma edentatum, ps: pachycondyla striata, po: pheidole obscurithorax, pr: pheidole radoszkowskii, p1: pheidole sp1. cs p1 pr ps po cg ee cr cc 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 discovery ability a ve ra g e a b u n d an ce ( lo g 10 x + 1 ) ta sales, et al. ant interactions and behavioral strategies262 species with low abundances (c. renggeri, c. genatus and p. striata) can be considered submissive. however, this does not necessarily imply absence of aggressiveness, only a lower competitive ability, as observed for c. renggeri (table 2). attack behavior frequencies lower than expected were registered for p. radoszkowskii, c. crassus, c. genatus and p. striata. we believe that for these camponotus this result can reflect their low abundance and discovery ability. in contrast, for p. striata the most probable explanation is its solitary foraging strategy, characteristic of the species. in the case of e. edentatum, the solitary foraging strategy also explains the lower than expected occurrence of avoidance behaviors (table 2). discussion the relationship between dominance and food discovery was a positive one and not a trade-off, at the rocky field studied. the discovery-dominance trade-off predicts that good discoverers are subordinate species, which maximize their rates of finding resources to access them before being dislodged by a behaviorally dominant species (vepsäläinen & pisarski, 1982; fellers, 1987; lebrun & feener, 2007). inversely, the ant assemblage here studied presents a set of ecologically dominant ant species that arrived first and controlled baits through the combination of numerical dominance and aggressive behavior, with the exception of one species that was not aggressive. species with the greatest discovery ability were those with the highest abundance at the baits. on the other hand, species with low discovery ability (bad discoverers) who are less abundant, visited a larger number of baits. one can therefore assume that the low discovery ability of these species could have been offset by exploitation of a broader area. since these species have low capacity to defend resources, they probably search for food over a larger area. in contrast, for the species with high discovery ability and abundance, we can hypothesize that the baits exploited by them were within their territory and thus were more quickly located and successfully defended. this positive relationship can also be a reflex of the typical environmental conditions of rocky fields. these areas are characterized by extreme daily fluctuations of temperature and relative humidity, plus the effects of winds and strong sunlight (pirani et al., 1994). besides, they are located at high altitudes (above 1,000 meters) and have soils with outcrops of quartzite rocks (guedes & orge, 1998). in extreme and hostile environments such as rocky fields, where food resources become scarce in certain periods (fowler et al., 1991), it is reasonable to assume that after spending energy to find resources, an ant species must take maximum advantage of it. also, we must consider that the relationships in rocky fields are more specialized, as shown for wasps, due to limitations in resource collection (clemente et al, 2013). therefore, after locating the resource, we noted that dominant species present a particular behavioral strategy to remain at the food source. this might also have been the reason why the average abundance of the species at the baits was not influenced by abiotic factors. pheidole radoszkowskii used the avoidance as behavioral strategy to remain at the baits. when faced with aggressive ants from other species, most of the time they responded with avoidance. actually, p. radoszkowskii exhibits asymmetry in the competitive relationship with other dominant species (perfecto, 1994). our data does not exclude their potential for aggression or coexistence. rather, they demonstrate its behavioral plasticity towards the species with which it co-occurs. further, p. radoszkowskii forms colonies with small populations (perfecto, 1994), which can explain the avoidance strategy observed in the present study. in species with small colonies, direct confrontation represents a greater cost than it does for species with more numerous colonies (carroll & jansen, 1973). therefore, avoidance a typical interference behavior of ants (fellers, 1987; yanoviak & kaspari, 2000; delsinne et al., 2007) seems to be more efficient to allow their use of the bait than attacking or giving up and seeking another resource. similarly, p. obscurithorax exhibited avoidance behaviors soon after locating the resource but became more aggressive after recruitment and the arrival of soldiers. storz and tschinkel (2004) reported a combination of foraging tactics of this species in function of the resource size. for small resources, the scouts carried the food back to the colony alone and only recruited when there was a larger resource. in the present study, the bait offered was hard to transport, thus requiring workers and soldiers recruitment to assure its use for a longer period. another strategy employed by p. obscurithorax to exploit the food resource was the use of tools. workers took small pebbles and pieces of leaves onto the plate and placed them in contact with the bait, then removed these materials and transported them back to the nest altogether with the food. the use of tools to carry resources that are not represented by discrete units assures obtaining approximately 10 times more food than direct transport (fellers & fellers, 1976). in the case of a subordinate species, this behavior is also utilized to assure later use of the food, since the parts of the resource covered become unavailable to dominant species (fellers & fellers, 1976). mass recruitment was the strategy presented by pheidole sp1 and c. sericea to dominate the bait. such strategy assured pheidole sp.1 the use of the resource towards c. crassus and c. renggeri, species with which it co-occurred most and which consequently presented low abundance. in general, the pheidole soldiers are recruited to carry the resource (mertl et al., 2010), but in the case of pheidole sp.1, we observed soldiers acting only for defense. sociobiology 61(3): 258-264 (september 2014) 263 the attacks exhibited by c. sericea combined with its lack of avoidance confirms this species’ high aggressiveness (longino, 2003). a considerable proportion of the attacks only consisted of turning the gaster up, in order to release small droplets of venom, which can be related to both offensive and defensive behavior (buren, 1959). therefore, its high abundance is due to a combination of aggressiveness and fast recruitment (longino, 2003). c. sericea, p. obscurithorax and p. radoszkowskii were considered ecologically dominants. this terminology is proposed by davidson (1998) who named as ecologically dominant ant species those which are behavioral (due to superior fight and/or recruitment abilities) and numerically dominant. we can suppose that the discovery-dominance trade-off was broken by the linked characteristics that define the ecological dominant status of these species. despite p. radoszkowskii being part of this relationship, this interpretation should be taken cautiously. its dominance at the baits was assured by their high discovery ability and abundance, but the behavioral strategy exhibited was avoidance, not aggressiveness. p. radoszkowskii demonstrates that others strategies could surpass the set of being fast and attack, and thus this is not the best strategy for all. in harsh circumstances each species has its own best strategy, which is also illustrated by seed disperser ant species at caatinga. in this case, highquality disperser ant species showed a strong preference for diaspores with highest elaiosome mass, transporting the seeds for longest distances, until their nests, whereas to the lowquality disperser ants, the best strategy was fed on elaiosomes in situ, and never transporting the seeds to their nests (leal et al., 2014). the competitive interactions recorded show a range of foraging strategies employed by different ant species composing an assemblage that guarantees exploitation of food resources for all of them. the nature of the competition and the ant behavioral strategies have interesting implications in understanding the species’ richness and composition of assemblages, especially in an environment where resources are scarce and ephemeral. acknowledgements the authors are grateful to r. s. camargo and k. delclaro for their constructive comments. also we are grateful to the fundação de apoio e amparo a pesquisa do estado de minas gerais (fapemig grant no. 00411/08), the national concil for scientific and technological development (cnpq) for a fellowship granted to the last author (process no. 307335/2009-7) and instituto estadual de florestas (ief) from minas gerais in special to clarice nascimento lantelme silva for technical support at ibitipoca park.. references andersen, a.n. 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(2000). community structure and the habitat template: ants in the tropical forest canopy and litter. oikos, 89: 259-266. doi: 10.13102/sociobiology.v65i4.3372sociobiology 65(4): 566-575 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 species richness and diversity in bee assemblages in a fragment of savanna (cerrado) at northeastern brazil introduction bees stand out as the dominant taxonomic group of pollinators in most geographic regions (williams et al., 2001). these bee populations depend on plant resources related to adult maintenance and offspring production, which include supplies of nectar and pollen (roubik, 1989; michener, 2007). some species require additional resources such as floral oils, floral fragrances or resins, as well as adequate nesting sites (roubik, 1989). the persistence of different populations of bees can be strongly affected by habitat quality, which can be evaluated, among other parameters, by the abundance abstract the conservation of the fauna of bees inhabiting the brazilian savanna is threatened due to changes in land use in the last decades. we investigated the composition, species richness and abundance of a bee assemblage in the vicinity of the chapada diamantina national park. in addition, we compiled data on composition and diversity from another bee assemblage located in the same portion of the cerrado, which was previously investigated by one of us almost 30 years ago, in order to produce a more complete panorama on beta diversity of bees in this region. we used a non-metric multidimensional scaling ordination analysis (nmds) to compare composition of bee assemblages from different types of open vegetation. we recorded 77 bee species (h’ = 2.95; j = 0.68), 42% of them were singletons. we collected slightly more than half of the species and 60% of the genera recorded in the bee assemblage studied three decades ago. h’ was significantly lower in our area than in the previous study (t = 8.588, p <0.001), but equitability (j) was very similar. several factors may contribute to these differences, including local differences in bee assemblage composition, differences in the probability of capturing the different species (many rare species), factors affecting the sampling itself, and perhaps species loss over the three decades separating the two studies. the magnitude of species loss is difficult to assess because the two studies were not carried out exactly in the same area and there were differences in sampling time and sampling effort. sociobiology an international journal on social insects cml aguiar1, eb santana2, cf martins3, f vivallo4, co santos5, gmm santos1 article history edited by eduardo almeida, usp, brazil received 24 april 2018 initial acceptance 30 june 2018 final acceptance 26 august 2018 publication date 11 october 2018 keywords apoidea, anthophila, bee community, bee abundance, biodiversity, chapada diamantina national park. corresponding author cândida m. l. aguiar departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina s/nº, novo horizonte 44036-900, feira de santana-ba, brasil. e-mail: candida.aguiar@gmail.com of available floral resources (rosa & ramalho, 2011). declines in species richness and abundance, as well as some substitution in the dominant species over the last decades have been reported in brazil (martins et al., 2013; cardoso & gonçalves, 2018). the brazilian savanna (cerrado) is a biodiversity hotspot (myers et al., 2000; silva & bates, 2002). in spite of its importance for the conservation of biodiversity, anthropic pressures on the cerrado have increased in recent decades, mainly due to the expansion of pasturelands and croplands, including the mechanized production of grains, soybean and maize (klink & machado, 2005; sano et al., 2010). the 1 universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil 2 ppg ecologia & evolução, universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil 3 universidade federal da paraíba (ufpb), joão pessoa, paraíba, brazil 4 museu nacional/universidade federal do rio de janeiro (ufrj), rio de janeiro, brazil 5 ppg ciências agrárias, universidade federal do recôncavo da bahia (ufrb), cruz das almas, bahia, brazil research article bees sociobiology 65(4): 566-575 (october, 2018) special issue 567 replacement of native vegetation promotes habitat loss and fragmentation, reduction of the local floristic diversity and in the abundance of floral resources available to the bees, as well as the reduction of cavities (e.g. tree hollows) available for nest construction. impacts on soil, such as those resulting from ploughing the soil (freitas et al., 2009), increase in pavement and changes in soil permeability (fortel et al., 2014) are also relevant, especially for the brood, since most bees nest under the soil surface (roubik, 1989). carvalho et al. (2009) pointed out the urgent need to understand how the land use in mosaics influences the persistence of animal and plant populations, as well as the maintenance of ecological processes. although the bee fauna of the southernmost part of the cerrado (19o to 24o s) is well sampled (silveira & campos, 1995; carvalho & bego, 1996; andena et al., 2005; 2012; and unpublished studies cited in faria & gonçalves, 2013), there are significant sampling gaps in the northern and middle portions of the cerrado domain. in this study we present new data on the bee assemblage of a fragment located in the middle portion of the cerrado, in the chapada diamantina, an area where there is a mosaic of phytophysiognomies (cerrado, caatinga, campos rupestres and forests) at short distances (harley, 1995). in addition, we compiled data on the composition and species richness from another bee assemblage located in the same portion of the cerrado at chapada diamantina region (12º 34’ 0’’ s; 41º 22’ 60’’ w, cerrado with elements of campos rupestres), which was previously investigated by one of us (martins, 1994), to yield a more complete panorama on beta diversity of bees in this region. these sites are located in the vicinity of a biodiversity protection area, the chapada diamantina national park (12° 20’ 12° 25’ s; 41° 35’ 41° 15’ w), and knowledge of local species richness can help in the establishment of land use policies and conservation strategies for the surrounding areas of the park. methods study area the study area is located in the municipality of palmeiras (altitude: 727 m), state of bahia, brazil. it is covered by cerrado (brazilian savanna vegetation). nearby there are other types of vegetation, mainly caatinga (seasonally dry forest) and campos rupestres (sandstone outcrop vegetation), which surround with this cerrado fragment. additionally, close to the cerrado there are different types of forests, forming a vegetation mosaic typical of the chapada diamantina region (harley, 1995). the climate is tropical humid, with rainy season from december to april in general, while the driest period is from august to november (jesus et al., 1983; nimer, 1989). the average rainfall ranges from 600 to 1000 mm/year, and the average temperature is 22° c (cei, 1994). the sampled area was divided into three sampling sites, i: teto verde (12° 26’ 03” s, 41° 29’ 22” w), ii: barranco (12° 26’ 19” s, 41° 30’ 11” w), and iii: coités (12° 25’ 50” s; 41° 30’ 20” w), ranging from 900 m to 1,700 m apart. in each site, three transects were chosen to sample the bees, totaling nine transects (see supplementary material 1 for geographic coordinates), each one measuring approximately 1,500 m in length and 6 m in width. the cerrado vegetation sampled was composed of herbaceous-shrub fields and sparse small trees (sites i and iii). trees were small to medium size on site ii. byrsonima sericea and byrsonima cydoniifolia (malpighiaceae) trees were very common in all three sites. signs of anthropic activities include the presence of nearby highways (br 242 and ba 480), some dirt roads, and smallscale clay extraction (site ii). this area suffers from burnings on native vegetation, as occurred in december 2015, and affected site i, near morro do pai inácio. data sampling we performed 17 samplings between 2013 and 2016 (table 1). in each sample, the bees were collected during two consecutive days, from 8:00 am to 4:00 pm, by two collectors, totaling 34 days and 408 h of sampling effort per collector. at each sampling, three transects were sampled, one at each site (i, ii, iii). the transect to be sampled in each subarea was randomly chosen, in order to distribute the collection effort among the nine transects. bees flying or visiting flowers were captured with entomological nets, according to sakagami et al. (1967). in each flowering plant, floral visitors were captured for 5 to 10 minutes. the bees were dry mounted and identified by one of us (f. vivallo) and other bee specialists employing the multi-family classification system used in michener (2007) and genusand species-level classification of moure et al. (2007). data analysis we used the shannon index (h’ = -σ pi x ln pi) to evaluate the diversity and the pielou index (j’ = h’/ log2s) to evaluate the uniformity of abundance distribution (magurran, 2011). we used a one-sample t-test proposed by hutcheson (zar, 1999) to investigate whether h’ value found in our area differed from that calculated for the area studied by martins (1994). species dominance was calculated using the kato index. this index considers as dominant the species whose value of the lower limit (li) of relative abundance is above the limit of dominance (ld). ld is obtained by the inverse of the total number of species captured multiplied by 100 (sakagami & matsumura, 1967). to evaluate whether the sample effort was sufficient to sample the species richness of the bee assemblage, we used chao and jackknife richness estimators (magurran, 2011). we used a non-metric multidimensional scaling ordination analysis (nmds) based on bray-curtis similarity index, to compare the similarity in the composition of bee assemblages from different types of open vegetation: cerrado (ceba1: lençóis, bahia (martins, 1994); ceba2: palmeiras, bahia (este estudo); cego1: iporá, goiás (santiago et al., cml aguiar, eb santana, cf martins, f vivallo, co santos, gmm santos – a bee assemblage in brazilian savanna568 2009); cema1: barreirinhas, maranhão (rêgo & albuquerque, 2012); cema2: chapadinha, maranhão (rêgo & albuquerque, 2012); cemg1: uberlândia, minas gerais (carvalho & bego, 1996); cemg2: paraopeba, minas gerais (silveira & campos, 1995), cesp1: corumbataí, são paulo (silveira & campos, 1995), cesp2: corumbataí, são paulo (andena et al., 2005), cesp3: santa rita do passa quatro, são paulo (andena et al., 2012); ceto1: esperantina, tocantins (santos et al., 2004)), campos rupestres (crba1: palmeiras, bahia (silva-pereira & santos, 2006); crmg1: ouro branco, minas gerais (araújo et al., 2006); crmg2: lavras novas, minas gerais (faria-mucci et al., 2003)), caatinga (caba1: casa nova, bahia (martins, 1994); caba2: castro alves, bahia (santos et al., 2013); caba3: itatim, bahia (aguiar & zanella, 2005); capb1 (são joão do cariri, paraíba (aguiar & martins, 1997); cape1: chã grande, pernambuco (milet-pinheiro & schlindwein, 2008); carn1: serra negra do norte, rio grande do norte (zanella, 2003)); and canga (cgmg1: ouro preto, minas gerais (araújo et al., 2006)). we constructed a matrix containing the number of species per bee genera in each of these 21 sites. we made fig 1a. species richness curves (estimators abundance data based, jackknife 2 and chao 1) in bee assemblages in a neotropical savanna (cerrado), brazil. this analysis using the package vegan version 2.4-3 (oksanen et al., 2017), and to plot the figure we used the ggplot2 package (wickham, 2009), both in the r software version 3.0.3 (r development core team, 2016). results the bee assemblage of this cerrado fragment was composed of 77 species, distributed in 42 genera (table 1). the number of species at each sampling point was 40 (site i), 43 (site ii) and 48 (site iii), and 43 species were recorded at a single sampling point (table 1). apidae had a significant contribution to the richness of species (74% of all species), especially centridini (s = 18) and meliponini (s = 10). the species diversity was h’ = 2.95 and the equability j = 0.68. the chao 1 and jackknife 2 estimators indicated that the expected richness for this area (130 and 138 species, respectively) is higher than the observed richness. the species richness curve showed an increasing slope, indicating that there are species not yet sampled in the assemblage (figs 1a, 1b). 300 250 200200 ci es 150 n um be r o f s pe c 100 n 50 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 lsamples ic+ 2013‐2016 (chao 1) ic‐ ic+ 1987‐1988 (chao 1) ic‐ ic+ 2013‐2016 (jackknife 2) ic‐ ic+ 1987‐1988 (jackknife 2) ic‐ sociobiology 65(4): 566-575 (october, 2018) special issue 569 fig 2. non-metric multidimensional scaling (nmds) ordination plot. s = 0.177. types of vegetation: caatinga (caba1; caba2; caba3; capb1; cape1; carn1), campos rupestres (crba1; crmg1; crmg2), canga (cgmg1), cerrado (ceba1; ceba2; cego1; cema1; cema2; cemg1; cemg2; cesp1; cesp2; cesp3; ceto1). the distribution of abundances was quite uneven among species. the vast majority of species were infrequent, 42% of them were singletons and 12% doubletons. few species were dominant: the introduced africanized honeybee apis mellifera linnaeus (20.5%), centris aenea lepeletier (14.5%), trigona spinipes (fabricius) (13.2%), scaptotrigona aff. postica (latreille) (11.5%), epicharis bicolor smith (4%), trigona hyalinata (lepeletier) (3.8%), epicharis analis lepeletier (3.2%), centris perforator smith (2.5%), and centris caxiensis ducke (2.1%). apidae showed the highest relative abundance (94% of individuals), while other bee families were poorly represented. the species a. mellifera, c. aenea, c. perforator and t. spinipes occurred in more than 50% of the samples, while most species (67 species) occurred in less than 25% of the samples (table 1). regarding the composition of the analyzed bee assemblages, the non-metric ordination analysis (nmds; s = 0.177) allowed to recognize two distinct groups (fig 2). the first grouping the bee assemblages from caatinga (ca), and a second one mixing bee assemblages from cerrado (ce), campos rupestres (cr) and canga (cg), which were not differentiated by type of vegetation, although this group differed from that of bee assemblages of the caatinga. discussion even with sampling extending for several years, we obtained a species richness curve that did not stabilize, which should be influenced by the occurrence of many rare species (williams et al., 2001), a similar finding of most surveys of fig 1b. species richness curves (estimators incidence data based, jackknife 1 and chao 2) in bee assemblages in a neotropical savanna (cerrado), brazil. 150 200 250 300 er  o f s pe ci es 0 50 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 n um be samples ic+ 2013‐2016 (chao 2) ic‐ ic+ 1987‐1988 (chao 2) ic‐ ic+ 2013‐2016 (jackknife 1) ic‐ ic+ 1987‐1988 (jackknife 1) ic‐ bee assemblages. many species (53%) were represented in only one of the three sampling points, indicating that sampling in a single site is quite biased to represent the local richness of species in bee assemblages, as pointed out by others cml aguiar, eb santana, cf martins, f vivallo, co santos, gmm santos – a bee assemblage in brazilian savanna570 (zanella, 2003; gonçalves et al., 2009). many species in bee assemblages are rare (singletons or doubletons), so the probability of capture them is small, even when there is a large sampling effort (williams et al., 2001). the comparison of the composition of this bee assemblage with that one located 19 km away previously investigated by one of us (martins, 1994) showed that each of them presents several exclusive elements (supplementary material 1). fifteen genera and 29 species were added to previous knowledge on the bee species richness of this portion of the cerrado. thus, the total number of bee species recorded for this cerrado at chapada diamantina, is at least 177 species (supplementary material 1). the analysis of beta diversity at genus level, which presents little taxonomic impediment, resulted in a total of 60 genera. most genera were represented by one or two species, but megachile, centris, augochloropsis and augochlora contributed with more than 10 species to the species richness (supplementary material 1). the assemblage recently sampled by us presented slightly more than half of the species and 60% of the genera recorded in the bee assemblage studied three decades ago (s = 147, h’ = 3.53, j = 0.71) (martins, 1994; supplementary material 1), despite the sampling effort in our study (34 days over the course of four years) was higher than that of martins (1994) (24 days in one year). the two bee assemblages here compared showed different diversity, h’ was significantly lower in our area (t = 8.588, p <0.001). on the other hand, the level of equitability (j) was very similar, indicating that differences in h’ values were mainly influenced by differences in species richness, as the abundance distribution was similar. it is noteworthy that some groups as the orchid bees (euglossini), anthidiini, megachile and its cleptoparasites (coelioxys) were much richer in the former study. on the other hand, oil collecting bees (centridini) were richer in our study site than in martins (1994), which may be related to the large local supply of floral oil by species of byrsonima (malpighiaceae) (aguiar et al., 2017). several factors may have contributed to these observed differences, including local differences in bee assemblage composition, even if they are close (see also williams et al., 2001; zanella, 2003; gonçalves et al., 2009), differences in the probability of capturing the different species, factors affecting the sampling itself, and perhaps species loss over the three decades separating the two studies. small populations, which are the majority of the components of both bee assemblages, are less likely to be sampled; although they may be resident species, they may not have been recorded. thus, the absence of many of the rare species collected by martins (1994; supplementary material 1) in current sampling may be a consequence to their rarity, as pointed out by martins et al. (2013) when they compared changes in the bee fauna over 40 years. in our case, the magnitude of species loss is difficult to assess because the two studies were not carried out exactly in the same area and there were differences in sampling time and sampling effort. there are large variations in the observed species richness among bee assemblages in the cerrado, as previously discussed by gonçalves et al. (2009), and faria and gonçalves (2013). bee assemblages composed of around 40 species were recorded in the northernmost portion (2o to 3o s) (rebêlo et al., 2003; rêgo & albuquerque, 2012), but santos et al. (2004) recorded 83 species in an area (5°20’ s) with a strong influence of livestock activity. on the other hand, in the southern portion of the cerrado (19° to 24° s), bee assemblages are generally composed of more than one hundred to almost two hundred species (carvalho & bego, 1996; menezes-pedro & camargo, 1991; silveira & campos, 1995; and unpublished studies cited in faria & gonçalves, 2013), although there are also some areas where species richness is lower (67-70 spp.) in this southernmost portion (andena et al., 2005; 2012). faria and gonçalves (2013) found that diversity and composition in brazilian bee assemblages were explained by different sets of abiotic variables. diversity (h’) and species richness were highest at places with higher temperature annual range. they also pointed out that the generic composition of bee assemblages varied in response to annual mean temperature and temperature seasonality, as well as to temperature annual range and annual precipitation. our results of the ordination analysis (nmds) reflected the differences in the composition of the bee faunas of cerrado and caatinga. zanella (2000, 2003) pointed out that the bee fauna of the caatinga presents several endemic elements, which differentiates this group of bee assemblages from those of other vegetations. the lack of differentiation among the bee assemblages of cerrado and campos rupestres is probably related to the proximity of these types of vegetation, since in some geographic regions, such as the chapada diamantina and other areas in the espinhaço moutain chain, campos rupestres occur in altitudes above of 900 m (giulietti & pirani, 1988), surrounded by lower areas covered by cerrado vegetation. thus, populations of many bee species can colonize neighboring areas in this mosaic of habitats. the sampling hiatus of bee assemblages in the northern and middle portions of the cerrado still do not allow a realistic picture of bee species diversity in this ecosystem. current knowledge suggests that there is an increase in the species richness of bees in the north-south direction of the cerrado. greater sampling effort should be undertaken in the non-sampled regions to increase knowledge on species richness and distribution, structure of assemblages, as well as to support bee conservation strategies in the face of the increasing degradation of the cerrado, due in particular to the expansion areas for agricultural crops and pastures. acknowledgments we are grateful to the bee specialists who identified some bee species collected in this research (antonio j. c. aguiar, fernando c.v. zanella, maria c. gaglianone and willian m. aguiar), and to emanuelle brito for assistance sociobiology 65(4): 566-575 (october, 2018) special issue 571 taxa n sites samples i ii iii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 andrenidae oxaeini oxaea flavescens klug 3 1 1 1 3 apidae apini apis mellifera linnaeus 172 39 58 75 8 37 2 4 6 3 10 3 3 9 2 18 13 12 42 bombini bombus morio (swederus) 4 2 2 1 1 2 centridini centris aenea lepeletier 122 12 15 95 1 1 1 81 10 1 1 5 21 centris caxiensis ducke 18 8 4 6 2 3 1 3 3 3 3 centris decolorata lepeletier 2 2 2 centris nitens lepeletier 1 1 1 centris varia (erichson) 1 1 1 centris tarsata smith 4 1 1 2 1 1 1 1 centris moerens (perty) 13 1 3 9 1 2 3 2 1 4 centris lutea friese 1 1 1 centris perforator smith 21 8 4 9 4 2 1 2 4 2 4 1 1 centris cf. spilopoda moure 8 4 1 3 3 4 1 centris tetrazona moure & seabra 8 1 3 4 1 3 1 3 centris sp.1 4 4 1 3 centris sp. 3 1 1 1 centris sp. 6 1 1 1 epicharis analis lepeletier 27 6 5 16 1 4 5 14 1 2 epicharis bicolor smith 34 15 5 14 18 14 1 1 epicharis cockerelli friese 4 1 1 2 1 1 2 epicharis flava friese 1 1 1 emphorini diadasia sp. 1 1 1 1 melitoma sp. 1 9 8 1 1 5 3 melitomella grisescens ducke 2 2 2 ericrocidini acanthopus excellens schrottky 3 3 2 1 mesoplia cf. friesei ducke 2 1 1 1 1 mesoplia rufipes (perty) 4 1 3 1 3 table 1. species composition and bee abundance in an assemblage in a neotropical savanna (cerrado), brazil. n: number of individuals; sample 1: oct 2013/2: nov 2013/3: dec 2013/4: jan 2014/5: feb 2014/6: mar 2014/7: sep 2014/ 8: nov 2014/ 9: feb 2015/ 10: mar 2015/ 11: apr 2015/ 12: aug 2015/ 13: oct 2015/ 14: jan 2016/ 15: mar 2016/ 16: may 2016/ 17: jul 2016. * species recorded in the area, in odor baits. in statistical analysis we also thank the national council for scientific and technological development, brazil (cnpq, no. 558228/2009-7, peld; 403774/2012-8, peld; 474065/2013-8) for financial support for this project. cnpq granted research fellowship to c.f. martins and g.m.m. santos. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nivel superior brasil (capes) finance code 001. this paper is part of the sigma project nº21565 mn/ufrj and the contribution number 22 from the hymn laboratório de hymenoptera/museu nacional of rio de janeiro. supplementary material doi: 10.13102/sociobiology.v65i4.3372.s2219 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3372/0 cml aguiar, eb santana, cf martins, f vivallo, co santos, gmm santos – a bee assemblage in brazilian savanna572 eucerini florilegus sp. 1 1 1 1 euglossini euglossa cordata (linnaeus) 2 * 1 1 1 1 euglossa melanotricha moure * * * * euglossa securigera dressler * * * * eulaema cingulata (fabricius) * * * * eulaema nigrita lepeletier 5 3 1 1 1 1 1 1 1 exomalopsini exomalopsis (phanomalopsis) sp. 1 1 1 1 exomalopsis (exomalopsis) sp. 2 1 1 1 meliponini frieseomelitta francoi (moure) 1 1 1 geotrigona mombuca (smith) 3 1 1 1 1 1 1 melipona quadrifasciata lepeletier 2 1 1 1 1 nannotrigona testaceicornis (lepeletier) 4 1 3 1 2 1 paratrigona incerta camargo & moure 7 1 4 2 4 2 1 partamona combinata pedro & camargo 2 2 1 1 scaptotrigona aff. postica (latreille) 97 10 18 69 7 1 59 1 23 3 2 1 tetragonisca sp. 1 1 1 1 trigona hyalinata (lepeletier) 32 32 23 8 1 trigona spinipes (fabricius) 111 25 44 42 7 28 1 6 3 12 6 7 13 28 protepeolini leiopodus abnormis (jörgensen) 1 1 1 tapinotaspidini lophopedia nigrispinis (vachal) 2 1 1 1 1 monoeca aff. mourei aguiar 1 1 1 tapinotaspoides sp. 1 2 1 1 1 1 tropidopedia nigrocarinata aguiar & melo 9 8 1 8 1 urbanapis diamantina aguiar & melo 9 9 1 7 1 xanthopedia sp. 1 5 4 1 1 4 tetrapediini tetrapedia amplitarsis friese 3 3 3 tetrapedia diversipes klug 4 4 3 1 xylocopini table 1. species composition and bee abundance in an assemblage in a neotropical savanna (cerrado), brazil. n: number of individuals; sample 1: oct 2013/2: nov 2013/3: dec 2013/4: jan 2014/5: feb 2014/6: mar 2014/7: sep 2014/ 8: nov 2014/ 9: feb 2015/ 10: mar 2015/ 11: apr 2015/ 12: aug 2015/ 13: oct 2015/ 14: jan 2016/ 15: mar 2016/ 16: may 2016/ 17: jul 2016. * species recorded in the area, in odor baits. (continuation) taxa n sites samples i ii iii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 sociobiology 65(4): 566-575 (october, 2018) special issue 573 ceratina (crewella) sp. 1 10 6 2 2 1 1 1 1 5 1 ceratina (crewella) sp. 2 1 1 1 ceratina (crewella) sp. 3 1 1 1 xylocopa cearensis ducke 4 4 2 2 xylocopa frontalis (olivier) 1 1 1 xylocopa subcyanea pérez 1 1 1 xylocopa sp. 2 1 1 1 colletidae colletes sp. 1 5 5 5 halictidae augochlorini augochlora (oxystoglossella) sp .3 5 2 2 1 1 2 1 1 augochlora (oxystoglossella) sp. 2 1 1 1 augochlora (augochlora) sp. 5 1 1 1 augochloropsis sp. 3 1 1 1 augochloropsis sp. 4 1 1 1 augochloropsis sp. 5 7 5 2 1 1 4 1 augochloropsis sp. 6 1 1 1 augochloropsis sp. 7 6 1 4 1 1 2 1 1 1 augochloropsis sp. 8 2 1 1 1 1 pseudaugochlora pandora (smith) 1 1 1 pseudaugochlora sp1 1 1 1 temnosoma cf. metallicum smith 1 1 1 halictini dialictus opacus (moure) 5 3 2 1 1 2 1 megachilidae anthidiini dicranthidium sp. 1 1 1 1 megachilini coelioxys (acrocoelioxys) sp. 1 1 1 1 megachile (pseudocentron) sp. 3 1 1 1 megachile (sayapis) sp. 1 1 1 1 megachile sp. 8 1 1 1 table 1. species composition and bee abundance in an assemblage in a neotropical savanna (cerrado), brazil. n: number of individuals; sample 1: oct 2013/2: nov 2013/3: dec 2013/4: jan 2014/5: feb 2014/6: mar 2014/7: sep 2014/ 8: nov 2014/ 9: feb 2015/ 10: mar 2015/ 11: apr 2015/ 12: aug 2015/ 13: oct 2015/ 14: jan 2016/ 15: mar 2016/ 16: may 2016/ 17: jul 2016. * species recorded in the area, in odor baits. 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(1999). biostatistical analysis. 4th ed. upper saddle river (nj): prentice hall, 663 p https://www.ncbi.nlm.nih.gov/labs/journals/environ-monit-assess/ http://www.springer.com/978-0-387-98140-6 http://www.springer.com/978-0-387-98140-6 https://www.ecologyandsociety.org/vol5/iss1/art7/ https://doi.org/10.1051/apido:2000148 https://doi.org/10.1051/apido:2000148 doi: 10.13102/sociobiology.v67i2.4517sociobiology 67(2): 232-238 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the red imported fire ant (solenopsis invicta), native from the parana river basin of south america, is a voracious consumer of numerous other arthropod species (nattrass et al., 2007). it also is an important agricultural and urban pest throughout most of the southeastern united states as well as in parts of several western states (wetterer, 2011). this invasive species has overtaken and eliminated many native ant species in most areas (lebrun et al., 2013). they first invaded mainland china in 2005 (zhang et al., 2013) and spread widely in southern china, causing serious damage to humans, animals and the ecological environment (kafle et al., 2013；ascunce et al., 2011). red imported fire ants are extremely toxic, and when the human body is smashed by red imported fire ants, it has pain like a fire, and then there will be blisters like burns. most people only feel pain and discomfort. abstract a comprehensive green worker ants control method that can be used to replace traditional chemical synthetic insecticides. in this study, the leaves and stems of gelsemium elegans were extracted with water as the solvent, and the bioactivity of g. elegans against worker ants was determined by the “water tube” method. the bioassay results of insecticidal activity showed that when the time was extended to the 10th day, the mortality of worker ants treated with g. elegans extract reached 55.00% (1/20 leaf extract), 46.67% (1/20 stem extract) and 45.00% (1 mg/ kg koumine). and the behavioral impact test results showed that the aggregation rate was reduced to 56.67% (1/100 leaf extract), 60.00% (1/100 stem extract) and 60.00% (0.5 mg/kg koumine); the climbing rate was reduced to 60.00% (1/100 leaf extract), 58.33% (1/100 stem extract) and 58.33% (0.5 mg/kg koumine). the effect on the walking ability of worker ants is obvious. the walking rate drops to 1.53 cm/s (1/100 leaf extract), 1.60 cm/s (1/100 stem extract) and 1.47 cm/s (0.5 mg/ kg koumine). the leaves, stem extracts and koumine components contained in g. elegans can affect their behavior and show insecticidal activity. g. elegans might be a good natural plant resource to control red imported fire ant. sociobiology an international journal on social insects q zheng, lp yang, sk lin, ql ma, dq qin, zx zhang article history edited by evandro nascimento silva, uefs, brazil received 30 may 2019 initial acceptance 22 january 2020 final acceptance 22 june 2020 publication date 30 june 2020 keywords gelsemium elegans benth, koumine, worker ant, bioactivity. corresponding author zx zhang south china agricultural university key laboratory of natural pesticide and chemical biology ministry of education guangzhou, 510642, china. e-mail: zdsys@scau.edu.cn however, a few people are allergic to toxic proteins in venom, which can cause anaphylactic shock and risk of death. traditional methods for managing the red imported fire ant are used insecticides or baits, but this might lead to groundwater contamination, non-target organism and other environmental considerations (vogt et al., 2002). now，with the improvement of people's quality of life, more consumers are inclined to solve pest problems safely and economically (huang et al., 2015). therefore, the search for safe, economical and effective prevention methods is the current priority. the use of chemical components from natural products in insect pest control is generally considered to be a safer alternative to the use of synthetic contact insecticides (chen, 2009). since naturally occurring chemicals have been found to have insecticidal activity, they have been widely used for pest control (appel et al., 2004). for example, the widely used plant essential oils have proven to have significant repellent south china agricultural university, key laboratory of natural pesticide & chemical biology, ministry of education, guangzhou, china research article ants insecticidal activity of the leaf and stem water extract of gelsemium elegans against solenopsis invicta sociobiology 67(2): 232-238 (june, 2020) 233 and toxic effects on ants，including camphor, turpentine, wintergreen, and such as d-limonene in citrus showed effective repellent or fumigation toxicity against the red imported fire ant (tang et al., 2013). gelsemium, a small genus of the loganiaceae family, includes three well-known species: gelsemium elegans benth. (g. elegans), native to china and southeast asia, gelsemium sempervirens ait. (g. sempervirens), and gelsemium rankinii small (g. rankinii), native to north america (liu et al., 2013). they have long been used by traditional folk medicine as an effective component in relieving pain and reducing anxiety (xu et al., 2012). despite its toxicity, the folks use it to resist tumors according to the theory of “combat poison with poison”, and there is also the effect of sedative pain, immune regulation, and rejuvenation of livestock. the primary active molecules of g. elegans are the alkaloids gelsemine, koumine, gelsenicine, and gelsevirine (liu et al., 2013). the purpose of this study was to determine the toxicity and behavioral inhibition of g. elegans to the red imported fire ant. in view of the extremely toxic nature of the whole plant, the leaves and stems of g. elegans were selected as natural sources to study the toxicity and behavioral inhibition of alkaloids on red imported fire ants. we hope to obtain a simple, economical and ecologically safe natural product to control red fire ants. materials and methods plant materials the g. elegans were collected from the insecticidal botanical garden at the south china agricultural university. the length of the stem was approximately 3-12 m. two parts of the g. elegans were used in the test: leaves and stem. the fresh and healthy vines were sent to the laboratory once collected. the dirt surface of the leaves was removed and the stems and leaves were separated carefully. the dried leaves and stems were gained by putting the fresh leaves and stems in a baking box at 40 °c for 5 h. chemicals koumine was purchased from jieshikang biotechnology co., ltd., qingdao, china. hplc-grade methanol was purchased from yiyang trading co., ltd., guangzhou, china. insects s. invicta colonies were collected from a suburb in guangzhou. worker ants and nest material were reared in the laboratory for bioassays in plastic containers (40 cm × 52 cm × 12 cm) coated with teflon emulsion on the top, in which a test tube (25 mm × 200 mm) filled with water and plugged with cotton was set up in order to provide water, and the ham sausage was used as a food source. the ants were kept in a comfortable environment at 25 ± 2 °c and 70 ± 10% rh until the test was completed. the major workers applied in the experiment were 4-5 mm in length (souto et al., 2012; huang et al., 2016; hu et al., 2017). preparation of plant extract and koumine the dried leaves and stems were broken up with a highspeed pulverizer and then collected by filtration using a standard test sieve (mesh 60, pore diameter 0.25 mm). the powdery dried plant material was submitted the extraction of compounds with pure water at room temperature, and the aqueous solution was pumped and filtered under vacuum. after filtration, we obtained an extraction solution of 1 g leaves dissolved in 20 ml (100 ml) water, which was expressed by 1/20 (1/100). then diluted with 1/100 of the leaf extract to get the concentration we need, such as 1/500, 1/5000. the same procedure as above was applied in order to obtain the same concentration of stem water extract. all extracts were stored at 4 °c. we used methanol as a solvent to dissolve the koumine to obtain a standard solution, diluted with water until the methanol content is less than 0.5%. further diluting was done to obtain 0.02, 0.05, 0.1, 0.5, 1 mg/kg of the standard aqueous solution of koumine. laboratory toxicity bioassays a toxic biological test was performed according to the method described by the predecessors (huang et al., 2015). worker ants were placed in a 250 ml beaker [71 mm (diameter) × 97 mm] in which the vertical wall was coated with fluon emulsion. the ants were deprived of water and food once placed in the beakers. after 6 hours of starvation, a test tube filled with extracts or standards and plugged with cotton was placed at the bottom of the beaker. all treatments were replicated thrice and each replicate included 20 worker ants. the worker ants were maintained at 25 ± 2 °c and relative humidity of 70 ± 10%. the same operation was used in the control group except that the extract or standard was changed to water and 0.5% methanol aqueous solution. the number of dead ants was recorded after 0.083, 1, 3, 5, 7 and 10 days after they were treated. the following formula was adopted to assess the mortality： mortality = (number of dead ants/number of total ants) × 100% behavior observation on aggregation ability of workers we reduced the concentration of the treatment to test the effect of the extract on the behavior of worker ants, thus eliminated the effect of the death of the worker on the evaluation results. the treatment of the worker ants was the same as the above steps. for the behavioral impact test of worker ants, high, medium and low concentrations were selected, 1/100, 1/500, 1/5000. from these three concentrations, the range of efficacy of the extract of g. elegans can be roughly estimated. when more than three worker ants gathered together, and the distance between each worker ant was less than 0.5 cm, we recorded this event as aggregation. the number of ants gathered was recorded after 0.083, 1, 3, 5, 7 and 10 days after they were treated. the following formula was adopted to assess the aggregation： q zheng, lp yang, sk lin, ql ma, dq qin, zx zhang – insecticidal activity of gelsemium elegans against solenopsis invicta234 aggregation level = (number of ants gathered/number of total ants) × 100% behavior observation on climbing ability of workers the concentration was similarly reduced to eliminate the effect of worker ants death on behavioral assessment, and the method of operation was the same as previously described. the worker ants were provoked by a bamboo stick (25 cm length). if the worker ants were able to climb more than 3 cm along the stick, we recorded the event as ants having climbing ability. the number of ants that could climb was recorded after 0.083, 1, 3, 5, 7 and 10 days after they were treated. the following formula was adopted to assess the climbing ability： climbing rate = (number of ants that can climb/number of total ants) × 100% behavior observation on walking ability of workers worker ants were treated with the same concentration and method as the above behavioral observation test, and the effects of the agents on their walking ability were observed at 0.083, 1, 3, 5, 7 and 10 days after they were treated. the worker ants were placed on the a4 cardboard marked with squares (square: 1 cm × 1 cm) and we recorded the length of the journey of the worker ants within 3 s on camera. the following formula was adopted to assess the walking speed： walking speed = (the length of walking distance of worker ants/three seconds) statistical analysis data were expressed as mean ± se after statistical analysis, and data charts were generated using graphpad prism 8 (graphpad software, usa). statistical analyses were carried out using spss 17.0 (spss inc., chicago). using the duncan-test to assess differences between the data, p < 0.05 was considered to be statistically significant. results insecticidal toxicity the insecticidal toxicity test showed that the leaves and stems of g. elegans had effective insecticidal activity against the worker ants. in the test, it can be found that the mortality of the treated worker ants gradually increased with time (fig 1). the mortality of worker ants in both control groups were less than 20%, which excluded the effect of worker ants physiological activity and methanol-water on the test results. for 1 d~10 d after treated, the mortality of worker ants increased from 6.67% to 55.00% (1/20 leaf extract), from 1.67% to 41.67% (1/100 leaf extract), from 0.00% to 13.33% ( 1/5000 leaf extract)；from 1.67% to 46.67% (1/20 stem extract), from 0.00% to 30.00% (1/100 stem extract), from 0.00% to 10.00% (1/5000 stem extract)；from 3.33% to 45.00% (1 mg/kg koumine), from 3.33% to 23.33% (0.1 mg/ kg koumine), from 0.00% to 11.67% (0.05 mg/kg koumine). however, on day 10，red fire ants treated with 1/100 leaf extract had a mortality of 40%, which was comparable to the mortality of worker ants treated with 1/20 stem extract or 1 mg/kg koumine. meanwhile, this result indicates that the leaf is slightly more toxic to the worker ants than the stem. on the 10th day of the experiment, there was a significant difference between the mortality of worker ants treated at different concentrations (leaf: f=44.46 p<0.001; stem: f=28.00 p<0.001; standard: f=28.62 p<0.001). fig 1. mortality of worker ants after fed with leaves extract (a), stems extract (b) and koumine (c). 1/20 means the extract that 1 g of plant material dissolved in 20 ml of water, and 1/100, 1/5000 were diluted by 1/20. each data point represents mean ± se of three replicate beakers each containing 20 ants. sociobiology 67(2): 232-238 (june, 2020) 235 impact on aggregating behavior the aggregation level of worker ants all decreased over time after treatment by extracts or standard (fig 2). for 1d~10d after treated, the aggregation level of worker ants decreased from 95.00% to 56.67% (1/100 leaf extract), from 100.00% to 66.67% (1/500 leaf extract), from 100.00% to 83.33% (1/5000 leaf extract); from 95.00% to 60.00% (1/100 stem extract), from 98.33% to 68.33% (1/500 stem extract), from 100.00% to 81.67% (1/5000 stem extract); from 95.00% to 58.33% (0.5 mg/kg koumine), from 98.33% to 75.00% (0.05 mg/kg koumine), from 100.00% to 83.33% (0.02 mg/kg koumine). from this result, it can be seen that the concentration of 1/5000 and 0.01 mg/kg has no effect on worker ants, and even 0.02 mg/kg is not effective. however, the effects of two concentrations of 1/100 and 1/500 on the aggregation behavior of worker ants were significantly higher than those in the control group. on the 10th day of the experiment, there was a significant difference between the aggregation level of worker ants treated at different concentrations (leaf: f=13.07 p=0.007; stem: f=11.73 p=0.008; standard: f=21.88 p=0.002). impact on climbing behavior after treatment, when the worker ants were picked up with a long bamboo stick, the climbing ability of the worker ants was significantly reduced (fig 3). as the treatment fig 2. effects on aggregation level of worker ants after fed with leaves extract (a)，stems extract (b) and koumine (c). 1/100 means the extract that 1g of plant material dissolved in 100 ml of water, and 1/500, 1/5000 were diluted by 1/100. each data point represents mean ± se of three replicate beakers each containing 20 ants. fig 3. effects on climbing rate of worker ants after fed with leaves extract (a), stems extract (b) and koumine (c). 1/100 means the extract that 1 g of plant material dissolved in 100 ml of water, and 1/500, 1/5000 were diluted by 1/100. each data point represents mean ± se of three replicate beakers each containing 20 ants. q zheng, lp yang, sk lin, ql ma, dq qin, zx zhang – insecticidal activity of gelsemium elegans against solenopsis invicta236 concentration increased, the inhibitory effect on the ability of worker ants to climb was also enhanced. the climbing rate of worker ants decreased from 86.67% to 60.00% (1/100 leaf extract), from 96.67% to 65.00% (1/500 leaf extract), from 100.00% to 83.33% (1/5000 leaf extract); from 93.33% to 58.33% (1/100 stem extract), from 98.33% to 71.67% (1/500 stem extract), from 100.00% to 85.00% (1/5000 stem extract); from 95.00% to 58.33% (0.5 mg/kg koumine), from 100.00% to 76.67% (0.05 mg/kg koumine), from 100.00% to 83.33% (0.02 mg/kg koumine). on the 10th day of the experiment, there was a significant difference between the climbing ability of worker ants treated at different concentrations (leaf: f=12.54 p=0.007; stem: f=11.29 p=0.009: standard: f=10.06 p=0.012). impact on walking ability figure 4 shows that both leaf and stem extracts have relatively high inhibitory activity on the walking of worker ants. before the treatment, the normal red fire ant’s walking speed is about 2.2~2.4 cm/s. on the 7th day of treatment, the walking ability of the worker ants tended to be stable. on day 10, the walking rate of treated worker ants decreased to 1.53 cm/s (1/100 leaf extract), 1.73 cm/s (1/500 leaf extract) and 2.03 cm/s (1/5000 leaf extraction); 1.60 cm/s (1/100 stem extract), 1.73 cm/s (1/500 stem extract) and 2.00 cm/s (1/5000 stem extract); 1.47 cm/s (0.5 mg/kg koumine), 1.63 cm/s (0.05 mg/kg koumine), 1.88 cm/s (0.02 mg/kg koumine). on the 10th day of the experiment, there was a significant difference between the walking ability of worker ants treated at different concentrations, except for standard treatment. (leaf: f=19.00 p=0.003; stem: f=8.62 p=0.017; standard: f=4.65 p=0.060). discussion the results showed that both the aqueous extracts of the leaves and stems of g. elegans had insecticidal activity against red imported fire ants, which could reduce the aggregation level, climbing ability and walking ability of red imported fire ants. we think this may be caused by the ingredients of the koumine contained in g.elegans. the main component of g. elegans is alkaloid, and the most abundant is koumine (xu et al., 2012). therefore, we also studied the insecticidal activity and behavioral effects of the koumine standards on red imported fire ants, and compared the effect of the aqueous extract of plant materials, the results showed that it has a good effect. the traditional method of controlling red imported fire ants uses chemical pesticides, which will pollute the environment and endanger people’s health. the use of chemicals from natural products in insect pest management is generally considered as a safer alternative to using synthetic contact insecticides (zhang et al., 2017). in our research team, certain components of some plants have been proved to have insecticidal activity against red imported fire ants. for example, cinnamaldehyde and eugenol contained in cinnamon have been found to have insecticidal activity against red imported fire ants (huang et al., 2015), pronephrium megacuspe (huang et al., 2016), michelia alba (qin et al., 2018), etc. their high concentration treatments resulted in 100% mortality of worker ants in 10 days, which is different from the lower mortality rate of our 1/20 high concentration treatment. compared with the previous research, the method used for the insecticidal activity test this time is different. most of the previous methods were to immerse plant insecticidal compounds in the soil where worker ants live, spray the whole body of worker ants or directly use plant residues to fumigate fire ants to test the insecticidal activity. and this time we used water as the extraction solvent, and used the “water tube” method to feed worker ants directly. fig 4. effects on walking rate of worker ants after fed with leaves extract (a), stems extract (b) and koumine (c). 1/100 means the extract that 1g of plant material dissolved in 100 ml of water, and 1/500, 1/5000 were diluted by 1/100. each data point represents mean ± se of three replicate beakers each containing 20 ants. sociobiology 67(2): 232-238 (june, 2020) 237 nevertheless, we still think that using g. elegans, a natural plant material, to control red imported fire ants has good potential. this lower mortality rate may better control red imported fire ants. we all know that queen ant lives on food fed by worker ants. the best way to kill queen ant is through worker ants. however, red fire ants are very vigilant. when the ants die quickly, the death information will quickly spread to the entire ant nest. the g. elegans extract will not cause a large number of red imported fire ants to die in a short time, so it will reduce the vigilance of red imported fire ants and may kill the queen ant. in addition, the main feature of g. elegans extract is that it does not require any chemical solvents. it uses water as an extraction solvent and can even through boil a pot of water to extract the plant material. it is convenient and low cost because the plant can be grown all year round. the significance of this research is to provide a new method to kill red imported fire ants. the new method is safer, more environment-friendly. the results of the insecticidal activity and behavioral effects reported in this study can provide a foundation and reference for future research on the control of red imported fire ants by g. elegans. the study has focused on koumine, a main insecticidal ingredient in g. elegans. nonetheless, more research is needed to see if there are other chemicals in g. elegans that might contribute to the toxicity or inhibition of behavior on red imported fire ants. acknowledgments this study was funded by national key research and development plan, china (no. 2018yfd0200300), modern agricultural industry technology system innovation team of guangdong province, china (no. 2018ml1129). references appel, a.g., gehret, m.j., & tanley, m.j. 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(2013). effects of hematoporphyrin monomethyl ether on worker behavior of red imported fire ant solenopsis invicta. sociobiology, 60(2): 169-173. doi: 10.13102/sociobiology.v60i2.169-173 doi: 10.13102/sociobiology.v62i1.76-81sociobiology 62(1): 76-81 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 survivorship of the formosan subterranean termite (isoptera: rhinotermitidae) in a hypoxic environment introduction the formosan subterranean termite, coptotermes formosanus shiraki (isoptera: rhinotermitidae), is a structural pest of major economic importance that was introduced into new orleans, louisiana from southeastern china following world war ii (la fage, 1987). in new orleans, c. formosanus populations are wellestablished and readily outcompete the native subterranean termites, reticulitermes spp. (isoptera: rhinotermitidae), as the primary structural pest in the greater new orleans area (su, 2003). it has been suggested that the city of new orleans has one of the highest termite pressures in north america (lax & osbrink, 2003). coptotermes formosanus is a cryptic species. it creates gallery systems as well as carton nests consisting of soil, wood, saliva, and frass below the soil surface, within trees, and inside tree stumps. excavation of c. formosanus foraging abstract the formosan subterranean termite, coptotermes formosanus shiraki, is a structural pest of major economic importance in the city of new orleans, louisiana. hurricane katrina made landfall along the united states gulf coast on august 29, 2005, inundating approximately 80% of the city. though termite colonies survived prolonged inundation, the survival mechanisms of colonies have yet to be fully understood. one hypothesis is that c. formosanus colonies survived within pockets of trapped air within their nesting system during this period of flooding. this hypothesis was tested by measuring mortality of groups of 20, 40, and 60 termites in airtight environments maintained at three different temperatures. groups of 20 termites maintained at 10, 21, and 32°c reached 100% mortality at 89.5, 52.0, and 3.5 days, respectively. groups of 40 termites maintained at 10, 21, and 32°c reached 100% mortality at 89.5, 51.0, and 3.5 days, respectively. groups of 60 termites maintained at 10, 21, and 32°c yielded 100% mortality at 57.5, 22.5, and 1.0 day, respectively. field colonies of c. formosanus established before hurricane katrina survived up to three weeks of flooding, and our findings suggest that it is possible for inundated colonies to survive this prolonged flooding by remaining within air pockets located in their nesting system until flood waters recede. sociobiology an international journal on social insects 1 city of new orleans mosquito, termite, and rodent control board, new orleans, la, usa 2 university of florida, fort lauderdale, fl, usa article history edited by og de souza, ufv, brazil received 30 may 2014 initial acceptance 19 june 2014 final acceptance 24 september 2014 keywords formosan subterranean termite, hurricane katrina, inundation, survival. corresponding author carrie b. cottone city of new orleans mosquito, termite and rodent control board 2100 leon c. simon drive new orleans, la 70122 e-mail: cbcottone@nola.gov galleries showed that branching subterranean galleries were connected to multiple nests consisting of cavities enclosed by carton material (king & spink, 1969). coptotermes formosanus readily infests live trees, creating voids and building carton material within them (la fage, 1987; osbrink et al., 1999). according to osbrink et al. (1999), these nests can be located both under the soil surface or meters above the ground level within tree trunks. on august 29, 2005, hurricane katrina made landfall along the united states gulf coast. subsequent levee breaches within new orleans resulted in approximately 80% of the city being inundated (www.fema.gov). in some areas, flooding lasted up to three weeks. cornelius et al. (2007) observed actively foraging termites within previously established inground monitoring stations that had been flooded for up to two weeks within months after the flood waters receded. owens et al. (2012) confirmed that at least some c. formosanus colonies research article termites cb cottone1, ny su2, r scheffrahn2, c riegel1 sociobiology 62(1): 76-81 (march, 2015) 77 present at established research sites before inundation were the same as those present following inundation. though termite colonies survived prolonged inundation, the survival mechanisms of colonies have yet to be fully understood. forschler and henderson (1995) determined that the lt50 value for inundated c. formosanus workers was 11.1 hours, and the lt90 value was 15.8 hours. field colonies of c. formosanus established before hurricane katrina survived up to three weeks of flooding, much longer than the survival capabilities observed of termites subjected to inundation bioassays. for c. formosanus colonies to survive this prolonged inundation, they must have had access to an oxygen source. the hypothesis tested in the current study is that c. formosanus colonies could have survived the flooding following hurricane katrina by exploiting oxygen trapped within their nesting system. it has been suggested that survival of c. formosanus during prolonged inundation may be due to hydrophobic properties of carton material (cornelius et al., 2007) and trapped air within voids of infested trees (osbrink et al., 2008). the number of termites and temperature within the nesting system could have influenced termite survivorship during inundation as well. this is because the greater the number of termites confined to a space, the faster the available oxygen would be consumed for respiration. temperature would have influenced survivorship, as termites exhibit increased respiration at relatively warmer temperatures (cotton, 1932). our objective was to determine if c. formosanus colonies could have survived the flooding following hurricane katrina by remaining in an airtight environment that contains a limited amount of oxygen for up to three weeks. materials and methods termites termites were collected from three field colonies in new orleans, louisiana. the first field colony was located n 29.92372, w -90.13063, the second colony was located n 30.01825, w -90.09747, and the third colony was located n 30.03189, w -90.07253. termites were collected from previously established milk crate traps (kleinpeter farms dairy, baton rouge, la) installed below the soil surface and filled with untreated pine (pinus sp.) stakes (2 by 4 by 28 cm in length). collected termites were transported and stored within corrugated cardboard moistened with distilled water in the laboratory at 24°c. within one week, termites were bioassayed. voucher specimens for all colonies were deposited in the nomtcb’s arthropod collection. termite survivorship in an airtight environment for each colony, there were three experimental groups and three control groups. the first group consisted of 20 termites per replicate, the second group consisted of 40 termites per replicate, and the third group consisted of 60 termites per replicate. each group consisted of 10% soldiers and 90% workers (undifferentiated larvae of at least the third instar), which is consistent with the soldier ratio observed in established field colonies (lax & osbrink, 2003). termites were maintained at three different temperatures: 10, 21, and 32°c. there were three replicates for each experimental and each control group for each temperature at which termites were maintained. bioassays were conducted within 9 ml glass vials, each containing 3 ml 3% agar to maintain moisture and two cylindrical pieces (2 mm diameter by 20 mm in length) of untreated wood as a food source. when the volumes of agar and wood resource were subtracted from the total volume of the vial, it was determined that the available air within each vial was 5.94 cm3. termites were added to their respective vials. the vials containing the experimental groups were topped with a screw cap and wrapped in parafilm (bemis, neenah, wi) to create an airtight environment. these vials served to replicate an environment similar to what may be observed within a pocket of air inside the termite nesting system during times of flooding. the vials containing the control groups were topped with a piece of stainless steel mesh (2 by 2 cm) (termimesh llc, austin, tx), which allowed for air circulation. mortality of termites maintained at 10 and 21°c was evaluated at 12 hour intervals until 100% mortality was reached. termites maintained at 32°c were observed at 6 hour intervals for mortality. data were corrected using abbott’s (1925) formula. pooled data were analyzed by probit analysis to determine the lethal time, in days, for 50% (lt50) and 90% (lt90) of the test population for all groups of termites maintained at the three different temperatures. mortality rates for groups of 20, 40, and 60 termites maintained at each temperature were compared to each other using analysis of variance (anova) (proc glm) and tukey’s honestly significant difference (hsd) test with a level of significance when α < 0.05. worker and soldier mortality rates at each temperature were compared using a paired t-test. mortality data of all termites maintained at each temperature were pooled and compared to each other using anova (proc glm). all statistical analyses were conducted using sas 9.1 (sas institute, 2003, cary, nc). results total mortality for all treatment groups was observed from 1.0 to 89.5 days, depending on temperature and the number of termites confined within each vial. percent mortality for all groups of termites maintained at three different temperatures was plotted over time (fig 1). probit analysis on groups of 20, 40, and 60 termites confined within airtight vials and maintained at three different temperatures yielded lt50 values that ranged from 0.3 and 23.0 days (table 1). mortality of the control groups maintained at all three temperatures ranged from zero to 5%. cb cottone, ny su, r scheffrahn, c riegel – survivorship of termites in a hypoxic environment78 mortality rates of groups of 20, 40, and 60 termites were significantly different at 10°c (f = 227.95, df = 2, p < 0.0001), 21°c (f = 125.57, df = 2, p < 0.0001), and 32°c (f = 7.86, df = 2, p < 0.0066). at 10 and 21°c, groups of 20 termites yielded the lowest mortality, while groups of 60 termites yielded the highest mortality. at 32°c, groups of 20 termites exhibited significantly lower mortality rates than that for groups of 60 termites, though mortality rates of groups of 40 termites were not significantly different than that of groups t ab le 1 . c om pa ri so n of le th al ti m es , l t 50 s an d l t 90 s (d ay s) , w ith th ei r as so ci at ed s lo pe s, 9 5% c l s, a nd p ea rs on x 2 v al ue s fr om g ro up s of 2 0, 40 , a nd 6 0 c . f or m os an us c on fin ed to a n ai rt ig ht e nv ir on m en t a nd m ai nt ai ne d at th re e di ff er en t t em pe ra tu re s. te m p n o. o f te rm ite s n sl op e ± se lt 50 95 % c l lt 90 95 % c l x 2 ( df ) p 10 20 32 ,2 20 2. 98 ± 0 .0 7 23 .0 0 22 .0 1 – 23 .9 6 61 .8 9 60 .2 2 – 63 .6 9 37 2. 14 ( 17 6) <0 .0 00 1 40 64 ,4 40 2. 51 ± 0 .1 2 20 .3 1 18 .0 6 – 22 .4 3 65 .9 6 63 .4 1 – 68 .7 1 92 1. 64 ( 17 6) <0 .0 00 1 60 62 ,1 00 3. 91 ± 0 .2 1 16 .8 0 15 .4 4 – 18 .0 2 35 .7 1 34 .2 7 – 37 .3 2 1, 50 5. 76 ( 11 2) <0 .0 00 1 21 20 18 ,7 20 2. 19 ± 0 .1 0 6. 99 5. 92 – 8 .0 6 26 .8 2 24 .4 4 – 29 .4 6 65 0. 10 ( 10 1) <0 .0 00 1 40 36 ,7 20 1. 88 ± 0 .0 7 4. 41 3. 70 – 5 .1 4 21 .1 8 19 .1 9 – 23 .3 4 76 8. 66 ( 99 ) <0 .0 00 1 60 24 ,3 00 1. 51 ± 0 .0 8 1. 92 1. 47 – 2 .4 2 13 .5 6 11 .6 3 – 15 .8 3 31 3. 02 ( 42 ) <0 .0 00 1 32 20 1, 26 0 3. 53 ± 0 .7 0 1. 14 0. 57 – 1 .7 1 2. 64 1. 76 – 6 .1 1 57 .6 1 (4 ) <0 .0 00 1 40 2, 52 0 2. 14 ± 0 .6 2 0. 43 0. 00 03 – 1 .2 8 1. 70 0. 16 – 5 .7 1 79 .4 5 (4 ) <0 .0 00 1 60 2, 16 0 3. 94 ± 0 .6 9 0. 27 0. 07 – 0 .3 8 0. 57 0. 39 – 2 .0 2 29 .4 0 (2 ) <0 .0 00 1 fig 1. percent mortality of termites maintained in an airtight environment at 10°c, 21°c, and 32°c plotted against time. closed circles represent mortality of groups of 20 termites. open circles represent mortality of groups of 40 termites. triangles represent mortality of groups of 60 termites. of 20 or 60 termites. workers showed significantly lower mortality rates than soldiers at 10°c (t = -14.26, df = 178, p < 0.0001) and at 21°c (t = -6.6, df = 103, p < 0.0001). at 32°c, worker and soldier mortality rates were not significantly different (t = -0.70, df = 6, p = 0.5119). analysis of variance conducted on pooled mortality of all groups of termites at each temperature showed a significant difference between mortality rates of termites maintained at 10, 21 and 32°c (f = 143.6, df = 2, p < 0.0001). termites maintained at 10°c exhibited significantly lower mortality than termites maintained at either 21 or 32°c. termites maintained at 32°c exhibited significantly higher mortality than termites maintained at either 10 or 21°c. sociobiology 62(1): 76-81 (march, 2015) 79 discussion significant differences of mortality rates between the experimental groups of termites confined within an airtight environment showed that the greater the ratio of available air space to the number of termites confined to that space, the greater the survivorship. at 10 and 21°c, groups of 20 termites, when compared to groups of 40 and 60 termites, survived for a significantly longer period. this was also observed for groups of 20 termites when compared to groups of 60 termites at 32°c. higher mortality for groups of 40 or 60 termites may be due to more termites consuming the available oxygen causing a lack of available oxygen, or from increasing levels of carbon dioxide produced through respiration. it was expected that soldiers would experience lower mortality rates than workers in a hypoxic environment. this is because workers pursue various tasks, including tunneling and feeding, thus expending more energy and increasing their oxygen requirements. however, our results showed otherwise. at 10 and 21°c, workers survived significantly longer than soldiers. at 32°c, no such difference between worker and soldier mortality was observed. it was also speculated that termites maintained at relatively lower temperatures would survive for longer periods than those maintained at relatively higher temperatures. this is because increased respiration by termites at relatively warmer temperatures reflects an increase of oxygen required by termites for metabolic activity (cotton, 1932). furthermore, it is known that termite physical activities such as foraging and wood consumption increase during months of relatively warmer air and soil temperature (fei & henderson, 2004; messenger and su, 2005; cornelius and osbrink, 2011; delaplane et al., 1991). results of this study confirmed our speculation. according to the data presented here, 50% of a group of 20 termites (i.e. 10 termites) can survive being confined to approximately 6 cm3 of air space for 23 days at 10°c, 7 days at 21°c, and 1 day at 32°c. ten percent of a group of 20 termites (i.e. two termites) can survive in this same space for almost 62 days at 10°c, 27 days at 21°c, and 3 days at 32°c. according to figures by cornelius and osbrink (2011), soil temperatures in new orleans, louisiana ranged from 25 to 35°c between july and october for two consecutive yrs. the temperature of saturated soil during the flooding that followed hurricane katrina is unknown, but it is possible that flood conditions reduced the soil temperature. therefore, termites inundated following the hurricane would likely be represented by workers and soldiers bioassayed at 21 and 32°c. between the temperatures of 32 and 21°c, the lt50s ranged from 1 to 7 days for 20 termites in a hypoxic environment. at these same temperatures, the lt90s ranged from 3 to 27 days. if the soil temperatures were lowered below 21°c during this flood event, termites within their nesting system could have survived for an even longer period. when considering subterranean termite colony mortality due to treatment, or in this case a flood event which may confine termites within pockets of air, the colony itself must be considered as a unit, rather than placing focus solely on the mortality of individual termites. even if only a portion of a colony survives, the colony itself may eventually recover, when more foragers and reproductives are produced (su & scheffrahn, 1990). a study of c. formosanus field colonies within city park revealed an initial reduction of c. formosanus populations after being inundated for more than two weeks (osbrink et al., 2008). however, this study showed populations rebounded the following year. this is indicative of a colony that experienced loss of individual termites due to inundation, but continued to produce foragers and increase its colony size. the lt90s derived from our data show that 10% of the test population can survive in approximately 6 cm3 of available air up to 27 days (3.8 weeks) at 21°c, which is longer than the two to three week inundation duration observed after hurricane katrina. during the flooding that followed hurricane katrina, termites not succumbed to rising water may have survived by remaining in pockets of air within their hydrophobic carton material. coptotermes formosanus readily builds carton nests within live trees and under the soil surface (osbrink et al., 1999). a study conducted by cornelius and osbrink (2010) revealed that termites allowed to live and feed within hollowed wood blocks were able to survive inundation as long as they were located within the hollowed core at the time of flooding. this is also indicative of termite colonies being able to survive by exploiting air pockets within their nesting system. whether c. formosanus survived inundation by this mechanism remains unknown, as carton material within tree voids and structures could not be observed during flooding. however, groups of termites can survive more than three weeks within an airtight environment, depending on temperature and the ratio of available space to the number of termites confined to that space. furthermore, c. formosanus individuals exist within subterranean nests and experience relatively low o2 and high co2 concentrations and are capable of slightly reducing their respiration rates in these environments (wheeler et al., 1996). this would also contribute to their survival capabilities in a hypoxic environment during times of flooding. survival mechanisms of insects inhabiting hypoxic environments vary. several species of insects reduce their metabolic rates and become quiescent when exposed to hypoxic environments. for example, intertidal root aphids, pemphigus trehernei foster, become quiescent when submerged (hoback & stanley, 2001). tiger beetle larvae of the species cicindela togata laferte reduce their metabolic rates to survive repeated flooding of their larval habitat along flood plains (hoback et al., 1998). based on our findings, the hypothesis that c. formosanus colonies can survive flooding by exploiting trapped air is accepted. it would be possible for inundated termites to survive the flooding following hurricane katrina by remaining within air pockets located in their nesting system until flood cb cottone, ny su, r scheffrahn, c riegel – survivorship of termites in a hypoxic environment80 waters receded, but only if their nesting system was sealed and provided sufficient protection from being filled with water. it is important to understand the capability of c. formosanus to survive flood conditions, which are not only city-wide and caused by natural disasters, but are more regularly localized within the city and caused by heavy rainfall. homeowners and individuals within the pest control community should understand that prolonged inundation will not eliminate c. formosanus colonies. acknowledgements the authors thank the following new orleans mosquito, termite, and rodent control board personnel for their 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(1996). comparative respiration and methane production rates in nearctic termites. journal of insect physiology, 42: 799-806. doi: 10.13102/sociobiology.v62i4.392sociobiology 62(4): 564-570 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 behavioral responses of apis mellifera adult workers to odors from healthy brood and diseased brood introduction chalkbrood disease is an invasive mycosis in apis mellifera ligustica (a. mellifera) produced by ascosphaera apis (a. apis), which mainly affects larvae (aronstein & murray, 2010). some colonies survive in areas that are seriously damaged by a. apis, which indicates the existence of tolerance in some genotypes of honey bees (wallner, 1999). hygienic behavior is the natural mechanism of disease resistance in honey bee colonies. one important aspect of hygienic behavior is the ability of individual bees to detect and respond to stimuli from diseased larvae early in the progression of the infection. the quick and efficient detection and removal of diseased larvae by these bees prevents the transmission of the disease throughout the colony. unhealthy brood emit disease-related odorants, which trigger hygienic bees to recognize and remove diseased brood (masterman et al., 1999; spivak et al., 2003). the key factor in the tolerance of a. mellifera strains to chalkbrood disease is the honey bee’s sensitive olfactory perception that triggers the hygienic behavior (francesco abstract studies of adult workers’ responses to infected brood were undertaken to isolate discrete volatile compounds that elicited honeybee hygienic behavior. using a freeze-killed brood assay, we determined that in healthy colonies adult workers recognized and emptied infected cells with a 95% clearance rate. spme-gc-ms results emptied >95% infected cells indicated differences in the composition and relative content of volatile compounds released by healthy and diseased brood. additionally, we determined that the main volatile compound released from the pathogen ascosphaera apis was phenethyl alcohol. the y-tube olfactometer indicated that 10to 20-day-old workers of healthy colonies, but only 15-day-old workers of diseased colonies, were significantly sensitive to differences in characteristic volatile compounds. this information could facilitate honey bee selection based on mechanisms that contribute to chalkbrood disease tolerance. sociobiology an international journal on social insects hx zhao1, q liang2, jh lee2, xf zhang1, wz huang1, hs chen 1, yx luo1 article history edited by michael hrncir, ufersa, brazil received 04 april 2014 initial acceptance 10 january 2015 final acceptance 04 june 2015 keywords apis mellifera ligustica, ascospheara apis, volatile compounds, olfactory, y-tube olfactometer. corresponding author yuexiong luo guangdong entomological institute guangzhou, 510260, china e-mail: lyxbee@126.com et al., 2004). in a dark hive, diseased brood emitted special odors, which appeared to be the most important source of recognition cues. volatile compounds mediated the capability of adult workers to recognize infested cells and empty them. nazzi et al. (2002) and nazzi and della (2004) indicated that infected brood emitted short-chain unsaturated hydrocarbons to induce honey bee hygienic behavior. masterman et al. (2001) suggested that the characteristic compounds of diseased brood inspired the behavioral response of adult workers. swanson et al. (2009) detected three volatile compounds from larvae infected with a. apis that were absent from healthy larvae. thus, we used the spme-gc-ms (solid phase microextractiongas chromatography and mass spectrometry) method to measure these differences of volatile compounds (swanson et al., 2009). previously, kraus (1990) indicated that adult workers produced an alarm pheromone repellent to varroa destructor (v. destructor). kraus (1993, 1994) used a y-tube olfactometer to measure mite behavior, which is triggered by the odors of cappedcells over those of other life stages. the odors of capped-cells research article bees 1 guangdong entomological institute, guangzhou, china 2 fujian agriculture and forestry university, fuzhou, china sociobiology 62(4): 564-570 (december, 2015) 565 belong to volatile compounds produced by larvae. swanson et al. (2009) indicated that chalkbrood-infected bee larvae emitted characteristic volatile compounds, which contained phenethyl acetate, 2-phenylethanol, and benzyl alcohol. the y-tube olfactometer bioassay was used to test the orientation behavior of adult workers to volatile compounds. our research aimed to measure the sensitivity of the workers olfactory perception to the characteristic volatile compounds of diseased brood. here, we tested the ability of individual hygienic and non-hygienic bees to discriminate healthy brood from diseased brood. colonies displaying diseased or healthy traits were tested for hygienic behavior using a freeze-killed brood assay (spivak & reuter, 1998a, 1998b), which is an indirect measure of a colony’s hygienic behavior. the number of dead pupae that were in the process of being removed (uncapped and/or partially removed), and the number completely removed from the cells were recorded every 24 h for the trial colonies. in the current study, only those colonies that had uncapped and removed more than 95% of the freeze-killed brood within 48 h were considered as hygienic behavior. we also used spme-gc-ms to measure the volatile compound differences of diseased brood and healthy brood. then, we used y-tube olfactometer to measure the olfactory responses of non-hygienic bees and hygienic bees. the paper aim to veritify behavioral responses of apis mellifera adult workers to odors from healthy brood and diseased brood. materials and methods biological materials experiments were conducted at fujian agriculture and forestry university during spring of the years 2011 and 2012. the six colonies of a. mellifera was divided into three infected -chalkbrood colonies and three healthy-larvae colonies. experiments freeze-killed brood assay measured the hygienic behavior we chose a sealed brood of white-eyed pupae containing approximately 300 cells on each side (5 × 6 cm) from a frame and froze it for 5 min in liquid nitrogen. we recorded the number of sealed cells and put the frame back in the colony. the tests were repeated on the same colony at least twice. collection of volatiles released by infested-brood and healthybrood cells semiochemicals released by infested and uninfected worker brood cells were studied by means of spme-gc-ms. we collected chalkbrood-infected larvae. four types of a. mellifera larvae were used: a) five healthy fifth instars; b) five chalkbrood-infected larvae; c) five chalkbrood-infected larvae fed an artificial diet with fungal mycelia; and d) fungal mycelia (mummies) from potato dextrose agar (pda) medium of a. apis. first, we prepared a larval diet that consisted of 50% royal jelly (guangdong entomological institute bee industry co., ltd, china), 6% d-glucose (amresco, usa), 6% d-fructose, 37% double distilled water, and 1% yeast extract by volume (aupinel et al., 2005). prior to adding the diet to each cell, using 24-well tissue culture plates (bd falcon™, usa) (evans, 2004; aupinel et al., 2005; gregorc & ellis, 2011), we homogeneously mixed and pre-warmed the food in an incubator 35ºc. on consecutive days, we transferred larvae to a clean culture plate containing fresh diet. we fed the larvae 350 µl of the diet daily (aupinel et al., 2007). for the diseased brood we maintained the 350 µl of larvae food in individual wells, but added 1.0 × 108/ ml a. apis spores (vandenberg & shimanuki, 1987). a. apis gradually infected the larvae, reaching from the abdomen to the head by 5 d. the pathogenic fungus of a. apis was cultured on pda medium for 5 days at 35ºc in darkness. pda medium contains potato 200 g, anhydrous dextrose glucose 20 g, agar 20 g, distilled water 1,000 ml (reynald et al., 2004). healthy brood and diseased brood were removed from the 24-well plate with sterile forceps and placed in flasks (50 ml) that were then sealed with foil. additionally, we analyzed fungal mycelia (mummies) from pda medium of a. apis. compounds released from healthy and diseased brood were studied by means of spme-gc-ms. the 75µm car/ pdms spme fiber coating was exposed to a headspace gas phase for 30 min in the incubator at 35°c before immediate desorption into the gc-ms apparatus. analytes were separated and analyzed by gc-ms using a varian 450 gas chromatograph with a mass selective detector (cp-sil 8 cb-ms 30 m×0.25 mm column, usa). the helium flow rate through the column was 1 ml/min in constant flow mode. the injector (270ºc worked in splitless mode. the initial column temperature was 40°c for 2 min, increasing from 40°c to 120ºc by 3ºc/min, and then rising to 250ºc at 5ºc/ min. the detector was held at 250ºc the electron-ionization mass spectra were obtained at 70 ev of ionization energy. detection was performed in a full scan mode from 29 to 600 a.m.u. after integration, the fraction of each component in the total ion current was calculated. the above process was repeated three times to measure the volatile compounds of larvae. y-tube olfactometer bioassay we tested the olfactory responses of different ages (1-,10-,15-, 20-, 25-, 30-, day-old workers) of honey bees to volatile compounds using a y-tube olfactometer bioassay. the olfactometer apparatus was situated inside an incubator (rt 30ºc, rh 70%, dark room). the mix of volatile compounds were placed in one of the sample holding chambers of the olfactometer while the other sample holding chamber remained empty as a clean air “blank”. all the different aged bees tested remained in situ for 20 min, after which time the sample hx zhao et al. – behavioral responses of apis mellifera adult workers to odors566 holding chambers were reversed to avoid any positional bias. clean holding containers were used for next bee samples. statistical analysis of data frequencies of honey bees responding to treatment vs. control stimuli were compared using the χ2 tests for equal proportions. “*” indicates significantly different at α=0.05 (0.01<p<0.05). results liquid nitrogen frozen brood we tested the ability of individual hygienic and nonhygienic colonies to discriminate healthy from diseased brood. the removal of freeze-killed brood was correlated with the removal rates of diseased brood, and colonies that uncapped and removed >95% of freeze-killed brood within 48 h tended to remove diseased brood rapidly. thus, we measured the clearance ratio of adult workers to remove the diseased brood. the frozen brood insert into the colonies to assess bee hygienic behavior after 48 h. three healthy-larvae colonies uncapped and removed more than 95% of the killed brood by 48 h after insertion into the colony (fig 1). however, in the same time period three diseased-brood colonies only uncapped and removed 45% of the killed brood. volatile compounds of diseased brood and healthy brood fig 1. hygienic behavior of honey bee, apis mellifera, adult workers in the healthy brood and diseased brood. fig 2. gas chromatography–mass spectrometry (gcms) total ion chromatography of healthy and diseasedhoney bee, apis mellifera, broods by solid phase microextraction (spme) method. a: gcms total ion chromatography of healthy brood by spme method. b: gcms total ion chromatography of diseased brood by spme method. sociobiology 62(4): 564-570 (december, 2015) 567 differences in the amount of compounds released by healthy and diseased broods were assessed by comparing the size of the corresponding peaks in spme-gc-ms analysis performed under the same conditions. both the chalkbroodinfected brood in the healthy colonies and the chalkbroodinfected brood from an artificial diet with fungal mycelia emitted compounds with some similar ingredients. the results indicated that the differences between volatile compounds of healthy brood and diseased brood are in composition and relative content, especially that of tert-butylbenzene, e-β-ocimene, cosmene, 2-menthene, 2-isopropenyltoluene, and 1,3,5,5-tetramethyl-3cyclohexadiene (fig 2). e-β-ocimene was the main volatile compound of healthy brood phase compared with diseased brood. e-β-ocimene made up 1/5th of the relative volatile content from the healthy brood phase, but was only1/10th of the relative content from the diseased brood phase. the amount of tert-butylbenzene emitted by diseased brood was approximately five times more than the amount emitted by healthy brood, and 2-isopropenyltoluene was three times higher from the healthy brood phase than diseased brood phase. the relative contents of 2-menthene, cosmene, and 1,3,5,5-tetramethyl-3-cyclohexadiene, also showed some differences. laboratory inoculation of pda with chalkbrood disease causing a. apis fungal hyphae, released γ-decalactone,2,5,5trimethyl-1-hexenbenzylalcohol, 2-(1,1-dimethylethyl)-3methyl-oxirane, phenethyl alcohol, phenethyl acetate, and benzyl alcohol (fig 3). y-tube olfactometer bioassay fig 3. gas chromatography–mass spectrometry (gcms) total ion chromatography of fungal mycelia (mummies) from pda medium of ascosphaeraapis by solid phase microextraction (spme) method. compared with blank coloies, the 10to 20-day-old workers of healthy colonies are significantly more sensitive to differences in characteristic volatiles compounds than workers of other ages, while only the 15-day-old workers of diseased colonies display a significantly heightened sensitivity to differences in characteristic volatiles compounds (fig 4). newly emerged workers have an immature olfactory sense that leads to a weaker sensitivity. honey bees take less than 3 min to reach the junction of a y-tube olfactometer. even with the variation observed, the open y-tube olfactometer is an efficient bioassay that showed workers could discriminate between the healthy larvae and diseased larvae. discussion the honey bee is a model to observe the olfactory system of insects and it was observed that some colonies perform hygienic behavior to eliminate diseased brood. the stimuli triggering hygienic was thought to be olfactory cues emanating from cells containing diseased brood (boecking & spivak, 1999), but the identities of these cues were still unknown. healthy colonies removed 95% frozen brood. however, hx zhao et al. – behavioral responses of apis mellifera adult workers to odors568 fig 4. olfactory responses of honey bees,apis mellifera,to odors from the characteristic volatile compounds and a blank. a: olfactory responses of different ages of workers in the healthy colony. b: olfactory responses of different ages of workers in the diseased colony. note: * indicated significant different, significant at α=0.05, (0.01<p<0.05). the diseased colonies only removed 45% frozen brood, and they did not display significant hygienic behavior. hygienic behavior is specifically defined as the detection and removal of 95% diseased larvae from the colony (rothenbuhler, 1964; wilson-rich et al., 2009). colonies that uncap and remove >95% of freeze-killed pupae within 48 h will rapidly remove diseased brood (swanson et al., 2009). olfactory stimuli guided the detection and removal of diseased larvae (masterman et al., 2001; gramacho & spivak, 2003; spivak et al., 2003). volatile compounds mediate olfactory stimuli. the diseased brood and healthy brood of a. mellifera released different volatile compounds that were distinguished by adult workers, and these differences affected the workers’ behavior. in the healthy colony, the detection of the diseased brood’s odor would trigger workers with even the lowest detection sensitivities, and they would rapidly begin uncapping and removing the diseased brood (palacio et al., 2010). in a darkened hive, odors emitted from diseased brood appeared to be the most important source of recognition cues. spivak and boecking (2001) indicated that olfactory cues mediated the tolerance of a. mellifera strains by triggering the hygienic behavior to clean mites, and that the chemical compounds induced the ability of the bees to recognize infested cells and empty them (spivak & downey, 1999). cells infested with mite emitted different volatile compounds than uninfected cells. martin et al. (2001) suggested that the honey bee’s hygienic behavior was triggered by the polar compounds of the mite cuticle. nazzi et al. (2001, 2004) indicated that there might be slight differences in the amount of some short-chain unsaturated hydrocarbons. martin et al. (2001) showed that mites emitted volatile compounds to induce bee hygienic behavior. masterman et al. (2001) suggested that olfactory cues from the diseased brood itself stimulates the behavioral response of bees. the body fluid of brood was very important as a stimulus for adult workers to remove treated brood (aumeier et al., 2002). in our experiment, we used the spme-gc-ms method to analyze the volatile compounds of healthy brood and diseased brood, which was infected with a. apis. our results indicated a difference in volatile compound composition and relative contents, especially of tert-butylbenzene and e-β-ocimene. this was the first report on tert-butylbenzene. e-β-ocimene had previously been shown to be emitted during the queens mating and to play a role in regulating worker ovary development (gilley et al., 2006). additionally, larvae emit e-β-ocimene, which acts as a hunger signal. the 1to 2-day-old larvae have a higher need for food than 3to 4-day-old larvae, so younger larvae produce more e-β-ocimene than older larvae (maisonnasse et al., 2009, 2010). although diseased brood and healthy brood released similar volatile compounds, they had significant differences, and they especially emitted different amounts of e-ß-ocimene. we propose that the young infected brood needed little food and did sociobiology 62(4): 564-570 (december, 2015) 569 not nurse, while the healthy brood needed more food and care. thus, e-ß-ocimene was emitted as a characteristic compound, which could be used to discriminate between a healthy brood and diseased brood. stimuli coming from both the diseased brood and the fungal pathogen, a. apis, were considered in our test. to discriminate which volatile compounds came from diseased brood, and were not emitted by a. apis, we measured the volatile compounds of a. apis in the artificial pda culture medium. completely different compounds were emitted. a. apis produced benzyl alcohol, phenethyl alcohol, and phenethyl acetate. these three compounds were reportedly emitted by diseased brood (swanson et al., 2009). the field bioassay of topical applications and paraffin larval dummies revealed that phenethyl acetate induce hygienic behavior (swanson et al., 2009). in our test, we detected phenethyl alcohol as the main volatile compound of a. apis, accounting for 1/4th of the relevant volatile compounds. however, the relative amount of benzyl alcohol and phenethyl acetate were <5%. in our test, we detected phenethyl alcohol and phenethyl acetate from diseased brood, but we could not detected benzyl alcohol from diseased brood. we found that diseased brood and healthy brood have significantly different volatile compounds, and the relative content of the main volatile compounds were also significantly different. to confirm that honey bees are able to recognize brood cells infested by a. apis, we used a y-tube olfactometer to test the olfactory responses of workers. we verified the existence different chemical compounds of healthy brood and diseased brood, and then identified, the chemical stimuli that induces the hygienic behavior of bees towards a. apis infested cells. actually, we chose five volatile compounds and analyzed their effects on the olfactory behavior of honey bees. the 10 to 20-day-old workers showed significant olfactory stimulated behavior in the healthy colony. it has been reported that 15-dayold workers performed hygienic activities (invernizzi & corbella 1999, arathi et al., 2000). moreover, 15-day-old workers rapidly initiated uncapping behaviors depending on the stimulus from a dead brood (arathi, 2000). middle-aged bees (12to 21-day-old) built and maintained the hive, and received and processed nectar (johnson, 2003, 2008a). they did not engage in nursing behavior (ben-shahar et al., 2002; ben-shahar, 2005). workers displayed age-related division of labor and natural behavioral plasticity (leconte & hefetz, 2008; ben-shahar, 2005). the change from cell cleaning to nurse and other in-hive tasks were performed by 21-day-old workers (whitfield, 1987). our research showed that 10to 20-day-old workers in hygienic colonies had the ability to discriminate characteristic odors. only the 15-day-old workers in the non-hygienic colony had sensitive enough olfactory perception to differentiate preferential odors. thus, 15-day-old workers showed a sensitive olfactory response, which correlated with the workers age. as a social insect, adult workers perform different tasks in keeping with temporal polyethism. the 10to 20-dayold workers of the healthy colonies could select five volatile compounds by y-tube olfactometer. these volatile compounds both detected in healthy brood and diseased brood, but they have the different relative contents. thus, we proposed adult workers of some colonies have olfactory perception sensitive enough to detect diseased brood. this information could facilitate the selection of honey bees with pronounced hygienic behavior based on mechanisms that contribute to chalkbrood disease tolerance. acknowledgments this work was supported by projects of the natural science foundation of china (31302041) and the modern bees industrial technology system (cars-45-kxj-7 and cars-45-syz-12). references aronstein ka, murray kd. 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(2009). genetic, individual, and group facilitation of disease resistance in insect societies. annual review of entomology, 54: 405-423. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2597sociobiology 65(2): 305-311 (june, 2018) longand short-term changes in social wasp community structure in an urban area introduction the social wasps are widely distributed throughout all continents, except antarctica. in brazil, they are represented primarily by the polistinae (carpenter & marques, 2001). brazilian paper wasps inhabit various environments, but show high degrees of association with urban areas, often constructing nests under or inside the walls of buildings (wenzel, 1998; alvarenga et al., 2010; torres et al., 2014; michelutti et al., 2013). the importance of studying social wasps and their diversity is emphasized by their ecological roles. for instance, social wasps may act as pollinators when visiting flowers (clemente et al., 2012), detritivores when foraging on decaying fruits (barbosa et al., 2014) and animal carcasses (moretti et al., 2011), and predators when foraging on agricultural pests (e.g., caterpillars) (elisei et al., 2010). as a result, social wasps are part of a complex intra and interspecific ecological web that is yet to be fully comprehended (menezes et al., 2014; virgínio et al., 2016). abstract the success of social wasps in anthropic environments is related to their ability to nest both in vegetation and human constructions, and, as humans modify their own environments, wasps community structure may shift as well. our aim was to assess the diversity of social wasps and their interactions with nesting substrates seasonally in an urban squares area in southeastern brazil, 15 years after the first diversity study in this area. we actively searched for nests in the rainy season between 2014 and 2015 and in the dry season of 2015. although social wasp species richness did not change since the 2000 assessment (13 species in 5 genera), the abundance decreased substantially. additionally, wasps showed a general trend of nesting on the man-made materials metal (n = 115, 60%) and concrete (n = 106, 36%), especially by the two most common species sampled: mischocyttarus cassununga and polistes versicolor. we suggest that abundance decrease may correspond to the reduction of green areas in the assayed locations. these results support the well-known importance of maintaining green areas in urban environments to promote the growth and conservation of diverse social wasp communities. sociobiology an international journal on social insects m detoni, bc barbosa, tt maciel, sjl dos santos, f prezoto article history edited by marcel hermes, ufla, brazil received 27 october 2017 initial acceptance 30 january 2018 final acceptance 30 january 2018 publication date 09 july 2018 keywords nesting, polistinae, synanthropism. corresponding author mateus fajardo de freitas salviato detoni laboratório de ecologia comportamental e bioacústica – labec universidade federal de juiz de fora campus universitário bairro martelos cep 36036-900 juiz de fora, minas gerais, brasil. e-mail: matedetoni@hotmail.com the nesting sites chosen by wasps are strongly influenced by predation and the weather (raposo-filho & rodrigues, 1984; souza et al., 2010). nesting on manmade substrates, in addition to natural ones, may be a useful strategy for wasp populations since their colonies may have higher developmental success as these locations may allow them to avoid their natural predators and take shelter from harsh weather conditions (jeanne, 1975; lima et al., 2000; alvarenga et al., 2010). studies with social wasp behavior and diversity in urban environments have been increasing in number in the last decade (e.g. alvarenga et al., 2010; jacques et al., 2012; naldoski, 2013; torres et al., 2014; see barbosa et al., 2016). they show the relation between many species of social wasps and their environments, such as how some species found in urban areas depend exclusively on natural vegetation, whereas other prefer artificial substrates (alvarenga et al., 2010; sinzato et al., 2011; castro et al., 2014; virgínio et al., 2016). moreover, as humans continue to change their laboratório de ecologia comportamental e bioacústica – labec, departamento de zoologia, universidade federal de juiz de fora, mg, brazil research article wasps mailto:matedetoni@hotmail.com m detoni, bc barbosa, tt maciel, sjl dos santos, f prezoto – changes in social wasp community structure306 environment, some species seem to change their nesting preferences as well (torres et al., 2014). other studies discuss these nesting tendencies in natural and urban areas (lima et al., 2000; alvarenga et al., 2010; maciel and barbosa, 2015), explaining the advantages of using both natural and man-made substrates. thus, we need to better understand the ecological relations between wasps and the urban environment. previous studies have begun comparing nesting preferences in natural vs. artificial environments. here, we build on that previous work to survey the diversity of social wasps, using urban locations surveyed in a study published 15 years prior to this one (lima et al., 2000). we compare the two sets of results to understand long-term changes in the social wasp community structure. we also investigated short-term seasonal changes in the social wasp community structure, by investigating population dynamics across two following seasons and studied the interactions between the species of social wasps and their nesting substrates, trying to establish a link between this relation and the group’s seasonality and diversity in order to better understand the ecology of social wasps in urban areas. material and methods area of study the study was carried out in the campus of universidade federal de juiz de fora (ufjf) (21º46’02.72’’s 43º22’34.9’’w 678 m asl), located within the urban perimeter of juiz de fora, minas gerais state, southeastern brazil. the climate in the area is characterized as humid subtropical, with dry winters (may to september) and rainy summers (october to april) (cwa), according to köppen-geiger (sá-júnior et al., 2012). the area has been recently classified by carvalho et al. (2014) as having a predominance of pinus elliottii engelm., showing a low diversity of plant species, and is considered an emergent ecosystem (fig 1). it may also be referred to as a novel ecosystem (maciel & barbosa, 2015). data sampling recording the wasp nests took place during one dry season (between december 2014 and february 2015), and one rainy season (between june and july 2015). we actively searched for colonies by inspecting man-made substrates (further classified into: asbestos, concrete, ceramic, metal, plastic, porcelain or stone), rocky outcrops, tree trunk cavities and canopies. when they were located, we photographed colonies, identified the material used as substrate, and collected 1-3 individuals with an entomological net. individuals were stored in labeled containers with 70% ethylic alcohol for later identification. for species identification, we used dichotomous keys suggested by richard (1978), hermes and kohler (2004), silveira (2008), andena et al. (2009) and carpenter and andena (2013). wasp samples were stored in the laboratório de ecologia comportamental e bioacústica (labec) of ufjf; one individual of each species was dry mounted for the voucher. fig 1. geographical representation of area of the campus of universidade federal de juiz de fora, delimited in red, in the urban perimeter of juiz de fora, minas gerais state, southeastern brazil. sociobiology 65(2): 305-311 (june, 2018) 307 data analysis species diversity was analyzed for rainy and dry season separately and combined using the shannon (h’) diversity index and the h’-based pielou (j’) evenness index. the past v.2.17c software (hammer et al., 2001) was used to perform diversity analyses. g-test was used to examine the difference in wealth and abundance between seasons; calculations were done with the bioestat software (v. 5.3). each species’ dominance was calculated and expressed as a percentage using the following formula: d= (i/t)*100, where i = total number of individuals of a given species and t = total number of individuals of all species collected. these percentages were then categorized as follows: eudominant > 10%; dominant = 5-10%; subdominant = 2-5%; recessive = 12%; or rare < 1% (friebe, 1983). in order to assess social wasp species richness in the area for each used method, we calculated the species’ rarefaction curves (sensu gotelli & colwell, 2001) through the software estimates 9 using 5000 randomizations. first and 2nd order non-parametric estimators of jackknife were used to project the maximum species richness that each method and area of study may reach. the software generates 5000 species accumulation curves, randomizing the sampling order; therefore, each point on the curve corresponds to the average of the accumulated richness for the 5000 curves and is associated to a standard deviation value. to design the bipartite graph among substrates and social wasps on the campus of ufjf, we used every colony in order to build an adjacency matrix for each season: a “quantitative matrix” that regards the frequency of nestsubstrate interactions for each wasp species through the r software (v.3.4.3). in order to assess general changes in the composition of the environment, we obtained satellite photographs of the campus area from the software google earth pro 7.3.0.3832 (32-bit) for the years of 2015 and 2010, which was the oldest record available. green areas were calculated in the same software using an area delimiting tool. additional data for previous years was obtained from vieira et al. (2016). results we identified a total of 13 species from five genera: mischocyttarus saussurre, 1853, polistes latreille, 1902, polybia lepeletier, 1836, protopolybia ducke, 1905 and synoeca de saussure, 1852 (table 1). species richness was significantly higher during the rainy season (n = 13 species) compared to the dry season (n = 8 species; g-test = 36.0685; p< 0.0002; table 1). the abundance of colonies was also higher during the rainy season (n = 197 colonies) compared to the dry season (n = 86 colonies) (283 total, average = 141.5; g-test = 366.8213; p< 0.0001). there were two eudominant species in the area, mischocyttarus cassununga (von ihering, 1903) and polistes versicolor (olivier, 1791), and one dominant, protopolybia exigua (de saussure, 1854). the remaining species were fit into subdominant, recessive or rare (table 1). tests with the jacknife estimators of 1st and 2nd order showed an estimation of 16 and 17 species (respectively) to the campus of ufjf. species frequency dominance rainy season dry season total mischocyttarus socialis (de saussure, 1854) 1 0 1 rare 0,4 mischocyttarus cassununga (von ihering, 1903) 100 49 149 eudominant 52,7 mischocyttarus drewseni saussure, 1954 10 3 13 subdominant 4,6 mischocyttarus wagnei (buysson, 1908) 1 0 1 rare 0,4 polistes ferreri (de saussure, 1853) 7 2 9 subdominant 3,2 polistes versicolor (olivier, 1791) 46 16 62 eudominant 21,9 polybia fastidiosuscula saussure, 1854 1 1 2 rare 0,7 polybia paulista (ihering, 1896) 1 1 2 rare 0,7 polybia platycephala richards, 1951 7 0 7 subdominant 2,5 polybia sp. 3 2 5 recessive 1,8 protopolybia exigua (de saussure, 1854) 16 12 28 dominant 9,9 protopolybia sedula (de saussure, 1854) 3 0 3 recessive 1,1 synoeca cyanea (fabricius, 1775) 1 0 1 rare 0,4 total 197 86 283 rainy season h´= 0,6678 d = 0,5076 j = 0,5995 dry season h´= 0,5682 d = 0,5823 j = 0,6292 total h´= 0,6477 d = 0,5265 j = 0,5814 table 1. species of social wasps sampled and values calculated for shannon’s index, dominance index and pielou’s index at the campus of universidade federal de juiz de fora, city of juiz de fora, minas gerais state, southeastern brazil, in the periods between december 2014 and january 2015 (rainy season) and between june and july 2015 (dry season). m detoni, bc barbosa, tt maciel, sjl dos santos, f prezoto – changes in social wasp community structure308 these results, along with the species rarefaction curve, are depicted in figure 2. regarding the nesting substrates, social wasps constructed nests on metal the most (115 nests, 40% of the total sampled colonies), followed by concrete (102 nests, 36%). the remainder of the nests (24%) was divided somewhat evenly among the remaining substrates. the eudominant species (m. cassununga and p. versicolor) have shown flexibility regarding the nesting substrates, found on six and five (respectively) of the eight substrates used by wasps on this study (figure 3). p. exigua, protopolybia sedula (de saussure, 1854) and synoeca cyanea (fabricius, 1775), on the other hand, exclusively used vegetation as their substrate. it is worth noting that the use of vegetation as nesting substrate was 43% lower in the dry season than in the rainy season. such reduction was also recorded for the species richness on this substrate, which decreased from six to only two species in the dry season. the investigation of the green areas within the campus between 2010 and 2015 showed that in 2010 the total green area was 496,966.9 m2, which corresponds to 36.9% of the total campus area (1,346,793.80 m2). the green area in 2015 was reduced to 334,004.8 m2, corresponding to 24.8% of the total campus area; a reduction of 12.1% of green area. additionally, vieira et al. (2016) described a 7.36% reduction of green areas in the campus between 2002 and 2010. by combining these data, we calculate that, between 2002 and 2015, green areas in the campus decreased in 18.56%. genera (mischocyttarus, protopolybia, polistes and polybia) and presented a single exclusive genus each (protonectarina in lima et al., 2000, synoeca in this study). these exclusive genera comprise very small portions of the samples (two colonies of protonectarina and one of synoeca) and show similarities in nesting habits (notably a preference for nesting almost exclusively in vegetation, according to richards & richards, 1951; wenzel, 1998). their presence may therefore represent rare occurrences rather than an actual change in the richness structure between those 15 years. the abundance of social wasps, on the other hand, has shown greater differences between the two studies in the area. while lima et al. (2000) sampled a mean 287.75 colonies per census, we found less than half this value (141.5 per census). this alarming decrease in the wasp populations may be a result of the progressive reduction of vegetation in the studied site. many tropical wasp species nest exclusively on vegetal substrate, and all benefit indirectly from the surrounding green areas, especially in urban areas (wenzel, 1998; alvarenga et al., 2010). although it cannot be concluded, we suggest that the significant decrease of vegetation (close to 20% in 15 years) may have played a major role in determining social wasp abundance in the area. our data seems to be in accordance with the richness of social wasps shown in environments with some degree of similarity to the campus. jacques et al. (2012) studied an urban area with integrated vegetation in southeastern brazil and sampled 21 species; however, only 7 genera and 13 of these species were recorded through the active nest search method, which is similar to our results (13 spp. in five genera). other diversity studies performed in natural areas show some variation regarding richness; for instance, in the atlantic rainforest, these values range between 10 (arab et al., 2010), 18 (santos et al., 2007) and 25 (hermes & köhler, 2004), highlighting the importance of urban areas, since the value we sampled (n=13) is within their range. similar to lima et al. (2000) we found that the abundance and species richness of social wasps is higher during the rainy season compared to the dry season. this is an evidence of a high colony cycle synchrony degree, as described for some independent-founding wasps (dantas de araujo, 1982). however, since 61.5% of the species were also present in the winter, our results agree with naldoski (2013), who suggests that the complexity of urban environments reduces the impact of seasons on the social wasp cycles. despite the differences in abundance and diversity between seasons, the ecological indices had low variation when values were compared across seasons and throughout the year. jacques et al. (2012) calculated diversity and dominance of social wasps in a different urban area in southeastern brazil, and their sample showed higher diversity (h’= 0.9814) and lesser dominance (d= 0.2581) than ours. this results suggests the fauna in the campus of universidade federal de juiz de fora is strongly dominated by a few species. indeed, fig 2. species rarefaction curves estimated from 5000 randomizations for the social wasps sampled in the campus of universidade federal de juiz de fora city of juiz de fora, minas gerais state, southeastern brazil, in the periods between december 2014 and january 2015 (rainy season) and between june and july 2015 (dry season). discussion comparing the diversity of wasps sampled in this study with the results found by lima et al. (2000) in the same area 15 years before provides various insights on how changes in the environmental structure of urban areas may affect the social wasp fauna. in terms of genera richness, both studies had very similar structures since they shared four sociobiology 65(2): 305-311 (june, 2018) 309 our samples showed two eudominant species (m. cassununga and p. versicolor) and a dominant one (protopolybia exigua), the rest being subdominant, recessive or rare. the fact that the structure of the index does not significantly differ between seasons suggests that highly dominant species are more adapted to persist throughout harsher (i.e., dryer) environmental conditions. the species m. cassununga and p. versicolor may be considered highly synanthropic, since they are more consistently abundant compared to other species (torres et al., 2014; castro et al., 2014). in our study, the swarming species (epiponini tribe) used larger substrate area to attach their large nests. moreover, the species of the epiponini tribe were recorded less often on man-made substrates, and some species were restricted to constructing nests on vegetation (jeanne, 1975; carpenter & marques, 2001). in other words, in an environment where green areas are receding (vieira et al., 2016), species capable of nesting preferably on artificial structures – here depicted by the eudominant m. cassununga and p. versicolor nesting on metal and concrete (see figure 3) – are more successful in occupying an urban environment. our results show that although the species richness of social wasps in the campus of ufjf has been relatively stable over the last 15 years, modifications on the campus’ physical structure, with more buildings and less vegetation, may be affecting the relative abundance of wasps and allowing certain species that are capable of nesting on man-made structures to dominate the community. this is clearly seen in the overall reduction of social wasp abundance over the past 15 years, as well as the decrease of species richness from rainy to dry seasons. altogether, these results highlight the importance of maintaining green areas in order to promote the growth and conservation of wasp populations. fig 3. the relation between wasps and different nesting substrates, involving 13 species of social wasps and eight types of substrate on the campus of universidade federal de juiz de fora, juiz de fora, minas gerais state, southeastern brazil. a rainy season (hot/humid) and b dry season (cold/dry). acknowledgements the authors would like to thank dr. jennifer jandt of the university of otago for her suggestions in the writing of this paper. we would also like to thank the universidade federal de juiz de fora (ufjf) and conselho nacional de pesquisa e desenvolvimento (cnpq) (f. prezoto 310713 / 2013-7) for financial support. references alvarenga rb, castro mm, santos-prezoto hh, prezoto f (2010). nesting of social wasps (hymenoptera, vespidae) in urban gardens in southeastern brazil. sociobiology 55: 445-452. arab a, cabrini i, de andrade cfs (2010). diversity of polistinae wasps (hymenoptera, vespidae) in fragments of atlantic rain forest with different levels of regeneration in southeastern brazil. sociobiology, 56 (2): 515-526. m detoni, bc barbosa, tt maciel, sjl dos santos, f prezoto – changes in social wasp community structure310 barbosa bc, detoni m, maciel tt, prezoto f (2016). studies of social wasp diversity in brazil: over 30 years of research, advancements and priorities. sociobiology, 63 (3): 858-880. doi: 1 0.13102/sociobiology.v63i3.1031 barbosa bc, paschoalini m, prezoto f (2014). temporal activity patterns and foraging behavior by social wasps (hymenoptera, polistinae) on fruits of mangifera indica l. 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michener, 2007). in addition, insects are associated to better fruit and seed productivity and development (kerr et al., 1996; imperatrizfonseca & nunes-silva, 2010; putra et al., 2014; silva et al., 2015), play an essential role for maintenance and balance of ecosystems. in this context, studies seek to measure the actual value of services to the environment that pollinators have (lonsdorf et al., 2009; gallai et al., 2009; giannini et al., 2015). however, the growth of agriculture and deforestation, driven by the intensification of agricultural practices (leblois et al., 2016), favors the emergence of pests and diseases, leading farmers to use pesticides to control insect pest populations abstract bees of genus apis are the main crop pollinators; however, the use of pesticides in agriculture may intoxicate them during foraging. in this study, we evaluated the toxic effects caused by difenoconazole (fungicide), fenpyroximate (acaricide) and mineral oil (adjuvant) used alone and associated (pesticide + adjuvant) on workers of apis mellifera l. bees were exposed to product doses recommended by manufacturers, orally and in contact on a contaminated surface in a controlled environment. all products presented low lethality, both in isolation and combination (except for difenoconazole via contact), however, they all showed toxic effects. the results showed that combination of pesticides with adjuvant augmented toxic effects. sociobiology an international journal on social insects dt leite1, rb sampaio1, co santos1, jn santos1, ed chambó2, cal carvalho1, gs sodré1 article history edited by solange augusto, ufu, brazil received 04 may 2018 initial acceptance 07 july 2018 final acceptance 05 september 2018 publication date 11 october 2018 keywords synergism, beekeeping, useful insects. corresponding author delzuite teles leite programa de pós-graduação em ciências agrárias universidade federal do recôncavo da bahia rua rui barbosa, 710 centro cep 44380-000, cruz das almas-ba, brasil. e-mail: delzuiteteles@hotmail.com (ecobichon, 2001; genersch, 2010; coupe et al., 2012), which, in turn, affects both food production and the environment (schreinemachers & tipraqsa, 2012; guedes et al., 2016). originally, the relationship between plant and pollinator was essentially positive; however, pollinators are suffering from the frequent use of pesticides in floral resources (blacquière et al., 2012; fürst et al., 2014; morais et al., 2018). bees are some of the main pollinating agents and have become the most affected individuals, due to the contact with contaminated sources that cause behavioral disorders and even death of individuals (sandrock et al., 2014; goulson, 2015). numerous factors can compromise colony development and perpetuate pests, parasites and pathogens (genersch, 2010). among them, the use of pesticides affects pests and bee species (della lucia et al., 2014; rondeau et al., 2014). 1 universidade federal do recôncavo da bahia, cruz das almas, bahia, brazil 2 instituto de natureza e cultura, universidade federal do amazonas, benjamin constant, amazonas, brazil research article bees dt leite, rb sampaio, co santos, jn santos, ed chambó, cal carvalho, gs sodré – toxicity of pesticides on apis mellifera l.738 studies on acaricide fenpyroximate (dahlgren et al., 2012; libyarlay et al., 2014) have reported its toxicity on apis mellifera l. syromyatnikov et al. (2017) and kinasih et al. (2017) also described the effects of the fungicide difenoconazole on bombus terrestris l. and trigona (tetragonula) laeviceps smith. fenpyroximate is an acaricide belonging to the chemical group of pyrazoles, classified as highly toxic and very dangerous to the environment. fungicide difenoconazole is a triazole, classified as extremely toxic and environmentally very dangerous. mineral oil is an aliphatic hydrocarbon indicated to several crops, classified as low toxic and not dangerous to the environment and it can be applied as an adjuvant added to pesticide syrup (mapa, 2018). studies have also proven that neurotoxic insecticides affect the immune system of the a. mellifera, favoring the onset of diseases (brandt et al., 2016) and showing that secondary effects can be as damaging as the lethal ones. however, toxicity caused by secondary effects is more difficult to diagnose, as it does not present immediate lethality and may further promote the spread of the active principle within the colony. in this context, this work aimed to analyze toxicity (on survival and secondary effects) of pesticides (fenpyroximate, difenoconazole and mineral oil) on a. mellifera when exposed to the contaminated surface of citrus leaves and the ingestion of candi paste, contaminated by these products. material and methods products used and description of bioassays bioassays were carried out to test the effects of the maximum recommended doses (table 1) of products for the phytosanitary control in citrus crops on a. mellifera. for that, the formulated products used were fenpyroximate (50 g/l of the active ingredient in the formulated product), difenoconazole (250 g/l of the active ingredient in the formulated product) and mineral oil (756 g/l of the active ingredient in the formulated product). the products were purchased at a commercial store. workers of a. mellifera were collected from three colonies kept in langstroth boxes in an apiary at the federal university of the recôncavo da bahia, cruz das almas, brazil. plastic cages (30 cm in diameter and 4 cm high) were made for bioassays with holes in the closed caps with voile fabric for air circulation for comfort of the bees and two lateral holes to fit the feeders adapted from centrifuge microtubes (one with water and another with the candi paste). bees were exposed to the products as follows: a) exposure through ingestion before being exposed to contaminated food, the bees were kept for three hours without feed. for each treatment, the recommended dose was added to 100 ml of honey and confectionery sugar homogenized with a glass stick in a becker to form the candi paste. then, the paste was offered to the bees in microtubes inside the cages for 96 h. b) contact exposure to contaminated surfaces (by pesticides) leaves of lemon citrus aurantifolia var. thaiti, collected from a plant without phytosanitary treatment, were immersed for 5 min in the solution with each pesticide, which was diluted in water as described in table 1 (adapted from carvalho et al., 2009). then, the leaves dried at room temperature for about two hours. as a control treatment, the leaves were immersed for 5 min in distilled water. assessment of toxicity (survival and secondary effects on bees) all treatments with different types of exposure were installed in chamber type b.o.d. at temperature (30 ± 5 ºc) and relative humidity (70 ± 5%) controlled, with absence of light. we evaluated the mortality of each bee at intervals of one, two, three, four, five, six, nine, 12, 15, 18, 21, 24, 30, 36, 42, 48, 60, 72 and 96 h after the beginning of the treatments (carvalho et al., 2009).the secondary effects were evaluated by means of bees observation: disorientation, paralysis, prostration, hyperexcitation, impaired motor coordination and agitation, according to cox and wilson (1984), and carvalho et al. (2009). statistical analysis a completely randomized design was used to measure the survival rate of the bees, with five treatments on different exposure methods (mineral oil, difenoconazole, fenpyroximate, difenoconazole + mineral oil, fenpyroximate active principle class dmr1 target organism chemical group difenoconazole fungicide 20 ml/100l colletotrichum gloeosporioides triazole fenpyroximate acaricide 100 ml/100l brevipalpus phoenicis pirazol mineral oil adjuvant 2 l/100l orthezia praelonga aliphatic hydrocarbons 1in accordance with ministry of agriculture, livestock and food supply (mapa), brazil. table 1. active principle, class, maximum dosages recommended by the manufacturer (dmr), target organism and chemical group of pesticides used in citrus orchards. sociobiology 65(4): 737-743 (october, 2018) special issue 739 + mineral oil) and with a control composed of distilled water with five repetitions. each repetition was composed of 10 bees, totaling 50 bees per treatment and 300 bees per experiment, from colonies installed in a box of the langstroth model. the data were submitted to the survival analysis using the survival package and submitted to statistical analysis in r® software (r development core team, 2016). kaplan-meier survival curves were generated to determine the proportion of surviving bees against times after application of pesticides by ingestion, contact with contaminated surface and topical contact. the log rank test was used to test the null hypothesis when the kaplan-meier curves were identical. results survival of bees the survival of bees submitted to ingestion treatments presented significant differences according to the log rank test of cox-mantel (χ2 = 8.8, gl = 5, p <0.0001), with 80% of survival 96 hours after exposure to the active principle difenoconazole. bees exposed only to fenpyroximate and difenoconazole + mineral oil had 94% and 92% survival rate, respectively, at 96 h of exposure. the survival of bees exposed to active principles mineral oil + fenpyroximate and only mineral oil were 92% and 84% at 72 and 60 h, respectively. bees that were not submitted to the active products had a survival rate of 94% (fig 1). fig 1. survival curves plotted from exposure time (hours) via ingestion until death of each bee (apis mellifera): difenoconazole + mineral oil (dfz+mo); mineral oil (mo); control (control); difenoconazole (dfz); mineral oil + fenpyroximate (mo+fpx); fenpyroximate (fpx); . curves indicate the median and 95%, respectively. dt leite, rb sampaio, co santos, jn santos, ed chambó, cal carvalho, gs sodré – toxicity of pesticides on apis mellifera l.740 the survival curve of the cox-mantel log rank test showed significant differences in the survival rates between bees that were submitted to different pesticides applied by contact (χ2 = 31.6, gl = 5, p <0.0001). there was mortality of 24% and 28% at 60 h after application of difenoconazole and mineral oil + fenpyroximate, respectively. at 40 and 96 h, mortality of bees exposed only to fenpyroximate and to mineral oil was 22% and 24%, respectively. the survival rate was lower for bees exposed to difenoconazole + 50% mineral oil at 96 h. the survival rate of control bees was 96% (fig 2). secondary effects on bees during the study, behavioral changes of bees were observed in all treatments, except for the control, where bees had the same behavior throughout the evaluation period. contact with mineral oil after 12 h of exposure to the product left the bees with impaired motor coordination, where the bees were unable to stay in the natural position and remained part of the time with the back to the ground. regardless of exposure type, difenoconazole showed effects after 15 h of evaluation, when workers of a. mellifera presented behaviors divergent to the control, such as agitation and changes in motor coordination. when difenoconazole was added to mineral oil, changes in motor coordination occurred after five hours of evaluation. for fenpyroximate, bees submitted to the contact with this acaricide had alterations in the motor coordination after 12 h of exposure. fenpyroximate added with mineral oil left fig 2. survival curves plotted from exposure time (hours) via contact until death of each bee (apis mellifera): difenoconazole (dfz); mineral oil + fenpyroximate (mo+fpx); fenpyroximate (fpx); difenoconazole + mineral oil (dfz+mo); mineral oil (mo); control (control). curves indicate the median and 95%, respectively. sociobiology 65(4): 737-743 (october, 2018) special issue 741 the bees at first agitated and later inactive (they did not show any reactions and remained stopped all the time). pesticides appeared to have repellent action, since bees exposed to the contaminated food moved away from the food during a certain time. in contrast, bees consumed the food naturally in the control treatment during the evaluations. discussion the application of difenoconazole, fenpyroximate and mineral oil in isolation had a little effect on bee survival or even in combination to the adjuvant (except difenoconazole via contact). however, secondary effects were evident. the products applied in combination with the adjuvant mineral oil had a faster and more noticeable action, causing agitation, changes in motor coordination, followed by prostration. adjuvants enhance penetration and fixation to improve efficiency by reducing dispersion (mullin et al., 2016). however, this combination has adverse physiological effects on non-target organisms, suggesting a negative point (mesnage et al., 2014; mullin, 2015). in this sense, mesnage et al. (2013) reported that mixing pesticides with adjuvants may alter toxicity of pesticides. generally, adjuvants are considered biologically inert and are not evaluated as agents with toxicological potential for non-target organisms, especially when they are combined to pesticides (ciarlo et al., 2012; mesnage et al., 2013). however, ciarlo et al. (2012) noted that adjuvants affect the olfactory ability of bees, which is essential for foraging. in our study, there was compromise of the search for food by the bees submitted to the treatments with pesticides. in addition, when pesticides were applied in combination, their effects were more pronounced. secondary effects can cause damage to adult bees, such as disorientation, which could compromise their return to the colony (ingram et al., 2015; silva et al., 2016). furthermore, bees could transport the chemical products to colonies, accumulating pesticides in the food, which suggests poisoning the larvae when fed with these contaminated products (johnson et al., 2010; martinello et al., 2017). difenoconazole was more toxic to bees exposed to contact when associated with the adjuvant. mineral oil may have augmented the effect of difenoconazole, since this adjuvant acts in the contact action mode (mapa, 2018), while difenoconazole is a systemic action compound with high residual effect (andrade junior & galbieri, 2014; balardin, 2015). on the other hand, difenoconazole was classified as little toxic for trigona (tetragonula) laeviceps smith by topical route (kinasih et al., 2017), suggesting that this fungicide used without addition of adjuvants is considered nontoxic for bee survival. nevertheless, the authors reported that this fungicide could cause secondary effects. syromyatnikov et al. (2017) found that difenoconazole affected mitochondrial respiration and consequently reduced energy production in flight muscles of bombus terrestris l. fenpyroximate applied in isolation or combination with the adjuvant did not significantly reduce bee survival in any of the contamination media evaluated. fenpyroximate is an acaricide classified as highly dangerous to the environment and belonging to pyrazole, which is a chemical group of neurotoxic action. however, in our study, high toxicity regarding the survival of a. mellifera was not observed. on the other hand, dahlgren et al. (2012) compared toxicity of fenpyroximate on workers and queens of a. mellifera, and reported that this acaricide was more toxic to bee workers than to queen. in our research, we also observed that bees exposed only to fenpyroximate showed changes in motor coordination; however, when exposed to fenpyroximate associated with mineral oil, the insects displayed agitation. li-byarlay et al. (2014) observed that the fenpyroximate could stimulate aggressiveness in bees of a. mellifera. bees could be, multiple times, exposed to the same active ingredient or more than one, which may affect the immune system by developing chronic problems, aggravating secondary effects (whitehorn et al., 2012; morais et al., 2018). exposure of bees to pesticides could become even more harmful, since there is the possibility of repeated, simultaneous and synergistic exposure among and between different chemical groups (luttik et al., 2012). in our study, despite low lethality of difenoconazole, fenpyroximate and mineral oil, except difenoconazole associated with mineral oil via contact, secondary effects were evident mainly when the products were associated with mineral oil. acknowledgements the authors thank coordenadoria de aperfeiçoamento de pessoal de nível superior (capes), conselho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação de amparo à pesquisa do estado da bahia (fapesb) for the financial support. references andrade junior, e.r. & galbieri, r. 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(2017). evaluation of the toxicity of fungicides to flight muscle mitochondria of bumblebee (bombus terrestris l.). pesticide biochemistry and physiology, 135: 41-46. doi: 10.1016/j.pestbp.2016.06.007 whitehorn, p.r., o’connor, s., wackers, f.l., & goulson, d. (2012). neonicotinoid pesticide reduces bumble bee colony growth and queen production. science, 336: 351-2 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4594sociobiology 67(1): 89-93 (march, 2020) introduction the honey bee (apis mellifera l.) (apidae: apini) is an eusocial bee species that is socioeconomically and environmentally important across the globe. apiculture produces highly valuable commercial products and is responsible for the pollination of more than 70% of the main crops grown across the globe (klein et al., 2006). honey bee colonies are affected by several pests, such as the greater wax moth (galleria mellonella l.) (lepidoptera: pyralidae), which can cause significant losses (charrière & imdorf, 1999; kwadha et al., 2017). moth caterpillars, especially between the 4th and 5th instars, can indirectly affect the survival of bees through the destruction of their wax combs, construction abstract honey bees (apis mellifera l.) have great global socioeconomic and environmental importance. however, the greater wax moth (galleria mellonella l.) is a pest that causes serious worldwide damage to honey bee colonies. good beekeeping practices and physical, chemical, or natural methods can be used to control wax moths. the use of natural products is a more sustainable option because of their lower toxicity to the environment and the colony. therefore, we evaluated the efficiency of four natural products for greater wax moth control: neem oil (azadirachta indica), eucalyptus oil (eucalyptus spp.), tobacco extract (nicotiana tabacum), and malagueta pepper extract (capsicum frutescens). we also evaluated their effects on adult bees and on the population growth of colonies. the 4th instar wax moths and adult bees were subjected to in vitro bioassays of different concentrations of the products. the results allowed us to establish a concentration for each product that was safe for the bees and effectively controlled the moth. then, we sprayed them on bee colonies to evaluate their effects on population growth. the neem and eucalyptus oils caused wax moth mortality at low concentrations, but did not affect colony population growth. however, they did have a toxic effect on adult bees. the tobacco and pepper extracts efficiently controlled the moth, but did not cause adult bee mortality or interfered with the population growth of the colonies. therefore, the tobacco and pepper extracts could efficientlycontrol the greater wax moth, without damaging honey bees. sociobiology an international journal on social insects dm telles, gm martineli, mf scaloppi, mp ferreira da luz, sm kadri, ro orsi article history edited by evandro n. silva, uefs, brazil received 05 july 2019 initial acceptance 28 january 2020 final acceptance 09 february 2020 publication date 18 april 2020 keywords beekeeping, pest, natural methods. corresponding author ricardo de oliveira orsi unesp-fmvz department of animal production 3780 universitaria avenue 18610034 botucatu-sp, brazil. e-mail: ricardo.orsi@unesp.br of galleries, and the consumption of stored food (kwadha et al., 2017). a deterioration in the wax combs can cause the colony to abscond and can reduce potential reutilization of the hive (fletcher, 1975). the use of a sanitary management through proper practice by the beekeeper can considerably reduce the incidence of the pest, and is an efficient way of keeping the moth population under control. there are also physical and chemical methods that can be used to control g. mellonella (büyükgüzel, 2009). however, physical and chemical methods are very expensive and may have toxic effects on the bees. furthermore, these methods may contaminate the bee products (bollhalder 1999; rortais et al., 2017). therefore, the use of natural products is a reasonable alternative for wax moth center of education, science and technology in rational beekeeping (nectar), college of veterinary medicine and animal sciences, unesp – são paulo state university, botucatu, brazil research article bees natural products can efficiently control the greater wax moth (lepidoptera: pyralidae), but are harmless to honey bees dm telles, gm martineli, mf scaloppi, mp ferreira da luz, s kadri, ro orsi – natural products can efficiently control the greater wax moth90 control (farghaly et al., 2017) because they generally have a low environmental contamination risk and there are few, if any, harmful residues in the bee products (schmutterer, 1990; el-wakeil, 2013). several oils and plant extracts have been shown to efficiently control wax moths (el-wakeil, 2013). neem oil (azadirachta indica) has been reported to be an important insecticide that is thought to be toxic tomore than 400 insect species due to the presence of a secondary metabolite, azadirachtin, which inhibits larval development (senthilnathan, 2015). eucalyptus oil (eucalyptus spp.) has eucaliptol as its main constituent and can also effectively control several crop pests, including alphitobius diaperinus (pinto júnior et al., 2010) and spodoptera frugiperda (souza et al., 2010). the malagueta pepper (capsicum frutescens) has several natural amides in its fruit composition that have insect repellent properties (maliszewska & tegowska 2012). among them, capsaicin acts as a neurotoxin that causes insect paralysis and death (iorizzi, 2000). tobacco extract (nicotiana tabacum) also has neurotoxic effects on some insect genera (taillebois et al., 2018). the use of natural products for wax moth control can be a more sustainable and safer option than chemical control. therefore, the aimsof this research were to evaluate the effects of neem oil, eucalyptus oil, malagueta pepper extract, and tobacco extract on greater wax moths; their toxicity to adult bees; and their effect on colony population growth. the results showed that at some concentrations, the natural products did not cause harmful effects to the bee colonies or to the adult bees and could efficiently control wax moths. materials and methods experimental site the experiment was conducted at a site located at the coordinates 22°50’ s and 48°25’ w, with an average altitude of 720 m. preparation of the natural products the natural products were neem oil, eucalyptus oil, tobacco extract, and malagueta pepper extract. these products were chosen based on their availability and low cost so that they would be accessible to beekeepers. the neem and eucalyptus oils were commercial brands (codipa® – 98% azadirachtin and bioessência – 100% oil). the tobacco extract was obtained by chopping 500 g of pure tobacco into pieces and diluting them in 2 lof boiling water. after 24 h, the solution was agitated and filtered. then, 200 ml of ethyl alcohol was added and the volume was made up to 2 l. the malagueta pepper extract was prepared by macerating 100 g of pepper in 2 lof water and leaving the mixture to settle for 24 h. then, the mixture was agitated and filtered to obtain the extract (guerra, 1985; santos et al., 1988). effect of the natural products on galleria mellonella larvae the 4th instar galleria mellonella larvae were deprived of food for 24 h before the trials. then, five larvae were placed in petri dishes with filter paper containing1.5 ml of one of the natural products. the products were diluted with distilled water in order to obtain the following concentrations: 0.0%, 0.5%, 1.0%, 2.5%, 5.0%, 10.0%, 20.0%, 40.0%, 80.0%, or 100.0%. the petri dishes were kept atroom temperature in the dark. the larval mortality was observed every 15 minutes for 4 h, which was16 assessments in total. each treatment was replicated five times. effects of the natural products onadult bees the lethal effect of the natural products on adult bees was evaluated by collecting 10 individuals from the central frame of a colony and immediately taking them to the laboratory. then, they were anesthetized with co2 and placedin plastic containers with filter paper. the filterpaper contained 2.0 ml of one of the natural products. each natural product had the following concentrations: 0.0%, 0.5%, 1.0%, 2.5%, 5.0%, 10.0%, 20.0%, 40.0%, 80.0%, or 100.0%. therefore, 400 bees were used in each replicate and there were five replicates. the container also had a feeder that provided the bees with sugar syrup (1:1) ad libitum. mortality was evaluated every 15 minutes over 4 h, what resulted in 16 counts in total. effects of the natural products on colony population development in vitro bioassays and adult bee and caterpillar analyses were used to establish a concentration for each product that was not lethal tothe bees but effectively controlled the moths. a total of 20 standardized colonies were used to evaluate any possible sub-lethal effects of the products on colony population growth. each colony had two central brood frames that were sprayed with 2 ml of one of the products or distilled water as the control. therefore, each product plus the control were applied tofour colonies. the concentration of each product was 0.5% neem oil, 0.5% eucalyptus oil, 0.5% tobacco extract, and 10% malagueta pepper extract. the brood frames contained an open brood and a capped brood area that were evaluated by a modified methodology proposed by al-tikrity et al. (1971). evaluation was done on a weekly basis for 35 days, i.e., six times in total. statistical analysis all statistical analysis was performed in r. measures of central tendency were used to compare the data. the data were normal and homocedastic. therefore, anova and tukey’s test were used to analyze any differences between the averages. the differences were considered statistically significant when p<0.05 (zar 1996). sociobiology 67(1): 89-93 (march, 2020) 91 results wax moth mortality the neem oil, eucalyptus oil, and tobacco extract led to wax moth mortality at all concentrations (p<0.05). however, the pepper extract only caused mortality at concentrations above 10% (p<0.05; fig 1). (0.5%), whereas the pepper extract caused pest mortality only at concentrations above 10%. negative effects were observed in adult bees when the two oil concentrations were higher than 5% (46% and 76% mortality, respectively, for the neem oil and the eucalyptus oil). in contrast, the tobacco and pepper extracts only caused bee mortality when used in concentrations above 80% and 100%, respectively. furthermore, the mortality rate was smaller than that caused by the oils (10–20% mortality). however, none of the extracts harmed colony population growth. the neem and eucalyptus oils, and the tobacco and pepper extracts efficiently controlled the greater wax moth. both the oils and the tobacco extract caused wax moth mortality at relatively low concentrations (0.5%), whereas the pepper extract only caused pest mortality at concentrations above 10%. previous studies have shown that neem oil is an efficient insecticide (abreu, 1998; souza & vendramim, 2000; vinuela, et al., 2000; senthil-nathan, 2015; chaudhary, 2017). it shows selective insect control because different insect groups have different resistance levels towards its mode of action (senthil-nathan, 2015). one of its action mechanisms is to block the synthesis of growth hormones (ecdysteroids), which implies that metamorphosis is interrupted in immature insects (isman, 2006). the higher susceptibility of the greater wax moth compared to the adult bees could be due to the fact that it belongs to the order lepidoptera, which are more susceptible to azadirachtin (zhong et al., 2017) and because it was at an immature growth phase (4th instar larvae) (schumutterer, 1990; moreira et al., 2008). despite the higher susceptibility of the wax moth, neem oil also had negative effects on adult bee survival when used at concentrations above 5% and these effects were dose-dependent. eucalyptus oil has also been shown to be an important natural insecticide and can efficiently control several crop pests (kim et al., 2003; pinto júnior et al., 2010; souza et al., 2010; fhati & shakarami, 2014). the oil, even at low concentrations, led to significant wax moth mortality, but also caused adult bee mortality when applied at concentrations above 5%. fig 1. greater wax moth (galleria mellonella) mortality by different natural products atvariousconcentrations (%). fig 2. adult honey beemortality for the different natural products atvarious concentrations (%). fig 3. population growth of a. mellifera colonies after using the different natural products. adult bee mortality and population development the effects of the products on adult bee mortality were observed and the results showed that eucalyptus and neem oil concentrations above 5% affected bee survival compared to the lower concentrations. however, only the pepper extract at 100% concentration and tobacco extract concentrations above 80% affected bee survival (p>0.05; fig 2). furthermore, applying the plant extracts to the colonies did not affect the open and closed breeding areas compared to the control (p>0.05; fig 3). discussion all four natural products efficiently controlled the greater wax moth. however, the oils and the tobacco extract caused wax moth mortality at relatively low concentrations dm telles, gm martineli, mf scaloppi, mp ferreira da luz, s kadri, ro orsi – natural products can efficiently control the greater wax moth92 the greater susceptibility of the greater wax moth compared to bees may also be due the larvae having a less developed chitin layer compared to adult individuals, which limits larvae physical protection against eucalyptus oil (chapman, 2013). in a similar way to neem oil, the eucalyptus oil led to significant mortality in bees when used at concentrations >5%, and its action was also dose dependent. the lack of harmful effects on colony population growth after the application of neem and eucalyptus oils may have been due to reduced exposure to the product because bee larvae remain individually housed within the alveoli. the 0.5% tobacco extract and the 10% pepper extract efficiently controlled the greater wax moth and did not cause bee mortality, even at high concentrations. they also did not influence colony population growth. the tobacco extract was only harmful to adult bees at doses >80% (safer levels compared to neem and eucalyptus oils). one of the main components of tobacco is nicotine, which is found in species of the genus nicotiana (isman, 2006). high doses of nicotine may affect larval survival and reduce the foraging activity of adult bees (singaravelan et al., 2006) because the compound acts on the nervous system. it competes with acetylcholine in the synapses of the axons and promotes the generation of new nerve impulses that lead to spasmodic contraction, convulsions, and insect death (taillebois et al., 2018). the alkaloid insecticides include nicotinoids and neonicotinoids, whose active ingredients are synthetic nicotine compounds. they are widely used to control several agricultural pests around the world (blacquierre et al., 2012). the pepper extract had significant effects on greater wax moth mortality when applied at concentrations equal to or greater than 10%. although the mortality rates of the pest occurred at higher concentrations of this extract than the other products, its use did not cause toxicity to adult bees, even when applied in high concentrations, and it did not interfere in colony population growth. among the compounds present in the fruits of the genus capsicum, the phenols capsaicin and dihydrocapsaicin, as well as diterpenoids, flavonoids, phenolic compounds, and saponins, have lethal, anti-feed and repellent effects on invertebrates (leete & louden, 1968; maliszewska & tegowska, 2012). the capsaicin lethal dose for apis mellifera bees is greater than 100 μg per individual (flesar et al., 2010). therefore, it is considered relatively non-toxic and is proving to be a safe compound that can be used in bee colonies. despite the low toxicity of the extract towards bees, there was a significant effect on mortality and the consequent control of the wax moth. it is possible that g. mellonella has a greater susceptibility to capsaicin (sarwar, 2015). these results show that the use of tobacco and pepper extracts is an effective alternative for wax moth control because they show reduced toxicity to bees compared to neem and eucalyptus oils and have a greater margin of safety. the compounds were extracted using ethyl alcohol as a solvent and distilled water was usedto further dilutethe mother solution composition, which may also have resulted in a lower toxic effect on moth larvae and adult bees compared to neem and eucalyptus oils. however, the increased toxicity of the neem and eucalyptus products towards bees may have been due to fact that the products were oils. the natural products based on neem, eucalyptus, tobacco, and pepper efficiently controlled the wax moth. however, the neem and eucalyptus oils have a lower margin of safety comparedto the pepper and tobacco extracts, and cause higher mortality among adult bees, even at low concentrations. therefore, extracts of tobacco and pepper are safer alternatives when attempting to control the greater wax moth while maintaining bee survival. acknowledgments this work was supported by fundação de amparo à pesquisa do estado de são paulo – fapesp (process 2013/07827-8). references abreu, j.h. 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(2017). effectiveness of the botanical insecticide azadirachtin against tirathaba rufivena (lepidoptera: pyralidae). florida entomologist, 100: 215-218. doi: 10.13102/sociobiology.v66i2.3448sociobiology 66(2): 306-315 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 trap-nesting bees from protected areas of atlantic forest, southeastern brazil introduction areas with exceptional concentrations of endemic species and high habitat loss (or high level of threats) are often referred as hotspots (myers et al., 2000). in brazil, the atlantic forest is one of these areas (mittermeier et al., 1999). among the various typologies of the atlantic forest, the ombrophylous dense is the most vigorous, heterogeneous and complex formation, characterized by the presence of upper strata with trees above 25 and 30m height, perennial and densely arranged (oliveira-filho & fontes, 2000). such heterogeneous environment has a high diversity of bee species (gonçalves & brandão, 2008). abstract the solitary bees that use preexisting cavities can be captured in trap-nests allowing to collect data on nesting biology and associated organisms. this man-made trap-nest facilitates the understanding of environmental components and landscape composition in the fauna of solitary bees. here, we aimed to increase the knowledge about trapnesting bee species from four protected areas atlantic forest in southeastern brazil and to test how abiotic local environmental components (temperature and rainfall) and forest cover affects the trap-nesting bee fauna. we recorded occupants from 847 nests founded by 17 bee species and seven cleptoparasite bees, associated to their host, summing 24 bee species sampled. the family with highest species richness was megachilidae, and the species with the largest number of founded nests was tetrapedia diversipes klug (apidae). diptera, coleoptera, and hymenoptera parasitized 15.2% of the founded nests. the period of highest nest occupation occurred between november and february, which correspond to the warmest and most humid months in the region. we found significant positive correlation between the number of nests and monthly accumulated rainfall. we verified that boraceia and ilhabela have the best status conservation based on native forest cover and we sampled the highest diversity of species in these areas. we improved the knowledge on trap-nesting bees communities from atlantic forest on new species sampled in this biome with their nesting biology and highlighted that rainfall influences positively the nest founding throughout the year and native forest cover influences diversity of species. sociobiology an international journal on social insects gd cordeiro1,3, s boff1,2, i alves-dos-santos3 article history edited by eduardo almeida, usp, brazil received 10 may 2018 initial acceptance 17 july 2018 final acceptance 14 december 2018 publication date 20 august 2019 keywords apiformes; artificial cavities; abiotic components; forest cover; tetrapedia diversipes. corresponding author guaraci duran cordeiro. universidade de são paulo departamento de ecologia instituto de biociências cidade universitária rua do matão, travessa 14, nº 321 cep: 05508-900, são paulo-sp, brasil. e-mail: guaradc@gmail.com worldwide there are around 20,250 bee species described and solitary bees comprise about 85% of the known species (ascher & pickering, 2018). solitary bees collect food, build and defend nest, and lay eggs without help from other nestmates. after accomplishing all those tasks, the female dies and therefore, there is no generation overlap (michener, 2007). most solitary bees nest in the ground, however many species use preexisting cavities to nest, such as bamboo canes (krombein, 1967; garófalo, 2000). the use of trap-nests to survey bees of an area results in more than a list of species only. such surveys can also provide information about the biology, nest materials, associated organisms, and even the influence of the abiotic 1 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, ribeirão preto, brazil 2 the university of milan, department of food, environmental and nutritional sciences, milan, italy 3 departamento de ecologia, instituto de biociências, universidade de são paulo, são paulo, brazil research article bees sociobiology 66(2): 306-315 (june, 2019) 307 local environmental components such as temperature and rainfall on nest colonization (camillo et al., 1995; tscharntke et al., 1998; jesus & garófalo, 2000; aguiar & garófalo, 2004; thiele, 2005; buschini, 2006; menezes et al., 2012). trapnests have been broadly used for sampling solitary bees since the extensive study by krombein (1967) in north america. after that, this technique has gained an application to agriculture as a means of increasing the solitary bee as crop pollinators (bosch & kemp, 2001; pitts-singer & cane, 2011; sedivy & dorn, 2014). this methodology also allowed to investigate the influence of landscape composition on trapnesting bee fauna (tscharntke et al., 1998; morato & campos 2000; steckel et al., 2014; stangler et al., 2016). however, nothing is known about the effects of landscape composition on this bee fauna in atlantic forest. in tropical countries trap-nesting bee species have been recorded in agroecosystems and forest fragments (camillo et al., 1995; morato & campos, 2000; viana et al., 2001; klein et al., 2002; alves-dos-santos, 2003; thiele, 2005; buschini, 2006; tylianakis et al., 2005; klein et al., 2006; pereira-peixoto et al., 2014; stangler et al., 2016). in atlantic forest, studies on the diversity of trap-nesting bee species and nesting biology were carried out mainly in semideciduous vegetation (garófalo, 2000; 2008; gazola & garófalo, 2009; rocha-filho & garófalo, 2015; oliveira & gonçalves, 2017) or forest urban remnants (alves-dos-santos, 2003; loyola & martins, 2006). therefore, the knowledge about trap-nesting bees from the ombrophilous dense vegetation in the atlantic forest still is scarce (menezes et al., 2012). correlating abiotic local environmental components and landscape conditions with trap-nest colonization and diversity can improve our understanding of how land use and planning decisions impact bees and pollination services (steffan-dewenter & schiele, 2008; pereira-peixoto et al., 2014). therefore, we aimed to increase the knowledge about trapnesting bee species from atlantic forest and to test how abiotic local environmental components such as temperature and rainfall and forest cover affects the trap-nesting bee fauna. for that, we dispose trap-nests in four protected areas of different conservation status along two years. material and methods study areas the study was carried out during two years from march 2007 to february 2009, in four protected areas of atlantic forest biome of ombrophilous dense vegetation in the state of são paulo, southeastern brazil (fig 1). (1) boraceia biological station (23º38’s 45º52’w; 750 to 900 m a.s.l.), with 16,450 ha, is located within the municipality of salesópolis. (2) neblinas park (23°45’s 46°09’w; 700 to 1,100 m a.s.l.), with 2,800 ha, is located within the municipalities of mogi das cruzes and bertioga. (3) ilhabela state park (23º47’s 45º21’w; 0 to 1,378 m a.s.l.), with 27,025 ha, is an island located in the municipality of ilhabela, which has elements of the ombrophilous dense vegetation, sandbanks, and mangrove. (4) serra da cantareira state park (23°22’s 46°36’w; 950 to 1,074 m a.s.l.), with 8,000 ha, is located within the municipalities of são paulo, guarulhos, mairiporã, and franco da rocha. the cantareira state park is a remnant fragment of atlantic rainforest located within the metropolitan region, just 12 km from the center of the são paulo city, with an intense interference of the metropolis. according to an updated köppen-geiger climate classification, the regional climate of the study areas is type cfa, characterized as humid subtropical, without a dry season, with a hot summer and average annual rainfall over 1400 mm (peel et al., 2007). fig 1. main reference cities and study sites in the four protected areas of atlantic forest domain, in the state of são paulo, brazil. bbs: boraceia biological station; np: neblinas park; isp: ilhabela state park; scp: serra da cantareira state park. gd cordeiro, s boff, i alves-dos-santos – trap-nesting bees from atlantic forest308 collection of trap-nests we sampled solitary bees in five trap-nests shelters (fig 2) containing a total of 1,000 trap-nests per study area. our trap-nests consisted of paper tubes (cardboard) housed in wood blocks and bamboo canes housed in plastic bottles. we used three sizes of paper tubes: tp1: 0.6 cm in diameter and 5.8 cm in length, tp2: 0.6 cm in diameter and 8.5 cm in length, and tg: 0.8 cm in diameter and 8.5 cm in length. the bamboo canes varied from 0.5 to 3.0 cm in diameter and from 8 to 25 cm in length. each shelter received a total of 200 trap-nests: 50 paper tubes of each size (tp1, tp2, and tg) and 50 bamboo canes. the use of different types of materials and cavity diameters are recommended because females choose cavities to build their nests based on preferred size and material (krombein, 1967; jesus & garófalo, 2000). we checked the trap-nests monthly, removed those used and filled, and took them to the laboratory, where they remained in room temperature into closed test-tube until the emergence of bees. each removed nest was replaced with an equivalent trap nest. as the individuals emerged, we fixed them with ethyl acetate vapor, and then pinned, labeled, and deposited them in the paulo nogueira neto entomological collection (cepann). landscape structure we measured the landscape spatial structure to assess the status conservation of the study areas. the identification of the land use types and the spatial arrangements of the landscape elements were carried out following metzger (2006). the following land uses were considered: native vegetation, reforestation (eucalyptus plantation), pastures, exposed soil, buildings, roads, and water surface. we measured the landscape structures from each sample unit delimited by the circumference of a circle with 1 km radius around the trapnest shelter as the medium point of the circumference using google earth pro® (version 7.3.1). this mapping is consistent with the dispersion and flight capacity of the most bee species (zurbuchen et al., 2010; benjamin et al., 2014). figures of landscape structure mapping around each sample unit are available in the supplementary material ( figs 1-4). data analyses we calculated correlations between climatic data, monthly average temperature and monthly accumulated rainfall separately with number of nests per month with the spearman coefficient (a = 0.05). we obtained climatic data from each municipality of the study areas at the website of instituto agronômico (http://www.ciiagro.sp.gov.br). to estimate the diversity of the trap-nests bees in the study areas we used the shannon (h’) and simpson (d) (magurran, 2004) indexes. we calculated evenness (ed) based on the simpson and shannon indexes, and pielou evenness (j’). the analyses were made in the program past 1.85 (hammer et al., 2001). results trap-nesting bees and associated organisms bees founded 1,201 nests in the four study areas. a total of 1,693 individual bees emerged from 814 nests emerged, which includes the offspring of hosts and cleptoparasitic bees together. from the remaining nests yielded non-bee parasites or lack of emergence due to complete mortality. the nests were founded by 17 bee species of three families: apidae (6 spp.), colletidae (1 sp.), and megachilidae (10 spp.). in addition to founders, seven species of cleptoparasitic bees of the families apidae (2 spp.) and megachilidae (5 spp.) occupied the nests. tetrapedia diversipes was the most abundant species and it was the only species found at all study areas, summing 88% of the founded nests. ilhabela had the highest richness of species (12 spp.), while cantareira had the lowest (3 spp.). neblinas had the highest values of diversity indexes shannon (h’=1.38) and simpson (d=0.62) diversity, but its species richness (8 spp.) was lower than in boraceia (11 spp.) and ilhabela (12 spp.) (table 1). more than 90% of the nests were founded in paper tubes, otherwise bamboo canes were used by a larger number of species (14 spp.). the bamboo canes with diameters between 0.8 and 1.3 cm were most frequently occupied, but preference varied among species. nest architectures of nesting species are available in supplementary material (supplementary material fig 5). fig 2. nesting shelter providing trap nests with bamboo canes housed in plastic bottle and paper tubes housed in wood blocks. sociobiology 66(2): 306-315 (june, 2019) 309 occupant species boraceia neblinas ilhabela cantareira n. nests n. indiv. ♂ ♀ tp1 tp2 tg bamboo canes apidae centris (h.) tarsata smith 4 1 44 49 119 95 24 1 1 16 31 coelioxoides waltheriae duckea 1* 41* 8* 55 35 20 eufriesea violaceae blanchard 1 1 3 3 1 euglossa anodorhynchi nemésiob 2 2 5 5 2 euglossa truncata rebêlo & moure 1 1 3 1 2 1 mesocheira bicolor fabriciusa 13* 17 7 10 tetrapedia diversipes klug 185 35 425 78 723 1346 819 527 324 371 22 6 tetrapedia sp. 9 9 22 14 8 9 colletidae hylaeus aff. brachyceratomerus moure b 1 1 2 2 1 megachilidae anthodioctes santosi urbanb 1 1 1 1 1 austrostelis iheringi schrottky a 1* 1 1 coelioxys (acrocoelioxys) sp.1a 1* 2 2 coelioxys (acrocoelioxys) sp.2a 1* 1 1 coelioxys (acrocoelioxys) sp.3a 1* 4 4 coelioxys (cyrtocoelioxys) sp.a 5* 5 5 megachile (a.) sussurans haliday 2 4 6 26 18 8 6 megachile (c.) pseudanthidioides moure b 1 1 6 6 1 megachile (chrysosarus) sp. 5 5 17 16 1 1 4 megachile (m.) benigna mitchellb 1 1 1 1 1 megachile (m.) maculata smithb 3 1 4 26 14 12 4 megachile (p.) nudiventris smithb 1 1 2 1 1 1 megachile (p.) bertonii schrottkyb 2 2 10 4 6 2 mielkeanthidium dissimile parizoto & urbanb 4 4 12 6 6 4 saranthidium musciforme schrottkyb 1 2 3 7 6 1 2 1 total of nests 199 46 482 87 814 1693 1061 632 335 379 38 62 total of species 11 8 12 3 shannon index (h’) 0.55 1.38 0.89 0.58 simpson index (d) 0.20 0.62 0.38 0.31 simpson evenness (ed) 0.16 0.49 0.20 0.59 shannon evenness (j’) 0.23 0.66 0.36 0.52 acleptoparasitic bees (see table 2 for respective hosts); bfirst time sampled in trap-nests; *number of attacked nests. table 1. number of nests and individuals of the bee species that used trap nests in four study areas sampled from march 2007 to february 2009. three sizes of paper tubes were used: tp1: 0.6 cm in diameter and 5.8 cm in length, tp2: 0.6 cm in diameter and 8.5 cm in length, and tg: 0.8 cm in diameter and 8.5 cm in length, bamboo canes varied from 0.5 to 3.0 cm in diameter and from 8 to 25 cm in length. the months with the largest number of founded nests were november and december (table 2), which correspond to the warmest and most humid season in southeastern brazil. we did not find a significant correlation between the number of nests and monthly average temperature, (parque das neblinas: r = 0.33, p = 0.10; cantareira: r = 0.35, p = 0.27; ilhabela: r = 0.38, p = 0.14; boraceia: r = 0.35, p = 0.08). otherwise, there were significant positive correlations between the number of nests and monthly accumulated rainfall in boraceia (r = 0.45, p = 0.02), ilhabela (r = 0.49, p = 0.05), and neblinas (r = 0.39, p = 0.05) except cantareira (r = -0.06, p = 0.86) (fig 3). there was complete mortality in 351 nests and in 151 nests the mortality was partial. in nests with partial mortality, we recorded 199 individuals. among the causes of death there were mainly fungal infestation at different development stages and natural enemies at the larval phase. in some nests, we observed dead pre-emergent adults, but we could not determine their cause of death. gd cordeiro, s boff, i alves-dos-santos – trap-nesting bees from atlantic forest310 fig 3. relationship between number of founded nests and monthly accumulated rainfall in four study areas. year 2007/2008/2009 2007/2008 month jan feb mar apr may jun jul aug sep oct nov dec centris (h.) tarsata smith -/1/1 28/17 1/1 eufriesea violaceae blanchard -/0/1 euglossa anodorhynchi nemésio 1/0 1/0 euglossa truncata rebêlo & moure 1/0 tetrapedia diversipes klug -/37/6 -/69/69 12/29/0 10/49 8/36 1/42 0/6 1/0 2/0 24/0 132/16 151/23 tetrapedia sp. 9/0/0 hylaeus aff. brachyceratomerus moure 1/0 anthodioctes santosi urban -/1/0 megachile (a.) sussurans haliday 0/1/0 0/1 1/0 0/2 1/0 megachile (c.) pseudanthidioides moure 0/0/1 megachile (chrysosarus) sp. -/1 0/1 2/0 1/0 megachile (m.) benigna mitchell 0/1 megachile (m.) maculate smith -/0/1 2/0 1/0 megachile (p.) nudiventris smith 0/1 megachile (p.) bertonii schrottky -/0/1 1/0 mielkeanthidium dissimile parizoto & urban -/0/4 saranthidium musciforme schrottky -/0/3 total of nests 46 149 52 60 45 44 6 2 2 31 197 180 total of species 3 7 4 2 2 2 1 2 1 6 5 6 table 2. number of nests founded by bees each month sampled from march 2007 to february 2009. (-) before starting the study. diptera, coleoptera, and hymenoptera (wasps and other bees) parasited 183 (15.2%) nests. among cleptoparasitic bees, coelioxoides waltheriae ducke was the most frequent species and parasitized only nests of t. diversipes. the most frequent non-bee cleptoparasites were two species of anthrax: anthrax hylaios marston and anthrax oedipus fabricius, most emerged from nests of t. diversipes (table 3). we recorded mites roubikia sp. (chaetodactylidae) in 118 nests of t. diversipes but no deaths were observed. sociobiology 66(2): 306-315 (june, 2019) 311 landscape structure in total 30.9 km² were measured in the four study areas. boraceia and ilhabela have the best status conservation considering the native forest cover around trap-nest shelters (table 4). boraceia has vegetation cover composed of 92% native forest without fragmentation; ilhabela has vegetation cover composed of 75% native forest and the remaining is urban areas; neblinas has a heterogeneous vegetation cover, composed of 57% native forest in different stages of succession and 40% eucalyptus plantations between 2 and 50 years old; cantareira has 65% native forest and the remaining is urban areas. boraceia neblinas ilhabela cantareira native forest 12.0 5.0 3.1 3.3 eucalyptus plantation 3.6 roads 0.2 0.2 buildings 0.01 0.01 1.0 1.8 water surface 0.8 0.1 total (km2) 13.0 8.9 4.1 5.1 % eucalyptus plantation 40.5 % roads 1.3 2.0 % buildings 0.1 0.1 25.4 35.0 % water surface 6.1 1.4 % total native forest 92.5 55.9 74.6 65.0 order family parasite species emerging individuals hosts species diptera bombilidae anthrax hylaios marston anthrax oedipus fabricius 29 4 25 13 1 7 tetrapedia diversipes centris tarsata uh tetrapedia diversipes megachile (chrysosarus) sp. uh coleoptera meloidae tetraonyx sexguttatus olivier 2 centris tarsata hymenoptera eulophidae leucospidae melittobia sp. leucospis egaia walker + 7 megachile sussurans megachile maculata leucospis manaica roman 3 uh apidae coelioxoides waltheriae ducke mesocheira bicolor fabricius 28 24 4 13 tetrapedia diversipes uh centris tarsata uh megachilidae austrostelis iheringi schrottky coelioxys (cyrtocoelioxys) sp. coelioxys (acrocoelioxys) sp.1 coelioxys (acrocoelioxys) sp.2 coelioxys (acrocoelioxys) sp.3 1 2 3 2 1 4 uh centris tarsata uh megachile bertonii megachile maculata uh uh: unknown host; +: many individuals in one nest. table 3. parasitic species and their respective host species in trap nests in four study areas from march 2007 to february 2009. table 4. measures (km2 and percentage) of the land-uses on the four study areas. discussion in the two years of sampling we caught 17 trap-nesting bee species, as well as seven cleptoparasite bees, associated to their host, summing a community of 24 bee species in the trap-nests. the number of species captured in the four areas studied ranged from three in cantareira to 12 in ilhabela. the diversity indexes of ilhabela and boraceia, with larger number of species (12 and 11 species, respectively), were not higher because of the lower evenness among these species. in both areas, as well as the other two areas, the dominance of t. diversipes was remarkable. ilhabela, boraceia, and neblinas had species richness higher or similar than other trap-nests studies in atlantic forest fragments of semideciduous vegetation which reached a maximum of 12 species (garófalo, 2000; 2008; gazola & garófalo, 2009; oliveira & gonçalves, 2017), forest urban remnants seven species (alves-dos-santos, 2003; loyola & martins, 2006), and seven species in araucaria forest (buschini, 2006). however, it was lower when compared to studies performed in other biomes, such as 25 species in cerrado (camillo et al., 1995) and 14 species in amazon (morato & campos, 2000). cantareira had lower number of species than aforementioned studies. however, as argued by oliveira and gonçalves (2017) comparisons between species richness obtained with trap-nests from different studies may be problematic because of the differences in sampling methodology, periods, the types, and arrangement of traps in the study areas. gd cordeiro, s boff, i alves-dos-santos – trap-nesting bees from atlantic forest312 we are confident about the degree of representativeness of the trap-nesting bees community in the study areas, because of the temporarily and spatially scale we performed. to obtain the data on this bee guild, we offered the traps for two years in the studied areas, while most studies using trap-nests method are performed in general for one year. this timescale showed some dynamic of the occupancy, as for example the low rate of nesting or activity during the winter, but not a complete stop. the activity of the trap-nesting bee species followed the typical flowering seasonality of tree species in atlantic forest, which occurs in the warmest and most humid months (morellato et al., 2000). consequently, in this period there are more resources to found nests. indeed, we found significant positive correlation between the number of nests and monthly accumulated rainfall in boraceia, ilhabela, and neblinas. the positive relationship between nesting frequency and rainfall was also reported in various studies (camillo et al., 1995; aguiar et al., 2005; thiele, 2005; buschini, 2006; gazola & garófalo, 2009). therefore, we highlighted that abiotic environmental components (rainfall) can influence number of nests of trap-nesting bees also in atlantic forest of ombrophylous dense vegetation. neblinas and cantareira are the least preserved fragments among the areas investigated here these localities have more than 30% of non-native forest areas. this factor probably influenced the lower species richness than wellpreserved areas, such as ilhabela and boraceia, because the values between 35-50% of non-native forest is considered the threshold that affects the richness of bee species and beeplant interaction networks (winfree et al., 2009; moreira et al., 2015; ferreira et al., 2015; saturni et al., 2016). moreover, cantareira is an isolated fragment surrounded by a metropolis, and this is pointed out that with increasing isolation of fragmented habitats the bee species richness declines significantly mainly due to the decrease in food sources and nesting sites (tscharntke et al., 1998; morato & campos, 2000; steckel et al., 2014; stangler et al., 2016). our results reinforce that ongoing fragmentation and deforestation affect diversity of trap-nesting bee communities even in dense vegetation as atlantic forest. associated organisms were abundant in the founded nests. while about 15% of founded nests were parasitized, the main mortality was caused by fungal infestation. parasites, especially parasitoids like melittobia and anthrax, attacked different species. however, parasites are common when cavities are offered in unnatural large numbers (sedivy & dorn, 2014). the natural enemies founded here belonged to the same families and genera as those found in the most studies throughout brazil (jesus & garófalo, 2000; alves-dos-santos, 2003; gazola & garófalo, 2009; aguiar & garófalo, 2004; aguiar et al., 2005; rocha-filho & garófalo, 2015). however, a lack of taxonomic knowledge on these insects has made it difficult to do a comprehensive comparison. the fungal infestation as one of the main causes of immature mortality of trap-nesting bees (antonini et al., 2003; camarotti-de-lima & martins, 2005; stangler et al., 2016), and the present study support this. this is probably due to the period of highest activity of bees coincides with high rainfall in the region. in the study areas, the annual rainfall is over 1,400 mm, one of the highest precipitation rates in south america (peel et al., 2007), consequently the air humidity is high which favors fungal infestation. otherwise, mites roubikia sp. (acari: chaetodactylidae) which emerged from t. diversipes exert a positive effect to control the fungi infestation (cordeiro et al., 2011). within solitary bees species using pre-existing cavities we found the patterns that are expected in general for insect communities: many rare species and few common or abundant species (magurran, 2004). indeed, there were one species dominant in all study sites: t. diversipes, which built more than 80% of the nests in the traps. probably the high occupation rate of this species in trap-nests results from its gregarious behavior and high rate of reuse by individuals emerged in the same site (alves-dos-santos et al., 2002; alves-dossantos, 2003). tetrapedia diversipes is commonly caught in trap-nests in atlantic forest fragments (camilo et al., 1995; garófalo, 2000; 2008; garófalo et al., 2004). by contrast, in other open biomes, centris species are usually those whom nest at highest frequency in trap-nests (camillo et al., 1995; morato & campos, 2000; buschini 2006). to conclude, we improved the knowledge on trapnesting bees and their associated organisms from atlantic forest. in addition, we showed that accumulated rainfall and forest cover play a role in trap-nesting bee community. rainfall influences positively the nest founding throughout the year and native forest cover influences diversity of species. acknowledgements we thank two reviewers for their very helpful suggestions. we appreciated the help in the laboratory and the field of ana luiza de oliveira nascimento, eduardo pinto, heber cavalheiro, mariana taniguchi, paola marchi, paulo césar fernandes, and tiago caetano. we thank carlos alberto garófalo, celso feitosa martins, evandro camillo, and solange augusto for the valuable suggestions for this manuscript. the following specialists identified the bees (apiformes): andré nemésio, daniele parizotto, danúncia urban, eduardo almeida, felipe vivallo, fernando silveira, leo correia da rocha, maria cristina gaglianone, and terry griswold; coleoptera: carlos campaner and simone rosa; diptera: carlos lamas. the são paulo research foundation (fapesp) granted a scholarship (07/51911-2) to gdcordeiro through the biota program (04/15801-0). the laboratory of bees at the university of são paulo provided us with infrastructure. instituto ecofututro, instituto florestal, and the museum of zoology of the university of são paulo for the permition for our research in their areas. sociobiology 66(2): 306-315 (june, 2019) 313 supplementary material h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfilemetadata/3448/0/2492 http://dx.doi.org/10.13102/sociobiology.v66i2.3448.s2492 references aguiar, c.m. & garófalo, c.a. 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(2009). a metaanalysis of bees’responses to anthropogenic disturbance. ecology, 90: 2068-76. doi: 10.1890/08-1245.1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2880sociobiology 65(3): 415-421 (september, 2018) density and spatial distribution of nests of ectatomma ruidum and pheidole fallax (hymenoptera: formicidae), as response to the recovery of coal mine areas introduction the assemblage of ant communities can be structured due to several factors such as vegetation structure and composition, microclimatic conditions, resource provision, altitude, latitude, site heterogeneity, density of nests and intra and interspecific competition (ryti & case, 1984, 1992; santamaria et al., 2009a). such factors generate random, aggregated or uniform distribution patterns (ryti & case, 1992), where each one responds to environmental constraints and/or biological interactions between ant colonies (dillier & wehner, 2004; schatz & lachaud, 2008). the spatial distribution of soil organisms is shown to be generally aggregated (clumped) and has shown to be related with heterogeneous habitat and scarce resources (sousasouto, viana-júnior & nascimento, 2013). in ants, uniform abstract in this study, the spatial pattern of two ant species of different feeding habits, ectatomma ruidum and pheidole fallax (hymenoptera: formicidae) was assessed in rehabilitated areas of “cerrejón” coal mine (colombia). we tested whether there is a relationship between spatial distribution pattern, age rehabilitation and temporal changes. three sites with different ages of rehabilitation (1, 9 and 20 years) and a secondary forest were sampled during dry and rainy seasons. within four plots (6 x 40m) per site, we located, counted and estimated the minimum distance among nests. our results indicated that the number of active nests varied according to sites and sampling season, e. ruidum had the highest density at both seasons, 166 nests ha-1 (forest) and 1333 nests ha-1 (9-y site). the nest density for p. fallax ranged between 125 and 625 nests ha-1 in the forest and the 20-y site, respectively, and at 1-year site ants were absent. our results indicated that the nest distribution strongly depended on the scale of observation. a uniform distribution pattern was also found, mainly at the local scale (plot level), while an aggregated and random distribution was found at the site level. we conclude that ant density responded mostly to seasonal changes (dry versus wet season). sociobiology an international journal on social insects y dominguez-haydar1, b gutierrez-rapalino1, jj jiménez2 article history edited by wesley dátilo, department of eco-ethology, instituto de ecología a.c., mexico received initial acceptance 05 february 2018 final acceptance 07 march 2018 publication date 22 june 2018 publication date 02 october 2018 keywords mine reclamation, ants, spatial distribution, renewal nest index, morisita index, nearestneighbor index. corresponding author yamileth dominguez-haydar facultad de ciencias básicas universidad del atlántico km 7 antigua vía puerto colombia barranquilla, colombia. e-mail: yamilethdominguez@mail.uniatlantico.edu.co distribution has been extensively referenced and corresponds to either intraand interspecific competition (ryti & case, 1992; redolfi et al., 2005). random patterns were related to homogeneous conditions, allowing organisms to be located in any available site and, thus, reducing intraspecific competition (soares & schoereder, 2001). in restored sites, research has focused mainly on ant composition and diversity, leaving aside the distribution patterns of the species, that can be related with microclimate requirements, successional processes, habitat stability and environmental constraints (holec, frouz & pokorný, 2006). in this study, we selected two species of ants, ectatomma ruidum (roger, 1860) and pheidole fallax (mayr, 1870) in order to analyze changes in pattern distributions in rehabilitated areas of cerrejón coal mine (la guajira, colombia). dominguezhaydar and armbrecht (2011) reported that these species 1 programa de biología, facultad de ciencias básicas, universidad del atlántico, barranquilla, colombia 2 araid researcher, department of biodiversity conservation and ecosystem restoration, pyrenean institute of ecology (ipe-csic) research article ants y dominguez-haydar, b gutierrez-rapalino, jj jiménez – nest distribution of e. ruidum and p. fallax in a coal mine416 were the most abundant in the sites with more rehabilitation time and in the forest without mine intervention. the forest surronding the mine is sub-xerophitic, plant species are caducipholy and loose the leaves during dry season. the season is determined by rainfall, two distinct rainfall peaks occur, a light one in april june, and a heavier one in october – december. this seasonality allows changes in resource availability (santos, dáttilo & presley, 2014), for this reason, it might change the distribution patterns of ants, which is relevant in restored sites. we expected that nest density responds to age of rehabilitation. therefore, nest density in areas with more time of rehabilitation will be more similar to forest than areas at the begining of rehabilitation. besides, areas with longer rehabilitation time as well as the forest, are more likely than expected by chance (ebc) to show other spatial pattern rather than random distribution. in the present study, we aimed at i) describing the spatial distribution of nests in the restored sites, ii) testing whether there is a relationship between the nest distribution, seasonality and the time after restoration. materials and methods study site the study was carried out in the cerrejón coal mine at la guajira, in northern colombia (11°3’n, 72°44’w 11°8’n, 72°37’w) between 200 and 240 m.a.s.l. (above sea level). the natural ecosystem surrounding the mine is a seasonal dry forest, with vegetation corresponding to subxerophytic and dry forest biotic zones. precipitation is mainly bimodal (april june and october – december) with an annual mean of 800 mm, evapotranspiration from 1000 to 1500 mm (gualdrón, 2011), and mean annual temperature of 27.5 ºc (isohyperthermy). during the sampling period (2014) a decrease in precipitation (407mm) and an increase in evaporation (2424 mm) was observed when compared with the historical trend of the last 10 years (cerrejón 2014). the rehabilitation protocol used in the mine consists of three stages: land geomorphological adequacy, topsoil stabilization with grasses (cenchrus ciliaris l) and revegetation with native species (domínguez-haydar & armbrecht, 2011; gualdrón, 2011; domínguez-haydar et al., 2018). due to the high climate variability at the study zone, we carried out two surveys, one in the dry season (march 2014) and the other one at the end of the rainy season (december 2014). we selected three areas with different times since rehabilitation (i.e., one, nine and twenty years old), and another area with a secondary forest not-intervened by mining. the forest is a compensation area, i.e., it is excluded for future mine use. the tree layer comprised species such as aspidosperma polyneuron müll.arg. and hura crepitans l. the shrub layer was comprised of cordia alba (jacq.) roem. and schult., machaerium sp. and mimosa arenosa; some exotic african grasses of the genera andropogon, brachiaria and panicum may occasionally be present (gualdrón, 2011). 1-y site have bare soil, dispersed patches of spontaneous grasses. without revegetation actions and without canopy cover. 9-y site, have high c. ciliaris and tree densities with large canopies (4 – 5 m) m. arenosa, a. macracantha, a. tortuosa, caesalpinia ebano h. karst. 20-y site have a mosaic of bare soil, some scattered patches of grasses and litter. m. arenosa, a. macracantha, caesalpinia ébano, p. juliflora, platymiscium pinnatum (jacq) dugand. (domínguez et al., 2018). species description ectatomma ruidum is a hunter ant with omnivorous feeding habits, and individual foraging on litter. the species is widely distributed in central and south america. they can be found in a wide range of sites, including altered sites by humans (fernández, 1991). pheidole fallax workers are generalist foragers (roux et al., 2013) with a diet that includes seeds (lôbo et al., 2011; gutierrez-rapalino & domínguez-haydar, 2017) and insects (jaffe, 1990). have individual foraging, each worker retrieving a small particle of food, and mass recruitment (many individuals cooperating) (itzkowitz & haley, 1983). this ants have neotropical distribution, i.e., the greater antilles, central america, colombia, venezuela and brazil (leal, wirth & tabarelli, 2007). spatial patterns and nests density obtaining independent areas of the same age of rehabilitation is a typical issue within mining zones. indeed, pseudoreplication is a common constraint researchers encounter as it is normally present within plots of different years (davies & gray, 2015). however, the large size of the study area (i.e. 20-y site have 70 ha) made it possible to separate plots for ensuring greater sampling independence. hence, within each area we established four plots (6 x 40m) with a minimal distance of 300 m from each other. in each plot we placed 27 tuna baits (2 g) in a 3 x 9 m grid on the soil to localize the nests. the ant workers that approached to the baits were followed to identify the nest entrances, which were pointed with flags. the total number of nests were counted and the distance to the closest nests was measured. data analysis nest density: based on the site type and the sampling season, the nest density of p. fallax and e. ruidum were compared using the non-parametric friedman test (with replicated blocked data and chi-square statistics), to compare sampling season, the sites were blocked and for analyzing differences among sites, the sampling season was blocked in the analysis (srivastava & jefferies, 1996; hollander et al., 2014). statistical analyses were performed in r (r development core team 2014). dispersion pattern: in order to determine how site recovery could affect spatial distribution of ant nests, we performed the clark and evans (1954) and morisita id (1959) sociobiology 65(3): 415-421 (september, 2018) 417 aggregation indices in the plots of recovered sites of 9 and 20 years old, as well as within the forest for both ant species. the drawback of using aggregation indices is that the actual spatial location of the nests is not not considered. thus, it does not provide additional information on the spatial distribution, other than the sampling unit size (rossi, lavelle & tondoh, 1995). plot level dispersion was tested with clark and evans (1954) nearest-neighbor index (r), this is defined as: , , where ro is the mean of the observed distances between nearest nest and re is the mean expected, r ranges from 0 to 2.15. the r index value is lower than 1 when the nests exhibit aggregated distribution, it approaches 1 when the distribution of the nests is random, and it is greater than 1 when the distribution is uniform. we evaluated the statistical significance of index values (r) using the standard normal variate z (clark & evans, 1954), , where se is the standard error of re, and , where n = number of nest and d is nest density. morisita id (1959) was used to determine site level dispersion pattern, this index is independent on the sample mean and the total number of individuals in samples, and it is given by: , where n is the number of sampling units (plots); x = number of nest found in the for the ith sampling unit. id is equals 1 for random distributions, is less than 1 for regular distributions and greater than 1 for clumped distributions. departure from randomness can be tested by z = (id – 1) / (2/nm 2) ½, where m corresponds to the mean of nest, random spatial distribution will be in case of 1.96 ≥ z ≥ -1.96. nest permanence: in order to establish the degree of temporal variation of the number of nests per area, we also calculated both a permanence and a renewal nest index according to cerdá et al. (2002) and redolfi et al. (2004). these indices were estimated counting the total number of nests in each sampling; including those that persisted from the previous sampling period. results nest density the number of nests of e. ruidum and p. fallax varied according to sites and sampling season. we found a total of 294 nests, with a range from 0 to 36 nests per plot. e. ruidum had the highest density in rainy season, with 1416.7 nests ha-1 in 9-y site, while p. fallax in dry season 583.3 nests ha-1 in 20-y site. we did not find both species e. ruidum and p. fallax in the 1-year site, neither in dry or wet seasons. in the 9-year site, the species were absent just in the dry season (fig 1). in general, we observed contrasting differences in the nest density of p. fallax and e. ruidum. the density of e. ruidum nests increased during rainy season, mainly in 9-y site (friedman test, c2 = 7.56, df = 1, p-value = 0.01). however, we did not find any significant differences among nest density of 9-y, 20-y and the forest (friedman test, c2 = 1.31, df = 2, p-value p > 0.1) (fig 1). p. fallax showed significant differences among sites (friedman test, c2 = 15.5 df = 2, p-value < 0.01), and 20-y site exhibited the highest density of nests, only two nests were found in the forest. for p. fallax, there were non-significant differences among seasons (friedman test, c2 = 1.16, df = 1, not significant). fig 1. box-plot of density of nest (log 10) of e. ruidum and p. fallax in three rehabilitated sites and a forest in “el cerrejon” mine. black = dry season, gray = wet season. y dominguez-haydar, b gutierrez-rapalino, jj jiménez – nest distribution of e. ruidum and p. fallax in a coal mine418 distribution patterns and temporal permanence of nests ants exhibited two types of spatial patterns. at the local scale, clark and evans (1954) nearest-neighbor index (r) showed a regular distribution for p. fallax in all sites, except in a plot in forest. while that e. ruidum showed mostly random distributions during wet season, however throughout the dry season the distribution was regular (table 1). in contrast, to site level morisita’s index showed that p. fallax nest were randomly distributed in both seasons and sites while a random distribution was found in the forest for e. ruidum only (table 2). climatic periods sites morisita (id) distribution z ectatomma ruidum dry season 1-y 0.000 9-y 0.000 20-y 0.000 forest 1.778 aggregated 5.224 * wet season 1-y 0.000 9-y 1.009 random 0.369 20-y 1.010 random 0.145 forest 2.635 aggregated 20.809 * pheidole fallax dry season 1-y 0.000 9-y 0.000 20-y 1.055 random 0.664 forest 2.000 random 1.414 wet season 1-y 0.000 9-y 1.333 random 1.061 20-y 1.022 random 0.270 forest * indicate significant differences (p < 0.01) table 2. spatial distribution of nests belonging to e. ruidum (e) and p. fallax,(p) at site scale, according to , morisita’s index (id). (z): test of significance. table 3. temporal permanence of e. ruidum y p. fallax nests in the study site. tn = total nests presented in the first sampling; (rn) remaining nest in second sample 1, nn: new nests that appear in sample 2, rmi = remaining index, rwi = renewal index. sites specie tn rn nn rmi rwi 9-y e. ruidum 114 0 114 0 100 p. fallax 9 0 9 0 100 20-y e. ruidum 40 1 38 2,5 95 p. fallax 35 5 30 14,3 85,7 forest e. ruidum 36 1 35 2,8 97 p. fallax 2 1 1 50 50 sites plot nearest neighbor n r z p-value distribution dry season 1-y 1-4 9-y 1-4 20-y 1 p-p 14 2.1 8.1 <0.01 regular 2 p-p 6 2 4.7 <0.01 regular 3 p-p 6 2 4.6 <0.01 regular 4 p-p 6 1.8 3.75 <0.01 regular forest 1 e-e 12 1.9 6.2 <0.01 regular 2 e-e 5 1.5 2.1 <0.05 regular 3 p-p 3 1 0.2 n.s random wet season 1-y 1-4 9-y 1 e-e 14 1.3 2.28 <0.01 regular 2 e-e 16 1.1 0.76 n.s random p-p 3 1.4 3.4 <0.01 regular 3 e-e 22 1.1 0.73 n.s random 4 e-e 22 0.9 -0.24 n.s random 20-y 1 p-p 10 1.5 2.37 <0.01 regular e-e 5 0.7 -0.95 n.s random 2 p-p 5 1.8 4.3 <0.01 regular e-e 6 0.8 -1 n.s random 3 p-p 5 1.8 3.4 <0.01 regular e-e 6 0.6 -1.8 n.s aggregated 4 p-p 3 2 5.9 <0.01 regular e-e 7 0.8 -0.5 n.s random forest 3 e-e 18 0.21 -2.5 <0.05 aggregated 4 e-e 3 1.2 1.9 <0.05 regular table 1. spatial distribution of nest for e. ruidum (e) and p. fallax, (p) at local scale. n: number of nest, r: clark & evans nearestneighbour index, z: test of significance. there is a high nest renewal for both species between the dry and rainy seasons, reaching values up to 85% and 100% (table 3). this index was lower in the forest and in 20-y site, and highest at 9-y site for both species. discussion overall, the density and permanence of p. fallax and e. ruidum nests responded positively with rehabilitation age in the cerrejon mine. the presence of these species has been related to areas with high vegetation cover, advanced rehabilitation stages and forest edges (torres, 1984a; domínguez-haydar & sociobiology 65(3): 415-421 (september, 2018) 419 armbrecht, 2011). additionally, for the first time, we described the density of p. fallax nests for mining areas. the density of nests found for e. ruidum was consistent with that found by santamaría et al. (2009a) in a previous study in other areas of the same mine, although, we detected lower nest density in all areas. the density was higher in areas with greater canopy cover and absent in uncovered areas. this also coincided with the absence of this specie in the area under rehabilitation for 1-y. this could be related with climatic differences between both studies. the sampling season had lower precipitation (cerrejón, 2014) that coincided with dry years of enso climatic perturbation (el niño southern oscillation effect). this also would explain the low abundance of p. fallax in contrast with a previous study (domínguez-haydar & armbrecht, 2011) where this ant is abundant in forests. the permanence of ant nests responded to seasonality, e. ruidum exhibited the major variability. highest density was found during rainy season, which coincides with the reproductive period of ants, and the establishment of new nests (levings & franks, 1982). from the results of the renewal index, a greater stability is deduced in the forest and in the 20-y site, unlike the 9-y site showed high variability due to absent of nests during dry season. the native vegetation at “el cerrejón” mine is principally deciduous, which allows direct light incidence able to increase the soil temperature for up to 45 oc during the dry season (domínguez-haydar, pers. obs.). thus, we noticed a reduction in the ants activities in those sites with less rehabilitation age and less canopy cover (domínguez-haydar & armbrecht, 2011). further, during the dry season resources such as seeds, invertebrates and extrafloral nectaries, which are important items in the diet of e. ruidum were diminished (passsera, lachaud & gomel, 1993; santamaría, armbrecht & lachaud, 2009b; santamaría, domínguez-haydar & armbrecht, 2009; gutierrez-rapalino & domínguez-haydar, 2017). p. fallax accumulate more seeds during dry season that e. ruidum (gutierrez-rapalino & domínguez-haydar, 2017). it is possible that this behavior helps to guarantee enough resources to p. fallax during dry season, and in consequence, greater permanence. p. fallax and e. ruidum showed different spatial patterns that varied among sites and scale. a comparison between seasons was not possible because the absent of e. ruidum in 9-y and 20-y during dry season. a regular pattern was obtained mostly at local scale and for p. fallax. a regular distribution pattern is the most common pattern in tropical forests, and is assumed in order to avoid interactions between ant colonies such as intraand inter-specific competition for space, resources and foraging areas (ryti & case, 1986, 1992; redolfi et al., 2005). soldiers of p. fallax have been reported in combats (torres, 1984b), therefore, competition may play an important role at local scale spatial distribution, as demonstrated for other soil invertebrates (jiménez, decaëns & rossi, 2006). however, for morisita analyses, random (in rehabilitated areas) and aggregated (in the forest) patterns were found when both species were included in the same analysis, it showed no evidence of interspecific competition. this is a contrasting result, considering that previous studies have documented ecological competition between p. fallax and other species of genus ectatomma as e. quadridens smith (1858) (fowler, 1994). e. quadridens obstructed the nests of p. fallax with sand grains, generating a uniform pattern in p. fallax. indicating that larger scales, other constrains different from biological competition are responsible of the spatial pattern observed. at local scale for e. ruidum, spatial patterns varied according to sites, with regular pattern observed mainly in the forest, while a random distribution prevailed in 9and 20-y sites. in the latter, this pattern was consistent for both scales and methods (nearest neighbor and id indices); this random distribution may be related with homogenous and suitable environmental conditions and diminishing of limiting factors as competition (caldeira et al., 2005; lőrinczi, 2011). the increase in nest density is a response to the suitable conditions present in the restored sites. as demonstrated in other studies, e. ruidum is a successful species in forested areas (santamaría, domínguez-haydar & armbrecht, 2009). on the other hand, the high renewal nest found in these sites, could prevent the development of stable competitive relations (lőrinczi, 2011) inducing aggregated or random patterns. however, this result differs from that reported in a previous study where a regular distribution was found at local scale (santamaría, domínguezhaydar & armbrecht, 2009). it is possible that differences in climatic conditions led to the high variability observed in spatial distribution. in contrast, with the random distribution observed in the 20-y and 9-y sites, an aggregated distribution was found in the forest which is related to higher habitat heterogeneity (fowler, 1994). therefore, we hypothesize that changes from random to aggregated patterns between the restored sites and the forest might be either, a response to more complex environmental conditions or an effect of other abiotic variables (as soil temperature and humidity) not analyzed in this study. due to the absence of both species in 1-y site, we posit that p. fallax and e. ruidum are indicators of intermediate and advances stage rehabilitation. however, the ant nests density responded to seasonal changes and there was not an increase with the rehabilitation time. the spatial pattern distributions changed for p. fallax in function of scale, whereas in e. ruidum existed an aggregated trend in the forest in both scales. however, we expected a shift from randomness to aggregation in response to more heterogeneous and complex environments, that shows that after 20 years were not yet those of the forest. the use of both indices (morisita and clark and evans) demonstrated this aggregation trend, in particular for e. ruidum. high nest removal led to a redistribution of the nest locations that contributes to seed dispersion in both seasons. in particular, p. y dominguez-haydar, b gutierrez-rapalino, jj jiménez – nest distribution of e. ruidum and p. fallax in a coal mine420 fallax accumulate viable seeds to germinate in nest and refuse piles (gutierrez-rapalino & domínguez-haydar, 2017). the emergence of new nests and the abandonment of the oldest also contributes with changes in chemical and physical (i.e. bioturbation) soil properties. soil heterogeneity is a desired attribute for ecological restoration (lane & bassirirad 2005, larkin, vivian-smith & zedler, 2006). acknowledgements this study was supported by a colciencias grant (colombia), (code: 1116-569-34827.) and “vicerrectoría de investigaciones extensión y proyección social” of universidad del atlántico. particular thanks are extended to “departamento de seguridad y gestión ambiental” of “cerrejón” for logistic assistance, to yair barros for field assistance, to dr. inge armbrecht for comments on an earlier draft of the manuscript and soraya villalobos for revision of the english language. authors contribution ydh, bgr, jjj conceived and designed the research; 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(1984b). niches and coexistence of ant communities in puerto rico: repeated patterns, biotropica. 16: 284-295. doi: 10.2307/2387937. doi: 10.13102/sociobiology.v61i2.136-144sociobiology 61(2): 136-144 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 response of ants to the leafhopper dalbulus quinquenotatus delong & nault (hemiptera: cicadellidae) and extrafloral nectaries following fire g. moya-raygoza¹, k.j. larsen² introduction ants (hymenoptera: formicidae) often live in mutualistic relationships with trophobiont insects that excrete honeydew, or with plants bearing extrafloral nectaries (efns) that produce nectar (hölldobler & wilson, 1990). some species of aphids, whiteflies, scale insects, mealybugs, treehoppers, and leafhoppers (hemiptera) live in facultative or obligatory mutualistic relationships with ants (way, 1963; buckley, 1987; blüthgen et al., 2006; gibb & cunningham, 2009; fagundes et al., 2013). in these associations, the insect provides honeydew, a sugary excretion of carbohydrates, amino acids, and water for the ants, whereas ants protect the hemipterans from natural enemies (delabie, 2001; heil & mckey, 2003; zhang et al., 2012; zhang et al., 2013). plants in over one hundred families bear efns that produce secretions rich in sugars, abstract previous investigations of mutualistic associations between ants and plants bearing extrafloral nectaries (efns) or between ants and trophobiont leafhoppers have studied these relationships separately, but nothing is known on how ant abundance responds to these two food resources occurring in the same habitat when that habitat is disturbed by fire. the objectives of this study are to document ant abundance with the trophobiont five-spotted gamagrass leafhopper, dalbulus quinquenotatus delong & nault, and with efns on trees of acacia pennatula (schlecht & cham.) benth. (fabaceae) that occur in the same habitat, and how ant abundance in both of these mutualisms is affected after disturbance by fire. this study was performed at several sites in central mexico where the perennial gamagrass tripsacum dactyloides l. (gramminae) and a. pennatula both occur. more ants were collected in association with the leafhopper d. quinquenotatus than with efns of a. pennatula. at sites where dry season fire occurred, new green leaves were produced by both t. dactyloides and a. pennatula after the burn. on these new leaves after fire, significantly more ants tended d. quinquenotatus leafhoppers on t. dactyloides than visited efns on a. pennatula. in burned sites the ants anoplolepis gracilipes smith, brachymyrmex obscurior forel and pheidole sp. live in association with the leafhoppers, whereas efns on a. pennatula were associated with the ants a. gracilipes, b. obscurior, camponotus sp., crematogaster sp. and solenopsis sp. sociobiology an international journal on social insects 1 universidad de guadalajara, cucba, jalisco, mexico. 2 department of biology, luther college, decorah, usa. article history edited by gilberto m m santos, uefs, brazil received 16 march 2014 initial acceptance 24 april 2014 final acceptance 16 may 2014 keywords acacia pennatula, five-spotted gamagrass leafhopper, tripsacum dactyloides, mutualism corresponding author gustavo moya-raygoza, ph.d. departamento de botánica y zoología cucba, universidad de guadalajara km 15.5 carretera guadalajara-nogales las agujas, zapopan, c.p. 45110 apdo. postal 139, jalisco, méxico e-mail: moyaraygoza@gmail.com amino acids, and lipids that attract ants, and in return these ants protect those plants from herbivores (gonzález-teuber & heil, 2009; byk & del-claro, 2011; marazzi et al., 2013; weber & keeler, 2013). ants are attracted to high quality sugar resources as food (heil & mckey, 2003). previous studies have shown that when both honeydew and extrafloral nectar are offered to ants, ants are more abundant at the honeydew rather than at exudates of efns (fiala, 1990; rashbrook et al., 1992; delclaro & oliveira, 1993; blüthgen et al., 2000; katayama et al., 2013). ants were more abundant tending the hemipterans, particularly when greater numbers of hemipterans are present because of the larger quantities of honeydew produced (katayama & suzuki, 2010). blüthgen et al. (2000) found greater numbers of ants at honeydew resources as opposed to efn resources because honeydew is apparently a higher quality research article ants sociobiology 61(2): 136-144 (june, 2014) 137 food resource, rich in amino acids. moreover, katayama and suzuki (2003) demonstrated that if an aphid colony increases in size, ants stop using efns and strengthen their mutualistic association with aphids. fire affects the growth of plants because some perennial species such as grasses and plants bearing efns quickly re-grow after disturbance occurs. new leaves formed after the plants burn are ready to be colonized directly or indirectly by ants, often attracted to food resources such as the honeydew produced by leafhoppers that feed on young grasses (moyaraygoza, 1995) or from nectar produced by efns (alvessilva & del-claro, 2013). ants respond to burned plants with efns or hemipterans in the same way. the abundance of ants increased on the shrub banisteriopsis campestris (a. juss.) which bears efns after fire, mainly because of concentrated extrafloral nectar (alves-silva & del claro, 2013). alvessilva (2011) and koptur et al. (2010) also found a richer ant community guarding plants from herbivory after fire because of the production of extrafloral nectar. similarly, higher numbers of ants were found tending the honeydew-producing fivespotted gamagrass leafhopper, dalbulus quinquenotatus delong & nault, after its host plant, the perennial gamagrass tripsacum dactyloides l. (gramminae), was burned (moyaraygoza, 1995). mutualisms between ants and efns-bearing plants and ants and trophobiont hemipterans have been investigated separately after disturbance by fire, but little is known how ant abundance responds to these two food resources when present in the same habitat. this study was performed in central mexico, where the perennial gamagrass t. dactyloides hosts d. quinquenotatus leafhoppers and trees of acacia pennatula (schlecht & cham.) benth. (fabaceae) with efns occur together in the same habitats (fig. 1a). these sites often are accidentally burned, and the fire often kills or drives away insects living on those plants. dalbulus quinquenotatus lives on the basal leaves of t. dactyloides in an obligatory mutualism with tending ants (larsen et al., 1991). ants tending d. quinquenotatus receive honeydew and protect this leafhopper from natural enemies (moya-raygoza & nault, 2000). in contrast, a. pennatula have efns and live in a mutualistic relationship with ants (moya-raygoza, 2005), providing nectar for the ants in return for protection from herbivory. fire is an important abiotic factor in mutualisms because it affects plant re-growth and the abundance of ants that depend on exudates produced indirectly by trophobiont insects and directly by efns. when fire consumes the foliage of both t. dactyloides and a. pennatula, the mutualisms involving ants with both species are temporarily disrupted. however, only a few days after being burned, new leaves of both plant species begin to re-grow (fig. 1b) and are soon colonized by d. quinquenotatus and ants in the case of t. dactyloides, or by ants visiting efns in the case of a. pennatula. measuring the total abundance of ants collecting honeydew from d. quinquenotatus and visiting efns resources before and after the host plants are burned helps us understand the ecological importance of mutualisms that can be strong driving forces for community organization (wimp & whitham, 2001). the objectives of this study are to document ant abundance with d. quinquenotatus leafhoppers and efns in the same habitat, and how ant abundance in both of these mutualisms is affected after disturbance to their habitat by fire. materials and methods study system nine field sites containing both t. dactyloides and a. pennatula were chosen for this study. each site had both species of plant present and covered an area of 0.05-0.25 ha. all sites were in the state of jalisco in central mexico. the sites were: 1) el arenal: 1,501 m elev, 20°46.032´n, 103°40.766´w; 2) los chorros: 1,371 m elev, 20°41.211´n, 103°41. 558´w; 3) san isidro: 1,266 m elev, 20°49.014´n, 103°20. 262´w; 4) agua caliente: 1,385 m elev, 20°25.770´n, 103°41.485´w; 5) cocula: 1,273 m elev, 20°25.595´n, 103°44.601´w; 6) san agustin: 1,638 m elev, 20°30.682´n, 103°28.796´w; 7) la mimila: 1,649 m elev, 20°44.411´n, 103°37.686´w; 8) el molino: 1,608 m elev, 20°23.938´n, 103°32.760´w; and 9) zapopan: 1,631 m elev, 20°44.283´n, 103°30.805´w (fig. 1c). the closest sites were 5.45 km apart (agua caliente and cocula) while the most distant sites (san isidro and cocula) were 60.44 km apart. all sites had similar habitat characteristics and belong to pine-oak ecosystem (rodríguez-trejo & myers, 2010). plants of both species live on steep slopes or beside roadways and grow on limestone soils (wilkes, 1972). the sites had similar vegetation consisting of a plant community containing t. dactyloides interspersed with a. pennatula trees and few other plants such lysilona sp. each t. dactyloides population was composed of scattered clumps consisting of clusters of stems. tripsacum dactyloides can use rhizomes to spread across the landscape and does not possess extrafloral nectaries. moreover, ants are present on t. dactyloides only when the plants are hosts for d. quinquenotatus leafhoppers as compared with plants without d. quinquenotatus (larsen, et al. 1991). all sites were sampled to determine the numbers of ants when leafhoppers and efns were available. acacia pennatula has actively secreting extrafloral nectaries on young leaves primarily from april to june (mcvaugh, 1987; moya-raygoza, 2005), whereas leafhoppers are present on t. dactyloides primarily during the wet season from june to september (moyaraygoza, 1995) when these habitats are not burned. we observed that when the habitats were burned, both plant species started to produce new green leaves within several days, and this altered the food resources available for visiting ants. fires generally occur from march to may towards the end of each dry season. the dry season in jalisco generally occurs from october to may and is characterized by lower rainfall, lower moya-raygoza and larsen ants, leafhoppers and efns after fire138 temperatures and shorter days as compared with the wet season which typically lasts from june to september (mosinoaleman & garcia, 1974). after burning, both honeydew and efn nectar food resources for ants are found in may and june within the same plant community. the highest nectar secretion rates have been documented from efns on young leaves of damaged plants (heil et al., 2004), while high numbers of d. quinquenotatus leafhoppers have been found on t. dactyloides after fire (moya-raygoza, 1995). no data were collected between october and april because ants do not visit either of these food resources during that time. the wet season begins in june, and no fires occur once the rains begin to fall. sampling we confirmed the presence of ants associated with efns of a. pennatula and d. quinquenotatus at each site. once these fires took place, we sampled ants on burned and unburned sites. we selected a. pennatula trees at each site and neighboring clumps of t. dactyloides. ten terminal branches on each selected a. pennatula tree and one basal leaf from each of ten different t. dactyloides clumps were randomly selected. terminal branches of a. pennatula were selected because the highest concentration of efns occurs on these branches, whereas basal leaves of t. dactyloides were selected because this is where the highest numbers of d. quinquenotatus are found. we collected all nymphs and adults of d. quinquenotatus leafhoppers and all tending ants from the basal 10 cm of each selected t. dactyloides stem. all efns were counted and ants collected from the terminal 10 cm of each selected a. pennatula branch. therefore ant abundance at each resource was quantified on one stem or branch for each of 10 separated plants of t. dactyloides and a. pennatula by site. we selected the same 10 cm surface on both plant species to have approximately the same area of food resource available for the ants. sampling at all sites was performed between 09:00 and 14:00 h, one site per day during the last week of may 2007, first week of june 2012, and the second week of september 2012. the arenal and los chorros sites were burned in may 2007, while the zapopan and los chorros sites were burned in june 2012. dalbulus quinquenotatus, efns and ants were sampled approximately one month after each fire. ants were sampled at these times because both extrafloral nectar produced by a. pennatula and honeydew produced by d. quinquenotatus was present. all collected insects were stored in 70% ethanol and returned to the lab for identification. analysis of deviance, using r.3.1.0 for windows (r project), was performed to evaluate the interaction (resource for ants, honeydew-extrafloral nectar × disturbance, fire-without fire) on the number of ants. this comparison included the ant abundance obtained on the three sampling dates. furthermore, the total number of ants tending d. quinquenotatus on t. dactyloides was compared vs the total number of ants on a. pennatula bearing efns with a wilcoxon test using spss 12 for windows. therefore a comparison of ant abundance at leafhoppers vs efns was conducted when combining both burned and unburned resources in the three sample dates. average and standard error were determined for the number of d. quinquenotatus nymphs and adults, tending ants, and efns for each burned and unburned site. fig 1. tritrophic interaction trophobiont five-spotted gamagrass leafhopper-ants-acacia pennatula. a) hillside in jalisco, mexico late in the dry season covered with t. dactyloides hosting d. quinquenotatus leafhoppers, interspersed with trees of a. pennatula bearing efns in unburned site. b) young green leaves growing on a. pennatula (left) and t. dactyloides (right) several days after being burned by fire. c) location of field sites containing both t. dactyloides and a. pennatula from the state of jalisco in central mexico. a b c sociobiology 61(2): 136-144 (june, 2014) 139 results ant species collected from burned t. dactyloides associated with the leafhopper d. quinquenotatus were anoplolepis gracilipes smith, pheidole sp., and brachymyrmex obscurior forel. ants found on unburned t. dactyloides tending d. quinquenotatus included a. gracilipes and b. obscurior. greater ant species richness was associated with a. pennatula. anoplolepis gracilipes, b. obscurior, camponotus sp., crematogaste sp. and solenopsis sp. were found at efns when a. pennatula was burned. ant taxa found visiting the efns of unburned a. pennatula included a. gracilipes, b. obscurior, crematogaster sp., dorymyrmex sp. and pheidole sp. we observed these ants differed in body size and likely collect and store honeydew or extrafloral nectar differently. new green leaves were produced by both t. dactyloides and a. pennatula after they were burned. disturbance by fire does not have the same effect on the numbers of ants tending d. quinquenotatus and visiting efns. we found an interaction between fire and plant species, and significantly more ants were found tending d. quinquenotatus leafhoppers on t. dactyloides than visiting efns (z = 7.63; p = 0.001). rapid colonization of new growth on t. dactyloides by ants and leafhoppers was observed after burning in the last week of may 2007. at this time only adult leafhoppers were observed in the two burned sites tended by a great number b. obscurior ants, while efns were visited by few ants of solenopsis sp. at the two burned sites (table 1 and fig. 2). in june 2012, leafhoppers were tended by pheidole sp. and a great number of nymphs were tended by great numbers of b. obscurior ants at the two 2012 burned sites (table 2 and fig. 3). near the end of the wet season in september 2012, four months after the june fire, a large number of leafhopper nymphs were tended by larger numbers of b. obscurior ants, while low numbers of a. gracilipes, camponotus sp., fig 2. average number (± standard error) of leafhoppers, ants tending d. quinquenotatus leafhoppers on t. dactyloides, efns, and ants visiting efns on a. pennatula from burned and unburned sites in jalisco, mexico in the last week of may 2007. crematogaster sp. and b. obscurior ants visited the efns at the two burned sites (table 3 and fig. 4). the number of ants tending leafhoppers was significantly higher than the number of ants found visiting efns of a. pennatula when combining both burned and unburned resources in the three sample dates (wilcoxon = 299.50; z = 3.04; p = 0.002). leafhoppers and ants were found together at the end of the dry season in may 2007 on the six unburned sites, while only in two of the six unburned sites ants visited the efns of a. pennatula (table 1 and fig. 2). in june 2012, at the end of the dry season, no ants or leafhoppers were found on the leaves of unburned t. dactyloides plants that were dried out (table 2 and fig. 3). in september 2012, at the end of the wet season, only in one of the four unburned sites ants visited the efns of a. pennatula, whereas in these four unburned sites ants tended the leafhoppers (table 3 and fig. 4). table 1. average number (± standard error) of dalbulus quinquenotatus nymphs, adults, and tending ants (and species of tending ant), acacia pennatula efns, and ants on 10 stems and 10 branches of t. dactyloides and a. pennatula respectively in burned (in may 2007) and unburned sites at locations in jalisco, mexico at the end of the dry season in may 2007. site ant/leafhopper interaction on tripsacum dactyloides ant/acacia interaction dalbulus quinquenotatus ants ant species a. pennatula efns ants ant speciesnymphs adults both resources burned 1. arenal 0 9.2 ± 1.7 7.7 ± 1.4 b. obscurior 5.6 ± 0.1 1.0 ± 0.4 solenopsis sp. 2. los chorros 0 1.4 ± 0.3 20.4 ± 5.7 b. obscurior 6.9 ± 0.3 0 both resources unburned 3. san isidro 3.9 ± 2.2 1.7 ± 0.7 15.1 ± 5.7 b. obscurior 5.8 ± 0.2 0 4. agua caliente 0.7 ± 0.5 0.5 ± 0.4 1.7 ± 1.3 b. obscurior 5.4 ± 0.1 1.0 ± 0.2 pheidole sp. 5. cocula 10.2 ± 1.2 1.2 ± 0.4 4.3 ± 0.8 b. obscurior 5.4 ± 0.1 0 6. san agustin 6.5 ± 3.3 1.9 ± 1.1 13.0 ± 5.1 b. obscurior 6.6 ± 0.1 0 7. la mimila 3.7 ± 1.9 1.1 ± 0.5 5.7 ± 4.3 b. obscurior 5.5 ± 0.2 0 8. el molino 2.0 ± 0.6 3.9 ± 1.2 12.1 ± 2.9 b. obscurior 6.6 ± 0.1 5.8 ± 1.4 b. obscurior moya-raygoza and larsen ants, leafhoppers and efns after fire140 table 2. average number (± standard error) of dalbulus quinquenotatus nymphs, adults, and tending ants (and species of tending ant), acacia pennatula efns, and ants on 10 stems and 10 branches of t. dactyloides and a. pennatula respectively in burned (in june 2012) and unburned sites at locations in jalisco, mexico in june 2012. site ant/leafhopper interaction on tripsacum dactyloides ant/acacia interaction dalbulus quinquenotatus ants ant species a. pennatula efns ants ant species nymphs adults both resources burned 1. zapopan 4.4 ± 2.3 2.4 ± 0.7 4.8 ± 1.8 pheidole sp. 5.6 ± 0.1 1.9 ± 0.2 a. gracilipes camponotus sp. 2. los chorros 37.0 ±17.9 1.1 ± 0.5 38.9 ± 16.2 b. obscurior 5.8 ± 0.3 1.5 ± 0.4 crematogaster sp. b. obscurior both resources unburned 3. san isidro 0 0 0 6.5 ± 0.4 2.5 ± 0.5 dorymyrmex sp. crematogaster sp. 4. san agustin 0 0 0 6.2 ± 0.4 0.3 ± 0.1 b. obscurior 5. la mimila 0 0 0 1.4 ± 0.7 1.1 ± 0.5 a. gracilipes 6. el arenal 0 0 0 6.3 ± 0.4 3.3 ± 0.9 a. gracilipes discussion the exudates honeydew and extrafloral nectar are key factors determining the abundance of ants when both food resources for ants are present (buckley, 1983; fiala, 1990; rashbrook et al., 1992; del-claro & oliveira, 1993; blüthgen et al., 2006; katayama et al., 2013). considering the abundance of ants tending the leafhopper d. quinquenotatus compared with the abundance of ants visiting efns, more ants were collected in association with d. quinquenotatus than with efns on a. pennatula. this finding is similar to the results of other studies (fiala, 1990; rashbrook et al., 1992; del-claro & oliveira, 1993; blüthgen et al., 2000; katayama & suzuki, 2003; katayama & suzuki, 2010; katayama et al., 2013) comparing ant abundance at honeydew-producing insects with plants with efns in non-disturbed conditions. in the rainforest canopy, ants are usually more abundant at honeydew than extrafloral nectar, as honeydew is apparently a more valuable resource to ants than nectar from efns (blüthgen et al., 2000). ants (camponotus sp.) also did not stop tending the honeydew-producing membracids (guayaquila xiphias fabricius) when an alternative efn sugar source was available on didymopanax vinosum (cham. & schltdl.), their host plant (del-claro & oliveira, 1993). recently katayama et al. (2013) demonstrated that the ant lasius japonicus santsci switches from visiting efns on the bean plant vicia faba l. to the aphid aphis craccivora koch, because the density and total food reward to ants from the aphids exceed that from efns. we ascribe the difference in abundance between ants visiting the leafhopper d. quinquenotatus and efns on a. pennatula to several factors. first, d. quinquenotatus leaffig 3. average number (± standard error) of leafhoppers, ants tending d. quinquenotatus leafhoppers on t. dactyloides, efns, and ants visiting efns on a. pennatula from burned and unburned sites in jalisco, mexico in the first week of june 2012. fig 4. average number (± standard error) of leafhoppers, ants tending d. quinquenotatus leafhoppers on t. dactyloides, efns, and ants visiting efns on a. pennatula from burned and unburned sites in jalisco, mexico in the second week of september 2012. sociobiology 61(2): 136-144 (june, 2014) 141 hoppers produce honeydew at a consistent rate (larsen et al.,1992), whereas efns are highly variable in nectar production over the course of a day, resulting in a less predictable resource for the ants. for example, nectar production is highly variable in the plant macaranga tanarious (l.) muell. arg. (heil et al., 2000). second, d. quinquenotatus is sedentary and gregarious (heady & nault 1985), resulting in a higher density of both nymphs and adult leafhoppers on the basal leaves of t. dactyloides. at higher leafhopper densities, more honeydew is produced in a concentrated area allowing easy collection by the ants. third, d. quinquenotatus responds to the stroking of their abdomen by antennae of tending ants by excreting and holding honeydew droplets until droplets are removed by ants (larsen et al., 1992). ant-tended dalbulus quinquenotatus leafhoppers secrete three to six times the volume of honeydew compared with other species of nonmyrmecophilous dalbulus leafhoppers (larsen et al., 1992), increasing the availability of honeydew for tending ants. in contrast, efns of a. pennatula do not respond to antennation by ants by increasing extrafloral nectar secretions. however, this is not universal as inga plants have been shown to increase nectar production in response to tending ants (bixenmann et al., 2011). fourth, d. quinquenotatus leafhoppers and their tending ants often live together in mud shelters made by tending ants on the basal leaves of the gamagrass. within these shelters, high densities of ants and leafhoppers occur and parasitism is reduced (moya-raygoza & larsen, 2008). these shelters help to increase the quantity of honeydew for tending ants by concentrating the leafhoppers, whereas a. pennatula does not provide shelters for ants in the form of big thorns as is found on other acacia species. providing shelter for members of the mutualism is important in establishing obligatory relationships (speight et al., 1999). fifth, the honeydew of myrmecophilous hemipterans contains melezitose that provide nitrogen and is a higher quality nectar than nectar from efns (cook & davidson, 2006). sixth, excess d. quinquenotatus leafhoppers are sometimes eaten by tending ants (moya-raygoza & nault, 2000), making the leafhoppers a high quality source of protein. ant colony growth and reproduction requires substantial quantities of protein (davidson et al., 2003). moreover, this d. quinquenotatus leafhopper-ant association is an obligate and highly specialized mutualism as compared with the more general and facultative ant-a. pennatula mutualism. moya-raygoza (2005) found that the ant b. obscurior visits active efns of a. pennatula but does not protect this species of acacia from herbivores. lack of protection by ants against herbivores is common among plants with efns (buckley, 1983; heads, 1986; oliveira et al., 1999; ruhren, 2003). in contrast, both moya-raygoza and nault (2000) and larsen et al. (2001) have shown that tending ants protect both nymph and adult d. quinquenotatus from predators. thus, this mutualism between d. quinquenotatus and ants is obligatory, as these leafhoppers apparently cannot live without tending ants. post-fire response both t. dactyloides and a. pennatula respond quickly to a fire event with new growth, producing young leaves ready to be colonized by herbivorous insects. previous studies conducted in the tropics have found that some species of plants respond to fire with vigorous growth, which can be colonized rapidly by herbivores (prada et al., 1995; vieira et al., 1996). we found that ants are adapted to colonize plants quickly after fire, taking advantage of new resources such as honeydew offered by d. quinquenotatus feeding on t. dactyloides and extrafloral nectar produced by efns of a. pennatula, resulting in the reestablishment of these mutualistic interactions only a few days after fire. we found more ants tending leafhoppers than visiting efns at burned sites where both t. dactyloides and a. pennatula were found. fire does not kill t. dactyloides, but instead stimulates the growth of new stems from t. dactyloides rhizomes. these new stems are the first food resources that appear within the table 3. average number (± standard error) of dalbulus quinquenotatus nymphs, adults, and tending ants (and species of tending ant), acacia pennatula efns, and ants on 10 stems and 10 branches of t. dactyloides and a. pennatula respectively in burned (in june 2012) and unburned sites at locations in jalisco, mexico at the end of the wet season in september 2012. site ant/leafhopper interaction on tripsacum dactyloides ant/acacia interaction dalbulus quinquenotatus ants ant species a. pennatula efns ants ant species nymphs adults both resources burned 1. zapopan 0.9 ± 0.7 0.5 ± 0.2 1.1 ± 0.5 a.gracilipes 5.5 ± 0.1 0.9 ± 0.2 a. gracilipes camponotus sp. 2. los chorros 9.5.0 ±2.1 1.0 ± 0.2 10.0 ± 1.3 b. obscurior 5.0 ± 0.2 0 both resources unburned 3. san isidro 15.1± 5.4 1.5± 0.5 14.3± 6.1 b. obscurior 5.6 ± 0.1 0 4. san agustín 8.9± 1.3 1.9± 0.3 1.4± 0.4 b. obscurior 5.4± 0.1 0 5. la mimila 1.2± 0.3 0.9± 0.3 1.4± 0.3 a. gracilipes 4.4 ± 0.2 0 6. el arenal 6.9± 2.8 3.9± 1.3 3.9± 1.4 a. gracilipes 4.9 ± 0.1 2.8 ± 0.6 a. gracilipes moya-raygoza and larsen ants, leafhoppers and efns after fire142 community and are quickly recolonized by d. quinquenotatus. these leafhoppers may come from contiguous unburned sites. these immigrant leafhoppers start to feed and produce large quantities of honeydew that attract large numbers of ants. the numbers of ants revealed this fast recolonization by leafhoppers and ants at sites where fire occurred in either may 2007 or june 2012. in contrast, in june 2012 no leafhoppers or ants were found on t. dactyloides leaves at unburned sites because those leaves were dried out. although efns at unburned sites were actively producing extrafloral nectar at that time, few ants were present. no previous studies have compared the ant abundance at leafhoppers and efns on fire-disturbed habitats when both resources are available at the same time. schowalter (2006), reported that ants and sap-sucking insects such as leafhoppers dominate early-successional tropical forests as they contain an abundance of young, succulent leaf tissue that favor sap-sucking hemipterans and tending ants. in north american grasslands, populations of some leafhopper species are significantly greater following fire due to immigration from unburned areas into rapidly growing burned areas (warren et al., 1987). previously, moya-raygoza (1995) found that d. quinquenotatus leafhoppers were found in larger numbers and tended by a greater number of ants in burned than unburned t. dactyloides colonies, because recently burned plants produce new young leaves with higher concentrations of nitrogen. similar results have been found in the interaction between ants and efn-bearing plants in other systems after disturbance. for example, pruned plants (conocarpus erectus l.) grew faster and produced higher numbers of extrafloral nectaries and attracted a higher density of ants (piovia-scott, 2011). leaf damage also increases the production of extrafloral nectar in different plants (heil et al., 2001). in another case, higher abundance of ants was found in the shrub b. campestris after fire because of a high concentration of extrafloral nectar (alves-silva & del-claro, 2013). similarly alvessilva (2011) and koptur et al. (2010) found a more diverse ant fauna guarding plants from herbivory after fire occurred due to the high production of extrafloral nectar. this is not surprising as ants are attracted to high quality sugar resources produced by plants with efns (heil & mckey, 2003). therefore, the availability of honeydew and extrafloral nectar to ants after fire is important because it can regulate ecological dominance, affecting the ant trophobiont and plant communities. greater numbers of ants tending leafhoppers may result in better protection of these honeydew producers by ants compared with the ant protection of plants with efns that can also occur in these fire-prone sites. moreover, colonization by ants after fire is important to initiate these mutualisms with both hemipterans and efns. our results highlight the importance of investigating mutualisms not only in paired species, but also among multiple mutualisms involving ants when a system is disturbed. acknowledgments we are grateful to miguel vasquez bolaños for the identification of some of the ant taxa. we also appreciate the comments and suggestions of two anonymous reviewers. references alves-sila, e. 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(2013). the ecological effects of ant-aphid mutualism on plants at a large spatial scale. sociobiology, 60: 236-241. doi: 10.13102/sociobiology.v60i3.236-241. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.9-20sociobiology 61(1): 9-20 (march, 2014) composition and diversity of ant species into leaf litter of two fragments of a semideciduous seasonal forest in the atlantic forest biome in barra do choça, bahia, brazil freitas, jms1,2,3, delabie, jhc1,2 & lacau, s1,2,3 introduction ants (hymenoptera: formicidae) form one of the most diverse and ecologically important insect groups in terms of their diverse and essential functions in terrestrial ecosystems (wilson & hölldobler, 1990; alonso & agosti, 2000). their predominance can be attributed in part to their eusocial nature, which favors their dispersal and successful occupation of new habitats (wilson & hölldobler, 2005). since the cretaceous period, these animals have demonstrated successful radiation throughout almost all terrestrial habitats, with numerical and biomass predominance in most of them (fernández & ospina, 2003; wilson & hölldobler, 2005). ant diversity in forest ecosystems is particularly high in the leaf litter (alonso & agosti, 2000; silva & brandão, 2010), although community composition is influenced by numerous factors, including the nature of the surrounding plant formations, soil composition and the local microclimate abstract we present here the results of a study of leaf litter ant diversity in remnant areas of semi-deciduous seasonal forests in the atlantic forest biome. standardized collections were made in 2011, using pitfall traps and winkler sacks in two fragments of native forest in the municipality of barra do choça in the micro-region of the planalto da conquista, in southwestern of the state of bahia, brazil. a total of 107 species from 37 ant genera and 9 subfamilies was collected. the observed richness was high, and the diversity indices (shannon-wiener) of the two fragments suggest that in spite of being strongly impacted by anthropogenic actions, they maintained high faunal diversity levels, similar to those observed in other original atlantic forest sites in state of bahia. analyses of the species accumulation curves (jackknife 2), however, indicated that survey effort was not sufficient to capture all of the species present. the high observed numbers of unique species, the shape of the species accumulation curves, and high values of estimated richness suggest that the survey areas were quite heterogeneous. these results provide new information concerning regional biodiversity that will be useful for continuing studies on fragmentation processes in the region. sociobiology an international journal on social insects 1 universidade estadual de santa cruz, ilhéus-ba, brazil. 2 laboratório de mirmecologia, ceplac/cepec/secen, ilhéus-ba, brazil. 3 universidade estadual do sudoeste da bahia, itapetinga-ba, brazil. article history edited by: gilberto m m santos, uefs, brazil received 05 november 2013 initial acceptance 13 december 2013 final acceptance 14 january 2014 keywords formicidae, planalto da conquista, atlantic forest, tropical forest corresponding author juliana martins da silva freitas univ. estadual do sudoeste da bahia laboratório de biossistemática animal itapetinga-ba, brazil e-mail:julliana.martins@yahoo.com.br (schowalter & sabin, 1991). ant community structures respond directly and quickly to both quantitative and qualitative environmental changes, and have therefore been the focus of stu-dies investigating the effects of environmental disturbances on ecological communities (veiga-ferreira et al., 2005; delabie et al., 2006, 2007). ants maintain numerous biotic associations with other organisms in their environments (wilson & hölldobler, 1990; rico-gray & oliveira, 2007), rapidly respond to habitat alterations such as fragmentation (peck et al., 1998; veiga-ferreira et al., 2005; delabie et al., 2006) and are relatively easily collected and identified (peck et al., 1998), making them ideal models for studying and monitoring global biodiversity and useful as bioindicators of disturbances caused by ecosystem size reductions the atlantic forest biome has been a focal area for environmental conservation efforts (dean, 2002). studies of atlantic forest biodiversity have almost exclusively foresearch article ants freitas, jms, delabie, jhc & lacau, s. leaf-litter ant communities in bahia atlantic forest, brazil 10 cused on ombrophilous forests on the coastal plains of brazil (ivanauskas & rodrigues, 2000; costa & mantovani, 1993; martins, 1993). however, a number of diverse ecosystems are found in this biome (brasil, 2000), including semi-deciduous and deciduous seasonal forests in the region of southwestern of the state of bahia, and some have been poorly studied (especially those situated more inland) (brasil, 2000). the semideciduous seasonal forest exhibits high biodiversity due to the confluence between atlantic forest, caatinga (dryland vegetation), and cerrado (neotropical savanna) biomes (soaresfilho, 2000; daniel & arruda, 2005; dean, 2002). they are highly threatened and have experienced critical levels of fragmentation due to agricultural expansion, pasture formation, and urban occupation, among other factors (campanili & prochnow, 2006). one of the most highly neglected regions in terms of studies of ant fauna diversity is the atlantic forest in the southwestern region of the state of bahia, principally the planalto da conquista. these vegetation formations are considered “inland atlantic forests of bahia” (classified as semideciduous seasonal forests) the present study was designed to examine the ant fauna of this region and characterize the composition and diversity of ant species in the leaf litter of two remnant forest fragments situated in the municipality of barra do choça, in the planalto da conquista, state of bahia state, brazil. materials and methods collection sites the surveys were performed in two areas of semideciduous seasonal forest: “remnant 1” (14°50’00”s 40°33’13”w; 86 hectares) and “remnant 2” (14°48’29”s 40°35’23”w; 62 hectares) (fig. 1). both fragments were located in the municipality of barra do choça, in the state of bahia, brazil, within the transition zone between dense ombrophilous forests and seasonal deciduous forest areas (ibge, 1993; 1997) in formations locally known as “mata de cipó” (soares-filho, 2000) ; between 20 and 50% of the trees there are large deciduous species (ibge, 1993; 1997; soares-filho, 2000). these once extensive native forest formations are currently represented only by remnant fragments that have experienced intense processes of environmental degradation from agro-pastoral activities and the selective extraction of commercially valuable trees (soares-filho, 2000; projeto mata atlântica interiorana da bahia, 2002; oliveira-filho et al., 1994). the fragments studied here are embedded within monoculture and pasture matrices, and their interiors demonstrate clear evidence of selective cutting and cattle trails. the regional climate is classified as high-elevation tropical (ibge, 1993; 1997), with a mean annual temperature of 19.8 ºc, and a mean annual rainfall rate of 734 mm. collection methodology ant collections were made in january and april/2011 using 47 winkler sacks and 47 pitfall traps (bestelmeyer et al., 2000) in each fragment (one winkler sack was destroyed in fragment 1, table 1) distributed at intervals of 30 m within an area of approximately 10 hectares – but always at least 100 m from the external edges of the fragment. a pitfall trap was installed at each collection point and left for two days. these traps consisted of cups of 7 cm diameter by 10 cm height containing only water and detergent. when the pitfall trap was removed, an additional sample of 1 m² of leaf litter was removed from the same site, passed through a sieve, and then processed in a winkler extractor for 48 hours (bestelmeyer et al., 2000). this standardized methodology was adapted from the ants of the leaf litter protocol of agosti & alonso (2001). biological material the biological material collected in the field was preserved in ethanol and then taken to the laboratório de mirmecologia (ceplac/cepec/secen) and laboratório de biossistemática animal (uesb/debi) where ant specimens were sorted out from the samples, mounted and identified to species level. the nomenclature follows bolton et al. (2011) and wilson (2003). representative materials of all of the species are deposited in the myrmecology laboratory collection (cpdc) under the reference number #5729. data analyses data was recorded using excel version 10 software (microsoft, 2007) which was used to calculate the relative frequencies of the species and their species richness for each different area and for each type of trap. estimates software version 8.2 (colwell, 1997) was used to generate species accumulation curves for each area and each type of trap in terms of the sampling effort employed (santos, 2003). the fig 1 satellite picture of the two forest remnant where the experiment was conducted. barra do choça, bahia, brazil (source: google earth, 2011). sociobiology 61(1): 9-20 (march, 2014) 11 estimated species richness was subsequently calculated for each area using the jackknife 2 index – an index based on the numbers of species that occur only once in a sample (singletons) and those occurring twice (doubletons). to determine if there were significant differences between the occurrences of ant species between the different fragments and between the different types of traps utilized, two-way analyses of variance (anova factorial) were performed using primer 5 software (clarke & gorley 2001). the shannon-wiener diversity index was used to calculate the alpha diversity of the two fragments using bioestat 5.3 software (ayres, 2011). this index was chosen as it gives the same weight to both rare and abundant species. the t test was used to determine if there were significant differences between the diversity index values (also using bioestat 5.3 software). results and discussion observed richness a synoptic list of the species collected in the present study, and their occurrences as a function of the collection areas and types of trap utilized, is presented in the appendix. a total of 107 ant species belonging to 36 genera and 9 subfamilies were observed, with 83 species found in fragment 1, and 67 in fragment 2 (table 1). these results indicated that the fragments analyzed retained relatively high faunal richnesses – even greater than reports for other areas of semideciduous forests in the atlantic forest biome (mentoni et al., 2011; dias et al., 2008, castillho et al., 2011). the only other study that has examined ant diversity in remnant seasonal semi-deciduous forests in the region around the planalto da conquista was undertaken in 2011 by sofia campiolo, ivan nascimento, and jacques delabie (personal communication, may 4, 2011). these investigators undertook collections during the dry season using winkler sacks in five areas relatively close to the present research site (in the municipalities of barra do choça, itambé, and vitória da conquista). their collection efforts were very similar to those of the present study, and they found between 47 and 86 species belonging to between 23 and 33 genera in the five fragments examined – results that are reasonably close to those of the present study (table 1). in both types of trap, fragment 1 demonstrated greater taxonomic richness than fragment 2 in terms of the species, genus, and subfamily levels. this result is somewhat surprising, as this fragment was 28% smaller than the other. in similar studies, and in accordance with the theory of island biogeography (macarthur and wilson, 1967), positive correlations have been found between species richness and fragment sizes (morini et al., 2007). one explanation for the observed discrepancy reported here could be that, in spite of the fact that the two fragments appeared to be phytosociologically similar (avaldo de oliveira soares filho, personal communication, january 10, 2011), the first fragment had more ecological niches available for ants. as such, and to better interpret the results, it would be interesting to quantify other diverse parameters in these forests fragments in future studies, such as the richness and diversity of their vegetation, their spatial structuring in terms of microhabitats, and available trophic resources. table 1 summary of the results found in two forest remnants at barra do choça, bahia, brazil. w: winkler sack, p: pitfall trap. remnant 1 remnant 2 w p w p number of samples 46 47 47 47 number of species occurrences 349 201 382 231 number of subfamilies 9 7 7 7 number of genus 31 28 22 22 number of species 66 54 50 44 number of estimated species (jackknife2) 109.6 94.9 66.6 73.3 singletons 25 26 11 19 doubletons 5 10 5 8 shannon-wiener 3.68 3.44 3.45 3.05 table 2 – variations fonts of anova test (two-way analyzes of variance factorial anova) for comparisons of samples collected in two forest remnants at barra do choça, bahia, brazil. ss degree of freedom ms f p intercept 6287.75 1 6287.75 140.653 0 place/trap 209.446 1 209.446 4.6852 0.03156 remnant 191.356 1 191.356 4.2805 0.03978 remnant/trap 7.813 1 7.813 0.1748 0.67634 error 9343.16 209 44.704 freitas, jms, delabie, jhc & lacau, s. leaf-litter ant communities in bahia atlantic forest, brazil 12 winkler sacks collected larger numbers of species in both fragments than did pitfall traps (table 1), although these differences were not statistically significant (f = 4.69 and p = 0.032, table 2). most published studies have shown that winkler sacks can collect greater numbers of species than most other techniques (sabu et al., 2011; vargas et al., 2009), although this assertion was recently challenged by souza et al., (2012) to the profit of pitfall traps. nevertheless, both techniques complement each other for maximum sampling efficiency (delabie et al., 2000). winkler sacks are most efficient for collecting smaller species with cryptic behavior and low activity levels (agosti et al, 2000), while pitfall traps are better for collecting large, active foraging ants that are not easily collected through winkler method, such as species of the genera camponotus, pachycondyla or odontomachus. winkler sacks also harvested higher mean numbers of species per sampling point than pitfall traps (table 3). these results are similar to those previously reported for native forest sites in the atlantic forest biome (marinho et al., 2002; suguituru et al., 2013). it is worth noting that there were differences between the two fragments studied here in terms of their mean species richness per sampling point, with this value being lower in fragment 2 (although these differences were not statistically significant) (table 3). table 3 -mean number of ant species (mean occurrence) observed in forest remnants in barra do choça, bahia, brazil. values in the main diagonal of the matrix are means ± 1 se. differences between the pairs of ant species are based on fisher post-hoctest. ns = not significant; * = p< 0.05; mw1 = species collected with winkler sacks in first forest remnant; mw2 = species collected with winkler sacks in second forest remnant; mp1 = species collected with pitfall traps in first forest remnant; mp2 = species collected with pitfall traps in second forest remnant; mw mw mp mp 1 2 1 2 mw 1 5.33±0.76 ns ns ns mw 2 7.64±1.13 * ns mp 1 3.72±0.65 ns mp 2 5.25±1.18 richness estimates richness accumulation curves for each type of trap for each area are shown in fig 2a and 2b. analyses of the accumulated richness curves indicated complete sampling of the ant fauna was not achieved in either type of trap, as none of the curves approached the asymptote. thus, projected richness values were always greater than observed values (table 1), which is the usual situation in biodiversity studies in the tropics (martins & santos, 1999; feitosa & ribeiro, 2005; baccaro et al., 2011; braga et al., 2010; leponce et al., 2004; delabie et al., 2007), since many rare species continue to be encountered even after extremely intense sampling efforts (santos, 2003). the high estimated richness values could be explained by the study areas possibly having heterogeneous species distributions, because, according to chao (1987), the greater the heterogeneity of the species’ spatial distributions, the greater will be the observed divergences between observed and expected richness. the large number of unique species encountered (table 1) corroborated the results of the observed richness curves and diversity estimators. unique species may be present in the area only as foragers (“tourist” species, according to belshaw & bolton, 1993), as rare species, as generalist species occasionally feeding in the locality, or as specialist that feed exclusively on plants occurring only in a single site in the study area. also, the surveys may have been undertaken using inadequate methodologies (particularly an issue with small populations) (novotny & basset, 2000). the proportions of unique species in the present work were always greater than 30% of the total numbers of species collected – reaching up to almost 50% in the case of pitfall traps in the first fragment. such high proportions of unique species are consistent with other biodiversity studies of arthropods in tropical regions (coddington et al., 2009) and with other ant studies undertaken in atlantic forest areas (pacheco et al., 2009). fig 2 species accumulation curves for ant species collected in winkler sacks (a) and pitfall traps (b) in two forest remnant in barra do choça, bahia, brazil. mw1: first forest remnant, mw2: second forest remnant, mp1: first forest remnant, mp2: second forest remnant, s: observed richness, jack 2: richness index estimated by jackknife 2. sociobiology 61(1): 9-20 (march, 2014) 13 analyses of faunal composition the richest subfamilies at the species level in fragment 1 were: myrmicinae (59% of the total number of species), formicinae (15.7%), and ponerinae (13.3%). the same tendency was observed at the genus level: myrmicinae (52.6% of the total number of genera), formicinae (13.2%), and ponerinae (10.5%) (appendix). the richest subfamilies at the species level in fragment 2 were: myrmicinae (58% of the total number of species encountered), ponerinae (14.5%), and formicinae (14.5%), while at the generic level, the richest subfamilies were: myrmicinae (44.4% of all of the genera encountered), formicinae (18.5%), and ponerinae (11.1%) (appendix). the observation that the subfamily myrmicinae was the richest in both fragments at both the genus and species levels, while the subfamilies formicinae and ponerinae demonstrated similar species richness, had been reported in other surveys of forested areas in the neotropical region (miranda et al., 2012). a particularly interesting find in fragment 1 was the capture of two specimens of ochetomyrmex (ochetomyrmex sp_lbsa_14010266) (very similar to ochetomyrmex semipolitus but possibly distinct) – a new record for this genus in the state of bahia, and extending the geographical distribution of this species by more than 2,380 km to the east (referring to the map given by fernandez, 2003). a new species of oxyepoecus santschi, 1923 was also encountered (six specimens in fragment 2), which is now being described (sebastien lacau, personal communication, june 4, 2012). another important collection was the capture of three specimens of monomorium delabiei fernandez, 2007 in fragment 2 – a species up to now only known from the holotype, described from guaratinga, bahia, brazil (fernandez, 2007). differences existed in the composition of the species encountered in the two study areas, with only 38% of the species common to both areas – indicating considerable differences in the compositions of their respective species communities (appendix). there was also a relationship between the global richness of each remnant at the species level and its degree of exclusivity, with fragment 1 being the most species rich (83 species) and having the most exclusive faunal composition (with 50.6% of the species and 36.8% of the genera being exclusive to that fragment), while fragment site 2 was the least rich (67 species) and had the least exclusive faunal composition (with 34.3% of the species and 7.6% of the genera being exclusive to that fragment). relative frequency and dominance to determine if the ant communities are organized into defined structural patterns, we examined the species relative frequencies in the two areas according to the types of traps in which they were caught. in the case of winkler sacks, it was observed that 10 species were responsible for half of the occurrences in fragment 1 (fig 3a), and eight species for half of the occurrences in fragment 2 (fig 3b). four of those species (hypoponera sp. 3, nylanderia sp. 2, gnamptogenys striatula and solenopsis sp. 6) only were common to both areas. in the case of the pitfall traps, eight species were responsible for half of the occurrences in fragment 1 (fig 3c) and five species in fragment 2 (fig 3d), with three species (pheidole radoszkowskii, gnamptogenys striatula, and nylanderia sp. 2) being encountered in both fragments. the overall results demonstrated that the three most frequent species (hypoponera sp. 3, octostruma sp. 1, and strumigenys sp. 4) were exclusively collected with winkler sacks, suggesting that they have cryptic lifestyles, and nest and forage below the leaf litter surface. the same types of frequency analyses were performed to determine the identities and relative frequencies of the genera responsible for 50% of the total occurrences in the collections. in the case of the winkler sacks, five genera (pheidole, solenopsis, hypoponera, nylanderia, and strumigenys) were responsible for 50% of the occurrences in fragment 1 (fig 4a), while four genera (solenopsis, hypoponera, nylanderia and pheidole) were responsible for this same percentage in fragment 2 (fig 4b). four genera (pheidole, solenopsis, hypoponera, and nylanderia) were encountered in both areas. in the case of the pitfall traps, three genera (pheidole, gnamptogenys, and ectatomma) were found to be responsible for 50% of the occurrences in fragment 1 (fig 4c), and four genera in fragment 2 (pheidole, pachycondyla, linepithema, and solenopsis) (fig 4d). only one genus (pheidole) was encountered in both fragments. these results emphasize the notable dominance of the genera pheidole, solenopsis, and nylanderia. the genus pheidole has been observed to be the most dominant in many studies of ant diversity, with two of its species consistently appearing among the most abundant arthropod representatives in the leaf litter. pheidole is the most diversified genus in the family formicidae (wilson, 2003) and its species are encountered in all soil microhabitats, have wide ranges of feeding habits (most are omnivores), and demonstrate great efficiency in recruiting workers to exploit many trophic resources (fernández, 2003). some pheidole species are rather aggressive in their relationships with competitors, are opportunists and can colonize a wide diversity of environments (wilson, 2003). the observed dominance of this genus in the present study therefore corroborates the recognition of this genus as the most abundant and diversified in the neotropical region (majer & delabie, 1994; marinho, et al. 2002; vasconcelos, 1999). in the same sense, the high abundance of solenopsis species observed in the present study confirms previous observations in the literature. some species of this genus are common throughout the world (fernandéz, 2003), principally in the leaf litter, with many of them being generalists in terms of their habitats and diets (fowler et al., 1991). these species freitas, jms, delabie, jhc & lacau, s. leaf-litter ant communities in bahia atlantic forest, brazil 14 fig 3 relative frequency of the species more collected in two forest remnant at barra do choça, bahia, brazil. (a) samples collected with winkler sacks in the first forest remnant; (b) samples collected with winkler sacks in the second forest remnant; (c) samples collected with pitfall traps in the first forest remnant; (d) samples collected with pitfall traps in the second forest remnant; fig 4 relative frequency of the most frequently collected genera in two forest remnants at barra do choça, bahia, brazil. (a) samples collected with winkler sacks in the first forest remnant; (b) samples collected with winkler sacks in the second forest remnant; (c) samples collected with pitfall traps in the first forest remnant; (d) samples collected with pitfall traps in the second forest remnant. sociobiology 61(1): 9-20 (march, 2014) 15 are also encountered with relatively high frequencies in agricultural areas (dias et al., 2008). the genus nylanderia was also very frequent in the present study, and previous publications have shown that the relatively small individuals that are characteristic of this genus are quite abundant (mentone, 2011), being terricolous or arborous, or occupying the leaf litter, in both natural and disturbed environments (fernández, 2003). diversity indices the alpha diversity estimates for the fragments, as calculated by the shannon and wiener index, can be found in table 1 and are comparable to other biodiversity studies of ants in the neotropical region (lopes et al., 2010; lutinski et al., 2008). the greatest alpha diversity was observed in fragment 1, corroborating the hypothesis that fragment 1 had the best phytosociological quality and favored the occurrence of a wider diversity of species. the situation in fragment 2 apparently represents a simplification of the original community structure. the differences between fragments were not, however, statistically significant (t = 1.3291; p = 0.1576). concluding comments the results obtained in the present study are totally original, and no similar research has previously been undertaken in the planalto da conquista region. it was observed that while the two fragments analyzed had both been subjected to anthropogenic modifications, they still maintained high natural faunal diversities typical of inland atlantic forest sites in state of bahia. the diversity and species richness observed in this study were actually greater than those reported in the literature for other semi-deciduous forests of the atlantic forest biome (mentoni et al., 2011; dias et al., 2008, castilho et al., 2011). when the two fragments were compared, it could be seen that the numbers of species, genera, and sub-families were greater in fragment 1. this result suggests that although the two fragments were superficially similar, the first was better preserved in terms of the ecological niches available for the ant fauna and therefore better reflected the original community structures of the formicidae in this ecosystem; the second fragment represented a greater simplification of the more complex original community. the results presented here will hopefully be useful to future conservation plans for remnant forest areas in the planalto da conquista, as ants can be easily used as biological indicators of the degradation (or preservation) of areas subjected to anthropogenic impacts (or management). additionally, it is hoped that this study will serve as a baseline for further investigations about regional biodiversity, as this region is desperately lacking this kind of information. acknowledgments we would like to thank drs. sofia campiolo and ivan nascimento allowing access to the unpublished ant data of the planalto da conquista experiment. we convey our thanks to the graduate and post-graduate students who assisted in data collection of this study; and to our friend dr. paulo sávio for the help in the statistical analysis. also we thank the granting bodies of cnpq, capes, 019/2013 fapesb and the pronex program, project secti-fapesb/ cnpq pnx 001/2009. references ayres, m. 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(1999) effects of forest disturbance on the structure of ground-foraging ant communities in central amafreitas, jms, delabie, jhc & lacau, s. leaf-litter ant communities in bahia atlantic forest, brazil 18 appendix list of ant species and their frequency of occurrence collected by two sampling methods and in two forest remnants in barra do choça, bahia, brazil: mw1 and mp1 represent the species collected with winkler sacks and pitfall traps in first forest remnant; mw2 and mp2 represent the species collected with winkler sacks and pitfall traps in the second forest remnant. subfamilies species mw1 mp1 mw2 mp2 dolichoderinae azteca sp. 1 1 7 azteca sp. 2 1 dolichoderus attelaboides (fabricius, 1775) 1 1 linepithema pulex wild, 2007 20 27 ecitoninae eciton burchelli westwood, 1842 1 labidus praedator (smith, 1858) 1 ectatomminae ectatomma edentatum roger 1863 1 7 ectatomma sp. 1 12 1 ectatomma sp. 3 10 gnamptogenys striatula mayr, 1884 19 20 19 25 formicinae acropyga guianensis weber, 1944 1 acropyga sp. 1 1 brachymyrmex patagonicus mayr, 1868 4 6 brachymyrmex sp. 1 7 brachymyrmex sp. 3 2 6 brachymyrmex sp. 4 8 brachymyrmex sp. 5 6 1 camponotus cingulatus mayr, 1862 7 4 camponotus novogranadensis mayr, 1870 14 8 5 5 camponotus renggeri emery, 1894 1 camponotus sp. 3 2 camponotus (tanaemyrmex) sp. 5 1 nylanderia sp. 1 5 1 2 1 nylanderia sp. 2 24 9 37 21 nylanderia sp. 4 2 nylanderia sp. 5 1 paratrechina longicornis (latreille, 1802) 1 heteroponerinae heteroponera mayri kempf, 1962 3 6 myrmicinae acanthognathus sp 1 1 acromyrmex aspersus (smith, 1858) 1 acromyrmex sp. 1 1 1 apterostigma pilosum mayr, 1865 3 zonia. biodiversity and conservation, 8: 409-420. veiga-ferreira, s., mayhé-nunes, a. j. & queiroz, j. m. 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(2003) the genus pheidole in the new world: a dominant, hyperdiverse ant genus. harvard university press. sociobiology 61(1): 9-20 (march, 2014) 19 myrmicinae (cont.) basiceros disciger (mayr, 1887) 1 carebara sp. 1 6 4 1 carebara sp. 2 4 crematogaster distans mayr, 1870 2 1 crematogaster sp. 2 1 5 1 crematogaster sp. 3 1 cyphomyrmex transversus emery, 1894 7 2 5 1 cyphomyrmex strigatus gp. sp. 1 1 hylomyrma balzani (emery, 1894) 18 7 19 3 megalomyrmex drifti kempf, 1961 1 megalomyrmex goeldii forel, 1912 7 8 monomorium delabiei fernández, 2007 1 myrmicocrypta sp. 1 1 myrmicocrypta sp. 2 4 ochetomyrmex sp. 1 (lbsa 40 10 266) 1 1 octostruma sp. 1 16 3 oxyepoecus myops albuquerque & brandão, 2009 5 oxyepoecus sp. 2 (lbsa_ 1 40 10 26 4) 4 2 pheidole radoszkowskii mayr, 1884 23 24 8 17 pheidole tristis gp. sp. 1 1 pheidole sp. 2 1 pheidole fallax gp. sp. 3 1 1 1 6 9 pheidole tristis gp. sp. 4 5 1 2 pheidole sp. 5 6 4 1 pheidole flavens gp. sp. 6 10 7 6 pheidole tristis gp. sp. 7 2 4 2 pheidole sp. 8 1 1 1 1 pheidole diligens gp. sp. 9 1 1 pheidole diligens gp. sp. 10 1 2 1 2 pheidole fallax gp. sp. 11 1 3 pheidole sp. 12 5 1 pheidole tristis gp. sp. 13 4 1 pheidole diligens gp. sp. 15 1 1 2 pheidole sp. 17 1 1 pheidole sp. 18 7 pheidole sp. 21 1 2 pheidole transversostriata mayr, 1887 1 1 procryptocerus hylaeus kempf, 1951 1 solenopsis sp. 3 2 2 solenopsis sp. 4 17 solenopsis sp. 6 1 3 2 22 1 3 solenopsis sp. 7 1 freitas, jms, delabie, jhc & lacau, s. leaf-litter ant communities in bahia atlantic forest, brazil 20 myrmicinae (cont.) solenopsis sp. 8 3 solenopsis sp. 9 8 6 14 3 solenopsis sp. 10 1 solenopsis sp. 11 1 solenopsis sp. 12 2 1 4 1 solenopsis sp. 13 18 2 5 1 solenopsis sp. 14 1 strumigenys appretiata (borgmeier, 1954) 2 1 strumigenys denticulata mayr, 1887 2 strumigenys sp. 1 2 strumigenys sp. 2 9 2 strumigenys sp. 3 5 1 strumigenys sp. 4 11 8 trachymyrmex sp. 1 1 wasmannia auropunctata (roger, 1863) 4 2 6 2 wasmannia iheringi forel, 1908 8 2 22 2 wasmannia sp. 3 4 ponerinae anochetus simoni emery, 1890 3 hypoponera foreli (mayr, 1887) 5 5 2 hypoponera sp. 1 3 hypoponera sp. 2 5 2 20 1 hypoponera sp. 3 24 20 hypoponera sp. 5 1 hypoponera sp. 6 9 hypoponera sp. 7 3 odontomachus chelifer (latreille, 1802) 9 8 odontomachus sp. 2 3 3 1 odontomachus sp. 3 1 pachycondyla crenata (roger, 1861) 1 pachycondyla moesta mayr, 1870 1 1 pachycondyla nr. magnifica 7 34 proceratiinae discothyrea sexarticulata borgmeier, 1954 1 22 1 pseudomyrmecinae pseudomyrmex tenuis (fabricius, 1804) 1 1 1 doi: 10.13102/sociobiology.v65i4.3442sociobiology 65(4): 558-565 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 searching for molecular markers to differentiate bombus terrestris (linnaeus) subspecies in the iberian peninsula introduction the average pollination dependency of crops was estimated to around 300 000 million of euros in 2009 (lautenbach et al., 2012). among the insect pollinators, the bumblebee bombus terrestris (linnaeus) is used in greenhouses all over the world because they are better pollinators than honeybees for crops as strawberry, tomato or melon, due to their large body and buzzing capacity. the economic value of this service, just for tomato, has been measured in 12 000 million of euros per year (velthuis & doorn, 2006). nevertheless, in the current scenario of bee decline, the introduction of non-native bombus species and subspecies without a proper legislation may affect local bumblebee biodiversity (goulson et al., abstract bumblebees (genus bombus latreille) are pollinator insects of great ecological and economic importance, which commercial use for pollination has increased since the 80s. however, the introduction of foreign bombus terrestris (linnaeus) has resulted in a decline of native bumblebee populations in japan, chile and argentina among others. to study the potential introgression of commercial b. terrestris into the iberian endemic subspecies bombus terrestris lusitanicus krüger it is necessary to find a precise molecular marker that differentiates both subspecies. for this purpose, comparative analyses were carried out between b. t. lusitanicus and bombus terrestris terrestris (linnaeus) from spain and from belgium by sequencing the nuclear genes elongation factor 1-α and arginine kinase and the mitochondrial gene 16s ribosomal rna, and genotyping with eleven microsatellite loci. no differentiation was observed at the nuclear level, but haplotypes found within the 16s sequence correlated with the morphological characterization of the subspecies. in a case study including individuals sampled before the establishment of bumblebee rearing companies and others from recent samplings, we detected hybrid individuals (those with non-matching morphological subspecies and 16s haplotype) more frequently in the south supporting the naturalization of commercial b. t. terrestris and introgression events between both subspecies. this marker should be used in iberian populations with the aim to support management and conservation actions in endemic populations of b. t. lusitanicus. sociobiology an international journal on social insects d cejas1, c ornosa2, i muñoz1, p de la rúa1 article history edited by cândida aguiar, uefs, brazil received 10 may 2018 initial acceptance 13 june 2018 final acceptance 03 august 2018 publication date 11 october 2018 keywords bumblebees, genetic diversity, argk, ef1, 16s, mitochondrial dna, iberian peninsula, microsatellites. corresponding author pilar de la rúa área de biología animal departamento de zoología y antropología física facultad de veterinaria universidad de murcia 30100 murcia, spain. e-mail: pdelarua@um.es 2015). such introductions suppose a risk for the conservation of endemic species and subspecies in many countries (lecocq et al., 2015) to the extent that invasions of commercial nonnative bumblebees have been reported as one of the 15 emerging issues for global conservation in 2017 (sutherland et al., 2016). in this sense, bombus terrestris (linnaeus) can leave greenhouses and colonize the environment (kraus et al., 2010), presenting some negative effects on native bees, for instance, displacing wild species while competing for resources such as pollen or nesting places (matsumura et al., 2004; aizen et al., 2018). this colonization might affect native species by spreading exotic diseases and parasites (whitehorn et al., 2013) and changing plant-pollinator interactions in non-native environments, impacting crops, native plants 1 departamento de zoología y antropología física, facultad de veterinaria, universidad de murcia, murcia, spain 2 departamento de biodiversidad, ecología y evolución, facultad de ciencias biológicas, universidad complutense, madrid, spain research article bees mailto:pdelarua@um.es sociobiology 65(4): 558-565 (october, 2018) special issue 559 and pollinators (morales et al., 2013; aizen et al., 2014). b. terrestris have also better competitive capacities such as large foraging ranges and a broad diet, and they emerge early in the season making them adaptable to different environments (matsumura et al., 2004). currently, the invasive distribution of b. terrestris is increasing due to direct human intervention. bumblebee introduction has impacted local bee populations in countries as chile, china, israel, japan, mexico, south africa, south korea, taiwan, and new zealand, where they are displacing native bee species to the edge of local extinction (inoue et al., 2008; schmid-hempel et al., 2014; acosta et al., 2016). the effect of reared b. terrestris in areas with consubspecific populations has been less investigated (lecocq et al., 2016). b. terrestris presents nine subspecies classified by their body hair color pattern and distribution range (rasmont et al., 2008). among them, bombus terrestris terrestris (linnaeus) and bombus terrestris dalmatinus dalla torre are the two most widely used in artificial rearing (velthuis & doorn, 2006). the endemic subspecies bombus terrestris lusitanicus krüger inhabits the iberian peninsula and reaches southern france where there is a natural contact zone with b. t. terrestris (ornosa & ortiz-sánchez, 2004). during the last years, b. t. terrestris has been also detected at the south of the peninsula (ornosa, 1996; vargas et al., 2013), mainly in almeria where more than 30 000 hectares of greenhouses are located. we hypothesize that the contact between the endemic and the introduced subspecies in this region leads to introgression events that may yield to a loss of genetic diversity, or even displacement of the local endemic subspecies by individuals of the commercial subspecies. the taxonomy of these two b. terrestris subspecies has been discussed before, but neither the barcoding approach (based on mitochondrial cox1 gene fragment variation) nor the cephalic labial gland secretions have been able to differentiate them (coppée et al., 2008; williams et al., 2012; lecocq et al., 2016). in this work, we aim to evaluate the sequence variation of two nuclear genes and one mitochondrial gene as potential subspecific markers to differentiate b. t. terrestris and b. t. lusitanicus. nuclear arginine kinase (argk) and elongation factor-1α (ef-1α) genes include introns that have been proven useful to differentiate bombus species (kawakita et al., 2003) and to characterize geographic variations (hines et al., 2006). the mitochondrial 16s ribosomal rna (16s) gene has a higher substitution rate than barcoding fragment cox1, what results useful to lower-level (genera and species) analysis on the hymenoptera (rokas et al., 2002; hines et al., 2006; cameron et al., 2007). furthermore, we have also used microsatellite loci developed by estoup et al. (1993, 1996) adequate to analyze the genetic structure of bumblebee populations (kraus et al., 2010, dreier et al., 2014, moreira et al., 2015). material and methods sampling preliminary sampling for the first experiment searching for molecular markers to differentiate b. terrestris subspecies in the iberian peninsula, we included 10 female individuals of b. t. lusitanicus and 20 b. t. terrestris sampled in 2011-2015. b. t. lusitanicus individuals were sampled along the bank of duero river (soria, spain), national park (n. p.) of guadarrama (madrid, spain) and the sierra nevada n. p. (granada, spain). b. t. terrestris individuals were sampled also in sierra nevada n. p. and in belgium (table s1). the species of every individual was confirmed through barcoding (murray et al., 2008), whereas the subspecies was determined through morphological characters. individuals were classified in three groups according to their subspecies and geographic origin: tls for b. t. lusitanicus from spain, tts for b. t. terrestris from spain and ttb for b. t. terrestris from belgium (table s1). samples were maintained in absolute ethanol and stored at -20 ºc in the laboratory until processed. case study in order to confirm the discriminative subspecific power of the 16s haplotypes we designed a case study by adding 123 individuals to the prior sampling: 48 b. t. lusitanicus individuals collected in spain between 1977 and 1985 before the settlement of bumblebee rearing companies plus 40 b. t. lusitanicus from different spanish locations, 29 b. t. terrestris (21 from the north of france and eight from sierra nevada n. p., spain) and six individuals considered morphologically as hybrids from the north of spain (huesca) and surroundings of sierra nevada n. p. (spain). these 75 individuals were collected between 2013 and 2015 (table 1 and table s2 in supplementary material) and their species status was also confirmed through barcoding (murray et al., 2008) table 1. subspecies, number of bombus terrestris individuals (n), country and years of sampling in the case study. distribution of 16s haplotypes detected is also shown. subspecies n country years 16s haplotype a g b. t. lusitanicus 48 spain 1977/1985 1 47 50 spain 2013/2015 7 43 total 98 8 90 b. t. terrestris 18 spain 2013/2014 7 11 10 belgium 2013 10 21 france 2015 21 total 49 38 11 hybrids 6 spain 2014 2 4 d cejas; c ornosa; i. muñoz; p. de la rúa – b. terrestris subspecies molecularly characterized in spain560 dna extraction, amplification and sequencing total dna was extracted from a hind leg of every individual following the protocol of ivanova et al. (2006). as a first step, the tissue was digested with proteinase k during six hours at 56 ºc in continuous shaking. puretaqtm readyto-go pcr beads (ge healthcare) were used for dna amplification. pcr profile consisted on initial denaturation at 94 ºc for 3 min followed by 36 cycles of denaturation at 94 ºc for 1 min, annealing temperature at 48 ºc (argk), 53 ºc (ef-1α) and 49 ºc (16s) for 1 min, elongation at 68 ºc (72 ºc for ef-1α) for 1 min and a final extension at 72 ºc for 10 min (hines et al., 2006). efficacy of pcr reactions was checked in 1.5% agarose gel stained with redsafe (ecogen). amplicons were sequenced using forward primers for ef-1α and argk fragments, and with both forward and reverse primers for the 16s fragment (secugen, madrid, spain). genotyping microsatellites loci were amplified in two pcr multiplex reactions: rb1 (b10, b11, b100, b96, b124 and b126), and rb2 (b118, b119, b121, b131 and b132), with 0.2 μm concentration for each primer. reactions also included mgcl2 (1.2 mm), dntps (0.3 mm), bsa (1.2 mg/ ml) and kappa dna polymerase (1.5 u, kapa biosystems®). pcr profiles for both multiplex were set following cejas et al. (personal information) as follows: initial denaturation at 95 ºc for 5 min followed by 30 cycles of denaturation at 92 ºc for 30 s, annealing temperature at 54 ºc for 30 s, elongation at 72 ºc for 30 s and a final extension at 72 ºc for 30 min. pcr products were sent to the servei central de suport a la investigació experimental (university of valencia, spain). allele scoring was performed using genemapper 4.8 (applied biosystems inc.) by comparing alleles with an internal size standard (genescan-500 liz, applied biosystems inc.). binsets were built to correct genetic analyzer errors. quality control was made manually. data analysis dna sequences were edited and aligned using geneious 7.1.3 (http://www.geneious.com, kearse et al., 2012). b. terrestris sequences of argk (af492888.1), ef-1α (af492955.1) (kawakita et al., 2003) and 16s (ay737386.1) (hines et al., 2006) genes were downloaded from genbank (http://www.ncbi.nlm.nih.gov/genbank/) and included for comparison purposes. mega6 (tamura et al., 2013) was used for sequence similarity analysis. individuals with less than nine microsatellites amplified were discarded. sibling workers from the same location inferred with colony 2.0.6.4 (wang, 2012) were excluded from further analyses. colony parameters were selected as default for a haplodiploid species. genetic parameters were calculated taken the three groups as three independent populations. genalex 6.5 (peakall & smouse, 2012) was used to obtain the number of alleles (na), effective number of alleles (ne), private number of alleles (np) and observed (ho) and expected (he) heterozygosity values. r program (r core team, 2013), with the adegenet package (jombart, 2008; jombart & ahmed, 2011) was used to perform a discriminant analysis of principal components (dapc), that is a multivariate method which allows probabilistic assignment of individuals to different clusters. information about the subspecies (b. t. lusitanicus or b. t. terrestris), country, year of sampling and 16s haplotype of each individual in both samplings (preliminary and case study) was used to calculate (1) kendall´s tau to estimate the correlation between subspecies and haplotype (hybrids were not taken into account, n = 147) and (2) pearson´s chi-squared test with yates continuity correction to analyze whether differences of 16s haplotype frequency in past and present samplings of b. t. lusitanicus were significant (n = 98). statistical analyses were carried in r with package stats version 3.4.3 (r core team, 2013). results sequence analyses in total, 23 argk, 27 ef-1α and 30 16s sequences were obtained in the preliminary study. after trimming the sequences, the size of the argk fragment was 755 bp long. seven point-mutations were detected (table 2): five transitions (4 a↔g and 1 c→t) and two transversions (1 c→g and 1 c→a), but none of them were subspecies or geographic specific. the fragment amplified of the ef-1α gene was 522 bp long. sequence alignment did not show any point mutations. the 16s trimmed fragment was 504 bp long. two point-mutations were detected (table 2): a transversion (a→t) and a transition (a→g). the transversion was only found in one b. t. terrestris individual (ttb.02), whereas the transition yielded two haplotypes that discriminate between the subspecies: individuals from the tls group (iberian b. t. lusitanicus) presented a guanine (haplotype-g) and those from ttb (belgian b. t. terrestris) presented an adenine (haplotype-a). in the tts group (b. t. terrestris from spain), both haplotypes were found in five individuals each. case study the final sequence set included 153 individuals (table 1, table s2 in supplementary material). in relation to b. t. lusitanicus 47 out of the 48 (97.92%) sampled in the 80s and 43 out of the 50 (86%) from the recent samplings presented the g-haplotype. the individual from the old sampling with g-haplotype was located at the north of spain near the pyrenees, whereas the individuals from the most recent surveys were distributed at the north (2), center (2) and south (3) of spain (fig s1 in supplementary material). all the french and belgian b. t. terrestris individuals (100%) presented the a-haplotype; however, seven of the 18 b. t. terrestris individuals sociobiology 65(4): 558-565 (october, 2018) special issue 561 (38.89%) recently sampled in spain showed the a-haplotype, all of them from southern spain (sierra nevada n. p.). individuals considered morphologically as hybrids presented both haplotypes: two bear a-haplotype and four g-haplotype. overall, 91.84% b. t. lusitanicus individuals showed g-haplotype, while 77.56% b. t. terrestris individuals showed a-haplotype. kendall´s tau coefficient showed a significant correlation between subspecies and haplotype (tau = 0.705, p < 2.2e-16). however, when comparing french and belgian b. t. terrestris populations with spanish b. t. lusitanicus from the 80s, the correlation between both variables is almost one (tau = 0.973, p < 2.2e-16). pearson´s chi-squared test for iberian b. t. lusitanicus populations showed no significant differences (p = 0.074) between old and current individuals in haplotype segregation. genotyping and clustering results one individual (ttb.05) was removed due to low amplification quality. two possible siblings (tts.01 and tts.03, p = 1) were detected, therefore one sample (tts.01) was randomly chosen and removed from posterior analyses (n = 28). values of the number of alleles and heterozygosity (table 3) were similar between tls and ttb and slightly higher in tts. the number of private alleles was different in the three groups, again higher in tts. bayesian information criterion (bic) calculated prior to the dapc yielded one as the most probable number of clusters. even though, dapc models with k = 2 and k = 3 were studied, but no correlation was found between the formed clusters with tls, tts and ttb groups (data not shown). discussion sequences of the two nuclear genes ef-1α and argk did not discriminate between the subspecies b. t. terrestris and b. t. lusitanicus in congruence with previous studies based on dna barcoding (williams et al., 2012) and the cephalic labial gland secretions (coppée et al., 2008; lecocq et al., 2016). though some diversity was found along the argk sequence, it did not allow grouping the individuals into the two morphologically described subspecies. these genes were selected because they were previously used by kawakita et al. (2003) and hines et al. (2006) for analyzing bombus intraspecificity. in fact, hines et al. (2006) were able to distinguish between bombus lucorum (linnaeus) and bombus table 2. point mutations found in the alignments of the two genes (argk and 16s). genbank sequences from bombus terrestris were concatenated and taken as reference (argk: af492888.1, 16s: ay737386.1) (bp = position of the mutation in base pairs; . = same nucleotide as in the reference sequence; ― = missing data). argk 16s bp 69 269 393 558 705 706 749 161 263 reference g c g c a g c a a tls.01 . . . . . . . g . tls.02 a . a . . . . g . tls.03 . . a . . . . g . tls.04 ― ― ― ― ― ― ― g . tls.05 a . . . . . . g . tls.06 . . . . . . . g . tls.07 . . . . . . . g . tls.08 . . . . . . . g . tls.09 a . a . . . . g . tls.10 a . a . . . . g . tts.01 . . . . . . . g . tts.02 a . a . . . . . . tts.03 a . a . . . . . . tts.04 a . . . . . . g . tts.05 ― ― ― ― ― ― ― . . tts.06 a t a . . . . g . tts.07 a . . . . . . . . tts.08 . . . . . . . . . tts.09 a . a . . . . g . tts.10 a . a . . . . g . ttb.01 a . . . . . . . . ttb.02 ― ― ― ― ― ― ― . . ttb.03 a . . . . . g . . ttb.04 ― ― ― ― ― ― ― . . ttb.05 ― ― ― ― ― ― ― . . ttb.06 ― ― ― ― ― ― ― . . ttb.07 . . . a g a . . g ttb.09 a . . . . . . ― . ttb.10 a . a . . . . . . group n na ne np ho he tls 10 5.182±0.749 3.606±0.556 5 0.543±0.075 0.619±0.078 tts 9 6.273±1.000 4.668±0.783 13 0.646±0.108 0.669±0.083 ttb 9 5.727±0.821 3.842±0.645 8 0.645±0.093 0.621±0.081 table 3. population genetic parameters across the three bombus terrestris groups (tls = b. t. lusitanicus from the spain; tts = b. t. terrestris from spain; and ttb = b. t. terrestris from belgium) based on eleven microsatellite loci (n = number of individuals, na = number of alleles, ne = number of effective alleles, np = number of privative alleles, ho = observed heterozygosity and he = expected heterozygosity). d cejas; c ornosa; i. muñoz; p. de la rúa – b. terrestris subspecies molecularly characterized in spain562 hypnorum (linnaeus) populations from europe and china. in the same sense, the dapc analysis based on microsatellite did not show a clustering agreement with the previously morphological defined groups. this low genetic differentiation between bombus subspecies based on microsatellite variation has been reported before in mainland europe (estoup et al., 1996; moreira et al., 2015), as a consequence of the high gene flow in b. terrestris populations, probably intensified due to contact and hybridization between commercial and wild b. terrestris populations. interestingly, the mitochondrial gene sequenced here showed a variation which allows discriminating two haplotypes. the unique haplotype of the mitochondrial gene 16s retrieved in the iberian individuals was not found in the belgian and french b. t. terrestris individuals used as a reference. furthermore, a significant correlation was found between haplotypes and subspecies in the case study, so it might be considered that g-haplotype identified b. t. lusitanicus and a-haplotype to b. t. terrestris. this is supported by the fact that all except one of the individuals collected between 1977 and 1985 before the establishment of the rearing companies and the start of the commercial use of b. terrestris (velthuis & doorn, 2006), showed the g-haplotype. the presence of individuals of b. t. terrestris in spain may be due to two situations: on one hand, a natural contact area at the north of the iberian peninsula may have been established given the overlapping distribution range of both subspecies in the pyrenees (ornosa & ortiz-sánchez, 2004; rasmont et al., 2008; ornosa et al., 2017); on the other hand the presence of b. t. terrestris in the south of the iberian peninsula may be due to individuals that have escaped from greenhouses where they are widely used for pollination of crops such as tomatoes (fig 1). the detection of hybrids and individuals with non-matching morphological subspecies and 16s haplotype is an evidence of subspecific introgression. the existence of individuals with b. t. terrestris phenotype and b. t. lusitanicus haplotype suggests that hybridization between commercially reared bumblebees and wild b. t. lusitanicus populations has occurred. the opposite case has also been observed: individuals with b. t. lusitanicus phenotype and b. t. terrestris haplotypes. given the maternal inheritance of the mitochondrial molecule we can deduce that both commercial queens and males have colonized the environment, which is still a discussed topic (velthuis & doorn, 2006; kraus et al., 2010, lecocq et al., 2015), meaning that b. t. terrestris is naturalized, for now, in the south of spain. fig 1. frequency of bombus terrestris lusitanicus, bombus terrestris terrestris and hybrids (those with morphological subspecies identification and 16s haplotype that did not match i. e. b. t. lusitanicus with a-haplotype and b. t. terrestris with g-haplotype) in the national park of sierra nevada (southern spain). size of the circles is proportional to the total number of individuals sampled at each location. the high density of greenhouses can be seen in the lower right corner of the figure. ortophotograph obtained from http://www.juntadeandalucia.es. sociobiology 65(4): 558-565 (october, 2018) special issue 563 b. terrestris subspecies usually do not interbreed due to the specificity of the hormones secreted by the male cephalic labial glands (coppée et al., 2008). yet, in the case of b. t. terrestris and b. t. lusitanicus, the secretions are chemically very similar (coppée et al., 2008) allowing more usual copulation between them than with other b. terrestris subspecies. in other attempts of colonizing new lands by b. terrestris (ings et al., 2010), the subspecies b. t. sassaricus stopped to be seen in the south of france two years after the end of its importation, and no hybrids were found although no molecular analyses were performed. in the case study, naturalized individuals of b. t. terrestris on the iberian peninsula may have colonized the environment in the south where the breeding companies are based and, given their ability to breed with b. t. lusitanicus, the number of hybrid individuals in this area may increase in the future. the effects that this hybridization is having on wild populations are unknown (facon et al., 2011), as are the effects that these introduced populations may have on native flora (aizen et al., 2018). however, according to the expansion models of lecocq et al. (2015), b. t. terrestris should not be able to continue its colonization on the iberian peninsula, as it is not suitable for its climatic conditions. future studies including more localities within the iberian peninsula and using preferably the 16s marker and other highly variable nuclear markers such as snps (single nucleotide polymorphisms) will show the degree of introduction of commercial b. terrestris over the territory, what is of conservation concern due to a possible impact on the genetic diversity of iberian populations or even to the displacement of the native wild populations. acknowledgments we would like to thank kevin maebe and guy smagghe for providing samples needed for this study, laura jara, vicente martínez and ana isabel asensio for helping with the analyses and carlos ruiz for productive discussion and two anonymous reviewers for comments on a previous version. sampling permissions were obtained from the corresponding authorities (national parks of sierra nevada and guadarrama). this work has been supported by the ministry of economy and competitiveness (biobombus project cgl2012-34897), inia-feder (rta2013-00042 c10-05) and fundación séneca (plan of science and technology of the region of murcia, grant of regional excellence 19908/germ/2015). irene muñoz is supported by fundación séneca (murcia, spain) through the post-doctoral fellowship “saavedra fajardo” (20036/sf/16). supplementary material doi: 10.13102/sociobiology.v65i4.3442.s2222 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3442/0 authors’ contribution pdlr and co designed the experiments. co organized the sampling and identified the taxonomic status of individuals. dc made the experimental work. data analysis and results interpretation were carried on by dc and im. first draft of this paper was redacted by dc, and final draft was revised and approved by pdlr, im and co. references acosta, a.l., giannini, t.c., imperatriz-fonseca, v.l. & saraiva, a.m. (2016). worldwide alien invasion: a methodological approach to forecast the potential spread of a highly invasive pollinator. plos one, 11: e0148295. doi: 10.1371/journal.pone.0148295 aizen, m.a., morales, c.l., vázquez, d.p., garibaldi, l.a., sáez, a. & harder, l.d. 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forti et al., 1993; bueno & campos-farinha, 1999). recent toxicological analysis of several active ingredients used for pest control reveal that in general inhibitors of cellular respiration meet the requirements for use in baits to control leaf-cutting ants (nagamoto et al., 2004; decio et al., 2013). hydramethylnon acts on insect cellular respiration by inhibiting electron transport system and consequently blocking atp production and decreasing mitochondrial oxygen sociobiology 60(2): 150-153 (2013) 151 consumption. metabolism disruption and subsequent decrease in atp result in delayed mortality by this active ingredient (bloomquist, 2010; irac, 2010). in view of this, the aim of the current work was to assess the toxicity of hydramethylnon to workers of atta sexdens rubropilosa forel. material and methods the a. sexdens rubropilosa workers used in the assays, whose body mass was about 20-25 mg, were randomly picked from a laboratory nest kept at centro de estudos de insetos sociais (instituto de biociências, unesp – univ. estadual paulista, campus de rio claro, sp) and some specimens were deposited in the coleção entomológica adolph hempel (instituto biológico, são paulo – sp, brazil). before the assays, nests were daily supplied with leaves of eucalyptus sp., oat seeds and occasionally with leaves of other plants such as hibiscus sp., ligustrum sp. or rose petals. fifty ants were put into five petri dishes (ten ants per dish) for each treatment. during the assays the ants were maintained on an artificial diet prepared with glucose (50 g/l), bacto-peptone (10 g/l), yeast extract (1.0 g/l) and agar (15 g/l) in distilled water (0.1 l) (bueno et al. 1997). the diet (0.5 g per dish) with hydramethylnon (experimental) or without (control) was offered daily on a small plastic cap. hydramethylnon dissolved either in acetone (ha) or in acetone and soy oil (9 ml of acetone per 1 ml of oil) (hao) was added to the artificial diet at concentrations of 1 µg/ml, 5 µg/ml, 10 µg/ml, 100 µg/ml, 200 µg/ml and 1000 µg/ml. three controls were established to verify that the hydramethylnon toxicity results were not biased by the chosen solvent: one group received the artificial diet (‘diet control’), another group received the artificial diet with acetone (‘acetone control’), and a third group received the artificial diet with acetone and soy oil (‘acetone + oil control’). acetone or the combination of acetone and soy oil were added at the same proportions as those used for the hydramethylnontreated groups. during the assays, ants were maintained in an incubator at temperature of 25 ± 1ºc and relative humidity ranging between 70-80% for maximum length of 25 d and the number of dead was registered daily. the survival average 50% (s50) was calculated and survival curves were compared by the computer-assisted software graph-pad tm using the logrank test (elandt-johnson & johnson 1980). results and discussion mortality rates of both controls ‘acetone’ and ‘acetone + oil’ did not significantly differ comparing to the group that was only fed on artificial diet, no solvent added (diet control), indicating that solvents did not affect the mortality of a. sexdens rubropilosa workers (tables 1 and 2). hydramethylnon dissolved in acetone resulted in decreased worker survival which was more drastic at concentrations of 200 µg/ml and 1000 µg/ml. ant survival median time was reduced from 14 d (acetone control) to 10 d (200 µg/ ml) and 9 d (1000 µg/ml). all ants were dead on the 19th day for the concentration of 100 µg/ml, 16th for 200 µg/ml and 15th for 1000 µg/ml (table 1). hydramethylnon dissolved in acetone + soy oil also resulted in decreased worker survival being more drastic at 100 µg/ml, 200 µg/ml and 1000 µg/ml. ant survival me-µg/ml, 200 µg/ml and 1000 µg/ml. ant survival me-g/ml, 200 µg/ml and 1000 µg/ml. ant survival me-µg/ml and 1000 µg/ml. ant survival me-g/ml and 1000 µg/ml. ant survival me-µg/ml. ant survival me-g/ml. ant survival median was reduced from 13 d (acetone + oil control) to only 6 d (100 µg/ml, 200 µg/ml and 1000 µg/ml). mortality of all ants occurred on the 13th day for concentration 100 µg/ml and on the 9th day for concentrations of 200 µg/ml and 1000 µg/ µl (table 2). soy oil in the diet made the toxicity of hydramethylnon to leaf-cutting ants more potent, a fact that was shown by the higher ant mortality rate in the treatment with hydramethylnon dissolved in acetone + oil in comparison with the ants treated with hydramethylnon dissolved only in acetone. few studies have revealed the mode of action of oils in insects. hewlett (1947) suggested that intoxication by oil was due to the mechanical action, interfering with breathing by blocking the spiracles. on the other hand, there are authors who believe that toxicity of oils is attributed to chemical action (singh et al. 1978) or may act both physically and chemically in the insect (obeng-ofori 1995). for taverner et al. (2001), oils can act in the insect nervous system by increasing the permeability of neuron membrane and thus affecting ion change and stressing excitability of neuron cells. however, in leafcutting ants, the oil allows an alternative via for the ingestion of oil-soluble active ingredients due to the feeding behavior of workers (bueno et al., 2008; decio et al., 2013). adult ants feed primarily on liquid. the ingested food moves into the infrabuccal cavity of workers, wich remain for 24h. solid parts of the food are retained in the cuticle folds and spines present on infrabuccal cavity and are subsequently discared in the trash (fowler et al., 1991; moreira et al., 2011). nevertheless, the liquid portion of the food passes after the opening post-pharyngeal gland, where occurs the separation of water-soluble compounds and oil-soluble compounds. the water-soluble compounds moving into the crop and oilsoluble compound enter the ducts of post-pharyngeal gland, where they are absorbed and transferred to the hemolymph and subsequently to the whole body (bueno et al., 2008). thus, recently decio et al. (2013) observed major part of soy oil from the diet is likely allocated in the lumen of post-pharyngeal glands where it will be metabolized and suggested that ant post-pharyngeal glands are involved in the metabolism of lipids. the current work demonstrated that hydramethylnon has a great potential as active ingredient to be incorporated in baits for leaf-cutting ant control especially because of its slow mode of action. this characteristic is fundamental for fc bueno, lc forti, oc bueno toxicity of hydramethylnon to leaf-cutting ants152 xdens rubropilosa forel (hymenoptera: formicidae) isoladas do formigueiro e alimentadas com dietas artificiais. an. soc. entom. bras., 26: 107-12. bueno, o.c. & campos-farinha a.e.c. (1999). as formigas domésticas. in: mariconi, f.a.m. (ed.). insetos e outros invasores de residência. piracicaba: fealq 135-180. bueno, o.c., bueno, f.c., diniz, e.a. & schneider, m.o. (2008). utilização de alimento pelas formigas-cortadeiras. in: vilela, f.c. et al. (eds). insetos sociais: da biologia à aplicação. viçosa: editora ufv 96-114. decio, p., silva-zacarin, e.c.m., bueno, f.c. & bueno o.c. (2013). toxicological and histopathological effects of hy-toxicological and histopathological effects of hydramethylnon on atta sexdens rubropilosa (hymenoptera: formicidae) workers. micron, 45: 22-31. doi: 10.1016/j. micron.201210.008. elandt-johnson, r. & johnson, n.l. (1980). survival models and data analysis. john wiley and sons, new york. etheridge, p. & phillips, f.t. (1976). laboratory evaluation of new inseticides and bait matrices for the control of leafcutting ants (hymenoptera, formicidae). bul. entom. res., 66: 569-78. forti, l.c., della lucia, t.m.c., yassu, w.k., bento, j.m.s. & pinhão, m.a.s. (1993). metodologias para experimentos com iscas granuladas para formigas cortadeiras. in: della lucia, t.m.c. (ed.). as formigas cortadeiras. viçosa: folha de obtaining ant-insecticidal baits of high efficiency since it allows that the workers live long enough to spread the active ingredient inside the colony. the concept of slow toxicity was firstly used to select active ingredients for controlling fire ants (stringer et al., 1964; williams, 1983; vander meer et al., 1985) and more recently it was adapted by nagamoto et al. (2004) for leaf-cutting ants. the selection based on this concept favour compounds that cause low mortality on the first 24 h after application (lower than 15%), but high mortality at the end of the experiment (above 90%). acknowledgements we thank fapesp (fundação de amparo a pesquisa do estado de são paulo) and cnpq (conselho nacional de desenvolvimento científico e tecnológico) for providing financial support for this research. we also thank two anonymous reviewers for valuable comments that improved this article. references bloomquist, j.r. insecticides: chemistries and characteristics. in: <www.ipmworld.umn.edu/chapters/bloomq.htm. acess: 20 de dezembro de 2010. bueno, o.c., morini, m.s.c., pagnocca, f.c., hebling, m.j.a. & silva, o.a. 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(1990). overview. in: applied myrmecology: a world perspective. vander meer, r.k., k. jaffe & a. cedeno (eds) san francisco: westview press 493-495. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4277sociobiology 67(1): 13-25 (march, 2020) introduction ants represent a group of social insects found in all terrestrial ecosystems, with the exception ofthe poles (hölldobler & wilson, 1990; fernández, 2003). in the tropics, these insects present the higher richness, abundance and number of endemic species (guenard et al., 2012; baccaro et al., 2015). formicidae is composed of 20 subfamilies, 474 genera, 16,029 species (antweb, 2018). the ubiquity of these organisms and their multiple interactions with other species make them one of the dominant insect families in tropical terrestrial ecosystems (campos, 2011; morreau & bell, 2013). formicids construct subterranean nests (soil and subsoil), as well as in the litter, tree cavities and plant roots (baccaro et al., 2015; billen et al., 2016). they live in symbiosis with other insects, plants, fungi and bacteria, play a vital role in abstract ants live under ideal microclimatic conditions for the development of microorganisms. as mechanisms to ensure the health of the colony and as a defense strategy, these insects developed exocrine glands that work in the production of antibiotics (chemical defense) and in the immune defense of the colony. this study aimed to describe the state-of-the-art on extraction methods, chemical characterization and the antibiotic potential of glandular secretions of ants. this is a review of the scientific literature between 1989 and 2017. a total of 52 articles were selected. these addressed the behavior, chemical characterization, the antimicrobial effect and evaluated methods of extraction. the most investigated genera are atta, acromyrmexand crematogaster. the glands most reported in the articles involving extraction of secretions were dufour, mandibular and metapleural. the most reported methods of extraction were gland maceration and extraction with (organic) solvents and direct extraction of the gland. most studies evaluated secretions with respect to ant behavior. there is a paucityin the literature about the chemical characterization of most glandular secretions of ants, as well as for most taxa. the same deficiency is observed with regard to prospecting the antibiotic and antifungal potential of these secretions. sociobiology an international journal on social insects c guarda, ja lutinski article history edited by evandro nascimento silva, uefs, brazil received 28 november 2019 initial acceptance 09 january 2020 final acceptance 10 january 2020 publication date 18 april 2020 keywords bioprospecting, metapleural gland, social insects, resistance to antibiotics, antimicrobial secretions. corresponding author junir a. lutinski programa de pós graduação em ciências da saúde (ppgcs) universidade comunitária da região de chapecó-unochapeco av. senador attílio fontana, 591-e, efapi cep: 89809-000, caixa postal: 1141 chapecó, santa catarina, brasil. e-mail: junir@unochapeco.edu.br enriching the soil in many ecosystems. when attacking plants or animals, they act as natural agricultural pest enemies and participate in a multitude of additional interactions that shape terrestrial ecosystems (morreau & bell, 2013). in brazil, 1,458 species have been cataloged, distributed in 111 genera, which means that the country has one of the largest diversity of these organisms in the world (baccaro et al., 2015). ants are social insects that live in colonies characterized by a dense aggregation of related individuals interacting with each other (tragust, 2016; penick et al., 2018). their activities are regulated by climatic factors, such as temperature and humidity (lutinski et al., 2017). these factors, within the colony, generateideal microenvironments for the development of symbiotic microorganisms or that expose the colony to potentially lethal diseases (hölldobler; wilson, 1990; tranter et al., 2015; penick et al., 2018). programa de pós graduação em ciências da saúde, universidade comunitária da região de chapecó-unochapeco, santa catarina, brazil review glandular secretions of ants (hymenoptera: formicidae): a review on extraction, chemical characterization and antibiotic potential c guarda, ja lutinski – glandular secretions of ants (hymenoptera: formicidae)14 as an adaptation and survival strategy, ants present mechanisms to defend their colonies (junqueira et al., 2014; penick et al., 2018). protection against pathogens can occur through behaviors, production of antibiotics (chemical defense) and immune defenses (yek & mueller, 2011; penick et al., 2018). ant colonies need to be kept clean. valuable resources such as stored foods need to be preserved and group members, including immature forms, have to be protected. these ecological and life history characteristics of insect societies probably increased the selective pressure to develop external immune defenses (tragust, 2016; penick et al., 2018). ants are known to have the largest number of exocrine glands, with 84 glands already described (billen & sobotník, 2015), most of which are used in defense against pathogens (yek & mueller, 2011; vander meer, 2012). the glands most cited in the scientific literature are metapleural, mandibular, venom gland and dufour (tragust, 2016; billen, 2017). the metapleural gland is of special importance (pech & billen, 2017), it is exclusive to ants and consists of pair structures located in the posterolateral region of the thorax (bot et al., 2002; junqueira et al., 2014, pech & billen, 2017). it is known mainly for the production of antibiotics capable of inhibiting fungi and bacteria that develop within the colonies (yek et al., 2012; tragust et al., 2013; tranter et al., 2015). its unique presence in ants suggests a decisive role in the origin and ecological success of these insects (hölldobler; wilson, 1990; ward, 2010, tranter et al., 2015). the metapleural gland is absent only in species of the genus camponotus, suggesting that its secretions are used in the recognition of colonies or species and in the marking of nest entrances (yek & mueller, 2011). the dufour and venom glands are abdominal exocrine glands which occur in association with the reproductive tract (hölldobler & wilson, 1990; zhou et al., 2018). the dufour gland produces trail, recruitment and mating pheromones (mitra, 2013). the venom gland releases alarm and trail pheromones and especially acts on the capture and defense of prey as an integral part in the topical application of venom (mitra, 2013; billen, 2017; zhou et al., 2018). the mandibular gland occurs in the anterior region of the head, near the basal margin of the compound eye (billen et al., 2016). it is responsible for secreting compounds that are part of an alarm and recruitment system, sex pheromones and antibiotic substances (billen et al., 2016; billen & al-khalifa, 2018). most studies with the metapleural, mandidular, venom and dufour glands have the objective of studying the evolutionary origin and morphology of the gland (yek & mueller, 2011; viera et al., 2011; pech & billen, 2017; billen, 2017; zhou et al., 2018). only a few ant species have been studied in detail to identify active chemical compounds and their function (ortius-lechner et al., 2000; yek & mueller, 2011; tranter et al., 2015, tragust, 2016). this provides support to state that ants depend on antimicrobial secretions for defense against pathogens (penick et al., 2018). however, if all or most species of ants produce secretions with antibiotic potential, this is not known, remaining a gap in the scientific literature. people are daily exposed to pathogenic microorganisms that have the potential to cause disease. the bacterial genera responsible for causing the highest frequency of nosocomial infections are escherichia castellani and chalmers, 1919, staphylococcus rosenbach 1884, enterobacter hormaeche and edwards 1960, klebsiella trevisan, 1885 and fungi of the genera candida berkin, 1923 and aspergillus micheli, 1729 (anvisa, 2004; maciel & cândido, 2010; almeida & farias, 2014). however, the increasing development of resistance of microorganisms to conventional antibiotics has become a worldwide concern (loureiro et al., 2016). given the above, there is a need to prospect new antibiotics for human needs. ants represent a promising and relatively unexplored alternative (melo & fortich, 2013; song et al., 2012; penick et al., 2018). in this context, the study aimed to describe the state-of-the-art on extraction methods, chemical characterization and the antibiotic potential of glandular secretions of ants. methodology characterization of the study in this study, we conducted an integrative review of the literature on what was scientifically produced on the extraction and use of glandular secretions of ants. the integrative review consists of the analysis of the theoretical and empirical literature (mendes et al., 2008; souza et al., 2010). the main objective of the integrative review is to provide an understanding of the phenomenon analyzed, with the potential to build a scientifically grounded knowledge in previous studies (mendes et al., 2008; souza et al., 2010). search, selection and inclusion of articles the search for articles was performed in capes journals portal, in an advanced search using the terms “gland secretion” and “ants”, without selecting a specific database. two articles found in the biblioteca virtual emsaúde (bvs) and three selected in the scientific electronic library online (scielo) were included because they directly met the criteria determined for analysis, as follows:attygalle et al. (1998), nascimento et al. (1996), marsaro-júnior et al. (2001), quinet et al. (2012) as well as melo and fortich (2013). time series included articles published between 1989 and 2017. the selection of articles was based on searches in the capes journals portal database. the search was carried out from march to june 2018. only articles were selected, not including theses, dissertations, monographs and abstracts. the selection included works published in national and international journals, in portuguese, spanish and english. all articles have been downloaded to an electronic directory in the portable document format (.pdf) extension and identified. in all, 329 articles were found. sociobiology 67(1): 13-25 (march, 2020) 15 the preliminary analysis was based on the reading of the titles, abstracts and keywords. according to the selection criteria the selected articles were those that presented in their scope: a) extraction of glandular secretions or ant compounds; b) chemical characterization of glandular secretions of ants and; c) test or application of ant secretions. from this preanalysis, we selected the articles of the review. articles that met the selection criteria were read in full. information was extracted and tabulated in a database in excel for windows (.xlsx) (microsoft inc., 2010). the information extracted was year of publication, scientific journal, authors, title, objective, study focus, taxon, studied gland, extraction method, chemical characterization and application. analysis data were explored for frequency and results presented through graphs and tables constructed in excel for windows (.xlsx) (microsoft inc., 2010). results in all, 52 articles were included in the review. there was an upward trend in the frequency of studies using substances extracted from ants during the evaluated period. the highest number was observed in 2012 (n = 7), followed by the year 2000 (n = 6). however, there was a fluctuation in the frequency in the period (figure 1). fig 1. historical series and frequency of the studies performed for the extraction of substances from ants, between 1989 and 2017. a total of 26 (50%) studies aimed to analyze behavior, 20 (38.46%) performed chemical characterization, six (11.54%) tested the antimicrobial effect and two (3.85%) evaluated extraction methods. only five of the articles selected were developed in brazil. the ant genera most cited in the reviewed articles were crematogaster lund, 1831 (9), followed by atta fabricius, 1804 (8), acromyrmex mayr, 1865 (6), solenopsis westwood, 1840 (5), cataglyphis foerster, 1850 (4), camponotus mayr, 1861 (3), pogonomyrmex mayr 1868(3) and polyergus latreille, 1804 (3) (appendix). the glands most frequently reported in the articles involving extraction of ant secretions were dufour (21.15%), metapleural (15.38%) and mandibular (11.53%). it is worth mentioning that many studies have reported a combination of glands, parts of the ant or the whole ant in the studies (table 1). c guarda, ja lutinski – glandular secretions of ants (hymenoptera: formicidae)16 the extraction methods most reported in the articles were maceration of the gland and extraction with solvents such as acetone, chloroform, dichloromethane, hexane, methanol, pentane, ethanol, direct extraction of the gland and maceration of the gland in water. in most articles, the main objective is to evaluate the behavior of ants. only 11.54% of the articles tested the antimicrobial effect of substances extracted from ants (table 2). discussion the majority (50%) of the selected articles analyzed the action of glandular substances on the behavioral interaction of ants. a minority evaluated antimicrobial activity and methods forextracting glandular secretions. the genera crematogaster, atta, acromyrmex and solenopsis were mentioned in the largest number of studies related to the extraction, identification and application of secretions. the most reported glands involving substance extraction were dufour, metapleural and mandibular. the most reported methods of extraction were maceration of the gland and extraction with (organic) solvents and direct extraction of the gland. ants present 85 exocrine glands secreting chemical substances (adams et al., 2012; billen & sobotník, 2015). the evolutionary origin suggests that most of these glands are used in defense against pathogens (yek & mueller, 2011; vander meer, 2012). the ecological niche and selective pressure may influence the amount and composition of the glandular secretions produced (yek & mueller, 2011; billen, 2017). glands and/or tissues frequency (n) percentage dufour 11 21.15 metapleural 8 15.38 mandibular 6 11.53 dufour and venom gland 5 9.61 venom gland 5 9.61 whole ant and trisected 4 7.69 post-pharyngeal 3 5.76 mandibular and gastric 2 3.84 whole ant, dufour and venom gland 1 1.92 head capsule 1 1.92 mandibular and postpharyngeal 1 1.92 mandibular, labial, dufour, venom gland and ovaries 1 1.92 mandibular and metapleural 1 1.92 mandibular, dufour and venom gland 1 1.92 intramandibular. 1 1.92 post-pharyngeal and dufour 1 1.92 table 1. glands and/or tissues used in the studies conducted for the extraction of substances from ants. table 2. articles with different methods of extraction and evaluations to test the origin and functions of substances extracted from ants. authors extractionmethod evaluation attygale et al. (1989); nascimento et al. (1996); leclercq et al. (2000); ortius et al. (2000); yek et al. (2012); quinet et al. (2012); liu et al. (2017) direct extraction from the gland repellent, antimicrobial and chemical. mori et al. (2000); ortius-lechner et al. (2003); hölldobler et al. (2004); rojas et al. (2004) maceration of the gland and extraction in water behavioral and chemical. cammaerts & cammaerts (1998); dahbi & lenoir (1998); marsaro-junior et al. (2001) maceration of the gland and extraction in acetone behavioral and antimicrobial lahav et al. (1998); lahav et al. (1999) maceration of the gland and extraction in chloroform and methanol compounds as recognition discriminators. d’ettore et al. (2000); ruando et al. (2005); dahbiet al. (2008) maceration of the gland and extraction in pentane behavior of aggression, repellent and chemical kohl et al. (2000) maceration of the gland and extraction in diethyl ether origin of the trail pheromone hölldobler et al. (2002); i’allemand et al. (2010); maceration of the gland and extraction in dichloromethane behavior of aggression and trail pheromone. vieira et al. (2012); fixed and stained glands histochemical cruz-lopez et al. (2000); fujiwara-tsujii et al. (2007); brindis et al. (2008); lommelena et al. (2008); nikbakhtzadeh et al. (2009); vander meeret al. (2010); rifflet et al. (2011); plowes et al. (2014); wang et al. (2014); martins et al. (2015); campos et al.(2016); norman et al. (2017) maceration of the gland and extraction in hexane response of workers to the extract of queens’ glands, behavior; chemical and the presence of egg marking pheromones sociobiology 67(1): 13-25 (march, 2020) 17 the same compound can be produced by different glands and have several functions. ants are able to use a unique mixture of compounds and varying concentrations to alter the function of their glandular substances (adams et al., 2012). however, the synergies and interactions of multiple glandular products are far from being fully understood. for these reasons, the products of the exocrine glands of the ants remain an exciting and challenging field of research (adams et al., 2012). in the secretion of the metapleural gland, more than 20 compounds have been isolated. most substances are acids (ortius-lechner et al., 2000; yek & mueller, 2011; yek et al., 2012; fernández-marin et al., 2015) and protein substances (yek et al., 2012), with antimicrobial function (yek et al., 2012; mendonça et al., 2009; pech & billen, 2017). the mandibular glands, venom gland and dufour gland also present a broad spectrum of chemical compounds with proven toxic, repellent and antimicrobial effects (mendonça et al., 2009; quinet et al., 2012; tragust, 2016). crematogaster ants have well developed metapleural glands (attygalle et al. 1989; vander meer, 2012; tranter et al., 2015). c. inflata that occurs in southeast asia has the largest known metapleural glands (measured by counting secretory cells) (billen et al., 2011). the amount of secretion produced by the metapleural gland and the greater ease of extraction due to size may justify the number of studies performed with crematogaster ants. the chemical characterization of the secretions of this gland (although different from attines) presents carboxylic and phenolic acid moieties associated with the antibiotic role (yek& mueller, 2011) and negative effects on predatory arthropods (vander meer, 2012). thus, species of crematogaster or other ants nesting in tree cavities, in myrmecophytic epiphytes and related habitats have a need for protection against pathogens and predators (vander meer, 2012). however, if the other ant taxa with similar ecological characteristics have antimicrobial glandular secretions and with particularities as to their composition, this has not been investigated yet. species of atta and acromyrmex (tribe attini) occur in the neotropical region and live in colonies formed by a large number of individuals and in obligatory mutualism with a genetically homogeneous culture. evidence indicates the evolution of individual immune defenses increased or more elaborated in the form of an active antimicrobial secretion of the metapleural gland (vander meer, 2012; tragust, 2016). ruelet al. (2013); maceration of the whole ant, glands and eggs, extraction in dichloromethane behavioral castella et al. (2010) thorax and abdomen, extraction in cacodylate sodium and calcium chloride antimicrobial liu et al. (2017) maceration of the gland and part of the ant, extraction in hexane chemical mendonça et al. (2009) synthetic substances antimicrobial castracani et al. (2003) maceration of the gland and extraction in solvent (not identified) chemical vieira et al. (2012) gland extraction, direct analysis chemical sainz-borgo et al. (2013) maceration of the head capsule and direct analysis behavioral hernandez et al. (1999) maceration of the head capsule and extraction in hexane behavioral of the mandibular gland secretion wood et al. (2002); wood et al. (2011) maceration of the head capsule and extraction in dichloromethane behavioral; repellent and chemical voegtle et al. (2008) maceration of the head capsule, thorax and gaster, extraction in methanol chemical jones et al. (2007); adams et al. (2012) maceration of the whole ant and trisected ant, extraction in methanol chemical in phylogenetic context laurent et al. (2003) maceration of the whole ant e extraction in dichloromethane and methanol chemical melo & fortich (2013) maceration of whole ant and extraction in ethanol antimicrobial jones et al. (2004) maceration of whole ant and extraction chemical song et al. (2012) ant powder and extraction in dichloromethane antimicrobial table 2. articles with different methods of extraction and evaluations to test the origin and functions of substances extracted from ants. (continuation) authors extractionmethod evaluation c guarda, ja lutinski – glandular secretions of ants (hymenoptera: formicidae)18 the metapleural gland that varies in size among species, and in many taxa, produces an antimicrobial secretion that flows freely on the cuticle or can be spread by movements of the legs and thorax in some species (holldobler & wilson 1990; yek & mueller 2011). for these reasons, fungus-growing ants have encouraged research aimed at identifying antimicrobial substances from gland secretions, with special attention to the metapleural gland. ants of the genus solenopsis live in colonies in the soil (vander meer, 2012). in the secretion of venom and metapleural glands of species of this genus were identified compounds used as external immune defense, in the individual protection of the other members of the group and developing descendants (vander meer, 2012). some species of this genus are known for their invading potential and called fire ants due to the burning sensation associated with their bites (vander meer, 2012; liu et al., 2017). identifying the action of the venom that can cause allergies in humans and testing different extraction methods have been the main objectives of the research conducted with solenopsis ants. venom alkaloids of ants of this genus are basic and have comprehensive physiological activities that include antimicrobial activity. in turn, the secretion of the metapleural gland produces acidic antibiotics (vander meer, 2012. yek & muller, 2011). the effects of the combination of acidic and basic compounds as antimicrobials need to be better understood. a higher frequency of selected articles addresses glandular secretions of ants with the objective of evaluating the origin and action of the pheromones in the intraand inter-specific behavior of the ants (cammaerts & cammaerts, 1998; jones et al., 2004; norman et al., 2017). the chemical characterization of the substances present in the secretions was the second goal that most motivated the investigation of glandular secretions of ants (castricani et al., 2003; wood et al., 2011; martins et al., 2015). antimicrobial activity of glandular secretions of ants appears as the third most cited objective in the literature consulted (attygale et al., 1989; quinet et al., 2012; song et al., 2012). evidence in the studies consulted suggests that investment in antimicrobial substances has evolved repeatedly in social strains and may be particularly important for species living in large, complex societies such as ants of the tribe attini (atta and acromyrmex), made up of large numbers of individuals living in obligatory mutualism with a homogeneous culture of fungi (penick et al., 2018). nevertheless, only a minority of species have been studied to identify the chemical composition and to test the antimicrobial effect of the secreted compounds (song et al., 2012; penick et al., 2018). it is observed the need to increase the number of ant species investigated (penick et al., 2018). the dufour gland is an exocrine gland that occurs associated with the sting apparatus, mainly produces compounds of lipid character, where the long chain hydrocarbons are among the main compounds. this gland plays a key role in the behavior of ant species. since many of the compounds produced are involved in the chemical communication mechanism of the species (serrão et al., 2015). the results found in this review indicate that most of the studies tested the secretions of the dufour gland in the behavioral interaction of ants. metapleural gland products have acidic characteristics, expressed as moieties of carboxylic acid or phenol (yek & mueller 2011; vander meer 2012). the antimicrobial activity of the metapleural gland secretion has been repeatedly demonstrated in several species of ants of different ant subfamilies (tragust, 2016; penick et al., 2018). in the present review, it is observed that the studies developed with the purpose of identifying substances with antibiotic potential used mainly secretions of the metapleural gland. the mandibular glands are typically formed by a reservoir in which glandular cells release their secretory products (billen & al-khalifa, 2017). the secretions of the mandibular gland of leaf-cutting ants contain alcohols and low molecular weight ketones that induce alarm behavior related to inter-colony recognition (mendonça et al., 2009). in the secretion produced by the mandibular gland of atta sexdens, inhibitory activities were confirmed against bacteria and fungi, including those that are resistant to conventional antibiotics (mendonça et al., 2009). it is observed that the studies using mandibular glands had a behavioral approach, chemical characterization and antimicrobial effect. the dissection of the glands and subsequent extraction with organic solvents, such as hexane, was the most used method among the selected studies. this technique allows the extraction of secretions in larger quantities, allowing the extraction of alkaloids and proteins successively from multiple ants, including those of smaller sizes like solenopsis (melo & fortich, 2013; liu et al., 2017). the direct extraction of the gland with capillaries or through suction with the use of needle and syringe allows the extraction of pure and individual secretions for direct use, although the quantity is quite limited and the method is laborious (liu et al., 2017). it was identified the use of this technique primarily for the chemical characterization of the secretions, in order to reduce possible contaminations that could interfere with the results. there is a lack in literature about the chemical characterization of most glandular secretions of ants, as well as for most taxa. the same lack is found with regard to prospecting the antibiotic and antifungal potential of such secretions. acknowledgements to the coordination for the improvement of higher education personnel (capes) and to unochapecó for the financial support to the research through the graduate support program of higher education community institutions (prosuc). sociobiology 67(1): 13-25 (march, 2020) 19 author journal taxon objective attygalle et al. (1989) journal of chemical ecology crematogaster physotothorax emery,1889 (c. deformis) to describe the chemical composition and function of the secretion of the metapleural gland. nascimento et al. (1996) journal of chemical ecology atta sexdens rubropilosa forel, 1908 a. cephalotes (linnaeus, 1758) to test the antimicrobial effect of secretions of the metapleural gland. cammaerts & cammaerts (1998) behavioural processes pheidolepallidula (nylander, 1849) to demonstrate the neighborhood marking on the nest entrance of p. pallidula. dahbi & lenoir (1998) behavioral ecology and sociobiology cataglyphisiberica (emery, 1906) to analyze the consequences of the separation of nests in the presence of hydrocarbons of the post-pharyngeal gland and the effect of the mixture of individuals after their separation. lahav et al. (1998) behavioral ecology and sociobiology cataglyphisniger (andre, 1881) to evaluate the role of hydrocarbons as recognition discriminators. lahav et al. (1999) naturwissenschaften cataglyphisniger (andre, 1881) to compare the composition of postpharyngeal gland secretions; investigate the biosynthesis and hydrocarbon exchange of this gland between queens and workers. hernandez et al. (1999) journal of chemical ecology atta laevigata (smith, 1858) to carry out chemical analysis of the secretion of the mandibular gland of the different castes of the ant a. laevigata. ortius-lechner et al. (2000) journal of chemical ecology acromyrmex octospinosus (forel, 1899) to perform chemical analysis of the secretions of the metapleural gland of acromyrmex octospinosus. leclercq et al. (2000) tetrahedron crematogaster brevispinosarochai to perform chemical analysis of the secretion of the dufour gland in crematogaster. d’ettore et al. (2000) chemoecology polyergus rufescens (latreille, 1798) to chemically characterize the secretions of the dufour gland in queen and worker of p. rufescens. leclercq et al. (2000) tetrahedron letters, crematogaster rochai forel, 1903 (crematogaster brevispinosarochai) to identify venom compounds in the secretions of the c. brevispinosarochai dufour gland. more et al. (2000) insectes sociaux polyergus rufescens (latreille, 1798) to verify the effect of the secretion of the main tissue and the mandibular gland of p. rufescens on the behavior of the workers of the host species. kohl et al. (2000) naturwissenschaften mayriella overbecki viehmeyer, 1925 identify whether the trail pheromone originates from the venom gland. cruz-lopez et al. (2001) journal of chemical ecology solenopsis geminata (fabricius, 1804) to evaluate the response of s. geminate workers to queen venom gland extract. marsaro-júnior et al. (2001) neotropical entomology atta sexdens rubropilosa forel, 1908 to investigate the effect of the secretion of the mandibular gland of a. sexdens on the germination of conidia of the fungus botrytiscinerea pers. wood et al. (2002) biochemical systematics and ecology crematogaster mimosa santschi, 1914 crematogaster nigriceps emery, 1897 and crematogaster gerstaeckeri sjostedti mayr, 1907 to identify the chemical composition of secretions of the mandibular gland of three crematogaster species. hölldobler et al. (2002) chemoecology metapone madagascarica gregg, 1958 and metapone quadridentata eguchi, 1998 to evaluate predatory behavior and communication in two species of metapone and to present the first record of a trail pheromone in this genus. laurent et al. (2003) tetrahedron letters crematogaster nigriceps emery, 1897 to investigate the defensive mechanisms in the genus crematogaster and to evaluate the composition of the secretion of the dufour gland. castricani et al. (2003) journal of mass spectrometry polyergus rufescens (latreille, 1798) chemical characterization of the p. rufens mandibular gland. table 1. authors, publication period, ant taxon used and objective of articles involving extraction of ant secretions in the period between 1989 and 2017. c guarda, ja lutinski – glandular secretions of ants (hymenoptera: formicidae)20 ortius-lechner et al. (2003) insectes sociaux acromyrmex octospinosus (forel, 1899) to test whether the offspring of more diverse workers can produce a more variable spectrum of compounds of metapleural glands. hölldobler et al. (2004) chemoecology pogonomyrmex barbatus (smith, 1858), pogonomyrmex rugosus emery, 1895 and pogonomyrmex maricopa wheeler, 1914. carry out a chemical analysis of the secretions of the dufour glands p.barbatus, p. rugorus and p. maricopa. rojas et al. (2004) physiologicalentomology solenopsis geminata (fabricius, 1804) to evaluate whether the difference in attractiveness of the queen with the workers results from a variation in the components of the recognition pheromone in s. germinata. jones et al. (2004) journal of chemical ecology camponotus mayr, 1861 to perform chemical analysis of the mandibular gland of nine species of camponotus. ruano et al. (2005) journal of insect physiology rossomyrmex minuchae tinaut, 1981 to evaluate the secretion of the dufour gland of r. minuchae, which has a repellent used to stop and pacify during nest usurpation. fujiwara-tsujii et al. (2006) zoological science camponotus obscuripes mayr, 1879 perform chemical analysis of secretions of the venom and dufour glands. jones et al. (2007) journal of natural products myrmicaria melanogaster emery, 1900 perform chemical analysis of the venom of m. melanogaster. brindis et al. (2008) neotropical entomology solenopsis geminata (fabricius, 1804) to evaluate behavioral differences caused by the secretion of the dufour gland and set a relationship between the chemical profile of the dufour gland and the different sizes of workers. dahbi et al. (2008) biochemical systematics and ecology cataglyphis viaticus crawley, 1920, cataglyphis mauritanicus (emery, 1906) to describe the chemical content of post pharyngeal and dufour glands of c. viaticus, c. mauritanicus. lommelenaet al. (2008) journal of insect physiolog gnamptogenys striatula mayr, 1884 to identify the chemical compounds on the surface of eggs of g. striatula and the secondary source of these compounds. voegtle et al. (2008) journal of chemical ecology camponotus irritibilis and camponotus quadrisectus (smith, f., 1858) to describe three compounds from the mandibular gland of c. quadrisectus and c. irritibilis. nikbakhtzadeh et al. (2009) toxicon pachycondyla sennaarensis (mayr, 1862) to characterize the secretions of venom glands of the ant p. sennaarensis. mendonça et al. (2009) antonie van leeuwenhoek atta fabricius, 1804 to perform antimicrobial tests using synthetic components of the main components of the mandibular and metapleural glands. vander meer et al. (2010) journal of chemical ecology solenopsis invicta buren, 1972 to isolate and identify the source of a component of the alarm pheromone of the fire ant worker. castella et al. (2010) insectes sociaux formica selysi bondroit, 1918 to evaluate whether the social or ecological variation at the colony level has an impact on the individuals’ immunological investment. i’allemand & witte (2010) biological invasions anoplolepis gracilipes (smith, f., 1857) to analyze the trail communication of a. gracilipes using methods established for p. longicornis. rifflet et al. (2011) plos one crematogaster striatula emery, 1892 to evaluate the toxicity of dufour gland secretions of c. striatula. wood et al. (2011) biochemical systematics and ecology cephalotes alfaroi (emery, 1890) and cephalotes cristatus (emery, 1890) chemical characterization of the mandibular gland. yek et al. (2012) proceedings of the royal society b acromyrmex octospinosus (reich, 1793) to analyze whether the secretions of the metapleural gland of a. octospinosus ants can be adjusted according to the fungal conidia to which they are exposed. appendix. authors, publication period, ant taxon used and objective of articles involving extraction of ant secretions in the period between 1989 and 2017. (continuation) author journal taxon objective sociobiology 67(1): 13-25 (march, 2020) 21 vieira et al. (2012) journal of chemical ecology trachymyrmex fuscus, atta laevigata (smith, 1858) and acromyrmex coronatus (fabricius, 1804) apterostigma pilosum mayr, 1865 and mycetarotes parallelus (emery, 1906) ectatomma brunneum smith, 1858 and pogonomyrmex naegeli forel, 1878 to identify the chemical composition of the metapleural gland of fungus-growing and non-growing ants. adams et al. (2012) biochemical systematics and ecology trachymyrmex forel, 1893 and sericomyrmex mayr, 1865 to perform chemical comparison of a set of species of sericomyrmex from trachymyrmex in a phylogenetic context. song et al. (2012) bioorganic & medicinal chemistry letters tetramorium, mayr, 1855 to test antimicrobial activity of extracts of ants of the genus tetramorium. vieira et al. (2012) plos one trachymyrmex fuscus, atta laevigata (smith, 1858) and acromyrmex coronatus (fabricius, 1804) apterostigma pilosum mayr, 1865 and mycetarotes parallelus (emery, 1906) ectatomma brunneum smith, 1858 and pogonomyrmex naegeli forel, 1878 to compare morphological differences in the metapleural gland of fungus-growing and non-growing ant species. quinet et al. (2012) the journal of venomous animals and toxins including tropical diseases cremetogaster distans mayr, 1870, crematogaster pygmaea forel, 1904 and crematogaster rochai forel, 1903 to test the antibacterial property of the venom of three species of crematogaster. ruel et al. (2013) naturwissenschaften aphaenogaster senilis mayr, 1853 to identify whether the eggs of a. senilis can act as means to prevent the development of future queens. sainz-borgo et al. (2013) revista de biologia tropical acromyrmex landolti (forel, 1885) to identify the origin of the recognition signal in the ant body and the effect of the isolation on the recognition ability. melo & fortich (2013) revista colombiana de ciências químico-farmacéuticas crematogaster and solenopsis to evaluate antimicrobial activity of extracts produced with crematogaster and solenopsis ants. plowes et al. (2014) journal of comparative physiology messorandrei and messorpergandei (mayr, 1886) to identify whether the venom gland of m. pergandei and m. andrei species is a source of chemical recruitment compounds. wang & chen (2015) journal of chemical ecology monomorium minimum (buckley, 1866) to evaluate whether the defensive secretions can contribute to m. minimum competitive ability. martins et al. (2015) chemoecology neoponera villosa (fabricius, 1804) to identify the compounds of the intramandibular gland and the cuticle. campos et al. (2016) journal of insect physiology atta opaciceps borgmeier, 1939 to identify the components of the venom gland of a. opaciceps involved in trail marking. liu et al. (2017) florida entomologist solenopsis invicta buren, 1972 to evaluate the efficacy in the of venom alkaloid extraction using different methods. norman et al. (2017) journal of chemical ecology atta colombica guérin-méneville, 1844, atta cephalotes (linnaeus, 1758), acromyrmex echinatior, ac. octospinosus), sericomyrmex amabilis, trachymyrmex cornetzi and apterostigma pilosum. to identify and compare the chemical composition of the alarm pheromones of two species of atta, two species of acromyrmex, two “higher” attines and the “lower” attine. table 1. authors, publication period, ant taxon used and objective of articles involving extraction of ant secretions in the period between 1989 and 2017. 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(2018). morphology and ultrastructure of dufour’s and venom glands in the ant camponotus japonicus mayr (hymenoptera: formicidae). micron, 104: 72-79. doi: 10.1016/j.micron.2017.10.011. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.4366sociobiology 67(3): 433-443 (september, 2020) introduction among other factors, the success of perfect social organization in insects is built on recognition among nestmates. this phenomenon is a determinant for avoidance of predators and parasites, or even loss of resources (mitra et al., 2014). by the detection and emission of chemical compounds, insects find mating partners, food, or prey; choose oviposition locations; defend themselves against predators; and organize their colonies, in the case of social insects (zarbin et al., 2009). compounds that form the basis of interaction between and among individuals of the same species are known as pheromones, while in different species, these compounds are called allelochemicals (gullan & cranston, 2012). abstract juvenile hormone (jh) is considered the main determinant of caste in social insects, though little is known about how this hormone acts in social wasps, especially the independent-founding species. the known relationship between jh titer and caste in the colony and we suggest a relationship among the effects of jh and the cuticular chemical profile. therefore, this study aimed to test the hypothesis that topical application of jh to larvae of different instars alters the cuticular chemical composition of newly emerged females of mischocyttarus consimilis (zikán), influencing the dynamics of colony. two techniques were used to evaluate the variation in cuticular chemical composition: fourier transform infrared photoacoustic spectroscopy and gas chromatography coupled to mass spectrometry (gc-ms). indeed, the application of jh did significantly alter the cuticular chemical composition of adult females that received treatment at the larval stage in comparison to control. the effects of jh were instar-dependent in that the results of topical application were significant when performed at third larval instar. overall, these results add evidence that caste determination may, at least in part may be pre-imaginal in species of independent-founding social wasps. sociobiology an international journal on social insects ef neves1,2,3, ts montagna1,2, lc santos-junior1,2, kb michelutti2,4, cal cardoso4, lhc andrade4, sm lima4, wf antonialli-junior1,2,4 article history edited by fábio nascimento, usp, brazil received 07 february 2019 initial acceptance 03 july 2019 final acceptance 10 july 2020 publication date 30 september 2020 keywords cuticular hydrocarbons; caste determination; vespidae; nestmate; chemical communication. corresponding author kamylla michelutti programa de pós-graduação em recursos naturais universidade estadual de mato grosso do sul cep: 79804-970, mato grosso do sul, brasil. e-mail: kamylla_michelutti@yahoo.com.br pheromones are chemical compounds produced by exocrine glands and are released from dermal structures. other compounds in the cuticle of insects can also act as pheromones, promoting interactions between individuals of the same species. these compounds are mainly represented by hydrocarbons produced in the oenocytes, absorbed by lipophorin and transported by hemolymph (howard & blomquist, 2005; blomquist & bagnères, 2010; gullan & cranston, 2012). surface pheromones express patterns that vary according to sex, caste, or age, as described by antonialli-junior et al. (2007) for the ant ectatomma vizottoi (almeida), by kather et al. (2011) for apis mellifera (l.), and by neves et al. (2012) for the social wasp mischocyttarus consimilis (zikán). 1 programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil 2 laboratório de ecologia comportamental, universidade estadual de mato grosso do sul, dourados-ms, brazil 3 instituto federal de educação ciência e tecnologia de mato grosso do sul, naviraí-ms, brazil 4 programa de pós-graduação em recursos naturais, universidade estadual de mato grosso do sul, dourados-ms, brazil research article wasps effect of larval topical application of juvenile hormone on cuticular chemical composition of mischocyttarus consimilis (vespidae: polistinae) females ef neves et al. – effect of juvenile hormone on mischocyttarus consimilis434 these chemical compounds are known as cuticular hydrocarbons (chcs), and they are regulated by exogenous and genetic mechanisms that act together to express a unique chemical profile that reflects the cuticle composition of each individual. the chemical profile can be acquired within the first few hours after emergence, as reported by panek et al. (2001) for polistes fuscatus (f.) and by neves et al. (2012) for m. consimilis. this chemical profile is specific to each individual, signaling, for example, the role of each worker within the colony (ferreira-caliman et al., 2010; kather et al., 2011). specifically, alpha females responsible for reproduction possess cuticular chemical compounds different from those found in workers, as described in studies of the wasp species polistes versicolor (olivier) (torres et al., 2014) and polistes ferreri (sassure) (soares et al., 2014). in addition, some chcs of social insect queens may induce sterility in workers, as seen in the study of van oystaeyen et al. (2014) with the wasp species vespula vulgaris, the buff-tailed bumblebee bombus terrestris and the desert ant cataglyphis iberica. on the other hand, an important inducer of caste determination in social insects is juvenile hormone (jh), which is present in higher titers in the hemolymph of queen wasps, compared to the titers in workers (giray et al., 2005; tibbetts & huang, 2010). in ants, different levels of jh also generate morphological differences between castes and subcastes (nijhout & wheeler, 1982). in bees, such as a. mellifera, jh titers are regulated by differential feeding at the larval phase, which originates queens and/or workers (laidlaw, 1992). similarly, in termites, different jh titers in worker larvae are responsible for the formation of presoldiers, soldiers, and nymphs (park & raina, 2003). in swarming wasps, kelstrup et al. (2014) suggested that the titers of jh prepare the ovaries for further development since the titer of jh of possible future queens increases in the absence of established queens. however, in independent-founding wasps with an almost imperceptible morphological differentiation between queens and workers, caste determination, which is primarily based on behavioral and physiological patterns, is poorly understood (weaver, 1996). in this case, caste determination is proposed to be post-imaginal (solís & strassmann, 1990). in these wasps, jh activity can affect the aggression of queens, prevent ovarian development of workers, and determine the guard and foraging activities in workers (robinson & vargo, 1997; giray et al., 2005). however, kelstrup et al. (2015) pointed out that the role of jh is not restricted since even founders of closely related wasps that share the same environment (polistes dominula and polistes smithii) have divergent endocrine profiles. nevertheless, some studies found evidence that, at least in part, caste determination in independent-founding wasps may be pre-imaginal, with jh playing an important role (gadagkar et al., 1990; judd et al., 2010). in polistes metricus (say, 1831), differential feeding at the larval phase produces larger adults with greater reproductive potential (rossi & hunt, 1988; judd et al., 2010). only one study has tested the effect of topical jh application in larvae of independentfounding wasps. it found a significant enhancement of the signals for pre-imaginal caste determination among this group of wasps (montagna et al., 2015). the relationship between jh titer and caste in the colony suggests a potential relationship also between the jh titer and the cuticular chemical profile in mischocyttarus species, as already evaluated in colonies of the wasp polistes dominula (christ, 1791) (sledge et al., 2004; izzo et al., 2010) and the ant myrmicaria eumenoides (lengyel et al., 2007). nonetheless, a few studies have reported on the relationship between the effects of jh titers in cuticular chemical profiles (lengyel et al., 2007; kelstrup et al., 2017; oliveira et al., 2017), especially in independent-founding paper wasps. therefore, the objective of this study was to experimentally treat larvae from different instars by topical application of jh and analysis the effect of this application in the chemical perfil of m. consimilis newly emerged females. by so doing, we tested the hypothesis that such treatment would alter the chemical cuticular profile of m. consimilis newly emerged females and prove an alignment between the endocrine system and chemical expression. material and methods treatments and collection of individuals experimental manipulation was performed using eight colonies of the eusocial wasp m. consimilis nested in rural areas in the municipality of dourados (mato grosso do sul state, brazil; 22º13’16”s, 54º48’20”w). these colonies were in the worker-producing phase (jeanne, 1972). all colonies were transferred to wooden artificial shelters, measuring 1.2 x 1.2 x 2.5 m and covered with tiles for thermoregulation, constructed as described by montagna et al. (2015). the interior of each shelter contained four movable boards, positioned horizontally 1.90 m off the ground, which were attached to transversal clapboard using a hinge that allowed individual rotation of 180 degrees. a single colony was transferred and attached to each board, as described by prezoto and machado (1999). the rotation of the boards enabled the topical application of jh (hormone iii code j2000, sigma-aldrich, protons cientifica ltda) by gravity, precisely onto the larvae selected for treatment. for maximal accuracy of jh application onto the larvae, an automatic 2 μl capacity micropipette was used, following the methodology of montagna et al. (2015). the concentration and volume of jh applied to the larvae followed the protocol used by montagna et al. (2015) who evaluated if the responses to the application of jh in larvae of m. consimilis would vary according to developmental stage, especially from the 3rd instar. the topical applications of jh were performed in the 3rd, 4th and 5th instars. the application protocol of 1 μg of jh in 1 μl of acetone, as described by shorter and tibbetts (2009), was adopted here. sociobiology 67(3): 433-443 (september, 2020) 435 a total of 460 larvae received jh application, 202 larvae of 3rd instar, 164 larvae of 4th instar and 94 of 5th instar. as a form of control, the development of 37 larvae that did not receive any type of treatment or manipulation was monitored. the population size of each colony, together with the very high mortality rate caused by manipulation, as reported by montagna et al. (2015), made it impossible to apply the pure solvent in individuals from the same colony in order to assess its effect in isolation. larval instar determination was performed by measuring the cephalic capsule of the larvae used in each treatment, based on an established rule (dyar, 1890). for instar control, a larva of the same age as treated larvae was removed from the nest for cephalic capsule measurement. previous studies have recognized five instars during the larval development of this species (michelutti et al., 2017). in order to facilitate application to the different larval instars and collect newly emerged females, daily nest mapping was performed on hexagon-printed paper (giannotti, 1998). as a result, it was possible to perform a daily monitoring of each treated larva/control, allowing us to detect the cell from which the adult emerged. each newly emerged female, treated, or not, during the larval stage, received a mark with a colored dot on the leg. then, when the females completed 12 days of life, they were collected, anesthetized and sacrificed by freezing for further analysis of the cuticular chemical profile. all the newly emerged females from the larvae under treatment, together with the same number from the group without any treatment, were collected, separated, sacrificed, and preserved by freezing, hence avoiding the use of any type of chemical fixative or preservative that might react with the chemical components of the cuticle. analysis of the effect of jh topical application on cuticular chemistry by fourier transform infrared photoacoustic spectroscopy the variation in cuticular chemical compounds was accessed by the nondestructive fourier transform infrared photoacoustic spectroscopy technique (ftir-pas). this technique has been successfully used to evaluate differences between groups, but without evaluation of compounds that might be responsible for the differences, and it has already been applied in studies with ants and social wasps (neves et al., 2012; torres et al., 2014). ftir-pas is faster and less expensive than gas chromatography, the method most commonly employed, and requires minimal sample preparation. it enables the analysis of opaque and solid materials, is nondestructive (michaelian, 2003), and can provide rapid and reliable results. our study complements the one performed by montagna et al. (2015), who evaluated ovarian development on the same samples used here; therefore, we standardized by analyzing the thorax of all females. these samples were preheated in a vacuum oven for 48h to minimize the absorption of moisture, which could interfere with the spectrum. infrared photoacoustic spectroscopy was used to measure the radiation absorbed by the samples in the mid-infrared region (4000 400 cm-1). this region is sensitive to vibrations of molecular chemical groups, enabling the identification of molecular radicals and the types of chemical bonds in the sample (smith, 1999). during the analysis, the system was purged with dry air to remove particles, water vapor, and co2, and the photoacoustic chamber coupled to the microphone was purged with helium gas to increase sensitivity. for each sample, a mean spectrum was obtained from 64 scans with resolution of 8 cm-1. possible differences among the cuticular chemical compositions of newly emerged females from the treated larvae were evaluated using discriminant function analysis based on peak intensities of the mean spectra (quinn & keough, 2005). using the same form of analysis, we compared the differences between the groups on a par. analysis of the effect of jh topical application on cuticular chemistry by gas chromatography coupled to mass spectrometry to complement the analysis and evaluate the qualitative and quantitative differences caused by jh application, the same thorax samples evaluated by ftir-pas were also analyzed by gas chromatography coupled to mass spectrometry (gcms). each sample was immersed in 2000 μl of hexane for 2 min. subsequently, the solute resulting from the extraction was dried in a fume hood and stored frozen for a maximum of 30 days. for the chromatographic analysis, each extract was dissolved in 200 μl of hexane. the samples were analyzed using a gas chromatograph (gc-2010 plus, shimadzu, kyoto, japan) equipped with a mass detector (gc-ms ultra 2010, shimadzu, kyoto, japan). separation was achieved with a fused silica db-5 capillary column (5% phenyl dimethylpolysiloxane, 30 m x 0.25 mm x 0.25 μm thickness; j & w, folsom, california). the analytical conditions were as follows: helium (99.99%) carrier gas at a flow rate of 1.0 ml min-1; 1 μl injection volume, splitless injection mode; initial oven temperature of 150 °c, followed by a ramp to 300 °c at 3 °c min-1, and holding the final temperature for 10 min; injector, detector, and transfer line temperatures of 220, 300, and 200 °c, respectively. ms was operated in scan mode with electron impact ionization voltage of 70 ev, mass band from m/z 45 to 600, and scan interval of 0.3 s. identification of the compounds was based on the calculated retention indexes (van den dool & kratz, 1963), employing a series of linear alkanes (c14-c38, sigma-aldrich, purity ≥ 90%), comparison with literature indexes (bonavitacougourdan et al., 1991; howard et al., 2001; bonckaert et al., 2012; costanzi et al., 2013; weiss et al., 2015), and mass spectral data from the equipment libraries (nist 21 and wiley 229) for interpretation of the spectra when no standard was available for comparison. we only considered the peaks that occurred in at least 30% of each of the groups of samples evaluated. ef neves et al. – effect of juvenile hormone on mischocyttarus consimilis436 the differences among the cuticular chemical compositions of newly emerged females from larvae under different treatments were evaluated by discriminant function analysis, considering the relative abundances of the peaks in the chromatograms, using all the peaks detected (quinn & keough, 2005) and systat 12 software. using the same form of analysis, we compared the differences between the groups on a par. results of all 460 larvae that received jh treatment in the larval phase, 202 corresponded to 3rd instar larvae, out of which only 29 larvae reached adulthood; of the 164 larvae of 4th instar, 34 reached adulthood, and of the 94 larvae that received jh application in the 5th instar, only 18 individuals completed their development to reach adulthood. analysis of the effect of jh topical application on cuticular chemistry by ftir-pas. visual inspection of the mean spectra obtained by ftir-pas suggested that differences among the studied groups were subtle (fig 1). however, discriminant analysis showed that the cuticular chemical profiles of the controls and newly emerged females from larvae that received topical applications of jh were significantly different (wilks’ lambda = 0.329; f = 2.847; p = 0.0012). the first canonical root explained 69% of the results, while the first and second roots together explained 84% of the results. fourteen peaks were analyzed, and five (at 1041, 1373, 1650, 2962, and 3093 cm-1) were significant for separation of the groups (figs 1 and 2; table 1). the analysis between groups, pair by pair, shows that only females, the larvae of which were applied with jh in the 3rd instar, and controls had significant difference in cuticle composition (f = 5.310; p = 0.027) and that functional group 8 (1650) n-h and/or c-n (amide ii) was/were the most important compound(s) for the separation of these two groups. analysis of the effect of jh topical application on cuticular chemistry by gas chromatography visual inspection of the chromatograms obtained by gc-ms (fig 3) showed that the groups receiving jh application at the third, fourth, and fifth larval instars presented fig 1. mean ftir-pas spectra of mischocyttarus consimilis thoraxes following jh application to different larval instars. arrows indicate the peaks used in the statistical analysis. fig 2. dispersion diagram of the results of discriminant analysis applied to the ftir-pas data, showing the two canonical roots of differentiation between the control group and the groups with application of juvenile hormone to different larval instars of mischocyttarus consimilis. sociobiology 67(3): 433-443 (september, 2020) 437 table 1. wavenumber, coefficients of the two canonical roots, functional group, and vibration mode of peaks identified in the mean ftir-pas spectra for wasps with jh application to different larval instars. peak wavenumber (cm-1) functional group vibration mode first canonical root second canonical root 1 1041 c-h bending in the plane 2.724 3.529 2 1079 c-h bending in the plane 3 1110 c-h bending in the plane 4 1157 c-h bending in the plane 5 1373 c-ch3 symmetrical bending -1.017 2.290 6 1450 ch2 scissors 7 1542 n-h and/or c-n (amide ii) balance in plane 8 1650 n-h and/or c-n (amide ii) plane bending and/or asymmetrical stretching 1.723 2.973 9 2854 ch2 symmetrical stretching 10 2877 ch-ch3 symmetrical stretching 11 2931 ch2 asymmetrical stretching -12 2962 ch3 asymmetrical stretching -0.890 4.841 13 3093 n-h harmonic bending 2.949 1.965 14 3425 n-h stretching fig 3. representative chromatograms for the control group and the groups with the 3rd, 4th, and 5th larval instars subjected to application of juvenile hormone, indicating the 10 compounds that were most statistically significant for separation of the groups. 1 = pentadecane; 2 = unknown; 3 = octadecane; 4 = unknown; 5 = 3-methylheneicosane; 6 = pentacosane; 7 = 9,13-dimethylpentacosane; 8 = y-methylheptacosane; 9 = 7-methylheptacosane; 10 = nonacosane. some compounds with greater intensity compared to the control group. for example, 7-methylheptacosane was more intense for the group that received jh at the 4th and 5th larval instar, while heptacosane and 3-methylpentacosane were more intense for all the treatments compared to the control group (fig 3). a total of 30 peaks were detected, of which 29 were identified (table 2). the statistical analysis showed that 16 peaks were significant for separation of the groups and that 10 stood out as being the most statistically significant (fig 3; tables 2 and 3). the discriminant analysis demonstrated that the cuticular chemical profiles of the control group and newly emerged females from larvae that received topical application of jh at different instars were significantly different (wilks’ lambda = 0.00; f = 2.83; p < 0.05). the first canonical root explained 71.6% of the results, and the first and second roots together explained 20% of the results (fig 4). ef neves et al. – effect of juvenile hormone on mischocyttarus consimilis438 in all samples that received jh treatment and in the control, higher levels of branched alkanes were found, followed by linear alkanes and a lower percentage of alkenes (fig 5). all identified compounds were shared by all types of samples (fig 5). the compound with the highest content in all types of samples was tricosadiene (table 2). major compounds found for specific treatment groups were as follows: 7-methylheptacosane (5th instar); heptacosane (4th, 5th instars and control); heneicosane (3rd instar); 2-methyloctacosane and 3-methyltriacontane (4th instar); 9,21-dimethyltritriacontane (3rd instar); 3-methylnonacosane (3rd and and 5th instars and control); 9-methylheneicosane (5rd instar and control) and nonacosane and 13-methylnonacosane (3rd and 4th instars) (table 2). the analysis between the groups, fig 4. dispersion diagram of the results of discriminant analysis applied to the gc-ms data, showing the two canonical roots of differentiation between the control group and the groups with application of juvenile hormone to different larval instars of mischocyttarus consimilis. fig 5. relative abundances of the compounds identified by gc-ms for the control group and the groups with application of juvenile hormone to different larval instars of mischocyttarus consimilis. pair by pair, shows that only the females, the larvae of which was applied with topical jh in the 3rd instar, and controls had significant difference in cuticular composition (f = 5.310; p = 0.027). in this analysis, heneicosane, 3-methylpentacosane, 13-methylnonacosane, 9,21-dimethyltritriacontane, and 11,15-dimethyltritriacontane were the most important compounds for the separation of these two groups. discussion the results demonstrated that topical application of jh at different larval stages significantly altered the cuticular composition of newly emerged females and that the results were instar-dependent. our findings complement the results of montagna et al. (2015) who found that an increase of jh titer at the larval stages altered the phenotype of adult females. in this sense, behavioral and morphological aspects are closely related to cuticular chemical composition (kelstrup et al., 2017; oliveira et al., 2017). indeed, kelstrup et al. (2014) also found a relationship between cuticular compounds and castes, age and reproductive status of females in the social wasp polybia micans. even though the effects of jh application found here were similar to those of studies such as kelstrup et al. (2014) and oliveira et al. (2017), some studies, such as kelstrup et al. (2015), report that the effects of jh may not be the same, even in closely related species. although the results of both techniques showed that cuticular chemical composition was significantly altered by the treatments, the techniques provided the significant difference between treatments at the 3rd and the control. montagna et al. (2015) also reported that 3rd instar was the larval stage for achieving the most significant results following application of jh. when jh application was performed at the 3rd instar, adult sociobiology 67(3): 433-443 (september, 2020) 439 females grew significantly larger, remained longer in the nest, and became less aggressive, compared to the control group and the other treatments. studies with social bees showed that topical application of jh leads to significant effects that are especially dependent on the instar to which the hormone is applied. in studies performed by hartfelder and rembold (1991) with the bee scaptotrigona postica depilis (latreille), it was observed that the period most sensitive to increased jh titer was between the fourth and fifth larval instars. in another work conducted by antonialli-junior and cruz-landim (2009) with a. mellifera, it was found that application of jh at the 3rd larval instar led to results that were more significant, leading compounds ecl 3rd instar 4th instar 5th instar control percentage (% ± standard deviation) octadecane 1800 0.24±0.21 0.11±0.09 0.22±0.23 0.43±0.75 3-methyloctadecane 1876 0.34±0.14 2.09±1.68 1.35±1.32 1.96±3.70 unknown 1970 1.21±1.28 0.32±0.35 2.06±5.66 0.31±0.42 x-methylnonadecane 1975 0.73±0.85 0.63±0.64 0.31±0.24 0.63±1.12 eicosane 2000 3.16±1.36 2.63±1.77 2.46±1.22 4.19±3.53 heneicosane 2100 6.45±5.631 1.97±1.81 0.72±0.34 0.71±0.63 9-methylheneicosane 2146 4.51±2.66 3.24±2.85 6.95±9.981 16.05±19.581 tricosadiene 2172 34.38±18.131 26.53±16.141 39.67±21.991 37.39±22.221 docosane 2200 1.44±0.44 1.19±0.58 2.10±1.29 2.09±1.01 tricosane 2300 2.86±1.45 2.72±1.69 3.78±2.36 2.99±2.63 9-methyltricosane 2331 0.54±0.49 0.48±0.57 0.45±0.29 0.57±0.43 pentacosane 2500 1.34±0.59 1.63±1.09 2.39±1.67 1.28±0.96 13-methylpentacosane 2538 0.47±0.27 0.36±0.50 0.33±0.28 0.31±0.33 5-methylpentacosane 2555 0.20±0.08 1.06±1.26 1.14±1.22 0.33±0.39 3-methylpentacosane 2567 2.05±1.71 1.99±2.50 0.56±0.85 0.22±0.45 5,9-dimethylpentacosane 2584 0.40±0.42 0.72±0.82 0.94±1.44 1.27±2.04 heptacosane 2700 2.39±1.78 5.06±7.011 4.06±5.55 2.26±2.10 7-methylheptacosane 2738 1.07±1.00 7.30±11.98 7.23±15.011 2.31±2.95 3-methylheptacosane 2778 6.17±4.991 4.82±6.19 5.45±4.531 6.05±5.591 14-methyloctacosane 2834 0.33±0.17 2.26±5.81 0.99±1.48 0.56±0.49 2-methyloctacosane 2859 0.91±1.28 7.50±10.671 1.58±1.60 0.45±0.66 4,8-dimethyloctacosane 2894 0.76±0.72 0.25±0.32 0.23±0.22 0.56±0.66 nonacosane 2900 5.51±4.161 5.03±3.431 4.07±3.27 4.28±2.58 13-methylnonacosane 2937 7.26±2.831 5.03±6.101 1.49±0.91 1.53±1.27 7-methylnonacosane 2942 0.95±0.34 0.23±0.39 0.48±0.43 1.52±3.80 3-methylnonacosane 2975 4.19±3.12 4.36±2.50 3.77±2.45 4.71±2.69 triacontene 2983 1.43±1.49 1.59±2.24 2.65±3.75 2.24±3.92 14-methyltriacontane 3035 1.18±2.34 1.83±1.84 0.84±0.99 0.47±0.67 3-methyltriacontane 3074 2.02±1.84 6.00±5.651 1.59±2.05 1.63±2.72 9,21-dimethyltritriacontane 3364 5.54±3.041 1.09±1.21 0.15±0.31 0.69±0.58 table 2. mean relative abundances and standard deviations for cuticular compounds identified by gc-ms in the control group and the groups with application of juvenile hormone to different larval instars of mischocyttarus consimilis. 1= major compounds. to greater preservation of the ovaries. similar findings have also been reported for other social hymenopterans, such as the ant harpegnathos saltator (jerdon), as demonstrated by penick et al. (2012), who concluded that application of jh at the 3rd and 4th larval instars resulted in adult females with greater propensity to become queens. the compound 3-methylheptacosane, although not major, presented a percentage 2-fold higher after jh treatment relative to control. indeed, this compound is important in females with greater hierarchical status in colonies, as reported by oi et al. (2015). in this study, the author demonstrated that this compound is used by queens of the species vespula vulgaris to mark their eggs and thus signal their presence to the workers. ef neves et al. – effect of juvenile hormone on mischocyttarus consimilis440 the analyses show that the significant differences found between jh treatments with the cuticle composition of the control females were between larvae with application of jh in the 3rd instar and, still, 4 compounds (heneicosane, 3-methylpentacosane, 13-methylnonacosane, 9,21-dimethyltritriacontane or 11,15-dimethyltritriacontane) were important for the separation between these two groups. of these four compounds, 3-methylpentacosane was found in higher levels in the cuticle of queens of polybia micans compared to the cuticle contents of workers (kelstrup et al., 2014), indicating its role in differentiating reproductive status within the wasp colony. on the other hand, 13-methylnonacosane was described in the study of soares et al. (2017) as one of the compounds that occurred in the cuticle of all studied wasp species of the genus mischocyttarus, table 3. compounds that presented statistically significant values, highlighting the 10 most significant ones for separation of the different larval instars that received jh treatment and control of the species mischocyttarus consimilis, as assessed by gc-ms. 1= 10 most significant compounds for separation of the groups. compounds calculated index root 1 root 2 octadecane1 1800 3.905 0.588 3-methyloctadecane1 1876 3.335 0.460 unknown 1970 1.858 1.955 x-methylnonadecane 1975 0.888 0.135 eicosane 2000 0.988 2.264 heneicosane1 2100 3.356 1.591 9-methylheneicosane1 2146 2.022 5.055 tricosadiene1 2172 9.763 8.481 docosane 2200 1.574 -0.691 9-methyltricosane 2331 -0.896 1.793 pentacosane 2500 2.924 -1.599 13-methylpentacosane 2538 1.308 3.162 5-methylpentacosane 2555 0.057 0.204 3-methylpentacosane 2567 1.963 1.066 5,9-dimethylpentacosane 2584 0.433 -2.122 heptacosane 2700 0.921 1.722 7-methylheptacosane1 2738 4.974 1.252 3-methylheptacosane1 2778 2.798 5.742 14-methyloctacosane1 2834 2.078 3.678 2-methyloctacosane 2859 0.724 1.480 4,8dimethyloctacosane1 2894 3.895 0.693 nonacosane 2900 -0.733 0.070 13-methylnonacosane 2937 -2.008 -0.426 7-methylnonacosane 2942 1.541 2.229 3-methylnonacosane1 2975 -4.364 0.275 triacontene 2983 1.291 0.291 14-methyltriacontane 3035 0.607 1.420 3-methyltriacontane 3074 0.033 -0.284 9,21-dimethyltritriacontane1 3364 5.312 1.644 suggesting its importance for colonies of these social wasps. linear alkane heneicosane is a key component in the recognition among nesting companions of vespa crabro (ruther et al., 2002). thus, of the four compounds, evidence suggests that at least 3 appear to mediate important interactions in the colonies of the species where they are present. kelstrup et al. (2017) also found a clear association between jh level, ovarian status and the proportions of hydrocarbon classes in b. longitarsus females. similarly, oliveira et al. (2017) demonstrated that vespula vulgaris workers treated with jh acquired a chc profile that became similar to that of the queen. all the samples showed a greater abundance of branched and linear alkanes and lower abundance of alkenes (fig 5) in agreement with the findings of sledge et al. (2001) in p. dominula, tannure-nascimento et al. (2007) in polistes satan (bequaert) and michelutti et al. (2017) in m. consimilis. methyl-branched compounds represented the majority of compounds and were also the most responsible for separating the groups. according to kather and martin (2015), this category of methyl-branched compounds, together with alkenes, consists of the most important compounds for the separation of groups of hymenoptera. gibbs (2002) pointed out that branched alkanes and alkenes are fundamentally important in chemical communication, whereas linear alkanes are compounds mainly responsible for protecting insects against desiccation. nonetheless, it is possible that linear alkanes might play a role in intraspecific recognition since lorenzi et al. (2004) showed that p. dominula workers less than a day old, when treated with linear alkane extracts, were not accepted by their nestmates, suggesting that these compounds may also be used as signals for intracolonial recognition. based on the results of this study, it can be concluded that the topical application of jh in different larval instars alters the chemical profile of newly emerged females in m. consimilis. the findings complement the results of the work performed by montagna et al. (2015) who reported that treatment of the larvae of this species at the 3rd instar led to larger females with greater potential to become gynes. therefore, the present results add further evidence that caste determination may, at least in part, be pre-imaginal in species of independent-founding social wasps. acknowledgments the authors thank: fundação de apoio ao desenvolvimento do ensino ciência e tecnologia do estado de mato grosso do sul (fundect) for doctoral scholarship granted to the first author (fundect/capes n° 03/2014). coordenação de aperfeiçoamento de pessoal de nível superior (capes) for doctoral scholarship granted to the second and fourth author. authors calc (grant number 311975/2018-6), lhca (grant number 305412/2014-0), sml (grant number 304029/20157) and wfaj (grant number 308182/2019-7) acknowledge research grants from conselho nacional de desenvolvimento sociobiology 67(3): 433-443 (september, 2020) 441 científico e tecnológico (cnpq). sisbio for authorization of the collect and of the transport of the specimens (sisbio license no.1748-1). author contributions erika f. neves and thiago s. montagna collection, data analysis; claudia a. l. cardoso, chemical data analysis; luis h. c. andrade and sandro m. lima, ftir-pas analysis; erika f. neves, luiz c. santos-junior and kamylla b. michelutti manuscript writing and william f. antonialli-junior, general supervision, discussion and writing of the data. the authors declare no conflict of interest. references antonialli-junior, w.f., lima, s.m., andrade, l.h.c. & suarez, y.r. 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(2009). feromônios de insetos: tecnologias e desafios para uma agricultura competitiva no brasil. química nova, 32: 722731. doi: 10.1590/s0100-40422009000300016 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.43-51sociobiology 61(1): 43-51 (march, 2014) evaluation of insects that exploit temporary protein resources emphasizing the action of ants (hymenoptera, formicidae) in a neotropical semi-deciduous forest lc santos-junior1,2, jm saraiva2, r silvestre1, wf antonialli-junior1,2 introduction the insects of the order hymenoptera have a wide diversity of habits and complex behaviors, culminating in the social organization of wasps, bees and ants (wilson, 1971; triplehorn & jonnson, 2011; rafael et al., 2012), in the tropics, ants have a strong presence in most terrestrial ecosystems (fittkau & klinge, 1973, erwin, 1989, stork, 1991, longino et al., 2002; ellwood & foster, 2004). these insects have broad geographic distributions and high species richness, forming one of the most ecologically successful groups (hölldobler & wilson, 1990; longino et al., 2002), and more than 2000 species are estimated to inhabit the neotropical region (fernández, 2000). the evolutionary success of ants is due to several aspects of social life, but especially the strategies for obtaining resources, particularly food, for their colonies. abstract the majority of the ants is opportunistic and generalist foragers, commonly feeding on vegetable secretions, seeds, and living or dead animal material. they may be present on any type of substrate even, occasionally on carcasses. this work, then, aimed to evaluate the action of insects, especially ants, in the exploitation of protein resources in forest environment. monthly collections were made over a year and, in each collection, were made observations during 12 consecutive hours. to simulate exposure of protein resources we used three types of baits, sardines, beef liver and chicken. to evaluate the importance of ants on protein resources for each type of bait there was a control replica with physical barrier to prevent their access. the ants were observed on all baits throughout the collection period. in total, the baits were visited by 34 species of ants. the main genus of ants to visit the baits were: pheidole, crematogaster and solenopsis. these results demonstrate that the presence of ants is important to ecological succession on temporary protein sources in forest environments interfering in the occurrence of other frequent groups in this type of resource, such as flies, for instance. the species that dominated the baits, when presents, were those that regardless of size and aggressiveness, presented mass recruitment and exploited the baits with large flow of individuals although some species that exhibit certain characteristics can locate the baits faster and eventually dominate them at some point, depending on the ants species that co-occur, the results for the sequence of colonization can be modified. sociobiology an international journal on social insects 1 universidade federal da grande dourados, dourados, mato grosso do sul, brazil. 2 universidade estadual do mato grosso do sul, mato grosso do sul, brazil. article history edited by: edilberto giannotti, unesp, brazil received 23 august 2013 initial acceptance 10 octuber 2013 final acceptance 12 december 2013 keywords formicidae, interspecific competition, foraging activity corresponding author luiz carlos santos junior post graduation program in entomology and biodiversity conservation universidade federal da grande dourados highway dourados/itahum, km 12 post office box 241 79804-970, dourados, ms, brazil e-mail: lc.santosjunior@yahoo.com.br some groups have a more specialized feeding mode, such as fungi cultivators (weber, 1972); others particularly prefer liquid food (delabie & fernández, 2003); and, mostly, ants are opportunistic and generalist foragers, commonly feeding on vegetable secretions, seeds, and living or dead animal material (fowler et al., 1991; kaspari, 2000). ants may be present on any type of substrate if conditions are favorable for foraging. according to clark and blom (1991), vertebrate or invertebrate carcasses, even if only occasionally, can be a source of additional food for ants that normally feed on seeds, for example. the decision made by an ant when locating a resource is to maximize the energy balance, in order to obtain a higher gain at low energetic cost for obtaining food, as predicted by the optimal foraging theory (sih, 1982 a, b, stephens & krebs 1986). due to restrictions on dominating and carrying research article ants lc santos-junior et al. exploration of protein resource by ants in a semi-deciduous forest44 resources, small ants should recruit other ants to ensure their domination after encountering a resource, avoiding the loss of that resource to a larger ant, or other animals (pearce-duvet & feener junior, 2010). additionally, the recruitment speed is directly related to the amount of resources that an ant can carry. therefore small ants should recruit faster than larger ants, since the smaller body size is satisfied quickly. the speed of food sources recruitment can be an important determinant of ant’s communities, since the evolutionary trade-off between exploitative and interference competition may be a key influence to the dominance of resources (davidson, 1998; parr & gibb, 2012). the intra or interspecific competition during the foraging activity occurs when individuals exploit similar resources (begon et al., 2006). the competition also occurs when there is an overlap of activity periods and areas for several species of ants that visit the same food source employing similar foraging strategies (petal, 1978; brandão et al., 2000; hölldobler & wilson, 1990). a species is competitively superior and considered dominant when it presents features that allow the monopoly of the resource, such as aggressive behavior or mass recruitment. the other species that co-occur with the dominant and do not have these characteristics are considered subordinate and usually have alternative strategies for obtaining resources (andersen, 1992; brandão et al., 2000). an example of these strategies is the infiltration behavior, in which some individuals of a subordinate species infiltrate among the dominants using a small fraction of the available resources (brandão et al., 2000; parr & gibb, 2010). these aggressive behaviors between individuals can lead in some resource domination cases by workers of one of the species preventing the access of others (brandão et al., 2000). the relationship between dominant and subordinate species can also be influenced by environmental factors. this influence can occur by direct physiological effects resulting from the tolerance of each species to microclimatic variations (de bie & hewitt, 1990). in particular, when competing species are subjected to a limiting condition, the dominant species may no longer use the resource as a way to reduce the physiological stress. consequently, subordinate species may take a risk under such adverse conditions and use the resource (bestelmeyer, 2000). in this sense, especially for ants, temperature is one of the most important factors, since the myrmecofauna is sensitive to desiccation. thus, high temperatures can influence the foraging strategy of ants and therefore their interactions on resource places (cerdá et al., 1997, 1998). therefore, this study aimed to evaluate the action of ants, especially the interspecific relationships, while visiting temporary protein sources in a forest environment. material and methods samples were collected monthly between june 2010 and july 2011 in a forest fragment of around 800,000 sqm (square meters). this area is semi-deciduous forest, according to the classification system of ibge, the brazilian institute of geography and statistics (veloso et al., 1991), and is located in dourados, mato grosso do sul (s 22º12’56.63” – w 54º 54’57.05”). the area was divided into 20 quadrats of 40,000 sqm. the quadrats were numbered from 1 to 20, and in each collection month, each quadrat was randomly assigned a number which then was removed to avoid repetition. samples were collected for 12 consecutive hours from 06:00 to 18:00. to evaluate the effect of ants under temporary protein sources in forest environments, 50g of each of three different baits were used at each collection point. the baits consisted of sardines, which are commonly used as bait for ant collections (benson & brandão, 1986; moutinho, 1991; brandão et al., 2000), as well as beef liver and chicken. at each collection point, the three types of baits were placed on disposable plates directly on the ground, 10m apart, in a straight line. the use of plates prevented access by some species of ants that can exploit baits beneath the plant litter. in this way, it was possible to monitor the interaction of all the species that occur only on the substrate. in order not to overestimate the occurrence and probable dominance of any species of ant, before the baits were installed at each site, a systematic search was made to avoid installing the bait on or near ant nests. to evaluate more specifically the action of ants at the baits, isolated control baits were installed, under the same conditions, with a physical barrier (colorless and odorless gel) around the plate, thus preventing access by any insect that was foraging on the soil. the consumption of baits was determined at the end of each collection, with the aid of an analytical scale, determined by the difference in weight between the beginning and the end of the period of exposure. occasional weight loss from drying was not taken into account. to evaluate whether the difference in consumption between the baits with and without barriers was significant, we applied a t test (using a 0.05% confidence level) to compare them. to evaluate whether the change in climatic conditions during the two seasons in the state of mato grosso do sul (zavatini, 1992) affected the richness and number of interactions between ant species that occurred on the baits, in the first 15 minutes of each hour of observation, temperature and relative humidity were measured with a hygrometer, and these data were evaluated by a pearson correlation test. throughout the observation period, the individual acts of behavioral interactions between different species of ants that co-occurred on the baits were quantified and qualified, according to the following parameters adapted by brandão et al. (2000): action: going forward = going toward an individual of another species with jaws open in an abrupt movement; biting = clasping body parts of the other individual with the jaws; exhibiting the stinger region or sting = turning the gaster downward from the abdomen; lifting the gaster = sociobiology 61(1): 43-51 (march, 2014) 45 shaking the gaster to expel pheromones; killing = attacks that resulted in the death of the individual attacked. reaction: staying on bait = the individual does not leave the bait even after attacked; escaping = the attacked individual leaves the bait quickly; exhibiting the stinger region or stinging the aggressor = the attacked individual displays the sting, and/or stings the attacker; lifting the gaster expelling pheromones = the attacked individual displays the gaster region, expelling toxic substances; fighting = the attacked individual defends itself, struggling with the attacker by using the jaw or other body parts; killing = the attacked individual, when defending itself, kills the attacker. the time that the species spent to locate and exploit the bait was quantified, as well as the number of individuals of each species present on the bait, in 1-minute intervals (flow of individuals). the mean flow was categorized as: weak flow: 3 to 10 individuals per minute; average flow: 11 to 30 individuals per minute; intense flow: more than 30 individuals per minute. the levels of ant aggression were categorized during interactions in values from 0-2 (0 = not aggressive; 1 = aggressive, 2 = very aggressive), taking into account the following parameters: not aggressive: they always fled and displayed no agonistic behavior; aggressive: they moved most of the time, but did not maintain any physical contact; very aggressive: they bit and/or killed, or even performed another aggressive act that caused injury to another individual. the types of interactions that mainly indicate the dominance or exclusion of other ants from the food source, according to brandão et al. (2000), were categorized as follows: dominated by being the only individual on the bait; dominated by being abundant; dominated by being aggressive; dominated by being abundant and aggressive; excluded other ants from the bait. to evaluate whether there was any relationship between the size of the species and the strategy adopted during the interactions between the species, the alitrunk of each individual collected on the bait was measured. according to brandão et al. (2000), this measurement is not affected by the individual’s physiological state, and is traditionally termed in taxonomic articles on ants as the measure of weber (wl). the sizes of the ants were categorized as: small: from 0.01 to 1.0 mm; average: from 1.01 to 2.0 mm; large: above 1.2 mm. all these parameters described above were correlated by a jaccard cluster analysis, to attempt to identify groups of ant species that adopt the same behavioral strategies during interactions on baits. after the interactions and/or consuming the baits, some foragers (one or two depending on the species) were collected while they were leaving the bait, and were placed in 70% ethanol for later identification at the genus level, using the keys of bolton (1994, 1995 and 2003); and at the species level, when possible, by comparing with standard specimens in the formicidae reference collection of the museum of myrmecology cepec/ceplac ilhéus, bahia. vouchers from this study were deposited in this collection under number # 5675. specimens of other insects were collected with forceps and/or brushes and stored in jars containing 70% ethanol for later identification to family level, with the aid of the dichotomous key of rafael et al. (2012) and by comparison with standard specimens in the entomological collection of the museum of biodiversity, ufgd/ms. results and discussion occurrence of insects in general the average consumption on baits without a barrier was 17.87g ± 5.45, and on baits with a barrier was 15.95g ± 5.86. the t test indicated no significant difference between these values (f= 1.41; p = 0.261). the presence or absence of ants, on these food sources, does not influence their consumption. baits where ants do not occur must be consumed by other groups of insects. during the months of collection, the mean temperature and relative humidity were 25.4°c ± 2.86% and 56.58% ± 14.16, respectively. there was no significant correlation between the consumption of baits and the temperature (f = 2.83; p = 0.163), or relative humidity (f= 2.68; p = 0.163), in both seasons. in general, the assembly of insects varied little on both types of baits (figs. 1 and 2). dipterans occurred most frequently on both baits (figs. 1 and 2). according to souza and linhares (1997), the insects most frequently evaluated in this type of substrate are the dipterans, especially the families calliphoridae, sarcophagidae and muscidae (oliveira-costa, 2003, 2008; gullan & cranston, 2008; pujol-luz et al., 2008). fly larvae compete intensely for resources on the carcasses in an attempt to consume the largest possible volume of food before the resource is exhausted (goodbrod & goff, 1990). although coleopterans are also an important group figure 1: relative frequency of the different insect orders that visited the 3 types of baits with physical barrier, exposed in forest areas between june/2010 to july/2011. lc santos-junior et al. exploration of protein resource by ants in a semi-deciduous forest46 that occurred in this type of resource according to the literature (oliveira-costa, 2003; rafael et al., 2012), they occurred in relatively low frequency compared to the other groups; as lepidoptera and orthoptera (figs. 1 and 2). souza et al. (2008) reported that insects such as coleopterans, even if frequent, most frequently visit protein sources only at the initial stage of putrefaction. cruz and vasconcelos (2006) discussed that the low occurrence of blattodea and orthoptera is associated with their feeding habits. most representatives of the latter group are phytophagous, occurring almost as accidental visitors (oliveiracosta, 2003). blattodea are opportunistic insects that exploit the most easily available resource. in general they are omnivorous, feeding on organic matter of any kind; however, they are sometimes also predators and attack other insects (triplehorn & jonnson, 2011). in this study, blattodea were present only on baits that were surrounded by a barrier (fig. 1) and hence without ants, indicating that the presence of ants seems to inhibit the action of this group. the baits with barriers were visited by insects of the orders diptera (75%), blattodea (11%), orthoptera (5%), coleoptera (2%) and lepidoptera (1%); the order hymenoptera was represented only by wasps (6%) (fig. 1). these results demonstrate that ants can inhibit the occurrence of wasps. moretti et al. (2011) demonstrated that these types of substrates may be an additional food source for the wasps; they observed wasps feeding directly on the baits and preying on adult insects. also for baits with barriers, coleoptera of the family staphylinidae and diptera of the family calliphoridae were very common. calliphorid flies occur in great abundance in this type of substrate, and are very common in manure and carrion (oliveira-costa, 2003; gullan & cranston, 2008; triplehorn & jonnson, 2011). the baits without barriers were visited by diptera (65%), hymenoptera (24%), orthoptera (6%), coleoptera (4%) and lepidoptera (1%) (fig. 2). on two occasions, baits without barriers were visited by spiders of the family lycosidae. according to centeno et al. (2002), and oliveira-costa (2008), this group of spiders plays an ecological role as predators of insects that belong to the cadaverous fauna. the frequency of occurrence of flies was about 10% lower on bait without barriers, which allowed ants to visit (table 1). even so, flies were the most frequent group, as described in trials such as those of souza and linhares (1997), oliveira-costa (2008), and rafael et al. (2012). still, this clear effect on the occurrence of flies should be analyzed with caution because it is a very common group in this type of resources besides being highly important to forensic entomology (oliveira-costa, 2003; pujol-luz et al., 2008). table 1.: frequencies relative (%) of occurrences of different orders and families of insects in the three types of baits exposed forested areas between the period june/2010 july/2011. insects baits (%) order family chicken sardine liver hymenoptera formicidae 34.73 33.22 32.04 vespidae 69.56 21.73 8.69 diptera calliphoridae 81.25 15.62 3.12 sarcophagidae 67.98 24.02 8.00 muscidae 80.01 15.96 4.03 syrphidae 66.66 20.00 13.33 coleoptera staphylinidae 87.09 12.9 3.22 scarabeidae 75.00 20.83 4.11 histeridae 96.87 3.12 0.00 orthoptera gryllidae 42.85 50.00 7.14 blattodea blattellidae 35.71 35.71 28.57 the most frequent insects on the sardine baits without barriers were members of the family gryllidae (50%); on beefliver baits, formicidae (32.04%); and histeridae (96.87%) were most frequent on chicken baits (table 1). as seen in table 1, the chicken bait was the most frequently visited overall. although subjective, the reason may be the strong odor emitted by the chicken bait on decomposition, compared to the others. occurrence of ants ants occurred on all types of baits throughout the collection period (table 2), although some species occurred more frequently on a certain type of bait than on another. however, one should take into account that the low occurrence of a species in different areas of collection may explain its low frequency on a specific type of bait. throughout the collection period, 34 ant species were observed (table 2); however, only 27 (80%) interacted with other species of ants. the other species were alone when visiting the baits, with no other ant species at that time. we quantified 194 behavioral acts during interactions between species (table 3). the most effective act involving action was biting (43.75%). figure 2: relative frequency of the different insect orders that visited the 3 types of baits without physical barrier exposed in forest areas between june/2010 to july/2011. sociobiology 61(1): 43-51 (march, 2014) 47 table 2.: relative frequency (%) of occurrence of different species of ants in each type of bait attractive exposed in the forest, between the period of the june/2010 july/2011. species baits chicken sardine liver subfamily: ponerinae odontomachus meinerti forel, 1905 1.29 0.00 0.00 odontomachus chelifer (latreille, 1802) 1.29 0.00 0.00 pachycondyla striata smith, 1858 3.89 2.12 0.00 pachycondyla verenae (forel, 1922) 5.19 4.25 1.92 pachycondyla villosa (fabricius, 1804) 11.68 8.51 3.84 subfamily: ectatomminae ectatomma brunneum f. smith, 1858 1.29 4.25 1.92 ectatomma tuberculatum f. smith, 1858 1.29 2.12 1.92 ectatomma permagnum forel, 1908 0.00 2.12 0.00 gnamptogenys sp. 0.00 2.12 5.76 subfamily: dolichoderinae azteca sp. 1.29 2.12 0.00 linepithema iniquum (mayr, 1870) 0.00 4.25 1.92 linepithema pulex wild, 2007 3.89 0.00 0.00 subfamily: formicinae camponotus crassus mayr, 1862 5.19 2.12 3.84 camponotus fastigatus roger, 1863 0.00 2.12 3.84 camponotus melanoticus emery, 1894 3.89 2.12 5.76 camponotus (myrmaphaenus) sp 0.00 2.12 3.84 camponotus sericeiventris guérin, 1838 2.59 0.00 0.00 nylanderia sp. 3.89 0.00 0.00 nylanderia guatemalensis (forel, 1885) 0.00 0.00 5.76 subfamily: ecitoninae labidus coecus (latreille, 1802) 0.00 2.12 1.92 subfamily: pseudomyrmecinae pseudomyrmex tenuis (fabricius, 1804) 2.59 6.38 1.92 subfamily: myrmicinae acromyrmex coronatus (fabricius, 1804) 2.59 0.00 0.00 atta sexdens rubropilosa forel, 1908 2.59 2.12 7.69 crematogaster nigropilosa mayr, 1870 3.89 8.51 0.00 crematogaster limata smith, 1858 6.49 10.63 5.76 pheidole oxyops forel, 1908 6.49 10.63 7.69 pheidole pubiventris mayr, 1887 7.79 10.63 5.76 pheidole radoszkowskii mayr, 1884 6.49 0.00 5.76 sericomyrmex sp. 2.59 0.00 1.92 solenopsis invicta buren, 1972 6.49 0.00 10.00 solenopsis sp. 0.00 0.00 7.69 trachymyrmex iheringi (emery, 1888) 2.59 2.12 0.00 trachymyrmex sp. 1.29 2.12 1.92 wasmannia scrobifera kempf, 1961 1.29 4.25 0.00 this behavior was also the most frequently described by brandão et al. (2000) and it seems to be one of the most effective behavioral strategies to dominate a resource. the most frequent act involving reaction was lifting the gaster (66.32%). according to longino (2003), species of the genus crematogaster exhibit their gaster, raising it in order to demonstrate that it is apparently larger than it actually is; or it can be used to eject formic acid or other substances as chemical defenses, depending on the situation and also on the species. table. 3: relative frequency of action and reaction behaviors executed by different ant species during interactions in the 3 types of baits exposed in forest areas between june/2010 to july/2011. action % reaction % biting 43.75 lifting the gaster 66.32 going forward 26.04 staying on bait 20.4 killing 13.54 escaping 13.26 expelling 17.7 the results demonstrate that as the frequency of interactions between species on baits increased, the consumption decreased (fig. 3 a, b and c). it seems that in most cases, the species opted to dominate the resource before exploiting it, which leads them to spend more time interacting with other species than consuming the resource. the correlations between the number of species of ants that visited the baits (f= 10.88; p= 0.030) and the number of interactions (f= 5.38; p= 0.01) with temperature were significant and positive in the rainy season. that is, the more the temperature increased, the more the number of visitor species increased, and consequently the number of interactions on baits increased as well (figura 4). the temperature, especially at the soil surface, is one of the factors that regulate the activity of foraging ground insects. at higher temperatures and favorable relative humidity, ants tend to increase their foraging activities and consequently the number of interactions between species also increases (hunt, 1974; levings, 1983; cerdá et al., 1997, 1998; dajoz, 2000). several species of the attini group visited and consumed the bait: atta sexdens rubropilosa, acromyrmex coronatus, sericomyrmex sp. and trachymyrmex sp. all of them carried pieces of bait to their colonies. however, these species are known to be restricted to feeding on fungi that grow on a composite substrate consisting mainly of plant material gathered by their workers (weber 1972; delabie & fernández, 2003). on the other hand, clark and blom (1991) stated that vertebrate carcasses may be an additional food source for ants that feed on seeds, for example, considering the frequency of availability of carcasses. conconi and rodríguez (1977) suggested that species of atta must feed on alternate materials such as meat. marques and del-claro (2006), also observed that ants of the genus atta was one of the most frequent visilc santos-junior et al. exploration of protein resource by ants in a semi-deciduous forest48 tors in inventory held in a cerrado area using sardine baits. here, the ants, regardless of species, required a mean of 4.1 ± 1.8 minutes to locate the bait, which was visited by a mean of 2.11 foragers per minute, regardless of the type of bait. species of the genera pheidole and crematogaster, and a. sexdens rubropilosa were the first to find and exploit resources in 33%, 27.77% and 13.88% of cases respectively, always with a mean flow of over 22 foragers per minute. species with mass recruitment and that forage in large flows are more likely to detect and numerically dominate food resources more rapidly, as noted by holldöbler and wilson (1990). brandão et al. (2000) argued that the order of arrival of species on the bait is not necessarily associated with their relative dominance, but rather with other factors such as proximity to the source of the nest, colony size, and foraging strategy. the jaccard cluster analysis (j = 0.92) indicated three distinct groups (fig. 5). group “a” included ants with a mean size of 1.24 ± 0.23 mm and a mean flow of 12 ± 7.0 foragers per minute. in this group 83% of the species were categorized as non-aggressive, unable to dominate the bait at any time. an exception in this group was azteca sp. which was considered aggressive; however, its mean flow was 2.1 ± 2 foragers per minute, which was weak according to the criteria used here. group b (fig. 5) included ants with a mean size of 3.28 ± 1.35 mm and a mean flow of 2 ± 0.5 foragers per minute. in this group, 95% of the ants were highly aggressive; however, they dominated the bait in only 5% of cases. in 60% of the cases in which species of this group dominated baits, it was because they were alone, as many species of poneromorphs that comprise this group forage individually (fig. 5). according to brandão et al. (2000), ants of this group, although they are large and generally aggressive predators, almost never dominate, and figure 3: (jaccard) grouping evaluating size, aggressiveness and average flow of different ant species during interactions in baits. a: medium-sized species, medium foraging flow, little aggressive and not dominant, b: large species, low foraging flow, very aggressive and not dominant, c: small species, intense foraging flow, not aggressive and dominant. figure 4: average consumption and total number of interactions per collection of different ant species in the 3 types of baits without physical barrier, exposed in forest areas between june/2010 to july/2011. sociobiology 61(1): 43-51 (march, 2014) 49 when they do, it is because they are the only ones present on the baits. their failure to dominate the bait when other species are present is due to their strategy of foraging individually. according to brandão et al. (2000), ants of the genus pachycondyla, for example, although they are relatively large, cannot monopolize baits and every time that they confront species that use group attack strategies, they are excluded from the baits. however, in 75% of the cases, although they may not dominate baits, they can remove relatively large pieces and carry them off, infiltrating between the dominant species. group “c” consisted of small ants (fig. 5) with a mean size of 0.80 ± 9.5mm and a mean flow of 35.45 ± 9.5 foragers per minute. in this group 81% of the species were not aggressive; however, they dominated baits in 85% of the cases. pheidole radoszkowskii, was a typical species of this group, which, when it occurred, dominated baits in 95% of the cases, maintaining a continuous and intense flow, according to the criteria used here. in particular, in 80% of cases where they were present, a. sexdens rubropilosa dominated the bait by being abundant and also by maintaining an intense flow. throughout the exposure period of baits it was possible to observe that depending on the time there was one species predominating in number in the bait, demonstrating that there is a succession of dominant species in these food sources, competition occurs more intensively when there is an overlap of activity periods and collection sites by several species of ants visiting the same food source (brandão et al. 2000). in this case, they can take very aggressive actions that may result in some cases of monopolization of the resource by workers of one species, preventing access by others. a species is considered dominant and competitively superior when it possesses features that allow it to monopolize a resource, such as aggressive behavior or mass recruitment. the other species that co-occur with the dominant species and do not possess these characteristics are considered subordinate and usually have alternative strategies for obtaining resources (andersen, 1992). the relationship between dominant and subordinate species may also be influenced by environmental factors. this influence may occur through a direct physiological effect resulting from the tolerance of each species to microclimate variations (bie & hewitt, 1990), such as temperature, because the myrmecofauna is sensitive to desiccation. high temperatures may influence the foraging strategy of ants and therefore their interactions at sites where resources are present (hunt, 1974; levings, 1983; cerdá et al., 1997 and 1998; dajoz, 2000). these results demonstrate that the presence of ants is important to ecological succession on temporary protein sources in forest environments interfering in the occurrence of other frequent groups in this type of resource. their presence may simply inhibit the presence of other insects, especially those that also forage on the ground, or even flies that avoid landing on the resource when there is an intense flow of ants exploiting it. another important action in this sense is when they prey on immature, especially of flies and adults of other species that detect and exploit this type of resource. the results show that there are three distinct groups of ants that can interact in this type of resource according to size, flow and aggressiveness toward other species. however, the ones that dominate the source are always those that arrive with less flow of individuals regardless of whether or not detecting the resource first than other species. therefore, depending on the ant species that co-occur, the results for the sequence of colonization can be modified. acknowledgments we thank the post-graduation program in entomology and biodiversity conservation ufgd, the coordination of improvement of higher education personnel (capes) for granting the scholarship to the first author, the national council of scientific and technological development (cnpq) for granting the scholarship to the fourth author. we also thank two anonymous reviewers for valuable comments that improved this article. references andersen, a.n. 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(1992). dinâmica climática no mato grosso do sul. geografia, 17: 65-91. doi: 10.13102/sociobiology.v66i1.3385sociobiology 66(1): 126-135 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 floral origin and physical and chemical characteristics of honey from africanized bees in apiaries of ubiratã and nova aurora, state of paraná introduction honey is a substance made mainly from the nectar of flowers, and because of the diversity of the brazilian flora, it may present peculiarities in its physical and chemical characteristics, depending on the type of plant that is part of its composition. different environments may lead to variations in honey characteristics due to floral species and soil characteristics, or due to seasonal factors such as temperature and rainfall (crane, 1990; marchini et al., 2004a). depending on the number of predominant floral species, honey may be classified as monofloral, bifloral or heterofloral. the latter, also called wild honey, has quite variable properties, depending on the environmental abstract physical and chemical characteristics of honey may vary due to the diversity of flora and soil characteristics, or seasonal factors. this study was carried out in two counties, nova aurora and ubiratã, located in the west and center-west regions of the state of paraná. the objective of the study was to verify if the physical and chemical parameters of apis mellifera (l.) honey are in accordance with the national standard, as well as to verify how the 21 samples collected in the two localities are grouped, based on the physical, chemical and pollen characteristics. honey was analyzed for sugar, ash, protein, moisture, color, electrical conductivity, formaldehyde index, diastase and viscosity, water activity and pollen content. samples of honey containing the dominant pollen types glycine max (l.) merr. and eucalyptus sp. formed groupings similar to those based on physical and chemical characteristics, however, the multivariate classification of honey samples in groups based on pollen types was not an efficient method to group samples of polyfloral honey. sociobiology an international journal on social insects es sekine1, eh takashiba3, ro bueno1, paa bueno1, mg caxambu1, mj sereia1, lc marchini4, accc moreti5, vaa toledo2 article history edited by eduardo almeida, usp, brazil received 27 april 2018 initial acceptance 16 july 2018 final acceptance 14 december 2018 publication date 25 april 2019 keywords apis mellifera; honey composition; physical and chemical parameters;. corresponding author es sekine departamento de biodiversidade e conservação da natureza (dabic) universidade tecnológica federal do paraná via rosalina maria dos santos nº 1233, cp: 271 cep: 87301-899 campo mourão-pr, brasil. e-mail: essekine@gmail.com conditions and the type of flower used, and it is not possible to generalize its characteristics (barth, 2004). for this reason, there is a need to compile data on the physical and chemical characteristics of honeys produced in the south and southeast regions of brazil, as there are indications that the standards established in the brazilian legislation (brasil, 2000) do not include all these honeys (barth et al., 2005). as the characteristics of honey depend on the species available for the supply of nectar, investigations aimed at identifying the plants used by bees in different regions of the country provide information that can be used in the management of hives and in the adoption of strategies for a better utilization of the flora with potential for honey production (marchini et al., 2004a; nogueira couto & couto, 2006). 1 universidade tecnológica federal do paraná – utfpr, campo mourão-pr, brazil 2 universidade estadual de maringá – uem, maringá-pr, brazil 3 universidade federal de santa maria – ufsm, santa maria-rs, brazil 4 escola superior de agricultura luiz de queiroz – esalq/usp, piracicaba-sp, brazil 5 instituto de zootecnia, nova odessa-sp, brazil research article bees sociobiology 66(1): 126-135 (march, 2019) 127 several studies have been carried out in the southern and southeastern brazil to investigate the floral origin of honey (bosco & luz, 2017; sekine et al., 2013) and its physicochemical characteristics (mendonça et al., 2008; sereia et al., 2011; barth et al., 2013; alves et al., 2011). this study aimed to analyze the floral origin, physical and chemical characteristics of samples of honey collected in two counties of paraná state at different times of the year and to verify whether physicochemical characteristics are grouped depending on the floral species used in honey production. material and methods obtaining samples of honey honey samples were collected in two apiaries (fig 1): apiary a, located in the county of nova aurora, state of paraná (24°31’50” s and 53°11’50” w) and apiary b, located in the county of ubiratã, state of paraná (24°29’41” s and 53°02’43” w). these counties are located in the phytogeographical domain of submontane semidecidous tropical forest (ibge, 2012). in both properties, beekeeping is an activity complementary to the family income, so the area surrounding the apiaries is occupied by agriculture and the properties have a forest fragment as legal reserve area of 13.65 ha and 2.39 ha for apiaries a and b, respectively in order to obtain the honey samples, three hives were randomly marked in each apiary. all hives were located near or inside the forest fragments. in each hive, we placed every month a frame with new bee-wax foundation without combs, or (when available) a frame with formed combs to obtain the honey produced by the bees during the period. inspection of hives was performed monthly to verify the existence of mature honey. to avoid sampling immature honey, honeycombs, with at least 75% of capped cells, were removed and centrifuged separately, in individual plastic bags, avoiding the honey mixture. after each harvest, the frames were replaced with other empty ones. the samples were transported to the laboratory in thermal boxes and kept under refrigeration for approximately six months. a total of 21 samples were taken for the two apiaries (nine from apiary a and 12 from apiary b) between december 2008 and may 2009. in april and may there was no mature honey in apiary a. physical and chemical analysis physical and chemical analyses were performed in triplicate for each sample. reducing sugars, total reducing sugars, sucrose, ash and protein were verified according to the methods adopted by the adolfo lutz institute (zenebon et al., 2008). the techniques used to determine the moisture content (atago co, 1988), color (vidal & fregosi, 1984), electrical conductivity (boe, 1986); ph, acidity, formaldehyde index (moraes & teixeira, 1998), diastase activity (c.a.c, 1990) and viscosity were done according to the methods cited by marchini et al. (2004a). the water activity (aw) determination was made directly using a water activity meter aqualab®. the analyses were carried out at the laboratory of useful insects of the department of entomology and acarology of the school of agriculture, luiz de queiroz-esalq/usp and at the chemistry and ecosystems laboratories of the federal technological university of paraná utfpr campo mourão. pollen analysis an aliquot of 10 g of each sample was diluted in distilled water and centrifuged for acetolysis (erdtman, 1952). the pollen analyses were: a) qualitative, by the identification of the pollen types by comparison with the collection of pollen slides of the plants collected in the region, and specialized literature; and b) quantitative, by the average count of 300 to 500 pollen grains on microscopy slides, in triplicate. pollen types were ############### #### # # # ######### ##### # ### # ### ######## # ###### ## ######### # # # # nova aurora ubiratãa b -53°53°10'53°20' 10 0 10 20km -53°53°10'53°20' 24°40' 24°30' 24°20' 24°40' 24°30' 24°20' # ##### ######## ## ## ### n centro-sul oeste centro ocidental noroeste norte central nortepioneiro centro oriental sudeste metropolitana de curitiba sudoeste fig 1. location of the sampling sites. a: apiary in nova aurora; b: apiary in ubiratã, state of paraná. es sekine et al. – physical-chemical characteristics and floral origin of honey128 classified into four frequency classes (louveaux et al., 1978; barth, 1989): dominant pollen (frequency over 45%), accessory pollen (from 16 to 45%), important isolated pollen (from 3 to 15%), occasional isolated pollen (frequency below 3%). data analysis we applied qualitative and quantitative descriptive statistical analysis and multivariate statistical analysis: clustering technique for the honey samples, using the statistical software r (2016). for the pollen data of the two areas, we calculated the sorensen similarity index (iss) (legendre & legendre, 1984; mueller-dombois & ellenberg, 1974; pielou, 1975), which is the most commonly used to compare qualitative floristic data between communities. mathematically, the index is defined as: iss = 2c/(a+b+c) × 100, where: a = number of species restricted to area a, b = number of species restricted to area b and c = number of species common to areas a and b. the results of the physical and chemical analyses of honey were compared between the sampling sites by clustering analysis using the euclidean distance by the upgma clustering method. in the cluster analysis, the mean euclidean distances for the properly standardized data were adopted as dissimilarity means. the clustering based on the pollen types found in honey was made from the presence and absence of pollen types using the jaccard coefficient by the upgma linkage method using the vegan package (oksanen, 2017) using the statistical software r. jaccard coefficient expresses the similarity between honey samples, based on the number of common pollen types. results physical and chemical characteristics there was predominance of the extra-light amber color (50%). light amber (27%) and white (23%) were also found in honey samples. the complete results of the physical and chemical parameters analyzed are listed in table 1. the parameters that have limits established by the brazilian legislation (diastase, moisture, ph, acidity, reducing sugars, sucrose and ash) (brasil, 2000) were met in the samples collected (table 1). only one of the samples contained moisture above the established values. pollen analysis in the honey samples, we identified 49 pollen types belonging to 25 families (table 2). two pollen types were not identified at the family level. the pollen types with higher frequency belong to the families fabaceae (34.9%), myrtaceae (11.7%) and piperaceae (11.4%) (figure 2a). 0 5 10 15 20 25 30 35 40 ar ec ac ea e as te ra ce ae br as si ca ce ae eu ph or bi ac ea e fa ba ce ae fl ac ou rt ia ce ae m im os ac ea e m yr ta ce ae o le ac ea e pi pe ra ce ae rh am na ce ae ro sa ce ae o th er % family 0 10 20 30 40 50 60 70 80 90 100 d ez d ez d ez ja n ja n ja n fe v/ m ar fe v/ m ar fe v/ m ar ab r ab r m ay % ■ apiary a □ apiary b 0 10 20 30 40 50 60 70 80 90 100 d ez d ez d ez ja n ja n ja n fe v/ m ar fe v/ m ar fe v/ m ar ab r ab r m ay % ■ apiary a □ apiary b 0 10 20 30 40 50 60 70 80 90 100 d ez d ez d ez ja n ja n ja n fe v/ m ar fe v/ m ar fe v/ m ar ab r ab r m ay % ■ apiary a □ apiary b fig 2. pollen types frequency in honey samples from two apiaries in the counties of ubiratã and nova aurora in the paraná state, brazil. a) frequency of the main families found in honey in the two apiaries; b, c and d, pollen frequency of the dominant species found in the two apiaries from december 2008 to may 2009, respectively, glycine max, eucalyptus spp., and piperaceae type. sociobiology 66(1): 126-135 (march, 2019) 129 a pi ar y m on th sa m pl e d ia st as e (g ot he ) co nd uti v (μ s. cm -1 ) m oi st ur e (% ) ph pr ot ei n % a ci di ty (m eq .k g1 ) fi 1 (m l. kg -1 ) a sh (% ) v is co s (m pa .s ) w a 2 a w tr s3 (% ) rs 4 (% ) su cr os e (% ) co lo r* * d p5 a d ec 1 24 .2 4 38 7. 33 18 .1 0 3. 51 0. 23 27 .1 1 10 .3 7 0. 43 25 45 .0 0 0. 54 76 .6 2 72 .2 9 4. 11 el a po lifl or al 2 14 .0 5 38 2. 00 19 .2 0 3. 59 0. 20 18 .7 4 8. 70 0. 07 10 30 .0 0 0. 60 81 .2 5 76 .4 5 4. 56 el a po lifl or al 3 24 .9 1 38 1. 67 18 .2 0 3. 47 0. 28 23 .7 2 9. 36 0. 07 15 35 .0 0 0. 62 78 .0 8 75 .3 5 2. 60 el a po lifl or al ja n 4 20 .2 9 38 2. 33 18 .4 0 3. 44 0. 34 32 .9 7 11 .0 5 0. 09 12 20 .0 0 0. 62 80 .1 9 74 .4 6 5. 44 el a po lifl or al 5 20 .6 8 38 3. 67 19 .2 0 3. 45 0. 34 31 .3 5 11 .4 0 0. 20 10 25 .0 0 0. 65 76 .8 6 74 .9 6 1. 80 la po lifl or al 6 33 .4 8 36 6. 67 17 .1 0 3. 49 0. 36 32 .1 7 12 .7 3 0. 20 26 70 .0 0 0. 56 79 .3 8 76 .1 1 3. 10 la po lifl or al m ar 7 16 .9 5 31 9. 67 19 .1 0 3. 42 0. 36 28 .7 1 8. 39 0. 16 11 30 .0 0 0. 60 77 .3 3 76 .4 8 0. 81 el a po lifl or al 8 31 .5 8 37 4. 00 17 .1 0 3. 49 0. 46 31 .4 3 10 .7 0 0. 17 31 15 .0 0 0. 58 78 .7 1 76 .9 0 1. 73 la pi pe ra ce ae 9 14 .7 4 30 1. 33 19 .1 0 3. 32 0. 27 27 .1 9 10 .0 7 0. 13 10 40 .0 0 0. 61 78 .0 6 74 .3 8 3. 49 el a po lifl or al m ea n a 22 .3 2 36 4. 30 18 .3 9 3. 46 0. 31 28 .1 5 10 .3 1 0. 17 17 01 .1 1 0. 60 78 .5 0 75 .2 6 3. 07 st an da rd d ev ia ti on 6. 91 31 .4 3 0. 85 0. 07 0. 08 4. 62 1. 37 0. 11 83 5. 15 0. 03 1. 55 1. 44 1. 49 b d ec 10 18 .3 3 10 6. 33 16 .7 0 3. 54 0. 25 17 .9 5 12 .4 2 0. 11 29 40 .0 0 0. 57 78 .6 4 74 .0 9 4. 33 w g ly ci ne m ax 11 23 .2 7 13 7. 77 16 .4 0 3. 53 0. 26 17 .7 6 10 .7 2 0. 06 41 15 .0 0 0. 56 77 .0 7 75 .7 6 1. 24 w g ly ci ne m ax 12 34 .0 2 12 0. 23 16 .7 0 3. 50 0. 26 21 .1 1 12 .4 0 0. 05 35 80 .0 0 0. 62 75 .4 4 74 .8 5 0. 56 w g lic in e m ax ja n 13 28 .4 5 14 1. 13 17 .1 0 3. 56 0. 27 23 .6 2 13 .5 7 0. 48 22 90 .0 0 0. 60 71 .4 2 69 .9 2 1. 43 w g ly ci ne m ax 14 36 .4 7 15 5. 17 16 .5 0 3. 52 0. 29 23 .1 1 13 .4 0 0. 08 34 05 .0 0 0. 56 76 .3 4 73 .0 2 3. 16 el a g ly ci ne m ax 15 24 .7 4 14 6. 70 17 .2 0 3. 55 0. 25 21 .4 7 12 .4 1 0. 07 22 20 .0 0 0. 63 77 .6 9 72 .3 8 5. 05 el a g ly ci ne m ax m ar 16 54 .0 2 34 2. 00 19 .0 0 3. 47 0. 22 43 .2 1 10 .3 7 0. 45 39 5. 00 0. 64 75 .4 2 71 .0 2 4. 18 el a po lifl or al 17 62 .2 5 34 2. 33 17 .9 0 3. 56 0. 33 25 .8 1 14 .0 8 0. 11 18 55 .0 0 0. 58 77 .0 3 72 .0 1 4. 77 el a po lifl or al 18 48 .6 7 31 7. 33 18 .1 0 3. 53 0. 34 25 .0 9 10 .3 7 0. 38 14 50 .0 0 0. 59 75 .6 8 72 .9 3 2. 61 el a po lifl or al a pr 19 37 .7 2 46 6. 00 18 .1 0 3. 63 0. 31 37 .1 8 10 .3 8 0. 14 13 80 .0 0 0. 59 78 .5 7 72 .5 7 5. 70 la eu ca ly pt us 20 48 .1 7 34 8. 33 21 .4 0 3. 48 0. 29 31 .8 3 12 .0 6 0. 08 46 5. 00 0. 65 76 .5 7 71 .2 3 5. 07 la po lifl or al m ay 21 46 .1 8 51 7. 00 17 .3 0 3. 56 0. 36 39 .2 6 9. 71 0. 46 19 60 .0 0 0. 61 79 .3 7 74 .7 4 4. 40 la eu ca ly pt us m ea n b 38 .5 2 26 1. 69 17 .7 0 3. 54 0. 29 27 .2 8 11 .8 2 0. 21 21 71 .2 5 0. 60 76 .6 0 72 .8 8 3. 54 st an da rd d ev ia ti on 13 .5 3 14 4. 31 1. 40 0. 04 0. 04 8. 55 1. 47 0. 18 11 77 .4 2 0. 03 2. 08 1. 74 1. 71 to ta l m ea n 32 .3 1 30 4. 97 17 .9 4 3. 51 0. 29 27 .6 0 11 .3 1 0. 19 20 11 .7 5 0. 60 77 .4 2 73 .7 7 3. 47 st an da rd d ev ia ti on 13 .6 1 12 3. 74 1. 23 0. 07 0. 06 7. 17 1. 49 0. 16 10 56 .9 5 0. 03 2. 12 1. 95 1. 53 st an da rd * m in . 8 m ax . 2 0 3. 34. 6 m ax . 5 0 m ax . 0 .6 m in . 6 5 m ax . 6 1 f or m ol in de x; 2 w at er a cti vi ty ; 3 to ta l r ed uc in g su ga r; 4 r ed uc in g su ga r, 5 d om in an t p ol le n (> 4 5% ); *b ra si l (2 00 0) ; * *e la = e xt ra l ig ht a m be r; l a = l ig ht a m be r; b = w hi te . t ab le 1 . p hy si ca l a nd c he m ic al p ar am et er s of 2 1 sa m pl es o f a pi s m el lif er a ho ne y, c ol le ct ed f ro m d ec em be r 20 08 to m ay 2 00 9 in th e co un ty o f n ov a a ur or a (a pi ar y a ) an d u bi ra tã (a pi ar y b ), st at e of p ar an á. es sekine et al. – physical-chemical characteristics and floral origin of honey130 the families with the highest number of pollen types represented were asteraceae (seven), euphorbiaceae, mimosaceae and myrtaceae (four types each). dominating pollen types (d) were found in several of the samples collected in apiary b, with glycine max (soybean) in the months of december and january, with frequencies between 51.4% and 90.6%. and eucalyptus spp. in the months of april (54.7%) and may (72.2%). in apiary a, the pollen of piperaceae was dominant (47.8%) in february (figure 2b, c, d). soybean pollen was also found as accessory pollen in seven other samples, piperaceae in four and eucalyptus in two samples. also found as accessory pollen, in at least one sample, pollen types of the genera baccharis, mikania, lonchocarpus and campomanesia and the species alchornea triplinervea (spreng.) müll.arg. parapiptadenia rigida (benth.) brenan (mimosaceae) appeared in all samples, but its contribution never reached more than 10% of the total of a sample. samples 5 and 7, both of apiary a, had higher number of pollen types (17). important isolated pollen (i) totaled 80 occurrences, from 30 pollen types. occasional isolated pollen (o) had 175 occurrences, from 50 pollen types. the similarity between the areas, measured from the pollen types present in the samples of honey, was 87%. cluster analysis cluster analysis based on the physical and chemical characteristics (figure 3a), showed two groups, evidencing the collection sites. one group consists mainly of samples of apiary a with four samples of apiary b, and the other group consisted mainly of samples of apiary b and only three samples of apiary a. the dendrogram formed by the pollen types presented high similarity (figure 3b), showing samples from the same site and month of collection, except for sample b19 and b21. fig 3. clusters obtained by the upgma method a) euclidean distance from physical and chemical characteristics and b) jaccard method from the pollen types. analyses made on samples of apis mellifera honey, collected from december 2008 to may 2009, in the counties of nova aurora and ubiratã, state of paraná. discussion the color of the honey samples indicates the presence of mineral content (bath & singh, 1999; finola et al., 2007). wild or polyfloral honey tends to exhibit a large color variation (almeida-anacleto & marchini, 2004). the samples analyzed here have a light color. in general, honey with light color has higher market value (boffo et al., 2012; silva et al., 2013; almeida-muradian et al., 2014). samples are within the ranges established in the brazilian legislation for all analyzed parameters. only one exceeded the maximum allowed for moisture (21.4%). the high moisture content is verified in honey harvested when the alveoli were not completely sealed. additionally, relative air humidity may also contribute to moisture in honey (faria, 1993; sereia et al., 2011). despite the care in collecting the samples with 75% of the alveoli covered, this sample had higher humidity. values lower than 18.5% usually indicate mature honey (marchini et al., 2004b). honey from partially capped cells often presents a high percentage of moisture (sanz et al., 1995). the stability of honey with moisture between 17 and 20% depends on a low content of microorganisms (malacalza et al., 2007). some parameters limited by brazilian legislation are indicative of honey quality. among them, the diastase index refers to alpha-amylase, the enzyme responsible for the digestion of starch. its origin is attributed to the salivary secretion of bees and is a parameter that gives indications of overheating. however, this is not always true because some types of honey naturally have less diastase than others, once the enzyme is added by the bees during the ripening of the nectar to the thicker consistency of honey. as some types of nectar are naturally thicker than others, they require less processing by the bee and, consequently, less diastase (white, 1994). the percentage of ash in honey expresses the content of mineral material, being a parameter widely used to check honey sociobiology 66(1): 126-135 (march, 2019) 131 quality (marchini et al., 2005). acidity is also an important component of honey, as it contributes to its stability, against the development of microorganisms (marchini et al., 2004b). brazilian legislation establishes a minimum of 65% of reducing sugars and a maximum of 6% of sucrose (brasil, 2000). in general, fructose and glucose account for about 80% of the total sugar content in honey, while sucrose and maltose represent about 10%. the balance of the different types of sugars results in differences in honey, such as viscosity, density and crystallization (white, 1975). however, the proportion of sugars may vary according to the botanical origin (nozal et al., 2005; de-melo et al., 2017; manzanares et al., 2017). with respect to the parameters not covered by the brazilian legislation, the percentage of protein can be used to detect honey adulteration, with 0.26% being the average value of the international standard (almeida-anacleto & marchini, 2004). however higher values were found in samples from different sources, reaching 1-2% (rebane & herodes 2008; demelo et al., 2017). the electrical conductivity is a parameter that can be used to determine the origin of honey (aganin, 1971) and is related to botanical origin, mineral content, ph, acidity, proteins and other substances present in honey (white jr, 1975; bogdanov, 1999). the electrical conductivity ranged from 106.33 to 517 μs.cm-1 in the presente study. higher values have been recorded in honey from different locations (sodré et al., 2003; sodré et al., 2007; almeida-anacleto & marchini; marchini et al., 2005; mendonça et al., 2008; alves et al., 2011). similar or lower values were also found (arruda et al., 2005; sereia et al., 2011). the values obtained for the formalin index ranged from 8.70 to 14.08 ml.kg-1. these values are similar to other studies (marchini et al., 2005; mendonça et al., 2008; sodré et al., 2003; almeida-anacleto & marchini, 2004; alves et al., 2011). viscosity and water activity also have no values established in brazilian legislation. the viscosity is mainly influenced by the percentage of water and the fructose and glucose ratio, honey with higher fructose content has lower viscosity, and also depends on temperature and floral origin. (fattori, 2004). in the present study viscosity values were between 395 and 4115, with the highest values observed in honeys with dominant pollen types glycine max and piperaceae. the values of water activity are between 0.54 and 0.65, with an average of 0.60 ± 0.03. under aerobic conditions, the inhibitory water activity is aw = 0.86, and substrates with aw = 0.6 are assured of microbial deterioration (almeidaanacleto, 2004). in general, the results of the physical and chemical parameters are quite variable between samples from different regions which makes difficult to establish a pattern related to the parameters not covered by the legislation. considering the pollen types present in the samples, the families asteraceae, euphorbiaceae, mimosaceae and myrtaceae were also frequent in other studies in the southern and southeastern regions, with similar vegetation (sereia et al., 2011; araujo et al., 2013; bosco & luz, 2017). the studied region is soybean producer (g. max) and six samples had this species as dominant pollen type. the two counties are located in agricultural areas, and the main crops are corn and soybeans. the genus eucalyptus was present in many samples. the contribution of species of this genus as dominant pollen in honey samples was verified in other studies in the southeast region (bastos et al., 2003; sodré et al., 2003; barth et al., 2005; luz et al., 2007; mendonça et al., 2008; silveira et al., 2012; araujo et al., 2013; barth et al., 2013). the increase of this pollen type in honey samples can be explained by the increase of reforested areas with species of eucalyptus. in the study area, it was verified that there are four species of eucalyptus, three that bloom between january and april and one that blooms in november. soybean flowering in the region occurs in the months of november, december and march (sekine et al., 2013). samples with these dominant pollen types were verified only in apiary b, which has smaller forested area. in apiary a, which has a larger forest remnant, polyfloral honey samples were collected. despite the predominance of agricultural crops in both properties, pollen analysis results reflect the greater diversity of this environment. the high similarity of pollen types shown in the dendrogram was expected, since the two apiaries belong to the same phytogeographic region at natural restoration stage. samples from apiary a, a1 and a3, contained piperaceae spp. as accessory pollen, and sample a2, lonchocarpus sp. and campomanesia spp. also as accessory pollen. samples a4, a5 and a6 had as accessory pollen arecaceae spp. and g. max. samples a7, a8 and a9 had in common several species, including piperaceae as dominant, accessory or important isolated pollen and acacia and p. rigida as important isolated pollen in the three samples. from apiary b, samples b19 and b21, had eucalyptus spp. as dominant pollen. samples b10 to b15 had in common g. max as dominant pollen and b17 to b20 g. max as accessory pollen. comparing the two dendrograms, it was observed that the clusters were not coincident, which shows that polyfloral honey samples are quite variable for physical and chemical characteristics. however, samples of the apiary b containing dominant pollen glycine max and piperaceae are grouped and present the highest values of viscosity and lower conductivity. marchini et al. (2004b) observed the influence of accessory and dominant pollen types on the physical and chemical characteristics of honey samples in the state of tocantins. in samples from the state of são paulo, it was also verified the grouping of physical and chemical characteristics in eucalyptus honey and wild honey (marchini et al., 2005). in another study also in são paulo state, it was verified the grouping of monofloral samples of orange, but the eucalyptus samples were distributed in several different groups (marchini et al., 2007). it was also verified the grouping of physical and chemical characteristics in moroccan unifloral honeys of eucalyptus, citrus and honeydew (terrab et al., 2003). es sekine et al. – physical-chemical characteristics and floral origin of honey132 family pollen type apiary a apiary b dec jan feb/mar dec jan feb/mar apr may 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 anacardiaceae mangifera indica o schinus terebinthifolius o i o o o i o arecaceae arecaceae type a a a i a o o i i o o o o asteraceae baccharis spp. a i i o i o i a o a i i bidens spp. o o o calyptocarpus biaristatus o o chromolaena pedunculosa o conyza bonariensis o o o mikania spp. i o a i i o senecio brasiliensis o o boraginaceae cordia ecalyculata o o o cordia trichotoma o brassicaceae indeterminada spp. i i i o o o caesalpinaceae bauhinia forficata i o o o o combretaceae combretum fruticosum o o o o euphorbiaceae alchornea triplinervea a i i o o o i o o o o o o bernardia pulchela o o i o o ricinus communis o o o o sebastiania brasiliensis i i o o o o i o fabaceae glicyne max o i i a a a o o o d d d d d d i a a a a i lonchocarpus sp. i a i i i i o i i o i i i o i flacourtiaceae casearia sylvestris o i o i i o lamiaceae hyptis mutabilis o o leonurus sibiricus o i o malvaceae wissadula subpeltata o o o o meliaceae melia azedarach o o o mimosaceae acacia type o o i i i o o o i o o o i leucaena leucocephala o o o o o o o mimosa sp. o o parapiptadenia rigida o i o o o o i i i o o o i o o i i o o i o myrtaceae campomanesia spp. o a i eucalyptus spp. o i o o o o o o o o o o i a a i i d i d hexaclamys edulis o i i o o myrcia type i table 2. pollen spectrum and frequency classes in 21 samples of apis mellifera honey collected at two locations in the counties of nova aurora (apiário a) and ubiratã (apiário b) from december 2008 to may 2009. sociobiology 66(1): 126-135 (march, 2019) 133 oleaceae ligustrum sp o o i o i o o o o o o piperaceae piperaceae type a i a o o o a d i o i o o i i a o rhamnaceae hovenia dulcis o i o rhamnaceae type a i o i o a o a rosaceae eriobotrya japonica o o o i o i prunus sellowii o o o rosaceae type o o o rubiaceae borreria sp. o o rutaceae zanthoxylum sp. i i i sapindaceae serjania spp. o o solanaceae solanaceae type o o solanum mauritianum o o solanum sanctaecatharinae o o o tiliaceae luehea divaricata o ni 1 i o i *d = dominant pollen (> 45,0%); a = accessory pollen (16,0 a 45,0%); i = important isolated pollen (3,0 a 15,0%); o = occasional isolated pollen (< 3,0%). ni = not identified. table 2. pollen spectrum and frequency classes in 21 samples of apis mellifera honey collected at two locations in the counties of nova aurora (apiário a) and ubiratã (apiário b) from december 2008 to may 2009. (continuation) family pollen type apiary a apiary b dec jan feb/mar dec jan feb/mar apr may 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 the geographic origin of the honey, collected in different localities with different types of vegetation can also influence the groupings of physical and chemical characteristics (santos et al., 2008) which was not verified in the present study. the similarities occurring in honey samples can be caused by the variability of floral species used in honey composition or due to the abiotic characteristics of the collection sites, which may influence the characteristics of the honey (crane, 1990; marchini et al., 2004b; barth, 2004). the samples form clusters of physical-chemical and pollen similarities related to the collection sites. samples of honey containing the dominant pollen g. max and eucalyptus were grouped according to physical and chemical characteristics, which shows the influence of the pollen type on the characteristics of honey, when the floral species present high representativeness. however, multivariate classification in groups based on pollen types formed no clusters in samples of polyfloral honey. acknowledgments the authors thank the beekeepers who made possible the collection of the samples used in this work. references aganin, a.f. 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(2008). procedimentos e determinações gerais. in: métodos físico-químicos para análise de alimentos. 4 ed. são paulo: instituto adolfo lutz, cap. 4, p. 125-130. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2843sociobiology 65(3): 397-402 (september, 2018) defensive strategies of a noctuid caterpillar in a myrmecophytic plant: are dyops larvae immune to azteca ants? introduction as most holometabolous insects, lepidoptera, one of the most diverse insect orders (aguiar et al., 2009; zhang, 2011), split their lives in distinct stages, enabling them to exploit different environments and diets. however, while adults are mobile and can easily escape from predators, lepidopteran larvae (caterpillars) present low mobility and usually feed on foliage, where vulnerability to natural enemies is high (bernays, 1997). together with birds, ants are the main predators of lepidopteran larvae (remmel et al., 2011; singer et al., 2012; sendoya & oliveira, 2017), and adults and larvae both exhibit a large array of strategies to avoid ant encounters (salazar & whitman, 2001; freitas, 1999; freitas & oliveira, 1992, 1996; machado & freitas, 2001; gentry & dyer, 2002; greeney et al., 2012; sendoya & oliveira, 2015, 2017). anti-predator strategies may involve gregariousness (turner & pitcher, 1986; lawrence, 1990; mcclure & despland abstract immature stages of insects are generally susceptive to their natural enemies, but many species developed defensive and evasive mechanisms to circumvent predation. gregarious larvae of the noctuid moth dyops cf. cuprescens feed on leaves of young cecropia pachystachya shrubs colonized by azteca ants. ants avoid contact with larval clusters, retreating to the nest when larvae are moving near the stems. provoked encounters revealed that dyops caterpillars present several specialized behaviors to avoid and overcome ant attacks, such as fleeing to under leaf, jumping off the leaf, curling and wriggling vigorously the anterior portion of the body, spitting droplets of oral fluids, or killing ants by pouncing them. these mechanisms allow the caterpillars to overcome ant attacks and consume leaves of ant-colonized plants. by feeding on a heavily protected plant, larvae can enjoy not only a competitor-free plant, but possibly also the enemy-free space created by the aggressive ants. sociobiology an international journal on social insects rr ramos1, avl freitas1, rb francini2 article history edited by evandro n. silva, uefs, brazil received 10 january 2018 initial acceptance 25 march 2018 final acceptance 31 may 2018 publication date 02 october 2018 keywords defensive behavior, larval behavior, insect-plant interaction, oral secretion, resource partitioning. corresponding author renato rogner ramos departamento de biologia animal instituto de biologia universidade estadual de campinas cep 13083-862, campinas-sp, brasil. e-mail: rognerramos@gmail.com 2011; greeney et al., 2012), coloration such as camouflage and aposematism (edmunds, 1974), morphological traits that make an enemy approach physically difficult, such as scoli, spines, and hairs (frost, 1959), construction of protective shelters using plant tissue and faeces (eubanks et al., 1997; freitas, 1999; freitas & oliveira 1992, 1996; machado & freitas, 2001; moraes et al., 2012), and the use of oral fluids to build circular barriers (devries, 1991). gregarious larvae of the moth dyops cf cuprescens hampson (noctuidae) use several species of urticaceae as larval hostplant (wiltshire, 1962, see also janzen and hallwachs, 2017, for pictures of early stages and host plant records), including cecropia pachystachya trécul (present study), a common myrmecophytic plant usually harboring colonies of azteca ants (formicidae: dolichoderinae) in their hollow stems (müller, 1881; ihering, 1907). azteca ants are known to prevent vines from climbing cecropia trunks (janzen, 1969; schupp, 1986; longino, 1991), and to attack 1 departamento de biologia animal and museu de zoologia, universidade estadual de campinas, são paulo, brazil 2 universidade católica de santos, são paulo, brazil research article ants rr ramos, av l freitas, rb francini – defensive strategies of dyops caterpillars against ants398 insect herbivores on leaves (davidson 2005; oliveira et al., 2015). therefore, given that encounters between dyops larvae and azteca ants are expected to occur frequently on cecropia foliage, we investigated the following questions: (1) do azteca ants attack dyops larvae feeding on cecropia leaves? (2) do dyops larvae present anti-predatory strategies to overcome ant attacks? material and methods the noctuid moth dyops cf. cuprescens is a species widely distributed in the neotropical region, occurring on the dry and humid central american forests, in the amazon and in the atlantic forest. their caterpillars are gregarious and were reported using host plants mainly of the family urticaceae (wiltshire, 1962; janzen & hallwachs, 2017; discover life, 2018). field observations and experiments were carried out in two areas of lowland dense ombrophilous forest (ibge, 2012) on coastal são paulo state, southeastern brazil: 1) most of the study was undertaken at the “vale do rio quilombo” (hereafter vq), santos municipality (further information in francini 2010). behavioral observations and simulated antcaterpillar encounters in this site were carried out from april to may 2017. 2). additional observations were carried out in the “parque estadual xixová-japui” (xj), são vicente municipality, from april to may 1992 (further information in freitas, 1996). encounters between dyops and azteca ants (n = 10) were provoked by gently removing one caterpillar (from 8 to 13 mm) from its cluster (n = 2 clusters) using a tweezer. the larva was then placed on the upper leaf surface of different individuals of c. pachystachya colonized by ants, categorized as follows: (1) the same plant where the larva was found (n = 4); (2) a different plant (same size and colonized by azteca ants), not infested by dyops larval clusters (n = 6). experimental plants were gently shaken before each provoked encounter to incite ant activity on leaves. ant-caterpillar interactions were recorded using two photographic cameras (nikon coolpix l5 and canon 7d) and two video cameras (go-pro hero 3 and sony dsc hx300). results larvae of dyops were always found in clusters containing 40 to 150 individuals (mean = 87.8, sd = 38.28, n = 7) on mature leaves of c. pachystachya (fig 1a, b). all observed clusters were located on young plants (1 to 2 m tall) colonized by azteca ants in both study sites, vq (n = 2 clusters) and xj (n = 5 clusters). in the beginning of our observations, despite the presence of larval clusters, all plants had most of their leaves intact. ants appeared not to be disturbed by the presence of larval clusters, keeping distance as caterpillars fed and moved freely on foliage. although larval clusters remained distant from the hollow cecropia stems harboring the ant colonies most of the time, encounters with ants inevitably occurred whenever a cluster moved from one leaf to another. on these occasions, the larval cluster walked through the petiole and plant stem to reach a new leaf (fig 1b). during these larval movements, ants retreated into the nest gallery and avoided contact with larvae. during the study period in vq, dyops-induced herbivory caused one azteca-occupied cecropia to have its leaves reduced from 10 to two in two weeks. a single observation of ant attack to a torn larva was observed, but the ant withdrew to the nest soon after the attack. behavioral strategies of dyops caterpillars to avoid ant attacks are summarized in table 1. nine out of 10 provoked encounters resulted in attacks by the ants (one larva was ignored on a plant hosting a cluster). evasive behavior was by far the most observed, with larvae moving to under leaf ceasing its movements (n = 6). on two occasions the caterpillars jumped off the leaf upon attack, and suspended themselves on the end of a silken thread (fig 1c). when attacked by ants (n = 7), dyops caterpillars exhibited a “beat reflex” (n = 3), curling and wriggling vigorously the anterior portion of the body to dislodge or keep ants away, and/or spit oral fluids (n = 4) that effectively repelled the ants (table 1). oral fluids are expelled as drops of a transparent liquid that turns dark behavior description n jumping off the leaf larva drops off the plant after successive bites or attacks, falling on the soil or on a lower leaf of the same plant, or hanging themselves by silken threads (see below). 2 dislodgment larva moves to under leaf ceasing its movements; the larva usually is not followed by the ants. 6 hanging from a silken thread the jumping larva can suspend itself by silken threads, eventually climbing back to leaf after a few minutes. 2 spiting oral fluids attacked larva raises the anterior portion of body and spits droplets of oral fluid towards the ants. 4 safety zone oral fluid falling on leaf surface forms moist patches that keep ants momentarily away (“safety perimeter”); ants touching these fluids showed several signs of disturbance such as body trembling, lethargy, rubbing the mandibles on leaf surface, and withdrawing stunned. 3 “beat reflex” curling and wriggling vigorously the anterior portion of the body can intimidate and temporarily keep ants away. 3 pounce pounce the ant simultaneously releasing oral fluid; ant dies almost instantly. 2 table 1. description of observed behavioral strategies of dyops larvae to avoid attacks by azteca ants on cecropia pachystachya (from 10 provoked encounters). n = total number of observations of each behavior based on the 10 provoked encounters. sociobiology 65(3): 397-402 (september, 2018) 399 after some minutes. these oral fluids falling on leaf surface form moist patches that keep ants away for several minutes, in a sort of “safety perimeter” (n = 3). ants soaked by these oral fluids vigorously cleaned their mandibles, antennae and head, and some of which died after few minutes. interestingly, ants that attached the caterpillar body ended up dying in few minutes (fig 1d) after biting them. in addition, two larvae were observed displaying a pounce behavior, simultaneously biting and spitting fluids towards the ant, causing immediate death of the aggressor (see additional data on table 1) (a video clip showing most of the anti-predator behaviors is available at the following link: https://youtu.be/yhyioqthfkg). see also a video as supplementary file: http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/2843/0 http://dx.doi.org/10.13102/sociobiology.v65i3.2843.s1892 discussion by living on a plant usually sheltering ant colonies, dyops caterpillars display a series of anti-predator behaviors, as observed in other species feeding on plants where encounters with ants are frequent (heads & lawton, 1985; bentley & benson, 1988; freitas & oliveira, 1992, 1996; oliveira & freitas, 2004; sendoya & oliveira, 2015, 2017; bächtold et al., 2012; moraes et al., 2012). most of the observed behaviors are common in non-myrmecophilous caterpillars, such as the “beat reflex”, biting, and jumping off the plant followed by hanging on a silken thread (heads & lawton, 1985; salazar & whitman, 2001; sendoya & oliveira, 2017). the behavior of spitting droplets of oral fluids against the aggressors is by far the most immediate antipredation strategy observed in dyops larvae. regurgitation is a common anti-predation behavior in caterpillars in general (freitas & oliveira, 1992; smedley et al., 1993; gentry & fig 1. a. cluster of about 150 larvae of dyops cf. cuprescens; b. a large larval cluster feeding on a nearly entire leaf (left arrow) after completely consuming a leaf (right arrow); note that a larval cluster must reach the main stem to reach a new leaf; c. a small larva (ca. of 1 cm) suspended by a silken thread after being attacked by ants; d. a medium sized larva (ca. of 3.5 cm) attacked by an ant that died later (white arrow); note the presence of brownish oral fluids beneath the larva. rr ramos, av l freitas, rb francini – defensive strategies of dyops caterpillars against ants400 dyer, 2002), and its effectiveness to deter ant attacks has been already tested (peterson et al., 1987; rostas & blassmann, 2009). in addition to the sub lethal effects observed when ants are soaked with oral fluids, dyops larvae also displayed a very specialized pounce behavior that cause immediate death of the aggressors. the observation of dead ants attached to the body of attacked caterpillars suggests that larval fluids can be poisonous to ants (see also collins, 2013), a defensive strategy that could be enhanced by gregariousness. however, further observations and experiments are needed to determine more precisely if the death of the ants attached to caterpillars’ bodies are caused by cuticle fluids, oral secretions, or bites by dyops larvae upon attack. in short, although the present study do not include chemical analysis of the oral fluid of dyops, the results strongly suggests that the oral fluids are not simple regurgitation of gut contents, as observed in other caterpillars and surely present noxious compounds that repel and protect the larvae against ants and other arthropod predators (peterson et al., 1987; rostas & blassmann, 2009). in addition to the potential improvement of chemicallybased ant deterrence, gregariousness is a defensive behavior by itself, where the group size and movement enhance the efficacy of defensive behaviors (lawrence, 1990; mcclure & despland, 2011; greeney et al., 2012). in fact, clusters of dyops larvae were observed freely moving through various parts of c. pachystachya without being disturbed by ants, which retreat to their nest in the stem galleries when caterpillars approach, or are displaced to patrol plant sectors away from dyops larval clusters. additional observations in both study sites showed that, except for dyops larvae, all other cecropia herbivores are eliminated from plants after azteca colonization. interestingly, another species of dyops (dyops cf. cuprescens (walker), was also observed in small cecropia plants colonized by azteca ants in the xj site. however, larvae of this species are isolated and build shelters by curling the leaf edge under the leaf blade to form a tube (see greeney & jones, 2003). further studies and experiments should be carried out to investigate if this species also present deterrence or other elaborate behaviors to escape from ant attacks. our field observations indicate that gregarious larvae of dyops can overcome the aggressiveness of azteca ants inhabiting c. pachystachya, making it possible to feed on an ant-defended plant (janzen, 1969; schupp, 1986). in addition, by using a protected plant, larvae can enjoy not only a competitor-free plant, but possibly also an enemy-free space created by the aggressive ant inhabitants (see price et al., 1980; jeffries & lawton, 1984; kaminski et al., 2010; dáttilo et al., 2016). acknowledgements we would like to thank dr. victor o. becker for helping with the identification of the dyops caterpillars. paulo s. oliveira, sebastian e. sendoya and lucas a. kaminski kindly revised the last version of the manuscript. avlf thanks the brazilian cnpq (grant 303834/2015-3), redelepsisbiota-brasil/cnpq (563332/2010-7) and fapesp (grant 2011/50225-3). references aguiar, a.p., dos santos, b.f., couri, m.s., rafael, j.a., costa, c., ide, s., duarte, m., grazia, j., schwertner, c.f., freitas, a.v.l. & azevedo, c.o. 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(2011). animal biodiversity: an outline of higher-level classification and survey of taxonomic richness. zootaxa, 3148: 1-237. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.119-132sociobiology 61(2): 119-132 (june, 2014) ant communities along a gradient of plant succession in mexican tropical coastal dunes p rojas1, c fragoso1, wp mackay2 introduction ants are social insects with important ecological functions. they influence ecosystems through soil bioturbation (lobry de bruyn & conacher, 1990), predation of invertebrates (gotwald, 1995) and mutualistic interactions with hundreds of plant species (jolivet, 1996). due to their high diversity, numerical and biomass dominance (fittkau & klinge, 1973, brown, 2000) and sensitivity to environmental changes (andersen, 1995), ants constitute an ideal group to inquire about patterns in community characterization. coastal dunes are complex and very dynamic environments that have been shaped by biological and physical processes like water and wind action (mclachlan, 1991). its high environmental heterogeneity is determined by distinct landforms and different plant communities (martínez et al., 2004). ant communities from coastal environments have been abstract most of mexican coastal dunes from the gulf of mexico have been severely disturbed by human activities. in the state of veracruz, the la mancha reserve is a very well preserved coastal community of sand dunes, where plant successional gradients are determined by topography. in this study we assessed species richness, diversity and faunal composition of ant assemblages in four plant physiognomies along a gradient of plant succession: grassland, shrub, deciduous forest and subdeciduous forest. using standardized and non-standardized sampling methods we found a total of 121 ant species distributed in 41 genera and seven subfamilies. grassland was the poorest site (21 species) and subdeciduous forest the richest (102 species). seven species, with records in ≥10% of samples, accounted 40.8% of total species occurrences: solenopsis molesta (21.6%), s. geminata (19.5%), azteca velox (14%), brachymyrmex sp. 1lm (11.7%), dorymyrmex bicolor (11.2%), camponotus planatus (11%) and pheidole susannae (10.7%). faunal composition between sites was highly different. nearly 40% of all species were found in a single site. in all sites but grassland we found high abundances of several species typical of disturbed ecosystems, indicating high levels of disturbance. a species similarity analysis clustered forests in one group and grassland and shrub in another, both groups separated by more than 60% of dissimilarity. similarity of ant assemblages suggests that deciduous and subdeciduous forests represent advanced stages of two different and independent successional paths. sociobiology an international journal on social insects 1 instituto de ecología a.c. (inecol). xalapa, méxico. 2 university of texas at el paso, el paso, usa. article history edited by kleber del-claro, ufu, brazil received 12 march 2014 initial acceptance 09 april 2014 final acceptance 12 may 2014 keywords ant assemblages, species richness, diversity, la mancha, méxico corresponding author patricia rojas instituto de ecología a.c. (inecol) carretera antigua a coatepec 351 el haya, xalapa 91070 veracruz, méxico. e-mail: patricia.rojas@inecol.mx poorly studied. however, the importance of these ecosystems for conservation of ants has been recently recognized (howe et al., 2010). communities from temperate and tropical coastal ecosystems are markedly different. in general, ant communities from temperate marine coasts have a low number of species and, independently of the number of sites included in a given locality, the number of species never surpass two dozen. for example, boomsma and de vries (1980) in the netherlands recorded only three species in sparsely vegetated sand dunes and grasslands, whereas studies in successional gradients from pioneer vegetation to mature forests carried out in finland (gallé, 1991) and spain (ruano et al.,1995), recorded 19 and 24 species respectively. in temperate dunes, species richness, abundance and equitability increases along vegetation succession, with higher values being found in sites with a more dense plant cover (boomsma & van loon, 1982; ruano et al., 1995). positive correlations have also been observed between research article ants p rojas et al ant communities in tropical coastal dunes 120 composition of ant assemblages and a more complex habitat (boomsma & van loon, 1982). these attributes of ant assemblages, however, are relatively independent of diversity and composition of vegetation, suggesting that in temperate coastal systems, plant succession stages are not coordinated with successional stages of ant communities (gallé, 1991). in tropical coastal dunes the number of ant species is more variable. studies in comparable vegetation mosaics, recorded from 22 to 92 species in cuba (fontenla, 1993, 1994), mexico (durou et al., 2002), and brazil (bonnet & lopes, 1993; texeira et al., 2005; vargas et al., 2007; cardoso et al., 2010). in these systems species richness varies between habitats, with higher values being found in more complex and heterogeneous habitats (fontenla, 1993; durou et al., 2002; vargas et al., 2007). faunal composition is another attribute of tropical coast ant assemblages that strongly varies with the type of vegetation (fontenla, 1993; cardoso et al., 2010) and plant cover (dorou et al., 2002). most of natural undisturbed coastal dunes of the mexican littoral zone of the gulf of mexico have disappeared due to extensive farming management, human settlements and touristic activities (moreno-casasola, 2006). however, the state of veracruz still harbors some well-preserved sites of coastal dunes that can enter inland up to 3 km. one of these sites is found within the ecological reserve of the centro de investigaciones costeras la mancha (cicolma). in the dunes of la mancha environmental gradients related to the force of the wind, sand movement, and depth of water table (ultimate mediated by topography), determine the establishment of different plant communities (moreno-casasola & vázquez, 2006). in this zone the vegetation follows a successional gradient, from pioneer plants and grasslands growing on the beach and young dunes, to deciduous and subdeciduous tropical forests established on older dunes. this last community constitutes the last remnant of this kind of forest in the gulf of mexico growing in sandy soils (moreno-casasola & travieso, 2006). several studies of ants have been conducted at la mancha, including numerous aspects of plant-ant interactions (rico-gray, 1989, 1993; mehltreter et al., 2003), evaluation of some invasive ants (fragoso & rojas, 2009), records in checklists (rojas 2001, 2011) and taxonomical studies (mackay et al., 2004). so far, no studies characterize the complete ant community in any vegetation type of this site have been published. the main objective of our study was to describe the ant communities of la mancha in four types of vegetation that represent a gradient of plant succession on coastal dunes. communities were characterized considering species richness, diversity, abundance and species composition. we hypothesized that richness and diversity would increase along the vegetation successional gradient; that each stage will have different faunal composition, and that differences will be higher between early and late successional stages. material and methods study area the study area is located at the centro de investigaciones costeras la mancha (cicolma) in the coast central region of veracruz state (96°22’40’’ w; 19°36’00’’ n) with an altitudinal range of 0-80 m elevation. the cicolma field station covers a total area of 83 ha. the zone is geologically young, shaped by miocene volcanic activity and by quaternary deposits (geissert, 1999). the weather is characterized by an annual average temperature of 25°c and an annual precipitation of 1500 mm, with the large amount of rains (78%) occurring during the rainy season of june-september. soils are unstructured luvic and calcaric arenosols (travieso & campos, 2006). sampling sites were established on four different successional stages of coastal dune vegetation: grassland, shrub, deciduous forest and subdeciduous forest (fig 1). grasslands and shrubs are found in semi stabilized dunes, with grasslands being found in upper dry parts of the dunes and shrubs on humid depressions, where water table is higher. tropical forests grow over stabilized dunes with subdeciduous forests being established on flat sites or sites with level relief, and deciduous forests located on more steep sites (moreno-casasola & travieso, 2006). grassland (g), (fig 1a) with a plant cover of nearly 40%, this community includes grasses and short shrubs that alternate with open spaces of bare sand. common species are trachypogon plumosus (humb. & bonpl. ex willd.) nees, andropogon glomeratus (walter) b.s.p. and chamaecrista chamaecristoides (colladon) greene (moreno-casasola & travieso, 2006). fig 1. la mancha plant physiognomies sampled in this study. a − grassland, b − shrub, c − deciduous forest, d − subdeciduous forest. sociobiology 61(2): 119-132 (june, 2014) 121 shrub (s), (fig 1b) medium size (2-3 m high) closed canopy shrub with a plant cover of 80% and with some isolated tress. predominant species are shrubs randia laetevirens standl., pluchea odorata (l.) cass. and mimosa chaetocarpa brandeg. (moreno-casasola & travieso, 2006). deciduous forest (df), (fig 1c) trees reach 12m height, with many deciduous species. most common species are bursera simaruba (l.) sarg., coccoloba barbadensis jacq. and ocotea cernua (nees) mez. the understory stratum is dominated by crossopetalum uragoga (jacq.) kuntze, chiococca alba (l.) hitchc. and randia aculeata l.; grasses and herbs are almost absent (castillo, 2006). subdeciduous forest (sf), (fig 1d) – the canopy (>20m height) is dominated by brosimum alicastrum sw., ficus cotinifolia kunth, cedrela odorata l. and enterolobium cyclocarpum (jacq.) griseb., whereas the lower canopy (6-15 m) is characterized by erithroxylum havanense jacq., nectandra salicifolia (kunth) nees and ocotea cernua. the species crossopetalum uragoga, schaefferia frutescens jacq. and hippocratea celastroides kunth predominate in the shrub stratum (castillo, 2006). ant sampling our sampling was performed only in one plot per vegetation type (no replicates); however we consider that the amount of traps and eight different sampling methods made this study valid for site comparisons. standardized sampling (ss). sampling of grassland, deciduous forest and subdeciduous forest was performed during 1992 whereas shrub was sampled two years later (1994). in each site, a 20x20m (400m2) plot was delimited and located at least 30m inside the respective vegetation type; the plot was a grid each five meters. sampling was made in the dry (may) and rainy (october) seasons. a total of 588 samples were obtained, using the following standardized sampling methods: pitfall traps (based on greenslade, 1964) (200 traps) in each site and plot 25 plastic container traps were set at distances of five meter intervals in a grid pattern. each container, with a volume of 250 ml and a diameter of 8 cm, was buried at ground level and filled 3/4 with 70% ethanol and a small quantity of commercial detergent. traps remained in the field five days. subterranean baits (200 baits) following the same grid pattern, 25 subterranean baits were set in each site and plot. each bait was buried at 20-30 cm depth, inside a plastic vial of 5 ml volume and with several holes in the walls (mackay & vinson, 1989); tuna fish and a mixture of honey and oatmeal were used as bait. traps were left buried for 48 hours. surface baits (72 baits) (see bestelmeyer et al., 2000) in each site and plot nine surface (three per row) baits were placed along three parallel rows separated 10 meters. in each row baits were separated 10 m each. a teaspoon of bait (the same type used in subterranean sampling) was placed within a 10cm diameter plastic petri dish, deposited aboveground and left for 5 hours (from 10:00 to 15:00 h). attracted ants were collected at one hour intervals. arboreal baits (70 baits) this method was used only in the two forests, because trees were nonexistent or very scarce in g and s plots, respectively. baits (same type used in former methods) were placed inside plastic containers of 150ml and tied at 1.5m height in different tree species with a minimal diameter of 20cm. baits were left for 5 hours (from 10:00 to 15:00h); attracted ants were collected at one hour intervals. twenty arboreal baits were placed in df and 15 in sf. leaf-litter samples (30 samples) (see bestelmeyer et al., 2000) this method was used in all sites except g, where no litter stratum was found. in each site five samples of litter (1m2) were collected and processed with berlese funnels until the litter was dry. samples were taken from the center and two meters inside the four corners of each plot. hand sampling (16 samples) in all sites, search and capture of ants was performed by two persons during 4h (8 hours per site). sampling included vegetation (epiphytes, foliage and hollow stems) and soil (litter, first centimeters of soil and within and under decaying logs). non standardized sampling (nss). it refers to any sample of ants obtained out of the ss protocol by hand sampling. ants were identified to genus level using bolton (1994); species were determined using specialized publications and revisions or by comparison with reference material from the ant collections of w.p. mackay and the laboratorio de invertebrados del suelo (inecol, xalapa). voucher specimens of all species were deposited in the latter collection. nomenclature follows bolton et al. (2007). data analysis seasonality differences in the number of species captured in rainy and dry season were analyzed with parametric t-test for dependent samples (when normality was fitted) and nonparametric wilcoxon matched pair test. analyses were performed for each site and the following ss methods: pitfall traps, subterranean baits, surface baits and arboreal baits. all these tests were performed using statistica (statsoft, 1999). seasonality differences for a given site (ss + nss samples) were compared considering 95% confidence intervals derived from rarefaction curves (mao tau, estimates program version 9, colwell, 2013). abundance in order to avoid over estimation of those species with large foraging areas or legionary habits, abundance was calculated as presence/absence data (occurrence) from ss samples. for a given species, abundance was calculated as frequency of occurrence (fo=ni/n x100, where ni is the number of samples where species i was found, and n is the total number of samples). the number of occurrences in samples was considered as an indirect measure of the relative p rojas et al ant communities in tropical coastal dunes 122 abundance of each species in each site (gottelli & colwell, 2011) and can be used to estimate abundance distribution of species along gradients (andersen, 1997). species richness and diversity considering ss data, species accumulation curves were generated as rarefaction curves (mao tau) and compared to estimated richness (chao 2 estimator) (gotelli & colwell, 2011) using estimates program (version 9, colwell, 2013). diversity was calculated using shannon and simpson indices; differences between vegetation types were evaluated using the t-tests of hutcheson (1970) and brower et al. (1998) for, respectively, shannon and simpson indices. these analyses were performed using past program (version 3, hammer et al., 2001). similarity similarity of the four sites was calculated following two approaches. in the first one a hierarchical similarity analysis of sites was made with the presence/absence data (ss and nss sampling) of each species in each site (121 species per four sites). variables were associated and clustered using kulczinski index (incidence-based) and upgma, respectively and represented as a similarity dendrogram; these analyses were performed using patn software (belbin, 1989). incidence-based sorensen index was also calculated for comparative purposes. in the second approach we used fo of each species obtained from ss sampling. the resultant species per site matrix (97 x 4) was analyzed using the morisitahorn index (abundance-based), from estimates (version 9, colwell, 2013). vertical distribution each species was assigned to one of the following vertical categories: soil only, vegetation only, both. assignment was made considering the more frequent localization of nests (personal observations) and by consulting natural history information available in current literature. results considering that no significant differences in number of species were found between dry and rainy seasons for all sites and sampling methods, except for pitfall trap data from g and sf (see table 1), and that similar numbers of species were collected during dry and rainy seasons (78 and 77 species, respectively) our results correspond to clumped data from both seasons. in the four sites 121 ant species of 41 genera and seven subfamilies were found. with the standardized sampling 97 species (80.2 % of total) were obtained from 1440 species occurrences from all sites; the remaining 24 species were captured using non standardized sampling. subfamilies with more species were myrmicinae (57 species) and formicinae (23); the genera with more species were camponotus (16 species), pheidole, pseudomyrmex (14 each), solenopsis and crematogaster (5 each) (appendix 1). the following seven species, with records in ≥10% of samples, accounted for 40.8% of total species occurrences: solenopsis molesta (21.6%), s. geminata (19.5%), azteca velox (14%), brachymyrmex sp. 1lm (11.7%), dorymyrmex bicolor (11.2%), camponotus planatus (11%) and pheidole susannae (10.7%). twenty eight species (23%) were repretable 1. average values and standard deviations (in brackets) in the number of ant species found in dry (xd) and rainy (xr) seasons at la mancha with four standardized sampling methods (ss). the last column shows values (and 95% confidence intervals) including all species in dry (nd) and rainy (nr) seasons collected by ss and non-standardized methods, nss. significant differences between seasons are in bolds (ns = no significant differences). pitfall (t-test) subterranean (wilcoxon) superficial (t-test) arboreal (wilcoxon) ss + nss grassland xd=3 (1.6) xr=1.6 (1.3) n=25 p=0.0007 xd=0.52 (0.59) xr=0.6 (0.65) n=25 ns xd=2.3 (1) xr=2.1 (1.3) n=9 ns not used nd=14 ± 5.1 nr=10 ± 3.6 shrub xd=6.2 (1.7) xr=6.4 (2.5) n=25 ns xd=0.16 (0.37) xr=0.2 (0.41) n=25 ns xd=3.8 (1.5) xr=3.8 (1.3) n=9 ns not used nd=29 ± 6.3 nr=38 ± 8.4 deciduous forest xd=2.7 (1.6) xr=2.5 (1.3) n=25 ns xd=0.88 (0.53) xr=0.80 (0.41) n=25 ns xd=4.1 (1.7) xr=4.7 (1.7) n=9 ns xd=1.9(1.1) xr=2(1) n=20 ns nd=26 ± 8.5 nr=34 ± 8.6 subdeciduous forest xd=2.5 (1.7) xr=4.2 (1.5) n=25 p=0.001 xd=0.36 (0.76) xr=0.44 (0.58) n=25 ns xd=3.8 (1.9) xr=3.2 (1.6) n=9 ns xd=1(0.8) xr=2(1.6) n=15 ns nd=50 ± 8.8 nr=44 ± 9.5 sociobiology 61(2): 119-132 (june, 2014) 123 sented only by a single record. species richness when considering only standardized sampling (ss), g was the site with the lowest species richness (15 species) whereas sf forest was the richest (62). df and s presented an intermediate value of richness (42 species each). estimates of expected species richness for each site (chao2 richness estimator) showed that g was the best sampled site (77% of expected number of species), followed by sf and df (64 and 59%, respectively). s turned to be the worst sampled site (56%) (fig 2 and table 2). when species captured with non-standardized sampling (nss) are added, sites followed the same order of species richness, although percentages of increase varied for each site. species richness values of g and sf were closer to those predicted by chao2 index, even exceeding the estimated total number of species (table 2). abundance the lowest number of occurrences was found in g (197, 13.7% of total). s was the site with the highest number of occurrences (454, 31.5% of total); however, only with pitfall traps data was this value significantly different from the other sites (anova, f3,196=52.88, p < 0.0001, tukey hsd test; xs= 6.30, ses= 0.31; xg= 2.34, seg= 0.22; xdf= 2.60, sedf= 0.21; xsf= 3.32, sesf= 0.25). the two forests showed intermediate occurrences values (deciduous: 393, 27.3%; subdeciduous: 396, 27.5%). on the basis of the five most abundant species from each site (table 3) communities were very different. noteworthy in g these five species, characterized by the presence of three dorymyrmex species, were not found in the two forests. alfig 2. species accumulation curves of ant species for each site. solid lines correspond to rarefaction curves (mao tau) of observed species richness; dashed lines correspond to chao2 predicted species richness. table 3. abundance expressed as percentage of occurrences in samples (fo) of the five most important ant species in each site (in bold). fo values of these species in the other sites are shown for comparison; a dash means absence of species. g = grassland; s = shrub; df = deciduous forest; sf = subdeciduous forest. g (n=122) s (n=132) df (n=172) sf (n=162) azteca velox 5.3 40.7 3.7 dorymyrmex bicolor 42.6 10.6 dorymyrmex smithi 22.9 dorymyrmex sp. aff. flavus 10.6 0.7 forelius pruinosus 19.7 15.1 brachymyrmex sp. 1 37.7 9.1 5.1 0.6 camponotus planatus 1.6 13.6 22.7 3.7 monomorium ebeninum 43.9 pheidole punctatissima 1.2 23.4 pheidole sp. 11lm 23.5 5.5 pheidole sp. 5lm 18.6 1.8 pheidole susannae 5.3 34.6 solenopsis molesta 0.8 29.5 30.8 21.0 solenopsis geminata 8.2 39.4 16.3 15.4 tetramorium spinosum 21.2 wasmannia auropunctata 0.8 7.0 22.2 though dorymyrmex sp. aff. flavus and d. bicolor appear also in s, they have lower abundances. in g and s these five species accounted for 82.7% and 45.8% of species occurrences, respectively. in df this group represented 56.5%, with the arboreal azteca velox the more abundant species. in sf forest this group includes only ground ants which accounted 47.7%, with four of these species characteristic of disturbed places. solenopsis geminata and s. molesta always appear in s, df and sf among the five more abundant species. diversity as expected, lower and higher diversity values were respectively observed in g and sf; however, diversity in s was higher than in df (shannon and simpson indices, table 4). in paired comparisons, all sites significantly differ in shannon and simpson indices (table 5), excepting differences between s and sf with simpson index. rank abundance plots (fig 3) also indicate that g was the less diverse assemblage (higher slope and shorter line) and that sf was the more diverse. even that s and df forest had the same number of species (ss sampling) the curve of s had a less steep slope indicating a higher evenness in the abundance of their species. long curves observed in sf, df and s reflect presence of many species with very low abundances. faunal composition appendix 1 (ss and nss data) show the list of all species found at la mancha, suggesting at first glance strong differences in faunal composition between sites. a similarity analysis of species presence/absence (kulczinski index) clustered forests in one group and g and s in another, both groups being separated by more than 60% table 2. observed (ss and ss+nss) and estimated (chao2) species richness of ants in the four sites. ss= standardized sampling; nss= non standardized sampling. ss ss+nss chao2 grassland 15 21 19.5 shrub 42 47 74.7 deciduous forest 42 50 70.9 subdeciduous forest 62 102 96.3 total 97 121 p rojas et al ant communities in tropical coastal dunes 124 of dissimilarity (fig 4). similar results were obtained when similarity was calculated with sorensen index (table 5). conversely, the morisita-horn index indicated a higher similarity of df and sf with s, leaving g as an isolated site. the number of species considered in morisita analysis was lower, because abundance data was obtained from standardized sampling only (table 5). table 4. ant diversity of the four sites studied obtained with two diversity indices. data from standardized sampling (ss). shannon (h´) mean (sd) simpson (1/d) mean (sd) grassland 2.06 (0.06) 6.09 (0.04) shrub 3.06 (0.04) 16.30 (0.09) deciduous forest 2.89 (0.05) 12.29 (0.09) subdeciduous forest 3.35 (0.06) 17.92 (0.17) site specificity at la mancha 48 ant species (ca. 40% of total) were found in a single site. sf had the largest number of exclusive species (39, 32.2%), whereas df and s had four exclusive species each (3.3%) and g only one species (0.8%). in contrast number of ubiquitous species (those found in all sites) was very low. only six species (5%) were found in the four vegetation types: brachymyrmex sp. 1lm, camponotus planatus, c. atriceps, cyphomyrmex rimosus, solenopsis geminata and s. molesta (appendix 1). vertical distribution soil-nesting species (69, 57%) dominated over plant-nesting species (48, 40%), with only four species nesting in both strata (3%) (appendix 1). five species in g were plant-nesting ants, but were low in abundance, and always captured with pitfall traps and soil baits; the remaining species (17, 76%) were soil dwellers. in s, very similar numbers of species nesting in soil (23, 49%) and vegetation (22, 47 %) were found, with two species nesting in both strata (4%). a similar situation was observed in df, 23 species (46%) nesting in soil, 25 (50%) in vegetation and two species (4%) in both strata. finally sf forest had, respectively, 56 (55%) and 42 (41%) soil and plant-nesting species, with four species (4%) inhabiting both strata. discussion in order to capture as many species as possible, ant communities were sampled in two seasons. sampling was not designed with the aim to compare seasonal patterns. accordingly, we conducted some statistical tests to confirm that all the ss data can be grouped. in general no significant differences were observed between rainy and dry season in the amount of species per sampling method and by site, excepting pitfall traps of g and sf. this can be explained by the dependence of this method on the foraging activity of ants (bestelmeyer, 2000). total species richness – the number of ant species en-fig 3. rank-abundance plot of ant species in each site. abundance is expressed on a log scale. fig 4. similarity between sites in function of presence/absence of ant species. scale shows values of dissimilarity (kulczinski index). g = grassland, s = shrub, df = deciduous forest, sf = subdeciduous forest. countered in the four vegetation types studied at la mancha was notably high, considering the relatively small area of the reserve (83ha, moreno-casasola & monroy, 2006). moreover, this number of species (121) make up nearly 34% of the total number of species recorded in the state of veracruz (rojas, 2011) and 13.7 % of all mexican ant species (vázquez-bolaños, 2011). comparatively, similar studies completed in tropical coastal ecosystems recorded lower values of species richness. for example in the brazilian “restinga” cardoso et al. (2010) reported 71 species along one transect of 6.5 km length, whereas vargas et al. (2007) recorded 92 species; similarly in mexico durou et al. (2002) found 96 species. the highest values found at la mancha can be explained because our study included two well-developed tropical forests. in spite that vargas et al. (2007) also sampled a tropical forest, their ant richness values were still lower than in la mancha. species richness and diversity along the vegetation gradient the four studied sites represent a successional plant gradient established on sand dunes, which also entails the stabilization of dunes. accordingly, the grassland with 21 ant species and low diversity values, represent the first stage in dune succession and it is the less complex environment with sociobiology 61(2): 119-132 (june, 2014) 125 only herbaceous vegetation alternating with areas of bare sand. in addition no litter layer exists and temperature differences between high and low coverture sites can be up to 11°c (moreno-casasola & travieso, 2006); thus scarcity of nesting sites for ants is not only restricted to vegetation, but also occurs in the soil. non structured sandy soils represent an unstable substrate for ants, and they would need to frequently reconstruct nest chambers and galleries (lubertazzi &tchinkel, 2003). the higher diversity and number of species (42) in the shrub zone, the second successional stage, can be explained by the presence of both herbs and bushes. these strata produce a litter layer that, even scarce as it was, offers suitable microsites for the arrival and settlement of more ant species. the two forests correspond to last successional stages, and although they have a well-developed arboreal, bush and litter strata, their plant diversity and environmental conditions are not the same (castillo, 2006) as was indicated by ant diversity and species richness. by being located in upper and steep places of stabilized dunes, deciduous forest is more exposed to wind, with a higher water runoff, and is consequently dryer. this translates into less plant cover, smaller tree height and fewer epiphytes. conversely the subdeciduous forest is located in a more humid flat and wind protected place; this causes a higher plant cover, taller trees and more epiphytes (novelo, 1978). correspondingly, the more stable subdeciduous forest harbor twofold ant species richness (102 species) than the stressed deciduous forest (50). it is widely recognized that in most of habitats, plant communities determine the physical structure of environment and therefore have a strong influence over the distribution and interactions of animal species (lawton, 1983; rosenzweig, 1995; tews et al., 2004). several studies completed in temperate coastal dunes (boomsma & van loon, 1982; dauber and wolters, 2005) and in tropical brazilian “restinga” (vargas et al., 2007) have shown that more heterogeneous environments correspond to late successional stages and harbor a more diverse fauna and a higher species rich ant communities. in general all of these studies conclude that this pattern is due to the higher amount of microhabitats and microclimates that in turn produces more availability of food and nest sites. the results of this study partially support our original hypothesis related to an increase of ant species richness along the plant successional gradient (g→s→df→sf). sites with the lower and higher species richness were, respectively, g and sf; however no significant differences in species richness were observed between the intermediate gradient stages, with estimated richness (chao 2) very similar in s and df, and even higher in the former. diversity values showed also the same pattern. on the basis of these results we propose that plant succession at la mancha has not been unidirectional, but that two independent paths have occurred after the development of shrubs from grasslands: the first path would be the successional change of shrubs into deciduous forest (s→df) in more stressed environmental conditions; alternatively the second path would be the change of shrubs towards subdeciduous forest (s→sf) in more stable environments. faunal composition through coastal plant succession, faunal composition of ant assemblages varies between temperate and tropical ecosystems. in temperate sites, species assemblages are very similar across vegetation physiognomies, as it has been shown by studies in coastal dunes (gallé, 1991; ruano et al., 1995) and grasslands (zorilla et al., 1986; dauber & wolters, 2005). independently of the successional stage, ant assemblages from these sites have not shown different number of species; instead the abundances of each species changes across the gradient. on the other hand, ant communities from tropical ecosystems show larger differences across vegetation successions. at la mancha, the four studied vegetation types presented different species assemblages as has been observed in other coastal dune vegetation studies (fontenla, 1993; durou et al., 2002; cardoso et al., 2010). considering its faunal composition the studied sites were divided into two separated groups. the first group included sites of earlier stages of succession (g and s) whereas the second one grouped the two forests (df and sf) corresponding to late successional stages. from the point of view of vegetation, at la mancha grasslands and shrubs also comprises a well differentiated group from deciduous and subdeciduous forests, sharing only 14% of plant species (castillo & travieso, 2006). ant membership in the first group was defined by the share of eight species typical of dry environments, in spite of differences in the number of species. among them dorymyrmex bicolor and forelius pruinosus are well adapted to xeric conditions and nest and forage on the soil of sunny sites, especially in grasslands (shattuck, 1992). tetramorium spinosum was another shared species widely distributed in arid zones of mexico, that also nest in exposed soil (rojas & fragoso, 1994). although they nest only in s, other shared species such as atta cephalotes and crematogaster crinosa were found foraging in both g and s habitats; this foraging strategy has been also recorded in other coastal environments, where ants living in adjacent forests use dunes and beaches as foraging areas (ruano et al., 1995). the remaining two species shared by g and s were pseudomyrmex brunneus and p. ejectus which table 5. species similarity values between grassland (g), shrub (s), deciduous forest (df) and subdeciduous forest (sf), calculated with three indices. diversity differences between sites are also indicated: †= significant differences (p < 0.005) comparing simpson index (brower et al., 1998). *= significant differences (p < 0.01) comparing shannon index (hutcheson, 1970). kulczinski sorensen morisita-horn g vs s†* 0.41 0.50 0.25 g vs df†* 0.76 0.20 0.09 g vs sf†* 0.65 0.19 0.05 s vs df†* 0.63 0.37 0.39 s vs sf* 0.49 0.44 0.34 df vs sf†* 0.34 0.58 0.37 p rojas et al ant communities in tropical coastal dunes 126 have been reported occurring sympatrically and nesting in dead twigs of woody and herbaceous plants (ward, 1985). we were unable to find nests of these species, but we observed them foraging in soil and plants at both sites. it remains to be demonstrated whether or not these species are adapted to live in harsh environments. each site, nevertheless, has some exclusive species. whereas g has only one unique species, the abundant ant dorymyrmex smithii which nests under dead stems of grasses, s had four exclusive species (neivamyrmex rugulosus, nesomyrmex wilda and two unidentified pheidole), all very low in abundance. the second group clustered both forests on the basis of 27 shared species (see appendix 1). the presence of a welldeveloped arboreal stratum determines that more than 50% of these species nest in trees. in spite of their faunal similarities, both forests were separated by species richness and by the amount of exclusive species. while sf contained 39 exclusive species (38% of their 102 species), in df only four species (all low abundant) were exclusive (8% of their 50 species). thus, ant fauna of df can be considered as an impoverished subset of sf with more microhabitats available to ants. interestingly in all sites, but g, ants typical of disturbed environments were found in high abundances. this was the case of solenopsis molesta, a generalist soil-nesting species (mackay & mackay, 2002), s. geminata, commonly found in disturbed ecosystems of the neotropics, and which have been found even penetrating tropical forests (risch & carroll, 1982; taber, 2000), and pheidole susannae which has been reported in disturbed habitats throughout the neotropics (wilson, 2003). unexpectedly, the higher number of ant species associated to disturbance was recorded in sf, currently considered as a functional forest in the last stages of succession (castillo, 2006). an explanation of this finding could be related that in the past botanists recognized this site as a strongly disturbed secondary forest (novelo, 1978); moreover gomez-pompa (as cited in paradowska & moreno-casasola, 2006) suggests that presence of useful trees is an indication that this forest underwent high disturbances in the past, even being used as orchard by prehispanic people. vertical distribution our results showed that 97% of species were found associated with a single vertical stratum (soil or vegetation) in agreement with other studies which show a high vertical segregation in ants (bruhl et al., 1998; yanoviak & kaspari, 2000). in the three sites with developed plant strata, vertical segregation was nearly 50%. considering that in our sampled forests canopy ants were under-sampled, this proportion should change once a detailed sampling of canopy is undertaken. remarkably, the only four species found nesting both in soil and vegetation were tramp and/or invasive species: paratrechina longicornis, pheidole punctattissima, s. geminata and tetramorium bicarinatum (kempf, 1972; mcglynn, 1999; wetterer, 2009). conclusions ant communities of tropical coastal environments have been, compared to other ecosystems, poorly studied in spite of their fragility and high risk of change due to climate change. in mexico this is the first study that characterized ant communities in this kind of ecosystem. considering that this country will probably be greatly affected in the future by climate change (international panel for climate change [ipcc], 2013), we expect that patterns obtained in this study will constitute a base line to evaluate future changes. although no continuous plant studies have been conducted in forests and shrubs, information is available on the changes that have occurred in dune grasslands over the last 20 years (alvarez-molina et al., 2012). this period of time corresponds to the time elapsed since we sampled these ant communities. considering that after 20 years dune grasslands have a higher coverture and more plant species typical of shrubs (alvarez-molina et al., 2012), we expect to find a similar trend in ant communities. we can also anticipate that no large changes will be observed in the two forests, as far as it seems that both ecosystems represent advanced stages in a successional process (castillo, 2006). changes in abundance of invasive ants however, could significantly influence species richness as it has been observed in other ecosystems (mcglynn, 1999). in this regard the recent record of tramp species monomorium pharaonis (pers. obs.) should be monitored. considering that sf harbor more than 84% of ant species richness of la mancha, and that it constitutes the last remnant of subdeciduous forest in the mexican gulf coasts (moreno-casasola & travieso, 2006) monitoring should be focused mainly at this site. acknowledgments to all the staff of the centro de investigaciones costeras la mancha (cicolma) for all its support. to araceli cartas, julián bueno and griselda camacho for their invaluable aid in the field. to maría luisa castillo for donating ant specimens. antonio angeles and martín de los santos help with the elaboration of data sheets and preparation of figures. finally we acknowledge two anonymous reviewers for their comments and suggestions that highly improved the manuscript. references alvarez-molina, l.l., martínez, m.l., pérez-maqueo, o., gallego-fernández, j.b. &. flores, p. 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(1986). structural characteristics of an ant community during succession. oikos, 47: 346-354. p rojas et al ant communities in tropical coastal dunes 130 appendix 1. number of records in samples of each ant species in the four sites studied at la mancha. the total number of samples are included in brackets. *= species captured with non-standardized sampling (nss). v= nesting in vegetation; s= nesting in soil; vs= nesting in vegetation and soil. species list grassland (n=122) shrub (n=132) deciduous forest (n=172) subdeciduous forest (n=162) dolichoderinae azteca forelii emery, 1893 v 0 0 * * azteca velox forel, 1899 v 0 7 70 6 dolichoderus diversus emery, 1894 v 0 0 1 * dolichoderus lutosus (smith, 1858) v 0 3 2 3 dorymyrmex bicolor wheeler, 1906 s 52 14 0 0 dorymyrmex smithi cole, 1936 s 28 0 0 0 dorymyrmex sp. aff. flavus s 13 1 0 0 forelius pruinosus (roger, 1863) s 24 20 0 0 ectatomminae ectatomma ruidum (roger, 1860) s 0 0 0 2 ecitoninae eciton burchelli parvispinum forel, 1899 s 0 0 0 * labidus coecus (latreille, 1802) s 0 0 0 1 labidus praedator (smith, 1858) s 0 0 0 * neivamyrmex opacithorax (emery, 1894) s 0 0 3 0 neivamyrmex pilosus (smith, 1858) s 0 0 0 * neivamyrmex rugulosus borgmeier, 1953 s 0 * 0 0 neivamyrmex swainsoni (shuckard, 1840) s 0 0 0 1 nomamyrmex esenbeckii wilsoni (santschi, 1920) s 0 * 0 1 formicinae acropyga smithii forel, 1893 s 0 0 0 * brachymyrmex heeri forel, 1874 s 0 * * 0 brachymyrmex sp. 1lm s 46 12 10 1 brachymyrmex sp. 2lm s 0 0 0 2 camponotus atriceps (smith, 1858) v 3 8 1 * camponotus cerberulus emery, 1920 v 0 0 0 * camponotus claviscapus forel, 1899 v 0 0 0 * camponotus coloratus forel, 1904 v 0 1 0 * camponotus coruscus (smith, 1862) v 0 0 0 1 camponotus etiolatus wheeler, 1904 v 0 0 1 * camponotus excisus mayr, 1870 v 0 0 0 1 camponotus linnaei forel, 1886 v 0 3 2 3 camponotus mucronatus hirsutinasus wheeler, 1934 v 0 3 13 * camponotus novogranadensis mayr, 1870 v 0 0 19 4 camponotus planatus roger, 1863 v 2 18 39 6 camponotus sericeiventris (guerin-meneville, 1838) v 0 0 1 7 camponotus conspicuus sharpi forel, 1893 v 0 0 0 * camponotus zoc forel, 1879 v 0 0 0 * camponotus sp. 1lm v 0 1 1 0 camponotus sp. 2 lm v 0 1 0 * myrmelachista skwarrae wheeler, 1934 v 0 0 1 * nylanderia steinheili (forel, 1893) s 0 8 22 1 paratrechina longicornis (latreille, 1802) sv 1 20 0 * myrmicinae atta cephalotes (linnaeus, 1758) s 4 21 0 0 atta mexicana (smith, 1858) s 0 0 0 4 cephalotes minutus (fabricius, 1804) v 0 3 0 5 cephalotes scutulatus (smith, 1867) v 0 * 10 * cephalotes umbraculatus (fabricius, 1804) v 0 0 10 1 crematogaster corvina mayr, 1870 v 0 0 1 0 crematogaster crinosa mayr, 1862 v * 27 0 0 crematogaster curvispinosa mayr, 1862 v 0 0 4 1 crematogaster torosa mayr, 1870 v 0 0 1 3 crematogaster sp. aff. curvispinosa v 0 0 0 1 cyphomyrmex costatus mann, 1922 s 0 0 0 * sociobiology 61(2): 119-132 (june, 2014) 131 cyphomyrmex rimosus (spinola, 1851) s * 8 13 6 megalomyrmex silvestri wheeler, 1909 s 0 0 0 * monomorium ebeninum forel, 1891 s * 58 0 * monomorium floricola (jerdon, 1851) v 0 0 0 16 mycetosoritis hartmanni (wheeler, 1907) s 0 0 1 0 mycocepurus curvispinosus mackay, 1998 s 0 0 0 3 mycocepurus smithii (forel, 1893) s 0 0 * 6 myrmicocrypta sp. s 0 0 0 1 nesomyrmex echinatinodis (forel, 1886) v 0 2 0 * nesomyrmex wilda (smith, 1943) v 0 1 0 0 pheidole punctatissima mayr, 1870 sv 0 0 2 38 pheidole susannae forel, 1886 s 0 7 * 56 pheidole sp. 1lm s 0 0 1 9 pheidole sp. 2lm s 0 0 2 2 pheidole sp. 3lm s 0 0 11 2 pheidole sp. 4lm s 0 0 32 3 pheidole sp. 5lm s 0 0 0 1 pheidole sp. 6lm s 0 2 0 1 pheidole sp. 7lm s 0 16 0 0 pheidole sp. 8lm s 0 0 * * pheidole sp. 9lm s 0 1 0 0 pheidole sp. 10lm s 0 31 0 9 pheidole sp. 11lm s 0 0 0 9 pheidole sp. 12lm s 0 0 2 0 rogeria belti mann, 1922 s 0 0 * 1 rogeria cuneola kugler, 1994 s 0 0 0 * sericomyrmex aztecus forel, 1855 s 0 0 0 9 solenopsis molesta (say, 1836) s 1 39 53 34 solenopsis geminata (fabricius, 1804) sv 10 52 28 25 solenopsis isopilis pacheco & mackay, 2013 s 0 0 1 10 solenopsis sp. aff. azteca s 0 0 0 * solenopsis sp. s 0 0 0 * strumigenys boneti brown, 1959 s 0 0 6 * strumigenys eggersi emery, 1890 s 0 0 0 * strumigenys elongata roger, 1863 s 0 0 0 1 strumigenys louisianae roger, 1863 s 0 1 0 1 strumigenys ludia mann, 1922 s 0 0 0 2 strumigenys nigrescens wheeler, 1911 s 0 0 0 * temnothorax subditivus (wheeler, 1903) s 0 2 3 * tetramorium bicarinatum (nylander, 1846) sv * 0 0 * tetramorium simillimum (smith, 1851) s * 0 0 * tetramorium spinosum (pergande, 1896) s * 28 0 0 trachymyrmex intermedius (forel, 1909) s 0 0 0 1 trachymyrmex sp. aff. saussurei s 6 22 0 6 wasmannia auropunctata (roger, 1863) s 1 0 12 36 xenomyrmex panamanus (wheeler, 1922) v 0 0 1 1 ponerinae hypoponera nitidula (emery, 1890) s 0 0 1 5 hypoponera opacior (forel, 1893) s 0 1 0 5 hypoponera sp. aff. vana s 0 0 * 5 odontomachus brunneus (patton, 1894) s 0 0 0 * odontomachus laticeps roger, 1861 s 0 0 0 1 pachycondyla crenata (roger, 1861) v 0 0 0 1 pachycondyla harpax (fabricius, 1804) s 0 0 3 8 pachycondyla stigma (fabricius, 1804) s 0 1 0 * pachycondyla villosa (fabricius, 1804) v 0 0 * 16 platythyrea punctata (smith, 1858) s 0 0 0 * pseudomyrmeciinae pseudomyrmex boopis (roger, 1863) s 0 0 0 1 pseudomyrmex brunneus (smith, 1877) v 3 3 0 0 pseudomyrmex cubaensis (forel, 1901) v 0 1 0 3 pseudomyrmex ejectus (smith, 1858) v 3 3 0 0 p rojas et al ant communities in tropical coastal dunes 132 pseudomyrmex elongatulus dalla torre, 1892 v 0 1 1 1 pseudomyrmex ferrugineus (smith, 1877) v 0 0 1 2 pseudomyrmex gracilis (fabricius, 1804) v 0 0 1 1 pseudomyrmex ita (forel, 1906) v 0 1 0 * pseudomyrmex oculatus (smith, 1855) v 0 0 0 * pseudomyrmex seminole ward, 1985 v 0 0 0 1 pseudomyrmex simplex (smith, 1877) v 0 1 0 * pseudomyrmex spiculus ward, 1989 v 0 0 1 * pseudomyrmex tenuissimus (emery, 1906) v 0 1 5 * pseudomyrmex sp. (pallens group) v 0 * 0 2 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 1-10 (2013) a review of the biology, ecology and behavior of velvety tree ants of north america r hoey-chamberlain, mk rust, jh klotz introduction the genus liometopum consists of 9 fossil and 8 extant species scattered over north america, europe and asia (www.antcat.org/catalog/329538). there are three north american species, found in the western u.s. and mexico: l. apiculatum mayr, l. luctuosum wheeler, and l. occidentale emery. the genus liometopum belongs to the subfamily dolichoderinae, which also includes other important structural pest ants such as the argentine ant, linepithema humile (mayr), and the odorous house ant, tapinoma sessile say. according to del toro et al. (2009), liometopum is only a “minor pest in and around rural housing areas” and “no severe damages to housing or property have been reported,” although the “uncomfortable bite and smell of this ant makes it a nuisance to affected residents.” however, structural damage has been reported for two species, l. luctuosum and l. occidentale (wheeler & wheeler, 1986; merickel & clark, 1994; gulmahamad, 1995; hedges, 1998; klotz et al., 2008). homeowners commonly complain of the odor associated with an infestation of velvety tree ants. abstract ants belonging to the genus liometopum are regionally distributed across north america, europe and asia. l. apiculatum mayr, l. luctuosum wheeler, and l. occidentale emery are found in western north america and are referred to as velvety tree ants. very little is known about the biology of these species, but they are similar. they are typically associated with trees and shrubs and are frequently found tending hemipterans. all three species are easily disturbed and resort to highly aggressive behaviors including the use of strong alarm odors. the following review is intended to summarize the literature regarding the biology and control of these species. special emphasis has been given to factors that might be important in their control and gaps in our current knowledge. sociobiology an international journal on social insects university of california riverside, riverside, ca, usa review article history edited by: kleber del-claro, ufu brazil received 24 october 2012 initial acceptance 27 november 2012 final acceptance 19 february 2013 keywords liometopum apiculatum, liometopum luctuosum, liometopum occidentale corresponding author michael k. rust department of entomology university of california riverside riverside, ca 92521-0314 e-mail: michael.rust@ucr.edu l. luctuosum and l. occidentale are often mistaken for carpenter ants (camponotus spp.) by homeowners and pest management professionals (pmps). this mistaken identity is due to morphological and behavioral characteristics they share with carpenter ants; namely polymorphic workers, a smooth convex thoracic profile, and the tendency to excavate wood (klotz et al., 2008). l. luctuosum are also often confused with the t. sessile since they have the same coloration, are similar in size, and produce an alarm pheromone with a very similar odor. consequently, their importance as structural pests may be greatly under reported, especially in california, oregon, and washington. all three north american species are easily disturbed and when aggravated, they resort to highly aggressive defensive behaviors, biting and releasing strong alarm odors from their anal glands (del toro et al., 2009). they produce a noxious alarm pheromone (described as smelling like rotten coconut or having an intense butyric acid-like odor). this pheromone consists of 6-methyl-5-hepten-2one, acetic acid, n-butyric acid, and 3-methylbutyric acid (isovaleric acid) (casnati et al., 1964). alarm pheromones r hoey-chamberlain, mk rust, jh klotz velvety tree ants of north america 2 of many dolichoderines seem to be less species-specific than olfactory sex attractants and other pheromones. for instance, the alarm pheromones of forelius pruinosus (roger), dolichoderus bispinosa (olivier), l. occidentale, and t. sessile will mutually affect one another. in l. occidentale this pheromone originates in the anal gland (wilson & pavan, 1959). in mexico, colonies of liometopum have been used as a food resource by people in rural areas for centuries. the immature stages of the reproductive caste, known as “escamoles” are consumed and are a high-quality source of protein, carbohydrates, and lipids. adult reproductives may also be consumed by humans during swarming, and worker brood is consumed when other stages are scarce (ramoselorduy & levieux, 1992). consequently, considerably more is known about the biology of l. apiculatum than any of the other species. taxonomy liometopum occidentale was originally described as liometopum microcephalum var. occidentale by emery (1895). wheeler (1905) relocated it to a variety of liometopum apiculatum. it was finally elevated to the species level by wheeler (1917) and remained there in a recent taxonomic review by del toro et al. (2009). liometopum luctuosum was originally named liometopum apiculatum subsp. luctuosum by wheeler (1905) and forel (1914). creighton (1950) relocated it to a subspecies of liometopum occidentale. it was elevated to the species level by wheeler & wheeler (1986), and subsequently confirmed by mackay et al. (1988) and del toro et al. (2009). liometopum apiculatum was first described by mayr (1870), who described the workers of this species. emery (1895) later described the queens of this species; wheeler (1905) described the males and wheeler & wheeler (1951) described the larvae. shattuck (1994) considered l. apiculatum a senior synonym of liometopum masonium. this species was also confirmed by del toro et al. (2009). caution should be exercised when examining the literature prior to the review by wheeler and wheeler (1986) in regards to l. luctuosum. however, l. luctuosum has only been reported in the southwestern u.s. and not mexico. workers of l. luctuosum are significantly smaller (n = 38, 0.91 3.53 mg) than l. occidentale (n=40, 1.44 5.24 mg). habitat/distribution liometopum apiculatum ants are found in arid and semi-arid regions of southwestern u.s. and mexico to quintana roo (del toro et al., 2009). they extend from colorado through texas, new mexico, southeastern arizona, and south into mexico (gulmahamad, 1995). they are usually found at elevations between 1000 and 2500 m, but their prime habitat is oak forests found around 2000 m. at higher elevations they are found in pinyon pine zones, up to the ponderosa pine and riparian zones; at lower elevations they inhabit creosote bush scrub and grasslands in microhabitats of clay, under rocks, boulders, and decaying logs (del toro et al., 2009). they have also been found in foothill meadows, deciduous canyon forests, pinyon-cedar woodlands, ponderosa pine-cedar-oak woodlands, and cottonwood-willow forests (mackay & mackay, 2002). at high elevations, their abundance decreases and they are replaced by l. luctuosum (del toro et al., 2009). altitude may play an important role in the distribution of liometopum. in regions of mexico explored by conconi et al. (1983b), l. luctuosum and l. apiculatum are only found between 2000 and 3000 m. although conditions below 1800 m looked favorable, they are absent. in the u.s., l. apiculatum is found from 1316 to 2438 m. liometopum luctuosum has been reported at elevations as low as 59 m (dr. laurel hansen, spokane community college, personal communication, sept. 6, 2012), but is typically found at elevations higher than 2400 m in more southern latitudes (wheeler & wheeler, 1986; del toro et al., 2009). the range of elevation of this species by state is as follows: wa: 59-724m, id: 664-786m, or: 277-454m, ca: 1280-1596m, and nv: 1372-2469m (dr. laurel hansen personal communication, sept. 6, 2012, personal collection) and above 2000 m in new mexico (mackay & mackay, 2002). their range extends from temperate habitats as far north as british columbia (canada), and to more arid habitats of central mexico and western texas. they inhabit pine, oak, douglas fir, and juniper forests, sagebrush, and highelevation riparian habitats (conconi et al., 1987a; clark & blom, 2007). this species is often strongly associated with but not limited to pine trees (del toro et al., 2009). liometopum occidentale is found from sea level to over 1840 m in coastal regions from southern washington to northern mexico (snelling & george, 1979; del toro et al., 2009; wang et al. 2010; dr. laurel hansen, personal communication, sept. 6, 2012). the range of elevation of this species also appears to depend on latitude with ants collected from locations in oregon as low as 7 m and up to 1700 m in california (wa: 31-142m, or: 7-348m, ca: 1421713m; dr. laurel hansen, personal communication, sept. 6, 2012, personal collection). they are the most common and dominant ant in oak and pine forests of southwestern u.s. (wheeler & wheeler, 1986; ward, 2005; del toro et al., 2009). they prefer to nest in the crevices of oaks, alders, elms, cottonwoods, and creosote, in soil, underneath bark of dead trees, and under rotten logs (cook, 1953). cook may be in error claiming that these are nests since we often see many, many adult workers in ‘resting places’ which are mistaken as nests, as noted by klotz et al. (2008). within their distribution range, the elevations at which liometopum are found decreases the further north the location. to definitively demonstrate that there is an effect of latitude on the elevation at which liometopum is distributed sociobiology 60(1): 1-10 (2013) 3 in north america, a larger collection of these ants must be made. nests nests of l. apiculatum are typically located underground and have a very distinctive structure. they fill hollowed-out chambers with a branched network of carton-like material made out of agglomerated soil and oral secretions until the entire nest resembles swiss cheese. within the nest as many as 3 or 4 large chambers containing this honeycombed carton-like material can be found. the carton-like material of this species is much finer than that of l. luctuosum. l. apiculatum tend to nest at higher densities than do l. luctuosum (conconi et al., 1987a). these ants are usually found nesting in dead logs, under stones, or in decaying stalks of yucca spp. (miller, 2007). they have also been collected in glass containers and rubber tires (del toro et al., 2009) and among the roots of various perennial xerophytes such as agave spp., opuntia spp., myrtillocactus geometrizans console, yucca filifera chabaud, senecio praecox dc., schinus molle l. or prosopis juliflora dc. (conconi et al., 1983b). in some habitats, the nests are deep under heavy boulders or large trees (wheeler, 1905; gregg, 1963a). the queen is always well protected and is usually in a remote place about 6-8 m from the largest chamber where the brood is stored. the chambers are connected by various galleries. liometopum luctuosum nest under rocks, decaying logs or at the base of large trees, or among the roots of trees such as quercus spp., juniperus spp. and pinus spp. (conconi et al., 1983b). they create carton nests similar in structure to l. apiculatum (del toro et al., 2009). similarly, the queen of l. luctuosum is well protected at a remote place about 6-8 m from the largest chamber where the brood is stored, and the chambers of the nest are connected by various galleries (conconi et al., 1987a). liometopum occidentale typically nest in soil, crevices of trees, and under the bark of dead trees (del toro et al., 2009). however, were never found by wang et al. (2010) leading to the speculation nests must lie deep under large boulders or among roots of large trees. to better understand the structure of their nests, more nests need to be excavated. colonies colonies of l. apiculatum are polydomous with segments of nests (or satellite colonies) scattered over the landscape (del toro et al., 2009). colonies are variable in size, ranging from a few hundred to hundreds of thousands of workers (gregg, 1963b; ramos-elorduy & levieux, 1992). colonies exploited for their brood by humans contain between 65,000 and 85,000 individuals, while undisturbed colonies may contain as many as 250,000 individuals. colonies remain useful for repeated brood collection by humans for 4 to 12 years (ramos-elorduy & levieux, 1992). colony foundation in l. apiculatum is by haplometrosis (non-cooperative), that is, a single fertile queen founds each colony (conconi et al., 1987a,b). colony foundation behavior is not uniform among founding queens. the time spent exploring, excavating, and removing excavated materials and waste are usually higher throughout the day, while oviposition, brood care, and inactivity increase at night (conconi et al., 1987b). founding queens prefer sites close to bodies of water; however, sites slightly further from water are more conducive to the establishment of a successful colony as persistently high humidity will result in the early death of a colony due to fungal invasion (conconi et al., 1983a). colony foundation in l. luctuosum is by pleometrosis (cooperative), in which 2 to 40 fertile queens found a single colony (conconi et al., 1987a). most colony foundation activities take place at night (conconi et al., 1987b). there is a division of labor among founder queens. however, this is not always divided the same for each foundation event. the amount of time dedicated to each activity by each queen varies with each colony foundation event. some queens are active in a variety of tasks. for example, some queens dedicate more time to brood care and others more time patrolling the nest area. the fewer ants founding together (3 or less), the more time spent per individual caring for brood, ovipositing, and exploring. trophallaxis, patrol activity, and inactivity decreases in these cases, but brood care still remains the primary activity of founding queens (conconi et al., 1987b). after finding no aggression between workers collected from significant distances apart and no territorial boundary, wang et al. (2010) speculated that l. occidentale colonies are large and polydomous. since they never found brood or queens, it is uncertain whether there are multiple queens within a nest, or whether each queen has some localized “sphere of exclusivity.” and it just seems unlikely that there could be just one queen that produces enough eggs to establish a colony that is one kilometer wide, they also speculated that l. occidentale are polygyne. colonies have been estimated to contain between 40,000 and 60,000 workers (ramos-elorduy & levieux, 1992; del toro et al., 2009). colony foundation of this species has not been studied as well as it has with the other two north american species. feeding liometopum apiculatum are opportunistic carnivores and granivores, and have also been observed foraging on dead insects, larger colonies being more predaceous (shapley, 1920). l. apiculatum also feeds on crustaceans, annelids, mollusks, dead vertebrates, animal droppings, and extrafloral nectar (velasco et al., 2007). these ants also obtain r hoey-chamberlain, mk rust, jh klotz velvety tree ants of north america 4 nectar or pollen from bear grass and substances from the outside of the ovaries of the flowers of century plants (agave scabra salm-dyck and a. chisosensis c.h.mull.) and spanish dagger (yucca spp.). workers have been attracted to various foods used as baits including apple sauce, sausage, vegetable soup, sugar water, and cookies. l. apiculatum have also been observed soliciting honeydew from insects including membracids (vanduzea segmentata (fowler)), aphids, and other ants (pogonomyrmex barbatus (smith, f.), camponotus sayi emery, and solenopsis xyloni mccook; van pelt, 1971). in some habitats the honeydew produced by hemipterans, cinara spp., dysmicoccus brevipes (cockerell) and saissetia oleae (oliver), are the main energy sources (velasco et al., 2007). in other words, hemipteran exudates make up the bulk of the diet of l. apiculatum (conconi et al., 1983b). their role in disrupting biological control has not been determined. liometopum occidentale are opportunistic omnivores (wheeler, 1905) and can often be found tending hemipterans and carrying prey insects back to the nest (gulmahamad, 1995). they readily attend hemipterans and are found in citrus groves, but their role in disrupting biological control has not been determined. their feeding preferences need to be studied to enable the development of an effective bait for pest control purposes. liometopum luctuosum is also an omnivore and has been observed feeding on secretions from both plants and insects such as aphids, membracids and scales, as well as miscellaneous foods such as meat, eggs, fruit and bread (conconi et al., 1987a). foraging activity ants of the genus liometopum lay down a trail pheromone that has an odor similar to butyric acid, but its chemical composition remains unknown. the source of trail pheromones in many species of dolichoderines is the pavan gland, a medioventral sac between the sixth and seventh abdominal sternites (pavan, 1955; billen, 1985). this gland may be the source of these pheromones in liometopum. all the dolichoderine trail pheromones tested so far have proved to be highly species-specific (wilson & pavan, 1959). dolichoderine foragers travel along trials in a manner described by shapley (1920) as “trail-running”, which we have observed as travelling rapidly along defined trails. the number of ants on these trails is governed largely by food and the speed of the ants by meteorological conditions. shapley (1920) noted that liometopum activity patterns are equally diurnal and nocturnal. l. apiculatum and l. occidentale are active at wide range of temperatures (8°c 38°c), humidity (5%-100%), wind, and light, but temperature is the most important factor affecting their activity. even in winter after a few warm days these ants have been observed foraging within a few feet of snow banks (shapley, 1920), an example of how imperative temperature is to foraging activity of these ants. liometopum appear to run at a speed very near the maximum speed possible under prevailing conditions, except at low temperatures. for temperatures below 15ºc, in which the activity level of these ants is very low, activity can be temporarily increased by exciting the ants into battle or by the discovery of food (shapley, 1920). foraging trails for l. apiculatum and l. occidentale are maintained over long intervals of time and even years (shapley, 1920). ants on these trails are for the most part unburdened with prey or objects, no matter if they are going toward or away from the nest. shapley (1920) suggests they are just patrolling, however, what is equally as likely is that these workers are carrying large amounts of honeydew or other liquid foods within their crop. liometopum apiculatum forage from march to september (mackay & mackay, 2002). workers forage almost exclusively on trails as wide as 2-3 cm on the soil surface, and when the temperature rises sharply at midday, they cease foraging and seek shelter under stones (conconi et al., 1983b; ramos-elorduy & levieux, 1992). the movement of this species is less erratic than l. occidentale at higher temperatures. an increase in temperature by 30°c changes the speed by 15 fold, increasing exponentially from 0.44 to 6.60 cm a second. there also appears to be little difference in the speed whether ants are moving towards or away from the nest, or between large and small workers during the summer months. however, after prolonged periods (two months or more) of low temperatures, the larger workers are faster than the small workers. also within a range of 14º to 38ºc there appears to be little effect of temperature on the number of ants on trails. maximal activity occurs between 12p.m. 12a.m during the summer months in southern alpine habitats such as mount wilson, ca (shapley, 1920). in natural environments, ants of this species forage in areas between 468 and 708 m2 (x = 612 m2); however, they only use between 16 to 30% of this area at any given time. the spatial distribution of the foraging areas for these species seems to be strongly correlated with the location of shrubs and trees infested by hemipterans (ramos-elorduy & levieux, 1992). liometopum occidentale form massive foraging trails that extend 60 m or more from the nest (gulmahamad, 1995; eckert & mallis, 1937), and can even be observed on hot days with temperatures between 24 and 38°c (tremper, 1971). ramos-elorduy & levieux, (1992) observed l. occidentale traveling mostly underground in very shallow galleries (1-2 cm deep), or in leaf litter, however, we have also observed massive above ground trails. according to ramos-elorduy and levieux (1992), l. occidentale in natural environments forage an areas as large as 2,000 m2, but they only utilize between 486 and 1198 m2 (x = 740 m2) of this area at any one time. this means that they are only using between 33 and 68% of this area, more than twice that of l. apiculatum. to reach a food source, l. luctuosum establish trails sociobiology 60(1): 1-10 (2013) 5 sometimes more than 100 m long. the galleries of these trails often run under leaf litter (conconi et al., 1983a,b). this protection could explain why these ants can be found trailing at almost any hour of the day (conconi et al., 1983b). pmps have stated they have observed both l. luctuosum and l. occidentale foraging during the day in great numbers in the spring and early summer, but around midsummer they switch to night time foraging. however, this has not been scientifically proven. interspecific interactions and nest-mate recognition liometopum are highly competitive, behaviorally dominant ants and play an ecologically similar role to the behaviorally dominant australian dolichoderines, anonychomyrma, papyrius and froggattella (andersen, 1997). ant communities often have a hierarchal order to them, with “submissive” ant species being more adaptable, and “territorial” species at the top of this hierarchy defending territories of varying sizes (petráková & schlaghamerský, 2011). ants fight in the spring when food is scarce and also fight to renew trails after winter pauses in activity (petráková & schlaghamerský, 2011). the interactions between liometopum spp. and native ants have been studied for l. microcephalum panzer, an old world species. l. microcpephalum is a behaviorally dominant ant species in europe and asia. this species builds nests several meters above ground in old living trees (especially oaks) and forage in other trees in the vicinity of the nest tree, similar to north american species of liometopum (petráková and schlaghamerský, 2011). some colonies are polydomous. these ants are very efficient hunters, but also tend aphids. l. microcephalum can be very aggressive toward other ant species, attacking by biting and spraying secretions that repels enemies, and initiating alarm behavior (petráková & schlaghamerský, 2011). aggressive behavior occurs close to the nest, on trails, on trees, and occasionally at food resources. l. microcephalum takes advantage of worker cooperation during aggressive interactions, a strategy used by smaller ants (petráková &schlaghamerský, 2011). aggressive intraspecific interactions are expected with all three north american species due to such similarity in ecology and behavior of this species. in tlaxco, tlaxcala, mexico, l. apiculatum was found to associate with fourteen species of hemiptera, including seven species of aphids (two in the cinara genus, anoecia cornicola (walsh), aphis lugentis williams, aphis solitaria (mcvicar baker), aphis helianthi monell, and aphis spp.), three species of scales (saissetia spp., s. olee (olivier), two species of pseudococcidae including dysmicoccus brevipes (cockerell)); one species of ortheziidae, and one species of dactylopiidae (eriococcus sp.) (velasco et al., 2007). yet another species of hemipteran they associate with are cochineal scales crassicoccus spp. that live on oak trees. competition between the invasive linepithema humile and l. occidentale has been documented (ward, 1987; holway, 1998; sanders et al., 2003). however, l. occidentale rarely co-exists with l. humile, perhaps because they are habitat or resource specialists that are found in such a wide range of habitats as l. humile or because they are weak competitors against invasive species (sanders et al., 2003). however, l. occidentale is the species of liometopum most affected by l. humile due to its “taxonomic and ecological similarities to linepithema humile in that “they are members of the same subfamily (dolichoderinae) and they are dominant, opportunistic, epigaeic (lives or forages primarily above ground) ants, with propensities to establish dense foraging trails, to tend hemiptera: sternorrhyncha, to move nest sites readily, and to forage and tend hemipterans under the same ambient conditions throughout the summer months (ward, 1987). ” this abundance of similarities makes them likely to compete in areas of co-occurrence. wang et al. (2010) tested the nest mate recognition of a limited number of l. occidentale colonies in james reserve and stunt ranch in southern california. results of this study showed that ants from sites separated by a kilometer or less were not aggressive to one another, while colonies separated by more than 100 km were aggressive to each other; although not every colony was always aggressive to every other one, even when arising from disjunct populations. wang et al. (2010) also test the aggression of l. occidentale toward other ant species, camponotus vicinus mayr, myrmecocystus ewarti snelling, m. testaceus snelling, pogonomyrmex subnitidus emery, solenopsis xyloni mccook and tapinoma sessile say, and found that they were highly aggressive towards all these species. l. luctuosum “has been reported as being a competitor of camponotus species in idaho, since both of them compete for similar nesting sites” (merickel & clark, 1994). however, l. luctuosum is the least studied of all the north american liometopum species. polymorphism polymorphism in l. apiculatum and l. luctuosum has been described as bimodal with diphasic allometry (conconi et al., 1987a). bimodal or biphasic allometry occurs when animals such as ants have structures that vary disproportionately with body size such as the heads and mandibles of soldiers and major and minor workers (grimaldi & engel, 2005). at some intermediate, boundary size, larger individuals have a disproportionately larger structure, but the structure is disproportionately small below this size (grimaldi & engel, 2005). the average live weight of the largest workers of l. apiculatum is about 3.24 mg and the average weight of the minor workers are half this, but the extremes of weight for majors and minors are probably in the ratio of four to one (shapely, 1920). the polymorphism of l. occidentale has not been described. r hoey-chamberlain, mk rust, jh klotz velvety tree ants of north america 6 reproduction immature stages of reproductives have been found in l. apiculatum nests from may to august, whereas the rest of the year the brood is of the worker caste (conconi et al., 1983a; del toro et al., 2009). males and gynes have been collected outside the nest from june to august and queens (likely founding queens) have been collected in july and august under stones and other landscape features (del toro et al., 2009). nuptial flights of this species occur during the day after a heavy rain during the months of april or may (conconi et al., 1983a). before a nuptial flight there is a great agitation of the workers, which leave the nest and run rapidly in a “zig-zag” fashion. the male and female alates leave the nest, but are less active. after a while the workers begin to bite the legs and wings of the alates, forcing them to climb the nearest plant. the workers continue to excite the alates with bites until they begin to beat their wings, and subsequently initiate flight one by one, not as a swarm. mating takes place in the air, and mated males and females fall to earth together, often still attached (conconi et al., 1983a). the life span of l. apiculatum queens is shorter than that of l. luctuosum queens (exact time difference not specified); however, their productivity (oviposition) is greater (conconi et al., 1987a). the annual productivity for an established colony (60 to 85,000 workers) of l. apiculatum is about 3-3.6 kg of brood per year (ramos-elorduy & levieux, 1992). oviposition by founding queens is large, however, only a small percentage reaches the adult stage of the f1 generation, partly because the smaller, “trophic” eggs are consumed as food. after laying her first batch of eggs, the queen delays laying more until the first eggs have developed into pupae. once the first workers emerge, the queen discontinues laying trophic eggs, which lowers the total amount of eggs laid but increases the proportion of viable eggs. eggs are laid all year round (conconi et al., 1983a). some virgin queens of l. apiculatum emerge from the nest, remove their wings, and dig a nest without mating. they will lay eggs, care for them, and eat them to survive. however, they only care for more recently laid eggs that have not turned yellow or dried out (conconi et al., 1983a). flights of l. occidentale reproductives have been observed throughout may (del toro et al., 2009). the annual productivity for a colony (40 to 60,000 workers) of this species is 2 to 2.8 kg of brood per year for 4-8 years (ramoselorduy & levieux, 1992). workers housed without a queen will also lay unfertilized eggs that are eaten or develop into males. reproductives of l. luctuosum have been observed flying in june and july and can be collected the day after the flight in large bodies of water or using a blacklight trap (del toro et al., 2009). the most productive colonies of liometopum are those that are more substantially surrounded by vegetation which likely contains honeydew excreting hemipterans. however, l. apiculatum appears to be more productive than l. occidentale even though l. apiculatum forages in a much smaller territory (ramos-elorduy & levieux, 1992). the reproductive life cycles of l. luctuosum and l. occidentale need further investigation. life cycle a study by conconi et al. (1983a) recorded the longevity of each of the reproductive castes and colony foundation of l. apiculatum. in this study, they studied the life cycle of this species under different conditions of humidity, temperature, and substrate. ant queens were placed either in glass tubes with moist cotton or in jars with soil, and were held at varying temperatures and relative humidity. observations of the time until different life cycle events occurred are summarized in table 1. observations of the longevity of the various reproductive castes of l. apiculatum are as follows: males lived 15 to 37 days, virgin queens lived 19 to 268 days and fertilized queens lived 17 to 316 days. however, the last estimate is incredibly short for a queen, so either these ants are highly polygynous or they live much longer in the wild. this study is a good start to give us an idea of the duration of different life cycle events and the life spans of each caste, however, further work is needed to understand the variability in time of these events under various conditions. no such studies have been conducted with l. occidentale or l. luctuosum. table 1. colony foundation of l. apiculatum modified from conconi et al. (1983a). glass tubes jar with soil 32°c/ 70-80% rh 26°c/ 40-50% rh event time (days) event time (days) first eggs 11.00 ± 2.3 first eggs 27.80 ± 6.9 first pupa 9.61 ± 2.1 first pupa 24.20 ± 6.2 first larva 9.84 ± 2.4 first larva 25.16 ± 7.8 first adults 28.23 ± 4.0 first adults 70.83 ± 11.3 total 28.23 ± 5.0 total 70.83 ± 11.4 associations and mutualisms beetles belonging to the family staphylinidae including sceptobius schmitti wasmann, dinardilla liometopi wasmann, dinardilla mexicana mann, and sceptobius dispar sharp have been found within the nests of l. apiculatum (gregg, 1963a; danoff burg, 1994). “dinardilla mexicana and s. dispar currently co-occupy l. apiculutiim nests on sociobiology 60(1): 1-10 (2013) 7 the eastern part of the mexican plateau, while s. schmitti and d. liometopi are found together in nests on the western part of the mexican plateau and then north to colorado. more collecting should be done in the central part of the mexican plateau to determine whether there is a zone of overlap befigure 1. insulation removed by l. luctusosum in a mountain home in the san bernardino mountains, ca. figure 2. damage to wood caused by workers of the l. occidentale. liometopum also have mutualistic relationships with both plants and other insects. one example is the mutualistic relationship of l. apiculatum with the cholla cactus opuntia imbricate (haw.). this species has been seen protecting the cacti from herbivores and seed predators as well as foraging on extrafloral nectars (miller, 2007). l. apiculatum tends the aphid cinara sp. 1 found on pinus rudis endl. and the aphid cinara sp.2 found on juniperus deppeana steud. (velasco et al., 2007). structural pests and their control when liometopum ants nest inside structures they can produce considerable amounts of frass consisting of chewed wood and insulation (fig. 1). the excavations of these ants, however, are of a finer texture than those of carpenter ants (fig. 2) and the frass is also much finer (fig. 3). attempts to eradicate nests indoors are often unsuccessful because foragers may congregate in hollows within insulation and wood, forming temporary ‘resting places,’ which are mistaken as nests (klotz et al., 2008). velvety tree ants are best managed by locating and treating all colonies around the structure (hedges, 1998). inspect for nests and foraging trails in or around wood such as stumps, trees, or landscape timbers (hansen & klotz, 1999). these ants are attracted to water-damaged wood, but nests can also be found in dry, sound wood and foam insulation. night inspections might be helpful during summer months as these ants are more active at night (klotz et al., 2008). a combination of baits and sprays should be used in an ipm program to successfully manage these ants. baits containing a sweet food attractant should be applied in or near foraging trails first, allowing time for the ants to carry the bait active ingredient into the nest. then dusts and sprays may be applied. dust formulations may be used by pmps to treat “resting” areas or satellite nests within structures (hedges, 1998; klotz et al., 2008). re-entry by undetected colonies outside a structure can be prevented with a perimeter spray around the foundation of the structure (hedges, 1998; klotz et al., 2008). tween these two geographical species groups” (danoff burg, 1994). the staphylinid liometoxenus was first described from specimens found foraging next to colonies of liometopum luctuosum and l. occidentale (kistner et al., 2002; del toro et al., 2009). a weevil liometophilus manni fall has also been discovered in the galleries of liometopum apiculatum in southern arizona and mexico (mann, 1914). the impact or role of these beetles on the ants is unknown. dinardilla and sceptobius beetles are also often observed alongside foragers or in nests of l. occidentale. sceptobius lativentris (fenyes) is only found with l. occidentale, this is unique among the sceptobiini (danoff burg, 1994). more collecting needs to be done in nests of l. occidentale to determine the current geographic distribution of s. lativentris (danoff burg, 1994). all members of the genus sceptobius are quick moving, long-legged beetles that interact with their ant hosts by running up to groups of ants, briefly grooming a number of them, and then running away to the periphery of the ant nest (danoff burg, 1994). dinardilla mexicana and d. liometopi interact actively and, in some cases, aggressively with some host ants. typically, one of these beetles approaches a stationary host ant from the side and begins grooming the ant’s legs with its mouthparts, after which the beetle mounts the ant and grooms its dorsum. this interaction can last from 3 minutes to 20 minutes and is likely used to spread the cuticular hydrocarbons that specify the colony odor from the ant to the beetle (danoff burg, 1994). the staphylinid liometoxenus was first described from specimens found foraging next to colonies of liometopum luctuosum and l. occidentale (kistner et al., 2002; del toro et al., 2009). r hoey-chamberlain, mk rust, jh klotz velvety tree ants of north america 8 a foraging trails outside the structure in the surrounding landscape, on utility lines, or on the trunks of trees should also be sprayed. trees in which the ants may be nesting are also target areas for treatment (klotz et al., 2008). access into the structure should be eliminated by trimming trees and shrubs that are in contact with the structure or wires and cables leading into the structure (klotz et al., 2008). entry points around windows, doors and fixtures should be sealed. ants located in dead wood are best eliminated by removing infested wood; however, this is not always possible. another treatment option is to drill and inject a small amount of insecticide dust labeled for this application into the galleries. ants located in firewood can be eliminated by discarding or burning the infested wood. never treat firewood with residual insecticide (hedges, 1998)! ants located in the soil under rocks and stones can be treated by thoroughly drenching the nest with residual insecticide (labeled for such use) using a compressed air sprayer (hedges, 1998). acknowledgments in part the research was supported by the carl strom western exterminator scholarship. we would like to thank drs. greg walker and les greenberg (uc riverside) and dr. laurel hansen (spokane community college) for their careful review of the manuscript and additional locality data. we also thank two anonymous reviewers for providing valuable comments that improved the article. references andersen, a.n. 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(1959). glandular sources and specificity of some chemical releasers of social behavior in dolichoderine ants. psyche, 66: 70-76. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.4251sociobiology 66(2): 247-256 (june, 2019) wood ant (formica polyctena) services and disservices in a danish apple plantation introduction the demand for ecofriendly and sustainable fruit production without pesticide residues is increasing. to achieve such production, new tools are needed to substitute current methods that are mainly based on synthetic chemicals (pesticides and artificial fertilizers). one way is to develop integrated pest management (ipm) based on biological rather than chemical solutions. due to their high numbers and being organized as a superorganism, ants can provide a number of services for commercial crops and plants in general. first, ants may prey on or deter arthropod pests (way & khoo, 1992; offenberg, 2014). this ant service is widely recognized. secondly, ants may provide nutrients to their host plants by depositing waste materials. this is well known from ant-plants living abstract ants possess properties that can be used to optimize plant production in agricultural systems. ant services can be herbivore and pathogen protection and fertilization of their plant partners. they may, however, also harm plants by facilitating antattended herbivorous homopterans. to assess whether wood ants can be used in ipm-systems to improve apple production, we transplanted wood ants into a danish apple plantation and tested whether ants (i) reduced the number of herbivores, (ii) led to higher amounts of leaf nutrients, (iii) controlled apple pathogens, (iv) increased homopteran abundance and (iv) whether these effects affected apple yields. during a two year study, we found that the wood ants significantly reduced the numbers of winter moth larvae, increased magnesium content in apple leaves (but did not affect 10 other nutrients), reduced the number of apples infected with apple brown rot and apple scab (on one apple variety) and increased aphid infections. in the first year, this led to higher apple production on ant trees, whereas ants had no effect on yields in the second year. it was evident that ants provided both services and disservices. if mutualistic ant-homopteran interactions can be disrupted, this would favor plant growth and open for the use of wood ants in sustainable plant management. we discuss how this may be accomplished. alternatively, ants may be used short term to knock down pest outbreaks (before building up homopteran populations) or used in crops that do not host ant-attended homopterans. sociobiology an international journal on social insects j offenberg, js nielsen, c damgaard article history edited by gilberto m. m. santos, uefs, brazil received 22 november 2018 initial acceptance 15 may 2019 final acceptance 15 may 2019 publication date 20 august 2019 keywords integrated pest management, winter moth, plant pathogens, fertilization, ant-aphid mutualism, apple diseases. corresponding author joachim offenberg department of bioscience aarhus university vejlsøvej 25, dk-8600 silkeborg. e-mail: joaf@bios.au.dk in close association with ants (rico-gray & oliveira, 2007), but recently it has been suggested that nutritional services may also take place in less specialized ant-plant partnerships, where ants do not nest in domatia, yet deposit fecal spots on plant tissue. these fecal spots may contain important nutrients that can be taken up directly by plant leaves (vidkjær et al., 2016; pinkalski et al., 2018). in addition, ants may enrich soils around plant roots with nutrients that originate from their waste deposits (folgarait, 1998; frouz et al., 2008; wagner & fleur nicklen, 2010). a potential second service from ants to plants may therefore be nutrient provisioning. lastly, ants may also provide plant services by reducing plant pathogen incidence. a number of studies have documented reduced plant pathogen incidence on host plants attended by ants compared to ant-free control plants. in a review of studies examining ant-plant-pathogen interactions, it was found that aarhus university, silkeborg, denmark research article ants j offenberg, js nielsen, c damgaard – ant services in apple plantations248 13 ant species were able to control at least 13 fungal and bacterial plant pathogens on 11 different plant species (j. offenberg, unpublished data – submitted to oikos). there can be several mechanisms behind pathogen protection. for example, ants may inhibit plant pathogens by eating fungal spores (letourneau, 1998; thornham et al., 2011), excreting antibiotics (peng & christian, 2005; gonzalez-teuber et al., 2014) or by deterring diseases vectors (letourneau, 1998; roux et al., 2011). taken together, the provision of these three types of services may lead to highly effective ant-based biocontrol programs. in some crops, ants have been shown to be more efficient than conventional control based on synthetic pesticides, and at lower costs (offenberg, 2015). however, not all ant activities are beneficial to plants. many ant species attend harmful honeydew-producing homopterans that feed on plant sap and can act as disease vectors (delabie, 2001). as ants improve hygiene and protect homopteran partners against natural enemies, homopteran populations may increase in numbers when ant-attended. therefore, ants can also exert indirect negative effects on plants (wäckers et al., 2017). a number of research activities aimed at developing techniques to disrupt ant-homopteran symbioses in commercial crops illustrate the importance of this disservice to agriculture. methods include blocking of ants’ access to plant canopies by placing sticky barriers around tree trunks or by providing sugar to ants via artificial feeders (nagy et al., 2013; nagy et al., 2015; wäckers et al., 2017). the latter will provide ants with a carbohydrate source that is an alternative to the honeydew produced by the attended homopterans. the presence of this alternative may alter ant behavior so they neglect their homopteran partners (nagy et al., 2015; wäckers et al., 2017) or even start to prey on them (way, 1954; offenberg, 2001). if ant-homopteran mutualism can be disrupted, beneficial ant services can be harvested without accompanying indirect negative effects. to assess the potential of an ant species as a biocontrol agent, it is thus important to map its services and disservices and to assess its combined net effect on plant performance. wood ants (formica spp.) have shown potential as biocontrol agents in temperate open field crops (way & khoo, 1992; nielsen et al., 2018). here, we present a study where we evaluated the services and disservices provided by wood ants (formica polyctena) that were transplanted into a danish apple plantation to control winter moth larvae (operophtera brumata). specifically, we tested the hypotheses that wood ants can (i) reduce herbivore abundance, (ii) fertilize host plants with nutrients, (iii) control plant pathogens, (iv) increase homopteran abundance and, lastly, that they via these effects can, (v) affect apple yields. methods study site the experiment was conducted in an organic apple orchard located near tirstrup in denmark (56° 18’ 36.9”n, 10° 41’ 28.1”e) in 2015 and 2016. the total size of the orchard was 1.3 ha and included approximately 3000 sevenyear-old row-arranged trees (3.5 m between rows and 1 m between trees within rows) of 11 varieties (though not all varieties were covered in our studies.). a two-meter wide vegetation buffer strip, consisting of non-hoed herbs and grasses, and two machinery tracks divided the 9 rows in two, with 4 rows to the west and 5 rows to the east. two additional vegetation buffer strips framed the study area to the west and the east. the main herbs and grasses were meadow-grass (poa sp.), dandelion (taraxacum sp.), orchard grass (dactylis glomerata), couch grass (elytrigia repens), common tansy (tanacetum vulgare), cow parsley (anthriscus sylvestris) and creeping thistle (cirsium arvense). drainpipes drained the area, but standing water was occasionally found in the lower parts of the plantation from fall to spring. first year study 2015 the experimental area used during the first year included 0.25 ha of the plantation covering nine rows of trees, each row holding one of five different varieties (holsteiner cox, alkmene, collina, angold and resista). trees that were severely damaged or recently re-grafted were excluded, and thus the total number of trees included in the study was 411. on 27-4 2015, eight wood ant (f. polyctena) mound fragments were transplanted into this area. the fragments were made from one large mound collected at løvenholm forest the same day by splitting the large mound into eight equally sized parts and providing each fragment with several ovipositing ant queens (nests are polygynous). the size of each fragment was approximately 90 l, and the spacing between them was 15-20 m, with four mounds placed in the central vegetation strip and two mounds in each of the two outer strips. in the first week after the transplantation, ants were fed with sugar dough (ambrosia®, 85 % sucrose, nordzucker) and water next to the mounds. after this, they were only offered sugar from feeders mounted on selected apple trees (sugar trees). feeders were made from a 50 ml centrifuge tube that was filled with 60-80 g sugar dough. five holes (ø=4mm) were made in the lid of each tube to allow ants access to the sugar and keep out larger animals. for more details on the wood ant transplantations and maps of the plantation, see nielsen et al. (2018). one sugar feeder was mounted with a metal wire to each of 34 trees at a height of approximately 1.2 m. trees with sugar feeders were selected by dividing each row of trees into four equally long segments, except one row with few trees that was divided into two segments. within each segment, one tree was randomly selected as a sugar tree. the 34 sugar trees were each paired with a neighboring control tree of similar appearance. ants were excluded from control trees by a sticky barrier (oecotak a5, oecos) applied around the trunk of the tree. all remaining trees in the plot were accessible to ants, but without sugar feeders. this design resulted in trees with variable numbers of ants, including trees without any ants. sociobiology 66(2): 247-256 (june, 2019) 249 on all the 411 trees in the plot, an ant density index was assessed for each tree. due to the high number of trees, ant densities were indexed as (i) no ants = index value 0, (ii) 1-10 ants = index value 1 or (iii) more than 10 ants per tree = index value 2, based on a 20-second inspection of each tree. this was done six times during the season (13-5, 22-5, 4-6, 116, 21-6, and 23-7). on four dates (4-6, 11-6, 21-6 and 23-7), the presence of aphids on each tree was assessed. the presence of at least one aphid colony on a tree was scored with the value 1, the absence of aphid colonies as 0, and the average value of the four dates was calculated for each tree. on 3-9, the number of apples was counted on each tree. during analyses, we tested the effect of the highest ant index value recorded on each tree during the six samplings (treated as a categorical variable) on the average aphid index and on the yield of apples (using the average ant index instead of the maximum ant index led to qualitatively similar results). second year study 2016 in the second year, we worked in another part of the plantation, approximately 50 meters south of the plot used in 2015. in april, we transplanted four f. polyctena ant mound fragments, each of about 180 l, into an approximately 0.11 hectare plot. two fragments were placed in each of the central and the eastern vegetation strips bordering the five eastern rows of apple trees. the four fragments were formed from one large mound collected from løvenholm forest, as described above (nielsen et al., 2018). mound material with ants was placed on the soil in the vegetation strips 3-4 meters from the nearest rows of apple trees. spacing between the two ant mounds on each side was approximately 20 m. the middle row of the five apple rows was not assessed, as trees in this row had been grafted the previous year. in each of the four remaining rows (with the varieties resista, holsteiner cox, collina and alkmene, respectively), 10 randomly selected trees were ant-excluded by providing them with sticky barriers as described above (ant-free trees), and another 10 trees were mounted with sugar feeders (sugar trees). we used the same type of feeders as in 2015, except we used a 40% organic sucrose solution plugged with a cotton plug that the ants could drink from (as we realized that the ants preferred sugar dissolved in water to sugar dough). all other trees within the four rows were freely accessible to ants, but without sugar feeders (the latter two types of trees commonly referred to as ant-trees). in the plot, there were between 31 and 37 trees of each variety. on all trees, we assessed the abundance of winter moth larvae, aphids, apples and fungal diseases and, lastly, we analyzed leaf chemistry on a subsample of trees to test whether ants fertilized the apple trees. on may 10, the number of operophtera brumata larvae was counted on each tree, and on july 28 the total number of shots and the number of shoots infested by green apple aphids (aphis pomi) were counted (we did not observe any rosy apple aphids, dysaphis plantaginea). in august, we sampled 20 leaves from each of the trees with sugar feeders and from each of the neighboring control trees without ants. leaves were sampled from the middle positions of shoots from the same year and dried at 60 degree celsius for 24 hours. the dried leaves were subsequently analyzed for their content of macro and micronutrients (n, p, k, ca, mg, s, fe, mn, zn, cu and b). during harvest, we counted the number of apples infected with brown rot (monilia fructigena) and apple scab as well as the number of first quality apples (eating apples; defined as apples without any damage, except up to three small scab marks) and second quality apples (juice apples; defined as apples with damage marks, but without holes in the apple skin). an apple was considered infected with scab if the apple contained more than three brown spots caused by venturia inaequalis, as this damage level means that the apple cannot be sold as an eating apple. disease incidence and apple yields were assessed on all trees. larval numbers, the proportion of aphid-infested shoots, yields of first and second quality apples, the proportion of apples infected with the two fungal diseases and leaf nutrients were subsequently compared between treatments. the sugar feeders on the sugar trees were assumed not to affect ant behavior, but only ant numbers (used to attract ants). there is no reason to believe ants behaved differently on trees with and without sugar feeders, as the ants foraging on both types of trees belonged to the same colony and, thus, were in the same nutritional condition (as ants perform collective foraging). statistical analyses the effects of ants (maximum ant index value for the 2015 data and treatment [ants vs no ants] for the 2016 data) were tested using approximate bayesian methods with the r package r-inla (rue et al., 2009). the response variables were either count data (apple yields, winter moth larvae), frequency data (aphid infections in 2015 and 2016, brown rot and scab infections), or concentrations (magnesium content). the count data were assumed to be distributed according to a zero-inflated negative binomial distribution, in which the additional zero values are generated by an independent process, and the log-transformed mean is modelled by a linear model of the fixed factors. the frequency data were assumed to be distributed according to a beta-binomial distribution, in which the logit-transformed mean probability is modelled by a linear model of the fixed factors. the concentrations were assumed to be normally distributed, where the mean is modelled by a linear model of the fixed factors. results first year study the presence of aphids on the trees was significantly affected by the maximum ant index value registered during the season, with a higher likelihood of aphid presence on trees j offenberg, js nielsen, c damgaard – ant services in apple plantations250 with more ants and without any aphids on trees where no ants were found (table 1; figure 1). this effect was consistent for all varieties. overall (across varieties and treatments), the highest number of trees infected with aphids was 7% during the third survey (21-6). apple yields were also significantly affected by the maximum ant index value, with all varieties showing higher yields on trees with more than 10 ants compared to trees with less than 10 ants (table 1; figure 2). when pooling all varieties, there was a 2.4-fold increase in apple yield on trees with more than 10 ants compared to trees with less or no ants. it should be noted, though, that yields were very low this year with an overall average of only 2.9 (± 0.27 se) apples per tree. table 1 shows the pooled effect with all varieties included. in this analysis, there was one significant interaction showing that the lowest ant index value of zero ants (index value 0) differed from the highest index value of >10 ants (index value 2) on collina trees. if, based on this single interaction, varieties were analyzed separately, a significant effect of ant presence was only seen on alkmene and a close to significant effect on resista. the lack of significance on the remaining varieties was probably due to the lowered sample sizes when varieties were analyzed separately. it should be noted, though, that the trend for higher yields with more ants is consistent across varieties (figure 2). table 1. the marginal posterior distributions of the effects of the fixed factors summarized by their 2.5%, 5%, 50%, 95% and 97.5% percentiles. the effects of ants on responses marked with two asterisks are considered two-tailed significant at the 5% level, as the quantiles within each response do not overlap 0, and responses marked with one asterisk are considered one-tailed significant, as only the upper 97.5% or the lower 2.5% overlap 0. response ant abundance variety mean sd 2.5% quantile 5% quantile 50% quantile 95% quantile 97.5% quantile 2015 aphids** max ant index (1-10 ants minus >10 ants category1) all varieties -0.66 0.29 -1.22 -1.13 -0.66 -0.18 -0.082 max ant index (0 ants minus >10 ants category) all varieties -0.66 0.26 -1.16 -1.08 -0.67 -0.22 -0.13 apple yield** max ant index (1-10 ants minus >10 ants category) all varieties -0.85 0.43 -1.7 -1.56 -0.84 -0.15 -0.016 max ant index (0 ants minus >10 ants category) all varieties -2.13 0.43 -2.99 -2.85 -2.13 -1.44 -1.32 2016 larvae** treatment (no ants minus ants2) all varieties 2.18 1.11 0.26 0.53 2.09 4.17 4.62 aphids** treatment (no ants minus ants) all varieties -1.27 0.42 -2.12 -1.98 -1.27 -0.6 -0.47 mg content* treatment (no ants minus ants) all varieties -0.0089 0.0045 -0.018 -0.016 -0.0089 -0.0014 0.0001 apples w brown rot treatment (no ants minus ants) alkmene & collina 0.6 0.31 -0.042 0.067 0.6 1.1 1.2 apples w scab* treatment (no ants minus ants) holsteiner cox 1.57 0.87 -0.026 0.21 1.52 3.07 3.4 first quality applesns treatment (no ants minus ants) all varieties -0.063 0.29 -0.64 -0.54 -0.062 0.41 0.5 second quality applesns treatment (no ants minus ants) all varieties 0.12 0.23 -0.35 -0.27 0.12 0.5 0.58 1 the high level ant index (>10 ants) is included in the intercept. 2 the treatment with ants is included in the intercept. fig 1. the mean (±se) proportion of trees infested with aphids in 2015 by maximum ant index. n = 141, 179, 91 trees, respectively for 0, 1-10 and >10 ants. second year study the number of o. brumata larvae was significantly reduced by ant presence, and this effect was consistent across all varieties (table 1; figure 3) with 4.4-fold more larvae found on control trees compared to trees with ants. again this year, sociobiology 66(2): 247-256 (june, 2019) 251 aphids were promoted by ant presence, as the average proportion of infested shoots was 2.5-fold higher on ant trees compared to the controls (table 1; figure 4). the total proportion of trees infected with aphids was 89%. regarding leaf nutrients, most nutrients were unaffected by ant presence, except magnesium. magnesium content was 5%, and significantly higher in leaves from ant trees compared to the controls (table 1 and figure 5). interestingly, fungal disease incidence was also affected by ant presence. only the varieties alkmene and collina were infected with brown rot. on these two varieties, brown rot incidence was 2.3-fold higher on control trees compared to trees with ants, and this difference was statistically significant (table 1 and figure 6). all apple varieties were infected with apple scab, but in this fig 2. mean (±se) number of apples by maximum ant index and apple variety. last group shows the overall mean of all varieties. n = alkmene 55, 30, 20; angold 8, 23, 14; collina 55, 40, 7; holsteiner cox 20, 70, 18; resista 3, 16, 32; overall mean 141, 179, 91 trees, respectively, for 0, 1-10 and >10 ants. fig 3. mean (±se) number of winter moth larvae (operophtera brumata) per tree by treatment in 2016. n = 96 trees with ants and 39 trees without ants. fig 4. mean (±se) proportion of aphid (aphis pomi) infested shoots per tree by treatment in 2016. n = 96 trees with ants and 39 trees without ants. case ant presence only led to a significant reduction on holsteiner cox (table 1, figure 7), though similar trends were observed on alkmene and collina. only resista, which is partly resistant to scab, showed a weak and non-significant trend in the opposite direction (figure 7). on holsteiner cox, the proportion of scabinfected apples was 11.4-fold higher on control trees compared to ant trees (table 1 and figure 7). there was no significant net effect of these services and disservices, as apple yields in 2016 were unaffected by ant presence; neither the number of first or second quality apples differed between treatments (table 1). total yield was 32.8 ± 1.86 se apples per tree in 2016, which was more than 11-fold higher than the 2015 yield (2.9 ± 0.27 se apples per tree). j offenberg, js nielsen, c damgaard – ant services in apple plantations252 discussion it was evident that wood ants provided both services and disservices in the apple plantation. ants reduced the number of winter moth larvae and the incidence of two fungal diseases and increased mg levels in the leaves of apple trees. during the first year, they also increased apple yield. on the other hand, ants also led to significantly more aphids on the apple trees. fig 5. mean (±se) magnesium content (%) in apple leaves by treatment. n = 35 trees with ants and 37 trees without ants. fig 6. mean (±se) proportion of apples infected with brown rot (monilia fructigena) by treatment. only apples on alkmene and collina are included, as the two other varieties were not infected by this pathogen. n = 49 trees with ants and 19 trees without ants. these results suggest that wood ants have the potential to be utilized as biocontrol agents in plantations if their mutualistic interaction with aphids can be broken or if they are used in systems that are not attacked by ant attended aphid species. alternatively, wood ants may be used curatively on a shortterm basis to combat pest outbreaks (e.g. winter moth larvae) before being removed from the plantation again in order to avoid subsequent build up in aphid numbers. fig 7. mean (±se) proportion of apples infected by scab by treatment and variety. note the difference in scale between the left (alkmene and collina) and right (holsteiner cox and resista) y-axes. n (trees) for no-ants and ant treatments, respectively: alkmene 9, 21; collina 10, 28; holsteiner cox 10, 21; resista 10, 26. sociobiology 66(2): 247-256 (june, 2019) 253 the anti-herbivore role played by ants is not surprising, as wood ants (f. rufa) are known to prey on winter moth larvae in forests (skinner & whittaker, 1981) as well as on a number of other pest insects, especially high-density epidemic species (way & khoo, 1992 and references therein). similarly to the data reported here, we showed in an earlier study that f. polyctena was able to reduce the number of winter moth larvae in the same apple plantation during the 2015 season on angold and holsteiner cox (nielsen et al., 2018). together, these results suggest that wood ants can be used to control winter moths in plantations. in addition, aphids were highly affected by ant presence, but in this case positively. trees with more ants were more likely to host aphids and showed a higher proportion of aphid infected shoots (table 1). these effects were pronounced, with ant trees experiencing several fold higher infection risks in 2015 (figure 2) and with a 2.4-fold higher proportion of shoots infected in 2016 (figure 5). this positive effect of ants on aphids was evident despite the fact that ants were fed with sugar in both years. this is surprising, as other studies have shown that sugar fed ants (lasius niger and l. grandis) are likely to decrease aphid tending and in this way open up for opportunities for aphid natural enemies to control the aphid populations (nagy et al., 2013; nagy et al., 2015; wäckers et al., 2017). in other cases, ants (oecophylla smaragdina and l. niger) may even start to prey on their aphid partners to utilize them as a source of protein when they are given alternative sources of sugar (way, 1954; offenberg, 2001). this may suggest that wood ants are more difficult to distract from aphid attendance than other ant species. we infer that more attractive sugar solutions than sucrose (at 85 and 40%) are required to distract wood ants from attending their aphid partners. it is known that different sugars and e.g. the addition of proteins or free amino acids to sugar solutions can substantially increase their attractiveness to ants (volkl et al., 1999; madsen et al., 2017). testing the attractiveness of different sugar concentrations, types of sugars and types of amino acids to wood ants may help to develop sugar solutions that can outcompete honeydew. in this way, we believe it is also possible to disrupt the wood ant-aphid mutualism. there is reason to believe that an optimized solution can outcompete honeydew, as there is already interspecific competition between aphids going on when it comes to ant attention. at the arrival of aphid species that produce superior honeydew, ants may neglect a previously otherwise attended aphid species because the latter now produce honeydew of inferior quality (compared to the honeydew from the new species) (cushman & addicott, 1989; tena et al., 2013). regarding nutrients, most leaf nutrients were unaffected by ant presence on the trees. however, we found increased levels of magnesium in the leaves of ant trees (table 1, figure 5). magnesium content increased from approximately 0.18% to almost 0.19% dry weight of leaves. both of these values are below the recommended level of 20% (gartnerirådgivningen, 2016), for which reason it is likely that the trees were limited by this nutrient. if this is the case and if ants were responsible for the observed increase, provisioning of nutrients could be another benefit resulting from ant attendance. however, more work is needed to uncover this potential service in more detail and to verify its cause. strangely, magnesium was the only nutrient out of 11 that was affected. whether ants in general increase levels of magnesium in their environment is interesting. increased magnesium levels have been observed in nests of atta laevigata, solenopsis invicta and l. niger, on the other hand, lower levels have been observed in nests of pogonomyrmes occidentalis (reviewed by folgarait, 1998). if the observed effect was, indeed, caused by ants, it is most likely a result of the deposition of ant fecal spots on the foliage, as the ground below ant and control trees was equally accessible to the ants in the plantation. if nutrients were absorbed via the soil, no difference between treatments was expected. uptake from foliage has been observed in weaver ants where nitrogen from their fecal spots was absorbed directly by coffee leaves (pinkalski et al., 2018). nitrogen, however, was not affected by ant presence in the current study. our study also found support for an ant-based plant pathogen defence, as we found lower levels of diseased apples on ant trees. significant effects on brown rot were found on both of the two varieties that were infected with this disease (alkmene and collina), whereas a significant effect on apple scab was found on holsteiner cox. in the cases where we detected significance, the magnitude of the effects was several-fold (>2-fold for brown rot and >11-fold for apple scab), whereas ants only lead to minor reductions of scab on alkmene and collina. only on resista did we find a weak non-significant trend in the opposite direction. this pattern may result from resista being partly resistant to apple scab (the name resista refers to apple scab resistance), seen by its lower infection rate compared to the other varieties (figure 7). the lack of significant effects on apple scab on alkmene and collina could be a result of low sample sizes and/or the fact that these two varieties were highly infected, with +40% of the apples carrying more than three apple scab spots. on these varieties, spot densities were high, which is why it requires more to reduce spot numbers to three spots or lower. the effects of ants may well have reduced spot numbers, but without being able to push numbers below three spots per apple. unfortunately, we did not count the number of spots on apples, but only divided apples into categories with less or more than three spots. however, it should be noted that despite a reduced pathogen incidence, this did not lead to significant effects on apple yields. the current results on pathogens are highly interesting, but more work is needed to assess whether effects are applicable, i.e. can lead to higher yields. it would be worthwhile to test the effect of wood ants in crops that are more susceptible to these two diseases, e.g. cherries and plums, which are plagued by brown rot to a greater extent than apples. despite significant effects on plant pathogens, we do not know the mechanism(s) behind these effects. in the case j offenberg, js nielsen, c damgaard – ant services in apple plantations254 of brown rot, the reduced incidence could result from ants deterring skin-damaging arthropods that would otherwise provide access points for infections. however, this fails to explain the reduced incidence of apple scab, as this fungus does not rely on skin damage to infect apples. instead, ant excretions could be a possible mechanism. f. polyctena excrete formic acid for defence and prey killing, and they use mellein in their trail pheromones (bestmann et al., 1992; morgan, 2008). both of these substances are known to possess antibiotic properties when applied in small dosages. formic acid has been suggested to be prophylactically applied by f. polyctena on nest material to control bacterial pathogens (sauerländer, 1961), and weaver ants control the entomopathogenic fungus metarhizium with this compound (tranter & hughes, 2015). similarly, mellein serve as an antiseptic when released in small amounts from the metapleural gland of crematogaster deformis (attygalle et al., 1989; morgan, 2008). thus, it is likely that f. polyctena excrete antibiotic substances (either for defence/attack or to mark their trails) that may end up on plant tissue, where they could potentially reduce pathogen spore germination and/or growth. it is known that formic acid inhibits brown rot. in petri dish cultures, brown rot growth was inhibited when 45% formic acid or crushed weaver ants (containing formic acid) were applied to the growth medium (zhang, 2017). in the plantation, we often observed f. polyctena spraying formic acid when disturbed. whether the ants also release formic acid in smaller doses on host plants is unknown. mellein, however, is likely to be deposited directly on apple trees when the ants use trail pheromones for navigation. the ants clearly provided both services and disservices in the apple plantation. during the first year, this resulted in increased apple yields on trees with the highest ant abundance. in the second year, however, the net effect did not affect apple production. possible explanations for this difference are unclear. in the second year, yields were more than 11 times higher than the low yield observed in 2015. it may be argued that ants are unable to increase yields during periods with high production, as there is no problem for the ants to fix in such periods. this could explain the lack of a positive effect in 2016. alternatively, the lack of an effect in 2016 could be due to higher numbers of aphids on the trees and, therefore, a build-up in the disservice. aphid densities were likely much higher in the second year, as the proportion of infected trees was 89% in 2016 compared to only 7% in 2015. unfortunately, we were unable to compare densities directly, as we did not count the numbers of colonies in 2015, but only whether trees were infected or not. in conclusion, we found support for all proposed hypotheses: (i) that ants reduce herbivore abundance (winter moth larvae), (ii) fertilize host plants (magnesium), (iii) control apple pathogens (brown rot and apple scab), (iv) increase homopteran abundance (aphis pomi) and that this (v) affects apple yields (in 2015). on the other hand, results are also inconclusive as (i) the net effect on yield was not consistent across years, and pathogen control was not consistent across apple varieties. this pinpoints that the current study must be considered a pilot study that needs spatial and temporal replication before we are able to recommend whether or not wood ants can be used in ipm approaches in fruit plantations. yet again, we feel confident to conclude that wood ants, at least under some conditions, provide positive effects on apples and probably also on other fruit crops. future studies may uncover whether ants should be used only temporarily during intense pest outbreaks in order to avoid a build-up of harmful homopterans, or whether wood ants are applicable in other crops that do not host ant-attended species. furthermore, future studies should focus on the development of sugar baits that can outcompete honeydew to the detriment of the antaphid mutualism. this may open the possibility of using wood ants long term (prophylactically), even in crops that are vulnerable to ant-attended pests. acknowledgement this study was supported by the gudp/icrofs organic rdd 2.2 project mothstop (34009-15-0984). authors' contributions j offenberg drafted the manuscript and contributed to fieldwork, conception, design, and interpretation. js nielsen contributed to fieldwork and design and cf damgaard performed the statistical analyses and contributed to interpretation and revisions of the manuscript. all authors have approved the final version of the manuscript. references attygalle, a.b., siegel, b., vostrowsky, o., bestmann, h.j. & maschwitz, u. 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(2017). the antimicrobial activities of oecophylla smaragdina. master degree master thesis, aarhus university. doi: 10.13102/sociobiology.v65i4.3472sociobiology 65(4): 686-695 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 bee diversity responses to forest and open areas in heterogeneous atlantic forest introduction over the years, human activity has modified about 43% of earth’s land surface (barnosky et al., 2012) by converting natural areas for several human activities, among them agriculture and urbanization (lambin et al., 2001). in brazil, this process is even harsher, with about a third of all land being converted, mostly for agriculture and cattle production (sparovek et al., 2010), endangering most, if not all, of the brazilian biomes (ferreira et al., 2012). the atlantic forest is one of these biomes, having lost more than 85% of its area (ribeiro et al., 2009) due to exploitation of forest resources abstract agriculture driven landscape changes have caused worldwide forest loss and fragmentation, severely affecting biodiversity and ecosystem services, amongst which pollination is remarkably important. bees are an essential pollinator group for forest plant populations and food production in tropical landscapes. they are also dependent on forested environments which are essential to maintaining their diversity and pollination services. we analysed bee diversity in forest patches and adjacent open areas to evaluate if bees can use complementary environments in heterogeneous altered tropical landscapes. the effect of landscape level heterogeneity and forest amount on bee diversity was also assessed. our hypothesis was that bee communities will be richer and more diverse in highly forested and heterogeneous landscapes when compared to areas dominated by few human-made environments, but due to supplementary foraging behaviors, they will be more abundant in open areas where flower availability is higher. we actively sampled bees visiting flowers within forest patches and in surrounding open areas between the cantareira and mantiqueira mountain ranges in são paulo, brazil. we found both higher bee richness and diversity in open areas than in forest patches, partially denying our initial hypothesis but supporting that bees are more abundant in open areas. we found strong indication that landscapes with a higher amount of forest and environmental heterogeneity can provide more resources for bees through resource complementation processes, maintaining their diversity in the landscape. the presence of forest patches close to crop and open areas is of utmost importance for the conservation of bees and pollination services with important consequences for land management in tropical environments. sociobiology an international journal on social insects ls nery1, jt takata1, bb camargo1, am chaves1, pa ferreira2, d boscolo1,2 article history edited by cândida aguiar, uefs, brazil eduardo almeida, usp, brazil received 10 may 2018 initial acceptance 16 july 2018 final acceptance 30 august 2018 publication date 11 october 2018 keywords pollination, landscape complementation, atlantic forest, landscape heterogeneity, brazil. corresponding author laura nery silva departamento de biologia faculdade de filosofia ciências e letras de ribeirão preto – ffclrp universidade de são paulo – usp cep 14040-901, ribeirão preto-sp, brasil. e-mail: laura.nery.silva@usp.br and land use activities for agriculture, pasture, forestry and urban expansion (dean & ferro, 1996), up to the point of being considered one of the 25 most endangered biodiversity hotspots of the world (myers et al., 2000). human driven land use changes can cause forest loss and fragmentation, changing landscape structure and leading to severe consequences for biodiversity and ecological processes (andrén, 1994; fahrig, 1998), compromising essential ecosystem services. one of the most threatened ecosystem services is crop pollination (potts et al., 2016), for habitat loss limits available resources for pollinators and fragmentation forces pollinators to change their foraging patterns, altering the topology of plant-pollinator 1 faculdade de filosofia ciências e letras de ribeirão preto (ffclrp), universidade de são paulo (usp), ribeirão preto, sp, brazil 2 programa de pós graduação em entomologia, universidade de são paulo (usp), ribeirão preto, sp, brazil research article bees sociobiology 65(4): 686-695 (october, 2018) special issue 687 interaction networks and consequently plant reproduction (brosi et al., 2008; van geert et al., 2010; moreira et al., 2015). bees are considered one of the most crucial pollinator groups, being undoubtedly related to the maintenance of plant diversity in tropical forests as well as food production in agroecosystems (bawa, 1990). pollination performed by bees also contributes to the world economy and human health, since this process is essential for at least 35% of the world’s food production (klein et al., 2007) and being responsible for providing different nutrients for human populations (eilers et al., 2011). for bees, habitat loss and fragmentation can increase the isolation of individuals and populations, affecting dispersion and foraging capacity (brosi et al., 2008; ferreira et al., 2015; boscolo et al., 2017). this isolation compromises bees’ chances to find mating partners, food, and nesting resources, with negative effects on population sizes and genetic diversity. when most populations are hindered like this, sharp decreases in abundance and richness of bee communities are expected (tonhasca et al., 2003; brosi et al., 2008; ferreira et al., 2015). habitat loss and fragmentation are being thus recognized as one of the top determinant factors for the general worldwide decline of bee populations detected during the last few years (potts et al., 2016). however, the magnitude of the impact of land use changes on bees can vary depending on the characteristics and requirements of each species (aizen & feisinger, 1994). the presence at the landscape level of enough natural areas such as forests is an important factor for the maintenance of bee diversity and pollination (brosi et al., 2008). landscapes with higher proportions of forest cover have a positive effect on bees species diversity (aizen & fiesinger, 1994; morato et al., 1999; tonhasca et al., 2003; brosi et al., 2008; brosi, 2009; ferreira et al., 2015; boscolo et al., 2017). the maintenance of forest patches in agricultural landscapes can even increase the diversity of flower visiting bees, especially of species considered flower and nesting specialists (carvalheiro et al., 2010). however, some species, such as social ground-nesting bees, may be able to nest and explore resources both in the forest and in open areas (ferreira et al., 2015), being less sensitive to environmental modifications. that plasticity in resource acquisition allows bees living in heterogeneous landscapes to explore resources in a wide range of environment, what must be especially true if additional food and nesting sites are available in non-native patches, such as in unmanaged pastures and open areas (steffan-dewenter et al., 2002). empirical studies have shown that in tropical regions altered by human activity, the maintenance of several different environments, instead of a single dominant anthropic land cover type, can favour many pollinator species (moreira et al., 2015; boscolo et al., 2017). depending on the requirements of each species, high landscape heterogeneity, namely the equitable presence of different types of land use and occupation, can increase the range of different environments used as habitat or source of complementary resources for bees (dunning et al., 1992; moreira et al., 2015, 2017). this complementation process is more likely to occur when different types of environments are near each other (lindgren et al., 2017), facilitating bee movements among these areas and consequently their ability to obtain resources, potentially increasing their fitness and population sizes (brosi et al., 2008). if this is the case, this opens a new range of environmental management possibilities to guarantee long term bee preservation along with human activities with associated ecosystem services in tropical environments. in order to develop strategies to conserve and increase bee abundance and diversity in altered landscapes, it is necessary to understand not only the requirements of these species regarding floral and nesting resources but also how landscape structure can affect their presence in different environments. our objective was to assess the influence of forest cover and landscape heterogeneity on the abundance and diversity of bees foraging within the forest and in adjacent open areas, which can be used as complimentary habitat in the face of forest loss. we hypothesize that bee communities will be richer and more diverse in highly forested and heterogeneous landscapes when compared to areas dominated by a few human-made environments with low forest cover. also, due to supplementation foraging behaviors, we expect bee abundance to be higher in open areas, where flowers are more abundant, than inside the forest. we expect to aid in the development of guidelines and strategies for the management of agricultural areas dependent on pollination services in order to reconcile the productivity of these areas with the preservation of more diverse pollinator communities, thus allowing the land occupation to be more sustainable. material and methods study area this study was developed at the region between cantareira and mantiqueira mountain ranges (são paulo, brazil), at the rural areas spanning from the city of itatiba (23º 01’ 00” s 46º 50’ 00” w) to igaratá (23º 12’ 00” s 46º 09’ 00” w) (fig 1). since 1994, the cantareira region belongs to the “são paulo city green belt” unesco biosphere reserve, being a key region to develop novel conservation approaches and sustainable development (jaeger, 2005). the region also contributes to water supply, providing to the most important water source for the são paulo city metropolitan region (whately & cunha, 2007). during the period of data sampling – november 2015 to march 2016 and november 2016 to march 2017 – the monthly precipitation at the region varied from 194.26 mm/ month to 281.16 mm/month (daee) with a mean annual temperature varying between 15-18 °c. ln silva, jt takata, bb camargo, am chaves, pa ferreira, d boscolo – diversity responses to forest and open areas688 the region is included within the atlantic forest biome and known to hold a great diversity of landscape structures with different degrees of anthropic intervention and forest fragmentation (ribeiro et al., 2009). land cover is very heterogeneous, including several types of forests, crops, commercial tree plantations, open areas and urbanized environments at different intensity levels (ibge, 1992). its complex of different heterogeneous landscape structures generates several levels of permeability and varying resource availability for bees, influencing the interaction between plants and these floral visitors. specifically, we focused on studying bees in both forest patches and adjacent open areas. sampled forests were composed of second growth dense ombrophilous forests in intermediate regeneration state (sifesp, 2009), presenting trees of up to 25m tall with a shrub understory (veloso et al., 1991). open areas encompassed unmanaged grassy pastures with only scattered trees, with the presence of asteraceae, poaceae and other herbaceous plants (s.1). all collected plant vouchers were deposited at the herbarium at ffclrp-usp. bee sampling to compare bee diversity in different environments, we sampled bees inside forest patches and in surrounding adjacent open areas. floral visiting bees were sampled actively with entomological nets. to delimit our search area, we installed inside each selected forest patch (see patch selection details below) a hexagonal plot with 25 meters sides, totalling 0.06 hectare sampled per forest site. to reduce edge effects all hexagons were installed at least 50 meters away from the forest edge. concurrently we delimited a region of similar area at the closest available open field surrounding the patch. in both environments we conducted recursive walks searching for flower visiting bees. within each hexagon, sequential 15 min of observations were made in all open flowers (up to two meters above ground) in sunny, hot days (20-31 °c), from 7:30 h to 14:00 h. we also sampled bees in open areas surrounding forest with the same searching method. bees were identified with the aid of experts to the most specific taxonomic level possible. fig 1. map of cantareira-mantiqueira mountain range region, são paulo state, brazil. the circles show the landscapes of 1 km radius with their respective types of environments indicated in figure 2. sociobiology 65(4): 686-695 (october, 2018) special issue 689 we conducted fieldwork at the season of the year in which flowering plants were most likely to be found (morelato lpc, personal communication), namely between november and february of 2015-16 and 2016-17. in each site, we sampled, at least, three times in each season. the use of these periods increased the chances of sampling a higher richness and abundance of flower visiting bees (nielsen & bascompte, 2007). sampling points selection and landscape pattern analysis land cover maps of the study area were generated from high resolution satellite images (esri, digitalglobe, geoeye, earthstar geographics, cnes/airbus ds, usda, usgs, aex, getmapping, aerogrid, ign, igp, swisstopo and gis user community) through remote sensing (rs) within geographic information systems (gis), using both manual and supervised classification at a scale of 1:5000 (executed by the spatial ecology and conservation laboratory-leec, unesp). the study region was classified into nine categories: water, forest, shrub, agriculture, forestry, open areas, wetland and anthropic (urban) areas (fig 2). subsequently, these areas were checked in the field to correct mapping errors so that in the end more than 90% of the region was correctly classified. from these maps, we selected 30 forest patches, with areas ranging between 15 and 25 ha and 24 open areas ranged from 0.73 to 26 ha. these forests were surrounded by several distinct anthropogenic environments (citrus, coffee, pasture, forestry, urban areas, etc.), with consequent variation in local vegetation structure that could influence local bee communities in different degrees. we purposely selected these 30 forest patches so that chances of high correlation between the amount of overall surrounding forest and landscape heterogeneity were reduced. bees were sampled both inside forest patches and in immediately adjacent open areas. to evaluate the influence of the composition of the surrounding landscape on flower visiting bees, we calculated two descriptive landscape metrics, forest cover and landscape heterogeneity. these measures were done within circular areas of varying radius surrounding sample plots. landscape pattern analysis was conducted with fragstat v.4 program (mcgarigal et al., 2012) to calculate forest cover and landscape heterogeneity (shannon-wiener index). the shannon-wiener index considers the number of different types of environments and its proportion in each landscape. if two landscapes are covered by exactly the same types of environments, that with the highest shannon-wiener value will be the one with the highest category evenness (mcgarigal et al., 2012). fig 2. mapped 1km radius landscapes. each colour represents an environment as indicated in the legend (complete class descriptions can be found in table s.2). percentages indicate the remaining amount of forest in each landscape. ln silva, jt takata, bb camargo, am chaves, pa ferreira, d boscolo – diversity responses to forest and open areas690 data analysis for each site of forest or open area, we estimated three bee community level variables: richness (number of species), abundance (total bee amount), from which we could also calculate the shannon diversity index. we calculated this index with the function diversity from the vegan package in r environment (team, 2017). the shannon index is defined as h’ = ∑i pi ln pi, where pi is the proportional abundance of species i and ln is the natural logarithm (oksanen et al., 2017). this index increases proportionally to the overall community diversity, being the sites with the highest species richness and equitability the most diverse and therefore represented by the highest index values. analysis of the effects of landscape patterns on bee diversity to evaluate the effects of landscape composition on bee diversity, we determined the spatial scale (landscape radius) in which linear models provided the best explanation of the response variables (steffan-dewenter et al., 2002). for that we considered buffers with a radius varying from 250m to 1000m in 250m intervals (steffan-dewenter et al., 2002; winfree et al., 2008) and then analysed it using simple linear regressions for each scale, having either forest cover or landscape heterogeneity as explanatory variables and each bee community descriptor as response variables. from these regressions, we obtained the r² values with their respective p-values (e.g., pearson, 1993; bergin et al., 2000; steffandewenter et al., 2002; moreira et al., 2015). the highest observed r² among all four scales for a given explanatory variable indicated the scale of effect to be used for each biological variable. after finding the scale of effect, we evaluated the influence of forest cover and landscape heterogeneity on bee richness, abundance, and diversity using simple linear regressions at the best selected scale for each variable. we used p values to check if the observed relationships were significant at a 0.05 alpha level and checked whether the parameters values were positive or negative to interpret the effect of landscape factors on biological variables. we then generated scatter plots for each response variable containing the observed data and model fit lines for both forest and open area sampling sites. this allowed us to directly compare the landscape effects on the communities observed in these two environments and check if they were the same. to effectively compare if the two sampling groups (forest and open areas) were significantly different from each other, we also conducted an analysis of variance for each bee community descriptor. we did all analyses in the r environment version 3.4.3. results we sampled 206 bee individuals within 54 species and morphotypes inside forest patches and 1875 bee individuals within 161 species and morphotypes in open areas surrounding these patches, totalling 2081 bee individuals in 176 species and morphotypes (table s. 3) with 39 common species between forest and open areas distributed within 4 of the 5 sampled families. these results show that in open areas we found about threefold the bee richness observed inside the forest. as expected for the selection of the scale of effect, the best explanation was found to be related to different scales for each response variable. bee community variables (shannon diversity index, richness, and abundance) inside the forest responded to forest cover in the scale of 750 m. in open areas, the best scale for these variables was at the 1000 m radius. for landscape heterogeneity, bee diversity best responded at the scale of 750 m for open areas and 1000 m for forest patches (fig 3). on the other hand, bee abundance responded to landscape heterogeneity at the 500 m, the smallest selected scale among all tested relationships (table s.4). models r² p forest cover a) bee shannon index in the forest ~ forest cover_750 0.05971 0.1931 a) bee shannon index in open areas ~ forest cover_1000 0.0964 0.1398 b) bee richness in the forest ~ forest cover_750 0.0252 0.4021 b) bee richness in open areas ~ forest cover_1000 0.1326 0.08018 c) bee abundance in the forest ~ forest cover_1000 0.03959 0.2918 c) bee abundance in open areas ~ forest cover_1000 0.1871 0.03476* landscape heterogeneity d) diversity in the forest ~ l. heterogeneity_1000 0.0217 0.4372 d) diversity in open areas ~ l.heterogeneity_750 0.01128 0.6213 e) richness in the forest ~ l. heterogeneity_1000 0.002946 0.7758 e) richness in open areas ~ l. heterogeneity_750 0.01714 0.542 f) abundance in the forest ~ l. heterogeneity_750 0.04273 0.2731 f) abundance in open areas ~ l. heterogeneity_500 0.06063 0.2461 table 1. linear regression results for bee richness, abundance and diversity (shannon index) inside the forest and in adjacent open areas. the table shows only the models selected with the highest r² results after landscape scale selection for the proportion of forest in landscape (f. cover) and landscape heterogeneity (l. heterogeneity) measured with the landscape shannon index. the letters “a” to “f” corresponds to the graphics of figure 2 with the same letters. significant p values are marked with *. sociobiology 65(4): 686-695 (october, 2018) special issue 691 using these variables at its best explanatory power, bee richness, abundance and diversity in open areas responded positively to forest cover. this response was more intense for bee richness and abundance than for diversity. however, we found the opposite response for bee diversity and richness inside the forest, with a negative effect of forest amount on these variables, which was more intense for bee diversity (fig 3 a, b). bee abundance did not respond to forest cover. differently, bee communities in open areas responded negatively to landscape heterogeneity for all three response variables, but more strongly for bee abundance (fig 3 f). fig 3. linear regression plots. dashed lines and hollow points are for open areas adjacent to forests and continuous line and black points correspond to forest patches. response for bee shannon index (bee diversity) in “a” and “d”; response for bee richness in “b” and “e”; and for bee abundance in “c” and “f”. graphs “a” and “b” show the effect of forest cover on bee richness at 750 m inside the forest and 1000 m in open areas. in “c” it is shown the forest cover effect on bee abundance at 1000 m for forest patches and open areas. however, “d” and “e” show the effect of landscape heterogeneity on bee diversity and richness at 1000 m in forest patches, and at 750 m in open areas, respectively. graph “f”, represents the effect of landscape heterogeneity on bee abundance at 750 m in forest patches and at 500 m in open areas. graphs “a” and “b” show that bee diversity is directly related to forest cover in open areas and is inversely related to forest cover in forest fragments. in “d” and “e”, bee diversity and richness were inversely related to increases in landscape heterogeneity in open areas, even though in forest fragments bee diversity increase when landscape heterogeneity is high. in “c” and “f” forest cover had a positive effect on bee abundance for both open areas and forest patches. landscape heterogeneity had a negative effect on this same response variable, for both open areas and forest patches. a b c d e f the only positive effect of landscape heterogeneity was on bee diversity sampled within the forest (fig 3 d), indicating that landscapes with more different available environments increases bee diversity inside the forest, but not outside. furthermore, the analysis of variance revealed that forest and open area sites were significantly different from each other for all three bee community variables, namely richness (f = 57.33, p < 0.0001), abundance (f = 59.86, p < 0.0001) and diversity (f = 47.78, p < 0.0001), with all variables having higher mean values always in open areas in comparison to forest interior. discussion we observed a distinct response pattern of bee communities between those sampled inside forest fragments and in surrounding open areas. most surprisingly was that, independently of the landscape context, open areas present more diverse and rich bee communities than those found inside the forest, with three times more species visiting flowers in the non-forested environment. these results deny our initial hypothesis for bee diversity and richness but corroborate it for their abundance. partially, the low levels of bee richness and diversity found inside the forest may be related to the sampled forest strata used in the current study. since we sampled bees that were foraging on flowers, the response depends directly on blooming intensity in the understory. however, flower abundance is known to be lower in the understory of mature forests, which usually presents reasonably closed canopy with little light input, hindering the growth and blooming of shrubs and herbaceous species closer to the ground (terborgh, 1985), where we were sampling. thus, sampling flower visitors in the understory of forest patches may indicate low species diversity, while in fact, sampling only this stratum may not ln silva, jt takata, bb camargo, am chaves, pa ferreira, d boscolo – diversity responses to forest and open areas692 allow forest bee communities to be fully assessed. in mature forests, blooming may be significantly higher in the well-lit canopy, according to the flowering season of the trees, and bees may be drawn there to forage (ramalho, 2004). to overcome this limitation, novel methods to sample flower visiting bees in the canopy for a wide range of trees must be developed. however, even with this limitation, the results for bee communities within and outside forest patches remain entirely comparable and have ecological significance and management value at the landscape level. what we found when considering landscape composition is that this higher overall bee diversity in open areas tends to increase along with higher amounts of surrounding forest at broader landscape scales (mainly 750 and 1000 m radius). this relationship between open areas and forest cover in the landscape can be understood as an effect of bees using open areas as an alternative, complementary habitat type (dunning et al., 1992; boscolo et al., 2017; lindgren et al., 2017). the hypothesis of habitat complementation, or likewise habitat supplementation, arises from the fact that open areas adjacent to forest fragments may offer a great amount of feeding resources, mainly due to a more constant and higher availability of flowers (e.g., asteraceae, myrtaceae) than in the understory of forest patches (moreira et al., 2015). thus, bees may leave the forest to forage in these adjacent areas. the presence of bees in these areas is a significant indication that, even though they are initially forest dwelling species, these animals also use the resources available outside the forest (brosi et al., 2008). for many atlantic forest bees, the forest is considered a high-quality habitat and has an essential role as source environment (brosi et al., 2008; tscharntke et al., 2012; ferreira et al., 2015), as it may offer a higher amount of adequate nesting sites. the availability of other kinds of resources, on the other hand, may be lower in the understory when compared to open areas. this attracts bees nesting in the forest to use alternative human made environments to fulfil their needs. species that nest above the ground, for instance, are highly dependent on forested areas for nesting and end up being more susceptible to forest loss when compared to species that nest in the ground (ferreira et al., 2015). for these species, the presence of forested habitat within foraging ranges is important to supply their needs, mainly due to enhanced nesting opportunities within the available forest patches (brosi et al., 2008). our results corroborate studies that elucidate the importance of maintaining high levels of forest quantity in the landscape in order to maintain biodiversity (fahrig et al., 2011; lindgren et al., 2017). open areas present different climatic conditions from forest patches, being drier and hotter than the forest understory (baudena et al., 2015). this may be a problem for species that are more sensitive to extreme climatic conditions (hilário et al., 2001). for these sensitive species, foraging outside the forest may be challenging, even if feeding resources are attractive in open areas, which are exposed to more light and have a higher amount of flower resources (terborgh, 1985; wender et al., 2004). in this scenario, higher amounts of forest in the landscape leads to lower isolation of remaining forest patches (fahrig, 2003), being extremely important for the maintenance of more sensitive bee species (brosi et al., 2008), for it reduces the distance and time needed to access open areas, making foraging there less demanding and more efficient (jha & vandermeer, 2010). landscapes composed of enough close by forest and open areas are then an advantageous situation for both sensitive species and those that can endure open areas conditions. resilient bees may be even more efficient in using both environments, exploring forest to forage and nest and adjacent open areas to forage for food. for bees that nest in the ground, open areas may even mean a source of nesting resources along with food (see ferreira et al., 2015). landscape configuration, i.e., the spatial distribution of forest patches and open areas, is thus of utmost importance for the conservation of bee diversity and abundance in the landscape (moreira et al., 2015; boscolo et al., 2017). this also means that the maintenance of forest and bee friendly non-forest environments in the landscape is favourable to plant pollination within forest patches and also for the provision of pollination ecosystem services for plants outside the forest, for example in agricultural areas (jha & vandermeer, 2010; moreira et al., 2015; boscolo et al., 2017; hipólito et al., 2018). highly forested landscapes may also be of ecological importance to provide other ecosystems services, such as climatic and water regulation and soil formation, among others (asner et al., 2004). for pollinators and pollination, the increase we found on species number not necessarily represents an improvement of environmental quality or ecosystem service provision. for example, the fragmentation of forest creates edges that receive a significant amount of sunlight and is an interesting environment for bees to forage due to more diversity of herbaceous flower resources (chacoff & aizen, 2006). however, that also favors the invasion of opportunistic and or exotic species (aizen et al., 2008). in the first moment the presence of these species increases richness, but in the long term these opportunistic species may be very competitive and represent a negative impact for forest communities and pollination within the remaining forest patches. likewise, the current rates of tropical forest replacement for anthropic open areas, mostly with agricultural purposes (gibbs et al., 2010), imposes an important challenge for the scientific argument of the need to maintain heterogeneous pollinator friendly landscapes. the economic pressure to acquire short term profits that dominates tropical landscape change processes usually leads to land management directives that hinder long-term maintenance of bees and associated pollination services. current agribusiness model tends to favor high yield but low durability or sustainability of the crops in tropical environments (landy et al., 1990), moving over sociobiology 65(4): 686-695 (october, 2018) special issue 693 native environments when production decreases due to environmental degradation. this constant movement of crop areas generates highly homogenized landscapes where pollination services will be lower due to pollinators debt. the yield of pollinator dependent crops has already been shown to be higher and to produce greater value for the landowner when the landscape has a more interspersed structure with native environments being closer and spatially alternated with crops (ricketts et al., 2004; klein et al., 2007; hipólito et al., 2018). this effects of the nearness between crops and native environments corroborates the idea that the presence of forest patches near crops can increase pollination (brosi et al., 2008). also, the presence of forests may favor the permanence of more sensitive species, which can offer specific more efficient pollinators for a particular crop or native plant species. in this scenario, our results give an important argument towards the ecological intensification of altered landscapes. we have strong indication that landscapes that have higher amount of forest and higher levels of environmental bee friendly heterogeneity can provide more food and nesting resources for bees, being more interesting for the maintenance of bee species and the ecological service of pollination they provide. acknowledgments we thank eduardo almeida, luciano elsinor lopes, sidnei mateus, leap-ffclrp-usp group, for assistance with fieldwork, species identification, and manuscript comments. cnpq for acm, bbc and lsn scholarships,, and for research funding (mcti/cnpq/universal 449740/2014-5), fapesp for jtt scholarship (process 2015/04973-9) and for funding the project “new sampling methods and statistical tools for biodiversity research: integrating animal movement ecology with population and community ecology”, fapesp (process 2013/50421-2), capes and ppgentomo-ffclrp-usp for paf scholarship. supplementary material doi: 10.13102/sociobiology.v65i4.3472.s2227 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3472/0 references aizen, m.a. & feinsinger, p. 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(2008). wild bee pollinators provide the majority of crop visitation across land–use gradients in new jersey and pennsylvania, usa. journal of applied ecology, 45: 793802. doi: 10.1111/j.1365-2664.2007.01418.x. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3568sociobiology 66(1): 179-185 (march, 2019) is the social wasp fauna in the tree canopy different from the understory? study of a particular area in the brazilian amazon rainforest introduction the polistinae social wasps are comprised of 26 genera and 958 species widely distributed in the neotropical region (pickett & carpenter, 2010). these wasps are important components of neotropical ecosystems due to their ubiquity and diversity, as well as their complex interactions with other organisms (silveira et al., 2012). the amazon rainforest is the largest biome in brazil, covering an area of 4,196,943 km2 within the brazilian territory, and is one of the most biodiverse places in the world, presenting the highest diversity of social wasps (silveira, 2002; somavilla et al., 2014; barbosa et al., 2016). silveira (2002) highlighted that 200 species have been recorded in the brazilian amazon, which represents about two-thirds of all brazilian polistinae fauna. however, somavilla and oliveira (2013) emphasized the need for more studies focusing on wasp distribution in the region. furthermore, the richness of social wasps in the brazilian amazon forest is still underestimated (somavilla & oliveira, 2017). abstract most studies about the insect community in rainforests only focus on the forest understory, and even though the rainforest canopy is one of the most fascinating and diverse environments, it remains poorly explored. therefore, we analyzed the difference between the social wasp composition in these two strata at the zf-2 station in the brazilian amazon rainforest, amazonas, manaus, using flight interception malaise traps, in the rainforest understory and canopy. we collected ninety-two species belonging to 18 genera; polybia was the richest genera (22 species), followed by mischocyttarus (14) and agelaia (13). fortyfour species were exclusively collected in the understory, twenty exclusively collected in the canopy, and twenty-eight in both strata. the understory was distinctly more diverse and more abundant than the canopy, while some rare or poorly collected species were only found in the canopy. we found a relationship between the species composition at the zf-2 station and the ducke reserve, manaus. therefore, we suggest using traps in canopy in the amazon biome as an effective method for collecting a higher diversity of social wasps. sociobiology an international journal on social insects a somavilla, rnm moraes junior, ja rafael article history edited by gilberto m. m. santos, uefs, brazil received 09 july 2018 initial acceptance 06 september 2018 final acceptance 14 october 2018 publication date 25 april 2019 keywords amazonas, malaise trap, polistinae wasps. corresponding author alexandre somvilla instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo nº 2936, cep: 69067-375 manaus, amazonas, brasil. e-mail: alexandresomavilla@gmail.com some studies about social wasps have been carried out in the brazilian amazon, as follows: maracá ecological station, roraima state with 36 species (raw, 1998) and four other locations in roraima with 85 species (barroso et al., 2017); caxiuanã reserve, pará state with 79 species (silveira, 2002) and 65 species (silva & silveira, 2009); serra do divisor national park, acre state with 20 species and three fragments close to rio branco with 36 species (morato et al., 2008; gomes et al., 2018); lakes of amapá, amapá state with 31 species (silveira et al., 2008); gurupi biological reserve, maranhão state with 38 species (somavilla et al., 2014); and three localities in north of rondônia state with 76 species (gomes, 2013). in the state of amazonas, six surveys have been carried out: mamirauá and alvarães reserves, with 46 and 42 species, respectively (silveira et al., 2008), jaú national park with 49 species (somavilla et al., 2015), madeira-purus rivers with 38 species (oliveira et al., 2015), embrapa-manaus with 52 species (somavilla et al., 2016), and ducke reserve with 103 species (somavilla & oliveira, 2017). instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus, amazonas, brazil research article wasps a somavilla, rnm moraes junior, ja rafael – social wasp fauna from canopy and understory amazon area180 despite the contributions of these works, somavilla et al. (2014) stated “there are many sample gaps in the amazon region and distribution and occurrence studies are necessary for improving this prior knowledge”. this may be due to limited exploration in the rainforest understory (somavilla et al., 2014). inventories from the amazon show that some polistinae species have been represented by one or a few specimens with all collections carried out in understories only, while tree canopies have been poorly explored by researchers. the previously mentioned studies from understories, and only two studies used the suspended trap method in tree canopies (roraima state and ducke reserve), but only as a complementary method and reaching low height, reaching at most 20 meters (barroso et al., 2017; somavilla & oliveira, 2017). the rainforest canopy is one of the most fascinating environments to develop studies about arthropod communities (erwin, 2013; nakamura et al., 2017). although the tropics hold the highest species diversity (erwin, 2013), many interactions in tropical forests are made in the canopies, due to the high number of species that inhabit canopies and the large amount of biomass that is generated in this stratum (nakamura et al., 2017). compared to other rainforest strata, the rainforest canopy presents higher illumination, temperatures, wind speeds, and relative humidity (nakamura et al., 2017). in this way, we asked: is the social wasp fauna different between the tree canopy and the understory? which environment/strata has the highest richness of social wasps? herein, we present a list of species and discuss the use of interception traps, malaise traps mounted in the canopy and understory, for collecting social wasps (polistinae) at the zf-2 station in the brazilian amazon rainforest. methods study area the study site is located inside one of the national institute for amazonian research (inpa) reserves tropical silviculture experimental station, at km 934 of br 174 on the road zf-2 (2°35’21”s – 60o06’55”w), herein called zf-2 station. this area is about 50 km north of manaus in amazonas, brazil and is managed by the project: the large-scale biosphereatmosphere experiment in amazonia (lba-inpa), which is the largest study about climate change in the amazon region. according to the köppen (1948) climate classification, the climate at the station is am type, with low annual thermic range and average monthly rainfall over 60 mm. the average annual temperature is 26.7 ºc, ranging between 23.3 ºc and 31.4 ºc, while average annual rainfall is 2286 mm and relative humidity is around 80%. the rainy season is from december to may, and the dry season from june to november (barbosa et al., 2015). the topography in this area is undulating with elevation ranging from 50 to 140 meters above sea level. wasp collection currently, brazil holds one of the largest research programs that uses towers as a standard structure to reach the tree canopy. such sampling tower is metallic and 40 m high, located inside a typical amazonian landscape, which is comprised of an ombrophilous dense forest with a canopy reaching 40 m, or even 50 m due to emergent trees. in this type of forest, it is difficult to distinguish between median and lower strata, but the mean canopy height in the amazon is 28.6 m (higuchi et al., 2009). a description of the flora at the research station can be found in martins et al. (2006). in the forest understory and canopy, we used a modified malaise trap gressit and gressit model (1972) that was 6-meter long with two collector vials (figure 1). in the canopy, the trap was mounted 32 meters from the ground in a metallic tower (6m wide x 6m length) to collect wasps and in understory on the forest floor. both traps were active for fifteen consecutive days each month, for a period of eight months between july 2016 to march 2017. the polistinae specimens were sorted and identified at the hymenoptera laboratory of the national institute of amazonian research (inpa). the vouchers were deposited into the inpa’s invertebrate collection. specimens were identified using the keys proposed by richards (1978), fig 1. sampling method at the zf-2 station (lba/inpa): (a) malaise trap in the understory and (b) malaise trap in the canopy at 32 m high. sociobiology 66(1): 179-185 (march, 2019) 181 carpenter and marques (2001), and carpenter (2004) and were compared to previously identified species from the inpa invertebrate collection. data analysis we used nonmetric multidimensional scaling (nmds) (minchin, 1987) to ordinate the inventories carried out in the brazilian amazon (see introduction topic) according to their species composition and to verify whether species displayed a preference for a particular area and used dissimilarity coefficient for the species composition from the zf-2 station compared to other inventories in the brazilian amazon. this analysis was conducted in r version 3.3.3. (r core team, 2017) using vegan package 2.4-0 (oksanen et al., 2016). for this analysis, we excluded the specimens identified as “morphospecies” or “varieties” and we used only the species collected in malaise or suspended traps, and we exclude the species of active search, light trap or attractive trap. results the survey from zf-2 station revealed a rich fauna of social wasps. we found 1.548 specimens in ninety-two species belonging to 18 genera (table 1). polybia lepeletier was the richest genera (22 species), followed by mischocyttarus de saussure (14) and agelaia lepeletier (13). together, these three genera comprised about 55% of the species listed. the only brazilian polistinae genera we did not collect from in the area were asteloeca raw, chartergus lepeletier, and protonectarina ducke, the first two not specious genera with three described species and protonectarina with occurrence only for the atlantic forest. in terms of specimens’ numbers collected, 870 were from understory and 678 from canopy. forty-four species were exclusively collected in the trap mounted in understory, twenty species were exclusively collected in the trap mounted in the tree canopy, and twenty-eight species were found in both traps (table 1). all species of agelaia, apoica, and pseudopolybia were collected in the understory. species of metapolybia, nectarinella, and synoeca were collected in the canopy. polybia species were collected in both strata. several species are new records for brazil or amazonas state: agelaia pallidiventris (new record for brazil), nectarinella manauara and protopolybia rugulosa (second time both species were collected for amazonian rainforest), a. flavipennis, polybia affinis, po. minarum, pr. nitida, pr. rotundata and pr. sedula (new records for amazonas state). the following specimens could not be determined to the species level: one species of agelaia, one species of polybia (myrapetra), and two species of mischocyttarus (haplometrobius). according to the dissimilarity analysis between the social wasps’ composition from different areas in the amazon, we found a relationship between the species composition of zf-2 station andducke reserve (figures 02, 03). the same relationship was found for alvarães and mamirauá reserve (am) and caxuanã reserve (pa). however, the composition of wasp fauna from the rio branco region (ac), serra do divisor national park (ac), maracá ecological station (rr), and lagos region (ap) were dissimilar from the zf-2 station. fig 2. first two nmds ordination axes considering social wasp species composition from inventories in the amazon region. legend: z (zf-2 station am), d (ducke reserve am), e (embrapa am), j (jaú national park am), p (purus-madeira rivers am), a (alvarães and mamirauá reserve am), m (maracá ecological station rr), t (tepequém, mocidade, viruá rr), r (rio branco region ac), s (serra do divisor national park ac), c (caxiuanã reserve pa), v (porto velho region ro), l (região dos lagos ap) and g (gurupi biological reserve ma). 1    social wasps species composition composition nmds axis 1 −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 0.8 dz e j p a m t r s c v l g n m d s a xi s 2  discussion in surveys conducted in the amazon rainforest, the diversity of social wasps is generally higher than in other biomes. surprisingly, we collected 92 species from the zf-2 station in only eight months, using two passive collection trap sin two strata (understory and canopy 32 meters high). the 92 species from the zf-2 station were distributed into 18 genera, mainly polybia (22 species), mischocyttarus (14), and agelaia (13). in other areas of amazonas state, as the ducke reserve, 103 social wasp species were collected from the understory stratum only, however, six types of collection methods were used during several years of collection and different methods. at ducke reserve, 19 genera were collected, mainly polybia (28 species), agelaia (12 species) and mischocyttarus (12 species). therefore, the results of species composition from both the zf-2 station and the ducke reserve were very similar. silva and silveira (2009) and somavilla et al. (2014) showed that fast inventories were efficient for sampling the most abundant species, recording three genera: agelaia, polybia and mischocyttarus. herein, we found, the same a somavilla, rnm moraes junior, ja rafael – social wasp fauna from canopy and understory amazon area182 most abundant genera, which constituted more than 50% of the specimens collected specimens. agelaia species usually form large colonies with millions of individuals (zucchi et al., 1995), and, consequently, are more likely to be captured (silva & silveira, 2009). mischocyttarus is the genus with more described species in social wasps, that can support the high diversity in this study. (silveira, 2002) and polybia has a very active foraging behavior, which facilitates the collection of these species in traps (carpenter & marques, 2001). most species of epiponini, belonging to apoica, brachygastra, clypearia, leipomeles, polybia, and protopolybia, were only collected in malaise traps. conversely, this method was less efficient for capturing polistes, a pattern that has also been reported by other researchers (silveira, 2002; silva & silveira, 2009). polistes nests at lower height and has usually small nests with few individuals, they are more difficult to be seen in forest (carpenter & marques, 2001). therefore, using only malaise traps to collect social wasps may underestimate the actual richness of polistes. conversely, there was a strong relationship of nesting in the forest canopy, as the rare species clypearia duckei, epipona tatua, and polybia depressa were only recorded in the canopy stratum at the zf-2 station and the ducke reserve (somavilla & oliveira, 2017). furthermore, richards (1978) recorded an e. tatua nest that was over 12 meters above ground in mato grosso, and somavilla (2013) photographed a nest above 20 meters at the ducke reserve. synoeca, and some brachygastra and polybia nests are also common in canopy, richards (1978) recorded a b. bilineolata nest above 13 meters above ground and somavilla et al. (2012) recorded a s. surinama and p. liliacea nest about 20 meters above ground. insects inhabiting the canopy of the amazon rainforest strata are poorly represented in collections, with many unknown specimens in these habitats (rafael & gorayeb, 1982). from inventories in the amazon, some polistinae species are represented by only one or a few specimens in the collections and occur in the tree canopy, a habitat that has been poorly explored by collectors. for example, in the two works they explored the canopy, 25 species in ducke reserve and seven in roraima state, manly agelaia, clypearia, metapolybia and polybia (barroso et al., 2017; somavilla & oliveira, 2017). likewise, there are few methods for collecting insects in these strata, that are often times costly and impractical. based on this observation and the need for collections in these environments, traps mounted in the canopy are efficient ways of collecting and improving knowledge about social wasp species, mainly in the amazon rainforest. furthermore, such methods should be used in conjunction with malaise traps mounted on the forest floor and active search. finally, our study shows that some species were only collected in the canopy (table 1). there are different methods for sampling social wasps, however, few studies have attempted to standardize these methods or establish comparable and adequate protocols to survey the fauna at a given site. one important factor to consider when implementing new social wasp sampling protocols is the distribution pattern of these organisms (silveira, 2002; somavilla et al., 2014). similarly, it is also important to determine the most efficient traps to collect the target group and dispose of them in a standardized manner (noll & gomes, 2009). through this study, we verified, that using traps in the upper forest canopy is very important for collecting social wasp species. furthermore, such method should be included in the collection protocol for the amazon biome.     fig 3. cluster dendrogram generated dissimilarity coefficient for the species composition from the zf-2 station compared to other inventories in the amazon region. legend: z (zf-2 station am), d (ducke reserve am), e (embrapa am), j (jaú national park am), p (purus-madeira rivers am), a (alvarães and mamirauá reserve am), m (maracá ecological station rr), t (tepequém, mocidade, viruá rr), r (rio branco region ac), s (serra do divisor national park ac), c (caxiuanã reserve pa), v (porto velho region ro), l (região dos lagos ap) and g (gurupi biological reserve ma). sociobiology 66(1): 179-185 (march, 2019) 183 the malaise trap in the understory captured more specimens and social wasp species when compared to the malaise trap in the canopy. probably because much of the foraging of social wasps happen in the understory, in addition to various feeding and nesting resources found near the forest floor (somavilla et al., 2012). in this study, we did not use the active search collection method. however, silveira (2002) concluded that the active search method was more efficient than traps, when they only used understory malaise trap. in order to determine which species were unique to the two traps, we note the importance of working with different stratum. in the amazon, where the forest canopy can reach great heights, using malaise traps in the forest canopy could be a good method for capturing social wasps. taxa malaise understory malaise canopy epiponini agelaia angulata (fabricius, 1804) x agelaia cajennensis (fabricius, 1798) x x agelaia centralis (cameron, 1907) x agelaia constructor (de saussure, 1854) x x agelaia flavipennis (ducke 1905) x agelaia fulvofasciata (degeer, 1773) x x agelaia hamiltoni (richards, 1978) x agelaia myrmecophila (ducke, 1905) x agelaia pallidiventris (richards, 1978) x agelaia pallipes (olivier, 1791) x x agelaia ornata (ducke, 1905) x agelaia testacea (fabricius, 1804) x x agelaia sp.1 x x angiopolybia pallens (lepeletier, 1836) x x angiopolybia paraensis (spinola, 1851) x apoica arborea de saussure, 1854 x apoica gelida van der vecht, 1898 x apoica pallens (fabricius, 1804) x apoica pallida (olivier, 1791) x apoica strigata richards, 1978 x apoica thoracica du buysson, 1906 x x brachygastra bilineolata spinola, 1841 x brachygastra lecheguana (latreille, 1824) x brachygastra scutellaris (fabricius, 1804) x chartergelus amazonicus richards, 1978 x chartergelus nigerrimus richards, 1978 x charterginus fulvus fox, 1898 x clypearia apicipennis (spinola, 1851) x clypearia duckei richards, 1978 x clypearia sulcata (de saussure, 1854) x x epipona tatua (cuvier, 1797) x leipomeles dorsata (fabricius, 1804) x leipomeles spilogastra cameron, 1912 x x metapolybia decorata (gribodo, 1896) x metapolybia nigra richards, 1978 x x metapolybia rufata richards, 1978 x metapolybia unilineata (r. von ihering, 1904) x nectarinella manauara silveira & santos jr., 2016 x parachartergus amazonensis (ducke, 1905) x parachartergus fraternus (gribodo, 1892) x x parachartergus richardsi willink, 1951 x x polybia belemensis richards, 1970 x polybia bifaciata de saussure, 1854 x polybia bistriata (fabricius, 1804) x x polybia depressa (ducke, 1905) x polybia dimidiata (olivier, 1792) x x polybia dimorpha richards, 1978 x x polybia emaciata lucas, 1879 x table 1. species of social wasps collected at the estação zf-2 (lba/inpa), as along with the method used to capture every species. malaise understory trap and malaise suspended trap. polybia incerta ducke, 1907 x polybia jurinei de saussure, 1854 x x polybia juruana r. von ihering, 1904 x polybia liliacea (fabricius, 1804) x x polybia minarum ducke, 1906 x polybia occidentalis (olivier, 1791) x x polybia parvulina richards, 1970 x polybia platycephala richards, 1951 x polybia procellosa zavattari, 1906 x x polybia rejecta (fabricius, 1798) x x polybia scrobalis richards, 1970 x x polybia signata ducke, 1905 x polybia singularis ducke, 1905 x x polybia striata (fabricius, 1787) x x polybia (myrapetra) sp. x protopolybia bituberculata silveira & carpenter, 1995 x protopolybia emortualis (de saussure, 1855) x x protopolybia exigua (de saussure, 1954) x protopolybia holoxantha (ducke, 1904) x protopolybia nitida (ducke, 1904) x protopolybia rotundata ducke, 1910 x protopolybia rugulosa ducke, 1907 x pseudopolybia compressa (de saussure, 1854) x pseudopolybia dificcilis (ducke, 1905) x pseudopolybia langi bequaert, 1944 x pseudopolybia vispiceps (de saussure, 1863) x synoeca surinama (linnaeus, 1767) x synoeca virginea (fabricius, 1804) x mischocyttarini mischocyttarus bertonii ducke, 1918 x mischocyttarus collaris (ducke, 1904) x mischocyttarus drewseni (de saussure, 1857) x mischocyttarus imitator (ducke, 1904) x mischocyttarus labiatus (fabricius, 1804) x mischocyttarus lecointei (ducke, 1898) x mischocyttarus metathoracicus (de saussure, 1854) x mischocyttarus punctatus (ducke, 1904) x x mischocyttarus smithii de saussure, 1853 x mischocyttarus surinamensis (de saussure, 1854) x x mischocyttarus synoecus richards, 1940 x mischocyttarus tomentosus zikán, 1935 x mischocyttarus (haplometrobius) sp.1 x mischocyttarus (haplometrobius) sp.2 x polistini polistes claripennis ducke, 1904 x x polistes versicolor (olivier, 1792) x total = 92species 72 47 taxa malaise understory malaise canopy epiponini a somavilla, rnm moraes junior, ja rafael – social wasp fauna from canopy and understory amazon area184 the similarities of social wasp species composition between zf-2 station and ducke reserve was not surprising since they are geographically close (50 km apart) and both areas have the same phytophysiognomy characteristics (ombrophilous dense and humid forest). in addition, these two sites, as well as some localities in roraima state, are the only places where canopy fauna was collected. the species composition between alvarães and mamirauá in amazonas and caxiuanã in pará were very similar, with both reserves located in lowland areas. the sites with different phytophysiognomies and that were geographically distant were the most dissimilar sites. the maracá ecological station with savanna formations, serra do divisor with more open and elevated vegetation, rio branco region and the amapá lake region, were the most dissimilar from the zf-2 station. collection efforts were discontinuous in these places. unfortunately, different methods and collection efforts in surveys are complicated, as silveira (2002) pointed out: “comparisons of local fauna by the use of information in collections always confront obstacles arising from unsystematic collecting methodologies. in general, appropriate information about the effort spent in finding a certain number of species is not available, and data about the relative abundance of the species are hardly recoverable”. we hope that this work stimulates other standardized surveys that explore the canopy fauna in the amazon region. finally, in this study we found that the social wasp fauna in the canopy is slightly different from the fauna of the understory, with many similar species in both forest strata. in addition, when comparing the strata, the understory had more diverse fauna than the canopy, while the canopy had some rare and poorly collected species. in this way, we suggest using traps suspended in the amazon biome to collect a high diversity of social wasps. in addition, continuous collection efforts are important for better sampling in a particular area. acknowledgments specimens were collected during a project coordinated by jar and financed by the conselho nacional de desenvolvimento científico e tecnológico (cnpq, process: 407623/2013-2) through the program “rede bia – biodiversidade de insetos na amazônia”. we sincerely thank cnpq for the postdoctoral scholarship (pdj – process number 150029/2017-9) of a. somavilla and research productivity fellowship of j.a. rafael, and marlon breno graça helped with analysis. references barbosa, b.c., 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(2017). social wasps (vespidae: polistinae) from ducke reserve, amazonas, brazil. sociobiology, 64: 125-129. doi: 10.13102/sociobiology.v64i1.1215 zucchi, r., sakagami, s.f., noll, f.b., mechi, m.r., mateus, s., baio, m.v. & shima, s.(1995). agelaiavicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of new york entomological society, 103: 129-137. doi: 10.13102/sociobiology.v62i4.364sociobiology 62(4): 506-512 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a new species and a new record of the ant genus stigmatomma roger (hymenoptera: formicidae) from india introduction based on the comparative study of mandible characters in worker caste, yoshimura and fisher (2012) revived stigmatomma from synonymy within amblyopone. it is widely distributed and currently represented by 63 living species and 2 fossil species (bolton, 2015). noteworthy contributions to this genus include, brown, 1960; taylor, 1979; morisita et al., 1989; terayama, 1989; dlussky et al., 1990; lattke, 1991; atanasov and dlussky, 1992; onoyama, 1999; xu, 2001; lacau and delabie, 2002; xu, 2006; ariaspenna, 2008; terayama, 2009; heterick, 2009; bharti and wachkoo, 2011 and xu, 2012. in india, stigmatomma is represented by 4 species (bharti, 2011; bolton, 2015), s. bellii (forel, 1990), s. rothneyi (forel, 1990), s. minutum forel, 1913 and s. boltoni (bharti & wachkoo, 2011) with recent exclusion of s. pertinax (baroni urbani, 1978) which has been transferred to bannapone (eguchi et al., 2015). here, we describe a new species s. xui sp. n. and also document a new record s. awa (xu & chu, 2012) from india, which we redescribe and formally transfer abstract a new species of the ant genus stigmatomma roger, 1859 collected from north-eastern himalaya is described: stigmatomma xui sp. n. another species stigmatomma awa (xu & chu, 2012) is also reported for the first time from india and is formally transferred from ambylopone to stigmatomma. a key is provided to distinguish the related species. sociobiology an international journal on social insects h bharti, js rilta article history edited by gilberto m. m. santos, uefs, brazil received 07 march 2014 initial acceptance 08 april 2014 final acceptance 13 september 2015 keywords amblyoponinae, stigmatomma, new species, ants, himalaya. corresponding author himender bharti department of zoology and environmental sciences, punjabi university, patiala 147002, india e-mail: himenderbharti@gmail.com from amblyopone to stigmatomma. an identification key to the known species of stigmatomma having eleven segmented antenna is provided herewith. materials and methods the specimens were collected through winkler’s extractor. these were preserved in 70% alcohol and later pinned as per standard procedure in ant taxonomy. the taxonomic analysis was conducted on a nikon smz 1500 stereo zoom microscope.for digital images, evolution mp digital camera was used on the same microscope with auto-montage (syncroscopy, division of synoptics, ltd.) software. later images were cleaned with adobe photoshop cs5 and helicon filter 5. holotype of new species has been deposited in puac (punjabi university patiala ant collection at department of zoology and environmental sciences, punjabi university, patiala, punjab, india) . measurements were recorded in millimeters on nikon smz 1500 stereo zoom microscope. standard measurements and indices follow xu and chu (2012). research article ants punjabi university, patiala, india sociobiology 62(4): 506-512 (december, 2015) 507 tl-total length: the total outstretched length of the individual, from the mandibular apex to the gastral apex. hl-head length: the straight-line length of the head in perfect full-face view, measured from the mid-point of the anterior clypeal margin to the midpoint of the occipital margin. in species where one or both of these margins is concave, the measurement is taken from the mid-point of a transverse line that spans the apices of the projecting portions. hw-head width: the maximum width of the head in full face view, excluding the eyes. sl-scape length: the straight-line length of the antennal scape, excluding the basal constriction or neck. ml-mandible length: the straight-line length of the mandible measured from apex to the lateral base. pw-pronotal width: the maximum width of the pronotum measured in dorsal view. wl-weber’s length: the diagonal length of the mesosoma in profile view, measured from the point at which the pronotum meets the cervical shield to the posterior basal angle of the metapleuron. pl-petiole length: the length of the petiole measured in profile from the anterior process to the posterior most point of the tergite, where it surrounds the gastral articulation. ph-petiole height: the height of the petiole measured in profile from the apex of the ventral (subpetiolar) process vertically to a line intersecting the dorsal most point of the node. dpw-dorsal petiole width: the maximum width of the petiole in dorsal view. gl: gaster length: the length of gaster in lateral view from the anterior most point of first gasteral segment to the posterior most point (excluding sting). ci-cephalic index = hw×100 / hl. si-scape index = sl×100 / hw. lpi-lateral petiole index = ph×100/pl. dpi-dorsal petiole index = dpw×100/pl. acronym of depository puac: punjabi university patiala, ant collection at department of zoology and environmental sciences, punjabi university, patiala, punjab, india. results genus diagnosis: head without mandible rectangular or trapezoidal mesosoma and gaster more or less cylindrical in form; mandibles elongate narrow, pointed and slightly curved at apex, middle to basal part of inner margin of mandible with triangular teeth arranged in two rows, or with more or less bifid teeth arranged in a single row, eye moderate to minute to vestigial, placed at the sides on above or below the mid length of head; antennae 11-12 segmented, filiform, the apex only slightly incrassate. mesosoma narrower than head, promesonotal suture distinct, mesosoma strongly constricted at promesonotal suture and divided in to almost two equal halaves; meso-metanotal sutue effaced; metanotum obliquely truncate posteriorly, basal portion passing into the apical portion by a more or less rounded curve, apical face of metanotum broadened , sides submargined; legs short, robust, tibiae of the posterior legs with two calcaria. petiole cubical, broadly attached to abiii, gaster narrow, not wider than the mesosoma, constriction between the basal two segments deep, giving the basal segment a nodiform appearance; sting exserted. description of new species stigmatomma xui sp. n. fig 1-3. stigmatomma xui sp. n.(worker):1. head in full face-veiw; 2. body in profile veiw; 3. body in dorsal veiw. type material: holotype (worker): india, east sikkim, rorathang, 27°11’49.91”n, 88°36’ 12.44”e, 587m,12.vi.2012, winkler (coll. joginder singh rilta). paratype worker with same data as of holotype [puac]. h bharti, js rilta – a new species and a new record of ant genus stigmatomma from india508 holotype measurements: tl: 1.87; hl: 0.43; hw: 0.34; sl: 0.22; ml: 0.29; pw: 0.21; wl: 0.46; pl: 0.18; ph: 0.19; dpw: 0.20; gl: 0.57; ci: 79; si: 64; lpi: 106; dpi: 112. paratype measurements: tl: 1.94; hl: 0.44; hw: 0.33; sl: 0.21; ml: 0.30; pw: 0.21; wl: 0.49; pl: 0.17; ph: 0.20; dpw: 0.21; gl: 0.54; ci: 75; si: 63; lpi: 117; dpi: 123. head: in full face view, head rectangular (ci:79); occipital margin weakly concave, occipital corners rounded; mandibles narrow and slender, outer margins almost straight except at the apical part with 8 teeth (2 apical and 2 basal teeth are simple; middle 4 are paired arranged in two rows); anterior margin of clypeus convex, with 6 dentiform setae which arise from flat cuticle; median setae fused at base; posttorular flange close to each other, covering antennal insertion; antennae 11 segmented; scape short, not reaching to posterior corner of head (si: 64); antennal segments 3-10 broader than long; segment 11 longer than broad; eyes absent (as seen under stereozoom optical microscope). mesosoma and petiole: in lateral view, mesosoma weakly convex; promesonotal suture distinct; meso-metanotal suture effaced. propodeal dorsum straight, posterolateral corner of propodeum rounded; petiole broadly attached to abiii, petiole quadrate in lateral view with anterior border almost straight, dorsal margin straight; subpetiolar process narrow, oblique with rounded anteroventral corner. in dorsal view, mesonotum constricted, propodeum slightly widened backward, propodeal declivity concave, petiole broader than long (dpi: 112), anterodorsal corner rounded. gaster: first gastral segment broader than long, broader than dorsal petiolar width, second gastral segment broader than the first gastral segment. sculpture: mandibles punctuated with rugosity. head densely punctuated, interfaces appear as micro-reticulation and opaque; dorsal surface of pronotum and mesonotum densely punctured. dorsum of propodeum sparsely punctured. lateral sides of mesonotum and metanotum superficially striated. petiole and gaster sparsely punctuated. pilosity and pubescence, pilosity sparse few erect or suberect hairs on mandibles, apical antennal segments and apex of gaster; dorsal surfaces of head and body with dense decumbent pubescence. tibiae with dense decumbent pubescence, but without suberect hairs. color: reddish brown; antennae, mandibles, legs and tip of gaster yellow. ecology: this species was collected by winkler’s extractor from an undisturbed dense forest. the thickness of leaf litter was about 4 inches. the floor of the forest receives limited sun light. the maximum recorded temperature of the area is 28°c with minimum -1°c and the region receives 325cm of rainfall per annum. etymology: the species is named in honor of prof. zhenghui xu. remarks: this new species most resembles s. sakaii (terayama, 1989), but can be distinguished from the latter by the presence of 6 dentiform setae which arise from flat cuticle; oblique subpetiolar process and rounded posterolateral corner of propodeum and anterodorsal corner of petiole, whilst s. sakaii is characterized by anterior clypeal margin with 8 dentiform setae which arise from tubercle-like cuticular projection; trapezoidal subpetiolar process; bluntly angled posterolateral corner of propodeum and anterodorsal corner of petiole. new record from india stigmatomma awa (xu & chu, 2012) new combination amblyopone awa xu & chu, 2012: 1192, figs. 51-56 (w. q.) china. fig 4-6. stigmatomma awa (worker): 4. head in full faceveiw; 5. body in profile veiw; 6. body in dorsal veiw. material examined: 7 workers: india: arunachal pradesh, lumla, 27°32’49.68”n, 91°43’ 56.99”e, 2800 m, 8.x.2013 winkler method (coll. joginder singh rilta). sociobiology 62(4): 506-512 (december, 2015) 509 1 worker india: arunachal pradesh, lumla, 27°32’49.68”n, 91°43’ 56.99”e, 2800 m, 13.x.2015 winkler method (coll. joginder singh rilta). tl: 6.37-6.71; hl: 0.79 0.86; hw: 0.670.74; sl: 0.410.46; ml: 0.55 0.62; pw: 0.38 0.41; wl: 0.98 1.13; pl: 0.36 0.38; ph: 0.400.43; dpw: 0.43 0.46; gl: 1.10 1.22; ci: 84.34 86.04; si: 61.1962.16; lpi: 111.12 113.15; dpi: 104.87121.05 (workers measured). head: in full-face view, head roughly trapezoidal, widened forward and longer than broad (ci:86.04); occipital margin weakly concave, occipital corners bluntly angled; lateral sides weakly convex, anterolateral corners acutely toothed; mandibles elongate, masticatory margin with a long apical tooth, a short subapical tooth; anterior clypeal margin with 8 dentiform setae, arranged in pairs; antennae short, 12-segmented, apices of scape reach to about 2/3rd of the distance from antennal socket to occipital corners (si:62.16), funiculi incrassate toward apex; eyes small with 3 facets, located behind the midpoint of the lateral sides of head. mesonotum and petiole: in lateral view, pronotum weakly convex, promesonotal suture distinctly notched, mesosoma short and convex, metanotal groove absent, propodeal dorsum straight about 2 times as long as declivity, posterodorsal corner rounded, declivity weakly convex; petiole trapezoidal, dorsal and anterior faces nearly straight, subpetiolar process roughly rectangular, with a large elliptical sub-transparent fenestra, ventral face straight, posteroventral corner rightly angled. in dorsal view, mesonotum constricted, propodeum slightly widened backward, propodeal declivity longitudinally concave; petiole broader than long, (dpi: 121.05) anterior and lateral sides weakly convex. gaster: first gastral segment broader than long, broader than dorsal petiolar width, second gastral segment broader than the first gastral segment. sculpture: mandible longitudinally striate, head densely punctured, interfaces appear as micro-reticulation; pronotum densely punctured, the narrow longitudinal middle strip without punctures, dorsum of mesonotum and propodeum densely punctured; lateral sides of mesonotum and metanotum finely longitudinally striate; petiole and gaster finely sparsely punctured. pilosity and pubescence: dorsal surface of head and body with sparse suberect short hairs, except two pairs of long hairs on anterior clypeal margin and dense decumbent pubescence; tibiae with dense decumbent pubescence, but without suberect hairs. colour: reddish brown, eyes black, antennae and legs yellowish brown. global distribution: palearctic region: china, tibet and india. remarks: stigmatomma awa (xu & chu, 2012) is reported here for the first time from india, earlier known from china and tibet. this species is remarkably different from the other known indian species with following combination of characters: mandibles with 7 teeth; anterior clypeal margin with 8 dentiform setae; eyes small, each with 3 facets; subpetiolar process with elliptical sub-transparent fenestra. this species was originally described in ambylopone and we formally place it in stigmatomma based on the diagnosis provided by yoshimura and fisher (2012). key to the known species of stigmatomma with eleven segmented antenna 1. anterolateral corners of head acutely toothed/genal teeth present (fig a), mesosoma smooth and shiny (fig c) (neotropical region: dominican republic, greater antilles, lesser antilles, type locality-puerto rico)…......... ..................................................... s. falcatum (lattke, 1991). – anterolateral corners of head not toothed/genal teeth absent (fig b), mesosoma variously sculptured from sparsely to densely punctuate (fig d)....................................……..…2 figs a-d. s. falcatum (lattke, 1991). a. anterolateral corners of head acutely toothed; b. anterolateral corners of head not toothed; c. mesosoma smooth and shiny; d. mesosoma variously sculptured from sparsely to densely punctuate. figs e & g. s. sakaii (terayama, 1989): e. head in full-face view; g. body in profile view. figs f & h. s. xui sp. n.: f. head in fullface view; h. body in profile view. h bharti, js rilta – a new species and a new record of ant genus stigmatomma from india510 2. in full face view, head trapezoidal in shape, masticatory margin of mandible with 5 set of paired teeth; in profile view (fig e), posterolateral corner of propodeum angulate, and anterodorsal corner of petiolar node bluntly angled; subpetiolar process trapezoidal (fig g) (palearctic region: japan; oriental region: china, type locality taiwan)...................................... s. sakaii (terayama, 1989). – in full face view, head rectangular in shape, masticatory margin of mandible with 4 set of paired teeth; in profile view (fig f), posterolateral corner of propodeum and anterodorsal corner of petiolar node rounded; subpetiolar process oblique (fig h) (oriental region: india)..................................................................... s. xui sp. n. fig 7-9. images illustrating the measurements used.7. head in fullface view with measuring lines for hl, hw, sl and ml. 8. body in lateral view with measuring lines for wl, ph, pl and gl. 9. body in dorsal view with measuring lines for pw and dpw. fig 10-13. stigmatomma sakaii (worker-image from antwiki):10. head in full face-veiw; 11. body in profile veiw; 12. body in dorsal veiw; 13. label. sociobiology 62(4): 506-512 (december, 2015) 511 fig 14-17. stigmatomma falcatum (worker-image from antwiki):14. head in full face-veiw; 15. body in profile veiw; 16. body in dorsal veiw; 17. 17. label of stigmatomma falcatum. acknowledgments sincere gratitude is due to prof. zhenghui xu, college of forestry, southwest forestry university, yunnan province, china for his valuable suggestions. financial assistance rendered by ministry of environment and forests (grant no. 14/35/2011-ers/re; 22018/41/2010-cs (tax)), govt. of india, new delhi is gratefully acknowledged. we also acknowledge govt. of arunachal pradesh and govt. of sikkim, department of forest, environment and wildlife management for granting permission to collect the material, and for other assistance supporting this research. finally, the authors wish to thank the team of antwiki for images of stigmatomma sakaii and falcatum. references arias-penna, t.m. 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(1991). studies of neotropical amblyopone erichson (hymenoptera: formicidae). contributions in science, 428: 1-7. morisita, m., kubota, m., onoyama, k., ogata, k., terayama, m, kondoh, m. & imai, h.t. (1989). a guide for the identification of japanese ants. 1. ponerinae, cerapachyinae, pseudomyrmecinae, dorylinae and leptanillinae. myrmecological society of japan, tokyo, 42 pp. onoyama, k. (1999). a new and a newly recorded species of the ant genus amblyopone (hymenopetra: formicidae) from japan. entomological science, 2: 157-161. taylor, r.w. (1979). melanesian ants of the genus amblyopone. australian journal of zoology, 26: 823-839. terayama, m. (1989). the ant tribe amblyoponini (hymenopetera, formicidae) of taiwan, with description of a new species. japanese journal of entomology, 57: 343-346. terayama, m. (2009). a synopsis of the family formicidae of taiwan (insecta: hymenoptera). research bulletin of kanto gakuen university, 17: 81-266. xu, z. 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(2012). a revision of male ants of the malagasy amblyoponinae (hymenoptera: formicidae) with resurrections of the genera stigmatomma and xymmer. plos one 7(3): e33325. doi:10.1371/journal.pone.0033325 doi: 10.13102/sociobiology.v66i1.3396sociobiology 66(1): 120-125 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 traditional knowledge and potential use of stingless bees (hymenoptera: meliponinae) in the manantlan sierra, jalisco, mexico introduction stingless bees or meliponines are eusocial bees that produce honey, wax, pollen and geopropoleo (singh, 2016) and that live in warm, tropical or subtropical regions around the world, up to 30º north and south (crane, 1994; michener, 2007). meliponines are relatively harmless because their stinger is atrophied, and so, their management is relatively abstract stingless bees (meliponines) play an important role in ecosystems; they pollinate different plant species, assist in the reproduction and conservation of floral biodiversity and their products can be obtained and sold, with the consequent economic benefit for stingless beekeepers. surveys were conducted to find out how much knowledge inhabitants of two marginalized communities of the manantlan sierra in jalisco, mexico, have on the use and exploitation of stingless bees. in addition, several stingless bee species of this region were captured and identified, and wild nests of those bees were located and recorded in their natural habitats. information about the knowledge and culture of stingless bees in the region was analyzed and based on that as well as on the most abundant species captured, those with more potential are suggested for management in a sustainable manner. unlike other areas of mexico where meliponiculture is practiced, in jalisco there is no record of traditional culture of meliponines. however, a certain level of knowledge and a high degree of interest was found among the respondents for engaging in keeping and managing stingless bees, mainly because their management does not involve the risk of stinging incidents. nine stingless bee species were identified in total. of these, the most abundant were scaptotrigona hellwegeri, friese, trigona fulviventris guérin-méneville, partomona bilineata schwarz, friseomelita nigra lepeletier and nannotrigona perilampoides cresson. it is recommended that studies are conducted to develop management practices for these bee species. the implementation of courses on how to keep these meliponines is also recommended, so that in the future, the inhabitants of these communities can benefit from the integral and sustainable use of stingless bees. sociobiology an international journal on social insects f contreras-escareño1, cm echazarreta g2, e guzmán-novoa3, jo macías-macías4 article history edited by solange augusto, ufu, brazil received 30 april 2018 initial acceptance 02 june 2018 final acceptance 02 december 2018 publication date 25 april 2019 keywords stingless bees, scaptotrigona hellwegeri, trigona fulviventris, partomona bilineata, nannotrigona perilampoides, friseomelita nigra, mexico. corresponding author francisca contreras escareño universidad de guadalajara. centro de investigaciones en abejas departamento de producción agrícola centro universitario de la costa sur independencia nacional nº 151 cp: 48900, autlán, jalisco, méxico. e-mail: francisca.contreras@academicos.udg.mx easy compared to honey bees that have a stinger enabled for their defense (crane, 1992; vit et al., 2013). meliponiculture or the cultivation of stingless bees was an activity of great importance for mesoamerican cultures before the arrival of spaniards to the americas. the mayans in particular, notably developed knowledge on how to manage and exploit different stingless bee species that inhabit the yucatan peninsula, mainly melipona beecheii bennet (weaver & weaver, 1981; 1 universidad de guadalajara, centro universitario de la costa sur, autlán, jalisco, méxico 2 universidad autónoma de yucatán, mérida yucatán, méxico 3 university of guelph, school of environmental sciences, ontario, canada 3 universidad de guadalajara, centro universitario del sur, ciudad guzmán, jalisco, méxico 4 universidad de guadalajara, centro universitario del sur, ciudad guzmán, jalisco, méxico research article bees sociobiology 66(1): 120-125 (march, 2019) 121 ruttner; 1988; echazarreta et al., 1997; quezada-euán, 2005; ramírez et al., 2014). the exploitation of stingless bees in mexico was very important until the demand for honey and wax was no longer supplied by these bees, but by the european honey bee (apis mellifera l), which was introduced to the americas during the seventeenth century. european honey bees quickly surpassed the production of stingless bees, which resulted in a decline in their culture and colonies (calkins & collage; 1975; gonzález, 1989). nowadays, except for their culture by mayan communities, the situation for stingless bees has worsened, due to the environmental deterioration caused by deforestation, competition for food resources with a mellifera, lack of knowledge of management techniques of the stingless bees colonies, as well as the abandonment of the field due to the lack of development opportunities. all this has led this activity to a sharp decline and risk of dissapearence (gonzález-acereto, 2008). the primordial use that the mayans gave to honey from meliponines was as a medicine for therapeutic purposes (quezada-euán & gonzález-acereto; 1994; echazarreta et al., 1997; vit, 2015; singh, 2016). on this regard, there is evidence of inhibitory effects of geopropoleo from stingless bee nests in some types of malignant cancer cells. geopropoleo has antioxidant activity, which could be part of the reason for its anticancer properties (milind et al., 2013). additionally, stingless bees are considered potential pollinators of wild plants and commercially important crops such as achiote (bixa orellana), chayote (sechium edule), coconut (cocos nucifera), carambola (averrhoa carambola), macadamia (macadamia integrifolia), mango (mangifera indica), strawberry (fragaria vesca), rambutan (nephelium lappaceum), tomato (lycopersicon esculentum), cucumber (cucumis sativus), avocado (persea americana), coffee (coffea arabica) and others that have been successfully pollinated by these bees (heard, 1999; slaa et al., 2006; giannini et al., 2012). stingless bees nest in cavities, preferably those of tree trunks (wille, 1983), which allows meliponine keepers to move them to locations near their homes, to keep them, protect them and to obtain their products. in this way, knowledge and tools to manage these bees have been generated over time, as well as ideas and beliefs related to religious festivals, which are still practiced in various settlements (gonzález-acereto, 1989; gonzález-acereto, 2008). meliponines belong to the subfamily meliponinae, which is composed of the meliponini tribes (ayala et al., 1996; ayala, 1999, michener, 2007). at present, about 500 species of stingless bees are known worldwide (crane, 1992; vit, 2015), 70% of which have been described for the americas, although only about 14 species of the melipona genus and 21 of the trigona genus are currently managed. in mexico, 46 species of meliponines have been described (ayala, 1999), and out of these, only the genera melipona and trigona and the subgenera scaptotrigona, plebeia and tetragonisca have been cultured. the richness of stingless bee species in mexico is threatened by problems of deforestation and changes of land use, which result in habitat loss derived from the reduction of nesting sites that are usually hollow trees (wille, 1983; 1993; ramírez et al., 2014; macias-macias et al., 2016). in the state of jalisco, there are no references to the use of stingless bees in the past, which is surprising, because these bees could provide economic and ecological benefits for inhabitants of these regions. therefore, there is opportunity to divulge and promote their use and exploitation (contreras, 2008). according to ayala (1999), 11 species of stingless bees can be found in jalisco. of these, two are endemic in temperate zones that are part of the neovolcanic axis of the sierra madre occidental, between the sierra del tigre, the nevado de colima national park and the sierra de manantlán (19°83’ 31. n,102° 98’31’’ 10°37’53’’ n-103°32’82’’ w and w, 19° 26’ 40’’ n-104°12’57’’ w, respectively). due to the importance and potential development of meliponiculture in the study region, an analysis of the knowledge and use of these types of bees by local inhabitants was conducted. additionally, different species of stingless bees were captured and identified in two communities of jalisco’s manantlan sierra. the data and information on traditional management, as well as the knowledge of the species that are most abundant in the region, will allow to suggest strategies for their sustainable use. materials and methods the study was carried out in the rural communities of cuzalapa and zenzontla, which are considered marginal zones within the manantlan sierra’s biosphere reserve (smbr). the smbr is located in southern coastal region of the state of jalisco. the community of cuzalapa is located between the coordinates 19°26’40” to 19°36’51” north and 104°12’57” to 109°22’49” west. cuzalapa belongs to the municipality of cuautitlán de garcía barragán with an altitude between 550 and 2,260 masl, and a rainfall between 1,500 and 1,700 mm. the climate is warm, with temperatures ranging from 18 to 22 °c, and it is the largest sub-basin within the smbr, with an area of 24,057 ha, of which 17,700 correspond to the reserve. it consists of 40% of valley area and 60% mountainous area, which makes it the largest flat agricultural area, with 307 ha of artificial irrigation and 1,893 ha of natural rain irrigation (jardel, 1992; vázquez et al., 1995). the community of zenzontla belongs to the municipality of tuxcacuesco, jalisco, and it is located northeast of the smbr witin the geographic coordinates 104°04’37” to 104°04’52” north, and 19°34’30” west. it has an area of 4,344 ha, with a semi-warm climate and average annual temperatures between 18 and 22 °c. it has an annual rainfall of 900 mm. this zone presents a very rugged topography with altitudes ranging from 800 masl at the edge of the ayuquila river that runs through zenzontla from northwest to southeast, up to 2,000 masl at the elevations to the north and south of the community (jardel, 1992; vázquez et al., 1995; delcombel et al., 1996). the geographic location of these zones can be seen in figure 1. f contreras-escareño, cm echazarreta g, e guzmán-novoa, jo macías-macías – traditional knowledge and potential use of stingless bees122 to obtain a diagnosis of the knowledge and use of stingless bees by local inhabitants, interviews were conducted according to the requirements for descriptive studies proposed by ibarra et al. (1988) and hernández et al. (1998). fifty questionnaires were applied between the two communities between november 2013 and february 2015. the selection of the interviewees was done completely at random, considering people over 18 years of age, including men and women. from the structured questionnaire, 13 questions were selected with which a descriptive analysis of the answers was carried out. in addition to the above, and with the aim of identifying the species of stingless bees present in the region, specimens were captured in two ways. the first way was to capture bees directly from the nests that were located with the aid of inhabitants of the two communities; nests locations were also recorded. the second way was with the help of an entomological net to trap bees that were on the flowers of plants near the trails that are used by the inhabitants of the area. bees were also collected in 26 transects of 1000 meters long, with a separation of 300 meters between them. transects were drawn at random in the forest areas of the two communities. the captured specimens were sacrificed in lethal chambers with ethyl acetate and mounted in entomological pins for their subsequent identification, which was made based on the keys and descriptions by ayala (1999) for stingless bees from mexico. results of all participants interviewed, 68% mentioned that they had some knowledge of stingless bees, either from talks with their grandparents or parents, or because they have direct contact with them. however, no evidence was found of traditional colony management of stingless bees that had been practiced in the past or in the present. the average age of the interviewees was 45 years, with young people showing little interest in these bees, opposite to older people. ten people had worked with honey bees (a. mellifera), eight with stingless bees and 16 with both. of 100% of the interviewees who knew about honey bees, 24 of them said that they had harvested honey from wild nests of stingless bees without keeping them or taking care of them, and they had never tried to capture a nest. the procedure they follow to obtain honey from stingless bees is to open a hole in the trunk of the tree where the nest is located. to do that, they use an ax or a machete, and in some cases, a chainsaw (usually they do not cut the tree). afterwards, they harvest the honey pots and place them in a container to later separate the honey from the wax. sixteen people claimed to have obtained between 250 and 1000 ml of honey per year in two harvest seasons (aprilmay and november-december). honey is always used for own consumption as a sweetener. despite the fact that stingless bees are not purposely kept and managed by the inhabitants of the manantlan sierra, 80% of respondents expressed enthusiasm and interest in knowing more and in learning about the cultivation and sustainable use of these bees. a total of 60 bee specimens were collected in the study sites, of which nine species were identified: s. hellwegeri, f. nigra, t. fulviventris, p. bilineata, n. perilampoides, lestrimelitta chamelensis, plebeia pulchra, plebeia moureana and plebeia parkeri. the number of specimens collected from each species, the name as they are known in the area and the percentage of total specimens are shown in table 1. fig 1. geographic location of the study areas in jalisco, mexico. scientific name number of specimens common name % of total s. hellwegeri 15 alazana 25 f. nigra 10 alitas blancas o zopilota 16.7 t. fulviventris 10 calzon cuero o jocoquillas 16.7 p. bilineata 10 negritos 16.7 n. perilampoides 5 doncellitas 8.3 l. chamelensis 4 limoncilla 6.7 p. pulchra 2 mosquitos 3.3 p. moureana 2 mosquitos 3.3 p. parkeri 2 mosquitos 3.3 total 60 100.0 table 1. scientific name, number of collected specimens, common name and percentage of all specimens collected for each stingless bee species in the communities of cuzalapa and zenzontla at the manantlan sierra in jalisco, mexico. sociobiology 66(1): 120-125 (march, 2019) 123 the number of wild nests of the different species that were located in the two communities can be seen in table 2. the bee species with the most individuals captured coincided with the species from which more wild nests were located, the majority of which were found housed in hollow trees. so, it was determined that the most abundant species with the greatest potential for cultivation are: s. hellwegeri, t. fulviventris, p. bilineata, f. nigra and n. perilampoides. it, and once harvested, leave the nest open, which results in its destruction due to the attack of various predators that inhabit the region (guadalupe alvarez, pers. com.). the same scenario has been reported from other regions, for example, hendrichs (1941), mentioned that in the state of guerrero, mexico, there were people known as “mieleros” who worked in an inadequate way. they destroyed colonies of stingless bees by knocking down trees where they nested and then, obtained the honey in a rustic way, which resulted in the product beign mixed with dead bees, pieces of wax and wood. likewise, nates-parra and rosso-londoño (2013), mention that in colombia, some species of meliponines are used in situ by honey hunters and local inhabitants to obtain several products. the fact that young people in cuzalapa and zenzontla show little interest in these bees is a situation that seems to be a constant at the national level. in places of mexico where meliponines are still kept, only adults are interested in preserving these traditions and in taking care of stingless bees to obtain their products (gonzález-acereto, 1989; reyesgonzález et al., 2014). however, the fact that 80% of the interviewees showed interest in knowing more about the care and management of these bees, demonstrates the potential they can have with the sustainable use of the most abundant species that were located in the region. the diversity of species recorded in this study can be considered as evidence that stingless bees are an important biotic resource that is poorly exploited in these communities. this warrants the dissemination of techniques to manage and exploit them, just like it is done in other regions of mexico (gonzález-acereto, 2008). although the inhabitants of these communities consider stingless bees to be very delicate, they also consider them harmless because they do not have a functional stinger. this represents an advantage for their culture and use, since they could have them in the yards of their houses without representing any danger to their families. in addition, with the sale of their products they would help support household expenses (rosas & medellín, 1996; nogueiraneto, 1997; gonzález-acereto, 2008). the most abundant species of stingless bees could be managed for the production of honey, geopropoleo, wax, pollen and could also be used as alternative pollinators for crops of economic importance (heard, 1999; slaa et al., 2006). in particular, honey may represent the product with the greatest potential for sale, since in other places (yucatán and puebla) it is marketed for therapeutic and medicinal purposes, to control different conditions of human health (medina, 1997; vit et al., 2015). in other regions of mexico, central america and south america, some of these bee species have been referenced as beign easy to adapt to artificial habitats, as well as for having good breeding characteristics and for an integral use of their products (nogueira-neto; 1997; gonzález-acereto, 2008). the above evidence suggests that there is potential for successfully managing stingless bees if meliponiculture is developed in the area of the southern coast of jalisco, mexico. species nests cuzalapa nests zenzontla total nests s. hellwegeri 6 8 14 t. fulviventris 6 4 10 p. bilineata 0 10 10 f nigra 7 2 9 n. perilampoides 3 2 5 total 22 26 48 table 2. species name and number of nests of stingless bees found in the communities of cuzalapa and zenzontla at the manantlan sierra in jalisco, mexico. discussion according to the surveys results, it is not possible to affirm that cultural and traditional management of meliponines has existed in the past in cuzalapa and zenzontla, jalisco. in mexico, the mayan culture developed a traditional system to keep, manage and exploit stingless bees, by housing their colonies in hollow trunks called jobones. the mayans were the only people who developed sophisticated methods of meliponiculture in the americas (ordetx & espina; 1966, weaver & weaver, 1981; ruttner, 1988; gonzalez-acereto, 2008). another place in mexico where meliponiculture is practiced is in the northern highlands of the state of puebla, where the nahuas cultivate the species scaptotrigona mexicana guérin-meneville, bees that they lodge in clay pots, which are placed one on top of the other and are sealed with ash moistened with water (márquez, 1994; gonzalez-acereto, 2008). in countries such as brazil, posey and camargo (1985) mention the names of 56 species of stingless bees that were recognized by the kayapó natives in the village of gorotire. of these, only nine species are considered “semi-domesticated” or managed in some way by the natives. the nests of these bees are continuously used year after year thanks to the fact that after they open one of them to extract a part of the honey, pollen, wax and brood, they are closed to avoid the attack of predators, which reflects an awareness of the conservation of bees, even if they are not technically cultivated. nogueiraneto (1997) also mentions several south american cultures that make use of native bees for the preservation and reconstruction of wild nests. this situation is different in cuzalapa and zenzontla, because the people who collect honey from a nest, usually do not care much about protecting f contreras-escareño, cm echazarreta g, e guzmán-novoa, jo macías-macías – traditional knowledge and potential use of stingless bees124 the knowledge about stingless bees of the inhabitants of the communities under study is very limited and empirical, unlike that of the mayas in the yucatan peninsula, and that of the nahuas from puebla’s north sierra. although there is no technical exploitation of stingless bees in the communities where this study was conducted, and although they are a resource that inhabitants exploit sporadically, there is interest in venturing into the use of these types of bees. this is why, it is necessary to carry out studies on aspects of the biology, nesting habits and foraging behavior of the most abundant species of stingless bees under the particular conditions of the manantlan sierra. once the above studies are conducted and specific management techniques are developed, the implementation of practical courses on how to keep and manage these bee species will be recommended. such strategy will establish meliponiculture as an alternative for the sustainable use of part of the region’s biodiversity, which may lead to the improvement of the quality of life of the smbr inhabitants. acknowledgments to the inhabitants of the communities of cuzalapa and zenzontla for their selfless collaboration and support in the collection of specimens in the field, especially from mr. guadalupe alvarez. to jorge gonzalez acereto and humberto moo valle for identifying the stingless bee species and to the university of guadalajara’s southern university center for funding this research. authors’ contribution f contreras-escareño and cm echazarreta g conceived the study and wrote the manuscript; f contreras-escareño, e guzmán-novoa, and jo macías-macías, assembled and analyzed the data. references ayala, r. 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(1983). biology of the stingless bees. annals review of entomology, 28: 41-64. doi: 10.13102/sociobiology.v61i3.338-340sociobiology 61(3): 338-340 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 first records of the myrmecophilous fungus laboulbenia camponoti batra (ascomycota: laboulbeniales) from the carpathian basin f báthori1, wp pfliegler2 & a tartally1 the order laboulbeniales comprises more than 2000 species in about 140 genera (santamaria, 2001; weir & blackwell, 2005; kirk et al., 2008). they are obligate ectoparasites of arthropods, and approximately 80% of the described laboulbeniales species parasitize coleoptera species (santamaria, 2001; henk et al., 2003; weir & blackwell, 2005). in the order hymenoptera, only ants are known to be hosts of certain species of laboulbeniales (espadaler & santamaria, 2003). thus far, four species of these fungi have been reported to be associated with ants in europe: rickia wasmannii cavara, 1899, is found in 15 countries on eight myrmica species; laboulbenia formicarium thaxt, 1908, in france, portugal and spain on two lasius species; laboulbenia camponoti batra, 1963, in bulgaria and spain on five camponotus species; and rickia lenoirii santamaria and espadaler, 2014, in greece and france on two messor species (herraiz & espadaler, 2007; lapeva-gjonova & santamaria, 2011; espadaler & santamaria, 2012; santamaria & espadaler, 2014). the effect of these ant parasitic fungi on their hosts is rather understudied except for the work of csata et al. (2014). abstract laboulbenia camponoti batra, 1963 (ascomycota: laboulbeniales), has been found on camponotus aethiops (latreille, 1798) (hymenoptera: formicidae) workers in the carpathian basin: in baziaş, caraş-severin (romania), and vienna (austria). vienna is the northernmost known locality of this fungus (48°12’ n). these new observations expand the area of l. camponoti from regions with mediterranean and subtropical climatic influences to the common borders of the continental and pannonian regions. these results show that camponotus samples from other climatic regions should be examined more closely for this fungal parasite. sociobiology an international journal on social insects 1 department of evolutionary zoology and human biology, university of debrecen, debrecen, hungary 2 department of genetics and applied microbiology, university of debrecen, debrecen, hungary article history edited by kleber del-claro, ufu brazil received 04 july 2014 initial acceptance 29 july 2014 final acceptance 27 august 2014 keywords austria, camponotus aethiops, central-europe, mycology, romania, social parasite corresponding author andrás tartally department of evolutionary zoology and human biology, university of debrecen egyetem tér 1, h-4032 debrecen, hungary e-mail: tartally.andras@science.unideb.hu tel: +36 52 316 666 extension 62349 they found that under laboratorial conditions the lifespan of myrmica scabrinodis nylander, 1846 individuals infected with r. wasmannii was significantly reduced in comparison with the lifespan of uninfected ants. moreover autoand allogrooming increased in infected nests. these facts support the parasitic character of ant-associated laboulbeniales fungi. only r. wasmannii has been reported among these four species in the carpathian basin (tartally et al., 2007). as camponotus aethiops (latreille, 1798) is a relatively common species in this region (csősz & al., 2011; pers. observ.), which is one of the known hosts of l. camponoti (espadaler & santamaria, 2012), we suspected the possibility to record l. camponoti from the carpathian basin. our aim was therefore to prove the presence of l. camponoti within this region by checking museum specimens of c. aethiops. though the other known (espadaler & santamaria, 2012) host ants (c. universitatis forel, 1890; c. pilicornis (roger, 1859); c. sylvaticus (olivier, 1792)) are not known from this region (csősz & al., 2011), we aimed to search for individuals among museum specimens from the carpathian basin. short note sociobiology 61(3): 338-340 (september 2014) 339 the ectoparasitic fungus l. camponoti was found for the first time in romania and austria (see: espadaler and santamaria, 2012 and references therein). the number of countries this fungus is recorded in is now increased from four to six: it has previously been found only in spain, bulgaria, turkey (for a review: espadaler and santamaria, 2012 and references therein) and india (batra, 1963). in its prior known localities, the mediterranean or subtropical climatic influence is strongly expressed. this may have led myrmecologists and mycologists to consider l. camponoti to be distributed solely in such climatic areas. however, the two newly recorded localities are in the common borders of the continental and pannonian regions (see: eea, 2011), and the new locality at vienna is the northernmost (48°12’ n) known latitude of l. camponoti in the world. these facts give a new picture of the potential distribution of this fungus. finding l. camponoti for a new region may call the attention of myrmecologists and mycologists to check camponotus specimens more intensively for the presence of this small and understudied fungus. materials and methods to reveal the presence of l. camponoti, all the specimens of camponotus aethiops (hymenoptera: formicidae) (workers, males, and queens) in the hymenoptera collection of the hungarian natural history museum were examined under an olympus szx9 stereomicroscope at magnifications of 12.6x-114x. no c. universitatis, c. pilicornis or c. sylvaticus specimens were found in this collection from the carpathian basin. pinned specimens of the host that were found to be infested were soaked in 70% ethanol for 5-12 hours and examined using transmissed light under a binocular microscope at 10x magnification. thalli were removed with an insect pin and cleared in lactic acid (12 hours) before being mounted in a pva-glycerol medium and photographed with an olympus digital camera through an olympus bx-40 microscope equipped with 40x and 100x lenses. measurements were taken with the manufacturer’s image acquisition software (dp controller). specimens are deposited in the fungi collection of the hungarian natural history museum on slides (inventory numbers: bp 105023, bp 105024). results and discussion more than 200 c. aethiops specimens were examined, originating from 34 parts of the carpathian basin (sites in hungary, romania, slovakia, austria, and serbia). only three specimens (less than 1.5% of the investigated samples) of c. aethiops workers were found to be parasitized by l. camponoti: two workers from vienna, austria (48°12’ n, 16°22’ e, 180 m a.s.l.), and one from baziaş, romania (44°48’ n, 21°23’ e, 85 m a.s.l.). the fungus grew from the cuticle of different body parts of the workers, mainly on the head and the legs (fig. 1-2). no infested queens or males were found. however, the numbers of queens and males in the museum collection were small. the number of thalli observed on infected camponotus specimens was relatively small. a dozen (mostly immature) thalli were found in two groups on an antenna of one specimen from vienna, while the other worker from the same location had only two immature thalli with developing perithecia (the spore-producing fruiting body of the fungus) on one leg. a single, mature thallus with visible spores was found on the head of the romanian specimen collected at baziaş (fig. 1). variation in the length and number of the sterile appendages was observable, as also noted in the species’ original description (batra, 1963), where explanations of life stages and morphology are also available. fig. 1. laboulbenia camponoti. a. group of thalli on antenna (vienna). b. young immature thallus (vienna). c. immature thallus with developing perithecium (vienna). d. mature thallus (baziaş). legend: p perithecium; sa – sterile appendages (their numbers show individual differences). f báthori et al. laboulbenia camponoti is reported from the carpathian basin340 the inconspicuous nature of l. camponoti has undoubtedly contributed to the scarcity of its distribution records. as illustrated by fig. 2., the thalli are very hard to locate, especially on older museum specimens with dust particles. determination of the fungus must be validated by light microscopy. because european camponotus species are usually large (see e.g. seifert, 2007), and therefore usually easily observed with the naked eye, myrmecologists rarely examine them by microscopy. however, these results demonstrate that a thorough examination of camponotus specimens from other climatic regions may reveal the presence of this little-known parasitic fungus. acknowledgments we would like to thank the numerous collectors whose work provided the samples that we examined. zoltán vas, curator, helped us in our work in the hymenoptera collection of the hungarian natural history museum. bf and at were supported by the ‘antlab’ marie curie career integration grant (of at) within the 7th european community framework programme. at was supported by a ‘bolyai jános’ scholarship of the hungarian academy of sciences (mta). references batra, s. w. t. (1963). some laboulbeniaceae (ascomycetes) on insects from india and indonesia. american journal of botany, 50: 986-992. csata, e., erős, k. & markó, b. (2014). effects of the ectoparasitic fungus rickia wasmannii on its ant host myrmica scabrinodis: changes in host mortality and behavior. insectes sociaux, 61: 247-252. doi: 10.1007/s00040-014-0349-3 csősz, s., markó, b. & gallé, l. (2011). the myrmecofauna (hymenoptera: formicidae) of hungary: an updated checklist. north-western journal of zoology, 7: 55-62. european environment agency (eea) (2011). biogeographic regions in europe. espadaler, x. & santamaria, s. (2012). ectoand endoparasitic fungi on ants from the holarctic region. psyche, 2012 (168478): 1-10. doi: 10.1155/2012/168478 henk, d.a., weir, a. & blackwell, m. (2003). laboulbeniopsis termitarius, an ectoparasite of termites newly recognized as a member of the laboulbeniomycetes. mycologia, 95: 561-564. herraiz, j.a. & espadaler, x. (2007). laboulbenia formicarum (ascomycota, laboulbeniales) reaches the mediterranean. sociobiology, 50: 449-455. lapeva-gjonova a. & santamaria, s. (2011). first record of laboulbeniales (ascomycota) on ants (hymenoptera: formicidae) in bulgaria. zoonotes, 22: 1-6. kirk, p.m., cannon, p.f., minter, d.w. & stalpers, j.a. (eds) (2008). ainsworth and bisby’s dictionary of the fungi (10th edition). cabi europe-uk, cromwell press, trowbridge, 771 p santamaria, s. (2001). los laboulbeniales, un grupo enigmático de hongos parásitos de insectos. lazaroa, 22: 3-19. santamaria, s., & espadaler, x. (2014). rickia lenoirii, a new ectoparasitic species, with comments on world laboulbeniales associated with ants. mycoscience (in press) doi: 10.1016/j. myc.2014.06.006 seifert, b. (2007). die ameisen mittelund nordeuropas. görlitz/tauer: lutra verlagsund vertriebsgesellschaft, 368 p tartally, a., szűcs, b. & ebsen, j.r. (2007). the first records of rickia wasmannii cavara, 1899, a myrmecophilous fungus, and its myrmica latreile, 1804 host ants in hungary and romania (ascomycetes: laboulbeniales, hymenoptera: formicidae) myrmecological news, 10: 123. weir, a., & blackwell, m. (2005). fungal biotrophic parasites of insects and other arthropods. in: f.e. vega & m. blackwell (eds.), insect-fungal associations: ecology and evolution (pp. 119-145). oxford: oxford university press. fig. 2. a laboulbenia camponoti individual on the scapus of a camponotus aethiops worker (vienna), the figure illustrates how meticulous it is to find this small fungus on a large camponotus individual, especially when dust on the host prevents easy recognition doi: 10.13102/sociobiology.v62i3.369sociobiology 62(3): 374-381 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 chemotaxonomic analysis of the venom composition within the ant genus strumigenys (hymenoptera: formicidae) in taiwan introduction the myrmicine ant tribe dacetini includes 9 genera and about 872 species largely distributed in the tropical and subtropical areas (bolton, 2000). dacetine ants are characterized by their pyriform head, their prey-seizure shaped mandibles and a singular spongiform tissue in the petiolar area (bolton, 1999, 2000). within the tribe, strumigenys and pyramica are the most speciose genera, containing 90% of total species (bolton, 2000). in taiwan, all the 27 species of dacetine ants belong to these two genera, but strumigenys ants received comparatively more attention by authors, especially in regard to taxonomy and phylogeny (lin & wu, 1998, 2001; hung et al., 2004). thirteen species of strumigenys are present across the island, nine of which being endemic (lin & wu, 1998, 2001). like all dacetini species, they are predators capturing small arthropods, mostly collembolans, by means of their specialized snap jaws (brown & wilson, 1959; abstract in taiwan, the ant genus strumigenys is represented by 13 species, nine of which being endemic to this island. classic morphological taxonomy can be complex and may lead to equivoque identification within this group. to clarify subtle species assignments, we investigated the venom composition of five strumigenys species, using spme extraction and gc/ms analyses, and searched for a suitable chemical marker. our results indicate that three out of the five species tested showed enough specificity in their chemical profiles to allow clear differentiation. however, the two remaining species could not be distinguished from each other on the basis of their venom composition. we further assessed the phylogenetic relationships between the five species, analyzing both morphological and chemical characters. our clusters revealed congruency between some species associations and suggested that the analysis of venom composition may apply, at least partially, to strumigenys chemosystematics. however, important discrepancies also appeared, signifying that selective pressures for chemical diversification have operated differentially during the speciation and dispersal processes within this genus in taiwan. sociobiology an international journal on social insects ch chien, cc lin article history edited by evandro do nascimento silva, uefs, brazil received 12 march 2014 initial acceptance 22 may 2014 final acceptance 15 september 2014 keywords dacetine ants, spme, venom, gc/ms corresponding author chung chi lin department of biology national changhua university of education 500-01 changhua , taiwan e-mail: cclin@cc.ncue.edu.tw masuko, 1984). colonies nest in rotten wood and under rocks of original or secondary forest litter. personal observations (c.-c. lin) indicate that they hardly contain more than 200 individuals in laboratory and that some species may be polygynous. however, because of their minute size and cryptic habits, field collections proved hazardous and some species are described from one specimen only (lin, 1998; lin & wu, 2001). consequently, their biology and social organization remain poorly investigated. in addition to these impediments, many taiwanese strumigenys species can display a great morphological resemblance, that can only be solved by subtle differences in the pilosity or other characters solely observed under electron microscope (lin, 1998, and see hereafter). this makes their identification complex even for specialists and strongly hampers the conduct of sound experimental studies. in ants, chemical analyses of the diverse exocrine secretions can be a valuable addition to the use of morphological characters to help for research article ants national changhua university of education, changhua city, taiwan sociobiology 62(3): 374-381 (september, 2015) 375 accurate species assignments. for instance, diagnostic suites of compounds have been identified in the secretions of the mandibular, postpharyngeal and dufour glands within the formicine ant genus cataglyphis (keegans et al., 1992; hefetz & lenoir, 1992; dahbi et al., 1996, 2008) and their specificity allowed the assessment of phylogenetic relationships that were in agreement with morphological data. in a same way, venom chemical composition also revealed its diagnostic power within the fire ants complex. similar solenopsis species and their hybrids can be accurately identified through their characteristic amounts of various 2,6-dialkyl piperidine (brand et al., 1973a,b; vander meer et al., 1985; vander meer and lofgren, 1990; dall’aglio-holvorcem et al., 2009) and δ1,6piperideine alkaloids (chen et al., 2010). more generally, an important diversification of both the function and chemistry of the venom has occurred in ants, which provides interesting material for chemotaxonomic analyses (hefetz, 1993). as part of our ongoing taxonomic investigation of the genus strumigenys in taiwan (lin, 1993, 1998; lin & wu, 2001), the composition of worker venom was investigated through gc/ms analyses in order to identify specific chemical markers for five species. we also assessed their phylogenetic relationships using classical taxonomy and finally compared the results obtained from both morphological and chemical analyses to evaluate the usefulness of our method for strumigenys chemosystematics. materials and methods ant collection and laboratory conditions colonies of strumigenys ants belonging to five species (s. chuchihensis, s. formosensis, s. liukueiensis, s. minutula and s. solifontis, respectively) were collected at four different localities in taiwan (table 1, fig 1) by hand searching. each colony contained about 50~150 workers, together with some developing brood and at least one queen. in the laboratory, they were reared in plastered nest, covered by a red plastic plate and kept at 25°c, 60% rh and a 13/11 ld photoperiod. they were fed every three days with collembolans, and water was provided ad libitum. morphological characters state definition the characters state definition and polarities used in this study largely followed prevailing theories of evolutionary change within the tribe dacetini (brown & wilson, 1959; baroni urbani & de andrade, 1994; bolton, 1999, 2000). the characters and character state codes used in the cladistic analysis are defined in table 2. characters include both those with simple binary state and multistate characters. all multistate characters were treated as unordered. if more than one state was present for a given taxon, the character was recorded as ‘0 & 1’ (meaning that the taxon had state 0 and 1). these characters were not weighted in the analysis. chemical analysis solid-phase-microextraction (spme). when applied to physically tiny ants, the chemotaxonomic techniques traditionally used to analyze their cuticular hydrocarbons or exocrine glands secretions require the dissection or sacrifice of dozens of workers, which may hamper any further laboratory experiments on the small and rare colonies of strumigenys. instead, we sampled the venom secreted by workers that were kept alive, using a 7 μm layer polydimethylsiloxane (pdms) fiber (supelco, sigma-aldrich, st louis, mo, usa). ants were initially immobilized on a special device made of foam (fig 2). when their abdomen is stimulated, strumigenys workers protrude their sting and droplets of venom stand out. the spme fiber was carefully thrust into the droplets to collect the venom. for each sample, we pooled the venom from three different species locale collection time colonies s. chuchihensis 1 2009/2011 1/ 1 s. minutula 2 2009 5 s. formosensis 2 2009 6 s. liukueiensis 3 2008/2011 1/ 3 s. solifontis 4 2009 3 table 1. summary of the respective dates and locales of collections for the five strumigenys species (cf. fig 1 for details of taiwan geography). fig 1. collecting sites of strumigenys ants. 1: fushan, ilan county; 2: pakuashan, changhua city; 3: chichi, nantou county; 4: shuisheliao, chiai county. fig 2. device used for ant immobilization (a) before spme venom extraction (b). ch chien, cc lin chemotaxonomy of strumigenys ants376 individuals. the spme fiber was then immediately desorbed in the injection port of a gas chromatograph. three samples were performed for each colony. gc/ms analyzes. venom composition was analyzed on a varian gc 3800 gas chromatograph coupled with varian saturn 2200 turbo mass (operating at 70ev, former varian inc. now agilent, palo alto, ca, usa). the gc/ms was fitted with a vf-5ms 5% phenyl 95% dimethylpolysiloxane capillary column (30 m x 0.25 mm). helium (purity 99.995%) was used as the carrier gas at 1.0 ml/min flow rate. the spme fiber was directly inserted into the injection port of gas chromatograph which was set at 2500c in split/splitless mode. the venom samples were run using the following temperature program from 1200c (5 min. initial hold) to 180°c at 10°c min-1(3 min. intermediate hold), then to 2400c at 10°cmin1 (10 min. second intermediate hold), and finally to 2800c at 50cmin-1 (6 min. final hold). the standard of the series n-alkane (c10~c22) was used to determine retention indexes (ri) (kováts, 1965). our objective in this study was to compare the chemical profiles of the five strumigenys species, not to provide absolute identifications of all of the different compounds that were found. statistical analysis all total ion chromatograms were processed through the ms workstation 6.9 (varian inc.). differences in the chemical profiles were assessed using principle component analysis (pca) for all peaks integrated. morphological and chemical patterns of the different species were further analyzed on separate clusters obtained from the respective coefficient of correlation matrices (single-link method, 1-pearson r). for the analysis, each chromatogram was divided into three sections while assigning a value to each peak according to its relative area (ra = target peak area / total peak area): peaks ranging from 0 to 10%, 10-50%, and >50% were assigned values of 1-3, respectively (modified from dahbi et al., 1996). all statistics were performed with statistica 7 (statsoft inc., tulsa, ok, usa). results morphological study the status of the characters for the various strumigenys species and the ensuing cluster analysis (fig 3) revealed three assemblies of species. a first clade includes the two species s. liukueiensis and s. solifontis. a second clade is composed of the two species s. chuchihensis and s. minutula. finally, placed at the root of the clustering, s .formosensis appears distinct from the other four species. our results match those obtained by lin (1998), who, analyzing a total 12 taiwanese and 5 japanese strumigenys ants, also classified these five species in three different groups, but in a slightly different way: the two species s. solifontis and s. liukueiensis were identically 1. number of teeth or denticles of apical fork of mandible (w, q): (0) 4; (1) 3; (2) 6. 2. preapical teeth of mandible (w, q): (0) spiniform; (1) reduced. 3. mandible shape (w, q): (0) hook like; (1) sickle like. 4. anterior clypeal margin (w, q): (0) transverse; (1) deeply concave medially. 5. long flagellate hair on margin of antennal acrobe (w, q): (0) absent; (1) present. 6. flagellate hair on genae (w, q): (0) absent; (1) present. 7. a row of erect hairs on occiput (w, q): (0) absent; (1) present. 8. pilosity type of carnium (w, q): (0) spatulate ; (1) erect; (2) flagellate; (3) spoon-shaped 9. hair on pronotal humeli (w): (0) absent; (1) flagellate; (2) erect; (3) columnar. 10. lateral marginal hair on mesonotum (w): (0) absent; (1) flagellate; (2) erect; (3) columnar. 11. flocculus hairs on pronotum (w): (0) absent; (1) sparse; (2) numerous. 12. sculpture on mesopleuron (w): (0) absent; (1) present. 13. sculpture on propodeum (w): (0) smooth; (1) microreticulate; (2) lacunous. 14. propodeal teeth (w, q): (0) well developed; (1) sub-spongiform ; (2) spongiform. 15. pilosity type on pedicel (w, q): (0) spatulate ; (1) erect; (2) flagellate; (3) spoon-shaped 16. density of hairs on fourth abdominal tergum (w, q): (0) sparse; (1) numerous. 17. mandiblular index, mi (q, w): (0) mi > 40; (1) mi < 40. 18. number of ommatidia (w): (0) > 10; (1) 6 ~ 10; (2) < 6. 19. ocellus (q): (0) large; (1) small. 20. mesonotum (q): (0) convex; (1) even. 21. pilosity type on tergum of gaster (w, q): (0) erect; (1) flagellate; (2) columnar; (3) spoon-shaped. 22. sculpture on lateral surfaces of pronotum (w): (0) smooth; (1) sculpture on part; (2) sculpture presnet on all. 23. total body length, tl (w): (0) > 3 mm; (1) 2 mm ~ 3 mm; (2) < 2 mm. table 2 morphological characters and their states used for the phylogenetic analysis of the genus strumigenys. (q: queen, w: worker). fig 3. cluster analysis based on the morphological characters. (single link, 1-pearson r). (s.for: s. formosensis; s.min: s. minutula; s.chu: s. chuchihensis; s.liu: s. liukueiensis; s.soli: s. solifontis). sociobiology 62(3): 374-381 (september, 2015) 377 the result of the cluster analysis using the assigned values for all integrated peaks is shown in fig 6. as in the morphological analysis (fig 3), three clades were revealed, but important discrepancies in their respective composition appeared. s. formosensis was clustered with s. solifontis, while s. chuchihensis and s. liukueiensis were associated, s. minutula being clearly distinguished from the other four species. clustered in a same solifontis group, confirming their close phylogenetic relation, whereas s. chuchihensis was included in their sister lewisi group. however, s. formosensis and s. minutula were both integrated in a distinct “tropical” group of strumigenys ants. later, in their description of the newlydiscovered s. chuchihensis, lin & wu (2001) finally considered this species closely related to those of the godeffroyi group, which includes, among others, s. minutula, s. solifontis and s. luikueiensis while s. formosensis was included in the mayri sister group of strumigenys species (bolton, 2000). chemical study for the five strumigenys species, a total of 16 constituents were detected in our chemical analysis of the workers venom composition (fig 4). according to their respective retention index and spectrum (table 3), we suggest that these compounds may be terpenes (and more precisely sesquiterpenes for most of them) but this will need further analyses to be clearly confirmed. nevertheless, they were present in sufficient quantities in our samples to allow for a comparison between species. despite important similarities as for the number and identity of constituents, as well as for the presence of a major peak (ra>50%) in all chemical profiles, some qualitative and quantitative differences between species were nonetheless revealed. first, none of each 16 compounds were commonly present in all venoms. in particular, the identity of the major constituents varies across the different profiles. the their secondary constituents (peaks 7 and 11, respectively). the pca analysis also shows that the chemical composition of the workers venom appears relatively constant for each of these three species. in comparison, the venoms of the two remaining species, s. liukueiensis and s. chuchihensis, show a distinct composition, but our chemical analysis was insufficient to distinguish them from each other. they both share the same major compound (peak 15), in similar proportions, and present several minor constituents in common. fig 4. gas chromatograms of venom revealing 16 different compounds across the five strumigenys species. fig 5. pca analysis based on the peaks’ relative areas (ras) (○: s. formosensis; ●: s. solifontis; ▲: s. minutula; △: s. chuchihensis; +: s. liukueiensis ). same is true concerning their respective abundance, ranging from 53.60% to 93.61% respectively (table 3). the pca illustrates the relative specificity of the various venom compositions (fig 5). three species present clearly distinct profiles. venom in s. minutula is mainly characterized by its major constituent (peak 14, absent in all other profiles), while s. formosensis and s. solifontis venoms share the same major compounds (peak 16) but in different proportions (table 3). these two species are further separated on the basis of fig 6. cluster analysis based on the chemical characters. (single link, 1-pearson r). (s.for: s. formosensis; s.min: s. minutula; s.chu: s. chuchihensis; s.liu: s. liukueiensis; s.soli: s. solifontis). ch chien, cc lin chemotaxonomy of strumigenys ants378 p ea k no . r t r a (% ) ± s .d . va lu es r i sp ec tr um s. fo rm os en si s 4 11 .9 2 1. 46 ± 0 .2 2 1 14 30 41 ( 40 ), 5 5 (1 00 ), 6 7 (1 9) , 7 9 (3 8) , 9 1 (3 7) , 1 07 ( 87 ), 1 21 ( 26 ), 1 33 (2 9) , 1 48 ( 49 ), 1 61 ( 22 ), 1 75 ( 4) , 1 89 ( 14 ), 2 17 ( 12 ), 2 46 ( 3) 7 12 .2 2 4. 03 ± 0 .3 4 1 14 48 41 ( 42 ), 5 5 (1 00 ), 6 7 (3 7) , 7 9 (5 0) , 9 1 (2 4) , 1 07 ( 37 ), 1 19 ( 17 ), 1 33 (1 9) , 1 47 ( 25 ), 1 61 ( 31 ), 1 75 ( 5) , 1 89 ( 12 ), 2 17 ( 9) , 2 46 ( 6) 15 13 .7 7 1. 37 ± 0 .0 7 1 15 34 41 ( 45 ), 5 5 (8 3) , 6 7 (2 6) , 7 9 (3 8) , 9 1 (4 7) , 1 07 ( 10 0) , 1 19 ( 47 ), 1 35 ( 23 ), 1 47 ( 31 ), 1 61 ( 13 ), 1 75 ( 5) , 1 89 ( 15 ), 2 17 ( 15 ), 2 46 ( 4) 16 14 .0 4 92 .1 2 ± 0. 19 3 15 47 41 ( 38 ), 5 5 (6 5) , 6 7 (2 1) , 7 9 (3 2) , 9 1 (3 3) , 1 07 ( 10 0) , 1 21 ( 47 ), 1 35 ( 23 ), 1 47 ( 21 ), 1 63 ( 17 ), 1 75 ( 4) , 1 89 ( 17 ), 2 17 ( 15 ), 2 46 ( 5) s. m in ut ul a 3 11 .8 8 1. 07 ± 0 .1 4 1 14 27 41 ( 50 ), 5 5 (3 1) , 6 9 (5 7) , 7 9 (9 1) , 9 1 (6 0) , 1 07 ( 64 ), 1 19 ( 42 ), 1 37 ( 37 ), 1 47 ( 25 ), 1 61 ( 11 ), 1 75 ( 2) , 1 89 ( 7) , 2 03 ( 13 ), 2 17 ( 2) 7 12 .2 0 0. 75 ± 0 .1 7 1 14 47 41 ( 30 ), 5 5 (1 00 ), 6 7 (3 4) , 7 9 (4 2) , 9 1 (2 0) , 1 07 ( 31 ), 1 19 ( 12 ), 1 33 (1 5) , 1 47 ( 22 ), 1 61 ( 23 ), 1 75 ( 2) , 1 89 ( 10 ), 2 17 ( 11 ), 2 46 ( 5) 9 12 .4 1 3. 84 ± 0 .3 6 1 14 59 41 ( 52 ), 5 5 (1 00 ), 6 7 (2 6) , 7 9 (5 8) , 9 3 (7 0) , 1 07 ( 43 ), 1 19 ( 23 ), 1 33 ( 23 ), 1 51 ( 32 ), 1 61 ( 4) , 1 75 ( 8) , 2 03 ( 13 ), 2 17 ( 1) , 2 32 ( 2) 14 13 .5 5 93 .6 1 ± 0. 77 3 15 23 41 ( 56 ), 5 5 (1 00 ), 6 7 (4 4) , 7 9 (7 6) , 9 1 (4 9) , 1 07 ( 70 ), 1 19 ( 43 ), 1 33 ( 26 ), 1 47 ( 26 ), 1 51 ( 35 ), 1 63 ( 15 ), 1 75 ( 6) , 1 89 ( 10 ), 2 17 ( 21 ), 2 46 ( 4) s. c hu ch ih en si s 2 11 .8 1 3. 23 ± 0 .3 4 1 14 23 41 ( 71 ), 5 5 (6 4) , 6 9 (3 2) , 7 9 (3 7) , 9 3 (5 7) , 1 07 ( 10 0) , 1 21 ( 93 ), 1 35 ( 42 ), 1 50 ( 36 ), 1 63 ( 37 ), 1 75 ( 7) , 1 89 ( 31 ), 2 17 ( 8) , 2 33 ( 8) , 2 46 ( 7) 6 12 .0 7 2. 78 ± 0 .0 6 1 14 38 41 ( 78 ), 5 5 (4 9) , 6 9 (3 5) , 7 9 (4 4) , 9 1 (6 8) , 1 07 ( 10 0) , 1 19 ( 80 ), 1 35 ( 15 ), 1 47 ( 37 ), 1 61 ( 9) , 1 75 ( 5) , 1 89 ( 5) , 2 03 ( 10 ), 2 17 ( 4) , 2 32 ( 4) 7 12 .2 3 18 .4 5 ± 1. 29 2 14 49 41 ( 31 ), 5 5 (1 00 ), 6 7 (3 7) , 7 9 (5 0) , 9 1 (2 5) , 1 07 ( 34 ), 1 19 ( 15 ), 1 33 (2 2) , 1 47 ( 30 ), 1 61 ( 30 ), 1 75 ( 4) , 1 89 ( 13 ), 2 17 ( 10 ), 2 46 ( 6) 12 12 .9 3 12 .4 9 ± 1. 05 2 14 91 41 ( 45 ), 5 5 (4 7) , 6 9 (1 8) , 7 9 (2 0) , 9 1 (4 4) , 1 07 ( 86 ), 1 21 ( 93 ), 1 35 ( 36 ), 1 47 ( 9) , 1 63 ( 10 0) , 1 75 ( 2) , 1 89 ( 14 ), 2 17 ( 8) , 2 31 ( 2) , 2 46 ( 4) 15 13 .8 0 53 .6 0 ± 2. 21 3 15 36 41 ( 43 ), 5 5 (6 4) , 6 7 (2 3) , 7 9 (3 0) , 9 1 (4 5) , 1 07 ( 10 0) , 1 19 ( 66 ), 1 33 ( 17 ), 1 47 ( 36 ), 1 63 ( 13 ), 1 75 ( 3) , 1 89 ( 12 ), 2 17 ( 13 ), 2 32 ( 1) , 2 46 ( 3) 16 14 .0 9 3. 97 ± 0 .4 1 1 15 50 41 ( 48 ), 5 5 (9 4) , 6 7 (2 8) , 7 9 (4 0) , 9 1 (4 9) , 1 07 ( 10 0) , 1 21 ( 38 ), 1 35 ( 23 ), 1 47 ( 24 ), 1 63 ( 14 ), 1 75 ( 4) , 1 89 ( 11 ), 2 17 ( 11 ), 2 46 ( 1) s. li uk ue ie ns is 2 11 .8 3 3. 84 ± 0 .5 2 1 14 24 41 ( 45 ), 5 5 (5 2) , 6 9 (2 3) , 7 9 (3 1) , 9 1 (5 3) , 1 07 ( 10 0) , 1 21 ( 82 ), 1 35 ( 38 ), 1 50 ( 32 ), 1 63 ( 40 ), 1 76 ( 3) , 1 89 ( 30 ), 2 17 ( 10 ), 2 33 ( 15 ), 2 47 ( 5) 7 12 .2 5 3. 18 ± 1 .5 7 1 14 50 41 ( 28 ), 5 5 (1 00 ), 6 7 (3 4) , 7 9 (4 1) , 9 5 (2 4) , 1 07 ( 30 ), 1 19 ( 9) , 1 33 (1 9) , 1 47 ( 19 ), 1 61 ( 24 ), 1 73 ( 4) , 1 89 ( 9) , 2 17 ( 10 ), 2 46 ( 4) 12 12 .9 5 14 .3 2 ± 0. 41 2 14 93 41 ( 44 ), 5 5 (5 1) , 6 9 (1 9) , 7 9 (2 4) , 9 1 (4 5) , 1 07 ( 95 ), 1 21 ( 87 ), 1 35 ( 35 ), 1 47 ( 9) , 1 63 ( 10 0) , 1 75 ( 2) , 1 89 ( 16 ), 2 17 ( 7) , 2 31 ( 2) , 2 46 ( 4) 13 13 .0 9 4. 76 ± 1 .6 0 1 15 01 41 ( 60 ), 5 7 (1 00 ), 7 1 (6 9) , 8 5 (4 5) , 9 1 (1 2) , 9 7 (1 0) 15 13 .8 2 61 .5 2 ± 2. 02 3 15 37 41 ( 39 ), 5 5 (6 8) , 6 7 (1 8) , 7 9 (3 0) , 9 1 (4 4) , 1 07 ( 10 0) , 1 19 ( 60 ), 1 33 ( 14 ), 1 47 ( 35 ), 1 63 ( 11 ), 1 75 ( 3) , 1 89 ( 10 ), 2 17 ( 11 ), 2 31 ( 1) , 2 46 ( 2) 16 14 .1 1 4. 16 ± 0 .3 1 1 15 51 41 ( 50 ), 5 5 (9 3) , 6 7 (3 6) , 7 9 (5 6) , 9 1 (6 6) , 1 07 ( 10 0) , 1 21 ( 42 ), 1 35 ( 24 ), 1 47 ( 19 ), 1 63 ( 14 ), 1 75 ( 3) , 1 89 ( 10 ), 2 17 ( 12 ), 2 46 ( 4) s. s ol ifo nt is 1 11 .2 5 1. 71 ± 0 .4 3 1 13 87 41 ( 74 ), 5 5 (2 8) , 6 9 (3 9) , 7 9 (4 6) , 9 1 (6 6) , 1 07 ( 10 0) , 1 21 ( 40 ), 1 33 ( 44 ), 1 47 ( 15 ), 1 61 ( 6) , 1 75 ( 13 ), 2 05 ( 6) 5 11 .9 8 1. 43 ± 0 .0 6 1 14 33 41 ( 38 ), 5 5 (1 00 ), 6 7 (2 1) , 7 9 (3 8) , 9 1 (3 6) , 1 07 ( 87 ), 1 21 ( 29 ), 1 33 ( 27 ), 1 48 ( 39 ), 1 61 ( 19 ), 1 75 ( 5) , 1 89 ( 8) , 2 17 ( 10 ) 8 12 .3 0 2. 80 ± 0 .6 7 1 14 53 41 ( 76 ), 5 5 (2 5) , 6 9 (3 3) , 7 9 (3 6) , 9 1 (5 5) , 1 07 ( 10 0) , 1 21 ( 37 ), 1 36 ( 15 ), 1 47 ( 22 ), 1 63 ( 10 ), 1 75 ( 4) , 1 89 ( 10 ), 2 03 ( 12 ), 2 17 ( 3) , 2 32 ( 4) 10 12 .5 7 0. 93 ± 0 .0 1 1 14 69 41 ( 53 ), 5 5 (8 2) , 6 7 (3 0) , 7 9 (3 7) , 9 1 (5 8) , 1 07 ( 10 0) , 1 21 ( 40 ), 1 33 ( 68 ), 1 49 ( 9) , 1 61 ( 6) , 1 75 ( 11 ), 1 89 ( 4) , 2 03 ( 4) , 2 17 ( 3) , 2 32 ( 4) 11 12 .7 8 15 .2 5 ± 1. 99 2 14 82 41 ( 39 ), 5 5 (6 5) , 6 7 (1 7) , 7 9 (3 4) , 9 1 (5 4) , 1 07 ( 10 0) , 1 21 ( 38 ), 1 33 ( 37 ), 1 49 ( 11 ), 1 61 ( 5) , 1 75 ( 10 ), 1 89 ( 1) , 2 03 ( 5) , 2 17 ( 2) , 2 32 ( 4) 15 13 .8 4 2. 59 ± 0 .2 1 1 15 38 41 ( 44 ), 5 5 (6 8) , 6 7 (2 3) , 7 9 (2 9) , 9 1 (4 6) , 1 07 ( 10 0) , 1 19 ( 63 ), 1 33 ( 15 ), 1 47 ( 35 ), 1 63 ( 12 ), 1 75 ( 3) , 1 89 ( 11 ), 2 17 ( 15 ), 2 46 ( 3) 16 14 .1 2 72 .1 7 ± 2. 11 3 15 51 41 ( 42 ), 5 5 (7 0) , 6 7 (2 4) , 7 9 (3 9) , 9 1 (4 1) , 1 07 ( 10 0) , 1 21 ( 42 ), 1 35 ( 24 ), 1 47 ( 18 ), 1 63 ( 14 ), 1 75 ( 3) , 1 89 ( 14 ), 2 17 ( 13 ), 2 46 ( 5) t ab le 3 r es ul ts o f th e g c /m s an al ys is f or t he fi ve s tr um ig en ys s pe ci es . ( p ea k no : pe ak n um be r, i n re fe re nc e to f ig . 4 ; r t: r et en tio n tim e; r a ± s .d . = m ea n re la tiv e ar ea ( % ) ± s. d .; r i: r et en tio n in de x) . sociobiology 62(3): 374-381 (september, 2015) 379 discussion taiwan relative geographic isolation and mountainous relief led to the development of an important endemism within its ant fauna (forel, 1912; terayama & kubota, 1989; lin, 1993, 1998). for instance, among dacetine ants, nine out of 13 species of strumigenys are only found on this island or surrounding islets. the present study aimed at providing a suitable chemical marker to elucidate subtle species assignments within this genus. for this purpose, we analyzed the venom composition of strumigenys workers that were maintained alive during the extraction process. we directly sampled droplets oozing from their extruded stings, which, as a result, contained a mixture of poison and dufour glands secretions. the gc/ms analysis suggested that most of the compounds revealed are terpenes, which are frequent constituents of ant exocrine secretions, often present in dufour gland (francke & schulz, 2010; morgan, 2008). the functions of dufour secretions are diverse and only fragmentarily understood but when experiments have been conducted, they revealed to act as semiochemicals rather than noxious secretions used for predation or colony defense (billen & morgan, 1998; morgan, 2008). in some species, they can be the medium for the trail pheromone (ritter et al., 1977; vander meer et al., 1981, 1988; alvarez et al., 1987), or used to mark a territory or delimit a home range (attygalle et al., 1983a,b). in others, the dufour gland can be the source of alarm and/ or propaganda substances (daloze et al., 1991; ruano et al., 2005). conversely to noxious secretions, these communicative chemicals are usually species-specific in their composition, hence subjected to selective pressures to diversify (hefetz, 1993). interestingly, our chemical analysis revealed that the venom of strumigenys workers offers diagnostic suites of constituents, at least for some species. three out of the five species used in this study (s. minutula, s. formosensis and s. solifontis) could be clearly separated in the pca analysis. they showed sufficient differences, both in the identity and relative abundance of their main compounds, to permit unambiguous species assignment. however, the remaining two species (s. chuchihensis and s. liukueiensis) presented similar chemical profiles and could not be distinguished on the basis of their venom composition. alternately, chemical convergence in exocrine secretions might occur when species share identical ecological conditions. more likely however, similar venom composition may reflect a lack of selective pressure for chemical diversification between sister species (hefetz, 1993; dahbi et al., 1996). in an attempt to clarify the situation, we assessed the phylogenetic relationships among the five strumigenys species. results of our morphological analysis are in agreement with those of earlier cladistic studies (lin, 1993, 1998; lin & wu, 2001; bolton, 1999, 2000), confirming the phylogenetic proximity between s. chuchihensis, s. liukueiensis, s. minutula and s. solifontis. we thus support the view that these four species may belong to the same “godeffroyi“ group, whereas s. formosensis appears rather different and should reasonably be considered as member of the distinct “mayri” group. these species associations are further supported by genetic analysis. hung et al. (2004) compared the size of internal transcribed spacer 2 (its2) rdna sequences to investigate the phylogenetic relationships within strumigenys ants in taiwan and japan. their results indicated that s. chuchihensis, s. minutula and s. solifontis belonged to a same clade, while s. formosensis was included into a distinct, monophyletic group, together with other species endemic to taiwan or surrounding islets. importantly, for this study s. chuchihensis and s. liukueiensis were sampled in distant locales, respectively in the northern and central part of taiwan (fig 1). previous collections concur to suggest that both endemic species occur in distinct areas and, altogether, evoke an allopatric speciation process between these two sister species (lin, 1993, 1998). were this hypothesis to be confirmed by additional biogeographic studies, the absence of ecological competition could imply a lack of any selective pressure to diversify their dufour gland secretions. the great similarity in their chemical profiles might therefore reflect the ancestral composition within the “godeffroyi“ group. this suggestion should be further tested on other semiochemicals, in particular cuticular hydrocarbons which are involved in nestmate recognition. a significant discrepancy between our two clusters concerns the chemical separation of s. minutula from the three other species of the “godeffroyi“ group. in the same time s. formosensis, though morphologically distinct, became closely associated with s. solifontis, and, at a lesser degree, with the two remaining “godeffroyi“ species. this important shift in species arrangements strongly suggests that ecological constraints have shaped the composition of dufour secretions in these strumigenys ants during their dispersal in taiwan (hefetz, 1993; dahbi et al., 1996). indeed, conversely to s. chuchihensis and s. liukueiensis, the three other species show a broader geographic repartition and can occur sympatrically on taiwan main island (lin, 1998). given their overlapping distributions and similar ecological niches, these closely related species have to maintain distinct exocrine compositions to preserve strict behavioral barriers, as illustrated by the pca analysis of the venom profiles. nevertheless, our understanding of the biogeographic history of strumigenys ants in taiwan is too fragmented to allow deducing the ancestral venom composition from these five chemical profiles, and further inferring the way they diversified. for instance, three species used in the present study are endemic to taiwan but belong to separate clades (s. chuchihensis and s. liukueiensis on one hand and s. formosensis on the other hand) and consequently do not share direct common ancestor. this provides interesting material to investigate the origin, dispersal, speciation and extinction processes within this genus in taiwan. from then on, it will be possible to speculate about the putative ancestral dufour composition and clarify the implications of our results in strumigenys chemosystematics. ch chien, cc lin chemotaxonomy of strumigenys ants380 finally, chemical analyses of workers venom secretions revealed to be a complementary but limited addition to the use of morphological characters for strumigenys taxonomy in taiwan. more experiments are needed to explore and compare the usefulness of other glandular secretions as phylogenetic indicators. references alvarez, f.m., vander meer, r.k. & lofgren, c.s. 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(1988). isolation of the trail recruitment pheromone of solenopsis invicta. journal of chemical ecology, 14: 825-838. vander meer, r.k. & lofgren, c.s. (1990). chemotaxonomy applied to fire ant systematics in the united states and south america. in r.k. vander meer, k. jaffe & a. cedeno (eds.), applied myrmecology: a world perspective (pp. 75–84). westview press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.2705sociobiology 66(1): 33-43 (march, 2019) ant community evolution according to aging in brazilian cocoa tree plantations introduction agricultural expansion is frequently blamed for significant reduction in local biodiversity (norris et al., 2010), although many species manage to survive in habitats that are disturbed or subjected to agricultural processes (lugo, 1988). however, a few agroforestry systems are rather similar to and act as natural forest ecosystems (michon & de foresta, abstract agriculture is frequently held accountable for the depletion of biotic diversity, although a few agroforestry systems support the conservation of a number of organisms. cocoa farming is noteworthy as an example of an agricultural activity that benefits or maintains species richness. however, the mechanism by which the biodiversity persists throughout the entire process of plant development remains obscure. in southeastern bahia, brazil, cacao tree plantations support the conservation of a large amount of organisms native to the atlantic forest, between them the ants. this study aims at recording the relationship between cocoa tree development and ant community structure. the experiment was carried out in a series of six cocoa tree plantations aged one, three, four, eight, fifteen and 33 years, distributed across the experimental grounds of the cocoa research center at ilhéus. 1,500 ant samples were collected using the sampling techniques: hand collection, honey and sardine baits, entomological blanket and “pitfall”. highest values for diversity and richness were reported in the 15-years-old cocoa plantation. no significant correlations between diversity, richness or plant age were reported. considering the faunistic composition, a statistical similarity was observed between the plantations close in age to one another. plant aging did not exert any influence on the diversity gradient and richness in the succession process of the ant community. in young plantations, there are low differences between the ants found on the ground and the ones found on the young cocoa trees. in older plantations, the ant community divides in two distinct assemblages on the ground and on the trees. the variations observed in the ant community along the plant development were likely caused by the structural organization of the dominant species mosaic. sociobiology an international journal on social insects es conceição1, tmc della lucia2, ao costa-neto3, es araujo4, eba koch5,6, jhc delabie6,7 article history edited by gilberto m. m. santos, uefs, brazil received 06 december 2017 initial acceptance 21 april 2018 final acceptance 01 november 2018 publication date 25 april 2019 keywords theobroma cacao, formicidae, diversity, tree growth, perennial crop, agroforestry. corresponding author eltamara souza da conceição departamento de ciências exatas e da terra universidade do estado da bahia (uneb) cp 59, cep 48.040-210 alagoinhas-ba, brasil. e-mail: econceicao@uneb.br 1995; delabie et al., 2007). in such areas, many species are able to survive in the landscape even after the native forest becomes locally extinct, particularly at times when the following characteristics come together: planted species, natural secondary vegetation and proximity to remnants of native vegetation (henriques, 2003; cassano et al., 2009). southeastern bahia is one of the largest cocoa growing regions in brazil, with the structure of the arboreal ant 1 departamento de ciências exatas e da terra, universidade do estado da bahia, bahia, brazil 2 departamento de biologia animal, universidade federal de viçosa. viçosa-mg, brazil 3 departamento de ciências biológicas, universidade estadual de feira de santana, feira de santana-ba, brazil 4 programa de pós-graduação em zoologia, universidade estadual de santa cruz, ilhéus-ba, brazil 5 programa de pós-graduação em ecologia e biomonitoramento, universidade federal da bahia, salvador-ba, brazil 6 laboratório de mirmecologia, cepec/ceplac, itabuna-ba, brazil 7 departamento de ciência agrárias e ambientais, universidade estadual de santa cruz, ilhéus-ba, brazil research article ants es conceição et al. – ant community evolution aging34 communities rather identical to that of the atlantic forest, because of the occurrence of the cacao trees and their shading trees, frequently associated with undergrowth (majer & delabie, 1993; roth et al., 1994; delabie et al., 2000; delabie & mariano, 2001; delabie et al., 2007; cassano et al., 2009; conceição et al., 2015). in this region, the “cabruca” (the traditional system of cocoa tree [theobroma cacao l., sterculiaceae] cultivation) and “derruba total” (integral knocked down trees) systems utilized in cocoa farming are examples of such kinds of agricultural management, “environmentally friendly”. “cabruca” involves planting the cocoa trees between indigenous trees which provide the necessary shade (delabie et al., 2007; cassano et al., 2009; schroth et al., 2004). but, according the same authors, in the “derruba total” system, exotic trees such like erythrina fusca lour. (fabaceae), are planted to ensure shading for the cacao trees. in both planting systems, therefore, there is a combination of planted species and secondary vegetation which supports and maintains local animal diversity, particularly ants (delabie et al., 2000; delabie & mariano, 2001), in a manner which is close to a secondary forest community (delabie et al., 2007). formicidae, the dominant invertebrates of tropical forests, contribute to maintain at low levels the populations of many other organisms being for that considered as “key components” (bihn et al., 2008); in addition to that many species are seen as ecosystem engineers as they are responsible in great part for mixing up mineral and organic soil components (folgarait, 1998). furthermore, they have other traits which highlight the pivotal role of this family in the ecosystems, an important reason for focusing on them in biodiversity studies. it is well established that environmental changes affect the ant community and other faunal components (andersen, 1990, 1997). ant population richness may be positively linked to vegetation density, as in the most heterogeneous habitats, there is a larger variety of nesting sites, food, microclimate and interspecific interactions (competition, predation, mutualism) than in the less complex habitats (corrêa et al., 2006). research conducted on diversity patterns is significant for developing strategies for species conservation. it also enables predicting the effects of environmental changes, such like fragmentation and simplification of habitats (retana & cerdá, 2000; graham et al., 2009). thus, studies done on the diversity patterns and population richness in agroforestry systems such like cocoa tree plantations may contribute to a clearer understanding of the variations that can occur throughout the plant development. a first assessment of the dominance consequences of certain species on the other ants has been done earlier, which appears as a function of plant ontogeny (conceição, 2015). in this new study, we intend to reveal the mechanisms responsible for this variation, according to the growth of the cocoa trees in the plantations of southeastern bahia, brazil. material and methods this study was carried out on the experimental grounds of the cocoa research center – cepec/ceplac (14º47’s, 39º02’w), in the municipality of ilhéus, state of bahia, brazil. located in the south-eastern coastal microregion of bahia, this area is characterized by a warm and humid climate of af-type (köppen, 1936), with an annual temperature ranging between 20 and 25 oc (santana et al., 2003); rain forest is the principal regional ecosystem, included in the mata atlântica (brazilian atlantic forest biome), with an annual regional rainfall from 2,000 to 2,400 mm on average and at an altitude of around 60 m a.s.l. (santana et al., 2003). areas of cocoa plantations were chosen in different developmental stages, planted under the “derruba total” system with erythrina sp. as shade; they were all subjected to similar agronomic management and edaphoclimatic conditions. the plants were located in the experimental blocks e, f, g and h (cepec nomenclature) (fig 1). the selected areas were planted with trees of 1, 3, 4, 8, 15 and 33 years of age, each of them with a surface of at least four hectares. fig 1. map of experimental areas of the cocoa research center (cepec), ilhéus, bahia, brazil (adapted from silva & melo (1968), identifying the blocks where the ants were sampled). the samples were collected between september 2008 and march 2009. three hundred cacao trees were randomly selected from within the planted areas. with at least 25m intervals between the trees, 50 trees were selected per planting age category, maintaining a minimum of 25m distance from the border. the canopy volume of cocoa trees was assessed using the measurements of the crown height and diameter to evaluate the observed changes according to plant ontogeny (fig 2). in each of the trees five conventional ant collection methods were applied: 1) sardine baits, 2) honey baits; 3) hand collection; 4) entomological sheet and 5) “pitfall” traps (bestelmeyer et al., 2000). a total of 1,500 samples were collected. the same collection method was applied, during the same period and conditions for each developmental plant stage. at the base of each plant, pitfall traps were installed for 24 hours to capture the ants, aiming to facilitate a comparison sociobiology 66(1): 33-43 (march, 2019) 35 with the arboreal fauna. the baits (sardine or honey) were put into containers (disposable plastic glasses) positioned at two extreme points on the branches of the canopy of each sampled cocoa tree. two hours later, they were taken down and the foragers were collected and packed into vials in 70% alcohol. hand collection involved the use of forceps to pick up the ants foraging on each tree trunk, up to 1.50 m in height, in 10 minutes of observation. finally, the trunk of each tree was subjected to 10 shakes and the formicidae were captured on an entomological sheet placed on the ground. after screening, all the biological material was identified with the help of the taxonomic keys and compared with the reference collection of the laboratory of myrmecology (cpdc acronym) at the cacao research center (cepec-ceplac). nomenclature followed bolton (2003), bolton et al. (2007), antcat (bolton, 2017) and antweb v6 13.3. the richness was estimated with the chao 2 index, thanks to the software estimates version 7.5 (colwell, 2005). the shannon-winner index was calculated using past (paleontological statistics) program, version 1.97 (hammer et al., 2001). to calculate the arboreal ant matrices, in each plant age class, the indexes of diversity and richness were assessed by combining the data of the litter sample, hand collection and honey and sardine baits per sample. calculation of the same indices was done separately for the epigeal ants. the similarity indices were determined using the past program version 1.97 (hammer et al., 2001), where the ant fauna found on trees were compared with that of soil at each class of age and subsequently its variation was observed throughout the course of the plant development. next, we performed a selection of models to verify which one (linear or nonlinear) best fits the relationship of similarity between ground-dwelling and arboreal ant communities and the age of planting. for the choice of the best model, the akaike criterion (aic) was used. the analyses were performed using software r v. 3.5.0 (r development core team 2018). in order to address the species composition, a multidimensional non-metric ordering (nmds) was performed. bray-curtis index was used, which is based on the frequency of occurrence of ant species in each one of the tree age classes. this analysis was performed in the software r v. 3.4.3 (r development core team 2018). then, a permutational multivariate analysis of variance (permanova) (anderson, 2001) was performed in which the presence/ absence of ant species in each one of the tree age classes was the response variable, while the predictor variable was tree age classes. we also performed the post-hoc comparison test to show which pairs of assemblages differ in composition. finally, we tested if there is variation in the number of ant species found per plant according the plantation ages, and if the difference between vegetation and soil varies, using two-way anova, followed by fisher´s pairwise multiple comparison tests. all these analyses were performed in the software r v. 3.4.3 (r development core team 2018). fig 2. comparative growth of cocoa tree plantations according to age, with 1, 3, 4, 8, 15, 33 years. results the commonest ant species in all the plant ages appear in bold in table 1. the number of occurrences varied greatly according to the developmental stages of the plants. regarding ground-dwelling ant species, both diversity and richness indexes were unexpectedly higher in younger cocoa trees (plantations of one, three and four-years-old, table 2). on the other hand, arboreal ants showed the highest diversity index on 15-years-old cocoa trees, whereas species richness was the lowest on one-year-old trees, the highest on 33-yearsold trees and intermediate on the other tree age classes (table 2). the diversity and richness indices showed no tendency either for growth or for decrease (table 2, in response to tree development). a greater similarity in the faunal composition was confirmed between the areas with 3and 4-years-old trees. the same was observed for both ground-dwelling and arboreal ant assemblages. in general, the younger areas stretch towards a similar composition (figs 3 and 4). soil and crown fauna show greater similarity when the cacao trees are younger. the pattern of distribution that best suited to explain the similarity between the assemblages of ground-dwelling and arboreal ants according to cocoa tree age follows the michaelis-menten’s model [y = 0.24835x / (-0.19347 + x)]. in the initial ages the similarity between the two strata is very high, and then it falls and continues stable along the aging of plantations (fig 5). permanova confirmed significant differences between plantations of different ages (table 3). the most important change is observed in the 8 years-old plantation which corresponds to the closure of the canopy cover. es conceição et al. – ant community evolution aging36 table 1. frequency of arboreal ant species (maximum: 50) found on cocoa trees in plantations of different ages. ilhéus, state of bahia, brazil. september 2008 to march 2009. bold highlights the commonest species. species age of the plantation (year) 1 3 4 8 15 33 atta cephalotes (linnaeus, 1758) 0 1 0 1 3 0 azteca chartifex forel, 1912 0 0 0 0 0 3 azteca paraensis borgmeier, 1937 0 6 10 0 6 9 brachymyrmex heeri forel 1874 3 4 2 7 3 7 brachymyrmex admotus mayr ,1887 13 6 3 1 3 0 brachymyrmex sp.1 1 0 0 0 0 0 brachymyrmex sp.2 4 4 0 0 0 0 brachymyrmex sp.3 0 0 0 1 0 0 camponotus atriceps (forma 1) (fr. smith, 1858) 0 0 0 0 6 0 camponotus atriceps (forma 2) (fr. smith, 1858) 0 0 0 0 1 0 camponotus balzani emery, 1894 0 0 0 0 1 0 camponotus bidens mayr, 1870 0 0 3 0 0 0 camponotus chartifex (fr. smith, 1860) 0 0 1 0 0 3 camponotus cingulatus (mayr, 1862) 0 1 3 0 0 1 camponotus fastigatus roger, 1863 2 9 12 4 11 2 camponotus sexguttatus (fabricius, 1793) 0 0 0 0 1 0 camponotus trapezoideus mayr, 1870 0 3 0 1 2 0 camponotus crassus mayr, 1862 4 10 19 4 11 1 camponotus (myrmobrachys) canescens mayr, 1870 0 0 0 0 1 2 camponotus (myrmobrachys) sp.1 0 1 0 0 0 3 camponotus (myrmobrachys) sp.2 0 0 1 2 0 0 camponotus (myrmobrachys) sp.3 0 0 0 0 2 0 camponotus (myrmobrachys) sp.4 0 0 0 0 2 0 cardiocondyla minutior (forel, 1899) 1 4 2 1 1 0 cardiocondyla obscurior (wheeler, 1929) 0 1 0 0 0 0 cephalotes angustus (mayr, 1862) 0 1 0 0 0 0 cephalotes atratus (linnaeus, 1758) 5 13 14 7 20 3 cephalotes goeldii (forel, 1912) 0 1 0 0 0 0 cephalotes maculatus (fr. smith, 1876) 0 1 1 0 1 0 cephalotes pallens (klug, 1824) 0 0 0 1 0 0 cephalotes pavonii (latreille, 1809) 0 3 0 1 4 0 cephalotes pusillus (klug, 1824) 0 0 2 0 1 0 cephalotes umbraculatus (fabricius, 1804) 0 1 0 0 0 0 crematogaster acuta (fabricius, 1804) 0 2 2 0 0 7 crematogaster carinata (mayr, 1862) 0 1 7 2 0 11 crematogaster curvispinosa mayr, 1862 2 6 5 18 1 11 crematogaster erecta mayr, 1866 2 24 20 12 22 0 crematogaster limata fr. smith, 1858 0 0 0 2 0 3 crematogaster longispina (forel, 1904) 0 7 9 1 2 9 crematogaster moelleri (forel, 1912) 0 0 0 0 0 6 crematogaster sp. near crucis 0 0 0 0 2 0 crematogaster tenuicula (forel, 1904) 0 0 0 0 0 3 crematogaster victima fr. smith, 1858 0 6 5 0 0 0 dolichoderus attelaboides (fabricius, 1775) 1 2 6 4 3 9 dolichoderus bidens (linnaeus, 1758) 0 0 0 0 7 0 sociobiology 66(1): 33-43 (march, 2019) 37 dolichoderus bispinosus (olivier, 1792) 1 9 3 0 0 3 dolichoderus diversus emery, 1894 1 0 0 1 0 0 dolichoderus imitator emery, 1894 0 0 0 2 0 0 dolichoderus lutosus (fr. smith, 1858) 2 3 0 0 1 1 dorymyrmex thoracicus (fr. smith, 1860) 0 0 0 0 1 0 ectatomma brunneum fr. smith, 1858 0 0 0 1 0 0 ectatomma permagnum forel, 1908 0 0 1 0 1 0 ectatomma tuberculatum (olivier, 1791) 0 0 0 26 22 15 gnamptogenys annulata mayr, 1887 0 0 0 0 0 1 hypoponera sp.1 0 0 0 1 0 0 hypoponera sp.5 0 0 0 0 1 0 linepithema humile (mayr, 1866) 2 3 0 2 5 1 linepithema neotropicum wild, 2007 31 16 10 12 3 3 megalomyrmex goeldii forel, 1912 0 0 0 0 0 1 monomorium floricola (jerdon, 1852) 2 25 16 16 31 1 mycocepurus smithi forel, 1893 2 0 1 0 0 0 nesomyrmex spininodis mayr, 1887 0 0 0 0 1 0 nesomyrmex asper (emery, 1896) 0 1 1 4 1 1 nylanderia fulva (mayr, 1862) 2 3 4 4 2 5 nylanderia guatemalensis (forel, 1885) 0 1 0 1 0 4 nylanderia sp.1 0 0 0 0 0 1 nylanderia sp.2 0 0 1 0 8 0 odontomachus haematodus (linnaeus, 1758) 0 0 0 0 1 0 odontomachus meinerti forel, 1905 0 0 0 0 0 1 neoponera crenata (roger, 1861) 0 2 1 2 0 0 neoponera inversa (f. smith, 1858) 0 0 0 0 8 13 neoponera moesta mayr, 1870 0 1 1 0 0 0 neoponera curvinodis (forel, 1899) 0 1 0 1 7 0 neoponera unidentata (mayr, 1862) 0 3 2 0 1 3 neoponera villosa (fabricius, 1804) 0 0 0 0 0 1 paratrechina longicornis (latreille, 1802) 0 0 0 0 5 0 pheidole diligens (smith, 1858) 0 0 1 0 0 0 pheidole flavida mayr, 1887 0 0 0 0 1 2 pheidole manuana wilson, 2003 1 1 1 0 0 1 pheidole midas wilson, 2003 0 0 0 0 1 1 pheidole nitidula emery, 1888 0 3 2 0 0 0 pheidole radoszkowskii mayr, 1884 1 1 0 0 0 0 pheidole sp.1 gp. fallax 3 6 2 3 4 0 pheidole sp.10 gp. fallax 0 0 0 0 0 1 pheidole sp.11 gp. fallax 0 0 0 0 1 0 pheidole sp.12 gp. fallax 0 0 0 0 1 1 pheidole sp.15 gp. flavens 0 0 0 0 3 1 pheidole sp.4 gp. flavens 0 0 0 2 3 0 pheidole sp.5 gp. fallax 0 0 0 0 0 1 pheidole sp.8 gp. flavens 0 0 0 0 0 3 table 1. frequency of arboreal ant species (maximum: 50) found on cocoa trees in plantations of different ages. ilhéus, state of bahia, brazil. september 2008 to march 2009. bold highlights the commonest species. (continuation) species age of the plantation (year) 1 3 4 8 15 33 es conceição et al. – ant community evolution aging38 age arboreal epigeic h’ chao 2 h’ chao 2 1 year 2.55 30.92 3.0 102.6 3 years 3.44 79.60 3.53 75.71 4 years 3.22 55.07 3.48 80.99 8 years 3.02 55.08 3.17 38.41 15 years 3.49 75.50 2.85 33.43 33 years 3.41 113.99 2.92 38.98 pheidole sp.9 gp. fallax 5 6 0 4 0 0 procryptocerus spiniperdus forel, 1899 0 0 0 0 2 1 pseudomyrmex elongatus (mayr, 1870) 0 1 0 0 0 0 pseudomyrmex gracilis (fabricius, 1804) 1 2 3 0 6 1 pseudomyrmex holmgreni wheeler, 1925 0 0 2 0 0 0 pseudomyrmex kuenckeli (emery, 1890) 0 0 1 0 0 0 pseudomyrmex oculatus (fr. smith, 1855) 0 0 1 1 3 1 pseudomyrmex pupa (forel, 1911) 0 1 0 0 1 0 pseudomyrmex sericeus (mayr, 1870) 0 1 0 0 0 0 pseudomyrmex sp.1 gp. pallidus 0 0 0 1 0 0 pseudomyrmex tenuis (fabricius, 1804) 0 0 0 0 0 1 pseudomyrmex termitarius (fr. smith, 1855) 0 5 4 3 8 0 sericomyrmex bondari borgmeier, 1937 0 0 0 0 0 1 solenopsis geminata (fabricius, 1804) 3 3 4 4 8 0 solenopsis saevissima (fr. smith, 1855) 0 0 0 0 0 1 solenopsis sp.1 0 0 0 0 1 0 solenopsis sp.2 0 2 1 1 8 3 solenopsis sp.3 0 3 4 7 2 5 strumigenys elongata roger, 1863 0 0 0 0 1 0 strumigenys spathula lattke and goitia, 1997 0 0 1 0 0 0 tetramorium simillimum (fr. smith, 1851) 0 0 0 0 3 0 wasmannia auropunctata (roger, 1863) 1 12 6 29 3 12 wasmannia rochai forel, 1912 0 1 0 1 2 0 number of trees investigated 50 50 50 50 50 50 number of species by age of the plantation 26 52 45 42 62 51 total number of species 113 table 1. frequency of arboreal ant species (maximum: 50) found on cocoa trees in plantations of different ages. ilhéus, state of bahia, brazil. september 2008 to march 2009. bold highlights the commonest species. (continuation) species age of the plantation (year) 1 3 4 8 15 33 table 2. diversity index (shannon wiener h’) and richness estimator (chao 2), assemblages of arboreal and epigeic ants in cocoa tree plantations of different ages. cepec experimental areas, ilhéus, bahia, brazil. september 2008 to march 2009. there are important variations in the ant richness according the cocoa tree increases (expressed by its height; fig 6a) and the cocoa tree canopy (fig 6b). basically, in the first stages, the number of ant species increases according to the age of the plantation, but when the cocoa tree canopies began to touch each other, the diversity of ground-dwelling ants decreases while the diversity of arboreal ants increases (fig 6). fig 3. dendrogram of similarity of arboreal ant assemblages in cocoa plantations according the age of the plantation (1 to 33 years old). cepec experimental areas, ilhéus, bahia, brazil. september 2008 to march 2009. jaccard´s coefficient sociobiology 66(1): 33-43 (march, 2019) 39 arboreal ants age (years) 1 3 4 8 15 33 1 0.004 0.0013 0.0015 <0.0001 <0.001 3 0.7381 0.7131 0.0181 0.0071 4 0.9733 0.0071 0.0025 8 0.0064 0.0023 15 0.7381 33 epigeic ants age (years) 1 3 4 8 15 33 1 <0.0001 <0.0001 0.2926 0.6735 0.3435 3 0.1557 <0.0001 <0.0001 <0.0001 4 <0.0001 <0.0001 <0.0001 8 0.5275 0.9161 15 0.5985 33 age (years) 1 3 4 8 15 33 1 0.0000629 0.0010204 0.6368319 0.0343438 0.0009982 3 0.9838423 0.0213842 0.4617723 0.9818033 4 0.1303235 0.8742881 1.0000000 8 0.7082823 0.1308828 15 0.8780006 33 table 3. post-hoc correlation of multiple comparisons showing the differences between the ant fauna composition according the age of the cocoa plantations. cepec experimental areas, ilhéus, bahia, brazil, from september 2008 to march 2009. p-values in bold are significant (<0.05). table 4. post-hoc fisher’s plsd comparisons showing the differences between the numbers of ant species per plant according to the age of the cocoa plantations. cepec experimental areas, ilhéus, bahia, brazil, september 2008 to march 2009. p-values in bold are significant (p < 0.05). fig 4. non-metric multidimensional ordering (nmds), ordering cocoa plantations (bray-curtis similarity) based on the frequency of occurrence of ant species in each one of the areas. cepec experimental areas, ilhéus, bahia, brazil, from september 2008 to march 2009. permanova p <0.001. we found significant differences in the average number of ant species recorded per plant according to the plantation age (f = 11.30, df = 5, p < 0.001) and stratum (f = 32.405, df = 1, p < 0.001), as well as a significant interaction between age and stratum (f = 11.901, df = 5, p < 0.001). in older and already producing plantations, the assemblages of ants living on the ground and on the trees are significantly different (fig 7). the fisher’s lsd test showed that the average number of ants living in the arboreal stratum was significantly lower in the one-year-old plantation than the other ages. in addition, 15 and 33 years-old plantations did not differ in their average number of species per plant, but presented a significantly larger number of ant species than plantations of intermediate ages (three to eight-years-old). the observed number of ants in the epigeic stratum was significantly lower in the one-year plantation compared to the three and four-years-old ones, however it was not significantly different from the other ages. the number of ants found was significantly higher for the intermediate ages (three and four-years-old) in relation to older plantations (table 4). es conceição et al. – ant community evolution aging40 discussion the ant species composition varies during succession, including the ones regarded as dominant in the mosaic of the cocoa trees such as the dolichoderines of the azteca genus and some other competitive species of the genera monomorium or crematogaster (majer et al., 1994; delabie et al., 2000; conceição et al., 2015). the wasmannia genus revealed a highly irregular frequency of occurrence, even more clearly for w. auropunctata, which is native to the neotropical region and recognized as invasive in other parts of the world (le breton et al., 2004; foucaud et al., 2010). the system of planting the cacao trees likely facilitates its population explosion while species of many other genera suffer from changes in land use. the cocoa tree plantations showed no evident variation between the diversity indexes in relation to the plantation age. no clear trend of increase or decrease was evident, even with respect to species richness, such as in other similar studies (majer & camer-pesci, 1991; oliveira et al., 2011). the agroecosystem seems to boost the ant diversity, independently of the developmental stage of the trees. the lack of any upward variation, as first expected, in these indices in the older plantations compared to the younger ones suggests a trend similar to that seen in tropical forests, in which the succession of the arboreal ant communities is observed practically only until 25 years post deforestation (neves et al., 2010). another noteworthy aspect is that the cacao tree crowns have no contact between them in the initial developmental years (fig 2), while the crown of the trees reach in contact at around 8 years of age. these observations may influence species richness, although exert no effect on the diversity index. in terms of richness, this trend, although significant, may have been rather weak, to such an extent as not to reflect the relationship between these indices and the tree age. fig 5. similarity between soil and arboreal ant assemblages according to cacao plantations age, southeastern bahia, brazil, september 2008 to march 2009. the dotted line corresponds to the correlation analysis. fig 6. influence of plant height (m) and crown volume (m3) of cocoa in the arboreal and epigeic ant richness in cocoa plantations. ilhéus, state of bahia, brazil. september 2008 to march 2009. lines represent supersmoother display to each strata group. fig 7. variation of the average number of ant species per plant in the arboreal stratum (green) and on the ground (grey) according the plantation ages. asterisks correspond to pair data significantly different. age (years) sociobiology 66(1): 33-43 (march, 2019) 41 the lowest diversity and richness indices values recorded in the one-year-old area definitely result from the straighter incidence of the sun on the ground, in comparison with all the others. ants are susceptible to microclimatic variations like solar radiation, which can be determinant in the survival of certain species (ríos-casanova, 2006). furthermore, species living in unfavorable environments most likely possess a better degree of physiological tolerance to variations of humidity and thermal amplitude, for example (hölldobler & wilson, 1990). the similarity observed in the indices of species diversity among the cocoa tree plantations of very different ages, like those with 3, 15 and 33-years-old, suggest some kind of resilience common to all the parcels in the whole cocoa farm. simultaneously, some species, either because of competition or stochastic factors, may not be present any longer in the environment, while others begin to arise into the community, inducing the observed variation between the diversity indexes. the ant community structure is dependent on the level of requireness or tolerance of each species, which in turn affects its distribution. the species composition pattern during succession differed among plants of very different ages, although some similarities were observed in the cocoa tree plantations that were close in age. thus, the assemblage structure which results from the advent of some species and the suppression of others, may have caused these variations (majer et al., 1994; sanders et al., 2007). these are natural oscillations that can also take place due to changes in the spatial distribution of certain populations, because although relatively stable over the time (medeiros et al., 1995), the arboreal ant mosaic gets continuously restructured depending on the development of the host tree and other factors, such as local microclimate. during the beginning of ecological succession in young plantations, the ant community varied greatly in composition with strong differences in the local distribution and in the abundance of arising species. during the later successional phases, however, a much less variation in the community composition was noted, similarly to what dauber and wolters (2005) observed in temperate grasslands. in young plantations, the ants living on the ground and on the cocoa trees belong to the same assemblage and there are only low differences between the species found on the ground and the ones found on the young trees. in older plantations, it exists at least three congruent phenomena which contribute to diverge the cocoa ant community in two distinct assemblages on one side, on the ground and on the other, on the trees: i) in plantations older than 8 years, the canopies of neighboring trees tend to be closer from one tree to another, which facilitates to the arboreal ants colonizing the habitat and allowing the mosaic of dominant ants to become organized (see majer et al., 1994); ii) the soil surface receives less and less direct sunlight according the plantation aging and; iii) there is low opportunities for ants living on and in the soil to tend mutualistic sap-sucking insects (delabie, 2001), which is an important element of the success of arboreal ants in general. the rareness of sunlight certainly contributes to decrease the productivity of the soil biota but not its richness, and is possibly responsible for the strong specialization observed in many organisms living in this stratum, such as extremely specialized predation, fungus-growers and scavengers. the cocoa tree leaf litter is regarded as the stratum at which more specialized and the larger number of ant species inhabit (delabie et al., 2000, 2007; delabie & mariano, 2001); the density of this stratum appears to have no effect either on the species diversity or richness (delabie & fowler, 1995). on the other hand, the organization of the arboreal species in function of the plant development is responsible for the mosaic of dominant ants which is typical of this kind of agroforestry (see majer et al., 1994). furthermore, the tree canopy may be colonized by an assemblage of typical arboreal species characteristic of the agro-ecosystem which distinctly diverges from the epigeal and edaphic faunal composition on its side closer from a forest assemblage (vasconcelos & vilhena, 2006; delabie et al., 2007). therefore, the conclusion drawn is that, among the cocoa tree plantations of the southeast of bahia, no gradient of diversity and richness of the ant community is evident that even temporarily follows the course of the development of the host plant. however, gradual changes are seen in the faunal composition, with a pattern of species being structured during the various developmental stages; this pattern is very different when the younger and older cocoa trees are compared. the alterations in the species distribution and regulation of the ant populations take place according to the mosaic of the dominant arboreal ants at all the plant developmental stages. this is a structured and organized phenomenon (see conceição et al., 2015), and is the most likely cause for the unequal variations observed in the species diversity and richness. acknowledgements thanks are due to ceplac’s staff, mainly josé raimundo maia dos santos and josé crispim soares do carmo for field assistance, as well as the trainees of myrmecology laboratory, flavia, alennay, brena and linsmara; 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(2006). species turnover and vertical partitioning of ant assemblages in the brazilian amazon: a comparison of forest and savannas. biotropica, 38: 100-106. doi: 10.1111/j.1744-7429.2006.00113.x 404 not found doi: 10.13102/sociobiology.v62i4.396sociobiology 62(4): 604-606 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 differential nest parasitism in three sympatric social wasps (hymenoptera: vespidae: polistes spp.) in the west indies introduction social wasps of the subfamily polistinae (hymenoptera: vespidae) are host to nest parasitoids from at least 14 families of insects (makino, 1985; nelson, 1968). social wasps with open-comb nests would seem especially vulnerable to parasitoid invasion. in most of the records compiled by makino (1985), the host is a species of polistes or mischocyttarus, whose nests are of this type. however, this bias may partly be due to the ease with which such parasitism is noted and the overall greater study devoted to these genera. wasps have a number of tactics to limit penetration by nest parasitoids. the presence of a parasitoid near the nest is likely to elicit prolonged, agitated patrolling of the comb surface by the wasps (lutz et al., 1984; pers. obs.), and starr (1990) and gadagkar (1991) suggest that constant vigilance and active repulsion by adult females are the most important guard against parasitism. if a parasitoid succeeds in laying eggs in the nest, most polistes and mischocyttarus make little apparent effort to remove parasitoid brood at any stage abstract jamaica’s three species of social wasps were found nesting together in a suburban area. their nests gave evidence of markedly different parasitoid loads in the sequence polistes crinitus > p. dorsalis > p. major. based on larval and pupal silk remains, the moth chalcoela pegasalis appears to be the main nest parasitoid at this locality. despite widely different parasitoid loads, the estimated per-nest production of adults was similar among the three species. this suggests a trade-off between investment in anti-parasitoid tactics and in other brood-care activities. sociobiology an international journal on social insects ck starr1, jm nelson2 article history edited by fernando barbosa noll, unesp, brazil received 13 april 2014 initial acceptance 11 june 2014 final acceptance 17 june 2015 keywords chalcoela pegasalis, jamaica, nest symbionts. corresponding author christopher k. starr dep’t of life sciences university of the west indies st augustine, trinidad & tobago e-mail: ckstarr@gmail.com (gadagkar, 1991). one apparent recourse is to excavate cells with parasitoid larvae and/or pupae, although we have seen evidence of this tactic in few species. nest parasitoids are very often made conspicuous by the larval silk and pupal cases that they leave behind, which may remain visible long after the wasps have left the nest. our observations over the years indicate that regions and localities can differ a great deal in the parasitoid load seen in nests, so that the wasps in some places are subject to heavy parasitoid pressure, while those in others are relatively parasitoid-free. what has not been examined up to now is whether cooccurring, closely-related species may differ in this respect. our purpose here is to compare nest-parasitoid load in three congeneric species at a single neotropical locality. materials and methods jamaica is an oceanic island with just three species of social wasps, all in the genus polistes (richards, 1978). one of us (cks) found all three species nesting abundantly research article wasps 1 university of the west indies, st augustine, trinidad & tobago 2 oral roberts university, tulsa, oklahoma, usa sociobiology 62(4): 604-606 (december, 2015) 605 on buildings and other human-made structures in suburban mona, near the city of kingston. nests are easily identifiable to species in the absence of wasps. among other features, the petiole is excentric in p. crinitus (felton) nests, centric is those of p. dorsalis (fabr.) and p. major beauvois, and the cells are notably larger in p. major than the other two species. the three species all nested in much the same places at our site, often interspersed, with no evident nest-site separation among them. all old nests that could conveniently be reached were collected from buildings on the university of the west indies campus, up to 100 nests per species. these were then examined in the laboratory with an ordinary dissecting microscope. cocoons spun by the wasp larvae just before pupating are closely applied to the cell walls (resembling a coat of varnish) and close the cell with a smooth cap. silk from parasitoids, in contrast, clutters the cell interior and renders it unserviceable for brood rearing. we counted a nest as parasitized if at least one cell was fouled by such foreign silk. adult wasps sometimes cut away parts of cells walls in an apparent attempt to remove the very tough, resistant parasitoid silk. we counted a nest as showing excavation if there was removal of at least a significant part of any cell wall. the number of larvae that have pupated (and presumably emerged as adults) in a given cell can be determined with confidence by dissecting the cell and counting the fecal pellets deposited in the cell base after the cocoon is spun. pellets are of fairly uniform size, and where there are two or (rarely) more in a cell base they are separated by a silk layer and therefore distinct (yamane & yamane, 1975). results and discussion nests of all three species varied widely in size in our samples from fewer than 25 to at least 250 cells. average nest size was significantly greater in p. dorsalis than p. crinitus and p. major (kruskal-wallis test, p<0.01), while the latter two showed no average difference (p>0.05) (table 1). (cks) found a high incidence of p. crinitus nests with evidence of nest parasitoids, while no p. major appeared affected. based on remains of larval and pupal silk, the predominant parasitoid appeared to be the widespread chalcoela pegasalis (walker) (lepidoptera: crambidae), previously recorded from p. crinitus (nelson, 1968). in contrast, no nest showed the pupae characteristic of pachysomoides (hymenoptera: ichneumonidae), although members of this genus are important parasitoids of several new world polistes spp. (nelson, 1968; makino, 1985). as seen in table 2, p. crinitus had the highest incidence of cells showing c. pegasalis infestation, p. major the lowest (x2>100, p<0.01), consistent with the relative fractions of nests with parasitoids in table 1. because the three species nest in much the same places at this one locality, it is reasonable to believe that they are equally exposed to searching nest parasitoids. nonetheless, the data show that p. crinitus colonies suffer the greatest impact and p. major the least. most cells of all species had at most one fecal pellet, with a very small number having two or three. mean percell production of adults differed significantly among species (kruskal-wallis test; p<0.01). combining nest size with percell production does not significantly alter this pattern. although species differ significantly in per-cell and per-nest production of adults, the differences were quite modest and showed no evident correlation with differences in parasitoid load. it is our interpretation that differences in losses of brood to parasitoids are offset by differences in another, unknown parameter to produce a trade-off. it is a plausible hypothesis that successful anti-parasitoid vigilance by adult females is so demanding that it markedly diminishes their attention to foraging and other aspects of brood-care. table 1. mean size (number of cells) of old nests, frequency of nests with parasitoid silk, and frequency of nests with evidence of cell excavation in three jamaican social wasps. further explanation in text. polistes crinitus n=100 polistes dorsalis n=100 polistes major n=74 mean size + sd 75.2+52.2 95.5+58.8 73.4+77.4 with silk 75 (75%) 21 (21%) 3 (4%) with excavation 24 (24%) 7 (7%) 7 (10%) the species show significant differences (p<0.01) in both the frequency of parasitized nests and frequency of nests with excavation (table 1; x2 = 108.4 and 13.7, respectively), although there is no significant difference between p. dorsalis and p. major in the latter parameter. consistent with this, at villa altagracia, san cristóbal, dominican republic one of us polistes crinitus n=7523 polistes dorsalis n=10,204 polistes major n=5428 cells with c. pegasalis silk 2042 (27.1%) 571 (5.6%) 27 (0.5%) per-cell production of adult wasps 0.69 0.72 0.59 table 2. frequency of cells with apparent chalcoela pegasalis silk and per-cell production of adult wasps in old nests of three jamaican social wasps. total numbers of cells below each species are from the nests enumerated in table 1. per-cell production of adults is based on counts of fecal pellets in cells bases; e.g. the 7523 cells of the p. crinitus nests contained an estimated 5203 pellets, a mean of 0.69 per cell. acknowledgements financial support for this study came from a university of the west indies travel grant to cks. we thank peter & tyra bacon for facilitation in jamaica and the journal’s reviewers for improving criticism. ck starr, jm nelson – polistes nest parasitoids606 references gadagkar, r. (1991). belonogaster, mischocyttarus, parapolybia and independent-founding ropalidia. in kg ross & rw matthews (eds.), the social biology of wasps. ithaca: cornell univ. press (pp 149-190). lutz, gg, strassmann, je, hughes, cr (1984). nest defense by the social wasps polistes exclamans and p. instabilis (hymenoptera: vespidae) against the parasitoid elasmus polistis (hymenoptera: chalcidoidea: eulophidae). entomological news, 95: 47-50. makino, s. (1985). list of parasitoids of polistine wasps. sphecos, 10: 19-25. nelson, j.m. (1968). parasites and symbionts of nests of polistes wasps. annals of the entomological society of america, 61: 1528-1539. richards, ow (1978). the social wasps of the americas, excluding the vespinae. london: british museum (natural history) 580 p. starr, ck (1990). holding the fort: colony defense in some primitively social wasps. in dl evans & jo schmidt (eds.), insect defenses. albany: state univ. of new york press (pp 421-463). yamane, sô, yamane, sk. (1975). [investigating methods for dead vespine nests (hymenoptera, vespidae).] seibutsu kyôzai (kikonia), 10: 18-39. doi: 10.13102/sociobiology.v65i4.3451sociobiology 65(4): 780-783 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the life histories of the "uruçu amarela" males (melipona flavolineata, apidae, meliponini) over the last 50 years, the life histories of male stingless bees (apidae: meliponini) have been documented in great detail (nogueira-neto, 1954; kerr et al., 1962; van veen et al., 1997; koffler et al., 2016; schorkopf, 2016), showing that male behaviors and reproductive strategies are diverse and very different of apini (engels & imperatrizfonseca, 1990; paxton, 2005). males leave the nests after they reach sexual maturity (van veen et al., 1997), before forming reproductive aggregations in specific sites (paxton, 2005). during the copula, the male attaches his genital capsule inside the female, resulting in a sterile individual (kerr et al., 1962; engels & imperatriz-fonseca, 1990). this short note combines several field observations and experiments that, together, help to understand the life histories of melipona flavolineata friese males. all observations took place at embrapa amazônia oriental, belém, pará state, brazil, between 2014 and 2017. abstract here we describe the life histories of adult males of the the amazonian stingless bee melipona flavolineata friese, commonly known as “uruçu amarela”. males reach sexual maturity inside nests, presenting seminal vesicles full of sperm cells and becoming able to fly at a mean age of 10 and 15 days, respectively. they aggregate twice in their lives, once before leaving the nest, and another at external congregation sites, by using their capacity to reach congregation sites dependent on morphological attributes, such as large eyes and elongated thorax. furthermore, we describe three atypical phenomena for meliponini males: m. flavolineata males have dimorphic color pattern; they lose their genital capsules, even when they fail to copulate; and penisless (sterile) males can stay alive for up to two days. the life history strategies of meliponini males have only just started to be told and provide many interesting questions for future studies. sociobiology an international journal on social insects jc veiga1, kl leão1, bwt coelho2, acm de queiroz3, c menezes3, fal contrera1 article history edited by denise alves, esalq-usp, brazil received 10 may 2018 initial acceptance 20 june 2018 final acceptance 16 august 2018 publication date 11 october 2018 keywords congregation sites, stingless bees, sterile males, dimorphism. corresponding author jamille costa veiga laboratório de biologia de abelhas instituto de ciências biológicas universidade federal do pará rua augusto corrêa nº 01 campus básico guamá cep 66075-110, belém, pará, brasil. e-mail: jal.cveiga@gmail.com inside the nests: intranidal behaviors, sexual maturity and the point of no return to study sexual maturation in m. flavolineata males, we used the total number of sperm cells in seminal vesicles as a proxy of a male’s sexual maturity, and evaluated its variation as a function of age (time since eclosure). males were held in minicolonies under laboratory conditions and sampled at different ages – from 0 to 25 days after emergence (esm1, table 1). we also tested the flight ability of each male through light stimulus (details in esm1). the sperm migration from the testicles to the seminal vesicles started between zero and five days, when we could observe a greater number of sperm bundles in the seminal vesicles, and a few free sperm cells. from 10 days, we could not find the bundles anymore, observing only the presence of individual sperm cells until 25 days (fig 1a). we found that males reach sexual maturity 1 laboratório de biologia e ecologia de abelhas, universidade federal do pará, belém, pará, brazil 2 coleção institucional do museu paraense emílio goeldi, belém, pará, brazil 3 laboratório de botânica embrapa amazônia oriental, belém, pará, brazil short note sociobiology 65(4): 780-783 (october, 2018) special issue 781 between 10 and 15 days after emergence, following which, total sperm cells was similar to subsequent age categories (kw-h (5;55) = 36.425; p < 0.001), and the increment in this number was followed by simultaneous improvement in flight ability (fig 1a). to investigate males’ intranidal behaviors and the age that males would leave the nests we marked 160 individuals with numbered and colored tags (40 worker bees from four different colonies). we observed three main activities performed by males inside the nests: walking-standing, self-grooming and nectar dehydration, as previously observed in other melipona species (van veen et al., 1997). we also observed males forming groups of at least three individuals with a mean age of 14 days. groups were observed near inner nest entrances, with a persistence of no longer than 48 h. after this, these individuals were never observed again inside the nests. outside of nests, we identified eight marked males gathering in aggregations, with a mean age of 20.5 days (± 1.41 s.d.). outside the nests: reaching the congregation sites melipona flavolineata reproductive aggregations can be formed at the entrance of conspecific nests, and sometimes are composed of hundreds of males (about 150 to 300 individuals; fig 2a-b). although male aggregations are only temporary, we observed that these aggregations remain present for several days or weeks between the months of july and december over three consecutive years (2013, 2014 and 2015). m. flavolineata seems to differ from other species of melipona, whose males are known to aggregate distant from nests and in some cases in groups of few individuals (sommeijer & de bruijn, 1995; santos et al., 2014), but whether such pattern sustains out of a meliponary context, it still need to be confirmed. aggregations are known to be a source of precopulatory selection in eusocial bees, selecting more competitive males in apis mellifera l. and scaptotrigona depillis (moure) (jaffé et al., 2010; koffler et al., 2016). based on this, we compared the body size of m. flavolineata males before and after the formation of congregation sites (corresponding to preand post-selection scenarios), as follows: (i) we first sampled males emerging as adults inside different nests (brood combs: pre-selection group; n = 90 obtained from ten nests); and (ii) after about 20 days of this first step, males were sampled from congregation sites (congregation: postselection group; n = 90 collected at random in five different congregation sites); finally (iii) all bees were measured in order to compare head and thorax attributes (details in esm1). we highlight that male origin was not controlled for the congregation group, which may have affected our results. however, we believe our sampling effort represented the variation in population once we obtained males from different congregation sites. we found differences in body size among males sampled before and after congregation formations: males sampled from brood combs showed higher median values and variation of interocular distance and intertegular distance, and lower median values of thorax length in relation to males sampled from congregation sites (fig 1b-e), despite not differing in other body size attributes such as head width, head length and tibia length (esm1, table 2). our results fig 1. development of sexual maturity of the "uruçu amarela" males, melipona flavolineata. here we show the phases of males’ life history and the variation of estimated number of total sperm cells in seminal vesicles as a function of males’ age in days; rank comparisons are indicated with letters (a); we also show the comparison of four body size attributes of melipona flavolineata males sampled from brood combs (n = 90) and congregation sites (n = 90), showing similarities in head width (b), and differences in interocular distance (c), thorax length (d) and intertegular distance (e). jc veiga, kl leão, bwt coelho, acm de queiroz, c menezes, fal contrera – the life of "uruçu amarela" males782 show that although all males had similar head sizes, the males sampled from congregation sites had shorter interocular distances, meaning they have larger eyes; they also had shorter intertegular distances and more elongate thoraxes than males sampled from brood combs, suggesting their body shape may be more aerodynamic. we also suggest that extremes were negatively selected as variation in body size was much lower in congregations. even though the brood comb males were not compared to males from the same colonies that successfully reached an aggregation, which would be ideal, our results corroborate with previous findings that meliponini males undergo pre-copulatory selection (koffler et al., 2016). atypical phenomena: color dimorphism and penisless males males of m. flavolineata exhibit dimorphic colorpatterning, including both light and dark morphs (fig 2). light morphs can be diagnosed by their yellow metasoma, yellow-ferruginous legs with blackish marks, and vertex and mesosoma with corn yellow setae. the decumbent setae on third to fifth metasomal tergum are bright yellow. all these patterns being similar to those of worker bees. the dark morph differs from light morphs in having black legs and metasoma, vertex and mesosoma with brownish-yellow setae. the decumbent setae on third to fifth metasomal tergum are brownish-yellow. we observed that the two morphs can emerge in the same colony. from a sample of twenty-one brood combs with preemergent pupae from twelve colonies, we observed of the 1,364 males emerging as adults, 78.15% were light morph and 21.85% were dark morph. the proportion of light and dark phenotypes produced in each brood comb varied and did not follow a pattern (esm1, table 3). according to pereboom and biesmeijer (2003) abdominal coloration in stingless bees might be involved in the regulation of body temperature in extreme thermal conditions. this suggests that, it is possible fig 2. melipona flavolineata reproductive aggregation placed at a congregation site near conspecific nests (a). detail of the aggregation showing males with different body color-patterns (b). melipona flavolineata dimorphic males, the light morph (left) and the dark morph (right). that variation in color-patterns of males of m. flavolineata has also a role in thermoregulation.to our knowledge, this is the first report of color dimorphism in male stingless bees. another atypical behavior was observed when we used males’ mating attempts as a proxy of sexual attractiveness of virgin queens in m. flavolineata under laboratory conditions (veiga et al., 2017). we observed two interesting behaviors: (i) m. flavolineata males permanently lose their genital capsules fig 3. atypical phenomena observed in melipona flavolineata males: (a) loss of male genital capsule without a successful copulation, (b) male genital capsules lost after mating failures, (c) male with complete reproductive apparatus, including the genital capsule (white arrow) and genital sterna (black arrows), and (d) a penisless male found at congregation sites, presenting only genital sterna (black arrows) and with absent genital capsule. sociobiology 65(4): 780-783 (october, 2018) special issue 783 when they copulate, also when they fail to do so (here failing in copulation means that males which attempted to copulate everted their genital capsule outside female’s body); and (ii) these penisless (sterile) males can stay alive, reengage in congregation sites and behave similarly to virgin males. when males failed in copulation, they simply everted their genital capsules, losing it in the air (fig 3a-b). we repeatedly observed such behavior under laboratory conditions, where 13.23% of 650 (in the first experiment), and 7.83% of 600 (in the second experiment) males permanently lost their genital capsules without a successful copulation. additionally, when we sampled reproductive aggregations, we found males lacking genital capsules (10% in the first congregation site, from a total of 50 sampled individuals; 5% in the second, from 60 samples, and 7.7% in the third, from 90 samples). penisless males stay alive on average for a further 2.62 days (±1.41 s.d.) and can be easily identified by the absence of the genital capsule (fig 3c-d). the life history of males of stingless bees has just started to be told. the main message is that their life histories are much more diverse than previously thought, and many aspects are yet to be described and understood. acknowledgements we would like to thank denyse cardoso da silva and rodriga de andrade e silva for their support in field work. we also thank dr. alistair campbell for language review, and the anonymous referees for insightful suggestions. we thank capes/embrapa (15/2014) for providing grants to jcv and kll. this research was funded by cnpq (400435/20144) through the pve 2014 project. we dedicate this work to the memory of prof. warwick e. kerr (1922 2018), a pioneer on studies of stingless bees. supplementary material doi: 10.13102/sociobiology.v65i4.3451.s2226 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3451/0 authors’ contributions jc veiga, fal contrera conceived the sampling designs; jc veiga, kl leão, bwt coelho collected and analyzed data; jc veiga, fal contrera wrote initial draft of the manuscript and all authors contributed to subsequent revisions and gave final approval for publication. references engels, w. & imperatriz-fonseca, v.l. (1990). caste development, reproductive strategies, and control of fertility in honey bees and stingless bees. in w. engels (ed.), social insects (pp. 167-230). berlin heidelberg: springer jaffé, r. & moritz, r.f.a. (2010). mating flights select for symmetry in honeybee drones (apis mellifera). naturwissenschaften, 97: 337-343. doi: 10.1007/s00114-0090638-2 kerr, w.e., zucchi, r., nakadaira, j.t. & butolo, j.e. (1962). reproduction in the social bees (hymenoptera: apidae). journal of the new york entomological society, 70: 265-276. koffler, s., meneses, h.m., kleinert, a. de m.p. & jaffé, r. (2016). competitive males have higher quality sperm in a monogamous social bee. bmc evolutionary biology, 16: 195. doi: 10.1186/s12862-016-0765-2 nogueira-neto, p. (1954). notas bionômicas sobre meliponíneos: iii–sobre a enxameagem. arquivos do museu nacional, 42: 419-451. paxton, r.j. (2005). male mating behaviour and mating systems of bees: an overview. apidologie, 36: 145-156. doi: 10.1051/apido: 2005007 pereboom, j.j.m. & biesmeijer, j.c. (2003). thermal constraints for stingless bee foragers: the importance of body size and coloration. oecologia, 42-50. doi: 10.1007/s00442-003-1324-2 santos, c.f., menezes, c., vollet-neto, a. & imperatrizfonseca, v.l. (2014). congregation sites and sleeping roost of male stingless bees (hymenoptera: apidae: meliponini). sociobiology, 61: 115-118. doi: 10.13102/ sociobiology.61i1.115-11 schorkopf, d.l. (2016). male meliponine bees (scaptotrigona aff. depilis) produce alarm pheromones to which workers respond with fight and males with flight. journal of comparative physiology. a, neuroethology, sensory, neural, and behavioral physiology, 202: 667-678. doi: 10.1007/s003 59-016-1109-9 sommeijer, m.j. & de bruijn, l.l.m. de (1995). drone congregations apart from the nest in melipona favosa. insectes sociaux, 42: 123-127. doi: 10.1007/bf01242448 van veen, j. w., sommeijer, m. j. & meeuwsen, f. (1997). behaviour of drones in melipona (apidae, meliponinae). insectes sociaux, 44: 435-447. doi: 10.1007/s000400050063 veiga, j.c., menezes, c. & contrera, f.a.l. (2017). insights into the role of age and social interactions on the sexual attractiveness of queens in an eusocial bee, melipona flavolineata (apidae, meliponini). naturwissenschaften, 104: 31. doi: 10.1007/s00114-017-1450-z _hlk519523577 _hlk519528295 _hlk519529539 _hlk519525982 _hlk519529630 _hlk519531514 doi: 10.13102/sociobiology.v67i2.4408sociobiology 67(2): 256-260 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction honey bees may visit a flower based on the floral color, size, patterning and social cues, and then were encouraged by the rewards of nectar or pollens (orban & plowright, 2014). however, the choice of nectar or pollens heavily depends on the colony nutritional deficiencies (hendriksma & shafir, 2016). nectar is foraged as the major energy resource of adult bees and the pollens provide the protein, lipids, vitamins and minerals for both adult bees and larvae, while the realization of pollination by bees is mainly based on their foraging behavior (brodschneider & crailsheim, 2010). the variations of available pollen resources are important for the health of honey bees due to the differences in nutrient composition (di pasquale et al., 2016). as the abstract bias foraging of pollen is general in different pollinators since various nutrition demanding, co-evolution and interaction of insect-plant. to clarify the preference of pollen foraging during sunflower blooming, the pollen foraging behaviors of apis mellifera linnaeus and apis cerana fabricius were observed. our results displayed that two summits of pollen foraging occurred in the morning before the ambient temperature climbed up to thirty-one degree centigrade and in the afternoon after the ambient temperature decreased below thirty-one degree centigrade, respectively. notably, the first foraging summit of apis cerana emerged one hour earlier than that of apis mellifera. these results imply that apis mellifera is less resistant to low temperature but more resistant to high temperature than apis cerana does. the colonies were surrounded by sunflowers with sporadic weeds, while only few maize dispersed over two hundred meters away. however, no more than forty percent of total pollens foraged by apis mellifera was from sunflower, and which was no more than twenty percent in apis cerana group. these results suggest that sunflower pollens are not the prior choice for both honey bee species, while the ratio of sunflower pollens foraged by apis mellifera is more than that of apis cerana does. sociobiology an international journal on social insects hp yang, j sun, p tang, cs ma, sd luo, j wu article history edited by solange augusto, ufu, brazil received 15 march 2019 initial acceptance 02 october 2019 final acceptance 30 march 2020 publication date 30 june 2020 keywords bias of pollens; sunflower; maize; weeds; apis mellifera; apis cerana. corresponding author yang huipeng, luo shudong, and wu jie key laboratory of pollinating insect biology of the ministry of agriculture institute of apicultural research chinese academy of agricultural sciences no. 1 beigou xiangshan, haidian district, 100093, beijing, china. e-mail: yanghuipeng@caas.cn / luoshudong@caas.cn / apis@vip.sina.com generalist pollinators, honey bees play a dominant role in pollination service due to the widely foraging spectrum of plant species and the plentiful foragers of a single nest (koski & ashman, 2015). the eastern honeybee, apis cerana fabricius (a. cerana), is native to asia and has been managed beekeeping at least 1700 years (chen et al., 2017). there are more than 2 million a. cerana colonies kept in china (chen et al., 2017). compared to apis mellifera linnaeus (a. mellifera), a. cerana is better in stress-tolerance, such as scattered floral resources, extreme weather conditions, long flight duration, effective hygienic behaviors and cooperative group-level defenses (park et al., 2015). therefore, a. cerana takes charge of an essential complement of a. mellifera on pollination service for both wild plants and intensive crops (park et al., 2015). key laboratory of pollinating insect biology of the ministry of agriculture, institute of apicultural research, chinese academy of agricultural sciences, beijing, china research article bees the ratio of sunflower pollens foraged by apis mellifera is more than that of apis cerana does during sunflower blooming sociobiology 67(2): 256-260 (june, 2020) 257 large-scale single species plantation of sunflower (helianthus annuus) or maize (zea mays) destroys the habitats and results in decline of pollinators (naug, 2009). nowadays, the seed production of sunflower mainly depends on the pollination service of honey bees (calderone, 2012), which are rewarded by both nectar and pollen (greenleaf & kremen, 2006). meanwhile, the maize, as an anemophilous crop, flowers at the same period and can also provide pollen (di pasquale et al., 2016). hence, the sunflower and the maize are main floral resources in july and august (di pasquale et al., 2016). however, the deficiency of histidine in maize pollens and phosphorus element in sunflower pollens, which are essential for honey bee health, results in dramatically reduced lifespan of workers (di pasquale et al., 2016; filipiak et al., 2017). furthermore, the clarification of nutritional requirements of different bee species will conduct the restoration of floral resource which develops diverse and nutritionally balanced plant communities (vaudo et al., 2015). in this current study, the colonies of a. mellifera and a. cerana were surrounded by intensive agrarian landscape of sunflower in bayannur of inner mongolia autonomous region of china, where the sunflowers occupied the largest plant area and the most extensive range. (http://www.bynr. gov.cn/bynchina/aboutbyn/famousbrandhigh/). in order to demonstrate the bias pollen foraging behavior of these two honey bee species, the daily activity of pollen foraging and pollen preference have been observed during the flowering period. materials and methods fields, surrounding vegetation and colonies this study was performed in an agrarian environment in bayannur of inner mongolia autonomous region of china (e107.40′38″, n40.58′28″ with altitude 1030 meters) from july 26th to august 26th of 2017 and 2018, where there were sunflower (92.9%) and maize (7.1%) crops during that time. besides, few xanthium and suaeda distributing as weeds at the edge of the sunflower and maize fields were flowering. due to the phenological consistency of these four plant species in the same habitat type, all pairwise comparisons were performed simultaneously. the colonies of a. mellifera were equalized to six frames of workers with one frame honey, while the colonies of a. cerana were equalized to three frames of workers with one frame honey. all the queens were half-year-old. for each bee species, five colonies were observed for foraging behavior. all the colonies were housed in standard langstroth hives and shipped to the location two weeks before the sunflower blooming. the apiary was surrounded by sunflowers. observation of foraging behavior the number of bees returning with pollen pellets and the amount of pollen pellets were observed from 6:00 to 20:00 during 3 continuous sunny days at the full-bloom stage of sunflower, respectively. to record the number of workers returning with pollens, a camera was fixed in front of the entrance of each hive. subsequently, the bees with pollen pellets were counted by eyes from the videos recorded by cameras at the first ten minutes of each hour. to obtain the pollen pellets collected by bees, pollen traps were set at the entrance of each colony (fig 1). the captured pollen pellets were collected at each hour. and then, the pollen pellets were dispersed sufficiently so as to be recorded as photos for further counting (fig 1). compared to the pollen pellets from the maize, xanthium and suaeda (m.x.s.), the pollen pellets from sunflower exhibited distinct color (fig 1). consequently, the photos were digitally analyzed via image pro plus software (version 6.0) for the distinguishing and counting the sunflower pollen pellets from the m.x.s.. meanwhile, the ambient temperature and humidity were recorded simultaneously by automatic recording meter (eliteck rh-4hc). fig 1. pollen foraging from sunflower (a), maize (b), xanthium (c) and suaeda (d) by honey bees. pollen pellets collection via pollen trap (e) and recording via camera (f). statistics the data was analyzed using the software prism (graphpad software, san diego, ca), and all data was expressed as the mean ± se (standard error). the results were analyzed using a two-way analysis of variance (anova), followed by a statistical significance determination using student’s t-test. hp yang, j sun, p tang, cs ma, sd luo, j wu – bias pollen collection of managed honeybee258 results daily activity of honey bees the daily activity of a. mellifera and a. cerana was detected firstly. the number of workers returning with pollen pellets displayed that two summits occurred in the morning and afternoon, respectively. the first pollen foraging summit and afternoon, respectively. the first pollen foraging summit of a. cerana happened between 8 and 10 o’clock, while it was one hour later in a. mellifera. and then, the foraging behavior decreased sharply until another summit occurred between 17 and 19 o’clock (fig 2a). meanwhile, the amount of pollen pellets collected by pollen traps exhibited the same tendency (fig 2b). during the observation period, the lowest temperature (13 degree centigrade) and highest humidity (94%) records happened at 6 o’clock, while the highest temperature record (36 degree centigrade) emerged at 12 o’clock and the lowest humidity record (23%) occurred at 15 o’clock (fig 3). bias of pollen foraging between a. mellifera and a. cerana as to the source of pollens, the ratio of sunflower pollen pellets were less than that of m.x.s. pollen pellets in both bee species (fig 4a). nevertheless, the ratio of sunflower pollens in a. mellifera was no more than forty percent, and which was no more than twenty percent in a. cerana (fig 4a). for each species, a. mellifera foraged more sunflower pollens before 9 o’clock and post 15 o’clock, while they foraged more m.x.s. pollens between 9 o’clock to 15 o’clock (fig 4b). in contrast, a. cerana foraged m.x.s. pollens more than that of sunflower during the entire observation period (fig 4b). discussion for bee species, the foraging of pollen and nectar is important for the colony development, while the behaviors are affected by floral resources and ambient temperature (aleixo et al., 2017). in the current study, the distinct differences of a. mellifera and a. cerana in foraging behavior and bias of pollens have been exhibited during the period of sunflower blooming with m.x.s as minor pollen resources and distributing sporadically. previously study shows that a. cerana starts foraging and reaches the peak of foraging activity earlier and at lower ambient temperatures than a. mellifera does, since a. mellifera demands higher thoracic temperature to forage than fig 2. the daily activity of honey bee foraging by worker counting (a) and pollen pellet counting (b). the ratio has been normalized to the maximum, respectively. five colonies for three continuous sunny days for each bee species. fig 3. the range of temperature and humidity of the apiary from sunrise to sunset for three continuous sunny days. sociobiology 67(2): 256-260 (june, 2020) 259 a. cerana does (tan et al., 2012). in this study, the increasing of foraging behavior was companied with the increasing of temperature, and the first summit in a. cerana happened one hour earlier than that of a. mellifera. however, the foraging behavior decreased sharply when the temperature was above 31 degree centigrade, and then a second foraging summit occurred in the later afternoon when the temperature went below 31 degree centigrade. what was more, the foraging behavior of a. cerana decreased earlier than that of a. mellifera. these results indicate that the a. mellifera is more tolerant to high temperature than a. cerana does. honey bees will weigh against the costs of time and energy with the gain of food (couvillon et al., 2015; danner et al., 2016). although the colonies of both a. mellifera and a. cerana were surrounded by mass flowering sunflowers and only few other flowering plants distributed sporadically in this current study, our results showed that more m.x.s. pollens were foraged. this phenomenon indicates that it is necessary for honey bees to forage m.x.s. pollens in spite of more time and energy demanded. the behaviors of foragers are mainly driven by the colony nutritional deficiencies so as to maintain the health of colonies (leonhardt & blüthgen, 2012; hendriksma & shafir, 2016). sunflower and maize are dominant pollen resource plants in summer, but their pollens are poorly nutrient for honey bees since the lower protein and essential amino acids content (nicolson & human, 2013; di pasquale et al., 2016). even though, a. mellifera foraging more pollens of maize (di pasquale et al., 2016). our results showed that both a. mellifera and a. cerana foraged more pollens of m.x.s. than that of sunflower, while a. cerana preferred foraging m.x.s. pollens more than a. mellifera did. hence, the bias foraging of pollens might be due to the serious deficiencies of nutrition. acknowledgements this work was supported financially by agricultural science and technology innovation program (caas-astip2017-iar) and the china agriculture research system (cars45). besides, the authors would like to thanks the croplife asia for partially financial support. authors’ contributions y.h.p., l.s.d. and w. j. conceived the study; y.h.p., s.j., t.p., m.c.s., l.s.d. performed the experiments; y.h.p. and l.s.d. analyzed the data and wrote this paper. references aleixo, k.p., menezes, c., imperatriz fonseca, v.l. & da silva, c.i. (2017). seasonal availability of floral resources and ambient temperature shape stingless bee foraging behavior (scaptotrigona aff. depilis). apidologie, 48: 117127. doi: 10.1007/s13592-016-0456-4. brodschneider, r. & crailsheim, k. (2010). nutrition and health in honey bees. apidologie, 41: 278-294. doi: 10.1051/ apido/2010012. calderone, n. w. (2012). insect pollinated crops, insect pollinators and us agriculture: trend analysis of aggregate data for the period 1992–2009. plos one, 7: e37235. doi: 10.1371/journal.pone.0037235. chen, c., liu, z., luo, y., xu, z., wang, s., zhang, x., dai, r., gao, j., chen, x., guo, h., wang, h., tang, j. & shi, w. (2017). managed honeybee colony losses of the eastern honeybee (apis cerana) in china (2011–2014). apidologie, 48: 692-702. doi: 10.1007/s13592-017-0514-6. couvillon, m. j., riddell pearce, f. c., accleton, c., fensome, k. a., quah, s. k. l., taylor, e. l. & ratnieks, f. l. w. (2015). fig 4. the ratio of pollen pellets foraged by workers for an entire day (a) and time sharing ratio of pollen pellets foraged by a. mellifera (b) and a. cerana (c). five colonies for three continuous sunny days for each bee species. hp yang, j sun, p tang, cs ma, sd luo, j wu – bias pollen collection of managed honeybee260 honey bee foraging distance depends on month and forage type. apidologie, 46: 61-70. doi: 10.1007/s13592-014-0302-5. danner, n., molitor, a. m., schiele, s., härtel, s. & steffandewenter, i. (2016). season and landscape composition affect pollen foraging distances and habitat use of honey bees. ecological applications, 26: 1920-1929. doi: 10.1890/15-1840.1. di pasquale, g., alaux, c., le conte, y., odoux, j.-f., pioz, m., vaissière, b. e., belzunces, l. p. & decourtye, a. 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(2015). bee nutrition and floral resource restoration. current opinion in insect science, 10: 133-141. doi: 10.1016/j. cois.2015.05.008. 477 furcotanilla, a new genus of the ant subfamily leptanillinae from china with descriptions of two new species of protanilla and p. rafflesi taylor (hymenoptera: formicidae) by zheng-hui xu1 abstract a new genus of the ant subfamily leptanillinae, furcotanilla gen. nov., discovered in southwestern china is described. the new genus is distributed in the oriental region and belongs to the tribe anomalomyrmini of leptanillinae. a key to the 4 known genera of leptanillinae of the world based on the worker caste is provided. two new species of protanilla collected from southwestern china, p. gengma sp. nov. and p. tibeta sp. nov., are described. the type-species of protanilla, p. rafflesi taylor, is also described based on the antweb images. a key to the 7 known species of protanilla of the world based on worker and queen castes is prepared. key words: hymenoptera, formicidae, leptanillinae, furcotanilla, new genus, new species. introduction after the establishment of protanilla taylor and anomalomyrma taylor (bolton, 1990, 1994), 6 new species of protanilla were described in the world (bolton, 1995, 2011; xu, 2002; xu & zhang, 2002; baroni urbani & de andrade, 2006; terayama, 2009). but the worker caste of anomalomyrma remained unknown. recently, borowiec et al. (2011) described 2 new species of anomalomyrma from the phillipines and malaysia, and the first discovery of the worker caste has provided sufficient knowledge for the identification of anomalomyrma. a new genus, furcotanilla gen. nov., based on the type species protanilla furcomandibula xu et zhang (2002) from southwestern china is erected. the new genus is distributed in the oriental region and belongs to the tribe 1 key laboratory of forest disaster warning and control in yunnan province, college of forestry, southwest forestry university, kunming, yunnan province 650224, china e-mail: xuzhenghui1962@163.com 478 sociobiolog y vol. 59, no. 2, 2012 anomalomyrmini of leptanillinae. in order to understand the differentiation between the new genus and other ones, a key to the 4 known genera of leptanillinae of the world based on the worker caste is provided. two new species of protanilla collected from southwestern china, p. gengma sp. nov. and p. tibeta sp. nov., are described. the type species of protanilla, p. rafflesi taylor, is also described based on the antweb images. a key to the 7 known species of protanilla of the world based on worker and queen castes is prepared. materials and methods the worker caste of protanilla gengma sp. nov. and p. tibeta sp. nov. were collected by the sample-plot method. descriptions and measurements were made under a xtb-1 stereo microscope with a micrometer. illustrations were made under a motic-700z stereo microscope with illustrative equipment. standard measurements and indices are as defined in bolton (1987) and xu & zhang (2002): tl-total length: the total outstretched length of the ant from the mandibular apex to the gastral apex. hl-head length: the length of the head proper, excluding the mandibles, measured in a straight line from the mid-point of the anterior clypeal margin to the mid-point of the occipital margin, in full-face view. in species where the occipital margin or the clypeal margin is concave, the measurement is taken from the mid-point of a transverse line spanning the anteriormost or posteriormost projecting points, respectively. hw-head width: the maximum width of the head in full face view, excluding the eyes. ci-cephalic index = hw×100 / hl. sl-scape length: the maximum straight line length of the antennal scape excluding the basal constriction or neck close to the condylar bulb. si-scape index = sl×100 / hw. ml-mandibular length: the straight-line length of the mandible from apex to the base. pw-pronotal width: the maximum width of the pronotum in dorsal view. al-alitrunk length: the diagonal length of the alitrunk in profile view 479 xu, z.-h. — a new genus of leptanillinae from china from the point at which the pronotum meets the cervical shield to the posterior base of the metapleuron. pnl-petiolar node length: with petiolar node in lateral view, the maximum longitudinal length of the node without its anterior and posterior peduncles. pnh-petiolar node height: with petiolar node in lateral view, the maximum vertical height of the node from summit to lowermost part of subpetiolar process. pnw-petiolar node width: the maximum width of the petiolar node in dorsal view. ppnl-postpetiolar node length: with postpetiolar node in lateral view, the maximum longitudinal length of the node without its anterior and posterior peduncles. ppnh-postpetiolar node height: with postpetiolar node in lateral view, the maximum vertical height of the node from summit to lowermost part of subpostpetiolar process. ppnw-postpetiolar node width: the maximum width of the postpetiolar node in dorsal view. the measurements of ml, pnl, pnh, pnw, ppnl, ppnh, and ppnw of protanilla rafflesi taylor are obtained from the antweb images according to the scale bar. all measurements are expressed in millimeters. the type specimens are deposited in the insect collection, southwest forestry university (swfu), kunming, yunnan province, china. key to known genera of leptanillinae of the world based on worker caste 1. mandibles subtriangular, masticatory margins shorter than or subequal to inner margins, the former with 3-5 teeth. antennal insertions very close to the anterior margin of the head. body very slender (old world tropics and temperate regions) (figs. 1-4) ....................... leptanilla emery, 1870 mandibles elongate, masticatory margins much longer than inner margins, the former with many more than 5 minute saw-like denticles or slender peg-like teeth. antennal insertions well behind the anterior margin of the head. body relatively robust ..............................................................................2 480 sociobiolog y vol. 59, no. 2, 2012 2. masticatory mrgins of mandibles with many minute saw-like denticles. in profile view, promesonotum high and strongly convex. metanotal groove deeply depressed. petiole broadly attached to the postpetiole (indo-australian) (figs. 5-8).......... .........anomalomyrma taylor, 1990 masticatory margins of mandibles with many slender peg-like teeth. in profile view, promesonotum low and weakly convex. metanotal groove shallowly notched. petiole narrowly attached to the postpetiole ...........3 3. in profile view, mandibles massive, lateroventral margin furcated and with 2 teeth. postpetiole broadly attached to anterior face of gaster. ventral face of postpetiole concave. in dorsal view, anterior margin of gaster deeply concave (oriental) (figs. 9-12) .. furcotanilla gen. nov. in profile view, mandibles slender, lateroventral margin not furcated and without teeth. postpetiole narrowly attached to anterior face of gaster. ventral face of postpetiole convex. in dorsal view, anterior margin of gaster almost straight (southern palaearctic, oriental, and indo-australian) (figs. 13-24) ...... ...................protanilla taylor, 1990. figs. 5-8: worker of anomalomyrma boltoni borowiec et al.; 5. head and body in profile view; 6. head in full face view; 7. mandible in dorsal view; 8. body in dorsal view. (drawn from images of borowiec et al., 2011. pilosity omitted). figs. 1-4: worker of leptanilla yunnanensis xu; 1. head and body in profile view; 2. head in full face view; 3. mandible in dorsal view; 4. body in dorsal view. (cited from xu, 2002. pilosity omitted). 481 xu, z.-h. — a new genus of leptanillinae from china description of new genus furcotanilla gen. nov. (figs. 9-12) type-species: protanilla furcomandibula xu et zhang gender: feminine. etymology: the name of the new genus is a combination of “furco-“, descriptive of the furcated mandibles found in the type-species, and the root “-tanilla”, commonly used in leptanillinae. diagnosis of worker: robust terrestrial leptanilline ants with the following combination of characters. head elongate and narrowed forward, anterolateral corners toothed, strongly constricted before the teeth. mandibles massive, elongate-triangular and strongly down-curved apically. lateroventral margin of mandible with a long tooth and a short tooth, look-like furcated. laterodorsal surface of mandible with a longitudinal groove. masticatory margin of mandible with numerous slender peg-like teeth, but crenulated at the down-curved apical third. apex of mandible with a long stout hair on the ventral face. clypeus trapezoidal, widened forward, anterior margin concave. antennal insertions well behind the anterior margin of head. antennae long, 12-segmented, scapes distinctly surpassed occipital corners, flagella filiform. eyes absent. mesonotum strongly constricted. promesonotal suture present. metanotal groove shallowly depressed. propodeal spiracles small and circular, lower-down on the side. metapleural bullae elongate elliptical, horizontal and close to the spiracles. petiole narrowly attached to postpetiole, petiolar node roughly square, subpetiolar process protruding, with large nearly circular semitransparent fenestra. 482 sociobiolog y vol. 59, no. 2, 2012 postpetiole broadly attached to the anterior face of gaster, ventral face of postpetiole deeply concave. lateral sides of the first gastral segment deeply and narrowly notched between the tergite and sternite at the anterior margin. in dorsal view, anterior margin of gaster deeply concave in order to accept the laterally compressed postpetiole. sting strong and extruding. cuticle smooth and shining. pilosity sparse. female and male: unknown. comparison: the new genus is close to protanilla taylor (figs. 13-24), but with the mandibles massive and furcated on the lateroventral margin; postpetiole broadly attached to the anterior face of gaster, ventral face of postpetiole deeply concave; anterior margin of gaster deeply concave in order to accept the postpetiole. systematic position: leptanillinae: anomalomyrmini. geographical range: oriental. discussion: xu & zhang (2002) described an interesting species protanilla furcomandibula from yunnan, china. it is a very special species with furcated mandibles, ventrally concaved postpetiole, broad articulation between postpetiole and gaster, and anteriorly concaved gaster, all these characters do not exist in the other species of protanilla. after a re-observation of the species, i realized that p. furcomandibula represented a new genus of leptanillinae, furcotanilla gen. nov., which has distinctly divided from protanilla. the type species, p. furcomandibula, is therefore transferred from protanilla to furfigs. 9-12: worker of furcotanilla furcomandibula (xu et zhang ); 9. head and body in profile view; 10. head in full face view; 11. mandible in dorsal view; 12. body in dorsal view. (cited from xu & zhang, 2002, slightly modified. pilosity omitted). 483 xu, z.-h. — a new genus of leptanillinae from china cotanilla. the new genus is distributed in the oriental region and belongs to the tribe anomalomyrmini of leptanillinae based on the elongate head, elongate mandibles, trapezoidal clypeus, well behind antennal insertions, elongate antennae, depressed metanotal groove, developed subpetiolar process, and relatively robust body. transference of species furcotanilla furcomandibula (xu et zhang) com. nov. (figs. 9-12) protanilla furcomandibula xu et zhang, 2002: 140-142, figs. 1-3. holotype worker and paratype worker, china: yunnan province, kunming, xishan forest park, huatingsi temple, 2250m, collected in a soil sample in conifer-broadleaf mixed forest, 2001.iii.31, zheng-hui xu leg., no.a00250. [holotype worker and paratype worker in swfu examined.] remarks: currently, furcotanilla furcomandibula (xu et zhang ) is the only known species in the new genus. according to the collection information, the species nests in the soil and forages on the ground in conifer-broadleaf mixed forest at altitudes around 2250m. key to known species of protanilla of the world based on worker and queen castes 1. in full face view, anterior margin of clypeus strongly concave. laterodorsal surface of mandible without longitudinal groove. in dorsal view, petiolar node laterally compressed and roughly elliptic, distinctly longer than broad. body bicolor, the middle portion black, the rest brownish yellow ...........2 in full face view, anterior margin of clypeus straight to weakly concave. laterodorsal surface of mandible with a longitudinal groove. in dorsal view, petiolar node anteroposteriorly compressed or square, as broad as long or broader than long. body concolor, uniformly yellowish brown or reddish brown ......................................................................................................3 2. in full face view, anterior 1/3 of the head distinctly narrowed forward. in profile view, anterodorsal corner of petiolar node roundly prominent. head brownish yellow. body small with tl 2.7-3.0 mm (xu, 2002: figs. 21-23) (china: yunnan province) ............................................. p. bicolor xu 484 sociobiolog y vol. 59, no. 2, 2012 in full face view, anterior 1/2 of the head distinctly narrowed forward. in profile view, anterodorsal corner of petiolar node rounded. head light black to blackish brown. body large with tl 4.1-4.5 mm (figs. 13-16) (china: yunnan province) ..................................................p. gengma sp. nov. 3. in dorsal view, petiolar node nearly square, as broad as long, and weakly narrowed backward. in profile view, anterior face of petiolar node vertical, postpetiolar node not inclined forward (terayama, 2009: figs. 113-118) (china: taiwan province) ...................................................... p. lini terayama in dorsal view, petiolar node anteroposteriorly compressed, distinctly broader than long, and weakly widened backward. in profile view, anterior face of petiolar node steeply sloped. postpetiolar node strongly inclined forward ..................................................................................................................4 4. in full face view, occipital margin almost straight. in dorsal view, anterior margin of gaster weakly concave (baroni urbani & de andrade, 2006: figs. 1-3) (sri lanka) ................... p. schoedli baroni urbani et de andrade in full face view, occipital margin weakly concave. in dorsal view, anterior margin of gaster straight ....................................................................................5 5. in full face view, anterior 1/2 of the head distinctly narrowed forward. in profile view, petiolar node relatively thin and roughly triangular, dorsal face very short, about 1/2 length of anterior face (xu, 2002: figs. 18-20) (china: yunnan province) ......................................................... p. concolor xu in full face view, anterior 1/3 of the head distinctly narrowed forward. in profile view, petiolar node relatively thick and roughly trapezoidal, dorsal face long, about as long as anterior face ..........................................................6 6. in profile view, posterodorsal corner of propodeum evenly convex. petiolar node strongly narrowed upward, anterior face nearly straight, dorsal face weakly convex, anterodorsal corner blunt (figs. 17-20) (china: tibet)… ...................................................................................................... p. tibeta sp. nov. in profile view, posterodorsal corner of propodeum strongly convex. petiolar node weakly narrowed upward, anterior face weakly concave, dorsal face strongly convex, anterodorsal corner roundly protruding (figs. 21-24) (singapore, malaysia) ...............................................................p. rafflesi taylor 485 xu, z.-h. — a new genus of leptanillinae from china descriptions of new species and protanilla rafflesi protanilla gengma sp. nov. (figs. 13-16) holotype worker: tl 4.1, hl 0.73, hw 0.60, ci 83, sl 0.68, si 113, ml 0.43, pw 0.48, al 1.20, pnl 0.38, pnh 0.48, pnw 0.26, ppnl 0.35, ppnh 0.48, ppnw 0.28. in full face view, head longer than broad, anterior 1/2 of the head distinctly narrowed forward. lateral sides evenly convex, anterolateral corners of head prominent and tooth-like. occipital margin weakly concave, occipital corners rounded. mandibles elongate and down-curved apically, dorsolateral surface without a longitudinal groove, basal corners roundly prominent, masticatory margin with about 21 peg-like teeth. clypeus nearly trapezoidal, anterior margin evenly concave. apex of labrum roundly convex, with 4 peg-like teeth. antennae 12-segmented, apices of scapes surpassed occipital corners by about 1/6 of its length, flagella segments 4-9 about as broad as long. in profile view, dorsum of pronotum nearly straight. promesonotal suture complete but not depressed. dorsum of mesonotum straight, slope down backward. metanotal groove moderately notched. dorsum of propodeum slightly convex, weakly slope down backward, posterodorsal corner evenly convex; declivity weakly convex, about 1/2 length of the dorsum. petiolar node nearly trapezoidal, anterior and posterior faces nearly straight, dorsal face roundly convex; anterodorsal corner slightly higher the posterodorsal corner, both are rounded. ventral face of petiole roundly convex, anterovenfigs. 13-16: worker of protanilla gengma sp. nov.; 13. head and body in profile view; 14. head in full face view; 15. mandible in dorsal view; 16. body in dorsal view. (pilosity omitted). 486 sociobiolog y vol. 59, no. 2, 2012 tral corner roundly extruding, with a circular semitransparent fenestra; posteroventral corner acutely angled. postpetiolar node strongly inclined forward, with dorsum roundly convex, anterodorsal corner bluntly prominent, anterior face straight; ventral face roundly convex, anteroventral corner bluntly angled. sting strong and extruding. in dorsal view, mesonotum strongly constricted. petiolar node nearly rectangular, longer than broad, length : width = 1.4:1, lateral sides evenly convex, anterior face nearly straight, posterior face roundly convex. postpetiolar node trapezoidal and widened backward, longer than broad, length : width = 1.3:1; lateral sides weakly convex, anterior face roundly convex, posterior face nearly straight. anterior margin of gaster straight. mandibles sparsely finely punctured. head and body smooth and shining. dorsa of head and gaster with abundant erect to suberect hairs and dense decumbent pubescence. alitrunk, petiole, and postpetiole with sparse erect to suberect hairs and abundant decumbent pubescence. scapes and tibiae with abundant suberect to subdecumbent hairs and dense decumbent pubescence. mouthparts with abundant longer hairs, apex of mandible with a stout long hair. head light black. mandibles, antennae, prothorax, legs, and gastral segments 2-4 yellowish brown. mesothorax, metathorax, propodeum, petiole, postpetiole, and first gastral segment black. paratype workers: tl 4.1-4.5, hl 0.70-0.78, hw 0.60-0.65, ci 83-87, sl 0.65-0.73, si 104-113, ml 0.43-0.48, pw 0.48-0.51, al 1.18-1.30, pnl 0.35-0.38, pnh 0.48-0.50, pnw 0.26-0.29, ppnl 0.33-0.35, ppnh 0.480.51, ppnw 0.28-0.30 (6 individuals measured.). as holotype, but head light black to blackish brown. holotype: worker, china: yunnan province, gengma county, mengding town, nantianmen, 1760m, collected from a soil sample in monsoon evergreen broadleaf forest, 2011.iii.10, hai-bin li leg., no. a11-29. paratypes: 6 workers, with the same data as holotype. comparison: this new species is close to p. bicolor xu, but in full face view, anterior 1/2 of the head distinctly narrowed forward; in profile view, anterodorsal corner of petiolar node rounded; head light black to blackish brown; body large with tl 4.1-4.5 mm. etymology: the new species is named after the type locality “gengma”. 487 xu, z.-h. — a new genus of leptanillinae from china protanilla tibeta sp. nov. (figs. 17-20) holotype worker: tl 2.6, hl 0.53, hw 0.40, ci 76, sl 0.43, si 106, ml 0.28, pw 0.33, al 0.75, pnl 0.18, pnh 0.30, pnw 0.23, ppnl 0.20, ppnh 0.30, ppnw 0.23. in full face view, head longer than broad, anterior 1/3 of the head distinctly narrowed forward. lateral sides weakly convex, anterolateral corners prominent and tooth-like, strongly concave before the teeth. occipital margin weakly concave. occipital corners roundly prominent. mandibles elongate and down-curved apically, laterodorsal surface with a longitudinal groove, basal corners roundly prominent, masticatory margin with about 12 peg-like teeth. clypeus roughly trapezoidal, anterior margin nearly straight. antennae 12-segmented, apices of scapes just reached to occipital corners, flagella segments 2-10 about as broad as long. in profile view, pronotum weakly convex. promesonotal suture complete but not depressed. dorsum of mesonotum straight and weakly slope down backward. metanotal groove shallowly depressed. dorsum of propodeum wea kly convex , p ostero dorsa l corner evenly convex ; declivity weakly convex, about 1/2 length of the dorsum. propodeal spiracle circular and small, lower down on the side. metapleural bulla elongate and roughly elliptical, close to the spiracle. petiolar node nearly trapezoidal, anterior face nearly straight, about as long as dorsal face, dorsal and posterior faces weakly convex; anterodorsal corner blunt, higher than posterodorsal corner, the latter rounded; ventral face oblique and straight, anteroventral process roughly square, with circular semitransparent fenestra. postpetiolar figs. 17-20: worker of protanilla tibeta sp. nov.; 17. head and body in profile view; 18. head in full face view; 19. mandible in dorsal view; 20. body in dorsal view. (pilosity omitted). 488 sociobiolog y vol. 59, no. 2, 2012 node strongly inclined forward, dorsum roundly convex, anterodorsal corner bluntly prominent, anterior face straight; ventral face roundly convex, anteroventral corner roundly protruding. sting strong and extruding. in dorsal view, petiolar node broader than long, width : length = 1.4:1, slightly widened backward; lateral sides weakly convex, anterior and posterior faces nearly straight. postpetiolar node about as broad as long, width : length = 1.1:1, weakly widened backward, lateral sides and anterior face roundly convex, posterior face nearly straight. anterior margin of gaster nearly straight. mandibles, head, and body smooth and shining. dorsa of head and body with sparse erect to suberect hairs and dense decumbent pubescence. scapes and tibiae with abundant subdecumbent short hairs and dense decumbent pubescence. mouthparts with abundant longer hairs, apex of mandible with a long stout hair on the ventral face. color reddish brown. mandibles, antennae, and legs brownish yellow. paratype worker: tl 2.7, hl 0.51, hw 0.40, ci 78, sl 0.40, si 100, ml 0.25, pw 0.33, al 0.83, pnl 0.18, pnh 0.31, pnw 0.23, ppnl 0.20, ppnh 0.30, ppnw 0.23 (1 individual measured.). as holotype. holotype: worker, china: tibet, medog county, damu town, damu village, 1200m, collected from a soil sample in the valley tropical rain forest, 2011.vii.20, leg. xia liu, no.a11-3925. paratype: 1 worker, with the same data as holotype, but collected from a ground sample, no.a11-3863. comparison: this new species is close to p. rafflesi taylor, but in profile view, posterodorsal corner of propodeum evenly convex; petiolar node strongly narrowed upward, anterior face nearly straight, dorsal face weakly convex, anterodorsal corner blunt. etymology: the new species is named after the type locality “tibet”. protanilla rafflesi taylor (figs. 21-24) holotype worker: tl 2.7, hl 0.52, hw 0.40, ci 77, sl 0.43, si 108, pw 0.36, al 0.80 (bolton, 1990); ml 0.26, pnl 0.17, pnh 0.29, pnw 0.24, ppnl 0.18, ppnh 0.33, ppnw 0.25. in full face view, head longer than broad, anterior 1/3 of the head distinctly narrowed forward. lateral sides weakly convex, anterolateral corners 489 xu, z.-h. — a new genus of leptanillinae from china prominent and tooth-like. occipital margin weakly concave, occipital corners roundly prominent. mandibles elongate and down-curved apically, dorsolateral surface with a longitudinal groove, basal corners rounded, masticatory margin with about 14 peg-like teeth. labrum with a pair of peg-like teeth. clypeus roughly trapezoidal, anterior margin slightly concave. antennae 12-segmented, scapes surpassed occipital corners by about 1/10 of its length, flagella segments 2-8 about as broad as long. in profile view, pronotum slightly convex. promesonotal suture complete but not depressed. dorsum of mesonotum straight, almost horizontal. metanotal groove widely shallowly notched. dorsum of propodeum straight, slightly slope down backward; posterodorsal corner roundly strongly convex; declivity weakly convex, about 1/2 length of the dorsum. propodeal spiracle small and circular, lower down on side. metapleural bulla elongate elliptical and horizontal, close to the spiracle. petiolar node roughly trapezoidal, weakly narrowed upward; anterior face weakly concave, dorsal face strongly convex, posterior face nearly straight; anterodorsal corner roundly protruding, higher than posterodorsal corner, the latter rounded; the shapes of ventral face and subpetiolar process are not clear because concealed by the hind coxa and femur. postpetiole moderately inclined forward, dorsal face roundly convex, anterior face weakly convex, anterodorsal corner roundly extruding ; anteroventral corner roundly protruding , posteroventral face weakly convex. sting strong and extruding. in dorsal view, petiolar node roughly rectang ular and slightly widened backward, distinctly broader than long, lateral sides weakly convex. postpetiolar node weakly widened backward, slightly wider than petiolar node, lateral sides weakly convex. figs. 21-24: worker of protanilla rafflesi taylor; 21. head and body in profile view; 22. head in full face view; 23. mandible in dorsal view; 24. body in dorsal view. (drawn from images of the antweb, california academy of sciences. pilosity omitted). 490 sociobiolog y vol. 59, no. 2, 2012 mandibles, head, and body smooth and shining. dorsa of head and body with sparse suberect hairs and abundant decumbent pubescence. scapes with sparse subdecumbent hairs and abundant decumbent pubescence. tibiae with abundant decumbent pubescence, but without standing hairs. mouthparts with abundant longer hairs, apex of mandible with a long stout hair. color brownish yellow. mandibles, antennae, and legs yellowish. holotype: worker, singapore: mcritchie, 1970.ix.1, d. h. murphy leg., deposited in the natural history museum, london (bolton, 1990). distribution: singapore and east malaysia (bolton, 1990). discussion: bolton (1990) assigned p. rafflesi taylor as the type-species for the establishment of the genus protanilla taylor. however, we sometimes encounter troubles when comparing an unknown species with p. rafflesi taylor because no complete description of the type-species has been published since 1990. recently, high quality images of the holotype specimen of p. rafflesi taylor were made available on the antweb of california academy of sciences. in order to facilitate the comparison, a complete description of p. rafflesi taylor is made based on the antweb images of the holotype specimen so as to consummate its taxonomic information. unfortunately, the lower portion of the petiole is concealed by the hind coxa and femur in the profile image, and the shapes of the ventral face and the subpetiolar process are kept unknown. acknowledgments this study is supported by the national natural science foundation of china (no. 30870333) and the key subject of forest protection of yunnan province. i thank miss xia liu (doctorate candidate of forest protection, beijing forestry university, beijing ) and mr. hai-bin li (student of forest protection, southwest forestry university, kunming ) for collecting the type specimens with me. i also thank california academy of sciences (usa) for the permission to use the images of protanilla rafflesi taylor from antweb. references baroni urbani, c. & de andrade, m.l. 2006. a new protanilla taylor, 1990 from sri lanka. myrmecologische nachrichten 8: 45-47. 491 xu, z.-h. — a new genus of leptanillinae from china bolton, b. 1987. a review of the solenopsis genus-group and revision of afrotropical monomorium mayr. bulletin of the british museum (natural history) (entomolog y) 54: 263-452. bolton, b. 1990. the higher classification of the ant subfamily leptanillinae. systematic entomolog y 15: 267-282. bolton, b. 1994. identification guide to the ant genera of the world. harvard university press, 222 pp. cambridge, massachusetts. bolton, b. 1995. a new general catalogue of the ants of the world. harvard university press, 504 pp. cambridge, massachusetts. bolton, b. 2011. an online catalog of the ants of the world. http://www.antcat.org/. borowiec, m. l., schulz, a, alpert, g. d. & banar, p. 2011. discovery of the worker caste and descriptions of two new species of anomalomyrma (hymenoptera: formicidae: leptanillinae) with unique abdominal morpholog y. zootaxa 2814: 1-14. emery, c. 1870. studi mirmecologici. bullettino della società entomologica italiana 2: 193-201. terayama, m. 2009. a synopsis of the family formicidae of taiwan. research bulletin of kanto gakuen university 17: 81-266. xu, z. 2002. a systematic study on the ant subfamily leptanillinae of china. acta entomologica sinica 45: 115-120. xu, z. & zhang, j. 2002. two new species of the ant subfamily leptanillinae from yunnan, china. acta zootaxonomica sinica 27: 139-144. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.99-104sociobiology 62(1): 99-104 (march, 2015) effect of magnetic field on the foraging rhythm and behavior of the swarm-founding paper wasp polybia paulista ihering (hymenoptera: vespidae) introduction homing and foraging abilities are of fundamental importance in social insects, because these activities are related to the search for food and/or material to construct their nest (spradbery, 1973). for successful foraging and homing, social insects must have good perception of environmental signals. this environmental perception allows animals to navigate and orient in space (mouritsen, 2001). multiple modalities are used in spatial orientation: vision, smell, and hearing, and detection of electric, gravitational and magnetic fields (mouritsen, 2001; wickelgren, 1996). the magnetic field of the earth provides animals with directional and positional information, even in darkness (wiltschko & wiltschko, 2006). many animals detect and use the geomagnetic field for orientation and navigation (wiltschko abstract the geomagnetic field can be used by insects for navigation and orientation, through different magnetoreception mechanisms. magnetic sensitivity is very well documented in honeybees, ants and termites, but few studies have examined this capability in social wasps. the present study analyzed the magnetic sensitivity of the paper wasp polybia paulista. the wasps’ behavior was analyzed in the normal geomagnetic field and in the presence of external magnetic fields generated by permanent magnets or by helmholtz coils. the frequency of foraging flights was measured in both conditions, and the behavior of the individuals on the nest surface was also analyzed. the magnetic field from the permanent magnet produced an increase in the frequency of departing foraging flights, and also the wasps grouped together on the nest surface in front of the magnet. the electromagnetic field created by the helmholtz coils also increased foraging flights, but individuals did not show grouping behavior. this helmholtz electromagnetic field induced wasp workers to perform “learning flights”. these results show for the first time that polybia paulista wasps are sensitive to magnetic fields, including it in the list of animal models to study magnetoreception and magnetic sensitivity. sociobiology an international journal on social insects mgc pereira-bomfim1; wf antonialli-junior1,2 & d acosta-avalos3 article history edited by gilberto m. m. santos, uefs, brazil received 10 july 2014 initial acceptance 25 october 2014 final acceptance 30 november 2014 keywords social wasp; foraging; homing; polistinae; epiponini corresponding author maria da graça c. pereira-bomfim programa de pós-graduação em entomologia e conservação da biodiversidade universidade federal da grande dourados faculdade de ciências biológicas e ambientais 241, 79804-970, dourados, mato grosso do sul, brazil e-mail: airambio@yahoo.com.br & wiltschko, 2006). in the case of insects, the social insects are the best studied in this respect. honeybees apis mellifera are highly sensitive to magnetic fields (kirschvink et al., 1997; walker & bitterman, 1989). there are two accepted models to explain magnetoreception in animals: the ferromagnetic hypothesis and the radical pair mechanism. the ferromagnetic hypothesis assumes that intracellular magnetic nanoparticles are responsible for transducing magnetic fields to biological signals through detection of magnetic torques at cellular mechanoreceptors (walker, 2008). the radical pair mechanism or light-dependent magnetoreception assumes that chemical reactions associated with the absorption of light can be modified by the presence of magnetic fields (ritz et al., 2010). in the case of social insects, only the ferromagnetic hypothesis has been explored, but the radical pair mechanism cannot be discarded. biomineralized magnetic material has been found in bees research article wasps 1 universidade federal da grande dourados, dourados, ms, brazil 2 universidade estadual de mato grosso do sul, dourados, ms, brazil 3 centro brasileiro de pesquisas físicas cbpf, rio de janeiro, rj, brazil mgc pereira-bomfim et al – effect of magnetic field on the polybia paulista foraging100 (desoil et al., 2005) and ants (acosta-avalos et al., 1999; esquivel et al., 1999; slowick & thorvilson, 1996; slowick et al., 1997), supporting the ferromagnetic hypothesis. magnetic material is deposited in hornet combs (stokroos et al., 2001). several studies have demonstrated the existence of magnetic sensitivity in bees (frier et al. 1996; lindauer & martin, 1972; wiltschko & wiltschko, 1995), ants (acostaavalos et al., 2001; anderson & vander meer, 1993; banks & srygley, 2003; çamlitepe & stradling, 1995; çamlitepe et al., 2005; jander & jander, 1998; kermarrec, 1981; klotz et al., 1997; riveros & srygley, 2008; rosengren & fortelius, 1986; sandoval et al., 2012). as far as we know, the only studies of magnetic sensitivity in wasps (vespidae) are in the subfamily vespinae (hornets; kisliuk & ishay, 1977; 1979). comb building in vespa orientalis is modified when the local magnetic field is increased by more than 60 times the corresponding local intensity; treated hornets built the nest comb with irregular cells, starting in the region of high intensity and continuing to construct in the direction of decreasing field intensity. this field intensity was lethal to both adults and larvae (kisliuk & ishay, 1977). in a different experiment, the same authors showed that the vertical component of the local geomagnetic field influences the wasps’ building orientation (kisliuk & ishay, 1979). these studies provided evidence that social wasps are sensitive to changes in the magnetic field. the present study investigated, for the first time, the magnetic sensitivity of social paper wasps (subfamily polistinae). we tested magnetic sensitivity in the swarm-founding paper wasp polybia paulista, through analysis of the effect of external magnetic fields on foraging and clustering behavior. material and methods eighteen colonies of polybia paulista (ihering, 1896) were used in the experiment; nine colonies were used as controls, and nine colonies were subjected to experimental external magnetic fields. the colonies were located in batayporã, mato grosso do sul, brazil (22º 17’ 96’’s, 53º 15’ 77’’w) and were analyzed during the period from march 2012 to january 2014. the geomagnetic field parameters in this region are: inclination -29.3°, declination -17.4°, horizontal intensity h = 0.19 oe, vertical component z = -0.11 oe and total intensity f = 0.22 oe (1 oe = 100 ut. oe is the acronym of oersted). to evaluate the change in foraging rhythm and colony behavior, the frequency of departing and homeward flights was measured, as well as the behavioral response of worker wasps located on the outer surface of the nest. to guarantee homogeneous experimental conditions in all the colonies, they were observed in the post-emergence stage (jeanne, 1972), because this stage has the largest number of individuals involved in foraging activities. each experimental session was conducted in daytime during the period of peak wasp activity, between 09:00 and 15:00 hs (andrade & prezoto, 2001; elisei et al., 2005; elpino-campos et al., 2007; lima & prezoto, 2003). the foraging rhythm was evaluated by observing the frequency of departing flights and homeward flights by individual wasps from the nest. the behavior of the individuals was observed in situ, in a sample for each colony, divided into two sessions of 15 minutes each. the first session (period a) corresponds to the colony in the presence of the normal geomagnetic field, without any altered magnetic field (amf). the second session (period b) corresponds to the colony in the presence of the normal geomagnetic field plus an amf. we sampled the behavior of wasps present on the outer surface of the nest during the trials, and observed if they executed grouping and “learning flights” (“ad libitum” sensu altmann, 1974). short time intervals in each session were used, to minimize the effects of changes in other abiotic factors that might have influenced the results. the local geomagnetic field was altered in two different ways. in four colonies used for the magnetic experiments, the magnetic field was altered using permanent magnets, fixed in the west side and suspended with a stick placed 10 cm away from the comb, generating a magnetic field with dipolar characteristics. the total intensity was 5.2 (± 3.1) oe being the higher value 13.9 oe and the lower 1.5 oe. the component with higher values were in the east-west direction being higher near to the magnet and with average value of 5.1 (± 2.9) oe, the component in the north-south direction showed a dipolar character being negative in the north side and positive in the south side (higher value of ±4.6 oe and lower value of ±0.3 oe, average value 0.07 (±1.5) oe). the vertical component had their values among -2.0 oe and +0.1 oe (average value 0.34 (± 0.56) oe). the average inclination was -1.6°±4.4°. the magnet generates a strong static magnetic field in the region of the comb and also a gradient in the eastwest direction (about 0.9 oe/cm). in the other five experimental colonies, the magnetic field was altered using helmholtz coils, following an adaptation of gonçalves et al. (2009). the apparatus consisted of a pair of helmholtz coils 30 cm in diameter oriented in the eastwest direction, each coil consisting of 46 spirals of cu wire 15 awg (cross section 1.5 mm2, resistance 0.01 ω), polished and wound in a 2cm-wide band. the pair of coils was connected to a digital electrical source, that provided a current of 1.16 a and a constant voltage of 15 v, generating a magnetic field almost uniform among the coils (total intensity 5.4 ± 0.8 oe). as can be seen, the variability in the intensity is lower compared with the magnet field. the same uniformity is observed in the west-east direction (average value of 5.2 ± 0.8 oe), the north-south direction (average value of 1.0 ± 0.9 oe) and the vertical component (average value 0.7 ± 0.9 oe). the average inclination was -6.9°±4.4°. we positioned each coil 2 cm from the comb edge (fig 1), and the comb edges were about 5.5 cm from the middle of the comb. in the middle of the comb the average magnetic field from the coils and the magnet was similar but in the case of the coils there was not a magnetic gradient. in this configuration the sociobiology 62(1): 99-104 (march, 2015) 101 helmholtz coil generated electric and magnetic fields at the surface of the comb. each coil is an equipotential with difference of electric potential of 15 v meaning that between both coils there was an electric field of about 100 v/m, that is similar to the atmospheric electric field (clarke et al., 2013). the magnetic field was monitored using a single axis magnetometer (globalmag, model tlmp-hall-050). to check only the effect of the amf and to exclude the effect of physical objects approaching the colonies, control observations were made for nine other colonies. four of them were studied using a sham magnet, consisting of a plastic object similar in color, shape and size to the permanent magnet. the sham magnet was positioned similarly to the permanent magnet, and the wasps’ behavioral response was observed during two 15-minute sessions, session 1 before the sham magnet was positioned in front of the colony, and session 2 with the sham magnet in front of the colony. the control experiments in the other five colonies were done with the pair of coils positioned as above, but in this case the digital electrical power source was turned off, and two 15-minute sessions were conducted as described above. to detect possible differences between the categories, the samples were grouped and compared using the mannwhitney test, with a 5% significance level. we compared the frequencies of departing and homeward flights measured in the experiments. results and discussion the total frequency of departing flights from the four colonies with the permanent magnet was 193 during session a and 235 during session b. in the control experiment, the frequency of departing flights was 84 in session 1 and 105 in session 2. the comparison between the experimental results fig 1. representation of the helmholtz coil apparatus with the variable-length pole, permitting the generation of altered magnetic field, in the region of the wasp nest. permits the conclusion that the increase in the intensity of the local magnetic field may have stimulated the foraging activity in the colony (fig 2). in the same group of four experimental colonies, the frequency of homeward flights was 233 in session a and 228 in session b. during session b, workers formed clusters on the outer surface of the nest (fig 3). the workers oriented themselves with the head and antenna pointing toward the perturbation source. kudô et al. (2003) observed that wasps respond to any potential source of threat by grouping outside the nest, orienting their heads and antenna toward the perturbation source. it is possible that this behavior is a defense strategy, fig 2. mann-whitney test of the number of departing flights and number of homeward flights performed by workers of the colonies of polybia paulista, during the experiments with the helmholtz coils (geomagnetic field h=6,00; p=0,014 and altered magnetic field h=6,06; p=0,014) and permanent magnet (geomagnetic field h=6,00; p=0,014 and altered magnetic field h=6,21; p=0,013). mgc pereira-bomfim et al – effect of magnetic field on the polybia paulista foraging102 perhaps indicating that the intensity of the magnetic field used in these experiments was interpreted as a source of threat. during session b the wasps remained still or moved only the antenna or the first pair of legs, and also walked randomly over the surface of the nest. this grouping behavior was not observed in the control experiments. therefore, the behavior can be interpreted as a result of the increase in the local magnetic field. in our experiments, the magnetic and electric fields generated by the helmholtz coils induced the workers to perform “learning flights” as a response to the presence of this altered field. during the learning flights the wasps acquire landmark information examining the location to which it will return (zeil et al., 1996). these flights were observed only during session b, with a mean number of 7.8 ± 2.6 (fig 4). for the five colonies studied with the helmholtz coils, the frequency of departing flights was 80 in session a and 108 in session b. the frequency of homeward flights was 93 in session a and 125 in session b. in the experiments with the five control colonies, the frequency of departing flights was 127 in session 1 and 111 in session 2, and the frequency of control homeward flights was 125 in session 1 and 134 in session 2. these results showed that the magnetic and electric field modified by the helmholtz coil also altered the wasps’ foraging behavior (fig 2). in these experiments, the grouping behavior described in the case of the permanent magnet was not observed, suggesting that the two sources of magnetic fields are perceived in different ways. the dissimilar effects of an oscillating and a static magnetic field was also observed by martin et al. (1989), who examined their influence on the execution of the honeybee “waggle dance”. they found that the rhythm of the dance was slower in the oscillating field and increased in the static magnetic field. fig 3. representation of the grouping behavior of polybia paulista workers (gw) on the nest surface during session a (i), before the application of the altered magnetic field with the permanent magnet (pm); and during session b (ii), with the permanent magnet in place. gw – grouping of wasps; wwasp. fig 4. number of “learning flights” performed by workers of the five colonies of polybia paulista, during the experiments with the helmholtz coils. a: normal geomagnetic field. b: altered magnetic field. the “learning flights” were not observed in all the control experiments with the helmholtz coils off. this kind of flight is typically performed by young workers as a way to acquire visual information to be used during foraging, and is less intense later in adult life (wei & dyer, 2009). according to wei et al. (2002), the performance of these flights by older individuals indicates a response to environmental uncertainty or change. this worker behavior may be the result of the presence of an electric field in the field generated by the helmholtz coil and that is not present in the permanent magnet, as it is known that bumblebees are able to detect electric field from flowers (clarke et al., 2013) and perhaps this ability is shared by wasps, or perhaps the wasps did not identify the magnetic field as a threat and tried to understand the magnetic field configuration better in order to use this information when it was time to return to the nest, recalibrating their orientation to the new local magnetic configuration. another behavior observed was a kind of disorientation shown by some workers returning to the nest and that were not present when the electromagnetic field was applied. apparently they were not able to land on the nest surface at the first attempt. however, the same behavior was observed in the control experiments, showing that the presence of the coils alters the landmarks around the nest, leading to a sudden disorientation in the wasps returning to the nest after foraging. the present results showed that the wasp polybia paulista is sensitive to modification of the local geomagnetic field by external sources, as has been described for other social insects including ants, bees and termites (wiltschko & wiltschko, 1995). the modified behaviors related to the presence of magnetic sociobiology 62(1): 99-104 (march, 2015) 103 fields were the frequency of foraging flights, the presence of “learning flights” and the grouping on the surface of the nest. the next question to be addressed is what mechanisms are involved in detecting the magnetic field. could it be magnetic nanoparticles, as predicted by the ferromagnetic hypothesis? or could radical pair reactions be occurring in the eyes or in ocelli? or perhaps another kind of magnetic field detection is combined with the gravitational field detection? recently, valkova and vacha (2012) discussed the possibility that honeybees use both mechanisms to detect the geomagnetic field. the stimulation of foraging flights by modified magnetic fields, as was observed in our study, is also intriguing. could the wasps correlate the intensity of the magnetic field with the appropriate time for foraging? the intensity of the geomagnetic field changes during the day (the daily variations), but the maximum increase is about 0.1 µt (skiles, 1985). kisliuk and ishay (1977) showed that magnetic fields of about 60 times the normal geomagnetic field are lethal to hornet workers. our study used magnetic fields of about 22 times the normal geomagnetic field, which were not lethal. in conclusion, magnetic or electric sensitivity has been demonstrated for the first time in polybia paulista wasps, adding this species to the list of animal models for studies of magnetoreception and electroreception. acknowledgments the authors thank janet w. reid (jwr associates) for the revision of the english text and yzel rondon súarez for their assistance with the statistical analysis. we are grateful to capes for a doctoral fellowship awarded to the first author. wfaj acknowledges his research grant from cnpq. daa acknowledges cnpq financial support. references acosta-avalos, d., wajnberg, e., oliveira, p.s., leal, l.l., farina, m. & esquivel, d.m.s. 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(1996). structure and function of learning flights in bees and wasps. the journal of experimental biology, 199: 245-252. doi: 10.13102/sociobiology.v66i3.4374sociobiology 66(3): 414-419 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 winter activity of ants in an urban area of western japan introduction in temperate regions, the foraging activity of ants generally declines during winter. ants are thermophilic and less resistant to cold temperature. most ant species exhibit peak activity at approximately 10℃–45℃, with dramatic decreases in the activity below 10℃ (hőlldobler & wilson, 1990). although rarely observed, prenolepis imparis (north america: talbot, 1943; fellers, 1989) and p. nitens (europe: lőrinczi, 2016) have been reported to be active during winter at approximately 2℃–3℃. in addition, formica polyctena exhibited higher foraging activity between mid-november and mid-march in slovakia (holecová et al., 2016). because the main islands of japan fall within a temperate region, ant activity in these areas is expected to be low during the cold winter season. to date, 296 ant species have been known abstract during winter, foraging activity of ants is considered low in temperate regions. winter activity of ground-dwelling ants was investigated using bait traps and quadrat sampling in an urban area of fukuoka city, western japan. six study sites were grouped into two categories: 4 open land types and 2 forest types. a total of 18 ant species were recorded between the end of january and beginning of march. the foraging activity of ants was generally low, except during relatively warm periods when the surface ground temperature was above 6 – 7°c or soil temperature was above 4 – 5°c. tetramorium tsushimae, messor aciculatus, and pheidole noda were the most abundant in the open land type, whereas nylanderia flavipes, p. noda, and crematogaster osakensis were the most abundant in the forest type. bait preference varied among the different species, e.g., p. noda preferred tuna over honey, whereas n. flavipes similarly responded to tuna and honey. this is the first detailed study on the relationship between temperature and ant activity in japanese mainland fauna. sociobiology an international journal on social insects s hosoishi1, mm rahman2, t murakami3, s-h park4, y kuboki1, k ogata1 article history edited by evandro n. silva, uefs, brazil received 17 february 2019 initial acceptance 04 august 2019 final acceptance 05 august 2019 publication date 14 november 2019 keywords bait preference, foraging activity, formicidae, indigenous ants, temperature. corresponding author s. hosoishi institute of tropical agriculture kyushu university motooka 744, nishi-ku fukuoka 8190395, japan e-mail: hosoishi@gmail.com from japan (terayama, 2014), however, no distinct “winter ants” have been reported from the japanese ant fauna. several studies on the seasonal activities of ants have been conducted in the areas of western and southern japan. in surveys from hiroshima focused on argentine ants, touyama et al. (2004) revealed that linepithema humile foraged in winter during the periods when temperatures were relatively warm. according to their study, l. humile did not forage when the ground surface temperature was below 10℃. working in yambaru (okinawa), suwabe et al. (2009) found that indigenous ant species have an activity peak between spring and early summer, whereas alien species have a peak between late summer and winter. yamane et al. (2004) examined seasonal changes of ants in amami-oshima using sugar syrup traps. they found that ant activity was low from december to march. okinawa and amami are located in the southern 1 institute of tropical agriculture, kyushu university, fukuoka, japan 2 department of entomology, faculty of agriculture, bangabandhu sheikh mujibur rahman agricultural university, gazipur, bangladesh 3 graduate education and research training program in decision science for sustainable society, kyushu university, fukuoka, japan 4 department of biological sciences and chemistry, kosin university, busan, republic of korea research article ants sociobiology 66(3): 414-419 (september, 2019) 415 region of japan, where the climate is relatively hot and wet even during winter. few comparable studies of indigenous ants have been conducted in mainland japan, although this region contains the majority of japan’s land area. within the japanese ant fauna, several invasive alien species have been reported in recent years (linepithema humile by sugiyama, 2000; okaue et al., 2007; solenopsis geminata by yamamoto & hosoishi, 2010; terayama et al., 2014). in 2017, the red imported fire ants (rifa) solenopsis invicta was found within the container yards of ports at several cities across japan (ministry of the environment, japan, 2018). despite these sightings, it seems unlikely that s. invicta has successfully colonized japan. however, the japanese ant fauna is continuously exposed to the hazards of a potential colonization due to frequent international transportations. because the ports are typically located near urban areas, introduced ant species may invade neighboring parks or residential areas. it is unclear whether the indigenous japanese ant species are competitively dominant over these invasive alien species, although north american winter ant prenolepis imparis could persist in spite of the presence of linepithema humile (holway et al., 2002). shipping containers are transported through the ports year-round, suggesting that alien ant species are also delivered to japan during the winter months, a season known to be unfavorable for indigenous ants. open green areas covered by grassland or shrubs can easily provide a breeding ground for those alien species. therefore, regular monitoring is required in green areas, such as parks and shrine, and residential areas to detect and prevent invasion of alien ant species. the aim of the present study was to assess the foraging activity of grounddwelling ants in western japan during the cold winter season. this information can contribute to the risk management of invasive alien ant species. materials and methods the studied urban areas are located in fukuoka city, in the western part of japan (fig 1). the elevation at the sites ranges from 5 to 61 m a.s.l. the mean annual temperature in fukuoka city is approximately 16.7℃ and mean annual precipitation is 1,694 mm (fukuoka prefecture, 2001). these study sites consisted of open land and forest types (table 1). island city central park (isl), hakozaki port memorial park (hpm), kyushu university hakozaki campus (kyu), and momochi central park (mmc) are located in the central residential area of fukuoka city. these four sites are artificial open spaces, where the peripheral area is predominantly grassland with planted trees and shrubs. kashi-gu shrine (kas) is a shrine founded in 724, with a woody conservation area behind the shrine. further, minami park (min) was established on a low hill side and features a woody conservation area densely covered with high broadleaved trees. the six study sites were grouped into one of the following two categories: open land types (isl, hfk, kyu and mmc) and forest types (kas and min). in their field surveys of ants, ogata (1998), park et al. (2014a, 2015b) recorded 15 species in hpm, 17 spp. in mmc, 10 spp. in kyu, 32 spp. in kas, and 25 spp. in min. our sampling followed the design proposed by lőrinczi (2016), but we did not use bait traps on trees owing to the lower number of planted trees at some study sites. bait traps and quadrat sampling were used to measure the foraging activity of ants. the sampling was conducted on 17 days between january 25 and march 13, 2018 in the six study sites (table 1). in western japan, these are the coldest months and ant activity is essentially low (touyama et al., 2004). for bait traps, we established three sets of baits, positioned ca. 10 m apart in all study sites. each set consisted of five baits along a line transect at ca. 3 m intervals. we set bait, a quarterteaspoon of tuna and honey, placed on paper filters (6 cm in diameter). tuna and honey were placed ca. 5 cm apart from each other. the bait placed on a paper attracts not only more dominant ants, but also smaller or less aggressive species due to the oil around and under the paper (bestelmeyer et al., 2000). in this study, we employed the baits on a paper to find those smaller or less aggressive ant species. for quadrat sampling, we established three observation plots of 50 cm × 50 cm in size, separated by ca. 15 m in all study sites. the number of workers of each ant species was recorded for each bait and quadrat over a 3-min period every hour for 5 consecutive hours from 10 a.m. to 3 p.m. surface ground temperature at 1 cm height above ground and soil temperature at 10 cm depth were measured with a digital thermometer (ad-5624, a&d co. ltd.). voucher ant specimens are preserved at the institute of tropical agriculture, kyushu university, fukuoka, japan. results and discussion among 2550 bait traps, only 77 traps (3.0%) attracted ants during the study period (tuna, 3.7% with 48/1275 traps; honey, 2.2% with 29/1275 traps). among 255 quadrat samplings, we observed foraging ants in 70 quadrats (27.4%). fig 1. map of the study sites in fukuoka city, western japan. isl, island-city central park; kas, kashi-gu shrine; hpm, hakozaki port memorial park; kyu, kyushu university hakozaki campus; mmc, momochi central park; min, minami park. s hosoishi, mm rahman, t murakami, s-h park, y kuboki, k ogata – winter activity of ants in western japan416 further, a total of 435 worker ants (bait, 218 individuals; quadrat, 217 individuals) were observed during this study, belonging to 14 genera and 18 species (table 2). the most abundant species at the open land types were tetramorium tsushimae, messor aciculatus, and pheidole noda. on the other hand, nylanderia flavipes, p. noda, and crematogaster osakensis were most abundant at the forest types. species isl hpm kas kyu min mmc dolichoderinae ochetellus glaber x tapinoma saohime x formicinae formica hayashi x formica japonica x lasius japonicus x nylanderia amia x x nylanderia flavipes x x myrmicinae aphaenogaster famelica x crematogaster osakensis x x messor aciculatus x x pheidole noda x x x x pristomyrmex punctatus x strumigenys lewisi x temnothorax congruus x temnothorax spinosior x tetramorium bicarinatum x tetramorium tsushimae x x x ponerinae brachyponera chinensis 　 x 　 　 　 x total number of species richness 6 3 3 5 5 6 table 2. occurrence of ants in winter season between january 25 and march 13, 2018. fig 2. abundance of foraging worker ants relative to surface ground temperature between january 25 and march 13, 2018. abundances of 18 ant species are plotted together. c od e st ud y si te l an d us e ty pe v eg et at io n ty pe c at eg or y g ps a re a (m ²) e st ab lis he d ag e su rv ey p er io d in 2 01 8 a nt s pe ci es is l is la nd -c ity c en tr al p ar k u rb an o pe n pa rk o rn am en ta l p la nt o pe n la nd 33 °3 9’ 52 ’’ n 13 5° 25 ’1 5’ ’e 10 0, 00 0 20 05 26 th j an ., 9t h, 2 7t h fe b. h pm h ak oz ak i p or t m em or ia l p ar k u rb an o pe n pa rk o rn am en ta l p la nt o pe n la nd 33 °3 8’ 02 ’’ n 13 0° 25 ’0 6’ ’e 10 ,0 51 19 73 25 th , 3 1s t j an ., 14 th f eb ., 6t h m ar . 15 c m m c m om oc hi c en tr al p ar k u rb an o pe n pa rk o rn am en ta l p la nt o pe n la nd 33 °3 5’ 17 ’’ n 13 0° 21 ’0 2’ ’e 40 ,0 13 19 88 8t h, 2 6t h fe b. 17 a, b, c k y u k yu sh u u ni ve rs ity , h ak oz ak i c am pu s u rb an o pe n ar ea o rn am en ta l p la nt o pe n la nd 33 °3 7’ 46 ’’ n 13 0° 25 ’3 3’ ’e 42 6, 00 0 19 11 13 rd , 2 1s t f eb ., 7t h m ar . 10 c m in m in am i p ar k u rb an fo re st p ar k e ve rg re en fo re st fo re st 33 °3 4’ 25 ’’ n 13 0° 23 ’1 4’ ’e 28 0, 36 9 19 41 23 rd f eb ., 13 th m ar . 25 a, b k a s k as hi -g u sh ri ne sh ri ne e ve rg re en fo re st fo re st 33 °3 9’ 12 ’’ n 13 0° 27 ’1 0’ ’e n a 72 4 1s t, 20 th f eb ., 2n d m ar . 32 c t ab le 1 . l oc at io n an d ch ar ac te ri st ic s of s ix s tu dy s ite s of u rb an a re a at f uk uo ka c ity , j ap an : a , o ga ta (1 99 8) ; b , p ar k et a l. (2 01 4a ); c , p ar k et a l. (2 01 4b ). sociobiology 66(3): 414-419 (september, 2019) 417 fig 3. winter activity patterns of the five most abundant ant species to surface ground temperature between january 25 and march 13, 2018. (a), tetramorium tsushimae; (b), messor aciculatus; (c), nylanderia flavipes; (d), pheidole noda; (e), crematogaster osakensis. used the relationship between foraging activity and surface ground temperature. the respective lowest surface ground temperature at which worker ants were active was as follows: t. tsushimae, 11℃–12℃ at the baits and 8℃–9℃ in the quadrats; p. noda, 7℃–8℃ at the baits and 7℃–8℃ in the quadrats; m. aciculatus, 11℃–12℃ at the baits and 6℃–7℃ in the quadrats; n. flavipes, 8℃–9℃ at the baits and no data available in the quadrats; c. osakensis, 13℃–14℃ at the baits and at 11℃–12 ℃ in the quadrats (fig 3). overall, the bait traps did not strongly attract ants during the studied winter season. bait preference slightly varied among the observed species. nylanderia flavipes did not show a distinct preference for tuna or honey baits (wilcoxon signed rank test, t = 16, p = 0.82, n = 8), whereas p. noda preferred tuna baits over honey baits (wilcoxon signed rank test, t = 0, p < 0.01, n = 10). the bait preferences were not available for the remaining species due to a lack of sufficient data. overall, the bait and survey data revealed low levels of winter foraging activity of the indigenous ants of western japan (fig 2). although no winter ants such as p. nitens and p. imparis have been found in fukuoka, western japan, some japanese ant species indicated the temperature-related foraging patterns in the area during winter. figure 3 shows the activity patterns observed for the five most abundant species during the study period. we could not detect distinct activity patterns for the remaining less abundant species due to a lack of sufficient data. foraging activities of worker ants were positively correlated with surface ground temperature (spearman’s rank correlation (n = 85), rs = 0.44, p < 0.01 for t. tsushimae; rs = 0.30, p < 0.01 for p. noda; rs = 0.31, p < 0.01 for m. aciculatus; rs = 0.37, p < 0.01 for n. flavipes; and rs = 0.36, p < 0.01 for c. osakensis). as our analyses did not show a significant relationship between foraging activity and soil temperature for n. flavipes and c. osakensis, we s hosoishi, mm rahman, t murakami, s-h park, y kuboki, k ogata – winter activity of ants in western japan418 the five most abundant species in this study are also the most common ant species observed in the study area during spring to autumn (ogata et al., 1998; park et al., 2014a, 2014b). the number of ant species observed at each study site represented ca. 20%–50% of the total documented fauna at the open land types and ca. 9%–20% of the species diversity documented at the forest types. although the sampling effort differed among the study sites, ant activities seemed to be higher at the open land types than at the forest types. these differences might be driven by the higher number of disturbance-associated species found in the open land types. foraging activity patterns of ants differed among species (fig 3). briese and macauley (1980) found a close relationship between foraging activity and soil surface temperature for harvester and non-harvester ants in semiarid australia. they documented active ant foraging at soil surface temperature above 10℃–15℃. some species, e.g., odontomachus sp. and camponotus sp. a became active above 10℃, whereas camponotus sp. b foraged above 17℃– 18℃ (briese and macauley 1980). similarly, our results indicate slight differences among species. in particular, t. tsushimae, m. aciculatus, p. noda and n. flavipes foraged at lower temperatures, whereas c. osakensis foraged at higher temperature (fig 3). the bait preferences of foraging ants depend on their nutritional needs and the condition in the nest. ant workers select carbohydrate-rich honey to accelerate their activity and collect proteinaceous tuna to develop broods (cook et al., 2010). it should be noted that we did not continuously observe each colony and the following discussion is based on the limited number of observations and mixed samples. the alates of n. flavipes are produced in may-june (japanese ant database group, 2008), suggesting that their colonies need proteinaceous foods during winter to spring. our study showed that n. flavipes equally preferred tuna and honey during the study period. few carbohydrate-rich foods are available during winter. honey was carried to worker ants to accelerate foraging and then tuna was provided for brood development. the ants’ foraging activities seem to be supported by a larger number of worker ants. pheidole noda distinctly preferred tuna over honey, suggesting that the colonies of this species were inclined to brood development using a smaller number of foraging workers during winter. generally, the quadrat observations revealed higher number of individuals than those observed using the bait traps. in the quadrat surveys, no foraging worker ants were observed carrying prey items in our study. however, lőrinczi (2016) found several foraging workers of p. nitens carrying small invertebrates such as springtails and potworms. we found several workers of t. tsushimae and ochetellus glaber on the ground or on stones during sunny days. blatrix et al. (2016) suggested a radiant-energy hypothesis, proposing that the radiant energy that heats foraging substrates is among the factors most constraining ant community diversity. both ant species, t. tsushimae and o. glaber, possibly use radiant energy for foraging outside during winter. invasive alien ant species exhibit temperature-related foraging patterns. linepithema humile was active at ground surface temperatures above 10℃ in hiroshima (touyama et al., 2004), s. invicta at ground surface temperature above 10℃ in texas (wuellner & saunders, 2003) or soil temperature above 15℃ in florida (porter & tschinkel, 1987), and s. geminata at ground surface temperature above 15℃ in texas (wuellner & saunders, 2003). the ministry of the environment, japan (2018) conducted the surveys using bait traps for alien ants in several japanese ports in february 2018, but they did not find the alien solenopsis species during winter sampling periods. the results of the present study support the finding of touyama et al. (2004) that ant activity is almost zero below 10℃ duirng winter in mainland japan. this suggests that colony budding or foraging outside the nest are presumably difficult for s. invicta and s. geminata during winter. if the two solenopsis spp. inhabiting shipping containers venture outside the nest during winter, they will encounter the five most abundant ant species reported in this study. local ant fauna and activities should be investigated in the cold winter season when ant activity is low. the ant fauna of western mainland japan is generally similar to that of eastern mainland japan, but the fauna is quite different in northeastern or southern mainland japan. the information provided by surveys such as those presented in this study will provide a foundational data set for the risk management of invasive alien ant species. acknowledgements we would like to thank anet members for encouragement. we would like to thank anonymous referees for reviewing the manuscript. this is a contribution from the fire ants research group, kyushu university, fukuoka, japan. we would like to thank enago (www.enago.jp) for the english language review. authors’ contribution sh, mmr, tm conceived and designed the study; shp, yk provided samples. sh, mmr performed experiments and analysis; sh analyzed the results. sh, mmr, ko led the writing. all authors read, discussed and approved the final manuscript. references bestelmeyer, b.t., agosti, d., alonso, l.e., brandão, c.r.f., brown jr., w.l., delabie, j.h.c. & silvestre. r. 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(in japanese). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.237-238sociobiology 61(2): 237-238 (june, 2014) presence of strepsiptera parasites in the independent-founding wasp, polistes satan k kudô1, k komatsu1, s mateus2, r zucchi2, fs nascimento2 parasites represent a significant proportion of the total biomass in natural ecosystems, particularly in insect societies (hughes et al., 2008). therefore, they are of great ecoethological significance. manipulative parasites are known to have the ability to redirect host relationships and extend the range of their habitats (lefevre et al., 2009). however, little is known about the abundance of parasites and pressures imposed through parasitism. although our understanding of social behavior has benefited greatly from the use of polistes wasps (reeve, 1991), the incidence of parasitism in this group has yet to be elucidated in detail. studies on parasitism in polistes wasps have mainly focused on the incidence and probable effects of parasitoids belonging to hymenoptera, lepidoptera and less common diptera (makino, 1985; yamane, 1996). these ectoparasitoids, which oviposit on cell walls, typically attach to pupal hosts and consume them, after which they pupate and leave. although the parasitoids of polistes wasps have received less attention in the last few decades, the abundance and ecological impact of the obligatory endoparasites, strepsiptera have been investigated at length in paper wasps (de oliveira & kogan, 1962; hughes et al., 2003; hughes et al., 2004; vannini et al., 2008; kathirithamby, 2009; manfredini abstract although the paper wasp genus, polistes, has been examined extensively, little is known about the occurrence of parasitism in this group. we detected the obligate parasitic insect group, strepsiptera in the gaster of polistes satan bequaert adult females. by dissecting 161 adult females from 24 colonies, we identified a total of four stylopized wasps in three colonies during the wet season. sociobiology an international journal on social insects 1 faculty of education, niigata university, niigata, japan. 2 universidade de são paulo (ffclrp), ribeirão preto, são paulo, brazil. article history edited by gilberto m m santos, uefs, brazil received 07 november 2013 initial acceptance 20 february 2014 final acceptance 06 march 2014 key words social wasp, interactions, parasitism corresponding author kazuyuki kudô laboratory of insect ecology faculty of education, niigata university niigata 950-2181 japan e-mail: kudok@ed.niigata-u.ac.jp et al., 2010; móczár & sziráki, 2011; beani et al., 2011; manfredini et al., 2013). cervo et al. (2000) hypothesized that the rapid spread of the introduced species p. dominulus christ in north america may be due to the lack of parasitization by strepsipterans. hughes et al. (2003, 2004) reported the prevalence of the parasite strepsiptera in some adult and immature polistes wasps. manfredini et al. (2013) recently examined susceptibility towards the parasite strepsiptera at both the individual (immune defense) and colony level, i.e. hygienic behavior (removal of diseased individuals by nestmates) in p. dominula christ. in the present study, we investigated the occurrence of parasitism by the strepsiptera in the neotropical paper wasp, p. satan. this species is an independent-founding wasp that is found in southeastern brazil (carpenter, 1996) and builds abundant nests in dark and sheltered places, such as inhabited human houses, stables, and the hollows of termite nests (richards, 1978). we recently determined the incidence of the hymenopterous parasitoid, pachysomoides sp. (ichneumonidae, cryptinae), in this species (kudô et al., 2013). in late july 2009 (dry season) and late march 2011 (wet season), 8 and 16 p. satan colonies, respectively, were collected at a farm near cajuru (47º15’s, 21º26’w) in são short note k kudô, et al strepsiptera parasites in a social wasp238 paulo state, brazil. a total of 63 and 98 adult females were collected from the dry (7.88±2.61 wasps/colony, mean±se) and wet colonies (6.13±1.71 wasps/colony). the gaster of all wasps collected was dissected under a binocular microscope to determine the existence of the parasite strepsiptera. we detected a total of four stylopized wasps in three colonies during the wet season. the number of strepsipterans was in one each of the three stylopized wasps, but two in the remaining stylopized wasp. although the overall percentage of stylopized wasps was only 2.5% (4 out of 161 wasps), we should have screened adults not only from nests, but also from foraging sites, as described by hughes et al. (2003, 2004). in addition, the dissection of all colony members including immature wasps is needed to evaluate the levels of parasitism by the strepsiptera in more detail in future studies. acknowledgments this study was supported in part by a grant-in-aid for young scientists (b) (no. 21770017) to k. kudô. we thank y. yamaguchi for his kind help during the field collection. references beani, l., dallai, r., mercati, d., cappa, f., giusti, f. & manfredini, f. (2011). when a parasite breaks all the rules of a colony: morphology and fate of wasps infected by a strepsiptera edoparasite. animal behavior, 82: 1305-1312. carpenter, j. m. (1996). distributional checklist of species of the genus polistes (hymenoptera: vespidae; polistinae, polistini). american museum novitates, 3188: 1-39. cervo, r., zacchi, f. & turillazzi, s. (2000). polistes dominulus (hymenoptera, vespidae) invading north america: some hypotheses for its rapid spread. insectes sociaux, 47: 155-157. de oliveira, s. j. & kogan, m. (1962). brazilian strepsiptera (insecta) parasitizing vespidae, with the description of three new species of xenos rossius, 1793 (stylopidae). memórias do instituto oswald cruz, 60: 1-11. hughes, d. p., beani, l., turillazzi, s. & kathirithamby, j. (2003). prevalence of the parasite strepsiptera in polistes as detected by dissection of immatures. insectes sociaux, 50: 62-68. hughes, d. p., kathirithamby, j. & beani, l. (2004). prevalence of the parasite strepsiptera in adult polistes wasps: field collections and literature overview. ethology, ecology and evolution, 16: 363-375. hughes, d. p., pierce, n. e. & boomsma, j. j. (2008). social insect symbionts: evolution in homeostatic fortresses. trends in ecology and evolution, 23: 672-677. kathirithamby, j. (2009). host-parasitoid association in strepsiptera. annual review of entomology, 54: 227-249. kudô, k., komatsu, k., konishi, k., mateus, s., zucchi, r. & nascimento, f. s. (2013). bionomic notes on pachysomoides sp. (hymenoptera: ichneumonidae), a parasitoid of the neotropical social wasp polistes satan bequaert (hymenoptera: vespidae). entomological science, 16: 360-362. lefevre, t., lebarbenchon, c., gauthier-clerc, m., misse, d., poulin, r. & thomas, f. (2009). the ecological significance of manipulative parasites. trends in ecology and evolution, 24: 41-48. makino, s. (1985). list of parasitoids of polistine wasps. sphecos, 10: 19-25. manfredini, f., massolo, a. & beani, l. (2010). a difficult choice for tiny pests: host-seeking behaviour in xenos vesparum triungulins. ethology, ecology and evolution, 22: 247-256. manfredini, f., grozinger, c. m. & beani, l. (2013). examining the “evolution of increased competitive ability” hypothesis in response to parasites and pathogens in the invasive paper wasp polistes dominula. naturwissenschaften 100: 219-228. móczár, l. & sziráki, g. (2011). observation of high degree stylopization of european paper wasp polistes dominula (christ, 1791) in hungary. natura somogyiensis, 19:229-234. reeve, h. k. (1991). polistes. in ross, k. g. & matthews, r. w. (eds.), the social biology of wasps (pp. 99-147). ithaca, ny, cornell university press. richards ow (1978). the social wasps of the americas, excluding the vespidae. london: british museum (natural history). 587 pp. vannini, l., carapelli, a., frati, f. & beani, l. (2008). nonsibling parasites (strepsiptera) develop together in the same paper wasp. parasitology, 135: 705-713. yamane, sô. (1996). ecological factors influencing the colony cycle of polistes wasps. in turillazzi, s. & west-eberhard, m. j. (eds.), the natural history and evolution of paper wasps (pp. 73-98). oxford, oxford university press. doi: 10.13102/sociobiology.v66i4.3576sociobiology 66(4): 610-613 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 social wasps as biological control agents against diaphania hyalinata (linnaeus, 1767) (lepidoptera, crambidae), a cucumber pest in amazonas, brazil diaphania hyalinata (linnaeus, 1767) (lepidoptera: crambidae), popularly known as “melonworm moth”, is considered one of the main pests of the cucumber (cucumis sativus l.) (gonring et al., 2002; 2003), and attacks other curcubitaceae species, such as: melon (cucumis melo l.), watermelon (citrullus lanatus (thunberg) matsumura & nakai), pumpkin (curcubita moschata duchesne) and zucchini (curcubita pepo l.) (fernandes, 1998; costa et al., 2008; santana júnior et al., 2012; panthi et al., 2017). adults deposit egg masses on leaf surfaces, where eggs hatch and larvae begin to feed on leaves, flowers, and fruits (santana júnior et al., 2012; panthi et al., 2017). in fruits, larvae feed on the surface or form galleries, damaging the cucumber pulp (fernandes, 1998) (figure 1). such attacks on fruit accelerate their decomposition due to penetration by saprophyte organisms, which completely rot the fruits and prevent their consumption (gonring et al., 2003; costa et al., 2008). depending on the intensity of infestation, the damage can reach 100% (melo et al., 2011). controlling abstract diaphania hyalinata (lepidoptera: crambidae), popularly known as “melonworm moth”, is considered a main pest of the cucumber, and depending on the intensity of infestation, the damage can reach 100%. herein, we report the predation of d. hyalinata larvae by seven social wasp species: brachygastra lecheguana, polybia dimidiata, polybia ignobilis, polybia liliacea, protopolybia minutissima, synoeca surinama and synoeca virginea, and predation of d. hyalinata pupae by polybia liliacea. we suggest that polybia liliacea, should be considered as a potential biological control for cucumbers, due to its high index of captured prey and intense foraging activity. sociobiology an international journal on social insects gm lourido, t mahlmann, a somavilla, kfg guerra article history edited by gilberto m.m. santos, uefs, brazil received 17 july 2018 initial acceptance 13 november 2019 final acceptance 20 november 2019 publication date 30 december 2019 keywords cucumis sativus, melonworm moth, polistinae, polybia liliacea, predation. corresponding author gilcélia melo lourido instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo, petrópolis cep: 69.067-375 manaus/am, brasil e-mail: gilourido@yahoo.com.br d. hyalinata is difficult because they are multivolt insects with high reproductive capacity and adapt to food resources that fluctuate in terms of quality and availability (guedes et al., 2010). pupae can form on cucumber leaves, soil, or adjacent plants, where the larvae form cocoons by winding a leaf section (capinera, 2004). although d. hyalinata is a widely studied species, due to its agricultural and economic importance, information about its predators is scarce. regarding predation by social wasps, there are only records of d. hyalinata larvae predation by polybia ingnobilis (haliday, 1836), which is the most common, and polybia scutellaris (white, 1841), which seems to be restricted to the atlantic forest (gonring et al., 2002; santana júnior et al., 2012; gonring et al., 2003; jacques et al., 2015). predatory wasps play an important role in the natural control of insect pests (picanço et al., 2012), as they search for and consume prey, adapt to environments, and compete for food, which make them highly efficient (santana júnior et al., 2012). according to prezoto et al. (2008), social wasps instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus, am, brazil short note sociobiology 66(4): 610-613 (december, 2019) 611 capture adult and/or immature insects to feed their larvae. the preference for preying upon lepidoptera is a good indicator that wasps could act as biological control agents for economically destructive caterpillar populations such as spodoptera frugiperda (smith, 1797), chlosyne lacinia saundersi (doubleday,1847), alabama argillacea (hübner, 1823), anticarsia gemmatalis hübner, 1818 and heliothis virescens (fabricius, 1777), which make up the most common pests abundantly found in small farms in neotropical environments such as brazil (prezoto et al., 2019). recently, in amazonas state, predatory activity of polistes canadensis (linnaeus, 1758) on plutella xylostella (linnaeus, 1758) has been observed at a small organic kale plantation (brassica oleracea l.) (montefusco et al., 2017). the importance of social wasps is recognized, however, little is known about their activity as agents of biological control for diaphania species (santana júnior et al., 2012). thus, we report predation of larvae, and, for the first time, pupae of d. hyalinata by social wasps. the present work is the result of collections, observations, and visual records (photos and films) made in january 2015, from a rural cucumber (cucumis sativus l.) plantation located approximately 45 km distant from rio preto da eva, amazonas, brazil, accessed by km 125 of am010 highway, and coordinates -2.842388°s, -59.438138°w. the area consists of a plantation with 2,000 cucumber plants, largely surrounded by primary dense ombrophilous forest, and entirely infested by d. hyalinata larvae and pupae. d. hyalinata and social wasps’ specimens were captured with entomological nets, and the vouchers deposited in the invertebrate collection of the national institute of amazonian research (inpa).the lepidopterans were raised in the laboratory and adults were identified based on arias and clavijo (2001). wasps were identified using richards (1978) and through comparisons with deposited and identified material from inpa’s collection. we observed brachygastra lecheguana (latreille, 1804), polybia dimidiata (olivier, 1792), polybia ignobilis (haliday, 1836), protopolybia minutissima (spinosa, 1851), synoeca surinama (linnaeus, 1767) and synoeca virginea (fabricius, 1804) in larvae only. the wasps were observed preying on fifth instar larvae during the capture, using their posterior legs to catch the larvae, and their mandibles during flight to stabilize the prey (figura 2a). featured, we observed polybia liliacea (fabricius, 1804) preying on larvae and pupae of d. hyalinata (figure 2a, b, c, d) (film supplementary), representing the first record of d. hyalinata pupa predation by social wasps.these pupae were found in large clusterson dried cucumber leaves, sharing the location (figure 2c). since we found no information about behavior of d. hyalinata pupae in the literature, we considered leaves as the pupation site. during foraging, wasps approached the pupation site and groped the leaves to locate the pupae (figure 2c), opened the cocoon, and captured the insect, using their posterior legs and mandibles, similar to larvae capture. apparently, d. hyalinata predation, as well as other agricultural pests of social wasps, do not present highly specialized relationships, and may in fact be more related to the original distribution of the social wasp species (carpenter & marques, 2001). herein, most species are typically from the amazon rainforest, except for brachygastra lecheguana and polybia ignobilis, which are widely distributed in the neotropics. this information is relevant since identifying and studying these predatory insects, especially in agricultural environments, are the first steps to identifying the best species to use in biological pest control (jacques et al., 2015). in this way, such information can support control strategies for d. hyalinata, associated with integrated pest management. acknowledgments. we thank the fundação de amparo à pesquisa do estado do amazonas (fapeam) for financial support trough proc. 062.02215/2014. fig 1. diaphania hyalinata larvae forming galleries inside the cucumber fruits. gm lourido, t mahlmann, a somavilla, kfg guerra – social wasps prey on cucumber moth pest in the amazon 612 references arias, q. & clavijo, j. 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(2012). natural biological control of diaphania spp. (lepidoptera: crambidae) by social wasps. sociobiology, 59: 561-571. supplementary file (film) http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/3576/0 1: polybia liliacea preying on diaphania hyalinata larvae. doi: 10.13102/sociobiology.v66i4.3576.s2103 2: polybia liliacea preying on diaphania hyalinata larvae. doi: 10.13102/sociobiology.v66i4.3576.s2658 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3338sociobiology 66(1): 113-119 (march, 2019) bionomic aspects of the solitary bee tetrapedia diversipes klug, 1810 (hymenoptera: apidae: tetrapediini) introduction the solitary bees comprise approximately 85% of the total bee species in the world (batra, 1984). in the habit of solitary life, females can exhibit complete independence, both in building and provisioning their nests, that is, there is no collaboration or division of labor between females, or between mothers and daughters (michener, 2007). among the bees with a solitary life style, there are those belonging to tetrapediini, which are restricted to the tropical regions of the americas and are distributed in two genera: tetrapedia and coelioxoides (alves-dos-santos et al., 2002). in brazil, the genus tetrapedia is represented by 28 species (moure, 2012), whose biology has been studied through the provision of artificial nests (trap nests) in the study areas because females nest in preexisting cavities. this method allows to obtain information about the bionomy of these bees, such as the material used in nest building, nest abstract the study aimed to describe bionomic aspects of tetrapedia diversipes klug, 1810 in order to allow the conservation or breeding of these bees. the nesting biology was studied using trap nests made with cardboard-paper tubes and plastic straws during the period between december 2014 and september 2015, in the bee unit of the federal university of ceará, brazil. a total of 135 nests were obtained with 593 brood cells, from which emerged 448 adults and individuals of two parasitic species. the proportion of females (n = 297) was higher than that of males (n = 151) resulting in a sex ratio of 1.97:1. the total mortality in the nests was 24.45% and the majority of deaths recorded occurred at the pupal stage (33.1%). during their reproductive life, females of this bee species were able to construct up to 16 brood cells. the trap-nests removal and transfer to the laboratory shortly after finished by the bees reduced the attack of natural enemies. sociobiology an international journal on social insects am cavalcante1, ci silva2, ams gomes1, gs pinto1, iga bomfim1, bm freitas1 article history edited by eduardo almeida, usp, brazil received 11 april 2018 initial acceptance 30 june 2018 final acceptance 14 december 2018 publication date 25 april 2019 keywords nest architecture, trap-nest, multivoltine bees. corresponding author arianne cavalcante universidade federal do ceará departamento de zootecnia laboratório de abelhas av. mister hull, s/nº, pici cep: 60455-760 fortaleza/ce, brasil. e-mail: arianne.mc@gmail.com architecture, sex ratio, natural enemies and pollen resources used in feeding the immature (alves-dos-santos, 2002; camillo, 2005; alves-dos-santos et al., 2007; neves et al., 2014). studies carried out in southeastern brazil using this technique allowed information on the behavior and nesting biology of some bee species belonging to tetrapedia (camillo, 2005; menezes et al., 2012; cordeiro et al., 2011; rochafilho & garófalo, 2015). however, the way these bees establish themselves in northeastern brazil is still poorly described (aguiar et al., 2005; neves et al., 2012). in this context, the aim of this work was to study the bionomy of the species tetrapedia diversipes klug, 1810 by the use of trap nests, considering aspects such as nesting rate; seasonality and nest architecture; development and emergence period; sex ratio (m: f); mortality rate and associated natural enemies. moreover, the knowledge of the bionomic aspects of this species will provide information for its conservation and the potential development of rational breeding of these bees 1 universidade federal do ceará, fortaleza, ceará, brazil 2 universidade de são paulo, butantã, são paulo, brazil research article bees am cavalcante, ams gomes, gs pinto, ci silva, iga bomfim, bm freitas – biology of nesting of tetrapedia diversipes114 for use in pollination services and for the development of management techniques for the maintenance of these native populations inserted in large urban centers. material and methods study area the study was carried out in the bee unit of the federal university of ceará, fortaleza (3°44’33.70 “s and 38°34’45.46” w), brazil. the climate of the region is warm subhumid tropical, with a rainy season from january to may. the mean annual precipitation is 1,338 mm and the mean temperatures vary between 26°c and 28°c. the study site is surrounded by urban areas and an altered, small fragment of forest with a vegetation characterized as vegetational complex of the coastal zone and perennial forest paludosa marítima (ipece, 2016). in this area it is possible to record the presence of native and exotic plant species. trap nests (tns) trap nests (tns) for tetrapedia diversipes nesting were made using two types of disposable cylindrical tubes. one type was made of black cardboard, measuring 5.0 mm in diameter, and the other type was made of transparent plastic straw, 4.5 mm in diameter. both tns were 12 cm long with one end closed with stingless bee wax. these nests were inserted into each of the 548 pre-existing cavities distributed along nine blocks of wood containing 6 to 10 cavities per line and were located more than 90 cm above the ground, where they were protected from rain and sun. the tns of cardboard were evaluated from december 2014 to september 2015, while plastic straw tns were installed in only one of the nine blocks used in the study; for this, 36 tns of cardboard were replaced by the transparent plastic straws which were evaluated only from june to october 2015, in order to observe the nesting behavior and the time taken in the construction of the brood cells. the tns were inspected daily and, when finished, they were removed and replaced by new empty ones. nests founded in cardboard were taken to the laboratory and were kept within a biochemical oxygen demand (b.o.d.) at 27°c until adults emerged. nests founded on straws were also placed in the b.o.d., but only until the larvae defecated. at this moment, immatures were transferred to artificial cells made of transparent biodegradable capsules to complete their development allowing observations on the development time of males and females. to determine the sex ratio both individuals emerging from cardboard tns and artificial cells were counted. one male and one female from each nest were killed and subsequently sent to a taxonomist to confirm the species identification; the other individuals were released in the wild at the nesting site. bees collected in this study are deposited in the bee unit entomological collection of the animal science department, at the federal university of ceará. number of nests built by each female of tetrapedia diversipes the number of nests founded by each female during their period of activity was counted through the numerical labeling of 80 bees done after their emergence from the tns (fig 1). for this, emerging bees were placed in falcon tubes and anesthetized by cooling in the freezer. after this procedure tags were fixed in the dorsal part of the thorax, between the wings, using super bonder loctite® glue brand. after being tagged, these bees were released near the place where the tns were installed. subsequently, it was observed whether these bees returned to nest in tns and how many nests were built by them. these observations were made daily in the morning and afternoon shifts for two hours of each monitoring period, totaling 456 hours of observation. nest architecture and time of immature development of tetrapedia diversipes after the emergence of the adult individuals, the tns were opened to determine the length, number of built cells, the presence of dead individuals, and the number of empty cells. the same procedure was performed with tns from which no individuals emerged after four months of incubation. in order to determine the immature development time, 24 nests built in the transparent plastic straws were opened 17 days after the nest was completed by the female when the larva present in the first cell had already defecated. these older larvae were placed in capsules measuring 24.6 mm in length and 7.7 mm in width (fig 2), as proposed by silva et al. (unpublished data). immatures were transferred according to the order arranged in the nests and deposited in b.o.d. to complete their development. observations of these capsules were performed daily. fig 1. tetrapedia diversipes female labelled with a numerical tag. bee unit, animal science department/ufc, fortaleza-ce, brazil 2015. sociobiology 66(1): 113-119 (march, 2019) 115 a mixture of pollen and oil in the interior of the cell, and after finishing to provisioning all the larval food, she laid a large egg on the food mass and closed the cell with a thin sand wall. this process was repeated in all subsequent cells, always from the furthest to the nearest to the nest entrance (n = 118). the time spent in building the first cell was three days in all observed tns, while for the other cells the female bee took from two to four days (n = 94). the total construction time in tns varied from three to fifteen days, according to the number of cells built per nest (n = 24). the number of nests established by each t. diversipes female was variable: out of the 80 tagged females 44 returned to the nests (55%), of which only 11% (n = 5) nested in the tns available. each monitored bee built from two to three nests during its reproductive life resulting in the production of 11 to 16 cells per female (table 1). nest architecture was observed in all nests obtained during the study. the nests were linear with brood cells built one after the other. the number of cells per nest ranged from one to eight, with a total of 593 cells for all studied nests. these cells were always arranged in a linear series and their lengths varied from 4.96 to 11.39 mm (= 7.66 ± 1.12). in the analyzed nests, the presence of vestibular cells, which corresponds to the empty space between the nest closure and the last brood cell, was observed in 48.15% of the nests (n = 65) and their length varied from 6.26 to 51.00 mm (= 31.71 ± 12.80). fig 2. a) transference of an immature tetrapedia diversipes from a nest built in plastic straw to a rearing capsule. b) larvae in the capsule identified according to its psoition inside the nest. bee unit, animal science department/ufc, fortaleza-ce, brazil 2015. data analysis nesting data were evaluated using descriptive statistics through tables and graphs generated in the microsoft excel software version 2010. the sex ratio was obtained dividing the number of females by the number of males and a chi-square test (x2) was used to analyze whether the proportion of males and females differed from that expected (1:1). in order to analyze the relationship between nesting ratio (%) and environmental variables (temperature, rainfall, and relative air humidity), the pearson correlation test was used in software r. precipitation had to be log-transformed to reach the normality. results nesting rate, seasonality and nest architecture during this study 135 nests of t. diversipes were founded, representing approximately 25% of the total number of tns (n = 548) available. the bee species centris analis and epanthidium tigrinum and two species of wasps, trypoxylon sp. and podium sp., also nested in the trap nests, but were not considered in this study. tetrapedia diversipes females occupied tns throughout the studied period, but nesting peak occurred from the rainy season to the beginning of the rainydry transition period, between march and june 2015 (fig 3). although the highest nesting rate occurred in march (5.29%) and june (3.83%), no significant correlations were observed between the monthly nesting rate (%) with temperature (r = -0.1635, p = 0.6517), monthly rainfall (r = 0.4285, p = 0.2166) (fig 3) and air humidity (r = 0.4954, p = 0.1454). brood cell construction was initiated after the t. diversipes female selected a cavity. then, the female began to build the first partition, in what would become the back of the nest, using a mix of sand grains collected near the nesting site, and an oily binder substance, the composition of which has not been determined. the cell partition, which is a structure built between two brood cells organized in a linear series, serves several purposes such as to insure each larva an adequate amount of food, to protect the immature from natural enemies and to indicate to the emerging bee the direction to leave the nest. after building the partition, the female made a series of foraging trips to collect and place a b fig 3. nesting rate of tetrapedia diversipes and environmental variables: a) mean temperature (°c) e b) monthly rainfall (mm) between december 2014 and september 2015. bee unit, animal science department/ufc, fortaleza-ce, brazil 2015. am cavalcante, ams gomes, gs pinto, ci silva, iga bomfim, bm freitas – biology of nesting of tetrapedia diversipes116 developmental period, emergency and sex ratio the time for immature development in t. diversipes was obtained from the larvae (n = 113) which were taken from the plastic straw tns (n = 24) and incubated inside the transparent biodegradable capsules. the immatures transferred to the capsules were in the last larval stage and took 20 to 32 days to become pupae. after becoming pupae, they took another 15 to 20 days to turn into pre-emergent adults, waiting only to complete the sexual dimorphism. the total development time of females varied from 40 to 66 days ( = 49 ± 5.6, n = 66), while in males this time ranged between 40 and 58 days ( = 47 ± 4.9, n = 31). out of the 593 brood cells built in the two types of tns studied, 448 adults of t. diversipes emerged, of which 151 were males (33.7%) and 297 were females (66.3%), resulting in a sex ratio 1:1.97 (m:f), differing significantly of the proportion 1: 1 (x2 = 47.58, p <0.0001). the emergence of females was higher than that of males in most of the studied period, with the exception of january, when there was no emergence. in contrast, the highest number of emergences for both sexes was observed in may, counting for 21.2% of registered emergencies (fig 4). mortality and associated natural enemies mortality recorded in nests of t. diversipes represented 24.45% (n = 145 cells) of the total number of cells constructed (n = 593). in the 145 cells where mortality was observed, immature developmental failure occurred in 67.6% of them (n = 98 cells), and this process was higher at the pupal stage, reaching 33.1% (n = 48 cells), followed by the phase of preemergent adults, when 20% (n = 29 cells) died. however, mortality reached only 4.5% (n = 21 cells) in the larval period. larvae or immatures that died in the cells due to the attack of natural enemies represented 31% (n = 45 cells) of the built cells, out of which 13.8% (n = 20 cells) were caused by ant attacks and 17.2% (n = 25 cells) due to fungi infection. however, the nests were already with fungi when they were opened and so we can not tell if they caused the death of the immature or if they came later. the mortality caused by cleptoparasites species was only 1.4% (n = 2 cells) caused by a bee species, coelioxys sp. (hymenoptera: megachilidae) and a species of diptera of the family bombyliidae. discussion data obtained in the present study showed that t. diversipes females nested during the whole study period (december to september). the higher percentage of nests was built in march (rainy season) and june (beginning of the dry season), probably due to the greater availability of resources provided by flowering plants in these months, such as dalechampia sp, whose pollen is used in the supply of the breeding cells (menezes et al., 2012). no significant relationship was found between the monthly nesting rate and the climate variables. similar data were recorded with the same bee species in the states of são paulo, rio de janeiro (se brazil) and bahia (ne brazil) by aguiar et al. (2005), camillo (2005), menezes et al. (2012), and rocha-filho and garófalo (2015). the studies carried out by alves-dos-santos et al. (2002) indicate that t. diversipes generations often reuse the nests in their birth places. however, these authors did not tag the emergent bees and did not provide data if founders of the new nests were actually bees that emerged from the same nests. although the nest reuse was confirmed for t. diversipes in the present study, it was found that only a fraction of the post-emergent females re-used nests. the low percentage of tns reused by the females of t. diversipes may be related to the interference caused by the handling of females and to the existence of tagged females t.n. founded by females total cells by females emerged bees total of emerged individuals mortality ♀ ♂ ♀ 20 2 13 7 4 11 2 ♀ 33 3 11 5 3 8 3 ♀ 54 2 11 6 2 8 3 ♀ 56 3 16 6 8 14 2 ♀ 77 2 12 5 3 8 4 mean ± sd 6 ± 0.84 4 ± 2.35 10 ± 2.68 3 ± 0.84 table 1. number of nests, brood cells built, and total offspring per female of the solitary bee tetrapedia diversipes between december 2014 and september 2015. bee unit, animal science department/ufc, fortaleza-ce, brazil 2015. fig 4. emergence of females and males of tetrapedia diversipes from january to november 2015. bee unit, animal science department/ ufc, fortaleza-ce, brazil 2015. sociobiology 66(1): 113-119 (march, 2019) 117 potential nesting sites, represented by natural cavities around the experimental area. however, the availability of these cavities was not investigated during this study. in respect to the nest architecture, the structure of each brood cell was built with a mixture of sand and oil, similar to that described by alves-dos-santos et al. (2002). the nests had one to eight cells, and this same pattern in number of cells per nest was reported by camillo (2005) and menezes et al. (2012). however, aguiar et al. (2005) observed a pattern of two to nine cells per nest of t. diversipes. these differences in the number of cells constructed per nest are possibly due to the size of the trap nest available to the bees and the size of the offspring a female bee produces during her reproductive life, as we observed by marking the newly emerged females. the total time for immature development in t. diversipes observed in the present study showed that males and females required a maximum time of 2 months to complete their cycle, suggesting that these individuals did not undergo diapause. this result differs from those obtained by alves-dos-santos et al. (2002) and aguiar et al. (2005), who recorded a longer development time of 6 to 8 months, inferring that in their studies immature specimens of t. diversipes presented pre-pupal diapause. it is worth noting that in the work of these authors, the nests of t. diversipes were kept at room temperature, and this probably contributed to the difference in the results obtained, since, in the present study, the nests were kept under constant temperature. according to aguiar and garófalo (2004), the occurrence of diapause in solitary bees is a protection mechanism to overcome the adverse conditions of a certain period, which does not occur within a controlled environment. the sex ratio may be influenced by the availability of resources in the environment and a greater proportion of either sex may be directly associated with this factor (marques & gaglianone, 2013). in this study, the sex ratio resulting from our tns showed higher production of females, differing from the results found in other surveys with t. diversipes, where males emerged in greater numbers (menezes et al., 2012; rocha-filho & garófalo, 2015). in other bee genus such as centris (mendes & rego, 2007) and xylocopa (pereira & garófalo, 2010), a more inclined sex ratio for females was also observed. according to michener (1974) females have the ability to control the sex of their offspring, but the factors that lead them to decide which egg will be fertilized is still unknown. the difference in the production of males and females observed in several bee species is usually attributed to high rates of parasitism (aguiar & martins 2002), a fact not observed in this study, and to several factors such as breeding mortality, seasonality, climate, availability of substrates and floral resources (michener, 1974; capaldi et al., 2007). according to torchio and tepedino (1980), the sex ratio can be considered an annual balance point, that is, if a greater investment is made in offspring in favor of a specific sex, this proportion must at some point be corrected in the following generations. according to pereira and garófalo (2010) this is an important characteristic when aiming at pollination because bee species with female-biased ratio can increase their populations quickly in the places where they are introduced. regarding the causes of bee mortality in the nests, they occurred due to failures in the development of the offspring and natural enemy attacks, which are classified according to their behavior in nest parasites, kleptoparasites or parasitoids (alves-dos-santos, 2002; oliveira et al., 2014). in the study of alves-dos-santos et al. (2007), the mortality rate among species of tetrapedia ranged from 20 to 40%, ratifying our findings. the highest incidence of mortality recorded for t. diversipes in the present study occurred at the pupal stages and at the end of the bee development, in the phase of preemergent adults. a higher number of deaths in immature stages was also reported by camillo (2005) and rochafilho and garófalo (2015), in studies with the same bee species. according to aguiar et al. (2013) the mortality of bees’ offspring in the early stages of development may be influenced by the manipulation and nest removal when they are transported to the laboratory. in the present study, the attack by cleptoparasites of t. diversipes was not an important mortality factor. nests parasitized by the bee coelioxys sp. (hymenoptera: megachilidae) and the bombyliidae (diptera) caused the death of only two individuals (1.4%). similar results were also found by aguiar and martins (2002) and menezes et al. (2012). although the kleptoparasitism associated with t. diversipes is frequently by coelioxoides (alves-dos-santos et al., 2007; rocha-filho et al., 2017), the occurrence of kleptoparasites of this genus was not observed in this study. however, the presence of coelioxys sp. (hymenoptera: megachilidae) in one of the tns was probably due to the presence of nests of centris analis (hymenoptera: centridine) in the study area. species of this same parasitoid were observed by camilo (2005) in t. diversipes, t. garofaloi, t. curvitasis and t. rugulosa nests. according to gerling and hermann, (1976) and boesi et al. (2009), females of bombyliidae most of the time, do not enter the nest of the female founder, they usually hover and oviposit at the entrance of the nest and when their larvae hatch find the host cells and feed in both larva and pupa and pollen. brood mortality due to fungi infection was diagnosed in studies carried out on other bee species nesting in trap nests (morato, 2001; camarotti-de-lima & martins, 2005; bernardino & gaglianone, 2008). according to roubik (1989), fungi can cause high mortality rates in nests of solitary bees, being considered a potential natural enemy of these bees. however, they were not a major problem in the present study, probably due to the controlled environmental conditions in which the nests were incubated. predatory ants were responsible for the death of 13.8% of the immature individuals of t. diversipes, attacking the trap nests and killing the larvae and plundering the pollen. am cavalcante, ams gomes, gs pinto, ci silva, iga bomfim, bm freitas – biology of nesting of tetrapedia diversipes118 predation of nests by ants was also recorded in studies by pinto (2005) and oliveira et al. (2014) using trap nests. finally, we can conclude that the use of cardboard trap nests and, in particular, those of plastic straw, made it possible to determine the time required to a t. diversipes female builds a brood cell, as well as the number of cells per nest and number of nests throughout its reproductive life. the removal of nests (tns) from the nesting site as soon as finished by the founder bee may contribute for a reduction in parasitism without compromising the habit of reusing nests by the bees, although in a low proportion. thus, this study amplifies the knowledge about the nesting biology of t. diversipes, but more studies on the bionomy of this species are necessary, aiming at the establishment of more accurate management techniques to this bee species. acknowledgment arianne m. cavalcante is grateful to patrícia dos santos vilhena of the faculty of philosophy, sciences and letters of ribeirão preto, university of são paulo (ffclrp/ usp) for identifying the tetrapedia specimes and capes for the granting of the masters’ scholarship. breno m. freitas thanks cnpq, brasília for the productivity grant in research (302934/2010-3). references aguiar, a.j.c. & martins, c.f. 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(2017). coelioxoides piscicauda sp. nov., a new cuckoo bee from southeastern brazil with a key to the species of coelioxoides cresson (hymenoptera: apidae). zootaxa, 4363: 535–543. doi: 10.11646/zootaxa.4363.4.5. torchio p. f. &amp; tepedino v. j. (1980). sex ratio, body size and seasonality in solitary bee, osmia lignaria propinqua cresson (hymenoptera: megachilidae). evolution, 34: 9931003. doi: 10.1111/j.1558-5646.1980.tb04037.x. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i2.183-189sociobiology 60(2): 183-189 (2013) differences of the daily flight activity rhythm in two neotropical stingless bees (hymenoptera, apidae) ms gouw, m gimenes introduction bees from meliponini tribe manifest elaborated social behaviour and are characterised by having perennial colonies, presenting morphological and behavioural differences in the female castes, and some workers forage on floral resources that are important for the colony, especially for feeding the offspring (michener, 2000). these foraging activities may occur at specific times of the day, as has been observed in studies on flight activities of several stingless bee species (hilário et al., 2003; pierrot & schlindwein, 2003; fidalgo & kleinert, 2007). flight activities may be related to a range of intrinsic and extrinsic factors. among the intrinsic factors, the colony’s reproductive phase may be related to flight activity. nunes-silva et al. (2010) observed that plebeia remota (holmberg) foragers collected more pollen in those months that corresponded to the colony’s reproductive season, generally the summer, and collected more nectar in the reproductive diapause (the winter months). abstract stingless bees are mainly tropical and subtropical, eusocial bees and perform activities that are both internal and external to the nest. this study aims to investigate and compare the daily flight activities of melipona scutellaris latreille and frieseomelitta doederleini (friese). the daily flight activities of the two meliponini species was regular for both initial and final activities and for preferential time of activity throughout the months, which may indicate the presence of a biological rhythm synchronized by the daily light-dark cycle. temperature, light intensity and relative humidity probably influenced the rhythms of activity during the day, in a way that may act as a modulator of bee rhythms. m. scutellaris was the larger and darker bees and showed earlier activities and in lower temperatures when compared to f. doederleini, which were smaller bees. sociobiology an international journal on social insects universidade estadual de feira de santana, feira de santana, bahia, brazil research article bees article history edited by: astrid de m. p. kleiner, usp, brazil received 20 february 2013 initial acceptance 30 march 2013 final acceptance 21 may 2013 keywords: biological rhythm, meliponini, melipona scutellaris, frieseomelitta doederleini corresponding author miriam gimenes departamento de ciências biológicas universidade estadual de feira de santana av. transnordestina s/n novo horizonte feira de santana, bahia, brazil 44036-900 e-mail: mgimenes@uefs.br bee flight activities at specific times of the day may be manifestations of rhythms resulting from the endogenous biological clock, as reported in several insect studies (koukkari & sothern, 2006). biological rhythms related to daily activity have been detected in scaptotrigona aff. depilis (moure) (bellusci & marques, 2001) and in frieseomelitta varia (lepeletier) (almeida, 2004; oda et al., 2007). extrinsic factors, particularly abiotic factors (temperature, light intensity and relative humidity), may also modulate the daily flight activities of eusocial bees. studies of the external daily activities of melipona asilvai moure (souza et al., 2006) and plebeia pugnax moure (in litt.) (hilário et al., 2001) have detected the effects of temperature leading to an increase and the effects of relative humidity leading to a decrease of bee activities. the interaction between the pattern of temperature change in the environment and the endothermic ability in bees has strong implications for when and where a particular bee can fly and thus to the resulting pattern of activity (willmer & stone, 2004). according to heinrich (1993), ambient temperature in relation to certain bee morphology ms gouw, m gimenes daily activity rhythm in stingless bees184 characteristics, such as colour and size, may, through the thermoregulatory mechanism, influence the timing of bee flight activity, so the smallest bees generally have a higher heat production rate per unit mass than the greatest bees. the timing of eusocial bee activity to collect resources may also be influenced by the time at which this resource is available in the environment (roubik, 1989). in this work, we studied the differences in the daily rhythm of flight activity of melipona scutellaris latreille and frieseomelitta doederleini (friese), and the influence of environmental and meteorological factors. we also investigated the relationship between the daily rhythm and body size of these two stingless bees. materials and methods studied species in order to conduct the study, we selected two colonies of bees from meliponini belonging to two different species: m. scutellaris and f. doederleini, located in an apiary in the village of pedra branca. bees from the two colonies were captured in areas near the apiary, where they were kept in wooden boxes covered with asbestos shingle, for at least one year. both nests were close to each other and on a wood shelf outdoors. the bees were not fed artificially during the study period. to analyse bee body size, measurements were made of the thorax width (inter-tegular width distance between wing bases) in 10 forager individuals from each species. according to cane (1987), this is the more reliable measure to consider bee body size. bee size classification was based on the parameters of roubik (1989) and michener (2000) and was statistically tested by analysis of variance (anova) and the tukey-kramer multiple comparison test. samples of the two meliponini species were collected and deposited in the johan becker entomological collection of the museu de zoologia da universidade estadual de feira de santana (mzfs). study area pedra branca (12º44’30’’s, 39º34’50’’w, at an altitude of 300 m) is located in santa terezinha town, in the foothills of the jibóia mountain range (bahia state, brazil). the predominant vegetation in pedra branca is the opened arboreal caatinga (dryland) with palm trees (sei, 2012). the santa terezinha town presents climate transition from sub-humid and dry to semi-arid, with average annual temperature ranging between 19oc and 27oc and annual rainfall from 800 to 1100 mm (sei, 2012). daily relative humidity and temperature data were collected with a digital thermo hygrometer and light intensity data was measured using a digital luximeter (lutron lx-107) at a distance of 100 cm from the soil, at the entrance to the studied colonies and in the nearest open air area. during the study period, the daily temperature varied from 19 to 36°c (26.5 ± 2.9°c, mean ± sd). the highest average temperature was recorded between november and january 2010 (28 ± 3.3°c) and the lowest average temperature was recorded in march 2010 (25 ± 1.9°c). the relative humidity varied from 39 to 99% (67 ± 13.7%) and the light intensity in the open air area from < 1 to 82,700 lux. sunrise and sunset times were obtained from the brazilian national observatory (http://euler.on.br/ephemeris/index.php). time of sunrise varied between 05:01 h (november 2009) and 06:00 h (july 2009) and time of sunset varied between 17:20 h (may 2010) and 18:13 h (january 2010). flight activity we quantified the flight activity of the colonies of the two species of studied bees by counting the bees that entered and exited the nest (without apparently carrying material), that entered with pollen, with resin on the corbiculae. observations and activity counts were conducted over three consecutive days in seven months (july, august, september and november 2009 and in january, march and may, 2010), from 04:00 to 19:00 h, for fifteen minutes in each hour of observation for each bee colony. for the two studied species, analyses of the daily activities external to the nest were carried out using the rayleigh test of circular statistical method (batschelet, 1980). preferential times for activities (or acrophases) were considered for values with the vector (r) significant above 0.7 (varying from 0 to 1, according to the dispersion of data) and p < 0.05. analyses were only carried out for values (number of individual bees) above 10. the daily activities of both colonies were compared using the watson-williams test (p < 0,05) (circular statistical method batschelet, 1980). results the two meliponini species had different average widths, 2.9 ± 0.11 mm for m. scutellaris, considered to be medium-small, and 1.3 ± 0.04 mm for f. doederleini, which is considered to be small (difference observed through anova and tukey-kramer multiple comparison test, p < 0.05). melipona scutellaris and f. doederleini occupied all the space within the wooden boxes and had many pots of food. additionally, in all the months of the study, the colonies of m. scutellaris and f. doederleini demonstrated a large number of total activity (entrance + exit) and entrance with pollen. in general, when compared to f. doederleini, m. scutellaris collected less resin (table 1). m. scutellaris and f. doederleini presented a higher number for total activity and entrance activity with pollen in january and march 2010. m. scutellaris also had a high number for bee entrance with pollen in august sociobiology 60(2): 183-189 (2013) 185 table 1. number of bee entrances and bee exits (total activity) and entrance activity with pollen and entrance activity with resin for melipona scutellaris and frieseomelitta doederleini , in the village of pedra branca (santa terezinha, bahia, brazil). months melipona scutellaris frieseomelitta doederleini (total) (pollen) (resin) (total) (pollen) (resin) july/09 3911 576 80 1231 196 565 aug/09 3054 2144 4 846 188 748 sept/09 3510 742 15 625 134 806 nov/09 6545 844 84 995 176 1309 jan/10 8804 1852 44 2115 508 2232 mar/10 12053 1431 75 1713 598 1860 may/10 1626 92 20 241 13 359 2009 (table 1). melipona scutellaris began their total daily flight activities (entrance + exit) and entrance activity with pollen between 04:00 (november) and 06:00 h (july) (fig 1) coinciding with the time of sunrise, which occurred earlier in november and later in july. furthermore, temperature values were higher in the former than in the later month. the initiation of total flight activities and entrance activity with pollen for f. doederleini occurred between 05:00 (november and january) and 08:00 h (july), being overall later than those of m. scutellaris. (fig 1). overall, throughout the study months, the temperature during the first flight activities of the two bee species varied from 19 to 26°c (average: 23 ± 1.4°c). m. scutellaris exited the nest with an li (light intensity) varying between < 1 to 290 lux at nest door and from < 1 to 4,500 lux in the open air. higher values occurred in the first activities of exiting the nest for f. doederleini. these bees exited the nest with an li varying between 10 to 4,300 lux at nest door and from 290 to 19,200 lux in the open air. in most of the observation months, the final times for entrance activity for m. scutellaris (17:00 – 18:00 h) coincided with sunset. these bees returned to the nest when the li was low, around 1 lux. the final times for the daily flight activities of f. doederleini occurred approximately one hour earlier than for the other species (fig 1), when the light intensity and temperature values were higher. a daily activity rhythm for bee entrance and bee exit in the nest (without visible material) was detected for m. scutellaris in most of the study months, with highest activity (or acrophases) occurring during the morning, between 06:10 h (november 2009) and 08:35 h (july 2009), with the exception of march 2010, when the acrophases were observed during the afternoon, varying from 12:45 h (exit) and 13:35 h (entrance) (table 2). during the observed days in march it was noticed a lot of rainfall. in relation to entrance activity with pollen, the acrophases for this species was in the morning, with times varying from 06:20 h (may 2010) to 08:50 h (july 2009) (table 2). f. doederleini’s acrophases for the activities of exit, entrance and entrance with pollen always occurred later than for m. scutellaris in all the study months and were mainly concentrated at the end of the morning (table 2). for entrance and exit activities, the acrophases varied from 08:00 h (september 2009) to 11:35 h (march 2010). preferential times for entrance activity with pollen occurred during the morning, varying from 08:50 h (january 2010) to 10:50 h (august 2009) (table 2). fig 1 number of individuals of melipona scutellaris and of frieseomelitta doederleini entering with pollen in the nest throughout the day (between 400 and 1800 h) from july 2009 to march 2010 in the village of pedra branca (santa terezinha, bahia). ms gouw, m gimenes daily activity rhythm in stingless bees186 table 2: acrophase times of exit and entrance activity (without visible material); entrance activity with pollen and entrance activity with resin for melipona scutellaris and frieseomelitta doederleini in the indicated months during 2009 and 2010, in the village of pedra branca (santa terezinha, bahia). bee/activity melipona scutellaris frieseomelitta doederleini exit entrance polen resin exit entrance polen resin jul/09 07:00 08:35 08:50 08:15 11:00 11:30 10:30 13:20 aug/09 * * 07:35 10:30 10:00 10:50 11:25 sep/09 07:20 * 06:45 08:05 10:45 08:00 10:15 12:25 nov/09 06:10 06:50 * 05:10 08:40 * 10:00 * jan/10 * * * 06:25 * * 08:50 * mar/10 12:45 13:35 08:00 07:00 11:35 10:40 12:50 may/10 07:05 07:25 06:20 11:00 12:10 *values for non significant mean vector (r)(below 0.7). during m. scutellaris acrophase times for the activities of exit, entrance and entrance with pollen, the temperature varied from 21 to 29°c (average 24 ± 2.0°c), the relative humidity from 56 to 99% (average 80 ± 9.8%) and the light intensity from 70 to 10,900 lux at nest door. for f. doederleini, the temperature varied from 24 to 29.3°c (average 27 ± 1.3°c), the relative humidity from 49 to 98% (average: 66 ± 12%) and the light intensity (nest door) from 180 to 6,900 lux. melipona scutellaris acrophases for entrance activity with resin varied from 05:10 h (november 2009) to 08:15 h (july 2009), while for f. doederleini this activity occurred between 11:25 h (august 2009) and 13:20 h (july 2009). discussion among the meliponini colonies studied in pedra branca regular flight activity times were observed, both for their first activities and in the acrophases of most activities. this regularity may be related to several factors that are both extrinsic (abiotic) and intrinsic (biotic) to the bees or to an association between these factors. several factors may determine the regularity of times for daily activities of bees. some authors explain the regularity of time of these organisms through time-space learning, which attributes learning to the ability to return to sources of food at regular times. in stingless bees, breed et al. (2002) state that trigona amalthea (olivier) is capable of associating location of food with its time of exposure. in addition, biesmeijer et al. (1998) observed that melipona spp.’s daily foraging activity is based on both previous experience, through which the bees are capable of retaining information about the external environment, and on social interactions within the colony. this regularity, both in times for the beginning and end of activities and, mainly, in the flight acrophases of meliponini species studied in pedra branca, may be related to the pres-in pedra branca, may be related to the pres-, may be related to the presence of a daily activity rhythm for the range of analysed activities, which occur at intervals of approximately 24 hours, at different times of the year. this daily activity rhythm may have an endogenous origin and may be a manifestation of the biological clock (dunlap et al., 2004). the endogenous character of meliponini species’ activity rhythm has been identified by maintaining colonies in constant laboratory conditions (bellusci & marques, 2001; almeida, 2004). biological rhythms may be synchronized or entrained by environmental cycles, such as the light/dark (dunlap et al., 2004). in pedra branca, the beginning and the end of m. scutellaris daily activities were associated with the hours of sunrise and sunset and generally occurred earlier in november, when the sun rises earlier, and later in july, when the sun rises later. thus, the daily light/dark cycle may function to entrain the rhythm of daily flight activity in these bees. the entrainment of daily bee activities by the light/dark cycle was also observed by bellusci and marques (2001) in scaptotrigona aff depilis foragers. few studies of the daily flight activity of bees in brazil have a chronobiological focus. hilário et al. (2003) detected the presence of daily rhythms in a range of activities in a m. bicolor lepeletier colony in são paulo, which occurred during the morning and differed according to the two seasons of the year earlier in summer and later in winter. in a forest in ubatuba (são paulo), fidalgo and kleinert (2007) observed acrophases for collecting pollen in m. rufiventris lepeletier (the majority being at the beginning of the morning) and suggested that this activity was related to time of sunrise. rhythms for daily flight activity which repeat at intervals of approximately 24 hours may allow organisms to anticipate and “prepare” themselves for favourable or unfavourable environmental changes (merrow et al., 2005) and are important in the ecological context, since bees may, for example, have foraging strategies, anticipating their visiting times in relation to the times of the opening of flowers and anthers (gimenes et al., 1993; 1996). meteorological factors, such as temperature and relasociobiology 60(2): 183-189 (2013) 187 tive humidity, may also modulate the bees´ external activities. the species of meliponini studied did not leave the nest before the temperature reached 19°c, but for m. scutellaris acrophase times generally occurred when the average temperature was around 24°c and the acrophases of f. doederleini occurred at higher temperature values (average 27°c). similar results have been obtained for frieseomelitta flight activity in other localities, such as in ribeirão preto (são paulo state) in a study of f. doederleini (under laboratory controlled conditions) (almeida, 2004) and of f. varia (under field conditions) (nevill et al., 2004). furthermore, positive correlation between bee activities of nest entrance with and without pollen and tempera-of nest entrance with and without pollen and temperaand temperature may be observed in the studies of souza et al. (2006), in relation to m. asilvai in pedra branca. however fidalgo and kleinert (2007) observed that in forest areas (são paulo) the effect of temperature and relative humidity was different depending on the activity of the bee. in this study the correlation between pollen collection and temperature was negative, but between nectar collection and temperature was positive. nates-parra and rodrigues (2011) also observed negative correlation between entrance activity with pollen of colonies of melipona eburnea friese and temperature in the dry and rainy seasons and a negative correlation between relative humidity and entrance with pollen in the rainy season. however, according to silva and ramalho (2011) the effect of relative humidity in the activities of stingless bees can be uncertain and casual, especially in humid tropical habitats. in this regard, we need more research about the effects of temperature and relative humidity in the flight activities stingless bees. the differences observed between daily flight activity and temperature values related to the two bee genera may be related to their different size and colour, since the larger and darker bees presented earlier onset and acrophase times when the temperature values were lower, compared to the smaller sized and lighter bees (f. doederleini). similar results were obtained by bruijn and sommeijer (1997) and teixeira and campos (2005), who worked with several meliponini species of different sizes. when studying different meliponini genera, biesmeijer and pereboom (2003) concluded that these bees’ thermal properties were significantly influenced by size and body colour, since the largest and darkest bees were able to regulate in lower temperature environments more easily than the smaller and lighter bees. m. scutellaris’s first and last activities generally occurred close to sunrise and sunset, when light intensity values were low. f. doederleini carried out their first and last activities at higher levels of light intensity, since this bee began its activities approximately one hour after m. scutellaris and finished its activities earlier. the effect of light intensity on flight activities has already been observed in other species of meliponini. when studying the trigona mosquito (=plebeia mosquito) (smith), lutz (1931) observed that light intensity interfered at the beginning and end of daily activities. further-furthermore, in ribeirão preto (sp), nevill et al. (2004) confirmed that this abiotic factor had a positive influence on the external activities of f. varia. according corbet et al. (1993) the daily activities of social bees would start at sunrise and would be more correlated with light intensity than the temperature. compared to the other months, the excessive rain observed during the march observations did not result in a reduction in the amount of activity of the studied bees. rain effect on flight activity of stingless bee could be different in other areas of northeast brazil. nascimento and nascimento (2012) observed in colonies of melipona asilvai moure, in a forest area (sergipe), that daily activity was about 90% lower in the rainy season and they suggest that the colony enters into seasonal diapause at this time of the year. however, in march, was observed a change in the acrophase times for total bee’s activities, which occurred later than in the other months. the acrophases for entrance activity with pollen, however, varied very little across the observation months. maintenance of the times for entrance activity with pollen in march may be related to the possible regular time of exposure of this resource on anthers. according to roubik (1989), in general, most plants present anther dehiscence at the beginning of the morning, which probably influenced the times the bees collected this resource. imperatriz-fonseca et al. (1985), working on the flight activity of plebeia remota (holmberg) in são paulo, confirmed that the availability of floral resources is an external factor which may positively influence flight activity and could be a signal to the bees to regulate their activities. abiotic factors, such as light intensity, temperature and humidity, and biotic factors, such as time of food exposure, may influence the flight activity times of meliponini species and may operate as modulating factors in the rhythm of activity. the direct response of the organism to an environmental stimulus allows it a flexibility that adds to the rigid entrainment mechanism, so that a modulating factor works in the fine adjustment of the synchronization for the biological rhythm (marques & waterhouse, 1994). according to dunlap et al. (2004), in some organisms, temperature cycles function as entraining agents on the biological clock, but more often they perform a modulating role on the biological rhythm, over entrainment by the light/dark cycle. the regularity of daily flight activities in meliponini species studied, as well as the maintenance of acrophases in bees with differences in external morphologies, from colonies at the same stage of development, strengthens the concept that these bees have a biological rhythm. acknowledgments the authors would like to thank dr. favízia f. de oliveira (universidade federal da bahia ufba), for identifying the bee species; mr. fernando basto de jesus for their ms gouw, m gimenes daily activity rhythm in stingless bees188 permission to work in the area with their bees; and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the masters study grant to msg. references almeida, g.f. (2004). estudo de componentes rítmicos detectados na colônia de frieseomelitta varia (hymenoptera, apidae, meliponini). msc dissertation. universidade de são paulo, são paulo, brasil. batschelet, e. (1980). circular statistics in biology. london: academic press. bellusci, s. & marques, m.d. (2001). circadian activity rhythm of the foragers of a eusocial bee (scaptotrigona aff depilis, hymenoptera, apidae, meliponinae) outside the nest. biol. rhythm res., 32: 117-124. biesmeijer, j.c., van nieuwstadt, m.g.l., lukács, s. & sommeijer, m.j. (1998). the role of internal and external information in foraging decisions of melipona workers (hymenoptera: meliponinae). behav. ecol. sociobiol., 42: 107-116. biesmeijer, j.c. & pereboom, j.j.m. (2003). thermal constraints for stingless bee foragers: the importance of body size and coloration. oecologia, 137: 42-50. breed, m.d., stocker, e.m., baumgartner, l.k. & vargas, s. (2002). time-place learning and the ecology of recruitment in a stingless bee, trigona amalthea (hymenoptera, apidea). apidologie, 33: 251-258. bruijn, l.l.m. & sommejer, m.j. (1997). colony foraging in different species of stingless bees (apidae, meliponini) and the regulation of individual nectar foraging. insectes soc., 44: 35-47. cane, l.h. (1987). estimation of bee size using intertegular span (apoidea). j. kans. entomol. soc., 60: 145-147. corbet, s. a., fussell, m., ake, r., fraser, a., gunson, c., savage, a. & smith, k. (1993). temperature and pollination activity of social bees. ecol. entomol., 18: 17-30. dunlap, j.c., loros, j.j. & decoursey, p.j. (2004). chronobiology: biological timekeeping. sunderlands, massachusetts: inc. publishers, sinauer associates. fidalgo, a.o. & kleinert, a.m.p. (2007). foraging behavior of melipona rufiventris lepeletier (apinae; meliponini) in ubatuba, sp, brazil. braz. j. biol, 67: 133-140. gimenes, m., benedito-silva, a.a. & marques, m.d. (1993). chronobiologic aspects of a coadaptive process: the interaction of ludwigia elegans flowers and its more frequent bee visitors. chronobiol., int., 10: 20-30. gimenes, m., benedito-silva, a.a. & marques, m.d. (1996). circadian rhythms of pollen and nectar collection by bees on the flowers of ludwigia elegans (onagraceae). biol. rhythm res., 27: 281-290. heinrich, b. (1993). the hot-blooded insects. strategies and mechanisms of thermoregulation. harvard university press, cambridge, massachusetts (usa). hilário, s.d., imperatriz-fonseca, v.l. & kleinert, a. (2001). responses to climatic factors by foragers of plebeia pugnax moure (in litt.) (apidae, meliponinae). rev. brasil. biol., 61: 191-196. hilário, s.d., gimenes, m. & imperatriz-fonseca, v.l. (2003). the influence of colony size in diel rhythms on flight activity of melipona bicolor lepeletier, 1836. in: g. melo & i. alves dos santos (eds.), apoidea neotropica: homenagem aos 90 anos de jesus santiago moure (pp 191-197). criciúma: inesc. imperatriz-fonseca, v.l., kleinert-giovannini, a. & pires, j.t. (1985). climate variations influence on the flight activity of plebeia remota holmberg (hymenoptera, apidae, meliponinae). rev. bras. entomol., 29: 427434. koukkari, w. & sothern, r. (2006). introducing biological rhythms. st. paul: springer (ed.), college of biological sciences. lutz, f.e. (1931). light as a factor in controlling the start of daily activity of a wren and stingless bees. the american museum of natural history, 468: 1-4. marques, m.d. & waterhouse, j.m. (1994). masking and the evolution of circadian rhythmicity. chronobiol. int., 11: 146155. merrow, m., spoelstral, k. & roenneberg, t. (2005). the circadian cycle: daily rhythms from behavior to genes. embo rep., 6: 930–935. michener, c.d. (2000), the bees of the world. baltimore: the johns hopkins university press. nascimento d.l. do & nascimento, f.s. 2012. extreme effects of season on the foraging activities and colony productivity of a stingless bee (melipona asilvai moure, 1971) in northeast brazil. psyche 2012, article id 267361, doi:10.1155/2012/267361. nates-parra, g. & rodríguez, c.a. (2011). forrajeo en colonias de melipona eburnea (hymenoptera: apidae) en el piedemonte llanero (meta, colombia). rev. colomb. entomol., 37: 121-127. nevill, a.m., teixeira, l.v., marques, m.d. & waterhouse, j.m. (2004). using covariance to unravel the effects of meteorological factors and daily and seasonal rhythms. biol. rhythm res., 35: 159-169. nunes-silva, p., hilário, s.d., santos filho, p.s. & imperatriz-fonseca, v.l.i. (2010). foraging activity in plebeia remota, a stingless bees specie, is influenced by the reproductive state of a colony. psyche, 2010, article id 241204, sociobiology 60(2): 183-189 (2013) 189 doi:10.1155/2010/2412041-16. oda, g.a., bellusci, s. & marques, m.d. (2007). daily rhythms related to distinct social tasks inside an eusocial bee colony. chronobiol. int., 24: 845-858. pierrot, l.m. & schlindwein, c. (2003). variation in daily flight activity and foraging patterns in colonies of urucu (apidae, meliponini). revta bras. zool., 20: 565-571. roubik, d.w. (1989). ecology and natural history of tropical bees. cambridge: cambridge university press. sei (2012). superintendência de estudos econômicos e sociais do estado da bahia. informações geográficas: clima e vegetação. http://.sei.ba.gov.br. accessed 24 october 2012. silva, m. d., ramalho, m. & rosa, j. f. (2011). p or que melipona scutellaris (hymenoptera, apidae) forrageia sob alta umidade relativa do ar? iheringia, série zoologia, 101: 131-137. souza, b.a., carvalho, c.a.l. & alves, r.m.c. (2006). flight activity of melipona asilvai (hymenoptera: apidae). braz. j. biol., 66: 731-737. teixeira, l.v. & campos, f.n.m. (2005). início da atividade de vôo em abelhas sem ferrão (hymenoptera, apidae): influência do tamanho da abelha e da temperatura ambiente. juiz de fora (mg). rev. bras. zool., 7: 195 – 202. willmer, p.g. & stone, g.n. (2004). behavioral, ecologi-behavioral, ecological, and physiological determinants of the activity patterns of bees. adv. study behav., 34: 347-466. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.341-344sociobiology 61(3): 341-344 (september 2014) new records, including a new species, of scuttle flies (diptera: phoridae) associated with leaf cutter ants (hymenoptera: formicidae) in brazil r. henry l. disney1 & marcos a. l. bragança2 introduction during 2012 malb and his colleagues cliver gomes, leandro silva, hendria martins and marcos teixeira collected diptera attacking or hovering in the vicinity of leaf cutter ants. the scuttle flies (phoridae) were preserved in 70% ethanol and sent to rhld for slide mounting and identification. the ants were identified by the brazilian team. the following species were obtained with the ant hosts indicated. the specimens are deposited in the collection of the museu de zoologia, universidade de são paulo, brazil (mzsp) and the university of cambridge museum of zoology (ucmz). genus allochaeta borgmeier the genotype is a. excedens described from the female only, from petropolis in brazil (borgmeier, 1924). in the same paper a. metatarsalis was described from the male only, from blumenau (santa catharina). later, from petropolis, a. longiciliata, known only from the female and a. propinqua known abstract among scuttle flies caught at colonies of leaf cutter ants were apterophora bragancai disney new species, and new host records for other species. sociobiology an international journal on social insects 1university of cambridge, department of zoology, cambridge, united kingdom 2universidade federal do tocantins, palmas, tocantins, brazil article history edited by evandro n. silva, uefs, brazil received 02 may 2014 initial acceptance 12 june 2014 final acceptance 26 june 2014 keywords new species, apterophora bragancai disney new species, taxonomy corresponding author dr henry l. disney department of zoology, university of cambridge downing street, cb2 3ej, u. k. e-mail: rhld2@hermes.cam.ac.uk only from the male were then added (borgmeier, 1926). however, subsequently these two were procured in copula, so a. propinqua was then synonymised with a. longiciliata (borgmeier, 1928). this served to emphasize the sexual dimorphism in this genus. subsequently a. senex, known from the female only was added from são paulo state, campinas (borgmeier & prado, 1975) and a. wallerae disney known from the male only was added from mexico (disney & bragança, 2000) along with possible males of a. excedens from viçosa, mg, brazil. in the present study further examples of these males were obtained (see below). borgmeier (1928) reported a. longiciliata soliciting food from its ant host, acromyrmex muticinodus. other reported hosts are the same species with acromyrmex niger smith (borgmeier, 1928), a. muticinodus with ac. niger (borgmeier, 1928), the putative males of a. excedens with atta laevigata smith and a. wallerae with atta cephalotes (linn.) (disney & bragança, 2000); and a. metatarsalis from a colony of the termite nasutitermes rippertii (rambur) (borgmeier, 1924). in the present study the following further records of the putative males of a. excedens were obtained (see below). short note r. h. l. disney & m. a. l. bragança new species of scuttle flies associated with leaf cutter ants342 material examined. 3 males, minas gerais state, viçosa, vii.2012, at atta sexdens (linn.) (marcos bragança, to-137). 4 males, minas gerais state, florestal, x.2012, at a. sexdens (cliver gomes, to-128, to-129). this species was previously reported with atta laevigata (see above). genus apterophora brues this genus closely resembles the large genus puliciphora. the flightless females differ only in having a longer proboscis. the males differ in having modified front tibiae. however, the form of these modifications ranges from a clearly distinct excavation of the ventral margin to an apical ventral projection. in the genus puliciphora modifications of the legs are not uncommon. thus several have swollen front basitarsi or these are otherwise modified (e.g. see puliciphora species b below). this genus may therefore eventually prove to be merely a subset of puliciphora. the species of apterophora are discussed by prado (1976), allowing identification of males. in our present state of knowledge, females can only be named when associated with their males. apterophora attophila borgmeier material examined. 3 males, minas gerais state, florestal, x.2012, at atta sexdens (linn.) (cliver gomes, to126, to-127, to-129). 1 male, minas gerais state, viçosa, vii.2012, at a. sexdens (marcos bragança, to-137). the type series was recorded with a. sexdens and prado (1976) added further records with the same ant along with records with eciton burchelli (westwood) and solenopsis geminata (f.); and also with the termite nasutiterms sp. apterophora borgmeieri prado material examined. 1 male, minas gerais state, viçosa, vii.2012, at atta sexdens (linn.) (marcos bragança, to-136). apterophora bragancai disney new species the fore tibia closely resembles that of a. caliginosa brues, but the longer anal tube and left hypandrial lobe of the hypopygium immediately distinguish the new species. male. postpedicels brown, tapered, 0.16 mm long and with about a dozen subcutaneous pit sensilla. palps brown, 0.24 mm long and 0.06 mm wide, with apical bristle 0.07 mm long. thorax brown. notopleuron with 3 bristles. scutellum with and anterior pair of fine hairs and a posterior pair of bristles. abdomen with brown tergites and paler brown venter. hypopygium as fig. 1. apart from brown patches in mid coaxa the legs are mainly yellow apart from the distal halves of the femora shading to brown. front tibia as fig. 2. front tarsus with posterodorsal hair palisades on segments 1 to 4. hairs below basal half of hind femur shorter than hairs of anteroventral row of outer half. wing 2.46 mm long. costal thicker than vein 3. costal index 0.65. costal ratios 0.55 : 1. haltere knob brown. material examined. holotype male, brazil, minas gerais state, viçosa, vii.2012, at atta sexdens (linn.) (marcos bragança, to-136) (5-162, mzsp). this fly was captured when it was flying over the ants along the foraging trail. genus mymosicarius borgmeier the females are keyed by disney, elizalde & folgarait (2006). mymosicarius grandicornis borgmeier material examined. 4 females, minas gerais state, viçosa, 14.iv.2012, at atta bisphaerica forel (m. bragança, to-132, to-133), 1 female, same locality and ant host, 28.vi.2012 (m. bragança, to-135), 4 females, same locality and host ant, ix, 2012 (hendria martins, to-140, to-141); 1 female, (jalapão) mateiros, 3.v.2012, at atta sexdens (linn.) (leandro silva, to-131). 11 females, minas gerais state, florestal, x.2012, at a. sexdens (cliver gomes, to-125-128, to-130). these records confirm previous records with these two ant hosts, which has also been recorded with a. laevigata (disney, elizalde & folgarait (2006). genus puliciphora dahl more than one hundred species are known in this cosmopolitan genus. species recognition has been based on the flightless females in the first instance and for most regions keys only exist for the females, those of the neotropical region being keyed by disney (2003). the boundaries of the genus are still not settled. the males of the following two species were obtained. these flies were captured when they were flying over the ants in a disturbed nest. puliciphora species a male. frons much wider than long and with 10 bristles level with or below anterior ocellus (4 supra antennals and 2-4 bristles). postpedicels brown, 1.0-1.1 mm wide and lacking sps vesicles. palps brown, 1.4 mm long, 0.3 mm wide and the longest bristles 0.9 mm long. thorax brown, but tending to be paler on sides. notopleuron with 3 bristles. scutellum with and anterior pair of small hairs and a posterior pair of bristles. abdomial tergites brown and venter gray. hypopygium as fig. 3, notably with a long anal tube relative to the length of the epandrium. legs pale dusky yellow. front tarsus with a posterodorsal hair palisade on segments 1-4. the 3 hairs sociobiology 61(3): 341-344 (september 2014) 343 below basal half of hind femur about as long as those of anteroventral row of outer half but clearly more robust. wings 1.1 mm long. costa about as wide as vein 3. costal index 0.6. costal ratios 0.37 : 1. haltere knob brownish gray. material examined.1 male, minas gerais state, viçosa, vi.2012, at atta sexdens (linn.) (m. bragança, to-138, mzsp). puliciphora species b male. frons brown, clearly wider than long and with 10 bristles below anterior ocellus. subglobose postpedicels light brown, 0.1 mm wide, with numerous sps vesicles. palps yellowish gray, 0.1-0.12 mm long, 0.04 mm wide and longest bristle 0.06-0.07 mm long. thorax brown, but paler at sides, with 3 bristles on notopleuron. scutellum with an anterior pair of small hairs and a posterior pair of bristles. abdominal terrgites brown and venter gray. hypopygium as fig 4. legs with slightly dusky but pale yellow femora and tibiae but tarsi all pale. front tarsus with posterodorsal hair palisades on segments 1-4 and basitarsus as fig. 5. hind femur with hairs below basal half shorter than those of anteroventral row of outer half. wings 1.5 mm long. costa about as wide as vein 3. costal index 0.55. costal ratios 0.7 : 1. haltere with pale stem and brown knob. material examined.1 male, minas gerais state, viçosa, vi.2012, at atta sexdens (linn.) (m. bragança, to-138, mzsp). a damaged specimen a distinctive male that has lost both its wings appears to be an undescribed species of the poorly defined genus macrocerides borgmeier or a similar genus subsequently distinguished from this genus. an intact specimen will be required to settle thee identity of this species. male. frons as fig. 6. postpedicels yellowish gray brown, 0.14 mm long and 0.25 mm greatest breadth. palps yellow, 0.12 mm long, 0.03 mm wide and longest bristle 0.05 mm long. thorax brown. notopleuron with 2 bristles. mesopleuron bare. scutellum with an anterior pair of small hairs and a posterior pair of bristles. abdominal tergites and venter brown. hypopygium as fig. 7. legs apart from brown on mid coxae yellow. front tarsus as fig. 8. hairs below basal half of hind femur shorter and finer than those of anteroventral row of outer half. material examined. 1 male, minas gerais state, florestal, x.2012, at trachymyrex sp. (cliver gomes, to-124, mzsp). this fly was captured when it was flying over the ants. acknowledgments malb thanks to cnpq and secretaria estadual de desenvolvimento econômico, ciência, tecnologia e inovação do tocantins, for the financial support to collect the phorid flies in several localities. rhld’s studies of phoridae are currently supported by grants from the balfour-browne trust fund (university of cambridge). we are grateful to terezinha della lucia, marco antonio oliveira for logistical support in localities of collect. we also thank to cliver gomes, leandro silva and hendria martins for helping to collect the flies. references borgmeier t (1924) novos generos e especies de phorideos do brasil. boletim do museu nacional do rio de janeiro, 1: 167-202. borgmeier t (1926) phorideos novos ou pouco conhecidos do brasil. boletim do museu nacional do rio de janeiro, 2(5), 39-52. borgmeier t (1928). nota prévia sobre alguns phorideos que parasitam formigas cortadeiras dos generos atta e acromyrmex. boletim biológico laboratorio de parasitologia, são paulo, 14: 119-26. borgmeier t, prado, ap do (1975) new or little-known neotropical phorid flies, with description of eight new genera (diptera, phoridae). studia entomologica, 18: 3-90. disney, r. h. l., (2003). seven new species of new world puliciphora dahl (diptera: phoridae) with a new key to the neotropical species. zootaxa, 162: 1-22. figs 1-2. apterophora bragancai male.1, left face of hypopygium; 2, front tibia. figs 3-5. puliciphora males. 3, species a, left face of hypopygium; 4-5, species b; 4, left face of hypopygium; 5, front basitarsus. figs 6-8. problem damaged specimen, male. 6, frontal view of head; 7, front tarsus; 8, left face of hypopygium. 1 2 543 6 7 8 r. h. l. disney & m. a. l. bragança new species of scuttle flies associated with leaf cutter ants344 disney, r. h. l. & bragança, a. l., (2000). two new species of phoridae (diptera) associated with leaf-cutter ants (hymenoptera: formicidae). sociobiology, 36: 33-39. disney, r. h. l., elizalde, l. & folgarait, p. j. (2006). new species and revision of myrmosicarius (diptera: phoridae) that parasitize leaf-cutter ants (hymenoptera: formicidae). sociobiology, 47: 771-809. prado, ap do (1976) records and descriptions of phorid flies, mainly of the neotropical region (diptera; phoridae). studia entomologica, 19: 561-609. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.329-336sociobiology 60(3): 329-336 (2013) understanding the complex structure of a plant-floral visitor network from different perspectives in coastal veracruz, mexico h hernández-yáñez1,2, n lara-rodríguez3, c díaz-castelazo1, w dáttilo4, v rico-gray1,4 introduction many interspecific interactions have been considered mutualisms, which in turn influence species’ fitness and community organization (thompson, 1999; rico-gray, 2001; kothamasi et al., 2010). for instance, pollination and seed dispersal by animals, and ant-plant mutualisms, are key ecological processes in many biological communities (vázquez et al., 2009). animal pollination is the basis of many terrestrial communities, without it many flowering plants could not reproduce, and without plants, most herbivorous and carnivorous animals would disappear (faegri & van der pijl, 1979; ollerton et al., 2011; burkle et al., 2013; tylianakis, 2013). it is then of the utmost importance to understand the basic structure of communities now that ecosystems are being massively destroyed. floral or pollination syndromes are sets of floral traits adapted to attract a certain subset of available abstract our premise was to understand the basic structure of the flower-flower visitor community at la mancha in veracruz, mexico. we used network analyses to study the structure of this community. in particular, to analyze, (1) if flower color and shape (“as a limited portion of the traditional floral syndromes definition”) were linked to the arrival of certain floral visitors, (2) if visits to flowers were generalist, specific and/or modular; and (3) which plant species, if any, in the core of the network could affect the stability of floral visitors. in order to analyze the organization of the plant-floral visitor community, we prepared network graphics using pajek, nestedness (as nodf) with aninhado, and modularity with the sa algorithm. the network obtained was nested suggesting that generalist species (with the most associations) were interacting with specialists (with fewer associations). furthermore, floral visitors (hymenoptera, diptera, lepidoptera and trochiilidae) did not exhibit a particular preference for a specific flower color or shape, each pollinator group visited most flowers/colors/shapes considered. the same was similar for all 14 resulting modules. as in other studies, we suggest that pollination leans to generalization rather than to specialization. we suggest that maybe seasonality/food resource could be the factors to analyze as the next step in floral visits which may be the answer to modularity in this seasonal ecosystem. sociobiology an international journal on social insects 1 instituto de ecología, xalapa, veracruz, mexico. 2 university of missouri, st. louis, mo, u.s.a. 3 universidad de alicante, san vicente del raspeig, alicante, spain. 4 universidad veracruzana, xalapa, veracruz, méxico. article history edited by kleber del-claro, ufu brazil received 07 march 2012 initial acceptance 26 april 2013 final acceptance 12 june 2013 keywords floral syndromes; flower visitors; modularity; mutualistic networks; pollination. corresponding author dr. victor rico-gray instituto de neuroetología universidad veracruzana calle luís castelazo s/n col. industrial-ánimas xalapa, veracruz 91190, méxico e-mails: vricogray@yahoo.com vrico@uv.mx pollinators (richards, 1997), and have provided some of the strongest support for pollinator driven floral evolution (danieli-silva et al., 2012; hirota et al., 2012). however, they could be misused, such that without previous observations a person could judge or ascertain which type of pollinator visits a certain plant species. faegri & van der pijl (1979) published a synthesis of this information, and thus established a paradigm in pollination ecology, although several doubts have arisen over its use (waser et al., 1996; hingston & mcquillan, 2000; gómez, 2002; olesen et al., 2007; ollerton et al., 2009). for instance, some authors do not perform close observations of plant floral visitors in the field (ollerton et al., 2009), or that such characteristics have been used to indicate that pollination tends to specialization (valdivia & hermann, 2006; but see waser et al., 1996). even though a highly criticized view (waser et al., 1996), some authors assume that specialization is the rule and generalization an research article wasps h hernández-yáñez, n lara-rodríguez, c díaz-castelazo, w dáttilo, v rico-gray plant-floral visitor network330 exception (kothamasi et al., 2010). although different studies discuss the existence of pollination syndromes (hingston & mcquillan, 2000; fenster et al., 2004; valdivia & hermann, 2009; danieli-silva et al., 2012), only a few consider communities instead of populations (ollerton et al., 2009). here we present a community based analysis of the plant-floral visitor system present at la mancha, on the coast of the state of veracruz, mexico. this is a seasonal environment (e.g.; rico-gray, 1993) where not all flowering plants produce flowers at the same time but rather in a sequence. for instance, not all flower colors and/or shapes are present all the time, thus flower visitors should visit available flowers. for example, which flowers do hummingbirds visit when no red-tubular flowers are available? what type of color and shape of flower are preferred by the different flower visitors? do the use of ‘traditional flower (pollination) syndromes help to organize associations in the community under study? (see methods: data analyses). for our community analysis we considered network metrics, such as nestedness, connectance, and niche overlap (guimarães & guimarães, 2006; guimarães et al., 2006, 2007; almeida-neto et al., 2008; dormann & gruber, 2009; díaz-castelazo et al., 2010), a modularity analysis (sa algorithm) (guimerà & amaral, 2005a, b), a core-periphery analysis (ucinet) (borgatti et al., 1999; borgatti & everett, 1999), and graphics (pajek) (de nooy et al., 2005). although some of these metrics (e.g., nestedness, conecctance) may seem unrelated to a pollination/pollination syndrome analysis, they do serve to characterize the community under study. in particular we were interested to determine (1) if flower color and shape (“as a limited part of the traditional floral syndromes definition”) were linked to the arrival of certain floral visitors, (2) if visits to flowers were generalistic, specific and/ or modular; and (3) which plant species, if any, in the core of the network could affect the stability of floral visitors. materials and methods study site field work was carried out at centro de investigaciones costeras la mancha (cicolma), located on the coast of the state of veracruz, mexico (19o 36' n, 96o 22' w; elevation < 100 m). the climate is warm and sub-humid; a rainy season occurs between june and september, total annual precipitation is ca. 1500 mm and mean annual temperature is 22o-26oc (soto & garcía, 1989). the major vegetation types are tropical dry and deciduous forests (with bursera simaruba (l.) sarg., cedrela odorata l., cnidoscolus herbaceus (l.) i. m. johnston, enterolobium cyclocarpum (jacq.) griseb.), sand dune vegetation (with ipomoea pes-caprae (l.) r. br., canavalia rosea (sw.) dc., chamaecrista chamaecristoides (colladon) greene, macroptilium atropurpureum (sessé et mociño) urban), mangrove forest (with rhizophora mangle l., laguncularia racemosa l., avicennia germinans l.), freshwater marsh (with typha domingensis l.), and flooded deciduous forest (with annona glabra l., pachira aquatica aubl. surrounding a freshwater lagoon) (castillo-campos & medina, 2000; moreno-casasola, 2006). to determine the associations among plants and their floral visitors (or potential pollinators), field work was done during five days per month between march 2007 and march 2008. we based sampling time in the size of the field station (64 ha) and the high variation within that area, which makes movements station relatively easy and fast. all observations were done walking along pre-established trails that covered the different vegetation associations present in the field station and surrounding area (rico-gray, 1993; díaz-castelazo et al., 2010, 2013), in periods of 15-20 min, between 08:00 and 16:00. we noted which plant species’ were flowering at the time and which animals were visiting them. an observation was registered as an interaction when a visitor (insect or bird) was seen feeding off a flower in a way that its behavior allowed for body to touch floral reproductive structures (stigma, anthers). to minimize weather effects, observations were not done during rain or strong windy periods. a limited number of plant samples were collected for identification at xal herbarium of instituto de ecología, a.c. (inecol) in xalapa, veracruz, mexico, we also used published floras (for instance, flora de veracruz). insect material was collected using nets and identified at inecol using the iexa collection. hummingbirds were identified by sight only. as cicolma is a restricted area for collection, we based our identifications on voucher specimens already deposited either in xal or iexa. furthermore, as this is a very seasonal environment, we considered that between-years comparisons may not be adequate since inter-year variations could render different results (rico-gray et al., 2012). data analysis data was organized using an excel spreadsheet (microsoft®). network graphics were done using pajek (de nooy et al., 2005). graphs helped to visualize the associations among nodes of two different (a, animals and p, plants) interactive (r) groups (jordano et al., 2003, 2006), such that r = r a x p, where r = 0 when no interactions is present between animal species i and plant species j, or = 1 when an interaction is present. adjacency matrix ‘r’ will be composed by ‘a + p’ nodes or species and k interactions or links (jordano et al., 2006). a nestedness analysis (nodf index see almeidanetoel al., 2008) was done using aninhado (guimarães & guimarães, 2006; www.guimaraes.bio.br). to determine if the degree of nestedness is higher than expected by chance alone, we compared the nestedness value of our network to 1,000 randomizations generated by null model ii (díaz-castelazo et al., 2010). in this null model, the probability of an sociobiology 60(3): 329-336 (2013) 331 interaction occurring is proportional to the level of generalization (mean number of interactions) of plant and animal species (bascompte et al., 2003). this allowed us to assess whether the value of nodf observed in the empirical network was higher than expected for networks of equal size and with similar heterogeneity in interactions among species. niche overlap and connectance were analyzed using r-bipartite (dormann & gruber, 2009). niche overlap was calculated using the mean similarity in interaction pattern between species i and k in both trophic levels (plants and plant-visitors) through connectance (c) is the proportion of possible interactions that are actually carried out within a network (jordano, 1987; olesen & jordano, 2002). modularity index (m) was done using the sa algorithm (guimerà et al., 2004; guimerà & amaral, 2005) to estimate the degree in which groups of species (animal and plants) interact more among each other than with other species in subgroups in the network. this analysis was based on a matrix using the following flower characteristics: flower symmetry (zygomorphic or actinomorphic), petals free or sympetalous, corolla tubular or not, and main flower color. the number of species sampled per flower color were: white 28 species, white/green 1, white/yellow 2, orange/yellow 2, yellow 19, pink 4, dark pink 1, lilac 9, lilac/white 1, beige 7, green/beige 1, green 3, red 5, burgundy 1, orange 1,purple 1, red/orange 1 specie. the above, of course, is a limited view of floral (pollination) syndromes, since (1) some of those characters may at times be correlated, and (2) it leaves out several traits that could be important, e.g., flower size, nectar characteristics or scent type. the software used is based on a heuristic process in order to find an optimum solution for the modularity function. the m index vary from 0 (extreme generalization) to 1.0 (extreme number of compartments) and decreases when the fraction of links among modules increases in the total network. high values of m indicate that different animal and plant species form modules that are semi-independent from other modules (rezende et al., 2009). to assess the significance of network modularity observed, we compared the obtained modularity value to 100 simulated networks generated by a null model (null model ii) that have equal size and with similar heterogeneity in interactions among species as the original network. after the first run, three modules were chosen at random within them one pair of species was also chosen at random, since there were three modules, we then established three pairs of species a, b, and c (each having a plant and an animal). to assess that our results were accurate, the algorithm was repeated 15 times (heuristic process) and we ‘followed’ the pair of species checking for possible module changes during the other 14 randomizations (rezende et al., 2009). the resulting sa modularity index m is a degree measure where a network is organized in modules with defined boundaries. also, to test if links within modules were organized in a nested pattern, we performed nestedness (nodf) analyses for those modules with links among four or more species (nine out of 14 modules) (also see dáttilo 2012; dáttilo et al. 2013). finally, analyses of species as core or peripheral components of the network were performed with ucinet for windows 6.0 (borgatti et al., 1999; analytic technologies inc.; http://www.analytictech.com/downloaduc6.htm), which performs categorical core/periphery analysis for bipartite graphs (borgatti et al., 1999; borgatti & everett, 1999). the core-periphery analysis is based on stochastic optimization processes, thus we performed 20 randomizations, obtaining the proportion of occurrences of a species within the core or the periphery for the entire set of randomizations. we obtained the flowering periods (months) for these species. figure 1. bipartite graph of a plant-flower visitor interaction network at la mancha, veracruz, mexico recorded from march 2007 to march 2008. nodes on the left correspond to flower visitors, whereas nodes on the right to plant species. lines ("links") connect interacting species. color code: orange = plants, red = butterflies (lepidoptera), yellow = wasps (hymenoptera), pink = hummingbirds (trochiilidae, green = flies (diptera). h hernández-yáñez, n lara-rodríguez, c díaz-castelazo, w dáttilo, v rico-gray plant-floral visitor network332 results we registered 392 interactions involving 89 plant species (there are more plant species in the station, for instance, wind-pollinated species) and 177 animal species [e.g., 29 flies (diptera), 73 butterflies (lepidoptera), 62 bees and wasps (hymenoptera), and three hummingbirds (trochilidae)] (appendices 1 and 2). the interaction matrix consisted of 266 species (fig. 1). the resulting network was significantly nested (nodf = 3.99, p < 0.01), and had a connectance of 0.02. niche overlap was 0.05 for plant species, whereas 0.04 for floral visitors. the modularity analysis produced 14 modules, grouped according to the pattern of interactions (fig. 2). five of these were not linked to other modules and contained less than four species, therefore were not used in nestedness analyses. this result shows that animal species were not associated to a given floral pattern within a module. moreover, a module may consist of different flower shapes and colors. similarly, animals within a module belong to different orders. the network was significantly modular (sa algorithm, m = 0.636, p < 0.0001, n = 100). the three pairs of species selected to follow within modules offered different results, pair ‘a’ was present in a module 100% of the program randomizations, pair ‘b’ was present in 27% of the randomizations, and pair ‘c’ was present in 80% of the randomizations. different results were obtained (heuristic program), and every time the program is run slight differences may be obtained, these results allow for a 69% of accuracy, considering that we used 21% out of the 14 modules. if we had “followed” a higher number of species our percentage of accuracy would have probably been greater. five of the 14 modules contained nine core plant species (bahunia divaricata, bidens pilosa, randia laetevirens, turnera ulmifolia, lantana camara, waltheria indica, cynanchum sp., tecoma stans, piscidia piscipula) (table 1), and within each module they were highly linked to different animal species. even though not simultaneously (rather one after the other), these nine species shared flowering during the 10 months of observations, offering permanent food resource. bees were the most common animals within modules. of which four species were present in the core of the network, apis mellifera, lasioglossum sp., trigona nigra and euglossa viridissima. when the nine modules were individually analyzed for nestedness, only one was significantly nested (nodf = 20.92, p < 0.01). discussion our results show a low degree of niche overlap (for both plants and visiting animal species in the network), high modularity (14 modules), low connectance, and significant nestedness. within modules we found flowers of different figure 2. network showing the modularity of a plant-flower visitor interaction network at la mancha, veracruz, mexico. each module has a different color (n= 14 modules/colors). each circle represents one plant or visitor species and is colored according to the color of its module. lines represent plant-visitor interactions. the majority of these modules are over-represented except for five of them that are formed only by two or three species. moreover, no module was exclusively represented by a particular group of colors or shape of flowers or any guild of plant-visitor, indicating that floral visitors did not exhibited flower preference in this study. sociobiology 60(3): 329-336 (2013) 333 colors and shapes, as well as a variety of visiting animals. the results of the core-periphery analysis show four core bee species (social or solitary bees) having the most interactions or links, suggesting they are super-generalist species. only one of the selected nine modules for nestedness analyses was indeed significantly nested. nine plant species constituted the core of the networks (table 1). thus, visitors obtain floral resources from core plants (the most visited) in different seasons, suggesting that modules may not be built around syndromes but instead because visitors need food they search and forage throughout the year. were interacting among them, influenced by the existence of super-generalist species. this suggests generalization among animal species, also supported by the idea that abundance of certain species affects the network’s nested pattern (vázquez et al., 2009, lange et al. 2013): mutualism in these species could be facultative. moreover, a nested structure could also be generated by abundance, spatial distribution and/or similar ecological processes, which can be expressed along a variety of temporal and/or spatial scales (lewinsohn & prado, 2006). all these may suggest that nestedness is not always the result of a coevolutionary process (kothamasi et al., 2010), as referred to many times in the past for flower-flower visitor interactions. furthermore, bees have been considered as common in low-latitude regions (olesen & jordano, 2002) and indeed they were the most common visitor in our study site. our results also suggest that bees were consistent in their visits throughout the day, which differs from the behavior of other animals (baldock et al., 2011). another consideration is the thin line between parasitism and the socalled facultative mutualists, which could easily cross it and become parasites if a floral visitor does not offer a reward to the plant, again exhibiting a tendency to generalization is some species (i.e., visit many species have many links). certain studies suggest extensive generalization as the rule (waser et al., 1996) rather than the exception (olesen & jordano, 2002), which in turn may trigger an increase in biodiversity as a consequence of plants and flower visitors searching for generalization among their mutualistic counterparts (kothamasi et al., 2010). furthermore, generalization could result from competition among flower visitors. when these are removed or displaced by competitors from preferred flower species, they have the option of returning after visits to alternative plants in bloom, thus the latter species benefit from displaced visitors (kothamasi et al., 2010), contributing to community coexistence and diversity. for instance, large pollination networks have modules whereas small networks do not, such that connectance could be the basis, modularity a complement of nestedness, and generalist species the glue between modules (bascompte & jordano, 2007). if connectance increases, the generalist core of species also increases and instead of a few species being the glue between modules these should disappear (bascompte & jordano, 2007). these authors, using data from different geographical areas and mostly collected in only one season and in different habitats, found that the biological contents of those modules tend to correspond to certain floral syndromes (bascompte & jordano, 2007). our data, however, was obtained from only one seasonal tropical community, exhibiting a long dry period (ca. 6-7 months), intermixed in a winter with cool strong northern winds (cold fronts or ‘nortes’), a short wet season (4-5 months), and many coexisting species. furthermore, these analyses differ in purpose, olesen and collaborators (2007) were searching in different networks for the presence of modules, and not to understand why these modules were present. if interactions were highly generalist then why have modules? our results suggest that species within modules species / months pp j d n o s a jl jn my ap bahuinia divaricata l. 1 x bidens pilosa l. 2 x x randia laetevirens standl. 3 x x turnera (ulmifolia l.) velutina presl 4 x x lantana camara l. 5 x x x waltheria indica l. 6 x x x cynanchum sp. 12 x tecoma stans (l.) juss. ex kunth 8 x x x x piscidia piscipula (l.) sarg. 6 x table 1. core of generalist plants species in the network and their flowering periods (based on xal herbarium specimens). pp = position of plant in the ordered matrix for nestedness. our results show low niche overlap for both groups (plants, animals), suggesting that each trophic level interacts with a different subset as its counterpart, which supports the high modularity result, and suggests that the same species’ were the niche of one and the other. the local level of species richness affects both the individuals and their interactions. if tropical habitats are species saturated, high species density should increase competition among them, reduce niche overlap and cause that these species should be more specialized in the long-run (olesen & jordano, 2002). although this may not be necessarily true due to flower color and shape, but it could be the result of competition for feeding resources (nectar, pollen). furthermore, the latter does not mean that forbidden interactions do not exist or were dismissed, different phenotypes could result from adaptation, neutral evolution and/or phylogenetic constraints, and these can influence mutualistic interactions (vázquez et al., 2009). the low overlap for flower visitors in our results (0.04) could also suggest the existence of competition, territoriality or very different feeding requirements. whereas the low overlap in plant species (0.05) is possible since visits were differential and there was no simultaneous overload to certain flower characteristics, supporting network modularity. that the network was significantly nested suggests that specialist species (those species with the least associations or links) were interacting with the generalists (those species with the most associations or links), and generalist species h hernández-yáñez, n lara-rodríguez, c díaz-castelazo, w dáttilo, v rico-gray plant-floral visitor network334 may not comply with the ‘established rules’ of floral syndromes, and other factors prevail. for instance, (1) competition, where some individuals are temporally displaced towards other flowers; (2) time of day (when observations were done), since plants vary throughout the day in the quantities of nectar and/or pollen offered (baldock et al., 2011); (3) plants could have nectar guides, suggesting special attractiveness for bees (free, 1970), or (4) have the correct phenology (available for any visitor). any structure forming compartments results from constraints of hosts and their visiting fauna (lewinsonh & prado, 2006). phenology is a very important constraint, since there is no way an interaction can exist if the two organisms do not have similar phenologies (jordano et al., 2003). not all links among flowers and visiting animals happen at the same time, and the impact of some of these interactions critically depend on time, which, on the other hand, allows for visits by other animals (even within a single day), e.g. shared-pollinators (baldock et al., 2011). lewinsohn and prado (2006) suggest the existence of mixed networks, i.e. nested compartments, although a nested network with compartments is not possible (lewinsohn & prado, 2006). our results suggest, however, a nested network with compartments, which may be possible if we consider the effect of temporal variation in the community (seasonality). moreover, the above studies were done under a variety of environmental conditions, which could be the answer to differences in results. the low connectance value obtained for our network supports the modularity analyses, indicating that not all possible interactions or links were enabled in the community. in pollination networks in general, connectance increases while network size decreases (olesen & jordano, 2002). what happens then with modularity? each analyzed module was very cohesive, whereas the opposite was obtained between modules. suggesting that species within a module were strongly interacting, while they were weakly interacting with species in other modules (jordano et al., 2006); this could explain the high modularity of the network. another explanation for the high modularity and nestedness values could be the super-generalist status of bee species in the network, also a common feature in modules. such ‘specialization’ suggested by our results could very well be the result of a phenological spatio-temporal synchronization of plants in modules, which is also supported by a lack of nestedness in practically all modules. there really was no specialization within modules, which could simply suggest the possible existence of preferences for quality and/or quantity of flower nectar, competition or phenology. in general, modularity could be explained by a combination of seasonality (phenology) and resource quality. beyond constraints elicited by plants and directing us to flower syndromes (bascompte, 2009), we suggest the importance of analyzing the temporal scale, which is probably the sole way to acknowledge what visitors do when preferred flowers are not available. when we review the flowering times of different species in the core of the network, they do not flower simultaneously rather they are distributed throughout the 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(1996). generalization in pollination systems, and why it matters. ecology, 77: 1043-1060. doi: 10.13102/sociobiology.v61i2.218-224sociobiology 61(2): 218-224 (june, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 reproductive status of the social wasp polistes versicolor (hymenoptera, vespidae) vo torres1, d sguarizi-antonio2, sm lima1, lhc andrade1 & wf antonialli-junior1 introduction an important feature for the ecological success of social insects is the division of labor among individuals in their colonies (wilson, 1985). for this reason, many investigators have devoted their efforts to elucidate the parameters that determine this division, especially the distinction and determination of the caste (robinson, 1992; o’donnell, 1995; o’donnell, 1998). the subfamily polistinae has characteristics that are important to understand how the social behavior has evolved in the wasps (ross & matthews, 1991). the degree of morphological differences among castes in this group can range from total absence (richards, 1971; strassmann et al., 2002) to sharp differences among castes (jeanne, 1991). this, indeed, may be a key feature in the evolution of social insects, since the presence of wide differentiation among castes indicates a higher degree of sociality (bourke, 1999). in the basal polistinae such as mischocyttarus and polistes, females are distinguished by their behavior, dominance hierarchy, degree of ovarian development and/or their abstract a fundamental feature in the evolution of social insects is the separation of castes, and the presence of wide differentiation between castes indicates a more advanced degree of sociability. in this study, we evaluated factors indicating the reproductive status of females in colonies of the social wasp polistes versicolor. the reproductive status of each female was examined by measuring nine morphometric characters, by tracing the cuticular chemical profile, by evidence of insemination and by recording the relative age. we conclude that p. versicolor colonies present 3 female groups according to cuticular chemical profile difference. the first group is made of females with filamentous ovarioles, typical of workers; the second one is females with intermediate ovarioles; and the third group is the group of the queens, which are older females, already inseminated and with the greatest degree of ovarian development. no significant external morphological differences were found among these female groups. therefore, despite the lack of significant morphological differences among females, there are other factors such as the chemical composition of the cuticula, which are indicative of the reproductive physiological condition of the female in the colony. sociobiology an international journal on social insects 1 universidade federal da grande dourados, dourados-ms, brazil. 2 universidade estadual de mato grosso do sul, dourados-ms, brazil. article history edited by marcel g hermes, ufl, brazil received 12 november 2013 initial acceptance 17 february 2014 final acceptance 06 march 2014 keywords cuticular hydrocarbons, polistinae ovarian development corresponding author viviana de oliveira torres progr. de pós-graduação em entomologia e conservação da biodiversidade univ. federal da grande dourados dourados, mato grosso do sul, brazil 79804-970 e-mail: vivianabio@yahoo.com.br reproductive physiology (röseler et al., 1985). the dominance status of the individual apparently initiates a physiological response that directly affects their ovarian development (wheeler, 1986). the queen shows the highest degree of ovarian development and, by using behavioral strategies to dominate all of the other females, she largely monopolizes reproduction while avoiding energy-consuming tasks such as foraging (jeanne, 1972; strassmann & meyer, 1983). evidently, on the lack of visible external traits, some other kind of detection mechanism, used by each member of the colony, is needed for the establishment and recognition of this hierarchy, and chemical communication is the most effective way to accomplish this recognition. among the compounds involved in this process are the cuticular hydrocarbons (chcs), which are a constituent of the lipid layer that coats the cuticle of insects, and have the primary functions of preventing desiccation (lockey, 1988) and creating a barrier against microorganisms (provost et al., 2008). chcs also act as contact pheromones, allowing conspecific individuals to identify each other, thus assisting in maintaining the colony structure, separating individuals according to their function research article wasps sociobiology 61(2): 218-224 (june, 2014) 219 in the colony, their physiological status and their hierarchical rank (provost et al., 2008), functioning, therefore, as a specific chemical signature of the individuals. sledge et al. (2001) and monnin (2006) noted that there is a strong correlation between reproductive status and chc profile of each individual in a colony of wasps. according to dapporto et al. (2005), in colonies of polistes dominula (christ) founded by a single female, the chc profile of the queen and its brood are different and, when one of those colonies becomes orphaned, a worker assumes the queen position, its ovaries develop, and acquires a chc profile similar to that of the original queen. maintenance of a reproductive monopoly by a queen is one of the goals reached by many social insects. in independent-founding species, it was believed that the queen maintains her reproductive status by using aggression toward other females; however, in recent decades many studies have demonstrated the importance and role of chcs in the communication among members of the colonies and in maintaining the status of the queen (bonavita-cougourdan et al., 1991; peeters et al., 1999; liebig et al., 2000; sledge et al., 2001; dapporto et al., 2005). this study is focused on analyzing the reproductive status of females of polistes versicolor (olivier) through examining morphological and reproductive physiological features and by tracing the chemical profiles of the cuticula. material and methods we collected 10 colonies of p. versicolor in the southern region of the state of mato grosso do sul, in the cities of dourados (22º13’16” s 54º48’20” w) and mundo novo (23º56’23” s 54º17’25”w). all of the females from each colony were evaluated for morphological, physiological and cuticular chemical profile analyses. the classification of the colonial stage are done according to the system proposed by jeanne (1972). after collection, the gaster of each female was individually fixed in an eppendorf containing absolute ethyl alcohol (99.8% pa) for later analysis of ovarian development, insemination and relative age. the remainder of the body was preserved by freezing, for subsequent morphometric measurements and analysis of the cuticular chemical profile. we performed nine morphometric measurements, modified from shima et al. (1994) and noll et al. (1997), in order to detect morphological diferences: head: width (hw), minimum interorbital distances (idx); mesosoma: width, length and height of mesoscutum (msw, msl and msh, respectively); metasoma: basal and apical heights of tergite 2 (t2bh and t2ah), length of tergite 2 (t2l); wing: partial length of the forewing (wl). the gaster was dissected under a zeiss binocular stereomicroscope for evaluation of the degree of ovarian development, insemination and relative age. the ovaries were classified according to the stage of development of the ovarioles, based on the observations of baio et al. (2004). for each female, the spermatheca was removed and put on a slide in a 1:1 solution of acid fucsina (1%) in order to determine the presence of sperm cells under a light microscope. the relative age of all adult females was determined, according to the pigmentation of the transverse apodeme across the hidden base of the fifth sternum, as follows: ly (light yellow), lb (light brown), db (dark brown) and ba (black). according to richards (1971) and west-eberhard (1973), this color sequence indicates a progression in the age of individuals, from younger (ly) to older females (ba). for analysis of the cuticular chemical profile, the thorax of each female was submitted to optical spectroscopy by fourier transform infrared photoacoustic spectroscopy (ftir-pas), after 48 hours in a vacuum oven, in order to minimize the water content. this technique was used by antonialli-junior et al. (2007 and 2008) and neves et al. (2012) and has proved reliable for assessing the chcs profiles of ants and wasps, even when compared to gas chromatography (ferreira et al., 2012). the ftir-pas technique measures the radiation absorbed by the sample. it is advantageous for application on very fragile objects, such as biological materials, because the lowintensity radiation does not destroy the sample. ftir-pas uses the infrared spectrum from 400 to 4000 cm-1 (silverstein et al., 2000; skoog et al., 2002), which is sensitive to the vibrations and rotations of molecular chemical groups, so it can identify and distinguish molecular radicals and some kinds of chemical bonds in the samples (smith, 1999). the resulting spectrum for each thorax was obtained from the mean of 64 spectra with a resolution of 8 cm-1, which were separated in absorption lines between 400 and 4000 cm-1, mostly those related to vibrations of chcs. the degree of ovarian development, morphometric data and the cuticular chemical profile were evaluated by stepwise discriminant analysis, which reveals the group of variables that better explain the groups evaluated in case of a difference. this is indicated by wilk’s lambda, a measure of the difference, if any, among the groups (quinn & keough, 2002). the chi-square test was performed to test the association between the relative age and the three groups of females (workers 1, workers 2 and queens). for all analyses, the variable was considered significant when the level reached was <0.05. results and discussion four kinds of ovarian development (fig. 1) were found in females of this species: type a, filamentous ovarioles without visibly developed oocytes; type b, ovarioles containing some oocytes in the initial stage of development; type c, ovarioles with moderately developed oocytes, some in the final phase of vitellogenesis; and type d, well-developed, longer ovariov.o. torres et al reproductive status of polistes versicolor220 lopment were also the inseminated ones. however, giannotti and machado (1999), gobbi et al. (2006) and murakami et al. (2009) analyzed several independent founding species, polistes lanio (fabricius), p. versicolor and mischocyttarus cassununga (von ihering) among them, and found six, five and five ovarian development patterns, respectively. other studies evaluating the relationship between the degree of ovarian development and the reproductive position occupied by the females were performed on parachartergus smithii (saussure) (mateus et al., 1997), pseudopolybia vespiceps (ducke) (shima et al., 1998), chartergellus communis (richards) (mateus et al., 1999), brachygastra lecheguana (latreille) (shima et al., 2000), parachartergus fraternus (gribodo) (mateus et al., 2004) and protopolybia chartegoides (gribodo) (felippotti et al., 2007), all of them species of epiponini. the colonies of p. versicolor contained on average 2.9 ± 1.72 inseminated females, demonstrating the potential of other females to replace the queen; however, only one female had an ovarian condition typical of a laying individual (fig. 1d). giannotti & machado (1999) and murakami et al. (2009) also reported more than one inseminated female in colonies of p. lanio and m. cassununga, respectively. in fact, the presence of more than one inseminated female in the colony, capable of reproducing, is a common feature in independent-founding species, as is the case for polistes and mischocyttarus. this fact shows that the distinction between reproductive and non-reproductive females is quite flexible and complex, depending on physiological, behavioral and ecological factors (murakami & shima, 2006). insemination of two or more females in the same colony can be a strategy to overcome problems encountered during the colony cycle, such as predation or parasitism, as suggested by murakami et al. (2009) for m. cassununga. gobbi et al. (2006) found that 75% of p. versicolor females in the aggregate and 85% in the foundation association were inseminated; therefore, insemination must occur before the formation of aggregates. the results from the analyzes of the cuticular chemical profile show that the 4 different types of ovarian development fig 1. different degrees of ovarian development found in females of polistes versicolor. a) type a, filamentous ovarioles without visible developed oocytes; b) type b, ovarioles containing some oocytes in the initial stage of development; c) type c, ovarioles with moderately developed oocytes, some in the final phase of vitellogenesis; d) type d, well-developed, longer ovario les, each containing two to several producing oocytes. les, each containing two to several producing oocytes. type d females were always inseminated. these four physiological conditions were described by baio et al. (2004) for brachygastra augusti (saussure). noll et al. (2004) also described these same conditions in the species apoica pallens (fabricius), charterginus fulvus (fox) and nectarinella championi (dover). in all of these species, the females with the most advanced ovarian devefig 2. mean curve for each group of mid-infrared absorption spectra of the thorax of polistes versicolor females, grouped according to cuticular chemical profile and indicating the significant peaks for separation of the groups. sociobiology 61(2): 218-224 (june, 2014) 221 degree are distributed among three distinct categories of females: a) workers 1, with filamentous ovarioles, type a; b) workers 2, with partially developed ovarioles, types b and c; and c) queens, with fully developed ovarioles, type d (fig. 1 and 2). these differences were significant (wilks’ lambda = 0.476, f = 6.303, p <0.001) (fig. 3). the spectra analyzed by ftir-pas showed significant differences among the cuticular chemical profiles, indicating seven significant peaks for the separation of females groups (fig. 2 and table 1). these compounds were linked to chitin (1238, 1523 e 2634 cm-1) and chcs (667, 1030, 1377 and 1450 cm-1) present in the female cuticle (table i). antoniallijunior et al. (2007) and neves et al. (2012) discuss the importance of these peaks for distinguishing the groups analysed. however, the most significant peaks for those groups were mainly those corresponding to the hydrocarbon band (fig. 2 and table 1). the first canonical root explained 93% of the results, and the second one the reimaning 7%, explaining together 100% of the results. therefore, it seems that every female within these three groups had a different physiological status within the colony, which leads to a difference in the cuticular chemical profile and probably in the recognition by other females of their position in the colony hierarchy. according to monnin (2006), there are correlations between reproductive status and the chc profile in social insects, and that differentiation is important for the establishment of a hierarchy in independent-founding species, as it is the case for many neotropical members of polistinae. in colonies of p. dominula founded by a single female, the chc profiles of the queen and workers were different (dapporto et al., 2005). bonckaert et al. (2012) investigated colonies of vespula vulgaris (linnaeus) and found that laying queens, queens in aggregate, virgin queens, and workers had different degrees of ovarian development, and this was correlated with their respective chcs profiles. the discriminant analysis of the 3 groups of females with different cuticular chemical profiles, however, showed no significant morphological differences (wilks’ lambda = 0.851, f = 3.182, p <0.05), indicating an absence of morphological differences among these groups. the absence of morphological differences among castes was also reported by giannotti & machado (1999) for p. lanio and by murakami et al. (2009) for m. cassununga, all of them independent-founding species. however, gobbi et al. (2006) observed that of p. versicolor female aggregate are significantly larger than first emerged females, foundress association and workers. tannure-nascimento et al. (2005) suggested that the morphological differences between reproductive and non-reproductive females of polistes satan (bequaert) during the colony cycle is due to seasonal nutritional differences. in fact, most studies describe the absence of morphological differences among castes in polistes and mischocyttarus (cumber, 1951; giannotti & machado, 1999; tannurenascimento et al., 2005; murakami et al., 2009), supporting the hypothesis of post-imaginal caste determination. however, studies such as those of gadagkar et al. (1991), keeping (2002), dapporto et al. (2008) and hunt et al. (2011) performed with ropalidia marginata (fabricius), belonogaster petiolata (degeer), polistes metricus (say) e p. dominula, respectively, show that, at least in part, the distinction of castes may be pre-imaginal. by analyzing the relative age of the females we found a higher frequency of young females during the post-emergence (pre-male) stage and, as the colony cycle advances, old females become more frequent during the post-emergence peak wave number (cm-1) canonical root 1 canonical root 2 functional group vibration model (1) 667 1.453 3.036 out-of-plane c-h (benzene) bending (2) 1030 3.185 -5.432 in plane c-h (benzene) bending (3) 1238 5.766 -5.481 -c-n stretching (4) 1377 -15.713 7.655 c-ch3 symmetric bending (5) 1450 2.659 1.669 c-ch2 and c-ch3 asymmetric bending scissors (6) 1523 3.216 -1.956 n-h bending (7) 2634 0.173 1.456 c-n and n-h overtone bending table 1. wave numbers, coefficients of the two canonical roots, functional groups, and vibrations of the peaks identified in the infrared absorption spectra of the thorax of the wasps, for analysis of the cuticular chemical profile. fig 3. dispersion diagram of the results of the stepwise discriminant analysis, showing the two canonical roots of differentiation of cuticular chemical profile in 3 different groups of females of polistes versicolor. v.o. torres et al reproductive status of polistes versicolor222 (post-male) and decline stages (fig. 4). the chi-square test (χ2 = 65.594, df = 6, p <0.05) indicated a relationship between the female age and the degree of ovarian development, in which the queens are always among the older females of the colony. the workers have a range of ages, which is probably related to the stage of the colony cycle. corroborating these results, baio et al. (2003), murakami et al. (2009) and felippotti et al. (2010), investigating colonies of metapolybia docilis (richards), m. cassununga and three species of clypearia respectively, reported that queens are among the older females in the colony, and that the presence of young and old females varies according to the stage of the colony cycle. murakami et al. (2009) observed that females with the most advanced ovarian development are older and are also more aggressive in the hierarchical ranking. all of these results agree with the system of gerontocracy (strassmann & meyer, 1983), common in independent-founding species, which means that, as the workers grow older, they are subject to more aggressive acts of from the dominant female. acknowledgments the authors thank janet w. reid (jwr associates) for the revision of the english text, and orlando t. silveira (museu paraense emílio goeldi) for the identification of the species. we are grateful to the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for an undergraduate fellowship awarded to the first author, and to capes for a doctoral fellowship to vo torres. wf antonialli-júnior acknowledges research grants warded by the cnpq. references antonialli-junior, w.f., lima, s.m., andrade, l.h.c. & súarez, y.r.. 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(1985). the sociogenesis of insect colonies. science, 228(4704): 1479-148. doi: 10.13102/sociobiology.v66i1.2881sociobiology 66(1): 52-60 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nesting biology of sympatric species of megachilidae bees in a conservation area in brazilian atlantic forest introduction megachilidae bees are distributed around the world with more than 4000 species described in 76 genera (michener, 2000). the new world species occurred from alaska to southern chile and argentina (raw, 2004) and most of them belong to the genus megachile, the leafcutter bees. one factor that probably contributes to the high species richness of this family is the use of nest building materials not explored by other groups of bees (litman et al., 2011). the use of leaf or petal fragments in the nest structure of megachilidae extends the interactions of these bees with plants beyond food sources. bees of different taxonomic groups use distinct types of material in the nest; species of the tribe megachilini characteristically use elongated pieces of leaves on the walls abstract megachilidae bees are important pollinators in the neotropical region, however information on the ecology and behavior of these species is still scarce. the objective of this study was to analyze the nesting biology of sympatric species in the união biological reserve, a remnant of atlantic forest in the southeastern brazil. our results indicated the occurrence of 17 species, representing significant richness compared to other areas in the atlantic forest. five sympatric species built ¾ of all nests found and the architecture of their nests was studied including, for the first time, nests of a species of megachile (ptilosarus). the use of petals or leaf fragments in the construction of the nests was observed for species of megachile (chrysosarus), confirming previous data. the nesting activity period in the trap-nests occurred mainly in the rainy season, with different peaks among the species. the results indicate that the distinctive characteristics of the species, such as the type of material used in the nests, the dimensions of cavities, and the asynchronous nesting period, could be important for the niche differentiation of these sympatric species, allowing the maintenance and the survival of the most abundant megachilidae populations in the area. sociobiology an international journal on social insects bns mello¹,², mc gaglianone¹ article history edited by cândida aguiar, uefs, brazil received 06 february 2018 initial acceptance 10 april 2018 final acceptance 08 november 2018 publication date 25 april 2019 keywords leafcutter bees, nest architecture, nesting activity, rebio união, solitary bees, trap-nests. corresponding author bruno nunes da silva mello universidade de são paulo faculdade de filosofia ciências e letras de ribeirão preto departamento de biologia av. bandeirantes nº 3900, monte alegre cep: 14040-901 ribeirão preto-sp, brasil. e-mail: brunomellouenf@gmail.com of the nests and leaf discs in the partitions between the cells and in the cell caps; in a different way, anthidiini bees use plant fibers or resin as the main component in nest construction (morato, 2001; alves-dos-santos, 2004; camarotti-de-lima & martins, 2005; litman et al., 2011). megachilidae females can nest in different places including preexisting cavities in plants or other substrates. this characteristic of life history allows the use of trap-nests as a sampling methodology, since natural nests are difficult to find (roubik, 1989; morato & martins, 2006). the use of trapnests also makes it possible to obtain more accurate information about nesting biology and the architecture and also about the building materials used in the nests (garófalo, 2000). for species distributed in brazil, some studies deal with the nesting biology of megachilidae (for example laroca, 1 universidade estadual do norte fluminense darcy ribeiro, laboratório de ciências ambientais, campus dos goytacazes-rj, brazil 2 universidade de são paulo, faculdade de filosofia, ciências e letras de ribeirão preto, departamento de biologia, ribeirão preto-sp, brazil research article bees sociobiology 66(1): 52-60 (march, 2019) 53 1971; laroca et al., 1987; martins & almeida, 1994; almeida et al. 1997; morato, 2001; morato, 2003; alves-dos-santos, 2004; zillikens & steiner, 2004; camarotti-de-lima & martins, 2005; cardoso & silveira, 2012; teixeira et al., 2011; marques & gaglianone, 2013; rocha-filho & garófalo, 2015; sabino & antonini, 2017), in different environments as distinct as forests and open or urban areas. none of them, however, analyzed the factors that would lead to the co-occurrence of species with similar needs of resources. to understand the ecological processes acting in the co-occurrence of these species, studies are required that address preferences for nesting sites, seasonality, architecture patterns, and the use of floral resources (blochtein & marques, 2003; kambli et al., 2017). these studies will be important to understand the interactions among the bees, including competition or facilitation and this information could help in the conservation of forest fragments, considering the great potential of these bees as pollinators. the present study aims to answer the following questions: does an ombrophilous forest in the brazilian atlantic forest have expressive richness of megachilidae species co-occurring? which factors may be allowing the cooccurrence of these sympatric species? material and methods study area the study was developed in a fragment of dense ombrophilous rain forest in the atlantic forest of brazil, which is an area of protection recently expanded to 7767.80 ha (reserva biológica união rj, 22°25’35” s; 42°2’4” w. the vegetation comprises stretches of lowland forest (up to 50 m altitude) and submontane forest, with regeneration areas in places formerly occupied by eucalyptus plantations (corymbia citriodora (hook) k.d. hill & l.a.s. johnson). the climate is predominantly humid tropical with an average annual temperature of 24 °c, precipitation of approximately 2200 mm/year. in the region, the wet season occurs from october to march and dry season from april to september (mma/icmbio, 2008). the vegetation comprises stretches of lowland forest (up to 50 m altitude) and submontane forest, with regeneration areas. the climate is predominantly humid tropical with an average annual temperature of 24 °c and precipitation of approximately 2200 mm/year (mma/icmbio, 2008). sampling procedure the bees were attracted to nest in trap-nests made of hollow bamboo canes inserted in p.e.t. bottles attached to wooden stakes 1.5 m from the ground and tubes of black cardboard inserted into wooden plates at the same height in tree branches. approximately 1440 trap-nests were offered simultaneously and monthly (120 in each sampling point), with diameters ranging from 6 to 20 mm and assorted lengths, from 60 to 215 mm (bamboo canes) and from 50 to 91 mm (cardboard tubes) distributed equally among the points. trapnests were installed at 12 sampling points, located at least 500 m apart between two closest points, and monitored from march 2008 to october 2010. at four of these points, another sampling period was carried out, from march 2012 to march 2013 using 480 trap-nests per month (120 in each point). the trap-nests were monitored monthly, and when the bees finished the activity, trap-nests were replaced by others of the same dimensions (diameter and length), always maintaining the same number of cavities in the sampling points. data analysis nests of the most abundant species throughout the study periods were analyzed in the laboratory to describe the architecture and material of construction. characterization of the nest architecture was performed through the types, dimensions, and forms of the elements that constitute the nests. data obtained were compared with other species of megachile (zilikens & steiner, 2004; aguiar et al., 2005; torretta et al., 2012; landry et al., 2014; torretta et al., 2014; rocha-filho & garófalo, 2015). the difference between diameters of the trap-nests used by bees of different species was tested by non-parametric kruskal-wallis and tukey test a posteriori (past 3.20, hammer et al., 2001). in a similar way, body size of the species was compared using the intertegular distance between the inner borders of the bee tegulae. the sex ratio was determined by calculating the proportion of the number of males in relation to the number of females considering the total of emergent during the study and differences of the expected (1:1) was tested through x2 test using program r (r development core team, 2017). results richness of bees in the study area bees of seventeen species of megachilidae nested in trap-nests located in dense ombrophilous forest and regenerated areas (table 1), corresponding to 17.7% of the bee nests found in the area. the five most abundant species (megachile (chrysosarus) pseudanthidioides moure, megachile (chrysosarus) sp., carloticola paraguayensis (schrottky), megachile (ptilosarus) sp. 1, and megachile (pseudocentron) nudiventris smith) represented 74.9% of the nests (table 1). period of nesting activity the nesting occurred mainly in the rainy season, with peaks of activity not overlapped among the most abundant species (fig 1). c. paraguayensis built nests almost exclusively in the rainy season, showing greater activity in february, but they also built one nest in the first and last month of dry season. megachile (chrysosarus) sp. built nests exclusively in the rainy season, showing greater activity in december-january. bns mello, mc gaglianone – nests of megachilidae in brazilian atlantic forest54 megachile (ptilosarus) sp. 1 presented two periods of activity in the year, with greater peak in march (beginning of the rainy season). m. nudiventris constructed nests throughout the year with small peaks in december, may and august. m. pseudanthidioides also built nests throughout the year, except in june and july, with periods of greater activity in octobernovember, periods corresponding to the beginning of the rainy season in the region (fig 1). nest architecture, nest substrate and materials of construction among the five most abundant species, c. paraguayensis (anthidiini) differed from the others through the use of resin mixed with sand or clay in the construction of the nests, while the megachile species used leaves and/or petals with or without clay. the nests of c. paraguayensis were constructed in the cardboard trap-nests, and formed a compact clay (n = 10) or sandy (n = 2) tube both bound together with clay (table 2), and the cells separated by buffers of the same material. the bottom table 1. number of nests of megachilidae constructed in trap-nests in a remnant of the atlantic forest in brazil (união biological reserve, rj, brazil). the five most abundant species are highlighted. *cleptoparasites; **number of nests where respective cleptoparasites emerged. species of megachilidae number of nests % nests anthidiini carloticola paraguayensis (schrottky) 14 11.38% hoplostelis nigritula (friese)* 02** hypanthidium divaricatum (smith) 02 1.62% hypanthidium foveolatum (alfken) 01 0.81% saranthidium marginata moure & urban 04 3.30% megachilini megachile (austromegachile) facialis vachal 01 0.81% megachile (chrysosarus) pseudanthidioides moure 28 22.8% megachile (chrysosarus) sp. 20 16.3% megachile (melanosarus) brasiliensis dalla torre 03 2.40% megachile (melanosarus) nigripennis spinola 01 0.81% megachile (moureapis) cf. benigna mitchell 01 0.81% megachile (moureapis) pleuralis vachal 02 1.62% megachile (moureapis) pseudopleuralis schrottky 02 1.62% megachile (pseudocentron) inscita mitchell 03 2.40% megachile (pseudocentron) nudiventris smith 14 11.38% megachile (pseudocentron) cf. subcingulata moure 02 1.62% megachile (ptilosarus) sp. 1 16 13.00% megachile (ptilosarus) sp. 2 09 7.33% coelioxis spp* 13** total nests 123 species richness (except cleptoparasites) 17 fig 1. nesting activity of megachilidae bees (number of nests constructed in trap-nests) in the união biological reserve, rj, brazil, from mar, 2008 to oct, 210 and from mar, 2012 to mar, 2013. of the cell was rounded and the wall smooth and aligned with the inner wall of the trap-nest. the female deposited clay and resin at the bottom of the trap-nest before constructing the first cell. the final cell, closest to the opening of the trap-nest, was considered as a vestibular cell, since it contained neither food nor eggs, and was filled with floral buds in 10 of the 12 nests analyzed. these buds were identified as belonging to one or more species of asteraceae and malpighiaceae, and a single type filled the vestibular cell of each nest. the two species of the subgenus m. (chrysosarus) used both bamboo and cardboard trap-nests. in the analyzed nests of m. (chrysosarus) pseudanthidioides (n = 20) and m. (chrysosarus) sp. (n = 19), the cells were constructed externally with leaf fragments and internally with agglutinated petals in a single linear series. these leaf materials were firmly attached to the clay tubes of the nests and were not attached to the trap-nest wall. the petals were deposited so as to form the inner wall of the cells. the cells started at the bottom of the trap-nest without any material deposited prior to construction of the first cell. in the bamboo canes cells of m. (chrysosarus) spp never filled completely the trap-nest, but there was a space between the final cell and the opening of the trap-nest. sociobiology 66(1): 52-60 (march, 2019) 55 table 2. most abundant megachilidae species in união biological reserve, rj, brazil. intertegular distance was used to compare body size among bees (m=male and f=female). evaluated attribute of nests: type (c=cardboard; b=bamboo cane) and diameter of occupied trap-nest, length of constructed nest, number of cells constructed per nest, and cell length (mean ± standard deviation). all measurements in mm. bee species intertegular distance m/f trap-nest type trap-nest diameter nest length cells per nest cell length carloticola paraguayensis 3.15±0.2/ 3.16±0.15 c (100%) 6-10 (8±1.0) n=12 57.9 to 90.1 (73.2±13.3) n=12 3 to 6 (4.5±1.0) 8.2 to 23.1 (12.8±4.2) n=26 megachile (chrysosarus) pseudanthidioides 3.17±0.2/ 3.46±0.2 c (75%) b (25%) 6-14.2 (8.5±3.0) n=17 16.1 to 111.6 (70.7±43.5) n=17 1 to 9 (4.5±1.7) 12 to 21.4 (15.2± 2) n=62 megachile (chrysosarus) sp. 3±0.1/ 3.51±0.1 c (60%) b (40%) 8-18.1 (10.8±3.6) n=19 32.5 to 153.0 (85.1±39.6) n=19 1 to 10 (4.9±2.2) 11.5 to 20.6 (15± 1.6) n=89 megachile (pseudocentron) nudiventris 3.37±0.1/ 3.47±0.2 b (100%) 10.3-16.8 (13.5±2.2) n=10 62.2 to 214.4 (130.5±46.5) n=10 4 to 9 (5.7±1.6) 16.8 to 29.8 (21.7±3.7) n=33 megachile (ptilosarus) sp. 1 2.2±0.15/ 2.46±0.15 c (100%) 6 (6±0.0) n=14 15.4 to 71.1 (53.3±17.4) n=14 1 to 8 (4.5±1.8) 7 to 15.3 (10±1.4) n=49 circular leaf discs formed the closure plugs and the cell partitions and elongated cuts of these materials were on the walls and bases of the cells, resulting in an approximately conical shape of the cell. m. (pseudocentron) nudiventris constructed the ten nests analyzed exclusively with leaf fragments, always in bamboo trap-nests. this species also constructed a linear series of cells, and in the majority of nests the female used all the available space of the trap-nest, and the nests length varied as indicated in table 2. all nests began with the first cell constructed directly on the bottom of the trap-nest through a tube of loosely arranged leaves. the side wall of the cells was formed by elongated pieces of leaf with the base folded inward, aiding closure of the base of the cell; the partitions consisted of circular discs of leaves. in three nests it was possible to observe the presence of leaves with different patterns of veins, parallel nerved and venation netted, and with different patterns of hairiness, indicating at least two distinct plant species as sources of leaf material for the same nest. m. (ptilosarus) sp. 1 used only cardboard trap-nests, and constructed nests with a linear array of cells and a thin layer of clay laterally lining the cells, externally. cells were constructed with leaf fragments, which were firmly attached to the clay on the outer face. in all nests the construction started at the bottom of the trap-nest. the leaf fragments of the base were disc shaped and on the wall the format was elongated. a single nest presented ring-shaped clay deposited outside the base of each cell on the leaf discs, holding them firmly attached. trap-nest diameter preferences the internal dimensions of the occupied trap nests are shown in table 2. c. paraguayensis used 8 mm cavities, on average, while m. pseudanthidioides occupied a wide range of diameters, but mainly 6 to 7.9 mm tubes (53% of nests were found in the 6 mm cavities). m. (chrysosarus) sp. used a larger proportion of larger cavities too (from 8 to 11.9 mm), being the only species in the group to occupy cavities larger than 18 mm in diameter. m. (pseudocentron) nudiventris used cavities larger than 10 mm (table 2, fig 2) and m. (ptilosarus) sp. 1 used only the smallest cavities (of 6 mm). body size and sex ratio the body sizes of the bees inferred by the intertegular distance showed that m. (ptilosarus) sp1 was the smallest species and m. (chrysosarus) sp the largest one. females were, in general, larger than males (table 2) and this difference was statistically significant for the two species of m. (chrysosarus) (m. sp: q = 8, p < 0.05, and m. pseudanthidioides: q = 5.5, p < 0.05) and for m. (ptilosarus) sp. 1 (q = 5.6, p < 0.05). males of m. (ptilosarus) sp. 1 were statistically smaller than the males of the other compared species. females of this species and of c. paraguayensis showed the smallest mean sizes, which differed from the other three species. the species with smaller average size of the females occupied the smaller cavities of trap-nests, whereas m. (pseudocentron) nudiventris, m. (chrysosarus) pseudanthidioides, and m. (chrysosarus) sp. occupied the largest cavities. all species, with the exception of m. (chrysosarus) sp., produced more females than males and sex ratios obtained for m. (chrysosarus) pseudanthidioides and m. (ptilosarus) sp.1 differed from the expected 1:1 (18♂:34♀, χ2 = 4.92 p = 0.02 and 12♂:27♀, χ2 = 5.77 p = 0.01, respectively). bns mello, mc gaglianone – nests of megachilidae in brazilian atlantic forest56 discussion megachilidae species sampled in the atlantic forest fragment studied has important representation in the community of bees that nest in cavities when compared to other communities sampled in brazil (garófalo et al., 2004), and more specifically in atlantic forest (aguiar et al., 2005; steiner et al., 2006; rocha-filho et al., 2017). the present study indicates that the união biological reserve is an important repository of megachilidae bees, an important group of pollinators in the neotropical region. this fact is possibly related to some factors such as the large extent of the forest fragment, environmental heterogeneity and wellpreserved areas at união biological reserve. the great relevance of this forest fragment has been also indicated for orchid bees (ramalho et al., 2009), vertebrates (araújo et al., 2008) and plant species (evaristo et al., 2011). five species constructed 3/4 of all megachilidae nests sampled during the study. analysis of the nest architecture of these five species showed three main types of materials used: resin mixed with clay or sand, exclusively leaf fragments, or leaf fragments and petals with or without clay. c. paraguayensis was the only studied species using resin as construction material. this behavior is widely known for anthidiini (roubik, 1989; muller, 1996; michener, 2000; camarotti-de-lima & martins, 2005), and the antimicrobial and repellent potential of resins is discussed as an advantage against predators or parasites (ghisalberti, 1979), despite the high energy cost to locate and handle this resource. the limitation of resin availability may be a preponderant factor in the distribution of c. paraguayensis nests. the study of the larval content of the cells (mello & gaglianone, unpublished data) indicated the presence of pollen grains of dalechampia sp. (euphorbiaceae), a species that presents floral resin, a rare feature among angiosperms and essential for these bees. this plant was observed as the most important pollen source for another bee, tetrapedia diversipes klug, in the same area (menezes et al., 2012). further studies are needed to understand the spatial and temporal distribution of nests and their relationship with pollen and resin supplying plants in different environments. unlike the use of resins, plant fragments are the most common construction material used by megachile species, the so-called leafcutter bees (roubik, 1989; raw, 2004). the use of leaf fragments, exclusively or adhered to the clay, as observed for the species of megachile (pseudocentron) and megachile (ptilosarus), respectively, in the present study, constitutes the behavior most widely performed by species of the genus. the use of petals, third type of the material used in the nest construction, was observed in this study only for species of megachile (chrysosarus), confirming previous studies with other species of the same subgenus (zillikens & steiner, 2004; laroca et al., 1992; torreta et al., 2014; rocha-filho & garófalo, 2015). the use of petals for the construction of rearing cells, substituting or together with leaves, was discussed by other authors as being related to the oral apparatus of these bees. species of megachile (chrysosarus) differ morphologically from species of other subgenus through the complete lack of cutting edges between the mandibular teeth (mitchell, 1943), which would be related to cutting more delicate material such as finer petals or leaves. the choice fig 2. percent of occupation of the trap-nests used by megachilidade bees in the rebio união, presented in diameter classes (mm). sociobiology 66(1): 52-60 (march, 2019) 57 between these different types of materials is probably related to availability in the environment and may vary with the plant phenology in the vicinity of the nests. the agglutination of vegetal fragments with clay resulted in the formation of a tube of firm walls in the studied nests of m. (ptilosarus) and megachile (chrysosarus). this architecture allows a more constant microclimate inside the nest, helping to maintain a favorable environment for the development of the immature bees. studies carried out for other species of the genus (zillikens & steiner, 2004; torreta et al., 2014) also verified the use of clay among the plant fragments structuring the nest. despite this similarity in the agglutination material of the plant fragments, megachile (ptilosarus) sp. 1 nests analyzed in this study were composed only of leaves, unlike the species of megachile (chrysosarus). to our knowledge, this is the first description of the architecture of nests of a species of ptilosarus. in addition to the building material, other characteristics of nest architecture are very important to understand the ecological role of these bees and their interactions with other bees that also use cavities and with their natural enemies. one of these characteristics can be the linear series of cells, which is related to the disposition of the cavity offered. as the study in natural cavities is hampered by the unpredictability of finding nests and by the camouflage, it is not known how flexible this characteristic is. however, the construction of vestibular cells, commonly observed in this linear structure, would be a strategy against parasite attack. in this case, an intruder entering the nest would initially reach the vestibular cell and, failing to find a larva or larval food, would possibly abandon it (morato, 2001; alves-dos-santos, 2004). the behavior reported in this paper for c. paraguayensis, filling the vestibular cells with floral buds, is apparently unheard of for megachilidae. this behavior potentially reduces parasitism, hindering the entry of the parasite into the nest or resulting in oviposition in an inappropriate place. filling the vestibular cell would lead the parasite to mistakenly oviposit on the buds, through the operculum of the cell, keeping the innermost cells safe. this interpretation however needs to be tested. in the study area, the use of floral buds was facilitated by the location of the c. paraguayensis nests in open areas, at the edge of the forest, where there are abundant herbs and climbers, with small flowers, flowering all year round and only a few meters from the trap-nests. other studies at sites with different conditions will allow analysis of whether there are relations between the availability of this resource and the abundance of the nests. the description of the internal dimensions of the nests of the studied species supplied new information, such as that related to the nests of c. paraguayensis, a rarely studied species and for which no descriptions of nests were found in the literature (a compilation of information obtained in the literature for the study taxa is presented in table 3). the low number of cells seems to be related to the large vestibular cell filled with floral buds, associated with the use of smaller nests, always with cardboard, which decreases the space for the construction of a larger number of rearing cells. bee species reference construction materials (% of studied nests) number of nests studiedleaves petals clay sand resin others carloticola paraguayensis (schrottky) this study 83.4 16.6 100 12 megachile (chrysosarus) catamarcensis schrottky torretta et al. 2014 x x 100 29 megachile (chrysosarus) guaranitica schrottky rocha-filho & garófalo 2016 100 100 26 megachile (chrysosarus) pseudanthidioides moure this study 100 100 100 17 megachile (chrysosarus) pseudanthidioides moure zilikens & steiner 2004 100 100 100 14 megachile (chrysosarus) sp. this study 100 100 100 19 megachile (pseudocentron) alleni mitchell landry et al. 2014 100 100 3.8 52 megachile (pseudocentron) gomphrenoides vachal torretta et al.2012 100 19 megachile (pseudocentron) inscita mitchell aguiar et al. 2005 100 11 megachile (pseudocentron) nudiventris smith this study 100 10 megachile (ptilosarus) sp. this study 100 100 14 table 3. materials used in the construction of megachilidae nests in união biological reserve. the numbers represent the frequency (%) of nests with the respective material; x represents the presence of the material but no information about frequency of use. bns mello, mc gaglianone – nests of megachilidae in brazilian atlantic forest58 our data showed that there is overlap in the period of nesting activity of the most abundant sympatric species in rebio união. however, species activity peaks are not coincident. among the species that use petals in cell construction, m. pseudanthidioides has a much longer nesting period and presents one of the peaks at the end of the rainy season (march), when m. (chrysosarus) sp is not active. allied to this temporal distinction in nesting activity, the floristic composition of the petal and leaf sources used in the nests of these two species is probably distinct given the high plant diversity and phenological patterns of the plants in the area (evaristo et al., 2011). in addition, the two species of m. (chrysosarus) occupy a greater proportion of cavities of different diameters, associated with significantly different body sizes. these characteristics result in non-overlapping ecological niches, a strategy that avoids competition between sympatric species that are closely related and share similar biological characteristics. the species that occupied mainly or exclusively the smallest cavity diameters, m. pseudanthidioides and m. (ptilosarus) sp. 1, also overlapped much of the activity period. however, the use of petals in nests of m. pseudanthidioides distinguishes the two species in relation to building materials and raises questions regarding the source of possibly distinct leaf fragments related to the different jaw forms observed in the two species. identification of sources of foliar resources for sympatric species in future studies would be of great relevance to clarify this issue. among the species that used the largest cavities, m. (pseudocentron) nudiventris stands out for occupying cavities above 14 mm in 60% of the nests studied. another distinctive characteristic of this species is the non-use of any binder material between the plant fragments on the cell walls. this species presents activity during most of the year, but with peaks not coincident with any other abundant species in the trap-nests, being the only species to nest in july, peak of the dry season in the region and the lowest temperatures. this fact suggests availability of resources throughout the year in the area, allowing the co-occurrence of different species of the megachilidae. m. nudiventris and m. (chrysosarus) sp. are the largest species in the present study. larger-sized females may have a competitive advantage over smaller species in relation to floral resources, as they could collect a larger volume of nectar and pollen to supply larger cells, in turn generating larger individuals (klostermeyer, et al., 1973; pyke, 1978). these species also produced nests with a higher number of cells, in comparison with the other abundant species in the area. evaluation of the floristic composition used as floral resources by these species could elucidate whether they present greater competitiveness related to the greater body size and greater productivity. the results indicate that the distinctive characteristics of the species, such as the type of material used in the nests, dimensions of the chosen cavities, and periods of nonoverlapping nesting activity, are important for delimitation of the niches of these sympatric species, enabling maintenance of their populations as the most abundant in the area. acknowledgements we would like to thank the chico mendes institute for biodiversity conservation (icmbio-mma) for permiting the study and whitson j.costa júnior for logistic help in união biological reserve; postgraduate program in ecology and natural resources/uenf and laboratory of ambiental sciences (lca/uenf) for logistical support; capes/procad (158/07) for the financial aid to the project; faperj and capes for granting scholarships for the first author. we thank dr. gabriel a. r. melo and dra. danuncia urban for identification of the bees; carolina rabelo de almeida for helping with the morphometry of the bees; and dr. carlos alberto garófalo and anonymous referees for the critical reading of the manuscript and suggestions. mcg is a pq scholarship holder of cnpq. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. references aguiar, c.m.l., garófalo, c.a. & almeida, g.f. 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(2004). nest architecture, life cycle and cleptoparasite of the neotropical leaf-cutting bee megachile (chrysosarus) pseudanthidioides moure (hymenoptera: megachilidae). journal of the kansas entomological society, 77: 193-202. doi: 10.2317/0310.29.1 doi: 10.13102/sociobiology.v62i3.427sociobiology 62(3): 334-339 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 flight and digging effort in leaf-cutting ant males and gynes introduction annually, mature atta colonies produce male and female winged ants that leave the colony to form new colonies and thereby, continue and perpetuate the species (hölldobler & wilson, 1990). the nuptial flight of leaf-cutting ants may occurs in the morning or afternoon according to species, controlled by the first rains starting the rainy season (staab & kleineidam, 2014 for atta vollenweideri), with the males forming “swarms” of 200 m in diameter and heights over 150 m above ground level for atta capiguara (amante, 1972). the flying distance varies according to species and the air flow speed. it is known that females of atta texana can fly at a speed of 5.33 ms-1, indicating a distance of 10.4 km (moser, 1967); atta sexdens at a speed of 1.57 ms-1, with a possible distance of 11.1 km (jutsum & quinlan, 1978), and field observations have shown a dispersion of queens of 9.6 km for the this species (cherrett, 1968). after the nuptial flight and copulation, a queen of atta sexdens digs a vertical tunnel of about abstract the nuptial flight and nest digging are high intensity activities which consume body reserves. the flight and digging effort was quantified by measuring the carbohydrate and total lipids content in males and females before and after the nuptial flight, and the queen’s digging effort during the foundation. the digging effort was quantified by experimentally stimulating the queens to dig a nest – one, two or three consecutive times – compared to the queens that did not dig. the colorimetric method was used to determine the soluble carbohydrates and extraction method of immersion was used to determine the total lipids. the results showed significant loss of carbohydrates and total lipids in males and females after the flight. on average the males contained 0.027 mg of soluble carbohydrates before the nuptial flight, and 0.005 mg after the nuptial flight, and the females contained 0.129 mg of soluble carbohydrates before the nuptial flight, and 0.079 mg after the nuptial flight. for the males the percentage of lipids decreased from 5.27±1.07% to 2.60±0.63% and for females from 36.46±4.86% to 32.62% after the nuptial flight. the digging effort of the queen caused a slight reduction in total carbohydrates, it was without digging 0.054 mg, normal digging 0.055 mg, double digging 0.045 mg (decrease of 20,22 %), and triple digging 0.044 mg (decrease of 20 %) per queen. based on our results we conclude that the carbohydrate content is the main energetic resource used for the nuptial flight and nest digging, for males and gynes of leaf-cutting ants. sociobiology an international journal on social insects ej silva, rs camargo, lc forti article history edited by kleber del-claro, ufu, brazil received 26 may 2014 initial acceptance 17 july 2014 final acceptance 10 september 2014 keywords ants, carbohydrates, lipids, social insects corresponding author roberto da silva camargo laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas unesp, 18603-970, caixa postal 237, botucatu, sp, brazil e-mail: camargobt@hotmail.com 15 cm and a chamber within which she is enclosed, caring for fungus culture and brood (autuori, 1942). the nest digging takes on average about 6 to 10 hours (autuori, 1942), with about 300 trips to stack the excavated soil by the queen, each trip lasting from 30 seconds to 30 minutes (ribeiro, 1972). because this activity is very intense, it is assumed that the energetic cost of the excavation is high. there is a few data available in the literature regarding the amount of energy required by the queen for digging the nests (camargo & forti, 2013 a). the energy required is removed from carbohydrate reserves, because the lipid content is spared when queen dug the nest (camargo & forti, 2013 b). the digging effort significantly affects the survival of queens of atta sexdens rubropilosa. at the first week after an extra digging effort, (successively digging of two or three nests) there is a higher mortality in queens. it can probably be stated that the mortality of the queens was caused by the depletion of body reserves (camargo et al., 2011). besides, in the claustral research article ants universidade estadual paulista unesp, botucatu, sp, brazil sociobiology 62(3): 334-339 (september, 2015) 335 phase, queens loose about 40% of their body weight after 60 days (della lucia et al., 1995), reaching minimum body mass before the first workers begin to forage. when foraging starts, the queen recovers her body mass, due to nourishment in the colony (della lucia et al., 1990, camargo et al., 2013). the goal of the present study was to measure the soluble carbohydrate and lipid contents of gynes and males consumed before and after the nuptial flight, and soluble carbohydrate consumed before and after the nest excavation by the queen. material and methods collecting atta sexdens rubropilosa females and males before and after the nuptial flight. we collected females and males (pre-nuptial flight) from three nests of a. sexdens rubropilosa over a mound of loose-soil before the nuptial flight. these insects (n = 40) were taken to the laboratory, immediately killed by freezing and analyzed for lipid content. fig 1. behavioral sequence of nest digging by the queen, illustrating the “turning point”, in other words, queen digs with head down, and when it can turn around into the chamber, it carries the soil pellet with head up until enclosure. the mated females (post-nuptial flight) were collected after shedding their wings and beginning to dig start their new colonies (n=20), and males after their death (n=20). after the collection, the queens were immediately stored in plastic containers (11 cm in diameter, 8 cm high), containing 1 cm of plaster at the bottom to keep the air moist. the queens and males were transported to the laboratório de insetos sociais-praga fca/unesp botucatu, sp, brazil, this travel took 30 minutes. digging effort this experimental set up was previously performed according to camargo et al. (2011) and camargo and forti (2013 b). one hour after the nuptial flight, the queens were placed in tubes filled with soil (25 cm high and 10 cm in diameter), removed at 60 cm in depth, at a density of 1.6 g/cm3. the queens did not start laying eggs before the experiments. four experimental series were performed, as follow: 1) without digging: queens did not dig at all, and were directly placed singly in a plastic container; ej silva, rs camargo, lc forti flight and digging effort in leaf-cutting ant males and gynes336 2) single digging: queens were allowed to dig in the tube filled with soil, until they sealed the excavated tunnel; 3) double digging: queens were allowed to dig twice in the tube filled with soil, by being immediately removed from the dug chamber and again met in a new digging tube, in which they started to excavate a second nest; 4) triple digging: queens were allowed to dig thrice in the tube filled with soil, by being met immediately in two new digging tubes after the first excavation. for all four groups, the queens were removed from the tubes and frozen in order to determine the soluble carbohydrates. in addition to the soluble carbohydrate content, the length of the excavated tunnel was determined, as well as the excavated chamber dimensions (length, width and height). the volume of excavated soil was also measured by direct weighing in a semi-analytical balance. the queens’ digging time was measured to assess their performance and effort. the queens’ digging time was observed in 3 ways: first – time spent in tunnel excavation, second – time spent in chamber excavation, and third – time spent in tunnel and chamber excavation, when the observer lost queen’s turning point (fig 1). this behavioral sequence was studied by fröhle and roces (2012). determination of carbohydrates the colorimetric determination of the soluble carbohydrates was achieved by the method of dubois et al. (1956), based on the reaction of carbohydrates, sulfuric acid and phenol. the reaction generates a yellow-orange color which is sensitive and stable. the absorbance was determined on a beckman du640 spectrophotometer. the blank received water in place of the extracts. glucose was used as standard (c=90.531abs + 3.7125, r2=0.992). the values were expressed as mg of total sugars. determining total lipid content males and females before and after the nuptial flight were killed by freezing and then subjected to the experimental procedure used by seal (2009). the procedure was as follow: 1) fresh mass of males and females was individually determined; 2) queens were dried for 24 hours at 70°c and their dry mass was determined; and 3) the lipid content was extracted with petroleum ether (bp 40-60°c) for 24 hours and the queens weighed again. this procedure was repeated for 72 hours of extraction. the percentage of total fat content was calculated using the formula: 100x (dm ffdm)/dm, where dm is the dry mass and ffdm is dry and fat free mass. the energy content of the ants was obtained by multiplying their lean mass by 18.87 j mg-1 and their fat mass by 39.33 j mg-1 (peakin, 1972). statistical analysis the total lipid and soluble carbohydrate content of males and queens before and after the nuptial flight and nest digging effort were compared using a paired t-test (α =0.05). the data of the excavation effort were subjected to the anova of repeated measures (α=0.05), comparing the variable total digging time, overall tunnel length and excavated soil of each experimental series. the analysis was performed using the sigmaplot 11.0 program. results nuptial flight effort the average fresh masses of male and females of a. sexdens rubropilosa before nuptial flight were 103.6±18.6 mg and 656.7±75.1 mg, whereas the masses of male and females after nuptial flight which had mated and flew were 154.3±7.9 mg and 631.2±38.8 mg, respectively (table 1). for males and females we verified a significant loss of soluble carbohydrate after the flight. on average the males contained 0.027 mg of soluble carbohydrates before the nuptial flight, and 0.005 mg after the nuptial flight (reduction of 81.48%) (t test=7.661, d.f.=18, p< 0.0001). on average the females contained 0.129 mg of soluble carbohydrates before the nuptial flight, and 0.079 mg after the nuptial flight (reduction of 38.76 %), differing significantly between them (t test, t=4.718, d.f.=18, p< 0.0001). lipids represented 36.46±4.86 % and 5.27±1.07% of the body mass of females and males before nuptial flight, respectively. this percentage decreased to 35.00±2.28% in females and 2.60±0.63% in males who mated and flew (table 1). as expected, significant differences were detected in the percentage of lipids of males before and after the nuptial flight (anova, f1;19=89.051, p<0.001). but, the percentage of lipids of females who had and had not flown were not significantly different (anova, f1;19=1.400, p=0.242). the energy content was 8563.24±1668.76 j for females before nuptial flight, 8413.37±802.56 j for females who had flown (table 1). male before nf (n=20) male after nf (n=20) female before nf (n=20) female after nf (n=20) fresh mass (mg) 103.68±18.61 154.29±7.96 656.72±75.05 631.18±38.81 dry mass (mg) 35.17±5,60 53.02±2.74 323.69±53.64 322.85±25.20 lean mass (mg) 33.31±5.32 51.63±2.54 203.68±25.75 209.39±11.63 lipid content (mg) 1.86±0.51 1.39±0.41 120,01±31.39 113.46±15.77 lipid percentage (%) 5.27±1.07 2.60±0.63 36.46±4.86 35.00±2.28 energy content (j) 8563.24±1668.76 8413.37±802.56 table 1. mean and standard deviation (m±sd) of masses (mg), percentage of fat and energy content (j) in the bodies of males and female of atta sexdens rubropilosa before and after nuptial flight. sociobiology 62(3): 334-339 (september, 2015) 337 digging effort in the single digging, the average time spent for the queens was of 383.21±59.01 (n=14) minutes to dig the tunnel, and 42.21±40.46 (n=14) minutes for the initial chamber. it was not possible to observe the turning point for some queens, they took an average of 523.75±164.79 (n=14) minutes to build the entire nest. in the double digging, the average spent time for the queens was of 310.35±137.94 (n=20) minutes to dig the tunnel, and 77.35±95.19 (n=20) minutes for the initial chamber. some went into claustral confinement without the observation of the queen’s turning point, thus the total digging time was of 305.0±184.59 (n=5) minutes to build the entire nest. in this experimental series, five queens did not do any digging. in the triple digging, the average spent time for the queens was of 331.21±169.90 (n=10) minutes to dig the tunnel, and 79.30±89.87 (n=10) minutes for the initial chamber. some of them enclosure without the observation of the queen’s turning point, thus the total digging time was of 347.67±73.21 (n=10) minutes to build the entire nest. in this experimental series, eight queens did no digging. the statistical analysis showed that there were differences at double digging (anova, f1;52=12,47, p<0.001), where the queen’s total digging time was higher at first than in the second digging time. moreover, there was difference in the triple digging (anova, f1;77=5.85, p<0.05), where the first founding nest in the triple digging experiment takes significantly longer than two following ones (tukey post test, first versus second, p<0.05; first versus third, p<0.01, second versus third, non significant). the overall of average removed soil dry mass was 35.66 ± 6.86 g for the single, 59.21±15.38 g for double and 87.90±16.98 for triple digging. there were significant differences among dry mass of soil excavated by the queen, being this amount higher for the triple digging, followed by double digging in the excavated soil (anova, f2;87=70,95, p<0.0001, tukey post test: first versus second, p<0.01; first versus third, p<0.01; second versus third, p<0.01). the overall tunnel length dug by the queens was 12.00± 2.02 cm for the single, 19.32±4.58 cm for double and 26.62±6.12 cm for triple digging. there were differences in the excavated tunnel length (anova: f2;87=51.84, p<0.0001, tukey post test: first versus second, p<0.01; first versus third, p<0.01; second versus third, p<0.01) queen’s digging effort caused a slight reduction in total carbohydrates when they excavated two and three nests. queens that did not dig presented 0.054 mg of total carbohydrates, and after a single digging, 0.055 mg. on the other hand, after double and triple digging they showed 0.045 mg and 0.044 mg, respectively, which represents a decrease of about 20,22 % of total carbohydrates (anova, f3;36= 3.1096, p=0.0376). experimental series tunnel chamber length height width single digging 12.0±2.02 3.93±0.71 2.11±0.38 2.78±0.38 double digging i 10.82±2.48 3.35±0.70 2.09±0.40 2.78±0.73 double digging ii 8.50±2.35 3.73±0.98 1.98±0.26 2.83±0.68 triple digging i 10.47±2.52 3.22±0.82 1.95±0.32 2.66±0.56 triple digging ii 8.43±1.99 2.95±0.54 1.83±0.34 2.23±0.44 triple digging iii 7.72±2.27 3.87±0.57 1.99±0.30 2.64±0.51 table 2. mean and standard deviation (cm) of tunnel length and dimensions of the initial chamber (cm) of initial nests of atta sexdens rubropilosa. discussion as expected, total soluble carbohydrate and lipid contents reduced in males and total soluble carbohydrate reduced in gynes of atta sexdens rubropilosa after the nuptial flight. this result is similar that found by jutsum and quinlan (1978) in atta sexdens. the authors found that 21% of the dry mass of the winged is carbohydrate, which is completely consumed after the nuptial flight. similarly, in a study of formica lugubris, it was demonstrated that carbohydrate (stored as glycogen) is the main energy source for the nuptial flight (passera et al., 1989). furthermore, both studies state that lipids are not used as energy for the females’ flight, as observed in our study (table 1) and in camargo & forti (2013 b). in cataglyphis cursor and iridomyrmex humilis, the males have higher carbohydrate content when compared with females (passera & keller, 1990). from these studies, it can be assumed that the first energy source to be depleted in the nuptial flight is the carbohydrate reserves. lipids and proteins degrade at slower rates when compared to carbohydrates, preserving these reserves for the activities after nuptial flight. it is known that the symbiotic fungus is a rich source of carbohydrates, especially glucose. according to silva et al. (2003), the glucose concentration in the fungus garden was of 27.7 mg.g-1, about two and a half times higher than that found in plant leaves (10.8 mg.g-1, for eucaliptus alba) and about six and a half times higher than that found in symbiotic fungi mycelia grown in culture medium (4.3 mg.g1). thus, the nourishment from the fungus garden allows the sexual forms to acquire sufficient reserves for the nuptial flight (fujihara et al., 2012), and the subsequent excavation of the nest by the founding queen, as showed in our study when queens dug two and three time. with regards to the digging effort, under the experimental conditions, leaf-cutting ant queens excavated the nest (table 2), as previously described in the literature (eidmann, 1935; cunha, 1968; camargo et al., 2011; fujihara et al., 2012), first a tunnel, and then a chamber (fig 1). the depth of the initial chamber, ranging from 8.5 to 15 cm (autuori, 1942; ribeiro, 1972). ej silva, rs camargo, lc forti flight and digging effort in leaf-cutting ant males and gynes338 our results show that carbohydrate content of the queens of atta sexdens rubropilosa were affected by the increased digging effort, in other words, by the successive excavations experimentally induced. additionally, camargo et al. (2013) found that a single worker (body weight 9.65±1.50 mg) dug on average 0.85±0.27 g of soil in 24 hours and consumed approximately 0.58±0.23 j, a significant energy cost by the workers. thus, there is a digging cost directly detected by the consumption of total carbohydrates. the queens dug 35.66 g in the single digging, and when subjected to triple excavation, dug about 87.90 g. furthermore, it is known that the oviposition rate is not affected by the digging effort (camargo et al., 2011), but it does accentuate mortality of these queens. the authors state that there is no clear justification for the queen’s mortality based on the depletion of body reserves. there may be two consequences of the successive effort. first, excessive cuticle abrasion due to the increasing digging, with associated water losses as described for harvesting ants (johnson, 2000; johnson & gibbs, 2004) and second, the accumulation of oxidative damage associated with the intense initial digging activity, as known for flying insects (magwere et al, 2006; sohal & buchan, 1981). based on our results it can be concluded that the amount of carbohydrates is the main energetic resource used for the nuptial flight for males and gynes, and digging nest for queens of leaf-cutting ants, when they dug two or three times. references amante, e. 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(2009). scaling of body weight and fat content in fungus gardening ant queens: does this explain why leafcutting ants found claustrally? insectes sociaux, 56: 135-141. doi: 10.1007/s00040-009-0002-8. silva, a., bacci, m., siqueira, c.g., bueno, o.c., pagnocca, f.c., hebling, m.j.a. (2003). survival of atta sexdens workers on different food sources. journal of insect physiology, 49: 307-313. doi: 10.1016/s0022-1910(03)00004-0. sohal, r.s. & buchan, p.b. (1981). relationship between physical activity and life span in the adult housefly, musca domestica. experimental gerontology, 16: 157-162. doi: 10.1016/0531-5565(81)90040-1. staab, m. & kleineidam, c.j. (2014). initiation of swarming behavior and synchronization of mating flights in the leaf-cutting ants atta vollenweideri forel, 1893 (hymenoptera: formicidae). myrmecological news, 19: 93-102. retrieved from: http:// www.myrmecologicalnews.org/cms/images/pdf/volume19/mn 19_93-102_non-printable.pdf. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.65-69sociobiology 62(1): 65-69 (march, 2015) no morphometric distinction between the host constrictotermes cyphergaster (silvestri) (isoptera: termitidae, nasutitermitinae) and its obligatory termitophile corotoca melantho schiødte (coleoptera: staphylinidae) introduction the controlled temperature and humidity within termite nests create an internal atmosphere that attracts other animals seeking shelter, protection and food (noirot & darlington, 2000). the nest may specially represent a food source for some inquiline termites and termitophile invertebrates (kistner, 1969). different species can live in association with termite nests, cohabiting with the host species or occupying nest cavities without direct contact with the host colony. kistner (1969) termed termitophiles the animals that live at least one phase of their life inside termite nests. in the brazilian savannah (cunha & brandão, 2000; cunha & morais, 2010; lopes & oliveira, 2005; costa et al., 2009; cristaldo et al., 2012) and brazilian amazonia (carrijo et al., 2012) different termitophile groups have been reported in termite nests: acarina, anura, araneae, blattaria, chilopoda, coleoptera, diplopoda, haplotaxida, heteroptera, hymenoptera, lepidoptera, orthoptera, opiliones and abstract different species may live in termite nests, cohabiting in close association with the host colony or occupying nest cavities without direct contact with the host. the strategy of termitophile organisms to become integrated into termite societies include appeasement through chemical, morphological and/or behavioral mimicry. we investigated the hypothesis that there is a morphological mimicry between the obligate termitophile corotoca melantho (coleoptera: staphylinidae) and workers of its termite host constrictotermes cyphergaster (isoptera: termitidae). pictures of thirty-one c. cyphergaster workers and c. melantho individuals were taken in top and side views and converted into thin-plate splines. four homologous landmarks and five semilandmarks (reference points) were marked on the head and abdomen of both species and digitized. the body shape of both species are morphometrically similar, so there is no discrimination between specimens of termitophile beetles and worker of termite hosts. body size of termite hosts is responsible for 20% to 30% of body shape variation, while the body size of termitophiles beetle affects near 50% to 60% body shape. however, termitophiles body shape had a greater variation than worker termites. this is the first study to compare morphological similarity among termites and termitophiles using morphometric geometry. our results indicated the existence of a morphological mimicry between c. cyphergaster and c. melantho. sociobiology an international journal on social insects hf cunha; js lima; lf souza; lga santos; jc nabout article history edited by reginaldo constantino, unb, brazil received 31 january 2014 initial acceptance 02 april 2014 final acceptance 12 november 2014 keywords coexistence, morphological mimicry, termite, termitophile corresponding author hélida ferreira da cunha ueg/ unucet/ br 153 fazenda barreiro do meio n° 3105 cep 75132-903, anápolis-go, brazil e-mail: cunhahf@ueg.br scorpiones. strategies of termitophiles integration into the social life of termites can include appeasement by chemical (wilson, 1971), morphological (kistner, 1969) or behavioral mimicry (kistner, 1979). hydrocarbons are used among social insects in species recognition, colony or castes (howard & blomquist, 2005) and are detected by antennal contact. the hydrocarbons of some social insect nest inquilines are very similar to that of their hosts (howard et al., 1982). some staphylinidae (coleoptera) species maintain mutualistic interactions with their hosts through chemical mimicry (rosa, 2012) and exocrine glands whose exudate can be licked by the workers, which, in turn, regurgitate stomach content for beetles (pasteels & kistner, 1971). some staphylinidae beetles have the ability of overlapping their abdomen on the thorax to reduce body length and develop physogastry (kistner, 1979; 1982). physogastry is the development of the reproductive and/or glandular system of termite hosts, in which the glands may produce chemical messages of termitophile acceptance (krikken, 2008). most termitophile physogastry beetles research article termites universidade estadual de goiás, unucet, anápolis, go, brazil hf cunha et al morphometric comparison between constrictotermes cyphergaster and corotoca melantho66 belong to the family staphylinidae (kistner, 1979; 1982). in this family, some species have cuticular hydrocarbons similar to those of their hosts (rosa, 2012). sometimes, physogastry is followed by a subsequent secondary sclerotization of some or all of the expanded membrane, as is the case for corotoca melantho, spirachtha eurymedusa and termitoiceus sp. nov. (seevers, 1957; kistner, 1982; jacobson & pasteels, 1985; kistner, 1990; cristaldo et al., 2012). the termitophiles have reduced body size and lateral abdominal projections, and seem to mimic the morphology of worker termites. kistner (1968, 1969) explained that the mimicry between termitophiles and termites is based on palpation and not on sight. krikken (2008) described several pronotum trichomes which presumably function in the communication among termitophiles and the termite hosts. in addition, the mentum morphology of both insect groups suggests a trophallaxis relationship. corotoca melantho is a species with ovoviviparous development (liebherr & kavanaugh, 1985), which may facilitate its survival within the termite nests, once females deposit larvae ready for pupation. the c. melantho have a short life cycle, and are thus quickly in touch with worker termites, from which they acquire food by trophallaxis (costa & vanin, 2010). staphylinidae beetles with appendages on the curved and physogastric abdomen are common among species that coexist with ants and termites (kistner, 1979). the termitophiles may request regurgitated food from the workers (costa & vanin, 2010), but may also feed on fecal matter and remains of dead animals (costa-lima, 1952). considering the interaction between the termite host and termitophile beetle, we investigate the hypothesis of a body shape similarity between the termitophile beetle corotoca melantho schiødte (coleoptera: staphylinidae) and workers of the termite constrictotermes cyphergaster (silvestri) (isoptera: termitidae). material and methods examined material twenty-four c. cyphergaster nests cohabited by c. melantho were collected in an area of cerrado sensu stricto, a savannah biome, at the parque estadual da serra de caldas (pescan). pescan is located between caldas novas and rio quente (17°46’56’’ s and 48°42’35’’ w) in the state of goiás, in the central region of brazil. the termite nests were removed from the trees, fragmented into small pieces and a sample of 10% was made to estimate the number of inhabiting individuals. the beetles were identified by comparison with the biological collection of the zoology museum of the university of são paulo (mzusp). the samples were fixed in 80% ethanol and stored in the laboratory at unucet/ ueg. morphometric data a total of 31 c. cyphergaster workers and 31 cohabiting c. melantho individuals were collected from 24 nests. pictures of all specimens were taken in top and side views, with a 3mp digital camera integrated into a stereomicroscope with 10x fixed eyepieces and 30x magnification. the pictures were saved as jpeg images with a 2048x1536 resolution and transformed into tps (thin-plate spline) files using tpsutil version 1.44 (rohlf, 2009a). four homologous landmarks represented as cartesian coordinates (x, y) were marked on the head of termites and beetles in top and side views (fig 1), and five semilandmarks (type 3 bookstein coordinates) were marked on the abdomen of termites and beetles in top and side views (fig 1). these semilandmarks are not truly homologous because in c. melantho the abdomen overrides the thorax. the landmarks and semilandmarks were digitized with the software tpsdig version 2.12 (rohlf, 2008b). the shape variables were obtained overlapping landmarks and semilandmarks through the general procrustes alignment using the software tpsrelw version 1.46 (rohlf, 2008c). this method calculates an average configuration (consensus) that minimizes the sum of squares of the distances between the points of each configuration and the reference configuration (landmarks). the deformation analysis obtained by tpsrelw generates a new set of variables – partial warps – calculated as the orthogonal coordinates of each eigenvector. the relative warp method is based on the principal component analysis of partial warp scores, run with α = 0 to give the same weight to partial warps at different scales, and results in uniform components. the thin-plate spline graphs were obtained using the software tpssplin version 1.20 (rohlf, 2004d). tps softwares (thin-plate splines) were obtained free of cost at the stony brook university morphometric website (http:// life.bio.sunysb.edu/morph). data analysis we used centroid size, which is the square root of the summed square distances between each anatomical landmark/ semilandmark and the shape’s centroid to compare body shapes. the average sizes of the centroid of the species (c. melantho and fig 1. distribution of landmarks and semilandmarks in side and top view for corotoca melantho and constrictotermes cyphegaster workers. four landmarks were marked on the basis of the antennas and the occipital area of the head in top view (a and b). four landmarks were marked on the basis of the antennas, postoccipital area, the mandible tip and vertex of the head in side view (c and d). five semilandmarks were marked representing the first and penultimate tergites and the apical tip of last tergite of the abdomen in top view (a and b). five semilandmarks were marked representing the first, middle and ultimate tergites and the middle sternite of the abdomen in side view (c and d). sociobiology 62(1): 65-69 (march, 2015) 67 the termitophile beetles are slightly smaller than worker termites. still, the difference in body size did not affect body shape for either species. body size of c. cyphergaster has no effect on body shape in top view (r2= 0.3378; f(gl=14, 16)= 0.5830; p= 0.8418) or in side view (r2= 0.2102; f(gl=14, 16)= 0.3041; p= 0.9847). body size of c. melantho has no effect on body shape in top view (r2= 0.5469; f(gl=14, 16)= 1.3790; p= 0.2664) or in side view (r2= 0.6299; f(gl=14, 16)= 1.9450; p= 0.1014). the body shape of both species is similar in side view (t2= 1.1516; df= 14, 47; p= 0.3317) and top view (t2= 1.2941; df= 14, 47; p= 0.2712). a total of 70.97% of the specimens were correctly classified in side view and 69.35% of specimens were correctly classified in top view, according to the discriminant analysis (fig 3). however, after applying the jackknife method, the accuracy in the classification of individuals into each group dropped to 50% and 56.45%, in top and side views, respectively. c. cyphergaster) were compared through a t-test (p<0.05). a regression analysis of the shape coordinates (dependent variable) and the centroid size (independent variable) (p<0.05) was carried out to evaluated if there is any effect of the size on the shape. to distinguish shape variables (in top and side views) from termitophile beetles and worker termite hosts a simple discriminant analysis was used, once a single function is needed to distinguish the two clusters. the parity of the means of the two groups was tested using hotelling’s t2 (p<0.05) (doornik & hansen, 2008) with 10000 permutations, to meet the multinormality assumption. the percentage of correctly classified individuals was estimated using the jackknife method to avoid bias in the initially classified group. the discriminant analysis carried out uses a linear model to visually confirm or reject the hypothesis that the two species are morphologically distinct (legendre & legendre, 2004). all statistical analyses were carried out using r (r development core team 2014). results there were in average 0.002 c. melantho individuals for each c. cyphergaster worker. a total of two, four or six specimens of termitophile beetles were found for 67% of the c. melantho recorded in the sampled nests. however, 18 termitophiles were found in one of the largest termite nests. the body shape, initially represented by the centroid size of c. cyphergaster workers and the termitophiles is similar in side view (tdf=60= 2.0707; p= 0.0431), but differed in top view (tdf=60= -1.1847; p= 0.2412). the thin plate splines show deformations in the variations of head and body shape (fig 2). fig 2. thin plate spline representation of principal trends (relative warps) in the variation of the body shape. grid show the deformation of the average shape in opposite directions of the first relative warps for corotoca melantho and constrictotermes cyphergaster in top view (a and b) and in lateral view (c and d). fig 3. frequency of discriminant scores for corotoca melantho (red) and constrictotermes cyphergaster (blue) in side view (a) and in top view (b). hf cunha et al morphometric comparison between constrictotermes cyphergaster and corotoca melantho68 acknowledgments our study has been continuously supported by different grants of the conselho nacional de desenvolvimento científico (cnpp n° 475484/2011-8), fundação de amparo à pesquisa do estado de goiás (fapeg) and coordenação de aperfeiçoamento de pessoal de nível superior (capes) (auxpe 2036/2013). jsl thanks cnpq by productivity grant (306719/2013-4). hfc thanks the university research and scientific production support program (probip/ueg). references carrijo, t.f., gonçalves, r.b. & santos, r.g. (2012). review of bees as guests in termite nests, with a new record of the communal bee, gaesochira obscura (smith, 1879) (hymenoptera, apidae), in nests of anoplotermes banksi emerson, 1925 (isoptera, termitidae, apicotermitinae). insectes sociaux, 59: 141-149, doi: 10.1007/s00040-012-0218-x costa. c. & vanin, s.a. (2010). coleoptera larval fauna associated with termite nests (isoptera) with emphasis on the bioluminescent termite nests from central brazil. psyche, 2010, article id 723947, 12 pages, doi:10.1155/2010/723947 costa, d.a., carvalho, r.a., lima-filho, g.f. & brandão d. (2009). inquilines and invertebrate fauna associated with termite nests of cornitermes cumulans (isoptera, termitidae) in the emas national park, mineiros, goiás, brazil. sociobiology, 53 (2b): 443-453. costa-lima, a. (1952). insetos do brasil, coleoptera, rio de janeiro: escola nacional de agronomia. (pp. 313-323). cristaldo, p.f., rosa, c.s., florencio, d.f., marins, a. & desouza, o. (2012). termitarium volume as a determinant of invasion by obligatory termitophiles and inquilines in the nests of constrictotermes cyphergaster (termitidae, nasutitermitinae). insectes sociaux, 59: 541-548. doi 10.1007/s00040-012-0249-3 cunha, h.f. & brandão, d. (2001). invertebrates associated with the neotropical termite constrictotermes cyphergaster (isoptera: termitidae, nasutitermitinae). sociobiology, 37: 593-599. cunha, h.f. & morais, p.p.a.m. (2010). relação espécie-área em cupinzeiros de pastagem, goiânia-go, brasil. entomobrasilis, 3: 60-63. doornik, j. a. & hansen, h. (2008). an omnibus test for univariate and multivariate normality. oxford bulletin of economics and statistics, 70: 927-939. doi: 10.1111/j.14680084.2008.00537.x howard, r.w., mcdaniel, d.c.a. & blomquist, l.j. (1982). chemical mimicry as an integrating mechanism for three termitophiles associated with reticulitermes virginicus (banks). psyche, 89: 157-67. discussion we found that the c. melantho population is higher in larger c. cyphergaster termite nests, as already described by cristaldo et al. 2012. rosa (2008) reported staphylinidae beetles as termitophiles in nests of 104 termite species. however, studies regarding termitophile staphylinidae in brazil are restricted to the borgmeier (1923, 1935, 1954, 1959 apud rosa, 2008), costa-lima (1952), cunha & brandão (2000), costa et al., (2009) and cristaldo et al. (2012). in the field, beetles were observed walking inside the nest, near termites, with no antagonistic reaction from its worker termite hosts (personal communication). the diversity of staphylinids observed to be cohabiting termite nests suggests the evolution of a specific interaction between some termite pairs and staphylinid species, which include adaptations such as morphological mimicry (our results) and chemical mimicry (rosa, 2012). no difference in c. cyphergaster workers and c. melantho body shape was observed in the morphometric analysis (see fig 2 and 3). the body shape can be represented by centroid size and shape variables (the partial warps and uniform components). the new shape variables, obtained by overlapping the nine landmarks and semilandmarks by procrustes alignment, showed that the termitophiles and workers of termite hosts are morphometrically similar. from 20% to 30% of the body shape variation is due to variation on termite host body size. however, body size for termitophiles affects from 50% to 60% of the body shape (see r2 of the regression between body size and body shape). the discriminant analysis confirmed that the specimens of termitophile beetles and termite host worker are indistinguishable (see fig 3). figure 3 shows higher variation among termitophiles than among the termite hosts. despite the visual difference on head shape between workers of termite hosts and termitophiles, the average shape is similar in both species (as corroborated by discriminant function). to our knowledge, morphometric geometric analysis has never been used to compare morphological similarity between termites and termitophiles. traditional morphometric estimates the variation and covariation of distance measures between pairs of points – length and width of morphological structures (monteiro & reis, 1999; zelditch et al., 2012). according to these authors, geometric morphometry enables analyzing the overall shape of the individual regardless of its size, and locates and describes the regions of shape changes, represented by anatomical points in homologous structures. although the results indicate that c. melantho can mimic the morphology of workers of c. cyphergaster, we cannot say that both species co-evolved, because such a statement would require a phylogeny analysis of both species in search of reciprocal adaptations. the interaction between c. melantho and c. cyphergaster should be investigated by additional analyses, such as behavioral experiments inside the nests and phylogenetic comparisons. sociobiology 62(1): 65-69 (march, 2015) 69 howard, r.w. & blomquist, g. (2005). ecological, behavioral, and biochemical aspects of insect hydrocarbons. annual review of entomology 50: 371-393. doi: 10.1146/annurev. ento.50.071803.130359 jacobson, h.r. & pasteels, j.m. (1985). a new termitophilous species of termitoptocinus silvestri from new guinea and a redescription of the genus (coleoptera, staphylinidae, aleocharinae, corotocini). indo-malayan zoology, 2: 319-323. kistner, d.h. (1968). revision of the african species of the termitophilous tribe corotocini (coleoptera: staphylinidae). i. a new genus and species from ovamboland and its zoogeographic significance. journal of the new york entomological society, 76: 213-221. kistner, d.h. (1969). the biology of termitophiles. in k. krishna & f.m. weesner (eds.), biology of termites. vol. i. (pp. 525-557). new york, academic press. kistner, d.h. (1970). australian termitophilous associated with microcerotermes (isoptera: amitermitinae). pacific insects, 12 : 9-15. kistner, d.h. (1979). social and evolutionary significance of social insect symbionts. in h.r. hermann (eds.), social insects, vol. 1. (pp. 339-413). academic press, new york, san francisco, and london. kistner, d.h. (1982). the social insects’ bestiary. in h.r. herman (ed.) social insects, vol. 3. (pp. 1-244). academic, new york. kistner, d.h. (1990). the integration of foreign insects into termite societies or why do termites tolerate foreign insects in their societies. sociobiology, 17: 191-215. krikken, j. (2008). two new species from kenya in the physogastric termitophilous genus termitoderus mateu 1966 (coleoptera scarabaeidae aphodiinae). tropical zoology, 21: 153-162. liebherr, j.k. & kavanaugh, d.h. (1985). ovoviviparity in carabid beetles of the genus pseudomorpha (insecta: coleoptera). journal of natural history, 19: 1079-1086. doi: 10.1080/00222938500770681. legendre, p. & legendre. l. (2004). numerical ecology. 3rd ed. amsterdan: elsevier science. lopes, s.m. & oliveira, e.h. (2005). espécie nova de ischnoptera burmeister, 1838 (blattaria: blattellidae: blattellinae) do estado de goiás, brasil, coletada em ninho de cupim. biota neotropica, 5: 71-74. doi.org/10.1590/s1676-06032005000100008 monteiro, l.r. & reis, s.f. (1999). princípios de morfometria geométrica. são paulo: holos 188p. noirot, c. & darlington, j.p.e.c. (2000). termite nests: architecture, regulation and defense, in t. abe, d.e. bignell & m. higashi (eds.), termites: evolution, sociality, symbioses, ecology. (pp. 121-139). netherlands: kluwer academic publishers. pasteels, j.m. & kistner, d.h. (1971). revision of the termitophilous subfamily trichopseniinae (coleoptera: staphylinidae). ii. the remainder of the genera with a representational study of the gland systems and a discussion of their relationships. miscellaneous publications of the entomological society of america, 7: 351399. r development core team (2014). r: a language and environment for statistical computing. available at: http:// www.r-project.org/. accessed date: 18.vi.2014. rohlf, f.j. (2009a). tpsutil version 1.44. stony brook, departament of ecology and evolution, state university of new york. available online at: http://life.bio.sunysb.edu/ morph accessed 30.v.2009. rohlf, f.j. (2008b). tpsdig version 2.12. stony brook, departament of ecology and evolution, state university of new york. available online at: http://life.bio.sunysb.edu/ morph accessed 30.v.2009. rohlf, f.j. (2008c). tpsrelw version 1.46. stony brook, departament of ecology and evolution, state university of new york. available online at: http://life.bio.sunysb.edu/morph accessed 30.v.2009. rohlf, f.j. (2004d). tpssplin version 1.20. stony brook, departament of ecology and evolution, state university of new york. available online at: http://life.bio.sunysb.edu/morph accessed 30.v.2009. rosa, c.s. (2008). interactions between termites (insecta: isoptera) and termitophiles. msc thesis (animal biology), universidade federal de viçosa, brazil. rosa, c.s. (2012). interspecific interactions in termite mounds. phd dissertation (entomology), universidade federal de viçosa, brazil. seevers, c.h. (1957). a monograph on the termitophilous staphylinidae (coleoptera). fieldiana: zoology, 40: 1-334. wilson, e.o. (1971). the insect societies. the belknap press of harvard university press, cambridge, mass. 548 p. zelditch, m.l., swiderski, d.l., & sheets, h.d. (2012). geometrics morphometrics for biologists: a primer. academic press of elsevier, 2nd. edition. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1):35-40 (2013) fumigant activity of eight plant essential oils against workers of red imported fire ant, solenopsis invicta l tang, yy sun, qp zhang, y zhou, n zhang, zx zhang introduction the red imported fire ant, solenopsis invcita buren, which causes severe damage to humans, animals, and the environment, is an important medical and agricultural pest in southern united states, as well as australia, the philippines, taiwan, and mainland china (zhang et al., 2007). the ant stings humans, pets, farm animals, and wildlife, as well as damaging farm, electrical equipments and irrigation systems. moreover, besides destroying crops and fruits directly or indirectly, they negatively affect the local biodiversity and cause nearly $5 billion losses in urban and agricultural areas yearly in the usa (cheng et al., 2008). common methods for managing the red imported fire ant are through insecticides or baits that are also a threat to human health and the environment because of their high toxicity of chemicals (vogt et al., 2002). reducing the use of abstract plant essential oils from eight plant species were tested for their insecticidal activities against the red imported fire ant, solenopsis invcita, by using a fumigation bioassay. this study reveals that the mortalities after treatment of the workers of red imported fire ants varied according to the classification of workers, oil type, dosage, and exposure time. among the essential oils tested, strong insecticidal activity was observed with the essential oils of camphor (cinnamomum camphora (l.) j. presl), artemisia annua (artemisia annua l.), eucalyptus (eucalyptus globulus labill.), mugwort (artemisia argyi h. lév. & vaniot), and wintergreen (ilex chinensis sims). ant mortalities from chrysanthemum oil (dendranthema indicum (l.) des moul.), turpentine oil (pinus massoniana lamb.), and forsythia oil (forsythia suspense (thunb.) vahl) treatments were significantly lower than those from the previously mentioned five essential oil treatments. this study showed that camphor, artemisia annua, eucalyptus, mugwort, and wintergreen oils may have potential to be used as substitutes for chemical insecticides. sociobiology an international journal on social insects south china agricultural university, wushan, guangzhou, people’s republic of china. research article ants article history edited by: gilberto m. m. santos, uefs, brazil received 06 december 2012 initial acceptance 16 february 2013 final acceptance 05 march 2013 keywords invasive ant, solenopsis invicta, control, plant essential oil, fumigation toxicity corresponding author zhi-xiang zhang key laboratory of natural pesticide and chemical biology, ministry of education, south china agricultural university, wushan, guangzhou 510642 people’s republic of china e-mail: zdsys@scau.educn synthetic contact insecticides has gained increasing interest in the research and development of alternative control tactics, such as fire ant fumigants (zhou et al., 2012). fumigation has a major function in insect pest elimination in stored products and in quarantine containers. studies on the toxicity of botanical essential oils on red imported fire ant have shown that various essential oils are repellent and/or toxic to the ant. citrus peels showed contact toxicity against red imported fire ants (sheppard, 1984). aromatic cedar mulch also showed repellent activity against red imported fire ants (thorvilson & rudd, 2001). vogt et al. (2002) tested mound drench formulations containing raw citrus peel extract (orange oil) on fire ants and found that cold-press citrus peel extract (‘orange oil’) can be effectively used as an organic alternative to conventional insecticides for drenching colonies. mint oil granules were proven to be toxic and repell tang, yy sun, qp zhang, y zhou, n zhang, zx zhang fumigant activity of essential oils against workers of fire ant36 lent to red imported fire ants, and all red imported fire ant mounds that were treated with mint oil granules were abandoned (appel et al., 2004). citronella oil was repellent and toxic to both argentine and red imported fire ants (wiltz et al., 2007). the essential oil from cinnamomum osmophloeum kaneh. leaves was reported to be toxic to red imported fire ants in open and closed exposure trials (cheng et al., 2008). callicarpenal and intermedeol were isolated from the leaves of american beautyberry (callicarpa americana l.) and japanese beautyberry (callicarpa japonica thunb.), and had repellent effects on red imported fire ants (chen et al., 2008). the over-the-counter essential oil products from china fyj were investigated for repellent effects against workers of red imported fire ants, and each of its six major components also showed repellent effects at various concentrations (chen, 2009). without prior exposure to air, vetiver oil showed a significant repellent effect to workers. however, the repellent effect of vetiver oil to previously exposed workers reduced at low concentrations (li et al., 2009). the repellent effects of five botanical essential oils, namely, cymbopogon nardus (l.) rendle, cinnamomum cassia (l.) c. presl, ilex pedunculosa miq., salvia sclarea l., and capsicum annuum l. on fire ant workers were evaluated using a y-tube olfactometer bioassay (wang et al., 2012). however, few researches existed on fumigant investigations related to plant essential oils against the red imported fire ant. zhou et al. (2012) tested the fumigant activity of methyl bromide and found that all red imported fire ants treated with methyl bromide above 22.52 g/m2 for 8 h died. plant essential oils of garlic (allium sativum l.), glove bud (eugenia caryophyllata thunb.), ajowan (trachyspermum ammi (l.) sprague), allspice (pimenta dioica (l.) merrill), caraway (carum carvi l.), dill (anethum graveolens l.), geranium (pelargonium graveolens l’ hér.), litsea (litsea cubeba (lour.) pers.) showed strong insecticidal activity after testing 59 oils on the japanese termite, reticulitermes speratus kolbe, by using a fumigation bioassay (park & shin, 2005; seo et al., 2009). formic acid has been tested in the laboratory for contact and fumigation toxicity against workers, alates, and queens of red imported fire ants (chen, 2012). because of being highly volatile, constituting a rich source of bioactive chemicals (chang et al., 2001 ), being safe as they are commonly used as fragrances and flavoring agents for foods and beverages (isman, 2000), plant essential oils were used as potential alternatives for chemical insecticides. we evaluated the fumigant activity of eight plant essential oils on red imported fire ant workers with a fumigation bioassay to determine the essential oils with efficient fumigant activity to red fire ant. materials and methods plant essential oils plant essential oils of camphor (cinnamomum camphora), wintergreen (ilex chinensis), eucalyptus (eucalyptus globulus), artemisia annua (artemisia annua), mugwort (artemisia argyi), chrysanthemum (dendranthema indicum), forsythia (forsythia suspense), and turpentine (pinus massoniana) were purchased from kangshen natural medicinal oil refinery (jiangxi, china), on january 12, 2012. insects s. invicta colonies were collected from the suburb of guangzhou and maintained in the laboratory for bioassays. the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor l.). a test tube (25 mm×200 mm), which was partially filled with water and plugged with cotton, was used as a water source. the ants were maintained in the laboratory at 25±2 c. fumigation toxicity bioassay the method of seo et al. (2009) was used with slight modifications to determine the effectiveness of controlling the red imported fire ant. a 1.5 ml centrifuge tube treated with the essential oil being tested was placed at the bottom lid of a glass cylinder (5 cm diameter×10 cm) with eight pin holes on the tube to allow the oil to vaporize into the bottle. the lid was then sealed. the vertical wall inside each glass cylinder was coated with a fluon emulsion and allowed to dry for 24 h to prevent the ants from escaping. we classified the fire ant workers as major workers (4.3 mm to 4.5 mm body-length, 1.0 mm to 1.1 mm head-width) and minor workers (2.8 mm to 3.0 mm body-length, 0.6 mm to 0.7 mm head-width). fifteen fire ant workers were placed at the bottom lid of the glass cylinder. the insects were maintained at 25±1°c and at a relative humidity of 80%. experiment 1 (cumulative mortalities were determined 12 and 24 h after testing at the concentration of 0.5, 1, 2, 3, and 5 mg/ centrifuge tube). experiment 2 (the cumulative mortalities were then determined 5, 8, 11, 13, and 24 h after testing with camphor, wintergreen, eucalyptus, artemisia annua, and mugwort oils at a concentration of 2 mg/centrifuge tube). all treatments were replicated three times. we used the following equations: statistical analysis for effect of oil type, dosage and exposure time on mortalities of the minor and major workers, data were transsociobiology 60(1): 35-40 (2013) 37 formed to arcsine square root values for three-way analysis of variance (anova) at p = 0.05 for the significance of main effects and various interactions. means were compared and separated by using the duncan-test. results when eight plant essential oils were bioassayed, the mortalities of minor and major workers varied significantly according to oil type, dosage, and exposure time (anova, p < 0.05). in addition, all of the interactions among the three factors were found to be significant (tables 1, 3). the results on mortality were shown in table 2 and table 4. significant differences were found among workers: developing from different oil types, exposed to different time and different dosage treatments (df = 28, f =7.623, f = 9.074, p = 0.0001). a 100% mortality was achieved in five out of eight essential oils, namely, camphor, artemisia annua, eucalyptus, mugwort, and turpentine oils, 12 h after treatment at 5 mg/ centrifuge tube to minor workers. wintergreen, chrysanthemum, and forsythia oil showed 100% fumigant activity 24 h after treatment. moreover, camphor, artemisia annua, eucalyptus, mugwort, and turpentine oils achieved 100% mortality 12 h after treatment at 5 mg/centrifuge tube to major workers. at a concentration of 5 mg/centrifuge tube, wintergreen, chrysanthemum, and forsythia oils showed 100% fumigant activity 24 h after treatment on major workers. plant essential oils with >80% mortality at 5 mg/filter paper were observed at lower concentrations. camphor, artemisia annua, and eucalyptus oils caused 100% mortality of minor workers at a concentration of 2 mg/centrifuge tube. in addition, wintergreen, mugwort, turpentine, and forsythia oils caused >80% mortality on minor workers at a concentration of 2 mg/centrifuge tube. for major workers, only camphor and eucalyptus oils caused >80% mortality at a concentration of 2 mg/centrifuge tube. after 24 h exposure to essential oils of camphor, artemisia annua, eucalyptus, and wintergreen at 1 mg/centrifuge tube, the insect mortalities were 100%, 100%, 80.00 ±6.67%, and 86.67 ±3.85% for minor workers and 60.00 ±6.67%, 60.00 ±6.67%, 53.33 ±6.67%, and 53.33 main effects interactions o d t o×d o×t d×t o×d×t df 7 4 1 28 7 4 28 f 224.339 734.465 628.156 14.829 42.245 13.402 7.623 p 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0: oil type, d: dosage, t: exposure time (p=0.05; df=239). table 1. analysis of variance (three-way anova) for main effects (oil type, dosage and exposure time) on mortality of minor workers, and interactions. table 2. mortality of minor workers of red imported fire ants caused by essential oils at different concentrations (mg/centr. tube) in the fumigation bioassay for 12. h and 24 h. * means sharing the same letters are not significantly different from each other (p>0.05, duncan test). essential oil mg/centrifuge tube mortality (%,mean±sem,n=3)* 12h 24h camphor 5 100.00 a 100.00 a 3 100.00 a 100.00 a 2 100.00 a 100.00 a 1 100.00 a 100.00 a 0.5 51.11±5.88 efghi 86.67±7.70 bc artemisia annua 5 100.00 a 100.00 a 3 100.00 a 100.00 a 2 100.00 a 100.00 a 1 80.00±10.18 cd 100.00 a 0.5 42.22±2.22 ghijkl 55.56±2.22 efgh eucalyptus 5 100.00 a 100.00 a 3 100.00 a 100.00 a 2 100.00 a 100.00 a 1 62.22±5.88 ef 80.00±6.67 cd 0.5 35.56±2.22 ijklm 48.89±2.22 efghijk wintergreen 5 66.67±7.70 de 100.00 a 3 53.33±6.67 efghi 100.00 a 2 33.33±3.85 jklm 100.00 a 1 15.56±4.44 nop 86.67±3.85 c 0.5 0.00 r 26.67±6.67 lmn mugwort 5 100 a 100.00 a 3 84.44±2.22 c 100.00 a 2 66.67±3.85d e 86.67±10.18 bc 1 31.11±2.22 klm 53.33±3.85 efghi 0.5 13.33±3.85 nopq 24.44±2.22 mno chrysanthemum 5 53.33±6.67 efghi 100.00 a 3 33.33±6.67 klm 93.33±6.67 ab 2 26.67±3.85 lmn 66.67±3.85 de 1 6.67±3.85 q 26.67±7.70 lmn 0.5 0.00 r 13.33±3.85 nopq turpentine 5 100 a 100.00 a 3 53.33±7.70 efghi 100.00 a 2 40.00±3.85 hijklm 80.00±7.70 cd 1 6.67±0.00 pq 40.00±3.85 hijklm 0.5 0.00 r 6.67±3.85 q forsythia 5 66.67±6.67 de 100.00 a 3 46.67±7.70 fghijk 100.00 a 2 33.33±3.85 jklm 86.67±3.85 c 1 13.33±6.67 opq 60.00±7.70 efg 0.5 0.00 r 0.00 r ck 0 0.00 o 0.0 o l tang, yy sun, qp zhang, y zhou, n zhang, zx zhang fumigant activity of essential oils against workers of fire ant38 main effects interactions o d t o×d o×t d×t o×d×t df 7 4 1 28 7 4 28 f 298.284 826.704 289.027 29.917 37.522 6.672 9.074 p 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0: oil type, d: dosage, t: exposure time (p=0.05; df=239). table 3. analysis of variance (three-way anova) for main effects (oi l type, dosage and exposure time) on mortality of major workers, and interactions. ±6.67% for major workers, respectively. however, these values decreased to 86.67 ±7.70%, 55.56 ±2.22%, 48.89 ±2.22%, and 26.67 ±6.67% for minor workers and 13.33 ±3.85%, 33.33 ±3.85%, 40.00 ±6.67%, and 20.00 ±6.67% for major workers, respectively, when the dosage decreased to 0.5 mg/centrifuge tube. the toxicities of camphor, artemisia annua, eucalyptus, wintergreen, and mugwort oils after treatment on minor and major workers at a concentration of 2 mg/centrifuge tube were able to control the red imported fire ants (figs. 1 and 2). fig. 1 showed that camphor, artemisia annua, and eucalyptus oils completely killed the minor workers after 11 h, whereas wintergreen and mugwort oils showed 26.67 ± 3.85% and 46.67 ± 6.67% mortality, respectively. however, when the exposure time increased to 24 h, the wintergreen oil caused 100% mortality to the fire ants. fig. 2 showed that the mortalities of major workers of red imported fire ant caused by camphor, eucalyptus, mugwort, wintergreen, and artemisia annua oils after 11 h were 73.33 ± 10.18%, 73.33 ± 6.67%, 40.00 ± 6.67%, 20 ± 3.85%, and 20 ± 6.67%, respectively, at a concentration of 2 mg/centrifuge tube. by contrast, at an exposure time of 24 h, the mortalities from the above five essential oils were 100%, 80 ± 7.70%, 46.67 ± 7.70%, 53.33 ± 6.67%, and 60 ± 6.67%, respectively. discussion this study showed that the mortalities of the workers of red imported fire ant after treatment varied according to the classification of workers, oil type, dosage, and exposure time. minor workers had a significantly higher mortality than major workers. the essential oils of camphor, artemisia annua, eucalyptus, mugwort, and wintergreen had the strong toxicity against red imported fire ants at a low concentration (p < 0.05). in addition, camphor, artemisia annua, and eucalyptus oils had a higher activity than wintergreen and mugwort oils (p < 0.05). however, the active ingredient of the oils necessitates further studies. essential oils, widely used as fragrances and flavors in the perfume and food industries, have long been reputed to repel, contact, fumigate insects, and control some important plant pathogens (koul et al., 2008). but the fumigant table 4. mortality of major workers of red imported fire ants caused by essential oils at different concentrations (mg/centr. tube) in the fumigation bioassay for 12 h and 24 h. essential oil mg/centrifuge tube mortality (%,mean±sem,n=3)* 12h 24h camphor 5 100.00 a 100.00 a 3 100.00 a 100.00 a 2 73.33±7.70 cd 100.00 a 1 53.33±7.70 efg 60.00±6.67 def 0.5 6.67±3.85 n 13.33±3.85 lmn artemisia annua 5 100.00 a 100.00 a 3 93.33±7.70 b 100.00 a 2 53.33±6.67 efg 73.33±6.67 cd 1 40.00±3.85 ghi 60.00±6.67 def 0.5 13.33±3.85 lmn 33.33±3.85 hijk eucalyptus 5 100.00 a 100.00 a 3 100.00 a 100.00 a 2 73.33±6.67 cd 84.44±4.44 c 1 40.00±6.67 ghi 53.33±6.67 efg 0.5 13.33±3.85 lmn 40.00±6.67 ghi wintergreen 5 26.67±7.70 ijkl 100.00 a 3 20.00±3.85 jklm 100.00 a 2 13.33±3.85 lmn 66.67±6.67 de 1 6.67±3.85 n 53.33±6.67 efg 0.5 0.00 o 20.00±6.67 jklm mugwort 5 100.00 a 100.00 a 3 66.67±10.18 de 100.00 a 2 40.00±6.67 ghi 46.67±7.70 fgh 1 26.67±7.70 ijkl 35.56±2.22 ghij 0.5 6.67±3085 n 13.33±3.85 lmn chrysanthemum 5 53.33±6.67efg 100.00 a 3 33.33±3.85 hijk 40.00±6.67 ghi 2 13.33±3.85 lmn 20.00±6.67 klm 1 6.67±3.85 n 11.11±2.22 mn 0.5 0.00 o 0.00 o turpentine 5 100.00 a 100.00 a 3 0.00 o 6.67±3.85 n 2 0.00 o 0.00 o 1 0.00 o 0.00 o 0.5 0.00 o 0.00 o forsythia 5 26.67±3.85 ijkl 100.00 a 3 6.67±3.85 n 13.33±3.85 lmn 2 0.00 o 0.00 o 1 0.00 o 0.00 o 0.5 0.00 o 0.00 o ck 0 0.00 o 0.0 o * means sharing the same letters are not significantly different from each other (p > 0.05, duncan test). sociobiology 60(1): 35-40 (2013) 39 figure 2. mortality of major workers of red imported fire ants treated with essential oils at 2 mg/centrifuge tube . activities of essential oils against red imported fire ant were rarely reported. zhou et al., (2011) tested the fumigant activity of methyl bromide and found that all red imported fire ant that were treated with methyl bromide above 22.52 g/m2 for 8 h died. plant essential oils from 29 plant species and components from garlic and glove bud oils, and plant essential oils from 30 plant species and components from ajowan, allspice, caraway, dill, geranium, and litsea oils were tested for their insecticidal activities against the japanese termite, r. speratus kolbe, by using a fumigation bioassay (park & shin 2005; seo et al., 2009). formic acid has been tested in the laboratory for contact and fumigation toxicity against workers, alates, and queens of red s. invicta (chen, 2012). the essential oils of camphor, artemisia annua, eucalyptus, mugwort, turpentine wintergreen, chrysanthemum, and forsythia showed effective toxicity against both minor and major workers of red imported fire ants. more specififigure 1. mortality of minor workers of red imported fire ants treated with essential oils at 2 mg/centrifuge tube. cally, camphor, artemisia annua, eucalyptus, mugwort, and wintergreen oils showed the strongest toxicity against red imported fire ants (p < 0.05). as a conclusion, this study showed that camphor, artemisia annua, eucalyptus, mugwort, and wintergreen oils may have potential as natural fumigants, and can be used as substitutes for chemical insecticides. moreover, the development of formulations with improved efficacy, stability and reduced costs necessitates further studies. acknowledgments this study was supported by the science and technology planning project of guangdong province, china (no.2012b020309004). references appel, a. g., m. j. gehret. & m. j. tanley. 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(2011). research on the suffocating efficacy of methyl bromide fumigation on the red imported fire ant solenopsis invicta buren. j. environ. entomol., 33: 70-73. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3772sociobiology 66(2): 227-238 (june, 2019) effects of fipronil on non-target ants and other invertebrates in a program for eradication of the argentine ant, linepithema humile introduction biological invasion is a global environmental problem that harms biodiversity and ecosystem function (clavero & garcia-berthou, 2005; mack et al., 2000). ants are some of the most successful invasive taxa in the world, with invasive ant species having become established on almost every continent (suarez et al., 2010). the success of these species is related to a suite of characteristics that favor interactions with humans (hoffmann et al., 2016; holway et al., 2002; mcglynn, 1999). these tramp species cause serious harm to the environment, agricultural productivity, human health, and the economy (holway et al., 2002; williams, 1994), as abstract pesticides are frequently used to eradicate invasive ant species, but pose ecological harm. previous studies assessed non-target effects only in terms of the increase or decrease of abundance or species richness after pesticide applications. positive effects of the release from pressure caused by invasive ant species have not been considered so far. to more accurately assess pesticide effects in the field, the nontarget effects of pesticides should be considered separately from the positive effects of such releases. here, we used monitoring data of ants and other invertebrates collected in a program for the eradication of the argentine ant, linepithema humile (mayr), using fipronil. first, we separately assessed the effects of l. humile abundance and fipronil exposure on non-target ants and other invertebrates using generalized linear models. the abundance of l. humile and the number of pesticide treatments were negatively associated with the total number of non-target individuals and taxonomic richness. we also noted negative relationships between the number of individuals of some ant species and other invertebrate taxonomic groups. the l. humile × pesticide interaction was significant, suggesting that the abundance of l. humile affected the level of impact of pesticide treatment on non-target fauna. second, we evaluated the dynamics of non-target ant communities for 3 years using principal response curve analyses. non-target ant communities treated with fipronil continuously for 3 years recovered little, whereas those treated for 1 year recovered to the level of the untreated and non-invaded environment. sociobiology an international journal on social insects y sakamoto1, ti hayashi1, mn inoue2, h ohnishi1, t kishimoto3, k goka1 article history edited by gilberto m. m. santos, uefs, brazil received 08 october 2018 initial acceptance 15 may 2019 final acceptance 15 may 2019 publication date 20 august 2019 keywords invasive alien species; pesticide impact; total number of individuals; species richness; community structure. corresponding author yoshiko sakamoto national institute for environmental studies 16-2 onogawa, tsukuba ibaraki 305-0053, japan. e-mail: sakamoto.yoshiko@nies.go.jp reflected by the fact that five ant species are listed among the world’s 100 worst invasive alien species (iucn issg, 2013). invasive ants are typically controlled with pesticides, such as in bait carriers (rabitsch, 2011; williams, 1994). pesticides have been used successfully in dozens of eradication programs targeting ant species, such as the little fire ant, wasmannia auropunctata, on santa fe island in the galápagos (abedrabbo, 1994; causton et al., 2005), the african big-headed ant, pheidole megacephala, and the tropical fire ant, solenopsis geminata, within kakadu national park, australia (hoffmann & o’connor, 2004), and the argentine ant, linepithema humile, on landfill islands in japan (sakamoto et al., 2017). however, the use of pesticides also harms non-target species (pisa et 1 national institute for environmental studies, tsukuba, ibaraki 305-0053, japan 2 tokyo university of agriculture and technology, fuchu, tokyo 183-8509 japan 3 museum of natural and environmental history, shizuoka, suruga-ku, shizuoka 422-8017, japan research article ants y sakamoto et al. – non-target effects of pesticide in invasive ant eradication program228 al., 2015; prasifka et al., 2005), as toxic baits can attract nontarget ants and other arthropods. fipronil, hydramethylnon, pyriproxyfen, and methoprene, commonly used in invasive ant eradication programs, pose risks (hoffmann et al., 2016). the impacts of these pesticides on non-target species in the field have been assessed only in terms of increases or decreases in the abundance or taxonomic richness of nontarget species after invasive ant control. plentovich et al. (2010) reported that hydramethylnon can be used to control s. geminata and tetramorium bicarinatum but also noted its negative effects on non-target ants, cockroaches, and crickets. by contrast, hoffmann (2010) documented the eradication of a small population of p. megacephala using hydramethylnon and the recovery of native ant abundance and species richness within the treated area. inoue et al. (2015) reported the shortterm recovery of non-target communities after application of fipronil to control l. humile and observed no non-target effects. however, the positive effects of release from pressure caused by invasive ant species have not been considered in previous research. to more accurately evaluate the effects of pesticides in the field, the non-target effects of pesticides must be considered separately from the positive effects of such releases. linepithema humile, a native of south america, is one of the most significant pest ant species worldwide (passera, 1994). in japan, it was first discovered in 1993 (sugiyama, 2000) and has since spread to 12 prefectures (national institute of environmental studies, 2014). since 2011, our group has conducted an eradication program using toxic baits containing fipronil (inoue et al., 2015), and we have successfully eradicated two tokyo populations on landfill islands (sakamoto et al., 2017). the aim of the eradication program is to protect the indigenous invertebrate communities from the invasive alien ant species. thus, non-target effects cannot be ignored when the method is applied to delicate natural areas. in this study, we evaluated the non-target effects of applying fipronil to eradicate l. humile in the two tokyo populations. first, we separately assessed the effects of the pesticide and l. humile on non-target ant and non-ant invertebrates using generalized linear models (glms). next, we used principal response curve (prc) analyses to evaluate the dynamics of the non-target communities after successful eradication and cessation of the pesticide applications. our findings will be useful for minimizing the risks to indigenous fauna as the eradication program moves to other areas. materials and methods study sites the tokai site is on a landfill island 370 m west of the oi container terminal, one of the largest international shipping ports in japan, where 8.5 ha was invaded by l. humile (fig 1a). the jonan site is on the landfill island jonan-jima, 1750 m southwest of the terminal, where 16 ha was invaded (fig 1a). pesticide fipronil is a phenyl pyrazole insecticide and a potent disrupter of the arthropod central nervous system via interference through the chloride channel regulated by γ-aminobutyric acid (rhône poulenc, 1996). fipronil acts slowly, allowing the pesticide in baits to be transferred from insect to insect (including queen and brood among social insects) by trophallaxis or contact (vail et al., 2003), resulting in reproductive inhibition in colonies. fipronil is effective at controlling invasive ant species, especially l. humile (klotz et al., 2007). pesticide treatment the eradication program began in april 2011 (inoue et al., 2015). to evaluate the effects of the program, we established three monitoring plots each at tokai (plots i–iii; fig 1b) and jonan (plots i–iii; fig 1c). we applied paste baits and sprays once a month (fig 2). the paste bait, aruzenchin ari ultra sugoto-taiji (50 mg l–1 fipronil; fumakilla, ltd., hiroshima, japan), was placed every 5 to 10 m along the streets and buildings. the bait was applied in a given month only if l. humile had been found in the same plot at any time during the previous 6 months. if we found brood or queens in vegetation or under pavement during bait application, we sprayed them with a solution of 50 mg l–1 fipronil (aruzenchin ari sugoto-taij ekizai, fumakilla, ltd.). the estimated total rate of active ingredient applied was 137 mg/ha at tokai and 1045 mg/ha at jonan over the 3 years. plot i at jonan was not treated in the first year for comparison with treated areas. fig 1. maps of (a) tokyo bay area and monitoring plots at (b) tokai and (c) jonan. solid line indicates transects in non-invaded plots (n, n); dashed line indicates transects in plots invaded by linepithema humile (i–iii, i–iii). squares indicate locations of sticky traps. sociobiology 66(2): 227-238 (june, 2019) 229 sampling of ant species and non-ant invertebrates to monitor the abundance of ant species and nonant invertebrates, sticky traps (8.8 cm × 19.5 cm × 2.2 cm; monitoring pp trap #j, kankyokiki co., ltd., osaka, japan) were placed every 50 m or so along the perimeter of invaded plots (plots i–iii and i–iii) and non-invaded, untreated plots (plot n in tokai and plot n in jonan; fig 1b, c). the traps were laid once a month from april 2011 to march 2014 in invaded plots and from april (jonan) or may (tokai) 2013 to march 2014 in non-invaded plots and were collected after 3 days. up to 108 trap points were set per month. captured ants and non-ant invertebrates were then identified and counted in the laboratory. ants were identified to species. the other invertebrates were identified to order, except for myriapoda and land snails, and coleoptera were identified to superfamily because of the variety of beetle feeding habits. we define all species except for l. humile as “non-target”. numerical and statistical analyses the effects of l. humile invasion and pesticide application on ants and non-ant invertebrates were statistically examined by two approaches. all analyses were conducted in r v. 3.1.1 software (r development core team, 2013). first, we used glms to examine the relationship of l. humile and pesticide treatment, and their interaction, with the total number of individuals and species or taxonomic richness (number of species or taxonomic groups) of nontargets captured by each trap (mccullagh & nelder, 1989). we created models in which the response variables were the log10(x + 1)-transformed total number of individuals of nontarget ant species or invertebrate taxonomic groups per trap or the integral number of non-target ant species or invertebrate taxonomic groups per trap. these models assumed a gaussian distribution in the response variable and used an identity-link function. the explanatory variables were the log10(number of l. humile per trap), number of pesticide treatments in the past 6 months, their interaction, and site (dummy variable). we were not interested in seasonal change, and so we selected the month with the largest number of individuals to avoid the effect of season. we therefore used the datasets of august 2011 and august 2013 in invaded plots (plots i–iii and i–iii) and that of august 2013 in non-invaded plots (plots n and n). we previously confirmed that the number of l. humile workers was not correlated with the number of pesticide treatments (r2 = 0.06). we also analyzed the relationships of the explanatory variables with the number of individuals of each non-target ant species or each invertebrate taxonomic group because those explanatory variables were associated with the total number of individuals and species richness in the above analyses. zero-inflated poisson regression models with the function zeroinfl from the pscl package (jackman, 2017) were used to analyze the relationships, because count data of the number of individuals of each species often include many zero observations. the explanatory variables and dataset were the same as above. we then tested whether the zero-inflated poisson regression model fit the data better than an ordinary poisson regression model by applying the vuong test (using the function vuong from the pscl package). we do not present results that could not be calculated owing to small sample sizes of species or taxonomic groups. we did not use bonferroni’s correction for multiple analyses because this would inflate the likelihood of a type ii error. instead, we used p < 0.025 for significance to decrease the likelihood of a type i error. second, to analyze the temporal dynamics of ant and non-ant invertebrate communities under pesticide treatment, we conducted prc analyses (van den brink & ter braak, 1999) using the vegan package (oksanen, 2013) of r. the prc method, which is based on the redundancy analysis ordination technique, can compare the temporal dynamics of treated communities with an arbitrarily prescribed “control” community (van den brink & ter braak, 1999). we performed the analyses of non-target ant and invertebrate community dynamics in plots i and ii in tokai from april 2011 to march 2014 with data from plot n (never invaded) as a control. in plot i the pesticide was discontinued after about 1 year, whereas in plot ii it was used for almost 3 years (fig 2). species abundance data were ln (10x + 1)-transformed to downfig 2. presence of linepithema humile and fipronil pesticide use history in each plot at tokai and jonan over 3 years. y sakamoto et al. – non-target effects of pesticide in invasive ant eradication program230 weight high abundance values (lepš & šmilauer, 2003). the significance of the overall treatment effect was tested using 1000 permutations and the first eigenvalue. the resulting prc diagram displays the regression coefficient (cdt, left axis) of the first principal component in the community pattern at each site d at each time t compared with the control, whose cdt is always zero by definition. an advantage of prc analysis is that it can detect taxon-level effects. the right axis indicates the species (or taxon) weight (bk). for a quantitative evaluation of prc, the quotient exp (cdt × bk) can be calculated for each species k at each site and each time. if the quotient is positive, species k is more abundant in the community than in the control. if it is negative, species k is less abundant. therefore, species k is more abundant if bk is on the same side of cdt on the vertical axis and is less abundant if bk is on the opposite side of cdt. the greater the value of the quotient, the more different the abundance of species k is between treatment and control. results the fauna table 1 shows the total numbers of ants and non-ant invertebrates caught by traps. in total, we collected 51,307 ants belonging to 35 species, including l. humile, and 41,324 non-target invertebrates. the last observations of l. humile were in december 2012 in tokai and december 2013 in jonan. in the treated plots (plots i, ii, iii, i, ii, and iii), l. humile was eradicated by the pesticide, as demonstrated with a statistical model (sakamoto et al., 2017). although the initial density of l. humile and number of fipronil treatments differed among plots (fig 2), the number of non-target ant individuals also decreased in the first year but started to recover after fipronil treatment ceased (fig 3). table 1. total numbers of each ant species and non-ant invertebrate taxonomic groups collected in monitoring traps at the tokai and jonan sites. taxa total tokai plots1 jonan plots1 i ii iii n i ii iii n ant species dolichoderinae linepithema humile 18628 74 1372 21 0 14626 218 2317 0 ochetellus glaber 330 237 63 0 15 2 1 12 0 technomyrmex gibbosus 8 0 5 0 0 2 0 1 0 formicinae camponotus japonicus 562 122 85 88 241 12 3 11 0 camponotus vitiosus 212 105 26 17 3 26 6 21 8 formica japonica 1750 546 227 137 180 13 5 216 426 lasius japonicus 50 6 9 2 1 9 7 4 12 lasius fuji 1 0 0 0 0 1 0 0 0 lasius productus 13 0 1 0 0 0 12 0 0 lasius sakagamii 18 4 0 7 0 4 1 2 0 lasius umbratus 1 0 0 0 1 0 0 0 0 nylanderia amia 284 108 3 37 1 17 3 2 113 nylanderia flavipes 35 5 1 8 10 5 6 0 0 paraparatrechina sakurae 1911 11 465 471 47 311 259 339 8 paratrechina longicornis 8 0 8 0 0 0 0 0 0 ponerinae brachyponera chinensis 1565 320 16 234 12 10 914 39 20 hypoponera opaciceps 1 0 0 0 0 0 0 1 0 myrmicinae aphaenogaster osimensis 2 2 0 0 0 0 0 0 0 crematogaster matsumurai 2145 685 31 1185 156 4 6 38 40 crematogaster osakensis 84 0 2 0 66 13 3 0 0 crematogaster teranishii 5 0 0 0 5 0 0 0 0 crematogaster vagula 7 0 0 0 7 0 0 0 0 myrmica kotokui 7 1 6 0 0 0 0 0 0 monomorium chinense 26 5 21 0 0 0 0 0 0 pheidole indica 1 1 0 0 0 0 0 0 0 sociobiology 66(2): 227-238 (june, 2019) 231 table 1. total numbers of each ant species and non-ant invertebrate taxonomic groups collected in monitoring traps at the tokai and jonan sites. (continuation) pheidole noda 1135 31 209 0 889 2 1 0 3 pristomyrmex punctatus 3339 41 246 112 173 32 3 2704 28 pyramica membranifera 1 0 1 0 0 0 0 0 0 solenopsis japonica 32 0 18 7 0 1 1 5 0 strumigenys lewisi 7 0 0 6 0 1 0 0 0 temnothorax anira 1 1 0 0 0 0 0 0 0 temnothorax congruus 190 66 45 24 0 14 13 25 3 temnothorax spinosior 5 3 2 0 0 0 0 0 0 tetramorium bicarinatum 4 0 0 0 0 0 4 0 0 tetramorium tsushimae 18939 1558 3974 2286 401 568 2151 6215 1786 total 51307 3932 6836 4642 2208 15673 3617 11952 2447 non-ant invertebrates (common name2) isopoda (sowbugs) 33389 1452 9084 9669 507 5442 1571 5600 64 myriapoda (centipedes/millipedes) 1343 171 155 581 8 103 122 184 19 araneae (spiders) 1242 193 198 297 24 169 181 165 15 orthoptera (grasshoppers) 239 25 35 28 9 73 13 44 12 dermaptera (earwigs) 1708 338 78 355 149 121 315 340 12 blattodea (cockroaches) 113 31 23 8 13 14 7 4 13 mantodea (mantis) 1 0 0 0 0 1 0 0 0 hemiptera (bugs) 820 89 96 306 19 94 100 107 9 coleoptera (beetles) byrrhoidea (pill beetles) 13 2 0 1 0 1 9 0 0 cantharoidea (soldier beetles) 3 0 1 0 0 0 1 1 0 caraboidea (ground beetles) 750 70 51 106 7 106 343 61 6 chrysomeloidea (longhorn beetles) 62 7 10 19 0 4 10 9 3 cucujoidea (darkling beetles) 326 67 84 81 7 11 50 24 2 curculionoidea (weevils) 618 63 89 107 6 61 70 212 10 dermestoidea (carpet beetles) 8 1 1 0 0 2 1 3 0 elateroidea (click beetles) 107 10 11 50 5 4 16 9 2 scarabaeoidea (gold beetles) 258 61 50 30 14 14 37 48 4 staphylinoidea (rove beetles) 80 18 6 11 0 2 14 27 2 land snails 244 5 74 56 25 14 21 46 3 total 41324 2603 10046 11705 793 6236 2881 6884 176 1 plots i–iii and i–iii were invaded by linepithema humile, and plots n and n were untreated, non-invaded plots. 2 representative example. effects of l. humile and pesticide on non-target species both l. humile abundance and fipronil treatment had negative associations with total number of ant individuals, ant species richness, total number of non-ant invertebrates, and non-ant invertebrate taxonomic richness (table 2). the l. humile × pesticide treatment interaction was significantly associated with total number of ant individuals. total number of non-ant invertebrates and taxonomic richness were affected by sites table 3 shows the relationships between l. humile abundance and fipronil treatment and the number of individuals in each ant species or each non-ant taxonomic group. modeling the data with zero-inflated poisson regression fit significantly better than (p < 0.025) or did not differ from the ordinary poisson regression model. in the majority of the species/ taxonomic groups, both l. humile and pesticide treatment were not significant in the zero-inflated part of the model. in the poisson part, the abundance of l. humile was negatively associated with the number of pristomyrmex punctatus and taxa total tokai plots1 jonan plots1 i ii iii n i ii iii n myrmicinae y sakamoto et al. – non-target effects of pesticide in invasive ant eradication program232 tetramorium tsushimae individuals, whereas the number of pesticide treatments was negatively associated with the number formica japonica, paraparatrechina sakurae, pheidole noda, p. punctatus, and t. tsushimae individuals. the interaction between the two variables was significantly associated with the number of p. punctatus and t. tsushimae. among the non-ant invertebrate groups, isopoda had negative relationships with both l. humile and fipronil treatment and blattodea had a negative relationship with pesticide treatment. some species and taxonomic groups were affected by site. community dynamics during chemical control and after eradication of l. humile the dynamics of non-target ant (fig 4) and non-ant invertebrate communities (fig 5) differed between pesticide usage histories. the deviations from the control were larger fig 3. mean total number of linepithema humile (red line) and non-target ants (blue line) per trap by month in plots invaded by l. humile (i–iii, i–iii) and untreated, non-invaded plots (n, n). sociobiology 66(2): 227-238 (june, 2019) 233 from spring to fall (march–november) and smaller in winter (december–february). the structures of non-target ant communities in plots i and ii clearly deviated from that in the non-invaded (control) plot (fig 4). the ant community structure in 3-year-treated plot ii responded to the treatment, in which the deviation from the control was larger in the second and third years than in the first year (fig 4a), whereas that in 1-year-treated plot i initially deviated from that in the non-invaded plot but was fig 5. principal response curve diagrams illustrating the shift of non-ant invertebrate communities over 3 years. (a) plot ii, where no pesticide was applied after october 2013, and (b) plot i, where no pesticide was applied after may 2012, relative to the control (untreated and noninvaded) plot over time. left axis, regression coefficient; right axis, taxon weights (only taxa with a score of >0.5 or <−0.5 are shown). the first canonical axis explains 29.72% (p < 0.001) of the total variation in plot ii and 62.97% (p < 0.001) in plot i. fig 4. principal response curve diagrams illustrating the shift of non-target ant communities over 3 years. (a) plot ii, where no pesticide was applied after october 2013, and (b) plot i, where no pesticide was applied after may 2012, relative to the control (untreated and non-invaded) plot over time. left axis, regression coefficient; right axis, species weights (only species with a score of >0.5 or <−0.5 are shown). the first canonical axis explains 56.63% (p < 0.001) of the total variation in plot ii and 50.96% (p < 0.001) in plot i. more similar in the third year (fig 4b). these results indicate that the ant community structure recovered about a year after the eradication program ended. the structures of non-ant invertebrate communities in plots i and ii did not deviate so clearly from that in the non-invaded (control) plot. the community structure in the 3-year-treated plot changed unidirectionally (i.e., isopoda decreased) with time (fig 5a), whereas that in the 1-year-treated plot did not respond clearly (fig 5b). discussion the number of l. humile was negatively associated with the total number of non-target ant individuals and species richness. as reported elsewhere in japan (miyake et al., 2002), in the usa (heller, 2004; suarez et al., 1998), and in europe (oliveras et al., 2005), l. humile reduces the diversity of indigenous ants. therefore, it is reasonable to conclude that the invasion by l. humile harmed non-target ants in our study area as well. our glm results for each ant species indicated decreases in p. punctatus and t. tsushimae abundance. the incidence of t. tsushimae was also reported to be clearly lower where l. humile had increased over time in several parks in japan (park et al., 2014). two mechanisms have been proposed to explain the displacement of indigenous ant fauna by l. humile invasion: exploitative competition and interference y sakamoto et al. – non-target effects of pesticide in invasive ant eradication program234 l. humile on other invertebrate taxonomic groups are also not universal, and various studies have reported negative, positive, or no relationship. factors underlying negative relationships may include direct feeding by l. humile on adult or immature organisms or spatial competition for limited habitats (cole et al., 1992; dreistadt et al., 1986), while factors underlying positive relationships may include feeding by invertebrates on dead and immature l. humile individuals or on the remains of prey items brought to the nest area by foraging l. humile (cole et al., 1992). negative effects of pesticide treatment were found in ants as well as in non-ant invertebrates. the greatest factor underlying the negative effects is that toxic baits are typically attractive to a wide range of non-target species (buczkowski, 2017). our glm analyses showed that the l. humile × pesticide treatment interaction also affected the total number of ants and non-ant invertebrates. this result can be interpreted in two ways, based on different biological scenarios. first, the effect of l. humile on indigenous invertebrates decreases when there is pesticide treatment, which kills the invasive ants. second, the effect of pesticide treatment on non-target fauna may decrease when the l. humile population is large and it may increase when the population is small. this idea reflects our observations and those of previous studies (abedrabbo, 1994; hoffmann & o’connor, 2004) that the invasive alien ants were eradicated first and non-target ants were not eradicated. this likely occurred because non-target fauna was deprived of opportunities to eat the bait because of greater consumption by l. humile (buczkowski & bennett, 2008; hoffmann, 2010; holway, 1999; human & gordon, 1996). we compared the prc results for 3 years in this study with those for only the first year in the previous study (inoue et al., 2015) in the same eradication program. it should be noted that the plots with low-density l. humile were selected for prc analyses and the control plot was untreated and never invaded in this study, whereas the plots with high-density l. humile were selected and the control plot was untreated but invaded in the previous study (inoue et al., 2015). inoue et al. (2015) concluded that non-target populations recovered within the first year of pesticide treatment, and they found no non-target effects of pesticide in the first year, we suspect because the number of l. humile was large. by contrast, our prc results showed that the non-target community structure recovered about a year after the eradication program ended. that is, until l. humile was eradicated, the negative effects of pesticides on non-targets increased as l. humile decreased, suggesting that non-target effects cannot be ignored. ensuring that indigenous ants and other invertebrates remain after pesticide treatment is crucial for ecosystem recovery after the eradication of invasive species. in fact, the invertebrate community recovered to a similar structure as in the non-invaded plot in this study. such recovery eventually can be achieved by the continuous recruitment of immigrants of indigenous species from non-invaded sites (holway, 1998). variable estimate se z-value p-value ant species total number of individuals intercept 1.911 linepithema humile –0.651 0.101 –6.443 <0.001 * pesticide treatment –0.127 0.027 –4.650 <0.001 * lh × pt1 0.137 0.036 3.850 <0.001 * site –0.262 0.126 –2.080 0.040 species richness intercept 4.155 linepithema humile –1.205 0.259 –4.654 <0.001 * pesticide treatment –0.326 0.070 –4.640 <0.001 * lh × pt1 0.161 0.091 1.762 0.081 site 0.300 0.324 0.927 0.356 non-ant invertebrates total number of individuals intercept 2.202 linepithema humile –0.499 0.080 –6.205 <0.001 * pesticide treatment –0.099 0.022 –4.539 <0.001 * lh × pt1 0.071 0.028 2.513 0.014 * site –0.333 0.101 –3.307 0.001 * taxonomic richness intercept 5.892 linepithema humile –1.143 0.283 –4.044 <0.001 * pesticide treatment –0.182 0.077 –2.367 0.020 * lh × pt1 0.022 0.100 0.216 0.829 site –1.724 0.353 –4.881 <0.001 * 1 linepithema humile × pesticide treatment interaction. * p < 0.025 table 2. results of generalized linear models examining the effects of linepithema humile, pesticide treatment, their interaction, and site on total number of individuals and species or taxonomic richness per trap. competition (holway, 1999; human & gordon, 1996). however, although negative associations were reported between several other ant species (i.e., f. japonica and crematogaster matsumurai) and l. humile in previous studies (miyake et al., 2002; park et al., 2014), no associations were observed between them in this study. the difference in results may be due in part to seasonal or temporal factors and/or small sample sizes. likewise, the number of l. humile was negatively associated with the total number of non-ant individuals and taxonomic richness. the analysis for each taxonomic group, however, showed that l. humile had negative associations with abundance of only isopods. almost all isopods we found were armadillidium vulgare, which can reproduce in urban areas (hornung et al., 2007). linepithema humile has been reported to cause both significant decreases (stanley & ward, 2012) and increases in isopod abundance (cole et al., 1992; human & gordon, 1997; walters & mackay, 2003). the impacts of sociobiology 66(2): 227-238 (june, 2019) 235 table 3. results of zero-inflated poisson regression models examining the effects of linepithema humile, pesticide treatment, their interaction, and site on the number of individuals of each ant species or non-ant invertebrate taxonomic group per trap. species/taxonomic group1 variable poisson model zero-inflated model estimate se z-value p-value estimate se z-value p-value ants formica japonica linepithema humile –38.02 na na na –89.79 448.9 –0.200 0.842 pesticide treatment –0.226 0.023 –10.03 <0.001 * 0.253 0.107 2.377 0.018 * lh × pt3 7.462 na na na 18.20 89.78 0.203 0.839 site –0.665 0.107 –6.194 <0.001 * –0.33 –0.687 0.492 paraparatrechina sakurae linepithema humile –24.22 na na na –108.3 379.9 –0.285 0.775 pesticide treatment –0.195 0.050 –3.885 <0.001 * –0.308 0.126 –2.443 0.015 * lh × pt3 4.96 na na na 21.43 75.97 0.282 0.778 site 0.02 0.230 0.099 0.921 –0.34 0.51 –0.655 0.513 pheidole noda linepithema humile –30.00 443.9 –0.068 0.946 –235.9 541.5 –0.436 0.663 pesticide treatment –1.419 0.143 –9.92 <0.001 * –18.84 na na na lh × pt3 9.967 88.77 0.112 0.911 107.7 na na na site –62.84 995.6 –0.063 0.950 –520.2 1201 –0.433 0.665 pristomyrmex punctatus linepithema humile –2.333 0.825 –2.827 0.005 * –1.4755 1.608 –0.918 0.359 pesticide treatment –0.769 0.234 –3.284 0.001 * –0.404 0.482 –0.838 0.402 lh × pt3 0.757 0.193 3.929 <0.001 * 0.493 0.365 1.352 0.176 site –3.100 1.197 –2.591 <0.001 * –2.233 2.507 –0.891 0.373 solenopsis japonica linepithema humile –50.56 6945 –0.007 0.994 58.39 1001000 0.000 1.000 pesticide treatment –9.585 387.2 –0.025 0.980 –7.570 na na na lh × pt3 10.50 1389 0.008 0.994 –11.82 200100 0.000 1.000 site 4.128 0.985 4.192 <0.001 * 7.438 19 0.383 0.702 tetramorium tsushimae linepithema humile –1.240 0.155 –8.022 <0.001 * 1.502 0.524 2.866 0.004 * pesticide treatment –0.116 0.008 –13.91 <0.001 * 0.164 0.135 1.214 0.225 lh × pt3 0.303 0.032 9.509 <0.001 * –0.298 0.166 –1.797 0.072 site –0.480 0.042 –11.54 <0.001 * 0.568 0.569 0.999 0.318 non-ant invertebrates isopoda (sowbugs2) linepithema humile –0.996 0.082 –12.09 <0.001 * 0.637 0.575 1.109 0.268 pesticide treatment –0.140 0.010 –13.63 <0.001 * 0.274 0.159 1.729 0.084 lh × pt3 –0.081 0.031 –2.581 0.010 * 0.059 0.175 0.340 0.734 site –0.292 0.054 –5.447 <0.001 * 1.497 0.657 2.279 0.023 * araneae (spiders2) linepithema humile –0.355 0.985 –0.360 0.719 2.042 1.690 1.208 0.227 pesticide treatment 0.155 0.080 1.949 0.051 0.242 0.381 0.634 0.526 lh × pt3 0.055 0.200 0.275 0.784 –0.359 0.351 –1.023 0.306 site –0.084 0.252 –0.333 0.739 1.953 1.048 1.864 0.062 orthoptera (grasshoppers2) linepithema humile 0.070 0.280 0.249 0.804 –10.785 47406 0.000 1.000 pesticide treatment –0.092 0.109 –0.839 0.401 0.336 0.198 1.699 0.089 lh × pt3 1.068 0.648 1.648 0.099 22.274 142.6 0.156 0.876 site 1.016 0.572 1.775 0.076 31.430 14269 0.002 0.998 however, our data revealed temporary negative effects of pesticides on non-target communities. it is also important to reduce the impacts on indigenous communities, because their restoration inhibits successful re-invasion into the ecological gap by invasive alien species (hoffmann, 2010; hoffmann & o’connor, 2004; plentovich et al., 2009; tschinkel & king, 2017). when an eradication program comes close to achieving success, the end should be judged by using a statistical model (sakamoto et al., 2017), for example, so as to avoid the unnecessary prolongation of pesticide treatment. moreover, in delicate infested habitats and in the presence of sensitive wildlife, traditional eradication methods with toxic baits may be inappropriate. to reduce the impacts on non-target fauna, target-specific approaches should be developed, such as using ribonucleic acid interference (campbell et al., 2015; gould, 2008) or prey-baiting (buczkowski, 2017). y sakamoto et al. – non-target effects of pesticide in invasive ant eradication program236 species/taxonomic group1 variable poisson model zero-inflated model estimate se z-value p-value estimate se z-value p-value blattodea (cockroaches2) linepithema humile –0.496 0.376 –1.318 0.188 –51.85 2490 –0.021 0.983 pesticide treatment –0.353 0.141 –2.508 0.012 * –0.054 0.276 –0.197 0.844 lh × pt3 1.744 0.707 2.467 0.014 * 23.71 146.9 0.161 0.872 site 0.842 0.653 1.290 0.197 20.75 716.3 0.029 0.977 hemiptera (bugs2) linepithema humile –0.687 0.379 –1.813 0.070 0.215 1.095 0.196 0.845 pesticide treatment –0.042 0.045 –0.925 0.355 0.187 0.187 1.001 0.317 lh × pt3 –0.051 0.115 –0.442 0.658 –0.034 0.289 –0.117 0.907 site –0.896 0.241 –3.723 <0.001 * –0.596 0.891 –0.670 0.503 caraboidea (ground beetles2) linepithema humile 0.053 0.268 0.196 0.844 –1.927 3502 –0.001 1.000 pesticide treatment –0.071 0.100 –0.708 0.479 0.190 0.200 0.948 0.343 lh × pt3 –0.305 0.186 –1.642 0.101 –0.323 700.4 0.000 1.000 site 0.802 0.499 1.608 0.108 14.747 617.5 0.024 0.981 cucujoidea (darkling beetles2) linepithema humile –0.595 0.616 –0.965 0.335 2.921 4624 0.001 0.999 pesticide treatment 0.287 0.183 1.563 0.118 10.39 116.8 0.089 0.929 lh × pt3 0.100 0.166 0.604 0.546 –0.710 924.8 –0.001 0.999 site –2.167 1.270 –1.706 0.088 0.616 2.0 0.308 0.758 curculionoidea (weevils2) linepithema humile 0.787 0.575 1.370 0.171 2.122 1.842 1.152 0.249 pesticide treatment 0.002 0.184 0.013 0.989 0.05 0.35 0.13 0.896 lh × pt3 –0.327 0.211 –1.550 0.121 –8.343 117.7 –0.071 0.943 site 0.808 0.721 1.120 0.263 5.612 4.35 1.291 0.197 elateroidea (click beetles2) linepithema humile –15.68 na na na –0.442 25990 0.000 1.000 pesticide treatment 41.77 na na na 12.12 393 0.031 0.975 lh × pt3 3.650 na na na 0.490 5198 0.000 1.000 site –3.188 0.920 –3.464 0.001 * –30.93 1661000 0.000 1.000 scarabaeoidea (gold beetles2) linepithema humile 0.143 0.336 0.426 0.670 0.050 0.723 0.069 0.945 pesticide treatment –0.030 0.168 –0.178 0.858 –0.037 0.391 –0.096 0.924 lh × pt3 –0.207 0.173 –1.195 0.232 –0.416 0.639 –0.650 0.515 site –0.763 0.769 –0.993 0.321 –1.932 2.128 –0.908 0.364 1 species or taxonomic groups that could not be calculated because of insufficient data are not shown. 2 representative example 3 linepithema humile × pesticide treatment interaction * p < 0.025 table 3. results of zero-inflated poisson regression models examining the effects of linepithema humile, pesticide treatment, their interaction, and site on the number of individuals of each ant species or non-ant invertebrate taxonomic group per trap. (continuation) acknowledgments we are grateful to takashi sugiyama and katsuo sugimaru (fumakilla ltd.), mitsuhiko toda and hideaki mori (japan wildlife research center), sachiko moriguchi (niigata university), and kazutaka suzuki, takuji nomura, and hiromoto agemori (national institute for environmental studies, nies) for conducting the eradication program and identifying species, and to makihiko ikegami and naoki h. kumagai (nies) for providing useful comments. we also greatly appreciate the support of fumakilla ltd. in providing its products. this research was supported by the environment research and technology development fund (no. 4-1401) of the ministry of the environment, japan. references abedrabbo, s. (1994). control of the little fire ant, wasmannia auropunctata, on santa fe island in the galapagos islands. in: d.f. williams (ed), exotic ants: biology, impact, and control of introduced species (pp. 219-227). boulder, colorado: westview press. buczkowski, g. 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(1994). exotic ants: biology, impact, and control of introduced species. boulder, colorado, usa: westview press. doi: 10.13102/sociobiology.v65i4.3375sociobiology 65(4): 630-639 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 different responses in geographic range shifts and increase of niche overlap in future climate scenario of the subspecies of melipona quadrifasciata lepeletier introduction pollination is considered a key element of global biodiversity conservation, because it provides a crucial ecosystem service, as without pollination, many plants would not be able to reproduce (klein et al., 2007). ollerton et al. (2011) estimated that approximately 87.5% of world’s flowering plants require animal pollination for fruit and seed production. pollination is also considered fundamental for food production because one-third of world’s leading food crops depends on animal pollination (klein et al., 2007). in brazil, giannini et al. (20015) demonstrated that about 89% of the crop species are pollinated by bees. abstract climate change is suggested to be one of the possible drivers of decline in pollinators. in this paper, we applied an ecological niche model to modeling distributional responses in face of climate changes for the subspecies of melipona quadrifasciata lepeletier. this species is divided into two subspecies based on difference in the yellow tergal stripes, which are continuous in m. q. quadrifasciata and interrupted in m. q. anthidioides. the geographic distribution of each subspecies is also distinct. m. q. quadrifasciata is found in colder regions in the southern states of brazil, whereas m. q. anthidioides is found in habitats with higher temperatures, suggesting that ecological features, such as adaption to distinct climatic conditions may take place. thus, the possibility of having different responses in geographic range shifts to future climate scenario would be expected. this study aimed to investigate the effects of climate changes on the distribution of the two m. quadrifasciata subspecies in brazil, using an ecological niche model by the maxent algorithm. our results indicate that the subspecies showed clear differences in geographic shift patterns and increased climate niche overlap in the future scenarios. m. q. anthidioides will have the potential for an increase of suitable climatic conditions in the atlantic forest, and towards the pampa biome, while m. q. quadrifasciata will suffer a reduction of adequate habitats in almost all of its current geographic distribution. given the potential adverse effects of climate changes for this subspecies, conservation actions are urgently needed to avoid that it goes extinct. sociobiology an international journal on social insects ko teixeira1, tcl silveira2, b harter-marques1 article history edited by denise alves, esalq-usp, brazil received 25 april 2018 initial acceptance 05 june 2018 final acceptance 21 august 2018 publication date 11 october 2018 keywords stingless bee, climate niche, habitat suitability, species distribution modeling, hybridization. corresponding author birgit harter-marques universidade do extremo sul catarinense programa de pós-graduação em ciências ambientais avenida universitária nº 1105 cep 88806-000, criciúma-sc, brasil. e-mail: bhm@unesc.net there is evidence of a global decline in pollinators, mainly honey bees (becher et al., 2013), which points to a more important role of wild bees as providers of this ecological service. wild bees, especially social stingless bees, are pollinators of many native plant species (imperatriz-fonseca et al., 2012) and effective crop pollinators (klein et al., 2007). among the stingless bees, the melipona genus has the largest number of species (moure et al., 2007), and can be found throughout the neotropical region, from mexico to misiones, argentina, with highest diversity in the amazon basin (silveira et al., 2002). so far, declines of melipona species in brazil have gone unnoticed, until three species (melipona bicolor schenkii gribodo, melipona marginata 1 universidade do extremo sul catarinense, programa de pós-graduação em ciências ambientais, criciúma, santa catarina, brazil 2 universidade federal de santa catarina, programa de pós-graduação em ecologia, florianópolis, santa catarina, brazil research article bees sociobiology 65(4): 630-639 (october, 2018) special issue 631 obscurior moure, melipona quadrifasciata quadrifasciata lepeletier) were reported to be threatened in the brazilian state of rio grande do sul (fontana et al., 2003). currently, there are eight melipona species reported to be threatened at national and/or state level (tossulino et al., 2006; icmbio, 2016). multiple anthropogenic pressures, including shortage of food sources and nesting sites due to habitat loss, aggressive agricultural practices and the spread of the alien species apis mellifera l., are the main factors responsible for the observed population declines (vanbergen et al., 2013). climate change also has been considered one of the possible drivers of the pollinators decline, because climatic variables provide general conditions for their occurrence and performance, according to their physiological limits (dupont et al., 2011). mainly temperature and precipitation have been shown to play important roles in plant-pollinator relationships and may provide critical insights to help explain the potential effects of climate change on these interactions (devoto et al., 2009). shifts in phenology induced by climate change have the potential to disrupt the temporal overlap between pollinators and their floral food resources. as pollinators will experiencing loss of some of their food plants, they are likely to suffer population declines, as relying on fewer plant species will expose them to lower overall densities of flowers and greater temporal and spatial variation in food supply (memmott et al., 2007). there are three general expectations for species’ responses to climate change: movement (shift their geographic ranges to environmental conditions within which they are able to maintain populations), adaptation (in terms of evolutionary change or of physiological acclimatization), or face extinction (holt, 1990). ecological niche modeling is an efficient and widely disseminated approach in conservationist studies to predict species’ spatial and evolutionary responses to the effects of global climate change via identification of their environmental requirements (soberón & nakamuna, 2009). in this paper, we apply an ecological niche model by the maxent algorithm to modeling distributional responses in the face of climate for the two subspecies of melipona quadrifasciata lepeletier. this species is one of the best-known social bee found in brazil, acting as a pollinator of several plant species and playing an important ecological role in the ecosystem (ramalho et al., 2004). in nature the species constructs the nest inside trees hollows (michener, 2007), so it is sensitive to forest fragmentation, especially when nesting sites are destroyed and availability of trophic resources is declined, compromising the maintenance of natural colonies. m. quadrifasciata has been divided into two subspecies: m. q. quadrifasciata and melipona q. anthidioides lepeletier (moure et al., 2007). the geographic distribution of each subspecies seems to be very distinct. m. q. quadrifasciata occurs in the southern states of brazil, from rio grande do sul to the southern são paulo state, mainly in cold regions, whereas m. q. anthidioides is found in habitats with higher temperatures from northeastern são paulo state to the northern part of bahia, and westwards to the western tip of minas gerais and central region of the goiás state (kerr, 1976). the main morphological difference between the two subspecies consists in the presence of continuous tergal stripes from the 2nd to the 5th segment in m. q. quadrifasciata and interrupted stripes in m. q. anthidioides (schwarz, 1948). nonetheless, there is a hybridization zone between the central region of são paulo state and the southern state of minas gerais, where intermediate patterns of tergal stripes can be found (kerr, 1976). furthermore, populations with an abdominal pattern identical to that of m. q. quadrifasciata could be found in the northeastern part of bahia state in warm regions with altitudes ranging from 500 to 700 m (batalha-filho et al., 2009). recently, molecular studies have been conducted on these species to identify and substantiate the maintenance of division into subspecies. while some studies have provided evidence that it is possible to recognize molecular differences between the two m. quadrifasciata subspecies, confirming the usefulness of the yellow metasomal stripes to identify them (waldschmidt et al., 2000; moretto & arias, 2005; souza et al., 2008; tavares et al., 2013), nascimento et al. (2010) did not reveal genetic structuring of m. quadrifasciata in function of the tergite stripe pattern. similar results were reported by batalhafilho et al. (2009) through pcr-rflp, and in phylogeographic studies of these bees (batalha-filho et al., 2010). the maintenance of distinct tergal strip patterns between m. q. quadrifasciata and m. q. anthidioides at different geographic regions together with the occurrence of a hybridization zone in the central region suggest that ecological features, such as adaption to distinct climatic conditions may take place (kerr, 1976). thus, the possibility of having different responses in geographic range shifts to future climate scenario would be expected. in view of this scenario, in the present study, we investigated the effects of climate changes on the distribution of the two m. quadrifasciata subspecies in brazil to answer two main questions: (i) are there differences in geographic shift patterns of the two subspecies to future climate scenario? (ii) is there an overlap of suitable habitats between them and, if so, how much will it be? therefore, the present study clarifies questions regarding geographic distribution, and climate niche overlap of m. quadrifasciata subspecies. material and methods occurrence data of subspecies of m. quadrifasciata were obtained from the following databases: the global biodiversity facility – gbif (www.gbif.org) and specieslink (www.splink.cria.org.br). the records that were not previously classified at the subspecies level were identified through available images from the collections and, when this was not possible, were classified according to the distribution map of batalha-filho et al. (2009). we excluded data that: (i) came from meliponaries (to avoid over-representation of some areas); (ii) were not georeferenced; and (iii) were impossible ko teixeira, tcl silveira, b harter-marques – future climate scenario of the subspecies of melipona quadrifasciata632 to classify to subspecies level. thus, 137 records were obtained for m. q. anthidioides and 68 records were obtained for m. q. quadrifasciata (fig 1). the environmental variables were obtained from the worldclim database (fick & hijmans, 2017) at a resolution of 10 arc-minute grid cells, what corresponds to 0.16 degrees or approximately 18 km in the tropics. the current climate model was based on the time series produced from 1970– 2000. future climate models were developed by the ipcc5ar5 fifth assessment report (field et al., 2014) in the scenario of greenhouse gases rcp8.5 to 2100. this scenario is considered the most pessimistic projection, predicting a global temperature rise by about 5 to 6 °c by 2100 and increase of extreme events such as heat waves and very intense rains (gent et al., 2011). it was chosen because we are currently on a trajectory closer to the rcp8.5 scenario than to the more moderate scenarios (peacock, 2012). considering the climatic conditions of the current subspecies distribution (tropical and subtropical environments and respective transition zones with seasonal temperature and high humidity (michener, 2007), 11 climatic variables were pre-selected. in order to avoid overfitting and reducing the predictive power of the model, variables that were correlated were excluded using variance inflation factor vif (dormann et al., 2013). we used the function vifstep included in the package usdm (naimi, 2015), using 10 as threshold. the final dataset of variables selected for the models were: annual average temperature (bio1), isothermality (bio3), annual temperature rate (bio7), precipitation of the wettest month (bio13), precipitation of the driest month (bio14), and seasonality of precipitation (bio15). a maximal entropy model (maxent) was used to model the current distributions of subspecies of m. quadrifasciata and to design their future habitat suitability. maxent is a machine learning approach which is based on maximum entropy algorithm (phillips et al., 2006). maxent has been show an adequate and widely method for modelling presence-only data (elith et al., 2006). this approach uses the presence-only data and background points (pseudoabsences), relating environmental predictors to construct suitability maps ranging from 0 (unsuitable) to 1 (suitable). in both subspecies we allowed linear, quadratic, product and hinge features between the habitat suitability values and each covariate (phillips & dudı´k, 2008). the projections were repeated 10 times, each selecting a different random sample of 80% when verifying the accuracy of the model in relation to the remaining 20% (phillips et al., 2006). the maxent algorithm performs better when the study area for the calibration model does not include areas outside the occurrence of species records (elith et al., 2011). therefore, considering the current distribution of the two subspecies, the obtained models were limited to the atlantic forest, pampa, and cerrado biomes. the models’ performances were evaluated by area under the receiver-operator curve (auc). to reduce sampling bias, a polarization layer was constructed, consisting of a map that incorporates sampling effort, that is, making the model less important for areas with high density of occurrence points and increasing the importance of regions with similar environmental characteristics, but which were not sampled (kramer-schadt et al., 2013). to characterize potential changes in the distributions of the two subspecies, we calculated the proportions of occupied grid cells in actual and projected scenarios in the atlantic forest, pampa, and cerrado delimitation. in order to produce the presence/absence maps to calculate the differences in occupied cells we first calculated the threshold which maximized the tss. we used the functions included in the presence absence package (freeman & moisen, 2008). the niche similarities and overlap between the two subspecies in present and future climatic scenarios was verified using the method proposed by broennimann et al. (2012), in a grid-environment space. this method uses core density functions to calculate the smoothed density of the number of occurrences and the available environments along the environmental axes of the principal components analysis (pca) and, based on these values, an occupancy index is estimated. afterwards, the schoener d-index was calculated, fig 1. spatial distribution of records of the two melipona quadrifasciata subspecies in brazil. grey circles represent the records of m. q. anthidioides, and black squares the m. q. quadrifasciata records. sociobiology 65(4): 630-639 (october, 2018) special issue 633 quantifying the niche overlap between the two subspecies (warren et al., 2008; broennimann et al., 2012). finally, two randomization tests were performed to evaluate the equivalence and niche similarity between the two subspecies. the equivalence test assesses whether niches are indistinguishable (warren et al., 2008). for this test, pseudo-replications were created, grouping the geographical areas of occurrences of the two subspecies and randomly dividing them into two groups; however, original sample sizes were maintained. for each of the pseudo-replications, we calculated the d-index and then contrasted the original d-value observed with a null distribution of 100 pseudoreplicated d-values. the hypothesis of niche equivalence was rejected if the probability of observed d falling in the null distribution was less than 0.05 (p < 0.05). the niche similarity test was used to question whether the occupied environmental space in one range is more similar or more different than the occupied environmental space in the other range than would be expected at random. for this test the distribution of the two subspecies in one interval was overlapped with the distribution in the other interval, randomly assigning a new location to each occurrence in the other range and, for each pseudo-repetition, index-d was calculated. this procedure was performed 100 times (from m. q. anthidioides to m. q. quadrifasciata and from m. q. quadrifasciata to m. q. anthidioides) to generate two new null distributions of d-values. the hypothesis of niche similarity was rejected if the probability of observed d falling in the null distribution was less than 0.05 in a two-column test (p < 0.05) (broennimann et al., 2012). results the auc values obtained for each model exceeded 0.7, with the highest value (0.785) occurring in the future model of m. q. anthidioides, which indicates a good agreement of the models and good predictive performance. our distribution model for the future scenarios of m. q. anthidioides suggested a clear trend of reduction in habitat suitability in the cerrado biome, and a potential increase of suitable climatic conditions in the atlantic forest, especially on the eastern coastal line from the state of santa catarina to the state of espírito santo. furthermore, the model predicted a shift from north to south towards the pampa biome (fig 2 a, b). fig 2. potential distribution of melipona quadrifasciata anthidioides (a. current, b. future) and melipona quadrifasciata quadrifasciata (c. current, d. future) in the atlantic forest, pampa, and cerrado biomes in brazil. ko teixeira, tcl silveira, b harter-marques – future climate scenario of the subspecies of melipona quadrifasciata634 meanwhile, the resulting distribution of m. q. quadrifasciata in future climatic scenarios showed a potential decrease in habitat suitability in almost all of its current distribution, with an increase of suitable landscapes only in the coastal region of bahia (fig 2 c, d). in addition, it is predicted to be an increase in suitability of m. q. anthidioides towards the potential distribution area of m. q. quadrifasciata (fig 2 b, d). the percentage of appropriate area occupation (with threshold of 0.5 for both subspecies) was 30.35% for m. q. anthidioides in the current model and increased to 31.75% in the future model. for m. q. quadrifasciata, the percentage of suitable area pixels was 16.36% in the current model and decreased to 13.74% in the future model. the current and future niches of the subspecies in the environmental space are defined by the first two axes of the pca, which explained 76.7% of the original climatic variation. the subspecies showed little overlap of the current niche (schoener’s d = 0.399, fig 3 d), and an increase in future niche overlap (schoener’s d = 0.406, fig 4 d). the current and future pcas showed a displacement of the centroid of the m. q. anthidioides niche (area with higher probability density of occurrence in the environmental space) towards bio3, bio14, and bio15 compared to the centroid of the m. q. quadrifasciata niche (fig 3 a, b, c and fig 4 a, b, c). the hypotheses of the current niche equivalence and similarity tests were not rejected (p = 1.00; p = 0.059; p = 0.079, respectively, fig 3 d, e, f). likewise, the hypotheses of future niche equivalence and similarity tests were not rejected (p = 1.00; p = 0.158; p = 0.119, respectively, fig 4 d, e, f). fig 3. current climatic niches occupied by the two subspecies of melipona quadrifasciata. niche occupied by m. q. anthidioides (a) and m. q. quadrifasciata (b) along the two-first axes of the pca (see (c) for details). grey shading represents the density of the occurrences of the subspecies by cell. the solid and dashed contour lines illustrate, respectively, 100% and 50% of the available environment. (c) contribution of the climate variables to the first-two axes of the pca (bio1: annual mean temperature, bio3: isothermality, bio7: annual temperature rate, bio13: precipitation of the wettest month, bio14: precipitation of the driest month, and bio15: precipitation seasonality), and contribution of the two-first axes to data variation. (d) histogram of the observed niche overlap d (d = 0.399) (bars with a diamond) and simulated niche overlaps (grey bars). (e) niche similarity of m. q. quadrifasciata to m. q. anthidioides, and (f) niche similarity of m. q. anthidioides to m. q. quadrifasciata. sociobiology 65(4): 630-639 (october, 2018) special issue 635 discussion according to our potential distribution models, temperature and precipitation (from the driest month and seasonality) are determinant factors for changes in distributions of the two subspecies studied. several studies on foraging activities of melipona species showed speciesspecific characteristics of flight in response to the variations in abiotic conditions (e.g. teixeira & campos, 2005), allowing interference on the geographic and ecological distribution of each species (pereboom & biesmejer, 2003). these studies conclude that air temperature and relative humidity directly affect the flight activity of melipona species with relatively large body sizes. scientific models of climate change for future climatic conditions indicate for the cerrado biome that, although rainfall tends to increase mainly in the form of more intense and extreme rainfall events, periods of drought will be longer and summers will become warmer with an increase in temperature of up to 4 °c by 2100 (juras, 2008). according to our models, as a consequence of the temperature increase and drought period predicted for the cerrado, m. q. anthidioides fig 4. future climatic niches occupied by the two subspecies of melipona quadrifasciata. niche occupied by m. q. anthidioides (a) and m. q. quadrifasciata (b) along the two-first axes of the pca (see (c) for details). grey shading represents the density of the occurrences of the subspecies by cell. the solid and dashed contour lines illustrate, respectively, 100% and 50% of the available (background) environment. (c) contribution of the climate variables to the first-two axes of the pca (bio1: annual mean temperature, bio3: isothermality, bio7: annual temperature rate, bio13: precipitation of the wettest month, bio14: precipitation of the driest month, and bio15: precipitation seasonality), and contribution of the two-first axes to data variation. (d) histogram of the observed niche overlap d (d = 0.406) (bars with a diamond) and simulated niche overlaps (grey bars). (e) niche similarity of m. q. quadrifasciata to m. q. anthidioides, and (f) niche similarity of m. q. anthidioides to m. q. quadrifasciata. ko teixeira, tcl silveira, b harter-marques – future climate scenario of the subspecies of melipona quadrifasciata636 will undergo a notable reduction in potential habitats in this biome, but will have a potential increase of geographical distribution in the atlantic forest, along almost the entire eastern coastline of brazil, while m. q. quadrifasciata will suffer a reduction of adequate habitats in almost all of its current geographic distribution. this finding reinforces the evidence of the existence of specific responses to variations in climatic conditions, as proposed by teixeira and campos (2005). furthermore, our results indicate that the two subspecies showed clear differences in geographic shift patterns, supporting the maintenance and acceptance of the division of m. quadrifasciata into two subspecies. regarding the disjunct populations with continuous bands similar to that of m. q. quadrifasciata in the northeast of brazil, our distribution models showed similar patterns in responses to climatic changes with m. q. anthidioides. waldschmidt et al. (2000) and batalha-filho (2010) showed that these northern populations with continuous tergal stripes and colonies of m. q. anthidioides were genetically similar, indicating that the existence of similar tergal band patterns does not determine that this group belongs to the subspecies m. q. quadrifasciata found in the southern part of brazil. furthermore, the similar pattern in responses to climate changes detected in our study, added to the low genetic variability among colonies of the two m. quadrifasciata groups found by these authors, suggests that both belong to the same subspecies, m. q. anthidioides. perhaps, the pattern of continuous bands of disjunct populations would be the result of the more recent differentiation within this group, resulting in a group adapted to higher temperatures and lower precipitation that characterizes the region of the northeast occupied by these populations. according to our distribution models, m. q. anthidioides will show an increase in distribution suitability towards the pampa biome. both subspecies show low current habitat availability in this biome, probably due to lack of adequate nesting sites, since melipona species build their nests in hollow trunks of trees that are not abundant in this region. this biome is formed by four main groups of natural field vegetation: campanha plateau, central depression, sul-rio-grandense plateau, and coastal plain, with herbaceous and shrub vegetation predominating and trees restricted to ‘capões’ and riverbanks (ibge, 2004). for this reason, the migration of m. q. anthidioides to this biome is unlikely, although in the future this region presents favorable climatic conditions for the subspecies. our distribution models clearly indicated that the future climate scenario will be more favorable to m. q. anthidioides when compared to m. q. quadrifasciata, as the first presented greater potential to expand its distribution through migration. this migration, according to our results, would lead to a 0.7% increase in the overlap of the potential distribution between the two subspecies. the increase in overlap, in turn, may lead to an increase in the size of the hybridization area currently existing between the two subspecies (batalha-filho et al., 2009). hybridization between populations may allow alleles from one genetic background to integrate into another if favored by selection (riesenberg, 2003). however, the net outcome of inter-population hybridization can be affected by several factors, such as the level of divergence between the populations and the rate of inbreeding in the populations (whitlock, 2000). in the present hybridization zones of the two subspecies of m. quadrifasciata, there are individuals with dorsal bands similar to one subspecies but with genetic markers of the other subspecies (waldschmidt et al., 2000; batalha-filho et al., 2009). in view of the future scenarios of the potential geographical distribution of the two subspecies (increase of suitable habitats of m. q. anthidioides, potential decrease in distribution for m. q. quadrifasciata, and increased climate niche overlap) it is possible that m. q. quadrifasciata, which is already in the red list of the endangered fauna of the state of rio grande do sul (fontana et al., 2003), will be at high risk of loss. due to the variation of some genetic markers, as rapd and rflp in the cytochrome b gene, social bees tend to have a differentiated genetic predisposition between their colonies in relation to their foraging behavior for example (oldroyd et al., 1992). the loss of m. q. quadrifasciata may lead to loss of differential responses presented by this subspecies, and, consequently, result in a reduction in the genetic variability of m. quadrifasciata groups. araújo et al. (2000) showed that populations of melipona are particularly sensitive to high genetic drift due to homozygosity in x0 sex determination locus, which generates diploid males that are either unviable or sterile, putting at risk the maintenance of the species. in this sense, to avoid that m. q. quadrifasciata goes extinct, conservation actions aiming to increase suitable areas for the expansion of this subspecies are urgently needed. acknowledgements authors thank anonymous reviewers for comments and suggestions, thaise sutil for her help in preparing the final version of the figures, and the brazilian coordination for the improvement of higher education personnel (capes) for financial support for the first author’s scholarship funding. authors’ contribution b harter-marques conceived and designed the study, and wrote the manuscript. ko teixeira obtained funding and wrote the manuscript. ko teixeira and tcl silveira assembled and analyzed the data. references araújo, e.d., diniz-filho, j.a.f. & oliveira, f.a. 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(2000). fixation of new alleles and the extinction of small populations: drift load, beneficial alleles, and sexual selection. evolution, 54: 1855-1861. doi: 10.1111/ j.0014-3820.2000.tb01232.x https://doi.org/10.1890/120126 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 56-64 (2013) the botanical profiles of dried bee pollen loads collected by apis mellifera (linnaeus) in brazil as freitas1, vas arruda2, lb almeida-muradian2, om barth1,3 introduction the marketing for apis mellifera (l). products is widely expanding in brazil in response to the increased exports in recent years. bee pollen production has gradually increased in response to the demand for dietary supplements and therapeutic products (barreto et al. 2005). these products result from the intensive pollination activity of the bees, which support the maintenance of the biodiversity of the pollen-producing flora (santos 2009). beekeeping has been widespread in several brazilian regions as an activity that supplements the income of small farmers while it has a low environmental impact and high importance for the social and the economic aspects of sustainability (vieira & resende, 2007). brazilian vegetation is geographically diverse and is divided into five macro-regions. the north region includes the largest tropical rainforest in the country such as a huge drainage area through the amazon basin. the northeast reabstract a total of 61 dried bee pollen samples collected in four brazilian macro-regions within 19 municipalities were analyzed aiming to identify the sources used by apis mellifera (l.) for pollen production and to enable a more accurate product certification. sample preparation followed the standard methodology, including washing the pollen grains with ethanol, then with water and homogenising the sediment in a water/glycerine solution for microscopic observation. pollen counts included at least 500 pollen grains per sample. only six samples, presenting a unique species or pollen type comprising more than 90% of the pollen sum, were considered monofloral, including ambrosia sp., cecropia sp., eucalyptus sp., fabaceae, mimosa scabrella (benth.) spp. and schinus sp. pollen types. the most frequent pollen types of the heterofloral pollen batches, based on a counting limit of 45%, included anadenanthera sp., asteraceae, brassica sp., caesalpiniaceae, cocos nucifera (l.) sp., mimosa caesalpiniaefolia (benth.) sp., mimosa verrucosa (benth.) sp., and myrcia sp. pollen types. this result may be related to the great diversity of the brazilian flora contributing to heterofloral pollen loads and honeys. sociobiology an international journal on social insects 1 universidade federal do rio de janeiro, rio de janeiro, brazil. 2 universidade de são paulo, são paulo, brazil. 3 fundação oswaldo cruz, instituto oswaldo cruz, rio de janeiro, brazil research article bees article history edited by: isabel alves-dos-santos, usp brazil received 25 december 2012 initial acceptance 27 february 2013 final acceptance 06 march 2013 keywords palynology, bee flora, pollen batches, brazil corresponding author alex da silva de freitas universidade fed. do rio de janeiro laboratório de palinologia departamento de geologia, ccmn rio de janeiro, rj, brazil e-mail: alexsilfre@gmail.com gion occurs in the eastward “bulge” of the country where there is a semiarid interior been largely given over to lowdensity vegetation. the southeast region is mainly an upland area with prevalence of high species diversity in the vegetation of the atlantic forest. the south region is the smallest macro-region, been distinct because of its temperate climate and the presence of araucaria forest. in the midwest region there is a dry forest (“cerrado”) characterized by drier climate and vegetation resistant to prolonged periods without rain, besides the humid “pantanal” areas (rizzini 1997). for many insects, and particularly for bees, pollen is the main source of the proteins and lipids required by larvae. pollen is essential to the normal growth and development of all individuals in a colony and for the reproduction and maintenance of the colony (marchini et al. 2006). pollen grains are collected on the corbicula of the hind legs of the bees during field activity and are stored in hive cells, forming the “bee bread” (moreti et al. 2002). the increasing variety and availability of apiary derivatives in the brazilian sociobiology 60(1): 56-64 (2013) 57 market, and international interest in these products encourage research to characterize the botanical origin of different pollen types collected by bees using palynological analysis for quality control (luz et al. 2007). the botanical origin of bee pollen loads is of extreme importance to a greater quality control, enabling better commercial value for the manufactured products. almeida-muradian et al. (2005) emphasized the importance of this quality control in targeting the commercial production of pollen products, including the flowering plants of each region and a wide range of physicochemical bee pollen analyses. melissopalinological analysis is an important tool for increasing knowledge about the botanical origins of bee pollen. regional data were presented by barth & luz (1998) with regard to pollen collection inside mangrove areas, barth (2004) in a review, modro et al. (2007), luz et al. (2007) in a forested area of rio de janeiro state, melo et al. (2009) in são paulo state, barth et al. (2009), luz et al. (2010) in minas gerais state, barth et al. (2010) with regard to the technical processing of pollen loads, luz et al. (2011) in espírito santo state, boff et al. (2011) in mato grosso do sul state, novais et al. (2009); novais et al. (2010); dórea et al. (2010); santos (2011) in bahia state and barth et al. (2011) in regions of venezuela. the present study aimed to investigate the botanical and geographical origins of bee pollen collected in four brazilian macro-regions. this study aimed to provide also results that will contribute to improve the certification of this bee product and the preservation of the native vegetation. materials and methods a total of 61 dried pollen load samples of a. mellifera collected by traps in four brazilian macro-regions, were analyzed using pollen analysis (figure 1). the procedure followed the methodology proposed by barth et al. (2010) using two grams of each sample of dried bee pollen homogenate stirring in 70% ethanol. centrifuge tubes were filled to 13 ml, and the material remained at rest for 30 min or overnight and was then submitted to sonication for five minutes to dissociate the pollen grains. after centrifugation, the samples containing a large amount of oil were submitted twice to ethanol extraction. the obtained sediment was diluted in a 1:1 mixture of water/ glycerine for 30 min. one drop of this well-homogenised pollen grain suspension was applied to a microscope slide and covered with a 24x24 mm cover glass. the stock pollen suspension was kept in glycerine at room temperature. two microscope slides, sealed with nail-lack, were prepared and more than 500 pollen grains were counted from each sample considering the two slides. samples were observed using light and polarized light microscopy. pollen figure.1 map of the five brazilian macro-region displaying the studied area with the municipalities collection. 1. rio grande do norte; 2. sergipe; 3. bahia; 4. espírito santo; 5. são paulo; 6. santa catarina; 7. rio grande do sul; 8. distrito federal; 9. mato grosso. as freitas, vas arruda, lb almeida-muradian, om barth botanical profiles of pollen loads collected by apis mellifera58 classes usually followed those established by zander (louveaux et al. 1978) meaning honey analysis pp (predominant pollen > 45%), ap (accessory pollen 15-45%), ip (important pollen 3-15%). these classes were used for qualitative and quantitative analyses of bee pollen loads in the present paper. samples were classified as monofloral or heterofloral batches according to their pollen grain percentages. samples were considered monofloral when presenting more than 90% of a unique pollen type. barth (1989), roubik & moreno (1991) and moreti et al. (2002) were used to aid in pollen identification. pollen morphology in the asteraceae family is not genus specific and was presented at the family ranking asteraceae. results the results of the palynological analysis showed a wide variety of pollen types found in each brazilian macroregion (tables 1, 2, 3). six of the 61 samples were monofloral. the monofloral samples were found in two samples from the southeast, two from the south and two from the midwest (tables 2 and 3). northeast region (table 1) – pollen from cocos nucifera was present in all 19 samples analyzed, with a concentration above 3%. this pollen type was present as a dominant pollen in seven samples (>45%), as an accessory pollen in ten samples and as an isolate pollen in two samples throughout the three states of the northeast region. pollen types of mimosa caesalpiniaefolia. (sample sergipe-7), m. scabrella (samples bahia-2, -5), m. verrucosa (samples sergipe-5, -6, -8, -10, -11), and myrcia sp. (samples sergipe-2, -3, -11) also exhibited a dominant pollen frequency. southeast region (table 2) – the unique dried bee pollen sample from espírito santo state was a monofloral sample of an unidentified plant of the fabaceae family was only found in that one sample. nine samples from pariquera açu in são paulo state all came from cocos nucifera and were very similar. the ribeirão preto municipality presented dominant pollen grain percentages of m. caesalpiniaefolia in six samples (são paulo-10, -11, -12, -13, -14, -15). schinus sp. was the main pollen type in two samples (são paulo-16, -18) and anadenanthera sp. in one sample only (são paulo-19). the other two samples (são paulo-17, -20) exhibited no dominant pollen types but had in common the pollen types of anadenanthera sp., eucalyptus sp., myrcia sp., and cecropia sp. pollen grains of the persea sp. type (avocado tree) were detected in only one sample (são paulo-17). taubaté municipality exhibited the m. caesalpiniaefolia (samples são paulo-21, -23) and m. scabrella (são paulo-22, -24) pollen types as dominant in two samples each, with the latter including a monofloral sample (são paulo-24). south region (table 3) – all samples analyzed from fraiburgo municipality (santa catarina-1, -2, -3, -4, -5, -6) were very similar, presenting a high percentage of asteraceae pollen grains as well as amaranthus/chenopodiaceae, brassica sp. and euphorbiaceae pollen grains in several samples. a caesalpiniaceae pollen type was dominant in campos novos (santa catarina-7), and m. caesalpiniaefolia was dominant in serra catarinense (santa catarina-8). five samples were analyzed from rio grande do sul state. three of these samples showed eucalyptus sp. pollen dominance in the municipalities of arvorezinha (rio grande do sul-4), santana do livramento (rio grande do sul-2) and são gabriel (rio grande do sul-5). the ambrosia sp. pollen type occurred in a sample from santana do livramento (rio grande do sul-1), and the brassica sp. pollen type was found in a sample from cruz alta (rio grande do sul-3), both of which were classified as monofloral. midwest region (table 3) midwestern brazil was represented by four samples. the distrito federal sample showed a high percentage of the schinus sp. pollen type, which was considered monofloral. three samples from mato grosso showed cecropia sp. pollen dominance, one of which (mato grosso-2) was considered monofloral. the most frequent or characteristic pollen types in the brazilian macro-regions of the present study can be summarised as follows: cocos nucifera (n = 19/19) is a typical pollen product in the brazilian northeast region (n = 19) and, pollen types of m. caesalpiniaefolia (n = 3), m. scabrella (n = 8) and m. verrucosa (n = 7) were common. pollen grains of asteraceae species were poorly represented in the samples of this macro-region. the main pollen types of the southeast region (n = 25) were asteraceae and myrcia sp. (n = 11/25 of each), such as m. caesalpiniaefolia (n = 12/25). the characteristic pollen types of the south macro-region (n = 13) were asteraceae (n = 10/13) and brassica sp. (n = 8/13). in the few samples of the midwest macro-region (n = 4) cecropia sp. (n = 4) pollen was detected in all samples, including in one monofloral sample primarily of schinus sp. pollen (n = 1). discussion northeast region – coconut trees grow frequently on seashores along the northeastern brazilian coastline (pires et al. 2004) and are naturally spread throughout the beaches. these trees are considered an important pollen source in neotropical regions (biesmeijer et al. 1992). cocos nucifera pollen grains were well represented in all samples of the three northeast states (rio grande do norte, sergipe and bahia). the pollen of this species was associated with m. verrucosa (“espinheiro preto”), a typical plant throughout the brazilian northeast (sodré et al. 2007), and mainly in samples of the state of sergipe. in addition, m. caesalpiniaefolia (“sabiá”) sociobiology 60(1): 56-64 (2013) 59 was found in association with cocos nucifera. novais et al. (2009) and dórea et al. (2010) highlight other mimosacea and arecaceae species for the bahia state. m. caesalpiniaefolia has a natural occurrence in the semi-arid brazilian region (carvalho 2007, queiroz, 2009) and has been introduced in more humid regions (ribaski et al. 2003) and is widely used in the reforestation of degraded areas (lorenzi 1998). caesalpiniaceae has been reported by several authors (absy & kerr, 1977; absy et al. 1980; kerr et al. 1986/1987; imperatriz-fonseca 1989; ramalho 1990) as an important food resource for native bees in different brazilian states. monofloral pollen loads of m. caesalpiniaefolia have also been collected in the state of rio de janeiro (luz et al. 2007). therefore it may be considered that the pollen of cocos and mimosa is very important in the northeast brazilian region. southeast region the fabaceae family is often visited by bees in search of nectar and pollen for the maintenance of the hive (moreti et al. 2007). a monofloral pollen batch of a fabaceae species came from espírito santo state. this species may be wild or a crop species, and the pollen morphology of several related species similar (carreira et al. 1996; moreti et al. 2007). the mimosaceae family comprises a large number of species of the brazilian flora that are of extreme significance for pollen and honey production. one batch with predominant pollen of the m. scabrella pollen type was obtained in the state of são paulo. the pollen grain morphology of this specie is not characteristic of a unique plant species but rather, groups of species (barth 1989). based on a survey of neotropical region, ramalho et al. (1990) reported that the m. scabrella pollen type was commonly collected by a. mellifera. the numerous monofloral batches of the cocos nucifera pollen type obtained in the state of são paulo were probably derived from the extensive coconut plantations in the region. surprisingly, these pollen grains dominated all samples from the munipality of pariquera açu, são paulo state, indicating that coconut plantations are significant for beekeeper activities. the attractive flowering of the wild trees or shrubs of anadenanthera sp. is extremely important for beekeepers as because, in addition to pollen and honey harvesting, these flowering plants draw hungry bees away from crop plantations, as of passiflora sp. such as those pollinated by bumblebees, where bees are not welcome (monika barth, personal information). south region the ambrosia sp. pollen type was characteristic of the state of rio grande do sul (rs). the anemophilous pollen of this genus, dispersed by the wind, is considered allergenic in this region and is suspected to have induced hay fever in several patients (vergamini et al. 2006). according to lorenzi (2008), species of the ambrosia genus have been found in agricultural areas of southern brazil and are considered a weed species. this pollen type is visited by a variety of insects. a monofloral pollen batch of eucalyptus sp. was also obtained in rio grande do sul state, but this genus is common throughout the brazilian regions where it is cultivated. pollen analyses of honey have certified the presence of eucalyptus sp. (barth et al. 2005; bastos et al. 2004; luz et al. 2007). the pollen morphology of brassica sp. which is similar in several species may be derived from crops (brassica napus l.) in the south of brazil (rosa et al. 2011) or from wild species occurring in field vegetation (moreti et al. 2002). midwest region another monofloral batch of winddispersed pollen was cecropia sp. from the region of mato grosso do sul. cecropia is a widespread small tree in brazil and produces small pollen that is very attractive to bees (luz et al. 2007). this genus was also observed by boff et al. (2011) as an accessory pollen type in analyzed pollen loads from southern pantanal in the state of mato grosso do sul (ms). bees have a “preference” for schinus sp. pollen. the main species, s. terebinthifolius (l.), has a worldwide distribution in tropical regions. the present monofloral sample was obtained in the brazilian federal district. the pollen of this genus occurs as an accessory percentage of the total pollen samples, including in honey samples (carreira & jardim, 1994). this genus is characteristic of drier vegetation (smith et al. 2004) and is widely sought by bees, especially when other species are not in the flower (baggio 1988). all dominant pollen types were present as accessory pollen in numerous samples. in addition, six non-dominant pollen types were identified as accessory pollen in the present study including pollen types of anadenanthera sp., brassica sp., caesalpiniaceae, cocos nucifera, m. caesalpiniaefolia and myrcia sp. several plant species and genera showed very similar pollen morphology to that of myrcia sp. (barth 1972). this pollen type occurs in pollen batches of nearly all brazilian states and was also highlighted by barth (1989) as an accessory pollen type in honey samples from bahia. from the data presented here, the great floristic diversity in brazil is apparent, as shown through the main pollen types identified in bee pollen loads. thus, there is a need for a comprehensive and detailed botanical certification program to increase the commercial value of pollen products. acknowledgements we thank the conselho nacional de desenvolvimento científico e tecnológico (cnpq) and the fundação de amparo à pesquisa do estado de são paulo (fapesp) for financial support and scholarships to the three last authors. two anonymous reviwers provided valuable comments that improved the article. as freitas, vas arruda, lb almeida-muradian, om barth botanical profiles of pollen loads collected by apis mellifera60 table 1. the pollen types identified in the pollen load samples of a. mellifera collected in northeast macro-region of brazil and their main botanical origins, considering a frequency of more than 3%. (samples were considered monofloral when presenting more than 90% of a unique pollen type). municipality brazilian macro-region = northeast pollen analysis and pollen types predominant botanical origin (representative pollen types) rio grande do norte-1 (natal) pp: cocos nucifera (60.2%) ap: mimosa verrucosa (36.0%) heterofloral sample with primary contributions from cocos nucifera and mimosa verrucosa rio grande do norte-2 (grupo santa luzia) pp: cocos nucifera (47.2%) ap: eucalyptus (25.0%) ip: mimosa caesalpiniaefolia (6.4%), myrcia (5.2%), mimosa scabrella (3.7%), asteraceae (3.4%), richardia (3.2%) heterofloral sample with primary contributions from cocos nucifera and eucalyptus sergipe-1 (povoado brejão) ap: mimosa scabrella (42.1%), cocos nucifera (35.4%), myrcia (17.5%) heterofloral sample with primary contributions from mimosa scabrella, cocos nucifera and myrcia sergipe-2 (povoado brejão) pp: myrcia (67.0%) ip: cocos nucifera (3.5%) heterofloral sample with a primary contribution from myrcia sergipe-3 (povoado brejão) pp: myrcia (55.5%) ap: mimosa scabrella (25.3%), cocos nucifera (17.2%) heterofloral sample with primary contributions from myrcia, mimosa scabrella and cocos nucifera sergipe-4 (povoado brejão) pp: cocos nucifera (47.5%) ap: myrcia (23.2%), mimosa scabrella (23.2%) heterofloral sample with primary contributions from cocos nucifera, myrcia and mimosa scabrella sergipe-5 pp: mimosa verrucosa (50.7%) ap: cocos nucifera (41.1%) ip: poaceae (8.0%) heterofloral sample with primary contributions from mimosa verrucosa and cocos nucifera sergipe-6 ap: mimosa verrucosa (42.8%), cocos nucifera (35.4%), myrcia (16.3%) ip: tapirira (5.4%) heterofloral sample with primary contributions from mimosa verrucosa, cocos nucifera and myrcia sergipe-7 pp: mimosa caesalpiniaefolia (50.1%) ap: cocos nucifera (41.3%) ip: myrcia (5.7%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia and cocos nucifera sergipe-8 ap: mimosa verrucosa (39.7%), cocos nucifera (36.8%), mimosa caesalpiniaefolia (15.7%) ip: myrcia (7.8%) heterofloral sample with primary contributions from mimosa verrucosa, cocos nucifera and mimosa caesalpiniaefolia sergipe-9 ap: cocos nucifera (35.1%), brassica (20.3%), myrcia (18.6%), mimosa verrucosa (16.5%) ip: tapirira (9.4%) heterofloral sample with primary contributions from cocos nucifera, brassica, myrcia and mimosa verrucosa sergipe-10 pp: mimosa verrucosa (56.0%) ap: cocos nucifera (29.3%) ip: brassica (14.8%) heterofloral sample with primary contributions from mimosa verrucosa and cocos nucifera sergipe-11 pp: cocos nucifera (53.3%); ap: mimosa verrucosa (39.9%) ip: myrcia (3.8%) heterofloral sample with primary contributions from cocos nucifera and mimosa verrucosa bahia-1 (canavieiras) pp: cocos nucifera (67.6%) ap: eucalyptus (31.3%) heterofloral sample with primary contributions from cocos nucifera and eucalyptus bahia-2 (canavieiras) pp:mimosa scabrella (59.0%) ap: cocos nucifera (41.0%) heterofloral sample with primary contributions from mimosa scabrella and cocos nucifera bahia-3 (canavieiras) pp: cocos nucifera (76.3%) ap: mimosa scabrella (18.4%) ip: eucalyptus (5.3%) heterofloral sample with primary contributions from cocos nucifera and mimosa scabrella bahia-4 (canavieiras) pp: cocos nucifera (85.5%) ip: vernonia (6.9%) heterofloral sample with a primary contribution from cocos nucifera bahia-5 (ilhéus) pp: mimosa scabrella (58.3%) ap: cocos nucifera (32.7%) ip: asteraceae (6.7%) heterofloral sample with primary contributions from mimosa scabrella and cocos nucifera bahia-6 (ilhéus) pp: asteraceae (81.5%) ip: cecropia (6.4%), mimosa scabrella (5.3%), cocos nucifera (4.5%) heterofloral sample with a primary contribution from asteraceae pp = predominant pollen (> 45%); ap = accessory pollen (15-45%); ip = isolate pollen (3-15%). sociobiology 60(1): 56-64 (2013) 61 table 2. the pollen types identified in the pollen load samples of a. mellifera collected in southeast macro-region of brazil and their main botanical origins, considering a frequency of more than 3%. (samples were considered monofloral when presenting more than 90% of a unique pollen type.) municipality brazilian macro-region = southeast pollen analysis and pollen types predominant botanical origin (representative pollen types) espírito santo pp: fabaceaefaboideae (90.6%) ip: myrcia (4.7%), eucalyptus (3.1%) monofloral sample of a fabaceaefaboideae species são paulo-1 (pariquera açu) pp: cocos nucifera (62.5%) ap: asteraceae (15.6%) ip: poaceae (12.5%), melastomataceae (9.4%) heterofloral sample with primary contributions from cocos nucifera, and asteraceae são paulo-2 (pariquera açu) pp: cocos nucifera (50.0%) ap: myrcia (22.2%), mimosa caesalpiniaefolia (19,4%) ip: mimosa scabrella (8.3%) heterofloral sample with primary contributions from cocos nucifera, myrcia and mimosa caesalpiniaefolia são paulo-3 (pariquera açu) pp: cocos nucifera (55.3%) ap: mimosa caesalpiniaefolia (27.8%), myrcia (16.9%) heterofloral sample with primary contributions from cocos nucifera mimosa caesalpiniaefolia and myrcia são paulo-4 (pariquera açu) pp: cocos nucifera (47.5%) ap: myrcia (17.0%), cecropia (16.4%) ip: mimosa caesalpiniaefolia (13.8%) heterofloral sample with primary contributions from cocos nucifera, myrcia, and cecropia são paulo-5 (pariquera açu) pp: cocos nucifera (81.2%) ip: ilex (6.2%), sebastiania (6.2%), asteraceae (5.3%), myrcia (4.2%), heterofloral sample with a primary contribution of cocos nucifera são paulo-6 (pariquera açu) pp: cocos nucifera (78.3%) ip: ilex (11.6%), asteraceae (5.8%), myrcia (4.3%) heterofloral sample with a primary contribution from cocos nucifera são paulo/sp 7 (pariquera açu) pp: cocos nucifera (57.7%) ip: cecropia (12.1%), ilex (9.1%), eucalyptus (4.8%), asteraceae (7.0%), myrcia (5.8%), unidentified (3.0%) heterofloral sample with a primary contribution from cocos nucifera são paulo-8 (pariquera açu) pp: cocos nucifera (68.3%) ip: eucalyptus (7.6%), myrcia (3.8%) heterofloral sample with a primary contribution from cocos nucifera são paulo-9 (pariquera açu) pp: cocos nucifera (58.2%) ap: cecropia (23.9%), mimosa caesalpiniaefolia (17.9%) ip: myrcia (3.8%) heterofloral sample with primary contributions from cocos nucifera, cecropia and mimosa caesalpiniaefolia são paulo-10 (ribeirão preto) pp: mimosa caesalpiniaefolia (57.9%) ap: asteraceae (26.8%) ip: poaceae (14.6%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia and asteraceae são paulo-11 (ribeirão preto) pp: mimosa caesalpiniaefolia (60.3%) ap: asteraceae (17.6%), poaceae (16.6%), ip: myrcia (3.8%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia, asteraceae and poaceae são paulo-12 (ribeirão preto) pp: mimosa caesalpiniaefolia (70.2%) ap: poaceae (18.2%) ip: asteraceae (11.6%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia and poaceae são paulo-13 (ribeirão preto) pp: mimosa caesalpiniaefolia (51.3%) ap: mimosa scabrella (15.0%) ip: poaceae (12.6%), euphorbiaceae (6.6%), fabaceae-faboideae (4.2%), heterofloral sample with primary contributions from mimosa caesalpiniaefolia and mimosa scabrella são paulo-14 (ribeirão preto) pp: mimosa caesalpiniaefolia (72.1%) ip: poaceae (12.5%), mimosa scabrella, (6.3%), asteraceae (4.7%), euphorbiaceae (4.4%) heterofloral sample with a primary contribution from mimosa caesalpiniaefolia são paulo-15 (ribeirão preto) pp: mimosa caesalpiniaefolia (64.5%) ap: poaceae (19.3%) ip: asteraceae (12.9%), euphorbiaceae (3.2%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia and poaceae são paulo-16 (ribeirão preto) pp: schinus (58.6%) ap: alternanthera (34.6%), asteraceae (3.0%) heterofloral sample with primary contributions from schinus and alternanthera são paulo-17 (ribeirão preto) ap: persea (25.9%), myrcia (20.3%), anadenanthera (18.6%), eucalyptus (18.4%) ip: cecropia (6.9%), tapirira (4.7%), asteraceae (3.5%) heterofloral sample with primary contributions from persea, myrcia, anadenanthera and eucalyptus são paulo-18 (ribeirão preto) pp: schinus (82.3%) ap: vernonia (15.3%) heterofloral sample with primary contributions from schinus and vernonia são paulo-19 (ribeirão preto) pp: anadenanthera (82.5%) ip: eucalyptus (10.0%), cecropia (5.7%) heterofloral sample with a primary contribution from anadenanthera são paulo-20 (ribeirão preto) ap: cecropia (43.2%) anadenanthera (33.9%) ip: eucalyptus (12.3%), myrcia (9.3%) heterofloral sample with primary contributions from cecropia and anadenanthera são paulo-21 (taubaté) pp: mimosa caesalpiniaefolia (46.1%) ap: mimosa scabrella (37.2%) ip: syagrus (10.2%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia and mimosa scabrella são paulo-22 (taubaté) pd: mimosa scabrella (80.0%)ip: malpighiaceae (5.9%) heterofloral sample with a primary contribution from mimosa scabrella são paulo-23 (taubaté) ap: mimosa caesalpiniaefolia (43.1%), antigonon leptopus (23.2%), eucalyptus (15.4%) pi: poaceae (11.0%), cecropia (7.3%) heterofloral sample with primary contributions from mimosa caesalpiniaefolia, antigonon leptopus and eucalyptus são paulo-24 (taubaté) pp: mimosa scabrella (92.3%) ip: antigonon leptopus (4.9%) monofloral sample of mimosa scabrella pp = predominant pollen (> 45%); ap = accessory pollen (15-45%); ip = isolate pollen (3-15%) as freitas, vas arruda, lb almeida-muradian, om barth botanical profiles of pollen loads collected by apis mellifera62 table 3. the pollen types identified in the pollen load samples of a. mellifera collected in south and midwest macro-region of brazil and their main botanical origins, considering a frequency of more than 3%. (samples were considered monofloral when presenting more than 90% of a unique pollen type). municipality brazilian macro-region = south pollen analysis and pollen types predominant botanical origin (representative pollen types) santa catarina-1 (fraiburgo) pp: asteraceae (54.2%); ap: brassica (36.4%); ip: amaranthus/chenopodiaceae (3.3%); euphorbiaceae (3.6%) heterofloral sample with primary contributions from asteraceae and brassica santa catarina-2 (fraiburgo) pp: asteraceae (76.2%) ap: brassica (20.0%) heterofloral sample with primary contributions from asteraceae and brassica santa catarina-3 (fraiburgo) pp: asteraceae (56.3%) ap: brassica (35.6%) ip: euphorbiaceae (6.1%) heterofloral sample with primary contributions from asteraceae and brassica santa catarina-4 (fraiburgo) pp: asteraceae (71.8%) ap: brassica (19.7%) ip: euphorbiaceae (7.0%) heterofloral sample with primary contributions from asteraceae and brassica santa catarina-5 (fraiburgo) pp: asteraceae (56.7%) ap: brassica (23.3%) ip: euphorbiacae (10.0%), amaranthus/ chenopodiaceae (6.7%), poaceae (3.3%) heterofloral sample with primary contributions from asteraceae and brassica santa catarina-6 (fraiburgo) pp: asteraceae (50.0%) ap: euphorbiaceae (26.6%), brassica (18.7%) ip: amaranthus/chenopodiaceae (4.7%) heterofloral sample with primary contributions from asteraceae, brassica and euphorbiaceae santa catarina-7 (campos novos) pp: caesalpiniaceae (63.1%) ip: asteraceae (13.6%), sebastiania (9.5%), apiaceae (8.5%), myrcia (3.8%) heterofloral sample with a primary contribution from caesalpiniaceae santa catarina-8 (serra catarinense) pp: mimosa caesalpiniaefolia (85.2%) ip: asteraceae (11.8%) heterofloral sample with a primary contribution from mimosa caesalpiniaefolia rio grande do sul-1 (santana do livramento) pp: ambrosia (99.4%) monofloral sample of ambrosia rio grande do sul-2 (santana do livramento) pp: eucalyptus (93.0%) ip: asteraceae (7.0%); monofloral sample of eucalyptus rio grande do sul-3 (cruz alta) pp: brassica (57.4%) ap: eucalyptus (42.6%) heterofloral sample with primary contributions from brassica and eucalyptus rio grande do sul-4 (arvorezinha) pp: eucalyptus (49.9%) ap: eupatorium (24.5%) ip: onagraceae (10.1%), ilex (6.5%), asteraceae (4.6%), myrcia (4.1%) heterofloral sample with primary contributions from eucalyptus and eupatorium rio grande do sul-5 (são gabriel) pp: eucalyptus (64.6%) ap: brassica napus (35.3%) heterofloral sample with primary contributions from eucalyptus and brassica napus distrito federal pp: schinus (92.1%) ip: eucalyptus (4.8%), cecropia (3.2%) monofloral sample of schinus mato grosso-1 (sinop) pp: cecropia (66.6%) ap: euphorbiaceae (26.7%); ip: poaceae (6.7%) heterofloral sample with primary contributions from cecropia and euphorbiaceae mato grosso-2 (sinop) pp: cecropia (97.1%) monofloral sample of cecropia mato grosso-3 (sinop) pp: cecropia (51.3%) ap: poaceae (44.3%) heterofloral sample with primary contributions from cecropia and poaceae pp = predominant pollen (> 45%); ap = accessory pollen (15-45%); ip = isolate pollen (3-15%). references absy, m.l. & kerr, w.e. 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(2009). relação entre a composição nutricional e a origem floral de pólen. rev. inst. adolfo lutz 68: 346-353. modro, a.f.h., message, d., luz, c.f.p. & neto, j.a.a.m. (2007). composição e qualidade do pólen apícola coletado em minas gerais. pesq. agrop. bras. 42: 1058-1065. moreti, a.c.c.c., fonseca, t.c., rodriguez, a.p.m., monteiro-hara, a.c.b.a. & barth, o.m. (2007). pólen das principais plantas da família fabaceae com aptidão forrageira e interesse apícola. rev. bras. bioc. 5: 396-398. moreti, a.c.c.c., marchini, l.c., souza, v.c. & rodrigues, r.r. (2002). atlas do pólen de plantas apícolas. papel virtual editora, rio de janeiro. novais, j.s., lima, l.c.l & santos, f.a.r. (2009). botanical affinity of pollen harvested by apis mellifera l. in a semiarid area from bahia, brazil, grana 48: 224-234. novais, j.s., lima, l.c.l & santos, f.a.r. 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(2003). sabiá (mimosa caesalpiniaefolia) árvore de múltiplo uso no brasil. colombo/ comunicado técnico 1-4. rizzini, c.t. (1997). tratado de fitogeografia do brasil. âmbito cultural edições, rio de janeiro. roubik, d.w & moreno, j.e.p. (1991). pollen and spores of barro colorado island. monographs in systematics botany. missouri: missouri botanical garden, st. louis. rosa, a.s., blochtein, b. & lima, d.k. (2011). honey contribution to canola pollination in southern brazil. sci. agr. 68: 255-259. doi: 10.1590/s0103-90162011000200018 smith, n., mori, s.a., henderson, a., stevenson, d.wm. & heald, s.v. (2004). flowering plants of the neotropics. the new york botanical garden, princeton and oxford. santos, c.s. (2009). apicultura uma alternativa na busca do desenvolvimento sustentável. rev. verde 4: 1-6. santos, f. a. r. (2011). identificação botânica do pólen apícola. magistra 23: 5-9. sodré, g., marchini, l.c., carvalho, c.a.l. & moreti, a.c.c.c. (2007). pollen analysis in honey samples from the two main producing regions in the brazilian northeast. an. acad. bras. ciênc. 79: 381-388. doi: 10.1590/s000137652007000300003 vergamini, s.m, zoppas, b.c.d.a., valencia-barrera, r.m. & fernández-gonzález, d. (2006). dinâmica aeropalinológica de gramineae na cidade de caxias do sul, rs. rev. bras. alerg. imunopat. 29: 14-17. vieira, a. & resende, r.b. (2007). o desafio de associar recursos e integrar competências para promover uma apicultura integrada e sustentável. in: souza, d.c. (ed.). apicultura – manual do agente de desenvolvimento rural brasília: sebrae. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3427sociobiology 66(2): 279-286 (june, 2019) pollen spectrum and trophic niche width of melipona scutellaris latreille, 1811 (hymenoptera: apidae) in highly urbanized and industrialized sites introduction meliponiculture is practiced throughout latin america and honey is the main product marketed. the rearing of stingless bees offers valuable and profitable resources, especially for family agriculture, as meliponiculture is a sustainable activity of low-cost as well as beneficial to the environment, such as pollination and reproduction of many agricultural and native plant species (wilms & wiechers, 1997; venturieri et al., 2012; quezada-euán et al., 2018). the rearing of stingless bees stands out in the northeastern brazil, mainly of melipona scutellaris latreille (silva & paz, 2012). this species plays an important role in maintaining natural vegetation, as well as helping to generate income for family farming (alves et al., 2012). abstract the floristic composition of an environment is important to ensure the trophic niche of bee species. melipona scutellaris latreille, is a typical stingless bee of atlantic rainforest sites in northeastern brazil, a region widely established in meliponaries for honey and pollen production. m. scutellaris is reared (meliponiculture) in rural and urban areas, where the species depends on the availability of different plants for nectar and pollen collection. in this study, we estimated food niche width, equitativity, and similarity between different colonies of m. scutellaris in highly urbanized and industrialized sites of the metropolitan region of salvador, bahia state, brazil. we analyzed pollen spectrum of 58 honey samples from six meliponaries, during 12 months. we identified 111 pollen types distributed in 28 plant families. the fabaceae family showed the highest diversity in pollen types (33.33% of the total) and mimosa caesalpiniifolia was the most frequent pollen type, found in 100% of the samples. m. scutellaris concentrated its foraging activity on a few trophic resources (h’ = 2.69 and j’ = 0.01) indicating a few melittophilous plant species belonging to the genera eucalyptus, mimosa, protium, serjania and tapirira, should be managed on a regional scale to favor meliponiculture with this native bee species. sociobiology an international journal on social insects br andrade1, as nascimento1, el franco2, dr santos1, rmo alves1, mapc costa1, cal carvalho1 article history edited by cândida aguiar, uefs, brazil received 09 may 2018 initial acceptance 20 june 2018 final acceptance 14 february 2019 publication date 20 august 2019 keywords stingless bee, melissopalynology, ecological indices, flora. corresponding author brunelle ramos andrade centro de ciências agrárias, ambientais e biológicas universidade federal do recôncavo da bahia (ufrb) rua rui barbosa, nº 710, centro cep 44380-000, cruz das almas-ba, brasil. e-mail: brunelle_05@hotmail.com products of stingless bees, especially honey, have a wide use in different industries, namely food, cosmetic, pharmaceutical and medical (guez et al., 2013). compared to honey from africanized bees (apis mellifera l.), melipona species produce an economically valuable honey, often from mixed sources of native floral (camargo et al., 2017), with distinct physical, chemical and biological features (abadio finco et al., 2010). the pollen analysis (melissopalynology) is an important tool for quality control of honey and management of honeybees and stingless bees colonies, since it allows to identify pollen types and plant species that contribute to the diet of these bees (barth et al., 2013; estevinho et al., 2016; souza et al., 2018). the pollen analysis is also a valuable tool for ecological studies on trophic niche and food preferences of social bees 1 universidade federal do recôncavo da bahia, cruz das almas-ba, brazil 2 universidade federal do vale do são francisco, senhor do bonfim-ba, brazil research article bees br andrade et al. – pollen spectrum and trophic niche width280 (kleinert et al., 2012; hilgert-moreira et al., 2013). studies in brazil have evaluated the niche width of stingless bees in the atlantic and amazonian forests (wilms & wiechers, 1997; ramalho et al., 2007; ferreira & absy, 2017; freitas et al., 2017; matos & santos, 2017), in the caatinga (santos et al., 2013; novais et al., 2014), cerrado (calaça et al., 2018), dunes (viana & kleinert, 2006), rocky fields (franco et al., 2009), and agricultural areas (lucas et al., 2017). this study is the first carried out on m. scutellaris trophic niche in an urbanized and industrialized site. we analyzed the pollen spectrum of regional honey samples to identify the main nectar sources for this species of stingless bee and evaluate variation in food niche width and similarity between colonies and meliponaries. we also aimed to generate data for the management of the regional melitophilous flora and thus contribute to meliponiculture development. material and methods study site and samples the samples were collected from five colonies of meliponaries (n=6) in highly urbanized and industrialized sites in the metropolitan region of salvador (mrs), bahia, brazil: salvador (i): s’12°51’32.4’’ and w’038°27’9.9’’; salvador (ii): s’12º49’58.7’’ and w’038º22’27.4’’; salvador (iii): s’12º51’28.3’’ and w’038º21’54.3’’; simões filho (iv): s’12º43’55.5’’ and w’038º23’51.6’’; lauro de freitas (v): s’12º50’38.1’’ and w’038º21’12.1’’; and dias d’ávila (vi): s’12º32’28.0’’ and w’038º21’42.3’’). all meliponaries were inserted into a fragment of the disturbed atlantic forest, in areas near and far from urban-industrial development such as: the landfill of salvador, the petrochemical complex of camaçari and the industrial center of aratu. the honey samples were collected monthly, from june 2015 to may 2016, totalizing 58 honey samples. we collected samples (approximately 200 g) from closed honey pots with disposable syringe. each sample was placed in plastic tubes, conditioned in thermal bags and sent for laboratorial analyses. sample preparation the honey samples were prepared using the method of louveaux et al. (1978), following modifications proposed by jones and bryant jr. (2004). to process each sample, 10 g of honey was weighed and dissolved in 10 ml of distilled water and 50 ml of 95% ethyl alcohol (etoh). the mixture was centrifuged (3000 rpm for 5 min) and the supernatant liquid was discarded. after centrifugation, the pollen sediment was dehydrated in glacial acetic acid and then subjected to the acetolysis process proposed by erdtman (1960). the resulting pellet was mounted between slides and cover slips with glycerinated gelatin (kisser, 1935), sealed with paraffin for further identification and pollen grain counts under the microscope. to identify pollen types in the samples, images were captured using an olympus® e-330 digital camera coupled to the olympus cx41 microscope. afterwards, pollen grains were compared with the collection of references of the federal university of recôncavo of bahia and specialized literature, such as barth (1989), roubik and moreno (1991) and punt et al. (2007). pollen analyses after identifying the pollen types, we counted up to 1,000 pollen grains per sample. the frequency class of pollen types proposed by louveaux et al. (1978) was used: predominant pollen pp (> 45% of total grains), secondary pollen sp (16 to 45%), important minor pollen imp (3 to 15%) and minor pollen mp (< 3%). we then established the relative frequency (%) of each pollen type between samples using the equation: f = (nij∕nj) x 100 where, f = relative frequency of pollen type i in sample j; ni = number of pollen grains of the pollen type i in the sample; n = total number of pollen grains in sample j. ecological indices trophic niche width and similarity we calculated the trophic niche width using the diversity index of shannon (1948) (h’= σ pk x ln pk), where pk is the proportion of each pollen type in the m. scutellaris diet. equitativity (or evenness) of pollen types was calculated using the index of pielou (1969) (j’ = h’/ h’max). the richness index (s) was obtained from the total number of pollen types identified in the honey samples and similarity between samples with the jaccard coefficient of similarity (cs). the analyses were performed using past software (paleontological statistics), version 2.17c (hammer et al., 2001). results we identified 111 pollen types distributed in 28 botanical families and 25 types did not have their pollen affinity determined (tables 1 and 2). in all meliponaries in the mrs, fabaceae showed the highest number of pollen types in honey samples, representing 33.33% of the total, followed by anacardiaceae (8.97%), arecaceae (8.97%) and euphorbiaceae (6.41%) (table 1). the relative frequency of pollen types of the samples also reveals the importance of some plant species for the study site. in meliponary salvador (i), pollen types m. caesalpiniifolia, miconia and solanum type were the most frequent among the samples, present in 100%, 89% and 78% respectively. in salvador (ii), the most frequent pollen types were acacia, eucalyptus and m. caesalpiniifolia, present in 100% of the samples. for meliponary salvador (iii) m. sociobiology 66(2): 279-286 (june, 2019) 281 ta bl e 1. p ol le n ty pe s id en tifi ed in h on ey s am pl es b y m el ip on a sc ut el la ri s fr om h ig hl y ur ba ni ze d an d in du st ri al iz ed s ite s in th e m et ro po lit an r eg io n of s al va do r, b ah ia , b ra zi l. f am ily p ol le n t yp es m on th ly s am pl in gs 01 02 03 04 05 06 07 08 09 10 11 12 a na ca rd ia ce ae sp on di as sp (5 ) a na ca rd ia ce ae ta pi ri ra sp (5 ) pp (1 ); s p (6 ) pp (2 ,5 ); sp (6 ) b ur se ra ce ae p ro tiu m sp (3 ,6 ) sp (1 ,4 ) c om br et ac ea e te rm in al ia sp (6 ) e up ho rb ia ce ae e up ho rb ia ce ae t yp e sp (6 ) fa ba ce ae a ca ci a sp (6 ) sp (2 ,6 ) sp (2 ) fa ba ce ae m im os a ca es al pi ni ifo lia sp (1 ,2 ) pp (2 ,5 ); sp (6 ) sp (1 ,2 ) sp (1 ,3 ) pp (3 ,6 ); sp (4 ) pp (1 ,2 ,3 ,6 ); sp (4 ) pp (3 ,4 ,6 ); sp (1 ,2 ,5 ) pp (1 ); sp (3 ,4 ,6 ) sp (5 ,6 ) pp (5 ) pp (5 ) fa ba ce ae fa ba ce ae t yp e 2 sp (6 ) l au ra ce ae p er se a am er ic an a sp (3 ) m al pi gh ia ce ae b yr so ni m a sp (3 ) sp (4 ) m el as to m at ac ea e m ic on ia pp (1 ) m el as to m at ac ea e ti bo uc hi na pp (4 ); sp (2 ,6 ) sp (2 ,4 ) sp (3 ,4 ) pp (5 ); sp (2 ,4 ) pp (6 ); sp (5 ) sp (6 ) sp (6 ) sp (4 ) sp (6 ) m yr ta ce ae e uc al yp tu s sp (4 ) sp (4 ) sp (3 ) m yr ta ce ae e ug en ia sp (5 ) m yr ta ce ae m yr ci a sp (1 ) sp (6 ) sp (2 ) sp (1 ,5 ) sp (1 ) pp (3 ) m yr ta ce ae p si di um sp (5 ) sp (5 ) sp (5 ) sp (6 ) sp (1 ,2 ,5 ) sp (2 ,6 ) sp (6 ) r ub ia ce ae b or re ri a sp (2 ) sp (3 ) r ub ia ce ae m itr ac ar pu s sp (2 ) sa pi nd ac ea e c up an ia sp (3 ) sp (5 ) so la na ce ae so la nu m t yp e sp (1 ) sp (6 ) pp (1 ) pp (1 ,5 ) sp (5 ) sp (1 ,5 ) sp (5 ,6 ) sp (5 ) so la na ce ae so la nu m p an ic ul at um pp (3 ); sp (4 ) pp (3 ,4 ); sp (2 ) pp (2 ) sp (2 ,4 ) sp (2 ,4 ) sp (3 ) p p – pr ed om in an t p ol le n (p p> 45 % o f to ta l p ol le n gr ai n) ; s p – se co nd ar y po lle n (4 5% >s p> 16 % ); m el ip on ar y: 1 – s al va do r (i ), 2 – sa lv ad or ( ii ), 3 – sa lv ad or ( ii i) , 4 – s im õe s fi lh o (i v ), 5 – l au ro d e fr ei ta s (v ) an d 6 – d ia s d ’á vi la (v i) . br andrade et al. – pollen spectrum and trophic niche width282 caesalpiniifolia (90%), serjania (70%) and tapirira (60%) were the most frequent. in simões filho (iv), the most frequent pollen types among the samples were acacia type, leucaena type, m. caesalpiniifolia, psidium, s. paniculatum and tibouchina, all present in 100% of the samples. in meliponary lauro de freitas (v), pollen types m. caesalpiniifolia (83%) table 2. number of honey samples of melipona scutellaris per meliponary and associated pollen types and plant families, in highly urbanized and industrialized sites in the metropolitan region of salvador, bahia, brazil. meliponary ̸ sites geographical coordinates number of samples pollen types family identified not identified* identified salvador (i) s’12°51’32.4’’; w’038°27’9.9’’ 09 42 6 17 salvador (ii) s’12º49’58.7’’; w’038º22’27.4’’ 08 38 5 15 salvador (iii) s’12º51’28.3’’; w’038º21’54.3’’ 10 32 6 14 simões filho (iv) s’12º43’55.5’’; w’038º23’51.6’’ 09 35 4 16 lauro de freitas (v) s’12º50’38.1’’; w’038º21’12.1’’ 12 27 4 11 dias d’ávila (vi) s’12º32’28.0’’; w’038º21’42.3’’ 10 28 2 14 *botanical affinity not identified. and solanum type (75%) were the most frequent, while in dias d’ávila (vi), m. caesalpiniifolia and tibouchina were the most frequent types, both representing 90% of the total, followed by protium (80%). mimosa caesalpiniifolia was frequent in the samples of all meliponary evidencing the importance of this plant species for the reared of melipona scutellaris. analyzing the meliponaries separately, we found that values of the trophic niche width ranged from h’ = 2.02 to h’ = 2.40, while equitativity ranged from j ‘= 0.55 to j’ = 0.72 (table 3). the richness index (s) was higher than 25 pollen types for all meliponaries. the highest richness values of pollen types were found in salvador (ii) and salvador table 3. trophic niche width (h’), equitativity (j’) and pollen type richness (s) in honey samples of melipona scutellaris from highly urbanized and industrialized sites in the metropolitan region of salvador, bahia, brazil. indices meliponaries salvador (i) salvador (ii) salvador (iii) lauro de freitas (iv) simões filho (v) dias d´ávila (vi) s 42 38 32 35 27 28 h´ 2.04 2.33 2.28 2.02 2.22 2.40 j´ 0.55 0.64 0.66 0.61 0.62 0.72 (iii) meliponaries, with 42 and 38 pollen types (tables 1 and 2). similarity between meliponaries was often low and below 0.3. the highest similarity index (cs = 0.37) occurred between salvador (i) and lauro de freitas (v), while the lowest index (cs = 0.18) occurred between salvador (iii) and simões filho (iv) (table 4). discussion the pollen analysis of honey samples from the metropolitan region of salvador, bahia, showed that m. scutellaris visits many plant species, represented in the pollen spectrum of the samples studied. meliponaries farther from the urban perimeter (simões filho iv, lauro de freitas v and dias d’ávila) presented higher diversity of pollen types explored by m. scutellaris; conversely meliponaries in the urban environment (salvador i, ii and iii) showed lower diversity of pollen types (table 1 and 2). this is possibly related to landscape fragmentation and consequent reduction of flora in large urban centers (elmqvist et al., 2016). the families anacardiaceae, arecaceae, euphorbiaceae and fabaceae were the most representative in our study. other authors (oliveira et al., 2009; ferreira & absy, 2015; souza et al., 2015; matos & santos, 2017) have also reported that species of these plant groups are important for cultivation of stingless bees. plant species of these families should receive more attention by beekeepers and researchers of stingless bees to identify and conserve species of these taxonomic groups, important suppliers of trophic resources for stingless bee. the pollen types of fabaceae family (mimosoideae) was the most diverse in honey samples of mrs. this was similar to results reported in other studies comparing floral resources used by m. scutellaris (matos & santos, 2017; sociobiology 66(2): 279-286 (june, 2019) 283 lucas et al., 2017), melipona subnitida ducke (meliponini) and a. mellifera (almeida-murandian et al., 2013). in the samples of all meliponaries studied, pollen type m. caesalpiniifolia occurred as predominant pollen (pd). mimosa caesalpiniifolia benth is considered an important source of nectar and pollen for bees and a mellitophilous plant species (carvalho, 2007; lima et al., 2008) thus it has potential to be used in beekeeping in the metropolitan region of salvador, due to its presence in the pollen spectrum of the honeys analyzed. this reinforces the use and conservation of this species in apicultural pasture of the studied region and other municipalities of bahia, as well as in other brazilian states. among the most frequent pollen types in the honeys of mrs, eucalyptus, m. caesalpiniifolia, protium and serjania stand out, and some species belong to these genera described as nectariferous plants (nascimento et al., 2015; matos & santos, 2017). thus, the conservation of species of these genera which are part of diet of m. scutellaris diet, is important for the success of meliponiculture in the mrs, as conservation of these species can increase the range of the trophic niche of m. scutellaris in the studied region contributing to flora and fauna conservation in this urban environment. in addition to the plants diversity, floral resources can increase stability of these pollinators in a given environment (venjakob et al., 2016). tibouchina was another very frequent pollen type in the samples and was identified and classified in different frequency classes (table 1). according to pirani and cortopassilaurino (1993), many tibouchina species are pollen and nectar sources used by stingless bees (meliponini) and a. mellifera. the tibouchina genus (melastomataceae) is distributed mainly in tropical and subtropical regions of the americas and encompasses approximately 350 species, 129 native to brazil, which occur naturally in the states of bahia, minas gerais, rio de janeiro and são paulo. tibouchina granulosa (desv.) cogn. is the most common species of this genus in brazilian cities (agostini & sazima, 2003; pirani & cortopassi-laurino, 1993). an important fact regarding the use of species of the tibouchina genus by honeybees is its flowering period, which occurs practically throughout the year. the tibouchina pollen type was identified in honey samples (table 1), indicating that this pollen type corresponds to a species with potential for use by m. scutellaris colonies settled in these places, because these plants are considered components of mellitophilous plant species for the maintenance of colonies. the identification of the solanum type and s. paniculatum type in the pollen spectrum of the analyzed honey samples does not present contribution to nectar volume of honey composition. the presence of these pollen grains in honey is possibly due to an involuntary addition, considering that bees visit several plant species and pollen grains attached to their bodies can be deposited in honey pots along with the nectar. according to dórea et al. (2017), solanaceae species are frequently found in urban areas, and are used as pollen sources by solitary and social bees. pollen types solanum and s. paniculatum have also been found in the pollen spectrum of meliponini honey in recent studies, such as barth et al. (2013), nascimento et al. (2015), souza et al. (2015) as well as matos and santos (2017). the index richness (s) reflects the amount of plant species visited during the foraging activity of m. scutellaris. meliponary salvador (iii), although inserted into an urban perimeter, had the highest richness index (42 pollen types) (table 3), indicating that the colonies may have been more exposed to variations in flowering (ramalho et al., 2007). another factor to be considered is the abundance of plant individuals of the same blooming species visited by the bees (m. scutellaris) to collect trophic resources in the area surrounding the meliponary. the bees may choose to collect trophic resources in certain preferential floral sources or search for resources in higher number of species to supply their energy and protein needs (silva et al., 2007; filipiak et al., 2017). on the other hand, meliponary lauro de freitas, far from the urban perimeter, had the lowest richness index (27 pollen types), indicating that bees concentrated foraging on local floral resources available throughout the year, which sufficiently supplied their food requirements. environmental factors affect the flowering periods (freitas et al., 2013) and in the foraging activity of bees (nascimento et al., 2012; oliveira et al., 2012). oliveira et al. (2009) observed in the amazon region that the frequency of rains promoted a diversification of pollen types due to low flowering. in this sense, diversity of pollen types explored by m. scutellaris in the mrs also suffers direct influence of the climatic factors, besides the factors related to landscape changes in the urban environment. ssa (i)* ssa (ii) ssa (iii) lauro de freitas (iv) simões filho (v) dias d’ ávila (vi) ssa (i) 0.23 0.27 0.28 0.18 0.25 ssa (ii) 0.29 0.35 0.21 0.22 ssa (iii) 0.37 0.29 0.30 lauro de freitas (iv) 0.29 0.31 simões filho (v) 0.21 dias d’ávila (vi) *sample data by location are available in table 2. ssa = salvador table 4. similarity matrix (coefficient of similarity of jaccard, cs) between meliponaries based on pollen types identified in melipona scutellaris honey from highly urbanized and industrialized sites in the metropolitan region of salvador, bahia, brazil. br andrade et al. – pollen spectrum and trophic niche width284 the variation in trophic niche width and equitativity values for the samples of each site studied (table 3) may be related to the number of samples and reveals the importance of the sampling period, which shows variation of the trophic niche in a given period. the largest range of the trophic niche was recorded for samples from meliponaries salvador (ii and iii) and simões filho (table 3). in general, the trophic niche width values found in our study is similar to results recorded for other bee species (carvalho et al., 1999; aguiar et al., 2013; sabino et al., 2016). there was a diversity of pollen types and oscillation in m. scutellaris trophic niche width in the different sites (meliponaries). in addition, meliponaries with higher diversity in pollen types in their samples also had a greater trophic niche width. however, some pollen types were more representative among samples such as m. caesalpiniifolia and tibouchina, indicative of relative concentration in the collection of floral resources by m. scutellaris in some plant species, which can be considered key species for the diet of these bees in the studied site. the low similarity in the resources explored by m. scutellaris in the mrs may have been influenced by the diversity of plants visited by the bees in different sites, thus evidencing the generalist foraging behavior of m. scutellaris, as described by ramalho et al. (2007). vegetation with several floral species is a determinant factor in the foraging activity of bees, when they do not have floral selectivity or preferences to resources in the environment (ramalho et al., 2007; vaudo et al., 2015; adgaba et al., 2017). velikova et al. (2000) report that a high density of resources in tropical habitats leads stingless bees (melipona) to forage in distances shorter than other bees do, with a maximum radius of 500 m. therefore, the pollen analysis of honey revealed diversity of plant species that can be explored by m. scutellaris for honey production. the contribution of the fabaceae family was remarkable in the results, as this family was the most frequent with higher richness of pollen types in the honey samples produced by m. scutellaris from mrs, showing that species of this family can be considered key plants for local meliponiculture, for example, m. caesalpiniifolia. the trophic niche width reveals that m. scutellaris bees explored various floral resources available in the environment, despite showing preference for m. caesalpiniifolia. based on the ecological indices of all meliponaries, m. scutellaris concentrated its foraging activity on a few trophic resources. therefore, we suggest the management on a regional scale of a few melittophilous plant species, belonging to the genus eucalyptus, m. caesalpiniifolia, protium, serjania and tapirira, to favor meliponiculture with this native bee species. acknowledgements this study was financed in part by the “coordenação de aperfeiçoamento de pessoal de nível superior brasil” (capes) finance code 001 and by the “fundação de amparo à pesquisa do estado da bahia” (fapesb) finance code pam0004/2014. we thank the “conselho nacional de desenvolvimento científico e tecnológico” (cnpq) by fellowship for calc (number 305885/2017-0). as nascimento wishes to thank capes for the postdoctoral fellowship pnpd20130760. references abadio finco, f.d.b., moura, l.l. & silva, i.g. 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(1997). floral resource partitioning between native melipona bees and the introduced africanized honey bee in the brazilian atlantic rain forest. apidologie, 28: 339-355. doi: 10.1051/apido:19970602 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i2.2844sociobiology 65(2): 291-298 (june, 2018) influence of food resource size on the foraging behavior of nasutitermes corniger (motschulsky) introduction termite foraging is a group activity composed of individual actions integrated with patterns organized towards a new food source, which involves communicating the location of the source to other members of the colony using chemical and tactile signals that stimulate the foragers to leave the nest and guide them to the discovered food (traniello & busher, 1985; traniello & leuthold, 2000; costa-leonardo, 2002; andara et al., 2004). this activity comprises recruitment and aggregation events of individuals that can change over time and, therefore, is a dynamic process governed by the nutritional requirements of the colony and quantity and/or quality of available food resources, abstract in general, termite foraging can be affected by physical and chemical factors linked to food. this study investigated if the wood length of eucalyptus grandis w. hill ex maiden, as a food resource, influences the behavior of foraging events of nasutitermes corniger (motschulsky). nests with mature and active colonies were collected in the field and transferred to glass cubes connected to a test arena under laboratory conditions. wooden blocks of e. grandis, with a 2.5 x 2.0 cm rectangular cross section, were offered to termites in three different lengths: 5, 10 and 15 cm. each test was repeated with 20 nests and lasted 60 minutes, when the following behavioral events and their duration were observed: initial exploration, initial recruitment and mass recruitment. at the end of each test, the quantities of termites (total, workers and soldiers) and gnawing workers were determined. the results show that longer blocks favored a higher occurrence of exploration and initial recruitment. however, the highest mass recruitment occurred with the 10 cm blocks. the length of the wood influenced the total number of termites recruited and gnawing workers; both were highest for the 10 cm blocks. there was no significant difference in relation to exploration time of the blocks and number of workers and soldiers recruited. therefore, we conclude that wood length is a factor that can affect n. corniger foraging. sociobiology an international journal on social insects ts souza1, vs gazal1,2, vj fernandes1, acc oliveira3, el aguiar-menezes1,2 article history edited by alexandre vasconcellos, ufpb, brazil received 11 january 2018 initial acceptance 19 march 2018 final acceptance 04 april 2018 publication date 09 july 2018 keywords arboreal termites, nasutitermitinae, eucalyptus grandis, foraging behavior, recruitment pattern. corresponding author thiago sampaio de souza postgraduate program in phytosanitary and applied biotechnology federal rural university of rio de janeiro rodovia br 465, km 7, bairro ufrrj zip code: 23897-000, seropédica rio de janeiro, brasil. e-mail: thiagosampaio.agro@gmail.com as observed in some species of the genus nasutitermes dudley (traniello & busher, 1985; traniello & leuthold, 2000; andara et al., 2004; gazal et al., 2014a, b). the arboreal termite nasutitermes corniger (motschulsky) is widely distributed in meso-america, from southern mexico to panama, and south america (atkinson & adams, 1997; torales, 2002; constantino, 2002; scheffrahn et al., 2005). in the last decades, n. corniger has become of great economic importance due to increasing reports of damage to residences in various brazilian and argentinian cities, which have confirmed its status as a pest (mill, 1991; menezes et al., 2000; constantino, 2002; costa-leonardo, 2002; fontes & milano, 2002; torales, 2002; albuquerque et al., 2012). 1 programa de pós-graduação em fitossanidade e biotecnologia aplicada, universidade federal rural do rio de janeiro (ufrrj), seropédica-rj, brazil 2 departamento entomologia e fitopatologia, instituto de ciências biológicas e da saúde, universidade federal rural do rio de janeiro (ufrrj), seropédica-rj, brazil 3 graduação em agronomia, universidade federal rural do rio de janeiro (ufrrj), seropédica-rj, brazil research article termites ts souza, vs gazal, vj fernandes, acc oliveira, el aguiar-menezes – termite recruitment affected by food size292 as urban pests, nasutitermes termites have been controlled using chemical insecticides on the nests or by removing the nests (united nations environment programme [unep], 2000; gerozisis et al., 2008), which are often high up in trees and difficult to access or sometimes impossible to locate and only the tunnels on walls of infested buildings are visible (menezes et al., 2000; fontes & millano, 2002). new technologies for termite control, which are more environmentally friendly, include a toxic bait system that is efficient for subterranean termites, especially against coptotermes formosanus shiraki and reticulitermes flavipes (kollar) (rhinotermitidae); however, the effectiveness of this system is still unknown for arboreal termites (su et al., 1995; united nations environment programme [unep], 2000; lee, 2002; su, 2002; potter, 2004; dow agrosciences, 2013). in this system, particularly in the sentricon™ colony elimination system, two pieces of untreated wood of equal size (approximately 2 cm x 20 cm) are placed in a tube-shaped artefact in the soil to monitor the insects. when the insects are detected on the wood, the pieces are substituted with bait impregnated with insecticide that inhibits insect growth, such as hexaflumuron (potter, 2004; ogg et al., 2006). su et al. (1995) described a prototype of a monitoring station that uses wooden pieces of picea sp. (pinaceae) that are approximately 2.5 x 4.0 x 28 cm and baited with hexaflumuron, which was found to be efficient at controlling subterranean termite colonies near buildings in florida. however, the lack of information about the mechanisms of food finding by termites, particularly for recruitment and orientation behavior, limits the use of a toxic bait system for nasutitermes (waller & la fage, 1987). despite the increasing importance of n. corniger as a pest, little is known about its foraging behavior in relation to exploration of food resources; although, there is a record that n. corniger selects wood based on the species and that it prefers the wood of eucalyptus grandis w. hill ex maiden (myrtaceae) more than pinus elliottii engelm (pinaceae) (gazal et al., 2010). in addition, the degree of decomposition (gazal et al., 2012) and chemical stimuli of wood (gazal et al., 2014a) also influence attraction, as well as substances of the salivary gland of n. corniger workers, which can have an aggregation or arrestant effect, and possibly pheromone substances in the feces that aid this species in the exploratory orientation of cellulosic substrates (gazal et al., 2014b). other factors can also affect the selection of a food resource by xylophagous termites, such as size, shape, volume and position of the food, surface area of the wood, presence of chemical substances in the food that can act as nutrients or allelochemicals, and density, hardness and humidity of the wood (usher & ocloo, 1974; howick, 1975; waller & la fage, 1987; gerozisis et al., 2008). in relation to size of the food resource, certain xylophagous termites prefer large logs or trees and others like small branches (wood, 1978). field tests conducted in ghana demonstrated that c. formosanus prefers to attack pieces of wood that have smaller volumes and larger surface areas (usher & ocloo, 1974; waller & la fage, 1987). in the laboratory, howick (1975) evaluated the preference of three termite species for different lengths of eucalyptus regnans f. muell. wood (with the same shape), which varied from 20 to 100 mm, and observed that coptotermes acinacicormis (froggatt) (rhinotermitidae) and nasutitermes exitiosus (hill) (termitidae) prefer longer pieces of eucalyptus, unlike mastotermes darwiniensis (froggatt) (mastotermitidae). cornelius and osbrink (2001) observed the effect of wooden pieces of picea sp., which had the same dimensions in cross section (3.5 x 1 cm) but varied in length (4 and 11 cm), on two species of subterranean termite and concluded that r. flavipes consumption was much higher for the longer pieces and that c. formosanus consumption was not affected by this factor. with the goal of selecting e. grandis wood of adequate size to use as monitoring bait for n. corniger, we verified if this physical factor influences the foraging behavior of this termite. material and methods collection of termite nests nests of n. corniger that had active and mature colonies (with the presence of winged individuals) and were 40 cm wide and 60 cm tall were removed from trees in the park parque frei leão vellozzo between december 2016 and may 2017. the park is an atlantic forest reserve maintained by the universidade federal do rio de janeiro (ufrj) that is located on the catalão peninsula in the municipality of rio de janeiro, rj, brazil (22°50’44”s, 43°13’19”w). taxonomic identification was made using the nest architecture described by thorne (1981) and collected soldiers (scheffrahn et al., 2005). the nests were collected in black, 100-liter plastic bags and placed in cardboard boxes to avoid them from being damaged during transport to the laboratory at the centro integrado de manejo de pragas (cimp) of the departamento de entomologia e fitopatologia at universidade federal rural do rio de janeiro (ufrrj), in the municipality of seropédica, rj, brazil. termite maintenance in the laboratory in the laboratory each collected nest was placed in a transparent glass cube (50.0 cm x 50.0 cm x 60.0 cm tall), supported on a styrofoam plate, containing a 5.0 cm layer of sterilized sand (fig 1). the termites had free access to the foraging arena through a silicone hose (ø = 8.0 mm; length = 10 cm) connected to a pvc pipe (black) inserted in the exit hole of the cube. each arena consisted of a glass plate bottom (50.0 x 40.0 cm) and perimeter wall (5.0 cm tall). the arena was placed on an acrylic tube (ø = 10.0 cm and length = 20.0 cm) so that the exit of the cube was at the same height as the top of the arena wall (gazal et al., 2010). to allow the termites to access the foraging arena, a glass ramp was connected from the end of the silicone hose to the arena. the ramp consisted sociobiology 65(2): 291-298 (june, 2018) 293 of two, transparent, glass plates (4.0 cm x 4.0 cm and 6.0 cm x 4.0 cm) fixed together with adhesive epoxy (durepoxi®); the longer plate was placed parallel to the termite exit. termites were prevented from escaping by placing transparent adhesive tape (5 cm wide) on the upper edges of the walls of the cube; the adhesive part was facing inward. pieces of moistened pinus sp. wood were placed in the arena as food. nearby, a pet bottle cap was placed that contained water to maintain the humidity of the foraging arena. the nests were maintained in a room, at a temperature of 25 ± 5ºc, relative humidity of 80 ± 5% and photoperiod of 10:14 hours (light:dark), at cimp/ufrrj. the pieces of wood and the sand in each box were moistened with distilled water each day. the experiments were conducted a week after the nests were brought to the laboratory. termite foraging arena setup the tests were conducted in the foraging arenas based on the methodology described by gazal et al. (2010). thirty minutes before the start of each test, the connection between the nest and arena was blocked with hydrophilic cotton to impede the termites from accessing the arena. then, the food was removed from the arena and replaced with the treatments (eucalyptus blocks that were 5, 10 and 15 cm long) on glass plates (5.0 x 4.0 cm, 10.0 x 4.0 cm and 15.0 x 4.0 cm, respectively). the treatments were placed in a situation of choice and equidistant from the termite access point to the arena; that is, the blocks were inside the arena, 19 cm from the base of the access ramp. for each test, the position of the treatments in the arena was randomized. fig 1. cube, for the colonies of nasutitermes corniger, connected to the foraging arena. ts souza, vs gazal, vj fernandes, acc oliveira, el aguiar-menezes – termite recruitment affected by food size294 bioassay three lengths of blocks of e. grandis wood (heartwood with 6 year old and 14% of humidity), the same size in cross section (2.5 cm x 2.0 cm), were tested: 5 cm, 10 cm and 15 cm. each test lasted 60 minutes and started when the access to the arena was reopened. the tests were conducted with 20 termite nests. during the test, the occurrence time of the following behavioral events was recorded with the id, of a chronometer (traniello, 1981; gazal ii, 2010): initial exploration (random arrival of the first soldier to the treatments); initial recruitment (arrival of the first worker); mass recruitment, which was when there was a continuous flow of workers to the substrate, demarcated by a trail on the glass plate of feces (mass arrival of workers to the treatments). the time to each behavioral event was measured, with the id, of a chronometer, from the beginning of the test to the corresponding event. the occurrence percentages of these events, for each treatment, were calculated by the total number of blocks with the occurrence event divided by the total number of blocks available in each treatment (n = 20), multiplied by 100. after 60 minutes of observation, the glass plates with the wood and respective recruited termites were removed. the number of termites present on each treatment, the number of termites (soldiers and workers) recruited to each plate of glass+treatment, and the number of gnawing workers (chewing on the wood) present on the treatments were recorded. statistical analysis the experimental design was of randomized blocks, where each nest represents a block, using 20 nests (20 blocks). the following dependent variables used to evaluate each treatment were: percentage of occurrence of each behavioral event (initial exploration, initial recruitment and mass recruitment) along the 20 nests tested, total number of termites recruited, time (in minutes) until the occurrence of each behavioral event, number of soldiers recruited, number of workers recruited, and number of workers gnawing wood. percentage data were obtained by summing the occurrence of the behavioral event in each repetition divided by the total number of repetitions (n = 20). the occurrence percentages of each behavioral event, for each treatment, were not transformed and compared using a chi-squared test. due to the absence of a normal distribution, the time data for each behavioral event and number of recruited workers were compared using the kruskal-wallis test (p < 0.05). the total number of recruited termites, the number of recruited soldiers and the number of gnawing workers met the assumptions of the analysis of variance (kolmogorov-smirnov and lilliefors at 5%) and was analyzed using anova and the averages were compared using tukey’s hsd test (p < 0.05). the data were analyzed by statistica® 10.0 and biostat® 5.3 programs. results nasutitermes corniger exhibited the behavior of initial exploration, initial recruitment and mass recruitment for all sizes of the e. grandis wood. however, the occurrence of initial exploration of the 10 and 15 cm blocks of wood was higher than the 5 cm blocks (test χ2 = 13.13; df = 2; p < 0.01). for the initial recruitment of the n. corniger workers, a higher occurrence for the 10 and 15 cm blocks was also observed when compared to the 5 cm blocks (test χ2 = 4.18; df = 2; p < 0.05). on the other hand, for mass recruitment of n. corniger workers the occurrence was higher for the 10 cm blocks (13/20) than the 5 cm (7/20) and 15 cm (6/20) blocks. for the 5 and 15 cm blocks, mass recruitment was similar (test χ2 = 16.82; df = 2; p < 0.01). the times for initial exploration, initial recruitment and mass recruitment of workers were not significantly different among the treatments (table 1). on average, initial exploration lasted 8.8 ± 0.5 min after the beginning of the test, initial recruitment lasted 17.7 ± 0.6 min, and mass recruitment lasted 34.9 ± 1.1 min. length of wood (cm) behavioral event1 initial exploration (min) initial recruitment (min) mass recruitment (min) 5.0 x 2.5 x 2.0 5.0 ± 0.4 a 10.5 ± 0.4 a 25.0 ± 1.1 a 10.0 x 2.5 x 2,0 11.0 ± 0.6 a 19.5 ± 0.5 a 42.4 ± 1.0 a 15.0 x 2.5 x 2.0 10.4 ± 0.6 a 23.1 ± 0.8 a 37.3 ± 1.2 a 1averages followed by the same letter in the column did not differ from each other by the kruskal-wallis test, p <0.05. table 1. elapsed time (average ± sd) from the beginning of the test to the occurrence of initial exploration, initial recruitment and mass recruitment exhibited by the workers of nasutitermes corniger (n = 20) on the wood of eucalyptus grandis offered in three lengths in a situation of choice during 60 minutes of observation under laboratory conditions. the total number of termites recruited to the 10 cm blocks (183.4 ± 6.2) was higher than the number for the 5 cm blocks (73.0 ± 8.6). however, the 15 cm blocks (111.2 ± 5.3) exhibited a recruitment of termites that was similar to the 5 cm blocks (f2.48 = 3.3; p < 0.05; fig 2). the total number of recruited soldiers to the e. grandis wood did not differ between the 10 cm (35.1 ± 1.6), 15 cm (30.2 ± 1.5) and 5 cm (16.8 ± 1.2) (f2.48 = 2.2; n.s.) blocks. the total number of recruited workers to the e. grandis wood for the 10 cm (148.4 ± 5.1), 15 cm (81.0 ± 4.2) and 5 cm (55.7 ± 7.3) blocks was similar (kruskal-wallis h2.48 = 4.7, n.s.). the total number of workers gnawing on the 10 cm (144.7 ± 4.9) e. grandis blocks was higher than the 5 cm (54.7 ± 7.0) blocks. however, for the wood that was 15 cm long the n. corniger workers exhibited a gnawing behavior that was similar to the 5 cm and 10 cm blocks (77.6 ± 4.1) (f2.48 = 3.6; p < 0.05; fig 3). sociobiology 65(2): 291-298 (june, 2018) 295 discussion independent of the size of the e. grandis wood, the three behavioral events (initial exploration, initial recruitment and mass recruitment of workers) were conducted by n. corniger while foraging for this food resource under laboratory conditions, which corroborates observations made by other authors for this species and other species of the genus (traniello, 1981; traniello & busher, 1985; arab & issa, 2000; gazal et al., 2010). in the study, mass recruitment of workers and some soldiers to the eucalyptus blocks occurred, which indicates the blocks were recognized as food, seeing that, according to traniello (1981), this behavioral event is only triggered when the food source is appropriate for consumption. in relation to the length of the blocks of e. grandis, initial exploration and initial recruitment of workers were more intense for the 10 cm (medium length) and 15 cm (longest) pieces, which suggests that these sizes of wood represent, at the same distance, more attractive food resources for n. corniger. it is possible that the individuals were directed by a higher concentration of volatiles released by the wood, but this chemical stimulus needs to be further investigated. mass recruitment of workers was greater for the medium length wood (10 cm), which allows us to infer that, after the termites begin to explore this food resource, discrimination occurs during the third stage of foraging. termites can measure the volume of a food source in a number of ways. for smaller food sources on the ground, some individuals need to walk or establish trails throughout the substrate so they can perceive the volume, since there is a difference in the threshold of perception for these insects in relation to variation in food size (lenz, 1994). howick (1975) studied how n. exitiosus consumed wooden blocks of e. regnans, which differed in length (20 to 100 x 1.5 x 2.5 mm), and found that consumption was greater as the length of the blocks increased. esenther (1970) and french et al. (1986) observed that r. flavipes modified its rate of consumption according to the size of the available food. studies of subterranean termites conducted by waller (1988; 1991) and lenz (1994) showed that the larger the food source, the greater the consumption, independent of the number of termites present. however, in the present work, we found that n. corniger clearly discriminated between the distinct sizes of wood, which was based on differences in total recruitment of termites and the number of gnawing workers. termite foraging is a group activity and composed of unified individual actions mediated by chemical and tactile stimuli (traniello & leuthold, 2000; costa-leonardo, 2002). the tactile stimuli come from the edges of solid objects present along the trail and of the food, and help the termites establish the trail of the colony to the food source (swoboda & miller, 2004). in the case of drywood termites, some species produce acoustic stimuli by drumming their head against the substrate or shaking their body as a mechanism to attract foragers and to assess the size of the food source (evans et al., 2005; 2007). hedlund and henderson (1999) verified that for c. formosanus the larger the size of the food source, the greater the consumption. in the present work, the shortest block (5 cm) had the lowest number of gnawing workers when compared to the medium length (10 cm) block; although, for the longest block (15 cm), the number of feeding workers was equivalent to the 5-cm block. this suggests that, for n. corniger, size is a factor that influences the exploration of a food source. however, apparently, starting at a certain size of the food source, the attributes responsible for recruitment would be reduced. on the other hand, the recruitment of cryptotermes domesticus (haviland) (kalotermitidae) workers is greater for smaller pieces of wood, a preference that could be related to food competition (evans et al., 2005). experiments conducted with the arboreal termites microcerotermes turneri (froggatt) (termitidae), nasutitermes graveoleus (hill) (termitidae) and fig 2. number of termites of nasutitermes corniger recruited (average ± sd) after 60 minutes of the test (n = 20) of three lengths of eucalyptus grandis wood: 1) 5.0 x 2.5 x 2.0 cm (size 1); 2) 10.0 x 2.5 x 2.0 cm (size 2); and 3) 15.0 x 2.5 x 2.0 cm (size 3). different letters indicate a significant difference between the treatments based on tukey’s hsd test, p < 0.05. fig 3. number of feeding nasutitermes corniger workers (average ± sd) after 60 min. of the test (n=20) for the wooden blocks eucalyptus grandis: 1) 5.0 x 2.5 x 2.0 cm (size 1); 2) 10.0 x 2.5 x 2.0 cm (size 2); and 3) 15.0 x 2.5 x 2.0 cm (size 3). different letters indicate a significant difference between the treatments based on tukey’s hsd test, p < 0.05. ts souza, vs gazal, vj fernandes, acc oliveira, el aguiar-menezes – termite recruitment affected by food size296 nasutitermes walkeri (hill) (termitidae), and two lengths of wood (2.5 cm and 10 cm), found that the three species preferred the longer pieces (gerozisis et al., 2008). in general, the present study found that wood of distinct sizes, which are potential food sources, present differences in attractiveness and can be discriminated by n. corniger. this discrimination occurs during the process of exploring the resource through a preference mechanism that results in differences in the total number of termites recruited and the number of workers feeding on the wood. wood size could play an important role in determining the food preference of n. corniger. future research should focus on quantifying this possible importance, as well as the role of the number (quantity) of wooden pieces in relation to n. corniger foraging. however, additional observations made while collecting the nests found that n. corniger forages in tunnels built on and a few centimeters below the soil surface, which allows us to infer that buried pieces of eucalyptus wood could be used as attractive bait. in addition, fontes and millano (2002) cite that, similar to subterranean termites, n. corniger can infest buildings through the soil. thus, this bait could aid in surveys of this species under field conditions, as occurs for some subterranean species, such as coptotermes gestroi (wasmann) (santos et al., 2010), and could also be used in monitoring stations in a toxic bait system to detect this termite in urban centers. based on the results of the laboratory tests, 10 cm pieces would be the most adequate size for this because they attracted the greatest mass recruitment. however, more studies are needed to support these hypotheses, since in addition to foraging behavior inherent to the species, other factors might interfere with termite foraging, such as edaphoclimatic conditions, presence of other food sources and predators, soil structure and humidity, and level of soil disturbance (hu & appel, 2004; souza et al., 2009; santos et al., 2010; dow agrosciences, 2013). acknowledgments we thank capes (coordenação de aperfeiçoamento de pessoal de nível superior) for the magister science scholarship to the first author. thanks also to angela iaffe (agronomic engineer of the horto florestal at ufrj) for acting as an intermediary with parque frei leão vellozzo so the termite colonies could be collected, flávio (employee of the staff of the park), the divisão de segurança (diseg) at ufrj, prof. joão vicente de figueiredo latorraca for allowing to use of the carpentry at the departamento de produtos florestais (dpf) at ufrrj, and we are also grateful to its carpentry staff. references albuquerque, a.c., matias, g.r.r.s., couto, a.a.v.o., oliveira, m.a.p. & vasconcellos, a. 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(1974). an investigation of stake size and shape in “graveyard” fields tests for termite resistance. ts souza, vs gazal, vj fernandes, acc oliveira, el aguiar-menezes – termite recruitment affected by food size298 journal of the institute of wood science, 9: 32-36. waller, d.a. (1988). host selection in subterranean termites: factors affecting choice (isoptera: rhinotermitidae). sociobiology, 14: 5-13. waller, d.a. (1991). feeding by reticulitermes spp. sociobiology, 19: 91-99. waller, d.a. & la fage, j.p. (1987). nutritional ecology of termites. in f. slansky jr. & j.g. rodriguez (eds.), nutritional ecology of insects, mites, spiders, and related invertebrates (pp. 487-532). new york: john wiley & sons. wood, t.g. (1978). food and feeding habits of termites. in m.v. brian (ed.), production ecology of ants and termites (pp. 55-80). london: cambridge university press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 11-19 (2013) tending-ants increase survivorship and reproductive success of calloconophora pugionata drietch (hemiptera, membracidae), a trophobiont herbivore of myrcia obovata o.berg (myrtales, myrtaceae) r fagundes1, 2, sp ribeiro2, k del-claro1 introduction nesting sites or food rewards provided by some plant species cause ants to protect it against herbivore damage (oliveira & freitas, 2004; rosumek et al., 2009; nahas et al., 2012). ant defense is a remarkable strategy and affects herbivore density on vegetation (janzen, 1966; del-claro et al., 1996; rosumek et al., 2009; nascimento & del-claro, 2010). these herbivores can be repelled or even predated by ants, and consequently are deprived of shelter, mates, food resources, nesting and oviposition sites (crutsinger & sanders, 2005; rosumek et al., 2009). nevertheless, some herbivores (sternorrhyncha, auchenorrhyncha and larvae of various lepidoptera species) are able to take advantage of ant presence and increase survival and reproduction rates abstract the trophic relations between ants and hemipterans are very common in the neotropical region, but rarely explored in dry montane ecosystems. given the diversity of outcomes of this type of interactions influenced by variation in biotic conditions (i.e. seasonality, spatial distribution, identity of species involved), new examples in different ecosystems can provide important data for a more general understanding of their impact in communities. we investigated the outcomes (direct benefits: survivorship and reproduction) of the relationship between the trophobiont herbivore calloconophora pugionata (membracidae) and its tending ants. the interaction occurs on myrcia obovata (myrtaceae), a common tree in montane forests and rupestrian fields of southeastern brazil, and has never been studied before. between 2008 and 2009, we selected and manipulated (ant-exclusion) trees in a pairwise experiment performed on plant branches infested by c. pugionata. this membracidae laid its eggs peculiarly on the leaf margins, a behaviour that increased egg survival even when ants were absent. all life stages of the hemipteran exhibited higher survival rates (two-fold) and increased fecundity (four-fold higher oviposition rates) when attended by ants. this study shows that this ant-hemipteran interaction occurs in dry montane biomes in a way that is similar to other tropical ecosystems in which ants protect the hemipterans against predators, thus increasing their survival and reproductive fitness. sociobiology an international journal on social insects 1 federal university of uberlândia, uberlândia, minas gerais, brazil 2 federal university of ouro preto, ouro preto, minas gerais, brazil research article ants article history edited by: gilberto m. m. santos, uefs brazil received 07 january 2013 initial acceptance 01 february 2013 final acceptance 18 february 2013 keywords ants, camponotus, interactions, mutualism, montane ecosystems corresponding author kleber del-claro laboratory of behavioral ecology and interactions federal university of uberlândia uberlândia, mg, brazil, 38400-902 +55 34 3218-2243 e-mail: delclaro@ufu.br in their patrolling (delabie, 2001; rico-gray & oliveira, 2007). ants and some species of sap-sucking hemipterans (membracidae, cicadellidae, psyllidae, fulgoridae, aphididae, coccidae and pseudococcidae) coevolved behavioural and morphological features to cooperate in non-obligate mutualistic interactions (beattie, 1985; hölldobler & wilson, 1990; delabie, 2001; lach et al., 2010). these relationships are named trophobiosis and involve the release of a nutritive secretion by hemipterans (honeydew) in exchange for ant protection against natural enemies (way, 1963; del-claro & oliveira, 1999, 2000). in some cases, the ants may provide an enemy-free space for its partners and host plants around the hemiptera aggregation (moreira & del-claro, 2005). r fagundes, sp ribeiro, k del-claro tending-ants and its trophobiont calloconophora pugionata 12 this type of mutualistic relationship can lead to increased reproductive fi tness for ants and hemipterans (byk & delclaro, 2011; delabie, 2001; del-claro & oliveira, 2000). the interaction between plants, sap-sucking herbivores and ants are an important model for a better understanding of ecological functions and interactions in communities because it explores the effects of positive interactions for species involved and mechanisms of species coexistence and coevolution, structure of food-webs and aspects of plant defenses against herbivores (thompson, 1999; bluthgen et al., 2000; del-caro & torezan-silingardi, 2009; rosumek et al., 2009). although well known for temperate regions and agricultural systems (delabie, 2001), the interactions between ants and hemipteran are poorly known in the tropics due to the high diversity of involved species (moreira & del-claro, 2005) and also for received attention in this issue only recently (styrsky & eubanks, 2006; rosumek et al., 2009). even though basically mutualistic, the trophobiosis may vary in time and type of ecosystem, and about the identity, abundance and behavior of the species involved, which results in differences in the outcomes of the interaction (rico-gray & oliveira, 2007). studies of new examples of trophobioses can help to understand the differences found in comparisons of the trophobioses outcomes in different environments and conditions (thompsom, 1999). myrcia obovata (o. berg) nied (myrtaceae) is a common small tree found in montane atlantic forests and rupestrian fields of southeastern brazil. this species serves as host plant for the sap-sucking herbivore c. pugionata drietch 1991 (hemiptera: membracidae, fig 1a). a previous study has shown that c. pugionata interacts with at least ten ant species, with camponotus rufi pes fabricius 1775 and camponotus crassus mayr 1962 as the most common ones (fagundes et al., 2012). infestation by c. pugionata occurs on branches, leaves and fruits. this treehopper lay masses of eggs on stems or leaf margins (fig. 1b). the genus calloconophora exhibits a high degree of parental care and does not usually interact with ants, which often act as predators (lin, 2006). in this study we investigated for the fi rst time in a tropical montane ecosystem the interaction between ants and a membracidae (c. pugionata). we evaluated the hypothesis that the interaction increases the fi tness of the treehopper: reproduction and survival. we also tested the prediction that eggs laid on the margins of the leaves are less predated than those placed on twigs, since this is a particular behavior of c. pugionata. finally, we evaluate the reduction in the number of predators as a mechanism of ant protection. we seek to demonstrate that the benefi ts of interaction with ants can be obtained by species that have low susceptibility to interact with ants, like c. pugionata. furthermore, we seek to expand the distribution of the occurrence of these types of interactions by including the tropical montane ecosystems like rupestrian fields, providing subsidies for comparisons between different tropical ecosystems. figure 1. calloconophora pugionata (membracidae) aggregation in myrcia obovata (myrtaceae) highlighting the life stages of the trophobiont hemipterans. a aggregation, tending by camponotus rufi pes. b egg masses on twigs and leaf margins (r fagundes, 2008). sociobiology 60(1): 11-19 (2013) 13 material and methods fieldwork was conducted in itacolomi state park (peit), in the mountains of the iron quadrangle in minas gerais state, southeastern brazil. the vegetation is composed of atlantic montane forest and rupestrian fields (campos rupestres). rupestrian fi elds consist of tortuous trees and shrubs immersed in open fi elds of grasses and rocky outcrop. this ecosystem presents high diversity of endemic species (giulietti et al., 1997) being part of cerrado (brazilian savannah) but related to areas with elevation higher than 1000 m, with acid soil and low nutrient availability (ribeiro & fernandes, 2000; guerra et al., 2011). the experiment was performed in an area of rupestrian fi elds at 1400m high (20°26’26” s, 43°30’52” w). ference: 0.3 ± 0.02 m, crown diameter: 2.0 ± 0.15 m). we selected individuals separated by a minimum distance of 5 meters, to keep independence, but most pairs distanced more than 10 meters apart. on each tree, we selected two branches at opposite sides of the plant infested with c. pugionata colonies with approximately the same number of individuals. we randomly assigned the branches as control or treatment. we added a strip of tape to the base of treatment branches and covered with a nontoxic sticky resin that prevents access by ants (tanglefoot ®). in addition, we removed nearby leaves that would get in contact with the treatment branches to ensure isolation. the experiment consisted of monitoring the change in the number of remaining membracids in response to the absence of ants. we monitored three life stages of membracids (egg mass, nymphs and adults). on the fi rst day of the experiment, all branches were free of manipulation and we assessed the abundance of ants and membracids. later, at the end of the afternoon, treatment branches received the ant exclusion resin. then, we recorded the number of remaining membracids every three days in each aggregation in control and treatment branches, totalling seven samples per experiment along 19 days. we counted the number of intact egg masses attached to branches and leaf margins in both treatment and control groups at the beginning and the end of the experimental period to determinate the importance of oviposition site for egg permanence. it was impossible to count each egg individually due to posture in dense masses. we estimated hemipteran survival based on changes in overall number of eggs, nymphs and adults. this method may overestimate the survival of eggs, because the dead eggs are replaced by new ovipositions. however, the comparison between experimental groups, allows us to assess the fi tness of the aggregation, because if the aggregation is not healthy enough to compensate losses or produce more individuals than it loses, the aggregation inevitably will perish. we quantifi ed the abundance of other arthropods counting those visiting the branches, treatment and control, in order to test ant repellence as a mechanism of protection. we classifi ed the visiting arthropods as predators or herbivores by morphology and behavior with direct observation. only arthropods observed feeding on individuals of c. pugionata colonies were recorded. this procedure may have underestimated the number of predators but gave us the confi dence to affi rm that those individuals counted defi nitely feed on c. pugionata. in addition, we recorded the abundance and species richness of tending (partners) and non-tending ants (visitors). the ants were classifi ed as non-tending and tending by direct observation of constant care of membracids and collection of honeydew. voucher specimens of membracids, and ants were figure 2. repeated measures anova of ant-exclusion experiments on egg masses(a), nymphs(b) and adults(c) of calloconophora pugionata (membracidae) in myrcia obovata (myrtaceae) over time. we conducted a manipulative experiment to evaluate the effect of tending ants on c. pugionata survival. we used 19 m. obovata individuals in january 2008 and 12 in january 2009. the plants shared similar architecture (mean ± standard deviation: height = 2.65 ± 0.15 m; trunk circumr fagundes, sp ribeiro, k del-claro tending-ants and its trophobiont calloconophora pugionata 14 collected at the end of the study period to confirm taxonomic identification using taxonomic keys and with the assistance of dr. rodrigo feitosa, an ant’s taxonomist. the specimens were deposited in the entomological collection of the laboratory of evolutionary ecology of canopy insects and natural succession of the federal university of ouro preto (ufop). we had all authorizations required to collect and research in brazilian natural reserves (ief uc09/009). data analysis all data were square root transformed to meet the model assumptions of normality and homoscedasticity. all graphs display non-transformed data. statistical analysis was performed in statistica 7.0. we used repeated measures anova to compare the variation in the number of membracids over time between control and treatment branches. we considered branches as fixed factors and days as repeated measures to the response variables: number of egg masses, nymphs and adults. we used fisher’s lsd paired comparisons as post-hoc test. we use repeated measure anova to evaluate the effect of the presence of ants and oviposition site (fixed factors) on egg survival of c. pugionata (response variable) over time (day 1 and day 21, repeated measures) in both years. were used ancova models to assess the relative importance of the presence of ants (fixed factor) and the number of days with adults present (continuous factor) on offspring survival (response variable). we used anova models to compare the variation in the number of predators of c. pugionata between experimental branches (with and without ants). values recorded on all sampling period were summed. we made comparisons using a generalized linear model, assuming a poisson distribution and log function. results we found 36.84 ± 9.86 (n=19) membracids per aggregation in treatment branches and 37.58 ± 7.30 (n=19) in control branches in 2008, and 60.00 ± 11.42 (n=12) and 38.83 ± 7.24 (n=12) in 2009 (mean ± standard error; number of samples). twelve ant species and morphospecies were observed tending c. pugionata (table 1). the most frequent species tending c. pugionata were camponotus rufipes (48% of ant occurrences in 2008 and 60% in 2009) and c. crassus (18% of occurrences in 2008 and 15% in 2009). the number of hemipterans per aggregation was higher in all life stages when c. pugionata was tended by ants (fig. 2). in most cases the number of individuals started higher in the treatment branch but ended lower, except for table 1. number of times an ant species was observed in different individual trees tending (td.) calloconophora pugionata (membracidae) or just visiting (vis.) myrcia obovata (myrtaceae). ants number of records 2008 2009 td. vis. td. vis. formicinae camponotus crassus mayr 1862 6 4 3 1 camponotus fastigatus roger 1863 1 2 0 0 camponotus novogranadensis mayr 1870 1 3 1 0 camponotus rufipes fabricius 1775 16 0 12 0 camponotus sp. 1 1 0 3 1 camponotus sp. 2 2 1 0 0 camponotus senex smith 1858 1 4 0 1 myrmicinae cephalotes pusillus klug 1824 1 0 0 0 crematogaster sp. 1 0 0 0 pheidole sp. 1 1 0 0 pseudomyrmecinae pseudomyrmex gracilis fabricius 1804 1 3 1 1 pseudomyrmex (gp pallidus) sp. smith 1855 1 1 0 0 “nymphs in 2008” for which the control aggregations had higher number of individuals, in both study years (table 2). in treatment branches the number of membracids always reduced over time. in control branches the number of membracids increased or remained constant over time. survival of eggs and nymphs was not influenced by the presence of adult membracids (ancova: eggs: ss=0.33, df=1.41, f=1.65, p=0.21; nymphs: ss=0.33, df=1.41, f=1.66; p=0.2). table 2. repeated measures anova for the number of calloconophora pugionata (membracidae) egg masses, nymphs, and adults between branch treatments (ants factor), days (repeated factor) and year in myrcia obovata (myrtaceae). source of variation df ss f p e gg m as s ants 1 0.03 0.03 0.87 year 1 2.35 1.97 0.17 ants*year 1 0.34 0.28 0.60 error 58 69.34 days 6 1.41 3.32 <0.01 days*ants 6 6.55 15.43 <0.01 days*year 6 1.08 2.54 0.02 days*ants*year 6 1.60 3.78 <0.01 error 348 24.63 y ou ng ants 1 8.95 4.96 0.03 year 1 11.37 6.30 0.01 ants*year 1 0.57 0.31 0.58 error 58 104.68 days 6 12.57 16.39 <0.01 days*ants 6 8.76 11.42 <0.01 days*year 6 1.42 1.85 0.09 days*ants*year 6 8.22 10.72 <0.01 error 348 44.48 a du lt ants 1 2.75 12.56 <0.01 year 1 1.71 7.79 0.01 ants*year 1 1.75 7.99 0.01 error 58 12.71 days 6 1.00 5.70 <0.01 days*ants 6 1.19 6.76 <0.01 days*year 6 0.60 3.40 <0.01 days*ants*year 6 0.20 1.15 0.33 error 348 10.19 sociobiology 60(1): 11-19 (2013) 15 the egg survival responded to the presence of ants, as well as fecundity of the female. but this response was different regarding the oviposition site and was not maintained over the years (table 2, fig. 2). in 2008, the number of intact eggs was lower when ants were excluded but only for those laid on the twigs. the eggs laid on the margins of the leaves did not respond to the absence of ants. in 2009, the number of intact eggs in both twigs and leaves increased in control branch but remain constant in treatment branch (table 3). eggs in leaf margin were always more numerous, including on ant absence (fig. 3). the abundance of c. pugionata predators was higher in treatment branches in both years (glz: predators: ants factor: wald. χ² = 28.65, df=1, p < 0.001;). although the overall number of predators has reduced by half in 2009, there was a difference between the experimental branches (glz: year factor: wald. χ² = 23.68, df=1, p < 0.001; ants*year: wald. χ² = 0.14, df=1, p < 0.001). as a result, arthropod predators are less frequent when ants are present, mainly in 2008 (fig. 4). spiders (mainly salticidae), wasps and braconid parasitoids were the most frequently observed predators of c. pugionata. the main type of damage to eggs was their drying out and typical opening marks from newly emerged parasitoids. discussion in recent decades, many studies have been published with examples of interactions between ants and hemipteran, but few in the neotropical region and most for the brazilian cerrado (buckley, 1987; oliveira & del-claro, 2005; rosumek et al., 2009). this study presents the interaction between the ants and the membracidae c. pugionata that occurs in montane ecosystems. the herbivore lives in myrcia obovata where it is assisted by several ant species, but most frequently by camponotus crassus and c. rufi pes. anthemipteran interactions are commonly multispecifi c for ant partnership which reduces the benefi t of the interaction, especially when the ant species is less aggressive (rico-gray & oliveira, 1987). these ant species is commonly found in association with hemipteran and plants in the cerrado (oliveira & brandão, 1991; shoereder et al., 2010) and rupestrian fi elds (guerra et al., 2011; fagundes et al., 2012), reinforcing the importance of camponotus as worldwide attendance of trophobiont insects. in this study, the interaction with ants is benefi cial at least to c. pugionata, which produces fewer eggs, dies or leave the plant when not associated with ants. the ants protect all hemipteran life stages against natural enemies (way, 1963; buckley, 1987; del-claro & oliveira, 2000), increasing their life expectancy and fertility while receiving honeydew as a reward (del-claro & oliveira, 2000). by becoming more aggressive while protecting the associated hemiptera, the ants attack and drive away anyone who comes close and reduce the occurrence of species such as the predators of hemiptera and even the host plant (way, 1963; del-claro & oliveira, 2000; rosumek et al. 2009). this repellent effect is common to several plant-ant-hemiptera systems (buckley & gullan, 1991; cushman & addicott, 1991; moreira & del-claro 2005) but can vary with time and with the species involved in the interaction (del-claro & oliveira, 2000; billick & tonkel, 2003). protection against natural enemies is considered the main benefi t received by arthropods interacting with ants in mutualistic relationships (rico-gray & oliveira, 2007). we did not evaluate the effects of the interaction to the ants, but such effort to collect exudate and protect the partner indicates the valuation of the resource by the ants (styrsky & eubanks, 2006). the honeydew is a constant source of food energy, predictable in time and space (delclaro & oliveira, 1993; blüthgen et al., 2000; davidson et al., 2003) which may increase ant aggregation survivorship and growth, as is the case with extrafl oral nectar (davidson et al., 2003; byk & del-claro, 2011). this type of resource is capable of replacing predation and scavenging, support entire colonies due to the high nutritional value and low energetic cost (davidson, 1997). therefore, when a kind of foraging is focused to a specifi c resource, such as in trophobiosis, it is defended at a high cost of energy (stadler & dixon, 1998a). so, it can be expected that the honeydew of c. pugionata is advantageous to its tending-ants. we observed in our study that camponotus rufi pes built its nests in the base of the stem of m. obovata, and in some cases built satellites nests made from leaves that housed aggregations c. pugionata. a similar observation was made for the system ants-guayaquila xiphias (oliveira et al., 2002). figure 3. number of egg masses per aggregation of calloconophora pugionata before and after exclusion of ants in treatment branch for both oviposition sites and years. r fagundes, sp ribeiro, k del-claro tending-ants and its trophobiont calloconophora pugionata 16 these authors suggested that the shelter-nest behavior was a response to the importance of honeydew for the ants. table 3. results of repeated measures anova for the comparisons of the number of egg masses between branch factor (control and treatment), period factor (before vs. after ant exclusion, repeated measure) and year (2008 and 2009). source of variation df ss f p year 1 84.85 4.37 0.04 branch 1 291.22 14.90 <0.01 egg position 1 13.67 0.70 0.40 year*branch 1 211.37 10.80 <0.01 year*egg position 1 13.81 0.71 0.40 branch*egg position 1 10.59 0.55 0.46 year*branch*egg position 1 4.97 0.26 0.61 error 116 2252.10 period 1 1975.89 105.50 <0.01 period*year 1 48.88 2.70 0.10 period*branch 1 239.73 1.32 <0.01 period*egg position 1 13.67 0.75 0.39 period*year*branch 1 176.23 9.70 <0.01 period*year*egg position 1 13.91 0.76 0.39 period*branch*egg position 1 10.59 0.58 0.45 period*year*branch*egg position 1 4.97 0.27 0.60 error 116 2106.69 the interaction with ants seems to be a good defense strategy for c. pugionata, but laying their eggs on the margins of the leaves, in addition to branches, increased egg survival at least in one studied year. temporal variability in interactions of ants and hemipteran have already been shown in other studies (bristow, 1984; cushman & whitham, 1989, 1991; del-claro & oliveira, 2000). the loss of the benefi ts of protection by ants over the years is attributed to the fl uctuations of the natural enemies of hemiptera partners (cushman and addicott, 1991). ants fail to defend or benefi t is too small for hemipterans when the predator population is low. in our study the non-effect of ant exclusion in 2009 coincides with a decrease in the amount of enemies of c. pugionata. the strategy of laying eggs on leaf margins is uncommon for hemiptera species (lin, 2006). eggs laid on leaf margins exhibited higher survival rates when compared to those placed on leaf branches, especially in the presence of ants. eggs laid on very crowded masses or long pellets in the branches may experience higher predation and parasitism due to the reduction in search time (tallamy & schaefer, 1997). the study of community ecology, focused on negative interactions between species, has led to the neglect of positive interactions and its importance in food webs (ohgushi, 2008; grinath et al., 2012). the effect of hemiptera-ant interaction can cause changes in community in terms of abunfigure 4. mean abundance of predators of c. pugionata in response to ant-exclusion in each study year. dance, distribution and survival of the associated arthropods (del-claro & oliveira, 2000; renault et al., 2005; styrsky & eubanks, 2006). on a larger scale, the effect of trophobiosis in the arthropod fauna from its host plant can affect the whole community (wimp & whitham, 2001; rosumek et al., 2009). fagundes et al. (2012) showed that the abundance of the ant species attendants of c. pugionata, camponotus crassus and c. rufi pes, are directly affected by the population size of c. pugionata, and the densifi cation of these species in the host plant leads to reduced diversity of ants, as well as the whole arthropod fauna from the host plant. as we know from other studies in different ecosystems, ants are key for the population growth of the trophobiont hemipteran and crucial to its survival (buckley, 1987; holldobler & wilson, 1990; delabie, 2001). the sap-sucking hemipteran has many natural enemies that keep its popusociobiology 60(1): 11-19 (2013) 17 lation low in natural environment (delabie 2001; del-claro, 2004). the association with ants provides an effective protection against these natural enemies in exchange for a food that has low cost to the hemipteran. the ants were important for the survival and fitness of c. pugionata, as is expected benefits for ants by highly energetic food provided by hemipterans. this interaction has a direct effect on the population parameters of the species involved increasing its life expectancy, growth and reproduction (bristow, 1991; delabie, 2001). additionally, these populations gain advantages in negative interactions such as competition for food (davidson, 2007; davidson et al., 2003). this high adaptive value of trophobioses may be important for the persistence of the populations involved, directly and indirectly, and ultimately to the maintenance of diversity (del-claro, 2004). trophobioses is distributed globally (rico-gray & oliveira, 2007) and occurs in almost all ecosystems (styrsky & eubanks, 2006; rosumek et al., 2009), but the interaction outcomes and its ecological consequences are variable in space and time (cushman e addicott, 1991; stadler & dixon, 2005) and some ecosystems are little explored, as tropical montane ecosystems (viana-silva & jacobi, 2012; guerra et al., 2011). in some cases, the interaction can be null for both or one of the parties (stadler & dixon, 1998a, b), alarming as to assume that all interactions between ants and hemipteran are mutualistic. only through studies in various conditions of space and time can predict the occurrence and degree of variability of these conditional interactions (cushmann & addicott, 1991). the hemipteran c. pugionata has a number of adaptations which suggest low interactivity with ants, as long hind legs, pigmentation for camouflage and parenting developed (lin, 2006). this features altogether suggests low interactivity and dependence on ants as protectors (rico-gray & oliveira, 2007). however, our results suggested that even in cases of low probability, the interaction can occur and have a mutualistic character. acknowledgments we thank pricila bonifacio and filipe paixão de lima for field assistance. the comments of cinthia borges da costa, jacques h. c. delabie, julio fontenele, ricardo idelfonso campos, rhainer guillermo nascimento ferreira and bruno madeira, and the two anonymous reviewers that improved the manuscript. this study was funded by cnpq (kdc 301248/2009-5/472046/2011-0; rfs mct– 027/2007) and fapemig. references beattie, a.j. 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(2001). biodiversity consequences of predation and host plant hybridization on an aphid– ant mutualism. ecology, 82: 440–452. doi: 10.1890/00129658. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3443sociobiology 66(2): 293-305 (june, 2019) continuous micro-environments associated orchid bees benefit from an atlantic forest remnant, paraná state, brazil introduction the tribe euglossini comprises more than 200 described species distributed in five genera with two of them being composed of cleptoparasitic species (dressler, 1982; cameron, 2004; nemésio, 2009). these bees are essential pollinators in the neotropical region and are responsible for pollination of several plants of economic and ecological importance (dressler, 1982; ramírez et al., 2002; milet-pinheiro & schlindwein, 2005; santos & absy, 2012; gianinni et al., 2015). moreover some orchid bee species are bioindicators of conserved areas and others are widely distributed in disturbed and fragmented environments (morato, 1994; peruquetti et al., 1999; aguiar & ganglianone, 2008). studies exploring abstract the fragmentation and habitat loss are the main causes of pollinators decline worldwide, however very little is known about the composition and distribution of neotropical pollinators along continuous micro-environments. to fill this gap, we carried out samplings of euglossini bees in a continuous area of forest with micro-environments of primary (remnant) and secondary (regeneration) forest of atlantic forest. we evaluated the differences in the composition and uniformity of orchid bees in different micro-environments, in order to characterize the responses of the local environmental changes in the attraction of bees to chemical traps. our results indicated that the composition and uniformity were similar between the two forest fragments studied here, although there are greater abundance of some species by micro-environments. we conclude that the characteristics of the sites in a continuous environment with primary and secondary forest do not seem to have an effect on the composition of the euglossini fauna, and that the chemical substances are complementary in the attractiveness of the orchid bee males. thus, our findings suggest that micro-environments in a continuous matrix near forest remnants can help to promote the reintegration of the orchid bee communities and contribute to the conservation of areas in process of forest regeneration. sociobiology an international journal on social insects ai sobreiro¹, lls peres², s boff¹,³ , ja henrique¹, vv alves jr¹ article history edited by eduardo almeida, usp, brazil received 09 may 2018 initial acceptance 01 july 2018 final acceptance 14 december 2018 publication date 20 august 2019 keywords conservation, diversity, euglossini, pollinators, primary versus secondary forest. corresponding author ana isabel sobreiro universidade federal da grande dourados laboratório de apicultura, unidade ii rodovia dourados – itahum, km 12 cep: 79.804-970, dourados-ms, brasil. e-mail: sobreiroanaisabel@gmail.com diversity of orchid bees at multiple scales (ecological and molecular level) are common in the neotropical region (raw, 1989; silveira et al., 2011; boff et al., 2014; gonçalves et al., 2014; eltz et al., 2015; giangarelli et al., 2015; penha et al., 2015; mccravy et al., 2016) because orchid bee males are easily attracted to chemical compounds (dodson et al., 1969). although a population expansion of few species has been noticed to south of north america (pemberton & wheeler, 2006; eltz et al., 2015; griswold et al., 2015), orchid bees are extremely diversified in central and south america (ramalho et al., 2009; cordeiro et al., 2012; aguiar & ganglianone, 2012; mccravy et al., 2016; botsch et al., 2017). in south america, the atlantic forest (af) is known to wide host bee diversity. approximately 50 species of euglossini have been 1 federal university of grande dourados, faculty of biological and environmental sciences, dourados, state of mato grosso do sul, brazil 2 federal university of lavras, department of entomology, lavras, state of minas gerais, brazil 3 the university of milan, department of food, environmental and nutritional sciences, milan, italy research article bees ai sobreiro et al. – micro-environments associated orchid bees benefit294 already recorded to this ecosystem (sofia et al., 2004; miletpinheiro & schlindwein, 2005; nemésio, 2009; cordeiro et al., 2012; gonçalves et al., 2014; oliveira et al., 2015; ferronato et al., 2017). although orchid bee fauna is considered to be diverse in the af, their communities seem to depend on the traits of local environment (moreira et al., 2015). historical process of land use and occupation in the af has deeply altered the habitats. from its original cover only 11.73% of its domain remains natural (ribeiro et al., 2009). several fragments are mostly surrounded by monoculture, pasture and cities (brasil, 2009). thus, af fragments are usually both small (< 50 ha) and isolated (ribeiro et al., 2009). although orchid bees are considered to be strong flyers, fragmentation may alter their dispersion range and thus affect local diversity, as a consequence, small forest fragments in urban areas may play the role as refuges for these bees (rosa et al., 2015; neame et al., 2012; storck-tonon et al., 2013; oliveira et al., 2015; fischer et al., 2016). studies show that euglossini populations are influenced by landscapes that surround the forest matrix. according to ferreira et al. (2013), antique urban fragments seem to offer the main conditions for the maintenance of orchid bee community: nest and food. in addition, small forest fragments, mainly in backyards, may serve as stepping stones between forest matrices. in another study, the authors evaluated the permeability of three matrices of arboreal crops (piassava palm, oil palm and rubber tree) and they found that the arboreal matrices are contributing to the landscape mosaic and the brazilian atlantic forest corridor (rosa et al., 2015). on the other hand, sugarcane crop that surround the forest matrix interferes with species dynamic, negatively affecting the richness and abundance, indicating that the forest edge functions as a barrier in the individuals flow (milet-pinheiro & schlindwein, 2005). although the studies were carried out in different crops and diversity scale (β and γ), they show that functional connectivity is an important factor for the reestablishment of the orchid bee populations. thus, the questions that still remain are: does forest regeneration surrounding to primary forests favor the connectivity between fragments? are anthropic landscapes (cities and monocultures) less impacting to the euglossini community in a forest matrix surrounded by forests in regeneration? to our knowledge, studies comparing pattern of orchid bee communities in stages of regeneration and environmental preservation in a continuous are scarce (moreira et al., 2015; boscolo et al., 2017; ferronato et al., 2017). in order to provide insights in this topic, we compared the attraction potential of orchid bees to chemical baits in multiple microenvironments distributed in continuous areas with primary and secondary forest. here we tested if different continuous microenvironments in primary or secondary forests affect: (i) the diversity indexes of orchid bees and, (ii) at temporal scale patterns of species distribution. we defined primary and secondary as vegetation heterogeneity at each sampling site, classified according to brazilian environmental standards (ibama, 1991; brasil, 1994). therefore, the present work investigated the differences in the composition and uniformity of orchid bees in different micro-environments, in order to characterize the response of local environment change in the bees attraction to chemical traps. material and methods study area the study was conducted in six different sampling sites selected in forest fragments of a continuous matrix with two distinct habitats: primary and secondary forests. all sampling sites (n = 6) were located at foz do iguaçu municipality in the refúgio biológico bela vista (rbv), western paraná state, brazil. the rbv reserve covers 1920 ha conservation unit which is part of the itaipu binacional complex, under a seasonal semi-deciduous forest. the area is surrounded by itaipu reservoir, alternate soybean and corn crops and it is 1300 m from city boundaries. this area includes fragments in advanced stages of regeneration and a part with native forest remnant. of these fragments we selected three sites placed at primary forest and other three sites placed at secondary forest (fig 1). sites characteristics and habitat complexity we used data from satellite google earth pro and global forest change dataset (hansen et al., 2013) to distribute sampling sites in different continuous micro-environments. the characteristics of the sites (table 1) were realized before starting the sampling. the size of each site was calculated using metric applications of the satellite programs (google earth pro and global forest change dataset). the distance from one site to another at each forest was established in an interval between 500-560 m (fig 1). georeferencing informations were digitized using software qgis version 2.18 (2016). the classification of vegetation according to brasil (1994) is provided in table 1. primary forest is all vegetal community with great biological diversity and minimal anthropic effects, not affecting original characteristics of forest structure (brasil, 1994). this forest structure favors the regeneration of late species and the formation of seed banks (brasil, 1994; clements, 1916), also known as “climax forest” or mature forest. in the study region, the forests are classified as seasonal semideciduous forest (ibama, 1991), one of the main formations of atlantic forest (brasil, 2010a). the national forest system determines primary formation such as natural forest (brasil, 2010b) or forest remnants. in this study, the primary forest is composed by forest remnant (native) and reforestation (which was performed with > 40 years) (itaipu, 1978; ziober & zanirato, 2014) (fig 1). sociobiology 66(2): 293-305 (june, 2019) 295 secondary forest is a habitat that has recovered after a great disturbance, resulting from natural processes of succession (brasil, 1994). usually young vegetation (< 15 years), consisting of open fields, high sunlight incidence and pioneering plant species with evident stages of regeneration (initial, intermediate and advanced) (clements, 1916; brasil, 1994). in this study, the secondary forest is composed by three stages of regeneration (initial, intermediate, and advanced), being a forest fragment that lost the original vegetation and it is naturally regenerating (fig 1). fig 1. location of the two forest fragments and each sampling site used in the study for the euglossini bee communities in paraná state, brazil. primary forest: sampling sites (nap, nrp and rep). secondary forest: sampling sites (ins, ims and ads). nrp = transition between native and reforested. table 1. enviromental gradient: geographic coordinates, elevation (m), estimated age of vegetation (years), size of sites (ha), stage of forest (brasil, 1994) and continuous sampling sites of primary and secondary forest, western region of paraná, southern brazil. primary forest secondary forest environmental gradient native forest (nap) transition between native and reforested (nrp) reforested (rep) initial stage of regeneration (ins) intermediate stage of regeneration (ims) advanced stage of regeneration (ads) geographic coordinates 25º26´2069”s 54º31´2932”o 25º26´3809”s 54º31´2721”o 25º26´5487”s 54º31´2466”o 25º27´3502”s 54º31´3147”o 25º27´2341”s 54º31´2849”o 25º27´2861”s 54º31´1164”o elevation (m) 251 246 247 224 224 238 age (years) > 40a > 40 a > 40 a ≤ 5 b ≤ 10 b ≤ 15 b size (ha) 315.70 --96.30 19.98 27.32 23.70 forest stage primary (remaining) primary (transition) primary (reforested) secondary (initial) secondary (intermediate) secondary (advanced) environmental aspects of sites native forest with difficult access, pristine native/ reforested transition plantations of seedlings native to the atlantic forest an open field covered by dense vines forest with few trees that were distant from each other, thereby forming clearings as woodland greenwood, forest and flooded environment a reference used to characterize the primary forest sites (itaipu, 1978). b reference used to characterize the secondary forest sites (ziober & zanirato, 2014). --division between nap and rep sites, cannot measure size (ha). ai sobreiro et al. – micro-environments associated orchid bees benefit296 survey male euglossini bees were sampled during 12 months from october 2013 to september 2014 at all sites (fig 1). at each site four bait traps were used, each one with a different fragrance (cineole, eugenol, methyl salicylate, and vanillin). the traps remained at each site for the entire months, covering after 12 months, 8640 hours (12 months x 30 days x 24 hours) of trap exposition. traps were fixed by a wooden support at a mean height of 1.5 m from the ground. the traps were visited monthly for the bee collections, cleaning the traps, and fragrances were refilled. the traps were made from plastic bottles (with modifications from the model of campos et al., 1989) (fig 2). at the bottom of each trap (bottle), it was add a solution containing the following proportions: water (200 ml), alcohol 92.6% (100 ml), two spoons of salt (5 g) and one spoon of neutral detergent (15 ml) (colorless and without flavor). the solution was refilled once a month. the essences were stored in small glass bottles and a small opening was made to pass a string where the fragrance would volatilize by capillarity. in the laboratory, the bees were cleaned, mounted in entomological pins and identified with the aid of specialist (r. b. gonçalves, federal university of paraná). data analysis to compare the diversity of the six sites we used the shannon-wiener diversity (h`). the dominance of the species at each site was calculated using berger-parker (d) and simpson (d-1) index (melo, 2008). lastly, evenness (j′) was used to define uniformity or homogeneity of species distribution at the sites (magurran, 2004). the rarefaction curve for the species richness of each studied site was obtained using 1000 randomizations, and it is commonly used to evaluate the sampling effort. in this study, we used the rarefaction curve to compare sampling effort among micro-environments (6 sites) and between two forests. the total sampling of study was represented by 72 samples, with 12 samples (one sample of bees per month during 12 months) at each site (total of 6 sites), i.e., 12 samples x 6 sites = 72 total samples. the detrended correspondence analysis (dca) was used to evaluate the relationship between the bee communities in the continuous micro-environments. fig 2. bait trap made with commercial 2000 ml plastic bottle. (a) nozzle with wire rod for support the suspended trap; (b) small glass bottle filled with chemical essence and a piece of string that served as wick for dispersion of essence; (c) two lateral openings of diameter equal with flaps for “landing area” of the bees; (d) six circular openings for draining excess water; (e) solution. modifications from the model of campos et al. (1989). sociobiology 66(2): 293-305 (june, 2019) 297 sørensen-dice index was used to compare similarities between sites and species richness. to perform the dca and rarefaction curve, we employed the vegan package (oksanen et al., 2018) using the software r version 3.4.1 (r development core team, 2017). this software was also used to realized the analysis of variance (anova) and to determine whether there were significant differences for: (i) species abundance per month; (ii) total abundance of species between forest types; (iii) species abundance among sites; (iv) species abundance by chemical essence. the specnumber function locates the number of species using the margin argument. if the experiment has more than one fragment or sampling site, it uses if the parameter groups and the function finds the total number of species for each parameter (hurlbert, 1971). we used this function to determine if the richness data were significant per month, forest types, among sites and chemical essence. in addition, this function was also used to calculate the diversity index, so we applied a test of anova to determine if there was significant difference. finally, aiming to compare the temporal distribution of the euglossini species in the two forests, we performed a histogram with all the species frequencies per sampled month. to make the histogram, we used the poncho.r function, and this analysis was performed with the aid of software r (r development core team, 2013). the poncho function is used to create histograms and multiple graphs of species distribution in a community in relation to environmental gradients or experimental variables (dambros, 2014). in the current study, the confidence interval was of 99%. results throughout the study, we collected 586 orchid bee males belonging to four genera and eight species. of these, 285 were collected in the primary forest and 301 specimens were collected in the secondary forest. the abundance was statistically different per month (df = 11, f = 3.987, p = 0.0002), although it did not vary significantly between forest types (df = 1, f = 0.0451, p = 0.832). the same orchid bee species (n = 8) were sampled in both primary and secondary forest. the highest abundance of orchid bees in the secondary forest was found at ads site (n = 162 individuals) and in the primary forest at rep site (n = 131 individuals). the site with the lowest abundance and diversity was at nrp with 62 individuals (6 spp.) and at ins with 66 individuals (7 spp.). the abundance did not vary significantly among the sites (df = 5, f = 0.9439, p = 0.4559). euglossa annectans dressler was the most abundant species in both primary and secondary fragment, with relative abundance of 70.52% (n = 201) and 42.19% (n = 127), respectively. eufriesea aff. auriceps (friese) was the less abundant species with 0.35% (n = 1) in the primary forest and 2.32% (n = 7) in the secondary forest. shannon-wiener index (h′) indicated the secondary forest with higher diversity (h′ = 1.66) than the primary forest (h′ = 1.08). the diversity was the highest at ads (h′ = 1.69) and the lowest at nrp (h′ = 0.80). regarding the bergerparker index (d), the dominance was higher in the primary forest fragment (d = 0.70) than in the secondary forest fragment (d = 0.41). among sites, the highest dominance was found at nrp (d = 0.79), probably due to the higher abundance of eg. annectans, whereas the lowest dominance was recorded at the ads site (d = 0.38). the simpson index was lower in the secondary forest (d = 0.75) than in the primary forest (d = 0.48). for the sampled sites, the simpson index at ads showed the lowest dominance (d = 0.77), while at nrp presented the highest dominance (d = 0.36). the evenness was higher in the secondary forest (j´ = 0.80) than in the primary forest (j´ = 0.52). among sites, the ins site obtained the maximum evenness (j′ = 0.83), followed by ads (j′ = 0.81). the sites with the lowest evenness were nap (j′ = 0.41) and nrp (j′ = 0.44) (table 2). species primary forest secondary forest nap nrp rep ins ims ads euglossa annectans dressler 72 49 80 24 41 62 euglossa cordata (linnaeus) 2 3 23 6 4 26 euglossa fimbriata moure 6 0 5 7 1 5 euglossa pleosticta dressler 1 6 14 12 6 25 eufriesea aff. auriceps (friese) 1 0 0 0 3 4 eufriesea violacea (blanchard) 8 1 7 14 11 27 eulaema nigrita lepeletier 1 1 1 2 6 9 exaerete smaragdina (guérin) 1 2 1 1 1 4 total of individuals 92 62 131 66 73 162 total of species 8 6 7 7 8 8 shannon-wiener (h′) 0.86 0.80 1.20 1.63 1.42 1.69 simpson (d-1) 0.37 0.36 0.58 0.76 0.64 0.77 berger-parker (d) 0.78 0.79 0.61 0.36 0.56 0.38 evenness (j′) 0.41 0.44 0.61 0.83 0.66 0.81 table 2. orchid bee species sampled with chemical compounds in continuous micro-environments of two forest fragments (primary and secondary) of atlantic forest, brazil. total abundance, divergence index (h′), dominance (d-1 and d), and evenness (j′). ai sobreiro et al. – micro-environments associated orchid bees benefit298 the rarefaction curve used to compare species richness suggests that the richness of the primary and secondary forest stabilized reaching the asymptote. being that in the primary forest stabilized at the 34th sample, while in the secondary forest stabilized at the 36th sample. on the other hand, comparing the rarefaction curve for the six sampling sites, the only site that approached of stabilizing was ads, while the other sites did not reach the total number of species richness for the micro-environment, sample adequacy was not sufficient at the nap, nrp, rep, ims, and ins sites. the sampling efficiency was 99.78 and the average estimated richness was 8.01 (fig 3). we verified that the distribution pattern of species per month in the secondary forest showed a higher frequency and abundance for all euglossini species, except eg. annectans which was most frequent and abundant in the primary forest. fig 3. rarefaction curve (1000 randomizations) for the species richness and individuals of orchid bees as a function of their abundance in the six sites of primary and secondary fragment of atlantic forest in paraná state, brazil. fig 4. temporal distribution histogram of euglossini bees in the primary and secondary atlantic forest fragment in paraná state, brazil from october 2013 to september 2014. this one was the only species that occurred during 11 months and the most abundant during 8 months in both forests, representing 55.97% (328 individuals) of the total number of sampled bees. the month with the highest abundance in the primary forest was september 2014 with 36.84% (105 individuals), while in the secondary forest august 2014 with 17.60% (53 individuals) presented the highest abundance. in october 2013, only ef. aff. auriceps and ef. violacea were recorded in the secondary forest fragment. in addition, anova indicated that the species richness did not change per month (df = 11, f = 1.713, p = 0.0703) and between forests types (df = 1, f = 3.16, p = 0.0765), although it varied significantly among sites (df = 5, f = 15.54, p = 0.00732 (fig 4). the detrended correspondence analysis (dca) revealed differences in the euglossini fauna ordination among the sampling sites (fig 5). the rep and ims sites are grouped by the occurrence pattern of species with diversity, dominance and evenness similar among the micro-environments. we observed that the primary forest sites (nap, nrp and rep) are grouped on the left side of the ordination and secondary forest sites (ins, ims and ads) are grouped on the right side of the ordination, connected by similarity between rep and ims. besides, it seems that eg. annectans is the species positioned closer to the primary forest grouped on the left side of the ordination (primary forest sites), as well as it is noticeable that ef. violacea is closer to the secondary forest sites on the right side of the ordination. euglossa cordata and eg. pleosticta were grouped among the geographically closest sites (rep, ins, and ads sites) with different conservation characteristics among the micro-environments (see fig 1) (fig 5). sociobiology 66(2): 293-305 (june, 2019) 299 the role of scents cineole was the most efficient scent in the species abundance attraction, accounting for 40.95% (240 individuals), followed by eugenol, which attracted 37.37% (219 individuals) of all sampled bees. in both forests, the vanillin and methyl salicylate traps attracted the lowest abundances with 17.74% (104 individuals) and 3.92% (23 individuals), respectively. in primary forest, the most attractant fragrance was eugenol with 47.01% (134 individuals) and in the secondary forest the fig 5. detrended correspondence analysis (dca) of the euglossini community at the sites (n = 6) in primary (nap, nrp and rep) and secondary forest fragment (ins, ims and ads) of atlantic forest in paraná state, brazil. according to sørensen-dice coefficients. fig 6. frequency of male euglossini bees captured using different chemical baits (cineole, eugenol, methyl salicylate and vanillin) within of primary and secondary atlantic forest fragment in paraná state, brazil. *methyl salicylate. cineole was more attractant with 43.85% (132 individuals). for micro-environments (sites), the eugenol was the most attractant essence with 10.75% (n = 63 individuals) at nap and 9.55% (n = 59 individuals) at rep. cineole attracted the highest abundance at ads (11.77%, n = 69 individuals), followed by nrp (6.82%, n = 40 individuals), ims (5.46%, n = 32 individuals) and ins (5.29%, n = 31 individuals). methyl salicylate was the less attractant scent with 0.17% (n = 1 individual) at ins and neither individual at nap (fig 6). ai sobreiro et al. – micro-environments associated orchid bees benefit300 the attractiveness of the essences was significantly different for the species richness (df = 3, f = 11.51, p = 3.84e-07) and diversity (df = 3, f = 6.043, p = 0.000531), but it was not different for the abundance (df = 3, f = 0.9479, p = 0.4203). we observed here that the essences present an attraction pattern for the euglossini genera, being that 89.79% of all the individuals of euglossa were attracted by eugenol and cineole, whereas 73.58% of all the individuals of eufriesea, eulaema, and exaerete were attracted by methyl salicylate and vanillin. in addition, the fragrances that attracted more species were methyl salicylate and vanillin with 7 species each one, followed by cineole and eugenol, which one attracted 6 species. discussion our analyzes showed that the species composition, abundance and indexes of diversity did not differ between the primary and secondary forest fragments. interestingly, the micro-environments of the primary and secondary forest seem to influence the preference of the species for conserved or altered sites. recently, ferronato et al. (2017) investigated the euglossini community in native and reforested forests and they observed that some species of orchid bees seem to indicate a better quality of habitat, besides the possible reintegration of the bee community from the reforested forests being favored by native forest. although studies comparing the richness and abundance of euglossini among several forest fragments are not scarce (sofia et al., 2004; aguiar et al., 2014; gonçalves et al., 2014; giangarelli et al., 2015; ferronato et al., 2017), very little is known about the distribution of these species in microenvironments, mainly comparing the micro-environments mosaic within the primary and secondary forest and if the distribution of the species affect the attractiveness of the essences in these different micro-environments. some studies suggest negative effects on the euglossini community in fragmented habitats (sofia et al., 2004; aguiar et al., 2014; gonçalves et al., 2014; giangarelli et al., 2015), however our results seem to indicate that fragmented habitats in process of regeneration can support the reintegration of the orchid bee community, as suggested by ferronato et al. (2017) for the restored forest areas. a possible evidence for this fact is the presence of ef. violacea in micro-environments of the primary and secondary forest, being more predominant in the forest in process of regeneration (secondary). this species was suggested as sensitive to fragment size reduction in studies involving small open areas or greenwoods and large forests (sofia & suzuki, 2004; giangarelli et al., 2009). our results seem to indicate that the euglossini species were little sensitive to fragmentation. this finding could be explained by the proximity of the secondary fragment to primary forest, and that the continuous micro-environments between primary and secondary forest cannot adversely affect the foraging dynamic of these bee species. it is worth emphasizing that the results presented here should be interpreted with caution, mainly due to the small geographic distance between the sampled fragments and that euglossini males could fly long distances in search of the display territory, not being restricted to a specific location until establishment of the territory (janzen, 1971; pokorny et al., 2014). the frequency and abundance for all euglossini species were higher in the secondary forest, except eg. annectans which predominated in the primary forest. an effect which may play an important role in orchid bees attraction is the scent volatilization of each compound (silva & rebêlo, 2002). bees in general can use scent as main driver to find resource. thus, if air flow is stronger in open areas than within the forests, the scent can be spread easier in the secondary forest (in process of regeneration) than within primary forest. although similar findings in different environments and previous studies have providing insights in this topic (raw, 1989; milet-pinheiro & schlindwein, 2005; nemésio & silveira, 2006), a more controlled experiment to understand the air flow effect in orchid bees attraction in these forest types is still missing. and too, factors such as wind speed, air temperatures and wind direction may cause different concentrations of odours dispersion (murlis et al., 1992), because the density between greenwoods and forests is different (raw, 1989; nemésio & silveira, 2006). concerning the predominance of eg. annectans in the primary forest, our findings corroborated with the studies of knoll and penatti (2012) and ferronato et al. (2017) suggesting that the species’ distribution may be related to the more humid and preserved regions of the atlantic forest. on the other hand, eg. cordata, eg. pleosticta, ef. violacea and el. nigrita were more abundant and more frequently occurred in the secondary forest, which may be due to responses to the micro-factors that do not play or are rare in primary forests (as temperature variation, sunlight incidence and relative humidity) (nemésio & silveira, 2006). eufriesea violacea was clearly predominant in the secondary forest, although this fragment had been altered and it is in process of natural regeneration, the preference of the species for this fragment may suggest environmental quality in forest recovery, as suggested by giangarelli et al. (2009). the number of ef. aff. auriceps recorded in this study was higher to those found in other studies in paraná state (santos & sofia, 2002; sofia et al., 2004; gonçalves et al., 2014), assuming that the more preserved conditions of the primary forest may favor the regeneration of the secondary forest in this study, influencing the ecological rebalancing of the euglossini community, and this condition could explain the record of ef. aff. auriceps, besides highlighting the importance of preserving small fragments for the euglossini community. it has been suggested that some species are indicators of disturbed environments, such as eg. cordata and el. nigrita (rebêlo & cabral, 1997; peruquetti et al., 1999; silva & rebêlo, 2002). however, our data confirm that forests in regeneration are important for the euglossini communities and sociobiology 66(2): 293-305 (june, 2019) 301 can have an effect on the micro-distribution (and maybe in the foraging behaviour). the current data are not congruent that eg. cordata and el. nigrita are indicators of disturbed forest in this study, as stressed by ramalho et al. (2009) and aguiar and ganglianone (2012), despite their tolerance to disturbed forest be evident (santos & sofia, 2002; aguiar & ganglianone, 2012; nemésio, 2013; giangarelli et al., 2015; mateus et al., 2015; ferronato et al., 2017; medeiros et al., 2017). this leads us to the question: do the different degrees of regeneration in continuous micro-environments not change the community of orchid bees or change to the point that negatively affect the community in the conserved sites of the primary forest? we highlight here that eg. annectans was more abundant in the preserved sites and it seems to indicate the positive effect of continuous microenvironments in regeneration on the community of orchid bees, considering the potential of the species to act as bioindicator of conserved habitats or with more success in recovery or reforested forests (aguiar & ganglianone, 2012; knoll & penatti, 2012; ferronato et al., 2017). in addition, the less preserved micro-environments had a lower abundance of this species, suggesting some restriction of the species in these sites, or that the euglossini species respond differently to local characteristics of micro-environments (moreira et al., 2015). as well as, the male euglossini bees perform their mating flights in sunlight open spaces (stern, 1991), which is another factor that could have contributed to the higher uniformity of species assemblages (except eg. annectans) at sites of less conserved continuous micro-environments of primary and secondary forest (rep, ads, ims and ins). when we compared the micro-environments sampled within the two forest types we found that species composition and uniformity indexes were different among microenvironments, this may occur because some euglossini species require different habitats to meet their specific foraging and nesting requirements (roubik, 1989; roubik & hanson, 2004). further, although the investigations involving the flight distance of euglossini are not conclusive (dressler, 1982; pokorny et al., 2014), the great flight capacity of some orchid bee species is evident (janzen, 1971; wikelski et al., 2010; nemésio, 2012), this potential of long-range flight can have contributed to the presence of almost all species in the micro-environments, which may be promoting pollen flow across the undisturbed and disturbed areas and contributing to the forest regeneration (gathmann & tscharntke, 2002; jha & dick, 2010). the occurrence of euglossini in continuous microenvironments can be interpreted as a possibility of gene flow among plants of the different micro-environments, considering the pollination potential of these bees, responsible for pollination of a wide variety of orchidaceae species and many plant species of other botanical families (dressler, 1982; cameron, 2004; roubik & hanson, 2004), and probably they are involved in success of regeneration favoring the forest recovery and maintenance of the ecological system (ferronato et al., 2017). the role of scents it is common the use of aromatic baits in studies carried out to attract the male euglossini bees (sofia & suzuki, 2004; silveira et al., 2011; gonçalves et al., 2014; giangarelli et al., 2015). scent traps offered for short periods is the most used method (aguiar & ganglianone, 2008; silveira et al., 2011; gonçalves et al., 2014; giangarelli et al., 2015; oliveirajunior et al., 2015), however the method that we used was of traps offered for long period (8640 hours/12 months), in order to maximize the efficiency of field samplings. according to storck-tonon et al. (2013), research on the euglossini fauna requires an efficient sampling effort for better estimates of species richness, including rare species. in this perspective, we highlight that even the volatility being an important factor in the attractiveness of individuals, the method that we used here (long period) presented as a positive point the efficiency in the sampling effort (99.78) and the estimation of richness in the primary (8 spp.) and secondary (8 spp.) forest near the ideal (8.01 spp.), which means that most of the species sensitive to the four essences used in this study were sampled. the essences most likely attracted very rare species or some species that are passing by the forest environments, thus attending to an efficient sampling method that it is not commonly employed. concerning the attractiveness of the essences, we found that there is a complementarity of the scents used and the euglossini genera attracted. the cineole and eugenol attracted approximately 90% of the total of euglossa species, and methyl salicylate and vanillin attracted approximately 75% of eufriesea, eulaema and exaerete individuals. this result showed that the variety of essences chosen may affect the diversity of euglossini species sampled. therefore, we emphasize the importance of cautiously evaluating the preference of the chemical compounds for euglossini, since multiple factors, besides the essences, can affect the attractiveness of the bees (stern, 1991; murlis et al., 1992; nemésio & silveira, 2006; oliveira-junior et al., 2015). here, we showed that continuous micro-environments seem to favor the reintegration of the euglossini community in forests in regeneration (secondary) close to primary forests. moreover, despite of temporal effect and euglossini fauna composition in different micro-environments, the primary and secondary forest do not seem to have an effect on the temporal variation and species richness. another important point is the complementarity of the essences in the attractiveness of euglossini genera, aiming that the choice of the compound can influence the composition and abundance of the species. although previous studies have focused on the responses of orchid bees to habitats and regions diversity (aguiar & ganglianone, 2012; aguiar et al., 2014; cordeiro et al., 2012; gonçalves et al., 2014; giangarelli et al., 2015; botsch et al., 2017; ferronato et al., 2017), few studies have investigated until now how micro-environments affect the structure and ai sobreiro et al. – micro-environments associated orchid bees benefit302 responses of these bees to local characteristics (abrahamczyk et al., 2011), especially between nearby fragments or in microenvironments that form a forest matrix. forest fragments are generally small and isolated (ribeiro et al., 2009) and in urban centers are restrict to green areas of different extensions which may affect the euglossini responses to forest structure of these green environments (ferreira et al., 2013), but our study showed that continuous micro-environments can favor the uniformity and reintegration of orchid bees, so we suggest that these facts should be considered in future actions of conservation and recovery of areas. acknowledgments we would also like to thank dr. yzel súarez rondon for his help with statistical calculations. thanks in particular to dr. josué raizer for the help in the analysis, graphics and statistics doubts. we thank veridiana araújo alves da costa pereira, rodolfo rubik, jorge borges dos santos, edson zanlorensi, and jarbas aguinaldo teixeira for their assistance in field colletions. we are grateful to the refúgio biológico bela vista (itaipu binacional) for providing transportation to the study sites and materials, as well as to coordenação de aperfeiçoamento de pessoal de nível superior (capes) for financial support, universidade federal da grande dourados (ufgd) for providing materials and instituto chico mendes de conservação da biodiversidade (icmbio-system) for permission to collect bees. authors’ contribution ai sobreiro, lls peres and vv alves-junior: experimental design. ai sobreiro and lls peres: fieldword and data collection. ai sobreiro: data analysis. ai sobreiro, lls pesres, s boff, jr amaral and vv alves-junior: writing of the manuscript. references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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(accessed date: 10 january, 2017). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.265-273sociobiology 61(3): 265-273 (september 2014) food competition mechanism between solenopsis invicta buren and tapinoma melanocephalum fabricus biqiu wu1,2, lei wang1, guangwen liang1, yongyue lu1, ling zeng1 introduction solenopsis invicta buren is a dangerous quarantine pest that significantly affects public safety, human health, husbandry, and the ecological environment in its invasion regions. s. invicta builds its nests in soil, workers may infiltrate and destroy infrastructure, especially underground wires, cables, and electrical equipment, at cost of millions of dollars; meanwhile, because of its aggressive, death of creatures also leads by fire ant (vinson, 2013). s. invicta has spread to and propagated in china, and occasionally threaten the people´s health (wang et al., 2013). s. invicta directly feeds on crops, livestock, and poultry and have thus caused huge economic losses in husbandry (lofgren et al., 1975; stewart & vinson, 1991; jetter et al., 2002). s. invicta also attacks young birds, spawn, calves of sea turtles and other reptiles, and small rodents (drees, 1994; giuliano et al., 1996; allen et al., 1997; abstract this study compared the amount of food resource depletion and interference competition at the individual and colony levels between solenopsis invicta and tapinoma melanocephalum in laboratory. the consumption of sausage, honey water, and mealworm by s. invicta colonies were of equal worker number was higher than that by t. melanocephalum colonies. however, the amounts of sausage, honey water, and mealworm depleted by s. invicta colonies were of equal worker biomass were lower than those by t. melanocephalum colonies. the consumption of sausage and mealworm by s. invicta colonies were of equal worker biomass were also significantly lower than that by t. melanocephalum colonies. individual-level interference competition between s. invicta and t. melanocephalum colonies in small space was intense. competition intensity and the mortality rate reached their maximum when the worker numbers of both colonies were equal. in any proportion, the mortality rate of t. melanocephalum reached over 80%, higher than that of s. invicta. s. invicta colonies were of equal worker biomass and number recruited more workers for colony-level interference competition and used more resources. but the death rates of s. invicta colonies were higher than those of t. melanocephalum colonies. the highly exploitative and interferencecompetitive of s. invicta in trails had restricted the foraging behavior and active region of t. melanocephalum. sociobiology an international journal on social insects 1 south china agricultural university, guangzhou, china. 2 guangxi province academy of agricultural sciences, guangxi, china. article history edited by jacques h. c. delabie, uesc, brazil received 15 deceber 2013 initial acceptance 15 may 2014 final acceptance 18 july 2014 keywords invasive species; interspecific coexistence; interspecific competition; experiments corresponding author ling zeng red imported fire ant research center south china agricultural university guangzhou 510642, china e-mail: zengling@scau.edu.cn parris et al., 2002; pascoe, 2002; allen et al., 2004). invasive ants significantly influence local ant populations, especially ant species with similar ecological characteristics (holway et al., 2002). for instance, s. invicta has replaced solenopsis geminata (f.) and solenopsis xyloni mccook in the north american ecological system through competitive exclusion after invasion (wilson and brown jr, 1958; porter et al., 1988; porter, 1992; porter and savignano, 1990). such replacement decreased the abundance and diversity of local ant colonies (porter & savignano, 1990) and even changed the coexistence mode of surviving local ant colonies in the biogeographic balance (gotelli & arnett, 2000). in 2004, s. invicta was found in wuchuan, guangdong province, indicating that the species had invaded the continental china (wang et al., 2013). the invasion of south china by s. invicta has significantly decreased the diversity of local ant communities research article ants bq wu et al food competition mechanism between ants266 (shen et al., 2007; wu et al., 2008). however, few studies have investigated the influence of s. invicta invasion on the dominant local species in guangdong, tapinoma melanocephalum (wu et al., 2008), as well as the coexistence and competition mechanisms between these species. this study examined the food resource depletion and interference competition at the individual and colony levels between s. invicta and t. melanocephalum laboratory populations to explore the competition and coexistence mechanisms of t. melanocephalum in response to s. invicta invasion in guangdong. materials and methods ant samples s. invicta and t. melanocephalum colonies were collected from polygyne colonies at wild grass ground or litchi orchard in southern china and maintained at the south china agricultural university and red imported fire ant research center, guangdong province. colonies of s. invicta and t. melanocephalum contained queens, workers and included immature at all developmental stages. these colonies were separated from the soil and placed in an open plastic box (23.5 cm length × 15.5 cm width × 9.0 cm hight) with small plastic boxes (8.5 cm length × 6.0 cm width × 5.0 cm hight) with wet plaster to serve as nest chambers, and then placed in floors at 28 °c, on a diet of 20% honey water (a test tube half full of honey water and plugged with cotton) and fresh mealworms (coleoptera: tenebrionidae) until needed for experiments. the inside walls of the bigger boxes were coated with the fluoropolymer resin, fluon (polytetrafluoroethylene, ici fluoropolymers, exon, pa) to prevent them from escaping. comparison between amounts of food resource depletion of s. invicta and t. melanocephalum test food the following were the food used in the tests: honey a high carbohydrate food resource (guangzhou baoshengyuan corporation); sausage a proteinand lipidrich artificial food resource (guangdong shuanghui food corporation); and mealworm (coleoptera: tenebrionidae) a high protein natural food resource (purchased from market). test method experimental colonies of s. invicta and t. melanocephalum were set up on both an equal worker biomass and equal worker number basis for average worker size varied among their forms (morrison et al., 2000). each experimental equal worker biomass colony contained 0.5 g of workers, 0.25 g of brood (included eggs, larvae and pupae) and two queens. each experimental equal worker number colony contained 1000 workers, 0.25g of brood and two queens. because counting live worker ants was not practical and the workers of t. melanocephalum were activity quickly, the method of workers added to colonies was operated like morrison (2000). but both ants were anaesthetized with ethyl ether before weigh with an electronic balance. we found that ethyl ether had no effect on ants in preliminary trials. each experimental colony occupied a plastic box (25 cm length × 18 cm width × 7 cm hight), equipped with a small plaster plastic box (8.5 cm length ×6 cm width × 5 cm hight) to serve as nest chambers, the sides of which were coated with fluon to prevent escapes. the mother colonies from which the experimental colonies were kept in the laboratory at 28°c for at least two weeks before experimental colony formation, on a diet of 20% honey water (a test tube half full of honey water and plugged with cotton) and fresh mealworms every day. the experimental colonies were placed in the laboratory at 28°c and 60% 75% rh, starved for 24 h before the beginning of the trials to produce a uniform state of hunger. each box containing an experimental colony was connected via a test tubing (1 cm inside diameter) to an adjacent (empty) box of the same dimensions. holes were drilled in the side of the box near the bottom to allow insertion of the tubing. 2.0g of sausage, mealworm, or 20% honey water (drop on the cotton) was placed in the test tube, the ants were allowed to forage for 24h, and then weighed the remaining food with an electronic balance. we conducted ten replicate trails for each food item, for each equivalent colony. test was stopped when the workers carried larvae and spawn to food, but this situation was rarely observed. portions of 2.0g of sausage, mealworm, and honey water were placed in a clean plastic tube, kept at the same conditions, but protected from the ants as evaporation loss control. individual level interference competition testing method healthy and similar size workers of s. invicta and t. melanocephalum were selected to carry out the interference competitive abilities at the individual level, and the agonistic interactions between s. invicta and t. melanocephalum were staged in small arenas. arenas consisted of a dry plastic petri dish (12 cm inside diameter) which was sterilized with 75% alcohol, washed with distilled water, with inner sides coated with fluon. ants were counted by allowing them to climb onto a small paint brush and then placing them into separate plastic boxes (23.5 cm length × 15.5 cm width × 9.0 cm hight) with inside walls coated with fluon. t. melanocephalum workers were placed into the plastic petri dish first, s. invicta workers were placed into the arena later. we chose five encounter ratios of s. invicta to t. melanocephalum, 1) 50 s. invicta sociobiology 61(3): 265-273 (september 2014) 267 workers to 10 t. melanocephalum workers (5:1); 2) 45 s. invicta workers to 15 t. melanocephalum workers (3:1); 3) 30 s. invicta workers to 30 t. melanocephalum workers (1:1); 4) 15 s. invicta workers to 45 t. melanocephalum workers (1:3); and 5) 10 s. invicta workers to 50 t. melanocephalum workers (1:5). the number of major s. invicta workers used in areas interaction in each encounter ratio accounted for 10%. we chose the t. melanocephalum workers in agonistic interactions were at the uniform size. the ants were observed for 3 h at 26ºc and 65% rh. interaction behavior was recorded, and the number of dead or mortally wounded workers was noted at the end of the trials. we conducted ten replicate trials for each encounter ratio. healthy individual ants (40 workers) of s. invicta and t. melanocephalum were independently placed in a clean and dry plastic petri dish; the number of dead workers was recorded to adjust the control death rate. this trail was conducted in ten replicates. calculation formula of adjusted death rate adjusted death rate (%) = (control survival rate % − treatment survival rate %) / control survival rate % × 100 colony level interference competition testing method the experimental colonies of both ants which deal with like 1.2.2 were evaluated in two pairwise comparisons. an equal worker biomass or an equal worker number colony of s. invicta was connected to an equal ‘size’ colony of t. melanocephalum via three intervening (empty) boxes by 10 cm length of tygon tubing (fig. 1). in the experimental setup, the boxes containing the ant colonies on each end are referred to as ’colony’ boxes; the empty boxes adjacent to each colony boxes are referred to as ‘adjacent’ boxes; and the connecting box in the middle is referred to as the ‘center’ box (morrison et al., 2000). 1.0 g of fresh mealworm and 0.5 g of sausage were placed in the center box and two adjacent boxes before the experiment, and then recorded the number of active and dead workers in the three empty boxes (center and both adjacent) when the ants foraging foods at 0.5 h, 24 h, 48 h, and 72 h, the number of dead workers and colonies, which invaded by another ants, in colony boxes when the ants foraging foods at 72 h were also noted. to statistically compare the survival rate of s. invicta and t. melanocephalum workers in the three empty boxes after 0.5 h min, 24 h, 48 h, and 72 h, the mortality of both ants in five boxes after 0.5 h, 24 h, 48 h, and 72 h, the number of invaded colonies at the end of trail. the interference competition between s. invicta and t. melanocephalum in each equal ‘size’ colony was conducted ten replicate trails. data processing the mean amount of resource acquired by each equal s. invicta and t. melanocephalum colonies was compared by t-test, the mean amount of resource acquired by s. invicta or t. melanocephalum was compared by one-way anova, for each type of resource. all pairwise comparisons of means were made by tukey method of multiple comparisons. the data were processed by spss 18.0. results comparison amounts of food resource depletion between s. invicta and t. melanocephalum resource consumption of the different food types varied in s. invicta and t. melanocephalum (table 1). when colonies were of equal worker number, s. invicta consumed significantly more sausage and honey water than did t. melanocephalum (p< 0.05). when colonies were of equal worker biomass, s. invicta consumed significantly less sausage and mealworm than did t. melanocephalum (p< 0.01).fig. 1. design of colony level interference competition experiments. table 1. amount of food resource depletion of s. invicta and t. melanocephalum after 24 h food resource colonies were of equal worker number colonies were of equal worker biomass t. melanocephalum s. invicta t. melanocephalum s. invicta sausage 0.020±0.001 0.027±0.003* 0.045±0.005 0.016±0.002** honey water 0.044±0.002 0.127±0.028* 0.065±0.004 0.054±0.007ns mealworm 0.018±0.001 0.019±0.002ns 0.033±0.003 0.015±0.001** note: “ns” indicates lack of significance. “*” and “**” represent significance between the quantities of food transferred by t. melanocephalum and s. invicta with equal worker number or biomass at the 0.05 and 0.01 probability levels, respectively (independent t-test). bq wu et al food competition mechanism between ants268 individual level interference competition in individual level competition, four interference conditions were observed: 1) t. melanocephalum workers voluntarily evading s. invicta to avoid fighting are referred to as ’ignore’; 2) when both ants encounter, they touch each other by antennae then avoiding rapidly are referred to as ‘contact’, we can see this condition frequent when t. melanocephalum workers less than s. invicta; 3) two or more native ants touch a invader ant by antennae, turn-back to eject defensive compounds from pygidial glands and avoiding quickly are referred to as ‘chemical defense’; 4) ants engaging in “fights” often in prolonged grappling with frequent biting and flexing their gaster in the direction of opponents are referred to as ‘physical aggression’, fighting usually occurred “group” attack (2 or more workers) by t. melanocephalum ants on single s. invicta ant, although this condition rarely saw in trails. there was no incidence of multiple invaders fighting lone native opponent. the individual-level interference competition between s. invicta and t. melanocephalum after 3 h were shown in table 2. the mortality rates of both ant workers were the lowest when the ratio of s. invicta to t. melanocephalum was 1:5. the mortality rates of both ant workers increased when the number of t. melanocephalum workers equal to s. invicta. when both ant workers were 30 (s. invicta to t. melanocephalum=1:1), the death rate of t. melanocephalum was 100%, and the mortality of s. invicta was more than 50%. the mortality rates of t. melanocephalum were over 99% in the ratio of 3:1 and 5:1 (s. invicta to t. melanocephalum), but no dead workers of s. invicta were observed. colony level interference competition proportion of active s. invicta workers in two adjacent boxes and center box to count the survival workers of s. invicta in each kind of food foraging in adjacent and center boxes, the number of survival workers appearing in each box accounts for the total workers (including dead and alive ants) which foraging in adjacent and center boxes as proportion of active s. invicta workers (table 3). when colonies were of equal worker biomass, sausage was used as the food resource, the active s. invicta workers in s. invicta colony adjacent box accounted for the maximum proportion (14.97%) at 0.5 h, and no workers were found in the center box and t. melanocephalum colony adjacent box. at 24 and 48 h, active s. invicta workers in the s. invicta colony adjacent box reached their maximum proportions (12.24% and 11.11%, respectively), which were significantly higher than those in t. melanocephalum colony adjacent box. at 72 h, active s. invicta workers in center box table 2. mortality rates of s. invicta and t. melanocephalum workers in individual level interference competition after 3 h. ant species confrontation ratio (s. invicta: t. melanocephalum) 1:5 1:3 1:1 3:1 5:1 s. invicta 0.15 0.22 0.57 0 0 t. melanocephalum 80.72 87.14 100 99.33 100 table 3. proportion of active s. invicta workers in the two adjacent boxes and center box. colonies were of equal worker biomass colonies were of equal worker number sausage mealworm sausage mealworm 0.5 h s. invicta colony adjacent box 14.97±13.56 a 16.54±8.87 a 14.98±11.94 a 16.22±9.65 a center box 0.00±0.00 b 0.85±2.68 b 0.00±0.00 b 0.00±0.00 b t. melanocephalum colony adjacent box 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b 24 h s. invicta colony adjacent box 12.24±4.08 a 13.37±4.28 a 13.61±6.02 a 10.15±4.24 a center box 7.63±3.21 b 9.18±4.32 b 4.74±5.68 b 10.91±2.91 a t. melanocephalum colony adjacent box 4.75±5.66 b 4.05±5.06 c 5.72±6.34 b 7.89±5.35 a 48 h s. invicta colony adjacent box 11.11±3.84 a 12.39±5.35 a 13.66±2.47 a 11.46±5.56 a center box 10.83±5.23 a 9.81±3.39 a 7.00±2.73 b 9.68±4.99 a t. melanocephalum colony adjacent box 6.07±4.63 b 5.25±4.97 b 7.86±5.20 b 7.04±3.28 a 72 h s. invicta colony adjacent box 11.49±3.18 a 12.25±7.36 a 12.74±4.73 a 12.93±3.32 a center box 11.89±2.84 a 5.32±5.95 b 7.62±6.26 ab 9.76±4.19 a t. melanocephalum colony adjacent box 4.65±5.01 b 7.31±5.28 b 5.45±5.79 b 5.19±4.60 b note: same-column means followed by the same letter are not significantly different at the 0.05 and 0.01 levels, respectively, as in table 4. sociobiology 61(3): 265-273 (september 2014) 269 reached their maximum proportion (11.89%), which was significantly higher than that in t. melanocephalum colony adjacent box. when mealworm was used as the food resource, active s. invicta workers in s. invicta colony adjacent box reached their maximum proportions and significantly higher than those in t. melanocephalum colony adjacent box at 0.5, 24, and 48 h. at 72 h, active s. invicta in workers in center box reached their maximum proportion (12.25%) and significantly higher than those in center box and t. melanocephalum colony adjacent box. when colonies were of equal worker number, sausage as the food resource, active s. invicta workers in s. invicta colony adjacent box had their maximum proportion at 0.5 h, and no workers were found in center box and t. melanocephalum colony adjacent box. at 24 and 48 h, active s. invicta workers in s. invicta colony adjacent box reached their maximum proportions (13.61% and 13.66%, respectively), significantly higher than those in center box and t. melanocephalum colony adjacent box. at 72 h, active s. invicta workers in s. invicta colony adjacent box reached their maximum proportion (12.74%), significantly higher than that in t. melanocephalum colony adjacent box. when mealworm was the food resource, active s. invicta workers in s. invicta colony adjacent box reached their maximum proportion (16.22 %) at 0.5 h, and no workers were found in the center box and t. melanocephalum colony adjacent box. at 24 h, active s. invicta workers in center box reached their maximum proportion (10.91%), indistinctively with those in center box and t. melanocephalum colony adjacent box. at 48 and 72 h, active s. invicta workers in s. invicta colony adjacent box reached their maximum proportions (11.46% and 12.93%, respectively), significantly higher than that in t. melanocephalum colony adjacent box. proportion of active t. melanocephalum workers in two adjacent boxes and center box the proportion of the active t. melanocephalum workers in which colonies were equivalent by worker biomass or number was counted and shown in table 4. when colonies were of equal worker biomass, sausage was used as the food resource, active t. melanocephalum workers were found only foraging in t. melanocephalum colony adjacent box at 0.5 h. active t. melanocephalum workers in t. melanocephalum colony adjacent box reached their maximum proportions (14.05%, 11.33%, and 12.06%, respectively) at 24, 48, and 72 h, therefore the minimum proportion of native ants foraging in s. invicta colony adjacent box was observed at the same time. at 24 and 48 h, the proportions of active t. melanocephalum workers in t. melanocephalum colony adjacent box were significantly higher than those in s. invicta colony adjacent box; at 72 h, the proportion of active t. melanocephalum workers in t. melanocephalum adjacent box was significantly higher than that in s. invicta colony adjacent box. when mealworm was used as the food resource, active t. melanocephalum workers were found only in t. melanocephalum colony adjacent box at 0.5 h. at 24 and 48 h, active t. melanocephalum workers in t. melanocephalum colony adjacent box reached their maximum proportions (14.13% and 8.92%, respectively), and the proportions of active t. melanocephalum workers in t. melanocephalum adjacent box were significantly higher than those in s. invicta colony adjacent box at 24 h. at 72 h, the proportions of active t. melanocephalum workers foraging in two adjacent and center boxes was non-significant. when colonies were of equal worker number, sausage was used as food resource, active t. melanocephalum workers table 4. proportion of active t. melanocephalum workers in two adjacent boxes and center box. colonies were of equal worker biomass colonies were of equal worker number sausage mealworm sausage mealworm 0.5h s. invicta colony adjacent box 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b center box 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b t. melanocephalum colony adjacent box 16.98±7.91 a 17.17±7.34 a 15.10±11.67 a 15.29±11.33 a 24h s. invicta colony adjacent box 3.08±4.50 b 2.87±5.30 b 2.91±5.04 b 0.60±1.88 b center box 6.40±5.03 b 5.09±6.01 b 6.48±7.04 b 10.03±4.88 a t. melanocephalum colony adjacent box 14.05±7.62 a 14.13±7.54 a 13.91±5.38 a 13.23±6.47 a 48h s. invicta colony adjacent box 3.31±3.71 b 5.28±7.51 a 4.24±5.74 b 1.07±3.38 b center box 9.14±6.82 a 7.85±7.29 a 3.63±4.79 b 8.55±6.37 a t. melanocephalum colony adjacent box 11.33±8.23 a 8.92±8.88 a 14.23±8.03 a 12.87±7.65 a 72h s. invicta colony adjacent box 3.38±4.62 b 7.21±11.66 a 3.11±5.12 b 2.05±4.41 b center box 7.29±6.62 ab 4.86±8.28 a 4.80±7.04 b 8.27±6.08 a t. melanocephalum colony adjacent box 12.06±8.89 a 4.86±8.28 a 14.61±5.19 a 12.48±8.19 a bq wu et al food competition mechanism between ants270 were found only in t. melanocephalum colony adjacent box at 0.5 h. at 24, 48, and 72 h, active t. melanocephalum workers in t. melanocephalum colony adjacent box reached their maximum proportions (13.91%, 14.23%, and 14.6%, respectively), significantly higher than that in center box and s. invicta colony adjacent box. when mealworm was used as food resource, active t. melanocephalum workers were found only in the t. melanocephalum colony adjacent box at 0.5 h. at 24, 48, and 72 h, active t. melanocephalum workers in t. melanocephalum colony adjacent box and center box reached their maximum proportions (13.23%, 12.87%, and 12.48%, respectively), significantly higher than that in s. invicta colony adjacent box. comparison of workers mortality rate in five boxes after 72 h although the case of invaders presenting to each other nest chamber can be saw in trails, intense fighting between s. invicta and t. melanocephalum was rarely observed, and ants usually establish their territories after 72 h (morrison et al., 2000). therefore, we recorded the workers mortality rate foraging in two colony boxes, two adjacent boxes, and center box after 72 h (table 5). when colonies were of equal worker biomass, in the case of sausage depletion by two ants, the mortality rates of both ants in t. melanocephalum colony box were the highest (>21%), followed by those in t. melanocephalum colony adjacent box and center box. the death rates of t. melanocephalum in s. invicta colony box and adjacent boxes were zero. in the case of mealworm depletion, the death rates of both ants in t. melanocephalum colony box were the highest, followed by those in t. melanocephalum colony adjacent box. the death rates of t. melanocephalum in center box, s. invicta colony adjacent box, and s. invicta colony box were zero. when colonies were of equal worker number, in the case of sausage depletion, the death rates of both ants in t. melanocephalum colony box were the highest (>22%). the death rate of t. melanocephalum workers in s. invicta colony box and s. invicta workers in t. melanocephalum colony adjacent box were the lowest. in the case of mealworm depletion, the death rates of both ants in t. melanocephalum colony box were the highest (>24%), followed by those in t. melanocephalum colony adjacent box and center box. the death rates of t. melanocephalum in s. invicta colony and adjacent boxes were zero. colonies of s. invicta and t. melanocephalum invading to each other nest chamber after 72 h we recorded and counted the colony number of s. invicta and t. melanocephalum workers invaded to opponent colony boxes after 72 h (fig. 2). when sausage was used as food resource, five s. invicta colonies (50% of all colonies in trails) which colonies were of equal worker biomass invaded to t. melanocephalum colony box, and no-one t. melanocephalum colony invaded to s. invicta colony box. nine s. invicta colonies (90% of all colonies in trails) which colonies were of equal worker number invaded to t. melanocephalum colony box, and six t. melanocephalum colonies (60% of all colonies in trails) invaded to s. invicta colony box. when mealworm was used as food resource, seven s. invicta colonies (70% of all colonies in trails) which colonies were of equal worker biomass, invaded t. melanocephalum colony box, while only one t. melanocephalum colony invaded s. invicta colony box. eight s. invicta colonies (80% of all colonies in trails) which colonies were of equal worker number, invaded t. melanocephalum colony box, while only two t. melanocephalum colonies invaded s. invicta colony box. these results indicate that s. invicta was more aggressive. table 5. comparison of workers mortality rate in five boxes after 72 h test box colonies were of equal worker biomass colonies were of equal worker number sausage mealworm sausage mealworm si tm si tm si tm si tm s. invicta colony box 31.29 0 21.55 0 27.88 0.96 28.64 0 s. invicta colony adjacent box 12.93 0 11.21 0 7.69 2.40 8.04 0 center box 2.72 2.04 5.60 0 3.85 1.44 4.02 2.01 t. melanocephalum colony adjacent box 5.44 2.72 3.88 9.05 1.92 3.37 2.01 4.52 t. melanocephalum colony box 21.09 21.77 28.88 19.83 22.12 28.37 24.12 26.63 note: si =s. invicta and tm = t. melanocephalum. fig. 2. colonies of s. invicta and t. melanocephalum invading to each other nest chamber after 72 h. sociobiology 61(3): 265-273 (september 2014) 271 discussion interspecific competition refers to the mutual interference or inhibition between two or more species. the essence of interspecific competition lies in the efficiency reduction of the reproduction, survival, growth, and other aspects of individuals of one species because of the exploitation or interference of common resources by individuals of another species. interspecific competition primarily refers to resource competition, namely, the mutually unfavorable effects of commonly exploiting scarce resources on biological individuals. resource competition can be classified into exploitation and interference competition. in exploitation competition, individuals of one species obtain more common resources than those of another species; in interference competition, individuals of one species limit or prevent individuals of another species from using the resources (reitz and trumble, 2002). interspecific competition is considered key in structuring local ant communities, and it has been described as the “hallmark of ant ecology” (cerda et al., 2013). local ants have been replaced by s. invicta because this species is highly exploitative and interference-competitive (porter and savignano, 1990; bhatkar et al., 1972; obin and vander meer, 1985; jones and phillips jr, 1987; hook and porter, 1990; jones and phillips jr, 1990; morrison, 2000, 1999, 2002). in this study, we found that food consumption of t. melanocephalum colonies which colonies were of equal worker number, was less than did s. invicta, t. melanocephalum workers (monomorphic, one-sized) are extremely small, 1.3 to 1.5 mm long (scheurer et al., 1998), only s. invicta worker (involving major and minor ants) was 3.24 times the average weight of t. melanocephalum may be responsible for the results. higher amount of food resource depleted by equal worker biomass t. melanocephalum colonies may be due to the number of t. melanocephalum workers more than s. invicta. in other words, one t. melanocephalum worker need less food than s. invicta to maintain its daily activities. intense fighting between s. invicta and t. melanocephalum was found in individual level interference competition in a small arena when worker number in both ants was equivalent or there were more workers of s. invicta than t. melanocephalum. s. invicta workers attacking t. melanocephalum usually by ‘physical aggression’, but t. melanocephalum worker would prefer to use ‘chemical defense’ to repel invader ants, which t. melanocephalum displayed alerting, alarm behavior, and the daubing of pygidial gland secretions (tomalski et al., 1987). t. melanocephalum ants would initiative attacking s. invicta when its workers were more than invaders, and a single s. invicta worker kept far away from t. melanocephalum ants to avoid attacked for application of the pygidial gland secretion to the legs or antennae of a foreign ant often resulted in a hindrance of movement or in the limbs adhering together (tomalski et al., 1987). however, mortality rate of t. melanocephalum was more than 80% in all treatments, s. invicta workers were more aggressive and its size (involving major and minor ants) bigger than t. melanocephalum may be the main reasons. in colony level interference competition, both ant colonies of equal worker biomass or number foraging in colony boxes and each colony adjacent box were more, and then foraging in father distance food resources. intense fighting in both ants was usually saw in t. melanocephalum colony box for native ant nest chamber intruded by s. invicta workers, but s. invicta paying its stronger aggressiveness for the higher mortality rate in this interference competition. in the indoor interference competition between s. invicta and t. melanocephalum, s. invicta recruited larger workers on food resources, intruded to t. melanocephalum colony box, indicated that s. invicta was highly exploitative and interference-competitive, which restricts the activity of t. melanocephalum. chemical defense used by t. melanocephalum repelled the exotic ant was a major reason to explain the highly mortality rate in s. invicta, and implied that t. melanocephalum was the native ant against with s. invicta in invaded region. the reasons can explain the phenomenon of coexistence between t. melanocephalum and s. invicta in invaded region in south china are as follow: 1) t. melanocephalum is opportunistic nester in places that sometimes remain habitable for only a few days or weeks (hölldobler and wilson, 1990), and highly adaptable in its nesting habits outdoors or indoors, the colonies occupy local sites include tufts of dead but temporarily moist grass, plant stems, and cavities beneath detritus in open, rapidly changing habitats (oster and wilson, 1978). indoors, the ant colonizes wall void or spaces between cabinetry and baseboards. it will also nest in potted plants (smith and whitman 1992) and the ant nest which abandon by s. invicta populations (we found in wild grass ground or litchi orchard in south china); 2) multiple queens may be spread out in multiple subcolonies, new colonies are probably formed by budding and there does not appear to be any infighting between members of different colonies or nests (smith and whitman 1992); 3) t. melanocephalum workers are favor many food resources, they are fond of honeydew and tend honeydew-excreting insects, and foraging on honeydew more efficiently than s. invicta (zheng and zhang, 2010) and t. melanocephalum workers are extremely small, a little food can maintain its daily activities; 5) t. melanocephalum has the habit of running rapidly and erratically when disturbed (li et al., 2008), using pygidial gland secretions to repel invaders; in addition, this native ant has higher tolerance to high temperature than s. invicta (zheng et al., 2007). s. invicta, the invaders, is foraging all year in guangdong province, has great capacity for plundering food resources, more aggressive, and a larger population, therefore they exhibits intense competitiveness than t. melanocephalum in our research. short-term invasions by s. invicta also bq wu et al food competition mechanism between ants272 significantly affect t. melanocephalum in simple habitats (unpublished). if s. invicta invaded in a shortage resource and vegetation over simplified habitat, for example in lawn (t. melanocephalum workers only can be saw 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[in chinese, english abstract]. journal of environmental entomology, 32: 312-317. doi: 10.13102/sociobiology.v67i1.4478sociobiology 67(1): 112-120 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the neotropical region has a rich fauna of social insects, as well as a great diversity of social wasps (vespidae, polistinae) (richards, 1978; carpenter & marques, 2001; noll, 2013). polistinae wasps are represented by around 940 species (noll, 2013; somavilla et al., 2014a) and the brazilian social wasps fauna (polistinae) is the richest in the world, with more than 300 species, 104 of them endemic from brazil (somavilla et al., 2014b). this subfamily is divided into four tribes: mischocyttarini (with one genus, mischocyttarus), polistini (with one genus, polistes), epiponini (19 genera) and ropalidiini, with no representatives in brazil (noll, 2013). abstract a survey of social wasps (vespidae, polistinae), common insects of neotropical fauna, which perform a great variety of ecosystemic services, was conducted for the first time in areas of the amazon forest in rondônia state. the state is part of the western amazon, a region harboring high biodiversity, which is under threat due to constant deforestation. three areas were sampled, and the wasps were actively collected, and an attractive liquid was sprayed onto the vegetation to bait the wasps. two thousand nine hundred and sixty-one wasps were sampled in all three areas, distributed in 72 species of 15 genera. thirtynine species were recorded for the first time in the state and three others (agelaia melanopyga cooper, brachygastra cooperi andena and carpenter and polybia diguetana du buysson) represents the first record for brazil. agelaia lepeletier was the most abundant genus in all areas and the greatest species richness was found for polybia lepeletier. the highest number of species was recorded in floresta nacional do jamari (51), followed by estação ecológica de cuniã (46) and forest fragment of universidade federal de rondônia (39). the latter also presented the highest number of different genera. the great diversity, mainly for epiponini, which represented 64 of the 72 species, can be attributed to location of the areas and methodology. sociobiology an international journal on social insects b gomes1, cs lima2, m silva3, fb noll3 article history edited by evandro nascimento silva, uefs, brazil received 19 april 2019 initial acceptance 27 september 2019 final acceptance 06 january 2020 publication date 18 april 2020 keywords epiponini, new records, rainforest, social insects, swarm-founding wasps. corresponding author marjorie da silva depto. zoologia e botânica instituto de biociências, letras e ciências exatas, ibilce/unesp rua cristóvão colombo nº 2265 jardim nazareth, cep: 15054-000 são josé do rio preto-sp, brasil. e-mail: marjoriebio@gmail.com social wasps perform important and priceless ecosystemic services. as predators of other insects, they are important in the maintenance of food chains and in biological control, since they feed on larvae that are crop pests such as tomato, coffee, corn, eucalyptus, citrus fruits and vegetables (carpenter & marques, 2001; souza et al., 2008, 2010a; brügger et al., 2019; prezoto et al., 2019). these wasps also act as pollinators (hunt et al., 1991; hermes & köler, 2006; santos et al., 2006; nadia et al., 2007) and are well known for symbiosis with ants (espelie & herman, 1988), honey insects (letourneau & choe, 1987) and birds (joyce, 1993). some species are still useful as indicators of environmental quality (souza et al., 2010b; urbini et al., 2010). 1 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo (usp), ribeirão preto-sp, brazil 2 universidade federal de rondônia (unir), campus josé ribeiro filho, porto velho-ro, brazil 3 instituto de biociências, letras e ciências exatas, universidade estadual paulista júlio de mesquita filho (unesp), são josé do rio preto-sp, brazil research article wasps high number of species of social wasps (hymenoptera, vespidae, polistinae) corroborates the great biodiversity of western amazon: a survey from rondônia, brazil sociobiology 67(1): 112-120 (march, 2020) 113 the brazilian amazon harbor the great diversity of polistinae wasps (silveira, 2002; silva & silveira, 2009), where 20 genera and about 200 species were recorded, representing 70% of the brazilian social wasps fauna (silveira, 2002). although this number seems very expressive, they are probably underestimated, given the extent of the amazon and the low number of surveys for social wasps in this biome: ilha de maracá – roraima (raw, 1998), caxiuanã – pará (silveira, 2002; silveira & silva, 2009), serra do divisor – acre (morato et al., 2008), porto acre, senador guiomard and rio branco – acre (gomes et al., 2018), dos lagos region – amapá (silveira et al., 2008), mamirauá and alvarães – amazonas (silveira et al., 2008), reserva ducke and jaú national park amazonas (somavilla et al., 2014b; 2015) and gurupi biological reserve – maranhão (somavilla et al., 2014a). there is also a bias in sampling, with most of the inventories (and consequently, the highest number of recorded species) concentrated in the amazonas state (barbosa et al., 2016). thus, further investigations should lead to an increase in recorded species. located in the northwestern of brazil, the state of rondônia is part of the western brazilian amazon (martins et al., 2014), which includes bolivia, colombia, ecuador, peru, and western brazil (finer et al., 2015). this region is one of the world’s last high-biodiversity wilderness areas (finer et al., 2015), characterized by large tracts of relatively intact humid tropical forest, which harbor an extraordinary species richness (bass et al., 2010; pointekowski et al., 2019). however, the western amazon has been suffering from constant deforestation (rosa et al., 2012; mayes et al., 2019). rondônia presents the third highest rate of deforestation and forest degradation of this biome, totaling 21% of the deforested areas of amazon from august 2014 to january 2016 (fonseca et al., 2016; pointekowski et al., 2019). the high index of deforestation in the state is a reflection of the expansion of the agriculture frontier towards northern brazil during the recent decades (souza jr. et al., 2013). the destruction of natural areas in the amazon, especially habitat fragmentation is the direct cause of the reduction of diversity due to severe abiotic and biotic changes (magnago et al., 2014; rocha et al., 2018). the only previous information regarding the social wasps of rondônia are the works of richards (1978), which is still the largest source of information on the distribution of epiponini and other neotropical polistinae, and raw (1988). in a current context of global climate change and accelerated species loss, the knowledge of biodiversity and the consequences of its destruction has become a major concern and challenge for the 21st century, since, as species disappear, we lose both known and unknown benefits they provide (gascon et al., 2015; troudet et al., 2017). despite the high abundance and richness in amazonian areas, knowledge about the diversity and biology of social wasps in the region is still underestimated. here, we presented the first survey of social wasps (polistinae) of rondônia state. material and methods three areas of amazon rainforest located in the northern state of rondônia, brazil were studied (fig 1): i – estação ecológica de cuniã (esec) with approximately 186.743,26 ha (icmbio, 2019a) is located between the cities of porto velho ro and humaita am (8º 05’ 24’’ s 63º 26’ 07’’ w); ii – floresta nacional do jamari (flona) with 222.114,24 ha (icmbio, 2019b) the region covers the districts of candeias do jamari, itapuã do oeste and cujubim ro (09º 00’ 00’’ s 62º 44’ 05’’ w); iii – forest fragment of universidade federal de rondônia (unir) in the capital porto velho (08º 49’ 53’’ s 63º 56’ 26’’ w), with approximately 50 ha (miranda, 2005). in the three areas, the vegetation is composed of dense ombrophilous forest and open ombrophilous forest. the climate is tropical humid, with an average temperature of 25 ºc, very rainy summer and winter a little drier. the relief is a little rough, and the average altitude is 240 meters (schlindwein et al., 2012; sedam, 2012). samples were performed from april 2010 to november 2011 in esec and, from december 2011 to december 2012 in flona and unir. wasps were collected actively (with the aid of an entomological net), follow the protocol developed by noll and gomes (2009), on trails or roads previously available in the forest. the protocol is based on the use of an attractive solution (50g of salt and 200g of sugar per liter of water), which was sprayed on the vegetation in five points equidistant 20 m. the points were observed individually for ten minutes and the liquid was reapplied every three hours of collection. fourteen collections of 12 hours (6:00 am to 06:00 pm) were made in each area, totaling 168 hours of work and 504 hours considering all areas. the collected specimens were stored in micro-tubes containing ethanol absolute. after identified, part of the collected material was deposited in the zoological collection of the museu paraense emílio goeldi, in belém, pará state, brazil (mischocyttarus) and in the hymenoptera collection of universidade estadual paulista “júlio de mesquita filho”, in são josé do rio preto, são paulo state, brazil. sampling adequacy was evaluated by the construction of a rarefaction curve for each area using the mao tau method (colwell et al., 2004). the non-parametric estimator jackknife 1 was used to compare the sample with the estimated richness since the species richness observed is often an additive estimator in relation to the real species richness (campos et al., 2013). both analyses were performed using past 3.24 (hammer et al., 2001). results a total of 72 species of 15 genera of social wasps were collected (table 1), most of them (17) sampled in the three areas, totalizing 2961 specimens. flona was the area with the highest number of species, 51, followed by esec, with 46 species and unir with 39 species. considering the number of b gomes, cs lima, m silva, fb noll – high number of species of social wasps in western amazon114 agelaia lepeletier, representing almost half (47.8%) of the total wasps sampled. the same occurred when each area was analyzed individually, with agelaia representing 46.8% of specimens sampled in flona, 58,7% in esec and 36% in unir. from the 72 species, 39 of them are new records for the state. other three species – agelaia melanopyga cooper 2000, brachygastra cooperi (richards, 1978), and polybia diguetana du buysson 1905 – were sampled for the first time in brazil. despite the high number of species sampled, jackknife values indicate that there were still species to be collected. however, the rarefaction curve (fig 2) tended to stabilize, indicating that the sampling effort was sufficient for a significant species registration. fig 2. rarefaction curve for species of social wasps (polistinae) collected in floresta nacional do jamari (flona), estação ecológica de cuniã (esec) and in the forest fragment of universidade federal de rondônia (unir). the x-axis represents the number of collections (1 to 14) and the y-axis represents the number of species sampled. the isolated symbols represent the richness estimated by the jackknife1 index, indicating that there were still species to be sampled for all areas. fig 1. satellite image of the areas sampled. a. floresta nacional do jamari (flona); b. estação ecológica de cuniã (esec); c. forest fragment of universidade federal de rondônia (unir). scale bar: 10km. source: googleearth. genera, 14 different genera were sampled in unir, followed by esec and flona, both with ten genera. four of the 15 genera sampled – apoica lepeletier, chartergellus bequaert, chartergus lepeletier, and pseudopolybia von dalla torre – were only found in unir. flona was also the area with the largest number of exclusive species (12), followed by unir (11) and esec (6). four of the six species of mischocyttarus de saussure were found only in esec. seventeen species were collected in all areas. polybia lepeletier and agelaia lepeletier were the most diverse genera, with 23 (one-third of species) and 13 species collected respectively. the greatest species richness for each area was also found for polybia, with 19 species in flona, 16 in unir and 14 in esec. despite the highest number of species of polybia, for all three areas, the most abundant genus was discussion from the surveys of social wasps carried out for the amazon biome, this survey sampled the highest number of epiponini species (64) and was the second in the number of polistinae species, 72 in total (the first was silveira, 2002 with 79 species sampled). three species were recorded for the first time in brazil. prior to this survey, the only distributional record for b. cooperi was the data of the holotype, in colombia (andena & carpenter, 2012). p. diguetana has already been recorded in bolivia, a country bordering rondônia. this species presents a broad distribution in other eastern amazon countries (peru, ecuador, and colômbia), venezuela, central america, méxico and southwestern of u.s.a. the presence of a. melanopyga in rondônia extends its distribution southernmost since the previous records are from central america, colombia and ecuador (richards, 1978; natural history laboratory iunh). sociobiology 67(1): 112-120 (march, 2020) 115 species flona esec unir total epiponini agelaia angulata (fabricius, 1804) 25 49 74 agelaia cajennensis (fabricius, 1798)1 13 21 21 55 agelaia centralis (cameron, 1907)1 15 53 68 agelaia fulvofasciata (degeer, 1773) 278 165 142 585 agelaia hamiltoni (richards, 1978) 117 223 7 347 agelaia lobipleura (richards, 1978)1 1 1 agelaia melanopyga cooper, 20002 1 1 agelaia myrmecophila (ducke, 1905)1 11 33 37 81 agelaia ornata (ducke, 1905)1 16 18 34 agelaia pallipes (olivier, 1791) 20 3 23 agelaia pallidiventris (richards, 1978)1 1 1 agelaia testacea (fabricius, 1804) 61 1 84 146 agelaia timida cooper, 20001 1 1 angiopolybia paraensis (spinola, 1851) 54 35 31 120 angiopolybia zischkai richards, 19781 85 190 198 473 apoica gelida van der vecht, 1973 1 1 brachygastra albula richards, 1978 3 3 brachygastra augusti (de saussure, 1854)1 2 1 3 brachygastra bilineolata spinola, 1841 8 2 10 brachygastra cooperi (richards, 1978)2 1 1 brachygastra lecheguana (latreille, 1824) 5 5 brachygastra scutellaris (fabricius, 1804) 5 1 1 7 chartergellus amazonicus (fabricius, 1804)1 30 30 chartergellus communis richards, 19781 1 1 chartergellus zonatus (spinola, 1851)1 1 1 charterginus fulvus fox, 18981 1 1 2 chartergus globiventris de saussure, 18541 2 2 leipomeles dorsata fabricius,1804 1 5 8 7 20 parachartergus flavofasciatus (cameron, 1906)1 4 4 15 23 parachartergus fraternus (gribodo, 1892)1 3 1 4 parachartergus lenkoi richards, 19781 1 2 3 parachartergus pseudoapicalis willink, 19591 1 1 2 parachartergus smithii (de saussure, 1854)1 1 1 2 polybia bifasciata de saussure, 18541 5 5 polybia bistriata (fabricius, 1804) 2 6 8 polybia catillifex moebius, 18561 4 9 13 polybia depressa (ducke, 1905)1 4 4 polybia diguetana buysson, 19052 1 1 polybia dimidiata (olivier, 1791) 3 5 2 10 polybia eberhardae cooper, 19931 4 20 24 polybia emaciata lucas,1879 19 23 42 polybia gorytoides gorytoides fox, 18991 13 3 17 33 polybia jurinei de saussure, 1854 39 1 5 45 polybia liliacea (fabricius, 1804) 42 4 2 48 polybia micans ducke, 19041 24 10 48 82 table 1. list of species and number of specimens of social wasps (vespidae, polistinae) collected in three areas of rondônia state, brazil. floresta nacional do jamari (flona); estação ecológica de cuniã (esec); forest fragment of universidade federal de rondônia (unir). b gomes, cs lima, m silva, fb noll – high number of species of social wasps in western amazon116 although many of the species found are common in other areas of the amazon, we also found species, such as agelaia pallidiventris (richards, 1978), with a single previous record for brazil in amazonas state. this species seems to be endemic to the western amazon, occurring also in colombia, ecuador and peru (richards, 1978; natural history laboratory iunh). for other species, the presence in rondonia helps to fill a gap in geographical distribution, as observed for chartergellus zonatus (spinola, 1851) and protopolybia rotundata ducke, 1910. geographic records for the former were from peru and pará state, in the north of brazil. the later, also represents the first record in northern brazil, since previous occurrence data were from french guiana and mato grosso state, in middle-west of brazil (richards, 1978; natural history laboratory iunh). the first occurrence in northern brazil was also found for parachartergus pseudoapicalis willink, 1959, previously recorded for the northeast, middlewest, south and southeastern regions of the country (richards, 1978; natural history laboratory iunh). agelaia was more abundant not only in this work but also in other regions of amazon forest (raw, 1988; silveira, 2002; silveira et al., 2008; morato et al., 2008; silva & silveira, 2009), probably by the habits of this genus that presents generalist and opportunistic species in relation to polybia parvulina richards, 19701 3 3 6 polybia platycephala richards, 1951 7 2 8 17 polybia procellosa dubitata ducke, 19101 1 1 polybia quadricincta saussure, 1854 3 3 polybia rejecta (fabricius, 1798) 79 58 59 196 polybia rufitarsis ducke, 19041 4 2 6 polybia scrobalis richards, 1970 1 5 6 polybia sericea (olivier, 1791) 1 1 polybia singularis ducke, 1909 7 1 8 polybia striata (fabricius, 1787) 59 4 1 64 polybia tinctipennis tinctipennis fox, 18981 24 1 1 26 protopolybia acutiscutis cameron, 1907 1 1 protopolybia chartergoides gribodo, 1891 1 1 protopolybiarotundata ducke, 19101 1 1 pseudopolybia compressa de saussure, 18641 1 1 pseudopolybia vespiceps de saussure, 1864 1 1 synoeca chalibea de saussure, 18521 14 1 15 synoeca surinama (linnaeus,1767) 8 3 11 synoeca virginea (fabricius, 1804) 96 18 1 115 mischocyttarini mischocyttarus flavicans flavicans (fabricius, 1804)1 1 1 mischocyttarus gomesi silveira, 2013 1 1 mischocyttarus interruptus richards, 19781 2 2 mischocyttarus labiatus (fabricius, 1804)1 13 2 3 18 mischocyttarus lecointei (ducke, 1904)1 2 1 3 mischocyttarus tomentosus zikan, 1935 6 6 polistini polistes canadensis linnaeus, 17581 4 4 polistes occipitalis ducke, 19041 1 1 species richness 51 46 39 species abundance 1195 958 808 2961 1 new records for rondonia; 2 new records for brazil. table 1. list of species and number of specimens of social wasps (vespidae, polistinae) collected in three areas of rondônia state, brazil. floresta nacional do jamari (flona); estação ecológica de cuniã (esec); forest fragment of universidade federal de rondônia (unir). (continuation) species flona esec unir total epiponini sociobiology 67(1): 112-120 (march, 2020) 117 the food and resources choice (oliveira et al., 2010). polybia was the richest genus in number of species, and also had been collected more frequently in other studies conducted in amazonia (silveira, 2002; silveira et al., 2008; morato et al., 2008; somavilla et al., 2014a; 2014b; 2015; gomes et al., 2018). once it is the genus of epiponini that presents the greatest number of species, with 58 already described (carpenter & kojima, 2002), this could be considered a predictable result. a sampling of a large diversity of polistinae can be attributed to more than one factor. one of them is the location of the collection areas in the western amazon, known for harboring great biodiversity (brown, 1991; hoorn et al., 2010; gomes et al., 2018). another factor is the methodology used. although the employment of only one type of capture method seems not to be able to conduct a comprehensive species inventory, due to the great diversity of ecological niches occupied by wasps (santos et al., 2007), the use of an attractive demonstrated to be a successful methodology, allowing the collection of more wasps than in previous surveys (raw, 1988; silveira, 2002; silveiraet al., 2008; moratoet al., 2008; silva & silveira, 2009). raw (1988) studying the activity of social wasps visiting various crops in two indigenous tribes in eastern rondonia, collected 13 species of which only five agelaia fulvofasciata (degeer, 1773), polybia bistriata (fabricius, 1804), p. jurinei de saussure, 1854, p. striata (fabricius, 1787) and synoeca surinama (linnaeus, 1767) were also sampled in this study. the author also collected epipona tatua, a species belonging to a genus not sampled in this survey, even with a much longer period of sampling and almost six times more species collected than raw. this fact is in line with data on the rarefaction curve and the richness index, suggesting that all the diversity of social wasps of the state was not accessed, reinforcing the need for further studies not only for rondônia but for amazonia and the entire neotropical region. an interesting result was the greater diversity in terms of the genera found in unir, with the presence of apoica, chartergellus, chartergus, and pseudopolybia registered only in this area. unir is the smallest area (around three and four times smaller than esec and flona, respectively) and the one with the greatest anthropogenic interference, with nearby buildings and constant flow of people and vehicles. studies conducted in areas with different levels of environmental preservation in the semideciduous atlantic forest, showed a great diversity of social wasps also in small fragments and degraded areas (noll & gomes, 2009; tanaka jr. & noll, 2011). the generalist characteristic of many species of social wasps in the choice of food and resources on foraging activities (raveret-richter, 2000; oliveira et al., 2010), and the flexibility in building nests according to the variety of ecological and climatic conditions (rodrigues, 1968; santos et al., 2007) allows some species to be well adapted to areas with anthropic interference (gomes & noll, 2009). nevertheless, consideration should also be given to the fact that anthropogenic environments provide resources, such as water, which may make it advantageous for wasps to nest near them. moreover, a smaller area is easier to be sampled more significantly than the larger ones. conclusion this is the first survey of social wasps carried out for the state of rondônia. among the 72 species collected, there were three new records for brazil. three areas were sampled and even in unir, the smallest one and under the anthropic influence, diversity was high, with some genera found only there, showing the importance of conservation of small forest fragments. moreover, most of the species were recorded for the first time in the state helping to fill in gaps in geographic distribution. nevertheless, large holes still remain in the geographic distribution for many species of social wasps in brazil, mainly in the amazon region, but also throughout the neotropical region. the absence of taxonomic and biogeographic data generates insufficient information to attribute any condition (such as “rare” or “threatened”) to the wasp species of the sampled areas. in a region of great diversity but also high indexes of deforestation, as western amazon, further studies become necessary and urgent. acknowledgments the authors thank john w. wenzel, james m. carpenter, orlando t. silveira, sidnei mateus, rogério b. lopes, raduan a. soleman, sérgio r. andena and luis f. f. gelin, for the support in the identification of the species. we also thank caio l. assunção, tony h. katsuragawa, moreno s. rodrigues and tainá kobs for the assistance in the collections. the specimens were collected under permits (sisbio nº 23197-1, 31901-1 and 31904-1). references andena, s.r. & carpenter, j.m. 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(2010). social paper wasps as bioindicators: a preliminar research with polistes dominulus (hymenoptera, vespidae) as a trace metal accumulator. chemosphere, 64: 697-703. doi: 10.1016/j. chemosphere. 2005.11.009. doi: 10.13102/sociobiology.v66i2.4331sociobiology 66(2): 198-208 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 exotic ants (hymenoptera, formicidae) invading mediterranean europe: a brief summary over about 200 years of documented introductions introduction introduction of exotic species is a global problem affecting human activities and whole natural ecosystems. exotic ants represent only a very tiny fraction of global ant diversity (mcglynn, 1999), and most of them tend not to spread outside the human-modified habitats where they first arrive (holway et al., 2002). nonetheless, many others have a significant ecological impact and may become invasive, even outside anthropogenic habitats, sparking serious concern and attention from researchers worldwide (holway et al., 2002; lach et al., 2010). five ant species are considered among one hundred worst alien species in the world, representing almost one third of the terrestrial invertebrates of the list abstract exotic ants have emerged as a relevant topic worldwide because of their remarkable impacts on native ecosystems and human activities. a first regional overview is given on the dozens of exotic ant species recorded in mediterranean europe since the end of the 19th century. about 40 exotic ant species, belonging to 17 genera and originating from 5 different biogeographical realms, are currently believed to be established in this region. the genera nylanderia and tetramorium are those hosting the larger proportion of species, while the afrotropical realm is the prevalent source of taxa. according to the available data, france, greece, italy and spain all host a high number of exotic species, which has increased at a dramatic rate during the last decades. on the other hand, mediterranean countries on the eastern part of the adriatic sea appear to be almost empty of exotic ants, perhaps due to both a lesser number of introductions and a lack of targeted investigations. neighboring countries of the region do not necessarily have more species in common than those geographically distant. very little is known on the intra-mediterranean or intrapalearctic introduction processes which probably occurred prior to the 19th century and on their influence on the current species distribution. the vast majority of the species that are currently established in the region are either restricted to indoor habitats or outdoor anthropogenic habitats, fewer of them were able to colonize semi-natural or natural habitats and very few are recognized as serious pests. sociobiology an international journal on social insects e schifani article history edited by jacques delabie, uesc, brazil received 15 december 2018 initial acceptance 11 april 2019 final acceptance 18 april 2019 publication date 20 august 2019 keywords alien species, invasive species, pest species, tramp species, globalization, biodiversity, conservation. corresponding author enrico schifani university of palermo section animal biology department of biological, chemical and pharmaceutical sciences and technologies (stebicef) via archirafi 18, i-90123, palermo, italy. e-mail: enrsc8@gmail.com produced by lowe et al. (2000). eradication of these species may unfortunately prove to be extremely difficult to achieve if populations are well-established (holway et al., 2002; hoffmann et al., 2011; hoffmann et al., 2016). in europe, alien terrestrial invertebrates represent one of the most numerous groups of organisms introduced, and are mostly represented by insects (roques et al., 2009). an exponential increase of the number of established species is currently being witnessed, and it is expected to continue in relation to globalization and expanding commerce (roques et al., 2009). climatic conditions made it so that only very few exotic ant species have become established outside heated buildings in central and northern europe (seifert, 2018). on the contrary, mediterranean europe has seen dozens of section animal biology, department of biological, chemical and pharmaceutical sciences and technologies (stebicef), university of palermo, italy review sociobiology 66(2): 198-208 (june, 2019) 199 exotic ant species establishing during the last 200 years in non-heated and even undisturbed natural ecosystems. the mediterranean basin was recognized as one of the twenty-five biodiversity hotspots worldwide (myers et al., 2000), and it is host to a rich ant fauna (borowiec, 2014). its invasion by the exotic argentine ant, linepithema humile (mayr, 1868), one of the worst invasive aliens in the world according to lowe et al. (2000), attracted alone dozens of studies, making l. humile the single most-studied ant species of the entire region (e.g. giraud et al., 2002; espadaler & gómez, 2003; gómez et al., 2003; rey & espadaler, 2004; blancafort & gómez, 2005; 2006; carpintero et al., 2005; 2014; jacquiery et al., 2005; oliveras et al., 2005a; 2005b; wetterer & wetterer, 2006; abril et al., 2007; 2010; 2013; carpintero & reyes-lópez, 2008; blight et al., 2009; 2010; 2012; roura pascual et al., 2009a; 2009b; 2010; pons et al., 2010; estany-tigerström et al., 2010; abril & gómez, 2011; angulo et al., 2011; diaz et al., 2014; centorame et al., 2017; queiroz & alvez, 2018). displacement of native ants and significant changes in the native ant communities are among the most commonly documented consequences of ant invasions, but in many cases notable effects have been also reported in regard to other invertebrates, vertebrates and plants (holldöbler & wilson, 1990; holway et al., 2002; lach et al., 2010). currently, records of exotic species in mediterranean europe come from a large number of different published studies, including only very few updated syntheses at national level (salata et al., 2017; blatrix et al., 2018). no comprehensive look at the exotic ants issue at regional or continental levels has ever been taken, while the same was done in case of organisms whose ecological impacts have been much less documented (e.g. nentwig, 2015). materials and methods a ‘practical’ definition of mediterranean europe was given: the sum the european territories of all the countries of the mediterranean region (see myers et al., 2000 for the region geographic definition), excluding the microstates (e.g. san marino), was considered (fig 1). following this definition, albania, bosnia and herzegovina, croatia, france, greece, italy, montenegro, portugal, slovenia, spain and turkey are the nations whose territories are included at least in part. these territories are not entirely characterized by a mediterranean climate, especially in the case of france, whose vast majority is not. however, since the non-mediterranean areas are usually not witnessing significant exotic ant invasion processes, the european territories of these nations are simplistically considered in their entirety instead of being divided in mediterranean and non-mediterranean areas. moreover, in order to facilitate the distinction of exotic from native species, especially in regard to the intra-mediterranean exotics, all the mediterranean islands that belong to these countries were excluded with the exceptions of the very large corsica, crete, sardinia and sicily. the hundreds of smaller islands of the mediterranean basin are in fact mostly littleknown from a myrmecological standpoint, and only very few were studied with particular regard to the exotic species (e.g. the balearic islands by gómez & espadaler, 2006). the existing published studies which contain data on exotic ant species in these areas were the main source of data for this study, particularly those which published the first records of certain taxa for a geographic area (nylander, 1856; emery, 1869; korlević, 1886; mantero, 1908; forel, 1911; emery, 1916; marchal, 1917; bondroit, 1918; paoli, 1920; santschi, 1925; zimmerman, 1934; frisque, 1935; donisthorpe, 1950; schmitz, 1950; ceballos, 1956; bernard, 1968; espadaler, 1979; süss, 1979; acosta & martinez, 1983; agosti & collingwood, 1987; bolton, 1987; ortiz & tinaut, 1987; kugler, 1988; poldi et al., 1995; collingwood & prince, 1998; espadaler, 1999; bračko, 2000; espadaler & collingwood, 2000; seifert, 2000; espadaler & espejo, 2002; seifert, 2003; reyes & espadaler, 2005; aktaç & kiran, 2006; galkowski, 2008; jucker et al., 2008; reyes-lópez et al., 2008; ugelviv et al., 2008; boieiro et al., 2009; casevitzweulersse & galkowski, 2009; legakis, 2011; borowiec & salata, 2012; obregón romero & reyes lópez, 2012; sanchez-garcia & espadaler, 2015; espadaler & pradera, 2016; salata et al., 2017; blatrix et al., 2018; espadaler et al., 2018; schifani & alicata, 2018). exotic ant species that quickly vanish from the areas where they are introduced are unlikely to have any significant impact on native ecosystems. therefore, only most likely established species were considered in this study. the eventual other published records of the same species after its first discovery in a region, as well as regional and national checklists, were consulted in order to determine if each species was to be considered probably still established or not, and which kind of habitats it had occupied. whenever no published indication could be found to confirm the surviving of an introduced population, the latter was considered extinct if fig 1. map of the mediterranean region. the areas not considered in this study are left blank. areas hosting no exotic ant species are marked in green, areas hosting a single exotic ant species are marked in yellow, areas hosting 4-7 exotic ant species are marked in orange, the remaining countries, hosting 14-21 exotic ant species, are marked in red. e schifani – exotic ants in mediterranean europe200 more than 15 years had passed without further records after the first, otherwise it was considered established unless published information explicitly suggested differently. finally, species only recorded at their very first arrival sites (e.g. most of those recorded by jucker et al., 2008) were not counted, as these introductions are mostly extremely temporary. antmaps (janicki et al., 2016) was also often consulted. results and discussion a total of 40 different species, belonging to 17 genera and four subfamilies, originating from five biogeographical realms, were found to be currently considered to be present as exotic in mediterranean europe and recorded since the second half of the 19th century. the prevalent genera are nylanderia emery, 1906 and tetramorium mayr, 1855 with 5 species each. the count also includes three provisionally unnamed morphospecies whose identity still needs to be ascertained (brachymyrmex sp. from france and nylanderia spp. from italy – see blatrix et al., 2018; schifani & alicata, 2018) and a dubious identification (tetramorium cf. simillimum (smith, f., 1851) from france – see blatrix et al., 2018). four relatively well-studied countries (france, greece, italy and spain) are characterized by four relatively rich lists of exotic species (table 1, fig 1), and similar incremental trends across the decades (fig 2). although some of the more recently introduced species may not manage to establish durable populations (thus lowering the ‘2000’ peaks in fig 2), the vast majority of them are already known as very successful tramp species across other regions of the world (see antmaps). france is distinguished among the four countries by its comparatively smaller number of exotic species established outdoor (only about 27% of the list, while 73% to 90% in the others), most-likely due to the fact that only a very small part of its territory belongs to the mediterranean climatic region. in these four countries, the exotic species number represent approximately around 4.5-7.5% of the entire myrmecofauna present. the afrotropical realm is always the most important source of species, while the rest of the species origins are mostly divided between the palearctic, indomalayan and neotropical realms (fig 3). such pattern is significantly different compared to the one at a global scale (mcglynn, 1999). it is important to note that many of these species are currently widespread across the globe due to human introductions, so that they may have easily been introduced to europe from areas outside their native range. five exotic species are actually shared by these four countries (hypoponera punctatissima (roger, 1859), linepithema humile, monomorium pharaonis (linnaeus, 1758), paratrechina longicornis (latreille, 1802), tetramorium bicarinatum (nylander, 1846)), while four others are present in only three of them (lasius neglectus van loon, boomsma & andrasfalvy, 1990, nylanderia jaegerskioeldi (mayr, 1904), pheidole indica mayr, 1879, strumigenys membranifera emery, 1869). in greece, the presence of l. neglectus was initially supposed to be incorrectly recorded due to misidentification with the native l. turcicus santschi, 1921 (borowiec & salata, 2012). later, the same authors reported to have collected material of both species (borowiec & salata, 2013; salata & borowiec, 2018) and the situation remained unresolved as the two may be conspecific (borowiec & salata, 2017a). in any case, l. neglectus was listed as a nonexotic species in greece lately (salata et al., 2017; salata & borowiec, 2018). the sørensen–dice coefficient was used in order to compare the list of these four species-rich countries (table 2). very different results were obtained if the comparison was made between all the exotic species of each country, only between those species that managed to establish outdoor or only between those able to colonize non-urban habitats. in any case, the coefficient values that were obtained do not show any strong biogeographic pattern of colonization: neighboring countries do not necessarily show a higher degree of similarity than geographically more distant countries do, suggesting that species often spread through multiple introduction events. portugal, whose fauna is usually regarded as relatively much less explored than that of neighboring spain (boieiro et al., 2009; borowiec & salata, 2017b) hosts a total of seven exotic species, all shared with spain with the exception of strumigenys silvestrii emery, 1906. the exotic ant fauna of turkish thrace (kiran & karaman, 2012), a relatively small region, only counts three species, all shared with neighboring greece, with the exception of n. vividula, which is probably absent from greece according to salata et al., 2017. only m. pharaonis, long regarded as the most ubiquitous household ant in the world (wetterer, 2010), is known in croatia (bračko, 2006), montenegro (karaman, 2011) and slovenia (bračko, 2007). fig 2. number of exotic ant species known in france (corsica, mainland), greece (crete, mainland, peloponnese), italy (mainland, sardinia, sicily) and spain (mainland) since the beginning of the 1900th century. 25 20 15 10 5 0 1900 1950 2000 2018 sociobiology 66(2): 198-208 (june, 2019) 201 species croatia france greece italy montenegro portugal slovenia spain turkish thrace aphaenogaster splendida (roger, 1859) roger, 1859 brachymyrmex sp. blatrix et al., 2018 brachymyrmex patagonicus mayr, 1868 espadaler & pradera, 2016 camponotus atriceps (smith, f., 1858) jucker et al., 2008 cardiocondyla emeryi forel, 1881 reyes-lópez et al., 2008 cardiocondyla mauritanica forel, 1890 seifert, 2003 seifert, 2003 ortiz & tinaut, 1987 cardiocondyla obscurior wheeler, w.m., 1929 sanchezgarcia & espadaler, 2015 hypoponera eduardi (forel, 1894) borowiec & salata, 2012 agosti & collingwood, 1987 hypoponera ergatandria (forel, 1893) bernard, 1968 hypoponera punctatissima (roger, 1859) bernard, 1968 agosti & collingwood, 1987 emery, 1916 collingwood & prince, 1998 ceballos, 1956 lasius neglectus van loon, boomsma & andrasfalvy, 1990 seifert, 2000 poldi et al., 1995 espadaler, 1999 lepisiota syriaca (andré, 1881) stitz, 1928 linepithema humile (mayr, 1868) marchal, 1917 bernard, 1968 paoli, 1920 schmitz, 1950 frisque, 1935 monomorium bicolor emery, 1877 agosti & collingwood, 1987 monomorium carbonarium (smith, f., 1858) galkowski, 2008 collingwood & prince, 1998 espadaler & collingwood, 2000 monomorium monomorium bolton, 1987 forel, 1911 monomorium pharaonis (linnaeus, 1758) korlevic, 1886 nylander, 1856 bolton, 1987 mantero, 1908 zimmermann, 1934 collingwood & prince, 1998 bračko, 2000 santschi, 1925 aktaç & kiran, 2006 nylanderia jaegerskioeldi (mayr, 1904) borowiec & salata, 2012 schifani & alicata, 2018 obregón romero & reyes lópez, 2012 espadaler & collingwood, 2000 nylanderia vividula (nylander, 1846) bernard, 1968 espadaler & collingwood, 2000 donisthorpe, 1950 nylanderia sp. 1 schifani & alicata, 2018 nylanderia sp. 2 schifani & alicata, 2018 table 1. exotic ant species established or seemingly established in france (corsica, mainland), greece (crete, mainland, peloponnese), italy (mainland, sardinia, sicily) and spain (mainland). the first confirmed record of the species in each country is presented on the right of the species name. species that have been found outdoors are marked in yellow, species that have also colonized areas outside urban contexts are marked in red. e schifani – exotic ants in mediterranean europe202 paratrechina longicornis (latreille, 1802) nylander, 1856 kugler, 1988 süss, 1979 espadaler & collingwood, 2000 pheidole anastasii (emery, 1896) bernard, 1968 pheidole bilimeki mayr, 1870 casevitzweulersse & galkowski, 2009 pheidole indica mayr, 1879 legakis, 2011 schifani & alicata, 2018 acosta & martinez, 1983 pheidole megacephala (fabricius, 1793) bernard, 1968 espadaler & pradera, 2016 plagiolepis alluaudi emery, 1894 blatrix et al., 2018 strumigenys membranifera emery, 1869 agosti & collingwood, 1987 emery, 1869 espadaler, 1979 strumigenys silvestrii emery, 1906 boieiro et al., 2009 tapinoma melanocephalum (fabricius, 1793) casevitzweulersse & galkowski, 2009 espadaler & espejo, 2002 technomyrmex difficilis forel, 1892 blatrix et al., 2018 technomyrmex pallipes (smith, f., 1876) jucker et al., 2008 technomyrmex vitiensis mann, 1921 blatrix et al., 2018 temnothorax longispinosum (roger, 1863) espadaler & collingwood, 2000 tetramorium bicarinatum (nylander, 1846) bondroit, 1918 salata et al., 2017 jucker et al., 2008 reyes & espadaler, 2005 tetramorium caldarium (roger, 1857) reyes & espadaler, 2005 tetramorium lanuginosum mayr, 1870 schifani & alicata, 2018 reyes & espadaler, 2005 tetramorium lucayanum wheeler, w.m., 1905 jucker et al., 2008 tetramorium cf. simillimum (smith, f., 1851) bernard, 1968 wasmannia auropunctata (roger, 1863) espadaler et al., 2018 species croatia france greece italy montenegro portugal slovenia spain turkish thrace table 1. exotic ant species established or seemingly established in france (corsica, mainland), greece (crete, mainland, peloponnese), italy (mainland, sardinia, sicily) and spain (mainland). the first confirmed record of the species in each country is presented on the right of the species name. species that have been found outdoors are marked in yellow, species that have also colonized areas outside urban contexts are marked in red. (continuation) sociobiology 66(2): 198-208 (june, 2019) 203 no exotic species is currently recorded in albania and bosnia and herzegovina. such condition of these countries, confirmed in the aforementioned relatively recent checklists, may partly be explained by a lack of targeted searches but may also be partly related to the recent history of the former yugoslavia, and certainly requires further investigations. no species were found to be considered exotic in any of the four large islands included in this study while at the same time being considered native in the respective country’s mainland or vice-versa. some species seem to differ in their invasion success across different countries examined: the same species may be restricted to indoor habitats in a country, be present outdoor but only in anthropogenic habitats in another, and to be present in semi-natural or even natural-habitats in a third one. such differences can be related to many factors, including environmental aspects and priority effects in the area where the species were introduced, genetic variability and consistence of the introduced population and time-related effects, since many invasion processes are known to be subject to a variety of lag phenomena (crooks, 2005). while only a small portion of the exotic ants listed in tab. 1 are considered significantly threatening to semi-natural and natural ecosystems in the mediterranean basin (lasius neglectus and linepithema humile above all), some others have aroused different levels of concern in different countries. for example, tetramorium bicarinatum, first recorded in the mediterranean region by bondroit (1918), is globally considered a low threat (wetterer, 2009), and usually restricted to indoor areas. however, it has lately started to be reported in outdoor urban contexts in spain (reyes & espadaler, 2005), italy (borowiec & salata, 2015; schifani & alicata, 2018) and greece (salata et al., 2017). moreover, in greece it was also considered by salata et al. (2017) among the three exotic species that displace native ants in the areas they occupy. in general, the few species that successfully invaded large areas of outdoor habitats in all the countries considered in this study are mostly distributed in areas characterized by a strictly mediterranean climate (e.g. blatrix et al., 2018). a significant exception is that of monomorium carbonarium (smith, f., 1858), whose presence is mostly distributed along the atlantic coast of france, while mostly along the mediterranean in iberia (miravete et al., 2013). given the long history of human activities and commerce across the mediterranean basin, many cases of old historical or pre-historical introductions have been detected for both animal and plant species. however, intramediterranean or intra-palearctic introductions of ants, usually much less obvious to detect than these of ants from other continents or latitudinal regions, actually represent a mostly unexplored topic. some cryptic species belonging to the tapinoma nigerrimum complex and the tetramorium caespitum complex were only recently recognize as exotic in some or all of the countries in this study after the latest taxonomic advances (seifert et al., 2017; wagner et al., 2017). however, the time of their introduction is unknown and thus they were excluded from the present analysis. another issue is that for a number of other mediterranean species, there is no precise knowledge about where their native range ends and where the exotic range begins. for example, the status of cardiocondyla mauritanica forel, 1890 in sicily is currently dubious (schifani & alicata, 2018): it is considered as an exotic species in greece (salata et al., 2017) and spain (reyes & espadaler, 2005; reyes-lópez et al., 2008), while as likely native to north africa (wetterer, 2012). in sicily, it is found in both natural and anthropogenic habitats, and many species appear to be naturally present in both sicily and north africa (alicata & schifani, 2019). monomorium subopacum (smith, f., 1858) was considered as exotic in greece by salata et al. (2017), while it is usually considered native in the mediterranean region, however it was removed in a more recent list by the same authors (salata & borowiec, 2018). fig 3. geographic origin of the exotic ant species of france (corsica, mainland), greece (crete, mainland, peloponnese), italy (mainland, sardinia, sicily) and spain (mainland). all species species established outdoor species established outside urban habitats f-g 31% 11% 0% f-i 30% 37% 80% f-s 46% 33% 60% g-i 55% 54% 0% g-s 51% 46% 13% s-i 60% 60% 44% table 2. sørensen-dice coefficient between exotic ant species present in france – f (corsica, mainland), greece – g (crete, mainland, peloponnese), italy – i (mainland, sicily, sardinia) and spain – s (mainland) as defined in this study and listed in table 1. e schifani – exotic ants in mediterranean europe204 aphaenogaster splendida (roger, 1859) may be exotic at least in some parts of the italian territory (schifani & alicata, 2018) and was only recently treated as an exotic species in crete (salata & borowiec, 2018). still, the definition of its native range remains unclear. similarly, monomorium monomorium bolton, 1987 is considered as exotic in greece (salata & borowiec, 2018) but it is usually considered native in mediterranean europe (e.g. antmaps). hypoponera eduardi is usually considered native to the mediterranean, but it is regarded as exotic in greece (salata et al., 2017) and turkey (kiran & karaman, 2012), although lapeva-gjonova and kiran (2012) treat it as a rare species attributing conservation value to an area of turkish thrace. another example can be provided by some forms related to lepisiota frauenfeldi (mayr, 1855), whose scattered distributions across the mediterranean basin may suggest some of these populations are introduced, especially given the vast exotic range attained by l. syriaca (andré, 1881) in greece according to salata et al. (2017) or the recent quick invasion of the canary islands by l. frauenfeldi cf. kantarensis (forel, 1911) (schifani et al., 2018). molecular data may allow to shed light into this complex and mostly unexplored issue in the future, as in the case of anochetus ghilianii forel, 1915 in southern spain (jowers et al., 2015). conclusions the numerical increase of exotic ant species established in mediterranean europe during the last decades is impressive, and so is also the increasing rate at which they continue to be introduced. nonetheless, other countries around the world are already dealing with much higher numbers of exotic species (e.g. deyrup et al., 2000). the balkan countries bordering the adriatic sea make an interesting exception in the region, which requires further investigation. it is impossible to predict which species are going to be successful invaders when they arrive, and the ongoing climate change further complicates this equation (bertelsmeier et al., 2015). moreover, existing data do not allow to attempt predictions on which species are going to be the next to be introduced in the region, especially that similarity between exotic ants assemblages is not always greater between neighboring countries. while our understanding of invasive ants has tremendously increased over the last decades, long-term effects and dynamics of the invasive populations are little-known since they are just starting to be observed (e.g. tartally et al., 2016). our knowledge also remains extremely scarce on those successful ants’ introductions that have most likely occurred across the mediterranean basin in historic and pre-historic times, and on their possible role in shaping the current faunas. the number of species which have caused serious damage and concerns in mediterranean europe remains relatively low for the time being. still, the current situation is concerning for the equilibrium and management of natural and anthropogenic ecosystems in the future. references abril, s., oliveras, j. & gómez, c. 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(1934). beitrag zur kenntnis der ameisenfauna suddalmatiens. abhandlungen der (k. k.) zoologischbotanischen gesellschaft in wien gegründet, 84: 1-65. doi: 10.13102/sociobiology.v65i4.3473sociobiology 65(4): 612-620 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 seasonal variation in bee-plant interactions in an inselberg in the atlantic forest in southeastern brazil introduction the fragmentation and degradation of natural and seminatural habitats and the consequent loss of plant species and pollinators are recurrent concerns in the literature (moreira et al., 2015; potts et al., 2016). predicting how interactions between plants and pollinators change in the face of biotic and abiotic factors is an important step in attempting to preserve forest ecosystems (sargent & ackerly, 2008). inselbergs are single or multiple rock outcrops occurring isolated in the surrounding flat landscape and they are considered among the priority areas for conservation (porembski & barthlott, 2000). the different microhabitats resulting from a marked microclimatic variation favor the establishment of different species in these environments (porembski et al., 1998), providing refuges for rare or endemic plant and animal species (santos abstract studies on bee-plant interactions are relevant to the understanding of temporal patterns in neotropical communities. in isolated habitats such as inselbergs little is yet known about the temporal dynamics in the availability of floral resources and interacting bee. in the present study, the objective is to verify the effect of seasonality on the bee-plant interaction in an atlantic forest inselberg in southeastern brazil. the bees were sampled monthly in the dry (april/2008-september/2008) and wet seasons (october/2008-march/2009) using an entomological net. a total of 322 bees of 33 species were captured on flowers of 34 species of plants during the year. bees richness was similar between seasons (22 species in the wet season and 21 in the dry season), but abundance was higher in the wet season (60% of individuals) and higher diversity occurred in the dry season. augochloropsis sp1 were the most abundant species and visited the largest number of plant species at each season. in the interaction network, plants with the highest degree were distinct between the seasons. the number of possible interactions was higher in the dry season compared to the wet season and connectance was similar; nestedness however varied between the seasons. the composition of plant and bees species was distinct between the seasons, as well as the interactions between them, mainly due to the alteration in the composition of the plant species and the change in the choice of the bees for the floral resources between the seasons. sociobiology an international journal on social insects mf marques, ms deprá, mc gaglianone article history edited by cândida aguiar, uefs, brazil received 10 may 2018 initial acceptance 26 june 2018 final acceptance 17 august 2018 publication date 11 october 2018 keywords floral visitors, seasonality, semidecidual forest, ecological networks. corresponding author marcelita frança marques universidade estadual do norte fluminense darcy ribeiro centro de biociências e biotecnologia laboratório de ciências ambientais avenida alberto lamego nº 2000 parque califórnia, cep 28013-602 campos dos goytacazes-rj, brasil. e-mail: marcelita_marques@yahoo.com.br et al., 1999; porembski & barthlott, 2000; silva, 2016). these ecosystems are subject to constant threats, including mineral extraction and wide agricultural practices in the surrounding areas (silva, 2016). the forest formations in inselberg areas can be determined by a complex system of gradients related to altitude, soil depth, water availability, and climatic variation, contributing to vegetation characterized by rupicolous communities and dry or savanna-like forests (gröger & huber, 2007). among the dry forests, the semi-deciduous seasonal forest in southeastern brazil is one of the most threatened and fragmented ecosystems in the world, consisting of small scattered fragments (santos et al., 2009; pimenta et al., 2011). this fact raises the importance of studying the plantpollinator interactions in this ecosystem since it guarantees the reproduction of plants and promotes their genetic diversity and also provides resources for animals (rech et al., 2014). programa de pós-graduação em ecologia e recursos naturais, universidade estadual do norte fluminense darcy ribeiro, centro de biociências e biotecnologia, laboratório de ciências ambientais, campos dos goytacazes, rio de janeiro, brazil research article bees sociobiology 65(4): 612-620 (october, 2018) special issue 613 these mutualistic interactions between bees and plants may vary on different spatial and temporal scales, according to environmental conditions (such as temperature, relative humidity and light intensity), and also to intrinsic characteristics of bees (activity and foraging periods) and plants biology (flowering period) (olesen et al., 2008; kleinert et al., 2009; burkle & alarcón, 2011; deprá & gaglianone, 2018). in the studies of the composition and distribution of floral resources, the inselbergs constitute an excellent model due to the geographic isolation and the endemism of species. the environmental variables may be associated to the different patterns of occurrence and distribution of plant resources, especially in forests with seasonal climates such as inselbergs composed of deciduous or semi-deciduous forests (morellato & leitão-filho, 1996; nunes et al., 2005; mauad et al., 2014). the approach of ecological networks and their metrics emerge as a contribution to the understanding of patterns in the structures of interactions among species, the fragility of these interactions, the impact of the environmental and anthropic effects, and the temporal and spatial variation of the species (memmott et al., 2004; silva et al., 2007; deprá & gaglianone, 2018). this network approach contributes to the planning required for the conservation and management of threatened environments (biesmeijer et al., 2005). studies on the evaluation of the community structure of bees and floral species in inselberg areas in brazil have been carried by aguiar and zanella (2005) and batalha-filho et al. (2007) in caatinga phytophysiognomies in the state of bahia, brazil. these authors approached the topic using traditional analytical tools of population and community studies. studies on bee-plant interactions in a unique habitat such as the inselbergs, with rare and endemic species and with seasonal vegetation that varies floral resources along the year, can bring relevant results for the understanding of the temporal dynamics of plant-pollinators in the atlantic forest. in addition they can provide knowledge to be used in conservation programs of this ecosystem. the present work aims to describe and analyze the seasonal variation in interactions between plants and bees in an atlantic forest inselberg. our hypothesis is that the key plants can be substituted between the seasons in the semidecidual seasonal forest, resulting in the changing in the availability of foraging resources that affects the bee-plant interactions. material and methods study area the samplings were carried out on semideciduous seasonal forest area, itaoca massif (21°48’ s 41°26’ w), a rocky outcrop of 900 ha at 420 m a.s.l., in southeastern brazil. this inselberg is isolated amidst the surrounding flat landscape and flooded areas surrounded by pastures, sugarcane plantations, and rock exploration activity. this area has a clear seasonal climate with low relative humidity comparing to other atlantic forest areas, and it is considered important for the floristic diversity of rio de janeiro state (pessanha et al., 2014) as a hotspot within the atlantic forest (murray-smith et al., 2008). the climate of the region is classified as aw (sensu köppen, 1948), characterized by tropical hot and humid with a dry winter (april to september) and wet summer (october to march). in the studied period (2008 and 2009), the amplitude of the abiotic values for the dry and wet seasons were: temperature 21.6 to 26.6 °c and 24.6 to 28.5 °c, relative humidity 74 to 78% and 74 to 82%, and monthly rainfall 3.5 to 63.8 mm and 51.6 to 526.2 mm, respectively (fig 1). climatic data was obtained from the weather station of campos/rj of national meteorological institute (inmet, 2017). sampling samplings of the floral visiting bees were carried out in the dry (april/2008 to september/2008) and wet seasons (october/2008 to march/2009) in the semi-deciduous seasonal forest vegetation, every 30 days, except in december when sampling was not possible due to impassable roads caused by intense rain. the sampling of bees was performed by a collector using an entomological net, from 7 am to 4 pm. all flowering plants, mainly herbs and shrubs, were inspected for up to three minutes in search of their floral visitors in a trail called locally fig 1. climatic conditions between april 2008 and march 2009 at the itaoca inselberg in the atlantic forest in southeastern brazil. dry season: white color; wet season: black color. temperature: square; relative humidity: circles; rainfall: bars. mf marques, ms deprá, mc gaglianone – seasonal variation in bee-plant interactions614 “tower trail” with 3000 meters long. arboreal plants were sampled using extensive netting up to 5 m in height. after capture, the bees were killed in flasks containing ethyl acetate and separated according to time interval and plant. in the laboratory, the bees were mounted with entomological pins, labeled, identified and deposited in the entomological collection in the setor de ecologia experimental of the laboratório de ciências ambientais of the universidade estadual do norte fluminense darcy ribeiro, in campos dos goytacazes, rj, brazil. identification of the plants was carried out by comparison with the reference material of the study area deposited in the herbário uenf (huenf) and confirmed by specialists. classification of angiosperms followed the system proposed by apg iii (2009). data analysis the diversity of the flower-visiting community was assessed by the shannon index (1-d) and dry and wet seasons were compared using the t-test diversity at the 5% level. equitability was obtained by the pielou index (j‘). these indices and tests were calculated using the past program 3.20 (hammer et al., 2001). from the data of visitation, adjacency matrices were constructed with the species of bees plotted on the rows and the plants in the columns, using the abundance of bees captured in each plant. two matrices were constructed considering the community of bees and plants visited in dry and wet season. from these matrices a tripartite graph was constructed for visualization of interactions between bees and plants in the dry and wet seasons. connectance, nestedness (nodf) and robustness (hl) network were analyzed using the bipartite package of program r 3.3.2 (r development core team, 2016), also used for the construction of networks. connectance (c=100 i/m) is the percentage of actually observed interactions (observed interactions=i) with respect to the total possible number of interactions in the network (m=bp, where b and p are the number of interacting bees and plants in the network, respectively) (jordano, 1987). the nestedness was based on overlap and decreasing fill – nodf and the higher value of this index means greater nestedness of the network (almeida-neto et al., 2008). nestedness occurs when specialist species tend to interact with more generalist species and these last one interact with each other (bascompte et al., 2003). the robustness hl was calculated to evaluate the resistance of the bees to secondary extinctions in relation to the random extinction of plants and this metrics calculates the area below the extinction curve generated by secondary extinctions (memmott et al., 2004). the differences in the networks between the seasons were quantified by the jaccard beta diversity index (jaccard beta diversity-jbd), which is defined as the turnover of the interactions between the networks (following novotny, 2009 and kemp et al., 2017). jbd is partitioned into the effects of the turnover of plant species (bp), floral visitor species (bh), both plant and floral visitor species (bph), and choices of floral visitor by plant species (bo) between the seasons. this index was calculated in program r 3.3.2 (r development core team, 2016). results in total, 322 bees from 33 species of the families apidae (18 species), halictidae (8), and megachilidae (7) were captured (table 1). the richness of bees was similar between the seasons, presenting 22 species of bees in the wet season and 21 in the dry season and among these, 10 species were sampled on flowers in both seasons. however, abundance was higher in the wet season (60% of individuals) compared to the dry season (40%). the bee diversity was higher in the dry season (h = 2.36) compared to the wet season (h = 2.08), and these values differ statistically (diversity h’ t-test: t = 2.068, p = 0.035). the equitability index was higher in the dry season (dry season j’: 0.77; wet season j’: 0.67) (table 3). the plants visited by these bees consisted of 34 species belonging to fabaceae (8 species), asteraceae (5), solanaceae (4), and malvaceae (2), in addition to 15 other families, each visited by one species (table 2). a total of 47% of the plant species were visited exclusively in the dry season, 29% exclusively in the wet season, and 24% were visited in both seasons. augochloropsis sp1, plebeia droryana (friese) and plebeia lucii moure were the most abundant bee species and occurred in both seasons. augochloropsis sp1 was particularly abundant in the wet season and interacted with a largest number of plant species in the dry season (5 in dry season, 8 in wet season and 10 in total). the second floral visitor most abundant and interacted with a largest number of plant in the dry season (6 in dry season, 3 in wet season and 8 in total) was p. droryana. in the wet season, p. lucii was the most abundant floral visitor and interacted with six plant species in total (2 in dry season and 4 in wet season). the plants with the highest degree were distinct between the seasons; austroeupatorium sp. and spermacoce verticillata l. showed the highest number of links during the wet season. crotalaria sp. and solanum hexandrum vell. were the most connected plants in the dry season. the number of possible interactions was higher in the dry season, 504 interactions (48 interactions observed), than in the wet season, 396 interactions (40 interactions observed) (fig 2). the connectance was similar between the both seasons (dry season: 0.095; wet season: 0.101). a higher nestedness was observed in the wet season (11.588) when compared to the dry season (3.933). the value of the robustness (hl) in the dry season was similar (0.617) to the wet season value (0.580) (table 3). rare interactions occurred in both seasons; 10 and 12 species of bees interacting with only one plant species each in the dry and wet seasons, respectively. sociobiology 65(4): 612-620 (october, 2018) special issue 615 bee family species acronym dry season wet season apidae apis mellifera linnaeus ap_me 0.5 bombus morio (swederus) bo_mo 4.7 centris flavifrons (fabricius) ce_fl 0.8 epicharis flava friese ep_fl 0.8 eufriesea surinamensis (linnaeus) eu_su 0.5 euglossa sp. eu_sp 1.0 eulaema cingulata (fabricius) eu_ci 1.6 eulaema nigrita lepeletier eu_ni 0.8 exomalopsis analis spinola ex_an 0.5 melissodes sp. me_sp 3.2 oxaea flavescens klug ox_fl 3.1 paratetrapedia fervida (smith) pa_fe 3.9 1.0 plebeia droryana (friese) pl_dr 33.6 11.9 plebeia lucii moure pl_lu 7.0 16.1 trigona spinipes (fabricius) tr_sp 3.1 1.0 xylocopa frontalis (olivier) xy_fr 1.6 xylocopa nigrocincta smith xy_ni 0.8 xylocopa ordinaria smith xy_or 0.8 halictidae ariphanarthra palpalis moure ar_pa 0.5 augochlora sp. aa_sp 0.5 augochlorella sp. ae_sp 9.4 13.0 augochloropsis sparsilis (vachal) au_sp 2.7 augochloropsis sp1 au_sp1 12.5 37.2 augochloropsis sp2 au_sp2 0.8 3.2 dialictus sp. di_sp 0.8 pseudaugochlora graminea (fabricius) ps_gr 1.6 megachilidae coelioxys sp. co_sp 0.5 hypanthidium divaricatum (smith) hy_di 3.1 0.5 hypanthidium foveolatum (alfken) hy_fo 2.2 3.7 megachile nudiventris smith me_nu 7.0 1.5 megachile pseudanthidioides moure me_ps 0.5 megachile sp1 me_sp1 0.5 megachile sp2 me_sp2 0.5 table 1. species composition (and acronyms) and relative abundance (%) of the bees captured in flowers in the dry and wet seasons on the itaoca inselberg in the atlantic forest in southeastern brazil. the species composition of bees and plants was distinct between the seasons, as well as the interactions between them. jaccard beta diversity (jbd) between wet and dry seasons networks was jbd=0.90. it was generated mostly by the turnover in plant species only (bp=0.30) and the changes in bees choices between the seasons (bo=0.26). the turnover of bees species only (bh=0.17) and the turnover of plant and bee species (bph=0.17) were less important. mf marques, ms deprá, mc gaglianone – seasonal variation in bee-plant interactions616 discussion the bee-plant interactions differ between the dry and wet seasons, mainly due to the alteration in the composition of the plant species and the change in the choice of the bees for the floral resources. changes in abundance and composition of flower resources between the seasons influenced the choices of plants by the bees. augochloropsis sp1 visited mainly flowers of austroeupatorium sp. in the wet season, and flowers of s. verticillata in the dry season, even when the two plants flowered in both seasons. in the wet season, p. droryana most frequently visited inga laurina (sw.). willd flowers and in the dry season, croton sp. and pavonia sidifolia kunth. kaiserbunbury et al. (2014) studying interactions in an inselberg also verified the importance of floristic composition in the structure of the bee-plant networks, similar to observed in this study. besides that, forbidden links need to be also considered in the discussion about the changes of interactions between the seasons. forbidden links are made up of links that do not occur due to phenotypic or temporal variation of the species in which, put simply, the species of bees and plants are not found together in time or space (jordano et al., 2003). plant family species acronym dry season wet season apocynaceae asclepias curassavica l. as_cu 1.6 asteraceae austroeupatorium sp. au_sp 7.0 41.8 bidens pilosa l. bi_pi 1.6 conyza sp. cn_sp 0.8 emilia sonchifolia (l.) dc. ex wight em_so 2.1 vernonanthura phosphorica (vell.) h.rob. ve_ph 0.8 2.1 boraginaceae cordia sp. co_sp 2.2 commelinaceae commelina benghalensis l. co_be 0.5 convolvulaceae jacquemontia aff. confusa meisn. ja_co 2.2 cucurbitaceae momordica charantia l. mo_ch 1.0 euphorbiaceae croton sp. cr_sp 10.9 fabaceae centrosema sp. ce_sp 0.5 chamaecrista sp. ch_sp 0.8 crotalaria sp. ct_sp 8.6 fabaceae caesalpinoideae sp1 fa_ca1 3.9 fabaceae caesalpinoideae sp2 fa_ca2 5.2 fabaceae caesalpinoideae sp3 fa_ca3 1.5 inga laurina (sw.) willd. in_la 6.2 piptadenia gonoacantha (mart.) j.f.macbr. pi_go 0.8 lamiaceae ocimum basilicum l. oc_ba 5.5 lecythidaceae lecythis sp. le_sp 3.1 malpighiaceae amorimia maritima (a.juss) w.r.anderson am_ma 4.6 malvaceae pavonia sidifolia kunth pa_si 10.9 sida rhombifolia l. si_rh 7.8 1.0 melastomataceae tibouchina sp. ti_sp 0.5 musaceae musa paradisiaca l. mu_pa 3.1 1.0 myrtaceae psidium sp. ps_sp 6.3 piperaceae peperomia rubricaulis (nees) a.dietr. pe_ru 4.6 rubiaceae spermacoce verticillata l. sp_ve 7.0 22.7 solanaceae aureliana fasciculata (vell.) sendtn. au_fa 1.0 solanum cordifolium dunal so_co 3.1 solanum hexandrum vell. so_he 6.3 0.5 solanum sp1 so_sp1 3.1 1.0 verbenaceae stachytarpheta cayennensis (rich.) vahl st_ca 1.6 3.2 table 2. species composition (and acronyms) of the plants visited by bees and relative abundance of their visitors in the dry and wet seasons on the itaoca inselberg in the atlantic forest in southeastern brazil. sociobiology 65(4): 612-620 (october, 2018) special issue 617 the interactions between bees and plants can vary on different spatial and temporal scales, influenced by several factors such as the flowering period of the plants, the activity period of the bees and competition (olesen et al., 2008; carstensen et al., 2014). the high diversity of bees found in the dry season reflects the greater uniformity in the distribution of abundance, since the richness was similar between the seasons and the network metrics seasonal sampling dry wet richness 21 bees-24 plants 22 bees-18 plants degree of bees (min-max) 1-8 1-5 degree of plants (min-max) 1-6 1-11 no possible interactions 504 396 no observed interactions 48 40 connectance 0.095 (9%) 0.101 (10%) nestedness (nodf) 3.933 11.588 robustness (hl) 0.617 0.580 table 3. metrics of interaction networks between bees and plants in the dry and wet seasons at itaoca inselberg in the atlantic forest in southeastern brazil. fig 2. bee-plant interactions in tropical atlantic forest at itaoca inselberg, southeastern brazil. the species are represented by bars: plant species in the center and bee species on bottom (wet season) and top (dry season). the size of links indicates the number of interactions. the acronyms of plants from left to right are: au_sp, ct_sp, sp_ve, so_he, so_co, si_rh, so_sp1, ve_ph, mu_pa, em_so, bi_pi, mo_ch, oc_ba, co_be, ps_sp, ce_sp, st_ca, as_cu, fa_ca2, cn_sp, fa_ca3, co_sp, in_la, ja_co, am_ma, cr_sp, ti_sp, ch_sp, pe_ru, fa_ca1, pi_go, le_sp, pa_si, au_fa. the acronyms are according to tables 1 and 2. abundance of bees was higher in the wet season. in this time, augochloropsis sp1 was responsible for 37% of visits on flowers, mainly austroeupatorium sp. (32%), and the dominance of this bee species and also of p. droryana and augochlorella sp. may explain the low diversity in the wet season. the composition of the bees and mainly of the plants varied strongly between the seasons, indicating that the majority of bee and plant species were replaced seasonally. the availability of flowering plants and the choice by the visitors influenced the temporal variation in their interactions. despite the distinct composition of bees and plants observed between the seasons, the values of the network metrics, such as connectivity and robustness, were not discrepant between them, which may indicate relative stability of the network topology. studies on network interactions of bees and plants point out that the topological metrics of the networks can remain stable despite variations in the composition of the species (olesen et al., 2008; petanidou et al., 2008; dupont et al., 2009), where the existing species may be replaced by other topologically similar species (dupont et al., 2009). on the other hand, o nestedness (nodf) differed between the seasons, demonstrating greater cohesion and resilience in wet season, despite both values were relatively low. in areas with marked seasonality, such as the studied mf marques, ms deprá, mc gaglianone – seasonal variation in bee-plant interactions618 area, these topological metrics were observed to be different between dry and wet seasons (santos et al., 2014), probably influenced by the seasonal character of the vegetation. especially the distinction in nestedness is probably related to different abundances of interactions between the seasons. the most nested network of the wet season was characterized by more generalist interactions composing the core of the network. in inselberg areas in mahe, the largest granitic island of the seychelles (indian ocean), the composition of the species and their abundance in the communities were the main determinants for the network architecture (kaiserbunbury et al., 2014). those authors emphasized that the floral composition determined the major changes in the patterns of network interactions and was mainly explained by the temporal variation. the most abundant species in both seasons was augochloropsis sp1 associated with its generalist behavior in the interaction with the largest number of plant species visited in both seasons, mainly in asteraceae. most species of this genus occur throughout the year and exhibit polylectic behavior, most frequently in species of asteraceae (mouga & krug, 2010; dec & mouga, 2014). other bee species common to the two seasons, p. droryana and p. lucii, were abundant and generalist in use of floral resources. this amplitude of the food niche, the activity throughout the year, and the greater abundance may be related to eusocial behavior of these stingless bees, besides colony permanence, and recruitment foraging habits (roubik, 1989). despite the similar behavior and corporal size, these two bee species presented differences in the visited plant species. this behavior would avoid competition in resource use, that can be an important factor for the coexistence of eussocial species. the bees of the xylocopa genus sampled in this study were observed only in the dry season in flowers of crotalaria sp. data obtained by bernardino and gaglianone (2008) in a coastal area in the same region also pointed out xylocopa bees is higher numbers in the dry season. flowers of crotalaria are considered important resources for bees due to their high concentration of nectar and availability of pollen from the anthesis to the total wilt of the flower (marques et al., 2013), both resources collected by females of xylocopa. the most abundant megachilidae species, hypanthidium divaricatum (smith), hypanthidium foveolatum (alfken) and megachile nudiventris smith, have been observed in the two seasons, mainly on flowers of asteraceae, convolvulaceae and myrtaceae. the species of megachilidae with smaller abundance were sampled only in the wet season. other studies in semi-deciduous seasonal forests in the state of rio de janeiro emphasized the nesting preference of these bees during the wet season (marques & gaglianone, 2008; teixeira et al., 2011). euglossina bees were sampled in both seasons, mainly euglossa and eufriesea in the wet season and eulaema in the dry season. aguiar and gaglianone (2011) sampled abundance peaks in both seasons to euglossina captured with aromatic baits in the same study area. the explanation for this could be related to flowering peaks, nesting activities and adult emergence (roubik & hanson, 2004). eufriesea surinamensis (linnaeus) is a seasonal species, with adult activity restricted to the wet season in the study area and in other semideciduous seasonal forests (aguiar & gaglianone, 2011). species of bees unique to the dry season or the wet season demonstrated specialist behavior, visiting only one or two plant species. most of these bee species were observed in plants that were common to both seasons, mainly austroeupatorium sp. and s. hexandrum. this is expected considering the model preferential attachment, since a new species is more likely to interact with species that already has many links in the network (barabási & albert, 1999; olesen et al., 2008). these seasonal variations of the interaction network may occur in accordance with natural cycles and fluctuations in flowering patterns of plants and in populations of floral visitors and can also be influenced by environmental conditions. future studies should investigate the causes of temporal variations in interactions between plants and pollinators, especially those that may be related to conservation of priority areas such as inselbergs, in order to identify actions that can minimize human impact and support conservation initiatives in these threatened ecosystem. acknowledgments the authors would like to thank inmet for the climatic data; dr. darren m. evans (newcastle universityuk) for providing the r code to beta diversity of interactions analysis; herbário uenf (huenf), herbário do jardim botânico do rio de janeiro (herbário rb) and msc. tatiane pereira de souza (uff-rj) for support in the identification of the plants; dr. gabriel augusto rodrigues de melo (ufprpr) for support in the identification of the bees; dr. willian moura de aguiar (ufes-ba) for help in the field; dr. ana paula madeira di beneditto (uenf-rj) for suggestions in a previous version of this work; faperj for scholarship to mfm; cnpq (pq2) and faperj (cne researcher) for support to mcg.this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. we are also grateful for the financial support given by rio rural/gef/bird. references aguiar, c.m.l. & zanella, f.c.v. 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(2011). biologia da nidificação de megachile (moureapis) benigna mitchell. entomobrasilis, 4: 92-99. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i4.3386sociobiology 65(4): 591-602 (october, 2018) special issue interaction network and niche analysis of natural enemy communities and their host bees (hymenoptera: apoidea) in fragments of cerrado and atlantic forest introduction of the approximately 20,000 known species of bees more than 85% are not social but solitary (batra, 1984) and among the solitary bees, 5% of them are species that nest in preformed cavities (krombein, 1967; batra, 1984). irrespective of being social or solitary, the bees form the most important group of pollinators providing a crucial ecosystem service through their role in the sexual reproduction of both wild plants and crops (klein et al., 2007; le féon et al., 2013). the abundance of many bee species has been declining for a variety of reasons, including agricultural intensification, which includes the loss of natural habitats, agricultural practices, floral resource availability and, increased pesticide and herbicide use (potts et al., 2010). this decline of species diversity has been shown to result in productivity decrease in many ecosystems (tilman et al., 2001) while the diverse abstract natural enemies are important components of solitary bee communities that nest in preexisting cavities because they act as a relevant mortality factor and can regulate host population growth. nevertheless, the natural enemyhost interaction remains poorly investigated. this research aimed to determine the composition of the community, the structure of the interaction network, and niche overlap and breadth of natural enemy species in areas of cerrado (brazilian savanna) and semideciduous seasonal forest (atlantic forest) in the state of são paulo, brazil. trap-nests made of black cardboard and bamboo canes were provided in the field and inspected monthly in each area, from august 2001 to july 2003 at cerrado and from june 2006 to may 2008 at the semideciduous seasonal forest. a modular structure in the interaction network was observed for both areas with the populations of natural enemies showing high degrees of specialization. this structure confers higher stability against disturbances than less specialized webs since these adversities spread more slowly through the network. the niche analysis showed low degrees of overlap for both, trophic and temporal, among the natural enemy populations. sociobiology an international journal on social insects r lima, d moure-oliveira, ca garófalo article history edited by solange augusto, ufu, brazil received 27 april 2018 initial acceptance 28 may 2018 final acceptance 21 august 2018 publication date 11 october 2018 keywords antagonistic interactions, cleptoparasite, cavity-nesters, parasitoid, solitary bees. corresponding author reinanda lima faculdade de filosofia, ciências e letras de ribeirão preto universidade de são paulo av. bandeirantes, 3900, cep 14040-901 ribeirão preto, são paulo, brasil. e-mail: reinanda-09lima@usp.br pollinator communities provide more stable and productive ecosystem services (rogers et al., 2014). among social species, the honey bees are the primarily managed pollinator in agriculture and although the increased use of them may mitigate the loss of pollination services caused by the decline of solitary bees, they cannot entirely substitute the contribution of solitary bees to crop pollination (winfree et al., 2008; garibaldi et al., 2013). therefore, a better knowledge of factors driving the population dynamics of solitary bees is essential for the future conservation of suitable habitats and ecological interactions (kearns et al., 1998). population dynamics can be driven by resources (bottomup) or by natural enemies (top-down). bottom-up factors mainly act as density-independent factors that limit population growth while top-down factors, i.e., natural enemies, can regulate population dynamics by positive density-dependent parasitism or predation (berryman, 2001). universidade de são paulo, ribeirão preto, são paulo, brazil research article bees r lima, d moure-oliveira, ca garófalo – interactions between natural enemies and their host bees592 therefore, natural enemies are important components associated with populations comprising the bee communities, responsible for many adult and immature deaths (rochafilho et al., 2017). despite this, while mutualistic interactions between plant-pollinator have received much attention, the antagonistic interaction between natural enemies and their host bees remain poorly investigated. in general, natural enemies of solitary bees are insects belonging to three orders, hymenoptera, diptera and coleoptera (krombein, 1967). depending on the resources used as food they can be classified as cleptoparasites or parasitoids; the first ones use the larval food provisioned by the host to feed their own immature, and the second ones employ the host immature as an alimentary resource to their offspring (roulston & goodell, 2011) among the bees used as hosts, the species that nest in preformed cavities comprise a relevant component of the trophic niche of several natural enemy species (krombein, 1967). these bee species perform a high variety of nesting behavior, use different substrates to nest, distinct floral sources to provision their nests, and show different reproductive phenologies (michener, 2007). this high diversity observed in this bee group relates with the variety of attack strategies, alimentary preferences, and phenology of many natural enemies and influence their niche breadth, once all these biological aspects represent one dimension of the multidimensional space proposed by hutchinson (1957) for ecological niches. therefore, to know the natural history of these natural enemies, their temporal niche, and hosts preferences is essential to understand the community structure and to propose methodologies for prevention of high mortalities rates of bees in conservational and management plans. so, the understanding this poorly studied natural enemy-host system added with the great diversity observed in both groups, host bees, and their natural enemies, makes them a good model for interaction networks and ecological niche approach. another essential component of the community structure, the interaction network, complement the niche study because it represents the interaction strength, their stability and how these interactions are organized among the species of the community (thébault & fontaine, 2010; pocock et al., 2016). also, this approach is important not only to understand the structure of interactions but also to identify patterns of responses to environmental changes (massol & petit, 2013; osorio et al., 2015). this study aimed to investigate the composition and structure of the interaction networks in two communities of natural enemies and their hosts, species of solitary bees that nest in preexisting cavities. also, this study investigates the diet breadth of each species of natural enemy and the overlap of trophic and temporal niches among these species. material and methods study areas the study was carried out in an area of cerrado (brazilian savanna) of the santa cecilia farm (henceforth referred as scf) (20°46’ s, 47°61’ w), municipality of patrocínio paulista, and in a remnant of semideciduous seasonal forest (atlantic forest) of the estação ecológica dos caetetus (henceforth referred as eec) (22°26’ s, 49°44’ w), municipality of gália and alvinlândia, both areas of the state of são paulo, brazil. the cerrado has an area of 98 ha that together with 49 ha of semideciduous seasonal forest form the protected area reserve of the scf. both areas, cerrado, and forest, are continuous and contiguous, and practically untouched for more than four decades (teixeira et al., 2004). our sampling was carried out in the cerrado area. according to köppen (1948), the local climate is classified as aw, with a cold and dry season from april to september and a hot and wet season from october to march. during the dry periods, the monthly temperature ranges from 17.4 °c to 22.3 °c and the precipitation from 0.2 to 122.9 mm, and during the hot seasons, the monthly temperature ranges from 21.1 to 23.3 °c and the precipitation from 29.3 to 363.8 mm. the sampling occurred from august 2001 to july 2003 in this area. the eec has an area of 2,178.84 ha. the local climate is classified as cwa (köppen, 1948), with temperatures below 18 ºc in winter and above 22 ºc in summer. the average annual precipitation was 1,700 mm (tabanez et al., 2005). the vegetation consists mostly of the well-preserved forest, including seasonal semideciduous and gallery forests, immersed in a matrix of agricultural land, containing coffee, pastures, and rubber tree (hevea brasiliensis l., euphorbiaceae) crops (tabanez et al., 2005). in this area, the sampling occurred from june 2006 to may 2008. data collection the nests were obtained using the trap-nest methodology proposed by camillo et al. (1995), where artificial cavities made of black cardboard and bamboo canes were provided in the field as nesting substrate. the cardboard tubes were 6 cm long × 0.6 cm diameter and 8 cm long × 0.8 cm diameter, with one end closed with the same material. these tubes were inserted into cavities drilled in wooden plates (length: 30 cm, height: 12 cm, thickness: 5.0 cm). the bamboo canes, with variable diameters (from 0.8 to 1.5 cm) and lengths (from 10 to 15 cm), were cut so that the nodal septum closed one end of the cane, and all sizes were not equally represented. the bamboo canes were inserted into three pvc tubes with a length of 25 cm and a diameter of 10 cm. at scf, three plates with 55 small tubes each, one plate with 20 large tubes, and nine sets of bamboo canes, each set containing 10 to 15 canes, were placed. the pvc tubes and the plates were hung from trees randomly chosen and sociobiology 65(4): 591-602 (october, 2018) special issue 593 positioned 1.80 m above the ground. at eec, the trap-nests were set in seven collection sites; each site had available 40 small tubes and 40 large tubes, and 120 bamboo canes. the trap-nests were put on supports and kept at the study sites fixed at the height of 1.5 m from the ground. to protect the trap-nests from the sun and the rain, the canes were put into pvc tubes, and each plate received a small cover of hard plastic. the traps were inspected monthly in each area, and completed nests were taken to the laboratory, replaced by empty tubes and kept at room temperature until the emergence of the adults. after the emergence, the nests were opened, and the contents were analyzed. voucher specimens of bees and natural enemies were deposited in the entomological collection of the departamento de biologia da faculdade de filosofia, ciências e letras de ribeirão preto, university of são paulo. diet breadth, niche overlap, and network analysis the interaction strength between natural enemies and their hosts was attributed based on the number of brood cells attacked by each parasite, once only one individual of natural enemy developed in each brood cell parasitized. the natural enemy melittobia sp. (hymenoptera: eulophidae) was not used in the analysis of niche breadth and overlap, and in the network analysis because it was not possible to attribute the interaction strength due to the strategy of attack performed by this species. the trophic niche breadth of the communities was calculated using three diversity indices for each natural enemy species: richness (s), that represents the total number of bee species used as host; shannon-wiener diversity index (h´), that represents the relation between the number and abundance of each bee species used as host through the formula ,where s is the total number of bee species used as host by the natural enemy, pi is the proportion of the number of the bee species i used as host in relation of the total number of host used, and the ln is the natural logarithm; and pielou’s evenness index, that indicates if the host bees were explored in a uniform way (j´ = 1) or no (j´ = 0). trophic and temporal niche overlap degree among all natural enemies species in each community, scf and eec, were calculated in the software timeoverlap version 1.0 (castro-arellano et al., 2010) using the pianka (1973) and czechanowski’s indices (feinsinger et al., 1981). utilizing the rosario algorithm, a null-model analysis based on 10,000 randomizations was performed to determine if the community overlap values found were different from that expected by chance using a two-tailed test and a significance level of 5%. to estimate the niche overlap, a matrix of absolute abundance of the number of brood cells attacked for each host species (trophic) or number of attacks observed monthly (temporal) for each natural enemy species were used. overlap degrees of trophic and temporal niches for each pair of natural enemies species were calculated with the schoener index (1986) using the formula implemented in the package spaa 0.2.2 for r version 3.4.3 (r core team, 2017), where i and h are the pair of natural enemy species compared, and pik and phk are the number of brood cell attacked for each host species k (trophic) or the total number of brood cells attacked in each month k (temporal). in order to describe the natural enemies-hosts interactions, a weighted network was built for each sampling local (scf and eec) from a matrix of absolute abundance in the software pajek version 5.03 (mrvar and batagelj, 2018) using the ‘kamadakawai free’ method, in which natural enemy and host species were connected to one another. the degree of specialization in the diet of the populations was calculated in the software r version 3.4.2 (r core team 2017) measuring the h2´ index (blüthgen et al., 2006) for each community. the significance of this index was estimated with a monte carlo procedure using a null model (patefield, 1981) with the generation of 1,000 random matrices. the nestedness of each network was calculated in the software nodf version 2.0 based on the weighted metric wnodf (almeida-neto & ulrich, 2011) . the significance of this index was estimated with a monte carlo procedure in which 1,000 random matrices were generated using a null model and the randomization algorithm rc (ulrich & gotelli, 2010; almeida-neto & ulrich, 2011). weighted modularity was calculated for each network using the ‘computemodules’ function in a bipartite package for r software version 3.4.4.3 (r core team, 2017), which uses the quanbiomo algorithm (q) for quantitative data matrices (dormann & strauss, 2014). the number of steps taken for the analysis was 10 x 105. modularity was tested against 1,000 null models using the method ‘r2d’, which yielded a score zq, equivalent to the z-score of a normal distribution; following the proposed by dormann and strauss (2014), values of zq above than 2 represents significant modularity. the modules formed by this analysis were represented in the network using different colors patterns. to observe the ‘network functional role’of each species of natural enemies and hosts, the ‘czvalues’ function based on species weighted (bascompte et al., 2006) was used to calculated the connection (c-values) and participation values (z-values), indicating the contribution among and within modules, respectively (olesen et al., 2007). using the critical values showed by olesen et al. (2007), 0.625 for c-value and 2.5 for z-value, the species were classified in specialists (low c and z), module hubs (low c and high z), connectors (high c and low z), and network hubs (high c and z). these three last classifications represent generalist organisms. species with no z-score (= na) were not shown in the graph. results at scf, 12 species of natural enemies attacked 49 nests of seven species of solitary bees. these natural enemies belong –si=1 pi ln pi s noih = 1 – 1/2 s k pik – phk r lima, d moure-oliveira, ca garófalo – interactions between natural enemies and their host bees594 to four insect orders, hymenoptera, diptera, coleoptera, and neuroptera. according to the attack behavior, more parasitoid species than cleptoparasites were sampled (table 1, fig 1). during the period of study, we found asymmetry in the phenology of the natural enemy species, as in 2003 when only coelioxoides exulans (holmberg) was sampled (fig 1). the natural enemies with the highest number of hosts were c. exulans (4 species), leucospis cayennensis westwood (3 species), anthrax hylaios marston and anthrax oedipus fabricius (2 species). these parasites presented higher richness and diversity of host bees, and c. exulans attacked the highest number of brood cells (n = 29) (tables 1 and 2). fig 1. phenology of the natural enemy species at cerrado of the santa cecília farm, sp, brazil. the sampling period was from august 2001 to july 2003. each adult emerged from one host brood cell. natural enemies code host bees code nn nc coleoptera (meloidae) nemognatha sp.b ne_sp centris analis ce_an 1 1 tetrapedia rugulosa te_ru 3 3 diptera (bombyliidae) anthrax aquilus a an_aq tetrapedia diversipes te_di 1 1 anthrax hylaios a an_hy tetrapedia diversipes 1 1 tetrapedia rugulosa 3 3 anthrax oedipus a an_oe tetrapedia diversipes 3 3 tetrapedia rugulosa 2 2 hymenoptera (apidae) coelioxoides exulans b cx_ex tetrapedia diversipes 3 3 tetrapedia curvitarsis te_cu 10 10 tetrapedia rugulosa 8 15 tetrapedia garofaloi te_ga 1 1 coelioxoides cf. waltheriae b cx_wa tetrapedia sp. te_sp 1 1 mesocheira bicolor b me_bi centris tarsata ce_ta 1 1 hymenoptera (eulophidae)* melittobia sp.* anthodioctes sp. 1 hymenoptera (leucospidae) leucospis cayennensis a le_ca centris analis 1 1 centris tarsata 2 2 tetrapedia diversipes 1 1 leucospis manaica a le_ma tetrapedia rugulosa 5 5 hymenoptera (vespoidea) mutillidae a muti tetrapedia rugulosa 1 1 neuroptera mantispidae a mant tetrapedia rugulosa 1 1 total 49 56 * species not used in the analysis of this study (see material and methods) table 1. natural enemies and their host bees that nested in trap-nests at cerrado of the santa cecília farm, sp, brazil, from august 2001 to july 2003. a parasitoid, b cleptoparasite, nn = number of attacked nests, nc = number of attacked brood cells, and code = code of species used in the network. sociobiology 65(4): 591-602 (october, 2018) special issue 595 natural enemy scf eec s h´ j´ s h´ j´ coleoptera (meloidae) nemognatha sp. 2 0.64 0.92 2 0.66 0.95 diptera (bombyliidae) anthrax aquilus 1 0 anthrax hylaios 2 0.56 0.81 anthrax oedipus 2 0.67 0.97 anthrax sp. 2 0.26 0.37 hymenoptera (apidae) coelioxoides exulans 4 1.06 0.76 1 0 coelioxoides cf. waltheriae 1 0 3 0.69 0.63 coelioxoides sp. 2 0.69 1.00 mesocheira bicolor 1 0 mesocheira sp. 2 0.60 0.86 hymenoptera (megachilidae) coelioxys sp. 4 1.21 0.88 hymenoptera (leucospidae) leucospis cayennensis 3 1.04 0.95 leucospis manaica 1 0 leucospis sp. 4 0.76 0.55 hymenoptera (vespoidea) mutillidae 1 0 lepidoptera pyralidae 3 0.96 0.87 neuroptera mantispidae 1 0 table 2. diversity indices, richness (s), shannon diversity (h´) and pielou’s evenness (j´), for each natural enemy species at cerrado of the santa cecília farm and semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil. the sampling period at scf was from august 2001 to july 2003, and from june 2006 to may 2008 at eec. fig 2. interaction network between natural enemies and their hosts that nested in trap-nests at cerrado of the santa cecília farm, sp, brazil, from august 2001 to july 2003. the circles represent the hosts, and the diamonds represent the natural enemy species. the thickness of the lines represents the interaction strength (number of parasitized brood cells). each color pattern indicates a distinct module. natural enemies and hosts’ codes displayed in table 1. the network was modular, comprised of five modules, and not nested, with the community of natural enemies showing high specialization (fig 2, table 3). the community of natural enemies showed low levels of trophic and temporal overlap. the paired analysis showed that only a few species of natural enemy overlap their trophic r lima, d moure-oliveira, ca garófalo – interactions between natural enemies and their host bees596 niches, highlighting the interaction observed between the bee fly a. hylaios and leucospis manaica roman (noih = 0.75). maximal values of trophic overlap (noih = 1) were observed in natural enemies with rare occurrence, as for mantispidae and mutillidae. paired temporal niche analysis did not show high overlap among the natural enemies populations, with similar phenology being observed only between few species, as for the cleptoparasite beetle nemognatha sp. and the parasitoid a. hylaios (noih = 0.75), and between the two bee fly species anthrax aquilus marston and a. oedipus (noih = 1) (tables 3 and 4). for both, natural enemies and hosts, all species of the community performed a specialist behavior in the network, showing low levels in the interaction strength between (c-values) and within modules (z-values). the parasitoid a. hylaios showed the highest connectivity levels within the natural enemies species (c-value = 0.5), while the bee tetrapedia rugulosa friese showed the highest within the host species (c-value = 0.48). no species showed z-values above 1.3 (fig 5, supplementary material 1). ne_ sp le_ca le_ma me_bi cx_ex mant muti an_oe an_hy an_aq cx_wa ne_sp 0 0.4 0 0 0 0 0.5 0.75 0.5 0 le_ca 0.25 0.25 0 0 0 0 0 0 0 0.25 le_ma 0.67 0 0 0 0 0 0 0.25 0 0.6 me_bi 0 0.5 0 0 0 0 0 0 0 0 cx_ex 0.52 0.1 0.52 0 0.03 0 0 0.03 0 0 mant 0.67 0 1 0 0.52 0 0 0.25 0 0 muti 0.67 0 1 0 0.52 1 0 0 0 0 an_oe 0.4 0.25 0.4 0 0.5 0.4 0.4 0.5 1 0 an_hy 0.67 0.25 0.75 0 0.62 0.75 0.75 0.65 0.5 0 an_aq 0 0.25 0 0 0.1 0 0 0.6 0.25 0 cx_wa 0 0 0 0 0 0 0 0 0 0 table 4. trophic (below) and temporal (above) niche overlap for each pair of natural enemy species at cerrado of the santa cecília farm, sp, brazil, from august 2001 to july 2003. highest values in bold. natural enemies’ codes displayed in table 1. interaction network trophic niche overlap temporal niche overlap wnodf q mod zq h2´ pianka czechanowski pianka czechanowski scf 19.40ns 0.32* 5 3.72 0.85*** 0.37** 0.29** 0.14* 0.11* eec 26.33** 0.31*** 4 11.41 0.63*** 0.46** 0.37** 0.18ns 0.14ns values significant for a * p < 0.05, ** p < 0.01, *** p < 0.001, and ns for non significant values. table 3. weighted nestedness (wnodf), weighted modularity (q) with the z-score (zq), number of modules shaped (mod), specialization degree (h2´), and trophic and temporal niche overlap metrics (pianka and czechanowski) calculated for the natural enemies at cerrado of the santa cecília farm (sampling from august 2001 to july 2003) and semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil (sampling from june 2006 to may 2008). at eec, nine species of natural enemies attacked 148 nests of seven species of solitary bees. the natural enemies belong to four orders, hymenoptera, diptera, coleoptera, and lepidoptera. according to the attack behavior, we found a higher number of cleptoparasites than parasitoids (table 2, fig 3). excepting pyralidae, all natural enemy species were sampled in both years of study (fig 3). the natural enemies with the highest number of host bees were coelioxys sp. (4 species), leucospis sp. (4 species), coelioxoides cf. waltheriae ducke (3 species) and pyralidae (3 species) (tables 2 and 5, fig 3). coelioxoides cf. waltheriae and nemognatha sp. were the cleptoparasites more frequent, attacking 66 and 56 cells respectively (table 5). the network presented a low degree of nestedness and a modular structure, with populations with a specialized diet, and four modules were formed in the network (table 3, fig 4). the analysis showed trophic niche overlap among the natural enemy populations. several species of natural enemies presented a high trophic overlap, as the interactions between c. exulans and anthrax sp. (noih = 0.93), nemognatha sp. and coelioxys sp. (noih = 0.86), and nemognatha sp. and coelioxoides sp. (noih = 0.86). the temporal analysis did not show overlap among the natural enemy populations, and in the paired analysis, the highest values were observed between coelioxys. sp. and leucospis. sp. (noih = 0.5), and nemognatha sp. and pyralidae (noih = 0.4) (tables 3 and 6). sociobiology 65(4): 591-602 (october, 2018) special issue 597 fig 3. phenology of the natural enemy species at the semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil, from june 2006 to may 2008. each adult emerged from one host brood cell. natural enemies code host bees code nn nc coleoptera (meloidae) nemognatha sp.b ne_sp tetrapedia diversipes te_di 17 20 tetrapedia sp. te_sp 30 35 diptera (bombyliidae) anthrax sp.a an_sp tetrapedia diversipes 12 13 tetrapedia sp. 1 1 hymenoptera (apidae) coelioxoides exulans b cx_ex tetrapedia diversipes 4 6 coelioxoides cf. waltheriae b cx_wa tetrapedia diversipes 43 50 tetrapedia rugulosa te_ru 2 4 tetrapedia sp. 9 12 coelioxoides sp.b cx_sp tetrapedia diversipes 3 4 tetrapedia sp. 3 4 mesocheira sp.b me_sp centris tarsata ce_ta 3 5 centris sp.2 ce_sp2 1 2 hymenoptera (megachilidae) coelioxys sp.b co_sp centris analis ce_an 1 1 centris tarsata 1 1 centris sp. ce_sp 1 1 centris sp.2 1 3 hymenoptera (leucospidae) leucospis sp.a le_sp centris analis 1 1 centris sp. 1 1 tetrapedia diversipes 6 6 lepidoptera pyralidae c pyra centris analis 1 1 tetrapedia diversipes 2 2 tetrapedia sp. 5 5 total 148 178 table 5. natural enemies and their host bees that nested in trap-nests at the semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil, from june 2006 to may 2008. a parasitoid, b cleptoparasite, c nest destroyer, nn = number of attacked nests, nc = number of attacked cells, and code = code of species used in the network. as observed for scf, all species of natural enemies and hosts exhibited a specialized behavior at eec, with low c and z-values. the cleptoparasite nemognatha sp. and the host tetrapedia diversipes klug showed the highest c-values, closed to 0.6, and no species showed z-values above 1.2 (fig 5, supplementary material 1). both communities were composed by similar groups of parasites and hosts. tetrapedia species were the most abundant host, while coelioxoides species were the most abundant natural enemy sampled. besides, centris and mesocheira bees, nemognatha beetles, leucospis wasps and anthrax beeflies were also important components of the two communities. r lima, d moure-oliveira, ca garófalo – interactions between natural enemies and their host bees598 le_sp co_sp pyra me_sp cx_wa cx_sp an_sp cx_ex ne_sp le_sp 0.5 0 0.29 0.11 0.1 0.24 0.17 0.13 co_sp 0.22 0 0.2 0.03 0 0 0.17 0.09 pyra 0.4 0.14 0.13 0.26 0.1 0.13 0 0.4 me_sp 0 0.45 0 0.11 0 0.29 0 0.11 cx_wa 0.67 0 0.47 0 0.08 0.39 0.04 0.33 cx_sp 0.5 0 0.79 0 0.68 0.29 0.1 0.12 an_sp 0.67 0 0.36 0 0.83 0.57 0 0.17 cx_ex 0.67 0 0.29 0 0.76 0.5 0.93 0.02 ne_sp 0.38 0 0.86 0 0.54 0.86 0,.43 0.36 fig 4. interactions network between natural enemies and their hosts that nested in trap-nests at the semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil, from june 2006 to may 2008. the circles represent the hosts, and the diamonds represent the natural enemy species. the thickness of the lines represents the interaction strength (number of parasitized brood cells). each color pattern indicates a distinct module. natural enemies and hosts’ codes displayed in table 5. table 6. trophic (below) and temporal (above) niche overlap for each pair of natural enemy species at the semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil, from june 2006 to may 2008. highest values in bold. natural enemies’ code displayed in table 1. discussion the communities here studied were composed of similar families and genera of natural enemies and hosts, and the natural enemies showed identical richness and diversity values of hosts used. it was also observed a modular network with high levels of specialization in the interactions between its component members, natural enemies, and their hosts. the modular structure is expected in antagonistic interactions where the predators, or natural enemies in this study, tend to optimize their attacks, and the preys, or hosts, tend to optimize their defenses, in an arms race, increasing the module formation in the web (thébault & fontaine, 2010). however, unlike what is reported here, high specialization degrees are not always observed in this network structure (araujo et al., 2018). organisms with preferences in the diet are more likely to occur in habitats structurally more diverse and poorly isolated (pereira-peixoto et al., 2016; araujo et al., 2018). both areas sampled in this study are surrounded by urban and agricultural landscapes, and despite the low diet breadth and the specialized performance observed in the network, the majority of the natural enemy species were reported attacking several host species, characterizing a generalist organism in an ecological concept. sociobiology 65(4): 591-602 (october, 2018) special issue 599 according to michener (2007), cleptoparasite bees usually attack nests of related lineages. this association between host-cleptoparasite would explain the attacks of the species of the genus coelioxoides for the nests of their sister genus tetrapedia, as had been reported by several authors (aguiar et al., 2005; gazola & garófalo, 2009; rocha-filho et al., 2017). the high abundance of coelioxoides parasites observed in both communities is related with the high abundance of their available host, tetrapedia bees, once parasites must synchronize their life cycles with their host’s life cycles (wcislo, 1987). among other species that attack closed phylogenetical taxon are the cuckoo bees of the genus coelioxys, cleptoparasites of many megachilidae bees (krombein, 1967). however, differently of the tetrapediini parasites and as observed in this study, coelioxys exhibited a less specialized behavior, parasitizing nests of others bee groups, as the species of the genus centris. this association with nests of the oil-collecting bees has also been reported by other authors such as morato et al. (1999), aguiar and martins (2002), gazola and garófalo (2009), araujo et al. (2018), oliveira and gonçalves (2017), rocha-filho et al. (2017) and araujo et al. (2018). species analyzed in this study and that are the natural enemies of many bees and wasps species, as anthrax and leucospis (krombein, 1967), showed a restrict diet breath due to the limited temporal niche. the same situation was observed for others parasites sampled, as the pyralidae moth and the mantisflies, who were reported parasitizing many insects groups (buys, 2008; hook et al., 2010), but in this study, they behave as a more specialized organism in the network analysis, attacking few host species. these aspects of the trophic and temporal niche contributed to the formation of a modular structure in the interaction network. it is worth mentioning that many of the natural enemies analyzed in this study use other groups as hosts, like eusocial bees, ground-nesting bees, and other insects; thus, in the present study was only evaluated a part of the trophic niche of some natural enemies. as other studies showed a modular pattern for this system even considering a broader niche breath for these natural enemies (araújo et al. 2018), we expected that our network structure maintain the modularity even adding others groups of host. under an individual and populational perspective, the niche specialization can favor the optimizing in the costbenefit relation, but also can increase the chance of extinction in face to environmental changes (biesmeijer et al., 2006). however, in a perspective of the community, the modular structure and the low connections for the nodes observed in our study indicate that the host-natural enemy interactions at scf and eec would present a high persistence and resilience against disturbances (thébault & fontaine, 2010). although the sampling effort, years and phytophysiognomy were different, the communities of scf and eec were structured by similar groups of natural enemies and host bees. likewise, other authors reported similar composition in distinct brazilian areas (aguiar & martins, 2002; gazola & garófalo, 2009; mesquita & augusto, 2011; oliveira & gonçalves, 2017; rocha-filho et al., 2017; araújo et al., 2018), which evidences a closed relationship between these parasites species and their host bees. evolutionary history and phylogenetic diversity are factors that strongly influence these ecological interactions (michener, 2007; staab et al., 2016; andreazzi et al., 2017). in conclusion, this study described the diversity of natural enemies and the network interaction structure in the community. preferences and seasonality strongly influenced the richness and abundance of these parasites and their interactions with each other and with their hosts. another point, the similarity observed among communities and time fig 5. connection (c-value) and participation (z-value) values for natural enemies (left) and hosts species (right) in the network of cerrado of the santa cecília farm, sp, brazil (black points) (sampling from august 2001 to july 2003) and of the semideciduous seasonal forest of the estação ecológica dos caetetus, sp, brazil (gray points) (sampling from june 2006 to may 2008). following olesen et al. (2007), the critical values (gray lines) for c is 0.625, and for z is 2.5. r lima, d moure-oliveira, ca garófalo – interactions between natural enemies and their host bees600 evidence the close relationship between these parasite species and their hosts. our study contributes to a better understanding of the complex and poorly studied natural enemy-host interaction, and provides relevant information on the diet breadth of these important components of the communities. acknowledgments the authors are grateful to j.c. serrano for the identification of the organisms, e.s.r. silva for technical support, the instituto florestal de são paulo for the permission to work at estação ecológica dos caetetus, the owners of the santa cecília farm who allowed us to have access to their land, and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the providing a phd fellowship to the first author (grant number 140135/20170) and a research scholarship to last author (process # 310 679//2014-1). the authors also thank cnpq and fapesp (process # 2010/10285-4), for financial support, research center on biodiversity and computing (biocomp), and the suggestions of the anonymous reviewers that improved the quality of the manuscript. supplementary material doi: 10.13102/sociobiology.v65i4.3386.s2216 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3386/0 references andreazzi, c.s., thompson, j.n., & guimarães jr, p.r. 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(1987). the roles of seasonality, host synchrony, and behaviour in the evolutions and distributions of nest parasites in the hymenoptera (insecta), with special reference to bees (apoidea). biological review, 62: 515-543. doi: 10.1111/j.1469-185x.1987.tb01640.x. winfree, r., williams, n.m., gaines, h., ascher, j.s. & kremen, c. (2008). wild bee pollinators provide the majority of crop visitation across land-use gradients in new jersey and pennsylvania, usa. journal of applied ecology, 45: 793802. doi: 10.1111/j.1365-2664.2007.01418.x _hlk507076757 doi: 10.13102/sociobiology.v65i4.3452sociobiology 65(4): 770-772 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sleeping behaviour of an oil-collecting bee, centris (paracentris) xanthomelaena moure & castro (hymenoptera: apidae: centridini) bee and wasp solitary species have been observed spending the night in dense clusters, and this behaviour is called sleeping aggregation (eg. linsley, 1962). this is suggested as a preliminary stage to social behaviour, since aggregation promotes temperature increase, and the vigilance against predators (evans & linsley, 1962). the sleeping aggregation was studied in some species of solitary bees as melissodes nigroaenea (smith) and melissoptila aff. bonaerensis holmberg (mahlmann et al., 2014), oxaea austera gerstaecker (oliveira & castro, 2002), augochlorella neglectula (cockerell) (wcislo, 2003), and idiomelissodes duplocincta (cockerell) (alcook, 1998). such aggregations had been reported for some species of centris as: centris smithii cresson, centris decolorata lepeletier, centris lanipes (fabricius) (starr & vélez, 2009), centris burgdorfi friese (sabino et al., 2017) and centris fuscata lepeletier (azevedo & faria jr, 2007). this behaviour can be described, generally, for males who spend the night primarily abstract sleep aggregations have been registered for some species of solitary bees and wasps. in this note we describe the aggregation behaviour of centris xanthomelaena moure & castro bees in their inactive periods. the dormitories were discovered close to the bee nesting sites, in a caatinga area. we monitored the males and females at the sleeping site for 16 consecutive days in july 2017. we observed that females of c. xanthomelaena spent the night outside their nests, in dry branches of mimosa tenuiflora (fabaceae). furthermore, males and females have shared the dormitories, and sometimes they formed mixed ones. both males and females flew around the nearest dormitories during all observation days. the data about the sleep behaviour of c. xanthomelaena will contribute to the understanding of these aspects within the centris genus. sociobiology an international journal on social insects hoj martins1, p oliveira-rebouças2, vs ferreira1 article history edited by solange augusto, ufu, brazil received 10 may 2018 initial acceptance 17 june 2018 final acceptance 21 august 2018 publication date 11 october 2018 keywords solitary bees, sleeping aggregation, dormitory. corresponding author herbeson ovidio de jesus martins campus de ciências agrárias colegiado de ciências biológicas universidade federal do vale do são francisco rodovia br 407, km 12 – lote 543 projeto de irrigação senador nilo coelho s/nº c1 cep 56300-990, petrolina-pe, brasil. e-mail: herbeson.bio@hotmail.com on plant branches, cavities or flowers (alves-dos-santos et al., 2009; starr & vélez, 2009; mahlmann et al., 2014; sabino et al., 2017). on the other hand, females can sleep inside their nests, as a strategy to protect their offspring (aguiar & gaglianone, 2003; martins et al., 2014), or atypically, outside them, as observed by sabino et al. (2017). there are few studies describing the nocturnal aspects and sleep aggregations of the centris species. thus, we described here the composition and aspects related to the sleeping roost of centris (paracentris) xanthomelaena moure and castro, which is an oil-collecting and ground-nesting solitary bee, endemic to the brazilian semiarid region. the sleeping roosts were identified in an area of caatinga at the campus de ciências agrárias of the universidade federal do vale do são francisco (cca/univasf) (9°19’44.2’’ s, 40°33’30.1w), petrolina, pernambuco state, brazil. the sleeping roosts were located close to four nesting sites, between 6 to 41 meters away from the nests. 1 universidade federal do vale do são francisco, petrolina, pernambuco, brazil 2 universidade estado da bahia, juazeiro, bahia, brazil short note sociobiology 65(4): 770-772 (october, 2018) special issue 771 in order to observe the activity in the dormitories, we captured and marked on the thorax (using non-toxic color pens, uni posca ®) six females and eight males in their sleeping site. after that, they were released. the sleeping roosts were monitored for 16 consecutive days, from 04:00 a.m. to 05:00 a.m. and from 06:00 p.m. to 08:00 p.m., in july 2017. voucher specimens were collected, pinned and sent to specialists for identification and deposition in the entomological collection of the museu nacional, rio de janeiro (mnrj). in this study, c. xanthomelaena females have displayed the behaviour of sleeping outside their nests. during the nesting activity, the females looked for places close to their nests and landed on dry branches of mimosa tenuiflora (fabaceae). we observed that the females overnighted at several points around the nesting sites. mimosa tenuiflora was the predominant plant in the area, but only six trees were used as dormitories by c. xanthomelaena specimens. the females always choose the same branch as dormitories throughout their nesting activity. nevertheless, there was alternation among dormitories. once selected, the same branch was used every day as a dormitory. thus, males and females shared the same branches. the sleeping aggregation was formed by three different ways: (1) only males (fig 2b), (2) only females (fig 1b), and (3) males and females together in mixed clusters (fig 1a). sometimes we observed females sleeping alone (fig 2c). the c. xanthomelaena females flew back to their dormitories, soon after the last trip from their nests, around 05:20 p.m. they have performed several flights and over flights around the trees, hovering over the branches, before they land at the definitive branch to spend the night, from 5:40 p.m. to 5:56 p.m. during this period, we have observed agonistic behaviour among individuals because there were collisions among males. after landing on the chosen branch, the females performed the body grooming activity, passing the front and middle legs over the head, wings, and rubbing the hind leg pair. males and females were grasping the substrate with the mandibles and legs, in order to keep the body held over the substrate. during the observer approach, the males exhibited the behaviour of keeping the hind legs raised (fig 2a), as seen in males of ancyloscelis males (alves-dos-santos, 1999), which could be a defensive behaviour. in the morning, the females left the dormitory at 04:50 a.m., before the sunrise, performing their first trip to collect resources before going back to their nests for the first time in the day. the males remained in the dormitories longer than the females, and left them around 05:30 a.m. unlike our register of c. xanthomelaena record, researchers have reported that centris females usually sleep in their nests as a sentinel behaviour (aguiar & gaglianone, 2003; rego et al., 2006; martins, et al., 2014). it may be useful to those females to defend their nests against the attack of natural enemies. the choice of the dormitories by c. xanthomelaena has been mainly associated with the proximity among trees and nesting sites and not associated with the plants that females use to collect floral resources. however, other studies on the centris dormitories have showed that some plants species, such as caesalpinia (starr & vélez, 2009) and krameria (sabino et al., 2017) were used as dormitories, as well as to collect floral resources. bee species, in general, have been reported forming clusters of individuals during the period of inactivity (linsley, 1962; alcook, 1998; alves-dos-santos et al., 2009), but in most cases these groups have been composed of males (alves-dos-santos et al., 2009; wcislo, 2003; mahlmann et al., 2014). likewise, we observed herein, c. burgdorfi females were also recorded sleeping outside their nests, on flowers and fruits of krameria tomentosa, and forming clusters of approximately 76 individuals, approximately (sabino et al., 2017). in addition, both males and females of c. xanthomelaena shared the same dormitories, sometimes mixed ones. mixed dormitories were also recorded by starr and vélez (2009) for the species c. decolorata, c. smithii and c. lanipes in anguilla. the data from the present study will contribute to the understanding of these aspects within the centris genus. future analyses will be needed to identify the pheromones associated with the dormitories and which aspects would influence the females and males to choose one of them. fig 1. main composition of sleeping aggregation on branches of mimosa tenuiflora in an caatinga area, petrolina, brazil, july 2017. (a) female and males sharing the same branches as dormitory, (b) sleeping aggregation composed only by females. hoj martins, p oliveira-rebouças, vs ferreira – bee sleeping aggregation772 acknowledgements the authors thank dr. felipe vivallo (museu de zoologia/universidade federal do rio de janeiro) for the identification of the species. references aguiar, c.m.l., gaglianone & m.c. (2003). nesting biology to centris (centris) aenea (hymenoptera, apidae, centridini). revista brasileira de zoologia, 20: 601-606. doi: 10.1590/s0101-81752003000400006 alcock, j. (1998). sleeping aggregations of the bee idiomelissodes duplocincta (cockerell) (hymenoptera: anthophorini) and their possible function journal of the kansas entomological society, 71: 74–84. alves-dos-santos, i. (1999). aspectos morfológicos e comportamentais dos machos de ancyloscelis latreille (anthophoridae, apoidea). revista brasileira de zoologia, 16, (supl. 2): 37–43. doi: 10.1590/s0101-81751999000600005 alves-dos-santos, i., gaglianone, m.c., naxara, s.r.c., engel, m.s. (2009). male sleeping aggregations of solitary oil-collecting bees in brazil (centridini, tapinotaspidini, and tetrapediini; hymenoptera: apidae). genetics and molecular research, 8: 515-524. azevedo, a.a., faria, l.r.r.jr (2007). nests of phacellodomus rufifrons (wied, 1821) (aves: furnariidae) as sleeping shelter for a solitary bee species (apidae: centridini) in southeastern brazil. lundiana, 8: 53-55. evans, h.e., linsley, e.g. (1960). notes on a sleeping aggregation of solitary bees and wasps. bulletin of the south california academy science, 59: 30-37. linsley, e.g. (1962). sleeping aggregations of aculeate hymenoptera – ii. annals of the entomological society of america, 55: 148–164. mahlmann, t., hipólito, j., oliveira, f.f. (2014). male sleeping aggregation of multiple eucerini bee genera (hymenoptera: apidae) in chapada diamantina, bahia, brazil. biodiversity data journal 2, 2: 15-56. doi: 10.3897/bdj.2.e1556 martins, c.f., peixoto, m.p., aguiar, c.m.l. (2014). plastic nesting behavior of centris (centris) flavifrons (hymenoptera: apidae: centridini) in an urban area. apidologie, 45: 156171. doi: 10.1007/s13592-013-0235-4 oliveira, f.f., castro, m.s. (2002). nota sobre o comportamento de agregação de machos de oxaea austera gerstaecker (hymenoptera, apoidea, oxaeinae) na caatinga do estado da bahia. brasil. revista brasileira de zoologia, 19: 301–303. rêgo, m.m.c., albuquerque, p.m.c., ramos, m.c., carreira, l. m. (2006). aspectos da biologia de nidificação de centris flavifrons (friese) (hymenoptera: apidae, centridini), um dos principais polinizadores do murici (byrsonima crassifolia l. kunth, malpighiaceae), no maranhão. neotropical entomology, 35: 579-587. doi: 10.1590/s1519-566x2006000500003 sabino, w.o., silva, c.i., alves-dos-santos, i. (2017). mating system and sleeping behaviour of the male and female centris (paracentris) burgdorfi friese. journal of insect behavior, 30: 103–118. doi: 10.1007/s10905-017-9600-x starr, c.k. & vélez, d. (2009). a dense daytime aggregation of solitary bees (hymenoptera: apidae: centridini) in the lesser antilles. journal of hymenoptera research, 18: 175–177. wcislo, w.t. (2003). a male sleeping roost of a sweat bee, augochlorella neglectula (ckll.) (hymenoptera: halictidae), in panamá. journal of the kansas entomological society, 76: 55–59. retrived from: http://www.jstor.org/stable/25086083 fig 2. behaviors observed in the sleeping aggregations on branches of mimosa tenuiflora branches in a caatinga area, petrolina, brazil, july 2017. (a) male with hind leg (red arrow) raised, (b) male sleeping aggregation, (c) female sleeping alone in a branch. _hlk517370902 _hlk513731096 _hlk513731822 sociobiology 65(4): october (2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 editorial special issue on bees undoubtedly bees are some of the most extensively researched group organisms on earth, and this might have to do with a long history of coexistence between these insects and humans. honey bees and other social bees have been exploited for food since prehistory, and their charms as highly elaborated societies have puzzled curious individuals, from naturalists, philosophers to scientists. currently, we stand at a stage of having documented the existence of approximately 20,000 species of bees worldwide (and expect that some few thousand more await to be taxonomically described), having characterized genomes of more than a handful of species, and having studied several facets of the basic as well as the applied biology of hundreds of species. in contrast to the impressive body of knowledge about bees nowadays, there exists a massive number of questions begging to be addressed too. this may be an apparent effect of the principle that “the more we learn, the more we realize how much we don’t know.” this is a quote attributed to albert einstein, who supposedly also had his concerns about bees! the present issue of sociobiology is dedicated to our beloved bees, to allow us all to learn more about them, but to realize how much we still do not know. in this special issue, we celebrate the 6th anniversary of the publication of the journal sociobiology in brazil. we are pleased to have a special issue on bees and to have its publication happening concurrently with the xii encontro sobre abelhas, an event that has allowed bee biologists from various parts of brazil to gather since 1994. this issue is composed of 32 papers, dealing with a diversity of topics that reflect the vigor of contemporary bee research. one contributed paper presents an extensive revision on the economic and cultural values of stingless bees among ethnic groups of tropical regions of the americas – in this case, a collaboration among researchers from four different countries provided examples of successful regional strategies in averting the cultural and economic loss in natural human heritage. other topics represented in the volume include comparative molecular cytogenetics in melipona, the identification of candidate reference genes for one orchid bee species, and the evaluation of the stability of gene expression across different developmental stages and a characterization of leucine-aminopeptidase a (lap-a) orthologs in the genome of bee species with varying levels of social organization, and the search for molecular markers to delimit subspecies of bees as well as the application of dna barcoding to investigate taxonomic questions within a highly polymorphic species. likewise, comparative analyses are employed to evaluate the intraspecific morphological variation in different contexts, including an investigation of the influence of drought periods on the morphological variation among populations using traditional and geometric morphometric techniques, and analyses of the chemical compounds that might have a pheromonal role in cephalic salivary gland and epicuticle. from an ecological perspective, there are questions of different natures that help to interpret the importance of characterizing local and regional assemblies of bees to explain aspects of the distribution of diversity through space and how this relates to the biology. these include the level of landscape heterogeneity regarding forest cover and an evaluation of the effect of this factor on bee diversity, the ecology of bee communities in natural and semi-natural habitats, and the analysis of the interaction between geographic range spatial niche overlap in future climate change scenarios. moreover, bee foraging behavior and diets are investigation topics discussed in this special issue as well as the effects of seasonality on the trophic niche. the general (and growing) interest for pollination is examined when talking about the role of bees as pollinators of agricultural crops and their impact on the productivity at large, and the effects of improved pollination efficiency in greenhouses. two important applications of basic knowledge about bee biology and their diets are investigation that relates the consumption of fermented artificial protein diet honey bees and its effect on the levels of hemolymph protein and vitellogenin, and the examination of the impact of pesticide toxicity on different species of stingless bees and the assessment of potential risks regarding their exposure to pesticides. five additional topics complete the range of subjects covered in this special issue: the robbing behavior and plasticity to obtain food resources in the eusocial stingless bees, the description of eusocial behavior in a sociobiology an international journal on social insects sociobiology 65(4): october (2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sweat bee (halictidae) species reared in the laboratory, nesting biology of solitary bees, behavior of males and the investigation of toxic metals in honey and propolis. the future of bee research looks prosperous and is clearly pointing out to very diverse directions. it is a pleasure for sociobiology to publish this special issue and, thus, allow the expression of the enthusiasm for bee research! we thank all authors and reviewers for this interesting and important special issue. last but not least, we would like to dedicate this special issue to dr. warwick kerr, whose academic legacy echoes in the work of several bee researchers throughout brazil and elsewhere. sincerely, cândida maria lima aguiar (uefs, brazil) denise araujo alves (esalq-usp, brazil) eduardo a. b. almeida (usp, brazil) kleber del-claro (ufu, brazil) solange cristina augusto (ufu, brazil) editors for this special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.2977sociobiology 66(1): 97-106 (march, 2019) characterizing honeybee cuticular hydrocarbons during foraging introduction when visiting a floral patch, honeybees have to decide to continue or abandon the feeding site. during this decision-making process, bees evaluate, among many aspects, the profitability of the food source in terms of sucrose concentration and reward rate, its distance from the hive, the difficulty to obtain food and its availability (seeley, 1995). it has been well studied in apis mellifera that foraging behavior is modulated by the profitability of the exploited nectar source: as food quality increases, so does the frequency of foraging visits and the intensity of nestmate recruitment within the hive; the interval in-between foraging visits is shortened, and the probability of abandoning the source decreases (núñez, 1970; núñez, 1982; seeley et al., 1991; von frisch, 1967). abstract honeybees (apis mellifera) adjust their time and effort during foraging activity. their metabolic rates together with body temperature rise while gathering profitable resources. these physiological changes may result in a differential cuticular profile, which in turn may bear communicational value. we evaluated if sucrose concentration of collected food affects the cuticular chemistry of honeybees during foraging. we trained bees to artificial feeders with high (2 m) and low (0.5 m) sucrose concentrations, and captured the active foragers for surface extraction of cuticular compounds. we sampled foragers just after feeding, before taking-off towards the hive, and upon landing at the hive entrance, before entering the hive. through gas chromatography-mass spectrometry analysis of cuticular extracts, we identified and quantified 48 compounds, including cuticular hydrocarbons (chcs) and volatiles associated with exocrine glands. we found that higher sucrose concentrations resulted in increased amounts of alkanes and alkenes in the surface extracts of foragers captured at the hive entrance, but not at the feeding site. our results suggest that the differences that have been reported for chcs in waggle-dancing honey bees can be already found once they return to the hive from profitable food sources. sociobiology an international journal on social insects ms balbuena1,2, a gonzález3*, wm farina1,2* article history edited by kleber del-claro, ufu, brazil. received 02 march 2018 initial acceptance 19 may 2018 final acceptance 16 june 2018 publication date 25 april 2019 keywords apis mellifera, food source exploitation, cuticular chemistry, chemical communication. corresponding author maría sol balbuena facultad de ciencias exactas y naturales, universidad de buenos aires pabellón ii, ciudad universitaria (c1428eha) ciudad autónoma de buenos aires, argentina. e-mail:msbalbuena@bg.fcen.uba.ar the productivity of the food source also correlates positively with the metabolic rate of the forager bee, and higher metabolic rates imply a higher motivational state in the bee (núñez & giurfa, 1996). indeed, the thoracic temperature of forager bees has been also used as a measure of foraging motivation (schmaranzer & stabentheiner, 1988; stabentheiner, 1996; stabentheiner & hagmüller, 1991; stabentheiner et al., 1995), and thermographic measurements have shown that thoracic temperature correlates with higher food quality, both at the feeding site (schmaranzer & stabentheiner, 1988; waddington, 1990) and inside the hive (farina & wainselboim, 2001; stabentheiner & hagmüller, 1991). when a forager bee returns to the hive from a profitable nectar source, it dances vigorously to communicate the discovered food source to its nestmates (von frisch, 1967). 1 laboratorio de insectos sociales, departamento de biodiversidad y biología experimental, facultad de ciencias exactas y naturales, universidad de buenos aires, ciudad autónoma de buenos aires, argentina 2 instituto de fisiología, biología molecular y neurociencias (ifibyne), conicet-universidad de buenos aires, buenos aires, argentina 3 laboratorio de ecología química, facultad de química, universidad de la república, montevideo, uruguay * senior co-authors research article bees ms balbuena, a gonzález, wm farina – cuticular chemistry in foraging honeybees98 this stereotypic display not only informs about distance and direction to the food source, but also facilitates the conveyance of chemical cues such as odors from the exploited food source (díaz et al., 2007; seeley, 1995; von frisch, 1967), as well as other signals produced during the waggle dance that may attract nestmates (dyer, 2002; grüter & farina, 2009; thom et al., 2007). the incoming forager also transfers the gathered crop contents through trophallaxis (mouth to mouth contacts) to receiver nestmates. both dance and trophallaxis affect the body temperature of the bees; waggle dancing increases the body temperature of the recruiting bee (stabentheiner, 1996; stabentheiner & hagmüller, 1991), and trophallaxis raises the body temperature of food receivers, which is also affected by unloading rate and body temperature of the donor bee (farina & wainselboim, 2001). changes in metabolic rates and/or thoracic temperatures of the active foragers may also promote a passive emission of cuticular hydrocarbons, which in turn may result in a chemical cue to promote foraging. indeed, four cuticular hydrocarbons (chcs), z-(9)-tricosene, tricosane, z-(9)-pentacosene and pentacosane, have been reported as putative semiochemicals emitted by waggledancing bees (thom et al., 2007). to mimic a situation that represents intensive dances, three of these compounds were artificially added into a hive, resulting in more foragers exiting the hive and visiting known food sources (gilley, 2014; gilley et al., 2012; thom et al., 2007). these four chcs were detected by sampling the abdominal surface of dancing bees, and despite their low volatility, they were also found in their surrounding headspace (thom et al., 2007). moreover, among several other compounds, the same hydrocarbons were reported not only in the headspace of foraging bees at the feeding site, but also in the cuticular extracts of these entire bees (schmitt et al., 2007), and have long been known as major cuticular hydrocarbons in forager honeybees (blomquist et al., 1980b). if chcs were chemical signals related to foraging motivation in honeybees, it might be expected that food source profitability modulates this signal, a possibility that has not yet been explored. this hypothesis rests on the fact that sucrose concentration modulates the foraging motivational state, which itself promotes changes in the metabolic rate and the body temperature, potentially promoting the emission of chcs. such differential emission of hydrocarbons might be detected not only within the social environment of the hive, but also in the foraging context. in this study, we characterized the cuticular hydrocarbon profiles of forager bees with respect to food source profitability. specifically, we analyzed the cuticular extracts of forager bees fed with different sucrose concentrations at two different stages during foraging: i) at the feeding site, and ii) once they land at the hive entrance. materials and methods study site and animals three colonies (h1, h2 and h3) of honeybees apis mellifera l. with a queen, brood and reserves were used. colonies h1 and h2 were reduced from ordinary commercial hives to four-frame langstroth hives, containing each about 10000 worker bees. these colonies were used for collecting forager bees at the feeding site. colony h3, with 3000-3500 honeybees, was housed in a two-frame observation hive, and was used for collecting foragers at the hive entrance, upon returning from the feeder. to prevent interference with other bee colonies, this hive was enclosed within a flight chamber (6 m length x 3 m wide x 2 m height), which consisted of a wooden structure with polyethylene mesh walls. the hive was located at one end of the chamber, allowing for 6 m direct flight from the feeder. except for the experimental periods, the flight chamber remained open and the bees could forage freely outside. hence, foragers from all beehives (h1 to h3) had access to a natural environment containing natural flowers. experiments were performed in spring 2012 (h1), summer 2013 (h2), and summer 2014 (h3) at the experimental field of universidad de buenos aires (argentina). chemical analyses of cuticular extracts were carried out at the universidad de la república in montevideo, uruguay. experimental procedure two experimental settings were employed; one for collecting forager bees at the feeding site (h1 and h2), and another for collecting them at the hive entrance (h3). non-foraging honeybees from inside the hive (h1 and h2, henceforth “hive bees”) were also captured and extracted for comparison purposes. to be sure that they were not foragers, these bees were caught from the center of the hive, in the surroundings of the brood area, where honeybees usually perform tasks as nurses or food processors (seeley, 1995). forager bees were trained to collect unscented 1 m sucrose solution at an artificial feeder located at either 10 (h1 and h2) or 6 m (h3) from the hive. once a foraging group of approximately 20-30 honeybees was established, unscented 0.5 m and 2 m sucrose solutions were offered in the artificial feeder. the two sucrose solutions were offered alternately, allowing 30 min with no feeding reward in between sucrose solution offerings. after changing the feeder solution, 15 min were allowed before capturing the bees. in this way, bees could make repeated trips from the hive to the feeder. each honeybee landing at the feeder was allowed to drink ad libitum and either captured before taking-off towards the hive (feeding site, h1 and h2) or color-marked on the wings with a dot of permanent marker (uni, mitsubishi pencil co., ltd, tokyo; usually used to mark queen bees) for later identification upon returning to the hive (h3). returning foragers were captured at the hive entrance, and the time elapsed during homewards flights were recorded. all captured bees were immediately sociobiology 66(1): 97-106 (march, 2019) 99 sacrificed with co2 and immersed in dichloromethane to extract cuticular compounds (see below). cuticular chemistry cuticular compounds were extracted in dichloromethane (dcm) at room temperature and under gentle hand stirring. in order to detect and quantify less abundant chcs such as methyl-branched alkanes or alkadienes, the bees were extracted in pooled samples consisting of groups of five bees for h1 and h2 (feeding site and hive bees), and three bees for h3 (hive entrance). in all cases, extractions were done in 1-dram screw-cap vials with either 3 or 2 ml of dcm (for samples containing 5 or 3 bees, respectively). after 1 min, the insects were removed and 100 µl of n-tridecane (0.503 mg/ml in hexane) were added as internal standard (is). the extracts were then concentrated to 1 ml under a stream of nitrogen for gc-ms analysis. chemical analyses were done by gas chromatography coupled to mass spectrometry (gc-ms), using a shimadzu qp-2010 gc-ms equipped with an at-5 ms column (alltech) (30 m × 0.25 mm, 0.25 μm), and operated with a constant carrier flow of 1 ml/min (he). the temperature of the gc oven was programmed from 70 °c (1 min) to 150 °c (1 min) at 10 °c/min, then raised to 300 °c at 5 °c/min, and held for 5 min at 300 °c. the injector temperature was 250 °c and the interphase temperature 310 °c. injection (1 μl) was in the splitless mode (sampling time 1 min), and mass spectra were acquired from m/z 30 to 350, except for the dmds-derivatized extracts, in which mass spectra were acquired from m/z 30 to 550 (70 ev, scan mode). for retention index calculations, a mixture of n-alkanes (100 ppm each, in hexane) was injected in the splitless mode, using the same temperature program. cuticular extracts were derivatized with dimethyl disulfide (dmds) for obtaining additional information for the unsaturated compounds. the extract (100 µl) was mixed in a v-shaped vial with 100 µl of dmds and 5 µl of an et2o solution of i2 (60 mg/ml). the reaction mixture was kept closed at 50 °c for 2 h, after which 200 µl of hexane and 100 µl of na2s2o3 (5% in water) were added. the organic layer was finally separated and concentrated to 50 µl under a stream of nitrogen for gc-ms analysis. statistical analysis for each analyzed sample, peak areas higher than 0.1% of the total ion chromatogram (tic) were considered for the analysis (excluding the area of the is). in addition, compounds were excluded if they were not present in at least 3 samples of any given treatment (hive bees, foragers fed with either 0.5 m or 2 m sucrose solution). in order to analyze the net amounts of all individual chcs (µg equivalents of is) in the cuticular extracts of the different treatments, we performed a principal component analysis (pca) (quinn & keough, 2002). in our data analysis, the principal components which explained at least 80% of the variance were then analyzed by manova, and the main components were analyzed by one-way anova/tukey hsd. in addition, chcs were grouped as alkanes and alkenes, and the added amounts for each group were compared among treatments using one-way anova/tukey hsd. finally, we also compared the amounts of the two alkanes and the two alkenes that have been reported as foraging promoters (thom et al., 2007). to reduce the risk of type 1 errors due to the multiple use of the same data, we corrected the significance thresholds using the bonferroni method (α´= a ⁄ k), with α = 0.05 and k = 4. thus, our significance threshold was α´= 0.0125. to compare flight time between foragers fed with 0.5 m or 2 m sucrose solutions we performed an anova. results we identified a total of 48 compounds by gc-ms (fig 1 and table 1), including chcs (9 through 48, except 13), as well as other compounds previously described in honeybees: 2-nonanol (1) and benzyl acetate (2), known as sting alarm pheromones (collins and blum 1982); geraniol (5), geranic acid (6) and farnesol (8), all related to the nasonov gland; and (z)-11-eicosen-1-ol (13), which has been reported in the sting gland (pickett et al. 1982; schmitt et al. 2007). we also found e-2-octenyl acetate (3), 2-nonanyl acetate (4) and e-2-decenyl acetate (7), which have not been reported in apis mellifera, and probably originate from plants previously visited by the bees. the identification of the compounds was based on mass spectra and retention index comparison with those of databases (table 1 and supp 1) (adams, 2007; el-sayed, 2014; linstrom & mallard, 2005). as previously reported (blomquist et al., 1980a; carlson et al., 1989; dani et al., 2004; francis et al., 1989; kather et al., 2011; mcdaniel et al., 1984), the chromatograms of cuticular extracts were dominated by n-alkanes (c23, c25, c27. peaks 17, 23 and 28, respectively) and (z)-11-eicosen-1-ol, including lower amounts of alkenes, alkadienes and methyl-branched alkanes (fig 1). dmds-derivatives of the alkenes showed double bond positions in c7 and c9 for lower molecular weight monoenes, while c8 and c10 unsaturations were found in longer chain alkenes. methyl-branched alkanes co-eluted as mixtures that were characterized by the higher abundance of diagnostic fragment ions indicating the position of the methyl branches in c11, c13 or c15 (table 1). we focused our quantitative analysis only on the chcs. the net amount of each chc was calculated from the tic peak areas as mg-equivalents of internal standard per bee, as shown in table 2 (see supp 2 for more details) for the different treatments, namely, hive bees, foragers fed with 0.5 m or 2 m sucrose solution concentrations. an initial pca analysis of chc net amounts was done for foragers captured at the feeding site (from hives h1 and h2), resulting in two principal components (pc) that represented 48.81 and 16.39% of the overall variance. ms balbuena, a gonzález, wm farina – cuticular chemistry in foraging honeybees100 the major contributing component (pc1) clearly segregated the two honeybee groups: hive bees and forager bees, with no obvious separation of forager bees fed with 0.5 or 2 m sucrose solutions (fig 2). although there was a significant interaction between the factors hive and treatment (manova: f10,120= 4.375, p < 0.01), lsd posthoc comparisons for individual pcs showed no interaction between these two factors in pc1. the graphic groupings observed in fig 2 for pc1 were corroborated by a significant difference in the composition of chcs for hive and forager bees (anova pc1: f = 121.22, df = 2, p < 0.01), and not for foragers fed with 0.5 or 2 m (tukey: hive bees vs. foragers: df = 67, p < 0.01; 0.5 m vs. 2 m: df = 67, p = 0.906, fig 2). fig 1. typical total ion chromatogram of cuticular extracts of apis mellifera foragers. the expanded portion (peaks 1 – 8) includes more volatile glandular compounds. peaks 9 through 48 correspond to cuticular hydrocarbons, except for the abundant (z)-11-eicosen-1-ol (13). see text for gc and ms conditions, tables 1 and s1 for characterization details, and table 2 for quantitative data (is: internal standard). peak1 compound(s) retention index2 diagnostic ions (%) s.i.3 exp. lit. 1 2-nonanol 1098 1098 129 (m+-ch 3 , 4), 126 (m+-h 2 o, 44), 45 (100), 31 (ch3o+, 2) 94 2 benzyl acetate 1166 1157 150 (m+, 30), 108 (100), 91 (60), 79 (33), 77 (19) 95 3 e-2-octenyl acetate 1208 1208 128 (m+-ch 2 co, 8), 110 (11), 43 (100) 94 4 2-nonanyl acetate 1232 n.a.4 126 (m+-ch 3 cooh, 11), 87 (m+-c 7 h 15 , 41), 43 (100) n.a. 5 geraniol 1254 1249 154 (m+, 1), 139 (m+-ch 3 , 3), 123 (m+-ch 2 oh, 11), 69 (100), 31 (ch3o+, 1) 94 6 geranic acid 1345 1342 168 (m+, 2), 150 (1), 123 (m+-co 2 h, 15), 100 (m+-c 5 h 8 ,20), 69 (100) 92 7 e-2-decenyl acetate 1404 1408 156 (m+-ch 2 co, 5), 138 (m+-ch 3 cooh, 6), 110 (15), 43 (100) 96 8 (e,z)-(2,6)-farnesol 1724 1722 136 (m+-c 5 h 10 o, 10), 69 (100) 92 9 9-nonadecene5 1874 1875 266 (m+, 13), 97 (86), 83 (98), 69 (84), 55 (100) dmds: 360 (m+), 187, 173 96 10 nonadecane 1898 1900 268 (m+, 3), 85 (53), 71 (70), 57 (100) 95 11 heneicosane 2100 2100 296 (m+, 2), 85 (56), 71 (71), 57 (100) 97 12 docosane 2199 2200 310 (m+, 2), 85 (58), 71 (74), 57 (100) 96 13 11-eicosen-1-ol 2265 2260 278 (m+-h 2 o, 8), 250 (2), 222 (2), 96 (84), 82 (100), 55 (89), 31 (ch 3 o+,5) dmds: 390 (m+), 217, 173 91 14 x,y-tricosadiene6 2268 n.a. 320 (m+, 15), 96 (94), 82 (79), 81 (95), 67 (100) n.a. 15 9-tricosene 2273 2272 322 (m+, 12), 97 (100), 83 (93), 69 (71) dmds: 416 (m+), 243, 173 95 16 7-tricosene 2280 2280 322 (m+, 11), 97 (100), 83 (95), 69 (78) dmds: 271, 145 96 17 tricosane 2300 2300 324 (m+, 5), 85 (66), 71 (84), 57 (100) 92 table 1. retention indices and mass spectrum diagnostic ions of chcs and glandular compounds in the cuticular extracts of honeybees (see supp 1 for full ion tables) sociobiology 66(1): 97-106 (march, 2019) 101 18 x-tetracosene 2374 2372 336 (m+, 13), 97 (100), 83 (97), 69 (74) n.a. 19 tetracosane 2400 2400 338 (m+, 2), 85 (59), 71 (75), 57 (100) 95 20 x,y-pentacosadiene 2470 n.a. 348 (m+, 23), 96 (100), 82 (97), 81 (93), 67 (82) n.a. 21 9-pentacosene 2474 2474 350 (m+, 8), 97 (100), 83 (83), 69 (67) dmds: 444 (m+), 271, 173 94 22 7-pentacosene 2481 2481 350 (m+, 7), 97 (100), 83 (81), 69 (69) dmds: 299, 145 94 23 pentacosane 2500 2500 352 (m+, 3), 85 (59), 71 (78), 57 (100) 94 24 13+11-me-pentacosane 2533 2533 351 (m+-ch 3 , 1), 225 (13-methyl, 4), 224 (13-methyl, 5), 197 (11-methyl, 3), 196 (11-methyl, 4), 169 (13-methyl, 7), 168 (13-methyl, 12), 85 (66), 71 (82), 57 (100) n.a. 25 hexacosane 2600 2600 366 (m+, 3), 85 (63), 71 (83), 57 (100) 93 26 9-heptacosene 2675 2675 378 (m+, 6), 97 (100), 83 (80), 69 (63) dmds: 299, 173 n.a. 27 7-heptacosene 2683 2683 378 (m+, 5), 97 (100), 83 (76), 69 (67) dmds: 327, 145 n.a. 28 heptacosane 2700 2700 380 (m+, 3), 85 (61), 71 (81), 57 (100) 91 29 13+11-me-heptacosane 2731 2733 379 (m+-ch 3 , 2), 253 (11-methyl, 3), 252 (11-methyl, 3), 225 (13-methyl, 5), 224 (13-methyl, 7), 197 (13-methyl, 5), 196 (13-methyl, 8), 183 (2), 169 (11-methyl, 6), 168 (11-methyl, 8), 85 (65), 71 (80), 57 (100) n.a. 30 octacosane 2799 2800 394 (m+, 2), 85 (64), 71 (80), 57 (100) 93 31 9-nonacosene 2878 2876 406 (m+, 7), 97 (100), 83 (78), 69 (60) dmds: 327, 173 n.a. 32 8-nonacosene 2882 n.a. 406 (m+, 8), 97 (100), 83 (77), 69 (60) dmds: 341, 159 n.a. 33 nonacosane 2900 2900 408 (m+, 2), 85 (62), 71 (81), 57 (100) 95 34 15+13+11-me-nonacosane 2931 n.a. 407 (m+-ch 3 , 1), 281 (11-methyl, 2), 280 (11-methyl, 2), 253 (13-methyl, 3), 252 (13-methyl, 4), 225 (15-methyl, 7), 224 (15-methyl, 10), 211 (1), 197 (13-methyl, 4), 196 (13-methyl, 7), 183 (3), 169 (11-methyl, 7), 168 (11-methyl, 7), 85 (64), 71 (80), 57 (100) n.a. 35 triacontane 3000 3000 422 (m+, 1), 85 (65), 71 (81), 57 (100) 92 36 x,y-hentriacontadiene 3063 3077 432 (m+, 8), 96 (100), 82 (85), 69 (71) n.a. 37 10-hentriacontene 3077 n.a. 434 (m+, 8), 97 (100), 83 (76), 69 (60) dmds: 341, 187 n.a. 38 8-hentriacontene 3084 3086 434 (m+, 8), 97 (100), 83 (78), 69 (62) dmds: 369, 159 n.a. 39 hentriacontane 3100 3100 436 (m+, 2), 85 (61), 71 (79), 57 (100) 92 40 15+13+11-mehentriacontane 3130 n.a. 435 (m+-ch 3 , 1), 309 (11-methyl, 1), 308 (11-methyl, 1), 281 (13-methyl, 4), 280 (13-methyl, 4), 253 (15-methyl, 4), 252 (15-methyl, 6), 225 (15-methyl, 4), 224 (15-methyl, 6), 197 (13-methyl, 5), 196 (13-methyl, 7), 169 (11-methyl, 6), 168 (11-methyl, 4), 85 (67), 71 (82), 57 (100) n.a. 41 x-dotriacontene 3180 n.a. 448 (m+, 4), 97 (100), 83 (81), 69 (62) n.a. 42 dotriacontane 3199 3200 450 (m+, 1), 85 (66), 71 (83), 57 (100) 94 43 x,y-tritriacontadiene 3258 n.a. 460 (m+, 10), 96 (100), 82 (84), 69 (73) n.a. 44 x-tritriacontene 3278 n.a. 462 (m+, 9), 97 (100), 83 (79), 69 (61) n.a. 45 y-tritriacontene 3285 n.a. 462 (m+, 7), 97 (100), 83 (75), 69 (59) n.a. 46 tritriacontane 3300 3300 464 (m+, 1), 85 (66), 71 (83), 57 (100) 93 47 x,y-pentatriacontadiene 3384 n.a. 488 (m+, 9), 96 (100), 82 (84), 69 (69) n.a. 48 x-pentatriacontene 3395 n.a. 490 (m+, 5), 97 (100), 83 (77), 69 (55) n.a. 1 peak numbers as in figure 1 2 experimental linear retention indices calculated according to (adams 2007). literature retention indices from (adams 2007; el-sayed 2014; herzner et al., 2013; linstrom and mallard 2005). 3 similarity index according to (adams 2007; linstrom and mallard 2005). 4 not available. 5 the geometry of double bonds in a. mellifera chcs was assumed to be z as previously reported (mcdaniel et al. 1984). 6 letters (x,y) indicate that the double bond position could not be determined unambiguously. table 1. retention indices and mass spectrum diagnostic ions of chcs and glandular compounds in the cuticular extracts of honeybees (see supp 1 for full ion tables). (continuation) peak1 compound(s) retention index2 diagnostic ions (%) s.i.3 exp. lit. ms balbuena, a gonzález, wm farina – cuticular chemistry in foraging honeybees102 we grouped the chcs into alkanes and alkenes, and compared their net amounts in extracts of forager bees captured at the feeding site, finding no differences with respect to the two rewarding treatments (alkanes: anova; f1,54= 69.719, p = 0.857; alkenes: anova; f1,54= 11.715, p = 0.948; fig 3a). similarly, the amounts of the individual chcs reported by thom et al. (2007) as semiochemicals involved in the waggle dance did not show differences in the cuticular extracts from forager bees collected at the feeding site after feeding on 2 m or 0.5 m sucrose solutions (anova; 9-tricosene: f1,36 = 1.238, p = 0.273; tricosane: f1,36 = 0.096, p = 0.7579; 9-pentacosene: f1,36 = 0.003, p = 0.9536; pentacosane: f1,36 = 0.058, p = 0.8109; fig 3b). when foragers fed with 0.5 m or 2 m sucrose solutions were allowed to fly back to the hive, the former took 13.2 ± 2.5 sec, while bees fed with high sucrose concentration flew faster, arriving after 7.2 ± 0.5 sec (anova: f1,68 = 5.24, p = 0.025). fig 2. relationship between cuticular hydrocarbons and sucrose concentration of hive bees and foragers. scores plot showing the relationship of cuticular hydrocarbons extracted from honeybees from different hives [diamonds: hive 1, spring 2012 (n = 5 samples per treatment); circles: hive 2, summer 2013 (n = 19 samples per treatment)], task groups (hive bees: white; forager bees: gray and black), and feeding treatments (gray: 0.5 m, black: 2 m). the two main principal components (pc) account for 48.81 and 16.39% of the overall data variance, respectively. fig 3. hydrocarbon amounts extracted from foragers captured at the feeding site. (a) net hydrocarbon amounts (µg equivalents of tridecane per bee) grouped as alkanes or alkenes, extracted from forager bees of different rewarding programs (light gray bars: 0.5 m; dark gray bars: 2 m). (b) net amounts of specific hydrocarbons (9-tricosene, tricosane, 9-pentacosene, and pentacosane) could be involved in the recruitment in forager bees. error bars represent standard error of the mean. asterisks indicate significant differences (p < 0.05). n.s. indicates no significant differences (p > 0.05). table 2. chcs for hive bees and foragers collected under different rewarding programs. numbers and color scale inside cells represent the amounts of each chc expressed as mg-equivalents of internal standard (mg eq is/bee). for more details see online resource 1. nd: not detected. hive entrance feeder reward hive bees pentatriacontene pentatriacontadiene tritriacontane tritriacontene tritriacontene tritriacontadiene dotriacontane dotriacontene me-hentriacontane hentriacontane 7-hentriacontene 9-hentriacontene hentriacontadiene triacontane me-nonacosane nonacosane 7-nonacosene 9-nonacosene octacosane me-heptacosane heptacosane 7-heptacosene 9-heptacosene hexacosane me-pentacosane pentacosane 7-pentacosene 9-pentacosene pentacosadiene tetracosane tetracosene tricosane 7-tricosene 9-tricosene tricosadiene docosane heneicosane nonadecane 9-nonadecene 2 m 0.5 m 2 m 0.5 m sucrose concentration nd nd nd nd nd nd nd nd 0.21 0.26 nd nd nd nd nd nd nd ndnd nd nd nd hydrocarbon amounts (mg eq is/bee) 0.24 0.20 0.11 0.47 0.51 0.15 3.13 0.23 1.11 0.20 6.22 0.32 0.51 0.58 0.17 8.90 0.76 0.54 0.63 0.92 9.11 0.53 0.36 0.79 3.95 5.22 7.91 0.22 0.40 0.13 2.10 10.06 1.41 0.22 0.15 0.73 0.32 0.48 3.58 1.03 13.98 0.13 1.15 1.38 5.62 1.68 16.53 0.38 1.45 3.41 1.29 14.86 0.73 0.73 2.33 0.98 8.43 0.48 0.30 0.44 2.22 3.48 6.20 0.19 0.17 0.78 5.49 0.84 0.75 0.26 0.17 0.73 0.32 0.45 3.37 0.97 13.77 0.16 1.19 5.60 1.67 16.72 0.39 1.52 3.36 1.29 15.28 0.75 0.80 2.29 0.83 8.78 0.51 0.33 0.48 2.32 3.65 6.51 0.18 0.21 0.14 0.85 5.82 0.82 0.81 0.80 0.25 1.52 0.93 1.00 6.14 1.63 21.27 0.32 2.05 8.83 2.24 22.02 0.60 1.47 3.49 0.89 13.50 0.88 0.51 1.20 0.93 6.16 0.24 0.48 0.41 2.54 3.13 3.47 0.26 0.75 4.61 0.51 0.370.41 0.72 4.86 0.85 0.24 3.70 3.31 2.59 0.42 0.22 0.48 6.39 0.83 1.21 0.60 0.90 14.64 1.06 3.72 1.75 0.61 25.52 2.88 9.98 2.55 0.49 24.79 2.16 7.01 1.08 1.10 1.52 0.07 0.95 sociobiology 66(1): 97-106 (march, 2019) 103 the chc profiles of bees captured at the hive entrance also showed some differences. pca analysis to compare chcs did not show a clear segregation between bees fed with 0.5 or 2 m sucrose solutions (supp 3). however, when grouping chcs into alkanes and alkenes, chc extracts from bees that returned from a feeder with higher sucrose reward showed higher net amounts of both, alkanes (anova; f1,28 = 6.595, p = 0.016) and alkenes (anova: f1,28 = 4.496, p = 0.043; fig 4a). this difference was also found for the individual alkanes (tricosane and pentacosane) reported as foraging promoters (thom et al., 2007), but not for the individual alkenes, which only showed a non-significant trend (anova: tricosane: f1,28 = 7,127, p = 0.012; pentacosane: f1,28 = 7.621, p = 0.010; 9-tricosene: f1,28= 2.477, p = 0.127; 9-pentacosene: f1,28= 1.73, p = 0.199; fig 4b). discussion in this study, we assessed whether the profitability of the exploited food source promotes changes in the cuticular hydrocarbon profile of honeybees during foraging. to do so, we analyzed the chcs of foragers fed with sucrose solutions of low and high concentrations. for comparison purposes, we also analyzed non-forager bees collected within the hive, and found that chc profiles of hive bees and foragers differ, independently of the hive from which the bees originated. in general, we found higher amounts of chcs in forager bees, but relatively more abundant high molecular weight chcs (> c31) in hive bees, as has been previously reported (del piccolo et al., 2010). in line with these results, task-related cuticular differences have been reported in apis mellifera (dani et al., 2004; kather et al., 2011), and at least in part, these differences are consistent with higher exposure of foraging bees to more variable environmental conditions such as temperature and humidity (heinrich, 1993), which differ from the highly controlled conditions experienced within the hive. when comparing the effect of sucrose concentration on the chcs of forager bees, we found that bees captured at the feeder, just after feeding and before taking-off back to the hive, showed similar chc profiles regardless of the concentration of the sucrose solution they had ingested. however, when the bees were captured just after arriving to the hive entrance, chc extracts from bees that returned from a highly profitable food source (2 m sucrose) contained more alkanes and alkenes than those that arrived from a poorer source (0.5 m sucrose). this difference was also observed for the two specific alkanes (n-tricosane and n-pentacosane) that have been reported as foraging promoters in dancing bees (thom et al., 2007). the two alkenes similarly reported, (z)-9tricosene and (z)-9-pentacosene, were found to be unaffected by food profitability, showing only a non-significant trend in line with that of the alkanes. it has been shown that honeybees increase their metabolic rates and thoracic temperature when collecting sugar solutions of higher concentrations and reward rates (schmaranzer & stabentheiner, 1988). metabolic rate and body temperature correlate with the motivational state of forager bees, which in turn depends on external stimuli such as food profitability (balderrama et al., 1992; moffatt, 2001; schmaranzer & stabentheiner, 1988). moreover, there is evidence that the flight velocity of foragers returning to the hive is higher when more profitable food (i.e. higher sugar concentration) is offered at the feeding site (von frisch & lindauer, 1955). in the same line, we found that bees fed with 0.5 m sucrose solution took about twice the time to return to the hive compared to bees fed with 2 m. due to the intense activity of the flight muscles, a bee flying back from a food source probably reaches higher temperatures if flying faster (von frisch & lindauer, 1955). if body temperature is at least partially responsible for the difference in the chc fig 4. hydrocarbon amounts extracted from bees captured at the hive entrance. (a) net hydrocarbon amounts (µg equivalents of tridecane per bee) grouped as alkanes or alkenes, extracted from forager bees of different rewarding programs (light gray bars: 0.5 m; dark gray bars: 2 m) captured upon arrival to the hive entrance (n = 15 for both sucrose concentrations). (b) net amounts of specific hydrocarbons (9-tricosene, tricosane, 9-pentacosene, and pentacosane) could be involved in the recruitment in forager bees. error bars represent standard error of the mean. asterisks above bars indicate significant differences (p < 0.05). n.s. indicates no significant differences (p > 0.05). ms balbuena, a gonzález, wm farina – cuticular chemistry in foraging honeybees104 profiles of bees returning from high and low profitable food sources, as we hypothesize here, this would explain that bees captured at the feeding site did not show the differences in chc profiles that were observed later, after flying back to the hive. in line with this argument, a comparison of net amounts of chcs extracted from bees captured before and after flying shows higher amounts for the later, regardless of the sucrose solution treatment. worth of note, our methodology involved capturing the bees and immediately extracting them, without freezing for later extraction to avoid neutralizing the possible effect of body temperature in the cuticular chemistry. these temperature changes would not be expected to upregulate chc biosynthesis, given that they occur within a very short time window. returning bees had to fly only 6 m in our experimental setup, and they did so right after feeding, so only a few seconds elapsed between feeding at the feeder and capture at the hive entrance. however, an increase in body temperature may affect the physicochemical properties of the honeybee cuticular envelope, for instance its density and viscosity, possibly making the chcs more available for surface extraction, but also possibly promoting their passive release (schmitt et al., 2007; thom et al., 2007). in fact, most of the chcs identified in our study have been previously described not only as components of cuticular extracts but also as volatiles sampled in the headspace of forager honeybees at the feeding site, thus indicating that chcs are slightly volatile (schmitt et al., 2007). a similar temperature effect on chc surface chemistry may also occur within the hive. it has been shown that during recruitment by waggle dancing, or when exchanging food with receiver nestmates, returning foragers adjust their thoracic temperatures in relation to the food source profitability (farina & wainselboim, 2001; stabentheiner, 1996; stabentheiner & hagmüller, 1991). moreover, four common honeybee chcs, two alkanes and two alkenes, have been reported on the body surface of waggle dancing honeybees (thom et al., 2007), and they are known to be perceived, learned and discriminated by workers (chaline et al., 2005; getz & smith, 1987). furthermore, when a subset of these compounds [(z)-9-tricosene, tricosane and pentacosane] were injected into the hive, the number of exiting bees increased, and the authors postulated that a passive emission of these compounds could be promoted by the high body temperature reached by the intense movement of the dancer bee (thom et al., 2007). indeed, thom et al. (2007) not only found chemical differences between dancer and non-dancer foragers returning from the same food source, but also between intense and less intense dancers. thus, our results are complementary with this notion, since we show that honeybees with different foraging motivational state present different chc profiles, but they do so even before entering the hive, and obviously before any possible temperature effect caused by the waggle dance. our results show that, when bees were captured at the hive entrance, some chcs (i.e. tricosane and pentacosane) are present in higher amounts in the surface extracts of foragers fed with high sugar concentration, compared to those from bees fed with low sugar concentration. this small but significant chemical difference in chc profiles could alert nestmates about the presence of good food sources, and may be enough to activate or reactivate unemployed foragers to forage. in the same line, thom and dornhaus (2007) suggest that volatile compounds of active foragers could promote an increase in foraging behavior. since chcs are only faintly volatile, body contacts may also be significant, and we have shown that simple body contacts with active foragers are enough to motivate experienced nestmate foragers to return to known feeding sites (balbuena et al., 2012). such social interactions are unrelated to dance-following or trophallaxis, and likely involve the perception of cuticular compounds. our results are in line with previous studies that suggest that cuticular chemistry may possess a signaling role during the recruitment process in honeybees, certainly secondary, but still potentially relevant. acknowledgments we thank héctor verna for technical assistance during the experimental period and carolina mengoni goñalons for helping us with the statistical analysis. we also thank anonymous referees for their comments. this study was partly supported by grants from agencia nacional de promoción científica y tecnológica (anpcyt), consejo nacional de investigaciones científicas y técnicas (conicet), universidad de buenos aires, and universidad de la república. references adams, r.p. 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(1991). collective decision-making in honey bees: how colonies choose among nectar sources. behavioral ecology and sociobiology, 28: 277-290. ms balbuena, a gonzález, wm farina – cuticular chemistry in foraging honeybees106 stabentheiner, a. (1996). effect of foraging distance on the thermal behaviour of honeybees during dancing, walking and trophallaxis. ethology, 102: 360-370. stabentheiner, a. & hagmüller, k. (1991). sweet food means “hot dancing” in honeybees. naturwissenschaften, 78: 471-473. stabentheiner, a., kovac, h. & hagmüller, k. (1995). thermal behavior of round and wagtail dancing honeybees. journal of comparative physiology b, 165: 433-444. thom, c. & dornhaus, a. (2007). preliminary report on the use of volatile compounds by foraging honey bees in the hive (hymenoptera: apidae: apis). entomologia generalis, 29: 299-304. doi: 10.1127/entom.gen/29/2007/299 thom, c., gilley, d.c., hooper, j. & esch, h.e. (2007). the scent of the waggle dance. plos biol, 5: e228. doi: 10.1371/ journal.pbio.0050228 von frisch, k. (1967). the dance language and orientation of bees. cambridge (massachusetts): the belknap press of harvard university press. 566 p. von frisch, k & lindauer, m. (1955). über die fluggeschwindigkeit der bienen und über ihre richtungsweisung bei seitenwind. naturwissenschaften, 42: 377–385. waddington, k.d. (1990). foraging profits and thoracic temperature of honey bees (apis mellifera). journal of comparative physiology b, 160: 325-329. conflict of interest: the authors declare that they have no conflict of interest. author contributions: msb, ag and wmf conceived and designed the experiments. msb performed the experiments. ag. performed chemical analysis. msb, ag and wmf performed data analysis. msb, ag and wmf drafted the manuscript. all authors revised and commented on the manuscript. supplementary files supplementary file 1. complete fragment ion profiles (> 2%) of the main compounds identified in surface extracts of honeybees. http://periodicos.uefs.br/index.php/sociobiology/rt/ suppfilemetadata/2977/0/1914 http://dx.doi.org/10.13102/sociobiology.v66i1.2977.s1914 supplementary file 2. quantitative analysis of chcs for hive bees and foragers collected under different rewarding programs. http://periodicos.uefs.br/index.php/sociobiology/rt/ suppfilemetadata/2977/0/1915 http://dx.doi.org/10.13102/sociobiology.v66i1.2977.s1915 supplementary file 3. relationship (scores plot) between chcs and fed sucrose concentration in honeybees upon arrival to the hive entrance (gray: 0.5 m, black: 2 m). the two main principal components (pc) account for 36.33 and 23.04% of the overall data variance, respectively. n = 15 for both treatments (manova: p = 0.191, f = 1.628). http://periodicos.uefs.br/index.php/sociobiology/rt/ suppfilemetadata/2977/0/1916 http://dx.doi.org/10.13102/sociobiology.v66i1.2977.s1916 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i4.4431sociobiology 66(4): 545-550 (december, 2019) myrmecophily and myrmecophagy of attacobius lavape (araneae: corinnidae) on solenopsis saevissima (hymenoptera: myrmicinae) introduction ants are social insects divided into castes from which workers are known for their capability to defend the colony against natural enemies (hölldobler & wilson, 1990). this altruistic behavior is justified by the high degree of relatedness among colony members (hamilton, 1964). thus, any strange organism that gets closer to the colony is considered a threat to be fought. however, many arthropod species (mainly insects) that live close to or inside the ant colonies, known as myrmecophiles, have to surpass this biological barrier (hölldobler & wilson, 1990). despite the risks of living with a group of so aggressive animals, the benefits a colony can abstract attacobius lavape, a small spider from the corinnidae family, has been recently described living inside a fire ant colony of solenopsis saevissima species in the municipality of morrinhos, south region of the goiás state, brazil. yet several aspects of this spider relationship with the host ant remain unknown. in this way, we performed an extension study to determine its local (morrinhos) and regional (latitudinal transect) occurrence. we also investigated if the spider uses the host ant as a feeding source. for this, we established arenas with a known number of young and adult ant individuals plus one spider and observed the feeding rate for some determined time. regarding local distribution, differently from most socially parasitic myrmecophiles, a. lavapes howed high local infestation, being found in 47% of the colonies in the sites where the spider occurred, and high transmission, being found in 42% of the 12 collection sites. regionally, among the 11 study sites, this species only occurred in the municipality of morrinhos, but its distribution still needs to be verified in the north region. attacobius lavape consumed eggs, larvae and pupae, confirming that the myrmecophily was explained by myrmecophagy. the spiders consumed eggs (not estimated), 4.45 ± 2.14 larvae and/or 3 ± 0.87 pupae per day. considering that the mean abundance was approximately seven spiders per colony (extent 1-23), we foresee an impact of 35 larvae and/or 21 pupae consumed per day in each hosting colony. the possibility of consuming sexualeggs, larvae and pupae classifies a. lavape as a potential agent of biological control of s. saevissima. sociobiology an international journal on social insects caf mendonça1, ma pesquero1, rsd carvalho2, fv arruda3 article history edited by kleber del-claro, ufu, brazil received 20 march 2019 initial acceptance 25 july 2019 final acceptance 02 october 2019 publication date 30 december 2019 keywords fire ant, inquiline, social parasite, biological control. corresponding author marcos antônio pesquero programa de pós-graduação em ambiente e sociedade universidade estadual de goiás rua rio de janeiro nº 99, centro cep 75650-000. morrinhos-go, brasil. e-mail: mapesq@ueg.br offer, such as food and protection against predators, should overcome the costs, otherwise the selection would not have favored this behavior (ceccarelli, 2013). myrmecophily occurs in only 41 spider species (~0.1% of all described species), belonging to 12 of the 112 described families (cushing, 1997, 2012). however, most of the studies focus on myrmecomorphy and myrmecophagy in spiders and little knowledge has been produced so far about myrmecophily, mainly the social integration and the spider impact to the colony fitness (cushing, 1997, 2012). while some species seem to be opportunistic hosts, others establish obligatory relationships, using several morphological, behavioral and chemical strategies for the integration into the host colony life (cushing, 1997, 2012). 1 programa de pós-graduação em ambiente e sociedade, universidade estadual de goiás, morrinhos, brazil 2 curso de graduação em ciências biológicas, universidade estadual de goiás, morrinhos, brazil 3 programa de pós-graduação em recursos naturais do cerrado, universidade estadual de goiás, anápolis, brazil research article ants caf mendonça, ma pesquero, rsd carvalho, fv arruda – myrmecophily of attacobius lavape on solenopsis saevissima546 the genus attacobius mello-leitão, 1925 (corinnidae) is composed of 16 known species (wsc, 2019), occurring from the central region of argentina up to the brazilian northeast region (bonaldo, 2000). myrmecophily is confirmed in three species of genus attacobius (attacobius luederwaldti (mello-leitão, 1923), attacobius attarum (roewer, 1935), and attacobius lavape bonaldo, pesquero and brescovit, 2018 (platnick & batista, 1995; erthal & tonhasca, 2001; ichinose et al., 2004; bonaldo et al., 2018; pereira-filho et al., 2018). however, the interaction incidences inside this group can be higher than registered so far, because other species of attacobius were collected through pitfall traps, yet without information on association with ants (bonaldo & brescovit, 2005). in the municipality of morrinhos, south region of the goiás state, we have found adults (males and females) and young a. lavape inside the solenopsis saevissima (f. smith, 1855) colonies. solenopsis saevissima has a wide distribution in south america and, as well as other 19 species belonging to the fire ants, it is known by its painful sting and mass attack (pitts et al., 2018). similar to the solenopsis invicta buren, 1972 species in the south region of the united states, when introduced in other regions, the fire ants can cause serious structural, ecological and public health problems (tschinkel, 2006). several fly species of the genus pseudacteon (diptera: phoridae) and orasema xanthopus (cameron, 1909) (hymenoptera: eucharitidae) are known parasitoids of s. saevissima (pesquero & dias, 2004, 2011), and the discovery of this new spider species expands the number of natural enemies with potential use in biological control programs. the mechanisms and adaptations involved in myrmecophily of a. lavape on s. saevissima, as well as the impact they cause on the hosting colony have not yet been described since the recent discovery of the spider a. lavape (bonaldo et al., 2018). therefore, we aim to describe the behaviors that allow myrmecophily and to verify the impact caused on the hosting colony. we seek to find if:i) the occurrence of a. lavape fits one of the three models of the distribution of social parasites (high infestation and low transmission, low infestation and high(ish) transmission, low infestation and very low transmission) proposed by thomas et al. (2005). ii) myrmecophily is a strategy for using the ant host as a food source (erthal & tonhasca, 2001). iii) attacobius lavape has plasticity in the use of host ant species as already documented for some spider species (cushing, 1997, 2012). material and methods local and regional distribution we performed the local collections during the transition from the rainy to the dry season (february to april) from 2016 to 2017, in 12 sites in the municipality of morrinhos, goiás, brazil (17°43’16’’s, 49°6’29’’w) within an approximate area of 59,673 ha. the distance between each point and its nearest neighbor was 6.99 ± 2.63 km. we have also performed 11 regional collections every 50 km, from morrinhos to são carlos, são paulo, brazil (22°0’55’’s, 47°53’28’’w). the study sites were chosen according to adequate characteristics for the occurrence of s. invicta, that is, humid soil with a predominance of low vegetation (pasture) and close to streams (lebrun et al., 2012). the predominant native vegetation in the study region is the cerrado lato sensu. the s. saevissima colonies found in the study sites were gradually transferred with a shovel to white plastic trays (50x30x7 cm) to verify the presence of a. lavape. we quantified the collecting areas (ha), the number of colonies found and the number of a. lavape individuals present in the colonies to estimate the local and regional occurrence of ants and spiders. we followed pitts et al. (2018) review for the identification of fire ants and bonaldo et al. (2018) for the identification of the inquiline spider. myrmecophagy we used 10 males and 10 females of a. lavape in the laboratory for predation tests (n = 20). the predation of a. lavape over s. saevissima was evaluated through a very small colony offer (10 larvae, 10 pupae, 40 adult workers, 2 males and 2 virgin queens) for an adult spider (male or female) per petri dish (10x1 cm) with lids on during 24 hours. the larvae and pupae offered to the spiders measured in average 1.82 ± 0.7 mm (n = 200) and 2.75 ± 0.45 mm of length (n = 200), respectively. the adult workers measured in average 4.15 ± 1.41 mm of total length. due to the difficulty of counting and sensibility to manipulation, the eggs were offered to the spiders together with 1 fertile queen and 10 workers. in this way, analyses of predation on eggs was quantified in the event occurrence rate (each time the spiders caught an eggs mass to eat) during one hour of observation for each spider (n = 20). larvae and pupae predation rates were compared by the u test of mann-whitney (ayres et al., 2007). hosting plasticity in order to verify the specificity of a. lavape myrmecophily at s. saevissima colony-level, we separately inserted 11 adult spiders (7 female and 4 male) in two plastic trays (30x15x10 cm) internally coated with fluon® containing other two s. saevissima colonies different from the spider original colony. we observed the spider behavior for 30 minutes or until it was accepted by the colony (we consider that there was acceptance when the spider joined the group of workers without being attacked), totaling 198 minutes of observation ad libitum sense (del-claro, 2010). since it was necessary to verify the specificity of a. lavape myrmecophily at the level of the fire ant species, we used the same previous procedures and 11 a. lavape individuals, but with two s. invicta colonies collected in the municipality of são carlos, são paulo state, totaling 203 minutes of observation. the acceptance frequency of a. lavape inside and among the fire ant species were compared by chi-square tests (χ2) for proportions equally expected and fischer exact probability, respectively, and the time it took the spiders to be accepted by the two fire ant species were compared by the u test of mannwhitney (ayres et al., 2007). the mean values presented in the text are accompanied by their standard deviations. sociobiology 66(4): 545-550 (december, 2019) 547 results local and regional distribution we inspected a total of 103 s. saevissima colonies in morrinhos (go) and the average density found in the 12 study sites was 12.95 ± 6.48 colonies/ha (2, 4, 5, 5, 6, 7, 7, 7, 8, 13, 15, 24 colonies). from the 108 spiders collected inside the colonies, 17 were males, 54 females, 31 young and 6 that escaped were not sexed. attacobius lavape occurred in five (42%) of the 12 sites visited, in 47.4 ± 25.7% of the colonies in the sites where it occurred, and in 24.3% of the 103 colonies found. in average, we found 6.75 ± 6.94 spiders/colony (1 to 23 individuals, n = 25). between morrinhos (go) and são carlos (sp), we opened 47 colonies of s. saevissima, but no spider was found. myrmecophagy we found no predation on adult workers, male, and virgin queen of s. saevissima offered to spiders during 24 hours. on the other hand, the spiders predated larvae and pupae using more larvae than pupae as food (4.45 ± 2.14 larvae/day vs. 3 ± 0.87 pupae/day, mann-whitney, u = 20, p = 0.004, n = 20). in 12 of the 20 repetitions of one-hour observations, the spiders collected eggs clutch from the workers and immediately started to ingest its content (see supplementary material online). hosting plasticity from the 11 adult spiders that were introduced in each of the two trays containing s. saevissima colonies (n = 22), 13 were accepted within 30 min of observation and 9 were not accepted (χ2 = 0.73, p = 0.39). from the 11 spiders introduced in each of the two s. invicta colonies, 15 were accepted and 7 were not accepted (χ2 = 2.9, p = 0.09). the frequency of spiders that were accepted by the s. saevissima and s. invicta colonies did not differ between themselves (59% vs. 68%, respectively. fischer’s exact test, p = 0.34). the average spent time for the spiders to be accepted by the colonies was 9 ± 6 min (n = 28) and did not differ between the species of fire ants (mann-whitney u = 53, p = 0.71, n = 28). inside the trays containing fire ant colonies, the spiders exhibited the behaviors of persecution, touching, avoidance, auto-grooming and stationary (table 1, figs 1, 2). after being accepted inside the colonies, the spiders used all the appendages during the contact with the ants, but mainly the first pairs of legs and the pedipalps, which were constantly taken to the chelicerae for auto-grooming. behavior frequency (%) persecution closely following the workers 26.94 touching contacting the workers through all the appendices 26.68 avoidance escaping and dodging the workers 19.95 auto-grooming passing the first pair of legs on the chelicerae 15.71 stationary no apparent movement and contact with the workers 10.72 table 1. frequency of behaviors exhibited by attacobius lavape inside fire ant colonies solenopsis saevissima and solenopsis invicta in laboratory. fig 1. attacobius lavape female closely following and touching the solenopsis saevissima workers in a colony maintained in laboratory. a-d: sequence of persecution behavior. caf mendonça, ma pesquero, rsd carvalho, fv arruda – myrmecophily of attacobius lavape on solenopsis saevissima548 discussion the small size of the a. lavape adults (total length of 3.27 mm for females and 3.04 mm for males; bonaldo et al., 2018) is a myrmecophilous trait that favors mobility within the colonies (cushing, 2012), and its myrmecophily on s. saevissima can be explained by myrmecophagy, for example with a. attarum on atta sexdens (linnaeus, 1758) (erthal & tonhasca, 2001). considering that the mean abundance was approximately seven spiders per colony (extent 1-23) and each spider consumed five larvae and/or three pupae per day, we foresee an impact of 28 larvae and/or pupae per day in each hosting colony, besides the eggs that have not been quantified. in addition to reducing colony fitness, it is still necessary to consider the negative spider impact on the production of new generations through eggs, larvae and pupae predation on sexual ant castes. regarding the growth rates of seven workers/day in s. invicta and solenopsis richteri forel, 1909 colonies up to five-month old (markin & dillier, 1971; markin et al., 1973), only one spider would be enough to inhibit the growth of an s. saevissima initial colony. thus, a. lavape presents a significant potential as s. saevissima biological control agent in sites where it becomes a plague. despite the integration of a. lavape with s. invicta observed in the specificity tests, the use of this spider in control programs in the united states still demands predation tests with colonies of solenopsis geminata (fabricius, 1804) (a native species of the country), and species of other genus and subfamilies. in fact, some species of myrmecophilous spiders may use up to three genera and five species different as hosts (cushing, 1997). together with other natural enemies (briano et al., 2012), a. lavape preys on fire ants and helps to keep a low colony density in south america. in the southern united states, where s. invicta was accidentally introduced without natural enemies (porter et al., 1997), the colony density is 16 times higher than s. saevissima density found in morrinhos (12.95 ± 6.48 colonies/ha), but the relationship of this low density to the presence of a. lavape needs to be further evaluated. the fact that the a. lavape occurs in 42% of the sites with s. saevissima and 47% of the colonies in the sites where it occurred demonstrates a “high local infestation and high regional transmission” pattern, different from the three distribution models of social parasite pointed by thomas et al. (2005). just as environmental disturbances may favor the population growth of s. invicta (lebrun et al., 2012), the large expansion of agribusiness and the consequent reduction of native vegetation in the southern region of goiás (costa & santos, 2010) may have favored the dispersal of s. saevissima and its guest a. lavape. the absence of a. lavape towards the são paulo state suggests endemism of the species in the morrinhos region. however, more collections towards the north, east and west regions of the municipality are necessary to confirm this hypothesis. the presence of very young individuals and adults of both sexes indicate that a. lavape reproduces inside the hosting ant colony, being a probably obligate guest (cushing, 1997). fig 2. attacobius lavape female on larvae and pupae, touching and being touched by the ants in a solenopsis saevissima colony maintained in laboratory. sociobiology 66(4): 545-550 (december, 2019) 549 despite the risks for the inquiline spider, such as to be identified during the mating and have its eggs destroyed by the ants (edmunds, 1978), the use of the colony interior as a reproduction site has already been observed for the eilica myrmecophila (simon, 1903) (in synonymy eilica puno) (gnaphosidae) spider and camponotus inca emery, 1903 ant (noonan, 1982). attacobius lavape behavior of closely following and touching the workers has also been observed with a. attarum on a. sexdens (erthal & tonhasca, 2001). this behavior suggests that the renewal or the restoration of the cuticle chemical profile by the spider is not regulated via biosynthesis, but by continuous exogenous transfer, indicating the use of chemical camouflage to remain in the hosting colony (dettner & liepert, 1994). when the varroa destructor anderson and trueman, 2000 mite was experimentally introduced in one colony of a different host bee species (apis cerana fabricius, 1793), it acquired a chemical profile similar to the new hosting species demonstrating chemical camouflage of the parasite mite (kather et al., 2015). in fact, no case of chemical mimicry by biosynthesis has been attributed to the myrmecophilous spiders, and the integration with the ant colonies occurs by the contact with the nest material, contact with the workers or by the predation of larvae and pupae (cushing, 2012), yet some cuticular hydrocarbon studies are necessary to confirm this hypothesis. supplementary material h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfiles/4431/0 acknowledgements two anonymous reviewers of the manuscript provided several insightful suggestions. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brazil (capes) finance code 001 (convênio n° 817164/2015 capes/proap and capes/fapeg nº 1656/2016). we also thank the prp/ueg and programa de concessão de bolsa de incentivo ao pesquisador (probipueg) for scholarship granted. references ayres, m., ayres, m. jr, ayres, d.l. & santos, a.a.s. 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(accessed: 25 jan, 2019). doi: 10.13102/sociobiology.v66i3.4376sociobiology 66(3): 500-507 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 colony breeding structure of reticulitermes (isoptera: rhinotermitidae) in northwest arkansas introduction the complicated life style of termites prevents traditional research methods from adequately assessing aspects of their biology. initially, a reticulitermes (isoptera; rhinotermitidae) subterranean termite colony is founded by a monogamous pair of winged alates, forming a simple family. this pair can be replaced by secondary reproductive descended from the original founding pair, changing the breeding structure of the colony to an extended family (vargo & husseneder, 2009). less commonly, colonies can consist of cohabiting individuals from unrelated reproductives. these are referred to as mixed families, likely caused by colony fusion (matsuura & nishida, 2001; deheer & vargo, 2004; fisher et al., 2004). while this structure is nearly impossible to determine from collecting and censusing colonies in the abstract termites, as social insects, have a complicated life cycle in which the colony breeding structure, that is the number of and origin of reproductives in a colony, can vary in relation to age and environmental factors. in this study, we used genetic methods to characterize the breeding structure of three species of reticulitermes from three sites in northwest arkansas and compared two habitats: undeveloped, forested sites and developed, agricultural sites. we found 57.1% of r. flavipes (kollar) in northwest arkansas (n = 28) were simple families, 39.3% were extended families and 3.6% were mixed families. similarly, for r. hageni banks (n = 23), we found 58.3% simple families, 33.3% extended families, and 8.3% mixed families. all of the r. virginicus (banks) samples (n = 5) were simple families. for r. flavipes and r. hageni, the percentage of extended families is intermediate to southeastern and northern usa populations, corresponding to the intermediate seasonality and climate in arkansas. the level of inbreeding in arkansas, estimated via f it , was relatively high and similar to northern populations of reticulitermes. there were significantly more extended family colonies at the developed site compared to the two undeveloped sites which contained more simple family colonies. this difference may occur as a strategy to cope with sparse resources in urban environments or as a consequence of different abiotic factors. sociobiology an international journal on social insects ma janowiecki1, ad tripodi2,4, el vargo1, al szalanski3 article history edited by paulo cristaldo, ufrpe, brazil received 21 february 2019 initial acceptance 17 april 2019 final acceptance 09 may 2019 publication date 14 november 2019 keywords termite, developed vs undeveloped, family structure, neotenics. corresponding author allen szalanski university of arkansas agri 301c, fayetteville, ar 72701. e-mail: aszalan@uark.edu field, microsatellite dna genotyping methods have been developed (vargo 2000; dronnet et al., 2004) and applied to reticulitermes termites, primarily along the east coast of the usa and europe (vargo & husseneder, 2009). in r. flavipes (kollar) populations examined in the southeastern usa, there are generally 75% simple families, 20% extended families, and 2% mixed families (vargo & husseneder, 2009). in populations of r. flavipes further north in the usa (i.e. virginia, delaware, massachusetts, nebraska), there are more extended families (41.4 – 71.4%) and higher levels of inbreeding that correlate to latitude and seasonal climatic variables (ab majid et al., 2013; vargo et al., 2013). breeding structure of r. hageni banks and r. virginicus (banks) have been determined in north and south carolina and are primarily simple families (>75%) with no mixed families (vargo & husseneder, 2009). however, in 1 department of entomology, texas a&m university, college station, texas, usa 2 usda ars pollinating insect research unit, logan, utah, usa 3 department of entomology, university of arkansas, fayetteville, arkansas, usa research article termites sociobiology 66(3): 500-507 (september, 2019) 501 r. virginicus this value may be elevated since this species undergoes asexual queen succession (aqs) (vargo et al., 2012). in this system, female secondary reproductives are produced parthenogenetically, and therefore maintain the genetic structure of a simple family (vargo et al., 2012). microsatellite genotyping workers to determine the breeding structure of a colony often cannot differentiate between simple and extended families in aqs species. although many studies have investigated subterranean termites in either developed or undeveloped settings, few have compared aspects of termite biology between these two environments. in its invasive range in france, dronnet et al. (2005) compared the genetic diversity of r. flavipes in urban and rural areas and found no differences. similarly, family structure was not found to be different in urban compared to rural areas for reticulitermes in the eastern usa (vargo & husseneder, 2009). in nebraska, there were more simple families of r. flavipes in developed sites compared to undeveloped sites that had more mixed families (ab majid et al., 2013; ab majid et al., 2018). in arkansas, three species of reticulitermes occur: r. flavipes, r. hageni, and r. virginicus (austin et al., 2004). these subterranean termites, especially r. flavipes, are economically important structural pests that cost americans approximately $11 billion annually for prevention, treatment, and repair of termite damage (su, 2002). in this study, we investigate family structure for these three species of reticulitermes from three sites in northwest arkansas. we also determined if there is a difference in family structure between developed and undeveloped areas. materials and methods samples termites were collected from three sites in northwest arkansas, usa (fig 1): lake wedington unit of the ozark st. francis national forest (osfnf) (abbreviated: lake wedington) (36.13128, -94.3946), lee creek area of osfnf (abbreviated: lee creek) (35.76183, -94.2738), and the university of arkansas agricultural research and extension center, fayetteville, ar (abbreviated: uark farm) (36.09908, -94.1659). sites were separated by 17.7 to 35.4 km from one another. all samples were collected between july 2014 and mar. 2016. each sample represents a colony and multiple individuals were collected per colony. in northwest arkansas, the mean annual temperature is 14.3 °c ranging from -4.5 to 32.1 °c, and the average annual precipitation is 1180 mm (fick & hijans, 2017). lake wedington was created in the 1930s through the northwest arkansas land use project that converted misused or idle farm land into recreational and national forest areas. from this location, 21 samples were collected from decaying logs (table 1). lee creek is located south of devil’s den state park, in osfnf. it is primarily secondary growth forest that was also developed in the 1930s through efforts of the civilian conservation corps (smith, 1992). twenty-one samples were collected at this location (table 1). the uark farm was established in 1888 under the hatch act of 1887, which provided research stations to land grant universities (strausberg, 1989; hillison, 1996). it contains mainly secondary pine forests that separate buildings and experimental agricultural plots. fourteen samples (table 1) were collected from both decaying logs and sentricon® termite colony elimination system bait stations (dow agrosciences, indianapolis, in, usa) containing untreated pieces of pine wood. fifty-six samples representing the three species and three localities were examined in this study (table 1). fig 1. sampling sites in northwest arkansas, usa. site r. flavipes r. hageni r. virginicus total lake wedington 10 10 1 21 lee creek 9 9 3 21 uark farm 9 4 1 14 totals 28 23 5 56 table 1. number of samples of each termite by location. land use per site was calculated in arcgis desktop v.10.5.1 (environmental systems research institute (esri), redlands, california, usa) using world_imagery base maps (source: esri, digitalglobe, geoeye, earthstar geographics, cnes/airbus ds, usda, usga, aerogrid, ign, and the gis user community, last updated september 2018). a buffer circle with a radius of 500 m was drawn around each sample location. within this area, different sections were manually drawn and scored as forest, water, road, agricultural, impervious surface, or building and the percentage of area was calculated for each (fig 2 a-c). each site was classified as “developed” or “undeveloped” according to the dominant (> 50%) land use within this radius. areas that were dominated by heavily manipulated land uses (e.g., roads, impervious surface, buildings, or agriculture) were classified as developed, whereas areas dominated by forest were classified as undeveloped. ma janowiecki et al. – reticulitermes breeding structure in arkansas502 all samples were collected into 95% ethanol and voucher specimens are deposited in the texas a&m university insect collection, college station, tx, usa (#740). morphological identification, when possible, used the keys of scheffrahn and su (1994) and was confirmed genetically using pcr-rflp (szalanski et al., 2003) and dna sequencing analysis using a region of the mtdna 16s gene per austin et al. (2004). mitochondrial dna sequencing genomic dna was extracted from the head from an individual specimen using a salting-out procedure with inhouse reagents (sambrook & russell, 2001). the 16s gene region of mtdna was amplified using the primers 16s: lr-j13007 (5’-ttacgctgttatccctaa-3’) (kambhampati & smith, 1995) and lr-n-13398 (5’-cgcctgtttatcaaa aacat-3’) (simon et al., 1994). the pcr reagents followed taylor et al. (1996) and the thermal cycler protocol consisted of 35 cycles of 94 °c for 45 s, 46 °c for 45 s, and 71 °c for 60s. samples were purified and concentrated with vwr centrifugal devices (vwr, radnor, pa) and sent to eurofins genomics (huntsville, al) for direct sequencing in both directions. consensus sequences were obtained from an alignment of sequences in both directions for each sample using geneious v6.1.8 (kearse et al., 2012). haplotypes were determined by comparing to an in-house database (als, unpublished), and genbank. microsatellite genotyping genomic dna was extracted individually from the heads of 20 worker termites from each sample using a saltingout procedure with in-house reagents (sambrook & russell, 2001) and stored at -20 °c. twelve total microsatellite loci (r. flavipes: 8 loci; r. hageni: 11 loci; r. virginicus: 7 loci) (vargo, 2000; dronnet et al., 2004) were amplified in a single multiplex of fluorescently-labeled primers (dye set: g5, applied biosystems, life technologies, grand island, ny, usa). each species had a unique pcr reaction mix that can be found in supplementary tables 1-3. the thermal cycler condition (modified from: vargo, 2000) consisted of a hot start at 95 °c followed by a touchdown program of six cycles at 94 °c (30s), 62 °c (30s), and 72 °c (30s) decreasing annealing temperature 1 °c per cycle; then 30 cycles of 94 °c (30s), 55 °c (30s), and 72 °c (30s); with a final extension at 72 °c for 5min. samples were genotyped at iowa state university dna facility fig 2. land use for a circle with a 500 m radius around each sampling site in northwest arkansas (a-c) summarized as a pie chart for each site (d-f). sociobiology 66(3): 500-507 (september, 2019) 503 (ames, ia, usa) using an abi prism 3730 dna analyzer (applied biosystems, life technologies, grand island, ny, usa) or in house with an abi 3500 genetic analyzer (applied biosystems, life technologies, grand island, ny, usa) with genescan 500 liz dye size standard (applied biosystems, life technologies, grand island, ny, usa). microsatellite analysis alleles were scored with the microsatellite plugin for geneious v6.1.8 (kearse et al., 2012). individuals and loci with more than 50% missing data were removed from analysis. to access if all samples represented unique colonies, genotypic differentiation between samples were calculated with genepop (raymond & rousset, 1995). following vargo (2003), samples from unique colonies had pairwise genotypic differentiation values that were significantly (p < 0.05) different than zero, whereas samples of the same colony yielded nonsignificant p-values. for any repeatedly sampled colony, only the first sample obtained was used for further analysis. within each colony, breeding structure was determined. alleles per locus were calculated and used to distinguish mixed family structure. a mixed family, defined by deheer and vargo (2004), contain more than four alleles at any one locus, a characteristic only possible with a colony containing two or more unrelated reproductives. remaining colonies that were not classified as mixed families were divided into simple and extended families based on whether they had genotypes consistent with a monogamous pair of reproductive, and if so, whether the genotype frequencies differed from the expected mendelian ratios (vargo, 2003). the genetic variation partitioning among the individual (i), colony (c), and total population (t) were calculated in fstat v. 2.9.3 (goudet, 2001) for each species separately. fic and fit estimate the level of inbreeding in individuals compared to others in the same colony and to others in the total population, respectively (vargo et al., 2013). fic was calculated for the extended families only because it is useful in estimating the number of reproductives present (vargo & husseneder, 2009). differences in family structure by location were calculated with a fisher exact probability test in r v. 3.4.1 (r core team 2013) because of the small sample size (i.e. more than 20% of cells are n < 5). graphs were also generated in r v. 3.4.1 (r core team 2013). results species identification for r. flavipes, there were seven unique 16s mtdna haplotypes from the 28 samples. for r. hageni, there were three unique 16s haplotypes in the 23 samples. and for r. virginicus, there were two 16s haplotypes in the 5 samples. within a species, haplotypes were not exclusive to geographic location collected (table 2). family structure a majority of pairwise genotypic differentiation values calculated for each species was significant, indicating that most samples represented unique colonies. to avoid sampling a single colony twice, eight samples with pairwise genotypic differentiation values close to zero were removed when comparing breeding structures between sites and species. six of these samples were r. flavipes and two were r. virginicus. no samples of r. hageni were sampled twice. combining all three species for the remaining samples (n=56), we observed 57.4% simple families, 37.7% extended families, and 4.9% mixed families (fig 3, table 3). all five colonies of r. virginicus collected were simple families. the fic values for extended family colonies of r. flavipes (0.055) and r. hageni (-0.082) were near zero, indicating that there were relatively high numbers of neotenics mating within the colony (~20; thorne et al., 1999). the relatively high values of fit for all three species indicate these colonies are inbred (table 3). family structure by site while there was no significant difference in the frequencies of different family types between sites for each species alone: r. flavipes (fisher exact probability test, p = 0.0638, n = 28), r. hageni (p = 0.2679, n = 23), r. virginicus (p = 1.00, n = 5); there is a significant difference in family structure type by site for all species combined (p = 0.012, n = 56). generally, there were more extended families at the uark farm and more simple families at lake wedington and lee creek (fig 3). there was no difference in family structure between lake wedington and lee creek for all three species combined (p = 0.1575). interestingly, all mixed families for r. flavipes and r. hageni occurred at one site: lake wedington (fig 3). 16s haplotype lake wedington lee creek uark farm total genbank accession number r. flavipes gg/ss 1 3 3 7 ay702099 r. flavipes j 0 0 1 1 ay441984 r. flavipes m 0 2 0 2 ay441987 r. flavipes mm 1 0 1 2 dq001965 r. flavipes p 6 2 2 10 ay538741 r. flavipes r 0 1 0 1 ay603499 r. flavipes tt 2 1 2 5 dq001972 r. hageni h1 7 8 2 17 ay257235 r. hageni h2 2 1 2 5 ay603505 r. hageni h4 1 0 0 1 eu484333 r. virginicus v1 1 2 0 3 ay257241 r. virginicus v15 0 1 1 2 eu259775 table 2. 16s mtdna haplotypes of each species by location. ma janowiecki et al. – reticulitermes breeding structure in arkansas504 discussion variation in breeding structure has been observed in different species and populations although the cause of this variation is unclear (vargo & husseneder, 2009). in r. flavipes, vargo et al. (2013) found this variation in breeding structure and inbreeding was correlated to mean annual temperature and seasonality. in cool, moist habitats, there were higher levels of inbreeding and more extended families (vargo et al., 2013). in arkansas, our results align with this large-scale model but we also found differences in family structure between sites. this pattern of family structure aligns with the land use analysis where lake wedington and lee creek are undeveloped with mainly forest (>96%) whereas the uark farm is a developed site that has a wider variety of land use and is primarily agricultural land (>80%) (fig 2 d-f). in undeveloped, forested sites, there are more simple families compared to developed sites that have more extended families. across these three species of reticulitermes in northwest arkansas, only 57.4% of the colonies sampled were simple families, 37.7% were extended families, and 4.9% (2 samples) were mixed families (fig 2, table 3). compared to previous studies, the percent of r. flavipes extended families is intermediate between southeastern (low percentage) and northern usa populations (high percentage) (table 3) which corresponds to the intermediate seasonality in arkansas (vargo & husseneder, 2009; vargo et al., 2013). for r. hageni, there are more extended families than was previously reported in north and south carolina (vargo & husseneder, 2009). in r. virginicus, the samples from northwest arkansas are similar in family structure to south carolina (table 3) (vargo & husseneder, 2009); although, this value may also be elevated because of the potential for asexual queen succession in this species (vargo et al., 2012). our methods may have been unable to distinguish between simple and extended families if parthenogenetic neotenics were present. for r. flavipes, the fic value for extended families is positive but close to zero and similar to what was observed in louisiana and massachusetts indicating many neotenics reproducing in the colony. the fit value estimated for r. flavipes in arkansas is relatively high and similar to more populations in tennessee and massachusetts indicating a similarly high level of inbreeding. for r. hageni and r. virginicus, the fit values are closer to the north carolina values, which for r. virginicus are opposite of the family structure percentages that were more similar to south carolina. species/population (n = no. of colonies) simple (percent) extended mixed families (percent) overall fitpercent fic reticulitermes flavipes central north carolina (n = 319) 78.4% 19.7% -0.209 1.9% 0.052 charleston, south carolina (n = 18) 72.2% 22.2% -0.140 5.6% 0.030 eastern massachusetts (n = 22) 27.3% 59.1% 0.097 13.6% 0.289 lincoln, nebraska (n = 8) 28.6% 0% na* 71.4% 0.166 nebraska (urban structures) (n = 20) 85.0% 0% na 15.0% 0.111 central tennessee (n = 48) nr† nr 0.260 nr 0.386 northwest arkansas (n = 28) 57.1% 39.3% 0.055 3.6% 0.250 new orleans, louisiana (n=20) 0% 50% 0.060 50% 0.362 reticulitermes hageni raleigh, north carolina (n = 15) 86.7% 13.3% -0.257 0% 0.357 charleston, south carolina (n = 21) 95.2% 4.8% na 0% 0.140 northwest arkansas (n = 23) 58.3% 33.3% -0.082 8.3% 0.394 reticulitermes virginicus raleigh, north carolina (n = 8) 75% 25% -0.332 0% 0.037 charleston, south carolina (n = 4) 100% 0% na 0% -0.04 northwest arkansas (n = 5) 100.0% 0.0% na 0% 0.241 *not applicable, †not reported table 3. comparison of family structure types and inbreeding coefficients to other studies. results from this study are shaded gray (modified from vargo and husseneder 2009, additional data from: ab majid et al. 2013, perdereau et al. 2015, and ab majid et al. 2018). sociobiology 66(3): 500-507 (september, 2019) 505 we found that the developed site, the uark farm, had relatively more extended family colonies of r. flavipes and r. hageni compared to the undeveloped sites (fig 3) corresponding to differences in land use (fig 2). this is the first report of this pattern of colonies of different family structure at developed vs. undeveloped sites. this stands in contrast to two studies in nebraska on undeveloped (ab majid et al., 2013) and developed (ab majid et al., 2018) sites that found no extended families at either site but generally more simple families at developed sites compared to mixed families on undeveloped sites. however, this study of undeveloped sites in nebraska represents a high outlier in the percent of mixed families (table 3) and has a small sample size (n = 8). as inferred from the genotypic differentiation, most samples represent unique colonies. there are several instances, however, where the same colony was sampled multiple times. two samples collected from sentricon® termite colony elimination system bait stations within several meters of each other at the uark farm both sampled the same colony. because they are near each other, this is not unexpected; however, it is interesting that only these two bait stations were foraged on by this colony, as there were multiple other bait stations in the same area that contained termites from different colonies. two samples from lee creek in fallen logs also represent the same colony and were collected within several meters from each other. these are also understandably the same colony due to the close proximity of sampling. overall, the basic spatial dynamics shown from these examples highlight an interesting aspect of termite biology that could be further investigated in future research to better understand how these species that appear to be inhabiting the same niche are able to coexist and partition space and resources. overall, we observed a relatively high percentage of extended families in northwest arkansas. this is more than what has been observed in much of the eastern usa with the exception of northern populations. we further found a significant difference in family structure between our sampling sites that can be described by the land use with undeveloped, forested sites having more simple families and developed, agricultural sites having more extended families. this may reflect different strategies dependent on the abundance of resources. in the developed site there are less suitable resources to colonize compared to the undeveloped site, making inheriting the natal nest as an extended family potentially more profitable than attempting to found a new colony. alternatively, this difference may be explained by adaptation to differences in temperature and moisture levels caused by urban heat islands or urban landscape management practices like irrigation, etc. fig 3. family structure (simple, extended, or mixed family) comparison for the three different species (r. flavipes, r. hageni, and r. virginicus) and three different sampling locations in northwest arkansas. ma janowiecki et al. – reticulitermes breeding structure in arkansas506 these results highlight the importance of understanding how termite biology shifts as they invade urban habitats. acknowledgements we wish to thank j. austin, j. demark, and d. johnson for providing samples. also thanks to c. trammel and c. aguero for their technical support and assistance. we thank m. ferguson and d. shoemate for assistance with gis analyses and anonymous referees for their comments. this research was supported in part by the university of arkansas, arkansas agricultural experiment station and the urban entomology endowment at texas a&m university. authors contribution maj, als, and adt conceived and designed the project. maj collected the data. adt, elv, and maj conducted the data analysis. maj wrote the manuscript with support from adt, als, and elv. supplementary material h t t p : / / p e r i o d i c o s . u e f s . b r / i n d e x . p h p / s o c i o b i o l o g y / r t / suppfiles/4376/0 http://dx.doi.org/10.13102/sociobiology.v66i3.4376.s2350 references ab majid, a.h., s.t. kamble, and n j. miller. 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(2013). clinal variation in colony breeding structure and level of inbreeding in the subterranean termites reticulitermes flavipes and r. grassei. molecular ecology, 22: 1447-1462. doi: 10.1111/mec.12166 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i3.313-317sociobiology 61(3): 313-317 (september 2014) genetic variability of stingless bees melipona mondury smith and melipona quadrifasciata lepeletier (hymenoptera: apidae) from a meliponary jr koser1, fo francisco2, g moretto1 introduction the strictly neotropical stingless bee genus melipona (hymenoptera: apidae) comprises more than sixty species in brazil (camargo & pedro, 2013) and is one of the most important insects for pollination in natural and in cultivated areas (heard, 1999; slaa et al., 2006). the species melipona (michmelia) mondury smith, 1863 inhabits the atlantic forest biome, from bahia to rio grande do sul states (camargo & pedro, 2013). the species melipona (melipona) quadrifasciata lepeletier, 1836 has a wide distribution in southern and southeastern brazil (camargo & pedro, 2013) where it is widely cultivated and especially valued in tomatoes production (santos et al., 2009; sarto et al., 2005). in addition, its propolis has medicinal properties (mercês et al., 2013). both species are endangered in several brazilian states (machado et al., 1998; marques et al., 2002; mikich & bérnils, 2004; santa catarina, 2011) due to habitat loss caused by deforestation (brosi et al., 2007; brown & oliveira, 2014). nowadays, the atlantic forest is extremely fragmented, and bee species suffer from the negative impacts of the interrupabstract the species of stingless bees melipona mondury smith and melipona quadrifasciata lepeletier are native to the atlantic forest. these species are sensitive to environmental changes and due to habitat loss they are endangered in several brazilian states. this study aimed to evaluate the genetic variability of populations of these two species at the meliponary of the regional university of blumenau through the use of heterospecific microsatellite primers. we collected one worker from 19 colonies of m. mondury and from 25 colonies of m. quadrifasciata. we found low levels of genetic variability for both species, which may be explained by queen philopatry, intraspecific reproductive parasitism, and/or artificial maintenance of hives. if natural populations of these species are also presenting low genetic variability they might be endangered. sociobiology an international journal on social insects 1 universidade regional de blumenau, blumenau, santa catarina, brazil. 2 instituto de biociências, universidade de são paulo, são paulo, são paulo, brazil. article history edited by astrid kleinert, usp, brazil received 13 march 2014 initial acceptance 24 april 2014 final acceptance 26 june 2014 keywords conservation, heterospecific primers, meliponini, microsatellites corresponding author jaqueline reginato koser departamento de ciências naturais universidade regional de blumenau blumenau, santa catarina, brazil e-mail: jaquelinekoser@gmail.com tion of gene flow and decrease in genetic diversity (freiria et al., 2012). one way to prevent local extinction of these bees is maintaining hives in free-foraging wooden boxes in meliponaries (apiaries for stingless bees). meliponiculture for crop pollination and for honey extraction has been an encouraging economic practice aligned with sustainable development and educational purposes in several countries (cortopassi-laurino et al., 2006). however, little is known about the genetic variability of hives of native bees in meliponaries (carvalho-zilse et al., 2009). such knowledge is essential for the development of conservation strategies and rational exploitation of native species (cortopassi-laurino et al., 2006; alves et al., 2011). our aim was to characterize the genetic variability of m. mondury and m. quadrifasciata maintained in the meliponary of the regional university of blumenau. material and methods we collected one worker from each of 19 colonies of m. mondury and from 25 colonies of m. quadrifasciata research article bees jr koser et al. genetic variability of melipona stingless bees 314 acquired from beekeepers from vale do itajaí region, in the santa catarina state, brazil and maintained currently at the meliponary of the regional university of blumenau (mrub) (fig. 1) localized at 26° 54’ 21.81’’ s 49° 04’ 48.53’’ w (fig.2). colonies of m. quadrifasciata have been maintained at mrub since 1998 and colonies of m. mondury since 2003. these hives were extracted directly from nature in geographically close cities and have not been artificially divided. cording to lopes et al. (2010b) for the mmo primers. pcr products were separated by electrophoresis in 12% polyacrylamide gels and stained with silver nitrate for visualization. the program arlequin v.3.5.1.3 (excoffier & lischer, 2010) was used to calculate allelic richness (â), observed heterozygosity (ho), expected heterozygosity (he), percentage of polymorphic loci (ppl) and fis with 10,000 permutations. hardy-weinberg equilibrium (hwe) and linkage disequilibrium (ld) were computed using genepop v.4.1.4 (rousset, 2008). p-values were adjusted with bonferroni correction (rice, 1989). the frequencies of null alleles were computed using cervus 3.0.6 (kalinowski et al., 2007). results genetic variability was low for m. mondury, but no inbreeding was detected (table 1). loci mbi254 and mmo19 deviated from hwe even after bonferroni correction (p< 0.0167). ld was detected between mbi254 and mmo19 after bonferroni correction (p= 0.0062). the presence of null alleles with a frequency of 29% was found in the locus mbi254, a heterospecific primer (table s1). the loci mmo19 and mmo21, both specific primers for m. mondury, were not affected by null alleles (table s1). melipona quadrifasciata also showed low genetic variability although higher than m. mondury (table 1). significant inbreeding was detected. primers developed for m. bicolor were more polymorphic in m. quadrifasciata than those developed for m. mondury (table s2). deviation from hwe was detected for the loci mbi218, mbi233 and mmo21 after bonferroni correction (p = 0.0000). ld was not detected (all p > 0.05). for m. quadrifasciata, the frequency of null alleles was higher than 25% in the following heterospecific primer pairs: mbi218, mbi233, mmo03, mmo11 and mmo21 (table s2). the loci mbi11, mbi88, mbi254 and mbi259 were not affected by null alleles (table s2). figure 1. meliponary of the regional university of blumenau (mrub). figure 2. localization of santa catarina state showing the vale do itajaí region. meliponary localization ( ● ). dna extraction was performed according to anderson and fuchs (1998). for both species, we used 10 microsatellite primer pairs developed for the species m. bicolor (mbi11, mbi28, mbi32, mbi33, mbi88, mbi218, mbi233, mbi254, mbi259 and mbi522) (peters et al., 1998) and 11 primer pairs developed for m. mondury (mmo03, mmo06, mmo08, mmo10, mmo11, mmo15, mmo19, mmo20, mmo21, mmo22 and mmo24) (lopes et al., 2010b). pcr reactions and the annealing temperatures were performed according to peters et al. (1998) for the mbi primers and actable 1. genetic variability for melipona mondury and melipona quadrifasciata based on microsatellite data. â: allelic richness; fis: inbreeding coefficient; he: expected heterozygosity; ho: observed heterozygosity; n: sample size; ppl: percentage of polymorphic loci. species n â ho he ppl fis p-value m. mondury 19 1.60 0.105 0.102 15% -0.00629 0.569307 m. quadrifasciata 25 2.22 0.129 0.189 50% 0.33493 0.000000 this article has supplementary material published and available on line doi: 10.13102/sociobiology.v61i3.313-317.s435 url: http://periodicos.uefs.br/ojs/index.php/sociobiology/article/ view/315 sociobiology 61(3): 313-317 (september 2014) 315 discussion our results indicate low genetic variability in the populations of m. mondury and m. quadrifasciata maintained at the mrub. low genetic variability in native stingless bees is also documented in studies using molecular markers such as rapd (tavares et al., 2001), mitochondrial polymorphism (brito et al., 2013), issr (inter single sequence repeats) (nascimento et al., 2010; miranda et al., 2012), and microsatellites (francisco et al., 2006; tavares et al., 2007; carvalho-zilse et al., 2009; francini et al., 2009; alves et al., 2011; duarte et al., 2011). a first explanation of this low variability could be the presence of null alleles. in this study, we found high values of null alleles only in one out of three polymorphic loci for m. mondury (mbi254) and in five out of nine polymorphic loci for m. quadrifasciata (mbi218, mbi233, mmo03, mmo11 and mmo21). in addition, null alleles might be responsible for the high number of monomorphic loci found in both species. the use of heterospecific primers may also be related to low genetic diversity. in the stingless bee plebeia remota, the same samples analyzed with heterospecific (francisco et al., 2006) and specific (francisco et al., 2013) primers showed high divergent values. the same result was found in melipona bees (lopes et al., 2010a). cross-species amplification is one of the advantages of microsatellites, and low variability should always be interpreted with caution when using heterospecific primers. however, data obtained from other populations/species with the same primer pairs we used show higher levels of genetic diversity (tables s3 and s4) suggesting that the low diversity we observed in this work are not due to heterospecific primers only. low variability may also be explained by the species natural biology. queens of most stingless bee species mate with a single male (monandric) (peters et al., 1999; palmer et al., 2002) and are known to nidify near maternal nests (nogueira-neto, 1954). their low dispersion increases genetic drift and inbreeding within sub-populations (hartl & clark, 2007). a special concern is the maintenance of alleles of the complementary sex determination system in small populations, because inbreeding can lead to the production of diploid males (cook & crozier, 1995; zayed, 2009; alves et al., 2011). for other stingless bee species, males are the dispersing sex (cameron et al., 2004; carvalho-zilse & kerr, 2004; francisco et al., 2013), but no data is available for m. mondury and m. quadrifasciata. nevertheless, the location of mrub near forest fragments might allow the mating of queens from the meliponaries with males from native forests in the surroundings, decreasing their inbreeding probability. managed populations of local bees can be considered a reservoir of genetic diversity if they can interbreed with wild populations (alves et al., 2011). however, to introduce bees from distant populations or another species that could hybridize with local populations might cause outbreeding depression (lynch, 1991; waser et al., 2000). recent data from wenseleers et al. (2011) showed intraspecific reproductive parasitism in m. scutellaris. if we speculate that this behavior occurs in other melipona species, colonies that were previously unrelated could become related if sibling queens take over these colonies. in these cases, genetic variability would decrease. another explanation to account for the low variability may be related to the artificial maintenance of hives through founder events and bottlenecks as already reported for apis mellifera (sheppard, 1988; schiff & sheppard, 1996; moritz et al., 2007; delaney et al., 2009; jaffé et al., 2010; meixner et al., 2010) and m. scutellaris (carvalho-zilse et al., 2009; alves et al., 2011). however, despite low variability, populations can be successfully maintained if a strong care is dispensed over the nests (alves et al., 2011). if natural populations of these species are also presenting low genetic variability they might be endangered. adaptation to environmental changes is dependent on the genetic variations that exist among members of a population. without variation, populations are more prone to extinction. therefore, the analysis of natural populations of these species in south brazil is crucial. detecting the male flight range is also important since they can prevent genetic isolation in fragmented populations. if meliponaries were able to assist in maintaining the genetic variability of natural populations, they could be used for research and reintroduction programs. acknowledgments we thank the universidade regional de blumenau furb for technical assistance. jr koser was supported by a scholarship from pipe/art 170. references alves, d.a., imperatriz-fonseca, v.l., francoy, t.m., santos-filho, p.s., billen, j., & wenseleers, t. 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(2009). bee genetics and conservation. apidologie, 40: 237-262. 493 field activity of reticulitermes grassei (isoptera: rhinotermitidae) in oak forests of the southern iberian peninsula by ana m. cárdenas1*, lourdes moyano2, patricia gallardo3 & juan m. hidalgo4 abstract this paper presents preliminary data on the field activity of reticulitermes grassei clément in oak forests of the southern iberian peninsula. recent research has provided information on the nature and intensity of termite damage to cork oaks (quercus suber, l.) in northern andalusia (spain). taking that information into account, the present study sought to determine annual field activity pattern in r. grassei, with a view to identifying more precisely the best time for applying control techniques. data were obtained from field monitoring experiments conducted over a complete one-year cycle using termite-specific baited traps. results for relative termite numbers at different periods indicated that forest activity was most intense in mid-summer, whilst the surface foraging area was greatest from late summer to early fall, peaking after the first autumnal rains. the findings of this study may help to enhance the efficacy of termite bait treatments in natural environments, since baits decay and lose effectiveness over time, and are also dispersed by the termites themselves. accurate information on peak termite activity periods would enable products to be applied in most favorable timing, thus optimizing the results of treatment. keywords: field activity, iberian peninsula, quercus suber, reticulitermes grassei, rhinotermitidae 1department of zoolog y. campus rabanales. university of córdoba. e-17071. córdoba (spain). e-mail: ba1cataa@uco.es 2department of zoolog y. campus rabanales. university of córdoba. e-17071. córdoba (spain). e-mail: b22moayl@uco.es 3departament of zoolog y. campus rabanales. university of córdoba. e-17071. córdoba (spain). e-mail: b42gatop@uco.e 4departament of zoolog y. campus rabanales. university of córdoba. e-17071. córdoba (spain). e-mail: zo2himaj@uco.ess 494 sociobiolog y vol. 59, no. 2, 2012 introduction termites are social insects that cause major damage to wooden structures in urban environments, but they also form part of natural ecosystems (haverty & sunden-bylehn 2000), where their most obvious role is in decomposition and in determining specific pathways for the subsequent humification and mineralization of residual materials (bignell & eggleton 2000). given the particularly cryptic behavior of termites, they tend to go unnoticed until the damage they cause has become highly manifest and their foraging territories are widespread. following eggleton (2000) and lenz et al. (2003), termites are generally classified by habits and habitats as follows: damp wood termites: inhabit and eat damp wood, especially stumps and fallen trees on the forest floor. dry wood termites: require no contact with the ground, live in trees, usually in the treetops, and tolerate low environmental humidity. subterranean termites: numerous in many parts of the world, living and reproducing in soil and feeding mainly on wood. some subterranean termites can build epigeal nests, and are major pests in towns and cities. mushroom growers: these termite species collect grass, leaves or wood, which are processed by basidiomycetes ‘symbionts’ grown in chambers inside the termites’ mound. through specific enzymes, fungi ‘digest’ plant debris taken by termites into the mound. four termite species are known in the iberian peninsula: two subterranean species (reticulitermes grassei clément and r. banyulensis clément), and two dry-wood species: kalotermes flavicollis (fabricius) and cryptotermes brevis (walker). of these, only r. grassei and k. flavicollis are found in andalusia, occurring both in natural environments and in urban areas, where they are responsible for extensive damage and significant economic losses. r. grassei colonies have been reported in most of the iberian peninsula, with the exception of a small north-eastern area (between catalonia, navarre and the ebro river basin) which is occupied by r. banyulensis. like other reticulitermes species, r. grassei usually eats the dead wood of old or senescent trees, and also attacks rural dwellings and urban structures such as buildings, bridges and dams. research has found that the termite is 495 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula a major pest in certain areas of andalusia, including palenciana (córdoba), where it has been recorded in over 33% of homes (gaju et al. 2002). although termites are also very common in most iberian forests, the presence of r. grassei has not so far been classed a pest problem in natural ecosystems. recently, however, our research group discovered a new type of damage affecting cork oaks (quercus suber l.) in southern iberian forests. the termite r. grassei was identified as the cause of sinuous galleries and adhesions that were hampering cork extraction, thus prompting economic losses (gallardo et al. 2010). numerous papers address termite biolog y, focussing on the number of instars, the definition of workers and the terminolog y used to differentiate castes. an overview of the literature on the life cycle of reticulitermes spp. can be found in lainé et al. (2003). however, no data are available on the field activity of this species in natural environments. most observations of foraging behavior and ecolog y in subterranean termites have been conducted in tropical forest environments (bignell & eggleton 2000). the pest management of subterranean termites is a complex issue: for many years, they were controlled by chemical barriers, which involved the abundant use of insecticides despite considerable toxicity both for humans and for the environment. newer, alternative termite-control methods require the placing of baits containing specific low-toxicity substances (usually chitin-synthesis inhibitors, verker & bravery 2000; 2004). successful control of r. grassei using baiting systems has been reported in urban areas of andalusia (alcaide 2010): termites feed on a lethal bait placed in a monitoring station, and thereafter spread the bait inside the mound, transferring it – through their trophallaxic behavior – to other members of the colony. clearly, control and treatment measures are most effective during periods of increased foraging and feeding activity, since more individuals are affected and the amount of insecticide ingested is higher. given its specificity and mode of application, this method might also be suitable for pest control in forest environments. nevertheless, attention should be paid to the environmental variables influencing the distribution, density and seasonal activity of subterranean termites (haagsma 1995; ramirez & lanfranco 2001); in temperate areas, fluctuations in temperature and humidity are more manifest in forest environments than in urban constructions. the 496 sociobiolog y vol. 59, no. 2, 2012 present study therefore sought to examine r. grassei field activity patterns in order to identify periods of peak activity, thus enabling the optimal timing of any control treatment required. materials and methods sampling area to study r. grassei field activity patterns, a pure cork-oak stand was selected, where termite damage (affecting 68.63% of trees) had previously been detected. the plot is located in central sierra morena (southern iberian peninsula), belongs to the “los baldíos” farm (utm coordinates 30s 03350944200826) and is part of the recovery management program linked to the construction of the breña ii dam (cárdenas et al. 2008). according to gallardo et al. (2010), the research area is of great ecological value, and comprises a gallery forest growing on the banks of the bejarano stream (a tributary of the river guadiato), and a mixed-oak forest dominated by quercus ilex l., q. suber l. and q. faginea lam., predominate, but also containing other tree species including pinus pinea l. and olea europaea var. sylvestris brot. the most representative scrubland species are genista spp., cistus salvifolius l. and daphne gnidium l. local soils are predominantly acid, and the climate is typically mediterranean. bioclimatologically, the area lies between the thermo-mediterranean and meso-mediterranean belts, with broad transitional zones (cárdenas & bach 1989). climate data (monthly average temperature and rainfall) recorded at the trassierra weather station (closest to the study area) during the sampling period (http://www.aemet.es 2011) are shown in fig. 1. field sampling field sampling was carried out from november 2009 to october 2010, using termite-specific baited traps. a survey of the different methods used for sampling for subterranean termites can be found in bignell & eggleton (2000). on the basis of this information, a sampling procedure similar to that used in earlier research (gallardo et al. 2010) was used, placing a set of baited traps at the foot of damaged trees. 497 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula forty trees were randomly selected in an area where termite damage had previously been observed. trees were marked (fig. 2) and geo-referenced (table 1). the average distance between sampled trees was 12.36 m ± 4.46 sd, and tree density for the study areas as a whole was 37 trees/ha. a total of 40 baited traps were placed in the study plot (one at the foot of each selected tree). each trap consisted of a container measuring 20 cm in diameter and 15 cm in height, open at the base and containing an untreated block of wood of roughly 10x5x5 cm that acted as starting bait. the trap also contained additional bait in the form of a pack of several previously-moistened strips of corrugated cardboard (25x5x0.5 cm). the trap was covered with a lid and was buried about 20 cm deep in the soil at the base of each tree; a little soil and straw were added. after around 35 days, the additional bait was removed and replaced. once in the laboratory, the corrugated cardboard strips were minutely examined to check for termites. workers and soldiers were isolated, identified and counted. a total of 10 samplings were performed over the total sampling period (table 2). spss and autocad programs were used for statistical analysis and graphic plotting, respectively. fig.1. monthly data of average temperature (ºc) and precipitation (mm) recorded at the meteorological station of trassierra (closest to the study area) during the sampling period. 498 sociobiolog y vol. 59, no. 2, 2012 results reticulitermes grassei field activity was analyzed in terms of changes in activity patterns and changes in the area of surface activity over time. these two parameters were considered independently of each other, on the basis of the sampling data recorded in table 3. field activity patterns to chart changes in r. grassei numbers over time, an analysis was made of monthly data for total termite numbers, and also for numbers of workers and soldiers captured during the sampling period. results are plotted in fig. 3. the graph clearly shows an extended period of peak surface activity. termite activity started in may and gradually increased, peaking in july and august, thereafter declining from late summer through fall, and reaching minimal values in winter due to extreme climate conditions. nevertheless, some workers were still observed in winter. fig. 2. general overview of the sampling area showing the sampled cork oaks (white colored and numbered trees). 499 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula activity curves for soldiers and workers considered independently ran parallel to those data for total termite activity, although workers started moving earlier and peak activity was recorded sooner than in soldiers; worker activity was also considerably greater. the caste ratio (soldiers/workers) remained constant at around 1/20 throughout the year. area of surface activity to track changes in the area of surface activity, the size of the area in which traps contained termites was monitored throughout the sampling period. table1. utm coordinates and tree reference (ref.) for the sampled cork oaks. tree (ref.) utm coordinates tree (ref.) utm coordinates tree (ref.) utm coordinates tree (ref.) utm coordinates 1 0335498 4201512 11 0335558 4201495 21 0335560 4201577 31 0335547 4201462 2 0335509 4201466 12 0335536 4201485 22 0335568 4201577 32 0335552 4201464 3 0335511 4201459 13 0335563 4201519 23 0335580 4201548 33 0335501 4201467 4 0335494 4201504 14 0335541 4201522 24 0335589 4201541 34 0335441 4201456 5 0335499 4201522 15 0335543 4201542 25 0335593 4201540 35 0335491 4201467 6 0335540 4201549 16 0335546 4201543 26 0335571 4201477 36 0335483 4201437 7 0335531 4201526 17 0335546 4201561 27 0335590 4201441 37 0335444 4201478 8 0335556 4201487 18 0335545 4201543 28 0335570 4201529 38 0335438 4201425 9 0335542 4201502 19 0335553 4201572 29 0335523 4201460 39 0335435 4201470 10 0335566 4201508 20 0335554 4201578 30 0335581 4201464 40 0335425 4201458 table 2. sampling dates. sampling number date sampling number date 1 06-11-2009 6 14-06-2010 2 03-12-2009 7 08-07-2010 3 03-02-2010 8 01-08-2010 4 23-03-2010 9 06-09-2010 5 18-05-2010 10 10-10-2010 500 sociobiolog y vol. 59, no. 2, 2012 ta bl e 3. n um be r o f t er m it es (w or ke rs : w a nd so ld ie rs : s ) c ap tu re d at e ac h sa m pl ed tr ee ( tr ee re f.) a nd sa m pl in g da te . tr ee n ov /0 9 d ec /0 9 fe b/ 10 m ar /1 0 m ay /1 0 ju n/ 10 ju l/ 10 a ug /1 0 se p/ 10 o ct /1 0 re f. s w s w s w s w s w s w s w s w s w s w 1 6 12 7 1 7 3 3 60 4 90 9 13 4 2 2 38 3 4 1 3 5 3 51 19 37 1 20 15 47 20 13 53 6 19 2 6 87 6 1 15 5 7 3 20 1 40 8 9 17 44 3 4 24 8 10 14 38 4 11 3 1 16 10 2 14 68 11 32 9 92 12 6 11 5 4 26 1 2 61 13 14 1 27 3 15 13 33 4 16 6 10 40 3 48 17 4 18 11 3 29 20 8 18 1 1 24 6 22 6 19 35 70 8 5 93 3 58 11 59 11 49 20 11 1 31 21 2 20 14 1 5 21 1 16 31 0 53 12 13 25 22 7 16 8 23 11 48 22 98 3 24 25 2 54 27 26 35 69 8 14 1 28 10 94 9 27 5 48 93 8 7 28 2 37 1 19 29 4 30 4 13 81 1 31 42 84 0 11 22 5 3 6 5 39 78 0 46 21 23 28 18 06 3 4 98 32 35 70 8 36 71 5 1 11 10 20 1 52 10 37 66 13 11 13 6 40 97 9 8 34 45 3 1 19 5 33 11 7 19 02 4 7 61 9 34 35 36 4 89 2 32 13 37 18 52 6 8 27 1 14 86 3 38 39 1 10 40 501 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula fig. 3. number of soldiers, workers and total termites captured monthly during the sampling period. the position of sampled trees in the study area is shown in fig. 4 a, whilst trees with termite-containing traps are shown in fig. 4 b, figs. 5 a and b, and figs. 6 a and b. at the start of the study (november 2009), termites were found on only three cork-oaks, but numbers progressively increased to peak at 24 trees in october 2010. overall data from all samples (table 3) show that termites were found at least once during the study period on 32 of the 40 trees sampled. the scatter plot of trees colonized by termites displayed no uniform or contagious distribution: in the first sampling (november 2009), for example, only three traps contained termites but the distance between them ranged between 5 and 37 m (trees 1-32 and 31-32, fig. 4 b). the number of traps containing r. grassei progressively increased over time (fig. 7), leading to an increase in the area of surface activity. even so, the number of termite-containing traps remained below ten throughout spring and up until midsummer. from july onward, the area of surface activity extended to 80% of the sampled trees (fig.6). the presence of termites in sampled trees was highly irregular. in some trees, termites once detected remained throughout the rest of the sampling period; this was the case of traps located in trees 19, 31 and 32, in which termites were found from november onward. in other traps, termites were recorded sporadically, with only one or two captures over the entire sampling period; for example, in trees 39 and 17 termites were found only in june and july, respectively. 502 sociobiolog y vol. 59, no. 2, 2012 fig. 4. a: overview of the sampling area; symbols and numbers correspond to each sampled cork oak. b: representation of the trees having termites in traps sampled in november december/2009. 503 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula fig. 5. representation of the trees having termites in traps. a: februarymarch/2010. b: may-june/2010. 504 sociobiolog y vol. 59, no. 2, 2012 fig. 6. representation of the trees with termites in traps sampled. a: julyaugust/2010. b: september-october/2010. 505 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula discussion termite treatments based on the use of specific baits, including chitin inhibitors, were first applied in the mid-1990s. a number of papers have reported on the application of these products (e.g. noviflumuron, hexaflumuron, diflubenzuron) for the treatment of several reticulitermes species, including r. grassei (getty et al. 2005; rojas et al. 2008). these products have a clearance time of between 2 and 34 months, depending on the concentration and the product used (alcaide 2010). even so, since baits decay and become less effective over time (due to desiccation, flooding….) (eggleton & bignell 1995), and since they are dispersed by the termites themselves, it is essential to establish the period of greatest termite activity in order to adjust the timing of product application and thus optimize the results of treatment. this study therefore aimed to determine r. grassei field activity patterns in order to pinpoint periods of peak activity and identify the environmental factors most likely to influence it. designing a sampling protocol to estimate population size, richness or abundance for soil/wood interface feeders such as termites poses a number of challenges (bignell & eggleton 2000). however, since research in this case focused not on these population parameters but on peak soil-surface activity, fig. 7. monthly number of traps containing termites. 506 sociobiolog y vol. 59, no. 2, 2012 a field experiment was designed to monitor termite activity over a full annual cycle. the design was based on information acquired during earlier research (gallardo et al. 2010) showing that r. grassei had already colonized the study area, causing damage to cork-oaks. observations in the new study therefore concentrated on changes in termite foraging behavior, often adapted to locating new food sources (traniello & leuthold 2000), rather than on dispersal or swarming strategies. search patterns have been analyzed in two species of reticulitermes, r. flavipes (reinherd et al. 1997) and r. santonensis (robson et al. 1995); mapping of foraging-gallery formation revealed a non-random search pattern, and showed that galleries were organized in such a way as to avoid overlaps. although there are no published data on changes in search behavior over time in subterranean termites, other issues – including the division of labor among workers during foraging, and the role of pheromones in foraging behavior – have been addressed in several reticulitermes species (traniello & leuthold 2000). in the present study, most individuals involved in foraging behavior were workers; only 5% were soldiers. this is consistent with findings reported by these authors who note that, in some termite species, soldiers play an organizational and defensive role in foraging activity. pomeroy (1983) drew attention to the relationship between termite distribution and certain environmental variables such as rainfall mean minimum temperature and soil differences. in addition, haagsma (1995) and ramirez & lanfranco (2001) suggest that the variable most influencing the distribution, density and seasonal activity of subterranean termites is humidity (related to rainfall and temperature). seasonality clearly affects both termite abundance and distribution (ohiagu 1979; wood et al. 1982; dibog et al. 1999). termites are generally less common in the surface layers of very dry soil, but overall population sizes may also fluctuate seasonally. the data obtained here confirm these findings, in that the greatest area of surface activity was recorded in early october, following the first autumn rains. peak termite numbers, however, were recorded in july, according to the thermophilous nature of this species. even in winter, when environmental conditions would appear to be unfavorable due to low temperatures and high humidity, some individuals remained active. lepage & darlington (2000) note that in seeking to maintain nest homeostasis, termite species face a number of environmental constraints: whilst other members the soil fauna, 507 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula such as earthworms, remain quiescent during the unfavorable period (dry season), termites remain active even in periods of disturbance, in order to protect the reproductive potential of the colony and ensure recovery when conditions become favorable. in summary, analysis of changes in population size and area of surface activity suggests that bait-based treatments for the control of r. grassei in southern iberian oak forests are best applied between july and october. nevertheless, as year-on-year variations may also occur (bignell et al. 1997; dibog et al. 1999), further long-term studies are required to pinpoint more accurately the optimal timing of treatment. acknowledgments we grateful to ingeniería y gestión del sur, ipa, s.l. for the financial support, dr. miguel gaju for his assistance in different aspects of this research, and mr. josé alcántara for his assistance with the graphs. references agencia española de meteorología. 2011. http://www.aemet.es/es/servidor-datos/accesodatos/listado-contenidos/detalles/series_climatologicas. alcaide, m.e. 2010. estudio del control integral de una plaga urbana de termitas en el sur de españa. tesis doctoral. universidad de córdoba. españa. 246 pp. bignell, d.e. & p. eggleton. 2000. termites in ecosystems. in: abe t., d.e. bignell & m. higashi (eds.), termites: evolution, sociality, symbioses, ecolog y. kluwer academic publisher. dordrecht. pp 365-387. bignell, d.e., p. eggleton, 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reticulitermes) using a baiting system: a case study in chatsworth, california, usa. in: lee c.y. & w.h. robinson (eds.), proceedings of the 5th international conference on urban pest. singapore, 10-13 july. pp 165-169. haagsma, k.a. 1995. colony size estimates, foraging trends, and physiological characteristics of the western subterranean termite (isoptera: rhinotermitidae). environmental entomolog y 24(6): 1520-1528. haverty, m. & a. sunden-bylehn. 2000. finding alternatives to persistent organic pollutants (pops) for termite management. international activities on persistent organic pollutants (pops) covered by the stockholm convention. stockholm, sueden. lainé v.l., v.l. lainé & d.j. wright. 2003. the life cycle of reticulitermes spp. (isoptera: rhinotermitidae): what do we know ?. bulletin of entomological research 93(4): 267-378. lenz, m., a. sunden-bylehn, b.l. thorne, v.r. lewis & m. haverty. 2003. finding alternatives to persistent organic pollutants (pops) for termite 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bosque 22(2): 77-84. 509 cárdenas, a.m. et al. — field activity of r. grassei in the iberian peninsula reinhard, j., h. hertel & m. kaib. 1997. systematic search for food in the subterraneantermite reticulitermes santonensis de feytaud (isoptera, rhinotermitidae). insectes sociaux 44: 147-158. robson, s.k., m.g. lesniak, r.v. kothandapani, j.f.a. traniello, b.l. thorne & v. fourcassié. 1995. nonrandom search geometry in subterranean termites. naturwissenschaften 82: 526-528. rojas, m.g., j.a. morales-ramos, e. lockwood, l. etheridge, j.b. carroll, m.c.h. coker & p.r. knight. 2008. areawide management of subterranean termites in south mississippi using baits. technical bulletin of agricultural research, service 1917: 1-44. traniello, j.f.a. & r.h. leuthold. 2000. behavior and ecolog y of foraging in termites. in: abe t., d.e. bignell & m. higashi (eds.), termites: evolution, sociality, symbiosis, ecolog y. kluwer academic publisher. the netherlands. pp 141-168. verker, r.h.j. & a.f. bravery. 2000. the uk termite erradication programme: justification and implementation. doc. no. irg/wp 00-10373. verker, r.h.j. & a.f. bravery. 2004. a case study from the uk of possible successful eradication of reticulitermes grassei. final workshop cost action e22 environmental optimization of wood protection lisboa-portugal, 22nd-23rd. wood, t.g., r.a. johnson, s. bacchus, m.o. shittu & j.m. anderson. 1982. abundance and distribution of termites (isoptera) in a riparian forest in the southern guinea savanna near mokwa, nigeria. geomorpholog y and ecolog y of tropical and subtropical regions 1: 139-148. doi: 10.13102/sociobiology.v62i1.70-75sociobiology 62(1): 70-75 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 soil-sampled termites in two contrasting ecosystems within the semiarid domain in northeastern brazil: abundance, biomass, and seasonal influences introduction termites are among the most abundant arthropods in tropical ecosystems, and their densities can exceed 8000 individuals.m-² (martius, 1994; bignell & eggleton, 2000). termites have very important functions in most terrestrial ecosystems due to their roles in decomposition processes and in energy and nutrient fluxes (matsumoto, 1976; bignell & eggleton, 2000; bandeira & vasconcellos, 2002). these organisms also influence the soil by redistributing material both horizontally and vertically and by increasing soil porosity (lee & wood, 1971; wood & sands, 1978; jones et al., 1994). it is estimated that up to 50% of the decomposition of abstract termites are important in tropical terrestrial ecosystems due to their role in fragmenting organic material and thus in energy and nutrient flows. the present study compared soilsampled termites in two contrasting ecosystems in the brazilian semiarid domain: humid highland forests (“brejo de altitude”) and seasonally dry tropical forests (“caatinga”). thirty soil core samples (12 l each), divided into three soil levels (a: 0-10; b:10-20; c: 20-30 cm), were analyzed in each area, 15 during the rainy season and 15 during the dry season. twentytwo termite species were encountered in the soil core samples, belonging to the termitidae (20 species) and rhinotermitidae (2 species). the abundance and biomass of termites in the humid highland forests were significantly greater during the rainy season (6080 individuals.m-² and 18.53 g.m-² fresh weight) than in the dry season (2215 individuals.m-² and 5.45 g.m-² fresh weight). the greatest abundance and biomass was encountered at the 0 and 20 cm depths. abundance and biomass did not differ significantly among the different seasons in the seasonally dry tropical forests (rainy season: 862 individuals.m-² and 1.4 g m-² fresh weight; dry season: 250 individuals.m-² and 0.45 g m-² fresh weight) nor among the different soil levels. the observed increase in termite numbers and biomass during the rainy season in the humid highland forest indicates that their decomposition activities are asymmetrical during the year. the quantitative similarity of termite numbers and fresh weights during the year in the seasonally dry tropical forest area suggests that other factors beyond climatic differences influence the abundance and biomass of these termites, at least at a local scale. sociobiology an international journal on social insects vfp araújo, mp silva, a vasconcellos article history edited by reginaldo constantino, unb, brazil received 20 april 2014 initial acceptance 21 june 2014 final acceptance 19 august 2014 keywords isoptera, vertical stratification, seasonally dry tropical forests, humid highland forests corresponding author alexandre vasconcellos laboratório de termitologia departamento de sistemática e ecologia centro de ciências exatas e da natureza universidade federal da paraíba 58051-900, joão pessoa, paraíba, brazil e-mail: avasconcellos@dse.ufpb.br plant organic detritus in tropical forests can be attributed to termites (bignell & eggleton, 2000). more water is retained in the soil in arid and semiarid ecosystems due to the activities of termites, which is believed to be directly reflected in vegetation structures and local primary productivity (holt & conventry, 1990; whitford, 1991). though few, quantitative studies at different times of the year in the same ecosystem have revealed seasonal variations in abundance and assemblage composition that may impact the temporal contributions of termites to processes such as organic decomposition (matsumoto, 1976; matsumoto & abe, 1979; collins, 1989; dibog et al., 1998; martius, 1998; vasconcellos, 2010). research article termites universidade federal da paraíba, joão pessoa, pb, brazil sociobiology 62(1): 70-75 (march, 2015) 71 seasonally dry tropical forests are characteristic of tropical environments with marked water restrictions during the dry season (dirzo et al., 2011). these dry forests occur widely in the semiarid region of northeastern brazil, which itself covers an area of approximately 735,000 km2 extending from 2° 54’ w to 17° 21’ s (andrade-lima, 1981; prado, 2003). this region is characterized by high temperatures and high evapotranspiration potentials that compound the effects of the low and irregular rainfall rates – resulting in low water availability in the soil for 7 to 11 months (andradelima, 1981). however, the regional landscape is actually a mosaic of different phytophysiognomies such as seasonally dry tropical arboreal, shrub, and spiny forests that are adapted to these very dry conditions (coimbra-filho & câmara, 1996). additionally, there are “islands” of highland humid forest within the semiarid region of northeastern brazil that are associated with landscapes that rise above 600 m, locally known as “brejos de altitude” (andrade-lima, 1982; tabarelli & santos, 2004). these highlands and plateaus offer more amenable environmental conditions, with greater soil and atmospheric humidity and denser vegetation cover (andradelima, 1966; tabarelli & santos, 2004; rodal & sales, 2008). as such, these unique landscapes function as biodiversity refuges, especially in the dry season, for organisms having a certain degree of mobility (andrade-lima, 1981). the present study sought to quantify the abundance, biomass, and vertical stratification of soil termites in two areas within the brazilian semiarid domain – one with seasonally dry tropical forest vegetation (caatinga), and the other with highland humid forest vegetation (brejos de altitude) – to determine the influences of the dry and rainy seasons on these arthropods. materials and methods the study sites were located in the fazenda almas private nature reserve (seasonally dry tropical forest) and in the mata do pau ferro state ecological reserve (highland humid forest) in paraiba state, brazil. the fazenda almas is a private protected area located in the municipality of são josé dos cordeiros (07°28’ s 36°52’ w) that encompasses 3,505 ha of seasonally dry tropical forest vegetation at altitudes ranging from 600 to 720 meters a.s.l. the mean annual precipitation, temperature, and humidity there are 560 mm ± 230 (sd), 25 oc, and 65% respectively (governo do estado da paraíba, 1985), with rainfall concentrated between february and april. the mata do pau ferro ecological reserve is located in the municipality of areia (6° 58’ s, 35° 42’ w) and encompasses 607 ha of ombrophilous forest communities at altitudes between 400 and 600 m a.s.l. (oliveira et al., 2006). it is situated within a micro-region of montane forests with mean annual rainfall, temperatures, and humidity of 1400 mm, 22 °c, and 85% respectively (embrapa, 1972). termites were collected in the mata do pau ferro site during december 1998 and january 1999 during the dry season, and in june and july 1999 during the rainy season. collections at the fazenda almas site were undertaken in may 2007 and november 2008 during the rainy and dry seasons respectively. the difference of the sampling period between the areas was about 8 years. however, for over 30 years, the areas have been free of human disturbance. fifteen collection points were established at minimum distances of 10 m from each other, spaced along a transect, and termites were collected using the methodology proposed by the tropical soil biology and fertility program (tsbf) (anderson & ingram, 1989), as modified by silva and bandeira (1999). blocks of soil (20 cm x 20 cm x 30 cm: 12 liters) were extracted at each collection point and divided into three strata: a (0-10 cm), b (10-20 cm), and c (20-30 cm), resulting in 45 samples each season (90 for each forest type). the three different soil strata were separated and broken up in plastic trays, and the termites encountered were manually collected and preserved in 75% alcohol. the specimens were removed to the laboratory to be counted and weighed (fresh weight in g.m-²). the specimens were subsequently deposited in the permanent collection of the termite laboratory at the federal university of paraiba. the abundance and biomass data were log (x+1) transformed to guarantee the normality and homogeneity of variance. the t-test was performed to compare abundance and biomass of the dry tropical forest and humid highland forest sites. analysis of variance (factorial anova), with the a posteriori tukey test, was performed to determine any significant differences between the seasons and between the different strata within each study area. the statistical tests were performed using statistic 5.0 software (statsoft, 1995). results twenty-two termite species were encountered overall, belonging to 13 genera comprising 20 species of the family termitidae and distributed among the subfamilies apicotermitinae (7 species), nasutitermitinae (6), syntermitinae (3), and termitinae (3), and two species of the family rhinotermitidae. only diversitermes diversimilis (silvestri) and amitermes amifer silvestri were encountered in both forest types (table 1). abundance (t test = 6,7; df = 178; p < 0.001 ) and biomass (t test = 5,7; df = 178; p < 0.001 ) were significantly higher in the humid highland forest. the fazenda almas site (seasonally dry tropical forest) yielded 10 termite species distributed among seven genera, of which eight species were encountered during the rainy season and seven during the dry season. the species anoplotermes sp. 3, grigiotermes sp. 2, ruptitermes silvestrii (emerson), and one unidentified species (of the family termitidae), were only encountered in the soil during the rainy season. constrictotermes cyphergaster (silvestri) and heterotermes sulcatus mathews, on the other hand, were only encountered during the dry season. amitermes amifer was predominant during both seasons in terms of both its abundance and biomass vfp araújo, mp silva, a vasconcellos – soil termites in the brazilian semiarid domain72 (table 1). during the rainy season, the mean abundance and biomass of the termites was 862 ± 532 individuals.m-² and 1.40 ± 1.02 g.m-² (fresh weight), respectively, while during the dry season these values were 250 ± 195 individuals.m-² and 0.45 ± 0.38 g.m-² (fresh weight) respectively. nonetheless, termite abundance and biomass did not differ significantly between the two seasons in this vegetation type (f1.84=1.91; p=0.17 and f1.84=0.93; p=0.33 respectively), nor between the different soil levels (f2.84=1.06; p=0.35 and f2.84=0.65; p=0.52 respectively). the same results were encountered when considering the interactive effects of the seasons and the soil strata on the termite fauna (f2.84=0.87; p=0.41 and f2.84=0.07; p=0.92 respectively) (fig 1). the mata do pau ferro site (humid highland forests) yielded 14 termite species distributed among 11 genera, of which nine were encountered during the rainy season and 12 in the dry season. nasutitermes jaraguae (holmgren) and neocapritermes sp. were encountered only during the rainy season, while five species were only observed during the dry season (diversitermes diversimiles, embiratermes neotenicus (holmgren), ibitermes inflatus vasconcellos, subulitermes microsoma (silvestri), and amitermes amifer). the termite species grigiotermes sp. 1 and embiratermes parvirostris constantino were predominant during both seasons, in terms of both their abundance and biomass (table 1). a total of 6080 ± 1477 individuals.m-² and 18.53 ± 6.18 g.m-² (fresh taxa mata do pau ferro fazenda almas abundance biomass layer abundance biomass layer feeding groupsrainy dry rainy dry rainy dry rainy dry termitidae apicotermitinae anoplotermes sp 1 234 189 0.44 0.36 a,b,c s anoplotermes sp.2 331 243 0.62 0.46 a,b,c s anoplotermes sp.3 6 0.01 b s grigiotermes sp. 1 1044 250 4.18 1.00 a,b,c s grigiotermes sp. 2 5 0.01 a s ruptitermes reconditus (silvestri) 332 38 1.23 0.14 b,c l ruptitermes silvestrii (emerson) 5 0.02 a l nasutitermitinae constrictotermes cyphergaster (silvestri) 9 0.02 a,b w diversitermes diversimiles (silvestri) 50 0.10 a 4 4 0.00 0.00 a,c w/l nasutitermes callimorphus mathews 226 23 0.58 0.06 a w nasutitermes jaraguae (snyder) 105 0.30 a,b w subulitermes microsoma (silvestri) 28 0.04 a s velocitermes sp. 306 2 0.38 0.00 a l syntermitinae embiratermes neotenicus (holmgren) 10 0.04 a,b w/s embiratermes parvirostris constantino 989 242 1.56 0.38 a,b,c s ibitermes inflatus vasconcellos 224 1.36 a,b s termitinae amitermes amifer silvestri 30 0.09 a,b 314 118 0.61 0.23 a,b,c w/s amitermes nordestinus melo and fontes 23 2 0.02 0.00 b,c w/s neocapritermes sp. 81 0.34 a,b s termitidae sp. 7 0.01 a rhinotermitidae heterotermes longiceps (snyder) 68 2 0.07 0.00 a,b w heterotermes sulcatus mathews 15 0.02 c w total 3648 1329 11.12 2.54 432 150 0.75 0.27 feeding groups: w= wood-feeders; s= soil-feeders; l= leaf-feeders; w/s= wood-soil interface feeders; w/l= wood-leaf interface feeder table 1 abundances (individuals.m-²) and biomasses (g fresh weight.m-²) of termites in two forest types within the brazilian semiarid domain: humid highland forest (“brejo de altitude” mata do pau ferro) and seasonally dry tropical forest (“caatinga” fazenda almas). sociobiology 62(1): 70-75 (march, 2015) 73 weight) were encountered during the rainy season, while 2215 ± 1099 individuals.m-² and 5.45 ± 3.04 g.m-² (fresh weight) were found during the dry season. termite abundance and biomass were both significantly greater during the rainy season (f1,84=7.21; p<0.01 and f1.84= 8.35; p<0.01 respectively), and vertical stratification was also identified (f2.84= 7.04; p<0.01 and f2.84= 3.13; p<0.05 respectively) (fig 1). however, when considering the interactive effects of the season and the soil strata on the termite fauna, it was observed that their abundances and biomasses did not vary significantly (f2.84=0.73; p=0.41 and f2.84=0.07; p=0.92 respectively). fig 1. biomasses (biomass density, g.m-2) and abundances (numerical density, individuals.m-2) of termites in different soil strata (a: 0-10 cm; b: 10-20 cm; c: 20-30 cm), in two forest types in the semiarid domain of northeastern brazil: seasonally dry tropical forest (“caatinga” fazenda almas) and thumid highland forest (“brejo de altitude” mata do pau ferro). discussion although they are both located within the brazilian semiarid domain, the dry tropical forest and humid highland forest sites demonstrate distinct climatic characteristics and consequently very little similarity between their termite species (two species), abundances, and biomasses. arthropods occupying the leaf litter/soil complex are generally influenced by the climatic characteristics of the host ecosystems (adis et al., 1989; harada & bandeira, 1994; dibog et al., 1998; pinheiro et al., 2002; doblas-miranda et al., 2007). the assemblage structure and foraging of termites in the neotropical region have been observed to be associated with climatic elements, especially temperature and rainfall regime, both in humid forests (torres & bandeira, 1985; cancello et al., 2014) and in semiarid ecosystems (moura et al., 2006; araújo et al., 2010), yet the humid forest sites show higher rates of biomass and abundance of termites. termite abundance in the soil at the dry tropical forests site was 862 individuals.m-2 during the rainy season and 250 individuals.m-2 during the dry season, while their biomass was 1.40 g.m-2 and 0.45 g.m-2 respectively. these values were larger than those reported by melo and bandeira (2004) for a number of microhabitats (soil, leaf litter, nests and wood) in another area of seasonally dry tropical forest experiencing anthropogenic pressure. in a study undertaken at the dry tropical forests site that considered only nest populations, vasconcellos et al. (2007) recorded abundance and biomass values of 278.2 individuals.m-2 and 0.9 g.m-2, respectively, for c. cyphergaster. by combining these values, the abundances and biomasses of termites in this seasonally dry tropical forest area appeared to oscillate between 428 and 610 individuals.m-2 and between 1.17 and 1.65 g.m-2 respectively. it is important to note that these values do not include other termite species that construct conspicuous nests in the area, or those that forage within tree trunks. the abundance and biomass of termites found only in the soil in the humid highland forest site were larger than those previously recorded in other ecosystems in the neotropical region (martius, 1994; bandeira & vasconcellos, 2002; vasconcellos, 2010). other quantitative methods, including mounds and arboreal nests, would be likely to increase abundance and biomass data. specifically in terms of the soil, the largest values recorded to the present time were 4331.54 individuals.m-2 and 7.65 g.m-2 fresh weights in an atlantic forest fragment in northeastern brazil (vasconcellos, 2010). even including estimated values for other microhabitats (such as nests and decomposing wood), termite populations in humid highland forest were still the highest so far reported for the neotropical region (bandeira & torres, 1985; martius, 1994; bandeira & harada, 1998; silva & bandeira, 1999; bignell & eggleton, 2000). the abundances and biomasses of termites at the dry tropical forests site did not demonstrate significant variations between the dry and rainy seasons. other studies undertaken in seasonally dry tropical forests in the region, however, demonstrate an effect on vertical distribution, foraging and abundance of insects in general (moura et al., 2006; araújo et al., 2010; vasconcellos et al., 2010a), including termites (melo & bandeira, 2004; vasconcellos et al., 2010b). apparently, other characteristics, such as food availability, soil moisture, and interspecific interactions, could minimize climatic effects on soil fauna in seasonally dry tropical forest ecosystems. the humid highland forest site, on vfp araújo, mp silva, a vasconcellos – soil termites in the brazilian semiarid domain74 the other hand, demonstrated significant variations in its termite population parameters between the dry and rainy seasons. although there have been very few publications focusing on seasonal variations of the termite fauna in humid tropical forests, eggleton and bignell (1995) suggested that abundance and biomass values in these ecosystems were greater in the rainy season. bandeira and harada (1998), however, reported higher values for these same parameters among soil termites during the dry season in the amazon forest. in relation to the depth of the soil samples, it was observed that the greatest abundances and biomasses were encountered in the top 20 cm profiles, independent of the forest type (walwork, 1970). according to peterson and luxton (1982), most of the edaphic biota is concentrated between 0 and 10 cm below the soil surface. this tendency does vary considerably, however, in different localities and when different taxa are considered (walwork, 1970; adis et al., 1989; dindal, 1990; harada & bandeira, 1994). a number of studies in the neotropical region have found that a 20 cm soil depth is the most adequate for collecting termites, especially in terms of species richness (vasconcellos, 2010; bandeira & vasconcellos, 2002; silva & bandeira, 1999). the observed increase in termite numbers and biomass during the rainy season in the humid highland forest indicates that their decomposition activities are asymmetrical during the year. the quantitative similarity of termite numbers and biomass during the year in the seasonally dry tropical forest area suggests that other factors, beyond climatic differences, influence the abundance and biomass of these termites, at least at a local scale. the quantitative differences observed between the two forest types reflect the effects of different environmental variables on ecosystems, in spite of the fact that they are both embedded in the semiarid brazilian domain. acknowledgements the authors are grateful to the braz family, owners of the rppn-fazenda almas, for logistical support during the research, and to nilton leite de sousa júnior for their input. this research was supported by cnpq/peld (520062/2006-0). references adis, j., ribeiro, e.f., morais, j.w. & cavalcante, e.t.s. 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(1978). the role of termites in ecosystems. in: m.v. brian (ed.), production ecology of ants and termites (pp. 245–292). cambridge: cambridge university press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4552sociobiology 67(1): 33-40 (march, 2020) introduction niche partitioning has often been used to explain ant species coexistence as well as ant community assemblages (blüthgen & feldhaar, 2010; camarota et al., 2016). however, the high number of ant species at the local scale (e.g., 86 species in 0.13 ha in northwestern australia and 30 species in 1 m2 of leaf litter in atlantic forest, brazil) (andersen, 1983; silva & brandão, 2010) has led ant ecologists to question the role of interspecific competition in ant species coexistence and propose alternative mechanisms for community ant assemblages (ribas & schoereder, 2002; cerdá et al., 2013). andersen (2008) proposed alternative explanations for the high diversity of ants at the local scale, based on sociability and modularity of their nests, which protect species abstract in this study, we investigated the mechanisms behind species coexistence and the relationships between functional diversity and species richness in ant assemblages in both forest and pasture habitats in the southwestern brazilian amazon. we addressed the specific question: what is the primary mechanism for species coexistence in forest and pasture habitats? according to the identified mechanism in each habitat, we had the following alternative expectations: (i) niche partitioning – we expected to observe a positive linear relationship between functional diversity and species richness, indicating a complementary relationship; or (ii) niche filtering – a positive constant asymptotic relation between functional diversity and species richness, indicating a functional redundant relationship. in total, we sampled 91 ant species, 82 species in a forest habitat and 16, in a pasture habitat. in the forest habitat, we identified niche filtering as the structuring mechanism of the ant assemblage, but we were unable to identify a clear mechanism in the pasture habitat. although the relationship between functional diversity and species richness was positive in both habitats, the relationship was weaker in the forest habitat, indicating a greater functional redundancy among the ant species in this habitat. our results reinforce the divergence of species coexistence mechanisms and ant assemblage structures in both natural and humanmodified habitats in the southwestern brazilian amazon. sociobiology an international journal on social insects as menezes1, fa schmidt1,2 article history edited by frederico neves, ufmg, brazil received 06 june 2019 initial acceptance 18 september 2019 final acceptance 19 december 2019 publication date 18 april 2020 keywords co-occurrence, morphological traits, niche, formicidae, resources. corresponding author andressa silvana oliveira de menezes universidade federal do acre campus br 364 km 04 cep: 69920-900, rio branco-ac, brasil. e-mail: andressasilvana@gmail.com against environmental harm (hölldobler & wilson, 2009). additionally, competitive interactions between species are highly variable depending on both temporal and spatial environmental variations. the dominance of resources and space by dominant ant species occurs in patches, which makes it possible to use these resources by subordinate species (andersen, 2008). thus, ant species could overcome the negative effects of interspecific competition. in this way, recent studies have suggested that niche filtering (similarities regarding the use of the resource) is the mechanism responsible for the coexistence of species in ant assemblages (arnan et al., 2011; fowler et al., 2013). ants are responsible for various functions in terrestrial ecosystems, which can be demonstrated by their morphological diversity that reflect their life histories such 1 universidade federal do acre, programa de pós-graduação em ecologia e manejo de recursos naturais. rio branco-ac, brazil 2 universidade federal do acre, centro de ciências biológicas e da natureza. rio branco-ac, brazil research article ants mechanisms of species coexistence and functional diversity of ant assemblages in forest and pasture habitats in southwestern brazilian amazon as menezes, fa schmidt – species coexistence and functional diversity of ant assemblages in the southwestern brazilian amazon34 as resource use, habitat preference, and foraging strategies (kaspari & weiser, 1999; silva & brandão, 2010; silva & brandão, 2014; schofield et al., 2016). thus, several studies have focused on functional diversity in ant assemblages based on the measurement of functional traits (bihn et al., 2010; arnan et al., 2014; silva & brandão, 2014; schofield et al., 2016; martello et al., 2018). given that niche partitioning promotes species coexistence that differ in the use of limiting resources and that niche filtering promotes the coexistence of species with similar niches, we can deduce specific expectations about the relationship between functional diversity and species richness in ant assemblages. first, in ant assemblages structured by niche partitioning, we expect there to be a linear and positive relationship between functional diversity and species richness. second, for ant assemblage structured by niche filtering, we expect the relationship between functional diversity and species richness to be positive, but when the relationship levels off, it achieves a constant asymptotic relationship. this has been interpreted to be a functionally redundant relationship (naeem et al., 2002). the selective action of the range of physical conditions, resources, and ecological interactions on the species set that make up an ecological community is reflected in the morphology and physiology of the species and on the assembly’s functional diversity (schofield et al., 2016). thus, we expect that ant species living in different environments will have distinct morphologies and physiologies, and consequently will exhibit different patterns of functional diversity (wiesher et al., 2012). besides forest habitats, the amazon region, due to conversion and fragmentation processes, contains large area of pastures. pastures are very different from forest habitats, specifically in relation to the abiotic conditions and resources available such as land cover, leaf litter soil cover, moisture, and biodiversity (fearnside, 2005; imazon, 2010; araújo et al., 2011). this is the case in the southwestern brazilian amazon, where ant assemblages in both forest and pasture habitats present very distinctive patterns in both fauna and species diversity (oliveira & schmidt, 2019). in this study, our main aim is to understand the mechanism (niche partitioning or niche filtering) for ant species coexistence and the implications of this mechanism for the relationship between functional diversity and species richness in two contrasting habitats (forest and pasture) in the southwestern brazilian amazon. therefore, we addressed the main question: what is the primary mechanism for species coexistence in forest and pasture habitats? according to the mechanism identified for each habitat, we had the following alternative expectations: (i) niche partitioning – we expected to observe a positive and linear relationship between functional diversity and the species richness, indicating a complementarity relationship; or (ii) niche filtering an asymptotic relationship between functional diversity and species richness, indicating a functional redundant relationship. material and methods study area the study was carried out in a forest fragment inside fazenda experimental catuaba (fec/ ufac, 10°04′s e 67°37′w) and in an adjacent pasture (fig 1). fec/ufac is 27 km away from rio branco, the capital of acre state. the size of the forest fragment is 1,200 ha, with vegetation of open rainforest with palm trees, bamboos and vines (medeiros et al., 2013). the surrounding area of fec/ufac is composed of pasture areas with exotic grasses and sparse remnants of palm and brazil nut trees (araújo & lani, 2012). sample design and ant identification ant sampling was carried out during the dry season (cemaden, 2016), from july to august 2016, when the average rainfall was 50 mm (inmet, 2015). in each habitat (forest and pasture), we employed 20 plots of 10 x 12 m, 60 meters apart from each other. the plots in the forest habitat were placed along the permanent sampling transect of biodiversity research program module (ppbio), while the plots distributed in the pasture habitat were 500 meters from the edge of the forest fragment (fig 1). in each plot, we offered five types of liquid food resources to the ants: 10 ml with h2o (distilled water), 20% amino acid (unflavored whey protein isolate), 20% carbohydrate (crystal sugar), 20% lipid (extra virgin olive oil), 1 % nacl, and dry cotton (control). we offered the resources to the ants using a cotton swab soaked in each type of resource and inserted in a 50 ml plastic centrifuge tube. each tube was arranged horizontally on the soil surface. we used liquid resources because observed patterns of resource use were not affected by the texture, shape or size of the resource, but depended only on the type of resource (fowler et al., 2013; kaspari et al., 2008). in each plot, we used five replicates of each resource type and control, creating a total of 30 centrifuge tubes. in each plot, we established transect lines every 2 m resulting in 30 subplots. at the vertices of each subplot, we randomly placed one of the 30 tubes with resources. we stored the resources in a styrofoam box (8 l) in order to avoid evaporation of the solution during the experiment. we placed the tubes at 08:30 a.m. and collected them after 3 hours of exposure (fowler et al., 2013). we transferred all ants present in the centrifuge tubes to 5 ml plastic tubes containing 96% alcohol, and subsequently separated, mounted and identified the ants in the laboratory. we conducted ant identification at the genus level using the taxonomic keys sourced from baccaro et al. (2015) and later sorted the ants into morpho-species. we identified ants at the species level by comparing our ants with specimens from the ant collection of the insect ecology laboratory of federal university of acre and with specimens from the ant collection of the ant systematic and biology laboratory of sociobiology 67(1): 33-40 (march, 2020) 35 federal university of paraná under the support provided by prof. dr. rodrigo feitosa and his research group. we deposited all voucher ant specimens in the ant collection of the insect ecology laboratory at ufac. statistical analysis we performed all analyses using the programming software r v. 3.2.2 (r development core team, 2015). we used specific packages as needed, which we describe below. what is the primary mechanism for species coexistence in forest and pasture habitats? to discern whether ant species coexistence in each habitat type is determined by niche partitioning or filtering, we tested whether niche overlap was greater among species that rarely co-occur or among species that frequently co-occur. for this, we used two matrices: one for niche overlap and one for species co-occurrence. in the niche overlap matrix, columns corresponded to the different types of resources offered (6), and the lines listed the species. the input values in the matrix recorded the ant species occurrence (number of tubes) in each resource category (n = 6). afterward, we ran a null model analysis using the ecosimr r package (gotelli et al., 2015). we compared the mean of the observed niche overlap to the expected mean of niche overlap, as obtained from 1000 randomized trials. thus, we tested the null hypothesis that the average of the observed niche overlap is lower than the average of the expected niche overlap at random (overlap below 5%) (gotelli & entsminger, 2004). we employed pianka’s index to obtain the niche overlap in resource use among paired species (pianka, 1973): fig 1. catuaba experimental farm ufac and são francisco farm, with 20 plots in each habitat type (forest and pasture), in the southwestern brazilian amazon. , where, pij and pik represent the proportion of resource i in relation to the total resources used by species j and species k. this index estimates the symmetric overlap of the categories (by habitat and resource) for each pair of species whose values range from 0 (no overlap) to 1 (total overlap) (pianka, 1973). we then compared the observed overlap index to the values obtained by the randomization of the original matrix (1,000) using the ra3 algorithm, that retains the observed niche width and randomizes the allocation of each use value for each different niche category (winemiller & pianka, 1990). we also constructed a species co-occurrence matrix for each habitat type (forest and pasture), calculating the number of combinations arranged in checkerboard space as menezes, fa schmidt – species coexistence and functional diversity of ant assemblages in the southwestern brazilian amazon36 (checkerboard units – cu) of a presence-and-absence matrix (gotelli, 2000), in the following way: cu = (ri – s) (rj – s), where s corresponds to the number of shared sites (containing both species), and ri and rj are the sum of the lines for species i and j. a higher c-score indicates that the species pair is more segregated with fewer shared sites (stone & roberts, 1990). we obtained the c-score index using the bipartite rpackage (dormann et al., 2008). next, for each species pair, we obtained the relationship between co-occurrence and niche overlap by applying a mantel’s test (douglas & endler, 1982), available in the vegan r-package (oksanen et al., 2017). we compared the observed value of the correlation coefficient (r) to the values obtained from 1,000 randomizations, using a 5% significance threshold. negative values of (r) indicate that species with similar niches are less likely to co-occur, suggesting that niche partitioning is the mechanism responsible for the coexistence of species in ant assemblages for each habitat type. on the other hand, positive values of (r) indicate that species with similar niches are more likely to co-occur, suggesting that niche filtering is the determinant factor of species coexistence in ant assemblages in each habitat type (fowler et al., 2013). alternative expectations for the relationship between functional diversity and species richness to evaluate the relationship between functional diversity and species richness, we measured eight morphological structures based on ant resource use and habitat preferences. according to silva and brandão (2010, 2014), trait indicators include: (i) weber length (wl) a body size indicator; (ii) head width (hw) based on the size of the mandible musculature; (iii) eye length (el) as important to search for food; (iv) interocular distance (id) since generalist species tend to have more distant eyes, positioned laterally; (v) posterior femoral length (fl) assuming that leg size can determine species distributions in leaf litter; (vi) scape length (sl) an important behavioral function; (vii) mandible length (ml) related to resource size; and (viii) clypeus length (cl) since species that depend on liquid resources tend to have a more developed clypeus. we conducted all measurements with a leica s8 apo stereomicroscope and using a micrometer lens. we used up to six workers per species (when available), to measure each morphological trait and obtain an average. however, for species with less than six workers sampled, we measured the number of workers available (from 1 to 5) (silva & brandão, 2010; martello et al., 2018). for polymorphic species, we only measured the smaller workers (del toro et al., 2015). we used an abundance matrix for each habitat (forest and pasture), where the lines corresponded to the plots, the columns indicated ant species identities, and the input values referred to the number of tubes that the species of ants visited in each plot. we also constructed a second matrix, which contained the traits in the columns and the species in the rows. after obtaining the averages for each morphological trait, we standardized the traits by dividing each by the weber’s length to reduce correlations with body size (martello et al., 2018). we transformed the distance measure between the eyes into a position measurement (hw-id) and then standardized the wl. we used the syncsa r-package (debastiani, 2018) to calculate the functional diversity index, using the rao. diversity function (rao’s quadratic entropy), based on a gower dissimilarity matrix calculated for rational values traits (pavoine et al., 2009) and equally weighted (swenson, 2014). this function calculates the square root of the onecomplement gower’s similarity index in order to keep the dissimilarity matrix with euclidean metric properties (debastiani, 2018). we used the rao quadratic diversity index as a metric to aggregate species abundance data and the functional differences between them (arnan et al., 2014). rao’s q varies between 0 and 1, where higher values indicate greater dissimilarity in community traits (arnan et al., 2014). we verified the relationship between species richness (explanatory variable) and functional diversity (response variable) through a simple linear model. in this model, we interpreted a high slope coefficient to be indicative of the niche partition expectation leading to a functional complementarity between species and interpreted a low slope coefficient as indicative of niche filtering expectation leading to functional redundancy. results ant fauna in total, 91 ant species were collected, belonging to 24 genera and distributed into six subfamilies. of all the subfamilies, only ponerinae was not recorded in pasture habitat. myrmicinae contained the highest number of species (61 species), followed by formicinae (17 species), dolichoderinae and ectatomminae (both with four species), ponerinae (three species), and pseudomyrmecinae (two species) (see table s1 at http://periodicos.uefs.br/index. php/sociobiology/rt/suppfiles/4552/0). the genera with the highest number of species was pheidole (28 species), followed by camponotus and crematogaster (both with 12 species), and solenopsis (eight species). in the forest habitat, 82 species of ants visited the centrifuge tubes, 75 of which were exclusive to this habitat. in the pasture habitat, only 16 species were recorded, of which only 9 were exclusive to it. only seven species of ants were recorded in both habitats: crematogaster tenuicula forel, dolichoderus inermis mackay, ectatomma brunneum smith, solenopsis invicta buren, s. saevissima (smith), s. sp. 2 and wasmannia auropunctata (roger). what is the primary mechanism for species coexistence in forest and pasture habitats? by employing pianka’s index and the c-score index, sociobiology 67(1): 33-40 (march, 2020) 37 we verified that the niche overlap is greater among species that frequently co-occur than those that do not frequently co-occur. in the results for paired ant species in the pasture habitat, many species did not have a niche overlap for resource use. in the forest habitat, many paired ant species overlapped, such as acromyrmex coronatus x pheidole sp. 17 and camponotus heathi x pheidole gr. dilligens sp. 3, with an overlap of (> 0.70), but there were other species such as camponotus heathi x pheidole biconstricta or pachycondyla crassenoda x pheidole sp. 16 that had a low overlap. the c-score index was between 0.001 to 0.05 in the pasture and 0.0014 to 0.05 in the forest, indicating that the co-occurrence between paired species is relatively high. the correlation between co-occurrence and niche overlap in the pasture was not significant (r = 0.047 and p = 0.308), while in the forest the correlation was positive and significant (r = 0.147 and p = 0.001) (fig 2). than in the forest habitat. therefore, in the pasture habitat there is a greater increase in functional diversity (rao’s q) with increasing of number of ant species than in the forest habitat. discussion our study was restricted by the timing of the sampling period (mornings) and the use of attractive baits, which can restrict the ant fauna, possibly leading to more limited perspective on ant species coexistence and functional diversity. however, our results are consistent with the findings of other studies on species coexistence (ribas & schoereder, 2002; cerdá et al., 2013) and niche partition versus niche filtering (fowler et al., 2013) in ant assemblages. despite these considerations, we demonstrate that in forest habitats (and not in pastures) the mechanism responsible for ant species coexistence is niche filtering. furthermore, the relationships between functional diversity and species richness are very different in forest vs. pasture ant assemblages. in the sections below, we offer possible explanations for these results and discuss their implications to ant assemblage structures in both habitat types. what is the primary mechanism for species coexistence in forest and pasture habitats? in the forest habitat, we identified niche filtering as the process responsible for the coexistence of ant assemblage species. still, ant assemblages in this habitat have similarities in resource use with high niche overlap, as co-occurring species can survive and remain in the assemblage. recent studies have indicated the importance of environmental filters for ant assemblages as an alternative explanation for the niche partitioning and the possible coexistence of species that have similar niches (wiescher et al., 2012; fowler et al., 2013). fig 2. relationship between niche overlap (pianka index) and spatial co-occurrence (c-score index), (p = 0.001, r = 0.147) for pairs of ant species sampled in the forest fragment at catuaba experimental farmufac, ac, brazil. alternative expectations for the relationship between functional diversity and species richness the species of ants in the pasture habitat had, on average, smaller morphological traits (except eye length) than those of ants in the forest environment (table s2 at http://periodicos. uefs.br/index.php/sociobiology/rt/suppfiles/4552/0). the relationship between functional diversity and species richness was positive in both habitats (forest and pasture). however, the slope of the relationship between functional diversity and species richness differed in each environment (fig 3). in the forest habitat, the relationship between species richness and functional diversity had a low slope, highlighting that an increase in ant species richness among plots is not tightly associated with an increase in functional diversity (rao’s q). in the pasture habitat, the coefficient of variation of the relationship between ant species richness and functional diversity was higher fig 3. relationship between functional diversity (rao’s q) and observed species richness in the plots of each habitat type (forest: intercept value = 0.29927, slope value = 0.00304 and pasture: intercept value = 0.05334, slope value = 0.02929), in southwestern brazilian amazon. as menezes, fa schmidt – species coexistence and functional diversity of ant assemblages in the southwestern brazilian amazon38 wiescher et al. (2012) reported that ant assemblages are governed by environmental conditions, and that adaptations to these conditions determine differences in the morphology and physiology of ant species. the modularity of ant nests, as well as ant sociality, protects them from possible adverse events since the caste division causes the queen to be fully protected in the nest, making her immune to external disturbances (andersen, 2008; hölldobler & wilson, 2009). in addition, species competition may cause ants to have different strategies regarding the use of similar resources that allow their coexistence in space, as well as at sites that dominant ants do not occupy (andersen, 2008). in the pasture habitat, we did not find a significant correlation between spatial co-occurrence and niche overlap. compared to forest habitat, we found few species of ants visiting the liquid resource tubes (16 species only). therefore, it is still unclear whether niche partitioning or filtering are the predominant structures for ant species in pasture habitat. our spatial co-occurrence data show that these few species are highly co-occurring and therefore they are not segregated in space. alternative expectations for the relationship between functional diversity and species richness functional redundancy is related to the stability of ecological communities (naeem et al., 2002), since high levels could either allow for ecosystem recovery or for random extinctions or disturbances when species loss does not translate to a loss in ecosystem functioning (naeem et al., 1995; fonseca & ganade, 2001; pillar et al., 2013; arnan et al., 2019). differences in the relationship between species richness and functional diversity by habitat type shows that functional redundancy is potentially higher in forest habitat. furthermore, the forest-pasture shifting to pasture not only has an effect on species richness but also on species functional role. therefore, we found that the high diversity of ant species in tropical forests leads to high functional redundancy for ant assemblages. as summarized above, in addition to being an important factor in the maintenance of ecological functioning, functional redundancy also indicates the existence of environmental filters in ecological communities (del toro et al., 2015). conclusion this study fills a gap in the research on the mechanisms behind ant species coexistence in forest and pasture habitats, contributing to knowledge on the relationship between functional diversity and ant species richness. in the forest habitat, niche filtering emerged as a key mechanism responsible for ant species coexistence. land cover change, such as forestpasture shifting in southwestern brazilian amazon, has been a serious environmental problem. in this study, we demonstrated that species loss led to a lack of functional redundancy, which is a key variable for managing ecosystem stability. however, for a more accurate understanding of the mechanism responsible for ant species coexistence, it is necessary to further analyze which environmental variables could act as ecological selective filters on ant species from regional pools to make up species set of ant assemblages at local scales. acknowledgments we thank to all the colleagues from ufac who helped in the field. we are grateful to r. feitosa and his team for identifying ant species. we thank e. morato, r. solar, r. silva and sendoya, s. and referees for their comments on previous manuscript versions. we are grateful to r. silva and f. martello for their support on statistical analyses, specifically regarding functional diversity. we are in debt with r. benzeev and m. báez for their kind review on english writing in the manuscript.this article is the result of the master’s thesis of the first author, which had financial support and grant from coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. references andersen, a.n. 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(1990). organization in natural assemblages of desert lizards and tropical fishes. ecological monographs, 60: 27-55. doi: 10.2307/1943025 doi: 10.13102/sociobiology.v62i1.88-91sociobiology 62(1): 88-91 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ambient temperature influences geographic changes in nest and colony size of polistes chinensis antennalis pérez (hymenoptera: vespidae) introduction climate is a major physical factor that determines the geological distributions and life histories of organisms. some climates generate different ecological traits among populations of the same species (e.g., angilletta, 2009). assessing adaptations to different climatic regimes helps us to understand the major selective pressures affecting organisms and to predict their reactions to global and local climatic changes in the environment (chown, 2001). in social insects, ambient temperature is an important physical factor that greatly affects insect activity (heinrich, 1993). differences in local ambient temperatures lead to differences in nesting activities (fucini et al., 2014), rate of development of immature stages (miyano, 1981), and duration of nesting periods (yamane, 1969), and intra-specific changes have been reported in the genus polistes (hymenoptera, vespidae). polistes paper wasps are independent-founding social wasps. a lone foundress builds an exposed nest and nurses the first brood of larvae until worker individuals emerge (pre-emergence period; reeve, 1991). after eclosion (post-emergence period), the adult individuals increase abstract in some polistes wasps, the foundresses build huge nests during the founding phase to improve the thermal condition of these nests. this implies that polistes wasps change their nesting manner in relation to ambient temperature. to test the hypothesis that nest size increases with latitude, colonies of polistes chinensis were collected from 11 locations. three nest parameters, the number of cells cell length and index of functional envelope, increased with latitude. the number of cells at the northernmost station was 60, which was 1.5 times more than in lower latitudes. cell length increased by approximately 4 mm from low to high latitudes, indicating that extra-building in p. chinensis is remarkable in adding new cells. the number of first broods was not correlated with latitude, whereas the number of second brood increased with latitude because of the numerous cells built at high latitudes. sociobiology an international journal on social insects s hozumi1, k kudô2, h katakura3, s yamane4 article history edited by fernando barbosa noll, unesp, brazil received 07 march 2014 initial acceptance 16 may 2014 final acceptance 13 september 2014 keywords paper wasp, polistes chinensis, geographic variation, nest architecture. corresponding author satoshi hozumi chigakukan secondary school mito 310-0914, japan e-mail: shoz@tokiwa.ac.jp colony production and produce as many reproductive gynes as possible (reproduction period). the wasps build an exposed comb, in which the construction of new cells and elongation of cells are commonly related to oviposition and the development of larvae, respectively (delaeurance’s law; delaeurance, 1957). however, species in different climates exhibit different nest-building activities and colony cycles. yamane (1972) showed that p. chinensis foundresses that inhabit the cool northern areas of japan (nesting duration 3.5–4 months) build many empty cells just before the emergence of the first adults and elongate the cells beyond the lengths of the pupae. in contrast, in the warm areas of japan, p. chinensis nesting activity is consistent with delaeurance’s law, and the nesting duration is approximately 6 months. a few previous studies have focused on the intra-specific changes in nest size, in relation to nest thermoregulation; i.e., adaptation to low temperature. yamane & kawamichi (1975) found that p. chinensis foundresses build larger nests in higher latitudinal regions and concluded that numerous and long cells function as air chambers that improve thermal conditions in the brood-rearing area of the nest, similar to the envelope of a vespine nest (functional envelope). this means research article wasps 1 chigakukan secondary school, mito 310-0914, japan 2 niigata university, niigata 950–2181, japan 3 hokkaido university, sapporo 060–0810, japan 4 ibaraki university, mito 310-8511, japan sociobiology 62(1): 88-91 (march, 2015) 89 that foundresses build more cells for nest thermoregulation to accelerate the development of the first batch of larvae (first brood) during the pre-emergence period. however, the observations were performed in only 2 locations, and thus further study is needed to understand the relationships between climate and the geographic variation in nest size. in this study, we set 2 goals. the first was to verify whether nest sizes increase geographically from low to high latitudes as yamane and kawamichi (1975) predicted. if p. chinensis foundresses change their nesting behavior according to ambient temperature, the number and the length of cells should increase with latitude. we collected p. chinensis nests at 11 locations across japan islands and examined the relationship between nest size and latitude. the second goal was to clarify the relationship between latitude and colony productivity. if foundresses invest more resources in nest building in higher latitudinal regions, the numbers of individuals in the first and second broods would be expected to decrease as a tradeoff. we investigated the numbers of individuals in first broods at 6 locations along a latitudinal gradient and calculated an index of functional envelope. in this study, we also discuss the difference in nesting behavior between lower and higher latitudinal areas. material and methods nest parameters in order to determine the latitudinal differences in nest size among the islands of japan, a total of 168 nests of p. chinensis antennalis were collected at the 11 locations shown in table 1 and fig. 1. two nest parameters, the number of cells and cell length, were measured for each nest. the measurement was performed as follows: number of cells, total number of cells was counted for each nest; cell length (mm), maximum value of the longest cell was measured for each nest with a vernier calliper to the nearest 0.1 mm. colony composition colony composition was investigated at 6 locations (st4, st7, st8, st9, st10, and st11) to determine latitudinal differences among colonies. the content of each cell (egg, young larva, old larva, pupa, or empty) was recorded for all nests immediately after nest collection. for this study, 1st to 3rd instar larvae were classified as young larvae, and 4th and 5th instar larvae were classified as old larvae. to examine the hypothesis of yamane and kawamichi (1975), we defined the index of functional envelope (ife) to be the nest volume per first brood, i.e., the entire nest volume per total number of individuals in the first brood. nest volume (ml) station locality latitude longitude mean annual temperature (˚c) date collected collector st1 fukuoka 33°34’n 130°23’e 17.0 19-21 may 2000 n. kumano st2 tsu 34°43’n 136°30’e 15.9 4-5 june 1997 k. kudô st3 toki 35˚21’n 137°11’e 15.8 7-8 june 1997 k. kudô st4 itako 35°56’n 140°34’e 14.5 9-10 june 1998, 10 june 1999 s. hozumi st5 tsurugi 36°27’n 136°37’e 14.3 16-17 june 1997 k. kudô st6 kanazawa 36°33’n 136°39’e 14.6 19 june and 7-8 july 1997 k. kudô st7 tanagura 37°01’n 140°22’e 11.5 8 june 1999 s. hozumi st8 mizusawa 39°08’n 141°08’e 10.7 8 june 1998, 19 june 1999 s. hozumi st9 shichinohe 40°41’n 141°08’e 10.4 4 july 1998, 3 july 1999 s. hozumi st10 kanagi 40°54’n 140°27’e 10.3 4 july 1998, 3 july 1999 s. hozumi st11 okushiri 42°08’n 139°29’e 9.7 21 july 1999, 18 july 2000 s. hozumi table 1. geographic positions and altitudes of localities where polistes chinesis nests were collected. fig 1. localities where nests of p. chinensis were collected. s hozumi, k kudô, h katakura, s yamane geographic changes in nest and colony size of polistes chinensis90 colony composition table 3 shows the colony composition. the number of individuals in the second brood and the number of empty cells increased significantly with latitude. the number of individuals in the first brood tended to decrease with latitude; however, there was no significant correlation. nest volume and ife increased significantly with latitude. was estimated as follows: all of the cells were filled with small granular glass beads (diameter 0.1 mm), and the volume of beads was measured with a graduated cylinder to the nearest 0.1 ml (see yamane et al., 1998). this measurement was repeated 3 times, and the values were averaged. statistics spearman’s rank correlation analysis was used to quantify the relationship between nest and colonial parameters and latitude. all analyses were performed with ezr ver. 1.22 (kanda, 2013) on a personal computer. results nest size variation table 2 shows the geographical variation in the number and length of cells. both parameters significantly increased with latitude (spearman’s rank correlation: number of cells, ρ = 0.6272, p < 0.05; cell length, ρ = 0.7671, p < 0.01). the number and length of cells were similar (approximately 40 and 22 mm, respectively) from st1 to st8, and the values increased until st11. cell length also increased significantly, but the range of increase was small; the maximum difference in mean value was 3.9 mm between st10 (23.9 mm) and st1 (20.0 mm). station n number of cells cell length (mm) mean median mean median st1 5 38.8 ± 5.3 38.0 20.0 ± 1.4 19.8 st2 14 36.6 ± 5.3 38.5 22.8 ± 1.1 22.7 st3 28 33.0 ± 6.0 33.0 22.0 ± 1.6 22.1 st4 18 37.3 ± 7.6 41.0 21.6 ± 0.9 22.0 st5 10 40.5 ± 7.1 37.0 22.0 ± 1.5 22.8 st6 5 38.0 ± 4.4 38.0 22.7 ± 0.9 21.3 st7 8 39.4 ± 5.4 41.0 21.8 ± 1.7 22.2 st8 20 39.8 ± 6.6 39.0 21.4 ± 0.8 21.5 st9 19 42.9 ± 4.0 43.0 22.9 ± 1.2 23.0 st10 16 45.4 ± 6.8 43.5 23.9 ± 1.4 24.1 st11 25 60.2 ± 15.8 61.0 23.0 ± 2.1 23.5 st 4 st 7 st 8 st 9 st 10 st 11 sp ea rm an ’s ra nk co rr el ati on n =1 9 n =8 n =1 9 n =1 8 n =1 3 n =2 4 ρ si gn ifi ca nc e fi rs t br oo d 14 .6 ± 4 .4 14 .0 ± 5 .6 12 .8 ± 5 .7 14 .2 ± 5 .1 12 .6 ± 2 .3 12 .4 ± 4 .7 (1 5. 0) (1 5. 0) (1 4. 0) (1 5. 0) (1 2. 0) (1 4. 0) -0 .6 78 9 p= 0. 13 81 se co nd b ro od 19 .8 ± 5 .6 15 .9 ± 5 .3 21 .1 ± 4 .6 23 .3 ± 7 .0 23 .1 ± 8 .8 38 .5 ± 1 3. 8 (2 1. 0) (1 8. 0) (2 1. 0) (2 3. 5) (2 2. 0) (4 0. 0) 0. 84 07 p< 0. 05 em pt y ce lls 4. 1 ± 3. 4 7. 9 ± 4. 9 6. 2 ± 4. 4 4. 6 ± 3. 7 9. 1 ± 6. 8 9. 4 ± 6. 1 (3 .5 ) (6 .0 ) (5 .0 ) (6 .0 ) (8 .0 ) (1 0. 0) 0. 89 86 p< 0. 05 n es t vo lu m e 6. 1 ± 1. 2 7. 0 ± 0. 4 6. 3 ± 1. 3 7. 2 ± 1. 1 7. 2 ± 0. 4 8. 7 ± 1. 5 (6 .0 ) (7 .1 ) (6 .0 ) (7 .2 ) (7 .2 ) (8 .5 ) 0. 94 28 p< 0. 05 in de x of fu nc tio na l en ve lo pe (i fe ) 0. 46 ± 0 .1 6 0. 59 ± 0 .2 6 0. 65 ± 0 .4 5 0. 77 ± 0 .8 0 0. 59 ± 0 .1 0 0. 84 ± 0 .4 2 (0 .4 1) (0 .4 9) (0 .4 9) (0 .5 0) (0 .5 8) (0 .6 8) 1. 00 0 p< 0. 00 5 t ab le 3 . m ea n nu m be rs o f i m m at ur e in di vi du al s (m ea n ± sd ) o f p ol is te s ch in en si s co lo ni es . i nd ex o f f un ct io na l e nv el op e (i fe ) w as c al cu la te d by d iv id in g th e ne st v ol um e by th e nu m be r o f t he fi rs t b ro od . p ar en th es es in di ca te m ed ia n of e ac h va lu e. table 2. mean (mean ± sd) number of cells and cell length of polistes chinensis nests collected from 11 localities. discussion the results of this study confirmed that foundresses in higher latitudinal regions built larger nests, based on the number of cells. the increase in the ife supports the hypothesis of yamane and kawamichi (1975) that the extra cells that enclose first broods may function as thermoregulatory air chambers during the pre-emergence period. latitude is often closely correlated with ambient temperature, and low ambient temperature is believed to influence the building activities of foundresses. the building of extra cells is known to increase cell temperatures (hozumi & yamane, 2001), which accelerates the rate of development of the first brood. the construction of a large number of new cells not only increases cell temperature but also sociobiology 62(1): 88-91 (march, 2015) 91 enables the colonies to rear more individuals during the short nesting period. because cell length greatly affects cell temperature, the thermal effect of the cells could further accelerate development of the immature stages. in this study, the maximum difference in cell length among the locations was only 4 mm. however, even a 4-mm elongation may increase cell temperatures by 1°c when the nest receives solar radiation (hozumi et al., 2008). nest size increased with latitude, whereas the numbers of individuals in the first brood were similar among locations. the numbers of individuals in the first brood were also similar to those reported in other studies, including p. chinensis from other areas in japan (miyano, 1980; yamane et al., 1998) and other polistes species (p. riparius, hozumi & yamane, 2008; p. biglumis, fucini et al., 2014). these results imply that a certain number of first brood individuals (ca. 14) are reared by foundresses during the founding phase, at least in the range of the study areas. however, the numbers of individuals from the second brood and the numbers of empty cells increased significantly with latitude. the foundresses continued to build new empty cells after laying the eggs of the first brood for nest thermoregulation and then began laying eggs to produce as many individuals as possible during the short nesting period. this indicates that p. chinensis foundresses at higher latitudes used more resources for nest thermoregulation and production of second broods instead of rearing more first broods. in conclusion, p. chinensis foundresses can adapt to local conditions by altering nest-building activities and colony production, and ambient temperature may significantly influence nesting activities. the building of extra cells is also seen in p. riparius, and the building activity (elongation of cells) is influenced by the local ambient temperature (hozumi & kudô, 2012). this implies that elongation of cells may occur in p. chinensis inhabiting cooler areas. on the building activities of number and length of cells, further studies are needed to assess whether variations in p. chinensis populations are due to phenotypic flexibility in response to environmental conditions or to genetic differences. acknowledgments we thank dr. n. kumano for kindly collected p. chinensis nests at fukuoka. we thank prof. s. f. mawatari for helpful discussion and encouragement during the course of this study. references angilletta, m.j. 2009. thermal adaptation: a theoretical and empirical synthesis. oxford univ press. p. 289. doi:10.1093/ acprof:oso/9780198570875.001.1 chown, s.l. 2001. physiological variation in insects: hierarchical levels and implications. journal of insect physiology, 47: 649-660. delaeurance, e.p. 1957. contribution à létude biologique des polistes (hyménoptères, vespidés). i. l’activité de construction. annales des sciences naturelles zoologie et biologie animale, 19: 91-222. fucini, s., uboni, a. & lorenzi, m.c. (2014). geographic variation in air temperature leads to intraspecific variability in the behavior and productivity of a eusocial insect. journal of insect behavior, doi: 10.1007/s10905-013-9436-y heinrich, b. (1993). hot blooded insects. harvard university press, cambridge, massachusetts. p. 601. hozumi, s. & k, kudô. (2012). adaptive nesting tactics in a paper wasp, polistes riparius, inhabiting cold climatic regions. sociobiology, 59: 1447–1458. hozumi, s. & yamane, s. (2001). incubation ability of the functional envelope in paper wasp nests hymenoptera, vespidae, polistes): i. field measurements of nest temperature using paper models. journal of ethology, 19: 39-46. doi:10.1007/ s101640170016 hozumi, s., yamane, s. & katakura, h. (2008). building of extra cells in the nests of paper wasps (hymenoptera; vespidae; polistes) as an adaptive measure in severely cold regions. sociobology, 51: 399-414. kanda, y. (2013). investigation of the freely available easyto-use software‘ezr’ for medical statistics. bone marrow transplantation, 48: 452-458. doi: 10.1038/bmt.2012.244 miyano s. (1980). life tables of colonies and workers in a paper wasp, polistes chinensis antennalis, in central japan (hymenoptera: vespidae). population ecology, 22: 69-88. miyano, s. (1981). brood development in polistes chinensis antennalis pérez i. seasonal variation of duration of immature stages and an experiment on thermal response of egg development. bulletin of the gifu prefectural museum, 2: 75-83. reeve, h.k. (1991). polistes. in: ross, k.g. & matthews, r.w. (eds). the social biology of wasps. (pp. 191–231). ithaca, ny: cornell university press. yamane, s. (1969). preliminary observations on the life history of two polistine wasps, polistes snelleni and p. biglumis in sapporo, northern japan. journal of the faculty of sciences of hokkaido university, ser. vi, zoology, 17: 78-105. yamane, s. (1972). life cycle and nest architecture of polistes wasps in the okushiri island, northern japan (hymenoptera, vespidae). journal of the faculty of sciences of hokkaido university, ser. vi, zoology, 18: 440-459. yamane, s., kudô, k., tajima, t., nihon’yanagi, k., shinoda, m., saito, k. & yamamoto, h., (1998). comparison of investment in nest construction by the foundresses of consubgeneric polistes wasps, p. (polistes) riparius and p. (p.) chinensis (hymenoptera, vespidae). journal of ethology, 16: 97-104. yamane, s. & kawamichi, t. (1975). bionomic comparison of polistes biglumis (hymenoptera, vespidae) at two different localities in hokkaido, northern japan, with reference to its probable adaptation to cold climate. kontyû, 43: 214-232. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(2): 169-173 (2013) doi: 10.13102/sociobiology.v60i2.169-173 effects of hematoporphyrin monomethyl ether on worker behavior of red imported fire ant solenopsis invicta zx zhang 1, 2 , y zhou 1, dm cheng 1, 3, * introduction the red imported fire ant, a pest newly introduced to mainland china in 2005 (zhang et al., 2007) and widely distributed in south china, threatens households, agriculture and wildlife. during foraging, worker ants leave a chemical pheromone trail to guide additional worker ants to the food source. these additional worker ants retrieve the food and return to the colony, also marking the pheromone trail laid down by the first group of ants (xu et al., 2007). the abilities to walk, grasp, aggregate and recognize food and water are important for forager ants or other worker ants that leave the nest. if they lose the ability to move and recognize, they will have difficulty living in the complicated external circumstances, which will directly cause a decrease in the food in their nest. this occurrence will eventually affect their population, and may even induce nestmates to become aggressive and bite each other. photosensitizer such as α-terthienyl (α-t) can affect abstract the effect of hematoporphyrin monomethyl ether (hmme) activated under visible light on worker behavior of solenopsis invcita was studied with the potter spray tower method. the results showed that greater than 10 mg/l hmme activated under visible light could reduce the walking, grasping, aggregation, and water and food recognition abilities of red imported fire worker ant significantly, but 100 mg/l hmme in darkness could not affect their abilities or behaviors significantly. therefore, hmme may be a potential novel insecticide that can be used as a substitute for toxic insecticides for controlling red imported fire ants. sociobiology an international journal on social insects 1 key laboratory of natural pesticide and chemical biology, ministry of education, south china agricultural university, guangzhou, china 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 department of plant protection, zhongkai university of agriculture and engineering, guangzhou, china research article ants article history edited by: yi-juan xu, scau, china received 13 march 2013 initial acceptance 24 april 2013 final acceptance 13 may 2013 keywords solenopsis invicta, red imported fire ant, hematoporphyrin monomethyl ether, behavior. corresponding author dongmei cheng key laboratory of natural pesticide and chemical biology, ministry of education south china agricultural university guangzhou, china, 510642 e-mail: zdsys@scau.edu.cn the walking behavior of worker ants and even kill them directly (yan et al., 2012; liu et al., 2011). however, α-t activated under uv light is not suitable for controlling worker ants because the worker ants leaving the nest usually wander under visible light. hematoporphyrin monomethyl ether (hmme), a novel photosensitizer activated under visible light, is the second generation of porphyrin-related photosensitizer; it consists of two monomer porphyrins, namely, 3-(1-methyloxyethyl)-8-(1-hydroxyethyl) deuteropor-phyrin ix and 8-(1-methyloxyethyl)3-(1-hydroxyethyl) deuteropor-phyrin ix, that are mutually locational isomers (chen et al., 2000). hmme possesses some excellent properties, such as strong photodynamic effect and fast removal from organs or cells. moreover, hmme has been reported for the treatment of some cancers (anderson et al., 2002; ascencio et al., 2007; moghissi et al., 2000; yoshihiro et al., 1993). hmme may be useful for controlling worker ants. to our knowledge, no research has been conducted on zx zhang, y zhou, dm cheng hmme effects on worker behavior of red imported fire ant170 the effect of hmme on red imported fire worker ants. this study aimed to investigate the effects of hmme activated under visible light on the behavior of red imported fire ants. materials and methods solenopsis invicta colonies were collected from the suburb of guangzhou and maintained in the laboratory for bioassays. the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor). a test tube (25 mm×200 mm), which was partially filled with water and plugged with cotton, was used as a water source. the ants were maintained in the laboratory at 25±2 °c. large worker ants used in the test were about 6 mm in length, whereas small worker ants were about 3 mm in length. hmme was provided by the institute of red and green photosensitizer (shanghai, china). the stock solution was prepared in ethanol at a concentration of 10 mg/ml and kept in the dark at -20 °c. a 75 w bromine tungsten lamp source (provided by zolix instrument co. ltd., beijing, china) provided spectral radiation from 350 nm to over 2500 nm, which falls within the entire visible range of wavelengths. the stock solution was reconstituted at 5, 10, 25, 50 and 100 mg/l in acetone–water mixtures (3/7, v/v). freshly prepared hmme solutions were kept from light at all times, except during actual measurement. hmme solution was applied to the worker ants with a potter spray tower (burkard manufacturing co. ltd., uk) using methods similar to those described by harris et al (1962). worker ants were placed in a glass petri dish (120 mm in diameter) whose vertical wall was coated with a fluon emulsion. the ants were then placed in the spray tower and sprayed with 2 ml hmme solution. the treated worker ants were transferred into a clean 500 ml beaker, whose vertical wall was coated with a fluon emulsion (the same as below), and then placed in darkness immediately and incubated at 25 °c for 30 h. they were then exposed to visible light emitted by the bromine tungsten lamp (20 cm in height above the 500 ml beaker) for 10 min. the treated worker ants were placed in darkness immediately and incubated at 25 °c for 1h, and then their behaviors were observed. each treatment was replicated three times, and each replicate included 30 to 40 worker ants. in the following methods used to observe the behavior of worker ants regarding water and food recognition, the worker ants were placed in darkness without food and water for 10 h before treatment. controls were similar to the above steps, except that 10 min of light treatment was replaced with 10 min of dark treatment. behavior observation on walking ability of worker ants worker ants were placed on an a4 paper. they were regarded as possessing walking ability if they could walk continuously for 10 cm and did not fall down. the formula was as follows: walking rate = number of worker ants possessing walking ability/number of worker ants per replicate × 100. behavior observation on worker ants’ aggregation worker ants were placed in a 500 ml beaker, and 20 min later, worker ant aggregation was observed. aggregation was considered present if over five worker ants gathered into an aggregate mass. the formula was as follows: aggregation rate = number of worker ants in aggregate mass/number of worker ants per replicate × 100. behavior observation on grasping ability of worker ants worker ants were placed on an a4 paper, and 10 s later, the a4 paper was turned over 180 degrees gently. they were regarded as possessing grasping ability if they would not fall down from the a4 paper. the formula was as follows: grasping rate = number of worker ants possessing grasping ability/number of worker ants per replicate × 100. behavior observation on water recognition of worker ants a water-soaked cotton ball (1 g) and 20 worker ants (the distance between the cotton ball and the ants was 25 cm) were placed on the midcourt line of a porcelain tray (20 cm × 30 cm × 5 cm) whose vertical wall was coated with a fluon emulsion (the same as below). then, the water drinking behavior of worker ant was observed within 30 min. worker ants were regarded as having water recognition ability if they continuously touched the cotton ball with their mouth for greater than 10 s. a worker ant was removed from the porcelain tray if it had drunk water. the formula was as follows: drinking water rate = number of worker ants drank water/number of worker ants per replicate × 100. behavior observation on food recognition of worker ants the treatment method was the same as that used for the behavior observation on the water recognition of worker ants, but the cotton ball was replaced with a dead larva of tenebrio molitor. worker ants were regarded as possessing food recognition ability if they continuously touched the dead larvae with their mouth for greater than 10 s. the formula was as follows: food recognition rate = number of worker ants could recognize the dead larvae/number of worker ants per replicate × 100. sociobiology 60(2): 169-173 (2013) 171 statistical analysis data were reported as means ± se based on three independent experiments. the percentage values were transformed into arc sin of square root of the percentages prior to the analysis, and three-factor anova with worker size, light treatment, and concentration as the main effects were conducted. moreover, the differences between the data were assessed by duncan’s multiple range test (sas 1989) with p < 0.05 regarded as statistically significant. the figures were produced using microsoft office excel 2007. results three-factor anova with worker size, light treatment, and concentration as the main effects, as well as two and three-way interactions between these effects, was conducted. results show that all three main effects and partial interactions were significant (table 1.). significant differences in walking, aggregation, grasping, and water and food recognition abilities were observed from the different treatment concentrations, light treatment, and size of the worker ants (table 2.). small and big worker ants possessed good walking, aggregation, and grasping abilities after treated with hmme in darkness. after treated with 5, 10, 25 and 50 mg/l hmme, the walking rates of small and big worker ants were greater than 96.0%. the walking rates were 86.67% and 98.89% at 100 mg/l, respectively (fig. 1a, table 2.). all the aggregation rates of small and big worker ants were greater than 92.0% (fig. 1b, table 2.), and all their grasping rates were greater than 82.0% at the treatment concentration in darkness (fig. 1c, table 2.). hmme activated under visible light could significantly affect the walking, aggregation, and grasping abilities of red imported worker fire ants. after treated with 10, 25, 50 and 100 mg/l hmme activated under visible light, the walking rates of small worker ants were 82.22%, 53.33%, 14.44% and 0.0%, respectively, and those of big worker ants were 92.22%, 81.11%, 34.44% and 0.0%, respectively, which were significantly different from the values obtained from the treatments in darkness (p < 0.05) (fig. 1a, table 2.). the aggregation rates of small worker ants were 70.83%, 50.83%, 26.67% and 2.50%, respectively, and those of big worker ants were 92.50%, 80.00%, 52.50% and 8.33%, respectively, which were significantly different from the values obtained from the treatments in darkness (p < 0.05) (fig. 1b, table 2.). the climbing rates of small worker ants were 45.56%, 21.11%, 2.22% and 0.0%, and those of big worker ants were 68.89%, 35.56%, 6.67%, and 0.0%, respectively, which were significantly different from the values obtained from the treatments in darkness (p < 0.05) (fig. 1c, table 2.). small and big worker ants possessed good water and food recognition abilities after treated with hmme in darkness. after treated with hmme at test concentration in darkness, the drinking water rates of small and big worker ants were greater than 73.00%, and their food recognition rates were greater than 90.00% (fig. 2, table 2.). the visible light-activated hmme could affect the water and food recognition abilities of worker ants significantly. after treated with 5, 10, 25, 50 and 100 mg/l hmme activated under visible light, the drinking water rates of small worker ants were 51.11%, 43.33%, 34.44%, 13.33% and 5.56%, respectively, and those of big worker ants were 84.44%, 78.89%, 61.11%, 32.22% and 14.44%, respectively, which were significantly different from the values obtained from the treatments in darkness (p < 0.05) (fig. 2a, table 2.). the food recognition rates of small worker ants were 80.00%, 56.67%, 44.44%, 20.00% and 12.22%, respectively, and those of big worker ants were 93.33%, 86.67%, 58.89%, 27.78% and 10.00%, respectively, which were significantly different from the values obtained from the treatments in darkness (p < 0.05) (fig. 2b, table 2.). fig. 1 the effect of visible light-activated hmme on walking ability (a), aggregation (b), and grasping ability (c) of worker ants. zx zhang, y zhou, dm cheng hmme effects on worker behavior of red imported fire ant172 discussion this study showed that hmme concentration greater than 10 mg/l activated under visible light could reduce the walking, grasping, aggregation, and water and food recognition abilities of red imported worker fire ants. however, 100 mg/l hmme in darkness does not affect these abilities or behaviors of worker ants. this finding suggests that hmme could be transmitted successfully in an ant nest, which is the key factor for effectively controlling red imported fire ants. hmme activated under visible light is a novel photosensitizer that has been effectively used for solid tumors, which means that it is relatively safe for humans. hmme is also safe for the environment because of its photodegradation characteristics. as a formulation for controlling red imported fire ants, hmme can be used as bait or as powder applied in the morning or late afternoon. thousands of worker ants living in one nest will then carry it into the nest, resulting in effectively decreasing the labor and photodegradation of hmme, and thus, producing good controlling effect. in conclusion, this study showed that hmme is a potential novel alternative for moderately and highly toxic insecticides to control red imported fire ant. reference anderson, g. s., miyagi, k., sampson, r. w. & sieber, f. 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(1993). photodynamic therapy (pdt) in early stage lung cancer. lung cancer, 9: 287-293. doi: 10.1016/0169-5002(93)90683-o. zhang, r., li, y., liu, n. & porter, s. d. (2007). an overview of the red imported fire ant (hymenoptera: formicidae) in mainland china. fla. entomol., 90: 723-731. http://journals. fcla.edu/flaent/article/view/75725/73383. fig. 2 the effect of visible light-activated hmme on water (a) and food (b) recognition of worker ants. sociobiology 60(2): 169-173 (2013) 173 ta bl e 1. a n o v a fo r m ai n fa ct or s th at a ff ec t t he b eh av io rs o f r ed im po rt ed fi re a nt s. fa ct or s w al ki ng a bi lit y a gg re ga tio n g ra sp in g ab ili ty w at er re co gn iti on a bi lit y fo od re co gn iti on a bi lit y f va lu es p va lu es f va lu es p va lu es f va lu es p va lu es f va lu es p va lu es f va lu es p va lu es a 23 5. 87 0 0. 00 01 82 .4 73 0. 00 01 20 4. 86 0 0. 00 01 96 .5 51 0. 00 01 88 .7 86 0. 00 01 b 99 0. 86 9 0. 00 01 38 4. 24 2 0. 00 01 15 05 .8 17 0. 00 01 60 7. 76 1 0. 00 01 66 8. 21 7 0. 00 01 c 22 .7 14 0. 00 01 20 .8 20 0. 00 01 30 .0 94 0. 00 01 12 9. 99 0 0. 00 01 18 .0 87 0. 00 01 a ×b 15 9. 55 4 0. 00 01 51 .7 79 0. 00 01 13 4. 49 7 0. 00 01 31 .0 30 0. 00 01 51 .7 49 0. 00 01 a ×c 2. 45 0 0. 04 68 0. 87 9 0. 50 23 2. 14 4 0. 07 61 1. 76 4 0. 13 83 3. 60 6 0. 00 75 b ×c 4. 23 3 0. 04 51 13 .7 27 0. 00 05 2. 49 6 0. 12 07 0. 98 1 0. 32 68 9. 76 6 0. 00 30 a ×b ×c 7. 79 0 0. 00 01 1. 41 8 0. 23 50 7. 29 0 0. 00 01 1. 01 0 0. 42 22 1. 30 1 0. 27 93 a : c on ce nt ra tio n, b : l ig ht tr ea tm en t, c : w or ke r s iz e (p =0 .0 5) ta bl e 2. in flu en ce s of d if fe re nt tr ea tm en ts o n be ha vi or s of re d im po rt ed fi re a nt s . tr ea tm en ts pe rc en ta ge (m ea ns ± se , % ) c on ce nt ra tio n (m g/ l ) l ig ht w or ke r s iz e w al ki ng a bi lit y a gg re ga tio n g ra sp in g ab ili ty w at er re co gn iti on fo od re co gn iti on 0 l ig ht sm al l 10 0. 00 ±0 .9 6a 98 .3 3± 3. 91 ab c 10 0. 00 ±0 .0 0a 95 .5 6± 1. 11 bc d 92 .2 2± 5. 09 bc de b ig 10 0. 00 ±0 .0 0a 99 .1 7± 1. 38 a 10 0. 00 ±0 .0 0a 98 .8 9± 1. 11 ab 96 .6 7± 1. 57 ab c d ar kn es s sm al l 10 0. 00 ±0 .9 6a 99 .1 7± 0. 72 a 10 0. 00 ±0 .0 0a 96 .6 7± 1. 92 ab c 97 .7 8± 0. 96 ab b ig 10 0. 00 ±0 .0 0a 99 .1 7± 1. 38 a 10 0. 00 ±0 .0 0a 10 0. 00 ±0 .0 0a 97 .7 8± 1. 11 ab 5 l ig ht sm al l 96 .6 7± 2. 48 b 90 .8 3± 5. 83 cd 84 .4 4± 2. 94 b 51 .1 1± 4. 01 ij 80 .0 0± 5. 98 f b ig 97 .7 8± 1. 57 ab 95 .8 3± 2. 47 ab c 96 .6 7± 1. 92 a 84 .4 4± 4. 01 ef g 93 .3 3± 1. 84 bc de d ar kn es s sm al l 98 .8 9± 0. 96 ab 99 .1 7± 0. 72 a 10 0. 00 ±0 .0 0a 90 .0 0± 3. 85 cd ef 95 .5 6± 0. 96 ab cd b ig 10 0. 00 ±0 .0 0a 98 .3 3± 1. 38 ab 10 0. 00 ±0 .0 0a 98 .8 9± 1. 11 ab 98 .8 9± 1. 84 a 10 l ig ht sm al l 82 .2 2± 10 .1 1d 70 .8 3± 4. 77 e 45 .5 6± 4. 01 d 43 .3 3± 1. 92 jk 56 .6 7± 4. 16 gh b ig 92 .2 2± 1. 84 c 92 .5 0± 3. 80 bc 68 .8 9± 5. 77 c 78 .8 9± 4. 84 fg 86 .6 7± 7. 86 ef d ar kn es s sm al l 98 .8 9± 0. 96 ab 97 .5 0± 1. 18 ab c 98 .8 9± 1. 11 a 88 .8 9± 4. 01 de f 93 .3 3± 2. 48 bc de b ig 96 .6 7± 1. 84 b 96 .6 7± 1. 86 ab c 98 .8 9± 1. 11 a 98 .8 9± 1. 11 ab 95 .5 6± 2. 22 ab cd 25 l ig ht sm al l 53 .3 3± 8. 80 e 50 .8 3± 6. 28 f 21 .1 1± 4. 84 e 34 .4 4± 4. 84 k 44 .4 4± 8. 37 h b ig 81 .1 1± 12 .1 1d 80 .0 0± 8. 28 de 35 .5 6± 6. 19 d 61 .1 1± 4. 84 hi 58 .8 9± 7. 43 g d ar kn es s sm al l 10 0. 00 ±0 .0 0a 97 .5 0± 1. 18 ab c 97 .7 8± 2. 22 a 86 .6 7± 1. 92 de f 97 .7 8± 1. 11 ab b ig 98 .8 9± 0. 96 ab 97 .5 0± 1. 18 ab c 98 .8 9± 1. 11 a 96 .6 7± 1. 92 ab c 96 .6 7± 1. 84 ab c 50 l ig ht sm al l 14 .4 4± 4. 81 g 26 .6 7± 7. 91 g 2. 22 ±4 .8 4g 13 .3 3± 3. 85 lm 20 .0 0± 2. 48 ij b ig 34 .4 4± 10 .2 3f 52 .5 0± 10 .3 7f 6. 67 ±1 .9 2f 32 .2 2± 2. 94 k 27 .7 8± 5. 30 i d ar kn es s sm al l 98 .8 9± 3. 64 ab 95 .0 0± 1. 18 ab c 98 .8 9± 1. 11 a 85 .5 6± 2. 94 ef g 96 .6 7± 1. 84 ab c b ig 10 0. 00 ±0 .0 0a 95 .0 0± 1. 86 ab c 98 .8 9± 1. 11 a 93 .3 3± 1. 92 cd e 94 .4 4± 1. 57 bc de 10 0 l ig ht sm al l 0. 00 ±0 .0 0h 2. 50 ±2 .5 0h 0. 00 ±0 .0 0g 5. 56 ±2 .2 2m 12 .2 2± 1. 11 j b ig 0. 00 ±0 .0 0h 8. 33 ±5 .0 7h 0. 00 ±0 .0 0g 14 .4 4± 2. 94 l 10 .0 0± 1. 92 j d ar kn es s sm al l 86 .6 7± 1. 92 cd 92 .5 0± 2. 50 bc 82 .2 2± 2. 94 b 73 .3 3± 5. 09 gh 90 .0 0± 1. 92 de f b ig 98 .8 9± 1. 11 ab 98 .3 3± 0. 83 ab c 10 0. 00 ±0 .0 0a 94 .4 4± 1. 11 cd e 91 .1 1± 1. 11 cd e a sh ar in g sa m e le tte rs m ea ns n ot s ig ni fic an tly d iff er en t f ro m e ac h ot he r ( p >0 .0 5, d un ca n’ s m ul tip le r an ge t es t) . doi: 10.13102/sociobiology.v61i4.470-477sociobiology 61(4): 470-477 (december,2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 utility of the its1 region for phylogenetic analysis in stingless bees: a case study of the endangered melipona yucatanica camargo, moure and roubik (apidae: meliponini) c ruiz1, w de j may-itzá2, jjg quezada-euán2, p de la rúa3 introduction molecular studies addressing the stingless bees are needed not only to support preliminary morphology-based hypotheses about phylogeny, population dynamics, species delimitation and evolution, but also to establish the basis of conservation programs for this group of bees (arias et al., 2006). towards these ends, a phylogeny of the stingless bee genus melipona has been published (ramírez et al., 2010) based on a multigene data set (nuclear: ef1-α, argk and rna pol-ii genes; mitochondrial: cox1 and rrna genes: 16s) approach. in that study 35 out of the 50 melipona species known-to-date were grouped in four subgenera previously described with morphological and ecological characters (camargo & pedro, 2007), but still some melipona species remain unassigned to a specific subgenus. abstract the internal transcribed spacers of the ribosomal rna gene have been recently proposed as an appropriate marker for genetic analysis of the molecular variation of stingless bees. herein we report the characterization of the complete its1 region in two populations (from mexico and guatemala) of the endangered melipona yucatanica camargo, moure and roubik. phylogenetic analyses showed low genetic variation between populations but defined a geographic structure with mexican and guatemalan specimens forming two well supported clades. low its1 genetic variation found between populations contrasts with high genetic variation found in other markers. phylogenetic analysis corroborates the inclusion of m. yucatanica within the subgenus melipona sensu stricto based on previous morphological studies. the results highlight the utility of the its1 for the characterization of stingless bee populations. sociobiology an international journal on social insects 1 universidad técnica particular de loja, loja, ecuador 2 universidad autónoma de yucatán, yucatán, méxico 3 universidad de murcia, murcia, españa article history edited by cândida maria l. aguiar, uefs, brazil received 29 may 2014 initial acceptance 23 july 2014 final acceptance 07 august 2014 keywords stingless bees, melipona, its1, phylogeny, mesoamerica. corresponding author pilar de la rúa área de biología animal, departamento de zoología y antropología física, facultad de veterinaria, campus de espinardo, universidad de murcia, 30100 murcia, españa e-mail: pdelarua@um.es other suitable markers for phylogeny and population genetic analyses are the internal transcribed spacers (its1 and its2) of the ribosomal genes (rrna). concerted evolution may have led to the homogeneity of repeat motifs within the its regions (hillis & dixon, 1991). therefore, they have been successfully applied for population and phylogenetic studies of some stingless bee species. in particular, in the tribe meliponini, fernandes-salomão et al. (2005) determined the complete its1 sequence from three melipona species and used partial sequences of eight species to infer its phylogenetic relationships. at the intraspecific level, the its1 region has demonstrated its usefulness for population analysis in the mexican species melipona beecheii bennett (mayitzá et al., 2009; 2012) and also in two brazilian species: melipona subnitida ducke (cruz et al., 2006) and melipona quinquefasciata lepeletier (pereira et al., 2009). research article bees sociobiology 61(4): 470-477 (december, 2014) 471 the mexican stingless bee melipona yucatanica camargo, moure and roubik is a small bee (8 mm in average) with less than 200 worker bees per colony (camargo et al., 1988). this species is associated with primary forest, and based on its patched distribution, various authors have suggested a recent fragmentation of m. yucatanica populations due to massive deforestation (camargo et al. 1988; ayala, 1999). preliminary molecular studies in mexican and guatemalan populations of m. yucatanica yielded different rflp patterns in the its2 region (de la rúa et al., 2007), suggesting allopatric speciation in populations geographically separated. furthermore, morphometric and bayesian analyses of the mitochondrial cox1 region and microsatellite loci revealed geographic differences between guatemalan and mexican populations, suggesting that m. yucatanica from méxico and guatemala could represent two distinct species (may-itzá et al., 2010). because of the rapid disappearance of its habitat and the difficulty to multiply colonies domestically, conservation measures to preserve m. yucatanica are urgently needed (may-itzá et al., 2010). the taxonomic position of this species based on morphological comparative studies have placed this species within the subgenus melipona s. str. (camargo & pedro, 2007), in relation to other south-american species (moure et al., 2007). however its taxonomic position has not been assessed on molecular grounds yet. herein, the entire its1 region of m. yucatanica has been sequenced and compared with available its1 (complete or 3’ end) from other melipona species as well as with previous results on m. yucatanica based on morphometry and molecular markers (de la rúa et al., 2007; may-itzá et al., 2010). we aimed to i) analyze the molecular diversity underlying this marker, and ii) test its phylogenetic signal to verify whether this species is included within the morphologically described subgenus melipona s. str. as suggested by camargo and pedro (2007). material and methods sampling feral colonies of m. yucatanica are difficult to find since they are restricted to preserved rain-forest, so we were only able to sample worker bees (10-20 worker bees per colony) from managed colonies located in two geographically distant mesoamerican regions where they naturally occur: seven in méxico and four in guatemala (table 1, fig 1). these locations showed notable climatic and geological differences: the pacific region of guatemala is a mountainous region, whereas the atlantic-caribbean region of méxico is a flat limestone area without mountainous zones. one specimen of each colony has been deposited in the insect collection of the department of zoology and physical anthropology (university of murcia, spain). dna extraction, amplification and sequencing dna was extracted from two worker bees per colony using the dneasy tissue kit (qiagen). the its1 region was amplified using the primers cas18sf1 and cas5p8sb1d fig 1. study area and sampling localities of melipona yucatanica in mesoamerican region. c ruiz, w de j may-itzá, jjg quezada-euán, p de la rúa characterization of its1 in m. yucatanica472 based on the conserved sequences of the 18s and 5.8s rdna respectively (ji et al., 2003). the amplification program consisted of 5 min. at 96 ºc, 34 cycles of 45 sec. at 96 ºc, 1 min. at 60 ºc and 1 min. at 72 ºc, and a final extension of 10 min. at 72 ºc. pcr reactions were performed with pcr beads puretaqtm ready-to-gotm (ge healthcare) in a thermocycler ptc-200 (biorad). pcr products were purified with qiaquick pcr purification kit (qiagen) before directly sequencing. sequencing was performed in both directions with the same pcr primers in an abi 3730 dna analyzer (applied biosystems) at the sequencing company secugen s. l. (madrid, spain). its1 sequences edited with the program mega 5 (tamura et al., 2011) were deposited in genbank with the accession numbers hq651726 (m. yucatanica from guatemala) and hq651727 (m. yucatanica from méxico). data analyses the its1 datasets used included the complete its1 gene for m. yucatanica, m. beecheii and melipona quadrifasciata lepeletier and partial sequences of its1 3’ end for other nine melipona species obtained from genbank (fernandessalomão et al., 2005; cruz et al., 2006; pereira et al., 2009). in order to compare its1 results with other markers, a multiplegene approach was carried out. sequence data of mitochondrial cox1 were obtained from genbank for m. yucatanica, and for the species with available its1 sequences (namely, m. quinquefasciata, m. beechei, melipona compressipes fabricius, m. quadrifasciata, melipona mandacaia smith, melipona bicolor lepeletier, melipona marginata lepeletier, melipona rufiventris lepeletier and melipona scutellaris latreille). separated and combined analyses were carried out. its1 sequences were aligned with mafft software (http://mafft.cbrc.jp/alignment/server/) using e-ins-i strategy. parameter of scoring matrix for nucleotide sequences was set to 20pam/ k = 2 as recommended when aligning related dna sequences (katoh & toh, 2008). its1 and cox1 genetic diversity in m. yucatanica and in other available melipona sequences were measure in dnasp (librado & rozas, 2009), genalex (peakall & smouse, 2012) and mega 5 (tamura et al., 2011) using various variability indices: number of genotypes, genotype diversity, variable sites, parsimony-informative sites, nucleotide diversity, and intraspecific genetic distance (k2p). phylogenetic analysis the best-fit evolutionary model for the bayesian analyses was determined with jmodeltest 0.1.1 (posada, 2008) using the akaike information criteria (aic). bayesian analyses were carried out using mrbayes version 3.1 (ronquist & huelsenbeck, 2003). separate runs were carried out with four simultaneous markov chains, each starting from a random tree. the analysis ran for 6 500 000 generations to allow runs to converge, and the chain was sampled every 100th generation. the first 14 000 trees were discarded as the “burn-in” before the chains converged on a stable value and the posterior probabilities of tree topology were determined from the remaining trees. also convergence was reassessed using the potential scale reduction factor (psrf, ronquist & huelsenbeck, 2003). results its1 and cox1 sequence variation the two m. yucatanica worker bees analyzed per colony showed the same its1 sequence, therefore only one sequence per colony was used in the subsequent analyses. the first 126 base pairs (bp) of the alignment correspond to the table 1. sampling localities of melipona yucatanica specimens in included in the study, geographical information and accession numbers of sequences in genbank. taxon id accession number country state locality geographical coordinates altitude myucmer1 hq651727 méxico yucatán lópez portillo 19.9351278 n -89.1879278 w 142 m myucmer2 kf564809 myucmer3 kf564810 myucmer4 kf564811 myucmer5 kf564812 méxico yucatán huntochac 19.8061861 n -89.5007389 w 80 m myucmer6 kf564813 myucmer7 kf564814 myucguat1 hq651726 guatemala jutiapa jutiapa 14.2980778 n -89.8970972 w 898 m myucguat2 kf564806 myucguat4 kf564807 guatemala santa rosa barberena 14.3072639 n -90.3564139 w 1220 m myucguat5 kf564808 sociobiology 61(4): 470-477 (december, 2014) 473 18s ribosomal region and the last 58 bp to the 5.8s, so the size of the its1 region was 1310-1312 bp in guatemalan and 1308 bp in mexican m. yucatanica. insertion-deletion (indel) events were observed in the alignment of the its1 sequence of m. yucatanica specimens from guatemala and méxico. two indels of 4 and 8 bp corresponded to a microsatellite locus showing different number of repeat motifs. this variation in the number of motifs was not detected among the specimens within each population but between them. an additional variation was detected among guatemalan m. yucatanica specimens, due to the presence of one insertion of two base pairs (tt) in the specimen 4 (myucguat4). in relation to other species, a large insertion of 80 bp was detected in m. quadrifasciata. there were three genotypes, one for mexican (n = 7, myucmer1-7) and two for guatemalan individuals (n = 1; myucguat4 and n = 3 myucguat1, 2 and 5). a higher number of unique genotypes was found in m. beechei, m. quinquefasciata and m. subnitida populations (table 2). the its1 nucleotide diversity (pi) was from 3 to 65 times lower for m. yucatanica than for the other three melipona species (table 2). this result contrasts with the nucleotide diversity and the genetic distances (k2p) values obtained for cox1, where m. yucatanica population had similar values or slightly higher (1.8 to 2.7 times higher) than those obtained for other available species (table 2). comparisons between markers showed that in m. yucatanica, its1 nucleotide diversity (pi = 0.0028) and genetic distance (d = 0.28%) is about five times lower than in cox1 (pi = 0.0136, d = 1.39%). on the other hand, in m. beecheii the comparison of both markers showed a different pattern, as the diversity and distance values are about 1.6 times higher with the its1 than with cox1 data (table 2). phylogenetic analyses the optimal model of nucleotide substitution, following aic criterion, was the transversional model (tvm). bayesian analysis of the complete its1 region, showed that this species is divided in two well supported clades (mexican and guatemalan specimens, p.p. = 1.0) defining a clear phylogeographic structure (fig 2), m. beecheii showed two clades with the same geographic structure, whereas m. subnitida and m. quinquefasciata showed a more complex pattern of population structure. m. yucatanica its1 sequences clustered with high support (p.p = 1.0) within the subgenus melipona s. str. together with m. quadrifasciata and m. subnitida. analysis of available cox1 sequences resulted in the same two well supported clades of mexican and guatemalan populations (fig 3). moreover the species m. yucatanica was also grouped with high support within the subgenus melipona s. str. the combined analysis of cox1 and its1 resulted in the same well supported clades (fig s1 in supplementary material). t ab le 2 . m ol ec ul ar d iv er si ty o f t he it s1 fr ag m en ts (c om pl et e an d pa rti al 3 ' a nd fr ag m en t) an d co x 1 (3 ' f ra gm en t) in fi ve m el ip on a sp ec ie s. d at a fr om m . y uc at an ic a in b ol d. d at a fr om o th er m el ip on a sp . o bt ai ne d fr om g en b an k. bp = l en gt h of t he f ra gm en t in b as e pa ir s, n = n um be r of i nd iv id ua ls , h = n um be r of g en ot yp es /h ap lo ty pe s, g d: g en ot yp e di ve rs ity , h d: h ap lo ty pe d iv er si ty , p i = nu cl eo tid e di ve rs ity , v = v ar ia bl e si te s, p ar = pa rs im on yin fo rm at iv e si te s, d = in tr as pe ci fic g en et ic d is ta nc e w ith k 2p ( in % ). d at as et it s1 c om pl et e (1 67 1 bp ) it s1 p ar ti al (6 94 b p) co x1 (5 80 b p) sp ec ie s bp n h g d pi v pa r d bp n h g d pi v pa r d bp n h h d pi v pa r d m el ip on a yu ca ta ni ca 14 68 -1 47 2 11 3 0. 25 0 0. 00 28 8 8 0. 3 59 560 1 11 2 0. 50 9 0. 00 09 1 1 0. 08 6 58 0 7 4 0. 81 0. 01 36 34 14 1. 39 m el ip on a be ec he ii 15 19 -1 58 2 10 9 0. 97 8 0. 01 23 9 40 20 1. 21 55 555 8 10 5 0. 84 4 0. 01 18 2 6 4 1. 04 58 0 46 18 0. 91 3 0. 00 74 4 25 16 0. 75 m el ip on a qu in qu ef as ci at a 57 060 9 9 9 1. 00 0. 01 81 65 10 2. 97 m el ip on a su bn iti da 59 061 2 13 13 1. 00 0. 06 01 8 18 5 44 7. 04 m el ip on a qu ad ri fa sc ia ta 58 0 14 5 31 0. 87 6 0. 00 50 9 55 32 0. 51 c ruiz, w de j may-itzá, jjg quezada-euán, p de la rúa characterization of its1 in m. yucatanica474 discussion phylogenetic analyses showed low genetic variation in its1 between populations but defined a geographic structure with mexican and guatemalan specimens forming two well supported clades. the characterization of the its1 region of the stingless bee m. yucatanica further evidenced that this region is longer in stingless bees than in other hymenoptera (pilgrim & pitts, 2006; taylor et al., 2006). in m. yucatanica its1 is within the range of that observed in other melipona species (from 1391 bp in m. quadrifasciata to 1940 in m. rufiventris, fernandes-salomão et al., 2005). the presence of insertions and deletions (indels) in this region is a common feature leading to sequence length variation, as it has been observed between mexican and guatemalan m. yucatanica specimens, and among brazilian populations of the species m. subnitida (cruz et al., 2006) and within m. quinquefasciata (pereira et al., 2009). the its1 clades separate between mexican and guatemalan populations showing the same phylogeographic pattern found in previous markers (its2, de la rúa et al., 2007; cox1 and microsatellites may-itzá et al., 2010). these results are congruent with the fact that both populations are located in opposite extremes of the species distribution range (ayala, 1999). however, an incongruent pattern of genetic variability was revealed when several markers were compared. populations of m. yucatanica showed lower levels of genetic diversity and genetic distance in the its1 region than those reported for other markers as cox1 and microsatellite data (may-itzá et al., 2010; table 2). its1 and cox1 have different mechanisms of evolution that have led to different patterns in intraspecific variations for various taxa (e.g., hansen et al., 2006; carlini et al., 2009; kornobis & pálsson, 2011). the low variation of its1 could therefore be explained by intraspecific rdna its1 homogenization due to the mechanisms of concerted evolution (hillis & dixon, 1991). however, this explanation cannot be extended to other melipona species. in m. beecheii, a species with wider geographic distribution, the opposite pattern was found with diversity and distance values of its1 higher than for cox1 data (may-itzá et al., 2010). moreover, when data are fig 2. bayesian analyses of the its1-complete region of several melipona species. population data of melipona yucatanica are indicated with a solid bar. available population data of several melipona species are indicated with empty bars. numbers above nodes show posterior probability ≥ 0.90. sociobiology 61(4): 470-477 (december, 2014) 475 compared between these two species, m. beecheii showed about four times higher divergence, genotype and nucleotide diversity of its1 (complete fragment) but almost two times lower values for cox1 data (table 2). unfortunately, there are no available data to compare this pattern with other melipona species; however, m. yucatanica showed a remarkably lower its1 divergence (from 12 to 80 times) and higher cox1 divergence (from 1.8 to 2.7 times) than all other melipona species analyzed (table 2). this observed distinct pattern of divergence between nuclear and mitochondrial markers in m. yucatanica populations in relation to other melipona species could be due to several causes which will require specific testing, among them: i) different efficiency of concerted evolution of its1 rdna between melipona species, as suggested for other taxa (armbruster & korte, 2006); ii) a complete replacement in one of the sampled populations of the original mitochondrial haplotype by introgression which led to higher levels of mitochondrial divergences. this hypothesis cannot be disregarded although until date, introgression events have not been described in melipona species yet; and iii) distinct population dynamics in relation to other the melipona species analyzed. further analyses including more markers and more m. yucatanica populations scattered across its distribution range are needed in order to understand the population dynamic of this endangered species. the phylogenetic signal obtained in the its1 bayesian analysis confirms the utility of this region as a potential phylogenetic marker for the genus melipona. this marker seems suitable for resolving population and subgenera divergences. in this sense, the phylogenetic analysis included m. yucatanica in the subgenus melipona s. str. together with other species as m. quadrifasciata and m. mandacaia. this molecular result confirms previous morphological studies (moure et al., 2007) and it is in accordance with recent molecular studies of the genus melipona (ramírez et al. 2010). in the light of recent numt dna descriptions in other melipona subspecies (cristiano et al., 2012; ruiz et al., 2013), the its1 region has demonstrated its potential as an appropriate marker for genetic studies in stingless bees. fig 3. bayesian analysis of the available cox1 gene from melipona species. numbers above nodes show posterior probability ≥ 0.90. population data of melipona yucatanica are indicated with a solid bar. available population data of several melipona species are indicated with empty bars. c ruiz, w de j may-itzá, jjg quezada-euán, p de la rúa characterization of its1 in m. yucatanica476 acknowledgements we thank eunice enríquez for her help with the guatemalan sampling and jorge gonzález acereto for the mexican samples. we also appreciate the input of the reviewers. this work has been supported by the bbva foundation, fondo de cooperación internacional en ciencia y tecnología unión europea – méxico (foncicyt) (project mutual 94293), and fondo sectorial de investigación para la educación (sepconacyt, project 103341). references armbruster, g.f.j. & korte, a. 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(2006). genetic and morphological comparisons of new and old world populations of spalangia species (hymenoptera: pteromalidae). ann. entomol. soc. am., 99: 799-808. doi: 0.1603/0013-8746(2006)99[799:gamcon]2.0.co;2 this article has online supplementary material at: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/428/0 doi: 10.13102/sociobiology.v61i4..s714 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i1.1-8sociobiology 61(1): 1-8 (march, 2014) an updated guide to the study of polyandry in social insects r jaffé 1. why study polyandry in social insects? understanding the adaptive significance of multiple mating by social insect queens (polyandry) has been a central goal of many social insect researchers for the past three decades (page & metcalf 1982; crozier & fjerdingstad 2001; boomsma et al. 2009; kraus & moritz 2010; palmer & oldroyd 2000). single paternity resulting from monandry (single mating) is currently regarded as a crucial precondition for the evolution of eusociality in the hymenoptera (ants, bees and wasps), since it maximizes genetic relatedness between colony members (boomsma 2009; hughes et al. 2008a). polyandry, on the other hand, dilutes the relatedness between group members because it generates half-sib families within a colony. in consequence, the benefits gained through inclusive fitness can be reduced (hamilton 1964), and may not outweigh the cost associated with the maintenance of sterile worker behaviors (page & metcalf 1982). polyandry has nevertheless evolved independently in ants, in bees and in wasps (hughes et al. 2008a). abstract in spite of the importance of understanding the adaptive significance of polyandry in the social hymenoptera (ants, bees and wasps), little consensus exists regarding the terminology employed, the use of different paternity estimates, the calculation of such estimates and their associated error measures, and the way paternity should be treated in comparative studies. here i summarize previous methodological contributions to the study of polyandry in social insects, hoping that such a compendium will serve as an updated guide to future researchers. i first revise the estimates describing queen mating behavior and paternity outcomes in polyandrous social insects, outlining appropriate methods for calculating them. i then address the errors associated to paternity estimates and explain how to account for them. finally i discuss in which cases paternity should be treated as a continuous or a categorical variable, and provide an insight into the distribution of paternity across the social hymenoptera. this technical review highlights the importance of standardizing research methods to prevent common errors, raise confidence in the reported data, and facilitate comparisons between studies, to help shed light into many unanswered questions. sociobiology an international journal on social insects instituto de biociências, universidade de são paulo (usp), são paulo-sp, brazil article history edited by: gilberto m m santos, uefs, brazil received 13 december 2013 initial acceptance 04 february 2014 final acceptance 04 march 2014 keywords effective paternity, multiple mating, multiple paternity, polyandry corresponding author rodolfo jaffé laboratório de abelhas departamento de ecologia instituto de biociências universidade de são paulo – usp rua do matão 321, 05508-090 são paulo-sp, brazil email: r.jaffe@ib.usp.br among the many hypotheses that have been proposed to explain the evolution of polyandry in the social hymenoptera, the genetic diversity or genetic variance hypothesis enjoys most current support (palmer & oldroyd 2000; crozier & fjerdingstad 2001). the increase in genetic diversity within colonies, resulting from the co-occurrence of worker offspring from different fathers, has been suggested as the most plausible explanation for the evolution and maintenance of polyandry in this group of insects. high genetic diversity among colony members has been shown to increase productivity and broaden tolerance to environmental changes (mattila & seeley 2007; oldroyd & fewell 2007), increase resistance to pathogens (hughes & boomsma 2006; seeley & tarpy 2007; schmidhempel 1998; baer & schmid-hempel 1999), and enhance an efficient division of labor (hughes et al. 2003; jaffé et al. 2007; smith et al. 2008). paternity frequency has been found to be negatively correlated with the number of queens per colony (hughes et al. 2008b; keller & reeve 1994), which suggests polyandry and polygyny (multiple queens) review r jaffé polyandry in social insects2 are alternative mechanisms to increase genetic diversity in social insect colonies. colony size, has also been found positively correlated to paternity frequency, indicating that larger colonies might profit more from genetic diversity (schmidhempel 1998; bourke 1999), or alternatively, that queens heading large colonies need to mate with several males to obtain enough sperm (sperm limitation hypothesis) (cole 1983; kraus et al. 2004; but see also jaffé et al. 2014). finally, a recent study found a negative association between paternity frequency and paternity biases, showing that queens of highly polyandrous species maximize genetic diversity by equalizing paternity (jaffé et al. 2012). in addition to increasing within-colony genetic diversity, polyandry causes the co-occurrence of different ejaculates in the female’s reproductive tract. this allows sexual selection to operate after copulation, either through the competition of ejaculates from different males to fertilize an egg (sperm competition) (simmons 2001) or through the ability of females to influence which sperm fertilize their eggs (cryptic female choice) (eberhard 1996). post-copulatory sexual selection is known to be a significant evolutionary force, shaping the evolution of male and female traits across taxa (andersson & simmons 2006). understanding the consequences of polyandry for the evolution of male and female traits is thus crucial to gain a complete understanding of the reproductive biology of social insects (kvarnemo and simmons 2013). moreover, this knowledge is essential to design effective breeding programs for commercial species. 2. an updated guide to the study of polyandry in social insects in spite of the importance of understanding the adaptive significance of polyandry in social insects, little consensus exists regarding the terminology employed, the use of different paternity estimates, the calculation of such estimates and their associated error measures, and the way paternity should be treated in comparative studies. methodological consensus and standardization is important, because it could prevent common errors, raise confidence in the reported data, and facilitate comparisons between studies. thus, my aim here is to gather and summarize previous methodological contributions to the study of polyandry in social insects, hoping that such a compendium will serve as an updated guide to future researchers. i first revise the estimates describing queen mating behavior and paternity outcomes in polyandrous social insects, providing definitions and estimation methods. i then address the errors associated to paternity estimates and explain how to account for them. finally, i discuss in which cases paternity should be treated as a continuous or a categorical variable, and provide an insight into the distribution of paternity across the social hymenoptera. 2.1. mating frequency, paternity frequency, effective paternity or paternity skew? paternity in the social hymenoptera is usually reported as observed and effective paternity. while observed paternity (k obs ), or paternity frequency, is the number of males siring offspring of a single queen, effective paternity (me ) is paternity weighted by the proportion of offspring sired by each male (nielsen et al. 2003). mating frequency, often confused with paternity frequency, measures the actual number of males that copulated with a single queen, even if some of them failed to sire any offspring (table 1). the distinction between these terms is important, because they reflect the outcome of different evolutionary processes. social insect research has focused on the study of effective paternity (me ), because this estimate reflects the average genetic relatedness among the workers of colonies headed by a single queen (pamilo 1993; boomsma & ratnieks 1996). effective paternity is indeed the most informative estimate for studies addressing the impact of polyandry on colony relatedness and reproductive conflicts (wenseleers & ratnieks 2006; sanetra & crozier 2001). however, effective paternity is not a good indicator of the actual number of males the queens mated with. observed paternity provides a more accurate estimate of the queen´s real mating frequency, even table 1: estimates describing queen mating behavior and paternity outcomes in polyandrous social insects. estimate definition estimation method copulation frequency number of times a single queen copulates * observation mating frequency number of males copulating with a single queen † observation insemination frequency number of males inseminating a single queen genotyping of sperm from spermatheca observed paternity (kobs ) or paternity frequency number of males fertilizing eggs and siring offspring of a single queen genotyping of eggs, pupae or worker offspring effective paternity (me ) observed paternity weighted by the proportion of offspring sired by each male. see ke3 estimate (nielsen et al. 2003) paternity skew degree of paternity bias among the offspring of polyandrous queens. see b-index (nonacs 2000) * note that a queen may copulate several times with the same male. † note that insemination may or may not be involved. ^ sociobiology 61(1): 1-8 (march, 2014) 3 though processes of post-copulatory sexual selection (sperm competition and cryptic female choice) might prevent some sperm from fertilizing eggs and siring offspring (simmons 2001), and thus the actual mating frequency of a queen might be higher than the observed paternity. because of this, observed paternity should always be provided along with effective paternity estimates. mating frequency and insemination frequency should also be provided if available, although they are usually more difficult to assess (baer 2011). the degree of paternity biases among the offspring of polyandrous females (paternity skew), is another key quantitative measure needed to address mechanisms of post-copulatory sexual selection and sexual conflict (den boer et al. 2010; jaffé et al. 2012; baer et al. 2006). levels of paternity skew may also be the outcome of kin-selection processes, as high paternity skew can bias paternity toward one or a few males, thus increasing genetic relatedness among the offspring of polyandrous queens (jaffé et al. 2012; cole 1983). providing paternity skew along with paternity estimates, is thus essential. 2.2. general methodological considerations and useful software paternity is most commonly deduced from molecular markers, by grouping offspring sharing the same father and assigning them to patrilines. a large body of data from studies employing genetic markers have accumulated during the past decade (boomsma et al. 2009), and this trend is likely to remain or increase with new technological developments that allow massively parallel and multiplexed sample sequencing (ellegren 2013; allendorf et al. 2010). studies estimating paternity from worker genotypes should be careful not to sample offspring from different colonies occurring together in one colony. for instance, there is growing awareness of “worker drifting” between colonies of social hymenoptera (lopez-vaamonde et al. 2004). a sample of honeybee workers collected at a nest entrance, for example, is usually composed of workers from the study colony as well as drifter workers from other colonies (neumann et al. 2000). depending on the genotypes of these samples, drifter workers could be misinterpreted as workers from a different patriline, thus inflating paternity estimates. it is therefore important to avoid sampling drifter workers, as this would simplify analyses and yield accurate paternity estimates. an easy way to avoid sampling drifter workers is to collect freshly emerged workers inside the colonies, or to sample pupae or even eggs (paxton et al. 2003). in cases where it is not possible to avoid sampling drifter offspring, or where colonies have more than one queen, sibship reconstruction analyses can be performed to assign workers into queens and patrilines within queens. a particularly useful software to perform this kind of analyses is colony, a free program implementing maximum likelihood method to assign sibship and parentage jointly, using individual multilocus genotypes at a number of co-dominant or dominant marker loci (jones & wang, 2010). colony can be found here: http://www.zsl.org/science/software/colony matesoft (moilanen et al. 2004) is a free software developed to estimate paternity statistics in haplodiploid organisms (like the social hymenoptera), based on co-dominant genetic marker data. the genetic data can be queen genotypes, genotypes of worker offspring from a single queen, or genotypes of sperm stored in the queen’s spermatheca. a particularly appealing feature of this program is that it can also deduce parental genotypes and provide a likelihood probability for alternative genotypes. matesoft can be found here: http://www. bi.ku.dk/staff/jspedersen/matesoft/ another approach to deduce paternity from worker genotypes is to estimate effective paternity based on the genetic relatedness between workers (rww). relatedness between worker offspring of a queen that mated with a single male is rww = 0.75, while relatedness between worker offspring of a highly polyandrous queen approaches rww = 0.25. hence, effective paternity (me ) can be obtained from the relatedness between the workers of a single queen, following pamilo (1993): me = 0.5 / (gww – 0.25), where gww is the pedigree relatedness. this approach assumes that the regression worker-worker relatedness (rww) is identical to the pedigree relatedness (gww), and hence should only be applied under no inbreeding. kingroup is a free open source program implementing a maximum likelihood approach to pedigree relationships reconstruction and kin group assignment (konovalov et al. 2004). it allows estimating relatedness bewteen offspring and includes a number of features originally found in the program kinship (goodnight & queller 1999), which is no longer updated and only runs in the classic macintosh os platform. kingroup can be found here: https://code.google.com/p/kingroup/ among the different skew indexes, the b-index (nonacs 2000) has been proposed as the standard estimate to be used in future studies, since it can be easily calculated from paternity data and shows very robust statistical properties (nonacs 2003). the b-index can be calculated using the skew calculator available here: https://www.eeb.ucla.edu/faculty/nonacs/ pi.html 2.3. non-detection and non-sampling errors paternity estimates from genetic data are affected by two main types of error: non-detection and non-sampling errors. the non-detection error (nde) is the probability of two fathering males having identical haplotypes by chance. nde is determined by the number of markers employed and their level of polymorphism and is an indicator of the resolution of these markers. it should always be reported along with paternity estimates to provide a quantitative measure of accuracy (high ndes imply a low detection power, and thus paternity estimates might be underestimated). nde can also be calcur jaffé polyandry in social insects4 lated when estimating the number of matrilines in a colony (or the number of reproductive females). formulae for calculating ndes are summarized in table 2. the non-sampling error (nse) estimates the number of males siring offspring remaining undetected because of an insufficient sampling. nse will be affected by the level of paternity skew, and hence needs to be estimated based on the paternity shares of each male and the number of workers analyzed. in species with an observed paternity kobs = 1, nse should be calculated to indicate the probability of failing to detect a second male, which fertilized some of the queen`s eggs but remained undetected because none of its offspring were sampled. a low nse would indicate that queens are indeed monandrous, as no further males remained undetected because of sampling effects. in species with an observed paternity kobs > 1, nse should be calculated to estimate the number of males remaining undetected due to insufficient sampling. to this end, a given frequency distribution (binomial, poisson, etc.) can be fitted to the real distribution of workers among patrilines. the expected frequencies for each category (number of sired workers) can then be computed, and the number of males remaining undetected because of an insufficient sampling estimated as the expected frequency for the zero or less than one category (cornuet & aries 1980; human et al. 2013). in this case, nse can be accounted for by adding the number of undetected males to observed paternity estimates. nielsen´s effective paternity estimate (nielsen et al. 2003) is already corrected for sample size, so there is no need for additional corrections for that estimator. approaches for calculating nses are summarized in table 2. 2.4. is paternity a continuous or a categorical variable? traditionally, paternity has been treated as a categorical variable in the social hymenoptera, with species being grouped into different paternity or polyandry categories. boomsma and ratnieks (1996) first proposed four paternity categories, based on the frequency of multiple paternity among study queens and the mean value of effective paternity in the study population. later studies proposed variations of these original categories, and employed either observed paternity (referred to as mating frequency), effective paternity or the frequency of multiple paternity among study queens, as grouping characteristics (see table 3). to date, however, there is no consensus on how many categories should be used, how to establish the limits between them or which characteristics to use for assigning species into paternity categories. a recent study retrieved paternity data for 87 polyandrous species of social hymenoptera (jaffé et al. 2012). this data set shows that paternity is not normally distributed, with nearly half of all polyandrous species (n = 46) showing mean observed paternities below 2 (fig. 1). two polyandry categories could be created based on this distribution: low polyandry (mean observed paternity below 2) and high polyandry (mean observed paternity above 2). by so doing, the high polyandry category would merge about half of all polyandrous species (n = 41), with mean observed paternities ranging from 2 to 55. clearly, informative variance would be lost by this grouping, as selective forces differ considerably between species with small colonies and queens that mate with a few males (such as bumble bees), and species with huge colonies and highly polyandrous queens (such as honeybees). nevertheless, such categorization based on the real frequency distribution of paternity across species is more parsimonious than the creation of categories based on arbitrary assumptions and lacking consensus across studies. table 2: non-detection and non-sampling errors. type of error level formula reference patriline non-detection population ( ∑qi 2 ) ( ∑ ri 2 )…( ∑ zi 2 ) boomsma & ratnieks 1996 patriline non-detection colony ( qi ) ( ri )…( zi ) † foster et al. 1999 matriline detection probability locus n/a € richards et al. 2005 patriline non-sampling for kobs = 1 colony (1 p)n ‡ foster et al. 1999 patriline non-sampling for kobs > 1 colony n/a £ human et al. 2013 * qi are the allele frequencies at the first locus, ri the allele frequencies at the second locus, and zi are the allele frequencies at the last locus. this calculation assumes all loci are unlinked and under hardy-weinberg equilibrium. † see corrections for ambiguity in identification of paternal alleles (foster et al. 1999). € not applicable. see different cases depending on the inheritance of distinct grandparental alleles (richards et al. 2005). ‡ p is the proportion of offspring sired by the second male (usually set to three values: p = 0.50, p = 0.25 and p = 0.10) and n is the number of worker offspring analyzed. £ not applicable. frequency distribution fitting. for an example see section 4.3.3.5 in (human et al. 2013). fig. 1: frequency distribution of mean observed paternity (kobs) in 87 polyandrous species of social hymenoptera (data taken from jaffé et al. 2012). sociobiology 61(1): 1-8 (march, 2014) 5 table 3: paternity categories as reported in the literature. category original description * reference single paternity double mating absent or very rare; population-wide effective mating frequency < 1.05 boomsma & ratnieks 1996 single-double paternity double mating occurs in ca 20%-50% of queens; effective mating frequency 1.05-1.25 single-multiple paternity mating frequency above two occurs regularly; effective mating frequency 1.4-2 multiple paternity mating frequency usually greater than two; effective mating frequency > 2 polyandry multiple mating oldroyd & fewell 2007 extreme polyandry mating number > 6 monandry n/a† hughes et al. 2008b facultative low polyandry effective mating frequencies of < 2 moderate polyandry effective mating frequencies of 2–10 extreme polyandry effective mating frequencies of > 10 monandry n/a† hughes et al. 2008afacultative low polyandry < 2 effective mates high polyandry > 2 effective mates singly mated n/a† boomsma et al. 2009 facultatively multiply mated usually ≥ 50% singly mated with a variable minority of queens mated to 2-5 males obligately multiply mated almost always ≥ 2 and often ≥ 5 matings per queen * note that the word “mating” in the original descriptions actually refer to paternity. † not applicable phylogenetic studies assessing the transition from monandry to polyandry could benefit from categorizing species by their paternity frequency to perform ancestral state reconstruction analyses (hughes et al. 2008a). similarly, studies aiming to detect adaptations to polyandry, or the consequences of polyandry for the evolution of other relevant traits, could profit from comparing the traits of interest between highly polyandrous, lowly-polyandrous and monandrous species (den boer et al. 2010). however, efforts aiming to detect biologically meaningful associations between paternity frequency and other continuous traits or ecological factors would maximize detection power and avoid losing meaningful variance by employing the actual paternity estimates, as most sexual selection studies do (simmons 2001). even though boomsma (2013) argued that facultative and obligate polyandry appear to be mutually exclusive lineagespecific syndromes, ecological factors could still have shaped the evolution of paternity frequency within as well as across clades (arnqvist & nilsson 2000). hence, treating paternity as a continuous variable could prove more informative to unravel such factors. 3. future perspectives the study of polyandry in social insects offers exciting opportunities for future research. efforts are still needed to understand, for example, how paternity skew has been shaped by the interplay between kin selection and sexual selection (jaffé et al. 2012). likewise, the mechanisms and adaptations by which queens and males influence paternity outcomes are still largely unknown (baer et al. 2001; den boer et al. 2009; den boer et al. 2010), because sexual selection has been considerably understudied in the social insects (boomsma et al. 2005). also, very little is known about the conflicts mediating paternity of sexual offspring (moritz et al. 2005; hughes and boomsma 2008), as most studies have analyzed paternity in worker offspring. finally, understanding the consequences of polyandry for the evolution of male and female traits could substantially improve current breeding programs of commercial species, such as honeybees. for instance, incorporating male selection into current honeybee breeding programs, which thus far focus exclusively on queen or colony traits (bienefeld et al. 2008), could substantially increase breeding efficiency by improving drone and sperm quality, assuring high queen mating success, and speeding up the whole process of selecting desirable traits. standardizing research methods could aid such future research efforts by preventing common errors, raising confidence in the reported data, and facilitating comparisons between studies. a first step towards this standardization could be to employ a similar terminology and to report comparable paternity estimates, along with their associated error measures. the unification of available software into a common open source platform such as r (r core team 2013), could also facilitate analyses as well as enhance collaborative work. finally, it is very important to make paternity data available through open access data bases or data repositories, so that they can be used in comparative studies and re-analyzed when new analytical tools become available. r jaffé polyandry in social insects6 acknowledgements i thank vera l. imperatriz-fonseca for general support, jacobus j. boomsma, sheina koffler, robin f. a. moritz and two anonimous referees for constructive criticism, and the fundação de amparo à pesquisa do estado de são paulo for funding (fapesp 2012/13200-5). references allendorf, f.w., hohenlohe, p.a. & luikart, g. 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(2006) comparative analysis of worker reproduction and policing in eusocial hymenoptera supports relatedness theory. american naturalist, 168 (6):e163-e179. doi:10.1086/508619 doi: 10.13102/sociobiology.v65i4.3428sociobiology 65(4): 766-769 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a scientifc note on validation of housekeeping genes for the primitively eusocial bee euglossa viridissima friese (apidae: euglossini) one of the most crucial factors for studies of gene expression of target genes is the procedure of normalization (kubista et al., 2006). for this purpose, reference genes (or house-keeping genes) are used, which are assumed to be constitutively expressed and hence their expression is expected to be stable and constant irrespective of developmental stage or any other experimental manipulation. studies of candidate gene expression, using quantitative real time pcr, often focus on the evaluation of differences in gene expression amongst developmental stages or within a stage amongst different tissues (lockett et al., 2016). candidates for suitable reference genes have been identified in several species of the apidae, e.g. honeybees (lourenço et al., 2008), bumble bees (horňáková et al., 2009; niu et al., 2014), and stingless bees (dallacqua et al., 2007). however, reference genes have not been described for orchid abstract studies on the expression of genes in different contexts are essential to our understanding of the functioning of organisms and their adaptations to the environment. gene expression studies require steps of normalization, which are done using the stable expression pattern of reference genes. for many different eusocial bees reference genes have been discovered, but not for the primitively eusocial euglossini bees. we used available genomic resources of euglossini species and the gene information of apis mellifera linnaeus to develop a set of reference genes for the primitive eusocial bee euglossa viridissima friese. we tested nine genes, in distinct developmental stages, using three different algorithms, to infer stability of gene expression. the tata-binding protein (tbp) and 14-3-3 epsilon were the most stable genes across all developmental stages. the strongest deviation in gene expression pattern occurred in pupae, which require a different set of genes for normalizing gene expression. sociobiology an international journal on social insects s boff1,2, a friedel1, a miertsch1, jjg quezada-euán3, rj paxton1,4, hmg lattorff1,4,5 article history edited by cândida aguiar, uefs, brazil received 09 may 2018 initial acceptance 08 june 2018 final acceptance 24 july 2018 publication date 11 october 2018 keywords developmental stage, gene expression, orchid bees, gene normalization. corresponding author samuel boff facoltá scienze agrarie e alimentari università degli studi di milano via celoria 2, 2133, milano, italy. e-mail: samboff@gmail.com bees, although transcriptome and genome data are available for a few of these species (woodard et al., 2011; kapheim et al., 2015; brand et al., 2017). here we identified a set of candidate reference genes for the orchid bee euglossa viridissima friese based on the transcriptome reference for euglossa cordata (linnaeus) (woodard et al., 2011) and annotated genes of apis mellifera linnaeus (elsik et al., 2015). we tested for stability of gene expression in individuals across different developmental stages (including adults). we choose nine candidate reference genes, namely ribosomal protein 28s (28s), actin, arginine kinase (argk), elongation factor 1a (ef-1a), 14-3-3 epsilon, glutathione s-transferase-1 (gst1), inositol 1,4,5-triphosphate receptors (itpr), ribosomal protein s18 (rps18) and tata-binding protein (tbp). for argk we used two different primer pairs, 1 institut für biologie, martin-luther-universität halle-wittenberg, halle (saale), germany 2 dipartimento di scienze per gli alimenti la nutrizione, l’ambiente, università degli studi di milano, milano, italy 3 departamento de apicultura tropical, campus ciencias biológicas y agropecuarias, universidad autónoma de yucatán, mérida-xmatkuil, mexico 4 german centre for integrative biodiversity research (idiv) halle-jena-leipzig, leipzig, germany 5 international centre of insect physiology and ecology (icipe), nairobi, kenya short note sociobiology 65(4): 766-769 (october, 2018) special issue 767 argk and argk_mod. tata-binding protein (tbp) is often considered stable in gene expression studies and is useful for gene normalization (yüzbaşioğlu et al., 2010). this gene is related to rna polymerase ii transcription factor. the 14-3-3 epsilon protein is abundantly expressed in the central nervous system (cns), modulating the activity of a number of kinases and ion channels in humans (berg et al., 2003). although the gene 14-3-3 epsilon is linked to several functions in flies (e.g. embryonic hatching, germ cell migration, gonad formation, wing venation and eye development, according to flybase), this gene was found to be consistently expressed in queens and workers of honeybees (santos & hartfelder, 2015) providing evidence for its suitability for gene normalization. raw reads of the e. cordata transcriptome (woodard et al., 2011) were mapped against the nucleotide sequences of a. mellifera representing the respective housekeeping genes using clc bio genomics workbench v7.5 (clc bio, arhus, denmark). read mapping was performed using the large gapped read mapping algorithm followed by local realignments. finally, consensus sequences were extracted and transferred to primer3 (untergrasser et al., 2012), which was used with default settings, except that the target product size was set at a maximum of 150 bp. an exception was actin with 271 bp (see locke et al., 2012). primer sequences can be found in table 1. a qiaxcel capillary electrophoresis system (accuracy to 3 bp) was used to confirm pcr product sizes based on the expected size. rna was extracted from whole bodied larvae (n = 9) and pupae (n = 8) and the abdomens from adult bees (n = 9) that were collected from14 nests (department of apiculture, uady, méxico). with exception of larvae, bees were morphologically sexed, and only females were used for our analysis. gene stability was based on reffinder. this software compares and ranks the results from four different algorithms (bestkeeper, delta ct, genorm and normfinder) and reports stability of the gene based on standard deviations of ct values (xie et al., 2012). since the bestkeeper algorithm uses the original ct values as input assuming normality of the data (although they are logarithmic in nature), it seems to indicate false differences (livak & schmittgen, 2001; khanlou & bockstaele, 2012; chen et al., 2016). here, we did not use bestkeeper in our analysis. the 10 primers designed to amplify 9 reference genes (table 1) each produced a single product of the expected size. the pcr efficiency for the primers ranged from 85 93.5% (mean ± s.d. = 1.78 ± 0.06). for larvae, the stability value (sv) of rps18 (1.68) and 14-3-3 epsilon (1.73) were smallest and thus, the most stable reference genes (fig 1a) indicated by the comprehensive ranking analysis. in pupal samples, argk, supported by both pairs of primers, argk (1.41) and argk_mod (1.86), and actin (4.12) were the most stable genes (fig 1b). in adult samples, the gene gst1 (1.86) was the most stable, followed by tbp (2.55) (fig 1c). when larvae, pupae and adults were analysed together, tbp (1.48) was the most stable gene, followed by 14-3-3 epsilon (2.78) (fig 1d). table 1. details of the reference genes designed developed and tested in samples of euglossa viridissima. actin, however, was not designed in this study (see locke et al., 2012 ). bp = base pairs. f = forward primer sequence and r = reverse primer sequence. at = annealing temperature. gene name and symbol amplicon size (bp) sequence at molecular function actin 271 f cgtgccgatagtattcttg rcttcgtcaccaacatagg 60 motility, morphological changes, cell division and intracellular movementφ arginine kinase (argk) 138 f-cccagctggtgaattcatcg r-tccaaaccagacagagtgct 60 arginine kinase activity⊗ arginine kinase modified (argk_mod) 143 f-atgctcccgacgctgaatc r-ggtcaagattgccaaggga 60 arginine kinase activity⊗ elongation factor f1 (ef1a) 176 f-ccaatttctggttggcacgg r-agaggaagacggagagcctt 58 catalyzes the delivery of aminoacyl-trna to the ribosome in a gtp-dependent manner⊕ 14-3-3 epsilon 118 f-ataagggcgtcgagaggaag r-cacgggagcagatgtttgtc 60 protein binding, protein heterodimerization activity⊗ glutathione s-transferase-1 (gst1) 141 f-tccaaagaaacgtgcactcg r-gccgtgccaatactttcgaa 58 ddt-dehydrochlorinase activity and glutathione transferase activity⊗ inositol 1,4,5-triphosphate receptor (itpr) 85 f-tgggtgacactccttcttca r-tgtcgtgcgtttcatcaaga 58 inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity⊗ ribosomal protein s18 (rps18) 87 f-attcttcgtgtcatgggcac r-gtaccgacgacctacacctt 60 rna binding and structural constituent of ribosome⊗ ribosomal protein (28s) 139 f-ctcccctagtagaacgtcgc r-cgacctgataccgtacgagt 60 rrna endonuclease activity and rrna n-glycosylase activity∗ tata-binding protein (tbp) 150 f-gggtgaagaagatggtgcag r-acaccgaccaaatctccagg 60 general rna polymerase ii transcription factor⊗ information source: *gene ontology (http://geneontology.org/), flybase (http://flybase.org/), φhennessey et al., 1993, ⊕marygold et al., 2017). s boff, a friedel, a miertsch, jjg quezada-euán, rj paxton, hmg lattorff – housekeeping genes for euglossa768 out of nine potential endogenous genes, we suggest two (tbp and 14-3-3 epsilon) to be used for controlling gene normalization and for assessing relative gene expression of target genes. it is important to state that another set of genes (tbp and rps18) was considered stable for immature samples. this may imply that alternatively, rps18 has reasonable applicability to analyze gene expression in non-adult bees. although, groups of most stable genes varied amongst different developmental stages, the genes tbp and 14-33 epsilon were among the three most stable in three of four (larvae, adult and all samples analyzed together) sets that we tested (see fig 1). thus, it might be of benefit to use these housekeeping genes to study gene expression in primitively eusocial species like euglossini bees, especially because recent studies showed that there are major dissimilarities between ages and environment (lockett et al., 2016), in different tissues (e.g. brain and abdomen), and between solitary and eusocial bee species with respect to transcriptional regulation (jones et al., 2017; kapheim et al., 2015). fig 1. stability values (geomean ranking values) of 9 reference genes according to reffinder. the most stable genes, those with lower values, are listed on the left and the least stable genes on the right. (a) larvae, (b) pupae, (c) adult (abdomen of females) and (d) all of individuals combined. epsilon = 14-3-3 epsilon; argkm = argk_mod. acknowledgement we thank anonymous referees, cnpq (conselho nacional de desenvolvimento científico e tecnológico) and the program science without borders brazil (ciências sem fronteiras) for financial support (sb). supplementary material doi: 10.13102/sociobiology.v65i4.3428.s2233 link: http://periodicos.uefs.br/index.php/sociobiology/rt/ suppfiles/3428/0 authors’ contribution sb, rjp conceived and designed the study; jjqe provided samples. sb, hmgl designed the primers. sb, af, am performed experiments and analysis; sb, hmgl analyzed the results. sb, hmgl led the writing. all authors read, discussed and approved the final manuscript. most stable genes least stable genes a b c 0 2 4 6 8 10 rp s1 8 ep sil on tb p ac tin 28 s ef 1a gs t1 itp r ar gk ar gk m g eo m ea n of ra nk in g va lu es 0 2 4 6 8 10 12 ar gk ar gk m ac tin rp s1 8 tb p ef 1a itp r gs t1 ep sil on 28 s g eo m ea n of ra nk in g va lu es 0 1 2 3 4 5 6 7 8 9 gs t1 tb p ep sil on ac tin rp s1 8 ar gk m ar gk ef 1a 28 s itp r g eo m ea n of ra nk in g va lu es 0 2 4 6 8 10 tb p ep sil on ac tin ef 1a gs t1 itp r ar gk _m od ar gk rp s1 8 28 s g eo m ea n of ra nk in g va lu es most stable genes least stable genes d ar gk mtb p tb p tb p tb p sociobiology 65(4): 766-769 (october, 2018) special issue 769 references berg, d., holzmann, c., & riess, o. 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(2010). assessment of housekeeping genes for use in normalization of real time pcr in skeletal muscle with chronic degenerative changes. experimental and molecular pathology, 88: 326329. doi: 10.1016/j.yexmp.2009.12.007 doi: 10.13102/sociobiology.v61i1.52-59sociobiology 61(1): 52-59 (march, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 studies on an enigmatic blepharidatta wheeler population (hymenoptera: formicidae) from the brazilian caatinga jc pereira1, jhc delabie3,4, lrs zanette1, y quinet1,2 introduction the myrmicine ant genus blepharidatta is a strictly neotropical group that was described by wheeler (1915) from workers of blepharidatta brasiliensis wheeler collected near belém (state of pará, brazil), in the amazon forest. together with the genus wasmannia, it forms the monophyletic tribe blepharidattini (wheeler & wheeler, 1991; bolton, 1995), which is considered to be a close relative of the fungus-growing ant tribe attini (schultz & meier, 1995). up to seven species are currently recognized (silva, 2007), but most of them are waiting for a formal taxonomic treatment or confirmation. based on morphological as well as behavioral data, only three species are formally recognized: b. brasiliensis found in the amazonian forest, blepharidatta conops kempf, an inhabitant of savanna-like formation from central brazil (cerrado), and an undescribed species (blepharidatta sp-ba, hereafter) known from the atlantic rainforest abstract blepharidatta is a rare neotropical ant genus formed by predatory species whose small colonies nest in soil or leaf-litter. a population of blepharidatta that presents affinities with blepharidatta conops kempf was found in the caatinga biome, at the “reserva particular do patrimônio natural serra das almas” (rppnsa), in crateús (state of ceará, brazil). the aim of our study was to obtain data on the nest architecture, size and composition of colonies, foraging behavior, and female castes morphology for this newly found population, and to compare it with other blepharidatta species, particularly with b. conops. the results show that blepharidatta sp. and b. conops share key features of their biology such as their basic nest architecture, diet and foraging behavior, and the presence of a single ergatoid queen with a phragmotic head. however, marked differences were also found in head and mesosoma morphology of the queen, nest architecture, colony size, and queen location in the nest. two alternative hypotheses are presented. the newly found blepharidatta population represents a new species, possibly endemic to the caatinga biome or it represents an extreme of the phenotypic variations observed among the populations forming b. conops. sociobiology an international journal on social insects 1 universidade federal do ceará, fortaleza, ceará, brazil 2 universidade estadual do ceará, fortaleza, ceará, brazil 3 universidade estadual de santa cruz, ilhéus-itabuna, bahia, brazil 4 ceplac/cepec centro de pesquisas do cacau, itabuna, bahia, brazil article history edited by gilberto m m santos, uefs, brazil received 02 october 2013 initial acceptance 28 november 2013 final acceptance 29 january 2014 keywords: northeastern brazil, semiarid environment, ants, blepharidattini, ergatoid queen corresponding author: yves quinet laboratório de entomologia instituto superior de ciências biomédicas – iscb universidade estadual do ceará fortaleza, ce, brazil, 60714-903 email: yvesq@terra.com.br of the state of bahia, eastern brazil (rabeling et al., 2006; brandão et al., 2008; cassano et al., 2009) (fig 1). all are ground-dwelling predatory species that nest in the ground or in the leaf-litter, with small monogynous or polygynous colonies (brandão et al., 2001; rabeling et al., 2006; silva, 2007). queens are ergatoid (sensu peeters, 1991, i.e. permanently wingless and worker-like) and, at least in b. conops, it is believed that the foundation of new colonies is by fission of established colonies (brandão et al., 2001). the best studied species is b. conops (diniz & brandão, 1997; diniz et al., 1998; brandão et al., 2001, 2008). b. conops colonies (up to 250 workers) live in simple nests with a single opening and a vertical tunnel with 2 cm of diameter and 20 cm deep that ends in a cone-shaped widening at the bottom. furthermore, all mature nests have a horizontal subsidiary chamber connected to the vertical tunnel through a narrow tunnel that opens at the mid-length of the vertical tunnel. it serves as a refuge for queen and brood when research article ants sociobiology 61(1): 52-59 (march, 2014) 53 nests are visited or inhabited by myrmecophiles or predators. queens of b. conops have a characteristic phragmotic head that together with the anterior slope of pronotum forms a frontal disk whose shape and dimensions fit the subsidiary chamber entrance and is used by the queen to block that entrance (brandão et al., 2001). the foragers patrol a roughly circular area around the nest opening, where they collect live and dead arthropods (mainly other ants) to feed their larvae (diniz & brandão, 1997). in the bottom area of the nest, prey is dismembered, chewed and fed to larvae via trophallaxis. the discarded remains are arranged in a ring around the nest opening and workers concealed in the nest entrance ambush arthropods, including other ants, attracted by the carcasses ring (diniz et al., 1998). the other known species of blepharidatta have different nesting habits: their smaller colonies (mean number of workers varying from 112 to 132) are found within the leaflitter, in natural cavities between leaves or in rotting branches (rabeling et al., 2006; silva, 2007). contrary to b. conops, the queens of b. brasiliensis and blepharidatta sp-ba do not have a phragmotic head (silva, 2007). colonies of b. conops occur in locally dense but widely scattered populations, with large areas of cerrado devoided of b. conops (diniz & brandão, 1997; brandão et al., 2001). according to brandão et al. (2001), this distribution pattern may be explained by the limited dispersal mode of ergatoid queens and by the type of nest foundation (fission of established colonies). such nest distribution may be characteristic of all blepharidatta species and probably explains why ant species of this genus are considered rare (brandão et al., 2001). here we present data on nest architecture, size and composition of colonies, foraging behavior, morphology (female castes) of a caatinga population of an unidentified species of blepharidatta (quinet & tavares, 2005). we compare our findings to the data available for other species of blepharidatta, particularly b. conops and discuss the identity of this potential new taxon material and methods study site the study was conducted from november 2011 to june 2012 in a 4.5ha area of the “reserva particular do patrimônio natural serra das almas rppnsa” (5º08’s, 40º51’w), a 6146ha protected area of deciduous thorny woodland vegetation (caatinga) in crateús (state of ceará, northeastern brazil, 5º10’s 40º40’w) (fig 1). nest architecture, size and composition of colonies twenty nine nests of blepharidatta sp. were located in the study area, using freshly killed termites as baits. any blepharidatta sp. worker that picked a termite was then followed back to its nest. nineteen nests were excavated to study nest architecture as well as the size and composition of colonies. before initiating a nest excavation, the maximum diameter of the nest opening and of the carcasses ring around it was recorded (see diniz & brandão, 1997). the carcasses forming the ring were collected, and a 30-cm deep trench was dug in order to obtain a 20-cm side cube with the nest opening in the middle of the fig. 1. distribution of known blepharidatta populations and study site location in the state of ceará (brazil) (rppnsa: reserva particular do patrimônio natural serra das almas). () blepharidatta brasiliensis; () blepharidatta conops; () blepharidatta sp-ba;  studied blepharidatta new population ceará crateús rppnsa caatinga cerrado amazon atlantic forest 0 500 1000 1500 km fortaleza jc pereira et al an enigmatic population of blepharidatta from northeastern brazil54 upper face. starting from one lateral side, the cube was sliced with a spatula until a nest chamber or a tunnel was found. the depth, maximum height and maximum diameter of each chamber were recorded, as well as the direction, diameter and length of each tunnel leading to a chamber. all biological material (workers, queen(s), male(s) and brood; invertebrate and vegetal fragments; myrmecophiles) found in chambers or tunnels was collected, and, whenever possible, its exact location in the nest was recorded. workers, queen(s), male(s), brood and myrmecophile organisms found in each nest were counted and fixed in ethanol 90%. diet and foraging behavior in order to obtain information on blepharidatta sp. diet, all invertebrate and vegetal fragments found in the carcasses ring and chambers of 10 excavated nests were analyzed. fragments were first separated in three categories: ants, other invertebrates and seeds. ant fragments were identified at least to genus level. fragments of other invertebrates were identified to order level. the diel foraging activity pattern was investigated by monitoring three colonies for a 24h period: the first from 12/16/2011 (10 a.m.) to 12/17/2011 (9 a.m.), the two others from 06/16/2012 (9 a.m.) to 06/17/2012 (8 a.m.). the nest opening of each colony was observed for 10min every hour and all ants that left or entered the nest were counted. in total, four hours of foraging activity were monitored per nest. soil surface temperature was recorded each time ant activity was measured. the density and foraging area size of blepharidatta sp. nests were assessed using a 144 m2 area (12 x 12 m) with a grid of 1m2 quadrats (fig 6). each quadrat was baited with sardine and checked 40 min later. baiting was repeated three times in three successive days. each quadrat whose bait was visited by blepharidatta sp. workers at least one time was then baited four times, on three consecutive days, with dead workers (or soldiers) of nasutitermes sp. all workers carrying termites back to the nest were followed and their path marked with pieces of plastic straw. body measurements the maximum transverse diameter of the frontal disk (phragmotic head plus anterior slope of the pronotum) of 10 blepharidatta sp. queens (one per nest) and the maximum head width of 54 workers (from 18 nests) were measured under a stereomicroscope with an ocular micrometer. a petri dish filled with fine white sand was used to correctly position the ants under a microscope. total body length of 10 queens (from the foremost part of the frontal disk to the tip of gaster, in individuals with outstretched body) and 20 workers (from the middle part of the clypeus to the tip of the gaster, in individuals with outstretched body) were also measured. voucher specimens of blepharidatta sp. are deposited at the myrmecological collection of the laboratório de entomologia, universidade estadual do ceará, in fortaleza, ce, brazil, at the myrmecological collection of the museu de zoologia of the universidade de são paulo [mzsp] in são paulo, sp, brazil, and at the myrmecological collection of the centro de pesquisas do cacau [cpdc], ceplac, in itabuna, ba, brazil. results nest architecture, size and composition of colonies all excavated nests (n=19) had a carcasses ring that surrounded the nest opening, with invertebrate fragments sometimes partially obstructing it. in three nests, however, the carcasses ring was inconspicuous, with very few invertebrate or vegetal fragments. carcasses rings were circular or elliptic, with maximum diameter varying from 5 to 20cm. mean fragment density in the carcasses ring was 8 (± 6.3) fragments/ cm2 (n=10; range = 1.7-19.1 fragments/cm2). all nests had only one circular (sometimes elliptic) nest opening whose mean diameter was 0.73 (± 0.17) cm (n=14; range: 0.5-1 cm). each nest entrance was connected to a tunnel with approximately the same mean diameter (0.74 ± 0.23 cm; n=15; range = 0.5–1.3 cm), that led to a first chamber (carcasses chamber), and ended in a bottom chamber (fig 2). in six nests, the tunnel was a straight and vertical structure connecting the nest opening to the carcasses and the bottom chambers (fig 2). in other seven nests, the tunnel had sloped parts, with, sometimes, a branching pattern (fig 2). the bottom chamber was located at a mean depth of 26.5 (± 6.3) cm (n=19; range = 15-40 cm). it housed the fig 2. schematic drawing of four blepharidatta sp. nests excavated from 11/2011 to 06//2012, at the “reserva particular do patrimônio natural serra das almas”, ce, brazil. r: carcasses ring; o: nest opening; t: tunnel; cc: carcasses chamber; bc: bottom chamber; uc: undefined chamber. 2 cm o cc bc r r t o cc bc r r 2 cm cc t t t 2 cm r r o cc t bc cc o cc bc r r 2 cm uc uc uc t t sociobiology 61(1): 52-59 (march, 2014) 55 queen and brood in most nests (n=8) where a queen was found (n=12). its mean maximum height and diameter were 2.3 (± 1.54) cm (n=12; range: 0.7-2.5 cm) and 3.3 (± 1.07) cm (n=12; range: 2-4 cm) respectively. a carcasses chamber, so called because it was full of carcasses, was found in most (14 out 19) nests. it was located very close to the nest opening (fig 2), at a mean depth of 4.9 (± 2.4) cm (n=14, range: 1-11 cm), and its mean maximum height and diameter were 1.9 (± 0.95) cm (n=12; range: 0.52.5 cm) and 2.8 (± 0.8) cm (n=12; range: 2-4 cm), respectively. in six nests, one to two additional carcasses chambers were found at a depth varying from 10 to 25 cm (fig 2). in one nest, up to five chambers were found (a carcasses chamber, a bottom chamber and three extra chambers), in addition to branching tunnels (fig 2). a possible subsidiary chamber, as defined by brandão et al. (2001) for b. conops, was found in one nest. this chamber was located at the mid-length of the vertical tunnel leading to the bottom chamber, at a depth of 13 cm, and contained a queen, brood and 15 males. queens were missing in seven nests, possibly because they escaped during the excavation process. all other nests (n=12) had a single queen, except one that had two queens. most queens (8 out 13) were found in the bottom chamber together with brood. in three remaining nests, the queen was found in the carcasses chamber (n=2) or in the subsidiary chamber (sensu brandão et al., 2001) (n=1). two queens could not be assigned to a specific nest location. a total of twenty five males were found, in chambers of four nests, together with the queen and brood. fifteen of them were found in the subsidiary chamber of one nest; in the three other nests, from one to five males were found in the bottom chamber. three myrmecophiles were frequently found in blepharidatta sp. nests, almost always in the bottom chamber: two species of the crustacean genus trichorhina buddelund (oniscidea, platyarthridae) and one cockroach species (corydiidae); we also noticed the frequent occurrence of the pseudoscorpion petterchernes brasiliensis heurtault (chernetidae), up to now recorded only from burrows of small mammals in the state of pernambuco, brazil (heurtault, 1986). average colony size (number of workers) was 193 (± 107.4) (n=19; range: 30-437). most colonies (13 out 19) ranged from 110 and 220 workers; a few (n=4) reached more than 300 workers (one of them had more than 400 workers) (fig 3). queen and worker morphology blepharidatta sp. queens are ergatoid and have an enlarged phragmotic head that, together with the anterior slope of the pronotum, form a nearly circular disk (fig 4) whose mean maximum transverse diameter is 1.68 (± 0.04) mm (n=10; range: 1.60-1.76 mm) (table 1). frontal disk cuticle is discretely reticulate-punctate, nearly smooth; head margin is strongly curved upwards, and stiff hairs protrude laterally from the perimeter of the disk (fig 4). total mean body length of queens is 4.96 (± 0.12) mm (n=10; range: 4.8-5.20 mm). blepharidatta sp. workers are monomorphic (mean body length: 3.79 ± 0.22 mm; n=20; range: 3.50-4.48 mm), and their mean maximum head width is 0.96 (± 0.05) mm (n=54; range: 0.84-1.04 mm) (table 1). fig 3. distribution (%) of colony size categories for 19 nests of blepharidatta sp. excavated from 11/2011 to 06/2012, at the “reserva particular do patrimônio natural serra das almas”, ce, brazil. fig 4. phragmotic head of blepharidatta conops (a) (photo by april nobile – www.antweb.org) and blepharidatta sp. (b) queen. 0.5 mm 0.5 mm b a table 1. mean maximum transverse diameter (mm) of queen (q) frontal disk and mean workers (w) maximum head width (mm) in different blepharidatta conops populations (sr, ba, sel, ch-g) and in the blepharidatta population found at the “reserva particular do patrimônio natural da serra das almas” (rppnsa), in crateús (state of ceará). sr: serranópolis (state of goiás); ba: balsas (state of maranhão); sel: selvíria (state of mato grosso do sul); ch-g: chapada dos guimarães (state of mato grosso). data within parenthesis refer to standard errors of the means. data source for b. conops: brandão et al. (2001). blepharidatta conops blepharidatta sp. sr ba sel ch-g rppnsa q 1.55 (± 0.03) n=12 1.2 (± 0.06) n=5 1.45 n =1 1.51 n=1 1.68 (± 0.04) n=10 w 0.87 (± 0.04) n=5 0.75 (± 0.05) n=5 0.78 n=1 --0.96 (± 0.05) n=54 jc pereira et al an enigmatic population of blepharidatta from northeastern brazil56 diet and foraging behavior in total, 13,576 fragments were found in the carcasses ring and chambers of 10 blepharidatta sp. nests. of these, 8,385 (62%) were from ants, 4,660 (34%) from others invertebrates, and 522 (4%) were whole seeds or pieces of seeds. most invertebrate and seed items were found in the carcasses ring (77% and 85% respectively); the rest was found in nest chambers, mostly in carcasses chambers. most ant fragments (93%) could be identified; 61% of the other invertebrate fragments and none of the seeds could be identified. ant carcasses included 41 species from 18 (or 19) genera belonging to seven subfamilies (table 2). three species accounted for 50% of all ant carcasses. the most common species was ectatomma muticum mayr (ectatomminae) (23%), followed by camponotus crassus mayr (formicinae) (16%) and labidus sp. nr. coecus (ecitoninae) (11%). other eight species (acromyrmex rugosus (smith), gnamptogenys striatula mayr, camponotus sp., cephalotes pusillus (klug), eciton sp., crematogaster sp., pheidole sp., odontomachus bauri emery) were frequent, accounting each for 3 to 7% of all ant carcasses. it is also worth mentioning the ecitoninae group since 18% of all ant carcasses were from eciton (3 species), labidus or neivamyrmex (4 species), nomamyrmex (1 species) and an unidentified genus (1 species). other invertebrate carcasses were from eight insect groups (blattodea [including termites], coleoptera, dermaptera, hemiptera, hymenoptera, lepidoptera, neuroptera, orthoptera), and two arachnid groups (araneae, scorpiones). however, the vast majority (92%) of carcasses were from four groups: hemiptera (37.5%), coleoptera (22.4%), araneae (19.4%) and termites (13%) (all termite fragments were heads of nasutitermes sp. soldiers). foraging activities of blepharidatta sp. are predominantly crepuscular, with two peaks of activity, the first one corresponding to the night/day transition period (5 a.m.-9 a.m.), the second one to the day/night transition period (4 p.m.-7 p.m.) (fig 5). at other periods, especially during the warmest period of the day (± 10 a.m.-3 p.m.), foraging activity stops almost entirely, at least at the nest opening (fig 5). however, at night, from 7 p.m. to 2 a.m., intense cleaning activity was observed in all nests, with workers carrying carcasses or sand particles out of the nest and depositing them on the carcasses ring, while others removed carcasses obstructing the nest opening and organized them on the carcasses ring. during the period when nests foraging activity was monitored (up to 12 hours of nest opening observation for the three nests), it was observed that the carcasses ring was frequently visited by invertebrates, including by ants that robbed carcasses and sometimes inspected blepharidatta sp. nest opening. however, none of these visitors was ambushed by blepharidatta sp. workers, contrary to what was observed in b. conops, whose workers, concealed inside the nest entrance, frequently ambush visitors (diniz et al., 1998). collective transport of large prey items was observed in blepharidatta sp. in one occasion. more than 12 workers table 2. list of subfamilies and genera identified in ant carcasses found in the carcasses ring and nest chambers of 10 blepharidatta sp. nests, with number of species found in each genus. subfamily genus species number dolichoderinae dolichoderus 1 dorymyrmex 1 ectatomminae ectatomma 1 gnamptogenys 1 ecitoninae eciton 3 labidus/neivamyrmex 4 nomamyrmex 1 unidentified genus 1 formicinae brachymyrmex 1 camponotus 5 myrmicinae acromyrmex 1 cephalotes 1 crematogaster 4 cyphomyrmex 1 pheidole 10 solenopsis 2 strumigenys 1 ponerinae odontomachus 1 pseudomyrmecinae pseudomyrmex 1 a b figure 5. daily cycle of activity of two blepharidatta sp. colonies (a, b) at the “reserva particular do patrimônio natural serra das almas”, ce, brazil. all ants exiting/entering the nest were counted during 10 minutes, during a 24-hour observation period (a: 16.12-17 .12.2011; b: 16.06-17.06.2012). bars: foraging ants/10 min; line with dots: soil surface temperature. sunrise: 5.30 h (onset of daylight: 6.00 h); sunset: 17.30 h (beginning of darkness: 18.00 h). the daily cycle of activity of the third monitored nest (16.06-17.06.2012), not shown here, had similar pattern. sociobiology 61(1): 52-59 (march, 2014) 57 were observed grasping and pulling the appendages of two still alive and fighting ectatomma muticum workers, trying to transport them to the nest. only two nests were found in the 144 m2 area mapped (three if a nest close to the area border is included) (fig 6), giving a nest density of 0.020 nest/m2. although some foragers were observed foraging at a distance of seven meters from the nest, the most common foraging distances observed were two to three meters (fig 6). blepharidatta sp. nest foraging area can therefore be described as a nearly circular area with a radius of ± 2.5m around the nest opening. discussion there is no doubt that b. conops and blepharidatta sp. belong to a group of closely related taxa since they share key features of their behavior, ecology and morphology, while at the same time both show fundamental differences with the other blepharidatta species living in tropical humid environments. b. conops and blepharidatta sp. are soil-dwelling species with nest opening surrounded by a carcasses ring (diniz et al., 1998), while b. brasiliensis and blepharidatta sp-ba nest in leaf-litter, and do not form carcasses ring (rabeling et al., 2006; silva, 2007). b. conops and blepharidatta sp. are monogynous species whose ergatoid queens have a phragmotic head (brandão et al., 2001), while b. brasiliensis and blepharidatta sp-ba have polygynous colonies, and queens do not have a phragmotic head (rabeling et al., 2006; silva, 2007). it is worth mentioning, however, that polygyny or monogyny of blepharidatta sp-ba remains a controversial question (silva, 2007). frontal disk diameter in queens, head width in workers and body length in queens and workers are almost identical in both species (table 1) (diniz et al., 1998; silva, 2007). on the other hand, body length in queens and workers of b. conops and blepharidatta sp. (± 5 mm for queens; ± 3 mm for workers) is almost three times that observed in b. brasiliensis and blepharidatta sp-ba (± 2mm and ± 1mm for queens and workers, respectively) (silva, 2007). blepharidatta sp. and b. conops have similar diet and foraging activity patterns, with two daily and mostly diurnal peaks of foraging activity separated by periods of inactivity, and a protein diet predominantly made of other ant species (diniz et al., 1998; brandão et al., 2008). in b. brasiliensis, foraging activity is predominantly nocturnal (rabeling et al., 2006). finally, the two groups of populations are found in completely different environments: rainforests (amazon and atlantic rainforest) for b. brasiliensis and blepharidatta spba (rabeling et al., 2006; silva, 2007); drier and savannalike environments (cerrado and caatinga) for b. conops and blepharidatta sp. (diniz et al., 1998; brandão et al., 2001). according to silva (2007), b. conops possesses more derived character states (e.g. phragmotic head of queens) when compared to other blephadatta species. considering the number of key behavioral and morphological features shared by b. conops and blepharidatta sp., it can be stated that blepharidatta sp. shares with b. conops this apomorphic condition. however, our study also shows the existence of many differences between the populations living in the cerrado biome (b. conops) and the population found in the caatinga biome (blepharidatta sp.). the most noticeable are those related to queen morphology. in blepharidatta sp., frontal disk cuticle is discretely reticulate-punctate, nearly smooth, while it is rugose with a strong relief composed of closely packed polygonal units delimited by ridges in b. conops (brandão et al., 2001) (fig 4). furthermore, other morphological differences, not detailed above, can be seen in the mesosoma sculpture, much stronger in the b. conops queen than in blepharidatta sp., in the humeral angle of the pronotum which is sharply angulated in blepharidatta sp. but rounded in b. conops. finally, the pilosity on the first gastral tergite of the queen is erect and short in blepharidatta sp. while it is subdecumbent and a little longer in b. conops. blepharidatta sp. and b. conops also differ in relation to nest architecture, colony size, location of queens and brood in nests and foraging behavior. blepharidatta sp. nests are deeper (± 26.5 cm; up to 40 cm in some nests) than in b. conops (up to 20 cm in mature nests) (diniz et al., 1998). conversely, the mean diameter of tunnels and nest opening is much greater in b. conops (2cm versus 0.7cm in blepharidatta sp.) (brandão et al., 2001). in most excavated nests of blepharidatta sp., 12 m 12 m : trail back to the nest with a bait (dead termite) : nest of blepharidatta sp. fig 6. mapping of blepharidatta sp. nests and of the trails used by foragers to go back to the nest with a bait (dead termite) in a 144 m2 (12 x 12 m) area with a grid of 1 m2 quadrats, at the “reserva particular do patrimônio natural serra das almas”, ce, brazil, in 01/2012. jc pereira et al an enigmatic population of blepharidatta from northeastern brazil58 there was one, or more, chambers located close to the nest opening and full of carcasses. the function of those carcasses chambers in blepharidatta sp. is probably to store prey carcasses before discarding them on the carcasses ring. no such chamber was found in b. conops nests. one of the most important differences in nest architecture between b. conops and blepharidatta sp. is the so-called subsidiary chamber. in b. conops, all mature nests have a subsidiary chambers used as refuge by the queen and the brood (diniz et al., 1998; brandão et al., 2001). only one blepharidatta sp. nest had a chamber full with brood, queen and males and whose features suggest a subsidiary chamber. more generally, blepharidatta sp. nests are more variable in structure than b. conops nests. in b. conops nests, there is only one tunnel, always vertical (diniz et al., 1998). in blepharidatta sp., the main tunnel varies from vertical to inclined. furthermore, the main tunnel can have branchings, the result being nests three-dimensionally more complex that those of b. conops. it cannot be excluded that the characteristics of the soil where the nests of blepharidatta sp. were built have a strong influence on nest architecture. however, such characteristics were not analyzed in the present study, nor in studies with b. conops (diniz et al., 1998). as in other blepharidatta ants, blepharidatta sp. colonies are small. however, the average number of workers in blepharidatta sp. colonies (193 ± 107, n=19) is higher than in b. conops (142 ± 57, n=19) (diniz et al., 1998), b. brasiliensis (132 ± 96, n=13) (rabeling et al., 2006) and blepharidatta spba (112 ± 13, n=2) (silva, 2007). one of the blepharidatta sp. nests had up to 437 workers. in b. conops, the largest known colony size is 248 workers (diniz et al., 1998). interestingly, two queens were found in a single blepharidatta sp. nest. this observation supports the hypothesis that in b. conops, the foundation of new colonies is through fission of established colonies (brandão et al., 2001). if this hypothesis is correct, there must be a time when two queens are present in the nest: the old resident queen and a young virgin queen. the location of queen and brood in nests is another difference between b. conops and blepharidatta sp. in most nests of blepharidatta sp., the queen and brood were found in the bottom chamber. in b. conops, they are generally found in the subsidiary chamber, but never in the bottom chamber of the nest, which is used as a place for prey dismembring and to temporarily store carcasses, before they are taken out of the nest (diniz et al., 1998; brandão et al., 2001). foraging behavior of blepharidatta sp. and b. conops is very similar. however, some differences were observed. according to diniz et al. (1998), the ants and other arthropods visiting the carcasses ring of nests “are frequently ambushed by b. conops workers concealed immediately inside the nest entrance”. such behavior was never observed in blepharidatta sp. another significant difference is the size of foraging area. in blepharidatta sp. the mean radius of foraging area (2.5 m) is nearly twice that observed in b. conops (1.5 m), while nest density seems to be much lower in blepharidatta sp. (0.02 nests/m2 against 0.2 nests/m2 in b. conops), at least in the investigated area (diniz et al., 1998). however, the influence of local factors on nest density, such as prey density or competition with other ant species that use similar resources can not be excluded. although it cannot be excluded that blepharidatta sp. is a species different from b. conops, it is obvious that both taxa are closely related and constitute a single evolutionary unit, with a branch that spread into the cerrado biome (savanna) and another into the caatinga biome (semi-arid). the traits that strongly suggest the occurrence of two distinct species are seen in the queen morphology, mainly on the frontal disk and mesosoma. on the other hand, it has been suggested that b. conops distribution in the cerrado biome is fragmented in a range of local populations, with size and sculpture of queen frontal disk varying from one population to the other (brandão et al., 2001). therefore, blepharidatta sp. could be a local variation of b. conops adapted to semi-arid conditions and consequently represent one extreme of the phenotypic variations observed in populations of b. conops. ant populations in species that have lost mating flight have low levels of gene flow and can evolve independently, as it could happen in fragmented populations of b. conops or its ancestor since the early quaternary. future studies should use cytogenetic and molecular tools to compare these blepharidatta populations (e.g. mariano et al., 2008; resende et al., 2010 with dinoponera lucida emery 1901). finally, our study extends the known geographic range of the blepharidatta group and the range of biomes where it is found, as it is the first time that the genus, until now found only in wet forests and savannas (cerrado), is reported for the neotropical semi-arid region. acknowledgments the authors are grateful to rodrigo dos santos machado feitosa (universidade federal do paraná) for the identification of ant carcasses. we are also grateful to leila aparecida souza (universidade estadual do ceará [uece]), sonia maria lopes (museu nacional – universidade federal do rio de janeiro) and mark harvey (western australian museum – australia) for the identification of the myrmecophile fauna (crustaceans, cockroaches and pseudoscorpions, respectively) found in blepharidatta sp. nests. we also thank uece for transport facilities as well as the “associação caatinga” and the staff of the rppn “serra das almas” for allowing us excellent field work conditions. jcp acknowledges her capes fellowship during her msc, and jhc his cnpq research grant. references bolton, b. 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(1991). the larva of blepharidatta (hymenoptera: formicidae). journal of the newyork entomological society, 99: 132-137. spatial distribution and .indd open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 20-29 (2013) spatial distribution and architecture of acromyrmex landolti forel (hymenoptera, formicidae) nests in pastures of southwestern bahia, brazil mr silva junior1, ma castellani1, aa moreira1, m d’esquivel2, lc forti3, s lacau2 introduction the ants (hymenoptera: formicidae) are insects which present a vast taxonomic diversity and occupy a great variety of ecological niche and frequently spread out and become dominant in all habitats of the globe (hölldobler & wilson, 1990; wilson & hölldobler, 2005). one of the biological characteristics which explains this extraordinary ecological success is partly due to its great capacity of modifying or exploring its environment for nests construction (hölldobler & wilson, 1990; passera & aron, 2005). the tribe attini groups the ants which cultivate fungi abstract ants of the genera atta and acromyrmex (myrmicinae: attini) are in general pests for agricultural and pasture areas. in southwestern bahia pastures, in brazil, acromyrmex landolti forel occurs at intermediate densities of 260 nests ha-1. in spite of its economic importance, there is not enough research on the bioecological aspects of such species. this paper aims to study the spatial distribution and the architectural pattern of a. landolti nests, as well as to establish the foundations for the improvement of strategies for controlling the pest. the research was carried out during the period of june 2011 and may 2012, in an area of pastures measuring 2.7 ha, in itapetinga, in bahia. twenty five nests were selected. the external mound area, height of the tower, distance between the tower and the external mound area, as well as the tower diameter were all registered. in order to describe the internal architecture, the same nests were completely excavated and five were molded in cement. the spatial distribution of the nests is of aggregated type. the towers height is 2.1 cm and the mean diameter is 1.2 cm. the amount of loose soil is of about 8.0 cm from the tower, with an mean area of 472.9±312.5 cm² and mean volume of 1.4 ± 0.9 l. the depth of the nests varies between 7.0 and 78.0 cm, with an mean of 33.2±21.29 cm. the nests have, in mean, 4.4±2.0 chambers, being two nests with just one chamber (8%) and another nest (4%) with 11 chambers. the highest frequency of chambers (73.6%) occurs in the first 5-10 cm depth. the closest chambers have tunnels of 0.5 cm in length, and 23% of the tunnels measure 1.0 cm in length, with up to 44.7% of the tunnels measuring up to 30 cm. the mean dimensions of the chambers are: width: 6.2 cm; height: 5.1 cm; and length: 5.7 cm. the majority of the chambers are located near the entrance holes. sociobiology an international journal on social insects 1 southwestern bahia state university uesb, vitória da conquista, ba, brazil 2 southwestern bahia state university uesb, itapetinga, ba, brazil 3 são paulo state university unesp/fca, botucatu, sp, brazil research article ants article history edited by: edilberto giannotti, unesp brazil received: 24 october 2012 initial acceptance: 21 november 2012 final acceptance: 11 march 2013 keywords negative binomial, chambers, depth, channels corresponding author maria aparecida castellani department of fitotecny and zootecny southwestern bahia state university box 95, vitória da conquista, ba brazil, 45.083-900 e-mail: castellani@uesb.edu.br. and are exclusive to the new world, which adds up to a total of 15 genera and 297 valid species (bolton et al., 2006; brandão et al., 2011). the genera which derive from this species, atta and acromyrmex, comprise the real leaf-cutting ants, because they use fresh parts of plants to cultivate their symbiotic fungi reaching the status of agricultural pest in silvicultural and pasture areas. the leaf cutting ants are highly specialized in selecting the plant substrate and in the construction of the nests, with greater structural complexity in the atta species (forti et al., 2011). among the species of the genus acromyrmex, which specialized in grass cutting, we can refer to acromyrmex sociobiology 60(1): 20-29 (2013) 21 landolti forel and acromyrmex balzani emery by its vast geographic distribution and damages caused to pastures. acromyrmex balzani was initially described as subspecies of a. landolti and later was taken to the category of species by fowler (1988). they are very similar species, in the morphological aspects of its workers and in the habits of foraging nest construction. such species construct relatively small nests, however, they generally occur in high densities, causing damage to pastures (gonçalves, 1961; 1967, mariconi et al., 1963; amante, 1967 a, b). in venezuela it was registered densities of 1,000 and 6,000 nests ha-1(labrador et al., 1972; espina & timaure, 1977) whereas in paraguay acromyrmex landolti fracticornis forel may reach a density of 4,400 nests ha-1 making it impossible for ranchers to breed cattle in the infested areas (fowler & robinson, 1977). in the state of bahia, brazil, 900 nests ha-1 were found for the specie a. balzani (lewis, 1975). the nests of leaf-cutting ants can present a casual or uniform spatial distribution in atta, in areas with smaller or bigger density rate of nests, respectively (caldeira et al., 2005). for nests of acromyrmex spp. casual distributions was already registered (cantarelli et al., 2006), while aggregated distribution was registered for a. balzani (caldato, 2010). ants of the genus acromyrmex build their nests in an even simpler way compared with the species of the genus atta, which usually has a small number of chambers. similarities and differences observed in the architecture of the nests, between genera of attini (weber, 1972), also occur between the acromyrmex species of the moellerius group. while most of these species have a typical tube straw surrounding the hole tunnel of the nest, with a unique vertical tunnel, interconnecting the various rounded chambers, the nests of acromyrmex heyeri (forel, 1899) present an amount of loose soil with two or three entrances, with several vertical tunnels connected to a unique chamber located at depth of approximately 40 cm (gonçalves, 1961). in a. balzani, mendes et al. (1992) found a total of six chambers and registered a maximum depth of 1.2 m, whereas silva et al. (2010) found a maximum of fourteen chambers, placed in a depth of 2 m, and the majority of an ellipsoid type. still regarding this species, the greatest amount of chambers are in the first 30 cm placed in the projection of the loose soil. the ant a. landolti build nests with two or three chambers overlapping and linked by a vertical gallery (weber, 1972). there is not enough research about the effects of the ant a. landolti to the conditions of the brazilian pastures. on the other hand, it is known that other acromyrmex species, specially a. balzani, share similarities in morphological aspects, its workers, foraging habit and nest construction. therefore, the aims of this paper were: a) to investigate the spatial distribution and architecture of nests of this species in pasture areas in southwestern bahia, brazil, and b) to compare the found characteristics with those already reported for other acromyrmex species. we hope to contribute to improve the foundations of management strategies of a. landolti nest spreading in southern bahia pastures. material and methods the experiment was carried out in the period from july 2011 to april 2012, in the farm “lagoa de alagoinhas”, in the municipality of itapetinga, bahia, in northeastern brazil. the farm is located at the height of 15°21’s and 40°17’w. it is a rural area mainly taken by extensive cattle pastures (brachiaria sp., poaceae) with 2.7 ha, having a sub-humid mean annual rain fall of 867 mm and mean temperature of and 25.4◦c. the area of investigation is part of the geomorphological unit known as depression of itabuna-itapetinga, where the type of greatest distribution is the “chernossolos argilúvicos órticos”, which correspond to the internal area of the depression, with soils which are imperfectly drained, shallow and rarely deeper than 70 cm. this soil also has high rates of silt and primary minerals (nacif, 2000). in order to carry out the research on spatial distribution, the nests of a. landolti were located, identified with stacks and geo-referenced by using a gps (global position system) device. then, a map was generated with the identification of each nest. the map was subdivided in samples of 10 m x 10 m making a total of 270 quadrants of 100 m². the data were submitted to an analysis of dispersion rates to verify which types of spatial distribution of the nests occur in the area. the indexes of dispersion used were the mean/ variance ratio or index of dispersion (i) and the index of dispersion of morisita (iδ) (davis, 1993). for the description of the external structure, twenty nests were randomly selected respecting the following criteria: 1) nest activity verified by means of the presence of the ants in the entrance holes and the presence of recent loose soil; 2) all selected nests were observed belonging to a set of nests locally organized in nucleus. the following nest characteristics were registered: the measurement of the area of loosen soil distance, height of the tower of the entrance hole to the center of the area of loose soil and its diameter. the amount of loose soil around each nest was collected, stored in plastic bags, identified and taken to the laboratory of animal biosystematics lbsa/uesb in order to establish their volumes by using a beaker. in order to study the internal architecture, the same nests for which external area was investigated, were excavated by means of the procedure described by moreira et al. (2003, 2004 a, b), plus five other nests (adding to a total of twenty five) selected following the two criteria mentioned above. the five extra nests were molded with cement. the excavation started with the opening of a trench, 140 cm long, 80 cm wide and having a depth of 100 cm, with the center of its side wards border at 25 cm from the entrance hole. afterwards, by using a compass, and having as a central point the ms rodrigues junior, ma castellani, aa moreira, m d’esquivel, lc forti, s lacau on the nests of acromyrmex landolti22 centre of the loose soil, the four cardinal points were marked by the use of fine cords 4 m long, fixed in stacks. the spatial location of the chambers were determined in relation to the axes x (east/west) and y (north/south), to the amount of loose soil as well as to the foraging hole. after applying neutral powder in the foraging hole, by using a manual device, the excavation was carried out from the entrance hole till the chambers and channels. the following measures of the chambers and channels were registered: height, width and length, position in relation to the axes x and y. the real volume of each chamber was determined by using a plastic bag which involved the whole room of the chamber internally, within which it was injected enough water to fill up all the internal space of the chamber, by means of a beaker. the estimated volume of the chamber was calculated according to the mathematical models of ellipsis and spherical forms. in order to make cement mould of the nests, the procedures described by moreira et al. (2004 a, b) were adopted. a mix of 5 kg of cement and 10 l of water was injected, by means of a funnel, in the nest entrance hole till the saturation point. after seven days, the nests were excavated and the measures of the chambers and tunnels were registered. the mean values and deviation rates were also calculated upon the data collected. results a total of 701 nests of a. landolti was registered in the study area (fig. 1). the obtained morisita index (iδ) was 1.53, the index of dispersion or mean/variance ratio (i) was 2.39, confirming a contagious distribution (binomial negative) that characterizes the aggregation of the nests. in each investigated nest of a. landolti it was found only one entrance tunnel, despite the existence of up to six towers interconnected. the mean height of towers was of 2.1±0.69 cm, with a minimum height of 1.0 cm and a maximum of 4.0 cm, while the mean diameter found was of 1.2±0.3 cm, with a minimum diameter of 0.7 cm and a maximum of 1.5 cm (table 1). the amount of loose soil was registered at a mean distance of 8.0±4.5 cm from the entrance to the nest tower, with a minimum distance of 2.0 cm, and a maximum of 20.0 cm. the area of the loose soil measured 472.9±312.5 cm², ranging from 116.3 cm² to 1,600.0 cm², with a mean volume of 1.4±0.9 l, with variation from 0.3 l to 4.8 l. the nests had a maximum depth of 78.0 cm and a minimum of 6.0 cm (table 2), with mean depth of 33.2±21.29 cm and 98% of the nests being less than 60 cm deep. in the 25 nests that were excavated, the mean number of the chambers found was of 4.4±2.0, two nests (8.0%) having only one chamber and one nest with 11(4.0%) chambers. the most of the nests with 11 (4.0%) have from 3 to 6 chambers (table 2). the chamber with the smallest depth was found at 0.5 cm below ground level. most of the chambers were encountered before 20 cm in depth (73.6%), with 33 of them built between 5 and 10 cm. no chamber was found between 30 and 35 cm of depth, and also between 60 and 70 cm (fig. 2). the mean width of the chambers was 6.2±7.2 cm (1.5-18.0 cm), the mean height 5.1±2.3 cm (1.5-11 cm) and the mean length 5.7± 2.8 cm (1.5-17 cm) (table 2). the biggest chamber measured 18.0 cm in width, 10.0 cm in height and 12.0 cm in length. on the other hand, the smaller chamber measured 1.5 cm long (table 2). as for the twenty nests marked with neutral powder, the maximum depth registered was 58 cm and the chambers found up to 45 cm in depth were full of fungi, brood and adults. all the chambers located up to 10 cm deep had eggs, larvae and adult ants and there were empty chambers only from this depth onwards. the chambers with soil were found from 25 cm deep onwards, except for the depth interval between 45 cm and 55 cm in depth (fig. 3). the closest chambers were connected by tunnels measuring 0.5 cm of length. we observed that 23% of the chambers had tunnels with 1.0 cm in length and 44.7% had tunnels measuring up to 3.0 cm of length. in total 85 tunnels were found connecting chambers (fig. 4). there was a great variety of real volume of the chambers, from 19.0 ml to 1,335.38 ml, establishing a standard deviation larger to its own mean (223 ± 295.4). the greatest similarity of the chambers, in general, occurred with the spherical form (table 3). however, considering only the four nests that were similar based on the number of chambers (21 in total), we observed that other forms of chambers seems to occur as evidenced by the expressive lack of fit of chamber geometry to the tested models (sphere or ellipsoid). as far as the spatial distribution of chambers is configure. 1. aggregated distribution map of the nests of acromyrmex landolti in the study area (2.7 ha) in itapetinga-ba, brazil, 2012 (15°21’3.87”s / 40°17’29.07”w). sociobiology 60(1): 20-29 (2013) 23 cerned, in relation to the axes x and y, the data show that in the majority of nests the chambers are near the entrance hole. furthermore, the same nests had chambers between the hole and the amount of loose soil, with chambers located before the foraging hole or even sidewards in relation to such hole position (fig. 5). discussion the aggregated spatial distribution of a. landolti nests found in this research was also verified for a. balzani in pastures of são paulo state, brazil (caldato, 2010). nevertheless, nest distribuition differed from the spatial distribution of acromymex spp. nests observed in argentina, which followed the poisson model (cantarelli et al., 2006). several factors influence directly or indirectly the spatial distribution of ant nests. for instance, the presence of competitors can be highlighted (hölldobler & wilson, 1990), habitat complexity, edaphic condition, climate change (fowler, 1983) as well as the composition and spatial distribution pattern of the existing vegetation in the nesting area (zanetti et al., 2000). in acromyrmex striatus roger, the influence of such factors is observed in the choice of type of soil by the recently fertilized females during the nest foundation, in the definition of success and in the abundance of the species. it was reported a higher density of colonies in areas of exhausted soil than in areas of natural fertile soil (diehl-fleig & rocha, 1998). in order to explain the trend towards aggregated distribution of nests of a. balzani, mendes et al. (1992) worked on the hypothesis that there are “marks” of positive conditions derived from the presence of types of grass preferred by this species, a factor that shows clear influence of vegetation cover in the spatial distribution of nests. the same hypothesis can be considered to explain the aggregation of nests of a. landolti. in support of this hipothesis we call attentions on the fact that the pastures in the figure. 2. frequency of the vertical distribution of the chambers in 25 nests of acromyrmex landolti (n = 110). itapetinga, ba, brazil, 2012. figure 3. percent (%) average of empty chambers with fungus and soil, and creates adult, 20 nests of acromyrmex landolti at different depths. itapetinga, ba, brazil, 2012. ms rodrigues junior, ma castellani, aa moreira, m d’esquivel, lc forti, s lacau on the nests of acromyrmex landolti24 southwest of bahia are not very uniform in the “varietal” composition of grass stands, besides being subject to disturbances, such as overgrazzing and regular fires can contribute to the presence of such “marks” with more adequate vegetation substrate. there was a great variety of dimensions in the external structure of nests. it was also confirmed the presence of an entrance hole in each nest, despite the existence of nests with up to six interconnected towers, what matches with other data for a. balzani (mendes et al., 1992; andrade, 1991, pimenta et al., 2007; silva et al., 2010). the typical tower found in a. landolti nests, is probably a mechanism that regulates the temperature and the internal humidity of the colony, thus protecting the nest entrance against changes of temperature and other climate factors, and invasion of predators (perdomo, 2008). the distance of the tower to the amount of loose soil in the nests of a. landolti was in mean (8.0±4.5 cm) smaller than the recorded mean for the nests of a. balzani, in botucatu, são paulo (caldato, 2010). moreover, the mean area figure 4. length distribution of tunnels 85 acromyrmex landolti 25 nests in itapetinga-ba, 2012. figure. 5. schematic drawing of the boundary area of loose soil, the arrangement of the intake tower and the chambers relative to loose soil nests of acromyrmex landolti: n01 and n16-chambers arranged between the area of loose soil and inlet pipe, n02, n12, n14 and n25 most chambers disposed before and sides of the tower. itapetinga, ba, brazil, 2012. sociobiology 60(1): 20-29 (2013) 25 of the loose soil was 472.9±312.5 cm², ranging from 116.3 cm² to 1,600.0 cm², with a mean volume of 1.4±0.9 l, with variation of 0.3 l to 4.8 l. in a general perspective, comparing our results with those of silva et al. (2010), we observe that the area and the volume of loose soil of the nests of a. landolti are respectively 73% and 52% smaller than those of a. balzani. manufacturers of bait pesticides use area of loose soil to calculate the doses of such pesticide to control cutting-ants from 8 to 10g of bait/m2 to atta and of 8 to 10 g/nest to acromyrmex, which has smaller nests. considering that, in mean, the area of loose soil of a. landolti was of 472.9 cm2, with only one nest with area greater than 1 m² (n3) (table 1), the recommendation implies over dosage of the product, leading to a non-usage of baits or to the return of the dosage to the amount of loose soil. this could increase the possibilities of adverse effects on the non-target mirmecofauna. nests of a. landolti are less deep in relation to those of a. balzani, with maximum depth of 78.0 cm, whereas for a. balzani there are records of nests of up to 210 cm (silva et al., 2010), 160 cm (caldato, 2010), 124 cm (mendes et al., 1992), 95 cm (pimenta et al., 2007) and 60 cm (gonçalves, 1961) of depth. similarly, the nests of a. landolti have their first chamber almost at the same level of the ground (from 1.5 to 9 cm deep in the soil), whereas in the case of a. balzani, the depth of the first chamber ranges from 4.0 (caldato, 2010) to 30 cm (silva et al., 2010), suggesting that the upper chamber of the nest is more superficial in the former species. the highest frequency of the chambers was registered in the first 20 cm of depth (fig. 2), and chambers between 30 and 35 cm were not found. between 60 and 65 cm and 65 to 70 cm, just one record since 98% of the nests presented depth inferior to 60 cm. the differences between the two species may be due to the specific preferences in edaphoclimatic conditions according to their nesting habits (forti et al., 2011), and to distinct local conditions for nesting in the study areas above cited. in nests of leaf-cutting ants, the number of chambers, as well as its occupation, varies as a function of many factors, such as age of the nest, site of nest construction, among others. it is possible that there exists a pattern for internal architecture of a. landolti nests, with three to six chambers, since 88% of excavated nests had in mean, 2.3 chambers (soares et al., 2006), while for a. balzani there are records varying from 3 to 6 and from 3 to 14 chambers with an mean of 8.6 chambers per nest (silva et al., 2010). caldato (2010) registered the polydomy in a. balzani calling each unit of a nest of a subnest, totaling, in some cases, eight subnests and until 33 chambers in a single nest. considering the close phylogenetic relationships between a. balzani and a. landolti, the hypothesis of polydomy nest must be investigated for a. landolti. it was observed that three of the studied nests of a. landolti had chambers interconnected between them by tunnels distinct of the vertical one. this suggests that this ant has a relative plasticity in the architecture of its nests, building structures adapted to provide better conditions for the colony. this result differs from other ones relative to several acromyrmex species that were reported to have nests with a unique vertical tunnel that interconnect the chambers (weber, 1972; mendes, 1990; soares et al., 2006; caldato, 2010). the differences in nest chamber dimensions show the inexistence of a pattern, the same result found for nests of a. balzani located in bahia (silva et al., 2010), são paulo and rio grande do sul (gonçalves, 1961), minas gerais (mendes et al., 1992) and goiás (pimenta et al., 2007). regarding the occupation of the chambers, those with fungi, eggs, pupas and adults were found up to 30 cm deep (fig. 3), while chambers containing soil where noted below 25 cm. no chamber containing trash was observed since this ant deposits the trash out of the nest, near the amount of loose soil. this result is in agreement with caldato (2010) for a. balzani, which presented the biggest amount of chambers in the first 30 cm, with predominance of fungi. the volume of the nest chambers of acromyrmex is very variable, not only within a species but also between species. in our study we found chambers of a. landolti with a minimum volume of 19 ml (nest 10) and maximum of 1,335 ml (nest 18), with mean of 91.5±65.0 ml (n10) and 509.3±436.9 ml (n18). silva et al., (2010), for a. balzani, with registers of bigger mean volume of 205.5±142.1 ml, with bigger volume 25 ml and smaller 31 ml. mendes et al. (1992), for the same species, estimated a volume of 910.1 ml, in mean, whereas pimenta et al. (2007) recorded 1435.8 ml for the bigger and 3.6 ml for the smaller chamber of a. balzani, resulting in a great difference in bigger volumes as well as for a. landolti. it was not possible to establish a pattern for the chambers of a. landolti based on the tested models (sphere and ellipsoid) since the relationship among real values and estimated ones were distant in 1.0 ml in the majority of cases. many authors consider the ellipsoid form as the most common one for a. balzani (mendes et al., 1992; pimenta et al., 2007; poderoso et al., 2009; silva et al., 2010). however, by observing the data presented for 21 chambers of 4 nests only 42,9% of the chambers presented similarity to the spherical form, characterizing the fact that other forms of chambers were found that are not adjusted to the models compared, therefore not allowing a possible estimate of a pattern. for most of the nests of a. landolti, the chambers are located between the foraging hole and the amount of loose soil (n01 and n16 of in fig. 5) or in the proximity of the foraging hole. however, it was observed the ocurrence of chambers radially distributed from the principal tunnel that presents an inclined orientation (n02 in figure 5) or situated before and laterally in relation to the entrance tunnel (n12, ms rodrigues junior, ma castellani, aa moreira, m d’esquivel, lc forti, s lacau on the nests of acromyrmex landolti26 table 1. distance from tower to loose soil (cm), tower height (cm), tower diameter (cm), loose soil area (cm²) and volume of loose soil (l) in twenty nests of acromyrmex landolti. itapetinga, ba, brazil, 2012. nests distance from tower to loose soil (cm) tower height (cm) tower diameter (cm) loose soil area (cm²) volume of loose soil (l) n1 09.5 1.5 1.5 432.0 1.3 n2 06.0 2.0 1.5 308.0 0.9 n3 06.0 2.0 1.0 1600.0 4.8 n4 10.0 1.5 1.3 300.0 1.0 n5 06.0 2.0 1.0 493.0 1.5 n6 06.0 2.5 1.0 608.0 1.8 n7 04.0 1.5 1.0 558.0 1.7 n8 04.0 2.0 1.5 441.0 1.3 n9 04.0 1.0 1.0 425.0 1.3 n10 08.5 2.5 1.5 720.0 2.2 n11 07.0 2.0 1.3 429.0 1.3 n12 17.0 2.0 1.0 116.3 0.3 n13 20.0 3.0 1.0 476.0 1.5 n14 05.0 2.5 1.5 180.0 0.5 n15 05.0 1.5 0.8 496.0 1.5 n16 12.0 4.0 1.5 198.0 0.6 n17 08.0 2.5 1.5 440.0 1.3 n18 12.0 3.0 1.0 700.0 2.1 n19 02.0 1.5 0.8 200.0 0.7 n20 07.0 2.0 0.7 338.0 1.2 mean 08.0 2.1 1.2 472.9 1.4 standard deviation 04.5 0.7 0.3 312.5 0.9 minimum 02.0 1.5 0.7 116.3 0.3 maximum 20.0 4.0 1.5 1600.0 4.8 table 2. mean, standard deviation (s), maximum (max.) and minimum (min.) of chamber dimensions (width, height and length), and depth of nests of acromyrmex landolti. itapetinga, ba, brazil, 2012. nests depth (cm) number of chambers width (cm) height (cm) length (cm) mean s max. min. mean s max. min. mean s max. min. n1 58 4 6.1 1.8 7.0 3.5 6.5 2.5 9.0 3.0 6.6 2.3 9.0 3.5 n2 40 5 5.4 2.3 8.0 3.0 3.4 0.9 5.0 3.0 6.5 2.5 9.0 3.5 n3 36 3 9.7 3.5 13.0 6.0 6.7 2.5 9.0 4.0 9.7 2.1 12.0 8.0 n4 58 6 4.8 1.2 7.0 3.5 4.0 1.3 6.0 2.5 5.1 2.0 7.5 2.5 n5 21 6 5.1 1.6 7.0 3.5 3.3 0.6 4.0 2.5 4.7 2.8 8.5 2.5 n6 23 4 6.5 2.5 10.0 4.0 5.8 2.6 8.0 3.0 5.0 1.8 7.0 3.0 n7 7 1 8.0 8.0 8.0 8.0 8.0 8.0 10.0 10.0 10.0 n8 8 1 7.0 7.0 7.0 8.0 8.0 8.0 9.0 9.0 9.0 n9 18 4 5.3 1.7 7.0 3.0 4.6 2.2 7.0 2.5 6.1 1.3 7.5 5.0 n10 50 4 5.5 2.9 9.0 2.0 4.0 1.7 5.0 1.5 5.1 1.7 7.0 3.5 n11 18 6 4.9 2.2 9.0 3.0 3.7 2.1 7.0 2.0 4.5 2.5 9.0 2.0 n12 42 11 4.8 1.5 8.0 3.0 4.0 1.0 5.0 2.0 4.4 1.4 6.0 1.5 n13 8 6 5.3 3.1 10.0 1.5 5.2 3.2 9.0 2.0 5.6 3.5 10.0 2.0 n14 16 3 6.2 2.5 9.0 4.5 6.7 2.1 9.0 5.0 5.8 2.3 8.0 3.5 n15 27 3 5.2 0.8 6.0 4.5 4.8 2.0 7.0 3.0 6.3 1.2 7.0 5.0 n16 6 3 7.3 4.5 12.0 3.0 6.0 3.6 10.0 3.0 4.7 3.1 8.0 2.0 n17 19 3 6.8 2.3 9.0 4.5 7.2 2.8 10.0 4.5 8.7 3.5 12.0 5.0 n18 30 6 8.0 1.4 10.0 6.0 6.5 2.4 11.0 4.0 9.0 4.4 17.0 5.0 n19 14 3 5.8 3.3 9.0 2.5 4.2 1.4 5.0 2.5 5.3 3.1 8.0 2.0 n20 23 6 5.7 1.5 7.0 3.0 4.9 1.7 6.0 2.5 5.8 2.3 8.0 2.0 n21 45 4 5.3 1.6 7.0 3.5 5.5 2.0 8.0 3.5 4.1 0.6 5.0 3.5 n22 78 3 4.7 0.6 5.0 4.0 4.8 1.0 6.0 4.0 3.3 0.6 4.0 3.0 n23 74 6 6.0 3.8 11.0 1.5 5.5 3.1 10.0 2.0 4.2 2.6 8.5 1.5 n24 56 4 6.8 4.6 12.0 2.0 6.6 3.4 11.0 3.0 5.6 3.7 10.0 1.5 n25 55 5 9.4 5.2 18.0 5.0 7.4 1.8 10,.0 5.0 6.9 3.2 12.0 3.5 sociobiology 60(1): 20-29 (2013) 27 table 3. real and estimated volume (ml) by geometric similarity of the sphere and ellipsoid and the relationship between real and estimated volume (v1/v2 and v1/v3), mean, standard deviation (s), maximum (max.) and minimum (min.) four nests of acromyrmex landolti. itapetinga, ba, brazil, 2012. nest chambers volume of real chambers (v1) ellipsoid volume (v2) sphere volume (v3) v1/v2 v1/v3 n2 1 141 75.4 95.3 1.9 1.5 2 289 99 133 2.9 2.2 3 218 167.6 179.6 1.3 1.2 4 34 16.5 16.6 2.1 2.0 5 47 18.8 19.4 2.5 2.4 mean 145.8 75.5 88.8 2.1 1.6 s 109.5 62.7 71.2 0.6 1.5 maximum 289 167.6 179.6 2.9 1.6 minimum 34 16.5 16.6 1.3 2.0 n5 1 32 13.1 14.1 2.4 2.3 2 30 13.7 14.1 2.2 2.1 3 40 18.8 19.4 2.1 2.1 4 196 124.6 143.8 1.6 1.4 5 183 117.3 133 1.6 1.4 6 51 27.5 29.5 1.9 1.7 mean 88.7 52.5 59 2 1.5 s 78.6 53.3 61.9 0.4 1.3 maximum 196 124.6 143.8 2.4 1.4 minimum 30 13.1 14.1 1.6 2.1 n10 1 96 62.8 65.4 1.5 1.5 2 176 148.4 167.1 1.2 1.1 3 19 5.5 6.7 3.5 2.8 4 75 78.5 79.4 1 0.9 mean 91.5 73.8 79.7 0.6 1.1 s 65 58.8 66.3 1.1 1.0 maximum 176 148.4 167.1 3.5 1.1 minimum 19 5.5 6.7 1 2.8 n18 1 172 91.6 95.3 1.9 1.8 2 660 345.6 381.7 1.9 1.7 3 1335 881.2 982.3 1.5 1.4 4 300 131.9 133 2.3 2.3 5 284 117.3 133 2.4 2.1 6 305 201.1 206.5 1.5 1.5 mean 509.3 294.8 322 1.9 1.6 s 436.9 301.5 339.3 0.4 1.3 maximum 1335 881.2 982.3 2.4 1.4 minimum 172 91.6 95.3 1.5 1.8 ms rodrigues junior, ma castellani, aa moreira, m d’esquivel, lc forti, s lacau on the nests of acromyrmex landolti28 n14 e n25 in figure 5), similarly to the result of soares et al. (2006) on a. rugosus rugosus. in a. balzani, there seems to have a pattern of spatial distribution of the chambers because they are located between the tower projections and the amount of loose soil, overlapping and connected by only one vertical channel (gonçalves 1961; pimenta et al., 2007; caldato, 2010). the research about morphometrics of the nests of a. landolti, showed that this species can alter the form of its nest constructions, maybe as an adaptation to local edaphoclimatic conditions or as a response to the age of the colony. it is also possible to draw a hypothesis of the effect of the edaphic conditions of the study area classified as “chernossolo argilúvico órtico” and characterized by its short depth, rarely being further than 70 cm, presenting a bad draining system, having high levels of silt and rich in primary minerals (nacif, 2000). this could have been the most important factor determining the charateristics of a. landolti nest structure. overall the data obtained show that the spatial distribution of a. landolti is of the aggregated type. the external nest architecture of a. landolti and a. balzani are very similar, since both of them have a typical tower surrounding the entrance with an amount of loose soil in the vicinity, making this a pattern. however, nests of a. landolti in its external and internal structures have dimensions different from other ant species of the same genus. acromyrmex balzani differs by having smaller size in several structural parts of the nest and less depth for nesting. also, these two species differ because a. landolti presents an internal architectural pattern relatively variable and distinct, with an opening tunnel that may bifurcate and connect more than two chambers. however, there is a similarity between their nests because they have a number of chambers relatively low. acknowledgments we thank the coordenação de aperfeiçoamento de pessoal de nível superior – capes, universidade estadual do sudoeste da bahia 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open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the cavity-nesting bee guild (apoidea) in a neotropical sandy coastal plain introduction bee populations are dependent of key resources related to their feeding, mating and nesting (westrich, 1996). the brazilian “restingas” (herbaceous-shrub and arboreal vegetation covering the sandy coastal plain) are under intense degradation, which has produced accentuated changes in the landscape and habitat loss (rocha et al., 2007). the loss of local biodiversity, primarily by fragmentation and habitat loss can reduce the availability of nesting sites and food resources, and threaten bee communities and pollination services (viana et al., 2012). abstract some solitary bees establish their nests in preexisting cavities. such nesting behavior facilitates the investigation of their life history, as well as the monitoring of their populations in natural, semi-natural and cropped habitats. this study aimed to evaluate the acceptance of artificial substrates by cavity-nesting bees in a heterogeneous landscape. we investigated the percentage of occupation of the different trap-nests, the monthly fluctuations in the nesting activity, offspring sex ratio, mortality and parasitism, in two phytophysiognomies: herbaceous-shrub restinga (site 1) and arboreal restinga (site 2). we used as trap-nests, bamboo canes, large and small straws of cardboard inserted into solid wooden blocks. five bee species established 193 nests, from which 386 adults emerged. centris tarsata smith was the most abundant species. large straws were significantly more occupied than small straws (χ² = 19.951; df = 1; p < 0.0001). offspring mortality rate for unknown reasons was significant different between sites, 11% (site 1) and 20% (site 2) (χ² = 4.203; df = 1; p = 0.04). the cavity-nesting bee guild had similar composition in both phytophysiognomies, there was a similar rate of occupation of trap-nests in both sites, as well as dominance of c. tarsata nests. offspring mortality and parasites attack rates seem to be the more distinctive aspects between the herbaceousshrub and arboreal restinga sampled. our study indicated that remnant fragments of coastal native habitats may be important nesting sites for the maintenance of bee populations, some of which have been indicated as candidates for management as pollinators of cultivated plants in brazil. sociobiology an international journal on social insects p oliveira-rebouças1,4, cml aguiar2,4, vs ferreira5, gs sodré3,4, cal carvalho3,4, m gimenes2 article history edited by solange augusto, ufu, brazil received 09 may 2018 initial acceptance 17 june 2018 final acceptance 21 august 2018 publication date 11 october 2018 keywords centris, euglossa, trap-nests, tetrapedia, nesting biology. corresponding author patricia luiza de oliveira rebouças departamento de tecnologia e ciências sociais universidade do estado da bahia rua edgar chastinet, s/nº, são geraldo cep 48905-680, juazeiro-ba, brasil. e-mail: preboucas@uneb.br several bee species nest in natural and artificial preexisting cavities (roubik, 1989). the trap-nests sampling technique was designed by krombein (1967) in order to obtain nests of these bees. since then, it has been used to answer questions related to breeding and nesting biology (garofalo et al., 2012). it also has allowed a management of bee pollinators in agricultural and natural landscapes, resulting in the increasing of the agricultural production (bosch & kemp, 2000; magalhães & freitas, 2013; yamamoto et al., 2014). some studies have investigated the nesting biology of solitary bee species inhabiting the atlantic coastal plain (viana et al., 2001; aguiar & martins, 2002; camarotti-de-lima 1 universidade do estado da bahia, juazeiro, bahia, brazil 2 universidade estadual de feira de santana, feira de santana, bahia, brazil 3 universidade federal do recôncavo da bahia, cruz das almas, bahia, brazil 4 ppgciências agrárias, universidade federal do recôncavo da bahia, cruz das almas, bahia, brazil 5 universidade federal do vale do são francisco, petrolina, pernambuco, brazil research article bees sociobiology 65(4): 706-713 (october, 2018) special issue 707 & martins, 2005; bernardino & gaglianone, 2008; martins et al., 2014). some of these cavity-nesting bee species, such as centris and xylocopa spp. are involved in pollination of crops and wild plants (gottsberger et al., 1988; buchmann, 2004; gaglianone et al., 2010; yamamoto et al., 2012; oliveira et al., 2013). this study aimed to evaluate the acceptance of artificial substrates (trap-nests) for nest by cavity-nesting bees in a heterogeneous landscape covered by different phytophysiognomies of ”restingas”. we investigated the percentage of occupation of the different trap-nests, the monthly fluctuations in the nesting activity of these species, as well as the sex ratio of the offspring and the incidence of mortality and parasitism of the brood cells. material and methods study area this study was carried out in a private reserve (rppndsa, reserva particular do patrimônio natural dunas de santo antônio; 12°27’s; 37°56’w), in mata de são joão, state of bahia, northeast of brazil. according to the classification of köppen, the local climate is tropical (af), hot and humid without dry seasons. the average temperature is between 21°c and 31 °c, and annual rainfall varies from 1,600 to 2,000 mm. the rainiest period occurs from april to june (sei, 1999). the phytophysiognomies of this restinga are similar to those described by cogliatti-carvalho et al. (2001), the herbaceous-shrub vegetation of the restingas are characterized by bare sand corridors, allowing a higher incidence of sunlight, while in the arboreal restinga, there is a greater density of woody vegetation, less sunlight incidence, increased availability of litter on the ground and high humidity. the study site is covered by an herbaceous-shrub vegetation in which humiria balsamifera, waltheria cinerescens, chamaecrista ramosa, chrysobalanus icaco, and cuphea brachiata were plants often found, and by arboreal vegetation, a kind of restinga forest, in which bowdichia virgilioides, stryphnodendron pulcherrimum, andira nitida and anacardium occidentale are common plant species and can exceed 12 m in height (queiroz, 2007). these plant species are the most important food resources for wild bees in sandy coastal plain of bahia, brazil (viana & kleinert, 2006). sampling the sampling was carried out in two sites 2.5 km distant from each other: one in the herbaceous-shrub vegetation (site 1), and other in arboreal vegetation (site 2). although the sampling points are located in different phytophysiognomies, they are not far from each other enough to suggest that these bee populations are distinct. the trap-nests were kept in steel shelves covered with a plastic tarpaulin. we used as trap-nests straws made of black cardboard inserted into solid wooden blocks, as well as bamboo canes following camillo et al. (1995), and aguiar and garofalo (2004). six blocks of wood perforated by holes were placed at each site. three blocks contained 60 holes each, in which black cardboard straws measuring 58 x 6 mm (ss = small cardboard straws) were inserted. three other blocks contained 56 holes each, in which trap-nests of 105 x 8 mm (ls = large cardboard straws) were placed. in addition, 18 hollow bamboo canes were arranged in groups containing six bamboo canes of variable lengths (90 to 220 mm) and diameters (8 to 16 mm). the trap-nests were inspected once a month, from november 2006 to november 2007. the trap-nests used by bees were collected and replaced by new ones. each nest was maintained in a glass tube at room temperature, and they were opened after the emergence of the imagos. the number of brood cells, the presence of dead individuals and their stage of development, as well as the presence of other insects in the brood cells were recorded. data analysis the χ² test was used to compare trap-nest occupation in each site and the occupied trap-nests of each size (zar, 2011). the χ² test goodness of fit tests was used to compare the following aspects between the two sites: length of trapnest and the frequency of occupation of the trap-nest (is the percentage the ratio between the total number of nests founded by the total number of nests available large and small card nests plus cane bamboo in each site), in order to check the extent to which the observed sex ratio deviated from the expected frequency (1m:1f), and to analyze differences in progeny sex ratio among nests of different size and nests from different sites . heterogeneity χ² test was used to analyze the rates of immature mortality and parasitism (zar, 2011). the mann-whitney test was used to monthly assess whether there were preferably in the two types of trap-nest offered, and also to check the difference on the development time between males and females emerged in the nest (zar, 2011). spearman correlation (rs) analyses were performed to assess monthly temperature and precipitation effects on the monthly frequency of new nests, being considered significant p <0.05 by the student’s t-test (zar, 2011). sörensen index (cs) was used to evaluate the degree of similarity in the species composition of the cavity-nesting guild in both sites (magurran, 2011). the analyzes were performed using r software (r development core team, 2016). results trap-nests occupation, species richness and abundance five bee species nested in the trap-nests (table 1). although the phytophysiognomies are different in the two sampling points, there was no significant difference in the total number of nests between these sites (χ² = 0.375; df = 1; p = 0.54). a similar species composition of the cavitynesting bee guild was found in the two sites (cs = 0.88). only tetrapedia diversipes klug occurred exclusively in the site 2. p oliveira-rebouças, cml aguiar, vs ferreira, gs sodré, cal carvalho, m gimenes – cavity-nesting bee guild in restinga708 the occupation rates of the trap-nests were also similar, 25% (site 1) and 28% (site 2). centris tarsata smith established the highest number of nests in both sites, 18% of the all nests in the site 1 and 20% in the site 2 (table 1). regarding to the occupation of the straws of different dimensions, the total occupation rate (the two sites together) was 36% to the large straws (122 nests established), and 11% to the small straws (39 nests). there was a statistically significant difference in the occupation rates of these straws of different dimensions (χ² = 19.951; df = 1; p < 0.001). centris analis fabricius, c. tarsata, and t. diversipes used both types of cardboard straws, while centris trigonoides lepeletier used only the large straws. c. tarsata established significantly more nests in large straws than in small ones (χ² = 82.6; df = 1; p < 0.001), while c. analis established a higher number of nests in small straws (χ² = 12.772; df = 1; p < 0.001). there was no significant difference in the occupation of large and small straws by t. diversipes (χ² = 3.469; df = 1; p > 0.05) (table 1). regarding to the occupation of bamboo canes, 89% of those that were made available to the bees were occupied. c. tarsata used mainly those from 10 to 13 mm diameter, while euglossa cordata linnaeus, occupied only those with 18 mm in diameter. bee species site 1 site 2 total ls ss bc total ls ss bc n i n i n i n i n i n i n i n i centris analis 15 26 5 5 10 21 15 15 15 15 centris tarsata 64 178 51 106 13 72 73 113 53 60 4 16 53 centris trigonoides 10 23 10 23 1 2 1 2 tetrapedia diversipes 12 19 2 4 10 15 euglossa cordata 2 1 2 1 1 9 1 9 total 91 228 66 134 10 21 15 73 102 158 56 66 29 30 17 62 table 1. number of nests provisioned (n) and number of imagos emerged (i) in restinga vegetation, bahia, brazil. site 1: herbaceous-shrub vegetation, site 2: arboreal vegetation. ls = large cardboard straws, ss = small cardboard straws, bc = bamboo canes. fig 1. number of nests established by bees in trap-nests, maximum and minimum temperature, and monthly pluviosity, in mata de são joão, state of bahia, brazil, from november 2006 to november 2007. fig 2. number of nests established by bee species in restinga vegetation. temporal distribution of nesting activity nesting activity in this restinga occurred mainly from december to june, with an abundance peak of nests in december (fig 1). the total number of nests established per month, was influenced by the nesting activity of c. tarsata, which established the highest number of nests. the temporal distribution of its nesting activity was more restricted (six months) than that of other species, such as c. analis, which had the longest nesting period (nine months) (fig 2). there was a significant positive correlation between minimum monthly temperature and the total number of nests built (rs = 0.71; p < 0.05), for the nests built by c. tarsata (rs = 0.62; p < 0.05) and c. trigonoides (rs = 0.64; p < 0.05), but not for the other species. there was no correlation between monthly rainfall and the total number of nests (rs = 0.03; p < 0.05). sociobiology 65(4): 706-713 (october, 2018) special issue 709 brood cells provisioned, emergence of imagos and sex ratio c. tarsata provisioned the highest number of brood cells, both in cardboard straws (n = 263 brood cell) and bamboo canes (n = 130) (table 2). the average number of brood cells per nest was 2.39 in cardboard nests, and 4.48 in bamboo canes. the number of brood cells varied in all types of trap-nests, with a maximum of 11 brood cells per nest in bamboo straws and seven in cardboards tubes. the most frequent number of brood cells per nest was one (21% of nests), three (22%) in cardboard tubes, and five (23%) and ten (17%) in bamboo straws. the brood cells were arranged linearly in the nests established in cardboard straws, while they tended to be oblique in bamboo canes. most c. analis nests (52%) had three brood cells, while in c. trigonoides nests four brood cells were more frequent (44%) and three brood cells were common in t. diversipes nests (38%). the interval between the collection of the nests and the emergence of imagos was quite variable. most c. trigonoides and t. diversipes immatures had a development time longer than 121 days (table 3). only c. tarsata had a significant difference on the development time between males (maximum of 50 days) and females (maximum of 54 days) (u = 10.734; p < 0.05). from the 554 brood cells provisioned by bees, 386 imagos emerged (table 2), so the emergence success was 70% in this habitat. in the nests built in bamboo canes, the emergence success was higher than 90% of the brood cells provisioned in the nests of c. tarsata and e. cordata. in the nests built by these bees in cardboard tubes, this percentage ranged between 50 and 66% (table 2). the sex ratio of c. tarsata offspring was significantly different from 1:1 (χ² = 18.91; p < 0.0001) in cardboard straws (4m: 1f). but the sex ratio of the offspring produced in bamboo canes did not differ significantly from 1:1 (table 2). bee species ♂ ♀ sr χ² eb bc c/n (n) (n) (n) (n) c. tarsata (cardboard straws) 143 35 4.1m:1f 18.910* 166 263 2.39 c. tarsata (bamboo canes) 63 66 1.0m:1f 0.004 125 130 4.48 c. analis 32 20 1.6m:1f 0.974 41 81 2.08 c. trigonoides 16 11 1.4m:1f 0.168 25 36 3.45 t. diversipes 16 3 5.3m:1f 3.606 19 33 2.75 e. cordata 5 6 1.0m:1f 10 11 1.10 total 275 141 386 554 * p < 0.05 table 2. number (n) of males and females of cavity-nesting bees produced in trap-nests (including imagos found dead inside brood cells), offspring sex ratio (sr), chi-square test for sr (χ²), number of imagos emerged (eb), number of brood cells (bc), and number of brood cells per bee nest (c/n). mortality emergences of natural enemies (anthrax sp., mesocheira bicolor fabricius, leucospis sp.) were recorded in 45 brood cells from these bee nests (table 4). bee offspring mortality rate for unknown reasons was 11% (site 1) and 20% (site 2) (table 4). there was a significant difference between the sampling sites (χ² = 4.203; df = 1; p < 0.05). the mortality rate in c. tarsata offspring was lower in the nests established in bamboo canes (4%) than in cardboard straws (19%) (table 4). the incidence of brood cell parasitism was higher at the arboreal vegetation (site 2 = 13%), than in the herbaceousshrubby vegetation (site 1 = 5%), significantly different (χ² = 4.629; df = 1; p < 0.05). c. tarsata had low rates of parasitism in cardboard straws and in bamboo canes, as well as in both sites (table 4). t. diversipes had high percentage of brood cells attacked by natural enemies (table 4). bee species time intervals (in days) 01 -3 0 31 -6 0 61 -9 0 91 -1 20 12 115 0 15 118 0 18 121 0 21 124 0 24 127 1 >2 71 n c. tarsata 150 142 291 c. analis 22 4 15 41 c. trigonoides 1 3 1 2 12 5 24 t. diversipes 1 3 2 4 3 3 16 e. cordata 1 7 1 9 table 3. number of imagos of cavity-nesting bees emerged from trap-nests per time intervals between the collection of the nests and the emergence. p oliveira-rebouças, cml aguiar, vs ferreira, gs sodré, cal carvalho, m gimenes – cavity-nesting bee guild in restinga710 discussion the cavity-nest bee guild in the rppn-dsa had few species, as well as in other coastal habitats in the northest of brazil, as sand dunes (viana et al., 2001), coastal savanna (coastal “tabuleiros”), and coastal rainforest (aguiar & martins, 2002). differences in species richness in cavitynesting guilds may be related to differences in the species composition of each bee assemblage, as they can also be influenced by sampling, since there are different probabilities of nesting females finding the trap-nests and accepting them as substrates of nesting. flower-visiting data from rppn-dsa have indicated that there are at least ten other cavity-nesting bee species in this area. the abundance of several of these cavity-nesting bees in flowers was low, suggesting that their populations are small (p. oliveira-rebouças, unpublished data). the absence of xylocopa species using the trap-nests was surprising, since they are usual components of the cavitynesting bees guild in the brazilian coastal plain (viana et al., 2001; aguiar & martins, 2002; viana & alves-dossantos, 2002; silva et al., 2015). five xylocopa species were collected visiting flowers in the rppn-dsa, and two of them (xylocopa cearensis ducke and xylocopa subcyanea pérez) were very abundant in this bee assemblage (p. oliveirarebouças, unpublished data). however, they did not use the trap-nests available, although there were adequate for their nesting (bamboo canes up to 220 mm in diameter). the percentage of trap-nests occupied was moderate, both in site 1 (25%) and site 2 (28%). these trap-nests occupation rates were higher than those recorded in other coastal habitats in northeast brazil. viana et al. (2001) reported that solitary bees used only 14% of the trap-nests available in a fragment of tropical sand dunes, and aguiar and martins (2002) recorded that bees occupied 7% of the trap-nests in a coastal savanna (tabuleiros vegetation). on the other hand, these authors recorded occupancy of 25% of trap-nests in a coastal rainforest. the trap-nests occupation rates by nesting bees can be influenced by several factors, such as the local availability of natural substrates for nesting (frankie et al., 1988; viana et al., 2001; silva & viana, 2002), the diversity of the trap-nests available (vandenberg, 1995), the degree of exposure to the sun (frankie et al., 1988), and the local availability of trophic resources (gathmann et al., 1994). these factors appear to have not interfered in the trapnest occupation rates between sites in the rppn-dsa, since there was no difference in the occupation of the trap-nests in herbaceous-shrub and arboreal phytophysiognomies. the abundance of bee nests established in each site (91 and 102 nests) in the restinga rppn-dsa was higher than those found in sand dunes (n = 62) (viana et al., 2001), in coastal rainforest (n = 19), in coastal savanna vegetation (n = 47), and in a mosaic of these two latter habitats (n = 69) (aguiar & martins, 2002). differences in the number of bee nests may be related to the number of sampled sites in each area, to the sampling effort, as well as to the local dominance of some species that can establish many nests. in the rppn-dsa, the high number of nests in both phytophysiognomies was due to the high nesting success of c. tarsata in these habitats. this high dominance of c. tarsata on artificial nesting substrates has been recorded in other habitats in northeast brazil, as coastal sand dunes (viana et al., 2001), coastal savanna (aguiar & martins, 2002), dry forest (“caatinga”) and semideciduous forest (aguiar & garófalo, 2004). these findings support the hypothesis raised by aguiar and garofalo (2004) about the high ability of c. tarsata populations to occupy open environments, with high temperatures and high insolation. probably, this factor contributed to the large number of nests founded by c. tarsata females in rppn-dsa, because much of the this area is covered by an herbaceous-shrub vegetation, which allows high incidence of sunlight. these solitary bees used different types and sizes of trap-nests in unequal proportions. in large straws there were higher number of nests, and they attracted more bee species than small straws, as was also observed in other habitats (aguiar & garofalo, 2004; pina & aguiar, 2011). c. tarsata showed more affinity for large cardboard straws, while c. analis used mainly small cardboard straws, similar to that observed in coastal savanna, where 54% of the c. tarsata nests were built in large straws and 88% of c. analis nests were in small straws (aguiar & martins, 2002). table 4. mortality percentage in bee brood cells due to unknown causes or parasitism. site 1: herbaceous-shrub vegetation, site 2: arboreal vegetation. nbc: total number of brood cells attacked by natural enemies (parasites). bee species unknown causes (%) parasitism (%) natural enemies nbc (n) site 1 site 2 total site 1 site 2 total c. tarsata (bamboo canes) 1.4 7.0 3.9 2.7 12.3 6.9 anthrax sp. mesocheira bicolor 1 8 c. tarsata (cardboard straws) 10.1 29.6 19.4 4.3 8.8 6.5 anthrax sp. mesocheira bicolor leucospis sp. 7 7 3 c. analis 20.5 37.8 28.4 6.8 10.8 8.6 leucospis sp. 7 c. trigonoides 29.4 27.8 5.9 5.6 leucospis sp. 2 t. diversipes 18.2 18.2 21.2 21.2 leucospis sp. anthrax sp. 6 1 e. cordata 50.0 9.1 33.3 33.3 anthrax sp. 3 sociobiology 65(4): 706-713 (october, 2018) special issue 711 we registered high amount of trap-nests occupied by solitary bees (especially c. tarsata) in the hottest months (summer) in the rppn-dsa, with a peak of abundance in december, when rainfall was low. in other studies, variations were observed between seasons and years. in coastal savanna, the highest number of nests was registered in summer (november), a rainy and hot season (aguiar & martins 2002). according to viana et al. (2001), in coastal sand dunes of northeastern brazil, the dominant species, c. tarsata, had abundance peak of nests in december (in summer, the dry season) in the first year, while a higher number of nests was found in the rainy months (autumn) in the second year. the fluctuations in the frequency of nesting can be related to intrinsic factors of the cavity-nesting species or to environmental factors, such as weather patterns, or the dynamic of floral resources availability (frankie et al., 1988). the number of brood cells built per nest was variable in all species, as reported in other habitats, for c. tarsata (aguiar & garófalo, 2004; buschini & wolff, 2006) and c. analis (jesus & garófalo, 2000). large variations in the number of brood cells were also found in different types of trap-nests (cardboard straws and bamboo canes) in the studied area. the number of brood cells built per nest, as well as the brood cells arrangement in nests, depend upon the size of the cavity. on the other hand, behavioral decisions of the female bees regarding time spent in the same cavity to produce offspring affect the number of brood cells produced by nest, since the more time invested in the same nest the more brood cells are expected to be produced. alternatively, distributing the reproductive effort in several nests should also have some effect on variability in the number of cells per nest (jesus & garofalo, 2000). the total sex ratio of c. tarsata offspring was biased to males, mainly in cardboard straws (4m:1f), but not in bamboo canes (1m:1f). bias to males have been recorded in some populations of c. tarsata (aguiar & martins 2002; aguiar & garófalo, 2004; buschini & wolff, 2006). however, in other populations, the sex ratio of the offspring was 1:1 (silva et al., 2001; aguiar & garófalo, 2004). several factors can affect the offspring sex ratio of cavity-nesting bees, as the length (alonso et al., 2012; gruber et al., 2011; stephen & osgood, 1965) and diameter of the cavities (rust 1998), the abundance of food resources in the environment, the foraging efficiency of nesting females (torchio & tepedino, 1980), and conditions of the mother (seidelmann et al., 2010). the mortality rate by unknown reasons varied among the species. c. tarsata offspring had low mortality in bamboo canes (< 4%), and higher in cardboard straws (19%). these values were lower than those in other habitats, as reported by aguiar and garófalo (2004), who recorded 41% in dry forest and 42% in a semideciduous forest in brazil. buschini and wolff (2006) found high rates of offspring mortality (58% to 70%) by unknown reasons in swamp habitat and grasslands in southern brazil. the mortality rate in c. analis offspring (35%) was moderate similar to that observed in agricultural areas in brazil (24-25%) (aguiar & pina, 2012). higher mortality rates were found in an urban area in southeastern brazil, 63% (jesus & garófalo, 2000) and 42% (couto & camillo, 2007). offspring mortality may be related to failures in development, environmental factors, such as temperature (jesus & garófalo, 2000; gazola & garófalo, 2009), changes in air humidity conditions (buschini & wolff, 2006), or even to the handling of the nests (aguiar & pina, 2012). although the phytophysiognomies sampled in the rppn-dsa were different, the cavity-nesting bee guild had a similar composition in the studied sites, there was a similar rate of occupation of trap-nests in both sites, as well as dominance of c. tarsata nests. mortality rates due to unknown causes and by parasite attack on the offspring of some cavity-nesting bee species seem to be the most distinctive aspect between herbaceous-shrub restinga and arboreal restinga phytophysiognomies sampled. another relevant aspect was the lack of records on both sites of some cavity-nesting species, collected in flowers, but did not use the available artificial nesting substrates, indicating that the trap-nest sampling method alone is not enough to adequately sample the species richness of the cavity-nesting bee guild. finally, our study indicated that remnant fragments of coastal native habitats may be refuges for the maintenance of bee populations, some of which, such as c. tarsata and c. analis, have been indicated as candidates for management as pollinators of cultivated plants in brazil. acknowledgments this study was financed in part by the “coordenação de aperfeiçoamento de pessoal de nível superior brasil” (capes) finance code 001. we thank “conselho nacional de desenvolvimento científico e tecnológico” (cnpq) for granting a scholarship to calc (number 305885/2017-0). references aguiar, c.m.l. & garófalo, c.a. 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(2011). biostatistical analysis. new jersey: prentice hall, 944 p. http://www.r-project.org/ _hlk518820966 _hlk510027675 doi: 10.13102/sociobiology.v66i3.4264sociobiology 66(3): 440-447 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 diversity of ants (hymenoptera: formicidae) in a sub-montane and sub-tropical cityscape of northeastern mexico introduction many urban areas around the world have rapidly grown and expanded into outlying rural areas, negatively transforming the regional environment and conditions for a variety of productive activities dependent on the availability and quality of natural resources, such as livestock grazing and crop production (gaviría-gutíerrez, 2014). urban ecosystems feature physical barriers, high temperatures, and warm wind streams as well as pollution and therefore represent artificial abstract the role of urban ecosystems in maintaining biodiversity and ecosystem services is highly variable because of the heterogeneity of habitats in human-used landscapes. we analyzed ant diversity in a sub-montane and sub-tropical urban area of northeastern mexico to determine the conservation value of this cityscape. ants were collected in 16 sites (each located at a 1 km2 quadrat) across the cityscape, including spaces at the periphery and urban center, during the rainy season (august to october) of 2015. to capture ants, in each site eight pitfall traps, with for different baiting treatments were installed along a 100m transect, and hand collections were performed. in total, 7,415 ant workers belonging to 32 species, 23 genera, 11 tribes, and 5 sub-families were collected. the richness and structure of the assemblages varied among the sampling sites. the compositional similarity also varied significantly among sampling sites, and unique species were found in four sites. each site showed an important and particular ant assemblage that differed from that of the other sites and the surrounding habitats in the cityscape. the results suggest that some sampling sites in the studied cityscape may contribute to the conservation of certain ant groups and invertebrate communities threatened by urban intensification. ultimately, our findings support the importance of conserved areas and green spaces for the conservation of native species in and near urban areas. sociobiology an international journal on social insects ma garcía-martínez1,2, v vanoye-eligio3, or leyva-ovalle1, p zetina-córdoba2, mj aguilar-méndez4,5, m rosas-mejía3 article history edited by evandro n. silva, uefs, brazil received 23 november 2018 initial acceptance 01 may 2019 final acceptance 16 june 2019 publication date 14 november 2019 keywords biodiversity management, humandominated landscape, urban ecosystem, native species. corresponding author madai rosas-mejía instituto de ecología aplicada universidad autónoma de tamaulipas división del golfo 356, col. libertad, cd. victoria 87019, tamaulipas, mexico. e-mail: marosas@docentes.uat.edu.mx environments (simonetti et al., 2010). the human-induced processes associated with urbanization may threaten biodiversity and affect ecosystem productivity, leading to the loss of habitats, biomass, and the carbon storage capacity of ecosystems (seto et al., 2012). several studies have suggested that the fauna inhabiting cityscapes may, in some cases, positively respond to human-induced changes, including the increased availability of certain resources or other beneficial environmental conditions. in fact, the settlement and dispersal of some fauna, such as invasive species, may be favored (uno et al., 2010). 1 universidad veracruzana, facultad de ciencias biológicas y agropecuarias región: orizaba–córdoba, amatlán de los reyes, veracruz, mexico 2 universidad politécnica de huatusco, huatusco, veracruz, mexico 3 instituto de ecología aplicada, universidad autónoma de tamaulipas, cd. victoria, tamaulipas, mexico 4 universidad de guanajuato, departamento de biología, división de ciencias naturales y exactas, guanajuato, mexico. 5 instituto politécnico nacional, unidad profesional interdisciplinaria de ingeniería campus guanajuato, silao de la victoria, guanajuato, mexico research article ants sociobiology 66(3): 440-447 (september, 2019) 441 currently, the spread of human settlements and infrastructure is accelerating across the globe, and some projections estimate that the worldwide urban population will reach almost 5 billion in 2030 (seto et al., 2012). the domination of the planet by urban ecosystems reveals the immediate need of evaluating the value of cityscapes and their associated fauna. such assessments are important for the management of urban fauna and for the generation of different management alternatives for urban microhabitats to avoid the disappearance of native species. urban ecosystems have been extensively studied in the field of ecology, and their importance for the conservation of flora and fauna is increasingly being recognized. the majority of studies have focused on the conservation value of cityscapes (alvey, 2006; shochat, 2010). several research studies on gray and green urban areas have highlighted the relative importance of urban or peri-urban habitats as biodiversity reservoirs based on the study of several common bioindicator groups, including ants, butterflies, beetles, and spiders (aronson, 2014; ramírez-restrepo, 2015; rocha & castaño, 2015). in general, as urban intensification increases, a significant and negative decrease in the diversity of native species is found along with an increase in the abundance of some generalist species (buczkowski & richmond, 2012). however, high levels of biodiversity have also been reported in green cities (aronson et al., 2014; fortel et al., 2014) with favorable temperatures and high resource availability due to the maintenance of natural and native vegetation (rocha & castaño-meneses, 2015). ideal organisms for biodiversity analysis in urban environments should perform various functions in the community, have a relatively short generation time (in order to assess sensitivity to environmental changes), and be abundant and relatively easy to collect (monteiro-júnior et al., 2015). based on these criteria, insects are good indicators of the impact of urbanization on biodiversity (jaganmohan et al., 2013). among the vast insect groups, ants (hymenoptera: formicidae) represent a suitable group for studying the effects of urbanization because of their important ecological role in ecosystems. in particular, ants form a highly diverse and abundant insect group with distinct habitat and nutrient preferences and have important effects on soil physical and chemical properties (calcaterra et al., 2010). usually, ants are abundant in urban zones, although some studies have indicated that urbanization has resulted in the permanent loss of up to 85% of ant species (buczkowski & krushelnycky, 2012; buczkowski & richmond, 2012). in several urban cityscapes, certain dominant and abundant ant species were found to damage the structures of buildings, contaminate stored food, cause direct harm to humans, nest in gardens, and be vectors of pathogens (olayamasmela, 2005). in recent decades, research has also been carried out on diverse topics surrounding the use of formicides in urban environments or inside or outside household, listing the effects on different species (cupul-magaña, 2009; taheri & reyes-lópez, 2018). other studies have evaluated variation in the diversity patterns of ant communities along of a gradient of urban disturbance (rocha & castaño-meneses, 2015), ant responses to urban environments (angilleta jr., 2007), ants as nosocomial vectors (rodovahlo et al., 2007), ant responses to insecticides, and ant management with repellents (mothapo & wossler, 2016). until now, little research has been carried out on the urban myrmeco fauna of mexico. one study detailed a rapid descriptive ecological assessment for testing the effect of urbanization on ant morphospecies (macgregor-fors et al., 2015), and several additional studies have reported on the importance of urban green spaces as reservoirs of ant diversity (rocha & castaño-meneses, 2015), the bait preferences of ants (rosas-mejía et al., 2015), and new species records (rosas-mejía et al., 2013; landero-torres et al., 2014). however, it is crucial to contribute toward current knowledge of the real influence of urbanization on ant species diversity at a local and landscape level. the present study was designed to analyze the ant diversity associated with 16 sampling sites in a cityscape of northeastern mexico surrounded by a submontane and sub-tropical thorn scrub area. in particular, we analyzed the changes in alpha, beta, and gamma diversity among ant assemblages to determine the conservation value of each sampling site within the cityscape. greater knowledge of ants and other groups of arthropods in cityscapes can improve our understanding on how the urbanization process affects these insects and will contribute to the formation of baseline management and planning strategies for the conservation of native species. materials and methods study area the study was conducted in the sub-montane and subtropical city of ciudad victoria, located in central tamaulipas between the parallels 23°47’21”and 23°41’47” n and the meridians 99°11’40” and 99°04’30” w at an elevation range of 295 to 495 m a.s.l. the city has a population of 305,155 inhabitants. the climate is sub-humid semi-warm throughout the year with a summer rainfall pattern. the average annual rainfall is 926 mm, and the average annual temperature is 24°c. the soil is mainly characterized as medium lithosol with a small proportion of medium-fine rendzina (inegi, 2012). the vegetation near the downtown area of the cityscape is dominated by ornamental and fruit trees. meanwhile, in the peripheral areas, native vegetation is present in different stages of degradation. the vegetation type is known as submontane and sub-tropical tamaulipan thorn scrub and it is mainly composed of large and medium-sized bushes ranging from 3 to 5 m in height (salazar-olivo & solis-rojas, 2015). site selection to establish sampling sites across the studied cityscape, we established the boundaries of ciudad victoria by digitizing ma garcía-martínez et al. – diversity of urban ants in northeastern mexico.442 the boundaries between urban and non-urban area on a previously generated land use/cover map of the study region belonging to an ongoing project (rosas in prep.). then, we randomly set a 1 × 1 km grid on the urban polygon of the city and established all quadrat centroids as the sampling sites. later, through ground truthing, we adjusted the position of each sampling site to the nearest public land where sampling was feasible (given that several points were originally set at inaccessible areas). the resulting number of sampling sites was 20, but for security reasons, several sites in the periurban areas of the city were discarded, reducing the number of sampling sites to 16 (fig 1). ant sampling ant sampling was carried out from august to october 2015 during the rainy season along a 100-m long transect in each sampling site. to capture ants, eight pitfall traps (300 ml plastic containers) were set along each transect, and two people also performed a direct visual search and hand collection. two pitfall traps had no bait. another two were baited with tuna, two with fruit, and two with honey. these traps were alternately set every 10 m along the length of each transect and were recovered after 48 h of exposure in the field. direct collections were performed for 60 min along each transect between 09:00 and 16:00 h. fig 1. location of the studied cityscape in ciudad victoria, tamaulipas, mexico. the location of the cityscape (white circle) within the state of tamaulipas (black polygon) is shown. the black circles indicate the sampling sites (larger size indicates greater species richness). all collected ants were preserved in 100% ethanol, and one to three of the collected specimens per sample that differed morphologically were dry mounted. the key of mackay and vinson (1989) was used to identify ant genera along with several additional keys for species identification depending on the genus involved. voucher specimens were deposited in the ant collection of the laboratorio de zoología of the instituto de ecología aplicada of the universidad autónoma de tamaulipas in ciudad victoria, tamaulipas, mexico. data analysis the number of species occurrences at each site during the three months of sampling was used as a measure of abundance (maximum abundance = 30). the inventory completeness for each site was calculated using the sample coverage estimator (ĉn), which is a less biased estimator of inventory completeness than non-parametric methods. this estimator indicates the proportion of the “total community” represented by the trapped species; when ĉn ≈ 100%, sampling is complete given the effort and utilized capture technique (chao & jost, 2012). values of ĉn were calculated using the inext package for r (hsieh et al. 2016). the comparison of species richness among sites is only ecologically appropriate for sites with a similar level of inventory completeness. therefore, the comparison of our estimates of species richness per site could be biased because of differences in inventory completeness. additionally, species richness is sensitive to variations in the number of singletons and doubletons (chao & jost, 2012). thus, we estimated the species richness for each site using coverage-based interand extrapolations in the inext package for r (hsieh et al., 2016). we considered 99.99% completeness as a reliable estimator of richness for all sites (chao & jost, 2012). to compare species richness among sites, we used 95% confidence intervals in which significant differences were indicated by non-overlapping confidence intervals (cumming et al., 2007). sociobiology 66(3): 440-447 (september, 2019) 443 to evaluate differences in species dominance and rarity and in community evenness among sites, ant abundance was represented by rank-abundance species curves or whittaker plots (magurran, 2004). we plotted the proportional abundance of each species, ordered from the most to the least abundant, to show differences in species dominance and rarity and in assemblage evenness among sites. to analyze beta diversity, we determined the compositional similarity among sites using the jaccard index. this index has values ranging from 0 (minimum similarity) to 100 (maximum similarity) (magurran, 2004). it only takes into account shared species and the presence/absence of species between sites (jost, 2006). the compositional similarity among sites was represented by a cluster analysis using the unweighted pair group method with arithmetic mean (upgma) linkage technique. for the pos thoc analysis, a similarity profile test (simprof) was used as a statistical test to compare similarity among assemblages in the primer software version 6.1.16 (clarke & gorley, 2006). the simprof test assumes that a real clustering of assemblages will be evidenced by an excess of smaller and/or larger similarities than expected under the null hypothesis that all assemblages are drawn from the same species assemblage (clarke et al., 2008). to quantify the contribution of each site to total diversity (i.e., gamma diversity), we calculated the average number of species absent from each site (beta diversity), which is defined as β=γ–α, where γ corresponds with the number of species historically recorded in the study area (gamma diversity) and α with the average number of species present in a given site (alpha diversity) (lande, 1996). this approach allows for a direct comparison between alpha and beta diversities in terms of numbers (or percentages) of species (crist et al., 2003). results in total, 7,415 ant workers belonging to 32 species, 23 genera, 11 tribes, and 5 sub-families were collected. the sub-family myrmicinae had the highest number of tribes, genera, and species. the genera crematogaster, pheidole, and solenopsis had the highest number of species (3 spp. each), followed by forelius, mycocepurus, and pseudomyrmex (2 spp.). the 17 remaining genera were represented by only one species. the average inventory completeness was 99.57% (range: 97.83–100%). considering all 16 sampling sites, the overall inventory completeness was 99.9%. with respect to species abundance, a total of 1,057 species occurrences were recorded in the entire sampling, ranging from 36 (s7) to 136 (s16). we observed that the assemblage structure changed across the sites (fig 2). considering proportional abundance, the general pattern indicated an increase in species dominance in sites s1, s2, s4, and s6 (fig 2). the dominant species in the studied cityscape were atta texana (buckley), forelius mccooki (mccook), paratrechina longicornis (latreille), pheidole bilimeki (mayr), p. dentata (mayr), solenopsis geminata (fabricius), and s. xyloni (mccook), with a relative abundance greater than 20%. with respect to observed species richness (fig 3), a minimum of four species were recorded in sites s1 and s2 and a maximum of 18 in site s16. the estimated species richness increased significantly from site s1 to site s16. fig 2. rank-abundance curves of the ant assemblages recorded in the sub-montane and sub-tropical cityscape of ciudad victoria, tamaulipas, mexico. only species with a relative abundance higher than 20% (above dashed line) in a given sampling period are shown. pl: paratrechina longicornis (latreille), sg: solenopsis geminata (fabricius), pd: pheidole dentata mayr, fm: forelius mccooki (mccook), at: atta texana (buckley), sx: solenopsis xyloni mccook, pb: pheidole bilimeki mayr. fig 3. comparison of ant species richness estimates for a rarefied and extrapolated sample with a size up to double the reference sample. differences are considered to be statistically significant when the 95% confidence intervals (c.i.) do not overlap; for overlapping intervals, no differences are assumed (α=0.05). the evaluation of compositional similarity using a cluster analysis and simprof test based on the jaccard index showed the significant separation of three assemblage clusters at a similarity of 7.34% (π=3.18, p=0.002) and 10.27% (π=2.62, p=0.003) (fig 4). the ant assemblage collected in site s6 was significantly separated from the assemblages collected in sites s15 and s7 and the remaining assemblages. ma garcía-martínez et al. – diversity of urban ants in northeastern mexico.444 regarding species turnover among sampling sites, 12.50% of total species were unique to a single site, and no single species was shared by all sites (fig 5). in addition, no single species was shared by 5, 10, 11, 12, 14 or 15 sites, although 3.12% of total collected species were shared by 13 sites. the species lasius niger (linnaeus), carebara urichi (wheeler), cyphomyrmex rimosus (spinola), and mycocepurus smithii (forel) were unique to sites s3, s6, s7, and s15, respectively. in the remaining sites, no unique species were found. demonstrated that the cityscape of ciudad victoria in northeastern may provide several and unique habitats for ants. overall, the alpha, beta, and gamma diversities of the studied assemblages seem to be high in comparison to those of other urban myrmecofaunas for the fig 6 (slipinski et al., 2012; ossola et al., 2015; rocha & castaño-meneses, 2015). a high proportion of native species and a relatively low proportion of exotic species (1.32%) were found in addition to high compositional dissimilarity among the different sampling sites. the conservation value of the studied cityscape likely rests on the fact that it is formed by diverse habitats that support a particular array of myrmeco fauna. therefore, cityscapes should be included in conservation strategies for terrestrial biodiversity based on their conservation value. notably, compared to previous studies on urban myrmeco fauna, we recorded a higher number of species (clarke et al., 2008; cupul-magaña, 2009; slipinski et al., 2012; ossola et al., 2015). we also recorded a higher proportion of native ants with respect to exotic ants, yet it is important to emphasize that the encountered exotic species of p. longicornis, l. niger, m. floricola (jerdon), and tapinoma melanocephalum (fabricius, 1793) are considered highly problematic at the worldwide level (pagad et al., 2015; bertelsmeier et al., 2017). further studies on urban ant assemblages should be performed to elucidate the relative influence (positive or negative) of different habitats and the landscape drivers of ant diversity in novel urban-influenced ecosystems. the sites of the studied cityscape varied significantly in their conservation value and associated ant diversity because of the presence of lowly (e.g., structurally complex parks and gardens) and highly urbanized areas (e.g., areas with high built cover). overall, we observed that species richness is highly variable among the sampled sites. particularly, sites s15 and s16, which are close to vast areas of sub-montane fig 4. dendrograms of the hierarchical clustering of urban ant assemblages recorded in the sub-montane and sub-tropical cityscape of ciudad victoria, tamaulipas, mexico. the dendrograms display as continuous lines the divisions for which the simprof test rejects the null hypothesis (assemblages in these groups have no further structure to explore); the groups of assemblages indicated by dashed lines were not separated by the simprof test (at p<0.05). fig 5. percentage of total species (n=32) that occur in the sub-montane and sub-tropical cityscape of ciudad victoria, tamaulipas, mexico. discussion the present study evidences the high variability of urban entomo fauna as a result of constant anthropogenic pressures (slipinski et al., 2012). in our study, we empirically fig 6. contribution of alpha and beta diversity to the gamma diversity of urban ant assemblages of the sub-montane and subtropical cityscape of ciudad victoria, tamaulipas, mexico. the gray bars represent alpha diversity per sampling site, the white bars beta diversity, and the dotted line mean alpha diversity. all diversities are expressed as a percentage of total recorded species (n=32). sociobiology 66(3): 440-447 (september, 2019) 445 and sub-tropical thorn scrub, showed a significantly higher species richness. in contrast, sites with high urban intensity or built cover, such as s5, had lower species richness. in human-dominated landscapes, it appears that the structure, of ant communities, changes as a result of interspecific competition and the exclusion of native species by introduced dominant species. the dominant species a. texana, f. mccooki, p. longicornis, p. bilimeki, p. dentata, s. geminata, and s. xyloni were particularly well represented in peri-urban sites (s1, s2, s3, s4) of the urbanized area near areas with native vegetation. these dominant species mainly exhibit generalist nesting and feeding habits, which provide greater plasticity, and are highly adaptable to areas with a high degree of anthropogenic disturbance. such generalist species may have some advantages over other specialist or native species in anthropized environments. their abundance patterns may be the result of construction activities during the ant sampling. thus, the dominant species mainly exhibited generalist nesting and feeding habits, which provide greater plasticity and enable adaption to areas with a high degree of anthropogenic disturbance (pećarevićet al., 2010). the presence of aggressive and dominant species in urban sites or in sites near conserved areas could interfere with the dispersal of arthropods living in the soil and adversely affect the composition of the arthropod community (lessard & buddle, 2005). however, in one of the sites, we found that the presence of l. niger, an introduced species that is highly common around the world, did not seem to cause an imbalance in the native myrmeco fauna, even though our analysis is not necessarily focused on the positive or negative effects of introduced species. it appears that most introduced ants remain confined to habitats modified by humans without altering the community of native ants (lester, 2005). one previous study suggested that urban areas connected and containing native vegetation have higher ant species richness and lower dominance compared to other urban sites in the city center (rocha & castaño-meneses, 2015). this was confirmed by our finding that three of the four species unique to particular sites had specialized feeding habits and were found in sites near native vegetation fragments. in contrast, urbanization, which results in changes in vegetation cover, habitat fragmentation, and environmental pollution, may negatively affect species richness (yasuda & koike, 2009). accordingly, a smaller number of species are expected in areas of greater urban influence. in the present case, a smaller number of species was indeed obtained in urban areas (s13 and s14), although the number of species (11 and 12, respectively), was still notable. the myrmecofauna found at these latter sites is common in anthropized environments and is composed a mixture of native and exotic species that show a high tolerance to human disturbances. in the analyses of compositional similarity, we observed a significant separation into three assemblage groups. site s6, which is located in a landscape composed of human settlements and infrastructure, differed significantly from site s16, which is located near a remnant of native vegetation (fig 1). this finding is concordant with the studies of kamura et al. (2007) and cupul-magaña (2009), where in changes were reported in the compositional similarity and structure of ant assemblages between sites located near mountain forest fragments and sites located near urban centers. one of the explanations for this phenomenon is the differential availability of the resources used by ants in urban areas and native forests (kim, 1992). in conclusion, this study provides one of the first standardized inventories of the myrmecofauna associated with a cityscape in mexico. the gamma diversity of ants in the studied cityscape is mainly determined by the beta diversity or high species turnover of ants among the sampling sites to the fig 6. each studied site holds an important and particular myrmeco fauna that differs from that of the other sites and surrounding habitats of the cityscape. the relatively high alpha and beta diversities may be shaped by the high habitat heterogeneity of the cityscape, even though urban intensification can also negatively affect the alpha diversity of ants and probably of another arthropods. therefore, some green areas in the cityscape may positively contribute toward the conservation of ant assemblages and probably other invertebrate communities that are threatened by urban intensification. these data support the importance of conserving green areas and of considering such areas in urban planning measures to conserve native species. we suggest that future research studies analyze the relative effects of the resources and environmental conditions associated with the different habitats present in the cityscapes of the sierra madre of tamaulipas, including those of transitional zones and wellconserved regions, to further determine the conservation value of novel urban-influenced ecosystems. acknowledgments the authors would like to thank dr. manuel lara villalón for kindly providing part of the equipment for identification used in this study. we thank the associate editor (evandro n. silva) and the anonymous reviewers for their valuable comments and suggestions, which were helpful in improving the quality of the article. authors’ contribution ma garcía-martínez and m rosas-mejía: designed the experiment, performed experiments, and analysis, wrote the paper. or leyva-ovalle and p zetina-córdoba helped in performing experiments and the preparation of the paper. v vanoye-eligio and mj aguilar-méndez: performed the analysis, wrote the paper. references alvey, a.a. 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(2009). the contribution of the bark of isolated trees as habitat for ants in an urban landscape. landscape and urban planning, 92: 276-281. doi: 10.1016/j. landurbplan.2009.05.008 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.189-197sociobiology 61(2): 189-197 (june, 2014) pollinator sharing in specialized bee pollination systems: a test with the synchronopatric lip flowers of centrosema benth (fabaceae) m ramalho1, m silva1,2, g carvalho2 introduction floral attributes have been protagonists in ample and controversial debates concerning adaptive changes and the mechanisms subjacent to flower-visitor interactions (e.g., herrera, 1996; ollerton et al., 2007). the roots of these debates reside, in large part, in the understanding that floral characteristics are adaptively flexible (endress, 1994), even when they involve presumably specialized pollination modes (tripp & manos 2008), as flower-visitor interactions are often scale and ecological context dependent (herrera, 2005; ollerton et al., 2007). in comparing extensive floral data (on floral traits and pollinators) from six communities around the world, ollerton et al. abstract bee-pollinated lip flowers of two synchronopatric species of centrosema were used as models to examine the influence of specialized pollination systems on the ecological mechanisms of pollinator sharing. regression analysis of bee abundances in the habitat on bee abundances on c. pubescens flowers was significant (r = 0.69; p = 0.001) and became very consistent and highly significant (r = 0.87; p = 0.00001) using a size threshold of bee pollinators longer than 15mm. these same relationships were not significant (p > 0.01), however, for c. brasilianum flowers. the structures of the two pollination systems also sustained the hypothesis of a size threshold for pollinators, although only the c. pubescens-bees interactions sustained the hypothesis of random interactions proportional to species abundances in the habitat. the flower visitor pools of the two plant species shared the same four main bee guilds: the pollinators centridini, xylocopa, and euglossini and the primary nectar robber oxaea. however, a significant divergence (p < 0.01) was detected between the two systems when the abundances and behaviors (pollinators or cheaters) of the main shared flower visitors were incorporated into the overall quantitative analysis (nmds). the flowers size differences are not significant (p > 0.05) and could not explain these divergences. particularly, the concentrations of the largest pollinators eulaema and xylocopa on c. pubescens flowers and the behavior shift of centridini bees that act as legitimate pollinators in c. pubescens and as nectar robbers in c. brasilianum are better understood as functional foraging responses triggered by the synchronopatry and by nectar volume differences (p = 0.001) between both lip flowers. paradoxically, the robbery activity of centridini bees arises as a supply side effect of smaller nectar volume in c. brasilianum flowers. sociobiology an international journal on social insects 1 universidade federal da bahia (ufba), salvador, ba, brazil. 2 faculdade de tecnologia e ciências, salvador, ba, brazil. article history edited by candida m l aguiar, uefs, brazil received 30 december 2013 initial acceptance 07 march 2014 final acceptance 21 may 2014 keywords nectar robber, size threshold , random interaction, bee pollinated flower corresponding author mauro ramalho laboratório de ecologia da polinização instituto de biologia universidade federal da bahia salvador, bahia, brazil cep: 40.210-730 e-mail: mrramauro@gmail.com (2009) concluded that the “pollination syndrome hypothesis”, as traditionally stated, did not properly describe the diversity of floral phenotypes and only poorly predicted plant species pollinators. these authors therefore recommended a ‘fresh look at how the traits of flowers and pollinators relate to visitation and pollen transfer’ in searching for appropriate descriptions of functional floral diversity. a question central to this debate is if other variables can be as important as morphological restrictions in structuring pollination systems. recent analyses of partnerships and the structures of flower-visitor webs have both indicated that morphological constraints continue to play a central role in the general theory of the organization of pollination systems. research article bees m ramalho et al pollinator sharing in synchronopatric lip flowers190 the relationship between proboscis lengths and nectar chamber depth, for example, sustains the hypothesis that size thresholds are important in regulating those interactions (stang et al., 2006). on the other hand, the pattern of “asymmetric specialization” that emerges from the webs (e.g., vázquez, 2005) is also better explained when both size constraints and species abundance are incorporated into theoretical models (stang et al., 2007). in this case, the following basic question directs the studies: are neutral mechanisms of random interactions proportional to species abundance sufficient to explain the asymmetric specialization (plants with specialized flowers frequently interacting with generalist visitors, and specialized floral visitors frequently interacting with generalized flowers) that has been observed in recent analyses of flower-visitor interaction webs (vázquez & aizen, 2004; vázquez, 2005; stang et al., 2007)? here we focus on the question of pollinator sharing in specialized pollination systems and have chosen as a model two synchronopatric species of centrosema (fabaceae) with specialized zygomorphic keel flowers. the highly zygomorphic keel flowers of fabaceae are often pollinated by bees (faegri & van der pijl, 1979; westerkamp, 1996; galloni et al., 2007). bee-pollinated keel flowers have nectar guides and produce nectar as the main floral reward for those insects, but only large bees with appropriate nectar-searching behaviors are able to provoke the mechanical exposition of the fertile pollination organs (van der pijl, 1954; gottsberger et al., 1988; lopes & machado 1996; etcheverry et al., 2003; ramalho & rosa, 2010). a distinctive floral trait of bee pollinated keel flowers is protection of their pollen from bee consumption (which would presumably be more effective in lip-flowers, as their keel is dorsally positioned in relation to the landing petal) (westerkamp, 1996). centrosema pubescens benth and centrosema brasilianum (l.) benth have large and very similar lip-flowers with notable bee-pollination traits (endress 1994). these two species show complete overlapping of their flowering periods and occur sympatrically along the tropical atlantic coast of brazil in herbaceous-shrub vegetation habitats (coastal “restingas” and sand dune areas). their synchronopatric and specialized flowers should largely share pollinators according to the predictions of both the hypothesis of morphological constraints and that of random interactions proportional to species abundance. in cases such as this, pollinator sharing would favor the coexistence of species with subtle differences in their floral biology due to the potential loss of significant quantities of pollen to hetero-specific flowers (jacobi et al., 2005). this study therefore assumed the following specific premises: first, the most shared groups of pollinators must be abundant in the habitat if divergences in floral biology of the two centrosema species have only small influences on the functional differentiation of the two pollination systems; second, subtle morpho-functional differences in flower traits should produce greater differences among pollinating bees than among nonpollinating bees (flower visitors). material and methods study area the present study was undertaken in “restinga” vegetation along the eastern tropical atlantic coast of brazil. this coastal restinga vegetation develops under regional influences of the “the tropical atlantic domain” (por, 1992). the field study site was located in the pituaçu metropolitan park (pmp) in the city of salvador, bahia state, brazil (13°00’ s; 38°30’ w). the pmp is dominated by secondary growths of open shrubby-forests with patches of exposed sand dunes, covering an area of 450 ha, at approximately 50m above sea level. the regional climate is af, according to köppen classification system, with a mean annual rainfall of approximately 1500mm, and mean monthly temperatures vary between 18°c and 22°c. there is no marked dry season, although the vegetation, which grows on sandy soils, is exposed to sporadic water deficits throughout the year, and there are many areas with only thin vegetation covers. plant species. centrosema pubescens benth. and centrosema brasilianum (l.) benth. occupy open vegetation areas (dominated by herbs and small shrubs) in tropical restinga and coastal sand dune sites. c. brasilianum is a procumbent herb with glabrous inflorescences; its flowers are bluish-purple with white-yellowish pale stripes (nectar guides) on the banner petal. c. pubescens is a climbing vine with velutinous inflorescences; its flowers are bluish-pale lilac with pink and creamy white stripes on the banner. the flag (or banner) serves as a large landing platform for the bees on both lip flowers and the corolla base is completely surrounded by a robust tubular calyx. the robust keel (in a dorsal position) surrounds and protects the fertile verticils and it must be displaced upwards by bees moving towards the corolla base. nectar is the main floral reward, and is well-protected at the base of the corolla by the tight juxtaposition of the petal bases (which form a rigid and very narrow passage to the nectar chamber). the nectar can be reached through the interiors of the flowers by robust bees with long proboscis (personal observation). the two centrosema species have complete overlapping flowering periods: c. brasilianum blooms year-round and c. pubescens from march to july (m. ramalho & m. silva unpublished). permanent 20m x 20m plots were delimited in high density patches for field experiments, sampling, and bee visitation observations. the observed numbers of open flowers/day were relatively low (5-10 flowers/m2/day and 10-15 flowers/m2/day of c.pubescens and c. brasilianum, respectively; m.silva & m. ramalho unpublished) even in these dense patches where the flowers of both species were sometimes intermingled. floral biology and morphology measures. the basic analyses of floral biology were performed following dafni (1992). stigma receptivity was tested with hydrogen peroxide, and nectar volumes were measured in ten flowers from five sociobiology 61(2): 189-197 (june, 2014) 191 different individuals of each centrosema species. flowers (n = 10) were bagged one day before opening and the nectar was withdrawn using micropipettes soon after 08:00 h. this time was standardized based on a posteriori characterization of the period of highest floral visitor activity. ten flowers from ten different individuals of each centrosema species were measured with an electronic digital caliper (accuracy 0.1mm): the length and width of the banner, the length of the keel and the depth of the corolla were measured in fresh flowers. in this latter case, it was measured the distance between the point of insertion of the banner’s spur and the internal base of the nectar chamber. bee sampling data. floral visitors were captured using hand-held insect nets during the periods of overlapping flowering of the two centrosema species (march to july) in two successive years. based on observational field data on floral biology (principally anthesis and anther dehiscence), the bees were intensively sampled on the flowers of both species from 07:00 h to 12:00 h (most of the flowers are senescent after 12:00 h) on a daily basis for 15 minutes/species every hour for 24 days (30 hours of sampling efforts) along the overlapping flowering period. visitor behavior on the flowers was observed simultaneously (and photographed for posterior analysis) during 15 minutes/h (totaling 15 hours) to characterize pollinators and non-pollinators. bees that behaved in discordance with the keel morphology (i.e., making holes in the corolla and accessing nectar from the outside of the flower or biting the anthers to collect pollen) were considered flower robbers (see inouye, 1980) and were separated into two categories (inouye, 1980): primary or secondary. primary flower robbers make perforations in the corolla in order to gain access to the nectar (or pollen), while secondary flower robbers take advantage of the perforations made by primary flower robbers. in general, visitors are called “cheaters” if they consume floral resources without entering into contact with the fertile verticils (inouye, 1980). the bee morpho-species were determined by consulting published keys and bee references in the pollination ecology laboratory (ecopol) and the bee biology and ecology laboratory (labea) at the biology institute of the universidade federal da bahia (ufba). the identifications of the centridini and euglossini bee species were confirmed by dr. fernando zanella (universidade federal da integração latino-americana unila, paraná, brasil) and dr. ednaldo l. das neves (faculdade jorge amado, bahia) respectively; the scientific names follow moure et al. (2007). all specimens were deposited in the ecopol. the centrosema species were identified by dr. luciano paganucci de queiroz of the universidade estadual de feira de santana bahia (uefs), and those specimens were deposited in the alexandre leal costa herbarium at the universidade federalda bahia (ufbaalch). data analysis. the mann-whitney non-parametric test (α = 0.05) was used to compare the quantities of nectar produced by c. pubescens and c. brasilianum. the t-test was used to compare flower measures between both species (gotelli & ellison 2004). in the global comparative analyses of the two pollination systems pseudo-species were created corresponding to two behavioral categories: robbers and pollinators. data concerning the composition and abundance of ‘pseudo-species’ visiting the flowers of six individual plants of c. pubescens and c. brasilianum were used in non-metric multidimensional scaling (nmds) based on a bray-curtis similarity matrix. analyses of similarity (anosim) of each plant species were performed to test the null hypothesis of equality in the composition and abundance of floral visitors of these pollination systems. detailed analyses of the contributions of each bee group to the observed dissimilarity (simper) were also performed. rare pseudo-species with frequencies equal or less than one were excluded from the analysis. the hypothesis of random interactions being proportional to species abundances (vázquez & aizen, 2004; vázquez, 2005;) was evaluated comparing bee abundances in the local habitat. the bee abundances in the local habitat (pmp) were estimated by bee sampling during one year period. using hand insect nets, the bees were captured on the flowers of all detected bee plants (sakagami et al., 1967) in a transect of 2.5km length, from 07:00 h to 17:00 h, totalizing 240h of sampling effort. linear regressions between the absolute abundance values of each bee species in the habitat and their respective ‘relative abundance’ on the flowers were estimated using graphpad instat 3 software (at a significance level of 0.01). the relative abundance of each bee species were estimated as follows: the abundance was transformed into a value between 0 and 1 (with 1= the maximum abundance observed on the flowers) by dividing the number of individuals on each flower by the total number of individuals of the most abundant bee species observed on both flowers. using values of relative abundance between 0 and 1 facilitate interpretations of the graphs in terms of the probability of interactions being proportional to species abundance. a significance level of 0.01 was used. as the flowers of centrosema are quite large and their nectar is well-protected at the base of the corolla, we assumed an a posteriori size threshold of floral visitors to test for size constraints on random interactions proportional to species abundances (stang et al., 2007). body size was used as a surrogate for proboscis length, as all of the pollinators observed belonged to the general category of long-tongued bees (michener, 2000). it is important to note that all members of the orchid bees or euglossini have proboscis longer than the other bee groups with similar body size. the orchid bees have very long proboscis (longer than ¾ of their body length) that are more or less proportional to their body size (with several exceptions): being, for example, up to 10mm long in species whose bodies are approximately 10mm long, and up to 40mm long in species larger than 20mm (roubik & hanson m ramalho et al pollinator sharing in synchronopatric lip flowers192 2004). five individuals of each bee species were measured to estimate their body lengths using an electronic digital caliper (accuracy 0.1mm). the distance between the top of the head (at the height of middle ocellus) and the end extremity of abdomen was measured in order to obtain a rough estimate of relative size for insertion of bees in the following body length categories: small bees <9.9mm; medium bees ≥ 10 < 15mm; large bees ≥ 15 < 20mm; very large bees ≥ 20mm. results the very similar zygomorphic lip-flowers of c. brasilianum and c. pubescens both offer nectar as the main reward to flower visitors. nectar is produced from the start of flower anthesis (during the night) until pre-senescence (near 12:00 h) in both species, thus being available from sunrise until noon. in synchrony with nectar availability, the stamen is receptive from early morning until floral senescence in both species. c. pubescens produced greater quantities of nectar (p < 0.01) than c. brasilianum (table 1), so that the availability of this resource to foragers is potentially greater on the flowers of the former species. active pollen harvests were made by very few non-pollinators that often visited the flowers after the peak of activity of the pollinators – so that pollen protection by the keel structure appears to be quite effective considering the legitimate visitors. the sizes of the flowers are very similar in the two species of centrosema and there are no significant differences in width (p = 0.45) and length (p = 0.67) of the banner and, mainly, in the depth of the corolla (p = 0.45). the difference between the flowers is observed only on the dimensions of the keel (p < 0.001), more robust in c. pubescens. the keel size probably does not modify the nectar accessibility by large bees (≥ 15 < 19.9 mm) or very large bees (≥ 20 mm), however, it could affect the behavior of small and medium sized bees on the flowers. a total of 489 flower visitors were sampled on the flowers of the two centrosema species, of which almost 98% and 27 species were bees (principally robust bees, with body sizes > 10mm; table 2). most of the bees collected nectar in the flowers of both centrosema species, with the exception of few pollen robbers. eighty-six percent of the observed bee species made the legitimate nectar harvests on c. pubescens flowers, as compared to only 46% on c. brasilianum. during legitimate visits in both flowers, pollinators typically landed on the ventral lip (banner) of the flower and forced their head and thorax towards the corolla base, displacing the keel upwards and triggering pollen deposition on their backs (nototribic pollination). they accessed the nectar by inserting their long tongues into the nectar chamber through a rigid and very narrow passage at the base of the corolla. this corolla structure therefore impedes legitimate nectar access by small bees (e.g., bees < 10mm). in terms of both species richness and abundances on flowers, the major groups of pollinators were large (body length > 15mm < 20mm) or very large bees (body length ≥ 20mm), all with long (e.g., xylocopa) or very long proboscis (eulaema, euglossini), as well as some medium-sized euglossini bees with very long proboscis (euglossa, euglossini). the non-pollinators were medium-sized nectar robber bees (oxaea) and small pollen robber bees (ceratina and augochloropsis). the roles of medium-size centridini bees varied with centrosema species (table 2). in contrast to legitimate visitors, nectar robbers always moved along the outside of the flower to the base of the perianth, where they would pierce the calyx to gain access to the nectar chamber. this type of behavior was often displayed by individuals of oxaea species on c. brasilianum flowers (14% of total flower visitors) and on c. pubescens flowers (17% of total flower visitors), and by centris and epicharis bees on c. brasilianum flowers. oxaea usually acted as a primary nectar robber, making holes in the calyx that could be used by secondary nectar robbers (centris, epicharis, ceratina, pseudaugochlora, and augochloropsis). ceratina, pseudaugochlora, and augochloropsis also acted as pollen robbers, harvesting it with their mouth parts directly from the anthers; ceratina bees were often the primary pollen robbers, punching holes in the anthers inside the keel that the other two groups would later take advantage of. a high similarity was seen between the two pollination systems, considering their sharing of higher taxa and functional bee groups. by contrast, considering the actual numtable 1. floral biology of two synchronopatric species of centrosema (fabaceae) in an coastal tropical restinga (brazil). flower measures (n = 10) are described in methods. character centrosema pubescens centrosema brasilianum anthesis (start-end) 00:00 h – 05:00 h 00:00 h – 02:00 h stigma receptivity (start-end) 05:00 h until senescence 02:00 h until senescence nectar volume (ml) 26 ± 4.20 14 ± 5.50 floral reward nectar nectar flower color bluish-pale lilac, with magenta and creamy-white stripes on the banner (nectar guide) bluish-purple, with whitish-yellow pale stripes on the banner (nectar guide) flower measures (mm) banner length 33.71 ± 2.16 36.4 ± 3.54 banner width 37.66 ± 2.79 38.85 ± 3.75 keel length 20.41 ± 1.75 16.34 ± 0.79 corolla depth 5.34 ± 0.31 5.46 ± 0.36 sociobiology 61(2): 189-197 (june, 2014) 193 table 2. abundance distributions (on the flowers and in the pmp habitat), size categories, and behaviors of the floral visitors to centrosema flowers: centrosema pubescens and centrosema brasilianum. the behavioral categories follow inouye (1980): r = primary flower robbers; rs = secondary flower robbers; p = pollinators; n.r. = not recorded. the size categories of the bees were based on body lengths: (•) small bees <9.9mm; (••) medium sized bees ≥10 <14.9mm; (••••) large bees ≥15 <19.9mm; (•••••••) very large bees ≥ 20mm. bee groups flower visitor abundance bee size categories habitat pmp c.pubescens c.brasilianum euglossini euglossa cordata (linnaeus, 1758) 87 17(p) 57(p) •• euglossa ignita smith, 1874 0 1(p) 0 •• euglossa securigera dressler, 1982 0 2(p) 0 •• eufriesia cf. mussitans fabricius, 1787 26 0 1(p) •••• eulaema cingulata moure 1950 13 12(p) 3(p) •••• eulaema flavescens friese, 1899 6 6(p) 0 ••••••• eulaema nigrita (lepeletier, 1841) 71 45(p) 10(p) •••• eulaema bombiformis niveofasciata friese, 1899 4 17(p) 0 ••••••• bombini bombus brevivillus franklin,1913 26 10(p) 3(p) •••• centridini centris (hemisiella) tarsata (smith, 1874) 15 1(p) 1(rs) •• centris (centris) flavifrons (fabricius, 1775) 14 1(p) 1(rs) •••• centris (centris) leprieuri (spinola, 1841) 48 0 8(rs) •• centris (trachina) fuscata (lepeletier, 1841) 65 2(p) 2(rs) •• epicharis (xanthepicharis) bicolor smith, 1854 2 1(p) 1(rs) •••• epicharis (epicharis) flava (friese, 1900) 11 15(p) 1(rs) •••• xylocopini xylocopa (megaxylocopa) frontalis (olivier, 1789) 131 85(p) 1(p) ••••••• xylocopa (neoxylocopa) nigrocincta smith,1854 15 3(p) 0 •••• xylocopa (neoxylocopa) suspecta moure & camargo, 1988 14 2(p) 1(p) •••• xylocopa (neoxylocopa) cearensis ducke, 1910 1 0 1(p) •••• xylocopa (neoxylocopa) grisescens lepeletier,1841 4 1(p) 0 ••••••• oxaeini oxaea flavescens klung, 1807 18 2(r) 13(r) •• oxaea sp.1 48 57(r) 10(r) •• ceratinini ceratina (crewella) sp.1 32(r) 24(r) • augochlorini pseudaugochlora pandora smith, 1853 50 0(rs) 6(rs) •• augochloropsis callichroa cockerell, 1900 37 4(rs) 6(rs) • exomalopsini exomalopsis sp.1 13 0 2(r) • ericrocidini acanthopus sp.1 2 3(p) 0 • other insects (n.r.) 8 (n.r.) 10 (n.r.) m ramalho et al pollinator sharing in synchronopatric lip flowers194 bers of species, only 46% of the pollinators (12/26) and 67% of the non-pollinators (4/6) were shared by both centrosema species. the percentage of shared bee species between the two flowers partially reflected sampling artifacts (the small numbers of sampled individuals of several species; table 2). the main differences between the systems, however, are related to the distributions of abundances of the large pollinators and the shifting behavior of a shared bee group (centridini). apparent size constraints can explain basic differences in the behaviors of bee visitors to the specialized lip flowers of centrosema (table 2) – with pollinators usually being larger than 15mm; non-pollinators were consistently less than 15mm long. among the medium-sized bees (10-15mm), only some euglossa species with very large proboscis (> 10mm) were abundant on flower and would legitimately access the nectar while performing pollination. the primary nectar flower robbers were medium-sized bees oxaea (< 15mm). the behaviors of medium-size centridini bees on the flowers were less predictable as they acted as pollinators of c. pubescens and as secondary nectar robbers of c. brasilianum. in this latter case, flower robbing was probably related to the lower nectar volume in c. brasilianum flowers. the nectar robber behavior of some larger centridini bees (two species of epicharis) probably was stimulated by easy access to nectar in c. brasilianum flowers that had been previously perforated by primary flower robbers (oxaea). the relationship between the relative abundance of bees on centrosema flowers and their abundance in the habitat (fig 1; see also table 2) was very consistent and extremely significant (r = 0.87; p < 0.00001) when the analyses were restricted to large bees (≥ 15mm) visiting c. pubescens flowers (fig 1a). although significant, a loss of consistency was seen in the relationship when all floral visitors to c. pubescens flowers were included, independent of their sizes (r = 0.69; p = 0.001). the abundance of bees in the habitat, however, was not predictive of their abundance on c. brasilianum flowers under any circumstances – whether considering a minimum size constraint of 15mm (fig 1b; p = 0.49; p = 0.04) or including all floral visitors in the analyses (r = 0.42; p = 0.034). the smaller nectar rewards and the activities of secondary flower robbers on c. brasilianum flowers seem to affect mainly large pollinators (e.g., eulaema nigrita and xylocopa frontalis) that probably shift to c. pubescens flowers with large nectar volume. considering the abundances and behaviors of floral visitors (pollinator or non-pollinator), the nmds analysis revealed two distinct structural and functional organizations of the pollination systems of c. brasilianum and c. pubescens (fig 2). analysis of similarity (anosim) confirmed significant differences between the pooled visiting bees (average dissimilarity = 83%; p = 0.011). quantitative analyses of the contributions of particular bee groups to the observed dissimilarity indicated that the abundance distributions of the very large eulaema and xylocopa bees and the medium-sized euglossa bees (with very large proboscides) were particularly fig. 1. relationships between abundance of bees (pollinators + nonpollinators) in the habitat (pmp) and on the flowers of centrosema: a) centrosema pubescens (r = 0.87; p < 0.00001); b) centrosema brasilianum (r = 0.49; p = 0.04). it is presented only the regression curves for the large bees + very large bees + euglossini (bees with very long proboscis). a b important to the ecological differentiation between the two pollination systems. the pollinating bees xylocopa frontalis, eulaema nigrita and eulaema meriana together, for example, were responsible for 27% of the observed dissimilarity between the two systems, while medium-sized pollinating bees euglossa contributed 25%; medium-sized centris bees contributed 4.2%, mainly due to the fact that they behave as pollinators or robbers depending on the centrosema species. altogether, the primary nectar or pollen robber bees oxaea and ceratina respectively, contributed for only 10% of the dissimilarity. sociobiology 61(2): 189-197 (june, 2014) 195 discussion similarity in floral biology and mainly synchronopatry expose the two centrosema species to share the main visiting bees and functional bee groups (camargo et al., 1984; roubik, 1989; michener, 2000): the huge carpenter bees of the genus xylocopa and the very large-tongued euglossini bees as the main pollinators; oxaea primary nectar robbers; ceratina primary pollen robbers; and the floral oil bees centridini whose role is dependent on centrosema species. in particular, the primary nectar and pollen robbers were bees evenly shared by both flowers, as expected, suggesting they are weakly responding to subtle floral differences between the centrosema species (e.g., flower color and keel size). on the other hand, the significant differences in the abundances of shared pivotal pollinators, mainly the large bees xylocopa and eulaema, and secondary nectar robbers, cannot be attributed to random effects only. the hypothesis of a size threshold modulating the interactions between nectar-flowers and consumers (stang et al., 2006) is mainly supported by the predictable interactions of c. pubescens with large and very large bees that have large or very large proboscis (xylocopa and eulaema species). these relationships also corroborate the influence of random interactions proportional to bee species abundances in the habitat (vázquez & aizen, 2004, vázquez, 2005; stang et al., 2007). the observed partnerships between pollinators and the very specialized centrosema lip-flowers should therefore be attributed to the action of two basic mechanisms: neutral random interactions driven by species’ abundances in the habitat, and interactions modulated by morphological constraints (i.e., bee size threshold). on the other hand, neither morphological constraints (e.g., proboscis x corolla length) nor neutral interactions (vázquez, 2005; stang et al., 2006) fully encompass the mechanisms responsible for the high concentrations of very large bees on c. pubescens flowers and the observed shifting of the behavior of centridini bees from legitimate c. pubescens pollinators to c. brasilianum nectar robbers. significant difference in keel size between both flowers doesn’t explain adequately the exploitation modes and sharing of these two nectar sources by the bees. if the keel provides resistance to visitors, it was expected some difference in the frequency distribution of mid-sized bees between the two centrosema species and a higher frequency of nectar thefts in c. pubescens flowers with the largest keel: only the first prediction is partially supported by the high abundance of euglossa cordata on c. brasilianum flowers. the abundance distributions of primary and secondary nectar robbers on both centrosema species are therefore not affected by the keel size. bees are notable for their ability to choose among nectar sources comparing foraging cost/benefit ratios (waddington, 1980; pyke, 1984). for instance, among coexisting bombus species, the largest bees with the largest proboscis tend to choose flowers with the longest corollas, from which they can collect more nectar more efficiently than smaller bees (e.g., harder, 1985; heinrich, 2004). if the bees were mainly responding to size restrictions, therefore, the largest bees should visit the flowers of both centrosema species with similar frequencies, in light of their similar conditions of nectar access (i.e., similar flower sizes and corolla lengths) and similar flower densities in the habitat (see methods). the same would not be true, however, in terms of floral nectar volume, and some large and very large-sized bees (e.g., xylocopa frontalis and eulaema species) likely choose c. pubescens flowers simply because they can obtain more nectar per visit. the difference in flower color between the centrosema species must be used for recognition and choice of c. pubescens flowers, with more nectar, by these large bees. despite the low contribution of secondary nectar robbers to the structural divergences between centrosema pollination systems (i.e., 4% dissimilarity), the centridini bees deserve attention because of the distinctive roles of “cheaters”on structuring flower-visitor webs (genini et al., 2010). centridini is one of the most abundant bee group in tropical coastal restinga and sand dunes (ramalho & silva, 2002; viana & kleinert, 2006; oliveira-rebouças & gimenes, 2011; rosa & ramalho, 2011), and therefore its low abundance on centrosema flowers would not sustain the hypothesis of random interactions proportional to abundances in the habitat (vázquez, 2005; vázquez & aizen, 2004). behaving as secondary nectar robbers in centrosema flowers is probably a response of centridini bees to a contingent relationship: encounters facilitated by the abundance of these bees in the habitat (ramalho & silva, 2002; rosa & ramalho, 2011) and by the long flowering periods of centrosema species. in light of the intense activity of primary flower robbers (oxaea spp) on both centrosema species (14% to 17% of total flower visitors on flowers), and particularly on c. pubescens, if secondary flower robbers were responding to access opportunities to nectar by preexisting perforations in the corolla, it would be expected that they would rob the flowers species centrosema pubescens centrosema brasilianum cb1 cb2 cb3 cb4 cb5 cb6 cp1 cp2 cp3 cp4 cp5 cp6 2d stress: 0,09 anosim p = 0,011 fig. 2. ordination diagram of the two pollination systems by nonmetric multidimensional scaling (nmds): cp = centrosema pubescens and cb = centrosema brasilianum. m ramalho et al pollinator sharing in synchronopatric lip flowers196 of both species or that the secondary robbery activity should be slightly higher in c. pubescens flowers. as such, it must not be by chance that changes from legitimate (pollinator) to illegitimate (secondary nectar robbery) visiting behavior mainly involve medium-sized centridini bees (=15mm) and c. brasilianum flowers with smaller nectar volumes. the high observed frequency of medium-sized euglossa bees, with very long tongues (proboscides >10mm) on c. brasilianum flowers provides indirect evidence that the body size/proboscis length ratio play a role in this relationship – as to maximize returns from the exploitation of the smaller nectar volumes in c. brasilianum flowers, medium-sized bees must have very long tongues (euglossa species) or be primary flower robbers (oxaea); being a secondary flower robber (centridini) would be the “best thing to do with a worst thing”, as they would be highly exposed to visit depleted flowers. in some circumstances, nectar robbing (even from specialized flowers) can be a very rewarding strategy for bees, depending on their ability to make adjustments in their foraging behaviors (zhang et al., 2011), and that is probably why cheaters are ubiquitous in mutualistic flower-visitors networks (genini et al., 2010). nectar robbing behavior could be stimulated by size restrictions triggered by subtle differences in corolla lengths among very similar zoophilous flowers and size differences between individuals visiting the same flower (urcelay et al., 2006; zhang et al., 2011) or by difference in nectar volume in centrosema species. paradoxically, in the relationship between the two centrosema species and centridini bees, secondary nectar robber seems to be more advantageous in the flowers with smaller nectar volume. in synthesis, pollinators sharing by the two centrosema species is potentialized by synchronopatry and modulated by pollinator choices between flower sources with different nectar volume and, apparently, by direct or indirect interactions among floral visitors, including flower robbers. the concentrations of the largest bee pollinators on c. pubescens flowers and the secondary nectar robber activity of medium-sized centridini bees on c. brasilianum flowers are foraging responses better understood by nectar volume differences than by differences in floral morphology per se. from the point of view of plant reproduction, nectar robbery has detrimental effects on maternal functions (e.g. seed set), depending on the species’ reproductive system (e.g., irwin et al., 2001). as such, by reducing the nectar volume, probably c. brasilianum is selecting for medium-sized bees with the largest proboscis (e.g. eulgossa) as its major pollinators, and therefore it should present some reproductive adjustment to compensate for the loss of large-bodied pollinators and the parallel-paradoxical increase in robbers activities. both centrosema species invest principally in cross-pollination, although the ratios of seeds/ ovules and seeds/fruits are both significantly smaller (p = 0.0001) in c. brasilianum than in c. pubescens (m. ramalho & m. silva unpublished), suggesting the first species is more adjusted to being visited and cross-pollinated mainly by a smaller number of large or very large bee species. references camargo, j.m.f., gottsberger, g. & silberbauer-gottsberger, i. 1984.on the phenology and flower visiting behavior of oxaea flavescens (klug) (oxaeinae, andrenidade, hymenoptera) in são paulo, brazil. beitrage zur biologie der pflanzen, 59: 159-179. dafni, a. 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(2011). behavioural differences between male and female carpenter bees in nectar robbing and its effect on reproductive success in glechoma longituba (lamiaceae). plant biology, 13 (suppl. 1): 25-32. doi: 10.1111/j.1438-8677.2009.00279.x. doi: 10.13102/sociobiology.v65i3.2885sociobiology 65(3): 422-432 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sequencing the ant fauna of a small island: can metagenomic analysis enable faster identification for routine ant surveys? introduction recent advances in sequencing technology (next generation sequencing or ngs) have expanded the possibilities of using dna barcoding to identify species in complex environmental surveys (telfer et al., 2015). using improved ngs techniques, often referred to as abstract all known ant species from a small western australian island were subjected to dna barcoding of the co1 gene, with a view to using the database to identify ants by next generation sequencing in subsequent, routine surveys. a further aim was to evaluate whether the data could be used to see if any new species had arrived on the island since the total fauna had been inventoried. of the 125 unique ant species then known from the island, 72 were successfully barcoded. those that were refractory to amplification were largely the result of sample age and/or contamination. following this base-line barcoding, ants were sampled from 14 regular sampling sites and ant sequences were obtained from the bulked ‘metagenomic soup’. prior to doing this, a parataxonomist had identified all ant species in the samples and returned them to the ‘soup’. successful identification for each site varied from 38% (sites 12 and 27) to 100% of species (site 10). comparison of the number of species recovered with the number of sequences obtained from each sample showed a positive correlation between the two variables. when a site had >1,000 sequences, the average recovery rate was 79%, which is in contrast to the lowest four recovery rates (site samples 12, 22, 26 and 27), which had fewer than 440 amplicon sequences. the ability to detect individuals that occur at low frequencies is also important. we analysed each site individually to determine if a species was detected and how that related to the proportion of individuals in the pooled sample. where a species was present at <4% of the total sample, it was only detected 10% of the time, indicating that adequate sequencing depth is critical to species recovery. we conclude that this technique was only partially successful in replacing conventional taxonomy and that it could have limited ability to detect incursions unless the new arrival is abundant. current barcoding is no longer limited to the co1 gene and other genes are characterised for identification of intractable groups where co1 does not provide appropriate levels of resolution. sociobiology an international journal on social insects jd majer1,2,3, ma castalanelli4, jl ledger4, nr gunawardene 2, be heterick2,3 article history edited by gilberto m. m. santos, uefs, brazil received 09 february 2018 initial acceptance 28 march 2018 final acceptance 10 abril 2018 publication date 02 october 2018 keywords next generation sequencing, barrow island, evaluation, biosecurity, ant incursion. corresponding author jonathan d. majer school of biological sciences university of western australia perth, wa 6009, australia e-mail: jonathan.majer@uwa.edu.au “metagenomics”, the effects of environmental change (e.g., deforestation, resource extraction, site development) can be examined at the level of whole ecosystems (e.g., beng et al., 2016; gibson et al., 2014; ji et al., 2013; smith et al., 2005; yang et al., 2014; yu et al., 2012). this can be achieved by establishing a baseline record of biodiversity within an ecosystem and then routinely comparing the 1 school of biological sciences, university of western australia, perth, wa 6009, australia 2 department of environment and agriculture, curtin university, perth, wa 6102, australia 3 western australian museum, perth, wa 6000, australia 4 ecodiagnostics pty. ltd., 46 48 banksia rd, welshpool wa 6106, australia research article ants sociobiology 65(3): 422-432 (september, 2018) 423 current biodiversity to that of the baseline, a procedure that is exemplified by the approaches of yu et al. (2012) and kocher et al. (2017). rather than sorting and separating specimens, dna is extracted from a batch or ‘soup’ of specimens from a field collection (often >100 specimens in a vial) and the dna sequenced using ngs techniques (pochon et al., 2013; yoon et al., 2016). this technique allows hundreds of different taxa to be sequenced simultaneously and the sequence data that is generated can be compared, as a batch, to the dna reference library. outputs from studies of this type provide huge datasets, and lead to a good understanding of sample diversity at a reasonable cost (ji et al., 2013). more importantly, previously unrecorded species that are rare, cryptic, or are invasive may be identified because their genetic signature differs from the reference database. the barrow island invertebrate surveillance project provided an opportunity to evaluate the application of this technique in an invertebrate species context, concentrating on the ant fauna. barrow island is western australia’s second largest offshore island; it is a class a reserve and also happens to be australia’s only land-based oil field. the presence of industrial interests on the island, and also its important conservation value, has meant that the island is not publicly accessible. this has resulted in the exclusion, or control and eradication of non-indigenous or invasive species. in 2009, chevron australia pty ltd and its joint venture participants undertook the construction of a liquefied natural gas plant on the island. one of the conditions under which approval for the plant was granted was the implementation of a rigorous biosecurity effort to ensure that no non-indigenous species (nis) were allowed to establish on the island and, if any new species were to be introduced, have a 0.8 probability of detection if they are present. to fulfill this condition a nonindigenous species surveillance program was implemented. if ngs procedures were to be used for diagnostics, it would be critical for the technique to be sensitive and reliable enough to detect previously unrecorded species. the aim of this pilot study is to evaluate whether this is achievable using a single gene. a series of systematic surveys on flora and fauna has been performed using purpose-designed sampling protocols in order to provide baseline data on the existing terrestrial invertebrate species on barrow island. as part of the fauna surveys, terrestrial invertebrates were sampled between 2005 and 2008 (majer et al., 2013). callan et al. (2011) initially recorded a total of 1,873 species and morphospecies, with subsequent surveys and taxonomic developments increasing the count to 2,670 species with 25 invertebrate species considered non-indigenous to bwi (thomas et al., 2017). the barrow island collection represents one of the few areas in australia where sampling of invertebrates has occurred before and after development. the ant species on barrow island are well-documented, totalling 125 (since upgraded to 129) species, none of which are endemic to the island (heterick, 2013). the presence of a voucher specimen library of dry and wet preserved ant species from the island enabled us to establish a reference dna barcoding library for the barrow island ants. dna was extracted and barcoded for specimens of each species to establish a dna reference database. as regular, repeated surveys of invertebrate species are still ongoing on barrow island, a pilot study was then conducted to test whether the ant specimens collected in subsequent samples could be verified using the ngs technique and the dna reference database. specifically, we evaluated the efficacy of universal forward primer ci-j-1718 and the reverse primers hco and ci-n-2191 in recovering and identifying multiple ant species within a trap using a roche’s 454 gs-junior metabarcoding approach. gs-junior was selected due to its ability (at the time) to sequence >400 bps per direction, and this was seen as a cost effective solution. we intended that the reference database of ant species barcodes could be used by future researchers to rapidly determine the species composition within samples taken from the field, a process that normally takes two weeks of a taxonomist’s time. in view of the fact that our surveys were designed to detect whether any nis had been introduced during the construction of the gas liquification plant, it was thought that the technique also might have the potential to identify non-indigenous ant species if present within the collection. the ngs procedure that we utilised has since been superseded by more refined techniques such as illumina mi and hi-seq. nevertheless, we consider it timely to report on our experiences with this procedure and to consider the feasibility of routinely using newer barcoding procedures for identifying ants and, ultimately, other invertebrates in bulk samples. methods invertebrate sampling: all ant species from the barrow island voucher specimen collection were identified morphologically by beh. specimens were vouchered between 2005 and 2008 at curtin university and are now curated at the western australian museum, with a duplicate set at the western australian department of primary industries and regional development. the majority of the voucher collection is dry-preserved, with some species requiring vouchers from more recent surveys due to the deterioration of the original voucher specimen. single specimens of all 125 ant species (appendix 1) were submitted for barcoding in order to provide baseline data for the ant fauna of the island. then, in order to test whether it is feasible to identify ants from unsorted trap samples, 14 of the barrow island sites were sampled in september 2013 using multiple trapping methods. these were: 1) night hand collection (nhc), a method whereby trained field workers collect ants by hand in the evening (this method typically yields low abundances of ant species but more cryptic diversity); 2) window trap (win), which is a water trap with jd majer, ma castalanelli, jl ledger, nr gunawardene, be heterick – ant bar coding for detecting incursions424 a perspex window that captures flying insects (one drawback with this method is that many ant species are attracted by the water); 3) suction samples (suc), a method whereby a garden blower/vacuum suctions small insects from low shrubs and branches and which usually yields high abundances of shrub-foraging ant species; and 4) barrier pitfall trap, bait trap and litter trap (bbl), a combination of three sampling methods that focus on ground-dwelling ants and often yield high abundances of a few species. for the purpose of this exercise, all samples from a site were combined. all other invertebrate groups were removed from the samples, and the ants were identified to morphospecies by a parataxonomist before being returned to 14 vials representing the bulked samples for each of the sampling sites. the specimens were preserved in 70% ethanol. barcoding procedure: a non-destructive dna extraction method, ande (castalanelli et al., 2010), was used to extract dna from the morphologically identified ant specimens and from the bulked, unsorted trap samples. amplification of the target barcoding region from individual ant specimens was performed using the cytochrome oxidase 1 (co1) primers outlined in table 1. following pcr amplification of the target region and subsequent dna sequencing, sequences were edited using geneious pro 8.0.3 (biomatters ltd) and aligned with the reference data set using geneious’ built-in alignment algorithm. geneious pro 8.0.3 was used to detect the presence of numts by translating each co1 sequence with the standard invertebrate and drosophila codes. forward and reverse sequences were manually edited, primer sequences removed, and the final quality checked. consensus sequences were used to interrogate all available public sequence databases to determine if the morphological and molecular results used to determine the identifications were congruent. the ngs run was conducted using roche gs junior (454). this ngs platform was selected due to its lower cost and its ability to generate sequence lengths >400bp per direction. the sequencing run was performed at the western australian state agriculture biotechnology centre (sabc). analysis of the ngs data was conducted using an ecodiagnostics pty. ltd. in-house bioinformatics pipeline which de-convoluted the dna sequences into individual site samples and then compared sets of sequences from each sample to the co1 reference database. results baseline ant data: a summary of the sample sequencing outcomes for the 126 ant species is shown in table 2 and sequences for each of these species are deposited in genbank. in total, 72 species were successfully dna barcoded and the remaining 53 were unsuccessful. five samples returned a dna barcode that was incongruent with the morphological result and were considered contaminated due to their molecular similarity to the barcode for a species of gastropod that is commonly found in some of the invertebrate samples. of the remaining 48 species that failed to generate a sequence age of the specimens was a possible reason for failure. for 13 species that failed to amplify, sequences from public databases (i.e., ncbi; genbank) were available and hence substituted for the failed amplification. primer sequence 5’ – 3’ ci-j-1718 gga gga ttt gga aat tga tta gtt cc ci-n-2191 ccc ggt aaa att aaa ata taa act tc lco ggt caa caa atc ata aag ata ttg g hco taa act tca ggg tga cca aaa aat ca table 1. cytochrome oxidase 1 primers used to generate dna barcodes for the reference database. dna originating from each site sample was amplified using the forward primer ci-j-1718 and the reverse primers hco and ci-n-2191, with the additional m13 sequences added to the 5` end of the forward and reverse primers. a second round of pcr amplification was performed to attach the roche lib-a adapters to the previous pcr product. a unique midtag barcode specific to each individual barrow island site sample was also incorporated, allowing multiple samples to be pooled together for sequencing on the gs junior. the gs junior sequencing run was set up as per the manufacturer’s instruction, and run for 200 flows. sequencing result count successfully sequenced 72 no sequence generated 53 sample contaminated 5 external public database sequence (e.g. ncbi) 13 total 125 table 2. summary of sample sequencing outcomes. pooled sample analysis: the ant species found in each of the 14 pooled samples and the species that were recovered by ngs from each site are shown in table 3. the sequencing run generated 42,098 high quality sequences; any sequences <400bp in length were removed, reducing the data set to 23,072 sequences from the 14 site samples. the number of sequences dramatically varied between site samples, with between 13 (site sample 12) and 10,046 (site sample 17; table 4) amplicon sequences being recovered. valid assignments were made when similarity of the ngs sequence to one of the reference species was greater than 95%. since the samples were taken in september 2013, which represents only a small time capsule of the total invertebrate surveillance effort, only 39 out of the 126 ant species were present within the 14 site samples (table 3). six species in the 14 site samples did not have a corresponding co1 reference sequence. despite several sites missing one to three reference species (table 4), the majority of site samples had >92% of their sequences assigned to a reference (table 4). sociobiology 65(3): 422-432 (september, 2018) 425 species/site id 9 10 11 12 14 16 17 20 21 22 23 24 26 27 anochetus rectangularis b camponotus capito m n m camponotus gibbinotus b m m m camponotus fieldeae b b m b m m b b m camponotus scratius b camponotus simpsoni m cardiocondyla atalanta m iridomyrmex anceps b b b m b b b b b iridomyrmex cephaloinclinus b iridomyrmex chasei m b m m iridomyrmex dromus n n n n iridomyrmex exsanguis b b m m b m b b iridomyrmex minor b b b b b b b m m b iridomyrmex mjobergi m iridomyrmex sanguineus m m m n m m m m m melophorus biroi m m melophorus paramorphomenus b melophorus hirsutipes b m meranoplus curvispina m m monomorium laeve m m m monomorium leae m monomorium sydneyense b b b b ochetellus flavipes b ochetellus sp. jdm 527 b b b m paratrechina longicornis b b pheidole turneri b pheidole variabilis b b b b b b m b b polyrhachis ammonoeides m b m polyrhachis inconspicua m m m m m polyrhachis seducta m polyrhachis sp. jdm 808 m m m n m m m m rhytidoponera crassinoda m m rhytidoponera taurus m m m tetramorium spininode m tetramorium striolatum complex m * m = morphological identification only; n = next generation sequencing detection only; b = both morphological identification and next generation sequencing detection table 3. comparison between morphological identification and molecular detection using next generation sequencing. the only exception was site sample 9, where the percentage of unassigned species was 31% (table 4). analysis of these unassigned sequences showed that in the majority of cases they clustered with camponotus, iridomyrmex, and polyrhachis clades but weren’t closely related to any particular species (>15% pairwise divergence from its closest neighbor). one issue known to occur during pcr when multiple templates are present is cross amplification of two species (hass et al., 2017), i.e. the front half is of one species the back half is of another. combined, they create a unique chimeric sequence that cannot be assigned to a reference. the term “recovery” is here defined as the number of species identified using the ngs barcoding approach compared to the number of morphologically identified species. recovery for each site varied from 38% (sites 12 and 27) to 100% (site 10; table 4). six sites had additional species that jd majer, ma castalanelli, jl ledger, nr gunawardene, be heterick – ant bar coding for detecting incursions426 were not recognised by morphological methods. of particular note were the highly similar species iridomyrmex exsanguis and iridomyrmex dromus (table 3). comparison of the number of species recovered with the number of sequences obtained from each trap sample shows a positive correlation between the two variables (fig 1). site id 9 10 11 12 14 16 17 20 21 22 23 24 26 27 no. of species identified morphologically 6 6 7 8 4 3 6 10 8 15 13 9 7 13 no. of species identified with ngs 6 6 6 3 2 3 6 7 7 7 7 7 3 5 no. of morphologically identified species in ngs result 5 6 6 3 2 2 5 7 5 7 6 6 3 5 no. of species missed by ngs 1 1 1 2 1 1 percentage of species identified with ngs 83 100 86 38 50 67 83 70 63 47 46 67 43 38 no. of sequences 2,431 1,498 651 13 876 3,796 10,046 481 1,871 256 434 308 26 385 no. of species missing a co1 reference 1 1 2 3 2 1 2 1 3 percentage of assigned sequences 69 95 96 100 97 97 97 95 97 95 98 93 96 96 table 4. overview of the ngs sequencing data, including the number of species present at each site and whether they were recovered by ngs. also shown are the outcomes of the ngs sequencing run output for each site, including the number of sequences that were assigned with > 95% similarity to a reference sequence. fig 1. number of 454 sequences generated per sample in relation to the percentage of species recovered. fig 2. sensitivity of the next generation sequencing technique, illustrated by showing the rate at which a species was detected in preserved barrow island ant material. the “hit” (light part of bar) shows the degree of success relative to the frequency at which that particular ant species occurred within the material. when a site had >1,000 sequences, the average recovery rate was 79%, which is in contrast to the lowest four recovery rates (site samples 12, 22, 26 and 27), which had fewer than 440 sequences. one exception was site 24, which produced a low number of sequences but still had 67% recovery rate (table 4). validating a highly sensitive technique with the ability to detect individuals that occur at low frequencies is one of the most important functions of any biosecurity venture. to examine the sensitivity of the ngs technique, we analysed each site individually to determine if a species was detected (hit) and how that related to the number of individuals per species (determined by dividing the number of species identified per site by the total number of individuals per site sample [termed species occurrence). figure 2 indicates that where a species was present at <4% of the total sample size, it was only detected 10% of the time. as the frequency at which a species occurred increased, so too did the rate at which that species was detected. the only exceptions were iridomyrmex minor at sites 24 and 26, iridomyrmex chasei at site 24, camponotus fieldeae at site 16, monomorium laeve at site 24, and polyrhachis ammonoeides at site 14. sociobiology 65(3): 422-432 (september, 2018) 427 discussion this study demonstrates the encouraging potential of ngs metabarcoding to characterise ant species from bulk trap samples, despite the study being resource-limited (only a single sequencing run was costed). a number of technical difficulties have been highlighted in this pilot study. these include variation in the number of sequences recovered between trap sample sites (13 to 10,046) and lack of sequencing depth, which clearly affects the ability to recover species. this can be largely overcome by the rapid development of improved technologies, and we acknowledge that the sequencing platform and chemistry used in this study has largely been superseded and discontinued. newer procedures will lead to significant improvements in the recovery of sequences from mixed trap samples that here varied between 38 and 100% for individual trap samples. another major factor that needs to be overcome is preferential amplification. future projects need to select priming sites that are either void of mutations or have minimal mutations and are shown to detect all intended target species in a comprehensive fashion. increased depth and more suitable primers may lead to the development of robust and practical monitoring methods with a very high diagnostic sensitivity and specificity. the occasional incongruence between species identified by ngs and morphological species was probably caused by contamination from unrelated taxa due to their molecular similarity to the barcode concerned. this probably arose in some of our samples because traces of gastropod dna, which were often found in our samples prior to removal of the ants, were preferentially amplified. these invertebrates were much larger than the ants and exuded a dna-rich slime. because of the nature of the project and its restricted resourcing, only three primer pairs could be used to try and generate sequences. these three primer pairs (table 1) are generalist primers that have been shown to successfully amplify genetic material from invertebrates (simon et al., 1994). however, experience has shown that 20% or more of the samples will be refractory to amplification due to primer mismatch, poor quality dna, and pcr inhibition. future work should involve the design of more specific primers and, if possible, fresh samples that haven’t been collected in pitfall traps. (pitfall trapped material reveals rapid degradation of dna and may also have high levels of contaminating dna present (castalanelli et al., 2011).) the variation in number of sequences between sites may be in part be attributed to the preservation of specimens in a lower grade of ethanol (i.e., 70%), which was used throughout the nis project for specimen preservation. this may have contributed to the lack of amplification success and also to technical difficulties in making the pcr products from each trap sample of similar molarity prior to ngs library preparation. species species recovery rate (%) number of mutations (26bp) number of mutations @ 5` (5bp) anochetus rectangularis 100 1 0 camponotus capito 50 3 1 camponotus gibbinotus 25 3 1 camponotus fieldeae 56 4 1 camponotus scratius 100 0 0 camponotus simpsoni 0 2 1 cardiocondyla atalanta 0 5 1 iridomyrmex anceps 89 3 1 iridomyrmex cephaloinclinus 100 2 1 iridomyrmex chasei 50 2 1 iridomyrmex exsanguis 63 na* missing datum iridomyrmex minor 80 na na iridomyrmex mjobergi 0 0 0 iridomyrmex sanguineus 50 2 1 melophorus biroi 0 na na melophorus paramorphomenus 100 4 1 melophorus hirsutipes 50 na missing datum meranoplus curvispina 0 4 1 monomorium laeve 0 3 2 monomorium leae 0 2 0 monomorium sydneyense 100 2 1 ochetellus flavipes 100 3 1 ochetellus sp. jdm 527 75 5 1 paratrechina longicornis 100 3 1 pheidole turneri 100 1 0 pheidole variabilis 78 1 0 polyrhachis ammonoeides 33 2 1 polyrhachis inconspicua 0 na missing datum polyrhachis seducta 0 na na polyrhachis sp. jdm 808 43 2 0 rhytidoponera crassinoda 0 3 1 rhytidoponera taurus 66 4 1 tetramorium spininode 100 1 0 tetramorium striolatum complex 100 0 0 * na indicates that no data was available to examine bind efficiency. table 5. comparison of species recovery over all sites and number of mutations within the ci-j-1715 priming site. the species that were not recovered are underlined. jd majer, ma castalanelli, jl ledger, nr gunawardene, be heterick – ant bar coding for detecting incursions428 lessons learned: this pilot study successfully generated a reference ant dna barcode database that is fundamental to the development of improved ngs metabarcoding and dna based individual specimen identification approaches. our investigation revealed a number of issues of which users should be aware and take care to address, namely: • age of material – try to use as fresh a sample as possible; • appropriate, high-grade preservative – use preservatives that maximise preservation of genetic material; • morphologically based taxonomy – underpin investigation with a sound taxonomic database; • contamination of samples by non-target taxa – be aware of other taxa, including plant material, that occur in the samples and how this could contaminate the dna; • cross amplification between related taxa – be aware of this possibility; and • cost of barcoding has to be considered at the outset of investigations such as this. returning to the original thrust behind this investigation, can barcoding using the co1 gene be used to detect ant incursions with an 80% confidence of detection? with recovery rates averaging only 79% and sometimes falling as low as 38%, the answer is no. however, though there are acknowledged gaps in the database that we have generated, these can be rectified with further study to increase the robustness of data interpretation and species identification. furthermore, barcoding is no longer limited to co1; more recently, other genes have been preferred for the intractable groups (e.g., see tables in purty & chatterjee, 2016). acknowledgements the gorgon gas development is operated by an australian subsidiary of chevron and is a joint venture of the australian subsidiaries of chevron (47.3%), exxonmobil (25%), shell (25%), osaka gas (1.25%), tokyo gas (1%) and jera (0.417%). the authors are grateful to chevron australia pty ltd for on-island support with logistics, and the gorgon joint venturers for financial support to undertake the surveillance component of this project. funding from curtin university was provided in order to undertake the barcoding of specimens. christopher taylor, morgan lythe, brad scanlon, and callum smithyman all assisted the project, either in the field and/or in the laboratory. references beng, k.c., tomlinson, k.w., shen, x.h., surget-groba, y., hughes, a.c., corlett, r.t. & silk, j.f. (2016). the utility of dna metabarcoding for studying the response of arthropod diversity and composition to land-use change in the tropics. scientific reports, 6: 24965. doi: 10.1038/srep24965 recovery and sensitivity: the ability to detect an individual accurately within a particular sample is one of the most important functions of biosecurity surveillance, regardless of whether it involves morphological or molecular techniques. therefore, we believe that the most important aspect of this pilot study is understanding recovery and sensitivity. underpinning this is the need for good baseline data from conventional morphological taxonomic approaches. as mentioned earlier, iridomyrmex exsanguis and iridomyrmex dromus were not distinguished by morphological methods (table 3). these two species are morphologically very similar, but seem to have a different nest structure and behaviour around the nest. physically, they can only be distinguished with difficulty. the iridomyrmex exsanguis worker is always pale yellow and has a noticeable propodeal angle, i.e., is truncate when viewed in profile. iridomyrmex dromus is commonly pale also, but the colour can range from depigmented yellow to almost black, and viewed in profile the propodeum lacks a noticeable angle, i.e., is not truncate. anyone considering using ngs procedures should be aware of this sort of subtlety in taxonomic differentiation. apart from the anomaly with site 24, these results suggest that an important contributor to recovery for a given sample is the number of sequences; namely, the more sequences, or greater sequencing depth, the greater the recovery. these results are congruent with other studies that used newer technologies. for instance, brandon-mong et al. (2015) evaluated the miseq (illumina) and showed that for lepidopteran specific primers, 106,070 sequences recovered 60%, which was increased to 80% when the number of sequences was increased to 685,208. the results presented here can generally be firstly explained by preferential amplification. while species occurrence is likely to cause some bias towards preferential amplification, it seems that primer design is the most probable cause. since full-length dna barcoding only allowed us to examine the primer binding site for ci-j-1715, only this section was scrutinised (table 5). the data suggest that as the mutations within the priming site increase, the chance of recovery decreases. the noteworthy examples where primers clearly contributed to good sequence recovery were anochetus rectangularis, camponotus scratius, pheidole turneri, tetramorium spininode, and tetramorium striolatum, all of which had either zero mutations or a single mutation and 100% recovery. in comparison, monomorium laeve and cardiocondyla atalanta, which had three and four mutated sites, respectively, failed to be recovered; more importantly monomorium laeve had two mutations that occurred within the 5` binding site which is the most important part of the priming site (table 5). interestingly, the species that were not recovered (table 5: underlined) tended to have mutations that ranged from two to five and a species occurrence of <7%; suggesting that priming mutations and rarity compounded the failure to recover a species. sociobiology 65(3): 422-432 (september, 2018) 429 biomatters ltd. geneious version 8.0.3. biomatters. available from http://www.geneious.com/ brandon-mong, g. j., gan h. m., sing, k. w., lee, p. s., lim, p. e. & wilson, j.j. 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(2013). a taxonomic overview and key to the ants of barrow island, western australia. records of the western australian museum, supplement, 83: 375-404. doi: 10.18195/issn.0313-122x.83.2013.375-404 ji, y, ashton, l., pedley s. m., edwards, d. p., tang, y., nakamura, a., kitching, r., dolman, p. m., woodcock, p., edwards, f. a. & larsen, t. h. (2013). reliable, verifiable and efficient monitoring of biodiversity via metabarcoding. ecology letters, 16: 1245-1257. doi: 10.1111/ele.12162 kocher, a., gantier, j. c., gaborit, p., zinger, l., holota, h., valiere, s., dusfour, i., girod, r., bañuls, a. l. & murienne, j. (2017). vector soup: high-throughput identification of neotropical phlebotomine sand flies using metabarcoding. molecular ecology resources, 17: 172-182. doi: 10.1111/1755-0998.12556 majer, j. d., callan, s. k., edwards, k., gunawardene, n. r. & taylor, c. k. (2013). baseline survey of the terrestrial invertebrate fauna of barrow island. records of the western australian museum, supplement, 83: 13-112. doi: 10.18195/ issn.0313-122x.83.2013.013-112 pochon, x., bott, n. j., smith, k. f. & wood, s. a. (2013). evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests. plos one, 8(9): e73935. doi: 10.1371/journal.pone.0073935 purty, r. s. & chatterjee, s. (2016). dna barcoding: an effective technique in molecular taxonomy. austin journal of biotechnology and bioengineering, 3: 1059. simon, c., frati, f., beckenbach, a., crespi, b., liu, h., & flook, p. (1994). evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. annals of the entomological society of america, 87: 651-701. doi: 10.1093/aesa/87.6.651 smith, m. a., fisher, b. l. & hebert p. d. n. (2005). dna barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of madagascar. philosophical transactions of the royal society of london, b biological sciences, 360: 1825-34. doi: 10.1098/rstb.2005.1714 telfer, a. c., young, m. r., quinn, j., perez, k., sobel, c. n., sones, j. e., levesque-beaudin, v., derbyshire, r., fernandeztriana, j., rougerie, r. & thevanayagam, a., (2015). biodiversity inventories in high gear: dna barcoding facilitates a rapid biotic survey of a temperate nature reserve. biodiversity data journal, 3: e6313. doi: 10.3897/bdj.3.e6313 thomas, m. l., gunawardene, n., horton, k., williams, a., o’connor, s., mckirdy, s. & van der merwe, j. (2017). many eyes on the ground: citizen science is an effective early detection tool for biosecurity. biological invasions, 19: 1-15. doi: 10.1007/s10530-017-1481-6 yang, c., wang, x., miller, j. a., de blécourt, m., ji, y, yang, c, harrison, r.d. & douglas, w. y. (2014). using metabarcoding to ask if easily collected soil and leaf-litter samples can be used as a general biodiversity indicator. ecological indicators, 46: 379-89. doi: 10.1016/j.ecolind.2014.06.028 yoon, t. h., kang, h. e., kang, c. k., lee, s. h., ahn, d. h., park, h. & kim, h. w. (2016). development of a cost-effective metabarcoding strategy for analysis of the marine phytoplankton community. peerj, 4: e2115. doi: 10.7717/peerj.2115 yu, d. w., ji, y., emerson, b.c., wang, x., ye, c., yang, c. & ding, z. (2012). biodiversity soup: metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring. methods in ecology and evolution, 3: 613623. doi: 10.1111/j.2041-210x.2012.00198.x jd majer, ma castalanelli, jl ledger, nr gunawardene, be heterick – ant bar coding for detecting incursions430 genus species/sub-species author sequence genbank accession number aenictus turneri forel pass tbc amblyopone sp. [queen] pass tbc anochetus rectangularis mayr pass tbc anochetus renatae shattuck & slipinska, 2012 failed arnoldius sp. jdm 433 failed brachyponera lutea (mayr) pass tbc camponotus capito mayr pass tbc camponotus donnellani shattuck & mcarthur failed; old camponotus evae zeuxis forel failed camponotus fieldeae forel pass tbc camponotus gibbinotus forel pass tbc camponotus lownei forel pass tbc camponotus rubiginosus complex sp. jdm 1158 failed; old camponotus scratius forel pass tbc camponotus simpsoni mcarthur pass tbc cardiocondyla atalanta forel pass tbc cardiocondyla nuda (mayr) contaminated carebara sp. jdm 1131 failed crematogaster laeviceps chasei forel pass tbc crematogaster queenslandica forel failed discothyrea clavicornis emery contaminated doleromyrma rottnestensis (wheeler) failed hypoponera queenslandensis (forel) pass tbc iridomyrmex agilis forel failed; old iridomyrmex anceps roger pass tbc iridomyrmex bicknelli emery pass tbc iridomyrmex cephaloinclinus shattuck pass tbc iridomyrmex chasei forel pass tbc iridomyrmex coeruleus heterick & shattuck pass tbc iridomyrmex difficilis heterick & shattuck pass tbc iridomyrmex discors forel pass tbc iridomyrmex dromus clark pass tbc iridomyrmex exsanguis forel pass tbc iridomyrmex gibbus heterick & shattuck failed; old iridomyrmex hartmeyeri forel failed iridomyrmex minor forel pass tbc iridomyrmex mjobergi forel pass tbc iridomyrmex sanguineus forel ncbi iridomyrmex suchieri heterick & shattuck pass tbc iridomyrmex tenuiceps heterick & shattuck pass tbc iridomyrmex xanthocoxa heterick & shattuck pass tbc leptanilla swani wheeler [males only] failed leptogenys tricosa taylor pass tbc leptogenys sp. jdm 1128 failed appendix 1. list of species supplied for dna barcoding, indicating which were successfully barcoded, which failed and species for which sequences were obtained from the ncbh public database. supplementary material sociobiology 65(3): 422-432 (september, 2018) 431 appendix 1. list of species supplied for dna barcoding, indicating which were successfully barcoded, which failed and species for which sequences were obtained from the ncbh public database. (continuation) genus species/sub-species author sequence genbank accession number lioponera brevis clark ncbi lioponera clarki crawley failed lioponera iovis (forel) pass tbc lioponera longitarsus mayr failed lioponera ruficornis clark pass tbc lioponera sp. jdm 942 pass tbc melophorus aeneovirens wheeler pass tbc melophorus biroi forel ncbi melophorus hirsutipes heterick, castalanelli & shattuck pass tbc melophorus ludius forel ncbi melophorus microtriches heterick, castalanelli & shattuck pass tbc melophorus paramorphomenus heterick, castalanelli & shattuck pass tbc melophorus parvimolaris heterick, castalanelli & shattuck pass tbc melophorus rufoniger heterick, castalanelli & shattuck pass tbc melophorus sulla forel ncbi melophorus teretinotus heterick, castalanelli & shattuck pass tbc melophorus turneri forel pass tbc meranoplus dimidiatus smith failed meranoplus fenestratus smith failed meranoplus mjobergi forel contaminated meranoplus curvispina forel pass tbc meranoplus sp. jdm 865 failed meranoplus sp. jdm 1133 failed monomorium antipodum (nw form) forel ncbi monomorium antipodum complex sp. jdm 717 contaminated monomorium arenarium heterick pass tbc monomorium disetigerum heterick failed; old monomorium euryodon heterick pass tbc monomorium fieldi forel ncbi monomorium insolescens wheeler ncbi monomorium laeve mayr ncbi monomorium leae forel pass tbc monomorium punctulatum heterick pass tbc monomorium rubriceps gp sp. jdm 1175 ncbi monomorium sydneyense forel pass tbc monomorium sydneyense complex sp. jdm 101 pass tbc nylanderia glabrior (forel) pass tbc ochetellus flavipes kirby pass tbc ochetellus sp. jdm 527 pass tbc odontomachus ruficeps smith pass tbc opisthopsis haddoni rufoniger forel ncbi jd majer, ma castalanelli, jl ledger, nr gunawardene, be heterick – ant bar coding for detecting incursions432 appendix 1. list of species supplied for dna barcoding, indicating which were successfully barcoded, which failed and species for which sequences were obtained from the ncbh public database. (continuation) genus species/sub-species author sequence genbank accession number paraparatrechina minutula forel pass tbc paraparatrechina minutula gp sp. jdm 916 failed; old paratrechina longicornis (latreille) pass tbc pheidole mjobergi forel pass tbc pheidole rugosula gregg pass tbc pheidole turneri forel pass tbc pheidole variabilis mayr pass tbc pheidole sp. jdm 684 pass tbc platythyrea sp. [male] pass tbc polyrhachis ammonoeides roger pass tbc polyrhachis bohemia kohout failed polyrhachis gravis clark failed; old polyrhachis inconspicua emery ncbi polyrhachis lata emery ncbi polyrhachis melaneura kohout failed polyrhachis seducta kohout pass tbc polyrhachis (campomyrma) sp. jdm 703 failed; old polyrhachis (campomyrma) sp. jdm 1009 pass tbc polyrhachis (campomyrma) sp. jdm 1010 pass tbc polyrhachis (chariomyrma) sp. jdm 807 failed polyrhachis (chariomyrma) sp. jdm 808 failed probolomyrmex latalongus shattuck, gunawardene & heterick, 2012 contaminated pseudoneoponera denticulata (kirby) pass tbc rhytidoponera convexa complex sp. jdm 1129 failed; old rhytidoponera crassinoda forel pass tbc rhytidoponera taurus forel pass tbc rhytidoponera tyloxys brown & douglas failed solenopsis belisarius forel failed solenopsis clarki crawley pass tbc stigmacros punctatissima mcareavey failed stigmacros termitoxena wheeler failed; old strumigenys emmae emery failed tapinoma minutum broomense forel pass tbc tapinoma sp. jdm 981 failed tetramorium cf. megalops bolton pass tbc tetramorium sjostedti forel pass tbc tetramorium spininode bolton pass tbc tetramorium striolatum complex pass tbc tetramorium striolatum complex sp. jdm 36 pass tbc tetraponera punctulata smith pass tbc zasphinctus duchaussoyi andré pass tbc open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.6173sociobiology 68(3): e6173 (september, 2021) introduction stingless bees form a monophyletic tribe of corbiculate bees with a few hundred recognized species of about 60 genera (rasmussen & cameron, 2010). they are highly eusocial bees that inhabit the tropical and sub-tropical regions of the world. stingless bees form perennial colonies with a single queen, a few hundred to several thousand workers, and a few hundred males (michener, 2007). to sustain their colony, foragers collect nectar, pollen, resin, mud, sap, honeydew, animal protein, fungal spores for nutrition, or nestbuilding materials (roubik, 1989; eltz et al., 2002). nectar is the principal source of carbohydrates from which bees obtain their energy (ramalho et al., 1991; nicolson, 2011). stingless bees are generalist foragers, i.e., explore diverse angiosperm abstract knowledge about floral resources is essential for bee management and conservation. pollen analysis of honey is the most traditional method for determining the nectar resources of a bee species. however, the collection of honey samples is difficult in cavity-nesting natural stingless bee colonies. furthermore, it is detrimental to the wild bee’s colony and may threaten their survivability. we analyzed adhered body surface pollen of incoming nectar foragers (which were smeared incidentally during nectar foraging) as an alternative method to determine nectariferous flora of tetragonula iridipennis in west bengal, india. by this method, we have identified 75 pollen types. the number of obtained pollen types was lower in the human-altered habitats of midnapore city (44 pollen types) than the semi-natural habitats of garhbeta (71 pollen types). excluding a few pollen types of non-nectariferous plants, most of the pollen types came from nectariferous plants of both crop and noncrop species. non-crop flowering plants (viz. ailanthus excelsa, borassus flabellifer, eucalyptus tereticornis, lannea coromandelica, peltophorum pterocarpum, and tectona grandis) provided a significant amount of nectar to the bee species and, therefore, play an important role in the conservation of the bee species. sociobiology an international journal on social insects sourabh bisui1, ujjwal layek2, prakash karmakar1 article history edited by evandro nascimento silva, uefs, brazil received 10 january 2021 inicial acceptance 18 june 2021 final acceptance 23 july 2021 publication date 13 august 2021 keywords human-altered habitat, nectar forager, non-crop plant, semi-natural habitat. corresponding author prakash karmakar department of botany & forestry, vidyasagar university midnapore 721102, india. e-mail: prakashbot1973@gmail.com taxa to collect nectar (ramalho et al., 1990; roubik & moreno patiño, 2009; vossler et al., 2014) and maintain high floral constancy (layek & karmakar, 2018a). while collecting nectar, some pollen grains incidentally adhered to the hairy body parts of the bees are transmitted inadvertently to the hives. after a meticulous investigation of pollen morphology, the origin of the plant resources collected by bees can be established (jones & jones, 2001). thus, pollen grains act as dependable ‘fingerprints’ of plants, evidenced in the colonies’ food materials and smeared on the foragers’ body surface (eltz et al., 2001). stingless bees performed as one of the most important pollinators of native plants and economic crops (slaa et al., 2006; rallanawannee & duangphakdee, 2019). therefore, stingless bees management is considered an effective way to enhance crop yield via increasing pollination 1 department of botany & forestry, vidyasagar university, midnapore721102, india 2 department of botany, rampurhat college, birbhum 731224, india research article bees determination of nectar resources through body surface pollen analysis: a study with the stingless bee tetragonula iridipennis smith (apidae: meliponini) in west bengal, india id mailto:prakashbot1973@gmail.com https://orcid.org/0000-0002-4725-8928 sourabh bisui, ujjwal layek, prakash karmakar – determination of nectar resources through body surface pollen analysis of stingless bee 2 services. knowledge of bee flora vis-à-vis bee diet is essential for sustainable meliponiculture, management, and proper conservation strategies for a sustainable livelihood in a region. the dammar bee, tetragonula iridipennis smith is the most abundant stingless bee in india, including west bengal. the management and utilization of the bee species for pollination purposes are also minimal. only a few people have utilized this bee species to pollinate crops like cucumber (kishan et al., 2017) and bitter gourd (bisui et al., 2020). inadequate development of meliponiculture using the bee species has several reasons, including scarcity of data about its foraging behavior for nectar resources. quantitatively, nectariferous plants were depicted through palynological analysis of pot honey (vijayakumar & jeyaraaj, 2016; layek & karmakar, 2018a). pollen analyses of honey (i.e., melissopalynology) have been used to accurately determine the botanical and geographic origin of honey (louveaux et al., 1978, von der ohe et al., 2004; ponnuchamy et al., 2014). but, the collection of honey samples from natural nests of stingless bees is difficult due to their cavity-nesting habit. although field observation was considered a way to determine bee-visited plants (layek & karmakar, 2016; roopa et al., 2017), this procedure has some impediments like spatio-temporal inaccessibility by foraging bees. moreover, the collection method (honey-sampling) may destroy natural nests and intimidates their survivability. therefore, it is essential to develop an alternative method for the determination of the nectar resources of the bee species. to contribute to the knowledge of the floral resources of t. iridipennis in semi-natural and human-altered areas of west bengal, the goals of the paper were to: (1) identify the nectar resources through body surface pollen analysis (2) estimate the relative nectar contribution of the bee-visited plants (3) provide additional information about the acceptability of the used method as an alternative to palynological analysis of honey. material and methods study areas the present work was conducted in garhbeta (22.86° n and 87.35° e) and midnapore town (22.43° n and 87.32° e) in paschim medinipur district of west bengal, india (fig 1). we considered the study sites in garhbeta as semi-natural areas (having dense vegetation and greater agricultural activities) and the study sites in midnapore town as human-altered areas (less vegetation and little or no agricultural activities). within the semi-natural areas of garhbeta, major cultivated crops were brassica juncea (l.) czern., coriandrum sativum l., fig 1. map showing sampling sites (circles). sociobiology 68(3): e6173 (september, 2021) 3 oryza sativa l., sesamum indicum l., solanum melongena l., solanum tuberosum l., triticum aestivum l., and some other cucurbits. several weeds like alternanthera spp., celosia argentea l., parthenium hysterophorus l., mimosa pudica l., sida spp., taraxacum officinale (l.) weber ex f. h. wigg, trianthema portulacastrum l., and tridax procumbens l. were grown in croplands as well as other uncultivated areas. a few tree species (acacia auriculiformis a. cunm. ex benth., ailanthus excelsa roxb., anacardium occidentale l., delonix regia (boj. ex hook.) raf., eucalyptus tereticornis sm., lannea coromandelica (houtt.) merr., peltophorum pterocarpum (dc.) k. heyne, shorea robusta roth, and tectona grandis l. f.) were also found. in addition to the above-mentioned tree species, the human-altered areas of midnapore town contain only a few non-crop flowering plants along the roadside and within the vidyasagar university campus. sampling colonies samples were collected from 7 colonies of t. iridipennis. four colonies (namely, a, b, c and d) were selected from garhbeta, and three colonies (namely, e, f and g) were selected from midnapore town. in garhbeta, the distance between two nearby selected colonies ranged from 0.63 km (between colony b and colony c) to 1.46 km (between colony a and colony d). in midnapore town, colony distance was 0.87 km (between colony e and colony f), 1.06 km (between colony e and colony g), and 1.62 km (between colony f and colony g). nesting substrates were building walls (colony b, c, e, and f), tree trunks of tectona grandis (colony d), and terminalia arjuna (roxb.) wight & arn. (colony a). the colony ‘g’ was within a broken wall and placed outside the window of the palynology and plant reproductive biology laboratory of vidyasagar university. the nests were oriented southward (colony b, c, and d), northwest (colony e and f), east (colony a), and westward (colony g) direction with an elevation of >2 m from the ground level. collection of nectar foragers samples were collected at three time-slots (8.00–10.00 h, 12.00–14.00 h, and 16.00–18.00 h) on a sampling day. to capture the incoming nectar foragers, we closed the entrance of the selected colonies for 5–10 minutes. among the incoming bees, we selected the bees who have swollen abdomen but without corbicular loads. we captured the bees by using clean forceps and kept them within a glass vial (single bee per vial) using 1 ml faa (formaldehyde, glacial acetic acid, 95% ethanol, and distilled water) a ratio of 5:5:50:40) solution. then bee container having faa solution was stirred to dislodge the pollen grains smeared to the bee’s body surface and removed the bees from the solution. during 2016–2019, month-wise samples were collected, and in each sampling day, we collected 10–20 samples. in this way, we collected 1413 samples (= bees) from garhbeta and 1319 samples from midnapore town (table 1). palynological analyses after shaking (to homogenize the pollen solution), 10 μl of solution were taken from the samples (pollen containing faa solution) by micropipette. the solution was slid on a clear glass slide. the non-acetolysed pollens present within the taken sample were examined under a light microscope. based on the morphology of the pollen types, the stock month number of captured bees garhbeta midnapore with body-surface pollen without bodysurface pollen total with body-surface pollen without bodysurface pollen total january 117 6 123 92 04 96 february 114 5 119 91 03 94 march 124 11 135 105 07 112 april 126 16 142 117 08 125 may 100 18 118 112 12 124 june 119 21 140 138 17 155 july 99 09 108 93 07 100 august 90 05 95 85 04 89 september 101 06 107 89 05 94 october 81 03 84 106 06 112 november 105 04 109 100 04 104 december 127 06 133 109 05 114 total 1303 110 1413 1237 82 1319 table 1. month wise sample size in semi-natural areas of garhbeta and human-altered areas of midnapore town in west bengal. sourabh bisui, ujjwal layek, prakash karmakar – determination of nectar resources through body surface pollen analysis of stingless bee 4 pollen samples (collected on a sampling day) were sorted into different sub-groups for further analysis. additionally, we counted the number of pollen grains suspended in the 10 μl solution. noticeably, we did not consider all the samples (to reduce experimental time and hardness) and counted only those samples (n = 166 for garhbeta, n = 150 for midnapore town) that were homogenous in pollen content. we repeated the counting procedure three times for a sample and estimated the average number of pollen grains in 10 μl. then, we calculated the total number of pollen grains present within the sample according to the initial volume of the solution. samples of each sub-group (having similar pollen types) were taken together for further analysis. samples were centrifuged for 10 min at 2500 rpm (1036 g). after discarding the supernatant, pollen sediment was used for palynological preparation through the acetolysis method (erdtman, 1960). the acetolysed pollen was mounted on a glass slide using glycerine jelly. then, pollen types were identify using the reference slides prepared from the local flora and with the help of published articles (pal & karmakar, 2013; layek & karmakar, 2016, 2018b; bisui et al., 2019; layek et al., 2020b) and an unpublished ph.d. thesis (ghosh, 2018). microscopy was done by using a nikon eclipse lv100 pol polarising microscope and leica dm 1000 ergo trinocular microscope. microphotographs of some pollen types were taken. pollen was classified according to the pollen type system of joosten and de klerk (2002) and de klerk and joosten (2007), which was based on morphological features. samples without any pollen content were not considered during the percentage calculation of identified pollen types. a few samples were heterogeneous, which means they have more than one pollen type. to calculate the occurrence of pollen types, we attributed a value of 1 for a particular pollen type per homogenous (containing one kind of pollen type) sample. for a heterogeneous sample, the given value was 1/x for each pollen type present within the sample. here, x is the number of pollen types present within this sample. we estimated the percentage of pollen types by considering the total number of pollen samples, month-wise and year-round. after the estimation of month-wise occurrence (%) of the pollen types, we classified the pollen types into four categories: very frequent (> 30%), frequent (10–30%), less frequent (3% to <10%), and rare (< 3%). floral morphology of some nectar contributing plants we selected a few nectariferous plants (based on pollen types within the analyzed samples) to collect data about their floral morphology. we considered flower size and shape, the position of anthers, the number of pollen grains per flower, and the pollen size of those plants. according to the length of the flower, we classified them into small-sized (< 1 cm), medium-sized (1–3 cm), and large-sized (> 3 cm). based on the multiplication values between polar diameter (pd) and equatorial diameter (ed), we classified the pollen grains into three size groups (layek & karmakar, 2018a; layek et al., 2020a): small-sized (pd x ed < 625 µm2), medium-sized (pd x ed 625–2500 µm2) and large-sized (pd x ed > 2500 µm2). statistical analysis statistical analyses of the samples were conducted to obtain the arithmetic mean and standard deviation. one-way anova and duncan’s multiple range test (dmrt) were used to analyze data. a generalized linear model (glm) with a poisson distribution and log link function was used to test if there was a difference in pollen richness among the studied colonies. results were treated as significant if p ≤ 0.05. we analyzed the monthly utilized pollen type’s similarity (agglomerative hierarchical clustering) using the jaccard similarity coefficient. these statistical analyses were performed using spss (16.0) statistical packages and microsoft excel. to check the extent to which the floral characters explained the number of pollen grains on the bee’s body surface, principal component analysis (pca) was performed using past software version 3.26 (hammer et al., 2001). results among the analyzed samples (1413 in garhbeta and 1319 in midnapore town), most of them contained pollen types (table 1). a total of 75 pollen types belonging to 39 plant families were identified (table 2). photomicrographs of some pollen types were depicted in fig 2. higher represented families were fabaceae (9 pollen types), asteraceae (5 pollen types), lamiaceae (4 pollen types), and myrtaceae (4 pollen types). all most all the obtained pollen types were indicating nectariferous plants. however, a few pollen types viz. capparis zeylanica, chenopodium album, holoptelea integrifolia, luffa aegyptiaca, ricinus communis, solanum sisymbriifolium, streblus asper, and trema orientalis came from non-nectariferous plants (particularly for this bee species). the issue of plant resource types is more likely to be species-specific, and some of these non-nectariferous plants (viz. luffa aegyptiaca, ricinus communis) may provide nectar to other insect species. major nectar contributed families (based on the occurrence of pollen types of nectariferous plants) were myrtaceae, fabaceae, lamiaceae, brassicaceae, arecaceae, and anacardiaceae. if we see the picture round the year, frequent pollen types obtained in semi-natural areas of garhbeta were eucalyptus tereticornis (14.51%), brassica juncea (11.34%), tectona grandis (9.62%), peltophorum pterocarpum (7.12%), borassus flabellifer (3.71%), lannea coromandelica (3.26%), and delonix regia (3.22%) (table 2). in human-altered areas of midnapore town, frequent pollen types were eucalyptus tereticornis (24.66%), peltophorum pterocarpum (13.20%), delonix regia (6.95%), tectona grandis (6.01%), borassus flabellifer (4.21%), and lannea coromandelica (4.61%). sociobiology 68(3): e6173 (september, 2021) 5 family pollen type occurrence (%) garhbeta midnapore acanthaceae hygrophila auriculata 0.38 justicia adhatoda 0.46 0.81 justicia simplex 0.23 aizoaceae trianthema portulacastrum 0.46 amaranthaceae chenopodium album 0.31 amaryllidaceae allium cepa 0.46 anacardiaceae lannea coromandelica 3.26 4.61 mangifera indica 1.00 1.61 spondias pinnata 0.61 apiaceae coriandrum sativum 2.55 apocynaceae alstonia scholaris 0.61 1.29 wrightia tinctoria 1.05 arecaceae borassus flabellifer 3.71 4.21 cocos nucifera 1.07 5.37 phoenix sylvestris 1.66 asteraceae chrysanthemum indicum 0.81 eupatorium odoratum 0.15 0.73 mikania scandens 1.53 2.18 tridax procumbens 1.07 1.37 xanthium strumarium 0.31 bignoniaceae millingtonia hortensis 0.46 brassicaceae brassica juncea 11.34 raphanus sativus 0.15 cannabaceae trema orientalis 0.90 2.12 capparaceae capparis zeylanica 0.04 convolvulaceae evolvulus nummularius 1.15 cornaceae alangium salviifolium 0.69 0.64 cucurbitaceae coccinia grandis 2.53 2.67 cucumis sativus 0.46 luffa aegyptiaca 0.65 0.40 momordica charantia 0.31 dipterocarpaceae shorea robusta 0.92 0.81 euphorbiaceae chrozophora rottleri 0.31 croton bonplandianus 1.61 1.05 ricinus communis 0.27 0.49 fabaceae acacia auriculiformis 2.30 3.47 albizia lebbeck 0.46 0.40 cassia fistula 0.15 0.32 dalbergia sissoo 0.31 0.57 delonix regia 3.22 6.95 lablab purpureus 0.46 leucaena leucocephala 0.38 0.57 millettia pinnata 0.46 peltophorum pterocarpum 7.12 13.20 lamiaceae ocimum tenuiflorum 0.38 0.65 salvia splendens 0.24 tectona grandis 9.62 6.01 vitex negundo 1.77 lythraceae lagerstroemia speciosa 0.32 malvaceae bombax ceiba 0.23 ceiba pentandra 1.00 grewia asiatica 0.38 meliaceae azadirachta indica 1.07 0.57 melia azedarach 0.84 swietenia mahagoni 0.15 0.40 menispermaceae tinospora cordifolia 0.92 0.89 moraceae streblus asper 0.23 moringaceae moringa oleifera 1.92 myrtaceae eucalyptus tereticornis 14.51 24.66 syzygium cumini 1.46 1.62 syzygium jambos 0.15 0.24 syzygium reticulatum 0.38 0.49 nyctaginaceae boerhavia diffusa 1.07 0.40 oxalidaceae oxalis corniculata 0.23 pedaliaceae sesamum indicum 0.69 phyllanthaceae bridelia retusa 2.53 0.81 rhamnaceae ziziphus mauritiana 0.69 0.40 rubiaceae meyna spinosa 0.08 rutaceae citrus type 0.38 0.16 sapindaceae sapindus mukorossi 0.08 sapotaceae mimusops elengi 0.23 1.62 simaroubaceae ailanthus excelsa 1.07 2.06 solanaceae petunia × alkinsiana 0.32 solanum sisymbriifolium 0.38 0.75 ulmaceae holoptelea integrifolia 0.81 table 2. year-round occurrence of different pollen types (obtained from bee’s body surface pollen analyses) in garhbeta and midnapore town. note: in bold pollen types came from non-nectariferous plants; others pollen types are indicating nectar sources. family pollen type occurrence (%) garhbeta midnapore sourabh bisui, ujjwal layek, prakash karmakar – determination of nectar resources through body surface pollen analysis of stingless bee 6 monthly pollen richness significantly differed among the studied colonies (glm, type iii: coefficient = -0.11, wald χ2 = 32.70, d.f. = 1, p < 0.001; intercept: coefficient = 2.56, wald χ2 = 1106.76, d.f. = 1, p < 0.001). in general, colonies within semi-natural areas of garhbeta have greater pollen richness than the colonies within human-altered areas of midnapore town. zone-wise, 71 pollen types were identified in semi-natural areas of garhbeta (table 3) and 44 pollen types in human-altered areas of midnapore town (table 4). important pollen types (including very frequent and frequent) were acacia auriculiformis, ailanthus excelsa, borassus flabellifer, brassica juncea, bridelia retusa, cocos nucifera, coriandrum sativum, croton bonplandianum, delonix regia, eucalyptus tereticornis, lannea coromandelica, moringa oleifera, peltophorum pterocarpum, phoenix sylvestris, syzygium cumini, tectona grandis, and vitex negundo. the number of pollen types obtained per month was also varied (fig 3). from september to december, the number of obtained pollen types was lesser than the rest of the months. monthly pollen type’s similarity analysis showed two clusters in both the studied areas (fig 4). pollen types from october to february were grouped into cluster 1, and the remaining (during march to september) constituted cluster 2. january and february (similarity index of 0.67 in garhbeta and 0.57 in midnapore town) and july and august (similarity index of 0.58 in garhbeta and 0.62 in midnapore town) periods showed higher pollen type similarity in both study areas. fig 2. micrographs of some pollen types obtained from body surface of t. iridipennis. 1. alangium salviifolium. 2. borassus flabellifer. 3. cocos nucifera. 4. coriandrum sativum. 5. eucalyptus tereticornis. 6. hygrophila auriculata. 7. justicia simplex. 8–9. leucaena leucocephala. 10–11. moringa oleifera. 12. peltophorum pterocarpum. 13. phoenix sylvestris. 14–15. tectona grandis. 16–17. tinospora cordifolia. 18. tridax procumbens. 19–20. ziziphus mauritiana. scale bars 10 µm. sociobiology 68(3): e6173 (september, 2021) 7 pollen content per bee’s body surface significantly differed among the studied plant species (f9, 306 = 152.52, p = 1.6e-107). foragers bore a higher number of pollen grains when they foraged on ailanthus excelsa, eucalyptus tereticornis, lannea coromandelica, and tectona grandis (table 5; fig 5). on the other hand, bees foraged on brassica juncea have low pollen content on their body surface. regarding the floral fig 3. month-wise richness of pollen types in garhbeta and midnapore town of west bengal. morphology of these plants, it was revealed that small-sized, dish or stellate or brush flowers having exposed anthers with small-sized pollen grains delivered a larger amount of pollen to the forager’s body surface. discussion at present, desertification of natural and semi-natural habitats because of deforestation is one of the most significant reasons for the loss of biodiversity and certain key pollinators, such as bees, in terrestrial ecosystems throughout the world (kevan, 1999; farig 2003; brown & paxton, 2009). in most cases, various human activities that cause deforestation eventually result in habitat fragmentation and are expected to negatively influence the population of a bee species (winfree et al., 2011; senapathi et al., 2015). however, bee species can even thrive in human-altered landscapes (sirohi et al., 2015). floral resources serve as critical components for maintaining bee fauna (laha et al., 2020). field surveillance, melissopalynological study, or gut content analysis are three well-known methods employed by scientists to identify nectar sources used by bee species. however, each of these methods has some limitations. in field observations and gut content analyses, it would be difficult to do quantitative estimations. melissopalynological analyses also have limitations like fig 4. dendrogram of utilized pollen type’s similarity. a. garhbeta. b. midnapore town. sourabh bisui, ujjwal layek, prakash karmakar – determination of nectar resources through body surface pollen analysis of stingless bee 8 month pollen types very frequent frequent less frequent rare january brassica juncea eucalyptus tereticornis ailanthus excelsa, coriandrum sativum, mangifera indica, moringa oleifera, phoenix sylvestris chenopodium album, holoptelea integrifolia, mikania scandens february brassica juncea, lannea coromandelica ailanthus excelsa, coriandrum sativum, eucalyptus tereticornis, holoptelea integrifolia, lablab purpureus, mangifera indica, moringa oleifera, phoenix sylvestris, spondias pinnata allium cepa, chenopodium album, mikania scandens, streblus asper march lannea coromandelica, syzygium cumini alangium salviifolium, albizia lebbeck, borassus flabellifer, ceiba pentandra, coccinia grandis, millettia pinnata, momordica charantia, shorea robusta, ziziphus mauritiana allium cepa, bombax ceiba, capparis zeylanica, cocos nucifera, dalbergia sissoo, grewia asiatica, justicia adhatoda, mangifera indica, melia azedarach, raphanus sativus, ricinus communis, spondias pinnata, tridax procumbens april borassus flabellifer, delonix regia alangium salviifolium, azadirachta indica, citrus type, coccinia grandis, croton bonplandianus, evolvulus nummularius, lannea coromandelica, melia azedarach, shorea robusta, syzygium cumini, tinospora cordifolia albizia lebbeck, bombax ceiba, cassia fistula, grewia asiatica, justicia adhatoda, oxalis corniculata, ricinus communis, spondias pinnata, syzygium jambos, trema orientalis, tridax procumbens, ziziphus mauritiana may croton bonplandianus, delonix regia, peltophorum pterocarpum, tectona grandis azadirachta indica, borassus flabellifer, coccinia grandis, evolvulus nummularius, melia azedarach, sesamum indicum, syzygium reticulatum, tridax procumbens cocos nucifera, justicia adhatoda, meyna spinosa, oxalis corniculata, tinospora cordifolia, solanum sisymbriifolium, trianthema portulacastrum, trema orientalis june delonix regia, peltophorum pterocarpum, tectona grandis boerhavia diffusa, coccinia grandis, cocos nucifera, evolvulus nummularius, sesamum indicum, trianthema portulacastrum chrozophora rottleri, croton bonplandianus, hygrophila auriculata, justicia simplex, solanum sisymbriifolium, tinospora cordifolia, trema orientalis july tectona grandis acacia auriculiformis, boerhavia diffusa, bridelia retusa, coccinia grandis, croton bonplandianus, mimusops elengi, peltophorum pterocarpum, tridax procumbens chrozophora rottleri, evolvulus nummularius, hygrophila auriculata, ocimum tenuiflorum, solanum sisymbriifolium, swietenia mahagoni, tinospora cordifolia, trema orientalis august tectona grandis peltophorum pterocarpum, vitex negundo acacia auriculiformis, bridelia retusa, coccinia grandis, cucumis sativus, tinospora cordifolia cocos nucifera, evolvulus nummularius, hygrophila auriculata, ocimum tenuiflorum, trema orientalis, tridax procumbens september acacia auriculiformis, bridelia retusa, peltophorum pterocarpum, vitex negundo boerhavia diffusa, eucalyptus tereticornis ocimum tenuiflorum, trema orientalis, tridax procumbens october eucalyptus tereticornis acacia auriculiformis, cucumis sativus, leucaena leucocephala, luffa aegyptiaca chromolaena odoratum november eucalyptus tereticornis brassica juncea alstonia scholaris, mikania scandens, millingtonia hortensis, xanthium strumarium luffa aegyptiaca december brassica juncea coriandrum sativum, eucalyptus tereticornis, moringa oleifera, phoenix sylvestris mikania scandens alstonia scholaris note: in bold pollen types came from non-nectariferous plants; others pollen types are indicating nectar sources. table 3. month-wise pollen types obtained from bee’s body surface pollen analysis in garhbeta, west bengal. difficult sample collection (from wild hives) and mode of collection, which is an important criterion for an accurate depiction of nectar sources (layek et al., 2020b). besides, the presence of over-represented and under-represented pollen grains in honey samples may again provide a faulty result. in the case of social bees of populated colonies, the method (analysis of pollen content on the bee’s body surface) is poorly destructive compared to killing a whole colony. furthermore, by utilizing this method, we determined the nectar contributing potential of the bee-visited plants. sociobiology 68(3): e6173 (september, 2021) 9 we can also eliminate the effect of over and underrepresentation of pollen types. pollen content on a bee’s body surface indicates the representation potential of a pollen type within honey samples. therefore, counting the bee’s body surface pollen may give information about the lessrepresented and over-represented pollen types. in this regard, we considered brassica juncea as a less-represented pollen type and ailanthus excelsa, eucalyptus tereticornis, lannea coromandelica, and tectona grandis as over-represented pollen types. over-representation of some pollen types mentioned above was also reported by several workers (layek & karmakar, 2018b). however, the over-representation of tectona grandis as a nectar source will be the first time report made by the present authors from west bengal. month pollen types very frequent frequent less frequent rare january eucalyptus tereticornis ailanthus excelsa chrysanthemum indicum, mangifera indica, mikania scandens petunia × alkinsiana february eucalyptus tereticornis, lannea coromandelica ailanthus excelsa mangifera indica, mikania scandens march borassus flabellifer, lannea coromandelica, syzygium cumini alangium salviifolium, albizia lebbeck, coccinia grandis, cocos nucifera, dalbergia sissoo, mangifera indica, shorea robusta, ziziphus mauritiana justicia adhatoda, ricinus communis, salvia splendens, tridax procumbens april borassus flabellifer, delonix regia alangium salviifolium, azadirachta indica, cassia fistula, coccinia grandis, cocos nucifera, justicia adhatoda, lannea coromandelica, shorea robusta, syzygium cumini albizia lebbeck, citrus type, croton bonplandianus, ricinus communis, syzygium jambos, tinospora cordifolia, tridax procumbens, ziziphus mauritiana, trema orientalis may delonix regia, peltophorum pterocarpum, tectona grandis borassus flabellifer, cocos nucifera, coccinia grandis, croton bonplandianus, justicia adhatoda, syzygium reticulatum, trema orientalis, tridax procumbens azadirachta indica, solanum sisymbriifolium, tinospora cordifolia june delonix regia, peltophorum pterocarpum, tectona grandis cocos nucifera, mimusops elengi, trema orientalis boerhavia diffusa, coccinia grandis, croton bonplandianus, solanum sisymbriifolium, tinospora cordifolia july tectona grandis peltophorum pterocarpum acacia auriculiformis, coccinia grandis, croton bonplandianus, lagerstroemia speciosa, mimusops elengi, ocimum tenuiflorum, swietenia mahagoni, trema orientalis boerhavia diffusa, bridelia retusa, solanum sisymbriifolium, tinospora cordifolia, tridax procumbens august acacia auriculiformis, peltophorum pterocarpum bridelia retusa, coccinia grandis, cocos nucifera, tectona grandis, trema orientalis, tridax procumbens, wrightia tinctoria boerhavia diffusa, ocimum tenuiflorum, tinospora cordifolia september peltophorum pterocarpum acacia auriculiformis bridelia retusa, eucalyptus tereticornis, ocimum tenuiflorum, trema orientalis, tridax procumbens, wrightia tinctoria boerhavia diffusa october eucalyptus tereticornis cocos nucifera acacia auriculiformis, chromolaena odoratum, leucaena leucocephala luffa aegyptiaca november eucalyptus tereticornis alstonia scholaris, chromolaena odoratum, cocos nucifera, mikania scandens luffa aegyptiaca december eucalyptus tereticornis cocos nucifera alstonia scholaris, chrysanthemum indicum, mikania scandens petunia × alkinsiana note: in bold pollen types came from non-nectariferous plants; others pollen types are indicating nectar sources. table 4. month-wise pollen types obtained from bee’s body surface pollen analysis in midnapore town, west bengal. sourabh bisui, ujjwal layek, prakash karmakar – determination of nectar resources through body surface pollen analysis of stingless bee 10 while considering the floral morphology of some low, medium, and over-represented pollen taxa, we provided a relation between flower shapes and pollen representation. in general, small dish/stellate flowers with exposed anthers and easily accessible short columns of nectar (ailanthus excelsa, lannea coromandelica, and tectona grandis) and brush flower (eucalyptus tereticornis) containing a copious amount of nectar along with exposed anthers having small-sized pollen grains favored over-representation. workers of t. iridipennis may visit a greater number of flowers in a single bout on these plants, and hairy body parts contact with a higher number of pollen grains than the plants bearing large-sized flowers with large-sized pollen grains in hidden anthers. besides the merits of the utilized method, there are some limitations: (i) absence of pollen grains on the body surface of some incoming bees, (ii) sometimes occurrence of more than one pollen type from a single incoming bee, and (iii) body surface of some captured bees contain pollen types of non-nectariferous but polleniferous plants. the absence of pollen on the bee’s body surface of a returning forager may have several reasons – (i) bee is a water forager, (ii) extrafloral nectar or another sugary liquid forager (iii) foraged exclusively over female flowers of some unisexual plant species (iv) the forager may be a nectar thief/robber. considering the fig 5. pca chart showing difference between body surface pollen content and floral characteristics. ac = acacia auriculiformis, bo = borassus flabellifer, br = brassica juncea, co = cocos nucifera, de = delonix regia, eu = eucalyptus tereticornis, la = lannea coromandelica, pe = peltophorum pterocarpum, te = tectona grandis; a = body surface pollen contents, b = flower size, c = flower shape, d = pollen presentation, e = pollen yield per flower, f = pollen size. plant species pollen content/bee’s body surface floral morphology flower size flower shape pollen presentation pollen yield/flower pollen size acacia auriculiformis 46.75d ± 10.70 small brush exposed low medium ailanthus excelsa 229.50a ± 69.45 small dish exposed medium large borassus flabellifer 41.08de ± 10.23 small tubular exposed low large brassica juncea 21.63e ± 9.14 medium cruciform exposed medium medium cocos nucifera 44.80de ± 15.00 small stellate exposed high medium delonix regia 38.39de ± 18.82 large flag exposed medium large eucalyptus tereticornis 192.32b ± 47.55 small brush exposed high small lannea coromandelica 180.24b ± 50.13 small stellate exposed medium small peltophorum pterocarpum 28.33de ± 9.20 medium dish exposed medium large tectona grandis 143.06c ± 41.47 medium dish exposed medium small values are given in mean ± standard deviation. different letters indicate significant differences (dmrt at 5%). table 5. pollen presentation (on bee’s body surface) of some nectariferous plants and their floral morphology. occurrence of more than one pollen type or non-nectariferous pollen types on a bee’s body surface, we inferred that it is due to foragers (i) shifting from one plant resource to another plant resource during its entire foraging span, (ii) picking up residual pollen from the hive environment, (iii) having air-born pollen from wind-pollinated plants adhering to their body, or (iv) visiting flowers of two or more plant species in a single bout. however, the first and second reasons are more agreeable for the occurrence of more than one pollen type or pollen types of non-nectariferous plants on bee’s body surface. regarding the obtained pollen types, most of them were common to the pollen types of polleniferous plants obtained in our previous study (bisui et al., 2019). the utilization of bee-visited plants as a source of both nectar and pollen has been regarded as a profitable foraging strategy and was previously documented for honey bees (layek et al., 2015; taha et al., 2019; layek & karmakar, 2020) and stingless bees (layek & karmakar, 2018a). few pollen types like alstonia scholaris, oxalis corniculata, and wrightia tinctoria are new record from west bengal. the presence of azadirachta indica pollen types indicated that the bee species might collect toxic rewards (pollen and nectar) from the sociobiology 68(3): e6173 (september, 2021) 11 plant species during its flowering period (march–april). an enormous collection of toxic rewards may lead to the collapse of the bee colonies. therefore, beekeepers need to precaution during this time period to avoid collecting these toxic rewards by managed bees. the bee species utilized a greater number of nectariferous plants in semi-natural areas of garhbeta than the human-altered areas of midnapore town. in the semi-natural areas of garhbeta, t. iridipennis vigorously utilized crop plants (e.g., brassica juncea) and non-crop plants (viz. borassus flabellifer, delonix regia, eucalyptus tereticornis, lannea coromandelica, peltophorum pterocarpum, and tectona grandis). however, within the human-altered areas of midnapore town, the bee species mainly depends on the non-crop flowering plants. progressive urbanization with increasing population density in humanaltered areas of midnapore town leads to a gradual decline in natural forest and agricultural fields. nevertheless, the trees mentioned above are present in the university premises and along the roadside and became available to the studied colonies. the maximum amount of nectar contributed family was myrtaceae, followed by fabaceae, lamiaceae, arecaceae, anacardiaceae, and brassicaceae. the nectar contribution of a plant family depends on the plant species’ nectar yielding potential and their abundance around the studied colonies (within their foraging range). the significance of these families (except lamiaceae) as a source of nectar was well established in india (vijaykumar & jeyaraaj, 2016; roopa et al., 2017) as well as outside the country (obregon & nates-parra, 2014; novais et al., 2015). the high nectar contributing potential of the family lamiaceae to the bee species is newly registered for west bengal. in addition, the most important member of this family is tectona grandis which served as a vital nectar source during monsoon (july–august), i.e., the dearth period for most of the bees in west bengal. therefore, the plant played an important role in the sustenance of the bee species. conclusions our results show that most incoming foragers (those are without any corbicular load and of the regarded as probably nectar forager) contain pollen types over the body surface. with the elimination of a few pollen types of non-nectariferous plants, the obtained pollen spectra can be regarded as nectariferous flora for a given bee species. therefore, bee’s body surface pollen analysis seems to be an effective alternative to the traditional methods (i.e., pollen analysis of honey and field observation) in regards to (i) accuracy of the determining nectariferous plants, (ii) avoiding honeysampling difficulties, (iii) avoiding the destruction of natural nests, and (iv) survivability of the bee species. this method allows characterizing a large number of plants foraged by the stingless bee (t. iridipennis). however, only a few plants (viz. borassus flabellifer, brassica juncea, delonix regia, eucalyptus tereticornis, lannea coromandelica, peltophorum pterocarpum, and tectona grandis) have contributed a greater amount of nectar and thereby played an important role in the conservation of the bee species. moreover, we found that the bee species utilized more diverse floral resources in semi-natural areas of garhbeta (utilize both crop and non-crop plants) than the human-altered areas of midnapore town (depends on mainly non-crop trees). thus, non-crop flowering plants play an important role in colony health and conservation of the bee species. finally, stingless bees act as key species in maintaining ecosystem function and ecosystem health; bee pasturage and nesting sites will maintain the proper bee growth and pollination services with the untamed diversity of flowering plants and crops. acknowledgments the first author is thankful to ugc for granting the rajiv gandhi national fellowship (f1-17.1/2013-14/rgnf2013-14-sc-wes41832/(sa-iii/website). we would also like to thank the authorities of vidyasagar university for providing the necessary laboratory facilities. we are indeed thankful to the usic section of vu 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(2011). native pollinators in anthropogenic habitats. annual review of ecology, evolution, and systematics, 42: 1-22. doi: 10.1146/ annurev-ecolsys-102710-145042 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i1.3378sociobiology 66(1): 81-87 (march, 2019) individual acclimatization of apis mellifera l. to the thermal homeostasis of the colony introduction the greatest economic value provided by bees is pollination, which helps to maintain the planet’s biodiversity and helps to increase global agricultural production. deprivation of this service can negatively affect the sexual reproduction and genetic diversity of plants, as well as compromise the food production and related products (klein et al., 2007). the monitoring of the pollination dependence of 141 brazilian agricultural crops – including crops for food, clothing, livestock and biofuel – showed that 85 crops depend on bee pollination. the estimated annual economic input of pollination get close to 12 billion of dollars (giannini et al., 2015). another study revealed that 68% of the 53 main brazilian cash crops are pollinator-dependent. moreover, the cultivated area (59%) and the monetary value (68%) of pollinator-dependent cash crops are higher than that of abstract bees play an important role in maintaining biodiversity by promoting the pollination of numerous plant species. recent global climate changes are affecting the average air temperature, thereby altering the biological processes of many species. the objective of this study was to evaluate the adaptation of apis mellifera l. bees to temperature increases and their responses to thermal homeostasis in the colony. research was performed at the federal university of paraíba laboratory of bees using three treatments: control, 33 °c and 40 °c. for the latter two treatments, colonies were kept in a 24 m² climate chamber with an opening at the hive entrance, giving the bees access to the outside environment. the following parameters were evaluated: difference between internal and external hivetemperature, thorax surface temperature and total protein concentration in the hemolymph. internal colony temperature varied according to the external hive temperature. nurse bees that care for larvae exhibited higher heat production, expressed as thorax surface temperature. total protein content in the hemolymph was highest in the 40 °c treatment and decreased with ambient temperature. external hive temperature influences internal hive temperature, and nurse bees have higher capacities for thermogenesis. sociobiology an international journal on social insects mv lima1, laf pascoal2, e paes saraiva3, ko soares3, jpaf queiroz1, a evangelista-rodrigues3 article history edited by kleber delclaro, ufu, brazil received 26 april 2018 initial acceptance 17 july 2018 final acceptance 24 july 2018 publication date 25 april 2019 keywords behavior, hemolymph, total protein, thermogenesis, thermoregulation. corresponding author marcos venâncio lima universidade federal do ceará av. mister hull, s/n pic fortaleza, ceará, brasil. e-mail: venancio.zootecnia@yahoo.com.br non-dependent (novais et al., 2016), which reinforces the importance of pollinators to the national economy. however, according to the intergovernmental panel on climate change (ipcc), the planet is undergoing climate change with significant effects on the environment, biological processes, human health, water resources, agriculture and biodiversity. the main change observed thus far is a significant increase in air temperature that will affect the permanence of many species in their habitats, leading to environmental imbalance. the ipcc report estimates that 20-30% of species will be at high risk of extinction in the case of a 2 to 3 °c rise in average global air temperature (ipcc, 2014). in brazil, climate change may reduce the probability of pollinator occurrence in up to 0.25 and will affect in up to 100% of the municipalities producers, depending on the crop species (giannini et al., 2017). individual bees are heterothermic insects because they are able to change between ectothermic and endothermic 1 universidade federal do ceará, fortaleza, brazil 2 universidade federal da paraíba, bananeiras, brazil 3 universidade federal da paraíba, areia, brazil research article bees mv lima, laf pascoal, e paes saraiva, ko soares, jpaf queiroz, a evangelista-rodrigues – individual acclimatization of apis mellifera l.82 states through endogenous thermogenic mechanisms, differing from entirely ectothermic insects (wilmer & stone, 2004; stabentheiner et al., 2012). the endothermic warm-up results from the contraction of flight muscles, a mechanism called “shivering thermogenesis” (heinrich, 1979; heinrich, 1993; heinrich & esch, 1994; heinrich, 1996). endothermy episodes in bees are often associated with activities outside the nest (kovac et al., 2010; kovac & stabentheiner, 2011) but social species use their fine thermoregulation ability to control the temperature of the nest or swarm, and for brood incubation (heinrich, 1981; southwick & heldmaier, 1987; heinrich, 1993; heinrich, 1996; bujok et al., 2002; seeley et al., 2003; stabentheiner et al., 2003; jones & oldroyd, 2007). the precise nest temperature control can be seen as one of the greatest innovations in bee biology, made possible due to the evolution of sociality (seeley, 2006). the thermal homeostasis of the colony is determined by polyandrous mating that establishes the genetic diversity of individuals as well as their responses to colony temperature maintenance, integrating behavioral and physiological mechanisms (jones et al., 2004). through these mechanisms, social bees can maintain the colony internal temperature between 33 °c and 36 °c, which is ideal for juvenile development and normal behavioral expression (winston, 2003). therefore, we intend to investigate the mechanisms of internal temperature control of apis mellifera colonies. materials and methods location and experimental procedures experiments were conducted at the laboratory of bees of the apiculture and sericulture sector, agricultural sciences center, universidade federal da paraíba, areia, brazil (6º 58” s, 35º 41” w and 574 m altitude). the climate is tropical semi-humid (as), with rainfall in the autumn and winter, according to the climatic classification of köppen (alvares et al., 2013). according to the brazilian national meteorological institute (inmet, 2018), the provisional climatological normals for the study location are as follows: mean annual atmospheric temperature of 22.5 ºc (maximum of 38.2 ºc and minimum of 19.6 ºc), mean annual wind speed of 3.0 m s-1, mean annual relative humidity of 82.6%, and cumulative annual rainfall of 1,359 mm. the animal model was the honeybee (apis mellifera l). initially, bee colonies were kept in hives manufactured from wood (2 cm thick), measuring 20 × 25 × 50 cm (width × height × length) and containing five frames with honeycomb wax. one side of the brood chamber was replaced by glass – to facilitate the fixation and reading of the thermometer by the observer – covered with black cardboard to promote insulation. as we intended to evaluate the influence of the environmental temperature over internal hive temperature, whether in a controlled (i.e., climatic chamber) or natural environment, no additional insulation was applied to the hives. eight beehives with similar population densities and queens less than one year old were selected. each beehive was considered as an experimental unit. the experimental period was comprised of 60 consecutive days between january and march 2016. four hives were subjected to different ambient temperatures (33 and 40 °c) inside a climatic chamber. there were 30 days of exposure at 33 °c, followed by 30 days of exposure at 40 °c. the other four hives were exposed to the natural variation of ambient temperature outside the climatic chamber (control group). the climatic chamber measured 24 m² area, built in masonry, with ceramic floor and pvc ceiling lining. the climatic chamber was heated by infrared lamps (250 w). the bottom board and the hive entrance were connected to an opening in the chamber wall. thus, bees of the hives placed inside the climatic chamber had free access to the outside, keeping their external activities such as disposing dump waste dump waste products and foraging. relation between internal and external hive temperature an analogical thermometer for maximum and minimum (incoterm, porto alegre, rs, brazil) was installed on the glass side of each brood chamber to monitor the internal (ti, °c) and external (ta, °c) hive temperatures. the temperature data were recorded from 7h00 to 19h00, with a three-day interval between sampling days. body surface temperature the body surface temperature (ts, °c) was measured with a thermal image camera (model flir tg165, flir® systems inc., oregon, usa,temperature range –25 to 380 °c, accuracy of ± 1.5%, resolution of 0.1 °c) that had been calibrated to an emissivity of 0.95. thermal images were recorded from bees that were standing and performing nursing work (i.e., feeding larvae) over the central brood frames. images were taken when the camera center point was above the thorax region. data collection was performed on four sampling days, with intervals of 15 days between evaluations. at each sampling day, thermal images were recorded from five bees of each experimental unit (i.e., beehives) of each treatment (control, 33 °c, and 40 °c) at each timeof day (8h00, 12h00 and 16h00). a total of 480 bees were monitored. determination of total protein in the hemolymph to evaluate the total protein concentrations in the hemolymph, a 40 cm2 section of closed brood comb was removed from each experimental unit – including control group, 33 °c and 40 °c treatments – after 15 days of exposure to the thermal environment. these brood combs were taken to anincubator chamber (bio-oxygen demand type, model sp-500, splabor, presidente prudente, sp, brazil) that had been calibrated to a temperature of 33 °c and humidity of 80%. sociobiology 66(1): 81-87 (march, 2019) 83 the brood combs remained inside the incubator chamber for 24 hours until the emergence of the bees. immediately after emergence, 100 bees from each experimental unit were marked on the thorax with organic blue ink and were returned to their original hives. three samplings were performed after 12 days, and 10 marked bees were recaptured at each sampling. for hemolymph extraction, the bees were previously anesthetized on ice for 10 minutes. a micropipette was introduced into the dorsal heart through the intersegmental membrane. the hemolymph was aspiratedand then transferred to 0.6 ml eppendorf tubes. three drops of a 0.1% phenylthiourea solution (aldrich-p7629, grade i, 98%, merck kgaa, darmstadt, germany) were added. samples were centrifuged at 1,400 rpm for 4 minutes at 4 °c, and the supernatant was removed and frozen at –20 °c. total protein quantification was performed by the bradford method (1976), and standard curve construction was performed using bovine serum albumin (bsa). quantification of samples was performed by spectrophotometry with a wavelength of 595 nm (cremonez et al., 1998). data analysis the study design used was fully randomized with a subdivided plot, where the main plot was the treatment (temperature ranges) and the subplots were the evaluation times. the data were analyzed using the general linear models procedure (glm) of the software statistical analysis system (statistical analysis system [sas], 1999). between treatment means were compared by the tukey test with a level of significance set in 5% for all variables. a regression analysis was performed to verify the relationship between internal hive temperature and external hive temperature (i.e. atmospheric temperature) using the originpro 8 software (originlab corporation ©, northampton, ma, usa). results external and internal hive temperatures the 12-hour variation of the internal and external hive temperature is shown in the table 1. the ta mean was 27.83 ± 0.08 °c (n = 2,080). the tiof the control group varied significantly (p < 0.05) as a function of the ta, as shown in fig 1. on the other hand, the beehives maintained at a constant temperature of 33 °c showed no temporal variation in ti, except for at 9h00 (34.35 ± 0.49 °c, n = 40, p < 0.05), when the lowest mean temperature was recorded (fig 2). there was no significant temporal variation in ti of the beehives submitted to the controlled thermal environment of 40 °c (37.68 ± 0.02 °c, n = 520, p > 0.05). body surface temperature treatments and time of day significantly affected (p < 0.05) the ts (fig 3). there was no significant difference between 33 °c and 40 °c treatments within each time of day. bees of the control group showed the lowest ts at all times of day. for thecontrol group and 33 °c treatment, temperatures time internal hive temperature (treatments) external hive temperature control group 33 °c 40 °c mean±se max min mean±se max min mean±se max min mean±se max min 7h00 28.9±1.59f 32.0 25.0 35.72±0.45a 37.0 35.0 37.4±0.57a 38.0 36.5 24.0±1.85g 33.0 21.0 8h00 29.2±1.57ef 32.0 26.0 35.77±0.53a 37.0 35.0 37.51±0.49a 38.0 37.0 25.75±2.68ef 35.0 22.0 9h00 29.89±1.70ed 35.0 26.5 34.35±4.26b 37.0 35.0 37.62±0.47a 38.0 36.0 28.2±2.97bc 36.0 23.0 10h00 30.29±1.70bdc 34.0 27.0 36.07±0.34a 38.0 35.0 37.4±0.56a 38.0 36.0 29.21±3.16bac 36.0 23.0 11h00 30.83±1.63ba 34.5 27.5 36.15±0.49a 38.0 36.0 37.35±0.65a 38.5 36.0 30.21±2.83a 36.0 24.0 12h00 30.86±1.61ba 34.0 28.0 36.22±0.54a 38.0 36.0 37.66±0.50a 38.5 37.0 30.1±3.18a 34.0 24.0 13h00 30.87±1.60ba 34.0 27.0 36.32±0.49a 38.0 36.0 37.85±0.36a 38.5 37.0 30.3±3.18a 34.0 23.0 14h00 31.05±1.68a 34.0 27.0 36.22±0.50a 38.0 36.0 37.77±0.50a 38.5 37.0 29.56±3.29ba 34.0 22.0 15h00 30.81±1.66ba 34.0 27.0 35.12±2.15a 38.0 36.0 37.8±0.40a 38.0 37.0 29.7±3.77a 33.0 22.0 16h00 30.62±1.56bac 33.0 27.0 36.07±0.47a 37.0 35.0 37.9±0.33a 38.0 37.0 28.03±2.84dc 32.0 22.0 17h00 30.1±1.33dc 32.5 27.0 36.1±0.30a 37.0 35.0 37.9±0.30a 38.0 37.0 26.65±2.43ed 29.0 22.0 18h00 29.71±1.22ed 32.0 26.0 36.07±0.42a 37.0 35.0 37.9±0.30a 38.0 37.0 25.45±1.73ef 29.0 21.0 19h00 29.39±1.24ef 32.0 26.0 36.05±0.22a 38.0 36.0 38.0±0.30a 38.0 38.0 25.05±2.35gf 29.0 21.0 values with different letters in the same column represent significant differences according to the tukey test (p <0.001) table 1. external and internal hive temperature of apis mellifera colonies exposed to natural environmental (control group) and to controlled temperatures of 33 and 40 °c. mv lima, laf pascoal, e paes saraiva, ko soares, jpaf queiroz, a evangelista-rodrigues – individual acclimatization of apis mellifera l.84 were lowest at 8h00, highest at 12h00 and intermediate at 16h00. for the 40 °c treatment,the lowest temperature was at 8h00 (32.21 ± 0.22 °c, n = 40), and no differences were observed between 12h00 (34.07 ± 0.31 °c, n = 40) and 16h00 (33.06 ± 0.21 °c, n = 40). among treatments, the bees at 33 °c and 40 °c had similar ts (p > 0.05), but their ti were higher at 40 °c (37.68 ± 0.08 °c, n = 520) than at 33 °c (35.88 ± 0.06 °c, n = 520). as measurements were taken from bees standing on central brood frames, our results suggest that because they are experiencing a higher temperature, they maintained low individual heat production in an attempt to cool the colony and not overheat the internal temperature and damage brood development. total protein concentration in the hemolymph the total protein content in bee hemolymph differed between all treatments (p < 0.05). as temperature increased, total protein content also increased, as shown in fig 4. the lowest concentration of total protein in the hemolymph was obtained from the bees of the control group (34.71 ± 1.43 µg ml-1, n = 24). the 33 °c treatment revealed an intermediate concentration of total protein (43.35 ± 2.07 µg ml-1, n = 12). in the 40 °c treatment bees, a higher (p < 0.05) concentration of total protein in the hemolymph was observed (54.48 ± 2.42 µg ml-1, n = 12). fig 2. temporal variation of the internal temperature (ti, ° c) of beehives submitted to a 33 °c thermal controlled environment. different letters indicate a significant difference (tukey test, p < 0.05). fig 1. internal hive temperature (ti, °c) of the control group as a function of the external hive temperature (ta, °c). fig 3. body surface temperature (ts, °c) at different treatment temperatures. different capital letters in the same time of day and different lowercase letters in the same treatment indicate a significant difference (tukey test, p < 0.05). fig 4. total protein content in the hemolymph of 12-day-old bees at different treatment temperatures. different letters indicate a significant difference (tukey test, p < 0.05). discussion the ideal internal temperature of a colony for brood development is between 33 °c and 36 °c (rosenkranz et al., 1992; hess, 1926; himmer, 1927; dunham, 1929; tautz et al., 2003). in the present study, colonies of the control group maintained mean ti below the ideal range. the 33 °c treatment colonies maintained ti within or slightly above the sociobiology 66(1): 81-87 (march, 2019) 85 ideal range, and the 40 °c colonies maintained ti above the ideal maximum at all times of day. a high ti with a low heat dissipation capacity can seriously damage a bee colony. according to himmer (1927), who studied the effect of temperature on pupating broods, few bees emerged at temperatures below 28 °c or above 38 °c. our results show that colonies of the control group reached 28.9 °c at 19h00, and the colonies of the 40 °c treatment reached 38 °c at 19h00, not exceeding the extreme range for pupae malformation. however, the neural malformation of pupae should be considered. the brood rearing temperature may affect the adult brain and the behavioralperformance of the bees, inside and outside the hive (tautz et al., 2003; groh et al., 2004). according to jones et al. (2005), bees pupated below 28 °c or above 36 °c will have a decreased capacity for memory and learning in the realization and recognition of foraging activities. consequently, the pollination by workers may be impaired. our results suggest that the elevation of the thoracic temperature occurred by two different adaptive mechanisms. in the control group, where the average ti remained below ideal range, the bees possibly activated the muscular heat production. thermogenesis by muscle vibration is important not only to preflight warm-up (heinrich, 1979; heinrich & esch, 1994; seeley et al., 2003) but also to improve the bee metabolism (roubik, 2012), control of the internal hive temperature and brood incubation (kronenberg & heller, 1982; heinrich, 1993; heinrich, 1996; kleinhenz et al., 2003). the heat production rates are caste-dependent; workers bees contribute more than drones and queens for hive thermoregulation (fahrenholz et al., 1989). when the internal hive temperature exceeded the ideal range for brood development (i.e., 33 and 40 ° c treatments), workers probably sought to intensify the convective heat loss by fanning over the brood area (southwick & heldmaier, 1987; southwick & moritz, 1987; starks & gilley, 1999). according to bordier et al. (2017), heat-challenged workers bees show a nurse-like profile as a result of increased expression of specific genes. the activation of fanning behavior and the ability of nest cooling by workers arepositively influenced by intracolonial genetic diversity (jones et al., 2004; graham et al., 2006; jones et al., 2007). adade and cruz-landim (2004) investigated the aging of the flying muscles of newly emerged bees, nurse bees and forager bees in the genus apis. they found that the muscle fibers of nurse bees have larger diametersthan those of newly emerged bees and forager bees. nurse bees, compared with newly emerged bees and forager bees, also have larger amounts of stored glycogen. these results indicate that nurse bees have the highest individual capacities for thermogenesis and contribute most to thermal homeostasis. adade and cruz-landim (2004) have shown that nurse bees have higher levels of activity and greater capacity to generate metabolic heat by contracting their thorax muscles and generating adenosine triphosphate. our data on concentrations of total protein in the hemolymph are in agreement with those of crailsheim (1992), who studied colonies throughout the year and observed that the amount of total protein in the hemolymph changes seasonally. this author observed that protein content is lower during the winter and tends to increase during the spring. protein content also decreases with age due to less use according to activity. forager bees perform locomotor activities and do not require protein. however, protein content is higher in young bees up to 12 days of age (nurse bees) and even higher with increasing temperature, as temperatures are higher in spring than in winter. the main function of hemolymph is the distribution of available nutrients from the digestion process and the receipt of metabolic products (rocha et al., 2003). the protein present in the hemolymph of nurse bees is metabolized as a storage protein (amdam and omholt, 2002). this storage remains in the fat body, which is a central metabolic organ for these insects (arrese & soulages, 2010). according to amdam and omholt (2002), protein stored by the fat body is used in several vital processes, such as the maintenance of tissues, and represents a source of energy through the catabolism of glycogenic amino acids that are released into the hemolymph when necessary. according to the results of the present study, the lower amount of protein in the hemolymph at lower temperatures can be explained by the immediate use of this nutrient by nurse bees for heat generation, or by storage to prolong the thermogenic capacity. this study measured the concentration of protein dispersed in the hemolymph, disregarding the fat body, which was discarded after sample centrifugation. acknowledgements this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. references adade, c.m. & cruz-landim, c. 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(2003). a biologia da abelha. porto alegre: magister, 276p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.4333sociobiology 66(2): 257-262 (june, 2019) geographic spread of solenopsis globularia (hymenoptera, formicidae) introduction several species of solenopsis have spread from their native ranges and have become exotic pests, most notably solenopsis geminata (fabricius) and solenopsis invicta buren, two neotropical species that have spread broadly to sites around the world (wetterer, 2011, 2013). here, i examine the geographic spread of a smaller, less conspicuous neotropical solenopsis species that has spread to diverse areas outside its native range: solenopsis globularia (smith). the genus solenopsis is often informally divided into two groups: “fire ants” and “thief ants.” fire ants are relatively large, highly polymorphic, and with a potent sting (e.g., s. geminata and s. invicta). thief ants are usually small, monomorphic, and not known to sting humans (e.g., solenopsis molesta buren). solenopsis globularia falls between these two categories; it is intermediate in size, mildly polymorphic, and not known to sting humans. abstract several species of solenopsis have spread beyond their native ranges and have become exotic pests, most notably solenopsis geminata (fabricius) and solenopsis invicta buren. here, i examine the geographic spread of a smaller, less conspicuous solenopsis species, solenopsis globularia (smith). i compiled s. globularia specimen records from >700 sites. i documented the earliest known s. globularia records for 59 geographic areas (countries, us states, and major west indian islands), including the following with no published records: anguilla, antigua, aruba, barbuda, bonaire, british virgin islands, congo, curaçao, dominica, martinique, montserrat, nevis, st kitts, st martin, san andrés island, senegal, tobago, and trinidad. solenopsis globularia has a broad distribution in the new world, from corrientes, argentina (28.4°s) in the south to craven county, north carolina (35.1°n) in the north. most s. globularia records come from islands. s. globularia may be invasive in some of its new world range, such as the galapagos islands. all populations of s. globularia outside the new world are probably exotic, introduced through human commerce, including populations on atlantic islands (ascension, cabo verde, st helena), pacific islands (hawaii, french polynesia, philippines), and africa (congo, ivory coast, senegal). on the cabo verde islands, off the coast of west africa, s. globularia is widespread on all nine inhabited islands. records from nine diverse sites in ivory coast indicates that s. globularia is well able to spread in continental africa as well. sociobiology an international journal on social insects jk wetterer article history edited by john lattke, ufpr, brazil received 17 december 2018 initial acceptance 11 february 2019 final acceptance 01 april 2019 publication date 02 august 2019 keywords biogeography, exotic range, geographic range, native range corresponding author james k. wetterer florida atlantic university wilkes honors college 5353 parkside drive jupiter, fl 33458, usa. e-mail: wetterer@fau.edu the taxonomy of the genus solenopsis has long been in disarray and most researchers do not even attempt to identify small solenopsis specimens to species level. solenopsis globularia, however, has distinctive morphology, allowing reliable positive identification. taxonomy and identification smith (1858) described myrmica globularia (= s. globularia) from brazil. the name globularia no doubt refers to its large “globular” post-petiole, one primary identifying feature. many other sub-specific taxa of s. globularia were subsequently described, based primarily on color (see creighton 1950), but researchers disagree over how to distinguish among these taxa. some authors simply identify specimens as s. globularia (s.l.) (morrison 1998), solenopsis cf. globularia (wetterer, 2011), or as belonging to the s. globularia complex (jacobs et al., 2011). wilkes honors college, florida atlantic university, jupiter, florida, usa research article ants jk wetterer – geographic spread of solenopsis globularia258 pacheco and mackay (2013) recently simplified the taxonomy of the globularia complex, synonymizing most subspecies. junior synonyms of s. globularia now include: s. globularia borinquenensis wheeler, s. globularia cubaensis wheeler, s. globularia littoralis creighton, s. globularia lucayensis curta forel, s. globularia mobilensis smith, s. globularia pacifica wheeler, s. globularia pacifica rubida wheeler, and s. globularia steinheili forel. pacheco and mackay (2013) raised to species level two members of the s. globularia complex: the darkercolored solenopsis desecheoensis mann known only from two specimens collected on desecheo island (an uninhabited islet west of puerto rico) and the lighter-colored solenopsis lucayensis wheeler, known only from nicholl’s town, bahamas. pacheco and mackay (2013) left open the possibility further collections will indicate that both s. desecheoensis and s. lucayensis are also junior synonyms of s. globularia. unfortunately, collecting more specimens of s. desecheoensis may be difficult because the island was used as a bombing range until 1952 and is now closed to the public because of the danger of unexploded ordnance. pacheco and mackay (2013) placed one additional species in the s. globularia complex: solenopsis bucki kempf, known only from a single specimen collected in erechim, rio grande do sul, brazil. pacheco and mackay (2013) wrote: “solenopsis bucki is easily separated by having a mandible with only three teeth that is elongate and nearly straight, while s. globularia has a mandible with four teeth on the masticatory border.” pacheco and mackay (2013) concluded that a combination of two characters was diagnostic of the s. globularia complex: postpetiole more than half as wide as the gaster and eyes of workers with at least 12 ommatidia. solenopsis loretana santschi, a species known from argentina and paraguay, has a similarly broad postpetiole , but it has much smaller eyes. pacheco and mackay (2013) wrote: “solenopsis loretana is easily differentiated from s. globularia as it has a small eye with only 3-5 ommatidia; s. globularia nearly always has 15-25 ommatidia.” counting ommatidia under a dissecting scope can be difficult, but counting them from well-focused microphotographs is much easier. from photographs of s. globularia posted on-line at antweb, i counted only 11-13 ommatidia on most specimens, including types. still, this eye size is much larger than that of most other small solenopsis species. methods using published and unpublished records, i documented the range of s. globularia. i obtained unpublished site records from museum specimens in the collections of the museum of comparative zoology (mcz) and the us national museum of natural history (usnm). in addition, i used on-line databases with collection information on specimens by antweb (www.antweb.org) and the global biodiversity information facility (www.gbif.org). i obtained geo-coordinates for collection sites from published references, specimen labels, maps, or geography web sites (e.g., earth.google.com, www.tageo.com, and www. fallingrain.com). if a site record listed a geographic region rather than a “point locale,” and i had no other record for this region, i used the coordinates of the largest town within the region or, in the case of small islands and natural areas, the center of the region. published records usually included collection dates. in a number of cases, publications did not include the collection dates for specimens, but i was able to determine the approximate date based on information on the collector’s travel dates or limit the date by the collector’s date of death. for example, wheeler (1913) recorded specimens of s. globularia cubaensis collected by j. c. gundlach (1810-1896) in cuba that necessarily pre-dated gundlach’s death in 1896. results i collected s. globularia in florida, el salvador, cabo verde, and on 30 west indian islands (anguilla, antigua, aruba, barbados, barbuda, bonaire, buck island, culebra, curaçao, dominica, grenada, guadeloupe, margarita, marie galante, martinique, montserrat, nevis, puerto rico, st croix, st kitts, st john, st lucia, st martin, st thomas, st vincent, san andrés, tobago, tortola, trinidad, vieques). my pinned s. globularia voucher specimens are deposited in the us national museum of natural history (usnm), my personal collection (jkwc), and the collection of john t. longino (jtlc) (see supplementary file). i compiled s. globularia specimen records from >700 sites (fig 1), and documented the earliest known s. globularia records for 59 geographic areas (countries, us states, and major west indian islands; tables 1-3), including the following with no published records: anguilla, antigua, aruba, barbuda, bonaire, british virgin islands, cabo verde, congo, curaçao, dominica, martinique, montserrat, nevis, st kitts, st martin, san andrés island, senegal, tobago, and trinidad (tables 1-3). collingwood and van harten’s (1993) report of solenopsis innota from the cabo verde islands off the coast of west africa, appears to be based on misidentification of s. globularia. santschi (1915) described solenopsis geminata innota santschi from gabon, liberia, and congo (zaire), but it is now considered a junior synonym of s. geminata (wheeler, 1922; creighton, 1950; trager, 1991). collingwood and van harten (1993) compared the cabo verde specimens with s. geminata and s. globularia noting “the enlarged petiole and postpetiole link it with s. globularia of south america. it differs from both species by its small eyes.” in 2003, i collected ants in cabo verde and found no solenopsis geminata, which is very polymorphic. instead, i found a solenopsis species with an enlarged petiole and post-petiole at 83 sites across all nine inhabited islands (see supplementary file). the hundreds of specimens i collected are only slightly polymorphic, and have the same range of body size and eye size as s. globularia. i conclude that collingwood and van sociobiology 66(2): 257-262 (june, 2019) 259 harten (1993) simply misidentified s. globularia in cabo verde as s. innota, apparently due to a misunderstanding about eye size. taylor (2015) presented photos of a specimen identified as s. globularia from brazzaville, republic of congo, which is almost black, much like s. desecheoensis, and darker than any s. globularia specimens i have seen, including those from cabo verde and ivory coast. discussion solenopsis globularia has a broad geographic distribution in the new world, from corrientes, argentina (28.4°s; calcaterra et al., 2010) in the south to craven county, north carolina (35.1°n; guénard et al., 2012) in the north (fig 1). solenopsis globularia appears to be particularly common on islands. for example, s. globularia is known from almost every major caribbean island (table 2), except jamaica. surprisingly, there are no s. globularia records from panama, honduras, and guyana. s. globularia may be exotic to the galapagos islands. although pezzatti et al. (1998) wrote that s. globularia was “definitely native” in the galapagos, herrera (2015) classified s. globularia in the galapagos as “introduced, questionable native.” remarkably, s. globularia has been reported from 30 different islands of the galapagos (herrera & roque-albedo, 2007). all populations of s. globularia outside the new world are probably exotic, introduced through human commerce, including populations on atlantic islands (ascension, cabo verde, st helena), pacific islands (hawaii, french polynesia, philippines), and africa (congo, ivory coast, senegal). on the cabo verde islands, off the coast of west africa, s. globularia is extremely widespread on all nine inhabited islands, primarily in gardens and in acacia stands. the common habit of nesting in planted flower beds may facilitate its ability to colonize new areas, carried along in potted plants. records from nine diverse sites in ivory coast (kouakou et al., 2018; l.m.m. kouakou pers. comm.) indicates that s. globularia is well able to spread in continental africa. in fact, s. globularia may already have an extensive but overlooked range in west africa. due to taxonomic difficulties of identifying small solenopsis species, published faunal inventories often include many solenopsis species identified only to genus. for example, jaffe and lattke (1994) listed eight solenopsis species in their surveys of west indian islands: solenopsis geminata fig 1. geographic distribution of solenopsis globularia records. map created using carto.com. table 1. earliest known continental records for solenopsis globularia in the new world. unpublished records include collector, source, and site. south america earliest record brazil ≤1858 (smith, 1858) french guiana 1868 (radoszkowsky, 1884) colombia ≤1912 (forel, 1912 as s. globularia lucayensis curta) surinam 1959 (kempf, 1961) venezuela 1977-1978 (lubin, 1985 as s. globularia group) paraguay 1993 (pacheco & mackay, 2013) argentina 2007-2008 (calcaterra et al., 2010) ecuador ≤2015 (reyes-puig & rios-alvear, 2015) central america costa rica ≤1908 (forel, 1908) nicaragua ≤1981 (van huis, 1981) guatemala 2009 (j.t. longino, antweb): 3.5km nw morales el salvador 2012 (wetterer et al., 2016) north america alabama 1926 (creighton, 1930 as s. globularia littoralis) mississippi 1928 (creighton, 1930 as s. globularia littoralis) mexico 1929 (creighton, 1930 as s. globularia littoralis) florida ≤1933 (smith, 1933 as s. globularia littoralis) georgia ≤1933 (smith, 1933 as s. globularia littoralis) south carolina ≤1933 (smith, 1933 as s. globularia littoralis) louisiana ≤1960 (moser & blum, 1960) north carolina ≤1962 (carter, 1962 as s. globularia littoralis) arizona 1999 (k. ross, pers. comm.): portal jk wetterer – geographic spread of solenopsis globularia260 often found foraging above ground; their comparatively large eyes seem well adapted for this habit. also, unlike other small solenopsis species, s. globularia is commonly collected at baited traps. levins et al. (1973) anecdotally noted aggressive interactions between solenopsis globularia and monomorium ebeninum at baits. although this species can be very common, there are few records of s. globularia as a pest species, e.g., as a pest inside hospitals (e.g., bragança & lima, 2010), perhaps nesting inside potted plants. genetic work is needed to determine whether solenopsis globularia, as currently defined, is a single, highly variable species or if it is a species complex. if solenopsis globularia is complex, finding consistent characters that define the boundaries among the species would be valuable. earliest record us virgin is. 1878 (forel, 1881 as s. steinheili) st vincent ≤1893 (forel, 1893) cuba ≤1896 (wheeler, 1913 as s. globularia cubaensis) grenada ≤1897 (forel, 1897) galapagos 1899 (wheeler, 1919 as s. globularia pacifica) haiti 1901 (wheeler & mann, 1914) puerto rico 1906 (wheeler, 1908 as s. globularia borinquenensis) st lucia 1919 (j.c. bradley, mcz): castries dominican rep. 1928 (menozzi & russo, 1930 as s. globularia borinquenensis) +antigua 1936 (r.e. blackwelder, usnm): no site isla de aves 1966 (pacheco & mackay, 2013) bahamas 1991-1993 (deyrup, 1994) barbados 1998 (wetterer et al., 2016) guadeloupe 1999 (galkowski, 2016) +tobago 2003 (j.k. wetterer, usnm): bon accord +trinidad 2003 (j.k. wetterer, usnm): st. augustine +curaçao 2004 (j.k. wetterer, usnm): piscadera +bonaire 2004 (g. van hoorn, mcz): lima +dominica 2004 (j.k. wetterer, usnm): roseau +british virgin is. 2005 (j.k. wetterer, usnm): brandy wine bay, tortola +anguilla 2006 (j.k. wetterer, usnm): meads bay +st martin 2006 (j.k. wetterer, usnm): baie nettle +aruba 2007 (j.k. wetterer, usnm): mon plaisir +barbuda 2007 (j.k. wetterer, usnm): rock bay +st kitts 2007 (j.k. wetterer, usnm): basseterre +nevis 2007 (j.k. wetterer, usnm): charlestown +montserrat 2007 (j.k. wetterer, usnm): carrs bay +martinique 2008 (j.k. wetterer, usnm): anse turin +san andrés 2015 (j.k. wetterer, usnm): san andrés table 2. earliest known records for solenopsis globularia on islands of the west indies and the galapagos. + = no previously published records. mcz = museum of comparative zoology. usnm = us national museum of natural history. plus seven unidentified species, one of which was almost certainly s. globularia. no doubt large numbers of s. globularia specimen records remain hidden in the long lists of unidentified solenopsis species recorded in similar faunal studies, particularly from south and central america. i expect that a perusal of unidentified solenopsis specimens in many museums would yield more s. globularia records. there has been little research on the ecology of solenopsis globularia. solenopsis globularia nests in soil and under rocks and logs, and is common on beaches (pacheco & mackay 2013). it is seldom found in dense forests (see supplementary file). in many respects, s. globularia is more like a very small fire ant, than a somewhat large thief ant. unlike most other small solenopsis species, s. globularia is table 3. earliest known records for solenopsis globularia outside the new world. + = no previously published records. continental earliest record +congo 2007 (taylor, 2015): brazzaville ivory coast 2014 (kouakou et al. 2018) +senegal 2016 (k. gomez; antweb kg03260): dakar island ascension island 1958 (duffey, 1964 as s. globularia steinheili) st helena ≤1976 (taylor, 1976) +cabo verde ≤1993 (collingwood & van harten, 1993 as s. innota) hawaii 2005 (wang, 2007) french polynesia 2006 (ramage, 2014) philippines 2008 (mabutol-afidchao, 2013) acknowledgments i thank m. wetterer for comments on this manuscript; l.m.m. kouakou and k. ross for unpublished site information, l.m.m. kouakou for providing photos of s. globularia from ivory coast, the national geographic society, national science foundation, and florida atlantic university for financial support. references bragança, m.a.l. & lima, j.d. 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(1919). expedition of the california academy of sciences to the galapagos islands, 1905-1906. part 14. the ants of the galapagos islands. proceedings of the california academy of sciences, (4) 2 (2): 259-297. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4481sociobiology 67(1): 41-47 (march, 2020) introduction solenopsis invicta (red imported fire ant, rifa) is an invasive pest native to south america (dai, 2015). it is widely distributed in china, the usa, new zealand, australia, and other parts of the world (ascunce et al., 2011). rifa has many characteristics, such as food mixed, diverse dispersal ways, fierce habits, and strong reproductive ability (lin, 2012). moreover, it is very aggressive to human beings and seriously affects the biodiversity, agriculture, and forestry production in the invaded area (huang et al., 2011). thus, rifa is considered as one of the 100 most dangerous invading pests in the world. at present, the main methods used to control rifa include plant quarantine, physical control, chemical control, and biological control (wang et al., 2005). among them, chemical control is the most widely used method. although chemical insecticides can achieve good control effect in a short time, they also bring a series of problems, such as environmental abstract this paper studied the fumigating activity of litsea cubeba oil and citral on solenopsis invicta, identified and analyzed the chemical constituents and volatile components of l. cubeba oil via solid-phase microextraction which were then identified via gas chromatography-mass spectrometry. the results showed that citral and (z)-3,7-dimethylocta-2,6-diena were the main components of l. cubeba oil, as well as its volatile compounds. according to the experimental results, l. cubeba oil and citral had good fumigating activity on workers, and also had significant inhibition on the walking ability and climbing ability of workers. at the same time, the effects of the two agentia on the fumigating activity and behavioral inhibition of microergate were stronger than those of macroergate. after treating with l. cubeba oil and citral for 24 hours, the walking rate and grasping rate of microergate were both 0 %. the results showed that l. cubeba oil and citral had good control effect on s. invicta. sociobiology an international journal on social insects cx xiao1, yt tan1, ff wang1, qh wu1, dq qin1, zx zhang1,2,3 article history edited by evandro nascimento silva, uefs, brazil received 20 april 2019 initial acceptance 21 january 2020 final acceptance 22 january 2020 publication date 18 april 2020 keywords solenopsis invicta, litsea cubeba oil, citral, fumigating activity. corresponding author zhixiang zhang key laboratory of natural pesticide and chemical biology of the ministry of education south china agricultural university, guangzhou, 510642, china e-mail: zdsys@scau.edu.cn pollution, impact on human and non-target organisms, and emergence of insecticide resistance (gao, 2010). therefore, in order to solve these problems, it is necessary to search for natural substances that are nontoxic to non-target organisms and can rapidly degrade in natural environment. essential oils are volatile aromatic substances extracted from the roots, stems, leaves, flowers, or fruits of a plant via physical or chemical methods (adorjan et al., 2010).they are widely used in medicine, chemical industry, food industry, and plant protection (zhu et al., 2009). they are also used as a fumigant, repellent, and contact insecticide (palacios et al., 2009; sfara et al., 2009).some plant essential oils have a repellent or toxic effect on some insects. the essential oils of callistemon citrinus leaves show repellent and fumigating effects on adults of callosobruchus maculatus (sohani et al., 2013). the essential oils of vitex negundo exhibit insecticidal activity against sitophilus zeamais (lu et al., 2009). volatile compounds of michelia alba leaves can control rifa populations (qin et al., 2018). 1 agricultural college of south china agricultural university, guangzhou, china 2 guangdong biological pesticide engineering technology research center, south china agricultural university, guangzhou, china 3 key laboratory of natural pesticide and chemical biology of the ministry of education, south china agricultural university, guangzhou, china research article ants the fumigating activity of litsea cubeba oil and citral on solenopsis invicta cx xiao, yt tan, ff wang, qh wu, dq qin, zx zhang – toxicity of botanical pesticide to solenopsis invicta42 litsea cubeba is a typical chinese perfume plant resource that is mainly found in the southern provinces and regions of the yangtze river to tibet. the main component of l. cubeba oil is citral, containing 60% ~ 80%, and the oil is mainly derived from l. cubeba fruits (zhao et al., 2010). several studies emphasized the strong antibacterial effect of l. cubeba oil on aspergillus flavus and fungi imperfecti (huang et al., 1994; yu et al., 2002; huang et al., 2005; zhou et al., 2013; li et al., 2016) and found that l. cubeba oil has strong antioxidant activity (zhang et al., 2017). wagan et al. (2016) found that l. cubeba oil has a strong repellent effect on monomorium pharaonis. zhang et al. (2017) found that the extract of l. cubeba fruits has strong fumigant and repellent activities on adult sitophilus zeamais, is harmless to human beings, does not pollute the environment, and has a pleasant smell. therefore, it can be used as a germicide and for pest control and crop disease prevention, and it has broad prospects for development. in the present study, we determined the fumigating activity of l. cubeba oil and citral on microergate and macroergate and analyzed the chemical and volatile constituents of l. cubeba oil. our results showed that l. cubeba oil and citral can be used to control rifa. materials and methods chemical reagents l. cubeba oil and citral were purchased from guangzhou suining biological instrument co., ltd., china. l. cubeba oil was stored in a refrigerator at 4 °c. the purity of citral was 95%. insects rifa colonies were collected from the campus of south china agricultural university. the ants and nest materials were placed into plastic boxes (40 cm × 52 cm × 12 cm), which were coated with teflon emulsion on the top. a test tube (25 mm × 200 mm) used as a water source was partially filled with water and plugged with cotton. mealworms (tenebrio molitors) were used as the food source. t. molitor larvae were purchased from the insect–fish market in guangzhou, fed with wheat bran, and kept in a dry indoor environment at 25 ± 2 °c. this study opted to test microergate and macroergate. the microergates and macroergates were approximately 3 and 5 mm in length, respectively (lin et al., 2005; zhang et al., 2007). chemical analysis via gas chromatography–mass spectrometry l. cubeba oil was diluted 2000 times with ethyl acetate. subsequently, 0.5 ìl of the sample was taken to be directly detected using an agilent 7890 gas chromatograph coupled with an agilent mass spectrometer detector. a hp-5(ms) capillary column (30 m × 0.25 mm × 0.25 ìm) was used to separate the compounds. mass spectra were obtained by electron ionization at 70 ev with a spectral range of 35-450 m/z. the injection temperature was 250 °c. the oven temperature was initially set at 40 °c for 1 min to 100 °c (10 °c/min) for 2 min, then to 200 °c (10 °c/min) for 2 min, and to 280 °c (15 °c/min) for 5 min. the detector was operated at 280 °c. helium was used as a carrier gas at a flow rate of 1 ml/min and split ratio of 10:1. the compounds were identified via mass spectroscopy. then, the spectra were compared with those from the computer mass libraries (chen et al., 2012). extraction of volatiles via solid-phase microextraction (spme) approximately 0.5 ml of essential oil was taken in a 50 ml headspace vial, sealed, and placed in a 35 °c water bath for 20 min. subsequently, spme fiber (50/30 ìm, car/dvb/ pdms) was inserted for head-space extraction for 20 min. the sampled heat at 250 °c was absorbed for 3 min. the injection temperature was 250 °c. the oven temperature was initially set at 40 °c for 1 min to 100 °c (10 °c/min) for 2 min and then to 200 °c (50 °c/min) for 2 min. the detector was operated at 280 °c. helium was used as a carrier gas at a flow rate of 1 ml/min and split ratio of 50:1. the compounds were identified via mass spectroscopy, and the spectra were then compared with those from computer mass libraries. toxicity bioassay of fumigant the fumigating activity of l. cubeba oil and citral was determined on the basis of preliminary experiments. the l. cubeba oil and citral were packed in 1.5 ml centrifuge tube (axygen biosciences, usa) with eight pinholes to make them vaporize outside. then, the centrifuge tube was placed at the bottom of the 250 ml rigid plastic cup. the fluon emulsion (guangzhou xingsheng street science and technology co., ltd., china) was evenly coated with the wall of each hard-plastic cup to below 2 cm and dried for 24 h to prevent the ants from escaping. twenty-five microergates and macroergates were placed into a plastic cup bottom and then sealed with plastic wrap and rubber band. the insects were placed at a temperature of 25 ± 2 °c and a relative humidity of 80%. two concentrations (4 and 8 ìl/tube) of essential oil and citral were applied to workers, respectively, and the cumulative mortalities were assessed after 4, 8, 12, 16, and 24 h of treatment. each treatment had three replicates, with 25 workers per replicate. behavior observation on walking ability of workers workers were treated in the same way as above. to observe the walking ability of the workers, the plastic cup was shaken to disperse the workers in the cup. then, videos of ants walking were recorded, and we watched the worker walking. if the workers could walk continuously for 3 s or more, they were considered to have walking ability. each treatment had three replicates, with 25 workers per replicate. the following equation was used to calculate the walking rate: sociobiology 67(1): 41-47 (march, 2020) 43 walking rate (%) = (number of workers that possessed walking ability/number of workers per replicate) × 100 behavior observation on grasping ability of workers workers were treated in the same way as above. to observe the grasping ability of the workers, the plastic cup was shaken to evenly distribute the workers at the bottom of the cup. after 10 s, the cup was gently rotated 180°. if the ants did not drop from the bottom of the cup, they were considered to have grasping ability. each treatment had three replicates, with 25 workers per replicate. the following equation was used to calculate the grasping rate: grasping rate (%) = (number of workers possessing grasping ability/number of workers per replicate) × 100 statistical analysis the data were represented as means ± se. statistical analysis was carried out by using spss 24.0 (spss inc., chicago). charts were created by using originpro 8. furthermore, the differences between data were compared by using tukey’s test, and values with p < 0.05 were regarded statistically significant. results figure 1 and tables 1 and 2 show that the chemical constituents of l. cubeba oil were mainly citral and (z)-3,7dimethylocta-2,6-diena (27.76% and 26.13%, respectively). the volatile constituents of l. cubeba oil were also mainly citral and (z)-3,7-dimethylocta-2,6-diena (22.43% and 29.34%, respectively). other compounds, such as d-limonene, (+)-2-bornanone, and eucalyptol, accounted for more than 40% of all the volatiles detected from the essential oil. fumigating toxicity bioassay figure 2 shows that the l. cubeba oil and citral had good fumigating activity to microergate and macroergate. with the increase of concentration and time of essential oil and citral, the fumigating activity increased. the mortality of microergate was higher than that of macroergate. after 24 h fig 1. gc-ms total ion chromatograms of chemical compositions (a) and volatile components (b) of l. cubeba oil. ×107 intensity intensity 7x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 + tic 扫扫 sample.d 12.30 12.82 16.4610.42 26.067.86 6.96 9.33 14.68 24.0422.21 counts vs. 扫扫扫扫 (min) 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 7x10 0 0.5 1 1.5 2 2.5 + tic 扫扫 spme.d 13.14 13.91 7.81 10.57 6.96 6.19 9.331.65 19.95 12.49 counts vs. 扫扫扫扫 (min) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a b min min ×107 in te ns ity in te ns ity cx xiao, yt tan, ff wang, qh wu, dq qin, zx zhang – toxicity of botanical pesticide to solenopsis invicta44 of treatment, the mortalities of l. cubeba oil to macroergate were 40% and 60%, and the mortalities of microergate were 40% and 90%. the mortalities of citral to macroergate were 36% and 76%, and the mortalities of microergate were 36% and 96%. this finding indicated that l. cubeba oil and citral had strong fumigating activity to microergate (f = 144.231, df = 5, p < 0.05; f = 243.559, df = 5, p < 0.05). during the first 4 h of treatment, the mortality rate of workers was low. walking ability bioassay figure 3 shows that l. cubeba oil and citral have good inhibitory effects on the walking ability of microergate and macroergate. with the increase of concentration and time of essential oil and citral, the inhibitory effect increased. after treatment with 2.67 ìl/ml and 5.33 ìl/ml of l. cubeba oil for 24 h, the walking rate of macroergate decreased to 60% and identified peaks retention time (min) formula compound relative content (%) 1 1.65 c3h6o acetone 0.6461 2 6.19 c10h16 (1s)-2,6,6-trimethylbicyclo [3.1.1]hept-2-ene 1.5081 3 6.34 c10h18 r(-)3,7-dimethyl-1,6-octadiene 0.4769 4 6.96 c10h18 dihydropinene 2.6908 5 7.81 c10h16 d-limonene 7.2376 6 9.33 c10h18o linalool 1.3316 7 10.57 c10h16o (+)-2-bornanone 6.4628 8 13.14 c10h16o (z)-3,7-dimethylocta-2,6-diena 29.3397 9 13.91 c10h16o citral 22.4322 10 19.95 c15h22 (+)-cuparene 1.4533 table 2. volatile components of litsea cubeba oil. identified peaks retention time (min) formula compound relative content (%) 1 6.96 c10h16 (1r)-(+)-á-pinene 0.1734 2 7.86 c10h18o eucalyptol 1.0972 3 9.33 c10h18o linalool 0.5738 4 10.42 c10h16o (+)-2-bornanone 2.6873 5 12.30 c10h16o (z)-3,7-dimethylocta-2,6-diena 26.1297 6 12.82 c10h16o citral 27.7618 7 14.68 c6h12o 3-hydroxy-2,3-dimethyl-1-butene 0.6092 8 16.21 c15h24 (+)-b-selinene 1.5495 9 16.46 c15h22 (+)-cuparene 2.7297 10 22.21 c16h22o4 dibutyl phthalate 0.2840 table 1. chemical compositions of litsea cubeba oil. fig 2. mortality of workers (a: macroergate, b: microergate) after treating for 24 hours of litsea cubeba oil and citral. ba sociobiology 67(1): 41-47 (march, 2020) 45 32% (f = 54.000, df = 5, p < 0.05), and that of microergate decreased to 32% and 16% (f = 48.000, df = 5, p < 0.05). after treatment with 2.67 ìl/ml and 5.33 ìl/ml of citral for 4–24 h, the walking rate of macroergate decreased to 44% and 16% (f = 27.000, df = 5, p < 0.05), and that of microergate decreased to 40% and 0% (f = 300.000, df = 5, p < 0.05). the walking rate of microergate was lower than that of macroergate. discussion our results showed that l. cubeba oil and citral have certain toxicity to rifa and have significant inhibitory effect on the walking and climbing ability of the workers. high concentration of essential oil and pure products had obvious toxic effects on rifa. rifas live in complex environments, and a large number of workers may appear in fields, lawns, and roadside habitats (tang et al., 2016). their small size and good grasping and walking abilities enable them to adapt to various environments. the ability of workers to avoid natural enemies and resist harsh environment can be reduced by lowering the ability of workers to grasp and walk. moreover, this process can significantly inhibit the escape and migration of ants, thereby seriously affecting their quality of life (li et al., 2015). our results showed that l. cubeba oil had strong inhibitory effect on the activity of workers and could effectively control rifa. in china, the natural resources of l. cubeba are very rich, and this plant has a wide range of applications. l. cubeba can be used as a medicine and flavoring agent and fig 3. effects on walking ability of workers (c: macroergate, d: microergate) after treating for 24 hours of litsea cubeba oil and citral. c d grasping ability bioassay figure 4 shows that l. cubeba oil and citral have good inhibitory effect on the climbing ability of microergate and macroergate. after treatment with 5.33 ìl/ml of l. cubeba oil and citral for 24 h, the climbing rate of macroergate was 20% and 16%, respectively, and that of microergate was 0%. the climbing rate of microergate was lower than that of macroergate, indicating that the inhibitory effect of microergate was more significant. moreover, with the increase of essential oil and citral concentration, the inhibitory effect increased (f = 75.000, df = 5, p < 0.05; f = 192.000, df = 5, p < 0.05). fig 4. effects on grasping ability of workers (c: macroergate, d: microergate) after treating for 24 hours of litsea cubeba oil and citral. c d cx xiao, yt tan, ff wang, qh wu, dq qin, zx zhang – toxicity of botanical pesticide to solenopsis invicta46 preservative in food (fang, 2007; chen et al., 2013). citral is the main component of l. cubeba oil. it can be extracted from essential oils, such as l. cubeba oil, lemon grass oil, and citrus oil (liu et al., 2013). citral can also be synthesized by appropriate methods to improve its volatility and stability (zhong et al., 2015). given the nature of l. cubeba oil and citral and their fumigating activity against rifa, people can apply them to their bodies or spray them around the house where rifas invade, thereby achieving certain insecticidal and repellent effects. in conclusion, l. cubeba oil and its main component, citral, have good feasibility and potential in the prevention and control of rifa and are sources of natural insecticides for the control of rifa. acknowledgment this research was supported by national key r&d program of china (2017yfd0201203); guangdong provincial special fund for modern 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(2009). progress in the research of plant essential oil. hunan agricultural sciences, 8: 86-89, 92. doi: 10.16498/j.cnki. hnnykx.2009.08.001 doi: 10.13102/sociobiology.v66i1.3577sociobiology 66(1): 166-178 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the queen of the paper wasp polistes jokahamae (hymenoptera: polistinae) is not aggressive but maintains her reproductive priority introduction eusocial insects are divided into two groups according to their caste characteristics: primitively and advanced eusocial groups (wilson, 1971; jeanne, 2003). the latter include hornets and honeybees and are characterized mainly by morphologically discernible differences between the queen and workers. in contrast, primitively eusocial groups, which include polistes (paper wasps) and bumble bees, are characterized by the absence of critical morphological differences between the queen and workers. the queen of an advanced eusocial group maintains her queen status (including reproductive priority) using the queen pheromone (wyatt, 2014), while the queen of a primitively eusocial group has abstract the behaviors performed on the nest by the foundress queen and workers of the paper wasp polistes jokahamae were observed in three colonies in the field and one colony in a cage set in the field. each queen was rarely ranked top in the dominance hierarchy determined by the pairwise dominance-subordinate interactions and did not display more frequent direct aggression toward the top-ranked worker than toward other workers. furthermore, the queen exhibited aggression less frequently than did the most aggressive workers in all four colonies. the dominance order among the workers was positively correlated with the emergence order, with older workers being more dominant. the queen laid eggs in a dominant or monopolized way; some dominant workers laid eggs in three colonies. these observations suggest that the queen maintained her queen status, including her reproductive priority, using signals rather than aggression. lateral vibrations (rapidly laterally vibrating the abdomen) and abdominal rubbing (rubbing the abdomen onto the comb) appeared to be candidate signals of the fertility or reproductive potential of the performer. lateral vibrations were performed only by the queen, and their frequency was positively correlated with the frequency of ovipositing. the queen and some dominant workers performed abdominal rubbing; the frequency was higher for the queen than for any of the dominant workers early in the colony’s development, but not later. although performers of abdominal rubbing were more likely to lay eggs than nonperformers, the frequency of abdominal rubbing was not a predictor of the frequency of ovipositing. sociobiology an international journal on social insects h yoshimura, j yamada, yy yamada article history edited by gilberto m. m. santos, uefs, brazil received 16 july 2018 initial acceptance 24 august 2018 final acceptance 14 october 2018 publication date 25 april 2019 keywords aggression, dominance hierarchy, signal, primitive eusociality, social insect. corresponding author yoshihiro y. yamada, insect ecology laboratory graduate school of bioresources mie university 1577 kurimamachiya-cho tsu, mie 514-8507, japan. e-mail: yamada-y@bio.mie-u.ac.jp been considered to achieve this using physical aggression (van doorn & heringa, 1986; monnin & peeters, 1999; jandt et al., 2014). the physical aggression usually includes biting with mandibles, rushing, chasing, and mounting on the backs of subordinate individuals. in primitively eusocial insects, aggression is also used to establish and maintain the dominance hierarchy among colony members, including the queen: higher-ranked individuals are more aggressive in that they perform aggressive behaviors more frequently (reeve, 1991; monnin & peeters, 1999). the queen (or replacement queen) in primitively eusocial insects is the most aggressive colony member, is ranked the highest in the dominance hierarchy and monopolizes reproduction (e.g., pardi, 1948; strassmann & meyer, 1983; reeve, 1991; ishikawa et al., 2011). insect ecology laboratory, graduate school of bioresources, mie university, tsu, japan research article wasps sociobiology 66(1): 166-178 (march, 2019) 167 moreover, the aggression is directed primarily toward individuals ranked immediately below the performers of the aggression in the dominance hierarchy (reeve, 1991; cant et al., 2006; ishikawa et al., 2010). the chemicals (cuticular hydrocarbons) on the surface and/or the face pattern function as signals of the bearer’s agonistic ability in some species, and the costs associated with fighting over rank are reduced (tibbetts & lindsay, 2008; dapporto et al., 2010a, 2010b; tibbetts et al., 2010). however, aggression still plays a primary role: the top-ranked wasp in the dominance hierarchy determined by aggression monopolizes oviposition. dapporto et al. (2010a) refer to a mild type of aggression as dominance behavior (probably examination of the chemical profile of cuticular hydrocarbons) and a harsh type of aggression as attack or aggression, but here, we use the term aggression for both actions. however, the rank in a dominance hierarchy determined by aggression may differ from that in a dominance hierarchy in reproduction (röseler, 1991); the former and latter hierarchies are often called social and reproductive dominance hierarchies, respectively. such a difference between reproductive and social dominance is found in two primitively eusocial species of the polistinae: ropalidia marginata and p. japonicas (chandrashekara & gadagkar, 1991; sumana & gadagkar, 2003; ishikawa et al., 2011). the queens of these two species are peaceful and not top-ranked in the social dominance hierarchy, but they do monopolize ovipositing. these species are considered to maintain the queen’s status by signaling her fertility, that is, by using an honest signal, as does the queen of advanced eusocial groups (keller & nonacs, 1993; peeters & liebig, 2009). the r. marginata queen uses a pheromone to maintain her queen status (mitra, 2014), which is considered an honest signal of her fertility. it is likely that the p. japonicas queen also uses a pheromone to maintain her status in the colony, since only the queen performs abdominal wagging (ishikawa et al., 2011), as does the r. marginata queen while releasing a pheromone. it is important to realize that the social dominance hierarchy of paper wasps has mostly been investigated for a foundress association established before the emergence of workers or for workers after the disappearance of the queen. to the best of our knowledge, among the species with colonies composed of one queen and her daughters (henceforth called queen-daughter colonies), the dominance ranks of individual colony members (including the foundress) have been revealed only in the following four species: polistes chinensis antennalis (morimoto, 1961), mischocyttarus cassununga (murakami & shima, 2010), and the above two species, r. marginata and p. japonicus. colonies of r. marginata may include several foundresses because a colony was founded by one or several foundresses (shakarad & gadagkar, 1995); observations are usually made of nests for which the number of foundresses is unknown. the foundress queen of p. chinensis and m. cassununga is ranked top in the social dominance hierarchy. the relatedness between the current and future colony members is higher in a queen-daughter colony than in a foundress association or an orphan colony if the foundress queen is mated once such monandry is common in paper wasps (strassmann, 2001). moreover, the queen in queen-daughter colonies is mated, while her daughters are unmated (at least at the early stage) and cannot produce females. as a result, as long as the queen is sufficiently fertile, unmated daughters benefit from working for the colony (bourke & franks, 1995). these characteristics of the queen-daughter colonies make the conflict between the queen and daughters more likely to be resolved using an honest signal of fertility rather than physical aggression. it would be interesting to determine how frequently a peaceful queen is found in queen-daughter colonies and what determines the peacefulness or aggressiveness of the queen in these colonies. these questions could be answered by characterizing the dominance hierarchies in queen-daughter colonies of many species. such information would also provide a deeper understanding of the evolution of the mechanisms underlying the establishment and maintenance of the queen’s status in the polistinae (jandt et al., 2014). we, therefore, investigated the mechanisms used to maintain the queen’s status in queen-daughter colonies of p. (megapolistes) jokahamae radoszkowski, 1887 (nee p. jadwigae) (vespidae: polistinae). polistes jokahamae queens usually found a nest solitarily and independently of conspecifics (kasuya, 1981). first, we determined whether the p. jokahamae queen maintains her status using aggression by addressing the following three questions: (1) whether the queen is the most aggressive and top-ranked individual in the social dominance hierarchy determined by aggression; (2) whether the queen directs aggression primarily toward the top-ranked worker in the social dominance hierarchy (the top-ranked worker becomes the primary egg-layer after the disappearance of the queen [miyano, 1991]); and (3) whether the queen is the exclusive (or primary) egg layer. we also wanted to identify any interactions between the emergence order and dominance rank in the dominance hierarchy since older workers are usually more dominant in temperate paper wasps (tsuji & tsuji, 2005). our present results showed that the queen was peaceful but still the exclusive or the primary egg layer (see "results"). we then explored the possibility that a behavior functions as an honest signal of the queen’s fertility. materials and methods biology of p. jokahamae polistes jokahamae is an independent, solitary founding paper wasp (kasuya, 1981) and is common on the islands of japan except for hokkaido. overwintered queens emerge from diapause in late march and begin to found a nest in mid-april to early may (in mie, japan). workers emerge in late may to july, followed by the emergence of males and gynes (potential queens of the next generation) (yoshimura & yamada, 2018). h yoshimura, j yamada, yy yamada – non-aggressive paper-wasp queen168 colonies and videoing four colonies of p. jokahamae were observed over five years on the mie university campus in tsu, mie, japan: 2010 (colony 3), 2011 (colony 5), 2013 (colony 6), and 2014 (colony 9) (table 1): observed colonies were numbered each year according to the order of discovery of them, but many colonies were lost before the appearance of reproductives. the observations began around the emergence of the first worker (table 1). we could easily distinguish between the queen and early-emerging workers from the differences in body size (yoshimura & yamada, 2018), the level of wing damage, and the size of the yellow patches on the abdomen; the queen has larger yellow patches than early-emerging workers (h. yoshimura & y.y. yamada, unpublished). colonies 3 and 9 were reared in a tent (pyramidal with a curved ceiling, 2.0 m × 1.5 m × 1.3 m) with mesh sheets on two sides facing each other. the tent windows were closed in colony 3, and sufficient wax moth larvae, honey, water, and dried trees for nest materials were provided for the wasps to collect ad libitum. the tent windows for colony 9 were opened to enable the wasps to forage outside the tent. colonies 5 and 6 were founded naturally on a lintel in greenhouses whose windows were kept open. honey and water were placed near the nest for these three colonies. the wasps were marked by attaching small pieces of differently colored photographic paper, which were labeled with numbers, to the mesonotum using instant glue (aron alpha® jerry type, toagosei, osaka, japan). the label paper was kept as light as possible by removing its chartaceous backing. the behavior of individual wasps on the nest was recorded by a digital video camera (hdc-tm 35 or v700m, panasonic, osaka, japan) every day from 6 a.m. to 6 p.m. for a period from the day of first worker emergence or colony discovery (table 1) until colony disappearance or august 20. video sessions were canceled when there was a strong wind or heavy rain. video analysis was performed for the worker phase (reeve, 1991) when the colony comprises the queen and workers. we analyzed several periods during which the colony comprised almost the same colony members (table 1). each analysis period typically lasted four to seven days, during which we analyzed the behavior of individual wasps for two to five days (usually every other day) (table 1). the behavior was analyzed for six consecutive hours (from 11 a.m. to 5 p.m.) on the days selected, although the video time did not reach this stablished time on some days due to bad weather or difficulties encountered while videoing (table 1). analysis of behavior and determination of rank in the dominance hierarchy aggression, ovipositing, and foraging behaviors were recorded by watching the videos, and their hourly frequencies were calculated. the aggressive behaviors included biting, rushing, and chasing: biting part (usually the head) of the opponent with the mandibles; rushing at the opponent, which sometimes resulted in the head touching part of the opponent; and chasing the opponent, which often occurred when the opponent was trying to escape the rushing. rushing and chasing were classified in the same category because these two behaviors were often confused. mounting a foreign wasp was sometimes observed, but not against other nestmates. individuals who exhibited or received each aggressive behavior were identified and recorded. when ovipositing, the female wasp first inserts her abdomen into a cell and then lays an egg on the cell wall. we observed that female wasps often inserted their abdomens into cells without laying an egg. we observed 172 cases in which it was possible to determine whether or not a focal wasp laid an egg after inserting her abdomen into the cell. the female laid an egg in 38 of the 41 cases in which colony date of first worker emergence or colony discovery date of first male emergence date of queen disappearance analysis period total analysis time (h) observation dates number of workers on each observation day 3 june 22 (2)a august 4 august 3 1 17.0 july 4, 6, 7, 9, 16 5 2 30.0 july 18, 20, 22, 24, 25 6 or 7 3 29.0 july 26, 27, 28, 29, 30 7 or 8 5 july 3 (4)a -b august 16 1 12.0 july 17, 18 4 2 11.0 july 28, 30 7 or 8 3 12.0 august 12, 14 12 6 june 24 (4)a august 11 july 21 1 7.7 july 3, 5 3 2 15.1 july 7, 9, 11 3 3 5.2 july 15, 17 3 4 8.5 july 18, 19, 20 3 9 june 11 october 9 july 18 1 24.0 june 16, 17, 19, 21 8 or 10 2 24.0 june 23, 25, 27, 29 12 3 20.0 july 1, 4, 5, 6 12 4 17.5 july 15, 16, 17 9 a some workers had already emerged when the colony was discovered. numbers in parentheses indicate the numbers of workers when colonies 3, 5, and 6 were discovered. b the colony collapsed due to an ant attack on august 16, just before the reproductives were assumed to emerge. table 1. information on colony development and analysis periods. sociobiology 66(1): 166-178 (march, 2019) 169 she was observed to keep her abdomen in the cell for ≥120 s, while no egg was laid in the 131 cases in which her abdomen remained in the cell for <120 s. thus, when we could not verify whether or not an egg was actually laid after the abdomen was inserted into a cell, we assumed that oviposition occurred only when the female kept her abdomen in a cell for ≥120s. lateral vibrations, rushing with flapping, and abdominal rubbing were recorded as candidate behaviors for a fertility signal, and the relationship between their frequencies and the frequency of ovipositing was analyzed. lateral vibrations refer to rapidly vibrating the abdomen laterally for a period of approximately 1 s. rushing with flapping refers to a focal wasp rushing at several wasps one after another on the nest while flapping (m = 22.8, sd = 39.9 s, max = 362 s, min = 9 s, n = 549). abdominal rubbing refers to rubbing the abdominal tip onto one or several cells one after another (m = 3.8 s, sd = 2.7 s, max = 33 s, min = 1 s, n = 249). the hourly frequency of each kind of behavior was obtained by dividing the total frequency observed during the daily analysis period (usually six hours) by the time during which the focal wasp stayed on the nest, that is, the daily analysis period minus the total foraging time, with each foraging time corresponding to the time span from leaving the nest to returning to it. the dominance rank was determined using two methods: (1) calculating the dominance index (di; premnath et al., 1990) and (2) calculating standings, corresponding to the recorded wins and losses in agonistic contests (i.e., pairwise dominancesubordinate interactions) for all possible pairs of wasps on the nest (ishikawa et al., 2010). these two methods were used because they might show different dominance ranks; however, as reported in the results section, this difference was quite small. episodes of all kinds of aggression were counted without weightings. when using standings, we assumed that for a pair of wasps, the wasp displaying aggression over the other with a greater frequency was dominant over the other. a wasp was then ranked according to the number of subordinate individuals for that wasp minus the number of dominant individuals over it. statistical analysis the hourly frequencies of each of five kinds of behavior (aggression, ovipositing, rushing with flapping, lateral vibrations, and abdominal rubbing) performed by the queen in a day were compared with those performed by the worker with the highest frequency of the corresponding behavior in individual colonies. a stratified wilcoxon signedrank test (an exact test for paired samples) was used, with the colony incorporated in a stratum variable (cytel, 2012) because the data did not conform to a normal distribution. to analyze the possible effect of time, the observation days were dichotomized into the early and late stages of the worker phase, which were defined as periods before and after the time point of two weeks before disappearance of the queen. the observed frequencies of a focal behavior were then compared between the early and late stages using the exact permutation test for two independent samples with strata (cytel, 2012). moreover, we used a binomial test in each analysis period to determine whether the queen directed aggression more intensively toward the highest-ranked worker or the worker with the highest frequency of aggression (these were often actually the same individual). in other words, we compared the observed frequency with the frequencies obtained if she randomly directed aggression toward other colony members. the relationship between the dominance rank and emergence order was also analyzed for each colony using the generalized cochran-mantel-haenszel test with the analysis period incorporated as a stratum variable (cytel, 2012), with the p values calculated using the exact method. this analysis revealed whether a relationship was present throughout all the analysis periods for each colony. some workers emerged on the same day and some workers had already emerged on the nest-discovery day (table 1); in such cases, they were assigned the same number (the mean number of a range of emergence orders for them). the above tests were implemented using statxact 10 software (cytel, cambridge, ma, usa), except the binomial test, which was conducted using ncss 11 (ncss statistical software, kaysville, ut, usa). statistical significance was assessed using the sequential bonferroni multiple-comparison method (holm, 1979) where necessary. when the relationship between the dominance rank and emergence order was analyzed, the relative ranks or orders among all colony members present on the nest were used to control for differences in the total number of colony members on the nest. relative ranks or orders were calculated using the expression (rank or order of a given worker 1)/(number of workers 1), which produced values of 0 and 1 for the firstand last-ranked or emerging wasps, respectively. we also examined the relationships between the hourly frequencies of ovipositing and those of lateral vibrations, abdominal rubbing, and rushing with flapping. this analysis was applied to (1) the queen of each colony and (2) all colony members; the latter procedures were not applied to lateral vibrations because only the queen performed them. the analysis was performed using a linear mixed model. the frequency of the focal behavior was incorporated into the model as a fixed factor, and the colonies and individuals (nesting in the colonies) were incorporated as random factors. regarding random factors, random intercepts or both random slopes against the frequency of the focal behavior and random intercepts were considered. the decision of which to choose was based on the aic values; consequently, the former was chosen except when analyzing rushing with flapping for the queen. the linear mixed model analysis was performed using the lme4 package in r version 3.4.1 (r foundation, 2017). unless we detected a significant relationship between the frequencies of oviposition and focal behavior, we explored whether performers of the focal behavior were more likely to oviposit than nonperformers. we compared the proportion (r) h yoshimura, j yamada, yy yamada – non-aggressive paper-wasp queen170 of ovipositing days among the days on which a focal wasp performed the focal behavior with the proportion (rʹ) of ovipositing days among the days on which the focal wasp did not. this analysis was performed both for the queens and for all colony members. unfortunately, this analysis did not detect a significant difference in any cases (not presented in the results); i.e., if a female performs the focal behavior on a given day, it does not necessarily indicate that she is likely to oviposit on that day. then, we compared the proportion (s) of those females who oviposited at least once among those females who performed the focal behavior at least once during four designated days with the proportion (sʹ) of the ovipositing females among those females who never performed the focal behavior during the four days. this analysis was applied to the last four observing days for adult females who stayed on the nest for at least four observing days. if a difference is detected, it indicates that focal behavior performers are more likely to oviposit than nonperformers, although they do not necessarily oviposit on the days when they perform it. the significance of the difference for the above two analyses was assessed by calculating exact probability values (cytel, 2012) after verifying the homogeneity of odds ratios r/(1 r): rʹ/(1 rʹ) or s/(1 s): sʹ/(1 sʹ) among the individuals or colonies; i.e., after verifying that different individuals or individuals of different colonies are similarly likely to oviposit colony total number analysis period frequency of oviposition cells reproductivesa queen workersb 3 42 5, 0 1 7 2 (1–1, 4–1) 2 14 8 (1–5, 4–3) 3 10 17 (1–5, 4–11, 6–1) 5 36 – 1 0 0 2 8 0 3 6 0 6 38 5, 4 1 2 0 2 2 1 (3–1) 3 0 0 4 0 0 9 48 1, 0 1 7 0 2 11 9 (2–5, 3–1, 6–1, 7–2) 3 14 16 (2–9, 7–3, 9–4) 4 35 3 (2–1, 9–1, 10–1) a left, males; right, females. colony 5 collapsed just before the reproductives were assumed to emerge. b numbers in parentheses are the number of eggs for each ovipositing worker. the numbers before and after each en dash indicate the emergence order of the workers and the frequency of oviposition, respectively. table 2. colony size and eggs laid. fig 1. comparison of the frequencies of five kinds of behavior between the queen (open) and the worker (gray) that performed the focal behavior most frequently. comparisons were made separately for the early and late stages of the worker phase. the p values were obtained using the stratified wilcoxon signed-rank test, and those for comparisons between the early and late stages for each behavior performed by the queen (q) and the worker (w) were as follows: aggression, 0.057 (q) and 0.063 (w); ovipositing, 0.114 (q) and 0.004(w); rushing with flapping, 0.170 (q) and 0.504 (w); lateral vibration, 0.418 (q); and abdominal rubbing, <0.001 (q) and 0.972 (w) (exact permutation test). when they perform the focal behavior. the individuals or colonies were incorporated as a stratum factor. the satxactⓡ software was used for the calculation. results colony characteristics the total numbers of cells constructed and workers emerging in individual colonies ranged from 36 to 48 and 11 to 22, respectively (table 2). one or several reproductives (males and gynes, i.e., females emerging after the emergence of the first males) emerged in each colony except colony 5, which produced no reproductives because the colony collapsed due to being attacked by ants just before the reproductives were assumed to emerge. in colony 3, the queen disappeared after the male emergence, while the queens of colonies 5, 6, and 9 disappeared before this (table 1); the queen of colony 5 disappeared when the colony collapsed. ovipositing individual queens performed 0-35 ovipositions during each analysis period (table 2). meanwhile, one to several workers began to oviposit in the individual colonies as the colony developed, except colony 5. individual workers performed up to 11 ovipositions during each analysis period, but their number was smaller than the ovipositions by the queen with the exception of analysis period 3 in colony 3. the hourly frequency of ovipositing was significantly higher for the queen than for the workers ovipositing most frequently among the workers early in the colony’s development, but not later (fig 1). workers that oviposited usually had a high ranking, but ovipositing was performed most frequently by 0,0 0,7 1,4 1 2 3 4 5 6 7 8 9 p = 0.013 p = 0.096 oviposition 0,0 9,0 18,0 1 2 3 4 5 6 7 8 9 p = 0.719 p = 0.002 rushing with flapping 0,0 3,0 6,0 1 2 3 4 5 6 7 8 9 p < 0.001 p = 0.004 lateral vibration 0,0 1,5 3,0 3 5 6 9 3 5 6 9 colony p = 0.001 p = 0.755 abdominal rubbing 0,0 3,0 6,0 1 2 3 4 5 6 7 8 9 p < 0.001 early p < 0.001 late aggression h ou rly fr eq ue nc y 6.0 3.0 0.0 1.4 0.7 0.0 18.0 9.0 0.0 6.0 3.0 0.0 3.0 1.5 0.0 sociobiology 66(1): 166-178 (march, 2019) 171 the secondor third-ranked worker rather than the top-ranked one (appendix s1). dominance hierarchy the queens were rarely ranked top in the dominance hierarchy during the individual analysis periods in the four colonies irrespective of whe ther the dominance rank was based on the di or standings for agonistic contests (fig 2). older workers were the more dominant workers during most of the analysis periods for the two kinds of dominance ranks based on the di and standings (generalized cochran-mantel-haenszel test: colonies 3, 5, and 9, p < 0.001 [di], p < 0.001[standings]; colony 6, p = 0.056 [di], p = 0.037 [standings]). however, the oldest worker often failed to achieve the highest rank. the queens were never the most frequent aggressors among the colony members during any analysis period (appendix s2); the hourly aggression frequency of the queen was significantly lower than that of the most aggressive worker (fig 1). higher-ranked workers often but not always performed aggressive behaviors more frequently (appendix s2). however, the most dominant worker often did not exhibit the highest frequency of aggression in any colony. the queen rarely displayed intensive aggression toward the top-ranked worker during any analysis period in the four colonies (fig 3). the frequency of aggression did not differ significantly from the assumption that she was randomly aggressive toward all workers (p > 0.05, binomial test; specific statistical results not presented), irrespective of whether the dominance rank was based on the di or standings. one exception was period 4 in colony 9 for the dominance rank based on standings (p < 0.001), during which the queen directed all aggressive behaviors toward the top-ranked worker; this individual was not top-ranked using the di. the queen also did not display intensive aggression toward the worker exhibiting the highest frequency of aggressive behaviors (p > 0.05, binomial test; specific statistical results not presented). a particularly interesting finding was that the queen often received aggression from many workers irrespective of their dominance ranks (fig 3). fig 2. relationship between the dominance rank and the emergence order. the dominance rank was based on the dominance index (di, solid) and standings (open). the dominance rank and emergence order are expressed according to the relative rank or order: the first and last ones are indicated by 0 and 1, respectively. the queen was designated by a relative emergence order of 0. relative emergence order r el at iv e do m in an ce r an k 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0,0 0,5 1,0 0,0 0,5 1,0 0,0 0,5 1,0 0,0 0,5 1,0 0,0 0,5 1,0 r el at iv e do m in an ce ra nk 0,0 0,5 1,0 0,0 0,5 1,0 0,0 0,5 1,0 relative emergence order 3–3 5–3 6–3 9–3 3–2 5–2 6–2 6–4 9–2 9–4 colony 3–1 colony 5–1 colony 6–1 colony 9–1 0.0 0.5 1.0 h yoshimura, j yamada, yy yamada – non-aggressive paper-wasp queen172 lateral vibrations, abdominal rubbing, and rushing with flapping lateral vibrations were performed only by the queen, never by workers (fig 1). the hourly frequency of ovipositions performed by the queen was significantly related to that of the lateral vibrations (χ2 = 6.6, df = 1, p = 0.010; fig 4). some high-ranked workers, as well as the queen, performed abdominal rubbing (appendix s1). the queen exhibited this behavior most frequently among the colony members early but not late in the colony’s development (fig 1). the frequency of the queen performing abdominal rubbing did not reflect her frequency of ovipositing (χ2 = 0.4, df = 1, p = 0.554; figures not presented). the same was true for the frequency of all nest members (χ2 = 0.0, df =1, p = 0.851; figures not presented). however, females who performed abdominal rubbing at least once during the four days were more likely to oviposit than nonperformers during that period (table 3). the frequency of rushing with flapping differed markedly between the colonies (fig 1, appendix s2), with no and very few events seen in colonies 9 and 3, respectively. fig 3. hourly frequencies (means and se values) of the aggressive behaviors that the queen exhibited toward (solid) and received from (open) individual workers, who are ordered according to their dominance ranks based on the di. the missing dominance rank for each analysis period indicates the queen’s rank. colony abdominal rubbing performers nonperformers 3 3/3 3/4 5 1/1 1/7 6 0/2 0/2 9 2/2 2/11 p valuesa 0.011 (1.0) table 3. comparison of proportions of ovipositing females among ones performing abdominal rubbing and those among nonperformers. rushing with flapping was mainly performed by dominant workers and the queen (appendix s2). however, the topranked worker did not always do this with the highest frequency among the workers, and many workers (including low-ranked ones) performed rushing with flapping during period 3 in colony 5. the hourly frequency for the worker performing rushing with flapping most often among the workers was h ou rly fr eq ue nc y of a gg re ss io n dominance rank 0,0 0,5 1 2 3 4 6 colony 3–1 0,0 0,5 1 3 4,5 4,5 6 7 8 3–2 0,0 0,2 2 3 4 5 6 7 8,5 8,5 3–3 0,0 0,5 1 3 4 5 colony 5–1 0,0 1,0 1 3 4 5 6 7,5 7,5 9 5–2 0,0 0,2 2 3 4 5,55,5 7 8 9 10 11 12 13 5–3 0,0 2,0 1 3 4 colony 6–1 0,0 1,0 1 2 4 6–2 0,0 5,0 1 2,5 2,5 6–3 0,0 1,0 1 2,5 2,5 4 5 6 8 9,5 9,5 11 colony 9–1 0,0 0,2 1 2 3 4 6 6 6 8 9 10 11 13 9–2 0,0 1,0 1 2 4 4 6 7 8 9 1 1 1 1 9–3 0,0 1,0 1,5 1,5 3 5 6–4 0,0 0,2 1 3 4 5 6 7 8 9 10 9–4 2 4 5.5 8 10 12 3 5.5 7 9 11 13 1 2 4.5 4.5 6 7 8 9 10 11 12.5 12.5 0.5 0.0 1.0 0.0 0.2 0.0 1.0 0.0 0.2 0.0 1.0 0.0 0.2 0.0 0.5 0.0 0.5 0.0 0.2 0.0 2.0 0.0 1.0 0.0 5.0 0.0 1.0 0.0 a exact test for comparison between the odds for performers and nonperformers; figure in parentheses indicates the p value for the exact test for homogeneity of odds ratios among different colonies. 4.5 4.5 8.5 8.5 7.5 7.5 2.5 2.5 1.5 1.5 2.5 2.5 9.5 9.5 sociobiology 66(1): 166-178 (march, 2019) 173 similar to that for the queen early in the colony’s development, but later, the former was higher than the latter (fig 1). none of the analyses detected a relationship between oviposition and rushing with flapping. it is unlikely that colony members can estimate who is likely to oviposit or how many eggs the ovipositor islikely to lay based on rushing with flapping. lateral vibrations are exhibited by many polistes paper wasps, including p. dominulus (brillet et al., 1999), polistes fuscatus (savoyard et al., 1998), and polistes instabilis (molina & o’donnell, 2009). the queen usually performs lateral vibrations with the highest frequency in the colonies of these species and is the only individual to perform them in the p. jokahamae colony. lateral vibrations have been assumed to have two main functions: (1) as a signal for communicating between larvae and adults; and (2) as a form of aggression in the dominance hierarchy (jeanne, 2009) since lateral vibrations are usually performed while feeding larvae or encountering other colony members. the p. jokahamae foundresses did not perform lateral vibrations under these situations, so lateral vibrations performed by the p. jokahamae foundresses do not appear to have such a function. moreover, the lateral vibration signal is mechanical, but it might also be chemical if odor release accompanies the vibrations. experiments are required to further clarify the characteristics and function of the lateral vibrations. performers of abdominal rubbing were more likely to oviposit than nonperformers, although the daily frequency of ovipositing was not related to that of abdominal rubbing. one plausible explanation is that the frequency of abdominal rubbing is related to the level of egg maturation. workers are thought not to be allowed to lay eggs easily in the presence of the queen, and even the queen does not oviposit unless cells suitable for oviposition, that is, empty cells or cells containing pupae or last-instar larvae, are available. as a result, the frequency of abdominal rubbing might not be related to that of ovipositing. abdominal rubbing appears to correspond to the abdominal wagging observed in p. japonicus (ishikawa et al., 2011) and the abdominal rubbing of the ventral surface of the abdominal tip on the nest observed in r. marginata (originally referred to as “rub abdomen” in r. marginata; bhadra et al., 2007, 2010; mitra & gadagkar, 2011). these behaviors appear to accompany the release and/or smearing of a chemical or queen pheromone on the cells of the nest, since it has been recently discovered that r. marginata queens maintain their queen status with the queen pheromone that is released during abdominal rubbing (see mitra, 2014 for a review). however, it should be noted that the similar behavior of abdominal stroking found in some other paper wasps has been suggested to have a different function (e.g., cervo & lorenzi, 1996; van hooser et al., 2002; lorenzi et al., 2011) the performer demonstrating her own status (e.g., queen or high rank in the dominance hierarchy) to the immatures. future further experiments are needed to clarify the function of the abdominal rubbing observed in p. jokahamae. lateral vibration is considered to be an honest signal of the queen’s fertility. in general, to keep a signal honest, displaying a signal should incur some cost (bradbury & vehrencamp, 2011). lateral vibration may be physically demanding. alternatively, the cost of lateral vibration may be ignorable, but workers may check the queen’s fertility by fig 4. relationship between hourly frequencies of ovipositing and lateral vibrations performed by the queen during one day. circles, squares, diamonds, and triangles indicate colonies 3, 5, 6, and 9, respectively. note that the queen of colony 6 performed neither ovipositing nor lateral vibration during one day on six occasions. discussion the p. jokahamae queen was not aggressive or ranked top in the dominance hierarchy determined by aggression, but she oviposited either monopolistically or dominantly. moreover, the queen did not direct aggression more frequently toward the most aggressive or top-ranked worker (a potential successor) in the social dominance hierarchy compared with the frequency obtained if she randomly directed aggression toward the other workers. these observations strongly suggest that the queen maintains her queen status without using aggression. instead, the queen is considered to maintain her status by demonstrating her fertility or reproductive potential via some signal. the most likely candidate signal is lateral vibration because the frequency of lateral vibrationis positively related to that of ovipositing. in addition, abdominal rubbing also appears to function as a signal. however, this signal only predicted who is likely to oviposit but not how many eggs the ovipositor is likely to lay. rushing with flapping is not considered to be involved in maintaining the queen’s status. miyano (1991) observed that the queen was not aggressive in a queen-daughter colony of p. jokahamae. however, the observation period in that study was only four hours, during which time the queen did not oviposit, and the workers originated from colonies different from the queen’s. therefore, our present study have disclosed for the first time that the p. jokahamae queen is not aggressive but she oviposits either monopolistically or dominantly. h ou rl y fr eq ue nc y of o vi po si tio ns hourly frequency of lateral vibrations 1.5 1.0 0.5 0.0 0.0 0.5 1.0 1.5 h yoshimura, j yamada, yy yamada – non-aggressive paper-wasp queen174 directly examining the numbers of eggs laid by the queen, as do p. dominulus workers (liebig et al., 2005), and/or by indirectly checking the profile of chemicals that may be released while performing abdominal rubbing or that of cuticular hydrocarbons (note that the queen often received aggression). in other words, workers do not absolutely rely on the signal, and then, they check the queen’s fertility in alternative ways. if workers find the signal dishonest, workers may cease being workers and start to prepare for ovipositing. whether workers check the queen’s fertility directly as well as receiving the signal related to her fertility is an interesting topic for future studies (see tibbetts & izzo, 2010). some dominant workers started ovipositing late in the colony’s development, but the queen did not hinder these ovipositing behaviors by using aggression. however, some eggs laid by workers were eaten by the queen (queen policing) and workers (worker policing), and some eggs laid by the queen were also eaten by workers. such behaviors have been analyzed in some paper wasps (saigo & tsuchida, 2004; liebig et al., 2005; dapporto et al., 2010a), but unfortunately, in the present study, it was not possible to identify all such events by observing the video or to perform a quantitative analysis. our research team has disclosed that the queen in queen-daughter colonies of two polistes species (p. japonicus and p. jokahamae) is peaceful and does not use a social dominance hierarchy to maintain her queen status. a particularly interesting finding is that polistes snelleni queens are also peaceful (yamasaki & tsuchida, 2014), which suggests that queen’s signals control the colony, although the dominance hierarchy needs to be investigated in detail. it is of great importance to determine whether aggression is involved in maintaining the queen’s status in queen-daughter colonies of other paper wasp species and to determine which signal is involved if aggression is not involved. acknowledgments we thank two anonymous referees for kind and helpful comments. authors’ contributions yy yamada conceived and designed the experiment. j yamada and h yoshimura carried out the fieldwork and assembled the data. yy yamada and h yoshimura performed the data analyses and wrote the manuscript. references bhadra, a., iyer, p.l., sumana, a., deshpande, s.a., ghosh, s. & gadagkar, r. 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(2018). the first brood emerges smaller, lighter, and with lower lipid stores in the paper wasp polistes jokahamae (hymenoptera: vespidae). insectes sociaux, 65: 473-481. doi: 10.1007/s00040-018-0636-5 sociobiology 66(1): 166-178 (march, 2019) 177 0,0 1,5 3,0 q 1 2 3 4 5 a colony 3–1 0,0 0,3 0,6 q 1 2 3.5 3.5 5 6 3–2 0,0 0,5 1,0 q 1 2 3 4 5 6 3–3 0,0 0,4 0,8 q 1 2 3 a a a colony 6–1 0,0 0,1 0,2 q 1 2 3 a a a 6–2 0,0 0,5 1,0 q 1 2.5 2.5 a a a 6–3 0,0 0,2 0,4 q 1.5 1.5 3 4 a a 6–4 0,0 0,4 0,8 q 1 2 3 4 a a colony 5–1 0,0 1,5 3,0 q 1 2 3 4 5 6.5 6.5 5–2 0,0 0,5 1,0 q 1 2 3 4.5 4.5 6 5–3 0,0 3,0 q 1 2.5 2.5 4 5 6 colony 9–1 0,0 1,5 3,0 q 1 2 3 4 6 6 6 9–2 0,0 2,0 4,0 q 1 2 3.5 3.5 5 6 9–3 0,0 1,5 3,0 q 1 2 3 4 5 6 9–4 h ou rly fr eq ue nc y of e ac h be ha vi or dominance rank appendix fig s1. hourly frequencies (means and se values) of ovipositing (solid), lateral vibrations (open), and abdominal rubbing (checked) performed by the queen (q) and the firstto sixth-ranked workers. dominance ranks were based on the dominance index (di). no workers performed lateral vibrations. the number following the colony number indicates the analysis period. the letter “a” on the x-axis indicates no corresponding worker. seventhand lower-ranked workers occasionally performed ovipositing and abdominal rubbing: ovipositing, 0.06 ± 0.06 (mean ± se) for the eighth-ranked worker during period 4 in colony 9; abdominal rubbing, 0.17 ± 0.00 for the eighth-ranked worker during period 3 in colony 5, and 0.08 ± 0.00 for the eighth-ranked worker during period 1 in colony 9. 3.0 1.5 0.0 0.6 0.3 0.0 1.0 0.5 0.0 0.8 0.4 0.0 0.2 0.1 0.0 1.0 0.5 0.0 0.4 0.2 0.0 0.8 0.4 0.0 3.0 1.5 0.0 1.0 0.5 0.0 3.0 0.0 3.0 1.5 0.0 4.0 2.0 0.0 3.0 1.5 0.0 h yoshimura, j yamada, yy yamada – non-aggressive paper-wasp queen178 fig s2. hourly frequencies (means and se values) of aggressive behaviors (solid) and rushing with flapping (open) performed by the queen (q) and workers, who are ordered according to their dominance ranks based on the di. appendix 0,0 3,0 6,0 q 1 2.5 2.5 4 5 6 7 8.5 8.5 10 a a 0,0 1,0 2,0 q 1 2 3 4 a a a a a a a a 0,0 2,0 4,0 q 1 2 3 4 5 6.5 6.5 8 a a a a 0,0 2,0 4,0 q 1 2 3 4.5 4.5 6 7 8 9 10 11 12 0,0 1,0 2,0 q 1 2 3 4 5 a a a 0,0 1,0 2,0 q 1 2 3.5 3.5 5 6 7 a 0,0 1,0 2,0 q 1 2 3 4 5 6 7.5 7.5 0,0 5,0 10,0 q 1 2 3 a 0,0 3,0 6,0 q 1 2 3 a 0,0 7,0 14,0 21,0 q 1 2 2 a 0,0 6,0 12,0 q 1.5 1.5 3 4 0,0 5,0 10,0 q q q q q q q q q q q q q 0,0 2,0 4,0 q 1 2 3 4 5 6 7 8 9 a a a 0,0 5,0 10,0 q 1 2 3 4 6 6 6 8 9 10 11 12 colony 6–1 6–2 6–3 6–4 colony 9–1 9–2 9–3 9–4 colony 3–1 3–2 3–3 colony 5–1 5–2 5–3 h ou rly fr eq ue nc y of e ac h be ha vi or dominance rank q 1 2 3.5 3.5 5 6 7 8 9 10 11.5 11.5 2.0 1.0 0.0 2.0 1.0 0.0 2.0 1.0 0.0 10.0 5.0 0.0 6.0 3.0 0.0 21.0 14.0 7.0 0.0 12.0 6.0 0.0 2.0 1.0 0.0 4.0 2.0 0.0 4.0 2.0 0.0 6.0 3.0 0.0 10.0 5.0 0.0 10.0 5.0 0.0 4.0 2.0 0.0 q dominance rank doi: 10.13102/sociobiology.v65i4.3475sociobiology 65(4): 654-661 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 leucine-aminopeptidase a (lap-a) encoding gene in apoidea: from genomic identification to functional insights based on gene expression introduction aminopeptidases (α-aminoacyl-peptide hydrolase, ec 3.4.11) are ion-dependent exopeptidases that catalyze the hydrolytic cleavage of amino acid residues on the n-terminal region of (poly) peptides. there are more than twenty classes of aminopeptidases, well conserved in structure, from bacteria to humans (sanderink et al., 1988; matsui et al., 2006; nandan & nampoothiri, 2017). these classes include: leucine(or leucyl-) aminopeptidase, membranal alanyl aminopeptidase, aminopeptidase a, aminopeptidase b, aminopeptidase n, aminopeptidase y, cistinyl aminopeptidase, tripeptidyl aminopeptidase, prolyl aminopeptidase, glutamyl aminopeptidase and aspartyl aminopeptidase. this classification is based on the type of substrate that each aminopeptidase hydrolyzes. for instance, abstract aminopeptidases are enzymes that cleave the n-terminal region of proteins and show structural conservation in prokaryotes and eukaryotes. we aimed to identify leucine-aminopeptidase a (lap-a) orthologs in the genome of bee species with different levels of social organization, and to explore the putative roles of this enzyme based on gene expression data. we identified a single gene for lap-a on chromosome 15 of apis mellifera l. and predicted orthologs in genomes of 11 bee species. we found evidence of lap-a expression in more than 50 bee species. in honeybee and other bees, lap-a transcripts were expressed in diverse tissues, including: brains, fat bodies, ovaries, testicles, integuments, and glands, on different developmental stages that spanned from embryogenesis to adult life. our findings on the transcriptional activity of lap-a are consistent with previously published data on enzymatic activity of lap-a in bees throughout the development in different tissues and in both sexes. the presence of lap-a gene in the apoidea genomes and its ubiquitous expression support housekeeping roles of this enzyme and broad-spectrum functions in bees, independent of their life styles. sociobiology an international journal on social insects l bataglia1,2, ic godoy2, ma del lama2, fmf nunes1,2 article history edited by cândida aguiar, uefs, brazil received 10 may 2018 initial acceptance 19 july 2018 final acceptance 22 august 2018 publication date 11 october 2018 keywords lap-a, bee, gene expression, aminopeptidase, pcr. corresponding author marco antonio del lama francis morais franco nunes departamento de genética e evolução universidade federal de são carlos rodovia washington luis, km 235 cep 13565-905 são carlos-sp, brasil. e-mail: madl dmdl@ufscar.br fmfn francis.nunes@ufscar.br the aminopeptidase n hydrolyzes alanine and leucine residues, while the aminopeptidase a hydrolyzes aspartate and glutamate (ferreira & terra, 1984; terra & ferreira, 1994). although aminopeptidases are conserved and well described as digestive enzymes, they can perform specific physiological roles in the processing and degradation of peptides depending on the organism and tissue (mcculloch et al., 1994). in humans, for example, the perturbed activity of aminopeptidases is related with aging and pathologies (taylor, 1993), such as hypertension (zee et al., 2018) and cancers (schreiber et al., 2018). in insects, aminopeptidases are also associated with the recognition of cry proteins (gómez et al., 2018). cry proteins, produced by gram-positive and entomopathogenic bacteria bacillus thuringiensis, are cleaved in the midgut to form active toxins. these toxins bind to specific receptors in 1 programa de pós-graduação em genética, universidade de são paulo, ribeirão preto, são paulo, brazil 2 universidade federal de são carlos, são paulo, brazil research article bees mailto:dmdl@ufscar.br mailto:francis.nunes@ufscar.br sociobiology 65(4): 654-661 (october, 2018) special issue 655 the midgut, aminopeptidases being one of these receptors, producing pores that lead to cell lysis and the death of the insect (ferré & van rie, 2002). also, some strategies for controlling insect pests are based on the screening for natural inhibitors of aminopeptidases (valencia et al., 2014). aminopeptidases are more active than carboxypeptidases in the gut of insects (terra & ferreira, 1994). the aminopeptidases of insects have molecular mass between 90 e 130 kda and present optimum activity when physiological ph ranges from 7.2 to 9.0. they have a large specificity to their substrate and, most of the time, are n aminopeptidases. the aminopeptidases in orthoptera, hemiptera and coleoptera adephaga are found in a soluble form, while aminopeptidases in coleoptera polyphaga, diptera and lepidoptera associate with the microvillar membrane of intestine cells (terra & ferreira, 1994). in hymenoptera, most studies involving aminopeptidases focused on wasps and bees. the activity of a single aminopeptidase was observed in the gut extract of the bee scaptotrigona bipunctata (lepeletier) (schumaker et al., 1993). in the wasp polistes versicolor (olivier), five aminopeptidases were found during the development of males and females (del lama & ferreira, 2003). aminopeptidases were also identified in 14 species of bees (apis mellifera l., melipona quadrifasciata lepeletier, melipona quinquefasciata lepeletier, melipona marginata lepeletier, melipona bicolor bicolor lepeletier (earlier named melipona nigra lepeletier), melipona scutellaris latreille, melipona rufiventris lepeletier, scaptotrigona xanthotricha moure, scaptotrigona postica latreille, scaptotrigona bipunctata (lepeletier), nannotrigona testaceicornis (lepeletier), schwarziana quadripunctata (lepeletier), plebeia droryana (friese) and friesella schrottkyi (friese)). in all these bee species, one aminopeptidase (named as group 1) was always active, while the activity of other aminopeptidases varied according to species (named as group 2, restricted to pupae with pigmented eyes) (del lama & mestriner, 1984). in honeybees (a. mellifera), enzymatic activities of four leucine-aminopeptidase (lap) were reported, suggesting the existence of four different lap-coding genes (del lama et al., 2001). lap-a was invariably detected in all developmental stages (embryos, larvae, pupae and adults) and in different tissues of each gender (digestive tract, reproductive tract, cerebral ganglia, thorax, antenna and malpighian tubules) while lap-d, lap-g and lap-p showed more specific spatiotemporal activity patterns (del lama et al., 2001). leucine-aminopeptidases (lap, ec 3.4.11.1) are cytosolic proteases (liew et al., 2013), also known as peptidase s and cytosol aminopeptidase (http://enzyme.expasy.org/ ec/3.4.11.1), belonging to the (metalloexo) peptidase family m17 (ncbi: cdd cd00433 and domain architecture id 10087321) (matsui et al., 2006). comparing results from two published studies, we clearly observed that enzymatic activities of the lap-a (del lama et al., 2001) and the aminopeptidase of the group 1 (del lama & mestriner, 1984) presented constitutive bands and similar gel electrophoretic patterns based on the hydrolysis of the same substrate (derivatives of beta-naphthylamine). together, the information reinforces that observed uniform gel bands found in both studies represent the activity of the same enzyme, i.e., lap-a. thus, this evidence leads us to hypothesize that both mrnas and enzymes resulting from lap-a gene expression have housekeeping roles in cells and broad-spectrum functions in bees. in this study, we aimed (i) to identify cytosolic lap-a orthologs in the genome of bee species with different levels of social organization, (ii) to verify if the lap-a gene expression profiles fit well to the previously reported lap-a enzymatic activity and (iii) to suggest putative roles of this enzyme based on literature information and gene expression data (presence of mrna) in different tissues and developmental stages of honeybees (a. mellifera) and other bees. materials and methods in silico analysis searches for a cytosolic leucine-aminopeptidase (lap-a) and other aminopeptidases encoding genes of honeybees were performed in the ncbi-genbank (http:// www.ncbi.nlm.nih.gov) database, using the key words “aminopeptidase”, “cytosol” and “a. mellifera”. resulting a. mellifera lap-a nucleotide and protein sequences were recovered in fasta format. genomic identification (using a. mellifera genome data, version amel4.5) and manual annotation of honeybee lap-a gene structure (exon/intron) were performed using blast (altschul et al., 1990) and artemis platform (rutherford et al., 2000), and served as guide for the design of specific lap-a primers (fig s1) used for gene expression analysis. a. mellifera lap-a protein sequence was also used for new blastp rounds against both genbank (ncbi) and beebase (http://hymenopteragenome. org/beebase/) databases, to search for orthologs in the genomes of other bees: apidae (apis florea fabricius, apis dorsata fabricius, m. quadrifasciata, eufriesea mexicana (mocsáry), bombus terrestris l., bombus impatiens cresson, habropoda laboriosa fabricius), megachilidae (megachile rotundata (fabricius)) and halictidae (lasioglossum albipes (fabricius), dufourea novaeangliae (robertson)). as validation criteria for orthologs, we considered the occurrence of reciprocal best blast hit with honeybee lap-a protein sequence, identity threshold >75% (mazza et al., 2009), highly significant e-value <1e-30 (nunes et al., 2004), and also the presence of the functional domain m17 peptidase (cd00433) (analyzed by the ncbi cd-search tool, https://www.ncbi.nlm.nih. gov/structure/cdd/wrpsb.cgi). in addition, digital expression based on blastn searches against rna-seq data (sra and tsa transcriptomic collections publicly available at ncbi) from many bees was performed. http://enzyme.expasy.org/ec/3.4.11.1) http://enzyme.expasy.org/ec/3.4.11.1) http://hymenopteragenome.org/beebase/) http://hymenopteragenome.org/beebase/) https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi) https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi) l bataglia, ic godoy, ma del lama, fmf nunes – leucine-aminopeptidase a in bees656 genomic dna isolation genomic dna (gdna) was isolated by the phenolchloroform method (sheppard & mcpheron, 1991) from thorax of an individual forager worker from each bee species: a. mellifera, partamona helleri (friese), eulaema nigrita lepeletier, euglossa cordata (linnaeus) and bombus brasiliensis lepeletier. the dna was then suspended in 50 µl of te 1x buffer (tris 10 mm, edta 0.1 mm, ph 8.0). samples were quantified spectrophotometrically using a nanodrop® nd-1000 spectrophotometer (nanodrop technologies), and diluted to 100 ng/µl (using te 1x buffer accordingly). total rna isolation and cdna synthesis the species a. mellifera whole bodies or dissected tissues (brain, fat body, integument, ovary, testicle) of individuals in immature stages of development (embryo, larvae and pupae) and adults, of both sexes, with females of both castes (queens and workers) were collected (according to table s1) and pooled into individual microtubes based on sample origin. total rna of each pooled sample was isolated using trizol method (invitrogen) following the manufacturer’s instructions. samples were quantified spectrophotometrically using a nanodrop® nd-1000 spectrophotometer (nanodrop technologies), and ~3 µg were treated with dnase i (invitrogen) to eliminate contaminant dna. dnase-treated samples were used as template to synthetize first strand cdna using superscript ii reverse transcriptase (invitrogen) and oligo(dt)12-18 primers (invitrogen) according to the manufacturer’s protocol. pcr for gene expression analysis presence or absence of the lap-a encoding gene expression was performed using end-point polymerase chain reaction (pcr). cdna samples were amplified through conventional pcr using specific lap-a primers, lap-a-f (forward): 5’-agaggctttaggagatgcag-3’ and lapa-r (reverse): 5’-gctggagtatctgccaaatg-3’. the reaction mixtures were prepared with 10 µl of taq pol master mix 2x green (cellco), 0.8 µl of each primer pair (10 pmols/ µl), 1 µl of cdna (dilution 1:10), in a final volume of 20 μl. amplification reactions were assayed in the veriti® 96-well thermal cycler (applied biosystems), as follows: first step of 94 °c for 1 minute; 30 cycles of 94 °c for 35 seconds, 56 ºc for 35 seconds and 72 ºc for 35 seconds; and a final step at 72 ºc for 5 minutes. the expression of the rpl32 reference gene (nm_001011587.1) (lourenço et al., 2008) was used to check the quality and the reliability of the cdna samples; except for the use of 30 cycles and 0.5 µl of each primer pair (10 pmols/ µl, rpl32-f: 5’-cgtcatatgttgccaactggt-3’ and rpl32-r: 5’-ttgagcacgttcaacaatgg-3’), all other conditions of pcr cycling and reaction mixtures to amplify the rpl32 gene were as those used for lap-a. amplification products of both genes were analyzed by electrophoresis in 2% agarose gels in 1x tbe buffer (89mm tris base, 89mm boric acid, 2 mm edta, ph 8.0) and stained with a unisafe dye solution (20,000x, uniscience), under conditions of 55 ma and 100 volts. the bands were visualized and documented using a kodak edas 290 system. each gel band volume (the sum of all pixel intensities within the band boundaries excluding background signs) was densitometrically measured by imagej version 1.52a software (abramoff et al., 2004), and lap-a/rpl32 ratio was calculated to normalize lap-a transcripts abundance. sequencing for sequencing, 1 μl dna sample (100 ng/µl) from a. mellifera, e. nigrita, p. helleri, b. brasiliensis and e. cordata was subjected to amplification, using 2.5 µl of dntp (250 mm), 2.5 µl of buffer 10x (invitrogen), 2.5 µl of mgcl2 (50 mm, invitrogen), 1 µl of each lap-a primer (10 pmols/µl, lap-a-f or ), 0.2 µl of taq platinum® (5 u/µl, invitrogen), in a final volume of 25 μl. amplification reactions were assayed in the veriti® 96-well thermal cycler (applied biosystems), as follows: first step of 95 °c for 3 minutes; 35 cycles of 95 °c for 30 seconds, 56 ºc for 45 seconds and 72 ºc for 45 seconds; and a final step at 72 ºc for 10 minutes. amplification products were analyzed by electrophoresis in 1% agarose gels in 1x tbe buffer and stained with a gel red solution (10000x, biotium), under conditions of 55 ma and 100 volts. the bands were visualized and documented using a molecular image gel doc™ xr+ photodocumenter (biorad) and image lag 4.0 software (bio-rad). concerning the dna sequencing, a single and intense band was obtained for a. mellifera. however, some unspecific bands were observed for other species. the bands compatible with the expected fragment size based on the lap-a gene fragment from a. mellifera (278 bp) were cut from the gel and placed in separate tubes with 200 µl of sterilized water. the tubes were heated at 70 °c for 30 minutes to partially melt the agarose and facilitate the passage of dna to water. from these samples, a second round of pcr were performed under the same conditions (cycle number and reagents), only modifying the annealing temperature to 62 °c, to increase specific amplification. the new amplification products were purified using 2 µl of exoprostartm (illustratm) enzyme and 8 μl of gdna amplified in each reaction. the purification reaction was incubated in the veriti® 96-well thermal cycler (applied biosystems) at 37 °c for one hour, at 80 °c for 15 minutes and at 20 °c for 5 minutes. next, an aliquot of this reaction was submitted to 1% agarose gel electrophoresis under the conditions cited above. purified pcr products that showed single and clear bands for each species were selected for sequencing in both directions. the sequencing reaction used 3 µl of sterilized water, 3 μl of buffer save money 2.5x, 1 μl of big dye terminator version 3.1 (thermo fisher scientific), 1 μl of forward or reverse lap-a primer and 2 μl of pcr purified product per well in the sequencing 96-well plate, with a final volume of 10 µl. the reaction was conducted in 40 cycles: 96 °c for 1 minute, 50 °c for 15 seconds, 62 °c sociobiology 65(4): 654-661 (october, 2018) special issue 657 for 15 seconds and 60 °c for 4 minutes. for precipitation, washing steps using 75% isopropanol (v/v) and 70% ethanol (v/v) were used. after centrifugations, the 96-well plate was incubated at 37 °c for 10 minutes and sent to laboratório de biotecnologia of fcav/unesp (jaboticabal, sp, brazil) for sequencing. the sequencing reactions were assayed in an abi 3700 automatic sequencer (applied biosystems) and the electropherograms were analyzed and edited in codoncode version 3.7.1 program (codoncode). results lap-a genomics we identified 22 potential aminopeptidases encoding genes in the a. mellifera genome and 50 splicing variants, distributed in eight of the sixteen chromosomes (haploid genome) (table s2). our analysis resulted in the identification of a single gene model noted as being an a. mellifera cytosolic leucine-aminopeptidase encoding gene (lap-a) (geneid: 552109 and genbank accession number: xm_016916769.1). lap-a gene locus is located in chromosome 15, and its fulllength mrna of 1,617 base pairs (bp) contains 46 bp of 5’utr, 86 bp of 3’utr, and 1,485 bp of coding region (cds) that generates a protein with 494 amino acids (genbank accession number: xp_016772258.1). it is worthwhile mentioning that the a. mellifera lap-a protein sequence has 53% identity (score: 494, query cover: 94%, e-value: 5e-67) with human cytosol aminopeptidase (np_056991.2). the honeybee lap-a sequences in fasta format were recovered from genbank, and served as a starting point to search for orthologs in genomes of other bee species (apoidea). we found predicted orthologs of lap-a from solitary to social species: apidae (a. dorsata, a. florea, m. quadrifasciata, e. mexicana, b. terrestris, b. impatiens, h. laboriosa), megachilidae (m. rotundata), and halictidae (l. albipes, d. novaeangliae) (table 1). in addition, a coherent evolutionary relationship between lap-a of bees was obtained (fig s2) when we aligned only central protein regions (~334-338 amino acid residues) encompassing their conserved peptidase_m17 domains (cd00433). using specific lap-a primers, we amplified fragments of 278 bp and 201 bp in length using a. mellifera gdna and cdna samples as template, respectively (fig. s1, and lanes 1 and 2 of fig s3). we also used these primers to amplify and sequence the lap-a gene from gdna samples of the following corbiculated bees: p. helleri, e. nigrita, e. cordata and b. brasiliensis. pcr products of a. mellifera lap-a were (re)sequenced only for validation, as its genome is already known. in general, the results of sequencing were incomplete, but satisfactory. sequencing of putative lap-a fragments from p. helleri and b. brasiliensis did not present sufficient quality and length for analyzes, while those sequences from a. mellifera, e. nigrita and e. cordata were reliable (see fig s3) and aligned with lap-a, as expected (table s3). species taxonomy (family/tribe) life style accession number identity in relation to a. mellifera e-value in relation to a. mellifera apis mellifera apidae/ apini advanced eusocial xp_016772258.1 100% 0.0 apis dorsata apidae/ apini advanced eusocial xp_006609318.1 99% 0.0 apis florea apidae/ apini advanced eusocial xp_012345156.1 98% 0.0 melipona quadrifasciata apidae/ meliponini advanced eusocial kox72754.1 91% 0.0 eufriesea mexicana apidae/ euglossini facultative simple eusocial xp_017752846.1 92% 0.0 bombus terrestris apidae/ bombini obligate simple eusocial xp_003401321.1 90% 0.0 bombus impatiens apidae/ bombini obligate simple eusocial xp_003484843.1 90% 0.0 habropoda laboriosa apidae/ anthophorini solitary xp_017799497.1 87% 0.0 megachile rotundata megachilidae/ megachilini solitary xp_012141098.1 85% 0.0 lasioglossum albipes halictidae/ halictini facultative simple eusocial lalb20051 (beebase id) 81% 0.0 dufourea novaeangliae halictidae/ rophitini solitary xp_015439447.1 85% 0.0 table 1. orthologs of apis mellifera lap-a protein sequence (xp_016772258.1), identified by blastp tool against genomic data of apoidea species available at ncbi or beebase. highlighted in bold, corbiculated bee species from apini, meliponini, euglossini and bombini tribes. l bataglia, ic godoy, ma del lama, fmf nunes – leucine-aminopeptidase a in bees658 lap-a mrna expression we explored lap-a transcriptional expression data in different species, tissues and developmental stages in order to gain functional insights and to verify the correspondence between mrna profiles and enzymatic activity. toward this end, two strategies were used: digital expression using blast searches against all currently tsa and sra (transcriptomic) data available for apoidea (table s4), and semiquantitative rt-pcr assays using honeybee cdna samples (fig 1 to fig 6). regarding digital expression, more than 50 different bee species belonging to six distinct taxonomic apoidea families (apidae, megachilidae, halictidae, melittidae, colletidae and andrenidae) presented some transcriptional evidence for lap-a (table s4), corroborating our hypothesis about its constitutive expression and general roles in bees. about rt-pcr data, the reference gene rpl32 provided us information about the quality and the reliability of the samples (fig s4 to fig s9), as well as served to normalize the abundance of lap-a transcripts. even though some fluctuations in lap-a expression are sometimes observed in the different analyzed contexts (fig s10), its presence was invariably found in all of them. embryogenesis our findings indicate that lap-a is expressed throughout the embryogenesis of both honeybee sexes (fig 1), increasing towards late embryogenesis (fig s10). rnaseq data also supported the expression of lap-a during the embryonic development of a. mellifera, apis cerana fabricius, bombus hunti greene and megalopta genalis meade-waldo (table s4). differentiation of reproductive organs lap-a transcripts were also detected in the ovaries and testicles during post-embryonic development of a. mellifera, when the differentiation of these reproductive tissues occurs (fig 4). in addition, rna-seq data also support expression of lap-a transcripts in ovaries of queens and workers undergoing distinct reproductive status in a. mellifera and b. terrestris, and in drone testicles of the same two species (table s4). fig 1. expression of lap-a (cdna, 201 bp) in male (drone) and female embryos throughout the embryogenesis (time interval in hours). in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. fig 2. expression of lap-a (cdna, 201 bp) in whole bodies of bipotent female larvae (l1 and l2, collected from worker brood cells) and worker larvae (l3 to pp3). in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. fig 3. expression of lap-a (cdna, 201 bp) in whole bodies of developing drone larvae (l1 to l5s3). in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. adults we verified a subtle differential expression of lap-a between brains and fat bodies of 7 days-old adult honeybee workers (fig 5, fig s10). we also observed lap-a expression in a. mellifera transcriptomic data from samples of abdomen, antennae, head, mushroom body, fat body, hypopharyngeal gland, second thoracic ganglia, sting gland, nasonov gland, muscle, mandibular gland, midgut, malpighian tubule collected in different sexes and castes (table s4). moreover, many bee species showed expression of lap-a in head, abdomen, thorax and antennae, or in whole body during the adult phase (table s4). fig 4. expression of lap-a (cdna, 201 bp) in drone testicles or queen ovaries during different developmental stages. in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. female drone drone queen post-embryonic development we observed the expression of lap-a during entire larval development of males and females of a. mellifera (fig 2 and fig 3), and data hint specific sex profiles (fig s10). from tsa/sra data, we detected expression of lap-a in larvae of a. mellifera, b. terrestris, b. hunti, m. rotundata and m. genalis, and in pupal tissues of a. mellifera, b. terrestris, b. hunti e m. genalis (table s4). sociobiology 65(4): 654-661 (october, 2018) special issue 659 integument lap-a is found to be expressed in the abdominal and thoracic integument (epidermis plus cuticle) of honeybee pupae (pw), pharate adults (pp) and adult workers (ne and for) (fig 6), showing a peak in pp (fig s10). lap is an important enzyme present in the gut of many insects (for review, see bozić et al., 2007). we detected transcripts of lap-a in developing larvae of both honeybee sexes (fig 2 and fig 3). it is in accordance with lap-a enzymatic activity found in larvae of 2-6 days (del lama et al., 2001). moreover, metalloaminopeptidase has been associated with the larval molting process in invertebrates (hong et al., 1993). it is tempting to speculate that this aminopeptidase may also act in the degradation of proteins that anchor the cuticle, facilitating apolysis and ecdysis (shedding of the old cuticle). furthermore, imaginal (pupal-to-adult) molting depends on the replacement of the old cuticle by a newly synthesized version. lap-a enzymatic activity was observed in young and pigmented pupae of honeybees (del lama et al., 2001). in addition, the peak of lap-a found in the pp integument (fig 6) coincides with the ecdysteroid peak that induces pupal cuticle apolysis and the onset of adult cuticle synthesis in a. mellifera (pinto et al., 2002; elias-neto et al., 2009). aminopeptidases were found in integument of drosophila melanogaster meigen (knowles & fristrom, 1967), and there are reports of an increase in lap concentration in the epidermis of arthropods during the molting process. evidence suggests that epidermal cells of bees secrete lap-a to the molting fluid that fills the ecdysial space (spearman, 1973). we detected lap-a transcripts during the differentiation of reproductive organs of a. mellifera (fig 4), as well as in females (queen and worker) ovaries and in male testicles of adult individuals of a. mellifera and b. terrestris (table s4). lap-a enzyme is active in reproductive tracts of adult drones (testes, mucus glands, ejaculatory bulb and seminal vesicles) and also in both virgin and inseminated queens (ovaries/oviduct and spermatheca) (del lama et al., 2001), corroborating our data. in the tick haemaphysalis longicornis neumann, lap was observed in different ovary cells responsible for the nutrition of developing oocytes (hatta et al., 2010). the same authors also silenced the expression of lap gene and observed a negative impact in reproduction due to reduced oviposition. the data suggest a role of lap in the metabolism of nutrients that support vitellogenesis. also, high expression of lap was detected in sperm proteome of d. melanogaster meigen (dorus et al., 2006). it is believed that lap is present in the acrosome of sperm, an organelle that contains many digestive enzymes involved in the process of egg fertilization (togo & morisawa, 2004). considering the high conservation of aminopeptidases, we assume that the lap functions (linked to reproductive processes) described above may be preserved in bees. in adults, the activity of lap-a was found in digestive tract, thorax, antennae, hemolymph, malpighian tubule and brain of a. mellifera (del lama et al., 2001). here, we showed lap-a mrna expression in dozens of tissues from different species of bees (fig 5, fig s10, table s4). together, our data reinforce the hypothesis of pervasive expression and housekeeping roles of this aminopeptidase in cells and broad-spectrum functions in bees. fig 6. expression of lap-a (cdna, 201 bp) in integument (thoracic and abdominal) of workers during different stages of development. in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. discussion here we identified lap-a orthologs in apoidea genomes and the tree topology generated (fig s2) fits well the proposal for bee phylogeny currently accepted (see kapheim et al., 2015). also, we found expression of lap-a in all conditions tested by semiquantitative rt-pcr assays using honeybee (a. mellifera) cdna samples and also found evidence of expression using blast searches against all currently tsa and sra (transcriptomic) data available for apoidea. together, our data support the coherence between mrna and enzyme activity profiles of lap-a (del lama & mestriner, 1984; del lama et al., 2001). below we suggested and discussed putative roles of this enzyme in different tissues and developmental stages of honeybees and other bees, based on literature information and gene expression data. the increasing of expression of lap-a towards late embryogenesis (fig 1) is probably related to eggshell break for larval hatching. to date, lap-a was the only active leucine-aminopeptidase found in eggs of a. mellifera, suggesting roles in yolk protein digestion (del lama et al., 2001). lap-a transcripts found during early embryogenesis of honeybees (0-6h, fig 1) may also indicate their maternal deposit and expression after genome activation, probably acting during cleavage divisions (pires et al., 2016). during larval stages, intense feeding requires digestive enzymes capable of hydrolyzing several protein substrates. fig 5. expression of lap-a (cdna, 201 bp) in 7-day-old worker brains and fat bodies. in m, molecular weight marker and in c-, negative control reactions with no added template (cdna). pcr products were visualized in agarose (2%) gel electrophoresis. for information of each sample, see table s1. l bataglia, ic godoy, ma del lama, fmf nunes – leucine-aminopeptidase a in bees660 concluding remarks the constant presence of lap-a mrna expression presented here are consistent with lap-a enzymatic activity previously published (del lama & mestriner, 1984; del lama et al., 2001). our data demonstrate that lap-a is a ubiquitously expressed gene at transcriptional level, with potential functions in many apoidea species. it is postulated that, in addition to the digestive role, aminopeptidases have metabolic roles, such as the regulation of circulating concentrations of amino acids and peptides in the body, maintenance of osmotic stability and delivery of amino acids for protein anabolism (see del lama & ferreira, 2003). also, lap-a plays roles in the regulation of synthesis and degradation of intracellular proteins (protein turnover) (miller, 1987; yen et al., 1980; bartling et al., 1992). our findings support the multifunctional aspects of this enzyme acting in distinct biological processes of bees, independent of their life styles. acknowledgments we are grateful to camilla v. pires, flávia c. p. freitas, tiago falcon, felipe martelli, fabiano abreu, natália hernandes, aline aleixo, tathyana mello and leonardo n. paula for providing rna samples, and also to katia ferreira, danielle l. lucena, thiago s. depintor, eduardo a. b. almeida, zilá l. p. simões and márcia m. g. bitondi for technical assistance. we would like to thank michael c. jaskot and fernando h. biase for correcting and improving language and style of the text, and also the two anonymous reviewers for their constructive comments and suggestions that greatly contributed to improving the final version of the manuscript. supplementary material doi: 10.13102/sociobiology.v65i4.3475.s2223 link: http://periodicos.uefs.br/index.php/sociobiology/rt/supp files/3475/0 authors’ contributions the study was conceived and coordinated by madl and fmfn. lb and icg conducted the lab experiments. lb, madl and fmfn analyzed the data and wrote the manuscript. all authors contributed to discussions of the data and edited the manuscript. all authors approved the final manuscript version. references abramoff, m.d., magalhães, p.j. & ram, s.j. 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(2018) gene variation of endoplasmic reticulum aminopeptidases 1 and 2, and risk of blood pressure progression and incident hypertension among 17,255 initially healthy women. international journal of genomics, 2018: 23 08585. doi: 10.1155/2018/2308585. _hlk513760046 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2849sociobiology 65(3): 515-523 (september, 2018) evaluating the efficiency of different sampling methods to survey social wasps (vespidae: polistinae) in an anthropized environment introduction social wasps occurring in brazil belong to the cosmopolitan polistinae, which are particularly speciose in the neotropics (silveira, 2002). three polistine tribes are found in brazil, with totals for the subfamily counting to as much as 21 genera and 343 species (hermes et al., 2017), corresponding to approximately 35% of the world fauna (prezoto et al., 2007). environmental structure directly affects the social wasp fauna (santos et al., 2007), since some species will only nest under specific vegetational conditions, such as shape and size of the leaves, trunk diameter and presence of thorns (cruz et abstract social wasps play important ecological roles, such as the natural biological control of other arthropods as well as major components of the flower-visiting insect guild. despite many studies focusing on the survey of these organisms in brazil, information on the community structure of polistines in anthropized environmets is still rare. the goals of the present study were: i) to survey the social wasp fauna in an anthropized area in the transition of cerrado and atlantic forest; ii) to investigate the efficiency of two sampling methods, namely active search for wasps and the use of attractive traps; iii) to investigate the performance of different attractive baits in the capture success of social wasps in the study area. sampling of social wasps was conducted by actively searching for individuals and by using attractive traps. a total of 40 species was recorded, with agelaia multipicta and agelaia vicina species being the most frequently collected with attractive traps and mischocyttarus cassununga by actively searching for wasps. in all analyses performed (except when comparing abundance of social wasps considering the molasses bait and the active search), actively searching for wasps was the best method. this is also highlighted by the fact that the time spent actively capturing polistines was considerably lower than the time (and costs) that the traps were left in the field. active search, as demonstrated by previous studies, remains as the best capturing methodology when surveying neotropical social wasps, either in natural or anthropized environments. sociobiology an international journal on social insects gc jacques1,2, ep pires1, mg hermes1, ldb faria1, mm souza3, lcp silveira1 article history edited by gilberto m. m. santos, uefs, brazil received 15 january 2018 initial acceptance 26 april 2018 final acceptance 08 may 2018 publication date 02 october 2018 keywords baits, diversity, neotropical, polistine wasps, survey. corresponding author gabriel de castro jacques departamento de entomologia universidade federal de lavras av. doutor sylvio menicucci nº 1001 kennedy, 37200-000, lavras-mg, brasil. e-mail: gabriel.jacques@ifmg.edu.br al., 2006). however, several species are highly sinantropic, nesting preferably in urban environments (michelutti et al., 2013; oliveira et al., 2017) due to higher prey and protected nesting site availability (prezoto et al., 2007). human disturbance, such as direct colony destruction, excessive use of insecticides (souza et al., 2012), and forest fragmentation strongly decrease nesting possibilities (souza et al., 2010; souza et al., 2014b). also, some species are sensitive to changes in abiotic factors (luminosity, temperature and humidity) that may be related to environmental degradation, making them good indicators of environmental quality (souza et al., 2010). in this context, biological surveys are essential to support conservation actions and programs (elpino-campos et al., 2007). since polistines are easily surveyed in tropical 1 universidade federal de lavras, minas gerais, brazil 2 instituto federal de educação, ciência e tecnologia de minas gerais, campus bambuí, minas gerais, brazil 3 instituto federal educação, ciência e tecnologia do sul de minas, campus inconfidentes, minas gerais, brazil research article wasp gc jacques et al. – evaluating the efficiency of different sampling methods to survey social wasps516 systems and are active during all seasons, and may be sampled in a short period of time (kumar et al., 2009), their study in a conservationist perspective is strongly recommended. there are a large number of studies on the diversity of social wasps in the last ten years (barbosa et al., 2016; souza et al., 2017), although there is still no standard in the duration of the study and methodology of data collection. the date of studies ranges from a few days to 144 months, with 12 months being the most usual (barbosa et al., 2016). for the collection methodology, there are several forms of capture such as the active search and the attractive traps, malaise, möerick and light traps (silva & silveira, 2009; jacques et al., 2015; gradinete & noll, 2013; locher et al., 2014; souza et al., 2015), but most studies use active search and attractive traps together (barbosa et al., 2016). this wide variety of methodologies and study time hampers the comparative analyzes between the different studies. in relation to the attractive traps, there is a great diversity of baits used as juices of mango, guava, passion fruit and orange, sardine stock and honey. this diversity of baits occurs mainly due to the lack of a study that tests the efficiency of the best bait for this method. in addition, there is a wide variation in the amount, arrangement and duration of the traps (souza & prezoto, 2006; elpino-campos et al., 2007; jacques et al., 2015; locher et al., 2014). the standardization of this methodology would optimize the time and monetary costs to establish these traps in the field. most of studies dealing with community structure of social wasps carried out in brazil are focused in natural environments (silveira, 2002; silva-pereira & santos, 2006; souza & prezoto, 2006; elpino-campos et al., 2007; santos et al., 2007; silveira et al., 2008; gomes & noll, 2009; silva & silveira, 2009; santos et al., 2009; arab et al., 2010; prezoto & clemente, 2010; souza et al., 2010; bonfim & antonialli junior, 2012; simões et al., 2012; souza et al., 2012; gradinete & noll, 2013; locher et al., 2014; souza et al., 2014a, 2014b; togni et al., 2014; somavilla et al., 2014, 2015; souza et al., 2015; brunismann et al., 2016; elisei et al., 2017). the polistine fauna of anthropized areas, however, is still poorly investigated (jacques et al., 2012, 2015; oliveira et al., 2017). with that in mind, the goals of the present study are: i) to survey the social wasp fauna in an anthropized area in the transition of cerrado and atlantic forest in southern minas gerais, brazil; ii) to investigate the efficiency of two sampling methods, namely active search for wasps and the use of attractive traps; and iii) to investigate the performance of different attractive baits in the capture success of social wasps in the study area. material and methods the study was conducted in the federal university of lavras (ufla), lavras, minas gerais, brazil, which is located at 21º 14’ 30” s and 45º 00’ 10” w. the altitude is approximatelly 918 m above sea level and the climate is characterized as cwa köppen, with rainy summers and dry winters. the mean annual pluviosity is 1,411 mm and the mean annual temperature is 19.3 ºc. the university´s campus has a total area of 508 ha, which is highly anthropized, with 117 ha of urban settlements and 207 ha of crops. from the remaining 184 ha, only 33.23 ha comprehend atlantic forest and cerrado fragments. collecting of social wasps in the study area was conducted from april 2014 to june 2015, by actively searching for individuals and with the aid of attractive traps. active searches for wasps were conducted in the entire area of the campus, totaling 14 field incursions of five hours each, totaling 70 hours of sampling effort (two collectors = 140 hours). flowering plants, logs, vegetation close to lakes, and buildings were inspected and the wasps collected with entomological nets (souza & prezoto, 2006; elpino-campos et al., 2007). attractive traps were made using plastic bottles with three triangular openings (2 x 2 x 2 cm) at approximately 10 cm from the base (souza & prezoto, 2006). four types of attractive baits were used: a) natural passion fruit juice (passiflora edulis f. flavicarpa deg.) prepared with 1 kg of fruit blended with 250g granulated sugar plus two liters of water; b) sardine stock, using two sardine cans mixed with two litters of water (souza & prezoto, 2006); c) pure honey; and d) 50% diluted sugar cane molasses (jacques et al., 2015). the traps were installed in seven different fragments of the forest remnants inside the campus, with 150 ml attractive substance placed in eight bottles each, totaling 32 traps per fragment. the traps were placed at 1.5 m from the ground and at 10 m apart from each other (fig 1). the traps stayed in the field for seven days, when the wasps were removed and preserved in 70% alcohol. social wasps were identified to species level using keys (richards, 1978; carpenter, 2004) and by specialists (dr. james m. carpenter, amnh, and mgh and mms coauthors of the study). the diversity of wasps was calculated with the shannonwiener (h’) index and the dominance with the berger-parker (dpb) index, using the software past (hammer et al., 2005). to evaluate sampling effort success we constructed species accumulation curves using the observed richness with a 95% confidence interval, under the bootstrap 1 estimator in the software estimates 9.1.0 (cowell, 2013). this estimator uses information from all collected species instead of restricting the analysis to rare species (santos, 2003). to test which bait was more successful regarding species richness and abundance, generalized linear mixed models (glmm) were performed assuming sampling date as the random variable, reducing the effects of sampling and repeated measures, and the different baits and active search as explicative variables. lastly, tukey contrast analyses with bonferroni correction (i.e., multiple comparisons of means) were employed as pairwise comparisons. poisson errors distribution was assumed for both models and statistical analyses were performed using the software r (r development coreteam, 2017). sociobiology 65(3): 515-523 (september, 2018) 517 another factor that may contribute to a higher richness of social wasps in urban areas is likely a methodological one, because dense vegetation and nest camouflage will certainly difficult sampling when actively searching for these insects inside forests (souza et al., 2010). also, the environmental structure of the study area is highly complex and heterogeneous, with human constructions, crops, and atlantic forest and cerrado fragments, which may favor coexistence of a higher number of species due to a greater offering of microhabitats and resources, as well as nesting materials and substrates (santos et al., 2007; souza et al., 2012). a high dominance of only few species was observed. agelaia vicina de sausurre and agelaia multipicta (haliday) were the most frequent species when considering only the attractive traps (40.16% and 23.0%, respectively). some species of agelaia are necrophagous, and were captured with the sardine stock in the present study, which may be used as an additional proteic resource offered to the larvae (souza & prezoto, 2006). also, they may establish colonies with up to one million adults (zucchi et al., 1995), which provides an enhanced foraging capacity and, consequently, better chances for one to capture specimens of agelaia (hunt et al., 2001). a high abundance of this taxon have already been reported in several studies in brazil (gomes & noll, 2009; arab et al., 2010; jacques et al., 2012; gradinete & noll, 2013; locher et al., 2014; togni et al., 2014; jacques et al., 2015). another species that was frequently trapped was polybia fastidiosuscula saussure, with 10.53% of relative frequency. since the traps were placed inside atlantic forest fragments, the occurrence of this taxon may indicate a good conservation status of this area (souza et al., 2010). despite occurring in different biomas and altitudes (souza et al., 2010, albuquerque et al., 2015), p. fastidiosuscula is particularly dominant above 1,500 meters high (souza et al., 2015). fig 1. distributional scheme of the attractive bait-traps used in the present study. results and discussion a total of 11 genera and 40 species of social wasps was collected during the study, with a total diversity index of 2.495 (table 1). the present collecting effort resulted in the fifth highest polistine richness considering similar studies in brazil (table 2) (barbosa et al., 2016), only behind those conducted in the amazon (silveira, 2002; silva & silveira, 2009; somavilla et al., 2014; somavilla et al., 2015). the high richness found herein may be a result of the high sinantropic affinity presented by several social wasp species (michelutti et al., 2013; oliveira et al., 2017), despite colony development and productivity being negatively affected by the urban environment (michelutti et al., 2013; torres et al., 2014). however, nesting in buildings reduces interspecific competition, predation by vertebrates, and offers better protection against climatic adversities (mcglynn, 2012; michelutti et al., 2013). fig 2. species accumulation curve for social wasps collected at anthropized environment using the observed species richness within a 95% confidence interval and the estimated species richness (bootstrap 1). gc jacques et al. – evaluating the efficiency of different sampling methods to survey social wasps518 table 1. richness, diversity and dominance of social wasp species collected at anthropized environment. taxon active search attractive traps totals nº of individuals frequency (%) nº of individuals frequency (%) nº of individuals frequency (%) agelaia centralis (cameron) 2 0.85 0 0.00 2 0.18 agelaia multipicta (haliday) 1 0.42 201 23.00 202 18.20 agelaia vicina de saussure 1 0.42 351 40.16 352 31.71 apoica pallens (fabricius) 5 2.12 9 1.03 14 1.26 brachygastra lecheguana (latreille) 3 1.27 3 0.34 6 0.54 clypearia augustior (ducke) 2 0.85 0 0.00 2 0.18 mischocyttarus ignotus zikán 9 3.81 4 0.46 13 1.17 mischocyttarus cassununga (r. von. ihering) 68 28.81 3 0.34 71 6.40 mischocyttarus drewseni de sausurre 3 1.27 5 0.57 8 0.72 mischocyttarus paraguayensis de willink 3 1.27 0 0.00 3 0.27 mischocyttarus labiatus fabricius 5 2.12 6 0.69 11 0.99 mischocyttarus rotundicolis (cameron) 1 0.42 0 0.00 1 0.09 mischocyttarus socialis (de saussure) 1 0.42 3 0.34 4 0.36 mischocyttarus sp. 2 0.85 0 0.00 2 0.18 parachartergus fraternus (griboldo) 1 0.42 0 0.00 1 0.09 polistes actaeon haliday 1 0.42 0 0.00 1 0.09 polistes billardieri (fabricius) 3 1.27 0 0.00 3 0.27 polistes cinerascens (de saussure) 1 0.42 0 0.00 1 0.09 polistes ferreri (de saussure) 1 0.42 4 0.46 5 0.45 polistes pacificus fabricius 1 0.42 0 0.00 1 0.09 polistes simillimus zikán 19 8.05 0 0.00 19 1.71 polistes subsericius (de saussure) 3 1.27 0 0.00 3 0.27 polistes versicolor (olivier) 8 3.39 3 0.34 11 0.99 polybia bifasciata de saussure 5 2.12 46 5.26 51 4.59 polybia bistriata (fabricius) 5 2.12 32 3.66 37 3.33 polybia chrysothorax (lichtenstein) 2 0.85 7 0.80 9 0.81 polybia fastidiosuscula (de saussure) 8 3.39 92 10.53 100 9.01 polybia ignobilis (haliday) 5 2.12 3 0.34 8 0.72 polybia jurinei de saussure 1 0.42 3 0.34 4 0.36 polybia minarum (ducke) 1 0.42 25 2.86 26 2.34 polybia occidentalis (olivier) 12 5.08 19 2.17 31 2.79 polybia paulista (r. von. ihering) 1 0.42 0 0.00 1 0.09 polybia platycephala (richards) 1 0.42 11 1.26 12 1.08 polybia punctata du buysson 2 0.85 35 4.00 37 3.33 polybia quadrincicta de saussure 2 0.85 0 0.00 2 0.18 polybia scutellaris (white) 14 5.93 0 0.00 14 1.26 polybia sericea (olivier) 1 0.42 0 0.00 1 0.09 protonectarina sylveirae (de saussure) 6 2.54 5 0.57 11 0.99 protopobybia sedula (de saussure) 22 9.32 0 0.00 22 1.98 synoeca cyanea (fabricius) 4 1.69 4 0.46 8 0.72 individual totals 236 874 1110 species richness (s`) 40 23 40 shannon-wiener index (h`) 2.871 1.956 2.495 berger-parker index (dpb) 0.288 0.4016 0.3171 sociobiology 65(3): 515-523 (september, 2018) 519 mischocyttarus cassununga (r. von. ihering) was the most sampled species by actively searching for these insects. it is a highly sinathropic taxon easily found nesting in human buildings (jacques et al., 2012; oliveira et al., 2017). this nesting behavior may have been adopted to avoid competition for resources with other social wasps, for an anthropized environment that harbor various crops may offer a considerable amount of prey itens (prezoto et al., 2007). the species accumulation discovery depicted in fig 2 (sobs = 40) shows that the species curve tends to an asymptote. also, the estimated number of species (bootstrap 1 – 44.21) lies inside the confidence interval of 95%, showing that our sampling effort was sufficient. despite the fact that the monthly active collecting effort may be considered low (ten hours per month), sampling throughout an entire year yelded results that may be considered a good approximation of the real biotic diversity within the study area (barbosa et al., 2016). all species captured by attractive traps were also sampled through active search (compare table 1 and table 3), highlighting the efficiency of this method. it is by far the most widely used sampling method to survey social wasps and the one with the higher sampling success regarding exclusive species (a species recorded by only one method) (barbosa et al., 2016; maciel et al., 2016), with a total of 17 exclusive species sampled herein. although, the use of other methodologies may reveal sampling success of exclusive species as well, for some have been exclusively recorded by other methods such as attractive traps (e.g. souza & prezoto, 2006; elpino-campos et al., 2007; jacques et al., 2012; loucher et al., 2014; souza et al., 2014b; jacques et al., 2015). also, important ecological and phenological information may be gathered by using different approaches to access community structure. several kinds of attractive baits have been used to access social wasp species richness, such as natural and industrialized fruit juices (passion fruit, orange, guava, mango, pineapple), honey and sardine stock (barbosa et al., 2016; maciel et al., 2016). this bait diversity may be a result of the lack of studies testing the efficiency of each of these attractions, which may vary depending on the environmental conditions as well (souza et al., 2012; souza et al., 2015). in the present study, species abundance recovered by the four baits and by active search were consistently different, demonstrating that active search was the most effective collecting method, followed by molasses, honey, sardine and passion fruit juice (table 4, fig 3). species richness also presented similar outcomes as ascribed for abundance (table 5, fig 4). finally, table 6 depicts contrast analyses comparing each collecting method in a pairwise way, revealing that only abundance retrieved by active search versus the use of molasses bait have not shown statistical differences. in all other cases (species abundance and richness), actively searching for social wasps was the most effective collecting methodology. authors ecological systems species richness silveira, 2002 amazon 79 silva & silveira, 2009 amazon 65 somavilla et al., 2014 amazon 58 somavilla et al., 2015 amazon 49 present study anthropized environment 40 souza et al., 2014a cerrado 38 souza & prezoto, 2006 cerrado and semidecidual forest 38 souza et al., 2010 riparian forest 36 brunismann et al., 2016 semidecidual forest 35 souza et al., 2015 semidecidual forest 34 simões et al., 2012 cerrado 32 locher et al., 2014 riparian forest 31 jacques et al., 2015 agroecosystem 29 elpino-campos et al., 2007 cerrado 29 souza et al., 2014b riparian forest 28 jacques et al., 2012 anthropized environment 26 prezoto & clemente, 2010 rock field 23 gradinete & noll, 2013 cerrado 22 togni et al., 2014 atlantic forest 21 santos et al., 2009 cerrado 19 bonfim & antonialli junior, 2012 riparian forest 18 santos et al., 2007 atlantic forest 18 oliveira et al., 2017 anthropized environment 18 santos et al., 2007 restinga 16 auad et al., 2010 grazing system 13 silva-pereira & santos, 2006 rock field 11 elisei et al., 2017 caatinga 10 arab et al., 2010 atlantic forest 10 santos et al., 2007 mangrove 8 gomes & noll, 2009 semidecidual forest 7 table 2. polistinae richness comparison among studies carried out in different ecological systems in brazil. fig 3. abundance of individuals of social wasps attracted by different baits and active search. gc jacques et al. – evaluating the efficiency of different sampling methods to survey social wasps520 fixed effect species abundance coefficients estimate std. error z-value pr(>|z|) (intercept) 2.2878 0.1464 15.628 <0.001 passionfruit -1.8556 0.2594 -7.152 <0.001 honey -0.9793 0.2476 -3.955 <0.001 molasses -0.6162 0.2450 -2.515 <0.05 sardine -1.0880 0.2486 -4.376 <0.001 random effects group variance std. dev. sampling date (intercept) 0.2475 0.4975 table 3. richness, diversity and dominance of social wasp species collected with different attractive baits at anthropized environment. taxon honey molasses sardine passion fruit juice nº of individuals frequency (%) nº of individuals frequency (%) nº of individuals frequency (%) nº of individuals frequency (%) agelaia multipicta (haliday) 43 18.30 57 16.86 85 42.08 16 16.16 agelaia vicina de saussure 73 31.06 130 38.46 109 53.96 39 39.39 apoica pallens (fabricius) 4 1.70 5 1.48 0 0.00 0 0.00 brachygastra lecheguana (latreille) 0 0.00 3 0.89 0 0.00 0 0.00 mischocyttarus ignotus zikán 1 0.43 3 0.89 0 0.00 0 0.00 mischocyttarus cassununga (r. von. ihering) 1 0.43 2 0.59 0 0.00 0 0.00 mischocyttarus drewseni de sausurre 2 0.85 3 0.89 0 0.00 0 0.00 mischocyttarus labiatus fabricius 0 0.00 6 1.78 0 0.00 0 0.00 mischocyttarus socialis (de saussure) 1 0.43 1 0.30 0 0.00 1 1.01 polistes ferreri (de saussure) 2 0.85 2 0.59 0 0.00 0 0.00 polistes versicolor (olivier) 1 0.43 2 0.59 0 0.00 0 0.00 polybia bifasciata de saussure 22 9.36 18 5.33 0 0.00 6 6.06 polybia bistriata (fabricius) 12 5.11 12 3.55 2 0.99 6 6.06 polybia chrysothorax (lichtenstein) 5 2.13 2 0.59 0 0.00 0 0.00 polybia fastidiosuscula (de saussure) 27 11.49 48 14.20 2 0.99 15 15.15 polybia ignobilis (haliday) 1 0.43 1 0.30 0 0.00 1 1.01 polybia jurinei de saussure 0 0.00 0 0.00 0 0.00 3 3.03 polybia minarum (ducke) 10 4.26 10 2.96 3 1.49 2 2.02 polybia occidentalis (olivier) 8 3.40 9 2.66 0 0.00 2 2.02 polybia platycephala (richards) 4 1.70 5 1.48 0 0.00 2 2.02 polybia punctata du buysson 16 6.81 16 4.73 1 0.50 2 2.02 protonectarina sylveirae (de saussure) 1 0.43 0 0.00 0 0.00 4 4.04 synoeca cyanea (fabricius) 1 0.43 3 0.89 0 0.00 0 0.00 individual totals 235 338 202 99 species richness (s`) 20 21 6 13 shannon-wiener index (h`) 2.193 2.084 0.8773 1.931 berger-parker index (dpb) 0.3106 0.3846 0.5396 0.3939 table 4. general linear mixed models on social wasps species abundance and different attractive baits and active search. sugar cane molasses is still a poorly explored attractive bait in studies surveying polistine wasps (jacques et al., 2015), but as shown above, was the most effective regarding species composition with 21 out of 23 taxa collected by traps. on the other hand, passion fruit juice traps captured only 13 species, with only one being exclusive to this attractive. sardine stock is a widely used attractive, with 71% os the studies conducted in the state of minas gerais making use of this product (maciel et al., 2016). however, almost all abundance and species richness captured by this attractive belong to only two species, a. multipicta and a. vicina, since these exhibit necrophagous habits (souza & prezoto, 2006). finally, when monetary values are compared, sugar cane molasses is a cheaper product when compared to honey, for example, which allied to its efficiency in the capture of social wasps makes it an ideal attractive bait for studies of this nature. sociobiology 65(3): 515-523 (september, 2018) 521 surveying organisms that may be influenced by anthropogenic activity provides the most valuable information when indicating priority areas for conservation. furthermore, when these organisms are easily accessible and sampled in a fast and efficient way, studies may be conducted within a reasonable amount of time. this is the case of neotropical social wasps. it is clear by our results that actively searching for polistines is far superior than using attractive baits: two collectors spent a total of 140 hours in the field, while traps remained in the field for a total of 1,176 hours (summing up all trap hours individually). all our analyses recovered active searching for wasps as the best method regarding species richness and abundance, despite using a somewhat more efficient and affordable attractive bait (sugar cane molasses). another factor that may affect our understanding of the real efficiency of attractive traps is that most studies conducted in brazil make use of different kinds of baits (maciel et al., 2016). also, field hours differ abruptly among analyzed studies. since no real statististical comparison among baits and field hours used in different studies may be accomplished, active searching for neotropical social wasps remains as the best surveying method for this group of organisms, as indicated by silveira (2002). fig 4. social wasps species richness attracted by different baits and active search. fixed effect species richness coefficients estimate std. error z-value pr(>|z|) (intercept) 1.3443 0.1188 11.313 <0.001 passionfruit -1.6397 0.2187 -7.496 <0.001 honey -0.7325 0.1854 -3.951 <0.001 molasses -0.5640 0.1816 -3.106 <0.01 sardine -1.6397 0.2187 -7.496 <0.001 random effects group variance std. dev. sampling date (intercept) 0.07649 0.2766 table 5. general linear mixed models on social wasps species richness and different attractive baits and active search. contrast analyses (tukey test) abundance p-value richness p-value active x honey <0.05 active x honey <0.001 active x molasses =0.58 active x molasses <0.05 active x passion fruit <0.001 active x passion fruit 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(1995). agelaia vicina, a swarm-founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137 doi: 10.13102/sociobiology.v65i4.3388sociobiology 65(4): 662-670 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the colour and the shape: morphological variation on a facultatively eusocial bee augochlora (augochlora) amphitrite (schrottky) introduction the tribe augochlorini beebe is a monophyletic bee lineage with 663 species distributed in 35 genera (gonçalves, 2016), known for the brilliant green coloration present in most of its representatives (engel, 2000). the tribe is restricted to the western hemisphere, being very abundant in tropical south america (engel, 2000; michener, 2007). augochlorine bees exhibit a large array of foraging and social behaviors, nest substrates and architecture (eickwort & sakagami, 1979; engel, 2000; schwarz et al., 2007). species may be solitary to primitively eusocial (danforth & eickwort, 1997; michener, 2007; gonçalves, 2016), nest on soil or decaying wood (eickwort & sakagami, 1979) or even be cleptoparasites (engel, 2013). some behavioral aspects, as nesting habits and division of labor, are frequently linked with morphological abstract augochlorine bees exhibit a large array of foraging and social behaviors, nest substrates and architecture. the huge diversity of behaviors is frequently linked with morphological traits. all levels of variation should be analyzed in order to provide a broader view of evolution. augochlora (augochlora) amphitrite schrottky occurs from northern of argentina to southeastern of brazil. the species nests in decaying wood and is facultatively eusocial. color variation and head polymorphism were already mentioned in the literature and the main goal of the present paper is to evaluate the morphological variation of the species. for this purpose, we examined 720 specimens and carried out qualitative and quantitative analyses with traditional morphometrics. other 25 augochlora species were studied and we propose a revised diagnosis for a. amphitrite. a remarkable color variation is described, there are three morphs: green, dark blue, and black. there are no geographical patterns linked with the color variation. we propose that odontochlora lethe schrottky and odontochlora styx schrottky are junior synonyms of odontochlora amphitrite schrottky. those names refer to black male and female occurring within a. amphitrite distribution. there is a continuous variation on size and shape of head. again, we do not find any relation of morphology with distribution. besides gena swelling, the adductor ridge of mandible is strongly developed on macrocephalic females. due variations showed, a. amphitrite is a bee candidate to be a model for studies to link morphology, function and behavior. sociobiology an international journal on social insects a lepeco, rb gonçalves article history edited by solange augusto, ufu, brazil received 27 april 2018 initial acceptance 12 july 2018 final acceptance 28 august 2018 publication date 11 october 2018 keywords behavior, morphology, nest, social, taxonomy. corresponding author rodrigo barbosa gonçalves departamento de zoologia universidade federal do paraná cx. postal 19020, cep 81531-980 curitiba, paraná, brasil. e-mail: goncalvesrb@gmail.com traits (eickwort, 1969). for example, substrate usage for nesting can be inferred by morphology, since females have developed ridges and teeth on mandible to excavate wood (eickwort, 1969). as another example, allometry on head growth for some species may be indicative of social interactions (sakagami & moure, 1965). head polymorphism is known for augochlora smith, corynurella eickwort, megalopta smith and rhinocorynura schrottky (sakagami & moure, 1965; eickwort, 1969; gonçalves, 2010; gonçalves & melo, 2012). females whose heads are enlarged, especially due to the swelling of vertex and gena have been denominated as macrocephalic (sakagami & moure, 1965; santos & silveira, 2009; gonçalves & melo, 2012). augochlora is the second most speciose genus of augochlorini with approximately 114 species (moure, 2012). the genus is a monophyletic lineage belonging to the universidade federal do paraná, curitiba, paraná, brazil research article bees sociobiology 65(4): 662-670 (october, 2018) special issue 663 augochlora clade (eickwort, 1969; engel, 2000; gonçalves, 2016; meira & gonçalves, 2018). the two extant subgenera use different nesting substrates. augochlora sensu stricto nest in decaying wood (eickwort 1969; wcislo et al., 2003; dalmazzo & roig-alsina, 2012, 2015) and was formerly considered solitary based on the study of stockhammer (1966), however recent studies indicate facultative primitively eusocial behavior for some species (wcislo et al., 2003; dalmazzo & roig-alsina, 2012, 2015). augochlora (oxystoglossella) species nest in the soil (eickwort, 1969), being the studied species considered primitively eusocial with some caste differentiation (eickwort & eickwort, 1972). macrocephalic females are known for augochlora (oxystoglossella) iphigenia holmberg (sakagami & moure, 1965; dalmazzo & roig-alsina, 2011) and for augochlora (oxystoglossella) empusa engel, hinojosa-díaz and bennett (engel et al., 2012). the augochlora species from southern argentina and uruguay were revised by dalmazzo and roigalsina (2011), beside this, no taxonomic revisions for the entire genus were taken so far. augochlora (augochlora) amphitrite schrottky occurs from northern argentina to southeastern brazil (moure, 2012). this resilient species is found in natural, rural and urban areas (gonçalves & melo, 2005; gonçalves et al., 2014; taura & laroca, 2001). the few studies with the species include the description of a mostly bilateral gynandromorphy by alvarez et al. (2014) and diet analysis and pollen host selection by dalmazzo and vossler (2015a; 2015b). the species was formerly described as odontochlora amphitrite schrottky, whose type specimen is probably lost (moure, 2012). the same occurs with other type specimens of curt schrottky (rasmussen et al., 2009), which may include names that correspond to morphological variations. despite that dalmazzo and roig-alsina (2011) were able to identify the species based on the original description and the distribution pattern. a. amphitrite possesses the typical spine on the first sternum and a longitudinal furrow on its scutellum (schrottky, 1909; dalmazzo & roig-alsina, 2011), a combination of features found in a small group of augochlora sensu stricto. the specimens are usually metallic green with blue and violet reflections (dalmazzo & roig-alsina, 2011). according to michener (2007), primitively eusocial behavior includes division of labor between foundresses and workers, which differs more distinctly in physiology and behavior than in morphology. a. amphitrite can also be considered facultative because single and multiple female nests have been found (dalmazzo & roig-alsina, 2012). the authors also recognized that morphological variation is related to social behavior, since some females never observed outside the nests were distinctly larger than foragers. in addition, they had allometrically enlarged heads and developed ovaries. on the other hand, the forager females had worn mandibles and wings, and undeveloped ovaries. still, intermediate states of wearing and ovary development were also observed in foragers and suggested a continuous variation. other studied augochlora sensu stricto. species probably present facultative primitively eusocial behavior, but morphological variation seems to be restricted to size (wcislo et al., 2003; dalmazzo & roig-alsina 2015). the main goal of the present paper is to evaluate the morphological variation of a. amphitrite. we described and studied the color variation and its putative relationship with distribution. macrocephalism is investigated with morphometrics to search for castes and for a relation to geographic distribution. material and methods we examined material from ‘coleção entomológica pe. jesus santiago moure, universidade federal do paraná’ (dzup). the augochlora collection with about 10,000 specimens was sorted to pull out the specimens of a. amphitrite. the species identification followed the diagnosis and redescription of dalmazzo and roig-alsina (2011), accompanied by search for additional characters. size and coloration were considered a priori as variation and not used to diagnose the species. structure terminology follows eickwort (1969) and michener (2007) except where we refer to the basal area of propodeum as the metapostnotum. we use the abbreviations t1, t2 etc., to denote the metasomal terga; s1, s2 etc., to denote metasomal sterna. sculpturing terminology follows harris (1979). in the course of identification, we studied 25 south american morphospecies from the dzup collection to refine the delineation of a. amphitrite. a sum of seven males from different regions and also four males from other species had their genitalia prepared to provide additional characters. males were softened in wet chamber from two to three days. genitalia were removed with entomological pins and clarified in 10% koh for 24 hours. the dissected structures were finally preserved in glycerol. we studied 720 specimens of a. amphitrite, 358 of them being measured and included in the morphometric analyses. the map for the distribution records was made with qgis 2.18.15 ‘las palmas’. photographs of specimens were taken with a nikon d700 and 107 mm sigma macro lens using helicon remote for controlling image capture. the same configuration was used for all images. all photographs were taken in manual mode, 1/160, 5.6, iso 100, ev 0.3. a white sheet was used as background and the illumination followed the system of kawada and buffington (2016). image stacking was made with helicon focus (version 6.0.18) – render method based on method c (pyramid). for color determination we followed the method of aguiar (2005). we use the color picker tool (sample average option) on gimp 2.8.16 (©the gimp team). the color sample was made on an area of 100x100 pixels, always on the same specimen portion. the corresponding rgb color code was recorded. a lepeco, rb gonçalves – augochlora amphitrite morphological variation664 measurements were made with leica stemi dv4 and a micrometric rule. the following measurements were taken: intertegular distance (it), taken at middle portion of tegulae; ocular distance, measured at eye notch (od); vertex length, in frontal view (vl); eye width, in lateral view (ew), taken from eye notch in an orthogonal line to the eye posterior margin; gena width, in lateral view (gw), in a continuation to the measurement of eye width; and mandible width (mw), taken at distal part of the adductor ridge. principal components analysis was carried out for the entire dataset and separately for subsets containing only specimens from the curitiba and palotina regions (paraná, brazil). first pca axis was considered as representing the overall body size. grouping tests (lda and manova) and linear regression were carried out. for this analysis we used as attributes the color and distribution. data was log transformed prior to the analysis. all analyses were carried out with r (r core team, 2017). the r packages ade4 (dray & dufour, 2007), ggfortify (horikoshi & tang, 2016), ggplot (wickham, 2010), mass (venables & ripley, 2002) and vegan (oksanen et al., 2018) were used for processing analysis and plot graphs. results taxonomy augochlora (augochlora) amphitrite (schrottky, 1909) (figs 1-5) odontochlora amphitrite schrottky, 1909: 142 (females from argentina, buenos aires and la plata, types whereabouts unknown). odontochlora styx schrottky, 1909: 143 (female from paraguay, alto paraná, puerto bertoni, type whereabouts unknown). new synonym. odontochlora thebe schrottky, 1909: 143–144 (male from argentina, buenos aires, type whereabouts unknown). decision for synonymy: dalmazzo & roig alsina (2011). odontochlora lethe schrottky, 1909: 144 (male from paraguay, alto paraná, puerto bertoni, type whereabouts unknown). new synonym. females of a. amphitrite can be distinguished from most other augochlora sensu stricto by the scutellum with a median longitudinal furrow which is strongly depressed posteriorly and by the s1 with a spiniform median projection. they can be further separated from similar species by the combination of the following features: clypeus mostly polished, microreticulate only on basal corners; supraclypeal area sparsely punctate and microreticulate; facial carina short, becoming a line above eye notch; distal portion of mandibular adductor ridge produced; metapostnotum with longitudinal, weakly sinuose, carinae; t1 punctate-puncticulate, with some scattered coarser punctures; t2 punctate-puncticulate, with few coarser punctures; and t3 puncticulate. males can be distinguished from other species of augochlora sensu stricto by the combination of the following features: clypeus mostly polished, microreticulate only on basal corners; supraclypeal area densely punctate and microreticulate, facial carina very short; scutellum with a median longitudinal furrow and with polished sublateral spots; mesepisternum areolate-rugulose anteriorly to mesepisternal furrow and punctate posteriorly to the furrow; metapostnotum fig 1. color variation of augochlora amphitrite. a. b. c. d. e. f. (bars correspond to 1mm). sociobiology 65(4): 662-670 (october, 2018) special issue 665 with longitudinal, weakly sinuose, carinae; tergal discs mostly punctate, with impunctate marginal zones; s1 slightly tuberculate; and genitalia wider than longer. color variation we found striking color variation on a. amphitrite (fig 1), with most examined material (n = 530; 69.86 %) being predominantly metallic green (rgb 79:174:87, fig 1a). the green morph is found along the entire species distribution (fig 2). some predominantly green specimens also exhibit a bluish iridescence (rgb 95:109:161, fig 1d), most frequently observed on their head and metasoma, these specimens can be found elsewhere. entirely dark blue (rgb 63:95:125, fig 1b, e) specimens (n = 119; 16.53%) are known from north of argentina and western paraná, brazil. the third morph, entirely black (n = 98; 13.61%) was found from the same collection series (rgb 71:74:66, fig 1c, f) and was also sampled in other localities, predominantly in paraná state (fig 2). the close similarity in structure, microsculpturing, pubescence and male genitalia supports the hypothesis that the color morphs belong to a single species. the size and shape variation described below was found in all three-color morphs. finally, the sympatric distribution of these morphs reinforces this hypothesis, and we therefore relied on it to propose the taxonomic synonymies presented above. fig 2. distribution records for augochlora amphitrite. color morphs are indicated by circle colors. fig 3. cephalic polymorphism in augochlora amphitrite. a) small female, b) macrocephalic female, c) left mandible of a small female, d) left mandible of a macrocephalic female. arrows indicate the mandibular adductor ridge. a and b, and c and d, respectively, at same scale (bars correspond to 1mm and 0.5mm respectively). schrottky (1909) described o. styx and o. lethe, black female and male, respectively forms from puerto bertoni (paraguay). their type locality is very close to known occurrences of the black color morph of a. amphitrite and the original descriptions agree closely with a. amphitrite. indeed, only color was used to separate o. styx from other augochlora and o. lethe from a. amphitrite in schrottky’s (1910) key. unfortunately, the whereabouts of the involved a lepeco, rb gonçalves – augochlora amphitrite morphological variation666 type specimens is unknown, but in the course of an ongoing genus revision we examined several species from argentina, paraguay and brazil and no entirely black morph deserving species status was found. morphometric variation females of a. amphitrite exhibit different body sizes and head shapes (fig 3). however, according to pca the variation is continuous and enlarged head females did not form a separated group (fig 4). those females are mainly distributed on the left upper corner of the pca graph. there is no association of morphometric variation with body color and distribution. as expected, the variables vectors show the same direction with pc1 axis showing a positive relation with to body size, however some structures are also variable on shape. the vector of gena length was strongly related to pc1 (fig 5a). the mandible width and vertex length were more associate with pc2 axis than other variables. however, they showed different trends, with mandible expansion of the adductor ridge associated to macrocephalic females, while the vertex increased with body size but not necessarily with any other morphological measurement females. mandible attributes are related to body size but the macrocephalic females are more dispersed from the regression line (fig 5b, upper right). according to these results the distal portion of the mandibular adductor ridge (fig 3 c-d) came out as an additional important feature of the macrocephalic females. fig 4. principal component analysis of augochlora amphitrite. vectors: intertegular distance (it); ocular distance (od); vertex length (vl); eye width (ew); gena width (gw); and mandible width (mw). fig 5. plot of pc1 gena and mandible width of augochlora amphitrite. regression line indicated in color. sociobiology 65(4): 662-670 (october, 2018) special issue 667 discussion color variation although most a. amphitrite specimens have a predominantly metallic green integument, the blue and black morphs are not rare, corresponding together to 30% of the sample. is noteworthy that one third of the analyzed specimens were taken from palotina, where black and dark blue specimens are remarkably common. dalmazzo and roigalsina (2011) already mentioned entirely blue specimens from central argentina. additionally, we found the dark blue morph in three series from the paraná state, brazil (guarapuava, palotina and telêmaco borba municipalities) and a single specimen from northern argentina (jujuy department). black specimens were found from several localities in brazil and also from argentina. the black color morph should occur in paraguay and it is plausible that the two names proposed by schrottky (1909) correspond to black morphs of a. amphitrite. the three-color morphs are not distinguished by any diagnostic feature, the male genital capsules are similar and the morphometric analyses do not separate the color morphs. lastly, a preservation artifact is discarded because the three morphs were sampled from a single locality (palotina) and preserved under the same conditions by the senior author. color difference was documented for several bee groups as intraspecific variation. as an example, carolan et al. (2012) studied three species of bombus latreille delimited with molecular data, but they found that coloration of the body setae was not useful to recognize the involved species, with a given color pattern being the same for different species. as another example, ferrari and melo (2014) studied some species of euglossa latreille with different color morphs and allopatric distributions. the authors had investigated the relationships of the specimens with molecular data and found that blue and green morphs corresponded to the same species (see also penha et al., 2015; frantine-silva et al., 2017). intraspecific color variation has already been documented for halictines, the most common variation involving the metasomal color of halictini thomson. the color morphs of halictus lutescens friese and lasioglossum apristum vachal are related to variation in body size (sakagami & okazawa, 1985; miyanaga et al., 1999). on the other hand, color morphs of lasioglossum politum schenck are linked with geographical distribution (murao & tadauchi, 2011). however, in augochlorini color variation is not necessarily related to distribution or behavior. previous cases have been reported by gonçalves (2017) for paroxystoglossa mourella gonçalves and by smith-pardo (2010) for neocorynura pseudobaccha cockerell and in both of them no relation with distribution and behavior was found. the blackish bees are unusual within halictinae, the previously known cases of black specimens on augochlorini coming from ariphanarthra palpalis moure, megalopta, neocorynura schrottky and pseudaugochlora michener. dark brown and black species can be found in most augochlorini lineages but no phylogenetic optimization was carried out to map the evolution of color within this bee clade. the black morph of a. amphitrite probably represents a derived condition. most of the examined species of augochlora are metallic green or blue, with few exceptions as the black and cupreous augochlora perimelas cockerell. the other species of the augochlora clade are metallic green, with some darkened specimens of augochlorella sandhouse (coelho, 2004). the visible green and blue colors result from light interaction with the integument microstructure (berthier, 2007; seago et al., 2009). on the other hand, the black color results from a melanization process, being an example, the complex regulation of melanin given by wittkopp and beldade (2009). the final insect color depends on a complex interaction of pigments and structural organization (ghiradella, 2009). the biochemical and genetic determination of the integument color can be even more complex and are an open field of investigation for bees. morphometric variation a. amphitrite exhibits a remarkable variation in body size and head shape (fig 3). the variation is continuous and does not correspond to true dimorphism among females, with a distinct macrocephalic morph as found in other groups such as rhinocorynura (gonçalves & melo, 2012). nevertheless, we use herein the term macrocephalic for the large females with enlarged gena and developed distal part of the mandibular adductor ridge. polymorphism in social species is usually associated with latitudinal and altitudinal gradients and also with seasonality (davison & field, 2016) but we did not find any pattern for a. amphitrite. division of labor involving females of a. amphitrite from the same nest was documented by dalmazzo and roigalsina (2012). the authors found that macrocephalic females stay inside the multi-female nests and probably act as dominant. this link of behavior, function and form has been widely documented for halictinae (richards & packer, 1996; soucy, 2002; richards et al., 2003) but no previous case involving augochlora sensu stricto was published. dalmazzo and roigalsina (2015) found that in augochlora phoemonoe schrottky there is no apparent structural differentiation between dominant and subordinate individuals, but body size and physiology differ between them. in augochlora istmii schwarz there is only size variation among nest mates (wcislo et al., 2003). more studies can reveal different degrees of morphological and behavior variation for species of augochlora. the swollen gena was already discussed as an important feature to identify macrocephalic forms in eusocial species of augochlorini (sakagami & moure 1965; santos & silveira, 2009). there is no study on head musculature for the tribe, but in ants the major forms with enlarged heads have developed muscles, including those for moving the mandibles a lepeco, rb gonçalves – augochlora amphitrite morphological variation668 (lillico-ouachour et al., 2018). the mandible is also modified in a. amphitrite (fig 3 c-d) with enlargement of the distal part of the adductor ridge in macrocephalic females. in addition to its role in nest construction, the mandible is also used in intranidal disputes, with important consequences on the dominance exerted by foundresses (pabalan et al., 2000; packer et al., 2003). females with enlarged heads in a. amphitrite probably have stronger musculature for mandible control, a hypothesis to be tested in further studies. other lineages of augochlorini have also intraspecific variation in head morphology. in megalopta, another group with wood nesting facultative eusocial species, the gena show different degrees of swelling and a genal projection of variable length is present (santos & silveira, 2009). in this bee tribe, the most remarkable cases of cephalic polymorphism come from the rhinocorynura group (gonçalves & melo, 2012). significant variation in size among females is observed within all species of rhinocorynura and in some species of its sister group (gonçalves, 2010). in the clade formed by rhinocorynura inflaticeps ducke + rhinocorynura vernoniae schrottky, the variation is also mostly continuous, but the females differ qualitatively: the larger ones have distinct lateral prongs in their clypeus. there is clearly a continuous variation among the larger females in the size of the lateral clypeal projection, but in the small females, the lateral projections are completely lacking. concluding remarks we presented a revised diagnosis for a. amphitrite to broader comparison with additional 21 species not studied by dalmazzo and roig-alsina (2011). the species revealed remarkable cases of color and shape polymorphisms. the color variation includes discrete morphs with unknown causes. the morphometric variation is probably related to nest interaction based on previous observations and do not follow any clear geographical pattern. based on the evidences from this study and from dalmazzo and roig-alsina (2012) a. amphitrite is considered facultative primitively eusocial and is a candidate to be a model for studies to link form, function and behavior within bees. acknowledgements we thank mauricio moura for the help with the statistical analysis, gabriel melo and john lattke for enlightenment discussions, and milagros dalmazzo for the comments on the manuscript. the manuscript title refers to the second foo fighters’ studio album. authors’ contribution rb gonçalves conceived the study. a lepeco performed the measurements, statistical analyses and specimen imaging. a. lepeco and rb gonçalves contributed equally to the writing of the manuscript. references aguiar, a. 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(africanized honeybee) with queens of different genetic origins introduction the hygienic behavior of honeybees is a natural resistance mechanism to the brood diseases and parasites (wilson-rich et al., 2009), characterized by uncapping and removal of dead and diseased brood, being an inherited characteristic (rothenbühler, 1964). the varroa destructor anderson and trueman mite (anderson & trueman, 2000) is an ectoparasite of brood and adult individuals of apis bees. since the v. destructor introduction in brazil for more than 30 years, infestation levels remain low and do not cause potential damage to colonies (junkes et al., 2007; wielewski et al., 2013; schafaschek et al., 2016). abstract the variables related to the varroa mite and the hygienic behavior in africanized honeybee colonies were evaluated from october 2013 to june 2014. three groups of different genetic origins were evaluated. one group consisted of queens from honeybee selection program of the maringá state university (maringá/pr). these queens were selected for royal jelly production by molecular markers for the expression of the mrjp3 protein. another group consisted of queens from the queen producer gian bejger (santa terezinha/sc). these queens were selected for hygienic behavior and monitored for varroa destructor anderson and trueman infestation. the last group was composed of queens from captured swarms/colonies that did not undergo any selection process and were chosen randomly in the apiary where the experiment was installed (irineópolis/sc). colonies with queens of maringá presented an increase in the invasion rate and total reproduction of varroa as there was reduction of hygienic behavior over the evaluation period. colonies with queens of santa terezinha presented the highest (p˂0.05) hygienic behavior with an average of 92.0%. this group presented the lowest (p˂0.05) total and effective reproduction of the mite (1.7 and 0.9 of total and fertile offspring, respectively). colonies with queens of irineópolis presented the lowest hygienic behavior (78.0%) (p˂0.05) and the highest total (12.6) (p˂0.05) and effective (5.3) (p˂0.05) reproduction of the mite. the use of selected queens, with hygienic behavior, interferes with the varroa population dynamics, contributing to the reduction of the invasion and total and effective reproduction rates of the mite. sociobiology an international journal on social insects tp schafaschek1, er hickel2, cal de oliveira3, vaa de toledo3 article history edited by cândida aguiar, uefs, brazil received 10 may 2018 initial acceptance 01 june 2018 final acceptance 15 april 2019 publication date 14 november 2019 keywords honeybee selection; parasite resistance; honey production corresponding author tânia patrícia schafaschek empresa de pesquisa agropecuária e extensão rural de santa catarina estação experimental de videira rua joão zardo, 1660, cep 89560-000 videira, santa catarina, brasil. e-mail: tanias@epagri.sc.gov.br several factors contribute to this condition, among them the presence of the africanized honeybee, which are more resistant than the european ones, mainly due to the reduced female fecundity of the v. destructor in the brood cells of africanized apis mellifera l. workers (rosenkranz et al., 2010). other important characteristics that limit the mite population are grooming behavior (moretto et al., 1993; moretto et al., 1995; nganso et al., 2017), mite mortality in adult honeybees (evans & spivak, 2010), removal of varroa-infested brood (guerra jr et al., 2000), descendant mites mortality (mondragón et al., 2006; rivera-marchand et al., 2012). the climatic conditions and the time of year 1 company of agricultural research and rural extension of santa catarina, videira experimental station, videira-sc, brazil 2 agricultural research and rural extension company of santa catarina, itajaí experimental station, itajaí-sc, brazil 3 department of animal science, maringá state university, maringá-pr, brazil research article bees open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.3444sociobiology 66(3): 448-456 (september, 2019) also influence the reproductive capacity of varroa (moretto et al. 1991; currie & tahmasbi, 2008), with mite females producing more offspring during pollen production than in other year seasons (mondragón et al., 2005). it is also found that there is a genetic association between the hygienic behavior and the infestation level in adult honeybees by v. destructor mite (arechavaleta-velasco et al., 2012; tsuruda et al., 2012). the africanized honeybees selection for the hygienic behavior characteristic decreases the total reproduction rate of the mite and, consequently, the infestation rate in adult honeybees (wielewski et al., 2012). some strains of honeybees have the ability to keep the mite population at low levels, apparently by reducing the reproductive success of mites in worker brood (harris et al., 2012). this behavior related to the removal of varroa-infested brood was described by harbo and harris (2009) as “varroa sensitive hygiene” (vsh). the two main characteristics of this behavior are the removal of infected brood and the high frequency of non-reproductive mites. recently has been found changes in the v. destructor reproduction rates in several regions of brazil that have been attributed to the change from the japanese haplotype (j) to the korean haplotype (k) of varroa (strapazzon et al., 2009). carneiro et al. (2007) found an increase in the reproductive capacity of v. destructor in state of santa catarina, where the mite fertile female percentage increased from 56% in the 1980s to 86% in 2005/2006. the difference in the female percentage that produced deutonymphs, that is, female offspring that can reach the adult stage was of 72% in 2005/2006, against 35% in 1986-1987. the recent arrival of the k haplotype in brazil (garrido et al., 2003; guerra et al., 2010) requires further investigation about the dynamics of this parasite-host interaction, as it is still little known whether the effects intensity caused by v. destructor depends on honeybee genotype, mite genotype or of the both (calderón et al., 2010). the objective of this research was to evaluate the relationship between the hygienic behavior traits, infestation and invasion rates, as well as the reproductive success of the v. destructor mite, in colonies of africanized honeybees with queens of different origins and selected for different traits. the hypotheses tested were: i. queens originated from a selection process present higher hygienic behavior; ii. the higher hygienic behavior interferes in the reproductive dynamics of v. destructor, reducing reproduction rates and consequently the infestation levels in adult honeybees. material and methods the experiment was carried out in an apiary located in the irineópolis municipality, in the state of santa catarina, brazil, at latitude 26°17’29” south and longitude 50°51’18” west, with an altitude of 762 m. the predominant climate in the region is the humid mesotherm, with mild summers (temperate or cfb of köppen). the predominant vegetation is mixed ombrophilous forest, with secondary vegetation and areas of agricultural activities. three groups of queens were evaluated, coming from different regions and selected by different processes for different traits. one group consisted of queens from honeybee selection program of the maringá state university, from maringá city, located in the northwest region of the paraná state, located at latitude 23°25’30” south and longitude 51°56’20” west, with altitude of 550 m and humid mesothermic climate, with hot summers (subtropical or cfa of köppen). these queens were selected for royal jelly production by molecular markers for the expression of the mrjp3 protein (baitala et al., 2010), showing a tendency of homozygosis for this locus (parpinelli et al., 2014) after years of selection. another group consisted of queens from the queen producer gian bejger of the santa terezinha municipality, in the state of santa catarina, located between the limits of the northern plateau of santa catarina and the upper itajaí valley, at latitude 26°46’43” south, longitude 50°00’29” west and altitude of 610 m, with predominant moist mesothermal climate with mild summers (temperate or cfb of köppen). these queens were selected for hygienic behavior and monitored for v. destructor infestation. the last group was composed of queens from captured swarms/colonies that did not undergo any selection process and were chosen randomly in the apiary where the experiment was installed (irineópolis / sc) described previously. for all groups evaluated, the queens were produced using the doolittle adapted method (1889). queens were mated in the air in local from origin of the groups using individual mating nucs. the queens were introduced in the experimental apiary from december 2012 to april 2013 in 40 standard langstroth hives with ten frames each, being 15 colonies from maringá/ pr, nine colonies from santa terezinha/sc and 16 colonies from irineópolis/sc. from these 40 colonies, those that survived the winter of 2013 were evaluated, being four from maringá, four from santa terezinha and 12 from irineópolis groups. in the region of the experimental apiary, the winter is from june 21 to september 21. the loss of many colonies in the winter of 2013, can be explained by a severe winter occurred, including the occurrence of snow, that is uncommon in the region. the establishment of the colonies with queens from other regions may have been difficult because an influence of climatic variables compared of the colonies with local queens, as verified by schafaschek et al. (2016). the introduction of the queen so late december to april, may have been difficult the initial development of this colonies because a shortage of flowering in this period in the region. therefore, many colonies from this group were lost in the winter. avoiding swarming and to provide space for colonies development, it was adopted in management the replacement of two to four combs with brood and feed by frames with new wax foundation, as the colonies presented total occupation tp schafaschek et al. – reproduction of varroa destructor and hygienic behavior450 of the nest combs. thus, the provision of space for the performance of the queen laying was provided according to the need of each evaluated genetic group, allowing the expression of its real development potential. the queen laying on the supers was avoided using queen-excluder screens. the supers were added according to the development of each colony and the nectar flow in the region. the evaluations were carried out monthly, from october 2013 to june 2014. the infestation percentage by v. destructor in adult honeybees, pupae invasion rate, mite total and effective reproduction, hygienic behavior of honeybees, total colony weight and honey productivity were evaluated. the v. destructor infestation in adult worker honeybees was evaluated using the method described by stort et al. (1981), in which approximately one hundred adult worker honeybees were collected from a comb and placed in a bottle with alcohol 70%. subsequently, stirred the contents and performed the separation of honeybees from the mites to evaluate the colony infestation, in percentage. the invasion rate in pupae was evaluated according to the method proposed by de jong and gonçalves (1981). this method included the retrieval of a comb of worker-capped brood from each colony and the removal of 100 worker pupae (50 from one side of the comb and 50 from another side) with brown eye and body at the beginning of pigmentation. adult female mites and their offspring (eggs, protonymphs and deutonymphs) in pupae and alveoli were analyzed by attached light and counted to obtain the invasion rate and the total and effective reproduction. the invasion rate in worker pupae was obtained by the formula: invasion rate (%) = (number of invaded pupae/number of analyzed pupae) x 100. total reproduction (rt), represented by the total number of offsprings produced by mite, was determined by the formula: rt = total number of offsprings/number of original adult female mites. the mite effective reproduction (re), that is, the number of viable progenies for reproduction, was determined by the formula: re = (number of deutonymphs + young adults)/nº of original adult mite females. the hygienic behavior, represented by the percentage of uncapped brood cells (%), was evaluated using the rothenbühler adapted method (1964). for this purpose, a comb containing pupae of workers corresponding to the pupal stage with pink eyes (17 to 18 days) was collected from each colony. a 5 x 6 cm comb section (approximately 100 capped brood-pupae of the comb) was cut and then frozen for 24 h. this section was photographed for later counting of the number of capped cells. after 24 h of freezing, the sections were returned to the respective colonies and, again, photographed 24 h after the return, for analysis of the uncapped wells. the colonies were weighed at night, when all worker honeybees were inside the hives. the hive weight was considered with the combs and the weight of the empty supers, feeder and queen excluder screen was discounted to calculate the weight of the colonies. the honey yield was evaluated after two harvests, carried out in november and december of 2013, being calculated by the weight difference between full and empty supers after extracting the honey. statistical analyzes of the differences in hygiene behavior traits, varroa invasion rate in worker pupae, total reproduction, effective reproduction and varroa infestation in adult honeybees according to the origin of the queen and the evaluation period as well as the interaction between these two factors were performed using the generalized linear models procedure, by proc genmod of sas version 9.3 (sas, 2012). the binomial distribution with logit binding function was admitted for the hygienic behavior and varroa invasion rate in worker pupae and the poisson distribution with log binding function was used for the total reproduction, effective reproduction and varroa infestation in adult honeybee traits. the means for these traits were compared by the t-test at the 5% level of significance. for this test the means were compared two by two. the colony weight data were submitted to variance analysis, implemented in proc glm of sas version 9.3 (sas, 2012), and linear regression curves were estimated as function of the evaluated months. the means for different groups of queens were compared by tukey test at the 5% level of significance. fig 1. invasion rate by varroa destructor in worker pupae (a) and hygienic behavior of apis mellifera bees (b) in colonies with queens from maringá/pr, from santa terezinha/sc and from irineópolis/sc. sociobiology 66(3): 448-456 (september, 2019) 451 results there was an interaction effect between the origin of the queen and the evaluation period for the varroa invasion rate in worker pupae (fig 1 a). the hygienic behavior showed effect of the origin of the queen and interaction between the origin of the queen and the time of evaluation (fig 1 b). there was also interaction between the origin of the queen and the evaluation time for the number of adult female mites in worker pupae cells. thus, this variable was not included in the evaluation model of total offspring number and the fertile offspring number. the number of adult mites had quadratic interaction with the total offspring number and linear interaction with the fertile offspring number. the total and effective reproduction had an effect of the origin of the queen for the maringá and santa terezinha groups and interaction effect between the origin of the queen and the evaluation period for the three groups evaluated. this interaction was quadratic for maringá (fig 2 a) and irineópolis (fig 2 c) groups and linear for santa terezinha (fig 2 b) group. fig 2. number of adult mites in worker cells. number of total offspring and number of fertile offspring of v. destructor in a. mellifera bee colonies with queens from maringá/pr (a), santa terezinha/sc (b) and irineópolis/sc (c). tp schafaschek et al. – reproduction of varroa destructor and hygienic behavior452 varroa infestation in adult honeybees had an isolated effect of the origin of queen and the evaluation period (fig 3). as expected, the number of mites detected in the samples was in function of the number of collected honeybees. so to control the sampling, the number of collected honeybees was considered as covariate in the model in order to improve accuracy and reduce model error. it was found that the lowest infestation rates occurred in november and december (fig 3). the mean infestation rates are presented in table 1. the hygienic behavior was related to the varroa invasion rate in pupae and mite total reproduction. however, this relationship presented different behavior for each queen group. it was found, by means of the equation derivation, that for the queens from maringá, when a decrease of the hygienic behavior began, near february of 2014, there was a marked increase of the varroa invasion rate in pupae and in the its total reproduction. effective reproduction also increased, but in a later period, as of may 2014. the mean for this variable over the period was 2.3 fertile offspring and the hygienic behavior of 84.0% (table 1). differently from the other groups of queens, the maringá group had a reduction of the mite effective reproduction until february of 2014 (fig 2 a). the santa terezinha group also presented an increase in the varroa invasion rate in pupae from the reduction of hygienic behavior (fig 1). however, they had high rates of hygienic behavior and remained stable throughout the period, with an average of 92.0%. they also presented the lowest (p˂0.05) values of total reproduction (1.7), and effective reproduction (0.9) of the mite (table 1). the irineópolis group presented the lowest mean (p˂0.05) of hygienic behavior (78.0%), occurring an increase in the invasion rate, with the decrease of this behavior. they presented maximum value of total and effective reproduction in february 2014 (fig 2 c), when the lowest values of hygienic behavior were observed (fig 1 c). this group presented the highest mean (p˂0.05) of total reproduction and the effective reproduction (table 1). table 1. mean ± standard error, for the analyzed variables, from october 2013 to june 2014, in colonies with queens from maringá/ pr, santa terezinha/sc and irineópolis/sc, in irineópolis, santa catarina state, brazil. varroa invasion rate in worker pupae (%) hygienic behavior (%) total reproduction (number of total descendants) effective reproduction (number of fertile offspring) varroa infestation in adult bees (%) maringá/pr 2.0±0.10 b¹ 84.0±0.06 b 5.7±0.09 b 2.3±0.14 b 4.9±0.07 b santa terezinha/sc 1.0±0.20 c 92.0±0.08 a 1.7±0.16 c 0.9±0.23 c 3.5±0.96 c irineópolis/sc 6.0±0..06 a 78.0±0.03 c 12.6±0.04 a 5.3±0.06 a 6.1±0.45 a ¹means followed by the same letter in the same column are not statistically different (p> 0.05). by the t test. fig 3. infestation by varroa destructor in apis mellifera bee colonies with queens from maringá/pr (a), santa terezinha/sc (b) and irineópolis/sc (c). as expected, there was an effect of the evaluation period on the colonies weight (fig 4), with a significant reduction in the weight from january. this was possibly due to the reduction in the bee population due to the decrease in the availability of food resources in the environment. the honey productivity was evaluated only for the groups from santa terezinha and irineópolis. the maringá group delayed the honey storage in the supers. this group presented a rapid development of the colony, driven by the high rate of laying of the queens. however, the use of the sociobiology 66(3): 448-456 (september, 2019) 453 queen excluder screens, in this case, provided little space for the colony development, causing high swarming rates in the colonies of this group. this fact did not allow sufficient workers to carry out nectar storage, and consequently, honey production was impaired. so, this group presenting an insufficient amount of honey and only at the end of the apicultural crop, that is, at the end of december and beginning of january. due to the shortage of flowering from this period, it was decided not to harvest the honey in the maringá group not to harm the survival of the colonies. there was no significant difference in honey productivity between santa terezinha and irineópolis groups. the mean honey production was 16.3 kg/colony for santa terezinha and 13.4 kg/colony for the irineópolis groups. more recently, carneiro et al. (2014) evidenced an increase of 22% in relation to carneiro et al. (2007), with a mean number of 3.2 descendants per reproductive cycle. besides, the beekeepers of the states of santa catarina and rio grande do sul are bringing thousands of european queen honeybees from argentina in last years, what can explain this arise in mite reproduction. the selection process of the santa terezinha group, based on high hygienic behavior and low varroa infestation, was probably the one that input the trait of reduced total and effective reproduction, associated to the high hygienic behavior of this group. the introduction of genetically selected queens in this experiment, contributed to the reduction of total and effective reproduction of mite, since the group of irineópolis queens, which did not undergo a rigorous selection process, presented a total and effective reproduction significantly high (12.6 and 5.3, respectively). this group also had the highest rates of infestation in adult honeybees (6.1%) (table 1). the results agree with wielewski et al. (2012), where the low total reproduction represented low invasion and infestation rates by varroa for the groups with selected africanized honey bee queens. harbo and harris (2001) also found that queens selected for decreased varroa mite reproduction maintain this trait even when free-mated with unselected drones, working with european honeybee. the finding that the maringá group presented reduction of the effective reproduction until the month of february of 2014, is possibly due to some traits of this group acquired by the selection process. despite the increase in hygienic behavior until the month of february, there was no reduction in total reproduction, except for effective reproduction. some important traits that limit the mite population in africanized a. mellifera are the grooming behavior, adult bees mite mortality (junkes et al., 2007) and descendants mite mortality (mondragón et al., 2006). the selection of honeybees for hygienic behavior possibly also selected for these other limiting traits for mite population in the colony. calderón et al. (2010) reported that the reduction of the varroa mite reproductive capacity is the most important factor in the tolerance of honeybees to this parasite. the limitation of mite growth population due to the reduction of its reproductive capacity was also found in colonies of european honey bee by locke and fries (2011). pinto et al. (2012) found a negative correlation between invasion rate and hygienic behavior in africanized honeybees from taubaté and viçosa, in the southeastern region of brazil, suggesting that colonies with higher hygienic behavior have the lowest rates of varroa invasion. another factor explaining these results is that the maringá group, from a honeybee selection program, has been working since 2007 with molecular markers for the expression of the mrjp3 protein involved in the royal jelly production (baitala et al., 2010). parpinelli et al. (2014) indicated that these honeybees tended to homozygosis for this locus. li et al. fig 4. weight of apis mellifera bee colonies regardless of the queens’ origin from october 2013 to june 2014. discussion the results found in this research are in agreement with wielewski et al. (2012), who found that the selection of africanized honeybees for hygienic behavior traits reduces the infestation rate in adult honeybees by varroa, by reducing the mite total reproduction. these authors obtained values of total reproduction of 1.02 individuals for the same lineage of the maringá group, evaluated in maringá / pr as well as infestation rates of 8.3% and invasion rates of 9.5%. however, in this experiment the mean value of total reproduction for maringá group was higher (5.7), infestation rates (4.9%), and invasion rates (2.0%) were lower than those found by wielewski et al. (2012). the total reproduction values obtained for the santa terezinha group (1.7) are similar to those detected by moretto et al. (1996), which found a total reproduction of 1.7 descendants evaluating colonies of africanized honey bee in the rio do sul municipality/sc, in the period of 1986/1987. carneiro et al. (2007), however, found changes in the v. destructor reproductive capacity in the state of santa catarina, after 20 years, where there was a 60% increase, with females leaving around 2.6 descendants in the region of blumenau/sc. tp schafaschek et al. – reproduction of varroa destructor and hygienic behavior454 (2008), analyzing the proteins of the hypopharyngeal glands of winter honeybees, selected for high royal jelly production, detected a great variety of proteins, similar to those found in royal jelly, especially mrjp3. mrjps are the main proteins present in the royal jelly secreted by the hypopharyngeal glands of the workers. studies on the influence of brood attractiveness on varroa invasion rate have shown that queen larvae and queen larvae extracts were significantly less attractive than worker and drone larvae and that the royal jelly presented a repellent effect (calderone et al. 2002). in general, the levels of varroa infestation in adult honeybees found in this study were low. however, it was found that the groups with selected queens, maringá group, selected by molecular markers for the mrjp protein expression and the santa terezinha group, selected for low v. destructor infestation and high hygienic behavior had the lowest rates of varroa infestation in adult honeybees, while the irineópolis group, with unselected queens, had the highest varroa infestation. the values found for maringá and santa terezinha groups confer with the infestation indexes found by strapazzon et al. (2009) and carneiro et al. (2014), in the region of blumenau/sc, which remained around 3.0% and 4.1%, respectively. the infestation levels observed in this research presented a reduction in the spring and summer months, increasing from the end of summer and reaching the highest levels in the autumn months for the three groups of evaluated queens (fig 3). the results are similar to those obtained by toledo and nogueira-couto (1996), who found that the infestation rate in adult africanized honeybees decreased from winter to spring decreased in the summer and increased again in the fall. this behavior was possibly due to the reduction of the honeybee population in the colonies in the following period at the end of the summer and during the winter, leading to a higher concentration of the mites on the remaining honeybees. the delay in the honey storage in colonies with queens from maringá may have been influenced by the management adopted with the use of queen excluder. the limited space in the nest due to the use of these screens may have caused the swarming of the colonies with higher posture capacity. this provided less population of foraging honeybees during the period of greater nectar flow, which occur from september to november, delaying the storage for the harvest. the results corroborate with faquinello et al. (2011) who found that superior genotypes can express themselves differently depending on the production system to which they are submitted. schafaschek et al. (2016) also found that queens from different regions had their development influenced by the adopted management and local climatic variables, with advantage to the adapted local queens. therefore, it is recommended the adoption of different management for colonies with queen from other regions. as well as, selected queens for high production and with high laying ability need be provided with ideal conditions for production, as space for laying and abundant food. this may contribute to the higher expression of productive potential of the selected strains. the results confirm the first tested hypothesis that the queens originated from a selection process present higher hygienic behavior. in the experiment the queens originated from selection processes (maringá/pr and santa terezinha/ sc) had the highest mean of hygienic behavior. the second hypothesis tested, that the higher hygienic behavior interferes in the reproductive dynamics of v. destructor, reducing reproduction rates and consequently the infestation levels in adult honeybees, was also confirmed. it was found that the high hygienic behavior promoted the reduction of the invasion, total and effective reproduction rates of the v. destructor mite and in the infestation levels in adult honeybees. acknowledgment to the beekeepers gian bejger and domingos bejger, for the donation of queens of one evaluated group. to the bee research group gpbee, of the maringá state university, for aid in queen production. to cnpq, processes numbers 479329/2009-5, 308283/2011 and 311663/2014-1, and fundação araucária protocol 15095 agreement 422, for the financial support. references anderson, d.l. & trueman, j.w.h. 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(2009). genetic, individual, and group facilitation of disease resistance in insect societies. annual review of entomology, 54: 405-423. doi: 10.1146/annurev.ento.53.103106.093301 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.225-233sociobiology 61(2): 225-233 (june, 2014) the social wasp fauna of a riparian forest in southeastern brazil (hymenoptera, vespidae) ga locher1, oc togni1, ot silveira2, e giannotti1 abstract introduction native forests have been exploited for years in a degrading manner, resulting in a number of environmental problems such as species extinctions, local climate change, soil erosion, siltation and eutrophication of watercourses (ferreira & dias, 2004). in order to monitor the effects of changes in the environment, to develop conservation strategies on a local scale, information on biodiversity of an area is essential. the concept of diversity can be divided into alpha, beta and gamma, namely the biological diversity in natural and changed communities, the biodiversity exchange rate between different communities, and the species richness of all the communities which integrate a landscape, respectively (moreno, 2001). an inventory of a local fauna which generates data on alpha diversity is the first step in conservation biology. brazil hosts a great diversity of social wasps, reaching a total of 319 species, about 33% of the social wasp species described worldwide (prezoto et al., 2007). due to this high diversity and the importance that has been assigned to this an inventory of social wasps was carried out on a monthly basis, from march 2010 to march 2011 in a section of riparian forest along the passa-cinco river in ipeúna, são paulo state, brazil. two active collecting methods (active collecting and point sampling using a liquid bait) and one passive method (baited pet bottle trap) were used. the results increased the data on the diversity of social wasps of the state and were used to compare richness, equitability and diversity obtained with several collecting methods which have been employed to social wasps. thirty-one species belonging to eight genera were recorded; the most abundant were agelaia vicina (de saussure) and agelaia pallipes (olivier). both species belong to the tribe epiponini, which was dominant in the sample. regarding sampling methods, the active collecting ones sampled the greatest richness value and the highest shannon-wiener diversity index and also presented the largest number of exclusive species. however, the other methods have also obtained exclusive species. sociobiology an international journal on social insects 1 instituto de biociências, universidade estadual paulista, rio claro, sp, brazil. 2 museu paraense emílio goeldi, belém, pa, brazil. article history edited by gilberto m m santos, uefs, brazil received 23 august 2013 initial acceptance 21 november 2013 final acceptance 23 march 2014 keywords richness, abundance, polistinae, attractive traps, active sampling corresponding author gabriela de almeida locher departamento de zoologia instituto de biociências universidade estadual paulista 13506-900 rio claro, sp, brazil e-mail: gabriela.locher@gmail.com group of insects, researches related to diversity of social wasps in this country are increasing in number and have covered different regions, environments and biomes (richards, 1978; rodrigues & machado, 1982; marques, 1989; marques et al., 1993; diniz & kitayama, 1994; mechi, 1996; santos, 1996; lima et al., 2000; raw, 2003; marques et al., 2005; mechi, 2005; melo et al., 2005; hermes & köhler, 2006; santos et al., 2006; silva-pereira & santos, 2006; souza & prezoto, 2006; elpino-campos et al., 2007; santos et al., 2007; morato et al., 2008; ribeiro junior, 2008; silveira et al., 2008; souza et al., 2008; gomes & noll, 2009; santos et al., 2009a; santos et al., 2009b; alvarenga et al., 2010; auad et al., 2010; lima et al., 2010; prezoto & clemente, 2010; santos & presley, 2010; souza et al., 2010; pereira & antonialli-junior, 2011; silva et al. 2011; souza et al., 2011; tanaka junior & noll, 2011; jacques et al., 2012; silveira et al., 2012; simões et al., 2012 ; auko & silvestre, 2013; grandinete & noll, 2013; togni et al. 2014). in the state of são paulo, studies on diversity of social wasps are still scarce, mainly focusing on the northwestern region, with studies in localities such as paulo de research article wasps ga locher et al the social wasp fauna of a riparian forest226 faria, pindorama and neves paulista (gomes & noll, 2009), patrocínio paulista (lima et al., 2010), magda, bebedouro, matão and barretos (tanaka junior & noll, 2011); on the central eastern region, with researches in rio claro (rodrigues & machado, 1982), luíz antônio, corumbataí (mechi, 1996), and santa rita do passa quatro (mechi, 2005); and finally at the littoral in ubatuba (togni et al., 2014). according to the above information, this study aimed to conduct an inventory of the diversity of social wasps in an area of riparian forest in the central eastern region of são paulo state, increasing the data on the diversity of social wasps of the state and to compare richness, equitability and diversity obtained through the several methods used. materials and methods social wasps (vespidae, polistinae) were sampled monthly from march 2010 to march 2011 along the western edge of a riparian forest fragment (semideciduous alluvial forest, according to rodrigues, 1999) by the margins of the passa-cinco river, in a landscape with the surrounding matrix composed by sugarcane plantations, in the rural municipality of ipeúna state of são paulo (22 ° 24’52 .55 “s, 47 ° 43’35 .55” w). the passa-cinco is one of the main rivers of the corumbataí river basin, in a region that presents a subtropical climate (köppen cwa type) characterized by dry winter and rainy summer, with an average temperature in the warmest month of up to 22˚c (palma-silva, 1999). originally, the watershed vegetation was mainly composed of semideciduous forests, and “cerrado” vegetation spots in smaller areas (koffler, 1993 as cited invalente & vettorazzi, 2002). according to valente and vettorazzi (2002) the sub-basin of passa-cinco river had in year 2000 51.72% of its surface occupied by pasture areas, 14.3% by sugarcane, 15.67% native forest, 10.5% planted forest, 0.74% cerrado, 0.5% urban areas and 6.47% by others. three different methods were used for sampling the wasps: baited pet bottle trap, active collecting, and point sampling using a liquid bait. the baited pet bottle trap method consisted in 10 attractive traps made with 2 l pet type bottles (polyethylene terephthalate bottles) installed along a 1000 meter trail bordered by riparian vegetation and sugarcane plantation. the traps were set on the edge of the vegetation at a height of approximately 1.5 meters (ribeiro junior, 2008), with a 100 meters interval between traps (togni et al., 2014). each bottle had four circular orifices at the middle, and contained 200 ml of attractive liquid (modified from souza & prezoto, 2006) consisting of natural industrialized guava juice and sugar solution (togni et al., 2014). after a week, traps were removed, the attractive liquid sifted and all social wasps encountered were fixed in 70% ethanol. active collecting (without the use of any attractive) consisted in active searching for individuals along the same 1000 meter trail mentioned before collected with an entomological net, at the time of greatest foraging activity between 10:00 am to 3:00 pm (see prezoto et al., 2008). rounds of active collecting were made at two different days each month by four independent collectors on the same transect where the bottle traps were placed. the collected individuals were associated with the nearest trap point for further analysis and comparisons. the method of point sampling using a liquid bait was made with a sucrose solution (1: 4, commercial sugar: water) with 2 cm³ of salt for each half liter of solution which was sprayed over the vegetation at 10 marked points along the sampling trail (with a distance of 100 meters between points). at least 30 minutes after spraying, active searching was performed by two independent collectors on the vegetation for 3 minutes with the aid of an entomological net (modified from liow et al., 2001; lima et al., 2010; tanaka junior & noll, 2011). the species relative abundances were computed by dividing the number of collected individuals of each species by the total abundance found (expressed as percentage) and the richness value is the total number of species encountered. the richness value obtained by each method was divided by the total richness sampled to evaluate the efficiency of each of them. we calculated the shannon-wiener index and equitability of samples obtained by each method according to the formulas from krebs (1998) in order to compare the diversity sampled by each of them. the rarefaction curve based on samples was obtained using the mao tau estimator (colwell et al., 2004) calculated with estimatesversion 8.2 (colwell, 2009) and used to compare the three methods based on the monthly richness sampling effort. the species accumulation curve for months of collection was calculated through the addition of new species found every month. to analyze the equality of samples obtained from each sampling method, the kruskal-wallis test (bioestat 5.0 version 5.0 (ayres et al., 2007)) was used. for the test, the raw abundance data of each species, and the relative abundances (percentage), were used. results and discussion social wasps community in ipeúna sp after 13 months of sampling activity in the rural area in ipeúna, são paulo state, 31 species of eight genera of social wasps were found. a total of 954 individuals was collected and 86.58% belonged to the epiponini tribe, while only 9.43% belonged to the tribe polistini and 3.98% to the tribe mischocyttarini (table 1). other studies have also noted the predominance of epiponini in the total abundance of social wasps found. representatives of this tribe constituted more than 70% in many studies conducted in brazil (santos, 1996; mechi, 2005; hermes & köhler, 2006; santos et al., 2006; sociobiology 61(2): 225-233 (june, 2014) 227 elpino-campos et al., 2007; morato et al., 2008; gomes & noll, 2009; santos et al., 2009b; silva & silveira, 2009; auad et al., 2010; alvarenga et al., 2010; lima et al., 2010; pereira & antonialli-junior, 2011; tanaka junior & noll, 2011; simões et al., 2012; grandinete & noll, 2013; togni et al., 2014). however, when only nests are the targets of surveys, the abundance of epiponini is often smaller, as observed in juiz de fora, minas gerais (lima et al., 2000), with only 11.03% of the colonies belonging to this tribe and in biritinga, bahia, were 67.43% of the total abundance found was of epiponini, yet only 25.71% of the nests found belonged to this tribe (santos & presley, 2010). this is probably due to greater depredation of epiponini nests by humans because of their large size, to destruction by rain and also by the greater facility of finding mischocittarini and polistini nests next to human constructions. differently from the other polistine tribes in the neotropics, epiponini is composed of swarming species that may construct large nests and consequently have greater abundance of individuals (jeanne, 1991; carpenter & marques, 2001), a factor that certainly concurs to the high occurrence of specimens from this group in active and passive collections, highlighting the importance of this tribe to the ecosystem studied. the species with greater abundances (table 1) were agelaia vicina (404 individuals, relative abundance = 42.35%) and a. pallipes (138 individuals, relative abundance = 14.47%). agelaia vicina is known as the polistine species with the largest nests and colonies, and zucchi et al. (1995) reported the existence of a colony with more than one million adult individuals and nest with over 7.5 million cells. oliveira et al. (2010) added that the high growth rate, large population and high number of queens provide nests with such proportions. the magnitude of these colonies certainly explains the larger abundance of individuals of this species in the present work, which might in fact be produced by just a few colonies in the area. oliveira et al. (2010) suggest that a. vicina should be considered in many environments as a keystone species, i.e. as populations which determine the stability (integrity and persistence over time) of a community through their activities and abundances (paine, 1969). in addition to having very large colonies and nests, a. vicina presents elevated offspring production rate, requiring therefore a large amount of prey, consisting of a large diversity of arthropods, and severely impacting their populations locally (oliveira et al., 2010). among the species belonging to the tribe mischocyttarini, that with the greatest abundance was mischocyttarus drewseni with 26 individuals collected, i.e. 2.73% of the total. m. drewseni, which is widely distributed in brazil (richards, 1978), rarely presents colonies with more than 30 individuals (jeanne, 1972), and was diagnosed by souza et al. (2010) as an indicator of degraded areas and strong human pressure in riparian forest of rio das mortes, in barroso, minas gerais. the species with the greatest abundance belonging to the polistini tribe was polistes versicolor with 37 specimens (3.88%). plt. versicolor is another species with a wide distribution in brazil (richards, 1978), which has been suggested as a biological control agent in certain crops, such as for control of the eucalyptus defoliator (elisei et al., 2010). a colony of plt. versicolor can collect around 4015 preys during a year, consisting in a majority (but not exclusively) of lepidopteran larvae (prezoto et al., 2006). among the species found in the area, only apoica pallens had not been recorded by richards (1978) in the state of são paulo, but it has been sampled in other inventories (rodrigues & machado, 1982; mechi, 1996; togni et al., 2014). while recorded by richards (1978) for são paulo, polistes ferreri and mischocyttarus mattogrossoensis were not found by other authors. these species may be rather easily misidentified due to close similarity with other more abundant species. the wasp m. mattogrossoensis is apparently restricted to cerrado areas (raw, 2003) as shown by the recorded occurrences in brazil: mato grosso (richards, 1978; diniz & kitayama, 1994; 1998), goiás (raw, 2003) and mato grosso do sul (grandinete & noll, 2013). however this species was also recorded in an urban environment in cruz das almas, bahia (marques et al., 1993). comparing the studies on diversity of social wasps carried out in the state of são paulo (rodrigues & machado, 1982; mechi, 1996; mechi, 2005; gomes & noll, 2009; lima et al., 2010; tanaka junior & noll, 2011; togni et al., 2014) a survey accomplished at rio claro (rodrigues & machado, 1982; in an environment comprised of eucalyptus plantations with secondary regrowth of semideciduous seasonal forest, approximately 25 kilometers away from ipeúna) showed the greatest richness (33 species), with the present study coming next with 31 species. the survey at rio claro, although using a quite distinct sampling method (active search for colonies during 13 years) and being performed in a area with a much larger area with 2230 hectares, showed 21 species in common with that of ipeúna. nine species (polybia bifasciata, pseudopolybia vespiceps, agelaia vicina, mischocyttarus mattogrossoensis, polistes acteon, plt. ferreri, plt. geminatus, plt. lanio and plt. subsericeus) were exclusive to ipeúna, whereas 12 species (polybia platycephala, protonectarina sylveirae, protopolybia exigua, p. sedula, parachartergus pseudapicalis, apoica flavissima, mischocyttarus araujoi, m. cerberus, m. labiatus, m. latior, polistes canadensis and plt. consobrinus) were exclusive to rio claro. despite these differences, it is important to emphasize that 63.63% of the species sampled more than 30 years ago in rio claro were found in ipeúna (even considering the distinctly smaller collection effort), thus showing a noticeable similarity between those social wasps communities. it is truly remarkable that even a small fragment of riparian vegetation with very strong anthropic influences due to nearby large plantations, has also obtained a very impressive species richness. it is noteworthy that the idea of high diversity in this degraded area, may in fact be indicative of ga locher et al the social wasp fauna of a riparian forest228 table 1. abundance of wasp species collected with the three methods (ac active collection, ps point sample using a liquid bait and bt attractive pet bottle traps), total abundance and relative abundance ( %) of species collected in passa cinco river’s riparian forest in ipeúna. (ac active collection, ps point sample, bt bottle traps). species ac ps bt total relative abundance agelaia multipicta (haliday, 1836) 1 2 0 3 0.31 agelaia pallipes (olivier, 1791) 20 32 86 138 14.47 agelaia vicina (de saussure, 1854) 159 93 152 404 42.35 apoica pallens (fabricius, 1804) 0 0 1 1 0.1 brachygastra augusti (de saussure, 1854) 1 1 0 2 0.21 brachygastra lecheguana (latreille, 1824) 3 1 0 4 0.42 polybia chrysothorax (lichtenstein, 1796) 4 0 10 14 1.47 polybia dimidiata (olivier, 1791) 33 5 17 55 5.77 polybia fastidiosuscula de saussure, 1854 0 9 32 41 4.3 polybia ignobilis (haliday, 1836) 19 4 23 46 4.82 polybia jurinei de saussure,1854 0 0 3 3 0.31 polybia gr. occidentalis sp 1 (olivier, 1791) 12 16 2 30 3.14 polybia gr. occidentalis sp 2 (olivier, 1791) 7 12 7 26 2.73 polybia paulista h. von ihering, 1896 10 10 3 23 2.41 polybia sericea (olivier, 1791) 12 5 3 20 2.1 polybia bifasciata de saussure, 1854 0 1 0 1 0.1 pseudopolybia vespiceps (de saussure, 1864) 7 4 0 11 1.15 synoeca cyanea (fabricius, 1775) 4 0 0 4 0.42 epiponini’s total 292 195 339 826 86.58 mischocyttarus cassununga (von ihering, 1903) 5 0 0 5 0.52 mischocyttarus drewseni de saussure, 1857 22 4 0 26 2.73 mischocyttarus mattogrossoensis zikán, 1935 3 0 0 3 0.31 mischocyttarus rotundicollis (cameron, 1912) 3 0 1 4 0.42 mischocyttarini’s total 33 4 1 38 3.98 polistes actaeon haliday, 1836 2 0 0 2 0.21 polistes billardieri fabricius, 1804 3 2 1 4 0.42 polistes cinerascens de saussure, 1854 6 0 0 8 0.84 polistes lanio (fabricius, 1775) 0 0 2 2 0.21 polistes ferreri de saussure, 1853 4 1 3 8 0.84 polistes simillimus zikán, 1951 7 5 3 15 1.57 polistes geminatus fox, 1898 4 0 0 4 0.42 polistes subsericeus de saussure, 1854 6 4 0 10 1.05 polistes versicolor (olivier, 1791) 6 14 17 37 3.88 polistini’s total 38 26 26 90 9.43 total abundance 363 225 366 954 100 total richness 26 20 18 31 exclusive richness 6 1 3 sociobiology 61(2): 225-233 (june, 2014) 229 an impoverishment of the social wasps fauna of the state of são paulo that may be happening for a long time, making thirty-one species appear to be a high diversity when compared with other inventories carried out in the state. comparison of sampling methods with regard to the methods used for sampling social wasps (active collecting, point sample using liquid bait, and baited bottle traps), a remarkable aspect to note was their large complementarities, with only 38.71% of the species being collected by all three methods (table 1). by using the active collection method, 363 individuals belonging to 26 species (seven genera) were obtained. six species were captured exclusively by this method: synoeca cyanea, mischocyttarus cassununga, m. mattogrossoensis, polistes actaeon, plt. cinerascens and plt. geminatus. among these species, plt. actaeon and m. cassununga have already been collected by attractive pet bottle traps by souza et. al. (2011) e jacques et al. (2012) respectively. synoeca cyanea, as well as m. cassununga were also previously sampled by passive trapping, actually with malaise trap by auad et al. (2010). the other species were captured in other studies with different active collecting methods, as active collecting with liquid bait (plt. geminatus (tanaka junior & noll, 2011), s. cyanea and m. cassununga (lima et al., 2010), m. mattogrossoensis (grandinete & noll, 2013)) and active collection in quadrants and point sampling (m. cassununga and plt. cinerascens, (souza & prezoto, 2006)). the method of point sampling using a liquid bait was responsible for the capture of 225 individuals belonging to 20 species (six genera), with only polybia bifasciata being exclusive. this species was previously captured by souza and prezoto (2006) with the method of point sampling, while togni (2009), jacques et al. (2012) and simões et al. (2012) obtained specimens using attractive traps. the method of baited pet bottle traps collected 366 specimens belonging to 18 species (five genera), of which apoica pallens, polybia jurinei and polistes lanio were exclusives to this technique. these species, however, had already been sampled by different methods in other studies: apoica pallens, though being a species with nocturne foraging habit (hunt et al., 1995), which would explain absence in the active collections during daytime, was otherwise captured actively during the day by mechi (1996), souza and prezoto (2006), elpino-campos et al. (2007), silveira et al.(2008), clemente (2009), silva and silveira (2009) and souza et al. (2011). polybia jurinei was found by active collection by souza and prezoto (2006), elpino-campos et al. (2007), silva and silveira (2009) and jacques et al. (2012); by active collection in flowering plants by mechi (1996; 2005), and by active collecting with the use of liquid bait by noll and gomes (2009), lima et al. (2010) and grandinete and noll (2013). polistes lanio was collected by active collection by simões et al. (2012) and by active collection while scanning flowering plants by mechi (1996; 2005). the most efficient method, regarding the number of species collected, was the active collection, responsible for capturing 83.87% of the species, followed by point sampling using a liquid bait (64.52%) and baited pet bottle traps (58.06%). when adding the samples obtained through active collection and point sample using liquid bait, quite different from the method that uses attractive pet bottle traps which is characteristically passive, 28 species were sampled (90.32% of total richness) with 13 being exclusive of those methods. the diversity index of shannon-wiener (h') for the total sample was 2.248 and the evenness (j’) was 0.655. comparing the methods employed regarding these indexes, it was noted that the highest shannon-wiener index was obtained by active collection (h‘ = 2.297), which is even higher than that obtained for the entire sample, whereas the lowest index was obtained by the attractive pet bottle traps method (h‘= 1.854). the equitability obtained in the area was very similar between active collection (j‘ = 0.705) and point sampling using a liquid bait (j’ = 0.717) and was lower for samples collected using attractive pet bottle traps (j ‘= 0.641). in this way, we note that the attractive pet bottle traps were responsible for less homogeneous samples regarding the species abundance, with a less equity in these samples and therefore a lower diversity index. when the methods are analyzed as a sole group regarding the difference of abundance sampled, there is no significant difference between them (h' = 5.202, df = 2, (p) kruskal-wallis = 0.0742) and the same occurs when using the species relative abundance of each method in the analysis (h' = 4.348, df = 2, (p) kruskal-wallis = 0.114). by comparison of the curves of species accumulation with the rarefaction curves (figure 1) it is possible to see that the curve of richness accumulation obtained through active collection is that closest to an asymptote, seeming to stabilize in the last four collection days. it is worth noting that the curve obtained for the rarefaction of baited bottle traps tends to an asymptote in a similar way to the curve of active collection, although with a smaller number of species than that obtained by the other techniques, indicating a false idea of good sampling. therefore, the method shown to be more efficient was the active collection, since it was responsible for the largest number of species obtained exclusively by one method, for the highest shannon-wiener index and a rarefaction curve compatible with the curve of expected species. this method had already been reported as the most efficient in respect of richness by other researches (souza & prezoto, 2006; elpinocampos et al., 2007; silva & silveira, 2009; souza et al., 2011; jacques et al., 2012; simões et al., 2012). the low equitabilities and richness obtained for attractive traps, indicate the need for additional sampling with other methods. however, the occurrence of exclusive species demonstrates the importance ga locher et al the social wasp fauna of a riparian forest230 of this technique. therefore the use of more than one collection method is indicated in conducting inventories of social wasps, as proven in other studies (silveira, 2002; souza & prezoto, 2006; ribeiro junior, 2008; clemente, 2009; togni, 2009; souza et al., 2011; jacques et al., 2012; simões et al., 2012). acknowledgments we thank the brazilian national council for scientific and technological development (cnpq) for financial support and the brazilian institute for the environment and renewable natural resources (sisbio / ibama) for the authorization for collection and transport of specimens, number 22250-1. references alvarenga, r.d., de castro, m.m., santos-prezoto, h.h. & prezoto, f. 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(2002). análise da estrutura da paisagem na bacia do rio corumbataí, sp. scientia forestalis, 62: 114-129. zucchi, r., sakagami, s.f., noll, f.b., mechi, m.r., mateus, s., baio, m.v. & shima, s.n. (1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3779sociobiology 66(2): 339-347 (june, 2019) morphometric changes in three species of euglossini (hymenoptera: apidae) in response to landscape structure introduction the progressive degradation of terrestrial ecosystems has brought several challenges to conservation proposals, especially because some organisms are sensitive to changes in the environment, such as habitat fragmentation. according to bennett and saunders (2010) such fragmentation is a threat to populations, for it may cause loss of genetic diversity through inbreeding, variations in microclimatic conditions and in food sources, which also affects birth and mortality rates, abstract habitat fragmentation and expansion of agricultural activities are anthropic factors that can strongly impact biodiversity. thus, sustainable practices of land use, such as agroforestry systems, are adopted with the proposal of improving environmental quality and restore ecological processes. in flying insects, habitat fragmentation may cause changes in the wing shape and size. therefore, we hypothesized that landscape structure would affect wing size and shape in three species of euglossini (eulaema atleticana nemésio, euglossa cordata (linnaeus) and euglossa ignita smith). the analysed specimens were collected in five areas, four forest areas with strong anthropic influence and an agroforestry system area. the results of the wing shape analysis have showed that the individuals of the three collected species within the agroforestry system diverge significantly (p<0.05) from those collected in the other areas. on the wings of eg. cordata and eg. ignita, differences in shape have occurred mainly in the medial region, which actively participates in the individual’s flying ability. the wing size has showed meaningful difference only to the population of eg. ignita (p=0,005). for eg. cordata and el. atleticana, there was a significant correlation (r<0.05) between the morphometric data and the landscape metrics, which shows a close relationship between these species and the forest cover. the wing shape and size pursue an important function for the individual’s ability in the environment, such as dispersion capacity and fertility rate, respectively. we concluded that the morphometric differences can reveal the existence of environmental stress for euglossine bees. therefore, the results contribute to establish morphometry variability as a tool for environmental quality monitoring. sociobiology an international journal on social insects mg ribeiro¹, wm aguiar¹, la nunes², ls carneiro³ article history edited by evandro n. silva, uefs, brazil received 13 october 2018 initial acceptance 20 november 2018 final acceptance 30 may 2019 publication date 20 august 2019 keywords agroforestry system, atlantic forest, environmental stress, fragmentation, geometric morphometrics, solitary bee. corresponding author mariléa g ribeiro universidade estadual de feira de santana programa de pós-graduação em modelagem em ciências da terra e do ambiente av. transnordestina s/nº, novo horizonte feira de santana, bahia, brasil. e-mail: lea-grmil@hotmail.com and may lead them to extinction. therefore, proposals such as agroforestry systems (safs) are presented as alternatives for the maintenance of environmental quality and sustainable use of forest ecosystems (torralba et al., 2016). safs consist of a land use technique in which agricultural species are grown among the native species of a forest ecosystem, exotic tree species or a mix of both. the main advantages of safs in an agricultural matrix, as compared to monocultures, is their more complex structure in terms of vegetation stratification, niche diversification and energy 1 programa de pós-graduação em modelagem em ciências da terra e do ambiente, universidade estadual de feira de santana, bahia, brazil 2 programa de pós-graduação em enfermagem e saúde, universidade estadual do sudoeste da bahia, jequié, bahia, brazil 3 laboratório de entomologia, departamento de ciências biológicas, universidade estadual de feira de santana, bahia, brazil research article bees mg ribeiro et al. – morphometric changes in three species of euglossini340 flows. this practice ensures the maintenance of ecosystem services and benefits for biodiversity (torralba et al., 2016); promotes the increase of carbon and biomass stocks in the soil (cardinael et al., 2017) and may contain a similar diversity of plant species to those of secondary forests (sistla et al., 2016). despite the benefits for the environment, the impacts of the safs for the biodiversity is a topic that needs further investigations, because studies indicate, for example, that this kind of land use impacts the soil microbiota (wang et al., 2017), it is stressful for ceroglossus chilensis (eschscholtz) beetles (benítez et al., 2008), and may also influence the richness and abundance of bees in the environment (kennedy et al., 2013). bees, in turn, are considered key organisms for the functioning of agroforestry systems (matos et al., 2013), however, studies show that these insects are affected by soil use practices (neves et al., 2012) and that one of the reasons for the decline of solitary bees, in the world, is the agriculture intensification (kremen, 2002). in the case of the euglossini tribe, a bee group which includes species with primitive social as well as solitary behavior (freiria et al., 2017), the evaluation of the effects of habitat fragmentation on the wing shape and size has showed interesting results (silva et al., 2009). bees of this tribe, also known as orchid bees are important pollinators and, therefore, they are considered important targets for native plant conservation policies (brosi, 2009) and for analysis of the effects of fragmentation (aguiar et al., 2015). only in the brazilian atlantic forest more than 60 species of euglossini have been described, and some species are endemic to this biome (nemésio, 2009). among bees, the shape and size of their bodies probably reflect the influence of environmental, geographic and seasonal barriers (skandalis et al., 2009). while the wing shape presents greater evolutionary restrictions and is directly associated to the aerodynamics of this structure (nunes et al., 2013), the body size has high levels of plasticity in response to the environment (ghosh et al., 2013) and confers several advantages to the individual. the survival, fecundity, success in mating rates will be higher if this individual is bigger (kingsolver & huey, 2008). holometabolous insects, such as bees, in the adulthood do not undergo morphological changes, therefore the variance in the body size may be a response to the natural selection, climatic fluctuations and the availability of resource in the larval stage (chown & gaston, 2010), while the variance in wing shape is a response to environmental variability due to the costs and benefits involved in dispersion on fragmented landscapes (hill et al., 1999). then, a promising tool in the impact analysis of environmental changes on populations is the use of morphometry, a technique which allows the closure of morphological data in a set of numerical and graphical data, with the purpose of expressing, testing hypothetical relationships and detecting patterns within the collected data (richtsmeier et al., 2002). it should be noted that compared to other techniques, such as molecular techniques, morphometry is an economical and effective technique, and, therefore, it has been widely used by biological sciences to support works in the areas of taxonomy and population analysis (francoy et al., 2012; nunes et al., 2013; dellicour et al., 2017). therefore, in view of the fragmentation of the atlantic forest, one of the world’s hotspots, due to its high biodiversity, which is endemic and endangered, and the expansion of agricultural practices in its territory, like safs, this study has aimed to test the hypothesis that the landscape structure affects wing shape and size of three species of euglossini (eulaema atleticana nemésio, euglossa cordata (linnaeus) e euglossa ignita smith). materials and methods study area the study was conducted within the limits of the environmental protection area of pratigi (apa pratigi), located in the state of bahia, brazil, between the coordinates 13º55’48”s and 38º57’13’’w. the region has an average annual temperature of 25 °c, regular and abundant rainfall and relative humidity around 80% to 90% (nascimento et al., 2007). the environmental protection area of pratigi (apa pratigi) is defined by law as an area of sustainable use and it aims to protect the remnants of the brazilian atlantic forest and associated ecosystems, in which extractive and agricultural activities are allowed in a controlled manner. thus, the landscape of the environmental protection area of pratigi can be described as intensely modified and fragmented, composed mainly of remnants of atlantic forest in different stages of regeneration, agroforestry systems, mangrove, restinga (coastal ecosystem with sandy soils and various vegetal communities) and pasture. sampling the specimens were collected at five sampling points. the landscape location and characteristics of each point are shown in table 1. the collections occurred between june 2014 to may 2015, through aromatic traps, according to the model proposed by aguiar and gaglianone, 2008. a cotton swab soaked with synthetic compounds (eucalyptol, methyl cinnamate, benzyl acetate, vanillin and methyl salicylate) was added into each trap in order to attract the males of euglossini. traps were set in the vegetation at 1.60m from the ground and remained in the place from 09:00am to 3:00pm. at the end of this period, the bees were sacrificed with ethyl acetate. in the laboratory, the specimens were assembled and identified through the identification keys (nemésio, 2009) and by comparison with the reference collection. thereafter, all the specimens were deposited in the entomological collection of the environmental studies laboratory of the universidade estadual de feira de santana (uefs). sociobiology 66(2): 339-347 (june, 2019) 341 morphometric analysis in order to quantify the variance in the wing shape and size we adopted geometric morphometrics methods. for this purpose, 22 to 30 individuals of the three most abundant species in the study area, el. atleticana, eg. cordata and eg. ignita, were selected out from a total of 423 individuals (table 2). initially, the right anterior wing of each individual was removed and fixed on a microscope slide. after that, wings were photographed in a stereomicroscope with a coupled camera, integrated with the leica application suite software. the images of each wing were converted to the tps format, through the tpsutill 1.60 program (rohlf, 2013). afterwards, there was an insertion of landmarks and semilandmarks in the wing veins, through the tpsdig.2 program (version 2.18) (rohlf, 2015). around the marginal cell, on the wings of the eg. ignita and eg. cordata specimens, 13 landmarks and 13 semilandmarks were inserted, whereas on the wings of el. atleticana, 10 landmarks and 15 semilandmarks were inserted (fig 1). data of semilandmarks were aligned afterwards, through the tpsrelw 1.49 program (rohlf, 2010). the statistical analysis started with the generalize procrustes analysis (gpa), which is characterized as a positional adjustment between all used wings. after the gpa, the wing shape of each individual was used to compare the variance of shape within and among populations (adams et al., 2004). the differences in shape, were analysed by using the multivariate statistical methods of canonical variable analysis (cva) and procrustes distance. the differences in shape were illustrated by deformation grids, generated by the thin-plate-spline technique (bookstein, 1989). these analyses were conducted in the morphoj program, version 1.06 (klingenberg, 2008). furthermore, the procrustes distance was used for the cluster analysis by the unweighted pair-group method with arithmetic mean (upgma), thus, the average values of shape served as a basis for assessing the degree of similarity between the analysed groups. this analysis was conducted in the past program, version 2.17 (hammer et al., 2001) and the results were represented in the mode of a dendrogram, produced from a bootstrap with 1000 repetitions. we used the centroid size data, which is a geometric measure, defined from the square root of the sum of the square distance of each anatomical landmark, from the centre of the shape to evaluate the wing size (monteiro & reis, 1999). the centroid size values were extracted from the morphoj program and submitted to a variance analysis (anova one way) in the past program. the relationship of the variance in the wing shape and size with the characteristics of the landscape was conducted through the mantel test, a statistical method used for the correlation of two distance or similarity matrices, through the past program (hammer et al., 2001). landscape classification the landscape metrics were calculated for an area of 5 km radius around the five collection points through the v_late 2.0 extension of the arcgis 10 program. this radius metric was estimated based on euglossini flight capacity as presented by pokorny et al. (2014). in turn, the landscape table 1. collection points, geographic coordinates and landscape composition around collection points, totalling an area of 5 km radius. collection points geographic coordinates altitude landscape composition area 1 13°41’07.3” s / 39°05’18.8” w 71 dense ombrophilous forest¹ (68,9%); restinga² (4%); mangroves (14,3%); pasture (1,2%). area 2 13°50’36.7” s / 39°17’21.5” w 230 dense ombrophilous forest of submontane¹ (60,5%); theobroma cacao l., syzygium aromaticum (l.) merr. & l.m.perry and bactris gasipaes kunth (33,7%); pasture (4,2%). area 3 13°54’50.4” s/ 39°27’23.9” w 470 dense ombrophilous forest of submontane¹ and dense ombrophilous forest of montane¹ (45,8%); theobroma cacao l.(10,1%); hevea brasiliensis muell. arg. and pasture (15,4%). area 4 13°53’51.8” s / 39°27’44.8” w 680 dense ombrophilous forest of montane¹ (43,4%); theobroma cacao l. and agriculture (41,6%); pasture (13,4%). area-saf 13°46’55.90”s 39°16’53.8” w 230 dense ombrophilous forest¹ (50%); musa paradisiaca l. (31,8%); theobroma cacao l. and forest (11,9%); pasture (5,7%). ¹ classification adopted by instituto brasileiro de geografia e estatística ibge (2012) to characterize the kinds of landscape, which can occur throughout atlantic forest. ² coastal ecosystem with sandy soils and diverse plant communities. species area 1 area 2 area 3 area 4 areasaf eulaema atleticana 31 26 30 26 29 euglossa cordata 30 28 29 28 26 euglossa ignita 29 29 28 22 32 table 2. number of individuals examined at each collection point. mg ribeiro et al. – morphometric changes in three species of euglossini342 metrics (with emphasis on forest fragment cover area) were obtained for three indexes: 1) total area; 2) total of core area, adopting 100m as an area subject to edge effect; 3) average size of the fragments (mcgarigal & ene, 2013) (table 3). therefore, the soil use and occupation were surveyed around the five collection points through the use of data in shape file format produced by the organization for conservation of the southern lowlands of bahia (oct) (see fig s1, supplementary material doi: 10.13102/sociobiology.v66i2.3779.s2234). fig 1. landmarks (points) and semilandmarks (circles) used for shape and size of the wing analysis of eulaema atleticana (a), euglossa cordata (b) and euglossa ignita (c). the wing cells were named according to the classification adopted by francoy et al (2012): marginal (m), 2nd submarginal (sub2), 3nd submarginal (sub3), 1st medial (med1), 2nd medial (med2), 2nd cubital (cub2). collection points total area of forest (ha) mean patch size (ha) total core area (ha) area 1 5,367 536.7 4,481 area 2 4,753 148.5 3,096 area 3 3,420 142.5 1,807 area 4 2,667 106.7 1,373 area-saf 3,928 178.5 2,208 table 3. landscape metric for an area of 5 km radius around the collection points. fig 2. scatter plot of the five populations of euglossa ignita in relation to the canonical variables cva 1 and cva 2, arranged in a cartesian plane and obtained from the wing shape. the deformation diagrams for the negative and positive sides of each axis are around the graph. sociobiology 66(2): 339-347 (june, 2019) 343 results the first three variables were necessary to explain more than 80% of the total variation in the canonical variable analysis (cva). the dispersion graph and the deformation grid have revealed important differences in the wing shape of populations of the three species, with emphasis on eg. ignita species and the individuals collected within the safs. the wing shape of the eg. ignita populations (fig 2) has diverged mainly between the following cells: 2nd medial, marginal and 3rd submarginal. for saf’s population, the deformation grid cva 1 has demonstrated that there was a reduction of the 2nd medial and the central portion of the marginal region, accompanied by an increase of the 3rd submarginal. correlation of matrixes r p euglossa cordata shape x altitude 0,04113 0,0991 size x altitude 0,02437 0,1984 shape x mean patch size 0,1121 0,0053* size x mean patch size 0,1296 0,0085* shape x total core area 0,07339 0,0104* size x total core area 0,08407 0,0116* eulaema atleticana shape x altitude 0,1579 0,3019 size x altitude 0,0378 0,1442 shape x mean patch size 0,0804 0,0339* size x mean patch size 0,1151 0,0164* shape x total core area 0,0627 0,0285* size x total core area 0,0885 0,0116* shape x total area of forest 0,0375 0,0883 size x total area of forest 0,0542 0,0349* *p< 0,05, significant table 4. mantel test used to compare the environmental data matrices and morphometric data (wing shape and size) of euglossa cordata and eulaema atleticana, with 5000 permutations. fig 3. dendrogram upgma obtained from the average values of the shape. (a) euglossa cordata, with cophenetic correlation of 95%; (b) euglossa ignita with cophenetic correlation of 86%, (c) eulaema atleticana, cophenetic correlation of 83%. from the values of the distance of procrustes (p<0,05), the differences of shape were meaningful for the three species. for eg. ignita and el. atleticana, the highest values of dissimilarity were found among the population of area 4 and of the saf (p=0,01 and p=0,007, respectively). for eg. cordata, were found among the population of the saf and area 3 (p=0,01). the results of the cluster analysis (fig 3) have demonstrated the wing shape of the collected populations in the saf differs significantly from the other places. these differences in the wing shape have put the safs population as an isolated group. the formed dendrogram for each species has showed the following cophenetic correlation: el. atleticana 83%, eg. cordata 95% and eg. ignita 86%. variations in the wing size, based on the centroid size, have been significant only for eg. ignita (anova, f=5,923 and p=0,00045) (fig 4). we have verified that the largest variation interval has been identified for area 4 and saf populations, while the smallest variation interval has been observed for the area 3. according to tukey’s range test, the area 3 population has been the only one that has differed statistically from the area 4 population (p=0.0248) and area 5 (p=0.0005) indicating the individuals with the lowest values for wing size. the mantel test (table 4) has demonstrated that the elevation is not a determining factor for differences in wing shape and size of the species, even though it is a geographic characteristic which varies significantly within the study area. although the value of p was significant, the value of r does not show that the shape and size of the wing are influenced by the landscape metrics. however, this data suggests that there is a trend of variation in the data, which may, in theory, be due to environmental variables. the wing shape and size of eg. cordata and el. atleticana have showed positive correlation with the mean fragment size and the total core area (p< 0,05), and the wing size of el. atleticana has showed correlation with the total area of forest. mg ribeiro et al. – morphometric changes in three species of euglossini344 abundance of plants used as a source of resources, leading to foraging farther and farther. during foraging, these bees will be exposed to temperature variations and food scarcity, factors that are capable for influencing aspects of the phenotype and ecology of these insects (whitman & agrawal, 2009). according to these authors, phenotypic plasticity guarantees to individuals an ecological niche amplitude and occupation of new spaces. in anthropogenic environments, such as areas with forest remnants, the percentages of incident light, humidity, temperature and wind speed are modified (bennett & sauders, 2010). in accordance with reed et al. (2011), the effects of these changes are commonly observed in species with brief life cycles and with high population and fertility rates, because they allow the appearance of morphological changes in a short period of time. in these circumstances, the insecta class is widely used in analyses of phenotypic plasticity (moczek, 2010). the interaction between environmental conditions (temperature, humidity, geographic location, altitude) and wing shape was identified in some groups of insects, namely: diptera (prudhomme et al., 2016), lepidoptera (bai et al., 2015), hymenoptera (silva et al., 2009), demonstrating that the wing shape is an indicator of stress during the development of these organisms. however, at the landscape level, investigations about the effects of landscape changes on wing shape are still scarce. silva et al. (2009) reported that specimens of euglossa pleosticta (dressler), which were collected in border and interior environments of the forest fragment, showed important differences in the wing symmetry of the individuals, which suggests that changes in wing shape were due to instability in larval development, as a result of climatic and anthropic factors operating over the environmental conditions within the border as well as in the core area of the fragment. outomuro et al. (2013) identified trithemis dragonflies collected in open areas had wings wider than those collected in forest areas, demonstrating that wider wings were a favourable condition in open areas. beyond the shape, the wing size of insects may also indicate the environmental conditions to which the individuals were exposed. thus, the occurrence of individuals with larger wings within the saf suggests they were able to forage over larger ranges, using the agricultural areas, or returned to the safs with a greater supply of resources. similar results were found by merckx and van dick (2006) for butterflies of the pararge aegeria (linnaeus) species. in assessing the larval development of this species in three landscapes (agricultural landscape, forest landscape and landscape with forest fragments), authors notice that larvae that developed in agricultural landscape had more thoracic mass and higher wing loading than individuals developed in the forest landscape. in insects, the wing size can be used as a proxy for the size body (nijhout & callier, 2015). for bees, the body size represents a greater ability in the environment, for it endow large individuals with high foraging capacity and fig 4. box plot and tukey’s range test for the centroid size of euglossa ignita. the horizontal lines represent the medians; the rectangles represent the interquartile range (25-75%), while the vertical lines inform the intervals of variance. by tukey’s range test, the same letters do not differ statistically from each other. discussion the morphometric analysis has demonstrated that the wing shape of the three species has varied significantly among the populations, especially those which were collected within the saf. this result indicates that the landscape may be causing these changes in the populations, since we have found a correlation between the landscape metrics and the wing shape and size of two species: eg. cordata and el. atleticana. furthermore, the medial region, which is known to perform a key role in the flying capacity of insects (johansson et al., 2009), it has undergone changes in eg. cordata and eg. ignita populations. conditions which are found during foraging, may be a possible reason for the existence of these changes, because changes in wing morphology may be related to the costs and benefits that are involved in the dispersion pattern in fragmented landscapes (hill, 1999). landscapes that differ in extent of fragmentation provide different selection processes for dispersion, therefore, to deal with these differences, organisms have as alternatives the genetic adaptation or the phenotypic plasticity (merckx & van dick, 2006). due to fragmentation, a new set of environmental conditions is generated, causing changes in ecosystem processes and occurrence of problems, such as edge effect. for euglossini bees which are dependent on various forest resources, the creation of open areas or the introduction of agricultural activity, as in saf, can cause a drop in the sociobiology 66(2): 339-347 (june, 2019) 345 mating success (kingsolver & huey, 2008). euglossini bees are benefited by the body size, as long as the males collect aromatic compounds in the plants, which are possibly used for the attraction of females, and consequently, they help in the dispersion of pollen. euglossini females find resin in the plants for building of the nests, as well as, nectar and pollen which are inserted into the nest for feeding the offspring. for this cause, euglossini bees tend to occupy larger fragments to guarantee the necessary resources for feeding and nesting (brosi, 2009). therefore, fragmentation and other changes in the landscape are problems that can impact strongly the populations of these bees. these impacts can reflect on the body size, which may indicate the decline of resources in the environment (renauld et al., 2016). some researches (benjamin et al., 2014; renauld et al., 2016) have pointed out that fragmentation and proliferation of agricultural areas affect the bee’s abundance regarding body size. thus, the persistence in a particular environment will depend on the dispersion potential of the species, therefore, bees with greater mobility will be less prone to the effects of habitat loss (jauker et al., 2013). considering that smaller habitat patches may have fewer resources, only those individuals with greater foraging capacity will be able to use the dispersed resources in the landscape. bees are pollinators of great importance in tropical areas, however, evaluations about the impact of the simplification of ecosystems on these taxa are still scarce. our study has demonstrated that the variations in the shape and size of euglossini bees are good indicators of environmental variance and the safs have made a significant influence on the morphological characteristics of these bees. thus, to consider the speed with which natural environments are converted into agricultural areas, the response of these pollinators, to these changes, becomes a fertile field for future researches and proposals for conservation of bees. acknowledgments we thank the foundation for research support of the state of bahia (fapesb) for granting the scholarship to the first author, as well as, the organization for conservation of the southern lowlands of bahia (oct) for their support in fieldworks. references adams, d.c., rohlf f.j. & slice e.d. 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(pp.1-63). usa: science publishers, enfield. doi: 10.13102/sociobiology.v65i4.3455sociobiology 65(4): 714-721 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 improved pollination efficiency and reduced honey bee colony decline in greenhouses by allowing access to the outside during part of the day introduction closed agricultural cultivation systems (greenhouses) were developed in order to optimize vegetable production and to obtain high quality products (muijzenberg, 1980). greenhouses allow the control of various climatic variables; they reduce the vulnerability of crops to insect pests through the use of screens or other types of barriers; also, irrigation and fertilization can be applied with greatly increased efficiency (van straten et al., 2010). however, more than 40% of the crops produced in greenhouses require pollinating agents (guerra-sanz, 2008), principally bees (klein et al., 2007), which do not easily adapt to these closed environments (nye, 1962; abrol, 2012). abstract although honey bees are efficient pollinators of many crops cultivated in greenhouses, it is difficult to maintain colony strength and consequently pollination efficiency. many bees die under greenhouse conditions and the colonies rapidly weaken. we examined the effect of adaptations to the hive entrance that allowed control of whether and when bees had access to the outside environment to see how it would affect pollination efficiency and colony condition in greenhouses with flowering cucumber (cucumis sativus) plants in comparison with colonies that remained constantly inside the greenhouse. we recorded the type and period of visitation to the cucumber flowers, numbers of honey bees entering and leaving the two-entrance hives and the effect of this type of management on the quantity of brood and food. five frame langstroth “nucleus” colonies were equipped with two 30 square centimeter entrances and two 3.0 cm diameter circular openings. allowing the bees to make visits outside the greenhouse in early morning with redirection of bees into the greenhouse at 8:30 a.m. did not reduce visitation to cucumber flowers in the greenhouse. maintaining colonies in the greenhouse reduced brood area and food stores. these losses were significantly reduced in colonies that had access to the outside during the early morning. another advantage of alternating access to the inside and the outside of the greenhouse was that there was less possibility of interactions between bees and people working on the crop; also, pesticide applications could be made without directly affecting foraging bees. sociobiology an international journal on social insects d nicodemo1, eb malheiros2, d de jong3, rhn couto2 article history edited by denise alves, esalq-usp, brazil received 10 may 2018 initial acceptance 18 june 2018 final acceptance 20 august 2018 publication date 11 october 2018 keywords apis mellifera, brood, cucumber, cucumis sativus, fruit set, honey. corresponding author daniel nicodemo universidade estadual paulista (unesp) faculdade de ciências agrárias e tecnológicas rod. comte. joão ribeiro de barros (sp 294) km 651, bairro das antas cep 17900-000, dracena-sp, brasil. e-mail: daniel.nicodemo@unesp.br bees are commonly used for greenhouse pollination of various crops, including tomatoes and bell peppers, which are normally pollinated by bumble bees, mainly bombus terrestris (l.) (free, 1993; velthuis & van doorn, 2006). stingless bees, such as tetragonisca angustula (latreille) and nannotrigona spp., have also been found to be efficient pollinators of tomatoes (del sarto et al., 2005), peppers (santos et al., 2008), strawberries (malagodi-braga & kleinert, 2004) and cucumbers (nicodemo e al., 2013). these stingless bees are considered effective and useful pollinators in greenhouses because they are generalist foragers, have a tendency for flower fidelity, do not attack agricultural workers who tend the crop, rarely abandon their nests and store food (slaa et al., 2006). 1 universidade estadual paulista (unesp), faculdade de ciências agrárias e tecnológicas, são paulo, brazil 2 universidade estadual paulista (unesp), faculdade de ciências agrárias e veterinárias, jaboticabal, são paulo, brazil 3 universidade de são paulo (usp), faculdade de medicina de ribeirão preto, são paulo, brazil research article bees sociobiology 65(4): 714-721 (october, 2018) special issue 715 though bombus spp. bees have been widely used in various countries for greenhouse pollination (velthuis & van doorn, 2006), they are not globally distributed and are considered a difficult to control exotic pest in some regions where they have been introduced (dafni et al., 2010). stingless bees, meliponini, have a more restricted distribution, and when they are introduced into a greenhouse environment, they need to adapt to foraging in a restricted area; also, they do not invariably visit the flowers that need pollination when under confinement (bomfim et al., 2014). given the current knowledge about honey bee management and the facility in finding beekeepers who have colonies available for pollination services practically throughout the world, honey bees have the potential to become an optimal solution for pollinating crops in greenhouses, if techniques can be improved to adapt these bees to foraging in closed environments (katayama, 1987; guerra-sanz, 2008). honey bees are the most widely used insects for pollination services of agricultural crops grown in open fields, since they have numerous foragers per colony that visit extensive areas, superior to many other species of bees (mcgregor, 1976; southwick & southwick, 1992; beekman & ratnieks, 2000; garibaldi et al., 2013; bartomeus et al., 2014). in addition, they forage under a broad ambient temperature range (abou-shaara, 2014) and have efficient communication (winston 1987), which favors the collection of large amounts of food resources necessary for the development of their colonies (couto & couto, 2006; brodschneider & crailsheim, 2010). however, their use on greenhouse crops is often limited because colonies in greenhouses normally decline rapidly (sabara & winston, 2003). given the advantages of using honey bees for pollination services and the problems that are still unresolved concerning adapting these bees for working in closed environments, we investigated whether allowing bees to alternatively forage inside and outside the greenhouse would allow efficient pollination, while maintaining colony integrity. material and methods greenhouse and crop details the work was carried out in ribeirão preto, são paulo state, brazil (21°10’02” s and 47°51’53” w), at altitude 546 m. cucumbers (cucumis sativus l.) cultivar aodai were planted from seed in trays containing planting substrate. after 20 days, the seedlings were transplanted to the three greenhouses, each with dimensions of 13 meters long x 8 meters wide, by 1.8 m tall, covered with low density polyethylene film with uv blocking and anti-aphid screening on the sides (totally enclosed). cucumber seedlings (n = 144) were planted in each of the three greenhouses with a spacing of 0.5 x 1.0 m. fertilizer was added to the irrigation water based on soil analysis and recommendations in iac technical bulletin 100 (raij et al., 1997). pest control was carried out using registered products for cucumber cultivation (mapa, 2018) at night, when there were no more bees working outside the hives. preparation and development of the colonies one week before they were introduced into the greenhouses, three colonies housed in five-frame standard langstroth hives were equalized in terms of brood and food comb areas. a comparison of the development of the colonies kept in the greenhouses was evaluated by mapping all the combs on the day the hives were introduced and again one day after they were removed from the greenhouses (al-tikrity et al., 1971). hive and outside temperature within the greenhouses were measured with two thermometers. one was placed in the greenhouse at 1.4 meters height and a second on the hive floor of the colonies in the treatment named 24h-normal, as described below. hive placement and management colonies of africanized honey bees (apis mellifera l.) were submitted to three different managements of hives. each greenhouse was supplied with a water source accessible to the bees. in the first, a five-frame hive was placed in a hive-sized cutout in the side screen of the greenhouse, at a height of 1.4 meters. in this way, the foraging bees could be directed to either inside or outside the greenhouse. these five-frame hives had two 30 cm² rectangular entrances, on opposite ends. at 2.5 cm above each of these two entrances, an additional opening 3.0 cm in diameter was made. a fine mesh metal screen funnel directed to the inside of the hive was placed in this opening, through which foragers bees could enter the hive but not leave it. the opening at the point of this screen funnel was about 0.5 cm in diameter (fig 1). in order to direct the bees to forage outside of the greenhouse, the lower entrance was blocked with a metal plate. those bees that were still foraging in the greenhouse could enter the hive via the circular upper entrance, which was always kept open. when the objective was to direct the bees to forage inside the greenhouse, the lower hive entrance that was directed towards the outside was blocked with a metal plate and the entrance to the inside was opened (fig 1). since the screen funnel point would be crushed and blocked by one of the comb frames, one deep frame was replaced with a shallow frame to allow space for the funnel. the hive then had four standard deep frames and one shallow frame. this management was called two-entrance hive. a second greenhouse was pollinated by bee colonies that went through an adaptation period aiming to induce the bees to reorient in the greenhouse. the hives were kept in a dark room at 30 ºc and 70% relative humidity for 72 hours before being introduced into the greenhouse. during this confinement period the hive cover was replaced with an 8-mesh screen and the single hive entrance closed. water and sucrose syrup (1:1 w/v) were supplied through this screen ad libitum. on the day that the hive was introduced into the d nicodemo, eb malheiros, d de jong, rhn couto – two-entrance hive for greenhouse pollination716 greenhouse, the screen top was removed and replaced with a normal wooden cover and the normal entrance was opened. the hive was placed 1.4 m above the greenhouse floor. this management was named 24 h-dark. the third greenhouse was given a hive that was introduced directly from the apiary and the management of this hive was entitled 24 h-normal. the colonies were placed inside the greenhouses after the cucumber plants had begun flowering. each of the three greenhouses received one colony, which remained there for 10 days. this process was repeated three times, so that each greenhouse received three colonies over 30 days. the hives with inside and outside entrances were manipulated at different times during each of the three 10-day periods. during the first 10-day period, the bees could visit the cucumber flowers from 6-12:30; during the second 10-day period, they had access to the flowers inside the greenhouse from 6-10:30. during the third 10-day period, the bees were directed towards the outside until 8:30. after 8:30, the bees were redirected to forage inside the greenhouse, until 12:30, when they were again allowed access only to the outside. we chose to carry out the bee visitation tests in the morning, as this is the period in which pollen grains are more viable compared to the afternoon period (nicodemo et al., 2012). flight activity visual counts of the number of bees entering and leaving the hive during five-minute intervals were made throughout the period of flight activity of the bees. a random blocks design (blocks = days) was used, with four repetitions per colony in partitions divided in time, with five treatments (colonies managed in different ways) in the partitions. visits to the cucumber flowers the frequency of bee visits was estimated during all the day by counting bees during 10 minutes each hour, from the first visit until bees stopped visiting, observed by walking through each greenhouse. a random blocks design (blocks = days) was used, with four repetitions per colony in partitions divided in time, with five treatments (colonies managed in different ways) in the partitions. cucumber pollination to determine fruit production with and without bee visits, 25 pistillate flowers were selected before anthesis and some were covered with fine mesh nylon bags during part of the day, allowing bee visitation only during one of four periods: 6:00-8:30, 6:10:30, 6:00-12:30, 6:00-18:00 (n = 5 for each period). the other five remained covered, preventing visitation. this was repeated during five days for each of the bee access regimes. the percentage fruit set and mean fruit weight were measured later. statistical analyses the data were submitted to analysis of variance. multiple comparisons were made using the tukey test, at a significance level of 5%, using sas software (sas institute, 2003). fig 1. (a) view from above of the two-entrance hive without cover and frames; (b) internal view of the usual entrance which is closed and an upper entrance with a fine mesh metal screen funnel, external view of the hive installed in the greenhouse with the usual entrance (c) open and (d) closed. a b c d sociobiology 65(4): 714-721 (october, 2018) special issue 717 results the bees quickly adapted to the two-entrance hives, including landing of foragers on the screens of the circular entrances, even when the normal entrance was available. the bees were not able to overcome the screen cones and were successfully directed to the inside or the outside of the greenhouses. fig 2. remaining comb area (%) of brood, pollen and honey in colonies introduced into greenhouses in which the bees had access to the outside or not during a period of 10 days. fig 3. mean temperature during the day within the greenhouse and within the hive (temperature probes on the floor of the hive and outside the hive at about 1.4 meters height). temperatures were recorded daily during 10 days. 18 20 22 24 26 28 30 32 34 36 38 0 2 4 6 8 10 12 14 16 18 20 22 te m pe ra tu re ( °c ) time (h) greenhouse beehive t em pe ra tu re (º c ) time (h) all of the colonies used in the greenhouses had a reduction in the comb areas occupied by bee brood, pollen and honey (fig 2). this reduction was less accentuated in the twoentrance colonies, which were able to forage to the outside during part of the day. the reduction in the brood area among the colonies that were completely confined to the greenhouses was over 70%, indicating that these colonies would have a drastic population decline in the following weeks if they were to remain in the greenhouse. bees were sometimes found to be lost within the greenhouses, independent of the management schedule; they kept hitting against the side screening or the plastic covering and did not return the colonies. the reduction in pollen in the comb was less in the colonies that had access to the outside earlier in the morning, with about 36% remaining after 10 days in the greenhouse. the greatest reduction (91%) in comb area with pollen was observed for the two-entrance hive that had the least access to outside the greenhouse. when comb area with honey was compared, the best performance was among bees with access to the outside, with 44–62% remaining. among the colonies completely confined in the greenhouse, less than 40% of the comb area with honey remained. inside hive temperatures ranged from 24.8 to 31.1 oc, compared to an ambient temperature of 18.8–37.7 oc (fig 3), demonstrating that these bees thermoregulated efficiently. the flow of bees entering and leaving the hives was greatest in the colonies that could forage to the outside early in the morning (fig 4). besides beginning to work about an hour earlier than the other colonies when they had access to the cucumber plants, they also visited the plants more intensively. fig 4. number of bees leaving (a) and entering (b) the hives introduced into greenhouses in which the bees had access to the outside or not during a period of 10 days. fig 2          0 10 20 30 40 50 60 70 80 90 100 6-12:30 6-10:30 8:30-12:30 24 h (dark) 24 h (normal) r em ai ni ng a re a (% ) access to the greenhouse (h) brood pollen honey             0 20 40 60 80 100 120 140 160 180 200 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 n um be r o f b ee s le av in g th e hi ve time (h) 6-12:30 6-10:30 8:30-12:30 24 h (dark) 24 h (normal) 0 20 40 60 80 100 120 140 160 180 200 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 n um be r o f b ee s en te ri ng th e hi ve time (h) 6-12:30 6-10:30 8:30-12:30 24 h (dark) 24 h (normal) a  b  d nicodemo, eb malheiros, d de jong, rhn couto – two-entrance hive for greenhouse pollination718 the number of bees that left the colonies to forage outside the greenhouse early in the morning, between 6 and 7:00 was 13.8 times greater than the mean number of bees that could only fly into the greenhouse. after the bees stopped collecting nectar and pollen from the cucumber flowers at about 15:00 (fig 5), the bees kept confined in the greenhouse no longer had access to period of bee access to the greenhouse period of availability of flowers to visitation of bees 6:00 – 8:30 6:00 – 10:30 6:00 – 12:30 6:00 – 18:00 6:00–12:30 90 90 90 80 6:00–10:30 90 100 90 90 8:30–12:30 – 90 100 90 24h (dark) 80 100 80 80 24h (normal) 80 90 80 70 outside food sources; however, they continued to fly in and out of the entrance. the number of bees returning to the hives after 16:00 was 25.2% of the total number of bees returning during the whole day. the number of bees flying out of the two-entrance colonies increased with the period of time available to forage outside the greenhouse. the total number of bees flying out of the hives among bees that could fly outside early in the morning and after 12:30 was 570% and 209% superior to the number of bees that left the colonies in hives that only had access to the outside after 12:30 and 10:30, respectively. these bees could collect food, resins and water from outside the greenhouse. when the main entrance was closed, the bees returned through the screen cone entrance within 30 minutes, both for bees coming from inside and outside the greenhouse. bees visited the flowers after initiation of the anthesis period, which began about 6:00 (nicodemo et al., 2012), with a gradual increase in the frequency of visits until 9:00 in all of the greenhouses. from then on, the number of bees on the flowers declined. comparing the different management schedules, the best performance was made by bees that had access to the outside early in the morning, especially when comparing pollen foraging on the cucumber flowers (fig 5c), which is fundamental to assure pollination. the bees from the colonies that were confined for three days in the dark before being introduced into the greenhouse made fewer visits to the cucumber flowers. some of the workers of these colonies were occupied removing bees that had died during the time that the colonies had been kept closed in the dark. all of the greenhouses had both bees that visited flowers and bees that incessantly flew against the screening, trying to get out. flowers that were not visited by bees did not produce cucumbers. the visitation by the bees until 10:30 allowed the obtaining of good fruit set rates (table 1). allowing bees to continue foraging until 12:30 did not increase fruit formation. in fact, less fruit was formed when the flowers were visited by the bees during the entire of anthesis period. fruit weight was greatest when the flowers were visited up to 12:30 (table 2); the heaviest fruits were obtained when the bees could visit from 8:30 to 12:30 and from anthesis until 10:30. table 1. cucumber fruit set (%) after four pre-established daily periods of visitation by bees that were maintained in hives that allowed access to the outside of the greenhouse during part of the day or not. half of the 24h hives were maintained in a dark room for 72 h before placing them in the greenhouse (= dark). fig 5. mean number of nectar collecting bees visiting staminate (a) and pistilate (b) and pollen collecting bees visiting staminate (c) cucumber flowers in the greenhouse. bees had access to the outside or not during a period of 10 days.      a        b          0 5 10 15 20 25 30 35 5 6 7 8 9 10 11 12 13 14 15 16n um be r o f b ee s co lle ct in g ne ct ar o f st am in at e fl ow er s time (h) 6‐12:30 6‐10:30 8:30‐12:30 24 h (dark) 24 h (normal) 0 1 2 3 4 5 6 7 8 9 10 5 6 7 8 9 10 11 12 13 14 15 16 n um be r o f b ee s co lle ct in g ne ct ar o f pi st ill at e fl ow er s time (h) 6 12 30 6 10 30 8 30 12 30 24 h (d k) 24 h ( l) c      0 5 10 15 20 25 30 35 40 45 5 6 7 8 9 10 11 12 13 14 15 n um be r o f b ee s co lle ct in g po lle n time (h) 6-12:30 6-10:30 8:30-12:30 24 h (dark) 24 h (normal) c      0 5 10 15 20 25 30 35 40 45 5 6 7 8 9 10 11 12 13 14 15 n um be r o f b ee s co lle ct in g po lle n time (h) 6-12:30 6-10:30 8:30-12:30 24 h (dark) 24 h (normal)      a        b          0 5 10 15 20 25 30 35 5 6 7 8 9 10 11 12 13 14 15 16n um be r o f b ee s co lle ct in g ne ct ar o f st am in at e fl ow er s time (h) 6‐12:30 6‐10:30 8:30‐12:30 24 h (dark) 24 h (normal) 0 1 2 3 4 5 6 7 8 9 10 5 6 7 8 9 10 11 12 13 14 15 16 n um be r o f b ee s co lle ct in g ne ct ar o f pi st ill at e fl ow er s time (h) 6 12 30 6 10 30 8 30 12 30 24 h (d k) 24 h ( l) sociobiology 65(4): 714-721 (october, 2018) special issue 719 allowing access to flowers until 8:30 was not sufficient to give satisfactory pollination; the fruits that came from flowers open to visitation until 10:30 were heavier, indicating that bees should be allowed to visit the flowers until this time. allowing bees to visit flowers during the whole time of anthesis did not result in heavier fruit. fruit was lighter when the bees visited the flowers in the greenhouse more than five hours per day. discussion besides helping overcome the reductions in brood, honey and pollen stores in the brood combs that occur in colonies confined to the greenhouse, the use of two-entrance hives could facilitate crop management, since the work can be done when the bees are foraging outside, without concern with getting stung. pesticides can also be applied without needing to remove the hives from the greenhouse if the bees are directed to leave the crop and the hive is covered to avoid direct contamination. inadequate diet, especially when it involves protein, impedes the full development of the hypopharyngeal glands of the bees. the nurse bees then cannot adequately feed the larvae (dustmann & ohe, 1988; crailshem et al., 1992). greenhouses can have cultivated areas from less than 100 m² to over 1,000 m²; however, even in the larger greenhouses, the nutritional necessities of the bees will rarely be met, meaning that the bees will have deficiencies (naug, 2009). using bees for pollination in greenhouses generally results in thermal stress for the colonies because greenhouses are normally hotter than the outside environment during the day (cermeño, 1979; sganzerla, 1995). consequently, the colonies will need to put extra effort into temperature regulation in order safeguard their brood. the higher temperatures are beneficial for some crops, such as peppers (sganzerla, 1995). however, for the bees the microclimate and the available food resources are normally not ideal. since bees are essential for production of many crops because of their role in pollination, the use of a two-entrance hive could help reduce stress in comparison with the normal option of maintaining the bees inside the greenhouse 24 hours per day. bees that are allocated to the work of cooling the colony could otherwise participate in the collecting and processing of pollen and nectar if the temperature inside the hive was less of an issue. if the bees do not have access to the outside, a water supply can be offered within the hive for the bees to use for evaporative cooling (couto & couto, 2006). an important factor to consider about the performance of the bees within greenhouses is the type of material used to cover these structures. the greenhouses that we used, similar to most used throughout the world, are covered with polyethylene plastic, which blocks ultraviolet light (diaz & fereres, 2007), which is important for bee navigation (edrich et al., 1979; guerra-sanz, 2008). the efficiency of the bees in the greenhouse can be negatively affected by a lack of or low intensity of ultraviolet light (dag & eisikowitch, 1995). the bees that had access to the outside early in the morning began to forage earlier than the bees in the other flight control regimes. the first hours of the day are especially important for bees that collect pollen (free, 1993), an essential diet component for brood production (herbert jr. et al., 1977; brodschneider & crailsheim, 2010). in nature, the bees mainly collect pollen early in the day, because that is when most flowers open and become available (mcgregor, 1976; couto & couto, 2006; reyes-carrillo et al., 2007). cucumbers plants of the aodai cultivar are monoecious and highly dependent on animal pollinators, especially honey bees (mcgregor, 1976; klein et al., 2007). other investigations have shown that honey bees are also efficient pollinators of cucumbers grown in greenhouses (nogueiracouto & calmona, 1993; nicodemo et al., 2013). although the effectiveness of honey bees as pollinating agents of cucumber was already known, our work brings contributions regarding the management of hives into greenhouses, in order to facilitate the maintenance of colonies and pest management of crops when hives are introduced to the greenhouses for pollination services. in our study, all of the management schemes helped produce cucumbers with an average weight above 350 g when flower visitation occurred up until 10:30. when bees were able to visit the flowers more than five hours per day, the period of bee activity inside the greenhouse period of availability of flowers to visitation of bees 6:00–8:30 6:00–10:30 6:00–12:30 6:00–18:00 6:00–12:30 272.8 ab* 370.1 aa 261.6 bb 274.9 bb 6:00–10:30 270.8 ab 375.2 aa 371.4 aa 375.9 aa 8:30–12:30 – 375.2 aa 372.8 aa 373.1 aa 24h (dark) 265.4 ab 363.1 aa 243.5 bb 244.1 cb 24h (normal) 261.9 ab 357.8 aa 244.0 bb 247.2 cb *means followed by the same letter (upper case in the row and lowercase in the column) were not significantly different based on the tukey test. table 2. average weight of cucumber fruits (g) in four pre-established daily periods of visitation of bees that were installed in hives that allowed access to the outside of the greenhouse during part of the day or not. d nicodemo, eb malheiros, d de jong, rhn couto – two-entrance hive for greenhouse pollination720 mean weight of the fruit was less than 300 g. excess visitation can induce the bees to remove pollen grains that have been deposited on the stigmas, whether intentional or not, as was found in a field experiment involving pollination of pumpkin (cucurbita maxima) in an area with 1,200 m² of plantings and 30 colonies of honey bees (nicodemo et al., 2009). acknowledgments the authors thank cnpq, for the scholarship and financial assistance. authors’ contribution rhnc collaborated with definition of the pollination methods. ddj collaborated with the definition of methods for evaluating the performance of beehives. ebm was responsible for defining the statistical design. dn was responsible for designing the beehive with two entrances and collecting the data. all authors participated in the analysis of the results and in article writing. references abou-shaara, h.f. 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(1987). the biology of the honey bee. cambridge: harvard university press. 281p ole_link1 ole_link2 _hlk519099685 _hlk519419437 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.3503sociobiology 66(2): 367-376 (june, 2019) variation in chemical composition of cuticular and nonpolar compounds of venom of apoica pallens and polistes versicolor introduction the hymenoptera is one of the largest orders of insects, which comprises social wasps, bees and ants, among others. in insects’ societies, it was necessary to evolve a mechanism to keep the cohesion between their members, mostly in the form of chemical compounds used as signals exchanged during interactions, called semiochemicals. among these compounds, the cuticular hydrocarbons (chcs), which are constituents of the lipid layer of insects’ cuticle, stand out (lockey, 1988). abstract although cuticular hydrocarbons and venom are important to the evolutionary success of social behavior, studies that investigated these compounds in tropical social wasps are rare. thus, the aim of this study was to compare the cuticular chemical composition and the nonpolar portion of venom of apoica pallens, a swarm-founding wasp and polistes versicolor an independent-founding wasp. gas chromatography coupled to mass spectrometry (gc/ms) technique was used. in the samples of a. pallens, 66 compounds were identified on the cuticle and 87 in venom, 13 are unique of the cuticle and 26 of venom. in the samples of p. versicolor, 85 compounds were identified on the cuticle and 60 in venom, 10 are exclusive of the cuticle and 5 of venom. the results show that, although they present different foundation types and organize in colonies with significantly different population number, the variation in chain length of compounds is relatively similar. in addition, in both types of samples of both species, the most representative class of compounds in content and number are the branched alkanes, which are recognized as the most effective during interactions between nestmates. however, there is greater similarity in content of shared compounds between samples of cuticle and venom of a. pallens, suggesting that because it is a species that is organized in more populous colonies, it may have a more elaborate signaling system based on volatile compounds of venom. sociobiology an international journal on social insects a mendonça1,3, kb michelutti2,3, cal cardoso2, wf antonialli junior1,2,3 article history edited by gilberto m. m. santos, uefs, brazil received 01 june 2018 initial acceptance 03 march 2019 final acceptance 03 march 2019 publication date 20 august 2019 keywords chemical signature; cuticular hydrocarbons; gc-ms; poison; social insect; wasp. corresponding author a mendonça universidade federal da grande dourados programa de pós-graduação em entomologia e conservação da biodiversidade rodovia dourados-itahum, km 12 cidade universitária, c.p. 533 cep 79804-970, dourados-ms, brasil. e-mail: angel_bio1@yahoo.com.br the chcs act as contact or surface pheromones, as already reported in some studies (abdalla et al., 2003; neves et al., 2012; bello et al., 2015), and as signals or clues for nestmates, allowing the identification of conspecifics, assisting in maintenance of colonial structure, distinguishing individuals according to their function, physiological status and hierarchical rank (provost et al., 2008), acting as a specific chemical signature of the individual. these surface pheromones are mainly composed of hydrocarbons, especially linear alkanes, branched alkanes and alkenes (devigne & biseau, 2012). the primary functions of 1 universidade federal da grande dourados, programa de pós-graduação em entomologia e conservação da biodiversidade, dourados-ms, brazil 2 universidade estadual de mato grosso do sul, programa de pós-graduação em recursos naturais, dourados-ms, brazil 3 laboratório de ecologia comportamental – labeco, universidade estadual de mato grosso do sul, dourados-ms, brazil research article wasps a mendonça et al. – chemical composition of cuticular and of venom of wasps368 chcs are to prevent water loss and act as a protective coating for insects (gibbs, 1998). in addition, they mediate intra and interspecific interactions between these insects (blomquist & bagneres, 2010). it is known that chcs vary significantly between species (antonialli-junior et al., 2008; ferreira et al., 2012; santos & nascimento, 2015), castes (nunes et al., 2009, ferreira-caliman et al., 2013) and nestmates (lorenzi et al., 2004; costanzi et al., 2013), besides signaling dominance and fertility (cuvillier-hot et al., 2001; van oystaeyen et al., 2014), and may also vary according to the insect’s age (abdalla et al., 2003; biseau et al., 2004; antonialli-junior et al., 2007; nunes et al., 2009). social insects are also able to synthesize compounds for venom production, which represents part of a mechanism to capture prey and defend their colonies, and can also act on communication, as described by mateus (2011), which assessed that the wasp parachartergus fraternus uses venom to mark a new site for colony foundation before the start of migration. therefore, it can be inferred that there are elements in venom composition that must act as signals exchanged during interactions between conspecifics, probably those lighter and volatile among all compounds found in venom. bruschini et al. (2006a) found pheromones in the volatile portion of p. dominula venom that play a communicative role by inducing alarm behavior. behavioral studies with venom extracts (bruschini et al., 2008) also revealed that p. dominula wasps are more stimulated by venom of workers than foundresses. in addition, post and jeanne (1984) evaluated the potential role of volatile components of venom of polistes female wasps as a sex pheromone, attractive to males. thus, the study on volatile components of venom is also important to understand better which type of compounds are used for exchange of signals between conspecifics in colonies of social wasps. apoica pallens (fabricius, 1804) is a social swarmfounding wasp of nocturnal habit and thus presents large compound eyes and ocelli that are adaptations to seeing in dark (schremmer, 1972). this species is found from mexico to northeastern argentina (richards, 1978) and their colonies have from hundreds to thousands of individuals. polistes versicolor (olivier, 1791) is an independent-founding wasp of diurnal habit, whose nests have a single uncovered comb attached to the substrate by a peduncle. their colonies are relatively small in number of individuals and are very common in urban areas, being abundant in south america, present from costa rica to southern brazil and argentina (richards, 1978). despite chcs and venom being recognized as important compounds to the evolutionary success of social behavior, and that there is a relatively significant number of studies about them, still are rare those that investigated the cuticular composition and nonpolar portion of venom (sledge et al., 1999; dani et al., 2000; bruschini et al., 2006b; bruschini et al., 2008; silva et al., 2016) in tropical social wasps species. thus, the aim of this study was to compare the chemical composition of cuticle and nonpolar portion of venom of apoica pallens, a swarm-founding wasp and polistes versicolor an independent-founding wasp. materials and methods samples collection and extraction of cuticular compounds and nonpolar compounds of venom a total of 110 females of a. pallens from a single colony were used, all workers with the same approximate age (older), determined by the method of apodeme coloring (richards, 1971; west-eberhard, 1973) and 310 females of p. versicolor from 3 colonies, all with the same approximate age. colonies of both species were collected in rural and forest areas in the surroundings of dourados-ms, brazil (22°14′38.8″s, 54°49′36.6″w) in february 2015 and april 2016, respectively. the collections were performed using plastic bags and ether, by involving their nests and subsequently detaching them from the substrate. then, the wasps were transferred to the laboratory, where they were anesthetized at low temperature for subsequent extraction. for extraction of chcs, 10 workers of each species, totaling 10 samples of each species were used. extraction of chcs of the whole individuals was performed without any kind of fixative. each sample was immersed in a glass container with 2 ml of hexane for 2 minutes. after solute removal, samples were dried under fume hood and frozen for a maximum of 30 days. for chromatographic analysis, each extract was solubilized in 200 µl of hexane (tedia, hplc grade). for characterization of the nonpolar portion of a. pallens venom, 10 readings were performed, each sample with the content of 10 venom reservoirs. for venom samples of p. versicolor a triplicate was performed, each with the content of 100 reservoirs. this number varied between the two species due to their body size, and consequently of their venom reservoir. the definition of number of reservoirs was performed using preliminary tests carried out on samples from each species. venom reservoir extraction was performed by dissection, in ultra-pure water in order to prevent that membrane compounds were extracted, with the aid of tweezers and stereomicroscope, and subsequent removal of glandular filaments and sting. to extract the venom, the reservoir was lightly pressed into a vial until the release of its content. during the entire procedure, all samples were kept on ice to prevent volatilization and degradation of lighter compounds. then, samples were subjected to extraction in hexane followed by ethyl acetate. for each sample, 200 μl of ultra-pure water and 200 µl of hexane (hplc grade) were added, followed by agitation for 30 seconds and rest for three minutes, later the phases were separated. in the aqueous fraction 500 μl of ethyl acetate (hplc grade) were added, followed by agitation for 30 seconds and rest for 10 hours. after phase separation, the ethyl acetate fraction was joined to the hexane fraction of each sample, dried under fume hood, and solubilized in 200 µl of hexane. sociobiology 66(2): 367-376 (june, 2019) 369 analysis of samples by gas chromatography coupled to mass spectrometry (gc/ms) samples were analyzed employing a gas chromatograph coupled to a mass spectrometer (gc-ms ultra 2010, shimadzu, kyoto, japan), using a fused silica capillary column db-5 (j & w, folsom, california, usa) with 30 m length x 0.25 mm diameter x 0.25 μm thickness. the analysis conditions, programming of column temperature and scanning parameters were the same described in the study by paula et al. (2018). the identification of compounds was performed employing the calculated retention index (van den dool & kratz, 1963), using a mixture of linear alkanes (c7-c40, sigma aldrich with purity ≥ 90%) and the linear alkanes were employed as standard for identification of compounds. these calculated indexes were compared with the literature retention indexes (brown et al., 1991; howard et al., 2001; zhu et al., 2006; moore et al., 2014; weiss et al., 2014; silva et al., 2016), associated with interpretation of mass spectra obtained for the samples and compared with databases (nist21 & wiley229). based on the methodology described by dapporto et al. (2004, 2005), the area of each peak of the chromatogram of each sample was transformed into percentage. in addition, the compounds representing less than 0.5% were not presented in the tables. the major compounds were considered those that represented at least 4% of the total relative area. to assess the relationship between cuticle and venom of these two species, a discriminant function analysis (dfa) was employed using only the compounds shared by the 4 groups, in which wilk’s lambda near 0 reveals that the groups do not overlap, i.e., are different, and values close to 1 show overlap. results in samples of a. pallens cuticle 74 peaks were detected, 66 of these were identified (89.2%) with carbonic chain length varying from c16 to c37. the five major compounds in these samples were 13-methylheptacosane (15.56%) heptacosane (13.09%); x-methylheptacosane (10.83%); 13-methylhentriacontane (8.32%); 13-methylpentacosane (7.17%) (table 1). the most numerous and abundant classes of compounds were branched alkanes representing 74.5% of compounds, linear alkanes 23.5% and alkenes 1.6%. in samples of chemical profile of venom of this species 97 peaks were detected, of these 87 were identified (89.7%) ranging from c15 to c37. the five major compounds were 13-methylheptacosane (9.73%), 13-methylpentacosane (7.65%), 11,15-dimethylpentacosane (5.24%), 6-methyloctacosane (4.67%), x-methylheptacosane (4.95%) (table 1). branched alkanes were the most significant regarding number of compounds and abundance, representing 77.5% of compounds, followed by linear alkanes 8%, and alkenes 7.5% (figs 1b and 2b). fig 1. mean percentage of different classes of compounds in a) cuticular chemical profile and b) venom chemical profile of apoica pallens and polistes versicolor. fig 2. number of compounds of the different classes in a) cuticular chemical profile and b) venom chemical profile of apoica pallens and polistes versicolor. a mendonça et al. – chemical composition of cuticular and of venom of wasps370 calculated index (kraftz) compounds apoica pallens cuticle apoica pallens venom polistes versicolor cuticle polistes versicolor venom percentage (%±standard deviation) 1500 pentadecane nd 0.36±0.61 0.04±0.02 4.91±4.53* 1568 (r)-(-)-mellein nd 0.63±1.48 nd nd 1600 hexadecane§ 0.02±0.02 0.19±0.60 0.02±0.01 4.74±2.08* 1658 x-methylhexadecane nd 3.52±4.31 nd nd 1662 heptadecadiene nd 0.29±0.69 0.04±0.05 2.36±2.18 1699 heptadecane nd 0.34±0.60 0.01±0.01 2.49±0.81 1734 4hidroximellein nd 0.58±1.64 nd nd 1744 x-methylheptadecane nd 0.65±1.47 nd nd 1774 3-methylheptadecane nd 0.58±1.45 nd nd 1793 octadecene nd 0.32±0.99 0.03±0.02 nd 1800 octadecane nd 0.06±0.09 0.11±0.03 11.75±1.48* 1822 2-methyl-6-undecenyl piperidine nd 3.60±3.51 nd nd 1826 x-methyloctadecane nd 1.05±2.70 0.03±0.05 4.11±1.93 1874 nonadecene 0.07±0.11 0.67±0.70 nd 1.33±0.17 1900 nonadecane nd nd 0.01±0.00 0.72±0.20 1918 x-methylnonadecane tr 0.56±0.71 nd nd 1926 x-methylnonadecane tr 0.01±0.04 nd 0.59±0.18 1952 x-methylnonadecane nd 2.39±4.50 nd nd 1967 2-methylnonadecane nd 2.91±5.63 nd 3.39±0.44 1978 3-methylnonadecane nd 3.58±3.12 nd nd 1982 eicosene nd 0.05±0.08 0.02±0.04 4.33±1.91* 2001 eicosane nd 0.16±0.29 0.02±0.00 1.00±0.30 2116 x-methylheneicosane nd nd 0.01±0.01 0.56±0.23 2131 7-;9-;11-methylheneicosane nd 2.24±2.60 0.02±0.03 0.64±1.11 2172 x-methylheneicosane nd nd 0.58±1.00 1.34±0.29 2273 tricosadiene§ 0.02±0.06 0.63±1.99 0.09±0.17 1.04±0.69 2278 tricosene§ 0.01±0.04 2.46±2.50 0.02±0.03 0.44±0.76 2300 tricosane§ 0.08±0.10 0.19±0.27 0.10±0.16 1.95±0.43 2394 x-tetracosene nd nd 0.01±0.00 0.56±0.32 2399 tetracosane§ 0.06±0.03 0.27±0.38 0.01±0.00 0.52±0.17 2426 x-methyltetracosane nd 3.73±3.03 0.02±0.04 1.77±2.30 2500 pentacosane§ 6.33±0.72 3.31±3.39 0.07±0.04 1.14±0.10 2534 13-methylpentacosane 7.17±3.4* 7.65±6.99* 0.03±0.01 nd 2539 7-methylpentacosane 1.05±0.54 0.91±0.91 nd nd 2552 5-methylpentacosane§ 0.55±0.65 2.57±4.68 0.23±0.03 2.91±0.94 2562 11.15-dimethylpentacosane 0.65±0.38 5.24±3.37* nd nd 2570 3-methylpentacosane§ 2.87±1.25 2.40±2.30 0.03±0.05 1.39±1.55 2599 hexacosane§ 1.12±0.26 0.12±0.11 0.02±0.01 0.89±0.50 2633 12-methylhesacosane 0.76±0.35 0.43±0.39 0.01±0.00 nd 2680 heptacosene 0.56±0.46 0.15±0.21 0.01±0.01 nd 2692 heptacosene 0.44±1.07 1.94±2.09 nd nd 2700 heptacosane§ 13.09±3.63* nd 1.12±0.27 2.29±1.02 2711 x-methylheptacosane nd 4.95±4.38* 0.02±0.04 1.1±0.69 2732 13-methylheptacosane§ 15.56±5.81* 9.74±9.41* 0.19±0.03 0.11±0.19 2742 7-methylheptacosane 5.3±6.71 1.42±1.43 0.04±0.01 nd 2763 x-methylheptacosane 1.41±0.79 0.47±0.89 nd nd table 1. percentage area of nonpolar compounds (> 0.5%) present in cuticle and venom of eusocial wasps apoica pallens and polistes versicolor. sociobiology 66(2): 367-376 (june, 2019) 371 2765 x-methylheptacosane 0.77±1.03 0.74±1.48 nd nd 2770 3-methylheptacosane§ 10.83±1.99* 3.13±3.06 2.43±0.52 4.27±2.14* 2800 octacosane§ 0.48±0.08 0.13±0.16 0.3±0.06 1.66±1.60 2830 14-. 13-. 10-methyloctacosane§ 0.78±0.20 0.28±0.24 0.17±0.05 3.58±2.35 2845 6-methyloctacosane nd 4.67±4.84* 0.01±0.01 nd 2850 x-methyloctacosane nd nd 0.03±0.04 0.57±0.51 2901 nonacosane§ 1.70±0.54 0.15±0.18 5.37±0.92* 3.04±2.10 2928 13-methylnonacosene § 3.03±0.55 0.77±0.80 2.34±0.58 2.26±1.10 2933 15-methylnonacosane 1.17±0.23 0.10±0.22 3.73±0.85* nd 2936 13-methylnonacosane 0.60±0.15 nd 0.22±0.04 nd 2941 7-methylnonacosane nd 0.03±0.07 0.61±0.17 0.10±0.18 2951 5-methylnonacosane nd nd 0.55±0.08 0.18±0.31 2966 9.13-dimethylnonacosane nd nd 0.98±0.33 0.04±0.08 2975 3-methylnonacosane§ 1.74±0.41 0.05±0.11 12.23±1.34* 4.16±2.23 2984 5.x-dimethylnonacosane nd 3.44±4.33 nd 0.15±0.26 3000 triacontane§ 0.11±0.10 0.07±0.17 0.19±0.04 1.81±1.67 3028 10-methyltriacontane 0.22±0.05 nd 1.03±0.21 nd 3100 hentriacontane§ 0.08±0.03 1.17±2.51 1.17±0.34 1.07±0.95 3119 11-. 13-methylhentriacontane nd nd 0.04±0.05 3.60±0.91 3128 13-methylhentriacontane§ 8.32±1.79* 0.80±0.84 26.07±3.45* 3.85±2.55 3137 9-+13-methylhentriacontane nd nd 1.05±0.25 nd 3153 x-methylhentriacontane nd nd 0.82±0.20 nd 3155 13.17-dimethylhentriacontane nd nd 2.86±0.81 0.87±1.51 3165 7.15-dimethylhentriacontane nd nd 0.87±0.24 nd 3160 11.19-dimethylhentriacontane 2.22±0.52 nd nd nd 3174 5.15-dimethylhentriacontane nd nd 1.56±0.7 nd 3202 dotriacontane 0.10±0.06 nd 0.92±0.47 0.76±0.74 3228 16-. 14-methyldocotriacontane 0.15±0.07 nd 1.85±0.32 nd 3240 x-methyldotriacontane nd 1.11±1.83 nd nd 3284 x.y-dimethyldotriacontane 0.84±1.18 nd nd nd 3329 15-methyltritriacontane§ 2.57±0.7 0.06±0.19 23.49±6.37* 2.23±0.17 3334 x-methyltritriacontane nd nd 0.81±0.22 nd 3356 11.21-dimethyltritriacontane 2.71±0.68 nd nd nd 3365 11.15-dimethyltritriacontane (11.15-dimec33 nd 0.55±0.99 0.43±0.12 nd 3401 tetratriacontane nd nd 0.68±0.31 nd 3523 17-. 13-methylpentatriacontane nd nd 0.75±0.11 nd 3745 13.23 dimethylheptatriacontane 0.91±0.3 0.07±0.14 nd nd * = major compounds; §= compounds present in all samples; tr: trace (<0.005%); nd: not detected table 1. percentage area of nonpolar compounds (> 0.5%) present in cuticle and venom of eusocial wasps apoica pallens and polistes versicolor. (continuation) the cuticle presented 13 unique compounds and the venom 26 (table 1), and these two groups share 43 compounds. in samples of p. versicolor cuticle 93 peaks were detected, 85 of these were identified (91.4%), with chain length ranging from c15 to c37. the five major compounds were nonacosane (5.37%), 15-methylnonacosane (3.73%), 3-methylnonacosane (12.23%), 13-methylhentriacontane (26.07%) and 15-methyltritriacontane (23.49%) (table 1). branched alkanes were the most significant in number of compounds and abundance, representing 89% of compounds, followed by linear alkanes 10.4%, and alkenes 0.4% (figs 1a and 2a). in the venom of this species 64 peaks were detected calculated index (kraftz) compounds apoica pallens cuticle apoica pallens venom polistes versicolor cuticle polistes versicolor venom percentage (%±standard deviation) a mendonça et al. – chemical composition of cuticular and of venom of wasps372 and 60 identified (93.8%), ranging from c15 to c33. the major compounds (table 1) were pentadecane (4.91%), hexadecane (4.74%), octadecane (11.75%), eicosene (4.33%) and 3-methylheptacosane (4.27%). branched alkanes were the most significant regarding number of compounds and abundance, representing 46.3% of compounds, followed by linear alkanes 42.3%, and alkenes 7.5% (figs 1b and 2b). in these samples, 10 are unique compounds of cuticle and 5 of venom (table 1), and both share 49 compounds. among cuticular samples of both species, 41 compounds are shared, 25 are unique of a. pallens and 44 of polistes versicolor. among the samples of venom, 43 compounds are shared, 44 are unique of a. pallens and 17 unique of p. versicolor. the discriminant analysis shows that there are significant differences between chemical composition of cuticle and venom of the two species (fig 3), with wilk’s lambda = 0.001, p < 0.001 and f = 205.12. in this analysis, the first canonical root explains 95% of the results. found for other species of epiponini, such as polybia paulista ranging from c19 to c36 (kudô et al., 2017) and protopolybia exigua ranging from c14 to c36 (silva et al., 2016). in the cuticular profile of p. versicolor, the identified compounds range from c15 to c37, differing from the p. dominula, whose compounds range from c21 to c35 (lorenzi et al., 2004), and p. fuscatus ranging from c21 to c33 (espelie et al., 1994). however, brito et al. (2015) identified in the cuticular profile of the same species compounds that range from c8 to c30, although, in this study, the authors also evaluated cuticular compounds of immature stages. the variation in chain length found in both species is relatively similar, thus, without considering the classes of compounds, the fact that one species builds populous colonies and the other small colonies, does not appear to have an influence on this aspect. in the chemical profile of a. pallens venom, 87 nonpolar compounds were found ranging from c15 to c37. however, in the venom of p. exigua, there is variation in chain length from c19 to c30 (silva et al., 2016). nonpolar compounds of polybioides raphigastra venom vary from c11 to c18 (sledge et al., 1999). in the venom of p. versicolor, 60 compounds were identified ranging from c15 to c37. bruschini et al. (2008) studying the volatile portion of venom from different castes of p. dominula found 42 compounds, but do not describe the chain length variation. samples of p. versicolor cuticle have 85 compounds and a. pallens 66. so, despite being an independent-founding species with relatively small colonies, its cuticular chemical composition seems to be more complex. greater diversity in the profile of a species of polistes wasp was described by lorenzi et al. (2014), which associate the complexity in the cuticular profile of individuals from different colonies of polistes biglumis to the presence of parasites. the increase in complexity of cuticular profile makes it difficult for parasites to break the intracolonial code of wasps. this fact proves the adaptive plasticity of chcs of wasps of the genus polistes towards the influences in social environment (lorenzi et al., 2014). it is likely that p. versicolor has greater susceptibility to parasitism because the occurrence of parasitism in this genus has been reported in several studies (dapporto et al., 2007; kudô et al., 2014; lorenzi et al., 2014; torres et al., 2016). in both species studied, the branched alkanes are those that occur in greater abundance and number of compounds on the cuticle and in venom, followed by linear alkanes and finally alkenes (figs 1 and 2). similarly, bonavita-cougourdan et al. (1991) studying polistes dominula also identified in the same order of abundance the compounds present on the cuticle of these wasps. it is known that branched alkanes seem to be more involved with signaling during intraspecific interactions (lommelen et al., 2006). dani et al. (2001) and lorenzi et al. (2014) emphasize the communicative role of these compounds, since this class presents a high molecular fig 3. dispersion diagram showing the differences in cuticular chemical profiles and chemical profile of nonpolar compounds of venom of apoica pallens and polistes versicolor wasps. wilk’s lambda = 0.001, p < 0.001 and f = 205.12. discussion the cuticle and venom of the two species differ in number and content of compounds, which was already expected (bruschini et al., 2006b; ferreira et al.,2012; khidr et al., 2013; soares et al., 2017) because they are different species. p. versicolor presented more compounds on the cuticle than in venom. however, in both species, both on the cuticle and in venom, the most important compounds in terms of abundance were the branched alkanes, moreover, the cuticle and venom of p. versicolor share more compounds than a. pallens. in the cuticular chemical profile of a. pallens, the compounds range from c16 to c37, and this variation in chain length of compounds from the cuticle is similar to those sociobiology 66(2): 367-376 (june, 2019) 373 complexity, exhibiting high potential to encode information (leconte & hefetz, 2008; blomquist & bagnères, 2010). in this context, some authors consider branched alkanes the main mediators of chemical interactions between nestmates (dani et al., 1996; murakami et al., 2015). alkenes, although appearing in smaller proportions than other compounds in the samples of both species, also seem to be more related to the exchange of signals during chemical communication (gibbs, 2002; menzel et al., 2017). on the other hand, linear alkanes seem to be more involved in providing a barrier to prevent water loss (armold & regnier, 1975; menzel et al., 2017), i.e. the impermeability of the cuticle. however, tannure-nascimento et al. (2007) studying polistes satan identified greater abundance of linear alkanes in this wasp cuticle, suggesting that these compounds are also important as signals to mediate interactions between nestmates. the role of these compounds in venom, therefore, needs to be evaluated. both in the samples of a. pallens and p. versicolor there was higher concentration of heavy compounds on the cuticle, and light compounds in venom, varying significantly both qualitatively and quantitatively. according to blomquist and bagnères (2010) compounds below c20 are volatile and, therefore, can act as signals sent and received at a particular distance; while those of molecular weight above c20 can act as surface pheromone (ishay et al., 1965). the relatively lighter compounds, according to blomquist and bagnères (2010) are the most volatiles, and some studies with social wasps (ishay et al., 1965; jeanne, 1981; sledge et al., 1999; dani et al., 2000; bruschini et al., 2006a, 2006b, 2008) demonstrated that colony defense is performed by a collective response of the volatile components of venom, which consequently act as alarm pheromones unleashing attacks and recruiting nestmates. thus, the presence of a significant number of nonpolar compounds in the venom of both species suggests that at least part of them might be involved in some kind of chemical signaling. indeed, post and jeanne (1984) assessed that volatile elements of the venom of three species of wasps are responsible for attracting and stimulating behavior of males, and that males of p. fuscatus respond to venom of polistes exclamans and vespula maculifrons, although the intensity of the response is smaller when compared to his own species, suggesting that at least some of the volatile components of venom are chemically similar and that these components can vary among species only in relative proportions. more compounds were identified in the samples of a. pallens venom than in those of p. versicolor. it seems likely that a. pallens uses these compounds more effectively to mediate communication between nestmates, especially for colony defense, since by having large populations these wasps probably use volatile compounds of venom to alert nestmates, once even the nest structure is greater. on the other hand, as p. versicolor colonies contain few individuals, they may not use as effectively the compounds of venom for this purpose, or at least these compounds do not need to act at relatively long distances. in this context, greater complexity in venom profile might indicate greater action during interactions enabling that information reaches a greater number of workers. p. versicolor shares qualitatively more compounds between samples of venom and cuticle than a. pallens. however, in fig 3, the results show greater overlap of data of a. pallens. in this sense, the greater overlap is explained because samples of a. pallens display greater similarity in the content of shared compounds. thus, for both species, the shared compounds suggest that they may be the most effective as signals exchanged during interactions between nestmates. indeed, the literature has demonstrated the importance of variation in content of compounds for intraspecific chemical communication of wasps (panek & gamboa, 2000; cotoneschi et al., 2009; bonelli et al., 2015). the greater overlap of data of a. pallens (fig 3), suggest that the same compounds used to mediate interactions on the cuticle might also be used in venom. therefore, due to being an evolutionary more derived species that is organized in more populous colonies, it may have a more elaborate signaling system. p. versicolor is an evolutionary less derived species whose colonies are relatively less populous and by this reason may have a more rudimentary alarm system (jeanne, 1982). in conclusion, this study showed that, despite being wasps of different foundation types that are organized in colonies with significantly different population number, the variation in chain length of compounds is relatively similar. in addition, in both types of samples of both species, the most important compounds are branched alkanes that are recognized as the most effective during interactions between nestmates. however, there is significantly greater similarity in content of shared compounds between samples of cuticle and venom of a. pallens, suggesting that the same compounds used to mediate interactions on the cuticle are also used in venom. therefore, because it is a species that is organized in more populous colonies, it may have a more elaborate signaling system based on volatile compounds of venom, although, indeed, behavioral tests are needed to prove it. aknowledments the authors thank fundação de apoio ao desenvolvimento do ensino. ciência e tecnologia do estado de mato grosso do sul (fundect) for doctoral scholarship granted to the first author (fundect/capes n° 03/2014). coordenação de aperfeiçoamento de pessoal de nível superior (capes) for doctoral scholarship granted to the second author and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for productivity scholarship (wfaj grant number 307998/2014-2), (calc grant number 310801/2015-0). sisbio for authorization of the collect and of the transport of the specimens (sisbio license no.1748-1). a mendonça et al. – chemical composition of cuticular and of venom of wasps374 authors’ contribution the original idea for this research was conceived by a mendonça and wf antonialli junior. a mendonça, kb michelutti and cal cardoso performed the experiments and analyzed the data. a mendonça drafted the manuscript. all the authors reviewed and approved the final manuscript. references abdalla, f. c., jones, g.r., morgan, e.d. & cruz-landim, c. 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(2006). development changes of cuticular hydrocarbons in chrysomya rufifacies larvae: potential for determining larval age. medical and veterinary entomology, 20: 438-444. doi: 10.13102/sociobiology.v60i2.204-209sociobiology 60(2): 204-209 (2013) social wasps on eugenia uniflora linnaeus (myrtaceae) plants in an urban area gk souza1, tg pikart1, gc jacques1, aa castro1, mm de souza2, je serrão1, jc zanuncio1 introduction social wasps (hymenoptera: vespidae) belong to the subfamilies polistinae, stenogastrinae and vespinae (schmitz & moritz, 1998). among these families, only polistinae can be found in the neotropical region, with approximately 319 species occurring in brazil (carpenter & marques, 2001). social wasps are an important part of food webs, preying on insects of the orders coleoptera, diptera, hemiptera, hymenoptera and lepidoptera (prezoto et al., 2005) in terrestrial ecosystems (richter, 2000; torezan-silingardi, 2011). studies of social wasps have focused on their habitat preferences (da cruz et al., 2006; de souza et al., 2010b), nest density (diniz & kitayama, 1994), seasonal number of colonies, nesting habits (strassmann et al., 1997; diniz & kitayama, 1998; santos et al., 2009a), insecticide selectivity (galvan et al., 2002; bacci et al., 2009), and floral visitation (da silva-pereira & santos, 2006). however, diversity of these insects in urban areas has not been well studied. floristic diversity and vegetation structure can deterabstract social wasps (hymenoptera: vespidae) of the subfamily polistinae can be effectively incorporated into ipm systems for urban forestry. this study, conducted in viçosa, minas gerais state, brazil, in may 2011, identified species of this group foraging on eugenia uniflora linnaeus (myrtaceae) plants. the study area was monitored once a week and data collected included daily activity pattern, diversity, dominance and overlap of temporal niches by social wasps. data analysis revealed that e. uniflora plants were visited by 217 individuals of 16 species of the subfamily polistinae. foraging behavior of social wasps bore no relationship with sampling time, but overlap of temporal niche was high. wasps were not observed damaging healthy fruits, but were probably searching for lonchaeidae and tephritidae larvae. this study highlights the need for conservation of predator diversity in order to provide a partial alternative to the environmentally degrading chemical pesticides currently used in urban forestry for pest control. sociobiology an international journal on social insects 1 universidade federal de viçosa, viçosa mg, brazil 2 instituto federal de educação, ciência e tecnologia sul de minas gerais, pouso redondo-mg, brazil research article wasps article history edited by: gilberto m. m. santos, uefs brazil received 23 february 2013 initial acceptance 30 march 2013 final acceptance 11 april 2013 keywords biological control, diversity, polistinae, urban forestry, vespidae corresponding author tiago georg pikart departamento de entomologia universidade federal de viçosa viçosa, minas gerais, brazil 36570-000 e-mail: tiago.florestal@gmail.com mine composition of social wasp communities (santos et al., 2007) by affecting foraging activity during their search for water, vegetable fiber, carbohydrates, and proteins (richter, 2000; elisei et al., 2010). social wasps, like other generalist organisms, forage predominantly on the most abundant resource, without preference and/or selective behavior (santos et al., 2007), but they can focus their resource collection activities on a small group of plants (aguiar & santos, 2007). their generalist condition allows them to have low dependence on specific or constant food resources (real, 1981). brazilian myrtaceae includes trees and shrubs with potential for fruit production and use in urban forestry (donadio & moro, 2004). eugenia uniflora linnaeus (myrtaceae) is a semi-deciduous tree native to the south and southeast regions of brazil used in urban forestry (alves et al., 2008). its popularity is mainly due to the pleasant and refreshing taste of its fruits, which resemble cherries. however, the occurrence of insect pests such as eugeniamyia dispar maia, mendonça & romanowski (diptera: cecidomyiidae) and anastrepha fraterculus (wiedemann) (diptera: tephritidae) (bierhals et sociobiology 60(2): 204-209 (2013) 205 al., 2012) has been discouraging the use of e. uniflora for landscaping and urban forestry in brazil. social wasps are important natural enemies of insect pests (prezoto et al., 2005; de souza et al., 2010a; picanço et al., 2010, 2011) and their diversity is similar or even higher in urban areas than in natural environments (jacques et al., 2012). in this study we identified the social wasp species foraging on e. uniflora (myrtaceae) plants in order to evaluate the potential of this group to provide biological control in an integrated pest management program. material and methods diversity of social wasps (vespidae: polistinae) on e. uniflora plants was evaluated at the universidade federal de viçosa (ufv) in an urban area of viçosa, minas gerais state, brazil, in may 2011. two people monitored four heavily fruiting e. uniflora plants used in the urban forestry of the university once a week between 9:00 h and 15:00 h over a period of four weeks, and living specimens of social wasps foraging on these plants were collected with an insect net and preserved in a vial with 92.8% ethanol. collected specimens were taken to the laboratory of biological control of insects at ufv, mounted and identified. collection data were used to analyze daily activity pattern, diversity, dominance and temporal niche overlap of social wasp species. the diversity of social wasps was calculated with the shannon index (shannon, 1948) using the formula h’ = σpkxlnpk. the evenness of visits by each wasp species to e. uniflora plants was calculated with the formula j’ = h’/h’max (pielou, 1969). the dominance index was calculated with berger parker (may, 1975) with the formula d = nmax/nt. the temporal niche overlap per pair of wasp species was determined by using the schoener index (schoener, 1986) with the formula noih = 1-1/2σk|pik-phk|d. species with very small numbers of individuals (n <10) were excluded from the analysis. a kolmogorov-smirnov test for two samples was used to evaluate interspecific differences between activity patterns per pair of social wasp species (siegel, 1956). results and discussion plants of e. uniflora were visited by 217 individuals of 16 species of social wasps of the subfamily polistinae (table 1). species diversity was similar to that of the atlantic forest (santos et al., 2007), but it was lower than that of cerrado (de souza & prezoto, 2006; elpino-campos et al., 2007) and amazon forest (silveira et al., 2008; silva & silveira, 2009) areas. the similarity between the fauna of these insects and the species commonly found in the urban area sampled and the atlantic forest region is important. viçosa is included in this biome, which shows that social wasps have a high capacity to adapt to urban environments. besides occurring in different ecosystems, differences in collection methods and sample sizes may have contributed to differences in diversity between this study and those from other regions. on the other hand, surveys considering only one plant species showed similar or lower diversity than that of the present study (de souza et al., 2010a; santos & presley, 2010; de souza et al., 2011), suggesting that social wasp diversity may be determined more by variation in tolerance levels between species than by habitat complexity (bomfim & antonialli junior, 2012). table 1. number of species and individuals of social wasps (hymenoptera: vespidae: polistinae) collected on eugenia uniflora (myrtaceae) fruits in viçosa, minas gerais state, brazil number of individuals 04 may 18 may 25 may 30 may total agelaia multipicta 4 14 20 10 48 agelaia vicina 2 0 0 0 2 brachygastra lecheguana 0 0 1 6 7 mischocyttarus atramentarius 1 0 0 0 1 mischocyttarus cassununga 7 6 8 8 29 mischocyttarus drewseni 0 0 1 0 1 polistes actaeon 0 1 3 0 4 polistes simillimus 1 1 6 3 11 polistes versicolor 9 5 4 3 21 polybia bifasciata 1 0 1 0 2 polybia fastidiosuscula 7 9 8 7 31 polybia ignobilis 1 0 0 0 1 polybia jurinei 1 0 0 0 1 polybia platycephala 5 5 23 18 51 polybia sericea 0 2 2 1 5 protopolybia exigua 0 1 1 0 2 total 39 44 78 56 217 species of the dipteran families lonchaeidae and tephritidae, and trigona spinipes (fabricius) (hymenoptera: apidae) foraged on e. uniflora fruits. apis mellifera linnaeus (hymenoptera: apidae) and t. spinipes visited flowers of this plant, but social wasps were not observed in these structures. these last insects can collect nectar to feed their larvae and adults (da silva et al., 2011), but this survey was conducted during the dry season, when nectar production could have been insufficient to attract them. social wasps have no specialized structures for transporting pollen (corbicula) as do a. mellifera and t. spinipes, which were visiting e. uniflora flowers to collect pollen. all social wasp species exploited mainly green fruits gk souza, tg pikart, gc jacques, aa castro, mm de souza, je serrão, jc zanuncio social wasps foraging on eugenia uniflora206 with damaged areas and pre-existing holes caused by t. spinipes, and by oviposition behavior of fruit flies, as observed in anacardium occidentale linnaeus (anacardiaceae) plantations (santos & presley, 2010), and on myrciaria sp. (myrtaceae) plants (de souza et al., 2010a). brachygastra lecheguana (latreille), polistes simillimus zikán, polistes versicolor (olivier), polybia ignobilis (haliday), polybia platycephala richards, and polybia sericea (olivier) prey on larval forms of coleopteran, dipteran, and lepidopteran pests of agricultural and forest crops (prezoto et al., 2005; prezoto et al., 2006; bichara et al., 2009; elisei et al., 2010). foraging behavior of social wasp species may be related to the presence of larvae of fruit flies in e. uniflora. polybia platycephala (n = 51, d= 0.235) and agelaia multipicta (haliday) (n= 48, d= 0.221) were the social wasp species with the higher values of frequency and for the dominance index, followed by polybia fastidiosuscula saussure (n= 31, d= 0.143), mischocyttarus cassununga (von ihering) (n= 29, d= 0.134), p. versicolor (n= 21, d= 0.097), p. simillimus (n= 11, d= 0.051), b. lecheguana (n= 7, d= 0.032), p. sericea (n= 5, d= 0.023), and polistes actaeon haliday (n= 4, d= 0.018) (tables 1 and 2). polybia bifasciata saussure, protopolybia exígua saussure, and agelaia vicina (saussure) (n= 2, d= 0.009), and mischocyttarus atramentarius zikán, mischocyttarus drewseni (saussure), p. ignobilis and polybia jurinei (saussure) (n= 1, d= 0.005) were less frequent (tables 1 and 2) and of accidental occurrence. the active collection of social wasps is an efficient process and can include most species in a survey (de souza & prezoto, 2006; de souza et al., 2011; jacques et al., 2012), but some of them, such as a. vicina and p. bifasciata, may only be collected in baited traps (de souza & prezoto, 2006; jacques et al., 2012), which may explain their low frequencies in this study. fig 1. temporal foraging activity of social wasps (hymenoptera: vespidae: polistinae) on eugenia uniflora (myrtaceae) fruits. viçosa, minas gerais state, brazil. the foraging behavior of social wasps showed no relationship with sampling time (fig 1), differing from that in a. occidentale plantations, where species of this group foraged more frequently between 09:00 h and 12:00 h, and from table 2. dominance (berger-parker) of social wasp species (hymenoptera: vespidae: polistinae) collected on eugenia uniflora (myrtaceae) fruits as function of time of the day and total period. viçosa, minas gerais state, brazil. 09:45 10:45 11:45 12:45 13:45 14:45 total agelaia multipicta 0.194 0.180 0.177 0.243 0.333 0.172 0.221 agelaia vicina 0.059 0.009 brachygastra lecheguana 0.029 0.054 0.048 0.069 0.032 mischocyttarus atramentarius 0.024 0.005 mischocyttarus cassununga 0.056 0.051 0.235 0.162 0.191 0.103 0.134 mischocyttarus drewseni 0.024 0.005 polistes actaeon 0.028 0.026 0.024 0.035 0.018 polistes simillinus 0.056 0.077 0.029 0.027 0.071 0.035 0.051 polistes versicolor 0.139 0.103 0.088 0.081 0.071 0.103 0.097 polybia bifasciata 0.026 0.029 0.009 polybia fastidiosuscula 0.222 0.128 0.177 0.108 0.071 0.172 0.143 polybia ignobilis 0.028 0.005 polybia jurinei 0.027 0.005 polybia platycephala 0.222 0.333 0.177 0.270 0.119 0.310 0.235 polybia sericea 0.056 0.077 0.023 protopolybia exigua 0.027 0.024 0.009 number of individuals 36 39 34 37 42 29 217 shannon-winner index (h´) 0.843 0.828 0.847 0.820 0.869 0.806 0.911 pielou evenness index (j’) 0.884 0.868 0.888 0.859 0.835 0.892 0.757 sociobiology 60(2): 204-209 (2013) 207 14:00 h to 16:00 h (santos & presley, 2010), or the higher foraging activity of the social wasp parachartergus fraternus (gribodo) on sunny days, between 12:00 h and 14:00 h (santos et al., 2009b). climatic factors, such as light, temperature and wind speed, directly affect the foraging behavior of social wasps (kasper et al., 2008; santos et al., 2009b; de castro et al., 2011), so changes in these factors during the collection periods may have affected the results. diversity and evenness values for social wasp species were also similar during all collecting periods (table 2). the temporal niche overlap between pairs of species of social wasps was high, 0.65-0.91 (schoener index) (table 3), with the highest value for p. fastidiosuscula and p. versicolor (noih index= 0.9109). only five of the 15 comparisons table 3. interspecific pairwise comparison of activity patterns of social wasps (species with more than 10 individuals) collected on eugenia uniflora (myrtaceae) fruits in viçosa, minas gerais state, brazil. values above the diagonal correspond to statistical significance by the kolmogorov-smirnov test for two samples (significant values in bold, p< 0.05). values below the diagonal correspond to temporal niche overlap between pairs of species of social wasps (schoener index). a. m. m. c. p. s. p. v. p. f. p. p. agelaia multipicta 0.8096 0.0122 0.0361 0.2090 0.6974 mischocyttarus cassununga 0.8297 0.2090 0.2090 0.9996 0.2090 polistes simillinus 0.8371 0.6834 0.2090 0.0361 0.0361 polistes versicolor 0.8065 0.6700 0.7879 0.2090 0.0361 polybia fastidiosuscula 0.7466 0.6607 0.7126 0.9109 0.2090 polybia platycephala 0.7990 0.6531 0.7825 0.8487 0.8229 between pairs of species differed statistically, that is, with low value of temporal niche overlap (table 3). these results are similar to those of social wasps sharing the same food resource (santos & presley, 2010), again suggesting a tendency of co-existence between species of this group. social wasps are important predators of insect herbivores and preserving their diversity can contribute significantly to reduced application of chemical pesticides in urban forestry. social wasps can be easily found in anthropized environments (zanette et al., 2005; alvarenga et al., 2010; jacques et al., 2012), but small changes like adding floral resources to gardens or even the creation of public squares will enhance their presence and efficiency. acknowledgements the authors thank dr. tobias o. de oliveira (museu paraense emílio goeldi, belém, pará) for the identification of social wasps species. to “conselho nac. de desenv. científico e tecnológico (cnpq)”, “coord. de aperfeiçoamento de pessoal de nível superior (capes)” and “fundação de amparo à pesquisa do estado de minas gerais (fapemig)” for financial support. global edico corrected and edited the english of this manuscript. we thank two anonymous reviewers for valuable comments that improved this article. references aguiar, c.m.l. & santos, g.m.m. 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(2011). predatory behavior of pachodynerus brevithorax (hymenoptera: vespidae, eumeninae) on endophytic herbivore beetles in the brazilian tropical savanna. sociobiology, 57: 181-189. zanette, l.r.s., martins, r.p. & ribeiro, s.p. (2005). effects of urbanization on neotropical wasp and bee assemblages in a brazilian metropolis. landscape urban plan., 71: 105-121. doi: 10.1016/j.landurbplan.2004.02.003 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v65i3.2718sociobiology 65(3): 471-481 (september, 2018) different physiognomies and the structure of euglossini bee (hymenoptera: apidae) communities introduction although the atlantic forest is one of the 25 hotspots of endemism and diversity of species in the world (myers et al., 2000), it is one of the most devastated and threatened ecosystems on the planet (ribeiro et al., 2009). the isolation of this area from the other two large blocks of south american wet forests, the amazon and the andean forests, together with other factors, including its extensive latitudinal distribution (>27 degrees of latitude) and wide altitudinal variation (from 0 to 2,700 m above sea level), imbues considerable landscape diversity (silva & casteleti, 2005). as a result, this forest presents a high degree of biodiversity. abstract our aim was to evaluate the occurrence of orchid bees in remnants of the atlantic forest. we sampled specimens from five regions of southeast brazil, covering three different physiognomies of the atlantic forest (mixed araucaria forests with high altitude fields, atlantic semi-deciduous forest and atlantic rain forest). the distances between the sampling sites ranged from 24 to 746 km. male orchid bees attracted by fragrances were actively sampled monthly during one year using entomological nets and aromatic traps. a total of 1,482 bees were captured, including four genera and at least 31 species. we observed differences in the abundance and richness of species sampled among sites. climatic variables, mostly relative humidity, explained twice more of the observed differences in the euglossini communities than simple spatial variation. our study found differences in the composition of euglossine bee communities as well as in their patterns of abundance and dominance among different vegetation formations. however, the level of pairwise similarity among the euglossini communities sampled, although highly variable, was not related to climatic factors or geographical distances between the sampling sites. the greatest dissimilarities in the composition of the orchid bee communities were observed between sites around 400 km from each other rather than among sites that were further apart. a possible explanation is that the sampled areas that were 400 km from each other were also highly dissimilar regarding climate, especially altitude. sociobiology an international journal on social insects cas machado1*, cp costa2*, tm francoy3 article history edited by cândida aguiar, uefs, brazil received 08 december 2017 initial acceptance 17 march 2018 final acceptance 23 may 2018 publication date 02 october 2018 keywords euglossini, physiognomies, atlantic forest, beta diversity, conservation. corresponding author tiago maurício francoy escola de artes, ciências e humanidades universidade de são paulo – usp av. arlindo bettio, 1000, vila guaraciaba cep 03828-000, são paulo-sp, brasil. e-mail: tfrancoy@usp.br as part of this rich biodiversity, euglossine bees (hymenoptera: apidae: euglossini), commonly known as orchid bees, have been widely sampled in different neotropical ecosystems in recent decades, mostly in fragments or remnants of the atlantic forest (e.g. rebêlo & garófalo, 1997; tonhasca et al., 2002; sofia et al., 2004; aguiar et al., 2014; rochafilho & garófalo, 2013, 2014; giangarelli et al., 2015; costa & francoy, 2017). some these studies have shown structural differences in euglossine bee communities from distinct biogeographical regions, and differences have usually been attributed mainly to historical factors (aguiar et al. 2014). however, comparative studies of orchid bee communities from different vegetation physiognomies have shown that regional 1 faculdade de filosofia, ciências e letras de ribeirão preto; universidade de são paulo, ribeirão preto, sp, brazil 2 faculdade de medicina ribeirão preto, universidade de são paulo, departamento de genética, ribeirão preto, sp, brazil 3 escola de artes, ciências e humanidades, universidade de são paulo, são paulo, brazil * the authors contributed equaly to this work research article bees cas machado, cp costa, tm francoy – beta diversity of orchid bees472 differences in community structure are also influenced by climatic, geomorphological, and/or vegetational parameters (nemésio & silveira, 2007; sydney et al., 2010; mattozo et al., 2011; aguiar et al., 2014; nemésio & vasconcelos, 2013; giangarelli et al., 2015; costa & francoy, 2017; medeiros et al., 2017; moreira et al., 2017). originally distributed throughout most of the brazilian coast and parts of the interior in the southern region of the country, the atlantic forest is composed of a series of rather diverse vegetation physiognomies (silva & casteleti, 2005), which includes the atlantic rain forest and the atlantic semi-deciduous forest (morellato & haddad, 2000; oliveirafilho & fontes, 2000). although the atlantic forest is mostly composed of these two typical vegetation physiognomies, são paulo state in southeast brazil has varied biogeographic components, including the atlantic component and elements of the biogeographic component of central brazil, the western component, constituting an unmatched mosaic of vegetation physiognomies. in this context, we examined the community structure of euglossine bees in three distinct vegetation types of the atlantic forest. we investigated the importance of the different vegetation types and different climatic conditions (temperature, humidity, precipitation) and geomorphological factors (altitude, latitude, longitude) on the species composition of those bee communities. materials and methods study sites and field sampling – sampling of male orchid bees was carried out in three different phytophysiognomies (araucaria mixed forests with high altitude fields, atlantic semi-deciduous forest and atlantic rain forest) from five remnants of atlantic forest located in the state of são paulo, in southeast brazil (appendix 1): 1campos do jordão (22º44’19” s, 45º30’32” w), araucaria mixed forests, is located in the serra da mantiqueira, at 1,628 meters of elevation. the study in this region was carried out in native forest parque estadual de campos do jordão (pecj). according to köoper’s classification, the climate is cwa, mesothermal with dry winter. it is characterized by steep slopes with scalloped cliffs covered by transition vegetation between high-altitude fields and moist araucaria forests. 2pindamonhangaba (22º55’50” s; 45º27’22” w), atlantic semi deciduous forests, is located at 567 meters of elevation. according to köoper’s classification, the climate is cwa, humid subtropical, presenting average annual temperature of 22.4 °c, with warm temperatures in summer and mild winter. as a transition zone, it has a prevalence of vegetation characteristic of a seasonal forest, with environments that are less humid than those are where dense rain forest develops. 3parque estadual do morro do diabo (pemd) (22°27′0″ s, 52°10′0″ w), atlantic semi deciduous forests, is located in the pontal do paranapanema, municipality of teodoro sampaio city, west of são paulo state, at 338 meters of elevation. according to köoper’s classification, the climate is cwa, dry weather with hot and humid summers. it presents average annual temperature of 21 °c, with warm temperatures in summer and mild winter. mean annual precipitation ranges from 1,100 to 1,300 mm. 4ubatuba (23°26’13” s; 45°04’08” w), atlantic rain forests, is located at sea level. according to köoper’s classification, the climate is af, tropical rainforest climate, with warm temperatures in summer and mild winter. mean annual precipitation is 2,650 mm, and even in the driest months, from june to august, the average monthly precipitation is never less than 80 mm. the vegetation has a high degree of plant species endemism, in the lower areas, trees tend to be robust and tall, whereas with increasing altitude, they tend to become thin and low; this occurs closer to the ocean. the trees of the slopes can grow to over 40 meters, due to the abundance of organic matter. 5parque estadual turístico do alto ribeira (petar) (24°27′36″ s, 48°36′0″ w), atlantic rain forests, is located in the south of são paulo state, in the serra de paranapiacaba, between the baixada do ribeira and the planalto atlântico. according to köoper’s classification, the climate is cfb, humid subtropical without dry season and with cool summer. mean annual precipitation ranges from 1,500 to 2,000 mm. once a month during one year, the bees were actively collected with entomological nets, from april 2014 to march 2015 in campos de jordão, pindamonhangaba and ubatuba, and from may 2014 to april 2015 in pemd and petar, by two collectors. specimens were collected on sunny days between 08:00 h and 15:00 h, in transects approximately 60 m in length. we chose chemicals traditionally used in similar studies, and that showed good attractiveness to euglossine males (e.g. uehara-prado & garófalo, 2006; rocha-filho & garófalo, 2013; 2014): benzyl acetate, eugenol, eucalyptol, methyl salicylate and vanillin. sampling was conducted using methodology reported by rebêlo and garófalo (1991). we used baits with scents made from cotton wool wrapped in gauze and tied with a string; they were fixed in the branches of trees at the height of 1.5 m above the ground, at a distance of 5 m apart. the fragrance in each of the cotton wads was replaced every 60 min. all individuals were killed in 96% ethanol and preserved in this solution for subsequent molecular analysis (unpublished data). all specimens were stored in our laboratory, “laboratório de genética e conservação de abelhas” – escola de artes, ciências e humanidades – universidade de são paulo, at -20° c. the identification of specimens was based on the keys published by kimsey (1979, 1982), dressler (1982a), rebêlo and moure (1995), oliveira (2006), faria jr and melo (2007), nemésio (2009), nemésio and engel (2012). we also followed the species distribution criteria presented in moure’s bee catalogue (moure et al., 2008). doubtfull identifications were confirmed by a specialist. data analysis descriptive indices were estimated, including shannonwiener diversity (h’), to quantify species diversity based on sociobiology 65(3): 471-481 (september, 2018) 473 the number of males collected, and the data were compared using hutcheson’s t-test (hutcheson, 1970), as recommended by magurran (2004) for comparing species assemblages. we also calculated simpson’s index (s’) (magurran, 2004) to estimate the probability of randomly collecting two individuals of the community that belong to different species. to determine uniformity, we calculated pielou’s measure of species evenness (j’) (pielou, 1969). the berger-parker dominance index (d) was calculated to species dominance (magurran, 1988). for comparing the communities either qualitatively or quantitatively, we used β-diversity measures by similarity coefficients. the sørensen (sørensen, 1948) coefficient was used to compare community composition between the study areas. the quantitative similarity coefficient of bray–curtis was used to analyze the similarity in the fauna of the two sampled areas based on the relative abundance of the males. the mantel test statistic was calculated to analyze the variation of species composition among pairs of sampling sites about the geographic and the climatic distances between these sites. the mantel test uses information about pairwise dissimilarities (1 similarity) among sites; thus the male abundance dissimilarity matrices utilized in the mantel tests were built using both the bray–curtis index (which takes species relative abundances into account) and the sørensen index (which relies on presence–absence data). the geographic and climatic (based on precipitation, temperature and humidity) distances were calculated using the euclidean distance between sites. we also ran partial mantel tests to analyze the influence of the climatic variables independent of site location (geography) and vice versa. climatic data were obtained from the centro integrado de informações agrometeorológicas – ciiagro. to determine if the climate or geographic variables of our study sites influenced the relative abundances of the different orchid-bee species, we performed a canonical correspondence analysis (cca). correlation tests were done to evaluate the associations among variables before performed cca. the significance of each climatic and spatial variable in the cca was evaluated using anova. all of the tests of our study were performed using the statistical package r version 3.3.3 (r development core team, 2017). results we sampled 1,482 males, of four genera and at least 31 species. from this total, 1426 males, from 29 species were sampled using scent baits. it is important to note that 56 individuals were sampled on flowers and eufriesea surinamensis and euglossa stellfeldi were sampled only on flowers but not on the scent baits. the most attractive scent was eucalyptol, which attracted around 85% of the total visits and 28 species of bees. vanillin was the second most attractive fragrance and eugenol was the third (appendix 2). in petar, bees visited only eucalyptol. it is noteworthy that in one of our collection points, campos do jordão, no bees belonging to the tribe euglossini were collected, despite monthly sampling efforts. due to this fact, all the comparisons will be made using only the other localities, where it was possible to sample the euglossini community. the abundance of individuals and the number of species were different among the areas, and they were also higher in the warmer months (november to march, fig 1). only two species were present in all four sites: euglossa fimbriata rebêlo & moure and eulaema nigrita fig 1. monthly fluctuation of the abundance of bees and species richness of the euglossini tribe as a function of temperature (in degrees celsius) in areas with different forest vegetation types in são paulo state, brazil: (a) petar: parque estadual turístico do alto ribeira; (b) pindamonhangaba; (c) pemd: parque estadual morro do diabo and (d) ubatuba. cas machado, cp costa, tm francoy – beta diversity of orchid bees474 lepeletier, and only seven other species were present in three sites: eufriesea violacea (blanchard), euglossa annectans dressler, euglossa cordata (linnaeus), euglossa leucotricha rebêlo & moure, euglossa pleosticta dressler, euglossa securigera dressler and euglossa truncata rebêlo & moure (table 1). when species diversity was quantified based on the number of collected individuals, the shannon diversity index (h’ index mean = 1.676, range = 0.881-2.523) was significantly different (p < 0.01) for the sampled communities when compared pair to pair, except between pemd and petar. likewise, the simpson index (s’) differed between the sampling sites (p < 0.01), indicating a lower diversity in the petar community (s’ = 0.418), while the ubatuba area presented the highest diversity (s’ = 0.897). the pielou’s evenness index (j’) demonstrated a lower evenness in pemd (j’ = 0.436), while pindamonhangaba was more uniform (j’ = 0.780). similarly, the dominant species representation, determined by the berger-parker dominance index, was high for the pemd community (d = 0.731), dominated by eg. pleosticta, accounting for 73.1% of all bees collected in this area. dominance was lower in ubatuba (d = 0.151), where the most abundant species, euglossa imperialis cockerell, represented 15% of all bees sampled at this location (table 2). species pindamonhangaba ubatuba pemd petar n % n % n % n % eufriesea dentilabris (mocsáry, 1897) 1 3.57 eufriesea mussitans (fabricius, 1787) 1 3.57 eufriesea smaragdina (perty, 1833) 1 0.17 1 0.14 eufriesea surinamensis (linnaeus, 1758) 1 0.17 eufriesea violacea (blanchard, 1840) 2 0.33 50 7.09 3 10.71 euglossa analis westwood, 1840 2 0.33 euglossa annectans dressler, 1982 1 0.70 1 0.17 36 5.11 euglossa clausi nemésio & engel, 2012 2 0.33 euglossa cordata (linnaeus, 1758) 38 26.57 75 12.38 17 2.41 euglossa crassipunctata moure, 1968 1 0.70 12 1.98 euglossa fimbriata rebêlo & moure, 1996 5 3.50 10 1.65 18 2.55 2 7.14 euglossa ignita smith, 1874 0 0.00 2 0.33 euglossa imperialis cockerell, 1922 6 4.20 92 15.18 euglossa iopoecila dressler, 1982 4 2.80 81 13.37 euglossa ioprosopa dressler, 1982 1 0.70 6 0.99 euglossa iopyrrha dressler, 1982 1 0.17 euglossa leucotricha rebêlo & moure, 1996 19 13.29 5 0.83 1 0.14 euglossa mandibularis friese, 1899 18 2.97 euglossa melanotricha moure, 1967 1 0.70 1 0.17 euglossa pleosticta dressler, 1982 14 9.79 70 11.55 516 73.19 euglossa roderici nemésio, 2009 1 0.70 44 7.26 euglossa sapphirina moure, 1968 0 0.00 56 9.24 euglossa securigera dressler. 1982 10 1.65 1 0.14 euglossa stellfeldi moure. 1947 2 1.40 euglossa townsendi cockerell. 1904 2 0.33 euglossa truncata rebêlo & moure, 1996 4 2.80 7 1.16 2 0.28 euglossa viridis (perty, 1833) 13 9.09 77 12.38 eulaema cingulata (fabricius, 1804) 1 0.70 9 1.49 eulaema nigrita lepeletier, 1841 31 21.68 19 3.14 55 7.80 21 75.00 eulaema seabrai moure, 1960 1 0.70 1 0.17 exaerete smaragdina (guérin, 1844) 1 0.17 8 1.13 total 143 100 606 100 705 100 28 100 table 1. euglossine bees collected in different sampling sites of the atlantic forest at são paulo state, brazil. pemd: parque estadual morro do diabo; petar: parque estadual turístico do alto ribeira. sociobiology 65(3): 471-481 (september, 2018) 475 sampling sites index diversity shannon (h’) simpson (s’) evenness (j’) bergerparker (d) pindamonhangaba 2.256 0.859 0.781 0.254 ubatuba 2.523 0.897 0.757 0.151 pemd 1.045 0.449 0.436 0.731 petar 0.881 0.418 0.547 0.750 the level of pairwise similarity among the sampled bee communities was low and showed considerable variation, ranging from 7.09 to 32.72 % (bray–curtis index mean = 17.96 %, table3) when considering the relative abundances of all species. according to this index, pindamonhangaba and ubatuba were the most similar areas, with 17 species in common. when considering the presence or absence of species, the pairwise values of similarity among the areas were high (sørensen index: mean = 42.24 %, range = 17.39-73.91%, table 3). however, for the sørensen index, pindamonhangaba and petar were the least similar areas regarding species composition; while for the bray–curtis index, petar and table 2. shannon’s diversity (h’), simpson’s diversity (s’), pielou’s evenness (j’) and berger-parker dominance (d) for the four study areas. pindamonhangaba ubatuba pemd petar pindamonhangaba 0.327 0.167 0.251 ubatuba 0.739 0.189 0.072 pemd 0.551 0.512 0.071 petar 0.173 0.181 0.375 table 3. sørensen index (bottom of the table) and bray–curtis index (top of the table) for the four study areas. pemd: parque estadual morro do diabo; petar: parque estadual turístico do alto ribeira. pemd were the least similar areas. further analyses were based solely on relative abundance data. similarity in bee species composition among the different pairs of study sites was not significant both as function of the climatic (mantel test: r = -0.028, p = 0.625) and geographic distances among these locations (r = 0.485, p = 0.16667). the greatest similarities in the composition of bee assemblages tended to be observed among pairs of sites located in similar physiognomies. our cca analyses revealed that 75.5% of the total variation in the bee species composition could be explained by a combination of humidity and altitude; whereas an additional 24.5% remained unexplained or represented stochastic variation. the model with two climatic variables was significant (anova: 1.546, p = 0.041) in explaining the observed variation based on the relative abundances of orchid bee species within our study region. it can be further seen in our cca ordination graph that the species matrix and the climatic variables had a similar spatial structure. pemd was relatively separated from the remaining sites regarding the composition of its fauna (fig 2). humidity tended to increase from interior to coastal regions; this may help explain the observed latitudinal differences in the community composition of the orchid bees, with several of these species occurring exclusively in some parts of the climatic gradients. fig 2. cca ordination plot of the sampling sites in relation to the orchid bee and climatic and geographic variables. pemd: parque estadual morro do diabo; petar: parque estadual turístico do alto ribeira. cas machado, cp costa, tm francoy – beta diversity of orchid bees476 discussion regarding euglossini, nemésio (2009) recognized around 54 species in the atlantic forest; however, new species continue to be described (e.g. nemésio, 2011; hinojosa-díaz et al., 2012; faria jr. & melo, 2012; nemésio & engel, 2012). assuming at least 62 orchid bee species inhabit this area, and 31 (approximately 50%) of these species were found in our study in a relatively small portion of the area, the sampled regions could be a refuge for a large number of euglossini species. these data are similar to the richness pattern of this group reported for southeastern brazil (nemésio, 2009). in contrast, campos do jordão was characterized by a lack euglossini bees, probably as a consequence of the local geoclimatic characteristics since this region is characterized by araucaria mixed forests with high altitude fields (for more details see costa & francoy, 2017). despite scarcity of information about orchid bee presence in araucaria forests, dias and buschini (2013) indicated that these bees have a low diversity and abundance in araucaria forest, being markedly smaller compared to other physiognomies. these authors (dias & buschini, 2013) sampled 35 male specimens belonging to two species, el. nigrita and eg. fimbriata, in a similar area of araucaria forest in paraná state. however, our area of araucaria forest in campos do jordão is located at an altitude of 1,628 m, what probably accounted to the absence of euglossini bees, since altitudinal variation can drastically reduce abundance patterns and alter the species compositions of orchid bee communities (abrahamczyk et al., 2011). our data suggest that altitude is an important factor that induces variation in several other geomorphological and climatic features and therefore, helps to modify species composition and abundance of orchid bees, even at small scales of variation, however, this result has to be tested in future studies. the efficiency of eucalyptol, or cineole, in attracting orchid bees is already well known (e.g. dressler, 1982b; rebêlo & garófalo, 1991; sofia et al., 2004; rocha-filho & garófalo, 2014). however, eugenol, which was very attractive to male euglossine in other sites (e.g. dressler, 1982b; rocha-filho & garófalo, 2014), was poor attractants our study. geographical variation in fragrance preference of male orchid bees are common and was also reported by others authors (e.g. pearson & dressler, 1985, sofia et al., 2004; farias et al., 2007; rocha-filho & garófalo, 2014). the distribution patterns of abundance in the sampled areas were similar to those of other studies made in other atlantic forest fragments; a few species had many individuals, and many species had few individuals (e.g., rebêlo & garófalo, 1997; sofia & suzuki, 2004; sofia et al., 2004; aguiar & gaglianone, 2008; ramalho et al., 2009; giangarelli et al., 2015; rocha-filho & garófalo, 2013). this tendency can be the result of the weak association of some species with the essences used (viana et al., 2002) or can be the distribution pattern of these communities (aguiar & gaglianone, 2008). this pattern was more evident in pemd, where individuals of eg. pleosticta represented 73.2% of the total, considered typical species of semi-deciduous areas. also, species richness of orchid bees was characterized by a north-south gradient, with a gradual reduction from north to south direction, similar to other studies (wittmann et al., 1988; sofia & suzuki, 2004; mattozo et al., 2011; cordeiro et al., 2013). ubatuba was the area with the highest species richness of all areas sampled, even after the equalization efforts with rarefaction techniques; meanwhile, petar, the area furthest south, had the lowest species richness and abundance of orchid bees. most of the sample areas included species with a wide distribution range along the north–south corridor of the atlantic forest, such as eg. cordata and el. nigrita, which occurs along the entire coast of brazil, as well as species that predominate in the southeastern region, such as eg. fimbriata and eg. pleosticta (cordeiro et al., 2013). several euglossine species found in the studied areas, such as eufriesea dentilabris (mocsáry), euglossa iopoecila dressler, euglossa stellfeldi moure and euglossa roderici nemésio, are endemic to the atlantic forest. el. nigrita and eg. fimbriata were the only two species found in all sample areas. also, the finding of el. nigrita in all the areas was also observed in other surveys made in the atlantic forest (e.g. farias et al., 2008; nemésio & silveira, 2010; aguiar et al., 2014). this species was abundant in areas in different conservation stages; it is tolerant to disturbances in the environment and vegetation (peruquetti et al., 1999; ramalho et al., 2009; aguiar & gaglianone, 2012). the great flight range could account for their prevalence in various areas (for more details see dressler, 1982b; roubik & hanson, 2004). the relative abundance of el. nigrita varied between 3 and 75%; meanwhile for eg. pleosticta, the predominant species in pemd, abundance ranged from 9 and 73%. this variation in abundance affects the similarity between areas since we found values between 0.07 and 0.32 for bray–curtis index, which considers the relative abundances of all species. a similar result was found for the sørensen index, which presented a high amplitude (0.17-0.74), due to the considerable variation in the species richness across the sampled areas. the values of richness and/or abundance of euglossine bees are in disagreement with those of previous studies in similar areas of the atlantic rain forests (singer & sazima, 2004; rocha-filho & garófalo, 2013, 2014), including the low richness of the eufriesea genus and the high abundance of two species, which frequently are recorded in semi deciduous forests (rocha-filho & garófalo, 2013), eg. pleosticta and eg. imperialis in the coastal region. however, these comparisons should be viewed with wariness, since the sample design used in the studies is often different. according to roubik (2001), reduced chances of resampling could reflect the dynamics of the bee communities on a short time scale. additionally, our results revealed that the level of pairwise similarity among the euglossini communities sampled, although highly variable, was not related to climatic or geographical distances sociobiology 65(3): 471-481 (september, 2018) 477 among the sampling sites. the greatest dissimilarities in the composition of the orchid bee communities were observed among areas distant around 400 km from each other rather than among the sites further apart. one reason for this fact could be that the sampled areas distant 400 km from each other were also highly dissimilar regarding climate-related variables, especially altitude. besides, despite orchid bees show the large foraging range, becoming strong flyers (janzen, 1971), several species of bees found in the ubatuba were not collected in the pindamonhangaba, although both areas are only 61 km apart and connected, largely, by areas of eucalyptus and forest remnants. in our study region, the atlantic forest is mostly characterized by three vegetation physiognomies, mixed araucaria forests with high altitude fields, atlantic rain forest and atlantic semi-deciduous forest, which include several natural gradients within their latitudinal and longitudinal extensions, as well as an altitudinal variation from the coast to the interior. according to lázaro and totland (2010), vegetation types and differences in the availability of key resources can be one of the drivers of change population patterns of pollinators. especially, in the neotropical region, the floral landscape is spatially and temporally heterogeneous for foraging bees, promoting considerable change in the abundance pattern and amount of brood in the nests for bees (smith et al., 2012). climatic variables, mostly relative humidity, explained twice the observed differences in the euglossini communities compared to spatial variation alone. relative humidity seems to be the most important abiotic factor for our study, which was also found in another study that found the preference of orchid bees for humid forests (roubik & hanson, 2004). we found to a great richness of orchid bees in the atlantic rain forest, even within a small spatial scale. this could be due to the high diversity of habitats, influenced by the wide geomorphological and climatic variation, besides the atlantic and occidental components, which extend throughout são paulo state, from the coast to the interior, constituting a mosaic of landscapes that probably favors the occurrence of euglossini in this region. different physiognomies and, consequently, the variation in the availability of key resources can change population patterns of orchid bees (see nemésio & silveira, 2007); a thorough analysis of resource availability in the sampling areas would be important to clarify this issue. in addition to the abiotic factors that can affect orchid bee assemblages, other components, such as competition with similar species, historical occurrences, and habitat homogeneity (roubik, 2001; tonhasca et al., 2002; roubik & hanson, 2004) could also influence euglossine communities. the cleptoparasitic euglossine bees, such as genus exaerete, involve additional biotic factors for their occurrence, such as the occurrence of host species in the genera eulaema and eufriesea (aguiar et al., 2014); however, these factors have not been evaluated in this study, but should be considered in future studies. our data reinforces the general pattern that the euglossine fauna of the atlantic forest changes according to the abiotic factors (nemésio & silveira, 2007; abrahamczyk et al., 2011; aguiar et al., 2014; nemésio & vasconcelos, 2013; giangarelli et al., 2015, medeiros et al., 2017). however, a lack of basic studies on euglossini biology impedes an objective evaluation of the real influence of these factors on community composition (nemésio & vasconcelos, 2013). nevertheless, our sampling area that can be considered floristically diverse, with drastic differences among physiognomies, suggests that variations in vegetation community composition can help to explain, at least in part, differences in the composition of euglossine communities. acknowledgments dr david de jong was extremely kind in helping us to improve the english of our manuscript. mr. josé carlos serrano kindly confirmed doubtful identifications of our specimens. we are also thankful to agência paulista de tecnologia dos agronegócios (apta-saa, sp), in particular, dr érica weinstein teixeira, for support and infrastructure for our sampling. we also thank ibama (brazilian institute of environment and renewable natural resources)/icmbiosisbio (institute chico mendes – mma) for permission to collect bees. financial support was provided by fapesp (process 2011/07857-9 to tmf and process 2013/02158-0 to cpc). references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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di na 1 1 2 e uf ri es ea s ur in am en si s e uf ri es ea v io la ce a 2 37 3 1 12 55 e ug lo ss a an al is 2 2 e ug lo ss a an ne ct an s 1 1 1 23 6 1 5 38 e ug lo ss a cl au si 2 2 e ug lo ss a co rd at a 1 37 65 17 1 1 1 12 3 e ug lo ss a cr as si pu nc ta ta 1 12 13 e ug lo ss a fim br ia ta 5 10 18 2 35 e ug lo ss a ig ni ta 2 2 e ug lo ss a im pe ri al is 2 3 74 1 7 1 1 89 e ug lo ss a io po ec ila 4 77 1 82 e ug lo ss a io pr os op a 1 6 7 e ug lo ss a io py rr ha 1 1 e ug lo ss a le uc ot ri ch a 18 5 1 24 e ug lo ss a m an di bu la ri s 1 5 10 16 e ug lo ss a m el an ot ri ch a 1 1 e ug lo ss a pl eo st ic ta 7 12 64 40 4 3 11 1 2 1 73 57 8 e ug lo ss a ro de ri ci 5 1 29 1 6 42 e ug lo ss a sa pp hi ri na 49 3 1 53 e ug lo ss a se cu ri ge ra 12 9 1 1 23 e ug lo ss a st el lfe ld i e ug lo ss a to w ns en di 1 1 2 e ug lo ss a tr un ca ta 3 5 2 1 11 e ug lo ss a vi ri di s 1 10 46 12 2 9 1 81 e ul ae m a ci ng ul at a 1 9 10 e ul ae m a ni gr ita 30 19 46 21 7 12 3 e ul ae m a se ab ra i 1 1 e xa er et e sm ar ag di na 1 5 2 8 a bu nd an ce 1 10 9 14 0 49 5 55 4 28 1 41 18 3 19 3 0 5 99 14 26 * r ic hn es s 1 5 3 16 24 10 5 1 10 3 2 6 2 0 5 5 29 ** *t he to ta l r ef er s on ly to th e in di vi du al s co lle ct ed th ro ug h th e us e of a ro m at ic tr ap s. ** t he to ta l r ef er s on ly to th e sp ec ie s co lle ct ed th ro ug h th e us e of a ro m at ic tr ap s. e uf ri es ea s ur in am en si sa nd e ug lo ss a st el lfe ld i w er e on ly c ol le ct ed in fl ow er s of p la nt s. 511 trajectory of waterand fat-soluble dyes in the grass-cutting ant atta capiguara (hymenoptera, formicidae): evaluation of infrabuccal cavity, post-pharyngeal glands and gaster by luiz carlos forti1, marcílio de souza silva1*, ricardo toshio fujihara1, nádia caldato1 & marise grecca garcia1 abstract the diet of leaf-cutting ants is based on cultivation of their symbiotic fungus, whose successful cultivation depends on the task of incorporation and handling of vegetable substrate. this task may cause the workers to be contaminated with toxic substances and thus decrease the survival of the colony. the objective of this study was to analyze the contamination of workers of atta capiguara as well as the dissemination trajectory of waterand fat-soluble substances. four colonies received non-toxic baits containing water-soluble dye rhodamine-b and three, non-toxic baits with fat-soluble dye sudan iii. the dye rhodamine-b stained the gaster in 40.31% of workers and showed no significant difference among castes. the sudan iii stained the infrabuccal cavity in 35.41%, post-pharyngeal glands of 24.22% and gaster in only 8.44% of the workers, with no significant difference among the castes. the watersoluble dye was spread in the body of workers through the digestive system while fat-soluble dye was diverted to the post-pharyngeal glands. keywords: digestive system; glands; leaf-cutting ants; manipulation; toxic baits introduction social insects, such as leaf-cutting ants, present a division of labor and complex behavioral activities, including foraging (hölldobler & wilson 1990). their principal food source comes from the cultivation of the symbiotic fungus leucoagaricus gong ylophorus with vegetal substrate (silva et al. 2003; santos et al. 2006). however, larger workers obtain part of their diet 1laboratório de insetos sociais-praga, defesa fitossanitária, fca/unesp, fazenda exp. lageado, rua josé barbosa de barros 1780, zip code 18610-307, po box 237, botucatu, sp, brazil. *corresponding author: agromss@hotmail.com 512 sociobiolog y vol. 59, no. 2, 2012 from liquids released during the cutting of leaves (peregrine & mudd 1975; quinlan & cherrett 1978; forti & andrade 1999). in practical terms, the incorporation of vegetal substrate into the fungus garden is closely related to the contamination of workers by insecticides formulated into granulated bait (andrade et al. 2002). this knowledge has generated a series of studies in the area of chemical control of leaf-cutting ants, but most are experiments with insecticides that aim to elucidate the dynamic of workers contamination (echols 1966; andrade 2002; pagnocca et al. 2006; santos et al. 2006; nagamoto et al. 2007). according to peregrine and cherrett (1976), many studies are performed that seek to discover different chemical substances with formicidal activity, but few focus on obtaining knowledge of their action mechanism. to this end, dyes are utilized as a tool to demonstrate not only the ingestion of plant sap by ants but also the dispersal of insecticides within their organism (peregrine et al. 1972; bueno et al. 2001; forti et al. 2007). furthermore, it should be emphasized that the digestive system of adult ants, when compared to that of other insects, presents some adaptations. anterior to the mouth opening, just after the glossa, is found the infrabuccal cavity, in which the solid material is retained by a filter of small bristles, where only the liquid or semi-solid foods pass to the proventriculus. the latter regulates the passage of food from the crop to the ventriculus, where digestion then occurs (fowler et al. 1991; chapman 1998; jesus & bueno 2007). at the end of the pharynx, near its transition with the esophagus, the ducts of the post-pharyngeal glands open dorsally, as found in formicidae and solitary wasps (delage-darchen 1976; strohm et al. 2007; bagnères & blomquist 2010). according to some authors, these glands are associated with the synthesis of lipids (peregrine et al. 1972; gama 1985; eelen 2006) and the accumulation of cuticular hydrocarbons during ontogenesis, important in the regulation and recognition of nest partners (soroker et al. 1994; bagnères & blomquist 2010). nevertheless, these glands remain little studied. the crop, although considered underdeveloped in the genus atta, is responsible for the storage of liquids (paul & roces 2003). nevertheless, some studies suggest the absence of trophalaxis among its workers, who did not present sharing of stored food (andrade 1997; andrade et al. 2002; bueno 2005). 513 forti, l.c. et al. —trajectory of dyes in grass-cutting ants the singular morphological characteristics of formicidae and the differentiated behavioral repertoire of grass-cutting ants, which process less substrate for incorporation into the symbiotic fungus, are factors to be evaluated in the dispersal of insecticides and supply of informations on the post-pharyngeal glands, whose functions still have not been completely elucidated. therefore, the present study aimed to analyze the contamination frequency of workers of a. capiguara, as well as the dissemination trajectory of waterand fatsoluble substances (in the form of dyes), through their digestive system and post-pharyngeal glands. material and methods collection of a. capiguara colonies young colonies of a. capiguara were collected from pasture areas in botucatu, sp, brazil (23°06'08''s, 48°25'28''w) and transported to the laboratório de insetos sociais-praga, fca/unesp, botucatu. they were raised in a closed system at a temperature of 24ºc ± 1 and relative air humidity of 70% ± 1, utilizing transparent plastic pots (1 cm of plaster at the bottom) to maintain the humidity of the colony. the vegetal materials used as substrate were leaves of sugarcane (saccharum officinarum) or tifton grass (cynodon spp.). before performing the experiments, the colonies were maintained without receiving leaves for 48 hours. preparation and offering of baits the baits containing water-soluble dye were prepared with 90% sugarcane leaf powder, 6% water and 4% rhodamine-b dye (vetec química fina). for the baits with fat-soluble dye, the proportion was 90% sugarcane leaf powder, 6% soy oil and 4% sudan iii (vetec química fina). after mixing, the ingredients were placed in disposable polyethylene bags with a small hole in the nozzle (2.5 mm ø), thereby formulating the bait filets. after 24 hours of drying at room temperature, the filets were cut into pellets 0.5 cm in length. four colonies received baits containing the water-soluble dye rhodamine-b whereas three received baits with the fat-soluble sudan iii dye, at the dose of 0.5g/colony. it should be emphasized that neither contained the active ingredient and that, after the baits had been offered for 30 hours, the fungus garden and the workers were stored in a freezer. 514 sociobiolog y vol. 59, no. 2, 2012 evaluation of dye digestion the workers of each colony were separated into three castes, according to the width of the cephalic capsule: gardeners (< 1.4 mm), generalists (1.4 – 2.1 mm) and foragers (> 2.1 mm). the colonies that received baits with rhodamine-b were evaluated only for staining of the gaster. with the aid of forceps, this was compressed on dried ribbons of pig intestine on account of their protein content, which enabled easy impregnation of the dye. after this procedure, the ribbons were placed under ultraviolet light, permitting a count of the number de individuals marked with the dye (forti et al. 2007). the workers of the colonies that received sudan iii baits were dissected stereoscopically to observe the infrabuccal cavity (ic), post-pharyngeal glands (ppg) and the gaster. data analysis after the period during which baits were offered to the workers (30 hours), these individuals were collected and counted. subsequently, the absolute frequency data of stained and unstained anatomical structures were submitted to the chi-square test (χ2), at 5% significance level, utilizing the program bioestat 5.0 (ayres et al. 2007). when the test revealed significance, a contingency table and partition analysis were completed to assess whether the castes differed as to the dye utilized (wateror fat-soluble). results when the dye rhodamine-b was utilized, the proportion of a. capiguara workers that presented stained versus unstained gaster structures (crop, ventriculus and rectum) was independent of the caste, that is, there was no significant difference among these types (table 1). of the total of 583 workers analyzed, 40.31% had a stained gaster, revealing that the water-soluble substance rhodamine-b was disseminated through the digestive tract of the workers. thus, it is supposed that substances toxic to ants such as some insecticides, for example, when water-soluble, can also contaminate the same by digestive pathways. taking into consideration the relative frequency of the data presented in table 1, the results show that 39.19% of the foragers had a stained gaster, the same occurring to 44.19% of generalists and 38.10% of gardeners. according to forti et al. (2007), the 515 forti, l.c. et al. —trajectory of dyes in grass-cutting ants highest level of contamination (about 50%) occurs in the first 24 hours in all sizes of workers, thus, justifying our assessment during 30 hours. in relation to the trajectory of sudan iii fat-soluble dye, out of a total of 545 workers dissected, only those with unstained ic presented a significant difference (table 2). of the 164 foragers dissected, 25.61% presented stained ic, 19.51% ppg and only 5.49%, a stained gaster. these percentages were obtained in relation to the total number of workers, since not all that had table 1proportion of a. capiguara workers by caste with gaster stained and unstained after application of baits with rhodamine-b dye. ns: not significant by chi-square test (p<0.05). structure proportion caste χ2 pforagers generalists gardeners gaster stained 87 76 72 1.00ns 0.3159 unstained 135 96 117 0.56ns 0.4505 general 1.57ns 0.4549 table 2 proportion of a. capiguara workers by caste with anatomical structures stained and unstained after application of baits with sudan iii dye. ic: infrabuccal cavity; ppg: post-pharyngeal glands. structure proportion castes χ2 pforagers generalists gardeners ic stained 42 51 100 3.22ns 0.0726 unstained 122ab 93a 137b 8.43* 0.0037 general 11.65* 0.0029 ppg stained 32 35 65 0.95ns 0.3272 unstained 132 109 172 2.34ns 0.1254 general 3.30ns 0.1913 gaster stained 9 12 25 0.80ns 0.3701 unstained 155 132 212 2.41ns 0.1204 general 3.21ns 0.2004 * frequencies followed by distinct letters on the line differ with each other by the chi-square test (p<0.05). ns: not significant by the chi-square test (p<0.05). table 3 absolute and relative frequency of a. capiguara workers presenting stained ppg and gaster, among those with ic stained by sudan iii. ic: infrabuccal cavity; ppg: post-pharyngeal glands; af: absolute frequency; rf: relative frequency. caste (ic stained) n ppg stained gaster stained only ic stained af rf (%) af rf (%) af rf (%) foragers 42 32 76.19 9 21.43 1 2.38 generalists 51 35 68.63 12 23.53 4 7.84 gardeners 100 65 65.00 25 25.00 10 10.00 total 193 132 68.39 46 23.84 15 7.77 516 sociobiolog y vol. 59, no. 2, 2012 stained ic also presented stained ppg, which also occurred with the gaster. among the generalists and gardeners, the same calculation was performed. the total data on stained workers demonstrate that not all individuals came into contact with the dye, or that only a percentage of them were contaminated. thus, the ic was most frequently stained structure among the castes (25.61%), with gardeners representing the highest proportion of stained individuals (42.19%). this category also presented the highest percentage of workers with stained ppg (27.43%) and gaster (10.55%). analysis of the workers that had stained ic also reveals which individuals had ppg and gaster stained (table 3). the workers with ic stained by sudan iii also presented stained ppg, suggesting that the fat-soluble dye was disseminated by ppg in the majority of workers that had come into contact with this dye. this structure must also be the contamination route of other fat-soluble substances that can be used as insecticides. probably, in the workers that presented only stained ic, which were the minority, the dye had not yet disseminated to the ppg and/or to the gaster, independently of the caste analyzed, due to its high solubility in lipids, which by ingesting the dye tended to present more stained ppg. discussion in relation to rhodamine-b, individuals were found with stained gaster in the three castes of a. capiguara workers, but without significant difference among them. water-soluble substances dissolved in plant sap can be absorbed by the digestive tract, as observed by andrade (1997) when studying the ingestion of rhodamine-b by workers of atta sexdens rubropilosa, by means of vegetal sap previously stained with this substance. this study verified that more than 50% of the workers evaluated had a stained gaster, in other words, a contaminated digestive system, but the absorption of dye was greater among the gardeners, who present greater specialization in licking or cutting stained leaf fragments, than among the foragers, due to the fact that in the act of cutting, the substrate absorbs less sap. although the objective of andrade (1997) had been to assess the ingestion of the vegetal sap utilized as substrate for the symbiotic fungus by the workers, the response found may be compared to that obtained in the present study, 517 forti, l.c. et al. —trajectory of dyes in grass-cutting ants since there was no significant difference among the castes. a contamination difference among the a. capiguara castes could not have been evidenced by the fact that this species processes less substrate and by having used bait, which is a solid substrate in contrast to the liquid sap substrate. despite this, other studies have also revealed staining in the gaster after the use of rhodamine-b such as that of forti et al. (2007), who analyzed the dispersal of the active formicidal ingredient sulfluramide, jointly with rhodamine-b as a tracer, and found 66.5% of a. sexdens rubropilosa workers stained in laboratory colonies and 40.9% in field colonies. bueno et al. (2001), also observed gaster staining among workers of a. sexdens by rhodamine-b, but concluded that the allogroming and self-grooming performed by the workers is responsible for displacing the dye. in relation to dispersal of fat-soluble substances, such as sudan iii dye, less than 50% of the workers were found to be contaminated in similar proportions among the three castes, as occurred with the employment of water-soluble dye. these results corroborate those reported by echols (1966), peregrine et al. (1972) and andrade (2002). the percentage of a. capiguara workers with a gaster stained by sudan iii was much smaller in relation to the other structures, in agreement with andrade (2002), who studied workers of acromyrmex subterraneus treated with baits without the active principle and stained with sudan black (fatsoluble). it was observed that 27.9% of the worker presented stained ppg and ic while only 9.1% possessed stained crop and ventriculus. it is probable that sudan iii dye requires a longer exposure of substrate stained by grass-cutting workers due to lesser frequency of manipulation. another reason may be related to the quantity of lipid substances present in the bait and its ingestion, given that the probability of ppg staining is proportional to the quantity of lipids present in these glands ( jesus & bueno 2007). according to vinson et al. (1980), the destination of lipid compounds in the digestive system is the ppg lumen, thus explaining the low presence of sudan iii dye in workers gaster. among such species as tapinoma melanocephalum, monomorium pharaonis, linepithema humile and paratrechina fulva, if the quantity of lipids ingested by the workers is large, this substance is stored in both the ppg and crop of the individuals. however, these lipids are not 518 sociobiolog y vol. 59, no. 2, 2012 transported to the ventriculus and after some time return via the esophagus until they reach the ppg ( jesus & bueno 2007). thus, water-soluble substances are disseminated in the workers organism through the digestive system whereas fat-soluble ones are directed to such glands as the ppg. what varies is the percentage of individuals contaminated due the substrate preference and manipulation time, according to the castes of each species. acknowledgments the authors are grateful to the school of agronomy (fca/unesp) for making this research possible and to “cnpq conselho nacional de desenvolvimento científico e tecnológico” for support to the first author (grant 150137/2004-4). references andrade, a.p.p. 1997. comportamento forrageiro e aprendizado de operárias de atta sexdens rubropilosa forel, 1908 (hymenoptera, formicidae) em condições de campo e laboratório. msc. thesis, universidade estadual paulista, botucatu, são paulo, brazil (unpublished). 100. andrade, a.p.p. 2002. biologia e taxonomia comparadas das subespécies de acromyrmex subterraneus forel, 1893 (hymenoptera, formicidae) e contaminação das operárias por iscas tóxicas. phd. thesis, universidade estadual paulista, botucatu, são paulo, brazil (unpublished). 168. andrade, a.p.p., l.c. forti, a.a. moreira, m.a.c. boaretto, v.m. ramos & c.a.o. matos. 2002. behavior of atta sexdens rubropilosa (hymenoptera: formicidae) workers during the preparation of the leaf substrate for symbiont fungus culture. sociobiolog y 40: 1-14. ayres, m., j.r.m. ayres, d.l. ayres & a.s. santos. 2007. bioestat 5.0: aplicações estatísticas nas áreas das ciências biológicas e médicas. instituto de desenvolvimento sustentável mamirauá. belém, pa. bagnères, a.g. & g.j. blomquist. 2010. site of synthesis, mechanism of transport and selective deposition of hydrocarbons. pp. 75-99. in: insect hydrocarbons g.j. blomquist & a.g. bagnères (eds.), university of nevada, reno. bueno, o.c. 2005. filtro infrabucal e glândula pós-faríngea da saúva-limão atta sexdens rubropilosa forel (hymenoptera: formicidae). rio claro. associate professor thesis, universidade estadual paulista, botucatu, são paulo, brazil (unpublished). 107. bueno, o.c., d. fresneau, o.m. schineider, c. silveira & f.c. bueno. 2001. fluxo de corantes hidrossolúveis e lipossolúveis no trato digestivo de operárias de atta sexdens l. 1758 (hymenoptera: formicidae). in: anais do xv encontro de mirmecologia, 519 forti, l.c. et al. —trajectory of dyes in grass-cutting ants londrina, pr, brasil. chapman, r.f. 1998. the insects: structure and function. cambridge university press, cambridge. 770 p. delage-darchen, b. 1976. les glandes post-pharyngiennes des fourmis. connaissances actuelles sur leur structure, leur fonctinnement, leur rôle. annals of biolog y 15(1-2): 63-76. echols, h.w. 1966. assimilation and transfer of mirex in colonies of texas leaf-cutting ants. journal of economic entomolog y 59: 1336-1338. eelen, d., l.w. børgesen & j. billen 2006. functional morpholog y of the postpharyngeal gland of queens and workers of the ant monomorium pharaonis (l.). acta zoologica 87(2): 101-111. forti, l.c. & a.p.p. andrade .1999. ingestão de líquidos por atta sexdens (l.) 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the postpharyngeal gland of female european beewolves (hymenoptera, crabronidae). arthropod structure & development 36(2): 113-122. vinson, s.b., s.a. phillips jr. & h.j. williams. 1980. the function of the post-pharyngeal glands of the red imported fire ant, solenopsis invicta, buren. journal of insect physiolog y 26(9): 645-650. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(2): 129-134 (2013) doi: 10.13102/sociobiology.v60i2.129-134 the spatial distribution of mtdna and phylogeographic analysis of the ant cardiocondyla kagutsuchi (hymenoptera: formicidae) in japan i okita1, k murase2, 3, t sato3, k kato4, a hosoda5, m terayama2, k masuko6 introduction many invasive ant species, such as the argentine ant or the red imported fire ant, are not desirable because they outcompete and eliminate native ants (suarez et al., 2008), as well as they cause serious agricultural damage and are harmful to humans (heinze et al., 2006, suarez et al., 2008). for such species, early detection and monitoring are important to the management of new invasions. however, the situation is not always simple. there are many invasive species that have been transferred without being noticed and have spread their distribution in new land. in such cases, to reveal their current distribution is important for the conservational actions regarding native species. cardiocondyla kagutsuchi terayama (c. kagutsuchi) is one of such species. the genus cardiocondyla is a common ant genus that belongs to the subfamily myrmicinae. it is abstract in this study, we investigated the geographical distribution of haplotypes of cardiocondyla kagutsuchi terayama in japan using coi/ii mitochondrial dna. we also examined their genealogy with c. kagutsuchi in other areas and their close relative species. four haplotypes were found. while two of them were found in a limited area (ishigaki and okinawa islands) separately, the others were distributed widely across honsyu, shikoku, and kyusyu areas in japan. the newly invaded area by c. kagutsuchi in japan was shizuoka prefecture. their haplotype of shizuoka were the same as the two haplotypes of the honsyu, shikoku, and kyusyu areas. the haplotype network showed that the two haplotypes were distant from each other. the distance between them was 33, even though the two haplotypes are distributed in the same area. from the phylogenetic tree that we constructed, we found that c. strigifrons was in the same clade as c. kagutsuchi. sociobiology an international journal on social insects 1 gifu university, gifu, gifu, japan 2 the university of tokyo, bunkyo-ku, tokyo, japan 3 tokyo university of agriculture and technology, fuchu, tokyo, japan 4 shizuoka prefectural research and coordination office, shizuoka, shizuoka, japan 5 hamamatsu gakuin university junior college, hamamatsu, shizuoka, japan 6 senshu university, kawasaki, kanagawa, japan research article ants article history edited by: yi-juan xu, scau – china received 15 november 2012 initial acceptance 20 february 2013 final acceptance 27 march 2013 keywords ants, cardiocondyla, male polymorphism, mitochondrial dna, spatial distribution corresponding author kaori murase graduate school of natural sciences nagoya city univesity yamanohata 1, mizuho-cho, mizuho-ku nagoya, aichi 467-8501, japan e-mail: kmurase@nsc.nagoya-cu.ac.jp an invasive ant group, and is commonly known as “stealthy invaders” (heinze et al., 2006). approximately 50 species are currently recognized as belonging to this genus, most of which are distributed in the old world tropics and subtropics, but a few of which occur in the temperate zone. some species are also found widely separated in north america and the pacific islands, as a result of human introduction. several species of this genus have a striking male polymorphism, with both winged and wingless forms (terayama, 1999; seifert, 2003; heinze et al., 2005; yamauchi et al., 2005). these males differ not only in morphology, but also in reproductive tactics (yamauchi & kawase, 1992; yamauchi & kinomura, 1993; heinze et al., 1998; anderson et al., 2003). recently, seifert (2003, 2008) revised the taxonomy of this genus as a result of a morphological study that used the morphometrical method. however, questions remain around i okita, k murase, t sato, k kato, a hosoda, m terayama, k masuko spatial distribution of cardiocondyla kagutsuchi in japan130 the status of cardiocondyla kagutsuchi terayama, 1999. this ant is known to have three morphologically different colony types: type 1 produces both winged and wingless (ergatoid) males in each colony; type 2 produces both short-winged (brachypterous) and wingless males; and type 3 produces only wingless males (yamauchi et al., 2005). consequently, yamauchi et al. (2005) renamed c. kagutsuchi sensu seifert (2003) as “c. kagutsuchi complex.” types 1 and 3 have been found in japan. type 1 occurs only on ishigaki island, the ryukyu islands, whereas type 3 is distributed from kanto district to okinawa island (terayama et al., 1992; terayama, 1999). terayama (1999) considered both types to be different species due to the difference in morphology of the male caste and karyotype of the worker caste (terayama, 1999). however, in the recent taxonomic revision of the genus, both types were considered conspecific due to the high morphological similarity between the worker castes (seifert, 2003; yoshimura et al., 2008). as mentioned above, there have been many studies about its classification or male polymorphism. such kind of studies is not good enough to understand all the aspects of the problems with invasive species. in the study of invasive species, it is important to estimate the invasion routes. when estimating the invasion routes, we need to know the haplotype distribution of the target species. thus, we need to know the haplotype distribution of c. kagutsuchi in order to promote the conservation biology of the native ant diversity in japan. however, there was no study about its haplotype distribution. the first purpose of this study is to collect this ant from across japan and investigate their mtdna sequences to reveal the distribution of their haplotypes in japan. the second purpose is to construct genealogical trees and a haplotype network to clarify their position in the genus cardiocondyla. according to “japanese ant image database” (http:// ant.edb.miyakyo-u.ac.jp/j/index.html), c. kagutsuchi is not distributed in the eastern japan without kanagawa prefecture. we, however, have collected a lot of colonies of c. kagutsuchi in shizuoka prefecture, located in the eastern japan, in the last few years. where do they come from? there has been no report of their existence in the area. they may have come from foreign countries. the third purpose is to infer the invasion routes of c. kagutsuchi in shizuoka prefecture, using haplotype distribution of mtdna across japan and c. kagutsuchi in foreign countries. materials and methods sampling we collected all samples during the species’ reproductive season (june and july) in 2010. this ant is known to inhabit open areas and nest in the soil (terayama, 1999); therefore, we searched for these ants in green areas, vegetable fields, parks, near the seashore, etc. in each locality, we spent at least one hour searching for the ant; if we did not find any ants within this time, we moved to a different place. colonies or foraging workers of c. kagutsuchi were collected from 27 localities between kanto and ishigaki islands in japan. nests were located by following foragers back to the nest entrance, and complete colonies and colony fragments were then collected by carefully digging up soil using a scoop. some nests were found in gaps between tiles and under stones. all samples were collected near the seashore, with the exception of those collected from shizuoka prefecture and ishigaki island. following collection, samples were stored in ethanol (99.5%). only workers were used for dna analysis. when we could sample male individuals in each locality, we examined their wing polymorphism. dna extraction, amplification, and purification a 890-bp fragment of coi/ii (including 58 base pairs of leucine trna), corresponding to positions 2284 to 3191 in the drosophila yakuba burla mitochondrial genome (clary & wolstenholme, 1985), was used for phylogenetic analysis. dna was extracted from individual workers using landry’s method (cheung et al., 1993). the dried pellet was eluted in purified water and preserved at –80°c until further analysis. we amplified coi/ii using the primers c1-j-2195 and c2-n-3661 (simon et al., 1994). polymerase chain reactions (pcrs) were performed using sybr premix ex taq (takara) and conducted in a mycycler (bio-rad) and mj mini gradient thermal cycler (bio-rad). pcr consisted of 34 cycles of denaturing at 95°c for 1 min, annealing at 59°c for 1 min, and extension at 72°c for 2 min, with the exception of the initial denaturing step at 95°c for 4 min. amplified dna was purified using a pcr purification kit (qiagen). all products were sequenced with the 3730xl dna analyzer (applied biosystems) using the primers c1-j-2195 and c2-n-3661 (simon et al., 1994). to obtain the complete sequences, some additional primers were also designed by nukui and okita (unpubl.): 3f [5′-cctttaattagaggatacac-3′] • and 4f [5′-ggcagataagtgcaaaggac-3′] (which were used with c1-j-2195). 1r [5′-ttctatagagtgattttggaggag-3′], • 2r [5′-ggtatacctctgagacc-3′] and 3r [5′-cagctcctatagagagaacatag-3′] (which were used with c2-n-3661). phylogenetic analyses we obtained 28 sequences and aligned these with a further 11 sequences obtained from genbank using the clustal w algorithm (thompson et al., 1994). the accession numbers of the 11 additional sequences were sociobiology 60(2): 129-134 (2013) 131 dq023083, dq023084, dq023085, dq023086, dq023087, dq023088, dq023091, dq023094, dq023102, dq023108, and dq023118 (heinze et al., 2005). six of the 11 sequences (dq023083, dq023084, dq023085, dq023086, dq023087, and dq023088) were sequences of c. kagutsuchi originating from hawaii, malaysia, and indonesia; the other 5 sequences (dq023091, dq023094, dq023102, dq023108, and dq023118) were sequences of c. mauritanica, c. minutior, c. obscurior, c. strigifrons, and c. wroughtonii, which were used as outgroups (heinze et al., 2005). phylogenetic relationships were inferred from the aligned coi/ii using a distance and bayesian analysis. a neighbor-joining tree (saitou & nei, 1987) was constructed using the kimura 2-parameter distance method (kimura, 1980) in mega5, with bootstrap values estimated from 5000 replicates. in the bayesian analysis, a single run consisted of 300,000 generations. we also constructed a haplotype network using tcs 1.21 (clement et al., 2000) with the haplotypes identified by the sequences described above. results we found four different haplotypes of c. kagutsuchi in japan. the distribution of each of the four haplotypes is shown in fig. 1. the number of sequences we obtained was 28, while the number of sampling locality was 27. this is because the two types of haplotype were found in sampling locality 5. the first was distributed in kanagawa (1 locality), shizuoka (4), aichi (1), wakayama (1), and hyogo (1) (honshu area); kagawa (1) (shikoku area); and miyazaki (1) and kagoshima (1) (kyushu area). the second was distributed in shizuoka (8 localities) (honshu area); tokushima (2), kagawa (1), and kochi (1) (shikoku area); and kumamoto (1) (kyushu area). the third was found only on okinawa island (2 localities), and the fourth was found only on ishigaki island (2 localities). the first and second haplotypes were distributed widely across the temperate zone in japan (honsyu, shikoku, and kyusyu areas) and, in some cases, were found very close together. in contrast, the third and fourth haplotypes were found in a limited area (a single island). we constructed a neighbor-joining tree and a bayesian tree to analyze the relationship between haplotypes and male polymorphism (figs. 2, 3). both trees showed that c. wroughtonii, c. obscurior, and c. minutior were outgroups, but c. strigifrons was in the same group as c. kagutsuchi. cardiocondyla mauritanica was also an outgroup according to the neighbor-joining tree, but was in the same group as c. kagutsuchi according to the bayesian tree. the accession numbers of the 4 new sequences were showed in each figure. we also constructed a haplotype network (fig. 4). it contained four haplotypes found in japan in this study. the distance between the haplotype 1 and 2 was 33. the distance between the haplotype 1 and 3 was two, the number is the smallest among all haplotype pairs. the distance between the haplotype 1 and 4 was 10. the distance between the haplotype 2 and 3 was 31. the distance between the haplotype 2 and 4 was 41, the number is the highest. the distance between the haplotype 3 and 4 was 10. fig. 1. distribution of c. kagutsuchi in japan. sampling localities are numbered from 1 to 27. the haplotype of c. kagutsuchi in each locality is indicated by the corresponding symbol. shizuoka prefecture is enlarged because many sampling localities are included in it. sampling locality 5 (in the enlarged part of the figure) is divided into 5a and 5b because the two types of haplotype were found. i okita, k murase, t sato, k kato, a hosoda, m terayama, k masuko spatial distribution of cardiocondyla kagutsuchi in japan132 fig. 3. consensus tree of cardiocondyla based on the mitochondrial coi/ii using mrbayes (300,000 generations). sample names are given in the same way as in fig 2. white circles indicate that both winged and wingless males are present (type 1, dimorphic), a white circle with asterisk is for both shortwinged and wingless males (type 2, dimorphic), and filled circles are for only wingless males (type 3, monomorphic), respectively. no males are known from c. kagutsuchi from mauna kea, hawaii and yomitanson, okinawa. the haplotype numbers in fig 1 are added in the right of the figure, with their accession numbers. fig. 2. neighbor-joining tree based on the mitochondrial coi/ii genes (890 bp) of the ant genus cardiocondyla. numbers indicate bootstrap values over 50% in 5000 pseudoreplications. each sample name is given in the following order: (1) species name; (2); sampling locality number in fig 1 if the sample is ours; (3) local area name; (4) prefecture or state name; (5) country name; and (6) accession number in genbank if the sample is in the data of heinze et al. (2005). the species names are as follows: k—c. kagutsuchi, s—c. strigifrons, ma—c. mauritanica, mi—c. minutior, o—c. obscurior, and w—c. wroughtonii. sampling locality 5 is divided into 5a and 5b because the two types of haplotype were found. the haplotype numbers in fig 1 are added in the right of the figure, with their accession numbers. k 1 yugawara kanagawa jpn k 2 atami shizuoka jpn k 5a yaizu shizuoka jpn k 7 iwata shizuoka jpn k 9 hamamatsu shizuoka jpn k 13 gamagori aichi jpn k 14 nachikatsuura wakayama jpn k 15 aioi hyogo jpn k 18 takamatsu kagawa jpn k 21 miyazaki miyazaki jpn k 23 kagoshima kagoshima jpn k 24 yomitanson okinawa jpn k 25 nanjo okinawa jpn k gombak selangor mys dq023084 k kuala_lumpur kwp mys dq023083 k 26 ishigaki okinawa jpn k 27 ishigaki okinawa jpn k gombak selangor mys dq023085 k kuala_lumpur kwp mys dq023086 k bogor java idn dq023087 s bedugul bali idn dq023108 k mauna_kea hawaii usa dq023088 k 3 shimoda shizuoka jpn k 4 shimizu shizuoka jpn k 5b yaizu shizuoka jpn k 6 fukuroi shizuoka jpn k 8 iwata shizuoka jpn k 10 hamamatsu shizuoka jpn k 11 hamamatsu shizuoka jpn k 12 hamamatsu shizuoka jpn k 16 naruto tokushima jpn k 17 anan tokushima jpn k 19 utazu kagawa jpn k 20 kochi kochi jpn k 22 yatsushiro kumamoto jpn ma ta_cenc gozo mlt dq023091 mi itabuna bahia bra dq023094 o ishigaki okinawa jpn dq023102 w nago okinawa jpn dq023118 88 99 78 78 78 59 94 100 65 100 97 96 100 100 0.01 haplotype 1 (ab723724) haplotype 3 (ab723726) haplotype 4 (ab723727) haplotype 2 (ab723725) o ishigaki okinawa jpn dq023102 w nago okinawa jpn dq023118 mi itabuna bahia bra dq023094 ma ta_cenc gozo mlt dq023091 0.54 s bedugul bali idn dq023108 0.79 0.98 k mauna kea hawaii usa dq023088 k 4 shimizu shizuoka jpn 1.00 k 26 ishigaki okinawa jpn k gombak selangor mys dq023085 1.00 k gombak selangor mys dq023084 0.69 0.58 0.73 k 9 hamamatsu shizuoka jpn k 24 yomitanson okinawa jpn (haplotype 3: ab723726) (haplotype 1: ab723724) * (haplotype 4: ab723727) (haplotype 2: ab723725) 1.00 0.1 sociobiology 60(2): 129-134 (2013) 133 discussion at first, we want to discuss the first purpose. the distributions of the four different haplotypes are shown in fig. 1. haplotypes 1 and 2 were distributed widely across japan, rather than being restricted to a single area, such as an island. this overlapped distribution pattern and this relatively low level of genetic variability across a wide area indicate that c. kagutsuchi is not native ant species in japan and it may have extended its distribution quickly. what caused this distribution pattern and this low variability? is it a result of this species being invasive, mating in its natal nest, or being a cryptic species, or is there some other reason? this would be a very interesting area for future study. we want to discuss the second purpose. the haplotype 1 and 2 were placed in distant clades in phylogenetic trees (figs. 2, 3) and they were considerably distant from each other in the haplotype network (fig. 4). this suggests that the haplotype 1 and 2 are originated from other localities. the haplotype 1 is a near relation of dq023083 and dq023084, which were sampled in malaysia, whereas the haplotype 2 is closely related to dq023088, which was sampled in hawaii. further, the haplotype 2 is more closely related to dq023108, c. strigifrons, than the other haplotypes of c. kagutsuchi. this might indicate that the two haplotypes are different species fig. 4. parsimonious network among four haplotypes. open circles indicate identified haplotypes, and the filled circle indicates a hypothetical haplotype. “h1” means haplotype 1 in fig 2, “h2” does haplotype 2, and so forth. the numbers between nodes indicate the number of nucleotide substitutions. 1 1 9 31 h2 h3 h1 h4 okinawa island ishigaki island honsyu, shikoku, and kyusyu areas honsyu, shikoku, and kyusyu areas 1 1 9 31 h2 h3 h1 h4 okinawa island ishigaki island honsyu, shikoku, and kyusyu areas honsyu, shikoku, and kyusyu areas rather than the same species. for the topic whether the two haplotypes are different species or not, their nuclear dna would be needed to be examined because of the possibility of their crossing. we want to discuss the third purpose. c. kagutsuchi that had expanded its distribution to the eastern japan was neither the foreign new genotype nor the one in isolated islands like okinawa: it was the haplotype 1 and 2. this result indicates that there is a strong possibility of its distribution expansion in honsyu area in japan. we should keep an eye on the routes it will use for its future expansion. most of the colonies in this study were sampled in its breeding season. in consequence, as for male dimorphism, type 1, which is regarded as c. kagutsuchi sensu terayama, 1999 and in which there is male dimorphism, was only found on ishigaki island and appeared as an independent clade in the phylogenetic analysis. for future study, whether male wing polymorphism and other ecological differences are related to genetic variations is an interesting issue. acknowledgments we thank h. nukui, h. iyozumi, k. kinomura, k. yamauchi, t. matsumoto, y. kawashima, f. ikeda, r. kawakami, h. nakada, and k. tsuchida for their helpful support. for letting us collect in their gardens and fields, we are also thankful to h. matsuya, m. matsuya, and the staff of arakawa garden shop, yuto cultural center, lakehamana garden park and takenaka garden shop. lastly, we are grateful to the night class teachers of iwata minami high school for deep understanding and support for our study. references anderson, c., cremer, s. & heinze, j. 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(1992). pheromonal manipulation of workers by a fighting male to kill his rival males in the ant cardiocondyla wroughtonii. naturwissenschaften, 79: 274– 276. doi: 10.1007/bf01175395 yamauchi, k. & kinomura, k. (1993). lethal fighting and reproductive strategies of dimorphic males in cardiocondyla ants (hymenoptera: formicidae). in t. inoue & s.k. yamane (eds.), evolution of insect societies (pp. 373–402). tokyo: hakuhinsha. yoshimura, m., kubota, m., onoyama, k. & ogata, k. (2008). taxonomic changes since the publication of japanese ant image database 2003. ari, 31:13–28. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.429-435sociobiology 60(4): 429-435 (2013) the odorant-binding protein obp11 gene shows different spatiotemporal roles in the olfactory system of apis mellifera ligustica and apis cerana cerana hx zhao1,2, yx luo2, jh lee3, xf zhang2, q liang3, xn zeng1 introduction for social insects such as the honeybee, olfactory language plays a critical role in colony life, with important functional roles for work within and outside of the hive. according to honeybee biology, newly emerged bees are developmentally immature (calderone, 1998), and they join a caste that mainly cleans cells while awaiting functional maturity (winston, 1987). from the ages of 4-12 days (seeley, 1982), the nursing caste feeds larvae or the queen for about 1 week (seeley, 1979). middle-aged bees (12-21 days old) build and maintain the nest, and receive and process nectar (johnson, 2003, 2008a). after 21 days, workers initiate tasks outside of the nest (foraging nectar and pollen, scouting, defending) (seeley, 1995; seeley & visscher, 2004; beekman et al., 2006; visscher, 2007). these behaviors are correlated abstract odorant-binding proteins participate in the olfactory system of the honeybee. apis mellifera ligustica and apis cerana cerana are species of honeybee that have different biological functions. the two species have diversified olfactory systems, with a. cerana displaying sensitive olfactory involvement in collecting nectar and pollen from small plants; and a. mellifera collecting from large nectariferous plants. we hypothesized that, given this difference in biological activity, the obp11 genes of a. mellifera and a. cerana may show different olfactory expression patterns. we cloned and sequenced the obp11 genes from a. mellifera (amobp11) and a. cerana (acobp11). using quantitative real-time pcr, we demonstrated that nurse workers, which have the highest olfactory sensitivity in the a. mellifera hive, have the highest expression of the amobp11 gene; whereas 1-dayemerged workers, which have the lowest olfactory sensitivity, have correspondingly low expression. however, the highest expression of the acobp11 gene is observed for foragers, which display the highest olfactory sensitivity in the a. cerana population. the obp11 protein from the two species is highly conserved, with an apparent molecular weight and predicted extracellular localization that is similar to other obp proteins. the expression of the obp11 gene in a. mellifera and a. cerana correlates with the different roles of the olfactory system for the two different species. these findings support the critical role of odorant-binding proteins in the honeybee olfactory system. sociobiology an international journal on social insects 1 south china agricultural university, guangzhou, china. 2 guangdong entomological institute, guangzhou , china. 3 fujian agriculture and forestry university, fuzhou, china. research article bees article history edited by: yi-juan xu, scau, china received 27 march 2013 initial acceptance 04 june 2013 final acceptance 01 july 2013 keywords apis mellifera ligustica, apis cerana cerana, odorant-binding proteins, realtime pcr corresponding author xinnian zeng south china agricultural university guangzhou, 510642, china e-mail: zengxn@scau.edu.cn with the function of the olfactory system, which mediates volatile signals to workers, as opposed to contact perception (maisonnasse, 2010). moritz and crewe suggested that the queen emits volatile pheromones to inhibit new queens or the ovary development of workers (moritz & crewe, 1991). honeybees have been observed as a model for the insect olfactory system (vanesa, 2009). the odorant-binding proteins (obps) of honeybees are the main functional proteins in the olfactory system. obps recognize and distinguish volatile compounds and then transport these compounds to olfactory receptors. honeybee obps are small, water soluble molecules, which are expressed as abundant extracellular proteins. the genome of the honeybee (honeybee genome sequencing consortium, 2006) contains 21 genes encoding obps (forêt & maleszka, 2006), each gene with a markedly different expression pattern. most of the obps are restricted hx zhao et al. different spatiotemporal roles in the olfactory system of honey bees430 to olfactory tissues, with particularly high expression in the antenna, while the obp11 gene is expressed exclusively in the antenna of adult bees (francesca et al., 2010). apis cerana cerana fabricius (a. cerana), is an important local species in china. a. cerana has a keen olfactory ability, foraging small and dispersed nectariferous plants, defending strongly against ectoparasitic v. destructor and chalkbrood disease, and providing tolerance for low environmental temperatures (sarah et al., 2010). on the other hand, apis mellifera ligustica spinola (a. mellifera) belongs to a species of honeybee that mainly forages large nectariferous plant and produces a number of bee products (royal jelly, pollen, propolis, etc). a. mellifera is easily infected with varroa destructor and chalkbrood disease. while a. cerana and a. mellifera are two important honeybee species that exhibit long-term evolutionary divergence (qiu et al., 2012), the colonies of a. cerana suffer less damage than those of a. mellifera from parasites and chalkbrood disease. because of its sensitive olfactory system, a. cerana recognizes and distinguishes dummy larvae more efficiently than a. mellifera. however, to our knowledge, there have been few direct comparisons between the molecular mechanisms of olfactory sensing of the two species. therefore, in this study, experimentation was performed to compare the obp11 genes of a. cerana and a. mellifera at the molecular level. we examined whether the obp11 gene expression pattern correlates with the different roles of the olfactory system in a. mellifera and a. cerana, thus providing a molecular basis for the differences in behavior of the two species. based on our findings, we propose that the obp11 gene expression patterns vary according to the divergent evolutionary behavior of two species. material and methods samples collection a. mellifera and a. cerana were fed in an experimental apiary of bee science at the college of bee science of fujian agriculture and forestry university during the spring of 20112012. individual insects were collected within 24h of emergence, marked with enamel paint on the thorax to identify them by age, and then introduced into the same healthy colony. when appropriate, antennae were harvested from a. mellifera and a. cerana. the samples were first collected into trizol reagent and then stored at -70oc until use. total rna extraction and cdna synthesis total rna was extracted from the antennae of sampled bees using trizol reagent according to the manual’s protocol (invitrogen, usa). subsequently, cdna was synthesized using the promega rt-pcr system according to the manual’s protocol(promega, usa). obp11 gene cloning and expression the primers used for amplifying the obp11 gene from a. mellifera and a. cerana were designed using primer primer 5.0 (table 1). pcr was performed using the following program: denaturation at 94ºc for 1 min; 35 cycles of 94ºc for 40 s, annealing at 57ºc for 50 s, and extension at 72ºc for 50 min; and a final extension at 72ºc for 7 min. pcr products were cloned into pgem-t vector (promega, usa) and transformed into e. coli dh5α. positive colonies were selected by identification with the restriction endonucleases ecor i and xhol i, and then the obp11 gene fragments were purified using a gel extraction kit (sangon, shanghai) and cloned into pet28a vector, which was digested with the same restriction endonucleases, to construct recombinant expression plasmids pet-28a-amobp11 and pet-28a-acobp11. we used iptg to induce expression of recombinant proteins in e. coli. primer sense and antisense sequences (5′–3′) purpose aobp11 gaattcatgaaagcagcagaaatttg / ctcgagtcacggagcaataaacgcta cdna isolation (reverse transcriptase pcr) bobp11 tctcgtttatggggaaatcagcgat / tccgtattccgtagcttcgacatcc expression analysis (real-time pcr) β-actin tgccaacactgtcctttctg / agaattgacccaccaatcca internal control table 1. oligonucleotide primers used for isolation and expression analysis of odorant-binding proteins of apis mellifera ligustica and apis cerana cerana real-time pcr (rt-pcr) antennae were separately collected at 1, 4, 10, 15, 20, 25 and 30 days of age, and total rna was extracted and reverse transcribed to synthesize cdna, in accordance with the promega manual. samples were stored at -70ºc until use. real-time pcr was performed using the applied biosystems steponeplus real-time pcr system with sybr green dye (promega) in 96-well plates (abi, usa). relative quantification analysis was performed according to cycle threshold values (ct) generated from the promega gotaq 2-step rtqpcr system (promega, usa). standard curves were prepared using a purified pcr product for the obp11 and β-actin genes. for each experiment, the endogenous β-actin g gene was analyzed in triplicate (internal control, shu et al., 2011) with a non-template reaction (negative control) and a water only reaction (blank control). relative quantification analysis was performed using the comparative standard method (schefe et al., 2006). statistical analysis quantitative data are presented as mean ± standard deviation (sd) for real-time pcr experiments. one-way anova (spss 17.0 statistical software) was used to analyze the different expression pattern of each gene at different ages. sociobiology 60(4): 429-435 (2013) 431 fig. 1 analysis of the sequences of the amobp11 and acobp11 genes. a-alignment of the amobp11 and acobp11 nucleic acid sequences. divergent nucleic acid sequences are circled. b-alignment of the deduced amobp11 and acobp11 protein sequences. divergent amino acid sequences are circled. c-alignment the cds protein sequences of 12obps (1-11 and 13) and amobp11 and acobp11. the conserved cysteine residues in this alignment are shaded in dark. results sequence analysis of the amobp11 and acobp11 genes to characterize the amobp11 and acobp11 genes, a 432 bp open reading frame was amplified and sequenced. the amobp11 and acobp11 genes have many similar characteristics with about 99.31% identity (fig. 1a). the amobp11 gene encodes a 144 amino acid protein that contains a 23 amino acid hydrophobic signal peptide at the n-terminus. the software signalp 4.0 (petersen et al., 2011) predicted a molecular weight of 16.6 kda and a pi of 5.06. the acobp11 gene encodes a 138 amino acid protein that has a predicted molecular weight of 16.2 kda and a predicted pi of 4.95 (http://web.expasy.org/cgi-bin/protparam/protparam). the deduced amino acid sequences of amobp11 and acobp11 were aligned using dnaman software (fig.1b). the alignment shows that the two sequences vary in sequence at the c-terminus, which is truncated by 6 cysteine residues for acobp11. using tmhmm2.0 posterior probabilities for sequences outside the transmembrane, amobp11 and acobp11, like the other obps, are predicted to be extracellular proteins. according to the kyte and doolittle method, amobp11 and acobp11 are hydrophilic in property; however, these proteins have some aliphatic amino acids, with an aliphatic index of 81.88 for acobp11 and 83.15 for amobp11, as well as four region of lipophilicity (http://web.expasy.org/protparam/). compared with 12 obps (1-11and 13), amobp11 and acobp11 have six conserved cysteine residues, which are shaded in dark (fig. 1c). heterologous expression of the amobp5 and acobp5 gene we cloned the amobp11 and acobp11 genes into plasmids, as verified by digestion with the restriction endonucleases ecor i and xhol i. subsequently, cohesive termini of the target gene were inserted into the expression vector pet-28a and transformed into e. coli bl21/rosetta competent cells. finally, we used 1 mm iptg to induce amobp11 and acobp11 gene expression. the electrophoretic bands corresponding to recombinant obp11 protein from pet-28a-amobp11-e.coli rosetta (amobp11) and pet-28a-amobp11-e.coli rosetta (acobp11) were verified to be approximately 16 kda on a 12% sds-page gel (fig.2). we used ultrasonic energy to release amobp11 and acobp11 protein, which formed an insoluble inclusion body. expression profiling of amobp11 and acobp11 genes by realtime pcr to further characterize the functions of the respective obp11 genes in each species of honeybee, we used real-time pcr to quantitatively assess expression levels. the pcr amplification efficiency for the obp11 gene was 94.8% (slope = -3.453). the efficiency for the β-actin gene was 96.4% (slope hx zhao et al. different spatiotemporal roles in the olfactory system of honey bees432 fig. 2 sds-page analysis of recombinant amobp11 (a) and acobp11 (b). a: lane m: protein molecular weight marker; lane 1: pet-28a+e.coli rosetta; lane 2: pet-28a-acobp11+e.coli rosetta without iptg to induce expression; lanes 3-5: pet-28a-acobp11+e. coli rosetta induced with iptg; lane 6-7: pet-28a-amobp11 +e.coli rosetta induced with iptg; lane 8: pet-28a-amobp11+e. coli rosetta without iptg; lane 9: e.coli rosetta; lane 10: pet-28a+e.coli rosetta. arrows show the obp11 expression proteins. fig.3 amobp11 gene expression levels in apis mellifera ligustica antennae across seven ages determined by qrt-pcr expression levels of the amobp11 gene were calculated relative to the control ß-actin gene using the standard curve method. bars on each column represent sd for three independent experiments. the amobp11 gene expression levels in 10-day-old and 15-day-old workers were significantly higher than others ages (f=5.269, p<0.01, n=3), and expression of the amobp11 gene in 1-day-old workers was the lowest. there were no significant differences between expression in 10-day-old and 15day-old workers, or others days workers (duncan’ anova test). = -3.412). this indicates that the relative standard curve method for real-time pcr analysis with sybr green dye is experimentally suitable. the transcript abundance was calculated based on the difference in threshold cycle (ct) values between the obp11 and β-actin gene transcripts. the amobp11 gene demonstrated the highest expression in the antennae of 10-day-old and 15-day-old workers, with nearly 200-fold increase. other days presented relatively lower expression (fig.3). in contrast, the acobp11 gene demonstrated expression that was low at 1 day with a slight increase up to 20 days. the highest expression was observed in 25-day-old and 30-day-old workers, which demonstrated approximately 9to 12-fold increase in expression as compared to 1-day-old workers (fig.4). fig.4 acobp11 gene expression levels in apis cerana cerana antennae across seven ages determined by qrt-pcr expression levels of the acobp11 gene were calculated relative to the control ß-actin gene using the standard curve method. bars on each column represent sd for three independent experiments. the acobp11 gene expression levels in 25-day-old and 30-day-old workers were significantly higher than others ages (f=6.507, p<0.01, n=3), and expression of the acobp11 gene in 1-day-old workers was the lowest. there were no significant differences between expression in 25-day-old and 30day-old workers, or between expression in 30-day-old and 10-dayold workers (duncan’ anova test). discussion in this study, we cloned and identified the acobp11 and amobp11 genes. based on our sequence analysis, we concluded that the obp11 genes of the two species have high similarity. the predicted protein sequences of amobp11 and acobp11 are conserved throughout the molecule, with the exception of one internal residue and six residues at the cterminus. furthermore, hydrophobicity analysis suggests that, similar to other honeybee obps, amobp11 and acobp11 are extracellular proteins. we heterologously expressed the amobp11 and acobp11 genes in e. coli and demonstrated an apparent molecular mass that agrees with the predicted values for the native honeybee proteins. as a social insect, the foragers of a. mellifera seek large nectariferous plants, while the a. cerana foragers seek small and dispersed nectariferous plants. therefore, the divergent behavior of a. mellifera and a. cerana foragers suggests a diversity of olfactory function. based on this diversity, we proposed that the expression of the obp11 gene may differ for a. mellifera and a. cerana. previously, forêt and maleszka (2006) indicated that the obp11 gene is expressed exclusively in the antennae of adult bees; thus, we collected different aged workers’ antennae to analyze developmental variations in obp11 gene expression. varying expression patterns of the obp11 gene in the life cycle of each species could lead to different behaviors within and outside of the colony. both amobp11 and acobp11 genes showed the lowest expression levels in 1-day-old workers. winston (1987) and calderone (1998) reported that newly emerged bees cannot fly or sting, and thus are developmentally immature (winston, sociobiology 60(4): 429-435 (2013) 433 1987; calderone, 1998). therefore, the low expression of the obp11 gene in the 1-day-old workers of both species correlates with reduced olfactory function. olfactory sensing for the two species is closely correlated with the function of the obp11 gene. our results demonstrate that the developmental and temporal expression profile of the amobp11 and acobp11 genes differs in the time and extent of induction after the first day of emergence. for a. mellifera, the obp11 gene expression peaks at 10 to 15 days of age in workers, with nearly 200-fold higher expression than at others ages; whereas for a. cerana, the obp11 gene, expression rises gradually with the highest expression in the 25-day-old workers. the different peaks of expression for the two species correlate with different behavioral functions in the life cycle of the honeybee. for most honeybee species, 10-day-old workers feed larvae, while 12to 21-day-old workers build and store and process food. at the peak of amobp11 gene expression (10 and 15 days old workers), the honeybees perform nurse-tasks, begin to secrete beeswax, and work to clean within the hive. while performing these in-hive tasks, the 10to 15-day-old workers are attracted by brood pheromones. for this reason, we propose that the amobp11 gene expression in the olfactory system is correlated with nursing tasks. on the other hand, at the peak of acobp11 expression (25 days of age) most worker bees begin to forage for nectar and pollen (johnson, 2008b, 2010). the later peak of acobp11 expression is similar to that of acer-asp2 (antenna special protein), which is expressed more highly in 27-day-old workers than at other ages (lee et al., 2008). acer-asp2 is related to odorant sensors that function in the collection of certain nectars and pollens (danty et al., 1997; li, et al., 2008). therefore, we propose that the acobp11 gene has a similar function in out-hive foraging, and that the difference in the temporal expression of the amobp11 and acobp11 genes might suggest different functional outcomes in sensing the intention of the obp11 proteins for the two species. the differences in the levels of induction of the amobp11 and acobp11 genes also may be indicative of diffe-rent functions. the amobp11 gene is induced nearly 200-fold at 10day-old workers, while the acobp11 gene is induced approximately 5-fold in 10-day-old workers; and 10to 12-fold at its peak induction in 25 to 30-day-old workers. further experiments are needed to assess why obvious expression differences of the amobp11 and acobp11 genes, and the obp11 gene show different roles in the olfactory system of a. mellifera and a. cerana. the olfactory stimuli that motivate honeybees may come from outside the hive or from within the hive, and differing exposure to each of these stimuli would undoubtedly have a profound effect, which is consistent with the obp expression and may determine the behavior of the honeybee. the olfactory system within the hive functions to motivate workers to perform different tasks based on the age of larvae (le conte, 2001, 2008; maisonnasse, 2009). larvae of different ages emit volatile compounds to adjust the behavior of the honeybees. young larvae emit e-ß-ocimene, a highly volatile pheromone that is dispersed within the colony to accelerate forage behavior of in-hive workers by the olfactory system (maisonnasse, 2010). brood ester pheromones motivate older workers to take care of the brood (slessor, 2005; pankiw, 2007; peters, 2010). newly emerging bees engage in some nursing behaviors too, whereas the others ages, such as middle-aged and 12-21 days old workers do not engage in nursing behavior (ben-shahar et al., 2002; ben-shahar, 2005). meanwhile, e-ß-ocimene is one of the monoterpene volatile organic compounds, which is emited by larvae to engage workers in nursing tasks. during the process of performing nursing tasks, obps play an important role in the olfactory system of the honeybee. obps assist e-ß-ocimene to transfer to the olfactory receptor, which elicits the corresponding behavior. the amobp11 gene belongs to one of the main antennae obps, the 10to 15-day-old workers have the highest expression of amobp11 gene. thus, we propose that the high amobp11 gene expression of 10to 15-day-old workers is correlated with sensitive olfactory reception of volatile brood compounds, which might encourage them to engage in nursing behaviors (maisonnasse, 2009). on the other hand, because the peak of acobp11 gene expression is in the foraging stage, it is likely that the function of this obp11 gene for a. cerana is more highly evolved to respond to a different set of olfactory stimuli, which are encountered outside of the hive. the olfactory system of a. cerana is known to be more sensitive than that of a. mellifera, especially in regards to the collection of honey and pollen. therefore, it is likely that the obp11 proteins of these two species might performing in the response to different sources of stimuli to determine the differing behavior of these two species at different stages. we conclude that the spatiotemporal expression patterns of the obp11 gene in a. mellifera and a. cerana suggests that this gene plays different roles in the olfactory sensitivity of workers. conclusions we demonstrated that the nurse workers, which have the highest olfactory sensitivity in the a. mellifera colony, have the highest expression of amobp11 gene; whereas 1-day-old workers, which have lowest olfactory sensitivity, have correspondingly low expression. however, the highest expression of acobp11 gene is observed for foragers, which display the highest olfactory sensitivity in the a. cerana population. acknowledgments this work was supported by projects of the modern bees industrial technology system (cars-45-kxj-7 and cars-45syz-12) from the ministry of agriculture of china and project of the insect behavior research (gjhz1140) from the department of education of guangdong province. hx zhao et al. different spatiotemporal roles in the olfactory system of honey bees434 references beekman m., fathke rl. & seeley td. 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(1987). the biology of the honey bee. harvard university press, cambridge. doi: 10.13102/sociobiology.v67i1.4432sociobiology 67(1): 80-88 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the great majority of plants on the planet depend on pollination by animals to produce fruit and seeds, and bees are the main agents involved in this process for both wild and cultivated plants (klein et al., 2006; ricketts et al., 2008). the economic value of pollination carried out by animals is estimated at usd 235-577 billion annually, with bees accounting for 90% of that value, but despite the existence of about 20,000 bee species in the world, only a few species such as those belonging to the genera apis and bombus, are bred for agricultural pollination (klein et al., 2006; magalhães & freitas, 2013; potts et al., 2016). however, a recent decline in the population of these bees in certain regions of the planet has drawn attention to the risk underlying the over dependence of abstract the recent decline in population of generalist bees such as those of the genera apis and bombus has shown the need to breed and manage a larger number of bee species. among the species with potential use for agricultural pollination in the neotropics, a peculiar small group has specialised in collecting floral oil. therefore, the aim of this study was to analyse the chemical profile and to identify the main constituents of the floral oil of the nance (byrsonima sericea), an abundant species in the northeast of brazil and widely used by oil-collecting bees. a sample of 400 flowers of the nance were collected between october 2017 and january 2018. the samples were derivatised (mstfa) and analysed by gas chromatography-mass spectrometry in a model 7890b gc gas chromatograph system coupled to a model 5977a msd mass spectrometer. the compounds were separated using an hp-5ms capillary column and identified by comparing the mass spectra with the national institute of standards and technology (nist) database, and by comparison of the retention indices (ri). from the chromatographic analysis, it was possible to identify 23 constituents, especially fatty acids and carboxylic acids. the results indicate the presence of tricosanoic acid, palmitic acid and heneicosanoic acid as the main constituents of the oil under study. there is still a need for studies that would better explain the relationship of these constituents with the bees that use the oil. sociobiology an international journal on social insects fl rosa1, abs barbosa1, ths rodrigues2, gj zocolo3, bm freitas1 article history edited by evandro nascimento silva, uefs, brazil received 22 march 2019 initial acceptance 21 january 2020 final acceptance 21 january 2020 publication date 18 april 2020 keywords solitary bees; floral lipids; byrsonima sericea, mass spectrometry. corresponding author felipe lima rosa campus do pici universidade federal do ceará – ufc av. humberto monte, s/n° cep 60356-001, fortaleza-ce, brasil. e-mail: limarosafelipe@gmail.com the world agricultural production to only a few bee species, and highlighted the need to breed and manage a larger number of bee species for crop pollination (ipbes, 2016). among the bee species with potential use for agricultural pollination in the neotropics, a peculiar small group has specialised in collecting floral oil for both nesting and feeding purposes (wnson et al., 1997; dotterl &vereecken, 2010). these bees, abundant in the region and known as oil-collecting bees, are non-social species and have evolved independently in five different groups belonging to two taxonomic families: melittinae (melittidae), ctenoplectrini, centridini, tapinotaspidini e tetrapediini (apidae) (alves dos santos et al., 2002; vinson et al., 2006; reis et al., 2007). due to their peculiarity of collecting floral oils, these bees present a tight relationship to plant species that provide this resource. 1 universidade federal do ceará (ufc), fortaleza, brazil 2 universidade estadual do vale do acaraú (uva), sobral, brazil 3 empresa brasileira de pesquisa agropecuária (embrapa), fortaleza, brazil research article bees chemical characterisation of the floral oil of the nance (byrsonima sericea): discovering the constituents used in reproduction by oil-collecting bees sociobiology 67(1): 80-88 (march, 2020) 81 eleven botanical families are known to have evolved and specialised in offering this reward to their pollinators. the supply of floral oils has been interpreted as a highly successful evolutionary strategy to enhance pollination since these lipids are produced especifically to increase the attractiveness of the flowers to the pollinators (buchmann, 1987; dotterl & schaffler, 2007). one such family is malpighiaceae. around 85% of its species occur in the neotropical region and bear floral characteristics in accordance to the mellitophilous pollination syndrome (lombello & forni-martins, 2003). in fact, cultivated species of this family, such as acerola (malpighia emarginata), wild species exploited by local populations, such as nance (byrsonima spp.), or by the fauna, such as byrsonima cydoniifolia, are totally dependent on oil-collecting bees to produce fruit (freitas et al., 1999; pereira & freitas, 2002; sazan et al., 2014). in addition, species belonging to other botanical families, such as cashew (anacardium occidentale), brazil nut (bertholettia excelsa) and sweet-passion-fruit (passiflora alata), although not exclusively dependent on these bees, also benefit from the pollination they carry out when visiting the flowers in search of pollen and/or nectar (freitas & paxton, 1998; ganglione et al., 2010; cavalcante et al., 2012). as such, species of oil-collecting bees are important pollinators of various plant species and crops in the neotropics. however, oil-collecting bees are neither bred rationally nor used for agricultural pollination. although studies of some species of the genus centris have shown that they are abundant and may even be managed in trap nests in areas of nance and acerola (pereira & freitas, 2002; magalhães & freitas, 2013), the dependence of these bees on floral oils for bulding their nests and feeding their brood has been seen as a limiting factor for their use on a larger scale and with other agricultural crops (freitas & pereira, 2004; lourenço, et al., 2019). therefore, knowledge of the chemical composition of floral oils may be important in order to get around this obstacle. studies of the chemical characteristics of the floral oils poduced by some plant species have pointed to a diverse combination of long-chain saturated and unsaturated fatty acids with or without β-acetoxy and monoor diglyceride substituents, in addition to the presence of a smaller number of other groups (dumri et al., 2008; capellari et al., 2011). the different chemical compounds present in these oils are probably related to differences in their attractiveness to pollinators, suggesting the existence of components in these rewards that are responsible for acting as mediators between the different species of plants and their pollinators. however, it is not yet known whether this mediation is carried out by the action of isolated compounds or if the characteristics of the mixture of compounds that play the main role in the attraction (cappellari et al., 2011; leonard & masek, 2014). while the components responsible for attracting pollinators play animportant role for the plants, those related to larval nutrition and nest building are crucial for bee reproduction (michener, 2000). studies aimed at knowledge of the chemical constituents present in floral oils are fundamental for a better understanding of how the plant-pollinator interactions work in this highly specialised system, as well as for generating information that may serve as a basis for the development of technologies which could aid in the conservation of both animal and plant species, in addition to facilitating the management and permanence of these bees in agricultural crops that benefit from their pollination services. therefore, the aim of the present study was to analyse the chemical profile and to identify the main constituents of the floral oil of byrsonima sericea, a species abundant in the northeast of brazil and widely used by oil-collecting bees (rosa et al., 2007; lourenço et al., 2019). material and methods the plant material was collected between october 2017 and january 2018, from three specimens of the nance (byrsonima sericea) that were in full bloom during this period in the pici campus belonging to the federal university of ceará (ufc). the nance species was identified at the prisco bezerra herbarium, also in ufc, from dried and mounted plant samples collected from the specimens studied. these plants are part of the local vegetation which is formed by a predominantly precoastal-plain forest with a semi-deciduous forest physiognomy (moro et al., 2015). according to the köppen classification (1948), the climate in the region is type aw’, hot sub-humid tropical, with a rainy period from january to may. samples of floral oil were obtained by collecting the nance inflorescences which are the type terminal racemic and measure between 7 and 11 cm in length. these inflorescences were protected in net bags before floral anthesis (opening of the floral buds), to prevent bees or other agents from removing the oil from the flowers. after anthesis of most of the inflorescence buds, they were cut with pruning shears in the region of the peduncle and placed in zipper bags, avoiding direct contact with any material that could be contaminated. then, the inflorescences were taken to the bee laboratory of the federal university of ceará located less than 1km away to the farthest sampled nance specimen for oil collection. the oil from each flower was collected directly from the epithelial glands (n = ± 4,000 glands, from of a total of 400 flowers) which were pierced using capillary tubes (10 μl capacity). the capillary tubes were filled with the oil by rubbing them against the glands (fig 1a) simulating the collecting behaviour of the bees. although this collecting procedure is laborious, it allows the obtention of pure oil. after the oil was collected in the capillary tubes, it was transferred to a 4 ml vial (fig 1b) and kept in a freezer at -14°c for chromatographic analysis. chromatographic analysis of the oil samples were performed at the multi-user natural product chemistry laboratory of embrapa agroindústria tropical. to characterise the compounds in the oil we collected, samples were derivatised fl rosa, abs barbosa, ths rodrigues, gj zocolo, bm freitas – chemical characterisation of the floral oil of the murici (byrsonima sericea)82 using the following procedure: the samples were first weighed and separated into aliquots of 10 mg which were then solubilised in 50 μl of ethyl acetate (c4h8o2), homogenised and placed in a rotary evaporator. once the solvent had evaporated, 200 μl of pyridine followed by 200 μl of the silylating agent, mstfa [n-methyl-n (trimethylsilyl) trifluoroacetamide] were added. the solution was then homogenised and kept for 30 min at 37°c. at the end of this period, the mixture was analysed by gas chromatographymass spectrometry (gcms). the derivatised samples were analysed by gcms using a model 7890b gc system gas chromatograph (agilent technologies, santa clara, calif., usa) coupled to a model 5977a msd mass spectrometer (agilent technologies, santa clara, calif., usa). the compounds were separated using a 30m x 250μm x 0.25μm hp-5ms capillary column (agilent j & w gc columns, santa clara, ca), with the samples injected in splitless mode, using helium as the entrainment gas at a flow rate of 1 ml min-1. the oven was programmed for floral-exudate analysis with a heating ramp of 50°c to 260°c at 4°c/min and maintained at 260°c for 15 min. the mass spectrometer was operated in scan mode (40-650 m/z) with electron impact (ei) at 70 ev. the total time of the analysis was 65.947 min. the retention indices (ri) of the compounds were obtained by co-injecting the samples with a mixture of c7c30 standard n-alkanes, and calculated as per van den dool & kratz, 1963 (adams, 2007). the derivatised compounds (mstfa) were identified by comparing the mass spectra with the national institute of standards and technology (nist) database, and by comparing the calculated retention indices with those found in the literature. fig 1. collecting oil from nance (byrsonima sericea) flowers. a) using a capillary tube (10 μl) for removing the sample directly from the epithelial gland. b) floral oil kept in a 4 ml vial. results the amount of pure oil obtained from the total number of nance (byrsonima sericea) flowers used in the study (n = 400) was 2.8829 g, with a mean of 0.0072 g per flower. the oil was translucent and yellowish in colour. by means of the chromatographic analysis carried out on the derivatised floral oil of byrsonima sericea, more than 50 chemical compounds were detected, but it was only possible to confirm the identification of 23 constituents (table 1). important among these were fatty acids (11) and carboxylic acids (6). the other constituents are fatty alcohols (2), ketones (2), diterpene (1) and aldehyde (1). the samples required 65.947 min on average for elucidation of all the structures. when the total ion chromatogram (tic) was evaluated (fig 2), the first 15 min of the chromatographic run were ignored. this range was disregarded because it only presented compounds formed from the silylating agents.the compounds that were identified, together with the analytical characteristics used for their identification, are listed in table 1, and numbered following the order of retention time (rt). tricosanoic acid, which is a long-chain fatty acid, presented the largest relative area. this compound eluted at a high retention time (57.83 min) and the electron impact fragmentation included a base m/z fragment of 411 and a less intense m/z of 75. the mass spectrometric analysis of this compound is presented in fig 3, and shows the similarities to the library that was used in the identification. as expected, the most representative chemical components of the oil under study were the fatty acids. the results indicate the presence of tricosanoic acid (38.18%), palmitic acid (4.62%) and heneicosanoic acid (4.21%) as major components (table 1). a less intense 43 m/z fragmentation signal was frequent in most of the structures in the analysed matrix. this signal is usually generated in the presence of an acetyl group in the chain. most of the constituents, especially the fatty acids, were identified in tms-derivative form due to the use of the silylating agent, and it is possible to find in their mass spectra the fragmentation pattern that includes the 75 m/z signal, which is a characteristic ion in this type of reaction. sociobiology 67(1): 80-88 (march, 2020) 83 table 1. chemical compounds identified in oils collected from nance (byrsonima sericea) flowers, with their respective retention times (rt in min), retention indices (experimental and from the literature) (ri), relative areas and representative ions (m/z). rt = retention time; ri_exp = experimental linear retention index; ri_lit = retention index found in the literature; *compounds derived from tms. peak compound rt (min) riexp rilit area match r. match representative ions (m/z) 1 hexanoic acid* 18.439 1299 1290 8.4 x 105 819 874 43, 75, 131, 173(bp), 230(m +.), 2 2-butenoic acid* 29.272 1658 1646 2.8 x 106 900 918 73, 133, 147, 199, 273(bp), 330(m +.) 3 2-pentadecanone 30.547 1704 1698 2.1 x 106 678 855 43, 58(bp), 71, 85, 226(m +.), 259 4 hexadecanal 33.588 1820 1818 1.0 x 107 890 926 43, 57, 68, 82(bp), 96, 109, 240(m +.) 5 2-heptadecanone 35.74 1906 1899 3.8 x 106 853 907 43, 58(bp), 71, 85, 254(m +.) 6 hexadecanoic acid, methylester 36.333 1931 1920 1.9 x 106 820 883 43, 55, 74(bp), 87, 143, 227.2, 270.3(m +.) 7 hexanedioic acid* 37.139 1964 1964 4.8 x 106 912 923 73, 75, 111, 141, 317.2(bp), 374(m +.) 8 geranyl linallol 38.767 2034 2034 6.6 x 106 812 882 41, 55, 69(bp), 81, 93, 107, 121, 290(m +.), 309 9 myristic acid* 39.084 2048 2085 7.3 x 105 780 866 43, 73, 75, 129, 285(bp), 342(m +.) 10 linoleic acid, methylester 40.249 2098 2098 9.9 x 106 936 942 41, 55, 67(bp), 81, 95, 263.2, 294.3(m +.) 11 citramalic acid* 40.732 2120 2127 1.1 x 106 694 746 73(bp), 115, 147, 299.2, 301, 331.2, 433.2, 490(m +.) 12 pentadecanoic acid* 41.545 2157 2184 1.1 x 106 840 880 73, 75, 129, 299.3(bp), 356(m +.) 13 9-hexadecenoic acid, (z)* 43.752 2260 2268 2.8 x 106 770 846 43, 73, 75, 129, 185, 311(bp), 335, 368(m +.) 14 nonanedioic acid* 43.966 2270 2272 5.5 x 106 806 896 73, 75, 129, 311.2, 359.2(bp), 416(m +.) 15 palmitic acid* 44.345 2288 2288 5.5 x 107 949 959 75, 129, 313.3(bp), 370(m +.) 16 oleic acid, (e)* 47.848 2462 2464 2.0 x 106 875 897 73, 75, 129, 339.3(bp), 396(m +.) 17 stearic acid* 48.379 2490 2486 2.6 x 107 934 935 75, 129, 341.3(bp), 398(m +.) 18 oleic acid, (z)* 49.351 2541 2466 2.2 x 107 812 815 75, 129, 155, 339.3(bp), 396(m +.) 19 heneicosanoic acid* 52.302 2696 2708 5.0 x 107 696 783 43, 75, 267.3, 365.3, 383.3(bp), 440(m +.) 20 docosanol* 54.605 2796 2784 9.4 x 106 874 902 43, 73, 75, 95, 267.3, 365.3, 383.3(bp), 440(m +.) 21 tricosanoic acid* 57.832 2906 2920 4.5 x 108 725 810 43, 57, 75, 125, 257.2, 295.3, 393.4, 411.4(bp), 468(m +.) 22 1-tetracosanol* 59.067 2940 2988 1.1 x 108 684 831 43, 57, 73, 75, 97, 125, 257.2, 295.3, 411.4(bp), 468(m +.) 23 tetracosanoic acid* 65.838 >3000 3088 1.6 x 106 860 909 43, 75, 129, 425.4(bp), 482(m +.) fig 2. total ion chromatogram of the floral oil of nance (byrsonima sericea) derivatised with tms silylating agent. white = compound present in the white sample, n.i = unidentified compound. it was not possible to identify some of the compounds present in the oil under study simply from a comparison with the nist library database and the retention indices. among these unidentified chemicals, three (fig 4) presented a relevant relative area compared to the others (17.35%, 19.59% and 33.38%). fl rosa, abs barbosa, ths rodrigues, gj zocolo, bm freitas – chemical characterisation of the floral oil of the murici (byrsonima sericea)84 the unknown compounds 1 and 2 showed a fragmentation pattern that included the base peak (bp) m/z of 117. this fragmentation pattern is commonly found in compounds identified as diacylglycerol. because this group of analytes has also been previously identified in samples of floral oils, it is believed that these unknown compounds may be isomers. however, following the identification criteria adopted in this work, the similarity when compared to the nist library database was low and their identification was therefore not reliable. to identify these components, a deeper investigation with more detailed spectroscopic techniques is necessary. fig 3. expansion of the total ion chromatogram (a) and similarity of the mass spectra of the compounds with the largest relative areas identified in the floral oil of nance (byrsonima sericea), showing the mass spectra from the nist library in blue. b) palmitic acid; c) heneicosanoic acid; d) ticosanoic acid. discussion the species of nance investigated in this study, byrsonima sericea, had a higher yield of floral oil than other previously studied species, perhaps due to a greater quantity of flowers produced per individual (rosa & ramalho, 2011; barônio et al., 2017). the availability of floral resources is one of the factors that can influence the nesting of bees that use this resource (scheper et al., 2015, dainese et al., 2018). in the particular case of oil-collecting bees, studies of nesting activity in bee species of the genus centris in trap nests have shown that they do not nest in the absence of this resource, with its abundance seeming to regulate the nesting rate (magalhães & freitas, 2013; lourenço et al., 2019). byrsonima sericea, in turn, appears to be a good source of this floral resource for these bees, since lourenço et al., 2019, working in the same area as the present research, saw c. analis nesting in trap nests throughout all six months of the study, with the peak of nest construction and brood production coinciding with the start of flowering in this plant species. it is not yet known what role many of the floral-oil chemical components found in this study play for the bees. however, manning (2001) suggests that fatty acids may be involved in energy metabolism, as well as the synthesis of fat reserves, glycogen and cell-membrane structure, which are important in the development, reproduction and nutrition of bees. sociobiology 67(1): 80-88 (march, 2020) 85 in the case of oil-collecting bees, this refers to larval nutrition, since adults do not use the oil as food (vogel, 1990; alvesdos-santos et al., 2007). however, nutrition at the larval stage is important in many solitary bee species, and can have serious consequences on the emergence rate, longevity, and reproductive capacity of the adult individual (t’ai & cane, 2002; radmacher & strohm, 2010). the literature indicates a combination of long-chain saturated and unsaturated fatty acids, with or without β-acetoxy substituents and monoor diglycerides, as the main constituents of floral oils, as well as the smaller presence of other groups (dumri et al., 2008; capellari et al., 2011). however, these fatty acids can undergo biotransformation due to reactions caused by enzymes bees add to the oil when using their mandibles to mix and place it in the cells inside their nests, giving rise to products that are different from those originally secreted by the plant and harvested by the bees (reis et al., 2007). in the present study, ion chromatograms showed that the compounds classified as fatty acids were those with the highest relative area, which would suggest the importance of these compounds in the floral oil of b. sericea and probably other plant species that produce floral oils. among other characteristics, these fatty acids varied in saturation and chain length, between 14 and 24 carbons. regarding the size of the carbon chain, tetradecanoic acid, or myristic acid as it is also known, was one of the shortest fatty acids found in the sample. this acid has a medium saturated carbon chain with 14 carbons and, together with linoleic acid, which was also identified, is mainly related to antimicrobial properties (desbois & smith, 2010). considering that oil-collecting bees usually nest in the soil or in natural wood cavities, the properties of these acids must be very important to the bees as they help keep the developmental environment of the brood protected from fungi and bacteria. tricosanoic acid was the most representative in the matrix, and together with tetracosanoic acid was the longest in relation to chain size. these constituents, due mainly to the size of their chain, required a high retention time to elucidate their structure; they are long-chain linear molecular compounds with the presence of 23 and 24 carbons respectively. there is no literature indicating the role of longchain fatty acids in bee reproduction, but nutritionally they are metabolised mainly during larval development, and constitute an important source of energy (cantrill et al., 1981). perhaps, from the quantities present in the nance oil, these acids may act as source of energy for the brood. various fatty acids found in the floral oil of b. sericea, such as tetradecanoic, palmitic, stearic, tricosanoic, oleic and linoleic acid, among others, appear to be common constituents of floral oils produced by other plant species. therefore, although in different concentrations, they are also present in the floral oil of phymatidium delicatulum, phymatidium tillandsioides, pterandra pyroidea, byrsonima crassifolia and diascia spp. (vison et al, 1997; reis et al., 2006; dumri et al., 2008; capellari et al., 2011). however, these constituents can vary between the different genera and botanical families. the family malpighiaceae, for example, includes genera that present distinct fatty acids and that can cause chemical variations among the floral oils (reis et al., 2007; renner & schaefer, 2010; capellari et al., 2011). these differences may be used to better attract and win the preference of their pollinators. in addition, some species seem to produce far more specific and unique fatty acids. reis et al. (2007) isolated the fatty acid (3r, 7r)-3,7-diacetoxy-docosanoic acid, named as birsonic acid, as the main constituent of the floral oil of b. intermedia. this acid was not isolated in any other study carried out with floral oils, and although the species studied here belongs to the same genus as that investigated by reis fig 4. mass spectra of the unidentified compounds. a) unknown 1, rt = 55.44 min; b) unknown 2, rt = 56.405 min; c) unknown 3, rt = 60.85 min. fl rosa, abs barbosa, ths rodrigues, gj zocolo, bm freitas – chemical characterisation of the floral oil of the murici (byrsonima sericea)86 et al. (2007), it was not possible to identify the presence of birsonic acid in our oil samples. this result can be due either to differences in the spectroscopic analysis used in the two studies, or, more likely, because this fatty acid is specific to b. intermedia. it was also possible to identify two ketones (2-pentadecanone and 2-heptadecanone) in the oil of b. sericea under analysis. it is known that ketones are not important for bee feeding, but are present in small amounts in some floral oils. this group of compounds may be involved in other functions related to bee nesting, since the oils are also used in preparing the nest cells (alves dos santos et al., 2002; melo & gaglianone, 2005). using mass spectrometry, it was also possible to confirm the presence of geranyl linallol in the oil under study. geranyl linallol is a volatile molecule that is derived from linallol, which in turn is an important terpenoid, commonly found in the composition of floral aromas (raguso, 2016; krug et al., 2018). floral aromas comprise a mixture of volatile compounds which are present in pollen, nectar, petals, sepals and other floral structures (farré-armengol et al., 2015), and which play an important role in the plant-animal relationship, being mainly involved in defensive ecological interactions against pathogens and predators and in attracting pollinators (war et al., 2012, song & ryu, 2013, fernandes et al., 2019). although volatiles have a great influence on the collection of floral resources, it is believed that in most cases, attraction of the visitors to the flowers is not due to one compound or class of compounds, but to a set of compounds belonging to different classes (reis et al., 2004; fernandes et al., 2019). however, considering that no other compound with a similar function was identified in the samples, it is possible that geranyl linallol is the only floral attractant in the oil of b. sericea. if other volatile compounds are also involved in attracting visitors to the flowers of this plant species, they must be present in other parts and products of the flowers. despite some of the unidentified compounds have characteristic fragmentation peaks that resemble diacylglycerol isomers, it is possible that they are artefacts or products of the derivatisation reaction. although derivatisation, especially silylation, is typically used to reduce polarity and alter the properties of the analyte to get better separation and improve the sensitivity of the method, in some cases this reaction may generate artefacts under the influence of the reagents, and may cause a reading error (moldoveuanu & david, 2018). in addition, it can be difficult to identify compounds in the analysed matrix simply by means of the spectrometric method used; the identification of these analytes is also hampered, as the mass spectra of many silylated compounds are not present in the usual libraries (moldoveuanu & david, 2018). a knowledge of food sources is essential for understanding the associations between bees and plants (lima et al., 2017). as such, awareness of the chemical composition of floral oils, an essential resource for nesting and brood feeding in oil-collecting bees, can allow measures to be taken for conserving bee species of this group by adopting practices that favour the presence of plant species that produces the specific oils they prefer, as well as developing techniques for agricultural pollination which employ the bees. this may involve cultivating the species that supply the floral oils, and developing artificial oils that include the chemical compounds responsible for attracting the bees, are involved in nest construction and possess the antimicrobial and nutritional properties for development of the brood, the minimum conditions required for these bee species to nest in agricultural areas. acknowledgement the authors are thankful to the national institute of science and technology-inct bionat, grant 465637/2014-0, brazil and the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – under finance code 001 for partly financing this research. breno m. freitas also thanks cnpq for a research productivity fellowships (#302934/2010-3). references adams, r.p. 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(2012). mechanisms of plant defense against insect herbivores. plant signaling and behavior, 7: 1306-1320. doi: 10.4161/bpsb.21663 doi: 10.13102/sociobiology.v66i3.3660sociobiology 66(3): 420-425 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 aphaenogaster ichnusa santschi, 1925, bona species, and redescription of aphaenogaster subterranea (latreille, 1798) (hymenoptera, formicidae) introduction aphaenogaster subterranea (latreille, 1798) is considered a common species in europe. the type specimens were collected around brive-la-gaillarde, corrèze, france. the identification of specimens from france was not considered problematic. however, specimens from corsica and the mediterranean coast show morphological characters that differ noticeably from those of specimens from the rest of france (including brive-la-gaillarde). these characters are (i) shorter and more triangular propodeal spines, (ii) a smoother cuticle, and (iii) a body color globally lighter. this combination of characters led to identification errors: specimens with propodeal spines particularly reduced are labeled as aphaenogaster pallida (nylander, 1849) in the andré collection at the muséum national d’histoire naturelle of paris (mnhn). these specimens, used as reference, led numerous authors to mention a. pallida from southern france and corsica in a range of publications (andré, 1883; de gaulle, 1908; emery, 1908; bernard, 1968, 1983; casevitzabstract morphological and molecular investigation conducted in france and the tyrrhenian islands reveal that aphaenogaster subterranea is composed of two distinct species. we propose to raise a. subterranea var. ichnusa santschi, 1925, described from sardinia, to the species status, a. ichnusa santschi, 1925 stat. nov. this species differs from a. subterranea by having shorter propodeal spines and a less sculptured cuticle. phylogenetic reconstruction using the mitochondrial marker coi shows that the two species form well separated clades, with a genetic distance (k2p) of 9.8 %. in the studied area, the two species are parapatric, a. ichnusa occurring in sardinia, corsica and the mediterranean area of continental france, and a. subterranea occurring north of this area. we propose a redescription of both species and designation of a neotype for a. subterranea. sociobiology an international journal on social insects c galkowski1, c aubert1,2, r blatrix1,2 article history edited by kleber del-claro, ufu, brazil received 01 august 2018 initial acceptance 06 december 2018 final acceptance 29 january 2019 publication date 14 november 2019 keywords ant taxonomy, coi, dna barcode, western palearctic, corsica, sardinia. corresponding author christophe galkowski 104 route de mounic, 33160 saint-aubin-de-médoc, france. e-mail: chris.gal@wanadoo.fr weulersse, 1990). those characters, albeit stable within the mediterranean population, were most often considered irrelevant in systematics. indeed, lighter individuals occur naturally in all populations and the sculpture of the cuticle varies to some extent within most species of the genus aphaenogaster mayr, 1853. the differences between mediterranean and non-mediterranean specimens could correspond to mere intraspecific variability, or could result from particular environmental conditions under mediterranean climate (ants tend to have a lighter color when developing at higher temperature). aphaenogaster subterranea has a broad distribution, from western europe to asia minor and caucasus, throughout which it has a rather homogeneous morphology. thus, the ancient authors described few varieties or sub-species, although they were quite prolific with this respect in other genera. interestingly, santschi (1925) described the taxon ichnusa as a mere variety of a. subterranea. the type specimen is from sorgono, sardinia. according to santschi, the variety ichnusa differ from the typical subterranea by its yellow1 antarea (www.antarea.fr), association for the study and mapping of the ants of france 2 cefe, cnrs, university of montpellier, university paul valéry montpellier 3, ephe, ird, montpellier, france research article ants sociobiology 66(3): 420-425 (september, 2019) 421 brownish color, by the seventh article of the funiculus more elongated and by the propodeal spines weaker. in his revision of the palearctic aphaenogaster, finzi (1930) did not find any difference in the size of the funiculus segments between the specimens of ichnusa and of subterranea he had access to. he only noticed, again, the small and triangular propodeal spines. baroni urbani (1971), in his catalog of the ants of italy, only reported the existence of the variety ichnusa, considered as endemic to sardinia. the type of a. subterranea var. ichnusa, of which high quality pictures can be viewed online thanks to the antweb project (available from http://www.antweb.org. accessed 4 july 2018. specimen casent0913132), shows the same combination of morphological characters as specimens from corsica and the mediterranean coast of france. we present a morphological and molecular analysis of specimens of a. subterranea collected in sardinia, corsica and continental france, showing that the taxon ichnusa should be considered as a valid species, distinct from a. subterranea sensu stricto. we propose hereafter the redescription of a. ichnusa stat. nov. and of a. subterranea, and the designation of a neotype for a. subterranea. material and methods morphometrics the following characters were measured (all measures are in mm): hl: maximum length of head, measured along a median line from vertex to anterior margin of clypeus. hw: maximum width of head, including the eyes. sl: maximum length of scape. the condylar bulb is excluded from the measurement.. ed: maximum diameter of the eye. ml: maximum length of mesosoma (alitrunk). mw: maximum width of mesosoma, measured on pronotum. psl: length of propodeal spine, measured from the center of the propodeal spiracle to the tip of the spine. pw: maximum width of petiole. pl: maximum length of petiole. ppw: maximum width of post-petiole. ppl: maximum length of post-petiole. from these measurements, four ratios were computed: cephalic index (hw*100/hl), scape index (sl*100/hl), mesosoma index (ml*100/mw), spine index (psl*100/ hl). using the maximum length of head as denominator, we intent to reduce the effect of inter-individual size variation. mitochondrial dna analysis new dna sequences for part of the gene coding for the cytochrome c oxidase subunit i (coi, 658 bp) were obtained either within the framework of the international barcode of life project (ibol) or at the center for evolutionary and functional ecology (cefe) in montpellier, france. in the latter case, each specimen was used entirely for dna extraction, whereas in the former case, only one leg was used and vouchers were stored at the cefe. after dna extraction, the coi fragments were amplified through polymerase chain reaction (pcr) using the primer pairs lepf1: 5’-attcaaccaatcataaagatat-3’ and lepr1: 5’-taaacttctggatgtccaaaaa-3’ (hebert et al., 2004). sanger dideoxy sequencing of the unpurified pcr amplicons was performed in both direction using the same primers as those used for the initial amplification. sequences were edited using codoncode aligner (codoncode corporation, dedham, ma, usa), and contigs were built from forward and reverse sequences generated for each gene. conflicting base calls were coded using the ambiguity code. sequences were aligned with muscle (edgar, 2004). alignments were inspected visually and edited manually using mesquite v. 3.31 (maddison and maddison, 2017) when they could be improved. genetic distances were estimated in mesquite using the kimura 2-parameter model (k2p). a partition scheme was defined with partitionfinder (lanfear et al., 2016) for each phylogenetic analysis, using the akaike information criteria for nucleotide substitution model selection. prior data blocks were defined by codon position. phylogenetic reconstructions were performed using bayesian inference with mrbayes v. 3.2.6 (ronquist et al., 2012). two analyses of four chains were run for 1 000 000 generations, sampling trees every 500 generations with 25 % burn-in samples discarded for each run. the phylogenetic tree was rooted using messor structor (latreille) as outgroup. results redescription of aphaenogaster subterranea (latreille, 1798) and designation of a neotype the type specimen of a. subterranea designated by latreille could not be found in the collection of the national museum of natural history (mnhn) in paris, france, and we consider it is lost. in order to fix a neotype and paraneotypes, we collected worker specimens from one nest at brive, terra typica of the species. the neotype is deposited at mnhn. nine workers from the same nest, designated as paraneotypes, are deposited at mnhn, from which a new neotype could easily be designated in case of loss or destruction of the neotype. ten workers of this same nest have also been deposited at the senckenberg museum für naturkunde of görlitz, germany. material examined for morphometrics: brive 45.169n 1.500e, 210 m, 02-vii-2008, forest edge. neotype of aphaenogaster subterranea. fumel 44.494n 0.960e, 341 m, 11-viii-2002, leaf litter. sauveterre de guyenne 44.707n 0.087w, 20-iv-1998, forest edge. saint sever 43.783n 0.526w, 17-vii-2004, leaf litter. mont de marsan 43.894n 0.497w, 18-vii-2004, urban parc. saint aubin de médoc 44.926n 0.730w, 12-vii-2002, garden. c galkowski, c aubert, r blatrix – redescription of aphaenogaster subterranea422 pauillac 45.217n 0.765w, 31-viii-2001, garden. hourtin 45.224n 1.167w, 12-iii-2002, leaf litter. duravel 44.517n 1.083e, 12-viii-2004, leaf litter. mauroux 44.452n 1.043e, 29-x-2017, leaf litter. châteauroux 46.825n 1.697e, vi1995, park. berzy le sec, 30-iv-2017 leg colindre. bethisy saint martin, 15-ix-2016 leg colindre. aigueblanche, 15viii-2016 leg colindre. puy mary, 11-vi-2015 leg lebas. redescription of the worker (table 1, fig 1) head brown, of the same color as the rest of the body, longer that wide, widening from mandibles to occiput. eyes rather small, c. 1/6 of head length, located lateraly at midlength between occiput and clypeus. scapes long, overreaching the occipital margin distinctly, very slightly arched, their diameter increasing from base to apex. funicular segments c. 1.5 times longer than wide, antennal club weakly pronounced, of four segments. clypeus almost smooth, with a few weak longitudinal carinae, anteriorly emarginate. frontal carinae short, frontal lobes narrow. head weakly sculptured, with a few striae extending from frontal carinae and around the eyes. space above antennal fossae weakly reticulate. the sculpture of the cuticule vanish towards the occiput, which is entirely smooth. mandibles strong, rounded, with a few longitudinal striae, masticatory margin armed with c. 4 relatively strong apical teeth followed by c. 4 basal denticles more or less distinct. the whole head is covered by long, semi-erect, setae. many semi-erect to semi-decumbent setae on the scape. aphaenogaster subterranea (n = 30) aphaenogaster ichnusa (n = 30) hl (mm) 1.056 ± 0.066 (0.935-1.131) 1.047 ± 0.116 (0.918-1.156) hw (mm) 0.962 ± 0.070 (0.816-1.029) 0.951 ± 0.112 (0.816-1.088) sl (mm) 1.021 ± 0.044 (0.918-1.054) 0.991 ± 0.068 (0.918-1.071) ed (mm) 0.185 ± 0.017 (0.153-0.204) 0.176 ± 0.020 (0.161-0.212) mw (mm) 0.572 ± 0.039 (0.510-0.612) 0.575 ± 0.064 (0.493-0.680) ml (mm) 1.390 ± 0.076 (1.207-1.462) 1.352 ± 0.140 (1.190-1.530) psl (mm) 0.193 ± 0.026 (0.153-0.217) 0.155 ± 0.033 (0.110-0.204) pl (mm) 0.402 ± 0.024 (0.357-0.425) 0.405 ± 0.037 (0.357-0.459) pw (mm) 0.209 ± 0.018 (0.170-0.230) 0.205 ± 0.018 (0.187-0.238) ppl (mm) 0.287 ± 0.021 (0.247-0.323) 0.265 ± 0.025 (0.221-0.306) ppw (mm) 0.271 ± 0.021 (0.221-0.289) 0.249 ± 0.023 (0.221-0.281) cephalic index hw*100/hl 91.1 ± 2.9 (86.2-94.7) 90.8 ± 1.1 (89.3-92.9) scape index sl*100/hl 96.8 ± 3.1 (93.3-103.6) 95.1 ± 5.0 (85.7-100.9) mesosoma index ml*100/mw 243.4 ± 7.0 (236.1-260.0) 235.2 ± 7.6 (222.9-247.2) spine index psl*100/hl 18.2 ± 1.6 (15.3-20.2) 14.7 ± 2.0 (12.1-19.0) table 1: morphometric characteristics of aphaenogaster subterranea and a. ichnusa. mean ± sd (min-max). characters in bold are the most discriminant. fig 1. illustrations of aphaenogaster subterranea and a. ichnusa stat. nov. showing the reduced propodeal spines and the less sculptured pronotum for the latter. mesosoma evenly brown, light brown or dark brown, with long erect setae, more numerous on pronotum and mesonotum. promesonotum very rounded, separated from propodeum by a guttershaped groove. propodeal spines clearly distinct, pointed shaped, oriented at c. 45°, of a length almost equal to the distance between their tips. cuticle weakly sculptured: sides of pronotum with weak longitudinal striae, space between wrinkles smooth or slightly irregular, summit of pronotum almost smooth. mesonotum a bit more sculptured, with sometimes a few transversal striae at the summit. mesopleuron et propodeum much more sculptured, with marked longitudinal ridges separated by a dense reticulation mainly visible on the lower part of the mesopleuron, summit of propodeum with transversal ridges. space between propodeal spines almost smooth with a few fine transversal striae. petiole and post-petiole brown. petiole with a long anterior peduncle, summit of node rounded, anterior and posterior faces convex. post-petiole rounded. cuticule smooth on nodes, slightly sculptured on the peduncle of the petiole and on the lower lateral parts of petiole and post-petiole. nodes of petiole and post-petiole with a few long semi-erect sociobiology 66(3): 420-425 (september, 2019) 423 setae directed backwards. gaster smooth and shiny, of the same color as mesosoma or sometimes slightly darker in the lighter individuals. all gaster segments with long erect or semi-erect setae, and with sparse pubescence made of thin and appressed hairs. appendices long, light brown, with many decumbent or semi-decumbent setae, and a few semi-erect setae on the interior border of femurs and tibias. distribution in france and tyrrhenian islands: fig 2. redescription of aphaenogaster ichnusa santschi 1925, stat. nov. we did not examine the type specimen directly but we viewed the high quality pictures of it accessible online thanks to the antweb project (available from http://www.antweb. org. accessed 4 july 2018. specimen casent0913132) material examined for morphometrics: agay 43.427n 6.846e, 83 m, 14-vii-2008, cracks in rock. saint raphael 43.443n 6.849e, 15-vii-2008, leaf litter. ile de port cros, 24-x-2015 leg séchet. montpellier, 01-vi2014 leg blatrix. banyuls 42.461n 3.089e, 19-iv-2006, leaf litter. fitou 42.873n 2.988e, 23-vi-2010. argelès sur mer, 29-iii-2017 leg lebas. corsica: piana 42.242n 8.654e, 18-vii-2011, under a stone. col de bavella 41.795n 9.226e, 23-vii-2009, forest. tizzano, 14-iv-2017 leg colindre. bonifacio 41.405n. 9.123e, 14vii-2013, under stone in forest. fig 2. distribution map of aphaenogaster subterranea and a. ichnusa stat. nov. in corsica, sardinia and continental france. blue circles: a. subterranea; red triangles: a. ichnusa; large black symbols: type localities for the two species; filled symbols: morphology and mitochondrial dna (coi) were investigated; empty symbols: only morphology was investigated. fig 3. phylogenetic reconstruction of specimens of aphaenogaster subterranea and a. ichnusa stat. nov. in corsica, sardinia and continental france, based on a bayesian approach using the cytochrome c oxidase subunit i gene (coi). posterior probabilities above 0.90 are indicated. c galkowski, c aubert, r blatrix – redescription of aphaenogaster subterranea424 sardinia: olbia 41.186n 9.328e, 24-vii-2014, leaf litter. tempio pausania 40.911n 9.094e, parc. bolotana 04-v-2011 leg verdinelli. buddusò 01-vi-2017 leg verdinelli. nuoro 01vi-2016 leg verdinelli. redescription of the worker (table 1, fig 1) whole body brown yellow, some nests contain darker individuals. head characters as for a. subterranea, but sculpture of cuticle finer, carinae on clypeus almost indistinguishable, and smooth space on the occiput larger. continental specimens may be more sculptured as those from corsica and sardinia. cuticular pattern on mesosoma as for a. subterranea, but weaker, summit of pronotum and mesonotum and space between propodeal spines entirely smooth. propodeal spines short, triangular, sometimes reduced to mere carinated angles in small individuals. the setae have the same distribution as for a. subterranea. petiole, gaster and appendices as for a. subterranea, setae on tibias finer and more decumbent. distribution in france and tyrrhenian islands: fig 2. mitochondrial dna analysis for this study, 34 new coi sequences were generated (22 by ibol and 12 by the cefe), with genbank accession numbers mh138330 to mh138350, mh138398 and mh630453 to mh630464. sequences are also publicly available on the dataset “ds-asub – aphaenogaster subterranea and aphaenogaster ichnusa” of the bold – barcode of life data systems (doi: dx.doi.org/10.5883/dsasub). the dataset included 658 characters. the two species form two well supported clades (fig 3) with a k2p genetic distance of 9.8 %. the average intraspecific genetic distance is 0.06 % for a. subterranea and 0.7 % for a. ichnusa. for a. ichnusa, the genetic distance between mainland france and the tyrrhenian islands (corsica and sardinia) is 1.2 %. only one specimen, from caussols, shows a disagreement between morphology and coi (fig 3). this specimen was collected on a meridional plateau at >1000 m asl, which hosts cold tolerant insect communities although it is close to the mediterranean cost. introgression between the two species is likely to explain this disagreement. conclusion schulz (1994) defined the a. subterranea species group which was considered to contain a dozen species. our study, along with others in progress (see salata and borowiec, 2018), reveal that this group is more diversified that previously thought. although our study focuses on france, which represents only a small part of the distribution range of the a. subterranea group, the two species here show roughly parapatric distributions that seem driven by climate. aphaenogaster ichnusa seems to be restricted to areas under the influence of the mediterranean climate whereas a. subterranea is found in more temperate environments. although the tyrrhenian islands are located in the mediterranean region, both a. subterranea and a. ichnusa are reported from sicily (schifani and alicata, 2018), and only a. ichnusa in corsica and sardinia. in sicily, a. subterranea is found above 1200 m asl and a. ichnusa at lower altitudes (schifani and alicata, 2018), confirming opposite climate preferences between the two species. our sampling includes a few sites on the transition zone between mediterranean and temperate climates where the two species are detected a few km apart. nevertheless, the two genetic clades are clearly distinct, suggesting efficient reproductive isolation although the two species may occur in sympatry in certain regions. acknowledgements we thank thierry noel (university of montpellier), thibaud decaëns and the members of the groupe naturaliste de l’université de montpellier (gnum) for their help in processing samples, paul hebert (university of guelph) for providing a privileged access to ibol sequencing facility, and marie-pierre dubois and the service des marqueurs génétiques en ecologie (cefe) for providing facilities for molecular biology bench work. many ant specimens were obtained through the antarea project (www.antarea.fr). this research was funded by grants from the fond de solidarité et de développement des initiatives etudiantes (fsdie) of the university of montpellier and from the office de l’environnement de la corse (oec). authors’ contribution cg designed and conducted the morphological analysis and drafted the manuscript. ca contributed to molecular analysis. rb designed and conducted the molecular analysis and contributed writing the manuscript. all authors approved the final version of the manuscript. references andré, e. 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(1994). aphaenogaster graeca nova species (hym: formicidae) aus dem olymp-gebirge (griechenland) und eine gliederung der gattung aphaenogaster. beitraege zur entomologie, 44: 417–429. doi: 10.13102/sociobiology.v66i3.4378sociobiology 66(3): 508-514 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the effect of forest fragmentation on polistinae introduction a forest fragment is defined as a natural vegetation area interrupted by natural or anthropic barriers (e.g., roads, villages, agricultural and forestal crops, pastures, mountains, lakes, dams) which are capable of significantly diminishing the flow of animals, floral resources, pollen and seeds (viana & tabanez, 1996), altering the biota (laurence et al., 2006; nascimento et al., 2006) and the habitat abiotic factors – and, consequently, the nesting places of social insects (schwarcz filho et al., 2004). this fragmentation process negatively affects different ecosystems, especially the atlantic rainforest, which has been suffering for thousands of years (dean, 1997). the atlantic rainforest, with its varied phyto physiognomies, is brazil’s oldest forest formation, having been established at least 70.000.000 years ago, in a practically abstract the forest fragmentation is caused by natural or anthropic actions, which affect negatively the biota and the environmental services rendered by biological diversity. however, there is little information on the reflex of these actions in many different groups of animals, such as social wasps, which are abundant and significantly present in neotropical environments, causing a major impact in the communities they live due to their role in food webs. as their natural enemies, wasps are important in the control of agricultural plagues; in the natural environment, they are nectar collectors, frequent flower visitors, and potential pollinators of many species of plants. these factors justify studies which would evaluate in what way the forest fragmentation acts on these insects biodiversity. this study was carried out in four fragments, each of a different size, located in the municipalities of inconfidentes and ouro fino, in the south of the state of minas gerais (brazil), where the phytophysiognomy is the montane semideciduous seasonal forest. the research was carried out between december 2016 and march 2018, with the same sampling collection for each fragment, totalizing 104 sampling days. in total, 28 species and 51 colonies were recorded in the four areas and a greater richness for the greatest fragment (f4). the conclusion reached was that the size and heterogeneity of the fragment have an important role in maintaining the richness of social wasps. sociobiology an international journal on social insects et bueno¹, mm souza¹, ma clemente2 article history edited by evandro n. silva, uefs, brazil received 22 february 2019 initial acceptance 29 may 2019 final acceptance 29 may 2019 publication date 14 november 2019 keywords atlantic rainforest; vespidae; epiponini; pseudopolybia; mischocyttarus. corresponding author marcos m. souza instituto federal de ciência e tecnologiasul de minas gerais campus inconfidentes praça tiradentes, nº416, centro cep. 37576-000 inconfidentes-mg, brasil. e-mail: marcos.souza@ifsuldeminas.edu.br continuous coverage along the coastal region. it extends from the northeast region until the state of rio grande do sul; its central portion is located in the sea mountain and mantiqueira mountain, comprising the states of são paulo, minas gerais, rio de janeiro and espírito santo (rizzini, 1997). this biome contains an abundance of ecosystems regarding biological diversities (myers et al., 2000), however, the vast majority of its reminiscent is in small fragments (viana & tabanez, 1996). in these fragments, the plant populations are comprised of few individuals of the same species, which generates a considerable percentage of endogamy and consequently a high probability of extinction of the local species (costa, 2003). however, these few reminiscents may work as stepping stones (linking points or ecological trampolines), which are small habiting areas scattered through the array which may help some species in the flow between fragments, since there is 1 instituto federal de ciência e tecnologiasul de minas gerais, inconfidentes-mg, brazil 2 universidade estadual paulista "júlio de mesquita filho", instituto de biociências, zoology department, rio claro-sp, brazil research article wasps sociobiology 66(3): 508-514 (september, 2019) 509 the occurrence of ecological corridors to allow these actions, which also reflects upon the fauna (viana & pinheiro, 1998). it is vital to understand how the animal populations behave when confronted with environmental fragmentation in order to establish actions that can minimize the negative effects of this process on the biota; however, there is little to no information on a variety of animals in this context, such as wasps, both the solitary (morato & campos, 2000) and the social ones (graça & somavilla, 2018). these insects are commonly known in brazil as marimbondos or cabas, and belong to the hymenoptera order, vespidae family, which is comprised of six subfamilies grouping solitary species (massarinae, eumeninae, and euparigiinae) and social species (stenogastrinae, vespinae, and polistinae) (carpenter & marques, 2001). there are, around the globe, 974 species of social wasps, from which 552 occur in the american continent and 346 in brazil (carpenter & anderna, 2013), where there is only the recording of the subfamily polistinae, with 26 described genera (carpenter & marques, 2001). they perform many environmental services in different natural and agricultural ecosystems, acting in the biological control as predators of many insects which are plagues in different cultures (carpenter & marques, 2001; prezoto et al., 2005; hunt, 2007; souza & zanuncio, 2012); they are potential pollinators of different species of angiosperms (clemente et al., 2012), constitute important floral visitors (santos et al., 2006; clemente et al., 2012), and can be used as bioindicators of the conservation level of the riparian forest (souza et al., 2010). studies that utilized the social wasps as ecological models to evaluate the impact of fragmentation are scarce, however, graça and somavilla (2018) have made an important contribution to the amazon biome, showing that the continuous vegetation areas are home to an abundance (common and rare), richness and diversity greater than when compared to the fragments highly affected by anthropic actions. considering the factors of abundance, richness, diversity, registration ease, colony monitoring, and constancy in the different seasons of the year, the social wasps show themselves as an interesting group to be evaluated regarding the forest fragmentation effects. therefore, it is expected that the largest forest fragments house a greater richness of social wasps, as well as a higher number of colonies. material and methods the study was performed from december 2016 to march 2018 in four forest fragments, each one with a different size (fig. 1, table 1) in the municipalities of inconfidentes (fragment f1, fragment f2, and fragment f3) and ouro fino (fragment f4), in the south of the state of minas gerais, brazil. for each area, 26 days of sampling were carried out, which adds up to 104 days of sample collection, in a total of 2096 hours of sampling. the areas have similar ecological characteristics (as shown in table 1) but differ in size, circularity and surrounding array. the researched areas are montane seasonal semideciduous forest, in the domain of the atlantic rainforest biome (oliveira filho, 2006). to record the species two methodologies were applied: the active search and the attractive traps. the first was used between 8 a.m. and 5 p.m. in the four seasons of the year, split in five days for each forest fragment in a total of 80 hours of sampling, performed by three researchers. this method was consisted of running across the pre-existant tracks in the border and interior of the forest, and the capturing of the insects was assisted by an entomological web. fig 1. location of the montane semideciduous seasonal forest fragments used to evaluate the impact of fragmentation on the social wasps’ communities in the south side of the state of minas gerais, in the southeast region of brazil. et bueno, mm souza, ma clemente – forest fragmentation and polistinae510 the second methodology, the attractive traps, was used with pet bottles, as suggested by souza et al. (2015a). ten unities were distributed for each fragment in three months – september and october 2017, and march 2018 – being kept in the field for seven consecutive days, in a total of 84 sampling days, being 21 for each fragment. the traps were distributed in sites indicated by the researcher, considering the highest possibility if collecting social wasps, such as forest border, close to water courses etc. the bottle-traps were placed in a straight line and hang in the vegetation at 1,5 meters from the ground, with a distance of 10 meters between them and utilizing as bait 250 ml of fresh passionfruit juice (souza et al., 2015a). at the end of the seventh day after the installation of the traps on the field, the material was sorted with a clamp and a sieve and subsequently accommodated in 70%-pure alcohol, in order to be possible to proceed with the assembling and identification. the species were stored in wet and dry methods in the biological collection of the federal institute of the south of minas gerais (ifsuldeminas), in the incondidentes campus, and registered on the website of the reference center for environmental information (cria). the identifications were performed with the help of taxonomic keys (carpenter & marques, 2001), and by comparison with the samples of the aforementioned biological collection performed by professor orlando tobias da silveira, ph.d., from the emílio goeldi museum (in belém, pará). in order to analyze the diversity, the researchers considered the number of colonies for each species (souza et al., 2010), adopting the shannon-weaver index; to evaluate the fauna similarities between the fragments, a grouping analysis was performed via the unweighted pair–group method using arithmetic means (upg-ma), through the jaccard similarity coefficient (krebs, 1998). results and discussion a total of 28 species and 21 colonies was registered, being the fragment f4 the one which presented greater richness, with 21 species (table 2), seven of them being exclusive (mischocyttarus atrametarius, mischocyttarus cassununga, mischocyttarus sp. 2, mischocyttarus wagneri, polybia minarum, polybia platycephala, pseudopolybia vespisceps); nevertheless, it presented a lower diversity level than f1. the fragment f4 has 336,367 m², being the largest area in the study; it also has hydric resources, and there is no agricultural activity or eucalyptus plantation in its array. these characteristics have a positive implication in the richness of wasps sampled (21 species), since they allow the establishment and survival of more species of social wasps, creating distinct microhabitats for the organisms and therefore providing more protection of predators and increasing the availability and diversity of food resources and nesting substrates (santos et al., 2007; francisco et al., 2018). another important factor is the size of the fragment f4, which is from 15 to 58% larger than the other areas (table 1), added to the heterogeneity of the environment, which is in intermediate regeneration, as discussed by some authors (morato & campos, 2000; santos et al., 2007). this fragment was also the only one in which the species pseudopolybia vespiceps was registered. it is considered a bioindicator of well-conserved riparian forest continuous areas (souza et al., 2010); its less significant ecological plasticity entails a restriction of environments with specific conditions, transforming the species in a specialist in specific habitats (cruz et al., 2006), which sustains the hypothesis of why the fragment f4 presents more richness. table 1. different ecological characteristics regarding size and pastoral agricultural activities in the forest fragments (f1, f2, f3, and f4) in which the social wasps’ communities were evaluated, in the south of the state of minas gerais, in the southeast region of brazil. fragment characteristics fragment f1 fragment f2 fragment f3 fragment f4 size (m²) 51179 53140 198295 336367 coordinates 22° 23’ 60” s, 46° 20’ 26” w 22° 22’ 21” s, 46° 19’ 34” w 22° 21’ 93” s, 46° 20’ 55” w 22° 20’,74” s, 46° 20’ 95” w circularity index 0,52 (highly elongated) 0,93 (rounded) 0,6 (elongated) 0,6 (elongated) presence of lentic and lotic water courses + + + + livestock activity + + + agricultural activity + eucalyptus cultivation + canopy formation + + + + average distance to other fragments (m) f2= 885 f3= 2350 f4= 4170 f1= 885 f3= 1277 f4= 3121 f1=2350 f2=1277 f4= 1391 f1= 4170 f2= 3121 f3= 1391 conservation level secondary forest in intermediate stage of regeneration secondary forest in intermediate stage of regeneration secondary forest in intermediate stage of regeneration secondary forest in intermediate stage of regeneration presence (+) and absence (-) sociobiology 66(3): 508-514 (september, 2019) 511 in the samples performed with the attractive traps, both agelaia multipicta and a. pallipes were registered in all the fragments. it is notable the abundance of a. pallipes (65 individuals) in the fragment f1, probably due to the smaller size of the fragment, increasing the probability of the trap being installed close to a colony, combined with the fact that the size of the area makes the foraging easier, since the range of action of the social wasps is on average 200 meters (gomes et al., 2007; bichara filho et al., 2009). in larger areas, the competition among the species from close taxons which explore the same resources tends to decrease and establish a similar population size (fox & fox, 2000). this behavior is verified in the fragment f4, where other three species were registered – a. multipicta, a. pallipes e a. vicina –, all in similar abundance, which is likely to show the decrease in the competition between the species of this genus. since it presents a larger area, the fragment f4 has a larger availability of hydric and food resources, in addition to a larger number of nesting sites. the abundance of species from the genus agelaia in diversity studies is a side effect of a large number of individuals in these colonies (hunt et al., 2001; graça & somavilla, 2018), as noted in other studies carried out in the state of minas gerais (souza et al., 2012; souza et al., 2015b). nevertheless, when the number of colonies (table 2) and shannon-weaver diversity index are considered – f1(2,22003), f2 (1,69574), f3 (1,84622) and f4 (1,95545), as calculated based on the number of colonies per species – fragment f1 was more relevant than f4, which may be an outcome of the fact that denser and higher vegetation areas hinder the registration of social wasps’ nests (silveira, 2002; souza & prezoto, 2006) due to the colonies being more cryptic (wenzel & carpenter, 1994), while smaller areas such as f1 experiment the opposite (souza & prezoto, 2006). this situation corroborates the study carried out by santos et al. (2009) which demonstrated that the abundance of species is not related to the number of nests found in each area. considering that the fragments are similar regarding their ecological characteristics but different in size, the method of average association was applied to evaluate the similarity of the social wasps’ fauna (fig 2). it was observed that f1 is very similar to f2, including their sizes (table 1), but they are different from f4, which behaved in a different way, resulting in seven exclusive species – even if f4 is more isolated from the other fragments regarding the other study areas, what can be explained due to the ability of the wasps to adapt to fragmentation better than bees (morato & campos, 2000). notithstanding, according to graça and somavilla (2018) in their study in the amazon rainforest, it was verified that many species have colonized not only continuous forest areas but also fragments; there is however a decrease in the number and abundance of species, which reveals a rupture in the structure of the fragmented community. thus, the researchers concluded that the forest fragmentation is likely to threaten abundant and rare species equally. five species were collected in all the four fragments (a. multipicta, a. pallipes, a. vicina, polistes versicolor, and polybia fastidiosuscula) and other three were common ground to at least three fragments (mischocyttarus drewseni, polistes cinerascens, polistes simillimus, polybia paulista, and synoeca cyanea). this suggests that these fragments once formed a continuous forest, and due to its fragmentation the entomofauna of some areas suffered more pressure and lost some species over the time. this is a consequence of the border effect (lovejoy et al., 1986), which alters the abiotic and biotic factors (lovejoy et al., 1986; wirth et al., 2007); as a result, many species are lost (woodroffe & ginsberg, 1998). in the study carried out by graça and somavilla (2018) many species were found in both forests, with an emphasis on agelaia fulvofasciata, agelaia testacea, agelaia pallipes, and polybia rejecta, genera also present in this study. the authors point out that even if the species are present both in the continuous and in the fragmented forest, there was a significant change in the absolute abundance values. these patterns show the fragility of the abundant and the common species, such as the genus agelaia, which is impacted by anthropic disturbances, being frequently suppressed (gaston, 2010). the smaller the fragment, the more severe the border effects manifest themselves, altering the microclimate on forest borders, which increases the probability of species invasion, eventually favoring opportunistic species with specialized habitat (devictor et al., 2008; nordén et al., 2013; matthews et al., 2014), thus limiting its pecial occurrence. another effect entailed by the fragment isolation is that many species are not able to percolate, hindering the chances of colonization (tischendorf & fahrig, 2000; blandón et al., 2016). under that perspective, the size of the fragment, as well as heterogeneity, seem relevant for the maintenance of the abundance of social wasps, as previously discussed by other authors (souza et al., 2010; souza et al., 2014). the conclusion the present study reaches is that the size of the fragment is a decisive factor concerning the richness, diversity, and abundance of social wasps. these insects may fig 2. index of similarity in the social wasps’ fauna in the four forest fragments in the municipalities of inconfidentes and ouro fino, in the south of the state of minas gerais in the southeast region of brazil. et bueno, mm souza, ma clemente – forest fragmentation and polistinae512 be used as ecological models to forecast the level of impact of fragmentation and vegetation area in resident communities. it is also noted the importance of the preservation of the existing areas and the incentive to increase the vegetation areas. references bichara-filho, c.c., santos, g.m.m., resende, j.j., cruz, j.d., gobbi, n. and machado, v.l.l. 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(1998). edge effects and the extinction of populations inside protected areas. science, 280: 2126–2128. doi: 10.1126/science.280.5372.2126 wright, s.j., stoner, k.e., beckman, n., corlett, r.t., dirzo, r., muller-landau, h.c., nunez-iturri, g., peres, c.a. and wang, b.c. (2007). the plight of large animals in tropical forests and the consequences for plant regeneration. biotropica, 39: 289–291. doi: 10.1111/j.1744-7429.2007.00293.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4562sociobiology 67(2): 301-307 (june, 2020) introduction the paper wasp mischocyttarus cerberus is widely distributed across brazil (richards, 1978; oliveira et al., 2017). one or a few inseminated females (foundresses or queens) initiate new nests, which give rise to generations of other females (workers and future queens) and males, as the cycle of the colony progresses. new colonies are established throughout the year (giannotti, 1998a). similar to other independent-founding wasps, no clear morphological caste differences are observed (see jeanne, 1980). however, the nestmates establish a reproductive-based division of labor depending upon dominance-subordination interactions. normally, only one queen exists per colony (poltronieri & rodrigues, 1976; giannotti, 1998a). it lays most of the eggs abstract mischocyttarus cerberus stands out as the most investigated species of eusocial paper wasp, in brazil. while the adult characteristics were relatively well reported in the earlier studies, very meager information was available regarding their immature stages. this study provides a general description of the immature morphology of the brood of m. cerberus, by studying the number of instars and analyzing the degree of influence exerted by some of the environmental factors on the individuals in the immature phases. this work involves a detailed study of 72 wasp colonies from rio claro and ribeirão preto. using the larvae drawn from 41 nests, the number of instars was calculated; besides, the degree to which a few environmental factors could affect the immature brood development was assessed in 31 nests. eggs showed patterns similar in terms of form and size to that of the species described earlier. the two ventral lobes, characteristic of the mischocyttarus larvae, were fully developed only in the fifth instar. based on the head measurements, we found that m. cerberus also express five larval instars, which is in agreement with reports published earlier for the most part of social wasps. besides, larvae took longer than eggs and pupae to develop. from the results of our study, we concluded that m. cerberus showed the typical developmental pattern in the immature stages of the genus. sociobiology an international journal on social insects rc da silva¹, ds assis¹, ar de souza¹, fs nascimento¹, e giannotti² article history edited by gilberto m. m. santos, uefs, brazil received 07 june 2019 initial acceptance 15 october 2019 final acceptance 07 april 2020 publication date 30 june 2020 keywords larval instars, larval development, brood description, mischocyttarini. corresponding author rafael carvalho da silva universidade de são paulo – usp faculdade de filosofia, ciências e letras de ribeirão preto departamento de biologia av. bandeirantes nº 3900, vila monte alegre, cep: 14040-900 ribeirão preto, são paulo, brasil. e-mail: rcsilva2812@usp.br and is the most aggressive female, often displays aggressive behaviors toward their nestmates (noda et al., 2001). the adult wasps are also known to be capable of defending their nests against ants (togni & giannotti, 2007; togni & giannotti, 2008). one common aspect of the biological and ecological studies described prior was that they focused only on the adult wasps, which seems to be the general pattern adopted in the research of eusocial insects (giannotti, 1998; togni & giannotti, 2007; togni & giannotti, 2008). however, paper wasp colonies include substantial numbers of immature brood, like eggs, larvae and pupae. these immature stages are observed in the nest in the most part of the colony cycle and the adults often exhibit interactions with them. a considerable duration of individuals` lives is spent in 1 universidade de são paulo (usp), faculdade de filosofia, ciências e letras de ribeirão preto, departamento de biologia, ribeirão preto-sp, brazil 2 universidade estadual paulista (unesp), instituto de biociências, departamento de zoologia, rio claro-sp, brazil research article wasps notes on brood morphology and development of the neotropical eusocial wasp mischocyttarus cerberus (hymenoptera, vespidae, polistinae) rc da silva, ds assis, ar de souza, fs nascimento, e giannotti – brood development on an eusocial wasp 302 the immature stages (giannotti & fieri, 1991; giannotti & machado, 1994; giannotti 1995; prezoto & gobbi, 2005; solis et al., 2012; cecílio et al., 2015; rocha & giannotti, 2016). the considerable presence of interspecific variation in the larval morphology implies its taxonomical significance (nelson, 1982; kojima, 1998; carpenter et al., 2000). larvae of mischocyttarus wasps were first described by having the first abdominal sternite with one, two or three lobes projected forward, besides their first abdominal spiracle is twice as big the remaining and mandibles have a single elongated tooth (richards, 1978). kojima (1998) was the first to study larvae of m. cerberus, however he studied only material coming from two nests and his notes regarded only to morphological aspects. despite of that, there is still a lack of detailed information regarding the immature stages of m. cerberus up to this moment. thus, we used laboratory and field observations of the paper wasp m. cerberus to describe (i) the general external morphology of the eggs, larvae and pupae, (ii) the number of larval instars and (iii) the duration of the immature stage in relation to temperature and nest phase variation. material and methods study location and wasp collection during the years of 1991-1993 and 2017, we examined a total of 72 m. cerberus field colonies located in the municipalities of rio claro (22°24’ s; 47°33’ w, at elevation 612 m) and ribeirão preto (21°05’ s, 47°50’ w, at elevation 531 m), são paulo state, southwest brazil. to assess the general external morphology and determine the number of larval instars, 41 field nests were collected, from which 177 eggs, 259 larvae, 31 pre-pupae (correspond to the individual in transition between fifth instar larvae and pupae) and 69 pupae were sampled. these immature individuals were preserved in 70% ethanol. to describe the duration of each immature stage, field observations were performed on a total of 31 naturally established colonies, out of which we followed the development of 404 eggs, 232 larvae and 284 pupae. the general external morphology of immature stages the eggs, larvae, pre-pupae and pupae were carefully studied under a binocular stereomicroscope (leica mz125). the immature stages were described based on the terminology used in the previous studies (kojima, 1998; cecílio et al., 2015; rocha & giannotti, 2016). the external morphometry of the immature stages each immature individual was carefully removed from its cell and measured under a stereomicroscope. for each egg, we measured the maximum width, whereas for each larva, pre-pupa and pupa, we measured the maximum width of the head capsule. after measuring all samples, mean values were calculated and pair-wise comparisons were carried to check whether the groups were different or not. the number of larval instars the number of larval instars was determined depending upon a visual analysis of the data on head width, plotted in a frequency-distribution graph. distinct peaks corresponding to distinct size frequency distribution peaks were categorized as instars. to validate our method, the head width data of the larvae were then tested according to dyar’s rule (dyar, 1890), which suggested that the larval head grew in geometrical progression, increasing its maximum width in the ratio of 1.44 at each ecdysis (parra & haddad, 1989). duration of the immature stages in relation to temperature and nest phase variations this party of the study was performed in rio claro. the climate classification of the area is mesothermic according to köppen (1948), which means that there are two well defined seasons along the year, a rainy (from october to march) and a dry (from april to september) season. the eggs, larvae and pupae present in each nest were mapped during regular visits (at 2to 10-day intervals) under natural conditions. thus, the overall duration (in days) of each immature stage was assessed during the months of the year when the investigations were done. the months were grouped as follows: december – february; march – may; june – august, and september – november. in the discussion, we explored the manner in which the results differed with respect whether the study period was cold and dry or rainy and sunny. besides that, duration of the immature stages was calculated based on the colonial stages of development (preand post-worker emergence). statistical analyses the cephalic capsule width was compared using a linear model (lm). in our model, we used the cephalic capsule width as the response variable and the m. cerberus life stages as the explanatory variable. we used a normal model, because it best fitted our hypothesis (weights aic: poisson < 0.0001; normal = 0.999). posteriorly, we used the tukey’s test for a post-hoc analysis with p correction, employing false rate discovery (fdr). besides that, the developmental time for each phase (egg, larvae and pupae) was compared based on the different month groups throughout the year using the kruskalwallis test. brood development time according to nest phase (pre and post-worker emergence) were compared through mann-whitney u test. statistical analyses were performed using the r and past version 3.20 software. results the general external morphology of the immature stages the eggs showed slight curvature, narrow at the base, fixed onto one of the nest cell wall angles and projecting into the nest cell lumen at a roughly 45°angle and coated with an adhesive secretion (fig 1a). the vermiform larvae are sociobiology 67(2): 301-307 (june, 2020) 303 soft-bodied, having sclerotized pale brown cephalic capsules, which became even darker in color through the molting cycle. the thoracic and abdominal segments are whitish, in numbers of 3 and 10, respectively (fig 1d). larvae are fixed by their distal end in the nest cell, until the third instar (fig 1b); from the fourth instar onwards they become free. the fifth instar larvae has two abdominal lobes, covered by small bristles (fig 1). the abdominal lobes are observed as small protuberances since the third instar, and they achieve full development only in the fifth instar (fig 1d). thus, only the last instar expresses the typical aspect of the mischocyttarus larvae, with clearly evident abdominal lobes, prominently projected forward. the diameter of the first spiracular atrium (sited between the second and third thoracic segments) is almost twice the diameter of the rest of the atria (fig 1d). the transversal anal slot is located in the last (anal) segment (fig 1d). the head is composed of the cephalic capsule and mouth parts, which are more highly sclerotized in the last larval instar (fig 1c). the cephalic capsule is brown in color compared to the rest of the larval body. the pre-pupa or pharate pupa is categorized as the late period of the fifth larval instar and characterized by the elongated body of the last-instar larva (fig 1e).through the transparent pre-pupal cuticle the reddish-brown compound eyes of the pupa can be observed to be fully developed (fig 1e). during the pre-pupal phase the abdominal lobes are projected rearward rather than forward. finally, in the pupal stage, the appendages remain free from the body and the wings are not fully distended until this stage is complete (fig 1e). at the beginning, the pupa is whitish in color with reddish-brown compound eyes, and gradually its body turns to be yellow and black. besides, the dark pigmentation appears to develop faster than the yellow pigmentation along the body. the external morphometry of the immature stages the linear model analysis revealed that the groups of eggs, larvae, pre-pupae and pupae differed in their width (f = 3365; p < 0.0001). generally, the head width increases progressively, as the wasps develop from one instar to the other (table 1). figure 1: a) example of one m. cerberus egg attached to a piece of nest material – white bar represents 0.5 mm; b) frontal view of one fifthinstar larvae with its abdominal lobes in evidence – black bar corresponds to 1.0 mm; c) frontal view of one fifth-instar larvae cephalic capsule – black bar represents 0.5 mm; d) lateral view of one fifth-instar larvae – white bar represents 0.5 mm; e) lateral view from pre-pupae with elongated body of fifth-instar larvae to a fully developed pupae before its emergence – black bar correspond to 5.0 mm. a1 to a10 – abdominal segments from 1 to 10; ab – abdomen; al – abdominal lobes; ar – antennal ring; as – abdominal slot; at – antenna; bt – bristles; cc – cephalic capsule; dwg – developed wings; ey – compound eyes; hd – head; la – labrum; lb – labium; lg – legs; lp – labial papilla;me – mentum; mp – maxillary papilla; ms – median suture; mt – mandible tooth; mx – maxilla; nwg – non-developed wings; pa – point of attachment; sp1 spiracule 1; t1 to t3– thoracic segments from 1 to 3; tb – temporal band; tp – tentorial pit; tx – thorax. rc da silva, ds assis, ar de souza, fs nascimento, e giannotti – brood development on an eusocial wasp 304 five larval instars were determined by the visual inspection of the distribution-graph indicating the measurements of the head width (fig 2). supporting these findings, the five larval instars proposed expressed significant differences based on the average of their head width when compared (f = 3365; p < 0.0001). finally, our data analyzed larval average growth rate according to dyar`s rule , we observed mean growth ratio among the instars = 1.44 (1.39 between l1 and l2, 1.40 between l2 and l3, 1.43 between l3 and l4 and 1.52 between l4 and l5). duration of the immature stages in relation to monthly temperature and nest phase variations the overall duration of each brood stage is presented in table 2. from the kruskal-wallis test it is clear that the eggs (h = 74.68, p < 0.001), larvae (h = 719.28, p < 0.001) and pupae (h = 24.37, p < 0.001) take different times to develop throughout the year. the sum of the mean values of the developmental times (eggs + larvae + pupae) are similar for the warmer and rainier months (from september to may), however, they are longer for the coldest and drier ones (from june to august) (table 2). the mean period of incubation of the eggs in the preworker emergence stage of the colonies of m. cerberus was significantly longer than in the post-emergence stage (mannwhiney test, u = 2072.5, p = 0.05). the respective times of duration of the egg incubation were 13.7±4.4 (9-22, n = 17) and 11.6±3.9 (7-28, n = 191) days. on the other hand, there was not significant difference between the larval period in the pre and the post-worker emergence stages in colonies (mann-whiney test, u = 879.0, p = 0.68). the respective times of duration of the larval periods were 30.2±9.7 (16-48, n = 15) and 30.5±11.9 (19-93, n = 111) days. the mean of pupal period in the pre-worker emergence was significantly longer than in the post-worker emergence (mann-whiney test, u= 1284.5, p = 0.001). the respective time of duration of the pupae was 24.2±4.6 (16-33, n = 15) and 19.6±4.5 (9-36, n = 111) days. n mean stand. dev median min max eggs 177 0.496 0.053 0.496 0.372 0.622 larvae 1 49 0.452 0.041 0.465 0.346 0.558 larvae 2 38 0.644 0.04 0.637 0.589 0.745 larvae 3 34 0.897 0.07 0.893 0.757 1.024 larvae 4 72 1.441 0.212 1.359 1.05 1.749 larvae 5 66 1.895 0.099 1.884 1.756 2.148 pre-pupae 31 1.97 0.084 1.984 1.769 2.139 pupae 69 2.438 0.155 2.48 1.718 2.666 table 1 – descriptive statistics of the widths (mm) of the eggs and the cephalic capsules of the larvae (l1 – l5), pre-pupae (pp) and the pupae (p) of mischocyttarus cerberus. fig 2. frequency distribution of the maximum widths of head capsules of m. cerberus larvae. l1: first instar; l2: second instar; l3: third instar; l4: fourth instar and l5: fifth instar. sociobiology 67(2): 301-307 (june, 2020) 305 discussion morphology of the immature stages the m. cerberus eggs are similar in appearance to those described earlier for the wasps from the same genus (giannotti & silva, 1993; cecilio et al., 2015; rocha & giannotti, 2016). as observed for m. drewseni (giannotti & trevisoli, 1993), only the last larval instar expressed the features of the mischocyttarus larvae (wheeler & wheeler, 1979), in which the abdominal lobes were fully evident and prominently projected forward. from the findings of several researchers, these structures may be involved in different functions. one hypothesis suggests that they could be used as sensorial structures, based on the amount of bristles covering them (rocha & giannotti, 2016). a second hypothesis proposes that they could play a role when the larvae are ingesting food, functioning as a mean to hold the solid food (reid, 1942; wheeler and wheeler, 1979). a third hypothesis proposes that these lobes could assist during the process of saliva transference (larvae – adult), by pumping the larval saliva into the adults when they solicit it (jeanne, 1972), in a behavior described as “lobe erection” (hunt, 1988). finally, according to giannotti & silva (1993), these lobes may help the larvae to sustain themselves inside the cells, preventing them from falling. the pattern of the cuticle changing from a lighter color to a blackish one during the pupal stage of development appears to be the general rule when compared to the species analyzed earlier (giannotti & silva, 1993; rocha & giannotti, 2016). determination of the morphometry of the immature brood and instars our findings concerning determination of the number of instars corresponded to previously published results for most of the mischocyttarus species, including m. cassununga (giannotti & silva, 1993), m. drewseni (giannotti & trevisoli 1993), m. latior (cecílio et al., 2014) and m. nomurae (rocha & giannotti, 2016). a similar pattern has also been identified for other social wasps from different genera, such as agelaia (giannotti, 1998b), brachygastra (machado et al., 1988), polistes (giannotti, 1995; prezoto & gobbi, 2005), polybia (solis et al., 2012) and protopolybia (silveira, 1994). in fact, the presence of five instars has been proposed as a pattern number for the larvae of polistinae and vespinae, whereas for the stenogastrinae the number may range from three to five (kojima, 1998). on the other hand, our results differed from the findings of silva (1984) and raposo-filho (1981), who determined only four instars for m. extinctus and m. atramentarius. some authors proposed that the number of larval instars might vary in accordance to the biotic and abiotic factors, such as the environmental temperature, hereditary traits or even nutritional frequency (parra & haddad, 1989). like in m. cerberus, it was verified that in polistes lanio (giannotti, 1995) the eggs are wider than the larvae of the first instar. on the other hand, these findings differed from the results reported for the other mischocyttarus, such as m. cassunuga (giannotti & fieri, 1991), m. drewseni (giannotti & trevisoli, 1993) and m. latior (cecílio et al., 2015), which months average duration in days of immature stages egg larvae pupae sum on mean values (september – november) rainfall average 102.0 mm temperature average 20.0ºc (10.2ºc 30.0ºc) 11.3 ± 3.4 (n = 100) 29.2 ± 9.0 (n = 45) 19.6 ± 3.6 (n = 29) 60.1 days (december – february) rainfall average 175.0 mm temperature average 20.9ºc (13.4ºc 28.1ºc) 10.6 ± 2.4 (n = 233) 30.4 ± 6.2 (n = 144) 19.5 ± 2.9 (n = 169) 60.5 days (march may) rainfall average 190,1 mm average temperature 19.9ºc (11.8ºc 27.5ºc) 13.5 ± 4.0 (n = 60) 34.4 ± 9.4 (n = 37) 20.0±3.9 (n = 82) 67.9 days (june – august) rainfall average 13.3 mm temperature average 18.2ºc (7.0ºc 29.3ºc) 20.9 ± 3.5 (n = 20) 69.7 ± 19.0 (n = 7) 30.3 ± 4.0 (n = 4) 120.9 days mean total duration 11.7 ± 3.7 31.9 ± 10.5 19.8 ± 3.5 table 2 average duration (in days) of the immature stages of mischocyttarus cerberus styx related to the seasons. rc da silva, ds assis, ar de souza, fs nascimento, e giannotti – brood development on an eusocial wasp 306 have eggs and first larvae instar of similar sizes. besides that, the head width dimensions of fifth larval instars and pre-pupae were statistically different, which is supported by the findings of rocha and giannotti (2016). a larger pre-pupal head size might be required for providing sufficient space for the head of the pupa growing below it. duration of the immature stages in relation to monthly temperature and nest phase variations on comparison with the data published earlier on the mischocyttarus spp., we noticed that the larval stage is the one that takes more time to develop (jeanne, 1972; litte, 1977,1979, 1981; dantas-de-araujo, 1980; raposo-filho, 1981; silva, 1984; giannotti & fieri, 1991; michelutti et al., 2014; cecilio et al., 2015). this is perhaps because larval development is affected by many more variables than only temperature, such as food availability, colony cycle and genetic factors (silva, 1984; giannotti & trevisoli, 1993; giannotti & machado, 1994; cecilio et al., 2015). on the other hand, the egg and pupal stages tend to be affected more by temperature throughout their developmental cycle. in other words, these two stages do not exhibit any dependence on the food provided by the foragers (cecilio et al., 2015). the average values of brood development did not differ in most of the studied months (except from june to august), which means that studies involving brood should be avoided during the dry and cold months, because they seem to be more sensible during this period. eggs and pupae seem to require more time to develop in pre-worker emergence nests rather than in post-worker nests, which contradicts with was previously found in other species of the same genus, where eggs develop faster in pre-worker emergence (m. latior: cecílio et al., 2015). this fact might be important for colony survival, taking into consideration that as fast as the brood develop, more adult females would be in the nest to help nest-foundress and less time the foundress would be alone (especially in the cases where only one foundress start the nest) (cecílio et al., 2015). on the other hand, it is possible to believe that it is not a problem for m. cerberus because cases of nest foundress association are also common (noda et al., 2001). in conclusion, the present study confirmed the presence of five larval instars in m. cerberus, which reinforces the concept of five instars as being the standard for the polistinae larvae. we also added more details to the early description given for the mature larvae, by kojima (1998). it is our hope that our data will prove useful for further studies which may involve different approaches. considering that different larval stages were morphologically identified here, chemical analyses of the cuticle appears to be a promising field, not fully explored yet, which can be used to confirm whether the different instars exhibit specific hydrocarbon patterns, so far the only study performed up to this moment was carried by michelutti et al. (2017). this could add another function for these chemical substances, widely used in adult-adult communication. finally, we believe that further studies relating to the differences in types and pattern of the bristles found on the abdominal lobes are required, to clarify whether they may be used as possible taxonomic characters for the mischocyttarus wasps. author’s contribution rcs, ars e eg planed, designed and executed experimental work, rcs, dsa and eg conducted data analyses, rcs, ars, fsn wrote the manuscript. acknowledgments the authors would like to thank an anonymous reviewer for its suggestions and helpful comments in the first version of the manuscript. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001 (rcs and ars) and fundação de amparo à pesquisa do estado de são paulo (fapesp) under grant d.s.a. 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(ed.). academic press, new york, 437 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.3741sociobiology 66(3): 491-499 (september, 2019) litter quality affects termite sheeting production and water infiltration in the soil introduction in terrestrial ecosystems, bioturbation by soil physical engineers (sensu jones et al., 1994) regulate several important ecological processes, such as those influencing soil fertility and water dynamic in soil (lavelle et al., 2006; bottinelli et al., 2015). in temperate and tropical humid ecosystems this role is mainly carried out by earthworms (lavelle et al., 1997). however, in tropical african and asian drylands, termites, and abstract this study aimed to understand the relationship between termite foraging activity and the ecological benefits derived from their activity in soil dynamics and water infiltration. a field study was carried out for six months, between pre-wet and wet seasons, with different food baits (elephant dung, acacia auriculiformis leaves, twigs and leaves of lantana camara as well as ficus religiosa, pterocarpus marsupium, prosopis juliflora, michelia champaca, azadirachta indica and hevea brasiliensis wood stakes) installed on the soil surface in a semi-deciduous forest in southern india. at the end of the experiment we determined bait consumption, water infiltration rate in soil, and the amount of soil sheetings covering the different baits. the initial infiltration rates under the baits were compared to those at the end of the experiment. three termite species, odontotermes obesus, o. feae and microtermes obesi, were found associated with some of the baits in the study area. among the different baits, elephant dung and acacia leaves were the most preferred and a positive relationship was observed between the quantity of soil sheetings and the bait consumption rate. termite preference for elephant dung and acacia leaves was also associated with higher water infiltration rates. however, this difference was only significant at the beginning of the experiment and no significant difference was measured once the steady state was reached. in conclusion, we showed that resource quality was of primary importance for soil sheeting production but that the influence of termites on water infiltration remained limited, most likely because of the low stability of their tunnels in the soil. sociobiology an international journal on social insects rr shanbhag1,2, a harit3,4, s cheik3,5,6, e chaudhary3,6, n bottinelli5,7, r sundararaj2, p jouquet3,5 article history edited by og desouza, ufv, brazil received 16 september 2018 initial acceptance 21 december 2018 final acceptance 23 march 2019 publication date 14 november 2019 keywords bait consumption, odontotermes spp., india, forest. corresponding author pascal jouquet umr iees, fest team 32 av. h. varagnat 93143 bondy, france e-mail: pascal.jouquet@ird.fr especially fungus-growing termites (macrotermitinae subfamily) are the key bioturbation agents (jouquet et al., 2016). in these environments, they directly or indirectly influence the availability of nutrient and water resources to “subordinate organisms” (sensu jouquet et al., 2006) from microorganisms to plants (e.g., wood & sands, 1978; schaefer & whitford, 1981; konaté et al., 1999; harry et al., 2001; jouquet et al., 2005; bignell, 2006; sileshi et al., 2010). the influence of termites on ecosystem functioning relies on their ability to 1 indian plywood industries research and training institute, bangalore, karnataka, india 2 institute of wood science and technology, bangalore, karnataka, india 3 indo-french cell for water science (ifcws), civil engineering department, indian institute of science, bangalore, karnataka, india 4 school of environmental sciences, mahatma gandhi university, kottayam, kerala, india 5 institute of ecology and environmental sciences (umr 242 iees paris, upmc, cnrs, ird, inra, upec, univ. p. diderot), institute of research for development, bondy, france 6 center for ecological sciences, indian institute of science, bangalore, karnataka, india 7 soils and fertilizers research institute (sfri), hanoi, vietnam research article termites rr shanbhag et al. – influence of termite foraging activity on soil functioning492 act as key decomposers of organic residues (collins, 1981; freyman et al., 2008, 2010; fatondji et al., 2009) and in the formation of biostructures (termite mounds and sheetings) and biopores (tunnels and below-ground chambers) which have specific physical, chemical and biological properties differing from the surrounding soil environment (holt & lepage, 2000; jouquet et al., 2011). termite mounds are often conspicuous features of tropical landscapes and, for this reason, their specific properties and abundance have been extensively studied (e.g., moe et al., 2009; horiuchi et al., 2014; joseph et al., 2014; sujada et al., 2014; jouquet et al., 2017; shanbhag et al., 2017; muvengwi et al., 2018). comparatively, the specific properties and abundance of soil sheetings have received much less attention, although it has been suggested that they represent several tons of soil ha-1 year-1 in some situations (rouland et al., 2003; ali et al., 2013; harit et al., 2017a). termites also influence soil functioning through the formation of foraging galleries which increase soil porosity (e.g., lee & wood, 1971; bottinelli et al., 2015) and consequently water infiltration (elkins et al., 1986; mando et al., 1996, 1999; léonard & rajot, 2001; mettrop et al., 2013) and water storage (lobry & conacher, 1990; ouédraogo et al., 2006; pringle et al., 2010). as a consequence, this impact of termites on soil porosity and water dynamics makes them important agents of agro-ecosystem functioning (mando & miedema, 1997; evans et al., 2011; jouquet et al., 2018). termites are considered “intended” or “extended soil engineers” since they manipulate soil functioning to protect themselves against desiccation and predators, to maintain a micro-climate within their constructions and/or to favor the growth of the vegetation they prefer to consume (jouquet et al., 2006). the close relationship between the ecological impacts of termites and their ecological needs has mainly been demonstrated in the context of termite nest constructions (mounds and fungus-growing chambers) (dangerfield et al., 1998; jouquet et al., 2002, 2003; turner, 2004). however, termites also adapt the amount and the properties of their sheetings depending on substrate physical and chemical properties and feeding preferences, with relatively long-lasting structures being most different to the parent soil material (harit et al., 2017b). for example, non-fungus growing termites prefer mammalian dung over alternative food items because of its higher c/n ratio (freyman et al., 2008) and consequently use a larger amount of soil to cover dung pats than plant litter (herrick & lal, 1996). as another example, a positive relationship was also measured in senegal between the quantity of sheetings and the amount of substrate consumed by fungusgrowing termites as well as the number of pores on the surface (rouland et al., 2003). consequently, the relationship between food preference, bioturbation (i.e., the formation of sheeting and galleries) and water infiltration in soil remains unexplored, especially in asia where the influence of fungus-growing termites on soil dynamic is poorly documented (jouquet et al., 2016). therefore, the aim of this study was to examine the links between termite feeding preferences and the production of sheetings and galleries in a tropical forest in southern india. we then investigated the relationship between resource quality, termite foraging activity and water infiltration in soil. our hypotheses were that (i) the more termites prefer a type of resource and the more they produce sheetings above-ground and galleries below-ground, and (ii) that higher resource consumption is associated to greater water infiltration in soil. materials and methods study site and experimental design the study was carried out in a park at the institute of wood science and technology (iwst), bangalore (13.0112° n, 77.5702° e) from february to august 2016. the study site is dominated by teak trees, which represent one of the major types of tree plantation in south india. the climate in the area is semi-arid with an average annual rainfall of 900 mm year-1, a mean annual temperature of 23.6 °c and a wet (july-october), a dry (november-april) and pre-wet season (may-june) (jouquet et al., 2015; cheik et al., 2018). situated at an elevation of 900 to 1000 m above sea level, the soil in the area is described as luvisol by fao (soil survey staff, 2014). the soil is reddish brown in color and has a loamy texture with ~19 and 36% of clay and sand, respectively, and a bulk density of ~ 0.9 g cm-3 and it is characterized by a good drainage (soil survey staff, 2014). it is moderately acidic with low organic carbon content (~1.3%) (cheik et al., 2018). the experiment consisted in the utilization of organic baits that were placed on the soil surface to attract termites. ten different substrates were used. elephant dung (‘ed’) was chosen for its attractiveness because termites are often observed below elephant dung in south indian forests (chaudhary and jouquet, com. pers.). lantana camara twigs (‘lt’) and leaves (‘ll’) were also used because lantana sp. is known to have secondary metabolites which act as feeding deterrents for termites (sousa & costa, 2012). this plant is also an invasive species in india and how termites participate to its degradation remains unknown (ramaswami & sukumar, 2014) and therefore an important question in this environment. acacia auriculiformis leaves (‘ac’) and different wood stakes (ficus religiosa (‘fi’), michelia champaca (‘mi’), hevea brasiliensis (‘hb’), prosopis juliflora (‘pj’), pterocarpus marsupium (‘pt’) and azadirachta indica (‘az’)) were also used because they constituted gradients of high quality resources, with higher n contents in leaves than in wood, and less to high density wood materials (d ~ 0.39, 0.43, 0.53, 0.63, 0.67 and 0.93 g cm-3, respectively) (shanbhag & sundararaj, 2013a,b). recently produced ed (< 2 days), recently fallen and nondecayed ac collected on the ground and cut pieces of lantana (lt, ~10 cm in length, and ll) were air-dried during 10 days before being placed on the soil surface while wood stakes (15 cm in length x 3.5 x 3.5 cm) were dried at 80 °c during 2 days. sociobiology 66(3): 491-499 (september, 2019) 493 all baits were weighed before the experiment (~200 g bait1 in average). since termite mounds were not visible in the study field, substrates were randomly distributed with a minimum distance between bait ~ 2 m. baits were also covered by plastic boxes (23 cm long x 17 cm wide x 8 cm height) performed with 2 mm holes on their sides. boxes were covered on their top to protect them from the rain but they were open on the ground to allow soil fauna to have access to the baits. five replicates were used per treatment and 5 boxes without baits were also installed on the soil surface for the control treatment. biodiversity and litter consumption at the end of the experiment, all baits were carefully removed. baits with termite activity were recorded. all active soil macrofauna under the baits but above the soil surface, including termites, were sampled and kept in vials containing 80% alcohol and further identified at the species or taxon level. soil fauna diversity was described by the number of species and morpho-species (taxon richness, r) and the shannon index (h’). soil sheetings covering the substrates were collected and placed in separate plastic bags along with the residual organic bait. both sheetings and bait materials, except wood samples, were dried at room temperature (~30°c) until they reached a constant weight, and then weighed and expressed as g soil sheeting g bait-1. wood stakes where dried at 80°c for two days. termite feeding preferences were evaluated by comparing the weight of the baits before and after the experiment. values were then converted to percentage weight loss. the amounts of soil translocated over the individual baits were also calculated. soil physical properties water infiltration rates were measured under the baits following the beerkan method (haverkamp et al., 1994; braud et al., 2005). a cylinder (diam 11 cm) was positioned at the soil surface and inserted to a depth of 1 cm to prevent lateral water loss. a fixed volume of water (100 ml, corresponding to a water depth of 1 cm) was initially poured into the cylinder, and the time needed for the water to infiltrate was recorded. as soon as the first volume had completely infiltrated, another equal volume of water was added to the cylinder and the time for this volume to infiltrate (cumulative time) was recorded. the procedure was repeated until steady state conditions were reached. a distinction was made between the infiltration rate at the beginning and end of the experiment. the first three recordings were considered as starting points and the average infiltration rate of the last three readings were considered for the last points. undisturbed soil cores were also sampled in the surrounding top-soil environment (0-5 cm depth) to confirm that initial water content, θi (m 3 m−3), was similar for all the treatments. the soil in the center of the cylinder was also sampled using a smaller cylinder (5.7 diam x 5 cm high). samples were weighed humid and after drying at 110°c for 48 hours. these values were used to measure soil bulk density and gravimetric water content at saturation. statistical analyses all statistics were calculated using r studio and r version 3.2.1. (r development core team, 2008). residues were tested for normality using the shapiro-wilk test and the homogeneity of variance was tested using levene’s test. a oneway analysis of variance (anova) was performed on normally distributed data sets and differences between means were tested with fischer least significant (lsd) tests. data were analyzed using kruskal-wallis chi-squared tests when residues failed to show normal distribution even after log-transformation. differences were considered significant at p < 0.05. results soil biodiversity the influence of the feeding baits on soil macrofauna biodiversity is shown in table 1. no significant difference in taxon richness and shannon index could be measured (kruskal-wallis test, p = 0.235 and 0.388 for r and h’, respectively). three termite species were found (odontotermes obesus, o. feae and microtermes obesi) in ac and ed but with a low occurrence (table 2). along with termites many different organisms were also found below all the baits, such as pseudo scorpions (n = 66), centipedes (n = 23), coleoptera (n = 16), as well as ants (n = 3), millipedes (n = 2) and snails (n = 2). bait preferences and soil sheeting production the bait consumption rates highlighted a clear preference for ed and to a lesser extent for leaves (no significant difference between ac and ll, p = 0.219), table 1. diversity indices (taxon richness r and shannon index h’) of the soil macrofauna for each treatment (elephant dung = ed, acacia auriculiformis leaves = ac, lantana camara twigs = lt and leaves = ll, and wood stakes of ficus religiosa = fi, pterocarpus marsupium = pt, prosopis juliflora = pj, michelia champaca = mi, azadirachta indica = az and hevea brasiliensis = hb). values in parenthesis are standard deviations (n = 5). treatments control ed ac ll lt fi pt pj mi az hb r 0.40 (0.55) 1.20 (0.84) 1.60 (1.14) 1.60 (0.55) 1.20 (0.84) 0.80 (0.84) 0.60 (0.55) 0.60 (0.89) 0.60 (0.89) 0.60 (0.55) 0.60 (0.89) h’ 0.00 (0.00) 0.27 (0.37) 0.36 (0.39) 0.28 (0.27) 0.21 (0.30) 0.14 (0.31) 0.00 (0.00) 0.14 (0.31) 0.14 (0.31) 0.00 (0.00) 0.14 (0.31) rr shanbhag et al. – influence of termite foraging activity on soil functioning494 that were consumed at a rate of 98.8% and 58%, respectively (anova, f9,40 = 23.34, p < 0.001) (figure 1). conversely, the amounts of bait consumed were very low for the different wood substrates (no significant difference between fi, pt, pj, mi, az and hb, p > 0.05). the consumption rates of lt were intermediate between those of ll and ac on one side and those for the wood substrates on the other side (p < 0.05 in all cases). the quantity of soil sheetings also varied between substrates (kruskal-wallis chi squared = 34.33, df = 9, p < 0.001; figure 2). ed, lt and ll were covered with 2.18 g soil g bait-1, while the wood substrates were covered with only 0.09 g soil g bait-1. intermediate values were measured for ac with 0.9 g soil g bait-1 (p > 0.05 in all cases, except with ed where p = 0.041). a positive relationship was found between the total amount of soil sheetings (in grams) and the bait consumption rate (in %) (y = 9.69 x; r² = 0.75; p < 0.001; figure 3). table 2. soil macrofauna (total number of individuals or occurrence for termites and ants) found on the soil surface and below the baits at the end of the experiment. baits were elephant dung (‘ed’), acacia auriculiformis leaves (‘ac’), lantana camara twigs (‘lt’) and leaves (‘ll’), and wood stakes of ficus religiosa (‘fi’), pterocarpus marsupium (‘pt’), prosopis juliflora (‘pj’), michelia champaca (‘mi’), azadirachta indica (‘az’) and hevea brasiliensis (‘hb’) and in the surrounding soil without bait (ctrl). taxon meanings: isoptera (sp.1 = odontotermes obesus; sp.2 = o. feae; sp.3 = microtermes obesi). coleoptera (sp1 = platypodidae sp.; sp.2 = bostrychidae sp.; sp.3 = cerambycidae sp.). hymenoptera (sp.1 = camponotus pennsylvanicus; sp.2 = monomorium minimum). diplopoda (sp.1 = millipedes). chilopoda (sp.1 = centipedes). arachinida (sp.1 = cheliferoidea sp.). mollusca (sp.1 = snail). number of individuals or occurrence of soil macrofauna isoptera coleoptera hymenoptera diplopoda chilopoda arachinida mollusca bait types sp.1 sp.2 sp.3 sp.1 sp.2 sp.3 sp. 1 sp.2 sp.1 sp.1 sp.1 sp.1 ed 1 3 1 6 ac 1 1 2 2 14 lt 1 1 3 9 ll 2 1 2 37 fi 3 2 2 pt 2 pj 2 1 1 mi 1 1 az 1 2 hb 2 1 2 3 ctrl 2 figure 1. baits preference and consumption by soil macrofauna, in % weight loss of the initial bait materials (elephant dung (‘ed’), acacia auriculiformis leaves (‘ac’), lantana camara twigs (‘lt’) and leaves (‘ll’), wood stakes of ficus religiosa (‘fi’), pterocarpus marsupium (‘pt’), prosopis juliflora (‘pj’), michelia champaca (‘mi’), azadirachta indica (‘az’) and hevea brasiliensis (‘hb’)). vertical bars are standard errors. histograms with the same letter are not significantly different at p = 0.05 (n = 5). figure 2. amount of soil sheetings (in grams) produced by termites on the different bait types (see legend of figure 1 for more details concerning the treatments). vertical bars are standard errors. histograms with the same letter are not significantly different at p = 0.05 (n = 5). sociobiology 66(3): 491-499 (september, 2019) 495 soil belowground properties termite activity did not have a significant effect on soil bulk density (0.93 g cm-3 in average; anova test, f10,44 = 1.47, p = 0.183) or soil moisture at saturation (f10,44 = 0.79, p = 0.637). however, water infiltration below the baits during the three first consecutive trials showed significant differences between treatments (f10,43 = 6.54, p < 0.001, figure 4). the highest infiltration rate was measured for ed with an infiltration rate of 26.8 ml sec-1 (p < 0.05 in all cases). no difference occurred between the other substrates with an average water infiltration rate of 4.5 ml sec-1 (p > 0.05 in all cases), except for ac with intermediate average values of 11.3 ml sec-1. figures 5a, b show that the infiltration rate at the beginning of the experiment was linearly related to the consumption rate and the amount of soil sheetings (y = 0.18 x + 3.02, r² = 0.46, p < 0.001; y = 0.01 x + 4.52, r² = 0.55, p < 0.001, for the consumption rate and the quantity of soil sheeting, respectively). however, the baits did not significantly influence the steady state infiltration rate (~ 0.96 ml sec-1 on average; kruskal-wallis chi-squared test = 8.76, df = 10, p = 0.555). figure 3. relationship between termite feeding preference (in % bait consumption) and the amount of soil translocated (in grams). see legend of figure 1 for more details concerning the treatments. the dashed line corresponds to the linear regression. figure 4. water infiltration rates of the soil below of the baits (ml water sec-1) at the beginning of the beerkan experiment. vertical bars are standard errors. histograms with the same letter are not significantly different at p = 0.05 (n = 5). see legend of figure 1 for more details concerning the treatments. figure 5a, b. relationships between water infiltration rate of the soil below of the baits at the beginning of the beerkan experiment (in mm sec-1) and soil macrofauna feeding preference (in % bait consumption) or the amount of soil sheetings (in kg soil). linear regressions are displayed. discussion impact of the baits on soil biodiversity in total, three termite species were identified (table 2). along with termites, several soil macrofauna were observed below the baits. some could be associated with litter degradation (e.g., snails, millipedes) while others were predators (e.g., ants, chilopods). unfortunately, due to the overall low number of soil macrofauna individuals and number of repetition (n = 5) we could not determine if some of the baits were more attractive than others (i.e. no significant rr shanbhag et al. – influence of termite foraging activity on soil functioning496 difference in r and h’). in addition, termites were not sampled with most the baits, although the presence of sheetings showed that ed, ac, ll and lt baits were attractive to them. since samples were collected only once after a period of six months, it is also possible that other termite species and/or other soil invertebrates were also involved in earlier decomposition stages of the baits but were no longer active when samples were collected (sundararaj et al., 2015). similarly, six months is a short period for wood decomposition (van geffen et al., 2010) and it is also possible that other termite species would have been found associated with wood baits if the experiment had lasted longer. termite feeding preference and soil translocation termites produce sheetings mainly to protect themselves from direct sunlight and predation while they forage (jouquet et al., 2006, 2015; harit et al., 2017b), and to excavate soil to construct below-ground galleries and nest chambers (harit et al., 2017a). termites do not always cover the food material on which they feed with sheetings but in this study a clear relationship was measured between the amount of sheetings produced over the feeding substrate and the amount of substrate that was consumed. this result therefore confirms the study of rouland et al. (2003) who found a positive relationship between the amount of millet cane consumed by termites and the quantity of soil sheetings. it is also in agreement with jouquet et al. (2015) and harit et al. (2017a) who showed that when termites prefer a food they invest more energy in the production of soil sheetings. soil translocation levels were the highest for ed samples since all the elephant dung was covered and filled in with sheetings. termites clearly preferred ed over the other baits, most likely because of its high c and n contents (~40 and 1 % in south indian woodlands, respectively; chaudhary com. pers.) (freymann et al., 2008). ed was also perhaps more attractive to termites because it is a mechanically and biochemically “preprocessed” substrate, after its passage through the elephant gut. indeed, it is likely that its high mass per volume ratio in comparison reduced the energy needed for termites, and their symbiotic fungi, to mechanically and biochemically break it down (he, 2013). termites then preferred feeding on leaves and to a lesser extent twigs but wooden baits were almost left untouched by termites during this experiment. this preference can easily be explained by the fact that leaves are less dense and enriched in n compared with twigs and wood (e.g. 2.4% and 0.62% of n in leaves and wood of acacia, respectively; snowdon et al., 2005). due to the low consumption of wood we could not confirm the results of shanbhag and sundararaj (2013a, b) who showed that odontotermes spp. termites prefer less dense wood over denser wood types. finally, this study also showed that termites can consume lantana camara, which is an invasive species in india where it is becoming a threat to biodiversity (priyanka & joshi, 2013). therefore, this study confirms the key role played by termites in the decomposition of litter and herbivore dung in tropical ecosystems (collins, 1981; freymann et al., 2008). it also suggests that l. camara is likely to be degraded by termites in south indian forests, despite the fact that it contains secondary metabolites that are used for biological control of forest pests, including termites (verma et al., 2009; yuan & hu, 2012). effects of termites on water infiltration in our experiment, the consumption rate and amount of soil sheetings were linearly related to the water infiltration rate at the beginning of the beerkan measurements. it is likely that the higher water infiltration in soil for the ed treatment resulted from the higher foraging activity of termites below ed. hence a larger number and/or diameter of galleries may have been produced in the area, as shown by the greater excavation of soil in the form of sheetings. our study confirmed the higher water infiltration rate associated with termite foraging activity (mando & miedema, 1997; léonard & rajot, 2001; léonard et al., 2004; kaiser et al., 2017). however, this higher water infiltration rate was only significant for the initial stages of the experiment and no significant differences were found in the later stages. consequently, this result suggests that the macropores produced by termites were unstable. it is likely that galleries were initially playing the role of preferential flow paths, improving water infiltration in the soil, but that they did not resist the successive applications of water on the soil and collapsed, leading to similar soil porosity for all the treatments. this scenario would explain why no difference in soil bulk density and soil humidity at saturation was measured at the end of the experiment. it also suggests that termite foraging activity has a rather limited impact on soil porosity after prolonged rains. these conclusions are, however, in contradiction with results found in africa where fungusgrowing termites increased soil porosity and water infiltration and retention (mando & miedema, 1997; konaté et al., 1999; léonard & rajot, 2001; léonard et al., 2004; kaiser et al., 2017). different soil properties between our study site and the study sites in africa could explain these differences. in africa, soils were mainly sandy with a high bulk density (> 1.5-2), while in our study site the soil was characterized by a very low bulk density (~ 0.9, shanbhag, pers. obs.) and a good water drainage, which is a characteristic of luvisol (soil survey staff, 2014). more research is therefore clearly needed to examine how the impact of termites, and especially the stability of their foraging galleries, vary with soil properties. conclusion the aim of this study was to gain a better understanding of the relationship between termite food preferences and the ecological benefits derived from their activity in terms of soil dynamics and water infiltration. a clear relationship was found between termite feeding preference, soil sheeting sociobiology 66(3): 491-499 (september, 2019) 497 production and water infiltration, at least in the initial stages of the experiment. thus, this confirms the need for new data concerning the link between plot-scale termite ecology and their impacts on ecosystem processes. the lack of evidence in the present study that termites affect water infiltration raises questions about the stability of termite foraging galleries and their overall role in regulating soil bulk density in luvisol. therefore, a perspective of this study would be to test the generality of this result in other soil pedological contexts (e.g., in compacted or poorly draining soils). finally, another key conclusion of this study is the ability of termites to consume lantana camara residues, which cannot be eaten by most other forest insects in india. acknowledgements authors are gratefully acknowledging the financial support provided by university grant commission, india (grant no. no. f.15-1/2014-15/pdfwm-2014-15-ge-kar-22426 (sa-ii) dated 30-jan-2015). we also thank the editor and the two anonymous reviewers for their useful suggestions on the draft script. this research was funded by the indo-french cell for water science (lmi ifcws/cefirse, ird) and the french national program ec2co-biohefect “macroflux”. references ali, i.g., sheridan, g., french, j.r.j. & ahmed, b.m. 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(2012). repellent, antifeedant, and toxic activities of lantana camara leaf extract against reticulitermes flavipes (isoptera: rhinotermitidae). journal of economic entomology, 105: 2115-2121. doi: 10.1603/ec12026 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.6246sociobiology 68(3): e6246 (september, 2021) introduction the building of nests by ants is a very costly activity in terms of energy and it has a key role in protecting the colony from predators and weather, as well as facilitating thermal regulation and helping the colony to fight pests and diseases (pie et al., 2004; tschinkel, 2015). in some cases, the nests may have chambers where there are larvae, pupae and eggs. they can be built into plant structures (roots, stem, leaves), on the soil surface, under the litter, or in deeper layers of soil reaching several meters in depth and can be formed of abstract the nests of ants neoponera have chambers that can also be occupied by other species of organisms that can be tenants, visitors or prey. however, few studies have considered the assemblage of the associated species and described their composition. this study aimed to describe the architecture and catalog the visitors and prey species found in neoponera verenae nests. talcum powder was pumped inside eight nests to mark the chambers and tunnels. the nests were then excavated to describe the architecture and obtain measurements of chambers. the associated species encountered in the nests were collected and identified allowing us to obtain new records of visiting (linepithema sp., cryptodesmid millipedes and neotropacarus sp.) and prey taxa (membracids, apid bees and springtails) of n. verenae. generally, nests had a single entrance hole and a depth of up to 42 cm. nest chambers were found with three basic forms, elliptical, hangers and boot. although studies show that this species can occupy abandoned nests of leaf-cutting ants, we found that the nests of n. verenae were more similar to those of ectatomma ants. indeed, we found one of the n. verenae nests was attached to a ectatomma edentatum nest, leading us to suggest that n. verenae may occupy abandoned nests or displace other ants to occupy them. sociobiology an international journal on social insects hugo r moleiro1, adolfo da silva-melo1,2, edilberto giannotti1 article history edited by jacques hubert charles delabie, uesc, brazil received 17 february 2021 initial acceptance 15 june 2021 final acceptance 19 july 2021 publication date 13 august 2021 keywords ants, hypogeic invertebrates, nest construction, predation. corresponding author hugo ribeiro moleiro universidade estadual paulista instituto de biociências, campus de rio claro departamento de zoologia avenida 24-a, 1515, bela vista cep: 13506-900, rio claro-sp, brasil. e-mail: hu.gomoleiro@hotmail.com various construction materials available in the environment (hölldobler & wilson, 1990; silva-melo & giannotti, 2010; römer et al., 2020). nests can also serve as habitat for a variety of animals, both invertebrates (pérez-lachaud & lachaud, 2014) and vertebrates (harris & savage, 2020). the fact that ants often present aggressive behaviors to defend their nests, makes nest interiors a safe location for other invertebrate species to take shelter (laakso & setälä, 1998; hughes et al., 2008). it can be said that many of these tenants, in some way, depend on ants at least during part of their life cycle (hölldobler 1 departamento de zoologia, instituto de biociências, universidade estadual paulista, rio claro, brazil 2 universidade federal do tocantins, campus universitário de araguaína, araguaína, brazil research article ants nest architecture and animals associated with neoponera verenae (forel) (formicidae, ponerinae) hugo r moleiro, adolfo da silva-melo, edilberto giannotti – nest architecture and associated animals with n. verenae2 & wilson, 1990). however, the relationships between the species that live inside these nests are not always harmonious. these habitats can present a complex network of interactions between tenants and those with hosts, which may include predations and parasitism (parmentier et al., 2016). currently, there are few studies showing which species of animals live with ants inside their nests (pérez-lachaud & lachaud, 2014; castaño-meneses et al., 2019; rocha et al., 2020; mota filho et al., 2021). some of these studies are limited to certain groups of animals such as mites (uppstrom & klompen, 2011), coleoptera (päivinen et al., 2002) and myriapoda (stoev & lapeva-gjonova, 2005). ponerinae is a subfamily with a variety of nest architectures and nesting sites, being able to live from the subsoil (silva-melo & giannotti, 2010; guimarães et al., 2018) to the canopy of forests (longino & nadkarni, 1990; camargo & oliveira, 2012). neoponera is a genus from this subfamily that often have nests located in hollow or decaying logs, healthy trees, epiphytes or underground (araujo et al., 2019). in general, they are simple nests, with few chambers, little branched and preventing the organization of complex structures of food storage and maintenance of immature (antonialli-junior et al., 2015). although some species may have nests with up to thousands of workers (leal & oliveira, 1995), most neoponera species have nests that rarely surpass 200 individuals (gobin et al., 2003; yagound et al., 2017). for this reason, it is expected to find fewer (although equally important and poorly understood) visitor species in comparison with those found in larger nests of other genera such as the formica (härkönen & sorvari, 2014). this difference is thought to be a result of the larger nests ants presenting a greater variety of microhabitats allowing more species to obtain shelter inside than small nest (wilson, 1971). species of the neoponera prey on small arthropods, although they may also act as scavengers (wild, 2005). they also feed on seed elaiosomes and can have role as seed dispersers when transporting them to their nests (horvitz, 1981). they have also been observed carrying liquid between the mandibles (hölldobler, 1985) and visiting extrafloral nectaries (byk & del-claro, 2010). many species of this genus are specialized in termite predation (mill, 1984; leal & oliveira, 1995), although there are also more generalist species in relation to the type of prey (lachaud et al., 1984, fresneau, 1985). neoponera verenae (forel) is a species that can be found from southern mexico to paraguay occupying a variety of habitats, from rain forests to fields and pastures (wild, 2005). however, almost all the information in the literature regarding this species appears under neoponera obscuricornis (emery) (wild, 2005), which for some time has generated a few uncertainties about the characteristics of these species, particularly in central and south america. previous studies have recorded this species occupying nests in tree trunks (traniello & hölldobler, 1984; araujo et al., 2019), small rotting branches (yagound et al., 2017), plant roots and clumps of grass in pastures (wild, 2005). however, there has been no detailed analysis of the architecture of these nests. n. verenae is a species limited to making only slight modifications in natural cavities or dens built by other animals to build their nests, however, due to the space limitations that this strategy entails, n. verenae often performs migrations in search of new nest sites (pezon et al., 2005). this study aimed to describe some architectural characteristics of n. verenae nests, such as the number of chambers, their dimensions, shapes and spatial arrangement, depth, number and diameter of the entrance holes, in addition to cataloging the visitors and prey species found in them. materials and methods eight n. verenae nests were collected from the campus of the são paulo state university (unesp), city of rio claro, são paulo (22° 23' 40" s/ 47° 32' 44" w). the nests were found within an area covering about 30 ha, where excavations were allowed without interfering with other research areas on the campus. in addition, locations were selected where it was possible to visually locate the workers that were foraging. some of these nests were already known from previous observations by the authors. other were located by searching grassy and wooded places where biscuits and sausage were spread to attract workers of the species. they were followed to the entrance of the nest, which was then marked with colored ribbons until the day of excavation of the nest. confirmation of the identification of the species took place after collecting the workers observed, prior to the excavation date. the identified workers are deposited in the entomological collection of the department of biodiversity (formerly the department of zoology) of the biosciences institute, in unesp – rio claro campus. nest excavation followed the methodology proposed by antonialli-junior and giannotti (1997; 2001), as also used by vieira et al. (2007) and silva-melo and giannotti (2010). only one nest (nv1) was filled with epoxy cement following tschinkel (2010). neutral talcum powder was pumped with the aid of a duster into the main entrance of the nests. dusters are commonly used to pump poison powder into the nests of atta and acromyrmex species and the same dusters were also effective for pumping neutral talc into the nests of n. verenae. this strategy facilitated the visualization of tunnels and how they are connected to the chambers (caldato et al., 2016). to describe the nest architecture, we cleared around the main entrance to remove the soil cylinder where the nest was found. after isolating this cylinder, it was carefully excavated in vertical layers from the edges to the center of the cylinder with the use of a sharp trowel, until reaching the nest chambers. in this way, it was possible to prevent possible organisms present in the vicinity of the nest from mixing with those inside. if it was noticed that the nest continued to a depth greater than that initially excavated, the process was resumed sociobiology 68(3): e6246 (september, 2021) 3 to a greater depth. the excavation was completed when it was noted that there were no more deep chambers or tunnels. during the excavation process, we measured the height, width and length of the chambers as well as the diameter of the inlet and/or outlet (antonialli-junior & giannotti, 1997; 2001; caldato et al., 2016) and we made a sketch, in scale, considering the shape and arrangement of the chambers and tunnels of each nest. in this study, we counted the n. verenae population in all nests except the nv1 and nv4 nests because in the first we used epoxy cement for the mold and ants were not collected from the second. the prey and visiting species were collected from the tunnels and chambers of the analyzed nests. were defined as prey the individuals of other species that were found paralyzed or disjointed, as well as their parts, in the nest chambers. as visitors were considered the organisms of other species that were found inside the nest and seemed not to have suffered any type of predation or attack. the visitor and prey species encountered in the nests were collected and identified. results the depth of n. verenae nests ranged between 6 cm and 42 cm ( = 25.125 cm, sd = 12.845 cm; n = 8). there was a single entrance/exit in all except for one nest (nv2) that had three holes. the diameter of the entrance holes ranged from 0.7 to 3.0 cm ( = 1.437 cm, sd = 0.773 cm; n = 8) (table 2). the nv1 nest is shaped like a boot and for presenting a single chamber, in a low depth and relatively small when compared to the chambers found in the other nests, it was deduced that it was in the initial stages of development. before being excavated this nest was filled with epoxy cement, which provided a three-dimensional mold (fig 1a). the nv2 nest was the only one with three entrances/ exits. the first chamber had two branches, one leading to another two chambers and the second leads to a single chamber. pupae were found just after the entrances to these lateral holes, they were also found in the chambers ii, iii and iv. larvae were observed in the chambers i, ii and iv. prey remains were found in chamber iv (table 2, fig 1b). the nv3 nest had at its entrance a vertical tunnel leading to two chambers. chamber i has an elliptical structure, whereas chamber ii is boot shaped and was constructed next to a root (fig 1c). the nv4 nest had an entrance/exit hole with a short tunnel that branches into two chambers lying on opposite sides, forming a hanger shape, with one side (chamber ii) being closest to the surface (fig 1d). the nv5 nest has a single entrance hole with a vertical tunnel leading to a chamber with a division and a branch leading to another two chambers. we did not record any tunnel connecting chamber ii. chamber iii is a large ellipsoid and at the base has a connection tunnel that could be for a chamber to be built later (fig 2a). fig 1. neoponera verenae architecture nests. a: nv1, b: nv2, c: nv3, d: nv4. scales 2 cm. the nest nv6 has the largest entrance diameter recorded and is connected to a short tunnel that leads to a boot shaped chamber (i), with larvae and pupae. chamber ii has a triangular fig 2. neoponera verenae's nest architecture. a: nv5, b: nv6, c: nv7, d: nv8. env: entrance to the chambers where neoponera verenae individuals were found. eee: entrance to the chambers where ectatomma edentatum individuals were found. scales 2 cm. hugo r moleiro, adolfo da silva-melo, edilberto giannotti – nest architecture and associated animals with n. verenae4 shape and at the base was earth mixed with dark colored organic matter. at the base of this chamber two tunnels extended vertically until they converge approximately 4 cm away from the chamber. chamber iii is reached after the junction of the tunnels and has the same shape as chamber ii but is smaller. chamber iii was filled with pupae and larvae (fig 2b). the nv7 nest has an entrance hole connected to a wide tunnel that leads to the chamber i and branches towards chambers ii, iii and iv. chamber v was built next to a root and in the center is filled with earth, like a division. chamber vi is a simple tunnel expansion, chamber vii has a slight lateral expansion and viii is slightly rectangular receiving the upper tunnel from the side, which exits the chamber on the opposite side to the chamber ix. waste was stored in chamber ix, which had a lot of decaying organic matter and associated animals (table 2, fig 2c). the nv8 nest, is similar to the hanger shape that was seen in nv4, however it was built next to a root. this nest has a hole with a tunnel leading to a chamber (inv), in the basal portion there were two tunnels, one goes toward the chamber iiinv, the second branches into a smaller chamber (iinv) and an ectatomma edentatum (roger) nest (fig 2d). chamber iiinv of nest nv8 is an expanded region in the vertical tunnel that branches horizontally giving access to the chambers vnv, vinv and viinv, the latter is branched, reaffirming the hanger structure. in this nest pupae were found at the end of the hook-shaped chamber ivnv. the larvae were found in chamber viinv. inside the nests where n. verenae individuals were counted, we found eggs in three of them (nv6, nv7 and nv8;  = 1,67 ± sd = 2,42; n = 6), larvae were present in all nests, except nv3 ( = 11,5 ± sd = 8,31; n = 6), pupae, in turn, were absent only from nv3 and nv5 nests ( = 33,17 ± sd = 30,23; n = 6), while males were only found in nv8 nest ( = 0,33 ± sd = 0,82; n = 6) and workers in all of them ( = 98,5 ± sd = 64,28; n = 6). no queens were found in any sample (table 1). half of the eight nests had associated organisms or fragments thereof, totaling thirteen different taxa, of which seven (53.8%) were classified as prey. these organisms were mostly arthropods but were also found in one of the nests (nv7) mollusks (gastropoda) and nematodes. this was also the nest with the highest number of taxa found. regarding arthropods, five distinct classes of this group were identified, nest egg larva pupa male worker collection date nv2 25 76 54 21/03/2014 nv3 98 01/03/2014 nv5 10 75 03/04/2014 nv6 1 13 31 226 03/04/2014 nv7 6 7 36 61 10/04/2014 nv8 3 14 56 2 77 10/04/2014 table 1. populations of neoponera verenae in six nests. mostly insects (orders hemiptera, hymenoptera, lepidoptera and coleoptera) found at different stages of development; but also, diplopoda, malacostraca (isopoda), entognatha (collembola) and arachnida (acarina) (table 2). discussion nest excavation followed by description and mapping enables the collection and counting of adult ants, their immatures and the other organisms present. this method is advantageous because it enables the collection of live specimens unlike the method used by tschinkel (2010), in which all inhabitants of the nest are killed to obtain the mold. in addition to the ants, other invertebrates were found in the nests of n. verenae. our analysis did not identify the ecological role that each taxon plays within the nest or the importance of ants to their life cycle and survival. however, it was possible distinguish between possible preys and other associated animals. a variety of different animal species have been found associated with the nests of other neoponera species, but some of the records made in this work are new to the group. as is the case with the presence of linepithema sp. in two of the nests collected, milipedes of the family cryptodesmidae, although there are records of presence of individuals of the same order (polydesmida) in nests of other species of ponerinae (castaño-meneses et al., 2019). the presence of nematodes in nests is generally associated with parasitism between them and the ants (poinar, 2012), even though its known that certain species of nematodes can also act as carriers of pathogens (ishaq et al., 2021), or even commensals (maschwitz et al., 2016) on certain ant species. however, records of nematodes in nests of n. verenae have not been found, even if rare records of their presence in nests of neoponera villosa (fabricius) are known (wheeler, 1928). the confirmed association between n. verenae ants and mites of the neotropacarus genus is also an unprecedented record. this genus is known for inhabit the surface of plants from different families (ferla & moraes, 2008; zhang, 2012; berton et al., 2019), which leads us to think that its presence inside a subterranean nest may be of something accidental, although it is impossible to affirm from our observations how these specimens reached this habitat. however, mites from other genera of the astigmata group, to which neotropacarus belongs, have already been found in nests of other ponerinae ants (castaño-meneses et al., 2015), including species of neoponera (araujo et al., 2019). the records of isopoda coexisting as tenants in nests of other species of neoponera (triplehorn & johnson, 2005) and also of other ponerinae ants (almeida & queiroz, 2015; castaño-meneses et al., 2019) are recurrent. the same can be said about the association with mites of the laelapidae family (castaño-meneses et al., 2015; rocha et al., 2020), including the record of this group within nests of n. verenae (lopes et al., 2014). sociobiology 68(3): e6246 (september, 2021) 5 t ab le 2 . c ha ra ct er is tic s of n . v er en ae n es ts a nd a ss oc ia te d an im al s fo un d in si de th em . h = h ei gh t, w = w id th , l = le ng th . n es t c ha m be rs d im en si on s (c m ) to ta l d ep th o f th e ne st s (c m ) e nt ra nc e ho le di am et er (c m ) n um be r of en tr an ce h ol es o rg an is m s pr es en ts in th e ne st i ii ii i iv v v i v ii v ii i ix n v1 h w l 4 1 to 3 2 6 0. 7 1 n v2 h w l 2 3 2 6 5 5 2 3. 5 14 2 3 3 35 1 3 p re y in se ct a: h em ip te ra (m em br ac id ae n ym ph ) n v3 h w l 0. 5 14 6 3. 5 10 4 16 1 1 n v4 h w l 1. 5 to 3 .5 40 3. 5 1. 5 to 2 .5 30 2. 5 11 1. 3 1 n v5 h w l 1. 5 3. 5 3 1. 5 2. 5 1. 5 8 5 12 25 1 1 v is ito r in se ct a: h ym en op te ra : f or m ic id ae (l in ep ith em a sp .) d ip lo po da : p ol yd es m id a (c ry pt od es m id ae ) n v6 h w l 2 to 3 2. 75 3 14 32 14 2. 2 2. 4 1. 5 42 3 1 n v7 h w l 1 3 ? 1 4 3 0. 8 1 ? 0. 8 1. 25 ? 6. 5 2. 5 8 1. 5 1. 5 4 1. 7 1. 5 5 1. 7 4 6 2. 5 2 5 33 2. 2 1 v is ito r n em at od a in se ct a: h ym en op te ra : f or m ic id ae (l in ep ith em a sp .) m al ac os tra ca : i so po da a ra ch ni da : m es os tig m at a (l ae la pi da e) ; sa rc op tif or m es : a ca rid ae (n eo tr op ac ar us s p. ) p re y m ol lu sc a: g as tro po da in se ct a: l ep id op te ra (l ar va e) , c ol eo pt er a (f ra gm en t), h ym en op te ra : u nd et er m in at ed (f ra gm en t); a pi da e (f ra gm en t) e nt og na th a: c ol le m bo la n v8 h w l 3 3. 5 7. 5 1 3 ? 2 2. 75 7 1 6 ? 2 3 2 2 3 6 3 6 7 33 1. 3 1 v is ito r d ip lo po da : p ol yd es m id a (c ry pt od es m id ae ) hugo r moleiro, adolfo da silva-melo, edilberto giannotti – nest architecture and associated animals with n. verenae6 our findings showed a clear lack of specialization in the diet of n. verenae. some of the prey species found inside the nest are similar to those from other neoponera species. lepidoptera larvae have been recorded as prey for n. verenae previously by longino (2010), also serving as food for other neoponera species, as neoponera apicalis (latreille) (fresneau, 1985). n. obscuricornis were observed preying on hemiptera nymphs (sujii et al., 2004), however predation on membracidae is something new, being known only in other groups of poneromorph ants (arias-penna, 2008), the same can be said in relation to the capture of apid bees (ariaspenna, 2008; ostwald et al., 2018) and other hymenopterans as ants (arias-penna, 2008; tofolo et al., 2011). published studies on neoponera nests located in cocoa plantations and on bromeliads have already pointed out the occurrence of springtails living in the nests (triplehorn & johnson, 2005; castaño-meneses et al., 2015; araujo et al., 2019). in addition, there are several records of other species of ponerinae preying on springtails (brandão et al., 2015), but this had not yet been registered for n. verenae. predation on beetles is known for n. apicalis (fresneau, 1985) and foraging ants of the species n. obscuricornis were observed attacking beetles in the brazilian cerrado (byk & del-claro, 2010). the architecture of n. verenae nests is very diverse, but we observed similarities between them. the nests are most often constructed with a single entrance orifice as well as those found by araujo et al. (2019) in cocoa agroforestry plantations – up to 3 cm in diameter, with vertical chambers up to a depth of 42 cm. however, delabie et al. (2008) reported that n. verenae ants can construct nests that may reach up to 150 cm in depth. nv8 has a tunnel between chambers iiinv and vnv that follows the path opened by a root, which may be the result of an opportunistic strategy adopted by the ants to take advantage of existing cracks and openings, thereby saving energy. this fact may explain why the nv8 nest does not present the architectural structures observed in the other nests. this nest was also connected to an ectatomma edentatum (roger) nest on a branch of a blind ending side chamber, which had ten workers, nine pupae and one larva. according to delabie et al. (2008), n. verenae can occupy the abandoned nests of leaf-cutter ants or termites. but when we compare the architectural structures of n. verenae nests with those of some fungi cultivating ants we saw that the nests of n. verenae are not similar to mycocepurus goeldii (forel) and mycocepurus smithii (forel) (rabeling et al., 2007), mycetagroicus inflatus (brandão & mayhé-nunes) (jesovnik et al., 2013), acromyrmex ambiguus (emery) (bollazzi & roces, 2007), and acromyrmex rugosus (smith) (verza et al., 2007), and totally different from atta bisphaerica (forel) (moreira et al., 2004). it is possible that n. verenae use nests built by other species of insects or ants. we believe that the nests of n. verenae are more similar to those of ectatomma planidens (borgmeier) (antonialli-jr & giannotti, 2001), ectatomma brunneum (smith) (lapola et al., 2003) and ectatomma vizottoi (almeida filho) (vieira et al., 2007). this similarity comes from the arrangement of the chambers along the light from the entrance/exit hole, but according to these authors these nests are very deep and have chambers with appendices. as the places where the nests were collected had little or no litter, none of the nests had an epigeic construction pattern, covered by litter, or using decomposing plant material (e.g. trunks, palm leaves or dry fruits) as nest substrate seen in other surveys on n. verenae (delabie et al., 2008; araujo et al., 2019). a further evidence of the plasticity of architecture found for nests of this species and the non-dependence of this type of microhabitat for nesting. the absence of a clearer construction pattern and the fact that one of the nests is attached to an e. edentatum nest leads us to suggest that n. verenae may occupy abandoned nests or even displace other ants to occupy part or all of them. to test this hypothesis, it is interesting to carry out studies that can follow the development of nests of n. verenae and verify the occurrence of more nests of this species attached to nests of other ants, or even of other organisms. acknowledgements the authors thank: dr. fernando josé zara and dr. jacques hubert charles delabie for ant identification, dr. carmem silvia fontanetti christofoletti for the identification of millipedes, dr. fabio akashi hernandes for identifying mites, dr. william fernando antonialli junior for helping to excavate nest nv 4, jaime roberto somera for the preparation of drawings and the anonymous referees for the notes and suggestions. the second author thanks the brazilian council for scientific and technological development for financial assistance. authors’ contribution conceptualization: asm, eg, hrm methodology: hrm, asm, eg validation: asm, hrm, eg formal analysis: asm investigation: hrm, asm, eg resources: eg, asm, hrm data curation: hrm, asm writing-original draft: hrm, asm, eg writing-review & editing: hrm, asm, eg visualization: hrm, asm, eg supervision: eg, asm project administration: eg, asm funding acquisition: asm, eg references almeida, f.s. & queiroz, j.m. 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(2012). neotropacarus bakeri (collyer, 1967) rediscovered (sarcoptiformes: acaridae). systematic and applied acarology, 17: 24 tw-target-text __fieldmark__525_1353340481 __fieldmark__572_3108162155 __fieldmark__527_2216515828 __fieldmark__542_3306811674 __fieldmark__511_1058472212 __fieldmark__2589_2822924566 __fieldmark__442_3339125622 __fieldmark__407_1051378900 __fieldmark__479_2822924566 __fieldmark__513_3012261980 __fieldmark__535_3287479944 __fieldmark__546_3740737740 tw-target-text1 __ddelink__4103_1058472212 __ddelink__1994_2147263316 __ddelink__1961_1421047838 __ddelink__1865_863292364 __ddelink__1957_169739897 doi: 10.13102/sociobiology.v66i3.4266sociobiology 66(3): 394-399 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 emerging vistas of remote sensing tools in pollination studies introduction pollination is the processes of transfer of pollen from the male to female flower being aided by insects as a major share although different agents like wind, water also play their role in pollination. around 35% of pollination process was reported to be directly dependent upon insects for pollination (klein et al., 2007; ollerton et al., 2011). bees are an important group of pollinators playing a vital role in the pollination, fruit and seed set of economically important agricultural crops (kremen et al., 2007). securing these essential and valuable pollination services by the bees is vital to achieve food security, preservation of biodiversity in agro-ecosystems (peh & lewis, 2012; winfree, 2013). bee decline is a global crisis as negatively influenced by indiscriminate usage of pesticides, loss of their natural habitats, spread of pests and pathogens and by invasive species (brown abstract with the growth of information and technology across the globe, remote sensing applications find a place in the ecological studies of pollinators. the utilization of remote sensing tools in understanding the ecosystem services rendered by the bee pollinators is reviewed here. we discussed how radar and radio telemetry techniques help to track individual bees, their foraging behaviour and density in relation to altered phenology of flowering crops in a landscape. satellite imagery tools represent a set of techniques used to assess landscape affected by anthropogenic factors that directly influence pollination ecology of bees. habitat modelling data were generated using tools like moderate resolution imaging spectroradiometer to monitoring invasive bee species that cause a threat to native bee fauna. we explained the utilization of unmanned aerial vehicles to map the floral resources that influence the density and incidence of pollinators. remote sensing tools were used to measure sequences of pollination events. sociobiology an international journal on social insects v krishnasamy1, r sundaraguru2, u amala3 article history edited by kleber del-claro, ufu, brazil received 24 november 2018 initial acceptance 11 june 2019 final acceptance 18 july 2019 publication date 14 november 2019 keywords lidar; pollinators; remote sensing; sensors; unmanned aerial vehicles. corresponding author venkateswaran krishnasamy associate professor, department of electronics and communication cmr institute of technology, 132 aecs layout, itpl main road kundalahalli, bengaluru 560037, india. e-mail: vvenkates05@gmail.com et al., 2009; potts et al., 2010). there is an immense need to conserve the bees for the valuable pollination service and to retain their diversity in native ecosystem. native bees being efficient pollinators with vast majority of them of ground nesting habit making their domestication and conservation a difficult phenomenon (rader et al., 2013). bees being active fliers and rapidly mobile in cropping ecosystem, traditional field observations are cumbersome to study their foraging behaviour in different crops (kimura et al., 2014). remote sensing tools were widely employed for its manless interventions in studying the movement, range and distribution of pollinators and floral traits. remote sensing is defined as the method of measuring an object or phenomenon without having a physical contact through the detection of reflected or emitted electromagnetic energy (mulla, 2013). remote sensing can provide space and time efficient observations and could be widely used in 1 associate professor, ece department, cmr institute of technology, bengaluru, karnataka, india 2 professor & head, ece department, sir. m. visvesvaraya institute of technology, bengaluru, karnataka, india 3 scientist (entomology), division of germplasm conservation and utilization. icar-national bureau of agricultural insect resources, bengaluru, karnataka india review sociobiology 66(3): 394-399 (september, 2019) 395 studying the ecosystem services rendered by the pollinators. this tool is widely employed in mapping vegetation structure, pest and disease monitoring, distribution of invasive or threatened species, measuring and monitoring biodiversity and to assess habitat suitability (dong et al., 2003; diao & wang, 2014). the present paper discusses the application of potential remote sensing tools used in pollinator ecology studies, such as mapping of floral habitats with pollinator abundance, tracking of bees to study invasive species in the target area, quantification of vegetation cover to understand the foraging behaviour of bees and tools used to predict pollination services in crops. radio telemetry in tracking bees the major challenge in utilization of solitary bees is their domestication. the vast majority of solitary bees are ground nesting in nature (vinson and frankie, 1991; cane, 1992; arathi et al.,2017) and identification of their nesting sites is of paramount importance. remote sensing tools were widely used to track the movement pattern of the bees so that their nesting sites and habitats could be well studied for their domestication. social bees were reported to be loaded with rfid tags (radio frequency identification) to study their movement pattern, flight distance, hive entries (streit et al., 2003; van geystelen, 2016). individual native bees could be tagged using sensor tags to identify their foraging area, distance between their habitats and foraging area, peak time of activity of the bees. designing a sensor tag relevant to the individual bee weight is vital to track the events in a foraging day of an individual bee. harmonic radars contain a rectifier circuit which generates an echo that equates to one half of the wavelength transmitted. this circuit will be installed in focal individual to be tracked to study its movement riley et al. 1996 used harmonic radars to track focal bees once they leave their nest to study their flight behaviour. focal bee of bombus terrestris audax focal worker bees were tracked by implanting radar transponder in its thorax and number of flights, total time taken for flight in its life time and distance covered during total flight period (woodgate et al., 2016) was recorded. osborne et al. (1999) used harmonic radars to study the foraging range and flower constancy of bumble bees. radars were reported to be efficient in tracking the flight path of bees over greater distances in an accurate manner (carreck et al., 1999). radio telemetry is a widely employed tool in tracking individual bees to study their foraging distance, floral preference, foraging range, habitat preference etc. vegetation and barriers create shadows areas with which tags are undetectable in radars (chapman et al., 2011). the presence of uneven topography blocks the solar radiation resulting in lower values of reflectance compared to areas unaffected by shadows (giles, 2001). the utilization of radio telemetry tools in tracking the movement of individual insects like carpenter bees, beetles, mormon crickets and migrating dragonflies were reported by researchers (hedin & ranius, 2002; sword et al., 2005; holland et al., 2006). this tool demands greater energy requirement and is widely used in larger sized bees like bumble bees and orchid bees (wikelski et al., 2010). incorporating a transmitter in the body of a small sized bees more than its body mass might affect its flight performance. designing a micro-transmitter suiting to the small body sized bees is the need of the hour so as to track its movement. micro-radio transmitter was attached to a male orchid bee exaerete frontalis to track its movement in a complex and forested environment (wikelski et al., 2010). radio telemetry tags were used to track the spatio-temporal movements of bumble bees, bombus terrestris (hagen et al., 2011). under indian conditions, native nomiinae bee, hoplonomia westwoodi is a buzz pollinator of major solanaceous crops like tomato, brinjal causing enhanced fruit and seed set (amala & shivalingaswamy, 2016). the bee prefers to construct its nest in managed mud pots/flower pots in backyard farms with minimal disturbance of soil (amala & shivalingaswamy, 2018). the nesting sites of h. westwoodi could be propagated in newer areas by splitting the brood from the mother nest and seeding in newer areas for its augmenting its nesting sites (amala et al., unpublished data). identification and tracking its nest to seed in newer areas to increase its abundance might help in enhanced pollination, fruit and seed set of different crops. designing a battery powered sensor tag for small sized native bees like h. westwoodi to track its movement, its nest location might aid in its domestication in artificial trap nests. studying bee foraging behaviour active tools like lidar (light detection and ranging) helps in predicting bee population mapping and migration pattern in response to change in vegetation type with respect to greenness, water content and photoperiod. it can track large scale movement of individuals without observer bias and to measure bee density across time and space (hoffman et al., 2007). vertical looking radars can detect moving bees in flight to assess their size, shape and wing beating frequency (chapman et al., 2011). custom made harmonic radars of less weight were used in bumble bees to understand its flight behaviour (riley, 1989). lidar derived maps correlate tree basal area and tree height to derive tree age and to locate the habitats of cavity nesting bees (martinuzzi et al., 2009). this would help in identifying the habitat suitability for a particular tree for solitary bees for nesting. time series of landscape data can help to understand how crop phenology affects the abundance and diversity of the bee pollinators in a given habitat. the flowering phenology of tropical tree tabebuia guayacan was obtained at broader scales using high spatial resolution satellite imagery which was marked by the higher abundance of small and medium sized bees (sánchez et al., 2011). landscape characterization using satellite imagery tools bees target areas with relatively higher floral abundance as different resources increase their foraging efficiency. the v krishnasamy, r sundaraguru, u amala – applications of remote sensing in pollination studies396 floral density of a landscape was positively correlated with its nesting preference and foraging pattern of the native bees (roulston & goodell, 2011). landscape simplification, heterogeneity and fragmentation as a major reason for the decline in pollinator abundance was reported by boscolo et al. 2017. the two-dimensional images of a landscape cover could be characterized using aerial photography or satellite imagery across spatial as well as temporal scales to assess the suitability of a landscape for pollinator abundance (strand et al., 2008; newton et al., 2009). brosi et al., 2007 reported that an analysis based on satellite imagery and landsat images revealed nil negative impact of small-scale isolation from forest segments over the bee richness and abundance with relatively higher abundance of stingless bees.the landscape data obtained using these tools not only provides information about the bee’s view on a suitability of habitat but also can serve as an advisory tool to the beekeepers to utilize certain areas specifically as foraging sites in a habitat. unmanned aerial vehicles in mapping floral areas diversification of agricultural landscape is vital in preserving native pollinators to protect their ecosystem services (landis, 2017). the amount of wild floral resources, type of floral resources and blooming period were positively correlated with the visits of the pollinators (tuell et al., 2008). quantification of wildflowers in a large area using manual counts is a challenging task but is a prerequisite to develop conservation strategies for bee pollinators. mapping the vegetation cover is a crucial step to implement conservation and restoration programmes (he et al., 2005). low resolution satellite imagery has been used over centuries to map the vegetation and to estimate the floral resources across a wider area (thorp et al., 2011). they are useful in monitoring trends in crop production, monitoring pest and disease incidence but the results are greatly influenced by the prevailing cloud cover (eberhardt et al., 2016). satellite imagery is a passive way of remote sensing that utilizes sensors to measure the energy that is reflected by or emitted from any matter. though satellite imagery tools were used for long term monitoring of an ecosystem, these tools have few disadvantages that affect their output like limited use of optical sensors during cloudy weather conditions, slow data dissemination, high cost of imagery and delayed initial image acquisition in case of non-pointable sensors. the disadvantage of satellite imagery tools was overcome by the use of unmanned aerial vehicles (uavs) borne with low weigh sensors with wireless receivers, small on-board global navigational satellite system (gnss), receivers to provide precise information on vegetation mapping with high resolution (anderson and gaston, 2013; zhang & kovacs, 2012). unmanned aerial vehicles (uav) borne with sensors along with image processing tools aid in identification and classification of floral resources to predict the occurrence and abundance of pollinators (lino et al., 2011). xavier et al. 2018 reported that an uav equipped with digital camera to capture the images of the floral area and the captures images were subjected supervised image classification to study the correlation between the floral counts and pollinator abundance. high spatial and temporal data especially photosynthetic active radiation (par) and leaf area index (lar), greening and browning of vegetation will directly help to assess the preference of bees for foraging in an agricultural landscape (nightingale et al., 2008). based on the par and lar data, the greenness of plants and trees and their blooming status could be assessed over a period of time to ascertain the availability of foraging sources for the bees (goward et al., 1995). greenness of an area is an important factor to study the plant-pollinator interaction. the greenness of target area referred as normalized difference vegetation index (ndvi) data could be quantified using advanced very high-resolution radiometer (avhrr) used simple linear regression slope for each pixel. the data provides information on interannual change rate in green up onset for certain time period which is a crucial data for migratory beekeepers as greenness of an area over a time period directly influence all plant-pollinator interaction (zhang et al., 2007). remote sensing tools in predicting pollination bee visitation although an important tool to measure successful pollination, all the flowers visited by the bees does not guarantee pollination. boff et al. (2018) reported that high visitation rate of oil bees increased the pollination efficiency of couepia uiti in pantanal wetland. quantification of pollination needs visual manual data collection which involves physical human movement amidst crop canopy in an ecosystem. the mobile insects are highly impacted by the human interferences while floral damage could be frequently encountered (eckert et al. 2010; brys & jacquemyn, 2012). remote sensing applications involving automated imaging and classification serves as a viable tool in observing bee visitation enabling better understanding of pollination events (galbraith et al., 2015). terrestrial laser scanning (tls) is a ground based active imaging method that utilizes three dimensional variations of the object by comparing it with the sequential terrestrial laser scans (bauer et al., 2005; rosser et al., 2005). tls system generates algorithms to understand the three-dimensional canopy structureswhich is a measure of vegetation structure and floral patch in a given ecosystem for successful pollination (malhi et al., 2018). multi temporal images using satellite imagery could measure floral density, pollinator occurrence near the target flowers over a time period. this automated imaging of pollinator density could be correlated with flowering phenology to predict bee visitation rates that in turn could be validated using visual pollinator counts (suetsugu & hayamizu, 2014). detection of invasive species invasive exotic bee species pose a potential threat to the native bee fauna by competitive displacement, may sociobiology 66(3): 394-399 (september, 2019) 397 compete for floral resources, spread pathogenic disease amidst the native fauna causing a loss to native diversity of the bees (freitas et al., 2009). invasive bees particularly might compete with native bee fauna for floral resources, thereby disrupting the plant pollinator mutualisms in any ecosystem (dohzono & yokoyama, 2010). remote sensing tools utilize habitat suitability models to follow the invasive species of bees in temporal and spatial scale to understand the impact of invasive species on native bee fauna. tracking of invasive bee species in any ecosystem is vital to prevent the entry and spread of exotic pests and pathogens in the native bee fauna. the spread of infective trypanosomatid pathogen, crithidia bombi in bombus sp. colonies was predicted using spatially explicit model with greater and consistent accuracy by otterstatter and thomson (2008). invasive bee species may compete with nest site requirement of native species and spatially and temporally deplete rewarding resources of native species (stout & morales, 2009). honeybee species, apis mellifera scutellata was introduced from south africa to brazil for an effort for breeding. the introduced bees escaped containment and bred with european honeybees resulted in hybridization. the “hybridized african honeybees (ahb)” were reported to be more aggressive in behaviour (franca et al., 1994) with its range expanded gradually to mexico, california and florida (lazaneo, 2002; szalanski & magnus, 2010; winston, 1992). the further spread and equilibrium range of ahb in united states was studied using satellitederived vegetation phenology data (nightingale et al., 2008). jarnevich et al., (2014) used habitat suitability models utilising vegetation covers captured using moderate resolution imaging spectroradiometer, land surface phenology data to develop vegetation continuous fields (vcf) product to study the distributional range expansion of africanized honeybees. conclusion land-use changes caused by anthropogenic factors attributing to loss of bee pollinators could be easily assessed using remote sensing tools is an unexplored area of research which would help in expansion of bee habitats. sensor tags and micro-transmitters were widely deployed to study the behaviour of large sized bees and other insects (pasquet et al., 2008; wikelski et al., 2006) where there is a need to design suitable remote sensing-based tags to track small or medium sized native buzz pollinating bees like h. westwoodi, amegilla zonata to conserve their habitats and to study their foraging pattern and movement between different crops. there is a vital need for the collaborative research between the pollination ecologists and remote sensing experts to devise suitable tools to monitor pollinators in cropping ecosystems for their conservation and utilization. references amala, u., & shivalingaswamy, t.m. 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(2012). the application of small unmanned aerial systems for precision agriculture: a review. precision agriculture, 13: 693-712. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4909sociobiology 67(2): 213-222 (june, 2020) introduction myrmecologists have developed many ingenious sampling methods to collect ants (garcía-martínez et al., 2018; lutinski et al., 2013; oliveira-santos et al., 2009; pacheco & vasconcelos, 2012; smith & tschinkel, 2009), and most of the collecting efforts have the objective of testing ecological questions (gotelli et al., 2011). however, because each sampling method can only record a portion of the community, even though they are used in the same space and time, the sampling method itself acts as a filter (lee et al., 2019). indeed, concerns about the choice of sampling methods abstract a key factor to study ants is the choice of an appropriate sampling method since distinct sampling methods can capture distinct ant fauna and, therefore, leading to bias in the interpretation and conclusion of the patterns observed. despite it is well known that the ant fauna is vertically stratified, some of the sampling methods cannot be used throughout the whole vertical stratum (e.g., fogging and winkler extractor). here, we compared and evaluated the complementarity of distinct sampling methods in ant surveys on canopy and forest floor in a tropical rainforest of mexico. sampling in 10 trees in canopy and on the forest floor around the trees using two baits (tuna and honey) and a standardized hand collecting, we found a total of 44 ant species, belonging to 17 genera and five subfamilies. ant species composition was different among sampling methods at both vertical strata. besides, hand collecting yielded higher ant species richness and more exclusive species than either bait at both vertical strata, but both tuna and honey baits also led to the detection of some, though fewer, exclusive ant species. the combination of hand collecting, tuna, and honey baits should thus be considered complementary tools for ant inventories, since using the three methods together yielded more complete inventories at both vertical strata. all methods tested here can be used on distinct vertical strata, ensuring that data in different vertical strata are comparable, allowing more reliable comparisons among these different habitats, i.e., vertical stratification. sociobiology an international journal on social insects r antoniazzi1, da ahuatzin1, j pelayo-martínez2, l ortiz-lozada2, m leponce3,4, w dáttilo1 article history edited by kleber del-claro, ufu, brazil received 10 december 2019 initial acceptance 16 january 2020 final acceptance 11 april 2020 publication date 30 june 2020 keywords canopy, ant survey, sampling method, arboreal ants, ground-dwelling ants, epigeic ants. corresponding author wesley dáttilo, reuber antoniazzi red de ecoetología instituto de ecología a.c. xalapa, veracruz, mexico. e-mail: wesley.dattilo@inecol.mx reuberjunior@gmail.com deserve special attention in the literature, and these works provide valuable information to be known before starting sampling (gotelli et al., 2011; lee et al., 2019; souza et al., 2016). specifically, comparisons between distinct sampling methods should help researchers to evaluate the trade-offs between sampling completeness and the costs and time required (souza et al., 2012). also, the choice of sampling method should be adapted to the scientific question under consideration to avoid bias in the interpretation of results and conclusions (lee et al., 2019). therefore, understanding the shortcomings and advantages of each sampling method is important for myrmecological investigations (gotelli et al., 2011). 1 red de ecoetología, instituto de ecología a.c., xalapa, veracruz, mexico 2 servicios especializados en estudios integrales ambientales, xalapa, mexico 3 biodiversity monitoring & assessment, royal belgian institute of natural sciences 4 evolutionary biology & ecology, université libre de bruxelles, brussels, belgium research article ants on the effectiveness of hand collection to complement baits when studying ant vertical stratification in tropical rainforests r antoniazzi, d ahuatzin; j pelayo-martínez; l ortiz-lozada; m leponce; w dáttilo – comprehensive ant sampling methods 214 hand collecting is one of the sampling methods that has been recognized as efficient to sample ants, often used to capture arboreal (adams et al., 2017; yanoviak & kaspari, 2000) and ground-dwelling ants (bestelmeyer et al., 2000; longino et al., 2002). this is one of the primeval sampling method to capture insects, requiring little equipment, as forceps or aspirators (packard, 1873). hand collecting can be more efficient for recording ants than most other sampling methods, including pitfall traps or baits (gotelli et al., 2011) and is often at least, if not more efficient than other sampling methods (ellison et al., 2007; pisarski et al., 1982; romero & jaffe, 1989). to obtain comparable samples, it is recommended that the ant survey be standardized in space and time (gotelli et al., 2011; kaspari, 1996). while comparisons between hand collecting and other sampling methods have received attention in the literature (gotelli et al., 2011), these comparisons are limited to a few habitats and circumstances; for example, among vertical strata. distinct vertical strata in tropical forests present differences in their abiotic conditions and, hence, in biotic communities that occupy these habitats (brühl et al., 1998; shaw, 2004). for example, ant assemblages from different vertical strata differ in their nutrient restrictions; ground assemblages have less access to carbohydrates, while canopy assemblages are more limited in protein (brandão et al., 2012; yanoviak & kaspari, 2000). accordingly, the use of honey baits (carbohydrate source), tuna baits (protein source) or a mix of both is widely used to sample ants (bestelmeyer et al., 2000; yanoviak & kaspari, 2000). also, the use of tuna and honey baits separately can affect the composition and abundance of sampled ants even in the same strata. for instance, myrmica species preferred honey whereas formicine genera preferred tuna bait in temperate environments (véle et al., 2009). moreover, it is known that baits are often dominated by few, resource-dominant species, that restrict the presence of subordinate or submissive species, both on the ground and in the canopy (bestelmeyer et al., 2000; camarota et al., 2018; carval et al., 2016). baits are widely used by myrmecologists, especially because they are less demanding in time and cost than other methods, such as pitfall traps or winkler (souza et al., 2012). even though baits may record fewer species than other sampling methods, they can be useful for detecting changes in ant assemblages among habitats (lopes & vasconcelos, 2008). therefore, it has been suggested that baits and hand collecting combined are a highly efficient way to sample ants, for instance, in the canopy (adams et al., 2017; yanoviak & kaspari, 2000). however, no study has tested the efficiency of such combination across vertical strata. many studies have performed intensive efforts to sample ants across vertical strata, from the forest floor to the treetops, i.e., vertical stratification of ants (brühl et al., 1998; wilkie et al., 2010), however, most of these studies use different sampling methods at each vertical stratum. if the ecological question does not involve comparison among vertical strata, but rather detect the total ant diversity, it is better to use as many sampling methods as possible (gotelli et al., 2011). on the other hand, when different sampling methods are used at different vertical strata, it is difficult or impossible to determine whether apparent differences in richness and composition among habitats are due to true differences or to differences in the ant fauna that each sampling method record (gotelli et al., 2011). when it is not possible to use several sampling methods in all habitats to be compared, it is recommended that fewer sampling methods, even if only one, be used in a standardized way across all sampling sites (steiner et al., 2005). accordingly, to compare habitats with distinct conditions, hand collecting is recommended instead, for instance, litter sampling or pitfall traps, as it can be used in sites that have no leaf litter or are rocky, steep or too disturbed by human and domestic animal traffic (gotelli et al., 2011), or on distinct vertical strata. specifically, it is impossible to use, for instance, fogging on the forest floor, due to the inherent limitations of these sampling methods. in contrast, hand collecting and baits are widely used to record ants at all vertical strata (adams et al., 2017; leponce et al., 2019). baits and hand collecting combined are a widely used sampling method to sample ants, especially in the canopy strata (e.g., adams et al., 2017; yanoviak & kaspari, 2000; yanoviak et al., 2008). therefore, it is necessary to use standardized sampling methods to compare ant fauna over space and time (king & porter, 2005; longino et al., 2002; lopes & vasconcelos, 2008; romero & jaffe, 1989; tista & fiedler, 2011), including across vertical strata. most comparisons between sampling methods are concentrated on the forest floor, without considering other vertical strata. here, we aimed to evaluate the effectiveness of the combination of hand collecting, honey bait, and tuna bait in recording ants on the forest floor and in the canopy of a mexican tropical rain forest. we expected that hand collecting would collect more ant species than tuna or honey baits because baits are known to collect few ant species per bait, e.g. due to competition (baccaro et al., 2010; bestelmeyer et al., 2000). unlike bait samples, hand collecting should allow sampling cryptic, submissive, and less abundant ants. we also expected that all sampling methods combined would improve the ant survey, leading to a better description of the ant community (longino & colwell, 1997) spread over distinct vertical strata. our results will provide a framework that emphasizes the reliability of the combination of hand collecting and baits to sample ants on the ground and in the canopy. method study area the study was conducted in a 130 ha fragment of tropical rainforest in the municipality of ixhuatán in the state of veracruz, mexico (18°2’22.99” n, 94°21’27.61” w, 20-60m a.s.l.). the fragment is inside a privately protected sociobiology 67(2): 213-222 (june, 2020) 215 area (área de protección y desarrollo de ceratozamia) established in 2015 by the braskem idesa company, as a management unit for wildlife conservation (retes lópez et al., 2010). the climate of the study area is warm and humid with a mean annual temperature of 27 °c and an annual rainfall of 1,800 mm. half of the vegetation of the fragment is composed of grasslands with isolated trees. the other half is composed of a remnant of secondary lowland tropical rainforest with characteristic species bursera simaruba (l.) sarg. (burseraceae), cecropia obtusifolia bertol. (urticaceae), coccoloba hondurensis lundell (polygonaceae), cupania dentata moc. & sessé ex dc. (sapindaceae), guazuma ulmifolia lam. (malvaceae), miconia argentea (sw.) dc. (melastomataceae) (ortiz-lozada et al., 2017). ant sampling we established four transects, covering most of the forest fragment, that were at least 100 m apart from each other. then, we selected 10 trees, at least 50 m apart, among the tallest and safest to climb. tree crowns (hereafter called “canopy strata”) were accessed using the ‘single rope climbing technique’ (perry, 1978). ants were also sampled on the forest floor around each climbed tree. the sampling in both strata was performed between 09:00 and 16:00 h in may 2016, at a rate of one tree per day (total sampling period= 10 days). we used three widely used sampling methods to collect ants, namely tuna bait, honey bait, and hand collection, to sample ants on the canopy and forest floor (e.g., adams et al., 2017; yanoviak & kaspari, 2000). the tuna and honey baits consisted of approximately one teaspoon of tuna and one teaspoon of honey. tuna and honey were added separately on plastic plates, for the ground samples, and in plastic pots, for the canopy samples. one tuna and one honey bait per tree were placed near the main fork, as high in the canopy as possible (10-15 m above the ground, depending on tree height) and on the forest floor, at the base of each tree. the baits were left for 60-90 min, then collected in individual plastic bags (bestelmeyer et al., 2000). ants were also collected by hand using forceps for 10 minutes at each stratum. in the canopy, ants were collected on the trunk and branches and leaves, and the forest floor, around each tree. all samples were stored in 70% ethanol and transported to the laboratory for sorting, mounting, and identification to the lowest taxonomic level, morphospecies or species when possible. we used a reference collection determined by the colección entomológica iexa (instituto de ecología a.c.), “antweb” (fisher, 2002), and “ants of costa rica” (longino, 2007). data analyses for all analyses, we only focused on comparisons of sampling methods within each vertical stratum, without comparing the canopy and forest floor, following our hypothesis. we performed ant species accumulation curves in relation to our sampling units (i.e., trees) for each method and stratum separately and for all sampling methods combined for each vertical stratum. to do this, we used rarefaction and extrapolation methods (doubling the sample size as suggested by chao et al. (2014)). we implemented these methods in r (r core team, 2017) using the package inext (hsieh et al., 2016). we used generalized linear mixed models, (glmm) (bolker et al., 2009) because our observations were not independent (hurlbert, 1984), to test whether the ant species richness differed among sampling methods, following the data exploration proposed by zuur et al. (2010). we built the structure of the fixed-effect model using ant species richness as a function of the sampling method and we used as the random effect the tree (sampling points, n = 10). we considered each tree (canopy and forest floor) as a random effect because each sampling point would present particular abiotic conditions, and biotic communities, due to the high diversity of organisms in tropical forests, even though in small scales (benson, 1985; dejean et al., 2008). all models followed the poisson distribution and logarithmic link function, frequently used for count data (o’hara & kotze, 2010; zuur & ieno, 2016). we examined the distribution of the model residuals to confirm the appropriateness of the poisson error distribution. to test the differences in species composition among the sampling methods, we performed a generalized linear multivariate model (glmmv) (wang et al., 2012) using a presence-absence data matrix and a binomial error distribution. we performed these analyses using the “mvabund” package (wang et al., 2012), r software (r core team, 2017). results with all sampling methods combined, we recorded 44 ant species belonging to 17 genera and five subfamilies (table 1); 23 species were recorded from the canopy and 37 on the forest floor. the subfamily myrmicinae represented the highest species richness (41%, 18 ant species), followed by pseudomyrmecinae (18%, eight ant species), dolichoderinae (16%, seven ant species), formicinae (14%, six ant species), and ponerinae (11%, five ant species) (table 1). we found that ant species composition differed between the sampling methods (hand collecting, tuna bait, honey bait) both in the canopy (glmmv: deviance = 63.39, p < 0.05) and on the forest floor (glmmv: deviance = 157.52, p < 0.001). using tuna bait, we recorded two and six exclusive species for canopy and forest floor, while using honey bait we found three and one exclusive species. using hand collecting, we recorded 17 and 21 exclusive species for canopy and forest floor (fig 1a-b). in the canopy, the total sampling coverage was 86.6%, hand collecting was 91.4%, tuna bait was 47.8%, and honey bait was 55 % (fig 1c). on the forest floor, the sampling coverage total was 86.6%, hand collecting was 88.2%, tuna bait was 32.6% and honey bait was 69.4% (fig 1d). r antoniazzi, d ahuatzin; j pelayo-martínez; l ortiz-lozada; m leponce; w dáttilo – comprehensive ant sampling methods 216 subfamily species canopy forest floor canopy forest floor hand tuna honey hand tuna honey total total collecting bait bait collecting bait bait dolichoderinae azteca alfari emery, 1893 3 3 azteca forelii emery, 1893 5 1 1 6 1 azteca instabilis (smith, 1862) azteca nigra forel, 1912 2 2 dolichoderus bispinosus (olivier, 1792) 2 9 1 2 10 dolichoderus lutosus (olivier, 1792) 1 1 linepithema sp1 2 2 formicinae camponotus brettesi forel, 1899 5 1 1 6 1 camponotus linnaei forel, 1886 1 1 1 1 camponotus mucronatus emery, 1890 1 2 1 2 camponotus novogranadensis mayr, 1870 1 1 1 4 3 4 camponotus planatus roger, 1863 3 3 nylanderia sp1 1 2 3 myrmicinae carebara sp1 1 1 cephalotes basalis (smith, 1876) 1 1 cephalotes minutus (fabricius, 1804) 2 2 cephalotes scutulatus (smith, 1867) 2 2 2 2 cephalotes umbraculatus (fabricius, 1804) 2 1 2 1 crematogaster curvispinosa mayr, 1862 1 1 1 1 crematogaster torosa mayr, 1870 1 1 nesomyrmex pleuriticus (kempf, 1959) 1 1 pheidole absurda forel, 1886 1 1 1 1 pheidole flavens roger, 1863 2 1 6 9 pheidole punctatissima mayr, 1870 1 1 pheidole simonsi wilson, 2003 1 1 pheidole susannae forel, 1886 2 1 1 4 pheidole sp1 1 1 solenopsis geminata (fabricius, 1804) 3 3 2 8 solenopsis sp1 1 1 trachymyrmex intermedius (forel, 1909) 1 1 2 wasmannia rochai forel, 1912 3 4 2 1 2 1 9 4 ponerinae neoponera carinulata (roger, 1861) 1 1 1 1 neoponera unidentata (mayr, 1862) 2 2 neoponera villosa (fabricius, 1804) 1 1 odontomachus ruginodis smith, 1937 1 1 pachycondyla harpax (fabricius, 1804) 2 2 pseudomyrmecinae pseudomyrmex boopis (roger, 1863) 6 1 7 pseudomyrmex cubaensis (forel, 1901) 1 1 pseudomyrmex elongatulus (dalla torre, 1892) 1 1 5 2 5 pseudomyrmex elongatus (mayr, 1870) 3 3 3 3 pseudomyrmex oculatus (smith, 1855) 1 1 1 1 pseudomyrmex gracilis (fabricius, 1804) 2 3 2 3 pseudomyrmex salvini (forel, 1899) 2 1 3 pseudomyrmex subater (wheeler & mann, 1914) 2 2 2 2 table 1. occurrence of ant species sampled with different sampling methods at the forest floor and canopy level of a fragment of lowland tropical forest, gulf of mexico. sociobiology 67(2): 213-222 (june, 2020) 217 ant species richness was higher using hand collecting (canopy, mean ± sd: 4 ± 2.36, forest floor: 5.2 ± 1.62) than tuna (canopy: 0.9 ± 0.57, forest floor: 1.6 ± 0.70) and honey baits (canopy: 0.3 ± 0.48, forest floor: 1.5 ± 0.85) at both vertical strata (canopy: χ2 = 31.6; df = 2, 26; p < 0.001; forest floor: χ2 = 46.55; df = 2, 26; p < 0.001) (fig 2). discussion we found that hand collecting recorded about four times more ant species in the canopy and on the forest floor than tuna or honey bait. each sampling method recorded different ant species composition, and although tuna and honey baits also recorded some exclusive ant species in both vertical strata, hand collecting was remarkably the sampling method with which most exclusive ant species were recorded. our findings allow us to infer that hand collecting is a suitable sampling method across vertical strata and using it together with baits yields a comprehensive inventory. important ecological questions have been addressed over the past few years using hand collecting in association with tuna and honey baits to sample ants, especially in the canopy strata (e.g., adams et al., 2017; yanoviak & kaspari, 2000; yanoviak et al., 2008). however, it had not been evaluated whether the use of these sampling methods combined increases the efficiency in sampling ants on distinct vertical strata. since we collected during the day on only 10 trees using hand collecting for 10 min on distinct vertical strata in addition to tuna and honey baits, we recorded a satisfactory number of ant species for canopy (23) and forest floor (37) in fig 1. venn diagrams of the comparisons of ant sampling methods in the canopy (a) and on the forest floor (b). sample-size-based rarefaction (solid lines) and extrapolation curves (dashed lines, up to double the sample size) of ant species diversity using different sample methods for the canopy (c) and the forest floor (d) of a tropical rainforest fragment, gulf of mexico. shaded regions represent the 95% confidence intervals. the total sampled ant species are denoted by a black circle sign, while the red circles represent the hand collecting method, the purple circles represent tuna and the green circle honey bait methods. r antoniazzi, d ahuatzin; j pelayo-martínez; l ortiz-lozada; m leponce; w dáttilo – comprehensive ant sampling methods 218 the tropical forest studied. certainly, other sampling methods, e.g. fogging in the canopy (longino et al., 2002) and winkler extractor on the forest floor (gotelli et al., 2011), may be more effective for capturing ant fauna in that habitat. however, fogging is time-consuming both in the field and the laboratory (adis et al., 1984), and it is difficult to determine from which vertical stratum the sampled ant came from, besides that it is not equally efficient in sampling all canopy microhabitats (e.g. ant fauna living in epiphytes; yanoviak et al., 2003). similarly, the use of the winkler extractor to sample grounddwelling ants represents an increase in cost and time compared to other sampling methods (souza et al., 2012). therefore, we have validated the combination of hand collecting, tuna, and honey baits to capture ants and to test ecological questions, such as vertical stratification of ants. sampling by hand collecting in the canopy is not always an easy task since it involved climbing (lowman et al., 2012), but the single rope technique, the use of canopy cranes and other methods can circumvent this limitation (basset et al., 2003). here, we found that hand collecting allows us to record most ant species richness in the canopy and on the forest floor, with a high rate of exclusive ant fig 2. box plots of the ant species richness total and per sampling method for canopy (top) and forest floor (bottom). sociobiology 67(2): 213-222 (june, 2020) 219 species, in comparison with tuna or honey baits. also, hand collecting can be easily standardized in terms of time and survey area when carried out by the same collector(s) in all treatments or habitats (gotelli et al., 2011). “collector effect” results from differences among collectors in their sampling efficiency in sampling ants (longino et al., 2002) and can be a serious confounding variable even if the collections were carried out over the same space and time. the collector effect can be accounted for using statistical tools, such as including the identity of the collector in the statistical model as a random effect. thus, it is possible to obtain the effect of the variables of interest in the statistical model independent of the undesirable effect of differences among collectors (crawley, 2013). this is useful because the sampling efficiency of hand collecting increases with more collectors and also more time devoted (morrison, 1996), allowing better comparisons between distinct sampling points (bestelmeyer et al., 2000). thus, the use of hand collecting to collect ants shows many advantages for the ant fauna survey in both vertical strata, mainly when compared to tuna or honey baits. despite we have found more species richness and more exclusive ant species with hand collecting, we also found exclusive ant species on baits. also, species composition was different among sampling methods. thus, the use of baits can improve the ant inventory on the canopy and forest floor. baits often attract only trophic generalists, a significant proportion of ant fauna worldwide (bestelmeyer et al., 2000), allowing to be used in most habitats to sample ants. besides, some factors can lead to sample fewer ant species on baits, such as competition by exclusion among resource dominant ant species (baccaro et al., 2010; ribeiro et al., 2013). in this context, many ant species can first find and occupy the baits, subsequently being removed by the dominant species, due to their most aggressiveness and recruitment rates (davidson, 1998). also, ant species exhibit preferences for carbohydrates or proteins, leading to preferences for distinct bait types, as honey or tuna (yanoviak & kaspari, 2000), being better mixing tuna and honey in one bait type, as widely used by myrmecologists (bestelmeyer et al., 2000; leponce et al., 2019; yanoviak et al., 2007). it is important to highlight that a potential limitation of our study related to the comparison of sampling methods is that the covered area where we searched ants using hand collecting could not be the same area under the influence of bait attractiveness. however, we emphasized here the comparison of such sampling methods in the tree crown and forest floor around these trees, despite the inherent characteristics of each sampling methods, and hence the difficulty in comparing them, even if in the same site, at the same time (lee et al., 2019). although the use of distinct sampling methods can be redundant in the ant fauna obtained from comprehensive surveys, it is known that each of them records certain ant fauna, e.g. winkler captures small ants vs. pitfall traps capture large ants, being complementary (parr & chown, 2001). specifically related to our findings, ant fauna recorded by tuna and honey bait was a subset of hand collecting species. however, these baits also recorded some exclusive ant species, corroborating the potential complementarity of these sampling methods to capture ants in the vertical strata studied. here, we emphasized that hand collecting, tuna, and honey baits combined can be used both in the canopy and on the forest floor, allowing comparisons between distinct vertical strata. besides, baits can be used to record ants in different microhabitats and periods and can provide information related to habitat use and activity patterns by ants on very fine scales (luque & reyeslópez, 2007). also, since the bait technique is widely used in several ant surveys (bestelmeyer et al., 2000), the use of this sampling method allows comparisons among previous studies. ground-based techniques using baits to sample ants on the canopy have been improved in recent years, such as the recently described “arboreal bait line technique” (leponce et al., 2019). this sampling method consists of hoisting the tuna and honey baits using a line strung by slingshot from the ground. using this sampling method it is possible to determine with precision height and vertical strata and, because it uses the same method along the whole vertical strata, data are comparable, though the number of species recorded is limited (for more detail, please see leponce et al., 2019). our results evidenced that baits are reliable to compare the distinct vertical strata, consistent with a previous study that compared ant fauna from distinct habitats using only baits (lopes & vasconcelos, 2008). in short, our results highlight the importance of comprehensive sampling methods, including hand collecting, tuna bait, and honey bait to record ants, on distinct vertical strata. while hand collecting was by far the more efficient sampling method to record ants in both vertical strata, tuna and honey baits also provided some exclusive ant species. finally, it is important to consider appropriate methodologies for answering ecological questions, e.g. using the same sampling methods when the interest is to compare different vertical strata or between any distinct habitats. acknowledgments we are thankful to tatiana joaqui, frederico neves and arleu viana-jr. for revising previous versions of the manuscript and to erick corro, brenda rattoni, pedro luna, felipe aoki, and joão penna for ideas and comments. we are also grateful to josé alberto toto hernández for support in the field. we thank roberto velasco (industrial director), antonio santos souza galvão (sustainability), ana luisa martínez lópez and ana paulina deméneghi calatayud (environment) and braskem idesa for the support provided to carry out this study in the ceratozamia area of protection and development. ra (cvu 771787) and da (cvu 701588) thank consejo nacianal de ciencia y tecnología (conacytmexico), which provided support to their ph.d. r antoniazzi, d ahuatzin; 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(2010). a protocol for data exploration to avoid common statistical problems. methods in ecology and evolution, 1(1), 3–14. doi: 10.1111/ j.2041-210x.2009.00001.x doi: 10.13102/sociobiology.v61i3.286-292sociobiology 61(3): 286-292 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 mutualistic relationships between the shield ant, meranoplus bicolor (guérin–méneville) (hymenoptera: formicidae) and honeydew–producing hemipterans in guava plantation i burikam, d kantha introduction the shield ant, meranoplus bicolor (guérin–méneville) (hymenoptera: formicidae), is a common ground nesting species of the subfamily myrmicinae, and is widely distributed throughout the entire oriental region (schödlh, 1998). the workers not only forage on dead arthropods as scavengers, but also collect honeydew as carbohydrate source from hemipterans, e.g. the cotton aphid, aphis gossypii glover (hemiptera: aphidae) and the striped mealybug, ferrisia virgata (cockerell) (hemiptera: pseudococcidae), in agricultural ecosystem. however, the trophobiotic relationships or mutualism between m. bicolor and honeydew–producing hemipterans are unknown. mutualism between ants and honeydew–producing hemipterans has been identified as a continuum of relationships ranging from mutualistic to antagonistic (stadler & dixon, 2005; billick et al., 2007), and hemipterans tending by abstract mutualistic relationships between the shield ant, meranoplus bicolor (guérin– méneville), and two species of hemipteran, aphis gossypii glover and ferrisia virgata (cockerell), were investigated in an unsprayed guava plot at kamphaeng saen, nakhon pathom, thailand. the reciprocal benefits were observed in both field and laboratory studies. m. bicolor activity coincided with peak seasonal activity of both hemipterans during june–august. we indicated two sets of support evidence in m. bicolor honeydew preference: (i) statistically higher value of adjusted honeydew weight collected by ant workers from a. gossypii compared with that from f. virgata (p–value = .005), and (ii) the higher value of the strength of effect (η2 = .62) in the total variance of multi-species association. the physical property on honeydew viscosity was discussed concerning ant preference. we used two–group, ant–tended and ant–excluded, between–subjects multivariate analysis of variance (manova) in order to show hemipteran benefits. both hemipteran populations increased in the ant–tended treatment, together with lesser amounts of two species of coccinellids, menochilus sexmaculatus (fabricius) and coccinella transversalis fabricius, and one species of syrphid fly, pseudodorus clavatus (fabricius), compared with the ant exclusion treatment (p–value <.001). the facultative mutualistic relationships of m. bicolor and the two hemipteran species were mentioned. sociobiology an international journal on social insects kasetsart university, nakhon pathom, thailand article history edited by jacques h. c. delabie, uesc, brazil received 10 deceber 2013 initial acceptance 19 may 2014 final acceptance 20 july 2014 keywords cotton aphid, striped mealybug, coccinellids, syrphid fly, psidium guajava corresponding author intawat burikam department of entomology faculty of agriculture at kamphaeng saen, kasetsart university nakhon pathom 73140, thailand e–mail: intawat.b@ku.ac.th ants are mostly facultative or opportunistic (delabie, 2001). generally, ants benefit from associations with hemipterans by obtaining carbohydrate–rich food source in the form of “honeydew” secreted from hemipterans (e.g.: nixon, 1951; way, 1963; hölldobler & wilson, 1990). specifically, the benefits to ants have been focused on the foraging behavior of worker ants (stadler & dixon, 2005; grover et al., 2007; kay et al., 2010). some have concentrated on fitness benefits in terms of ant colony growth (grover et al., 2007; helms & vinson, 2008; wilder et al., 2011). in return the benefits, ants may reduce hemipteran contamination of their waste products, removing dead individuals, protecting natural enemies, and transport hemipterans to new feeding sites, resulting in the abundance of hemipteran populations (e.g.: way, 1963; nielsen et al., 2010; stadler & dixon, 2005). ants exploit hemipterans not only for their honeydew, but also as a protein source when foraging on them as a common prey (buckley, 1987; hölldobler & wilson, 1990; delabie, 2001). however, this research article ants sociobiology 61(3): 286-292 (september 2014) 287 type of antagonistic relationships will not be treated here; we are looking at a concrete evidence of mutually benefits among both partners. in this study we verified, in both field and laboratory experiments, the reciprocal benefits of m. bicolor and two species of honeydew–producing hemipterans, a. gossypii and f. virgata. we concentrated for over three–month period in the guava plantation of horticulture department, kasetsart university, nakhon pathom, thailand, observing the mutualism of ant–hemipterans including the abundance of natural enemies, mainly predators. we tested three hypotheses: (i) ants receiving benefits in terms of honeydew from mutualistic associations in guava agroecosystem; (ii) ants protecting hemipterans from natural enemies therefore the densities of natural enemies decrease in the presence of ants; and (iii) in consequence of the two hypotheses mentioned earlier, resulting in the increments of hemipteran densities in ant–hemipteran associations compared with the ant–exclusion arrangement. materials & methods study species the study was conducted during april–december 2012 in the unsprayed varietal collection plots (varieties: phant si thong, kim ju, and vhan pi roon), consisting of 336 guava trees, psidium guajava, of horticulture department, faculty of agriculture at kamphaeng saen, kasetsart university, thailand (14.0358 ºn, 99.9826 ºe). the predominant ground–nesting ant species in the study area was the native m. bicolor, with only a few colonies of the invasive ant species the tropical fire–ant, solenopsis geminata (fabricius) near the perimeter of the plantation. the honeydew–producing hemipterans were a. gossypii and f. virgata. the natural enemies, mainly predators, were two species of coccinellid beetles, menochilus sexmaculatus (fabricius) and coccinella transversalis fabricius (coleoptera: coccinellidae), and one species of syrphid fly, pseudodorus clavatus (fabricius) (diptera: syrphidae). ant benefit and honeydew preference the direct benefit of m. bicolor was obtained by weighing a certain number of foraging ants, and then calculating the difference of weight gain between foragers descending and ascending the guava branches. we measured weight gains of m. bicolor after visiting hemipteran colonies as honeydew receiving. we randomly chose foraging ants from the field to weigh for honeydew loading; 50 ant foragers ascending the guava branch before reaching hemipteran colonies, and the other 50 individuals descending the branch with full load of honeydew. honeydew loads were measured from the weight differences of ants filled with honeydew and ants ascending the guava branch. individual worker of m. bicolor was captured in an empty hard gelatin capsule (size 0; outer diameter 7.65 mm, height 21.7 mm, and volume 0.68 ml of torpac inc., nj), and shortly after, the capsule containing the arrested ant was weighed on a digital balance. the actual ant weight was obtained from the subtraction of the capsule weight. we weighed, from field collected, two sets (n = 200) foraging ants visiting a. gossypii, and one set (n = 100) of ants visiting f. virgata. the honeydew loads were confirmed with the laboratory set up by feeding of m. bicolor workers with honeydew. a set of field collected workers (n = 100) leaving their nests for foraging were randomly chosen, holding in captivity for 24 hours without food, and subsequently captured inside the gelatin capsule for weighing. after weighing, half of the 24–hour arrested m. bicolor was offered with guava leaves occupied by honeydew exudates of a. gossypii, and the other half of ants with honeydew from f. virgata. the ants were allowed to feed on honeydew until they either refused to feed or left the guava leave. all m. bicolor workers were weighed for the second time in order to obtain honeydew loads before releasing back to their former habitats. hemipteran benefits we randomly selected 30 guava trees, age 6 years old, approximately 1.65 m in height and 2.5–2.75 m in diameter from the pesticide–free guava plot as our study units. one of two similar branches was randomly chosen from each selected tree to perform ant–exclusion treatment, using sticky barrier around the base of the branch covering 20 cm in length. the target branch was first wrapped around with plastic wrap, and then applied with generic horticultural glue (colorless and odorless). the objective of the gluey barrier is to prevent ants and other crawling insects from reaching hemipteran colonies at the guava shoots, allowing only the entering of air–borne insects, including winged aphids, mealybug crawlers, ladybugs, and syrphid flies. the barriers were examined periodically, and reapplied the glue as required, in order to maintain the effectiveness as ant barriers throughout the experimental period. the other branch was left unmanipulated as the ant– presence treatment. there was the total of 60 experimental units. this ant–exclusion/presence experiment was started in may, beginning with equal numbers of both a. gossypii and f. virgata between the two treatments on the same guava tree. insect observations were made during peak seasonal activities of both hemipterans and their natural enemies in june– august 2012. on each chosen guava tree, we randomly selected one terminal shoot from the total of 3–5 shoots of each experimental unit, in order to make observations. all terminal shoot belonging to each experimental unit had an equal chance to be picked on each data collection day. the number of hemipterans: a. gossypii, f. virgata; and larvae of predators: m. sexmaculatus, c. transversalis, and p. clavatus, occupying the branch terminal side of 30 cm in length of both presence i burikam, d kantha meranoplus bicolor–hemipteran mutualism in guava288 and absence of m. bicolor were counted at various intervals throughout the duration of the experiment from april–december 2012. we counted the insects at interval of 3–5 days, with the total of 7 times per month during june–august, coincided with the peak activities of both hemipterans and their predators, and every 15 days in other months. however, the observation data or multivariate responses of the five dependent variables were derived from the average of 7 times x 3 months = 21 field observations during peak activities of the insects in june–august 2012. we recorded the number of m. bicolor moving up or down (bidirectional) passed a fixed point on the treatment branch with no gluey barrier for 3–min period, to ascertain ant activity throughout the overall experimental period from april–december 2012. all observations in the field were done during 08:30–11:30 hours. statistical analyses to answer the question on the difference of honeydew weights or ant’s honeydew preference between m. bicolor collecting a. gossypii honeydew compared with those of f. virgata, we used analysis of covariance (ancova) of ibm spss statistics (verma, 2013; meyer et al., 2013). body weight of m. bicolor workers with empty stomach (24–hour unfed workers) or weight before receiving honeydew was treated as covariate, and the criterion variable or dependent variable was ant weight after eating honeydew from each hemipteran species. the analysis of covariance approach was used in order to adjust the initial variations of m. bicolor worker size. the honeydew–producing hemipteran benefits were demonstrated by interference of ants, predominantly m. bicolor, with sticky barrier applying around the base of the main branch in order to exclude the ant. the abundance of hemipterans and natural enemies were compared between presence and absence of m. bicolor. we anticipated more hemipterans and less natural enemies in the ant–attended guava branches. most studies of ant–hemipteran interactions included either ant or hemipteran removals from the study plants, and made comparisons with the unmanipulated partners. the conclusions, in general, relied on statistical analysis by the uses of univariate analysis of variance (anova), which concentrated on one dependent variable, with attempts to make findings from multiple analyses of anova (e.g.: flatt & weisser, 2000; billick et al., 2007; daane et al., 2007; mgocheki & addison, 2009; styrsky & eubanks, 2010). herein we used multivariate analysis of variance (manova) of ibm spss statistics (meyer et al., 2013; rencher & christensen, 2012), in order to draw one solid conclusion of ant–hemipteran mutualism based on the comparison of five dependent variables from two groups, presence and absence of m. bicolor on guava branches. these five dependent variables or multivariate responses were number of insects: i.e. nymphs and adults of a. gossypii; nymphs and adults of f. virgata; larvae of m. sexmaculatus; larvae of c. transversalis; and larvae of p. clavatus. all insect counts were transformed into log (y + 1) format; where y = number of insect, in order to agree with statistical assumptions. several outputs were requested from the manova analysis of ibm spss. box’s test of equality of covariance matrices expected to see if the dependent variable covariance matrices are equal across the levels of the presence–absence of m. bicolor. bartlett’s test of sphericity was demanded to ascertain sufficient correlation between dependent measures in order to proceed with the analysis. the core manova output was inquired for the multivariate null hypothesis evaluation of no differences between presence and absence of m. bicolor on the composite dependent (number of insects) variate. when the multivariate test is statistically significant, we can proceed with some assessments of each dependent variable. we performed the tests of between–subjects effects to evaluate the statistical significance of each dependent variable separately. bonferroni–corrected alpha level was applied to avoid alpha inflation in order to evaluate these presence and absence of m. bicolor effects. we divided .05 by the number of anovas and obtained .05/5 or a bonferroni-corrected alpha level of .01. results and discussion m. bicolor generally foraged on honeydew of hemipterans as carbohydrate source throughout the year in guava plantation at kamphaeng saen. monthly averages (± se) of m. bicolor activity from april–december 2012 are presented in fig 1. m. bicolor activity coincided with population fluctuations of both hemipterans (a. gossypii, and f. virgata), with peaks seasonal activities in june–august (fig 1). there were very high correlation coefficients (r’s) between ant activity and either a. gossypii or f. virgata density at r =.97 (p–value < .001; n = 9) and r = .93 (p–value < .001; n = 9), respectively. m. bicolor dominated the other ground–nesting ant species, solenopsis geminata, in the studied guava plot, although s. geminata has been considered as one of the most invasive ant species worldwide (wetterer, 2011), but not in this guava ecosystem with history of pesticide applications. there were no s. geminata workers observed on the experimental guava trees. the tolerance to pesticides of m. bicolor was probably due to the protection of long fine hair covering the entire body (schödlh, 1998), together with the defensive behavior of meranoplus by curling up the body and feigned dead when disturbed (hölldobler, 1988). ant benefit and honeydew preference in the studied guava plantation, foragers of m. bicolor leaving their nests weighed approximately 2.48 mg (se = .08; n = 150). after visiting hemipteran colonies, m. bicolor with honeydew loaded, descending the branch back to their nests weighed on average 8.69 mg (se = 0.1; n = 150). the sociobiology 61(3): 286-292 (september 2014) 289 honeydew loading is about 6.21 mg (8.69 − 2.48) or roughly estimate around 2.5–fold (6.21 ÷ 2.48) of the mean forager weight departure from their nests. the weighting capacity of m. bicolor workers was reconfirmed in a confined study of laboratory feeding of ant workers to different kinds of honeydew from both hemipteran species. after 24 hours in captivity, m. bicolor workers weighed 3.61 mg (se = 0.12; n = 100) on average. we selected larger workers with more tolerance and easier for seizing in order to withstand the 24– hour starvation before obtaining honeydew. these workers were fully fed with honeydew from different hemipteran species, and later weighed approximately 10.70 mg (se = 0.15; n = 100). the overall expected value of honeydew loading is 7.09 mg (10.70 – 3.61), with an estimate of 2.96–fold (7.09 ÷ 3.61) of the average worker weight after 24 hour in caging. the former 2.5–fold honeydew loading from field foragers was slightly lesser; this was probably due to the offering of honeydew by trophallaxis among workers before returning to their nests (pfeiffer & linsenmair, 2007). one–way between–subjects ancova assessing the difference of honeydew loadings from two hemipteran species of m. bicolor workers showed that the covariate effect or weight of 24–hour captured m. bicolor before honeydew feeding was statistically significant, f (1, 97) = 786.297, p–value < .001. moreover, a statistically significant effect of honeydew source, from either a. gossypii or f. virgata honeydew, was obtained, f (1, 97) = 8.387, p–value = .005. mean weight of ants before eating honeydew of f. virgata group was higher than that of a. gossypii group, leading to higher full up honeydew loading from f. virgata compared with that from a. gossypii (fig 2). however, the use of ancova approach removed the covariate effect and unveiled the reversal outcome. mean weight of m. bicolor plus honeydew from a. gossypii was significantly higher when corrected for weight prior to receiving honeydew (adjusted mean = 10.85; se = .072; 95% ci = 10.707–10.992) than mean weight of worker ant with honeydew loaded from f. virgata (adjusted mean = 10.55; se = .072; 95% ci = 10.408–10.693) (fig 2). this could indicate that m. bicolor workers prefer honeydew from a. gossypii to that from f. virgata. ants are expected to concentrate their honeydew collection activities on hemipteran species offering higher reward in terms of both quantitative and qualitative effects. hemipteran species that produce larger amount of honeydew, or having honeydew with the presence of preferred sugars or amino acids should be more attractive to certain ant species (cushman, 1991; völkl et al., 1999; yao, 2014). ant preference for particular sugars in hemipteran honeydew can be species specific (blüthgen & fiedler, 2004). several ant species react strongly to honeydew that holds large amounts of melezitose (völkl et al., 1999), while others prefer sucrose to melezitose (blüthgen & fiedler, 2004). on the other hand, a. gossypii honeydew consisted of mainly sucrose, fructose, and erlose (lawo et al., 2009), with no appearance of melezitose. honeydew composition of f. virgata is unknown; however, some studies of mealybugs’ honeydew show composition of fructose, glucose, sucrose, and small amounts of melezitose and raffinose, together with a variety of amino acids (gray, 1952; salama & rizk, 1969). another difference in honeydew quality beside the composition of sugars and amino acids is a physical property specific� cally honeydew viscosity. in our study, honeydew excreted by f. virgata was more viscous than that by a. gossypii, which their honeydew seemed to be watery liquid. a study in argentine ant showed that workers fed eightfold longer on gel sucrose composition, and removed fivefold less sucrose than workers feeding on liquid sucrose (silverman & roulston, 2001). the later would agree with lesser amounts of honeydew loading of m. bicolor from f. virgata than that from a. gossypii in this study. fig 1. monthly average of meranoplus bicolor activity (± se, vertical line), and hemipteran densities (ferrisia virgata and aphis gossypii) from 30 ant–tended guava branches at kamphaeng saen, nakhon pathom, thailand in year 2012. fig 2. mean weight of meranoplus bicolor workers (mg) with empty stomach (24–hour without food), mean weight with honeydew loading, and adjusted mean weight from different hemipteran species, aphis gossypii and ferrisia virgata. value on top of column chart indicates data label; different letters followed mean values represent statistically significant differences (p–values ≤ .005). i burikam, d kantha meranoplus bicolor–hemipteran mutualism in guava290 hemipteran benefits a two–group between–subjects manova was done on logarithmic transformed data [log (y + 1); y = observation data] of five dependent variables: no. of a. gossypii; no. of f. virgata; no. of m. sexmaculatus larvae; no. of c. transversalis larvae; and no. of p. clavatus larvae. the independent variable or treatment was the presence-absence of ants, particularly m. bicolor, in guava plantation. there were two treatments, i.e. ant–tended and ant–excluded. in general, the ant–excluded treatment with sticky barrier was quite effective against m. bicolor, the slow–moving ant species. even though some ants could accidentally reach the colonies of hemipterans on the exclusion treatment from adjacent branches due to the contact with nearby branches via wind blowing, however, these ants could not return back to their nests or could not be able to recruit additional ant foragers. the sample consisted of 60 guava branches divided into equal amounts of presence and absence of m. bicolor. the output of box’s test of equality of covariance matrices was statistically significant (box’s m = 69.998; p-value < .001), showing that the dependent variable covariance matrices were not equal across the levels of the presence–absence of m. bicolor. therefore, pillai’s trace was used to evaluate all multivariate effects (meyer et al., 2013). bartlett’s test of sphericity was statistically significant (approximate chicsquare = 99.838; pcvalue < .001), indicating sufficient correlation between the dependent variables to proceed with the manova. using pillai’s trace as the criteria, the combined dependent variable was significantly affected by the presencecabsence of m. bicolor, pillai’s trace = .807, f (5, 54) = 45.244, p-value < .001. there were reliable multivariate differences between ant–tended and ant–excluded treatments on the combined dependent variate. the partial eta squared = .807 (partial η2), equivalent to the full eta squared (η2) in this two–group design (levine & hullett, 2002), indicating that we had a very high proportion of the total variance (.807, or about 81%) explained by the activity of m. bicolor. each dependent measure or each observed insect density was assessed individually in order to determine the strength of the statistically significant multivariate effect. the result of the tests of the univariate effects is shown in table 1. we had statistically significance univariate effects on all dependent variables (table 1; p-values < .001). of all insect species under investigation, a. gossypii provided the highest effect size (η2 = .62), while m. sexmaculatus had highest effect size in terms of natural enemies (η2 = .55) (table 1). the descriptive information for the univariate analysis is presented in fig 3; providing each dependent measure’s observed means, and total averages obtaining from 30 guava trees in the study. the presence of m. bicolor–tended hemipterans had a considerable impact on insect populations not only hemipteran themselves, but also their natural enemies. on ant–tended treatment, we detected higher densities of both hemipteran species, together with lesser amounts of all natural enemies compared with the ant–excluded treatment (fig 3). there was more abundant in density of roughly 7.6–fold [{antilog (1.824) – 1} ÷ {antilog (1.002) – 1} or 68.50 ÷ 9.05 = 7.57] of a. gosypii than f. virgata from 30 guava trees in the study (fig 3). in general, we would say that m. bicolor preferred to associated with a. gossypii more than f. virgata, in this meaning the preference of honeydew collecting, as indicated by higher value of the strength of effect or effect size (levine & hullett, 2002; meyers et al., 2013), i.e. η2 = .62 and η2 = .52, respectively (table 1). this could be the second evidence in supporting the previous study of honeydew preference of 24– hour captured m. bicolor. among the three natural enemies or predators, m. sexmaculatus had more strength of effect (η2 = .55), i.e. would be more effective predator, than the other two competitors (η2s = .39, and .32) in this m. bicolor–hemipteran association (table 1). even though the surphid fly, p. clavatus, was more abundant than the other two coccinellid predators, but its appearance in the guava plot was restrict to june till august. in addition, there were no p. clavatus larvae found preying on the striped mealybug, f. virgata, in our study. the mutualistic relationships or trophobiotic interactions between either a. gossypii or f. virgata with ants have been classified as facultative and very common phenomenon by delabie (2001). there are two main reasons from this study in supporting the above mentioned: firstly, both hemipteran species are polyphagous and cosmopolitan species (blackman & eastop, 2000; da silva-torres et al., 2013), any mutualistic relationship with ants should be opportunistic or facultative rather than obligatory; and secondly, m. bicolor is the most common species of the genus meranoplus in the oriental region (schödlh, 1998), and is widely distributed as ground nesting species in disturbed habitats of agricultural fig 3. effect of meranoplus bicolor-exclusion on hemipterans (aphis gossypii and ferrisia virgata) and their natural enemies (menochilus sexmaculatus, coccinella transversalis and pseudodorus clavatus). different letters on top of dark columns represent statistically significant differences (p–values < .001); numbers on top of clear columns indicate data labels of total average or grand mean from 30 guava trees. sociobiology 61(3): 286-292 (september 2014) 291 ecosystem, therefore the acquiring for food in the vicinity should be by selection of most abundant resources. this study showed that ant attending had a considerable effect on hemipterous pest densities in guava plantation. there were more individuals of hemipterans in the ant tending guava branches, together with lesser amounts of natural enemies mainly predators because of ant guarding activities. in return the benefit, ants received carbohydrate sources in terms of honeydew from both hemipterans. in general, the results of this ant exclusion experiment using gluey barrier are agreed with previous studies done in fruit orchards; e.g. cherry (stutz & schmidt-entling, 2011), apple (stewart-jones et al., 2008; miñarro et al., 2010; nagy et al., 2013); and in vineyards (mgocheki & addison, 2009). in conclusion, mutualistic relationships between m. bicolor and honeydew–producing hemipterans were revealed. m. bicolor preferably collected honeydew of a. gossypii more than that of f. virgata, because it was not only easier to find, i.e. more abundant, but also more ingestible, due to the physical property of watery liquid. other alluring properties of honeydew to ants could be honeydew composition in terms of sugars and amino acids, which needed further investigations. the ant–exclusion promoted an increment in predator densities, and thus leading to a tentatively conservation biological control of hemipterous pests in guava agroecosystem. acknowledgment we thank horticulture department of the faculty of agriculture at kampkaeng saen, kasetsart university, particularly unaroj boonprakob and kriengsak thaipong, for their support in field experiment. financial support was partially offered to junior author by the graduate school, kasetsart university. references billick, i., hammer, s., reithel, j.s. & abbot, p. 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(2014). costs and constraints in aphid–ant mutualism. ecological research, 29:383–391. doi:10.1007/s11284014-1151-4 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.4610sociobiology 68(1): e-4610 (march, 2021) the need for ecological indicators the need for organisms that rapidly indicate the complex anthropogenic habitat transformations is decisive in this fastchanging world. indicators are living organisms that are easily monitored and whose status reflects or predicts the conditions of the environment where they are found (landres et al., 1988; siddig et al., 2016). indicators can be separated into three categories corresponding to their main applications: (i) environmental, when responding to e.g. chemical compounds; (ii) ecological, related to disturbances such as habitat fragmentation, and (iii) as biodiversity estimators (mcgeoch, 1998). a particular species can be considered an indicator, but taxa hierarchically placed above species are preferable because no single species should be expected to indicate an entire ecosystem condition, and also because many factors unrelated to the degradation of ecological integrity may affect the population status of an indicator species (carignan & abstract orchid bees have been considered as good ecological indicators of habitat disturbances but recently añino et al. (2019. sociobiology, 66: 194-197) highlighted reasons why the euglossini role as indicators should be reevaluated. despite agreeing with some points raised by them, we present an alternative view for the use of orchid bees as indicators. for us, the main problematic issues are: (i) the authors do not present a clear definition of ecological indicator, including its role as an indirect measure of the biota response to disturbed environments; (ii) they do not properly acknowledge the relative good taxonomic status of orchid bees when compared to the remaining bees; (iii) and they do not distinguish the use of particular euglossini species as indicators in certain circumstances. in spite of some knowledge gaps, we argue that euglossini are good candidates to be ecological indicators in tropical forests, maybe the best candidates among all the bees. sociobiology an international journal on social insects rodrigo barbosa gonçalves1, luiz roberto ribeiro faria2 article history edited by gilberto m. m. santos, uefs, brazil received 19 july 2019 initial acceptance 25 september 2020 final acceptance 25 september 2020 publication date 22 february 2021 keywords bioindicator, fragmentation, pollinator, neotropics, wild bees. corresponding author rodrigo barbosa gonçalves departamento de zoologia universidade federal do paraná cx. postal 19020, cep 81531-980 curitiba, paraná, brasil. e‑mail: goncalvesrb@gmail.com villeard, 2002). several groups of invertebrates and vertebrates have been proposed as indicators based on established selection frameworks or on other justifications (siddig et al., 2016). the criteria for selection of indicators have been extensively discussed in several papers and rely on the particular objective and scale of the measured disturbance (landres et al., 1988; mcgeoch, 1998; carignan & villeard, 2002; siddig et al., 2016). reyes-novelo et al. (2009) compiled the seven most common criteria for selection of ecological indicators and discussed their application to bees. the authors conclude that wild bees can be used as indicators, but further validation studies are necessary. independently of the selection framework, bees have been used as ecological indicators especially for being a keystone group due to its main role as pollinator of wild and crop plants (kevan, 1999; garibaldi et al., 2013). the public concern about the decline of bees is increasing worldwide and the group can be considered as a flagship for conservation (fortel et al., 2014). 1 universidade federal do paraná, departamento de zoologia, curitiba-pr, brazil 2 universidade federal da integração latino-americana, instituto latino-americano de ciências da vida e da natureza, foz do iguaçu-pr, brazil opinion in euglossini we trust as ecological indicators: a reply to añino et al. (2019) rb gonçalves, lrr faria – euglossini as ecological indicators2 a particular group of corbiculate bees, the orchid bees (tribe euglossini), have been used as ecological indicators in the neotropical region. there are about 230 species of euglossini, distributed among five monophyletic genera (moure et al., 2007; ramírez et al., 2010). this group occurs from southern united states to argentina, but most of its diversity is concentrated in neotropical forests (dressler, 1982; roubik & hanson, 2004). much of the existing knowledge on these bees, especially that generated by structured inventories, was enhanced by the discovery and use of synthetic chemical compounds to attract adult males (dodson et al., 1969). the justifications for the use of euglossini as indicators are missing but can rely on their responses to fragmentation (e.g. allen et al., 2019) and also on the relative good knowledge of their biology, taxonomy and distribution when compared with bees as a whole (allen et al., 2019). still, the presence and relative abundance of particular species such as eufriesea violacea (blanchard, 1840), euglossa marianae nemésio, 2011 and eulaema nigrita lepeletier, 1841 have been considered as indicative of habitat quality (e.g. tonhasca et al., 2002; nemésio, 2009; faria & melo, 2012; gonçalves et al., 2014). recently, añino et al. (2019) highlighted some reasons why euglossini should be reevaluated as indicators, based on criticisms contextualized under the seven established selection criteria reviewed by reyes-novelo et al. (2009). here we argue against some aspects of their argumentation, concluding that this tribe meets the criteria to be considered as ecological indicator. the seven selection criteria for ecological indicator applied to euglossini 1. the taxonomy of the group must be well known and stable so that the species can be identified reliably. añino et al. (2019) mention the lack of taxonomical security for the female identification of most species as the main problem, an argument also raised for criterion 2. in fact, euglossini taxonomy is based almost entirely on males since they are consistently more abundant on museum collections (e.g. nemésio, 2012a). this male dependency is caused by the use of chemicals for attracting and sampling individuals, an important aspect regarding the easiness of sampling (criterion 4). as virtually all euglossini sampling protocols are based on chemical compounds to attract males, the ability to identify females is not necessary to the use of these bees as ecological indicators according to criteria 1, 4 and 6. in other words, euglossini males are the candidates to be ecological indicators. on the other hand, orchid bees nesting biology and flower preference relies on female identification and the emergence of males is currently necessary to a proper species identification. in the case of euglossine males, identification keys are available for all the four genera, besides aglae lepeletier & serville that is monospecific. the most intimidating scenario regards the genus euglossa latreille (ca. 120 species) that lacks a comprehensive revision. however, the situation is not the same for all the subgenus and species groups and some of them could almost be considered as “solved”, mainly in some particular regions. we believe the best scenario is for the brazilian atlantic forest, whose entire fauna was studied in an extensive monograph (nemésio, 2009), besides robust regional identification keys (e.g. nemésio, 2011) and taxonomic papers focusing on particular species group of euglossa (e.g. faria & melo, 2012; nemésio, 2012b). these considerations are even more relevant when we consider that using some euglossine groups as indicators is much more feasible than using others (see criterion 4 and 6). so, the dependence of taxonomic keys would diminish in these specific taxa, making this problem much more treatable. finally, even with all the existing caveats on euglossine taxonomy, only a few other bee tribes (e.g. anthidiini, centridini, emphorini, eucerini, meliponini, tapinotaspidini) have such a good taxonomic knowledge accumulated to the neotropical region as a whole, but other selection criteria were not fully addressed to these groups, especially criterion 6. 2. biology and lifestyle must be well known. lifestyles are rather uniform within bee tribes, however euglossini comprizes solitary, communal, primitively eusocial nest builders and also cleptoparasite species (michener, 2007). as pointed out by añino et al., studies on several aspects of biology are lacking for most euglossine species. this is true, but it is not a particularity of these bees since this is an expected pattern for most invertebrates. again, the comparison with the scenario for other tribes requires to realize that the biology of euglossini can be considered as “well known”, as, for example, the observation that about 10% of its species were recorded and studied with trap-nests (costa & gonçalves, 2019). few other bee tribes have higher percentage of studied species. still, there is a significant amount of information on the plants these bees visit for pollen, nectar, resin and aromatic compounds, besides nesting biology (including information on parasites), distribution of species along altitudinal gradients, etc. (synthesized mainly by dressler, 1982; ramírez et al., 2002; cameron, 2004; roubik & hanson, 2004). an important issue (see criterion 1) regards that establishing meaningful links of the information on natural history for males and females critically depend on the correct identification of females (see nemesio, 2012a). but even with this caveat, when existing biological data for euglossini is compared with information for other bee tribes in the neotropical region, perhaps only centridini and meliponini have similar overall knowledge. 3. the group should be composed of well-defined and rich trophic guild that should be important in the structure and functioning of ecosystems. as for the bees as a whole, euglossini is a well-defined trophic guild of pollinators and matches very well with this criterion. the particular plant hosts for most species are unknown and we agree with añino and colleagues that more attention should be given to this subject. however, we do not agree that the ability of some euglossine to thrive in the absence of its orchid mutualists (pemberton & wheeler, 2006) hampers their classification as sociobiology 68(1): e-4610 (march, 2021) 3 indicators, especially according to this particular criterion of selection. after all, how particular is this observation? and even though euglossine bees could succeed without orchids, they will need to visit plants to collect other resources. also, a trophic narrow specialization is not necessarily a requisite to fit in this criterion. the relation of orchid bees and orchids are usually misunderstood and the importance of orchid bees as pollinators and/or flower visitors cannot be neglected. as pointed out by roubik and hanson (2004), orchid bees have been recorded foraging for nectar on flowers in 51 plant families, but how specific these interactions are among bees and plants have not been examined in detail. studies focusing on the foraging behavior of some euglossine species have also revealed that a significative number of plants are visited by these bees. a high diversity of plants, 50 pollen types belonging to 20 botanical families, was used by females of euglossa cordata (linnaeus, 1758) for provisioning their brood cells (ferreiracaliman et al., 2018). these authors also discussed that even if their study have been carried out in an area where invasive plants were abundant, euglossa cordata preferred to forage on native plants to collect floral resources. silva et al. (2012) recorded that the flora visited by eulaema nigrita is compound by 40 species from 19 botanical families, even if the species exhibited some preference for plants with poricidal anthers. and it should be highlighted that bees were collected during the flowering period of passiflora alata, the main source of nectar for this bee where the study was carried out. expressive numbers were also found for euglossa annectans dressler, 1982 (74 pollen types from 24 families, during a three-year fieldwork; cortopassi-laurino et al., 2009) and euglossa atroveneta dressler, 1978 (74 plant species detected in larval provisions of 51 nests during a single year; arriaga & hernández, 1998), in studies based on samples of larval provisions. all these data would allow the conclusion that euglossa females may act as pollinators of many forest species (cortopassi-laurino et al., 2009). when pollen is collected from males, results are also relevant. in a study carried out in the chocó region, ospina-torres et al. (2015) gathered pollen grains attached to the body of 352 males of at least 22 euglossine species, and found that these males were associated to 84 plant species, but dependent of a small group of them. they also discussed a nested pattern in this relation, as several specialists bees and plants have interacted with a small group of generalists (both bees and plants). extreme mutualisms appear not to be the main pattern on plant-pollinator interactions, as interactions seem to be much more asymmetrical (e.g. olesen & jordano, 2002). some particular evidence from euglossine x euglossophilous plants also suggests so (e.g. borrell, 2005). and all the matter on orchid bees becomes even more important if we consider that this tribe comprises 20-30% of the total bee species in various neotropical forests (see roubik & hanson, 2004). we believe that studies focusing on the plants visited by those species with greater potential to indication (see criterion 6) should be prioritized, as a way to attack this impediment in a faster and more targeted manner. 4. organisms should be easily captured, manipulated and observed; the study of the group should not jeopardize its conservation. it was noted by añino and collaborators that the chemical compounds facilitate the collection of male orchid bees, a practical resource especially in rapid assessments of indication programs. the relative easiness to sample males should be considered as a positive aspect that enables the use of the group as indicator despite the uncertainty about females frequency (see nemesio, 2012a). bees as a whole are sampled in flowers with hand-nets or caught with bowl traps, a hard task in tropical forests, and none alternative bee group has a particular sampling method so efficient as the scents are for euglossines. the hand-netting (an active sampling method) and baittraps (a passive method) are two methods for capture, and while the first is considered more effective (nemésio & vasconcelos, 2014), traps allow a higher number of replications, which maximizes the sampling effort (knoll & penatti, 2012). the sufficient replication is a guideline to biological sampling in environmental licensing studies (ferraz, 2012), favoring the use of bait-traps. the highlighted problem with hand-netting is the bias caused by the skills of the collector, an issue that can be avoided by a training phase (but see discussion on nemésio, 2012a). the attraction of orchid bees to baits, allied to the overall size of these bees, makes the sampling easier when compared to hand-netting bees in flowers under active search. in the case of bait-traps, sydney and gonçalves (2015: p. 35) concluded that “alternative baited trap designs do not have much influence on the richness and abundance parameters (except for the landing platform), thus making most studies directly comparable”. still, performance of baittraps should be replicated in other landscapes for validating the prediction of sydney and gonçalves (2015). with regards on collection jeopardizing euglossine conservation, there are no studies focusing on the actual impacts of samplings on the populations of orchid bees (nemésio, 2012a). 5. the geographical distribution of the group should be broad, including different habitats, allowing the use of a variety of experimental designs and comparisons. orchid bees present a broad geographical distribution along the new world, occurring from southern united states to argentina (michener, 2007). the southern limit of their distribution is not so well defined, with a record of a species of euglossa near buenos aires (martín garcía island; roigalsina, 2008). regarding the western limits in southern america, trans-andean distributions seem to be restricted to lower latitudes (e.g. dick et al., 2004), but the extension of geographical distributions within genus and species widely varies (see roubik & hanson, 2004). we conclude that the distribution of orchid bees is broad enough to consider this criterion as applicable to the group. however, it is important to highlight that the abundance and richness of euglossini is greater on tropical forests of central and south america than rb gonçalves, lrr faria – euglossini as ecological indicators4 on the northern and the southern portions of their distribution (michener, 2007), restricting where they (and which species) can be effectively sampled in indication studies. we agree with añino et al. (2019) that understanding the distribution of orchid bees is a complex matter. but, again, in a comparative way: are there so many other groups of bees with so predictable distributions? is this a reason to set aside any possibility of using bees or orchid bees as indicators? we believe that one of the main questions in añino and colleagues’ argumentation, the lack of definition of a specific scale and context for indication, is particularly important for this criterion. one species, regarded as a promising indicator in a specific context, is not necessarily an indicator in other. previous authors agreed that the spatial scale should be always evaluated when selecting an indicator (carignan & villeard, 2002; siddig et al., 2016). in the case of particular species indicating disturbances, a simple example comes from euglossa fimbriata moure, 1968. within the atlantic forest this species could be assumed to be rare in the deep interior of the forest (nemésio, 2009), suggesting a kind of negative relation with forested areas. on the other hand, in a different context, the brazilian cerrado, the species shows a clear tendency to be positive related to the savanna proportion in the landscape (moreira et al., 2017). even if these statements could both suggest a general relation with more open areas, the potential of the species as indicators is clearly different in these two specific contexts. considering the spatial scale for indication, it could also help to define how broad a species distribution has to be for being a useful indicator. for spatially restricted contexts, species with restricted distributions could even be more appropriate. we also point that, whenever possible, eventual shifts between the historic and the current distribution of species should be considered to state their roles as bioindicators. euglossa marianae could be used as an example, as its historic distribution is regarded to be much broader than the current. old records presented by nemésio (2009) [as euglossa analis], nemésio (2011) as well as faria and melo (2012), state that the species used to occur from southern bahia to northern são paulo state, mostly in litorean areas. several recent inventories carried out within this region (e.g. ramalho et al., 2009), in forest fragments with very distinct sizes, have shown that the species is currently found only in fragments larger than 3,200 ha, with most records from preserved fragments larger than 6,000 ha (see discussion in coswosk et al., 2018). añino and colleagues also claimed the impossibility of accurately defining the distribution ranges of many species as they (i) are not attracted by chemical baits, and (ii) are able to fly long distances to find the baits, moving across different habitats. dealing with the point (i) is much simpler: these species should not be considered in bioindication, in the same way as those presenting a striking geographical structuration in scent preference, and in this latter case, the use of the species should be restricted to the spatial context where this issue is controllable. the point (ii) is knotty, especially if the bioindication uses parameters of euglossine assemblages, as the existence of directional artificial stimuli from scents could homogenize euglossine assemblages (see milet-pinheiro & schlindwein, 2005; ramalho et al., 2013; coswosk et al., 2018). however, the existence of such a directional stimuli could be, in theory, even reinforcing the possibility of using orchid bees as bioindicators. considering the example of euglossa marianae (and also other species from the analis species group) within the brazilian atlantic forest, even though they are able to fly long distances to reach the baits, these bees continue to be strongly associated to the inner sites of large forests (tonhasca et al., 2002; nemésio & silveira, 2006; coswosk et al., 2018). 6. species should tend to specialize in a particular habitat, so that they are sensitive to habitat degradation and regeneration. we believe this is a decisive criterion for indicator selection. añino et al. mentioned two main problems, (i) population stability and rare species hampering the interpretation of the responses of orchid bees, and (ii) studies showing none or positive responses to disturbances. in the case of population stability, the authors cited the study of roubik (2001), conducted in a natural area that was not subject to intense disturbance. but this example can be taken as indicative that populations do not oscillate that high under undisturbed conditions, an important background to orchid bees selection as indicators. it would then be necessary to advocate the realization of studies focusing on the population stability of species in disturbed environments, in order to allow the necessary comparisons, before simply discarding the possibility of using orchid bees as indicators. we agree that rare species individually could not be good indicators of disturbance since their abundance can be expected to naturally fluctuate and they can be expected to disproportionately respond to habitat fragmentation (see ramalho et al., 2013). but in this specific case, using assemblages of several species could tend to minimize the effect the presence of particular species. the unexpected relations between euglossini diversity and fragmentation mentioned by añino et al. should be contextualized. botsch et al. (2017) found that while the abundance and species richness of orchid bees were not different in fragmented and contiguous forest, the community composition and evenness really were, and one of their conclusions was “these results demonstrate the conservation value of continuous forest, given the differences in community composition between continuous forest and fragments, greater community evenness in continuous forest, and a trend toward greater β-diversity” (botsch et al., 2017: p. 639). robust evidence on this matter was recently provided by allen et al. (2019), which stated that euglossine bees can be useful as indicators of the impacts of human disturbance. they even suggested (p. 752) that “orchid bee abundance is a simple measure that can be monitored by conservation managers without the need for much analytical expertise or even species identification. although this should not be used in isolation, it could serve as a useful warning flag of negative impacts of disturbance”. sociobiology 68(1): e-4610 (march, 2021) 5 we also evaluate that añino et al. have not assessed the matter of using particular euglossini species as indicators of disturbation. two examples are eulaema nigrita and eufriesea violacea, if the first is commonly considered as an indicator of habitat perturbation, while the latter is an indicator of habitat quality in fragments of atlantic forest in southern brazil (gonçalves et al., 2014). but it is important to highlight here the question of the spatial scale/ context (see above), as there is no evidence of the effects of habitat disturbance on eulaema nigrita in the brazilian cerrado (silva & de marco jr, 2014). we discussed above the role of euglossa marianae as an indicator of large preserved forests in the brazilian atlantic forest (nemesio, 2009; faria & melo, 2012). this species is still a good indicator in another context, although quite related to fragment size, the edge effects. two studies carried out in large fragments of brazilian atlantic forest (nemésio & silveira, 2006 [as e. analis]; coswosk et al., 2018) brought evidence showing that this species avoids the edge of the forest. additional discussion on other candidates for indication is presented by nemésio (2012a). all these examples have shown that even with their great potential to exploit resources due high fight capacity (pokorny et al., 2015), orchid bees species are able to respond to alterations in habitats. 7. the group must have species with potential economic importance. the main economic importance of bees regards pollination. as far as we know there is no reported case of euglossini as the main pollinator of crop plants, differently from other bee groups (giannini et al., 2015). there are some records on the importance of orchid bees as the main pollinators of brazilian nut (bertholletia excelsa, lecythidaceae) (mori & boeke, 1987; maués et al., 2002), pollinators of mangaba fruits (hancornia speciosa, apocynaceae) (oliveira et al., 2014) and minor pollinators of passion fruit (passiflora spp., passifloraceae) (gaglianone et al., 2014). however, the indirect effect of pollination service on natural systems should not be discarded. the long known extraordinary flight ability of euglossine bees (e.g. wikelski et al., 2010), allied with their trapline foraging behavior (e.g. ackerman et al., 1982), make these bees an important element for promoting outcrossing among plants with natural low densities (janzen, 1971). euglossini are good ecological indicators the selection of an ecological indicator is subject to the study purpose and related parameters such as spatial extent and habitat predictors, and no specific group can be considered a universal indicator (siddig et al., 2016). these parameters are variable under different scenarios and should be stressed before or even after the use of indicators, when interpreting the results. we believe that it is possible to assume that orchid bees collectively match the seven selection criteria and should be considered as ecological indicator of disturbances in tropical forests. the main reasons that support their use are the ease of sampling, established taxonomy and sufficient knowledge of the most general patterns regarding the responses to disturbances. one can argue that these criteria may not be fully addressed by the tribe, but (i) biological knowledge is never to be considered as complete; (ii) not necessarily all criteria should be completely matched; and (iii) the relative knowledge on euglossine bees, when compared to alternative candidates within wild bees, can be a desirable proxy for selection. still, particular species can also be secondarily used to indicate habitat alterations and they could also be subject to the seven selection criteria. for example, taxonomy (criterion 1), that can be problematic in the case of cryptic species and/ or some particular species groups (e.g. the euglossa cordata group). in brazil the installation of enterprises or activity potentially harmful to the environment such as highways, hydropower plant and mining, must undertake environmental licensing with a preparation of environmental studies. the practical choice of indicators deals with the enterprise kind, the preliminary assessment, and the need for informative groups including particular species and higher taxa (ferraz, 2012), and this choice is made by the inspection agencies and the consultant companies. terrestrial invertebrates are important groups to be assessed especially in enterprises regarding vegetation suppression. in amazon and atlantic forests orchid bees have already been used as indicators in such environmental studies together with other groups such as ants and frugivorous butterflies. in the specific case of approval of new dams, empirical evidence suggests that these bees should be considered in environmental studies before approving such enterprises (storck-tonon & peres, 2017). academic researchers should be aware of the pros and cons of the use of the group as ecological indicators since they have the qualifications to be competent consultants in environmental studies (silveira et al., 2010). the points raised by añino et al. (2019) are certainly relevant and we agree with some of them. however, the present rebuttal offered an alternative view for the use of orchid bees as ecological indicators. we wonder to continue the debate they have proposed about the use of orchid bees as ecological indicators and all opinions should be respectfully considered. anyway, the debate is a healthy way to contextualize the existing data and searching for a solid basis for euglossinebased indication, centered in scientific evidence. acknowledgements we thank josé vicente da silva for comments on environmental licensing and indicators choice and léo correia da rocha filho for his comments on the manuscript. contributions of authors rbg and lrrf equally contributed to the arguments presented here and wrote the final version together. rb gonçalves, lrr faria – euglossini as ecological indicators6 references ackerman, j.d. 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(2010). large-range movements of neotropical orchid bees observed via radio telemetry. plos one, 5: e10738. doi: 10.1371/journal.pone.0010738 doi: 10.13102/sociobiology.v67i2.4855sociobiology 67(2): 322-325 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ericrocidini comprise a tribe of cleptoparasite bees composed of 11 genera, mostly with neotropical distribution (snelling & brooks, 1985; moure & melo, 2007). these bees are almost exclusively related to centris fabricius nests (snelling & brooks, 1985; rozen & buchmann, 1990;rochafilho et al., 2009), except mesoplia rufipes (perty) and mesonychium asteria (smith) which have been respectively reported as cleptoparasites (rozen, 1969;hiller & wittmann, 1994; gaglianone, 2005; rocha-filho et al., 2008) and probable cleptoparasites (gaglianone, 2005; rocha-filho et al., 2008) of epicharis klug species. despite the large number of species belonging to this tribe, the associations between cleptoparasites and hosts in this group still poorly known. the genus mesonychium lepeletier and serville, is the second most diverse of ericrocidini, with nine species (moure & melo, 2007). this genus is widespread distributed abstract mesonychium asteria (smith) is a cleptoparasitic bee with occurrence restricted to south america. in this study, we provide new information related to the host association and cleptoparasitic behavior of this species in nests of centris xanthomelaena moure and castro. observations were conducted at the nesting sites of c. xanthomelaena in a caatinga area of pernambuco state, brazil. females of m. asteria were observed performing overflying in nest aggregations and attacking some nests. we have confirmed the cleptoparasitic association with the emergence of adult m. asteria from the host bee nests, and also by the presence of its larva on the brood cells. sociobiology an international journal on social insects hoj martins1, pl oliveira-rebouças2, vs ferreira1 article history edited by evandro nascimento silva, uefs, brazil received 01 november 2019 initial acceptance 07 january 2020 final acceptance 08 january 2020 publication date 30 june 2020 keywords caatinga; centris (paracentris); host association; solitary bee. corresponding author herbeson ovidio de jesus martins universidade federal do vale do são francisco campus de ciências agrárias, colegiado de ciências biológicas, rodovia br 407, km 12 – lote 543 projeto de irrigação senador nilo coelho s/nº c1 cep: 56300-990 petrolina-pe, brasil. e-mail: herbeson.bio@hotmail.com in the high and/or dry regions in south america (michener, 1979; snelling & brooks, 1985; silveira et al., 2002). little is known about the biology of this genus and its association with hosts, with m. asteria and mesonychium jenseni (friese, 1906) the only two species with their host records available in the literature (see rocha-filho et al., 2009 and references therein). from this study, we provide new information related to the cleptoparasitic behavior of m. asteria in nests of centris (paracentris) xanthomelaena moure and castro, a groundnest bee endemic of caatinga. observations on m. asteria were carried out at the nesting sites of c. xanthomelaena in a caatinga area at the campus de ciências agrárias of the universidade federal do vale do são francisco (cca/ univasf) (9°19’44.2”s, 40°33’30.1”w), petrolina, pernambuco state, brazil. the climate is dry and hot semiarid (bsh according to köpen 1 universidade federal do vale do são francisco, petrolina, brazil 2 universidade do estado da bahia, juazeiro, brazil host records and cleptoparasitic behavior of the cuckoo bee mesonychium asteria (smith) (apidae, ericrocidini) in nests of centris xanthomelaena moure & castro (apidae, centridini) short note sociobiology 67(2): 322-325 (june, 2020) 323 classification) with low rainfall mostly from february to april (alvares et al., 2013). the monitoring of the nests was done from may to june 2015 and from july to november 2017. field observations were conducted between 04:50 a.m. and 06:00 p.m. voucher specimens were sent to bee taxonomists for identification and then they were deposited in the entomological collection of the national museum in rio de janeiro (mnrj). mesonychium asteria was the main natural enemy of the nests of c. xanthomelaena. we identified two behaviors performed by females of m. asteria: (1) overflying of nesting sites, and (2) invasion of nests (fig 1a). cleptoparasitic females visited the nest site at least twice a day, once during the morning and another during the afternoon. besides that, females of m. asteria flyover near the ground patrolling any hole in the ravine. after finding an active nest of c. xanthomelaena, the female of m. asteria either invaded it immediately or landed close to the nest entrance waiting until the host left temporarily the nest, for about 10/15 min, to invade it (fig 1b, c). mesonychium asteria spent between 50 seconds and 14 minutes inside the host nests (n = 6). after landing close to the nest entrance, the females externalized their ovipositors immediately. during the period of invasion we sometimes observed a buzz inside the nest. after parasitized, the nests were quite frequently visited, and the overfly behavior was suppressed. fig 1. behavior of the female of mesonychium asteria. (a) cleptoparasite bee invading nest, (b), (c) female waiting close to the nest entrance, (d) m. asteria visiting flower of rhaphiodon echinus close to nest site. hoj martins, pl oliveira-rebouças, vs ferreira – host records and cleptoparasitic behavior of the cuckoo bee 324 it indicated that m. asteria might be able to memorize their parasite nests, as reported to other cleptoparasite bees (coville et al., 1983). in three occasions, the female of m. asteria entered the nest while the host bee was inside and the cleptoparasite was thrown out of the nest. eleven adults of m. asteria emerged from nine nests. the developing time for these adults ranged from 159 to 227 days. four larvae of a natural enemy, probably m. asteria, based on larval development of bees from the same tribe (vinson et al., 1987; rozen et al., 2011), were observed in open brood cells of four different nests. in one brood cell a first instar larval stage was found above the pollen mass with no presence of the host bee’s egg or larvae (fig 2a). larva from the first instar stage had a prognathous head, head capsule pigmented and sclerotized with an elongate mandible, and the chorion attached to its end (fig 2b), as visualized on other first instar larva of ericrocidini tribe (vinson et al., 1987; rozen et al., 2011). in three brood cells we observed a well-developed larva, already with the cocoon. the cocoon of m. asteria has a silken fibrous consistency (fig 2c), that is different from the cocoon of c. xanthomelaena. in addition, we also observed m. asteria and c. xanthomelaena sharing the same source of nectar: poincianella microphylla (fabaceae) and rhaphiodon echinus (lamiaceae) (fig 1d). despite of the genus mesonychium have been reported as a parasite of nests from the subgenus centris (paracentris) cameron, (rocha-filho et al., 2009), this study is the first to confirm the association of both. for the genus centris, only centris (centris) pulchra moure, oliveira and viana, was reported as host of m. asteria (rocha-filho et al., 2009). however, mahlmann and oliveira (2012) pointed to a possible error in the identification of m. asteria in their study, which makes the present study unprecedented in the registration of m. asteria as a nest cleptoparasite for the genus centris. further, the records of m. asteria which indicated association with epicharis nests, were based on indirect evidence of parasitism in nests of epicharis nigrita (friese) and epicharis icolour smith, (gaglianone, 2005; rocha-filho et al., 2008), because there were not records of emergence of m. asteria from these possible host bee brood cells. finally, it is probable that m. asteria has a preference for c. xanthomelaena nests, since in the same area two other species of centris (centris) fabricius, and one of centris (trachina) klug, are nesting, however no cases of invasion or emergence of m. asteria from their nests was observed. this fact may be associated first with the body similarity of the parasite species with its host, as well as brood cell size. beyond that, the evidence of parasite and host sharing the same food source may explain the m. asteria preference for c. xanthomelaena nests. acknowledgements the authors thank dr. felipe vivallo (museu nacional/ ufrj) for the identification of the species and for comments and suggestions in the earlier version of this note. we also thank matthew white ellis for the advice on english language. references alvares, c.a., stape, j.l., sentelhas, p.c., gonçalves, j.l.m. & sparovek, g.(2013). köppen’s climate classification map for brazil. meteorologische zeitschrift, 22: 711-728. doi: 10.1127/ 0941-2948/2013/0507 coville, r., frankie, g. & vinson, s. (1983). nests of centris segregata (hymenoptera: anthophoridae) with a review of the nesting habits of the genus. journal of the kansas entomological society, 56: 109-122. gaglianone, m.c. (2005). nesting biology, seasonality, and flower hosts of epicharis nigrita (friese, 1900) (hymenoptera: apidae: centridini), with a comparative analysis for the genus. studies on neotropical fauna and environment, 40:191-200. doi: 10.1080/01650520500250145 hiller, b. & wittmann, d. (1994). seasonality, nesting biology and mating behavior of the oil-collecting bee epicharis dejeanii (anthophoridae, centridini). biociências, 2: 107-124. mahlmann, t. & oliveira, f.f. (2012). a new species of centris (centris) (fabricius) from northeastern brazil, with taxonomic notes on c. (c.) pulchra moure, oliveira & viana (hymenoptera, apidae). zookeys, 255:4 9-65. doi: 10.3897/ zookeys.255.4303 michener, c.d. (1979). biogeography of the bees. annals of the missouri botanical garden, 66: 277-347. fig 2. larvae and parasitized cells feature.(a) parasitized cell, (b) first instar larvae, (c) mesonychium asteria cocoon. sociobiology 67(2): 322-325 (june, 2020) 325 moure, j.s. & castro, m.s. (2001). uma nova espécie de centris fabricius (hymenoptera, apoidea, anthophoridae) do nordeste do brasil. revista brasileira de zoologia, 18: 329-333. moure, j.s. & melo, g.a.r. (2007). ericrocidini cockerell & atkins, 1902. in moure, j.s., urban, d. & melo, g.a.r. catalogue of bees (hymenoptera, apoidea) in the neotropical region (pp. 158-166). sociedade brasileira de entomologia, curitiba, brasil. rocha-filho, l.c., silva, c.i., gaglianone, m.c. & augusto, s.c. (2008). nesting behavior and natural enemies of epicharis (epicharis) bicolor smith 1854 (hymenoptera: apidae). tropical zoology, 21: 227-242. rocha-filho, l.c., morato, e. & melo, g.a. (2009). new host records of aglaomelissa duckei and a compilation of host associations of ericrocidini bees (hymenoptera: apidae). zoologia, 26: 299-304. rozen, j.g. (1969). the larvae of the anthophoridae (hymenoptera: apoidea). part 3. the melectini, ericrocidini, and rhathymini. american museum novitates, 2382: 1-24. rozen, j.g. & buchmann, s.l. (1990). nesting biology and immature stages of the bees centris caesalpiniae, c. pallida, and the cleptoparasite ericrocis lata (hymenoptera: apoidea: anthophoridae). american museum novitates, 2985: 1-30. rozen, j.g., vinson, s.b., coville, r.e. & frankie, g.w. (2011). biology of the cleptoparasitic bee mesoplia sapphirina (ericrocidini) and its host centris flavofasciata (centridini) (apidae: apinae). american museum novitates, 3723: 1-36. silveira, f.a., melo, g.a.r. & almeida, e.a.b. (2002). abelhas brasileiras: sistemática e identificação. belo horizonte, brasil, 253 p smith, f. (1854). catalogue of hymenopterous insects in the collection of the british museum. part 2. apidae. london: british museum, 199-465. snelling, r.r. & brooks, r.w. (1985). a review of the genera of cleptoparasitic bees of the tribe ericrocidini (hymenoptera: anthophoridae). contributions in science, natural history museum of los angeles county, 369: 1–34. vinson, s.b., frankie, g.w. & coville, r.e. (1987). nesting habits of centris flavofasciata friese (hymenoptera: apoidea: anthophoridae) in costa rica. journal of the kansas entomological society, 60: 249-263. doi: 10.13102/sociobiology.v66i1.3446sociobiology 66(1): 190-193 (march, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 foraging activity of xylocopa cearensis (ducke) in sand dune landscape bees foraging strategy is affected by the local distribution and abundance of flower resources, mainly nectar and pollen. the solitary bee xylocopa cearensis (ducke) is large (>10 mm) and long-flying (pasquet et al., 2008) and thus is more likely to move between patchy sand dunes of restinga vegetation (pinto et al., 1984), which is naturally fragmented and associated to the atlantic forest domain. this ecosystem combines patchy bushy vegetation and harsh environmental conditions (high speed winds, intense solar radiation and salinity) that can be influential to the foraging range of x. cearensis which is locally abundant and the main pollinator of many plants (viana et al., 2002; viana et al., 2006). bee´s essential resources (food, mates and nesting material) must be achieved within foraging range from their nests, causing a tradeoff between trophic resource profit and energy cost to bridge some temporal and spatial distance. abstract bees foraging strategy is affected by the distribution and abundance of flower resources, mainly nectar and pollen. homing time of female xylocopa cearensis (ducke) bees to their nests was assessed through a simple translocation method. the hypothesis addressed was that resource distribution in the landscape level influences bee homing time. the study area comprises about 300ha in a sand dune field with patchy shrubs in salvador, bahia, brazil. the mean homing time after translocation was 60min (sd = 4.36min; n = 03), except for one bee that did not returned. the translocation technique was successfully applied to large solitary bees, since they do return to their own nest and can be easily recognized when arriving. also, a bee returned carrying pollen, what suggests foraging activity after translocations. results evidence landscape functional connectivity since bees were able to move through local habitats. further studies should address movement cost tradeoffs and its consequences on bee diversity conservation. sociobiology an international journal on social insects fo silva1,4, bf viana2,4, d boscolo3,4, rl santos2,4 article history edited by solange augusto, ufu, brazil received 10 may 2018 initial acceptance 11 june 2018 final acceptance 11 november 2018 publication date 25 april 2019 keywords bee conservation; flight distance; translocation experiment; functional connectivity. corresponding author fabiana oliveira da silva universidade federal de sergipe campus do sertão rodovia engenheiro jorge neto km 0, silos cep 49680-000, nossa senhora da glória sergipe, brasil. e-mail: fabia714@ufs.br this study addresses the hypothesis that both clumped food resources and nesting sites distribution may influence interpatch movement of x. cearensis within a landscape. we used translocation experiments to assess the flight range and the homing-time of female bees of x. cearensis to their nests. the study area comprises 300ha in a sand dune field in salvador, bahia (12o55`07.19”s and 38o19`03.78”o), brazil (fig 1). according to köppen classification, the local climate is tropical hot, humid, with annual average temperature of 25.8 ºc, mean relative humidity of 81%, and monthly rainfall of 175.03 mm. nests were located following a previous study (silva & viana, 2002) and new records. from seven nesting sites, active nests were selected by chance and the spontaneous flight of individual female bees were recorded. during observations mean temperature was 31.95 ºc (sd = 2.35), ur was 62.63% (sd = 2.70) and cloudiness ranged from sunny 1 universidade federal de sergipe – campus do sertão, nossa senhora da glória, sergipe, brazil 2 programa de pós-graduação em ecologia e biomonitoramento, universidade federal da bahia, instituto de biologia, salvador, bahia, brazil 3 universidade de são paulo – faculdade de filosofia ciências e letras, ribeirão preto, são paulo, brazil 4 national institute of science and technology in interdisciplinary and transdisciplinary studies in ecology and evolution (inct in-tree) short note sociobiology 66(1): 190-193 (march, 2019) 191 to > 70%, during three days, from january 11th to 13th, 2009, between 05:25 am and 01:30 pm. based on microclimate and flight activity data, the translocation experiments were performed on april 20th and july 2nd, 12th and 20th, 2009, between 05:30 am and 09:30 am, during the period of highest bee foraging activity. translocations were performed from nests located at 12 nesting sites (table 1), with mean distance of 490.16 m (sd = 362.50; range = 7.07 to 942.23 m). bees were collected at nest entrance using a piece of a transparent plastic tube (aprox 10 -15 cm long x 2 cm wide), wide enough for the individual bee to walk inside of it, and closed with plastic lids. the nest entrance and thorax of female bees were marked with white nail polish before translocation, becoming distinguishable at returning. bees were translocated to three distances (200 m, 400 m and 600 m) in randomly chosen directions, comprising the most common spectrum of flight range, but yet below the maximum spontaneous displacement to allow nest returning (pasquet et al., 2008). also, distances allowed bees to forage only in restinga area, preventing them to be translocated into anthropogenous surrounding habitats. a return event was recorded when it came back to the nest, but if no return was detected after 24 hours of release, it was recorded as no return. to be sure that only active females were translocated we first inducted females to fly out of the nest by slightly attacking against the nested branch with a wooden stick. we recorded the time of bee capture, the time of release after translocation, and the time the bee returned to the original nest (detected through continuous focal observation). for each distance four female bees were translocated, each taken from a different nest entrance and mostly from different nest sites (table 1). data were analyzed using the observed homing times after release, which were compared using a one-tailed t-test and a cox proportional hazards survival analysis (cox, 1972) using survival package (r version 3.1.2 software). the explanatory variable was the translocation distance and the dependent variable was the homing time and return success rate (proportion of returned individuals) per unit time for each translocation distance. bees that did not return were considered as right censored (incomplete) data (cox, 1972). spontaneous female flights from natural nests started from 5:41 to 9:23 min and finished from 12:05 13:20 pm, with higher intensity from dusk to 10:30 am. foraging trips ranged from 1 min to 58 min, with an average homing time of 19.62 min (sd = 15.74, n = 41 foraging trips). similarly, camillo et al., (1986) found that flight duration of x. suspecta females varied from 5 to 37 minutes (18.3 ± 9.2 min), although daily flights occurred throughout the day, with a higher frequency between 4 and 5 pm. the mean homing time after translocation, in all distances was greater than spontaneous flight and one female did not return after 400 m translocation, indicating that bees may have faced harsher conditions or longer homing distances with the translocation than expected for natural flights. just one bee translocated at 400 m returned carrying pollen, suggesting that foraging activity is possible after translocations, since none carried any pollen before release. these large solitary bees were table 1. main results from translocation experiments from natural nests of xylocopa cearensis, in sand dunes, salvador, bahia. distance (m) nest site nest location (utm 24s) number of nests number of bees % of returning mean time (sd) time range (min. and max.) 50 n5(2)** 573893 and 8571829 4 4 100 n9 574099 and 8572535 31.5 (39.37) 7-89 n15 573919 and 8572542 200 n7 573857 and 8571759 4 4 100 n6 573862 and 8571754 n8 573849 and 8571768 28 (21.57) 5-57 n16 573963 and 8572612 400 n2 573820 and 8571850 4 4 75 n7* 573857 and 8571759 49.3 (44.02) n11 574039 and 8572528 6-94 n17 573940 and 8572693 600 n2 573820 and 8571850 4 4 100 n3 573823 and 8571824 62 (25.73) 35-93 n10 574095 and 8572581 n15 573919 and 8572542 * female did not return ** two bees were taken from two different nests fo silva, bf viana, d boscolo, rl santos – foraging activity in sand dune192 fig 1. location of the study site: a – in relation to brazil, b – bahia state and c – and geographical range of the environmental protection area of abaeté sand dunes (1.800 ha) showing the types of natural habitats of the landscape. translocation experiments were performed in nests located in the northern part (upper area), comprising 300 ha (map: eduardo f. moreira) a b c successfully translocated, since they returned to their nest and could be easily recognized when arriving. although tests show no clear relation between translocation distance and homing time, the preliminary results provide experimental evidence of the functional connectivity within restinga landscape, since bees were able to overcome local interpatch distances (fig 1), although with lower efficiency than spontaneous flights. the decrease in probability and higher return time is expected when foragers are released in distant areas they are not familiar with. there was a tendency of bees to return earlier when released from 50 and 200 m distances than when released further away from their nests, although all tested distances comprise their common range (fig 2). also, it is evidence that bees may forage across local habitats. previous studies on foraging paths of x. cearensis in shrub patches, found that bees stay longer and visit more flowers in a single patch because resources are clumped (costa et al., 2002; pigozzo et al., 2007; ramalho & rosa, 2010; freire & pigozzo, 2014), because of high individual or flower density than other species (viana et al., 2002; viana & kleinert, 2006). in the case which at least part of the individuals of the bee population has small foraging range, then local habitat structure and their interpatch distances are very meaningful for conservation purposes. therefore, maintaining the diversity of habitat patches in sand dunes within a landscape level is essential to retain bee diversity and pollination services. further studies should include more individuals and greater distances such as 1000 m to provide evidence that the harsh environment and clumped floral resources, may favor short foraging distances as found for other bees (gathmann & tscharntke, 2002; zurbuchen et al., 2010). studies addressing such tradeoff and its consequences on movement patterns and service delivery for xylocopa bees in restinga environment are still needed. sociobiology 66(1): 190-193 (march, 2019) 193 acknowledgments thanks to the valuable contribution of labea team to field work. fos thanks for the scholarship from the brazilian national research council (cnpq/capes) and the graduate program in ecology and biomonitoring (ecobio) and to jorge santana from parque das dunas for logistic support. db thanks for the scholarship from the brazilian coordination for the improvement of higher education personnel (capes). references camillo, e., garófalo, c.a. & muccillo, g. (1986). on the bionomics of xylocopa suspecta (moure) in southern brazil: nest construction and biological cycle (hym., anthophoridae). revista brasileira de biologia, 46: 383-393. costa, c.b.n., costa, j.a.s., rodarte, a.t.a. & jacobi, c. m. (2002). comportamento de forrageio de xylocopa (neoxylocopa) cearensis ducke, 1910 (apidae) em waltheria cinerascens a. st. hil. (sterculiaceae) em dunas costeiras (apa do abaeté, salvador, bahia, brasil). sitientibus, série ciências biológicas, 2: 23-28. cox, d. r. (1972). regression models and life-tables. journal of the royal statistical society, 34: 187-220. freire, n.f. & pigozzo, c.m. (2014). comportamento de forrageio da xylocopa cearensis ducke, 1910, em população de comolia ovalifolia dc trina em um ambiente de restinga, salvador-ba. candombá, 10: 33-41. retrived from: http:// r e v i s t a s . u n i j o r g e . e d u . b r / c a n d o m b a / 2 0 1 4 v 1 0 n 1 / p d f / candomba_2014_pag33.pdf. gathmann, a. & tscharntke, t. (2002). foraging range of solitary bees. journal of animal ecology, 71: 757-764. doi: 10.1046/j.1365-2656.2002.00641.x. pasquet, r.s., peltier, a., hufford, m.b., oudin, e., saulnier, j., paul, l., knudsen, j.t., herren, h.r. & gepts, p. (2008). long-distance pollen flow assessment through evaluation of pollinator foraging range suggests transgene escape distances. proceedings of the national academy of science, 105: 1345613461. doi: 10.1073/pnas.0806040105. pinto, g.l.p., bautista, h.p. & ferreira, j.d.c.a. (1984). a restinga do litoral nordeste do estado da bahia. in l.d. lacerda (eds.), restingas: origem, estrutura e processos,(pp.195-216). rio de janeiro: comissão editorial da universidade federal fluminense. pigozzo, c.m., neves, e.l., jacobi, c.m. & viana, b.f. (2007). comportamento de forrageamento de xylocopa (neoxylocopa) cearensis ducke (hymenoptera: apidae, xylocopini) em uma população de cuphea brachiata koehne (lythraceae). neotropical entomology, 36: 652-656. doi: 10. 1590/s1519-566x2007000500003. ramalho, m. & rosa, j.f. (2010). ecologia da interação entre as pequenas flores de quilha de stylosanthes viscosa sw. (faboideae) e as grandes abelhas xylocopa (neoxylocopa) cearensis ducke, 1910 (apoidea, hymenoptera), em duna tropical. biota neotropica, 10: 93-100. doi: 10.1590/s167606032010000300010. silva, f.o, viana, b.f. (2005). ninhos de xylocopa (neoxylocopa) cearenses ducke, 1910 (hymenoptera, anthophoridae) nas dunas litorâneas do abaeté, salvador, bahia. revista nordestina de zoologia, 2: 3-31. silva, f.o. & viana, b.f. (2002). distribuição de ninhos de abelhas xylocopa (hymenoptera: apidae) em uma área de dunas litorâneas. neotropical entomology, 31: 661-664. doi: 10.1590/s1519-566x2002000400025. viana, b.f., kleinert, a.m.p. & silva, f.o. (2002). ecologia xylocopa (neoxylocopa) cearensis (hym., anthophoridae) nas dunas litorâneas de abaeté, salvador, bahia. iheringia, série zoologia, 92: 47-57. doi: 10.1590/s0073-47212002000400007. viana, b.f., silva, f.o. & kleinert, a.m.p. (2006). a flora apícola de uma área restrita de dunas litorâneas, abaeté, salvador, bahia. revista brasileira de botânica, 29: 13-25. doi: 10.1590/s0100-84042006000100003. viana, b.f. & kleinert, a.m.p. (2006). structure of beeflower system in the coastal sand dune of abaeté, northeastern brazil. revista brasileira de entomologia, 50: 53-63. doi: 10.1590/s0085-56262006000100008. fig 2. homing times of individual females of x. cearensis after translocation according to translocation distance, estimated from the cox proportional hazards model. time limit used was 120 m for better graph visualization, as no bee took more than this time to return and exploratory tests performed using 240 and 280 m did not change the results. (n = 17 female bees, including one that did not return at 400 m release). data are not statistically significant because of low sample size, but two groups with different mean return time can be seen, one with the two shorter distances and shorter times and the other with the longer distances and times. time after releas (minutes) re tu rn p ro ba bi lit y 1197 correlation of the nest density and the number of workers in bait traps for fire ants (solenopsis invicta) in southern china by yong-yue lu, lei wang*, yijuan xu, ling zeng & ningdong li abstract the relationship between solenopsis invicta nest density and the number of fire ant workers in bait traps and percentages of traps capturing ants were investigated in the waste land of wuchuan, guangdong, south china. the results showed that fire ant nest density is positively correlated with the number of workers captured in traps, and could be described by n=60.53lnd +348.0d+421.1. the workers exceeded 200 and 300 in bait traps while the density of fire ant nests was over 0.023 and 0.084 ind./m2, respectively. the percentages of traps capturing ants were also positively correlated with fire ant nest density and fit by pe=1/(1+e0.9694-309.85d). when the nest density was over 0.018 ind./m2, over 99% of traps captured fire ant workers. n=8.8796e0.0346pe was the fitting line for worker amount and trap percentage. the workers per trap were about 50, 100, and 200 when the trap percentages were 50%, 70% and 90%, respectively. key words: fire ants, solenopsis invicta, nest density, bait traps introduction the red imported fire ant, solenopsis invicta, is a serious economic pest. since its introduction into the u.s., the ant has infested most of the southern states (williams et al. 2003). to date, solenopsis invicta is also seen in australia, new zealand, mainland china and taiwan (mccubbin & weiner 2002, pascoe 2002, zeng et al. 2005). red imported fire ants can cause many problems to red imported fire ant research center, south china agricultural university, guangzhou 510642, p.r. china corresponding author: lu yong-yue red imported fire ant research center south china agricultural university guangzhou 510642, p.r. china email: luyong yue@scau.edu.cn , insectlu@163.com *joint first author 1198 sociobiolog y vol. 59, no. 3, 2012 human health, agriculture and native animals. it can cause 12.2-26.1% potato loss, and numbers of the fire ants are negatively correlated with potato and soybean seed yields (adams et al. 1988, apperson & powell 1983). the red imported fire ant can also feed on flowers and developing fruits of citrus, and destroy young citrus trees (banks et al. 1991). because of its painful and allergenic stings, fire ants are a serious health threat to some people. high density of fire ant populations may make lawns lose their value (lofgren et al. 1975). however, sometimes the fire ant is also considered as a beneficial insect in many crops. fire ants can prey on boll weevils in cotton (sterling 1978), and citrus leafminers in citrus trees (zappalà et al. 2007). although pesticides may harm nontarget animals, chemicals are effective control methods reducing the population of fire ants. chemicals are formulation as drenches and baits (kemp et al. 2000), and baiting is an effective control measure for fire ants (lofgren & weidhass 1972). before bait application, the amount and density of fire ant nests and workers must be surveyed and obtained accurately. this work takes much time and manpower, especially in large areas with high fire ant density. oi et al. (2004) utilized a gis system to map the bait stations in fish farms, and the map can indicate the locations of fire ant nests. that method can save 43% of time compared to walking surveys. the objective of our study is to reveal the relationships among nest density, the amount of worker ants captured by bait trap, and the percentage of bait traps capturing ants. a calculation method is then designed to calculate the percentage of bait traps capturing ants to estimate the density of fire ant nests and workers in the surveyed area. this will allow more efficient surveying of fire ants. materials and methods study sites and sampling we conducted this study during april and june, 2005 in wuchuan, guangdong, china. the habitat surveyed was wasteland. we chose five sites for the experiment, where densities of fire ant mound were different covering in total 2.4 hm2. the experimental sites were divided into 16 blocks with one block of about 1500 m2. nest densities were recorded by visual observation. no. of fire ant workers were sampled by using bait traps. the sampling method is 1199 lu, y.-l. et al. — correlation of fire ant nest density with bait traps similar to huang et al. (2011). at each block, 25ml plastic vials containing a 5 mm thick piece of sausage was placed on the ground surface for 30 min. the number of bait traps depended on the area of site. more than 15 bait traps were put in each block. the ants captured in traps were dipped into soapy water and counted later. the amount of traps which did not trap any ants was recorded. at the same time, the number of solenopsis invicta active nests was counted in each block. inspections were performed twice each month, and five times in total. statistical analysis nest density was calculated through the nest number divided by the acreage of one block. no. of fire ant workers per bait trap was the mean of the workers captured by all traps in each block. percentage of bait traps capturing fire ants in each block was presented by the ratio of the trap numbers with and without fire ants. all statistical analyses were conducted using spss, version 14.0 (spss inc., chicago, il, usa). fig. 1. correlation of nest density with number of worker captured by bait traps. n=60.5257lnd +348.0473d+421.1183, r=0.9454, n=80, df=79, f=319.8, p<0.01. n means no. of workers captured by bait traps, and d means nest density in this function 1200 sociobiolog y vol. 59, no. 3, 2012 table 1. nest density, worker amount per bait trap, and percentage of bait traps capturing workers at wasteland, southern china. nest density ind./m2 d no. of workers per bait trap n percent of baits capturing workers pe nest density ind./m2 d no. of workers per bait trap n percent of baits capturing workers pe 0 0 0 0.02800 212.7 100 0.00099 13.8 15.0 0.02889 202.5 94.4 0.00105 27.0 21.1 0.03000 234.5 100 0.00167 21.4 33.3 0.03188 243.2 93.8 0.00182 18.3 31.3 0.04000 202.4 100 0.00200 20.1 50.0 0.04333 199.5 100 0.00214 29.5 57.1 0.04420 223.2 94.4 0.00222 47.2 40.7 0.05000 292.5 91.7 0.00227 51.7 45.5 0.05000 274.4 100 0.00286 68.9 61.9 0.05195 252.3 93.3 0.00333 52.9 73.3 0.05250 282.8 100 0.00441 90.2 84.6 0.05500 190.8 91.7 0.00446 84.7 65.0 0.05733 269.5 100 0.00452 70.2 66.7 0.05778 244.3 92.3 0.00500 78.3 81.3 0.05810 279.3 100 0.00519 115.2 53.8 0.06063 254.1 100 0.00556 100.1 71.4 0.06200 265.3 100 0.00667 72.3 57.1 0.06417 312.4 91.7 0.00706 122.3 76.5 0.06815 303.4 92.3 0.00752 142.4 84.6 0.07417 279.3 100.0 0.00830 113.5 76.9 0.07600 233.3 100 0.00833 194.4 58.3 0.08000 265.6 100 0.00859 162.3 75.0 0.08417 265.5 91.7 0.01000 214.2 84.6 0.08667 204.3 100.0 0.01048 150.5 90.5 0.08917 231.5 91.7 0.01064 195.8 94.4 0.09200 302.1 100 0.01200 175.3 90.9 0.09333 365.8 100 0.01311 203.6 100 0.09714 346.7 100 0.01350 164.1 93.8 0.09926 354.4 84.6 0.01364 140.5 86.7 0.10000 332.0 100 0.01417 203.2 100 0.10519 367.8 92.3 0.01773 244.6 95.0 0.11500 318.4 91.7 0.01852 186.3 80.0 0.12100 289.2 100 0.02000 233.2 100 0.12667 299.6 100 0.02229 182.2 93.8 0.13000 386.3 91.7 0.02500 208.3 84.6 0.13143 353.6 100 0.02500 169.5 100 0.13704 362.3 92.3 0.02620 178.4 94.4 0.13778 395.6 100 0.02679 264.6 100 0.13833 344.2 100 0.02750 226.9 91.7 0.14000 388.4 100 1201 lu, y.-l. et al. — correlation of fire ant nest density with bait traps results 80 groups of data were obtained over 3 months and listed in table 1. fire ant nest density is presented from low to high. fig.1 shows that the interdependence of density of solenopsis invicta nests and mean number of ants in traps. the best fit line, a natural logarithm function, n=60.53lnd +348.1d+421.1(r=0.9454, n=80, df=79, f=319.8, p<0.01), predicted the amount of workers were captured in traps as the fire ant nest density increased. the relationship suggested that growth speed of amount of ants was the fastest when the nest density varied from 0 to 0.005 ind./m2, and ant amount was over 100 ind. per trap. the growth rate decreased fewer than 2% when density of nests was over 0.017 ind./m2. a bait trap can capture more than 200 and 300 workers on average when the nest densities were over 0.023 and 0.084 ind./m2, respectively. fig. 2 shows that the higher percentages of bait traps capturing ants were associated with higher fire ant nest density. more than 50% of the traps of the captured ants occurred in mounds with a density of 0.0032 mounds/m2. the growth percentage of the trap capturing ants was very fast. when the fig. 2. correlation of nest density with baits capturing workers. pe=1/(1+exp(0.9694-309.85d)), r=0.9124, n=80, df=79, f=387.5, p<0.01. pe means percent of baits capturing workers, and d means nest density in this function. 1202 sociobiolog y vol. 59, no. 3, 2012 density was over 0.018 nest/m2, the lowest percent of traps capturing ants was nearly 90%. in other words, nearly all traps can capture fire ants. and the best fit line was pe=1/(1+exp(0.9694-309.85d)) (r=0.9124, n=80, df=79, f=387.5, p<0.01). the dynamics of worker number captured by bait trap with percent of baits capturing workers varied (fig.3). the function, n=8.8796e0.0346pe (r=0.8974, n=80, df=79, f=137.8, p<0.01) could describe the relationship between the two variables. first, the number of worker ants per trap increased gradually with trap percent progressing under 50% by which they increased sharply with trap percent over 70~80%. the worker amounts were about 50, 100, and 200 ants per trap when the trap percent were 50%, 70% and 90%, respectively. discussion the results indicate a correlation between the density of fire ant nests, amount of foraging workers per trap, and percentage of bait traps capturing ants. we can estimate the amount of fire ant nests, and workers in surveyed area by bait trap sampling according to the functions described. the results fig. 3 correlation of worker number captured by bait trap with baits capturing workers. n=8.8796e0.0346pe, r=0.8974, n=80, df=79, f=137.8, p<0.01. n means no. of workers captured by bait traps, and pe means percent of baits capturing workers in this function. 1203 lu, y.-l. et al. — correlation of fire ant nest density with bait traps can be not only be used in monitoring fire ants, but also in controlling them. estimating fire ant density can guide how much pesticide bait should be used, and promote the efficiency of pesticide utilization. bait trap sampling is an important method in ant sampling (briano et al. 2002), and it can be utilized to determine whether an area contains active fire ant mounds (oi et al. 2004). currently, pesticide bait is still an effective control method for fire ants (williams et al. 2001), especially in countries and regions newly invaded by fire ants. before baits are spread, the population of fire ants must be surveyed and located. the time required to perform a walking survey to marked nests was 2.8 person-h (oi et al. 2004). in comparison, the bait trap sampling method only required 1 person-h. significant time can be saved by using the bait trap sampling method when controlling a large fire ant population. this method is faster than that decribed by oi et al (2004). in sampling, advanced technologies are needed. bao et al.(2011) designed a new hotdog-baited trap (b-trap) to detect red imported fire ants under field conditions in taiwan and demonstrated the b-trap was more efficientthan other methods. there needs to be a simple index to assess the occurrence and severity of fire ants, especially for pest management enterprises. adams et al. (1986) reported that the economically significant threshold of fire ants is 80 fire ant workers per trap in average. in our investigation, about 50% and 100% of traps could capture ants when the mean density exceeded 0.0032, and 0.02 ind. per m2, respectively. this observation suggests that the percentage and density are key data in evaluating whether fire ants are widespread or severe. our research could aid development of a standard in classifying hazard rating of solenopsis invicta. acknowledgments we would like to thank jun huang and shenlei li for observations and records. our study was supported by national natural science foundation of china (award# 30900942) and the national basic research program of china (award# 2009cb119200). references adams, c.t., w.a. banks & c.s. lofgren. 1988. red imported fire ant (hymenoptera: formicidae): correlation of ant density with damage to two cultivars of potatoes (solanum tuberosum l.). j. eco. ent. 81(3):905-909. 1204 sociobiolog y vol. 59, no. 3, 2012 apperson, c.s., e.e. powell.1983. correlation of the red imported fire ant (hymenoptera: formicidae) with reduced soybean yields in north carolina. j. eco. ent. 76(2):259263. banks, w.a., c.t. adams & c.s. lofgren. 1991. damage to young citrus trees by the red imported fire ant (hymenoptera: formicidae). j. eco. ent. 84(1):241-246. briano, j.a., d.f. williams, d.h. oi & l.r. davis jr. 2002. field host range of the fire ant pathogens thelohania solenopsae (microsporida: thelohaniidae) and vairimorpha invictae (microsporida: burenellidae) in south america. bio. control. 24(2002):98-102. huang, j., y.j. xu, y.y. lu & g.w. liang. 2011. changes to the spatial distribution of ageratum conyzoides (asterales:asteraceae) due to red imported fire ant solenopsis invicta (hymenoptera: formicidae) in china. j. insect behav. 24(4):307-316. kemp, s.f., r.d. deshazo, j.e. moffitt, d.f. williams & w.a. buhner 2nd. 2000. expanding habitat of the imported fire ant (solenopsis invicta): a public health concern. j. allerg y clin. immunol. 105(4):683-91. lofgren, c.s., w.a. banks & b.m. glancey. 1975. biolog y and control of imported fire ants. ann. re. ent. 20:1-30. lofgren, c.s., d.e. weidhaas. 1972. on the eradication of imported fire ant: a theoretical appraisal. bul. ent. soc. am. 18:17-20. mccubbin, k.i., j.m. weiner. 2002. fire ant in australia: a new medical and ecological hazard. med. j. aust. 176(11):518-519. oi, d.h., c.a. watson & d.f. williams. 2004. monitoring and management of red imported fire ants in a tropical fish farm. flo. ent. 87(4):522-527. pascoe, a. 2002. strategies for managing incursions of exotic animals to new zealand. micronesia. 6:129-135. sterling, w.l. 1978. fortuitous biological suppression of the boll weevil by the red imported fire ant. env. ent. 7:564-568. williams, d.f., d.h. oi, s.d. porter, r.m. pereira & j.a. briano. 2003. biological control of imported fire ants (hymenoptera: formicidae). am. ent. 49(3):150-163. williams, d.f., h.l. collins & d.h. oi. 2001. the red imported fire ant (hymenoptera: formicidae): an historical perspective of treatment programs and the development of chemical baits for control. am. entomol. 46:146-159. zappalà , l., m.a. hoy & r.d. cave. 2007. interactions between the red imported fire ant, the citrus leafminer, and its parasitoid ageniaspis citricola (hymenoptera: encyrtidae): laboratory and field evaluations. biocontrol sci. techn. 17: 353-363. zeng, l., y.y. lu, x.f. he, w.q. zhang & g.w. liang. 2005. identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan, guangdong. chin. bul. ent. 42(2):144-148. bao, s.z., l. kafle & c.j. shih. 2011. a new baited trap for monitoring solenopsis invicta (formicidae: hymenoptera) in taiwan. appl. entomol. zool. 46:165-169. doi: 10.13102/sociobiology.v67i1.4503sociobiology 67(1): 74-79 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction honey bees have an instinct behavior of removing infested, diseased or dead broods, clean their cells and take these diseased or dead broods out of their hives, which is called hygienic behavior of honey bees (rothenbuhler, 1964 a,b). hygienic behavior in the honey bee species a. mellifera was noticed and reported in earlier years (park, 1936). honey bees with a higher hygienic behavior level in their colonies are more resistant towards two brood diseases i-e american foul brood (spivak, 1996; spivak & reuter, 2001) and chalkbrood (invernizzi et al., 2011). selectively bred honey bees for hygienic behavior shows higher level of hygienic behavior while non selective bred honey bees show lower level of hygienic behavior (masterman et al., 2000). hygienic behavior is one of the most desirable traits for selective breeding of the honey bees by the breeders (spivak, 1996). abstract indigenous and exotic honey bee species were evaluated for their hygienic behavior in the climatic condition of peshawar khyber pakhtunkhwa, pakistan. colonies of equal strength from indigenous (apis cerana) and exotic (apis mellifera) species were selected for the study. the same colonies were tested in two seasons. sealed brood were killed with different methods i.e pin killed and freeze killed. the uncapping of cells and brood removal was recorded at different intervals. significant differences were recorded between hygienic behavior of both species of honey bees. apis cerana showed significantly superior hygienic behavior than apis mellifera in both seasons. at different intervals in both species significant differences were recorded. a significant difference was recorded after 12 and 24 hours between the species in both seasons. no significant differences were recorded after 48 hours in both species. from the study it is concluded that indigenous honey bee species has superior hygienic behavior than exotic species. sociobiology an international journal on social insects m shakeel1, h ali2, s ahmad1 article history edited by evandro nascimento silva, uefs, brazil received 26 may 2019 initial acceptance 17 december 2019 final acceptance 07 january 2020 publication date 18 april 2020 keywords pin killed, freeze killed, brood removal, uncapping. corresponding author hussain ali entomology section agricultural research institute tarnab peshawar, khyber pakhtunkhwa, pakistan. e-mail: hussaintanha@yahoo.com natural physical defense mechanism, i-e grooming and hygienic behaviors of a. mellifera and a. cerana, play a main role in their protection against brood diseases and varroa mites (boeking & spivak, 1999).the extent of damage caused by varroa to its original host a. cerana is less as compared to western honey bee a. mellifera colonies ( jong et al., 1982). the main reason for lower varroa mite population in a. cerana colonies is their behavior of efficiently attacking and killing the introduced varroa mite individuals in their colonies (peng et al., 1987). in a. cerana colony, the varroa mites prefer to infest drone cells while in a. mellifera colonies the varroa mites have adapted to infest both drone and worker cells, and this adaptation to reproduce in worker cells in a. mellifera has led to greater damages due to disease transmission from varroa that ultimately lead to lower bee population (koeniger et al., 1981, 1983). application of pesticides/acaricides for controlling varroa mites in the 1 department of entomology, faculty of crop protection sciences, the university of agriculture peshawar, pakistan 2 entomology section, agricultural research institute tarnab, peshawar, khyber pakhtunkhwa, pakistan research article bees comparison of hygienic behavior of exotic honey bee apis mellifera l. and indigenous honey bee apis cerana of pakistan sociobiology 67(1): 74-79 (march, 2020) 75 honey bees hive cause contamination of honey with chemical residues and resistance to these pesticides by varroa mites (lodesani et al., 1992& 1995). selective breeding of honey bees for higher level of hygienic behavior by the breeders may act as an alternate to pesticide usage for varroa mite infestation and other brood diseases in honey bee hives (spivak, 1996). the comparison of african honey bee a. mellifera scutellata with the different hybrids of european honey bee a. mellifera showed that the first mentioned honey bees showed higher levels of hygienic behavior than the second one (nganso et al., 2017). studies showed that a. cerana showed higher level of hygienic behavior in removing artificially killed broods in a specific area of comb than a. mellifera in two seasons of southern part of china (lin et al., 2016). our experiments in this research aim to compare the level of hygienic behavior of exotic honey bees a. mellifera and native honey bees a. cerana of pakistan in different seasons, and provide a baseline for future related studies. materials and methods selection of study site and determination of hygienic behavior of a. mellifera and a. cerana: experiments on hygienic behavior of a. mellifera and a. cerana were conducted on apiaries located at medicinal plants garden of khyber pakhtunkhwa forest department. strong honey bee hives consisted of langstroth design with 10 frames of combs were selected for conducting experiments in the spring season of march and summer season of june, 2018. all the colonies were having a fertile queen, workers, broods, honey and pollen. honey bee colonies were healthy and strong. there was no sign of any disease in the colonies. frames with broods covered area were selected. experiments on hygienic behavior were conducted on two methods i-e freeze killing of broods (reuter & spivak, 1998)) and pin killing of brood assay (newton and ostasiewski, 1986). freeze killing assay broods of a. mellifera and a. cerana were killed by liquid nitrogen treatment to find out difference between their rates of dead brood removal in the spring season of march, 2018. 5 hives of honey bees of both species were selected. one frame having most of the sealed brood cells present was selected from each hive and was tagged. an area on each frame with most of the sealed brood cells was selected by pressing with a hardboard cylinder with a diameter of 6.5 cm and height of 8 cm. about 10 ml of liquid nitrogen was poured in to cylinder through volumetric flask for freeze killing the broods present in area covered by hardboard cylinder, containing a total of 150 cells. the cylinder was removed till the liquid nitrogen was fully evaporated. the frames were returned back to their respective hives and were kept at their original position. after 12 hours, 24 hours and 48 hours the data were recorded by counting the dead eradicated and remaining broods by honey bees. the parameter of number of dead broods removed during 24 hours was considered as hygienic behavior of both species of honey bees. pin killed brood assay for determination of hygienic behavior of a. mellifera and a. cerana pin killed brood assay was conducted in summer season of june, 2018. a total of 5 hives were selected from each species of honey bees for conducting experiment. the frame having most of the sealed brood cells was selected from each hive. area having the most of sealed brood cells was selected and was pressed through hardboard cylinder (covering a total of 150 cells). the brood present in the area covered by hardboard cylinder was killed through a sterile metal pin. the frames were then returned back to their original position at their respective hives. the number of dead broods removed by both species of honey bees in their hives during 12, 24 and 48 hours were recoded. statistical data analysis the total number of cells that was covered under hardboard cylinder in both species was 150 capped brood cells. the data was recorded by comparing the freeze uncapped dead brood removed cells with capped dead brood cells in hives of both species. the percentage of uncapped dead broods removed in both species of honey bees after 12 hrs, 24 hrs, 48 hrs was calculated. the percentage of uncapped dead broods removed was compared with capped dead broods to evaluate hygienic behavior of a. mellifera and a. cerana. for comparison of means of both variables (capped and uncapped cells) student’s t test was used for correlation and significance of means. all the statistical analyses were conducted through statistical package spss (version 20.0). results freeze killed brood assays the results in fig 1 shows that there is significant difference (p<0.05) of hygienic behavior level between a. mellifera and a. cerana in the spring season. the trend of hygienic behavior of a. cerana was significantly (p<0.05) higher than a. mellifera and the number of uncapped cells and number of dead brood eradication during 12 hrs, 24 hrs was higher. no significant differences were recorded after 48 hrs in both exotic and indigenous species. similar kind of behavior was recorded in the summer season as well (fig 2). a. cerana showed significantly (p<0.05) higher hygienic behavior than a. mellifera and the number of uncapped cells and number of dead brood removal during 12 hrs, 24 hrs was higher. both species showed similar hygienic behavior and no significant differences were recoded after 48 hrs. m shakeel, h ali, s ahmad – comparison of bees hygienic behavior 76 pin killed brood assays the results in fig 3 shows the comparison of hygienic behavior level between a. mellifera and a. cerana in spring season by pin kill brood assay method. a significantly (p<0.05) lower number of uncapped cells and dead brood removal was recoded in a. mellifera than a. cerana after 12 and 24 hours. however, no differences were recorded after 48 hours. fig 4. comparison of hygienic behavior of a. mellifera and a. cerana in pin killed brood assay during summer season. means followed by same letter are non-significant at 5% significant level. fig 1. comparison of hygienic behavior of a. mellifera and a. cerana in freeze killed brood assay during spring season. means followed by same letter are non-significant at 5% significant level. fig 2. comparison of hygienic behavior of a. mellifera and a. cerana in freeze killed brood assay during summer season. means followed by same letter are non-significant at 5% significant level. fig 3. comparison of hygienic behavior of a. mellifera and a. cerana in pin killed brood assay during spring season. means followed by same letter are non-significant at 5% significant level. discussion results of freeze killed brood assay show that overall hygienic behavior of a. mellifera was slightly lower than a. cerana in both spring and summer seasons, however a. cerana showed slightly higher hygienic behavior than a. mellifera in spring than summer season. a. cerana expresses higher level of hygienic behavior than a. mellifera in autumn season than spring season in uncapping and removal of dead broods during freeze killed brood assay (lin et al., 2016). hygienic behavior is expressed variably among different species, subspecies and races of honey bees irrespective of different seasons and environmental conditions (liliaet al., 2016; spivak & galliam, 1998 a, b; spivak & reuter, 1998; rasolofoarivao et al, 2015; athreya & reddy; 2013). our results show that a. cerana hygienic behavior is higher in spring than summer season. this difference between both species may be attributed to the higher floral resources and lower temperature in spring as compared to summer season. our results are in conformity with (lin et al., 2016) in which a. cerana showed higher level of hygienic behavior compared to a. mellifera. bee population in hive didn’t have effect on their hygienic behavior which shows that higher efficiency of hygienic behavior is instinct to a. cerana in comparison to a. mellifera (lin et al., 2016). there are genetic reasons for phenotypic expression of the hygienic behavior (moritz, 1988). the instinct nature of higher level of hygienic behavior in a. cerana is the presence of seven genetic loci controlling hygienic behavior (lapidge et al., 2002). all the colonies selected for assessing hygienic behavior of a. cerana and a. mellifera were naturally bred and the queen naturally mated in our experiment. the results suggested that slight difference of bee population in colony did not have significant effect on their hygienic behavior in both species of honey bees. since a. cerana is more efficient at removing the dead broods from their cells, the prevalence of deadly viruses like deformed wing virus and sac brood virus carried by varroa destructor is lower in a. cerana colonies in during the summer seasons the assays were repeated and similar results like springs was recorded fig 4. both species showed no significant differences after 48 hours in their hygienic behavior but they showed significant (p<0.05) difference after 24 and 12 hours. a. cerana hygienic behavior was significantly higher after 12 and 24 hours in removal of dead brood and uncapped cells. sociobiology 67(1): 74-79 (march, 2020) 77 comparison to a. mellifera (highfield et al., 2009; mondet et al., 2016). boecking and drescher, (1992, 1999) demonstrated that africanized bees were more efficient in hygienic behavior as compared to western honey bees and removed broods faster in plastic combs than wax combs. our results are in conformity with (palacio et al., 1996, 2000; spivak & downey, 1998) research demonstrating that honey bees were faster at removing pin killed broods as compared to freeze killed broods. the reason for this behavior may be due to easy detection of pin killed broods because of the hole made in the wax seal of the cell having brood by honey bees through olfaction of pheromones released by broods (palacio et al., 1996). the indigenous honey bee species of pakistan, a. cerana showed significant higher hygienic behavior than the exotic species untill 24 hours. this could also be the reason explaining less pests and diseases attack in a. cerana than in a. mellifera. these are confirmed by some earlier studies of (lint et al., 2016). fig a. freeze killing of broods with liquid nitrogen 1 and 2 apis mellifera, 3 and 4 apiscerana 1 2 3 4 fig a. freeze killing of broods with liquid nitrogen 1 and 2 apis mellifera, 3 and 4 apis cerana. fig b. pin killing of broods in the selected area 1 and 2 apis mellifera, 3 and 4 apis cerana. 2 3 1 4 fig b. pin killing of broods in the selected area 1 and 2 apis mellifera, 3 and 4 apiscerana m shakeel, h ali, s ahmad – comparison of bees hygienic behavior 78 acknowledgments thanks to the reviewers for their valuable comments on the manuscript. special thanks to forest department khyber pakhtunkhwa, peshawar for providing a. cerana for the experiment. authors’ contributions muhammad shakeel, hussain ali and sajjad ahamd planned the experiments, ms and ha conducted the experiments, sa and ha analyzed the data, ms and hs wrote the text. sa checked the version and corrected the text. ha submitted the paper. references athreya, s.v.r. & reddy, m.s. 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(2009). deformed wing virus implicated in overwintering honeybee colony losses. applied and environmental microbiology, 75: 7212-7220. doi: 10.1128/aem.02227-09 jong, d. e., morse, r.a. & eickwort, g.c.(1982). mite pests of honey bees. annual review of entomology, 27: 229-252. doi: 10.1146/annurev.en.27.010182.001305 koeniger, n., koeniger, g. a, & wijyagunasekara, n.h.p. (1981). observations on the adaptation of varroa jacobsoni to its natural host apis cerana in sri lanka. apidologie, 12: 37-40. koeniger, n.,koeniger, g., & e. a-dlkfibenardo, m. (1983). observations on mites of the asian honeybee species (apis cerana, apis dorsata, apis florea). apidologie, 14: 197-204. de guzman, l.i., thomas e.r., frake, a.m. & kirrane, m.j. (2015). brood removal influences fall of varroa destructor in honey bee colonies. journal of apicultural research, 54: 216225. doi: 10.1080/00218839.2015.1117294 lin, z., page, p., li, l., qin, y., zhang, y., hu, f., neumann, p., zheng, h. & dietemann, v. (2016). go east for better honey bee health: apis cerana is faster at hygienic behavior than a. mellifera. plos one, 11: e0162647. doi: 10.1371/ journal.pone.0162647 lodesani, m., pellacani, a., bergomi, s., carpana, e., rabitti, t., & lasagni, p. (1992). residue determination from some products used against varroa infestation in bees. apidologie, 23: 257-272. lodesani, m., colombo, m., & sperafico, m. (1995). in effectiveness of apistan treatment against the mite varroa jacobsoni oud. in several districts of lombardy (italy). apidologie, 26: 67-72 masterman, r., smith, b.h., & spivak, m. (2000). brood odor discrimination abilities in hygienic honey bees (apis mellifera l.) using proboscis extension reflex conditioning. journal of insect behavior, 13: 87-101. mondet, f., kim, s.h., de miranda, j.r., beslay, d., le conte, y., & mercer, a.r. (2016). specific cues associated with honey bee social defense against varroa destructor infested brood. science reports, 6: 25444. doi: 10.1038/ srep25444. nganso, b.t., fombong, a.t., yusuf, a.a., pirk, c.w.w, stuhl, c., & torto, b. (2017). hygienic and grooming behaviors in african and european honeybees new damage categories in varroa destructor. plos one, 12: e0179329. doi: 10.1371/ journal.pone.0179329 park, o.w. (1936). disease resistance and american foulbrood. american bee journal. 76: 12-15. palacio, m.a., figini, e., rodriguez, e.m., rufinengo, s., del hoyo, m. l. & bedascarrasbure, e. (1996). selección para comportamiento higiénico en una población de apis mellifera, in: anales del v congreso iberolatinoamericano de apiculture mercedes, uruguay, 148-150 p. palacio, m.a., figini, e., rodriguez, e.m., rufinengo, s., bedascarrasbure e., del hoyo, m.l. (2000). changes in a population of apis mellifera selected for its hygienic behavior. apidologie, 31: 471-478. doi: 10.1051/apido:2000139 peng, y.s.c., fang, y., xu, & gel, s. (1987). the resistance mechanism of the asian honey bee, apis cerana fabr., to an ectoparasitic mite, varroa jacobsoni oudemans. journal of invertebrate pathology, 49: 54-60. doi: 10.1016/0022-2011 (87)90125-x rasolofoarivao, h., delatte, h., raveloson ravaomanarivo, l.h., reynaud, b., & clémencet, j. (2015). assessing hygienic behavior of apis mellifera unicolor (hymenoptera: apidae), the endemic honey bee from madagascar. genetics and molecular research, 14: 5879-5889. doi: 10.4238/2015.june.1.5 reuter, g.s., & spivak, m. (1998). a simple assay for honey bee hygienic behavior. bee culture, 126: 23-25. rothenbuhler, w. (1964a). behavior genetics of nest cleaning in honey bees. iv. responses of f1 and backcross generations to disease-killed brood. american zoologist, 4: 111-123. retrieved from http://www.jstor.org/stable/3881284 sociobiology 67(1): 74-79 (march, 2020) 79 rothenbuhler, w.c. (1964b). behavior genetics of nest cleaning in honeybees. i. responses of four inbred lines to disease killed brood. animal behavior, 12: 578-583. doi: 10.1016/00033472(64)90082-x spivak, m. (1996). honeybee hygienic behavior and defense against varroa jacobsoni, apidologie, 27: 245-260. doi: 10.10 51/apido:19960407 spivak, m. & downey, d.l. (1998). field assays for hygienic behavior in disease resistance in honey bees (apidae: hymenoptera). journal of economic entomology, 91: 64-70. doi: 10.1093/jee/91.1.64 spivak, m. & reuter, g.s. (1998). performance of hygienic honey bee colonies in a commercial apiary. apidologie, 29: 291-302. doi: 10.1051/apido:19980308 spivak m. & reuter g.s. (2001). resistance to american foulbrood disease by honey bee colonies apis mellifera bred for hygienic behavior. apidologie, 32: 555-565. doi: 10.1051/ apido:2001103 doi: 10.13102/sociobiology.v67i1.4381sociobiology 67(1): 126-128 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the asiatic honeybee (apis cerana) is an important pollinator to wild plants and crops and plays a critical role in the agricultural ecology system in china (su & chen, 2009). nevertheless, chinese honeybee colonies have recently suffered heavy losses due to the effects of biotic and abiotic factors (diao et al., 2018). the small hive beetle (aethinatumida murray, coleoptera: nitidulidae) (shb) is an invasive species which has been identified in apis mellifera colonies from several regions around the world (hood, 2004; neumann & elzen, 2004; cervancia et al., 2016; al toufailia et al., 2017; muli et al., 2018), and in spite of this, little is known about shb in a. cerana in china. suspected shb was first found in the front of beehive by beekeepers in guangdong province on october 25, 2017. then, we collected shb samples from three apiaries with similar symptoms to further confirm shb infected a. cerana abstract we report the infestation of small hive beetle, aethina tumida, in a honeybee, apis cerana, in south china. this is the first record for domestic chinese honey bee infested with small hive beetle. sociobiology an international journal on social insects hx zhao1, sa yang2, jl liu3, wz huang1, ch ji4, q ren4, xs xia1, cs hou2 article history edited by evandro nascimento silva, uefs, brazil received 25 february 2019 initial acceptance 19 april 2019 final acceptance 07 january 2020 publication date 18 april 2020 keywords aethina tumida, small hive beetle, asian honeybee, apis cerana, china. corresponding author hou chun sheng institute of apicultural research chinese academy of agricultural sciences beijing, 100093, china. e-mail: houchunsheng@caas.cn colonies (fig 1). the three honeybee apiaries were located in luhe county, shanwei city, guangdong province, china (23°11’ n, 115°33’ e). we collected these samples and identified them using morphologic characteristics as described by neumann et al. (2013). dna was extracted from shb samples using dna kit (tiangen biotech co., ltd., bj, china) following the manufacturer’s instructions. pcr amplifications were carried out in 25 µl volume containing a pair primer: forward, 5’ ggtggatcttcagttgatttagc 3’ and reverse, tcagctgggggataaaattg 3’ (neumann et al., 2013). the pcr products were run in 1.2% agarose gel stained with gvii (biomec, bj, china) and then were sent to sequence by shanghai sangonbio-tech (shanghai, china). all colonies from three apiaries were visually observed through classical shb infection symptoms (neumann & elzen, 2004). any possible shb including larvae and adults 1 guangdong key laboratory of animal conservation and resource utilization, guangdong public laboratory of wild animal conservation and utilization, guangdong institute of applied biological resources, guangdong academy of science, guangzhou, china 2 institute of apicultural research, chinese academy of agricultural sciences, beijing, china 3 south china agricultural university, guangzhou, china 4 chongqing academy of animal science, chongqing, china first detection of small hive beetle aethina tumida murray (coleoptera: nitidulidae) infesting eastern honeybee, apis cerana fabricius (hymenoptera: apidae), in china short note sociobiology 67(1): 126-128 (march, 2020) 127 were collected and brought to our laboratory for taxonomic and molecular identification. as shown in fig 2a-2d, the morphologic characteristics of the larvae and adult shbs were consistent with those of aethina tumida as demonstrated by neumann et al. (2013). in order to further confirm shb at the molecular level, we amplified a target fragment with known primer-pair and found it was in accordance with expected size, 1009 bp (fig 2e). our study indicates that shb has invaded a. cerana colonies in china and this result was in accordance with that of cervancia et al. (2016), which found shb in a. cerana colonies in the philippines. however, the origin of shb associated to a. cerana colonies in china is still unknown. here, we provided the first report of shb infesting a. cerana colonies in china and the second record in asia after the philippines in 2014. this demonstrated that shb is expanding fig 1. shb larvae aggregating on the honeybee (a. cerana) comb. a, shb invading honey bee colony (a. cerana); b, magnification of the area indicated in a. fig 2. identified the shb using morphologic characteristics and molecular method. a, larva shb; b, pupae shb; c, adult shb; d, ventral view of shb; e, molecular identification of shb. hx zhao, sa yang, ja liu, wz huang, ch ji, q ren, xs xia, cs hou – detection the small hive beetle in apiscerana128 its host range, bringing as a likely result in the reduction of colony population. thus, china must be in alert state and look for ways to prevent the diffusion of this invasive pest. acknowledgements the authors are grateful to the beekeeper, qiu ziyu, for providing the samples and help to inspect the presence of shb of apiary. we gratefully acknowledge financial support of the national natural science foundationof china (no. 31811530276, 31572471) and the earmarked fund for china agriculture research system (cars-44-syz11), gdas special project of science and technology development (2018gdascx-0107). disclosure the authors declare no conflict of interest. references al toufailia, h., alves, d.a., de c bená, d., bento, j.ms., iwanicki, n.s., cline, a.r., ellis, j.d. & ratnieks, f.l. (2017). first record of small hive beetle, aethina tumida murray, in south america. journal of apicultural research, 1:76-80. doi: 10.1080/00218839.2017.1284476. cervancia, c.r., de guzman, l.i., polintan, e.a., dupo, a.l. and locsin, a.a. (2016).current status of small hive beetle infestation in the philippines. journal of apicultural research, 6:74-77. doi: 10.1080/00218839.2016.1194053. diao, q.y., li, b.b., zhao, h.x., wu, y.y., guo, r., dai, p.l., chen, d.f., wang, q. & hou, c.s. (2018). enhancement of chronic bee paralysis virus levels in honeybees acute exposed to imidacloprid: a chinese case study. science ofthe total environment, 630:487-494. doi: 10.1016/j. scitotenv.2018.02.258. hood, w. m. (2004). the small hive beetle, aethina tumida: a review. bee world, 3: 51-59. doi: 10.1080/000 5772x. 2004.11099624. muli, e., kilonzo, j. & sookar, p. (2018). small hive beetle infestations in apis mellifera unicolor colonies in mauritius island, mauritius. bee world, 2:44-45. doi: 10.1080/00057 72x.2018.1434751. neumann, p. & elzen, p.j. (2004). the biology of the small hive beetle (aethina tumida, coleoptera: nitidulidae): gaps in our knowledge of an invasive species. apidologie, 35: 229247. doi: 10.1051/apido:2004010. neumann, p., evans, j.d., pettis, j.s., pirk, c.w.w., schäfer, m.o., tanner, g. & ellis, j.d. (2013). standard methods forsmall hive beetle research. journal of apicultural research, 4: 1-32. doi: 10.3896/ibra.1.52.4.19. su, s.k. & chen, s.l. (2009). the beekeeping and ecology. journal of bee, 1: 8-10. doi: 10.3969/j.issn.1003-9139.2009.01.036. doi: 10.13102/sociobiology.v65i4.3379sociobiology 65(4): 640-644 (october, 2018) special issue open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 toxic metals in the crude propolis and its transfer rate to the ethanolic extract introduction crude propolis (cp) is a resinous material harvesting by the bees surrounding the hives, with important protecting role for a bee colony. it has a strong and characteristic aroma, representing a complex set of substances (55% resins and balsams, 30% waxes, 10% volatile oils and about 5% pollen) and mechanical impurities (banskota et al., 2001; sonmez et al., 2005), that depends on the geographic region, botanical source and bee specie (finger et al., 2014). this bee product has used since ancient time, due the several biological and therapeutic properties, being widely used as antibacterial, antiviral, antitumor, antifungal, antioxidant, immunomodulatory, presenting many other biological activities (orsi et al., 2006; sforcin, 2007; kunimasa et al., 2011; canale et al., 2017). among the various substances present in its composition, the largest groups of isolated compounds are that of flavonoids, being also aromatic aldehydes, phenolic acids, organic acids, abstract concentrations of six toxic metals (ni, cr, hg, cd, pb and sn) in 106 samples of brazilian crude propolis and the transfer rate of these contaminants to ethanolic extract of propolis were evaluated by atomic absorption spectrophotometry. the results show the presence of all the analyzed metals in the samples of crude propolis of são paulo and minas gerais states. regarding the transfer of these metals to ethanolic extract of propolis, a significant reduction was observed for all metals analyzed. the crude propolis can be considered as an indicator of toxic metals in the environment and the reduction observed in the ethanolic extract of propolis makes the product safe for consumption. sociobiology an international journal on social insects ro orsi, dcb barros, rcm silva, jv queiroz, wlp araújo, aj shinohara article history edited by kleber delclaro, ufu, brazil received 26 april 2018 initial acceptance 04 july 2018 final acceptance 13 september 2018 publication date 11 october 2018 keywords beekeeping, environment, food safety, toxicity. corresponding author ricardo de oliveira orsi faculdade de medicina veterinária e zootecnia fmvz campus botucatu, distrito de rubião junior s/nº , caixa postal 560 cep:18618-970, botucatu-sp, brasil. e-mail: ricardo.orsi@unesp.br vitamins, amino acids, minerals, among others (banskota et al., 2001; falcão et al., 2010; huang et al., 2014). in addition, the cp has a high content of minerals, such as aluminum, vanadium, iron, calcium, silicon, manganese, among others (marcucci et al., 2001). as a natural product, the cp can be subject to the presence of toxic elements in its composition, being directly associated with the environmental pollution of the anthropic origin around the apiaries (gong et al., 2012; bonvehí & bermejo, 2013). the contamination of the environment by toxic metals is a constant concern of the society, arising from industrialization and urbanization (arslan & arikan, 2013). cp collected from hives installed in apiaries near contaminated areas may present potentially toxic metals in their composition. finger et al. (2014) found high levels of cadmium, chromium and lead in crude propolis from different regions of paraná state brazil, allowing the identification of specific areas with environmental contamination. cp can be considered an indicator of environmental contamination, college of veterinary medicine and animal sciences, são paulo state university (unesp), botucatu, sp, brazil research article bees mailto:orsi@fmvz.unesp.br sociobiology 65(4): 640-644 (october, 2018) special issue 641 as observed by several authors that investigated the serbian, spanish, macedonia, italian, among others propolis (bonvehí & bermejo, 2013; popov et al., 2017; tosic et al., 2017). however, data about the transfer rate of the metals toxics presents in the cp to ethanolic extract of propolis (eep) is scarce in scientific literature. depending on the toxic metals concentration on the eep, the biological activity of the extract can be affected (bonvehí & bermejo, 2013). the principal form of consumption of the propolis occurs in the form of extract and derived products (canale et al., 2017), and knowing the safety of this product for consumption is important. thus, the goal of this study was to evaluate the presence of six toxic metals (ni, cr, hg, cd, pb and sn) in 106 samples of brazilian crude propolis (cp) and the transfer rate of these contaminants to the ethanolic extract of propolis (eep). materials and methods sample collection samples of cp were collected in apiaries located in rio grande do norte (rn) (n = 3), pernambuco (pe) (n = 1), ceará (ce) (n = 1), goiás (go) (n = 4), tocantins (to) (n = 1), rio grande do sul (rs) (n = 9), mato grosso (mt) (n = 3), minas gerais (mg) (n = 7), são paulo (sp) (n = 50), paraná (pr) (n = 15), and santa catarina (sc) (n = 12) states, totaling 106 samples from 79 cities (fig 1). the harvested propolis kept in a freezer until analysis. samples were provided by beekeepers. preparation and analysis of potentially toxic metals in crude propolis and alcoholic extract each cp samples was crushed and homogenized with amortar and pistil. then, 1 gram was separated and homogenized again. subsequently, 100 mg of each sample was placed in “pyrex 50 ml” test tubes and then nitric acid and perchloric acid were added in a ratio of 9: 1. for the eep preparation, 30 g of the cp homogenized was diluted in 100 ml of the ethanol 70% (orsi et al., 2000). the solutions remained under the light, under frequent agitation, for seven days. after this period, the solutions were gravity filtered with a commercial 40 mesh paper filter. then, 1 ml of each eep sample was homogenized and placed in “pyrex 50 ml” test tube, and nitric acid and perchloric acid were added in a ratio of 9: 1. the cp and eep samples (residue was not analyzed) were then placed in “tecnal model te-040/25” thermostatic digestion block exhaust hood and heated at 250 ºc for two and a half hours for digestion and elimination of organic matter. after this period, the samples were resuspended in distilled water, the volume was completed to 25 ml. the qualitative and quantitative analyses were carried out with an atomic absorption spectrophotometry, according to the methodology described by sarruge and haag (1974), with equipment of the brand “varian”, model “spectro 12/1475”. the calibration, for the spectrophotometric analyses, was performed with standard solutions, according to each metal analyzed. the detection limits, in mgkg-1 of pb, ni, hg, cr, cd and sn were 0.025; 0.005; 0.01; 0.004; 0.002; 0.03 respectively. the samples analyses were performed in duplicate. for the calculation of the concentrationof metals found in the cp and eep, the following formula was used: metal concentration = [(reading metal blank reading) × vol]/ sample (100 mg cp or 1 ml eep) statistical analysis for the quantitative variables observed in the samples, the results were compared by analysis of variance (anova) with kruskal-wallis test for comparing means at the 5% level of significance (zar, 1996). results how the honeybees collect the vegetal resin to produce the propolis (cp), this product may present contamination coming from the environmental, as toxic metals. in this work, we observed that the cp produced in different regions of brazil shows toxic metals depending on the collect place (table 1). lead content in crude propolis ranged from 0.70 ± 0.8 (minas gerais state) to 6.88 ± 8.8 mgkg-1 (são paulo state), and after processing ranged from 0.10 ± 0.0 to 1.93 ± 1.1 mgl-1, with reduction average of 91.90%. the nickel content in crude propolis ranged from 0.10 ± 0.0 (paraná and rio grande do sul states) to 42.50 ± 0.0 mgkg-1 (tocantins state). after the processing, the eep nickel presence do not observed. fig 1. samples number of cp collected in the brazilian states. rio grande do norte (rn), pernambuco (pe), ceará (ce), goiás (go), tocantins (to), rio grande do sul (rs), mato grosso (mt), minas gerais (mg), são paulo (sp), paraná (pr) and santa catarina (sc) state (from https://pt.depositphotos.com/8347343/stock-photo-mapofbrazil-with-states.html.). ro orsi, dcb barros, rcm silva, jv queiroz, wlp araújo, aj shinohara – toxic metals in the crude propolis642 the mercury content in crude propolis ranged from 0.10 ± 0.0 (tocantins state) to 1.20 ± 0.3 mgkg-1 (rio grande do norte state); after processing ranged from 0.30 ± 0.0 to 0.50 ± 0.1 mgl-1, with reduction this element from 48.28% to 100.00%. the chromium content in crude propolis ranged from 0.10 ± 0.0 (paraná state) to 20.25 ± 3.6 mgkg-1 (são paulo state), however, after the processing this element was found in the eep only in the samples of são paulo state at 0.15 ± 0.1 mgl-1. the cadmium content in crude propolis ranged from 0.24 ± 0.1 (paraná state) to 0.92 ± 0.3 mgkg-1 (rio grande do sul state), and after processing ranged from 0.13 ± 0.1 to 0.20 ± 0.0 mgl1. tin content in crude propolis ranged from 1.80 ± 0.0 (ceará state) to 52.60 ± 10.7 mgkg-1 (são paulo state), and after processing ranged from 0.10 ± 0.0 to 3.68 ± 4.3 mgl-1, the percentage of reduction ranged from 71.81 to 100%. discussion evaluating the brazilian states, we can observe that the all toxic metals analyzed were found in são paulo and minas gerais, showing strong correlation with the industrialization of the southeastern region of the country. according to finger et al. (2014), some potentially toxic metals are found in waste from chemical and electronic industries, especially the burning of fossilfuels such as petroleum. the increased presence of metals in cp may be indicative of contaminated environment, how verified by other authors (bonvehí & bermejo, 2013; popov et al., 2017; tosic et al., 2017). the incorporation of toxic metals into propolis may also be associated with several factors, such as the industrial activity, with the liberation of particles that can remain suspended in the air; the indiscriminate use of fertilizers and the practice of irrigation with contaminated water (alloway, 1990; liu et al., 2009; sawidis et al., 2011). however, even areas of less anthropogenic contact and industrial activies are also subject to contamination, due to the transportation of metals, mainly through atmospheric (steinnes et al., 1997), as observed for other states evaluated, at where the toxic metal contents varied (table 1). the deposition of these potentially toxic metals in propolis can occur either by gravity or by precipitation. thus, water droplets would transport the particles to the impact areas, which would be areas where apis mellifera l. bees collect plant resins for propolis. however, there is a possibility of transport of these molecules to waterways, after primary transport by rainfall (gibbs, 1973). the movement of these pollutants can occur due to atmospheric circulation of particles, and soil deposition may occur in areas far from the pollutant source sites (onder & dursun, 2006). thus, the deposition forms in the plants or soil can be natural, by atmospheric, precipitation or anthropogenic, through irrigation or fertilization; therefore, when they are fixed in plant resins, the material used to make propolis, will be collected by a. mellifera bees. toxic metals, because they are not biodegradable, can accumulate in living tissues along the food chain reaching humans mainly through food. many organisms that feed on organic matter or natural resources from existing plants such as pollen and nectar can to absorb these toxic elements and thus be a potential risk to human health (aguiar et al., 2002), being able to alter the cellular structures, enzymes and replace metal cofactors of enzymatic activities, essentials to the metabolism of living organisms. thus, the excess or lack of these elements can lead to disorders in the body, and in extreme cases, to death (cain et al., 2004). these elements can be introduced into living tissue through water, food, respiration and even the skin itself. however, 90% of the intake of toxic metals and other contaminants occur through food consumption.thus, the consumption of propolis as a beneficial natural product for humans can become a source of contamination of toxic metals to those who use it (cain et al., 2004). glinski (2000) suggests that the contamination of bee products with toxic metals poses a serious health risk to the population since it can promote a suppression of the immune system, toxic to the organism and, consequently, diseases. however, the mainly way of human consumption is in the form of extracts or manufactured products (finger et al., 2014; canale et al., 2017), making it important the knowledge of the dynamics of transfer of this metals to products consumed by humans or animals. then, we produced the ethanolic extract of propolis (eep) of each cp sample and evaluated what would be the concentration of these after a simple extraction and filtration process. we verified that there was a significant reduction in the concentration of all the metals analyzed in the eep, ranging from 24.24 to 100.00%. this fact shows that the process of filtration and separation of the filtrate from the sludge eliminates some of the metals found in cp, reducing the risk of contamination by metals considered as toxics, and making the product safe for consumption. during the preparation of cp by the honeybees, the natural occurrence of pollen from flowers occurs, as well as bees wax. thus, the presence of hydrocarbons and fatty acids that presents in the cp shows low solubility in the ethanol and could be a source of retention to metals after the processing (banskota et al., 2001; vishchur et al., 2016). also, according to bezerra et al. (2009) there is also an affinity to toxic metals for the cellulose, that is presente in the filter paper; however, it is not possible to infer that the amount of metals absorbed and concentrated in the propolis ground was due to the extraction process by the filter. the cp can be considered as an indicator of toxic metals in the environment and the reduction observed in the epp makes the product safe for consumption. sociobiology 65(4): 640-644 (october, 2018) special issue 643 acknowledgments this work was supported by fundação de amparo à pesquisa do estado de são paulo – fapesp (process 04/03575-5). authors’ contribution ro orsi, conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing – original draft, writing – review & editing; dcb barros, conceptualization, data curation, formal analysis, investigation, methodology, resources, validation, visualization, writing – review & editing; rcm silva, conceptualization, data curation, formal analysis, investigation, methodology, resources, validation, visualization, writing – review & editing; jv queiroz, conceptualization, formal analysis, software, visualization, writing – review & editing; wlp araújo, conceptualization, formal analysis, software, visualization, writing – review & editing; aj shinohara, conceptualization, data curation, formal analysis, methodology, resources, software, validation, visualization, writing – review & editing. references aguiar, m.r.m.p., novaes a.c. & guarino a.w.s. 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(2013). element content of propolis collected from different areas of south spain. metal sp mg rn ce pe to go mt pr sc rs pb cp 6.88±8.8 0.70±0.8 2.47±1.4 0.90±0.0 lod lod 3.3±2.5 3.50±4.0 1.02±1.7 1.80±1.0 1.15±1.1 eep 1.93±1.1 0.13±0.1 0.20±0.0 0.10±0.0 lod lod 1.20±1.3 0.85±1.1 0.30±0.3 0.35±0.2 0.60±0.4 red 71.95% 81.43% 91.90% 88.89% lod lod 63.64% 75.71% 70.59% 80.56% 47.83% ni cp 4.97±1.9 0.40±0.0 lod lod lod 42.50±0.0 lod lod 0.10±0.0 1.85±1.1 0.10±0.0 eep lod lod lod lod lod lod lod lod lod lod lod red 100.00% 100.00% lod lod lod 100.00% lod lod 100.00% 100.00% 100.00% hg cp 0.92±0.7 0.66±0.3 1.20±0.3 lod lod 0.10±0.0 lod lod lod 1.04+0.3 0.58±0.5 eep lod 0.50+0.1 lod lod lod lod lod lod lod lod 0.30+0.0 red 100.00% 24.24% 100.00% lod lod 100.00% lod lod lod 100.00% 48.28% cr cp 20.25±3.6 0.40±0.0 lod lod lod 18.00±0.0 lod lod 0.10±0.0 lod lod eep 0.15+0.1 lod lod lod lod lod lod lod lod lod lod red 99.26% 100.00% lod lod lod 100.00% lod lod 100.00% lod lod cd cp 0.53±0.5 0.40±0.3 0.80±0.0 lod lod lod 0.50±0.0 0.50±0.6 0.24±0.1 0.66±0.4 0.92±0.3 eep lod 0.17±0.1 lod lod lod lod lod lod lod 0.20±0.0 0.13±0.1 red 100.00% 57.50% 100.00% lod lod lod 100.00% 100.00% 100.00% 69.70% 85.87% sn cp 52.6±10.7 8.78±4.2 7.20±5.7 1.80±0.0 6.40±0.0 9.30±0.0 3.13±1.4 2.30±0.0 8.22±5.5 12.9±9.9 10.5±6.7 eep 3.68±4.3 1.00±0.1 0.10±0.0 lod lod lod 0.20±0.0 0.6±0.0 1.59±0.9 1.95±1.2 2.96±0.6 red 93.00% 88.61% 98.61% 100.00% 100.00% 100.00% 93.61% 73.91% 80.66% 84.88% 71.81% cp: crude propolis, eep: ethanolic extract of propolis, red: percentage of reduction of metals, calculated by difference of the metals contents in the cp and the respectively eep. *comparing the averages which showed contamination levels. lod: limits of detection. abbreviatures of brazilian states: sp são paulo, mg minas gerais, rn rio grande do norte, ce ceará, pe pernambuco, to tocantins, go goiás, mt mato grosso, pr paraná, sc santa catarina, rs rio grande do sul. table 1.presence of toxic metals in the crude propolis (mgkg-1) and ethanolic extract (mgl-1) samples of propolis and reduction percentage of these metals in the extract. ro orsi, dcb barros, rcm silva, jv queiroz, wlp araújo, aj shinohara – toxic metals in the crude propolis644 environmental monitoring and assessment, 185: 6035-6047. doi: 10.1007/s10661-012-3004-3 cain, d.j., luoma, s.n. & wallace, w.g. 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(1996). biostatistical analysis. new jersey: pretince hall, 718 p https://doi.org/10.1126/science.180.4081.71 https://doi.org/10.1016/j.foodchem.2012.02.127 http://dx.doi.org/10.1093/ecam/nep024 http://dx.doi.org/10.1093/ecam/nep024 https://doi.org/10.1016/s0378-8741(00)00326-3 https://doi.org/10.1016/j.envpol.2011.08.008 https://doi.org/10.1016/j.jep.2007.05.012 https://doi.org/10.1016/j.jep.2007.05.012 https://doi.org/10.1016/j.jep.2005.06.035 abstract materials_and_methods sample_collection_and_preparation fractional_precipitation_and_resuspensio 549 influence of cave size and presence of bat guano on ant visitation by wesley dáttilo1*, ricardo e. vicente1, rafael v. nunes2 & rodrigo m. feitosa3 abstract this is the first study which evaluated the influence of cave size and presence of bat guano in ant visitation in brazilian caves. we provide a list of the ants associated with 27 caves in northeastern brazil, an area situated in the transition between cerrado (brazilian savanna) and amazon domain. the study was conducted between january and august 2010. we recorded 24 ant species inserted into 12 genera, 10 tribes, and six subfamilies. the size of the cave and the presence of guano did not influence the richness of ants, and most of the caves had single species. camponotus atriceps was the species with the larger distribution, being collected in five caves. in addition, we discuss geographic distribution of records and possible ecological roles of ants in cave environments. key words: biospeleolog y, cavernicolous; competition; invertebrates. introduction despite the fact that brazil has one of the most valuable and diversified speleological patrimonies in the world (santos et al. 2002), cave fauna inventories are rare (dessen et al. 1980). until 1994, there were 76 known vertebrate species and 537 invertebrate species inhabiting brazilian caves (pinto-da-rocha 1995). these organisms can be classified into three categories: (1) trogloxenes, which spend part of their life into the cave but return to the exterior to finish their life cycle; (2) troglophiles, which have established populations and can finish their life cycle both in the exterior and interior of 1 departament of ecolog y and botany, insect-plant interactions lab., universidade federal de mato grosso, 78060-900. cuiabá, mato grosso, brazil. 2 departament of biolog y and zoolog y, universidade federal de mato grosso, 78060-900. cuiabá, mt, brazil. 3 department of entomolog y, museu de zoologia da universidade de são paulo, 04263-000, são paulo, sp, brazil. *email: wdattilo@hotmail.com 550 sociobiolog y vol. 59, no. 2, 2012 the cave, and (3) troglobites, which are restricted to the cave environment and only finish their life cycle inside the cave (holsinger & culver 1988). one of the most important features of caves is the absence of light or low light incidence and a high environmental stability (culver 1982, howarth 1983). therefore, with the absence of photosynthetic organisms, invertebrates are responsible for major richness and abundance in almost all cave ecosystems. troglophiles can be considered the most frequent kind of organism in cave habitats (trajano 1987, trajano & gnaspini-netto 1991, ferreira & horta 2001). although ants are frequently cited inhabiting caves, there are no records of troglobite ant species in these habitats. in most of the records, ant fauna are found far from the cave entrance. in addition, ant species found in caves are usually common in other ecosystems and have wide geographic distribution. the presence of ants has been considered by author as accidental in most cases (wilso 1962, tinaut & lópez 2001, dáttilo et al. 2010). some authors attribute the ant entry in caves to foraging, being associated with bat feces (guano) (ferreira & martins 1999a, 1999b, ferreira et al. 2000, roncin & deharveng 2003, santana et al. 2010). the ants collect fresh guano, and carry it back to the nest where it is used as food (moulds 2006). these statements lead us to suppose that in the caves where guano is present, the ant visitation is higher than in the caves that this resource is not found. furthermore, as the species-area relationship is one of the more established patterns in ecolog y (macarthur & wilson 1967) and has been evidenced in cave environments (culver et al. 2004), we also suppose that the number of species of ants will increase as the size of the caves grows. finally, we provide a species list of ants associated with different caves in the southern of maranhão state, northeastern brazil and we discuss geographic distribution of records and possible ecological roles of ants in cave environments. material and methods study area we developed this work in 27 caves in the municipality of estreito, located in the marker between states of maranhão and tocantins in january and august 2010. this region is inserted into a transition zone between “cerrado” (brazilian savanna) and amazon biomes at the tocantins river basin. the 551 dattilo, w. et al. — factors influencing ant visitation to caves physiognomy surrounding the caves can be classified as cerrado sensu stricto. for detailed descriptions of this classification see the classic studies of eiten (1972) and ribeiro & walter (1998). the mean annual temperature is 26.1 ºc, with mean precipitation of 1718 mm fitting in a tropical rain pattern where the rainy period corresponds to 80% of annual precipitation (ceste 2004). the class of soil that predominates in the region is neosoil, quartzarenic, in flat topography and sandy sediment cover and alterations in rocks of quartz and sandstone (reatto et al. 1998). table 1. number, geographic coordinates, length (m), height (m), presence (p) or absence (a) of guano and number of installed pitfalls in studied caves in municipality of estreito, maranhão, brazil. cave coordinates length (m) height (m) guano n° of pitfalls 1 6°03'57" s, 47°30'06" w 5.3 1.8 (a) 2 2 6°43'17'' s, 47°28'07'' w 6 2.5 (a) 2 3 6°51'45" s, 47°28'07" w 6.2 3.5 (p) 1 4 6°50'45'' s, 47°32'00'' w 6.2 1.7 (a) 2 5 6°51'45'' s, 47°32'00'' w 6.2 2 (p) 2 6 6°32'09'' s, 47°28'46'' w 7 1.3 (a) 2 7 6°51'48" s, 47°30'58" w 7 1.6 (a) 2 8 6°51'48'' s, 47°30'50'' w 7 1 (a) 2 9 6°51'36" s, 47°31'08" w 7.4 1.4 (a) 1 10 6°52'36'' s, 47°31'08'' w 7.4 1 (a) 2 11 6°54'36'' s, 47°31'08'' w 7.4 4 (p) 2 12 6°52'20'' s, 47°29'53'' w 8 1.5 (p) 2 13 6°53'28" s, 47°29'53" w 8 1 (p) 2 14 6°44'23'' s, 47°23'20'' w 8 1.5 (p) 2 15 6°48'20'' s, 47°31'12'' w 9 1.5 (a) 2 16 6°42'22'' s, 47°29'43'' w 10.3 1 (p) 2 17 6°55'52'' s, 47°22'50'' w 11.6 1.5 (a) 2 18 6°51'45'' s, 47°27'48'' w 15 1.5 (a) 3 19 6°49'20'' s, 47°32'11'' w 16 1 (p) 3 20 6°22'12'' s, 47°32'31'' w 17 2 (a) 3 21 6°42'17'' s, 47°28'07'' w 17.5 2 (a) 3 22 6°41'18'' s, 47°27'06'' w 19.5 1 (p) 4 23 6°42'17" s, 47°29'07" w 19.5 2.5 (p) 4 24 6°55'31'' s, 47°30'58'' w 20 1.5 (a) 4 25 6°53'31'' s, 47°29'58'' w 20 1.7 (p) 4 26 6°53'31" s, 47°29'58" w 20 2 (p) 4 27 6°40'46'' s, 47°29'24'' w 21.3 3.3 (a) 4 552 sociobiolog y vol. 59, no. 2, 2012 data collection and analysis we used two kinds of sampling : pitfall traps and manual collection. pitfalls were made with 500 ml plastic cups containing a 150 ml solution composed of 70% alcohol and detergent. the pitfalls remained in the caves for 48 hours. the number of installed pitfalls ranged among caves as a function of the size of cave (table 1). manual collections were made in the installation and removal of pitfall traps. the ants collected in all caves were identified through comparisons with the available collection of the museu de zoologia of universidade de são paulo, brazil (mzsp). all collected ants were deposited in the setor de entomologia of coleção zoológica of universidade federal de mato grosso, brazil (cemt). to assess whether the presence of bat guano influenced the number of ants species we noted the data for bat guano presence/absence of each cave. for this, we used non-parametric mann-whitney u test. to evaluate if the cave area influenced the number of ants species we measures on each cave: major length, also called development and major height (table 1). spearman correlation was performed to verify if there is relation within richness and size of the cave. all tests were made using systat 8.0 (wilkinson 1998). results we found 24 ant species inserted into 12 genera, 10 tribes and six subfamilies. myrmicinae was the subfamily that had most taxa recorded with 14 species. the most abundant genera were camponotus and pheidole, represented by five and six species respectively. additionally, 81.4% of caves (n= 22) had only a single species, and camponotus atriceps (smith 1858) was present in five of 27 sampled caves (table 2). twelve of the 27 caves had guano. the presence of guano did not influence ant richness (mann-whitney, z(u)= 1.073, p= 0.283). the average size (length x height) of the caves was 16.4 m² ± 13.2; however, ant richness was not related with the cave size (spearman’s correlation, rs= -0.191, t= -0.976, p= 0.338). therefore, neither guano or cave size are modulating resources of ant richness in these environments. 553 dattilo, w. et al. — factors influencing ant visitation to caves table 2. taxonomic classification, author, year, number of cave of occurrence, geographical distribution (geo. dist.) and respective references of ant fauna collected in municipality of estreito, maranhão state, northeastern brazil in january and august 2010. *geographical distributions was determined only for ants identified to species level. geographic distribution: (neo) neotropical, (sou) south america, (am) american continent. references: (1) lapola et al. 2003, (2) giraud et al. 2000, (3) brandão 1991, (4) solis et al. 2010, (5) dáttilo et al. 2010, (6) naves 1985, (7) maes & mackay 1993. taxon, author, year cave geo.dist* references subfamily dolichoderinae tribe dolichoderini dolichoderus lund, 1831 dolichoderus sp.1 23 subfamily ecitoninae tribe ecitonini eciton latreille, 1804 eciton sp.1 7 subfamily ectatomminae tribe ectatommini ectatomma brunneum smith, 1858 22 neo 1 gnamptogenys striatula mayr, 1884 9 neo 2 subfamily formicinae tribe camponotini camponotus mayr, 1866 camponotus sp.1 5 camponotus sp.2 12 camponotus sp.3 9 camponotus atriceps mayr, 1862 4, 11, 14, 15, 26 neo 3 camponotus vittatus forel, 1904 10 neo 4 subfamily myrmicinae tribe attini acromyrmex mayr, 1865 acromyrmex hystrix (latreille, 1802) 27 sou 5 acromyrmex sp.1 13 tribe cephalotini cephalotes atratus (linnaeus, 1758) 16, 23 neo 3 554 sociobiolog y vol. 59, no. 2, 2012 discussion this is the first study we are aware of where the influence of the size of the cave and the presence of guano on the richness of ants in brazilian caves was evaluated. among the ants identified to species level, all have a wide geographic distribution (table 2) and some of them occur in many kinds of habitats (eg. pheidole obscurithorax naves 1985) including urban ones, such as camponotus vittatus forel 1904 (naves 1985, rodovalho et al. 2005). according to roncin et al. (2001), the absolute majority of ant species that table 2 (continued). taxonomic classification, author, year, number of cave of occurrence, geographical distribution (geo. dist.) and respective references of ant fauna collected in municipality of estreito, maranhão state, northeastern brazil in january and august 2010. *geographical distributions was determined only for ants identified to species level. geographic distribution: (neo) neotropical, (sou) south america, (am) american continent. references: (1) lapola et al. 2003, (2) giraud et al. 2000, (3) brandão 1991, (4) solis et al. 2010, (5) dáttilo et al. 2010, (6) naves 1985, (7) maes & mackay 1993. taxon, author, year cave geo.dist* references tribe crematogastrini crematogaster lund, 1831 crematogaster sp.1 24 crematogaster sp.2 25 crematogaster sp.3 13 tribe pheidolini pheidole westwood, 1839 pheidole sp.1 17, 21 pheidole sp.2 8* pheidole sp.3 18 pheidole sp.4 9 pheidole sp.5 3 pheidole obscurithorax naves, 1985 2, 19 am 6 tribe solenopsidini solenopsis westwood, 1840 solenopsis sp.1 14 solenopsis sp.2 6, 20 subfamily ponerinae tribe ponerini odontomachus opaciventris forel, 1899 1, 3 neo 7 555 dattilo, w. et al. — factors influencing ant visitation to caves occur inside caves are also found in environments outside the cave. the low number of ant species by cave was already found by other authors in northeastern and southern brazil (ferreira & horta 2001, silva et al. 2005, silva & ferreira 2009a, 2009b, santana et al. 2010). the low number of species in all caves and its lack of relationship with the studied variables (presence of guano and size of the cave), added to wide geographical distributions of found species, emphasizes that the presence of the ants in these caves is accidental, which has been reported several times (wilson 1962, tinaut & lópez 2001, dáttilo et al. 2010). it is noteworthy that the size of the caves in this study are small, though some authors claim that the ants associated with caves can be found only at the entrance of the cave (ferreira & martins 1999a, 1999b, jordão 2003, santana et al. 2010, dáttilo et al. 2010). the species-area relationship is one of the most studied patterns in ecolog y and used in different systems (macarthur & wilson 1963, vasconcelos et al. 2006, lozano-zambrano et al. 2009), but it does not apply to cave environments. this pattern is based on larger areas that include a high variety and availability of habitats (macarthur & wilson 1967), which should not apply to a cave, since its environmental conditions are constant (belles 1987) and the resources are usually scarce and unpredictable (christiansen 1965). thus, considering only the size of the cave, there isn’t an expected increase in species richness of ants in this environment. additionally, in some cases, the presence of guano may become a limiting factor for the increase in richness and abundance of ants and other invertebrate predators, since many organisms associated with this type of substrate are an important funding source for predators (gnaspini-neto 1989, ferreira & martins 1999a, 1999b, ferreira et al. 2000, santana et al. 2010). however, in this study the presence of guano didn’t influence the richness of ants in the caves. this probably occurred because the caves were small and the accumulation of guano wasn’t large enough to support several species of ants. besides, the competition between the ants would limit the increase in richness of the ants in caves where the guano was present. the colonization and foraging of many arthropods including ants in caves mainly occurs due the favorable and constant environmental conditions inside the cave (bellés 1987). despite these favorable conditions, the availability of food resources may negatively affect spatial distribution and 556 sociobiolog y vol. 59, no. 2, 2012 diversity of ant fauna inside caves (poulson & culver 1969). this may occur because resources in caves are usually scarce and unpredictable, which, over long periods of time, would require morphological and physiological adaptations (christiansen 1965) that ants do not have. also, it is probable that high rates of competition due to the scarcity of food resources allow few species to establish inside caves generating the “one ant species for one cave” pattern, related in our study. these are two good reasons why wilson (1962) considers that ants and other social insects can not be a “real” troglobyte. the probability of existence of troglobyte ants has generated discussion among researches for a long time, mainly with respect to the absence of gene flow within cave populations (wilson 1962, tinaut & lópez 2001, roncin & deharveng 2003). some researchers suggest that some ant species are probably troglobytes, such as leptogenys khammouanensis roncin & deharveng 2003 (tinaut & lópez 2001) and hypoponera ragusai (emery 1894) (roncin & deharveng 2003), mainly due to the constant presence of nests of both species in cave interiors and males and females being apterous. however, there is the possibility that these species, being subterranean, simply prefer cryptic habitats, for instance, rodent galleries (decu et al. 1998, tinaut & lópez 2001). additionally, the scarcity of resources faced by subterranean ants is a good parallel for us to understand the difficulties that a troglobyte ant would find in this kind of habitat (deharveng & bedos 2000). despite the difficulty of categorizing these insects in the three cave fauna categories of organisms, ants may have an important role in cave ecosystems seeing as they may promote the transitions of nutrients between the exterior and interior of cave. further studies that characterize the ecolog y and faunistic composition of brazilian caves are recommended for conservation and management of these unique habitats. acknowledgements we thank thiago izzo for careful statistical support and jéssica falcão for comments on earlier versions of this manuscript. we also thank capes for the masters fellowship to wd and rvn. rmf thanks fapesp for a phd. fellowship (nº 2011/24160-1), and cnpq for a dti fellowship to rev (nº 381261/2011-5). 557 dattilo, w. et al. — factors influencing ant visitation to caves references bellés, x. 1987. fauna cavernicola i intersticial de la península ibérica i les illes balears. mallorca: consell superior d’investigacions científiques. 207 pp. brandão, c.r.f. 1991. adendos ao catálogo abreviado das formigas da região neotropical (hymenoptera: formicidae). rev. bras. entomol. 35: 319-412. ceste, e. 2004. estudo de impacto ambiental-relatório de impacto ambiental do ahe estreito. rio de janeiro: ceste consórcio estreito de energia. 235 pp. christiansen, k.a. 1965. behavior and form in the evolution of cave collembola. evolution 19: 529-533. culver, d.c. 1982. cave life. massachusets: harvard university press. 189 pp. dáttilo, w.f.c., r.e. vicente, r.v. nunes, & m.s.g. carvalho. 2010. primeiro registro da quenquém 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cornelius 2002; amburgey 1977; johnson & others 2011). according to su (1982) mortality should nott be the sole basis on which insecticidesare evaluated, rather a behavioral responses should also be considered, due to the nature of termites to effectively seal off or avoid treated areas. when a standard laboratory termite test (american wood protection association e1-09) was conducted in 2-3forest products department, mississippi state university, p.o. box 9820 mississippi state, ms 39762 1graduate research assistant, email: tjohnson@cfr.msstate.edu 2assistant professor, email: skitchens@cfr.msstate.edu 3professor emeritus, email: terryamburgey@yahoo.com 1460 sociobiolog y vol. 59, no. 4, 2012 september of 2011 utilizing a single colony of reticulitermes sp. exposed to organosilane treated wafers, behavioral changes and color phenomena were observed ( johnson & others 2011) as well significant results in regard to specimen weight losses as well as termite mortality ( johnson 2012). results of that study indicated termite mortality that was potentially caused by the bacterium serratia marcescens (bizio), though there was no known introduction of the bacteria to the termite test specimens. observations made by johnson & others (2011) closely matched observations made in a study by debach and mcomie (1939) in which termites exposed to s. marcescens exhibited varying red colorations post mortem and characteristic behavior changes. s. marcescens is known to have pathogenic effects when introduced to many termite species (debach & mcomie 1939; khan & others 1977; connick jr. & others 2001; osbrink & others 2001); however, s. marcescens is also a facultative anaerobe that is a symbiont of the anaerobic protozoa within the termite gut (adams & boopathy 2005). the common presence of s. marcescens in the digestive tracts of many insects is found to be nonpathogenic due to the lack of invasive power for the bacterium to penetrate the mid-gut wall (burges & hussey 1971). when something disrupts the symbiotic relationships of a gut bacteria however, (e.g., termites treated with immuno-suppressing compounds) the termites’ immune defense response is suppressed (connick & others 2001; osbrink & others 2001). johnson & others (2011) hypothesized that a pathogenic response of s. marcescens, hosted by the termites, might be triggered by the organosilane compound through the disruption of symbiotic protozoa present in the gut of termites, though termite cadavers of the study were not analyzed for positive identification of the presence of the bacterium. isolated strains of s. marcescens were used in a study on c. formosanus (connick & others 2001), yielding high termite mortality rates, and red features observed post mortem. both members of the family rhinotermitidae, reticulitermes and coptotermes are classified as “lower termites”, which harbor dense populations of gut protists (ohkuma & others 2001). s. marcescens is specifically documented to be present in the gut of c. formosanus and is credited to causing septicemia (blood poisoning ) in c. formosanus (adams & boopathy 2001; osbrink & others 2001. the purpose of this study was to determine if similar phenomena occurred by 1461 johnson, t.e. et al. — continuation study of subterranean termites the introduction of an organosilane treated food source to c. formosanus, as occurred with native termite species ( johnson & others 2011). methods the awpa e1-09 standard method for laboratory evaluation to determine resistance to subterranean termites was observed for this test (awpa 2011a). termites (c. formosanus) were collected from pine-veneer bucket traps at the mississippi agricultural and forestry experiment station in mcneill, ms (awpa hazard zone 5) (fig. 1). coptotermes formosanus (shiraki) isthe primary termite species at this site and test organisms were identified based on physical attributes. southern yellow pine (pinus spp.) sapwood wafers vacuum-treated with formulations of the organosilane 3-(trimethoxysilyl)propyldimethyl octadecyl ammonium chloride, (si-quat), were used as the food source for worker termites. the si-quat used in this study is widely known for its antimicrobial properties (kemper & others 2005; isquith & others 1972; hayes & white 1984; monticello & others 2009). non-treated pine sapwood wafers served as controls. the treatments are listed in tablea 1. non-sterile test chambers were constructed from french-square bottles containing 150-ml of pool filter sand and 20-ml. of deionized water (deviation from the standard 30-ml.). test wafers were placed in contact with the sand in each test chamber and 1-g of termites (9% soldiers) was added to each chamber (deviation from the standard 400 termites). the duration of this test was twenty-eight days. results and discussion some mortality was documented with termites exposed to treated wood wafers during an initial inspection fourteen days into the study. final results at the end of the testing period showed significantly less weight loss of test wafers among all treatment groups in comparison to the untreated control group. the 2.5% si-plus treatment group had the least weight lossleast feeding and highest block rating of all treatment groups. as shown in table 2, block ratings were higher for all treatment groups (indicating resistance to termite attack) than the untreated control group ratings. termite mortality was significant among all treatment groups with heavy/ complete mortality in the 2.5% ai treatment groups. no unusual observations were made on termite 1462 sociobiolog y vol. 59, no. 4, 2012 behavior in the present study as were made in past studies with reticulitermes sp. ( johnson & others 2011). one possible explanation is the documented tolerance of c. formosanus to chemical treatments in regards to susceptibility. in a study conducted by su (1990) in which both c. formosanus and reticulitermes flavipes (kollar) were utilized to evaluate eleven soil termiticides, it was concluded that r. flavipes was more susceptible to all termiticides used. for many wood preservatives, certain retentions must be attained for suitable use in exposure areas subject to formosan termite activity (awpa 2011b) due to higher tolerance of the formosan. summary the primary goal of the present study was to examine feeding of c. formosanus on organosilane-treated wood wafers. treatment of wood wafers with the organosilane compound showed increased termite resistance over nontreated wafers, and high termite mortality. though high levels of resistance and mortality were reported, observations of worker termite behavior and post mortem appearance did not yield abnormal results as were observed with reticulitermes sp. perhaps studies conducted utilizing higher concentrations of the organosilane 3-(trimethoxysilyl)-propyldimethyl octadecyl ammonium chloride should be conducted in an attempt to provoke a pathogenic response of the gut bacteria, s. marcescens. literature cited adams, l. & r. boopathy 2005. isolation and characterization of enteric bacteria from the hindgut of formosan termite. bioresource technolog y 96:1592-1598. amburgey, t.l. 1977. factors influencing termite feeding on brown-rotted wood. sociobiolog y 3:3-12. 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(http://www.aegisasia.com/pdfnew/improved_control_of_microbial_exposure_a_30_ month_field.pdf ) monticello, r.a., s.c. kitchens & t.l. amburgey 2009. a commercial, registered, and durable antimicrobial available for use on wood and wood products. proceedings, third international coating wood and wood composites conference: durable and sustainable-today and beyond. charlotte, nc. ohkuma, m., s. noda, y. hongoh & t. kudo 2001. coevolution of symbiotic systems of termites and their gut microorganisms. riken rev 41:73-74 osbrink, w.l.a., k.s. williams, w.j. connick jr., m.s. wright & a . r . l a x 2 0 0 1 . virulence of bacteria associated with the formosan subterranean termite (isoptera: rhinotermitidae) in new orleans, la environmental entomolog y 30(2):443-448 rosahn, p.d. & c.k. hu 1933. pathogenicity of serratia marcescens (bacillus prodigiosus). proceedings of the society of experimental biolog y and medicine 30:1326-1327 su, n.y., m. tamashiro, j.r. yates & m.i. haverty 1982. effect of behavior on the evaluation of insecticides for prevention of or remedial control of the formosan subterranean termite. journal of economic entomolog y 75(2):188-193 su, n.y. & r.h. scheffrahn 1990. comparison of eleven soil termiticides against the formosan subterranean termite and eastern subterranean termite (isoptera: rhinotermitidae). journal of economic entomolog y 83(5):1918-1924 1464 sociobiolog y vol. 59, no. 4, 2012 yu, v.l. 1979. serratia marcescens: historical perspective and clinical review. new england journal of medicine 300: 887-893 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.4968sociobiology 67(3): 401-407 (september, 2020) introduction johan christian fabricius (1745–1808) was one of the most important entomologists of the 18th century, having named nearly 10,000 species of animals and established the basis for the modern insect classification (zimsen, 1964; tuxen, 1967). born in tönder, south denmark, fabricius had a very liberal upbringing for his time (tuxen, 1967). when he was seventeen, he went to copenhagen and later to uppsala (sweden) where he remained for two years studying with carl nilsson linnaeus (1707–1778) (tuxen, 1967). from linnaeus’ work fabricius learned that "the systematic order with which the study of sciences ought to be pursued" and during his stay in sweden, he began the study of insects (papavero, 1971). in 1764, he returned to copenhagen and there he laid the foundations of his “genera insectorum” (fabricius, 1776), based on the small collection he possessed (papavero, 1971). abstract in this paper the primary types of centris bees described by the danish entomologist johan christian fabricius were studied. the primary types of c. flavifrons, c. analis, c. furcata, c. haemorrhoidalis, c. lanipes, c. longimana, c. similis, c. tabaniformis, and c. versicolor were analyzed, providing notes on their current status and depository. in addition, some photographs of selected species as well as morphological characteristics to recognize all fabricius’ centris bees are also provided. sociobiology an international journal on social insects f vivallo article history edited by candida aguiar, uefs, brazil received 14 january 2020 initial acceptance 16 june 2020 final acceptance 22 july 2020 publication date 30 september, 2020 keywords anthophila, centridini, neotropical region, solitary bees, taxonomy. corresponding author felipe vivallo hymn laboratório de hymenoptera departamento de entomologia museu nacional universidade federal do rio de janeiro quinta da boa vista, são cristóvão cep: 20940-040, rio de janeiro-rj, brasil. e-mail: fvivallo@yahoo.com in 1765, fabricius went to leipzig (germany) to study economy. there he was zealously engaged in writing his “systema entomologiae” (fabricius, 1775), and in collecting plants and insects of the neighborhood (papavero, 1971). in 1767, fabricius went to edinburgh (scotland) and later to london (england). there, he was part of the most intimate circle of the swedish botanist daniel solander (1733–1782), at that time located at the british museum (currently the natural history museum, london, nhmuk). solander introduced fabricius to the scientific clubs and to the british naturalist and botanist joseph banks (1743–1820). thanks to the friendship with banks, fabricius established contact with other important naturalists who allowed him access to their important libraries and insect collections. during that time, he identified and described the insects of his colleagues, at the same time that his “systema entomologiae” gained ground considerably, as well as his own insect collection which was sent to copenhagen (papavero, 1971). museu nacional, universidade federal do rio de janeiro, rio de janeiro-rj, brazil research article bees the nominal species of the bee genus centris described by johan christian fabricius (hymenoptera: apidae) f vivallo – the fabricius’ centris bees402 between 1772 and 1775 fabricius spent the winters in copenhagen and the summers in london. his friends banks and solander had returned from their voyage around the world, and had brought numerous specimens, especially insects (papavero, 1971). in 1775, fabricius published his work “systema entomologiae”, which included the descriptions of the bees apis flavifrons, a. haemorrhoidalis, a. lanipes and a. versicolor. all these species would be later transferred to the genus centris fabricius, 1804 that he would create subsequently. between 1798 and 1804, fabricius went to copenhagen every spring to describe the many new insects that his friends and students count ove ramel sehestedt and the danish zoologist niels tønder lund (1749–1809) accumulated (papavero, 1971). in 1804, fabricius published “systema piezatorum” which included several new species of insects from different parts of the world. in that book he described the genus centris, including the species c. longimana and c. tabaniformis. between the bees described, fabricius also proposed bombus furcatus, b. similis and anthophora analis as new species. all these also currently placed in the genus centris. fabricius’ hymenoptera collection according to papavero (1971) fabricius’ own collection remained in kiel (germany) after his death. in 1950, it was transferred from the zoologischen museum kiel (zmk) to the natural history museum of denmark (nhmd, formerly the zoological museum of the university of copenhagen zmuc), where is now kept as a long term loan from the zmk (tuxen 1967). the fabricius’ collection is still arranged in the order of his monographs (zimsen, 1964). the hymenoptera collection consists of 24 boxes and it is no longer in fabricius’ original boxes, but the name label, however, has not been removed from the insects (zimsen, 1964). fabricius’ hymenopterous is the collection in which the insects have most often been rearranged in the past, and this fact does not make the work easier for future specialists (zimsen, 1964). it is not clear who was the collector of some of the fabricius’ centris bees. in c. analis, c. furcata, c. longimana, c. similis, and c. tabaniformis he only cited it as “schmidt”. according to belkin et al. (1965) those specimens were collected by the surgeon johan christian schmidt, an employed in saint croix, then a danish possession in the west indies. belkin et al. (1965) pointed out that the majority the mosquitoes species described by fabricius’s and collected by schmidt do not occur in the island of saint croix or in the lesser antilles and furthermore the locality “america meridionali” probably indicates the south american continent. this also may apply to some of the bees described by fabricius, like c. furcata, c. longimana and c. similis. schmidt visited several west indian islands, certain places in south america mainland, such as essequibo in guyana (zimsen, 1964). therefore, all of the south american species cited as having been collected by him can with certainty be considered as coming from the vicinity of the named locality. in “systema entomologie” fabricius (1775) mentioned “rohr” as one of the collectors of some of the specimens he studied. julius philip benjamin von rohr (1735–1792) was a naturalist and medical who went to the west indies in 1757 and again in 1783 (zimsen, 1964). by order of the government of that time, he made a zoological journey to puerto rico, jamaica, the lesser antilles and the nearest countries along the coast of south america, mainly cayenne, french guiana (zimsen, 1964). from that trip he sent to denmark a large collection of insects that were studied later. the specimens of the collection of count ove ramel sehestedt are an important part of the fabricius collection, and many of its types are very well preserved (zimsen, 1964). this collection includes many tropical insects that came to fabricius’ collection through danish officers sent to the colonies of denmark in guinea (now ghana), tranquebar (now india) and to the west indies. sehestedt combined his collection with that of lund, and this latter was the one with the right of possession. due to his unexpected death on the way to norway, the collection was sold by his widow to the danish state (zimsen, 1964). sehestedt and lund were in contact with several tropical collectors, many of whom sent their insects to fabricius. of the approximately 1500 new taxa described by fabricius (1775) in “systema entomologie”, one third was from joseph banks’s collection, including the type specimen of c. flavifrons. banks (1743–1820) was a british naturalist and his collection, including the large number of fabrician types, went to the british museum via the london linnean society. the banks collection is currently housed at the nhmuk. the neotropical bee species described by fabricius were originally studied by moure (1960). however, in this article is presented additional information on the species belong to the genus centris which complement that provided by moure (1960). material and methods the primary types were personally studied. all labels are yellowish white (due to the effect of time) and rectangular, and the data contained on them is black, handwritten or printed, unless otherwise indicated. the specific features of the labels, like coloration or type of writing are presented in squared brackets ([]). the morphological terminology follows michener (2007). results recognition of fabricius’ primary types according to moure (1960) the primary types of centris longimana and c. similis were in the zoological museum of the university of copenhagen (now nhmd), while those of c. analis, c. haemorrhoidalis and c. lanipes were in the museum of kiel (zmk). however, all these sociobiology 67(3): 401-407 (september, 2020) 403 types are currently housed at nhmd. the primary type of c. flavifrons in housed in the nhmuk at the banks’ collection. fabricius did not indicate the sex of the species described nor the number of specimens he used on each description. his type specimens can be recognized by his handwritten labels (see fig 1 in ruta 2013 and fig 1, plate 19 in horn & kahle 1936). systematics genus centris fabricius, 1804 centris (centris) fabricius, 1804 centris flavifrons (fabricius, 1775) apis flavifrons fabricius, 1775: 383. type data this species was described based on an unknown number of specimens collected in an undetermined locality in brazil (“brasilia”). fabricius (1775) did not indicate the sex of the specimen(s) he studied, but moure (1960) found a single male in the banks collection, assuming it was the holotype. the specimen is currently housed at nhmuk and it has the following data label: [circular white label without text]\ [black-rimmed] apis flavifrons. fabr. sp. nel.[?] n°41 [handwritten]\ bmnh(e) #668695 [printed] (nhmuk). type locality brazil (“brasilia”) [probably near rio de janeiro, see moure (1960)]. comments despite the antiquity of the specimen, it is very wellconserved. centris flavifrons is widely distributed in the neotropical region, ranging from argentina to mexico. it has different phenotypes that were formally proposed as new nominal species, subspecies or varieties, which are now considered junior synonyms (see moure et al., 2007). this species has yellowish pubescence on mesoscutum and mesoscutellum, and an intertegular band of blackish hairs, sometimes covering almost completely the dorsal surface of the mesosoma, or very reduced, almost absent. females have the trimmal angle rounded and well-developed (fig 14 in snelling, 1984). males have a narrow band of whitish pubescence on the distal margin of terga 2 to 4 interrupted in the middle or if complete, then with shorter hairs in the middle (snelling, 1984). this species has metander males which have the clypeus smooth and polished with scatter punctures, and the black marks of clypeus extending onto the disc (snelling, 1984). centris haemorrhoidalis (fabricius, 1775) apis haemorrhoidalis fabricius, 1775: 386. figs. 1, 2 type data fabricius did not indicate the sex or how many specimens he used to describe this species. moure (1960) designated the lectotype choosing a female now housed at nhmd. the specimen has the following data label: haemorrhoidalis. [handwritten]\[white label with black rim] lectotype centris haemorrhoidalis fabricius [handwritten] det. j. s. moure 19 [printed] 58 [handwritten] (nhmd). a headless male paralectotype without labels is also housed at nhmd. type locality fabricius (1775) indicated the type locality as “america”, but moure (1960) cited it as “americae insulis” [probably greater antilles, see moure (1960)]. comments fabricius (1775) did not mention the collector of the type specimens. this species has been recorded in cuba, grenada, jamaica, puerto rico (moure et al., 2007), british virgin islands, hispaniola island and united states virgin islands (genaro & franz 2008). both sexes have blackish pubescence covering the body almost completely, except on distal terga and sterna. females have blackish metasoma with greenish or bluish metallic reflections, except terga 4 to 6 orange (figs 1, 2). males’ metasoma is similar, except for figs 1-2. centris haemorrhoidalis (fabricius, 1775) (lectotype female). fig 1: habitus, dorsal view (scale bar 1 mm). fig 2: habitus, lateral view (scale bar 1 mm). f vivallo – the fabricius’ centris bees404 a yellowish spot on lateral sides of tergum 2 and the orange terga extend from segments 4 to 7 (figs. 3, 4). centris tabaniformis fabricius, 1804 centris tabaniformis fabricius, 1804: 358. junior synonym of c. haemorrhoidalis (moure 1960). figs. 3, 4 type data this nominal species was described based on at least two males collected in an undetermined locality cited as “america meridionali”. both specimens are housed at nhmd, one of them designated the lectotype by moure (1960). the specimen has the following data label: c. tabaniformis [indecipherable writing] schmidt lectotype♂ haemorrhoidalis (f.) moure 1958 x [handwritten\ [red label] type [printed]\ [white label] zmuc 00241527 [printed] (nhmd). type locality “america meridionali”’. comments the type specimen was collected by schmidt and belonged to the sehestedt collection. moure (1960) considered that “america meridionalis” corresponds to a continental site in south america, specifically guyana. nevertheless, for the label of the type of c. tabaniformis he interpreted that it was a mistake for “america meridionalis insulis” (lesser antilles), since at that time he had only seen specimens from islands of that region. centris versicolor (fabricius, 1775) apis versicolor fabricius, 1775: 386. figs. 5, 6 type data this species was proposed based on an unknown number of specimens collected in an undetermined locality in america. three females of two different species compose the type series. moure (1960) designated lectotype the specimen better conserved and the only one with original fabricius’ label. the specimen has the following data label: versicolor [handwritten]\ [white label with black rim] lectotype centris versicolor [handwritten] det. j. s. moure 19 [printed] 58 [handwritten] (nhmd). the paralectotypes are badly damaged, they belong to c. smithii cresson, 1879 and they do not have labels. type locality america [probably lesser antilles, see moure 1960]. comments the type specimens were collected by julius rohr, who also collected the primary types of c. lanipes. both sexes figs 3-4. centris tabaniformis fabricius, 1804 (lectotype male). fig 3: habitus, dorsal view (scale bar 1 mm). fig 4: habitus, lateral view (scale bar 1 mm). of this species have the mandibles blackish. females have the mesosoma covered by orange to light orange pubescence as well as the scopa, and the metasoma has the first three terga and the anterior half of the fourth with bluish metallic reflections. the rest of the terga are orange and covered by yellow hairs (figs. 5, 6). males have similar pubescence than females. centris (hemisiella) moure, 1945 centris lanipes (fabricius, 1775) apis lanipes fabricius, 1775: 386. figs. 7, 8 type data fabricius proposed this species based on an unknown number of specimens collected in an undetermined locality in america. the type series was composed at least by four females and one male currently housed at nhmd. in 1960, moure designated one of the females as the lectotype, choosing the specimen with original fabricius’ label. the lectotype has the following data label: lanipes [handwritten]\ [white label with black rim] lectotype centris lanipes f ♀ [handwritten] det. j.s. moure 19 [printed] 58 [handwritten] (nhmd). one of the females was collected in brazil and it represents a different species. the rest of the paralectotypes do not have labels and are in general badly damage. sociobiology 67(3): 401-407 (september, 2020) 405 type locality america [probably greater antilles, see moure 1960]. comments the type specimens were collected by julius rohr, who also collected the primary types of c. versicolor. this species is only known from the type specimens. genaro & franz (2008) cited it in several caribbean islands, but unfortunately as result of a misidentification. the mesoscutum and mesoscutellum are covered by light orange pubescence, darker in the scopa; the basitarsus of middle legs has orange hairs; the clypeus does not have a longitudinal raised line, and the metasoma is uniformly orange (figs 7, 8). additional information on the morphology of this species can be found in moure (1960). centris (heterocentris) cockerell, 1899 centris analis (fabricius, 1804) anthophora analis fabricius, 1804: 375. type data this species was described based on an unknown number of males. two syntypes were found at nhmd and the one collected by schmidt was designated by moure (1960) as the lectotype. the specimen has the following data label: a: analis ex am: mer: schmidt lectotype c. (heterocentris) ♂ moure 58 x [handwritten]\ [red label] type [printed]\ [white label] zmuc 00241561 [printed] (zmc). the paralectotype was not studied. type locality “amer. mer.” [according to moure (1960) probably from guiana]. comments the type specimen was collected by schmidt and it belonged to the collection of niels tønder lund. moure wrote the word “lectotype” directly on the original fabricius’ label. the females of this species have the lower corner of the pronotum with a few yellow to orange, long, coarse and simple hairs. males have the mandible tridentate with the innermost teeth small and close to each other, and a yellow spot on the anteroapical surface of the scape. this species was correctly interpreted by snelling (1984) and thiele (2003). centris (melanocentris) friese, 1901 centris furcata (fabricius, 1804) bombus furcatus fabricius, 1804: 350. figs 5-6. centris versicolor (fabricius, 1775) (lectotype female). fig 5: habitus, dorsal view (scale bar 1 mm). fig 6: habitus, lateral view (scale bar 1 mm). figs 7-8. centris lanipes (fabricius, 1775) (lectotype female). fig 7: habitus, dorsal view (scale bar 1 mm). fig 8: habitus, lateral view (scale bar 1 mm). f vivallo – the fabricius’ centris bees406 the primary type of this species was recently studied so it seems not necessary to cite the same information here. for information about the label as well as notes of the morphology of the species see vivallo (2016). centris (trachina) klug, 1810 centris longimana fabricius, 1804 centris longimana fabricius, 1804: 356. type data this species was proposed based on an undetermined number of specimens collected in “america meridionali”. the type series, currently housed at nhmd was studied by moure (1960) who designated a female as the lectotype. the specimen has the following data label: c: longimana ex am: mer: schmidt holotype ♀ moure 1954 x [handwritten]\ [red label] type [printed] (nhmd). according to moure (1960) this specimen, along with the male that compose the type series belonged to the sehestedt’s collection. type locality “america meridionali” [according to moure (1960) probably guiana]. comment females have the interantennal and paraocular areas with yellowish pubescence; the vertex, mesoscutum and mesoscutellum with the base of the hairs grayish or whitish and dark brown apex; the labrum and the mandibles mostly yellow, and the clypeus dark brown with a relatively narrow yellow inverted t-like stain. males have the mesosoma with brownish pubescence, with slightly lighter hairs on mesoscutellum; the clypeus relatively convex (in lateral view) with two large dark brown spots projected downwards and surpassing the upper half. centris similis (fabricius, 1804) bombus similis fabricius, 1804: 351. type data fabricius (1804) described this species based on two females from “america meridionali”. one of the syntypes, currently housed at nhmd was studied by moure (1960) designating it as the lectotype. the specimen has the following data label: bo similis ex am: mer: schmidt x [handwritten]\ [red label] type [printed]\ lectotye [printed] centris similis (f) [handwritten] j. s. moure [printed] 1958 [handwritten] (nhmd). according to moure (1960) there is an additional specimen of the type series, now paralectotype, housed at nhmd but it was not examined during the development of this article. type locality “america meridionali” [according to moure (1960) probably guiana]. comments the specimen was collected by schmidt, the same collector of the primary types of c. analis and c. furcata. the specimen belonged to the lund’s collection. morphological characters to identify this species can be found in snelling (1984). discussion all the fabricius’ species were described in the same very short manner: the name, an extremely short diagnosis, and a reference to the locality and collector. unfortunately, he generally mentioned the locality in the broadest sense: “america”, “america meridionali” or “america insulis”, which turn almost impossible to determine the real locality where the specimens were collected. due the way in that fabricius described his species the direct study of their primary types is fundamental to recognize them, as was originally done by moure (1960) and now in the present paper. this also applies to other old entomologist, like amédée louis michel lepeletier de saint-fargeau (1770‒1845), heinrich friese (1860‒1948) and theodore dru alison cockerell (1866‒1948), three of the most important melittologist that worked with bees from the new world and whose species can only be identified studying their type specimens. some of the species described by fabricius are very common and occur widely in the neotropical region, like centris analis, c. longimana and c. similis. however, other species, like c. lanipes and c. versicolor have been misidentified for a long time, originating incorrect information about their bionomy and distributional range. the primary types of the centris bees described by fabricius are well curated and in a very good condition, despite their antiquity. this fact is very important for the taxonomists that will need to study these and other type specimens in the future. acknowledgments i thank lars vilhelmsen, jesper birkedal schmidt, mikkel høgh post (nhmd) and michael ohl (museum für naturkunde, germany) for their help during the development of this article. i also thank the anonymous reviewers for helping in improving it. financial support was provided by conselho nacional de desenvolvimento científico e tecnológico (cnpq, grant 444320/2014-8), brazil. this paper is part of the sigma project nº21565 mn/ufrj and the contribution number 48 from the hymn. sociobiology 67(3): 401-407 (september, 2020) 407 references belkin, j.n., schick, r.x. & heinemann, s.j. 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(1964). the type material of j.c. fabricius. munksgaard, copenhagen. 656 p. doi: 10.13102/sociobiology.v61i3.300-306sociobiology 61(3): 300-306 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 polygyny, inbreeding and wingless males in the malagasy ant cardiocondyla shuckardi forel (hymenoptera, formicidae) j heinze,1 a schrempf,1 t wanke1, h rakotondrazafy,2 t rakotondranaivo2, b fisher2,3 introduction the myrmicine ant genus cardiocondyla comprises about 100 species of minute to small ants that are widely distributed throughout africa, europe, and asia (seifert, 2003). colony composition and the reproductive behavior of queens and males vary tremendously among species, making cardiocondyla an ideal system to investigate the evolution of social structures and alternative reproductive tactics in ants. because of its ancestral male diphenism with winged disperser males and wingless, “ergatoid” males (kugler, 1983), cardiocondyla has become a model system for the investigation of alternative reproductive tactics in male ants (e.g., oettler et al., 2010; schwander & leimar, 2011). winged males resemble typical males of other ant species in morphology, reproductive physiology, and behavior: they are peaceful, disperse shortly after eclosion, and use their limited sperm supply to inseminate young queens from other colonies. wingless males, in contrast have strong shear or sickle-shaped mandibles, are relatively long-lived (several abstract the ant genus cardiocondyla exhibits a fascinating diversity of its reproductive biology, with winged and wingless males, long-winged and short-winged queens, strict monogyny and facultative polygyny with or without queen fighting. here we report on the previously unstudied malagasy ant c. shuckardi. we describe the nesting habits, male morphology and colony structure of this species. furthermore, based on the genotypes from three microsatellite loci we document a very high incidence of sib-mating. sociobiology an international journal on social insects 1 universität regensburg, biologie i, 93040 regensburg, germany. 2 madagascar biodiversity center, pbzt tsimbazaza, 101 antananarivo, madagascar. 3 california academy of sciences, san francisco, usa. article history edited by gilberto m m santos, uefs, brazil received 30 january 2014 initial acceptance 05 april 2014 final acceptance 08 july 2014 keywords inbreeding, reproductive tactics, ergatoid males, colony structure, madagascar. corresponding author: jürgen heinze universität regensburg, biologie i, 93040, regensburg, germany e-mail: juergen.heinze@ur.de weeks to one year, yamauchi et al., 2006) and, uniquely among social hymenoptera, have lifelong spermatogenesis (heinze & hölldobler, 1993). wingless males rarely if ever disperse, and in many species engage in lethal fighting with rivals and attempt to monopolize mating with all female sexuals emerging from their natal nests (stuart et al., 1987; kinomura & yamauchi, 1987; heinze et al., 1998). within cardiocondyla, winged disperser males have been lost convergently in at least two clades, and in several species fighting among wingless males has been replaced by territoriality or mutual tolerance (frohschammer & heinze, 2009; lenoir et al., 2007). queen behavior is similarly variable. facultative polygyny, i.e., the peaceful coexistence of several fertile queens per nest, appears to be the ancestral state, from which queen-queen fighting (yamauchi et al., 2007; heinze & weber, 2011) and obligate monogyny (a single queen per nest) have evolved (schrempf & heinze, 2007). previous investigations have focused on the social organization and life history of species of southeast asian or central european origin (kugler, 1983; kinomura & yaresearch article ants sociobiology 61(3): 300-306 (september 2014) 301 mauchi, 1987; heinze et al., 1998; lenoir et al., 2007), but little is known about the african “c. shuckardi group” (sensu seifert, 2003). this group is of particular interest as it is phylogenetically situated between the c. nuda clade (with facultative polygyny and male fighting at least in c. mauritanica and c. kagutsuchi) and a clade consisting of several monogynous species with only wingless, mutually tolerant males (oettler et al., 2010). one of the six presently recognized species of the c. shuckardi group, c. venustula wheeler, 1908 has recently been studied in populations introduced to kaua’i (hawai’i; frohschammer & heinze, 2009) and puerto rico (j. heinze and susanne jacobs, unpubl.), and in a native population in south africa (heinze et al., 2013). as yet, only wingless males have been found, with some particularly large males from south africa combining the typical morphology of wingless males with the presence of ocelli and vestigial wings without otherwise approaching the morphology of typical winged ant males. c. venustula males appear to defend small territories and fight against males that intrude into their home range. colonies were at least temporarily polygynous, in contrast to wheeler’s report on monogyny (wheeler, 1908). c. shuckardi forel, 1891 was described from imerina, the central highlands of madagascar. according to b. seifert (pers. comm.) it appears to be restricted to this island. records of “c. shuckardi” from other parts of africa (samways, 1983; van hamburg et al., 2004; hita garcia et al., 2009; kone et al., 2012), the arab peninsula (collingwood & agosti, 1996) and iran (ghahari & collingwood, 2011) are probably misidentifications of other species of the c. shuckardi group owing to the often extremely close morphological similarities among species of cardiocondyla (e.g., seifert, 2003, 2008). here we describe the results of a field study on the occurrence and colony composition of c. shuckardi on the outskirts of antananarivo, madagascar, and describe the wingless males of this species. material and methods study area we identified potentially suitable collecting sites from previous records of c. shuckardi captured in malaise and pitfall traps as listed on www.antweb.org. in march 2012, we visited four sites: the garden of the madagascar biodiversity centre at tsimbazaza, antananarivo (18° 55’ 57” s, 47° 31’ 31.8” e, 1284 m), a sandy threshing floor and paths in front of the palace of ilafy (18° 51’ 14.5” s, 47° 33’ 56.8” e, 1356 m), the edge of a eucalypt plantation at ambohidrabiby (18° 45’ 55” s, 47° 36’ 37” e, 1381 m), and a gravel path branching off route nationale 2 at mandraka park (18° 54’ 20” s, 47° 53’ 35” e, 1369 m). we followed foragers of c. shuckardi to their nest entrances in the soil. nests were carefully excavated, adults and brood were collected into plastic vials with an aspirator. all specimens were transferred to the madagascar biodiversity centre in antananarivo, and censused under a binocular microscope. ovaries of queens were dissected following buschinger and alloway (1978). population genetics we investigated the suitability of primers previously developed to amplify seven microsatellite loci in other species of cardiocondyla (ce2-3a, ce2-4a, ce2-4e, ce25d, ce2-12d, lenoir et al., 2005; card8 and card21, schrempf et al., 2005) for the determination of the genetic structure of c. shuckardi colonies. though only three loci exhibited some variability, we analyzed the genotypes of 4 to 20 workers from each of 14 colonies (total 143 workers) and 1 to 6 dealate queens from 11 colonies (total 31 queens) from ilavy, 10 workers and 4 or 5 queens from each of two colonies from mandraka, and 10 workers and 5 queens each from one colony from ambohidrabiby and one colony from tsimbazaza to obtain an estimate of colony and population structure of this ant (233 total individuals). specimens included both old and young queens, most of which still had wings when collected. we could not determine the genotypes at all loci in some individuals, and three individuals were removed because their ce2-12d genotypes appeared to have alleles not found anywhere else. we used relatedness 4.02 (goodnight & queller, 1999) to estimate inbreeding coefficients and nestmate relatedness in colonies from ilafy. confidence intervals were obtained by jackknifing by loci. from the inbreeding coefficient we calculated the frequency of sib-mating following suzuki and iwasa (1980). morphology of males six males of c. shuckardi were mounted on points and inspected under a wild m10 binocular microscope with a wild 1.6x planapochromatic objective at a magnification of 160-200x. we measured head width, scape length, eye diameter, mesonotum width and length (weber’s length), petiole width, and postpetiole width. in addition, we counted the number of funicular segments. results occurrence and colony composition c. shuckardi appears to be a common ant in degraded, open patches of grassland, in sandy areas along the edges of unpaved paths, and in parks and gardens in the central highlands of madagascar. nests were particularly dense and easy to locate in the regularly watered gardens of madagascar biodiversity centre and near a parched ditch at ilafy. solitary workers were seen foraging over distances of more than two meters, and in a few cases we also observed pairs of workers engaged in tandem running, a typical behavior for cardiocondyla (wilson, 1959; heinze et al., 2006). colony nests j heinze et al sociobiology of the ant cardiocondyla shuckardi302 consisted of pea-sized chambers in sandy soil, under stones, roots, or between pebbles in the upper 15 cm of the ground. most nest entrances were surrounded by conspicuous middens of corpses of other ants, predominantly pheidole. in total, we censused 62 colonies (32 from ilafy, 16 from tsimbazaza, 10 from ambohidrabiby, 4 from mandraka). individual nests contained between one and seven dealate queens and up to 85 workers. in the most intensively studied site, ilafy, six nests were queenless, 11 had one queen, and the remaining 15 nests had two to seven queens (26 queenright colonies: median, quartiles 1, 2, 4). nests contained 10 to 80 workers (median, quartiles, 30.5, 22, 40). colony composition was similar in the other localities. upon collection, 14 colonies contained 1 to 10 winged female sexuals, seven colonies contained a single wingless male each, and one colony contained two wingless males. we observed numerous solitary, wingless queens moving outside the nest, suggesting that at least a fraction of young queens disperse after mating and shedding their wings in their natal nests. dissection showed that the ovaries of all queens in two multi-queen colonies (all five queens from a colony from tsimbazaza, five of 12 queens from a colony from ambohidrabiby) contained developing and mature eggs and had a filled spermatheca. from ovarian status it appeared that three or four of these 10 queens had only recently begun to produce eggs. the ovaries of the other queens contained clearly visible corpora lutea and/or two or three mature eggs, suggesting that they had been fertile for a longer period. multi-queen colonies therefore appear to be truly polygynous. population genetics only three of the seven tested cardiocondyla primers were suitable for the genetic analysis of colony structure. in 183 workers and 50 queens from 18 colonies, we found seven alleles at ce2-3a (82 – 94bp), 5 alleles at ce2-12d (143, 159, 163, 165, 167) and two alleles at card8 (128 and 130bp). the number and variability of loci allows for only a crude analysis of colony structure. nevertheless, our analysis clearly suggests a high frequency of inbreeding (table 1). in the most intensively studied population, ilafy, observed heterozygosities of workers and queens were much lower than expected heterozygosities at all three loci, resulting in inbreeding coefficients fis of 1.000 at card 8, 0.808 at ce2-3a, and 0.559 at ce2-12d (overall fis 0.685 ± se 0.133, corresponding to an average percentage of sib-mating of 89.7%). the presence of null alleles cannot be ruled out for card 8. ignoring this locus, the inbreeding coefficients at ce2-3a and ce-12d alone give an average percentage of sib-mating of 94.4% and 83.5%, respectively. a similar excess of homozygotes was observed in colonies from the other collection sites (table 1). eleven of 18 colonies contained worker genotypes that were not compatible with single mating by a single queen. several genotype combinations suggest the coexistence of several matrilines rather than multiple queen matings. for example, one worker and one queen from the mandraka 2 colony had a genotype at all three loci that did not overlap with those of other workers and queens. average nestmate relatedness in the 14 colonies from ilafy was 0.784 ± 0.112, ranging in individual colonies from 0.487 ± 0.079 to 1 ± 0 in colonies in which all workers had the same genotype at all loci. excluding the almost invariable locus card8 relatedness did not change the result (0.787 ± 0.098, range 0.475 ± 0.069 to 1± 0). relatedness among workers was 0.766 ± 0.108, ranging from 0.356 ± 0.0.082 to 1 ± 0 (excluding card 8 0.771 ± 0.095; range 0.353 ± 0.069 to 1 ± 0). the high inbreeding coefficient and the frequent occurrence of more than two worker genotypes per colony suggests that this value does not reflect monogyny and monandry. instead, many colonies appear to be composed of several inbred lineages of workers. in contrast, queens showed much less variation than workers: all but two queens from ilafy had the same homozygous genotype at loci card8 and ce2-3a and nestmate queens had one or two different genotypes at ce2-12d. queen relatedness in the five multi-queen colonies was 0.949 ± 0.034 (excluding card 8 0.848 ± 0.076). morphology of males wingless males closely resemble those of c. venustula in morphology, coloration, and size (fig 1). compared to wingless males of other cardiocondyla species, but similar to those of c. venustula, wingless males of c. shuckardi are relatively large (weber’s length 0.62–0.67 mm; thorax width 0.35–0.37 mm). they have large heads (head width 0.49– 0.53mm) with strongly sclerotized mandibles and relatively small eyes (eye diameter 0.10–0.11 mm). the antennae resemble those of workers (scape length 0.39–0.41) and the funiculus consists of 10 to 11 segments (fig. 2), with the typical fusion of segments previously reported from other wingless cardiocondyla males (seifert, 2003). the pronotal shoulders are well-developed but appear to be less angular than in c. venustula from south africa (fig. 1). petiole and postpetiole width ranged from 0.16–0.19 mm and 0.25–0.28 mm, respectively, with a median ratio between petiole and postpetiole width of 0.65. several males showed injuries such as missing legs (as in the c. venustula male in fig. 1), and one male was found decapitated. this suggests that wingless males of c. shuckardi engage in fights for the monopolization of mating with nestmate female sexuals similar to those engaged in by their counterparts in other species. discussion cardiocondyla shuckardi appears to be rather common in the madagascar highlands. nest density was particularly high in places with regularly high humidity, i.e., in gardens or along dry ditches. this matches findings from other sociobiology 61(3): 300-306 (september 2014) 303 cardiocondyla species, which build nests in humid patches in xeric environments, i.e., on sandy river banks or beaches (seifert, 2003; lenoir, 2006; oettler et al., 2010). we collected the colonies from cavities in the uppermost 10 cm of the soil after heavy rains, but it is likely that the ants move deeper underground during drier periods. for example, c. elegans nests reach to a depth of 40 cm (lenoir, 2006) and nest chambers of the desert species c. ulianini were found as deep as 1.5 m (marikovsky & yakushkin, 1974). colonies of c. shuckardi were small, with fewer than 100 workers, as is typical for cardiocondyla (heinze, 1999; oettler et al., 2010). nevertheless, because of the high density of nests, foragers of c. shuckardi appeared to be among the most abundant ants in our collecting sites. the species presumably plays a considerable role in anthropogenically disturbed, open habitat, such as plantations and gardens. its ecology therefore appears to resemble that of c. venustula in south africa (e.g., samways, 1983; van hamburg et al., 2004). our study clarifies two important aspects of the reproductive biology of this species. first, its colonies are at least temporarily facultatively polygynous, and second, males are wingless and resemble those of the related species c. venustula in size and morphology (frohschammer and heinze 2009). in the studied specimens of c. shuckardi, pronotal shoulders appeared to be less angular than in the presently available males of c. venustula, but because of the limited sample, the large variation of c. venustula males (heinze et al., 2013) and the lack of males from other species of the c. shuckardi group, it would be premature to define universal diagnostic features. injuries in the examined males of c. shuckardi and the simultaneous presence of two males in field colonies suggest that males may attack and damage young rivals but do not always engage in lethal fighting with other adult males. hence, male behavior appears to be similar to that of c. venustula and c. mauritanica males (frohschammer and heinze, 2009; heinze et al., 1993). whether c. shuckardi males defend small “territories” within the nests against other males, as observed in c. venustula (frohschammer and heinze, 2009), remains to be determined. as in other species of this genus, analyzing the genetic structure of colonies and populations of c. shuckardi was difficult because of the extremely low variability of genetic markers. only three of seven microsatellite loci were variable to some extent. this obviously prevents us from making conclusions about queen mating frequencies or fine-scaled population and colony structure. nevertheless, the available data clearly suggest that inbreeding is extremely common in c. shuckardi. from the inbreeding coefficient we estimate that more than 80% of all matings involve full sibs. this matches the condition in monogynous c. batesii, c. elegans, and c. nigra, where 50–80% of all matings involve brothers and sisters (schrempf et al., 2005; lenoir et al., 2007, schrempf, 2014). dissection data and the co-occurrence of up to four different worker genotypes in colonies of c. shuckardi indicate that several mothers may contribute to the offspring of single colonies. furthermore, the genotypes indicate that individuals may occasionally be exchanged among nests. one queen and one worker from mandraka had a multilocus-genotype different from those of all other studied nestmates, suggesting the adoption of alien queens as in c. elegans (lenoir et al., 2007). the combination of frequent inbreeding, polygyny, and queen or worker adoption results in a high nestmate relatedness value, which presumably would be much lower if corrected for inbreeding (e.g., pamilo, 1985). however, such a correction is not yet possible in c. shuckardi, as the mating frequency of queens is unknown and single males may mate with multiple queens. regardless of the exact value of relatedness, the genotype patterns suggest that queens mate in their natal nests with brothers and then disperse and seek adoption in other nests, in a manner similar to what has been observed in related species (schrempf et al., 2005; 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(2007). queen-queen competition and reproductive skew in a cardiocondyla ant. insectes sociaux,. 54: 268–274. doi: 10.1007/ s00040-007-0941-x table 1 genotypes of workers and queens of the ant cardiocondyla shuckardi from 18 colonies from four populations on madagascar (ilafy, ambohidrabiby, tsimbazaza, mandraka) at three microsatellite loci (1a:card8, ce2-3a; 1b: ce2-12d). for each locus, the number of workers / queens with a certain genotype is given. table 1a card8 ce2-3a 128/ 128 128/ 130 130/ 130 82/84 84/84 84/86 84/94 86/86 86/90 86/92 86/94 88/88 90/90 92/92 92/94 94/94 ilafy 1 19/6 15/6 1/0 2/0 1/0 ilafy 2 10/4 9/4 ilafy 3 9/3 1/0 13/3 6/0 1/0 ilafy 4 19/5 1/0 5/4 2/0 1/0 ilafy 5 10/5 1/0 9/5 ilafy 6 10/10/ ilafy 7 9/1 9/1 ilafy 8 10/1 10/1 ilafy 9 8/1 8/1 ilafy 10 4/4/ilafy 11 10/1 4/1 6/0 ilafy 12 10/1 8/1 2/0 ilafy 13 8/3 8/3 ilafy 14 4/4/ambo 8/5 1/0 1/0 1/0 1/3 3/2 3/0 1/0 1/0 tsimb 1 10/5 10/5 mand 1 10/5 1/0 1/1 1/1 6/3 mand 2 1/1 9/3 1/1 0/1 0/1 6/1 3/0 j heinze et al sociobiology of the ant cardiocondyla shuckardi306 table 1 b ce2-12d 143/143 143/159 159/159 159/167 163/163 163/165 165/165 165/167 167/167 ilafy 1 2/2 8/3 1/0 1/0 ilafy 2 1/0 3/0 3/2 ilafy 3 2/0 1/1 16/2 ilafy 4 1/0 4/0 ¾ ilafy 5 10/5 ilafy 6 3/7/ilafy 7 3/0 2/0 3/1 ilafy 8 10/1 ilafy 9 1/0 2/1 5/0 ilafy 10 1/1/1/1/ilafy 11 10/1 ilafy 12 4/1 6/0 ilafy 13 2/0 2/1 3/2 ilafy 14 4/ambo 4/5 tsimb 1 8/2 mand 1 7/3 1/1 1/1 mand 2 4/0 3/2 2/1 1/1 doi: 10.13102/sociobiology.v66i4.4596sociobiology 66(4): 614-618 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the ants taxon is characterized by great variability in body size, which plays an important role for their survival (bugrova, 2010; bernadou et al., 2016; bishop et al., 2016; kramer et al., 2016; wills et al., 2018). ants’ body sizes are conditioned upon factors both intrinsic (evolutionary constraints, genetic factors, social environment, colony age, need in colony defense and foragers) and extrinsic (physical environment, food availability and nutrition, anthropogenic disturbances and environmental pollution) (kaspari, 2005; johansson & gibb, 2012; grześ et al., 2015; bernadou et al., 2016; kramer et al., 2016; purcell et al., 2016; wills et al., 2018). these factors interact with one another (wills et al., 2018). significant differences in ant sizes exist between castes within a given nest. in the case of polymorphic species, it often occurs that the workers, too, differ greatly and can be designated as minor and major workers. each type of such workers performs different activities (hölldobler & wilson, 1990). smaller differences in size can be found also in workers of monomorphic species (grześ et al., 2016). physiological differentiation of workers can be the reason for a specific division of labor (fénéron et al., 1996), and this division is one of the bases for ants’ successful organization (hölldobler & wilson, 1990). abstract the division of functions among ant workers and their mutual cooperation is one of the reasons for ants’ success. the activities that workers perform in the nest can be divided by age or morphology. we studied the body size of workers of the wood ant formica polyctena as a function of their activity. our results show that workers exploiting protein baits were larger than workers attracted to carbohydrate baits. the biggest of all were workers located at the upper parts of the nest, which shows the importance of nest defense and maintenance. it also points that the distribution of functions does not have to be given only by workers age. work division based on several mechanisms may be advantageous for colony functioning. sociobiology an international journal on social insects a véle1, r modlinger2 article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 08 july 2019 initial acceptance 18 september 2019 final acceptance 18 november 2019 publication date 30 december 2019 keywords bait, food, formica, labor, nest. corresponding author adam véle forestry and game management research institute strnady 136, cz – 252 02 jíloviště, czech republic. e-mail: adam.vele@centrum.cz the european red wood ant formica polyctena förster, 1850 is a monomorphic wood ant species with north-palearctic distribution. the colonies are generally polygynous, frequently with more than 1 million workers. they collect honeydew, forage for arthropod prey and scavenge (horstmann, 1972; czechowski et al., 2002). f. polyctena is listed on the red list of threatened species, but also it is used for biological protection against forest pests (adlung, 1966; iucn, 1996) we examine the question as to whether body size in workers of the f. polyctena is a determinant of the activities that they perform. specifically, we investigate whether there are differences among hunting workers, workers that collect honeydew, and workers laboring in the upper parts of the nest. our study was conducted in a forest in the north of the czech republic (50°58’30.02”n, 15°13’09.34”e) at an altitude of 440 m a.s.l. the forest is dominated by norway spruce (picea abies). the research was conducted in august 2016. we randomly selected 20 separately located nests. the smallest distance between the nests was 50 m and nests belonged to different colonies. we placed baits consisting of sugar (honey) and protein-rich (tuna fish) foods on paper plates at a distance of 5 m from each nest. each bait contained 1 forestry and game management research institute, strnady, czech republic 2 faculty of forestry and wood sciences, czech university of life sciences prague, czech republic short note body size of wood ant workers affects their work division sociobiology 66(4): 614-618 (december, 2019) 615 1 cm3 of each type of food. after 10 min, we collected five workers from each type of bait and five workers from the upper parts of the nests (15 individuals per nest). for all captured workers, we measured the dimensions: eye length, head width, pronotum width, mandible width, and hind femur length (fig 1). the measurement was performed using a binocular magnifier with 45× magnification. the differences between the measured parameters according to work division were determined using one-way anova nested within the nest. in a statistically significant morphological parameter, the significance of differences by work division was also compared using treatment contrasts (crawley, 2013; pekár & brabec 2009). all analyses were performed in the r 3.3.2 environment (r core team 2016). fig 1. measured parts of the bodies of formica polyctena workers: a) mandible width, b) head and pronotum width, c) femur length, d) eye length. all morphological parameters measured in ants acquired at different capture points (tuna, honey, nest) were statistically significantly different (anova: df = 2, p < 0.001). body size values were highest at the nest, and individuals of somewhat smaller sizes were found on the tuna. the smallest parameters were those of individuals on the honey bait. these differences were statistically significant in all cases (contrast: p < 0.001, fig 2). our results may be explained by the degree of specialization, which differs between worker individuals (otto, 1958; horstmann, 1973). the larger body size of wood ant workers gathering protein food vis-à-vis those collecting honeydew has been described by rosengren & sundström (1987). there may be several possible causes for this fact. larger workers are able to catch and transfer large prey (batchelor, 2012), they move faster and further from the nest (rissing, 1982; wright et al., 2000), they have better orientation ability (bernstein & bernstein, 1969), they are more resistant against unfavourable meteorological conditions (kay & rissing, 2005), and more aggressive and better fighters (batchelor et al., 2012). all these features are advantage, because the occurrence of prey at the territory is random and may not be in places, where there are numerous ants (sundström, 1993; yao, 2012). workers at low-density sites may more commonly meet up with competing ants, which leads to a reduction in the number of the smaller workers employed for foraging (savolainen & vepsalainen, 1989; kay & rissing, 2005). conversely, the sources of honeydew are stable and well attended (del-claro & oliveira, 1993). a véle, r modlinger – body size and work division616 a unique finding is that wood ant workers working outside the nest are smaller than workers on the nest. it is known that the wood ants are divided into groups working inside and outside the nests and that these groups differ in age, physiology, and behaviour (rosengren, 1977). the higher aggression of large workers is advantageous in defending the nest (kay & rissing, 2005; batchelor, 2012). a loss of large workers is more costly for the colony (kay & rissing, 2005). the probability of loss is high when ants are moving on the territory, and especially at its edges (wilson, 1971). to date, however, work division, as a life history trait of wood ants, has been regarded as a typical example of age polytheism (younger workers are found in nests, older individuals work outside the nest) (see rosengren, 1977). our results on specific data prove the presumption of otto (1958) that the division of functions need not be given only by age polyethism. acknowledgements we would like to thank anonymous referees for their helpful comments. our contribution was supported by the ministry of agriculture of the czech republic, institutional support [grant number mze-ro0118]. the study was also supported by the internal grant agency of faculty of forestry and wood sciences czech university of life sciences in prague – project iga fld czu b10/17. rm is supported financially by extemit k, no. cz. 02. 1.01/ 0.0/0.0/15_003/0000433 financed by op rde during the preparation of the manuscript. authors’ contribution a véle contributed to fieldwork, conception and design. r modlinger performed the statistical analyses. both authors contributed to interpretation and to revisions of the manuscript. fig 2. overview of measured dimensions (head, pronotum and mandible width, femur and eye length) of formica polyctena workers in dependence on their work division (tuna – workers gathering protein food, honey workers collecting honeydew, nest – workers at the upper parts of nests). horizontal line in the middle of the box is median, whiskers represent 1.5 times the inter-quartile range. one hundred workers were used for measurement in every group. sociobiology 66(4): 614-618 (december, 2019) 617 references adlung, k.g., a critical evaluation of the european research on use of red wood ants (formica rufa group) for the protection of forests against harmful insects. zeitschrift für angewandte entomologie, 57: 167-189. batchelor, t.p., santini, g. & briffa, m. 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(2012). seasonal trends in honeydew-foraging strategies in the red wood ant formica yessensis (hymenoptera: formicidae). sociobiology, 59: 1351-1363. doi: 10.13102/sociobiology.v59i4.519. doi: 10.13102/sociobiology.v65i3.3263sociobiology 65(3): 456-462 (september, 2018) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the effect of nest size and species identity on plant selection in acromyrmex leaf-cutting ants introduction leaf-cutting ants, atta forel and acromyrmex mayr, are considered the dominant herbivores of the neotropical region (hölldobler & wilson, 1990). the workers cut, collect and carry plant fragments to the nest where they grow a symbiotic fungus, the main food source of larvae (cherrett, 1989). these ants forage on a large number of plant species, but generally most of their diet consists of a few selected ones (rockwood & hubbel, 1987; franzel & farji-brener, 2000). there are external and intrinsic factors that can influence the diet composition of leaf-cutting ants. on one hand, the number and amount of plant species harvested depend on their diversity, nutritional quality, chemical/physical defenses, abundance and spatio-temporal distribution (nichols-orians & schultz, 1990; franzel & farji-brener, 2000; farji-brener, 2001; wirth et al., 2003; lanan, 2014). however, diet composition also depends on intrinsic characteristics of the colony, such as size and age of the nests (wetterer, 1994; kost et al., 2005). while the external factors that modeled leaf-cutting abstract leaf-cutting ants are key organisms because their role as primary consumers and potential agricultural pests. however, their foraging ecology was mostly studied as response of extrinsic factors such as climate and plant species traits. we evaluated the effects of intrinsic factors (i. e., nest size and species identity) on the foraging behavior in two species of acromyrmex mayr leaf-cutting ants. while large and small nests of a. lobicornis emery showed similar feeding behavior, small nests of a. striatus roger harvested a greater percent of the plant species available in their foraging area and showed a higher level of selectivity than larger nests. we discussed some probable reasons for this pattern. our results highlight the relevance of intrinsic factors and species characteristics to explain changes in the foraging ecology of leaf-cutting ants as colony growth. sociobiology an international journal on social insects le jofré1, ai medina1, ag farji-brener2, mm moglia1 article history edited by ricardo solar, ufmg, brazil received 26 march 2018 initial acceptance 20 june 2018 final acceptance 20 july 2018 publication date 02 october 2018 keywords acromyrmex, ant nests, chaco, foraging behavior. corresponding author laura elizabeth jofré universidad nacional de san luis área de zoología ejercito de los andes nº 950, d5700hhw, san luis, argentina. e-mail: lauelijofre@gmail.com ant diet has been largely studied (see a review in hölldobler & wilson, 2011), those related with the colony status and species identity remain less understood. some authors suggest that the foraging ecology of small or juvenile colonies differs from large or mature colonies (wetterer, 1994; kost et al., 2005). for example, in some atta species small colonies forage mainly herbs near their nests, while larger colonies forage more distant woody species (wetterer, 1994; kost et al., 2005). however, most of these comparisons between trophic behavior and nest size have been made on the genus atta, being less known for acromyrmex. moreover, species characteristics such as their worker density and the complexity of the trail system may also influence on their foraging ecology. we complemented this information analyzing whether acromyrmex species also change their diet as they growth. the knowledge whether small and adult leaf-cutting ant colonies differ in their foraging ecology will provide theoretical and applied useful information. first, this info will help to better understand the relationship between the ontogeny of herbivore populations and their foraging 1 universidad nacional de san luis, san luis, argentina 2 laboratorio de investigaciones en hormigas (liho), inibioma-conicet-universidad nacional del comahue, rio negro, argentina research article ants sociobiology 65(3):456-462 (september, 2018) 457 preferences. second, and because these ants are considered one of the major pests of forestry and agricultural lands, the info of how diet changes as nest growths will be vital to applied adequate management practices. acromyrmex striatus emery and a. lobicornis roger are two of the most common leaf-cutting ant species in argentina (farji-brener & ruggiero, 1994). these species are good model to evaluate the effect of colony size and species identity on their diet because live in simpatry but differ in some characteristics. concretely, a. lobicornis constructs nests with an external nest-mound (farji-brener, 2000; bollazzi et al., 2008) that house about 10.000 workers (jofré & medina, 2012) and build well defined trails to forage. in contrast, a. striatus constructs relatively smaller subterranean nests without a nest-mound, in areas of low vegetation cover and their workers forage at close distances from the nest without a defined network trail system (boneto, 1959; diehl-fleig, 1995). in addition, both species consume a high percentage of species available in the environment (armani & quirán, 2007; nobua-behrmann, 2014), and their degree of selectivity or opportunism depends on the temporal space fluctuations of the resources (farji-brener & protomastro, 1992; pilati et al., 1997; franzel & farji-brener, 2000; armani & quirán, 2007; nobua-behrmann, 2014). the objective of this work was to evaluate the effect of nest size and species identity on the foraging behavior of two leaf-cutting ant species. specifically, we tested whether diet composition, plant richness, niche breadth and degree of selectivity depends on nest size, species identity and/or the interaction between them. materials and methods study area the nests of a. lobicornis and a. striatus were randomly selected in a natural reserve of san luis, argentina (33° 07’ s y 66° 03’ w). the area has 340 ha, with an average altitude of 850 amsl. the average annual temperature in january (summer) is 25 °c and 9 °c in july (winter); the mean annual rainfall is about 600 mm (del vitto et al., 1994). the vegetation is represented by species belonging to the phytogeographic province of chaco, chaqueño serrano district (cabrera & willink, 1980). this nature reserve was affected by overgrazing, fire, and logging. due to these disturbances native plant species typical of chaco serrano as well as exotic species are common in the area (del vitto et al., 1994; chébez, 2006). the dominant native species are lithraea molleoides, prosopis caldenia, vachellia caven, celtis ehrenbergiana, briza subaristata, eragrostis lugens, bouteloua curtipendula, schizachyrium plumigerum, bothriochloa springfieldii mixed with exotic species such as rosa rubiginosa, ulmus sp., robinia pseudoacacia and cynodon sp. (del vitto et al., 1994; chébez, 2006). nest size we consider nest size as a proxy of colony growth because for leaf-cutting ants, nests size is considered a good estimator of the colony age (fowler, 1977; vieira-neto et al., 2016). colony size influences the number and caste morphology of workers as well as the distance where the colony search for resources (hölldobler & wilson, 2011). we consider the nesting characteristics of each species to separate small from large nests: the size of nest-mound in a. lobicornis and the number of entrance holes in a. striatus. these measurements were carried out in a total of 28 nests (14 of each species). to differentiate small from large nests in a. lobicornis we used the criteria described in farji-brener et al. (2003), where small colonies were those with nest-mound diameters (φ) ≤ 70 cm and large with φ ≥ 100 cm. for a. striatus, diehl-fleig (1995) states that number of entrance holes of nest are good estimator of the age of colony. using this criterion, small colonies were divided into those with up to 2 entrance holes and large colonies with more than 5 entrance holes. consequently, each size category was composed of 7 nests for each species. diet composition and resource availability during the summer of 2013-2014 (decembermarch), the season of higher ant activity and plant abundance, we determined the diet composition and resource availability in 28 colonies of both species of acromyrmex. diet composition was determined by collecting of the vegetal material carried by the workers during 5 minutes in the hours of maximum foraging activity. this collection was carried out to complete 8 samples per nest on two non-consecutive days (a total of 224 diet samples for both species). the collected material was placed in paper bags and taken to laboratory for analysis under a stereo microscope. this procedure is widely employed to determine diet composition and foraging preferences of leaf cutting-ants (pilati et al., 1997; franzel & farji-brener, 2000; farji-brener 2001; behrmann, 2014; farji-brener & tadey, 2017). each fragment present in the diet was identified at the plant species or genus level with the help of a reference collection of plants from the study site. we identified of 6250 plant fragments carried by a. lobicornis and 2169 by a. striatus, (approximately 85 % to species level) since ant workers collect entire leaves or parts of flowers and fruit. subsequently, the relative frequency of each plant species in the diet (di) in each colony was estimated as the number of fragments per plant species divided the number of total carried fragments. in the same sampling period, the resource availability (oi) was determined within the foraging area. circular foraging areas were estimated using an average length of foraging trails, which represent the radius of the foraging circumference area. in a. striatus the average distances le jofré, ai medina, ag farji-brener, mm moglia – foraging ecology and nest size in leaf-cutting ants458 ps is traveled by the forage workers were used, since this species does not build cleared trails. therefore, the forage areas were estimated in 1520 m2 for a. lobicornis and 78.5 m2 for a. striatus. to estimate the availabity of plant resources for each colony we randomly located 10 square plots within each foraging area following the methodology of hays et al. (1981). five squares of 1 m2 (subdivided in 10 x 10 cm) were used to determine the frequency of herbs and 5 squares of (subdivided in 20 x 20 cm) to determine the frequency of woody species. the relative frequency of each species per m2 was estimated as the number of subdivisions where the species was present divided the number of total subdivisions. the mean value of the 10 squares was used to determine the relative frequency (oi) of each plant species on the foraging area. niche breadth and foraging selectivity the relative frequencies of each plant species in foraging area (oi) and their relative frequency in the diet (di) were used to estimate the trophic breadth and ant selectivity for each plant species. feinsinger’s niche breadth index (ps, feinsinger et al., 1981) was used to evaluate the trophic breadth. this index estimates (in %) the overlap’s degree between the frequencies’ distribution of the resources in the diet with the distribution of frequencies of resources in foraging area as: ( )[ ]∑= 100.,min ii dops where oi, is the relative frequency of each plant species in foraging area, and di represent the relative frequency of each plant species in the diet. the ps index takes values between 0 and 100, being the minimum trophic breadth or specialism and maximum trophic breadth or generalism, respectively (feinsinger et al., 1981). to determine which plant species was selectively consumed we used the ivlev index (1961): ii ii od od is + − = the is index takes values between -1 and 1 being maximum rejection or maximum selectivity, respectively. values close to -1 indicate that the species is in a lower proportion in the diet than its availability in the environment (rejection). values near 0 indicate that the proportion of species in the diet and in the environment is similar (opportunism). while values close to one 1 indicate that the species is in a higher proportion in the diet than in the environment (selectivity). we subjectively consider that a plant species was consumed selectively when it is was ≥ 0.7. therefore, the percentage of the diet composed of species consumed selectively was calculated summing the percent of all the plant species in the diet with an is ≥ 0.7(appendix 1 and appendix 2). data analysis we calculated the species richness in the diet and in the foraging area for each nest size. these data were compared between nest sizes of each species using either independent t-tests or its non-parametric equivalent as appropriate. data were analyzed using r program v.3.2.2 (r core team, 2015). in addition, plant species from diet and resources were separated into herbaceous and woody species, estimating the average percentage collected in nests of different sizes. to determine the dependence of nest size and the presence in the diet of herbaceous and woody species we used a chi-square test. to compare the niche breadth (ps) and percentages of the selectively foraged species (is ≥ 0.7) between the nest sizes of each species we used a two-way anova for each response variable. nest size and ant species were considered fixed factors. anova assumption was checked. post-hoc comparisons were performed using tukey tests. data were analyzed using statistica 8.0 ®. finally, permutational multivariate analysis of variance (permanova), with 999 permutations, was carried out for analyze on the effect of nest size and ant species identity on diet composition. data were analyzed using r program v.3.2.2 (r core team, 2015). results plant richness and diet composition acromyrmex striatus and a. lobicornis used a large portion of the available plant resources but showed some differences depending on the nest size. in a. striatus, small nests harvested 36 of 83 plant species available in their foraging area (43%). the most frequently harvested plant species were celtis ehrenbergiana, cynodon sp., tripodanthus flagellaris, oxalis sp. and vachellia caven which represents 62% of the species consumed by small nests (appendix 3). conversely, large nests of a. striatus harvested only 10 of 51 plant species available in their foraging area (20%). the most harvested plant species were cynodon sp. and prosopis caldenia representing 85% of their total diet (appendix 3). accordingly, the species richness consumed per nest was higher in small nests (9 ± 4) than in large nests (4 ± 1), (w = 1, p = 0.003). on the other hand, the areas where the small nests were found showed a greater mean number of species available (t = -2.7, df = 11, p = 0.02, see fig 1). the percentage represented by herbaceous and woody species in the diet of a. striatus showed similar values according to the size of their nests. both small nests and large nests collected a larger proportion of woody species (woody: 48.5% small nests and 66% large nests). while the herbaceous species reached values between 30 and 35% (χ2 = 2.5, df = 1, p = 0.1; fig 2). the foraging areas showed a high percentage of woody plants in both, large and small nests (χ2 = 1.4, df = 1, p = 0.2, fig 2) (see table 1). sociobiology 65(3):456-462 (september, 2018) 459 in a. lobicornis, small nests harvested 49 of 82 plant species available in their foraging area (60%).the most frequently harvested species were celtis ehrenbergiana, cynodon sp., heterotheca subaxillaris, schkuhria pinnata representing approximately 45% of the total of the diet (appendix 4). on the other hand, large nests harvested 52 of 87 plants species available in their foraging area (60%). the most commonly consumed vegetable items were cynodon sp. and rosa rubiginosa, which accounted for 28% of the total diet. the rest of the species consumed in small and large nests contributed less than 6% to the total diet (appendix 4). the richness of plant species consumed per nest and those available in the foraging area were similar between large and small colonies (all p > 0.4) (fig 3). the percentage of herbaceous and woody species harvested by a. lobicornis was similar in the small nests (approximately 45%), while the large nests presented higher values of woody species in their diet (60% woody vs. 30% herbaceous) (χ2 = 4.6, df = 1, p = 0.03, fig 4). foraging areas showed a higher percentage of herbaceous species in both types of nests (χ2 = 0, df = 1, p = 1, fig 4) (see table 1). finally, diet composition was similar between nest sizes and ant species (permanova, all p > 0.40, table 2). acromyrmex striatus acromyrmex lobicornis small nests large nests small nests large nests sp. richness consumed 9 ± 4a 4 ± 1b 14 ± 4c 14 ± 4c sp. richness foraging area 26 ± 5a 16 ± 7b 28 ± 5c 30 ± 3c niche breadth (ps) 52 ± 18a 29 ± 15b 32 ± 9c 30 ± 7c % spp selec. cons. 47 ± 29a 12 ± 20b 47 ± 16c 54 ± 18c df ss ms f r2 p specie 1 114281 114281 214.5 0.91 0.40 size 1 117478 117478 220.5 0.94 0.40 interaction 1 -119623 -119623 -224.6 -0.96 0.81 residuals 24 12785 533 0.102 0.10 total 27 124920 1 table 1. summary of the main results of the species richness consumed, species richness in the foraging area, niche breadth (ps) and percentage of species consumed selectively for small nests and large of a. lobicornis y a. striatus. the values correspond to the mean ± sd. different letters in the rows indicate statistically significant differences (p < 0.05) for each species of leaf-cutting ants. fig 1. average species richness (± sd) in the diet and in foraging area of a. striatus. different letters indicate significant statistical differences (p < 0.05). fig 2. relative proportion (%) of woody (w) and herbaceous (h) species present in the diet and in the offer of small and large nests of a. striatus. others: unidentified species in the diet. niche breadth and foraging selectivity the niche breadth (ps) indexes varied between nests sizes depending the ant species (f 1, 24 = 6.3, p = 0.02) (fig 5a, appendix 5). the ps of a. striatus was significantly higher in large nests (52 ± 18) than in small ones (29 ± 15), suggesting that small nests of a. striatus are mostly specialists. but a. lobicornis presented similar ps values between small nests (32 ± 9) and large ones (30 ± 7) (table 1, appendix 5). the percentage of species harvested selectively varied between nests sizes depending the ant species (f1, 24 = 6.8, p = 0.015) (fig 5b, appendix 5). small colonies of a. striatus were more selective than large ones. small colonies selectively harvested the 47 % ± 29 (mean ± sd) of his diet, whereas large colonies only the 12 % ± 20. conversely, large and small colonies of a. lobicornis showed a similar percent of species harvested selectively (table 1, appendix 5). table 2. results of permanova from nest size and ant species identity on diet composition. le jofré, ai medina, ag farji-brener, mm moglia – foraging ecology and nest size in leaf-cutting ants460 discussion it is known that the foraging ecology of leaf-cutting ants is affected by extrinsic factors, such as plant defense traits (nichols-orians & schultz, 1990; wirth et al., 2003). here we demonstrated intrinsic factors, such as nest size and species identity, can also affect the foraging patterns in this ant group. despite that diet composition was similar among nest sizes and species identity, both species showed different foraging behaviors depending on nest size. while small and large nests of a. lobicornis showed similar number of species harvested, percent of species harvested selectively and niche amplitude, small nests of a. striatus showed differences between these three descriptors of foraging behavior regarding large nests. small colonies of a. striatus showed higher values of niche breadth (ps = 52 ±18) whereas their diet consisted of 47% fig 3. average species richness (± sd) in the diet and in the foraging area of a. lobicornis. fig 5. graphical representation of the interaction between nest size (small and large nests) and species of leaf-cutting ants (as and al) for the variables (a) niche breadth (ps) and (b) species consumed selectively (%). as: acromyrmex striatus, al: a. lobicornis. different letters mean statistical significant differences (p < 0.05). fig 4. relative proportion (%) of woody (w) and herbaceous (h) species present in the diet and in the offer of small and large nests of a. lobicornis. others: unidentified species in the diet. equal letters indicate significant differences (p < 0.05). (±29) of species selectively harvested, suggesting high selectivity in small than in larger nests. in other words, in a. striatus the selectivity and the importance of selected plant species in its diet decrease as colony growths. one possible explanation is that the higher selectivity of small nests is consequence of a low capacity to deal against chemical and physical plant defenses in earlier stages of the colony ontogeny. future works should test this hypothesis. the great flexibility in the foraging of this ant species found in other studies may support this hypothesis, since high variability of diet may help to compensate the negative effect of chemical plant defenses on their fungi culture (farji-brener & protomastro, 1992; nobua-behrmann, 2014). conversely, large and small nest of a. lobicornis showed similar niche breadth values (ps = 32 and 30, respectively) suggesting selectivity for both nest sizes (table 1). these results are similar to those found in different regions of argentina where a. lobicornis showed high selectivity in summer (franzel & farji-brener, 1992; nobua-bhermann, 2014). sociobiology 65(3):456-462 (september, 2018) 461 an open question is why a. lobicornis do not change their selectivity level as the colony growths. in other words, which characteristics differ between the studied species that can be associated to a change in the selectivity as nest grows in a. striatus but not in a. lobicornis. these ants have different thermal tolerance ranges that result in temporally separated foraging activities avoiding interference competition (nobuabehrmann et al., 2017). on the one hand leaf-cutting ants have different nesting and digging behavior at the same soil temperature regimes (thermic or hyperthermic soil) (bollazi et al., 2008). on the other hand, a. lobicornis has some typical characteristics of a dominant species, such as big colonies (stuble et. al. 2017, nobua-behrmann et al., 2017). a better thermoregulation capacity and a larger worker density make a. lobicornis more ecologically dominant than a. striatus. this suggests that the high selectivity of a. striatus in the most vulnerable stages of growth can help this subordinate species to coexist with a superior competitor. foraging selectivity involves cost and benefits. to be selective is a time and energy demanding activity, particularly costly in climatically rigorous environments such as in our sample region. however, this cost could be compensated by the nutritional quality of the selected food. we suggest that the balance between costs and benefits that stimulate the change of foraging strategy as the colony growth depends also on intrinsic characteristics of the ant species. moreover, our results highlight the relevance of intrinsic factors to explain changes in the foraging ecology of leaf-cutting ants as colony growth. supplementary material http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/3263/0 http://dx.doi.org/10.13102/sociobiology.v65i3.3263.s2210 acknowledgments we thank the people who collaborated in the field samplings and the staff of the “wildlife conservation center la florida” of the province of san luis. references armani, a. & quirán, e. 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(2003). herbivory of leaf-cutting ants: a case study on atta colombica in the tropical rainforest of panama, berlin: springer verlag, 230 p. doi: 10.13102/sociobiology.v67i2.5084sociobiology 67(2): 330-334 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 arthropods are typically sexually dimorphic, and hermaphroditism is very rare among them (narita et al., 2010). however, developmental defects, which occur at low frequencies under natural conditions, may lead to morphologically anomalous individuals with both female and male traits (narita et al., 2010). a distinction can be made between cases in which male and female traits are uniformly combined (intersexes) and those in which chimeric mosaics occur (gynandromorphs sensu lato), and this distinction can either be made on a phenotypic or genetic basis (narita et al., 2010). narita et al. (2010) reviewed several mechanisms that may lead to the development of these forms in arthropods, most of the existing knowledge being based on drosophila fallén, 1823 (diptera: drosophilidae). in ants (hymenoptera: formicidae), different kinds of morphologically intermediate forms between castes or sexes (sex mosaics) have been described. among them, ergatoids, abstract ergatandromorphism is the result of an aberrant development in which part of the body of a social insect shows the traits of the worker caste, while the other resembles a male. it is considered a specific case of gynandromorphism. specimens with these characteristics have rarely been collected in different ant lineages across the world. here, we provide the first description of ergatandromorphism in the ant myrmica lobulicornis nylander, 1857: an ergatandromorphous specimen was recovered during an arthropod sampling campaign across altitudinal and ecological gradients on the italian alps (stelvio national park), together with 480 workers and 4 queens of the same species, which expressed the normal phenotype. sociobiology an international journal on social insects e schifani1, c castracani1, fa spotti1, d giannetti1, m ghizzoni1, m gobbi2, l pedrotti3, da grasso1, a mori1 article history edited by evandro nascimento silva, uefs, brazil received 11 march 2020 initial acceptance 08 april 2020 final acceptance 09 april 2020 publication date 30 june 2020 keywords bilateral mosaic, gynandromorph, developmental defects, italian alps, stelvio national park. corresponding author enrico schifani, cristina castracani department of chemistry, life sciences & environmental sustainability, university of parma, parco area delle scienze, 11/a, 43124 parma, italy. e-mails: enrsc8@gmail.com cristina.castracani@unipr.it which represent a subcategory of ergatogynes (workerqueen), are the only ones who possess a functional role within ant colonies and their occurrence may be frequent in some species (peeters, 1991; molet et al., 2012). on the other hand, other ergatogynes (intercastes), as well as the chimeric mosaics (queen-male: gynandromorphs sensu stricto, workermale: ergatandromorphs or androergatomorphs, soldiermale: dinergatandromorphs), are typically very rare hybrid phenotypes with no functional role (donisthorpe, 1929; berndt & kremer, 1983; peeters, 1991; silva & feitosa, 2019). all these aberrant forms have been seen as an opportunity to understand the development and evolution of castes and the mechanisms for sex determination in social insects (e.g. donisthorpe, 1929; yang & abouheif, 2011). ergatandromorphism has been reported as a phylogenetically widespread phenomenon occurring in tens of ant species (creighton, 1928; donisthorpe, 1929; 1938; 1939; 1 department of chemistry, life sciences & environmental sustainability, university of parma, parma, italy 2 section of invertebrate zoology and hydrobiology, muse-science museum of trento, trento, italy 3 stelvio national park, bormio, italy ergatandromorphism in the ant myrmica lobulicornis nylander, 1857 (formicidae: myrmicinae) short note sociobiology 67(2): 330-334 (june, 2020) 331 wheeler, 1914; 1937; parapura, 1972; torossian, 1974; berndt & kremer, 1982; crosland et al., 1988; kremer & berndt, 1986; kinomura & yamauchi, 1994; heinze & trenkle, 1997; yosuke, 2008; yoshizawa et al., 2009; de campos et al., 2011; yang & abouheif, 2011; skvarla & dowling, 2014). normally, they occur unfrequently, but in a few cases, they were discovered in large numbers (donisthorpe, 1946; kinomura & yamauchi, 1994; yoshizawa et al., 2009). under laboratory conditions, they may be obtained by causing heat shocks during egg development (berndt & kremer, 1982). phenotypically, ergatandromorphs are usually characterized by a bilateral mosaic development which affects at least the head, and often parts of the mesosoma, while one of the sexes is overall prevalent. usually, the reproductive system of gynandromorphs or ergatandromorphs is only that of one of the two sexes (cokendolpher & francke, 1983; heinze & trenkle, 1997; yoshizawa et al., 2009). however, their reproductive capabilities remain unclear and there are very few reports on the behavior of these forms (e.g. torossian, 1974; yoshizawa et al., 2009). here, we report the discovery of the first case of ergatandromorphism in the myrmicine ant myrmica lobulicornis nylander, 1857 (formicidae: myrmicinae), a species whose taxonomic identity was recognized recently (seifert, 2005; see also radchenko & elmes, 2010; jansen et al., 2010; guillem et al., 2016). its lineage is estimated to have differentiated from its closest relatives quite recently (jansen et al., 2010). today, m. lobulicornis is associated with montane and alpine environments across europe, in the alps occurring between 1,000 and 2,700 m asl in sunny grasslands and pastures (seifert, 2018). in italy, relatively few data on its distribution were published so far (radchenko & elmes, 2010; sielezniew et al., 2010; glaser et al., 2011; schifani & alicata, 2018). standardized surveys covering the italian ant fauna remain very rare (e.g. castracani et al., 2010; spotti et al. 2010; 2015; gibb et al. 2017). during 2017 and 2018, the second step of a mid-term extensive survey of alpine arthropod and bird faunas was carried out in the protected area of the stelvio national park (lombardy sector), italian alps, in co-ordination and with the same survey design of the other three italian alpine national parks and in cooperation with muse-science museum of trento. terrestrial arthropods were sampled using pitfall traps, baited with a standard mixture of wine-vinegar and salt (latella et al., 2019), across an altitudinal and ecological gradient. as a result, a total of 480 workers, 4 queens and 1 ergatandromorph of m. lobulicornis were collected from approximately 1,400 to 2,400 m asl. the ergatandromorph is stored in the myrmecology lab collection at the university of parma (italy), and has the following collection data: valle messi, sondrio, 46°17’55.1”n, 10°31’09.4”e, 2045 m asl, southexposed, festucetum variae grassland with shrubs and scree, 29.v-14.vi.2018, trap code 6.2.4. a total of 26 workers were also collected from the same plot. the ergatandromorphous specimen was photographed using a canon 6d reflex and mp-e 65mm f/2.8 1‒5× macro photo lens. helicon focus was then used to fuse images taken at different focal planes into single images with greater depth of field. most of the body of the ergatandromorph is notably worker-like (fig 1, 2), while only the right side of the head is clearly that of a male. in comparison with the left side, the right one is characterized by a blackish color, a smaller mandible, a male-like antenna (unfortunately partly broken), a much larger eye, two ocelli, a different surface sculpture and a different development of the frons (fig 2). morphometric characteristics respectively of the male-like and worker-like fig 1. myrmica lobulicornis ergatandromorph specimen (whole body). up to down: dorsal view; profile (left) view, profile (right) view. scale bar: 0.5 mm. e schifani et al. – ergatandromorphism in myrmica lobulicornis332 parts of the head follow the standard proportions of the two castes (seifert, 2005; 2018; radchenko & elmes, 2010). however, the mesosoma is abnormal, although still mostly worker-like (fig 1, most evident in profile view). as the worker-like phenotype is prevalent, it is unsurprising that the specimen does not possess the male genitalia, which are particularly large and evident in myrmica males. in conclusion, the specimen morphology follows the common aspect of ergatandromorph ants: a bilateral mosaic in the head, while one sexual phenotype prevails in the rest of the body. while gynandromorphs or ergatandromorphs have already been reported in few other myrmica species (e.g. donisthorpe, 1929), most of the old records appear unreliable due to the huge amount of taxonomic changes that the genus went through, and the occurrence of these forms in m. lobulicornis has never been reported before (see radchenko & elmes, 2010). however, it is worth noting that meinert (1861) reported a case of grynandromorphism in m. lobicornis nylander, 1846, which is morphologically very close to m. lobulicornis and was not considered a separate taxon at that time (seifert, 2005; radchenko & elmes, 2010; seifert, 2018). acknowledgements this work was supported by stelvio national park as part of the mid-term monitoring of alpine faunistic biodiversity in relation to climate change. the work was granted by the italian ministry for environment, land and sea protection and co-financed by autonomous province of trento. alessandro gugiatti and paolo belotti supervised and managed field work for survey of epigean arthropod fauna, teresa boscolo and michael bernasconi (muse-science museum of trento, italy) sorted the formicidae collected by pitfall traps. this work was also supported by grants from the university of parma (fil-2019) assigned to d.a. grasso and a. mori and benefited from the equipment and framework of the comp-hub initiative, funded by the “departments of excellence” program of the italian ministry for education, university and research (miur, 2018–2022). authors contribution e. schifani performed the taxonomic part and drafted the first version of the manuscript. d. giannetti provided high quality pictures of the ergatandromorph. c. castracani, f.a. spotti and m. ghizzoni directed and coordinated the processing of the ant specimens. m. gobbi and l. pedrotti conceived and organized the sampling campaign. l. pedrotti, d.a. grasso and a. mori were responsible of funding acquisition. all authors contributed to draft the final version of the manuscript. references berndt, k.p. & kremer, g. 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(2008). a bilateral gynandromorph in the pirate ant polyergus samurai. ari, 31: 63-67. 1205 nestmate recognition and cuticular hydrocarbons of two sympatric species of reticulitermes in japan (isoptera: rhinotermitidae) by yoko takematsu1* & kohei kambara2 abstract the nestmate recognition of two sympatric species, r. kanmonensis and r. speratus, was investigated in terms of agonistic behavior and trophallactic behavior. r. speratus showed strong agonistic behavior against different species, but no trophallactic contact with them. however, agonistic behavior against different colonies of the same species was weak, and trophallactic exchange of food was observed. on the other hand, r. kanmonensis showed strong agonistic behavior not only against different species but also against different colonies of the same species, and trophallactic contact was absent. these results indicate that r. kanmonensis does not exhibit colony fusion, unlike r. speratus, which is known to exhibit colony fusion. this marked difference in the occurrence of colony fusion can be related to the difference in the distribution pattern of the two species. cuticular hydrocarbons of both species were also analyzed. relatively high hydrocarbon homogeneity was observed among colonies in r. kanmonensis compared to r. speratus. key words: nestmate recognition; cuticular hydrocarbons; agonistic behavior; trophallaxis; colony fusion; sympatric distribution; invasive species. introduction genus reticulitermes is one of the common genera that cause serious damage to woodwork in temperate regions. r. speratus is widely distributed throughout the japanese mainland and had been known as the only species of this genus. however, it was found that another species, r. kanmonensis, 1department of biological and environmental science, faculty of agriculture, yamaguchi university, yoshida 1677-1, yamaguchi 753-8515, japan 2the united graduate school of agricultural sciences, tottori university, tottori 680-8553, japan *corresponding author. present address: department of biological and environmental sciences, yamaguchi university, 1677-1 yoshida, yamaguchi, 753-8515, japan; e-mail: takematu@yamaguchi-u. ac.jp 1206 sociobiolog y vol. 59, no. 4, 2012 is distributed sympatrically with r. speratus in southernmost honshu and is thought to be an alien species (kitade & matsumoto 1993; takematsu 1999; morimoto 2000). since the first report of this species as r. flaviceps by nawa (1911), it has not been distinguished from r. speratus morphologically and has been controlled using the same method as for r. speratus. however, takematsu (1999) presented the characters distinguishing this species from r. speratus, and since then the two species have been treated separately in the ecological study of reticulitermes. kambara & takematsu (2009) showed that r. kanmonensis tend to choose less favorable habitats (less damaged wood) when coexisting with r. speratus, and this fact predicted that even living woods and houses are vulnerable to termite damage by r. kanmonensis. in order to control this species more effectively, it is important to clarify the ecological features of r. kanmonensis. kitade & hayashi (2002) reported the detailed distribution range of this species. it is remarkable that the distribution area of r. kanmonensis is restricted to the coastal areas on both sides of the kanmon strait located between honshu and kyushu islands, and that this species has not expanded its distribution range for a century since the first record in 1911 (nawa 1911; nawa 1912a,1912b; nawa 1917; kitade & matsumoto 1993; takematsu 1999; morimoto 2000). within the whole distribution area, r. kanmonensis and r. speratus were distributed sympatrically. however, the local distribution of the two species was not homogeneous; the distribution ratio of some local areas was strongly biased toward r. kanmonensis (kitade & hayashi 2002; kambara & takematsu 2009). why did r. kanmonensis show such a biased distribution pattern without expanding their distribution range? here we consider whether one of the reasons for the distribution pattern might be associated with the interspecific and intraspecific interactions between the two species. kitade et al. (2004) reported the highly aggregated distribution of incipient colonies of r. kanmonensis in a small area and the lack of mitochondrial haplotype variation within a mature nest, and they assumed the absence of worker mingling and the strong intraspecific competition among colonies. in the present study, we investigate the recognitions for nestmate and nonnestmate of r. kanmonensis and r. speratus in terms of the agonistic behavior and trophallactic behavior. we also analyze the cuticular hydrocarbons 1207 takematsu, y. & k. kambara — nestmate recognition in sympatric reticulitermes sp. of both species, which are reported to play a key role as cues for nestmate recognition, and discuss the relationship between nestmate recognition and cuticular hydrocarbon composition. materials and methods termites four collecting sites were chosen according to the local distribution ratio of the two species: r. kanmonensis-area (site1: distribution ratio of r. kanmonensis was 100%), r. kanmonensis dominant-area (site2: distribution ratio of r. kanmonensis was 85%), r. speratus dominant-area (site3: distribution ratio of r. kanmonensis was 35%), and r. speratus-area (site4: distribution ratio of r. kanmonensis was 0%). sites 1-3 were in a forest in ejio park, onoda (131°12’e, 34°2’n), yamaguchi prefecture. site 4 was in yoshida, yamaguchi (131°27’e, 34°9’n), yamaguchi prefecture. three colonies for each species were collected from each site, respectively. bioassay bioassays were conducted using a plastic container (82mm diameter x 46mm height) with a sheet of filter paper at 25°c. to stain the termite gut for discriminating the test individuals and target ones, termites of each colony were divided into two groups and were fed a filter paper stained by red food coloring and blue food coloring respectively for 24 hrs. thirty workers were introduced into the test arena as test individuals, and after 3 hrs a target individual was introduced into the arena. survival and occurrence of body color change of the target individual were recorded after 24hrs. in these bioassays, we regarded the death of target individual as “agonistic behavior” (i.e. non-nestmate recognition) and body color change of a target individual as “trophallaxis behavior” (i.e. nestmate recognition). the following three experiments were carried out in order to evaluate nestmate and non-nestmate recognitions for r. kanmonensis as well as for r. speratus. experiment 1: recognition between individuals from the same colony was tested. both test individuals and target ones were sampled from the same colony in each trial. for r. kanmonensis, one colony collected from each of 1208 sociobiolog y vol. 59, no. 4, 2012 sites 1 to 3 was used. one colony was used from site 4 for r. speratus. every trial was replicated 15 times. experiment 2: recognition between individuals from different colonies of the same species was tested. test individuals and target ones were sampled from the same species but different colonies in each trial. colonies collected from sites 1 to 3 were used for the tests of r. kanmonensis and for r. speratus samples from site 4 were used. in each site, 3 colonies were used for 6 possible combinations of test/target sets. every set was replicated 3 times, and in total 18 replication were done for each site. experiment 3: recognition between individuals from the different species was tested. test individuals and target ones were sampled from different species in each trial. for the tests of r. kanmonensis, individuals of one colony of r. kanmonensis collected from each of sites 1 to 3 were used as test individuals and individuals of one colony of r. speratus collected from each of sites 2 to 4 were used as target individuals. in each site, every trial was replicated 5 times and in total 15 replications were done. for the tests of r. speratus, individuals of a colony of r. speratus collected from site 4 were used as test individuals and individuals of one colony of r. kanmonensis from each of sites 1 to 3 were used as target individuals. every trial was replicated 10 times. gc analysis cuticular hydrocarbons were extracted from 3 colonies of r. kanmonensis from sites 1 to 3 respectively and 3 colonies of r. speratus from sites 2 to 4 respectively. three replications were done for each colony except the colony of r. kanmonensis from site 3 which admitted no replication due to a lack of individuals. cuticular hydrocarbons were extracted by immersing 100 workers in 2 ml of n-hexane for 5 min. after evaporation of the hexane, the extracts were redissolved in 1 to 10 µl of n-hexane for gas chromatography (gc). the gas chromatography was conducted by a shimadzu gc-14b (shimadzu, kyoto, japan) equipped with a flame ionization detector (fid) and db-1ht capillary column (frontier lab; length 30m, film thickness, 0.1 µm). helium was used as the carrier gas. the injection temperature was programmed at 65°c for 0.01 min and then raised at 30°c/min to 355°c, with a final hold for 26.3 min. the oven temperature was kept at 60°c for 0.1min and then raised at 20°c/min to 200°c, 10°c/min to 275°c, 5°c/ min to 355°c, with a final hold for 10 min. the detection temperature was 1209 takematsu, y. & k. kambara — nestmate recognition in sympatric reticulitermes sp. 355°c. hydrocarbons were identified according to the results of the gc-ms analysis reported in takematsu & yamaoka (1999) and quantified as the percent of the total hydrocarbon component. the differences of cuticular hydrocarbons within a colony and within a site were analyzed using principal component analysis (pca), employing the component ratio of cuticular hydrocarbons. the calculations were performed using the excel-toukei software program (social survey research information co., ltd., 2006). results bioassay numbers of survival and trophallaxis of target individuals of r. kanmonensis and r. speratus are shown in table 1. in the assay for the individuals from the same colony (experiment 1), all target individuals of both r. kanmonensis and r. speratus survived, and occurrences of trophallaxis were more than 86%. these facts indicated that both r. kanmonensis and r. speratus have strong nestmate recognition for individuals from the same colony. conversely, in the assay for the individuals from the different species (experiment 3), most individuals of both r. kanmonensis and r. speratus were dead and no trophaltable 1. number of survivors and trophallaxis of target individuals of r. kanmonensis and r. speratus. experiment species site n number of surviving targets (%) number of color changed targets (%) experiment1 r. kanmonensis site 1 15 15 (100.0) 15 (100.0) (same colony) r. kanmonensis site 2 15 15 (100.0) 15 (100.0) r. kanmonensis site 3 15 15 (100.0) 13 (86.7) r. speratus site 4 15 15 (100.0) 14 (93.3) experiment 2 r. kanmonensis site 1 18 7 (38.9) 3 (16.7) (different colony) r. kanmonensis site 2 18 9 (50.0) 2 (11.1) r. kanmonensis site 3 18 3 (16.7) 0 (0.0) r. speratus site 4 18 18 (100.0) 8 (44.4) experiment 3 r. kanmonensis site 1 15 0 (0.0) 0 (0.0) (different species) r. kanmonensis site 2 15 2 (13.3) 0 (0.0) r. kanmonensis site 3 15 1 (6.7) 0 (0.0) r. speratus site 4 10 0 (0.0) 0 (0.0) 1210 sociobiolog y vol. 59, no. 4, 2012 lactic behavior was observed. these indicated that both species have strong agonistic behavior against the different species. on the other hand, in the assay for the individuals from the different colonies of the same species (experiment 2), the results showed a marked difference between the two species. in the case of r. speratus, all target individuals survived, and the ratio of trophallactic behavior was 44.4%. while, in the case of r. kanmonensis, more than 50% of target individuals were dead and only a few target individuals changed their body color: especially in site 3 (r. speratus dominant-area), the survival rate of target individuals of r. kanmonensis was remarkably low and no trophallactic behavior was observed. gc analysis from r. kanmonensis, 21 components of hydrocarbons were identified and quantified. from r. speratus, 22 components of hydrocarbons were identified and quantified. cuticular hydrocarbon components of both species were different (takematsu & yamaoka 1999). nine substances were in fig. 1 pca evaluating the differences of cuticular hydrocarbons within and among colonies of r. kanmonensis based on the proportion of 20 major chc components. a-c show the colony of each site; solid diamond, solid circle and solid triangle show site 1, 2 and 3 respectively. 1211 takematsu, y. & k. kambara — nestmate recognition in sympatric reticulitermes sp. common. the results of the pca were plotted using the first and second principal component axes. fig. 1 shows the result of the pca evaluating the differences of cuticular hydrocarbons within and among colonies of r. kanmonensis based on the proportion of 21 components. the first principal component (pc1) explained 32.08% of the total variance and the second principal component (pc2) explained 25.92% of the total variance. fig. 2 shows the result of the pca evaluating the differences of cuticular hydrocarbons within and among colonies of r. speratus based on the proportion of 22 components. the first principal component (pc1) explained 43.28% of the total variance and the second principal component (pc2) explained 15.99% of the total variance. in both species, within each colony there was a very similar cuticular hydrocarbon profile, and they could be separable with other colonies. on the other hand, variations among colonies within a site of r. kanmonensis (fig. fig. 2 pca evaluating the differences of cuticular hydrocarbons within and among colonies of r. speratus based on the proportion of 20 major chc components. a-c show the colony of each site; solid circle, solid triangle and solid square show site 2, 3 and 4 respectively. 1212 sociobiolog y vol. 59, no. 4, 2012 1) were much smaller than those of site 4 for r. speratus (fig. 2 solid squares), which was used in the bioassays. discussion r. kanmonensis and r speratus are distributed sympatrically and r. kanmonensis is thought to be an alien species having been introduced into the native distribution range of r. speratus. r. speratus showed strong agonistic behavior against different species and no trophallactic contact with them. however, agonistic behavior against different colonies of the same species was weak and trophallactic behaviors were often observed. on the other hand, r. kanmonensis, a sympatric species with r. speratus, showed strong agonistic behavior against different colonies of the same species as well as of different species and no trophallactic contact was observed. these results suggest that r. speratus has colony fusion as already described in matsuura & nishda (2001), while r. kanmonensis so strongly exclude non-nestmate individuals that colony fusion can not occur. kitade et al. (2004) showed the lack of mitochondrial haplotype variation in r. kanmonensis within a mature nest and the aggregated distribution of founding colonies nested in small branches. this indicated that worker mingling did not occur in r. kanmonensis and founding colonies were under a strong intraspecific competition with mature colonies, resulting in a low survival rate of incipient colonies. in agreement with these results of kitade et al. (2004), our data showed strong intraspecific competition and at the same time a lack of colony fusion in the case of r. kanmonensis. for some reticulitermes species, occurrences of colony fusion have been reported, and it is known that the frequency of the phenomenon is highly variable depending on populations (bulmer & traniello 2002; deheer & vargo 2004,2008; leniaud et al. 2009; matsuura & nishida 2001; nobre et al. 2008; perdereau et al. 2010a). perdereau et al. (2010a), among others, investigated the social organization of r. flavipes with a special interest in invading species and reported that colony fusion occurred more frequently in introduced populations of france than in native populations of north america. the occurrence of colony fusion may be thought of as a key to lowering the cost for the intraspecific competition. this provides one possible mode of ecological advantage. in invaded habitats, colony fusion can be understood 1213 takematsu, y. & k. kambara — nestmate recognition in sympatric reticulitermes sp. as a mechanism that is favorable for the invasive success to attaining high worker densities and interspecific dominance. in many social insects, shifts in the social organization have already been observed and thought to contribute to invasive success (as was summarized by perdereau et al. 2010a). leniaud et al. (2009) also reported unicoloniality and low intraspecific aggression of r. urbis, an invading species in france, resulting in invasive success in the introduced region. since this is a case of r. kanmonensis being introduced into ranges where another ecologically more potent species is dominant, then it might have been thought that r. kanmonensis without the ability of colony fusion is unlikely to survive in such ecologically recessive environments. however, r. kanmonensis persisted for more than a century in such an area. the present result suggests that this long-term persistence was attained without resort to the ability of colony fusion. in this respect, it is to be noted that r. kanmonensis has a specific flexibility in the choice of habitats; that is r. kanmonensis tends to choose less favorable habitats (houses or living woods) when coexisting with r. speratus (kambara & takematsu 2009). to date, the original distribution of r. kanmonensis is not clear. it may be of interest to clarify the original distribution of r. kanmonensis and the social organization in that area. cuticular hydrocarbons are generally considered as an important cue for interspecific and colony recognition (clément & bagnères 1998). it has been well accepted that the cuticular hydrocarbon compositions are different qualitatively at the specific level, whereas there are quantitative differences at the intraspecific level (howard 1993; haverty et al. 1997). perdereau et al. (2010b) investigated the cuticular hydrocarbons of native and introduced populations of r. flavipes and reported the remarkable hydrocarbon homogeneity observed within the introduced populations compared to the native populations due to a reduction of genetic diversity through a genetic bottleneck or a selective process for the less common alleles of recognition. perdereau et al. (2010b) also assumed that the homogeneity of cuticular hydrocarbons in introduced populations of r. flavipes could explain the lack of intraspecific aggression and, indirectly, the high rate of colony fusion within these introduced populations the present study shows the homogeneity of cuticular hydrocarbons in r. kanmonensis compared to r. speratus. this phenomenon can be explained 1214 sociobiolog y vol. 59, no. 4, 2012 as a reduction of genetic diversity in the invading species as reported in perdereau et al. (2010b). however, based on the results of agonistic behavior, r. kanmonensis, an invading species, showed strong intraspecific competition in spite of the homogeneity of cuticular hydrocarbons. therefore in the present study, the intensity of agonistic behavior seems to depend not only on cuticular hydrocarbon-related factors but on other features of each species such as ecological or genetic traits. acknowledgments we thank dr. o. kitade (faculty of science, ibaraki university) for useful and valuable comments on field sampling. this study is supported by kakenhi (no. 16580039). references bulmer, m.s. & j.f.a. traniello 2002. lack of aggression and spatial association of colony members in reticulitermes flavipes. j. insect behav. 15: 121–126. clément, j.l. & a.g. bagnères 1998. nestmate recognition in termites. in: r.k. vander meer, m.d. breed, k.e. espelie & m.l. winston (eds.), pheromone communication in social insects. ants, wasps, bees, and termites. westview press, boulder, co, us: 126–155. deheer, c.j. & e.l. vargo 2004. colony genetic organization and colony fusion in the termite reticulitermes flavipes as revealed by foraging patterns over time and space. mol. ecol. 13: 431–441. deheer, c.j. & e.l. vargo 2008. strong mitochondrial dna similarity but low relatedness at microsatellite loci among families within fused colonies of the termite reticulitermes flavipes. insectes sociaux 55: 190–199. kambara, k. & y. takematsu 2009. field habitat selection of two coexistence species of reticulitermes, r. speratus and r. kanmonensis (isoptera, rhinotermitidae). sociobiology 54: 65–75. kitade, o. & y. hayashi 2002. localized distribution of an alien termite reticulitermes kanmonensis (isoptera: rhinotermitidae). entomological science 5: 197–201. kitade, o., y. hayashi, y. kikuchi & s. kawarasaki 2004. distribution and composition of colony founding associations of a subterranean termite, reticulitermes kanmonensis. entomological science 7: 1–8. kitade, o. & t. matsumoto 1993. symbiotic protistan faunae of reticulitermes (isoptera: rhinotermitidae) in the japan archipelago. sociobiolog y 23: 135–153. howard, r.w. 1993. cuticular hydrocarbons and chemical communication. in: d.w. stanley-samuelson & d.r. nelson (eds.), insect lipids: chemistry, biochemistry and biolog y. university of nebraska press, lincoln, ne, us: 179–226. 1215 takematsu, y. & k. kambara — nestmate recognition in sympatric reticulitermes sp. haverty, m.i., m.s. collins, l.j. nelson & b.l. thorne 1997. cuticular hydrocarbons of termites of the british virgin islands. j. chem. ecol. 23: 927–964. leniaud, l., a. pichon, p. uva & a.g. bagnres 2009. unicoloniality in reticulitermes urbis: a novel feature in a potentially invasive termite species. bulletin of entomological research, 99: 1–10. matsuura, k. & t. nishida 2001. colony fusion in a termite: what makes the society “open”? insectes sociaux 48: 378–383. morimoto, k. 2000. termite. in: japan termite control association (ed.), termite and control. japan termite control association, tokyo: 1–126. nawa, u. 1911. on “kiashi-shiroari” (reticulitermes flaviceps). konchu sekai 16: 33. nawa, u. 1912a. on a termite that emerges earlier. konchu sekai 15: 194–195. nawa, u. 1912b. nuptial flight of termite that emerges earlier. konchu sekai 17: 154. nawa, u. 1917. distribution of “kanmon-shiroari”. konchu sekai 21: 72–73. nobre, t., l. nunes & d.e. bignell 2008. colony interactions in reticulitermes grassei population assessed by molecular genetic methods. insectes sociaux 55: 66–73. perdereau, e., a.g. bagnères, s. dupont & f. dedeine 2010a. high occurrence of colony fusion in a european population of the american termite reticulitermes flavipes. insectes socaux 57:393–402. perdereau, e., f. dedeine, j.p. christidès & a.g. bagnères 2010b. variations in worker cuticular hydrocarbons and soldier isoprenoid defensive secretions within and among introduced and native populations of the subterranean termite, reticulitermes flavipes. j. chem. ecol. 36: 1189–1198. takematsu, y. 1999. the genus reticulitermes (isoptera: rhinotermitidae) in japan, with description of a new species. entomological science 2: 231–244. takematsu, y. & r. yamaoka 1999. cuticular hydrocarbons of reticulitermes (isoptera: rhinotermitidae) in japan and neighboring countries as chemotaxonomic characters. applied entomolog y and zoolog y 34: 179–188. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.23-27sociobiology 62(1): 23-27 (march, 2015) the influence of environmental complexity on the worker morphometry of ant assemblages introduction body size is the main phenotypic characteristic of organisms, because it strongly affects the way they interact with the environment. ants are organisms with wide intra and interspecific variability in body size (kaspari & weiser, 1999; geraghty et al., 2007, hurlbert et al., 2008). the size-grain hypothesis predicts that small animals perceive the terrestrial surface more rugose than large animals (kaspari & weiser, 1999). a benefit of being small would be exploiting food and shelter available in the interstices of the environment (kaspari & weiser, 1999; espadaler & gómez, 2001; hurlbert et al., 2008). furthermore, large ant species tend to have proportionally longer legs (kaspari & weiser, 1999), which allows them to move abstract the objective of the present study was to test whether environmental complexity influences the morphology of leaf litter worker ants, as predicted by the size-grain hypothesis. we collected data from three types of vegetation (shrubby, shrubbyarboreal, and arboreal) in restinga da marambaia, southeastern brazil. the shrubby vegetation had a very superficial leaf litter compared to the other two vegetation types. we measured head width, body length, and femur length of the ants collected in each vegetation type. we used average head width (hw) as a proxy for body size. the shrubby-arboreal and arboreal vegetation types were assumed to represent more rugose environments than the shrubby vegetation. leg length allometry was observed in each and all vegetation types. we did not find significant differences in body size and allometry of ant assemblage among vegetation types. hence, the size-grain hypothesis was corroborated only for leg allometry, but it did not predict a general environmental influence on ant morphometry. sociobiology an international journal on social insects lxt chevalier1, ab vargas1,2, jm queiroz1 article history edited by gilberto m. m. santos, uefs, brazil received 14 july 2014 initial acceptance 04 september 2014 final acceptance 25 september 2014 keywords size-grain hypothesis, environmental body size, complexity, restinga vegetation. corresponding author jarbas m. queiroz departamento de ciências ambientais, instituto de florestas, universidade federal rural do rio de janeiro br 465, km 7, seropédica, 23897-000, rj, brazil e-mail: jarquiz@gmail.com faster in planar environments, and, hence, makes it easier for them to find resources (hurlbert et al., 2008; gibb & parr, 2013). small ant species forage more efficiently in rugose environments than in planar environments (farji-brener et al., 2004; sarty et al., 2006). if small ants have more advantages in rugose environments, we could expect the ant community to have a larger proportion of small-bodied species in more rugose environments. a study carried out in african savannas did not corroborate this prediction (parr et al., 2003). however, farji-brener (2004) suggested that this may have occurred due to the use of environments whose rugosity gradient has been created by recent factors and that new tests should be restricted to natural gradients of environmental rugosity. in brazil, restinga environments are very heterogeneous in terms of vegetation: it is possible to find from forest sites to sites with sparsely distributed herbs on sand banks (menezes research article ants 1 universidade federal rural do rio de janeiro, rj, brazil 2 centro universitário de volta redonda (unifoa), rj, brazil lxt chevalier, ab vargas, jm queiroz environmental complexity and ant morphometry24 & araújo, 2005). different vegetation types have different soil cover, which creates environments with different degrees of leaf litter cover (vargas et al., 2007). the amount of leaf litter affects ant species richness (vargas et al., 2007; bastos & harada, 2011) and sites with differences in leaf litter can be used to carry out natural experiments aimed at testing the size-grain hypothesis. hence, the objective of the present study was to test whether the restinga ant communities with different types of vegetation differ in terms of worker ant morphometry. we aimed at testing three predictions: (1) the larger the body size of an ant species, the higher the ratio between leg length and body size; (2) rugose environments should have ant species with smaller body size than planar environments; (3) environmental rugosity influences the relationship between leg size and body size. material and methods study area we collected the data in two sampling events: one in the dry season (august, 2004) and the other in the rainy season (march, 2005), in three vegetation types in restinga da marambaia (23°03’ s 44°03’ w), state of rio de janeiro, southeastern brazil. the first vegetation (shrubby) is homogeneous with predominance of allagoptera arenaria (gomes) o. kuntze (arecaceae) and has no or superficial leaf litter, composed mainly of leaves of this palm. the second vegetation (shrubby-arboreal) is more heterogeneous, denser, and has higher plant richness than the first; its soil is covered by leaf litter. the third vegetation (arboreal) is a forest with a plant cover less dense than that of the second vegetation. it is the most heterogeneous vegetation, with the highest plant richness, and tallest trees. it has high density of bromeliads in the understory and deep leaf litter cover (menezes & araújo, 2005). the shrubby vegetation was considered a planar or less rugose environment, because its leaf litter cover is thinner and more superficial than that of the other vegetation types. the shrubby-arboreal and arboreal vegetation types had more and deeper leaf litter cover; hence, they were considered rugose environments (vargas et al., 2007). sampling procedures in each vegetation type, we marked three 1,200-m2 plots (40 x 30 m), 100 m to 200 m apart from each other. the plots in the shrubby vegetation were ca. 500 m apart from the plots of the shrubby-arboreal vegetation, and the plots of both of these vegetation types were 1,000 m apart from the arboreal vegetation. in each plot, we set up 20 pitfall traps, at 10 m from each other, which remained open for 48 h in the field (see vargas et al., 2007). morphometry data we measured head width and femur length, in millimeters, of three specimens of each ant species, whenever possible. head width is considered a precise standard measurement of body size (hölldobler & wilson, 1990; kaspari & weiser, 1999) and femur length is a good proxy for total leg length. data analysis in total, we analyzed 84 out of the 92 species collected by vargas et al. (2007). to test the prediction that body size influences the ratio between leg length and body size, we used average head width (hw) as an independent variable and the ratio between femur length (fl) and hw as a dependent variable in a linear regression. to test whether environmental rugosity influenced the morphometry of worker ants, we compared hw and the relationship between hw and fl among the three vegetation types with a non-parametric analysis of variance. all analyses were performed in systat 8.0. results head width (hw) explained a significant part of the variation observed in the femur length/head width ratio for both the total set of species (r2 = 0.1027; p = 0.003; figure 1) and for each of vegetation type separately (shrubby, r2 = 0.102; p fig 1. relationship between head width and femur length/head width ratio (p = 0.003) for the ant fauna of restinga da marambaia, state of rio de janeiro, southeastern brazil. = 0.005; shrubby-arboreal, r2 = 0.115; p = 0.007; arboreal, r2 = 0.137; p = 0.002; figure 2). the average ant head width for the total dataset was 0.873 mm (standard error = 0.064). the average head width was 0.944 mm (standard error = 0.119) for the shrubby vegetation, 0.911 mm (standard error = 0.079) for the shrubby-arboreal, and 0.820 mm (standard error = 0.065) for the arboreal vegetation. average body size did not differ significantly among vegetation types (kruskal-wallis, u = 0.418; p = 0.811; figure 3). in addition, environmental rugosity did not sociobiology 62(1): 23-27 (march, 2015) 25 influence the fl/hw ratio (kruskal-wallis, u = 0.210; p = 0.900; figure 4). discussion the three vegetation types analyzed in the present study differed from each other in ant species richness. the most complex vegetation type with highest plant diversity has also the richest ant fauna (vargas et al., 2007). our results confirmed allometry in leg size (first prediction) for the entire ant fauna and each vegetation separately. larger ants had disproportionally longer legs than smaller ants, which corroborates previous studies (kaspari & weiser, 1999; espadaler & gómez, 2001; parr et al., 2003). farji-brener (2004) suggested that size comparisons of ant species should be done between environments with natural differences in rugosity. this is the case of the vegetation types studied by us, which, due to variations in the amount of leaf litter, formed a rugosity gradient (vargas et al., 2007). according to the predictions of the size-grain hypothesis, we expected that the most complex environment, which corresponded to the vegetation type with highest plant diversity and highest amount of leaf litter, would have an ant fauna with smaller average body size than the vegetation type with lower amount of leaf litter. in addition, environmental rugosity would also affect the relationship between leg length and body size; more rugose environments would tend to be occupied by species with proportionally shorter legs (kaspari & weisser, 1999). however, we did not find a relationship between workers morphometry and environmental rugosity, because no significant difference in average body size among vegetation types was detected: environmental rugosity did not affect leg allometry either. parr et al. (2003) carried out similar tests in african savannas and found the highest abundance of small ants in the more planar environment, with the lowest amount of leaf litter, contrary to their expectations. conversely, gibb and parr (2013) found a relationship between environment rugosity and body size, as expected, but not in all situations tested. fig 2. relationship between ant head width and femur length/head width ratio in each vegetation type in restinga da marambaia, state of rio de janeiro, southeastern brazil: (a) shrubby vegetation (p = 0.005), (b) shrubby-arboreal vegetation (p = 0.007), and (c) arboreal vegetation (p = 0.002). fig 3. head width (hw) of ants in different plant vegetation types in restinga da marambaia, state of rio de janeiro, southeastern brazil. fig 4. femur length/head width ratio (fl/hw) of ants in different vegetation types in restinga da marambaia, state of rio de janeiro, southeastern brazil. lxt chevalier, ab vargas, jm queiroz environmental complexity and ant morphometry26 parr et al. (2003) rejected the hypothesis that the ant sampling method (pitfall traps) might have influenced the results. however, pitfall traps tend to capture larger species than other sampling methods (vargas et al., 2009). very small species, which do not move much, may not be captured in pitfalls; hence, there might be a bias in the result of size comparisons between faunas (vargas et al., 2009). it would be important to test this hypothesis with the same morphometric analyses used in the present study, but with samples obtained with different methods. the lack of significant differences in ant size observed in the studied communities in different restinga vegetation types may have resulted from large species with relatively short legs that can succeed in rugose environments and from small species with long legs that can succeed in planar environments (gibb & parr, 2013). in addition, it is important to consider a potential influence of intraspecific variations on those tests. for example, in the same colony of atta robusta or camponotus crassus, which are species found in the restinga, worker ants with different body sizes could explore resources of different sizes and forage in environments with different rugosity, and, therefore, increase the total success of the colony (farji-brener et al., 2004; longino, 2005). besides a. robusta and c. crassus, other species such as odontomachus chelifer and trachymyrmex atlanticus have intraspecific variations in body measurements. the lack of consensus about the influence of the environment on the morphometry of leaf litter worker ants suggests that other factors may influence the results (chow & parr, 2004). other morphometric data could also be added to comparative analyses on environmental influence (silva & brandão, 2011). however, ants with different body sizes use the habitat in different ways and can explore different resources (kaspari & weisser, 1999). the lack of differences in ant morphometry among environments can simply mean that the species in the richest community shares the resources in the rugose environment, which has the highest amount of leaf litter, according to its characteristics; larger ants can forage on the leaf litter, whereas smaller ants walk in the space within the leaf litter. in spite of interpreting these differences as potential spatial segregation among species, we should consider that they might explain the higher species diversity observed in more complex environments. acknowledgments we thank the comments of anonymous reviewers, and caex-marambaia (brazilian army), for the permission to work in the restinga da marambaia area. jmq received a scholarship from faperj (proc.101.472/2010). references bastos, a. h. s. & harada, a. y. 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(2009). teste da regra de equivalência energética para formigas de serapilheira: efeitos de diferentes métodos de estimativa de abundância em floresta ombrófila. neotropical. entomology, 38: 867-870. doi: 10.1590/s1519-566x2009000600023. yanoviak, s. p. & kaspari, m. 2000. community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 256-266. doi: 10.1034/j.1600-0706.2000.890206.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.6159sociobiology 68(2): e6159 (june, 2021) termites play important roles in ecosystems, especially as decomposers and soil engineers (bignell & eggleton, 2000; holt & lepage, 2000). termites can also cause serious structural damage to buildings, crops, and plantation forests (su & scheffrahn, 2000; rouland-lefèvre, 2010). some termite species have been introduced outside their native ranges, where they have become invasive, and caused substantial economic, and possible ecological impacts (evans, 2010; evans et al., 2013). evans et al. (2013) recognized 28 invasive termite species and found that they all shared three characteristics: they eat wood, nest in wood, and easily generate secondary reproductives. to date, seven termite species, which are considered to have been introduced, are established in japan: zootermopsis nevadensis (hagen), incisitermes immigrans (snyder), i. minor (hagen), i. abstract some termites have been introduced outside their native ranges, and it has been suggested that both wood-eating and wood-nesting lifestyles are critical for the transportation of propagules. here, we present the first record of the soil-feeding and soil-nesting termite pericapritermes nitobei (isoptera: termitidae) in the motobu peninsula on the northern part of okinawa island in the central ryukyus. the fact that previous extensive surveys of termites in the ryukyu archipelago did not detect p. nitobei in the central ryukyus, including the motobu peninsula, and its discontinuous distribution, suggest that p. nitobei has been recently introduced into the motobu peninsula of okinawa island from the yaeyama islands, similar to the fungus-growing and soilnesting termite odontotermes formosanus (isoptera: termitidae). together with o. formosanus in the southern part of okinawa island, our findings raise the possibility that non-wood-feeding and non-wood-nesting termites can be introduced outside their native ranges by human activities. sociobiology an international journal on social insects tomonari nozaki1,3, masaru hojo2,4, toshihisa yashiro3*, kenji matsuura3 article history edited by paulo cristaldo, ufrpe, brazil received 24 december 2020 initial acceptance 16 march 2021 final acceptance 08 april 2021 publication date 31 may 2021 keywords biological invasions, invasive termites, pericapritermes nitobei, social insects, the ryukyu archipelago. corresponding author tomonari nozaki laboratory of evolutionary genomics, center for the development of new model organisms, national institute for basic biology okazaki 444-8585, japan e-mail: tomonari64.1.8@gmail.com schwarzi (banks), coptotermes formosanus shiraki, c. gestroi (wasmann), and reticulitermes kanmonensis takematsu (morimoto & ishii, 2000; kitade & hayashi, 2002; ohmura & tokoro, 2003; ide et al., 2016; yashiro et al., 2018). although two of these termite species (i. schwarzi and r. kanmonensis) have not been listed as invasive by evans et al. (2013), they also have the above-mentioned three characteristics of invasive termites (luykx, 1986; kitade et al., 2011). the ryukyu archipelago of japan is located in the subtropical zone of the western pacific ocean between mainland japan and taiwan. faunistically, the ryukyu archipelago is classified into three regions: the northern, central, and southern ryukyus, based on their relation to the tokara and kerama gaps (fig 1a, kimoto, 1967; ota, 1998; motokawa, 2000). it has been known that the higher 1 laboratory of evolutionary genomics, national institute for basic biology, japan 2 global education institute, university of the ryukyus, japan 3 laboratory of insect ecology, graduate school of agriculture, kyoto university, japan 4 national cheng kung university, taiwan *present address: division of agro-environment research, kyushu okinawa agricultural research center, naro, koshi, japan short note first record of the soil-feeding higher termites (isoptera: termitidae) from okinawa island, the central ryukyus, japan id https://orcid.org/0000-0003-4358-8118 tomonari nozaki, masaru hojo, toshihisa yashiro, kenji matsuura – first record of pericapritermes on okinawa island in japan2 termites (family termitidae) of the ryukyu archipelago are distributed only in the southern ryukyus, including ishigaki and iriomote islands, which are part of the yaeyama islands with one exception, the fungus-growing termite odontotermes formosanus (shiraki), which is distributed not only on the yaeyama islands, but also on the okinawa islands in the central ryukyus (ikehara, 1966; yasuda et al., 2000). it is suggested that the o. formosanus found on the okinawa islands has been introduced from the yaeyama islands (presumably from iriomote island), based on its discontinuous and restricted distribution on okinawa island (hojo, 2019). in this study, we report the unusual findings of a population of the soil-feeding higher termite pericapritermes nitobei (shiraki) (isoptera: termitidae) on the northern part of okinawa island in the central ryukyus, as this species is native to the yaeyama islands and other regions (taiwan, mainland china, and southeast asia) (ikehara, 1966; yasuda et al., 2000; krishna et al., 2013). on february 6th, 2017, we found a termite colony having soldiers with a twisted left mandible and curved right mandible (i.e., asymmetric snapping mandibles) from the soil in the motobu peninsula in the northern part of okinawa island (fig 1b). based on the soldier morphology, termites from that colony were suggested to belong to pericapritermes (bourguignon et al., 2008). this genus contains only one known species in the ryukyu archipelago, p. nitobei (ikehara, 1966; yasuda et al., 2000). during the second survey conducted on june 3rd and 4th, 2019, we successfully collected six colonies of pericapritermes from the soil and observed egg piles in three of the six colonies (table 1). these colonies were at least 100 m apart; the distribution was limited in the roadside forest (fig 1c). the collected termites were fixed in absolute ethanol and brought back to the laboratory. in order to confirm the identity of the termite species collected in the motobu peninsula and to clarify its relationship to other pericapritermes species, we performed a phylogenetic analysis based on the sequences of the mitochondrial cytochrome c oxidase subunit ii (coii) gene. one randomly chosen worker from the six colonies of the termite from the motobu peninsula was used for the phylogenetic analysis. termite dna was extracted from the head using the dneasy prep kit (qiagen) according to the manufacturer’s protocol and used as a template for polymerase chain reaction (pcr). a fragment of coii was amplified by pcr using the primer set, modified a-tleu (5′-cagataagtgcattggattt-3′) (miura et al., 1998) and b-tlys (5′gtttaagagaccagtacttg -3′) (liu & beckenbach, 1992). the pcr amplification was performed in 20 μl total reaction volumes containing 10 μl of primestar hs dna polymerase (takara), 0.6 μl of each primer (10 μm), 1 μl of template dna, and 7.8 μl of high-purity water. the pcr cycling conditions consisted of 35 cycles of denaturation (98°c, 10 s), annealing (50°c, 5 s), and extension (72°c, 1 min). amplified double-stranded dna was purified using a qiaquick pcr purification kit (qiagen) and then sent to an outside sequence service (fasmac inc., kanagawa, japan). we obtained 684 bp coii sequences. consensus sequences were aligned using the clustalx algorithm (thompson et al., 1997) from the bioedit 7.0.4.1 sequence editor (hall, 1999) and corrected manually. the coii sequences of p. nitobei in its native range (including ishigaki island, iriomote island, taiwan, and mainland china) (genbank accession numbers (gb#) ab109517, mh194575, and mw073093–mw073099) and other termitid species (gb# ab109518, dq442140, dq442217, kp026278, and ky224616) were also used in the analysis. bayesian inference was performed using mrbayes 3.1.2 (ronquist et al., 2003) with the gtr + g model selected by the hierarchical likelihood ratio test (hlrt) in mrmodeltest 2.3 (nylander et al., 2004). homallotermes eleanorae emerson (gb# dq442140) was used as an outgroup. the phylogenetic analysis methods have been described in a previous report (yashiro et al., 2010). additionally, we analyzed the obtained nuclear internal transcribed spacer 2 (its2) sequences (264 bp) of the termite from the motobu peninsula and the its2 sequences of p. nitobei in its native range (gb#mw067661– mw067667). primer sequences and pcr conditions are detailed in a previous report (maryańska-nadachowska et al., 2010), and pcr product purification and sequencing were conducted as described above. the coii and its2 sequences obtained in this study were deposited in the ddbj/embl/ genbank nucleotide sequence databases (gb# mw073087– mw073092 and mw067655–mw067660). all of the mitochondrial coii sequences of individuals from the six colonies obtained from the motobu peninsula of okinawa island were identical to each other (gb# mw073087–mw073092), and to one of two haplotypes of p. nitobei on ishigaki and iriomote of the yaeyama islands (gb# mw073093–mw073096, and mw073098) (fig 2). therefore, the termite collected from the motobu peninsula were phylogenetically identified as p. nitobei. phylogenetic trees based on mitochondrial coii sequences showed the monophyly of p. nitobei, including individuals from the motobu peninsula, the yaeyama islands, taiwan, and mainland china populations (bayesian posterior probability = 0.93), where individuals from the six colonies from the motobu peninsula formed a clade with those from the yaeyama islands (bayesian posterior probability = 0.91) (fig 2). in addition, all of the nuclear its2 sequences of individuals from 12 colonies of p. nitobei in the ryukyu archipelago (six in the motobu peninsula, one on ishigaki island, and five on iriomote island) were identical to each other (gb# mw067655–mw067666), but these sequences were different from the its2 sequence of p. nitobei in taiwan (gb# mw067667) (98% sequence identity). considering the fact that the previous extensive surveys of termites in the ryukyu archipelago (including the motobu peninsula) did not detect p. nitobei on okinawa island (fig 1a, ikehara 1966; yasuda et al., 2000), and its discontinuous distribution, approximately 350 km east of the sociobiology 68(2): e6159 (june, 2021) 3 fig 1. the soil feeding termite collected in the motobu peninsula of okinawa island. (a) the map of the ryukyu archipelago. (b) a photo of the termite, which was phylogenetically identified as pericapritermes nitobei. the inset is an enlarged view of soldier head with a twisted left mandible and curved right mandible. (c) the map showing motobu peninsula and the collection sites in the survey on june 2019. filled and open circles indicate the presence and absence of the termite, respectively. maps were drawn by the “maptools” in the r software (version 3.6.2; http://cran.r-project.org/) with the data provided by national land numerical information, ministry of land, infrastructure, transport and tourism, japan (https://nlftp.mlit.go.jp/index.html). tomonari nozaki, masaru hojo, toshihisa yashiro, kenji matsuura – first record of pericapritermes on okinawa island in japan4 previous easternmost record of the species (ishigaki island, the yaeyama islands) (fig 1a, ikehara, 1966; yasuda et al., 2000; krishna et al., 2013), our results strongly suggest that p. nitobei in the motobu peninsula has been recently introduced from the yaeyama islands. we found the soil feeding higher termite p. nitobei in the motobu peninsula in the central ryukyus in 2017 and 2019, and observed egg piles, indicating the establishment of a population. the termite is most likely to have been recently introduced from the yaeyama islands in the southern ryukyus, as is suspected for the fungus-growing higher termite o. formosanus on okinawa island (hojo, 2019). interestingly, both are soil-nesting species (chiu et al., 2015; chiu et al., 2018). trade in live plants with soil is an important pathway facilitating long-distance invasions of soil-borne insects (meurisse et al., 2019). so far, several tree and shrub species have been introduced from the yaeyama islands into public parks on okinawa island, as well as in the motobu peninsula (ocean expo. commemorative park management foundation (1997)); therefore, soil transferred with live plants is one of the possible introduction pathways for these termites. our finding raised the possibility that not only woodfeeding and wood-nesting termites, but also soil-feeding termites can be introduced and established outside their native ranges by human activities. although many aspects of its biology (including the capability of producing secondary reproductives) and its impact on other organisms in the ecosystem remains to be investigated, the population of p. nitobei on the motobu peninsula of okinawa island, together with the population of o. formosanus on okinawa island, would provide a promising model system for the study of biological invasion by non-wood-feeding and non-woodnesting termites. fig 2. bayesian phylogenetic tree of pericapritermes termites based on mitochondrial coii sequences, including the soil feeding termite in the motobu peninsula, which was identified as p. nitobei (presented in bold). the corresponding posterior probabilities (≥ 0.70) are shown by the branches. genbank accession numbers are shown in parentheses. sociobiology 68(2): e6159 (june, 2021) 5 acknowledgments we thank s. shigenobu for the experimental equipment and the space, and n. idogawa, w. yao, and k. watanabe for sample collection. we also appreciate m. ohkuma for providing information about p. nitobei on iriomote island, and anonymous reviewers for critical review of the manuscript. this study was supported by funding from the japan society for the promotion of science to t.n. 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(in japanese with english summary). doi: 10.20848/kontyu.3.4_139 doi: 10.13102/sociobiology.v62i1.92-98sociobiology 62(1): 92-98 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 record of parasitoids in nests of social wasps (hymenoptera: vespidae: polistinae) introduction polistinae wasps primarily use plant materials to build their nests and can be divided into two groups according to nesting behavior and architecture: independentand swarmfounding species (carpenter, 1991; wenzel, 1998). independentfounding species (polistini, mischocyttarini and some old world ropalidiini) build unprotected combs that are small in size with few brood cells (jeanne, 1980; carpenter & marques, 2001). swarming-founding species (epiponini and most ropalidiini) in turn builds large nests with thousands of cells protected by an envelope (jeanne, 1980; carpenter & marques, 2001). paper wasp (hymenoptera: vespidae: polistinae) nests are frequently invaded by natural parasitoids of eggs, larvae and pupae. the nest of a social wasp is an environment rich in resources for many predators and parasitoids, which might be attracted and can cause high costs to the colony (soares et al., 2006). larvae and pupae are the target of many of these natural enemies including ants, birds, and parasitoid hymenopterans (makino, 1985; yamane, 1996; clouse, 2001). despite the abstract the aim of this study was to record the parasitoid species found in social wasps nests sampled in different localities in brazil. we sampled nests of mischocyttarus cassununga, mischocyttarus consimilis, mischocyttarus imitator, polistes canadensis, polistes cinerascens, polistes versicolor, angiopolybia pallens, leipomeles spilogastra, polybia jurinei and two indeterminate species of mischocyttarus. thus, we observed that nests of m. cassununga, m. imitator and mischocyttarus (phi) sp.1 were parasitized by toechorychus guarapuavus (ichneumonidae) and nests of m. consimilis, m. imitator and mischocyttarus sp.2 were parasitized by toechorychus fluminensis (ichneumonidae). nests of p. versicolor and p. cinerascens were parasitized by elasmus polistis (eulophidae) and nest of p. canadensis was parasitized by simenota depressa (trigonalidae); nest of a. pallens and l. spilogastra, was infested by brachymeria sp.1 and brachymeria sp.2 (chalcididae), respectively. nests of m. cassununga and polybia jurinei were parasitized by megaselia scalaris (phoridae). sociobiology an international journal on social insects 1instituto nacional de pesquisas da amazônia, petrópolis, manaus, am, brazil 2universidade federal de são carlos, são carlos, sp, brazil 3universidade estadual de santa cruz, ilhéus, ba, brazil article history edited by gilberto m. m. santos, uefs, brazil received 19 march 2014 initial acceptance 23 may 2014 final acceptance 25 september 2014 keywords epiponini, eusocial, mischocyttarini, paper wasps, parasitoids, polistini. corresponding author alexandre somavilla coord. de biodiversidade, instituto nacional de pesquisas da amazônia av. andré araújo, 2936, petrópolis, 69067-375, manaus, am, brazil e-mail: alexandre.s@hotmail.com paucity of studies on the subject, the attack by hymenopterans parasitoids might be one of the main causes of mortality among social wasps in the early stages of development (wenzel, 1998). in spite of that, there are few attempts to reporting parasitism in these social wasps (makino, 1985; yamane, 1996; clouse, 2001). consequently there are few records concerning natural enemies of paper wasps and how harmful such natural enemies might be for a wasp colony. to brazil, soares et al. (2006) reported a species of pachysomoides sp. (ichneumonidae) and megaselia scalaris (diptera, phoridae) parasitizing mischocyttarus cassununga (von ihering) nests in minas gerais; dorfey and köhler (2011) reported elasmus polistis berks, 1971 parasitizing polistes versicolor (oliver, 1792) nests in rio grande do sul; and trindade et al. (2012) and santos and noll (2013) reported the occurrence of seminota marginata (westwood, 1874) in apoica flavissima (van der vecht, 1973) nests in são paulo. the aim of this study was to record parasitoids found in social wasps nests, sampled during several years of survey in different localities in brazil. research article wasps a somavilla1,4, k schoeninger1, af carvalho2, rst menezes3, ma del lama2, ma costa3, ml oliveira1 sociobiology 62(1): 92-98 (march, 2015) 93 material and methods during several collection trips, between 2009 and 2013, across brazilian biomes as atlantic rainforest (gramado [rs], sinimbu [rs], ibirapitanga [ba], maricá [rj], ubatuba [sp], viçosa [mg]), pantanal (dourados [ms]), caatinga (jacobina [ba]) and amazonia (manaquiri [am], manaus [am], rorainópolis [rr]) we removed entire nests of polistine species from the field and separated all adults from the offspring. the later were put in plastic recipients covered by voile tissue and maintained in laboratory to remove individuals from the emerged social wasps. the combs containing offspring were maintained during approximately 40 days at 28°c and relative humidity of 70% + 5%, in a biochemical oxygen demand (bod) incubator and monitored daily until adults emerged. after the 40 days, we verified the emergence of polistines and parasitoids as well as the nests were open for counting of cells. however, for the nest of angiopolybia pallens (olivier, 1792) and mischocyttarus consimilis zikán, 1949, this procedure could not be performed since the nest were damaged. most of the individuals (hosts and parasitoids) were fixed in absolute ethanol and some samples were pinned and are deposited in the coleção zoológica de invertebrados of the instituto nacional de pesquisas da amazônia (inpa) and in the laboratório de genética evolutiva de himenópteros of the universidade federal de são carlos (lgeh-ufscar). voucher nests were deposited in both collections as well. specimens were identified using the following identification keys: richards (1978) and carpenter and marques (2001) for social wasps, burks (1960) and boucek (1992) for parasitoid wasps and brown (2010) for phoridae. results we sampled two nests of mischocyttarus cassununga (r. von ihering, 1903), one nest from viçosa, in the state of minas gerais and one nest from ubatuba, in the state of são paulo; one nest of mischocyttarus consimilis zikán, 1949 from dourados, in the state of mato grosso do sul; two nests of mischocyttarus imitator (ducke, 1904) (fig 1a, 1b), one nest from viçosa, in the state of minas gerais and other from maricá, in the state of rio de janeiro; one nest of mischocyttarus (phi) sp.1 from jacobina, in the state of bahia; one abandoned nest of mischocyttarus sp.2 from manaus, in the state of amazonas, five nests of polistes canadensis (linnaeus, 1758) (fig 2a, 2b, 2c) from manaquiri, amazonas, one nest of polistes cinerascens de saussure, 1854 from sinimbu, in the state of rio grande do sul; two nests of polistes versicolor (olivier, 1791) (fig 3a, 3b) from gramado and sinimbu, rio grande do sul; one nest of angiopolybia pallens (lepeletier, 1836) (fig 4a, 4b) from ibirapitanga, bahia; one nest of leipomeles spilogastra cameron, 1912 (fig 5a, 5b) from rorainópolis, in the state of roraima and one nest of polybia jurinei de saussure, 1854 from manaus, amazonas (table 1). fig 1. (a) mischocyttarus imitator nest; (b) mischocyttarus imitator habitus; (c) toechorychus guarapuavus habitus. fig 2. (a, b) polistes canadensis nests; (c) polistes canadensis habitus; (d, e) two color forms to seminota depressa. fig 3. (a) polistes versicolor nest; (b) polistes versicolor habitus; (c) elasmus polistis habitus. the abovementioned nests were infected by hymenopterans or by dipterans parasitoids, as follows (see also table 1): (1) mischocyttarini: m. cassununga was parasitized by toechorychus guarapuavus tedesco, 2013 (ichneumonidae) and megaselia scalaris (loew, 1866) (diptera: phoridae), m. consimilis was parasitized by toechorychus fluminensis tedesco, 2013 (ichneumonidae) and m. imitator was parasitized by t. guarapuavus (fig 1c) and t. fluminensis. (2) polistini: nests of p. cinerascens and p. versicolor were parasitized by elasmus polistis burks, 1971 (fig 3c) (eulophidae) and nests of p. canadensis were parasitized by seminota depressa degeer, 1773 (fig 2d, 2e) (trigonalidae). a somavilla et al. parasitoids in nests of social wasps94 t ab le 1 . r ec or ds o f pa ra si to id s in n es ts o f so ci al w as ps s am pl ed in d if fe re nt lo ca lit ie s of b ra zi l. n um be r of e m er ge d ad ul t s oc ia l w as ps f ro m th e ne st ( h os t n ); n um be r of pa ra si to id s em er ge d fr om th e ne st (p ar as ito id n ); c om b an d nu m be r o f c el ls fr om th e co m b (c om b / c el l n ). h os t pa ra si to id h os t n pa ra si to id n c om b/ ce ll n l oc al ity m is ch oc yt ta ru s ca ss un un ga ( r . v on ih er in g, 1 90 3) to ec ho ry ch us g ua ra pu av us t ed es co , 2 01 3 (i ch ne um on id ae ) 52 30 ( 30 ♂ ) 1/ 14 9 v iç os a, m g : u fv , ( 20 º4 5’ s, 4 2º 52 ’w ) m is ch oc yt ta ru s ca ss un un ga (r . v on ih er in g, 1 90 3) m eg as el ia s ca la ri s l oe w , 1 86 6 (p ho ri da e) 03 50 1/ 23 u ba tu ba , s p: r pp n a ng el in r ai nf or es t ( 23 º2 6’ s, 45 º0 4’ w ) m is ch oc yt ta ru s co ns im ili s z ik án , 1 94 1 to ec ho ry ch us fl um in en si s te de sc o, 2 01 3 (i ch ne um on id ae ) 05 01 ( 01 ♂ ) 1/ d ou ra do s, m s (2 2° 13 ’s , 5 4° 51 ’w ) m is ch oc yt ta ru s im ita to r (d uc ke , 1 79 2) to ec ho ry ch us g ua ra pu av us t ed es co , 2 01 3 (i ch ne um on id ae ) 35 04 ( 04 ♂ ) 1/ 10 4 v iç os a, m g : m at a do p ar aí so ( 20 º4 5’ s, 4 2º 52 ’w ) m is ch oc yt ta ru s im ita to r (d uc ke , 1 79 2) to ec ho ry ch us fl um in en si s te de sc o, 2 01 3 (i ch ne um on id ae ) 06 03 ( 03 ♂ ) 1/ 45 m ar ic á, r j: e st ra da d o e sp ra ia do ( 22 °5 5’ s, 4 2° 47 ’w ) m is ch oc yt ta ru s (p hi ) s p. 1 to ec ho ry ch us g ua ra pu av us t ed es co , 2 01 3 (i ch ne um on id ae ) 08 02 ( 02 ♂ ) 1/ 12 4 ja co bi na , b a ( 11 °1 1’ s, 4 0° 29 ’ w ) m is ch oc yt ta ru s sp .2 ( ab an do ne d ne st ) to ec ho ry ch us fl um in en si s te de sc o, 2 01 3 (i ch ne um on id ae ) 00 01 ( 01 ♂ ) 1/ 12 m an au s, a m : d uc ke r es er ve ( 02 º5 5’ s, 5 9º 58 ’w ) p ol is te s ca na de ns is ( l in na eu s, 1 75 8) se m in ot a de pr es sa d eg ee r, 17 73 ( tr ig on al id ae ) 41 09 ( 09 ♂ ) 5/ 13 1 m an aq ui ri , a m : b r 31 9 (3 °2 6’ s, 6 0° 26 ’w ). p ol is te s ci ne ra sc en s de s au ss ur e, 1 85 4 e la sm us p ol is tis b er ks , 1 97 1 (e ul op hi da e) 16 14 ( 12 ♀ , 0 2♂ ) 1/ 15 0 si ni m bu , r s: r pp n u n is c ( 29 °2 3’ s, 5 2° 32 ’w ) p ol is te s ve rs ic ol or ( o liv ie r, 17 92 ) e la sm us p ol is tis b er ks , 1 97 1 (e ul op hi da e) 12 1 35 7 (2 40 ♀ , 1 17 ♂ ) 1/ 69 6 g ra m ad o, r s (2 9º 22 ’s , 5 0º 52 ’w ) p ol is te s ve rs ic ol or ( o liv ie r, 17 92 ) e la sm us p ol is tis b er ks , 1 97 1 (e ul op hi da e) 52 43 9 (2 58 ♀ , 1 81 ♂ ) 1/ 95 0 si ni m bu , r s: r pp n u n is c ( 29 °2 3’ s, 5 2° 32 ’w ) a ng io po ly bi a pa lle ns (o liv ie r, 17 92 ) b ra ch ym er ia s p. 1 (c ha lc id id ae ) 82 8 11 ( 11 ♀ ) 6/ ib ir ap ita ng a, b a : b r 10 1 (1 4º 09 ’s , 5 9º 22 ’w ) le ip om el es s pi lo ga st ra ( c am er on , 1 91 2) b ra ch ym er ia s p. 2 (c ha lc id id ae ) 15 04 ( 04 ♀ ) 3/ 16 5 r or ai nó po lis , r r : b r 1 74 ( 00 º5 6’ n , 6 0º 25 ’w ) p ol yb ia ju ri ne i d e sa us su re , 1 85 4 m eg as el ia s ca la ri s l oe w , 1 86 6 (p ho ri da e) 11 02 1/ 68 m an au s, a m : d uc ke r es er ve ( 02 º5 5’ s, 5 9º 58 ’w ) sociobiology 62(1): 92-98 (march, 2015) 95 the polistinae wasps might be more susceptible to the attack of parasitoids when living in colonies with absence of brood keepers while foraging (clouse, 1997; 2001). another factor that may contribute to the attack of parasitoids is the absence of a nest-protecting envelope. polistes and mischocyttarus are independent-founding groups with remarkable non-enveloped nests. epiponini species in turn shows swarm-founding behavior build nest envelopes that protect fragile brood. strassmann (1981) suggests that swarm-founding species are less susceptible to parasitism once adults protect constantly the nest, preventing the invasion of possible enemies. thus, the difference of parasitoidism between species with enveloped and species with non-enveloped nests and its significance must be further investigated. most ichneumonidae are ecto or endoparasitoids of arthropods, usually attacking immature instars of holometabolous insects of the order lepidoptera, coleoptera, diptera and neuroptera (clausen, 1940; askew, 1971; hanson & gauld, 1995). these insects are considered important in biological control since they are parasitoids that always kill the host (kumagai & graf, 2000). there are few reports of ichneumonidae parasitizing paper wasp nests and this lack of information is mostly due to the great difficult in detecting symptoms of parasitism in nests of these social insects. until now, there are records only for mischocyttarus, polistes and dolichovespula rohwer. species of toechorychus townes (ichneumonidae) are apparently parasitoids in nests of vespidae and pupae of lepidoptera, but host records were previously known only for two species: toechorychus abactus (cresson, 1874) and toechorychus cassunungae (brauns, 1905) are known to attack species of mischocyttarus (brauns, 1905; bertoni, 1911; costalima, 1962; makino, 1985) and toechorychus albimaculatus (taschenberg, 1876) attack p. canadensis (makino, 1985) seminota (trigonalidae) has been reported as a parasitoid of some genera of social wasps such as apoica lepeletier, mischocyttarus, parachartergus r. von ihering, polistes and pseudopolybia von dalla torre (makino, 1985; weinstein & austin, 1991; carmean & kimsey, 1998) but little is known on the biology of these associations being that weinstein and austin (1991) considered paper wasps as the secondary host of trigonalidae. on the other hand, santos and noll (2013) observing the emergence of parasitoid seminota marginata (westwood, 1874) in one nest of apoica flavissima van der vecht, 1972 suggested that social wasps may be both primary and secondary hosts of such parasitoids as they extract and chew vegetable fiber. we observed that individuals of s. depressa which emerged from the nests of p. canadensis had variations regarding the pigmentation on the integument (fig 2d, 2e), one totally black with blackened wings and the other with black head and mesosoma, brown metasoma, and hyaline wings. the size and pattern of bristles and score were similar in both forms. elasmus westwood, 1833 species are in mostly a parasitoid of lepidoptera, although some hymenoptera, particularly braconidae and ichneumonidae have also been recorded as hosts (gibson, 1993; graham, 1995). e. polistis fig 4. (a) angiopolybia pallens nest; (b) angiopolybia pallens habitus; (c) brachymeria sp.1 habitus. fig 5. (a) leipomeles spilogastra nest; (b) leipomeles spilogastra habitus; (c) brachymeria sp.2 habitus. (3) epiponini: nests of a. pallens (fig 4a) and l. spilogastra (fig. 5a) were parasitized by brachymeria sp.1 (fig 4c) and brachymeria sp.2 (fig 5c) (chalcididae) respectively, and in the nest of p. jurinei was recorded parasitism by megaselia scalaris (phoridae). furthermore, we observed a dominance of males from species seminota and toechorychus which emerged from mischocyttarus and polistes nests, respectively, compared with elasmus and brachymeria which there was dominance of emerging females. individuals of chalcididae started to emerge two days after collection of the nests extending to the seventh day. individuals of ichneumonidae started to emerge seven days after collection of the nests extending to the twelfth day. for individuals of seminota depressa and elasmus polistis the emergency period took place over thirty days. for megaselia scalaris, we were not able to register the emergency period. discussion in this study we reported parasitism in seven nests of five mischocyttarus de saussure species, eight nests of three species of polistes latreille and only in three nests of three epiponini species. moreover, it was possible to verify a higher number of parasitoids per nest in polistes and mischocyttarus when compared to epiponini (table 1). social insects such as a somavilla et al. parasitoids in nests of social wasps96 was reported in nests of p. versicolor (dorfey & köhler, 2011). we report for the first time e. polistis parasitizing polistes cinerascens. we could observe that a large amount of e. polistis (796) emerged from two differents nests of p. versicolor and this can be explained due to the size of referred nest that was large and contained 1.646 cells. in nests of epiponini we observed parasitism by two species of brachymeria westwood, 1829 (chalcididae), which are primary or hyperparasitoids of lepidoptera (mostly young pupae) and only one species of diptera (mostly mature larvae) (fernández & sharkey, 2006). many neotropical brachymeria are associated to social wasp nests while some parasitize moths developing in wasp nests. social wasps known as hosts for brachymeria belong to the genera agelaia lepeletier, angiopolybia araujo, brachygastra petry, chartergus lepeletier, metapolybia ducke, polistes, polybia lepeletier and synoeca de saussure (boucek, 1992). in this study we reported for the first time brachymeria parasitizing leipomeles spilongastra. megaselia scalaris (phoridae) is a cosmopolitan insect that is primarily a detritivore and might act as facultative predator of immature hosts (disney & berghof, 2005). it has been recorded the presence of eggs and larvae of this dipteran in honeycombs of apis mellifera linnaeus, 1758 resulting in the nest abandonment (ronna, 1936). this fly has already been reported in nests of mischocyttarus cerberus ducke 1918, protopolybia acutiscutis (cameron, 1907), polybia occidentalis (olivier, 1791) and polybia simillima smith, 1862, causing serious injuries to the colonies (young, 1984; giannotti, 1998; london & jeanne, 1998). in this study we reported for the first time megaselia scalaris parasitizing polybia jurinei. with regard to the sex ratio of parasitoids that emerged from nests of social wasps, this can be influenced by many factors, such as environmental conditions and the body size of hosts (townes, 1958; hamilton, 1967; taylor & stern, 1971; boldt et al., 1973; vinson, 1997). eggs deposited in hosts with larger body size can give rise to females descendants (as observed in this study to elasmus and brachymeria) since they require more resources to their development and consequently eggs deposited in hosts with smaller body size can give rise to male descendants (as observed in this study to toechorychus and seminota) (taylor & stern, 1971; boldt et al., 1973). strassmann (1981) suggested that males typically emerge first and can wait for the females emerge to mate. moreover, it is possible the occurrence of females egglaying in another pupa from the same nest in which it has emerged (macom & landolt, 1995), which contributes to a high number of parasitoids in the same nest as observed in this study. concluding remarks in this study we reported for first time toechorychus guarapuavus and toechorychus fluminensis parasitizing nests of mischocyttarus cassununga, mischocyttarus consimilis and mischocyttarus imitator; elasmus polistis parasitizing polistes cinerascens and brachymeria sp. parasitizing a nest of leipomeles spilongastra. we also reported for the first time megaselia scalaris parasitizing polybia jurinei. however, further studies involving more species should bring to light about relationships between neotropical paper wasps and their parasitoids. acknowledgments we sincerely thank to reserva ducke (manaus, am), rppn angelin rainforest (ubatuba, sp) and rppn unisc (sinimbu, rs) for providing sampling permissions in their areas. to conselho nacional de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado do amazonas (fapeam), fundação de amparo à pesquisa do estado de são paulo (fapesp, process 2011/13391-2) and fundação de amparo à pesquisa do estado da bahia (fapesb, n° bol0494/2011) for scholarship supports. thanks are also to elijah talamas (usda-sel) for his suggestions on an earlier version of the manuscript. we also thank to coleção entomológica de santa cruz do sul for dates about the parasitoids from rio grande do sul. to dr. fernando b. noll (unesp) for the help in the identification of trigonalidae and some social wasps, dr. alexandre aguiar (ufes) and bernardo santos for the help in the identification of ichneumonidae, dr. marcelo tavares and bruno cancián (ufes) for the help in the identification of chalcididae and dayana alves da silva (uems) by providing the nest and individuals of mischocyttarus consimilis. references arab, a., pietrobon, t.a.o., britto, f.b., rocha, t., santos, l., barbieri, e.f. & fowler, h.g. 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(1998). a generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae: vespinae, polistinae). american museum novitates, 3224: 1-39. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.4437sociobiology 66(3): 523-526 (september, 2019) nest association between camponotus fallax (hymenoptera: formicidae) and vespa crabro (hymenoptera: vespidae) in central iberian peninsula diverse authors have reported a large number of cases about interactions between formicidae and vespidae (e.g. rusina, 2011). most interactions are related with cases of predation by ants on wasp nests or directly on solitary species (e.g. schatz et al., 2006). these cases are more frequent in tropical areas than in temperate areas (jeanne, 1979). the observations in europe are mostly related to attacks by ants on paper wasp (polistes sp.) nests (e.g.turillazzi & ugolini, 1979; rusina, 2011; kozyra & baraniak, 2016; pérez-bote & mora-rubio, 2018). paper wasps build open and exposed nests that resemble an upside-down umbrella, whereas vespa nests are protected by a carton envelope with a single opening towards the outside. the biological function of this envelope is to protect the colony and maintain an adequate microclimate, constant humidity and temperature (klingner et al., 2005), being specially suggested as an adaptation to reduce ant predation (jeanne, 1975). other interactions between these groups have been described as mutualisms or commensalisms, being especially common in tropical areas. thereby, several authors have reported that some species of arboreal wasps abstract because social wasps often defend their nests by inflicting painful stings or bites, some animals associate with them looking for protection against potential predators. some neotropical vespids are known to maintain associations with other insect and vertebrate taxa, such as birds and bats, however there are not previous records about associations between ants and hornets in europe. in this study, we reported the first case of association between the arboreal ant camponotus fallax and the european hornet vespa crabro in europe. the observations were made in central spain, where two colonies of c. fallax were found in two different avian nest-boxes inhabited by v. crabro when spotless starlings finished their breeding season. the reasons of this possible association are also discussed. sociobiology an international journal on social insects c mora-rubio, jl pérez-bote, j muriel article history edited by evandro n. silva, uefs, brazil received 26 march 2019 initial acceptance 04 august 2019 final acceptance 05 august 2019 publication date 14 november 2019 keywords camponotus fallax, ants, european hornet, vespa crabro, nest association. corresponding author carlos mora-rubio department of zoology university of extremadura av. elvas s/nº, 06006, badajoz, spain. e-mail: cmorarub@alumnos.unex.es (e.g. polybia rejecta and synoeca virginea) build their nests close to azteca chartifex nests in french guiana and brazil (somavilla et al., 2013; souza et al., 2013; leguen et al., 2015). this association supposes a benefit for both species (reciprocal protection from attacks of birds and other ants), so it could be considered as a mutualism. moreover, other study carried out by corbara and co-authors (2018) in french guiana showed that nests of the arboreal wasp protopolybia emortualis are found close to those of dolichoredus bidens, an aggressive arboreal ant. this species protects wasps from attacks of army ants, which means a benefit for them but no for ants, so this association seems to be a kind of commensalism. as far as we know, it has not been previously recorded in europe any case that demonstrates a possible relationship between ants and european hornets, vespa crabro (l., 1758). therefore, observations reported here might be the first that demonstrates a relationship between these insects. the observations took place on november 23rd and 24th, 2018, in a nest-box population of spotless starling (sturnus unicolor, temminck 1820) located in central spain department of zoology, university of extremadura, badajoz, spain short note c mora-rubio, jl pérez-bote, j muriel – nest association betweencamponotus fallax and vespa crabro524 (soto del real, madrid; lat. 40.451820, long. -3.452038, alt. 920-940 m. above sea level). our study area is a holmoakdehesa ecosystem covered by a deciduous wood land of quercus pyrenaica (willd) and fraxinus angustifolius (tourn. ex l.), with intercalations of acer monspessulanum (l.) and crataegus monogyna (jacq.), with abundant open areas used by a low load of bovine and equine cattle. this area exhibits a continental mediterranean climate (köppengeiger climate classification: csb category (reviewed in peel et al., 2007)) with hot and dry summers (muriel et al., 2015). this starling colony consists of 246 nest-boxes and is located about 1.5-2.5 kilometres from soto del real. during both days, revisions of spotless starling nest-boxes were made in order to count how many of them were occupied by nests of european hornets. in two out of the seventeen nest-boxes (11.67%) that contained hornet nests from the current year, it could be observed how a great number of camponotus fallax (nylander, 1856) specimens occupied their tops, just below the carton envelope. both nest-boxes (separated from each other by a distance of 636.91 m) were hooked on branches of two specimens of q. pyrenaica (lat. 40.7472077; long. -3.8147455; alt. 924 m.and lat. 40.7479135; long. -3.8084156, alt. 925 m.), being located 3 metres above ground level, approximately. as a matter of fact, formicids appeared within the protective carton envelope, between the upper wall of the nest-box and the first comb of the nest. the first nest was occupied by several v. crabro specimens, whereas the second one was unoccupied, presenting a poor condition (incomplete combs and envelope). the nest deterioration could be a consequence of the destruction by starlings, which want to take possession of the nest-box after the reproductive cycle of v. crabro. in turn, the first nest contained a higher quantity of c. fallax specimens than the second one. in fact, in the first nest we found reproductive specimens (winged ants), larvae and workers (ca.250) (fig 1a), whereas in the second nest only workers and very few larvae (ca. 25) were found (fig 1b). this fact could be related to the abandonment of the nest by hornets or the cleaning of the nest-box by starlings. the occupation of avian nest-boxes by ants has been previously reported in europe. thus, lambrechts and schatz (2014) observed, in a study carried out in southern france, that some parid nest-boxes (1.9% of bird nest-boxes in a rural woodland plot, and 16.7% in an urban plot) were occupied by c. fallax. taking into account previous studies, the case presented here does not seem to be related to any kind of predation (no ants were observed in the combs of the nest) or mutualism (there is no clear benefit for hornets as result of this relationship). however, it must be considered that c. fallax is a species with nocturnal habits (yamamoto & del-claro, 2008) and that the revisions were made during the day. still, it is possible that c. fallax takes advantage of the protection and refuge that the nest-box and the hornet nest offer, or even of the microclimate generated by the envelope and the metabolism of pupae, larvae and adult of european hornets (schmolz et al., 1995; klingner et al., 2005). thus, this association could be considered a kind of commensalism, similar to that described by corbara and co-authors (2018), but resulting in a benefit for ants, not for hornets. in addition, ants could be using the honeydew produced by larvae as a source of food, thereby resulting in a case of trophobiosis. this mutualistic relationship is common between ants and insects ‘homoptera’ (delabie, 2001), and there are also reports of some species of mutillidae that take advantage of the honeydew produced by the larvae of some polistes wasps species (e.g. uboni & lorenzini, 2013; morarubio & pérez-bote, 2018). on the other hand, it is very likely that hornets were unaware of the existence of ants in their own nest, because c. fallax specimens were found within the outer envelope. because of that, this relationship between ants and hornets could be described too as a case of plesiobiosis [original term introduced by wheeler (1901)]. fig 1. a: adults (workers and reproductive specimens) and larvae of camponotus fallax in the first nest. b: workers on the second nest. sociobiology 66(3): 523-526 (september, 2019) 525 it is defined as the occasional or regular closeness between nests of two colonies of social insects, without biological interdependence (low or no contact between both of them) (kanizsai et al., 2013). this kind of association is common between ants (czechowski & vepsäläinen, 1999; kanizsai et al., 2013; kvifte et al., 2017), but there are no observations of this relationship between ants and hornets. thus, this would be the first report of plesiobiosis between these two groups. this interspecific tolerance could be a consequence of similar cuticular hydrocarbon profiles between c. fallax and v. crabro, since different studies have shown that several heterospecific insects are able to integrate into social insect colonies thanks to these surface hydrocarbons which play an important role in nestmate recognition (howard et al., 1980; vander meer et al., 1989; singer & espelie, 1992; layton & espelie, 1994). we report here the first european record on association between ants (c. fallax) and european hornets (v. crabro). we have exposed all the possible interactions that could explain this association between both species throughout the text, but further studies should carry out more precise monitoring in order to determine what kind of interaction is really happening between these common hymenopterans of our forests. acknowledgements we are very grateful to dr. diego gil (mncn-csic) for allowing us to sample the nest-box colony of spotless starlings (s. unicolor), which he has been monitoring since 2001; 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(2008). natural history and foraging behavior of the carpenter ant camponotus sericeiventris guérin, 1838 (formicinae, campotonini) in the brazilian tropical savanna. acta ethologica, 11: 55-65. doi: 10.1007/s10211-008-0041-6 1253 bee visitors of quaresmeira flowers (tibouchina granulosa cogn.) in the region of dourados (ms-brasil) by anna kátia brizola-bonacina1, valeska marques arruda2, valter vieira alvesjunior1, josé chaud-netto3 & leandro pereira polatto3 abstract this research was developed in the center-urban area at the city of dourados (ms), in tibouchina granulosa trees, a plant popularly known as “quaresmeira” in brazil. the floral visitors of this species were recorded in three daily periods: 7 8 a.m., 1 – 2 p.m. and 4 – 5 p. m. bee visitors of those plants were collected with an entomological net when they landed on the flowers. the bees were anesthetized in a closed camera containing etila acetate, conserved in dietrich´s fixative and, soon afterwards, transferred to 70% ethyl alcohol for subsequent identification. almost 300 specimens of africanized apis mellifera, trigona spinipes and tetragonisca angustula were collected. the presence of these three species of bees in the flowers of t. granulosa suggests that they can be considered the main floral visitors of that vegetal species. t. angustula workers did not visit the flowers of t. granulosa between 7 and 8 a.m., but they were the main floral visitors from 4 to 5 p.m. the workers of t. spinipes presented a very aggressive behavior against the africanized a. mellifera workers on the quaresmeira flowers, defending the food sources with strength and efficiency and provoking a drastic reduction in the number of honeybee visits between 1 and 2 p.m. nevertheless, they did not interfere in the opportunistic activity of foraging by t. angustula workers, mainly between 4 and 5 p.m. the most important result of this research was the detection of the competitive exclusion principle between a. mellifera and 1faculdade de ciências biológicas e ambientais, universidade federal da grande dourados, rodovia douradositahum, km 12, cep 79.804-970, dourados-ms, brasil. e-mail: annabonacina@ufgd. edu.br 2faculdade de sinop (fasipe), avenida magda cassia pissinati 69, cep 78550-000, sinop-mt, brasil. 3departamento de biologia, instituto de biociências,unesp, avenida 24 a 1515, caixa postal 199, cep 13506-900, rio claro-sp, brasil. 1254 sociobiolog y vol. 59, no. 4, 2012 t. spinipes species in relation to the foraging behavior, when they exploit the floral resources of t. granulosa. keywords: quaresmeira, tibouchina granulosa, foraging behavior, bees, competitive exclusion principle. introduction the species tibouchina granulosa (cogn.) belongs to the melastomataceae family, being popularly known as quaresmeira. it is a characteristic plant of atlantic pluvial forest and blooms twice a year (from july to august and from december to march). their flowers present coloration that varies from rose to purple. the tree grows to between 8 and 12 meters in height, with a trunk of 30-40 cm diameter and pubescent tough leaves in both faces (lorenzi 1992). in the city of dourados (ms) it is used in forestation and ornamentation of the main streets. one of the most important subjects in research on conservation of biodiversity is the study of the interactions between floral visitants and vegetal species in natural ecosystems. according to kearns and inouye (1997), the loss of pollination is as severe and harmful in any ecosystem as the physical alterations of the habitat, but, frequently, it is generally considered of minor importance in relation to the loss of species. in tropical regions such as brazil, the main pollen vectors are bees (silberbauer-gottsberger & gottsberger 1988, bawa 1990, renner & feil 1993, rincón et al. 1999, barbola et al. 2000, aguiar & santos 2007), simply because these organisms are strictly dependent on floral resources for their survival (roubik 1989, michener 2007). brazil is a very rich environment in species of social stingless bees of the subfamily meliponinae. the life cycle and the social regulation of these bee species are intrinsically dependent on a very good brood feeding during all the developmental phases, and also during adult life. one of the main advantages of the socialization is the development of efficient mechanisms of communication among the members of the colony, which provide conditions to explore the habitat and to collect information on alternative sources of foraging. this ability improves their capacity to use the floral resources at a given moment, as well as to recruit additional foragers to seek the source of food that is being explored (beekman et al. 2007, díaz et al. 2007). this strateg y makes possible the selective exploration of more 1255 brizola-bonacina, a.k. et al. — bee visitors to quaresmeira flowers in brazil profitable sources in a frequently unstable natural environment (von frisch 1967, seeley 1995), because the food sources are frequently ephemeral and dispersed, although they can be diversified in some habitats. the communication systems, still little studied for most species, are also quite diversified. each of many behavioral characteristics can be potentially changed in order to improve the strateg y for exploitation of the floral resources and also the individual and collective alimentary economy (ramalho et al. 1991). therefore, the foraging efficiency of social bees depends on the influence of many abiotic factors, as well as of conditions of the habitat, as the space and temporary distribution of food sources (sherman & visscher 2002, dornhaus & chittka 2004, pasquet et al. 2008). the profitability of the alimentary sources and the amount of food stocked in the nest are also essential factors in the regulation of the foraging activity of the colony (seeley 1989, pírez & farina 2004, grüter & farina 2007). for this reason, the stingless bees present foraging strategies that vary from individual and opportunists to collective and monopolists. apis mellifera is an exotic species, of relatively recent introduction in the american continent. after the importation of african honeybees, a. m. scutellata (lepeletier 1836), from south africa and tanzania to brazil in 1956 (kerr 1967), a new organism which resulted from crossings among these bees and four european subspecies of honeybees was produced (ruttner 1986, stort & gonçalves 1994, pereira & chaud-netto 2005). these poly-hybrid bees presented scutellata-like characteristics, such as reproductive, foraging and defensive behaviors. for this reason they received the name of africanized honeybees (diniz et al. 2003). the relationships between these bees and the neotropical environment surprised researchers all over the world, as they have high adaptability to variable ecological conditions, produce a great number of swarms, and their workers are very efficient foragers, competing for floral resources with many species of native bees (pereira & chaud-netto 2005, traveset & richardson 2006, carbonari et al. 2009, brizolabonacina 2009). the scientific community considers scarce the knowledge on the influence of the honey bee on the brazilian flora. furthermore, minimal information is available on the interference of the natural sources of floral resources and environmental factors in the foraging activity of this eusocial bee. the effi1256 sociobiolog y vol. 59, no. 4, 2012 cient system of communication and the fast population growth of a. mellifera make this bee a dominant floral visitor in natural environments and also in agriculture areas, especially in the tropics. nevertheless, its presence can produce a potentially negative effect on the reproduction of many native plants, which are not adapted to pollination by this bee. the workers of a. mellifera consume all the floral resources of these vegetable species and, consequently, this harmful behavior reduces the activity of the native pollinators (traveset & richardson 2006, carbonari et al. 2009). on the other hand, many native plants already have their reproductive effectiveness maximized as a result of the intense activity of pollination of the honey bee in their flowers. the relationships between floral visitors and angiosperms are based on exchange of rewards. most of the time, a floral visit is motivated by the food offer or, in other words, the vegetal species offer attractives, as nectar and pollen, and receive the benefits of pollination (pesson 1984). complex interactions between floral visitors and the vegetable species which are visited can be observed in different environments. the visitants developed varied strategies to explore the floral resources produced, including the exploitation of different plant species (pleasants 1980), the search for nectar and/or pollen at different times of the day or even in different periods of a determined season (ginsberg 1983, koptur et al. 1988, morato & campos 2000), and foraging in different patches of resources (carpenter 1979) and at different densities in the patches ( johnson & hubbell 1974, ginsberg 1983). the objective of this research was to get information on the species of bees that visit the flowers of t. granulosa, as well as the interactions among these organisms in relation to the use of the alimentary resources produced by this species. material and methods the bees were collected with an entomological net in trees of t. granulosa (quaresmeira) in the center-urban area of the city of dourados (ms). three intervals of one hour each were established for the collections: the first from 7 to 8 a.m., the second from 1 to 2 p.m. and the third from 4 to 5 p.m. during the time elapsed in each interval the bees foraging the flowers of t. granulosa were collected, anesthetized in a camera containing ethyl acetate, and grouped considering the collection interval. soon afterwards the specimens were fixed 1257 brizola-bonacina, a.k. et al. — bee visitors to quaresmeira flowers in brazil in dietrich (beçak & paulete 1976), for 12 hours. after the fixation they were conserved in ethyl alcohol to 70% for subsequent selection and identification in species level, with base in morphologic characters. the number of bees collected in the flowers of t. granulosa was recorded, and the relative frequencies obtained for each species were calculated, using formula (ni / n) x 100 where, ni = number of collected bees of a certain species and n = total number of collected bees. the number of visitors of each bee species to the flowers of t. granulosa was used to perform a faunistic analysis of abundance, setting a 5% confidence interval (ci) and 1% probability (p) (kaps & lamberson, 2004). for this finality the floral visitors were classified in one of the following classes of abundance (silveira neto et al. 1976): 1. very abundant in this case, the number of visitors of a certain species was higher than the upper limit of the ci at 1% p level; 2. abundant – the number of individuals observed was between the upper limits of the ci at 5% and 1% p level; 3. common –the number of visitors recorded on the flowers was within the ci at 5% p level; 4. dispersed – the number of visitors was within the lower limits of the ci at 5% and 1% p level; rare – in this case, the number of visitors was smaller than the lower limit of the ci at 1% p level. the results of this analysis permit to determine the characteristics of the floral visitors and their importance to the flowers of t. granulosa. to verify whether the environmental factors were related to the foraging activity of the floral visitors the pearson´s correlation test was applied to the data. the main objective of this analysis is to determine the functional dependency between the foraging activity and each abiotic variable. the spss program for windows was used to determine the confidence intervals and to perform the pearson’s correlation tests. the frequencies of visitors of the species observed on the flowers of t. granulosa were calculated and the g test of independence was used to compare the data. for this purpose the software bioestat 5.0 (ayres et al. 2007) was used. results and discussion workers of three bee species [apis mellifera (l. 1758), trigona spinipes (fabr. 1793) and tetragonisca angustula (l. 1911)] foraged the flowers and 1258 sociobiolog y vol. 59, no. 4, 2012 were considered the predominant visitors of tibouchina granulosa. in total, 294 bees were collected throughout the experiment (table 1). the relationship among the species of bees collected in the flowers of t. granulosa during the daily intervals of observation can be observed in fig. 1. an analysis of that figure revealed that the frequency of bees on the flowers of t. granulosa varied according to the period of the day and the behavior of each visitor species. the workers of t. spinipes were the most aggressive visitors in the food source. in the first daily interval of observations, a. mellifera was more abundant in number of individuals. in brazil, this species is represented mainly by the fig. 1. relationships among the species of bees collected on the flowers of t. granulosa during the daily intervals of observation. table 1. bees collected on the flowers of t. granulosa in three daily intervals of observation. daily intervals bees apis mellifera trigona spinipes tetragonisca angustula 7 to 8 a.m. 51 37 0 1 to 2 p.m. 7 82 27 4 to 5 p.m. 23 16 51 total 81 135 78 1259 brizola-bonacina, a.k. et al. — bee visitors to quaresmeira flowers in brazil africanized honeybees, a polyhybrid bee resulting of crossings among four subspecies of european origin (a.m.ligustica, a.m.caucasica, a.m.mellifera and a. m. iberica) and the african subspecies, a. m. scutellata (ruttner 1986, stort & gonçalves 1994, pereira & chaud-netto 2005). according to free (1980), africanized honeybees are extremely efficient in the recruitment of foragers, and rapidly attract a great number of collectors for the exploitation of food sources. in the second daily interval of collections the most representative bee species observed in the flowers of t. granulosa was t. spinipes (“irapuá”). the workers of this species presented a very aggressive behavior against the africanized a. mellifera workers on the quaresmeira flowers, defending the food sources with strength and efficiency, and provoking a drastic reduction in the number of honeybee visits between 1 and 2 p.m. the workers of africanized honeybees practically were expelled from the food sources in this period. nevertheless, the workers of t. spinipes did not interfere in the opportunistic activity of foraging by t. angustula workers, mainly between 4 and 5 p.m. according to johnson & hubbell (1974), the recruitment system of trigona workers is not as efficient as the observed in a. mellifera, but they compensate for this apparent disadvantage with an extremely aggressive behavior in the sources of food. this peculiar behavior, to the detriment of the great number of “rivals”, can vary from an alarm posture to direct corporal confrontations. engels & engels (1980) noted many aggressive exhibitions of meliponids in relation to foragers of a. mellifera, in the exploitation of floral resources. in all the cases they observed that the stingless bees were victorious, as some honeybee workers had their wings damaged and most of them could not fly after the confrontations. in the third daily interval of observation and collection, the workers of t. angustula (“jataí”) were more abundant. these small-sized meliponids do not enter in direct competition with the larger bees by the floral resource that is being explored. they act only as collectors of the remaining pollen found in the floral structures, resulting from the foraging activity of other organisms which visited the same source of food before them (laroca 1970). there was a significant negative correlation between the number of visits (n.v.) performed by workers of t. spinipes and relative air humidity (r.h.) values (r = 0.9532; t = 31.54; df = 1), but not with the temperature (t) 1260 sociobiolog y vol. 59, no. 4, 2012 (r = 0.4003; t = 0.4369; df = 1). for t. angustula there was a non-significant correlation between n.v. and r.h. (r = 0.0243; t = 0.0243; df = 1), and also between n.v. and t (r = 0.7683; t = 12.00; df = 1), although this last value obtained for t is very close to the critical value of significance (12.70) at 2.5% of probability indicated in the table for t values, in the bicaudal test (kaps & lamberson 2004). in the case of apis mellifera there was a significant negative correlation between n.v. and t (r = 0.9868; t = 60.99; df = 1), but a non-significant positive correlation between n.v. and r.h. (r = 0.7716; t = 12.13; df = 1). the effect of both climatic variables on the foraging activity of t. spinipes, t. angustula and a. mellifera workers was clearly different. the bees usually forage more intensely when the relative humidity of the air is low and the environmental temperature is high. the results obtained for t. spinipes in this research are similar to those registered by hilário et al. (2000) and kasper et al. (2008), in what concerns to the relationship between food collection and relative humidity of the air. on the other hand, although the correlation between the number of flower visits of t. spinipes and the temperature has not been significant, there was a tendency of increase in the n.v. in higher temperatures. in the case of a. mellifera there was a decrease in the n.v. under higher temperatures and lower values of r.h. and an increase in the n.v. in opposite conditions. these results are different from those obtained in other research (e.g. coelho 1991, polatto et al. 2012). the results of the correlation tests performed in this research represent an argument in favor of the occurrence of competitive exclusion between t. spinipes and a. mellifera regarding food exploitation: the foraging activity of the africanized honeybees was moved to less favorable schedules, particularly the beginning and the end of the day, to avoid the trophic overlapping with t. spinipes in the middle of the day, considered the period in which the environmental conditions are more favorable. the frequency of visits of t. spinipes workers to the flowers of t. granulosa (45.92%) was greater in relation to the frequencies recorded for workers of a. mellifera (27.55%) and t. angustula (26.53%) (g = 155.34; p< 0.0001; df= 4). renner (1983) observed 34 species of plants of the family melastomataceae, common in neotropical communities (atlantic forest and amazonian forest), and verified that all of them were visited by species of trigona (5 species and 4 subspecies, respectively). 1261 brizola-bonacina, a.k. et al. — bee visitors to quaresmeira flowers in brazil in the present research, considering the classes of abundance proposed by silveira neto et al. (1976), t. spinipes was classified as a very abundant species in relation to a. mellifera and t. angustula, considered two common species. in relation to the foraging behavior, the three species were classified as constant organisms. t. spinipes was the dominant species on the flowers of t. granulosa. the predominance of t. spinipes, followed by a. mellifera and t. angustula, three bee species with eusocial lifestyle, may be a consequence of the large number of members in their colonies and their generalist habits. vieira & marchini (2009) studied the diversity of bees in a brazilian cerrado area in the city of cassilândia, mato grosso do sul, and observed that the two most abundant species were also a. mellifera and t. spinipes. in a particular environment, the most abundant alimentary sources are efficiently exploited by these bees, and they generally forage in groups (kerr et al. 1981, wilms et al. 1996, johnson & steiner 2000). the workers of apis and trigona established, amongst themselves, a competition relationship for exclusion in relation to the foraging behavior, when they explore the floral resources of t. granulosa. in other words, the presence of one of them on the flowers drastically reduced the presence of the other (fig. 1). the way bees explore floral sources of pollen and nectar depends on relative selection pressures regarding the bee, the flower and also the environment. the environmental characteristics and the time that the food sources stay available to the visitor are fundamental variables in the consumers’ economy. in an environment with previsible resources in time and space, specialization can present advantages in relation to the search and use of food (macarthur & pianka 1966). specialization eventually brings a competitive advantage in the use of specific resources. on the other hand, the generalist alimentary habit makes possible the adaptation of the species to the variations in the food offered, besides those due to the competitors’ presence (morse 1980). the social apids maintain a great colonial biomass for a long period and, for this reason, they cannot specialize (michener 1979). additionally, they present morphologic and functional characteristics (for instance, the corbicle and a long proboscis) that favor the generalist alimentary habit (michener et al. 1978, loken 1981). for this reason they are predisposed to interspecific interactions in the use of the floral sources of food. 1262 sociobiolog y vol. 59, no. 4, 2012 there is some indirect evidence that competition alters the method of use of the floral sources among apidae (inouye 1978, heinrich 1978, 1979, pyke 1982). the foragers of some species of meliponids exhibit aggressive and monopolist behaviors during the bloomed periods of the year ( johnson & hubbell 1974, 1975, hubbell & johnson 1978), and some species which form great colonies make demarcations with pheromones in the area where the colony is acting, with the apparent purpose of avoiding the installation of new nests (hubbell & johnson 1977). koeniger & vorwohl (1979) observed that, among four species of apidae (apis dorsata, a. florea, a. cerana and trigona iridipennis), the smallest of them (t. iridipennis) exhibited aggressive behavior in relation to the other three species. those authors admitted that this behavior may be related to the smaller area of food exploration by the colony of t. iridipennis, and would have the function of preserving the foraging space of this species. they also observed a larger diversification of t. iridipennis in relation to food collection. this peculiarity apparently attests to the efficiency of that bee to defend its feeding territory. the hypothesis postulated by koeniger and vorwohl establishes a certain difficulty for the analysis of the coexistence among meliponid species. for instance, trigonini, the larger species which form great colonies and, therefore, have a larger action area, exhibit aggressive and monopolist behaviors ( johnson & hubbell 1975, hubbell & johnson 1978, roubik & johnson 1982), while the smaller species avoid direct contact with the larger ones and are passive and opportunist in relation to the gathering of floral resources. engel & dingemans-bakels (1980) noticed that small species of stingless bees, as plebeia minima, tetragonisca spp. and trigona jaty foraged some types of flowers which were not visited by larger bees, and suggested the existence of some alimentary specialization related with the size of the species. conclusions the data obtained in this research allowed the following conclusions: (1) the capture of bees in foraging activity on the flowers of t. granulosa indicates the possibility of effective pollination; (2) the floral resources are collected by the bees a. mellifera, t. spinipes and t. angustula; (3) the constant presence of apis and trigona on the flowers of t. granulosa indicates that this vegetable species is an important food source for them; (4) of the bees 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biological invasions as disruptors of plant reproductive mutualisms. trends in ecolog y & evolution 21: 208-216. vieira, g. h. c. & l. c. marchini 2009. diversity of bees (hymenoptera: apoidea) in a cerrado area in the city of cassilândia, mato grosso do sul, brazil. sociobiolog y 53: 597–607. 1267 brizola-bonacina, a.k. et al. — bee visitors to quaresmeira flowers in brazil wilms, w., v. l. imperatriz-fonseca & w. engels 1996. resource partitioning between highly eusocial bees and possible impact of the introduced africanized honey bee on native stingless bees in the brazilian rainforest. studies on neotropical fauna and environment 31: 137–151. 1375 litter as an important resource determining the diversity of epigeic ants in the south-central part of bahia state, brazil by karine santana carvalho 1*, ivan cardoso do nascimento1, jacques hubert charles delabie2, juliana zina1, ana lúcia biggi de souza1, elmo borges de azevedo koch1, marcos augusto ferraz carneiro1& anselmo souza santos1. abstract this study evaluates the richness and composition of the epigeic ant fauna in two caatinga areas (site 1: brejo novo and 2: frizuba) within a transitional region (between the caatinga and the decidual atlantic forest) in the municipality of jequié, state of bahia, brazil. ants were sampled using pitfall traps and winkler extractor method in 50 randomly chosen points, totalizing a sampling area of 12.5 ha per site. overall, we identified 60 species belongingto 27 genera. the most speciose genera were pheidole, camponotus, and solenopsis (with five species each) followed by wasmannia and cephalotes (four species each). pheidole sp1. was the most frequent species (occurring in approximately 60% of the samples). since site 1 did not possess a litter layer (and therefore could not be sampled by the winkler extractor) comparisons between the two areas were made using only the data provided by the pitfalltrap method. the simpson diversity indexes calculated for sites 1 and 2 were 0.87 and 0.89, respectively, and showed no statistically significant differences (p ≤ 0.417). the jaccard coefficient revealed only 35.5% similarity in species composition between the two areas. the results suggest that the presence of litter contributed to increased species richness (the winkler method added 28 species in the sum total of species collected) and “shaped” a distinct faunal composition of the area. the present study is the first ant fauna inventory in the region and reveals an unexplored conservation potential for the decidual forest and the caatinga of northeastern brazil. departamento de ciências biológicas, universidade estadual do sudoeste da bahia, avenida josé moreira sobreira, s/n, jequiezinho, 45200-000, jequié, ba, brazil. ksczool@yahoo.com.bricardoso@ hotmail.comjuzina74@gmail.comanabiggi@bol.com.brelmoborges@gmail.comafc.marcos@yahoo. com.branselmo144@gmail.com laboratório de mirmecologia, convênio ceplac/uesc, cepec, c.p. 7, 45600-000 itabuna, ba, braziljacques.delabie@gmail.com *to whom correspondences should be addressed 1376 sociobiolog y vol. 59, no. 4, 2012 key-words: epigeic ant fauna, atlantic rainforest, caatinga, conservation, diversity introduction there are approximately 3,000 described species of ants in the neotropics (bolton 2003). however, it is estimated that the same amount still remains to be discovered or described (bolton 2003). thus, studies of ant communities are an important tool for conservation of biodiversity, especially in brazil, a country recognized as “mega-diverse”(brandon et al. 2005, mma 2008), harboring approximately 13.2% of the world’s biota (lewinsohn & prado 2006). in this context, the caatinga deserves to be highlighted as it is the main domain of northeastern brazil, covering about 7% of the country (leal et al. 2003b).although the ant fauna of the caatinga has been recently receiving more attention, as indicated by the publication of studies conducted in this domain (e.g. brandão, 1995 in the states of bahia and piaui; quinet & tavares 2005; nunes et al., 2008 in the state of ceará; leal, 2003a, 2003b in the states of alagoas and sergipe; santos et al., 1999, silva, 2011, soares et al. 2003 and oliveira et al., 2009 in the state of bahia), transitional areas between the caatinga and the atlantic rainforest or the caatinga and the cerrado are still under sampled. one reason for the small number of studies in these areas lies in the fact that few are located in protected areas and most of them are already largely impacted by human activities. moreover, questions about transitional areas that pervade current studies on priority areas for conservation (i.e. are levels of endemism and species concentration higher in transitional areas?) remain unknown for many animal groups (kark et al. 2007). the state of bahia is a perfect locality to study transitional areas, since it harbors three different domains (caatinga, cerrado and atlantic rainforest). however, except for the literature cited above, the ant fauna of the state is still under sampled and this situation is even more dramatic for the transitional zones, which, with the exception ofthe studies published by soares et al. (2003) and carvalho et al. (2004), with ant communities in forests under the influence of the caatinga, are neglected by the scientific community. surveys focusing on basic ecological parameters, such as composition, richness, and abundance of species, are essential not only to understand the 1377 carvalho, k. s, et al. — litter as an important resource and the diversity of ants ecological traits that modeled a community, but also to subsidize conservation acts. in this sense, in order to increase the existing knowledge on the ant fauna inhabiting transitional areas we compared the epigeic ant communities in areas between the caatinga and the atlantic rainforest in the central south of bahia state. materials and methods study area: samples were collected in february 2003 in sites in the municipality of jequié, ba, brazil. the region of jequié is located on the watershed of the contas river and is marked by different phyto-physiognomies of atlantic rainforest (deciduous forest) and caatinga. in the region there are patches of caatinga vegetation which provide a great variety of landscapes, remarkable biological richness, and certain amounts of endemism (ibama 2011). the areas used for collections were randomly chosen and named by their location as follows: site 1 brejo novo (13º56’34.5”s; 40º06’31.6”w) and site 2 frizuba (13°55’33.81’’ s; 40°01’24.66’’ w), 10 km distant from one another. ant samples: the sampling of ants followed the standard protocol (all protocol: ants of the leaf litter, agosti et al., 2000) for soil and litter species. fifty points were randomly selected fromthe sampling area in each site (brejo novo and frizuba). each point was separated from the others by intervals of 50 m, totalizing an area of 12.5 ha per site (for details see agosti & alonso, 2000). at each sampling point a pitfall trap was installed containing water and detergent-based solution, which remained in the field for 24 hours. after this period, the specimens were fixed in alcohol 70%and taken to the laboratory for further identification. at the same points, samples were taken fromthe leaf litter of a 1m2 area, sieved and separated into bags for the extraction of the specimens by means of winkler extractors. due to a lack of litter to be extracted by the winkler method in frizuba, this method was carried out only in brejo novo. the extractors remained active for 48 hours. during this period the ants migrated into the containers with alcohol 70% attached to 1378 sociobiolog y vol. 59, no. 4, 2012 the base of the extractors. the specimens were mounted and identified according to bolton (2003). the identification of ants was carried out at the laboratory of invertebrate zoolog y in the universidade estadual do sudoeste da bahia, uesb, municipality of jequié, state of bahia, brazil. the species identifications were conducted using taxonomic keys and by comparison with the collections of the laboratório de mirmecologia of the centro de pesquisa do cacau cepec/ceplac in itabuna, state of bahia.feeding habit and nesting sites were categorized according to the literature available. data analysis: the expected richness was calculated based on the estimators chao i, jackknife i, and bootstrap. the composition and diversity of species between the different sites were evaluated by using the diversity index (simpson) and the similarity index ( jaccard). in order to calculate the diversity index, the frequency levels of the species were used instead of the abundance levels, due to differences in the efficiency of recruitment among ant species. when comparing the richness and the similarity between ant species, we considered only the results obtained with the pitfall trap, since this method was implemented in both sites. astudent t test was used to compare the values of the diversity index obtained for both studied sites. we considered significant values of p<0.05. data analyzes was carried out using the softwares estimates (version 7.5.2) and systat (version 12). results in total, we surveyed 60 species of ants belonging to 27 genera and seven subfamilies (table 1). the most common subfamily, with over half of all species of ants collected (63.3%) was myrmicinae. the second most common subfamily was formicinae (15%) followed by ponerinae (8.3% of species) (table 1). the most diverse genera were pheidole, camponotus, and solenopsis (with five species each) followed by wasmanniaandcephalotes (four species) (table 1). 1379 carvalho, k. s, et al. — litter as an important resource and the diversity of ants pheidole sp1.was the most frequent species in the samples, representing more than half of all species surveyed in site 1and 2. solenopsis sp.1 and fig. 1. nesting site of ants collected in two sites (brejo novo and frizuba) in the municipality of jequié, ba, brazil. fig. 2. feeding habits of ants collected in two areas (brejo novo and frizuba) in the region of jequié, ba, brazil. 1380 sociobiolog y vol. 59, no. 4, 2012 nylanderia sp.1 also presented high values of frequency of occurrence (table 1, see appendix.). in general, the species of ants recorded in both sites nested more frequently in the soil than in the vegetation (figure 1). microhabitats used for nesting may be rotting trunks, fruit and seeds in the litter. the frequency of feeding habits was similar between both sites, being the omnivore habit more common than the carnivore and herbivorehabits (figure 2). species richness: in site 1 (brejo novo) 58 species were recordedby both methods combined (pitfall traps and winkler). over half (65.5%) of all species collected in brejo novo were recorded with the winkler method (1m2 litter).the estimated number of species ranged from 69.9 (±0) to 89.5 (± 15.9). considering the separate methods, with thepitfall traps, the estimated richness ranged from 21.7 (± 0) to 29.5 (± 10.5), and for the winkler method the values ranged from 58.18 (± 0) to 75.6 (± 15.1) (table 2). these values were higher than the richness observed. in site 2 (frizuba), 25 species were recordedandthe estimators of richness ranged from 28.0 (± 0) to 32.8 (± 2.9) (table 2). also, the richness estimators showed higher values than the observed richness. diversity and similarity in species composition: the results of the simpson diversity index calculated for sites 1 and 2showed no statistically significant differences (p =0.42) (table 1, appendix). the correlation coefficient of jaccard revealed only 35.5% of similarity in species composition between the two studied sites. discussion the diversity of formicidae recorded in the two sites surveyed in this study follows the same pattern already known for other neotropical domains, with the myrmicinae subfamily being more diverse thanall other subfamilies (bolton et al. 2005). with the exception of camponotus (that belongs to the second most common subfamily in this study) all genera recorded in the present study that presenta high number of species richness belong to the myrmicinae subfamily, such as pheidole, solenopsis, and crematogaster. according to wilson (1976) these genera may be considered to be prevalent 1381 carvalho, k. s, et al. — litter as an important resource and the diversity of ants worldwide. the high number of species, as well as a variety of adaptations that allows the species of these genera to occupy different environments, may be the factors responsible for their abundance and prevalence in most neotropical ant communities (wilson 1976). when compared with the ant fauna of the caatinga, the species richness recorded in the present study can be considered relatively high (santos et al. 1999: 50 species; oliveira et al., 2009: 32 species). however it is equivalent to thatfound by leal (2003) (61) and lower when compared to the resultsof silva (2011) (143 species). nevertheless, differences related to sampling methods should be taken into account. additionally, as the values of observed richness showed, the total richness was not reached in both studied sites. more sampling effort should be implemented as this would probably increase the number of recorded species. on the other hand, this result is very common in neotropical environments for insect fauna. when compared with the ant fauna of other transitional zones in the state, the present study showed the highest number of species (carvalho et al., 2004: 55 species belonging to 23 genera). the deciduous forest (mata de cipó) is characterized by shrubby understory trees and xerophytic species and occurs in areaswhere thereinfall is usually between 800-1000 mm/year and which havewell-defined rainy and dry periods (thomas 2003). although this physiognomy is subject to a high degree of degradation, according to carvalho et al. (2004), transitional areas between the caatinga and the deciduous forest revealed an interesting biological diversity with a probable endemism. most species of ants found in the two areas followed the same pattern: omnivorous (or generalists) with nests in the soil. theseresults may be related to the sampling methods, since the combination of pitfall traps and winkler is considered the best way to capture soil and leaf litter ants (agosti & alonso 2000). the predominantly generalist life, however, may be related to food shortages in highly seasonal environments, such as the caatinga. similarly, the low frequency of species that cultivate fungus (attini) is probably related to the lack of sufficientvegetation to maintainthe colonies of these ants (leal 2003). structural differences related to vegetation traits may explain the low values of similarity between the two studied sites despite the short distance between them. the spatial heterogeneity between the sites may be due to the 1382 sociobiolog y vol. 59, no. 4, 2012 presence (site 1) and absence (site 2) of litter, a resource that, according to hölldobler and wilson (1990), increases the number of available habitats for nesting and foraging. this statement is supported by the fact that the total richness recorded in brejo novo was obtained in 1m2 litter. these results corroborate other studies of ants in tropical forests, where the heterogeneous distribution of resources such as litter is one of the most important factors in determining the foraging, nesting and diversity of these insects (mcglynn & kirksey 2000; armbrecht et al.2004; mcglynn 2006; bastos & harada 2011). although this study has not quantified or measured vegetation structure, we can infer that the species of ants found in two areas reflect the composition of the ant fauna of the region and can be correlated with the premise of the literature, that species diversity increases with increasing environmental complexity, since more complex environments support a greater diversity of ecological niches, resulting in a greater amount of foraging and nesting sites, reducing the competition between coexisting species. in fact, the presence of litter contributed both to increase the species richness, since the winkler method added 28 species (or 46.6%) tothe total sum of species collected, and “shaped” a differentiated faunal area in sites that lacked this feature. the present study represents the first survey of ants in a transitional zone between the caatinga and the atlantic rainforest in the center south of the state of bahia and one of few published data about ecotonal areas in the country. using ants as ecological indicators, we suggest that there may be an expressive potential for studies in transitional areas, and also that these areas may represent important spots for conservation, harboring high species richness levels. acknowledgments to the universidadeestadual do sudoeste da bahia, for financing this study, the staff of the laboratório de mirmecologia ceplac/cepec in itabuna for the technical and logistic support in field collections, especially to jose raimundo maia and jose crispin. topronex (prnx 0011/2009) for financial support and to annebaldissen for the english version. to andre teixeira da silva for suggestions and assistance in the statistical analyzes. 1383 carvalho, k. s, et al. — litter as an important resource and the diversity of ants references agosti, d. & l. alonso. 2000. the all protocol: a standard protocol for the collection of ground-dwelling ants. in: agosti, d., j. majer; l. alonso & t.r. schultz (ed.). ants: standard methods for measuring and monitoring biodiversity, pp. 204-206. smithsonian institute press, washington, usa. agosti, d., j. majer; l. alonso & t.r. schuttz. 2000. sampling ground-dwelling ants: case studies from the worlds’ rain forests. 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(ed.) caxixuanã: desafios para a conservação de uma floresta nacional na amazônia. p. 525-536. editora museu paraense emílio goeldi,belém, br bolton, b. 2003.synopsis and classification of formicidae.pp.370.memoirs of the american museum entomological institute, gainnesville, florida, usa. bolton, b., g. alpert, p.s. ward & p. naskrecki 2005.bolton’s catalogue of the word. harvard university press, london, uk. brandão, c.r.f. 1995. formigas dos cerrados e caatingas. pp. 147. universidade de são paulo. – usp, são paulo, brazil. brandon, k., c.a.b. fonseca, a.b. rylands, & j.m.c. silva. 2005. conservação brasileira: desafios e oportunidades. megadiversidade1: 7-13. carvalho, k.s., a.l.b. souza, m.s. pereira, c.p. sampaio & j.h.c. delabie. 2004. comunidade de formigas epígeas no ecótono mata de cipó, domínio da mata atlântica, ba, brasil. acta biologicaleopoldensia. 249-257. holldobler, b. & e.o. wilson. 1990. the ants. pp.746. bellknap press, cambridge, uk. ibama .2011. http ://www.ibama .g ov.br/e cossistema s/caating a .htm . acesso em 01/06/2012 kark, s., t.f. allnutt, n. levin, l.l. manne&p.h. williams. 2007. the role of transitional areas as avian biodiversity centres. global ecolog y and biogeography. 16:187-196. http:// dx.doi.org/10.1111/j.14668238.2006.00274. leal, i.r. 2003a. dispersão de sementes por formigas na caatinga. in: i.r. leal, m. tabarelli& j.m.c. silva (ed.) ecologia e conservação da caatinga pp. 593-624. editora universitária ufpe, recife, brazil. leal, i.r. 2003. diversidade de formigas em diferentes unidades de paisagem caatinga. in: i.r. leal, m. tabarelli& j.m.c. silva (ed.) ecologia e conservação da caatinga. pp. 235-461.editora universitária ufpe, recife, brazil. lewinsohn, t. m. & p.i. prado. 2006. síntese do conhecimento atual da biodiversidade brasileira. in: t. m. lewinsohn (ed.) avaliação do estado do conhecimento da biodiversidade brasileira. pp. 21-1009, brasília, df, brazil. mma. 2008. livro vermelho da fauna brasileira ameaçada de extinção. in: machado abm, 1384 sociobiolog y vol. 59, no. 4, 2012 drummond, g.m.; paglia, a.p. (ed.) pp.1420..ministério do meio ambiente brasilia, df, fundação biodiversitas, belo horizonte, mg, brazil. mcglynn, t.p. 2006. ants on the move: resource limitation of a litter-nesting ant community in costa rica. biotropica. 38:419-427. mcglynn, t.p. & s.e. kirksey. 2000. the efects of food presentation and micro-habitat upon resource monopoly in a ground-foranging ant (hymenoptera: formicidae) community. revista de biologia tropical 48: 629-642. nunes, l.a.p.l., j.a.a. filho& r.i.q. menezes. 2008. recolonização da fauna edáfica em áreas de caatinga submetidas a queimadas. revista caatinga 21(3) : 214-220. oliveira, g.v.f., c. batista; k.s. silva, f.r.s. martins & m.m. corrêa. 2009. diversidade de formigas de áreas preservadas e em regeneração de caatinga da floresta nacional contendas do sincorá, sudoeste da bahia. ix congresso de ecologia do brasil, são lourenço, mg, brazil. quinet, y.,& a.a. tavares. 2005. formigas (hymenoptera:formicidae) da área reserva serra das almas, ceará, brasil. in: f.s. araujo, m.j.n. rodal& m.r.v. barbosa (ed.) análise das variações da biodiversidade do bioma caatinga: suporte a estratégias regionais de conservação. pp. 329-350. ministério do meio ambiente, brasília, df, brazil. santos, g.m.m., j.h.c. delabie& j.j. resende. 1999. caracterização da mirmecofuna (hymenoptera: formicidae) associada à vegetação periférica de inselbergs (caatinga arbórea estacional semi-decídua) em itatim bahia, brazil. sitientibus: série ciências biológicas 20: 33-43. silva, e.m. 2011. estrutura da comunidade de formigas (hymenoptera: formicidae) associada a duas fisionomias de caatinga em milagres, bahia, brasil. pp. 47..msc dissertação. universidade estadual de feira de santana, bahia, brazil. soares, i.m.f., a.a. santos, d. gomes, j.h.c. delabie, & i.f. castro. 2003. comunidades de formigas (hymenoptera: formicidae) em uma “ilha” de floresta ombrófila serrana em região da caatinga (ba, brasil). actabiologicaleopoldensia 25: 197-204. thomas w.w. 2003. natural vegetation types in southern bahia. in: prado p.i., landau e.c., moura r.t., pinto l.p.s., fonseca g.a.b., alger k. (orgs.) corredor debiodiversidade da mata atlântica do sul da bahia. publicação em cd-rom, ilhéus, iesb/ ci / cabs / ufmg / unicamp. wilson, e. o. 1976. which are the most prevalent ant genera? studia entomolog y. 19: 187-200. the appendix with both tables begins on page 1385. 1385 carvalho, k. s, et al. — litter as an important resource and the diversity of ants frequency of occurrence(%) subfamily/species site 1 site 2 w p p ecitoninae labidus coecus 0.0 2.0 2.0 odontomachus bauri 8.0 0.0 4.0 odontomachus sp.1 0.0 6.0 0.0 ectatominae ectatomma suzanae 0.0 0.0 12.0 ectatomma sp.1 6.0 50.0 0.0 formicinae brachymyrmex sp.1 2.0 0.0 0.0 camponotus (myrmobrachys) sp.1 12.0 42.0 0.0 camponotus crassus 2.0 0.0 0.0 campontus sp.1 12.0 42.0 4.0 camponotus sp.2 4.0 16.0 2.0 camponotus sp3 0.0 4.0 0.0 nylanderia sp.1 32.0 16.0 30.0 nylanderia sp.2 6.0 0.0 4.0 nylanderia sp.3 2.0 0.0 0.0 myrmicinae acromyrmex subterraneus brunneus 0.0 6.0 2.0 atta cephalotes 0.0 2.0 0.0 cephalotes pusillus 2.0 0.0 0.0 cephalolotes persimilis 0.0 2.0 0.0 cephalotes sp.1 8.0 0.0 2.0 cephalotes sp.2 2.0 0.0 0.0 crematogaster sp.1 6.0 10.0 12.0 crematogastersp.2 2.0 0.0 0.0 crematogaster sp.3 6.0 0.0 0.0 dorymyrmex sp.1 0.0 26.0 10.0 table 1. frequency of occurrence of epigeic ant species collected at two sites in the municipality of jequié, ba, brazil. site 1 = brejo novo, site 2 = frizuba,w = winklermethod, and p = pitfall traps method. 1386 sociobiolog y vol. 59, no. 4, 2012 pheidole sp.4grupo diligens 12.0 0.0 4.0 pheidole sp.5grupo tristis 6.0 0.0 0.0 pyramica denticulate 14.0 0.0 0.0 rogeria besucheti 2.0 0.0 0.0 rogeria blanda 2.0 0.0 0.0 rogeria sp.1 2.0 0.0 0.0 solenopsis globularia 2.0 0.0 0.0 solenopsis sp.1 30.0 20.0 44.0 solenopsis sp.2 4.0 0.0 8.0 solenopsis sp.3 0.0 0.0 2.0 solenpsis (diplorhoptrum) sp.2 2.0 0.0 0.0 strumigenys mandibulata 2.0 0.0 0.0 strumigenys sp.1 0.0 0.0 2.0 wasmannia auropunctata (roger, 1863) 2.0 0.0 0.0 wasmannia sp.1 36.0 0.0 10.0 wasmannia sp.2 4.0 0.0 2.0 wasmannia sp.3 2.0 0.0 0.0 cerapachynae cerapachyssp.1 0.0 2.0 0.0 ponerinae anochetus simony 4.0 0.0 0.0 hypoponera sp.2 2.0 0.0 0.0 hypoponerasp.1 2.0 0.0 0.0 hypoponera sp.3 4.0 0.0 0.0 thaumatomymex sp. prox. mutilatus 4.0 0.0 0.0 proceratinae 0.0 0.0 0.0 discothyrea sexarticulata 4.0 0.0 0.0 pseudomyrmecinae pseudomyrmex oculatus 2.0 0.0 0.0 pseudomyrmex sp.1 4.0 2.0 6.0 l. table 1 (continued). frequency of occurrence of epigeic ant species collected at two sites in the municipality of jequié, ba, brazil. site 1 = brejo novo, site 2 = frizuba,w = winklermethod, and p = pitfall traps method. 1387 carvalho, k. s, et al. — litter as an important resource and the diversity of ants brejo novo frizuba sampling method pitfall winkler combined pitfall observed richness (mao tau) 19 48 58 25 estimated richness: chao i 29.50 ± 8.8 75.60 ± 15.1 89.5 ± 15.9 29.6 ± 4.4 jackknife i 24.73 ± 2.98 71.52 ± 7.95 85.44 ± 7.4 32.8 ±2.9 bootstrap 21.78± 0 58.18 ±0 69.98 ±0 28.7 ±0 simpson diversity index 0.87 0.89 table 2. richness (estimators and indexes) of epigeic ant species in two sites in the municipality of jequié, ba, brazil. cyphomyrmex transversus 2.0 0.0 2.0 hylomyrma sp 0.0 0.0 6.0 nesomyrmex sp.1 0.0 2.0 0.0 nesomyrmex itinerans 2.0 0.0 0.0 basiceros rugifera(mayr, 1887) 10.0 0.0 0.0 basiceros stenognatha 2.0 0.0 0.0 basiceros jheringi 2.0 0.0 0.0 pheidole sp.1 0.0 58.0 56.0 pheidole sp.2 4.0 12.0 22.0 pheidole sp.3grupo diligens 18.0 0.0 0.0 table 1 (continued). frequency of occurrence of epigeic ant species collected at two sites in the municipality of jequié, ba, brazil. site 1 = brejo novo, site 2 = frizuba,w = winklermethod, and p = pitfall traps method. doi: 10.13102/sociobiology.v61i4.478-482sociobiology 61(4): 478-482 (december, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 population structure of melipona subnitida ducke (hymenoptera: apidae: meliponini) at the southern limit of its distribution based on geometric morphometrics of forewings cbs lima¹, la nunes², mf ribeiro³ , cal de carvalho¹ introduction stingless bees form an important group of pollinators in the neotropics, playing a significant role on the maintenance of ecosystems and agricultural production (heard, 1999; slaa et al., 2006). the stingless bee melipona subnitida ducke, locally known as "jandaira", is endemic to the caatinga (zanella, 2000), one of the biomes found in the northeastern brazil, which is characterized by a semiarid climate and xerophylous vegetation (andrade-lima, 1981). m. subnitida is found mainly in the states of bahia, ceará, rio grande do norte and paraiba (martins, 2002; camargo & pedro, 2007; webbee, 2014), playing an important role on pollination of cultivated plants, as well as on honey and wax production (cruz et al., 2005). abstract bees provide fundamental services to humanity, and many researchers have been concerned about the rapid loss of genetic diversity that these organisms have been suffering. the stingless bee melipona subnitida ducke is endemic to northeastern brazil and has high potential for the production of honey and wax; it is also an important pollinator in the caatinga biome. populations of m. subnitida have increasingly declined due to predatory extractivism and habitat destruction. however, knowledge about its population structure could give insights on strategies for monitoring and conservation of this species. here we collected workers from nine sites located at the southern limit of the species distribution and employed geometric morphometric techniques on their forewings in search of covariance between sampling site and wing morphology. a very significant correlation between both variables was observed, indicating that the divergence among the sampled populations of m. subnitida was due to geographical distance among the sampling sites and, hence, suggesting the formation of different groups of populations along the studied geographical zone, each one with specific characteristics. since m. subnitida habitat has been increasingly fragmented thus hindering the genetic flow among populations, our findings will contribute to the formulation of management and conservation plans for this species in order to preserve its genetic diversity and, therefore, to collaborate to the generation of income for beekeepers in meliponiculture programs. sociobiology an international journal on social insects 1 universidade federal do recôncavo da bahia, cruz das almas, ba, brazil 2 universidade estadual do sudoeste da bahia, jequié, ba, brazil 3 empresa brasileira de pesquisa agropecuária, petrolina, pe, brazil article history edited by cândida maria l. aguiar, uefs, brazil received 22 august 2014 initial acceptance 10 september 2014 final acceptance 06 november 2014 keywords meliponiculture, jandaira bee, manova, procrustes superposition, shape. corresponding author cândida beatriz da silva lima universidade federal do recôncavo da bahia, rua ruy barbosa, 710, centro, cruz das almas/ba, brazil e-mail: candidab.lima26@gmail.com the predatory exploitation of nests of native bees by humans has become a major concern for scientists (brown & paxton, 2009), but deforestation, agriculture intensification and introduction/spread of exotic competing bee species are considered the main threats to most indigenous species (freitas et al., 2009). on the other hand, knowledge about the population structure of these organisms has been considered critical for management and conservation plans of endangered species (nunes et al., 2008; bonatti et al., 2014). the employment of geometric morphometrics on identification and assessment of population diversity/structure has been proven efficient (breuker et al., 2006; wappler et al., 2012). geometric morphometrics allows detecting relationships between animal populations and their geographic locations through a rigorous analysis of variations in the research article bees sociobiology 61(4): 478-482 (december, 2014) 479 shape of a model structure. the most common application of the geometric morphometrics is the identification of landmark configurations in several morphological characters (klingenberg, 2002). cartesian coordinates provide the relative positions of each point, and, therefore, they make possible the reconstruction of shape and the identification of shape variations (rohlf & marcus, 1993; rohlf, 1998). this method is very sensitive to subtle variations and, hence being suitable for detection of groups and subgroups (francoy et al., 2011). studies on population structure and geographic variation of bees have been carried out based on morphometric data comparing races or populations (ferreira et al., 2011; lima junior et al., 2012; nunes et al., 2013). those analyses are usually based on wing characters due to their high heritability and because they are strongly affected by the environment (diniz-filho & bini, 1994). a possible disadvantage of this technique is that the biological structures that will be studied need to be completely undamaged. however, such disadvantage is rarely a critical factor (lyra et al., 2010). considering the ecological and economical importance of stingless bees for the neotropics and the necessity of studies on the population structure of endemic species of the caatinga biome, the aim of this study was to determine the population diversity of m. subnitida from sites located at the southern limit of its distribution based on geometric morphometrics of forewings. material and methods we collected 630 workers from 63 colonies of m. subnitida in nine localities of northeastern brazil, from august to december 2012 and in july 2013 (table 1). we removed the bees’ forewings, placed them between microscope slides, and photographed them with a digital camera coupled to a stereomicroscope for the analyses of venation. we transformed the photographs in the software tpsutil 1.40 (rohlf, 2008a). we inserted ten landmarks (fig. 1) at the vein fig 1 right forewing of melipona subnitida showing the 10 landmarks located at wing vein intersections, which were used in the morphometric analysis. results in the pca applied to the populations of m. subnitida, the first four principal components explained 62.96% of the total variation among individuals of different communities: pc1 = 20.61%, pc2 = 16.26%, pc3 = 14.20%, and pc4 = 11.88%. we represented the group distribution in a bidimensional space, using the scores of the first two components, to test for dispersal among groups. based on the mahalanobis and procrustes distances, the largest difference was observed between the populations from exu and água branca (305 km apart) whereas the highest morphological proximity occurred between the populations from água branca and mata grande (30 km apart) (table 2). based on a dendrogram (upgma; fig. 2), we observed morphological differences between the populations from exu, passira and taquaritinga do norte, which were grouped in a table 1. origin and geographic location of the melipona subnitida samples at the southern limit of its distribution natural in brazil. nnumber of samples. intersections of each forewing using tpsdig version 2.17 (rohlf, 2008b). we used the data obtained as variables for multivariate analyses, such as principal component analysis (pca), canonical correlation analysis, mahalanobis distance (d²), and procrustes distance in the software morphoj 1.03 (klingenberg, 2011). we also made cluster analyses based on upgma (unweighted pair group method with arithmetic mean) and a mantel test based on geographic distance, and size and shape of bee wings in the software past 2.17. sites/ state n latitude (s) longitude (w) altitude (m) climate / relief água branca/alagoas 9 9º10’24.7’’ 37°51’41.9’’ 380 semiarid/ massive cumaru/pernambuco 6 8°1’58.55’’ 35°45’3.11’’ 348 semiarid/ low hills exu/pernambuco 3 7°20’22.66’’ 39°54’58.54’’ 887 semiarid/low mountains joá/paulo afonso/ bahia 6 09º31’08.8’’ 38°25’36.7’’ 243 semiarid/ upland mata grande/alagoas 12 09o11’09.3’’ 37°50’09.8’’ 424 semiarid/ massive passira/pernambuco 5 7°55’37.85’’ 35°30’14.01’’ 160 semiarid/ high massive riacho das almas/pernambuco 6 8°3’40.79’’ 35°49’9.62’’ 413 semiarid/ low hills são josé/ paulo afonso /bahia 8 09º39’04.8’’ 38º22’43.2’’ 243 semiarid/upland taquaritinga do norte/pernambuco 8 7°56’14.15’’ 36°7’05.7’’ 785 tropical/massive cbs lima, la nunes, mf ribeiro, cal de carvalho geometric morphometrics of melipona subnitida forewing480 discussion the geometrical morphometrics of forewings was very efficient in detecting variability among the populations and, hence, in unveiling the population structure of m. subnitida. the results of the morphometric analysis pointed to morphological variability in wing shape among populations along their geographic distribution. this variation was associated with geographic distance among the sampling sites and is also probably related to the environmental variability among sampling localities. the populations from exu and água branca showed the highest index of morphometric divergence, with a dissimilarity value of 3.68, which may be explained by an interaction between geographic distance and climatic/relief differences between regions. exu is characterized by a semi-arid climate: warm in the summer and cold in the winter; it is located between massifs and low mountain ranges (300 – 800 m height); on the other hand, água branca is characterized by a tropical semi-arid climate with summer rains, and it is located between massifs and high mountain ranges (650 – 1,000 m height) (cprm, 2014). the less divergent populations were those of água branca and mata grande, both located in the state of alagoas (dissimilarity value = 1.33). these areas are geographically close to each other (3 km) and have similar climate, vegetation, and relief. in the cluster analysis, the formation of groups reflected morphological differences between the populations and, in general, we found a correlation between forewing morphology and geographical distance. however, the populations from exu and passira, which are more than 300 km apart, curiously were found to form a distinct group, indicating morphological similarities between the two far apart populations. these similarities might be a result of the similar environmental conditions found in exu and passira, which, despite the distance, present suchlike vegetation and relief (cpmr, 2014). fig 2 dendrogram generated by upgma with the average morphometric distances between melipona subnitida colonies from different localities. more isolated branch, standing out from populations of other regions, with a correlation index (p< 0.0001), which shows that the divergence between population is highly significant. the correlation between shape, size, altitude, and geographic distance among 63 m. subnitida colonies compared with a mantel test (table 3) indicated a positive correlation between wing shape and geographic distance among all populations (p < 0.01). the other correlations had high p-values, which points to no relationship among variables. there was variation in the shape of the forewings among m. subnitida populations, which is reflected in the formation of groups. table 2. mahalanobis distances (lower half of the matrix) and procrustes distance (upper half of the matrix) among populations calculated from the canonical correlation analysis. sites: ab: água branca; cu: cumaru; ex: exu; jo: joá; mt: mata grande; pa: passira; ra: riacho das almas; sj: são josé; tn: taquaritinga do norte. table 3. mantel test used to compare matrices of shape, size, altitude, and geographic distance, based on wing measurements of melipona subnitida with 5,000 permutations. ns not significant; * significant ab cu ex jo mt pa ra sj tn ab 0.017 0.020 0.015 0.008 0.019 0.017 0.015 0.014 cu 2.93 0.015 0.012 0.014 0.015 0.010 0.013 0.016 ex 3.68 2.54 0.019 0.017 0.014 0.017 0.014 0.015 jo 2.96 1.78 3.08 0.012 0.019 0.013 0.011 0.016 mt 1.33 2.32 3.27 2.37 0.017 0.018 0.010 0.017 pa 3.37 2.17 2.84 2.52 2.77 0.017 0.012 0.017 ra 3.21 1.68 2.85 2.11 3.10 2.66 0.015 0.013 sj 2.87 1.94 2.63 1.83 2.13 1.71 2.31 0.016 tn 2.47 2.65 2.48 2.87 2.72 3.10 2.41 2.70 compared matrices r² p shape x altitude 0.009 0.473ns shape x geographic distance 0.506 0.008* size x shape 0.282 0.972ns size x altitude 0.072 0.537ns size x geographic distance 0.045 0.512ns sociobiology 61(4): 478-482 (december, 2014) 481 brasileira de pesquisa agropecuária (embrapa) (cod. seg 02.11.01.029.00.00) for the financial support, coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarship (c. b. da s. lima) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) (proc. 305228/2013-7),. references andrade-lima, d. 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(2011). morphometric analysis of populations of centris aenea lepeletier (hymenoptera: apidae) from northeastern brazil. neotrop. entomol., 40: 97-102. we have previously found similar results when studying population diversity in melipona quadrifasciata anthidioides lepeletier from different regions of bahia state (nunes et al., 2008), observing that the populations that were located far from each other showed morphological divergence. mendes et al. (2007) observed morphological differences between nannotrigona testaceicornis (lepeletier) groups from urban and rural areas in the municipality of uberlândia, state of minas gerais, in the southeast of brazil. these authors associated these differences with selective pressures in the urban area and with a higher probability of genetic variability in the rural area. once the temperature is a factor that can influence the insect populations and distribution (easterling et al., 2000; damos & savopoulou-soultani, 2012), and considering the expected impacts of climate warming on stingless bees (saraiva et al., 2012; giannini et al., 2012), the characterization of population structures of m. subnitida will help to preserve its genetic diversity, increasing the likelihood of conserving lineages more adapted to the expected climatic changes, with potential impacts on pollination. melipona subnitida is narrowly distributed and it is currently found in fragmented and geographically-isolated environments (zanella, 2000; bonatti et al., 2014). the strong relationship between geographical distance and morphological variability found in our study indicates a lack of genetic flow among populations that are located far from each other or even among those that would be able to crossbreed if in favorable environmental conditions, corroborating the importance of conserving nesting sites and food sources. this finding will contribute to the formulation of management and conservation plans for m. subnitida in order to preserve its population diversity and, therefore, to collaborate to the generation of income in meliponiculture programs. in a broader sense, our results highlight the importance of characterizing the population structure of the stingless bees, because they help to identify differentiated populations, which need more human intervention as a way to decrease the rates of inbreeding. conclusion there is a correlation between sampling site and wing shape in m. subnitida populations inhabiting the limit of the species distribution, indicating that the divergence among the sampled populations is due to geographical distance among the sampling sites and, hence, suggesting the formation of characterized groups of populations along the studied zone. acknowledgements the authors thank cherre s. b. da silva for the helpful suggestions for the manuscript, the beekeepers for providing samples of bees, and the anonymous reviewers for their criticisms and suggestions to the manuscript, the empresa cbs lima, la nunes, mf ribeiro, cal de carvalho geometric morphometrics of melipona subnitida forewing482 freitas, b.m., imperatriz-fonseca, v.l., medina, l.m., kleinert, a. de m. p., galetto, l., nates-parra, g. & quezadaeuán, j.j.g. 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(2000). the bees of the caatinga (hymenoptera, apoidea, apiformes): a species list and comparative notes regarding their distribution. apidologie, 31: 579-592. doi: 10.1051/apido:2000148. doi: 10.13102/sociobiology.v66i3.4338sociobiology 66(3): 400-407 (september, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a worker-like female of myrmica sabuleti meinert, 1861 (hymenoptera: formicidae: myrmicinae) in a pitfall trap with five mermithids (nematoda: mermithidae) protruding from the gaster introduction nematodes of 10 families are known as ant parasites worldwide, with mermithidae as the largest and most conspicuous one (summarized by poinar, 1975; poinar, 2012). they occasionally can be found either by dissecting the gaster of a host ant or because they attract attention when leaving their host. sometimes, mermithids considerably change the morphology of the (female or male) hosts compared with unparasitized conspecifics, forming so-called parasitogenic phenotypes (e.g. kloft, 1949; czechowski et al., 2007a; csősz & majoros, 2009; csősz, 2012; poinar, 2012; de bekker et al., 2018). wheeler (1928) distinguished three discrete aberrant forms of female infested ants, mermithergates (resembling workers), gynaecoid mermithergates (intermorphs), and abstract a worker-like female of myrmica sabuleti meinert, 1861, pitfall-trapped near jena, germany, in late summer 2016, was infested by five postparasitic juvenile mermithids. they poked out of the ant´s gaster as a trail of seven filaments of various lengths. apart from its swollen gaster, the ant differed from conspecifics in several morphometric parameters. using both morphological and molecular techniques, the parasite family mermithidae was confirmed. our stray find raises multiple questions concerning the genus and species identity of the parasite, its biology, and the infestation rate of the host ant population. more mermithid awareness by the various researchers working with myrmica will help, but directed fieldwork, experimental life-history research, and molecular studies are needed to emancipate progress in ant-mermithid research from serendipity. sociobiology an international journal on social insects fm steiner1†, g köhler2†, b seifert3, w arthofer1, bc schlick-steiner1‡, a buschinger4‡ article history edited by kleber del-claro, ufu, brazil received 30 december 2018 initial acceptance 23 april 2019 final acceptance 27 april 2019 publication date 14 november 2019 keywords ant parasite, nematode, gregarious parasitism, 18s, coi, ant morphometrics. corresponding author florian m. steiner university of innsbruck molecular ecology group department of ecology technikerstr, 25, 6020 innsbruck, austria. e-mail: florian.m.steiner@uibk.ac.at mermithogynes (resembling gynes). csősz and majoros (2009) hypothesized that all infested adult females originate from gyne-destined larvae even if resembling workers. mermithids in ants have been noticed for a long time, with the first report dating back to 1747 (poinar, 2012). nevertheless, knowledge is still scarce, and the number of reliably documented instances of mermithids in ants was estimated at just “> 50” (lachaud & perez-lachaud, 2015). particularly, the life history of most ant mermithids has remained mysterious, and even for the best analyzed systems, crucial questions remain open (e.g., kaiser, 1986; poinar et al., 2007). all resolved instances have in common that the parasite enters its host as preparasitic juvenile, grows in the gaster, and leaves the gaster as postparasitic juvenile, causing its host’s death (poinar, 2012). most frequently, a single mermithid per ant occurs, densely coiled up within the gaster 1 molecular ecology group, department of ecology, university of innsbruck, innsbruck, austria 2 institute of ecology and evolution, friedrich schiller university of jena, jena, germany 3 section pterygota, senckenberg museum of natural history, görlitz, germany 4 reinheim, germany † these authors contributed equally as first authors ‡ these authors contributed equally as last authors research article ants sociobiology 66(3): 400-407 (september, 2019) 401 and invisible from outside; the highest number of individuals in a single ant was nine (poinar et al., 2007). several species of the myrmicine genus myrmica have been reported as hosts of mermithids: m. gallienii bondroit, 1920, m. rubra (linnaeus, 1758), m. ruginodis nylander, 1846, m. rugulosa nylander, 1846, m. sabuleti meinert, 1861, m. scabrinodis nylander, 1846, and m. schencki viereck, 1903 (czechowski et al., 2007a; b and references therein). interpreting these host records, of which several date back to the first half of the 20th century, needs be done with a grain of salt, though, because the morphological changes of ants parasitized by mermithids often make species identification difficult (and have even resulted in erroneous species descriptions; csősz, 2012; borowiec & salata, 2015) and because the species-level identification of myrmica has remained difficult, even when not parasitized (radchenko & elmes, 2010; seifert, 2018). here, we describe a case of gregarious parasitism with five mermithids protruding from the gaster of a pitfalltrapped worker-like myrmica ant. based on morphometric and molecular-genetic analyses of the host and the parasite, respectively, we discuss questions about parasite taxonomy, parasite biology, and the host’s infestation rate. like in many other instances of mermithids in ants, many questions remain open – more directed research efforts from various directions will be needed to solve the many exciting questions in this field. material and methods sampling as part of a 2012-2017 monitoring project for studying arthropod communities by the institute of ecology and evolution (friedrich-schiller-university of jena), a pitfall-trapping campaign was conducted in the leutra valley near jena/thuringia, germany; for details, see köhler (2018) and autorenkollektiv (2019). briefly, a total of 72 pitfall traps were exposed in groups of four traps (at the corners of 1.4 × 1.4 m squares) along three transects from mid april to mid october. the traps used were hard plastic tubes with an opening diameter of 4.7 cm and a depth of 9 cm; the trapping liquid was a 1,2-propandiol:water = 3:1 (v/v) mixture plus a dash of liquid soap to reduce surface tension and a dash of quinine sulfate powder to repel small mammals. the infested myrmica specimen was trapped on a xerothermous, south-exposed (22° inclination) cherry meadow orchard with bromus-rich grassland, partly surrounded by a sparse pine forest (50° 52’ 15’’ n / 11° 33’ 48’’ e, 240 m above sea level) during trap exposure 19.ix.-05.x.2016. this ant specimen was the only ant of the monitoring project for which parasite infestation was detected; upon sorting, it was transferred to 70% ethanol. the parasitized ant host and one parasite individual were deposited in the ant and nematode collection, respectively, of senckenberg museum für naturkunde görlitz, germany. measurements and photographs the externally visible mermithid filaments were measured with a zeiss stereomicroscope (sm xx) with measuring ocular using 12.5× and 50× magnification. photographs were taken using a zeiss stereomicroscope (stemi 305) with integrated hd ip wi-fi camera and transmitting light (institute of ecology and evolution, university of jena). later, the ant was dissected in tap water in a wax bowl under a wild m5 dissecting microscope at 25× magnification using two watchmaker forceps dumont no. 5. after dissection, the entire mermithids were measured and photographs taken under 50× magnification. during further handling, one worm got lost. identifications host species identification was done by (a) using the characters in the key of seifert (2018) to exclude less similar species of the myrmica scabrinodis and m. sabuleti species complexes, (b) running the infected specimen as wild-card in a two-class linear discriminant analysis (lda) comparing nine m. bibikoffi kutter, 1963 workers from three sites in germany and switzerland and 77 m. sabuleti workers from 34 sites in the whole westpalaearctic range, and (c) running the same individuals in a principal component analysis (pca). the stereomicroscopic investigation methods, the removal of allometric variance (rav), and the set of 19 investigated characters used in lda and pca are described in seifert et al. (2014). lda and pca were run using the spss 16.0 software package (ibm, usa). for morphological identification of the nematodes, two of the five individuals were processed into glycerin (poinar, 1975) and studied with a nikon smz-10 r stereoscopic microscope and a nikon opti-phot compound microscope at magnifications of up to 800× (g. poinar, corvallis/oregon, usa). for molecular identification, one nematode was analyzed via the 18s ribosomal dna (18srdna) and the cytochrome c oxidase i (coi) marker. nematode dna was extracted using the qiaamp dna micro kit (qiagen, germany) following the instructions of the manufacturer. 18s pcr was performed in 1× quantitect probe pcr mastermix containing 0.2 µm nf1 primer, 0.2 µm 18sr2b primer (both porazinska et al., 2009), and 2 µl dna extract in 10 µl total volume. coi pcr reactions contained 1× mytaq buffer (bioline, uk), 0.2 µm nem_coi_f primer, 0.2 µm nem_ coi_r primer (malysheva et al., 2016), 0.25 u mytaq (bioline), and 1 µl dna extract in 10 µl total volume. the thermal profile for 18s was 10 min initial denaturation at 95 °c, followed by 35 cycles of 30 s at 95 °c, 30 s at 58 °c, 1 min at 72 °c, and a final elongation of 10 min at 72 °c; the profile for coi was as in malysheva et al. (2016). presence of pcr product was verified by gel electrophoresis, and 1 µl amplicon was cloned with the clonejet pcr cloning kit (thermo) following the instructions of the manufacturer. insert size was determined by pcr with the vector primers provided with the kit. plasmid dna of three colonies with fm steiner et al. – five mermithid parasites in a female myrmica ant 402 correct insert size was extracted by alkaline lysis (sambrook et al., 2001) and sanger sequenced using the forward vector primer. the obtained sequence was analyzed with the basic local alignment search tool (blast), which searches for regions of similarity between sequences of a query sequence and the ncbi genbank database. because for coi, default settings did not yield any plausible result, “optimize for more dissimilar sequences (discontiguous megablast)” was chosen. results host ant there are six myrmica species known from central europe (seifert, 2018) to which the nematode host might possibly belong: m. sabuleti, m. bibikoffi, m. scabrinodis, m. lonae finzi, 1926, m. vandeli bondroit, 1920, and m. curvithorax bondroit, 1920. the latter four species can be clearly excluded by simple eye inspection based on structure of basal scape and head sculpture as described in seifert (2018). regarding the former two species, normal workers of myrmica sabuleti without teratological or parasite-induced changes of morphology are easily separable from its temporary social parasite myrmica bibikoffi by different rav-corrected body ratios. these discriminators are, according to an lda with stepwise character reduction, a smaller postpetiole width index ppw/cs1050, a smaller postpetiolar hair length index pphl/ cs1050, a lower postocular distance index pooc/cs1050, a larger frontal carinae index fl/fr1050, and a shorter spine length index sp/cs1050. however, nematode infection is known in myrmica to result in more massive petiole and postpetiole, a smaller frontal carinae index, and more diverging propodeal spines (czechowski et al., 2007a; csősz & majoros, 2009). this could make the identification of the host specimen problematic. considering the five diagnostic characters mentioned above and running the leutra host specimen as wild card in a twoclass lda comparing m. bibikoffi and m. sabuleti, the host was allocated to myrmica sabuleti with p=0.611. furthermore, the infested specimen was clearly allocated to m. sabuleti in a pca using these five characters, but it was the most m. bibikoffi-like specimen within the m. sabuleti cluster (fig 1) due to its increased width and height of waist segments and the lower frontal carinae index (table 1). the just low likelihood of belonging to m. bibikoffi is also supported by the rarity of this species in general and the fact that it has never been found in the leutra valley, which has been intensively studied myrmecologically since the 1970s (seifert, 1982). fig 1. bivariate plot of the score of the 1st factor of a principal component analysis (pca) and of the score of a linear discriminant analysis (lda) of the nematode-infested specimen (black rhomb), of nine myrmica bibikoffi workers (grey dots) and of 77 m. sabuleti workers (grey rhombs) from the whole w-palaearctic range. the infested specimen was run in the lda as wild-card, that is, without imposing a hypothesis. the set of originally 19 characters was reduced to the five most diagnostic ones in both analyses. table 1. morphometric data (average ± standard deviations, minimum and maximum values in square brackets) of workers of myrmica bibikoffi, of the nematode-infested m. sabuleti worker from leutra, and of normal m. sabuleti workers from its whole palaearctic range. the data are corrected in the allometric space. the data for pphl, metsp, and metl are based on a reduced sample of only 77 specimens. for definitions of the morphometric characters, see seifert et al. (2014). bibikoffi (n=9) sabuleti leutra (n=1) sabuleti (n=313) cs [µm] 1154 ± 76 [1062,1257] 1165 1158 ± 65 [ 942,1322] cl/cw (1150) 1.042 ± 0.020 [0.996,1.064] 1.055 1.031 ± 0.015 [0.990,1.074] sl/cs (1150) 0.810 ± 0.013 [0.795,0.833] 0.848 0.802 ± 0.017 [0.761,0.851] sw/sl (1150) 0.179 ± 0.008 [0.137,0.179] 0.173 0.199 ± 0.017 [0.159,0.245] pooc/cl (1150) 0.444 ± 0.004 [0.440,0.450] 0.424 0.432 ± 0.009 [0.408,0.456] eye/cs (1150) 0.194 ± 0.007 [0.183,0.202] 0.200 0.195 ± 0.006 [0.181,0.220] fl/cs (1150) 0.459 ± 0.013 [0.443,0.484] 0.464 0.457 ± 0.012 [0.421,0.489] fr/cs (1150) 0.327 ± 0.017 [0.312,0.357] 0.325 0.300 ± 0.014 [0.262,0.356] fl/fr (1150) 1.405 ± 0.040 [1.333,1.457] 1.423 1.530 ± 0.076 [1.306,1.739] pew/cs (1150) 0.321 ± 0.025 [0.300,0.370] 0.321 0.280 ± 0.011 [0.253,0.318] ppw/cs (1150) 0.461 ± 0.031 [0.430,0.520] 0.482 0.399 ± 0.016 [0.351,0.453] peh/cs (1150) 0.364 ± 0.015 [0.345,0.391] 0.365 0.335 ± 0.012 [0.307,0.373] pel/cs (1150) 0.499 ± 0.024 [0.465,0.528] 0.533 0.479 ± 0.014 [0.441,0.523] sp/cs (1150) 0.386 ± 0.021 [0.345,0.419] 0.445 0.398 ± 0.023 [0.303,0.455] pphl/cs (1150) 0.211 ± 0.010 [0.198,0.227] 0.164 0.168 ± 0.013 [0.115,0.197] metl/cs (1150) 0.239 ± 0.016 [0.214,0.259] 0.245 0.228 ± 0.010 [0.194,0.256] metsp/cs (1150) 0.207 ± 0.029 [0.147,0.243] 0.187 0.180 ± 0.018 [0.147,0.250] sociobiology 66(3): 400-407 (september, 2019) 403 the infested female myrmica ant was a mermithergate according to the categorisation by wheeler (1928) and had no ocelli. it possessed a slightly thickened and longer gaster (~2.5 mm) compared with unparasitized workers from the same trap (~2.0 mm). mermithid parasites at the myrmica worker’s back end, seven filaments of various lengths protruded from the gaster, of the same yellowbrown color as the ant itself, presumably because of storage in the same trap liquid (fig 2a). the nematodes had penetrated the intersegmental membranes between gastral segments three and four (= abdominal segments six and seven), rupturing both tergites and sternites distinctly. the worm parts were more or less straight and rod-like, and of two worms, both anterior and posterior ends were visible outside the ant´s gaster, whereas of three worms, only one end (one front and two rear ends) were visible before dissection. the coiled-up parts of the worms were partially visible through the cuticle up to the second gaster segment (fig 2b). three filaments were rising more ventrally and rather short (0.9/1.2/2.0 mm), whereas four filaments were rising more dorsally and much longer (3.5/4.8/5.1/6.5 mm), with the longest exceeding the ant size (~5.5 mm) (fig 2a). their diameters were between 0.13-0.19 mm at the base and 0.11-0.15 mm at the ends, narrowing only slightly. after removal of the tergites and sternites, the parts of the worms inside the ant were found coiled and had remained pale, contrasting the yellow-brown parts exposed to the pitfall fluid (fig 2c). a total of five mermithids could be confirmed, with the largest reaching 12.0 mm total length (fig 2d). only small remains of the ant´s internal organs were discernible and not clearly identifiable. morphologically, the nematodes were identified as postparasitic juvenile mermithids (family mermithidae). the genus can only be determined in adults (g. poinar, unpubl., using the key in poinar, 1975). this morphological identification was corroborated by sequencing 363 basepairs of the 18sr dna (genbank accession number mh793873) and 563 basepairs of coi (genbank accession number mk256737). the 18s blast search yielded several hits with 100% query cover, 99% identity match, and e=0 to the genera agamermis and hexamermis; the coi blast search yielded hits with 98% query cover, 73% identity match, and e=10-81 to the same genera. a b c d fig 2. the infested myrmica female and its parasites. (a) two workers of myrmica sabuleti, on the left infested by mermithids (total ant body length ~5.5 mm, gaster length ~2.5 mm, length of visible part of longest mermithid filament ~6.5 mm, diameters at base 0.13-0.19 mm and at the ends 0.11-0.15 mm), on the right an uninfested conspecific. (b) through the myrmica gaster, parts of the mermithids are visible. (c) the dissected myrmica gaster shows the compact juvenile mermithids. (d) the five juvenile mermithids of various lengths found in the infested myrmica (length of longest mermithid 12.0 mm). (photograph (a) by a. ebeling & i. wolf; photographs (b-d) by a. buschinger). fm steiner et al. – five mermithid parasites in a female myrmica ant 404 discussion our record of myrmica sabuleti, identified using morphometrics, as host of five postparasitic mermithids, identified using morphology and molecular genetics, was a stray find during a pitfall-trapping campaign with research aims other than ant mermithid infection. multiple questions about parasite taxonomy, parasite biology, and the infestation rate of the host population can now be addressed, albeit mostly without definitively answering them. what is the genus and species identity of the parasite? the morphological identification of postparasitic juveniles, at which stage mermithids leave their ant hosts, is not possible to species nor to genus (poinar, 1975). mermithid species from ants have thus often remained unidentified on morphological grounds (e.g., mcinnes & tschinkel, 1996; csősz & majoros, 2009; o’grady & breen, 2011; laciny et al., 2017), and the genus and species identity of mermithidae in myrmica has never been resolved so far (poinar, 2012). dna-sequence-based identification can be a valuable tool (poinar et al., 2007). the sequences we established are similar to ones from agamermis and hexamermis, which build a monophylum within the mermithidae (park et al., 2011; kubo et al., 2016). both agamermis and hexamermis have been reported from a broad range of insect hosts (e.g., achinelly & camino, 2008; stubbins et al., 2016; and references therein) but, as far as we know, not from ants (poinar, 2012 and web of science queries on 20 november 2018). however, despite the high similarity to agamermis and hexamermis, uncertainty remains, in that of the safely identified recent mermithid genera found in ants (poinar, 2012), two (allomermis, pheromermis) are represented in genbank (nucleotide query of 21 november 2018) but not the other four (agamomermis, camponotimermis, comanimermis, meximermis). this uncertainty is typical of current mermithid research – a lack of sequence data from safely identified specimens of the mermithid species at question and thus a lack of comparison has often impeded taxonomic resolution even when molecular analyses were undertaken (hornets: villemant et al., 2015; ants: laciny, 2017; moths: kumar et al., 2018; bumble bees: tripodi & strange, 2018). a comprehensive reference dna sequence database of the mermithid species identified so far in ants would allow improved taxonomic resolution for postparasitic individuals and thus access to the knowledge established for those parasites. at which developmental stage of the ant did the parasite infest it? the female m. sabuleti presented here showed similar differences from nonparasitized conspecifics to those described by csősz and majoros (2009) for mermithogenic myrmica gallienii and to those described by czechowski et al. (2007a) for mermithogenic m. sabuleti. these differences (i.e., not the enlarged gaster) allow us to conclude that the parasites entered the ant before the ant was adult, in line with findings that mermithids that enter ant larvae can be carried over to the adult stage (wheeler, 1901). likely, the ovipositing mermithid was close to the ant nest, thus resulting in multiple infections – hexamermis adults, for example, produce eggs in the soil, and from these eggs hatch pre-infective stages that search the surroundings for hosts (poinar & gyrisco, 1962). what is the relevance of the high number of five parasites in the same host? in most ants parasitized, a single mermithid is found. the highest available numbers of mermithids per ant individual are four in myrmica (csősz & majoros, 2009), five in solenopsis and lasius (mcinnes & tschinkel, 1996; o’grady & breen, 2011), and nine in solenopsis (poinar et al., 2007). in all these instances, large numbers of parasitized ants were screened and the higher the number of individuals per ant, the rarer it was detected. our find is among the highest numbers of mermithid per ant host recorded, but just a single infected worker was found. this combination is improbable (but obviously not impossible) if the frequency distribution of multiple infections (2, 3, 4, etc. nematodes per ant) in the m. sabuleti population leutra is the same as in those other studies. series of infected m. sabuleti from the population analyzed here will be needed to interpret our current find of five parasites as typical or atypical of this particular relationship. did the parasite start emerging from the host before or after the host was pitfall-trapped? our find of a pitfall-trapped host with parasites protruding from its gaster could mean that the parasites started emerging before the ant got caught (pre-trapping) or just after contact with the trapping liquid (post-trapping). pre-trapping emergence may be possible, in that the process of emerging from the ant was reported to take, for example, one hour (after which the ant hosts lived for a further hour; o’grady & breen, 2011). in support of pre-trapping emergence may be seen that the worms were shaped rod-like and did not, as normally the case, form a tangle – the rodlike posture may have resulted from the ant walking some distance with the worms trailing behind. alternatively, the rod shape may have resulted from death in the trapping liquid. this aspect could be tested by killing living mermithids in diluted 1,2-propandiol. walking with such a voluminous trail appears difficult, but before contact with the liquid, the worms may have been much thinner – mermithids were reported to gain volume once in contact with water by imbibing it (poinar et al., 2007). not in line with pre-trapping emergence is the report by kaiser (1986) that the ants remain still during the parasites’ emergence and only after completed emergence run around hastily before they die. post-trapping emergence would be in line with reports for various ant mermithids that contact of the host with water sociobiology 66(3): 400-407 (september, 2019) 405 was necessary to trigger emergence of the parasite (crawley & baylis, 1921; kaiser, 1986; maeyama et al., 1994; poinar et al., 2007; o’grady & breen, 2011). from many parasitized grasshoppers pitfall-trapped in the same liquid used here, not a single emergence of the mermithids was observed (g. köhler, unpubl.), but this may not be in conflict with post-trapping emergence in this ant case because the mermithids of the two hosts may have different emergence triggers. experiments with living, infected m. sabuleti from the population analyzed here will be needed to solve this question. did the parasite trigger water-seeking behavior in the host? whenever the parasites started emerging from the host, the ant may have been trapped because it fell into the pitfall trap by chance or because the parasite triggered its host to seek a moist environment. such behavioral manipulation has been postulated for several mermithids in ants (kaiser, 1986; mayeama et al., 1994; poinar et al., 2007). a potential adaptive value of seeking a moist environment appears somewhat problematic in a dry habitat during a dry period (total rainfall measured by a climate station 6 km from the habitat was 16.1 mm during the trapping period 19.ix.05.x.2016; jena university of applied science, unpubl.) but cannot be refuted entirely. avoiding exposure to the dry conditions above surface may actually be more adaptive for the parasites. experimental evidence is needed. what is the infestation rate of the host population? the literature record holds both high infestation rates in some ant nests of selected host populations (mcinnes & tschinkel, 1996; czechowski et al., 2007b; csősz & majoros, 2009) and difficulties in finding mermithids in ant populations infested in a previous year (reviewed by o’grady & breen, 2011). at the population level, this means clumped spatial distribution with temporal variation. our find of a single parasitized worker out of thousands myrmica workers trapped in the habitat analyzed mid april to mid october 2016 (g. köhler, unpubl.) may thus mean that the m. sabuleti population has a low infestation rate. alternatively, it might have a high infestation rate but no trap may have been close to a strongly infested nest or infested ants may be influenced by their parasites to rather stay in their nests during dry periods (see previous section). to characterize the infestation rate of the analyzed population, but also those of other myrmica populations, dedicated screening of many living individuals from many nests will be needed, ideally from multiple years, as done recently in a single-year survey of thousands of bumble bees at the continent scale (tripodi & strange, 2018) and in a multiple-year survey of tens of thousands of hornets at the district scale (villemant et al., 2015). if combined with genetic approaches, such studies would also help to shed some light on the entirely understudied population genetics of nematode parasites in general (cole & viney, 2018). conclusion while a lot of research effort has been directed at many of the parasitic organisms infesting myrmica species (witek et al., 2014), data on mermithids are scarce. as myrmica ants are important study systems for various disciplines (e.g., radchenko & elmes, 2010; seifert, 2018), including the study of lycaenid butterflies and socially parasitic ants (e.g., barbero et al., 2012; tartally et al., 2019), awareness of this phenomenon by the various researchers working with myrmica may bring to light further instances of mermithid infections, possibly with living individuals. however, additionally to mermithid awareness when analyzing other aspects of myrmica ants, directed and dedicated efforts in fieldwork, experimental life-history research, and moleculargenetic analyses will be needed to reduce the dependence of mermithid research in myrmica and ants generally on chance. acknowledgments for managing the pitfall-trapping campaign, to winfried voigt, sylvia creutzburg, and student helpers; for taking photographs, to anne ebeling and ilka wolf (all jena, germany); for identifying the nematode parasites morphologically and valuable comments on an earlier version of the manuscript, to george poinar (corvallis, usa); for mycological expertise, to heinrich dörfelt (halle, germany), alexander schmidt (göttingen, germany), and martin kirchmair (innsbruck, austria); for assistance in the molecular laboratory, to elisabeth zangerl (innsbruck, austria); for access to weather data, to jena university of applied science (jena, germany); for help with literature search, to alice laciny (vienna, austria) and ian will (orland, florida); for constructive input, to three anonymous reviewers. the research project (fe: 02.0234/2003/lrb) in jena from 2012-2017 was funded by the bundesanstalt für straßenwesen (bast), bergisch gladbach, germany. authors’ contributions gk noticed the host ant when sorting pitfall-trapped insects. gk and ab measured and photodocumented the nematodes. ab dissected the host ant. bs analyzed the host ant and other myrmica ants using morphometrics and ran statistics on the data. fms, wa, and bcs-s designed the molecular analyses of the nematodes and interpreted the data. all authors wrote the paper. references achinelly, m.f. & camino, n.b. 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(2018). rarely reported, widely distributed, and unexpectedly diverse: molecular characterization of mermithid nematodes (nematoda: mermithidae) infecting bumble bees (hymenoptera: apidae: bombus) in the usa. parasitology, 145: 1558-1563. doi: 10.1017/s0031182018000410 villemant, c., zuccon, d., rome, q., muller, f., poinar, g.o. & justine, j.l. (2015). can parasites halt the invader? mermithid nematodes parasitizing the yellow-legged asian hornet in france. peerj, 3: article e947. doi: 10.7717/peerj.947 wheeler, w.m. (1901). the parasitic origin of macroërgates among ants. the american naturalist, 35: 877-886. wheeler, w.m. (1928). mermis parasitism and intercastes among ants. journal of experimental zoology, 50: 165-237. witek, m., barbero, f. & markó, b. (2014). myrmica ants host highly diverse parasitic communities: from social parasites to microbes. insectes sociaux, 61: 307-323. doi: 10.1007/ s00040-014-0362-6 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.423-427sociobiology 61(4): 423-427 (december, 2014) nesting sites, nest density and spatial distribution of melipona colimana ayala (hymenoptera: apidae: meliponini) in two highland zones of western, mexico introduction stingless bees or meliponini is a group of bees with great biological and morphological diversity distributed mainly in the tropical and subtropical regions of the world (michener, 2000) and 250 species have been described in south and central america (camargo & pedro, 1992; nogueira-neto, 1997; michener, 2000). in mexico, 46 species of stingless bees have been identified; two of them are classified as endemic to the mountain ranges of western mexico: melipona colimana ayala and melipona fasciata latreille (ayala, 1999). m. colimana endemism is linked to the mesophyll mountain forest in elevations over 1000 meters above sea level, in the geographical zones that correspond to the manantlan mountain range, the national park nevado of colima and the tigre mountain range (ayala, 1999). usually abstract sociobiology an international journal on social insects jo macías-macías¹, jjg quezada-euán², jm tapia-gonzalez¹, f contreras-escareño³ article history edited by cândida maria l. aguiar, uefs, brazil received 23 september 2014 initial acceptance 13 november 2014 final acceptance 04 december 2014 keywords melipona colimana, nest density, spatial distribution, quercus laurina, jalisco, méxico. corresponding author josé octavio macias-macias. universidad de guadalajara. centro universitario del sur. departamento de desarrollo regional. enrique arreola silva av. 883. cd. guzmán, jalisco. mexico. cp. 49000 e-mail: joseoc@cusur.udg.mx stingless bees use diverse lodgings to establish their nests, as gaps and cavities in trees, electricity poles, under house´s roofs and abandoned termites and wasps’ nests. (nogueiraneto, 1997, roubik, 2006). for that reason, it is hard to acknowledge which are the places m. colimana sets its nest, therefore there is a scientific interest in studying and get to know the places it uses for this purpose in its origin habitat, which will provide us new information about the nesting sites of stingless bees in temperate weather. on the other hand, the conservation of stingless bees is important because they take part in the ecological interactions that contribute to the biodiversity maintenance, acting as pollinators of different plant species, and because they can act as indicators of environmental disturbances (brown & albrecht, 2001; imperatriz-fonseca, 2002; slaa et al., 2006). forest exploitation has negatively affected the presence of research article bees 1 universidad de guadalajara. centro universitario del sur, méxico 2 universidad autónoma de yucatán, mexico 3 universidad de guadalajara. centro universitario de la costa sur, méxico melipona colimana ayala is endemic to the temperate forests of western mexico and may be in conservation risk due to forest exploitation. differences between the density of nests, nesting sites and spatial distribution in two places with different levels of human disturbance were established. a preserved (p) and a disturbed area (d) were identified: the forest had not been exploited for more than 18 years in the p zone, while there had been recent forest exploitation of d zone in less than two years. it was determined that nesting sites, nest density and the number of potential nest sites were predominant in the p zone. in total, 27 of 30 colonies were found on oak trees (quercus laurina) with a diameter at breast height of 183.4 ± 34.21 cm which shows a close relationship of this bee species with this type of tree. a positive correlation between the dbh of the nesting sites in relation to the trees with nests and the presence of cavities was found. the nests are distributed in the form of aggregates in p and d zones (r = 0.31 and 0.39) with a density of 0.17 haˉ¹ and 0.04 haˉ¹ colonies respectively. forestry exploitation seems to be affecting wild populations since the trees that bees use as nesting sites are destroyed in d zone. jo macías-macías, jjg quezada-euán, jm tapia-gonzalez, f contreras-escareño nesting habits of melipona colimana in higland zones of mexico424 stingless bee nests and their density, often leading to their disappearance (cannon et al., 1994; brown & albrecht, 2001; venturieri, 2002; eltz et al., 2003; samejima et al., 2004). in this regard, m. colimana might be in conservation risk since the species original habitat is a zone where there is commercial exploitation of forest resources, especially oak trees (quercus spp) and pines (pinus spp) (comision nacional forestal [conafor], 2010). the purpose of this work is to know the type of tree that m.colimana prefers to nest, and evaluate nest density, nesting sites and spatial distribution in two zones with different level of human disturbance; to infer if deforestation could affect the presence of this species. material and methods study site the observations were carried out in the halo mountain range in southern jalisco, mexico (18° 58’ 00” n, 102° 59’ 45” w, 1600 meters above sea level), in the surroundings of san isidro village. the territory of this zone is 132, 644 km², in which 79,055 km² are used for forestry. vegetation is mainly mountain mesophyll forest, made up of various useful timber-yielding species such as oak and pine trees and other species such as abies religiosa, lysoma apaculcensis, betula pendula, cornus disciflora and litzea glucesens (instituto nacional de estadistica geografia e informatica [inegi]; 2005, carranza, 2008). the weather is characterized by sub-humid temperate with summer rain (relative humidity 69%). the average temperature in the last 10 years was 69.08 °f (min. 52.23 °f and max. 79.88 °f) with an average rainfall of 930mm (min. 120 mm and max. 1230 mm) (comision nacional del agua [conagua], 2011). m. colimana is a 9.5 mm long bee, with black integument, yellow marks and orange pubescence; it is a species morphologically close to m. fasciata with the difference that m. colimana has black terga and yellow apical segments (ayala, 1999). two wild populations of m. colimana colonies were found in two contiguous areas with different degree of human perturbation. the first area was a disturbed zone (d) there had been recent forest exploitation in less than two years. in the second area, the forest had not been exploited for more than 18 years (p). nesting sites and nest density in order to find wild nests, fourteen 1000-meter-long transects were established with a distance of 200m between each other. a 100 x 100 meter quadrant was traced around every 100 meters of each transect; in this area, wild colonies were located and recorded. the geographical position of each nest and the scientific name of the tree, where the nest was located, were recorded. the total study area and the nesting density per hectare were calculated with the geographical position data and the arcview 8.3 software. in order to obtain numerical data from the oak trees, which the bees used as a nesting site, a 17.84-meter-long string-stick was placed every 100 meters along the transects. the string was spun around in circles making a circumference that covered a measuring area of 1000 m². one hectare per measured transect was covered with this method (instituto nacional de investigaciones forestales agricolas y pecuarias [inifap], 2006). the number of oak trees, the diameter at breast height (dbh), and the number of oak trees with holes were counted at each measuring station. afterwards, there was a comparison of this data in both areas p and d by using a t-test for the diameter at breast height (dbh) of the nesting sites; and a chi-square goodness-of-fit test (x²) for the number of nests, the trees with cavities (potential nest sites) and trees without cavities. the relationship between the dbh of trees with nests and the ones with cavities was evaluated with a correlation. to make a comparison with other tree types that exist in the region and the ones m. colimana prefers, a record of other tree species was made for every 100 oak trees found in each zone. the statistical software statgraphics plus® (1999) was also used for this statistical analysis. spatial distribution the nearest neighbour distance (clark & evans, 1954) was used in order to determine the distribution pattern in the nesting sites. the comparison of average distances between the closest neighbour nest that was observed (xra) and the average distances in the closest neighbour nest that was expected (xre), which would be obtained if the nests were placed randomly. clark and evan’s index is calculated as follows (krebs, 1998): r= xra/xre. if the r value >1, the spatial distribution pattern is regular or uniform, if r<1 the organisms are forming aggregations, and if r=1 the distribution is random. results the total study area was 594 hectares from which 280 were registered in the search of nests. thirty wild nests were located: 24 in the p area and six in the d area. 27 nests were found in oak trees q. laurina; the other three nests were found in other tree species: l. apaculcensis, c. disciflora and l. glucesens in the p area. the average diameter at breast height of trees with nests was 169.20 ± 42.74 in the p area and 197.16 ± 56.27 in the d area. there were statistical differences between the two zones in the number of nests (x² = 10.8; p<0.05, df= 1, 29; 0.05 = 3.84), the number of q. laurina trees (x² = 568; p<0.05, df= 1, 1286; 0.05 = 3.84), the oak trees dbh (t=35.14, df=1, 1285, p<0.05) and the number of oak trees with cavities (x² = 23.52; p<0.05, df= 1, 67; 0.05 = 3.84).the p area had the highest values in almost sociobiology 61(4): 423-427 (december, 2014) 425 all parameters, except that in the d area the number of oak trees was higher, but with a dbh lower than the p area. the number of oak trees without nests, their diameter at breast height, the number of trees with cavities and the density of nests in every zone are shown in table 1. for every 100 trees of q. laurina there is an average 8.5 of other trees in the p area. in the d area there were 2.6 other trees for every 100 q. laurina that had no cavities in their trunks. in both areas there is a relationship between the nesting sites dbh (oak trees), the nests in trees and tree holes availability in them. (ji²= 62.244, df=1, 58 p<0.01 y ji²= 252.456, df=1, 282 p<0.01). nests presented aggregated distribution in both areas (p= 0.39 y d=0.31). the number of nests and their spatial distribution can be seen in fig. 1. discussion many stingless bee species are opportunistic and take advantage of several trees’ cavities for nesting (hubbell & johnson 1977; roubik, 1989). m. colimana, should be closely related to the q. laurina trees with cavities, since it is one of the main species in this region and it is very common to have natural trunk cavities in old trees (cuevas et al., 2004). meliponini species have a natural tendency of taking advantage of the arboreal species predominant in their place of origin, such as melipona subnitida ducke, melipona asilvai moure, and melipona quadrifasciata lepeletier in brazil, which nesting in commiphora lepthophoeos, table 1. number of oak trees without nests, diameter at breast height, number of trees with cavities and density of the nests in the zone preserved (p) and disturbed (d). fig 1. the spatial distribution of melipona colimana wild nests in the study area. every o mark indicates the position of a nest in the disturbed (d) area (n=6) and the ʘ mark indicates a nest in preserved (p) area (n=24). caesalpinia pyramidalis and caryocar brasiliense trees (martins et al., 2001; antonini & martins, 2003). if there is a strong preference for a predominant tree to nesting in the distribution area, it would leave the bees in a risky situation as the absence of these tree species would directly affect bee populations. unfortunately, for m. colimana, the oldest q. laurina trees having higher biomass are the most useful ones for companies dedicated to vegetal coal production (reyes, 2012), so the activities arising from this industry may be decreasing the nesting site availability. the activity of the forest exploitation of zone d seems to be also affecting the presence of different trees to q. laurina, given that the low number of trees that are not oak trees, reflects the negative impact that has been held in the zone. it is observed that the p zone was the one with the highest number of nests, dbh and q. laurina trees with cavities that might be a consequence of having no recent forest exploitation in the zone, which has allowed the subsistence of thicker oak trees. in forests habitats stingless bees have been found making their nests mainly in trees over 60 cm in diameter (brown & albrecht 2001; antonini & martins 2003; eltz et al., 2003; fierro et al., 2012) thus, a higher dbh and quantity of oak trees with available holes might have been a factor for the p zone to have a higher number of nests of m. colimana. despite the higher number of oak trees found in d zone compared to the p zone, these are not useful for bees, since they are young trees which do not have any cavities in their structure. the number of nests found per hectare in both zones was low compared to what was reported by other authors. roubik (2006) estimates that the usual colonies of melipona and trigona quantity per hectare is from 2 to 6, while in works that were carried out in rainy and broken up forest areas, the general density of stingless bees wild nests was 8.4 and 6.7 nests per haˉ¹ (batista et al., 2001; eltz et al., 2002). as the same way, antonini & martins (2003), found a density of 3 nests per haˉ¹ of m. quadrifasciata in a study conducted in a brazilian savanna, which is a high nest density compared with the density nests found in m. colimana. although this low nest density found in m. colimana is the first report about stingless bees in a temperate climate, the results suggest that wild populations of this species might be endangered by forest exploitation. the m. colimana nests were found forming aggregations in both study zones, which coincides with the report about other stingless bees species such as scaptotrigona. pectoralis dalla torre, frieseomelitta nigra cresson, trigona. fulviventris guérin, tetragonisca angustula latreille and nannotrigona testaceicornis lepeletier (slaa, 2002; santos, 2006). since stingless bees nest on tree’s hollow spaces, its density and spatial distribution depends on the presence and distribution of the trees that are used as nesting site (batista et al, 2001; santos, 2006), in this case the nest distribution of m. colimana depended on the distribution of q. laurina trees. despite not having replicas of these observations, this information can give us sign that forest exploitation could be affecting wild nesting of m. colimana, resulting in a different letters denote statistic differences p<0.05 study zone number of oak trees diameter at breast height (cm) number of trees with cavities density of nests (haˉ¹) zone p 216 a 116.22 ± 40.91 a 54 a 0.17 a zone d 1071 b 26.24 ± 32.83 b 14 b 0.04 b jo macías-macías, jjg quezada-euán, jm tapia-gonzalez, f contreras-escareño nesting habits of melipona colimana in higland zones of mexico426 lower number of trees which bees can use as nesting sites. it has been found that the anthropogenic activity, directly or indirectly, affects density, diversity and spatial distribution of stingless bee communities (brown & albrecht, 2001, hsiang et al., 2001; moreno & cardoso 2002; samejima et al., 2004), therefore, it would be important to do another study with more replicas to get data that show us in a conclusive way that the forest exploitation of q. laurina can put in risk the presence of m. colimana nests in its original habitat, which is why it is important to emphasize its protection. acknowledgements especially to angelica f.s. this article is part of j. o. macias macias research as an agriculture and livestock science phd scholarship student in ccba-uady sponsored by conacyt-promep. thanks to bernardo soto, to the community of san isidro and gustavo alcázar oceguera for his invaluable help and knowledge about the stingless bees’ location. to the cusur research coordination (university of guadalajara) and to the apiculture students for their help in the fieldwork. references antonini, y. & martins p.r. 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(2006). stingless bees in applied pollination: practice and perspectives. apidologie 37: 293-315. doi 10.1051/apido: 2006022 statgraphics plus (1999). for windows 4.1 copyrigth©, by statistical graphics corp, all rights reserved. venturieri, c.g. (2002). exploração florestal e impacto sobre abelhas indigenas sem ferrão. in: imperatriz-fonseca vl (ed). best management practices in agricultura for sustainable use and conservation of pollinators. agreement fao sao paulo university. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.541-546sociobiology 61(4): 541-546 (december 2014) defensive repertoire of the stingless bee melipona flavolineata friese (hymenoptera: apidae) introduction the meliponini bees are a diverse group of social insects comprising over 400 species (michener, 2000; camargo & pedro, 2007). these bees are called stingless bees as a result of their loss of the sting apparatus and consequent inability to defend themselves by stinging, as commonly observed in other bees (wilson, 1971). despite the lack of sting, the meliponini bees have developed a series of defensive mechanisms, including biting and resin deposition among others, which are triggered and modulated by inter and intraspecific chemical and visual stimuli (wilson, 1971; wittmann et al., 1990; schorkopf et al., 2009). stingless bee colonies store a great amount of abstract despite the loss of the sting apparatus, meliponini (stingless) bees have not lost their ability to defend themselves. several defensive strategies have been described for the group, including biting and resin deposition. defensive behavior can be mediated by chemical communication, for example through the use of alarm pheromones. the stingless bee species melipona flavolineata friese is an important species for meliponiculture in brazil, especially in the amazon region. in order to improve the current management methods for the species, this study aimed to describe the range of defensive strategies used by the stingless bee m. flavolineata towards inter and intraspecific chemical signals known to trigger defensive responses in related species, namely the head secretions of the robber bee lestrimelitta limao (smith) and the mandibular gland extract of conspecifics m. flavolineata workers. the stimuli provoked different defensive reactions. the head secretions of the robber bee repelled returning foragers, elicited the enclosing of the nest entrance tube with batumen balls and the agglomeration of workers outside the box. in contrast, the mandibular gland extract elicited aggression towards the pheromone deposition site, transport of resin and generalised agitated flights. our results confirm the role of the mandibular gland as a source of alarm pheromone in this species and the chemical triggering of a specific defensive response to the known cleptoparasite l. limao. sociobiology an international journal on social insects tm nunes1, lg von zuben1, l costa2, gc venturieri2 article history edited by denise araujo alves, esalq, usp, brazil received 30 september 2014 initial acceptance 04 november 2014 final acceptance 04 december 2014 keywords meliponini, pheromones, mandibular gland, lestrimelitta limao corresponding author túlio m. nunes nppns, departamento de física e química, fcfrp, universidade de são paulo, sp, brazil e-mail: tulionunes@usp.br resources which have to be defended from a variety of predators and parasites, including many species of insects and also vertebrates (nogueira-neto, 1997). the diversity of defence mechanisms described for the group is vast and ranges from narrow nest entrances to chemical weapons (roubik et al., 1987; couvillon et al., 2008). the most commonly described defensive behavior is the specific biting of hairs and vulnerable body regions such as the eyes and ears (wilson, 1971; wittmann, 1985). biting behavior is usually linked to distressful sounds (wilson 1971). another characteristic defensive mechanism is the deposition of plant resins on the potential predator (greco et al., 2010). resin deposits are a common feature on different species of stingless bees’ nests and in the presence of a disturbance research article bees 1 universidade de são paulo, sp, brazil 2 empresa brasileira de pesquisa agropecuária, embrapa amazônia oriental, belém, pa, brazil tm nunes, lg von zuben, l costa, gc venturieri defensive repertoire of melipona flavolineata 542 the bees carry this material with their legs or mandibles and deposit it on the predator (sakagami, 1982). still, some species make use of caustic chemical secretions as described in the genus oxytrigona (roubik et al., 1987). among natural enemies of stingless bees are the kleptobiotic stingless bees belonging to the lestrimelitta genus. the robber bees invade other stingless bee species’ nests and pillage their reserves, carrying wax, honey, pollen and mostly larval food from recently enclosed reproductive cells (sakagami et al., 1993). the stingless bees have developed a range of defensive behaviors against the attack of lestrimelitta species (wittmann et al., 1990; nunes et al., 2008; grüter et al., 2012). some species respond nonaggressively to the attack of lestrimelitta bees by hiding at the periphery of the nest and underneath brood combs (michener, 1946; sakagami, et al., 1993). other species strongly react to the attacks, biting the legs and wings of the invasive species, depositing resin and closing the nest entrance (wittmann, 1985; nunes et al., 2008). beyond the defensive mechanisms triggered by interspecific stimuli, intraspecific chemical communication is also thought to initiate aggressive responses (cruz & lópez et al., 2007; schorkopf et al., 2009). the mandibular glands of stingless bees are regarded as a source of intraspecific communication substances eliciting alarm responses (smith & roubik 1983; cruz & lópez et al., 2007; schorkopf et al., 2009). the species analysed showed highly volatile gland contents (schorkopf et al., 2009). in the presence of its secretion, workers attacked the pheromone source, avoided the food sources and exhibited agitated behavior, which consists in vertical flights at high speed and intense buzzing sounds (smith & roubik 1983; johnson et al., 1985; cruz & lópez et al., 2007; schorkopf et al., 2009). the stingless beekeeping is a rapidly growing activity in the tropical areas of the globe (cortopassi-laurino et al., 2006; contrera et al., 2011). these bees provide a wide range of economic opportunities such as the extraction of natural products like honey and resin and also improve the production of local crops through pollination (venturieri, 2008; oliveira et al., 2012). in the brazilian amazon region, the management of the stingless bee melipona flavolineata friese is an economic alternative for small land holders (venturieri et al., 2003; magalhães & venturieri, 2010). even though its economic value has been shown, the correct management of this species requires detailed behavioural analysis. understanding the means by which the species defends itself from a wide range of predators and parasites is essential to develop this culture in scale. we thus analysed the range of defensive reaction of the stingless bee m. flavolineata, evaluating workers’ responses towards inter and intraspecific chemical signals known for triggering defensive response in related species, the head secretions of robber bees (lestrimelitta limao (smith)) and conspecific mandibular glands contents. materials and methods bees and study site the tests were conducted at “cocal do tauá”, municipality of santo antônio do tauá, pará, brazil (1°05’17.54”s 48°15’20.82”o) in august 2014, between 9:00 and 14:00h. for the tests, we used 14 colonies of m. flavolineata conditioned in wooden boxes specially designed for this species (venturieri, 2008). behavioral tests to evaluate the defensive responses of m. flavolineata workers colonies were randomly allocated to three groups: (1) l. limao head extract (2) m. flavolineata mandibular gland extract and (3) pure solvent (dichloromethane), the latter to avoid any ambiguous result due to a reaction against the solvent odor. the l. limao group consisted of an extract of macerated l. limao heads (three bee-equivalent, i.e. the amount of compound used for each colony was equivalent to the content of three workers of l. limao) in dichloromethane. in the mandibular gland group, colonies were treated with an extract of m. flavolineata dissected mandibular glands (four gland-equivalent). each treatment was applied directly at the entrance tube. following the treatment, workers behavior repertoire was recorded for 5 minutes. in order to evaluate the repellence effect of each treatment group, the number of foragers entering the nest was recorded, if no bees entered the nest during this time, we recorded the time the first forager returned. the defensive response of workers was evaluated by recording the total number of bees exiting the nest and gathering at the nest entrance around the treatment site. also, the presence or absence of the following behavioral patterns has been recorded: attacks to the extract deposition site, presence of workers carrying resin in their hind legs and overall agitation (i.e. workers increasing the flight speed, performing vertical flights and noticeable buzzing sound). after 10 minutes from the treatment, the colony was opened in order to evaluate its general state, presence of resin and batumen balls deposits. statistical analyses in order to analyse the number of workers entering the nest five minutes following the treatment and the number of workers leaving the nest and gathering at its entrance, a generalized linear model (glm) with poisson error distribution was used to assess differences among treatments, with the level of statistical significance established at α < 0.05. the number of workers entering the nest and total number of workers gathering at the box were regarded as dependent variables and treatment (with three levels: solvent control, mandibular gland and l. limao head extract) as categorical sociobiology 61(4): 541-546 (december 2014) 543 independent variable. the multiple pair-wise comparisons among treatments were made using tukey contrasts. results and discussion workers of m. flavolineata showed a clearly distinct behavioral pattern in response to the control group and treatments (the cephalic extract of the robber bee and the mandibular glands extract). workers in the control group did not present any visible change in the behavior after the solvent was deposited at the nest entrance, showing no generalized agitation and workers returning normally to the nest. the comparison between colonies treated with the cephalic extract of l. limao and the control group showed a significantly lower number of workers returning to the nest in five minutes following the treatment indicating a repellent effect of the cephalic extract (poisson glm: z = 7.50, p<0.001, n = 10; fig 1). in the colonies treated with the cephalic extract of l. limao when compared to the control group (poisson glm: z = 7.78, p<0.001, n = 10; fig 2). the crowding workers displayed intense vibration of the thorax and wings and frequent rubbing of the last pair of legs to the tip of the abdomen (fig 3). after a few minutes of this behavioral display, the workers ceased the vibration and returned to the interior of the nest. ten minutes after the start of the treatment, observations inside the colonies showed workers dragging small batumen balls (1-5mm) to the entrance tube. batumen balls deposits were observed in all the nests and usually took place on the side of the internal entrance tube aperture and were associated with ventilation openings (fig 4). the deposition of batumen balls at the nest entrance as a reaction to l. limao attacks have been observed many times during natural raids (venturieri, pers. observation). the entrance blockage using batumen balls has also been described for the species melipona paraensis ducke and this artefact was found inside of the nest of the tropical species melipona seminigra merrilae cockerell and melipona crinita moure & kerr (portugal-araujo, 1978) showing that this defensive strategy is shared by other species of this genus. the colonies treated with the mandibular gland extract showed a different response from those in the control group or treated with the cephalic extract of the robber bee. the number of bees returning to the nest during five minutes following the treatment decreased in this group when compared to the control group (poisson glm: z = 3.34, p<0.001, n = 9; fig 1). this reduction however was smaller than the one observed in the colonies treated with the cephalic extract of the robber bee (poisson glm: z = 4.40, p<0.001, n = 9; fig 1). the workers returning to the hive were not repelled by the pheromone source but instead became agitated performing fig. 1 – box-and-whisker plots of the number of melipona flavolineata workers entering the nest during five minutes following the treatment. box plots show the median, 25&75% percentiles. whiskers show all data excluding outliers (dots). the duration of the repellent effect varied across the colonies and the maximum latency observed was 8 minutes until the first worker entered the nest. following the repellent effect, the workers from inside the nest initiated an intense vibration and a conspicuous buzzing sound. next, the workers started to leave the nest in great numbers and to agglomerate in the box surrounding its entrance (i.e. the site of the extract deposition). the maximum number of workers observed crowding at the nest entrance was 223 in one colony treated with the cephalic extract of the robber bee. there was a significantly higher number of workers crowding at the nest fig. 2 – box-and-whisker plots of the number of melipona flavolineata workers gathering outside the nest box during five minutes following the treatment. box plots show the median, 25&75% percentiles. whiskers show all data excluding outliers (dots). tm nunes, lg von zuben, l costa, gc venturieri defensive repertoire of melipona flavolineata 544 quick angular flights and an intense buzzing sound. following the treatment with the mandibular gland extract, several workers left the nest and stayed outside the box, gathering around the nest entrance. the maximum number of workers agglomerated outside the box near its entrance was higher when compared to the control group (poisson glm: z = 4.55, p<0.001, n = 9; fig 2) but significantly lower in comparison with the robber bee cephalic extract group (poisson glm: z = -13.95, p<0.001, n = 9; fig 2). generalized agitation with workers flying quickly in angular flights and noticeable buzzing sounds occurred after both treatments but not in the control (table 1). some behavioural displays could only be observed in colonies treated with the mandibular gland extract, for example workers leaving the hive carrying resin in their corbicula and similar behaviour happening in neighbouring colonies (table 1, fig 5). similarly, attacks to the pheromone site only were observed in the mandibular gland extract group (table 1). the results described here demonstrate the repellent effect of m. flavolineata workers in the presence of cephalic secretions of l. limao. the avoidance of the nest under attack fig 3 – workers of melipona flavolineata gathering outside the nest box surrounding the entrance after the deposition of l. limao cephalic extract. fig 4 – batumen balls deposit inside a melipona flavolineata nest. table 1 – percentage of melipona flavolineata colonies responding to the different pheromone treatments. ce, cephalic extracts of the robber bee l. limao. gm, mandibular gland extracts of conspecifics m. flavolineata workers. fig 5 – workers of melipona flavolineata carrying resin in their corbicula after the treatment with mandibular gland extract of by returning foragers has been reported for different species of stingless bees (michener 1946; kerr 1951; sakagami & laroca, 1963). the tests demonstrated that the behavioral responses are mediated by interspecific chemical signals which are secreted by the cephalic glands of the robber bee. the cephalic extract contains chemical secretions of the mandibular glands and the labial glands. the composition of the two glands differs substantially and the source of the repellent effect remains to be investigated (von zuben, 2012). the volatility of the robber bee cephalic secretions together with the gradual shift from avoidance of the secretion source to a defensive response (i.e. workers agitated around the entrance) suggests a dose-dependent reaction towards the robber bee compounds. three bee-equivalent extracts had a repellent effect. the highly volatile aspect of the compounds results in a quick decrease of its quantity at the treatment site. a few minutes following the treatment with the cephalic extract, the reaction observed changed from hiding and avoiding the source of the stimuli to generalized agitation, noticeable buzzing and workers leaving the colony and gathering at the treatment site. this shift in the behavior suggests that workers of m. flavolineata can use different defence strategies against control treatment behaviour ce mg generalised agitation 80 75 attack to pheromone site 75 workers leaving the nest carrying resin 100 workers from neighbouring colonies leaving the nest carrying resin 100 resin deposits inside of the nest 100 100 100 batumen ball deposits inside of the nest 100 100 100 sociobiology 61(4): 541-546 (december 2014) 545 l. limao attacks in response to varying concentration of the secretions. the concentration of the cephalic extract at the moment it was applied mimics the presence of a substantial number of robber bee workers in the entrance tube which is avoided by the hosts. smaller amount of this secretion reflects the presence of a few individuals and trigger more aggressive reaction from the hosts. the host species could stop an attack with relatively few casualties in the presence of a small group of the robber bees but not in the presence of a large group. avoiding a battle in the presence of a large number of invaders results in the loss of storage resources but it prevents a significant worker loss, which are necessary for the hive re-construction after the attack. the mixed defensive strategy to avoid large groups of invaders and to attack small groups might be associated with a better cost-benefit for this specie than the strategy to always fight observed in some other species (e.g. tetragonisca angustula (latreille)). the tests with co-specific mandibular gland secretions suggest its use as an alarm pheromone for this species. attacks of the pheromone source, agitated angular flights and workers leaving the nest with resin on the hind legs shows the use of these glandular compounds in triggering defensive responses. the results for this species corroborate the idea of mandibular gland as the source of alarm pheromones in stingless bees (smith & roubik, 1983; cruz & lópez et al., 2007; schorkopf et al., 2009). there were clear differences between the responses towards inter and intraspecific chemical secretions. while the interspecific signals (i.e. head extract of the robber bee) provoked a repellent effect, the intraspecific pheromone (mandibular gland extract) elicited generalised agitation of foragers arriving in the nest. moreover, there were specific responses in the intraspecific mandibular gland extract group such as workers carrying resin that could not be observed in the robber bee extract group. the differences described support the idea that workers of m. flavolineata do not use alarm pheromones from mandibular gland during the attack of l. limao, but instead react directly towards the heterospecific signals. acknowledgments the authors are thankful for the financial support from bionorte (cnpq/fapespa) provided to gcv and fapesp (tmn proc. 2011/22991-3). references camargo, j.m.f. & pedro, s.r.m. 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(1990). robber bees (lestrimelitta limao) and their host chemical and visual cues in nest defense by trigona (tetragonisca) angustula (apidae: meliponinae). j. chem. ecol. 16: 631-641. doi: 10.1007/bf01021793. doi: 10.13102/sociobiology.v67i1.4727sociobiology 67(1): 26-32 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction herbivory is a biotic interaction between plants and animals that has a variety of consequences for plants, from partial leaf loss to the death of whole organisms (herrera & pellmyr, 2002). herbivorous insects have diverse relationships with plants, spending most of their lives or whole lifecycles on abstract to reduce herbivory, plants bearing extrafloral nectaries interact with ants and attract them by providing food. as plant bodyguards, ants respond to the resource provision and, using their antennae, detect chemical messages from the host plants that help them to locate herbivores. ants can also use their vision to explore the environment; however, information is lacking on how interactions between visual signs and the availability of extrafloral nectar affect ant aggressiveness near resources. we addressed the following question in this study: does the ants’ ability to visualize potential herbivores enhance their aggression under a constant provision of a high-quality food source? using an experimental approach within the semiarid intertropical region of tehuacan-cuicatlán (mexico), we manipulated the availability of food sources by constantly offering artificial nectaries on the shrub prosopis laevigata (fabaceae). over two time periods (day and night), we tested how the presence of a high-quality food source affected ant aggressiveness to herbivores. therefore, we offered dummy caterpillars and counted the number of marks left by enemy attacks. overall the attack rate was extremely high: 84.25% of the dummy caterpillars were injured. ants were responsible for 86.22% of the marks left by enemies, and their aggression increased during the day, especially towards caterpillars in trees with high-quality food sources. during the night, ants probably rely mostly on their antennae to detect potential herbivores; therefore, their ability to detect dummy caterpillars was greater during the day. we show that, besides nectar quality and availability, visualizing herbivores may enhance ant aggressiveness. sociobiology an international journal on social insects jcc pena 1,2, p luna3, f aoki-gonçalves2, mfc jacobo4, tm patiño4, ks morales4, mv vázquez4, jh gárcia-chavez4, w dáttilo3 article history edited by gilberto m. m. santos, uefs, brazil received 17 september 2019 initial acceptance 19 october 2019 final acceptance 11 november 2019 publication date 18 april 2020 keywords ant-plant-herbivore interactions; artificial nectaries; semiarid environments; dummy caterpillars; multitrophic interactions; enemy free space. corresponding author wesley dáttilo red de ecoetología instituto de ecología a.c. antigua carretera a coatepec 351, el haya xalapa, veracruz, cp 91070, mexico. e-mail: wdattilo@hotmail.com wesley.dattilo@inecol.mx one plant individual and using a variety of strategies to acquire nutrients (strauss & zangerl, 2002). over the course of their evolution, plants have developed strategies to avoid or reduce damage by herbivores (del-claro et al., 2016), in instance the association between ants and extrafloral nectaries, which are nectar-producing glands located in plant structures, such as leaves, fruits, and branches, not related to pollination (heil, 2015). 1 spatial ecology and conservation lab (leec), department of ecology, instituto de biociências, universidade estadual paulista, rio claro-sp, brazil 2 red de ambiente y sustentabilidad,instituto de ecología, a.c., xalapa, mexico 3 red de ecoetología, instituto de ecología a.c., xalapa, mexico 4 facultad de ciencias biológicas, benemérita universidad autónoma de puebla, puebla, mexico research article ants i can see you: temporal variation in ant aggressiveness towards herbivores under continuous provision of highor low-quality food sources sociobiology 67(1): 26-32 (march, 2020) 27 extrafloral nectar is rich in carbohydrates, amino acids, lipids, and other organic components, and it attracts a great variety of visitors (del-claro et al., 2016; lange et al., 2017). by providing extrafloral nectar, plants feed ants in exchange for protection against potential herbivores (koptur, 1984; campos & camacho, 2014; dáttilo et al., 2017; lange et al., 2017). however, this relationship is subject to environmental constraints (e.g. temperature and humidity) and the specific characteristics of ants, plants and their natural enemies (e.g. period of activity of ants and nectar quality) (gottsberger et al., 1984; falcão et al., 2014;anjos et al., 2017;lange et al., 2017). the quality and quantity of resources provided by extrafloral nectaries differ among plant species and individuals and are influenced by factors such as the time of day, nutritional input and changes in the environment, e.g. wind temperature and relative humidity (gottsberger et al., 1984; rico-gray & oliveira, 2007; lange et al., 2017). for instance, extrafloral nectaries of the shrub prosopis laevigata (fabaceae) are more productive at night (when the abundance of herbivores is higher), thus increasing nearby ant activity (dáttilo et al., 2015). moreover, the presence of extrafloral nectaries affects the composition and frequency with which ant species forage on plants (dáttilo et al., 2014; anjos et al., 2017). it has also been observed that the activity and quality of extrafloral nectaries vary with ant activity and, therefore, with the hour of the day (lange et al., 2017). plants can also increase nectar production near leaves attacked by herbivorous insects, which leads to an increase in the local recruitment of ants (ness, 2003). in this way, the variation in extrafloral nectaries’ productivity and the quality of the resource provided affects ant activity, aggressiveness and, consequently, performance as plant defenders (anjos et al., 2017; fagundes et al., 2017; lange et al., 2017; flores-flores et al., 2018). ants detect chemical signals, such as pheromone and non-pheromone compounds, using their antennae when exploring the environment, to locate their nest or to locate a food source (knaden & graham, 2015). when interacting with plants, ants are influenced by volatile and non-volatile compounds in the nectar produced by extrafloral nectaries or produced by plants under attack from herbivorous insects (herbivore-induced plant volatiles) (nelson et al., 2019). however, ants also use visual stimuli when foraging (knaden & graham, 2015) and to identify their position in relation to their nest, including landmarks, panoramic characteristics and other visual cues (graham & cheng, 2009; buehlmann et al., 2018; fernandes et al., 2018; freas et al., 2018).the ants’ visual system is related to their habit; nocturnal species have a greater number of photoreceptors (greiner et al., 2007), which capture more light to the detriment of visual accuracy (land, 1997; yilmaz et al., 2014), whereas diurnal ants benefit from higher optic resolution, as light is not limited (yilmaz et al., 2014). in fact, it has already been demonstrated that ant species can identify and attack potential herbivores, differentiating caterpillar models and dead caterpillars from cubes made of plasticine (leles et al., 2017). although it is known that visual and, mainly, chemical signals assist ants when foraging and exploring the environment (knaden & graham, 2015), there is still a lack of information on whether, and how, the interactions between chemical (e.g. detection of food with antennae) and visual (e.g. visualization of a herbivorous insect) stimuli influence ants’ performance in protecting plants against herbivores. in semiarid regions, extrafloral nectaries tend to be more active at night, when environmental conditions are more suitable for herbivores to forage and plants require more protection (fitzpatrick et al., 2014; dáttilo et al., 2015). however, ants are constantly foraging on plants during the day, despite the presence of extrafloral nectaries, since microclimatic characteristics are more suitable above than on the ground (fitzpatrick et al., 2014; luna et al., 2016). higher temperatures also affect nectar production – extrafloral nectaries are usually less active during the day when plants suffer from water stress, which may cause loss of nectar due to evaporation (falcão et al., 2014; dáttilo et al., 2015). during the dry season, nutrients, and especially water, are limiting factors for both ants and plants (rico-gray et al., 2006). therefore, under these limiting conditions, plants alter their nectar provisioning; they can increase the nectar quality, enhancing the ants’ protection when the cost of losing tissue is higher (pringle et al., 2013; lange et al., 2017) or reduce their investment in indirect defences – nectar provisioning – and increase investment in direct defences, such as trichomes (yamawo et al., 2012; calixto et al., 2015). either way, ants are subject to the plants’ responses to environmental conditions and nectar production; therefore, one may expect ant aggression towards potential herbivores located close to sources of food, such as extrafloral nectaries, to increase in drier environments and/or seasons. in the semiarid tehuacan-cuicatlán valley (mexico), it has been shown that there is a turnover of ant species on the same individual of the shrub p. laevigata between day and night periods; therefore, each species is associated with a different quality and amount of resource, since p. laevigata’s extrafloral nectaries are mostly active at night (dáttilo et al., 2015). in semiarid regions, nectaries and herbivores are more active at night; therefore, ant species foraging during this period are more effective plant defenders (flores-flores et al., 2018). however, considering that ants can also visually detect potential herbivores (knaden & graham, 2015; leles et al., 2017) and the differences in visual accuracy between nocturnal and diurnal species (yilmaz et al., 2014), how would the constant provision of high-quality food sources over day-night periods affect ant aggressiveness? would the aggressiveness of ants be enhanced by the interaction between chemical (detection of food with antennae) and visual (visualization of a potential herbivore during the day) stimuli? the aim of our study was to evaluate the effects of a constant source of food during day-night periods on jcc pena et al. – food source quality influences on ant aggressiveness 28 ant aggressiveness towards potential herbivores (dummy caterpillars) in a population of p. laevigata. we assessed (1) the difference in the frequency of attacks by ants on dummy caterpillars between shrubs with constant sources of highand low-quality food and (2) the difference in the frequency of attacks between day and night. we predicted that, since the abundance of ants and their aggression increases in plants with higher quality nectar (anjos et al., 2017; flores-flores et al., 2018) and during the day ants can visually detect potential herbivores (yilmaz et al., 2014; leles et al., 2017), we hypothesised we would observe the highest frequency of attacks on dummy caterpillars in trees with a higher quality food source during the day. material and methods study area the study was developed within the limits of the helia bravo hollis botanic garden (97w 27’ 29”, 18n 19’ 55”), located in the municipality of zapotitlán salinas (puebla, mexico), part of the biosphere reserve of tehuacán-cuicatlán (fig 1a). the area is located within the mexican xerophytic region with pronounced seasonality, with a long dry season between november and may and a rainy season between june and october. the mean annual temperature is 18–22°c and mean total annual precipitation is 400mm. experimental design the study was conducted in april 2019 in an area dominated by the thorny shrub prosopis laevigata (fabaceae) (locally known as mezquitera) (fig 1a). we randomly selected 40 trees separated by at least eight meters to avoid the same ant colonies foraging in more than one sampled plant. in each shrub, we fixed 10 artificial nectaries, represented by 1.5 ml microtubes, on ten different branches approximately 1.60 m above the ground and separated from each other by at least 30 cm. p. laevigata individuals were divided into two treatment groups in a pair wise design. twenty shrubs received artificial nectaries containing a high-quality food source: a solution of water and honeybee nectar (similar to flores-flores et al., 2018) with 30% sugar, measured in brix degrees. the artificial nectaries of the remaining 20 shrubs were filled with water, hereafter, a low-quality food source. the use of dummy caterpillars has the potential to provide insights into the influence of a constant provision of resources on ants’ responses towards potential herbivores. in general, models made of plasticine are distributed over the branches or leaves to resemble lepidoptera larvae feeding on plants (low et al., 2014; roslin et al., 2017). when trying to feed on or attack the dummy caterpillars, different animal groups leave distinct marks, allowing their identity to a coarse, but informative, taxonomic level (e.g. birds, reptiles or chewing insects) (low et al., 2014) (fig 1b). these models have already provided useful information about the effects of plant and vegetation structure (leles et al., 2017; frey et al., 2018) and caterpillar characteristics (hossie & sherratt, 2012) on the relationships between caterpillars and their natural enemies. ants have been shown to be responsible for most of the attacks on dummy caterpillars (roslin et al., 2017), evidence that caterpillar models are efficient tools to assess the ants’ response towards herbivorous arthropods (leles et al., 2017). fig 1. a. study area within the helia bravo hollis botanic garden (zapotitlán salinas, puebla, mexico), highlighting the area dominated by the thorny shrubprosopis laevigata (fabaceae). b. dummy caterpillar with a mark left by an ant species. c. lepidoptera larvae from the geometridae family observed in the study area. sociobiology 67(1): 26-32 (march, 2020) 29 to evaluate the effects of food resource quality on ant aggressiveness towards potential herbivores, we fixed 10 dummy caterpillars (totaling 400 caterpillars) made of non-toxic green plasticine (dixon comercializadora s.a., tultitlan, mexico) to each shrub (fig 1b). we modeled the caterpillars by pressing the plasticine against a metal lemon squeezer, then cutting it while measuring with a scale ruler to ensure that all caterpillars were the same length and circumference (3.5 x 0.5 mm, respectively). caterpillars resembled lepidoptera larvae from the geometridae family (fig 1c) and were fixed in a looping position (as roslin et al., 2017) using instant glue (kola loka®, dupont, wilmington, usa) on the same branches as and 10 cm away from artificial nectaries. caterpillars were affixed at 7 am on the first day, and we performed four visits after every 12 hours, at 7 pm (to monitor the attacks made by natural enemies during the day) and 7 am (to monitor night-time attacks), totaling 48 hours of the experiment. during each visit, we renewed the contents of the artificial nectaries and counted the number of marks left on each dummy caterpillar by potential natural enemies (hereafter, frequency of attacks). we identified them as ants or other organisms using guidelines and available literature (low et al., 2014; roslin et al., 2017). since we wanted to measure differences in the ant’s aggressiveness towards potential invaders between day and night, the caterpillars were not retrieved after being attacked for the first time so we could obtain a cumulative attack rate per caterpillar and per treatment. data analysis we compared the frequency of attacks by ants on dummy caterpillars during the day and night in shrubs with lowand high-quality food sources using a generalized linear mixed-effects models with the r software (r core team, 2018). we considered the frequency of attacks by ants in each treatment as dependent variables and the periods of the day (day and night) and food source quality treatments (each shrub represented a random block)as fixed factors. as we counted the number of marks on each caterpillar, we used a poisson error distribution with a logarithmic transformation. furthermore, we measured the explained deviance using wald χ2 test. results we observed marks left by natural enemies in 337 (84.25%) dummy caterpillars at least once during the whole experiment (two caterpillars could not be retrieved). considering the cumulative attack rate, we observed 624 marks left by natural enemies (mean ± sd = 15.60 ± 3.88 marks per shrub),and ants were responsible for 538 (13.45 ± 4.90 marks per shrub) or 86.22% (fig 2). the remaining marks (86, 13.78%) were left by other animal groups, such as birds, reptiles, mammals and other arthropods (e.g. parasitic wasps) (fig 2). considering treatments, we did not observe differences between the frequency of attacks by ants on caterpillars in shrubs with high(264 marks) and low-quality resources (274 marks) (χ2 = 0.14, df = 1, p = 0.7) (table 1). however, the frequency of attacks by ants on caterpillars was higher during the day (χ2 = 25.56, df = 3, p< 0.0001) (table 1, fig 3). in shrubs with low-quality resources, we observed a high number of attack marks during the first 24 hours, followed by a clear decrease (fig 3). when assessing the interaction between fixed factors, we observed that the frequency of attacks by ants against dummy caterpillars was higher during the day in trees with high-quality nectar (χ2 = 12.01, df =1, p = 0.007) (fig 3). fig 2. proportion of attack marks left by ants and other natural enemies on dummy caterpillars near artificial nectaries fixed on the branches of the shrub prosopis laevigata (fabaceae) during day and night and with different food source quality treatments over a 48-hour period. treatment frequency of attacks by ants during the day frequency of attacks by ants during the night total high-quality food source 164 100 264 low-quality food source 142 132 274 table 1. frequency of attacks by ants on dummy caterpillars near artificial nectaries fixed on the branches of the shrub prosopis laevigata during day and night and under different food source quality treatments over a 48-hourperiod. discussion we found that the interactions between detecting a food source and visual signs enhanced ants’ aggressiveness jcc pena et al. – food source quality influences on ant aggressiveness 30 towards potential invaders. our data shows that ants foraging during the day, which are attracted by artificial nectaries with a high-quality food source, might be able to visually detect dummy caterpillars. as expected, the ant’s aggressiveness near these resources was higher compared with ants near the artificial nectaries filled with water. even with resources ad libidum, ants continuously attacked potential herbivores when they were able to visually detect them. however, nocturnal ants probably rely more on chemical signals from their host plant and the invader, picked up using their antennae, to detect herbivores; therefore, they were attracted by our artificial nectaries but did not efficiently detect the dummy caterpillars. in arid environments, resources are transient, and territorial and competitive animals, such as ants, will protect them against invaders (rico-gray, 1989). therefore, not only were the artificial nectaries containing a high-quality food source considered valuable by the ants, but also those with a low-quality food source, as they were a source of water; in the tehuacán cuicatlán valley, the dry season started eight months before our experiment. furthermore, a turnover of dominant ant species over day-night periods has been demonstrated in p. leavigata; the dominant ant species during the day was camponotus rubritorax, while c. atriceps was dominant at night (dáttilo et al., 2015). at the beginning of our experiment, each ant species was attracted by the new source of water in the trees with a low-quality food source, which probably explains the high number of marks during the first 24 hours. however, the high-quality food source contained honeybee nectar (i.e. fructose), rich in carbohydrates and amino acids as well as water, making it a better food resource and increasing its attractiveness to ants and, thus, the aggressiveness of ants against potential invaders (grover et al., 2007; anjos et al., 2017; flores-flores et al., 2018). carbohydrate scarcity has been shown to reduce ant activity and aggressiveness, and access to carbohydrates and protein-rich resources contribute to their competitive behavior (grover et al., 2007; passos & leal, 2019). therefore, the ants initially defended both resources, but only the high-quality food source maintained a higher level of aggression over the entire 48-hour period. ant species have different morphological adaptations to the availability of light – diurnal species have higher optical accuracy, whereas the eyes of nocturnal species capture light more efficiently (land, 1997; greiner et al., 2007; yilmaz et al., 2014). the 60% difference in the attack rate between day and night on the shrubs with high-quality nectar is indicative of the ability of c. rubritorax to visually detect the dummy caterpillars. it is likely c. atriceps detected our caterpillars using their antennae when exploring the environment near the artificial nectaries at night, not having enough visual accuracy to efficiently detect the dummy caterpillars by sight. possibly, caterpillars were attacked more on the first night because they were first detected then as an unknown strange object. by the following night, they were ignored by the ants, which is evidentfrom the reduced frequency of attacks in the shrubs with both highand low-quality food sources. therefore, we have demonstrated that, when the ants were exploiting the high-quality food sources, their ability to visually detect potential herbivores enhanced their aggressiveness, since the frequency of attacks only remained constantly high during the day. more studies are required to confirm if there are morphological and physiological differences in the eyes of the two species since evidence has shown that c. rubrithorax is adapted to activity in the day and c. atriceps at night (floresflores et al., 2018). the lower frequency of attacks at night in trees with a high-quality food source could also be related to the activity of p. laevigata’s extrafloral nectaries. c. atriceps, which forages mostly at night, has several small sources of nectar available, as its foraging time coincides with the activity of p. laevigata’s extrafloral nectaries (dáttilo et al., 2015). consequently, during the day, ants such as c. rubritorax do not have the same resources from p. laevigata available and probably concentrated more on our artificial nectaries. therefore, higher numbers of ants near the artificial nectaries and their ability to visually detect the dummy caterpillars enhanced c. rubritorax’s aggressiveness against potential herbivores. we observed an extremely high proportion of attacked caterpillars during the whole experiment: 84.25% in only two days. studies conducted in different environments, such as tropical savanna, urban area and tropical forest, that left models exposed for over four days observed attack marks on only 11.7, 16.2 and 20.9% of caterpillars, respectively (moreno & ferro, 2012; leles et al., 2017; frey et al., 2018). therefore, our study demonstrated that caterpillar models can be used efficiently to explore the relationships between ants, the resources provided by plant species and herbivorous insects. this method can be applied when other resources are available, for example, to assess the relationship between ants fig 3. variation on the frequency of attacks by ants on each dummy caterpillar (mean 3.36 ± 2.49 marks per caterpillar on each shrub) near artificial nectaries fixed on the branches of 40 individuals of the shrub prosopis laevigata (fabaceae) during day and night and under different food source quality treatments over a 48-hourperiod. sociobiology 67(1): 26-32 (march, 2020) 31 and potential invaders when plants are flowering or fructifying. more studies are needed to explore if this higher predation rate can also be observed in other systems or environments. we have demonstrated that the interaction between detecting a high-quality food source and visual signs enhances ant aggressiveness towards potential herbivores. in shrubs with high-quality artificial nectaries, the frequency of attacks remained higher during the day, indicating that ants invest more energy defending a nutritive food source than water, and their ability to visualize a potential herbivore may enhance their effectiveness as plant defenders. during the night, ants probably detected our caterpillars using their antennae and had grown accustomed to the strange object by the following night. therefore, not only resource availability and quality but also visual signs, modulate ant-plant-herbivore interactions. in addition, both visual cues and chemical signals influence the ants’ behaviour. acknowledgements we thank the helia bravo hollis botanic garden administration. jc castro pena was supported by são paulo research foundation (fapesp) (grants: 2018/00107-3, 2018/ 22215-2). references anjos, d.v., caserio, b., rezende, f.t., ribeiro, s.p., delclaro, k., fagundes, r. 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(2014). eye structure, activity rhythms, and visually-driven behavior are tuned to visual niche in ants. frontiers in behavioral neuroscience, 8: 1-9. doi: 10.3389/fnbeh.2014.00205 doi: 10.13102/sociobiology.v61i4.554-559sociobiology 61(4): 554-559 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 rescue of stingless bee (hymenoptera: apidae: meliponini) nests: an important form of mitigating impacts caused by deforestation introduction pollinators, and especially bees, are responsible for the production of fruits and seed crops that are essential to guarantee human food resources, as well as in the maintenance of worldwide economy (tepedino, 1979; slaa et al., 2006; klein et al., 2007). unfortunately, pollinator diversity and abundance has decreased worldwide, due to deforestation, habitat fragmentation and the use of pesticides in agriculture (garibaldi et al., 2011; gonzález-varo et al., 2013). in natural habitats, the lack of pollinators detrimentally affects wild plant reproducibility, thereby causing local extinction, and adversely affecting other dependent species (allen-wandell et al., 1998; biesmeijer et al., 2006; steffan-dewenter et al., 2006; ramírez et al., 2011). stingless bees (meliponini) have been identified as important pollinators in both natural environments and crops (imperatriz-fonseca et al., 2006; slaa et al., 2006). hence their preservation is of the utmost importance for the sustenance abstract as stingless bees are important pollinators of wild and cultivated plants, their preservation is of vital importance to sustain the global ecosystem and to safeguard human food resources. the construction of large dams for the production of energy involves the removal of wide extents of riparian vegetation, where many species of bees, especially meliponini, build their nests. the rescue of bee colonies is essential, not only in the conservation of pollinators, but also in the use of these colonies in meliponiculture and biological research. the aim of this work was to describe the procedures used in the rescue of stingless bee colonies at the time of deforestation, prior to initiating construction of a large dam in the madeira river (amazon basin, brazil). with simple equipment and widely known methods of meliponiculture 287 stingless bee nests were rescued, of which 15.7% were reallocated and 26.5% perished. the remaining 57.8% recovered well and were donated to local stingless beekeepers. the rescue of meliponini nests during deforestation, besides resulting in the conservation of numerous colonies of various species, also contributes to the generation of environmental and social benefits. sociobiology an international journal on social insects l costa¹, rm franco¹, lf guimarães¹, a vollet-neto¹,², fr silva¹,², gd cordeiro¹,² article history edited by denise araujo alves, esalq-usp, brazil received 01 october 2014 initial acceptance 10 november 2014 final acceptance 10 december 2014 keywords fauna rescue, pollinators, dam, amazon basin, meliponiculture. corresponding author guaraci duran cordeiro universidade de são paulo departamento de biologia, ffclrp av. bandeirantes, 3900, ribeirão preto 14040-901, são paulo, brazil e-mail: guaradc@usp.br of ecosystems and food resources worldwide. many species of meliponini are especially vulnerable to environmental degradation. in melipona illiger bees, for example, inbreeding can lead to decline or even the extinction of native bee populations through the presence of diploid males (kerr, 1987; carvalho, 2001; alves et al., 2011; francini et al., 2012). meliponini bees inhabit tropical and subtropical regions of the world, their diversity and abundance reaching the highest expression in the amazon basin (michener, 2007; camargo & pedro, 2012). thus, this region has become an essential area for research and conservation. in brazil, as in other tropical countries worldwide, economic growth has given rise to the construction of large infrastructure projects, especially power plants. the construction of large dams for the production of energy involves the removal of wide extents of riparian vegetation prior to the formation of reservoirs, thereby causing damage to the entire aquatic and riparian environment (junk & mello, 1990). riparian research article bees 1 arcadis logos, divisão de meio ambiente, são paulo, brazil 2 universidade de são paulo, são paulo, brazil sociobiology 61(4): 554-559 (december 2014) 555 environments are especially important ecosystems for wildlife, where many bee species, especially meliponini, find the appropriate conditions for nesting (roubik, 1989, 2006; camargo, 1994). in this context, the inclusion of stingless bee rescue programs in infrastructure projects, such as power plant construction, is an effective way of mitigating environmental damage caused by deforestation. furthermore, the rescue of stingless bees nests, besides providing an unusual opportunity of data sampling for research in various aspects of meliponini biology, can be a source of colonies appropriate for meliponiculture, an important activity in sustainable land use and environmental education (kerr et al., 1996; souza et al., 2012). the aim of this study was to describe plausible procedures for rescuing stingless bee nests, and to discuss the possibilities for improvements in deforestation activities. data was also presented on the various bee-species found during deforestation, prior to building the santo antonio hydroelectric power plant on the margin of the madeira river, rondonia state, brazil. material and methods study area and equipment used the rescue of stingless bee nests was undertaken within the south-western amazon basin in the state of rondonia, northern brazil, on the left margin of the madeira river (8° 47′ 29.49″ s and 63° 58′ 58.5″ w). this area was undergoing intense deforestation prior to construction of the santo antonio hydroelectric power plant. the area consists of 1,620ha of preserved riparian forest. the regional climate is equatorial af (köppen, 1948), with an average annual rainfall of 2,300mm, and an average annual temperature of 26°c. the intense dry season lasts from may to august. rescue was carried out between august 2010 and october 2011. the rescue teams consisted of a specialist in stingless beekeeping (meliponiculture) and two field assistants with experience of working in forest environments. one of the field assistants latter was a chainsaw operator. all the members used personal protective equipment for fauna rescue. the tools used were those traditionally employed in meliponiculture for handling colonies (nogueira-neto, 1997) (table 1). beekeeper suits were also used for protection against defensive species. a four-wheel-drive pickup was the means of access to areas of deforestation and the transportation of rescued colonies. search and rescue of nests the search lasted eight hours a day in four distinct situations: coincident with deforestation (forefront) during cutting; lugging to storage; log stacking; and the final stage, loading for transportation out. in all of the situations, active search went ahead. binoculars were used in the search for nests, while simultaneously everybody was on the look out for bees among logs and branches. on coming across a nest, the surrounding area was taped off. gps data of the located colonies, photos and samples of worker bees (n~10) in alcohol 96%, were collected for species identification. after localizing and signalizing the colonies, in accordance with nesting biology, a decision was taken as to whether to transfer to a beehive or to leave wherever the colony had been originally found, in the original tree trunk or branch, in a termite nest, or an external nest. vertical modular hives, adapted from venturieri (2004) and carvalho-zilse et al. (2005) were used. these came in two sizes: inner space 12x12x7 cm, for small colonies, and inner space 20 x 20 x 7cm, for larger ones. cube-shaped boxes, inner space 40 x 40 x 40cm, were used for species in which nest architecture was not adjustable to vertical boxes. table 1. list of basic material for one stingless bees rescue team (three people). basic tool for the rescue of meliponini bees description quantity chainsaw 1 bucksaw 1 hatchet 1 metal wedges 2 crowbar 1 sledgehammer 1 stone chisel 1 machete 1 knife 1 painting spatula 1 3” paintbrush 1 syringe >20ml material of continuous use plastic bottles 500ml material of continuous use plastic bag >1l material of continuous use plastic tray 2 insect aspirator 1 samples containers material of continuous use alcohol 96% material of continuous use 5l bottle with water 2 beekeeper suit 3 wood stapler 1 metal net of fine mesh material of continuous use strong scissors 1 stingless bees’ hives material of continuous use striped warning tape material of continuous use adhesive tape material of continuous use rope >10 m binoculars 1 camera 1 gps 1 l costa, rm franco, lf guimarães, a vollet-neto, fr silva, gd cordeiro – rescue of stingless bee nests to mitigate deforestation impacts556 if the decision was to do the transference to a beehive, the trunk or branch was opened with a chain saw, according to instructions by nogueira-neto (1997) and coletto-silva (2005), but with adaptations. the transference of colonies associated with termitaria and external nests was with stone chisels, hammers, matches and knives. pollen and honey pots were not transferred to beehives, so as to avoid parasites. only the light colored, mature brood combs (pupal stage and the last larval stage) and cerumen were transferred. food storage pots were placed in plastic bags or boxes and stored in a refrigerator. pollen and honey were used for feeding the colonies later on. brood combs containing larval food were discarded. after an interval of approximately 24 hours, usually on the following day, the colonies that had been transferred to beehives were sealed in, by closing the entrance, and then transported to our field base, which was 3 to 32km from the rescue areas, for a period of observation and care. this was done at the end of the day, in order to capture the maximum number of forager workers in transit. during the observation and care period (c. one month), the colonies were nourished with sugar syrup (or their own honey) and their own pollen. internal and external feeders were used for sugar syrup (kerr et al., 1996; nogueira-neto, 1997), whereas only internal ones were for pollen. cerumen, i.e., a mixture of bee wax and resin used for building nest structures, was collected, washed and returned to the nests for reutilization by the workers. vinegar traps were employed against phoridae fly infestation (see nogueira-neto (1997) for details of parasite control methods). in the case of colonies to be left within their original log, the nest size was first estimated according to the species in question, whereupon the log was trimmed at both ends with a chainsaw. afterwards, these were sealed by way of a thin metal mesh, to so avoid the entrance of phoridae flies and the exit of bees. in cases of species associated with termites or those with external nests, and depending on the possibility, the entire structure was detached from the tree. these colonies were transported to our field base or re-allocated in the nearby forest at a level higher than the final water level of the reservoir. the decision for the right procedure depended on the biology of the species found, or in another words, nesting habits, behavior, chances of survival in a bee hive, and the size of the trunk or external nest. the colonies transported to the field base after the recuperation period, were donated to those local, experienced stingless beekeepers who presented the necessary conditions for giving special care. bee samples were later identified by dr. silvia r. m. pedro (ffclrp, universidade de são paulo, brazil). results and discussion throughout the deforestation process, 416 colonies of stingless bees were found. from these, bee-specimens were collected from 118 nests (28% of the total found), comprising 36 species of 15 of the 33 known neotropical genera of meliponini (table 2). it shows that the number of species in the area is certainly higher and the rescue of meliponini is essential during deforestation activities, not only in the amazon region, but also in other tropical forests. being present at the forefront of deforestation was an important step in the rescue of stingless bees, since the impact of large trees falling to the ground often resulted in cracks in the hollow branches or trunks where nests were located. most of the meliponini nests found were in branches (up to 20m high). since brood combs, as well as honey and pollen pots, were seriously damaged after the fall, colonies had to be quickly transferred to beehives. as external nests (trigona jurine) and those in termitaria (mainly partamona schwarz) were also often severely damaged with the fall, there was no other choice, but to attempt transferring them to a beehive, as well. a special problem in the forefront of deforestation, especially in the case of melipona bees, was the destruction of colonies by deforestation workers to collect honey. the workers knew from tradition that melipona bees are harmless and produce good honey. in this case, the presence and orientation given by the rescue team were fundamental in preserving colonies of a harmless species. there was also evidence of colonies of melipona in cracked branches, some days after felling, having fallen prey to eira barbara linnaeus (mustelidae) and potos flavus schreber (procyonidae), mammal species common in the area, but of minor problem. the attacks of these animals were also recorded by nogueira-neto (1997). at the second stage in the deforestation process, during lugging to storage, colonies, which were not evident at the time of cutting, were discovered, whereupon machine operators usually gave aid in signalizing. however, upon removal from one place to another, a further mishap appeared. several colonies presented problems with strong insolation, honey fermentation and infestation by phoridae and hermetia latreille. in this case, transfer to a beehive was considered to be the best, immediate, option. during the process of stacking, the rescue team was able to avoid loosing species with cryptic behavior, such as plebeia schwarz. in the final stage, when the logs were being loaded for transportation, colonies, such as of tetragonisca moure, nannotrigona cockerell, scaptotrigona moure, frieseomelitta ihering, and trigona jurine, were still found. considering all the stages in the deforestation process, 416 stingless bee nests were found. however, 31% of these had been destroyed (n = 129) either during felling, in attempts to abstract honey, or by parasites. however, the remainder (69%, n = 287) presented mature brood combs and queens and could consequently be recuperated. as regards species of the genera melipona, scaptotrigona, tetragona lepeletier & serville, frieseomelitta, nannotrigona, and tetragonisca, there were no relevant problems for most following rescue with the methods used for transference to beehives. the importance of the non-transference of brood sociobiology 61(4): 554-559 (december 2014) 557 combs containing larval food and storage pots to the beehives becomes evident, since the problem of parasite infestation, especially by phoridae flies, was thus avoided. most species recovered well, when transferred to beehives and brought to our field base for the period of observation and care. in the case of the presence of phoridae or hermetia flies, the problem was solved by using vinegar traps. this technique is widely known in meliponiculture (nogueira-neto, 1997). as 166 colonies, 57.8% of all those rescued, were considered to be in good condition, these were donated to local stingless beekeepers. in the case of some species, transfer to beehives compensated little, due to hardy defense and sensitivity to rescue, leading to perishing after disturbance. this occurred in the case of ptilotrigona lurida smith, trigona truculenta almeida and other species of trigona, mainly those with external nests. in the case of these species, oxytrigona spp., table 2. species identified during the meliponini rescue at santo antônio dam, madeira river, brazil. species n. nests substratum cephalotrigona femorata (smith, 1854) 9 trunk/branch frieseomelitta silvestrii (friese, 1902) 1 trunk/branch frieseomelitta trichocerata moure, 1990 7 trunk/branch melipona (michmelia) brachychaeta moure, 1950 2 trunk/branch melipona (michmelia) seminigra abunensis cockerell, 1912 5 trunk/branch melipona (michmelia) sp. 1 (gr. rufiventris) 1 trunk/branch melipona (michmelia) sp. 2 (gr. melanoventer) 1 trunk/branch nannotrigona melanocera (schwarz, 1938) 3 trunk/branch oxytrigona cf. flaveola (friese, 1900) 3 trunk/branch oxytrigona obscura (friese, 1900) 1 trunk/branch partamona ailyae camargo, 1980 7 termitaria/trunk partamona batesi pedro & camargo, 2003 5 termitaria partamona sp. 1 termitaria partamona testacea (klug, 1807) 4 termitaria partamona vicina camargo, 1980 1 termitaria plebeia alvarengai moure, 1994 1 trunk/branch ptilotrigona lurida (smith, 1854) 8 trunk/branch scaptotrigona polysticta moure, 1950 1 trunk/branch scaptotrigona sp. 1 1 trunk/branch scaptotrigona sp. 2 1 trunk/branch scaptotrigona sp. 3 3 trunk/branch scaptotrigona sp. 4 (gr. bipunctata) 1 trunk/branch tetragona clavipes (fabricius, 1804) 9 trunk/branch tetragona essequiboensis (schwarz, 1940) 1 trunk/branch tetragona goettei (friese, 1900) 4 trunk/branch tetragona truncata moure, 1971 1 trunk/branch tetragonisca angustula (latreille, 1811) 14 trunk/branch trichotrigona sp. n 1 * trigona branneri cockerell, 1912 2 external trigona chanchamayoensis schwarz, 1948 1 external trigona crassipes (fabricius, 1793) 5 trunk/branch trigona dallatorreana friese, 1900 1 external trigona guianae cockerell, 1910 5 trunk/branch trigona pallens (fabricius, 1798) 1 trunk/branch trigona truculenta almeida, 1984 1 trunk trigona williana friese, 1900 5 trunk/branch l costa, rm franco, lf guimarães, a vollet-neto, fr silva, gd cordeiro – rescue of stingless bee nests to mitigate deforestation impacts558 due to their defensiveness, and colonies attached to or inside very large logs, they were considered preferable to reallocate the entire colony, as it was, to the nearby forest with the aid of deforestation machinery, up to the very margin of the future reservoir. we reallocated 15.7% of the rescued colonies (n = 45), including mainly p. lurida, trigona spp., partamona spp. and oxytrigona spp. however, attention is called to the difficulty in finding a safe place to leave the colonies, since besides the possibility of exceptional flooding, there is the lack of adequate and appropriate programs to monitor colony survival. furthermore, as already pointed out, most colonies were severely damaged during felling, with frequent parasite infestation. as regards colonies brought to the field base for care and observation, 26.5% (n = 76), among species with external nests, colonies left inside logs and termitaria, perished, most after one to two months due to phoridae fly infestation. these factors led to suppose that the probability of reallocated colony survival would be low. thus, apart from the destiny chosen for rescued colonies (re-allocation or donation to stingless beekeepers or research centers), a monitoring program is called for as a way of evaluating colony survival and guiding future rescue action. as part of this monitoring process, and in the case of donations to beekeepers, it is also important to promote training courses for updating knowledge (fig 1). furthermore, it is necessary to have a larger number of available rescue teams, at least one per forefront of deforestation, for improving results. stinglessbee rescue, concomitant with deforestation, is imperative as a means of aiding in the conservation of pollinators, for providing singular opportunities for data sampling on stingless bee biology, and in support of meliponiculture. by using the simple methods employed in meliponiculture, the rescue of meliponini can mitigate the various environmental impacts caused by deforestation, besides generating social and cultural benefits. acknowledgments we are very grateful to dr. silvia r. m. pedro for species identification, felipe a.s l. contrera and giorgio c. venturieri for suggestions on the manuscript, arcadis logos (sandra favorito, laerte viola, beatris beça, and alex aurani) for all the support during rescue, and to all field colleagues. references allen-wardell, g., bernhardt, p., bitner, r., burquez, a., buchmann, s., cane, j.h., cox, p. a., 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(2004) meliponicultura i: caixa racional de criação. comunicado técnico embrapa 123: 1-3. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i2.171-177sociobiology 61(2): 171-177 (june, 2014) coexistence patterns between ants and spiders in grassland habitats am rákóczi, f samu introduction ants have immense and complex effects on ecosystems because of their sheer abundance, biomass and the complex interactions in which they are involved (hölldobler & wilson, 1990). ants possess various forceful defence mechanisms such as formic-acid, aggressive attack, stings, and social defence (wilson, 1976; yanoviak & kaspari, 2000). defence makes ants best avoided by most predators, which presents them as ideal models for mimics among arthropods (schowalter, 2006), or makes them a food best suited for specialist predators. ant associations that have developed in many arthropod taxa fall into three categories: myrmecomorphy, myrmecophagy and myrmecophily. myrmecomorphs are ant-mimicking species which have acquired morphological and/or behavioural similarity to ants, myrmecophagous species are ant-eaters that specialise in subduing ant prey. here since only those association types occurred in our study area we only consider the ant-eating and ant-mimicking species and do not deal with the third type of ant associated spiders, the abstract the ecological importance of both ants and spiders is well known, as well as the relationship between certain spiders and ants. the two main strategies ˗ myrmecomorphy (ant-mimicking) and myrmecophagy (ant-eating) ˗ that connect spiders to ants have been mostly studied at the behavioural level. however, less is known about how these relationships manifest at the ecological level by shaping the distribution of populations and assemblages. our question was how ant-mimicking and ant-eating spiders associate with ant genera as revealed by field co-occurrence patterns. for both spider groups we examined strength and specificity of the association, and how it is affected by ant size and defence strategy. to study spider-ant association patterns we carried out pitfall sampling on the dolomitic sas hill located in budapest, hungary. spiders and ants were collected at eight grassland locations by operating five pitfalls/location continuously for two years. to find co-occurrence patterns, two approaches were used: correlation analyses to uncover possible spider-ant pairs, and null-model analyses (c-score) to show negative associations. these alternative statistical methods revealed consistent co-occurrence patterns. associations were generally broad, not specific to exact ant genera. ant-eating spiders showed a stronger association with ants. both ant-mimicking and ant-eating spiders associated more strongly with formicine ants species with formic acid or anal gland secretions, and had neutral association with myrmicine ants species with stings and cuticle defences. sociobiology an international journal on social insects centre for agricultural research, hungarian academy of sciences, budapest, hungary. article history edited by gilberto m m santos, uefs, brazil received 21 february 2014 initial acceptance 21 march 2014 final acceptance 10 may 2014 keywords sas hill, species co-occurrence, correlation, mimicry, myrmecomorphy, myrmecophagy corresponding author ferenc samu plant protection institute centre for agricultural research hungarian academy of sciences postal address: po. box 102, budapest h-1525 hungary e-mail: feri.samu@gmail.com myrmecophils, which are highly integrated into host colonies (cushing, 2012; pekar et al., 2012). spiders can use one or more of these strategies, making spider-ant relationship a complex system to observe (mciver & stonedahl, 1993; cushing, 1997). ant associates can be found in various spider families (salticidae, gnaphosidae, theridiide, zodariidae, liocraniidae, linyphiidae) (cushing, 1997; pekar, 2004b). ant associated spiders have many morphological and behavioural adaptations. in ant-mimicking species body shape often resembles three body regions, legs are long and slender and there may be cuticle modifications present that resemble mandibles, compound eyes or sting. the movement of ant-mimics frequently becomes ant-like, including holding forelegs like antennae (reiskind, 1977; ceccarelli, 2008). ant association may also manifest in special foraging and predatory strategies, most tangible in specialist ant-eaters, like zodarion spp. (pekar, 2004). spider-ant relationship is also shaped by ants, which are the models of mimicry and/or potential prey. such a relationship is logically influenced by ant size and also by defence research article ants rákóczi & samu: spider-ant coexistence patterns172 type ants possess (holway, 1999; feener, 2000). ants concerned in the present study fall into two main categories: ants that rely on cuticular structures, sting and ants that mostly rely on the use of formic acid or gland secretions. these coincide with two broader taxonomic groups, being either “myrmicine” (myrmicinae subfamily) or “formicine” (formicinae and dolichoderinae subfamilies) ants (edwards et al., 1974; shattuck, 1992; bolton, 2003). myrmicine ants have thick cuticle and cuticle structures, such as spines (also present in some formicinae, but not present in the genera included in the present study); they possess a distinct postpetiole and a functional sting is always present, while in the formicine group, species armour is different, lack both postpetiole and sting; their defence is based on the use of their mandibles and on toxin exuded from the tips of their abdomens (hermann, 1969; edwards et al., 1974). we treated these taxonomic groups as representing two different defence types, because such modifications are important selective factors for both predators and mimics. although ant associations have been mostly studied through the resulting morphological and behavioural modifications, it also has an ecological context, because ant models should be present in the same microhabitat, and have direct or indirect ecological interactions that are related to co-occurrence (edmunds, 1978). direct trophic connection may exist between ants and spiders, but ants may also influence spiders indirectly through their ecological impact, e.g. aphid tending (renault et al., 2005; sanders & van veen, 2012). in recent years connection between spiders and ants has gained more and more attention in behavioural, morphological and evolutionary studies (cushing, 1997; pekar, 2004b; pekar, 2004a; nelson & jackson, 2009; cushing, 2012; nelson & jackson, 2012), but the ecological patterns observable in the field has to be examined for a complex view on ant-spider relationship. analysing seasonally divided datasets from 40 pitfalls in a grassland ecosystem we tried to answer the following questions: (i) is there any non-neutral co-occurrence pattern between ant associated spiders and ants? (ii) how specific is the association between ant associated spiders and ants? (iii) is the strength of the relationship different between spider strategies and is it influenced by spider and ant size and ant defence type? material and methods study area our field study took place on the top area of sas hill nature reserve, budapest (47°28’48.68”n, 19° 1’1.22”e), between 2010 and 2012. this is a grassland covered dolomitic hill, a refuge for many rare spider species (szinetár et al., 2012), and has been a nature reserve since 1958. arachnological research at sas hill has an especially rich tradition (balogh, 1935; samu & szinetár, 2000; rákóczi & samu, 2012; szinetár et al., 2012). these studies made us notice the especially high number of ant associated spider species, which reaches 14 species with the present study (szinetár et al., 2012). contrary to spiders the ant fauna of the hill have not been previously studied and published neither on generic or specific level. from hungary 126 species of ants in 34 genera are known (csősz et al., 2011). collection of ants and spiders was made in eight dry dolomitic grassland patches scattered on the 35 ha area of the hill. botanically they belonged to open and closed dolomitic dry grasslands, with festuca pallens host as a characteristic grass species. detailed habitat description and co-ordinates are given in szinetár et al. (2012). sampling we collected spiders and ants by pitfall trapping. pitfall traps containing 40% ethylene-glycol with a small drop of liquid soap, had 7.5 cm diameter openings and a laminated plate was applied c. 3 cm higher than the surface as a cover (kádár & samu, 2006). pitfall trap sampling lasted from 29 april 2010 to 24 may 2012. traps were emptied fortnightly, except in winter when, depending on the weather, the traps were emptied c. every four weeks. each location was sampled with five traps in a linear transect with 2 m between traps. collected samples were placed in 70% alcohol; both spiders and ants were sorted and identified under a stereomicroscope. adult spiders were determined to species, while ants were determined to genera. voucher specimens were placed in the collection of the plant protection institute, centre for agricultural research, hungarian academy of sciences. we used several determination keys for spiders (loksa, 1969; loksa, 1972; roberts, 1995; nentwig et al., 2013), and for ants (somfai, 1959; czechowski et al., 2012). the nomenclature of spiders followed the world spider catalogue (platnick, 2013). data classification and analysis the co-occurrence of spiders and ants was examined at two different levels, for which two datasets were derived from raw data: ‘trap’ level dataset contained summarized data of a given pitfall trap over all emptying occasions (n = 40 datasets); ‘trap-season’ level datasets contained summarized data of a given pitfall trap for a season of a year. in the latter datasets we placed winter catches (that represented fewer animals) into autumn or spring, with the division date 1 january, resulting in 7 seasons: 2010 spring, 2010 summer, 2010 autumn, 2011spring, 2011summer, 2011autumn and 2012 spring (n = 280 datasets). in each approach spider species data and ant generic data were used. we assessed the relationship between spiders and ants based on various, biologically meaningful classifications. ants were classified by average size in a genera; and by their taxonomic type also related to defence type: myrmicine (cuticular defence, sting) or formicine (formic acid or gland secretions) (bettini et al., 1978; bolton, 2003). we considered only workers. mean worker size was taken from the literature (somfai, 1959). size difference between dimorphic worker classes was not small in all cases. dimorphism was taken into consideration by calculating mean size from the worker classes. list of ant genera, their classification and mean size are given in table 1. spiders were divided into two groups based on their association type to ants: ant-eating “myrmecophages” and ant-mimicking “myrmecomorphs”, derived from data in sociobiology 61(2): 171-177 (june, 2014) 173 the literature (cushing, 1997; pekar, 2004; platnick, 2010; pekar & jarab, 2011; cushing, 2012; nentwig et al., 2013), and the average size in each species was also considered; spiwas also considered; spider classification and size are given in table 2. in the statistical analyses we have included only species/ genera where more than five individuals were found during the study. we used spearman correlation to reveal positive or negative correlation between counts of individuals of ant genera and spider species. a non-parametric approach was used because of the skewed distribution of counts (many 0 values and some high counts). ant and spider related factors that influence the strength of correlation were analysed by linear mixed model. the model included spearman correlation coefficient values as response variable, spider strategy, ant defence type, average ant and spider size in given genus/species as explanatory variables, and to control for the non-independence of values within genus or species, spider species and ant genera were added to the model as random factors (faraway, 2005). specificity of the relationship (measured as the number of significant correlations) was analysed by nominal logistic analysis. analyses were carried out by r 2.15.2 (r core team, 2013). we used co-occurrence analysis to detect possible nonrandom patterns in presence absence matrices, comparing them to matrices generated by randomization. analysis was carried out by ecosim’s (build 021605) co-occurrence module (gotelli & entsminger, 2010). we used location by taxon presenceabsence matrices, where location datasets were either trap or trap-season, and taxon was (i) only ant genera; (ii) only ant associated spider species; (iii) both ants and ant associated spiders. the co-occurrence analysis searches for checkerboard units (cu), which are 2x2 sub matrices in the original presence-absence matrix. the number of cus for a species pair is the number of localities where only one of the species occurs, i.e. their occurrence is mutually exclusive (stone & roberts, 1990). for a given species-pair the negative association is represented by a large number of cus in every possible habitat combination. the average number of cus for all the possible species combination is the checkerboard score (c-score), which is a measure of negative association in the community (stone & roberts, 1990; gotelli, 2000; gotelli & entsminger, 2010). the null-model matrices are monte-carlo randomizations of the original matrix. the average of such randomized c-score values represent the case without biological interactions, higher observed c-score values than that indicate negative, while lower observed values indicate positive associations between the species. results quantitative results during the whole sampling period we emptied the 40 traps 40 times. in total 10,230 ant specimens and 751 ant associated spiders were found. the total number of ant genera was 13 (table 1), the ant associated spiders were represented by 11 species (table 2). most ant associated spiders were relatively rare, the majority representing the ant-eating strategy. a single ant-eating species, z. rubidum, made up nearly 90% of all ant associated spiders, and it meant a very high, 16% dominance among all spiders. genus abbrev. (5 character) subfamily morph mean size (mm) no. of indiv. bothriomyrmex bothr. dolichoderinae formicine 2.5 13 tapinoma tapin. dolichoderinae formicine 3.0 5550 camponotus campo. formicinae formicine 10.0 1596 formica formi. formicinae formicine 7.0 1074 lasius lasiu. formicinae formicine 3.0 1179 plagiolepis plagi. formicinae formicine 1.5 210 leptothorax lepto. mirmicinae myrmicine 2.5 12 messor messo mirmicinae myrmicine 8.5 112 myrmecina myrme. mirmicinae myrmicine 5.5 3 myrmica myrmi. mirmicinae myrmicine 5.5 185 solenopsis solen. mirmicinae myrmicine 1.5 20 temnothorax temno. mirmicinae myrmicine 2.5 33 tetramorium tetra. mirmicinae myrmicine 2.5 243 species name family association type no. of indiv. % of σaa mean size (mm) callilepis schuszteri simon gnaphosidae ant-eater 15 2.0 5.2 euryopis quinqueguttata koch theridiidae ant-eater 2 0.3 2.3 harpactea hombergi (scopoli) dysderidae ant-mimic 1 0.1 4.8 micaria dives (lucas) gnaphosidae ant-mimic 6 0.8 3.1 micaria formicaria (sundevall) gnaphosidae ant-mimic 2 0.3 6.6 micaria pulicaria (sundevall) gnaphosidae ant-mimic 1 0.1 4.3 micaria silesiaca koch gnaphosidae ant-mimic 1 0.1 4.3 phrurolithus festivus (koch) corinnidae ant-mimic 8 1.1 2.7 phrurolithus szilyi herman corinnidae ant-mimic 42 5.6 2.3 synageles hilarulus (koch) salticidae ant-mimic 2 0.3 3.0 zodarion rubidum simon zodariidae ant-eater 671 89.3 3.5 all spiders 4,051 all ant associated spiders (σaa) 751 σaa as % of all spiders 18.5 table 1. list of ant genera in the present study. subfamily and grouping according to morphs are given, together with mean worker size and number of specimens caught in the study. table 2. list of ant associated (aa) spider species on sas hill. number of individuals refers to total catch during the period. catches of aa species also expressed as % of all aa (σaa). as a reference total number of spider individuals (including non aa) and total number of aa spiders caught are given. the mean size of each species is also given. rákóczi & samu: spider-ant coexistence patterns174 correlation analysis spearman correlation analyses were performed on the trap-season dataset. there was a strong correlation between the overall number of ants and ant associated spiders (ρ = 0.65, p < 0.0001). correlation was also calculated at the functional grouping levels. spider ant association types showed no correlation with myrmicine ants (ant-eating spiders: ρ = 0.025, p = 0.68; ant-mimicking spiders: ρ = 0.024, p = 0.69), but correlation with formicine ants was significant and of similar strength for both spider groups (ant-eating spiders: ρ = 0.55, p < 0.0001, ant-mimicking spiders: ρ = 0.54, p < 0.0001). correlation analysis between individual spider species and ant genera was also performed (table 3). analysing the pattern of significant correlations, it is clear that the association of spiders is broader than ant genera, because all spider species were significantly positively associated with more than one ant genus (table 3). analysing the number of significant correlations of the spider species in a nominal logistic model including spider strategy, ant type, average ant and spider size as explanatory variables, ant size proved to be marginally significant (wald test: χ2 = 4.052, df = 1, p < 0.04), the spider association became more frequent with increasing ant size. ant type proved to be highly significant (wald test: χ2 = 13.70 df = 1, p < 0.0002), with much more significant associations of spiders with formicine ants. we also wanted to know how the strength of associations was dependent on spider and ant strategies and average spider and ant size. we tested a linear mixed model on spearman correlation coefficients, which had normal distribution (kolmogorov-smirnov test, d = 0.131, ns). the model included spider strategy, ant defence type and ant and spider size as explanatory variables, and spider species and ant genus as random factors. spider size was marginally significant, with smaller spiders correlating more with ants (f = 21.51, df = 1, p = 0.044); spider strategy was also marginally significant, with ant-eaters more strongly associated with ants (f = 22.83, df = 1, p = 0.041). the most important factor proved to be ant defence type showing a much higher correlation of spiders to the formicine group than to myrmicine (f = 12.92, df = 1, p = 0.005). co-occurrence analysis the co-occurrence analysis revealed positive association in the ant-spider assemblage. we made simulations on data of “just spider”, “just ant” and “ant+spider” assemblages. observed c-scores were consistently lower than simulated ones, as measured by standard effect size (s.e.s.) in the spider-ant assemblage, meaning that on average the mixed assemblage is more associated than pure taxa assemblages (table 5). considering specific species pairs, the number of cus is a measure of negative association. higher number of cus was found between myrmicine ants and ant associated spiders. in z. rubidum we found no cu with any of the ant genera. the cu pattern of spider-ant species pairs is given in table 5. discussion the main purpose of the present study was to reveal if known ant associated spiders respond to the distribution of ants in an ecologically measurable way. the results certainly support the hypothesis, that non-random co-occurrence patterns exist in the field between ants and ant associated spiders. although associations were rather broad, they were influenced by spider and ant characteristics, from which ant defence type seemed to be the most important. for our purposes the sas hill in budapest proved to be a very good location where we could sample 11 ant associated species. this is important, because most ant associated species are relatively rare (cushing, 1997; pekar, 2004b; pekar, 2004a; nelson & jackson, 2009; cushing, 2012; nelson & jackson, 2012), and to study their ecology and relations to other taxa is therefore not easy. measuring the association pattern indicated by ants and ant associated spiders first of all gave us the result that associations are not at the lowest taxonomic resolution of the present study (spider species and ant genera), but at the higher spider/ant micaria dives phrurolithus festivus phrurolithus szilyi callilepis schuszteri ♣ zodarion rubidum ♣ lepto. ■ -0.04 0.1 0 0.21 ●● 0.17 ● messo. ■ -0.04 0.03 0.13 0.02 -0.06 myrmi. ■ 0.01 0.07 -0.11 -0.1 0 solen. ■ 0.1 -0.05 0.14 -0.05 0.01 temno. ■ 0.02 0.09 0.01 0 0.13 tetra. ■ 0.07 -0.01 0.01 -0.04 0 bothr. -0.04 0.11 0.02 -0.04 -0.01 campo. 0.28 ●● 0.15 ● 0.24 ●● 0.23 ●● 0.37 ●●● formi. 0.17 ●● 0.15 ● 0.20 ●● 0.23 ●● 0.53 ●●●● lasiu. 0.11 0.11 0.16 ● 0.01 0.54 ●●●● plagi. 0.21 ●● 0.09 0.18 ● 0.14 ● 0.29 ●● tapin. 0.19 ● 0.13 0.32 ●●● 0.09 0.34 ●●● table 3. spearman correlation coefficients (ϱ) of ant associated spiders and ant genera in the trap-season dataset. row header contains ant genera, (abbreviated names, c.f. table 2). ■ marked ants are myrmicine, unmarked ones are formicine ants. spiders marked with ♣ are ant-eaters, unmarked ones are ant-mimics. the number of ● symbols marks the strength of the correlation (denoted by: ● – 0.10.19, ●● – 0.2-0.29, ●●● – 0.3-0.39, ●●●● >0.4). all marked correlations were significant at p<0.05. sociobiology 61(2): 171-177 (june, 2014) 175 spider/ant micaria dives phrurolithus szilyi phrurolithus festivus callilepis schuszteri ♣ zodarion rubidum ♣ lepto.■ 6 ●● 0 2 6 ●● 0 messo.■ 3 2 8 ●●● 0 0 myrmi. ■ 5 ● 5 ● 2 6 ●● 0 solen. ■ 4 ● 4 ● 9 ●●● 4 ● 0 temno. ■ 8 ●●● 2 0 6 ●● 0 tetra. ■ 0 0 0 0 0 bothr. 3 6 ●● 8 ●●● 6 ●● 0 campo. 0 0 0 0 0 formi. 0 0 0 0 0 lasiu. 0 0 0 0 0 plagi. 0 0 0 0 0 tapin. 0 0 0 0 0 level of functional and morphological groups. at these higher levels the two statistical methods, measuring positive and negative associations, gave congruent results. our results are also in agreement with observations about the moderately narrow diet of ant-eating spiders. these spiders are specialised on consuming not a single ant species, but rather a broader spectrum of species, such as genera or subfamily (pekar, 2004; pekar et al., 2012). based on spider response to olfactory cues produced only in a narrow range of genera, in a recent study cardenas et al. (2012) argue that z. rubidum previously thought as an “ant generalist” in fact, preys mostly on the genera lasius and formica. our field results support this notion. it was proved that spider and ant size are marginally significant factors in the association, most probably for different reasons in ant-mimicking and ant-eating spiders. on one hand, preying on ants is risky because of the defences, which favours a higher spider/ant size ratio, but for the same reason large ant-eaters can be less associated with ants because they may have a broader diet. on the other hand, ants below a certain size might not be preferred, because preying on them results in lower profit (less nutrition in preferred body parts) compared to cost (pekar, 2004b; pekar et al., 2010; cushing, 2012). in z. rubidum we know from case studies that the spider shows preference for similar sized or larger ants (pekar, 2004b). probably for other ant-eaters, size ratio with prey plays similar role. in ant mimics size plays important role because appropriate size enchases the accuracy of the table 5. the number of checkerboard units (cu) of ant associated spiders and ant genera in the trap-season dataset. row header contains ant genera (names abbreviated, c.f. table 2). ■ marked ants are myrmicine, unmarked ones are formicine ants. spiders marked with ♣ are ant-eaters, unmarked ones are ant-mimics. numbers show the number of cus observed for the species pair. the number of cus is visually represented by the number of ● symbols (no symbol – <4, ● – 4-5, ●● – 6-7, ●●● – 8-9). mimic, making it more effective. the strongest pattern found was, that both ant-mimicking and ant eating spiders showed positive association with formicine ants and neutral or negative association with myrmicine ants, as it was confirmed by both statistical approaches. in ant-mimicking spiders the reason could be that the numerical dominance of formicine ants makes these ants a better model for batesian-mimicry (schowalter, 2006). the strength of the association was the strongest in ant-eating spiders, where the reason for co-occurrence pattern could be the difference how they are able to cope with different defences: thick cuticle, propodeal spikes and sting with neurotoxins vs. formic acid (blum, 1992; bolton, 2003). the aim of the present study was to reveal spider-ant association patterns from a field study. this is an alternative and complementary approach to laboratory studies, where preference is tested under highly artificial circumstances, and relatively little can be said about their realisation in the field. the specificity of spider-ant association proved to be relatively broad in the field, with ant associated spiders correlating with more than one ant genera. in the present study we uncovered a non-random pattern of co-occurrence, where possibly for different reasons for ant-mimicking and ant-eating spiders the most substantial pattern was a stronger association with fomicine than with mymicine ants. acknowledgements the authors are grateful to erika botos, kinga fetykó, éva szita, gábor lőrinczi and zsolt lang for their contribution in this research. we thank stano pekar for detailed comments on a previous version of the manuscript. we are indebted to sándor csősz for reviewing the manuscript before submission and giving us insightful advises. we thank four anonymous referees for their comments and criticisms on the manuscript. the project was supported by otka grant k81971, by the colleagues of sas hill nature reserve and by pál kézdy from the directorate of duna-ipoly national park. composition trap s.e.s. trap p trap (c-score) trapseason s.e.s. trapseason p trapseason (c-score) only spider -1.8 0.02 3.42 -1.1 0.13 15.53 only ant -2.1 0.01 2.31 -3.2 0.0002 11.82 spider + ant -3.2 0.0002 1.67 -3.5 0.0001 10.04 table 4. observed c-scores, standard effect size (s. e. s.) and significance level of co-occurrence analysis on trap and trap season datasets. rákóczi & samu: spider-ant coexistence patterns176 references balogh, j. i. 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(2012). a sas-hegy pókfaunája ii. a sas-hegy faunakutatásának 80 éve a hegyről kimutatott pókfajok kommentált listája [spider fauna of mt sas-hegy ii. 80 years of fauna research on mt sas-hegy, with the annotated list of spiders]. rosalia, 8: 333-362. wilson, e. o. (1976). organization of colony defense in ant pheidole dentata mayr (hymenoptera-formicidae). behavioral ecology and sociobiology, 1: 63-81. doi: 10.1007/bf00299953 yanoviak, s.p. & m. kaspari. (2000). community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 259-266. doi: 10.1034/j.1600-0706.2000.890206.x. doi: 10.13102/sociobiology.v66i4.3590sociobiology 66(4): 582-591 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 impacts of fire in social wasps community in an area of regenerating brazilian savanna introduction in some ecosystems such as seasonally dry forest (pinarda & huffmana, 1997), mixed temperated forest (abrams, 1992) and brazilian savanna (miranda et al., 2002), fire is one of the most common natural perturbations and is an important element in the dynamics of these ecosystems (hobbs & huenneke, 1992). fire occurrence is influenced by meteorological conditions and climatic changes; however, according to mistry (1998), the principal cause of fire in the brazilian savanna is through agricultural activities. brazilian savanna mostly occurs at tropical latitudes and have their existence directly linked to the rain cycle abstract fire is one of the most common natural harassments and it is characterized as an important component of the systems, although little studied regarding the influence of this event in social wasps communities. one month after the end of the collections, the area of regenerating brazilian savanna of the present study undertook a fire in exactly half of the points sampled, which motivated one further year of sampling. the aim of this study was to compare the social wasps fauna collected before and after the fire, indicating a possible impact on their populations. in the first year of collection, before the fire, 21 species (seven genera) were sampled with a total amount of 297 individuals. in the second year, after the fire, 14 species were collected (reduction of 33,33% in richness) and six genera, with an abundance of 153 wasp individuals of vespidae (loss of 48,48% of individuals). the area 1 in the first year, presented an abundance of 182 individuals (61,27%) and 16 species (76,19%) and the area 2, 115 (38,73%) and 19 species (90,47%). for the area 1 in the second year, 74 individuals (48,36%) and 12 species (85,71%) were captured and for the area 2 there were 79 individuals (51,64%) distributed among nine species (64,28%). however, we did not find significant values for both the abundance and for the richness among the areas and the years of sampling. nine species were not registered in the second year, although two presented their registration only after the fire. sociobiology an international journal on social insects ma clemente1, hf ceridório2, drs mendes1, r guevara3, ot silveira4, e giannotti1, hr moleiro1, km vieira5 article history edited by gilberto m. m. santos, uefs, brazil received 18 july 2018 initial acceptance 15 may 2019 final acceptance 29 october 2019 publication date 30 december 2019 keywords inventory, polistinae, conservation, richness corresponding author mateus aparecido clemente instituto federal de ciência e tecnologia sudeste minas gerais, campus barbacena rua monsenhor josé augusto nº 204 36205-018, são josé, barbacena/mg, brasil. e-mail: mateus1981@gmail.com (andrade, 2008) and fire (miranda et al., 2002). the savanna is a huge biome of south america and is considered a “hotspot” because of its biodiversity and its high degree of degradation (mittermeier et al., 2005). in brazil, the savanna extends over the states of mato grosso, mato grosso do sul, goiás, minas gerais, piauí, the federal district, tocantins and part of the states of bahia, ceará, maranhão, paraná, rondônia and são paulo. the main natural cause for burnings in the savanna is lightning strikes occurring with higher intensity in the first rains, when vegetation is still dry (ramos-neto & pivello, 2000). fires frequently alters the regeneration rate of many woody species, reduces their density through the mortality of small individuals (hoffmann, 1996; 2000; medeiros & 1 universidade estadual paulista júlio de mesquita filho, instituto de biociências, zoology department, rio claro-sp, brazil 2 centro universitário hermínio ometto – uniararas, araras-sp, brazil 3 red de biología evolutiva, instituto de ecología ac, inecol, xalapa, méxico 4 coordenação de zoologia, museu paraense emílio goeldi, campus de pesquisas, belém-pa, brazil 5 universidade federal de mato grosso do sul, centro de ciências biológicas e da saúde, programa de pós graduação em ecologia e conservação, campo grande-ms, brazil research article wasps sociobiology 66(4): 582-591 (december, 2019) 583 miranda, 2005) and favors the proliferation of herbs. fires can also alter the pattern of flowering of some species (prada et al., 1995), the tenacity of leaves (vieira et al., 1996) and in general, fires increase the availability of food for the herbivores during the dry season (rodrigues, 1996), due to these bloom after fire and tender leaves. insects are important in the colonization of the burned areas once fire can attract a wide range of arthropods through their perception of smoke and high temperatures; many of them use the burned trees for breeding and larvae that are born feed on dead wood (lyon et al., 2000). insects are the most abundant and diverse group of animals in the terrestrial environment and particularly, the hymenoptera is one of the largest orders with around 130 thousand species (rafael et al., 2012). among hymenoptera, wasps are highlighted because they provide different environmental services as floral visitors (heithaus, 1979; barros, 1998; silva-pereira & santos, 2006; hermes & köhler, 2006) and pollinators (barros, 1998), being also important predators mainly of the larvae of lepidoptera, thus being one of the main top-down drivers of herbivory. in other words wasps are important biological control agents (prezoto, 1999). despite social wasps present a considerable share of the desirable characteristics of a bioindicator group little research has been conducted on this topic. as far as we are aware of, there are only three studies exploring the use of wasps as bioindicators: two studies investigated the concentration of heavy metals in larvae, pupae and adults of two species of wasps in europe (urbini et al., 2006), and the third one was conducted in brazil by souza et al. (2010) who investigated the use of wasps as bioindicators of conserved riparian woods of rio das mortes, city of barroso, minas gerais. of the thirty six species registered by souza et al. (2010) pseudopolybia vespiceps (saussure) and polybia fastidiosuscula (saussure) were considered as indicators of conserved forest, while mischocyttarus drewseni (saussure) was an indicator of highly impacted areas. in this study we explored the potential of the community of social wasps as bioindicators of perturbation by fire in the brazilian savanna. we took advantage of a fire that consumed roughly half of an area that was being surveyed to investigate the richness of social wasps. this unfortunate incident gave us the opportunity to investigate how the community of social wasps reutilize and recolonize burn down areas therefore proving an indication of ecosystem recovery. in particular we aimed to compare the community of social wasps before and after the fire. material and methods study site the studied site was an area of regenerating brazilian savanna about 25 ha limits at the south with the são paulo state university (universidade estadual paulista – unesp) and at the northeast with the state forest edmundo navarro de andrade – feena. the area presents a history of successive impacts, but it was observed a regeneration stage before the fire. the climate in the region, according to the classification of köppen, is of the cwa type (tropical with two well defined seasons), characterized by drought in winter and average temperature in the hottest month above 22°c. the dominant vegetation at the study site is characterized by the dominance of grove species such as the brazilian savanna and herbaceous species such as from the families poaceae, fabaceae and asteraceae (personal communication) and arboreous species such as from the families myrtaceae, asteraceae, melastomataceae, fabaceae (potascheff et al., 2010), the site presents an aspect of woods, absence of sub-woods and trees of uniform size (cardoso-leite et al., 2004) being that the area of studies presents approximately 250.000 m2 (fig 1). initial survey was planned along 10 sampling points spaced at least 100 meters from each other. all points were visited every other month, from september 2012 to july 2013 and wasps were caught with attractive traps and by active search. just after july 2013, approximately 140.000 m2 area caught fire with a considerable loss of the vegetation community what motivated a further year of work to address the impact of fire on the wasp community. thus, the study site was divided in two areas, area 1 (points from 1 to 5) that remained unaffected by fire and area 2 (points 6 to 10) that was burned (fig 1). we resumed our sampling campaign 45 days after the end of the burning and kept the sampling campaign from september 2013 to july 2014. methods of sampling attractive traps – 10 points were marked, being five at about 1.5 m above the soil and five in the level canopy (about 5-9 meters from the soil). each trap was made of a 2 liters pet container, placed at 100 meters from each other. in each container there were made four circular openings with about 3 cm in diameter and baited with 200 ml of attractive juice (concentrated juice of passion fruit with sugar). the samples were collected with the aid of sieve and tweezers and fixed in 70% alcohol containers of the collection type (locher et al., 2014). active search – a team of three trained people in all collections applying the same effort. the search for wasps in through the area along existing trails and when found, wasps were collected with the help of an entomological net. wasps were killed in a death chamber containing ether and latter fixed in 70% alcohol. active search was conducted from 9:00 to 16:00 that covers the peak of foraging activity of most wasps. identification and destination of the collected material samples were identified by genders and species with the help of dichotomous keys (richards, 1978; carpenter & marques, 2001) and comparison with reference material at the collection of social wasps of the zoology department. voucher specimens were deposited in the collection of invertebrates of the emilio goeldi museum of pará (mpeg). ma clemente et al. – impacts of fire in social wasps in a brazilian savanna584 data analysis the programs used in the analysis were past – version 1.49 (hammer et al., 2001), bioestat version 5.0 (ayres et al., 2007) and resources of the free program r development core team (2009). in order to compare if there were significant differences in abundance and species richness between the pre and post fire period, we pooled by adding the abundance of each species in every collection point before and after the fire. we used the total abundance at each point and the species richness observed at each point as response variables to test if the fire had affected these community metrics. we used generalized linear model on the program r, where abundance and richness were the response variables in separated models and fire with two levels (preand post-fire) was the explanatory variable. both models were fitted with the gamma distribution and the inverse link function to meet model assumptions. to verify if the number of collections was enough for a sampling of the social wasps community in the areas of this study and between the pre and post fire periods, the rarefaction method by the program r was used. to estimate the number of species in the pre and post fire period, the jackknife 1 and 2 estimators were used. the non-metric multidimensional scale (mds) was used for the evaluation of inter-local differences of area 1 (points from 1 to 5) without burning in the first year (a1/sq), area 2 (points from 6 to 10) without burning in the first year (a2/sq), area 1 without burning in the second year (a1/sq), area 2 with burning in the second year (a2/q). the aim was to verify the similarity among the points in these areas and the confection of the mds figure was performed in the program r using the mass package. the mds is a method that takes as basis the proximity of objects, subjected to stimulus used to produce a spatial representation of them (härdle & simar, 2007). in this case, the objects are the pre-defined points in each area and the stimulus was the fire that occurred in one of the sites. the proximity expresses the similarity among these points. the mds is a technique of dimensional reduction, once its aim is to find a set of points in low dimension (usually two dimensions) which reflect the configuration of the data in high dimension. results in the first year of collection, 21 species were sampled, distributed among seven genera and a total of 297 individuals. in the second year, after a case of fire, only 14 species were collected (33.3% reduction in species richness) and six genera, with a total abundance of 153 wasps (48.5% less than in previous year) (table 1). the abundance of social wasps was reduced significantly by 48% after the fire (x2 = 8.1, df = 1, p =0.005 ). in the same way, species richness was reduced by 49% after the fire (x2 = 17; df = 1; p< 0.001). rarefaction curves (fig 2) showed that in the pre and post fire periods the sampling effort was sufficient to estimate accurately the composition of the wasp community in the study site. the jackknife 1 and 2 estimators predicted 25.2 and 24.8 species respectively for the pre-fire period what means that we recorded between 83.4% and 84.8% of the actual species fig 1. map of distribution of collection points in the regenerating savanna in the campus of unesp rio claro and the respective areas in function of the fire event. sociobiology 66(4): 582-591 (december, 2019) 585 composition of the wasp community in the study site. for the post-fire period, jackknife1 and 2 estimators predicted 18.4 and 19.8 species respectively what means that we sampled between 76.2% and 70.1% of the actual species richness left after the burn, respectively. 1st year 2nd year species area 1 area 2 total area 1 area 2 total wo/burn wo/burn wo/burn burn area 1 area 2 agelaia multipicta 6 4 10 10 agelaia pallipes 125 53 178 52 50 102 280 agelaia vicina 8 8 16 16 apoica pallida 1 1 2 2 brachygastra lecheguana 1 3 4 4 polybia chrysothorax 9 1 10 1 1 11 polybia dimidiata 6 5 11 5 4 9 20 polybia fastidiosuscula 1 2 3 3 3 6 polybia ignobilis 10 14 24 1 2 3 27 polybia minarum 1 1 1 polybia occidentalis 3 3 2 2 4 7 polybia paulista 5 5 1 1 6 polybia sericea 2 1 3 2 1 3 6 polybia jurinei 1 4 5 2 2 7 protonectarina sylveirae 1 1 1 synoeca cyanea 1 1 2 1 6 7 9 mischocyttarus cassununga 1 1 1 mischocyttarus drewseni 1 1 10 10 11 mischocyttarus mattogrossoensis 4 6 10 10 mischocyttarus rotundicollis 1 1 1 mischocyttarus tricolor 1 1 2 2 mischocyttarus montei 2 2 2 polistes lanio 3 4 7 3 3 10 total abundance 182 115 297 74 79 153 450 total richness 16 19 21 12 9 14 23 table 1. abundance of social wasps in areas 1 (without burning) and 2 (without burning) in the first year and areas 1 (without burning) and 2 (with burning) in the second year in a fragment of brazilian savanna, rio claro, brazil. fig 2. rarefaction curve based in the sampling (rarefaction calculated with the program r) for the periods before and after the fire. area 1 y1 area 2 y1 area 1 y2 area 2 y2 area 1 y1 h=0.1 p = 0.871 h = 1.7 p = 0.191 h = 1.7 p = 0.196 area 2 y1 h = 1.9 p = 0.172 h = 3.5 p = 0.062 h = 5.5 p = 0.019 area 1 y2 h = 3.1 p = 0.078 h = 1.1 p = 0.297 h<0.1 p = 0.935 area 2 y2 h = 3.7 p = 0.054 h = 0.2 p = 0.631 h=0.1 p = 0.809 table 2. summary of kruskal-wallis tests employed to compared species richness (upper triangle) and abundance (lower triangle). when broken down by areas and years we observed in the first year, in area 1 total abundance of 182 individuals (61.3%) distributed in 16 species (76.2%) while in area 2 there were 115 individuals (38.7%) and 19 species (90.5%) and there were no significant differences between the two areas in year one in abundance and species richness (table 2). cross comparison of areas and years showed a significant 52.6% reduction in the species richness of area 2 after the burn, ma clemente et al. – impacts of fire in social wasps in a brazilian savanna586 and a marginally not significant 36.8% difference (p = 0.06) when comparing area 2 before the burn with area one after the fire. in abundance we observed a marginally not significant 36.8% difference between years in the unburned area, and a just marginally not significant 56.5% difference in abundance between area 1 before the fire and area two after the fire (table 2 and fig 3). the non-metric multidimensional scaling (mds) showed that area 1 and 2 before the fire had the wasps communities with the highest degree of resemblance in composition and structure as the polygons that define them greatly overlapped (fig 4). in contrast area 2 before and after the burn represent the most distant polygons suggesting considerable differences in fig 3. distribution of the species of social wasps in areas 1 and 2 before and after the fire in a brazilian savanna fragment in rio claro. fig 4. multidimensional scale (mds) non metric for the evaluation of interlocal differences in area 1 without burning in the first year (a1/sq), area 2 without burning in the first year (a2/sq), area 1 without burning in the second year (a1/sq), area 2 with burning in the second year (a2/q). composition and structure of the recorded communities. this analysis also suggests either an indirect effect of fire on the wasp community of the non-burn area or simply caught the yearly variability in the community, since there a notorios spatial separation of the polygons define area 1 before and after the burn. agelaia pallipes (olivier) represented 59.9% (178) of the individuals collected in the first inventory and 66.7% (102) in the second, being the most abundant species. the second most representative species was polybia ignobilis (olivier), with 24 individuals (8.1%) in the first inventory and mischocyttarus drewseni, with 10 individuals (6.5%) in the second. the other species presented a frequency below 6.0% in the two years of sampling. sociobiology 66(4): 582-591 (december, 2019) 587 a. multipicta (haliday), a. vicina (saussure), apoica pallida (olivier), poly. minarum (ducke), protonectarina sylveirae (saussure), m. mattogrossoensis (zikán), m. rotundicollis (cameron), m. tricolor (richards) and m. montei (zikán) were the species that were not observed after the fire, representing a loss of 42.8% of the species registered for the first year (fig 5). however, two species (brachygastra lecheguana (latreille) and m. cassununga (r. von ihering) were not registered in the first year of collection, but were sampled after the fire in the area (table 1 and fig 5). in the first month of collection after the fire (september 2013), we saw a nest of b. lecheguana still active, although affected by the burning. however, in the following five sampling campaigns the nest was observed to decline and there were no further capture of individuals of this specie. regarding m. cassununga, only one individual was collected during all the collection period, in the month of november. fig 5. distribution of species of social wasps in the regenerating savanna in rio claro. gray letters in the first year indicate the species that disappeared in the post-fire period and those that are presented in gray for the second year were registered only in the post-fire. discussion in the brazilian literature, few studies emphasize the impact of fire on the community of social wasp, even when they had occurred during inventories and at most it is related only as an eventuality during the collection period. thus, a deep discussion over this subject is very much limited by the lack of robust data on this issue. a significant reduction in the number of species and abundance before and after fire in this study was observed. the losses of 33.3% species and 48.5% in abundance after the fire are similar to the data of locher et al. (2014) in ipeúna, são paulo. in the cane field, where the burning of the straw occurred, there was a decrease of 31.4% in the abundance and 18.2% in the richness. in the riparian forest, where burning had not occurred, but closer to the sugar cane monoculture, there were 19.5% less records and 25% less species observed. corroborating the present study we cannot distinguish between indirect fire effects and annual variation. this same relationship was observed by locher et al. (2014) in a panel of riparian forest and plantation of sugar cane. the author reported that six months after the start of the collections there was the burning of sugarcane straw. when analyzing the abundances of captured species of the three previous months and the three months after the fire, it was observed that there was no significant difference among the samples of the riparian forest and those from the sugarcane plantation. however, after the burning of the straw of sugarcane, there were different species of social wasps, both at the edge of the riparian forest, as in the sugarcane plantation, despite the richness was less than three months after the fire. which eliminating this environment, there was a decrease in the abundance of individuals from the second month after the firing, factor that according to the author can be correlated with the reduction of protected environments and trapped in the planting area, and therefore an increased migration into the woods, making it difficult to collect these, both in the sugarcane plantation, as in the edge of the riparian forest. rarefaction curves showed that in the pre and post fire periods the sampling effort was sufficient to estimate accurately the composition of the wasp community in the study site. however, it is important to outline that the collections were finalized in cold months (may and june 2014), what can explain the lower number of individuals and species sampled. ma clemente et al. – impacts of fire in social wasps in a brazilian savanna588 the air temperature during a fire can vary from 85°c to 840°c, while the soil temperatures range from 29°c to 55°c at 1 cm depth. for species of social wasps, nests can be built on the abaxial surface of leaves, in human constructions (polistes, mischocyttarus, apoica, some species of polybia), directly on the trunk of a tree (synoeca), caught in vegetable branches (brachygastra and polybia), hidden in cavities, such as holes in the trunks of trees or soil (some species of agelaia and polybia) (carpenter & marques, 2001). all these genera present in this study were vulnerable in the area of the fire due to high temperatures in both the vegetation and on the ground. the variation in soil temperature below 5 cm depth is almost zero, reaching a maximum of 3°c, without occurrence of fire (miranda et al., 1993). castro neves & miranda (1996) showed that after a fire in an off-course change of the albedo and heat flow in soil increased the amplitude of soil temperature of about 30°c to 1 cm deep, 10°c 5 cm deep, while the depth of 10 cm did not changed. due to the a. pallipes nesting habit, it is possible that the burning did not reach their nests as they are found in such abandoned chambers of the genera ant nests atta, in armadillo tunnels, among the roots of trees, hollow trunks and various artificial constructions, generating greater protection (zucchi et al., 1995; noll et al., 1997). however, these effects are short-term, since the vegetation tends to recover quickly. the heat also influences altering the flowering pattern of some species (prada et al., 1995), toughness of leaves (vieira et al., 1996) and particularly enhancing the availability of food for herbivores during the dry season (rodrigues, 1996). the feeding of social wasps is based on proteins from the capture of insects and other arthropods, nectar carbohydrates and exudates of hemiptera, besides cellular contents and water (gobbi & machado, 1985). by using a range of features present in the environment, such as water, vegetable fiber, nectar and prey, social wasps reveal an opportunistic character, they return to places with large supply of resources or food, in search for the optimization of foraging and decrease in the search effort (raveret-richter, 2000). thus, this change in food availability may favor the social wasps and maintain populations even after the fire. as well as the organisms, resources are also affected differently by the fire. consequently, specialist species (dependent on one or a few types of food) can be more subject to local extinction. as for generalist species, the limitation of some resources has few negative effects on populations, since they can be replaced by another (frizzo et al., 2011). clemente et al. (2013) in their study of interaction networks of social wasps and different species of plants, noted that the network was more complex in the riparian forests, presenting a higher number of species and individuals and a larger number of links between them. the degree of specialization of the network was more general in the riparian forests when compared to other phytophysiognomy studied, rupestrian field, which presents more strict vegetation characteristics. interactions in the rupestrian field tended to specialization, with higher chances of local extinctions. an environment such as the one of the present study, after firing, can have considerably affected specialist species, which leads to decrease in abundance or even local extinction of these species. lawton (1983) and santos et al. (2007) reported that environments with more complex structure enable the establishment and survival of more species of social wasps. the vegetation exerts considerable influence on the social wasps communities as it provides support for foundation of nests and food resources, and indirectly affects these communities by variations caused in temperature, air humidity and amount of ambient shadow. the species of social wasps that nest only on certain conditions, select the locations of their nests by the density and types of vegetation, whether open or closed, as well as the shape and arrangement of leaves and other plant structures (machado, 1982; santos & gobbi, 1998). thus, the alteration of the environment by the fire episode is a limiting factor for some species which explains the lower richness and abundance when compared to the first year. the study performed by chaibub (2013) aimed to compare the abundance, richness and diversity of social wasps with the work done by elpino-campos et al. (2007), in the same area from 2003 to 2004 where there were three fires during the collection period. the aim was to see whether the reduction of anthropic action (fire) over time would generate a significant increase in the diversity and abundance of social wasps. the diversity of species found in ccpiu (itororó ecological reserve and hunt and fishing club) before the fire was higher (1.063), compared to 10 years after (0.916). there was not the capture of unique species in this new study. also, there was no difference in the abundance of individuals and the richness of species in the samples before and ten years after the fire (paired t = 0.174, p = 0.872, paired t = 4.045, p = 0.027, respectively), and between the different seasons of the year (paired t = 0.174, p = 0.872; paired t = 4.045, p = 0.027, respectively), although a greater abundance of individuals 10 years after the fire had been demonstrated. chaibub (2013) reports that with the current surveillance of the ccpiu reserve, fires, frequent in the savanna biome (oliveira & marquis, 2002), but not in an anthropic way, became rare or null. even with this increased protection of the area and the rare burning events, the study elpino-campos et al. (2007) sampled ten species more than the ones in chaibub (2013). a. pallipes showed the highest abundance in regenerating savanna, with approximately 60% of individuals collected in the first year and more than 64% in the second, which corroborates in the study by gomes and noll (2009). the large representativity of a. pallipes in the regenerating savanna, an area with a high degree of degradation, possibly occurs because this species is less sensitive regarding the environmental degradation, since it nests in cavities in the soil (noll et al., 1997). the same applies to the second year where the highest frequency is of this species over others. sociobiology 66(4): 582-591 (december, 2019) 589 a. vicina was not registered in the second year, which can be explained since this species needs hollow tree trunks or large natural cavities such as caves, since it is the species that builds the largest colonies, representatives of the subfamily polistinae, with its population reaching more than one million individuals and a great capacity for foraging (zucchi et al., 1995; oliveira et al., 2010). a. pallipes has wide distribution, from costa rica to argentina and paraguay (richards, 1978). the great variety of habitats to its nest foundation gives a. pallipes greater plasticity, which explains the higher frequency in the regenerating savanna, an environment with high anthropogenic impact and degradation (zucchi et al., 1995; noll et al., 1997). a. pallipes, is possibly less sensitive in relation to environmental degradation (noll et al., 1997). in a study conducted in four cities in the northwest of the state of são paulo, a. pallipes was present in all study areas (tanakajunior & noll, 2011), also being representative in other studies such as gomes and noll (2009). species like poly. sericea (olivier), poly. paulista (h. von ihering), b. lecheguana and poly. ignobilis (haliday) present a wide range of ecological tolerance than other species and are usually dominant in open ecosystems, with strict environmental conditions (santos et al., 2007). in this study, these species, even with less frequency, were able to hold on to the second year. especially b. lecheguana that was observed in the first month of post-fire collection, but the nest had deceased due to fire and more individuals were not observed in the remaining months. in the study by souza et al. (2010), through multivariate analysis of indication value (species indicator value), obtained through the monte carlo test, in order to assess the ecological relationship between social wasps and the different faces in the area of study revealed that poly. fastidiosuscula (saussure), was present only in preserved areas, however, for m. drewseni the indication value was for degraded areas. in this study, poly. fastidiosuscula presented the same abundance (n = 3) before and after the fire. but m. drewseni, corroborating souza et al. (2010), presented only one individual in the first year and after the fire were ten where degradation was bigger. rodrigues (1996) and vieira et al. (1996) report that resettlement may be of two types, endogenous or exogenous. endogenous resettlement is carried out by individuals who survived the fire, either by taking refuge in shelters, nests, or move temporarily to adjacent areas and when resettling in the area their offspring spreads. exogenous recolonization is characterized by the death of local individuals and establishment of immigrant individuals, usually coming from adjacent localities that were not affected by fire (marini-filho, 2000). this area of regenerating savanna is bordered on the south by the universidade estadual paulista unesp and on the northeast by the state forest edmundo navarro de andrade (cardoso-leite et al., 2004). in the lower part of the area, there is a river that forms a swampy wood. (plattinetijúnior, 1979). these two vegetation areas may have served as a refuge for social wasp species or feeding the burned area with species. however it is difficult to determine how long the animal community recovered from the impacts of fire. what the different studies seem to show is that apparently these recovery processes seem to be more rapid for the invertebrate fauna (vasconcelos et al., 2008) than for the small vertebrates fauna (fariaet al., 2004; henriques et al., 2006). once noted the considerable impact on the richness and abundance of social wasps after the fire, monitoring in the long term is important to verify that the restoration of fauna occurs. these aspects motivate most studies in this area in the long term. acknowledgments to professor dr. reinaldo monteiro from the department of bothanics of são paulo state university (unesp, rio claro, brazil) for his classification of the areas. to capes higher education personnel improvement coordination for the financial support in the doctorate and the sandwich period in mexico. to the inecol (institute of ecology). references abrams, m.d. 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(1995). agelaia vicina, a swarmfounding polistinae with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137. doi: 10.13102/sociobiology.v61i3.324-331sociobiology 61(3): 324-331 (september 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 termite assemblages in dry tropical forests of northeastern brazil: are termites bioindicators of environmental disturbances? ab viana junior1, vb souza2, yt reis1, ap marques-costa1 introduction termites are among the most abundant insects in tropical ecosystems (bignell & eggleton, 2000). they are considered ‘ecosystem engineers’ because they have the ability to greatly modify their own habitats and to alter the structure of the ecosystems in which they live (jones et al., 1994; ferreira et al., 2011). the ecological importance of termites is based on several aspects, which have been observed in arid and semi-arid ecosystems. they participate in the decomposition and in the flow of carbon and nutrients (bignell & eggleton, 2000; bandeira & vasconcellos, 2002), moving particles at different depths (jouquet et al., 2011), increasing soil porosity (holt & lepage, 2000), and consequently increasing water retention, affecting directly the vegetation structure and the local primary proabstract termites exhibit several characteristics that emphasize their potential as bioindicators of habitat quality for use in environmental monitoring studies, but little is known about this group in vegetations of semi-arid regions of brazil. the present study was conducted in three areas of caatinga under different levels of anthropogenic disturbance, in the high backwoods of sergipe state, aiming to verify whether termite communities create different groups associated with the conservation of the area, by analyzing richness, abundance, and composition. twelve transects of 65 x 2 m were set up in each area, where each one consisted of five plots of 5 x 2 m, making it possible to collect termites in all potential nesting and foraging sites. five feeding groups of termites were sampled: (wf) wood-feeders, (sf) soil-feeders, (swf) soil/wood interface-feeders, (lf) litter-foragers, and (spf) specialized-feeders. soil samples were collected from each plot in order to measure the environmental variables particle size, moisture percentage, and soil ph. overall, richness and abundance were significantly different in the three studied areas. wood-feeders were the most dominant in number of species and number of encounters collected at all sites, whereas the composition of termites in each area, given the environmental disturbances, was distinct. the environmental variables reinforced that the areas are different in terms of degree of conservation. the agreement between environmental variables and ecological data for species composition fortifies the potential of termites as biological indicators of habitat quality in areas of caatinga of northeastern brazil. sociobiology an international journal on social insects 1 universidade federal de sergipe, são cristóvão, se, brazil. 2 universidade tiradentes, aracaju, se, brazil. article history edited by alexandre vasconcellos, ufpb-brazil received 08 february 2014 initial acceptance 23 march 2014 final acceptance 09 june 2014 keywords bioindicator; caatinga; environmental variables; feeding groups; isoptera. corresponding author arleu barbosa viana-junior universidade federal de sergipe programa de pós-grad. em ecologia e conservação av. marechal rondon, s/n jardim rosa elze são cristóvão, se, brazil 49100-000 e-mail: arleubarbosa@yahoo.com.br ductivity (nash & whitford, 1995). for these reasons, they are considered key organisms to maintaining the structure and functional integrity of ecosystems (holt & coventry, 1990; whitford, 1991) and have been considered for ecological monitoring analysis (brown jr, 1997). termites had a score of 20, on a scale from 0 to 24 established by brown jr (1991), in analysis of potential bioindicators using some animal groups (being after butterflies and ants). this score was given following the attributes such as taxonomic and ecological diversity, easy identification, widespread geographic distribution, functional importance, sedentarism, and good response to disturbances (brown jr, 1991; 1997). however, still little is known about the effects of environmental disturbances in areas of caatinga affecting termites. it is common knowledge that caatinga, despite being research article termites sociobiology 61(3): 324-331 (september 2014) 325 a brazilian endemic biome, has been changed over decades. it used to occupy an area of about 840,000 km2 (santos et al., 2011), corresponding to 54% of the northeastern region and 11% of the national territory (alves et al., 2009). currently, about 45.3% of its area is degraded, being ranked as the third brazilian biome most changed by humans, after the atlantic forest and the cerrado (leal et al., 2005), and it may have its status changed to the second most disturbed, according to castelletti et al. (2003). caatinga biome presents only eleven strictly protected areas, which correspond to less than 1% of the biome under legal protection, being the biome with fewer and lesser extent of protected areas in the country (leal et al., 2005). this study was conducted in three areas under different levels of anthropogenic disturbance, in two municipalities of sergipe state, aiming to verify if termite communities are associated with areas of different levels of conservation. some abiotic variables were also evaluated, in order to check, for example, if there was some sort of association between these variables and the established environmental structures. also, it was investigated if termites could reflect, on their community composition, the variation of the habitat, as observed in other environments, like in humid (eggleton et al., 2002; jones et al., 2003) and dry tropical forest (vasconcellos et al., 2010; alves et al., 2011). material and methods study site this study was conducted from april to may 2012 and from november 2012 to january 2013, in two municipalities of the state of sergipe, northeastern brazil: at the protected area named monumento natural grota do angico (9°39’s/37°41’w), in poço redondo, and at the permanent protection area named fazenda são pedro (10°02›s/37°24›w), in porto da folha. both areas are characterized as dry tropical forests, with predominance of xeric vegetation, presence of cacti [pilosocereus gounellei (a. weber ex. k. schum.) bly. ex. rowl., melocactus zehntneri (britton & rose) luetzelb, selenicereus grandiflorus (l.) britton & rose], shrubs and trees [poincianella pyramidalis (tul.) queiroz, aspidosperma pyrifolium mart., sideroxylon obtusifolium (humb. ex. roem. & schult.) td penn], and bromeliads [bromelia laciniosa mart. ex. schult., bromelia pinguin l.]. the average annual temperature goes up to 26°c, with annual average rainfall of 550 mm, being the period of rain concentrated from march to june. the choice of study areas was based according the land use and historic conservation; thus, the areas were classified as: a1, abandoned pasture area, characterized by sparse vegetation with predominance of herbaceous and shrubs plants, with presence of cattle; a2, area in regeneration process for five years, located in the monumento natural grota do angico, characterized by being more heterogeneous than the area a1, with presence of trees 4-6 m high, and presence of bromeliads; and a3, located at the fazenda são pedro, without disturbance for over 30 years, characterized by dense vegetation, with trees 20 m high, and presence of bromeliads. termite sampling a protocol similar to that described by jones and eggleton (2000) was applied to each sampling site. twelve transects of 65 x 2 m were installed in each area, subdivided into five plots of 5 x 2 m, spaced from each other by 10 m and alternating to the right and left, for a total sample of 60 spots and area of 600 m2/area. distances of 100 m between each transect and 50 m from the edge of the fragment was maintained. the number of plots where a given species was present was used as estimation for relative abundance (jones, 2000; bignell & eggleton, 2000; oliveira et al., 2013). as standard time scale, each plot was explored for 1h/person, making it possible to collect termites in all potential nesting and foraging sites. the specimens were identified to generic level with the aid of identification keys (constantino, 1999), and to species level by comparisons with samples previously identified and housed at the collection of the laboratório de agricultura e pragas florestais of the universidade federal de sergipe, and at the museu de zoologia da universidade de são paulo (mzusp). feeding groups all genera and species identified were classified into five feeding groups, according to the classification suggested by swift and bignell (2001), and to information in the literature (gontijo & domingos, 1991; desouza & brown, 1994; mélo & bandeira, 2004; reis & cancello, 2007; vasconcellos et al., 2010; alves et al., 2011), namely: (wf) wood-feeders (termites feeding on wood and wood litter, including dead branches still attached to trees); (sf) soil-feeders (termites feeding deliberately on mineral soil, with higher proportions of soil organic matter and silica, and lower proportions of recognizable plant tissue than in other groups); (swf) soil/ wood interface-feeders (termites feeding in highly decayed wood which has become friable and soul-like, or predominantly within soil under logs or soil plastered on the surface or inside of rotting logs or mixed with leaf litter in stilt-root complexes); (lf) litter-foragers (termites foraging on leaves and small woody items, often taken back and stored temporarily in the nest); and (spf) specialized-feeders (species of termites that feed on fungi, algae, lichens on the bark of trees, manure and vertebrate carcasses). abiotic variables the following abiotic variables related with soil were analyzed: particle size, moisture, and ph. for particle size ab viana junior et al. termites assemblages in the caatinga of brazil326 analysis, 300 grams of soil were collected from each plot using an auger, totaling 30 samples/area. all samples were taken to the instituto de tecnologia e pesquisa de sergipe (itps), where the percentages of sand, silt and clay were measured. in each plot, moisture and soil ph measurements were taken using a ph meter (ph instrutherm 2500). statistical analyses an one-way anova with tukey’s test a posteriori was conducted to verify significant differences in relative abundance and mean richness per transect between the three areas sampled. species richness was estimated for each site using the non-parametric richness estimator jacknife1 (colwell & coddington, 1994), considered one of the best tools to estimate this parameter (palmer, 1990; walther & moore, 2005). based on samples from each plot, accumulation curves were constructed, with 1000 randomizations to compare richness between sampling sites. to analyze the species composition, a non-metric multidimensional scaling (nmds), with data ordered by transects, and a matrix of presence/absence of the species, were performed. an analysis of similarity (anosim) was carried out with a significance level of 95%, to verify the existence of significant differences in species composition between sampling sites; jaccard similarity index was used to measure distance (muellerdombois & ellenberg, 1974; hammer et al., 2001). furthermore, a principal component analysis (pca) with the abiotic data was used to verify if there was spatial segregation between the respective areas (clarke & warwick, 2001), for which the data were logarithmized. in the pca, the variable sand was excluded due to high correlation with silt and clay (0.86 and 0.79, respectively). this procedure is advisable when there is a strong correlation between the variables analyzed, causing no loss of information (clarke & warkick, 2001). to verify that the variables differ statistically between the areas, an one-way anova was performed with all five environmental variables (tukey post hoc test, p < 0.05), and with the values of the first pca components. the statistical software r (r development core team, 2008) was used to make anova and tukey’s test. the richness accumulation curve was performed using the program estimates 9.1.0 (colwell, 2009). the nmds, anosim and the pca were performed with the aid of statistical software past (hammer et al., 2001). results one hundred and eighty samples of termites, classified in three families, 12 genera and 16 species, were found at the experimental sites. termitidae was the most abundant and richest family, with 14 species collected, followed by kalotermitidae and rhinotermitidae, with one species each. only three species were common for all areas, namely: nasutitermes macrocephalus (silvestri), heterotermes sulcatus (mathews), and amitermes amifer silvestri. two of the sixteen species collected were exclusive of the area a1 (anoplotermes sp. and amitermes sp.), and five of the area a3 (rugitermes sp., ruptitermes sp., cylindrotermes sp., inquilinitermes fur (silvestri), and microcerotermes cf. indistinctus mathews (table 1). significant differences in relative abundance (f2, 33=12.70; p < 0.01), and in the richness of termites (f2, 33 = 10.04; p < 0.01), were found among the studied areas (fig. 1). from the values obtained by the non-parametric estimator jacknife1, the estimated species richness was close to the observed (table 1). from the observation of non-overlapping confidence intervals, it was found a difference in the richness between the areas a1 and a3 (fig. 2). in relation to feeding groups, the wood-feeders were the most dominant in number of species and number of encounters collected at all areas, representing about 50% of the total fauna found. the most conserved area (a3) was the only one where all groups were found (fig. 3). the nmds analysis showed differences in the species composition of termites when the sites a1 and a3 were compared (fig. 4), while anosim showed significant difference between all the three areas (table 2). the richness and relative abundance of termites varied positively with the degree of conservation of the area, i.e. the more conserved the area, more abundant and diverse was the termite community. the level of disturbance significantly altered the composition of species and the feeding groups present. the abiotic factors associated with soil analyses (particle size, moisture, and ph) showed significant difference in at least one area (table 3). separation between sampling areas was evident through the principal component analysis (pca) (fig. 5), indicating that such areas can be considered different; anova of the first components showed significant differences between the studied areas (f2, 15 = 20.25; p < 0.001). discussion results found by araujo (1970) and constantino (1998) showed termitidae as the richest family of isoptera with regards to number of species, abundance, and diversity in ecological terms, which is consistent with the results found here. in another hand, kalotermitidae, a family that includes all drywoods and some dampwoods termites that do not require soil contact to survive, presented low abundance in the areas sampled. in this family, different species have different temperature and moisture requirements, but generally speaking, they inhabit and eat various types of dead wood (cancello, 1996) and may nest in the treetops, hindering their sampling (roisin et al., 2006), which could explain the lack of specimens in our samples. sociobiology 61(3): 324-331 (september 2014) 327 table 1. termites collected in three areas of caatinga, sergipe state, brazil: a1 (pasture area), a2 (scrub forest), and a3 (arboreal forest); feeding groups: wf (wood-feeders), sf (soil-feeders), lf (litter foragers), swf (soil/wood interface-feeders), spf (specializedfeeders). species area a1 area a2 area a3 # of species found feeding group kalotermitidae rugitermes sp. 0 0 2 2 wf rhinotermitidae heterotermes sulcatus (mathews) 10 18 8 36 wf termitidae apicotermitinae anoplotermes sp. 4 0 0 4 sf ruptitermes sp. 0 0 4 4 spf nasutitermitinae constrictotermes cyphergaster (silvestri) 0 5 7 12 spf diversitermes sp. 0 5 14 19 lf nasutitermes corniger (motschulsky) 0 3 4 7 wf nasutitermes macrocephalus (silvestri) 1 8 2 11 wf termitinae amitermes amifer silvestri 7 11 25 43 swf amitermes nordestinus mélo & fontes 4 5 0 9 swf amitermes sp. 1 0 0 1 swf cylindrotermes sp. 0 0 2 2 wf inquilinitermes fur (silvestri) 0 0 1 1 sf microcerotermes cf. exiguus (hagen) 0 1 8 9 wf microcerotermes cf. indistinctus mathews 0 0 11 11 wf termes sp. 0 2 7 9 swf richness 6 9 13 16 number of encounters (relative abundance) 27 58 95 180 estimated richness (jackknife 1) 7.97±2.76 9.98±1.96 13.98±1.96 fig 1. (a) richness average; and (b) abundance per transect in three areas of caatinga, sergipe state, brazil. different letters indicate significant differences given by the tukey test (p<0.05). kalotermitidae can also be absent in a disturbed environment, as it would have the number of trees and dead wood reduced, decreasing the likely nesting sites of the group (vasconcellos et al., 2010). even considering richness and relative abundance between the different areas analyzed, some species were common and had relatively high frequency. the assemblages of termites include a great relative abundance of h. sulcatus, which appears to be one of the most important species to the wood cycle in dry areas and it is very resistant to disturbance (alves et al., 2011). melo and bandeira (2007) measured the influence of this species in wood consumption in the caatinga, and found that it was a generalist species that could consume wood in different stages of decomposition, even when already attacked by other species of termites. the species a. amifer has been recorded in the caatinga (alves et al., 2011; vasconcellos et al., 2010), atlantic forest, and cerrado (mélo & bandeira, 2004), and may be considered of wide distribution. n. macrocephalus is also considered to have a wide distable 2. analysis of similarity (anosim) for the species composition of termites communities sampled in three areas (a1, a2, and a3) of caatinga, sergipe state, brazil. area r-value p-value a1 x a2 0.14 0.01 a1 x a3 0.39 <0.01 a2 x a3 0.36 <0.01 ab viana junior et al. termites assemblages in the caatinga of brazil328 tribution, since it was recorded in all three fragments sampled in this study, being already previously recorded in areas of caatinga (mélo & bandeira, 2004), amazon, atlantic forest, and cerrado (constantino, 2005; alves et al., 2011; souza et al., 2012). to date, there is no work with regards to its biology or ecology. from the results of relative abundance, average richness per transect, and the accumulation curve, it is possible to suggest that the richness and abundance of termites can be changed by altering habitat, being directly related to the conservation of the area, in agreement with results observed in other researches on termites, in savannah (dosso et al., 2012; cunha & orlando, 2011; carrijo et al., 2009; brandão & souza, 1998), humid tropical forests (ackerman et al., 2009; eggleton et al., 1996; desouza & brown, 1994; bandeira & torres, 1985) or caatinga (alves et al., 2011; vasconcellos et al., 2010; bandeira et al., 2003). fig 2. accumulation species curves estimated for termites with confidence interval of 95%, in three areas of caatinga, sergipe state, brazil, with different levels of disturbance. area a1 (crosses); area a2 (open circles), and area a3 (closed circles). fig 3. termite species richness and relative abundance (encounters) by feeding group, in three areas of caatinga under different levels of disturbance, sergipe state, brazil. a1, abandoned pasture area; a2, regeneration area; a3, conservation area. feeding groups: wf, wood-feeders; sf, soil-feeders; lf, litter-feeders; swf, soil/wood interface-feeders; spf, specialized-feeders. fig 4. analysis of non-metric multidimensional scaling (nmds) for the species composition of termites in three areas of caatinga, sergipe state, brazil: area a1 (crosses); area a2 (open circles), and area a3 (closed circles). fig 5. principal component analysis (pca) for environmental variables in three areas of caatinga, sergipe state, brazil: area a1 (crosses); area a2 (open circles), and area a3 (closed circles). the abundance of wood-feeders, when compared to other groups, is already a result well discussed in the literature (souza et al., 2012; alves et al., 2011; vasconcellos et al., 2005). soil-feeders and soil/wood interface-feeders are more sensitive to environmental disturbances and natural weather fluctuations than wood-feeders in humid forests (desouza & brown, 1994; bandeira et al., 2003), but also, the small amount of organic matter in the soil (consequence of low leaf productivity) could be a contributing factor to the low abundance of those groups in the caatinga environment (mélo & bandeira, 2004). however, the wood-feeders were the group most affected by disturbance in the cerrado and caatinga environments (carrijo et al., 2009; vasconcellos et al., 2010), as shown in the present study. the variation in species composition in the three areas studied corroborates the hypothesis that anthropogenic changes sociobiology 61(3): 324-331 (september 2014) 329 (including deforestation and/or land use) may be responsible for changes in the availability of plant material and/or ecological niches for species (junqueira et al., 2008). as a result, a direct and positive relationship between the local termite diversity and the conservation status of the area could be assumed, i.e., termites responded to disturbances occurred in caatinga sites of the high backwoods of sergipe, mapped through the differences in richness, abundance and composition of species in the areas sampled. alves et al. (2011) analyzing three areas of caatinga quite similar with regards to disturbance levels, observed, through a pca (using eleven environmental variables), no significant difference on termites assemblage composition, whereas vasconcellos et al. (2010) found that areas with different levels of disturbance presented different communities of termites. thus, the results presented here, combined with data from literature, reinforce the potential of termites as biological indicators of environmental quality in the areas of caatinga, which could be applied to other ecosystems. acknowledgments we would like to thank drs. alexandre vasconcellos (ufpb) and genésio ribeiro (ufs) for the important contributions on the master’s dissertation of the first author, which generated this paper; to dr. maurício rocha (mzusp) for the identification of termites specimens included in this study. we also thank to dr. ana paula albano araújo for help in the statistical analysis; to dr. mirian watts and john watts for their suggestions and revision of the paper; and to rony peterson, sidieris da costa, and brisa marina, for their essential help during fieldwork. we also thank the funding agency capes for the master’s scholarship given to the first author. references ackerman, i. l., constantino, r., gauch jr., h. g., lehmann, j.; riha, s. j. & fernandes, e.c.m. 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(1991). subterranean termites and long-term productivity of desert range lands. sociobiology, 19: 235-242. doi: 10.13102/sociobiology.v66i2.3478sociobiology 66(2): 358-366 (june, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 physiological selectivity of insecticides from different chemical groups and cuticle thickness of protonectarina sylveirae (saussure, 1854) and brachygastra lecheguana (latreille, 1824) (hymenoptera: vespidae) introduction fall armyworm spodoptera frugiperda (smith & abbot, 1797) (lepidoptera: noctuidae) is a polyphagous pest capable of causing significant damage in maize, zea mays l., soy, glycine max l. and cotton, gossypium hirsitum l. (nagoshi, 2009; bueno et al., 2011; aguierre et al., 2016). s. frugiperda damage has been reported in north america, central america, brazil, argentina, the united states, and recently in africa (prowell et al., 2004; clark et al., 2007; casmuz et al., 2010; tindo et al., 2017). s. frugiperda caterpillars can occur on maize throughout the plant canopy. hatched caterpillars scrape the leaf limb epidermis and can cut the collar in the next stages. in the late stages, s. frugiperda damages the cartridge and may damage abstract the use of insecticides more toxic to pest than to natural enemies may favor the conservation of these beneficial organisms. this study aimed to evaluate the physiological selectivity of insecticides from different chemical groups to vespidae protonectarina sylveirae (saussure, 1854) and brachygastra lecheguana (latreille, 1824) and cuticle thickness in order to evaluate the tolerance between the two species to the same insecticides. we immersed maize leaves (10 x 10 cm) in insecticide solution in dose (100%), subdose (50%) and distilled water (control) for 5 seconds. we dried the leaves in the shade and subsequently packed them in petri dishes. the 20 wasps, per plate, received 10% honey and the plates were closed with fine organza fabric and elasticated. after 24 hours, we evaluated the percentage of dead wasps. alpha-cypermethrin insecticide was highly toxic to p. sylveirae and b. lecheguana in dose (100%) and subdose (50%). novaluron, chlorantraniliprole, spinosad and indoxacarb insecticides were poorly toxic to p. sylveirae and b. lecheguana in dose (100%) and subdose (50%). the high toxicity of insecticides methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin to p. sylveirae corresponded to a smaller cuticle thickness. therefore, novaluron, chlorantraniliprole, spinosad and indoxacarb insecticides showed physiological selectivity to p. sylveirae and b. lecheguana. sociobiology an international journal on social insects ws soares¹, smd júnior¹, iw da silva², a plata-rueda², ea souza², fl fernandes² article history edited by gilberto m. m. santos. uefs, brazil received 11 may 2018 initial acceptance 02 october 2018 final acceptance 15 may 2015 publication date 20 august 2019 keywords anatomical characterization, neurotoxic insecticide, tolerance, toxicity. corresponding author walyson silva soares instituto de ciências agrárias universidade federal de uberlândia (ufu) cep 38400-902, uberlândia-mg, brasil. e-mail: walysonagronomia@gmail.com the maize ear during severe infestations. economic damage has already been reported for $ 3 billion a year in africa (rodriguez-del-bosque et al., 2011; jeger et al., 2017; maiga et al., 2017). controlling this pest can occur by cultural, chemical and biological (trichogramma pretiosum) methods and using plants with different expressions of bacillus thurigiencis (bt) proteins (bt) (capineira, 2005; balestrin & bordin, 2016; bortolotto et al., 2016; roel et al., 2017). among the alternative methods, chemical control may be effective to control s. frugiperda, helicoverpa zea (boddie, 1850) and agrotis ipsilon (hufnagel, 1766) (lepidoptera: noctuidae), as shown by blanco et al. (2014). however, overuse of insecticides has resulted in the development of resistant populations and negative impacts on natural enemies (yu et al., 2003; romeis et al., 2006; ahmad & arif, 2010). 1 universidade federal de uberlândia (ufu), uberlândia-mg, brazil 2 universidade federal de viçosa (ufv), rio paranaíba-mg, brazil research article wasps sociobiology 66(2): 358-366 (june, 2019) 359 beneficial organisms of agricultural importance are important for reducing pest population. biological control with predatory wasps polybia ignobolis (haliday, 1836), vespula shidai (linneaus, 1758), vespula vulgaris (linneaus, 1758) (picanço et al., 2010), protonectarina sylveirae (saussure, 1854) and brachygastra lecheguana (latreille, 1824) (hymenoptera: vespidae) is a viable method for controlling lepidoptera insects (ross & matthews, 1991; miranda et al., 1998; gonring et al., 2002; gonring et al., 2003; pereira et al., 2007a; fernandes et al., 2008; picanço et al., 2011; ghoneim, 2014; saraiva et al., 2017). conservation of predatory wasps in integrated pest management (ipm) programs becomes essential, because this organism has a great importance to regulating the population dynamics of insect pests (pereira et al., 2007a). the strategies to conserve natural enemies is to use insecticides with physiological selectivity. selectivity may be physiological when using insecticides more toxic to pest than to natural enemies (pedigo, 1999). physiological selectivity of the insecticides from the anthranilic diamide group (chlorantraniliprole) to p. sylveirae, b. lecheguana and polybia sp., growth regulator (buprofezin) in hymenoptera insects was observed by fernandes et al. (2013) and araujo et al. (2017). high toxicity of pyrethroids (deltamethrin) in dose (100%) and subdose (50%) to p. sylveirae and b. lecheguana was observed by bacci et al. (2009). the use of the recommended dose (100%) for pest control allows the evaluation of the impact of these products on predatory wasps. already the use of the subdose (50%) allows to evaluate the impact of insecticides when half of their concentrations have been decomposed (suinaga et al., 1996). the potential toxicity of insecticides against natural enemies may correlate with their physicochemical characteristics as well as thickness and chemical composition of the wasp cuticle (leite et al., 1998; gusmão et al., 2000; katagi, 2001). on the other hand, the tolerance of wasps to insecticides may be associated with the lower penetration rate of these products to the integument (bacci et al., 2006). biochemical and physiological studies to elucidate the mechanism of physiological selectivity to predatory wasps are unknown (bacci et al., 2006). thus, this study aimed to evaluate the physiological selectivity of insecticides from different chemical groups, with the respective dose (100%) and subdose (50%) for maize, p. sylveirae and b. lecheguana wasps, and cuticle thickness of predatory wasps in order to evaluate the tolerance between the two species with the same insecticides. material and methods we conducted the study at the integrated pest management laboratory of the federal university of viçosa (ufv) rio paranaíba campus from november 2017 to january 2018. p. sylveirae and b. lecheguana wasps were collected in nests on the campus. the nests were in trees and shrubs. later the specimens were packed in plastic bags and sent to ufv campus viçosa under the care of dr. paulo sérgio fiuza (curator and taxonomist of the ufv museum – viçosa) to identify the species. we used insecticides registered to control s. frugiperda in z. mays (mapa, 2017) (table 1). the concentration of the active ingredients used in the bioassay corresponds to 100 and 50% of the dose recorded in the mapa (2017). subdose (50%) was used to verify whether the insecticide was selective when degraded by half its concentration (100%). the experimental design was completely randomized, in a 10 x 2 x 2 factorial scheme (insecticide x concentration of active ingredient x species) with 4 replicates. the experimental plots consisted of 20 wasps of each species (p. sylveirae and b. lecheguana). insecticides (table 1) were diluted in distilled water with adhesive spreader (polyester and silicone copolymer 1000 g l-1). water plus adhesive spreader was the control treatment. maize leaves were cut (10 x 10 cm) and immersed in the insecticide broth for five seconds for each treatment (bacci et al., 2001; galvan et al., 2002). the leaves were then dried in the shade for thirty minutes and packed in petri dishes (9 cm in diameter and 2 cm in height). we prepared a solution with 10% honey to feed 20 wasps per dish. the solution with honey was placed in the lateral walls of the dishes to avoid contact with insecticides. the dishes were covered with fine organza fabric and elasticated. each petri dish, containing the insects, was conditioned in b.o.d (biochemical oxygen demand) at 25 ± 1ºc, 70 ± 1% relative humidity and 12-hour photoperiod. after 24 hours, we evaluated the percentage of dead wasps. the wasps were considered dead when they did not move. subsequently, mortality rates were corrected using the abbott’s formula (abbott, 1925). the insecticides were classified as non-selective or highly toxic (mortalities between 100-70%), moderately selective or moderately toxic (mortalities between 69-30%) and selective or poorly toxic (mortality between 29-0%) (bacci et al., 2006). mortality data were transformed into rank noise (conover & iman, 1981) to perform analysis of variance at p ≤ 0.05 and then the means were compared using the skottknott grouping test ap ≤ 0.05 using the program speed stat 1.0 (mundstock, 2017). anatomy of the abdomen cuticle of p. sylveirae and b. lecheguana wasp species were captured in nests located on the ufv campus rio paranaíba on banana and hibiscus plants. for the experiments, we used four wasps (p. sylveirae and b. lecheguana) and two blocks, each block containing two abdomens. we performed anatomical sections on the abdomens of adult p. sylveirae and b. lecheguana females to verify, through cuticle thickness, the tolerance of insects to insecticides. ws soares et al. – physiological selectivity of insecticides and cuticle thickness360 insecticides chemical group commercial dose (ml ha-1or g ha-1) concentration (mg i.a. ha-1) molecular weight (g mol-1) solubility (mg l-1 or g l-1) indoxacarb 150 ce oxadiazines 400 60 527.8 0.2 alpha-cypermethrin 100 fs pyrethroid 50 5 416.3 2.06 deltamethrin 25 ce 200 5 505.2 1.3 x 10-6 chlorantraniliprole 200 sc anthranilic 125 25 483.15 1.023 methomyl+novaluron 440 + 35 ce oxime methyl carbamate + benzoylurea 500 220 + 17,5 162.20 + 492.7 54.7 novaluron 100 ce benzoylurea 400 40 492.7 3 x 10-6 lambda-cyhalothrin + chlorantraniliprole 50 + 100 sc pyrethroid + anthranilamide 150 7,5 + 15 449.9 6.3 x 10-6 chlorfenapyr 240 sc pyrazole analogue 750 180 407,6 5,28 spinosad 480 sc spinosyns 100 48 732.0 (spinosyn a) +746.0 (spinosyn d) 235 + 0.332 table 1. chemical group, commercial dose, concentration of active ingredient, molecular weight and solubility of the insecticides registered for maize crop, in the control of the caterpillar spodoptera frugiperda. the integument of the wasps’ abdomen was transferred to the zamboni fixative solution (stefanini, 1967) remaining for 24 hours in a vacuum chamber. we submitted the collected material to dehydration to the growing series in ethanol (70, 80, 90 and 95%) for 1 hour. after dehydration, the infiltration occurred on leica historesin and kept in the refrigerator for 24 hours. subsequently, they were added to histomolds with a historesin solution plus leica polymerizer in a 15:1 ml ratio in an oven at 55ºc for 24 hours. historesin blocks containing the integument were sectioned at 3 μm thickness using the rotary microtome. historesin sections were transferred to a vessel with water in order to expand them and avoid historesin overlap over the cuticle coming from the abdomen of the wasps. the sections were collected on 12 glass slides per species, totaling 24 slides. we placed them into the hotplate for slide cutting setting. the tissue attached to the slide was stained with toluidine blue. after staining, the coverslips were placed on the cuttings, fixed with permount, causing permanent slides. the stained slides were photographed under the microscope olympus cx 41 coupled to the nikon d3100 camera. we edited the images (micrographs) and adjusted contrast, white balance, balance and scale insertion in the photoshop cc program. results insecticide significant interaction for insecticide and species (f9.120 = 2.95, p ≤ 0.003) occurred, insecticide and dose (100 and 50%) (f9.120 = 3.79, p < 0.001) for wasp mortality p. sylveirae and b. lecheguana. insecticides (f9.120 = 90.11, p ≤ 0.001), species (f1.120 = 14.91, p < 0.001) and dose (f1.120 = 120.30, p ≤ 0.001) were also significant. insecticide interaction, dose and species (f9.120 = 0.805, p = 0.6127) and species and dose interaction (f9.120 = 0.223, p = 0.6376) were not significant. table 2. mortality (%) of protonectarina sylveirae (p) and brachygastra lecheguana (b) wasps observed after immersion of maize leaves in insecticide broth with the respective doses recommended for maize crop and subdose, in the control of spodoptera frugiperda. insecticides mortality (%) (mean ± se)¹ species n p. sylveirae b. lecheguana means methomyl + novaluron 16 80.6 ± 4.9aa 53.1 ± 6.7bb 66.9 ± 5.4 chlorantraniliprole + lambda-cyhalothrin 16 79.4 ± 5.6aa 53.8 ± 8.2bb 66.5 ± 5.8 deltamethrin 16 71.3 ± 5.3aa 49.4 ± 7.7bb 60.3 ± 5.3 novaluron 16 10.0 ± 3.3ca 4.4 ± 1.7da 7.2 ± 1.9 chlorantraniliprole 16 6.3 ± 2.4ca 13.1 ± 6.4ca 9.7 ± 3.4 spinosad 16 15.0 ± 5.4ca 14.4 ± 5.4ca 14.7 ± 3.7 chlorfenapyr 16 25.0 ± 6.4ba 15.0 ± 5.0ca 20.0 ± 4.1 alpha-cypermethrin 16 78.8 ± 5.2aa 73.5 ± 6.5aa 76.2 ± 4.1 indoxacarb 16 13.6 ± 4.3ca 18.8 ± 8.5ca 16.2 ± 4.6 control 16 0.0 ± 0.0da 0.0 ± 0.0da 0.0 ± 0.0 means 38.2 ± 11.0 29.5 ± 8.0 ¹means followed by upper case letter in the column (comparison between insecticides) and, lowercase in the line (comparison between species), did not differ by the skott-knott skin test p ≤ 0.05. alpha-cypermethrin insecticide was highly toxic (nonselective) to p. sylveirae and b. lecheguana wasps in dose (100%) and subdose (50%) (table 2). methanyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin were highly toxic to p. sylveirae and moderately toxic to b. lecheguana in dose (100%). novaluron, chlorantraniliprole, spinosad and indoxacarb insecticides were poorly toxic (selective) to p. sylveirae and b. lecheguana in dose and subdose (table 2 and 3). sociobiology 66(2): 358-366 (june, 2019) 361 b. lecheguana was the most tolerant species to methomyl + novaluron, chlorantraniliprole + lambdacyhalothrin and deltamethrin. p. sylveirae and b. lecheguana showed similar tolerance to novaluron, chlorantraniliprole, spinosad, chlorfenapyr, alpha-cypermethrin and indoxacarb (table 2). alpha-cypermethrin caused similar mortality among wasp at half the dose. regarding the other insecticides, dose was more toxic to wasps than subdose (table 3). subdose of insecticides resulted in lower wasp mortality in all evaluated treatments compared to the recommended dose (table 3). subdoses of ethanyl + novaluron, chlorantraniliprole + lambdacyhalothrin and deltamethrin were moderately toxic to p. sylveirae and b. lecheguana. anatomy of the abdomen cuticle of p. sylveirae and b. lecheguana b. lecheguana showed higher cuticle thickness than p. sylveirae (fig 1 a and b). the high toxicity of insecticides methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin to p. sylveirae corresponded graphically to the smaller cuticle thickness of this species (fig 1a). table 3. wasp mortality (%) observed after maize leaf immersion in insecticide broth with the respective doses recommended (100%) for maize crop and subdose (50%), in the control of spodoptera frugiperda. insecticides mortality (%) (mean ± se)¹ dose (%) n 100 50 means methomyl + novaluron 16 79.4 ± 4.9aa 54.4 ± 7.4bb 66.9 ± 5.4 chlorantraniliprole + lambda-cyhalothrin 16 80.0 ± 5.7aa 53.1 ± 7.8bb 66.5 ± 5.8 deltamethrin 16 73.8 ± 5.4aa 46.9 ± 6.4bb 60.3 ± 5.3 novaluron 16 13.1 ± 2.3ca 1.3 ± 0.8cb 7.2 ± 1.9 chlorantraniliprole 16 18.8 ± 5.2ca 0.6 ± 0.6cb 9.7 ± 3.4 spinosad 16 27.5 ± 3.2ba 1.9 ± 1.3cb 14.7 ± 3.7 chlorfenapyr 16 31.3 ± 5.0ba 8.8 ± 3.6cb 20.0 ± 4.1 alpha-cypermethrin 16 79.4 ± 7.4aa 73.1 ± 3.7aa 76.2 ± 4.1 indoxacarb 16 26.3 ± 7.0ba 6.3 ± 3.7cb 16.2 ± 4.6 control 16 0.0 0 ± da 0.0± 0 ca 0.0 ± 0 means 42.9 ± 9.7 24.6 ± 9.0 ¹means followed by upper case letter in the column (comparison between insecticides) and lowercase in the line (comparison of recommended dose and subdose for each insecticide), did not differ by the skott-knott skin test p ≤ 0.05. fig 1. corrected mortality (%) and cuticle thickness of the protonectarina sylveirae and brachygastra lecheguana wasps at the recommended dose (a) and histological cutting of the abdomen of p. sylveirae and b. lecheguana, emphasizing the cuticle (b). yellow arrows in the histological cutting of the abdomen indicate the cuticle thickness of the species. discussion neurotoxic insecticides showed variable toxicity to p. sylveirae and b. lecheguana in dose (100%) and subdose (50%). the most toxic insecticides to wasps were alpha-cypermethrin, followed by methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin. deltamethrin was highly toxic in dose (100%) and subdose (50%) to p. sylveirae and b. lecheguana (100% mortality) in the physiological selectivity study by galvan et al. (2002). similarly, this insecticide was toxic to p. sylveirae in dose and subdose (bacci et al., 2009) and highly toxic (84% mortality) to the polybia sericea wasp (olivier, 1791) (hymenoptera: vespidae) (santos et al., 2003). few studies have reported the physiological selectivity of methomyl + novaluron to predatory wasps. liu et al. (2016) observed high toxicity of methomyl to telenomus remus (nixon, 1937) (hymenoptera: scelionidae), parasitoids of s. frugiperda eggs. in this study, the high toxicity caused by alphacypermethrin to two predatory wasp species continued with half the doses. therefore, besides the high impact of this insecticide at application, this effect persists even after the decomposition of half of the active principles (bacci et al., 2006). ws soares et al. – physiological selectivity of insecticides and cuticle thickness362 the high mortality rate of p. sylveirae and b. lecheguana caused by alpha-cypermethrin, methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin may be due to the penetration rate of the insecticide in the insect integument. this is a result of the relationship between the insecticide affinity with the thickness and chemical composition of the cuticle and the compound solubility (yu, 2008). thus, more lipophilic compounds are inversely proportional to solubility and tend to penetrate to a greater extent in the insect’s body, given the similarity with the cuticle, which was the insecticide deltamethrin case (less than 0.002 ppm solubility in water) (leite et al., 1998; gusmão et al., 2000). however, low penetration rate of insecticides from the diamide group, oxadiazine, spinosyns and pyrazole analogue to wasp cuticle may be the explanation of the physiological selectivity (salgado et al., 1998; wing et al., 1998; jeschke, 2016; cotton institute of mato grosso do sul, 2016). the other hypothesis related to physicochemical properties of insecticides (katagi, 2001). the main physicochemical properties are molecular weight and polarity. the lower the molecular weight and polarity the higher the insecticide penetration in the insect (stock & holloway, 1993; leite et al., 1998; pereira et al., 2014). this may explain the high mortality rate due to the molecular weight of alpha-cypermethrin (416.3 g mol-1) and deltamethrin (505.21 g mol-1). spinosad (spinosad a = 731.98 g mol-1, spinosad d = 746.0 g mol-1) resulted in lower predatory wasp mortality. (tomlin, 1995; thompson et al., 1999; pesticide properties database [ppdb], 2018). the highest toxicity of the methomyl + novaluron and chlorantraniliprole + lambda-cyhalothrin mixtures occurred due to methomyl and lambda-cyhalothrin in the mixture. these had a synergistic effect. chlorantraniliprole has higher larvicidal activity and novaluron is a growth regulator of young phases, having little effect on adults (cordova et al., 2006; xu et al., 2017). lambda-cyhalothrin is a pyrethroid of insect contact and has a broad spectrum of action, being toxic to several lepidoptera and coleoptera insect groups and natural enemies (ruberson & tilman, 1999; santos et al., 2007; palmquist, 2012). the anthranilic diamide (chlorantraniliprole) was poorly toxic to b. lecheguana and p. sylveirae, probably due to the greater affinity with the lepidoptera ryanodine receptors, which justifies the selectivity of this group to b. lecheguana and p. sylveirae adults (jeschke, 2016; araujo et al., 2017). in addition, low toxicity may also relate to the increased rate of metabolism of the compound by wasps compared to the pest or target changing of the insecticide against natural enemies (yu, 1987). fernandes et al. (2013) observed this response with p. sylveire, b. lecheguana and polybia sp. the low toxicity of novaluron to the wasps may be because it acts during the young phase, preventing the cuticle formation in the larval stage (desneux et al., 2007; jeschke, 2016). this study targeted adult insects. b. lecheguana and p. sylveirae wasps showed low mortality when submitted to the spinosad, chlorfenapyr and indoxacarb treatment. the mechanism that elucidates the physiological selectivity of these insecticides to p. sylveirae and b. lecheguana wasps are not well understood due to lack of biochemical and physiological studies. araujo et al. (2017) found that indoxacarb and spinosad caused low mortality of solenopsis saevissima (smith, 1855) (hymenoptera: formicidae). spinosad selectivity has been reported for most predators. similarly, the probable physiological selectivity of indoxacarb may be related to the low bioactivation of esterases and/or transferases enzymes in detoxification, which has already been observed in some parasitoids, chrysopodes and coccinellidae (willian et al., 2003; zhao et al., 2003; campos et al., 2011; pereira et al., 2014). anatomy of the abdomen cuticle of p. sylveirae and b. lecheguana b. lecheguana tolerance compared to p. sylveirae can be observed with the treatment methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin. these insecticides were less toxic (49 – 54%) for b. lecheguana and more toxic (71 – 80%) for p. sylveira. deltamethrin was highly toxic to b. lecheguana and p. sylveirae causing 100% mortality (galvan et al., 2002). b. lecheguana tolerance to deltamethrin compared to p. sylveirae differs from that obtained by galvan et al. (2002) and crespo et al. (2002). results show that the cuticle thickness of b. lecheguana is twice as thick as p. sylveirae. methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin, and deltamethrin insecticides applied to p. sylveirae wasps showed higher mortality compared to b. lecheguana. the greater the thickness the greater the difficulty of penetration, mainly by the physical and chemical barrier that may have higher wax concentration, making it difficult to penetrate insecticides. several studies have shown lower penetration of β-cypermethrin, permethrin, deltamethrin and lambda-cyhalothrin due to the cuticle thickness of bactrocera dorsalis hendel (diptera: tephritidae), anopheles funestus (giles, 1900) (diptera: culicide), helicoverpa armigera (hubner, 1827) (lepidoptera: noctuidae) cimex lectularius (linnaeus, 1758) (hemiptera: cimicidae) and spodoptera litura (fabricius, 1775) (lepidoptera: noctuidae) (ahmad et al., 2006; liu et al., 2009; wood et al., 2010; lin et al., 2012; lilly et al., 2016). final remark the present study provided important practical information to improve ipm systems using insecticides novaluron, chlorantraniliprole, espinosade, clorfernapir and indoxacarbe that presented physiological selectivity to protonectarina sylveirae and brachygastra lecheguana. however, the insecticide alpha-cypermethrin remained highly toxic even in the subdose (50%) and the insecticides methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin in the dose (100%) were not selective to the sociobiology 66(2): 358-366 (june, 2019) 363 wasps p. sylveirae and b. lecheguana. which demonstrates the need to develop effective products for pest and less toxic to the natural enemy. on the other hand, b. lecheguana wasp showed higher cuticle thickness and was more tolerant to insecticides methomyl + novaluron, chlorantraniliprole + lambda-cyhalothrin and deltamethrin compared to p. sylveirae. authors’ contribution designed the experiments (fl fernandes), carried out the fieldwork (smd junior and a plata-rueda), performed the laboratory work (ea souza and iw da silva), carried statistical analysis and wrote the manuscript (ws soares). acknowledgements we thank to conselho nacional de desenvolvimento científico e tecnológico (cnpq), coordenação de aperfeiçoamento de pessoal de nível superior” (capes) and fundação de amparo à pesquisa do estado de minas gerais (fapemig) for financial support. references aguierre, l.a., juarez, a.h., flores, m., cerna, e., landeros, j., fíras, g.a. & harris, m.k. 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(2003). voltagedependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite dcjw. neurotoxicology, 24: 83-96. doi: 10.1016/s0161813x(02)00112-2 1217 refinement of methods for sexing instars and caste members in neotermes koshunensis (isoptera, kalotermitidae) by yasushi miyaguni 1,2, koji sugio 3, kazuki tsuji 4 abstract exact sexing of individuals is important to an understanding of termite societies. we found that the female-specific sexual character of the elongated seventh sternite, known in other termites, is useful for distinguishing the sex of individuals of the dry-wood termite neotermes koshunensis (shiraki) from the fifth instar onward. the method described is highly efficient at sexing instars, nymphs, soldiers and alates. keywords: dry-wood termites, kalotermitidae, neotermes koshunensis, morpholog y, sexual dimorphism. introduction insect societies among the hymenoptera and isoptera (termites) divide labor between reproductive and non-reproductive castes, whereas in hymenopteran societies males almost never play any social role. in termite societies males and females are assumed to share the same social roles (krishna 1969, holldöbler & wilson 1990, roisin 2000). however, a growing body of empirical evidence suggests that this premise in termites does not always hold true, and caste-specific sex biases have been reported (roisin 2000). for a better understanding of the roles of the sexes in termite social systems, we need more empirical data on various termite taxa. with this aim, the sexing of individual termites is indispensable. however, the small morphological differences between male and female termites often make morphological sexing difficult. \1environmental science and conservation biolog y, united graduate school of agricultural sciences, kagoshima university, kagoshima 890-8580, japan 2corresponding author: e-mail: neotenic_of_termite@yahoo.co.jp 3department of science education, faculty of education, university of the ryukyus, okinawa 9030213, japan 4entomological laboratory, faculty of agriculture, university of the ryukyus, okinawa 903-0213, japan 1218 sociobiolog y vol. 59, no. 4, 2012 alates (imagos) of many termites show a female-specific morphological character, viz., a widely extended seventh sternite that covers most or all of the eighth and ninth sternites (weesner 1969). in some species, immature females also show similar sexual characteristics that can be used for sexing (thompson & synder 1920, mensa-bonsu 1976, myles & chang 1984, roisin 1992). zimet and stuart (1982) described the sexual dimorphism in immature stages of reticulitermes flavipes (rhinotermitidae), providing currently the most useful information for sexing immature stages of termites. however, they did not identify the stage at which this character becomes practical for sexing. so far no study has examined the validity of this sexing method in every developmental stage of immatures. we did so by observation of the sternite structure in all immature stages of neotermes koshunensis (kalotermitidae), followed by confirmation of sex by gonadal dissection. materials and methods n. koshunensis is distributed from taiwan to okinawa (ikehara 1966). a colony of n. koshunensis collected on the main island of okinawa in september 2009 was reared in the laboratory (in the dark at ambient temperature) using a plastic box with pine woods as food for about 2 months before the study. a preliminary examination showed that alates of this species show sexual dimorphism in the sternites (y. miyaguni, unpublished data). for every instar, nymphal stage and alates, we used the morpholog y of the sternites to classify individuals into putative females and putative males under a stereomicroscope (×40 magnification at maximum). we then dissected 30 putative females and 30 putative males of each instar and caste to determine the true sex through inspection of the gonads (thompson and synder 1920). instars and castes were defined according to katoh et al. (2007). the sternite structures were recorded on a digital camera attached to the stereomicroscope (olympus e-system e-330; olympus corporation, tokyo, japan). results the sexual dimorphism of nymphs was very clear, especially in pre-alates, which could be sexed under a magnifying glass (×10). sixth instar females showed considerable individual variation in the degree of sternite development, ranging from only a slight extension to near complete coverage of the 1219 miyaguni, y. et al. — methods for sexing in neotermes koshuensis eighth sternite (fig. 1). the sexual difference in soldiers was similar to that of sixth instars. it was less conspicuous in fifth instars (figs. 1, 2). despite this variation, dissections revealed that our sexing of individuals using this morphological indicator was 100% accurate in those developmental stages and castes (table 1). some fourth instar females did not show a sexual character and were misclassified as males, although we correctly determined the sex of other females (table 1). there was a significant difference in the proportion of misidentified individuals between fifth and fourth instars (fisher’s exact test, p < 0.001). all third instars were classified as males by our criterion, as this instar showed no sexual difference in the sternite configuration (table 1). in first and second instars, the gonads were indistinguishable between fig. 1. the illustration of the sternite structures of females in n. koshunensis. numerals on illustration show the sternite numbers. 3l 6l, third to sixth instar; pre-a, pre-alate nymph; a, alate. 1220 sociobiolog y vol. 59, no. 4, 2012 testes and ovaries, and therefore it was not possible to determine the sex. thus, sexing using the seventh sternite was highly accurate in all stages from the fifth instar onward. discussion the sexual characteristics of n. koshunensis females begin to develop from the fourth instar, but the sexual difference in the sternites from the fifth instar on becomes conspicuous enough to be informative as a morphological indicator of sex (table 1, figs. 1, 2). in termite species that have a linear caste developmental pathway, the timing or stage at which the wing buds begin to develop differs among individuals (roisin 2000, katoh et al 2007). the variation that we found in the seventh sternite morpholog y in the fourth to sixth table 1. the result of sexing based on sexual dimorphism. instars & castes n true sex proportion misidentified male female putative male 3rd instar 30 20 10 0.3 4th instar 30 18 12 0.4 5th instar 30 30 0 0.0 6th instar 30 30 0 0.0 early nymph 30 30 0 0.0 pre-alate nymph 30 30 0 0.0 alate 30 30 0 0.0 soldier 30 30 0 0.0 putative female 3rd instar 4th instar 30 0 30 0.0 5th instar 30 0 30 0.0 6th instar 30 0 30 0.0 early nymph 30 0 30 0.0 pre-alate nymph 30 0 30 0.0 alate 30 0 30 0.0 soldier 30 0 30 0.0 1221 miyaguni, y. et al. — methods for sexing in neotermes koshuensis instar females of n. koshunensis may reflect a similar individual difference in development. although the generality of our finding in n. koshunensis is still to be confirmed in other colonies, our study indicates that sternite morpholog y can be used for sexing some younger stages. acknowledgments we thank h. tatsuta, e. vargo, h. tanaka, s. dobata and m. k. hojo. who kindly gave us invaluable advice during the course of the study. fig. 2. the illustration of the sternite structures of males in n. koshunensis. numerals on illustration show the sternite numbers. 3l 6l, third to sixth instar; pre-a, pre-alate nymph; a, alate. 1222 sociobiolog y vol. 59, no. 4, 2012 references hölldobler, b. & e. o. wilson 1990. the ants. cambridge, ma. harvard univ. press. 732 p. ikehara, s. 1966. distribution of termites in ryukyu archipelago. bulletin of arts & science division, university of the ryukyus, (mathematics & natural sciences). 9: 49-178. katoh, h., t. matsumoto & t. miura 2007. alate differentiation and compound-eye development in the dry-wood termite neotermes koshunensis (isoptera, kalotermitidae). insectes sociaux 54: 11-19. krishna, k. 1969. introduction. pp. 1-18. in: krishna, k. & f. m. weesner (eds.). biolog y of termites vol.1. academic press. new york. mensa-bonsu, a. 1976. the production and elimination of supplementary reproductives in porotermes adamsoni (froggatt) (isoptera, hodotermitidae). insectes sociaux 23: 113-154. myles, t. g. & f. chang 1984. the caste system and caste mechanisms of neotermes connexus (isoptera: kalotermitidae). sociobiolog y 9 (3): 163-319. roisin, y. 1992. development of non-reproductive castes in the neotropical termite genera cornitermes, embiratermes and rhynchotermes (isoptera, nasutitermitinae). insectes sociaux 39: 313-324. roisin y. 2000. diversity and evolution of caste patterns. pp. 95-119. in: t. abe, d. e. bignell & m. higashi (eds.). termites: evolution, sociality, symbioses, ecolog y. kluwer academic publishers. dordrecht. thompson, c. b. & t. e. synder 1920. the “third form,” the wingless reproductive type of termites: reticulitermes and prorhinotermes. journal of morpholog y. 34 (3): 591633. weesner, f. m., 1969. external anotomy pp. 19-47. in: krishna, k. & f. m. weesner (eds.). biolog y of termites vol.1. academic press. new york. zimet, m. & a. m. stuart 1982. sexual dimorphism in the immature stages of the termite, reticulitermes flavipes (isoptera: rhinotermitidae). sociobiolog y 7 (1): 1-7 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4614sociobiology 67(2): 261-267 (june, 2020) introduction studies on the nesting biology of wild bee species that have the potential to be managed as crop pollinators are an important step towards the development of management system for these bee species (bosch & kemp, 2002). centris analis fabricius is one of the wild bee species that have risen more interest as manageable pollinator in brazil, given its abundance and great ability to colonize artificial nesting substrates (jesus & garófalo, 2000; gazola & garófalo, 2009; oliveira & schlindwein, 2009; pina & aguiar, 2011; martins abstract the ideal cavity dimensions for neotropical cavity-nesting bees with the potential to be managed as pollinators have not been getting proper attention. we investigated whether the occupancy of trap-nests by centris analis fabricius and tetrapedia diversipes klug, and other nesting aspects, are affected by trap-nest length. the used trap-nests were cardboard tubes measuring 5, 10, 15 and 20 cm in length, and ø 8 mm. the percentage of occupied trap-nests of 10 cm by c. analis was higher than that of the 5 cm ones (χ2=11.17, gl=1, p<0.001). on the other hand, there was not difference between the occupation of 10 and 15 cm long trap-nests (χ2=0.51, gl=1, p=0.48), and between those measuring 15 and 20 cm long (χ2=1.36, gl=1, p=0.24). t. diversipes occupied a smaller number of 5 cm trap-nests than the 10 cm (χ2=1.52, gl=1, p=0.22), as well as that the 15 cm were more occupied than the 10 cm trap-nests (χ2=4.23, gl=1, p=0.04); moreover, there was not difference between the occupation of 15 and 20 cm trap-nests (χ2=0.28, gl=1, p=0.59). both species showed higher percentages of dead immatures in nests set in the shortest trap-nests, whereas these mortality percentages were lower in the longest ones. by taking into consideration that there was not significant difference in many of the assessed parameters in comparison to values recorded for 15 and 20 cm long trap-nests, it seems likely to recommend the adoption of 10 cm long trap-nests for c. analis reproduction in agricultural sites that depend on the pollination service provided by this bee species. sociobiology an international journal on social insects co santos1, pec peixoto2, cml aguiar3 article history edited by astrid kleinert, usp, brazil received 18 july 2019 initial acceptance 28 january 2020 final acceptance 08 april 2020 publication date 30 june 2020 keywords nesting biology, cavity-nesting bees, crop pollinator. corresponding author claudia oliveira dos santos programa de pós-graduação em ecologia e evolução (ppgecoevol/uefs) universidade estadual de feira de santana av. transnordestina s/nº, novo horizonte cep 44036-900, feira de santana-ba, brasil. e-mail: cauoliver2@yahoo.com.br et al., 2012; magalhães & freitas, 2012). knowledge about the biology of this species has been growing significantly; there are data about the local abundance and time distribution of the nesting activity of populations living in different landscapes, including crop areas (oliveira & schlindwein, 2009; pina & aguiar, 2011; martins et al, 2012; magalhães & freitas 2012), as well as about nest architecture, nesting behavior (jesus & garófalo, 2000) and the attack of natural enemies to brood cells (gazola & garófalo, 2003). on the other hand, other nesting aspects, such as the ideal dimensions of nesting cavities, have been poorly investigated (alonso et al., 2011). 1 pós-graduação em ecologia e evolução, universidade estadual de feira de santana (uefs), bahia, brazil 2 universidade federal de minas gerais (ufmg), belo horizonte, minas gerais, brazil 3 universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil research article bees cavity length affects the occupation of trap-nests by centris analis and tetrapedia diversipes (hymenoptera: apidae) co santos, pec peixoto, cml aguiar – cavity length affects the occupation of trap-nests262 many aspects of nesting biology can be influenced by the diameter or the length of the used cavities, such as the choice for the cavity (bosch, 1994a; bosch, 1994b; rust, 1998), the number of brood cells produced per nest (bosch, 1994a; bosch, 1994b; alonso et al., 2011), sex ratio (stephen & osgood, 1965; torchio & tepedino, 1980; bosch, 1994b; o’neill et al., 2010; gruber et al. 2011; alonso et al., 2011; seildelmann et al., 2015) and the mortality ratio of immatures (aguiar & pina, 2012; seildelmann et al., 2015). alonso et al. (2011) have assessed the effect of subtle variations (5.5 to 7.0 cm) in trap-nest length on nesting-cavity selection by c. analis females, on the number of brood cells per nest and on sex ratio. other studies focused on testing longer lengths are necessary in order to produce a more consolidated database capable of subsidizing the management of this crop pollinator. the relationship between sex ratio and nesting-cavity dimensions is quite important for centris bees, since only females collect floral resources and provide pollination services to oil-flowers and pollen-flowers. c. analis females are bigger than males (jesus & garófalo, 2000); therefore, their production could be limited in small-diameter cavities, similar to what has been observed in other bee species that show male production bias in narrow cavities (stephen & osgood, 1965; bosch, 1994; o’neill et al., 2010; gruber et al. 2011; seildelmann et al., 2015). accordingly, it is essential knowing the cavity dimensions capable of producing more females in order to be successful in managing centris populations for crop pollination. the aim of the present study was to investigate whether the occupancy of trap-nests by two oil-bee species, c. analis and tetrapedia diversipes klug, and other nesting biology aspects, such as sex ratio and mortality of the offspring, are affected by different trap-nest lengths. materials and methods study site sampling was carried out at twoagricultural areas, dmq (maria quitéria district) (12°16’00” s/ 38°58’00” w) and dhu (humildes district) (12°20’08” s/ 38°51’17” w), feira de santana municipality, bahia state, brazil. the prevailing soil use in dmq is the cultivation of temporary crops (beans, guandu-beans, maize) and of acerola trees (malpighia emarginata) in small properties (family farming). the sampling procedure in dhu was carried out in chácara bocaiúvas (23.4 ha), which grows organic horticultural products. sampling – a sampling point was installed in each site. each sampling point consisted of 16 wooden blocks measuring varying thickness (from 4 cm to 19 cm); each wooden block had 60 cavities. the used trap-nests were cardboard straws, which were closed with cardboard caps in their rear tips. these straws were inserted into cavities drilled lengthwise in the wooden blocks (camillo et al., 1995). the cardboard straws had 8-mm internal diameter, following pina and aguiar (2011), who reported a high nesting frequency of c. analis in trap-nests of this diameter, and 4 different lengths (5, 10, 15 and 20 cm). in all cases, the entire cardboard straw was sheltered inside the wooden block, except for 1 cm in the rear tip of it. each site had four blocks with 240 trap-nests of each of the assessed lengths. the 16 blocks in each location were grouped on steel shelves protected by plastic tarpaulin, based on aguiar et al. (2005). the trap-nests were inspected once a month, from october 2011 to september 2012, with an otoscope. nests presenting concluded closing walls were removed and taken to the entomology laboratory of the universidade estadual de feira de santana (uefs) to be daily observed until the emergence of adults, who were pinned, dry-mounted, separated by sex and taxonomically identified. data analysis to evaluate if the occupation of trap-nests changed according to trap-nest length, we recorded for each trap-nest available (n=240) if it was occupied. then, a chi-square test was applied to assess whether the frequency of occupations (response variable) changed among trap-nestsof different lengths (explanatory variable). in case we found a significant difference among the four trap-nests length, we performed subsequent pairwise comparisons (using chi-square tests) between trap-nests of similar lengths (i.e. 5 and 10 cm, 10 and 15 cm and 15 and 20 cm). a generalized least squares model was performed to assess whether the number of provisioned brood cells changed due to trap-nest length. this model considered the number of brood cells in each trap-nest as the response variable and trapnest length as explanatory variable. the variance of residues was adjusted according to each trap-nest length category, in order to achieve a better fit the model to the collected data. one analysis was carried out for each bee species. the same model structure described above was repeated to test whether the number of emerged adults changed due to trapnest length, but we replaced the number of brood cells by the number of emerged adults. for both analyses considering the number of brood cells and emerged adults, only data of c. analis collected in dmq were analyzed, given the low trapnest occupation recorded in dhm. for t. diversipes only data collected in dhu and for traps nests with 10, 15 and 20 cm were analyzed, given the low trap-nest occupation recorded in dmq and for nests with 5 cm. these analyses were carried out in the nlme package of the r software (r development core team, 2004). a chi-square test was applied to verify whether the number of males and females (sex ratio) of the offspring differed from an expected proportion of 1:1. to calculate the expected proportion for each sex, we divided the total number of offspring by two. to evaluate if sex ratio changed according to trap-nest length, we performed a general linear model. for this model we considered the number of males in relation to the total number of individuals in each trap-nest sociobiology 67(2): 261-267 (june, 2020) 263 as the response variable and trap-nest length as the predictor variable. the general linear model was not carried for t. diversipes due to the low sample size for trap-nests of 5, 10 and 20 cm length. a generalized linear model with poisson error distribution was performed to assess whether the number of brood cells with dead individuals changed due to trap-nest length. the number of dead individuals was considered as the response variable and trap-nest length was considered the explanatory variable. this analysis was only applied to c. analis in the dmq site, given the low nesting frequency observed in the dhm site. regarding t. diversipes, it was not possible to perform this analysis because offspring mortality was very low. generalized linear models with poisson error distribution were used to assess the association between the number of occupied trap-nests on every month of the year and the mean temperature, humidity and rainfall. weather variables were obtained in the weather station of the universidade estadual de feira de santana, located in the same municipality as the sampling area. nine models were built (tables 4 and 5) and site and species were maintained as predictor variables in most of them (the exception occurred for the null model). we opted to keep site and species in most models because we were interested in evaluate if the number of occupied cells varied in response to climatic variables independent of site and species. the akaike information criterion was used to select the most parsimonious model to explain the relationship between number of occupied cavities and the climatic variables. the significance of the selected model was calculated using a maximum likelihood ratio test. for this, the selected model was compared with a null model that retained only species and site as predictor variables. all analyses were performed in the r software (r development core team, 2004). results 1. occupation of pre-existing cavities the number of nests established by bees in the dmq site (acerola orchard) was almost twice the number recorded for the site used for diversified crops (dhu) (table 1). c. analis was the dominant species in the dmq site, either when it comes to number of established nests (91% of all bee nests) or number of brood cells (n=460). on the other hand, this species recorded low frequency in the dhu site, whereas t. diversipes established 80% of all bee nests (202 brood cells) in it. other cavity-nesting bee species (centris tarsata smith and megachile spp) have established few nests in both sites (table 1). the percentage of occupied trap-nests by c. analis differed between trap-nests of different lengths (χ2=17.85, df=3, p<0.001, table 1). the proportion of occupied trapnests recorded for the 10 cm trap-nests was higher than that of the 5 cm ones (χ2=11.17, df=1, p<0.001), but there was not difference in the proportion of occupied nests between 10 and 15 cm trap-nests (χ2=0.51, df=1, p=0.48) and between the 15 and 20 cm ones (χ2=1.36, df=1, p=0.24). the percentage of nests established by c. analis (n=84) in dmq for nests with 5, 10, 15 and 20 cm were respectively 7%, 30%, 37% and 26%, indicating that the occupation of 5 cm trap-nests was the smallest, while the occupation was similar among trap-nests of 10, 15 and 20 cm. bee species trap-nest length 5cm 10cm 15cm 20cm centris analis 6 25 31 22 site dmq centris tarsata 2 4 megachile (acentron) sp. 4 1 megachile (sayapis) sp. 5 1 total 6 27 36 23 % cavities occupied 2.5 11.2 15.0 9.5 centris analis 2 2 centris tarsata 1 1 site dhu tetrapedia diversipes 1 5 15 19 megachile (tylomegachile) sp. 3 2 megachile sp. 1 1 megachile sp. 2 1 total 1 8 17 24 % cavities occupied 0.4 3.3 7.1 10.4 table 1. number of nests established by solitary bees in trap-nests in two agricultural areas (dmq and dhu), feira de santana, ba, brazil. most brood cells of c. analis in the dmq site were found in the longest cavities, 15 cm (40%) and 20 cm (30%), whereas the smallest number of brood cells (3%) was observed in the shortest trap-nests (5cm long ones). the number of built brood cells changed according tonest length (generalized least squares model: f(2,75)=18.54, p<0.001). the mean number of brood cells varied from 2.6 in 5 cm nests to 6.2 in 20 cm nests (figure 1). some cavities were not fully occupied by brood cells in trap-nests at all tested lengths. variation in the space occupied by brood cells reached 44% of the cavity in 20 cm long trap-nests and 69% in the 5cm long trap-nests. for t. diversipes, the percentage of occupied trap-nests also differed among trap-nests of different lengths (χ2=22.12, df=3, p<0.001). there was no difference in occupation between 5 and 10 cm trap-nests (χ2=1.52, df=1, p=0.22), but the percentage of occupied trap-nest was higher for 15 cm trap-nests in comparison to the 10 cm ones (χ2=4.23, df=1, p=0.04). there was no difference in occupation between the 15 and 20 cm trap-nests (χ2=0.28, df=1, p=0.59). percentage of nests established by t. diversipes (n=40) for nests with 5, 10, co santos, pec peixoto, cml aguiar – cavity length affects the occupation of trap-nests264 15 and 20 cm were respectively 2.5%, 12.5%, 37.5% and 47.5%. therefore, the smallest occupation in t. diversipes occurred for trap-nests with 5 and 10 cm, while the greatest occupation occurred for trap-nests with 15 and 20 cm. most brood cells of t. diversipes were built in longer cavities, 20 cm (55%) and 15 cm (33%), whereas the shortest trap-nests (5 cm) only sheltered 1.5% of the brood cells built by this bee species. the mean number of cells per nest established in cavities presenting different lengths varied from 3.0 in the only 5 cm nest that was occupied (although not included in the statistical analysis) to 5.8 in 20 cm nests (figure 1). the occupied space by brood cells varied from 2627% of the trap-nest (in 20 and 15 cm long straws) to 60% of them (in 5 cm long straws). the mean number of brood cells provisioned by t. diversipes did not differ due to trap-nest length (generalized least squares model: f(2,36)=2.36, p=0.11). for t. diversipes, the number of emerged adults did not differ due to trap-nest length (generalized least squares model: f(2,36)=1.93, p=0.16). the incidence of attacks from natural enemies to the nests was low. only 0.2% of c. analis brood cells in the dmq site was lost, due to the attack by the cleptoparasitic bee, mesocheira bicolor (fabricius) (hymenoptera, apidae), whereas there was not any attack to c. analis nests in the dhu site. it was observed that 7% of t. diversipes brood cells were lost because of attack by the cleptoparasitic bee, coelioxoides sp (hymenoptera, apidae). fig 1. mean number of brood cells provisioned in trap-nests of different lengths. centris analis (= dark gray bars), tetrapedia diversipes (= light gray bars). bee species/ site number of dead immatures % of brood cells e l p a 5cm 10cm 15cm 20cm centris analis/ dmq 17 42 2 17 75 28 13 6 centris analis/ dhu 2 3 0 0 0 11 50 0 tetrapedia diversipes/dhu 4 12 1 0 100 14 3 8 2. immature mortality, emerged adults and parasitism the mortality percentage recorded for c. analis offspring due to unknown causes reached 17% of the brood cells (n=460) assessed in the dmq site and 29% in dhu (n=17). there was immature death in 8% of the brood cells (n=202) in t. diversipes nests. mortality percentages were higher at the larval stage in both species (table 2). both species showed higher immature mortality percentage in nests established in the shortest trap-nests (5 cm long), whereas the mortality percentages were lower in the longest trap-nest (20 cm) (table 2). the number of immature individuals of c. analis that died differed among trap-nests with different lengths in the dmq site (χ2 = 20.64, df= 3, p < 0.001). the number of dead immatures gradually decreased from the 5 cm to the 20 cm long trap-nests (figure 2). the number of emerged adults of c. analis was higher in the 15 and 20 cm nests than in the 10 cm ones (generalized least squares model: f(2,75)=7.29, p=0.001; figure 3). table 2. number of immatures of centris analis and tetrapedia diversipes dead in the nests and percentage of brood cells containing dead immatures in cavities of different lengths (5, 10, 15, 20 cm). sites = dmq, dhu. e = egg, l = larvae, p = pupae, a = adult fig 2. mean number of dead immatures of centris analis in trapnests of different lengths. 3. sex ratio the sex ratio of c. analis offspring in the dmq site was 0.9m:1f, which was similar to the expected proportion of 1:1(χ2=0.69, df=1, p=0.40). there was no difference in the sex ratio of c. analis offspring produced in trap-nests presenting different lengths (general linear model: f(2,47)=1.35, p=0.27) (table 3). the sex ratio of t. diversipes offspring was 0.4 m:1f, which was significantly different from the expected proportion of 1:1 (χ2=5.45, df=1, p=0.02). sociobiology 67(2): 261-267 (june, 2020) 265 4. nesting activity and climatic factors there was nesting activity by solitary bees throughout most the year. c. analis mainly nested from september to march and recorded low nesting activity from april to august. t. diversipes kept its nesting activity from january to march and from june to september, except for july. bee nesting activity was associated with climatic factors. according to the akaike information criterion, the model based on temperature and rainfall effects was the most parsimonious (table 4). according to this model, the number of occupied trap-nests in each month of the year increased as the mean bee species/site trap-nest length males females centris analis/ dmq 5cm 02 03 10cm 22 21 15cm 48 46 20cm 24 34 centris analis/ dhu 5cm 0 0 10cm 3 1 15cm 2 2 20cm 0 0 tetrapedia diversipes/ dhu 5cm 0 0 10cm 4 7 15cm 13 17 20cm 13 14 table 3. number of males and females of centris analis and tetrapedia diversipes produced in trap-nests with different lengths. sites = dmq, dhu models aicc df daicc wi site + species+ temperature + rainfall 162.7 7 0 0.83 site + species + air humidity + temperature + rainfall 166.1 8 3.4 0.15 site + species + air humidity + temperature 171.9 7 9.2 0.008 site + species+ temperature 172.5 6 9.9 0.006 site + species + rainfall 176.3 6 13.6 <0.001 site + species 177.6 5 15.0 <0.001 site + species +air humidity 179.4 6 16.7 <0.001 site + species + air humidity + rainfall 179.4 7 16.8 <0.001 null 233.1 1 70.4 <0.001 (daicc represents the difference between the aicc value of model i and the aicc value of the most parsimonious model; wi is the weight of akaike model i). aicc / bias corrected version of the akaike information criterion. table 4. summary of generalized linear models with poisson distribution, which describe the relationship between number of occupied nests, temperature and precipitation, using the study site and bee genera as covariates. the models are classified in ascending order of aicc values. fig 3. mean number of emerged adults in trap-nests of different lengths. centris analis (= dark gray bars), tetrapedia diversipes (= light gray bars). monthly temperature and rainfall also increased (χ2 = 21.22, df = 2, p = 0.001). this analysis indicated the presence of two outliers with extremely high number of occupied nests. after removing them, the most parsimonious model indicated that the number of occupied nests were related to temperature and air humidity variations (χ2 = 9.5, df = 2, p = 0.009; table 5). the number of occupied trap-nests increased as temperature (b = 0.1) and humidity (b = 0.06) also increased. models aicc df daicc wi site + species + air humidity + temperature 113.5 7 0.0 0.33893 site + species + temperature 114.0 6 0.5 0.26567 site + species + temperature + rainfall 114.8 7 1.3 0.17358 site + species + air humidity + temperature + rainfall 116.5 8 3.0 0.07616 site + species 116.5 5 3.0 0.07443 site + species + air humidity 117.6 6 4.1 0.04314 site + species + rainfall 119.2 6 5.7 0.01993 site + species + air humidity + rainfall 121.0 7 7.5 0.00811 null 131.6 1 18.1 <0.001 (daicc represents the difference between the aicc value of model i and the aicc value of the most parsimonious model; wi is the weight of akaike model i). aicc / bias corrected version of the akaike information criterion. table 5. summary of generalized linear models with poisson distribution, which describe the relationship between number of occupied nests, temperature and precipitation, using the study site and bee genera as covariates. in this case, two outliers were removed in relation to the results shown in table 4. the models are classified in ascending order of aicc values. co santos, pec peixoto, cml aguiar – cavity length affects the occupation of trap-nests266 discussion the oil bee c. analis accepted a wide range of cavity lengths (from 5 to 20 cm) as nesting substrate; however, the shortest trap-nests (5 cm) were clearly less attractive than the longest ones (10 cm or longer). the present study did not show any preferential occupancy between the longest trap-nests (10, 15 and 20 cm), and this outcome suggests that they are all appropriate for c. analis nesting. similarly, alonso et al. (2011) did not find significant differences in occupancy by nesting females of c. analis in wooden blocks presenting small length differences (5.5, 6.0, 6.5 and 7.0 cm), although the shortest trap-nests recorded higher occupation in a third study site. in addition, the number of brood cells produced by c. analis was larger in longer cavities. when there is a low incidence of parasitism, as reported in this study, the occupation of longer cavities, where a female can produce a larger number of brood cells, seems to be an advantage, since it would lead to lower costs with the selection and establishment of new nests. on the other hand, in a different scenario, with a higher incidence of parasitism, could be advantageous to spread offspring over several nests, because some cleptoparasites can attack the same nest several times (gazola & garófalo, 2003; aguiar, unpublished data). the oil bee t. diversipes also showed trend of preferring to nest in longer trap-nests. in these straws, females left a considerable portion of the cavity empty. this strategy can play a role, protecting the brood cells against natural enemies. the mortality percentage recorded for c. analis offspring due to unknown causes suggests that there are not considerable losses of such offspring in these sites, similar to previous observation in dmq and in nesting areas close to it (aguiar & pina, 2012; aguiar et al., 2013). on the other hand, jesus and garófalo (2000), gazola and garófalo (2003), as well as couto and camillo (2007) reported great brood cell losses for this species in an urban area. factors causing the death of immature tropical solitary bees, who nest in trap-nests, remain poorly investigated. some researchers suggest that high temperatures influence immature mortality (frankie et al., 1988; jesus & garófalo, 2000; gazola & garófalo, 2003). however, couto and camillo (2007) did not find evidences about the influence of temperature on c. analis offspring mortality when they compared mortality percentages in nests established in shady vs. sunny areas. aguiar et al. (2013) have suggested that nest management during its removal from the field, as well as during nest transportation to the laboratory – which, sometimes means long distances -, could also have some influence on mortality. bosch and kemp (2001) previously reported the importance of handling the nests to decrease immature mortality incidence, because the inappropriate handling of osmia lignaria say (megachilidae) nests can take larvae away from their provisions and cause death due to starvation. results have shown low mortality in the longest trapnests, although t. diversipes and c. analis align the brood cells in the trap-nests (jesus & garófalo, 2000; camillo et al., 1995), a fact that could result in difficulty for individuals reared at the bottom of the nest to reach the exit. this outcome suggests that longer cavities would not limit the survival of their offspring. on the other hand, both bee species showed higher mortality percentages in the shortest trap-nests (5 cm). the sex ratio of c. analis offspring produced in trapnests of different lengths showed no differences; however, it is necessary to consider that the number of nests recorded in 5 cm long trap-nests, as well as the number of emerged adults, was too small, so they were not included in the analysis. on the other hand, alonso et al. (2011) have concluded that cavity length has influenced offspring sex ratio, since there was male-biased sex ratio in the two shortest trap-nests (5.5 and 6.0 cm), whereas the sex ratio was not significantly different from 1:1 in the two longest ones (6.5 and 7.0 cm). the nesting activity of c. analis was intense in summer and decreased from may to august, as previously reported for dmq (pina & aguiar, 2011). based on results in the present study, temperature and rainfall have influenced the frequency of nesting in these cavity-nest bees. the low offspring production from mid-fall and throughout the winter must be considered when planning c. analis management for crop pollination, as it would result in low availability of female pollinators. finally, the present study points out that longer trapnests (10 cm or longer) are the best options to produce c. analis and t. diversipes offspring, since they have good acceptance by nesting females, and there is low offspring mortality in nests established in these longer cavities. it seems plausible recommending the adoption of 10 cm trap-nests for c. analis breeding in agricultural areas that depend on its pollination service, if one taken into account the higher costs and logistics impairments to produce wooden blocks at the appropriate thickness to shelter the longest trap-nests (longer than 10 cm), as well as the lack of significant difference in many of the assessed parameters in comparison to the parameters recorded for the 15 and 20 cm long cavities. acknowledgments we thank the national council for scientific and technological development, brazil (cnpq, proc. no. 475715/2008-0, no. 562518/2010-0), and the fundação de amparo à pesquisa do estado da bahia (fapesb, to app0042/2009) for financial support for this project. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. c.s. oliveira received msc scholarship from capes. references aguiar, c.m.l., garófalo, c.a., almeida, g.f. 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(1998). the effects of cavity diameter and length on the nesting biology of osmia lignaria propinqua cresson (hym.: megachilidae). journal of hymenoptera research, 7: 84-93. seidelmann k, bienasch a, pröhl, f. (2015). the impact of nest tube dimensions on reproduction parameters in a cavity nesting solitary bee, osmia bicornis (hymenoptera: megachilidae). apidologie 47: 114-122. doi: 10.1007/s13592015-0380-z stephen, w.p. & osgood, c.e. (1965). influence of tunnel size and nesting medium on sex ratios in a leaf-cutter bee, megachile rotundata. journal of economic entomology, 58: 965-968 torchio, p.f. & tepedino, v.j. (1980). sex ratio, body size and seasonality in a solitary bee, osmia lignaria propinqua cresson (hymenoptera: megachilidae). evolution, 34: 993-1003. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i2.4303sociobiology 66(2): 381-388 (june, 2019) occurrence and nesting behavior of social wasps in an anthropized environment introduction urban development can be classified as one of the main reasons for local extinction rates, which, consequently, eliminates a large number of native species (czech et al., 2000; marzluff, 2001), or, at least, implies in the decreasing of overall diversity (faeth et al., 2011). the urbanization impact can be severe, causing losses on resource availability (both food and nesting sites), pollution increasing, microclimate alteration, affecting local biodiversity and pollination services (grimm et al., 2008; grab et al., 2019). abundance, abundance, species richness and evenness are affected by urbanization (shochat et al., 2010). abstract some effort has been made concerning the measurement of the social biodiversity of wasps in brazil. however, few approaches were made regarding how these taxa have adapted their nesting behavior to anthropic mixed environments. thus, the present work aimed to survey the occurrence of social wasps in an anthropic area and their relationship with different types of nesting substrates. increasing the knowledge of social wasps in anthropic areas may allow us to develop strategies for their conservation and management. twenty long-term surveys were made at the universidade de são paulo, ribeirão preto campus, são paulo state. during the searches, we collected information about wasp species and nesting substrate. a total of 20 species of eight genera were identified, and a total of 431 active colonies were registered. epiponini was the tribe that expressed the most species richness. on the other hand, mischocyttarini was represented by more active colonies. mischocyttarus cerberus had a remarkable greater number of colonies, whether compared to the other species which agreed with the idea of preference of anthropic environments by independent founding wasps. nesting behavior was associated with eight substrate categories. in general, we observed that some species may express certain plasticity regarding to their nesting substrate preference, whereas some expressed certain specificity. these findings may be related to the fact that some species are less sensitive to urban areas and then express a plastic behavior concerning to where they start their nests. facing the increase of current urbanization process and, consequently, habitat loss, this type of study contributes toward a better understanding of how these insects are affected and behave in altered environment. sociobiology an international journal on social insects rc da silva, as prato, ds assis, fs nascimento article history edited by marcel hermes, ufla, brazil received 03 december 2018 initial acceptance 26 february 2019 final acceptance 29 march 2019 publication date 20 august 2019 keywords synanthropism; biodiversity of wasps; polistinae wasps; nesting substrate. corresponding author rafael carvalho da silva universidade de são paulo – usp faculdade de filosofia, ciências e letras de ribeirão preto, departamento de biologia av. bandeirantes nº 3900, vila monte alegre cep 14040-900, ribeirão preto-sp, brasil. e-mail: rcswasp@gmail.com even though insects are found to be spread worldwide, few approaches have analyzed the urbanization effect over them, especially in neotropical regions (lima et al., 2000; mcintyre, 2000; de barros alvarenga et al., 2010; detoni et al., 2018). information concerning how wasps respond to urbanization is almost scarce, especially for the social species (detoni et al., 2018), which perform different ecological roles over the environments where they live, acting as predators of different agricultural pests (oliveira et al., 2017b; barbosa et al., 2018), as detritivores of decaying fruits or animal carcasses (barbosa et al., 2014; clemente et al., 2012), as fruit damagers (de souza et al., 2010a), as pollinators (somavilla & köhler, 2012), and even like environmental quality indicators (de souza et al., 2010b). universidade de são paulo (usp), faculdade de filosofia, ciências e letras de ribeirão preto, departamento de biologia, ribeirão preto-sp, brazil research article wasps rc da silva, as prato, ds assis, fs nascimento – social wasps in anthropized environment382 among studies that have been previously carried out, it was suggested that some social wasps belonging to polistinae (hymenoptera, vespidae) seem to be well adapted to urban areas, and that they are able to nest in different types of human-made structures (de barros alvarenga et al., 2010; oliveira et al., 2017a). this interaction can be understood based on the availability of structures in such areas, which enable wasps to get protection from unfavorable weather. in addition, any threat related to predation and competition for nesting substrates (lima et al., 2000; prezoto et al., 2007) is reduced. metal, concrete, ceramic, stone, plastic, porcelain, asbestos, synthetic materials, and wood are highlighted as the substrates chosen by wasps in urban areas (lima et al., 2000; detoni et al., 2018). interestingly, some species may stock their nests according to what is available in the environment. in summary, it is known that there are some species which are capable of nesting in both urban and natural areas, on the other hand, there are others which are limited to one or another (lima et al., 2000; de barros alvarenga, 2010; oliveira et al., 2017a). these wasps are known because of their diversity in neotropical regions, comprising almost a thousand of species described (pickett & carpenter, 2010). basically, these wasps start building their nests by using two different strategies, which permit their classification in: independent founding wasps (polistes and mischocyttarus) or swarm-founding wasps (epiponini) (jeanne, 1980; wenzel, 1998). brazilian social wasp biodiversity is actually well known for several areas with different vegetal formation, such as brazilian savanna and semideciduous mountain forest (de souza & prezoto, 2006), campos rupestres (silva-pereira & santos, 2006), mangrove and resting (santos et al., 2007), semideciduous seasonal forest (gomes & noll, 2009), dense and open upland forest, upland forest dense downland, closed and open flooded forest, high and low white-sand forest (somavilla et al., 2015) and montane humid forest (somavilla et al., 2017). though, there are still few approaches regarding such types of study in anthropized and agricultural-urbanized places (lima et al., 2000; de barros alvarenga et al., 2010; oliveira et al., 2017a; detoni et al., 2018). consequently, biodiversity and nesting habits of polistinae social wasps remain relatively little known in such areas (oliveira et al., 2017a). thus, to fill these gaps, we present here information regarding to surveys in an agricultural-urbanized mixed area in the interior of são paulo state, brazil. understanding specific details about the social wasp’s occurrence across mosaic-like environment may contribute for better knowledge about the particular ecological characteristics of wasps (de souza et al., 2010b), in addition, and to develop different strategies of conservation and management of these insects. material and methods study area the present study was conducted at the university of são paulo, usp, campus of ribeirão preto, brazil. the city is situated in the northeast part of the state of são paulo, brazil (21° 05’ s, 47° 50’ w, 531 m altitude). the campus has an area of 574.75 ha, with more than the half of its area combined by buildings and a wooded area composed of exotic and native vegetation (barth, 2006), characterized as semideciduos seasonal forest (fragoso & varanda, 2010). it is surrounded in its northern border by a large sugarcane plantation (fig 1). the weather regime is characterized by a strong seasonality, with a rainy summer and a dry winter. the major part of the rainfall (80%) occurs from october to march. on the other hand, the lower incidence of rainfall is registered from june to august (5%) (ipt, 2000). data collection nest recordings were obtained from april to september in 2016. we made 21 discontinuous surveys during the period (21 days of data collection), and we searched for nests in 108 building and surrounding gardens across the studied area (fig 1). the searches were always performed in two periods: from 08:00h to 11:00h and from 14:00h to 17:00h. a total of 126 hours (six hours per day) of inspection effort based on active search method was carried out, which was commonly used in similar studies (de souza & prezoto, 2006; oliveira et al., 2017a). during the searches, we registered the name of species, the number of nests for each species and finally the types of substrate used by them for nesting. once a nest was found, information was included in an excel spreadsheet (see supplementary material doi: 10.13102/sociobiology. v66i2.4303.s2280). when necessary, samples were collected with an entomological net and then they were archived in the entomological collection of the laboratório de comportamento e ecologia de insetos sociais from faculdade de filosofia, ciências e letras de ribeirão preto, university of são paulo, usp, campus of ribeirão preto, sp, brazil – sp. species identification was done by using taxonomic keys of richards (1978) and carpenter and marques (2001). comparisons were also made with identified specimens deposited in the entomological collection of the laboratory, and finally, some sampled material was confirmed by specialists. nesting substrate categories were based on eight types as follows: 1 – glass; 2 – masonry; 3 – metal; 4 – paper; 5 – plant material; 6 – plastic; 7 – wood or lastly 8 – n/a (not applicable to others). we performed a compilation including wasp species and their substrates, and the results were then plotted in a network of interactions, which allowed us to verify for any substrate preference in general and also for each species. statistical analysis data analysis were performed in the r software v.3.4.3. first, for measuring the diversity of wasps in each substrate we used the shannon-wiener (h) index, and for evenness we used the pielou index (j). we chose the shannon-wiener sociobiology 66(2): 381-388 (june, 2019) 383 index as a diversity measure because the index uses both abundance and evenness of species. moreover, the pielou index showed how equal the species are nesting in the substrate (magurran, 2004). both indexes were used for each substrate with sampled colonies. we used the package vegan v.2.4.4 (oksanen et al., 2007). we analyzed the species specificity index with the package ‘bipartite’ v.2.08. this index calculates the interactions between the parts, the value ranges from 0 to 1, where 0 is low specificity and 1 higher specificity. furthermore, we used the values of proportional similarity as suggested by feinsinger et al. (1981). these values are calculated in the interaction between the availability of resources and their use by the species (dormann et al., 2008). the differences among the richness in the substrates were tested with the g test. the g test was used to evaluate the values observed, and to test these values with the expected value. results and discussion a total of 431 active colonies were registered during the collection months and 20 species of social wasps were found, which were allocated in eight genera (table 1). a similar results were also found by oliveira et al. (2017a) and lima and colleagues (2000) in previous surveys also performed in urban areas. the wasp richness data found in our study are partially in accordance with what was found in another area with similar anthropic characteristics. for instance, detoni et al. (2018) studied an urban perimeter associated mainly to pinus ellioti in southestern brazil and registered 13 species of wasps, distributed in five genera. some species that we found along our survey were also sampled in a previous study, associated to sugarcane field (barbosa et al., 2018), which reinforce the importance of social wasp conservation in such environments. interestingly, although we had performed a survey in an urbanized area, we obtained relatively the same richness of species as togni et al. (2014) who collected wasps in a nature reserve located in atlantic forest biome. mischocyttarus and polybia highlighted as the most represented by species in the studied area, both with six species each, while in contrast, some genera were represented by only one species (table 1). the fact that mischocyttarus is the richest genus of social wasps (around 240 species), out of which 117 occur in brazil and polybia being the genus with most species described within epiponini might support the high diversity of both genus in the present study. results concerning richness of mischocyttarus and polybia had previously been presented in anthropized environments surrounded by atlantic forest fragments and pinus elliotti (oliveira et al., 2017a; detoni et al, 2018) and either in agricultural environments (de castro jacques et al., 2015). it has been proposed that this sinanthropism might be selected due to the lower rates of vertebrate predation and an interspecific dispute over sources (see mcglynn, 2012), although living in disturbed areas may affect colony performance (michelutti et al., 2013). in our work, mischocyttarus cerberus was the species with the highest number of nests found. similar results were registered by elpino-campos et al. (2007) and oliveira et al. (2017a), besides that, this species was found in six out of eight substrates; nevertheless, it seemed to prefer nesting over masonry substrate rather than other substrates. in general, we registered more nests of independent founding wasps (mischocyttarus) than swarming-founding wasps, which differs from studies performed in more preserved areas where epiponini is more frequent (togni et al., 2014). it should be understood considering that the studied place contains plenty of buildings and adjacent vegetation, which corroborates with the high degree of synanthropism proposed fig 1. geographic representation of the studied area through the campus of university of são paulo – ribeirão preto. rc da silva, as prato, ds assis, fs nascimento – social wasps in anthropized environment384 between the genus of independent founding wasps and urban areas (lima et al., 2000; de barros alvarenga et al., 2010; oliveira et al., 2017a). moreover, nests of primitively eusocial wasps normally are smaller, which consequently attract less human attention (smith, 2004, elpino-campos et al., 2007). several nests of mischocyttarus were found in places where they would not be easily noticed, for example, underneath air conditioners (personal observation). curiously we sampled only one polistes species in the studied area, which represents another group of wasp which is normally found associated to anthropized areas (detoni et al., 2018; .maciel et al., 2016). among the 431 colonies registered, the most used nesting substrate was masonry in 42% of the records, which may correlate with the greater availability of this substrate in the studied area. secondly, metal substrate was represented with 32.71%, followed by wood substrate with 13.92% of nest found; the fourth position was occupied by plant material with 3.71%, followed by glass substrate with 3.25%, and by plastic substrate which was represented by 3.25% of the results, then by n/a category 0.7%, and lastly by paper substrate with 0.46% (fig 2, table 3). our results agreed with those found by detoni et al. (2018), regarding the most used nesting substrate by wasps in urban areas. on the other hand, this result might have been affected by each nesting substrate type availability rate in the area rather than preference, but it has not been directly verified. the shannon index ranged from 0.637 to 1.906, n/a and plant material, respectively. the plant material had the major diversity (shannon index = 1.906), followed by man-made buildings (shannon index = 1.857), in which the glass had lower shannon diversity (0.657). the evenness of the paper substrate was 1, being more even than all substrates, and the lowest value was the glass substrate with 0.597. all the results are in table 2. some species were all specific to their substrate and obtained specificity equal to 1 (agelaia pallipes, brachygastra augusti, metapolybia sp.2, mischocyttarus montei, polybia ignobilis, polybia sp.1, polybia sp.2, protonectarina sylveirae, protopolybia exigua and protopolybia sedula). however for some it may be related to the low number of nests registered and not specificity per se (table1). nevertheless, other species remain above 0.420 of specificity. mischocyttarus sp.1 showed the lowest specificity with 0.426. the more common sampled species, m. cerberus, had a specificity of 0.52 (fig 2). tabel 1. species, number of colonies found of each species and relative frequency of nests sampled along the survey. species number of active colonies % mischocyttarus cerberus ducke, 1918 172 39.91 mischocyttarus metathoracicus (de saussure, 1854) 97 22.51 mischocyttarus cassununga (r. vonihering, 1903) 39 9.05 polybia occidentalis (olivier, 1792) 31 7.19 polistes versicolor (olivier, 1792) 28 6.50 polybia paulista h. von. ihering,1896 24 5.57 mischocyttarus sp.1 9 2.09 mischocyttarus montei zikán 1949 7 1.62 polybia ignobilis (haliday, 1836) 4 0.93 protopolybia exigua (de saussure, 1854) 4 0.93 polybia fastidiosuscula de saussure, 1854 3 0.71 metapolybia miltoni andena & carpenter, 2011 2 0.46 protopolybia sedula (de saussure, 1854) 2 0.47 mischocyttarus sp.2 2 0.47 metapolybia sp.1 1 0.23 agelaia pallipes (olivier, 1792) 1 0.23 brachygastra augusti (de saussure, 1854) 1 0.23 protonectarina sylveirae (de saussure, 1854) 1 0.23 polybia sp.1 2 0.47 polybia sp.2 1 0.23 species richness 20 431 100 table 2. diversity index to survey. s = number of observed species, h’ = shannon-wiener index and j’ = pielou evenness index. substrate s h’ j’ masonry 14 1.86 0.7 wood 9 1.79 0.82 plant material 8 1.91 0.92 metal 8 1.42 0.68 plastic 6 1.74 0.97 glass 3 0.66 0.6 paper 2 0.69 1 n/a 2 0.64 0.92 sociobiology 66(2): 381-388 (june, 2019) 385 among the species found during the study, it was possible to notice that some of them showed a certain plasticity in relation to the substrate used (figure 2). for instance, m. cerberus, mischocyttarus metathoracicus and polybia occidentalis were registered in almost all types of nesting substrate, but mischocyttarus montei showed less plasticity in relation to the substrate chosen, being registered only in metal substrates. the apparent preference of the wasp for nesting in masonry might be related to the greater offer of this substrate in comparison to the others, and this fact reinforces the idea of a good adaptation of this species in urban environments (virgínio et al., 2016). in sequence, the second category most used by wasps was metal, this preference was probably related with a phenomenon known as “heat islands” (lola et al., 2013), the accumulated heat over the metallic superficies provides suitable conditions for the colonies to develop, it was previously shown in studies with social insects (virgínio et al., 2016). we believe that collection method chosen for sampling the fauna did not affected the number of species found, because normally the active search method is the most used in similar studies, and according to souza et al. (2016), depending on the situation, it is the most effective method to sample the richness of an area whether compared with others. on the other hand, some authors believe that the use of more than one collection method makes possible to get better information about the biodiversity (auad et al., 2010, souza et al., 2011). even though we had not searched for social wasps in the surrounding areas, based on previous data from the same region, it is possible to suggest that some genera might be more tolerant to the urbanization process expressing a kind of behavior nesting plasticity (polybia, mischocyttarus and polistes), because they have been previously sampled in natural areas and in ours, which is an anthropized one, whereas others were not registered in our study (gomes & noll, 2009; locher et al., 2014). urban environments may contribute in a positive way to increase the chances of social wasps survival, since they provide suitable areas for nesting, and confer protection against bad weather. moreover, they represent places where there is less competition for resources among many species, and even predation rates by natural enemies may be lower (prezoto et al., 2007). finally, considering the generalist diet of social wasps, it was suggested that their presence near sugarcane areas can be beneficial, because these insects may perform natural biological control against different sugarcane pests, such as diatraea saccharalis, mocis latipes and spodoptera frugiperda (barbosa et al., 2018). due to the increasing urbanization and reduction of natural vegetation in many environments, the elucidation through basic studies of how well adapted the species of social wasps are, and consequently their success to nest in altered spaces, contribute to help us to understand clearly the interactions between those insects and the environment. thus, the present study represents one of the few attempts carried out up to this moment about the biodiversity of social wasp fauna in areas occupied almost exclusively by human activities. in this way, future studies gathering data for polistinae wasps in this type of mixed areas may help us to understand how wasps adapt their behavior and lifestyle toward a changing environment. fig 2. relation between wasps and different nesting substrate, involving 20 wasp species and eight types of substrates on the university of são paulo campus of ribeirão preto, brazil. rc da silva, as prato, ds assis, fs nascimento – social wasps in anthropized environment386 besides that, it would fill gaps about the distribution of species along brazilian territory. finally, they would be important for a better understand regarding how some species are being more affected by human disturbance over their natural nesting areas. campus of the universidade de ribeirão preto, usp, brazil. apiacta, 41(2). clemente, m.a., lange, d., del-claro, k., prezoto, f., campos, n.r. & barbosa, b.c. 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(2018). long-and short-term changes in social wasp community structure in an urban area. sociobiology, 65: 305-311. doi: 10.13102/sociobiology. v65i2.2597 dormann, c. f., gruber, b., & fründ, j. (2008). introducing the bipartite package: analysing ecological networks. interaction, 1(0.2413793) elpino-campos, á., del-claro, k. & prezoto, f. (2007). diversity of social wasps (hymenoptera: vespidae) in cerrado fragments of uberlândia, minas gerais state, brazil. neotropical entomology, 36: 685-692. faeth, s. h., bang, c., saari, s. (2011). urban biodiversity: patterns and mechanisms. annals of the new york academy of sciences, 1223(1): 69-81. doi: 10.1111/j.1749-6632.2010. 05925.x pair of substrates g value pvalue masonry x paper 11.252 0.001 masonry x plastic 3.985 0.046 masonry x glass 8.733 0.003 masonry x n/a 11.252 0.001 wood x paper 4.818 0.028 wood x n/a 4.818 0.028 plant material x paper 3.854 0.049 plant material x n/a 3.854 0.049 metal x n/a 4.818 0.028 metal x paper 4.818 0.028 table 3. pairwise comparison regarding the richness of species found among substrates. acknowledgments the authors would like to thank dr. alexandre somavilla of the instituto nacional de pesquisas da amazônia for his suggestions and helpfull comments in the first version of this manuscript. besides, we would like to thank prof. dr. sergio ricardo andena of the universidade estadual de feira de santana for checking and confirming some samples. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001 (r.c.s.) conselho nacional de desenvolvimento científico e tecnológico (cnpq) – a.p.s. 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(1998). a generic key to the nests of hornets, yellow jackets, and paper wasps worldwide (vespidae: vespinae, polistinae). american museum novitates, 10024: 1-39. 633 impact of rainfall on the nesting activity of solenopsis invcta in south china by yong yue lu, dongchuan hao & guangwen liang* abstract through field observations we found that workers of solenopsis invcta would make prompt repair to the nest after the rainfall as the exits were often blocked with soils in the rain. therefore, after each rain, the mounds were significantly higher but no significant change was found in diameter. the growth of the mound volume has a very close relationship with the rainfall and its duration. the longer the rain lasted, the greater the rainfall was, the greater the nest volume grew. the results showed that the volume of mounds was not changing constantly during the year, and it was quite different in different seasons. in november each year, the fire ants grew less active as the temperature decreased, and in the following december to february, the number of fire ant nests remained at a very low level. when april began, many new nests appeared as the temperatures rose and the rainfall increased. from may to july, it rained, and some extinct and new mounds appeared at the same time. the total number of active mounds increased significantly. between august and october, when the temperature was relatively stable with not much rain, the mound number was maintained at a stable level. key words: solenopsis invicta; nesting behavior; rainfall introduction rainfall has a great impact on many aspects for fire ants such as foraging, reproduction and spread (porter & tschinkel 1987; cokendolpher & francke 1985; morrison et al. 2004). rain can reduce ants’ foraging activity by 40%. rainfall can affect the ants’ recruitment by blocking the underground channels and interfering with pheromones (porter & tscinkel 1987), thereby reducing the competitiveness of fire ants. the number of foraging worker ants red imported fire ant research center, south china agricultural university, guangzhou 510640, china * corresponding author, e-mail address: gwliang@scau.edu.cn 634 sociobiolog y vol. 59, no. 3, 2012 is positively correlated with the monthly rainfall, monthly mean temperature, monthly minimum temperature, monthly maximum temperature and monthly minimum relative humidity, but negatively correlated with the mean pressure and all these correlations were significant (xi et al. 2010). rainfall increased soil moisture and thus impacted various grades of all instars of fire ants living in the nests. some studies have shown that rainfall has minimal impact on the workers’ mortality (hood & tschinkel 1990), but drought greatly influenced the mortality rate of the young ants (wang 2005). ants are distributed in tropical and subtropical regions, where there are frequent rainfalls all the year round. the study of the effect of rainfall on fire ants’ nest-building activity is of great importance for monitoring the occurrence and spread of the ants’ populations. this study investigated the relationship between rainfall and the number of mounds to evaluate the effect of rainfall on the ants’ nest-building behavior. materials and methods test site this test was conducted on a grassland in the zengcheng base of south china agricultural university, and the grass and green belt in guangzhou university. observation of nest-repairing behavior after rainfall after rainfall in the field, ants’ nest repair process was observed and recorded in detail. the relationship between rainfall and the change of mound size in early april, we selected ten active nests in the experiment and measured the length, width, height to calculate the volume. then slender bamboo sticks marked with a scale were inserted at the top of and around the nest. after each rainfall, the height and width changes were recorded. the weather data was provided by the local weather bureau to identify the daily rainfall during the experiment. calculation method for mound volume the nest volume can be calculated as s v = 4/3π∙a/2∙b/2∙ht. in these equations, a is the length of the mound, b is its width and ht is its height (tschinkel 1993). 635 lu, y. et al. — impact of rainfall on fire ant nesting activity the mound changes throughout the year from december 2007 to november 2008, we measured the length, width and height of the tested nests which were free from interference at the end of every month, and calculated the volume according to the formula. meanwhile we recorded the number of active mounds, extinct mounds and new mounds. statistical analysis all statistical analyses were conducted using spss, version 14.0 (spss inc., chicago, il, usa). results nest-repair process through field observations we found that workers would make prompt repair to the nest after the rainfall as the exits were often blocked with soils in the rain. they would start work when it began to rain mildly, and a few workers could also be found making some urgent repairs when it rained hard. but the large-scale repair was carried out within 12h after the rain. immediately after rainfall, there were only several exit-holes on the surface of the mound and just a few ants were repairing. afterwards more and more workers carried soil particles to the top of the mounds, making more and more exit-holes. about one hour after the rain, there were crowded ants and exit-holes on the surface of the mound. ants carried soil particles from the bottom or inside to the surface of the mound, piling the soil particles at the surroundings of the exit-holes, making the exit-holes more chimney-like. then the workers constructed a cover on all the chimney-like exit-holes, which increased the height of the mounds constantly. therefore, after each rain, the mounds were significantly higher but no significant change was found in diameter. the greater damage the rainfall caused to the nests, the more repair efforts the ants made to the nests, and the greater the volume growth was. the relationship between rainfall and mound growth from march 30 to april 5 the rain lasted for five days with a total rainfall of 24.6ml, and the mound growth was 90% at this period. during this time, the temperature of guangzhou rose from 18.8 °c in march to 23.3 °c on april 5, so the temperature range was relatively large. meanwhile this rainfall 636 sociobiolog y vol. 59, no. 3, 2012 was the largest since the beginning of 2008, therefore after the rain many new nests appeared and the volume of the nests was also growing rapidly, which showed significant change. between april 6 and april 10, there was no rain and no new nest was found. from april 11 to 17, the rain lasted for four days with a total rainfall of 8.7ml, and mound growth was 11.07%. between april 18 and 22, the rain lasted for three days with a total rainfall of 85.9ml, and an increase of 37.43% was found in the volume of mounds. from april 23 to 26, it rained just one day, with a rainfall of 1.6mm, so the volume of mounds did not change significantly, (2.6%) and would have been difficult to see only from the exterior without accurate measurement. from the above, we can see the growth of the mound volume has a very close relationship with the rainfall and its duration. the longer the rain lasted, and the greater the rainfall was, the greater the nest volume grew (table 1). the dynamics of mound volume the results showed that the volume of mounds was not changing constantly during the year, and it was quite different in different seasons: (1) from march to april, the mounds for the ant populations could not be easily found after a cold dry winter. the number of the ants trapped during this period was only 100-130 per bottle, but the temperature began to rise during this time, so after the rain the mounds would be easily found the there would be emergence of many new nests. (2) from october to february, because of the cold weather, the ants were relatively inactive in its activities including the mound-repair work, so when the rainfall was little or even none, the mound growth was not obvious. between february and october in 2008, it rained hard for several tines in guangzhou, so the volume of the mounds was significantly reduced because of the ants did not completely fix the mounds. (3) from may to september, which was a period with high temperature and increased rainfalls, the ants became active so the number of ants trapped was 360 per bottle. during this time the ants’ mound-repair work was prompt and forceful, so in these months, with the rainfall stimulation, the mounds grew very obviously fast. however, in june 2008, in which it rained for 23 days with a rainfall of 1136.9mm, and each rain was hard so the mounds grew smaller and smaller because of the persistent rain erosion. in this period, the height of mounds decreased very significantly than before the rainfalls (table 2). 637 lu, y. et al. — impact of rainfall on fire ant nesting activity dynamics of mound numbers in november each year, the fire ants grew less active as the temperature decreased. the number of nests reduced a lot for the rain erosion. and in the following december to february, the number of fire ant nests remained at a very low level. when april began, many new nests appeared as the temperatures rose and the rainfall increased. from may to july, it rained, and some extinct and new mounds appeared at the same time. the total number of active mounds increased a lot more than before. between august and october, table 1 the relationship between mound growth and major climatic factors. date average temperature ( °c) average relative humidity (%) rainy days total rainfall (mm) average growth amount of mounds (cm3) growth rate (%) 3.30~4.05 18.17 91.71 5 24.6 6636.72±1296.37 90 4.06~4.10 25.12 83.4 0 0.5 0 0 4.11~4.17 24.03 85.14 4 8.7 1465.41±504.39 11.07 4.18~4.22 24.42 86.6 3 85.9 3971.28±612.04 37.43 4.23~4.26 20.63 75.5 1 1.6 407.72±128.55 2.6 4.27~4.30 20.9 84.5 2 41.3 4337.07±128.55 20.07 table 2 .the relationship between mound growth and climatic factors. month mo nth l y aver a g e temperature ( °c) mo nth l y aver a g e relative hum id it y (%) total rainfall (mm) rainy days average growth of mounds (cm3) jan 12.6 71.39 82.6 9 0 feb 11.1 68.24 63.9 9 -1914.68±1034.82 mar 18.8 75.48 79.5 11 6064.59±1171.55 apr 22.4 84.8 154.9 15 16818.2±2669.9 may 24.5 84.23 357.7 17 18876.06±2678.11 jun 26 89.27 1136.9 23 -2176.77±1334.55 jul 27.7 83.94 306.1 21 5478.93±2787.37 aug 27.8 82.71 131.1 13 1789.13±387.77 sep 27.3 80.64 103.2 11 1208.56±808.19 oct 25 79.48 180.1 6 -1308.09±433.21 nov 18.8 70.6 90.3 3 0 dec 16.6 70.94 14.1 2 0 638 sociobiolog y vol. 59, no. 3, 2012 when the temperature was relatively stable with not much rain, the mound number was maintained at a stable level (fig. 1). discussion ants are mostly distributed in tropical and subtropical regions (morrison et al. 2004), where there are frequent rainfalls all year, and especially humid and rainy springs, which cause certain difficulties for ant prevention and treatment. but spring and summer is a period of rapid propagation for fire ants, if we gave up prevention and control work in this period, the fire ants will thrive and expand greatly (porter & tschinkel 1987; cokendolpher & francke 1985), which will also add a huge challenge to ant prevention and control. therefore, the full understanding of ant population changes during the rainy season is the key to developing effective prevention measures. according to the previous report, mound height and volume were negatively correlated with temperature, and mound height was positively correlated with rainfall. the results of these studies indicate that in late summer, rainfall triggers mound rebuilding and repair but not a significant increase in size, which occurs as temperatures decrease into fall (vogt & smith 2007). our field investigation found that when the rainfall intensity was 12.5mm/12h (i.e. heavy rain) few workers would even go out, but the rains with very little intensity (e.g. drizzling ) had no obvious impact on the ants’ mounds and outdoor activities. however, if the rainfall was heavy enough to damage the fig. 1 annual dynamics of of s. invicta mounds 639 lu, y. et al. — impact of rainfall on fire ant nesting activity mounds, the rain erosion would reduce the height of the mounds, and then ants will repair the nests, making the mounds larger than before the rain. there was no obvious change in the nest length and width, but the height change is obvious. in addition, from april to july, after each rainfall many new mounds were found, which may be due to nest-moving and nest-dividing (zhao et al. 2009), or the peak of the nuptial flights at this time (xu et al. 2009). in winter when the ants are relatively inactive, although the ants make some repair work to the mounds after the rain, the size of mounds did not increase obviously, and was sometimes even smaller than before the rainfall. during the period without rainfall, no obvious change was found in the volume of the mounds. so we can see that rainfall is a key external factor for the ants’ mound volume change. we can predict that rainfalls are affecting not only the nest size but also the population growth of fire ants, by comparing the annual activity patterns of ants and the curves of their nests, taking into account the significant linear relationship between volume and ant population. acknowledgments our study was supported by the ph.d. programs ministry of education of china (no. 20104404110018). references cokendolpher, j.e. & o. francke 1985. temperature preferences of four species of fire ants (hymenoptera: formicidae: solenopsis). psyche 92 (1):91-102. hood, w.g. & w.r. tschinkel 1990. desiccation resistance in arboreal and terrestrial ants. physiol entomol 15 (1):23-35. morrison, l.w., s.d. porter, e. daniels & m.d. korzukhin 2004. potential global range expansion of the invasive fire ant, solenopsis invicta. biological invasions 6 (2):183191. poter, s.d. & w.r. tschinkel 1987. foraging in solenopsis invicta (hymenoptera: formicidae): effects of weather and season. environ entomol 16 (3):802-808. poter, s.d. & w.r. tschinkel 1993. fire ant thermal preferences: behavioral control of growth and metabolism. behav ecol sociobiol 32 (5):321-329. wang, f.x., f.r. lin & j.q. zhu 2005. quarantin and control of red imported fire ant. plant quarantine 19(3):150-153. xi, y.b., j.q. jiang, y.j. xu & y.y. lu 2010. temporal food searching dynamics of red imported fire ants (rifa) in litchi orchard and their influential factors. journal of anhui agricultural university, 67-70. 640 sociobiolog y vol. 59, no. 3, 2012 xu, y.j., j. huang, y.y. lu, l. zeng & g.w. liang 2009. observation of nuptial flights of the red imported fire ant, solenopsis invicta (hymenoptera: formicidae) in mainland china. sociobiolog y 54(3): 831-840. zhao, j., p.s. zhong, t. huang & ss zhang 2009. impact of precipitation on behavior of solenopsis invicta buren colony. chin j vector bio and control 20(6):542-544. vogt, j.t. & w.a. smith 2007. effects of simulated and natural rainfall on summer mound construction by imported fire ants (hymenoptera: formicidae). sociobiolog y 50 (2):379-390. bharti3 mar.indd 805 pseudolasius machhediensis, a new ant species from indian himalaya (hymenoptera: formicidae) by himender bharti, irfan gul & yash paul sharma abstract pseudolasius machhediensis sp. nov. is described from india. this marks the second species of this genus reported in india, with only pseudolasius familiaris (f. smith) described earlier. an identification key to worker caste is provided to separate the indian species. keywords: pseudolasius, ants, new species, indian himalaya. introduction the genus pseudolasius emery, 1887 is currently represented by 45 species and 15 subspecies from the world (bolton 2012). the pseudolasius ants are known for polymorphic worker caste, with most of the species having major and minor workers (lapolla 2004). pseudolasius awaits a global taxonomic revision, however, important taxonomic contributions to this genus include: emery, 1911 (all species key); menozzi, 1924 (afrotropical species key); wu, j. & wang, c. 1995 (china species key); xu, 1997 (china species key); zhou, 2001 (china, guangxi species key); lapolla, 2004 (afrotropical species key). during the course of present study one new species has been recorded from himalaya and this differs considerably from already described species of this genus. prior to this study, only one species had been recorded from india (bharti 2011). with the current addition 2 species signify the genus pseudolasius from india. a key for the worker caste of indian species has been provided. materials and methods the specimens were collected by handpicking method. the taxonomic analysis was conducted on a nikon smz 1500 stereo zoom microscope. department of zoolog y and environmental sciences, punjabi university, patiala147002, india e-mails: himenderbharti@gmail.com; irfangulhhh@gmail.com 806 sociobiolog y vol. 59, no. 3, 2012 for digital images, an mp evolution digital camera was used on the same microscope with auto-montage (syncroscopy, division of synoptics, ltd.) software. later, images were cleaned as per requirement with adobe photoshop cs5. holotype and paratypes of the species have been deposited in pupac, punjabi university patiala ant collection, patiala. morphological terminolog y for measurements (given in millimeters) includes: hlhead length: the length of the head capsule excluding the mandibles, measured in full face view in a straight line from the middle of the anterior clypeal margin to the middle of the occipital margin. hwhead width: the maximum width of the head in full face view behind the eyes. slscape length: the maximum straight line length of the scape, excluding the basal constriction or neck that occurs just distal of the condylar bulb. cicephalic index: hw/hl×100. si – scape index: sl/hw×100. description pseudolasius machhediensis sp. nov. (figs. 1-6) holotype, major worker: india, jammu & kashmir, machhedi, 32.72364n, 75.669464e, 2130m above msl, 15.xi.2008. paratypes: 2 major workers, 7 minor workers, same data as holotype; 2 major workers, 27 minor workers, india, himachal pradesh, gangradi, 32.844098n, 77.151283e, 1600m above msl, 05.xii.2010; 2 minor workers, india, himachal pradesh, kulu, patlikul town, 32.074n, 77.088e, 1200m above msl, 29.v.1999; hand picking (coll. irfan gul & himender bharti). description of major worker (figs. 1-3): worker measurements: hl 1.25-1.36(1.36); hw 1.15-1.40(1.40); sl 1.13-1.25(1.20). (n=5) head: head subquadrate [ci= 92-103(103)]; occipital margin weakly emarginate; occipital corners round; lateral sides subparallel, narrowing towards the anterior margin; anterior clypeal margin broadly round, somewhat transverse in the median portion; eyes small, round, situated infront of the 807 bharti, h. et al. — a new species of pseudolasius middle of sides of head; mandibles triangular, the masticatory margin with 6 prominent teeth, the 3rd apical tooth smaller than the 2nd and 4th, the 4th tooth larger than the 5th and 6th teeth; frontal area small, triangular; frontal carinae short, parallel; antennae slender,12 segmented, scape fairly long, distinctly surpassing the occipital margin [si= 85-100(85)], last segment of the funiculus slightly longer than the two preceding segments taken together. mesosoma and petiole: mesosoma with weakly convex promesonotal dorsum, broadest across humeral angles; pronotum convex with the sides round, mesonotum and metanotum with the sides somewhat flat; promesonotal and fig. 1. pseudolasius machhediensis sp. nov., major worker 1. head, dorsal view. 808 sociobiolog y vol. 59, no. 3, 2012 figs. 2-3: pseudolasius machhediensis sp. nov., 2. body, lateral view. 3. body, dorsal view. 809 bharti, h. et al. — a new species of pseudolasius mesometanotal sutures quite distinct; petiolar node with the sides more or less round, apex thin and more or less emarginate; legs stout and fairly long. gaster: gaster long , oval, narrowed infront and behind, pointed at apex. sculpture: head roughly smooth except for the microreticulated sculpture visible at the higher magnification; mandibles distinctly rugulose more so towards the apex; punctures and tubercles scattered all over the body; clypeus smooth and much more shiny; sculpture of mesosoma as in head; abdomen smooth and somewhat shiny. pilosity: body clothed with silky yellow pubescent hairs and longer and shorter outstanding hairs; pubescence is more on head and mesosoma. color: the species is pale yellow in color but in some specimens the head is darker and the abdomen brown; the teeth and eyes are black and the legs yellow. description of minor worker (figs. 4-6): worker measurements: hl 0.8-1.0(0.84); hw 0.72-1.02(0.77); sl 0.721.1(0.8). (n=28) head: head roughly square [ci= 89-102(91)]; occipital margin straight to feebly excised in some specimens; occipital corners round; lateral sides more or less parallel; anterior clypeal margin broadly rounded; eyes small, round, situated infront of the middle of sides of head; mandibles triangular, the masticatory margin with 6 prominent teeth, the 3rd apical tooth smaller than the 2nd and 4th, the 4th tooth larger than the 5th and 6th teeth; frontal area small, triangular; frontal carinae short, nearly parallel; antennae slender,12 segmented, scape fairly long, distinctly surpassing the occipital margin [si= 100-107(104)], last segment of the funiculus slightly longer than the two preceding segments taken together. mesosoma and petiole: mesosoma with weakly convex promesonotal dorsum, broadest across humeral angles; pronotum convex with the sides round, mesonotum and metanotum with the sides somewhat flat; promesonotal and mesometanotal sutures quite distinct; petiolar node with the sides more or less straight, apex thin and emarginate; legs stout and fairly long. gaster: gaster long, oval, narrowed in front and behind, pointed at apex. 810 sociobiolog y vol. 59, no. 3, 2012 sculpture: the sculpture is same as that of the major workers. the minors are more shining all over; the whole body is much smoother than the major workers; mandibles distinctly rugulose all over; punctures and tubercles scattered all over the body; clypeus smooth and shiny. pilosity: longer and shorter outstanding hairs scattered all over the body; the minor workers are quite deficient of pubescence, the pubescence is not as much as in case of major workers and is denser on the head. color: the color varies from light yellow to pale yellow with a darker abdomen; the teeth and eyes are black. fig.4. pseudolasius machhediensis sp. nov., minor worker. head, dorsal view. 811 bharti, h. et al. — a new species of pseudolasius distribution and habitat: the species has been collected from north-west himalaya, in jammu and kashmir and himachal pradesh. the species inhabits moderately wet and figs. 5-6: pseudolasius machhediensis sp. nov., minor worker. 5. body, lateral view; fig 6. body, dorsal view. 812 sociobiolog y vol. 59, no. 3, 2012 lighted forested areas with scarce undergrowth and in all the cases specimens were collected under stones by hand picking. etymology: the species is named after the type locality, machhedi. remarks: (based on major workers) the species is significantly different from all other reports of this genus. pseudolasius machhediensis sp. nov. somewhat resembles the chinese pseudolasius bidenticlypeus xu, 1997 but can be easily distinguished from the latter. in pseudolasius machhediensis sp. nov. the 4th tooth is larger than the 5th and 6th teeth and the clypeus is smooth without any denticle, whereas in pseudolasius bidenticlypeus the 4th tooth is smaller and the clypeus is with one blunt denticle at each side. it is different from pseudolasius familiaris (f. smith), the only species reported from india and can be easily separated from it, as in pseudolasius familiaris the teeth are five in number with 4th and 5th combined, but in pseudolasius machhediensis sp. nov., the teeth are six in number with the 5th and 6th combined, the head is weakly emarginate and the antennae are much longer. key to the known species of pseudolasius from india based on major worker caste mandibles armed with 5 teeth, the basal two combined; antennae somewhat short ………………………………….……...… pseudolasius familiaris (f. smith) mandibles armed with 6 teeth, the basal two combined; antennae long, distinctly surpassing the occipital margin ............................................................... ....................................................................pseudolasius machhediensis sp. nov. acknowledgements financial assistance rendered by department of science and technolog y (grant no. sr/so/as-65/2007), govt. of india, new delhi is gratefully acknowledged. references bharti, h. 2011. list of indian ants (hymenoptera: formicidae). halteres 3: 79-87. bolton, b. 2012. bolton’s catalogue and synopsis, in http://gap.entclub.org/ “ version: 1 january 2012. 813 bharti, h. et al. — a new species of pseudolasius emery, c. 1887. catalogo delle formiche esistenti nelle collezioni del museo civico di genova. parte terza. formiche della regione indo-malese e dell’australia. [part]. ann. mus. civ. stor. nat 4: 241-256. emery, c. 1911. fragments myrmécologiques. ann. soc. entomol. belg 55: 213-225 lapolla, j. s. 2004. taxonomic review of the ant genus pseudolasius in the afrotropical region. journal of the new york entomological society 112: 97-105. menozzi, c. 1924. alcune nuove formiche africane. . ann. mus. civ. stor. nat. giacomo doria 51: 225-227. wu, j. & c. wang 1995. the ants of china. beijing : china forestry publishing house, 214 pp. xu, z. 1997. a taxonomic study of the ant genus pseudolasius emery in china. zoological research 18: 1-6. zhou, s. y. 2001. ants of guangxi. guangxi normal university press, guilin, china. 255 pp. 561 natural biological control of diaphania spp. (lepidoptera: crambidae) by social wasps by paulo antônio santana júnior1, alfredo henrique rocha gonring2, marcelo coutinho picanço1*, rodrigo soares ramos1, júlio cláudio martins1 & dalton de oliveira ferreira1 abstract the social wasps (hymenoptera: vespidae) are important agents of biological control for agricultural pests. diaphania hyalinata l. and diaphania nitidalis cramer (lepidoptera: crambidae) are among the main pests of plants in the cucurbitaceae family. although the importance of social wasps is acknowledged, little is known about their activity as biological control agents in diaphania spp. thus, this work aimed to study the natural biological control of the caterpillars d. hyalinata and d. nitidalis by social wasps. we studied the natural biological control of caterpillars of d. hyalinata and d. nitidalis on cucumber hybrids sprint 440 ii and vlasstar. the main predators of diaphania caterpillars were the social wasps, followed by diptera: syrphidae; hemiptera: anthocoridae; coleoptera: coccinellidae, anthicidae; neuroptera: chrysopidae and arachnida: araneae. predation of d. hyalinata caterpillars by social wasps was high from the second to fifth instar. the predation of d. nitidalis caterpillars by social wasps was high from the second to fourth instar. there was no predation by social wasps on the first instar larvae of diaphania spp. the cucumber hybrids did not influence the predation of diaphania spp. by social wasps. the main social wasp predator of diaphania spp. was polybia ignobilis (haliday). also, we observed the social wasp polybia scutellaris (white) preying on d. hyalinata but at low intensity. key words: social insects, vespidae, diaphania hyalinata, diaphania nitidalis, cucurbitaceae, predators. introduction the family vespidae has social and solitary species. the social vespidae belong to three subfamilies: stenogastrinae, polistinae and vespinae (car1 federal university of viçosa, department of entomolog y, 36570-000, viçosa, mg, brazil, 2dupont do brasil s.a. *e-mail: picanço@ufv.br 562 sociobiolog y vol. 59, no. 2, 2012 penter 1991). stenogastrinae is the smallest of social subfamilies and it has about 50 species and six genera. it occurs in asia and oceania (carpenter & kojima 1997). the subfamily polistinae has more than 900 described species and it occurs in europe and the americas (carpenter et al. 1996). the vespinae have about 60 species and 40 genera and occur in the northern hemisphere (o’donnell 1998). the colonies of social vespidae are arranged in nests that have few or hundreds of individuals. in the social organization of individuals vespidae belongs to castes. in this social organization there is a division of labor that allows greater efficiency in caring for their offspring and in locating and gathering resources. in the nests there are one or few queens, sterile workers (who usually do not differ morphologically from queens), young (eggs, larvae and pupae) and drones. the queens have reproductive function and control of the colony. the workers look after the colony and collect the resources used in the nests. the drones mate with queens during nuptial flights and afterwards they die (richter 2000, kasper et al. 2004). during foraging , vespidae workers collect water, plant fibers, carbohydrates and proteins. water and fiber are used for nest building. the carbohydrate sources used by vespidae are: nectar, fruit pulp and plant sap. the nectar can be converted into honey. honey is a source of food reserves. the adult workers chew their prey and feed the larvae by trophallaxis. as adults are unable to eat solid food the larvae regurgitate a solution containing carbohydrates and protein for the feeding of adult workers (richter 2000, kasper et al. 2004). the food consumed by the social vespidae is important in maintaining the colonies, especially protein intake. the proteins used by the social vespidae are obtained mainly by predation on insects by their workers. their main prey are insects of the order diptera, hemiptera, hymenoptera and lepidoptera (gobbi & machado 1986, richter 2000). thus, the social vespidae are considered important agents of biological pest control (picanço et al. 2010, picanço et al. 2011). among the major pests of cucurbitaceae (pumpkin, squash, luffa plant, bur cucumber, cucumber, melon and watermelon) are diaphania hyalinata l. and diaphania nitidalis cramer (lepidoptera: crambidae) (bacci et al. 2006). the caterpillars of these species cause damage to plants by feeding on leaves, stems, flowers and fruits. these pests occur in south america (gonring et al. 2003a, 2003b), central america 563 santana, p.a. et al. — biological control of diaphania spp. by social wasps (king & saunders 1984) and north america (penã et al. 1987). although the importance of social wasps is acknowledged, little is known about their activity as biological control agents of diaphania spp. thus, this work aimed to study the natural biological control in the caterpillars of d. hyalinata and d. nitidalis by social vespidae. material and methods this work was conducted in a cucumber crop in viçosa (20°48'45"s, 42°56'15"w, altitude of 660m), minas gerais state, brazil from april to july 2000. in the cultivation of cucumber hybrids sprint ii 440 (dark green fruit for fresh consumption) and vlasstar (light green fruit for fresh consumption or as pickles) were used. the experimental design was randomized blocks with five replications. the repetition had six rows of nine plants. thus, each repetition had 54 plants. no pesticides were applied to the crop, and conventional cultivation practices were employed, according to filgueira (2000). in each repetition, 20 plants were infested with caterpillars. the plants were about 60 days old when they were infested. ten of these plants were infested with d. hyalinata and the other 10 plants were infested with d. nitidalis. the plants were subdivided into five groups of two plants. each plant in the group was infested with 10 larvae from one of five larval instars. therefore, each repetition received 100 larvae of each diaphania species. the infestation was carried out in the third leaf from the top of the branch. those plants received a tag identifying the species and instar of diaphania with which it was infested. the caterpillars used were obtained from laboratory rearing. this creation was conducted according to pratissoli et al. (2008). during each instar, the mortality of diaphania caterpillars was monitored. from sunrise to sunset we evaluated the causes of death to each caterpillar. predators observed preying on caterpillars were recorded according to their morphospecies. in unmarked plants, individuals of the predator morphospecies were collected and assembled. later on, after the change of instar, the caterpillars that were not dead were collected in plastic pots of 500 ml. the pots were taken to laboratory for evaluation of parasitized caterpillars. the emerged parasitoids were collected. in the laboratory the caterpillars were reared according pratissoli et al. (2008). specimens of predators and parasitoids were identified in the entomological museum of the federal university 564 sociobiolog y vol. 59, no. 2, 2012 of viçosa. the average mortality of diaphania caterpillars were calculated from the experimental data. we also calculated the 95% confidence intervals of mortality rates. results and discussion predators were the group of natural enemy that caused higher mortality in diaphania spp. caterpillars. these natural enemies were responsible for 98% of the natural mortality of larvae of d. hyalinata and d. nitidalis (fig. 1a). the observation of a higher natural biological control in diaphania by predators than by parasitoids is due to several factors. among the factors that contribute to the high efficiency of the predators are: search capability, consumption of prey, adaptability to the environment and competitiveness. the increased competitiveness of predators makes them often prefer to consume parasitized prey (reis jr. et al. 2000), or they consume larvae of parasitoids within herbivorous insects (leite et al. 2001). we observed that the main group of predators of diaphania spp. caterpillars were the social vespidae, followed by diptera: syrphidae; hemiptera: anthocoridae; coleoptera: coccinelidae, anthicidae; neuroptera: chrysopidae and arachnida: araneae. we also observed parasitism on caterpillars by diptera: tachinidae and hymenoptera: braconidae (fig. 1b). the superiority of natural control in diaphania spp. by the social vespidae is likely due to their social organization, search capabilities and competitiveness. the social organization of vespidae enables the division of labor between the components of the colony. this fact allows the wasps to locate their prey more efficiently and they capture a greater number of prey. other authors found that the social vespidae are also the most important predators of caterpillars such as ascia monuste (godart) (pieridae) (picanço et al. 2010), leucoptera coffeella (guérin-méneville) (lyonetiidae) (pereira et al. 2007a, pereira et al. 2007b) and tuta absoluta (meyrick) (gelechiidae) (picanço et al. 2011). due to the importance of social vespidae as natural enemies of diaphania spp. caterpillars their populations should be preserved or even increased in crops. among the practices for preserving these predators are: the application of insecticides with physiological selectivity, the selective use of pesticides and reduced applications. the use of insecticides that show physiological selectivity is important because these products are effective in controlling pests and 565 santana, p.a. et al. — biological control of diaphania spp. by social wasps they are somewhat toxic to natural enemies. in this context, picanço et al. (1998) found that deltamethrin, permethrin and trichlorfon show ecological selectivity in favor of the social vespidae. the selective use of insecticides reduces the contact of natural enemies with these products. for the social wasps this can be achieved by the apfig. 1. (a) factors of mortality and (b) predators of diaphania spp. caterpillars. brackets indicate 95% confidence interval. 566 sociobiolog y vol. 59, no. 2, 2012 plication of pesticides during periods of low visitation of these predators to crops such as during times of lower air temperature (picanço et al. 2010 picanço et al. 2011). the reduction of pesticide use can be obtained by the use of decision-making systems to control application of pesticides (bacci et al. 2008). vespidae populations can be increased by providing food for these predators by planting flowering plants next to crops (paula et al. 2004), preserving the forests that have trees that are the main nesting sites of the wasps (picanço et al. 2010, picanço et al. 2011) and transferring of vespidae nests to crop sites (prezoto & machado 1999). predation of d. hyalinata by social wasps was high for 2nd, 3rd, 4th and 5th instar caterpillars. the predation of d. nitidalis by social wasps was high for 2nd, 3rd and 4th instars. there was no predation of first instar larvae in either diaphania species by social wasps (fig. 2a and 2b). the caterpillars increase the body mass as they grow and the wasps preferntially prey on the larger caterpillars, which represent a greater food resource and are more easily located. corroborating this hypothesis picanço et al. (2010) and picanço et al. (2011) also observed that vespidae do not catch first instar larvae of a. monuste and t. absoluta. among the fifth instar caterpillars, there was higher predation in d. hyalinata than d. nitidalis (figs. 2a and 2b). this was possibly due to the location of these two species of diaphania caterpillars during their larval instars. caterpillars of d. hyalinata in all instars remain on the leaves, and are thus exposed to predation by social wasps (gonring et al. 2003b). conversely, d. nitidalis caterpillars in the last instar migrate into the interior of the fruit (gonring et al. 2003a) thereby minimizing exposure to predation by social vespidae. the type of cucumber hybrid did not influence predation of d. hyalinata and d.nitidalis caterpillars by social wasps (fig. 3a). this is possibly due to the fact that these predators primarily use chemical and visual cues in prey location and selection of prey. corroborating this hypothesis picanço et al. (2011) found that social vespidae use visual and tactile signals in location and selection of t. absoluta larvae on tomato plants. the main species of social vespidae we observed preying on larvae of d. hyalinata and d.nitidalis was polybia ignobilis (haliday). we also observed the wasp polybia scutellaris (white) preying on larvae of d. hyalinata at low 567 santana, p.a. et al. — biological control of diaphania spp. by social wasps intensity, and this wasp was not observed preying d. nitidalis (fig. 3b). the predominance of predation of diaphania by p. ignobilis is possibly due to the abundance of this wasp in various vegetation in both natural and cultivated areas (hermes & köhler 2006, elpino-campos et al. 2007, picanço et al. fig. 2. mortality in the larval instars of (a) diaphania hyalinata and (b) diaphania nitidalis caused by social wasps. brackets indicate 95% confidence interval. 568 sociobiolog y vol. 59, no. 2, 2012 2010). p. scutellaris occurs mainly in areas close to forests (machado & parra 1984). moreover, p. ignobilis has a large foraging area which has a radius greater than 500m (raw 1998) while the foraging area of p. scutellaris has a radius of 150m (machado & parra 1984). the colonies of p. ignobilis have more workers than p. scutellaris (raw 1998, santos et al. 2007). as seen in fig. 3. mortality of diaphania hyalinata and diaphania nitidalis caused by wasps as a function of (a) cucumber hybrids and (b) species of vespidae. brackets indicate 95% confidence interval. 569 santana, p.a. et al. — biological control of diaphania spp. by social wasps this work for diaphania spp., gomes et al. (2007) (for fly larvae) and picanço et al. (2010) (for a. monuste caterpillars) observed high efficiency of insect predation by p. ignobilis. in conclusion, our results demonstrate the importance of social wasps as natural enemies of d. hyalinata and d. nitidalis and allowed the identification of key species o f vespidae predators of these pests. this knowledge should be considered when planning pest management programs in order to preserve these predators and maximize their performance in biological pest control. acknowledgment we wish to thank capes, cnpq and fapemig for their financial support. references bacci, l., m.c. picanço, a.h.r. gonring, r.n.c. guedes & a.l.b. crespo 2006. critical yield components and key loss factors of tropical cucumber crops. crop protection 25: 1117-1125. bacci, l., m.c. picanço, m.f. moura, a.a. semeão, f.l. fernandes & e.g.f. morais 2008. sampling plan for thrips (thysanoptera: thripidae) on cucumber. neotropical entomolog y 37: 582-590. carpenter, j.m. 1991, phylogenetic relationships and the origin of social behavior in the vespidae. in: ross, k.g. & r.w. matthews. the social biolog y of wasps. ithaca: cornell university. carpenter, j.m. & j. kojima 1997. checklist of the subfamily stenogastrinae (hymenoptera: vespidae). journal of the new york entomological society 104: 21–36. carpenter, j.m., j.w. wenzel & j. kojima 1996. sinonymy of the genus occipitalia richards, 1978, with clypearia de saussure, 1854 (hymenoptera: vespidae, polistinae, epiponini). journal of hymenoptera research 5: 157165. elpino-campos, a., k. del-claro & f. prezoto 2007. diversity of social wasps (hymenoptera, vespidae) in cerrado fragments of uberlândia, minas gerais state, brazil. neotropical entomolog y 36: 685-692. filgueira, f.a.r. 2000. novo manual de olericultura: agrotecnologia moderna na produção e comercialização de hortaliças. viçosa: universidade federal de viçosa. gobbi, n. & v.l.l. machado 1986. material capturado e utilizado na alimentação de polybia (trichothorax) ignobilis (haliday, 1836) (hymenoptera, vespidae). anais da sociedade entomológica do brasil 15: 117-124. gomes, l., g. gomes, h.g. oliveira, j.j. morlin jr, i.c. desuó, i.m. silva, s.n. shima & c.j.v. zuben 2007. foraging by polybia (trichothorax) ignobilis (hymenoptera, vespidae) on flies at animal carcasses. revista brasileira de entomologia 51: 389-393. 570 sociobiolog y vol. 59, no. 2, 2012 gonring, a.h.r., m.c. picanço, j.c. zanuncio, m. puiatti & a.a. semeão 2003a. natural biological control and key mortality factors of the pickleworm, diaphania nitidalis stoll (lepidoptera: pyralidae), in cucumber. biological agriculture and horticulture 20: 365-380. gonring, a.h.r., m.c. picanço, r.n.c. guedes & e.m. silva 2003b. natural biological control and key mortality factors of diaphania hyalinata (lepidoptera: pyralidae) in cucumber. biocontrol science and technolog y13: 361-366. hermes, m.g. & a. köhler. 2006. the flower-visiting wasps (hymenoptera, vespidae, polistinae) in two areas of rio grande do sul state, southern brazil. revista brasileira de entomologia 50: 268-274. kasper, m.l., a.f. reeson, s.j.b. cooper, k.d. perry & a.d. austin 2004. assessment of prey overlap between a native (polistes humilis) and an introduced (vespula germanica) social wasp using morpholog y and phylogenetic analyses of 16s rdna. molecular ecolog y 13: 2037-2048. king, a.b.s. & j.l. saunders 1984. the invertebrate pests of annual food crops in central america. london: overseas development administration. leite, g.l.d., i.r. oliveira, r.n.c. guedes & m.c. picanço 2001. comportamento de predação de protonectarina sylveirae (saussure) (hymenoptera: vespidae) em mostarda. agro-ciencia 17: 93-96. machado, v.l.l. & j.r.p. parra 1984. capacidade de retorno ao ninho de operárias de polybia (myrapetra) scutellaris (white, 1841). anais da sociedade entomológica do brasil 13: 13-18. o’donnell s. 1998. reproductive caste determination in eusocial wasps (hymenoptera: vespidae). annual review of entomolog y 43: 323-346. paula, s.v., m.c. picanço, i.r. oliveira, m.r. gusmão 2004. controle de broqueadores de frutos de tomateiro com uso de faixas de culturas circundantes. bioscience journal 20: 33-39. penã, j.e., v.h. waddill & k.d. elsey 1987. survey of native parasites of the pickleworm, diaphania nitidalis stoll, and melonworm, diaphania hyalinata (l.) (lepidoptera: pyralidae), in southern and central florida. environmental entomolog y 16: 1062– 1066. pereira, e.j.g., m.c. picanço, l. bacci, a.l.b crespo & r.n.c. guedes 2007a. seasonal mortality factors of the coffee leafminer, leucoptera coffeella. bulletin of entomological research 97: 421-432. pereira, e.j.g., m.c. picanço, l. bacci, t.m.c. della lucia, e.m. silva, f.l. fernandes 2007b. natural mortality factors of leucoptera coffeella (lepidoptera: lyonetiidae) on coffea arabica. biocontrol science and technolog y 17: 441-455. picanço, m.c, l.j. ribeiro, g.l.d. leite, m.r. gusmão 1998. seletividade de inseticidas a polybia ignobilis (haliday) (hymenoptera: vespidae) predador de ascia monuste orseis (godart) (lepidoptera: pieridae). anais da sociedade entomológica do brasil 27: 85-90. 571 santana, p.a. et al. — biological control of diaphania spp. by social wasps picanço, m.c., i.r. oliveira, j.f. rosado, f.m. silva, p.c. gontijo & r.s. silva 2010. natural biological control of ascia monuste by the social wasp polybia ignobilis (hymenoptera: vespidae). sociobiolog y 55: 67-76. picanço, m.c., l. bacci, r.b. queiroz, g.a. silva, m.m.m. miranda, g.l.d. leite & f.a. suinaga 2011. social wasp predators of tuta absoluta. sociobiolog y 58: 621-634. pratissoli, d., r.a. polanczyk, a.m. holtz, t. tamanhoni, f.n. celestino & r.c. borges filho 2008. influência do substrato alimentar sobre o desenvolvimento de diaphania hyalinata l. (lepidoptera: crambidae). neotropical entomolog y 37:361-364. prezoto, f. & v.l.l.machado 1999. transferência de colônias de vespas (polistes simillimus zikan, 1951) (hymenoptera, vespidae) para abrigos artificiais e sua manutenção em uma cultura de zea mays. revista brasileira de entomologia 43: 239-241. raw, a. 1998. population densities and biomass of neotropical social wasps (hymenoptera, vespidae) related to colony size, hunting range and wasps size. revista brasileira de zoologia 15: 815-822. reis jr., r., o. desouza, e.f. vilela 2000. predators impairing the natural biological control of parasitoids. anais da sociedade entomológica do brasil. neotropical entomolog y 29: 507-514. richter, m.r. social wasp (hymenoptera: vespidae) foraging behavior 2000. annual review of entomolog y 45: 121–150. santos, g.m.m., c.c. bichara filho, j.j. resende, j.d. cruz & o.m. marques 2007. diversity and community structure of social wasps (hymenoptera, vespidae) in three ecosystems in itaparica island, bahia state, brazil. neotropical entomolog y 36: 180-185. 885 two new species of the genus leptogenys from guangxi, china (hymenoptera: formicidae) by shanyi zhou1,2, yuan chen1, zhilin chen1, pining zhou3, daxiong ban3 & ming ying huang3 abstract two new species of the ant genus leptogenys roger, 1861 from china are described, i. e. l. laeviterga sp. nov. and l. rufida sp. nov.. l. laeviterga is close to l. strena zhou, but mandibles without tooth; anterior margin of clypeus truncate and with a pair of denticles in the middle; meso-propodeal suture deeply depressed. l. rufida is close to l. confucii forel, but dorsum of head in front of the eyes finely punctuate; anterior margin of clypeus rounded and with a pair of denticles in the middle; body brownish red. a key to all known species of the genus from china based on the worker caste is provided. introduction the ant genus leptogenys roger, 1861 is a large genus in the family formicidae, of which 217 species and 31 subspecies or indeterminate species (varieties or races, etc.) have been described in the world. among the described species, 53 distribute in the ethiopian region, 42 in the oriental region, 31 in the indo-australian region, 30 in the neotropical region, and the remain species in australasian, malagasy and nearctic regions, no species in palaearctic region (bolton, 2011). in china, 17 species have been recorded before this study (wheeler, 1930; terayama, 1990; tang & wang, 1995; wu & wang, 1995; xu, 1996, 2000; zhou, 2001). when we studied the ant fauna of daming moutain national nature reserve of guangxi, two new species of the genus were discovered. descriptions of the new species are as below. the type specimens are deposited in the insect collection, guangxi 1 college of life sciences, guangxi normal university, guilin 541004, china. e-mail: syzhou5612@yahoo.com.cn 2 key laboratory of ecolog y of rare and endangered species and environmental protection, ministry of education, guilin, 541004, china. 3administrative bureau of daming mountain national nature reserve, nanning, 530114, china. 886 sociobiolog y vol. 59, no. 3, 2012 normal university, guilin, china. a key to all known species of the genus from china based on worker caste is provided. measurements and indices following bolton, 1975: total length (tl). the total outstretched length of the individual, from the mandibular apex to the gastral apex. head length (hl). the straight-line length of the head in perfect full-face view, measured from the mid-point of the anterior clypeal margin to the midpoint of the occipital margin. head width (hw). the maximum width of the head measured behind the eyes in full-face view. cephalic index (ci). hw×100/ hl. scape length (sl). the straight-line length of the antennal scape, excluding the basal constriction or neck. scape index (si). sl×100/ hw. pronotal width (pw). the maximum width of the pronotum measured in dorsal view. petiole height (ph). the height of the petiole measured in profile from the apex of the ventral (subpetiolar) process vertically to a line intersecting the dorsalmost point of the node. petiole length (pl). the length of the petiole from the anterior process to the posteriormost point of the tergite, where it surrounds the gastral articulation. lateral petiole index (lpi). ph×100/ pl. dorsal petiole width (dpw). the maximum width of the petiole in dorsal view. dorsal petiole index (dpi). dpw×100/ pl. all measurements are expressed in millimeters. key to the known species of leptogenys from china based on the worker caste 1 masticatory margin of mandible with 3 or more than 3 teeth. head nearly square .....................................................................................................................2 masticatory margin of mandible without or with only 1 tooth. head 887 zhou, s. et al. — two new species of leptogenys from guangxi, china elongate, distinctly longer than broad ............................................................5 2 inner margin of mandible without tooth .......................................................3 inner margin of mandible with tooth .............................................................4 3 masticatory margin of mandible with 3 teeth, in dorsal view petiolar node trapezoidal (china: yunnan; myanmar) ................ l. crassicornis emery masticatory margin of mandible with 5 teeth, in dorsal view petiolar node subsemicircular (china: hunan, guangxi) ........................ l. strena zhou 4 inner margin of mandible with 1 tooth. in dorsal view petiolar node semicircular. head and alitrunk black (china: yunnan) .........l. huangdii xu inner margin of mandible with 3 or more than 3 teeth. in dorsal view petiolar node crescent-shaped. head and alitrunk dark reddish brown (china: yunnan; myanmar; india) .................................................. l. birmana forel 5 head smooth and shining ..................................................................................6 head longitudinally striate or densely punctuate...................................... 11 6 scape of antenna surpassing occipital corner by half of its length (china: yunnan) ..........................................................................................l. pangui xu scape of antenna surpassing occipital corner by less than half of its length .....................................................................................................................7 7 head, alitrunk and gaster without metallic luster ........................................8 head, alitrunk and gaster with blue or purple metallic luster ...................9 8 anterior margin of clypeus not truncate, without denticle in the middle(china: zhejiang, hunan; vietnam; myanmar; sri lanka) .......... ............................................................................................. l. peuqueti (andre) anterior margin of clypeus truncate, with a pair of denticles in the middle (china: guangxi) ..........................................................l. laeviterga sp. nov. 9 anterior margin of clypeus truncate (china: yunnan, guangxi, taiwan; india; sri lanka; philippines) ....................................... l. chinensis (mayr) anterior margin of clypeus extruding ......................................................... 10 10 body black. subpetiolar process rounded at apex (china: yunnan, hunan, guangxi, hong kong, macao; myanmar; india; bangladesh) ................... .............................................................................................. ..l. minchinii forel body dark yellowish brown. subpetiolar process triangular, angled at apex (china:taiwan; japan) ......................................................... l. confucii forel 11 head longitudinally striate ............................................................................ 12 head densely punctate ................................................................................... 13 888 sociobiolog y vol. 59, no. 3, 2012 12 clypeus with longitudinal central carina (china: yunnan, guangdong, guangxi, taiwan; myanmar; india; sikkim; sri lanka; malaysia) ............ ....................................................................................... l. diminuta (f. smith) clypeus without longitudinal central carina (china: yunnan, guizhou, hunan, guangxi, fujian, taiwan, hong kong ; myanmar; sikkim; india) ........................................................................................... l. kitteli (mayr) 13 masticatory margin of mandible as long as inner margin. masticatory margin with1 small tooth in the middle (china: yunnan) .......................... .... ............................................................................................... l. zhuangzii xu masticatory margin of mandible shorter than inner margin. masticatory margin without tooth in the middle ............................................................ 14 14 anterior margin of clypeus truncate ........................................................... 15 anterior margin of clypeus extruded ........................................................... 17 15 anterior margin of clypeus without denticle in the middle (china: guangxi) ........................................................................l. huapingensis zhou anterior margin of clypeus with a pair of denticles in the middle ........ 16 16 head, alitrunk, and petiole densely and coarsely punctate (china: guangxi) ............................................................................ .l. binghamii forel dorsum of head in front of the eyes finely punctuate, alitrunk and petiole smooth and shining (china: guangxi) ............................ l. rufida sp.nov. 17 masticatory and inner margins of mandible separated by a blunt angle. (china : yunnan) .......................................................................l. mengzii xu masticatory margin of mandible rounded into inner margin................. 18 18 dorsum of propodeum a little longer than declivity, 2nd segment of flagellum of antenna longer than the 3rd segment (china: guangxi) ........... ............................................................................................l. hezhouensis zhou dorsum of propodeum about 3 times as long as declivity, 2nd and 3rd segments of flagellum of antenna about equal (china: yunnan) ...l. laozii xu descriptions of the new species leptogenys laeviterga sp. nov. figs. 1−3 holotype worker. tl 8.8, hl 1.67, hw 1.00, ci 59, sl 1.90, si 190, 889 zhou, s. et al. — two new species of leptogenys from guangxi, china figs. 1−6 leptogenys workers. 1−3 l. laeviterga sp. nov. 4−6 l. rufida sp. nov. 1, 4. head in full-face view 2, 5. body in profile view 3, 6. alitrunk in dorsal view 890 sociobiolog y vol. 59, no. 3, 2012 pw0.95, al 2.75, ed 0.25, ml 0.90, pl 0.80, ph 0.87, dpw 0.62, lpi 108, dpi 77. head longer than broad, lateral sides parallel, occipital margin straight, occipital carina distinct. mandibles elongate, with only apical tooth. clypeus acutely carinate, its anterior margin truncate, and with a pair of denticles in the middle. eyes moderately large, flat, placed in front of the midline of the lateral sides of the head. antennal scapes stout, about 1/3 of its length surpassed beyond the occipital margin. alitrunk slender, in profile view pro-mesonotum convex, pro-mesonotal suture and meso-propodeal suture distinct, metanotal groove deeply depressed. dorsum of propodeum straight, about twice as long as declivity, its posterodorsal corner rounded. petiolar node anteroposteriorly compressed, with anterior face evenly convex, posterior face straight, dorsum rounded. in dorsal view, the node sub-semicircular, longer than broad. subpetiolar process moderately large, triangular. legs long. gaster stout, longer than alitrunk, constriction between 1st and 2nd gastral segments distinct. sting long. mandibles and clypeus longitudinally striate. antennal scapes and flagella shagreened. head, alitrunk, petiole and gaster smooth and shining. the lower 1/3 of mesopleura and sides of propodeum longitudinally striate. declivity of propodeum transversely striate. head, alitrunk, petiole and gaster with abundant soft erect hairs, which combined with abundant short suberect hairs on head, antenna and legs. colored blackish red. mandibles and legs paler, brownish red. paratype worker. tl 8.3, hl 1.70, hw 1.02, ci 60, sl 1.90, si 186, pw 1.00, al 2.85, ed 0.27, ml 0.92, pl 0.75, ph 0.87, dpw 0.67, lpi 116, dpi 89. paratype worker. daming mountain national nature reserve, n23.49, e 108.43, 1230m, may 29.2011. yuan chen; paratype, 1 worker, data as holotype, but in may 21.2011. shanyi zhou. queen and male are unkown. ecolog y. unkown. etymolog y. this species is named after its shining gaster. this new species is close to l. strena zhou, but mandibles without tooth; anterior margin of clypeus truncate, and with a pair of denticles in the middle; meso-propodeal suture deeply depressed. it distinguished from l. lucidula emery by the lower 1/3 of mesopleura, sides of propodeum longitudinally 891 zhou, s. et al. — two new species of leptogenys from guangxi, china striate, and declivity of propodeum transversely striate. leptogenys rufida sp. nov. figs. 4−6 holotype worker. tl 5.6, hl 1.12, hw 0.75, ci 67, sl 1.12, si 149, pw 0.70, al 1.80, ed 0.20, ml 0.55, pl 0.65, ph 0.60, dpw 0.42, lpi 92, dpi 64. head roughly rectangular, distinctly longer than broad. occipital margin straight, occipital carina distinct. occipital corners rounded. lateral sides of head almost straight. mandibles slender, inner margin without tooth, masticatory margin with only 1 apical tooth, basal corner rounded. clypeus with sharp longitudinal central carina, anterior margin rounded, with a pair of denticles in the middle. scape of antenna surpassed occipital corner by about 1/4 of its length. segments of flagellum longer than broad, the 2nd and 3rd joints about equal. in profile view dorsum of pronotum feebly convex, mesonotum a little lower than pronotum, pro-mesonotal suture distinct; metanotal groove depressed but not deep. dorsum of propodeum slightly convex, about 3 times as long as declivity, declivity weakly convex. in profile view petiolar node trapezoid, declined forward to the anterior face, posterodorsal angle bluntly extruding, dorsal face evenly convex, posterior face straight. in dorsal view the node longer than broad, narrowed forward. subpetiolar process small, nearly square, posteroventral corner acute. constriction between the two basal gastral segments distinct. mandibles smooth and shining, clypeus longitudinally striate. scapes of antennae densely punctuate. dorsum of head in front of the eyes densely punctuate, occipital border and occipital corners smooth. dorsum of alitrunk, petiole and gaster smooth and shining. mesopleura and lateral sides of propodeum irregularly rugose. dorsum of head and body with abundant long erect hairs, short suberect hairs and decumbent pubescence. scapes and tibiae with abundant subdecumbent hairs and dense pubescence. head, alitrunk, petiole and gaster brownish red. apex of antennae, legs and apex of gaster paler, yellowish red. paratype worker. tl 6.4, hl 1.25, hw 0.80, ci 64, sl 1.25, si 156, pw 0.75, al 2.0, ed 0.25, ml 0.57, pl 0.70, ph 0.60, dpw 0.47, lpi 85, dpi 67. holotype worker, daming mountain national nature reserve, n23.49, 892 sociobiolog y vol. 59, no. 3, 2012 e 108.43, 1230m, may 29.2011. yuan chen; paratype: 1 worker, tianmu mountain nature reserve, zhejiang province of china. august 2.2011. zhilin chen. queen and male are unkown. ecolog y. unkown. etymolog y. this species is named after its red color. this new species is close to l. confucii forel, but with dorsum of head in front of the eyes finely punctuate, anterior margin of clypeus rounded, with a pair of denticles in the middle; body brownish red. acknowledgments this work is supported by the national natural science foundation of china (project no. 30770258, 31071971), the natural science foundation of guangxi (project no. 2010gxnsfe013004) and foundation of the key laboratory of ecolog y of rare and endangered species and environmental protection, ministry of education, guangxi normal university (project no. 1101z002). references bolton, b. 1975. a revision of the ant genus leptogenys roger in the ethiopian region, with a review of the malagasy species. bulletin of the british museum (natural history) (entomolog y) 31: 235-305. bolton, b. 2011. catalogue of species-group taxa. http://gap.entclub.org/contact.html. (accessed date: 1 march, 2011). tang, j., s. li, e.y.huang & b.y. zhang. 1995. economic insect fauna of china. fasc. 47. hymenoptera: formicidae (1). beijing : science press, 134 pp. terayama, m. 1990. a list of ponerinae of taiwan (hymenoptera: formicidae) . bulletin of toho gakuen. 4: 25-49. wheeler, w. m. 1930. a list of the known chinese ants. peking natural history bulletin 5: 53-81. wu, j. & c.l. wang 1995. the ants of china. beijing : china forestry publishing house. 214 pp. xu, z. h. 1996. a taxonomic study on the ant genus leptogenys (hymenoptera: formicidae) in china. journal of yunnan university. 11(4): 222-227. xu, z. h. 2000. five new species and one new record species of the ant genus leptogenys from yunnan province, china. entomologia sinica. 7(2): 117-126. zhou, s. y. 2001. ants of guangxi. guilin: guangxi normal university press, 255 pp. 521 effect of soil water content on toxicity of fipronil against solenopsis invicta by jie wang, qi yang, haixiang huang, he zhang, juan hu & yijuan xu* abstract this study evaluated the effect of fipronil on the survival of fire ant workers with different doses and soil water contents and further examined the persistent effect of the same dose of powder at 10%, 50% and 90% soil water content. the results showed that mortality was positively correlated to the dosage. this result indicated that the survival rates of workers treated by powder at different rsw (relative soil water content) were significantly different (p <0.01). at the rsw of 10% and 20%, the survival rates of workers were 40.67 and 49.00 respectively, which showed no obvious difference from other treatments but were lower than the control. the survival rate decreased sharply when the rsw was 90%, and was obviously lower than that of treatments at moderate (30-50%) rsw. the contact powder showed worst persistent effect when the soil water content was 10%, but at the soil water content of 50% and 90%, the lethal effect of the powder was higher and was more persistent. key words: solenopsis invicta, contact powder, soil water content, mortality rate, persistent effect introduction the red imported fire ant, solenopsis invicta buren, is a dangerous pest native to sub-amazonian south america, and invaded the southern united states due to quarantine negligence in the early twentieth century. s. invicta feeds variously, breeds rapidly, has ferocious and competitive habits, and does harm to human health, public safety, agriculture and forestry production and the ecological environment of the invaded region. it is listed as one of the world’s most dangerous 100 invasive pests (zeng et al. 2005). at the end of red imported fire ant research center, south china agricultural university, guangzhou 510642, china *corresponding author’s email: xuyijuan@yahoo.com 522 sociobiolog y vol. 59, no. 2, 2012 2004 serious harm caused by s. invicta was found in wuchuan, guangdong province which marked the successful invasion and colonization of this dangerous pest in china (zeng et al. 2005). ants are distributed in tropical and subtropical regions, where there are frequent rainfalls all year round, especially in the humid and rainy spring. spring and summer are the active breeding seasons for s. invicta. rainfall will not reduce the activity of fire ants, but induces nest moving and division for the ants, which will lead to the rapid increase in the number of fire ant nests (zhao et al. 2009). frequent rain also causes some difficulties for prevention and treatment, thus the traditional prevention work will not be conducted in this season. however, if the prevention and control work of this period are not maintained, s. invicta will thrive and create favorable conditions for expansion, which will add to the huge challenge of fire ant prevention and control. large-scale application of baits to control s. invicta is the most effective method. bait can help control s. invicta at a low level chronically (banks 1990). to take full advantage of bait and pesticides in the control of s. invicta in urban areas, a two-step method was recommended: first, baiting is used for large area processing, and then contact toxicity of insecticides is employed to deal with the remnants of ant mounds individually (drees et al. 2000). contact insecticides are commonly used in controlling red imported fire ants by individual mound treatment s(chen 2006; appel & woody 1990). fipronil was one of the most popular contact powders introduced for the control of s. invicta (sparks & diffie 1998; collins & callcott 1998; barr & best 2003), and was considered the ideal pesticide for s. invicta in frequent irrigation areas (greenberg et al. 2003). however, in the rainy spring and summer, rain erosion, soaking and humidity makes pharmaceutical degradation failure occur more frequently (krushelnycky et al. 2005). after each rainfall newly repaired nests are clearly identifiable, which may be convenient for the prevention and treatment of s. invicta in spring and summer, combined with the use of moisture-proof types of bait. it was reported that fipronil proved to have greater efficacy in the appropriate soil moisture conditions than dry soil conditions (zhuang et al. 2007). therefore, a full understanding about the effect of the amount of soil water content on 523 wang, j. et al. — effect of soil water content on toxicity of fipronil contact powder efficiency will help to facilitate fire ant control techniques after rainfalls in spring and summer. materials and methods insects and tested powder s. invicta workers were randomly collected from ten polyg yne colonies in the campus of south china agricultural university. the ants were analyzed within 2 weeks of capture. the social form of s. invicta was confirmed by the number of queens present in each colony (porter 1992). more than two queens were found in each polyg yne colony. ants were collected directly from the mounds with a gardening trowel and placed in plastic boxes. the upper inner edge of each box was lined with talcum powder to prevent escape. the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor l.). a test tube (25mm×200 mm), which was filled partially with water and plugged with cotton, was used as a water source. ants were maintained in the laboratory at 25 ± 2°c. contact powder (0.1% fipronil) was provided by entomolog y institute of guangdong, china. calculation of the soil water content for powder-soil mixture sandy soil was selected for the bioassay and was heated for 24 h at a temperature of 100°c. the formula for the calculation of soil water content was as follows: relative soil water content (rsw) =soil actual water content (saw)/ soil saturated water content (ssw)×100%: to prepare the soil with different rsw for powder-soil mixture, 500 g of sand plus powder was placed into plastic sample boxes (20 cm×15cm×15cm), and water was mixed with the sand according to the formula. before testing, there was a 5 min wait for the water to fully penetrate the sand. lethal effect of contact powder on s. invcta workers at different dose glass jars (6.5cm bottom diameter, 5.0cm diameter, 12cm height) were filled with the soil with 50% relative water content which contained contact powder against the fire ants. powder contents of 0.02g/150g sand, 0.08g/150g sand, 0.14g/150g sand, 0.20g/150g sand, and 0.26g/150g sand were used 524 sociobiolog y vol. 59, no. 2, 2012 for the test. 100 worker ants were put into each jar, whose edge was wiped with talcum powder to prevent escape of workers. five repeated tests were conducted for each dose. the drug content for the control was 0. we checked the mortality of workers and recorded the data 24 hours after the workers were put in. lethal effect of contact powder on s. invcta workers at different rsw different glass jars were filled with soil whose powder content was 0.26g/150g sand, and the relative water contents of the soil were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% for the test. 100 worker ants were put into each jar, whose edge was wiped with talcum powder to prevent escape of workers. five repeated tests were conducted for each rsw. two controls were set and the drug content was 0 while the relative water contents were 10% and 90% respectively. we checked the mortality of workers and recorded the data 24 hours after the workers were put in. persistent lethal effect of the contact powder on s. invcta workers soil with relative water contents of 10%, 50%, 90% and powder content of 0.26g/150g net sand was prepared in glass jars. the jars were sealed with plastic film to prevent excessive evaporation of water, and the date labels were tagged. 5 replicates were conducted for each rsw. these soil preparation steps were exactly followed for five days for the following bioassay. 100 worker ants were put into each of the glass jars with prepared soil, and the edge of talcum powder was wiped to prevent escape on the last day. we checked the mortality of workers and recorded the data 24 hours after the workers were put in. statistical analysis variations in the survival of s. invicta with varied rsw were analyzed using analysis of variance. t-test for paired data was used to compare the mortality of workers at different rsw (between 50% and 10% or 90%). linear regression was used to determine the relationship between the survival and powder’s weight. all the statistical analyses were conducted using the spss13.0 software package. 525 wang, j. et al. — effect of soil water content on toxicity of fipronil results lethal effect of contact powder on s. invcta workers at different dose the results indicated a strong correlation between the survival rate and powder’s concentration when rsw was 50% (fig. 1). the survival rates of the workers (y) in the powder treated soil were best predicted by fitting the linear model: y=-14.38x+107.02 (n=5, r=0.98625) in this model, x signifies the powder’s concentration. the survival rates were significantly and positively correlated to the powder’s concentration (p<0.01). lethal effect of contact powder on s. invcta workers at different rsw this result indicated that the survivals of worker treated by powder at different rsw were significantly different (p<0.01). when the rsw was 10% and 20%, survival of workers was 40.67 and 49.00 respectively, which were not obviously different from other treatments but lower than the control. the survival rate decreased sharply when the rsw was 90%, and was obviously lower than that of treatments at moderate (30-50%) rsw (table 1). fig. 1 lethal effect of contact powder on s. invcta workers at different dose 526 sociobiolog y vol. 59, no. 2, 2012 persistent lethal effect of the contact powder on s. invcta workers the result confirmed that the control efficacy of powder was stronger under the condition of the high (50% or 90%) rsw compared with the low (10%) rsw (for 50% rsw: t=-4.610, p=0.006; for 90% rsw: t=-7.201, p=0.001). in addition, the mortality of workers was lower than 10% after 2 days’ treatment at low (10%) rsw. while at high (50% or 90%) rsw, the mortality of workers was higher than 80% after 2 days’ treatment (fig. 2). discussion chemical control of s. invicta is easily affected by moist conditions and rainfall because the bait formulations have a propensity to degrade when wetted (kafle et al. 2009). therefore, the development of fire ant baits that fig. 2 persistent lethal effect of the contact powder on s. invcta workers table 1. lethal effect of contact powder on s. invcta workers at different rsw. rsw n survival (mean+ se) 10% 20% 30% 40% 50% 60% 70% 80% 90% ck 1 ck 2 5 5 5 5 5 5 5 5 5 5 5 40.67+4.57bc 49.00+3.81bc 65.00+7.70c 63.00+1.46c 64.33+4.39c 51.67+8.33bc 29.00+10.22bc 23.67+13.57bc 21.67+1.52b 99.00+0.67a 97.00+1.34a ck1 and ck2 mean no drug content while the relative water contents were 10% and 90% respectively. means in the same column followed by the same small letter are not significantly different (lsd) at the level of 0.05 527 wang, j. et al. — effect of soil water content on toxicity of fipronil are resistant to high humidity or water can increase the efficacy of chemical control (kafle et al. 2010). in this study, we observed that the moist soil condition may be helpful to the powder control effect. with the same relative water content in the soil, the amount of drug powder has a linear correlation with its lethal effect. when the relative water content in the soil is higher than 70%, the net powder showed a better lethal effect at the same dose, while the lethal effect becomes poor at the relatively low water content in soil (10% for example). when the dose is 0.26g/150g sand, the net lethal effect is almost lost and the worker mortality dropped to about 10% two days after the red ant powder trial was conducted in the indoor conditions. when the worker ants were directly exposed to the powder of 0.1% fipronil, the median lethal time for the workers was 20.06 h (zhuang et al. 2007). as our tests found, when the red ant powder was mixed with higher relative water content (50%) of the soil, the powder achieved the best efficacy two days later. we also found that the mortality rate of the workers is relatively high in the dry soil, probably because low humidity conditions are not suitable for the survival of the red imported fire ants. drought has a certain impact on s. invicta reproduction and spread. in addition, extreme humidity like rainfall will reduce 40% of ants’ foraging activity. rainfall will block the underground channels and interfere with pheromones, which will also affect recruitment for the fire ants (porter & tschinkel 1987). a prior study ruled out temperature factors on the survival rate of s. invicta (porter 1988), at constant temperature conditions, the soil moisture content and the amount of red powder have a great impact on the mortality of ant workers (hadley 1994). of course, these conclusions are drawn according to indoor experiments, so further verifications by field trials are needed. for social prevention and treatment of invasive insects like the red imported fire ant (gentz 2009), the use of bait (levy et al. 1974; williams 1983) and beauveria bassiana (stimac et al. 1993) were also employed in addition to the powder, among which the bait was more vulnerable to moisture, and the fungi can play a more significant control role in high humidity conditions. therefore, further studies should be carried out about the combined effects of powder and fungi in high humidity conditions, and the bait’s water-proof efficiency could be improved, which would significantly improve fire ant control techniques during the spring and summer rainy season. 528 sociobiolog y vol. 59, no. 2, 2012 acknowledgments this work is supported by the specialized research fund for the doctoral program of higher education of china (20104404110018). references appel, a.g. & l.g. woody 1990. individual mound treatment for rapid control of fire ants, pp. 248-251. in: bode, l.e., j.l. hazen & d.g. chasin [eds.], pesticide formulations and application systems, astm stp 1078, vol. 10. philadelphia, pa. banks, w. 1990. chemical control of the imported fire ants. applied myrmecolog y, ment of crabs. office of research and development report epa-600/3-76-007. us epa, washington, dc 596-603. barr, с.l. & r. best 2003. comparison of different formulations of broadcast fipronil for the control of red imported fire ants. result demonstration handbook 1999-2003, tex. ag. extension serv, bryan, tx. chen, j. 2006. digging behavior of solenopsis invicta workers when exposed to contact insecticides. journal of economic entomolog y 99:634-640. collins, h. & a. callcott 1998. fipronil: an ultra-low-dose bait toxicant for control of red imported fire ants (hymenoptera: formicidae). florida entomologist 407-415. drees, b.m., c.l. barr, s.b. vinson, r.e. gold, m.e. merchant & n. riggs 2000. managing imported fire ants in urban areas. tex. agric. ext. serv. b 6043. gentz, m. 2009. a review of chemical control options for invasive social insects in island ecosystems. journal of applied entomolog y 133, 229-235. greenberg, l., d. reierson & m.k. rust 2003. fipronil trials in california against the red imported fire ant, solenopsis invicta buren, using sugar water consumption and mound counts as measures of ant abundance. journal of agricultural and urban entomolog y 20, 221-233. hadley, n.f. 1994. water relations of terrestrial arthropods. academic press, san diego, ca. kafle, l., w. wenjer, r.k. meer, h. yiyou & s. chengjen 2009. microencapsulated bait: does it work with red imported fire ants, solenopsis invicta (hymenoptera: formicidae)? sociobiolog y 53, 729-737. kafle, l., w.j. wu & c.j. shih 2010. a new fire ant (hymenoptera: formicidae) bait base carrier for moist conditions. pest management science 66(10):1082-1088. krushelnycky, p.d., l.l. loope & n.j. reimer 2005. the ecolog y, policy, and management of ants in hawaii. proceedings of the hawaiian entomological society 37: 1-25. levy, r., j. carroll, y. chiù & w. banks 1974. toxicity of chemical baits against the red imported fire ant, solenopsis invicta. florida entomologist 155-159. porter, s.d. 1988. impact of temperature on colony growth and developmental rates of the ant, solenopsis invicta. journal of insect physiolog y 34, 1127-1133. porter, s.d. 1992. frequency and distribution of polyg yne fire ants (hymenoptera: formicidae) in florida. florida entomologist 248-257. 529 wang, j. et al. — effect of soil water content on toxicity of fipronil porter, s.d. & w.r . tschinkel 1987. foraging in solenopsis invicta (hymenoptera: formicidae): effects of weather and season. environmental entomolog y 16, 802808. sparks, b. & s. diffie 1998. evaluation of broadcast treatments of fipronil for control of red imported fire ants in georgia, pp. 159-162. i:n shanklin, d. (ed), proceedings imported fire ant research conference, hot springs, ar. stimac, j.l., r.m. pereira, s.b. alves & l.a. wood 1993. mortality in laboratory colonies of solenopsis invicta (hymenoptera: formicidae) treated with beauveria bassiana (deuteromycetes). journal of economic entomolog y 86, 1083-1087. williams, d.f. 1983. the development of toxic baits for the control of the imported fire ant. florida entomologist 66 162-172. zhuang, t.y., w.j. tian, x.n. li, c.x. wang , s.h. wu & l. wang 2007. efficacy of pyragne in controlling the red fire ant, solenopsis invicta. chinese bulletin of entomolog y 44: 746-748. zhao, j., p.s. zhong, t. huang & s.s. zhang 2009. impact of precipitation on behavior of solenopsis invicta buren colony. chinese journal of vector biolog y and control 20:542-544. zeng, l., y.y. lu, x.f. he, w.q. zhang & g.w. liang 2005. identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan, guangdong. chinese bulletin of entomolog y 42: 44-48. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i4.4581sociobiology 67(4): 599-603 (december, 2020) the employment of chemicals such as terpenes to attract male orchid bees since the late 1960s (dodson et al., 1969; williams & whitten, 1983) has led to several studies into the taxonomy and biogeography of euglossines. however, among the dozens of pure chemical substances produced by the industry, one salt, in particular, has stood out in the discovery of unusual species of the tribe: this is methyl cinnamate, a commercially available crystal and the most powerful chemical bait for aglae caerulea lepeletier de saint-fargeau and audinet-serville in the neotropics (williams & dodson, 1972; morato, 2001; anjos-silva et al., 2006; martins et al., 2016; anjos-silva, 2019a,b). several authors pointed out that the diversity of species in the gallery forests in the cerrado is a consequence of recurrent connections between the amazon and atlantic forests during the last glaciation periods. these events caused gallery forests in the cerrado to present similar diversity of species to that of the east of the amazon forest and the north of abstract several authors have suggested that gallery forests play an essential role as mesic corridors, opening the way to the colonization of the cerrado by forest-dependent species with ranges centered in the neighboring amazon and atlantic forests. one such species is the cleptoparasitic aglae caerulea lepeletier de saint-fargeau & audinet-serville, which has had its geographical distribution expanded by various records made in gallery forests in areas of that biome. we report here the occurrence of a. caerulea in a gallery forest located in santo antônio do leverger municipality, in mato grosso, brazil. eleven males were collected in the pantanal of barão de melgaço, in mato grosso, brazil. the results presented reinforce the need for further research to understanding better the distributional range limits of a. caerulea in southern south america, now that the species has been recorded for the first time almost 30 km from the flooded plains of the pantanal. sociobiology an international journal on social insects mh schorn de souza1,2, jds figueiredo2, jc cunha2, so pains2, mt brito2, fa labaig2, ca garófalo3, ej anjos-silva1,2 article history edited by cândida aguiar, uefs, brazil received 13 june 2019 initial acceptance 25 july 2019 final acceptance 20 july 2020 publication date 28 december 2020 keywords neotropics, hymenoptera, amazon forest, gallery forest, cerrado. corresponding author evandson j. anjos-silva https://orcid.org/0000-0003-0952-8437 unemat bee collection av. são joão nº 563, bloco superior ii cep 78200-000, cáceres-mt, brasil. e-mail: evandson@unemat.br the atlantic forest (oliveira-filho & ratter, 1995; costa, 2003; wang et al., 2004; fouquet et al., 2012; batalhafilho et al., 2013; sobral-souza et al., 2015). some authors have highlighted that gallery forests play an important role as mesic corridors, opening the way to colonization of the cerrado by forest-dependent species with ranges centered in the neighboring amazon and atlantic forests (sick, 1966; willis, 1992; moura and schlindwein, 2009; silva et al., 2013; martins et al., 2016). given the importance of gallery forests as alternative dispersion paths for several species, including a. caerulea, this study aimed to verify the occurrence of that species in a gallery forest located between the cerrado, pantanal and araguaia regions, in mato grosso state. the present study was conducted in a gallery forest located at the instituto federal de educação, ciência e tecnologia de mato grosso (ifmt), são vicente range (15°49’21.42” s; 55°25’06.36” w), in the santo antônio do leverger municipality, mato grosso, brazil (fig 1). the region is characterized by 1 programa de pós-graduação bionorte rede de biodiversidade e biotecnologia da amazônia legal, campus avançado do museu paraense emílio goeldi (mpeg-mctic), cuiabá-mt, brazil 2 laboratório de abelhas e vespas neotropicais, departamento de biologia, universidade do estado de mato grosso, cáceres-mt, brazil 3 departamento de biologia, faculdade de filosofia, ciências e letras de ribeirão preto-usp, ribeirão preto-sp, brazil range expansion of the cleptoparasitic orchid bee aglae caerulea in the pantanal of mato grosso, brazil short note mh schorn de souza et al. – expansion range of aglae caerulea in the pantanal600 a tropical savanna climate, aw type on the köppen-geiger climate classification system (peel et al. 2007). to check the occurrence or not of a. caerulea males in the study area, two plastic bottle traps containing methyl cinnamate were made available in the area from 28 may to 6 july. the trap consisted of a 500 ml plastic bottle with two side openings and a cotton pad containing methyl cinnamate hanging inside. since these traps were not inspected daily, the bottom of the bottle was filled with 250 ml of 96% ethyl alcohol to avoid the decomposition of specimens that could be attracted. the first inspection was made on 26 june, and the second occurred on 6 july when the study was finished. the males caught in the traps were removed, pinned, and housed in the universidade do estado de mato grosso unemat bee collection (ejas 90.002 90.013) of the laboratory of neotropical bees and wasps (labeve), cáceres municipality, mato grosso state, brazil. the geographical distribution data of a. caerulea presented in this study are based on the recent revision of occurrence records for this species (anjos-silva, 2019a,b), together with the specimens housed in the unemat bee collection. besides, the data made available from the global biodiversity information facility (gbif) (www.gbif.org), discover life bee species guide and world checklist (ascher & pickering, 2018) (http://www.discoverlife.org), moure’s bee catalogue (moure et al., 2012), and cria’s species link (www.splink.org.br) were also used (supplementary material 1). eleven a. caerulea males were caught at the beginning of the dry season in the paraguay-araguaia-tocantins divide at the northeast border of the pantanal (fig 1). of the collected males, eight males were removed from the traps during the first inspection of them, and the three remaining males were found when the traps were removed from the field. this study adds new occurrence points for a. caerulea in the neotropics (see supplementary material). considering that cleptoparasitic species have extraordinary dispersal ability and travel several kilometers in forests in a single day (wikelski et al., 2010; pokorny et al., 2015), it seems reasonable to assume that a. caerulea could be found in the southern portion of the pantanal sub-regions. concerning literature reviews, the results lead us to conclude that the são vicente range is the second most abundant area in the neotropics for a. caerulea (table 1), an area situated precisely between the amazon, paraguay, and araguaia-tocantins divide. reports of this species for the pantanal, therefore, may be influenced by (1) vegetation types and microclimates similar to amazon and atlantic forests (oliveira-filho & ratter, 1995; sobral-souza et al., 2015), (2) the formation of ecological corridors of gallery forests throughout the são vicente range region, situated in the same complex of parallel mountain ranges that also reaches the parque nacional da chapada dos guimarães, the location of the first record of this species for the cerrado domain (anjossilva et al., 2006), (3) the proximity to the flooded areas by fig 1. map of the amazon-paraguay divide, with locations of the previous records of a. caerulea (black circle) and the first a. caerulea record points in pantanal (black star), exactly between the sub-basin drainages of the paraguay river (hydrographic basin of rio da prata) and araguaia river (hydrographic basin of rio tocantins (ibge, 2000). the physiographic limits of the pantanal are indicated in hachured lines. sociobiology 67(4): 599-603 (december, 2020) 601 the cuiabá river and tributaries in the pantanal of barão de melgaço, less than 30 km from the flooded plain, (4) by the presence of its hostess species eulaema nigrita lepeletier, 1846, considered abundant when compared to other occurrence data for hostess and their parasites in studied orchid bee assemblages (williams & dodson, 1972; otero & sandino, 2003; anjos-silva et al., 2006; hentrich et al., 2007; abrahamczyk et al., 2011; silva et al., 2013; santos junior et al., 2014; figueiredo et al., 2015; martins et al., 2016, anjos-silva et al., 2019a,b). the register of a. caerulea exactly between the subbasins of the paraguay and araguaia rivers (ibge, 2000) reinforces the link between the a. caerulea populations in two of the largest brazilian river basins with populations in the western part of the northeast coastal basins. the results presented here demonstrate the need for further research to understand better the distributional range limits of a. caerulea in southern south america, now that the species has been recorded for the second time (anjos-silva, 2019a,b) almost 30 km from the flooded plains of the pantanal. acknowledgments mhss is grateful to the postgraduate program in biodiversity and biotechnology of the rede bionorte at the university of the state of mato grosso, to the research coordinator of the federal institute of education, science, and technology of mato grosso (ifmt), and são vicente campus for the logistical support during the collections. support was provided by fapemat (project number 334721/2008, 737955/2012, 222842/2015). table 1. occurrence records for aglae caerulea lepeletier de saint-fargeau & audinet-serville, according to altitude (elevation, m), habitat associations, chemical attractants, and data relative to the abundance of these cleptoparasitic species. study areas country long. lat. alt. attractants a. caerulea habitat references alto paraíso de goiás br -47.6088 -14.3152 1230 mc 1 ce silva et al., 2013 aragua, portachuelo ve -61.2547 9.0516 1200 flight 1 df gonzález, 1996 aragua, guamita ve -60.3613 9.0080 725 flight 1 df gonzález, 1996 aragua, el limón ve -67.5722 10.06 450 flight 1 df gonzález, 1996 aragua, via choroni ve -67.0581 10.5611 200 flight 1 df gonzález, 1996 aragua, moyobamba pe -76.5830 -6.0430 954 * 1 af abrahamczyk et al., 2011 silvânia br -48.4917 -16.498 920 mc 1 ce silva et al., 2013 santo antônio do leverger, são vicente range br -55.4058 -15.8297 750 mc 11 gf this study aragua, guamita ve -60.3613 9.0080 725 flight 1 df gonzález, 1996 pq. nacional chapada dos guimarães br -55.9867 -15.405 598 mc 8 gf anjos-silva et al., 2006 buena vista bo -63.6360 -17.516 424 * 1 df abrahamczyk et al., 2011 parque estadual do mirador br -45.8891 -6.6167 313 mc 2 gf martins et al., 2016 parque estadual cristalino br -55.8067 -9.4667 278 mc, bb 18 af figueiredo et al., 2015 parque nacional da serra do divisor br -73.7186 -7.4519 266 mc 1 af morato, 2001 parque nacional da serra do divisor br -73.6577 -7.4408 244 mc 1 af morato, 2001 cotriguaçu, são nicolau farm br -58.2161 -9.8623 235 mc 1 af schorn de souza (unpublished data) estação ecológica de serra das araras br -57.2000 -15.650 225 flight 1♀ ce anjos-silva, 2019a,b parque nacional da serra do divisor br -72.8577 -8.4052 207 mc 1 af morato, 2001 pijuayal pe -74.1920 -8.0900 159 * 1 af abrahamczyk et al., 2011 inselberg-station les nouragues fg -52.6833 4.0833 120 flight1 1 af hentrich et al., 2007 porto velho, teotônio waterfall br -64.0530 -8.8750 83 ms, eg 2 af santos junior et al., 2014 guaimía, anchicayá river co -76.9500 3.7667 73 ci, ms, sk 2 af otero & sandino, 2003 dawa, dawa field station gy -58.6597 6.3038 35 mc 3 af williams & dodson, 1972 mc: methyl cinnamate; bb: benzyl benzoate; ci: 1,8 cineole; ms: methyl salicylate; sk: skatole; eg: eugenol. af: amazon forest; gf: gallery forest; ce: cerrado; df: decidual forest; *no data; 1♂ observed after collecting floral scent on flowers of anthurium rubrinervium (link.) g. don 1839 (araceae) mh schorn de souza et al. – expansion range of aglae caerulea in the pantanal602 authors' contributions ejas and mhss conceived and designed the research; mhss, jdsf, jcc, sop, fal, and ejas collected, organized, and analysed the data; ejas, mhss and cag interpreted the data and wrote the manuscript. all authors read and approved the final manuscript. references abrahamczyk, s., gottleuber, p., matauschek, c. & kessler, m. 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(1992). zoogeographical origins of eastern brazilian birds. ornitologia neotropical, 3, 1-15. supplementary material doi: 10.13102/sociobiology.v67i4.4581.s2449 http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/4581/0 s1. distribution of 74 occurrences for aglae caerulea for the neotropical region, considered in the present research. 1483 plants used by bees as pollen sources in the brazilian “cerrado” by daniela de almeida-anacleto1; luís carlos marchini1*; augusta carolina de camargo; carmello moreti2 & vinicius castro souza3 abstract bee flora is the set of plants that supply food to bees in a given region. the recognition of the plants used by bees for the collection of pollen in order to enable adequate handling by the beekeeper and the improvement of the bee pasture are considered of great importance for a good beekeeping. some bee pollen samples were obtained monthly during one year from pollen collectors installed in five beehives in two different areas (“cerradão” and “cerrado” sensu stricto) in pirassununga, sp, brazil. it was noted that species of the asteraceae family were intensely visited by the apis mellifera bees, indicating the importance of them for an eventual bee flora recomposition. alternativelly bees visited nearby areas, using mainly eucalyptus sp. and citrus sp. for the collection of pollen. key words: apis mellifera, food, protein, bee plant introduction bee pollen is the result of the agglutination of flower pollen made by bees by means of the addition of salivary substances and small quantities of nectar or honey (brasil 2001). pollen besides being important for the nutrition of bees due to its physicochemical composition (funari et al. 2003; barreto et al. 2005; marchini et al. 2006). their quality and composition can change adult bee behavior, their fecundity, life span, brood area size, honey stock, colony population and food larval quality (toth et al. 2005; human et al. 2007, matttila & otis 2007) . 1departamento de entomologia e acarologia, esalq/usp, av. pádua dias, 11caixa postal 09, cep 13418-190, piracicaba, sp, brazil 2instituto de zootecnia/apta/saa, nova odessa, sp, brazil 3laboratório de sistemática vegetal, esalq/usppiracicaba, sp, brazil corresponding author: lcmarchi@usp.br. departamento de entomologia e acarologia, esalq/usp, av. pádua dias, 11caixa postal 09, cep 13418-190, piracicaba, sp, brazil 1484 sociobiolog y vol. 59, no. 4, 2012 beekeepers can harvest part of the pollen collected by the worker bees, and for that they use collectors, traps or pollen hunters which withdraw their pollen loads in a pollen basket before the bees penetrate the beehive (barreto et al. 2005). bee pollen can be used as food complement by humans and animals. the nutritional importance of the pollen for humans is recognized, as being a protein source of high biological value, also containing carbohydrates, lipids and minerals in its composition. pollen does not only contain antioxidant vitamins (a, c and e), but also those of the complex b and vitamin d (oliveira et al. 2009, melo & almeida-muradian 2010; lopes et al. 2011). the botanical origin of the pollen is of extreme importance for the definition of its physicochemical composition, and it is necessary to know the pollen spectrum of the collected product in order to establish the relation between the pollen composition, its physical characteristics and its botanical origin. according to modro et al. (2007) the nutritional components are correlated to the frequency of the specific pollen types and indicate the importance of the collection from several pollen sources for the maintenance of the richest and balanced diets. the objective of the present study was to identify the pollen sources used by the bees, aiming at a more appropriate handling of the local “cerrado” bee vegetation and to provide to beekeepers alternative botanical species for the recomposition of the bee flora. material and methods this research was carried out in remaining “cerrado” areas of the campus of the university of são paulo, pirassununga county, state of são paulo, brazil, located within the coordinates 21o 57 ’ 02 ” s, 47o 27 ’ 50 ” w and 630 m above sea level. the total area of the campus contains around 2.300 ha, occupied by different faces of the “cerrado” ecosystem, also known as “savannah”, located besides areas of cultivation and pastures. the preserved forests (“cerrado” sensu stricto, “cerradão” and riparian forest) correspond to 30 % of the total area (about 705 ha), while the pastures and cropland areas make up approximately 70 % of the total area (1.620 ha). two different areas were evaluated: 1“cerradão” with approximately 56 ha, 1485 almeida-anacleto, d. de — plants used by bees being a secondary forest, where the anthropic action has been controlled over more than 20 years, without fire incidence during this period. in this area the soil is classified as a dark-red latosol (pirassununga-plio-pleistocene formation), textures varying from clayey to sandy-clay, with the canopy of the vegetation reaching heights of 15 to 20 m; 2“cerrado” sensu stricto, characterized by a shrubby flora, with at most 6m height, including lianas and several herbaceous species. this fragment has around 93 ha and the soil is a red-yellow latosol with textures varying from clayey-sandy to sandy. fina & monteiro (2009) observed a quite varied floristic composition in the same “cerradão” area with 730 individuals distributed among 33 families, 60 genera and 80 species. the richest families are myrtaceae, lauraceae and euphorbiaceae; and the most important species are copaifera langsdorfi, anadenanthera falcata and siparuna guianensis and, according to the literature, several of these plants are sources of nectar and pollen for different bee species. a total of 5 swarms (in each area) of apis mellifera were collected, transferred to langstroth beehives, and maintained in the same place of origin. during one year, pollen collections were performed at the entrance of the beehives; collectors of pollen were set, monthly, at the entry of the hives. pollen samples were homogenized and 1 g of each was taken for preparation of slides. for slide preparation (3 replicates per sample), the acetolysis method (erdtman 1952) was adopted, which consists of the chemical treatment of pollen grains, removing their intine, cytoplasm and adhering substances, fossilizing them artificially in order to make the exine most transparent and more appropriate for the study of pollen details. the identification of the pollen types was made based on referential slides made with the pollen of the plants collected in the areas and also in slides already existent in the laboratory of the apiculture sector of the esalq/usp, piracicaba, sp, brazil, in which the work was carried out. qualitative analysis – through this analysis the botanical species were determined (or pollen types), considering morphological aspects of the grains comparing them with referential slides. quantitative analysis – was performed by means of counting 300 to 500 grains of pollen per sample and grouping them by botanical species and/ or pollen types. the pollen types were grouped in four classes of frequency. in 1486 sociobiolog y vol. 59, no. 4, 2012 other words, dominant pollen (> 45%) (dp), accessory pollen (15 to 45%) (ap), isolated important pollen (3 to 15%) (iip) and isolated occasional pollen (< 3%) (iop) (louveaux et al. 1978). with the data of pollen types from the “cerradão” and “cerrado” sensu stricto), the similarity index (si) of sorensen was calculated according to silveira neto et al. (1976), by the expression: si = 2j/(a+b), where: j= number of collected pollen types common to both: “cerradão” and “cerrado” sensu stricto. a = number of collected pollen types observed in the samples from the “cerradão” b = number of collected pollen types observed in the samples from the “cerrado” sensu stricto. si varies from 0 to 1, the closer to 1, more similar are the communities and the closer to 0, less similar they are. results and discussion with the quantitative analyses of the samples of the pollen loads it was possible to demonstrate the importance of the plant species in the production of honey, classifying them in the above mentioned frequency classes dp, ap, iip, and iop (tables 1 and 2). the following types of dp (figure 1) were observed: baccharis sp. (asteraceae), baccharis dracunculifolia (asteraceae), brachiaria sp. (poaceae) and asteraceae type only in the “cerradão”; alternanthera ficoides (amaranthaceae), eucalyptus sp. (myrtaceae) and vernonia polyanthes (asteraceae) only in the “cerrado” sensu stricto, and eupatorium sp. (asteraceae), didymopanax vinosum (araliaceae), mikania sp. (asteraceae) and citrus sp. (rutaceae) in both areas. it was also noted that bees visited nearby areas with eucalyptus sp. and citrus sp. for pollen collection. the most frequent plant species evaluated through the pollen from the collectors installed in the beehives of the “cerradão” were: mikania sp., which bloomed during four months of the collections; and citrus sp.1 and didymopanax vinosum, for three months. for the “cerrado” sensu stricto: eupatorium sp. and didymopanax vinosum, appeared during five months of the collections; citrus sp.1, for four months and mikania sp., alternanthera ficoides and wissadula subpetala, three months. 1487 almeida-anacleto, d. de — plants used by bees botanical specie or pollen type pollen spectrum/month jan. feb. mar. apr. may jun july ago sept oct nov dec amaranthaceae alternanthera ficoides ap araliaceae didymopanax vinosum dp iop iip arecaeae astrocaryum aculeatissimum iop asteraceae baccharis dracunculifolia dp asteraceae baccharis sp. dp asteraceae bidens gardnerii iip iip asteraceae eupatorium sp. dp dp asteraceae mikania sp. ap ap dp iop asteraceae vernonia cognata iip coniferae pinus sp. ap euphorbiaceaealchornea sp dp euphorbiaceae croton floribundus ap e u p h o r b i a c e a e c r o t o n urucurana ap fabaceae-caesalpinioidea caesalpinia sp. iop fabaceae-mimosoidea piptadenia moniliformis iop lamiaceae hyptis eriophylla iip malvaceae wissadula subpetala ap myrtaceae eucalyptus sp1. iip myrtaceae eugenia aurata iop myrtaceae eugenia bimarginata ap piperaceae – piper sp. ap dp poaceae brachiaria sp. ap dp rutaceae citrus sp1. iop iop dp solanaceae solanum sp. ap asteraceae type dp ap brassicaceae type iip poaceae type iop rubiaceae type iip rutaceae type iip dp = dominant pollen (> 45%), ap = accessory pollen (15 to 45%), iip = isolated important pollen (3 to 15%) and iop = isolated occasional pollen (< 3%). -no pollen or pollen type table 1. pollen spectrum of the pollen samples, originating from the collectors installed from the beehives of the "cerradão" area, pirassununga, sp, brazil. 1488 sociobiolog y vol. 59, no. 4, 2012 table 2 pollen spectrum of the pollen samples, originating from the collectors installed from the beehives of the “cerrado” sensu stricto area, pirassununga, sp, brazil. botanical specie or pollen type pollen spectrum/month jan. feb. mar. apr. may jun july ago sept oct nov dec amaranthaceae alternanthera ficoides ap dp ap araliaceae didymopanax vinosum iop iip ap iop dp are caeae astrocar yum aculeatissimum iop iop asteraceae eupatorium sp. dp dp iop dp dp asteraceae mikania hirsutissima ap asteraceae mikania sp. dp ap iop asteraceae montanoa sp. ap asteraceae vernonia cognata ap asteraceae vernonia polyanthes dp asteraceae vigueira sp. ap bombacaceae eriotheca gracilips iop convolvulaceae merremia macrocalix iip euphorbiaceae croton floribundus iip iip euphorbiaceae croton urucurana ap fa b a c e a e -mim o s o i d e a piptadenia moniliformis iip malvaceae sida sp. iip malvaceae wissadula subpetala iip iip iop myrtaceae eucalyptus sp1 dp ap poaceae brachiaria sp. ap iip poaceae zea mays iip rutaceae citrus sp1. iop iip dp iop sapindaceae serjania sp. solanaceae solanum sp. ap brassicaceae type iip malvastrum type iip myrcia type ap dp = dominant pollen (> 45%), ap = accessory pollen (15 to 45%), iip = isolated important pollen (3 to 15%) and iop = isolated occasional pollen (< 3%). -no pollen or pollen type 1489 almeida-anacleto, d. de — plants used by bees 1a lte rn at he ra fi co id es (a m ar an th ac ea e) , 2 -b ac ch ar is sp ., 3 -b ac ch ar is dr ac un cu lif ol ia (a st er ac ea e) , 4 -b ra ch ia ri a sp . ( po ac eae ), 5bi de ns g ar dn er ii (a st er ac ea e) , 6 -c itr us sp 1. ( r ut ac ea e) , 7 -c ro to n flo ri bu nd us , 8 -c ro to n ur uc ur an a (e up ho rb ia ce ae ), 9d id ym op an ax vi no su m (a ra lia ce ae ), 10 -e uc al yp tu s s p1 . ( m yr ta ce ae ), 11 -e ug en ia bi m ar gi na ta (m yr ta ce ae ), 12 -e up at or iu m sp ( a st er ac ea e) , 1 3m ik an ia s p. , 1 4m ik an ia h ir su tis sim a (a st er ac ea e) , 1 5m on ta no a sp . ( a st er ac ea e) , 1 6 a lch or ne a sp (e up ho rb ia ce ae ), 17 -s ol an um sp . ( so la na ce ae ), 18 -v er no ni a co gn at a, 1 9ve rn on ia p ol ya nt he s ( a st er ac ea e) , 2 0v ig ue ir a sp . (a st er ac ea e) , 2 1w iss ad ul a su bp et al a (m al va ce ae ) an d 22 m yr ci a ty pe (m yr ta ce ae ). fi g. 1 . p ol le n ty pe s w hi ch a pp ea re d as d om in an t o r ac ce ss or y po lle n in sa m pl es o f t he p ol le n lo ad s c ol le ct ed in a re a of “c er ra dã o” a nd “c er ra do ” se ns u str ic to o f p ir as su nu ng a, s p, b ra zi l. 1490 sociobiolog y vol. 59, no. 4, 2012 comparing the two areas, 15 pollen types occurred in both: three belonging to the asteraceae family, two to the euphorbiaceae and one of each of the following families, amaranthaceae, araliaceae, arecaceae, brassicaceae, fabaceae, malvaceae, myrtaceae, poaceae, rutaceae and solanaceae. the other 25 identified pollen types (tables 1 and 2) were observed either in the “cerrado” sensu stricto or in the “cerradão”. the similarity index of sorensen for the two areas was the same, is = 0.545, which indicates that the collection of pollen by bees has a medium similarity between the two forms of vegetation. for other bush areas, located in different brazilian states, the similarity index was very low for plants visited by solitary bees and stingless bees (moreti et al. 2006). in the distribution of the pollen types by family (figure 2), it is noticed that the greatest diversity of species belongs to the asteraceae family with 12 pollen types, followed by myrtaceae with four. costa (2002unpublished data) investigating pollen sources used by worker bees of apis mellifera in cruz das almas, ba, brazil, also observed the asteraceae as the family with the greatest number of pollen types and the same was observed by almeidaanacleto (2007) in piracicaba, sp, brazil. in ten pollen samples of the south region of brazil, almeida-muradian et al. (2005) observed that the most frequent families of plants were arecaceae, asteraceae and myrtaceae, with two families of plants coincident with the observed ones in the present study. in “cerrado” areas of cassilândia, ms, brazil differently than in the here studied area, the malpighiaceae and the fabaceae (mimosoideae) were the families with the greatest number of species of importance for the bees (vieira et al. 2008). through the analyses of the studied pollen samples, a great participation of isolated and occasional pollen types, was found in several samples. barth (1970) mentions that these species have little importance, when considering the quantity of nectar and pollen supplied by the plant; however, when the interest is the origin and geographical provenance of the samples the presence of these pollen types becomes significant. conclusion species of the asteraceae family are intensely visited by the apis mellifera bees for pollen collection in the são paulo state, both in the “cerrado” sensu 1491 almeida-anacleto, d. de — plants used by bees stricto and in the “cerradão” areas, indicating that this family is very important for a recomposition of the bee flora. it was noted that besides the existing plants occurring in the “cerrado” sensu stricto and in the “cerradão” areas, the bees visited nearby areas where the beehives were installed, making use mainly of eucalyptus sp. and citrus sp., for pollen collection. acknowledgments the authors thank fapesp for financial support fig. 2. number of pollen types by botanical family observed in the samples of pollen loads collected in beehives of apis mellifera in the “cerrado” areas of pirassununga, sp, brazil 1492 sociobiolog y vol. 59, no. 4, 2012 references almeida-anacleto, d. 2007. recursos alimentares, desenvolvimento das colônias e caracterísiticas físico químicas, microbiológicas e polínicas de mel.. tese (doutorado em ciências)curso de pós graduação em ciências, escola superior de agricultura “luiz de 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http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.5089sociobiology 67(3): 337-342 (september, 2020) introduction the family phoridae belonging to the order diptera, represent a group of insects with a great diversity of lifestyle during its immature phase, being mostly parasitoids (bragança, 2016). most of these parasitoids stand out for attacking ants of the genus atta and acromyrmex, known as leaf-cutting ants. ants of these genera harm diverse cultures of importance, both forest and agricultural at any stage of development (zanetti, 2000). phorid parasitoids stand out among the natural enemies of leaf-cutting ants, making use of workers as a host for their immature stage. adults oviposit specific parts of ants’ bodies. thus, the larvae feed on the internal content of the parts where the oviposition occurred, culminating in the death of the ant. the geographical distribution, both of hosts and parasitoids, occurs throughout the american continent, where abstracts leaf-cutting ants of the genus atta are considered one of the main forest and agricultural pests. currently, chemical control using granulated baits is considered the most efficient, however, with the inadequate adoption of management it can harm non-target organisms and the agro-ecosystem. phorid parasitoids have been studied so that they can be used as an alternative management strategy for leaf-cutting ants. the use of phorids as part of the management strategy in biological control, despite being still very incipient, may in the future become a safer alternative for the control of leaf-cutting ants. thus, the objective was to report the occurrence of parasitoids of the family phoridae associated with leaf-cutting ants of the genus atta in the municipality of cruz das almas. during the months of april to july, three environments were studied, being: pasture area, eucalyptus plantation and a fragment of atlantic forest. in each of these environments, three colonies of two species of ants atta sexdens sexdens and atta laevigata were selected. each collection consisted of six hours of observation, divided into eight periods of 45 minutes. observations were made on the trails and scouts consecutively, always starting with the scout. a total of 112 phorids belonging to two genera were collected, being 64 to genus eibesfeldtphora and 48 the genus myrmosicarius. sociobiology an international journal on social insects td souza1, rc pereira2 article history edited by evandro n. silva, uefs, brazil received 26 march 2020 initial acceptance 08 august 2020 final acceptance 08 august 2020 publication date 30 september 2020 keywords eibesfeldtphora, myrmosicarius, parasitoid, phoridae. corresponding author tamires doroteo de souza department of plant health universidade estadual paulista júlio de mesquita filho – unesp jaboticabal, são paulo, brasil. e-mail: tamiresdoroteo@gmail.com 28 species of ants are parasitized by at least 70 species of phorids (bragança, 2011). in the case of phorids of the genera eibesfeldtphora and myrmosicarius, the preferred place for oviposition is the head, where the larvae feed on the ant’s head mass. for this reason, they are called decapitating flies (porter & powes, 2018; porter, 1997). these flies generally attack ants in their tracks, causing a great impact on the behavior of the anthill (bragança et al., 2003). the mere presence of these flies on the trails leads to the interruption of the cutting activity of the plant material, as the workers flee to the nest in order to prevent the action of the parasitoids. mortality can occur from 1 to 6% in ants, in addition to having a significant impact on changing the behavior of all anthills (bragança, 2011). parasitoids can be species specific or attack more than one ant species (uribe et al., 2014). environmental variables such as temperature and humidity, in addition to biotic 1 department of plant health, universidade estadual paulista júlio de mesquita filho – unesp, jaboticabal-sp, brazil 2 professor of the department of forest engineering, federal universidade do reconcâvo da bahia-ufrb, cruz das almasba, brazil research article ants first record of the occurrence and abundance of phorids (diptera: phoridae) associated with leaf-cutting ants of the genus atta (hymenoptera: formicidae) in the municipality of cruz das almas-ba td souza, rc pereira – phorid parasitoids associated to leaf-cutting ants338 factors, such as ant traffic, seem to be limiting factors for the performance of these parasitoids. studies show that some species prefer to oviposit in environmental conditions with high temperatures and low humidity. such conditions, biotic or abiotic, are responsible for the successful performance of these parasitoids and the development of their immature phase (calcaterra et al., 2005; bragança et al., 2008). the discovery of these adverse effects on the work of ants demonstrates a possible contribution of these parasitoids to the management of leaf-cutting ants. however, taking into account the economic importance of these pest insects, the objective of the work is to report the occurrence of phorids (parasitoids) in species of ants of the genus atta in the municipality of cruz das almas-ba, evaluating the influence of abiotic factors (temperature and relative humidity and difference in living) and biotics (ants traffic in scouts and trails) in the activity of these parasitoids. materials and methods study area the study was conducted in the municipality of cruz das almas-ba, with latitude 12º 40 ‘12” s, longitude 39º 06’ 07” w and altitude of 220 m, located 120 km from the capital salvador (inmet, 2019). the region’s climate predominates high temperature and relative humidity according to the köppen-geiger classification (1936), varying between 17 to 38 ° c and average annual precipitation of 1.136 mm. the vegetation is classified as forest dense ombrophilous, having a high diversity in plant species, and the soils are classified as yellow latosol and yellow argisol with a sandy-clay-sandy texture (ibge,2016). selection and characterization of areas three areas with different vegetation were selected, the first being a pasture area with no shading. the second area was a eucalyptus ssp. forest, approximately 22 years old, with little shading. and the third is a fragment of atlantic forest with high shading. the choice of anthills in each area was defined by assessing the activity rhythm, by counting the ants on the trail, thus selecting the anthills that had their most intense activities. after the choice, the anthills were marked, isolated and each anthill was identified as a repetition, totaling three anthills per treatment. data collection the parasitoids were collected from april to july 2017 and the number of collections each month ranged from two to three, with a duration of six hours of observation divided into eight 45-minute intervals (gomes, 2011). in each interval, observations were made in the scouts and trails, starting at 8:00 am and ending at 5:00 pm. in the observation periods where there was no activity of ants, disturbances were caused in the colonies to instigate the workers to leave and thus attract the phorids. after the departure of the first workers, the time required for the appearance of the phorids was counted, totaling 216 hours of field observation. foraging activity was quantified on the scout and on the trail 50 cm from the scout by means of a manual counter, counting the number of foragers and collectors in the first 15 minutes of each interval. the remaining 30 minutes were dedicated to the observation of the phorids. the phorids that attacked the ants around the colony (trails and scouts) were collected with the aid of plastic containers (2.5 x 8.5 cm). at the end of each observation interval, the number of phorids collected was recorded, labeled, conditioned in 70% gl alcohol and were taken to the entomology laboratory of the federal university of reconceive the bahia (ufrb) to carry out the identification of biological material. in the field, in addition to the foraging rhythm of the ants and the activity of the phorids, the behavior of the ants in the face of attacks by the parasitoid (defense mechanism) was also observed. in each observation, the time of collection of the phorids, temperature and the relative humidity of the air were checked. meteorological data were obtained from the national institute of meteorology (inmet, 2017), based on local stations. abundance of parasitoids to determine the abundance of parasitoids, a 10 x 8.5 cm adhesive trap was impregnated with entomological glue on both sides (fig 1). the traps remained in the field for 24 hours. four traps were placed in each colony, two cards on the foraging trail and two on the scout’s entrance. the phorids captured by the traps were collected and taken to the entomology laboratory of the federal university of reconceive the bahia (ufrb), counted and conditioned in 70% gl alcohol. ant identification specimens of ants from each of the anthills observed were taken to the entomology laboratory at the federal university of reconceive the bahia and identified using the key from della lucia (1993). phorid identification the identification of the genders was made based on the work key of uribe et al. (2014). to confirm the genera, each specimen collected was taken to the biology laboratory of the federal university of reconceive the bahia (ufrb), placed in a petri dish and photographed with the aid of a stereomicroscope (leica ezd4) using the application suite application, the photos of each of the phorids were sent to a specialist at the national university of colombia. sociobiology 67(3): 337-342 (september, 2020) 339 statistical analysis the total abundance of phorids in the different areas and months of collections was compared using anova with a randomized block design and the means compared by the tukey test at 5% significance. climatic data were compared with pearson’s correlation coefficient, comparing the two variables (temperature and abundance of phorids) (sas university, 2002). results and discussion a total of 112 specimens of phorids were collected, 71% of the genus eibesfeldtphora and 29% of the genus myrmosicarius, associated with atta sexdens sexdens and atta laevigata. only one specimen of the genus myrmosicarius was found associated with atta sexdens sedens (table 1). the abundance of phorids between the collection areas differs statistically only in the natural area (fragment of atlantic forest) when compared to the other areas (pasture area and eucalyptus). the two agricultural areas will not differ statistically (f (2,318) = 4.09; p = 0.46). the total number of phorids captured per area is 10 in the natural area, in the eucalyptus planting area 39 and in the pasture area 44 (fig 2). the sampling made with the use of adhesive traps in the period of 24 hours, results in a total of 53 individuals collected, being 19 of the genus myrmosicarius (fig 3) and 34 of the genus eibesfeldtphora (fig 4). there is no significant difference in the abundance of phorids when comparing the two sampling methods performed in this work (observation and trap). in the method using traps it is possible to notice the preference of the attack for the activity areas of the ants, being more abundant in the trails (68.7%) and scouts (31.3%) (fig 2). the number of phorids is influenced by the traffic of ants, in all study areas. table 1. number of individuals of eibesfeldtphora and myrmosicarius (diptera: phoridae) in different species of atta, in cruz das almasba, brazil. species of ants phoridae genera eibesfeldtphora myrmosicarius atta sexdens sexdens 41 1 atta laevigata 23 47 total 64 48 fig 1. (a) details and distribution of the traps, (b) phorids captured from the genus myrmosicarius, (c) phorids captured from the genus eibesfeldtphora. fig 2. percentage of phorids collected in each activity zone (scout and trails). td souza, rc pereira – phorid parasitoids associated to leaf-cutting ants340 considering the three areas, phorids of only two genera are identified, being eibesfeldtphora and myrmosicarius, with a greater occurrence of the genus eibesfeldtphora. regarding the climatic factors evaluated, the correlation is significant only for temperature, and the abundance of phorids has a strong to very strong correlation (pearson r = 0.97; n = 8; p = 0.02). the relative humidity of the air does not show a correlation and is therefore not significant (pearson r = -0.57; n = 8; p = 0.08). only two genera of parasitoids were collected when attacking the workers of a. laevigata and a. sexdens sexdens. the results will indicate parasitoid preferences among ant species, having a direct relationship with the place of occurrence, since eibesfeldtphora tonhascai (brown, 2001) and myrmosicarius grandicornis (borgmeier, 1928), have already been reported as parasitoids of a. sexdens sexdens in the states from minas gerais, goiás and rio de janeiro (tonhasca et al., 2001; silva et al., 2008; souza, 2013). however, there are no reports in the literature of the occurrence of these parasitoids in the state of bahia, which allows us to affirm that the attacks of this phorid on atta in different latitudes suggest a process of adaptation to parasitism in a new host (pereira, 2001). fig 3. species collected from the genus myrmosicarius. important variations in the occurrence of these phorids can happen throughout the year and in certain environments (elizalde, 2011; silva et al., 2008; guillade et al., 2011), showing these clear dynamics between the species of ants. it is observed that the experiment shows the preference of some genera of phorids for a given environment, with the largest number of phorids found in areas with less dense vegetation and with a higher incidence of sunlight. elizalde and folgarait (2010), carried out studies where they associated species of phorids with specific habitats, and the results showed the preference of myrmosicarius for closed environments and eibesfeldtphora in open environments or, for areas with some type of culture. the greatest abundance of phorids is obtained in the pasture area. gomes (2011) reports that there is a greater number of parasitoid flies in areas with a higher incidence of light, as they are insects with daytime activity. this fact justifies the number of captured phorids being greater in areas with little vegetation. bragança et al. (2008) performing laboratory tests on ants of the genus atta where they were subjected to three different proportions of light to verify its influence on the action of parasitoids, the luminosities were high (simulating daytime light), medium (simulating dusk and dawn) and absence of light (simulating night). they note that there were only attacks by parasitic phorids at the high light level, suggesting that some species of parasitic phorids fig 4. species collected from the genus eibesfeldtphora. sociobiology 67(3): 337-342 (september, 2020) 341 are only active during the day and not active during dawn, dusk or at night, thus concluding that the visual stimulus may be an essential factor for the location of the host. the collection techniques used prove to be efficient to assess the abundance of phorids in the three study areas. despite the lower number of phorids being collected in the natural area, in contrast to the results obtained by galvão (2016), the detection of the presence of these flies in specific environments, already shows their activities in any environment. the rhythm of activity and the behavior of the attack against the species of ants in each environment may have made it difficult to visualize the phorids in the natural landscape area (bragança et al., 2008). eucalyptus plantations and pasture areas create an environment that facilitates the visualization of parasitoids since, it is believed that this is the main means of interaction between host and parasitoid (bailez, 2016). at first, the number of captured phorids seems to have been low, especially of the genus myrmosicarius. gomes (2011) states that the duration of sampling, in addition to the time and time of year in which these surveys are carried out, can influence the abundance of phorids, which in fact would justify the low number of captured parasitoids, and the study in question it was driven most of the time in rainy season. phorids of the genus myrmosicarius do not seem to be affected by environmental changes, as well as their hosts (wirth et al., 2007), whereas those of the genus eibesfeldtphora, are more susceptible to climate changes, due to their greater sensitivity to light, having preference for brighter environments (bragança, 2011). the abundance of parasitoids being negatively influenced by temperature, it is assumed that these phorids are literally linked to light and are more efficient in dry periods (martins, 2015). all species of ants studied have a defense mechanism against the action of parasitoids, such as changing the cutting time and protection of the body where the parasitoids can oviposit. during foraging. this behavior has been mentioned in other studies with other species of ants of the same genus, in order to hinder the action of parasitoids, since they prefer the size of the ants, always attacking larger ants (soldiers) (feener & brown 1993; tonhasca, 1996; bragança et al., 2009). linksvayer et al. (2002), studied the way that the smaller workers defend the bigger workers against oviposition during the daytime, and called this hitchhiking mechanism, which means “hitchhiking”. it is not yet known for certain whether it is really a defense mechanism against the parasitoid or if it has some other function, however during field observations it was possible to notice this behavior when it had the presence of phorids. the number of phorids is influenced by the traffic of ants in all areas of study, due to this, the hypothesis arises that the abundance of phorids may be associated with the release of allelochemicals, because when a disturbance was made in the colonies, instigating the output of ants also increased the number of captured phorids. communication between workers (smaller ants) and soldiers, as if it were a request for help, involves chemicals called alarm pheromone, which triggers the defense behavior in the colony. the constituent is composed of volatile substances which can be one of the main factors that attract phorids. as it is a mechanism that has not been studied yet, further studies are necessary between the pheromone of leaf-cutting ants and phorids. however, according to the observation of this work, everything indicates that it may be among the main interaction routes, in addition to visualization. conclusion this work was the first report of parasitoid phorids associated with ants of the genus atta in the recôncavo da bahia. the abundance of these parasitoids may prove to be a control method for leaf-cutting ants, however, the results found suggest the need for future studies, which can understand the role of these natural enemies of leaf-cutting ants in different habitats. references bailez, o. (2016). estratégias e táticas na interação forídeoformiga. oecologia australis, 20: 322-331. doi: 10.4257/ oeco.2016.2003.01 bragança, m.a.l., della lucia, t.m.c., tonhasca, j.a. (2003). first record of phorid parasitoids (diptera: phoridae) of the leaf-cutting ant atta bisphaerica forel (hymenoptera: formicidae). neotropical entomology, 32: 169-171. doi: 10.15 90/s1519-566x20030 00100028 bragança, m.a.l. (2007). perspectiva da contribuição de forídeos parasitoides no manejo de formigas cortadeiras. biológico, 69:177-181. recovered from: http://www.biologico.sp.gov. br/ uploa ds/docs/bio/ suplementos/v69_supl_2/p177-181.pdf bragança, m.a.l., souza, l.m.d., nogueira, c.a., della lucia, t.m.c. (2008). parasitism by neodohrniphora spp. malloch (diptera, phoridae) on workers of atta sexdens rubropilosa forel (hymenoptera, formicidae). revista brasileira de entomologia, 52: 300-302. doi: 10.1590/s008556262008000200011 bragança, m.a.l., nogueira, c.a., souza, l.m., della lucia, t.m.c. (2009). superparasitism and host discrimination by neodhorniphora elongata (díptera: phoridae), a parasitoid of the leaf-cutting ant atta sexdens rubropilosa (hymenoptera: formicidae). sociobiology, 54: 907-918. bragança, m.a.l. (2011). parasitoides de formigas-cortadeiras. formigas cortadeiras: da bioecologia ao manejo. viçosa: editora ufv, 343p. bragança, m.a.l. (2016). phorid flies parasitizing leafcutting ants: their occurrence, parasitism rates, biology and the first account of multiparasitism. sociobiology, 63: 1015-1021. doi: 10.13102/sociobiology.v63i4.1077 calcaterra, l.a., porter, s.d., briano, j.a. (2005). distribution td souza, rc pereira – phorid parasitoids associated to leaf-cutting ants342 and abundance of fire ant decapitating flies (díptera: phoridae: pseudacteon) in three regions of southern south america. annals of the entomological society of america, 98: 85-95. doi: 10.1603/0013-8746(2005)098[0085: daaofa]2.0.co;2 della lucia, t.m.c. & fowler, h.g. (1993). as formigas cortadeiras. in: della lucia tmc. as formigas cortadeiras: da bioecologia ao manejo. viçosa: editora ufv, 3p. elizalde, l. & folgarait, p.j. (2010). host diversity and environmental variables as determinants of the species richness of the parasitoids of leaf-cutting ants. journal of biogeography, 37: 2305-2316. doi: 10.1111/j.1365-2699.2010. 02361.x elizalde, l. & folgarait, p.j. (2011). biological attributes of argentinian phorid parasitoids (insecta: diptera: phoridae) of leaf-cutting ants. journal of natural history, 45: 2701-2723. doi: 10.1080/00222933.2011.602478 feener, d.h. & brown, b.v. (1993). oviposition behavior of an ant-parasitizing fly, neodohrniphora curvinervis (diptera: phoridae), and defense behavior by its leaf-cutting ant host atta cephalotes (hymenoptera: formicidae). journal of insect behavior, 6: 675-688. doi: 10.1007%252fbf01201669 galvão, a.r.a. (2016). parasitismo natural e abundância de forídeos parasitoides de atta sexdens (linnaeus, 1758) (hymenoptera: formicidae) em áreas de vegetação natural e agrícolas. 82p. dissertação (mestrado em ciências e tecnologias agropecuárias). universidade estadual do norte fluminense darcy ribeiro. instituto nacional de meterologia, http://www.inmet.gov.br/ portal/ (accessed july 2017). instituto brasileiro de geografia estatística, https://ibge.gov. br/ (accessed july 2017). gomes, d.s. (2011). ecologia de parasitoides (diptera: phoridae) de atta robusta borgmeier, 1939 (hymenopter: formicidae) em ambiente de restinga. 62p. (dissertação). curso de ciências biológicas. instituto de biologia. universidade federal rural do rio de janeiro. guillade, a.c., & folgarait, p.j. 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(1997). rearing the decapitating fly pseudacteon tricuspis (diptera: phoridae) in imported fire ants (hymenoptera: formicidae) from the united states. journal of economic entomology, 90: 135138. porter, s. d. & plowes, r. m. (2018). rearing and biology of the decapitating fly pseudacteon bifidus (diptera: phoridae): a parasitoid of tropical fire ants. florida entomologist, 101: 265-272. doi:10.1653/024.101.0218 silva, v.s.g., bailez, o., viana-bailez, a.m., tonhasca, j.r. a. & della lucia, t.m.c. (2008). survey of neodohrniphora spp. (diptera: phoridae) at colonies of atta sexdens rubropilosa (forel) and specificity of attack behavior in relation to their hosts. bulletin of entomological research, 98: 203-206. doi: 10.1017/s0007485307005548 souza, l.r.r. (2013). influência do tamanho das operárias e da sazonalidade no parasitismo das saúvas atta sexdens e atta laevigata (hymenoptera: formicidae) por moscas da família phoridae. 43f. dissertação (mestrado em ecologia de ecótonos). universidade federal do tocontins. porto nacional. tonhasca, a.j.r., bragança, m.a.l. & erthal, m. (2001). parasitism and biology of myrmosicarius grandicornis (diptera, phoridae) in relationship to its host, the leafcutting ant atta sexdens (hymenoptera, formicidae). insectes sociaux, 48: 154-158. doi: 10.1007/pl00001759 tonhasca, j.a. (1996). interactions between a parasitic fly, neodohrniphora declinata (diptera: phoridae), and its host, the leaf-cutting atta sexdens rubropillosa (hymenoptera: formicidae). ecotropica, 2: 157-164. uribe, s., brown, b.v., bragança, m.a.l., queiroz, j.m. & nogueira, c. a. (2014). new species of eibesfeldtphora disney (diptera: phoridae) and a new key to the genus. zootaxa, 3814: 443-450. doi: 10.11646/zootaxa.3814.3.11 wirth, r., meyer, s.t., almeida, w.r., araújo, m.v., barbosa, v.s. & leal, i.r. (2007). increasing densities of leaf-cutting ants (atta spp.) with proximity to the edge in a brazilian atlantic forest. journal of tropical ecology, 23: 501-505. doi: 10.1017/s0266467407004221 zanetti, r., vilela, e. f., zanuncio, j. c., leite, h. g. & freitas, g. d. (2000). influência da espécie cultivada e da vegetação nativa circundante na densidade de sauveiros em eucaliptais. pesquisa agropecuária brasileira, 35: 1911-1918. doi: 10.15 90/s0100-204x200000 1000001 67 hyenism in ants: non-target ants profit from polyergus rufescens raids (hymenoptera: formicidae) by pavel pech abstract the presence of slavemaker ants alters the structure of the ant community. the influence of slavemakers on non-host ants is believed to be indirect, via changing competitive interactions among the ants. according to my observation myrmica sabuleti (and probably also lasius alienus) profited from a polyergus rufescens raid against a formica fusca nest. both non-host species collected f. fusca corpses. these carcasses were used as food in the case of m. sabuleti at least. m. sabuleti profited substantially from the polyergus raid because the dry biomass weight of the collected f. fusca carcasses was approximately half of the dry biomass weight of all m. sabuleti workers. key words: ants, formicidae, profit, slavery, social parasitism it has been shown that the structure of an ant community is affected by both top-competitors and slavemakers (puntilla et al. 1996). the influence of slavemakers on non-host species is considered to be indirect, taking affect via changing competitive interactions among the ants. the socially parasitic ant polyergus rufescens (latreille, 1798) is a slave-maker who uses ants from the subgenus serviformica spp. as a host. in this paper i refer to the direct nutrient benefit gained by non-host ants from polyergus rufescens raiding. i observed a raid of p. rufescens on a formica fusca (linnaeus, 1758) nest on the green of havraníky (a village in south moravia) in the afternoon of 31.5.2011. the nests were approximately 15 m apart. the raid was already in progress when i arrived: the dead queen and many dead and dying f. fusca workers lay on the surface of the attacked nest and polyergus workers were carrying pupae away into their own nest. several f. fusca workers that had escaped the attack stormed about, in some cases carrying a rescued pupae. university of hradec králové, faculty of science, rokitanského 62, 500 03, czech republic e-mail: pavelpech1@centrum.cz 68 sociobiolog y vol. 59, no. 1, 2012 there was a nest of myrmica sabuleti (meinert, 1861) in the vicinity of the attacked f. fusca nest (approximately 20 cm away). m. sabuleti workers appeared to increase their activity levels as the number of polyergus workers on the attacked nest decreased. many m. sabuleti workers came to the attacked nest and transported carcasses into their own nest. moreover, m. sabuleti workers behaved very agressively towards f. fusca workers that had escaped the p. rufescens raid and attacked them with open mandibles. nevertheless, the f. fusca moved much faster than the m. sabuleti and escaped in all the cases that i observed. during the next day there were still some f. fusca carcasses on the plundered f. fusca nest. the m. sabuleti paid no attention to these carcasses. instead i observed several lasius alienus (förster, 1850) workers transporting them away. i dug up and searched all the nests in the site: the attacked f. fusca nest, m. sabuleti nest and l. alienus nests. there were 127 workers, 8 pupae, and 27 larvae of m. sabuleti, and 27 dead workers of f. fusca in the m. sabuleti nest. most f. fusca carcasses had been placed in a chamber with m. sabuleti larvae and these corpses were partly cut into pieces. the l. alienus nest contained only 3 l. alienus workers; it was not a proper nest but rather a temporary station. there was 12 dead f. fusca workers placed in the entrance and a further stock of corpses above the entrance of the station. there were 14 carcasses of f. fusca workers and 1 queen still on the surface of the former f. fusca nest. i found nothing inside this nest except 5 forgotten f. fusca pupae. i weighed the dry biomass of m. sabuleti workers and the carcasses of f. fusca. the total weight of the 27 f. fusca workers was 0.0558 g and the total weight of the 127 m. sabuleti workers was 0.1194 g. it seems that m. sabuleti benefited substantially: the weight of f. fusca carcasses in m. sabuleti nest was approximately half of the weight of all the m. sabuleti workers. it was not possible to make the same calculation in the case of l. alienus, because it was not clear which nest the station belonged to. conclusions non-host ants can benefit from the presence of slavemaker ants indirectly, through the reduction of competition (punttila et al. 1996), or directly, 69pech, p. — non-target ants profit from polyergus raids through the consumption of the victims of slavemakers' raids. independently of this observation it is known that ants are the most efficient of all invertebrate carcass scavengers (fellers & fellers 1982; retana et al. 1991), and it is probable that non-host ants collect most of the victims of slavemaker raids. clearly the supply of this food to non-host ants is occasional only. on the other hand, with regard to the range and frequecy of polyergus raids, the amount of food gained is not negligible. the observation of the aggressive behavior of m. sabuleti towards escaping f. fusca is interesting. i have never observed or read about such aggressiveness of m. sabuleti to f. fusca. the close vicinity of both nests confirms a non-conflict relationship between both nests of these species. it seems that the aggressive behavior of m. sabuleti to f. fusca is related to the unfavorable situation of the f. fusca colony attacked by polyergus. acknowledgments i wish to thank c. steer and i. oulehlová for improving the english version of the manuscript. references fellers, g.m., & j.h. fellers. 1982. scavenging rates of invertebrates in an eastern deciduous forest. american midland naturalist 107: 389-392. mori, a., c. castracani, d.a. grasso, f. le moli, & r. visicchio 2001. glandular sources of recruitment, trail, and propaganda semiochemicals in the slave-making ant polyergus rufescens. etholog y, ecolog y and evolution 13: 361-372. punttila, p., y. haila, & h. tukia 1996. ant communities in taiga clearcuts: habitat effects and species interactions. ecography 19: 16-28. retana, j., x. cerda & x. espadaler 1991. arthropod corpses in a temperate grassland: a limited supply. holarctic ecolog y 14: 63-67. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4438sociobiology 67(1): 59-64 (march, 2020) introduction ants comprise 5% of the world’s one hundred worst invasive alien species and of the seventeen land invertebrates listed, five (28%) are ants (vander meer & milne, 2017). among the most problematic invasive species, the asian needle ant, monomorium chinensis, santschi and the yellow crazy ant, anoplolepis gracilipes, smith, cause devastating environmental and urban problems all over the world and area threat to local biodiversity (zheng et al., 2008). current control measures of these species include perimeter spraying or direct application to nest sites for successful control. however, this can result in insecticidal run-off and environmental contamination often poisons other non-target organisms (welzel & choe, 2016). kafle et al. (2010) stated that the solid bait design was not ideal due to a tendency to become excessively hydrated or dehydrated and abstract the current control measures used against common household ants in urban and agricultural settings include perimeter insecticide applications. these often have the potential to cause problems of poisoning non-target organisms, due to the insecticidal runoff and environmental contamination. a gel-baiting technique is the most effective tool to control ants with less insecticides released into the environment. in this study two commercial gel baits; the boric acid (2.5% boric acid) and the fipronil (0.01% fipronil) baits, were evaluated against laboratory made baits (lab baits). the lab baits, consisted of: 1.5% boric + fructose (f1.5), 1.5% boric acid + fructose + molasses (m1.5), 3% boric acid + fructose (f3), and 3% boric + fructose + molasses (m3) were evaluated based on preference and mortality rates of the common household ant species: the asian needle ant, monomorium chinensis, santschi, and the yellow crazy ant, anoplolepis gracilipes, smith, under laboratory conditions. an inconsistent preference was observed between species and different baits; however, the fipronil bait and the lab bait m3, were preferred more by both ant species compared to the other baits tested. lab bait m3 also had a faster killing speed than the boric acid bait and the lab bait f3.based on the results it was concluded that lab bait m3 was a more efficient ant bait and is a potential alternative control measure to the current commercial baits. sociobiology an international journal on social insects ln kafle, ac neupane, ym wang, sr gangai article history edited by evandro nascimento silva, uefs, brazil received 26 march 2019 initial acceptance 07 january 2020 final acceptance 31 january 2020 publication date 18 april 2020 keywords bait, boric acid, monomorium chinensis, anoplolepis gracilipes, preference. corresponding author samantha rose gangai department of tropical agriculture and international cooperation national pingtung university of science and technology 1, sheufu rd, neipu, pingtung 91201, taiwan. e-mail: chamandah@gmail.com needing frequent maintenance. considering that most baits contain toxic substances, they are therefore environmentally hazardous and arising concern to world wide health agencies and environment authorities (kafle & shih, 2013). fipronil is a broad use insecticide used to control ants and other insect pests. it is highly toxic to sea and freshwater vertebrates and invertebrates. furthermore, fipronil has been found to be highly toxic to some birds, although non-toxic to ducks. it has also been found as highly toxic to honey bees, but non-toxic to earthworms (jackson et al., 2009). fibronil is frequently found in urban waterways and aquatic systems in amounts exceeding the lc50 values and therefore there is concern for its potential impact on non-target organisms, as has been reported in california (welzel & choe, 2016). similarly, boric acid is a pesticide that can also be found in nature and has been used for ant control since the early 1900’s. boric acid is slow acting and non-repellent department of tropical agriculture and international cooperation, national pingtung university of science and technology, pingtung, taiwan research article ants development of new boric acid gel baits for use on invasive ants (hymenoptera: formicidae) ln kafle, ac neupane, ym wang, sr gangai – development of new boric acid gel baits 60 thereby enhancing long-term ingestion (klotz & moss, 1996: klotz & williams, 1996). boric acid and sodium salts can be used to control insects, spiders, mites, algae, moulds, fungi, and weeds. the delayed activity of boric acid promotes a thorough distribution of the active ingredient within the nest, leading to the death of the entire colony (klotz & williams, 1996). commercial ant baits with boric acid as an active ingredient typically use concentrations of > 5% (klotz et al., 2000). during this study, we formulated a new composition of ant bait with boric acid as an active ingredient (ai). in order to determine an effective range of boric acid, we compared different lab baits with two concentrations of boric acid (1.5% and 3%) against two commercial ant baits; being them fipronil (0.014%) and boric acid (2.5%). the study focused on (1) evaluating the feeding preferences for lab baits against commercial baits, and (2) comparing the efficacy of the most preferred lab bait against commercial baits. the results of this study aim to develop new gel baits with a specific percentage of boric acid as the active ingredient (ai) for effective control of common household ants; m. chinensis and a. gracilipes. materials and methods the source of ants polygyne colonies of the asian needle ant, monomorium chinensis, santschi and the yellow crazy ant, anoplolepis gracilipes, smith, were obtained by excavation from field populations located around the national pingtung university of science and technology campus, pingtung, taiwan. the ants were separated from the soil using the water drip method, as described by kafle et al. (2008, 2009), and chen (2007), and reared under ambient laboratory conditions (27±1 °c and 50±3% rh), photoperiod of 14:10 h (l:d) and placed on a standard diet of water, meal worm larvae, peanut butter, and 10% sugar water at least one week before the tests. the test population, usually made up of the major workers, were isolated from the main population and starved for 24 hours before each test was conducted. preparation of the laboratory baits a stock solution made up of 25% boric acid was prepared by heating25g of boric acid powder (99%) (shimada chemical works, taipei, taiwan) and75gof fructose (sunright foods corporation, taipei, taiwan) at a temperature of 140ºc for 15 minutes, in order to completely dissolve the boric acid in the fructose. after cooling the mixture, 6g of the stock solution was added to 19g of either fructose or molasses to make up1.5% boric baits. similarly, to make up 3% boric baits, 13g of fructose or molasses was added to 12g of stock solution for 25g in each lab bait. the specific ratios of each component contained in the lab baits are shown in table 1. commercial baits two popular commercial ant baits used in taiwan were compared for efficacy against the laboratory baits: the boric acid bait (2.5% boric acid, chung tai sing chemical industry company limited, hsinchu, taiwan) and the fipronil bait (0.01% fipronil, family consumer products company limited, miaoli, taiwan). the baits were bought at the local market. preference tests in order to evaluate the feeding preferencesof the m. chinensis and a. gracilipes on the four laboratory baits (table 1) and two commercial baits, similar studies undertaken by kafle et al. (2010) and klotz (2000) were adapted for this test. acrylic rectangular foraging stations (18.5cm x 10.5cm x 4cm (l x w x h) were set up and modified to suit the purpose of the experiment. fluon was applied to the inner vertical surfaces to prevent ants from escaping the container. the test baits were arranged side by side on parafilm squares (1 cm x 1 cm) along one end of the setup and water was not provided during the test period. an artificial cardboard nest (2 cm x 2 cm) setup, as an artificial ant nest, were placed 1cm away from the inner wall and10cm away from the baits. the feeding preferences were recorded on 60-minute videos. these observations were reviewed and interval counts were done at 5 minute frames. ant-to-bait counts were determined by the number of ants actually feeding on the baits or foraging on the individual parafilm squares for at least 10 seconds. during the test period any dead ant was removed and replaced with live ants from the isolated population. this was to maintain the test population during the study period. a hundred m. chinensis and fifty a. gracilipes workers from the test populations were used in each individual test. each test was replicated four times under the laboratory conditions at temperatures of 25 ± 3ºc and rh 52 ± 3% and 14:10h l:d photoperiods. mortality tests according to the results of the preference tests, the lab baits f3 and m3 had the highest preferences used in the mortality test. rectangular foraging stations similar to those used in the preference tests were set as described by klotz et al. (2000) and kafle et al. (2010), and modified for this experiment. table 1. ratio of each constituent used in new laboratory bait preparations. constituent (%) lab baits* f1.5 f3 m1.5 m3 fructose 94 88 0 0 molasses 0 0 94 88 stock solution (25% boric acid) 6 12 6 12 total vol. (%) 100 100 100 100 *f = fructose, m = molasses, 1.5 = 1.5% boric acid and 3 = 3% boric acid sociobiology 67(1): 59-64 (march, 2020) 61 fluon was applied to the inner vertical surfaces to prevent ants from escaping. a single test bait per feeding arena was placed at one end of the foraging arena on a parafilm strip 1cm away from the vertical wall. an artificial cardboard nest was placed 10cm away from the bait at the opposite end of the foraging arena 1cm away from the inner wall. water was provided during the mortality tests and mealworm larvae were provided as food in the control sets and in all mortality tests. efficacies were compared across the baits; the boric acid, fipronil, lab bait f3, and lab bait m3, in tests similar to those described by kafle et al. (2010). observations of the test were recorded at 3-hour intervals for the first 24 hours and every 6 hours thereafter, until a hundred percent mortality was reached. thirty worker ants from the test populations of both species were used and each test was replicated four times under laboratory conditions;at temperatures of 25 ± 3ºc and rh 52 ± 3% and 14:10h l:d photoperiods. data collection and analysis each test setup was randomized and the data was sorted and compared on an excel spreadsheet using the procedures described by vander meer (2017) and kafle et al. (2010). the lethal time (lt50) was calculated through probit analysis using statplus (2017) and the means were compared using snk of sas (2017). results preference tests two fructose based lab baits, the f1.5 and f3 (table 1), were compared with the two commercial baits, fipronil and boric acid, to determine the preference of m. chinensis and a. gracilipes. the percentage of m. chinensis observed foraging on the fipronil bait was significantly higher than those observed on the rest of tested baits; however, the number of ants observed on the lab bait f1.5, the boric acid bait and the lab bait f3 were not significantly different (f = 6.2, p < 0.01) (table 2). the number of a. gracilipes observed foraging on the lab bait f3 was significantly higher than the other baits tested. the boric acid bait had the next highest preference. however, the number of a. gracilipes observed foraging on the two commercial baits and the lab bait f1.5 were not significantly different (f = 1.49, p < 0.27) (table 2). similarly, the number of m. chinensis observed foraging on the fipronil bait was significantly higher than the other three baits. however, the number of ants observed on the lab bait m1.5 and fipronil bait or lab bait m3 and the boric acid bait were not significantly different (f = 12.84, p < 0.01) (table 3). the number of m. chinensis observed foraging on the fipronil bait was statistically higher than the other three baits tested. however, there was no significant difference observed on the percentage of ants on all the baits tested (f = 0.14, p < 0.93) (table 3). the preference tests showed that lab bait m3 was more preferred by m. chinensis than the fructose based baits, while a. gracilipes preferred both the lab f3 and lab m3 baits. therefore, only the lab baits f3 and m3 were evaluated against the two commercial baits in the mortality tests. baits tested no. of ants foraging (mean ± se)* monomorium chinensis anoplolepis gracilipes boric acid bait (2.5% boric acid) 8.26 ± 0.37b 12 ± 3.72a fipronil bait (0.01% fipronil) 12.25 ± 1.12a 8.25 ± 1.38a lab bait 3f (5% boric acid) 7.00 ± 0.93b 8.25 ± 1.11a lab bait 4f (10% boric acid) 5.25 ± 0.8b 15.25 ± 3.68b *means within the same column followed by the same letter are not significantly different (p<0.05) (snk test, sas, 2017). table 2. preference of monomorium chinensis and anoplolepis gracilipes on new fructose and commercial baits under laboratory conditions. table 3. preference of monomorium chinensis and anoplolepis gracilipes on new molasses and commercial baits under laboratory conditions. baits tested no. of ants foraging (mean ± se)* monomorium chinensis anoplolepis gracilipes boric acid bait (2.5% boric acid) 5.74 ± 0.97b 10.75 ± 4.25a fipronil bait (0.01% fipronil) 13.09 ± 2.88a 13 ± 3.24a lab bait 3m (5% boric acid) 7.82 ± 3.39b 9.75 ± 4.11a lab bait 4m (10% boric acid) 12.19 ± 4.77a 12.25 ± 3.97a *means within the same column followed by the same letter are not significantly different (p<0.05) (snk test, sas, 2017). mortality tests mortality of m. chinensis by lab baits f3 and m3 against two commercialwere compared with the two commercial baits, fipronil and boric acid under the laboratory conditions. the percentage of m. chinensis killed by the lab baits(f3 and m3) fipronil and boric acid baits were not significantly different at 6 hat and 12 hat (6 hat: f= 1.07, p < 0.41; 12 hat: f = 2.61, p < 0.08) (table 4). at 24 hat, the percentage of ants killed by the fipronil bait was significantly higher than the lab bait m3 and the control. however, there was no significant difference in the percentage of ants killed by the boric acid, fipronil baits and the m3 or the boric acid bait, and the lab baits f3 and m3 (f = 7.81, p < 0.01) (table 4). the mortality rate of the m. chinensis by the lab bait m3, fipronil and boric acid baits were significantly higher than the control at 48, 72 and 84 hat. however, the percentage of ants killed by all three baits was not significantly different from each other. it was observed that all baits could kill up to 100% of m. chinensis within 84 hat. at 72 hat, the lab bait f3 killed 100% ants (48 hat: f = 8.83, p < 0.01; 72 hat: f = ln kafle, ac neupane, ym wang, sr gangai – development of new boric acid gel baits 62 194.62, p < 0.01; 84 hat: f = 2255.53, p < 0.01). based on the lt50 results, fipronil was the fastest killing bait, followed by the boric acid bait the lab bait m3 and then the lab bait f3 (table 4). mortality of a. gracilipes by lab baits f3 and m3 against two commercial were compared with the two commercial baits, fipronil and boric acid under the laboratory conditions. the percentage of a. gracilipes killed by the fipronil bait was significantly higher than the f3, m3 and boric acid baits at 6 hat and 12 hat. however, the percentage of ants killed by the boric acid bait and the lab baits f3 and m3 were not significantly different (6 hat: f = 136.08, p < 0.01; 12 hat: f = 29.76, p < 0.01) (table 5). observation timeα ant mortality (%) (mean ± se)* boric acid bait fipronil bait lab bait f3 lab bait m3 control 6 hat 2.62±1.02a 5.96±2.57a 0±0a 3.33±3.33a 2.50±1.60a 12 hat 12.82±4.40a 26.94±5.51a 0.81±0.81a 25.61±15.48a 2.50±1.60a 24 hat 46.38±11.86ab 73.86±9.96a 5.63±2.63b 44.96±17.25ab 6.67±1.36b 48 hat 68.11±13.19a 86.10±7.60a 59.73±13.58a 62.83±13.02a 20.84±2.50b 72 hat 93.58±4.05a 96.75±1.90a 100±0a 97.80±2.21a 24.17±1.60b 84 hat 100±0a 100±0a 100±0a 100±0a 24.17±1.60b lt50 β (h) 27.67 18.23 39.01 27.92 *means within the same row followed by the same letter (lower case) are not significantly different (p < 0.05) (snk test, sas, 2017) α hat = hours after treatment β lt50 values (h) were determined by probit analysis (statplus, 2017) f3 = 3% boric acid m3 = 3% boric acid observation timeα ant mortality (%) (mean ± se)* boric acid bait fipronil bait lab bait f3 lab bait m3 control 6 hat 3.70±0.80c 78.60±5.57a 1.62±0.93c 14.93±1.59b 6.67±1.36bc 12 hat 13.17±1.95c 90.32±4.62a 1.62±0.93c 43.56±13.87b 10.83±1.56c 24 hat 98.49±1.51a 100±0a 1.62±0.93c 97.14±2.86a 20.83±2.85b 48 hat 100±0a 100±0a 76.15±3.50b 98.57±1.43a 25.83±3.44c 72 hat 100.0±0a 100.0±0a 100.0±0a 100.0±0a 26.67±3.04b lt50 β 16.63 3.49 36.25 12.44 * means within the same row followed by the same letter (lower case) are not significantly different (p < 0.05) (snk test, sas, 2017) α hat = hours after treatment β lt50 values (h) were determined by probit analysis (statplus, 2017) f3 = 3% boric acid m3 = 3% boric acid table 4. mortality of m. chinensis by lab baits f3 and m3 against two commercial baits under laboratory conditions. at 24 hat, the percentage of ants killed by the fipronil bait was significantly higher than the other three baits tested. however, the percentage of ants killed by the boric acid, fipronil and the lab bait f3 were not significant different (f = 600.52, p < 0.01) (table 5). similarly, at 48 hat, the percentage of a. gracilipes killed by the fipronil and boric acid baits reached 100% and was significantly higher than lab baits f3 and m3. however, the percentage of ants killed by the boric acid and fipronil baits and lab bait m3 was not significantly different (f = 296.33, p < 0.01) (table 5). table 5. mortality of a. gracilipes by lab baits f3and m3 against two commercial baits under laboratory conditions. the percentage of a. gracilipes killed by the lab baits f3 and m3, and fipronil and boric acid baits reached 100% of ants at 72 hat and the percentage of ants killed by all tested baits was significantly higher than the control (f = 580.94, p < 0.01). based on the lt50 value the fipronil bait was the fastest killing bait of m. chinensis and a. gracilipes, followed by the lab bait m3, the boric acid bait and the lab bait f3 (table 5). discussion molasses and fructose are two of the most popular natural sugars used in insect control. a similar study undertaken by ulloa-chacon and jaramillo (2003) emphasized that toxic baits prepared with insecticides added to a sugar solution were attractive to the ghost ant and appropriate for use when sociobiology 67(1): 59-64 (march, 2020) 63 implemented in trials for the control of various species, similar to the argentine ant (iridomyrmex humilis mayr). during the preference tests on the molasses baits, a higher percentage of ants were observed to be actively foraging on the lab baits m1.5 and m3. the ants were attracted to the molasses over the fructose mainly due to the sucrose content, and thus both the m. chinensis and a. gracilipes were lured when it was used in these baits (binkley & wolfrom, 1953; curtin, 1983; saric et al., 2016). both m. chinensis and a. gracilipes are generalist feeders that seek out sources of carbohydrates, lipids and proteins as food sources (vanderwoude et al., 2006). therefore, it was observed how both species in this study actively fed on the sugar based baits. they were able to exploit their food resources by communicating with odour trails and rapid recruitment (sparks, 2015) in a similar way. they also may have been able to communicate danger when workers were stuck in the baits. during the study, it was observed that when workers became stuck in the bait and were left there for a while, other ants also ceased feeding. this behavior was observed in both m. chinensis and a. gracilipes. bluthgen and fiedler (2004a) observed the preference for sugars and amino acids of the nectarivorous ants in an australian tropical rain forest. using artificial nectar solutions, the feeding behavior of fifty-one ant species on these solutions was recorded. the results stated that preferences among carbohydrates were principally consistent between ant species. a very significant observation was that many of the ant species preferred: sugar, glucose, fructose or sucrose solutions containing mixtures of amino acids, such as: alanine; arginine, asparagine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and other substances in natural nectar over pure sugar solutions. these results correspond with previous studies thus confirming the variability in amino acids and carbohydrates proposes to play a key role in ant preferences and nutrition (bluthgen & fiedler, 2004b; bluthgen et al., 2004;vander meer et al., 1995; lanza, 1993). in a study on the bait preferences and toxicity of insecticides to white-footed ants, technomyrmex albipes, warner (2003) noted that fructose was found to be more significant than the nectar of brownea sp., or other sugars, while both the nectar and fructose of clerodendrum myricoides were significantly favored more than glucose and sucrose. this is evidence that different species of ants have a specific preference for sugar types. therefore, the results varied in the toxicity tests. all baits achieved 100% mortality by 72 hat. the killing speed of lab bait m3 was faster than the boric acid bait and the lab bait f3. a main reason for this may be the ingredients used in the baits. the lab bait f3 contained just fructose and boric acid. however, lab bait m3 contained fructose, molasses and boric acid, which may have had an impact on the feeding preference and thus the results. the killing speed of lab baits f3 and m3 against m. chinensis and a. gracilipes were faster than the boric acid bait and slower than the fipronil bait. it is assumed this is due to the different active ingredients used in each of the baits. the fipronil bait used in this study contained 0.01% active ingredient. fipronil is a sweet smelling poison that also aided it as a preference. it is toxic to insects by contact or ingestion (jackson et al., 2009). thus, is used in many products when a delayed kill is desired. fipronil disrupts the insect central nervous system and causes the hyperexcitation of nerves and muscles resulting in death. klotz et al. (2000) presumes that although there is little information available concerning the physiological mode of action of boric acid on insects, it has been shown that borate ions form strong complexes with sugar alcohols, and other organic functional groups. harper et al. (2012) also suggest that boron may be involved in the disruption of intercellular adhesion because saturated boric acid solutions can be used to dissociate cells. some advantages of using boric acid as an active ingredient in ant baits are the delayed activity and solubility in water. at a low concentration, boric acid is slowacting and less likely to be repellent (klotz & williams, 1996). as federal and state laws become more stringent on the use of residual insecticide spray, these baiting techniques may provide an effective control strategy (welzel & choe, 2016). the test formulations proved that the boric acid based molasses bait (m3) was an efficient formula. the results of this study showed, i) the new lab bait m3 is a potential alternative to current commercial gel baits, and ii) boric acid can be used as an alternative for the hazardous pesticide like fipronil, as the active ingredient in ant gel baits. acknowledgement this study was supported by the ministry of science and technology (most 106-2313-b-020-002 project), taiwan. the authors are grateful to the department of tropical agriculture and international cooperation, national pingtung university of science and technology for providing the laboratory facilities. author’s contribution the study concept and design: l. kafle and y. m. wang, the acquisition of data: sr gangai, analysis and interpretation of data: a.c. neupane, s.r gangai and l. kafle, manuscript preparation: a.c. neupane and l. kafle, and critical revision: l. kafle, a. c. neupane and y. m. wang. references binkley, w.w. & wolfrom, m.l. (1953). composition of cane juice and cane final molasses. advances in carbohydrate chemistry: scientific report series no. 15 (8):1 -25. bluthgen, n. & fiedler, k. (2004a). preferences for sugars and amino acids and their conditionality in a diverse nectarln kafle, ac neupane, ym wang, sr gangai – development of new boric acid gel baits 64 feeding ant community. journal of animal ecology, 73:155– 166. doi: 10.1111/j.1365-2656.2004.00789.x bluthgen, n. & fiedler, k. (2004b). competition for composition: lessons from nectar-feeding ant communities. ecology, 85: 1479-1485. doi: 10.1890/03-0430 bluthgen, n., gottsberger, g. & fiedler, k. (2004). sugar and amino acid composition of ant-attended nectar and honeydew sources from an australian rainforest. austral ecology, 29: 418-429. doi: 10.1111/j.1442-9993.2004.01380.x chen, j. (2007). advancement on techniques for the separation and maintenance of the red imported fire ant colonies. insect science, 14: 14. doi: 10.1111/j.1744-7917.2007.00120.x curtin, l.v. (1983). molasses – general considerations. molasses in animal nutrition, national feed ingredients association, west des moines, iowa. pp.111. harper, b., gervais, j.a., buhl, k. & stone, d. (2012). boric acid technical fact sheet. national pesticide information center, oregon state university extension services. http:// npic.orst.edu/factsheets/archive/borictech.html. (accessed date: 28 december, 2017). jackson, d., cornell, c.b., luukinen, b., buhl, b. & stone, d. (2009). fipronil general fact sheet. national pesticide information center, oregon state university extension services. http://npic.orst.edu/factsheets/archive/fiptech.html. (accessed date: 28 december, 2017). kafle, l., wu, w.j., vandermeer r.k. & shih, c.j. (2008). simplified approaches to determine the attractant preference of solenopsis invicta (hymenoptera: formicidae). applied entomology and zoology, 43 (3): 383-390. doi: 10.1303/ aez.2008.383 kafle, l., wu, w.j., vandermeer r.k., huang, y.y. & shih, c.j. (2009). microencapsulated bait: does it work with red imported fire ant, solenopsis invicta buren (hymenoptera: formicidae)? sociobiology, 53(3): 729-737. kafle, l., wu, w.j. & shih c.j. (2010). a new fire ant (hym.: formicidae) bait base carrier for moist conditions. pest management science, 66(10): 10828. doi: 10.1002/ps.1981 kafle, l. & shih, c.j. (2012). determining the most effective concentration of cypermethrin and the appropriate carrier particle size for fire ant (hymenoptera: formicidae) bait. pest management science, 68(3) 394-398. doi: 10.1002/ps.2275 kafle, l. & shih, c.j. (2013). toxicity and repellency of compounds from clove (syzygium aromaticum) to red imported fire ants solenopsis invicta (hymenoptera: formicidae). journal of economic entomology, 106 (1):131-135. doi: 10.1603/ec12230 klotz, j.h. & moss, j.i. (1996). oral toxicity of a boric acid sucrose water bait to florida carpenter ants (hymenoptera: formicidae). journal of entomological science, 31 (1): 9–12. doi: 10.18474/0749-8004-46.2.89 klotz, j.h. & williams, d.f. (1996). new approach to boric acid ant baits. the ipm practitioner, (8): 1-4. klotz, j.h., greenberg, l., amrhein, c. & rust, m.k. (2000). toxicity and repellency of borate-sucrose water baits to argentine ants (hym.: formicidae). journal of economic entomology, 93 (4): 1256-1258. doi: 10.1603/0022-0493-93.4.1256 lanza, j., vargo, e.l., pulim, s. & chang, y.z. (1993). preferences of the fire ants solenopsis invicta and s. geminate (hymenoptera. formicidae) for amino acid and sugar components of extrafloral nectars. environmental entomology, 22: 411-417. doi: 10.1093/ee/22.2.411 saric, l.c., filipcev, b.v., simurina, o.d., plavsic, d.v., saric, b.m., lazarevic, j.m. & milovanovic, i.l. (2016). sugar beet molasses: property and applications in osmotic dehydration of fruits and vegetables. food and feed research, 43 (2): 135-144. doi: 10.5937/ffr1602135š sas institute. (2017). sas user’s guide: statistics, version 8. sas institute, inc., cary, nc. sparks, k. (2015). molecular phylogenetics of australian monomorium. a thesis for degree of doctor of philosophy. school of biological sciences, ua, australia. statplus. (2017). analysist soft.version 5.4. statplus house inc. ca, usa. ulloa-chacon, p. & jaramillo, g. 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(2003). bait preferences and toxicity of insecticides to white-footed ants technomyrmex albipes (hymenoptera: formicidae). a thesis for degree of master of science. graduate school of the university of florida, usa, 59 p. zheng, j.h., mao, r. q. & zhang, r. j. (2008). competitive interactions between the yellow crazy ant and the red imported fire ant (hymenoptera: formicidae). journal of entomological science, 43 (3): 331-336. doi: 10.18474/0749-8004-43.3.331 1229 botanical origin of protein sources used by honeybees (apis mellifera) in an atlantic forest by talita antonia da silveira1*; maria emilene correia-oliveira1; augusta c. c. c. moreti2; ivani pozar otsuk2; luís carlos marchini1 abstract productive and reproductive traits of beehives are influenced by climate and food availability in the region where the bees are reared or maintained, thus honey and pollen storage, egg-laying conditions of the queen as well as comb occupation are subject to seasonal variations. the present study was conducted in the apiary of the department of entomology and acarology, college of agriculture luiz de queiróz, esalq/usp, in the municipality of piracicaba, in an area containing fruit trees, ornamental plants and a fragment of a native forest. the objective was to identify protein sources used by honeybees (apis mellifera) over a whole year (2010-2011) in remnants of the atlantic forest, information that can be used in the conservation and restoration of degraded areas. for sample preparation, the acetolysis method was adopted (eredtman 1952) and the quantitative analysis was performed by counting successive samples of 900 grains per sample which were grouped by botanical species and/or pollen types. the results show that the bees used various plant types in the area, including ruderal species, to maintain their colonies. apis mellifera seeks food sources in all plants in the surroundings of the apiary, including herbaceous, shrubs, trees, native or introduced. eucalyptus sp. played an important role as a food source in all seasons due to its wide availability around the apiary and its high flower production. the most frequent pollen types (greater than 10% of the sample) were anadenanthera sp., acacia sp, miconia sp. and eucalyptus sp. in winter; philodendron sp., mikania cordifolia, parthenium and eucalyptus sp. in spring ; alternanthera ficoidea, chamissoa altissima and eucalyptus sp. in summer; philodendron sp., raphanus sp. and eucalyptus sp. in autumn. key words: pallinolog y, pollen, beekeeping. 1programa de pós-graduação em entomologia e acarologia da escola superior de agricultura “luiz de queiroz”, universidade de são paulo, 13418-900, piracicaba, são paulo, brasil. 2instituto de zootecnia, secretaria de agricultura e abastecimento, 13460-000, nova odessa, são paulo, brasil *corresponding author: tali_aband@hotmail.com 1230 sociobiolog y vol. 59, no. 4, 2012 introduction pollen is the male gamete of flowers of angiosperms, necessary for egg fertilization and, therefore, for seed formation (raven 2001). it also serves as a reward for floral pollinators (schlindwein et al. 2005), mainly bees, which use pollen as a protein source of their diets (zerbo et al. 2001). honeybees, apis mellifera, use pollen as a food source in all developmental stages of the colony (basim et al. 2006), therefore, protein content and availability the pollen are essential factors for the colony (zerbo et al. 2001). pollen is the sole protein source (roubik 1989) necessary for the proper functioning of the hypopharyngeal gland in worker bees (crailsheim 1990). pollen grains vary in shape, size, color, appearance, morpholog y and can, thus, be used to identify plant gender and species (schmidt & buchmann 1993; almeida-muradian et al. 2005). pollen grain identification is made through its outer wall, chemically stable and morphologically varied, which allows the identification of various pollen types through the pollen analysis (salgado-labouriau 1973, barth 2004). this identification can be used as indicator of the geographical and botanical origin of honey (barth 2004), since the pollen types found in these products are considered “fingerprints” of bee foraging habits (wittmann & schlindwein 1995). knowledge of bee foraging habits can be an important tool for beekeeping development (pearson & braiden 1990), determining the site of apiary implantation and honey production (ashman et al. 2004; biesmeijer et al. 2006). this type of study can also be used for reforestation and conservation of areas and for the establishment of ecological corridors (modro 2009). this work aimed to identify the plant species used as protein sources by honeybees (apis mellifera) in remnants of the atlantic forest, information that can be used in conservation and restoration programs of degraded areas. materials and methods experimental area samples were collected at the beginning and end of each of the four seasons during a whole year between 2010 and 2011, at the apiary located in the department of entomolog y and acarolog y the college of agriculture “luiz de queiroz”, near a fragment of the atlantic forest, in the municipal1231 silveira, t.a. et al. — protein sources used by honeybees in atlantic forest ity of piracicaba (22º52’33”s, 47º38’30”w, altitude 546m), são paulo state, brazil. collection of samples five hives were selected under similar population conditions and in the first and last weeks of each season (spring, summer, autumn and winter), we installed pollen collectors that allowed the entrance of the bees, but intercepted the pollen collected, which fell into a tray and remained until it was removed. pollen analysis we used the acetolysis method (erdtman 1952) for identification of pollen types. the method forces the exit of the cellular content of the pollen grain, leaving only the cell wall. afterwards, slides were mounted on glycerinated gelatin. the grain characterization was made by comparison with a reference slide collection of the department of entomolog y and acarolog y of the college of agriculture “luiz de queiroz” (esalq/usp) and the specialized literature (barth 1970 a, b, c, moreti et al. 2002, roubik & moreno 1991). after the grain identification, we carried out a quantitative analysis by counting 300 grains per slide, 900 per sample (analysis made in triplicate). the quantitative analysis aims to determine the proportion of contribution of each pollen type, identifying and grouping it according to its occurrence as dominant pollen (dp), when it accounts for more than 45% of total pollen grains counted; accessory pollen (ap, 16-45%) and isolated pollen (ip, up to 15%). the ip was subdivided into significant isolated pollen (sip, 3-15%) and occasional isolated pollen (oip, less than 3% of the grains observed) (louveaux et al. 1978). the main pollen types were photomicrographed through a zeiss microscope coupled to a digital camera. the experimental design was completely randomized with five replicates (represented by hives). data were analyzed using the mixed procedure, sas (statistical analysis system), to determine the structure of variance and covariance matrix. the significance level for the variance analysis was 5%. the data were transformed to log (x+1) to achieve homoscedasticity in the variance analysis. 1232 sociobiolog y vol. 59, no. 4, 2012 results and discussion in the study period, 68 pollen types belonging to 19 botanical taxa were found (table 1). modro (2011) conducted a study in the same place and found similar results with regard to plant families, however, the author observed a total of 81 taxa. this difference can be explained due to the seasonality of some plant species and the presence of cropped areas in the region of collection. the main pollen types classified in terms of statistical significance were: in spring, mikania cordifolia and eucalyptus sp; in summer, alternanthera ficoidea (dominant); in fall, eucalyptus sp as accessory pollen, and in winter there was no statistical difference among the pollen types (table 1). according to renner (1968), most plant species have flowers that do not produce pollen or nectar throughout the day, but only at certain hours. thus, bee activity, food type and increased collection time, according to butler (1945); moffett & paker (1953), bennett & renner (1961), depend on the following characteristics: genetic trait of the hive, the amount of nectar available, sugar content in flowers, time of day, environmental factors and plant species. therefore, variations in collection sites greatly affect the collection of floral resources by bees. the families fabaceae, asteraceae and arecaceae showed the greatest number of species in winter, arecaceae and asteraceae in spring and asteraceae in autumn. the main pollen species were: acacia sp. and anadenanthera sp. (fabaceae), alternanthera ficoidea and chamissoa altissima (amaranthaceae), philodendron sp. (araceae), eucalyptus sp. (myrtaceae), mikania cordifolia and parthenium sp. (asteraceae) and raphanus sp. (brassicaceae). the difference in numbers of pollen types along the seasons shows the generalist habit of these bees, however, there seems to have a preference for some plant species that appear in all seasons such as eucalyptus, raphanus, astrocaryum and philodendron, although none of them are dominant. the preference of apis mellifera for eucalyptus pollen has been observed by other authors, whenever eucalyptus was present in the vicinity of the apiary (almeida-anacleto 2007 and carvalho et al. 1999, in piracicaba, são paulo state; modro 2006 and barreto 1999, in viçosa, minas gerais state; luz et al. 2007 in engenheiro paulo de frontin, rio de janeiro state, and moreti et al. 2011 in pindamonhangaba, são paulo state – all municipalities in brazil ). 1233 silveira, t.a. et al. — protein sources used by honeybees in atlantic forest table 1 average percentage of pollen types collected by apis mellifera in piracicaba, são paulo state, brazil, in the four seasons in 2010-2011. means followed by different capital letters in the columns differ by tukey-kramer (p <0.05). 1means in parentheses are averages transformed to log (x+1) # statistical data on produced pollen. dp -dominant pollen; ap – assistant pollen, sip – significant isolated pollen, oip occasional isolated pollen (x -no collection). family species season spring summer autumn winter amaranthaceae amaranthus sp. x x x 1.46±8.54 oip (0.36±0.24)a alternanthera ficoidea 3.70±14.76 sip (0.27±0.42)ab 59.89±6.61 dp (1.63±0.18)a 4.39±6.08 sip (0.69±0.17)b x chamissoa altissima x 16.88±10.61 ap (1.24±0.19)ab x x anacardiaceae tapirira sp. x x 2.13±14.78 oip (0.49±0.42)ab x araceae type1 2.10±8.53 oip (0.42±0.24)ab x 1.71±8.53 oip (0.43±0.24)ab 1.77±8.67 oip (0.41±0.24)a philodendron sp. 20.04±6.61 ap (1.01±0.19)ab 5.55±6.69 sip (0.68±0.19)ab 14.97±8.65 sip (0.89±0.24)ab 9.85±6.72 sip (0.72±0.19)a arecaceae type 1 1.00±10.45 oip (0.28±0.30)ab x 5.31±8.94 sip (0.67±0.24)ab 2.34±8.66 oip (0.47±0.24)a astrocaryum sp. 4.46±8.53 sip (0.59±0.24)ab 2.99±10.44 oip (0.54±0.30)ab 6.78±6.12 sip (0.66±0.17)ab 1.91±6.73 oip (0.33±0.19)a asteraceae bidens pilosa x x 0.87±6.74 pio (0.26±0.19)b 0.25±14.77 pio (0.08±0.42)a mikania cordifolia 34.10±7.56 ap (1.48±0.21)a x 5.22±8.53 sip (0.59±0.24)ab 1.05±10.71 oip (0.31±0.29)a parthenium sp. 30.96±10.45 ap (1.05±0.30)ab x 4.70±6.13 sip (0.64±0.17)ab 0.15±10.45 oip (0.04±0.30)a type 1 x x 3.04±10.71 sip (0.60±0.29)ab x sonchus oleraceus x x 0.55±8.67 oip (0.18±0.24)b x brassicaceae raphanus sp. 1.62±10.44 oip (0.36±0.30)ab 1.69±10.44 oip (0.36±0.30)b 21.62±5.03 ap (1.07±0.14)ab 0.87±7.46 oip (0.37±0.21)a cucurbitaceae momordica sp. x x 1.54±8.67 oip (0.39±0.24)ab 0.42±10.71 oip (0.14±0.29)a fabaceae acacia sp. x x 0.70±10.69 oip (0.06±0.29)b 17.59±8.54 ap (1.11±0.24)a anadenanthera sp. x x x 16.59±8.54 ap (0.12±0.24)a caesalpinia peltophoroides x x x 1.22±8.54 oip (0.34±0.24)a mimosa caesalpinifolia 8.40±7.39 sip (0.77±0.21)ab x x 0.04±10.45 oip (0.04±0.30)a 1234 sociobiolog y vol. 59, no. 4, 2012 quantitatively, the eucalyptus sp. grains showed some frequency in almost all seasons. this may be associated with the availability of these resources in the collection site and the high pollen production of this plant species that makes this type of resource more easily collected by bees. this fact is consistent with bawa (1983) and castro (1994) who state that the attractiveness of flowering can be influenced by the amount of pollen produced, the concentration and abundance of flowers, number of competing insects, the distance between the flowering sites and nest, and innate preference for the species. however, table 1 (continued). family species season spring summer autumn winter fabaceae type1 x x 1.43±10.45 oip (0.38±0.30)ab x lythraceae lagerstroemia indica 2.32±8.54 oip (0.51±0.24)ab x x x malvaceae paquira sp. x x x 0.15±10.45 oip (0.06±0.30)a dombeya sp. x 0.27±10.45 oip (0.11±0.30)b 0.72±8.53 oip (0.20±0.24)b x melastomataceae miconia sp. x x x 12.39±10.45 sip (1.13±0.30)a morus nigra 9.24±8.53 sip (0.98±0.24)ab 6.08±7.39 sip (0.63±0.21)ab 1.29±10.44 oip (0.30±0.30)ab myrtaceae eucalyptus sp. 31.34±5.03 ap (1.12±0.14)a 14.83±4.79 pii (0.87±0.13)ab 37.52±4.79 ap (1.53±0.13)a 18.79±5.66 ap (0.99±0.16)a piperaceae piper sp. 2.32±7.47 oip (0.42±0.21)ab 1.89±7.47 oip (0.40±0.21)b x 6.02±8.65 sip (0.75±0.24)a poaceae type 2 0.51±6.13 oip (0.16±0.11)b x x x oryza sp. x x 2.11±10.71 oip (0.46±0.29)ab x rutaceae citrus sp. x x 1.03±10.45 oip (0.28±0.30)ab x prunus persica x 8.14±8.54 sip (0.87±0.24)ab x x sapindaceae x x x 0.29±10.71 oip (0.11±0.29)b x solanaceae type solanaceae 0.96±10.45 oip (0.28±0.30)ab 4.54±10.45 sip (0.74±0.30)ab x x scrophulariaceae scopania sp. x 5.48±10.45 sip (0.57±0.30)ab x x 1235 silveira, t.a. et al. — protein sources used by honeybees in atlantic forest these factors are still subject to variation such as flower size, relative humidity, soil moisture, temperature, altitude, time and daylight duration. this generalist habit can occur due to the protein need of the colony, since the protein content may vary with the botanical origin. according to rouston et al. (2000), protein variation may not be directly related to the attractiveness of pollinators, since the pollen of zoophilic species is not richer in proteins than in anemophilous species. according to schmidt & buchmann (1993) bees collect pollen from many plant species and thus have good nutritional balance and high dilution of toxic potential of alkaloids and other toxins. the summer was the only season that showed dominance of a plant species, despite the 12 different types found. bees sought preferably the species alternanthera ficoidea, which is an herbaceous plant originating from the americas (mears 1977), showing the importance of plants often considered weeds for honey production (modro 2011). it is interesting to observe that this same plant appears as sip in spring and summer, which indicates that it could become an important bee forager in other seasons. the preference for this pollen type, which, at times, appears in small amounts changing to dominant in others, may be related to the energ y bees spend to seek other food sources, since this species occurs in abundance in the surroundings of the apiary (modro, et al. 2007; modro, et. al. 2011). pollen types that had a frequency lower than 10% may be considered secondary, because ramalho et al., (1985) believe that sources between 1 and 10% of pollen have little attractiveness as food source. these pollen types, however, may be useful as a suplement to food needs of the colony and become important for maintaining balance at other times of year. conclusion apis mellifera bees seek food sources in all plants in the surroundings of the apiary, regardless of the plant type, whether herbaceous, a shrub, a tree, native or introduced. the eucalyptus sp played an important role as bee forager in all seasons due to its wide availability in the vicinity of the apiary and its large production of flowers. the most frequent pollen types (greater than 10% of the sample) were: anadenanthera sp, acacia sp, miconia sp and eucalyptus sp during winter; 1236 sociobiolog y vol. 59, no. 4, 2012 philodendron sp, mikania cordifolia, parthenium sp and eucalyptus sp in spring ; alternanthera ficoidea, chamissoa altissima and eucalyptus sp in summer; philodendron sp, raphanus sp and eucalyptus sp in autumn. acknowledgment we wish to thank fapesp and capes for the financial support to the research project. references almeida-anacleto, d. de. 2007. recursos alimentares, desenvolvimento das colônias e características físico químicas, microbiológicas e polínicas de mel e cargas de pólen de meliponíneos, do município de piracicaba, estado de são paulo. 133 p. tese (doutorado em entomologia) escola superior de agricultura “luiz de queiroz”, universidade de são paulo, piracicaba. almeida-muradian, l.b., l.c. pamplona, s. coimbra & o.m. barth 2005. chemical composition and botanical evaluation of dried bee pollen pellets. journal of food composition and analysis 18: 105-111. ashman, t.l., t.m. knight, j.a. steets, p. amarasekare, m. burd, d.r. campbell, m.r. dudash, m.o. johnston, s.j. mazer, r.j. mitchell, m.t. morgan, w.g. wilson 2004. pollen limitation of plant reproduction: ecological and evolutionary causes and consequences. ecolog y 85: 2408–2421. barth, o.m. 2004. melissopalynolog y in brazil: a review of pollen analysis of honeys, propolis and pollen loads of bees. scientia agricola 61: 342-350. barth, o.m. 1970a. análise microscópica de algumas amostras de mel. 1 pólen dominante. anais academia brasileira de ciências 42, 2: 351-366. barth, o.m. 1970b. análise microscópica de algumas amostras de mel. 2 pólen acessório. anais academia brasileira de ciências 42, 3: 571-590. barth, o.m. 1970c. análise microscópica de algumas amostras de mel. 3 pólen isolado. anais academia brasileira de ciências 42, 4: 748-772. barreto, l.m.r.c. 1999. levantamento florístico e polínico e estudo melissopalinológico durante a principal safra da microrregião homogênea da zona da mata de viçosa, mg. 87p. dissertação (mestrado em entomologia) – universidade federal de viçosa, viçosa. basim, e., h. basim, m. özcan 2006. antibacterial activities of turkish pollen and propolis extracts against plant bacterial pathogens. journal of food engineering, oxon, 77, 4: 992-996. bawa, k.s. 1983. patterns of flowering in tropical plants. in.: jones, g.e.;little,r.j. 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(ed.). the hive and the honeybee. hamilton: dadant & sons, 927-988. schlindwein, c., d. wittmann, c.f. martins, a. hamm, j.a. siqueira, d. schiffler, d. & i.c. machado 2005. pollination of campanula rapunculus l.(campanulaceae): how much pollen flows into pollination and into reproduction of oligolectic pollinators? plant systematics and evolution 250: 147-156. wittmann, d. & c. schlindwein 1995. melittophilus plants, their pollen and flower visiting bees in southern brazil.1. loasaceae. biociências 3: 19-34. zerbo, a.c., r.l.m.s. moraes, & m.r. brochetto-braga 2001. protein requirements in larvae and adults of scaptotrigona postica (hymenoptera: apidia, meliponinae): midgut proteolytic activity and pollen digestion. comp biochem physiol 129: 139–147. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4865sociobiology 67(2): 281-291 (june, 2020) introduction agricultural production is intrinsically dependent upon the ecosystem services that the insects provide absolutely free of charge (zhang et al., 2007). above 70% of the world’s principal crops, enjoy the benefits of getting pollinated by the animals that visit the flowers (klein et al., 2007). globally, the value of biotic pollination-dependent crops was estimated to be us$ 235-577 billion annually (intergovernmental science-policy platform on biodiversity and ecosystem services [ipbes], 2016). in brazil, in 2018, this value was estimated at roughly us$ 43 billion for the biotic pollinationabstract agricultural landscapes sometimes include natural habitats which can support the ecosystem by enhancing the pollination of crops, thus boosting the productivity. this research was conducted between may and july 2017, in the municipality of tangará da serra, mato grosso, brazil, to assess the cerrado from the perspective of it being a crucial habitat to sustain the sunflower-pollinating bees (helianthus annuus l.). the bees were sampled using entomological nets and pan traps, in specifically marked out plots (20 m x 150 m), in the cerrado, and in a sunflower crop, at different distances from the cerrado border. the assessment was done in terms of the composition and species richness, abundance of individuals and the mass (g) of the sunflower chapters exposed and isolated from the floral visitors. while species richness showed no differences between the cerrado and sunflower crop, a difference was observed for abundance, with more numbers of individuals in the sunflower crop, most likely because of the food source supply. in the sunflower crop, the bee diversity decreased proportionally as the distance from the border increased. the seed mass of the sunflower chapters was significantly higher in the flowers open to visitors than in those of the isolated chapters open for visitation. from the results, it was evident that the bees present in the cerrado visit the sunflower crop to gather pollen and nectar, and thus assist in cross-pollinating them and raising the productivity. sociobiology an international journal on social insects mls almeida1, gs carvalho1, jr novais2, d storck-tonon3, ml oliveira4, t mahlmann4, ds nogueira4, mjb pereira1,3 article history edited by kleber del-claro, ufu, brasil received 06 november 2019 initial acceptance 28 february 2020 final acceptance 23 march 2020 publication date 30 june 2020 keywords native bees, ecosystem services, pollination, brazilian savanna. corresponding author mayra layra dos santos almeida programa de pós-graduação em ecologia e conservação universidade do estado de mato grosso rod. mt 358, km 07 caixa postal 287, cep: 78.300-000, jardim aeroporto tangará da serra, mato grosso, brasil. e-mail: mayyralayra@gmail.com dependent crops (plataforma brasileira de biodiversidade e serviços ecossistêmicos [bpbes], 2019). bees (hymenoptera, apoidea) play a vital role in the pollination process, because they are completely dependent upon floral resources (klein et al., 2007; potts et al., 2016). however, over the last 50 years there has been a general global reduction in these insects in agricultural landscapes (potts et al., 2010; cameron et al., 2011). this drop in the number of pollinators may be directly linked to the considerable decline in the natural habitats, in terms of quality and quantity (benton et al., 2003; lindgren et al., 2018), which is due to the conversion of native vegetation to specific crops (potts et al., 2010; cameron et al., 2011). 1 programa de pós-graduação em ecologia e conservação, universidade do estado de mato grosso-unemat, nova xavantina-mt, brazil 2 universidade do estado de mato grosso-unemat, tangará da serra-mt, brazil 3 programa de pós-graduação stricto sensu em ambiente e sistemas de produção agrícola, universidade do estado de mato grosso-unemat, tangará da serra-mt, brazil 4 instituto nacional de pesquisas da amazônia-inpa, coordenação de biodiversidade, manaus-am, brazil research article bees contribution of the cerrado as habitat for sunflower pollinating bees mls almeida et al. – cerrado as habitat for sunflower pollinating bees282 keeping this scenario, research has been developed with a focus on the conservation of natural habitats close to the cultivated areas as a good alternative for the sustenance of bees, as well as maintenance of the ecosystem by pollination of the crops (ricketts et al., 2008; carvalheiro et al., 2010; lindgren et al., 2018). this concept, which several researchers still largely consider a challenge, is constructed on the principle that most of the pollinators that visit crops, particularly the bees, use natural remnants near the culture to nest and explore floral resources (ricketts et al., 2008). the bees in these habitats were collected using simple and replicable sampling methods that can be easily adapted to a wide range of research works (gotelli & colwell, 2001; roulston et al., 2007). the common methods used most often for insect sampling are active and passive collection, termed complementary methods (moreira et al., 2016). as pollinator diversity may be related to floristic heterogeneity in a specific region (roulston & goodell, 2011), it is anticipated that more diverse habitats will offer better pollinator service in adjacent agricultural areas (garibaldi et al., 2011). in this context, possibly the brazilian cerrado in the surroundings of agricultural areas is a habitat favorable to the bees and to the service of pollination of cultures. this biome is composed of a mosaic of vegetation types, from the wide-open fields to the savannas and forests; besides, it also has a high floral diversity (oliveira-filho & ratter, 2002; silva et al., 2015). its physiognomic features may promote the presence of several species because of their propensity for interchange among the vegetation types (almeida & louzada, 2009; gries et al., 2012). despite the importance of native areas, our knowledge about the contribution of bees that live in these areas for crop pollination is still limited and, as far as we know, no study has been carried out to investigate the importance of cerrado as a pollinator reservoir in the state of mato grosso, the largest sunflower producer in brazil. sunflower ranks prominently among the pollinator-dependent crops. it is a plant naturally possessing self-incompatibility and depends upon bees as the predominant pollinators (free, 1993; hevia et al., 2016). the bees forage the sunflower chapters and help by transferring the pollen from one plant to another, thus boosting the seed production. but, over time, sunflower hybrids have been selected to minimize this pollinatordependence (greenleaf & kremen, 2006; sun et al., 2012). however, this percentage of pollinator dependence on seed production varies between the hybrids or sunflower varieties (mallinger & prasifka, 2017). in brazil, and specifically in the state of mato grosso, the country’s largest sunflower producer, many farmers believe that some sunflower varieties, such as helio250, have a low dependence on pollinators. thus, our work aimed to evaluate determined to assess the part played by the cerrado as a natural habitat for the bees, which offers the ecosystem the service of sunflower pollination and the contribution of the pollinators towards the production of linoleic sunflower seeds. this study can help to answer some significant questions, such as whether the richness and abundance of bees declines with the distance from the natural habitat (closed); whether the distance of the natural habitat influences the sunflower yield; whether the visit of the bees to the linoleic sunflower chapters boosts the seed weight and whether the canopy richness and abundance vary with the sampling technique. material and methods study area the study was conducted at the aparecida da serra farm (14°18’36.64”s, 57°44’47.00”w), in the municipality of tangará da serra, mato grosso state, brazil. the climate in that region is classified as rainy tropical (aw), according to the köppen geiger classification, with an annual average rainfall of ~ 1,860 mm and annual temperature of around 24º c. the region experiences a rainy (october to may) and a dry season (june to september) (dallacort et al., 2011). the study area has 4,450 ha of cerrado sensu stricto, with expressive floristic diversity, that is maintained as legal reserve rl. the rl represents approximately 56% of the total area of the farm and is bordered by 151.66 ha of sunflower. the hélio: h250 (linoleic) variety of sunflower used is the one normally recommended for crop in brazil, bolivia and paraguay. in brazil, the helio: h250 crop is cultivated under conditions of a second crop, in the production system that adopts the soybean-sunflower succession. the sunflower was cultivated following the conventional method, and included the addition of herbicides, fungicides and insecticides. sampling of bees the bees were sampled from may 2017, at the height of the sunflower bloom (10 days) in eight plots (20 x 150 m), four in the cerrado and four in the cultivated sunflower crop. the plots were any one of these distances (50, 150, 300 and 600 m) from the cerrado border and were demarcated in the direction of the interior border of the cerrado and the interior border of the crop. five traps were set up, 30 m from each other. these traps were composed of two plastic trays (30 cm long x 23 cm wide), one blue and one yellow painted with uv-reflecting paint (sprl, spray-color) (moreira et al., 2016), fixed to a wooden stake to 1.90 m above the soil level, being the identical height of the sunflower chapters. this methodology was adapted, following the methodology advocated by hevia et al. (2016). a similar pattern of traps was followed in the cerrado (fig 1). each tray contained the following ingredients: water 1l, liquid soap 1 teaspoon, and salt (nacl) 1 teaspoon, to trap the insects. the traps were left open for 24 hours during the peak of the sunflower flowering season. according to westphal et al. (2008) this methodology is popular in studies which compare the pollinator communities in different places or environments. the bees trapped in these pans were collected daily and placed in 70% alcohol, in containers. sociobiology 67(2): 281-291 (june, 2020) 283 active collections, using the entomological nets, were also performed in all eight plots (20 x 150 m), four in the cerrado and four in the sunflower crop, in transects installed 100 m distance from pan traps. sampling was done in the plots corresponding to one of these distances (50, 150, 300 and 600 m) using the traps directed towards the interior border of the cerrado and interior edge of the crop in both the environments (cerrado and crop). collection was performed between 7 am and 2 pm, alternately, one day in the cerrado and one day in the crop. this time was selected as it marked the interval of the most intense floral visitation by the bees in the sunflower crop (free 1993). to implement this technique, three collectors spent 10 minutes in each plot to collect the bees. the collection time was timed to ensure that the sampling effort was done in approximately equal lengths of time. the bees were trapped using the net, beginning at the 50 m plot and ending at the 600 m plot in about 40 minutes. after a 20-minute interval, the same route was once again traversed (in the reverse direction). the specimens collected were identified to the lowest possible taxonomic level (genus and species) by the bee taxonomy specialists. some of the specimens were deposited in the inpa invertebrate collection, manausam, in the didactic collection of the mato grosso state university-cpeda research center, tangará da serra, mato grosso, brazil. fig 1. schematic representation of bee sampling using blue and yellow pan traps, set up at 50, 150, 300 and 600m from the border to the interior of the cerrado and from the edge to the interior of the sunflower crops. exclusion experiment an exclusion experiment was done to study the contribution made by the flower visitors to self-sustain the sunflower pollination. first, 20 plants were selected at 4 points viz., 50, 150, 300 and 600 m away from the cerrado border. at each point, 20 flower buds were isolated using white voile tissue (1 mm mesh) during the reproductive phase (r) of the sunflower, in the pre-flowering (r3-r4) stage. when the flowering ended (phase: r6-r7) the bags were removed to facilitate full seed development (ball et al., 1992). at about 40 days after flowering, when the seeds achieved full maturity (phase: r9), the sunflower chapters were collected and manually processed. next, in order to remove the moisture, the seeds were placed in a greenhouse at 60º c for 48 hours. the seeds of each chapter were then weighed in a precision analytical balance, to four decimal places. data analysis the normality of the data was tested, after which different statistical tests were applied. rarefaction analysis based on individuals was done to compare the patterns of species richness and sample effort in the cerrado and the crop. the two environments were compared based on a visual evaluation of the 95% ci (confidence interval) overlap of the rarefaction curves, implemented in estimates 7.5 (colwell, 2009). the student’s t test was used to compare (i) total species richness and (ii) total abundance of the individuals of the cerrado and that of the crop. the species composition of each environment was evaluated using the principal component analysis (pcoa), and the bray-curtis similarity index. the difference in species composition between the cerrado and that of the sunflower crop was tested using the permutative multivariate variance analysis (permanova) (anderson, 2001). mls almeida et al. – cerrado as habitat for sunflower pollinating bees284 the generalized linear model-glm (with poisson error distribution) was used to estimate the effect exerted by distance and habitat type (explanatory variables) on the richness and abundance of the bees (response variables) using the vegan package (oksanen et al., 2019). subsequently, the analysis of variance (anova) followed by tukey’s posthoc test were done to compare the differences in richness and abundance between the points (distance) of each habitat using the agricolae package (mendiburu, 2019). the anova followed by the tukey post hoc test were also performed to compare the seed weight of the chapters under the open and closed conditions for the pollinators. to evaluate the relationship between weight and distance, linear regression (lm), followed by anova were used. the efficiency of the collection methods was assessed with the kruskal-wallis (kw) test, since the data failed to meet the normality assumptions. the analyses were performed using the r 3.2.4 package (the development core team, 2017). results in this study, 901 individuals belonging to 31 genera and 54 species of bees were collected (table 1). among these, 680 individuals belonging to 25 genera and 46 species were collected from the sunflower crop and 221 individuals belonging to 28 genera and 50 species from the cerrado. species such as nannotrigona melanocera (schwarz), oxytrigona flaveola (friese), pseudaugochlora flammula (almeida), were recorded for the first time in the cerrado matogrossense. apis mellifera l. was abundant in the sunflower crop and cerrado (331 and 63 individuals, respectively) and represented more than 40% of the total number of bees collected. among the wild bees collected in the crop, melipona quinquefasciata (lepeletier) (n = 54) was the species with the highest abundance, followed by fig 2. accumulation curves of the species based on the individuals for the bee community in the cerrado and sunflower crop. the dotted lines are 95% ci, which indicate no significant difference between the two environments. geotrigona gr.mombuca (smith) (n = 36). of the 54 species collected in the area under study, 37 were found in both the sunflower crop and the cerrado. the species accumulation curve for the cerrado revealed no inclination to stabilize, suggesting that more species could be collected in this area. for the sunflower crop, the curve appeared to stabilize from 500 individuals (fig 2). the mean richness was significantly equivalent between both the environments (t = -1.148; p = 0.09; fig 3a) however, the number of individuals differed, with greater abundance being observed in the sunflower crop (t = -4.87; p = 0.0001; fig 3b). the results of the permanova test showed a significant difference in the species composition identified in the cerrado habitat from that found in the sunflower crop (f = 3.96; p = 0.001), as shown by pcoa (fig 4). fig 3. average and standard deviation of the wealth (a) and abundance (b) of bees in the cerrado and sunflower crop. means followed by different letters indicate significant difference (test t; p <0.05). sociobiology 67(2): 281-291 (june, 2020) 285 cerrado habitat sunflower culture distance (m) species -600 -300 -150 -50 50 150 300 600 total c ol le ct p ol le n an d n ec ta r apis mellifera (linnaeus, 1758) 8 4 20 31 102 89 45 95 394 augochlora sp.1 2 1 1 4 augochlora sp. 2 2 1 3 augochloropsis sp. 1 3 2 2 7 augochloropsis sp. 2 1 3 4 augochloropsis sp. 3 2 2 augochloropsis sp. 4 1 3 4 eulaema cingulata (fabricius, 1804) 1 3 4 8 eulaema nigrita (lepeletier, 1841) 1 6 4 11 exomalopsis sp. 1 3 4 exomalopsis analis spinola, 1853 2 7 9 exomalopsis fulvofasciata smith, 1879 2 2 2 12 18 geotrigona gr.mombuca (smith, 1863) 2 2 5 5 15 21 50 lasioglossum (dialictus) sp. 1 1 1 4 6 lasioglossum (dialictus) sp. 2 5 1 2 5 1 14 lasioglossum (dialictus) sp. 3 1 3 4 lasioglossum (dialictus) sp. 4 1 1 megachile sp. 1 2 7 3 1 13 megachile sp. 2 1 2 3 megachile sp. 3 3 5 8 megachile sp. 4 2 2 melipona quinquefasciata lepeletier, 1836 3 3 3 25 29 63 melissodes nigroaenea (smith, 1854) 1 4 4 6 15 melissoptila sp. 2 2 nannotrigona melanocera (schwarz,1938) 1 1 1 9 12 oxaea sp. 1 1 1 7 9 oxaea sp. 2 4 2 2 8 oxytrigona flaveola (friese, 1900) 2 1 9 12 pereirapis sp. 1 1 paratrigona lineata (lepeletier, 1836) 2 4 2 1 15 3 27 plebeia sp. 1 4 5 pseudaugochlora flammula almeida, 2008 1 1 thectochlora alaris (vachal, 1904) 1 1 1 3 trigona guianae cockerell, 1910 1 7 6 17 31 trigonisca sp. 2 4 1 1 8 xylocopa sp. 1 2 3 8 1 13 28 c ol le ct p ol le n an d o il centris aenea lepeletier, 1841 2 5 1 8 centris cf. fuscata lepeletier, 1841 1 3 4 centris cf. spilopoda moure, 1969 1 2 3 9 2 17 centris flavifrons (fabricius, 1775) 1 2 1 4 centris lutea friese,1899 2 2 centris nitens lepeletier, 1841 2 2 centris scopipes friese, 1899 1 6 1 2 10 centris tarsata smith, 1874 1 2 4 5 12 epicharis cf. analis lepeletier, 1841 1 7 4 12 epicharis cockerelli friese, 1900 1 2 3 5 11 epicharis inhering friese, 1899 1 1 1 1 4 epicharis cf. analis lepeletier, 1841 1 7 4 12 abundance of individuals 17 42 73 90 214 283 66 116 901 species richness 8 21 26 31 23 33 8 5 54 table 1. richness and abundance of bees found at distances of 50, 150, 300 and 600m from the cerrado habitats and sunflower crops in tangará da serra-mt. mls almeida et al. – cerrado as habitat for sunflower pollinating bees286 the factor distance from the border was seen to influence the richness and abundance of the bees in the cerrado (r2 = 0.53; p = 0.0001) and sunflower crop (r2 = 0.40; p = 0.0013). in the sunflower crop, the number of species was significantly higher at 150 m (tukey: p = 0.0012; fig 5a) and abundance was greater at 50 and 150 m (tukey: p = 0.0018; fig 5b). the points located at the extremes (300 and 600m) revealed a lower degree of richness and abundance of individuals in both the cerrado and sunflower crop (fig 5). methods of bee sampling the number of bees captured by the passive method was notably more than the number of species captured by the active method (f = 12.23; p = 0.0012; fig 6a). for abundance, the passive method was revealed to be more effective than the active one, in bee sampling (f = 14.26; p = 0.0016; fig 6b). fig 4. principal component coordination order (pcoa) of the bees sampled in the cerrado (closed symbols) and sunflower (open symbols) at different distances, based on the bray-curtis similarity. fig 5. average and standard deviations of the wealth (a) and abundance of bees (b) in the cerrado and sunflower crops at different distances from the border. means followed by different letters in the same habitat indicate significant difference by the tukey test (p <0.05). fig 6. number of species (a) and individuals (b) sampled using the active collection method (entomological net) and passive traps (pan traps). means followed by different letters show significant difference (kw p < 0.05). sociobiology 67(2): 281-291 (june, 2020) 287 however, while the specimens belonging to family megachilidae were captured solely by the passive method, the species centris cf. fuscata (lepeletier) were captured only by the active method. the active collection involved 210 hours of sampling effort. exclusion experiment the lack of pollinators in the sunflower chapters directly affected the crop yield. the exclusion experiment revealed that the total mass of the seeds was significantly higher in 49% of the open chapters for floral visitors, compared to the isolated chapters (tukey: f = 30.77; p = 0.0019; fig 7a). the distance from the sunflower crop to the cerrado border also affected the seed weight of this crop (r2 = 0.25; p = 0.001), which was significantly higher at the point installed at 150 m from the cerrado (tukey: f = 4.999, p = 0.0007; fig 7b). discussion in this study, the intense richness of the wild bee species observed reiterates that the cerrado bordering the agricultural tracts acts as a vital habitat for insects, because this ecosystem contains great structural diversity and supports one of the richest flora in the world (mendonça et al., 2008). to survive and forage in the agricultural terrains, wild bees must be able to occupy supportive habitats that provide suitable nesting areas and adequate food sources (westrich, 1996) the whole year through. the cerrado, being composed of a vegetation mosaic (oliveira-filho & ratter, 2002; silva et al., 2015) meets these requirements that according to ricketts et al. (2008) and garibaldi et al. (2016) are essential for maintaining bees and pollinating ecosystem service in adjacent crops. this study shows a higher abundance of bees in the sunflower crop, which offers additional support for the hypothesis that crops having mass flowering, (like the sunflower), may temporarily decrease the number of bees in the adjacent forests (montero-castaño et al., 2016). the reason for this is likely due to the abundance of pollen and nectar the sunflower crop offers which attracts several bee species, inducing them to leave their natural habitat and forage in the cultivated areas. it is noteworthy that sunflower is a monoculture of temporary resource, which does not provide all the nutritional requirements of the bees (naug, 2009); therefore, the maintenance of these bees in the cultivated environment is conditioned by the quality and quantity of the natural habitats that surround the crop (montero-castaño et al., 2016). results found in this search followed the pattern revealed in other studies on the sunflower pollinators, indicating that the exotic bee a. mellifera was the most abundant floral visitor (carvalheiro et al., 2011; pisanty et al., 2014; sardiñas & kremen, 2015; hevia et al., 2016). besides mass flowering, colony size and food demand are some other factors that could explain the predominance of these insects in the crop. a. mellifera requires large quantities of stored food to meet the needs of its extensive colonies (rollin et al., 2013). in consonance with previous studies (ricketts et al., 2008; carvalheiro et al., 2010, 2011) the distance negatively affected the richness and abundance of bees in the cerrado, but in sunflower crop effect was not negative. probably this result is associated with the irregular development of sunflower plants in the first 50 m of the crop. at this distance, many chapters were destroyed due to herbivory and, according to jacobsen and raguso (2018) flowers destroyed by the herbivores reduces the floral display, which the pollinating insects ignore, as they represent poor food resources. as they received fewer floral visitors, the seed weight of the sunflower chapters was also affected and unexpectedly larger in the point installed at 150 m distance from the cerrado. in this point, the plants displayed uniformity in the developmental pattern and the sunflower chapters were morphologically more attractive; this resulted in a higher visitation rate and, therefore, greater seed yield. fig 7. weight of the sunflower seeds with and without the bee visitors (a) at the different distances of 50, 150, 300 and 600 m (b) from the cerrado border. means followed by different letters indicate significant difference by the tukey test (p <0.05). mls almeida et al. – cerrado as habitat for sunflower pollinating bees288 in the cerrado, as well as in the sunflower crops, the points found at the extremes revealed lower levels of both richness and abundance of bees. native bees prefer to forage plants near their nests, and in this study we found nests of these bees on the edge of the cerrado, justifying the greater richness and abundance at the points closest to it. le feon et al. (2013) report that the borders of native areas may offer one or more vital habitats for a variety of wild bee species in the agricultural terrains, mostly when related to a mass flowering crop, such as sunflower. more numbers of bees could be collected in the cerrado and in the sunflower crops using the passive trapping method with pan traps than with the active entomological net. this could be due to the attraction (toler et al., 2005; wilson et al., 2008) and bee preference for particular colors (gumbert & kunze, 2001; heneberg & bogusch, 2014). in melitophilia, the colors yellow and blue (chittka & thomson, 2004) indicate the presence and quality of the resources, like nectar and pollen, for the bees (chittka & thomson, 2004). this is the plausible reason for the findings in this study for the effective use of pan traps. the active collection, including the sampling of less numbers of species even, must be done alongside the passive method, as species like c. fuscata could be trapped using the entomological net alone. the experiments conducted including and lacking the involvement of the floral visitors demonstrated that the high productivity of the mass of linoleic sunflower seeds is pollinator-dependent. in this study the 49% increase in the weight of the chapters open to pollinators compared to that of the closed ones, highlights the dependence of the sunflower by the pollinators. mallinger et al. (2018) documented a similar result revealing a 45% increase in the seed produced from the chapters open to the bee visitors. both results may encourage greater endeavor in wild bee conservation, especially of those species recognized for enhancing crop quality and the commercial value (klatt et al., 2013). from this study, it is evident that the conservation of the cerrado abutting onto a sunflower crop enhanced the crop productivity by the action of the pollinating bees. as these bees collected the pollen and nectar, they promoted crosspollination and thus boosted the yield of the linoleic cultivar h250. this proves that the maintenance of the brazilian cerrado directly contributes to bee conservation, as these insects are critical to the sunflower production, including that of the linoleic cultivar. acknowledgments the authors express their gratitude to josé victor alves ferreira for valuable assistance during the collections, to camila volff for planning the experimental design, and to the franciosi group for making the area available and supporting the research. they also thank rede-bioagro project for funding the research and the coordination for the improvement of higher education personnel (capes) for granting the scholarship and providing financial assistance through proap. mls almeida; gs carvalho; jr novais; d storck-tonon; ml oliveira; t mahlmann; ds nogueira; mjb pereira. author contributions design of the work: mls almeida, gs carvalho, mjb pereira; drafting the work: mls almeida, gs carvalho, jr novais, d storcktonon, ml oliveira, t mahlmann, ds nogueir, mjb pereira; data collect: mls almeida, gs carvalho, jr novais; analysis / interpretation of data: mls almeida; gs carvalho, d storcktonon; oversight and leadership responsibility for the research: mls almeida; gs carvalho, d storcktonon,; taxonomy: ml oliveira, t mahlmann, ds nogueira; final approval of the version to be published: mls almeida, gs carvalho, jr novais, d storck-tonon, ml oliveira, t mahlmann, ds nogueir, mjb pereira. references almeida ssp, louzada jnc (2009). estrutura da comunidade de scarabaeinae (scarabaeidae: coleoptera) em fitofisionomias do cerrado e sua importância para a conservação. neotropical entomology, 38: 32-43. anderson mj (2001). a new method for non parametric multivariate analysis of variance. austral ecology, 26: 32-46. doi: 10.1111/j.1442-9993.2001.01070.pp.x ball st, campbell gs, konzak cf (1992). pollination bags affect wheat spike temperature. crop science, 32: 1155-1159. benton tg, vickery ja, wilson jd (2003). farmland biodiversity: is habitat heterogeneity the key? trends in ecology and evolution, 18: 182-88. doi: 10.1016/s0169-5347(03)00011-9 brittain c, kremen c, klein am (2013). biodiversity buffers pollination from changes in environmental conditions. global change biology, 19: 540-547. doi: 10.1111/gcb.12043 cameron sa, lozier jd, strange jp, koch jb, cordes n, solter lf, griswold tl (2011). patterns of widespread decline in north american bumble bees. pnas usa, 108: 662-667. doi: 10.1073/pnas.1014743108 carvalheiro lg, seymour cl, veldtman rn, susan w (2010). pollination services decline with distance from natural habitat even in biodiversity rich areas. journal of applied ecology, 47: 810-820. doi: 10.1111/j.1365-2664.2010.01829.x carvalheiro lg, veldtman r, shenkute ag, tesfay gb, pirk cww, donaldson js, nicolson sw (2011). natural and withinfarmland biodiversity enhances crop productivity ecology letters, 14: 251-259. doi: 10.1111/j.1461-0248.2010.01579.x sociobiology 67(2): 281-291 (june, 2020) 289 chittka l, thomson jd (2004). cognitive ecology of pollination: animal behavior and floral evolution. 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known worldwide (bolton, 2012). prionopelta kraepelini, reported here marks the first record of genus prionopelta from india. it is one of the most widely distributed species in the genus, being found from sumatra and peninsular malaysia east through the philippines and micronesia to samoa (shattuck, 2008). forel (1905) described prionopelta kraepelini based on worker and queen castes; shattuck (2008) carried the revision of prionopelta in the indo-pacific region. here we report its hitherto un-described male caste. the discovery is significant in backdrop that specimens were collected only in fragmented habitat of shivalik. therefore, further molecular studies are required to reveal the status of prionopelta kraepelini whether it is an introduced, tramp or native species. materials and methods the specimens were collected by hand picking in foothills of indian himalaya, the shivalik range. the taxonomic analysis was conducted on nikon smz 1500 stereomicroscope. for digital images, mp evolution digital camera department of zoology and environmental sciences, punjabi university, patiala – 147002 india e-mails: himenderbharti@gmail.com; aijaz_shoorida@yahoo.co.in 816 sociobiolog y vol. 59, no. 3, 2012 was used on the same microscope with auto-montage (syncroscopy, division of synoptics, ltd.) software. later, images were cleaned with adobe photoshop cs5. morphological definitions for measurements (accurate to 0.01 mm) include: tltotal outstretched length of a specimen, from mandibular apex to gastral apex. hlmaximum head length in full-face (dorsal) view, measured from the anterior-most point of the clypeal margin to the posterior-most point of the head proper. hwmaximum head width in full-face (dorsal) view. sllength of the scape (first antennal segment) excluding the basal neck and condyle. elmaximum measurable eye length of facetted part of eye. wlmesosomal length measured from the anterior surface of the pronotum proper (excluding the collar) to the posterior extension of the propodeal lobes. plmidline length of the petiolar node (excluding the anterior peduncle) in dorsal view. pwwidth of the petiolar node in dorsal view. t1wwidth of first gastral (third abdominal) tergite in dorsal view. cicephalic index: hw/hl x 100. pipetiolar index: pw/pl x 100. sscape index: sl/hw x 100. systematics prionopelta kraepelini forel (figs. 1-9) prionopelta kraepelini forel, 1905: 3 (w.q.) indonesia ( java). see also: shattuck, 2008: 23. global distribution caroline islands, fiji, indonesia, malaysia, philippines, samoa, sumatra and taiwan. 817 bharti, h & a. a. wachkoo — first record of prionopelta kraepelini worker diagnosis (figs. 1-3) sculpturing on dorsum of pronotum consisting of fine punctations which contrast markedly with widely spaced foveae on mesonotum and propodeum, the foveae on the propodeum varying in density across its width (weakest medially, stronger laterally). head width less than 0.48mm. petiole relatively narrow, petw less than 0.21 (shattuck 2008). worker measurements tl 1.78-2.07; hl 0.47-0.50; hw 0.36-0.39; sl 0.25-0.28; wl 0.55-0.59; fig. 1. dorsal view of the head of a worker of prionopelta kraepel 818 sociobiolog y vol. 59, no. 3, 2012 pl 0.14-0.16; pw 0.19-0.22; t1w 0.29-0.33. indices: ci 74.59-78.73; si 66.36-72.79; pi 133.71-142.31 (n=9). description of male (figs. 4-6) head as broad as long, including the large convex compound eyes. mandibles slender, curved, strap-like, the apex simple and acute; their tips overlap and the entire blades are tucked away under the clypeus in such a way that only figs. 2-3, dorsal and lateral views of prionopelta kraepelini workers. 819 bharti, h & a. a. wachkoo — first record of prionopelta kraepelini their external margins show externally along the anterior clypeal border that frequently carries serially arranged denticles homologous to that of worker. antennae slender, 13 segmented; antennal scrobe absent. notauli present and distinct; forming a complete y. mesepimeron without posterodorsal (epimeral) lobe. jugal lobe of hind wing absent. claws simple. petiolar node in general shape as in worker, but more slender. subpetiolar process reduced and fenestra within it almost absent in contrast to conspecific workers. gaster elongated; terminalia retracted; pygidium broadly rounded. fig. 4. dorsal view of the head of the male of prionopelta kraepelini. 820 sociobiolog y vol. 59, no. 3, 2012 color blackish, darker than in corresponding workers; sculpture foveate, foveae much better developed than in workers, coarser over the head and figs. 5-6. dorsal and lateral view of the male of prionopelta kraepelini. 821 bharti, h & a. a. wachkoo — first record of prionopelta kraepelini mesosoma, fine on gaster. pilosity dense, reclinate, suberect; longest on anteriormost part of clypeus and apex of gaster; longer than in workers. male measurements: tl 2.43; hl 0.48; hw 0.49; sl 0.15; el 0.21; wl 0.68; pl 0.18; pw 0.20; t1w 0.31. indices: ci 102.08; si 30.61; pi 111.11 (n=1). local distribution and habitat: this species gives the impression that at it is uncommon in the shivalik range of northwest himalaya. this is a cryptic species and was encountered under stones found in fragmented habitats and along roadside. apparently, it was not collected from forested habitats of the region. material examined himachal pradesh: andretta, 940m, 1 , 11.vi.2010; dattal, 940m, 4 , 16.vi.2010; nagabari, 420m, 2 , 1 , 18.vi.2009. jammu & kashmir: jasrota, 400m, 2 , 28.vii.2010 (coll. aijaz a. wachkoo). remarks: prionopelta kraepelini is recorded for the first time from india. the male of this species is also described for the first time. acknowledgment financial assistance rendered by ministry of environment and forests (grant no. 14/10/2007-ers/re), govt. of india, new delhi is gratefully acknowledged. references bolton, b. 2012. bolton’s catalogue and synopsis version: 1 january 2012. downloaded from http://gap.entclub.org/ on 10 january 2012. bolton, b., g. alpert, p. s. ward & p. naskrecki 2007. bolton’s catalogue of ants of the world: 1758-2005 [cd-rom]. harvard university press, cambridge, massachusetts. forel, a. 1905. ameisen aus java. gesammelt von prof. karl kraepelin 1904. mitteilungen aus dem naturhistorischen museum in hamburg 22: 1-26. shattuck, s. o. 2008. revision of the ant genus prionopelta (hymenoptera: formicidae) in the indo-pacific region. zootaxa 1846: 21-34. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i4.5658sociobiology 67(4): 501-507 (december, 2020) introduction increased habitat fragmentation and habitat loss, and the consequent decline in species richness, due to anthropogenic activities can have direct and indirect effects on ecological processes, such as diaspore removal by ants (myrmecochory) (christianini et al., 2007; bieber et al., 2014). highlighted among the direct effects are changes in the composition of ant assemblages, which could involve a decrease or loss of highquality diaspore removing species and increased removal by generalist species, with a negative effect on removal success (zelikova & breed, 2008; leal et al., 2014). however, there are cases where anthropogenic disorders can be related to abstract diaspore removal by ants is a crucial stage for successful myrmecochory and can be directly or indirectly affected by natural or anthropic changes to environments. among the consequences of such changes is variation in habitat attributes, such as changes in conditions and resources and, consequently, decreased diaspore removal or even the loss of this ecological process. the aim of this study was to assess whether canopy and litter cover affect diaspore removal by ants in the cerrado. we considered canopy and litter cover as proxies of humidity and temperature and evaluated whether changes in these environmental conditions could affect diaspore removal by ants. we hypothesized that the greater the canopy and litter cover (higher humidity and lower temperature), the smaller the number of diaspores removed by ants, since cerrado is an open environment. we tested this hypothesis by establishing three classes of cover for each proxy: low, intermediate, and high. we placed artificial diaspores under each cover class and quantified the number of diaspores removed. we found that variation in canopy and litter cover did not affect the number of diaspores removed by ants in studied areas of cerrado sensu stricto. we suggest that variation in habitat attributes in natural environments were less important for diaspore removal than in modified areas. our results indicate that understanding the processes and habitat attributes involved in diaspore removal by ants is important for conserving the cerrado. sociobiology an international journal on social insects ma rabelo1, ma angotti2,3, gs silva1, ac reis1, cr ribas2 article history edited by kleber del-claro, ufu, brazil received 22 july 2020 initial acceptance 14 september 2020 final acceptance 09 november 2020 publication date 28 december 2020 keywords temperature, humidity, vegetation cover, diaspore removal, myrmecochory, conservation. corresponding author mariana azevedo rabelo https://orcid.org/0000-0003-1425-0337 ppg em ecologia aplicada, setor de ecologia e conservação, laboratório de ecologia de formigas, instituto de ciências naturais, departamento de biologia, universidade federal de lavras po box 3037, cep 37200-900, lavras, minas gerais, brasil. e-mail: rabeloama@gmail.com variance in the general composition of dispersing ants, but not to their abundance and to the service provided by high-quality removers (oliveira et al., 2019). among indirect effects, decreased diaspore dispersion can affect the distribution and structure of vegetation (del toro & ribbons, 2019), and contribute to diaspore predation by other species (santana et al., 2013). for ants, the main indirect effects of habitat disturbance are associated with effects on habitat structure, microclimate, resource availability and competitive interactions (andersen, 2018). therefore, environmental alterations initiate changes in ecological dynamics and interactions among species and between species and their environment (oliver et al., 2016). ants assume an 1 universidade federal de lavras, programa de pós-graduação em ecologia aplicada, departamento de biologia, lavras-mg, brazil 2 universidade federal de lavras, laboratório de ecologia de formigas, departamento de biologia, lavras-mg, brazil 3 instituto federal de educação, ciência e tecnologia de mato grosso do sul – campus ponta porã, ponta porã, mato grosso do sul, brazil. research article ants canopy and litter cover do not alter diaspore removal by ants in the cerrado ma rabelo, ma angotti, gs silva, ac reis, cr ribas – diaspore removal by ants in the cerrado502 important role in diaspore dispersion since they act not only as primary dispersers, but also as secondary dispersers for nonmyrmecochorous diaspores (anjos et al., 2020). the removal of diaspores is possibly one of the most important steps in the dispersion process, as the type of interaction can be crucial for the viability, germination and establishment of the seedling (gallego et al., 2014; magalhães et al., 2018). most research on myrmecochory, either in natural or modified habitats, has identified factors that explain dispersal distance (andersen & morrison, 1998; gómez & espadaler, 2013), and size, quality, and quantity of removed diaspores (leal et al., 2014). however, little has been discussed about how environmental conditions, that are linked to environmental variation, can influence the process of diaspore dispersion (warren et al., 2012; del toro & ribbons, 2019). finding and removing diaspores is crucial for successful myrmecochory and so it is important that the ant assemblage of a given area is capable of performing this ecological process. at a local scale, however, habitat heterogeneity and complexity may influence ant species richness. ant species richness responds to microclimatic factors (kaspari et al., 2004; weiser et al., 2010), with temperature being one of the most relevant to the distribution of species, and they can influence foraging behaviors related to the physiological tolerances of species (fitzpatrick et al., 2014). changes in habitat complexity, such as variation in vegetation structure (gibb & parr, 2010), canopy and litter cover and tree density, affect the richness and composition of ant assemblages (neves et al., 2013). increased canopy cover decreases the incidence of sunlight and, consequently, alters the microclimate, which influences the availability, quality, and quantity of resources (kaspari et al., 2004). shaded areas can provide more suitable conditions for arthropod communities due to a more stable microclimate and higher resource availability (levings, 1982). however, myrmechocory can be favored in open and dry environments, and higher removal rates are expected in these environments where this interaction has coevolved more often (lengyel et al., 2009; lengyel et al., 2010). ants are considered the main removers of nonmyrmecochorous diaspores and a great portion of ground-dwelling ant communities are involved in this process (pizo & oliveira, 2001; passos & oliveira, 2003). changes in the amount of litter cover, the main component of the epigeic stratum (yanoviak & kaspari, 2000), can affect the diversity and distribution of ground dwelling ants (cardoso & schoereder, 2014). such changes in litter cover can also affect microclimatic conditions, such as temperature, humidity, and solar incidence (ahuatzin et al., 2019). the rate of diaspore removal by ants can be 10% higher in cerrado areas than in cultivated areas (rocha-ortega et al., 2017), which reinforces the importance of this biome in the maintenance of ecological processes (rabello et al., 2018). how changes in habitat heterogeneity and complexity affect diversity of ant assemblages is well described in the literature (palfi et al., 2017; queiroz et al., 2017). nevertheless, how habitat heterogeneity and complexity affect the ecological functions performed by ants, especially the removal and dispersion of diaspores, is not yet clear. consequently, the relationships among temperature, humidity and diaspore removal are not well explored. considering that environmental anthropization can change vegetation cover and microclimatic conditions, it is likely that such environmental changes could also negatively affect the ecological process of diaspore removal by ants. in this context, the aim of our work was to evaluate whether changes in environmental conditions related to humidity and temperature (estimated by canopy and litter cover) could affect diaspore removal by ants in natural habitats. we hypothesized that the greater the canopy and litter cover, the smaller the number of diaspores removed by ants, since cerrado is an open environment. methods study area the study was carried out on march 2016 in an area of cerrado sensu stricto (15º 26’ 00” s, 44º 49’ 19” w), characterized by heterogeneous habitats, composed of herbaceous, grasses, shrubs and scattered trees that provide different vegetation structures (oliveira-filho & ratter, 2002). the area is part of área de proteção ambiental do rio pandeiros (apa rio pandeiros), municipality of januária, northern minas gerais state, brazil, considered the largest conservation unit in the state, with 396.060,407 hectares. the climate of the area is semiarid with a mean annual temperature between 18.4 and 30.9 ºc and a mean annual precipitation of 903.0 mm (jardim & moura, 2018). the region is under many environmental pressures involving anthropogenic activities, such as vegetation loss, fire, monocultures (eucalyptus), pasture, and charcoal production (nunes et al., 2009). canopy and litter measurements to determine whether the number of diaspores removed by ants is influenced by environmental conditions and resources, we considered canopy and litter cover as proxies for such environmental characteristics, temperature and humidity. litter cover was measured as the percentage of covered soil (organic matter, twigs, sticks, leaves, and diaspores), considering only visual covering, and not measuring litter heterogeneity or volume. to do this we threw a 25 x 25 cm wire quadrat, that was subdivided into four quadrants, onto the soil and estimated the percentage of covered soil in the quadrat (modified from queiroz et al., 2013). we measured canopy cover by estimating the percentage of shaded quadrats using a concave forest densiometer (terrages). we established three classes of covering for each proxy to guarantee natural variation of measurements: 0 to 33% for low cover (lc), 34 to 66% for intermediate cover (ic), and 67 to 100% for high cover (hc). we then selected 15 sample points for each class, ensuring that they were 20 meters apart from each other, such that each independent variable, canopy and litter cover, had 45 sample points. sociobiology 67(4): 501-507 (december, 2020) 503 diaspore removal we placed 20 artificial diaspores at each sample point, which were plastic beads (0.03 g and 2 mm diameter) covered with an attractive paste (75% hydrogenated vegetable fat, 7% casein, 5% flavorless maltodextrin, 4.8% fructose, 4.7% glucose, 3% calcium carbonate and 0.5 % sucrose) (based on raimundo et al., 2004; bieber et al., 2014; rabello et al., 2015). the diaspore’s size allows both small and large ant species to perform diaspore removal (pizo & oliveira, 2001; leal et al., 2014; anjos et al., 2020). in regard of the attractive portion of artificial diaspores, its composition is chemically similar to natural ones that are attractive to ants. ants are attracted to diaspores with different proportions of lipids, either for myrmechocorous, or non-myrmechocorous diaspores (pizo & oliveira, 2001; leal et al., 2014). we used artificial diaspores since we could not find any plant species with enough arillate diaspores (nonmyrmecochoric) to perform the study. nonetheless, studies on diaspore removal and dispersion have shown that artificial diaspores are suitable alternatives for studies of myrmecochory (bieber et al., 2014; angotti et al., 2018), since they allow the replication of experiments and maintain the uniformity of physical and qualitative characteristics of diaspores. we provided a total of 300 artificial diaspores for each sampled class (lc, ic, and hc). both for litter and canopy covers. the diaspores were made available from 07h00 to 10h00 am at sample points that were protected by wire mesh (20 x 20 cm, 1.5 cm mesh) to avoid removal or predation by other invertebrates and vertebrates (pizo & oliveira, 2001; rabello et al., 2015; angotti et al., 2018). we quantified the number of diaspores at each point at the end of the exposure time to calculate the quantity removed by ants for each cover class. statistical analyses we built generalized linear models (glm’s), with quasibinomial error distribution, for each variable (canopy and litter cover) separately to evaluate the proportion of diaspores removed by ants in the three established classes cover ((lc, ic, and hc) (crawley, 2013). the proportion of removed diaspores (dependent variable) was related to canopy and litter cover (independent variables) in two independent models. we performed a residual analysis to verify the distribution and fit of the models, considering a significance probability of p < 0.05 for all analyses. all analyses were performed with r software (r development core team 2015). results of the 300 artificial diaspores provided for each canopy cover class, 253 diaspores were removed for lc, 278 for ic, and 231 for hc. of the 300 provided for each litter cover class, 246 were removed for lc, 243 for ic, and 228 for hc (table 1). however, the proportion of diaspores removed by ants classes of covering % seeds removed mean sd canopy cover low cover (lc) 84.33 16.86 5.65 intermediate cover (ic) 92.67 18.53 4.24 high cover (hc) 77 15.40 0 litter cover low cover (lc) 81.66 15.20 4.94 intermediate cover (ic) 81 16.20 0 high cover (hc) 76 16.40 2.82 table 1. variation in the removal of diaspores by ants by classes of canopy and litter cover: 0 to 33% (lc), 34 to 66% (ic) and 67 to 100% (hc) in cerrado stricto sensu areas. fig 1. proportion of artificial diaspores removed by ants under different (a) canopy cover class and (b) litter cover class in cerrado areas. was neither affected by canopy cover (f = 1.2; df = 42; p = 0.3) (fig 1a) nor litter cover (f = 0.1; df = 42; p = 0.9) (fig 1b). thus, our hypothesis, that areas with greater canopy cover (proxy for temperature and humidity) and greater litter cover (proxy for temperature, humidity, and resources) would have smaller diaspore removal, was rejected. ma rabelo, ma angotti, gs silva, ac reis, cr ribas – diaspore removal by ants in the cerrado504 discussion among the many changes that can occur with environmental conditions, we investigated those related to how canopy and litter cover affect the removal of nonmyrmecocoric diaspores by ants in natural areas of cerrado. we found both canopy and litter cover to have no effect on diaspore removal, which differed from our predictions. at first, our results show that the percentage of canopy and litter cover does not interfere quantitatively in the removal of diaspores. this reinforces the importance of ants as one of the main secondary removers of diaspores in the cerrado soil (christianini et al., 2012; magalhães et al., 2018). however, we cannot affirm that variations in canopy and litter cover, considered here as a proxy for temperature, humidity and resource, interfere in the quality of this process, as we do not collect the ants that performed the removals to find out if there is variation in the composition and the quality of the removing species. although we did not find an influence of the different coverages in the removal, the amount of diaspores removed was greater at the points where we evaluated canopy coverage than at the points evaluated for litter cover. what reinforces that ants possibly have different responses according to the environmental condition evaluated (weiser et al., 2010). we observed that areas with higher and lower proportions of litter cover had similar diaspore removal. this finding contradicts our prediction since variation in litter cover did not have an effect on the ability of ants to find and remove diaspores in our study. assessments of the seed dispersal process in natural and post-disturbance environments point to an increase in the rate of removal by ants in more open environments (andersen & morrison, 1998; batisda & tavalera, 2002), with the impacts on the ant community being greater in these environments than indoors (andersen, 2018). the rate of removal of diaspores and the composition of species of removing ants in savanna environments varies between open and closed areas (andersen & morrison, 1998), with different land uses (rabello et al., 2018) and different phytophysiognomies (gallegos et al., 2014; magalhães et al., 2018). unlike our study, these studies evaluate and compare the dispersion process between different phytophysiognomies or in environments that have suffered some type of disturbance, where the conditions of the habitat are markedly different and can compromise the service of diaspore removal (leal et al., 2014). habitats that have gone through changes in land use and, consequently, changes in their attributes, show negative effects on diaspore removal by ants, such as over a gradient of tree cover (rabello et al., 2018). the conversion of native forests into pasture, planted forests, and agroecosystems is highlighted as one of the main causes for declines in ecological functions (philpott & armbrecth, 2006; rabello et al., 2018). but is important to point out that in natural open environments, like cerrado, higher removal rates are expected in habitats with smaller canopy and litter cover. an important factor to be considered is that the microclimate conditions provided by the different canopy and litter cover can be within the thermal tolerance range of the removing species. the effects of microclimate variation, such as rising temperatures, have different effects on the ant community and depend on the species’ thermal tolerance (roeder et al., 2018). it is worth mentioning that we evaluated (although indirectly) the removal in relation to variations in conditions that occur in the area along years, differently from microclimate variations that occur throughout the day. possibly, if we followed the removal along the daily temperature variation, we could verify if there are effects on the rate of diaspores removal throughout the day (beamount et al., 2009; angotti et al., 2018), but this is not our intend in this study. biotic conditions and microclimate variations have daily and seasonal variation and can interfere with interactions between species and resources, with foraging activity directly related to temperature (hemmings & andrew, 2017). hemmings and andrew (2017) found that the body temperature of the ants showed no difference when they were on the ground and a nearby trunk, but varied between summer and winter. this suggests to us that perhaps the seasonal temperature variation is more important to alter the seed removal activity than the microclimate variation in a natural environment and with the same phytophysiognomy, which would explain the lack of effect of canopy and litter cover in our results. it is well studied that habitat heterogeneity is an important factor for the composition of ant assemblages (neves et al., 2013), and in some cases habitat differences have greater influences on community composition than on species richness (pacheco & vasconcelos, 2012). therefore, studies on the composition of diaspore remover assemblages can indicate the quality of ant removers and their possible role in successful diaspore dispersion (rabelo et al., submitted). the variation in the proportion of canopy opening can contribute to the variation in the composition of the ant community in the area and in the quality of the removing species (leal et al., 2014). the lack of influence of canopy and litter cover on the removal found in our study, may be related to the adaptation of ants to variations in local conditions and microclimate. as a mosaic of phytophysiognomies, and with continuous or discontinuous canopy cover (ribeiro & walter, 1998), the cerrado provides variation in soil shading and the amount of litter, as observed in our study. in addition, interactions between ants and diaspores are generally opportunistic, being carried out by a range of omnivorous, carnivorous and fungivorous species (christianini et al., 2012). thus, the interactions between the ants and the diaspores we offer may have been opportunistic and been performed by high-quality removing species (leal, et al., 2014), such as ectatomma edentatum (roger, 1863), ectatomma opaciventre (roger, 1861) or ectatomma brunneum (smith, 1858), which have been collected in other studies in the same region (rabelo et al., in submitted). because these species are larger, more agile and forage alone, they are more capable of traveling through different substrates. for smaller species, sociobiology 67(4): 501-507 (december, 2020) 505 such as pheidole jelskii (mayr, 1884) and pheidole capillata (emery, 1906) (rabelo et al., in submitted), litter thickness usually acts as a barrier (leaves, sticks, and fruits), hindering the foraging activity of ants (farji-brener et al., 2004) and, thus, decreasing diaspore transport. therefore, we highlight the importance of conserving the integrity of natural environments so that a full understanding of their processes and habitat attributes involved with diaspore removal by ants can be obtained. we also suggest further investigations of the interactions between seasonal variance and microhabitats. studies of natural habitats may help us to understand the success of ecological functions and to identify the habitat attributes that are most important for preserving in order to guarantee ecological functions in both natural and modified habitats. acknowledgements we thank the employees at área de proteção ambiental do rio pandeiros for their accommodation support during field work, and to members of laboratório de ecologia de formigas for discussions and contributions to this work. we thanks to suggestions from two anonymous referress in the previous version. this study was funded by the project apq03593-12 “desenvolvimento de ferramenta para priorização de descomissionamento de pequenas centrais hidrelétricas (phc) no estado de minas gerais e estudo de caso para a pch pandeiros”, which was a partnership between fundação de amparo à pesquisa de minas gerais (fapemig) and companhia energética de minas gerais s. a. 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(2010). canopy and litter ant assemblages share similar climate–species density relationships. biology letters, 6: 769-772. doi: 10.1098/rsbl. 2010.0151 1269 ecology of vespidae (hymenoptera)predators in coffea arabica plantations by marcelo coutinho picanço1, ivênio rubens de oliveira2, flávio lemes fernandes3*, hermínia emília prieto martinez4, leandro bacci5 & ézio marquez da silva3 abstract social vespidae exhibit control of leucoptera coffeella (lepidoptera: lyonetiidae) in brazil. the objective was to determine the ideal unit for sampling of predaceous vespidae in coffee crops in the vegetative and reproductive phases. this research was conducted in two coffee plantations in viçosa, mg. the factors being studied were: crop phase, canopy thirds, branch type, exhibition side of the plant to solar light and the position of the leaf on the branch. the number of predation mines by vespidae on all the leaves of each evaluated plant was recorded. in coffee plants in the vegetative phase the best sampling unit of the vespidae was the 5th or 6th pair of leaves on the primary plagiotropic branches of the median third of the canopy. in coffee plants already in the reproductive phase the best unit for sampling vespidae were leaves on the third apical of the 4th or 6th pair of leaves on primary plagiotropic branches on the plant face exposed to the sun in the afternoon period or on the median third on the 5th pair of leaves of the plant face exposed to the sun in the afternoon period. key words: social insects, coffea arabica, predator, sampling, coffee leaf miner, leucoptera coffeella. 1departamento de entomologia, universidade federal de viçosa, campus de viçosa, s/n, 36570-000 viçosa, mg, brasil 2empresa brasileira de pesquisa agropecuária, embrapa tabuleiros costeiros, av. beira mar, 3250 sementeira49025-040 aracaju, se, brasil 3instituto de ciências agrárias, programa de pós-graduação em produção vegetal, universidade federal de viçosa, campus de rio paranaíba, 38810-000 rio paranaíba, mg, brasil 4departamento de fitotecnia, universidade federal de viçosa, campus de viçosa, s/n, 36570-000 viçosa, mg, brasil 5universidade federal de sergipe, centro de ciências biológicas e da saúde, departamento de engenharia agronômica, av. marechal rondon, s/n, cidade universitária prof. “josé aloísio de campos” jardim rosa elze, 49100-000 aracaju, se, brasil author for correspondence: flaviofernandes@ufv.br 1270 sociobiolog y vol. 59, no. 4, 2012 introduction social vespidae have nests built in trees or in the ground. egg-laying is executed by the founding queen of the nest which due to its dominant activity obstructs the egg-laying or even destroys the eggs produced by other queens. the workers have protective, nest expansion, raising newborn (eggs, larvae and pupas) and foraging roles.foraging is carried out during the day to collect nectar and other insects. these sources will be used as food for larvae and adults (giannotti et al. 1995; raveret richter 2000). social vespidae capture insects of several orders, mainly lepidoptera larvae (matsuura 1968; akre 1982; caron & schaefer 1986; raveret richter 1990). vespidae predators have an important role in the natural control of populations of pest-insects in forest and crops like coffee. coffee crops shelter a great diversity of vespidae predators like: brachygastra lecheguana latr., eumenes sp., polistes versicolor (oliv.), polybia occidentalis oliv., polybia paulista ihering, polybia scutellaris white, protonectarina sylverae saussure, protopolybia spp. and synoeca cyanea l. these social wasps exercise great control on one of the main pests of this crop, the coffee leaf miner leucoptera coffeella (guer.mènev.) (lepidoptera: lyonetiidae) (nogueira neto 1940; fernandes et al. 2008; fernandes et al. 2009). during the day the adults of these wasps fly over the coffee plants searching for leaves that have mines made by l. coffeella caterpillars. when the wasps locate a mine they check if it has caterpillars inside. if it does, they tear the mine open with their oral appliance, capture the caterpillars, grind them and transport them to their nests. so, mines that suffer predation show tears which makes the evaluation of the populations of these predator insects possible (pereira et al. 2007a,b). one of the relevant aspects in field studies of social vespidae is determining appropriate place of occurrence to evaluate their populations. these studies are of great importance in basic ecolog y works as well as in applied ecolog y (tibbetts 2007; fernandes et al. 2008). in selecting the place of occurrence to be used in the evaluation of natural populations it is necessary that it be representative and makes fast sampling possible. according to the criterion for representativeness the best place of occurrence is that which correlates more with absolute density. however, this criterion is often not used in determining the sampling unit to create 1271 picanco, m.c. et al. —ecolog y of vespidae predators in coffea arabica sampling plans for social vespidae. this is due to the difficulty in determining the absolute density of these insects, especially in large plants like the coffee plant that, due to its many leaves, makes determining their absolute densities difficult (gusmão et al. 2004; bacci et al. 2006; moura et al. 2007). in evaluations of social vespidae populations it is possible to evaluate the number of nests or the adult population. in the evaluation of the adult population the applicable variables are density (obtained by direct counting or traps) or predation rate. in the evaluation of these insects as predators, the use of predation rate is the most appropriate form of monitoring these populations in natural environments (fernandes et al. 2008). as such, this work had as its aim the determining of the place of occurrence to assist in studies of social vespidae predators in coffee crops in the vegetative and reproductive phases. materials and methods this inquiry was carried out in two c. arabica fields on the universidade federal de viçosa, viçosa, minas gerais state, brazil. this period was used because of the greater attack frequency of l. coffeella on coffee plants and also because the predation rate of this insect by social vespidae is greater (pereira et al. 2007a,b). the crops were not irrigated and the cultural treatments were carried out as described by zambolim (2001). the plants were of the “catuaí” variety and the spacing used was 3 x 1 m. the plants in these crops were in both the vegetative phase and the reproductive phase. in the crop in the vegetative phase the plants were nearly 19 months old at the beginning of the evaluations and occupied about 30 ha. the crop in the reproductive phase of the was nearly seven years old and occupied around 70 ha. the vegetative phase of the coffee plant does not produce fruit, but at about 30 months old it produces its first harvest (plant in reproductive phase). the factors being studied were the phases of crop (vegetative or reproductive), thirds of the canopy (apical, median or basal), type of branch (primary or secondary plagiotropic), exposure of the plant to solar light (exposure of the face of the plant to the sun during the morning or afternoon periods) and the positions of the leaf on the branch (1st to the 8th pair of leaves totally expanded from the apex of the branch). in each crop the number of mines that received predation by adult social vespidae on all the leaves of each evaluated 1272 sociobiolog y vol. 59, no. 4, 2012 plant were recorded. for each one of these leaves the canopy thirds, the type of branch, the exposure of the plant to solar light and the position of the leaf on the branch were recorded. thirty four plants were evaluated in the crop in the vegetative phase and 43 plants in the crop in the reproductive phase. this was the maximum number possible to evaluate using 10 people, since the plants had up to 5,000 leaves and each person was able to evaluate one plant per day. the evaluated plants were apced equally in the crop in order to obtain systematized sampling points (bacca et al. 2006; moura et al. 2007). absolute densities were calculated (number of mines that received predation by plant) and the relative densities in each sampling unit (number of mines that received predation/sampling unit). in order to select the ideal unit for sampling of the social vespidae the criteria of representativeness and sampling speed were used. based on the criterion of speed, samples that presented at least a 20% frequency so that localization was fast were selected. by the criterion of representativeness samples whose relative densities correlated with absolute density were selected. for all of these, pearson correlations were calculated between absolute and relative densities in each sampling unit. ideal samples were considered those that presented a significant correlation in the t test (p<0.05). when more than one sampling unit presented significant correlation, the relative densities were subjected to the simple linear regression analysis of p<0.05 as a function of absolute densities. an ideal sample was considered as one whose curve presented a greater inclination. these proceedings were proposed by podoler & rogers (1975) to select the phase or factor of mortality better represented by the variation of total mortality in the studies of ecological life charts. as such, in the present work the use of these statistical methods in selecting a unit to create a sampling plan based on the criterion of representativeness is proposed, since it is intended to select which sampling unit better represents the variation in absolute density. results plants in vegetative phase positive and significant correlations were verified (p<0.05) between absolute densities with relative densities of vespidae predators in the three thirds of the canopy. the regression curve for predation on leaves in the median 1273 picanco, m.c. et al. —ecolog y of vespidae predators in coffea arabica third presented a greater inclination than the curves of the apical and basal thirds (table 1). therefore, in the vegetative phase coffee crops the sampling of social vespidae predators should be carried out on leaves from the median third of the canopy. table 1. pearson’s correlation between the absolute density of vespidae predators (mines predates/ leaf ) with the relative densities in the sampling units and regression curve of these relative densities as a function of the absolute densities in vegetative phase coffee plants. sampling unit pearson’s correlation regression curve r t p intercept inclination r2 f p canopy apical 0.6 4.47 <0.0001 0.002 0.91 (0.71-1.11) 0.4 19.95 <0.0001 median 0.9 9.15 <0.0001 -0.013 1.75 (1.56-1.94) 0.7 83.95 <0.0001 basal 0.6 4.4 <0.0001 0.003 0.72 (0.56-0.88) 0.4 19.36 0.0001 plant face exposed to the sun morning period 0.8 7.42 <0.0001 -0.23 0.80 (0.70-0.91) 0.6 55 <0.0001 afternoon period 0.6 4.22 0.0001 2.08 0.86 (0.66-1.07) 0.4 17.84 0.0002 type of branch primary plagiotropic 0.9 10.4 <0.0001 0.01 1.23 (1.11-1.35) 0.8 108 <0.0001 secondary plagiotropic 0.2 1.17 0.1264 0 0.11 (0.01-0.21) 0 1.36 0.2529 position of the leaf on the branch 1st 0.1 0.57 0.2852 * * * * * 2nd 0 0.24 0.4062 * * * * * 3rd 0.5 3.54 0.0006 -1.75 0.72 (0.52-0.92) 0.3 12.52 0.0013 4th 0.5 2.93 0.0031 -0.17 0.93 (0.61-1.25) 0.2 8.61 0.0061 5th 0.6 3.76 0.0003 0.09 2.06 (1.51-2.61) 0.3 14.12 0.0007 6th 0.4 2.11 0.0215 2.98 1.46 (0.76-2.16) 0.1 4.44 0.043 7th 0.6 3.97 0.0002 -1.03 4.12 (3.08-5.16) 0.3 15.77 0.0004 8th 0.2 1.11 0.1372 * * * * * * regression analysis was not carried out since the sampling unit had already been selected by the correlation analysis. 1274 sociobiolog y vol. 59, no. 4, 2012 regarding the sun exposure of the plant, verification of the two surfaces of the plant presented a positive and significant correlation (p<0.05) to total predation, and its regression curves presented similar inclinations (table 1). so, in vegetative phase coffee crops the sampling of the social vespidae predators can be done on either surface of the plant. among the types of branches of the plant, it was determined that only the predation that occurred on the primary plagiotropic branches presented positive and significant correlation (p<0.05) with the total predation (table 1). so, in vegetative phase coffee crops the sampling of the social vespidae predators must be carried out on leaves of the primary plagiotropic branches. among the pairs of leaves of the branch, positive and significant correlations were verified (p<0.05) among predation that took place in the 3rd, 4th, 5th, 6th and 7th pairs of insertion of the leaves in the branches with total predation in the median third of the canopy. however, leaves from the 7th pair were not selected because fast sampling was not possible to present less than a 20% frequency. the predation regression curves in the 5th and 6th pairs as a function of total predation presented greater inclinations than the curves of the 3rd and 4th pairs (table 1). therefore, in vegetative phase coffee crops the sampling of social vespidae predators must be carried out on leaves of the 5th or 6th pair. plants in reproductive phase positive and significant correlations were observed (p<0.05) between absolute density to the total l. coffeella predation by vespidae with predation table 2. pearson’s correlations between the absolute density of predaceous vespidae (mines/leaf ) on the plant with the relative densities in the canopy thirds and regression curves of these relative densities as a function of the absolute density in reproductive phase coffee plants. sampling unit pearson’s correlation regression curve r t p intercept inclination r2 f p thirds of the canopy apical 0.86 10.79 <0.0001 0.0078 0.93 (0.81-1.05) 0.6 61.94 <0.0001 median 0.89 12.76 <0.0001 -0.0039 1.05 (0.96-1.14) 0.8 127.3 <0.0001 basal 0.9 12.86 <0.0001 -0.001 0.88 (0.80-0.97) 0.8 115.9 <0.0001 1275 picanco, m.c. et al. —ecolog y of vespidae predators in coffea arabica intensities in the three thirds of the canopy. the regression curves of the relative densities of the apical and median thirds presented the greatest inclinations (table 2). therefore in reproductive phase coffee crops the sampling of social vespidae predators must be carried out on apical or medium third leaves. on the apical third of the canopy, the leaves exposed to the sun in the afternoon period better represented l. coffeella predation by social vespidae. in this canopy third the primary plagiotropic branches better represented l. coffeella predation by vespidae. the 4th and 6th leaf pairs also better represented table 3. pearson’s correlations between the absolute density of predaceous vespidae (mines/leaf ) on the apical third of the canopy with the relative densities in the sampling units and regression curve of these densities relative to the function of absolute densities in reproductive phase coffee plants. sampling unit pearson’s correlation regression curve r t p intercept inclination r2 f p plant face exposed to the sun morning period 0.8 7.36 <0.0001 -0.07 0.82 (0.71-0.93) 0.6 54.15 <0.0001 afternoon period 0.9 10.9 <0.0001 -0.46 1.23 (1.12-1.35) 0.8 118.9 <0.0001 type of branch primary plagiotropic 1 23.5 <0.0001 -0.23 1.27 (1.21-1.32) 0.9 552.9 <0.0001 secondary plagiotropic 0.6 4.53 <0.0001 0.48 0.78 (0.61-0.95) 0.4 20.51 <0.0001 position of the leaf on the branch 1st 0.4 2.45 0.0092 0.51 0.21 (0.13-0.30) 0.1 6.04 0.0183 2nd 0.8 7.44 <0.0001 -0.35 0.66 (0.57-0.75) 0.6 55.33 <0.0001 3rd 0.8 9.39 <0.0001 -1.92 1.57(1.40-1.74) 0.7 55.33 <0.0001 4th 0.9 10.7 <0.0001 -1.68 2.11 (1.92-2.31) 0.7 114.7 <0.0001 5th 0.8 8.18 <0.0001 -0.54 1.24 (1.09-1.40) 0.6 66.92 <0.0001 6th 0.5 3.78 0.0003 2.09 1.85 (1.46-2.24) 0.4 22.23 <0.0001 7th 0.6 4.71 <0.0001 2.43 1.18 (0.87-1.49) 0.3 14.3 0.0005 8th 0.6 2.5 0.0592 * * * * * * regression analysis was not carried out since the sampling unit had already been selected by the correlation analysis. 1276 sociobiolog y vol. 59, no. 4, 2012 l. coffeella predation by vespidae (table 3). therefore, in leaves from the apical third of reproductive phase coffee plants the sampling of social vespidae must be carried out in the 4th or 6th leaf pairs of primary plagiotropic branches plant surfaces exposed to the sun in the afternoon period. in the medium third of the canopy the leaves exposed in the sun in the afternoon period better represented l. coffeella predation by vespidae. in this canopy third, leaves of the primary plagiotropic branches as well as the secondary plagiotropic branches represented similar l. coffeella predation by vespidae. the leaves of the 5th pair better represented l. coffeella predation by vespidae (table 4). therefore, in leaves from the median third of reproductive phase coffee plants the sampling of social vespidae must be carried out in the 5th pair of leaves in plants exposed to the sun in the afternoon period. therefore, in coffee plants in the vegetative phase the best units for sampling social vespidae were the 5th or 6th pair of leaves of primary plagiotropic branches of the median third of the canopy. in reproductive phase coffee plants the best unit for sampling social vespidae was in leaves of the apical table 4. pearson’s correlations between the absolute density of predaceous vespidae (mines/leaf ) on the median third of the canopy with the relative densities in the sampling units and regression curve of these densities relative to the function of absolute densities in reproductive phase coffee plants. sampling unit pearson’s correlation regression curve r t p intercept inclination r2 f p plant face exposed to the sun morning period 0.71 6.38 <0.0001 0.10 0.75 (0.63-0.87) 0.50 40.63 <0.0001 afternoon period 0.75 7.17 <0.0001 0.11 1.14 (0.98-1.29) 0.49 37.18 <0.0001 type of branch primary plagiotropic 0.45 2.73 0.0053 1.86 0.72 (0.46-0.98) 0.20 7.45 0.0105 secondary plagiotropic 0.72 5.95 <0.0001 0.52 0.77 (0.64-0.90) 0.53 35.41 <0.0001 position of the leaf on the branch 1st 0.52 3.86 0.0002 0.03 0.15 (0.12-0.20) 0.27 14.91 0.0004 2nd 0.58 4.61 <0.0001 0.21 0.28 (0.22-0.34) 0.34 21.29 <0.0001 3rd 0.78 7.94 <0.0001 0.48 0.73 (0.63-0.82) 0.61 63.04 <0.0001 4th 0.85 10.46 <0.0001 0.82 1.47 (1.17-1.77) 0.37 24.18 <0.0001 5th 0.61 4.92 <0.0001 -0.29 2.02 (1.79-2.24) 0.66 78.78 <0.0001 6th 0.81 8.88 <0.0001 0.44 1.22 (0.96-1.48) 0.35 22.20 <0.0001 7th 0.59 4.71 <0.0001 -2.14 0.90 (4.36-5.44) 0.68 81.62 <0.0001 8th 0.82 3.03 0.0601 * * * * * *regression analysis was not carried out since the sampling unit had already been selected by the correlation analysis. 1277 picanco, m.c. et al. —ecolog y of vespidae predators in coffea arabica third in the 4th or 6th pair of leaves of primary plagiotropic branches in plants exposed to the sun in the afternoon period or in the median third in the 5th pair of leaves of the plant exposed to the sun in the afternoon period. discussion the preference for monitoring specific places of occurrence of social vespidae predation is due to the fact that these better represent the absolute density fluctuations of these insects on the plant. therefore, in plants with greater predation by vespidae greater densities of this insect will occur in these sampling units or vice-versa. as such, it is possible to infer that the factors that affect the absolute densities of these insects in the plants will also affect its relative densities in the sampling units selected. this fact does not take place with equal intensity in other parts of the plant. therefore, factors like climatic elements, prey density, toxins, nutrients and allelochemicals present in the leaves in the ideal vespidae sampling units must have similar influence to what occurred in the plant as a whole (bechinski & pedigo 1982; schuster 1998; pedigo & rice 2006). therefore, factors like weather or climate should have different impacts on vespidae in the different canopy thirds. these impacts should be more intense in the apex of the plants due to the leaves being more exposed to rain and extreme temperatures. inversely, the basal third where the leaves are less exposed to weather climates should be the opposite. therefore the fact of the canopy median third being the ideal place for vespidae sampling in vegetative phase as well as in reproductive phase plants should be related to the greatest density of l. coffeella caterpillars or intermediate exposure of vespidae to weather climate conditions in this part of the plant (villacorta 1980; carracedo et al. 1991; nestel et al. 1994). in general the leaves exposed to the sun in the afternoon period were better for sampling vespidae than those exposed to the sun in the morning. therefore, in leaves exposed to the sun in the morning, the variation of relative predator wasp densities differs from the rest of the plant. among the factors that can influence this response are the effects of air temperature and luminosity on the flight activity of vespidae. verification of greater flight activity of these insects in periods of higher temperature and luminosity was carried out and 1278 sociobiolog y vol. 59, no. 4, 2012 it is a fact that this takes place in the afternoon period (ghule et al. 1989; leite et al. 2001). in general, the leaves of the primary plagiotropic branches were better for sampling vespidae than the secondary plagiotropic branches. such results could be associated with the fact that secondary plagiotropic branches are more exposed to the weather because of being located in the most external part of the plant, which has a negative influence both on vespidae and on its prey (pereira et al. 2007a,b). on the whole, the ideal unit for sampling vespidae was located on leaves from the 4th to 6th pair. this unit, which is different from those recommended in the works of villacorta & tornero (1982); villacorta & gutierrez (1989); bearzoti & aquino (1994); villacorta & wilson (1994) based on studies about the evaluation of samples adequate for the evaluation of the nutrient content on the leaves. therefore the factors that influence vespidae density should be different from what affects the nutrient contents in the leaves. a possible mechanism for this phenomenon could be the previously mentioned higher incidence of toxins on older leaves like those present at the end of the branch or even the greater exposure of the leaves of the initial portion of the branches to weather (caixeta et al. 2004). in summary, our study provides important information for understanding the ecolog y of predation by wasps in coffee crops, in addition to proposing methodolog y for evaluation of vespidae predator populations in these agroecosystems. acknowledgments this research received financial support from capes, cnpq, fapemig, pnpd/café. we thank dra. enedina sacramento and dr. jeff oar for critically reading a draft of the manuscript and the manuscript referees were greatly appreciated. references akre r.d. 1982. social wasps. in: hermann, h. 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invasion from other habitats or expansion from forest gaps (hymenoptera: formicidae) by marcelo s. madureira1, tathiana g. sobrinho2 & josé h. schoereder2,3 abstract in a fragmented brazilian landscape, 24 species of ant, which are considered to be open-area specialists, occur exclusively in the pasture areas around the forest remnant (matrix). in this paper, we propose possible theoretical explanations for the occurrence of these exclusively matrix species, and suggest that these species originally occurred in forest gaps. we also determine whether these species occur in another type of open vegetation, the cerrado (brazilian savanna). ants were collected from ten forest gaps within three forest remnants. ant species sampled in forest gaps were compared to ant species collected from the cerrado. the aim here was to determine whether there were any similarities between the two sets of species, and also to collect information about the origin of matrix ant species. in the forest gaps, we sampled 44 species of ant. of these, 11 species were also found to occur in matrix areas and eight species in the cerrado vegetation. two scenarios could explain this result: (i) exotic ant species of open biomes migrate to, and establish in, the matrix; or (ii) the species that currently occur exclusively in the matrix areas are originally from forest gaps and have increased their distribution following the fragmentation event. we discuss reasons to support these scenarios as well as their implications for other ecological and conservation processes. keywords: disturbance, formicidae, habitat fragmentation, invasion, landscape ecolog y. 1 departamento de entomologia, universidade federal de viçosa, viçosa-mg, cep 36570-000 brazil. 2 departamento de biologia geral, universidade federal de viçosa, viçosa-mg, cep 36570-000 brazil. 3 corresponding author mail: jschoere@ufv.br 1138 sociobiolog y vol. 59, no. 4, 2012 introduction habitat fragmentation is the loss of continuous habitats, resulting in the formation of one or several smaller and isolated remnants (lovejoy et al. 1986, desouza et al. 2001, laurance 2008), surrounded by matrix habitats (i.e. pastures, crops and plantation), which can strongly influence the connectivity between fragment (turner 1996, didham 1997, ricketts 2001, sobrinho et al. 2003). fragmentation is considered to be one of the main processes responsible for the loss of biodiversity in tropical ecosystems (didham et al. 1996, turner & corlett 1996, majer et al. 1997, davies & margules 1998, vasconcelos & delabie 2000) and, therefore, has been frequently studied over the past few decades. fragmentation affects biodiversity and species composition through several processes, such as area reduction (desouza & brown 1994, carvalho & vasconcelos 1999) and an increase in isolation among remnants (hanski et al. 1994, ricketts et al. 2001), as well as through secondary effects, such as edge and shape effects (murcia 1995, sobrinho & schoereder 2007) and invasion by exotic species from the surrounding matrix (sobrinho et al. 2003, schmidt et al. 2008). the creation of edges between remnants and matrix areas can lead to changes in species number, population abundance and species composition (murcia 1995, zheng & chen 2000, sobrinho & schoereder 2007), because it increases the ration between perimeter and area (desouza et al. 2001), consequently increasing the proportion of habitats exposed to the matrix (kapos et al. 1997). edge creation causes microclimatic changes, increasing the incidence of winds and solar radiation and reducing the humidity in the strip that extends from the edge until approximately 100 m into the core (lovejoy et al. 1986, laurance & yensen 1991, murcia 1995, laurance 1997, turton & freiburger 1997). in this way, edge creation can cause alterations in communities of several animal taxa, including birds, mammals and invertebrates (lovejoy et al. 1986, fowler et al. 1993, brown & hutchings 1997, didham 1997, didham et al. 1998) and specifically ants (sobrinho & schoereder 2007). the influence of edge creation on communities depends on the type of matrix (laurence 2008), because this habitat type can influence fragment connectivity (ricketts 2001, donald & evans 2006, laurence 2008). matrices 1139 madureira, m.s. et al. — why do ant species occur in the matrix but not the forest? that are significantly different from the pristine vegetation and microclimate tend to be more hostile for native species (sodhi et al. 2005), decreasing their permeability into such areas. therefore, the matrix type can influence the nature and magnitude of edge effects in forest fragments (laurance 2008). some researchers report the existence of typical animal species in open areas (hölldobler & wilson 1990), which are adapted to drier and sunnier environments, in contrast to the humid and dark core remnant (harper et al. 2005). therefore, it is expected that fauna specialists will exist in matrix areas and that these species will also be present in edge habitats. in addition, these species might also occur more frequently in small remnants, which are more affected by edge effects (sobrinho & schoereder 2007). by contrast, typical open-area species might also occur in gaps in the remnant core independently of remnant area, although this pattern has not yet been investigated. in the fragmented landscape in the viçosa region, southeastern brazil, several studies have been carried out to verify the effects of habitat fragmentation on ant communities. in these studies, ant diversity and composition has responded to area, isolation, edge, shape and matrix effects (sobrinho et al. 2003, schoereder et al. 2004a, schoereder et al. 2004b, ribas et al. 2005, sobrinho & schoereder 2007). however, as these papers did not sample from forest gaps, the effect of such environments on the composition and diversity of ant species within this landscape remains unknown. in the above-described landscape, ants were sampled for three consecutive years from 18 remnants. in ten pasture matrix locations surrounding the remnants, ants were sampled during one of these years. although the remnants had been sampled more exhaustively than had the matrix pastures, sobrinho et al. (2003) reported the occurrence of 24 ant species that occurred exclusively in the matrix, and these were considered to be open-area specialists. in the current paper, we propose possible theoretical explanations for the occurrence of exclusive ant species in matrix pastures surrounding forest remnants in the viçosa region. we hypothesize that these species also occur in forest gaps and also verify whether these species occur in other open vegetation types, such as the cerrado (brazilian savanna). these hypotheses might also apply to other areas that have undergone fragmentation, because they propose explanations of the changes in not only species richness, but also species composition. 1140 sociobiolog y vol. 59, no. 4, 2012 methods study area the study was carried out in the viçosa region, southeastern brazil (20° 45' s, 42° 50'w). this region was covered by forest up until the 20th century, when an accelerated process of fragmentation began. the pristine forest was fragmented and intermingled with pastures and coffee plantations mainly during the 1930s and 1940s (gomes 1975). the remaining forest vegetation has been restricted to a few patches, particularly on hilltops. from the 1960s onwards, agriculture diminished in the region and several forest areas regenerated into secondary forests. currently, there is a mosaic of forest remnants in the region, with areas varying from three to 300 ha, forming an ideal system to study the effects of fragmentation. in addition, there is a large amount of available information about the species richness and composition of the fragments and matrix. sampling procedure to collect the ant species, ten forest gaps were sampled in the three largest forest remnants chosen based on the ‘nestedness’ observed in the species distribution in these remnants (personal observation). the larger remnants have more species than the smaller ones (macarthur & wilson 1967, desouza & brown 1994) and have most of the species that are also found in the smaller fragments. therefore, the larger remnants are the best representatives of species richness and composition in the study area. three gaps were sampled in each of the first two fragments (areas), whereas in the third largest remnant (300 ha), four gaps were sampled. to collect ants, five pitfall traps were placed in the centre of each gap. the pitfall traps were plastic containers (diameter 20 cm, height 15 cm), with an inner chamber containing the bait, in this case honey and sardine. the ants attracted to the bait fell into the pitfall and died in a 70% alcoholic solution. after 48 h, the solution was removed and the ants were taken to the laboratory. the collected ants were sorted, mounted and identified to genera using identification keys (bolton 1994). some species of ants were identified by comparison with the collection of the laboratório de ecologia de comunidades of the universidade federal de viçosa, where voucher specimens were deposited. 1141 madureira, m.s. et al. — why do ant species occur in the matrix but not the forest? the species collected in former studies (sobrinho et al. 2003, schoereder et al. 2004a, schoereder et al. 2004b, ribas et al. 2005, sobrinho & schoereder 2007) in the same region were classified into three groups: (i) exclusively matrix species; (ii) exclusively forest species; and (iii) species common to both the matrix and the forest. in addition, data regarding the occurrence of ant species in another vegetation type, the cerrado, were also obtained (serra do cipó and paraopeba; ribas et al. 2003). this vegetation is an open vegetation type that occurs approximately 200 km from the forests sampled. the ant species sampled in forest gaps were compared to the above lists, to determine whether there were any similarities among them, as well as information about the origin of the matrix species. results the results presented here might not conform to some of those from previous studies in the same region, because the collection underwent a thorough revision by an ant specialist, who split some species and grouped others. we sampled 44 ant species from the ten forest gaps in the three remnants (table 1). the revision of the list reported by sobrinho et al. (2003) resulted in 48 ant species in the matrices sampled. after our sampling, 10 of these 48 species remained exclusive to the matrix, as they were not found in any other vegetation sample (table 2). in addition, five species occurred only in cerrado vegetation and matrix (camponotus genatus, cephalotes minutus, pheidole sp. 7, pogonomyrmex naegeli and pseudomyrmex tenuis), and two only in forest gaps and matrix (apterostigma gr. pilosum sp. 1 and crematogaster sp. 6) (fig. 1). as we did not consider species that occurred either only in cerrado or in all three habitats (cerrado, forest gaps and matrix), these data were not included. discussion we propose two possible explanations of our result: (i) exotic ant species of open biomes have migrated and established in the viçosa region; and/or (ii) the species that now occur exclusively in the matrix areas were originally from forest gaps and increased their distribution following fragmentation. the forest fragmentation in the studied region might have enabled inva1142 sociobiolog y vol. 59, no. 4, 2012 sion by exotic ant species from open biomes, such as the cerrado. this disturbance has created vegetation types that are structurally similar to the cerrado vegetation, such as open fields and pastures. therefore, after fragmentation, species from cerrado might have invaded the viçosa region. this hypothesized migration can explain the existence of those matrix species that were found in both the matrix and the cerrado, but not in the remnants. the above scenario can lead to two further developments: the invading subfamily species myrmicinae (cont.) crematogaster sp. 4 crematogaster sp. 6 megalomyrmex goeldii megalomyrmex iheringi pheidole sp. 13 pheidole sp. 17 pheidole sp. 20 pheidole sp. 24 pheidole sp. 25 pheidole sp. 40 solenopsis sp. 1 solenopsis sp. 2 solenopsis sp. 5 trachymyrmex sp.1 trachymyrmex sp. 2 wasmannia sp. 1 wasmannia sp. 2 ponerinae odontomachus chelifer pachycondyla lenis pachycondyla magnifica pachycondyla striata pseudomyrmecinae pseudomyrmex sp. 1 table 1. ant species collected in ten forest gaps in three forest remnants in the viçosa region, brazil. subfamily species dolichoderinae azteca sp. 3 linepithema sp. 5 ecitoninae nomamyrmex sp. 1 ectatomminae ectatomma edentatum formicinae brachymyrmex pr. depilis camponotus arboreus camponotus atriceps camponotus balzani camponotus cingulatus camponotus crassus camponotus melanoticus camponotus. novogranadensis camponotus rufipes camponotus sp. 2 camponotus (tanaemyrmex) sp. 7 camponotus sp. 10 myrmelachista sp. 1 myrmicinae acromyrmex landolti landolti acromyrmex rugosus rugosus apterostigma gr. pilosum sp. 1 atta sexdens rubropilosa crematogaster sp. 3 1143 madureira, m.s. et al. — why do ant species occur in the matrix but not the forest? table 2. ant species collected in the matrix of the viçosa region, brazil. asterisks represent exclusive matrix ant species, which were not found in forest remnants, forest gaps or cerrado vegetation (paraopeba e santana do riacho-mg). matrix ant species forest gaps cerrado dolichoderinae linepithema sp. 2 x ecitoninae labidus praedator x x formicinae brachymyrmex prox. depilis x x x brachymyrmex prox. coactus* camponotus cingulatus x x camponotus rufipes x x x camponotus novogranadensis x x x camponotus melanoticus x x x camponotus genatus x camponotus crassus x x x nylanderia pr. fulva x paratrechina sp. 2* myrmicinae apterostigma gr. pilosum sp. 1 x atta sexdens rubropilosa x cardiocondyla emeryi* cephalotes minutus x crematogaster abstinens* crematogaster evallans* crematogaster sp. 6 x cyphomyrmex transversus x mycetarotes sp. 2* mycocepurus goeldii* mycocepurus smithii x trachymyrmex sp. 3 x trachymyrmex sp. 4 x wasmannia auropunctata x pheidole sp. 1 x 1144 sociobiolog y vol. 59, no. 4, 2012 ant species might (i) remain restricted to the matrix or (ii) invade the matrix and subsequently migrate to the forest gap. the environmental structure and composition, as well as their temporal modification, affect the distribution, abundance, composition and dynamics of species. habitat fragmentation can facilitate the establishment of exotic species and modifications of the vegetation, soil and humidity can guarantee the dispersal of these species in an altered environment (suarez et al. 1998). table 2 (continued). ant species collected in the matrix of the viçosa region, brazil. asterisks represent exclusive matrix ant species, which were not found in forest remnants, forest gaps or cerrado vegetation (paraopeba e santana do riacho-mg). matrix ant species forest gaps cerrado pheidole sp. 2 x pheidole sp. 3 x pheidole sp. 4* pheidole brevicona* pheidole sp. 6 x pheidole sp. 7 x pheidole gr. flavens sp. 8 x pheidole sp. 9 x pheidole sp. 10 x pogonomyrmex naegeli x solenopsis saevissima x ectatomminae ectatomma bruneum x x ectatomma edentatum x x x ectatomma permagnum x x ponerinae anochetus diegensis x odontomachus chelifer x x x odontomachus haematodus* pachycondyla harpax x pachycondyla marginata x pachycondyla striata x x x pseudomyrmecinae pseudomyrmex tenuis x 1145 madureira, m.s. et al. — why do ant species occur in the matrix but not the forest? the fragmentation of ecosystems can decrease population densities, causing local extinctions and leading to a reduction of negative interactions (desouza et al. 2001). this process can increase the invasibility and establishment by exotic species. in our study region, formation of matrix (pastures) can facilitate the establishment of exotic species because solar radiation, humidity and vegetation are similar to the supposed source habitats of such species. therefore, it is possible that invader ants in the matrix established in the forest gap of the studied remnant, in a similar way that argentine ants are able to invade matrix and remnant forest in the usa (suarez et al. 1998). by contrast, in fragmented amazonian landscapes, where the matrix is also formed by pastures, gascon et al. (1999) found that approximately 24 species (19% of the total) occur exclusively in matrix but that these species did not invade forest remnants. if invasion really occurs, it might represent a problem to conservation strategies. in addition, the invasion could lead to an increase in competition between native and alien ant species and, if the latter are more competitive, they could exclude native species. the exclusion of native species might have direct fig ure 1.: venn diagram representing the occurrence of sampled species in the studied environments. 1146 sociobiolog y vol. 59, no. 4, 2012 or indirect consequences for other species, given that ants generally maintain several ecological interactions with various organisms. for example, human & gordon (1996) showed that, since it invaded the usa, the argentine ant linepithema humile has competitively excluded several native ant species. in relation to the two species in that occur in both forest gaps and matrix pastures, we suggest that these species were already present in forest gaps of pristine forests in viçosa. therefore, the fragmentation event was equivalent to an increase in forest gap areas that facilitated the increase in population size and distribution of these typical gap species in the matrix. evidence in support of (or against) this hypothesis could be obtained by analyzing the regional distribution of these species. further studies would be important to clarify the composition ant species of open areas near to viçosa. if the exclusively matrix species do not exist in such areas, one can assume that these species are endemic to viçosa. this endemism would be supporting evidence for the hypothesis that species exclusively found in the matrix already occurred in pristine forest gaps and then invaded the matrix. an unexpected result was that there was no overlap in species sampled from forest gaps and cerrado vegetation (fig. 1). it might be that both invasion and migration of gap species occurred simultaneously after the fragmentation. therefore, some species would have come from forest gaps and invaded the matrix, and others would have come from open vegetation, such as cerrado, and migrated to the matrix of the viçosa region. this process might be continuous as there is no evidence to suggest that it has come to an end. finally, it is vital to stress the importance of knowing not only the species richness, but also the community composition of a habitat, because different ant species may indicate what ecological processes occur after habitat fragmentation. therefore, the species composition might be more important from a conservation point of view. acknowledgments we are indebted to carlos f. sperber for comments on the first draft of the manuscript, and to fredson vieira e silva, who helped us during the sampling. the authors were supported by cnpq grants, and fapemig gave support for the project. 1147 madureira, m.s. et al. — why do ant species occur in the matrix but not the forest? references bolton, b. 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(eds.). tropical forest remnants: ecolog y, management and conservation of fragmented communities. university of chicago press, chicago. vasconcelos, h. l. & j. h. c. delabie 2000. ground ant communities from central amazonia forest fragments. sampling ground-dwelling ants: case studies from the world’s rain forests. in d. agosti, j. majer, l. alonso, t. schultz (eds) curtin university school of environmental biolog y bulletin no. 18. perth, australia. zheng, d. & j. chen 2000. edge effects in fragmented landscapes: a generic model for delineating area of edge influences (d-aei). ecological modelling, 132: 175-190. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.435-440sociobiology 61(4): 435-440 (december, 2014) waste management in the stingless bee melipona beecheii bennett (hymenoptera: apidae) lam medina1, ag hart2, flw ratnieks3 introduction social living can cause problems which are not faced by non-social organisms, or exacerbates problems that are easily dealt with by solitary dwellers. in particular, waste poses a serious problem for social insects living within enclosed nests (bot et al., 2001; hart & ratnieks, 2001, 2002a; jackson & hart, 2009). localizing waste such as in a midden heap (e.g. atta colombica guérin-méneville, hart & ratnieks, 2002a; messor barbarus (linnaeus), anderson & ratnieks, 2000), or removing waste regularly (e.g. defecating outside the nest and undertaking behavior in apis mellifera linnaeus, visscher, 1983; seeley, 1985) are common strategies to reduce waste building and its negative impact. abstract waste management is important in insect societies because waste can be hazardous to adults, brood and food stores. the general organization of waste management and the influence of task partitioning, division of labor and age polyethism on waste processing were studied in three colonies of the tropical american stingless bee melipona beecheii bennett in yucatán, mexico. waste generated in the colony (feces, old brood cells, cocoons, dead adults and brood) was collected by workers throughout the nest and taken to specific waste dumps within the nest. during the day, workers based at the waste dumps formed waste pellets, which they directly transferred in 93% of cases, to other workers who subsequently removed them from the nest. this is an example of task partitioning and is hypothesized to improve nest hygiene as it has been found in leafcutting ants, atta. to investigate division of labor and age polyethism we marked a cohort of 144 emerging workers. workers forming waste pellets were on average 31.2±6.5 days old (x±sd, n= 40, range of 18-45 days). the life span of m. beecheii workers was 49.0±14.0 days (n= 144). there was no difference in the life span of workers which formed (52.2±11.6 days, n= 40) or did not form (49.9±11.5 days, n= 97) waste pellets, suggesting that waste work did not increase mortality. although waste was probably not hazardous to adults and brood, because the dumps are located outside the brood chamber, its presence inside the nests can attract phorid flies and predators, which can harm the colony. sociobiology an international journal on social insects 1 universidad autónoma de yucatán, mérida, yucatán, méxico 2 university of gloucestershire, gloucestershire, u.k. 3 university of sussex, brighton, u.k. article history edited by denise araujo alves, esalq, usp, brazil received 22 august 2014 initial acceptance 10 september 2014 final acceptance 06 november 2014 keywords stingless bees, nest hygiene, task partitioning, division of labor age polyethism. corresponding author luis a. medina medina departamento de apicultura, facultad de medicina veterinaria y zootecnia, universidad autónoma de yucatán, apartado postal 4-116 itzimná, c. p. 97100, mérida, yucatán, méxico. e-mail: mmedina@uady.mx waste management is often a combination of material transport and work organization and adaptations to either can improve effectiveness and efficiency (e.g. hart & ratnieks, 2001; hart & ratnieks, 2002a; hart et al., 2002a). work organization includes the organization of both tasks and the workforce performing them. division of labor has proved to be an important component of work organization in social insect colonies (robinson, 1992; gordon, 1996). task partitioning (how tasks are divided into subtasks) is also proving to be a powerful organizational principle (jeanne, 1986; ratnieks & anderson, 1999; anderson & ratnieks, 2000; anderson et al., 2001; hart & ratnieks, 2002b; hart et al., 2002a, b; hart, 2013). task partitioning describes research article bees lam medina, ag hart, flw ratnieks waste management in melipona beecheii436 situations where a single task, such as foraging, is divided into sequentially linked sub-tasks with a flow of material between them. it has been shown to be important in waste management in leafcutting ants atta. workers from “clean” parts of the nest, the fungus garden chambers, drop waste outside the “unclean” waste chambers where dedicated waste chamber workers collect it. the two-stage task partitioning (waste transporters coupled with waste chamber workers) with indirect transfer of waste, via caches outside the waste chambers, is hypothesized to help maintain nest hygiene by isolating the waste chambers (hart & ratnieks, 2001). waste management has not been extensively studied in social insects (hart & ratnieks, 2002a). one group which has received some attention are the stingless bees (meliponini). stingless bee waste comprises feces, old brood cells, cocoons and both dead adults and brood (eltz et al., 2001) and waste handling strategies are varied. workers of melipona favosa (fabricius) deposit waste in “waste dump areas” within the nest (where workers also defecate) before removing it (sommeijer, 1984; bruijn et al., 1989) whereas in some species, e.g. cephalotrigona capitata (smith) and trigona spinipes (fabricius), waste accumulates, eventually forming a large mass (scutellum), which can help to regulate nest temperature (michener, 1974, 2000; sakagami, 1982). in melipona compressipes (fabricius) and melipona scutellaris latreille, kerr and kerr (1999) reported that waste (cocoon + feces + wax) are removed from the nests and dropped at a distance of 1 to 45 m according to a gaussian distribution which reduces the cost of energy and the attraction of predators and pests. a detailed study of work organization in waste management (division of labor, task partitioning, spatial management and age polyethism) was carried out on the stingless bee melipona beecheii bennett. this species ranges from mexico to costa rica (ayala, 1999; van veen & arce, 1999) and colonies typically contain 500 to 2500 workers with a single singly-mated queen (van veen & arce, 1999; paxton et al., 2001). natural nests are built in tree hollows and have an entrance hole that connects to the nest cavity. within the cavity, the brood area consists of multiple horizontal combs of wax cells covered by layers of involucrum (van veen & arce, 1999). outside that there are many egg-shaped pots made from cerumen (wille & michener, 1973; van veen & arce, 1999) which are used for honey and pollen storage. material and methods study site and study colonies the study was carried out at the department of apiculture of the campus of biological and animal husbandry sciences, autonomous university of yucatan, merida, mexico, from march to september 2001 during the main nectar flow of “tsitsilche” (gymnopodium floribundum rolfe, polygonaceae) and many different fabaceae species (echazarreta et al., 1997). three healthy queenright colonies of m. beecheii were studied which originally were housed in log hives. two months before the study, colonies were transferred into observation nests consisting of a box (30x25x8 cm) with wooden sides and glass top and bottom. this allowed observation of intranidal behavior. colonies comprised c.900 workers of different ages, 2-4 brood combs and more than 20 honey and pollen pots surrounding the brood area. general waste dump features all colonies had two clear areas in which waste was dumped. the waste dump areas were 8-15 cm away from the brood area, outside the involucrum. no other waste dump areas were formed and the original dumps remained in the same place during the study period. task partitioning one waste dump area in each colony was continually observed for 15 min before switching to the second waste dump area. each dump was observed for a total of c. 170 h. individuals that were molding waste into pellets were considered “waste dump workers”. this behavior was characterized by the worker’s body movements when trying to separate small portions of the waste material from the dump (bruijn et al., 1989). workers that went to a waste dump to perform a specific activity (e.g. defecation, grooming or dropping material), but did not manipulate the waste to form pellets for removal, were not considered waste dump workers (in contrast to bruijn et al. (1989) who classified all bees performing activities at the dumps as waste workers). workers forming waste pellets were followed to determine the pellet’s destination. there were only two destinations. a pellet was either transferred to another worker who removed it from the nest, or it was taken outside the nest directly by the worker that formed it. task partitioning occurred when a pellet was transferred to another worker. pellets weighing an average of 18 mg were carried in the mandibles of the workers which represents 25.7% of the weight of the workers (x=70 mg). division of labor, age polyethism and worker longevity three mature brood combs from one colony were taken from the original log nest and kept in an incubator. all workers that emerged from 28 may to 5 june were individually marked with a numbered tag (opalithplättchen; 2.1 mg each tag which represents 3% of the weight of the workers) on the notum and introduced into an observation nest formed from the original colony. 144 workers in total were marked and introduced successfully. these marked bees allowed sociobiology 61(4): 435-440 (december, 2014) 437 detailed data on the ages of workers performing waste–related activities to be collected and to determine whether wasterelated activities affect worker longevity. they also enabled us to determine whether division of labor into waste workers and non-waste workers was occurring. individuals processing waste (e.g. collecting waste and dropping waste at the dumps to form waste caches, molding waste to form pellets) and workers releasing the waste pellets from the nests were recorded and their ages of workers performing these tasks and their longevity by a daily census were determined. data analysis the proportions of pellets transferred to other workers, which then removed the pellets from the nest, were compared in the three colonies using a chi-square contingency test (zar, 1999). the life span and survival curves of workers which had or had not formed pellets were compared using logrank test (machin et al., 2006). all mean values are expressed ± one standard deviations (sd). statistical analysis were performed using spss 11.0 for windows (spss inc., norusis, 2002). results general waste dump features waste management in m. beecheii was a three-stage process. first, workers collected waste from all over the nest, especially from the brood chamber, and deposited it in the waste dumps. this activity was observed during both day and night. second, workers at the dumps molded waste into pellets during the day. finally, and again only during the day, waste pellets were removed from the nest. the average number of waste pellets formed per day in the study nests was 25.3±8.1 (x±sd, n = 40 days study), which represents 454.9±145.9 mg of waste (pellets weighed 18.0±11.1 mg, x±sd, n= 45 pellets). task partitioning the destinations of 416 waste pellets were observed in the three study colonies (nest 1, n = 112; nest 2, n = 165; nest 3, n = 109). in total, 386 workers (92.8%) observed forming pellets subsequently transferred them to other workers which removed the pellets from the nest. there were no significant differences in the occurrence of task partitioning between the study colonies (cross-colony comparison: chi squared = 1.17, df= 2, p= 0.56, table 1). overall, 92.8% of all waste pellets formed by waste dump workers were transferred directly (from mandibles to mandibles) to another worker, and 7.2% were taken outside by the waste dump worker without transfer. when waste dump workers took waste outside without transfer (i.e. when task partitioning was absent), it was not because transfer partners were unavailable. in the marked cohort of workers, the only workers that formed pellets and subsequently removed them from the nest were workers that were close to switching tasks from pellet forming to flying outside the nest with waste (figs 1b and c). table 1. number (%) of waste pellets formed and transferred between waste dump workers and individuals removing pellets from the nest. nest 1 nest 2 nest 3 total with transfer 112 (91.0%) 165 (94.3%) 109 (92.4%) 386 (92.8%) without transfer 11 (9.0%) 10 (5.7%) 9 (7.6%) 30 (7.2 %) cross-colony comparison: χ2= 1.17, df= 2, p= 0.56 division of labor, age polyethism and worker longevity workers that collected waste within the nest (mainly in the brood area) and then placed it at the waste dump areas were 20.7±5.7 days old (x±sd, n= 46) with a range of 10-31 days (fig 1a). workers engaged in this task were removing wax from old brood combs, destroying old brood cells and involucrum, removing dead brood and collecting dead adults. workers then carried the waste to the dump areas where they dropped it to form waste caches. all workers performing duties inside the colonies defecated only at the two waste dumps areas, so feces do not have to be collected and carried out to these areas and other forms of waste were carried out to these places to form the wastes dumps. thirty-three percent of the age-marked cohorts were observed collecting waste and taking it to the dump areas. the age at which individuals first formed waste pellets was 31.2±6.5 days (x±sd, n= 40, range of 18-45 days). in total, 29.2% of the age-marked cohort engaged in this task (fig 1b). workers that started processing waste material at the dumps and forming waste pellets performed this task for about 2.5 days (range of 1-15 days) and were later observed flying outside the nest to remove the waste pellets at 34.5±5.9 days old (x±sd, n= 13) with a range of 29 to 59 days (fig 1c). however, only 10% of the age-marked cohort removed waste pellets from the nest. these workers were very active, removing waste pellets during the day at a high rate (some handled 4-5 waste pellets within an hour) until all waste was removed from the nest. although we did not record the distance flown before waste pellets were released, total flight durations were short (x±sd = 21.7±11.1 s, n = 65 flights). the first foraging flights in the cohort of age-marked m. beecheii workers were performed at 41.7±7.1 days old (x±sd, n= 58) with a range of 22 to 63 days (fig 1d). lam medina, ag hart, flw ratnieks waste management in melipona beecheii438 span of workers which had (52.2 ± 11.6 days, n = 40) or had not (49.9±11.5 days, n= 97) formed pellets. discussion our results show that waste disposal in m. beecheii is subject to partitioned organization, encompassing spatial management, division of labor with age polyethism, and task partitioning. waste management had three distinct stages connected by task partitioning with both direct and indirect transfer of waste between workers, and this pattern occurred consistently across all three study colonies. younger workers collected waste around the nest and transferred it to the waste dump areas where it formed a cache (a case of task partitioning with indirect transfer; ratnieks & anderson 1999). the percentage (33%) of worker bees collecting waste and taking it to the dump areas indicates that this is a common but not universal activity in the worker age polyethism schedule. much of the waste in the dumps was feces, which was not transferred but deposited directly by defecating workers. waste in the dump areas was then fashioned into discrete pellets by older workers. most pellets, 93%, were transferred directly by a dump worker to another worker, who was on average even older, who flew from the nest to dump the pellet. the other pellets, 7%, were removed from the nest without transfer. waste management has not been extensively studied in social insects. however, the system found in m. beecheii is similar to that found in the leafcutting ant, atta cephalotes linnaeus, where the transfer of waste to disposal zones is also subject to task partitioning. in a. cephalotes, waste is stored in specialized chambers in the underground nest that are distinct from the chambers where the ants grow the fungus gardens that they depend on for food. fungus garden workers deposit waste just outside the waste chambers, which they do not enter. dedicated waste chamber workers retrieve the waste and carry it into the chamber. thus, indirect transfer (coupled with division of labor and nest compartmentalization) enables effective isolation of hazardous waste (hart & ratnieks, 2001). good hygiene is important in leafcutting ants (and other fungus growing ants) because of the presence of the vulnerable fungus on which the ants depend. in m. beecheii, waste may not be as hazardous. possibly reflecting this, leafcutting ants working in waste chambers never become foragers, but in m. beecheii all waste dump workers eventually became foragers. task partitioning can have ergonomic advantages. in m. beecheii, de-coupling pellet making from pellet disposal allows workers to become specialized at each task (albeit only for a short period), which is likely to improve efficiency. this may be the advantage of task partitioning in this case. a potential cost of task partitioning with direct transfer is that the two task groups may become out of phase, such that members of one or other groups must wait for transfer partners. one way to reduce this problem is to introduce indirect transfer via a cache (hart et al., 2002a). however, in m. beecheii, introducing the life span of the age-marked cohort was 49.0±14.0 days (x±sd, n = 144) with a range of 3 to 71 days (last marked-worker seen alive, fig 1a). there was no significant difference (x2 = 1.373, p= 0.354, logrank test) in the life fig 1. survival curve (line, a) and frequency (%) of workers of melipona beecheii performing tasks related with the collection of waste (a), processing (molding) the waste at dump areas (b), releasing waste outside the nest (c) and foraging (d). age is given in days after emergence. sociobiology 61(4): 435-440 (december, 2014) 439 a further cache of waste pellets (in addition to the initial waste dump areas) is not likely to improve nest hygiene, since waste will be more distributed around the colony (increasing the area it could contaminate) and might not be collected. in order to limit the distribution of waste, workers must either transfer pellets directly to other workers who will subsequently remove the pellets from the nest or remove waste pellets themselves. we did not record the destination of waste outside the nest, but return flights were short (mean 21.7 seconds) compared with nectar and pollen collecting flights (15.6 and 25.1 minutes respectively; biesmeijer & toth, 1998) and it is likely that waste was dropped at a distance comparable to other melipona species (e.g. 18m in m. compressipes and 31m in m. scutellaris; kerr & kerr 1999). dumping waste close to the nest is typical in stingless bees. some species, like lestrimellita limao (smith) and lestrimellita niitkib ayala, simply drop waste in front of the nest entrance and in frieseomellita varia (lepeletier) waste is carried less than 1m from the nest (kerr & kerr, 1999). carrying waste away from the nest has a number of possible advantages. waste can reduce the space available for food storage or brood rearing and in species where waste accumulates in the nest, the thickness of waste increased with nest age (in trigona corvina cockerell a total thickness of 11-24 cm is found around the sides and base of the nest; michener, 1974). additionally, waste removal may help to avoid attracting parasitic phorid flies (in m. compressipes, larvae and pupae of the phorid fly megaselia scalaris (loew) were found only in the waste (kerr, 1996) and vertebrates (e.g. eira barbara (linnaeus) [mustelidae]; kerr & kerr 1999), both of which can attack and destroy the colony. waste may also provide a substrate on which potentially harmful microorganisms can thrive. division of labour in waste management was agedependent. workers collecting waste were younger (mean age, 20.7 days old) than workers processing (31.2 days old) and releasing waste pellets (34.5 days old), and also performed brood chamber duties such as wax removal. this pattern of multi-tasking is found in other species. for example, in m. favosa (sommeijer, 1984) and melipona bicolor lepeletier (bego, 1983), young workers (<20 days) both collected waste and performed brood chamber tasks such as building and provisioning cells. in addition to temporal polyethism, the schedule of waste management tasks in m. beecheii exhibited a strong spatial pattern. young workers first performed tasks within the brood chamber (waste collection), later graduating to tasks outside of the brood chamber (waste pellet forming) before finally performing tasks outside the nest (waste removal and foraging). this is an excellent example of the “conveyor belt” model (schmid-hempel, 1998), in which individuals move from safe tasks inside the nest to more hazardous tasks outside the nest as they age. it also mirrors the situation in honey bees, a. mellifera (seeley, 1982) in which workers carry out a range of tasks that co-occur spatially in the nest. seeley (1982) commented that stingless bees, with their distinctive nest layout, could be an important taxon to further investigate this. large societies, both human and insect, struggle with how to collect and dispose their waste (doan, 1998; dijkema et al., 2000; bot et al., 2001; hart & ratnieks, 2002a). effective waste management needs to encompass collection, processing and eventual disposal. in human society these together prevent waste building up, thereby reducing negative effects such as odor, unsightliness and disease-carrying vermin. similar to human waste management, we found that in the nests of m. beecheii, waste was collected daily and taken to an intermediate processing site (the waste dump areas), where it was processed and then disposed of. this insect management system probably prevents waste accumulation and consequent attraction of “vermin” (e.g. phorid flies and predators) in the same way that human waste management does. social organisms from widely different taxa often face similar problems for example, maintaining social hygiene and managing waste. waste management in social insects is currently understudied and we suggest that further studies need to be carried out to investigate this potentially vital component of social life. the stingless bees provide a priori examples of waste management diversity and this, combined with the ease with which they can be cultured and studied in the laboratory, give them the potential to become a model group for waste management and nest hygiene in social insects studies. acknowledgements l.m. medina thanks h. moo-valle and wr angulo for their personal support during fieldwork. the work of l.m. medina was supported by promep-mexico phd studentship. the study complies with the current laws of mexico. references anderson, c., franks, n.r. & mcshea, d.w. 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(1999). biostatistical analysis. new jersey: prentice hall, 663 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5305sociobiology 68(1): e5305 (march, 2021) introduction the modification of natural habitats is considered the main cause of the current loss of bee diversity (gonzálezvaro et al., 2013; collado et al., 2019). on a global scale, native bees are threatened mainly due to human disturbances caused by deforestation, intensification of agriculture and introduction of exotic species (review in winfree et al., 2009). although some species of bees are able to live and find resources in anthropized areas, such as urban fragments (martins et al., 2013), environmental anthropization tends to have a deleterious effect on the community of these insects (zanette et al., 2005). because of this, a growing number of studies have investigated how anthropic factors influence the abstract in the present study we inventoried the diversity of eusocial bees (hymenoptera: apidae) in preserved and anthropized areas of a tropical dry forest in the parque da sapucaia (montes claros, minas gerais, brazil). we tested the hypothesis that the diversity of bee species would: 1) be greater in the preserved areas, 2) respond positively to the structure of the vegetation and 3) decrease during the dry season. we sampled eusocial bee species in 18 plots of 10 x 10 m distributed throughout the park, being nine plots in anthropized areas and nine plots in areas with preserved vegetation. in total we recorded 382 individuals and eight species of eusocial bees. the most abundant species was oxytrigona tataira (smith) (n = 233) and the most common species was trigona spinipes (fabricius) recorded in 72.2% of the plots. as expected, we found that eusocial bee diversity (shannon diversity) was higher in preserved plots than in anthropized plots. tree species richness positively affected bee species richness and abundance, while tree abundance positively influenced the bee abundance and tree height positively affected the bee shannon diversity, corroborating our expectations. on the other hand, we detected no differences in the diversity of eusocial bees between dry and rainy seasons. our findings suggest that both natural (vegetation structure) and anthropogenic (habitat modification) factors are important predictors of the diversity of eusocial bee species in tropical dry forests. sociobiology an international journal on social insects priscila santos gonçalves1; walter santos de araújo2 article history edited by evandro n. silva, uefs, brazil received 14 may 2020 initial acceptance 06 december 2020 final acceptance 15 december 2020 publication date 26 march 2021 keywords eusocial insects, meliponini, pollination, plant-animal interaction, stingless bee. corresponding author walter s. araújo departamento de biologia geral centro de ciências biológicas e da saúde universidade estadual de montes claros av. professor rui braga, vila mauricéia, 39401-089, montes claros, mg, brasil. e-mail: walterbioaraujo@gmail.com diversity of native bees in tropical regions (liow et al., 2001; zanette et al., 2005; martins et al., 2013; ferreira et al., 2015; stein et al., 2018). fragmentation of natural habitats and environmental changes in the remaining fragments can affect bee pollination processes (taki et al., 2007; gonzález-varo et al., 2013). this is worrying because bees are the most important pollinators of several human food crops (klein et al., 2007; potts et al., 2010). these losses in pollination occur because the modification of natural habitats impoverishes local bee faunas (nemésio & silveira, 2010; aguiar & gaglianone, 2012), and also affects species composition (martins et al., 2013). the negative effects of anthropization on local bee communities tends to occur because the change in habitat characteristics leads to the 1 programa de pós-graduação em biodiversidade e uso dos recursos naturais, universidade estadual de montes claros, montes claros-mg, brazil 2 departamento de biologia geral, centro de ciências biológicas e da saúde, universidade estadual de montes claros, montes claros-mg, brazil research article bees diversity of eusocial bees in natural and anthropized areas of a tropical dry forest in the parque da sapucaia (montes claros, minas gerais, brazil) id mailto:walterbioaraujo@gmail.com�abstract https://orcid.org/0000-0003-0157-6151 ps gonçalves; ws araújo – eusocial bees in a tropical dry forest2 loss of more sensitive species and also those that need a larger area of foraging (ferreira et al., 2015). on the other hand, there is also evidence that anthropized habitats (for example, forests in early succession or recently disturbed) may have a high diversity of bee species (romey et al., 2007; pengelly & cartar, 2010; taki et al., 2013). in these cases, high diversity is generally associated with changes in faunistic composition and dominance of tolerant species in communities of anthropized environments (rubene et al., 2015). often these changes in the composition of bee communities occur through the colonization of exotic or super generalist species, which can also compromise the quality of the pollination service (valido et al., 2019). another factor that can affect the distribution of bees is the structure of vegetation (wu et al., 2018). there is evidence that the richness and abundance of bees is positively related to the richness of plants and the abundance of potential nesting resources (grundel et al., 2010). thus, the more heterogeneous and diverse the vegetation, the greater the variety of resources offered, and consequently the greater the diversity of bee species (tews et al., 2004; wu et al., 2018). in addition, the type of vegetation is also an important determinant of the composition of the bee community (sydenham et al., 2016; wu et al., 2018), because the greater the composition of the plant community, the greater the dissimilarity of the bee community (alvarenga et al., 2020). thus, understanding how the structure of the vegetation influences the diversity of bees is an important step for planning the conservation of bees, especially in vegetations that have been little studied, such as the tropical dry forest, one of the least studied forests on the globe (miles et al., 2016). tropical dry forests, also called seasonal deciduous forests, are distributed across the globe and in brazil they have a wide occurrence in the northern region of minas gerais (dupin et al., 2018). this type of vegetation is under the domain of a strong seasonal climatic regime alternating between rainy and dry periods (murphy & lugo, 1986). the pronounced seasonal climate associated with the occurrence of deciduous plant species causes a high deciduousness rate in the tropical dry forests in the dry season, with more than 50% of plants losing their leaves (sanchez-azofeifa et al., 2005). the strong deciduousness of the tropical dry forest, in addition to resulting in seasonal changes in the cycling of organic matter and microclimate conditions in the forests, also influences the distribution of animal species that use plant resources (costa & araújo, 2019). insects, such as bees, which feed on plant resources (e.g., nectar or pollen), are likely to be affected by the strong seasonal regime in deciduousness of tropical dry forests. in the present study we inventoried the diversity of eusocial bees (hymenoptera: apidae) in preserved and anthropized areas of a tropical dry forest in the northern region of minas gerais, brazil. thus, we investigate the following questions: i) does bee diversity differ between preserved and anthropized areas of tropical dry forest? ii) is there an effect of the vegetation structure on the diversity of bees? iii) does the diversity of bee species vary seasonally? our expectations are that the diversity of bee species will be greater in the preserved areas, respond positively to the structure of the vegetation and decrease during the deciduous period (dry season). materials and methods study area the study was performed in the parque da sapucaia, located in the municipality of montes claros, northern region of minas gerais state, brazil (16°44’36.7”s; 43°54’3.49”w). the climate of the region is tropical semi-arid (as of köppen), with hot summers and dry winters and an annual average temperature of 24.1°c (alvares et al., 2013). the park has an area of 30.2 hectares predominantly composed by tropical dry forest (santos et al., 2007), where the elevation ranges from 690 to 872 m. due to the use of the park for visitation and tourism activities, there are parts of the vegetation that have been degraded through undergrowth pruning, tree clearing and canopy opening, for the construction of trails and recreation areas (costa & araújo, 2019). however, most of the park area has relatively well-preserved natural vegetation. sampling sampling was carried out in six bi-monthly collections distributed between april 2017 and february 2018, being three collections in the dry season and three collections in the rainy season. the collections were performed in 18 plots of 10 x 10 m distributed throughout the park, being nine plots in anthropized areas and nine plots in areas with preserved vegetation. the anthropized areas were identified based on signs of human intervention, such as selective vegetation cutting and the presence of buildings (e.g., paved trails, cable car and kiosks) and garbage deposits. on the other hand, the preserved vegetation areas were characterized by pristine vegetation, with no detectable evidence of human intervention. in each plot, all plants with a circumference equal to or greater than 15 cm (measured 1.5 m above ground) were sampled to measure the vegetation structure (species richness, abundance and height of trees). the eusocial bee sampling was performed using pet bottle traps with mead bait (water, sugar and yeast with 24 hours of fermentation), being used one trap for three hours in each plot. all specimens collected were referred to the laboratory of interactions, ecology and biodiversity (lieb) of universidade estadual de montes claros (unimontes). the sorting and identification of insects was realized according silveira et al. (2002). data analyses we used generalized linear models (glm’s) to test the effect of habitat type (anthropized vs. preserved) and vegetation structure variables (tree species richness, tree abundance and tree height) on the eusocial bee diversity. to characterize the diversity of bees we measured the species sociobiology 68(1): e5305 (march, 2021) 3 richness, abundance and shannon-wienner diversity index each sampled plot. additionally, we also used glm’s to compare the species richness, abundance and shannon diversity between the two seasons (dry vs. rainy seasons). we applied a quasi-poisson distribution of errors to the models. finally, we used a multivariate analysis to test the differences in the composition of bee species between anthropized and preserved plots. firstly, we used non-metric multidimensional scaling (nmds) to order the samples based on the bray-curtis similarity index. subsequently, a non-parametric permutation procedure (anosim) was applied with 1,000 permutations, also based on the bray-curtis index, to test the significance of the groups formed in the nmds (hammer et al., 2001). the values of p and r were obtained and the similarity patterns between the studied plots were determined. all analyses were performed in the r statistical software version 3.6.1 (r development core team, 2020). results in total we recorded 382 individuals and eight species of eusocial bees (table 1). the most abundant species were oxytrigona tataira (smith) (n = 233), trigona spinipes (fabricius) (n = 80) and tetragonisca angustula (latreille) (n = 32). the most common species were t. spinipes recorded in 72.2% of the plots and t. angustula recorded in 66.7% of plots. we also detected the exotic species apis mellifera linnaeus, which was the fourth most abundant species (n = 24) and the third most common (occurring in 61.1% of plots) (table 1). of all collected bees, 202 individuals (mean 20.2 ±43.8) and five species (mean 2.2 ±1.2) were recorded in anthropized plots, whereas in the preserved plots we recorded 180 individuals (mean 18.0 ±30.2) and seven species (mean 2.7 ±1.4) (table 1). we found no effects of habitat type on bee species richness and abundance, but average shannon diversity values were higher in preserved plots than in anthropized plots (table 2; figure 1). on the other hand, the composition of bee community did not differ between preserved and anthropized plots (anosim: stress 0.159; r = 0.021, p = 0.359). fig 1. comparison of species richness (a), abundance (b) and shannon diversity (c) of eusocial bees sampled in anthropized and preserved plots in a tropical dry forest of parque da sapucaia (montes claros, mg, brazil). table 1. abundance and frequency of occurrence of eusocial bee species in the anthropized and preserved areas of parque da sapucaia (montes claros, mg, brazil). species anthropized plots preserved plots total n % n % n % apis mellifera linnaeus 7 27.8 17 33.3 24 61.1 nannotrigona testaceicornis (lepeletier) 0 0.0 1 5.6 1 5.6 oxytrigona tataira (smith) 144 27.8 89 16.7 233 44.4 plebeia droryana (friese) 4 5.6 0 0.0 4 5.6 plebeia remota (holmberg) 0 0.0 6 5.6 6 5.6 tetragonisca angustula (latreille) 11 22.2 21 44.4 32 66.7 trigona hyalinata (lepeletier) 0 0.0 2 11.1 2 11.1 trigona spinipes (fabricius) 36 38.9 44 33.3 80 72.2 total 202 180 382 ps gonçalves; ws araújo – eusocial bees in a tropical dry forest4 vegetation structure variables affected significantly the eusocial bee diversity (table 2; figure 2). tree species richness positively affected the bee species richness (figure 2a) and abundance (figure 2b), while tree abundance positively influenced the bee abundance (figure 2c) and tree height positively affected shannon diversity (figure 2d). on the other hand, no differences were detected in the species richness, abundance and shannon diversity of eusocial bees between dry and rainy seasons (table 3). response variable explanatory variable df resid. dev f p bee species richness habitat type 16 6.8980 2.5358 0.1353 tree species richness 15 3.7784 15.4778 0.0017 tree abundance 14 3.7327 0.2269 0.6417 tree height 13 2.9127 4.0687 0.0648 bee abundance habitat type 16 689.28 0.0683 0.7979 tree species richness 15 461.39 12.2787 0.0039 tree abundance 14 290.88 9.1866 0.0096 tree height 13 278.88 0.6466 0.4358 bee shannon diversity habitat type 16 4.6867 4.9306 0.0448 tree species richness 15 4.3376 2.1983 0.1620 tree abundance 14 3.9204 2.6269 0.1291 tree height 13 2.7099 7.6216 0.0162 table 2. results of generalized linear models showing the effects of habitat type (anthropized vs. preserved) and vegetation structure (tree species richness, abundance and height) on the species richness, abundance and shannon diversity of eusocial bees sampled in a tropical dry forest of parque da sapucaia (montes claros, mg, brazil). fig 2. effect of the vegetation structure variables on the eusocial bee diversity variables in a tropical dry forest of parque da sapucaia (montes claros, mg, brazil). a) effect of tree species richness on the bee species richness; b) effect of tree species richness on the bee abundance; c) effect of tree abundance on the bee abundance; d) effect of tree height on the bee shannon diversity. sociobiology 68(1): e5305 (march, 2021) 5 discussion the diversity of species recorded in the parque do sapucaia can be considered low when compared to other studies in similar vegetation. for example, alvarenga et al. (2020) recorded a total of 96 species of bees in areas of tropical dry forest of the parque estadual da mata seca, minas gerais. in other study, milet-pinheiro and schlindwein (2008) recorded 79 species of bees in a transition area between dry and semi-deciduous forests in pernambuco. a part of these differences in the number of species are probably due to the distinct sampling efforts and methods, which varied between studies. in addition, these studies also sampled not only eusocial bees, and when only this bee group is compared the differences are smaller (22 species recorded by alvarenga et al., 2020 and only three species recorded by milet-pinheiro & schlindwein, 2008). thus, considering eusocial bees, our results corroborate the pattern of low diversity in dry forest areas, when compared to other brazilian vegetation (zanella, 2000; milet-pinheiro & schlindwein, 2008). the most abundant and frequent native species in the parque sapucaia were o. tataira, t. spinipes and t. angustula. the species o. tataira is considered a generalist species tolerant of anthropized environments (souza et al., 2015). the species t. spinipes is considered one of the most aggressive species of the meliponini tribe and usually builds its nests in higher tree branches (souza et al., 2015). the bees of species t. angustula are natural pollinators of plants of neotropical regions, with occurrence in all brazilian territory (pedro, 2014). also noteworthy is the invasive species a. mellifera that occurred in both anthropized and preserved areas of the park. this naturalized species of european origin tends to have a low efficiency in the pollination of native plants, due to being a highly generalist species (hung et al., 2018). we found that the diversity (shannon diversity index) of bee species was higher in preserved areas than in anthropized areas, corroborating our expectations. there was also a trend towards greater species richness and abundance in the preserved plots (see figure 1), although we have not found significant results. we believe that the lower values of shannon diversity in anthropized areas is a combination of the lowest species richness and the greatest dominance of generalist species in anthropized areas. in the anthropized plots we recorded a lower total number of species and average number of species per plot than in preserved environments. furthermore, in the anthropized areas there was a much greater dominance of the o. tataira species, which represented 71% of the individuals, while in the preserved environments the dominance was 49%. according to souza et al. (2015) this species is widely distributed in brazil, being frequent in anthropized habitats, such as urban environments. our results corroborate previous studies indicating that habitat modification has negative effects on the diversity of native bee communities (winfree et al., 2009; ferreira et al., 2015; stein et al., 2018). our findings show that the community of bees also responds positively to the structure of vegetation, corroborating recent studies (e.g. wu et al., 2018; alvarenga et al., 2020). as expected, we found a positive effect of tree species richness on the abundance and species richness of bees. these findings support previous evidences that plant species diversity is an important indicator of diversity of floral resources for the exploitation of bees (ebeling et al., 2008; plascencia & philpott, 2017). our results also showed that the tree abundance and tree height influenced positively the abundance and shannon diversity of bee communities, respectively. a larger quantity and a larger size of trees in the vegetation may be an indication of greater availability of sites for nesting (grundel et al., 2010). corroborating our results, alvarenga et al. (2020) found a positive effect of tree height on bee diversity in another area of brazilian tropical dry forest. contradicting our expectations, eusocial bee diversity did not vary between the dry and rainy seasons. this result is probably due to the generalist species found in the study being active all year, both in the dry season and in the rainy season. exemplifying this the bees t. spinipes and t. angustula, which were the most common bees in the present study, are very generalist social bees (ricketts et al., 2008; williams et al., 2010). these species may also use different resources in the environment, such as open or forest environments, because are able to use the resources available in the vicinity of their nests (garibaldi et al., 2014). these results indicate a constancy of generalist species in different seasons, corroborating previous studies (oliveira et al., 2010; gostinski et al., 2016). in summary, we conclude that both natural (vegetation structure) and anthropogenic (habitat modification) factors affect the diversity of eusocial bee species registered in the parque da sapucaia. our findings reveal that maintaining the state of conservation and the vegetation structure is essential for the conservation of bee fauna in the tropical dry table 3. results of generalized linear models showing the effects of sampling season (dry vs. rainy seasons) on the species richness, abundance and shannon diversity of eusocial bees sampled in a tropical dry forest of parque da sapucaia (montes claros, mg, brazil). response variable dry season rainy season df resid. dev f p bee species richness 3.7 (±2.1) 3.0 (±1.0) 4 2.9607 0.2644 0.6342 bee abundance 28.0 (±32.0) 89.3 (±139.2) 4 501.74 0.5238 0.5093 bee shannon diversity 0.8 (±0.3) 0.6 (±0.4) 4 0.83349 0.6882 0.4534 ps gonçalves; ws araújo – eusocial bees in a tropical dry forest6 forest. in recent years the tropical dry forests in the northern of minas gerais have suffered considerable deforestation due to the increase in urban zones, agriculture and pastures (dupin et al., 2018). the intensification of land use and the modification of natural landscapes can lead to the extinction of many species of active bees and compromise the quality of ecological pollination services (potts et al., 2010; tylianakis, 2013). little is known about the bee fauna in the tropical dry forests (alvarenga et al., 2020), which reveals the importance of carrying out further studies, especially in the northern of minas gerais. therefore, future studies can investigate whether these patterns are recurrent in other tropical dry forest areas in the region, as well as compare the diversity of bees between preserved and anthropized environments in other types of vegetation. acknowledgments we wish to thank to professor santos d’angelo neto (in memoriam) for the plant species’ identification; to the colleagues of the laboratory of interactions, ecology and biodiversity – lieb/unimontes for their help during field sampling; to the cnpq for the fellowship granted to the first author (ic-cnpq); to the secretaria municipal do meio ambiente de montes claros (semma) for the collection license, and to the unimontes for its logistical support. authors contribution p.s.g.: conceptualization, methodology, investigation, writing & reviewing w.s.a.: conceptualization, methodology, formal analysis, writing & reviewing references alvarenga, a.s., silveira, f.a., dos santos júnior, j.e., de novais, s.m.a., quesada, m. & neves, f.s. 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(2005). effects of urbanization on neotropical wasp and bee assemblages in a brazilian metropolis. landscape and urban planning, 71: 105-121. doi: 10.1016/j.landurbplan.2004.02.003. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i2.135-144sociobiology 60(2): 135-144 (2013) molecular phylogeny of the ant subfamily formicinae (hymenoptera, formicidae) from china based on mitochondrial genes zl chen1, sy zhou1, dd ye1, y chen1, cw lu1 introduction ants are one of the most successful groups of eusocial insects. they act as an important part of the animal biomass in tropical rainforests and occupy key positions in many terrestrial environments (wilson & hölldobler 2005). resolving the phylogeny of major ant lineages is vital for understanding the factors contributing to their success. previous studies based on morphological (baroni urbani et al. 1992, bolton 2003), fossil-based (grimaldi et al. 1997, dlussky 1999, ward & brady 2003, bolton 2003), and molecular (astruc et al. 2004, saux et al. 2004, ward & brady 2003,ward & downie 2005, ward et al. 2005, brady et al. 2006, moreau et al. 2006, ouellette et al. 2006) data provided useful framework for understanding the relationships among ant subfamilies. however, relationships among genera within the subfamilies are not well understood. in addition, the genus-level phylogeny and classification of ant subfamilies remain controversial in many respects. formicinae is one of the most abundant ant subfamilies abstract to resolve long-standing discrepancies in the relationships among genera within the ant subfamily formicinae, a phylogenetic study of chinese formicine ants based on three mitochondria genes (cyt b, coi, coii) was conducted. phylogenetic trees obtained in the current study are consistent with several previously reported trees based on morphology, and specifically confirm and reinforce the classifications made by bolton (1994). the tribes lasiini, formicini, plagiolepidini and camponotini are strongly supported, while oecophyllini has moderate support despite being consistent across all analyses. we have also established that the genus camponotus and polyrhachis are indeed not monophyletic. additionally, we found strong evidence for polyrhachis paracamponota, as described by wu and wang in 1991, to be corrected as camponotus based on molecular, morphological and behavioral data. sociobiology an international journal on social insects 1 college of life sciences, guangxi normal university, guilin, china research article ants article history edited by: gilberto m. m. santos, uefs brazil received 26 december 2012 initial acceptance 19 february 2013 final acceptance 08 april 2013 keywords ant phylogeny; formicidae; cyt b, coi, coii corresponding author: shan-yi zhou college of life sciences guangxi normal university guilin, 541004, china. e-mail: syzhou5612@yahoo.com.cn in the holarctic (wilson 1955). according to bolton (2012), formicinae includes 49 extant genera and over 3700 species and subspecies in the world. although the subfamily includes a large number of abundant and ecologically important species that are often subjected to ecological and sociobiological studies, little is known about their phylogeny. although there are several classifications based on a variety of morphological characteristics, such as sexual traits and larval morphology (wheeler 1922, emery 1925, wheeler & wheeler 1985, agosti 1991, bolton 1994, 2003), the tribes or genus-groups represent artificial assemblages and are used inconsistently by different myrmecologists or even by the same myrmecologist at different times. in particular, some aspects of worker morphology show a strong tendency towards convergence, making it challenging to infer phylogenetic relationships from morphological characteristics alone (ward 2007). indeed, bolton has acknowledged that some tribes in his tribal arrangements would likely need to be re-evaluated (bolton 2003). no molecular phylogenetic study has been performed on the subfamily formicinae in china to date. this study zl chen, sy zhou, dd ye, y chen, cw lu molecular phylogeny of formicinae from china136 aimed to establish molecular relationships among formicinae members relative to previously established frameworks and to take a deeper look into species level relationships within more ambiguous assemblages. this was done by obtaining sequences of the mitochondrial genes cytochrome b (cyt b), cytochrome oxidase subunit 1 (coi) and cytochrome oxidase subunit 2 (coii) and comparing them using bayesian inference (bi) (nylander 2004), maximum parsimony (mp) and neighbour joining (nj) (swofford 2002). materials and methods taxon sampling in this study, a total of 47 species representing 14 genera from five tribes were selected to test the groups suggested by the tribal structure and dendrograms of wheeler (1922), emery (1925), wheeler and wheeler (1985), agosti (1991), and bolton (1994, 2003). cerapachys sulcinodis from the subfamily cerapachyinae and radoszkowskius oculata from the family mutillidae were added as outgroups. apart from r. oculata, all other vouchers of formicinae and c. sulcinodis, consisting of nestmate specimens from the same collection event have been deposited in the collection of guangxi normal university. detailed information of the species studied is listed in appendix 1. dna extraction, pcr, and sequencing alignment total genomic dna was extracted from ground whole workers, of which the gasters were removed to minimize contamination from gut bacteria, using standard ctab methods (slightly modified from navarro et al. 1999). dna sequence data from three protein-coding mitochondrial genes, namely cyt b, coi, and coii, were obtained using conventional pcr methods (villesen et al. 2004, ward & downie 2005). the sequences and positions on the mitochondrial dna of the primers used for pcr and sequencing are shown in table 1. the primers j2791 and h3665 were used to amplify fragments of mitochondrial dna that correspond to the 3’ end of coi, its, and trna-leucine and the 5’ end of coii. fragments were sequenced in both directions, and the resulting chronograms were assembled and edited using dnastar (bioinformatics pioneer dnastar, inc., wi). sequence for each gene fragment was aligned using clustalx v.1.83 (thompson et al. 1997). sites from the intergenic spacer (its) and trna-leucine were not used in the analyses. all new dna sequences generated in this study were submitted to the ncbi genbank database. sequence data of the outgroup r. oculata was obtained via genbank direct submission by wei, s.j. and chen, x.x. all genbank accession numbers related to this study are listed in appendix 1. phylogenetic analyses reconstruction of phylogenetic relationships among taxa was conducted using nj, mp, and bi methods. nj analysis was performed using paup* version 4.0b10 (ppc) (swofford 2002). estimates of nodal support on distance trees were obtained using bootstrap analyses (1000 replications). mp analysis was also unweighted and performed using paup* version 4.0b10 (ppc) (swofford 2002). it involved the use of a heuristic search with random sequence addition (10 replicates each) and the tbr branch-swapping algorithm. bayesian phylogenetics was used to estimate tree topology using mrbayes v.3.1.2 (ronquist & huelsenbeck 2003). data were partitioned by gene to yield a total of three data partitions, and the best-fitting model for each partition was selected using mrmodeltest v. 2.2 (nylander 2004) under akaike information criteria (posada & buckley 2004). results dna sequence composition table 2 shows the nucleotide content and substitution of three fragment sequences. the final data matrix contained 1830 characters (1049 variable sites, 897 parsimony-informative sites, 152 singleton sites) from the following gene fragments: cyt b-447 characters (270 variable sites, 232 parsimony-informative sites, 38 singleton sites), coi-825 aligned characters (433 variable sites, 379 parsimony-informative sites, 54 singleton sites), and coii-558 characters (341 variable sites, 289 parsimony-informative sites, 52 singleton designation sequence (5’–3’) position reference cb-11400 tatgtactacchtgaggdcaaatatc 9381-9406 modified from folmer et al. 1994 cb11884 attacaccncctaatttattaggrat 9840-9865 modified from folmer et al. 1994 lco1490 ggtcaacaaatcataaagatattgg 117-141 modified from folmer et al. 1994 hco2198 taaacttcagggtgaccaaaaaatca 700-726 modified from folmer et al. 1994 j2791 atacchcgdcgataytcaga 1300-1319 modified from chiotis et al. 2000 co-r tcrtgraagaagattatta 1650-1668 this study co-f cttttattaaaaathaacac 1586-1605 this study h3665 ccacaratttcwgaacattg 2177-2196 modified from chiotis et al. 2000 table 1. sequences of primmer used in this study. position refers to coordinates in the solenopsis invicta mitochondrion complete genome, genbank accession numbers: hq215540. primer combinations are as follows, with the forward primer listed wrst for each pair: cb-11400–cb-11884, lco1490–hco2198, j2791–h3665, j2791–coi-r, co-f–h3665. sociobiology 60(2): 135-144 (2013) 137 sites). the base composition of these three fragments varied among the studied species. on average, the base composition was: t 40.8%, c 17.8%, a 31.9%, and g 9.5%, with a strong at bias (72.7%) as is commonly found in other insect mitochondrial genomes (vogler & pearson 1996). the a+t contents of the third, second and first codon position from the three fragments were 84.2%, 66.2%, and 67.4%, respectively. the transitions of nucleotide substitution were more common than transversion with a transition. numerically, the transversion between a and t was the highest among the four types of nucleotide transversions, whereas the transition between c and t was the highest of the two types of nucleotide transitions. amino acid composition and substitution saturation the complete 1830 nucleotide sequence encoded 610 amino acids of 20 different types. leucine (leu) was the most frequent (13.53%) followed by isoleucine (ile) (13.30%). cysteine (cys) was the least frequent, with a constant content of 0.29%. all three protein-coding genes were tested for saturation. these were achieved by plotting the numbers of observed substitutions versus the uncorrected p-distance estimates. the scattergrams (fig. 1) show that tv increased along the uncorrected p-distance and ts reached saturation between certain pairs of taxa. phylogenetic trees phylogenetic analyses (figs. 2 to 4) showed that the outgroups c. sulcinodis and r. oculata were well-resolved from the formicinae taxa at the base of the trees with high confidence values (0.94 bayesian posterior probability (pp), 100% nj bootstrap, 99% mp bootstrap). as shown in figure 5e (this figure was synthesized from figs. 2 to 4 ), all consensus trees strongly indicated that the 14 genera of formicinae could be divided into five lineages, which we labeled as clades i-v, and consisted of genera from the tribes lasiini, formicini, oecophyllini, plagiolepidini and camponotini, respectively. our findings are consistent with morphological classifications of bolton (1994) (figs. 5e and 5f). clade i included four genera: lasius, nylanderia, prenolepis, pseudolasius (1.0 pp, 84% nj bootstrap, 54% mp bootstrap). pseudolasius appeared to be a sister group of 0 50 100 150 200 250 300 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 p-distance t s o r t v v a l u e s ts tv genes cs v pi s nuleotide content (%) nuleotide substitution t c a g a+t ii si sv r coi (825) 392 433 379 54 40.8 18.3 30.1 10.7 70.9 664 78 83 0.94 coii (558) 217 341 289 52 40.7 16.6 35.2 7.4 75.9 440 52 66 0.79 cyt b (447) 177 270 232 38 40.7 18.4 31.2 9.7 71.9 349 45 52 0.88 total (1830) 781 1049 879 152 40.8 17.8 31.9 9.5 72.7 1454 175 200 0.87 (lasius + (nylanderia + prenolepis)) in all three trees. these analyses showed that nylanderia is a sister genus of prenolepis with very strong support (1.0 pp, 90% nj bootstrap, 89% mp bootstrap). a supported clade of ((formica + polyergus) + (proformica + cataglyphis)) (1.0 pp, 73% nj bootstrap, 73% mp bootstrap) forms clade ii. our analyses showed formica as a sister genus of polyergus (1.0 pp, 97% nj bootstrap, 97% mp bootstrap), and proformica as a sister genus of cataglyphis with very strong support (1.0 pp, 97% nj bootstrap, 91% mp bootstrap) in all trees. clade iii included only one species (oecophylla smaragdina) and was placed as a sister group to clade ii. although this species was not supported by strong bootstrap values (0.58 pp, 54% nj bootstrap, 16% mp bootstrap), it was a consistent feature in all reconstructions. clade iv comprised of three genera: anoplolepis, a sister group to (plagiolepis + lepisiota). the genus plagiolepis and lepisiota also formed a sister group with good support in all trees. clade v included camponotus and polyrhachis with very strong support (1.0 pp, 100% nj bootstrap, 87% mp bootstrap). however the species-level phylogeny of the genera remains unresolved except for the distinct subclade of fig.1. scatterplots showing the number of substitutions (y-axes; ts, transitions; tv, transversions) versus uncorrected p-distance (x-axes) at each codon position. table 2. the content and substation of nucleotide sequences. cs, conserved sites; v, variable sites; pi, parsimony-informative sites; s, singleton sites; ii, identical pairs; si, transitional pairs; sv, transversional pairs; r, ts/tv. zl chen, sy zhou, dd ye, y chen, cw lu molecular phylogeny of formicinae from china138 (c. mitis + (c. vanispinus + (c. jianghuaensis + c. albosparsus))). c. singularis is a sister species of other species of the genus camponotus (including polyrhachis paracamponota, excluding c. yiningensis) with very strong support (98% nj bootstrap) in the nj tree (fig. 3) and modest support (67% mp bootstrap) in the mp tree (fig. 2). however, in the bi tree (fig. 4), c. parius first clustered with c. wasmanni with strong support (1.0 pp) and then as a sister group of c. sinfig. 2. maximum-parsimony (mp) consensus tree from 1000 bootstrap replicates, obtained from 48 species of the concatenated sequences of the cytb gene (447 bp), coi gene (825 bp) and coii gene (558 bp), with cerapachy sulcinodis and radoszkowskius oculata as the outgroups. gularis plus the rest of the species of camponotus (including p. paracamponota, excluding c. yiningensis). c. yiningensis was tightly associated with polyrhachis with very strong support (1.0 pp, 100% nj bootstrap, 87% mp bootstrap), and further studies on its status are needed. the species p. paracamponota clustered with camponotus, and was distinct from polyrhachis. sociobiology 60(2): 135-144 (2013) 139 fi g. 3 . n ei gh bo rjo in in g (n j) c on se ns us tr ee f ro m 1 00 0 bo ot st ra p re pl ic at es , o bt ai ne d fr om 4 8 sp ec ie s of th e co nc at en at ed s eq ue nc es o f th e c yt b g en e (4 47 b p) , c o i g en e (8 25 b p) a nd c o ii ge ne (5 58 b p) , w ith c er ap ac hy s ul ci no di s an d r ad os zk ow sk iu s oc ul at a as th e ou tg ro up s. fi g. 4 . b ay es ia n (b i) m aj or ity -r ul e co ns en su s tr ee , o bt ai ne d fr om 4 8 sp ec ie s of th e co nc at en at ed s eq ue nc es o f th e c yt b g en e (4 47 b p) , c o i ge ne ( 82 5 bp ) an d c o ii g en e (5 58 b p) th re e pa rt iti on s al l u nd er th e sa m e be st -fi t m od el (g t r +i +g ) s el ec tin g by a ic in m od el te st , w ith c er ap ac hy s ul ci no di s an d r ad os zk ow sk iu s oc ul at a as th e ou tg ro up s. zl chen, sy zhou, dd ye, y chen, cw lu molecular phylogeny of formicinae from china140 lasius, prenolepis, nylanderia and lasius were placed and formed the tribe lasiini, but disagrees with that of bolton (2003), in which the genera plagiolepis and lepisiota were added to form the tribe plagiolepidini. in addition, these four genera formed a strongly supported group in all trees, especially in the case of the sister genus relationship between nylanderia and prenolepis (1.0pp, 90% nj bootstrap, 99% mp bootstrap). these results are consistent with those of previous morphological (emery 1925, wheeler & wheeler 1953, trager 1984) and molecular studies (brady et al. 2006), however, in the study of moreau et al. (2006), the genus plagiolepis, pseudolasius and prenolepis emerges first, followed by lasius along with other two genera. besides the study by lapolla et al. (2010) in which prenolepis was treated as being paraphyletic to the group. in addition, monophyly of the genus lasius was strongly supported (0.99 pp, 90% nj bootstrap, 99% mp bootstrap). the results for clade ii are consistent with those of previous studies (bolton 1994, 2003) (figs. 5e, 5f and 5b). genera of the tribe formicini share the following morphological features (bolton 1994): 12-segmented antennae, antennal fig. 5 classifications of formicine genera based on the schemes of: (a) wheeler wm 1922; (b) bolton 2003; (c) wheeler, wm et al. 1985; (d) agosti 1991; (e) this study; (f) bolton 1994. {nb: only positions for species of interest in this phylogeny are noted; there are changes in classifications of other genera which are not being used in this study }. discussion results of the phylogenetic relationships of formicinae in this study (figs. 2 to 4, 5e) showed both similarities and differences compared with those of previous studies (fig. 5a-5d, 5f). surprisingly, results of our molecular phylogenetic trees have better fit with the morphological cladogram of bolton (1994), with which they are congruent, than with that of bolton (2003). clade i is best characterized morphologically with the worker alitrunk not conspicuously constricted or otherwise specialized and the mesonotum typically convex in profile view. the workers of lasius, nylanderia and prenolepis shared the following morphological characters (bolton 1994): mandibles roughly triangular with four to seven teeth, antennae 12-segmented, the torula close to but not touching the posterior clypeal margin. a propodeal spiracle present at or near the declivity of the propodeum, and the petiolar node in profile usually inclined forward, with a short anterior face and much longer posterior face. these data support the earlier hypothesis proposed by bolton in 1994, into which pseudosociobiology 60(2): 135-144 (2013) 141 sockets situated close to the posterior clypeal margin. orifices of propodeal spiracle oval, elliptical, or as elongated slits and near-vertical or inclined from the vertical. all of these analyses provided strong support for the two sister-group relationships of (formica + polyergus) and (proformica + cataglyphis), which is consistent with the molecular studies of moreau et al. (2006). in clade iii, the genus oecophylla was separated as a distinct lineage. this result is well supported by previous morphological studies (wheeler 1922, wheeler & wheeler 1985, bolton 1994, 2003) (fig. 5), which showed oecophylla as the tribe oecophllini. in our molecular phylogeny, oecophylla appears to be a sister of formicini but with low bootstrap support (0.58 pp, 0.54% nj bootstrap, 16% mp bootstrap). however, this topology is in agreement with that of moreau et al. (2006). wilson and taylor (1964) also suggested that oecophylla and clade ii cannot be given much credence considering the separate placement in morphologically and parsimony-based phylogenies, as well as its current geographical separation. however, fossil evidence indicate that oecophylla previously occurred in europe, suggesting that these genera may have shared a common ancestor. clade iv is a well supported clade consisting of members from the tribe plagiolepidini (anoplolepis + (plagiolepis + lepisiota)) (0.95 pp, 82% nj bootstrap, 53% mp bootstrap). bolton (1994) had previously placed the three genera into the tribe plagiolepidini based on a morphological study (fig. 5f) and the current study is the first to arrive at the same placement based on molecular phylogenetics. this tribe is distinguished by the following features: worker with 11-segmented antennae, antennal sockets fused with the posterior clypeal margin, and palp formula of 6,4. surprisingly, bolton (2003) proposed the genus plagiolepis and lepisiota to be included in the tribe plagiolepidini (fig. 5b). although bolton (2003) represents a more comprehensive summary of ant morphological characters assembled to date than his previous treatment (bolton 1994), it is likely that this reflects a genuine conflict between morphology and molecular data. clade v is strongly supported in all trees (1.0 pp, 100% nj bootstrap, 87% mp bootstrap) and consists of camponotus and polyrhachis. this result is in agreement with previous morphological (wheeler 1922, emery 1925b, wheeler & wheeler 1985, bolton 1994, 2003) (figs. 5) and molecular studies (astruc et al. 2004, brady et al. 2006, moreau et al. 2006). the tribe camponotini can be characterized by its 12segmented antennae, with antennal sockets situated far behind the posterior clypeal margin, and a palp formula of 6,4. camponotus is however a paraphyletic group, as is noted in other studies (brady et al. 1999, astruc et al. 2004, brady et al. 2006). camponotus yiningensis has been placed outside of the genus camponotus, which has been confirmed not to be monophyletic (brady et al. 1999, 2000; astruc et al. 2004, brady et al. 2006). morphological characters also reflected close, and sometimes overlapping, relationships between camponotus and polyrhachis. for instance, many species of camponotus acquired distinctive spines, and many species of polyrhachis have camber-shaped alitrunks. the species polyrhachis paracamponota was first described by wang and wu in 1991 based on a single holotype worker which possesses pronotal spines, and was placed in the genus polyrhachis. but having pronotal spines is very common in camponotus and polyrhachis, this morphological character could not be used for distinguishing between the two genera. the original descriptions exact match with the morphological character of the genus camponotus. in our opinion, the authorships also had the same idea, so this species be named “paracamponota”. besides, this species has polymorphic workers, and they have been observed to tunnel into the soil for subterranean nesting. in contrast, the workers of polyrhachis are exclusively monomorphic, and can only use existing cavities in the soil or under stones for nesting, but never excavate tunnels themselves. our phylogenetic reconstruction indicated that this species is associated with camponotus, and is clearly separated from polyrhachis. as such, there is strong evidence from morphological, behavioristic and molecular data that polyrhachis paracamponota should be placed as a member of camponotus. conclusion in conclusion, our study of the phylogenetic relationship of formicinae from china based on sequences from three protein-coding mitochondrial genes (cyt b, coi, coii) confirms and reinforces the findings of previous morphological studies (bolton 1994). the tribes lasiini (pseudolasius, prenolepis, paratrechina, lasius), formicini (formica, cataglyphis, proformica, polyergus), plagiolepidini (lepisiota, plagiolepis, anoplolepis), and camponotini (camponotus, polyrhachis) are strongly supported, while oecophyllini has moderate support despite being consistent across all analyses. we have also established that the genus camponotus and polyrhachis are indeed not monophyletic. additionally, evidence from molecular, morphological and behavioral data indicates that polyhachis paracamponota should be corrected as camponotus. acknowledgments we sincerely thank professor yu-feng xu (national taiwan normal university, taiwan), dr. jun-hao tang (national university of singapore) and dr. john r. fellowes (kadoorie farm and botanic garden, hong kong) for reviewing the english text. we thank two anonymous reviewers for helpful comments on the manuscript. thanks also to chao-tai wei (guangxi normal university) for providing us with some ant materials, de-long zeng (guangxi normal university) for helpful assistance and comments on phylogeny analysis. this study was supported by the national natural science zl chen, sy zhou, dd ye, y chen, cw lu molecular phylogeny of formicinae from china142 foundation of china (project nos. 30770258 and 31071971), foundation of the key laboratory of ecology of rare and endangered species and environmental protection, ministry of education, guangxi normal university. references agosti, d. 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(2005). the rise of the ants: a phylogenetic and ecological explanation. proc. nat. acad. sci. usa, 102: 7411-7414. zl chen, sy zhou, dd ye, y chen, cw lu molecular phylogeny of formicinae from china144 appendix 1 species collection locality voucher specimen genbank accession numbers cyt b coi coi & coii lepisiota xichangensis jingxi, guangxi gxjx0006 jq681097 jq681046 jq680992 plagiolepis manczshurica helan mt, inner mongolia nmhl0422 jq681098 jq681047 jq680993 plagiolepis rothneyi xiangtou mt, guangdong gdxt0122 jq681099 jq681048 jq680994 anoplolepis gracilipes beiliu, guangxi gxbl0001 jq681100 jq681049 jq680995 anoplolepis sp. bohai, yunnan ynbh0003 jq681101 jq681050 jq680996 pseudolasius cibdelus jingxi, guangxi gxjx0031 jq681102 jq681051 jq680997 pseudolasius similus jingxi, guangxi nmhl0269 jq681103 jq681052 jq680998 prenolepis sphingthorax jingxi, guangxi gxjx0144 jq681104 jq681053 jq680999 cataglyphis aenescens heze, shandong shandong_70 jq681105 hq619705 jq681000 cataglyphis sp. yangling, shanxi sxyl0007 jq681106 jq681054 jq681001 formica candida xiaowutai mt, hebei hebei_50 jq681107 hq619704 jq681002 formica longicepes helan mt, inner mongolia nmhl0227 jq681108 jq681055 jq681003 formica cunicularia xiaowutai mt, hebei hebei_307 jq681109 hq619714 jq681004 formica lemani xiaowutai mt, hebei hebei_251 jq681110 hq619712 jq681005 proformica mongolica helan mt, inner mongolia nmhl0045 jq681111 jq681056 jq681006 proformica jacoti xiaowutai mt, hebei hbxw0039 jq681112 jq681057 jq681007 nylanderia flavipes heze, shandong sdhz0104 jq681113 jq681058 jq681008 nylanderia vividula guilin, guangxi gxgl0111 jq681149 jq681093 jq681044 nylanderia bourbonica jingxi, guangxi gxjx0022 jq681114 jq681059 jq681009 lasius niger xiaowutai mt, hebei hbxw0263 jq681115 jq681060 jq681010 lasius flavus helan mt, inner mongolia nmhl0320 jq681116 jq681061 jq681011 lasius fuliginosus xiaowutai mt, hebei hbxw0266 jq681117 jq681062 jq681012 lasius alienus helan mt, inner mongolia nmhl0316 jq681118 jq681063 jq681013 oecophylla smaragdina xiangtou mt, guangdong gdxt0104 jq681119 jq681064 jq681014 polyrhachis illaudata jingxi, guangxi gxjx0141 jq681120 jq681065 jq681015 polyrhachis halidayi jingxi, guangxi gdjx0024 jq681121 jq681066 jq681016 polyrhachis rastellata rong’an, guangxi gxra0045 jq681122 jq681067 jq681017 polyrhachis dives beiliu, guangxi gxgl0099 jq681123 jq681068 jq681018 polyrhachis jianghuaensis beiliu, guangxi gxbl0006 jq681124 jq681069 jq681019 polyrhachis paracampponota jingxi, guangxi gxjx0009 jq681125 jq681070 jq681020 camponotus variegatus jingxi, guangxi gxjx0155 jq681126 jq681071 jq681021 camponotus herculeanus helan mt, inner mongolia nmhl0273 jq681127 jq681072 jq681022 camponotus albosparsus jingxi, guangxi gxjx0130 jq681128 jq681073 jq681023 camponotus vanispinus jingxi, guangxi gxjx0007 jq681129 jq681074 jq681024 camponotus wasmanni xiangtou mt, guangdong gdxt0102 jq681130 jq681075 jq681025 camponotus dolendus jingxi, guangxi gxjx0036 jq681131 jq681076 jq681026 camponotus jianghuaensis rong’an, guangxi gxra0010 jq681132 jq681077 jq681027 camponotus mitis bohai, yunnan ynbh0111 jq681133 jq681078 jq681028 camponotus helvus jingxi, guangxi gxjx0015 jq681134 jq681079 jq681029 camponotus yiningensis jingxi, guangxi gxjx0013 jq681135 jq681080 jq681030 camponotus albivillosus helan mt, inner mongolia nmhl2122 jq681136 jq681081 jq681031 camponotus lasiselene jingxi, guangxi gxjx0012 jq681137 jq681082 jq681032 camponotus parius beiliu, guangxi gxbl0009 jq681138 jq681083 jq681033 camponotus singularis beiliu, guangxi gxbl0008 jq681139 jq681084 jq681034 camponotus sp. 1 jingxi, guangxi gxjx0017 jq681140 jq681085 jq681035 camponotus sp. 2 jingxi, guangxi gxjx0123 jq681141 jq681086 jq681036 polyergus samurai beiliu, guangxi gxbl0212 jq681142 jq681087 jq681037 out-group cerapachys sulcinodis beiliu, guangxi gxbl0095 jq681145 jq681090 jq681040 radoszkowskius oculata from genbank nc_014485 nc_014485 nc_014485 doi: 10.13102/sociobiology.v62i1.34-38sociobiology 62(1): 34-38 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 absence of the parasite escovopsis in fungus garden pellets carried by gynes of atta sexdens introduction leaf-cutting ants in the genera atta fabricius and acromyrmex mayr (hymenoptera: formicidae) cultivate a mutualistic fungus for food. the ants maintain the fungal cultivar (leucoagaricus gongylophorus (moller) singer; basidiomycota: agaricales) in a structure named the fungus garden, composed of fungal mycelium and plant material collected by workers. fungus gardens are threatened by the specialized microfungal parasite escovopsis muchovej & della lucia (ascomycota: hypocreales), which negatively impacts the development of the colony. this parasite is highly virulent and may overgrow the ant gardens leading to the death of the colony (currie et al., 1999; currie, 2001a). it is suggested that microorganisms, especially antibioticsecreting actinobacteria found on the integument of workers protect the fungus gardens against this parasite (currie, 2001b; mueller et al., 2008; caldera et al., 2009; barke et al., 2011). abstract before the mating flight, the gyne of leaf-cutting ants takes a small pellet of their mutualistic fungus garden to start fungus culture in its new colony by vertically transmitting it. this mutualism is threatened by the specialized microfungal parasite escovopsis, which is exclusively associated with the ant’s fungus gardens. evidences suggest that escovopsis transmission between colonies is horizontal, i.e. the parasite is transferred between established nests. however, such studies analyzed a relatively small number of fungal pellets or were restricted to a few ant colonies. additionally, there is a report of rapid parasite dispersion, compatible with a winged vectored mechanism, suggesting that there is also vertical transmission. herein, we carried out a complementary study on the possibility of vertical transmission of escovopsis by sampling a large number of fungus pellets from gynes of atta sexdens, a species not previously studied from this perspective. gynes were collected during their mating flights in 2009 and 2010, and were left in moist chambers upon fungus regurgitation. each pellet was inoculated on potato dextrose agar and incubated at 25 °c, resulting in prevalence of the mutualistic cultivar, low proportions of other fungal species, and absence of escovopsis. thus, our study consolidates the results of previous reports that escovopsis vertical transmission does not occur or is negligible, thus enabling the characterization of this parasite transmission as horizontal. future studies on escovopsis transmission mechanisms may explain why, although horizontal, it seems to be as fast as the transmission mediated by winged vectors. sociobiology an international journal on social insects sm moreira1; a rodrigues2; lc forti1; ns nagamoto1 article history edited by rodrigo m. feitosa, ufpr, brazil received 31 january 2014 initial acceptance 23 march 2014 final acceptance 11 february 2015 keywords fungus growing ants, transmission, mating flight, parasitism. corresponding author nilson satoru nagamoto depto. proteção vegetal, fca, unesp 18.610-307, botucatu, sp, brazil. pob 237 e-mail: nsnagamoto@yahoo.com before leaving for the mating flight gynes collect a small fungus garden fragment and stores it in the infrabuccal pocket, which is located in the oral cavity of the ant. then, males and gynes from different leaf-cutting ant colonies simultaneously leave their parental nest to mate during the mating flight. once fertilized gyne reach the ground, they remove their wings and start the foundation of a new colony. the fungal pellet is then regurgitated by the gyne who starts the culturing of the new incipient fungus garden. this mode of transmission of the fungal cultivar is considered vertical (from parental to offspring colony). in atta, the gyne is responsible for the excavation of a tunnel and the first nest chamber in which she remains underground for about two months (claustral nest founding). after this period, the first emerged workers reopen the tunnel that connects to the ground surface (autuori, 1941; moser, 1967). much is known about the interaction between leafcutting ants and their mutualistic fungus, however, little research article ants 1 universidade estadual paulista (unesp), botucatu, sp, brazil 2 universidade estadual paulista (unesp), rio claro, sp, brazil sociobiology 62(1): 34-38 (march, 2015) 35 is known about escovopsis biology, especially its mode of transmission (currie, 2001b; yek et al., 2012). two studies, currie et al. (1999) and pagnocca et al. (2008) evaluated the transmission of the parasite between colonies. these reports indicate that no vertical transmission appears to occur (table 1). currie et al. (1999) evaluated pellets of gynes in their mating flight and in the fungus gardens of colonies in the claustral phase. however, the sampling size was relatively small and the study was restricted to one ant species (atta colombica guérin-méneville in panama). on the other hand, a second study evaluated the integument and fungal pellets of gynes collected immediately before the mating flight (when gynes were leaving the parental colony). although, pagnocca et al. (2008) used a larger sampling size in comparison to the currie et al.’s (1999) study (table 1) only one colony of atta laevigata f. smith and two colonies of atta capiguara gonçalves were sampled, in brazil. in addition, the variation of escovopsis prevalence between ant species and locations are aspects that prompt for additional studies to extend the existing data on the mode of transmission of this parasite (currie, 2001a; yek et al., 2012). considering that escovopsis was not found in air samples collected next to the ant colonies, and because its spores are typically slimy when mature (wet spores), it is inferred that it is horizontally transmitted (between established nests) through the migration of commensal arthropods, or via some other unknown mechanism (currie, 2001b; poulsen & currie, 2006; yek et al., 2012). moreover, multiple lines of evidence indicate this parasite appears to have a close relationship with fungus growing ant colonies because: (i) it was only found in the fungus gardens and waste material (muchovej & della lucia, 1990; seifert et al., 1995; currie et al., 1999; rodrigues et al., 2014); (ii) phylogenetic studies showed that specific escovopsis lineages are specialized on specific lineages of the ant cultivar (currie et al., 2003; gerardo et al., 2006a,b); and (iii) additional energy is necessary to grow the actinobacteria which are considered as a defense mechanism against this parasite, and the cultivation of such actinobacteria is enhanced in response to escovopsis infection (poulsen et al., 2003). interestingly, mikheyev (2008) demonstrated that a recent colonization by acromyrmex octospinosus reich in the guadeloupe island was probably initiated by a single fertilized gyne or colony derived from the continent. surprisingly, escovopsis was found in 12% of the established colonies on the island and worker ants were covered with white blooms of actinobacteria on their integuments. in addition, a study on young colonies of atta colombica (currie et al., 1999) indicates that shortly after the reopening of the tunnel, the fungus gardens of these colonies were infected by escovopsis. therefore, it may be considered that (i) the mechanisms of horizontal transmission of the parasite are quick and efficient, or (ii) vertical transmission is possible. because escovopsis is a specialized parasite (currie et al., 1999, 2003; poulsen & currie, 2006; yek et al., 2012), it can hypothetically exploit the fastest and most efficient mode of transmission, i.e. vertical transmission. this hypothesis table 1. studies involving the isolation of filamentous fungi from leaf-cutting ants during the mating flight period. study n sample type ant species year of collection site currie et al. (1999) 38 gyne pellets, after the mating flight* atta colombica 1998 gamboa, panama 8 gardens of incipient laboratory colonies** (aseptic rearing) atta colombica 1998 gamboa, panama 22 gardens of incipient field colonies** (in claustral phase) atta colombica 1998 gamboa, panama pagnocca et al. (2008) 144 gyne pellets + cuticle, before the mating flight* atta laevigata (1 colony) 2006 + 2007 santana farm, botucatu, brazil 120 gyne pellets + cuticle, before the mating flight* atta capiguara (2 colonies) 2006 + 2007 this work 267 gyne pellets, after the mating flight* atta sexdens 2009 + 2010 lageado farm, botucatu, brazil * isolation attempts were carried out in the same day of the mating flight. ** age of few weeks. may be considered plausible because it explains the result of mikheyev’s (2008). similarly, in systems of figs and their symbionts, the specific phytonematode schistonchus cobb is transmitted rapidly and efficiently by its winged vector (fig wasp, pollinator of figs): this transmission is vertical, permitting a colonization of new figs upon the arrival of this pollinator (vovlas et al., 1992; krishnan et al., 2010). thus, an additional investigation of the presence of escovopsis in fungal pellet, the most likely location for this parasite to be present in case of vertical transmission (currie et al., 1999), is critical. in the present work we investigate if escovopsis utilizes the mode of vertical transmission by carrying out a larger survey of atta sexdens linnaeus gyne pellets and comparing our results to existing studies. we focused our work on atta sexdens because it is one of the most common and sm moreira; a rodrigues; lc forti; ns nagamoto absence of the parasite escovopsis in gynes of atta sexdens36 widespread leaf-cutting ant species in latin america (solomon et al., 2008), and due to the fact that the gynes of this species have not been investigated for the presence of escovopsis. material and methods for gyne collection, we selected a small site (c.a. 1.3 ha) in a eucalyptus spp. plantation at lageado farm (22°49’53.25” s and 48º25’24.22” w), in botucatu, sp, brazil. this area did not contain any mature atta nests, but it was being used as an intense landing site of fertilized gynes. atta gynes can fly up to several km in nuptial flights (moser, 1967; jutsum & quinlan, 1978; ns nagamoto, personal obs.); thus, gynes collected in our small area actually came from surrounding areas (fragments of native forest, and plantation areas for such crops as cereals, grasses, eucalyptus l’heritier, and citrus linnaeus). in undisturbed forests, the atta spp. colony density is about 0.23 ha-1, lower than the trend in anthropically disturbed areas (wirth et al., 2007 and references included); thus, potentially dozens or hundreds of mature a. sexdens nests had contributed gynes to the collection area. this neighborhood includes some study areas where escovopsis was previously found: in a small colony of rodrigues et al. (2014) and in another work (ns nagamoto et al., unpublished data), it was demonstrated that one of the colonies studied by pagnocca et al. (2008), denominated “atta capiguara colony 1”, was indeed infected with this parasite. additionally, based on the long persistence of these ants’ colonization in these areas (ns nagamoto, personal obs.), it was inferred that the rate of colonies infected by escovopsis was probably high. gynes were sampled in two consecutive years: (i) on october 21th, 2009, 147 gynes were collected, of which 127 pellets were used, and (ii) on october 31th, 2010, 150 gynes, of which 140 pellets were used (some gynes did not regurgitate pellets; a few pellets were lost to contamination by bacteria; or no fungus grew on pellets). sampling was carried out after the mating flight, when gynes were still walking on the soil, or just after they started the excavation of the first tunnel. ants were collected with tweezers and placed into sterile disposable petri dishes that were wrapped in plastic bags. in the laboratory, gynes were transferred to a wet chamber (fig 1a), which consisted of a petri dish of 60 x 12 mm, that was placed in a bottom of a larger petri dish of 100 x 16 mm, with addition of 20 ml of water agar (as a source of moisture) between the plates, and covered by the 100 mm petri plate cover; and then, sealed with pvc film. after 24 hours, the regurgitated pellets were aseptically inoculated in petri dishes containing potato dextrose agar (pda, acumedia) supplemented with 50 mg l-1 of penicillin g and streptomycin sulfate (sigma) according to currie et al. (1999). plates were sealed with pvc film and incubated at 25 °c in the dark. plates were visually inspected over 21 days and once a fungus was detected on the pellets, we subcultured it in pda in order to obtain pure cultures. filamentous fungi identification was performed using morphological markers as stated in the classical taxonomic keys (domsch et al., 1980; samson et al., 2000). specific literature was used for morphological identification of the mutualistic fungus (pagnocca et al., 2001) and escovopsis (muchovej & della lucia, 1990; seifert et al., 1995). fig 1. general aspect of the wet chamber used in the study. moist chambers were made from petri dish of 60 x 12 submerged in an another dish of 100 x 16 mm containing water-agar (medium dyed red for contrast for picture) to promote regurgitation of pellets of the mutualistic fungus (a); the mutualistic fungus (b); filamentous fungi isolated from the pellet (c); parasitic fungus escovopsis, isolated from a colony of atta sexdens (d). for b, c and d, the culture medium is pda (potato dextrose agar). results and discussion in accord with previous studies of other atta species (currie et al., 1999; pagnocca et al., 2008), our research supports the hypothesis that escovopsis (fig 1d) is not found in the pellets of atta sexdens gynes (fig 2). as expected, l. gongylophorus (fig 1b) was recovered in a high proportion of the pellets collected in the two year sampling (70.1% in 2009 and 85.0% in 2010). in addition, other filamentous fungi (fig 1c) were isolated from pellets but at lower proportions (29.9% in 2009 and 15.0% in 2010) when compared to the mutualistic fungus. fig 2. prevalence of filamentous fungi in pellets extracted from gynes of atta sexdens collected in the mating flights of 2009 (n = 127 pellets) and 2010 (n = 140). the specialized parasite fungus escovopsis was not found in the survey. sociobiology 62(1): 34-38 (march, 2015) 37 the upper half of the garden, where escovopsis is less likely to be found (currie, 2001a). if these explanations are correct, even if escovopsis is present in a colony, this parasite would not be transported by gynes when they leave for their nuptial flight. this may explain why the present work and other studies (currie et al., 1999; pagnocca et al., 2008) have not detected escovopsis in fungal pellets. thus, in contrast with the low efficiency of horizontal transmission of non-specialized parasites, such as metarhizium, which are widely distributed in the environment (hughes et al. 2004), it is indicated that there may be highly effective specific mechanisms for horizontal transfer of escovopsis, which is only found in colonies of fungus-growing ants. future studies that investigate the horizontal transmission of escovopsis should clarify why the phenomenon appears to occur as rapidly as transmission mediated by winged vectors, and specify the mechanisms that augment transmission efficiency. acknowledgments we would like to thank cnpq (conselho nacional de desenvolvimento científico e tecnológico) for a scholarship granted to the first author, and the research productivity fellowship granted to lc forti (grant 301718/2013-0). we also wish to thank rmm adams and one anonymous referee for their valuable contributions to this manuscript. references autuori, m. (1941). contribuição para o conhecimento da saúva (atta spp. – hymenoptera: formicidae). i – evolução do sauveiro (atta sexdens rubropilosa forel, 1908). arquivos do instituto biológico, 12: 197-228. barke, j., seipke, r.f., yu, d.w. & hutchings, m.i. (2011). a mutualistic microbiome: how do fungus-growing ants select their antibiotic-producing bacteria? communicative and integrative biology, 4: 41-43. retrieved from http:// www.landesbioscience.com/journals/cib/barkecib4-1.pdf (accessed 01.20.2013) caldera, e.j., poulsen, m., suen, g. & currie, c.r. (2009). insect symbioses: a case study of past, present, and future fungusgrowing ant research. environmental entomology, 38: 78-92. doi: 10.1603/022.038.0110 currie, c.r. (2001a). prevalence and impact of a virulent parasite on a tripartite mutualism. oecologia, 128: 99-106. currie, c.r. (2001b). a community of ants, fungi, and bacteria: a multilateral approach to studying symbiosis. annual review of microbiology, 55: 357-380. currie, c.r., wong, b., stuart, a.e., schultz, t.r., rehner, s.a., mueller, u.g., sung, g., spatafora, j.w. & straus, n.a. (2003). ancient tripartite coevolution in the attine ant– microbe symbiosis. science, 299: 386-388. pagnocca et al. (2008) also found a significant presence of other filamentous fungi in the gyne’s infrabuccal pellets, indicating these microorganisms may be accidentally acquired together with the fungal garden fragments. in both studies, the number of pellets where the fungal mutualist was recovered was higher than the number of pellets with the presence of non-mutualistic fungi. many fungi such as cladosporium link found in pellets of a. capiguara and a. laevigata (pagnocca et al., 2008) are fast growing, and may be found in lower proportions in the fungal pellets when compared with the mutualistic fungus. however, such fungi still can easily overgrow the ant cultivar in the absence of the ants’ care but may not impact the successful initiation of a new garden by the gyne (poulsen & currie, 2006). our results corroborate those of previous studies, thus indicating that vertical transmission of escovopsis does not exist or, if it exists, is negligible. both horizontal and vertical transmission can be used by some specific parasites to infect a host population (fries & camazine, 2001). however, as to parasites that possess specific mechanisms such as chemotaxis , vertical transmission is more plausible: in fig systems, the plant parasitic nematode schistonchus presents chemotaxis to its vector (fig wasp, pollinator of figs) and thus accomplishes its vertical transmission by this mechanism (krishnan et al., 2010). thus, this example contrasts with the non-vertical transmission of escovopsis because it also presents chemotaxis, in this case, to the mutualistic fungus of leaf-cutting ants (gerardo et al., 2006a), and since this mutualist is vertically transmitted by winged gynes. horizontal transmission is expected in parasites that are generalist and widespread in the environment, such as metarhizium sorokin, which causes only sporadic horizontal infection in leaf-cutting ant workers (hughes et al. 2004). in contrast, taking into account that escovopsis quickly pursued the acromyrmex dispersion in islands (mikheyev, 2008), has shown fine evolutionary coupling to the mutualistic fungus (gerardo et al., 2006b), presents chemotaxis (gerardo et al., 2006a), is found only in fungus growing ant nests (currie et al., 1999, 2001a,b; rodrigues et al., 2014) and in their dumps (currie et al. 2001a), and is not transmitted by air (currie et al., 1999, 2001b) – non-vertical transmission was not the most expected result. additionally, the bacterium paenibacillus larvae white, that causes the specific disease american foulbrood in apis linnaeus bee larvae, is transmitted vertically and horizontally (fries & camazine, 2001), in contrast to the lack of vertical transmission of escovopsis. therefore, for now, the mode of escovopsis transmission may be better explained by emphasizing some specific mechanisms, rather than the general patterns of parasite transmission. leafcutting ant workers can detect fungal pathogens, such as escovopsis, when performing their grooming behavior (currie & stuart, 2001; yek et al., 2012); therefore, it is likely that gynes can also achieve this detection when gathering material to form their infrabuccal pellet. thus, gynes may be capable of selectively exploring the healthiest portions of fungus garden, focusing on sm moreira; a rodrigues; lc forti; ns nagamoto absence of the parasite escovopsis in gynes of atta sexdens38 currie, c.r., muller u.g. & malloch d. (1999). the agricultural pathology of ant fungus gardens. proceedings of the national academy of sciences usa., 96: 7998-8002. currie, c.r. & stuart, a.e. (2001). weeding and grooming of pathogens in agriculture by ants. proceedings of the royal society b, 268: 1033-1039. domsch, k.h., gams, w. & anderson, t.h. (1980). compendium of soil fungi. london: academic press. fries, i. & camazine, s. (2001). implications of horizontal and vertical pathogen transmission for honey bee epidemiology. apidologie, 32: 199-214. gerardo, n.m., jacobs, s.r., currie, c.r. & mueller, u.g. (2006a). ancient host-pathogen associations maintained by specificity of chemotaxis and antibiosis. plos biololgy, 4(8): e235. doi:10.1371/journal.pbio.0040235 gerardo, n.m., mueller, u.g. & currie, c.r. (2006b). complex host-pathogen coevolution in the apterostigma fungus-growing ant-microbe symbiosis. bmc evolutionary biology, 6: 88. hughes, w.o.h., thomsen, l., eilenberg, j. & boomsma, j.j. (2004). diversity of entomopathogenic fungi near leafcutting ant nests in a neotropical forest, with particular reference to metarhizium anisopliae var. anisopliae. journal of invertebrate pathology, 85: 46-53. jutsum, a.r. & quinlan, r.j. (1978). flight and substrate utilisation in laboratory-reared males of atta sexdens. journal of insect physiology, 24: 821-825. krishnan, a., muralidharan, s., sharma, l. & borges, r.m. (2010). a hitchhiker’s guide to a crowded syconium: how do fig nematodes find the right ride? functional ecology, 24: 741–749. doi: 10.1111/j.1365-2435.2010.01696.x mikheyev, a.s. (2008). history, genetics and pathology of a leaf-cutting ant introduction: a case study of the guadeloupe invasion. biological invasions, 10: 467-473. moser, j.c. (1967). mating activities of atta texana (hymenoptera: formicidae). insectes sociaux, 14: 295-312. muchovej, j.j. & della lucia, t.m.c. (1990). escovopsis, a new genus from leaf-cutting ant nests to replace phialocladus nomen invalidum. mycotaxon, 37: 191-195. mueller, u.g., dash, d., rabeling, c. & rodrigues, a. (2008). coevolution between attine ants and actinomycete bacteria: a reevaluation. evolution, 62: 2894-2912. pagnocca, f.c., bacci, m., fungaro, m. h., bueno, o. c., hebling, m. j., sant'anna & a., capelari, m. (2001). rapd analysis of the sexual state and sterile mycelium of the fungus cultivated by the leaf-cutting ant acromyrmex hispidus fallax. mycological research, 105: 173-176. pagnocca, f.c., rodrigues, a., nagamoto, n.s. & bacci, m. jr. (2008). yeasts and filamentous fungi carried by the gynes of leafcutting ants. antonie van leeuwenhoek, 94: 517-526. poulsen, m., bot, a.n.m., currie, c.r., nielsen, m.g. & boomsma, j.j. (2003). within colony transmission and the cost of a mutualistic bacterium in the leaf-cutting ant acromyrmex octospinosus. functional ecology, 17: 260-269. poulsen, m. & currie, c.r. (2006). complexity of insect-fungal associations: exploring the influence of microorganisms on attine ant-fungus symbiosis. in k. bourtzis & t.a. miller (eds.). insect symbiosis, volume 2 (pp. 57-77). newbury: crc press. rodrigues, a., passarini, m.r.z., ferro, m., nagamoto, n.s., forti, l.c., bacci, m. jr, sette, l.d. & pagnocca, f.c. (2014). fungal communities in nest soils of leaf-cutting ants. journal of basic microbiology, 54: 1186-1196. doi: 10.1002/ jobm.201200458 samson, r.a., hoekstra, e.s. & frisvad, j.c. (2000). introduction to food-airborne fungi (6th edn). baarn: centraalbureau voor schimmelcultures. seifert, k.a., samson, r.a. & chapela, i.h. (1995). escovopsis aspergilloides, a rediscovered hyphomycete from leaf-cutting ant nests. mycologia, 87: 407-413. solomon, s.e., bacci, m. jr, martins, j. jr, vinha, g.g. & mueller, u.g. (2008). paleodistributions and comparative molecular phylogeography of leafcutter ants (atta spp.) provide new insight into the origins of amazonian diversity. plos one, 3(7): e2738. doi:10.1371/journal.pone.0002738 vovlas, n., inserra, r.n., & greco, n. (1992). schistonchus caprifici parasitizing caprifig (ficus carica sylvestris) florets and the relationship with its fig wasp (blastophaga psenes) vector. nematologica, 38, 1: 215-226. wirth, r., meyer, s.t., almeida, w.r., araújo, m.v., barbosa, v.s. & leal, i.r. (2007). increasing densities of leaf-cutting ants (atta spp.) with proximity to the edge in a brazilian atlantic forest. journal of tropical ecology, 23: 501-505. yek, s.h., boomsma, j.j. & poulsen m. (2012). towards a better understanding of the evolution of specialized parasites of fungus-growing ant crops. psyche, volume 2012, article id 239392. doi:10.1155/2012/239392. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.523-528sociobiology 61(4): 523-528 (december, 2014) influence of experience on homing ability of foragers of melipona mandacaia smith (hymenoptera: apidae: meliponini) introduction stingless bees fly in order to collect the necessary food resources (pollen and nectar) and nest-building materials (resin, mud, etc.), and they need to travel certain distances for that (roubik, 1989). in fact, bees are faced constantly with the task of navigating back to their nests from remote food sources and this is called ‘homing ability’. honey bees are the most studied bees concerning these aspects. they evolved several methods for doing this, such as compass-direct ‘vector’ flights, use of learned land marks, and cognitive maps based on spatial memory and on two dimensional snapshots of the surroundings (anderson, 1977; cartwright & collet, 1981, 1983; gould, 1986; capaldi & dyer, 1999; menzel et al., 2005; menzel & giurfa, 2006; reynolds et al., 2007; menzel et al., 2012; cheeseman et al., 2014). nevertheless, for stingless bees, up to the abstract the distance a bee can fly to collect food is quite relevant, among other aspects, for successful pollination. however, studies on this aspect concerning stingless bees usually do not take into consideration their homing ability. the objectives of this study were to verify the maximum distance that foragers of melipona mandacaia smith can fly, and whether experience is relevant for their homing ability in a caatinga region of northeast brazil. five colonies were used to collect foragers. these were marked and released starting from 100 m from their nests and at every 100 m up to a maximum distance on which there would be no bee returning to the nest. to evaluate the influence of experience, after being marked, another group of bees was put back into colonies, collected again after eight days and released in five distances only (500, 1,000, 1,500, 2,000 and 2,500 m). in both experiments, as the distance increased, the returning success of the bees decreased significantly. in fact, there was a significant negative correlation between their returning success and the distances they were released. the maximum distance a translocate bee returned to its hive was 2,700 m. the percentage of success was very high for bees released at 500 and 1,000 m (100% and 77%, respectively), suggesting this is the common flight range for the species. in most cases, average percentage of success was significantly higher for experienced bees than for other bees reinforcing the idea that experience is quite relevant for homing ability. sociobiology an international journal on social insects f rodrigues¹,², mf ribeiro³ article history edited by denise araujo alves, esalq-usp, brazil received 30 september 2014 initial acceptance 11 november 2014 final acceptance 09 december 2014 keywords stingless bees, flight range, flight experience. corresponding author francimária rodrigues universidade federal rural do semiárido, ufersa departamento de ciências animais, dcan prédio do dcan ii, lado oeste av. francisco mota, 572 bairro costa e silva, 59.625-900 mossoró, rn, brazil e-mail: francimaria.rodrigues@ufersa.edu.br moment, no studies concerning homing ability have been performed. investigations have been restricted to foraging activity, recruitment, flight range and maximum flight distances. the distances traveled by foragers depend on several factors, as density and seasonality of food source, as well as the bee species (dornhaus et al., 2006), physiology and body size (araujo et al., 2004; greenleaf et al., 2007). moreover, other aspects, isolated or together, may also affect their flight, as internal colony conditions and climatic factors (hilário et al., 2000). flight range have been the object of some studies in a few stingless bee species. in trigona corvina cockerell, partamona aff. cupira (smith), tetragonisca angustula (latreille) and nannotrigona testaceicornis (lepeletier), the maximum distances reached by bees varied from 623 to 853 m (van nieuwstadt & ruano iraheta, 1996). among bees of the melipona illiger genus, the maximum distances were research article bees 1 universidade federal do vale do são francisco, petrolina, pe, brazil 2 universidade federal rural do semiárido (ufersa), mossoró, rn, brazil 3 empresa brasileira de pesquisa agropecuária (embrapa), petrolina, pe, brazil f rodrigues, mf ribeiro – homing ability of melipona mandacaia524 estimated in 2,000 m for melipona bicolor lepeletier and melipona scutellaris latreille (araújo et al., 2004), and 2,100 m for melipona mandacaia smith (kuhn-neto et al., 2009). the method generally used for such estimations is the training of bees up to a food source. another method is the capture and recapture of bees, which also allows obtaining information on the maximum flight distance that bees are able to travel to forage (roubik & aluja, 1983). there are no studies on the maximum distance a m. mandacaia forager can fly using this specific methodology. moreover, except by the study of kuhn-neto et al. (2009), there is no other information concerning the maximum flight distance of this species, specially taking into consideration the experience of bees, which is probably an important factor for homing ability, as demonstrated for honeybees. m. mandacaia is a very important species for the meliponiculture of petrolina, pernambuco state, and juazeiro, bahia state, in the northeastern region of brazil, being mainly used for honey production (ribeiro et al., 2012). however, it is relatively little studied, especially concerning its potential for pollination services. in this way, the present study was carried out with the objectives of verifying the maximum distances foragers can fly, their homing ability and the influence of experience on this process. material and methods the experiments were performed at embrapa semiárido (09°4’17.53”s 40°19’ 10.24” w) in an area of 2,100 ha, at 42 km from the city of petrolina (pernambuco state), a semiarid region in northeast brazil. the vegetation is typical of hiperxerophile “caatinga” (zanella, 2000), a type of savanna. the plants are used to low precipitation, that is restrict to a few months of the year, intense hours of sun and high temperatures, and many of them loose their leaves during the drought period. m. mandacaia, popularly known as ‘mandaçaia’, occurs naturally in this bioma and it is distributed along the são francisco river, in the states of bahia, ceará, paraíba, pernambuco e piauí (batalha-filho et al., 2011). maintenance of colonies five colonies of m. mandacaia were used in the experiments. the colonies were installed in hives kept at the entomology sector of embrapa semiárido, in a room maintained at ambient temperature (around 27 oc). bees had free access to the external environment through a plastic tube in the wall. supplementary food (apis mellifera linnaeus honey) was provided in average every eight days, according to a usual beekeeping practice. in the days of the experiments, the colonies did not receive any food. two experiments were performed from august 2011 to june 2012, which are described below, totaling 11 consecutive months. taking into account the possible effects of climate during the experimental period, the release of bees (as explained bellow) occurred in similar conditions of weather and daytime. thus, bees were released between 8 a.m. to 10 a.m., with average temperature of 27 oc and relative humidity of 65%. in days considered unfavorable (i.e., cloudy, windy, rainy, or with much different conditions concerning temperature and humidity) the release did not happen. experiment 1: homing ability of mixed foragers’ group (experienced and inexperienced bees) in order to be sure only foragers would be used in the experiment, bees were collected at the nest entrance with an insect aspirator when they were arriving from the field. afterwards, they were placed in acrylic cages (20 x 20 x 20 cm) containing food (a. mellifera honey), being one cage for each colony. these bees were then marked on the thorax with plastic nontoxic paint (one color for each investigated distance). soon after, they were put into wooden boxes and were kept there up to the following day, when they were released. initially, because we could not differentiate experienced from inexperienced bees, mixed foragers groups were used. therefore, mixed foragers groups of 25 bees from each colony were released at distance intervals of 100 m from their nest, up to the distance where no bee returned, i.e. from 100 m, 200 m and so on, up to 2,800 m. the distances from the releasing points were measured with a gps in a straight line in relation to the nests’ entrances (fig 1). for registering the bees that returned to their nests, small wooden boxes were placed where the original hives were. those observation boxes had a transparent glass cover, which allowed the observer to check the number and color of bees that returned. the original hives were kept closed throughout the day in order to avoid that other foragers (not marked) returned to the observation boxes. thus, marked bees were used only once, and for each evaluated distance, new marked bees were used. the percentage of success was calculated considering the number of bees released, and the number of bees that returned to their nests. experiment 2: homing ability of experienced foragers during the previous experiment, it was observed that sometimes the bees did not arrive. however, with repetitions of the same distances, other bees were able to return to their nests. therefore, in order to test the hypothesis that more experienced bees were more capable of recognizing the areas where they were released, and so could find the route back to their nests more easily, another experiment was carried out. the collection and marking of bees was done according to the same methodology already described. nevertheless, after being marked, the bees were put back into their own colonies. after eight days, these bees were collected again and placed into wooden boxes with sociobiology 61(4): 523-528 (december, 2014) 525 food. they were kept there until the next day, when they were released. in this way, these bees had in common eight days of flight experience. however, because we could not collect all marked bees, since some could have died, the number of bees released was defined according to the number of bees that we were able to collect on that day. the registration of returning bees was performed in the same way, but only five distances were tested (500 m; 1,000 m; 1,500 m; 2,000 m; 2,500 m) due to the lower availability of the bees. at the end of the experiment there were two groups of bees that were compared: a first group, where the experience was not considered (and therefore, could include bees with and/or without experience, i.e., a “mixed group”), and a second group, where only bees with experience were tested (“experienced group”). statistical analysis in order to verify if the colonies were statistically different, the kruskal-wallis test was applied. to assess the differences found for the returning success between the two groups of bees (“mixed group” and “experienced group”), a chi-square was applied. the relation between the returning success of the bees and the distance they traveled was tested using a linear regression (zar, 2010). results experiment 1: homing ability of mixed foragers group (experienced and inexperienced bees) this experiment used 3,225 individuals for releasing, being 25 bees from each colony in each distance. however, some colonies did not reach the same maximum distance (2,700 m), but smaller ones. thus, for each evaluated distance, in average 125 bees were released, but from 2,400 m, a smaller number of bees could be released (75-100 bees). nevertheless, colonies did not show differences among themselves (kruskal-wallis, p= 0.463; n= 27 distances) and for this reason the data related to the returning success of the bees were analyzed together. the number of bees that returned to their nests decreased gradually with increasing distance. as mentioned above, they reached a maximum distance of 2,700 m (fig 2). in general, bees had low success in coming back to their nests when released up to the distance of 2,300 m, 2,500 m and 2,700 m (4% of success, for these distances). it was clear that with increasing distance the percentage of success decreased, and there was a strong correlation between these two factors (fig 2). indeed, there was a highly significant negative correlation between the returning success of the bees and the distance from where they were released (spearman ranking correlation, rho= -0.937, p= 0.000, n= 27 distances; fig 2). experiment 2: homing ability of experienced foragers in this experiment, 113 individuals were released, being 12-35 bees in each distance, according to the availability of bees, as mentioned above. the returning success of the bees reached 100% when they were released up to 500 m (fig 2). even at the distance of 1,000 m the percentage of success was quite high (77%), reinforcing the idea that experience must be important for their homing ability. this percentage was twice as big as the one found for bees of the previous experiment (fig 2). fig 1. geographical location of the distances where the melipona mandacaia bees were released. font: laboratory of geoprocessing, embrapa semiárido. f rodrigues, mf ribeiro – homing ability of melipona mandacaia526 when the two groups of bees (“mixed group” and “experienced group”) were compared, most the results were higher when experienced bees were used (table 1). the only exception was for 2,000 m. thus, both groups of bees were very different in returning success (p values were highly significant at the level of 0.001, table 1). in the same way, as the distance increased, the returning success decreased significantly, showing a negative correlation (rho= -0.949, p= 0.014, n= 5 distances; fig 2). kuhn-neto et al. (2009) studied the flight of m. mandacaia through the training of foragers to the food source. they verified that the maximum distance reached was 2,100 m for larger bees, and 1,560 m for the smaller ones. this confirms what was found by nieuwstadt and iraheta (1996) when studying the relationship between the size of some bees (trigona corvina, partamona aff. cupira, tetragonisca angustula and nannotrigona testaceicornis) and their foraging range. the authors emphasized that the maximum distances obtained in the experiment of capture and recapture increased ca. 300 m in comparison to the experiment that used the artificial feeder. in the present study, with another methodology, m. mandacaia bees traveled larger distances (2,700 m) than in the study of khun-neto et al. (2009), i.e., from 2,100 to 1,560 m. we did not analyzed the size of the bees. however, it is possible that the discrepancy between the results found by us and by khun-neto et al. (2009) were due to the different methods used. on the other hand, both methods present limitations. the feeder method could underestimate the flight range since a feeder would not be as attractive for the bee as a flower. in addition, the method of releasing bees could fail when bees do not know the location where they are released (niewstadt & ihareta, 1996). another fact to be considered is that m. mandacaia is a stingless bee endemic from the “caatinga” region (zanella, 2000; batalha-filho et al., 2011) and this bioma is characterized by a low density of natural sources and a prolonged drought (drumond et al., 2000). thus, plant physiognomy and biology could force the bees to fly longer distances to feed. although both localities of the experiments (from khun-neto et al, 2009, and our study) are in “caatinga” areas, it is possible that in our case the region presented harder conditions (as the extreme drought of the last years). capaldi and dyer (1999), studying the homing ability of a. mellifera, concluded that several factors influence the performance of bees, as for example their learning in relation to nest location and place where they were released, as well as whether they are experienced in foraging. in fact, the data found in the present study with m. mandacaia suggest that flight experience is indeed important to bees for homing ability. sánchez et al. (2007) studied foraging experience in scaptotrigona mexicana (guérin) and found that more experienced bees tend to change to other food sources more easily than less experienced bees. when analyzing the effect of experience on the distance reached by s. mexicana, they observed that indeed the experience and not the distance of the feeders was the most relevant factor that affected the choice for the food source. these authors concluded that foraging experience could be an advantage for the colony since it allows the exploitation of new food sources, as contributes for diminishing the competition among foragers. the experience is usually neglected in flight distances studies of stingless bees. the homing ability of an experienced bee in relation to the ability of a naive (or less experienced) fig 2. percentages of success of melipona mandacaia bees returning to their nests in relation to the distance from where they were released for the “experienced group” and “mixed group”. table 1. comparison between the number of bees released and returning success of the bees (as well the percentage of success) for the two groups of melipona mandacaia bees (“mixed group” and “experienced group”), and statistical analysis (χc2 and p values, chisquare test), for the different distances. discussion roubik and aluja (1983) performed studies using a similar method applied in the present study and found that melipona fasciata latreille bees returned to their nests when released at the distance of 2,100 m, and cephalotrigona capitata (smith) returned from 1,500 m. these authors observed that there was a relation between the head size and the distance the bees could fly. moreover, they also made an estimative through regression tests, and verified that the maximum distances would be 2,400 m and 1,700 m respectively. distances (m) number of bees released number of returned bees percentage of success (%) xc2 (p values) mg eg mg eg mg eg 500 125 12 65 12 52.0 100.0 15.2 (p<<0.001) 1,000 125 13 37 10 29.6 77.0 21.1 (p<<0.001) 1,500 125 35 19 10 15.2 28.6 4.1 (p<<0.001) 2,000 125 33 22 4 17.7 12.1 1.0 (p<<0.001) 2,500 100 20 2 4 2.0 20.0 14.7 (p<<0.001) mg: “mixed group”; eg: “experienced group”. sociobiology 61(4): 523-528 (december, 2014) 527 one is remarkable in our results. in fact, the present study is the first one to investigate this factor. in addition, as it was observed, the returning success increased about 30% when more experienced bees were released at 500 m, according to table 1. in our experiments, we found that the returning success of the bees was higher for “experienced bees” than for the “mixed group” in all distances, except for 2,000 m (table 1). for this distance, the result was opposite, and the reason could be that, by chance, the bees released in that distance had a longer experience in the “mixed group” than in the other group. this question remains to be clarified. however, it was remarkable that in all other tested distances the number of succeeded bees was significantly higher for the “experienced group”. the mechanisms by which bees find their way back to their nests were not investigated in this study. however, it is possible that stingless bees use the same learning tools as honeybees, such as landmarks and spatial memory, as previously mentioned. in bumblebees, it was observed that bees that were released presented a ‘circling’ behavior: flying on circles over the release site (goulson & stout, 2001). the same was registered in our experiments and probably was used for initial orientation of the bees. conclusions although the maximum flight distance reached for m. mandacaia was 2,700 m, it was outstanding that all bees released at 500 m were able to return to their nests, and even at 1,000 m the large majority returned, suggesting that these distances are part of the common flight range of foragers. as mentioned by nogueira-neto (1997), studies on flight distances of stingless bees are relevant since they provide information on flight range of bees in relation to availability of food sources and possibilities for productivity. thus, the results presented in this study may be useful in crop pollination programs since the distance of hives in relation to cultivated areas may influence pollination efficiency, and consequently, productivity of crops. moreover, adequate bee pasture should be included in the common flight range of bees in order to guarantee their production. finally, our results demonstrated for the first time that experience can limit or improve the homing ability of stingless bees, and this aspect should be considered in future investigations. acknowledgments to juliara reis braga, cândida beatriz da silva lima and emison marcelino borges, for helping during data collection; to the beekeeper francisco camilo de sousa, for the donation of some colonies used in the experiments and honey and pollen used for feeding the bees; to ms.c. tatiana ayako taura, for helping with the satellite images; to dr. airton torres carvalho, for critical reading of the manuscript; to dr. marlon da silva garrido, dr. paulo gustavo serafim de carvalho, dr. sérgio dias hilário and one anonymous referee, for the valuable suggestions, erica m. t de alencar, for language advices; to coordenação de aperfeiçoamento de pessoal de nível superior (capes), for the post-graduation scholarship to f. rodrigues; to empresa brasileira de pesquisa agropecuária (embrapa semiárido) (cod. seg 02.11.01.029.00.00) for the facilities. references anderson, a.m. (1977) a model for landmark learning in the honey-bee. j. comp. physiol. 114: 335-355. doi: 10.1007/ bf00657328 araujo, e.d., costa, m., chaud-netto. j. & fowler, h.g. 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(2010). biostatistical analysis, 5th ed., new jersey: prentice hall, 943 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.415-422sociobiology 61(4): 415-422 (december, 2014) tree species used for nesting by stingless bees (hymenoptera: apidae: meliponini) in the atlantic rain forest (brazil): availability or selectivity introduction probably, the abundance of stingless bees (meliponini) is constrained by floral resources (hubbell & johnson, 1977; eltz et al., 2002) and/or by nesting sites (oliveira et al., 1995; samejima et al., 2004; teixeira & viana, 2005). in these eusocial bees, the choice of nesting site has profound influence on the longevity and reproductive success of colonies (hubbell & johnson, 1977) due to the limited flight range (araújo et al., 2004) and progressive swarm (nogueira-neto, 1997). the stingless bees use several substrates for nesting (nogueira-neto, 1997); however, the majority depends on preexisting cavities such as tree hollows, and few species build exposed nests (roubik, 1989; michener, 2000; antonini & martins, 2003; batista et al., 2003; martins et al., 2004; silva et al., 2013). the knowledge about tree species used by abstract the stingless bees (meliponini) are numerically dominant in tropical forests and most species depend on preexisting cavities for nesting, mainly tree hollows. however, it is still incipient the knowledge about basic characteristics of forest trees used for nesting. the basic questions addressed in this study include: would appropriate hollows be restricted to a few tree species? would there be selectivity in the use of tree hollows in the forest? these issues are addressed from the comparison of usage patterns among forest trees in different stages of forest regeneration in the atlantic forest (michelin reserve in northeastern brazil). among 89 nests (from six species) found in tree hollows, in a sampled area of 32 ha of forests, 78.7% were associated with live plants and 21.3% to dead trees. this result does not support the hypothesis of selectivity for living trees, considering the high rate of living trees: dead trees (40:1). nests were sampled from 41 tree species of 31 genera and 22 plant families. meliponini species showed no differential association with any tree species. the absence of selectivity of tree species as nesting site is probably due to the high diversity of trees per hectare of atlantic rainforest. the stingless bees also showed no selectivity for wood hardness, therefore the potential durability of tree hollows probably exerts weak selective pressure on bees, or at least the hardness variation range of trees used for nesting has no important influence on reproductive success of the colonies of stingless bees. sociobiology an international journal on social insects md silva1,2, m ramalho1 article history edited by vera l. imperatriz-fonseca, ibusp, brazil received 15 september 2014 initial acceptance 06 november 2014 final acceptance 08 december 2014 keywords stingless bees, preexisting cavities, arboreal substrates. corresponding author marilia dantas silva rua waldemar mascarenhas, s/nº portão (estrada velha da chesf) governador mangabeira, bahia, brazil 44.350-000 e-mail: ailirambio@hotmail.com stingless bees for nesting is still incipient (moreno & cardoso, 1997; aguilar-monge, 1999; martins et al., 2004; kleinert, 2006) and it is not known if these bees have shown some preference for nesting trees. according to nogueira-neto et al. (1986), some species of stingless bees may have specificity in the use of trees for nesting, which is corroborated by data sampled under low diversity of nesting trees (batista et al., 2003; martins et al., 2004; teixeira & viana, 2005; serra et al., 2009). however, the very high diversity and very low population densities of trees in tropical forests should favor no selectivity of nesting sites: for instance in the atlantic rainforest, the tree diversity can reach over 400 species of trees per hectare (see guedes et al., 2005). as an example, stingless bees would not present specificities when choosing trees for nesting in tropical dipterocarp forests and they would be opportunistic, although the tree hollows could be research article bees 1 universidade federal da bahia, salvador, ba, brazil 2 instituto federal de educação, ciência e tecnologia baiano, governador mangabeira, ba, brazil md silva, m ramalho tree species for nesting of stingless bees 416 selected by their characteristics, such as size (eltz et al., 2002). in the tropical forests of central america, hubbell and johnson (1977) observed low selectivity in the using of tree hollows and argued that the availability of nesting substrates would be higher than the density of stingless bee nests in the forest habitats. the hypothesis that tree hollow availability exceed the demand for nesting by stingless bees is supported by the low density of nests (2.8 nests/ha) compared to the high density of trees potentially suitable for nesting in the atlantic rain forest (silva et al., 2013). estimates of availability and use of nesting substrates are necessary for understanding the role of choice or preferences in structuring communities of stingless bees in forests (kleinert, 2006; silva et al., 2013). the basic questions addressed in this study include: would availability of suitable hollows for nesting be restricted to a few tree species? would there be selectivity in the use of tree hollows in the forest? this study compares the tree species used by the stingless bee community at different stages of forest regeneration in the atlantic rainforest. it is assumed that the availability of tree hollows with suitable sizes varies between stages of forest regeneration, affecting choice opportunities, according to the species-specific needs of stingless bees. if the availability and selectivity of cavities are important, the communities of stingless bees must change between the stages of forest regeneration. material and methods the present study was conducted in the michelin ecological reserve mer (13°50′s, 39°15′w) in the brazilian atlantic rainforest in northeastern brazil. the mer encompasses 3,096 ha of tropical rainforest at altitudes from 160 to 327 masl. the native forest forms a mosaic with rubber (hevea brasiliensis muell. arg., euphorbiaceae) plantations. the mer forest areas experienced severe anthropogenic impacts prior to 2004, generating a mosaic of forest fragments at different stages of regeneration. at the present time, the preserved nuclear areas of the largest fragments have attained a mature old growth stage of regeneration, with canopy heights of more than 20 m and many trees with circumferences >190 cm at breast height, as well as numerous old growth trees >300 cm in circumference reaching more than 30 m height. there are also extensive patches of forest at early stages of regeneration (with lower canopies and no old growth trees and with shrub and herbaceous plant cover in the understory), mainly at the edges of the largest fragments (flesher, 2006). two categories of forested habitats were discriminated to verify and compare the nest density and meliponini species richness: mature old growth forest and early stages of regeneration (is = initial state and as = advanced state). four replicates of the two forest categories were sampled in each of the four largest mer forest fragments. a total of 64 25x25 m plots was established and sampled in each of the four replicates (total area of 4 ha/replicate), for an overall total of 16 ha for each of the two forest categories (silva et al., 2013). all the trees within each plot were visually inspected in search of nests, with special attention being paid to large trees with circumferences at breast height (cbh) >190 cm – in which stingless bee nests tend to be concentrated in forest habitats (eltz et al., 2002; batista et al., 2003). the botanical material was collected for preparation of exsiccates and identification of tree species. to determine the wood density (hardness) of nesting trees (with meliponini nests), samples of 2.0 x 2.0 cm were collected from the tree trunk at breast height (cbh = 1.30 m): one block near to the core and the other close to the bark. the mercury porosimetry technique was used to estimate the wood dry mass, according to the method of vital (1984). the vegetation structure was measured using the t-square method (sutherland, 2006). twenty random points were drawn/replica and the distance from the point to the nearest individual (x) and its distance to the nearest neighbor on orthogonal were used to estimate the density of living trees (all trees, with or without nests) in the following perimeter categories (circumference at breast height-cbh = 1.30 cm): (1) 21-50 cm; (2) 51-80 cm; (3) 81-110 cm and (4) above 110 cm. the t-square technique was not used to estimate the density of dead trees because it is not suitable for events with very low frequency (sutherland, 2006). in this case, ten plots of 25x25 m (randomly chosen) were established for density measurement in each of the four replicates/forest stage and all the dead trees were counted (according to the perimeter categories used in the samplings of living trees). the density of living trees was calculated using the program ecological methodology, 2nd ed. (kenney & krebs, 2000), while the density of dead trees was obtained by dividing the number of trees by the area sampled in each habitat category. in addition to the density and size of nesting trees, both stages were compared in relation to the hardness of the wood. the “permutational multivariate analysis of variance” test permanova (anderson, 2005) was used for data analysis because the assumptions of homoscedasticity (levene test) and normality (kolmogorov-smirnov test) were not satisfied. the tests were run on graphpad instat and spss (spss 13.0 for windows, spss inc., chicago, il, usa) software. three dependent variables (total abundance, richness and wood hardness of nesting trees) were tested in relation to a single factor: stage of forest regeneration in two levels (is = initial state and as = advanced state). the bray-curtis measure was used with untransformed data for comparing both stages of forest regeneration at a significance level of 0.05 (anderson 2005). the nonparametric correlation test (spearman) was applied in two situations: 1) to estimate the relationship between the number of trees with nests and the number of largest trees (cbh> 80cm) per unit area of each habitat category (is and as); 2) to assess the relationship between the size of trees-cbh (or tree hardness) and richness and abundance of meliponini nests. the tests were run on graphpad instat 3.05 software (graphpad-software, 1998) at a significance level of 0.05. selectivity was used here as synonymous for preference that would be detected when a used category of sociobiology 61(4): 415-422 (december, 2014) 417 nesting trees (size or hardness) is higher than its availability in local forest habitat. the similarity of the nesting trees between species of stingless bees was estimated by cluster analysis (braycurtis coefficient) and data were run on past program (palaeontological statistics, see. 1.81). results and discussion of the total of 118 nests of stingless bees found in mer, 75.4% were in trees, 9.4% amid the rocky substrate, 7.6% inside termite nests and 7.6% in soil and slopes. among 89 nests found in tree hollows, 78.7% were found in living trees and 21.3% in dead trees. nests of five meliponini species were observed in hollows of living trees, of which only scaptotrigona xanthotricha moure was not found in dead trees (table 1). batista et al. (2003) found 5.4% of the nests of stingless bees in dead trees in a disturbed area of atlantic rainforest. in a recent review, cortopassi-laurino et al. (2009) found that living trees predominate largely over dead trees as nesting substrates to stingless bees. roubik (1989) has also argued that the stingless bees should occupy most durable substrates, which provide good physical protection in the forest, therefore living trees. although these data and arguments have internal coherence, they often lack a measure of availability of cavities in dead and living trees for testing preference or selectivity. in the atlantic rainforest of mer, the density of living trees is about 40 times higher than the density of dead trees and, therefore, these data do not support the hypothesis that stingless bees would avoid dead trees (table 2). alternatively, we should consider that many nests found in dead trees were established while the trees were still alive. the living trees with nests corresponded to 59 individuals and 42 species (table 3; fig 1). only one nest was found in most tree individuals and species and a maximum of 3-4 nests were associated to six tree species. this overall framework suggests that availability of cavities is common table 1. characteristics of stingless bees’ nests found in tree hollows in atlantic rainforest (michelin ecological reserve): advanced stage (or old growth mature; as) and initial stage (is) of forest regeneration; circumference at breath height (cbh)= 1.30m. in many tree species and trees selectivity by stingless bees is very low or nonexistent in this forest habitat. among the 194 tree species recorded in mer (rochasantos & talora, 2012) 41 (21%) showed meliponini nests, therefore apparently the appropriate tree hollows for these bees are well spread in the flora. if we consider the relatively high values of alpha and beta tree diversity in the atlantic rainforest (e.g, guedes et al., 2005), the data on mer refute the argument of hubbell and johnson (1977) that most of the nests of stingless bees would be found in relatively few species of trees out of the total available in any vegetation. in a disturbed area of atlantic rainforest, with depressed richness of trees, batista et al. (2003) found only 18 tree species with nests of stingless bees; while eltz et al. (2003) recorded nests in 38 species of trees in lowland dipterocarp forests, malaysia. in areas of caatinga, castro (2001), martins et al. (2004), teixeira and viana (2005) and souza et al. (2008) found most nests of stingless bees in only five tree species (caesalpinia pyramidalis tul.; commiphora leptophloeos mart. j.b. gillett.; schinopsis brasiliensis engl.; copaifera coriacea mart. and amburana cearensis schwacke & taub.). in savannah-cerrado areas, kerr (1971), aquino et al. (2007), antonini and martins (2003) and serra et al. (2009) found a predominance of nests in the species caryocar brasiliense cambess., qualea parviflora mart. and salvertia convallariaeodora a. st.-hil.. in mixed forest of araucaria, witter and colleagues (2010) reported certain table 2. density of living and dead trees (trees/ha) in the initial (is) and advanced (as) stages of forest regeneration distributed by circumference at breast height (cbh): cbh 1: 21-50 cm; cbh 2: 51-80 cm; cbh 3: 81-110 cm and cbh 4: above 110 cm). cbh living trees dead trees as is as is 1 536.5 599.7 8.50 5.00 2 257.1 165.1 7.25 9.25 3 92.9 61.2 8.25 9.75 4 96.3 41.1 4.25 2.50 number of nests as is average height height range cbh mean cbh range nesting trees melipona scutellaris latreille 6 5 8.4m (±4.2m) 2.5-13m 125cm (±39.7cm) 59-197cm living and dead plebeia droryana (friese) 2 85.5cm (±62.9cm) 41-130cm 55.5cm (±14.8cm) 45-66cm living and dead scaptotrigona bipunctata (lepeletier) 9 5 5.1m (±5.1m) 171cm-15m 120.1cm (±34.1cm) 70-180cm living and dead scaptotrigona xanthotricha (lepeletier) 13 8 3.2m (±4.2m) 25cm-13m 127.2cm (±34.8cm) 76-180cm living tetragonisca angustula (latreille) 14 20 3.1m (±4.7m) 1cm-11m 119.3cm (±85.3cm) 41-232cm living and dead trigona fuscipennis friese 1 4.5m 119.3cm living md silva, m ramalho tree species for nesting of stingless bees 418 table 3. tree species most used as nesting substrate by stingless bees in initial and advanced stages of forest regeneration. ecological group: t = tolerant to shade; i = intolerant to shade; nc = not classified (lorenzi, 2002a; 2002b; 2009; rocha-santos & talora, 2012). s.b (scaptotrigona bipunctata); s.x (scaptotrigona xanthotricha); t.a (tetragonisca angustula); m.s (melipona scutellaris) and t.f (trigona fuscipennis). (*) = sample not identified. family tree species ecological group and hardness number of nests meliponini species is as anacardiaceae thyrsodium spruceanum salzm. ex benth. t (0.41 g/cm) 2 1 t.a; s.b apocynaceae symphonia globulifera l.f. t (0.14 g/cm) 1 0 t.a araliaceae dendropanax bahiensis fiaschi. nc (0.35 g/cm) 0 1 s.x burseraceae protium icicariba (dc.) marchand. t (0.53 g/cm) 0 3 s.x; m.s burseraceae protium sp. nc(0.64 g/cm) 2 0 m.s; t.a chrysobalanaceae licania hypoleuca benth. t (*) 1 0 t.f clusiaceae aspidosperma c.f. spruceanum benth. ex müll.arg. t (0.38 g/cm) 1 0 t.a clusiaceae clusiacea sp.2 nc (0.69 g/cm) 1 0 s.b clusiaceae clusiacea sp.1 nc(0.60 g/cm) 0 1 s.x clusiaceae garcinia macrophylla mart. t (0.17 g/cm) 1 0 s.x clusiaceae tabernaemontana flavicans willd. ex roem. & schult. t (*) 0 1 m.s cunoniaceae lamanonia ternata vell. t (0.66 g/cm) 1 0 s.x elaeocarpaceae sloanea guianensis (aubl.) benth. t (0.83 g/cm) 2 1 p.d elaeocarpaceae sloanea obtusifolia (moric.) k. schum. i (0.15 g/cm) 0 1 t.a euphorbiaceae pera glabrata (schott) poepp. ex baill. i (0.23 g/cm) 0 1 t.a fabaceae albizia pedicellaris (dc.) l.rico. i (0.091 g/cm) 0 1 s.b fabaceae arapatiella psilophylla (harms) r.s.cowan. t (0.13 g/cm) 1 0 s.x fabaceae inga capitata desv. t (0.63 g/cm) 1 0 t.a fabaceae inga edulis mart. t (0.70 g/cm) 1 0 t.a fabaceae inga thibaudiana dc. i (0.86 g/cm) 1 0 m.s fabaceae inga sp.1 nc(0.78 g/cm) 1 0 s.b fabaceae peltogyne confertiflora (mart. ex hayne) benth. t (0.74 g/cm) 0 1 m.s fabaceae stryphnodendron pulcherrimum willd. hochr. i (0.69 g/cm) 4 0 s.x fabaceae swartzia polita (r.s.cowan) torke t (0.79 g/cm) 0 1 s.b fabaceae swartzia sp.1 nc(0.89 g/cm) 0 1 s.x hypericaceae vismia guianensis (aubl.) choisy i (0.90 g/cm) 1 0 t.a hypericaceae vismia guianensis (aubl.) choisy i (0.90 g/cm) 1 0 t.a icacinaceae emmotum nitens miers. t (0.83 g/cm) 0 1 t.a lauraceae ocotea cf. canaliculata (rich.) mez i (0.64 g/cm) 1 0 s.b lauraceae ocotea longifolia kunth. t (0.43 g/cm) 1 0 m.s melastomataceae henriettea succosa (aubl.) dc. t (0.75 g/cm) 1 1 t.a melastomataceae tibouchina sp. nc(*) 1 0 s.x meliaceae trichilia lepidota mart. t (0.31 g/cm) 0 3 t.a; s.b moraceae artocarpus heterophyllus lamk. t (0.92 g/cm) 1 0 t.a myrsinaceae myrsine sp.1 t (0.62 g/cm) 0 1 t.a myristicaceae virola gardneri (a.dc.) warb t (*) 0 1 s.x peraceae pogonophora schomburgkiana miers ex benth. i (*) 1 0 t.a phyllanthaceae amanoa guianensis aubl. i (0.79 g/cm) 0 1 t.a rubiaceae psycotria carthagenensis jacq. t (0.86 g/cm) 0 2 t.a; s.x salicaceae casearia sp.1 nc (0.84 g/cm) 0 1 s.x sapotaceae pouteria venosa (mart.) baehni. t (0.93 g/cm) 0 1 s.x urticaceae pourouma velutina mart. ex miq. i (0.16 g/cm) 2 0 t.a; s.x sociobiology 61(4): 415-422 (december, 2014) 419 nesting specificity by melipona bicolor schencki gribodo in a few species of lauraceae (59.52%). in urban area in the center of curitiba city (paraná state, brazil), taura and laroca (1991) also found a greater number of nests of stingless bees in only two tree species, jacaranda mimosaefolia d. don. and platanus sp. a common denominator to these studies with stingless bees in non-forested habitats is the dominance of some tree species and/ or the availability of tree hollows suitable for nesting in a small proportion of arboreal flora. by analogy, the plant family with highest frequency of stingless bee nests in mer was fabaceae (table 3) that was also highlighted in the review of nesting trees by cortopassi-laurino et al. (2009). in mer, fabaceae surpasses the others in richness and abundance in all stages of forest regeneration (rocha-santos & talora, 2012), and this predisposes its use as nesting trees by stingless bees: this usage reflects availability and not preference or selectivity. often, the authors who have studied stingless bee communities in forest habitats just point out the use of “suitable tree hollows for nesting” (mainly with apparent suitable size), regardless of tree species (hubbell & johnson, 1977; oliveira et al., 1995; eltz et al., 2003). when the availability of tree hollows is widely distributed in the tree flora, as in the mer rainforest, the apparent tree selectivity by meliponini disappears. trees used as nesting sites by meliponini have wood density (or hardness) ranging from 0.13 to 0.93 g/cm³ and about 70% of these trees have wood hardness above 0.6 g/cm³. the hardness had no significant relationship with nest abundance (p=0.4210) and richness (p=0.2779) of stingless bees (fig 2). there is no significant variation in the distribution of total nests related to trees’ hardness, fig 1. abundance (a) and richness (b) of tree species used for nesting by stingless bees in each replica forest habitat in advanced stage (as) and initial stage (is) of regeneration in the michelin ecological reserve (mer). however, plebeia droryana (friese) and melipona scutellaris latreille often used trees with higher densities (0.86 g/cm³ and 0.75 g/cm³, respectively), while tetragnonisca angustula (latreille) often occupies cavities in trees with low hardness (below 0.55 g/cm³). in these cases, we can rule out the effect of trees size used for nesting, as a confounding variable, mainly in the case of t. angustula, a generalist species able to use small hollows (silva et al., 2013, 2014). it was detected a difference in hardness of nesting trees between the two stages of forest regeneration (p=0.0002), in a first analysis of the four replicates. however, one of the replicas was detected as an outlier, with very low values of hardness in ‘is’ (0.13g/cm3) in comparison with the others replicas of ‘is’ stage of forest regeneration (around or above 0.5 g/cm3) and, a posteriori, it was excluded from the analysis. with this procedure, the differences in wood hardness disappeared between the two stages of fig 2. correlation between the abundance of nests (a), species richness of stingless bees (b) regarding the hardness of trees that were found with nest of stingless bees. forest (p >0.05). this result support the previous argument of similar hollow availability in trees regardless of the wood hardness or ecological group (e.g., tolerant or intolerant to shade; table 3). moreover, one nest per tree/family with varying wood hardness was the predominant pattern in both forest stages of regeneration (table 3), indicating that the hardness per se would not be relevant in the process of hollow formation and availability for meliponini. in summary, probably the hardness and durability of hollows in the trees have not exerted significant selective pressure on stingless bees in the rainforest, or at least the hardness variation range has had no influence on colonies reproductive success and longevity. md silva, m ramalho tree species for nesting of stingless bees 420 the average cbh of nesting trees in mer was 132.3cm (±66.1cm). the cbh had negative significant relationship (p=0.0224, r=-0.4953) with nest abundance and no significant relationship with stingless bee richness (p=0.1072) (fig 3). the lower occurrence of nests in larger trees should be a sampling artifact, however, reflecting the pattern of availability of cbhs categories, i.e. the great reduction in abundance of larger trees (table 2). in fact, the variation in the abundance of nests of stingless bees is not significant (p=0.944) between the largest categories 3 and 4 of cbh. samejima et al. (2004) indicated that the low density of stingless bee nests in disturbed areas of forest would be related primarily to the absence of largest trees for nesting (cbh above 150cm or diameter above 24cm). hubbell and johnson (1977) argued that the colonization of secondary forests by stingless bees would depend on the tree size, and the initial stage of forest regeneration should be colonized primarily by small bees and later by larger ones. the small nesting trees should be less fig 3. correlation between the abundance of nests (a), species richness of stingless bees (b) and abundance of nests of melipona scutellaris (ms), scaptotrigona xanthotricha (sx) and s. bipunctata (sb) (c) regarding the cbh (circumference at breast height) of trees that were found with nest of stingless bees. fig 4. similarity among meliponini species in use of plant families. braycurtis coefficient. caption: plebeia droryana (p.d); trigona fuscipennis (t.f); scaptotrigona bipunctata (s.b); melipona scutellaris (m.s); scaptotrigona xanthotricha (s.x); tetragonisca angustula (t.a). accessible to meliponini species with large colonial biomass (hubbell & johnson, 1977; roubik, 1983; samejima et al., 2004). sampling with trap nests also supports the argument that different species might respond to different thresholds of minimum size of cavities in the forest (silva et al., 2014). a positive relationship between stingless bees body size and minimum diameter of nesting trees was also detected (kleinert 2006), probably because the minimum biomass of colonies would also be lower in smaller species of stingless bees. however, in mer rainforest, the stingless bees with very different body sizes that would fit the profile of “large colonial biomass” (s. xanthotricha, s. bipunctata and m. scutellaris) show no significant variation (p=0.2666, r=-02540) in the occupancy of trees with different cbhs (fig 3c). likewise, the use of tree hollow sizes does not group the stingless bee taxa or sizes; on the contrary, affinities are random (fig 4). for instance, stingless bee species with more sampled nests also used a higher number of tree hollows and tend to have greater nesting overlap (e.g. t. angustula and s. xanthotricha). these results support the argument of weak selectivity for different hollow sizes among different species of stingless bees. the lack of variation in stingless bee richness with cbh trees (fig 3) and the lack of differences (species and abundance) between stages of forest regeneration support the argument that trees above a cbh´s threshold (or hollow size threshold) are used and shared by most stingless bee species in local communities. for example, trees above 80cm of cbh (table 2) or trap nests above 3l (silva et al., 2014) were used by stingless bees with wide range of body sizes and colonial biomasses. if there is any influence of the diameter of the trees on the stingless bees’ community in mer, it is unlikely that this factor is sufficient to explain the absence of spatial dynamics sociobiology 61(4): 415-422 (december, 2014) 421 in the community between stages of forest regeneration. even if the longevity of trees and tendency to develop hollows were influenced by the type of wood (e.g. hardness, chemicals, fiber, etc.) or by characteristics of tree growth (tolerant or intolerant to shade; table 3), there are not evidences that these attributes have effective expression on nesting site availability and usage by stingless bees of atlantic rainforest in mer, contrary to suppositions made by several authors. of the total number of nesting trees in mer, 51.6% were found in the early forest stage of regeneration and 49.4% in the old growth stage of forest regeneration. the differences were not significant between the two stages regarding the abundance of living trees with nests (p=0.9170) and richness of nesting trees (p=0.8326). according to silva and colleagues (2013) the abundance of nests and the species richness of stingless bees were also similar in both stage of forest regeneration. probably, there is no variation in the availability of suitable tree hollows per unit area between the two different stages of forest regeneration. there are contrasting approaches and mainly conflicting interpretations about the relative importance of pioneer or slow growth trees as nesting substrate for meliponini in tropical forests (hubbell & johnson, 1977; batista 2003; eltz et al., 2003; samejima et al., 2004). often, a higher wood hardness of slow growth trees (e.g. tolerant to shade) corresponds to a higher mechanical strength and natural wood durability (florsheim, 1992), which would mean less exposure to fungi and insects and weathering action (burger & richter, 1991). however, the similar availability of hollows on pioneer and slow growth trees is likely a key determinant of abundance and spatial distribution of stingless bees nests in forests at different stages of regeneration, and not the physical characteristics of these trees per se. alternatively, the range of variation in tree hollow availability between the stages of forest is well above the demand by the bees’ community. for example, hubbell and johnson (1977) estimated that the stingless bees should occupy 34% of tree hollows available in a tropical dry forest in costa rica. the low proportion of nests per trees with suitable sizes (1 nest per 100 trees with cbh >60cm), in atlantic rainforest of mer (silva et al., 2013) also supports that availability of tree hollows overcomes the stingless bees demands in both stage of forest regeneration. the mer data support the general argument of oversupply of arboreal substrates for nesting in the forest for stingless bees, suggesting access control mechanisms operating at the community level that would also explain the low nest density (2.8 nests/ha; silva et al., 2013). on the other hand, selectivity for tree species does not exist or at least is not relevant in spatial structuring (or temporal) of communities of stingless bees in this rainforest. the extreme disruption of forest habitats, accompanied by extensive deforestation and savannization of landscapes, however, tends to reduce the diversity of trees and change considerably the pattern of tree hollows supply, exposing stingless bees to greater convergence in the use of nesting substrates. in this process, generalist species in using cavities with higher swarm rates should become dominant in the communities, as probably is happening with t. angustula (silva et al., 2013, 2014). in such a scenario, the management of stingless bees in the forest should be closely associated with the management of diversity of trees for regeneration of tropical rainforests. acknowledgements to the pollination ecology lab staff (ecopol-ib/ ufba) for helping with field work. to dr. lazaro benedito da silva laboratory of plant morphology and anatomy (ib/ufba) for helping in determining the wood density of the trees. to michelin for logistical support. to capes for the first author’s doctoral scholarship. to cnpq (processes no. 481113/478271/2008 and 474313/2011-5) and fapesb (apr0114/2006) for research support. references aguilar-monge, i. 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(1984). métodos de determinação da densidade da madeira. boletim técnico 1. viçosa: sociedade de investigações florestais, 21 p. witter, s., lopes l.a., lisboa, b.b., blochtein, b., mondin, c.a. & imperatriz-fonseca, v.l. (2010). ninhos da abelha guaraipo (melipona bicolor schencki), espécie ameaçada, em remanescente de mata com araucária no rio grande do sul. série técnica fepagro 5, 37 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.4973sociobiology 68(1): e4973 (march, 2021) introduction brazil has become an important global trader of raw materials in the mineral sector, including bauxite, whose main reserves occur within the legal amazon (ibram 2010). in light of law enforcement and acquisition of environmental abstract environmental impact studies often involve monitoring and using bioindicators to evaluate the restoration stage of impacted areas. we aimed to assess ant assemblages’ response to the ecological succession of previously disturbed areas in the brazilian amazon. we sampled epigeic ant assemblages in five bauxite mining areas, representing different restoration stages, and compared them with two pristine areas. we also compared trends in species richness at the same mine site investigated 14 years earlier. ten pitfall traps and four winkler samples of litter were taken along a 100-m transect in each area. we expected that ant species richness would increase with the amelioration in habitat condition (i.e., environmental surrogates of ecological succession, including litter depth, soil penetrability, the circumference of trees, the distance of trees to adjacent trees, and percentage of ground cover). we also compared the efficacy of both sampling methods. due to more significant sampling effort, pitfall traps captured more ant species than winkler sacks. however, winkler samples’ addition allowed the collection of more cryptic species than by pitfall traps alone. we sampled a total of 129 ant species, with increases in ant species richness in more mature rehabilitation. nevertheless, similarity analysis indicated a significant difference between ant assemblages of rehabilitated areas and pristine ones. assemblages differed mainly by the presence of specialist and rare species, found only in pristine plots. rehabilitated areas exhibited a significant increase in tree circumference as they reached more ecologically advanced stages, which contributed to increasing ant species richness. these trends and comparison with the earlier study indicate that although there are favorable increases in ant species richness, in terms of species composition, rehabilitated areas were far from achieving an ant assemblage composition or environmental status that closely resembles pristine areas. sociobiology an international journal on social insects geraldo wilson fernandes¹, tate c. lana², carla r. ribas², josé henrique schoereder2, ricardo solar1, eduardo g. cordeiro², jonathan d. majer3, jacques hubert c. delabie4, evaldo ferreira vilela2 article history edited by evandro n. silva, uefs, brazil received 18 january 2020 initial acceptance 13 april 2020 final acceptance 15 march 2021 publication date 26 march 2021 keywords amazonian ants, trombetas, biomonitoring, biodiversity, ecological indicators, land rehabilitation. corresponding author geraldo wilson fernandes departamento de genética, ecologia e evolução, instituto de ciências biológicas universidade federal de minas gerais av. pres. antônio carlos nº 6627 pampulha, cep: 31270-901 belo horizonte-mg, brasil. e-mail: gw.fernandes@gmail.com certificates (lamb et al. 2005), mining companies have developed ecological restoration programs that aim to restore vegetation that resembles the forest, ecologically and visually (parrotta & knowles 1999). because of high levels of ecosystem impacts during mining operations (peterson & heemskerk 2001), restoration needs to develop complex 1 departamento de genética, ecologia & evolução, instituto de ciências biológicas, universidade federal de minas gerais, belo horizonte-mg, brazil 2 programa de pós-graduação em entomologia, universidade federal de viçosa, minas gerais, brazil 3 school of molecular and life sciences, curtin university of technology, perth, western australia 4 departamento de biologia geral, universidade federal de viçosa, minas gerais, brazil. centro de pesquisas do cacau – ceplac, bahia, brazil research article ants changes in epigaeic ant assemblage structure in the amazon during successional processes after bauxite mining id https://orcid.org/0000-0003-1559-6049 geraldo wilson fernandes et al. – ant communities after mining2 knowledge associated with the mining process, including restoration with native species and implementing long-term monitoring processes (e.g., fernandes et al., 2010; kollman et al., 2016). despite law enforcement and efforts by the industries involved, the monitoring of restoration programs in forest ecosystems has mainly been carried out from a dendrological perspective (kollman et al., 2016). most environmental components measured relate to the observed changes in factors such as habitat structure and plant biomass (reis & kageyama, 2003; but see fernandes et al., 2010). this simplistic approach underestimates meaningful ecological interactions that can potentially restore biodiversity and ecosystem services through natural succession processes, i.e., interactions necessary to re-establish ecosystem functioning (stanturf et al., 2001; harris et al., 2006); for a review, see kollman et al., 2016. detailed studies on recolonization of soil biota and distribution of species after mining operations can provide management programs with more robust information on how ecological processes evolve to achieve a sustainable stage (kollman et al., 2016). in this case, a common approach is the use of bioindicators that can reflect the restored sites’ environmental conditions (ribas et al., 2012; schmidt et al., 2013, donoso, 2017). some target ant species play specific ecological roles in the soil (andersen & sparling, 1997; bisevac & majer, 1999; passos & oliveira, 2003; 2004; donoso & ramon, 2009; schmidt et al., 2013), and their presence after mining operations can indicate the stage of ecological succession and soil regeneration (majer, 1983; underwood & fisher, 2006; ottonetti & tucci, 2006; ribas et al., 2012). most of the physical-chemical processes and crucial biological interactions for habitat reestablishment occur in the soil, where most ant species forage (schmidt et al., 2013). however, colonization and establishment of these species will be dependent on local environmental characteristics, as well as the management practices developed after mining, e.g., by planting a mix of attractive species that is favorable to the soil biota (majer, 1983; andersen & majer, 2004; dos santos alves et al., 2011). a widely used restoration management procedure in brazil is the return of topsoil after mining activities have ceased (parrotta & knowles, 1999). therefore, monitoring the response of ant assemblages to environmental restoration has to involve sampling sensitive parameters that reflect natural succession and permit evaluation of success and adoption of local management practices (ribas et al., 2012). disturbance in tropical rain forests directly influences ant assemblages’ structure (kaspari, 1996; dos santos alves et al., 2011). in contrast, the return of this faunistic group to a disturbed area increases the number of local processes and interactions needed for the ecosystem’s sustainability. an earlier 1992 study at the same mine-site considered here (majer, 1996) sampled ants in three forest reference sites and ten rehabilitation areas, ranging from 0-11 years of age. we built upon this study using some sites between 3-13 years older than those from the earlier study. we aimed to determine how and if ant community structure of restored areas following bauxite mining converges towards the structure level found in pristine adjacent forests with the increased passage of time. we hypothesize that 1) there would be an increase in ant species richness with the improvement of the environmental parameters according to restoration time, and 2) ant species composition would change as a response to ecological succession since some groups of species respond in distinctive ways to environmental parameters (underwood & fisher, 2006). materials and methods study site the study area is in the district of porto trombetas, 65 km from oriximiná (western region of pará state, brazil, eastern amazon) (fig 1), and 100 km west of the trombetas river confluence with the amazon river, northern brazil. fig 1. left: geographical localization of porto trombetas, north of brazil, pará state. right: study areas inserted in a hypsometric map. sociobiology 68(1): e4973 (march, 2021) 3 we carried out the study restoration areas of trombetas bauxite mines, operated by mineração rio do norte s.a. (hereafter, mrn) and situated in areas of tropical, pristine forest. since the 1980s, the restoration program used by mrn has involved topsoil replacement, application of litter and triturated wood, and planting mixed stands of native forest species, aiming to restore approximately 100 ha/year (parrotta & knowles 1999). the vegetation is characteristic equatorial evergreen rainforest. according to köppen, the local climate is am (tropical monsoonal), with well-established dry (winter) and wet (summer) seasons (see parrotta & knowles 1999). mean annual rainfall at porto trombetas in past years (1970–1993) was 2185.64 mm, whereas the mean maximum and minimum temperatures were 34.6 and 19.9 °c (see parrotta & knowles 1999). during our study period (undertaken between may 20 and june 20, 2006), the maximum and minimum temperatures in porto trombetas were respectively 32.5°c and 22.0°c; 476.5 mm of rainfall fell in may 2006, and 133.0 mm of rain fell in june 2006 (cptec / inpe). sampling design we selected two areas of undisturbed forest as controls (a plateau and a flank) and five areas with different ages since restoration (4 to 26-year-old rehabilitation areas) (table 1). within each area, a 100 m-long transect was arbitrarily set for sampling ants and measuring habitat structure. the latter included tree spacing and circumference at breast height, percentage soil cover and depth, litter depth and dry weight of litter, all commonly used as surrogate explanatory variables for ecological succession. we considered epigeic ants as all those ants foraging or nesting on the soil surface or in the litter. we used pitfall traps and winkler extractors to survey the ant fauna. combining these two methods yields a good sample, and they are the two most efficient techniques recommended for studying epigeic ants (bestelmeyer et al., 2000; delabie et al., 2000; brown & mathews, 2016). we installed ten pitfall traps, consisting of cups of 7 cm diameter, 200 ml volume, containing 30 ml of 70% ethanol, in the ground every 10 m along the 100 m transect. traps remained for seven consecutive days in each area. along the same transect, we sift 1 m² litter samples taken every 25 m, producing a total of four samples per plot. the litter was sieved and placed in winkler sacks that we left in winkler extractors for 72 hours outside the laboratory. this process allows sieved litter material to dry and ants to migrate in search of other habitats, falling into the vial of ethanol attached to the bottom of the extractor. after the sampling procedure, we sorted all material, identified it, and deposited voucher specimens at ceplac (comissão executiva do plano da lavoura cacaueira), bahia, brazil. we used various habitat variables as evidence of forest structural changes along the restoration chronosequence (a surrogate of forest succession). we considered these variables influential on ant community richness and composition (andersen, 2000; yanoviak & kaspari, 2000; ribas & schoereder, 2007; schmidt et al., 2013; solar et al., 2016a, see also paolucci et al., 2016). we obtained an average of tree spacing (distance) and tree circumference at breast height from the four nearest trees at each sampling point. we took other measures inside a 1 m2 frame placed around each trap before installation. we visually estimated soil cover percentage within each quadrat. we measured litter depth in each corner of the quadrat, with the mean providing an average litter depth per quadrat. at each corner, we measured soil penetrability by using a metal stick with standard pressure, obtaining the average of these four penetration measures. statistical analyses aiming to test sampling efficiency and using two distinct sampling methods and different sampling efforts between them, we built a species accumulation curve that calculated the expected richness for random samples of data. we used the chao 2 index as recommended by colwell and coddington (1994) as the best estimate for incidence-based richness. to test the influence of environmental parameters on ant species richness, we built a complete model. we used the mean values of tree spacing, tree circumference at breast height, percentage of soil cover, percentage of litter, and soil compression (using data for ten pitfall traps combined). winkler samples do not appear in the model as they were randomly taken in each area, not following the same statistical design as the pitfall trap samples. after evaluating the variables’ contribution, we simplified the model by removing non-significant variables until reaching the minimum suitable table 1. location and rehabilitation time for five mined areas and two pristine areas surveyed for epigeic ants in 2006 in the district of porto trombetas, pará state, brazil. the species richness of ants found in these areas is also shown. areas latitude longitude restoration time (years) total richness plateau forest 1°45’ 044’’ s 56°22’ 611’’w pristine 34 flank forest 1°41’ 795’’s 56°23’ 564’’w pristine 53 rehabilitated 2002 1°37’ 911’’s 56°26’ 675’’w 4 32 rehabilitated 1999 1°40’ 765’’s 56°26’ 378’’w 7 40 rehabilitated 1992 1°39’ 855’’s 56°25’ 639’’w 14 39 rehabilitated 1981 1°41’ 360’’s 56°23’ 379’’w 25 39 rehabilitated 1980 1°40’ 248’’s 56°23’ 481’’w 26 47 geraldo wilson fernandes et al. – ant communities after mining4 model. we fitted multiple regression models and tested them using poisson errors. we performed all analyses using the r development core team software. we compared the differences in species composition in ant assemblages between rehabilitated and native plots using non-metric multidimensional scale (nmds) multivariate analysis. we prepared a binary matrix (ant species absence or presence in each area) and calculated the dissimilarities between these areas by the jaccard index. the second step was a one-way analysis of similarity (one-way anosim) that established whether there were significant differences in species composition between plots (r-value and p<0.01) (clarke & green 1988). a posteriori, we built a table for ant species relative abundance. we considered all 14 samples in each area (10 pitfall traps and four winkler samples). we calculated the number of records (maximum 14 in each area) to represent each species’ relative abundance. results we recorded 129 ant species, representing 40 genera and eight subfamilies (table 1). myrmicinae was the richest subfamily, while pheidole was the richest genus, with 15 species, followed by solenopsis (10 spp.), crematogaster (7 spp.), strumigenys and pseudomyrmex (7 spp. each). solenopsis sp.1 presented the highest relative abundance (number of species occurrences among all samples) in rehabilitated and pristine areas. strumigenys denticulata was also widespread, both in rehabilitated and pristine plots. crematogaster tenuicola, nylanderia steinheili, crematogaster brasiliensis and ectatomma brunneum were the most common species. they were only found in rehabilitated areas, not found in any plateau or flank samples. mayaponera constricta and pachycondyla harpax were also widely distributed in the rehabilitated plots but were also found less frequently in pristine areas. we found 26 species exclusively in pristine areas, including species of the genera apterostigma, cyphomyrmex, discothyrea, thaumatomyrmex, eciton, gnamptogenys, basiceros, blepharidatta and strumigenys (table 2). we observed a tendency for an asymptote in the accumulation curve of total species sampled in the study. after combining all samples, the chao 2 index indicated a sampling efficiency of 74% (129 species observed), giving an expected number of 173 species (fig 2). pitfall traps captured 111 ant species, while winkler extractors collected 69 ant species, of which 17 we sampled exclusively by this method. in total, pitfall trap and winkler extractor samples differed by 37.7 % in terms of ant species composition (jaccard; p=0.006). winkler extraction captured four species of the genus hypoponera (including the species h. foreli), strumigenys denticulata and s. trudifera. gnamptogenys horni was found three times more frequently in pitfall traps than by winkler extraction, while pheidole sp.3 and acromyrmex sp.2 were only captured by pitfall trapping. fig 2. cumulative species number by accumulation curve and cumulative sampling effort. the dotted line represents the estimated species richness by chao 2 index. the continuous black line is the effort of all samples – coleman curve. the continuous gray line shows the sum of species collected in rehabilitated areas, whereas the black dashed line shows collected species in pristine plots (control). sociobiology 68(1): e4973 (march, 2021) 5 means for the environmental parameters are shown in table 3. none of these parameters explained ant species richness. rehabilitated plot means showed increased tree circumference toward a higher advanced stage close to values in pristine areas. this trend was positively correlated with increasing distance (spatial distribution) between trees (r=0.93, p=0.003). litter depth was significantly thicker in pristine areas and showed a high value in some of the younger rehabilitated plots (table 3). soil penetrability varied between rehabilitated areas, and pristine ones exhibited higher values. regarding soil litter cover, all plots except the seven yr-old plot exhibited complete coverage of soil (table 3) 2002 1999 1992 1981 1980 plateau flank years since rehabilitation 4 7 14 20 21 pristine pristine soil cover (%) 85.1+0.11a 69.5+0.23b 92.3+0.09a 94.7+0.08a 96.8+0.04a 89.5+0,12a 93.0+0.10a litter thickness (cm) 11.7+4.94a 5.4+2.61c 8.2+1.41b 7.9+2.12b 7.3+1.28b 12.0+2.51a 11.3+3.43a soil penetrability (cm) 10.2+4.86a 9.8+2.42a 10.8+2.51a 11.9+1.99a 7.1+2.01b 16.97+2.16 c 16.2+4.79 c distance to nearest trees (cm) 166.0+41.96a 175.8+44.12a 206.6+56.46b 196.5+45.35b 200.9+53.00b 315.2+85.29c 299.5+121.55c circumference at breast height tree circumference (cm) 18.9+3.14a 20.1+4.63a 30.4+6.74b 41.1+11.33c 31.03+9.24b 63.9+45.91e 53.6+22.65 d table 3. environmental characteristics (mean + sd) of rehabilitated and pristine areas. numbers with similar letters indicate no significant differences between means (see materials and methods). discussion the high richness of ant genera and morphospecies found in this study confirms previous studies showing that the amazon forest exhibits a very rich ant community (majer, 1996; majer & delabie, 1994). compared to inventory studies in tropical rainforests, our combined sampling effort of both methods was sufficient for a satisfactory survey of the community (129 spp, 74% of efficiency) (see solar et al., 2016b). we expected an increase of ant species richness in older successional stages associated with increases in environmental parameters (ribas et al., 2003, donoso et al., 2013). however, unlike in other studies in disturbed areas (ottonetti & tucci, 2006; vasconcelos et al., 2000; schmidt et al., 2013), this was not the case. however, the ant community structure was significantly distinct between areas (see solar et al., 2016a), mainly due to the presence of specific and sensitive species. the natural history and function of these ant species can tell us much about the ecological state and health of the areas where they occur (see bihn et al., 2010; leal et al., 2012; schmidt et al., 2013; solar et al., 2016b; donoso 2017). that could be the case of ant species exclusively found in pristine areas, such as basiceros balzani and blepharidata brasiliensis, associated with undisturbed forest (vasconcelos et al., 2000). also of relevance are gnamptogenys horni and strumigenys trudifera, endemic from the brazilian amazon (kempf & brown, 1969), discothyrea, with a low tolerance to disturbance (brown, 1957), and thaumatomyrmex, all of which are exclusive of tropical native areas (cerdá & dejean, 2011). moreover, the two species of army ants, eciton burchellii and neivamyrmex swainsonii, which prefer wetter and more pristine areas (levings, 1983), were only found in native areas. in this case, humidity, lower temperature oscillation, higher litter thickness, among other environmental conditions in the forest, probably favor their biological requirements over conditions in rehabilitated plots. fig 3. diagram of multivariate analysis of nmds (non-metric multidimensional scale). pristine areas are represented by n = plateau and f = flank, whereas rehabilitated areas are represented by the number of years since restoration (jaccard index, stress = 0.13). the increase in circumference and spacing of trees in rehabilitated plots towards or near values in pristine areas indicates a positive trend in ecological succession (ruiz-jaen & aide, 2005). in the early 1980s, mrn started a restoration program with a mix of native species of trees. this process favors the establishment of early growth trees. still, due to the short life-span of pioneer species (10-20 years), there would be no certainty about whether succession will proceed towards the mature forest for many years. in our study, a more sustainable succession is expected to be occurring because of the presence of climax species in rehabilitated areas, including bertholletia excelsa (lecythidaceae), stryphnodendron guianensis (fabaceae), sclerolobium paniculata (fabaceae), geraldo wilson fernandes et al. – ant communities after mining6 tapirira guianensis (anacardiaceae), and bowdichia virgilioides (fabaceae) (gwf. pers. obs). also, these rehabilitated areas may be enriched with propagules coming from nearby native vegetation. secondary forests can support their characteristic ant fauna, probably containing pioneer and/or generalist ant species and rare and more specific ones. furthermore, the presence of well-developed and established plants of rapidgrowth, like cecropia (e.g., in the four-year-old area), resulting in a thicker litter layer formed by fallen leaves, which is as thick as in the two native plots. these facts mean that even the young rehabilitated plots can support a rich epigeic ant community. despite a higher number of pitfall trap units than winkler samples, the latter method allowed us to sample smaller and cryptic species of the genera carebara, cyphomyrmex, discothyrea, hypoponera and strumigenys. pitfall traps collected exclusive species and, as expected, more active and larger ones (olson, 1991; orsolon-souza, 2011), including all species of the genus pseudomyrmex which mainly forage on plants. in our study, winkler extractors could have their efficacy impeded due to the atypical rainfall, with rainfall rates being much higher than 17 years ago (mrn data). lassau and hochuli (2004), using only pitfall traps, argued that a range of possible biases might accompany pitfall trapping as a sampling technique in structurally complex areas. in our study, pitfall traps and winkler extractors were complementary in sampling epigeic ants in structurally more complex native areas. despite the lower effort devoted to winkler samples, the number of ant species sampled by the two methods was sufficient. since the effectiveness of methods differs, we suggest future brief surveys must consider a combination of both methodologies, allowing a more accurate census of ant species in structurally complex areas of amazonia. table 4 compares the ant species richness values in rehabilitation between the earlier (majer, 1996) and the current study. the comparison focuses on species richness since the morphospecies code numbers are not standardized across the two studies. also, the earlier study data include arboreal ants, so values in sites where the tree stratum is well developed will tend to be relatively higher in majer’s (1996) study. with these limitations in mind, the values seem reasonably comparable in the younger sites, with similar values for the youngest two areas being obtained in both studies (table 4). although richness in the oldest three sites was lower, or in the range of majer’s (1996) 9-14-year-old sites, we remember that the earlier study included arboreal species, thus making richness in the pristine sites 62 to 75% higher. by analogy, the 14-26-year-old sites in the current study might be performing well in terms of species richness, supporting ant richness close to that of pristine sites. as in the earlier study, the multivariate analysis indicates that there is still a significant difference between rehabilitated and pristine sites in terms of species composition. unfortunately, it is impossible to compare this degree of difference between the earlier and the current study due to different analytical procedures. some studies have evaluated ant’s efficacy as bioindicators, but mining areas are poorly represented (ribas et al., 2012; schmidt et al., 2013). despite changes in the ant fauna composition between rehabilitation stages (schmidt et al., 2013), and even though 26 years is considered a relatively long time for restoration, these areas still have a valuable conservation value (e.g., rozendaal et al., 2019). thus, despite aiming to develop restoration of a forest characteristic of the area, succession still needs more time to achieve a full recovery in these previously impacted areas (see fernandes et al., 2010, also see rozendaal et al., 2019). these areas lack some species that indicate a healthy, stable, and advanced ecological stage found in native areas. the adjacent pristine areas play a fundamental role as the source of rare species’ propagules for the colonization of areas under the ecological successional process. hence, for reliable information on species diversity and ecosystem function, long-lasting monitoring is needed in rehabilitated and native areas (see bihn et al., 2010). acknowledgments we thank two anonymous reviewers for their valuable comments on earlier versions of this ms. we also thank kevin cazelles for his support in map design. financial aid was provided by the programa de pós-graduação em entomologia (ufv), cnpq, planta ltda, and fapemig, while mrn provided field logistics. age of rehabilitation (yr) 1992 study current study 0 4 0.3 8 4 32 5 46 6 43 7 41 40 7 41 8 46 9 55 10 46 11 44 14 39 25 39 26 47 plateau 54 34 flank 70 53 table 4. comparison of ant species richness values in rehabilitation from the earlier 1992 study (majer 1996) with values obtained in the current study. sociobiology 68(1): e4973 (march, 2021) 7 species 4 yrs 7 yrs 14 yrs 25 yrs 26 yrs flank plateau occurrence subfamily dolichoderinae dorymyrmex sp. 1 0 1 0 0 0 0 0 1.02 subfamily dorylinae acanthostichus brevicornis (emery) 0 0 1 0 0 1 0 2.04 eciton burchelli (westwood) 0 0 0 0 0 2 0 2.04 labidus spininodis (emery) 1 0 0 0 0 1 1 3.06 neivamyrmex sp. 1 0 0 0 1 0 1 0 2.04 neivamyrmex swainsonii (=fallax) shuckard 0 0 0 0 0 1 0 1.02 nomamyrmex esenbeckii (santschi) 0 0 1 0 0 0 0 1.02 subfamily ectatomminae ectatomma brunneum (smith) 3 8 0 3 2 0 0 16.33 ectatomma suzanae (almeida) 0 0 0 2 0 2 0 4.08 ectatomma tuberculatum (olivier) 0 0 0 0 2 0 0 2.04 gnamptogenys horni (santschi) 0 0 0 0 1 2 6 9.18 gnamptogenys moelleri (forel) 0 0 1 1 0 1 0 3.06 gnamptogenys striatula (mayr) 0 0 0 0 0 1 0 1.02 gnamptogenys sulcata (smith) 0 0 0 1 0 1 0 2.04 subfamily formicinae brachymyrmex sp. 1 0 8 0 0 0 0 0 8.16 brachymyrmex sp. 2 0 0 0 0 1 0 0 1.02 camponotus atriceps (smith) 1 0 0 0 0 0 0 1.02 camponotus fastigatus (roger) 0 2 0 1 0 0 0 3.06 camponotus leydigi (forel) 0 1 0 0 0 0 0 1.02 camponotus melanoticus (emery) 0 5 0 0 0 0 0 5.10 camponotus novogranadensis (mayr) 0 8 2 0 1 0 0 11.22 camponotus rufipes (fabricius) 0 8 0 0 1 0 0 9.18 gigantiops destructor (fabricius) 0 0 1 0 1 0 0 2.04 nylanderia fulva (mayr) 1 3 0 0 0 0 0 4.08 nylanderia guatemalensis (forel) 0 1 2 0 1 0 0 4.08 nylanderia sp. 1 2 5 1 2 0 1 0 11.22 nylanderia sp. 2 2 6 3 0 2 0 0 13.27 nylanderia sp. 3 0 3 4 2 2 1 0 12.24 nylanderia steinheili (forel) 2 1 4 2 1 0 0 10.20 subfamily myrmicinae acromyrmex sp. 1 0 0 1 0 0 1 0 2.04 acromyrmex sp. 2 0 0 0 0 0 1 4 5.10 apterostigma sp. 1 0 0 0 0 0 0 1 1.02 apterostigma sp. 2 0 0 0 0 0 1 0 1.02 atta sexdens (forel) 1 2 0 0 0 0 0 3.06 basiceros balzani (emery) 0 0 0 0 0 3 0 3.06 blepharidatta brasiliensis (wheeler) 0 0 0 0 0 1 2 3.06 carebara sp. 1 0 0 0 0 1 0 0 1.02* centromyrmex gigas (forel) 0 0 0 0 1 0 0 1.02 cephalotes sp. 1 0 0 1 0 0 0 0 1.02 cephalotes sp. 2 0 2 1 0 0 0 0 3.06 cephalotes sp. 3 0 0 0 0 0 1 0 1.02 crematogaster brasiliensis (mayr) 5 0 0 4 8 0 0 17.35 table 2. ant species found within each sample area and total species richness. the last column represents the relative percentage of occurrence of each species among all 98 traps. in this column, asterisks represent ant species sampled exclusively by winkler extractors. geraldo wilson fernandes et al. – ant communities after mining8 crematogaster curvispinosa (mayr) 1 0 0 0 0 0 0 1.02* crematogaster evallans (forel) 0 0 0 2 0 0 0 2.04 crematogaster flavosensitiva (longino) 0 0 2 0 0 0 1 3.06 crematogaster limata (smith) 0 0 3 2 3 0 0 8.16 crematogaster stollii (longino) 0 0 0 0 0 0 1 1.02 crematogaster tenuicula (forel) 2 3 5 12 6 0 0 28.57 cyphomyrmex costatus (mann) 0 0 0 0 0 0 1 1.02* cyphomyrmex rimosus (mayr) 2 3 0 0 0 0 0 5.10 cyphomyrmex sp. 1 0 1 0 0 1 0 2 4.08 cyphomyrmex sp. 2 1 0 0 0 0 0 0 1.02 cyphomyrmex sp. 3 0 0 0 1 1 0 0 2.04* cyphomyrmex sp. 4 0 0 0 2 1 0 0 3.06 myrmicocrypta sp. 1 0 0 0 0 1 0 0 1.02 ochetomyrmex sp. 1 0 0 3 1 2 1 1 8.16 pheidole jeannei (wilson) 0 0 1 0 0 3 0 4.08 pheidole midas (wilson) 0 0 1 0 1 1 1 4.08 pheidole sp. 1 gp fallax 0 0 1 3 0 0 0 4.08 pheidole sp. 2 gp fallax 2 0 0 0 0 0 0 2.04 pheidole sp. 3 gp diligens 0 0 2 0 1 1 4 8.16* pheidole sp. 4 gp diligens 0 0 0 0 0 1 0 1.02 pheidole sp. 5 gp fallax 0 0 0 0 0 1 0 1.02 pheidole sp. 6 gp gertrudae 1 1 0 0 0 2 0 4.08 pheidole sp. 14 0 0 0 0 0 1 0 1.02 pheidole sp. 16 0 1 0 0 0 0 0 1.02 pheidole sp. 17 2 0 0 0 0 2 0 4.08 pheidole sp. 19 0 0 0 0 1 0 1 2.04 pheidole sp. 24 0 0 0 0 0 1 0 1.02* pheidole sp. 25 0 0 0 0 1 0 0 1.02 pheidole sp. 29 gp gertrudae 0 0 1 0 0 1 1 3.06* rogeria sp. 1 0 4 0 4 2 1 1 12.24 sericomyrmex sp. 1 0 0 3 7 0 0 1 11.22 sericomyrmex sp. 2 0 0 1 0 1 0 1 3.06 solenopsis geminata (fabricius) 1 0 0 0 0 0 0 1.02 solenopsis sp. 1 2 7 11 3 3 1 4 31.63 solenopsis sp. 2 0 0 4 1 3 1 0 9.18 solenopsis sp. 3 0 0 0 0 0 1 0 1.02 solenopsis sp. 4 1 0 0 0 0 0 0 1.02 solenopsis sp. 5 0 0 2 0 0 0 0 2.04 solenopsis sp. 6 0 0 1 0 0 0 0 1.02 solenopsis sp. 7 1 0 0 0 1 0 0 2.04 solenopsis sp. 8 0 0 0 1 0 1 1 3.06* solenopsis sp. 9 1 0 0 0 0 0 0 1.02 strumigenys sp.2 0 0 0 1 0 0 0 1.02 strumigenys cordovensis (mayr) 0 0 0 1 0 0 0 1.02* strumigenys denticulata (mayr) 2 2 3 1 4 2 3 17.35 strumigenys elongata (roger) 0 0 0 0 0 1 0 1.02* species 4 yrs 7 yrs 14 yrs 25 yrs 26 yrs flank plateau occurrence subfamily myrmicinae table 2. ant species found within each sample area and total species richness. the last column represents the relative percentage of occurrence of each species among all 98 traps. in this column, asterisks represent ant species sampled exclusively by winkler extractors. (continuation) sociobiology 68(1): e4973 (march, 2021) 9 mycetomoellerius farinosus (emery) 0 0 0 4 1 0 0 5.10 mycetomoellerius sp. 1 0 0 0 0 0 2 0 2.04 strumigenys trudifera (smith) 0 0 0 0 0 1 2 3.06* strumigenys sp. 1 2 1 0 0 1 3 1 8.16* strumigenys sp. 6 0 0 0 0 0 1 0 1.02 wasmannia auropunctata (roger) 1 13 3 0 0 0 1 18.37 wasmannia sp. 2 0 1 2 0 0 0 0 3.06 wasmannia sp. 3 0 1 0 0 0 0 0 1.02 subfamily ponerinae anochetus diegensis (forel) 0 0 0 1 0 0 1 2.04 anochetus horridus (kempf) 0 0 0 2 0 2 0 4.08 anochetus mayri (emery) 0 5 0 2 1 0 0 8.16 anochetus sp. prox. bispinosus (smith) 0 0 0 1 1 0 0 2.04* anochetus sp. 1 0 0 0 0 1 0 1 2.04 anochetus sp. 2 0 0 0 1 0 0 0 1.02* hypoponera foreli (mayr) 0 2 6 5 2 3 1 19.39 hypoponera sp. 1 0 1 0 0 1 3 3 8.16* hypoponera sp. 2 2 5 1 2 2 3 2 17.35 hypoponera sp. 3 1 1 0 5 0 0 0 7.14 hypoponera sp. 4 0 0 0 0 1 2 2 5.10* hypoponera sp. 5 3 0 2 0 1 1 1 8.16 leptogenys sp. 1 0 0 2 0 0 1 1 4.08 mayaponera arhuaca (forel) 1 2 0 1 0 0 0 4.08 mayaponera constricta (mayr) 6 1 1 6 5 0 1 20.41 neoponera apicalis (latreille) 2 0 0 0 3 0 0 5.10 neoponera verenae (forel) 0 0 7 0 0 0 0 7.14 odontomachus allolabis (kempf) 0 0 0 1 0 0 0 1.02 odontomachus bauri (emery) 0 8 0 0 0 0 0 8.16 odontomachus caelatus (brown) 0 0 0 0 0 2 0 2.04 odontomachus haematodus (linnaeus) 3 0 0 1 2 1 0 7.14 odontomachus meinerti (forel) 0 0 0 0 0 0 1 1.02 pachycondyla crassinoda (latreille) 0 0 1 0 5 0 0 6.12 pachycondyla harpax (fabricius) 1 2 4 5 0 1 0 13.27 thaumatomyrmex sp. 1 0 0 0 0 0 0 1 1.02 subfamily proceratiinae discothyrea sp. 1 0 0 0 0 0 2 0 2.04* subfamily pseudomyrmecinae pseudomyrmex boopis (roger) 0 0 0 0 1 0 0 1.02 pseudomyrmex filiformis (fabricius) 0 0 0 0 0 1 0 1.02 pseudomyrmex gracilis (fabricius) 0 0 0 0 1 0 0 1.02 pseudomyrmex oculatus (smith) 0 1 0 0 0 0 0 1.02 pseudomyrmex termitarius (smith) 0 2 0 0 0 0 0 2.04 pseudomyrmex sp. 1 gp pallidus (smith) 0 0 0 0 0 1 0 1.02 pseudomyrmex sp. 2 gp pallidus (smith) 0 1 0 0 0 0 0 1.02 total species richness 32 40 39 39 47 53 34 species 4 yrs 7 yrs 14 yrs 25 yrs 26 yrs flank plateau occurrence subfamily myrmicinae table 2. ant species found within each sample area and total species richness. the last column represents the relative percentage of occurrence of each species among all 98 traps. in this column, asterisks represent ant species sampled exclusively by winkler extractors. (continuation) geraldo wilson fernandes et al. – ant communities after mining10 authors' contributions gwf: conceptualization, methodology, resources, data curation, writing & reviewing tcl: conceptualization, formal analysis, investigation, data curation, writing & reviewing jdm: conceptualization, methodology, formal analysis, investigation, data curation, writing & reviewing efv: conceptualization, methodology, formal analysis, resources, writing & reviewing crc: formal analysis, writing & reviewing rs: formal analysis, writing & reviewing egc: formal analysis jhs: formal analysis jhcd: data curation, writing & reviewing references andersen, a.n. & majer, j.d. (2004). ants show the way down under: invertebrates as bioindicators in land management. frontiers in ecology and the environment, 2: 291-298. andersen, a. n. & sparling, g. p. 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(2000). community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos, 89: 259-266. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i4.4926sociobiology 67(4): 545-553 (december, 2020) introduction bees are prolific pollinators, being responsible for the pollination of most wild and cultivated plants (ipbes, 2016). but recent events have caused significant losses of apis mellifera colonies, the most common pollinator used for agricultural purpose throughout the world. the generalized use of a. mellifera as pollinators has produced an overdependence of most crops on this bee species, pointing to the need to diversify the species of bees used for agricultural pollination. therefore, recent studies with other species of apis have been intensified in eastern parts of the globe to identify different pollinators capable of being used efficiently in an agricultural setting. alternatively, western nations have focused mainly on species of the genera megachile, nomia, osmia, centris, bombus and stingless bees (magalhães & freitas, 2013; putra et al., 2014; sterk et al., 2016; brunet & syed, 2017; cane et al., 2017). abstract bees are prolific pollinators and are responsible for the pollination of most wild and cultivated plants. this study aimed to learn about the flight activity of the stingless bee plebeia aff. flavocincta in tropical conditions as a parameter to evaluate the general state of the colonies, and to investigate the role of food resources and environmental factors in their flight activities. we recorded the worker flight activity (exit-when they leave the hive; trash-when they discard litter; entry with pollen; and entry without pollen) of four colonies throughout the year and monitored biotic and abiotic conditions that could affect the bee´s external activities such as blooming and meteorological conditions. results show that colonies remained active throughout the year and presented two major periods of external activity; one corresponded to greater food supply in the foraging area, and the other correlated with the time of year when fewer food resources were available. in addition, the four flight activities were all strongly correlated with each other. we concluded that in tropical conditions external activities of p. aff. flavocincta are regulated mainly by food availability rather than the prevailing weather conditions. we have found flight activities are an important indicator of the general health of the colony. sociobiology an international journal on social insects abs barbosa, hm meneses, fl rosa, bm freitas article history edited by evandro nascimento silva, uefs, brazil received 17 december 2019 initial acceptance 26 october 2020 final acceptance 28 november 2020 publication date 28 december 2020 keywords beekeeping; colony health; foraging activities; meliponini; tropical bees. corresponding author artur bruno barbosa http://orcid.org/0000-0001-7144-1055 campus do pici universidade federal do ceará – ufc avenida humberto monte, s/n° cep 60356-001, fortaleza-ce, brasil. e-mail: artur_bruno140@hotmail.com the stingless bees are the most common group of native bees found on the american continent. due to the large number of species with marked morphological and functional diversities and feeding habits, these bees have become excellent pollinators of wild native flora and have recently attracted interest in agricultural pollination (kakutani et al., 1993; kerr, 1996; nogueira-neto, 1997; amano et al., 2000; slaa et al., 2006; bomfim et al., 2015). however, studies within this group of bees have been concentrated on medium and large species of the genera melipona, scaptotrigona and trigona. smaller bees have not been included, such as those of the genus plebeia (cruz et al., 2004; del-sarto et al., 2005; greco et al., 2011; bomfim et al., 2015). the genus plebeia is characterized by small bees, usually between 3 and 7.5 mm (michener, 2000), which are widely distributed across the tropics and subtropics of the american continent. members of this genus can be found from southern mexico to northern argentina. in brazil, 21 species universidade federal do ceará – ufc, fortaleza, ce, brazil research article bees flight activity of the stingless bee plebeia aff. flavocincta in tropical conditions as an indicator of the general health of the colony abs barbosa, hm meneses, fl rosa, bm freitas – flight activity of plebeia aff. flavocincta indicates colony health546 of this genus are cataloged (moure et al., 2011; ascher and pickering, 2020). these bees have a lower demand for food resources due to their small size. they present an extremely generalist foraging strategy and demonstrate adaptive plasticity to environmental conditions, which allows them to have a broad geographic distribution and the potential to pollinate small flowers usually not visited by larger bee species (hrncir & maiasilva, 2013; lemos, 2014; silva et al., 2019). to maximize performance, it is important that the colonies are active, strong, and maintain a high reproductive rate to generate greater pollen demand and stimulate foraging behavior in flowers, subsequently pollinating them in the process (ramalho et al., 1985; nunes-silva et al., 2010; pick & blochtein, 2002a). activities of the bees either inside the nests or in flight are good indicators of the pollination potential from the colony at any given moment (nunes-silva et al., 2013; lichtenberg et al., 2010; filmer, 1932; lemos, 2014). while the bees’ activities within the nest reflects the status of the colony, the colony in turn depends directly on the external flight activity of those bees responsible for obtaining food, water, materials for building and maintaining the nest, and waste disposal (polatto et al., 2014). therefore, flight activities, including foraging patterns and pollination potential, qualifies as an effective parameter to evaluate the general state of bee colonies. these methods are used frequently and outlined in many publications (heard, 1999; pick & blochtein, 2002b; nunes-silva, 2013). environmental factors are a major influence on flight activity. in addition to food availability precipitation, temperature, wind speed and relative humidity have a direct effect on the external activity patterns of the colony (heard & hendrikz, 1993; hilário et al., 2001; hilário et al., 2007b; bartomeus et al., 2011). usually external conditions have a greater impact on foraging frequency than food availability. this tends to benefit bees that can act in broader temperature and humidity ranges (larger bees), which tend to have an advantage in foraging over smaller bees (kleinert et al., 2009). therefore, it is expected that small bees such as the ones of the genus plebeia have their foraging activities limited to favorable weather conditions when compared to larger bees. in fact, studies concentrated in southern brazil show that 60% relative humidity is ideal for peak activity of plebeia emerina and plebeia saiqui. minimum temperatures for foraging in p. saiqui is 11 °c, but 16 °c in p. emerina and plebeia remota (kleinert-giovaninni, 1982; pick & blochtein, 2002b). kleinert-giovaninni (1982) and hilário et al. (2007a) examined wind speed and found that p. emerina and p. remota restrict activities with winds above 4 m/s and 5.5 m/s, respectively. studies also show that in regions of high latitude, bees of the genus plebeia present reproductive diapause and decrease or interrupt the colony’s external activities due to the unfavorable field conditions in the cold months of the year (juliani, 1967; pick & blochtein, 2002b). nonetheless, there are few studies on species of the genus plebeia in tropical conditions. thus, we tested the hypothesis that for the stingless bee plebeia aff. flavocincta the general state of a colony can be assessed through external activities, which are affected by food availability. material and methods study area this study was conducted with the stingless bee plebeia aff. flavocincta between september 2017 and august 2018 in the bee unit (campus do pici universidade federal do ceará), located in the city of fortaleza, ceará (03° 43’ 02”s e 38° 32’ 35”w and 16 m of altitude) (ipece, 2017). the bee unit is located in urban remnants of a tableland forest vegetation (diogo et al., 2014). the characteristic climate of fortaleza is warm tropical sub-humid, with a rainy season comprising the months of january to may according to the köppen climate classification (ipece, 2017). the meteorological parameters were obtained through the automated meteorological station of the department of agricultural engineering (ufc), located approximately 750 m from the bee unit. the average annual rainfall during the study period was 1516.9 mm with an average temperature of 28.0 °c. the minimum and maximum annual temperature averages were 23.0 and 31.8 °c, respectively. relative air humidity was 73.5%, insolation was 2,974.7 hours per year and average wind speed was 3.4 m/s. flight activity the flight activities of the bee p. aff. flavocincta were recorded for 5 consecutive days every month in four colonies kept in rational hives between september 2017 and august 2018. bees entering and leaving a hive were quantified by manual counters for 10 minutes every hour, while there was natural light (6 am to 6 pm), as proposed by oliveira (1973). the observations were made at the nest entrance for the duration of the daily flight activities. this totaled 2,880 observations, and a sampling effort of 480 hours. the flight behavior of bees was classified in the following categories: exit-when they left to forage; trash-when they came out with materials to be discarded; entry with pollen-when they entered with a visible pollen load in the pollen basket; and lastly entry without pollen-when they entered the colony without apparent pollen load. statistical analysis due to the non-normal distribution of the data, nonparametric analyzes were used. the number of bees performing the flight activities (bee exit, trash, entry with pollen and entry without pollen) was analyzed with a kruskal-wallis test, followed by a dunn test with bonferroni’s adjustment at a significance level of 5% (p < 0.05). this was used to compare sociobiology 67(4): 545-553 (december, 2020) 547 the different periods of activity throughout the year and to test for temporal variation (dry and rainy season). in addition, a spearman correlation test was performed to evaluate possible correlations between climatic parameters (temperature, relative humidity, wind speed and precipitation) and flight activities (bee exit, trash, entry with pollen and entry without pollen). results the colonies of p. aff. flavocincta remained active throughout the year, with no interruption of foraging flights (fig 1). the range of daily flight activity lasted from 6 am to 6 pm, with this pattern being consistent throughout the year. thus, the daily amplitude achieved was 12 hours each day throughout the year. according to the correlation analysis performed to identify the relationship between meteorological parameters and flight activities, the meteorological parameters that had a significant effect on the bees’ behaviors were temperature, relative humidity and wind speed. there was no correlation between flight activities and precipitation. however, it is worth noting that during the rainy season there were no great precipitations at the moments the observations were carried out. temperature had a positive but weak correlation with all behavioral parameters (table 1). the bees p. aff. flavocincta began flight activities at 06:00 each morning, with temperatures around 23 °c. humidity had little influence on behaviors, only impacting trash removal. wind speed also presented weak correlation with flight activities. there was no correlation with precipitation and flight activities. fig 1. behavioral parameters of flight activity of plebeia aff. flavocincta throughout the months of the year; exit (ext); trash (tra); entry with pollen (ewp); entry without pollen (ewop). values corresponding to ten minutes of observation. table 1. spearman correlation analysis relating the influence of meteorological factors (temperature, relative humidity, wind speed and precipitation) to the flight activities of plebeia aff. flavocincta; behavioral parameters (bp); temperature (temp); relative humidity (rh); wind speed (ws); precipitation (prec); entry with pollen (ewp); entry without pollen (ewop). bp rh ws prec exit trash ewp ewop temp -0.6803** 0.4294* -0.6255** 0.3063* 0.3612* 0.2704* 0.3695* rh -0.6176** 0.6521** -0.1517 -0.2963* -0.0109 -0.228 ws -0.3207* 0.3353* 0.4714* 0.2383* 0.3631* prec -0.1235 -0.1478 -0.0042 -0.1954 exit 0.8565** 0.7759** 0.9764** trash 0.7634** 0.8668** ewp 0.7510** *p<0.05 **p<0.001 based on the data observed in fig 1, it was possible to distinguish two major periods of external activity: a period of high flight activity (period 1), which corresponded to the months of september to march; and a period of low flight activity (period 2), which corresponded to the months of april to august. when the periods of abundance (september to march) and shortage (from april to august) of foraging resources were evaluated, there was a significant difference for all recorded activities (table 2). flight activity differed temporally. for exit behaviors the period of greatest activity was between the months of september and april, while for the hygienic behaviors the greatest activity was recorded between september and february. the period from august to april had the highest abs barbosa, hm meneses, fl rosa, bm freitas – flight activity of plebeia aff. flavocincta indicates colony health548 activity for entry with pollen. conversely, the period from september to march had the highest amount of entry without pollen (table 3). these data complement the finding of two distinct periods (period 1 large flow of bees, period 2 reduced flow of bees) in the annual pattern of flight activities of p. aff. flavocincta under the study conditions. months ext tra ewp ewop sep 444.90 a 60.20 a 109.85 ab 467.50 a oct 385.95 a 40.25 ab 121.37 a 334.55 abc nov 294.70 abc 40.40 ab 135.25 a 415.37 abc dec 313.42 ab 48.32 ab 160.85 a 425.55 abc jan 311.80 abc 35.00 abc 76.20 abc 245.40 abc feb 412.60 a 34.85 abcd 146.40 a 379.55 abc mar 262.75 abc 20.10 bcde 114.35 ab 344.50 abc apr 205.90 abcd 10.95 cde 61.10 abcd 125.35 bcd may 100.30 cd 7.00 de 27.80 cd 114.40 cd jun 48.60 d 4.75 e 15.90 d 63.30 d jul 49.80 d 2.10 e 11.55 d 68.95 d aug 107.70 bcd 16.90 cde 73.50 abcd 196.75 bcd values corresponding to ten minutes of observation. table 2. average number of bees, followed by standard error, performing the behavioral parameters of flight activity of plebeia aff. flavocincta from september to march and april to august in fortaleza/ce. behavioral parameters average ± standard error sep mar apr aug exit 346.36±7.52a 102.46±7.48b trash 39.83±1.22a 8.34±0.64b entry with pollen 123.74±2.87a 37.97±2.69b entry without pollen 372.25±9.80a 113.75±7.27b difference between letters indicate p <0.05. values corresponding to ten minutes of observation. table 3. behavioral parameters of flight activity of plebeia aff. flavocincta among the months of the year; level of significance (p); exit (ext); trash (tra); entry with pollen (ewp); entry without pollen (ewop). fig 2. average number of plebeia aff. flavocincta bees by colony in flight activity at different times of the day throughout the year. values corresponding to ten minutes of observation. as for bee flights throughout the day, all activities began with a few workers at 06:00 in the morning and the number of bees gradually increased until reaching their peak between 09:00 and 10:00 am. at that moment, the maximum number exiting was 48 bees/min. the maximum entry with pollen was 9 bees/min and entry with nectar or water was 24 bees/min (fig 2). the exception was the hygienic behavior of throwing away debris produced (trash), whose peak activity was 4 bees/min, between 10:00 am and 12:00 pm (fig 2). after the peak number of bees recorded, the number of individuals gradually decreased, though it remained high through 04:00 pm. after 04:00 pm activity decreased rapidly (fig 2). it is important to emphasize that the sun sets around 06:00 pm each day at the latitude in which this study was conducted. in the northeast region of brazil, and in ceará in particular, the year is usually divided into two seasons which are dry and rainy. this is determined based on the regular occurrence of rainfall, directly influencing food availability. in the coastal region of ceará where this experiment was conducted the resource shortage period usually refers to the rainy season and the resource abundance period refers to the dry season, unlike what occurs in inland areas. however, in the comparison of dry and rainy seasons there was no significant difference (p<0.05) in the average number of individuals sociobiology 67(4): 545-553 (december, 2020) 549 performing flight activities in the hours of the day, except at 5 pm, when there were more bees performing flight activities in the rainy season. this is despite a similar pattern observed for the annual analysis presented in fig 2, including the highest flight activity occurring between 09:00 and 10:00 am in both seasons (fig 3). overall, there were more bees foraging in the dry season than in the rainy season, however there was no significant difference between them. fig 3. average number of individuals of plebeia aff. flavocincta that performed flight activity in relation to time during the dry and rainy season; exit (ext); trash (tra); entry with pollen (ewp); entry without pollen (ewop). values corresponding to ten minutes of observation. discussion our findings with data of daily flight amplitude throughout the year (fig 1) contrast with studies involving bees of the genus plebeia in temperate regions. in the state of rio grande do sul, brazil, p. saiqui exhibit a diapause in periods of scarce resources, and its daytime activity is limited from 08:00 am to 05:00 pm during periods of the year when foraging should be of high priority (pick & blochtein, 2002b). this difference in species behavior probably occurs due to the climatic contrast between the regions. factors such as temperature and humidity may limit colony flight activities, thus changing foraging time in these bees. studies with species of genus plebeia show that limitations of flight activities by climatic factors are mainly related to low temperatures (kleinert-giovaninni, 1982; pick & blochtein, 2002b). p. saiqui has a minimum recorded temperature for initial daily flight activities of 11 °c (pick & blochtein, 2002b) and p. emerina was not observed leaving the hive at temperatures below 16 °c (kleinert-giovaninni, 1982). however, this limitation does not occur in places where the minimum temperatures are higher like those seen in tropical regions. yet, flight activities of p. aff. flavocincta only began at a minimum temperature of 23.4 °c and did not reach the foraging peak until the temperature reached 28 °c at 09:00 am (fig 2). therefore, this plebeia species appears to be much more sensitive to low temperatures than other species of the same genus occurring in regions with milder temperatures. additionally, the small difference between minimum and maximum temperatures required for external nest activities may explain the poor correlations observed between temperature and flight activities of p. aff. flavocincta, which is unlike what is observed in other species of the same genus. the mean annual relative humidity (73.5%) in our study is within the optimal parameters recorded for p. saiqui (40 to 79%) (pick & blotchein, 2002b), while the p. emerina bees have a maximum humidity tolerance of 70% (kleinertgiovaninni, 1982). this demonstrates that bees of p. aff. flavocincta are more tolerant to high humidity than other bees of the same genus. lower relative humidity rates in tropical climates are usually tied to higher temperatures, and therefore may be a cumulative effect of two environmental parameters in this response. the average wind speed recorded during the year was 3.4 m/s. pick and blochtein (2002b) noticed that p. saiqui showed activity for the values of 0 m/s and 9 m/s, encompassing all of the values observed during this experiment. however, for p. emerina it was found that its activities were restricted at speeds exceeding 4 m/s (kleinert-giovaninni, 1982). in the area of the present study, the wind speed is usually low abs barbosa, hm meneses, fl rosa, bm freitas – flight activity of plebeia aff. flavocincta indicates colony health550 with no major variations. despite the small size of p. aff. flavocincta, this parameter did not constitute a limiting factor to the external activities of the nest. in studies evaluating the impact of precipitation on bee flying activities it is generally found that rain causes disruption in external movement (oliveira, 1973; iwama, 1977; kleinertgiovannini, 1982) or declines in movements (michener 1974; kleinert-giovannini & imperatriz-fonseca; 1986, hilário et al., 2001). however, nogueira-neto et al. (1959) observed a. mellifera, plebeia sp. and nannotrigona testaceicornis collecting pollen and nectar in rainy conditions. similar conditions were observed for meliponula nebulata by kajobe and echazarreta (2005). we observed similar behavior in this study for p. aff. flavocincta, when workers collected pollen during light rain. thus, all climatic parameters measured in our study were within the amplitude tolerated by plebeia bees and none of them limited the flight activity of p. aff. flavocincta bees. our analyses showed two major periods of external activities in p. aff. flavocincta colonies: one of great bee activity occurring from september to april (period 1); and other of less external activities by the bees from may to august (period 2). period 1 comprised the months of greater availability of food resources in the study area, when the main plant species present at the site were blooming during dry season (sep-nov) and also the flowering of most plant species occurring in the beginning and middle of the rainy season (jan-may). the second period (period 2), corresponded with the transition period between the rainy season and the dry season, when we found fewer food resources available which is a critical factor for the external activity of bees (polatto et al., 2014). thus, the population within the colony was likely larger during period 1, largely due to the relationship between the general health of the colony and the amount of flight activity quantified (hilário et al., 2000). we therefore find it is possible to recognize this period of greatest abundance and flight activity as being ideal for splitting colonies. the differentiation between periods influences both foraging activities by means of association with resource availability and internal hive activities such as egg laying and colony development (moo-valle et al., 2001; hofstede & sommeijer, 2006). this is due to collection rates of different trophic resources, implying a lower or higher rate of food storage, which is essential for the provisioning of brood cells and subsequent growth of the colony population. in the study area there is a great diversity of plant species that flourish throughout the year (diogo et al., 2014). this area is in a degraded remnant of tableland forest, but maintains a floristic diversity of plant species that respond physiologically in different ways according to seasonality. in addition, the variety of plant species that comprise the diet of p. aff. flavocincta impacts its survival strategy. these bees use different floral resources during different periods, potentializing the use of these plants, thus facilitating the maintenance of the population throughout the year (hrncir & maia-silva, 2013). based on what is observed in fig 2, there is a difference in flight behavior of bees of the genus plebeia as a function of region. in colder locations bees of the plebeia genus are influenced by the period of year in both the peak of flight activities and the maximum amplitude of flight hours (pick & blochtein, 2002b). in the observed pattern the peaks of bees exiting the hive and entering with pollen are similar. this indicates a strategy already observed for other species of stingless bees which imposes a greater effort of collection when there is a greater supply of resources of high nutritional value (maiasilva et al., 2015). variations in peak activity are observed in different species of bees, as well as species in the same genus and within the same species (pick & blochtein, 2002b). there was no difference between flight behaviors in the dry and rainy seasons, which was likely due to the peaks in collection of trophic resources (table 2) occurring in both periods. this leads to a similarity in the means of flight activity in the two seasons. the workers of p. aff. flavocincta effectively utilized flowering characteristics of both the dry and rainy season. in each of these periods there was a peak in flowering which allowed the colonies to maintain their populations. therefore, limitations of colony activities were only presented during times when the supply of trophic resources in the field decreased drastically. this could be correlated to periods of transition between the rainy and dry seasons. this period would require more artificial feeding, and preferably, growing other plant species that could provide natural resources during this time of the year. finally, our study aimed to investigate the flight activity of the stingless bee p. aff. flavocincta in tropical conditions throughout the year as a parameter to evaluate the general state of the colonies and to investigate the role of food resources and environmental factors in their flight activities. we found that colonies of p. aff. flavocincta remain active throughout the year and do not present diapause even in periods of food shortage. therefore, they can be used for both honey production and agricultural pollination throughout the year. we also found that most external activities occur during the period of higher food availability and is not linked to the rainy or dry season, but to a long period of blooming events running through both seasons. while p. aff. flavocincta seems to be dependent on higher temperatures to fully perform their activities outside the nest, its external activities were little affected by the environmental conditions. this is probably because meteorological conditions observed in tropical regions vary little throughout the year. thus, unlike what has been observed in other geographic regions and with other bee species, external activities of p. aff. flavocincta is regulated by food availability rather than prevailing weather conditions. furthermore, we found that flight activities are all strongly correlated with each other. this confirms the importance of the evaluation of flight activities as an indicator of the general health of the colony because it reflects the situation inside the nest in relation to the storage of resources, hygienic behavior and population size. sociobiology 67(4): 545-553 (december, 2020) 551 acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. breno m. freitas is thankful to cnpq for a research in productivity sponsorship (#308948/2016-5). authors contribution abs barbosa conceptualization, methodology, validation, data curation, visualization and writing hm meneses methodology, validation, investigation, formal analysis, data curation, visualization and writing fl rosa methodology, validation, investigation, writing and visualization bm freitas conceptualization, methodology, validation, resources, writing and project administration references amano, k., nemoto, t. & heard, t.a. 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(2013). stingless bees, melipona fasciculata, as efficient pollinators of eggplant (solanum melongena) in greenhouses. apidologie, 44: 537-546. doi: 10.1007/s13592-013-0204-y oliveira, m.a.c. (1973). um método para avaliação das atividades de vôo em plebeia saiqui (friese) (hymenoptera, meliponinae). boletim de zoologia e biologia, 30: 625-631. pick, r.a. & blochtein, b. (2002a). atividades de coleta e origem floral do pólen armazenado em colônias de plebeia saiqui (holmberg) (hymenoptera, apidae, meliponinae) no sul do brasil. revista brasileira de zoologia, 19: 289-300. pick, r.a. & blochtein, b. (2002b). atividades de vôo de plebeia saiqui (holmberg) (hymenoptera, apidae, meliponini) durante o período de postura da rainha e em diapausa. revista brasileira de zoologia, 19: 827-839. polatto, l.p., chaud-netto, j. & alves-junior, v.v. (2014). influence of abiotic factors and floral resource availability on daily foraging activity of bees. journal of insect behavior, 27: 593-612. doi: 10.1007/s10905-014-9452-6 sociobiology 67(4): 545-553 (december, 2020) 553 putra, r. e., permana, a. d. & kinasih, i. (2014). application of asiatic honey bees (apis cerana) and stingless bees (trigona laeviceps) as pollinator agents of hot pepper (capsicum annuum l.) at local indonesia farm system. psyche: a journal of entomology. doi: 10.1155/2014/687979 ramalho, m., imperatriz-fonseca, v.l., kleinekt-giovannini, a. & cortopassl-laurino, m. (1985). exploitation of floral resources by plebeia remota holmberg (apidae, meliponinae). apidologie, 16: 307-330. silva, j.g., meneses, h.m. & freitas, b.m. (2019). foraging behavior of the small-sized stingless bee plebeia aff. flavocincta. revista ciência agronômica, 50: 484-492. doi: 10.5935/18066690.20190057 slaa, e.j., chaves, l.a.s., malagodi-braga, k.s. & hofstede, f.e. (2006). stingless bees in applied pollination: practice and perspectives. apidologie, 37: 293-315. doi: 10.1051/ apido: 2006022 sterk, g., peters, b., gao, z. & zumkier, u. (2016). largescale monitoring of effects of clothianidin-dressed osr seeds on pollinating insects in northern germany: effects on large earth bumble bees (bombus terrestris). ecotoxicology, 25: 1666-1678. doi: 10.1007/s10646-016-1730-y open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4759sociobiology 67(2): 247-255 (june, 2020) introduction there is an estimation of 25,000 to 30,000 bee species in the world, of which 85% show solitary living habit (roubik, 1989; griswold et al., 1995). although solitary bees are very well represented in terms of numbers, studies regarding their nesting habits and biological cycle are scarce and restricted to few species. however, many of those bees have potential for agriculture as pollinators, as in the case of nomia melanderi, peponapis pruinosa and some species of the genera xylocopa abstract about 85% of pollinating bees are solitary, but few are used for agricultural pollination. the anthidiini tribe looks very promising in brazil, in the light of ecosystem utility, but it is still a scientificaly poorly researched species. in order to fill this gap, studies on the bionomics of these species may collaborate on future efforts for the use of epanthidium tigrinum as a pollinator. the presented research results aims to describe the bionomy and the biological cycle of the bee e. tigrinum (schrottky, 1905) in pre-existing cavities. conventional trap-nests inserted in wooden blocks were placed in covered spaces and daily observations of spontaneous nesting as well as individually tagged bees and nests incubated in b.o.d. were carried out during 11 months in fortaleza-ce. the results showed that the competition with other species of bees for the pre-existing cavities and the lack of food resources or nest building materials are limiting factors for the establishment of e. tigrinum populations. however, once these problems were remediated, the bees showed multivoltine behavior, nesting throughout the experimental period. data from 16 nests studied showed that bee foraging began on average at 7:50 a.m., foragers collected pollen in the morning and resin in the afternoon, took on average 15 days to complete a nest and there was a negative correlation with the temperature for the number of nests and for the number of cells constructed. the offspring emergency mean time was 43 days, the sexual ratio was 1.2: 1 (n = 66 emerged bees) and the mortality rate was 18.5%. the number of nests constructed per female ranged from 1 to 4 with an average size of 7.97 mm. it is concluded that e. tigrinum shows potential to rational rearing in trap nests aiming crop pollination. sociobiology an international journal on social insects ams gomes1, ci silva2, am cavalcante1, eem rocha1, bm freitas1 article history edited by evandro nascimento silva, uefs, brazil received 28 september 2019 initial acceptance 28 january 2020 final acceptance 09 february 2020 publication date 30 june 2020 keywords solitary bee, anthidiini, biological cycle, bee rearing. corresponding author angela m. s. gomes laboratório de abelhas departamento de zootecnia centro de ciências agrárias universidade federal do ceará cep 60.356-000, fortaleza-ce, brasil. e-mail: angela_msgomes@yahoo.com.br and centris, efficient pollinators in crops such as passion fruit (passiflora spp.) (freitas & oliveira-filho, 2003; silva et al., 2007),west indian cherry (malpighiae marginata dc) (vilhena & augusto, 2007; magalhães & freitas, 2013), cashew (anacardium occidentale l.) (freitas & paxton, 1996) and cucurbits (shuler et al., 2005; julier & roulston, 2009). in the megachilidae family some species like osmia spp and megachile rotundata, are already used in the pollination of crops such as pear (pyrus communis l.), apple (malus domestica borkh), almonds (prunus amygdalus dulcis), cherry 1 universidade federal do ceará, fortaleza, ceará, brazil 2 universidade de são paulo, butantã, são paulo, brazil research article bees bionomy and nesting behavior of the bee epanthidium tigrinum (schrottky, 1905) (hymenoptera: megachilidae) in trap-nests ams gomes, ci silva, am cavalcante, eem rocha, bm freitas – biology and nesting behavior of epanthidium tigrinum248 (prunusavium l.), apricot (prunus armeniaca l.), peach (prunus persica (l.) batsch), and alfalfa (medicago sativa l.) in the european union, canada, united states and japan (matsumoto et al., 2009; pitts-singer & cane, 2011; sedivy & dorn, 2013; sheffield, 2014). the megachilidae family is a very diverse group of bees. occurring worldwide, such bees can be easily differentiated by their distinct morphology, presenting a ventral scopa as the family´s main characteristic, and therefore the absence of scopa on the hind legs (silveira et al., 2002; michener, 2007). megachilidae is comprised of two subfamilies: the fideliinae and the megachilinae. megachilinae is composed by the tribes osmiini, dyoxini, lithurgini, anthidiini, and megachilini with over 4,000 species described, mostly presenting solitary behavior (nates-parra & gonzalez, 2000; michener, 2007). in brazil, the megachilidae family is represented by the subfamily megachilinae, moreover, from their tribes, only lithurgini, anthidiini, and megachilini hold described species in the country (silveira et al., 2002; michener, 2007). among those, anthidiini assembles a diversified group, which occurs in all continents, with a wide geographical distribution. despite that, locally, they often show little expressiveness of diversity and abundance (silveira et al., 2002; michener, 2007). the anthidiini build their nests in pre-existing cavities in wood, soil, or exposed on rocks, branches or leaves (morato & fields, 2000; michener, 2007). although there are still few biological and ecological data on anthidiini in brazil (camarotti-de-lima & martins, 2005), it is known that building material used by these bees in their nests are very diversified, and varies according to the genera and species. some bees gather plant fibers in order to be used for coating their cells; some, utilize resin, often cobbled with other materials, such as pebbles, leaf pieces or clay. despite the little knowledge about the nesting habits of those bees, and moreover, the information on that regard are exclusively based on groups that have built nests in trap-nests (alves-dossantos, 2004; alves-dos-santoset al., 2004; camarotti-delima & martins, 2005), yet a few biological and ecological data are related to the anthidiini in brazil (camarotti-delima & martins, 2005). an interesting group of anthidiini bees for further studies is the one whose species use resins in building their nests.among them, bees of the genus epanthidium, with 23 neotropical species (urban & moure, 2012). the resin, used by those bees is secreted from plants, collected still in a viscous state, and moreover, carried in the female’s mandibles to the nest. despite the little knowledge about their chemical characteristics, it is known that these resins possess fungicide and bactericide properties (alves-dos-santos et al., 2004). however, there is little information about the bionomy and nesting behavior of these species and how to handle them in nurseries. therefore, aiming to contribute to the knowledge and potential breeding and management of anthidiini, the present study aims to describe the bionomy and nesting behavior of the epanthidium trigrinum bee (schrottky, 1905) (hymenoptera -megachilidae) in trap-nests. material and methods experimental area the experiment was conducted at the bee unit of the department of animal science, at the federal university of ceará (ufc), in the city of fortaleza (latitude: 3°44’33.70”s; longitude: 38°34’45.46”), from december 2014 to october 2015. the local climate is characterized as aw’ by the köppen classification (1948), sub-humid hot tropical, rainy season from january through may, average rainfall of 1338.0 mm and average temperatures that variety of 26°c to 28°c (ipece, 2015). the area has a small portion of tableland and forest surrounding the place where some species of native and exotic fruit trees are found, such as coconut (cocos nucifera l.), mango (mangifera indica l.), cashew (anacardium occidentale l.), west indian cherry (malpighiae marginata dc) and nance (byrsonima crassifolia l.). trap-nests and sampling the trap-nests were made of black cardboard paper with 12 cm of length. initially, only nests with 5.5 mm diameter were used. however, due to the occurrence of high competition with other species of bees and wasps, nests with smaller diameters, 4.5 mm, were used. these nests were distributed in six wooden blocks, totaling 354 cavities, and then, installed in structures where they stayed protected from rain and direct sun rays. the trap-nests (tn) were inspected every day and when nests were finished, they were removed, labeled and stored individually in transparent plastic tubes, then closed with voile and cork stopper. the tubes containing tns were taken to the bee laboratory at the ufc bee unit, where they were kept inside a b.o.d. (biochemical oxygen demand) at 27°c and 60% relative humidity until the emergence of adults. at every observation, the nests removed to the lab were replaced by empty ones with the same diameter to assure the constant availability of nests. aiming to avoid robbing and abandonment of the nests of e.tigrinum, as well as to attract bees to the nesting place, a mixture of resin and wax of meliponinae was provided, same material used to close trap-nests. this mixture was extracted from the nests of bees melipona subnitida and scaptotrigona sp. nov. present in the meliponary at the bee unit. the material was available to the bees soon after being collected, by distributing small portions around the trap-nests. nesting behavior five females of e. tigrinum were marked with nontoxic paint as soon as they had begun building the nest. the bees were captured with plastic vials at the entrance of the trapsociobiology 67(2): 247-255 (june, 2020) 249 nests, then placed in a freezer until dormancy in order to be marked, and released afterward to go back to their nests. each bee was observed for an interval of five days. observations were performed during the whole day, starting at 5:00 am and ending at 6:00 pm, in order to assess the behavior of those bees, regarding the time they start foraging, the permanence time inside nest, in the field, the resources collected, and the time taken to finish a cell. subsequently, after the end of the observations, the bees received new identification, consisting of a numbered tag that was made of a4 sheet paper. the tags were fixed in the dorsal part of the thorax, between the wings, using super bonder loctite® glue brand. then the bees continued to be watched as to be determined the total of nests that each female have established; the number and the size of cells constructed; the time of cells construction and offspring emergence; the sex ratio, mortality and parasitism rate. statistical analysis the descriptive statistics were used for nesting data, seasonality, total of nests settled by each female, number and size of cells built, building time, emergence, sex ratio, mortality and parasitism rate. in order to determine the relation between climatic factors (temperature, humidity, precipitation and wind speed) with the departure and arrival of bees, and the number of nests and cells built, the spearman correlation test was used because data did not show normality. the statistical program past 2.17 was used for performing the analysis. results seasonality and nesting seasonality during all period of experiments, the trap-nests were occupied by bees epanthidium tigrinum. however, during the first months, the bees hardly ever completed their biological cycle because most of them abandoned the nests without finishing any cell. competition with other bee species for nesting sites and the robbery of nesting resources by other bees have been observed as the cause of a small number of nests during this period. aiming to overcome these problems, we replaced the trap nests used for narrower ones, only 4.5 mm in diameter in order to hinder the nesting of bees centris analis, the main competitor for nesting sites, and offered to the e. tigrinum females resin collected from nests of meliponine bees, preventing robbery of nests from each other. these procedures allowed the bees to nest, build their cells and complete their nesting cycle. there was no significant correlation (p˂0.5) of the precipitation and temperature with the occupation of nests, even though the highest occurrence of e. tigrinum females was observed during the months of july and august, after the rainy season (fig 1). when assessing the correlation between climatic factors with the number of nests established and with the total of cells built, a negative correlation was observed with temperature (rs=-0.639; p = 0.034) for the total of nests and cells (rs = -0.702; p = 0.016) (fig 2). fig 1. nesting of bees epanthidium tigrinum in trap-nests in function of temperature and pluvial precipitation, december 2014 through october 2014, at the bee unit of the department of animal science of the federal university of ceará. fig 2. correlation between temperature and (a) total number of nests established, and (b) total number of cells built by bees epanthidium tigrinum in trap-nests, at the bee unit of the department of animal science of the federal university of ceará, december 2014 through october 2015. ams gomes, ci silva, am cavalcante, eem rocha, bm freitas – biology and nesting behavior of epanthidium tigrinum250 nesting during the experimental period, 16 nests were completed, belonging to 10 different bees, with the distribution varying from 1 to 4 nests per bee (1.6 ± 0.97). from those nests, 67 individuals emerged (37 males and 30 females) (table 1) resulting in a sex ratio of 1.2:1. there was no occurrence of parasites in the observed nestsand the non-development of larvae was the main cause of mortality, resulting in a mortality rate of 18.5%. another factor responsible for the mortality was the attack of ants that occurred in two nests. the females built their nests in a linear fashion following the shape of the trap-nests and using mainly resin, which sometimes was mixed with sand or clay because these nests were constructed inside trap nests previously occupied by wasps or bees (centris analis and tetrapedia diversipes). the bees spent one day to build each cell, starting the collection of resin in the afternoon and collecting pollen and nectar in the following morning, before oviposition. when each cell was completed, the cycle restarted. in five nests we observed the occurrence of vestibular cells. these structures are empty cells which are larger than a regular one and located between the last provisioned cell and the nest closure. in the present study, the vestibular cell measured an average size of 28.66 ± 9.10 mm. the bees took in average 15.44 ± 5.93 days to complete one nest. from the first cells built, i.e., the more internal ones, emerged males, and from the more external cells emerged females, being the females the first ones to emerge. the sizes of cells varied regardless the bee’s sex, and measured an average size of 7.97±1.31 mm. the offspring took an average time of 43.69 ± 4.64 days to emerge. bees number of cells bees emerged construction duration (days) emergence duration (days) closing distance (mm) vestibule (mm) ♂ ♀ 1 6 1 4 11 46 24.67 1 6 4 0 19 48 38.62 17.61 2 5 2 2 11 43 20.35 2 5 2 2 17 43 20.91 29.75 3 5 4 0 15 49 23.30 42.44 3 9 1 4 17 44 21.13 5 9 5 3 25 40 31.62 29.06 6 6 2 3 25 36 17.56 24.45 7 6 3 3 15 43 20.35 8 6 3 3 12 49 13.98 10 7 3 3 14 43 23.51 10 2 1 0 7 49 64.75 10 2 2 0 15 45 26.52 10 2 1 1 7 44 21.02 11 2 1 1 11 45 26.31 12 3 2 1 26 32 31.31 total 37 30 nesting behavior the observed e. tigrinum bees started foraging, on average, at 07:50 am ± 30 min. in the morning hours those bees collected pollen and nectar, and resin in the afternoon hours. such collecting behavior was influenced by the temperature and relative air humidity, as well as the stage of nest building, since the bees have always started nest building by collecting resin, regardless of the time and definite nesting location. the bees remained in activity until 4:00 pm, although once a bee has finished activity as late as4:51 pm. the bees remained inside their nests between field trips for an average 9 ± 5 min, and in the field, they spent in average 19 ± 7 min, varying according to the resource collected. the moment of greatest bee activity occurred at 2:00 pm, which coincided with the greatest flight movement to collect resin, with a higher constancy of flight during the rest of the hours (fig 3). a behavior that has affected considerably the time bees executed their tasks along the day was the change of resource collection, which according to the observations, is the moment of oviposition and generally coincides with the last trip for pollen collection. but, in some cases, it took place after the first or second trip to collect resin. at the time of oviposition, the bees have come to spend over an hour inside the nest, and after that the female left and returned to nest a few times, making turns around its entrance but not flying. eventually, the bees table 1. data of nests of epanthidium tigrinum collected in trap-nests, from december 2014 to october 2015, at the bee unit of the department of animal science of the federal university of ceará. sociobiology 67(2): 247-255 (june, 2020) 251 returned to inside the nest and positioned themselves very close to the entrance, sometimes placingthe antennae out of the nest, and remained like this until reassuming the foraging flights. the morning flights of e. tigrinum for pollen collection have taken over an hour on some of the observation days. the collected pollen, loaded on the abdominal scopes, was very noticeable due to the yellowish coloration.in two days, two of the bees spent over 2 hours in the field. the time bees spent in the field has varied throughout the sampling period. every day, the first foraging flight was the longest one when comparing to the other foraging flights of the day, varying from 15 min to 2 hours and 30 min. in rainy days, the foraging behavior was affected with foraging flights much longer than usual. in case the bees were still in the field when the rain started, they would not return to the nests until the rain stops and the sun shines again. fig 3. flight activities of epanthidium tigrinum bees and gathering of resources in trap-nests at the bee unit, department of animal sciences of federal university of ceará, december 2014 through october 2015; a) departure of bees from nest; b) pollen arrival; c) resin arrival; d) nectar/ water arrival; e) turnings at the nest’ entrance. ams gomes, ci silva, am cavalcante, eem rocha, bm freitas – biology and nesting behavior of epanthidium tigrinum252 the behavior observed when these bees returned from the field is described as follows: first, they entered straight into the nest with the head facing in, then they came out backwards with the abdomen facing out. after that, the bees made a turn without flying and returned into the nest still with pollen in the scopa, but this time with the abdomen towards the inside of the nest.then,they unloaded the resource inside the cell. the sequence of movements entering and leaving the nest and “turning” backwards inside the nest only happened when the bees collected pollen, and it would take from 1 up to 5 min to be completed. flights for resin collection were shorter and took 30 min at the most, when bees were going to the field in search of this resource. however, in most cases, they returned empty and ended up collecting the supplied resin in a way that the flights were even shorter, varying from 1 up to 5 min only. the permanence of the bees inside the nest varied greatly, generally 1 to 11 min, although some have stayed inside the nest up to 42 min between flights. observations suggested that the individuals were collecting resin and quickly depositing it inside the nest and returning to collect more resin, only shaping it at the end of foraging hours,when they had collected all the resin they need for that day. only in a few cases the bees have slightly worked on the resin between the collecting flights. resin collection was easily noticeable because the bees carried the resin held in the mandibles in shapes of bulky pellets. pollen collection showed a negative correlation with humidity and rain precipitation (table 2). resin collection was not influenced, since the bees used mainly the supplied resin of meliponines, which was placed on the proximities of trap-nests in a covered place, which hindered the interaction of those bees with the environment during foraging. table 2. correlation of bee departure, pollen arrival, resin arrival, nectar/water arrival, and turning behavior with climatic variables of bees epanthidium tigrinumin trap-nests, at the bee unit of the federal university of ceará, december 2014 through october 2015. where rs is the value of correlation and p is the significance. temperature humidity precipitation wind speed rs p rs p rs p rs p exit 0.201 0.335 -0.319 0.120 -0.154 0.462 0.003 0.990 pollen 0.340 0.097 -0.477 0.016 -0.481 0.015 -0.301 0.143 resin -0.062 0.767 0.126 0.548 0.260 0.210 0.186 0.372 nectar/water -0.131 0.531 -0.045 0.830 -0.071 0.735 0.205 0.326 turning 0.348 0.088 -0.481 0.015 -0.440 0.028 -0.297 0.149 discussion seasonality and nesting most of the studies about the megachilidae species had been carried out in the northern hemisphere and have found seasonal species which individuals only are active during a short period of the year and the offspring show diapause, emerging as adults only when conditions are favorable again (torchio & tepedino, 1980; pitts-singer & cane, 2011; rinehart et al, 2013). in brazil, seasonality has already been seen for some species of megachilidae such as megachile (melanosarus) nigripennis spinola (marques & gaglianone, 2013), m. benigna mitchell, and m. maculata smith (cardoso & silveira, 2011), anthodioctes lunatus smith (camarottide-lima & martins, 2005) and anthodioctes megachiloides holmberg (alves-dos-santos, 2004) at greater latitudes, however morato (2001) suggests that anthodioctes moratoi, in the amazon region, nests throughout all year. this study demonstrates that the species epanthidium tigrinum remained active during the whole experimental period of 11 months varying only in number of nests constructed, and in quantity of cells built per nest. therefore, there is no seasonality for this species under the studied conditions. some studies suggest that seasonality in bees can be associated to the availability of specific resources for nest building, adult feeding and larvae provisioning (viana et al., 2001; mendes & rêgo, 2007). in this study, the first factor may have been relevant, because few of the observed bees completed nest building and showed high abandonment rate in the beginning of the study. in one case, a bee has robbed all material collected by another bee of the same species, which has resulted in the abandonment of both nests. the nidification, permanency of these individuals in nests and successfully conclusion of them, was only possible when the resin was provided close to the trap-nests. henceforth, it was possible to see many bees collecting this resin, both the marked bees that were observed and others of the same species that were not nesting in the trap-nests. these behaviors perhaps suggest the lack of plants that provide resin in the area, which makes nesting unviable for these bees. in this way, the availability of vegetal resin would be, indeed, a limiting resource for the establishment and maintenance of populations of this species in an area and constitute an important factor when considering the rational breeding of these bees the sex ratio in a bee species may vary and is associated with conditions of food availability or other local factors (marques & gaglianone, 2013). a work performed by sugiura (1994) with anthidium septem spinosum lepeletier found that mother bees of this species are able to manipulate, as an adaptive way, the sex ratio of descendants in relation to sociobiology 67(2): 247-255 (june, 2020) 253 their ability to invest in the offspring. in this work, the offspring position inside the nest is also described, demonstrating that males are reared in most internal cells while females are produced in the most external ones, as also observed in the present study. the author reports that this distribution is directly related to the reproductive life of these bees because larger males have a higher capacity for reproduction and require greater parental investment. the lower ratio of females’ production in comparison with males have also been reported for other bee species such as megachile (moureapis) benigna studied by teixeira et al. (2011) and megachile (pseudocentron) gomphrenoides (torretta et al., 2012). also, some nests of epanthidium tigrinum presented production of a single sex, fact already reported for some species of megachile, such as m. (austromegachile) orbiculata mitchell (morato, 2003), m. (moureapis) anthidioides radoszkowski (cardoso & silveira, 2003) and m. (moureapis) maculata smith 1853 (cardoso & silveira, 2011). the bees of the genus epanthidium are in the group of the anthidiini, which collect resin for building their nests. during the experimental period, before resin was supplied, an odd material was noticed being collected by those bees and used along with resin to build and close some nests; however, it was not possible to have this material analyzed. after supplying with resin, their nests started to be built basically from this material. this may suggest that, depending on the quantity of resin available, these bees may use other mixed materials to build their cells. cilla and rolón (2012) described the nests of epanthidium aff. sanguineum that made use of clay, resin and vegetal fibers in their construction. another important factor noticed was that some of these bees have built their cells in trap-nests previously occupied by bees centris analis and tetrapedia diversipes. cilla and rolón (2012b) also reported the occupancy of unoccupied nest of centris muralis burmeister by bees epanthidium aff. sanguineum. this behavior suggests a flexibility of these females in relation to the choice of nesting sites, not having specific requirements of places and conditions. nesting behavior apparently, environmental variations, resources availability, type of resource used, and intrinsic differences of the species influence the nesting behavior of megachilidae species. the bees e. tigrinum have collected pollen and nectar in the morning, and resins in the afternoon. such behavior optimizes usages of resources in the area, as the surrounding plants provide pollen, a limited resource, early in the morning and, once removed, there is no new supply until the next day. on the other hand, the resin becomes more malleable, therefore, easy to handle and be transported during the hottest hours of the day, which justifies collection only from noon on. moreover, at noon, there is not enough pollen available in the field anymore. in this study, while the bees e. tigrinum were making short flights for collecting resin and long flights for collecting both pollen and nectar, therefore spending more time in the nest when returning from the flights for pollen and nectar than from the flights for resin, alves-dos-santos (2004) reports that anthodioctes megachiloides holmberg, in são paulo, made longer flights when collecting resin and spent more time inside the nest; whereas when collecting pollen, the flights were shorter and the permanence inside nest, shorter. the unlimited supply of resin we offered very close to the nests of e. tigrinum has made this resource as much, or more available, and much more accessible and of easier collection than the resins that a. megachiloides had to search and collect in the surrounding vegetation of their nests. finally, the results presented in this study show the possibility of rearing epanthidium tigrinum bees in a rational way regarding the management and maintenance of the species in the desired area, pointing to crucial aspects to success such as the availability of resins and cavity size to prevent competitors. the establishment of managed populations of this bee will allow the further development of studies of the species, and a better understanding of the tribe anthidiini. conclusions the availability of resources for nest building and cell provisioning are limiting factors directly related to the obtainment of nests of epanthidium tigrinum and to the establishment of this bee species. the supply of stingless bee resin is an efficient alternative to meet the nesting needs of the epanthidium tigrinum bee in resource-limited settings. the competition with other species that nest in preexisting cavities can be avoided using nests of smaller diameters, suitable for epanthidium tigrinum, but not for larger species. the little demand for nesting places, polylectic behavior, non-existence of seasonality, and little or no predation by enemies and parasites, make it possible to establish nurseries of epanthidium tigrinum. there is still a large deficit of biological information about the tribe anthidiini, so that further studies are needed for future research aiming to extend the collected information. acknowledgements to the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – under finance code 001 for partly financing this research; professor dr. danúncia urban forgiving her time for the bees species identification in the study; dr. isabel alves dos santos for providing part of the reference material used in this work. breno m. freitas thanks cnpq for a productivity research sponsorship (302934/2010-3). ams gomes, ci silva, am cavalcante, eem rocha, bm freitas – biology and nesting behavior of epanthidium tigrinum254 references alves-dos-santos, i. 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(2001). diversidade e sazonalidade de abelhas solitárias (hymenoptera: apoidea) em dunas no nordeste do brasil. neotropical entomology, 30: 245-251. doi: 10.1590/s1519-566x2001000200006. vilhena, a.m.g.f. & augusto, s.c.(2007). polinizadores da aceroleira malpighiae marginata dc (malpighiaceae) em área de cerrado no triângulo mineiro. bioscience journal, 23: 14-23. 149 comparison of the efficacy of eleven soil termiticides on the formosan subterranean termite (coptotermes formosanus) by izumi fujimoto*, akio enoki, shuji itakura and hiromi tanaka abstract using the tunneling test, the standard test method recommended by the jwpa ( jwps-ts-s), the minimum effective concentrations of 11 chemicals, known as major soil termiticides in japan, were determined. addition of the emulsifying agent, tween 80, to the low water solubility chemicals among the termiticides yielded a 2to 8-fold increase in their termiticidal efficacies. in contrast, addition of tween 80 to the high water solubility chemicals produced no such enhancement of their termiticidal efficacies. in the confined test, in the case of the repellent-type chemicals of the 11 chemicals, all the test termites (10 workers and 2 soldiers) became intoxicated or paralyzed and moribund within one hour of exposure to the minimum concentration fulfilling the criteria of the jwpa standard test method. in the case of non-repellent type chemicals, all the workers became moribund within 2 hours of exposure to the minimum concentrations fulfilling the criteria of the standard test method; in contrast, it took 4 – 24 hours before the soldier termites became moribund. key words: coptotermes formosanus shiraki, termiticide, jwpa standard test method, tunneling test, confined test introduction formosan subterranean termites are considered as one of the most destructive insect pests of wooden structures throughout the world, especially in areas with warm, humid climates (kuriachan and gold 1998). control of subterranean termites has always been an important concern of homeowners and the pest control industry. for the past 60 years, termite control has mainly relied on termiticide treatments (schokecht et al. 1994; ibrahim et al. 2003). chlordane had nearly monopolized the termiticide market of japan faculty of agriculture, kinki university, nara 631-8505, japan *corresponding author; email: felixfuji@mvf.biglobe.ne.jp 150 sociobiolog y vol. 59, no. 1, 2012 until the first half of the 1980’s. however, this chemical was designated as a special chemical substance owing to its characteristics of not being readily degradable and showing high bioaccumulation and chronic toxicity, and was actually banned by an act passed in 1986 on the examination of chemicals and regulation of manufacture. organophosphates such as chlorpyrifos or phoxim began to be hastily employed as a substitute for chlordane, and were widely used. termite damage recurred frequently within 5 years of the treatment in chlorpyrifosor phoxim-treated houses, especially in areas of inhabitation of c. formosanus. the unusually short duration of the termiticidal performance of these chemicals became an issue of concern (tsunoda et al. 1989). further, many lawsuits for health reasons were filed by the inhabitants of chlorpyrifostreated houses, as well as by those of the neighboring houses. in an attempt to overcome these problems with chlorpyrifos and phoxim, the rates of disappearance of these chemicals under various conditions and the minimum treatment concentrations of the two termiticides required to fulfill the criteria of jwpa standard no.13 (1992) were investigated (enoki et al. 1995). these experiments demonstrated that more than 70% of the chlorpyrifos or phoxim disappeared, mainly by evaporation, within a year from the soil underneath the floors of houses treated with chlorpyrifos or phoxim in kyoto, nara or osaka. also, the minimum treatment concentration of chlorpyrifos or phoxim required to fulfill the criteria of the jwpa standard was in the range of 0.05% to 0.1%. in those days, the treatment concentration of chlorpyrifos or phoxim for soil treatment defined by the specification of each chemical was 1%. therefore, it is almost impossible to obtain protection against termite attacks for 5 years of wood-constructions built on ground soil treated with 1% solutions of chlorpyrifos or phoxim, because of the rapid evaporation of these chemicals from the soil. thus, measurements of the decay rate of a termiticide at the treatment site and the minimum treatment concentrations of the chemicals for complete prevention of termite attacks are essential for the proper use of termiticides, that is, use of optimal concentrations that would provide adequate protection against termite damage, while not posing a threat to the environment or to the health of living beings. recently, numerous types of termiticidal formulations, consisting of a variety of chemicals, have been developed as substitutes for organophosphates, and are already available commercially. it has been established that examination 151 fujimoto, i. et al. — efficacy of termiticides on c. formosanus of the boring activity of subterranean termites in treated soil is essential for evaluation of the termiticidal efficacy of a soil treatment chemical (tamashiro et al.1987; su et al.1997). in this study, to determine the minimum treatment concentrations of 11 chemicals which are widely used as soil termiticides in japan fulfilling the criteria of the jwps-ts-s, the termiticidal efficacies of the major chemicals were evaluated by laboratory tests. materials and methods test termites the subterranean termites, coptotermes formasanus shiraki, were used for the laboratory experiments. all the termites were collected from a laboratory colony maintained at the department of applied biological chemistry, faculty of agriculture, kinki university. test chemicals all the chemicals tested were of technical grade: permethrin, bifenthrin, etofenprox, silafluofen, dinotefuran, clothianidin, imidacloprid, thiamethoxam, fipronil, chlorfenapyr and chlorantraniliprole. preparation of test samples a certain weight (mg ) of the chemical was dissolved in ethanol to obtain a given volume (undiluted solution). the solution was diluted with ethanol to the prescribed concentrations (diluted solutions). a certain weight (mg ) of tween 80 (tokyo chemical industry co., ltd. gr) was dissolved in a given volume (ml) of the undiluted solution; this solution was diluted to 50 times with distilled water to obtain an emulsion. tunneling test the tunneling test was carried out in accordance with the jwps-ts-s. sandy loam sieved through 20 meshes was heat-sterilized at 60°c until its weight became constant, to obtain the non-chemically treated dried soil. twelve g of the dried soil was placed in a grass beaker (100 ml, 5 mm inside diameter). three ml of the dilute solution of a chemical or emulsion of the chemical was added to the dried sandy loam in the glass beaker and mixed thoroughly. the glass beaker was allowed to stand in a thermostat at 40°c for 28 days. three ml of distilled water was added to the soil in the beaker every 7 days. after 28 days, the beaker was taken out from the thermostat 152 sociobiolog y vol. 59, no. 1, 2012 and three ml of distilled water was added to the treated soil in the beaker. the moistened soil was tightly stuffed into the transparent part of a glass tube (15 mm inside diameter; 5 cm length of the transparent zone) with both the ends made of frosted glass for sliding contact. the glass tube was connected to two glass cylinders at both ends. wood flakes (ca. 5g ) were put into one of the two cylinders. untreated soil (ca. 20% moisture content) was placed in another cylinder, as shown in fig. 1. one hundred worker and 10 soldier termites were introduced into the untreated soil in the cylinder. the assembled unit was incubated at 28°c in the dark for 3 weeks. after this test period, the boring distance (gallery) by the termites was measured and the degree of boring caused by the pests was rated on the following scale. degree of boring 0: no boring 1: boring distance under 1 cm 2: boring distance under 2 cm 3: boring distance under 3 cm 4: boring distance under 4 cm 5: boring distance over 4 cm fig.1. apparatus for tunneling test. 153 fujimoto, i. et al. — efficacy of termiticides on c. formosanus if all the test termites were dead before the end of the test period, the time elapsed (in days) was recorded. in order for the test chemicals fulfilling the criteria of the jwps-ts-s, the degree of boring on the scale described above needs to be ranked 0 or 1. however, the test chemical is still considered to fulfill the criteria of the standard when the ranking of the degree of boring on the scale is 2, but all the test termites are dead within a week. five replicates were prepared for each sample. confined test twenty g of dried sandy loam was placed in a glass beaker (100 ml; 52 mm inside diameter). five ml of the dilute solution of a chemical or emulsion of the chemical was added to the soil in the beaker and mixed thoroughly. the beaker was allowed to stand in a thermostat at 40°c for 28 days. five ml of distilled water was added to the soil in the beaker every 7 days. after 28 days, the beaker was taken out from the thermostat. distilled water was added to the treated soil in the beaker to obtain a moisture content of ca. 23%. the surface of the soil in the beaker was pressed and leveled off with the disk-shaped end (1.5 cm diameter) of a glass rod. ten worker and two soldier termites were introduced into the soil in the beaker, as shown fig. 2. the beaker was maintained at 28°c in the dark. the change in the activity of the termites was visually assessed every one hour for the first eight hours, and then every 24 hours. the times required for a 100% moribund rate (no ambulation) and 100% dead rate (no movement at all) of the termites were assessed. three replicates were prepared for each sample. results and discussion tunneling test the average distances and the average degrees of boring in the tunneling test of the 11 chemicals are shown in table 1 3. termites penetrated the fig.2. apparatus for confined test. 154 sociobiolog y vol. 59, no. 1, 2012 untreated soil in the glass tube and reached the wood flakes in the opposite cylinder within 5 to 6 hours (table 1). the termites also penetrated the soil layer containing 1,250 ppm (treated conc.; 5,000 ppm) of tween 80 (w/w) and reached the wood flakes within 5 to 6 hours. the minimum effective concentrations in the soil of pyrethroids, permethrin, bifenthrin, etofenprox and silafluofen required to fulfill the criteria of the jwps-ts-s were 50 ppm (treatment conc.; 200 ppm), 12.5 ppm (50 ppm), 200 ppm (800 ppm) and 375 ppm (1,500 ppm), respectively (table 1). when tween 80 was added to each of the four pyrethroids at 5 to 10 times the amount of each pyrethroid, the minimum effective concentrations decreased to 25 ppm (treatment conc.; 100 pm), 6.25 ppm (25 ppm), 100 ppm (400 ppm) and 200 ppm (800 ppm), table 1. termiticidal efficacy of 4 pyrethroids, permethrin, bifenthrin, etofenprox, and silafluofen, in the tunneling test ( jwps-ts-s). chemicals conc. in soil (ppm) mean boring distance (mm) mean boring degree notes permethrin + tween 80 25 + 0 27 3 all the termites were dead within 17 days 50 + 0 4 1 all the termites were dead within 11 days 25 + 125 3 1 all the termites were dead within 11 days bifenthrin + tween 80 6.25 + 0 16 2 all the termites were dead within 11 days 12.5 + 0 3 1 all the termites were dead within 11 days 6.25 + 62.5 1 1 all the termites were dead within 11 days etofenprox + tween 80 100 + 0 23 3 all the termites were dead within 12 days 200 + 0 5 1 all the termites were dead within 12 days 100 + 500 1 1 all the termites were dead within 11 days silafluofen + tween 80 200 + 0 15 2 all the termites were dead within 11 days 375 + 0 9 1 all the termites were dead within 11 days 200 + 1,000 4 1 all the termites were dead within 12 days 375 + 125 9 1 all the termites were dead within 11 days tween 80 1,250 50 5 termites reached the wood flakes within 5 to 6 hours untreated 50 5 termites reached the wood flakes within 5 to 6 hours 155 fujimoto, i. et al. — efficacy of termiticides on c. formosanus respectively (table 1). no dead termites were found in the borings in the treated soil (fig. 3). the water solubility levels of permethrin, bifenthrin, etofenprox and silafluofen were 0.2 ppm (30°c), <0.1ppb (23°c), 0.02ppm (20°c) and 0.001 ppm (20°c), respectively. the treated soil layer contained 3 ml of water. therefore, the concentrations of the chemicals in the soil layer water were supposed to be lower than their minimum effective concentrations. thus, the low water solubility pyrethroids were emulsified with the emulsifying agent, tween 80, which raised the termiticidal efficacy of the chemicals by about two fold of that of the corresponding chemical in the absence of the emulsifying agent. in addition, it took more than 11 days for the pyrethroids at the minimum effective concentrations to kill all the test termites in the tunneling tests of the chemicals (table 1). the minimum effective concentrations of the 4 neonicotinoids, dinotefuran, clothianidin, imidacloprid and thiamethoxam, required to fulfill the table 2. termiticidal efficacy of 4 neonicotinoids, dinotefuran, clothianidin, imidacloprid, and thiamethoxam, in the tunneling test. ( jwps-ts-s). chemicals conc. in soil (ppm) mean boring distance (mm) mean boring degree (avg.) notes dinotefuran + tween 80 12.5 + 0 33 4 all the termites were dead within 4 days. 25 + 0 11 2 all the termites were dead within 4 days. 12.5 + 125 31 4 all the termites were dead within 4 days clothianidin + tween 80 12.5 + 0 26 3 all the termites were dead within 3 days 25 + 0 18 2 all the termites were dead within 3 days 12.5 + 125 24 3 all the termites were dead within 3 days imidacloprid + tween 80 25 + 0 22 3 all the termites were dead within 4 days 37.5 + 0 10 2 all the termites were dead within 4 days 25 + 125 22 3 all the termites were dead within 4 days thiamethoxam + tween 80 50 + 0 26 3 all the termites were dead within 3 days 100 + 0 16 2 all the termites were dead within 3 days 50 + 250 24 3 all the termites were dead within 3 days 156 sociobiolog y vol. 59, no. 1, 2012 criteria of the jwps-ts-s were 25 ppm (treatment conc.; 100 ppm), 25 ppm (100 ppm), 37.5 ppm (150 ppm) and 100 ppm (400 ppm) , respectively (table 2). no enhancement of the efficacy was obtained with the addition of tween 80 to the neonicotinoids. the water solubility levels of dinotefuran, clothianidin, imidacloprid and thiamethoxam are 40,000 ppm (20°c), 327 ppm (20°c), 510 ppm (20°c) an 4,100 ppm (25°c), respectively. each of the chemicals was supposed as dissolving completely in the soil-layer water, fig. 3. typical appearance of the soil in the tunneling test of a repellent rermiticide. no dead termites were found in the boring. fig. 4. typical appearance of the soil in the tunneling test of a non-repellent termiticide. about 20 dead termites were found in the boring. 157 fujimoto, i. et al. — efficacy of termiticides on c. formosanus yielding concentrations higher than the minimum-effective concentrations. thus, the addition of an emulsifying agent to such high water solublity chemicals, which dissolve completely in the water existing in the soil layer, exerts no influence on the efficacy of the chemicals. all the test termites were dead within 3 or 4 days. several dead termites were usually found inside the borings in the treated soil layer (fig. 4). the minimum effective concentrations of fipronil, chlorfenapyr and chlorantraniliprole required to fulfill the criteria of the jwps-ts-s were 75 ppm (treated conc.; 300 ppm), 2,000 ppm (8,000 ppm) and 250 ppm (1,000 ppm) , respectively (table 3). when tween 80 was added to fipronil or chlorantraniliprole at 10 or 4 times the amount, the minimum effective concentrations of the chemicals decreased to 45 ppm (treatment conc.: 180 ppm) and 125 ppm (500 ppm), respectively. in the presence of tween 80 at 3 to 5 times concentration of chlorfenapyr, the minimum effective concentration of chlorfenapyr significantly decreased to 250 ppm (1,000 ppm), one-eighth table 3. termiticidal efficacy of fipronil, chlorfenapyr and chlorantraniliprole in the tunneling test. ( jwps-ts-s). chemicals conc. in soil (ppm) mean boring distance (mm) mean boring degree (avg.) notes fipronil + tween 80 45 + 0 31 4 all the termites were dead within 2 days. 75 + 0 11 2 all the termites were dead within 2 days. 45 + 225 22 3 all the termites were dead within 2 days. 45 + 450 12 2 all the termites were dead within 2 days. chlorfenapyr + tween 80 250 + 0 42 5 all the termites were dead within 2 days. 2,000 + 0 19 2 all the termites were dead within 2 days. 250 + 250 24 3 all the termites were dead within 2 days. 250 + 500 20 3 all the termites were dead within 2 days. 250 + 750 18 2 all the termites were dead within 2 days. 250 + 1,250 4 1 all the termites were dead within 2 days. chlorantraniliprole + tween 80 125 + 0 26 3 all the termites were dead within 3 days 250 + 0 13 2 all the termites were dead within 3 days 125 + 500 3 1 all the termites were dead within 3 days 158 sociobiolog y vol. 59, no. 1, 2012 of that in the absence of tween 80. the water solubility levels of fipronil, chlorfenapyr and chlorantraniliprole are 2.4 ppm(20°c), 0.12ppm(25°c) or 1ppm(20°c), respectively. therefore, the concentration in the soil-layer water was supposed to be lower than its minimum effective concentration. thus, the emulsification of low water solubility chemicals with tween 80 raises their termiticidal efficacy. all the test termites were dead within 2 or 3 days. several dead termites were usually observed in the borings in the treated soil layer (fig. 5). confined test after ten worker and two soldier termites were introduced into the soil with 23% moisture content treated with each of the chemicals, the activities of the termites was assessed (tables 4 6). the activities of all the test termites were maintained for more than 24 hours in the soil containing 625 ppm or 1,250 ppm of tween 80 (table 4). in the tunneling test, termites penetrated the soil layer containing 1,250 ppm of tween 80 and reached the wood flakes within 5 to 6 hours (table 1). thus, the presence of tween 80 at 1,250 ppm in the soil had no efficacy on the termites. all the termites in the soils treated with the minimum effective concentrations of the 4 pyrethroids required to fulfill the criteria of the jwps-ts-s in the tunneling tests were seriously intoxicated or paralyzed, stopped walking within one hour, and were dead fig. 5. typical appearance of the soil in the tunneling test of a non-repellent termiticide. about 18 dead termites were found in the boring. 159 fujimoto, i. et al. — efficacy of termiticides on c. formosanus table 4. time required for a 100% moribund or dead rate after treatment with the 4 pyrethroids, permthirin, bifenthrin, etofenprox, and silafluofen, in the confined test. chemicals conc. in soil (ppm) time required for 100% moribund or dead rate (hr.) moribund dead permethrin + tween 80 25 + 0 worker 1 48 soldier 1 24 50 + 0 worker 1 48 soldier 1 24 25 + 125 worker 1 48 soldier 1 24 bifenthrin + tween 80 6.25 + 0 worker 2 24 soldier 2 24 12.5 + 0 worker 1 24 soldier 1 24 6.25 + 62.5 worker 1 24 soldier 1 24 etofenprox + tween 80 100 + 0 worker 1 48 soldier 1 24 200 + 0 worker 1 48 soldier 1 24 100 + 500 worker 1 48 soldier 1 24 silafluofen + tween 80 200 + 0 worker 1 24 soldier 1 24 375 + 0 worker 1 24 soldier 1 24 200 + 1,000 worker 1 24 soldier 1 24 tween 80 625 worker >24 soldier >24 1,250 worker >24 soldier >24 untreated worker >24 soldier >24 160 sociobiolog y vol. 59, no. 1, 2012 within one day or two days (table 4). the time required for a 100% moribund rate of the worker termites was the same as that for the soldier termites. this indicates that these pyrethroids penetrate into the termite bodies through their skin, because the soldier termites of c. formosanus are unable to ingest materials orally by themselves. the time periods required for a 100% moribund rate of the test termites in soil containing 25 ppm of permethrin, 6.25 ppm of bifenthrin, 100 ppm of etofenprox and 200 ppm of silafluofen were only 1 hour, 1 hour, 2 hours and 1 hour, respectively, (table 4) although none of the concentrations fulfilled the criteria for the tunneling test results (table 1). the time period required for a 100% dead rate of the test termites in soil treated with the minimum effective concentrations of permetnrin, bifenthrin, etofenprox and silafluofen was 1 day or 2 days (table 4), while that in the tunneling test was in the range of 11 to 17 days (table 1). furthermore, not even one dead termite usually existed in the borings excavated by the termites in the soils treated with the four pyrethroids (fig 2). that is, all the termites were dead and observed outside the treated soils in the tunneling tests. these findings suggest that the four pyrethroids showed a high repellent efficacy against c. formosanus. the time required for a 100% moribund rate of the worker termites in soil treated with the minimum effective concentrations of dinotefuran, clothianidin, imidaclroprid and thiamethoxam required to fulfill the criteria of the jwps-ts-s for the tunneling test was 2 hours, while that of the soldiers was 5 hours, 6 hours, 6 hours and 7 hours, respectively (table 5). thus, the time required for the soldiers was 3 to 3.5 times that for the workers. these findings indicate that a large portion of the total amount of each chemical incorporated into the workers was ingested orally from the dissolved water in the soil in which the chemicals were completely dissolved. the time required for a 100% dead rate of the termites in the soil treated with the minimum effective concentration of each neonicotinoid required to fulfill the criteria of the jwps-ts-s for the tunneling test was 1 day or 2 days (table 5), while that in the soil containing the same concentration as that in the confined test was 3 or 4 days (table 2). in the tunneling tests, several dead termites were usually observed in the borings excavated by the termites (fig. 4). these findings indicate that these neonicotinoids had no repellent effect against the termites. imidaclroprid has also been reported to be a non-repellent type of 161 fujimoto, i. et al. — efficacy of termiticides on c. formosanus termiticide (ishizaka, 2004). the time required for a 100% moribund rate of the workers in soil containing 12.5 ppm of dinotefuran, 12.5 ppm of clothianidin, 25 ppm of imidaclroprid or 50 ppm of thiamethoxam was 3 hours. in the tunneling tests using the same concentrations of the neonicotinoids, the test termites dug a tunnel with a length of more than 20 mm in the treated soil, although all the test termites in the tunneling tests were dead within 4 days (table 2). termites seem to dig tunnels in untreated soil and to advance table 5. time required for a 100% moribund or dead rate after treatment with the 4 neonicotinoids, dinotefuran, clothianidin, imidacloprid, and thiamethoxam, in the confined test. chemicals conc. in soil (ppm) time required for 100% moribund or dead rate (hr.) moribund dead dinotefuran + tween 80 12.5 + 0 worker 3 48 soldier 10 48 25 + 0 worker 2 48 soldier 5 24 12.5 + 125 worker 3 48 soldier 10 48 clothianidin + tween 80 12.5 + 0 worker 3 48 soldier 6 24 25 + 0 worker 2 48 soldier 6 24 12.5 + 125 worker 3 48 soldier 6 24 imidacloprid + tween 80 25 + 0 worker 3 48 soldier 8 48 37.5 + 0 worker 2 48 soldier 6 48 25 + 125 worker 3 48 soldier 8 48 thiamethoxam + tween 80 50 + 0 worker 3 48 soldier 7 24 100 + 0 worker 2 24 soldier 7 24 50 + 250 worker 3 48 soldier 7 24 162 sociobiolog y vol. 59, no. 1, 2012 by 8 to 10 mm per hour, because the termites penetrated the untreated soil layer over a length of about 5 cm and reached the wood flakes in the opposite cylinder within 5 to 6 hours (table 1). these findings indicate that all the test workers were probably intoxicated or paralyzed within 2 hours in soil treated with the minimum effective concentration of a non-repellent type of chemical, required to meet the criteria of the jwps-ts-s for the tunneling test. the time required for a 100% moribund rate of the workers in soil treated with fipronil, chlorfenapyr and chlorantraniliprole at the minimum effective concentration required for fulfillment of the criteria of the jwps-ts-s for the tunneling test was 2 hours (table 6). this time period was the same as that for each of the neonicotinoids which exhibited no repellent effect against the termites. the value for the soldiers was 4 hours or 24 hours. the three chemicals were insoluble in water (ca.4.6 ml), existing in the soil in table 6. time required for a 100% moribund or dead rate after treatment with fipronil, chlorfenapyr and chlorantraniliprole in the confined test. chemicals conc. in soil (ppm) time required for 100% moribund or dead rate (hr.) moribund dead fipronil + tween 80 45 + 0 worker 3 24 soldier 5 24 75 + 0 worker 2 24 soldier 4 24 45 + 450 worker 2 24 soldier 4 24 chlorfenapyr + tween 80 250 + 0 worker 4 6 soldier 7 8 250 + 250 worker 3 6 soldier 6 7 250 + 750 worker 2 6 soldier 4 7 chlorantraniliprole + tween 80 125 + 0 worker 3 72 soldier 24 48 250 + 0 worker 2 72 soldier 24 48 125 + 500 worker 2 72 soldier 24 48 163 fujimoto, i. et al. — efficacy of termiticides on c. formosanus the confined tests. emulsification of each of the chemicals with tween 80 enhanced the termiticidal activity of each by 2to 8-fold as compared with the efficacies in the absence of the emulsifying agent, as mentioned above (table 3). these findings indicate that a large portion of the total amount of each chemical incorporated into the worker termites was ingested orally when the chemicals were emulsified. the time required for a 100% dead rate of the test termites in soil treated with fipronil, chlorfenapyr and chlorantraniliprole at the minimum treatment concentration required for fulfillment of the criteria of the jwps-ts-s for the tunneling test was 1 day, 6 hours and 3 days, respectively (table 6), while that in the tunneling test was 2 or 3 days (table 3). several dead termites usually existed in the borings excavated by the termites in the tunneling tests (fig. 5). these findings indicate that the three chemicals had no repellent effect against the termites. fipronil has also been reported as a non-repellent termiticide (ibrahim et al., 2003). the results of this study suggest that the water solubility of termiticides affects their termiticidal efficacy. addition of an emulsifying agent improved the efficacy of the low water solubility chemicals, showing the possibility of the efficacy being improved depending on the formulation employed. in another study at the field site in kagoshima prefecture, soil treatment with the emulsion of the chemicals (3 l/m2) at the minimum effective concentrations needed to fulfill the criteria of the jwps-ts-s for the tunneling test could protect against termite attacks for at least 5 years. references enoki, a., k. ueshima, s. itakura, and h. tanaka (1995) termiticidal efficacy of organophosphates (ii), rate of disappearance of organophosphates in soil and wood. mem. foc. agr. kinki univ. 28:1 9 (in japanease). ibrahim, s. a., g. henderson, and h. fei (2003) toxity, repellency, and horizontal transmission of fipronil in the formosan subterranean termite (isoptera: rhinotermitidae). j. econ. entomol. 96: 461 467. ishizaka, k. (2004) new effective methods with a non-repellent termiticide, imidacloprid. mokuzaihozone 30: 259 264 (in japanease). kuriachan, i., and r. e. gold (1998) evaluation of the ability of reticulitermes flavipes (kollar), a subterranean termite (isoptera: rhinotermitidae) to differentiate between termiticide treated and untreated soils in laboratory tests. sociobiolog y 32: 151 166. schoknecht, u., d. rudolf, and h. hertel (1994) termite control with microencapsulated permethrin. pestc. sci. 40: 49 55. 164 sociobiolog y vol. 59, no. 1, 2012 su, n. y., v. chew, g. s. wheeler, and r. h. scheffrahn (1997) comparison of tunneling responses into insecticide-treated soil by field population and laboratory groups of subterranean termites (isoptera: rhinotermitidae). j. econ. entomol. 90: 503 509. tamashiro, m., j. r. yates, and r. h. ebesu (1987) the formosan subterranean termite in hawaii: problems and control, pp. 15 22 in m. tamashiro and n-y. su [eds.], biolog y and control of the formosan subterranean termite. college of tropical agriculture ＆human resources, university of hawaii, honolulu. ext. series 083 tsunoda, k. , t. yoshimura, and k. nishimoto (1989) effect of accelerated ageing on the termiticidal performance of organophosphates (2). soil burial. material u. organismen 24(1): 17 25. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.5443sociobiology 68(4): e5443 (december, 2021) introduction trehalose is a non-reducing disaccharide with two glycosidically linked glucose units present in all forms of life except mammals, including plants, bacteria, fungi, yeast, abstract trehalose provides the main energy source for the physiological activities of insects, especially in adverse conditions. trehalase is the only enzyme that hydrolyzes trehalose, therefore it is important to clarify the distribution and expression of trehalase under adverse conditions such as high temperatures and starvation. here, we have cloned the trehalase genes and investigated their expression in different tissues, at multiple development stages, and with the treatments of high temperature and starvation in bombus lantschouensis, which is considered to be one of the most commercially viable native species in china. the results suggest that the membrane-bound (bltre-2) cdna has an open reading frame of 1986 nucleotides, which encodes a protein of 662 amino acids, and two putative transmembrane domains. qrt-pcr analysis indicated that bltre-2 was expressed in 10 tissues and at nine development stages, with the highest expression in general in 30-day-old workers, and in ovarian tissue in particular. the expression of bltre-1 for 15-day-old workers which were exposed to a pre-treatment of 45°c increased over the first 5 h and subsequently decreased over time. in contrast the expression of bltre-2 consistently decreased over time. the highest expression levels of bltre-1 and bltre-2 were observed the newly emerged adult workers when starved for 16-20 h. these results indicate that bltre-2 may be part of the carbohydrate metabolism of the bumblebee, and that bltre-1 is a key gene regulating energy metabolism and providing trehalose when exposed to a high temperature. both bltre-1 and bltre-2 may balance trehalose and provide energy when b. lantschouensis is starved. sociobiology an international journal on social insects jiamin qin1,2, feng liu3, jie wu1, shaoyu he4, muhammad imran5, wen lou3, hongmei li-byarlay6, shudong luo1 article history edited by marco antonio costa, uesc, brazil received 24 may 2020 initial acceptance 22 november 2020 final acceptance 26 june 2021 publication date 19 november 2021 keywords bombus lantschouensis, trehalase, sequence analysis, stress conditions, gene expression. corresponding author shudong luo key laboratory for insect-pollinator biology institute of apicultural research, caas beijing 100093, china. e-mail: luoshudong@caas.cn nematodes, and insects (elbein et al., 2003; thompson, 2003; argüelles, 2014; nardelli et al., 2019). in these organisms, trehalose plays an important role in protecting proteins and cellular membranes during unfavorable environmental conditions such as heat, desiccation, dehydration, freezing, 1 institute of apicultural research, chinese academy of agricultural sciences, key laboratory of pollinating insect biology, ministry of agriculture and rural affairs, beijing, china 2 sericulture and apiculture research institute, yunnan academy of agricultural sciences, mengzi, china 3 apiculture institute of jiangxi province, nanchang, china 4 eastern bee research institute, yunnan agricultural university, kunming, china 5 department of entomology, the university of poonch rawalakot, azad jammu and kashmir, pakistan 6 agricultural research and development program, department of agriculture and life science, central state university, wilberforce, oh, united states research article bees molecular characterization and gene expression of trehalase in the bumblebee, bombus lantschouensis (hymenoptera: apidae) mailto:luoshudong@caas.cn jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis2 hyperosmosis, anhydrobiosis, and oxidation (wyatt, 1967; crowe et al., 1992; thompson, 2003; mitsumasu et al., 2010). as a major blood sugar of insects, trehalose is present in the hemolymph of larvae, pupae, and adults (alumot et al., 1969; becker et al., 1996; thompson, 2003). trehalose is the main metabolic source for physiological activity, such as growth, development (łopieńska-biernat et al., 2018), energy metabolism (thompson, 2003), anhydrobiosis (mitsumasu et al., 2010), feeding behavior (tang et al., 2014; yasugi et al., 2017), chitin synthesis (tang et al., 2012, 2016, 2017, 2018; shen et al., 2017; zhang et al., 2017), and diapause (kamei et al., 2011; yang et al., 2013; wang et al., 2018). on the other hand, it plays key roles in adverse circumstances, such as starvation (tang et al., 2014; shukla et al., 2014; liu et al., 2016). in insects, trehalase (α-glucoside-1-glucohydrolase, ec 3.2.1.28) is the only key enzyme that catalyzes the conversion of one molecule of trehalose to two molecules of glucose utilized through glycolysis (clegg & evans, 1961; tatun et al., 2008). two types of trehalase (soluble and membrane-bound) and corresponding genes (tre-1 and tre2) have been cloned in a variety of insects (qin et al., 2015; liu et al., 2016; shukla et al., 2016; zhao et al., 2016). both forms contain signature motifs (pggrfefyywdsy and qwdypnawpp) and a highly conserved glycine-rich region (ggggey). tre-1 and tre-2 break down the intracellular and extracellular trehalose, respectively (shukla et al., 2014; liu et al., 2016). the tre-1 gene, which was first cloned from tenebrio molitor in 1992, is mainly found in the digestive and circulatory systems (takiguchi et al., 1992). tre-2 contains a putative transmembrane domain (takiguchi et al., 1992; tang et al., 2008; gu et al., 2009; forcella et al., 2010) and is mostly identified in the muscle (mitsumasu et al., 2005). recent studies have indicated that only tre-1 and tre-2 are found in the majority of insects, such as apolygus lucorum (tan et al., 2014), helicoverpa armigera (ai et al., 2018), spodoptera exigua (tang et al., 2008), omphisa fuscidentalis (tatun et al., 2008), drosophila melanogaster (shukla et al., 2016), cnaphalocrocis medinalis (tian et al., 2016), and bemisia tabaci (wang et al., 2014). multiple tre1 have been cloned in insects, including harmonia axyridis (tang et al., 2014; shi et al., 2016), locusta migratoria (liu et al., 2016), nilaparvata lugens (zhao et al., 2016), tribolium castaneum (tang et al., 2016), and leptinotarsa decemlineata (shi et al., 2016). in addition, trehalase gene is expressed in various tissues inducing the diapause of silkworm eggs and improving the cold resistance (kamei et al., 2011; tan et al., 2014). generally, two types of trehalase, tre-1 and tre-2 are involved in various physiological functions such as longdistance flight metabolism, chitin synthesis during molting, energy metabolism, muscular movement, reproduction, and cold tolerance (chen et al., 2010; tang et al., 2012; zhang et al., 2012; shi et al., 2019). bumblebees (apidae, bombus latreille), important pollinators of many endangered alpine plants and agricultural crops, play a major role in maintaining biodiversity of ecosystems and agricultural production (williams & osborne, 2009; gunnarsson & federsel, 2014). many species of bumblebees (such as bombus terrestris, b. impatiens, b. occidentalis, and b. ignitus) have been used for commercial crop pollination (velthuis & van doorn, 2006). b. lantschouensis (hymenoptera: apidae), once mistaken for b. hypocrita, is widely distributed in the north china and is a native bumblebee species used in commercial applications – it is recognized in chinese agriculture to be an excellent pollinator (an et al., 2014). there is not much information is available on the function, structure, tissue distribution, and expression patterns of the trehalase gene in b. lantschouensis even though trehalase has been studied in honeybees (alumot et al., 1969; brandt & huber, 1979; lee et al., 2007; łopieńska-biernat et al., 2018). understanding the fundamental molecular characteristics and function of trehalase will enable us to improve the population and health of b. lantschouensis. in the present study, we cloned and characterized the tre-2 gene from b. lantschouensis and measured patterns of expression in tissues at different chronological ages. in addition, we investigated the expression patterns of both tre1 and tre-2 in b. lantschouensis at 45°c and under starvation conditions. materials and methods bumblebees newly mated queens of the bumblebee b. lantschouensis were collected from the hebei province of china. these queens were reared under controlled climatic conditions at a temperature of 29 ± 0.5°c and 60±5% relative humidity in continuous darkness. queens and their newly emerging workers were fed a 50% sucrose solution and pollen (from brassica campestris l. and prunus armenica l.). to ensure the adult bumblebees were the same age, all new workers were marked on the thorax with a white mark within 1 h. the workers used in this study were 15 days old but particular exceptions, which are noted later on. all the samples were taken from three colonies, and each colony was treated as an independent replicate. rna extraction, cdna synthesis, and pcr the total rna was extracted from the sample with trizol (invitrogen, carlsbad, ca, usa). first-strand cdna synthesis was synthesized from 1 μg of rna using a transcriptor first strand cdna synthesis kit (takara, dalian, china), and the undiluted first-strand cdna was used as the template for the polymerase chain reaction (pcr). conserved district fragment amplification to obtain a conservative fragment of bltre-2 from b. lantschouensis, a pair of degenerate primers, tre-2-f and tre-2-r (table 1), were designed based on the conserved sociobiology 68(4): e5443 (december, 2021) 3 amino acid sequences of the known tre-2 in b. terrestris and b. impatiens (genbank accession nos. xp_003393687 and xp_003490073). the pcr amplification reaction system contained 2 μl cdna template, 1 μl of each primer (10 μmol/l) and 12.5 μl of the pcr mix, and finally was topped to 25 μl volume with nuclease-free water. pcr conditions were as follows: 94°c for 5 min, 35 amplification cycles of 94°c for 40 s, 50°c for 30 s, and 72°c for 1 min, and then an extension at 72°c for 7 min. the pcr products were electrophoresed for 10 min under 200 volts and then excised from agarose gels (1%) and purified with dna purification kits (version 2.0, takara). purified pcr products (4.2 μl) were ligated into 0.8 μl pmd-19t vectors using 5 μl solution i at 16°c. the ligated constructs were transformed into tran1-t1 competent cells and cultured for 1 h at 37°c. we selected an ampicillin-resistant clone and sub-cultured in 600 μl lb liquid medium to obtain the optimum amount of the expression vector, which was sequenced using m13forward and m13-reverse. to complete the 5’ and 3’ ends, a transcriptor first strand cdna synthesis kit (takara) was used to synthesize 5’-race and 3’-race first-strand cdna according to the manufacturer’s instruction. specific primers 5’-ogsp and 5’op, 5’-igsp, and 5’-ip for 5’-race, and 3’-ogsp and 3’-op, 3’-igsp, and 3’-ip for 3’-race (table 1) were synthesized based on the cdna sequence of the pcr fragment. the first pcr amplification reaction system contained 2.5 μl cdna template, 1 μl of each primer (10 μmol/l) (5’-ogsp and 5’-op/3’-ogsp and 3’-op), 1 μl dntp mix, 2.5 μl 10×ex buffer, 0.4 μl ex taq hs, and was finally topped to 25 μl volume with nuclease-free water. pcr was performed under the following conditions: 94°c for 2 min, 35 amplification cycles of 94°c for 15 s, 60°c for 30 s, and 72°c for 40 s, and an extension at 72°c for 10 min. the products of the first pcr were diluted one-fold and then used as a template (2.5 μl) for the second pcr. the second pcr had the same reagent and content as the first pcr except for the primers (5’-igsp and 5’-ip/3’-igsp and 3’-ip) and was performed under the following conditions: 2 min at 94°c followed by 30 cycles of 15 s at 94°c, 30 s at 60°c, and 40 s at 72°c, and then 10 min at 72°c. protein and cdna sequence analyses the cdna sequence of bltre-2 was analyzed to determine its similarity with tre-2 genes of b. terrestris (xp_003393687) and b. impatiens (xp_003490073) deposited in genbank by using bioedit 7.0.9. the bltre-2 cdna sequence was deposited in the ncbi genbank (accession number mz292465). the signal peptides, molecular mass and theoretical isoelectric point (pi), n-glycosylation sites and transmembrane helices of the deduced amino acid sequences of bltre-2 were predicted by using the signalp 4.1 server, the expasy compute pi/mw tool (https://web. expasy.org/compute_pi/), the netnglyc 4.0 server, and the tmhmm server v. 2.0, respectively. the deduced amino acid sequences of bltre-2 were compared with trehalase genes of other species available in genbank. multiple alignments of trehalase genes were conducted using the software clustal x2. a phylogenetic tree was constructed based on the amino acid sequences by using the neighbor-joining (nj) algorithm in mega 6.06. the reliability of the branching was tested using the bootstrap method (1,000 replications). primer uses primer name primer sequence (5’-3’) the part fragment tre-2-f ggactggaaaagagcggtca tre-2-r ggtttcagatcgtcctggcacgatt 3’-race 3’-ogsp gttcgacgatttggaacgtc 3’-op taccgtcgttccactagtgattt 3’-igsp atacgcggaacgtaattggc 3’-ip cgcggatcctccactagtgatttcactatagg 5’-race 5’-ogsp ggtcgaacgtgttgttgatg 5’-op catggctacatgctgacagccta 5’-igsp gatctggttcctggtcggtg 5’-ip cgcggatccacagcctactgatgatcagtcgatg qrt-pcr tre-1-f1 tre-1-r1 agtgagtttgccttctgg gaggtctcgtgctgttga tre-2-f1 tcctcgttcgtttgtgacga tre-2-r1 tttggccaattacgttccgc reference gene actin-88-f gcgcgacattaaggagaaac actin-88-r ccatacccaggaaggaaggt table 1. primers used in this study. jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis4 bltre-2 expression in different tissues and different chronological ages to clarify the distribution of bltre-2 in b. lantschouensis, we dissected the workers in the ice-cold lysis buffer on ice. ten tissues (antennae, head, muscle, leg, wing, integument, midgut, malpighian tubule, fat body, and ovary) were collected from workers aged 15 days and placed separately into pcr tubes for rna extraction. to get enough samples for rna extraction, 3 to 15 workers were dissected (table 2). larvae, pupae, and adult workers aged 0 (new workers), 5, 10, 15, 20, 25, and 30 days old were dissected to investigate the relative expression of bltre-2 at different chronological ages. a sample of workers of the same age from each of three different colonies was dissected to extract rna from the whole body, and this triplicated. to determine the absolute copy of the target transcript, the cdna template was diluted (10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7) to gradient concentrations and then used to generate a standard curve. the qrt-pcr amplification reaction system contained 2 μl cdna template, 0.8μl of each primer (10 μmol/l), 10 μl sybr® premix ex taqtm ii, and 0.4 μl rox reference dye and was finally topped to 20 μl volume with ddh2o. the amplification conditions were as follows: 94°c for 30 s followed by 40 cycles of 94°c for 5 s and 60°c for 30 s. each sample was replicated three times. actin-88 gene (table 1) served as an endogenous reference gene for the determination of targeted mrna for its continuously expressed in bumblebee (li et al., 2010). completed, rna was extracted from three workers exposed to the same treatment. to investigate the relative expression of the bltre-1 and bltre-2 genes, we designed two pairs of specific primers: tre-1-f1 and tre-1-r1; tre-2-f1 and tre2-r1 (table 1) based on the conserved amino acid sequences of the two known forms of trehalase gene in b. lantschouensis (bltre-1: genbank accession nos. kj025078; bltre-2: genbank accession nos. mz292465), and the primers of the reference gene actin-88-f and actin-88-r (li et al., 2010; qin et al., 2015). the qrt-pcr reactions were conducted using an mx3000 qpcr system (agilent, usa) with buffers at 94°c for 30 s in 1 cycle; 94°c for 5 s and 60°c for 20 s in 40 cycles with a melt curve over a temperature ranging from 55 to 90°c. in each reaction, 25 μl of final volume was produced containing 10 μl of the sybr® premix ex taqtm ii, 2 μl of cdna sample, 0.8 μl of primer (10 μmol/l), 0.4 μl of rox reference dye, and 11 μl of rnase-free and dnaase-free h2o, according to the manufacturer’s instructions of sybr® premix ex taqtm ii kit (takara, dalian, china). all samples were run in triplicate. the qrt-pcr values of the focal genes were normalized using the actin-88 gene. statistical analyses transcript quantifications were calculated using the 2-δδct (livak & schmittgen, 2001). the lowest expression levels in different tissues and at different chronological ages were stated as 1. all data were analyzed using lsd tests of one-way anova in spss 13.0. results cloning and characterization analyses of bltre-2 the bltre-2 is 4,051 bp long, including an open reading frame (1,989 bp), a 5’-untranslated region (utr) (1,307 bp), and a 3’-utr (1,684 bp). the bltre-2 transcript encoded a protein of 662 amino acids (about 76.81 kda and an estimated pi of 5.94). the amino acids contained two signature motifs, pggrfrefyywdsy (residues 180-193) and qwdypnawpp (481-490), a highly conserved glycinerich region, ggggey (545-550), a signal peptide of 30 amino acids and a cleavage site (cya-st) between 30 and 31 (fig 1), and five putative n-glycosylation sites (amino acids 79, 276, 352, 386, and 527) suspected to be a glycoprotein. residues 13-31 and 598-620 comprised two putative transmembrane domains, mllsaaflallvvapcyas qvmtgilalvislaagfigmvvy (fig 1). the deduced amino acid sequence of trehalase from b. lantschouensis was aligned with the corresponding sequences of another insect trehalases (fig 2). bltre-2 is most similar to the other hymenopterans, such as bttre-2 (b. terrestris), bitre-2 (b. impatiens), amtre-2 (a. mellifera) and aftre-2 (a. florea) (table 3). it is also similar to tre-1 and tre-2 from harpegnathos saltator, s. exigua, bombyx mori, o. fuscidentalis, n. lugens, b. tabaci, and a. lucorum (table 3). tissue name number of individuals chronological age number of individuals antennae 15 larva 3 head 5 pupa 3 muscle 3 day 0 worker 3 leg 5 day 5 worker 3 wing 15 day 10 worker 3 integument 5 day 15 worker 3 midgut 15 day 20 worker 3 malpighian tubule 15 day 25 worker 3 fat body ovary 15 15 day 30 worker 3 table 2. the information of the number of individuals in each biological repetition for different tissues and chronological ages of b. lantschouensis. bltre-1 and bltre-2 expression at 45°c and during starvation in this study, 15-days old workers were exposed to 0, 1, 2, 3, 4, and 5 h at 45°c, representing the temperature treatments for our experiments. for the starvation treatments, the newly emerged (0 day) adult workers were starved for 0, 4, 8, 12, 16, 20, and 24 h. when the treatments were sociobiology 68(4): e5443 (december, 2021) 5 the alignment of multiple sequences indicated that the insect tre-2 gene is highly conserved, particularly in the middle of the putative catalytic domain (fig 2). in addition, we used the amino acid sequences of selected trehalase genes to construct a phylogenetic tree, which shows that bltre-2 has a higher identity with other tre-2 genes in insects. the entire tre-2 gene clustered together as a subgroup, and the tre-1 gene clustered into another subgroup (fig 3). table 3. the similarity comparison between amino acid sequences of trehalase genes from b. lantschouensis and other insects. species names gene names genbank no. similarity to bltre-1 similarity to bltre-2 bombus lantschouensis bltre-1 bltre-2 kj025078 mz292465 – 55.7 % 55.7 % – bombus terrestris bttre-1 bttre-2 xp_003400853 xp_003393687 99.5 % 56.0 % 55.2 % 98.9 % bombus impatiens bitre-1 bitre-2 xp_003491166 xp_003490073 99.0 % 55.1 % 54.9 % 97.5 % apis mellifera amtre-1 amtre-2 xp_393963 baf81545 88.3 % 59.6 % 54.5 % 84.2 % apis florea aftre-1 aftre-2 xp_003695047 xp_003696950 87.4 % 56.1 % 53.9 % 91.8 % harpegnathos saltator hstre-1 hstre-2 efn81352 efn85130 72.1 % 56.2 % 53.5 % 85.8 % spodoptera exigua setre-1 setre-2 aby8628 abu95354 58.5 % 56.2 % 54.1 % 69.4 % bombyx mori bmtre-1 bmtre-2 np_001037458 np_001036910 60.4 % 57.3 % 51.7 % 68.6 % omphisa fuscidentalis oftre-1 oftre-2 abo20846 abo20845 59.9 % 56.0 % 53.8 % 67.5 % nilaparvata lugens nltre-1 nltre-2 acn85420 acn85421 60.9 % 54.2 % 54.4 % 72.9 % bemisia tabaci bttre-1 bttre-2 afv79626 afv79627 57.1 % 57.7 % 50.4 % 70.4 % apolygus lucorum altre-1 altre-2 agk89789 agl34007 58.3 % 58.0 % 65.0 % 71.2 % bltre-2 expression in different tissues and at different chronological ages the qrt-pcr results detected the expression of bltre-2 in 10 tissues and at various chronological ages of b. lantschouensis. the bltre-2 had the highest expression levels in the ovary, followed by the midgut, antennae, muscle, malpighian tubule, integument, wings, head, and fat bodies, with the lowest expression level in legs (fig 4a). the bltre-2 gene expression in the larval stage is higher than that in the pupal stage (p = 0.011). in addition, analysis of different chronological ages revealed that bltre-2 had the lowest expression in 0-day-old workers, and highest expression in 30-day-old workers. from day 0 to 15, the gene expression increased gradually (fig 4b). bltre-1 and bltre-2 expression at 45°c and during starvation the expression of bltre-1 increased as temperature treatment time increased, reaching the highest level at 3 h, and then declined progressively (fig 5a). bltre-2 expression declined with treatment time in the first 3 h, and then it increased at the 4 h time point (fig 5b). overall, the results showed that in the 45°c treatments, the change of expression patterns differ between bltre-1 and bltre-2. in the starvation experiment, both bltre-1 and bltre-2 had the highest expression levels in adult bees starved for 16 and 20 h, as compared to other time points (fig 5c, d). the expression levels of bltre-1 in bees starved for 4 to 12 h and 24 h did not differ significantly from that of bees starved for 0 h (fig 5c). however, the expression level of bltre-2 in starved adult b. lantschouensis was higher than that in individuals starved for 0 h, and its expression increased with starvation time. jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis6 1 catcgttatcttgcgaattcgaagcaaaagaggaacttgttaaaagaagggaagtaa 58 aggagacaagagagaagaagagggattgtgtgaaagggtcgaccggtggagaaaaaa 115 atggcttggagctgcacgcgctgcggttcgacgaatatgctgctgagtgctgcgttcctc 1 m a w s c t r c g s t n m l l s a a f l 175 gcgcttctcgtcgttgctccgtgttacgctagcacagagaaggcaagctacgtgaaaccg 21 a l l v v a p c y a s t e k a s y v k p 235 cctccgtgtcagagcgatatttactgccatggcgagctgctgcacacgatacagatggcc 41 p p c q s d i y c h g e l l h t i q m a 295 tcgatctacaaggactcgaagacgttcgtcgacatgaagatgaaattctcgccgaacgag 61 s i y k d s k t f v d m k m k f s p n e 355 acgctgctcctatttcgcgaattcatggaaagcgtgaatcaaacaccgaccaggaaccag 81 t l l l f r e f m e s v n q t p t r n q 415 atcgaacaattcatcaacaacacgttcgaccaagaaggatccgagttcgaggaatggaac 101 i e q f i n n t f d q e g s e f e e w n 475 ccagtggactggaccagccaaccgaagtttcttaacaaaatccacgatcacgatcttcgc 121 p v d w t s q p k f l n k i h d h d l r 535 aaatttgcctctgatttgaaccaaatttggaaaatgttgggacgaaagatgaaagacgac 141 k f a s d l n q i w k m l g r k m k d d 595 gtgcgggtcaacgaggatcgatattccatcatctacgtgccgaatccggtgatcgtgccc 161 v r v n e d r y s i i y v p n p v i v p 655 ggcggccgattccgcgagttctactactgggactcgtactggatcgtgaaagggctgctg 181 g g r f r e f y y w d s y w i v k g l l 715 ctttcggagatgtacaccaccgtcaaaggaatgttaaccaatttcgtctctctggtggac 201 l s e m y t t v k g m l t n f v s l v d 775 aagatcggtttcatcccgaacggaggcagaatctactacgctaggagatctcagcctccc 221 k i g f i p n g g r i y y a r r s q p p 835 atgttgattcctatggtcgaagagtatctgaaggtgaccatcgactacaaatgcctggag 241 m l i p m v e e y l k v t i d y k c l e 895 gataaccttcaccttctagagaaggagtttgaattttggatgaccaataggacggtggac 261 d n l h l l e k e f e f w m t n r t v d 955 gttgaagtggatggagtgaagtacactttagccagattcttcgaggagtcttcgggacct 281 v e v d g v k y t l a r f f e e s s g p 1015 cgaccagaatcctacaaagaggattacctgaccagccaaagttttcgcacgaacgaagag 301 r p e s y k e d y l t s q s f r t n e e 1075 aaggacaactattacgcggaattgaagaccgcggccgagtccggctgggacttttctagt 321 k d n y y a e l k t a a e s g w d f s s 1135 cgatggttcatactagacggcacgaacaaaggtaacctgacgaacttgaaaacgagatac 341 r w f i l d g t n k g n l t n l k t r y 1195 attgtccccgtggacttgaattcgataatatatcgaaacgcgcagctgctagaacagtac 361 i v p v d l n s i i y r n a q l l e q y 1255 aatcaaaggatgggcaacgagaccaaggccgcgtattaccggaaaagagcggaggactgg 381 n q r m g n e t k a a y y r k r a e d w 1315 aaaagagcggtcacggccgtactgtggcacgatgaagtcggtgcttggctcgactacgat 401 k r a v t a v l w h d e v g a w l d y d 1375 ttactgaacgacatcaaaagagattatttttatccgacgaacgttctgccgctttggacc 421 l l n d i k r d y f y p t n v l p l w t 1435 gattgttacgacatcgcaaagagagaggaatacatagcgaaggtgctcaagtatctagag 441 d c y d i a k r e e y i a k v l k y l e 1495 aaaaatcaaataatgttaaatttgggcggtataccgaccaccctcgaacactctggtgaa 461 k n q i m l n l g g i p t t l e h s g e fig 1. nucleotide and deduced amino acid sequences of bltre-2. the numbers on the left are the positions of nucleotides and amino acids in the sequences; underlined amino acid residues represent the signal peptide; the cleavage site is indicated by an arrow; the n-glycosylation sites are indicated by a box; the highly conserved glycine-rich region is shaded gray and the trehalase signature motif is shaded red; transmembrane domains are gray and boxed. sociobiology 68(4): e5443 (december, 2021) 7 1555 caatgggattacccgaatgcctggccgcccttgcaatactttgtcatcatgtcgttgaat 481 q w d y p n a w p p l q y f v i m s l n 1615 aacaccggagacccgtgggcgcagaggctcgcctacgagatcagccaacgatgggttcgc 501 n t g d p w a q r l a y e i s q r w v r 1675 agcaactggaaggcgttcaacgagacgcacagcatgttcgagaagtatgacgccacggta 521 s n w k a f n e t h s m f e k y d a t v 1735 tcaggcggtcacggaggtggcggtgagtacgaggtgcaactaggtttcggttggagcaac 541 s g g h g g g g e y e v q l g f g w s n 1795 gggatcatcatggacttgctgaacaagtacggagatagactgacagccgaaattttcctc 561 g i i m d l l n k y g d r l t a e i f l 1855 gccatagtgcagagcttggcccctccagccgtcgtcgtttcgaccgccggtcaagtgatg 581 a i v q s l a p p a v v v s t a g q v m 1915 accggtattctcgccctcgtaatatcgttggccgcgggattcatcggaatggtggtttac 601 t g i l a l v i s l a a g f i g m v v y 1975 aaaaggcgacactactatgttcctggaccatcgacgatgccaaacaagagaaaagtgatc 621 k r r h y y v p g p s t m p n k r k v i 2035 tcaccgaccggaaacgtttatcgaaagaggatcgcctacactgaattgaaggacatgaac 641 s p t g n v y r k r i a y t e l k d m n 2095 aatgattgacgaccgttgctcttttagaaagccgattcgtttaaagagactcttaaagcg 661 n d * 2155 caacgagagcacaagaagaccggggataggataaaaggagcgcagacgcaaaaaggacac 2215 caactagaaaccagaaaccgtcgattactgactgatcgcgatcaagatcgaccgtgaacc 2275 aaccaacgaagatcgtcgctttcgattttctcgccaaaggctagaaagcttggtaacaat 2335 tcgtgtgcggtttcagatcgtcctggcacgattcgactacgcgaacatcatcgccgttga 2395 ttcgcgcacgcatctatcgcggtgaatcttccgactcgaacgtttttacggtcggatatt 2455 cgtaacaaaagttcgcacgttggtcgtgcgtgatcacgtccgtctcaagttctttttctc 2515 tttctcggttttcaattcggtaagtggatcgcgcgcgctacattcgcggggaaggtgata 2575 cggtagcacggtgacacgttaacgactctttgtaggacaactctgtcgctgatagactaa 2635 agcgcgaaaaatcgacttctccgaaggaaaacgttcgagaagagacggcagtgcggaacg 2695 atttggaacgcgacacagcacggcactaaaaaccgctggaaaccgtgcgtctcgctcgcc 2755 aagcttcgccgcgacttcgacgggtgtccgctcgcgtcgtgggctcccccactttaaccc 2815 ttcccttccactacgcgctcggcctaattacacgacagttatcttcattgcaagcacgcg 2875 cttatccttctcgaattaccttataatttcgaaatacactggcgacgagcgaaactcgca 2935 aaattgaacacgaatgatggtgtgaagagctgtgcaaggatcggaacgagcaacaacgtg 2995 cgtgcgttgaagctcggatgaagaatgtgcaactttgcgaccaagctttgtggcacgtac 3055 ttccacgttcattgtttataggtgagtttagataatcgattctgtagataaaaatgtcgt 3115 atctttgaggatatttttgtataaagagcagcttgtacgtttagtgaaagtcgtgttctt 3175 attgctatttaccatccgggataattcgcttgaacgttattatcgacttgtataaacgag 3235 tcgcgcaaagttgccgcaaggtgcgcattgctatccggaaacggtgacgagatgaacgat 3295 caattaccgacgacgagatttcgcaagaaccggtcttcgtgctttgtatccgtctaattg 3355 taaaagatgcatcgagctatcctcgttcgtttgtgacgaccgaatcgaaacaagagacgt 3415 cgtttctaatccgaactctatgctcgctcaacttttcttgtaaatagtaatacgtgtttt 3475 cgttgttcgacgatttggaacgtcgatacgcggaacgtaattggccaaacgcataagaca 3535 atttgaactgttacttctactataatcgttgtttatagacagatttacgtatcaaaaagc 3595 tcgaacgaacaaaattttgcacggccacggaacgagttattgacgaaccgtcgtcaaagc 3655 gcgtcaaccgacgaatcgaaaatcgactggttcgagactgaagaacgttaccgcgatatc 3715 tgcatcttccgctaatcgcgatcgatcgccggccgacgagcatcgaccaacgagcaccgc 3775 gtcttaatgatttttgacactgttagttttattgtgagcgacgcgaatattatatatata 3835 cagatatatatatatagatagatagatagttaggtagatagatagagaattttgtgtttg 3895 cgagtgaacgaaaaatcggtcgagagccgacgaagaatgatcgcggttgatcctatgagg 3955 aaatttaccgcatcacgaaggatgacgacgcggtcggatggactcgatcgttagctgcga 4015 ctaaaaaaaaaaaaaaaaaacctatagtgaaatcact fig 1. nucleotide and deduced amino acid sequences of bltre-2. (continuation) jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis8 fig 2. comparison of the amino acid sequences of tre in b. lantschouensis with those in other orthologs. letters on the left and numbers on the right are the gene names and positions of amino acids on the sequences, respectively. the conserved and similar amino acid residues are labeled in black and gray backgrounds, respectively. the highly conserved glycine-rich regions of trehalase are indicated by a double underline.     bltre-2 b. lantschouensis mawsc-trcgstnmllsaa--flallvvapcyastekasyvkpppcqsdiychgellhtiqma 60 bttre-2 b. terrestris mawsc-trcgstnmllsaa--flallvvapcyastekasyvkpppcqsdiychgellhtiqma 60 bitre-2 b. impatiens macsc-trcgstnmllsav--flaflvvapcyastekasyvkpppcqsdiychgellhtiqma 60 amtre-2 a. mellifera masscsircgsrnilvnaaatflallvvlrcfanae-----kpspcqsdvycrgellhtiqma 58 aftre-2 a. florea masscsircgsrnilvnaattflallvvlrcfanae-----kpppcqsdvycrgellhtiqma 58 bltre-1 b. lantschouensis --------mssglliavgvigliaaltdaasighas----vkatdcyseiyctgellktvqls 51 bttre-1 b. terrestris --------mpsglliavgvigliaaltdaasighas----vkatdcyseiyctgellktvqls 51 bitre-1 b. impatiens --------mpsglliavgvigliaaltdaasighas----vkatdcyseiyctgellktvqls 51 amtre-1 a. mellifera --------mmpglfaflgva-liasltdaasirran----rkamdcyseiyctgellktiqla 50 aftre-1 a. florea -------mqaagvfaflgva-liasltdaasirras----rkamdcyseiyctgellktiqla 51 bltre-2 b. lantschouensis siykdsktfvdmkmkfspnetlllfrefmesvnqtptrnqieqfinntfdqegsefeewnpvd 123 bttre-2 b. terrestris siykdsktfvdmkmkfspnetlllfrefmesvnqtptrnqieqfinntfdqegsefeewnpvd 123 bitre-2 b. impatiens siykdsktfvdmkmkyspnetlllfrefmervdqaptrnqieqfinntfdqegsefeewnpvd 123 amtre-2 a. mellifera siykdsktfvdmkmkrppdetlksfrefmerheqmptryqierfvndtfdpegsefedwdpdd 121 aftre-2 a. florea siykdsktfvdmkmkhpphetlklfrefmdrhdqmptrhqierfvndtfdpegsefeewdpdd 121 bltre-1 b. lantschouensis niysdsktfvdlqqindpeitlanfyelmketnnkptksqliqyvnenfis-sselvnwtlsd 113 bttre-1 b. terrestris niysdsktfvdlqqindpeitlanfyelmketnnkptksqliqyvnenfis-sselvnwtlsd 113 bitre-1 b. impatiens niysdsktfvdlqqindpeitlanfyelmketnnkptksqltqyvnenfva-snelvnwtlsd 113 amtre-1 a. mellifera eifpdsktfvdlhqmndpeitlsnfyslmnetgnkpsksqlaryvnenfas-snelvnwtlpd 112 aftre-1 a. florea eifpdsktfvdlhqindpeitlsnfyslmnetgnkpsksqltryvnenfas-snelvnwtlsd 113 bltre-2 b. lantschouensis wtsqpkflnkihdhdlrkfasdlnqiwkmlgrkmkddvrvnedrysiiyvpnpvivpggrfre 186 bttre-2 b. terrestris wtsqpkflnkihdhdlrkfasdlnqiwkmlgrkmkddvrvnedrysiiyvpnpvivpggrfre 186 bitre-2 b. impatiens wtsqpkflnkihdhdlrkfasdlnqiwkmlgrkmkddvrinedrysiiyvpnpvivpggrfre 186 amtre-2 a. mellifera wtfrpkflsrildddlrnfaselngiwkmlgrkmkddvrvneelysiiyvphpvivpggrfre 184 aftre-2 a. florea wtfrpkflsrildddlrnfasdlnsiwkmlgrkmkddvrvneelysiiyvpnpvivpggrfre 184 bltre-1 b. lantschouensis wtnnpsilqriqepkyyewakdlneiwkklarkvnpevarqpdrhsliyvpngliipggrfke 176 bttre-1 b. terrestris wtnnpsilqriqepkyyewakdlneiwkklarkvnpevarqpdrhsliyvpngliipggrfke 176 bitre-1 b. impatiens wtnnpsilqriqepkyyewvkdlneiwkklarkvnpevarqpdrhsliyvpngliipggrfke 176 amtre-1 a. mellifera wtespsilkrineakyrewakhlneiwkelarkinpevaeyperhsliyvdngfivpggrfke 175 aftre-1 a. florea wtenpsilkrineakyrewakhlneiwkelarkinpevaeyperhsliyvnngfivpggrfke 176 bltre-2 b. lantschouensis fyywdsywivkglllsemyttvkgmltnfvslvdkigfipnggriyyarrsqppmlipmveey 249 bttre-2 b. terrestris fyywdsywivkglllsemyttvkgmltnfvslvdkigfipnggriyyarrsqppmlipmveey 249 bitre-2 b. impatiens fyywdsywivkglllsemyttvkgmltnfvslvdkigfipnggriyyarrsqppmlipmveey 249 amtre-2 a. mellifera fyywdsywivkglllsemyttvkgmltnfvslvdkigfipnggriyytmrsqppmlipmvdey 247 aftre-2 a. florea fyywdsywivkglllsemyttvkgmlsnfvslvdkiglipnggriyyvmrsqppmlismvdey 247 bltre-1 b. lantschouensis fyywdsywvieglllsdmyqtargmidnflymvqkygfipnggriyylmrsqpplihlmvsky 239 sociobiology 68(4): e5443 (december, 2021) 9 bttre-1 b. terrestris fyywdsywvieglllsdmyqtargmidnflymvqkygfipnggriyylmrsqpplihlmvsky 239 bitre-1 b. impatiens fyywdsywvieglllsdmyqtargmidnflymvqkygfipnggriyylmrsqpplihlmvsky 239 amtre-1 a. mellifera fyywdsywvieglllsdmyqtargmidnflymvkkygfipnggriyylmrsqppllhlmvsry 238 aftre-1 a. florea fyywdsywvieglllcdmyqtargmidnflymvkkygfipnggriyylmrsqppllhlmvsry 239 bltre-2 b. lantschouensis lkvtidykclednlhllekefefwmtnrtvdvevdgvkytlarffeessgprpesykedylts 312 bttre-2 b. terrestris lkvtndytwlednlhllekefefwmtnrtvdvevdgvkytlarffeessgprpesykedylts 312 bitre-2 b. impatiens lkvtndykwlednlhllekefefwmtnrtvdvevdgvrytlarffeessgprpesykedylts 312 amtre-2 a. mellifera lkithdyewlennlyllekefdfwmtnrtveievdgvnyvlaryneqssgprpesykedylts 310 aftre-2 a. florea lktthdyewlennlyllekefdfwmtnrtveievdgvnyvmaryneessgprpesykedylts 310 bltre-1 b. lantschouensis ldftgdydylrkviptlesefafwqqkrmidvkkngrtykmghyavnstrprpesyredyeqa 302 bttre-1 b. terrestris ldftgdydylrkviptlesefafwqqkrmidvkkngrtykmghyavnstrprpesyredyeqa 302 bitre-1 b. impatiens ldftgdydylrkviptlesefafwqqkrmidvkkngrtykmghyavnstrprpesyredyeqa 302 amtre-1 a. mellifera ldftgdydylrsiistletefsfwqrekmidvekdgkiykmahyvvnstsprpesyredylma 301 aftre-1 a. florea ldftgdydylrsiistletefsfwqrekmidvekdgkiykmahymvnstsprpesyredylma 302 bltre-2 b. lantschouensis qsfrtneekdnyyaelktaaesgwdfssrwfildgtn-kgnltnlktryivpvdlnsiiyrna 374 bttre-2 b. terrestris qsfrtneekdnyyaelktaaesgwdfssrwfildgtn-kgnltnlktryivpvdlnsiiyrna 374 bitre-2 b. impatiens qsfrtneekdnyyaelktaaesgwdfssrwfildgtn-kgnltnlktryiipvdlnsiiyrna 374 amtre-2 a. mellifera qsfrtneekdnyyselktaaesgwdfssrwfildgtn-kgnltnlktryiipvdlnsiiyrna 372 aftre-2 a. florea qsfrtneekdnyyaelktaaesgwdfssrwfildgtn-kgnltnlktryiipvdlntiiyrna 372 bltre-1 b. lantschouensis qllpe-ksrdffynnikagaesgwdfsnrwciadnnnrtlsllnistqhiipvdlnailqqna 364 bttre-1 b. terrestris qllpe-ksrdffynnikagaesgwdfsnrwciadnnnrtlsllnistqhiipvdlnailqqna 364 bitre-1 b. impatiens qllpe-ksrdffynnikagaesgwdfsnrwciadnnnrtlsllnistqhiipvdlnailqqna 364 amtre-1 a. mellifera qripe-ksrdffynnikagaesgwdfsnrwfirnnnsstlslynistqyiipvdlnailqqna 363 aftre-1 a. florea qripe-ksrdxfynnikagaesgwdfsnrwfirnnnssalslynistqyiipvdlnailqqna 364 bltre-2 b. lantschouensis qlleqynqrmgnetkaayyrkraedwkravtavlwhdevgawldydllndikrdyfyptnvlp 437 bttre-2 b. terrestris qlleqynqrmgnetkaayyrkraedwkravtavlwhdevgawldydllndikrdyfyptnvlp 437 bitre-2 b. impatiens qlleqynqrmgnetkaayyrkraedwkravtavlwhdevgawldydllndikrdyfyptnvlp 437 amtre-2 a. mellifera vllaqynqrmgneskvayyqkraaewkraiqavlwhdevgawldydilndikrdyfyptnilp 435 aftre-2 a. florea mllakynqrmgneskvayyqkraaewkraitavlwheevgvwldydmlndikrdyfyptnilp 435 bltre-1 b. lantschouensis rllgefhsllgnnaksqyyhkvasqlqmaidnvlwneeegtwldydmknekprhafypsnlap 427 bttre-1 b. terrestris rllgefhsllgnnaksqyyhkvasqlqmaidnvlwneeegtwldydmknekprhafypsnlap 427 bitre-1 b. impatiens rllgefhsllgnnaksqyyhkvasqlqmaidnvlwneeegtwldydmknakprhafypsnlap 427 amtre-1 a. mellifera rllgefhtllgnnaksqyyqkiasqlqtaidnilwneadgiwldydlknqrprhmfypsnlap 426 aftre-1 a. florea rllgefhtllgnnaksqyyqkiasqlqtaidnvlwneadgiwldydmknqrprhmfypsnlap 427 bltre-2 b. lantschouensis lwtdcydiakreeyiakvlkyleknqimlnlggipttlehsgeqwdypnawpplqyfvimsln 500 bttre-2 b. terrestris lwtdcydiakreeyiakvlkyleknqimlnlggipttlehsgeqwdypnawpplqyfvimsln 500 bitre-2 b. impatiens lwtdcydiakreeyiakvlkyleknqimlnlggipttlehsgeqwdypnawpplqyfvimsln 500 amtre-2 a. mellifera lwtdcydiakreeyvskvlkyleknkimlnlggipttlehsgeqwdypnawpplqyfvimaln 498 aftre-2 a. florea lwtdcydlakreeyvskvlkyleknkimlnlggipstlehsgeqwdypnawpplqyfvimaln 498 fig 2. comparison of the amino acid sequences of tre in b. lantschouensis with those in other orthologs. (continuation) jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis10 bltre-1 b. lantschouensis lytrsynrlqrkryalsivkylktqnidtflggtptslnytgeqwdfpnawpplqsfivmgly 490 bttre-1 b. terrestris lytrsynrlqreryalsivkylktqnidtflggtptslnytgeqwdfpnawpplqsfivmgly 490 bitre-1 b. impatiens lytrsynrlqreryalsivkylktqnidtflggtptslnytgeqwdfpnawpplqsfivmgly 490 amtre-1 a. mellifera lytksynrgqreyygaatlrylksqnidnffggtptslnhtgeqwdfpnawpplqsfivmglh 489 aftre-1 a. florea lytksynrgqrehygattlrylksqnidsffggtptslnhtgeqwdfpnawpplqsfivmglh 490 bltre-2 b. lantschouensis ntgdpwaqrlayeisqrwvrsnwkafnethsmfekydatvsgghggggeyevqlgfgwsngii 563 bttre-2 b. terrestris ntgdpwaqrlayeisqrwvrsnwkafnethsmfekydatvsgghggggeyevqlgfgwsngii 563 bitre-2 b. impatiens ntgdpwaqrlayeisqrwvrsnwkafnethsmfekydatvsgghggggeyevqlgfgwsngii 563 amtre-2 a. mellifera ktedpwaqrlayeiserwvrsnykaynethsmfekydatvsgghggggeyevqlgfgwsngvi 561 aftre-2 a. florea ntedpwaqrlayeiserwvrsnykaynethsmfekydatvsgghggggeyevqlgfgwsngvi 561 bltre-1 b. lantschouensis wtgveeavnfahelafrwlgsnyagyveykemfekydsltpgksggggeydvqsgfgwangvv 553 bttre-1 b. terrestris wtgveeavnfahelafrwlgsnyagyveykemfekydsltpgksggggeydvqsgfgwtngvv 553 bitre-1 b. impatiens wtgveeavnfahelafrwlgsnyagyveykemfekydsltpgksggggeydvqsgfgwtngvv 553 amtre-1 a. mellifera wtgvreamdfahelafrwlaanyagyketgqmfekydsivpgqgggggeynvqtgfgwtngvv 552 aftre-1 a. florea wteareamdfaqelafrwlsanyagyketgqmfekydsivpgqgggggeynvqtgfgwtngvv 553 ggggey bltre-2 b. lantschouensis mdllnkygdrltaei-flaivqslappavvvs-tagqvmtgilalvislaagfigmvvykrrh 624 bttre-2 b. terrestris mdllnkygdrltaed-rfvivqslappavvvs-tagqvmtgilalvislaagfigmvvykrrh 624 bitre-2 b. impatiens mdllnkygdrltaed-rfvivqslappavvvvstagqvmtgilalvislaagfigmvvykrrh 625 amtre-2 a. mellifera mdllnrygdkltaedrfvatfhsnstpqpvvvstagqvmtgilalvislaagfig-------616 aftre-2 a. florea ldllnrygdkltaedrfvatfhsnstpqpvvvstagqvmtgilalvislaagfigmvvykrrh 624 bltre-1 b. lantschouensis leflntfpnikvkeisyindintenrq-----------------------------------580 bttre-1 b. terrestris leflntfpnikvkeisyindinteirq-----------------------------------580 bitre-1 b. impatiens leflntfpnikvkeisyindinteirq-----------------------------------580 amtre-1 a. mellifera leflntfssikvrevgyeddl-teveq-----------------------------------578 aftre-1 a. florea leflntfstikvrevgyeddl-teveq-----------------------------------579 bltre-2 b. lantschouensis yvpgpstmpnkrkvisptgnvyrkriaytelkdmnnd 662 bttre-2 b. terrestris yvpgpstmpnkrkvisptgnvyrkriaytelkdmnnd 662 bitre-2 b. impatiens yvpgpstmpnkrkvisptgnvyrkriaytelkdmnnd 663 amtre-2 a. mellifera ----------------------kmrcannaaq----626 aftre-2 a. florea yvpgpstmpnkrkvispsgnlyrkriaytelkdmnny 662 bltre-1 b. lantschouensis ------------------------------------- bttre-1 b. terrestris ------------------------------------- bitre-1 b. impatiens ------------------------------------- amtre-1 a. mellifera ------------------------------------- aftre-1 a. florea -------------------------------------   fig 2. comparison of the amino acid sequences of tre in b. lantschouensis with those in other orthologs. (continuation) sociobiology 68(4): e5443 (december, 2021) 11 48 fig 3. phylogenetic analysis of trehalase amino acid sequences from various species. b. lantschouensis (bltre-1: kj025078); b. terrestris (bttre-1: xp_003400853; bttre-2: xp_003393687); b. impatiens (bitre-1: xp_003491166; bitre-2: xp_003490073); a. florea (aftre-1: xp_003695047; aftre-2: xp_003696950); a. mellifera (amtre-1: xp_393963; amtre-2: baf81545); acyrthosiphon pisum (aptre-1: xp_001956264; aptre-2: xp_001949459); laodelphax striatella (lstre-1: afl03409; lstre-2: afl03410); a. lucorum (altre-1: agk89789; altre-2: agl34007); b. mori (bmtre-1: np_001037458; bmtre-2: np_001036910); s. frugiperda (sftre-1: abe27189; sftre-2: acf94698); l. migratoria (lmtre-1: acp28173); t. molitor (tmtre-1: ago32658); megachile rotundata (mrtre-1: xp_003705482). discussion in our study, we cloned the bltre-2 gene from b. lantschouensis using the homologous cloning and race techniques. the deduced amino acid sequence shared similarities with the tre-1 and tre-2 genes from various species, including a signal peptide leader, a glycine-rich region (ggggey), two signature motifs (pggrfrefyywdsy and qwdypnawpp), and putative n-glycosylation sites (santos et al., 2012). functions of the most conservative residues and regions remain unknown. additionally, only one transmembrane domain has been found in most insects, including b. mori (mitsumasu et al., 2005), nasonia vitripennis (tang et al., 2012), n. lugens (gu et al., 2009), o. fuscidentalis (tatun et al., 2008), s. exigua (chen et al., 2010), t. castaneum (tang et al., 2012), l. migratoria (liu et al., 2016), and c. medinalis (tian et al., 2016). however, the bltre-2 gene contained two putative transmembrane domains, mllsaaflallvvapcyas and qvmtgilalvislaagfigmvvy (fig 1), which are like those of a. mellifera (lee et al., 2007), laodelphax striatellus (zhang et al., 2010), spodoptera frugiperda (silva et al., 2009), jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis12 and aedes aegypti (tang et al., 2012). however, previous research suggested that bltre-1 gene have no transmembrane domain (qin et al., 2015). in this study, although the proteins encoded by bltre-2 gene showed obvious similarity to b. terrestris (98.9%), b. impatiens (97.5%), and a. mellifera (84.2%), the protein encoded by bltre-2 gene only showed 55.7% similarity to those encoded by bltre-1 gene (table 3). in insects, tre-1 and tre-2 are involved in many physiological processes (mitsumasu et al., 2005; shi et al., 2019). su et al. (1994) indicated that tre-2 can help transport sugars into oocytes of b. mori. furthermore, it has been shown that trehalose, glycogen, and glucose can be stored in growing oocytes during the period of vitellogenesis of rhodnius prolixus, (santos et al., 2012). similarly, in b. tabaci and r. prolixus, the expression levels of tre-2 were much higher in the ovary than in other tissues (santos et al., 2012; wang et al., 2014). in our study, the bltre-2 gene had the highest expression levels in the ovary and then in the midgut of b. lantschouensis (fig 4a). these findings suggest that the bltre-2 gene may play an important role in providing materials and energy for oocyte development in b. lantschouensis. the tre-2 gene is distributed differently in different tissues in insects, and this may be linked to its function. previous studies have shown that the tre-2 gene has higher expression in the integument of spodoptera litura (zou et al., 2013), the wing bud of n. lugens (zhang et al., 2017), and the gut of s. exigua (tang et al., 2008) and bactrocera dorsalis (xie et al., 2013) as compared with other tissues. fig 4. the relative expression levels of bltre-2 in different tissues (a) and chronological ages (b) of b. lantschouensis. an: antennae; he: head; mu: muscles; le: legs; wi: wings; in: integument; mg: midgut; mt: malpighian tubules; fb: fat body; ov: ovary; la: larva; pu: pupa; d0-d30: day 0 to day 30 worker. each value represents mean ± s.d., and different letters above bars indicate a significant difference (p-0.05). fig 5. the relative expression levels of two trehalase genes under adverse conditions. the bltre-1 and bltre-2 relative expression in workers which were exposed to 45°c ambient temperature for 0, 1, 2, 3, 4 and 5 h (a, b). the bltre-1 and bltre-2 relative expression in workers which were starved for 0, 4, 8, 12, 16, 20 and 24 h (c, d). each value represents the mean ± s.d., and different letters above the bars indicate significant differences (p-0.05). sociobiology 68(4): e5443 (december, 2021) 13 in b. lantschouensis, the bltre-2 gene may be involved in providing energy to the chitin synthesis process in the midgut or in supporting peristaltic movement of the midgut. these results suggest that the bltre-2 gene may perform specific functions in different tissues. our study found, for the first time, that bltre-2 has the highest expression level in 30-day-old worker bees (fig 4b), so we can assume that this expression level of bltre-2 is associated with the biological behavior of b. lantschouensis. older workers may depend mainly on bltre-2 to break down extracellular trehalose (mainly from food). next, the expression of bltre-2 was significantly higher in the larvae than in the pupae. it is possible that bltre-2 expression is involved in the feeding and development of larvae. in b. dorsalis, tre-2 was found to be highly expressed in metabolic tissues at both the adult and larval stages (xie et al., 2013). the results also revealed that expression of bltre-2 in 0to 20-day-old workers first increased and then declined with age (fig 4b). this result may be due to the social division of labor in b. lantschouensis colonies. both younger and older workers are engaged in various activities, such as helping the queen secrete wax, nesting, and nursing, and strong workers take part in foraging and guarding. previous studies suggested that insect trehalase, including tre-1 and tre-2 play critical roles in energy supply, growth, metamorphosis, stress recovery, chitin synthesis, and flight by catalyzing the hydrolysis of trehalose to glucose in insects (wyatt, 1967; thompson, 2003; shukla et al., 2014). insects adapt to changes in environmental temperature and maintain their energy by regulating their own body temperature. a high temperature not only breaks the moisture balance of the internal environment and interferes with normal metabolism but it also causes body temperature to rise and affects enzyme activity and protein function in insects (du et al., 2007). previous studies showed that trehalase optimizes temperature in the range of 40–65°c (zou et al., 2013; shukla et al., 2014; youngjin & yonggyun, 2017). at a high temperature, the parasitic nematode aphelenchoides besseyi improves its resistance by upregulating the trehalase gene (chen et al., 2016). in our study, the expression levels of bltre-1 and bltre-2 were, respectively, higher and lower than at 0 h under the 45°c treatment conditions. we hold the opinion that the activity of soluble trehalase was high at 45°c because trehalose was gradually hydrolyzed into glucose and used for stress recovery. bumblebees maintain vital activities by accumulating trehalose through soluble trehalase catalysis in such high-temperature conditions. by comparison, the expression level of bltre-2 was lower than that of bltre-1 for the 45°c treatment. this result suggests that bltre-1 may be involved in providing energy for physiological activity and that bltre-2 expression maybe constrained at a high temperature. trehalose is a feedback-regulating substance involved in the feeding behavior and nutrient intake of insects (wyatt, 1967). trehalose provides energy when insects are starving; thus, it has a role in the regulation of insect functions under starvation conditions (tang et al., 2014; youngjin & yonggyun, 2017). in h. axyridis adults, the stored food reserves can provide energy to sustain vital activities for 8 h, but energy limitations have a direct impact on the desire to find food (tang et al., 2014). our results showed no significant difference in the expression levels of both bltre-1 and bltre-2 in b. lantschouensis adults starved for 4 to 12 h as compared with those starved for 0 h, which may be because the stored food reserves were able to provide energy to sustain vital activities for 12 h in adults. in addition, the expression levels of bltre-1 and bltre-2 increased in adults starved for 16 to 20 h, which suggests that trehalose stores were degraded by trehalase. bltre-1 and bltre-2 function to facilitate the uptake and utilization of trehalose from blood and food, respectively (yaginuma et al., 1996). result of the present study show that bltre-1 is a key gene involved in regulating energy metabolism and providing glucose at a high temperature. bltre-1 and bltre-2 might balance trehalose and provide energy during periods of starvation. our study sheds light on the molecular function and gene expression of trehalase in b. lantschouensis, which adds further important information about the characteristics of this gene in the physiology and development of bumble bees in china. different native populations in different regions of china may display different types of adaptations to coldness, which is associated with trehalase expression. we provide new knowledge to assist future selection and breeding of b. lantschouensis in china. furthermore, the tre-1 and tre-2 genes participate in energy metabolism during developmental and physiological activities in various insects, which provides a reference for the protection and utilization of pollination insects. acknowledgments this work was supported by the national natural science foundation of china (31201858), the central public interest scientific institution basal research fund (y2018pt66 and y2021xk16), and key research and development project of jiangxi province (20192bbh80003). h.l.-b. is supported by the u.s. department of agriculture – national institute of food and agriculture (usda nifa award # ni181445xxxxg007). authors contribution jmq: conceptualization, investigation, validation, methodology, writing. fl: investigation, methodology. jw: supervision, project administration. syh: supervision. mi: review, resources. wl: resources. hml: writing. sdl: conceptualization, funding acquisition, supervision, project administration, writing. jiamin qin et al. – molecular characterization and gene expression of trehalase bombus lantschouensis14 references ai, d., cheng, s. h., chang, h. t., yang, t., wang, g. r. & yu, c. h. 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(2013). cdna cloning and characterization of two trehalases from spodoptera litura (lepidoptera: noctuidade). genetics and molecular research, 12: 901-915. doi: 10.4238/2013. april.2.7. ole_link4 ole_link1 ole_link25 ole_link28 ole_link12 ole_link13 ole_link11 _hlk33969697 _hlk33969716 ole_link3 ole_link2 ole_link16 ole_link17 _hlk33969816 ole_link8 ole_link7 _hlk33969624 ole_link23 ole_link21 ole_link22 ole_link29 ole_link30 ole_link70 ole_link71 _hlk64285825 ole_link45 ole_link95 ole_link96 ole_link61 ole_link63 ole_link64 ole_link65 ole_link44 ole_link47 ole_link46 _hlk64906452 71 effects of honeydew of phenacoccus solenopsis on foliar foraging by solenopsis invcta (hymenoptera: formicidae) by ai-ming zhou, yong-yue lu, ling zeng, yi-juan xu* & guang-wen liang* abstract the olfactory response of fire ants to plant leaves, mealybugs and the honeydew excreted by mealybugs was tested with a y-tube olfactometer. the foraging activities of fire ants on three plants were also measured. our results showed that plant leaves and mealybugs alone had no significant attraction to the fire ant workers, while fire ants could be obviously attracted by honeydew. the selection rate of fire ants on honeydew of hibiscus rosa-sinensis, cotton (gossypium spp.) and tomato (solanum lycopersicum) was 60.22%, 57.45% and 64.29% respectively. when mealybugs were present on plants, fire ant workers foraged more frequently on the plants than controls (p<0.05). as to different plants, fire ants preferred foraging on tomato (66.3 per plant) to hibiscus rosa-sinensis (50.4 per plant) and cotton (45.1 per plant). however, there was no significant difference in foraging frequency of fire ants on the three kinds of plant, with 24.9, 22.9 and 32.3 ants foraging per five minutes respectively. key words: solenopsis invicta, phenacoccus solenopsis, honeydew, preference, foraging activity. introduction the excretions of homopterans, called honeydew, are rich in hydrocarbons, amino acids, protein and water (way 1963, fischer et al. 2002), and held a special attraction to ant species (way 1963, buckley 1987). ants attend honey-producing homopteran insects by reducing not only the predation and parasitism by natural enemies but also the risk of fungal infection. in return, the ants obtain excreted honeydew from homopteran insects as food (way 1954, banks & macaulay 1967, tilles & wood 1982, yao et al. 2000, standler & dixon 1998). red imported fire ant research center, south china agricultural university, guangzhou 510642, china *corresponding authors. email: xuyijuan@yahoo.com, gwliang@scau.edu.cn 72 sociobiolog y vol. 59, no. 1, 2012 fire ants have invaded a variety of ecosystems and habitats (vinson 1997, holway et al. 2002), and established a variety of mutual-beneficial relations with honey-producing homopteran insects, such as aphids and scale insects which excrete honeydew to attract fire ants to forage, while the homopterans benefit by having the fire ants ward off potential predators (eubanks 2001, eubanks et al. 2002, harvey & eubanks 2004, kaplan & eubanks 2005). our preliminary results of field investigations show that the honeydew produced by phenacoccus solenopsis, a new invasive pest in south china (wu & zhang, 2009) has a strong attraction for the red imported fire ant, solenopsis invcita. but the mutual relationship between the fire ant and mealybug remains unknown. this study tested the effects of honeydew of p. solenopsis produced in three plants on foliar foraging by s. invcta indoors, which would be helpful for understanding the relationship between these two invasive species. materials and methods insects s. invcta colonies and p. solenopsis were collected from the suburb of guangzhou and maintained in the laboratory for bioassays. the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor l.). a test tube (25 mm×200 mm), which was filled partially with water and plugged with cotton, was used as a water source. p. solenopsis was fed on h. rosa-sinensis. ants and mealybugs were maintained in the laboratory at 25 ± 2°c. plants we grew cotton and tomato seedlings whose seeds were purchased from the local market and p. solenopsis seedlings were purchased directly from a nursery. all seedlings of the three plants were used for bioassay at the height of 55-60cm. olfactometer the olfactometer mainly consisted of the following accessories which were connected with the odorless silicone tube: circulating water pumps, y-tube tube boom, distilled water bottles, and odor source bottles. the length and diameter of the main and side arms were 19cm and 2.5cm respectively, with 73 zhou, a-i. et al — effects of honeydew on fire ant foraging 75° as the angle of two sides. gauze was fixed in the side arms to block the path to the odor bottles (fig. 1). bioassay olfactory response test the tested component was put into one of the odor source bottles, and distilled water was put into the other one as a control. 100 fire ant workers were induced to the main arm, and the number of workers which went through one or the other of the side arms and stayed for 30 seconds (regarded as reaction to the odor) were recorded for ten minutes after the beginning of the test. 10 replicates were used for each treatment. 95% ethanol was used to clean the olfactometer after each replicate, then it was washed with distilled water, and dried. the two side arms were exchanged in turn for treatments and control. the odor sources tested in the experiments were as follows: (1) five mealybugs, one of each instar; (2) one leaf (approximately 38 cm2) of each plant species with or without (3) honeydew of p. solenopsis. to collect the honeydew, we induced five 3rd instar mealybugs on the leaves in a petri dish, and we wrapped the petiole with moist cotton. mealybugs were removed after 24h and the leaves with honeydew were used for the olfactory response test. selection rate (%) was considered to be = (number of ants selecting the treatment odor / total number of ants in in control and treatment recordings) × 100% fig. 1 sketch map of y-tube olfactometer 74 sociobiolog y vol. 59, no. 1, 2012 foraging activity of s. invcta plant seedlings were cultivated in plastic pots of uniform size (diameter of upper bottom and lower bottom were 17cm and 12cm respectively, with the height as 14cm). the ektexine was painted with fluon to prevent ants from climbing up to plants from the outer surface of pots. 30 3rd instar mealybugs were induced to the seedling leaves, and a certain amount of vaseline was deposited on the base of the plants to prevent escape by the mealybugs. we placed the pot and ant’s artificial nest in a big plastic box (50cm×40cm×16cm) after the mealybugs colonized on the plants for 24h. one gram (about 950-1000 individuals) workers and one queen were placed in a plastic box (19cm×12cm×8cm) as an artificial nest, with water supplied everyday. when the bioassay began, the artificial nest was placed in the big box, and a plastic hose was used to build a bridge between the ants' nest and plant seedling for the foraging of workers. 24 hours later, we counted the number of the ants foraging on the seedlings. meanwhile, we recorded the number of ants passing the “foraging bridge” per five minutes. ten replicates were conducted. statistical analysis t-test for paired data was used to compare the selection rate of workers between the treatment and control. variations of foraging activity of workers among three plants were analyzed using analysis of variance. all the statistical analyses were conducted using the spss13.0 software package. results olfactory response of s. invicta compared with control, we found that all of the leaves of three plants tested had no obvious attraction on the fire ant workers (p>0.05). the results in fig. 2 indicated that the selection rate in both of the treatment and control for the same plant were nearly 50%. compared with controls, all nymphs and adults of p. solenopsis had no significant attraction for the fire ant workers (p>0.05). the results in fig. 3 indicated that the selection rate in both of the treatment and control in the same plant were nearly 50%. however, the honeydew excreted by the mealybugs attracted the fire ants significantly (p<0.05), while no difference showed in treatments among the three plant species. the selection rate of workers on the treatment and control 75 zhou, a-i. et al — effects of honeydew on fire ant foraging fig. 2 selectivity of fire ants to three plant species leaves (ns on bars indicates no significant difference between the treatment and control in the same plant) fig. 3 selectivity of fire ants to mealybugs (ns on bars indicates no significant difference between the treatment and control in the same stage of p. solenopsis) 76 sociobiolog y vol. 59, no. 1, 2012 in h. rosa-sinensis, g. spp., and s. lycopersicum were 60.22% and 39.78%, 57.45% and 42.55%, and 64.29%and 35.71% respectively (fig. 4). foraging activity of s. invcta foliar foraging numbers of fire ants increased more than 2-fold when mealbugs were present compared with control (fig. 5; p>0.05). numbers of foraging workers on the tomato plants was significantly more than on cotton and hibiscus when p. solenopsis was present. the number of foraging workers in the treatment and control on h. rosa-sinensis, g. spp., s. lycopersicum were 50.4/seedling and 16.6/seedling, 45.1/seedling and 12.5/seedling, and 66.3/ seedling and 14.4/seedling respectively (fig. 5). there was a significant difference in foraging frequency of fire ants when mealybugs were present compared to when they were absent (fig. 6; p>0.05), while there was no difference among the three plant species treatments. the number of foraging workers in the treatment and control on h. rosa-sinensis, g. spp., s. lycopersicum were 24.9 and 8.6 per five minutes, and 22.9 and 10.1/ seedling, per five minutes, 32.3/seedling and 9.7 per five minutes respectively (fig. 6). fig. 4 selectivity of fire ants to honeydew in three plants (* on bars indicates significant difference between the treatment and control in the same plant) 77 zhou, a-i. et al — effects of honeydew on fire ant foraging discussion we conclude that what attracted the fire ants was the honeydew excreted by p. solenopsis but not the mealybugs themselves. honeydew also facilitated foliar foraging by s.invcta, while the foraging preference on tomatoes indicated fig. 5 effects of mealybugs on quantity of foraging ants on plants (* on bars indicates significant difference between the treatment and control in the same plant) fig. 6 effects of mealybugs on foraging frequency of foraging ants (* on bars indicates significant difference between the treatment and control in the same plant) 78 sociobiolog y vol. 59, no. 1, 2012 that the preference of p. solenopsis for this species might affect the relationship between the fire ant and mealybug (p. solenopsis preferred to feed on tomato and excreted more honeydew, which supplied more food for the fire ants). our results corroborate precious reports that honeydew could stimulate the foraging activity of fire ants and more workers were attracted to cottons with higher density of aphids (kaplan & eubanks 2005). homopteran honeydew contains a variety of carbohydrates such as monosaccharides, disaccharides and polysaccharides. the polysaccharides can include pine trisaccharide, raffinose, and melezitose which may have some special activity on invasive ants (völkl et al. 1999). the chemical difference among the honeydew produced on different plants may also explain the fact that fire ants foraged more on tomato than the other two plants. therefore, further studies on honeydew and nectar composition will contribute to a clearer understanding of the interactions between fire ants and p. solenopsis. acknowledgments this study was supported by the national natural science foundation of china (no. 31101498). references banks, c.j. & e.d.m. macaulay 1967. effects of aphis fabae scop. and of its attendant ants and insect predators on yields of field beans (vicia faba l.). ann. appl. biol. 60:445–453. buckley, r.c. 1987. interactions involving plants, homoptera, and ants. annu. rev. ecol. syst. 18: 111–135 eubanks, m.d. 2001. estimates of the direct and indirect effects of red imported fire ants on biological control. biological control 21:35–43. eubanks, m.d., s.a. blackwell, c.j. parrish, z.d. delamar & h. hull-sanders 2002. intraguild predation of beneficial arthropods in cotton by red imported fire ants. environmental entomolog y 31:1168–1174. fischer, m.k., w.völkl, r . schopf & k.h. hoffmann 2002.age-specific patterns in honeydew production and honeydew composition in the aphid metopeurum fuscoviride: implications for ant-attendance. j. insect physiol., 48 (3):319—326. harvey, c.t. & m.d. eubanks 2004. effect of habitat complexity on biological control by the red imported fire ant (hymenoptera: formicidae) in collards. biological control 29:348–358. holway, d.a., l. lach, a.v. suarez, n.d. tsutsui & t.j. case 2002. the causes and consequences of ant invasions. annual review of ecolog y and systematics 33:181–233. 79 zhou, a-i. et al — effects of honeydew on fire ant foraging kaplan, i. & m.d. eubanks 2005. aphids alter the community-wide impact of fire ants. ecolog y 86: 1640–1649. stadler, b. & a.f.g. dixon 1998. costs of ant attendance for aphids. j. anim. ecol 67:454–459 tilles, d.a. & d.l. wood 1982. the influence of carpenter ant (camponotus modec) (hymenoptera: formicidae) attendance on the development and survival of aphids (cinara spp.) (homoptera: aphididae) in a giant sequoia forest. can. entomol 114:1133–1142 völkl, w., j. woodring, m. fischer, m.w. lorenz & k.h. hoffmann 1999. ant-aphid mutualisms: the impact of honeydew production and honeydew sugar composition on ant preferences. oecologia 118: 483-491 vison, s.b. 1997. invasion of the red imported fire ant (hymenoptera:formicidae) spread ,biolog y and impact. american entomologist 43(1):23-29 way, m.j. 1954. studies on the association of the ant oecophylla longinoda (latr.)(formicidae) with the scale insect saissetia zanzibarensis williams (coccidae). bull. entomol. res. 45:113–134 way, m.j. 1963. mutualism between ants and honeydew producing homoptera. annu. rev. entomol. 8:307–344. wu s.a. & r.z. zhang 2009. a new invasive pest, phenacoccus solenopsis, threatening seriously to cotton production. chinese bulletin of entomolog y 46: (1)159-162 yao, i., h. shibao & s. akimoto 2000. costs and benefits of ant attendance to the drepanosiphid aphid tuberculatus quercicola. oikos 89:3–10 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.6166sociobiology 68(2): e6166 (june, 2021) ants are among the most abundant groups of terrestrial invertebrates. ants have a wide variety of nesting sites, feeding habits, and trophic interactions (kaspari, 2000), and are the subject of basic and applied research. however, the biology of most species remains unknown, urging descriptive studies (krell, 2004; greene, 2005). dinoponera lucida emery, 1901 is a poneromorph ant endemic to the atlantic forest (peixoto et al., 2010; simon et al., 2020), endangered (en) according to the brazilian red list (mma, 2014; icmbio, 2018). dinoponera lucida is a forest-specialist solitary forager with no recruitment, a typical abstract ants present a wide variety of nesting sites, feeding habits, and trophic interactions, but the biology of most species remains unknown. dinoponera lucida is a poneromorph ant forest-specialist and solitary forager, endemic to the brazil’s atlantic forest. herein we describe foraging activities, guard and maintenance of the nests, orientation mode, and intraspecific interactions performed by d. lucida. we found three nests distant from each other at least 8.5 m, and the mean reached distance by a worker was 3.8 m. the workers showed colony fidelity and random forage in their territory. we observed two non-agonistic interactions between workers from the same nest, and two agonistic interactions between foraging workers from different nests. the low frequency of agonistic interactions suggests that workers from different nests are unlikely to forage in the same area. our results expand the knowledge on ants’ natural history through data on foraging activities, guard and maintenance of the nests, orientation mode and intraspecific interactions. sociobiology an international journal on social insects cássio zocca1,2,3, flávio curbani3,4,5, rodrigo b ferreira1,2,6, cecília waichert3,6, tathiana g sobrinho6,7, ana c srbek-araujo3,8,9 article history edited by evandro n. silva, uefs, brazil received 04 january 2021 initial acceptance 17 april 2021 final acceptance 21 april 2021 publication date 14 june 2021 keywords foraging behavior, orientation sense, guard, maintenance, intraspecific interactions. corresponding author cássio zocca instituto nacional da mata atlântica av. josé ruschi, 04, cep 29650-000 santa teresa, espírito santo, brasil. e-mail: zoccabio@hotmail.com behavior of dinoponera species (planqué et al., 2010; araujo et al., 2015; curbani et al., 2021). few ecological studies are available (see peixoto et al., 2008, 2010), but, as a threatened species, nesting and foraging data are crucial for conservation plans. herein, we add further information on intraspecific nonagonistic and interspecific agonistic interactions, guarding, and maintenance of the nests, foraging activities and orientation mode performed by d. lucida. we carried out observations from september 29th to october 2nd, 2017, in the reserva natural vale (22.711 ha), municipality of linhares, northern of espírito santo 1 instituto nacional da mata atlântica, santa teresa-es, brazil 2 projeto bromeligenous, instituto marcos daniel, vitória-es, brazil 3 programa de pós-graduação em ecologia de ecossistemas, universidade vila velha, vila velha-es, brazil 4 departamento de tecnologia industrial, universidade federal do espírito santo, vitória-es, brazil 5 projeto caiman, instituto marcos daniel, vitória-es, brazil 6 programa de pós-graduação em biologia animal, universidade federal do espírito santo, vitória-es, brazil 7 departamento de ciências agrárias e biológicas, universidade federal do espírito santo, são mateus-es, brazil 8 programa de pós-graduação em ciência animal, universidade vila velha, vila velha-es, brazil 9 instituto serradical de pesquisa e conservação, belo horizonte-mg, brazil short note a day in the life of the giant ant dinoponera lucida emery, 1901 (hymenoptera, formicidae): records of activities and intraspecific interactions id mailto:zoccabio@hotmail.com https://orcid.org/0000-0001-6457-1505 cássio zocca, flávio curbani, rodrigo b ferreira, cecília waichert, tathiana g sobrinho, ana c srbek-araujo – natural history of dinoponera lucida2 state, southeastern of brazil. we observed d. lucida workers in foraging activity for 48 hours (cumulative sampling), continuously from 6 am to 6 pm for four days, from three distinct nests in a 30 m x 30 m plot in a lowland coastal forest (19º 09’ 14.5” s, 40º 04’ 14.0” w). the solitary workers of d. lucida foraged for food around the nests, covering an estimated area of 11 m2 (based on mean radius reached by workers). workers do not appear to have specific pattern displacement or preference for any area. they randomly forage in their territory making a sinuous route while touching the leaf litter with antennae. we did not observe workers entering in nests other than their own nests, nor aggressive interactions between nestmates. dinoponera lucida had colony fidelity throughout sampling. workers returned straight to their original nests after capturing prey. dinoponera gigantea (perty, 1833) had similar returning behavior (fourcassié & oliveira, 2002). it is likely that d. lucida has returning strategy that uses directional fidelity within a home range, such as a sense of visual orientation. in our field interventions, we removed the leaf litter excluding chemical tracks and visual cues, as proposed by fresneau (1985) for neoponera apicalis (latreille, 1802). however, this removal did not change the course of d. lucida workers. this may indicate that d. lucida uses fixed visual cues in the environment that go beyond the leaf litter landmarks, such as trunks, branches, roots and shrubs, being important to define its stereotyped routes. in the dinoponera genus the use of visual cues has already been reported to d. gigantea (fourcassié et al., 1999) and d. quadriceps (azevedo et al., 2014). it is known that solitary forager ants learn individual stereotyped routes to increase their navigation efficiency (wystrach et al., 2011b), to obtain a food source (fresneau, 1985) or to return to the nest (wystrach et al., 2011a). forest ants, as d. lucida workers in foraging activity can have highly stereotyped routes between the nest and the feeding locations (niven, 2007). this orientation strategy was also reported for d. gigantea (fourcassié & oliveira, 2002) and d. quadriceps (azevedo et al., 2014). dinoponera lucida displayed permanent guard and maintenance of their nest. we observed sentinel workers at the nest’s openings demonstrating guarding behavior (fig 1a). permanent nest guarding and maintenance were previously reported for d. lucida (peixoto et al., 2010) and d. quadriceps (medeiros et al., 2016). probably, guarding activity inhibits non-nestmates intruders to access the nest. in addition, the workers also performed nest opening maintenance, such as removal of leaves, sticks, soil pellets and fallen plant fragments. the maintenance of nests was more evident on the third sampling day, with an increase in the number of workers at nest openings after rain (fig 1b). this behavior seems to be an immediate response of the workers of d. lucida to the environmental changes that could lead to risk-altering nest’s functions. we observed two non-agonistic interactions between workers from the same nest. in the first interaction, two workers displayed rapid antennation, moving away and continued solitary foraging. in the second interaction, workers cooperated in the transport of a seed to the nest. the first worker carrying a seed (swartzia myrtifolia var. elegans; fig 2a), dropped it when founded another worker, and returned to forage. immediately, the second worker took the seed and returned to the nest (fig 2b). in addition, we observed that the second worker foraged the leaf litter unlikely to follow a predefined route to find the nestmate. generally, poneromorph ants do not cooperate during foraging (fourcassié & oliveira, 2002; araújo & rodrigues, 2006), but cooperation in d. lucida should not be a rare behavior. labor division, and thus the observed cooperation between workers in the species, seem to follow age polyethism (individual age) (peixoto et al., 2008). we observed two agonistic interactions, both between foraging workers from nests 1 and 2 that occurred about 4 m from the openings of their nests (fig 3). the interactions lasted 1 to 5 minutes. on both occasions, we observed the movements and behavior typical of agonistic interactions described for d. lucida and congeners, such as antennal fig 1. dinoponera lucida nest opening at reserva natural vale, state of espírito santo, brazil. (a) guard activity by a worker-sentinel, (b) workers in nest maintenance after raining. sociobiology 68(2): e6166 (june, 2021) 3 boxing, gaster bending, bite in the legs and attempts to sting the opponent. at the end of the agonistic interactions, the workers moved away without any apparent damage and returned to forage. agonistic encounters observed herein for d. lucida were similar to other dinoponera species (fourcassié & oliveira, 2002; peixoto et al., 2008). peixoto et al. (2010) observed that the maximum distance reached by d. lucida workers in foraging activity was inversely related to the density of nests in the area, which indicates a strategy to minimize agonistic interactions. we found nests distant from each other at least 8.5 m (the distance between nests 1 and 2) and the mean reached distance by a worker was 3.8 ± 0.4 m (mean ± standard error). the low frequency of agonistic encounters may be due to the fact that workers from different nests usually do not forage in the same area. our results expand knowledge about natural history of d. lucida through data on foraging activities, guarding and maintenance of the nests, orientation mode and intraspecific interactions. however, descriptive data on dinoponera species are still scarce. in addition, we suggest that long-term monitoring is feasible to assess natural history aspects of d. lucida, improving knowledge about the lifestyle of poneromorph ants. acknowledgements we are grateful to josé simplício dos santos for the assistance during sampling. we thank reserva natural vale for the support to field activities. authors’ contributions cz: conceptualization, methodology, investigation, and writing fc: conceptualization, methodology, investigation, and writing rbf: conceptualization, methodology, and writing tgs: writing cw: writing acsa: conceptualization, methodology, and writing references araujo, a., medeiros, j.c., azevedo, d.l.o., medeiros, i.a., neto, w.a.s. & garcia, d. (2015). poneromorfas sem rainhas – dinoponera: aspectos ecológico-comportamentais. in: as formigas poneromorfas do brasil (eds. delabie, j.h.c., feitosa, r.m., serrão, j.e., mariano, c.s.f. & majer, j.d.). editus, ilhéus, pp. 237-246. araújo, a. & rodrigues, z. (2006). foraging behavior of the queenless ant dinoponera quadriceps santschi (hymenoptera: formicidae). neotropical entomology, 35: 159-164. doi: 10.1590/ s1519-566x2006000200002 azevedo, d.l.o., medeiros, j.c. & araújo, a. (2014). adjustments in the time, distance and direction of foraging in dinoponera quadriceps workers. journal of insect behavior, 27: 177-191. doi: 10.1007/s10905-013-9412-6 curbani, f., zocca, c., ferreira, r.b., waichert, c., sobrinho, t.g. & srbek-araujo, a.c. (2021). litter surface temperature: a driving factor affecting foraging activity in dinoponera lucida (hymenoptera: formicidae). sociobiology, 68: e6030. doi: 10.13102/sociobiology.v68i1.6030 fig 2. dinoponera lucida workers at the reserva natural vale, state of espírito santo, brazil. (a) seed of swartzia myrtifolia var elegans transported by a worker, (b) foraging workers from the same nest in cooperative interaction to transport the seed. fig 3. agonistic interaction between two dinoponera lucida workers from different nests at the reserva natural vale, state of espírito santo, brazil. cássio zocca, flávio curbani, rodrigo b ferreira, cecília waichert, tathiana g sobrinho, ana c srbek-araujo – natural history of dinoponera lucida4 fourcassié, v., henriques, a. & fontella, c. (1999). route fidelity and spatial orientation in the ant dinoponera gigantea (hymenoptera, formicidae) in a primary forest: a preliminary study. sociobiology, 34: 505-524. fourcassié, v. & oliveira, p.s. 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(2010). basic ecological information about the threatened ant dinoponera lucida emery (hymenoptera: formicidae: ponerinae), aiming its effective long-term conservation. in: species diversity and extinction (ed. tepper, g.h.). nova science publishers, inc., new york., pp. 183-213. peixoto, a.v., campiolo, s., lemes, t.n., delabie, j.h.c. & hora, r.r. (2008). comportamento e estrutura reprodutiva da formiga dinoponera lucida emery (hymenoptera, formicidae). revista brasileira de entomologia, 52: 88-94. doi: 10.1590/ s0085-56262008000100016 planqué, r., van den berg, j.b. & franks, n.r. (2010). recruitment strategies and colony size in ants. plos one, 5: e11664. doi: 10.1371/journal.pone.0011664 simon, s.s., schoereder, j.h. & teixeira, m.c. 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(2011b). views, landmarks, and routes: how do desert ants negotiate an obstacle course? journal of comparative physiology a: neuroethology, sensory, neural, and behavioral physiology, 197: 167-179. doi: 10.1007/s00359-010-0597-2 _hlk69755599 235 nest structure of the neotropical social wasp mischocyttarus baconi (hymenoptera: vespidae) by andrea a. scobie1 & christopher k. starr2 abstract the uncovered, single-comb nests of mischocyttarus baconi starr have short, stout, centric petioles. we describe nest size, cell size, petiole dimensions, and the position of the petiole. in a sample of 91 nests, the comb comprised up to 198 cells. cells had a mean side-to-side diameter at the mouth of 3.4 mm. the petiole had a mean length of 2.9 and a modal width of 2.0 mm. the degree of excentricity was determined for a sample of nests of at least 20 cells. as expected, those attached to vertical substrates (wall nests) showed greater excentricity than those attached to horizontal substrates (ceiling nests). introduction mischocyttarus is a genus of about 250 known species of independentfounding social wasps restricted to the new world (silveira 2008). in common with other independent-founding polistines, mischocyttarus nests typically each consist of a single uncovered comb of cells (gadagkar 1991). mischocyttarus baconi starr is found throughout the island of trinidad, west indies, where it often nests in and on buildings. the wasps very closely resemble the sympatric m. alfkenii (ducke), but the two build distinct nests (o’connor et al 2011). that of m. baconi is made of darker carton with the petiole attached at or near the middle of the comb (centric), while that of m. alfkenii is of lighter carton with the petiole attached at one edge of the comb (excentric). silveira (2008) places m. alfkenii (and by implication m. baconi) in a species group with seven other species within the subgenus phi. richards’s (1978) brief notes on nest form indicate that centric-petiole and excentric-petiole nests are each typical of several species in this species-group. dep’t of life sciences, univ. of the west indies, st augustine, trinidad & tobago 1present address: dep’t of biolog y, univ. of the southern caribbean, maracas st joseph, trinidad & tobago; an8scobie@yahoo.com 2 ckstarr@gmail.com 236 sociobiolog y vol. 59, no. 1, 2012 materials and methods our core study locality was the university of the west indies campus at st augustine, trinidad & tobago (10º39’n 61º24’w). in order to estimate the frequency distribution of final nest size, we collected all abandoned m. baconi nests from under concrete eaves of a large building at the core locality. our descriptive notes from these are supplemented by 28 active nests and from abandoned nests from other sites nearby. most nests were attached to either a horizontal (henceforth “ceiling”) or a vertical (henceforth “wall”) surface. before removal, we marked the tops of wall nests. fig. 1. map of nest z to illustrate calculation of mean side-to-side cell diameter and degree of excentricity. in this example, mean cell diameter is the summed length of the three lines (from edge to edge of the nest) divided by 23. intersecting diameter lines show the center, while • marks the position of the petiole. excentricity is the ratio of the distance (in cell diameters) between the center and the petiole to the distance between the center and the edge cell. where the petiole is not on a midline cell, we place it on the nearest cell wall or cell center along a midline in order to calculate excentricity. in nest z, excentricity = 2.5/4 = 0.625. 237 scobie, a.a. & c.k. starr — nest structure of mischocyttarus baconi in a sample of eight nests, we measured the width of the comb along three midlines (fig. 1). these figures were then used to calculate mean side-to-side cell diameter. we determined the degree of excentricity of all larger (≥ 20 cells) ceiling and wall nests according to two indices, in each of which a nest with the petiole attached to a peripheral cell has maximum excentricity: a) that of karsai & pénzes (1996), and b) a simpler alternative (fig. 1). results and discussion m. baconi builds a nest of medium-brown carton. the comb is usually very roughly circular and lies approximately parallel to the substrate, where this is flat. fig. 2 shows the array of nest sizes in our sample of 91 abandoned nests from the core locality. these had a range of 1-198 cells, with a median of 12 and mean of 26.5. the overall pattern, then, is of a large fraction of very small nests (most of which presumably failed without producing adult brood) and a smaller fraction of relatively large, successful nests. in measuring cell widths of eight nests, we found that one nest had unusually narrow cells. the mean cell width of the other seven nests was 3.4 mm. the petiole had a modal length of 3 mm (table 1). petiole width was variable, and often irregular within individual petioles. estimates to the nearest half-millimeter gave a modal width of 2 mm. fig. 2. size (number of cells) of 91 mischocyttarus baconi nests in descending order. 238 sociobiolog y vol. 59, no. 1, 2012 of 95 nests under concrete eaves at the core locality, 29 (31%) were attached to ceilings and 66 (69%) to walls, although both types of surface were abundantly available, and each nest had room to expand on all sides. the degree of excentricity, according to the index shown in fig. 1, of the 29 larger nests is shown in table 1. the data show substantially greater excentricity in wall nests. furthermore, fully centric nests (excenticity = 0) were found only on ceilings, and fully excentric nests (excentricity = 1) were found only on walls. in particular, combs from both types of nests tended to be roughly circular to about the same degree. in all wall nests, the petiole was higher than the comb center, although often not directly above the center. aside from the attachment position of the petiole, wall and ceiling nests showed one grossstructural difference: the cells of wall nests tended to open slightly downward, not perpendicular to the substrate. in gross structure, the nest of m. baconi resembles those of some other members of its species-group and subgenus. fig. 2 shows the size-frequency distribution of abandoned nests, consistent with the hypothesis of a very high failure rate early in the colony cycle, followed by a time of lower risk. the results show a distinct preference for walls over ceilings as a table 1. structural parameters of 26 nests of mischocyttarus baconi. a ceiling nest is attached below a horizontal substrate, while a wall nest is attached to a vertical substrate. comb excentricity, with a value between 0 and 1, is according to an index described in the text and illustrated in fig. 1. nest number petiole excentricity no. of cells length (mm) ceiling nests n=11 a 22 4 0.33 j 24 3 0.67 c 35 0.25 o 42 2 0.25 b2 52 2 0.33 b 53 0.38 c2 63 1 0.25 d2 64 0 y2 68 3 0 p 72 3 0.20 z2 94 3.5 0.30 mean 53.5 2.7 0.27 wall nests n=19 p2 21 2 1 n2 23 1 w 23 3 0.50 k 26 0.50 x2 27 0.67 y 38 4 1 l 40 3 0.67 v2 43 3 0.67 n 45 2 0.67 a3 48 4 0.25 x 50 3 0.25 m 52 2 0.67 z 55 2 0.63 r 56 2 1 r2 59 3 0.63 q 70 3 0.50 u2 86 4 0.50 s2 140 5 0.27 t2 198 4 0.36 mean 57.9 3.1 0.62 239 scobie, a.a. & c.k. starr — nest structure of mischocyttarus baconi nesting surface. in our experience, most independent-founding polistines that commonly nest on or in buildings much prefer horizontal or near-horizontal surfaces. it is not plain why m. baconi should tend to prefer walls or why most other species appear to prefer ceilings. the greater mean excentricity of walls nests evidently arises through a simple tendency to add new cells on the lower part of the perimeter. this would appear to make good mechanical sense, as a measure of excentricity in wall nests is expected to confer some stability against the shearing force of the nest’s weight. for much the same reason, centricity is expected in ceiling nests if the comb is parallel to the surface. acknowledgments the main part of this paper is drawn from the first author’s mphil thesis at the university of the west indies. we thank istván karsai and john wenzel for discussion and lee-ann beddoe for graphic assistance. references gadagkar, r. 1991. belonogaster, mischocyttarus, parapolybia and independent-founding ropalidia. pp. 149-190, in: ross, k.w. & r.w. matthews (eds.), the social biolog y of wasps. ithaca: cornell univ. press. karsai, s. & z. pénzes 1996. intraspecific variation in the comb structure of polistes dominulus: parameters, maturation, nest size and cell arrangement. insectes sociaux 43:277-296. o’connor, t., c.k. starr & s.a. cameron 2011. the neotropical social wasp mischocyttarus “alfkenii” ducke (hymenoptera: vespidae) is a pair of ethospecies. systematic entomology 36:446-452. richards, o.w. 1978. the social wasps of the americas, excluding the vespinae. london: british museum (natural history) 580 pp. silveira, o.t. 2008. phylogeny of wasps of the genus mischocyttarus de saussure (hymenoptera, vespidae, polistinae). revista brasileira de entomologia 52:510-549. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.4345sociobiology 66(3): 467-474 (september, 2019) pollination of grewia asiatica (malvaceae) by megachile cephalotes (hymenoptera: megachilidae): male vs. female pollination introduction in pollinator-dependent crops, high productivity in terms of quantity and quality of fruit and seed set is the function of good pollination (burd, 1994). bee potentially contribute to global food security by improving shape, size and post-harvest features (e.g. shelf life, firmness, color and sugar contents etc.) of many commercially grown fruits and vegetables (klatt, 2013). the vast majority of pollinator species is wild, including more than 20,000 species of bees (potts et al., 2016). the foraging behavior of pollinators (e.g. visitation frequency, visitation rate, seek for pollen and nectar and abstract the difference in behavior and morphology of male and female solitary bees-owing to different roles they performmay alter their pollination effectiveness. the present study was aimed to study the difference in foraging behavior of male and female megachile cephalotes smith (megachilidae) and its ultimate impact on pollination efficiency in terms of pollen deposition, fruit setting and post-harvest characters of phalsa crop, grewia asiatica l. (malvaceae). the effect of different environmental factors (light intensity, temperature, relative humidity and wind velocity) on foraging behavior was also studied. visitation frequency, visitation rate and pollen deposition of females were significantly higher than that of males. female individuals had also an edge over male counterparts by having longer body and proboscis lengths. males continued their activity throughout the day in low abundance without notable fluctuation while females attained their peak at 12:00 pm followed by a gradual decline until 4:00 pm. the maximum average fruit weight was recorded for female m. cephalotes pollinated fruits followed by open and male pollinated fruits. the shelf life in terms of post-harvest fruit weight and wrinkling was notably lower and more gradual in female pollinated fruits than open and male pollinated fruits. our results suggest that female m. cephalotes are better pollinators of g. asiatica in terms of its reproductive success and post-harvest parameters than the males. future studies should consider the biology and ecology of m. cephalotes with special focus on its artificial nesting. sociobiology an international journal on social insects w akram1, a sajjad1, s ali1, ma farooqi1, g mujtaba1, m ali2, a ahmad1 article history edited by celso martins, ufpb, brazil received 23 january 2019 initial acceptance 23 april 2019 final acceptance 05 august 2019 publication date 14 november 2019 keywords phalsa, bees, pollination, post-harvest qualities, foraging behavior. corresponding author asif sajjad department of entomology university college of agriculture and environmental sciences the islamia university of bahawalpur, bahawalpur, pakistan. e-mail: asifbinsajjad@gmail.com nectar robbing) can significantly affect pollen deposition on stigma (ohara & higashi, 1994; kudo, 2003). the foraging behavior of female solitary bees is better studied so far. however, some recent studies have shown considerable differences in life history, foraging behavior and ecology of male counterpart of the same species (neeman et al., 2006; cane, 2010; pascarella, 2010). although both the sexes feed on floral nectar in order to meet their own caloric need, their floral diet may also be as dissimilar as the diet of different bee species (roswell et al., 2019). females construct, maintain and defend their nests besides collecting pollen to provision young while males primarily search for suitable mates (willmer & stone, 2004). 1 department of entomology, university college of agriculture and environmental sciences, the islamia university of bahawalpur, bahawalpur, pakistan 2 department of entomology, muhammad nawaz shreef university of agriculture multan, pakistan research article bees w akram, a sajjad, s ali, ma farooqi, g mujtaba, m ali, a ahmad – male vs. female pollination468 therefore, both may differ in their foraging and pollination efficiencies (neeman et al., 2006). female solitary bees usually forage more efficiently and consequently pollinate better than males (o’toole & raw, 1991). males solitary bees however, contribute more to long-distance pollen flow (neeman, et al., 2006). according to recent studies, fruit set -in terms of quantitative and qualitative parameters including post-harvest characteristicsis more important predictor of pollination efficiency than pollen deposition (wang et al., 2017). this is due to certain pollination limitations e.g. each stigma attains an asymptote after certain number of pollen grains (sorensen & webber, 1997), ovules attract pollen grains regardless of whether they are pre-pollinated or not (falque et al., 1995) and deposition of non-viable and heterospecific pollen grains (bellusci et al., 2020). since male and female solitary bees vary in foraging behavior and morphology, both are likely to deposit pollen grains in different ways. females solitary bees are likely to deposit more viable pollen grains as they can exploit anthesis timing better and have shorter foraging distance and high visitation frequency than males (neeman et al., 2006). similarly, the amount of heterospecific pollen grains deposited on stigma by both the sexes may also vary as both show marked niche breadth and niche overlap in foraging resources (roswell et al., 2019). several species of megachile ( latreille, 1802) (hymenoptera: megachilidae) bees have been reported as efficient crop pollinators in indian subcontinents including mgegachile cephalotes (abrol et al., 2017a; singh et al., 2017). the females of m. cephalotes have unbranched and spirally grooved hairs (scopa) on their abdominal sternum which make them morphologically efficient pollinator than the males (kumar, 2015). the present study aimed to investigate the foraging behavior of male and female m. cephalotes on phalsa crop, and how efficiently they pollinate phalsa plants in terms of pollen deposition, fruit setting and post-harvest characters. the effect of different environmental factors (light intensity, temperature, relative humidity and wind velocity) on foraging behavior was also studied. materials and methods study site and plant species: the study was conducted at the horticultural research farm of the university college of agriculture and environmental sciences, baghdad-ul-jadeed campus, the islamia university of bahawalpur (iub), punjab, pakistan (29° 22’ 36.59”n 71° 45’ 44.04”e; 181 meters above sea level). the climate of the region is classified as subtropical with hot summers and cold winters. the mean daily minimum and maximum temperature reach 15°c and 30°c, in winter, and 20°c and 35°c, in summer, respectively. the average annual rainfall is 400 mm (parc, 1980). the experimental plant species was phalsa, grewia asiatica l. (malvaceae) sown in an area of 0.2 hectares containing 53 individuals. the nearby places include a planted forest, some waste land and buildings. phalsa is a fruit bearing shrub (can grow up to 12 feet) native to central america but is planted on commercial basis in pakistan (ullah et al., 2012; singh et al., 2015). yellow flowers (two centimeters in length) are borne in densely crowded axillary cymes. fresh and dry fruits of phalsa are consumed in human diet and leaves are used as forage for livestock (dev et al., 2017). in the indian subcontinent, phalsa individuals bloom in february and the fruits ripen during april to june (kumar et al., 2014). phalsa is self-compatible but cross-pollination by insects can significantly improve the reproductive success (randhawa & dass, 1962). the study was conducted from mid-february to end-may 2018 (i.e. from flowering to harvesting). flower visitor censuses and foraging behavior first, we conducted a three days-long survey of floral visitors in three widely isolated (>8 km) orchards (table 3) in order to assess the pollinators profile of g. asiatica in bahawalpur: fisheries complex (29° 23’ 15.4176’’ n, 71° 37’ 59.2284’’ e), lal-suhanra forest (29° 19’ 2.2116’’ n, 71° 54’ 16.9056’’ e) and iub campus (29° 22’ 36.588’’ n, 71° 45’ 44.0424’’ e). one collector was deployed to collect floral visitors randomly using a hand collection net for 4 hours (8-9 am, 10-11 am, 4-5 pm and 6-7 pm local time) in each orchard. in this way the total sampling efforts were 12 hours in all the three orchards. the collected pollinators other than m. cephalotes were identified to family level by using taxonomic keys proposed by borror et al. (1981). the body and proboscis length of both the female and male m. cephalotes was measured with a vernier caliper (n= 15 each sex). we only focused m. cephalotes for further studies at iub campus: visitation frequency (number of bees visiting an inflorescence of casually 30 flowers during two minutes; n=40 in each census), visitation rate (number of flowers visited by an individual during 120 seconds; n=40 bee individuals in each census) and stay time (time spent in handling a flower by an individual during a visit; n=120 in each census) throughout the flowering season with weekly intervals (total 6 census). a stop watch was used to record these observations. during each census, observations were made at 10:00, 12:00, 14:00, and 16:00 hours along the day. data regarding environmental factors such as temperature, relative humidity, wind speed and light intensity were recorded before each census with the help of thermo-hygrometer, anemometer and lux meter, respectively. pollinator effectiveness pollinator effectiveness was estimated in terms of pollen deposition, quantified after single visits of m. cephalotes on previously bagged flowers of g. asiatica. when m. cephalotes was abundant on the plants, the flowers were made available sociobiology 66(3): 467-474 (september, 2019) 469 to receive single visits. once a flower has been visited by a female or a male bee, stigma was removed using a sharp blade. a stereoscopic microscope with 40× magnification was used to count pollen grains. twenty such observations were made for each female and male m. cephalotes on twenty different plants in four splits i.e. five observations in each consecutive day for each sex. to test the pollination efficiency of female and male m. cephalotes in terms of plant reproductive success, other floral buds were caged with nylon mesh bags 24 hours before they opened. they were un-caged during the peak activity time of m. cephalotes and re-caged once a single visit had been made by male or female bee. ten such flowers on ten different plants were observed each for female and male m. cephalotes. the resultant fruit set and weight was measured as a function of their pollination efficiency. twenty openpollinated (unrestricted insect visitation) flowers and twenty caged (no insect visitation) flowers were also maintained on twenty different plants. post-harvest qualities the resulting fruits were harvested just at the start of ripening (i.e. 34 days after pollination) when their color was light pink. harvested fruits were weighed using an electronicbalance with 6 hours of post-harvest interval until 78 hours. since phalsa is a highly perishable fruit, the qualitative parameters including change in shape and color were also recorded at 6 hours of interval for 78 hours. the change in shape was recorded by visually attributing five ranks from tight skin to completely wrinkled skin. the change in color was carefully recorded using an rbg color chart i.e. ranged from deep pink to violet ret. data analysis in order to compare visitation frequency, visitation rate, stay time, pollen deposition, body length and proboscis length of male and female bees, the data were subjected to man-whitney u test (alpha 0.05) as it followed nonnormal distribution. one-way anova was applied to see the significant difference between average fruit weight of male, female and open pollinated fruits followed by tukey’s post hoc test at alpha 0.05. one-way anova was also applied to see the significant difference between postharvest fruit weight loss (%) of male, female and open pollinated fruits at each observation hour (alpha 0.05). we did not include selfpollination in the analysis as all the fruits were aborted in selfpollination treatment. chi-square test was used to verify whether fruit wrinkling and color significantly changed over observation dates. in order to know how much the variation in foraging behavior (i.e. stay time, visitation rate and visitation frequency) of female and male m. cephalotes is explained by environment factors (temperature, relative humidity, wind speed and light intensity), multiple linear regression analysis was performed. the linear regression’s f-test was applied with null hypothesis that model explains zero variance in dependent variables (foraging behavior). in order to see the significant environmental predictors of foraging behavior, their regression coefficients were compared at alpha 0.05 by using t-statistics. results a total of 458 individuals belonging to 18 species in three orders visited the flowers of g. asiatica i.e. 13 bees (hymenoptera), 2 butterflies, 1 moth (lepidoptera) and 2 flies (diptera). megachile cephalotes comprised 65% of the total floral visitors’ abundance on g. asiatica at all the three widely isolated locations in bahawalpur i.e. fisheries complex, lal-suhanra forest and iub campus. this highlights the importance of m. cephalotes as a most frequent floral visitor of g. asiatica in bahawalpur (table 1). locations total pollinator abundance m. cephalotes abundance percent m. cephalotes abundance fisheries complex 21 11 52.38 lal suhanra forest 24 12 50.00 iub campus 458 300 65.50 table 1. relative abundance of m. cephalotes with other floral visitors at three widely isolated locations in bahawalpur. the comparison of foraging behavior and morphological features of male and female m. cephalotes is presented in table 2. there was no significant difference between male and female m. cephalotes in terms of stay time. however, visitation frequency, visitation rate and pollen deposition (2.06±0.14 individuals per branch per 2 minutes; 13.70±0.50 flowers per 2 minutes; 639.35±3.17 pollen grains per stigma, respectively) of females were significantly higher than that of males (0.44±0.06 individuals per branch per 120 seconds; 13.42±0.67 flowers per 2 minutes; 12.05±1.19 pollen grains per stigma, respectively). regarding morphological features –important for cross pollinationfemale individuals had an edge over males by having longer body and proboscis lengths i.e. body length of 13.01±0.16 mm and 10.13±0.10 mm for female and male; proboscis length of 5.36±0.09 mm and 4.38±0.06 mm for female and male. the results of multiple linear regression revealed that the models had very low values of r2 and therefore did not explain much of the variation in foraging behavior (stay time, visitation frequency and visitation rate) on account of environmental factors however, they were significant (f-test; p<0.001) except the visitation frequency of male m. cephalotes (table 3). the t-statistics showed that visitation frequency of both the sexes largely remained unaffected with environmental factors except for light intensity which was w akram, a sajjad, s ali, ma farooqi, g mujtaba, m ali, a ahmad – male vs. female pollination470 positively associated with the females. wind significantly reduced the stay time in both the sexes while it improved the visitation rate in male bees. light intensity reduced the stay time in males and improved the visitation rate in females. temperature reduced the stay time in females and improved visitation rate of males (table 3). the activity of both the male and female m. cephalotes started somewhere between 9:00 am to 10:00 am. males continued their activity in low abundance without any notable fluctuation across the observation intervals. however, females attained their peak abundance at 12:00 pm followed by a gradual decline until 4:00 pm (fig 1). table 2. comparison of foraging behavior and morphological features of female and male m. cephalotes using man-whitney u test at alpha 0.05 (n = number of observations). results of man-whitney u test foraging behavior morphological features stay time n = 360 visitation rate n = 120 visitation frequency n = 120 pollen deposition n = 20 body length n = 15 proboscis length n = 5 female (mean of ranks) 1777.21 71.6 200.61 15.03 11.5 4 male (mean of ranks) 183.29 48.9 159.89 5.46 4 1.5 mann-whitney u 62609 4476 50141 8.5 0 0 pvalue 0.431 <0.001 <0.001 <0.001 <0.001 0.007 z-value -0.78601 -5.1271 -5.2792 -5.1714 .-4.6455 .-2.5067 table 3. multiple regression analysis between foraging behaviors of female and male m. cephalotes and environmental factors (m=male, f=female, t=temperature, r.h=relative humidity, w.s= wind speed, l.i=light intensity). gender model f p-value r2 stay time m y = 2.776 -0.047(t.) +0.149(r.h)* -0.058(w.s)* -0.005(l.i)* 17.516 <0.001 0.165 f y= 15.513 -0.305(t.)* -0.045(r.h) -0.098(w.s)* -0.001(l.i) 8.530 <0.001 0.088 visitation frequency m y = -2.9 +0.0629(t.) +0.038 (r.h) -0.022(w.s) +0(l.i) 0.977 0.423 0.033 f y= -0.991 +0.04(t.) -0.093 (r.h) -0.017(w.s) +0.007(l.i)* 6.815 <0.001 0.192 visitation rate m y= -37.557 +1.319(t.) -0.11 (r.h) +0.935(w.s)* +0.009(l.i) 6.237 <0.001 0.178 f y = -57.76 +1.499(t.)* +0.293 (r.h) -0.056(w.s) +0.016(l.i)* 6.162 <0.001 0.177 *significant predictors of behavior using t-statistics at alpha 0.05 open, female and male pollinated fruits were different in terms of their weight (d.f=2, f=8.84, p=0.001). the maximum average weight was recorded for female m. cephalotes pollinated fruits followed by open and male pollinated fruits which were also non-significant (fig 2). 0 1 2 3 4 10:00 am 12:00 pm 2:00 pm 4:00 pm in di vi du al s pe r pl an t p er 1 20 s ec on ds female male fig 1. diurnal dynamic pattern of female and male m. cephalotes in terms of average individuals per plant per 120 seconds. percent loss in weight was significantly lower in female pollinated fruits than that of open and male pollinated fruits at 6 hours after harvest (d.f. =2, p=0.012, f=5.20) and 12 (d.f. =2, p=0.001, f=9.82). remaining observations from 18 to 78 h after harvest showed non-significant difference in weight loss. 0.58 b 0.73 a 0.52 b 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 open female male a ve ra ge w ei gh t ( g) fig 2. comparison of female, male and open pollinated fruits in terms of their weight. means sharing similar letters are statistically non-significant at alpha 0.05. sociobiology 66(3): 467-474 (september, 2019) 471 the post-harvest decline in fruit weight was notably lower and more gradual in female pollinated fruits than open and male pollinated fruits. the fruits completely dried out at 54 h in case of open and male pollination whereas at 66 h in case of female pollination (fig 3). in case of post-harvest wrinkling, as compared to male and open pollination, female pollinated fruits did not show any wrinkling until 18 h after harvest whilst overall wrinkling remained lower in female pollinated fruits than open and male pollinated fruits until 60 h. (fig 4). fruit wrinkling significantly increased (p<0.001) with the increase in postharvest time in open pollinated, male pollinated and female pollinated fruits (χ2=292, 325, 306, respectively) however, fruit color significantly changed (p<0.001) with the increase in post-harvest time in female pollinated fruits alone (χ2=186). fig 4. the increase in fruit wrinkling with successive post-harvest observation in case of open, female and male pollination local names scientific names no. of visits of megachile cephalotes leh cirsium arvense 1 maskeet prosopis glandulosa 3 akk calotropis procera 1 kindiari carthamus persicus 1 ipleiple leucaena leucocephala 1 parkinsonia parkinsonia aculeata 3 janasa alhagi graecorum 9 bukan phyla nodiflora 1 alfalfa medicago sativa 4 frash tamarix aphylla 2 kikar acacia nilotica 1 kheera cucumis sativus 3 jand prosopis cineraria 2 table 4. visitation of m. cephalotes on alternate host plants at iub campus. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 6 12 18 24 30 36 42 48 54 60 66 72 78 w ei gh t (g ) hours open female male fig 3. fruit weight loss among open, female and male pollinated fruits after 6 to 78 hours of harvest. 0 1 2 3 4 5 6 6 12 18 24 30 36 42 48 54 60 66 72 78 w rin kl e (1 -5 r an k) hours open female male discussion in this study we have shown that m. cephalotes was the most abundant (65% of total abundance of 18 species) floral visitor of g. asiatica in the study sites whilst its females were the more frequent and efficient pollinators. phalsa flowers offer a rich source of energy and attract several pollinator populations e.g. including most abundant megachile, apis and xylocopa species (abrol, 1992). kumar et al. (2017) also recorded 21 flower visitors on phalsa in india. grewia asciatica therefore, shows a generalized pollination system (ollerton et al., 2007) yet m. cephalotes being the most abundant visitor, might be leading the system towards cryptic specialization (niemirski & zych, 2011). in the present study, we, for the first time, gave the empirical evidence of how the male and female m. cephalotes along with open and caged treatments affect the qualitative parameters of phalsa, signifying female m. cephalotes as the best choice under local conditions. visitation frequency and pollen deposition of female bees was significantly higher than male bees in this study. visitation frequency of male and female bees is the function of their relative abundance (i.e. proportion) in given space and time. the male-female ratio in megachile bees is a highly variable phenomenon depending upon a number of physical factors e.g. size and diameter of nesting tunnel and distance of nest from foraging resource (stephen & osgood, 1965; peterson & roitberg, 2006). moreover, females usually fly for shorter distances thereby increasing their visitation frequency and relative foraging efficiency while males contribute more to long-distance flow of pollen (neeman, et al., 2006). moreover, males in our study, foraged primarily on nectar and continued their activity in low abundance without any notable fluctuation across the observation intervals. male solitary bees perform the result of survey of alternate host plants at iub campus is shown in table 4. we observed 13 plant species which received at least one visit of male or female m. cephalotes. the maximum visits were recorded on alhagi graecorum (fabaceae). w akram, a sajjad, s ali, ma farooqi, g mujtaba, m ali, a ahmad – male vs. female pollination472 at least two co-occurring activities i.e. nectar foraging and mate searching while females focus mainly on foraging for collecting pollen for their larvae (neeman, et al., 2006). that is why males remained stable over day while females peaked at resource available time. pollen deposition in a single visit is predicted by a number of morphological and behavioral factors exhibited by bees. in m. cephalotes, females are larger in size than males besides having a number of scopal hairs on the ventral surface of their abdomen. this makes them a better pollen depositor than the males which are smaller in size with fewer scopal hairs (bzdyk, 2012; kumar, 2015). in the present study, female body length was in agreement with kumar (2015) who reported female body length of 12.36-13.52 mm and male body length of 10.47-12.02 mm. there was no significant difference between male and female m. cephalotes in terms of stay time. female m. cephalotes is larger in size having longer proboscis than males. moreover, females have to build nests, collect pollen for their young ones and lay eggs (eickwort, 1975; peterson & roitberg, 2006). therefore, to meet their energy and dietary requirements they have to uptake more nectar than the males (feuerbacher et al., 2003). previous studies have shown that bees with long proboscis ingest more volume of nectar than short proboscis (harder, 1983). this suggests that no matter the stay time of both the sexes is similar, but female bees might ingest larger amount of nectar during a visit than the male did. supporting this idea, danforth (1990) reported that difference in food requirements between sexes starts right from the brood stage i.e. female offspring of calliopsis persimilis (megachilidae) receive three trips of pollen whereas male offspring receive two trips of pollen. the comparison of male and female bees in their foraging response towards different environmental factors is rarely studied. in case of solitary bees ‘m. lanata and m. bicolor’ without giving the sex identityabrol and kapil (1986) and abrol (1987) found that visitation frequency was positively associated with light intensity, air temperature and solar radiation. in our case, visitation frequency of both the sexes of m. cephalotes largely remained unaffected with environmental factors except for light intensity which was positively associated with the females. this finding is in line with szabo and smith (1972) who also found a positive relationship between light intensity and visitation frequency of female m. rotundata. how stay time and visitation rate of megachile bees are affected by weather factors also remains unclear. szabo and smith (1972) showered that foraging activity of females m. rotundata was positively correlated with light intensity and temperature combined. in our study, wind speed significantly decreased the stay time in both the sexes while it improved the visitation rate in male bees. similarly, light intensity negatively influenced the stay time in males and positively influenced visitation rate in females. temperature negatively influenced the stay time in females and positively influenced the visitation rate of males. these findings are being reported for the first time for m. cephalotes and have implications for future studies and conservation programs. females in our study attained their peak abundance at 12:00 pm followed by a gradual decline until 4:00 pm. the activity of pollinators is mostly predicted by the anthesis, the time when large amount of nectar and pollen resources are available (varassin et al., 2001). kumar et al. (2017) noticed that anthesis lasted from 1130 to 1300 h in closely related grewia flavescens in india. they also noticed the maximum pollen viability and stigmatic receptivity at the time of anthesis. results of present study showed females of m. cephalotes were the better pollinators than males. a single visit of females produced significantly greater fruit weight than that of male and open pollinated fruits, which were also statistically similar. greater fruit weight in case of female m. cephalotes is certainly due to higher number of pollen grains deposited by them as compared to males. ‘why did the unrestricted open pollination result in lower fruit weight than the female m. cephalotes pollination?’ can be justified in terms of pollen limitation. there are two pollination limitations associated with pollen grains i.e. quantity (number) and quality of pollen grains (aizen & harder, 2007). the seed set increases with the number of pollen grains deposited on stigma and attains an asymptote after certain number of pollen grains (sorensen & webber, 1997). the number of pollen grains dropped on stigma is not merely the indicator of good seed set as ovules attract pollen grains in the similar way regardless of whether they are pre-pollinated or not (falque et al., 1995). moreover, viability of pollen grains and number of heterospecific pollen grains also affect seed set in angiosperms (bellusci et al., 2010). usually effective pollination has fortunate consequences on the post-harvested qualitative attributes like color, nutrient profile, size, firmness and overall shelf life of several fruits i.e. strawberry, feijoa, apples and almonds (patterson, 1990; brittain et al., 2014; klatt et al., 2014a; abrol et al., 2017b; colak et al., 2017). these post-harvest qualities have strong link with the market value of fruits and economy of farmers (klatt et al., 2014b). conclusion megachile cephalotes was the most abundant pollinator of phalsa in bahawalpur. females of m. cephalotes were the better pollinators than males in terms of pollen deposition, fruit weight, fruit size and shelf life (i.e. loss of fruit weight, shrinkage, and degradation in color). we also documented for the first time how abiotic factors including environment temperature, relative humidity, wind speed and light intensity affect the foraging behavior (stay time, visitation rate and frequency) of both female and male m. cephalotes. sociobiology 66(3): 467-474 (september, 2019) 473 references abrol, d.p. & kapil, r.p. 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(2017). evaluation of pollinator effectiveness based on pollen deposition and seed production in a gynodieocious alpine plant, cyananthus delavayi. ecology and evolution. 7:8156–8160. doi: 10.1002/ece3.3391 willmer, p.g. & stone, g.n. (2004). behavioral, ecological, and physiological determinants of the activity patterns of bees. advances in the study of behavior, 34: 347–466. doi: 10.1016/s0065-3454(04)34009-x doi: 10.13102/sociobiology.v62i2.328-330sociobiology 62(2): 328-330 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant castes from intercastes: much ado about nothing introduction the first explanation on the origin of ant soldiers is due to emery (1894) and it was further developed and supported by wilson (1954). according to this supposition some ant species first evolved a polymorphic sterile caste comprising minor and major workers with intermediate forms. later, natural selection favored disappearance of the intermediates and stabilization of the majors as a separate caste, the soldiers. this hypothesis can be tested (falsified) by the observation of typical non-worker (i.e. gyne or male) traits in soldiers. baroni urbani and passera (1996) suggested that ant soldiers have been directly selected from gynes and not from workers. this hypothesis can be falsified by the observation of typical worker or male traits in soldiers. baroni urbani and passera’s explanation was rejected by ward (1997) and reaffirmed by baroni urbani (1998) with the backing of new supporting examples. nobody ever criticized the arguments of baroni urbani (1998) but this paper had a strong, unpredictable effect on the subsequent ant literature: the term soldier was virtually banned from publication and substituted by “major worker” or by generic statements of worker polymorphism. this attitude suggests, without taking the risk of saying it explicitly, that soldiers are a worker subcaste and not a separate caste as everybody agreed before. abstract a recent hypothesis on the origin of ant soldiers by molet et al. (2012) is shown to be misleading since it is based on unfair, incomplete literature information and to be unscientific since it is not falsifiable. keywords unfair citation, ant castes, ant soldiers. sociobiology an international journal on social insects c baroni urbani article history edited by gilberto m. m. santos, uefs, brazil received 28 october 2014 initial acceptance 29 november 2014 final acceptance 05 december 2014 corresponding author cesare baroni urbani department of environmental sciences biogeography, university of basle, st. johannes-vorstadt 10 ch-4056 basle, switzerland e-mail: cesare.baroni-urbani@unibas.ch caste nomenclature the term soldier, first introduced in entomology by heer (1852) to describe a peculiar ant morphology, became established for a separate ant caste and accepted by the most influential authors in the literature ranging from mayr, emery, forel, wheeler, santschi to kempf, brown jr., wilson (e.g. 1971: 138 [where soldiers are termed as “a full caste”], and 1976: 355 [distinguishing “three female castes”]), and bolton (1995 [where soldiers are listed as soldiers, when known, for all ants], but not bolton, 2003 [where only polymorphic workers are listed for all genera where soldiers were listed in his previous publication]). by analogy with ants, the term soldier was successfully used also for termites (e.g. korb, 2008), aphids (e.g. shibao et al., 2003), and thrips (e.g. chapman et al., 2002). a bizarre consequence of the common, undeclared soldier refutation policy among ants is that, in contrast with the above entomological literature, the use of the term soldier appears to have been lately excluded from current literature (i.e. after baroni urbani, 1998), even for genera and species where it had been consistently used before (e.g. shattuck, 1999 [all australian ants]; bolton, 2003 [all ants]; wilson, 2003 [pheidole]; fernández, 2004 [carebara]; merti & traniello, 2009 [pheidole]; and several popular websites such as e.g. short note university of basle, st. johannes-vorstadt , basle, switzerland sociobiology 62(2): 328-330 (june, 2015) 329 discover life, 2014 [cephalotes]; encyclopedia of life, 2014 [family formicidae and a number of ant genera]; tree of life, 2014 [cephalotini]; wikipedia, 2014 [pheidole]). discussion only recently, ant students started again to use the term soldier. one of these new papers re-using this term (molet et al., 2012) will be discussed in the following since it contains erroneous statements needing correction. for example, the authors write properly that often, in ant literature, the term soldier has been wrongly applied to both, a true, discrete soldier caste and to large, polymorphic workers. but, in taking on credit for this idea they dismiss a number of important papers. first, molet et al. (l.c.: 336) go so far as to summarily reject both recent, documented contributions to the origin of ant soldiers by baroni urbani and passera (1996) and by ward (1997) since they “have been compromised by semantic problems in distinguishing soldiers and major workers”. as a matter of fact this criticism applies to ward (1997) only; baroni urbani and passera (1996: 223) wrote: “we do not believe that the largest individuals of continuously polymorphic species [i.e. major workers] are true soldiers”. not only, but molet et al. (l.c.) omitted mention to baroni urbani (1998), i.e. to an 18 pages study entirely devoted to ant castes as their 13 pages paper. in this study baroni urbani (p. 317) made the distinction between soldiers and major worker even clearer and gave credit for first understanding this difference to andré (1885), i.e. to a book published over 120 years before molet et al. (2012). molet et al., based on their presumed discovery of the difference between major workers and soldiers, find also a solomon solution to the dilemma on the worker or gyne origin of soldiers and propose a new hypothesis. their answer to the problem is that ant soldiers evolved neither from workers nor from gynes, but “from rare intercastes… erratically produced by colonies through environmental or genetic perturbations” (p. 328). this hypothetical need of pathological, rare intercastes producing soldiers remains entirely gratuitous and unnecessary since it offers no operational advantages on the previously available hypotheses. on the contrary, since it is impossible to design a test to falsify it, this supposition cannot be considered as a scientific hypothesis (popper, 1977). finally, molet et al. (l.c.) deserve credit for correctly using the term soldier but repeatedly use another erroneous, self-contradictory name i.e. “winged queen” instead of the correct “winged gyne”. queen is a function, not a caste name and all ant queens are wingless, by definition. references andré, e. (1885). les fourmis. hachette, paris. baroni urbani, c. (1998). the number of castes in ants, where major is smaller than minor and queens wear the shield of the soldiers. insectes sociaux, 45: 315-333. baroni urbani, c. & l. passera. (1996). origin of ant soldiers. nature, 383: 223. doi: 10.1038/383223a0. bolton, b. (1995). a new general catalogue of the ants of the world. harvard university press., cambridge, mass. bolton, b. (2003). synopsis and classification of the formicidae. memoirs of the american entomological institute, 71: 370 pp. chapman, t. w., kranz, b. d., bejah, k.-l., morris, d. c., schwarz, m. p. & crespi, b. j. (2002). the evolution of soldier reproduction in social thrips. behavioral ecology, 13: 519-525. discover life (2014). cephalotes pallens. http://www. discoverlife.org/20/q?search= cephalotes+pallens. accessed october 22, 2014. emery, c. (1894). die entstehung und ausbildung des arbeiterstandes bei den ameisen. biologisches zentrablatt., 14: 53-59. encyclopedia of life (2014). formicidae. http://eol.org/ pages/699/details. accessed october 22, 2014. fernández, f. (2004). the american species of the myrmicine ant genus carebara westwood (hymenoptera: formicidae). caldasia, 26: 191-238. heer, o. (1852). ueber die haus-ameise madeiras. naturforschenden gesellschaft, zürich, 24pp. korb, j. (2008). termites, hemimetabolous diploid white ants? frontiers in zoology, 5: 9 pp. doi: 10.1186/1742-9994-5-15. merti, a. l. & j. f. a. traniello. (2009). behavioral evolution in the major worker subcaste of twig-nesting pheidole (hymenoptera: formicidae): does morphological specialization influence task plasticity? behavioral ecology and sociobiology, 63: 1411-1426. doi 10.1007/s00265-009-0797-3. molet, m., d. e. wheeler, & c. peeters. (2012). evolution of novel mosaic castes in ants: modularity, phenotypic plasticity, and colonial buffering. american naturalist, 180: 328-341. popper, k. r. (1977). the logic of scientific discovery. taylor and francis, london & new york. shattuck, s. o. (1999). australian ants. their biology and identification. csiro publishing, collingwood, victoria. shibao, h., lee, j.-m., kutsukake, m. & fukatsu, t. (2003). aphid soldier differentiation: density acts on both embryos and newborn nymphs. naturwissenschaften, 90: 501-504. c baroni urbani – defending ant soldiers and their origins330 tree of life (2014). cephalotini. http://tolweb.org/ cephalotini/22434. accessed october 22, 2014. ward, p. s. (1997). ant soldiers are not modified queens. nature, 385: 494-495. doi: 10.1038/385494b0. wikipedia [english version] (2014). pheidole. https:// en.wikipedia.org/wiki/pheidole. accessed october 22. 2014. wilson, e. o. (1954). a new interpretation of the frequency curves associated with ant polymorphism. insectes sociaux, 1: 75-80. doi: 10.1007/bf02223154. wilson, e. o. (1976). a social ethogram of the neotropical arboreal ant zacryptocerus varians (fr. smith). animal behavior, 24: 354-363. wilson, e. o. (1971). the insect societies. the belknap press of harvard university press, cambridge, mass. wilson, e. o. (2003). pheidole in the new world. a dominant, hyperdiverse ant genus. harvard university press, cambridge, mass. 641 foraging populations of tube building termites, gnathamitermes perplexus (banks), associated with termiticide experiments in southern arizona (isoptera: termitidae) by javier g. miguelena¹ and paul b. baker¹‚² abstract in the southwestern desert region of arizona, a common non-structure invading species is the tube building termite gnathamitermes perplexus. it is a valuable species from an ecological point of view due to its role in the decomposition of dead wood in desert environments and its capacity to enrich and aerate vast quantities of soil. since it has no economic importance, very little is known of the effects of termite control measures on it. however, g. perplexus is likely exposed to termiticides used to manage more damaging termite species with which it co-occurs. since most common termiticides have relatively generalized modes of action, we hypothesized that g. perplexus populations would decrease significantly as a result of termiticide application. the results reported here are part of a larger study in which we were primarily interested in evaluating foraging termite populations of heterotermes aureus, associated with circular grids that were treated with termiticides. termites were collected monthly from 9 plots located at the santa rita experimental range (pima co., az) over a 3-year period. plots were equally and randomly assigned to three treatments: a control and two insecticide treatments of either fipronil (termidor®, basf) or chlorfenapyr (phantom®, basf). within the chlorfenapyr (phantom®) treatment, we saw a significant increase in termite foraging populations after termiticide application. this effect showed a spatial pattern in which more termites were found near the center of plots. however, the number of g. perplexus collected in the fipronil (termidor®) plots was reduced with respect to controls. this reduction in the number of termites also showed a spatial distribution with the decrease in termite numbers being stronger near the center of plots within the treated zone. ¹ university of arizona, tucson, az 85721. email: pbaker@ag.arizona.edu ² corresponding author 642 sociobiolog y vol. 59, no. 3, 2012 key words: heterotermes aureus, gnathamitermes perplexus, termidor®, phantom®, fipronil, chlorfenapyr introduction worldwide, subterranean termites offer significant ecological services as part of the detritus cycle. however, some species can also cause extensive damage to wooden structures as well as wood and cellulose products in temperate and tropical areas. in fact, the combination of damage and control/repair costs attributed to subterranean termites in the united states alone was estimated to cost consumers at least $us 1.5 billion (su and scheffrahn 1990). non-structure invading termites can be associated with, and occur in, the same areas as economically important termites. as a result, pest management actions might be inadvertently taken against benign or even beneficial termites. this is true for the tube building termite gnathamitermes perplexus (banks), which belongs to the family termitidae and is found in the desert southwest including southern arizona, southern nevada, southern california, far-western texas, and northern mexico (la fage 1976; jones and nutting 1979). its primary role in the ecosystems it inhabits is as a decomposer, feeding on a variety of cellulose materials. g. perplexus builds above-ground, sheet-like foraging shelters made of soil particles cemented together with saliva. under the shelter of these structures, it eats away the weathered portion of dead wood and other cellulose materials, sometimes consuming desert shrubs and grasses completely and leaving only hollow earthen tubes (light 1934). g. perplexus plays an important role in the sonoran desert ecosystem by aerating the soil horizon (la fage 1974). it has been estimated that this termite species moves over 500 kg/ha of soil every year, and in the process, it changes the soil composition by incorporating more clay into it and enriching it with carbon, nitrogen and other nutrients (nutting et al. 1987). although g. perplexus workers might be similar to those of other species, they are easily distinguished from the co-occurring species heterotermes aureus (snyder) by their size and coloration (baker and marchosky 2005). g. perplexus is not a serious economic pest as it does not cause structural damage. homeowners may on occasion encounter their mud sheets covering the remains of dead cacti in contact with the soil or at the base of trees or fence posts. since g. perplexus generally does not invade structures, this species is es643 miguelena, j.g. & p.b. baker — foraging populations of tube building termites sentially a non-target organism for the application of termiticides. although the impact of commercial termiticides on g. perplexus has not been studied previously, it is likely that their populations and foraging activity could be negatively impacted by many of the pest management actions directed to other termite pest species. this would result in the loss of the valuable ecological services that this species provides. here we report the measurements of g. perplexus foraging populations collected over 3 years as part of a study on the effects of fipronil (termidor®) and chlorfenapyr (phantom®) as termiticides applied to soil. both insecticides have a broad spectrum of action, are non-repellent termiticides and their active ingredients are of relatively recent discovery. fipronil is a neurotoxin that acts by blocking gaba-regulated chloride channels, while chlorfenapyr is a pro-insecticide that uncouples oxidative phosphorylation by disrupting the production of atp (pegido and rice 2006). they are registered for termite management and their use for this purpose is widespread. the active ingredients of both termiticides are also found in many other insecticide formulations due to their relatively generalized modes of action. besides evaluating the effects of these insecticides on termite density, we were also able to make some observations on the temporal variation of g. perplexus foraging activity. materials and methods field site the study was conducted at three distinct locations at the santa rita experimental range ≈ 40 km south of tucson, arizona, usa (elevation 984 m) (baker et al. 2010). this area was previously utilized for foraging studies by haverty and nutting (1975) and described by jones et al. (1987). the results reported here are part of a larger study designed to evaluate the effects of two insecticides, fipronil and chlorfenapyr on foraging termite populations of the economically important termite h. aureus. we first surveyed the research area looking for places heavily infested with h. aureus. then, several transects consisting of 10 to 15 termite collecting stations were placed in a 1.03 km2 area during the spring of 2004. each collection station consisted of three corrugated cardboard rolls (0.04 x 1.0-m strip of cr 30 x 250 b-flute sf cardboard, tucson container corp, tucson, arizona, usa) wrapped around a piece of ash wood (fraxinus sp.) (7.5 x 2.5 x 1.25-cm). cardboard 644 sociobiolog y vol. 59, no. 3, 2012 rolls were placed within a piece of 0.15-m diameter x 0.15-m long pvc pipe and covered with a concrete brick (20.3 x 15.2 x 2.5-cm). the surrounding collection stations were visually inspected approximately every four weeks for the presence of termites. once a station was determined to contain several hundred termites, a circular feeding grid was constructed around it. each grid consisted of 50 collection stations surrounding the central infested station and placed equidistantly (≈ 3.13-m apart) along the circumference on five circles or rings with increasing radii of 1.5, 2.0, 4.0, 7.0 and 10.0m; respectively. each ring was labeled a-e with the center station designated by x (see fig. 1). the three cardboard rolls contained within each of the 51 collecting stations in each experimental grid were labeled to link them to the corresponding feeding station. three research grids (replicates or separate sites) were established for each insecticide treatment. in some cases, it took several months ( july 2004 through march 2005) to establish a research grid due to temporal termite population fluctuations. visual inspections of all fig. 1circular field plot design used where termite stations were placed in a grid formation by concentric rings of increasing size. 645 miguelena, j.g. & p.b. baker — foraging populations of tube building termites stations were conducted monthly from the end of 2004 thru mid-2008 and termites were counted in each station. termite-infested cardboard rolls inside stations were replaced with new ones and were taken to our laboratory where the termites within were counted. termiticides the termiticides chlorfenapyr (phantom® 2 sc) and fipronil (termidor® sc) (basf corporation), were applied at the rate of 0.125 and 0.06 on november 14, 2005, and september 19, 2006, respectively to each of 3 plots. control plots (n=3) received only water on november 14, 2005. the applications were made by johnston’s pest solutions, a licensed pest control company. applications were made by the standard application procedure of rod and trench at 4 gallons per 10 liner foot per trench between the b and c rings (see fig. 1). the results presented here reflect only the g. perplexus counts. in general, the experimental design reproduces the effect of using these termiticides as a barrier treatment in an area where termites are already present. the grid design employed was especially designed to determine the spatial magnitude termiticide effects. since the termiticides used are non-repellent, termites are expected to come in contact with treated soil and die. changes in the distribution of termites can also be the result of indirect effects of the termiticide treatment on termite foraging patterns. for example, if none of the termite foragers traveling in a given direction returns, this might discourage further movement in that direction. data analysis we used the total termite count for each station as a starting point for statistical analyses. this consists of the sum of the termites visually estimated inside each station in the field and those counted from inside cardboard rolls. termite activity was recorded before and after treatment for all plots. in order to use the control grids as a baseline representing the natural termite activity, we averaged the termite counts of the 3 replicate grids for each treatment by month. then, we calculated the difference between treated and control plots (difference = termiticide control) for each month for both termiticide treatments. we used 2-sample t tests to compare the mean difference between control and treatment grids before and after treatment. this analysis was performed for whole grids, and subsets of them including the treatment zone (the center station or x and the a and b rings), and each 646 sociobiolog y vol. 59, no. 3, 2012 one of the rings outside it (c, d and e). a square root transformation was used whenever necessary to improve the normality and homogeneity of variance of the data. if homogeneity of variance could not be achieved through transformation, a welch’s anova was calculated instead. since collections for fipronil and chlorfenapyr grids were done at different times, their data were compared to different subsets of the monthly data for the controls. additionally, we looked for spatial patterns in g. perplexus density after treatment. we considered termite density as the average number of termites per station within a given area. both occupied and unoccupied stations were considered for this calculation. if the effects observed are caused by the termiticides, we would expect them to be stronger inside the treatment area and decrease with distance from it. we compared the mean termite densities of the different areas within plots with non-parametric kruskal-wallis tests. this test was used because termite density data was highly skewed due to the high frequency of low termite counts. whenever significant differences were found, a pair wise mann-whitney test with a bonferroni correction was calculated. results and discussion both h. aureus and g. perplexus foragers were collected in monitoring stations at all nine sites. however, the two species never occupied the same station at the same time. in addition, g. perplexus foragers represented only the 6.94% (58,854 out of 847,685 in total) of the termites collected from all sites. although the average number of g. perplexus in control plots for any given month was low (124.04 individuals per plot), a few explosive increases in foraging population numbers were observed in the fall (fig. 2). it might be relevant that these population increases happened in the late summer, when soil moisture is higher as a result of rainfall associated with the monsoon. the highest number of g. perplexus found in a single plot (2,565 individuals) was collected in november of 2005 from one of the fipronil plots before treatment. the number of stations within each plot occupied simultaneously by g. perplexus was also considerably low. in control plots, which represent natural termite populations, averages of 3.3 stations out of the 51 that make up the grid were occupied any given month. as with total termite counts, station occupancy was quite variable and it was particularly low during the 647 miguelena, j.g. & p.b. baker — foraging populations of tube building termites winter months. the maximum number of stations occupied within one plot simultaneously was 25, but this was a unique event. strong variation from one month to the next was evident and was likely related to weather or other environmental abiotic factors such as soil moisture, since the patterns of g. perplexus fluctuation where similar among untreated plots (fig.2). this suggests a factor that affects all g. perplexus populations in the region simultaneously. soil moisture and temperature have been previously shown to be determinant on the foraging activity of g. perplexus (la fage et al. 1976). taking these natural fluctuations into account, we considered that a decrease in termite activity after treatment does not necessarily indicate an effect caused by the termiticides. instead, we looked at changes in termite activity with respect to termite counts from the control plots. if a termiticide had an effect on termite activity, we would expect to see greater differences between treatment and control plots after application. when whole experimental grids are considered, the number of g. perplexus collected in the chlorfenapyr plots significantly increased with respect to the controls after treatment. although an increase in the number of foraging g. fig. 2 – average number of g. perplexus workers collected in all stations by month. the dates of application of the two treatments are marked with vertical lines of the corresponding pattern. 648 sociobiolog y vol. 59, no. 3, 2012 perplexus was observed in all the rings that make up a plot, no significant differences were found when c, d, and e rings were considered independently (table 1). this positive effect of chlorfenapyr on g. perplexus foraging populations could be explained by a decrease in the populations of the competing termite h. aureus. it has been established that these two species show interference competition and even aggression in the wild ( jones and trosset 1991). we will consider this possibility in future work including data on h. aureus populations. when we compared the number of termites per station in different zones of the experimental grid, a spatial pattern was apparent. after treatment with chlorfenapyr, termite numbers were higher in the treatment zone and they decreased with further distance from it. this kind of pattern was not present before application (fig. 3). although differences in g. perplexus density among different zones were not significant (kruskal-wallis test, h=5.091, hc=6.469, p=0.165), this certainly reinforces the idea that the application of chlorfenapyr was somehow advantageous for these termites. the plots treated with fipronil showed a rapid decrease in g. perplexus foraging populations immediately after treatment. this change can be clearly table 1 – mean difference in the number of termites collected between chlorfenapyr (phantom®) designated plots and untreated controls before and after treatment. mean negative values indicate lower termite abundance with respect to control plots. tests were performed for the whole experimental grid as well as subsets. in some cases, a cubic root transformation was used to improve the normality and homogeneity of variance of the data. differences between the numbers of g. perplexus collected from chlorfenapyr and control grids before treatment after treatment area considered n mean ± se mean ± se test statistic p test performed treatment zone 25 12.4 4.9 36.4 23.2 t=-0.18 0.8619 2-sample t test on cubic root transformed data c ring 25 14.3 15.7 25.0 12.7 t=0.32 0.7501 d ring 25 -9.8 30.1 26.2 15.1 t=-1.37 0.1831 e ring 25 -1.1 19.4 45.0 20.3 t=-1.84 0.0782 whole grid 25 -23.0 46.1 132.6 50.5 t=2.28 0.0328+ 2-sample t test + differences are significant but in the opposite direction than would be expected (the number of termites in clorfenapyr grids increases with respect to controls after treatment) . 649 miguelena, j.g. & p.b. baker — foraging populations of tube building termites appreciated when only the treatment zone is considered (fig 4). inside it, g. perplexus numbers dropped to zero after treatment and did not recover afterwards. a significant decrease with respect to the controls was observed when whole plots where considered, as well as with the treatment zone (table 2). for the c-ring, directly next to the treatment zone, results were suggestive of an effect although not significant (p=0.0614, see table 2). although g. perplexus were not completely eliminated from the outermost area of the fig. 4 average number of g. perplexus workers collected by month for the stations inside the treatment zone (a , b rings and x) for each treatment. the dates of application of the two treatments are marked with vertical lines of the corresponding pattern. fig. 3 g.perplexus density (workers per station) before and after treatment with chlorfenapyr (phantom®). results are shown for different zones within the grid: the treatment zone and each of the rings external to it. the standard error of the mean was used for error bars. 650 sociobiolog y vol. 59, no. 3, 2012 grid (the e ring ), their numbers decreased significantly with respect to the controls (table 2). a spatial pattern for the density of g. perplexus was also evident as a result of fipronil application (fig. 5). after treatment, termites were almost entirely absent from the treatment zone and the c ring. in contrast, the d and e rings had considerable numbers of termites present. the e ring, which is external to fig. 5 termite density (workers per station) before and after treatment with fipronil (termidor®). results are shown for different zones within the grid: the treatment zone and each of the rings outside. the standard error of the mean was used for the error bars. different letters are used to show significant differences in medians when these are present (determined with post-hoc mann-whitney tests with a bonferroni correction). notice the different scales used in the vertical axes of the graphs. table 2 mean difference in the number of termites collected between fipronil (termidor®) designated plots and untreated controls before and after treatment. mean negative values indicate greater termite abundance with respect to control plots. a cubic root transformation was used to improve the normality and homogeneity of variance of the data. also, a welch’s anova was used when the data lacked homogeneity of variance after transformation. differences between the numbers of g. perplexus collected from fipronil and control grids before treatment after treatment area considered n mean ± se mean ± se test statistic p test performed treatment zone 18 62.2 31.0 -7.0 3.3 f=7.70 0.0284* welch’s anova on cubic root transformed datac ring 27 65.0 50.9 -8.7 4.2 f=5.07 0.0614 d ring 27 -20.6 17.3 -23.3 15.1 t=1.20 0.2416 2-sample t test on cubic root transformed data e ring 27 162.5 91.4 18.8 17.5 t=2.10 0.0457* whole plot 27 269.0 156.9 -17.0 26.5 t=2.06 0.0496* * number of termites collected decreased significantly with respect to controls after fipronil treatment. 651 miguelena, j.g. & p.b. baker — foraging populations of tube building termites all others, had the highest density of g. perplexus, although this was still lower than the termite density in the same area before treatment. the differences between the numbers of termites collected in different rings after treatment were significant (kruskal-wallis test, h=10.89, hc=31.57, p=0.0123) and they delimited two distinct areas in the termite grid (fig.5). this pattern could be explained by the capacity of fipronil for horizontal transfer between termites. in trials performed with the subterranean termite reticulitermes hesperus, termite mortality was delayed up to one week at low doses which allowed for the transfer of fipronil to termites that were not directly exposed (saran and rust 2007). in the same study, it was determined that termites could transport fipronil to a distance of at least 3 meters from the exposure site. our results suggest that fipronil could have an effect on g. perplexus at 6 meters or more from the site of application. in summary, these results show that fipronil has a strong negative effect on g. perplexus when applied as described. applications of this termiticide would affect g. perplexus in the direct proximity of structures, and would also have an effect on termite populations in the surrounding areas, although to a lesser extent. it remains to be tested whether this is a direct result of the insecticidal activity of fipronil or is caused by a disruption of the normal movement patterns of g. perplexus. on the other hand, chlorfenapyr seems to slightly favor g. perplexus populations, probably as a result of the elimination of competing termites. in general, the use of chlorfenapyr would allow the persistence of g. perplexus and the preservation of the ecological services that this termite provides. acknowledgments we would like to thank basf for their support of this project. the authors also want to thank phil labbe for technical assistance with data collection and michael haverty and michael riehle for reviewing earlier drafts of the manuscript. this article reports the results of research only. mention of a proprietary product does not constitute an endorsement or a recommendation by the usda for its use. 652 sociobiolog y vol. 59, no. 3, 2012 references baker, p. b. & r. marchosky. 2005. arizona termites of economic importance. university of arizona publication #az1369. 1-19. baker, p., r. marchosky, d. cox & e. roper. 2010. evaluation of small impasse termite barrier registered plots around utility penetrations and vertical walls against two subterranean termites, heterotermes aureus and gnathamitermes perplexus (isoptera) in southern arizona. sociobiolog y 55:339-352. haverty, m. i. & w. l. nutting. 1975. density, dispersion & composition of desert termite foraging populations and their relationship to superficial dead wood. environ. entomol. 4:480-486. jones, s. c., m. w. trosset & w. l. nutting. 1987. biotic and abiotic influences on foraging of heterotermes aureus (snyder)(isoptera: rhinotermitidae). environ. entomol. 16:791-795. jones, s. c. & w. l. nutting. 1989. foraging ecolog y of subterranean termites in the sonoran desert. special biotic relationships in the arid southwest. university of new mexico press, albuquerque: 79-106. jones, s. & m. trosset. 1991. interference competition in desert subterranean termites. entomologia experimentalis et applicata 61:83-90. la fage, j. p. 1974. environmental factors controlling the foraging behavior of a desert subterranean termite, gnathamitermes perplexus (banks). university of arizona. la fage, j., m. haverty & w. nutting. 1976. environmental factors correlated with foraging behavior of a desert subterranean termite, gnathamitermes perplexus (banks)(isoptera: termitidae). sociobiolog y 2: 155-169. light, s. 1934. the desert termites of the genus amitermes. termites and termite control. university of california press, berkeley, calif 795:199-205. nutting, w. l., m. i. haverty & j. p. la fage. 1973. foraging behavior of two species of subterranean termites in the sonoran desert of arizona. proc. vii congress iussi 298-301. nutting, w., m. haverty & j. lafage. 1987. physical and chemical alteration of soil by two subterranean termite species in sonoran desert grassland. journal of arid environments 12:233-239. pedigo, l. p. & m. e. rice. 2006. entomolog y and pest management. 5th edition. prentice hall. saran, r. k. & m. k. rust. 2007. toxicity, uptake & transfer efficiency of fipronil in western subterranean termite (isoptera: rhinotermitidae). journal of economic entomolog y 100:495-508. su, n. y. & r. h. scheffrahn. 1990. economically important termites in the united states and their control. sociobiolog y 17:77-94. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4597sociobiology 67(1): 1-12 (march, 2020) introduction the atlantic forest is considered one of the main conservation hotspots (mayers et al., 2000). this biome is recognized as one of the richest and most threatened on the planet, with greater biodiversity and rate of endemism of fauna and flora species in process of degradation and extinction (myers et al., 2000; fundação sos mata atlântica & conservação internacional, 2005). this biome comprises a set of forest formations and associated ecosystems that include: dense ombrophilous forests, mixed ombrophilous forest, open ombrophilous forest, semideciduous forest, deciduous seasonal forest, restingas (coastal vegetation), mangroves, altitudinal grasslands and countryside swamps, and northeastern forest enclaves, with climatic and altimetric variations in the different latitudes (schäffer & prochnow, 2002). abstract this study was developed aiming to compile data concerning the occurrence and distribution of social wasp species from the brazilian atlantic forest, as well as recording exclusive and rare species. for this purpose, we compiled studies from the specialized literature and created a table containing the species occurrence in the different states which present the phytogeographic domain. a total of 170 species was recorded, corresponding to almost a half of the richness of social wasps in brazil, including 50 restricted occurence, with highlight to rio de janeiro state, which nevertheless is insufficiently sampled. this way, from this work, it is concluded that brazilian atlantic forest must be seen as an important refuge for polistinae. sociobiology an international journal on social insects mm souza¹, gs teofilo-guedes¹, lr milani², asb de souza³, pp gomes³ article history edited by gilberto m. m. santos, uefs, brazil received 09 july 2019 initial acceptance 27 september 2019 final acceptance 15 october 2019 publication date 18 april 2020 keywords mischocyttarus, polybia, polistes. corresponding author patricia p gomes instituto federal de educação, ciência e tecnologia do paraná campus umuarama rodovia pr 323, km 310. cep 87507-014 parque industrial umuarama-pr, brasil. e-mail: patricia.gomes@ifpr.edu.br in brazil, the original atlantic forest represented an area of 1,306,421 km2, approximately 15% of the national territory. the successive economic cycles, based on the exploitation of natural resources over the years, have caused a high destruction of the original forest. as consequence, there was a drastic reduction of forests, forming isolated fragments (castro et al., 2015). nowadays, studies indicate the deforestation of 29,075 hectares in the 17 brazilian states, an increase of 57.7% over the previous period (20142015) (fundação sos mata atlântica & instituto nacional de pesquisas espaciais, 2017). these aspects should stimulate research involving the biota of the atlantic forest. however, there are still few taxa with relevant scientific knowledge for the biome, and among invertebrates, only butterflies have an abundance of information (fundação sos mata atlântica & conservação internacional, 2005; pinto et al., 2006). this shows the importance 1 instituto federal de educação, ciência e tecnologia sul de minas, campus inconfidentes, minas gerais, brazil 2 partner biologist at instituto federal de educação, ciência e tecnologia sul de minas, campus inconfidentes the zoology lab, minas gerais, brazil 3 instituto federal de educação, ciência e tecnologia do paraná, campus umuarama, paraná, brazil review social wasps (vespidae: polistinae) from the brazilian atlantic forest mm souza, gs teofilo-guedes, lr milani, asb de souza, pp gomes – social wasps from the brazilian atlantic forest2 of studying the other groups of invertebrates that inhabit the atlantic forest, such as the social wasps. these animals are characteristic of neotropical insect fauna (silveira, 2002), being the brazilian fauna the most diverse in the world with 347 species distributed in three tribes: mischocyttarini (mischocyttarus and 117 species), polistini (polistes and 38 species) and epiponini (19 genera and 164 species) (carpenter & marques, 2001; carpenter, 2004; carpenter & andena, 2013). the taxonomic catalog of brazilian fauna brings some data about the polistinae. in according with the digital catalog, epiponini, mischocyttarini and polistini tribes present records for the north, northeast, midwest, southeast and south regions. however, there is not information about endemism for the three tribes (hermes et al., 2019). social wasps are fundamental in the communities in which they inhabit, since they participate in a singular way of the food webs. they act as predators or in the transport of pollen grains, being part of the pollinator community of some plant species (hermes & köhler, 2006; sühs et al., 2009; somavilla & köhler, 2012). some species still act as bioindicators of environmental quality in natural or conservation areas (souza et al., 2010). systematic studies on the social wasp community in the atlantic forest date back to the first half of the 20th century, with the european naturalists joseph francisco zikán in the itatiaia national park and adolpho ducke in different areas of southeastern brazil (richards, 1978). later, owain w. richards published the book “the social wasps of america: excluding the vespinae” that became reference for neotropical vespid taxonomists. after nearly 40 years, about 79 inventories were performed (barbosa et al., 2016a; jacques et al., 2018), sampling different ecosystems, such as areas of primary evergreen forests (souza et al., 2012), urban environments (oliveira et al., 2017) and agricultural areas (jacques et al., 2015). based on the locations where these surveys were conducted, it is possible to observe a gap on the knowledge of the social wasp community for the atlantic forest, because many areas have not yet been sampled (barbosa et al., 2016a; souza et al., 2017). therefore, it is not possible to determine precisely the number of species of social vespids for this biome, nor the occurrence and distribution of rare or endemic species (de souza et al., 2012). therefore, the goal of this study was to gather information on the social wasp community in the ecosystems of the atlantic forest biome in brazil, in order to group the distribution data, to produce a list of species for the biome, and to register endemic and rare species. also, social wasps’ similarity comparisons among the different phytophysiognomies of the studied biome were carried out. material and methods in the present study the following phytophysiognomies of the atlantic forest were considered: dense ombrophilous forests; mixed ombrophilous forest (semideciduous forest associated with araucaria forest); open ombrophilous forest; semideciduous seasonal forest; deciduous seasonal forest (also known as mata seca); altitudinal grasslands; areas of pioneer formations, known as mangroves, sandbanks, saline fields and alluvial areas; vegetative refuges; ecological tension areas; countryside swamps and northeastern forest enclaves, represented by disjunctions of dense ombrophilous forests, open ombrophilous forest, semideciduous seasonal forest, deciduous seasonal forest and native vegetation of the coastal and oceanic islands (brasil, 2008). it should be emphasized that the rupestrian fields and the dry woods (deciduous seasonal forest) may be associated with other biomes (oliveira-filho, 2006). therefore, in this study, were considered only the studies where these phytophysiognomies are in areas of atlantic forest. the files used as reference in the present study were obtained through the following research platforms: scielo, scopus, google scholar and research gate. articles and books with works on diversity were analyzed, as well as descriptions of new species carried out in different phytophysiognomies of the atlantic forest biome, including associated agricultural ecosystems (table 1), not considering works of ethology or of any other nature not related previously in the text. in the studies of lima et al. (2010) and tanaka et al. (2011) (table 1), different municipalities were sampled in são paulo state. however, only those located in the atlantic forest domain, according to the map proposed by the ministry of the environment, were considered (brasil, 2008). besides the material obtained in the research platforms, the book “the social wasps of america” (richards, 1978) was used considering only the species that had geographic reference about their locality of collection in the respective state, and disregarding the others. this is because, in many cases, only the state in which the species was collected is mentioned, so that it is not possible to define if the occurrence of the same was associated to the biome of the present study. inventory study data were also included,, one carried out in the machado river environmental protection area (21°47’53.93” s, 46°7’29.34” w), south of minas gerais, under sisbio collecting permits (63914-1) and ief-mg (062/2018), which began in september 2018, and another in ilha grande national park (23°41′51″s, 54°0′35″w), northwest of paraná, under sisbio collecting permits (sisbio 65047-2), which began in february 2019. both studies are using active search for species registration with the use of entomological net to collect the specimens. the material is being sorted, assembled and identified in the zoology laboratory of ifsuldeminas, campus inconfidentes, minas gerais. the species of conflicting identification are being sent to dr. orlando tobias da silveira, emílio goeldi museum, belém, pará, for taxonomic confirmation. a binary matrix, with presence–absence data of species on each region, was used for clustering analysis, using the similarity matrix obtained by jaccard’s community coefficient (sj), with the dendrogram constructed from the jaccard sociobiology 67(1): 1-12 (march, 2020) 3 table 1. list of inventories studies and description of new species of social wasps carried out in different phytophysiognomies of atlantic forest domain in brazil, informing authors and year of publication, state where they were carried out and number of species. authors and year of publication phytophysiognomy state number of species rodrigues & machado, 1982 semideciduous seasonal forest são paulo 33 lorenzato (1985) agrosystem associated with mixed forest santa catarina 12 raw (2000) deciduous seasonal forest bahia 01 hermes & kohler (2006) mixed forest rio grande do sul 25 souza & prezoto (2006) semideciduous seasonal forest minas gerais 38 santos et al. (2007) ombrophilous forest, coastal vegetation and mangrove bahia 21 sühs et al. (2009) deciduous seasonal forest rio grande do sul 25 auad et al. (2010) agrosystem associated with semideciduous seasonal forest minas gerais 13 gomes & noll (2010) semideciduous seasonal forest são paulo 12 lima et al. (2010) semideciduous seasonal forest são paulo 19 menezes et al. (2010) agrosystem associated with ombrophilous forest bahia 01 prezoto & clemente (2010) seasonal forest associated with rupestrian fields minas gerais 23 souza et al. (2010) semideciduous seasonal forest associated with rupestrian fields minas gerais 32 souza et al. (2010) semideciduous seasonal forest minas gerais 36 tanaka et al. (2011) semideciduous seasonal forest são paulo 29 menezes et al. (2011) ombrophilous forest bahia 1 bonfim & antonialli-júnior (2012) semideciduous seasonal forest mato grosso do sul 18 de souza et al. (2012) semideciduous seasonal forest associated with agrosystem minas gerais 8 souza et al., 2012 seasonal forest associated with rupestrian fields minas gerais 32 jacques et al. (2012) semideciduous seasonal forest minas gerais 26 souza et al. (2012) semideciduous seasonal forest and evergreen forest minas gerais 38 clemente et al. (2013) semideciduous seasonal forest associated with rupestrian fields minas gerais 15 togni et al. (2014) semideciduous seasonal and ombrophilous forests são paulo 31 albuquerque et al. (2015) semideciduous seasonal forest minas gerais 34 locher et al. (2014) semideciduous seasonal forest associated with monoculture são paulo 31 freitas et al. (2015) semideciduous seasonal forest associated with agrosystem minas gerais 19 klein et al. (2015) semideciduous seasonal forest associated withagrosystem rio grande do sul 15 souza et al. (2015a) mixed forest and altitudinal grasslands minas gerais 22 souza et al. (2015b) semideciduous seasonal forest minas gerais 34 souza et al. (2015c) semideciduous seasonal, ombrophilous and deciduous seasonal forests minas gerais e rio de janeiro 05 aragão & andena (2016) ombrophilous forest bahia 29 barbosa et al. (2016b) semideciduous seasonal forest minas gerais 36 brunismann et al. 2016 deciduous seasonal forest minas gerais 37 virgínio et al. (2016) ombrophilous forest rio grande do norte 20 lopes & menezes, 2017 ombrophilous forest bahia 01 barbosa et al. (2018) ombrophilous forest alagoas 02 jacques et al. (2018) semideciduous seasonal forest associated with urban area minas gerais 40 souza et al. (2018) mixed forest and rupestrian fields minas gerais 11 ribeiro et al. (2019) ombrophilous forest rio de janeiro 29 mm souza, gs teofilo-guedes, lr milani, asb de souza, pp gomes – social wasps from the brazilian atlantic forest4 coefficient data by the upgma (unweighted pair group method with arithmetic mean) clustering method. all data were processed in the softwares past 2.1 (hammer et al., 2001) and fitopac 2.1 (shepherd, 2010). results and discussion a total of 170 social wasp species belonging to 18 genera was recorded in the different phytophysiognomies of atlantic forest biome in brazil (table 2). this richness makes up about 48% of the polistinae fauna of brazil, which currently comprehends 346 species (carpenter & anderna, 2013; lopes & menezes, 2017), highlighting the importance of this biome in maintaining social wasp diversity in the country. however, there is no information for the biome in goiás, sergipe, pernambuco and paraíba states. the jaccard similarity index (table 3) ranged from 0 (when the state of alagoas was compared with the following states: rio grande do sul, são paulo, paraná, santa catarina and rio grande do norte) to 0.50427 (when the comparison was made between the states of são paulo and minas gerais). according to kent and coker (1992), values greater than or equal to 0.5 indicate high similarity. the greatest similarity between são paulo and minas gerais states (fig 1), may be due to two factors: (1) in function of the phytophysiognomy semideciduous forest, which constitutes the largest forest cover in both states; and (2) these states have been the best sampled for vespids, since together they account for 65% of the studies in the atlantic forest of brazil. the similarity test also revealed that states form groupings by region (fig 1). this possibly occurs due to climatic and phytophysiognomic factors, however, the espírito santo state differed from the other southeastern states, resembling more to bahia state. it is also necessary to consider that extreme climatic events, such as glaciations, and consequent expansion and reduction of tropical forests, could form barriers and refuges and thus affect the distribution of vespidae fauna in brazil. this has already been discussed for the genus synoeca (menezes et al., 2017), showing that espírito santo and bahia states have genetically similar populations, which differ from other southeastern states. this may also occur with other genera of vespids, which would support the similarity patterns found in the present study. the genus mischocyttarus presented the largest number of restricted species (n=35) (table 2) and much of this representativeness is the result of two studies published by zikán (1935; 1949) in the itatiaia national park. both studies have taxonomic bias, totaling 109 described species, many of which were no longer recorded after these publications. recently another inventory for social wasps was published by ribeiro et al. (2019) for the itatiaia national park. however, only five species of the genus were recorded, all of wide geographic occurrence. in the year 2018, two species of mischocyttarus identified by zikán in 1935 and 1949 in the itatiaia national park were found in other localities of minas gerais. a mischocyttarus table 3. similarity matrix (jaccard) of social wasps among different states of brazil * rs sp mg rj ba es pr sc rn al ms rs 1 sp 0,30769 1 mg 0,22936 0,50427 1 rj 0,18889 0,28448 0,37121 1 ba 0,11111 0,23077 0,21008 0,15842 1 es 0,22414 0,24176 0,28319 0,2967 0,28571 1 pr 0,325 0,21795 0,16514 0,16279 0,125 0,20755 1 sc 0,33333 0,2439 0,19643 0,21591 0,18644 0,31481 0,2619 1 rn 0,125 0,17568 0,14423 0,12195 0,17021 0,19149 0,1875 0,125 1 al 0 0 0,009524 0,012821 0,02439 0,02381 0 0 0 1 ms 0,175 0,2027 0,14151 0,11905 0,16327 0,13725 0,21212 0,11905 0,28 0,055556 1 *rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul. fig 1. dendrogram from the jaccard similarity index of social wasps among different states of the country (rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul). sociobiology 67(1): 1-12 (march, 2020) 5 interjectus (zikán, 1935) colony was recorded in the machado river environmental protection area, a semideciduous forest in south of the state, and mischocyttarus proximus (zikán, 1949) in the serra do papagaio state park (souza et al., 2018), in altitudinal grassland area. in this place, it was also recorded an unpublished species for brazil, mischocyttarus anthracinus richards, until then with occurrence only in paraguay. from these informations, the itatiaia national park and the serra do papagaio state park, conservation units of the serra da mantiqueira complex, are considered priority areas for the conservation of atlantic forest vespids. besides these conservation units, other regions also have rare species, that is, restricted to a single area inserted in the biome. in minas gerais: rio doce state park (mischocyttarus annulatus) (souza et al., 2012); serra de são josé environmental protection area in the municipality of tiradentes (polistes davillae) (souza et al., 2012); municipality of barroso (mischocyttarus artifex) (souza & prezoto, 2006), pandeiros river wildlife refuge (chartergellus communis and chartergus globiventris) (brunismann et al., 2016). in são paulo: the municipalities of magda, matão and barretos (brachygastra mouleae) (tanaka & noll, 2011), municipality of araçatuba (mischocyttarus aracatubaensis) (richards, 1978), municipalities of magda and bebedouro (mischocyttarus paulistanus) (tanaka & noll, 2011) and municipality of jundiaí (polistes niger) (richards, 1978). in bahia: ecological reserve michelin, municipality of igrapiúna (agelaia cajennensis and apoica pallida) (aragão & andena, 2016), municipality of itabuna(epipona media) (menezes et al., 2010) and the municipality of ilhéus (synoeca ilheensis) (lopes & menezes, 2017). in espírito santo: (mischocyttarus carbonarius carbonarius, mischocyttarus capichaba, mischocyttarus carinulatus and polistes deceptor) (richards, 1978). there is no information about exact location. in paraná: (mischocyttarus claretianus and mischocyttarus curitybanus) (richards, 1978). there is no information about exact location. in rio de janeiro: all species were recorded in the itatiaia national park (metapolybia bromelicula, mischocyttarus alternatus, mischocyttarus brackmanni, mischocyttarus buyssoni, mischocyttarus cabauna, mischocyttarus confirmatus, mischocyttarus confusoides, mischocyttarus costalimai, mischocyttarus crypticus, mischocyttarus cryptobius, mischocyttarus declaratus, mischocyttarus extinctus, mischocyttarus infrastrigatus, mischocyttarus itatiayaensis, mischocyttarus mimicus, mischocyttarus mutator, mischocyttarus similatus, mischocyttarus travassosi, polybia emaciata and polybia tinctipennis) (richards, 1978). in mato grosso do sul: municipality of batayporã, between são paulo and paraná states (polistes brefissus) (bonfim & antonialli júnior, 2012). the state of rio de janeiro has the highest number of species of restricted occurrence (n=20), followed by minas gerais (n=06), bahia, são paulo and espírito santo (n=04), paraná (n=02), mato grosso do sul (n=01). this result is due to the collection effort performed by zikán over a period of 26 years, in the first half of the 20th century, in the itatiaia national park (academia itatiaiense de história, 2015), but may also be a reflection of the forest cover, either by extension or conservation degree, of this conservation unit. these data reinforce the importance of this area for the conservation of vespids in brazil. several studies carried out in different areas of the atlantic forest commonly record occurrences of unpublished species for their respective states (souza & prezoto, 2006; menezes et al., 2010; 2011; souza et al., 2012, 2015c, 2018; brunismann et al., 2016; ribeiro et al., 2019). this shows that the fauna of vespids of the biome is possibly greater than already known. this is because most of the studies are restricted to the southeast of the country, mainly são paulo, minas gerais and rio de janeiro (fig 2). fig 2. localities where diversity studies were carried out, new records or description of new species of social wasps in atlantic forest ecosystems. source: ministério do meio ambiente, 2008. mm souza, gs teofilo-guedes, lr milani, asb de souza, pp gomes – social wasps from the brazilian atlantic forest6 table 2. species of social wasps recorded in different states of brazil: rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul (presence 1/absence 0) (+ rare species / restricted occurrence*). species of social wasps 170 rs sp mg rj ba es pr sc rn al ms agelaia angulata (fabricius, 1804) 0 1 1 0 1 0 0 0 0 0 0 agelaia cajennensis (fabricius, 1798) + 0 0 0 0 1 0 0 0 0 0 0 agelaia centralis (cameron, 1907) 0 1 1 0 0 0 0 0 0 0 0 agelaia multipicta (haliday, 1836) 1 1 1 1 0 0 0 0 0 0 0 agelaia myrmecophila (ducke, 1905) 0 0 1 0 1 0 0 0 0 0 0 agelaia pallipes (olivier, 1791) 0 1 1 0 0 0 0 0 1 0 1 agelaia vicina (de saussure, 1854) 1 1 1 0 0 0 0 0 0 0 0 angiopolybia pallens (lepeletier, 1836) 0 1 0 0 1 1 0 1 0 0 0 apoica flavissima van der vecht, 1973 0 1 0 1 0 0 0 0 0 0 0 apoica gelida van der vecht, 1973 0 0 1 0 0 0 0 0 1 0 0 apoica pallens (fabricius, 1804) 0 1 1 0 1 0 0 0 0 0 1 apoica pallida (oliver, 1791) + 0 0 0 0 1 0 0 0 0 0 0 apoica thoracica du buysson, 1906 0 0 1 0 0 1 0 0 0 0 0 brachygastra augusti (de saussure, 1854) 1 1 1 0 0 0 1 0 0 0 1 brachygastra lecheguana (latreille, 1824) 1 1 1 1 0 1 1 1 1 0 0 brachygastra moebiana (de saussure, 1867) 0 1 1 0 0 0 0 0 0 0 0 brachygastra mouleae richards, 1978 +/* 0 1 0 0 0 0 0 0 0 0 0 chartergellus communis richards, 1978 + 0 0 1 0 0 0 0 0 0 0 0 chartergus globiventris de saussure, 1854 + 0 0 1 0 0 0 0 0 0 0 0 clypearia angustior ducke, 1906 0 0 1 1 0 0 0 0 0 0 0 epipona media cooper, 2002 + 0 0 0 0 1 0 0 0 0 0 0 epipona tatua (cuvier, 1797) 0 0 1 0 0 1 0 0 0 0 0 leipomeles dorsata (fabricius, 1804) 0 0 0 0 1 1 0 0 0 0 0 metapolybia bromelicula araújo, 1945 +/* 0 0 0 1 0 0 0 0 0 0 0 metapolybia cingulata (fabricius, 1804) 0 1 1 0 1 0 0 0 0 0 0 metapolybia decorata (gribodo, 1896)* 0 0 0 1 1 0 0 0 0 0 0 metapolybia docilis richards, 1978 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus anthracinus richards, 1945 +/* 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus adjectus zikán, 1935 * 0 0 0 1 0 0 0 1 0 0 0 mischocyttarus annulatus richards, 1978 + 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus alternatus zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus aracatubaensis zikán, 1949 +/* 0 1 0 0 0 0 0 0 0 0 0 mischocyttarus araujoi zikán, 1949 * 0 1 1 1 0 0 0 0 0 0 0 mischocyttarus artifex (ducke, 1914) + 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus bahiae richards, 1945* 0 0 1 0 1 0 0 0 0 0 0 mischocyttarus bahiaensis zikán, 1949 0 1 1 0 1 0 0 0 0 0 0 mischocyttarus bertonii ducke, 1918 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus brackmanni zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus buyssoni (ducke, 1906) * 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus cabauna zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus carbonarius carbonarius de saussure, 1854 + 0 0 0 0 0 1 0 0 0 0 0 mischocyttarus capichaba zikán, 1949 +/* 0 0 0 0 0 1 0 0 0 0 0 mischocyttarus carinulatus zikán, 1949+/* 0 0 0 0 0 1 0 0 0 0 0 mischocyttarus cassununga (r. von ihering, 1903) 1 1 1 1 0 0 0 0 0 0 0 mischocyttarus catharinaensis zikán, 1949 +/* 0 0 0 0 0 0 0 1 0 0 0 sociobiology 67(1): 1-12 (march, 2020) 7 mischocyttarus cerberus ducke, 1918 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus claretianus zikán, 1949 +/* 0 0 0 0 0 0 1 0 0 0 0 mischocyttarus confirmatus zikán, 1935 + 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus confusoides zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus confusus zikán, 1935 * 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus costalimai zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus crypticus zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus cryptobius zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus curitybanus zikán, 1949 + 0 1 0 0 0 0 1 0 0 0 0 mischocyttarus declaratus zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus davillae richards, 1978 + 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus drewseni de saussure, 1857 1 1 1 1 0 1 0 1 0 1 1 mischocyttarus extinctus zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus flavoscutellatus zikán, 1935 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus fluminensis zikán, 1949 * 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus frontalis (fox, 1898) 0 0 1 0 0 0 1 0 0 0 0 mischocyttarus funerulus zikán, 1935 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus garbei zikán, 1935 * 0 0 0 1 0 1 0 0 0 0 0 mischocyttarus giffordi raw, 1985 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus hoffmanni zikán, 1949 * 1 1 0 0 0 0 0 1 0 0 0 mischocyttarus ignotus zikán, 1949 1 1 1 0 0 0 0 0 0 0 0 mischocyttarus iheringi zikán, 1935 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus infrastrigatus zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus interjectus zikán, 1935 * 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus itatiayaensis zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus labiatus (fabricius, 1804) 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus latior (fox, 1898) 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus marginatus (fox, 1898) 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus mattogrossoensis zikán, 1935 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus mirificus zikán, 1935 0 0 1 1 0 0 0 0 0 0 0 mischocyttarus mimicus zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus montei zikán, 1949 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus mourei zikán, 1949 * 0 0 1 0 0 0 1 1 0 0 0 mischocyttarus mutator zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus nomurae richards, 1978 + 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus paraguayensis zikán, 1935 0 1 1 0 0 0 1 1 0 0 0 mischocyttarus parallellogrammus zikán, 1935 * 0 1 1 1 0 0 0 1 0 0 0 mischocyttarus paulistanus zikán, 1935 +/* 0 1 0 0 0 0 0 0 0 0 0 mischocyttarus plaumanni zikán, 1949 + 0 0 0 0 0 0 0 1 0 0 0 mischocyttarus proximus zikán, 1949 +/* 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus punctatus (ducke, 1904) 0 0 1 0 0 0 0 0 0 0 0 mischocyttarus riograndensis richards, 1978 * 1 0 0 0 0 0 0 0 0 0 0 mischocyttarus richardsi zikán, 1949 + 0 0 0 0 0 0 0 1 0 0 0 mischocyttarus rotundicollis (cameron, 1912) 1 1 1 1 1 1 0 1 0 0 0 mischocyttarus santacruzi raw, 2000 0 0 0 0 1 0 0 0 0 0 0 mischocyttarus saussurei zikán, 1949 * 0 1 1 0 0 0 0 0 0 0 0 species of social wasps 170 rs sp mg rj ba es pr sc rn al ms table 2. species of social wasps recorded in different states of brazil: rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul (presence 1/absence 0) (+ rare species / endemics *). (continuation) mm souza, gs teofilo-guedes, lr milani, asb de souza, pp gomes – social wasps from the brazilian atlantic forest8 mischocyttarus scitulus zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus similatus zikán, 1935 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus socialis (de saussure, 1854) 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus souzalopesi zikán, 1949 +/* 0 1 0 0 0 0 0 0 0 0 0 mischocyttarus tomentosus zikán, 1935 0 0 0 0 1 1 0 0 0 0 0 mischocyttarus travassosi zikán, 1949 +/* 0 0 0 1 0 0 0 0 0 0 0 mischocyttarus tricolor richards, 1945 0 1 1 0 0 0 0 0 0 0 0 mischocyttarus wagneri (du buysson, 1908) 0 1 1 1 0 0 0 0 0 0 0 mischocyttarus wygodzinskyi zikán, 1949 * 0 1 1 1 0 0 0 0 0 0 0 mischocyttarus ypiranguensis da fonseca, 1926 0 1 1 0 0 0 0 0 0 0 0 parachartergus fraternus (gribodo, 1892) 0 1 1 0 0 0 0 0 0 0 0 parachartergus pseudapicalis willink, 1959 0 1 0 1 1 0 0 1 0 0 0 parachartergus smithii (de saussure, 1854) 0 1 1 0 0 0 0 0 0 0 0 parachartergus wagneri du buysson, 1904 * 0 0 1 1 0 0 0 0 0 0 0 polistes actaeon haliday, 1836 1 1 1 0 0 1 0 1 0 0 0 polistes bicolor lepeletier, 1836 + 0 0 1 0 0 0 0 0 0 0 0 polistes biguttatus haliday, 1836 1 0 0 1 0 0 1 1 0 0 0 polistes billardieri fabricius, 1804 1 1 1 0 1 0 0 0 1 0 1 polistes binotatus de saussure, 1853 + 0 0 0 1 0 0 0 0 0 0 0 polistes brevifissus richards, 1978 + 0 0 0 0 0 0 0 0 0 0 1 polistes canadensis (linnaeus, 1758) 0 1 1 0 1 0 0 0 1 0 0 polistes carnifex (fabricius, 1775) 0 1 1 1 0 1 1 0 1 0 0 polistes cavapyta de saussure, 1853 1 0 0 0 0 0 0 0 0 0 0 polistes cavapytiformis richards, 1978 1 0 1 0 0 0 0 0 0 0 0 polistes cinerascens de saussure, 1854 1 1 1 1 0 1 1 1 0 0 1 polistes consobrinus de saussure, 1858 * 1 1 0 0 0 0 1 0 0 0 0 polistes davillae richards, 1978 + 0 0 1 0 0 0 0 0 0 0 0 polistes deceptor schulz, 1905 + 0 0 0 0 0 1 0 0 0 0 0 polistes ferreri de saussure, 1853 1 1 1 0 0 0 1 0 0 0 0 polistes geminatus geminatus fox, 1898 0 1 1 0 0 0 0 0 0 0 1 polistes goeldii ducke, 1904 0 0 1 0 0 0 0 0 0 0 0 polistes lanio (fabricius, 1775) 1 1 1 1 0 1 1 0 0 0 0 polistes melanossoma de saussure, 1853 0 0 1 0 1 1 0 1 0 0 0 polistes niger brèthes, 1903 + 0 1 0 0 0 0 0 0 0 0 0 polistes occipitalis ducke, 1904 0 0 1 1 0 1 0 0 0 0 0 polistes pacificus flavopictus ducke, 1918 * 0 0 1 1 0 1 0 0 0 0 0 polistes pacificus pacificus fabricius, 1804 1 0 1 0 0 0 0 0 0 0 0 polistes satan bequaert, 1940 0 0 1 1 0 0 0 0 0 0 0 polistes simillimus zikán, 1948 1 1 1 0 0 0 1 1 0 0 1 polistes subsericeus de saussure, 1854 0 1 1 1 0 0 0 0 0 0 1 polistes versicolor (olivier, 1792) 1 1 1 1 0 1 0 1 1 0 1 polybia bifasciata de saussure, 1854 0 1 1 1 0 1 0 0 0 0 0 polybia bistriata (fabricius, 1804) 0 0 1 0 1 1 0 0 0 1 0 polybia brunnea (curtis, 1844) * 0 0 0 1 0 0 1 0 0 0 0 polybia catillifex moebius, 1856 0 1 0 1 1 0 0 0 0 0 0 polybia chrysothorax (lichtenstein, 1796) 0 1 1 1 1 1 1 0 1 0 1 species of social wasps 170 rs sp mg rj ba es pr sc rn al ms table 2. species of social wasps recorded in different states of brazil: rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul (presence 1/absence 0) (+ rare species / endemics *). (continuation) sociobiology 67(1): 1-12 (march, 2020) 9 polybia dimidiata (olivier, 1792) 0 1 1 1 0 1 0 0 0 0 0 polybia emaciata lucas, 1879 + 0 0 0 1 0 0 0 0 0 0 0 polybia erythrothorax richards, 1978 0 0 1 1 0 0 0 0 0 0 0 polybia fastidiosuscula de saussure, 1854 1 1 1 1 0 1 0 1 0 0 0 polybia flavifrons hecuba richards, 1951 0 0 1 1 0 0 0 1 0 0 0 polybia flavitincta fox, 1898 0 0 0 0 1 0 0 0 0 0 0 polybia ignobilis (haliday, 1836) 1 1 1 1 1 1 1 1 1 0 1 polybia jurinei de saussure, 1854 0 1 1 1 1 1 0 0 0 0 1 polybia liliacea (fabricius, 1804) 0 0 1 0 0 0 0 0 0 0 0 polybia lugubris de saussure, 1854 * 0 0 1 0 1 1 0 1 0 0 0 polybia minarum ducke, 1906 1 1 1 1 0 0 0 1 0 0 0 polybia occidentalis occidentalis (olivier, 1791) 0 1 1 1 1 1 0 0 1 0 1 polybia paulista h. von ihering, 1896 0 1 1 1 1 0 1 0 0 0 1 polybia platycephala sylvestris richards, 1978 1 1 1 1 1 0 0 0 0 0 0 polybia procellosa ducke, 1910 0 0 0 0 1 0 0 0 0 0 0 polybia punctata du buysson, 1908 * 0 0 1 1 0 1 0 1 0 0 0 polybia quadrincicta de saussure, 1854 0 1 1 1 0 0 0 0 0 0 0 polybia rejecta (fabricius, 1798) 0 0 1 1 1 1 0 0 1 0 0 polybia ruficeps xanthops schrottky, 1902 0 1 1 0 0 0 0 0 1 0 1 polybia rufitarsis ducke, 1904 0 0 0 0 1 0 0 0 0 0 0 polybia scutellaris (white, 1841) 1 0 1 1 0 1 1 0 0 0 0 polybia sericea (olivier, 1792) 1 1 1 1 1 1 1 1 1 0 1 polybia signata ducke, 1910 0 0 1 0 1 0 0 0 0 0 0 polybia striata (fabricius, 1787) 0 0 1 1 0 1 0 0 0 0 0 polybia tinctipennis fox, 1898 0 0 0 1 0 0 0 0 0 0 0 protonectarina sylveirae (de saussure) 1854 1 1 1 1 1 1 1 1 0 0 0 protopolybia exigua exigua (de saussure, 1854) 0 1 1 1 1 1 1 1 1 0 0 protopolybia sedula (de saussure, 1854) 0 1 1 1 1 1 0 1 0 0 0 pseudopolybia vespiceps (de saussure, 1863) 0 1 1 1 0 1 0 0 0 0 0 synoeca cyanea (fabricius, 1775) 1 1 1 1 1 1 1 1 0 0 0 synoeca ilheensis lopes & menenez, 2017 * 0 0 0 0 1 0 0 0 0 0 0 synoeca septentrionalis richards, 1978 * 0 0 0 0 1 1 0 0 0 0 0 synoeca surinama (linnaeus, 1767) 0 1 1 1 0 0 0 0 1 0 0 total of species for state 30 75 108 80 40 41 23 30 15 2 27 species of social wasps 170 rs sp mg rj ba es pr sc rn al ms table 2. species of social wasps recorded in different states of brazil: rs = rio grande do sul; sp = são paulo; mg = minas gerais; rj = rio de janeiro; ba = bahia; es = espírito santo; pr = paraná; sc = santa catarina; rn = rio grande do norte; al = alagoas; ms = mato grosso sul (presence 1/absence 0) (+ rare species / endemics *). (continuation) fifty species of social wasps have occurrence restricted to the atlantic forest, that is, are considered endemic for this phytogeographical domain (table 2 *), being 27 of occurrence restricted to a single state (table 2 +/*). the state of rio de janeiro has 31 spp, followed by minas gerais (16), são paulo (10), espírito santo and santa catarina (07), bahia (05), paraná and rio grande do sul (03). the rate of endemism of minas gerais is a result of the large number of studies carried out in its territory and its heterogeneity of ecosystems (souza et al., 2017). it is concluded with the present study that the atlantic forest biome constitutes an important refuge for the conservation of the brazilian species of social wasps. however, other studies are still necessary because there are regions without information or sub-sampled. acknowledgments to sisbio and ief-mg for granting the licenses; to the employees of icmbio for the logistics of parna of ilha grande, paraná; to the employees fernando and pedro from ief-mg of the city of machado, mg; to ifsuldeminas campus machado and inconfidentes for the transportation and logistics at the collection of the apa of rio machado bay; and to the students that assisted at data collection. mm souza, gs teofilo-guedes, lr milani, asb de souza, pp gomes – social wasps from the brazilian atlantic forest10 references academia itatiaiense de história. 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(1949). o gênero mischocyttarus saussure (hymenoptera, vespidae), com a descrição de 82 espécies novas. boletim do parque nacional do itatiaia, 1: 1-125. doi: 10.13102/sociobiology.v61i4.570-572sociobiology 61(4): 570-572 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 a scientific note on the presence of functional tibia for pollen transportation in the robber bee lestrimelitta limao smith (hymenoptera: apidae: meliponini) lg von zuben1,, tm nunes2 the robber stingless bees (genus lestrimelitta) have an unusual foraging ecology. there is no record of these bees visiting flowers, but all record suggests that they gather their food exclusively by raiding other stingless bees colonies (nogueira-neto, 1970; wille, 1983; sakagami et al., 1993). through mass attacks the workers rob all kind of nest resources such as pollen, honey, cerumen, and especially larval food (nogueira-neto, 1970; wille, 1983; sakagami, et al., 1993). the uncommon foraging strategy of this genus influenced the evolution of unique behavioral and morphological traits in robber stingless bee workers (sakagami et al., 1993). one of these traits, considered an adaptation for the cleptoparasitic behavior, is the absence of functional corbicula in the workers of lestrimelitta species (kerr, 1951; sakagami and laroca, 1963; wille, 1983; michener, 2000). according to these authors, the corbicula of robber bees’ workers is highly modified and lost its role for transporting pollen. in order to transport the pollen, the abstract stingless bees belonging to the lestrimelitta genus shows a unique foraging ecology for the group. instead of collecting food from flowers, these bees steal their food from other stingless bees’ hives. associated with this ecological feature there are morphological characteristics such as the modification of the tibia and loss of corbicula. the highly modified hind tibia is considered not functional for all species of this genus. however, observations of the pillaging attacks under controlled environment allowed us to verify workers carrying pollen using the hind tibia. this scientific note is the first record of pollen transportation on the legs by stingless bees from lestrimelitta genus. this observation contradicts the current assumption that robber bees’ tibia have missed their total function as a result of the cleptoparasitic behavior. sociobiology an international journal on social insects 1 universidade de são paulo, ffclrp, riberão preto, sp, brazil. 2 universidade de são paulo, fcfrp, riberão preto, sp, brazil. article history edited by cândida maria l. aguiar, uefs, brazil received 30 september 2014 initial acceptance 03 november 2014 final acceptance 30 november 2014 keywords meliponini, cleptoparasitism, corbicula corresponding author lucas garcia von zuben departamento de biologia, ffclrp universidade de são paulo riberão preto, sp, brazil e-mail: lucaszuben@gmail.com workers secrete a liquid, that dissolves the pollen and then swallow it, transporting it in the crop, mixed with nectar (kerr,1951; sakagami ad laroca, 1963; wille, 1983; roubik, 1989). to study the cleptoparistic behavior of lestrimelitta limao smith we placed one colony of this species in a greenhouse (6,4m x 9m x 3,5m) along with three potential hosts’ species: tetragonisca angustula (latreille), frieseomelitta varia (lepeletier) and nannotrigona testaceicornis (lepeletier). the species used in this study were collected at ribeirão preto, são paulo state, brazil, identified by a specialist in stingless bees (dr. sidnei mateus) and samples kept in the collection of the laboratory of animal behavior of ffclrp (university of são paulo). we also placed a variety of flowering plants and artificial feeders (filled with a water/sucrose solution 50% v/v) in order to offer food sources to the flower visiting species. the closed environment allowed the detailed observation of l. limao raids since the very beginning of the attacks. the greenhouse observations were conducted from 2 to 3 hours short note sociobiology 61(4): 570-572 (december 2014) 571 daily during one year and were carried out at the university of são paulo campus ribeirão preto, brazil. during our study, fifteen attacks to the host colonies were witnessed. among several details of l. limao attacks observed, we recorded a specific behavior that contradicts the current knowledge about the biology of the robber bee. during one raid to a f. varia colony it was possible to observe numerous robber bee workers carrying the pollen from the host nest in their hind tibia (fig 1c). the workers were observed manipulating pollen straight from the pollen pots (fig 1b) and some specimens of lestrimelitta were collected in order to confirm the resource transported. this observation is of interest since it shows that even though the tibia of robber bees workers are highly modified and lack the corbicula, the ability to transport pollen using this structure is maintained. interestingly, in different raids towards other host species it was possible to observe the robber bee workers transporting pollen in their crop, as described by kerr (1951). these results show that in certain conditions l. limao workers use both strategies to transport pollen back to their colonies. the pollen stored in the nest differs from the pollen in the flowers, since the former is manipulated by workers and in some species are fermented by the action of microorganisms (menezes et al. 2012). the consistency of the stored pollen also differs from the freshly collected one, this difference could explain the transport strategy chosen by the robber bee, but this hypothesis remains to be tested. the consistency of the stored pollen also has interspecific differences. while in f. varia the stored pollen is dry and unfermented, in scaptotrigona spp the pollen has a pasty consistency and acid smell as a result of fermentation (menezes et al. 2012). therefore, the ability to use different strategies to transport pollen can allow l. limao workers to explore a wide range of host species. workers of all stingless bees are able to carry pollen in their crop during the provisioning of brood cells and also during the reproductive nest swarming (nogueira-neto 1954; zucchi et al., 1999). moreover, some nonparasitic bees (e.g. ceratina and xylocopa) are similarly able to transport pollen on the crop and in their scopa (roubik 1989) showing that this ability of resource transportation on the legs and in the crop is not restricted to meliponini bees. our observation also demonstrates the potential of closed environments, such as greenhouses, to study details of the cleptoparasitic behavior that are difficult or sometimes impossible to study under natural conditions. acknowledgements the authors are grateful to dr. cristiano menezes for the figure of tetragonisca angustula attack and valuable comments to the text and to dr. sidnei mateus for the help with identification of the bees. we are also grateful to the reviewers for their valuable comments on the manuscript. references kerr, w. e. (1951). bases para o estudo da genética de populações dos hymenoptera em geral e dos apinae sociais em particular. an. esc. super. agric. luiz de queiroz, 8: 219-354. menezes, c., vollet neto, a., & imperatriz fonseca, v. l. (2012). a method for harvesting unfermented pollen from stingless bees (hymenoptera, apidae, meliponini). j. apicult. res., 51(3): 240. doi:10.3896/ibra.1.51.3.04 fig 1. pillage of stingless bees nests by the robber bee lestrimelitta limao. (a) aggressive reaction of the host tetragonisca angustula. (b) workers collecting pollen in a frieseomelitta varia nest attack. (c) a lestrimelitta limao worker carrying pollen at the hind tibia during its raid. lg von zuben, tm nunes pollen transportation in lestrimelitta limao572 michener, c. d. (2000). the bees of the world (vol. 1). baltimore: the johns hopkins university press. nogueira-neto, p. (1954). notas bionômicas sobre meliponíneos: iii – sobre a enxameagem. arq mus nac, l42: 419–451. nogueira-neto, p. (1970) behavior problems related to the pillages made by some parasitic stingless bees (meliponinae, apidae). development and evolution of behavior: essays in memory of tc schneirla, 416-434. roubik, d.w. (1989) ecology and natural history of tropical bees. cambridge: cambridge university press. doi: 10.1126/ science.248.4958.1026 sakagami, s. f., & laroca, s. (1963). additional observations on the habits of the cleptobiotic stingless bees, the genus lestrimelitta friese (hymenoptera, apoidea). j. fac. sci. hokkaido univ. series zoology, 15(2): 319-339. sakagami, s. f., roubik, d. w., & zucchi, r. (1993). ethology of the robber stingless bee, lestrimelitta limao (hymenoptera: apidae). sociobiology, 21: 237–277 wille a. (1983) biology of the stingless bees. annu. rev. entomol, 28: 41-64. doi: 10.1146/annurev.en.28.010183.000353 zucchi, r., silva-matos, e. d., nogueira ferreira, f. h., & garcia azevedo, g. (1999). on the cell provisioning and oviposition process (pop) of the stingless bees-nomenclature reappraisal and evolutionary considerations (hymenoptera, apidae, meliponinae). sociobiology, 34: 65-86. 573 the effects of temperature on the foraging activity of red imported fire ant workers (hymenoptera: formicidae) in south china by yong-yue lu*, lei wang, ling zeng & yi-juan xu abstract in this study, we investigated the effects of temperature of the ambient air, ground surface, and the soil at 5-cm deep on the foraging activity of the workers of red imported fire ant, solenopsis invicta buren, in south china with the method of bait traps. significant correlations were observed between the temperature and the foraging activity of fire ants. specifically, when the ambient temperature was above 20 °c, the fire ants foraged actively, and the activity reached a maximum when the ambient temperature was between 25 to 33 °c. but the foraging activity decreased as the ambient temperature rose higher than 34 °c. moreover, fire ants were found to forage at maximal rates with the soil surface temperature between 27 to 40 °c. the ants started foraging when the soil temperature at 5-cm deep was between 16 to 48 °c, while 28~37 °c was the optimal temperature for the foraging activity. the extreme temperature thresholds for foraging of the ambient air, soil-surface, and soil at 5-cm deep were 11 °c/44 °c, 10 °c/57 °c, and 12 °c/48 °c respectively. introduction the red imported fire ant, solenopsis invicta, is an important invasive species. it originated from south america and was introduced into the united sates of america (usa) in 1930s (vazquez et al. 2003). currently, fire ants inhabit about 129.5 million ha. in south usa and puerto rico (williams et al. 2003). it was estimated that fire ants caused more than $1-billion of economic loss each year in the southern states of usa (wojcik et al. 2001). in china, this pest was first detected on september 28, 2004 (zeng et al. red imported fire ant research center, south china agricultural university, guangzhou 510640, china * correspondence address:luyong yue@scau.edu.cn 574 sociobiolog y vol. 59, no. 2, 2012 2005), although lu et al. (2008) inferred that the first fire ant infestation probably occurred as early as in the 1990s. the amount of food collected by workers is critical for the colony’s maintenance and reproduction (bernstein 1979). their forging activity is affected by many environmental factors, such as humidity, soil moisture, daylight, and temperature, among which temperature serving as a primary factor (porter & tschinkel 1987). more specifically, fire ants foraged when the temperature of the soil 2-cm deep was from 15 to 43 °c in florida (porter & tschinkel 1987), while low temperature counteracted the ants’ forging (lofgren et al. 1964). in addition, the time of the year when the fire ants reach their maximal forging activity varied from region to region (vogt et al. 2003). quick adaptation to a new environment is an important characteristic of invasive species. fire ants have shown rapid acclimatization when they were exposed to low temperatures (xu et al. 2009). they can also develop quick adaptation and resistance to desiccation (phillips et al. 1996; xu et al. 2009). furthermore, the climate and environment of china are greatly different from that of south america and the usa. therefore, we hypothesized that the fire ants would show significant differences in foraging activity in china. to test this hypothesis, we first investigated the temperature thresholds for the foraging activity of s. invicta in south china in this study. materials and methods study site this study was conducted from january to july of 2006 in wuchuan, guangdong, china. the size of study site was 4800 m2 (60 m×80 m). more than 95% of the area was covered with grassy weeds. mound density the social type of red imported fire ants at the study site was determined as polyg yne. the active mounds were video-recorded every time before bait traps were set. ant worker sampling worker ants were sampled at bait traps. a total of 12 bait traps were set, with the distance of 6 m between every two traps. early morning (6am), afternoon (2pm), and dusk (6pm) were chosen for ant collection. the sampling 575 lu, y.-y. et al. — effects of temperature on fire ant foraging activity method was based on the previously reported one by briano et al. (2002) with modifications. the bait trap contained a 30 ml plastic vial with a 5-mm thick sausage inside. the traps were retrieved 30 min after they were set on the ground surface. all bait traps were shaded with white plates to keep cool and protect the fire ants from disturbance. trapped ants were dipped into soapy water and counted. the numbers of captured ants were counted for further analysis. ant samples were collected four times in each month. in order to keep the traps at the same position every time, colorful flags were used to mark their positions. ambient air temperature in shade (≈1 m above ground level), unshaded soil-surface temperature, and soil temperature at 5-cm deep were measured at the same time when the traps were set. the temperature taken into further analysis was the mean of those at 6am, 2pm, and 6pm. the number of ant workers per trap was the mean of those captured by the 36 trap vials collected each day. the foraging was considered as active when the per-trap number of worker ants was over 30 individuals. statistical analysis the correlations and models between the number of worker ants and the temperatures were analyzed by using statistical analysis system (sas) (sas institute, 1985). results dynamics of active mound density the active mound densities varied slightly from january to july of 2011 (fig.1). the minimum density was observed in february with 77.1 ants per ha, while the maximum was observed in june with 102.1 ants per ha. effects of temperature on the foraging of worker ants the numbers of red imported fire ant workers captured in baited vials at different temperatures are listed in table 1. from january to july in 2006, the ambient temperature ranged from 11.0 to 35.0 °c; the soil surface temperature ranged from 13.0 to 44.0 °c; and the soil temperature at 5-cm deep ranged from 17.0 to 39.0 °c. the number of workers captured indicated that the foraging was active when the ambient temperature, the soil surface temperature, and the soil temperature at 5-cm deep were above 20 °c, 22 576 sociobiolog y vol. 59, no. 2, 2012 fig. 1 dynamics of the active mound density from june to july of 2011. fig. 2 dynamics of red imported fire ant workers captured in baited vials at different ambient temperatures. each point represents the average number of ants captured per bait trap (n=12). 577 lu, y.-y. et al. — effects of temperature on fire ant foraging activity ta bl e 1. n um be rs o f r ed im po rt ed fi re a nt w or ke rs c ap tu re d in b ai te d vi al s a t d iff er en t t em pe ra tu re s. a m bi en t te m pe ra tu re (° c ) so il su rf ac e te m pe ra tu re (° c ) so il te m pe ra tu re at 5 cm d ep th (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se a m bi en t te m pe ra tu re (° c ) so il su rf ac e te m pe ra tu re ( °c )s oi l t em pe ra tu re at 5 cm d ee p (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se 11 .0 13 .0 17 .0 1. 20 ± 0. 95 27 .0 33 .0 28 .0 10 8. 83 ± 22 .6 0 11 .0 13 .0 17 .0 1. 80 ± 0. 75 27 .0 33 .0 30 .0 11 7. 50 ± 17 .6 8 11 .0 14 .0 17 .0 3. 60 ± 1. 56 27 .0 34 .0 30 .0 14 1. 33 ± 19 .7 3 12 .0 15 .0 18 .0 4. 40 ± 1. 76 27 .0 34 .0 30 .0 18 7. 00 ± 38 .9 4 12 .0 15 .0 18 .0 5. 80 ± 1. 44 28 .0 29 .0 30 .0 11 9. 00 ± 21 .2 7 12 .0 15 .0 18 .0 6. 00 ± 0. 91 28 .0 29 .0 28 .0 11 5. 17 ± 28 .1 5 12 .0 15 .0 19 .0 6. 40 ± 3. 93 28 .0 29 .0 28 .0 99 .3 3± 10 .2 9 12 .0 15 .0 19 .0 5. 60 ± 4. 09 28 .0 28 .0 29 .0 13 0. 17 ± 30 .2 8 12 .0 15 .0 18 .0 5. 80 ± 4. 97 28 .0 30 .0 29 .0 13 5. 17 ± 46 .1 6 12 .0 14 .0 17 .0 3. 40 ± 2. 02 28 .0 31 .0 33 .0 22 5. 50 ± 35 .2 6 12 .0 14 .0 16 .0 16 .0 0± 1. 08 28 .0 29 .0 31 .0 15 5. 17 ± 19 .7 3 12 .5 14 .0 16 .0 16 .0 0± 1. 73 29 .0 31 .0 28 .0 92 .6 7± 8. 68 13 .0 14 .0 16 .0 7. 83 ± 3. 66 29 .0 31 .0 29 .0 10 3. 33 ± 6. 95 13 .0 16 .0 19 .0 7. 60 ± 2. 02 29 .0 32 .0 29 .0 11 2. 17 ± 20 .5 8 13 .0 16 .0 20 .0 13 .4 0± 4. 37 29 .0 31 .0 31 .0 13 3. 00 ± 15 .1 4 14 .0 15 .0 17 .0 9. 83 ± 0. 99 29 .0 31 .0 30 .0 12 6. 83 ± 23 .8 8 14 .0 16 .0 16 .0 16 .0 0± 3. 80 29 .0 30 .0 32 .0 12 2. 67 ± 7. 43 15 .0 15 .0 17 .0 11 .6 7± 1. 70 29 .0 30 .0 29 .0 10 4. 50 ± 19 .5 2 15 .0 18 .0 17 .0 17 .0 0± 0. 88 29 .0 30 .0 29 .0 18 4. 67 ± 21 .9 1 17 .0 17 .0 17 .0 17 .1 7± 1. 39 29 .0 30 .0 31 .0 20 0. 33 ± 13 .6 3 17 .0 18 .0 18 .0 19 .6 7± 4. 99 30 .0 32 .0 30 .0 10 5. 50 ± 3. 20 17 .0 20 .0 19 .0 15 .1 7± 5. 05 30 .0 32 .0 30 .0 91 .3 3± 5. 65 17 .0 22 .0 18 .0 18 .0 0± 2. 29 30 .0 32 .0 31 .0 11 4. 17 ± 8. 40 17 .0 18 .0 19 .5 19 .5 0± 2. 53 30 .0 31 .0 31 .0 10 9. 00 ± 8. 35 18 .0 19 .0 19 .0 22 .5 0± 2. 50 30 .0 33 .0 32 .0 15 2. 17 ± 13 .9 7 578 sociobiolog y vol. 59, no. 2, 2012 ta bl e 1. n um be rs o f r ed im po rt ed fi re a nt w or ke rs c ap tu re d in b ai te d vi al s a t d iff er en t t em pe ra tu re s ( co nt in ue d) . a m bi en t te m pe ra tu re (° c ) so il su rf ac e te m pe ra tu re (° c ) so il te m pe ra tu re at 5 cm d ep th (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se a m bi en t te m pe ra tu re (° c ) so il su rf ac e te m pe ra tu re ( °c )s oi l t em pe ra tu re at 5 cm d ee p (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se 18 .0 20 .0 19 .0 19 .0 0± 6. 41 30 .0 30 .0 30 .0 14 5. 83 ± 20 .3 8 18 .0 22 .0 19 .5 19 .5 0± 4. 52 30 .0 32 .0 32 .0 25 1. 67 ± 16 .5 6 18 .0 19 .0 20 .0 20 .0 0± 6. 19 30 .0 33 .0 30 .0 14 2. 67 ± 18 .9 8 19 .0 20 .0 18 .0 24 .0 0± 2. 42 30 .0 35 .0 35 .0 23 7. 17 ± 14 .9 6 19 .0 20 .0 21 .0 26 .6 7± 6. 97 31 .0 33 .0 31 .0 12 9. 00 ± 9. 72 19 .0 25 .0 21 .0 21 .0 0± 4. 90 31 .0 34 .0 31 .0 11 9. 00 ± 14 .7 7 19 .0 25 .0 22 .0 22 .0 0± 3. 61 31 .0 34 .0 32 .0 12 0. 50 ± 6. 56 19 .0 22 .0 21 .5 21 .5 0± 2. 06 31 .0 34 .0 33 .0 10 5. 50 ± 15 .1 5 19 .0 21 .0 21 .0 21 .0 0± 6. 94 31 .0 33 .0 31 .0 15 1. 17 ± 24 .4 0 20 .0 22 .0 19 .0 30 .0 0± 3. 46 31 .0 33 .0 34 .0 13 8. 33 ± 11 .2 9 20 .0 20 .0 19 .0 32 .3 3± 3. 52 31 .0 34 .0 32 .0 10 7. 83 ± 18 .6 0 20 .0 23 .0 22 .0 70 .3 3± 18 .1 1 31 .0 37 .0 36 .0 11 7. 50 ± 23 .3 2 21 .0 23 .0 19 .0 37 .0 0± 4. 56 31 .0 33 .0 34 .0 15 8. 83 ± 11 .2 0 21 .0 23 .0 20 .0 42 .8 3± 5. 48 31 .0 32 .0 30 .0 18 9. 83 ± 22 .2 6 21 .0 22 .0 20 .0 44 .8 3± 6. 57 31 .0 33 .0 33 .0 20 3. 17 ± 16 .1 1 21 .0 22 .0 23 .0 78 .0 0± 18 .2 2 31 .0 32 .0 32 .0 26 9. 17 ± 31 .4 2 21 .0 21 .0 22 .0 71 .6 7± 17 .0 9 31 .0 33 .0 32 .0 23 4. 00 ± 9. 74 21 .0 21 .0 22 .0 73 .3 3± 17 .5 0 31 .0 33 .0 32 .0 22 0. 67 ± 10 .5 8 22 .0 24 .0 23 .0 86 .8 3± 16 .4 1 31 .0 35 .0 31 .0 15 3. 50 ± 23 .0 4 22 .0 22 .0 24 .0 78 .6 7± 10 .7 0 31 .0 37 .0 32 .0 17 0. 83 ± 20 .4 3 22 .0 24 .0 25 .0 79 .8 3± 16 .3 6 32 .0 35 .0 33 .0 12 9. 83 ± 14 .7 3 23 .0 25 .0 27 .0 88 .0 0± 20 .0 5 32 .0 35 .0 33 .0 13 5. 83 ± 24 .7 7 23 .0 24 .0 26 .0 81 .1 7± 18 .3 8 32 .0 36 .0 35 .0 16 6. 67 ± 13 .9 9 24 .0 25 .0 26 .0 89 .8 3± 22 .3 0 32 .0 36 .0 33 .0 11 7. 00 ± 26 .0 1 24 .0 26 .0 24 .0 91 .6 7± 22 .0 4 32 .0 37 .0 36 .0 11 3. 50 ± 25 .0 7 24 .0 28 .0 28 .0 88 .6 7± 19 .1 8 32 .0 35 .0 31 .0 13 8. 17 ± 22 .9 4 579 lu, y.-y. et al. — effects of temperature on fire ant foraging activity ta bl e 1 n um be rs o f r ed im po rt ed fi re a nt w or ke rs c ap tu re d in b ai te d vi al s a t d iff er en t t em pe ra tu re s ( co nt nu ed ). a m bi en t te m pe ra tu re (° c ) s oi l s ur fa ce te m pe ra tu re (° c ) so il te m pe ra tu re at 5 cm d ep th (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se a m bi en t te m pe ra tu re (° c ) so il su rf ac e te m pe ra tu re ( °c )s oi l t em pe ra tu re at 5 cm d ee p (° c ) w or ke rs c ap tu re d pe r b ai te d vi al ± se 24 .0 26 .0 26 .0 85 .3 3± 17 .0 3 32 .0 36 .0 36 .0 18 2. 83 ± 10 .6 1 25 .0 25 .0 25 .0 77 .5 0± 2. 15 32 .0 34 .0 31 .0 20 1. 00 ± 21 .1 2 25 .0 26 .0 27 .0 79 .0 0± 4. 62 32 .0 34 .0 32 .0 20 7. 17 ± 26 .8 2 25 .0 26 .0 27 .0 10 4. 17 ± 24 .5 6 32 .0 35 .0 33 .0 18 8. 17 ± 35 .9 3 25 .0 26 .0 27 .0 94 .1 7± 3. 53 32 .0 45 .0 36 .0 16 4. 67 ± 49 .8 3 25 .0 26 .0 28 .0 83 .1 7± 17 .1 6 33 .0 37 .0 34 .0 12 7. 00 ± 20 .2 3 25 .0 27 .0 29 .0 92 .1 7± 20 .9 7 33 .0 38 .0 36 .0 10 8. 83 ± 6. 53 26 .0 26 .0 26 .0 84 .6 7± 3. 79 33 .0 38 .0 37 .0 98 .5 0± 23 .3 9 26 .0 27 .0 28 .0 85 .0 0± 5. 14 33 .0 38 .0 37 .0 96 .0 0± 22 .6 2 26 .0 28 .0 29 .0 93 .1 7± 16 .7 2 33 .0 40 .0 37 .0 12 2. 67 ± 24 .2 6 26 .0 26 .0 27 .0 92 .6 7± 25 .1 7 33 .0 40 .0 34 .0 13 7. 33 ± 36 .2 8 26 .0 29 .0 31 .0 11 1. 33 ± 16 .6 4 33 .0 42 .0 36 .0 12 6. 00 ± 34 .4 3 26 .0 28 .0 30 .0 10 5. 17 ± 13 .1 6 33 .0 46 .0 37 .0 12 3. 67 ± 36 .0 3 26 .0 30 .0 29 .0 17 8. 17 ± 21 .0 2 34 .0 39 .0 35 .0 78 .1 7± 17 .2 5 27 .0 28 .0 29 .0 96 .0 0± 10 .2 7 34 .0 39 .0 36 .0 86 .0 0± 9. 05 27 .0 29 .0 28 .0 10 9. 00 ± 16 .4 1 34 .0 37 .0 33 .0 12 9. 17 ± 15 .6 3 27 .0 30 .0 33 .0 12 7. 33 ± 21 .4 3 34 .0 39 .0 36 .0 11 9. 17 ± 13 .3 9 27 .0 28 .0 28 .0 16 8. 83 ± 14 .4 1 34 .0 42 .0 37 .0 11 3. 67 ± 29 .0 5 27 .0 28 .0 29 .0 95 .5 0± 46 .8 6 34 .0 43 .0 38 .0 10 6. 17 ± 21 .9 5 27 .0 29 .0 25 .0 96 .3 3± 10 .5 6 35 .0 43 .0 38 .0 10 8. 00 ± 25 .7 5 27 .0 31 .0 27 .0 99 .1 7± 7. 02 35 .0 44 .0 39 .0 89 .8 3± 21 .9 9 * t em pe ra tu re w as th e m ea n of th os e m ea su re d th re e tim es a d ay , a nd th e nu m be rs o f a nt s w er e th e m ea n of th os e ca pt ur ed b y 36 b ai te d vi al s. 580 sociobiolog y vol. 59, no. 2, 2012 °c, and 19 °c respectively. statistical analyses revealed high significance of the effects of these three temperatures on the foraging activity of fire ants. specifically, the indices of correlations between the number of worker ants and the ambient temperature, soil surface temperature, and soil temperature at 5-cm deep were 0.8131 (p<0.01, n=144), 0.7253 (p<0.01, n=144), and 0.8051 (p<0.01, n=144), respectively. a function, y=662677.5/(1+34030.4exp(-(-0.3764x+0.0315x2 0.000556x3))), (f=111.5, p=0.01) was used to test the relationship between different ambient temperatures. y is the average number of fire ant workers per trap, and x is the temperature in the three cases. as shown in fig. 2, the fire ant became active in foraging when the ambient temperature was between 11 and 44 °c. the function for the effects of ground surface temperature is: y=1245.2/ (1+168886.5exp(-(0.5725x-0.007683x2-0.000013x3))) (f=99.5, p=0.01). no ants were captured when the soil-surface temperature was below 10 °c, and foragers were observed even when the temperature was as high as 57 fig. 3 dynamics of red imported fire ant workers captured in baited vials at different soil surface temperatures. each point represents the average number of ants captured per bait trap (n=12). 581 lu, y.-y. et al. — effects of temperature on fire ant foraging activity °c (fig.3). these results are consistent with previous report of porter and tschinkel (1987). the best predictor for fire ant foraging activity was the soil temperature at 5-cm deep. the fit function is: y=226944.82/(1+94298.3exp(-(0.1373x+0.01997 x2-0.000363x3))) (f=114.4, p=0.01). no workers were captured when the soil temperature at 5-cm deep was below 12 °c or above 48 °c (fig.4). discussion the foraging of s. invicta worker ants was noted when the soil temperature at 5-cm deep was from 12 to 48 °c, and when the ground surface temperature was between 10 and 57 °c. this suggests that the minimum foraging temperature for s. invicta worker ants is lower in china than that in florida, usa (porter & tschinkel 1987) , where the low soil temperature at 2-cm deep was 15 °c. the critical minimum and maximum ambient temperatures for fire ant foraging activity were 11 °cand 44 °c, respectively. indeed, the average ambient temperature in the winters of guangdong province is usufig. 4 dynamics of red imported fire ant workers captured in baited vials at different soil temperatures at 5-cm deep. each point represents the average number of ants captured per bait trap. 582 sociobiolog y vol. 59, no. 2, 2012 ally higher than 11.2 °c (guangdong meteorological administration, www. grmc.gov.cn), indicating that the fire ant foraging can occur throughout the whole year in this region. it is widely accepted that temperature is the most significant predictor for the foraging activity of fire ants (porter & tschinkel, 1987; vogt et al. 2003). as was shown in this study, different surroundings with varied temperatures may serve as an important factor for the ants to have different critical minimum and maximum temperatures for foraging. this is consistent with a previous report that field and laboratory imported fire ants had significantly different maximum foraging temperatures (57 vs 50°c) (drees et al. 2007). toxic bait is an effective method to control fire ants (lofgren & weidhass, 1972). however, it is critical to choose the right time to set the baits, to not only kill the ants most efficiently, but also prevent the baits from being damaged by other animals. in this study, we observed that the most active foraging occurred when the air temperature was at 25-33°c. therefore, toxic baits should be set when the temperature is in this range for maximal effects. acknowledgments we would like to thank ningdong li, and jun huang for sample collection and surveys. this work is supported by national basic research program of china (award# 2009cb119206) and national natural science foundation of china (award# 305712427). references drees, b.b.m., b. summerlin & s.b. vinson 2007. foraging activity and temperature relationship for the red imported fire ant. southwestern entomologist, 32(3):149155. bernstein, a.1979. ruth. schedules of foraging activity in species of ants. journal of animal ecolog y, 48(3):921-930. briano, j.a., williams, d.f., d.h. oi & l.r. j. davis 2001. field host range of the fire ant pathogens thelohania solenopsae (microsporida: thelohaniidae) and vairimorpha invictae (microsporida: burenellidae) in south america. biological control., 24(2002):98102. lofgren, c.s. & d.e. weidhaas 1972. on the eradication of imported fire ants: a theoretical appraisal. bulletin of entomolog y society american, 18:17-20. lofgren, c.s., bartless, f.j., c.e.j. stringer & w.a. banks 1964. imported fire ant toxic bait studies: further tests with granulated mirex-soybean oil bait. journal economic entomolog y, 57(5): 695-698. 583 lu, y.-y. et al. — effects of temperature on fire ant foraging activity lu, y.y., g.w. liang & l. zeng 2008. study on expansion pattern of red imported fire ant, solenopsis invicta buren,in south china. scientia agricultura sinica, 41(4):10531063. phillips, s.a., j.a. rafael & h.g. thorvilson 1996. desiccation resistance in populations of the red imported fire ant (hymenoptera: formicidae). environmental entomolog y, 25(2): 460-464. porter, d.s. & r.w. tschinkel 1987. foraging in solenopsis invicta (hymenoptera: formicidae): effects of weather and season. enviromental entomolog y, 16(3):802-808. sas institute. 1985. sas user of guide: statistics, version 5. sas institute, cary, nc. vazquez, r.j., s.d. porter & j.a. briano 2006. field release and establishment of the decapitating fly pseudacteon curvatus on red imported fire ants in florida. biocontrol, 51(2):207-216. vogt, j.t., smith, w.a., r.a. grantham & r. wright 2003. effects of temperature and season on foraging activity of red imported fire ants (hymenoptera: formicidae) in oklahoma. environmental entomolog y, 32(3):447-451. williams, d.f., oi, d.h., s.d. porter & m. roberto 2003. pereira, and juan a. briano. biological control of imported fire ants (hymenoptera: formicidae). american entomologist, 49(3):144-155. wojcik, d.p., craig, r.a., richard, j.b., elizabeth, a.f., p. j. donald & r.s. lutz 2001. red imported fire ants: impact on biodiversity. american entomologist, 47(1):16-23. xu, y.j., lu, y.y., huang, j., l. zeng & g.w., liang 2009. cold hardiness of natural populations of the red imported fire ant, solenopsis invicta buren (hymenoptera: formicidae) in shenzhen, guangdong. acta entomologica sinica, 52(9): 974-983. xu y.j., l., zeng, y.y. lu & g.w. liang 2009. effect of soil humidity on the survival of solenopsis invicta buren workers, 56(4):367-373. zeng l., y.y., lu, x.f., he, w.q. zhang & g.w. liang 2005. identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan, guangdong. chinese bulletin of entomolog y, 42(2):144-148. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i3.3463sociobiology 66(3): 457-466 (september, 2019) bees (hymenoptera, apoidea) in an ecotonal cerrado-amazon region in brazil introduction an ecotone is a transition area between two biomes, characterized by the presence of faunal and floral components from both biomes. therefore, faunal inventories performed in an ecotonal area may reveal important findings related to the limits of distribution of species that are endemic to either one of the biomes considered in this transitional area. in the sourthern border of the brazilian amazon, there is an extensive ecotonal region (also known as ´zone of ecological tension’), approximately 4,500 km long, between the two largest south american biomes, the cerrado savanna and the amazon forest (marimon et al., 2006). bees from this region, which comprise the most important pollinator group (klein et al., 2007; ollerton et al., 2011), are poorly known in this transitional area. previous to the present study, this group abstract little is known about the composition of the bee community in the cerrado-amazon transition area. herein, we present the results of a bee survey done within the municipality of conceição do araguaia, in the state of pará. six fragments were sampled twice (once in the dry season and once in the rainy season) using three methods of collecting: arboreal pitfalls with urine, scent traps, and pan traps. we recorded 67 bee species, distributed in 28 genera and eight tribes. except for partamona chapadicola, which is endemic to the cerrado biome, the remaining species we sampled occur in the amazonian forest. of those, 15 species are considered endemic to the amazonian biome (four euglossini, eight meliponini, one paratetrapedia, one xylocopa and one augochlorini), while 27 occur in forest and areas of cerrado (seven euglossini, fifteen meliponini, two paratetrapedia, one xylocopa and two augochlorini). among 53 species sampled (disregarding the euglossini species which were sampled almost exclusively with scent), 41 were captured with arboreal pitfall traps. of those, 20 species were from the meliponini tribe. sociobiology an international journal on social insects rps almeida¹, fv arruda², dp silva³, bwt coelho4 article history edited by cândida aguiar, uefs, brazil eduardo almeida, usp, brazil received 11 may 2018 initial acceptance 16 july 2018 final acceptance 14 december 2018 publication date 14 november 2019 keywords ecotonal region; scent traps; pitfall traps; pan trap. corresponding author rony peterson santos almeida museu paraense emílio goeldi (mpeg) programa de pós-graduação em zoologia coordenação de ciências da terra e ecologia (cocte), belém-pa, brasil. e-mail: rony__peterson@hotmail.com has been studied in only two localities, the bico-do-papagaio region, at the northern portion of the state of tocantins (santos et al., 2004), and the municipality of ribeirão cascalheira, in the northeastern of the state of mato grosso (oliveira-junior et al., 2015). in the first region, the sampling consisted of monthly, systematic sampling of flower visitors during the course of one year. the sampling in the other study area consisted of a rapid survey of orchid bees using baited traps and chemical baits with hand nets. the standard sampling method for bee inventory consists of capturing the bees visiting flowers using entomological nets (sakagami et al., 1967). however, net sampling demands a huge sampling effort, mainly in forested area with high canopy height. other sampling methods that are often used to capture bees consist of using passive traps to sample these organisms. one example, the pan/bowl trap, (cane et al., 1 museu paraense emílio goeldi (mpeg), programa de pós-graduação em zoologia (ppgzool), coordenação de ciências da terra e ecologia (cocte), belém-pa, brazil 2 universidade estadual de goiás, programa de pós-graduação em recursos naturais do cerrado (renac), anápolis-go, brazil 3 instituto federal goiano, rodovia geraldo silva nascimento, urutaí-go, brazil 4 museu paraense emílio goeldi (mpeg), coleção entomológica, belém-pa, brazil research article bees rps almeida et al. – bees in an ecotonal cerrado458 2000; roulston et al., 2007; wilson et al., 2008; joshi et al., 2015) consists of plastic plates painted with colors attractive to bees (usually blue, yellow, and white) and filled with water and soap droplets to decrease the water’s surface tension and avoid the specimens’ escape. another passive method, specifically designed for sampling orchid bees, uses scent/ baited traps made with pet bottles (nemésio, 2012; nemésio & vasconcelos, 2014). these insects are easily attracted to synthetic compounds that mimic floral fragrances, making field studies easy to conduct. although such traps are widely used, their effectiveness has been questioned recently (see nemésio & vasconcelos, 2014), after it was shown that scent baits allowed researchers to sample a different composition of species than active sampling. nonetheless, given the resource limitations and logistic difficulties in conducting field research, passive sampling of orchid bees with scent traps are still widely used. here, we also employed for the first time the use of an unusual passive sampling method for bees that is already commonly used to sample ants: urine traps. urine contains nitrogen and salts (kaspari et al., 2009), which make it very attractive to insects. the use of urine in arboreal pitfall traps is common in ant inventories (see more at maravalhas & vasconcelos, 2014). here we employed it to sample bees in multiple ecotonal transects simultaneously, along with other sampling methods usually used in bee inventories. therefore, since our goal was to perform rapid bee samplings in an ecotonal area between the amazon forest and the brazilian cerrado savanna, here we used the different passive sampling methods we described before to sample bees in multiple ecotonal areas of the amazon forest with the cerrado. the current study describes the composition of the bee community in the amazonia-cerrado ecotone in southeast pará. here, we discuss the distribution of each sampled species and also evaluate whether the ecotone is the limit of the species’ geographic distribution. although our sampling was selective to some degree, it contributes to the knowledge of the bee fauna of this region where knowledge on this group is scarce and that it also undergoing through rapid deforestation. since insect data is usually compromised by knowledge gaps, especially the wallacean shortfall (e.g. lack of knowledge on the biogeographic distribution of species; diniz-filho et al., 2010; cardoso et al., 2011), additional sampling is always welcomed to fight other biological data shortfalls (hortal et al., 2015). materials and methods study site we performed this study in the municipality of conceição do araguaia, in the southeastern portion of pará state (figure 1). six native vegetation fragments, varying in distance from 4 to 27 km, were sampled (site 1, 8°10’41.32”s 49°21’52.60”w; site 2, 7°55’35.21”s 49°24’10.49”w; site 3, 7°59’30.39”s 49°24’25.13”w; site 4, 8°00’01.25”s 49°22’12.49”w; site 5, 8°03’43.69”s 49°20’54.07”w; site 6, 8°02’33.44”s 49°27’34.66”w). the area sampled had high environmental complexity, due to its location in an ecotonal region between the cerrado savanna and the amazon forest, formed by seasonal forest/ombrophilous forest (haidar et al., 2013). the mean altitude of this region is 240 m, and the climate has two well-defined seasons, with the dry season extending from may to september and the wet season spanning october to april. the mean temperature is around 28ºc and the mean annual precipitation is 1,200 mm, according brazilian national institute of meteorology (inmet). bee sampling we sampled each fragment twice, once in the dry season (june 6-14 of 2017), and once in the wet season (november 6-14 of 2017). in each sampled area, we set an 80 m transect, at least 20 m from the fragment’s border to minimize the border effect (fisher, 1999). we sampled at five locations along each transect, each 20 m apart. in each sampling location, we set a group of traps (arboreal pitfalls with urine, a scent trap, and several pan traps) as described below. arboreal pitfalls with urine we installed four arboreal pitfall traps in tree canopies at locations between 1.5 and 3 m in height. the pitfall traps were consisted of 100 ml plastic cups, fixed to tree branches with wires. each trap was filled with a mixture of water (70%), human urine (30%), and soap droplets. the sampled bees were a by-product of this sampling protocol intended to sample ants. the pitfall traps remained in trees for a total of 48h in each of the six sampling locations. scent traps we used scent traps made of pet bottles with two openings (adapted from campos et al., 1989; sofia & suzuki, 2004). the traps were placed 1.7 m from the ground and fixed to tree branches with wire. we used five scents, one per sampling location in each one of the six areas. the scents used (benzyl acetate, methyl cinnamate, cineol, eugenol, and vanillin) were selected because they are commonly used to sample euglossini bees in field surveys due to their similarity to plant chemicals, or as attractants of male specimens (silva & de marco, 2014). scent traps were left out for seven days, revisited every 48 h to remove specimens from traps and replenish scents. pan traps we used colored pan traps filled with water and soap, commonly used to sample bees (krug & alves-dos-santos, 2008; hall, 2016; silva et al., 2017). we placed four (yellow, blue, orange, and green) 22 cm traps in each sampling location to increase the diversity of sampled insects. traps were placed on the ground spaced 20 cm apart from each other, forming a square. these traps were left at each location for 48 h. sociobiology 66(3): 457-466 (september, 2019) 459 sampled specimens pinning and identification bees were removed from the tubes, pinned, and labeled. species were identified using identification keys found in the supplemental material (supplementary material) and compared by bwtc with the identified specimens already deposited in the entomological collection from museu paraense emílio goeldi (mpeg). the sampled specimens received the collection numbers mpeg 03032032–03032076, and mpeg 03032797–03033191 and were deposited in the entomological collection of mpeg. the sampling license was granted to us by the secreataria de meio ambiente (semas) from the state of pará: capture, sampling, and transportation of wildlife (au nº3459/2017, process number 2016/39794). results in this study, we sampled 1,411 specimens belonging to 67 species, 28 genera, and 2 bee families (apidae and halictidae). in table 1, the species we sampled are listed, along with their abundance in each different sampling method we tested: arboreal urine pitfalls, scent traps, and pan traps. only one species, stilbochlora eickworti (engel, brooks & yanega, 1997), had not been previously reported for pará (moure et al., 2012). a total of 882 male orchid bees from 14 different species representing four of the euglossine genera were sampled: euglossa with eight species, eulaema with four, eufriesea, and exaerete represented by a single species. the most abundant species were eulaema nigrita lepeletier, 1841 (54.1% of the total amount of orchid bees sampled), el. cingulata (fabricius, 1804) (33.7 %) and euglossa ignita smith, 1874 (8.4 %). the remaining 11 species made up just 3.8 % of the total bees sampled. stingless bees were the second most abundant group sampled, with 481 specimens from 29 species, and 14 genera. the most common species was trigona aff. fuscipennis friese, 1900, with 285 specimens (59.3% of the total amount of stingless bees sampled), followed by tetragona clavipes (fabricius, 1804) (40 specimens; 8.3%) and trigona guiane cockerell, 1910 (37; 7.7%). the remaining species made up 24.3 % of the total of stingless bees sampled. fig 1. location of the six bee collecting areas in a cerrado and amazon forest ecotone in the municipality of conceição do araguaia, southeast of pará, brazil. rps almeida et al. – bees in an ecotonal cerrado460 the remaining species of apidae sampled in our inventory are distributed among the genera apis (1), paratetrapedia (4 species), ceratina (3), xylocopa (3), and tetrapedia (2). the three types of traps sampled only 24 specimens of halictidae, representing 11 species and morphospecies from five genera: augochlora (2), augochloropsis (5), megalopta (2), megaloptidia (1) and stilbochlora (1). the highest species richness was observed for the arboreal urine pitfalls, with which we sampled a total of 43 species, being twenty of meliponini, representing 11 genera. euglossa intersecta dressler, 1982 was the only orchid bee only sampled with the urine pitfalls. the scent traps showed the second highest richness, with 28 species sampled. the lowest species richness was recorded by the pan traps, with which only 15 species were captured. table 1. list of bee species sampled in six sites and using three different traps (arboreal pitfalls with urine, scent trap and pan traps) in a cerrado and amazon forest ecotone in the municipality of conceição do araguaia, southeast of pará, brazil. (n = abundance of bees). taxon scent trap pan trap pitfall n site n site n site apidae apini apis mellifera linnaeus, 1758 1 2 1 2 1 6 ceratinini ceratina sp.1 0 1 2 1 6 ceratina sp.2 0 0 1 6 ceratina sp.3 0 0 3 6 euglossini eufriesea concava (friese, 1899) 1 1 0 0 euglossa avicula dressler, 1982 2 5;6 0 0 euglossa decorata smith, 1874 1 6 0 0 euglossa ignita smith, 1874 74 1;2;3;4;5;6 0 0 euglossa intersecta latreille, 1817 0 0 2 5;6 euglossa liopoda dressler, 1982 1 6 1 4 0 euglossa orellana roubik, 2004 2 5;6 0 0 euglossa piliventris guérin, 1844 1 6 0 0 euglossa townsendi cockerell, 1904 1 5 0 0 eulaema cingulata (fabricius, 1804) 297 1;2;3;4;5;6 0 0 eulaema meriana (olivier, 1789) 9 1;3;4;5 0 0 eulaema mocsaryi (friese, 1899) 1 4 0 0 eulaema nigrita lepeletier, 1841 476 1;2;3;4;5;6 0 1 6 exaerete smaragdina (guérin, 1844) 12 2;4;5;6 0 0 meliponini aparatrigona impunctata (ducke, 1916) 1 4 0 0 celetrigona longicornis (friese, 1903) 0 0 2 2 cephalotrigona capitata (smith, 1854) 0 1 6 0 frieseomelitta longipes (smith, 1854) 0 0 1 2 frieseomelitta sp.1 1 1 0 0 melipona seminigra friese, 1903 0 0 2 4 melipona flavolineata friese, 1900 0 4 4;6 8 4 nannotrigona punctata (smith, 1854) 0 0 2 6 nannotrigona schultzei (friese, 1901) 0 0 3 6 oxytrigona sp.1 0 0 1 4 partamona chapadicola pedro & camargo, 2003 1 2 2 6 15 3;4;6 plebeia alvarengai moure, 1994 0 5 6 2 5 plebeia minima (gribodo, 1893) 0 0 1 6 plebeia sp.2 0 0 4 3;4 scaptotrigona postica (latreille, 1807) 11 1;6 1 5 0 scaptotrigona tubiba (smith, 1863) 4 2;3 0 0 scaptotrigona sp.1 1 1 0 0 sociobiology 66(3): 457-466 (september, 2019) 461 discussion the number of euglossini species we found (14 species) is similar to the number sampled by oliveira-junior et al. (2015) in a transitional forest at ribeirão cascalheira, in northeastern mato grosso state (16 species). five of the species we sampled were also sampled in the study by oliveira-junior et al. (2015): el. nigrita, el. cingulata (fabricius, 1804), el. mocsary (friese, 1899), eg. ignita and exaerete smaragdina (guérin, 1844). the first and second most abundant species, el. nigrita and el. cingulata, respectively, were the same in both inventories. meliponini scaura latitarsis (friese, 1900) 0 0 2 6 tetragona beebei (schwarz, 1938) 1 5 0 19 4;5;6 tetragona clavipes (fabricius, 1804) 0 0 40 2;3;4;6 tetragona truncata moure, 1971 0 0 1 4 tetragonisca angustula (latreille, 1811) 0 0 1 6 trigona branneri cockerell, 1912 0 0 1 6 trigona chanchamayoensis schwarz, 1948 0 1 5 7 6 trigona dallatorreana friese, 1900 1 5 1 2 0 trigona guianae cockerell, 1910 0 5 4;6 32 2;4;6 trigona hypogea silvestri, 1902 3 2 0 0 trigona pallens (fabricius, 1798) 1 4 0 0 trigona aff. fuscipennis friese, 1900 233 1;2;3;4;5;6 7 2;6 52 2;4;6 tapinotaspidini paratetrapedia flaveola aguiar & melo, 2011 0 0 1 6 paratetrapedia lugubris (cresson, 1878) 0 0 1 4 paratetrapedia testacea (smith, 1854) 0 0 1 4 paratetrapedia sp.01 0 0 1 4 tetrapediini tetrapedia sp.1 0 0 1 6 tetrapedia sp.2 0 0 2 2 xylocopini xylocopa aurulenta (fabricius, 1804) 0 0 6 2;3;5 xylocopa nigrocincta smith, 1854 0 0 1 2 xylocopa suspecta moure & camargo, 1988 0 0 1 6 halictidae augochlorini augochlora sp.1 0 0 2 1;3 augochlora sp.2 0 0 1 3 augochloropsis sp.1 0 0 2 4;6 augochloropsis sp.2 1 2 1 4 1 4 augochloropsis sp.3 1 3 0 0 augochloropsis sp.4 0 1 4 2 6 augochloropsis sp.5 0 0 1 6 megalopta amoena (spinola, 1853) 3 5 0 0 megalopta sodalis (vachal, 1904) 0 0 4 4;6 megaloptidia sp.1 0 0 2 3 stilbochlora eickworti (engel, brooks & yanega, 1997) 0 2 4;6 0 richness 28 15 43 exclusive species 19 2 31 abundance 1142 34 235 table 1. list of bee species sampled in six sites and using three different traps (arboreal pitfalls with urine, scent trap and pan traps) in a cerrado and amazon forest ecotone in the municipality of conceição do araguaia, southeast of pará, brazil. (n = abundance of bees). (continuation) taxon scent trap pan trap pitfall n site n site n site rps almeida et al. – bees in an ecotonal cerrado462 all orchid bee species sampled here, in addition to those listed by oliveira-junior et al., (2015), were recorded for brazilian amazon. among the 23 species (euglossa gr. purpura was not included), six (26.1% of the total of species) are endemic to the amazonian biome (nemésio & silveira, 2007): euglossa bidentata dressler, 1982, eg. chalybeata friese, 1925, eg. intersecta, eg. orellana roubik, 2004, eg. piliventris guérin, 1844 and el. mocsaryi; and two species, eufriesea concava (friese, 1899) and euglossa bursigera moure, 1970 are found in the amazonian basin and central america (but see moure et al., 2012 for a discussion regarding the distribution of ef. concava). thus, the cerradoamazon ecotone is expected to be the southern limit of their distributions. six species, among the 23 listed, occur also in atlantic forest domain (nemésio & silveira, 2007; sydney et al., 2010): eufriesea surinamensis (linnaeus, 1758), euglossa cordata (linnaeus, 1758), eg. liopoda dressler, 1982, eg. pleosticta dressler, 1982, eg. securigera dressler, 1982 and eg. avicula dressler, 1982. among the 23 species of orchid bees sampled in the amazonia-cerrado ecotone, nine were recorded from cerrado in the state of minas gerais: eg. cordata, eg. decorata smith, 1874, eg. imperialis, eg. securigera, eg. pleosticta, eg. townsendi, el. cingulata, el. nigrita, and ex. smaragdina. additionally, the species el. bombiformis (packard, 1869), el. cingulata, el. meriana (olivier, 1789), el. nigrita and euglossa ignita were recorded in the inventory carried out by vital et al. (2016) within the cerrado biome in the state of tocantins. therefore, 52.2% of the euglossine species sampled in the ecotonal region also occur in cerrado. except for p. chapadicola pedro and camargo, 2003, which occurs from the southeastern region of pará to up to the state of piauí in both the cerrado and caatinga biome (pedro & camargo, 2003), the remaining 24 species of stingless bees sampled in our study are present in the amazonian forest (rebêlo et al., 2003; moure et al., 2012; pedro, 2014). among these 24 species, six (25.0% of the total of species) are endemic to the amazonian biome: nannotrigona punctata (smith, 1854), n. schultzei (friese, 1901), tetragona beebei (schwarz, 1938), plebeia alvarengai moure, 1994, p. minima (gribodo, 1893) and friesiomellita longipes (smith, 1854). thus, the cerrado-amazon ecotone is the southern limit of the distributions of these endemic species, and to aparatrigona impunctata (ducke, 1916), which occurs in central america and the amazonian basin. this region is also likely the distribution limit of melipona seminigra friese, 1903 and m. flavolineata friese, 1900, because these species require large cavities in large trees for nesting (pioker-hara, 2011). in the state of tocantins, these species are present in the cerrado only because they are reared by humans (costa-neto et al., 2016). among the 24 species of meliponini bees sampled in the amazonia-cerrado ecotone we inventoried, five species were recorded in cerrado from state of goiás (santiago et al., 2009; moure et al., 2012): trigona chanchamayoensis schwarz, 1948, celetrigona longicornis (friese, 1903), trigona pallens (fabricius, 1798), t. branneri cockerell, 1912 and tetragona truncata moure, 1971. the species trigona guianae cockerell, 1910 is found in the states of tocantins, ceará, and paraiba (moure et al., 2012); and t. dalatorreana friese, 1900 is found in the ecotone area in the state of tocantins (santos et al., 2004). furthermore, eight species are widely distributed in brazil (moure et al., 2012), inhabiting both forest and cerrado (andena et al., 2005; santiago et al., 2009; imperatriz-fonseca et al., 2011; pioker-hara, 2011): cephalotrigona capitata (smith, 1854), s. postica (latreille, 1807), s. tubiba (smith, 1863), scaura latitarsis (friese, 1900), tetragonisca angustula (latreille, 1811), t. clavipes, t. hypogea silvestri, 1902 and t. aff. fuscipennis. therefore, about 62.5% of meliponini species sampled in this ecotone region occur in cerrado. the bees from the paratetrapedia genus are especially diverse in the amazon forest (aguiar & melo, 2011). among the three species of this genus we sampled and identified, only p. testacea (smith, 1854) is endemic to the amazon basin, occurring mainly in the eastern amazon (aguiar & melo, 2011). the species p. lugubris (cresson, 1878) has a panneotropical distribution, while p. flaveola aguiar and melo, 2011 is a widespread species distributed throughout brazil. we identified three species of xylocopa: x. nigrocincta smith, 1854, x. suspecta moure and camargo, 1988 with widely distribution (moure et al., 2012), and x. aurulenta (fabricius, 1804), which is restricted to the amazonian domain. we also recorded three species of sweat bees. two of them are widely distributed: megalopta amoena (spinola, 1853) occurs from guatemala to the state of são paulo in brazil, while m. sodalis (vachal, 1904) occurs from venezuela to the brazilian state of santa catarina. however, stilbochlora eickworti (engel, brooks & yanega, 1997) is strictly an amazonian species. only five species sampled in this study were among those listed by santos et al. (2004): el. nigrita, m. seminigra, t. dalatorreana, t. pallens, and p. testacea. this small proportion of overlap in sampled species is likely due to differences in the methodologies used in both the surveys. in total, santos et al. (2004) captured 83 species from 38 genera and five families. in this inventory, we expected to sample specimens from at least three different families of bees: apidae, halictidae, and andrenidae. the lack of andrenidae species is probably due to the poor sampling efficiency of the pan traps. the efficiency of pan traps is not well studied, but may be dependent on the local vegetation type, water availability, and both type and placement of traps (gonçalves et al., 2012). previous studies have reached different conclusions about the performance of this sampling method. krug and alvesdos-santos (2008), gonçalves and oliveira (2013), ayala (2014), perillo et al. (2017) for instance, reported capture efficiency. however, gonçalves et al. (2012) reported very poor performance of these traps in atlantic forest. sociobiology 66(3): 457-466 (september, 2019) 463 out of the 23 genera of stingless bees reported for pará (pedro, 2014), 48.8% were captured with arboreal urine pitfall. based on our results, we encourage the use of this trap as complementary sampling method for bee inventories, especially in northern brazil, where there is high species richness of stingless bees (oliveira et al., 1995). we suggest this sampling method may help overcome the difficulty in sampling them passively in forested areas, especially with pan traps. the arboreal urine pitfall traps also seem to be efficient in sampling specimens of paratetrapedia, tetrapedia, xilocopa, ceratina, and halictidae bees. in summary, the bee community in the transitional cerrado-amazon area we sampled contains fifteen endemic species from the amazonia region, one species endemic to the cerrado as well as 27 species which occur in forest and areas of cerrado. we suggest that future studies employing arboreal urine pitfalls are needed to properly evaluate the sampling efficiency of urine pitfall traps, especially for sampling meliponini bees. acknowledgement the authors are grateful for the financial support provided by coordenação de apoio de pessoal do ensino superior (capes) for the ph.d. scholarships provided (rpsa and fva). the authors would also like to thank dbo engenharia (in the name of ricardo a.p. pires) and field support provided during the surveys, especially, andréa c.r. santos, camila a. lima, crizanto b. carvalho, evellyn b. freitas, josé silonardo p. oliveira and nathane q. costa. we are also grateful to livia p. prado and joudellys a. silva for the suggestions in the manuscript, carol peretz for review of english and micael r. parreira for help with the map. author contributions rpsa collected the specimens and wrote the text; fva collected the specimens and wrote the text; dps wrote the text; e bwtc identified the specimens and wrote the text. references aguiar, a.j.c. & melo, g.a.r. 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(1948). stingless bees (meliponidae) of the western hemisphere. bulletin of the american museum of natural historty, 90: 1–546. doi: 10.13102/sociobiology.v62i1.128-131sociobiology 62(1): 128-131 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 new records of the dorylinae ant genus cheliomyrmex for the brazilian amazon basin introduction the dorylinae has currently 18 valid genera, among them the neotropical army ants, including the genera eciton latreille, 1804; labidus jurine, 1807; neivamyrmex borgmeier, 1894; nomamyrmex borgmeier, 1939 and cheliomyrmex mayr, 1870. these ants are dominant predators in tropical and subtropical terrestrial ecosystems (brady et al., 2014). cheliomyrmex are the most cryptic army ants of the new world (berghoff & franks, 2007). these ants are not usually collected with standard sampling techniques (agosti et al., 2000). fortunately, the males of dorylinae army ants are often sampled in light traps (see kempf, 1975; nascimento et al., 2011). most of these ants have subterranean habits, and little is known about their biology. chance encounters are still one of the most valuable opportunities to learn more about the occurrence and ecology of such army ants (berghoff & franks, 2007). cheliomyrmex have its polymorphism associated with labor division (eg. abstract the members of dorylinae are dominant predators in tropical and subtropical terrestrial ecosystems. the most cryptic army ant genus of the new world is cheliomyrmex. the only species recognized until now for the amazon brazilian basin is c. andicola, recorded in the state of acre. we provided the first records of cheliomyrmex megalonyx to brazil in the amazon basin. the major worker of cheliomyrmex megalonyx was collected in the state of rondônia while the minor was found in the state of amazonas. these records suggest that more efforts are needed to reveal the diversity, distribution and biology of these cryptic ants. sociobiology an international journal on social insects io fernandes¹, jlp souza¹,4, jhc delabie2, f fernández3 article history edited by evandro do nascimento silva, uefs, brazil received 03 september 2014 initial acceptance 10 october 2014 final acceptance 20 october 2014 keywords cheliomyrmex megalonyx, army ants, amazon rain forest, biogeography, cryptic ants. corresponding author itanna oliveira fernandes instituto nacional de pesquisas da amazônia inpa, coordenação em biodiversidade cbio. av. andré araújo, 2936, petrópolis, 69011970, c. p. 2223. manaus, am, brazil e-mail: itanna.fernandes@gmail.com major and minor workers) (hölldobler & wilson, 1990). the gradual size and the morphological transition in workerpolymorphic is demonstrable in the mandible and head of the workers (gotwald & kupiec, 1975). cheliomyrmex occurs from southern mexico to bolivia, including the brazilian amazon (kempf, 1972; 1975; watkins, 1976; berghoff & franks, 2007) with four valid species: c. andicola emery, 1894, c. audax santschi, 1921, c. megalonyx, wheeler, 1921 and c. morosus (smith, 1859). the only species recorded so far in the brazilian amazon basin is c. andicola, with records in the state of acre (kempf, 1972; 1975; watkins, 1976). the elevation of the state of acre ranges from 135-609m above sea level, so the occurrence of c. andicola indicates that this species is not restricted to the andean highlands, as pointed out by kempf (1975). we provide hereafter the first record of c. megalonyx to brazil in the amazon basin. the major and minor workers were sampled by winkler extractor (alonso & agosti, 2000), in santo antônio hydroelectric short note 1 instituto nacional de pesquisas da amazônia – inpa, manaus, am, brazil 2 laboratório de mirmecologia uesc cepec/ ceplac, ilhéus, ba, brazil 3 universidad nacional de colombia, carrera, bogotá, colombia 4 centro de estudos integrados da biodiversidade amazônica cenbam, manaus, am, brazil sociobiology 62(1): 128-131 (march, 2015) 129 plant, in the state of rondônia and near of benjamin constant city, during the biosbrasil project (moreira et al., 2013), in the state of amazonas, respectively. vouchers are deposited in instituto nacional de pesquisas da amazônia (inpa) at manaus and centro de pesquisas do cacau (cpdc) at ilhéus. the morphological terminology follows wheeler (1921) and gotwald and kupiec (1975). the species was identified using a zeiss discovery v12 stereomicroscope. digital images and measurements were made using a leica m165c stereomicroscope with a digital camera under a white light lamp. all measurements are given in millimeters, and the abbreviations used are: hw: head width. in full-face view, the maximum width of head posterior to compound eyes. hl: head length. in full-face view, the midline distance from level of maximum posterior projection of posterior margin of head to level of most anterior projection of anterior clypeal margin. sl: scape length. in frontal view, measured from apex of first antennal segment to base, excluding the basal condyle. wl: weber’s length. in lateral view, the diagonal length of mesosoma in profile, from the midpoint of the anterior pronotal declivity to posterior margin of the metapleuron. pw: pronotal width. in dorsal view, the maximum width of pronotum, measured from side to side. nhl: nodal height lateral. in lateral view, the distance from lower edge of petiolar sternite to apex of petiolar tergite (node), taken as a vertical measurement perpendicular to the longitudinal axis of the petiole. nll: nodal length lateral. in lateral view, the maximum longitudinal distance between anterior and posterior extremes of petiolar node, excluding anterior and posterior condyles. gl: gaster length. in lateral view, the maximum length of gaster. tl: total length. in lateral view, the summed length of hl, wl, nll, and gl. material examined: brazil, rondônia, santo antônio, ilha da pedra, km 0.5, 09°17’45”s 64°60’83”w, 20.x.2013, leg. i. o. fernandes, 1 major worker (inpa) (fig 1a-c); amazonas, benjamin constant, projeto biobrasil/gef/unep, 4°23’ 0”s 70°1’53”w, leg r. zanetti & n. dias, 1 minor worker (cpdc). the specimens were compared with the type material housed at national museum natural history (nmnh) of smithsonian institution (w. m. mann 1954 collection). three cotypes of major worker and six minor workers (non-type w. m wheeler collection) labeled “kartabo, b. g. jul. ago. 1920 cheliomyrmex megalonyx wheeler from british guiana” were studied. measurements of major workers (n=4) (type material between parentheses): hl: 1.240 (1.144-1.248); hw: 1.342 (1.445-1.636); sl: 0.681 (0.702-0.810); wl: 1.993 (2.099-2.196); pw: 0.764 (0.789-0.816); nhl: 0.391 (0.408-0.435); nll: 0.365 (0.397-0.415); gl: 1.770 (2.113-2.240); tl: 5.368 (5.857-5.995). measurements of minor workers (n=7) (non-type w. m. wheeler collection): hl: 1.210 (1.117-1.201); hw: 1.330 (1.289-1.312); sl: 0.670 (0.624-0.648); wl: 1.901 (1.715-1.810); pw: 0.751 (0.605-0.687; nhl: 0.381 (0.311-0.350); nll: 0.390 (0.318-0.340); gl: 1.763 (1.567-1.600); tl: 5.264 (4.717-4.951). minor and major workers are similar, but the minor worker has the following differences: mandibles subtriangular, apical tooth and 1st subapical tooth separated by diminutive teeth, and 2nd subapical tooth slightly distinguishable. we found initially some difficulties to differentiate between the major workers of c. megalonyx and c. morosus. the species are separated by variable characters, such as the sixth flagellar segment broader than long or longer than broad (watkins, 1976) and the position of the teeth (wheeler, 1921). after the exam of the type material, was clear the separation of these two species by the metapleuron area, well marked by oblique striae in c. megalonyx. fig 1. major worker of cheliomyrmex megalonyx, (a) lateral view, (b) full-face view and (c) lateral view of metapleuron and petiole. io fernandes et al new records of cheliomyrmex to brazil130 the area where the major worker was sampled in rondônia is included in a program of conservation monitoring supported by the santo antônio energia hydroelectric plant, and for the first time in four years of monitoring the water level had risen 19m above the maximum limit. cheliomyrmex megalonyx was collected on the 0.5 km (ilha de pedras) of santo antônio, and due to the elevation of the river water, the groundwater was also climbing, perhaps forcing some species to the surface. following the elevations of the river water, no additional specimens were encountered. cheliomyrmex megalonyx was previously recorded from guyana and venezuela (wheeler, 1921; kempf, 1972; watkins, 1976). we expanded its occurrence in more than 1.500 km, and also provided a map with the occurrences of the others species of the genus (fig 2). this first record of c. megalonyx on the amazon basin reinforces that efforts need to be increased to reveal the real distribution of cryptic ants. acknowledgments the first author (iof) kindly acknowledges support from inpa, smithsonian institution (nmnh), fapeam (edital 018/2013 pró-excelência), cbio and the pro-equipamentos project capes/ dcen – mct. thanks to inpa for expenses for the fourth author for a visit to inpa in manaus. the authors acknowledge their grants from capes/cnpq/pdse (iof), cnpq (jhcd) and cnpq/fapeam (jlps). this study was supported by the wildlife conservation program of santo antônio energia, the concessionaire responsible for building and operating the hydroelectric plant. (iof) kindly acknowledge the help of paulo v. cruz with photoshop program. fig 2. sites with occurrence of cheliomyrmex spp. in south america. berghoff, s. m. & franks, n. r. (2007). first record of the army ant cheliomyrmex morosus in panama and its high associate diversity. biotropica, 39: 771–777. doi: 10.1111/j.1744-7429.2007.00302.x. brady, s. g., fisher, b. l., schultz, t. r., & ward, p. s. (2014). the rise of army ants and their relative diversification of specialized predatory dorylinae ants. bmc evolutionary biology, 14: 93. doi:10.1186/1471-2148-14-93. gotwald jr., w. h & kupiec, b. m. (1975). taxonomic implication of doryline worker and morphology: cheliomyrmex morosus (hymenoptera: formicidae). annals of the entomological society of america, 68: 961–971. references alonso, l. e. & agosti, d. 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(2013). biodiversidade de solo em sistemas de uso da terra na amazônia ocidental. pp. 293-319. in: c.a. peres, j. barlow, t.a. gardner & i.c.g. vieira (orgs.). conservacão da biodiversidade em paisagens antropizadas do brasil, série pesquisa n. 220, editora ufpr, curitiba, paraná. isbn 978-85-65888-21-9. nascimento, i. c., delabie, j.h.c. & della lucia, t. (2011). phenology of mating flight in ecitoninae (hymenoptera: formicidae) in a brazilian atlantic forest location. annales de la société entomologique de france, 47(1-2): 112-118. doi: 10.1080/00379271.2011.10697702. watkins, j. f. i. (1976). the identification and distribution of new world army ants (dorylinae: formicidae). markham press fund of baylor university press, waco, texas 76703. 1–102. wheeler, w. m. (1921). observations on army ants of british guiana. proceedings of the american academy of arts and science. 56: 291-328. 1281 identification of the tandem running pheromone in diacamma sp. from japan (hymenoptera, formicidae) by n. fujiwara-tsujii1,4*, k. tokunaga2, t. akino2, k. tsuji3 & r. yamaoka2 abstract the japanese queenless ponerine ant diacamma sp. from japan employs tandem running during nest relocation, in which a leader ant guides nestmate followers one at a time. we replicated this process by presenting one entire abdominal part of a leader, except for the petiole to followers. when the abdominal part had been rinsed with n-hexane, however, it attracted significantly fewer followers. this suggests that chemicals on the leader’s abdominal part evoke tandem running. dissection of abdominal major exocrine glands revealed that the dufour’s gland was the source of this chemical signal. the chemicals were eluted in the hydrocarbon fraction by silica-gel column chromatography, and the quantitatively major component was estimated as heptadecene (c 17:1 ) through gas chromatograph-mass spectrometer (gcms) analysis. the position of the double bond was estimated to be between the 8th and 9th carbons through analysis of the epoxidized compound. only (z)-isomers of 8-heptadecene evoked tandem running in the followers. we identified the tandem running pheromone of this ant species to be (z)-8heptadecene. (163) keywords: tandem running, nest emigration, chemical communication, formicidae, ( )-8-heptadecene. introduction in many ant species, scout ants recruit their nestmates to newly discovered food sources and nesting sites, and also to battle sites at territorial borders. a variety of communication mechanisms comprise recruitment behavior of each 1venture laboratory, kyoto institute of technolog y, kyoto, 606-8585, japan 2chemical ecolog y laboratory, kyoto institute of technolog y, matsugasaki, kyoto, 606-8585, japan 3faculty of agriculture, university of the ryukyus, okinawa, 903-0213, japan 4present address: laboratory of insect behavior , national institute of agrobiological sciences (nias), ohwashi, tsukuba, ibaraki 305-8634, japan *corresponding author. e-mail: naoki99@affrc.go.jp 1282 sociobiolog y vol. 59, no. 4, 2012 ant species (hölldobler & wilson 1990). one behavior is known as tandem running, which is generally considered as one of the most primitive of these recruitment systems (wilson 1971). in this system, each scout (leader) ant recruits only one nestmate at a time (hölldobler & traniello 1980a; traniello & hölldobler 1984). the tandem running seems to be triggered by a signal from the leader’s gaster, which induces a following behavior when perceived by a nearby worker (follower). the signal is known as the tandem running pheromone. followers are always strongly attracted to the dorsal parts of abdominal segments or hind legs of leaders. in some ant species, the sources of tandem running pheromone have been reported to exude from exocrine glands (such as the pygidial or poison gland; möglich 1974; hölldobler & traniello 1980b). nevertheless, chemical identification has not yet been completed for the tandem running pheromones in any ant species. tandem running recruitment is also observed in the japanese queenless ponerine ants diacamma sp. from japan (fukumoto & abe 1983). they are very sensitive to environmental changes of nesting sites, and often move them in conjunction with tandem running recruitment. this sensitivity enabled us to develop a bioassay system to evaluate the tandem running activity in the laboratory and preliminary observation confirmed that a single fresh abdomen dissected from a leader evoked this behavior. the behavior was not evoked after the abdomen was rinsed with n-hexane. this suggests that the leader secretes the tandem running pheromone on abdominal surface, the compounds of which are soluble in n-hexane. this study sought to identify the glandular source of tandem running pheromone and its chemical structure in diacamma sp. from japan. material and methods ant colonies five test colonies of diacamma sp. from japan, each comprised of one mated worker (gamergate), 56-120 unmated sterile workers and broods, were collected during 2007-2009 in sueyoshi park, naha, on the main island of okinawa, japan. they were kept separately in small plastic cases (artificial nest, 110 × 75 × 25 mm, with a plaster floor) and placed at one end of a larger plastic container (foraging field, 640 × 380 × 90 mm). a red transparent 1283 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. plastic sheet was placed on the artificial nests to keep them dark. fresh water and mealworms were supplied every two days. inducing nest emigration nest emigration was artificially induced by removing the red plastic sheet and exposing the artificial nest to overhead (50 cm) light from a fluorescent lamp (100-w), as described by traniello & hölldobler (1984). prior to that, another plastic case of the same size with a plastic sheet was placed at the opposite end of the foraging field as a new nest. behavioral bioassay a total of 10 to 15 foragers from five colonies were individually killed by freezing, and their entire abdominal part, except for the petiole, were collected. these segments were used as dummies to imitate the leaders in the behavioral bioassay for tandem running. after artificially inducing nest emigration, each isolated segment [i] was picked up with forceps and gently inserted between a leader and follower of a tandem pair and moved away from the former. we counted the numbers of ants that responded by following the dummy for at least five seconds. in the same way, we tested segments extracted with 1 ml of n-hexane for 5 min [ii], as well as ones that had been re-treated with hexane extract [iii]. we conducted this bioassay sequentially with [i], [ii] and [iii] at least seven times using different tandem pairs. the recovery effect of samples re-treated to n-hexane extracted abdominal segment was used to evaluate its tandem following activity. the exocrine gland of the tandem running pheromone to identify the glandular sources of the tandem running pheromone, we dissected three major exocrine glands the dufour’s gland, poison gland and pygidial gland from ten workers, and immersed each one in 500µl of n-hexane for 5 min. each glandular extract was concentrated to an adequate volume, from which 0.1 ant equivalents (ae) each were tested. the bioassay was also conducted sequentially as described above at least 12 times using different tandem pairs. extraction and fractionation of tandem following pheromone thirty dufour’s gland extracts were chromatographed on 1 g of silica-gel (230-400 mesh, merck) and successively eluted with 3 ml each of n-hexane 1284 sociobiolog y vol. 59, no. 4, 2012 and dichloromethane (apolar and polar fraction). the n-hexane eluate was further chromatographed on 1 g of silica-gel (230-400 mesh, merck), impregnated with 10% agno 3 , and successively eluted with 3 ml of n-hexane and 15 % diethylether-in-hexane (e/h) to separate saturated hydrocarbons and unsaturated hydrocarbons, respectively. all these fractions were used in the behavioral bioassays as test samples. chemical analysis active fractions were subjected to analysis with a shimadzu gc-14a gas chromatograph equipped with a db-1ht apolar capillary column (15 m × 0.25 mm id × 0.1 µm film thickness, j&w scientific), and a flame ionization detector (fid). injection was made by splitless mode at 250°c for 1 min. helium was used as the carrier gas at a column head pressure of 200 kpa. the gc oven temperature was held at 80°c for 1 min, increased from 80°c to 280°c at a rate of 20°c per min, and then held at the final temperature for 5 min. fid was set at 250°c. major qualitative components in these active fractions were analyzed by a shimadzu gc-ms system qp-5000. a polar capillary column (db-wax, j&w scientific: 30 m × 0.25 mm id × 0.25 µm film thickness) was directly coupled to the mass spectrometer, and the carrier gas was helium at a column head pressure of 100 kpa. injection was made by splitless mode at 230°c for 1 min. the gc oven temperature program was held at 60°c for 1 min, increased from 60 to 160°c at a rate of 10°c per min, and held at the final temperature for 5 min. the 70ev ei spectra were recorded at a rate of 0.5 s per scan. determination of pheromone structure to determine the double bond position, the unsaturated hydrocarbon fraction was epoxydized with m-chloroperbenzoic acid (swern, 1953). the resultant epoxides were analyzed by gc-ms. the mass spectra of the epoxides demonstrated characteristic diagnostic ions, so we could determine the position of the double bond. eand zisomers (contributed by dr. tetsu ando of the tokyo university of agriculture and technolog y) were used to determine the geometric configuration of the active monoenyl hydrocarbons. these isomers were also used in the behavioral bioassay as test samples. 1285 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. results glandular source of tandem following pheromone during tandem running, followers continuously orientated to the abdomens of the leaders. when isolated intact abdominal segments of leaders were placed within pairs of ants, they were followed in 17 of 20 trials (85%) (fig. 1). after extracting the abdominal segment with n-hexane, however, only 5 of 20 ants (25%) followed the extracted segment. in contrast, 14 of 20 followers (70%) followed the segment when the extract had been re-applied. the number of ants that followed the segments extracted with n-hexane was significantly smaller than that for intact segments and those that had been re-treated with extract (fisher’s exact test with bonferroni correction for multiple testing, p<0.016). fig.1. proportion of the ants following intact, n-hexane extracted and re-treated n-hexane extract abdomens (n=15 for each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016). 1286 sociobiolog y vol. 59, no. 4, 2012 fig.2. (a) proportion of ants following intact, n-hexane extracted and re-treated dufour’s gland (dg) extract abdomens (n=15 each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016) (b) proportion of ants following intact, n-hexane extracted and re-treated pygidial gland (pg) extract abdomens (n=16, 12, 21 for each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016). (c) proportion of ants following intact, n-hexane extracted and re-treated poison gland (pog) extract abdomens (n=16, 15, 15 for each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016). fig. 2 compares the tandem running responses to dufour’s gland, poison gland and pygidial gland. when testing the dufour’s gland extract, 12 of 15 (80.0%), 3 of 15 (20.0%) and 10 of 15 (66.7%) followers kept following the segments which were intact, extracted and treated with the dufour’s gland extract, respectively (fisher’s exact test with bonferroni correction for multiple testing, p<0.016). in contrast, 13 of 16 (81.3%), 6 of 12 (50%) and 7 of 21 (33.3%) followers kept following the segments that were intact, extracted and treated with pygidial gland extract, while 14 of 16 (87.5%), 4 of 15 (26.7%) and 3 of 12 (25%) followers kept following the segments that were intact, extracted and treated with the poison gland extract, respectively. neither pygidial gland nor poison gland extract had higher numbers of followers compared to extracted segments (fisher’s exact test with bonferroni correction for multiple testing ). 1287 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. identification of the tandem running pheromone tandem running activity was compared between apolar and polar fractions obtained from the dufour’s gland extract (fig. 3). the apolar fraction induced tandem running much the same as intact abdomens (fisher’s exact test with bonferroni correction for multiple testing, p<0.016); 25 of 32 (78.1%) followers kept following the intact segment, and 4 of 27 (14.8%) and 31 of 54 (57.4%) followers kept following the extracted and treated ones, respectively. the polar fraction of dufour’s gland extract treated abdomen showed less activity. ants followed 19 of 49 polar-fraction treated abdomens (38.8%), 25 of 32 intact ones (78.1%) and 1 of 20 n-hexane extracted abdomens (5.0%). we also checked the additive or synergistic effect of the polar fraction to the apolar fraction’s activity, however, no significant effect was confirmed. ants followed 9 of 15 apolar-fraction treated abdomens (60%), 10 of 15 re-mixed apolarand polar-fraction treated abdomens (66.7%), 26 of 30 intact abdomens (86.7%) and 8 of 30 n-hexane extracted abdomens (26.7%). hydrocarbon compounds in the apolar fraction were further separated into saturated and unsaturated hydrocarbons to confirm their tandem running activity (fig. 4). for the unsaturated hydrocarbon fraction, resultant tandem running activity was confirmed to be much the same as that of intact abdomens (fisher’s exact test with bonferroni correction for multiple testing ). ants followed 12 of 20 intact abdomens (60.0%), 3 of 20 n-hexane extracted ones (15%) and 13 of 20 unsaturated-fraction treated ones (65.0%). the saturated hydrocarbon-fraction treated abdomen showed less tandem running activity. ants followed 7 of 8 intact abdomens (87.5%), 1 of 7 n-hexane extracted ones (14.3%) and 2 of 7 saturated hydrocarbon-fraction treated ones (28.6%). the unsaturated hydrocarbon fraction contained one major quantitative peak compound (x), for which the kovats’ retention index was 1667 on an apolar capillary column, and several minor compounds (fig. 5-a). the mass spectrum of compound x showed a typical alkene fragmentation (m/z 69, 55 and 41 as base peak ion), and presented a molecular ion at m/z 238. we therefore estimated it as heptadecene (c 17:1 ). the epoxidized compound x presented the retention index as 1850 on an apolar capillary column, and gave two characteristic fragment ions at m/z 141 and m/z 155 (fig. 5-b). this indicated the position of the double bond to be between the 8th and 9th carbon, and the compound x was identified as 8-heptadecene. 1288 sociobiolog y vol. 59, no. 4, 2012 fig.3. (a) proportion of ants following intact, n-hexane extracted and re-treated apolar fraction of dufour’s gland extract abdomens (n=32, 27, 54 for each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016).(b) proportion of ants following intact, n-hexane extracted and re-treated saturated hydrocarbon fraction abdomens (n=8, 7, 7 for each test) 1289 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. fig.4. (a) proportion of ants following intact, n-hexane extracted and re-treated unsaturated hydrocarbon fraction abdomens (n=20 for each test). (b) proportion of ants following intact, n-hexane extracted and re-treated saturated hydrocarbon fraction abdomens (n=20 for each test). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016). 1290 sociobiolog y vol. 59, no. 4, 2012 fig. 6 shows tandem running activity of the synthesized (e)and (z)isomer of 8-heptadecene. ants followed only the (z)isomer of 8-heptadecene (fisher’s exact test with bonferroni correction for multiple testing, p<0.016). ants followed 9 of 10 intact abdomens (90.0%), 2 of 10 extracted ones (20.0%) and 6 of 10 (z)-8-heptadecene treated ones (60.0%). the (e)-isomer of 8-heptadecene did not possess tandem running activity. ants followed 5 of 5 intact abdomens (100%), 2 of 14 extracted ones (14.3%) but 0 of 5 polarfraction treated ones (0%). (z)-8-heptadecene was identified as the tandem running pheromone of diacamma sp. from japan. fig.5. (a) gas chromatogram of unsaturated hydrocarbon fraction from dufour’s gland . (b) mass spectrum of peak x. important fragment ions were observed at m/z 141 and m/z 155. 1291 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. fig.6. proportion of ants following intact, n-hexane extracted and re-treated eand zisomer of 8-heptadecene abdomens (n=10 for each test of e-isomer, n=5, 14 and 5 for zisomer). bars with the same letter are not significantly different by fisher’s exact test with bonferroni correction for multiple testing (p<0.016). 1292 sociobiolog y vol. 59, no. 4, 2012 discussion we confirmed that tandem running behavior of follower ants was mediated by chemical compounds on the abdominal surface of leader ants, and that the compounds are soluble in n-hexane. the glandular source of this chemical bond between the tandem pair was identified as the dufour’s gland. the most potent activity was confirmed from the apolar fraction of this gland; no additive or synergistic effect was observed from the polar fractions. the chemical structure of tandem running pheromone was revealed to be unsaturated hydrocarbon, and analytical data revealed the quantitatively major component of the unsaturated hydrocarbon fraction as 8-heptadecene. synthesized (z)-isomer of 8-heptadecene possessed tandem running activity equal to that of the isolated abdomen, but no attractive effect was observed when the abdomen was treated by e-isomer. we identified the tandem running pheromone of this ant as (z)-8-heptadecene as it contributed greatly to the tandem running activity. some possibility of the contribution of minor components remains. some reports have already revealed that particular exocrine glands or organs such as poison gland, pygidial gland and hind legs, secrete tandem running pheromone (möglich 1974; hölldobler & traniello 1980b; maschwitz et al. 1986), but the active compound had not been identified. this is the first identification report of the tandem running pheromone of ants that is confirmed with the use of synthetic chemical compound that induced tandem running behavior. almost all recruitment pheromones of ants are reported to be derived from abdominal exocrine glands, including the dufour’s gland. in the subfamily ponerinae, it is the poison gland and pygidial gland that have been identified as the source of tandem running pheromone (möglich 1974; traniello & hölldobler 1984). diacamma sp. from japan belongs to the ponerinae, but the source of tandem running pheromone in this species is dufour’s gland. during our bioassays, there was no characteristic behavior observed when the follower ant was presented the abdominal part treated with pygidial gland extract. however, when the followers were presented an abdominal part treated with poison gland extract, they showed aggressive or escape behavior. these two exocrine glands do not contribute to communication related to tandem running. 1293 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. it is interesting that all the compounds so far identified as pheromone in the dufour’s gland belong to the terpenoid class; (z, e)-α-farnesene (solenopsis invicta, vander meer et al. 1988), all-trans-geranylgeranyl acetate and geranylgeraniol (ectatomma ruidum, bestmann et al. 1995) and a mixture of 4-methylgeranyl esters of fatty acids identified as trail pheromone (gnamptogenys striatula, blatrix et al. 2002). (z)-8-heptadecene is the first example of non-terpenoid recruitment pheromone secreted by the dufour’s gland. compared to other already reported recruitment pheromones, the polarity of (z)-8-heptadecene is low, because it does not include any polar function group in its structure. during the bioassay, we observed the characteristic behavior of the leader’s application of the pheromone on the surface of their abdomen with their hind legs. their body surfaces are known to be covered with cuticular hydrocarbons (chcs) (howard 1993). both chcs and tandem running pheromone are apolar, so are expected to have more affinity with each other than to the other reported recruitment pheromones that are mainly polar (morgan 2009). this chemical nature of (z)-8-heptadecene might contribute to the endurance of the effect of tandem running pheromone. in most ant species, the recruitment pheromone is secreted from a single exocrine gland, but multiple glands may be involved in some cases (vander meer et al. 1998). in diacamma sp. from japan, only dufour’s gland possessed activity, however, we should confirm whether other exocrine glands possess additional and/or synergic effect. it has been suggested that tandem running behavior could serve as a preadaptation for the evolution of chemical mass recruitment (möglich et al. 1974). this is because it engenders movement of only a single ant at a time, and requires only a tandem running pheromone for the tandem pair. no orientation pheromones are needed. other much more complex recruitment systems use sustainable chemical trails, and can attract many other workers to a food source, new nest site, or attack of intruders (vander meer et al. 1981; vander meer et al. 1988; vander meer et al. 1990; hölldobler & wilson 1978). recruitment and orientation pheromone components and use of those pheromones differ in species. detailed analysis of differences in recruitment systems among ants could reveal more about possible steps in the evolution of ant recruitment systems. 1294 sociobiolog y vol. 59, no. 4, 2012 acknowledgments this work was partially supported by a grant-in-aid for scientific research on priority areas, “emergence of adaptive motor function through interaction between body, brain, and environment,” from the japanese ministry of education, culture, sports, science and technolog y. this work was supported in part by grants-in-aid from the japan ministry of education, science and culture (20033015, 21247006 to k. tsuji). references bestmann, h.-j., e. janssen, f. kern, b. liepold, b. hölldobler & t. boveri 1995. all-transgeranylgeranyl acetate and geranylgeraniol, recruitment pheromone components in the dufour gland of poneromorph ant ectatomma ruidum. naturwissenschaften 82: 334-336. blatrix, r., c. schulz, p. jaisson, w. francke & a. hefetz 2002. trail pheromone of the ponerine ant gnamptogenys striatula: 4-methylgeranylesters from dufour’s gland. journal of chemical ecolog y 28: 2557-2567. fukumoto, y. & t. abe 1983. social organization of colony movement in the tropical ponerine ant, diacamma rugosum (le guillou). journal of etholog y 1: 101-108. hölldobler, b. & e.o. wilson 1978. the multiple recruitment systems of the african weaver ant oecophyla longinoda (latreille) (hymenoptera: formicidae). annals of the entomological society of america 76: 235-238. hölldobler, b. & e.o. wilson 1990. the ants. harvard university press, cambridge, ma. hölldobler, b. & j.f.a. traniello 1980a. tandem running pheromone in ponerine ants. naturwissenschaften 67: 360. hölldobler, b. & j.f.a. traniello 1980b. the pygidial gland and chemical recruitment communication in pachycondyla (=termitopone) laevigata. journal of chemical ecolog y 6: 883-893. howard, r.w. 1993. cuticular hydrocarbons and chemical communication. in: stanleysamuelson, d.w. & d.r. nelson (eds.) insect lipids: chemistry, biochemistry and biolog y. university of nebraska press, lincoln, ne: 179–226. maschwitz, u., k. jessen & s. knecht 1986. tandem recruitment and trail laying in the ponerine ant diacamma rugosum: signal analysis. etholog y 71: 20-41. möglich, m., u. maschwitz & b. hölldobler 1974. tandem calling : a new signal in ant communication. science 186: 1046-1047. morgan, e.d. 2009. trail pheromone of ants. physiological entomolog y 34: 1-17. swarn, d. 1953. epoxidation and hydroxylation of ethylenic compounds with organic peracids. organic reaction 7: 378-380. 1295 fujiwara-tsujii, n. et al. — tandem running pheromone in diacamma sp. traniello, j.f.a. & b. hölldobler 1984. chemical communication during tandem running in pachycondyla obscuricornis (hymenoptera: formicidae). journal of chemical ecolog y 10: 783-794. vander meer, r.k., f.d. williams & c.s. lofgren 1981. hydrocarbon components of the trail recruitment pheromone of the red imported fire ant, solenopsis invicta. tetrahedron letters 22: 1651-1654. vander meer, r.k., f.m alvarez & c.s. lofgren 1988. isolation of the trail recruitment pheromone of solenopsis invicta. journal of chemical ecolog y 14: 825-838. vander meer, r.k., c.s. lofgren & f.m alvarez 1990. the orientation inducer pheromone of the fire ant solenopsis invicta. physiological entomolog y 15: 483-488. vander meer, r.k. & l. morel 1998. pheromone directed behavior in ants. in: vander meer, r.k., m.d. breed, m.l. winston & k.e. espelie (eds.) pheromone communication in social insects: ants, wasps, bees, and termites. westview press, boulder, colorado: 176-182. wilson, e.o. 1971. the insect societies. belknap press of har vard univ. press, cambridge. 1509 fumigant toxicity of mentha arviensis leaves extracts on coptotermes heimi, heterotermes indicola and their gut f flagellates by naveeda akhtar qureshi1, muhammad zahid qureshi2 ,muhammad athar3 , anwar malik4 & aziz ullah5 abstract the extract of mentha arviensis (mint) has been found to have considerable application against microbial diseases. the present study is designed to check the insecticidal properties of mentha arviensis. crude extract of the leaves of mentha arviensis was obtained in benzene-ethanol (2:1) solvent using soxhelt apparatus and dried extract was applied (25mg, 50mg and 100 mg ) against termite workers, soldiers and their gut flagellates of coptotermes heimi and heterotermes indicola. a significant increase in mortality of termite workers, soldiers and their gut flagellates was observed depending upon a lethal dose over time, in both termite species. it, however, took a relatively longer time period to achieve 100% mortality of flagellates than their respective hosts. the dose dependent death of flagellates over time also indicates the mortality of flagellates was found to be associated with fumes originated from the extract of the plants as in starvation, termites and their associated flagellates can survive for three to four days and also their was no mortality of workers and their flagellates, in control. thus mentha arviensis extract can be safely used to control termites and other pests. key words: pest control, termites, gut flagellates, mint plant 1laboratory of entomolog y and parasitolog y, department of animal sciences, faculty of biological sciences, quaid-i-azam university 45320, islamabad, pakistan e-mail: naveedaqresh@gmail.com 2department of chemistry bhauddin zakaryia university, multan, pakistan 3department of chemistry, government college university, lahore, pakistan 4&5 departments of zoolog y, government college university, lahore, pakistan *correspondence to: naveeda akhtar qureshi1, department of animal sciences, faculty of biological sciences, quaid-i-azam university 45320, islamabad, pakistane-mail naveedaqresh@gmail.com 1510 sociobiolog y vol. 59, no. 4, 2012 introduction termites are small insects (isoptera, insecta) are worldwide and recorded firstly in japan in the 9th century ( jtca 1998). globally, termites are a big problem in both the agricultural and urban areas as they cause significant damages to crops, plants, buildings and woodwork and account for considerable economic damage. in order to make cellulose digested and ready for assimilation the termites live in strong mutualistic relationship with a variety of hindgut inhabiting endomicrobes (flagellates, bacteria and archea). these endomicrobes mainly flagellates produce the enzyme cellulase, which acts to render the plant celluloses suitable for assimilation (breznak 2000). it is logical therefore to presume that the termites cannot live without their flagellate fauna. in other words, to control the termite infestation, effectively one should focus on the symbionts living within their gut (stingl et al. 2004, 2005; ikeda et al. 2007; ohkuma 2008), the elimination of gut fauna will cause starvation and then ultimately casualty of termites (honigberg 1970; brune 2003). in practice termite infestations have been controlled by using synthetic insecticides such as; dieldrin, chlordane and heptachlor (ward et al. 2009; jamil et al. 2005; logan et al.1990). it was estimated that an about us$ 0.8-1.0 billion per year is used to prevent and control termite infestations, ( fushiki 1998; tsunoda 2003). these insecticides cause serious environmental hazards, if there are any leakages after heavy rain and have strong pungent odors (logan et al. 1990; martius 1998). these are a source of many disorders e.g., chlordane cause cancer, chlorpyriphos, dimethoate, and endosulfan cause genotoxicity ( jamil et al. 2005). polychlorinated biphenyls and organochlorine pesticides (e.g. αand γ-chlordane, p, p′-ddt (dichlorodiphenyltrichloroethane), p, p′-dde (dichloro diphenyl dichloro ethylene), methoxychlor, and pentachlorophenol, persist in carpets and cause leukemia and neuroses in children. alongside these drugs are also fatal for reproductive and immune system e.g. one popular termiticides chlordane proved to be causing cancer and has been banned in some countries. therefore, these are not recommended for indoor applications (ward et al. 2009; hatlelid et al. 2004; ramos & rojas 2005; gautam & henderson 2008). the use of many pesticides has therefore been banned in many countries (robinson 2005). considering the 1511 qureshi, n.a. et al.— fumigant toxicity of mentha arviensis toxigenic nature of synthetic pesticides, termiticides that are plant-derived organic chemicals are being explored which would be environmentally safe to use e.g. the plants like polygonal hydropiper, progesterone parviflorus, alaska yellow, red ceder, tung tree, pine resins red wood etc. the extracts of the tung tree and pine resin, has been reported to have anti-termite properties (bläske & hertel 2001; nunes et al. 2004). polygonal hydropiper, progesterone parviflorus, alaska yellow, red ceder and red wood strongly show anti feedant and toxic activities against termites (tellez et al. 2001; hennon et al. 2007). solvent and aqueous extract of plants such as diospyros, polygonal, eucalyptus, dalbergia, morus, neem etc. have termiticidal activities. in this reference, monoterpenoides isolated from flourensia cernua have proven to be strongly effective for termite control. matias (2005) explained insect pest repellent activities of menthol propylene glycol carbonate and its analogs also exhibit anti-inflammatory and anti-antigenic effects on human. similarly chemicals like menthol, α-terpineol, p-menthone, menthol acetate and others were isolated from mentha arvensis (deschamps et al. 2006; brito et al. 2007). menthol, the main component of mentha arviensis, has been used for centuries in medicines, proven to have anti algeric, anti-ulcer antispasmodic and antimicrobial activities (nascimento et al. 2009). menthol shows competitive inhibition for acetylcholine esterase produced by insects (lee et al. 2001). besides, it has been used as insecticides, a cooling agent, as food and in cosmetics. menthone, another constituent of mentha arviensis has been found to have antibacterial (kurita and koike 1982), anti-allergic (arakawa et al. 1992) and spasmolytic properties (gamez et al. 1990). the extract of mentha arviensis has fumigant toxicity and proven affective against sitophilus oryzae l, a rice weevil (lee et al. 2001). matias (2006) compared the structure of menthol and menthol propylene glycol carbonate (mr-8), the latter found to be more effective for pest control. the joint fao/who expert committee on food additives ( jecfa) reconfirmed the safety of menthol and its derivatives for use as an ingredient in food and cosmetics assessment conducted in 1999 (matias 2006). owing to the advantageous properties of mentha arviensis, the present research work is designed to investigate the toxigenic nature of mint plant (mentha arviensis) extract against termites and their entozoic flagellate populations. 1512 sociobiolog y vol. 59, no. 4, 2012 materials and methods extraction process dry leaves of mentha arviensis were ground and the extract was prepared using benzeneethanol solution (2:1) as a solvent following the method of vogel (1964). dry leaves (20 gm) were taken in a soxhelt extraction flask containing 300 ml of solvent. it was then connected with the extraction apparatus and the distilled water was allowed to flow into the condenser. heating was adjusted to boiling temperature. the extraction cycle was carried out at least 6 times per hour. after 4-5 hours extraction, the flask were then removed and allowed to cool. insect procurement and maintenance termite workers and soldiers of species coptotermes heimi (wasman) and heterotermes indicola (wasman) were collected from old trees of government college university gardens, lahore. they were kept for at least 1 week in glass jars containing water soaked filter papers at 30oc in an oven to clear their intestines from debris and other wood particles following the method of ramos and rojas (2005) with some modifications. dosage the solid extract in three different dosage concentrations (25 mg, 50 mg and 100 mg ) was sprinkled at the base of glass jars of 15 cm height and 7cm diameter.it was partitioned from rest of the space in the jars by a narrow iron net placed at 10cm height from base so that fumes can pass through the net. 100 termite workers of c. heimi were placed on the net so that they were not in direct contect with the crystals of mentha arviensis leaves extract. a small piece of filter paper for food source and water soaked cotton plug to keep humidity and water supplied to the termites were placed in the jar, on the net along with termite workers and tightened the jar with a lid. same sets of experiments were desinged for soliders of c. heimi and also for workers and soldiers of h. indicola (n =100 each). the fumigation effect of the extract on termite workers, soldiers and their intestinal flagellates was studied over 1to 6 hours of exposure. three replicates of each concentration were run. control preparations were run in parallel without the application of the extract. 1513 qureshi, n.a. et al.— fumigant toxicity of mentha arviensis physical observations of termites: termites workers and soldiers in each set of experiment were observed with magnifying glass for six hours. mortality percentage was calculated after each hour. gut flagellates: ten termites from each treatment group workers , soldiers and controls of both termite species were dissected to study the effect of the extract on gut flagellate population. for the study of flagellate population workers and soldiers of termites were decapitated and their entire guts were pulled out using a dissecting needle. the guts were dissected and the contents were squeezed out in a drop of 0.2 % saline solution on cover glasses. the contents were allowed to spread on the cover glass. the procedure was carried out under a stereo zoom microscope (olympus). the cover glasses were then inverted on improving neubaur’s chambers and the flagellates were counted under in four w squares (1 mm2) of the chamber at low magnification. the arithmetic mean of flagellates was calculated by adding the number of flagellates in all the four areas and dividing it by 4. the diameter of the spread smear was also measured. area of the smear was calculated according to the formula πr2. the total number of flagellates in a smear was calculated by using the following formula. total number of flagellates = average number of flagellates in 1 w area or 1mm2 × total area of the smear in mm. dead termites were also disected to study the survival of their flagellates. data analysis data were analyzed using two-way analysis of variance (anova) and means were separated using tukey’s hsd quantile function, using spss 10.0. all tests were conducted at á = 0.05 levels. results and discussion presently, the effect of mint extract was investigated to control termite infestation.the two castes of termites i.e. workers and soldiers were forced to live in an environment of 25mg, 50 mg and 100 mg dosage concentrations for a period of 6 hours. hundred percent mortalities of workers and soldiers were observed in a period of 3hours and there was also a significant decrease in flagellates population residing in the gut of both workers and soldiers but the hundered percent mortality in flagellates population was achieved within 1514 sociobiolog y vol. 59, no. 4, 2012 6 hours as was observed in the gut of dead termites . maximum mortality (100%) of workers and soldiers was recorded at 3, 2 and 1 hours subsequently on exposure to 25 mg, 50 mg and 100 mg of extract respectively( p < 0.05). fig. 1 (a-f) effect of different concentrations of mentha arviensis leaves extracts on coptotermes heimi and its entozoic flagellates. 1515 qureshi, n.a. et al.— fumigant toxicity of mentha arviensis whereas no mortality of workers or soldiers occurred in the control groups(fig 1(e & f)). the flagellates population in the guts of soldiers and workers were also recorded and their 100% mortality was obtained in 6, 3, and 2 hours fig. 2 (a-f) effect of different concentrations of mentha arviensis leaves extracts on heterotermes indicola and its entozoic flagellates. 1516 sociobiolog y vol. 59, no. 4, 2012 on exposure to 25 mg, 50 mg and 100 mg of extract respectively (p < 0.05) . after three hours although the termites had died but their gut flagellallates were still surviving upto six hours. an insignificant decrease in the flagellate population was observed in the control groups (fig. 1(a to d)). h. indicola also behave in a same manner as c.heimi on expoure to different concentrations of mentha arviensis extract and underwent 100% mortality at 25 mg, 50 mg and 100 mg of extract respectively at 3, 2 and 1 hours (p < 0.05). control non-exposed workers, soldiers were not affected (fig. 2 (a & b)) and their gut flagellates showed 100% mortality at 6, 3 and 2 hours after exposure to increasing concentrations of dose (p < 0.05) in the control groups no sinificant mortality was observed (fig. 2 (c to d)). the mean population of flagellates in both termite species, decreased dose-dependently in a very similar dose-dependent pattern, the mortality percentages of termite workers and soldiers increased. on the whole, the effect of the extract on termites and their flagellate fauna was quite possibly due to the vapors of its volatile components. the termites rapidly became less active and senseless on exposure to the mint extract indicating the vapors of the extract was to be toxic to the termites. the low percentage of extracts affects the flagellates earlier than their host termites. thus the termites at low concentration of doze underwent death as a consequence of the mortality of their flagellate protozoans. this appears to be true because termites and flagellates have developed a mutualistic type of association with each other and therefore in a stravation or defaunated worker termites can survive for more than two days and subsequently die of starvation, without flagellates (honigberg 1970). the gc-ms analysis of mentha arviensis revealed that it mainly composed of menthol (63.2%), menthone (13.1%), limonene (1.5%), β pinene (0.7%), α pinene (0.6 %), and linalool (0.2%). among these menthone showed 8.1 times more insecticidal properties and has an inhibitory effect on acetylcholineterase in rice weevil (lee at al. 2001). in the present investigations the fumigant toxicity of mentha arviensis leaves extracts found to have anti-termitic and antiprotozoal activity and since it has been reported by us fda as gras (generally recognized as safe) chemical agent (nan-yao su and scheffrahn, 1998) that is why can be a good substitute for the synthetic termitcidal and the antiproozoan drugs. 1517 qureshi, n.a. et al.— fumigant toxicity of mentha arviensis the present study shows that the extract of, mentha arviensis can be an effective natural anti-termite drug and its use would lessen the risk of environmental contamination as compared to the synthetic insecticides. acknowledgments the 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sociobiolog y 41: 27-36. vogel, a.i. 1964. a textbook of practical organic chemistry, including qualitative organic analysis. london (longmans, green and co.). ward, m.h., colt, j.s., metayer, c., gunier, r.b., lubin, j. & v. crouse. 2009. residential exposure to polychlorinated biphenyls and organochlorine pesticides and risk of childhood leukemia. environ. health perspective 117:1007-1013. doi: 10.13102/sociobiology.v67i4.5558sociobiology 67(4): 492-500 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction tropical forest canopies are known to support a high animal diversity. particularly arthropods can be very diverse and abundant (klimes et al., 2012). ants account for 20 to 60 % of total arthropod biomass of tropical forest canopy invertebrates (floren et al., 2014), and therefore, represent one of the most abundant and ecologically important animal groups in tropical regions (hölldobler & wilson, 1990; lach et al., 2010). ants are also widely considered as keystone species due to the important ecological role they play in many ecosystems (del toro et al., 2012). in tropical forest canopy, ants often exercise a high predation pressure and thus significantly influence structure and dynamics of arboreal arthropod communities (philpott & armbrecht, 2006). ant are also abstract ants constitute an important part of arboreal arthropod biomass in rainforests. nevertheless, there are only a few methods which permit a rapid assessment of these insects in the canopy layer. this study aims at evaluating the efficiency of a new variant type of pitfall trap i.e. “the funnel trap”, to sample arboreal ants in a secondary and gallery forest in lamto reserve (côte d’ivoire). this method was compared to standard arboreal pitfall trap and beating. in total, the 3 methods yielded 7072 ant workers belonging to 43 species, 14 genera and 5 subfamilies. tree beating recorded the highest ant’s numerical abundance (3670 workers), with 27 species, 12 genera and 3 subfamilies followed by the “funnel trap” that yielded 2800 ant workers, with 23 species belonging to 12 genera and 5 subfamilies. finally, arboreal pitfall traps caught the lowest individual with 602 ant workers from 20 species belonging to 9 genera and 3 subfamilies. the composition of species which are caught by arboreal pitfall trap and “funnel trap” was similar at 53 percent. tree beating showed a distinct species composition compared to arboreal pitfall trap and “funnel trap”. the “funnel trap” could be a fast and efficient way to quickly assess ant-biodiversity in forest canopies and agroecosystems as it looks like a nondestructive sampling method. sociobiology an international journal on social insects cd yodé1,2, k dosso2, lmm kouakou2 , k yeo2, w dekoninck3, s konate2, pk kouassi1 article history edited by gilberto m. m. santos, uefs, brazil received 06 july 2020 initial acceptance 07 september 2020 final acceptance 16 november 2020 publication date 28 december 2020 keywords pitfall trap, “funnel” trap, lamto reserve, biodiversity, habitat structure, tree canopy. corresponding author christine dakélé yodé felix houphouët boigny university research station in ecology of lamto scientific reserve bp 28 n’douci, côte d’ivoire. e-mail: christineyode@gmail.com used as indicator taxa for ecological surveys because they are relatively easy to sample in soil litter (alonso & agosti, 2000). despite this importance, the arboreal ant community remains poorly known for some region in tropics, mainly because the difficulty for scientists to find appropriate sampling method to sample either at three meters above the ground. for example, methods to sample arboreal ants such as the spikesand-belt method, single-rope technique, baited pitfall traps and so forth, are methods for which one needs an appropriate logistic to access to the canopy and for the installation of all sampling equipments (basset et al., 1997). nevertheless, few techniques such as the canopy fogging and beating do not require a lot of equipment in the canopy (castaño-meneses, 2014). currently, pitfall traps (including its variants) are often used for arboreal ant sampling (powell et al., 2011; chapin & smith, 2019). 1 felix houphouët boigny university, (ufr biosciences), zoology-biology of animal laboratory, abidjan, côte d’ivoire 2 nangui abrogoua university, ufr des sciences de la nature (ufr sn), research station in ecology of lamto, abidjan, côte d’ivoire 3 royal belgian institute of natural sciences, od taxonomy and phylogeny, brussels, belgium research article ants evaluating efficiency of different sampling methods for arboreal ants (hymenoptera: formicidae) in a west african forest-savanna mosaic sociobiology 67(4): 492-500 (december, 2020) 493 although, pitfall trapping is a well-recognized ant sampling technique, an important difficulty remains the installation of these traps above three meters high in the canopy. usually this installation requires climbing which is difficult and risky. hence, to sample canopy biota rigorous inventory techniques need to be developed. these techniques should be simple, fast and allow researchers to optimize their sampling in the canopy (yusah et al., 2012; yusah et al., 2018; leponce et al., 2019). here, this study reports the results of arboreal ant diversity surveys using different sampling methods in a tropical forest-savanna mosaic habitat at lamto scientific reserve (côte d’ivoire). overall, it aims at establishing a database on arboreal ant species richness in côte d’ivoire. indeed, the arboreal ant community remains poorly known and the only existing studies go back to the seventies (delage-darchen, 1970, 1971, 1972, 1973, 1974; levieux, 1976). this study aims at testing the efficiency of a new variant of pitfall trap, the “funnel trap” to collect arboreal ants in secondary and gallery forest of lamto scientific reserve. this trap was compared with arboreal baited pitfall trap and beating of tree leaves, by analysing the differences in numbers of ant workers, species richness and species composition. methods study site the study was conducted in lamto scientific reserve, located in central côte d’ivoire at 6°13’/6°25’ n and 4°97’/5°01’ w. the annual precipitation range is 1000–1500 mm/year while the mean monthly temperature is about 27°c. lamto scientific reserve contains a great heterogenous vegetation (abbadie et al., 2006) characterized by a forestsavanna mosaic habitat. the study was carried out in the gallery forest located at the border of bandama river, and the secondary forest that resulted from an experimental bush fire exclusion since 1962 (abbadie et al., 2006; gnaoré et al., 2018). sampling design and identification ants were sampled in three 100 × 50 m plots in gallery forests and secondary forest. on each plot, 20 trees with a circumference at breast height ≥ 32 cm were examined. overall samplings were carried out on 120 trees spread over 6 plots in all. on each plot, three sampling methods were used to collect arboreal ants: canopy beating, arboreal pitfall trap (agosti & alonso, 2000; underwood & fisher 2006; yeo et al ., 2013) and the “funnel trap”, a modified variant of the arboreal pitfall trap (fig 1). fig 1. “funnel trap” model. arboreal pitfall trap: these traps consisted of plastic cups (7.5 cm in diameter, 10.5 cm deep) with water, detergent and baited with tuna (ribas et al., 2003). they were placed on the central axis of the trees at least at 5 m above the ground thanks to a point. they were installed using a ladder. pitfall traps remained in service on the trees during 48 h. the “funnel trap”: the latter was made from empty bottles of 33 cl of mineral water or soda, rope and a bamboo shaft. bottles were cut in half at 8 cm from the top edge. next, the upper part of the bottle was placed upside down in the second part in order to have a funnel. these two parts were attached to their joints with a transparent adhesive tape. finally, two small holes were made at 1cm of the top edge of the bottle to insert a fine nylon thread whose length corresponds to the height at which the trap can be attached to the tree. to prevent the thread from mixing, it was previously wind up on small pieces of wood 7 cm. finally, tuna bait was placed inside the bottle with a little water and was hoisted at 5 meters into the trees using a bamboo of at least 5m height (fig 2). the traps remained in service on during 48 hours. the sampling was carried out on 120 trees. cd yodé, k dosso, lmm kouakou, k yeo, w dekoninck, s konate, pk kouassi – the “funnel trap”: a new sampling method for arboreal ants494 canopy beating: this method consisted in collecting the ants by beating the foliage of the lowest branches of the trees (between 2m and 3m high). the foliage of the trees was beaten in two sequences of 5 beats. each sequence was followed by the gathering of the ants that fell on the canvas. in total the foliage was beaten ten times at the same point. also, it had 20 collection points on a plot, and therefore 120 collection points in all. fig 2. installation of “funnel trap” in tree canopy. 10 0 m 50 m fig 3. scheme of the sampling plot in the lamto scientific reserve, côte d’ivoire. arboreal pitfall traps and funnel traps were installed on the same trees. as for the beating, they were generally carried out on the same trees or in thickets directly connected to the trees in which the traps were installed (fig 3). ants were identified to genus level using the guide of the genera to fisher and bolton (2016) with a leica mz6 microscope. at species level, the keys of bolton (1980, 1982 and 1987); rigato, 2016), and reliable digital keys (antweb.org) were used. when species-level identification was impossible, distinct specimens were sorted according to morphospecies. morphospecies were numbered according to the ant reference collection for côte d’ivoire at lamto ecological research station. the specimens were added to lamto ecological research station collection. data analysis samples of each plot were pooled to obtain a total of 120 samples for each sampling method. taxonomic structure (subfamily), species richness, abundance, and species composition were compared for each sampling method. the chao 2 index species richness estimator was calculated to extrapolate the species richness from our data. sample coverages were determined to estimate sampling efficiency based on different sampling method using estimates v.9.1.0. comparative analysis was carried out on the species richness and numerical abundance of the ants collected by the different trap types. kruskall-wallis and man-whitney pairwise comparison tests were used to test the differences across the trap types using species richness and numerical abundance. the comparison of species composition was conducted calculating jaccard similarity index. results general results overall, the three combined methods yielded 7072 ant workers belonging to 43 species, 14 genera and 5 subfamilies. the subfamilies were dolichoderinae, myrmicinae, formicinae, ponerinea and pseudomyrmicinae. all five subfamilies were collected with the funnel trap, whereas only three out of five subfamilies were collected with arboreal pitfall traps and beating, respectively (fig 4). sampling efficiency table 1 shows that the sample coverage varied between 71 and 93 %, illustrating that all three methods were suitable to investigate arboreal ant community. observed species observed species estimated species (chao 2) sampling coverage unique species doubletons arboreal pitfall 20 21,49 93% 4 3 funnel trap 23 32,26 71% 8 2 beating 27 31,17 86% 7 4 table 1. numbers of species caught by the trapping methods during study. sociobiology 67(4): 492-500 (december, 2020) 495 accumulation curves evolved towards asymptotic lines for all sampling methods. however, the chao 2 estimated species accumulation curves for “funnel trap” and beating increased steadily with sampling size (fig 5). in addition, the tree beating method collected the highest number of species (27 species), followed by funnel trap (23 species) and arboreal pitfall trap (20 species). funnel traps caught more unique species (8 species) followed by beating (7 species) and arboreal pitfall (4 species) (table 1). species richness and diversity tree beating recorded the highest number of ant species (27 species) with 3670 workers, followed by the “funnel trap” (23 species) for 2800 workers. arboreal pitfall traps caught the lowest number of ant species (20 species) and workers (602). the mean ant species number found in traps differed significantly between the 3 sampling methods (kruskallfig 4. ant species distribution within the different subfamilies encountered for each sampling method. fig 5. species accumulation curves of the three sampling methods. wallis: x 2 = 89.66; df = 2; p = 0.0001). tree beating method recorded the highest mean number of ant species (2.59 species/trap) followed by “funnel trap” method (1.63 species/ trap) and arboreal pitfall trap method (0.98 species/trap). the man-whitney pairwise comparison test showed that “funnel trap” caught more ant species than arboreal pitfall trap (u = 4670; p < 0.001). on the other hand, tree beating method caught more ant species than “funnel trap” (u = 4216; p < 0.0001) and arboreal pitfall trap (u = 2335; p < 0.001) (such as fig 6). the three sampling methods yielded a high diversity, but the value of the simpson diversity index was higher for the beating method (0.89), followed by that of the funnel trap (0.86) and finally the arboreal pitfall trap (0.84). on the other hand, evenness values were low, with e=0.34 for the beating and e=0.36 for both funnel trap and arboreal pitfall trap, respectively (table 2). cd yodé, k dosso, lmm kouakou, k yeo, w dekoninck, s konate, pk kouassi – the “funnel trap”: a new sampling method for arboreal ants496 ant abundance and species composition overall, ant abundance (individuals) varied significantly among each sampling method (anova of kruskal-wallis test: x 2 = 85.23; df = 2; p = 0.0001; such as fig 7). on average, the tree beating method (29.93 individuals/trap) and “funnel trap” (23.33 individuals/trap) caught the highest numbers of ant workers, whilst arboreal pitfall trap caught 5.02 individuals/trap. mann-whitney pairwise comparison test indicated that the funnel trap caught more ant workers than the arboreal pitfall trap (u = 3931; p = 0.001). however, the tree beating method caught more ant workers (individuals) than the funnel trap (u = 5686; p = 0.004), and arboreal pitfall trap (u = 2307; p = 0.001). globally, beating yielded a different species composition that funnel trap and arboreal pitfall trap. otherwise, jaccard similarity index showed that the arboreal pitfall trap and funnel trap had approximately a similar species composition with a similarity percentage at 53% of shared species. tree beating shared 30% species with arboreal pitfall trap and 22% with the funnel trap (table 3). however, funnel trap caught species that were not found in the arboreal pitfall trap and beating (table 4). fig 7. ants numerical abundance mean (± se) collected in different traps (numerical abundance per sample). fig 6. sample species richness of each trap. species richness simpson index evenness beating 27 0,89 0.34 funnel trap 23 0,88 0.36 arboreal pitfall 20 0,86 0.36 table 2. measure of diversity in the different sampling method. sociobiology 67(4): 492-500 (december, 2020) 497 discussion several studies already focused on the comparison of different sampling techniques to collect arboreal ant (kaspari, 2000; yusah et al., 2012; garcia-martinez, 2018; leponce et al., 2019). here, we demonstrate that the “funnel trap” can also subfamily espèces arboreal pitfall (n=120) funnel trap (n=120) beating (n=120) formicinae camponotus puberulus emery,1897 0 10 0 camponotus compressiscapus andré, 1889 0 62 0 camponotus solon forel, 1886 3 26 0 camponotus acvapimensis mayr, 1862 0 0 2 camponutus maculatus fabrius, 1782 2 47 0 lepisiota sp.1 92 181 18 oecophylla longinoda latreille, 1802 122 1538 488 plagiolepis alluaudi emery, 1894 0 0 90 plagiolepis sp.2 0 0 21 plagiolepis sp.3 0 0 3 polyrachis sp.1 0 1 4 dolichoderinae tapinoma lugubre santschi, 1917 0 9 0 tapinoma sp.1 0 0 2 tapinoma sp.2 0 0 16 myrmicinae cataulacus traegaohdi santschi, 1914 2 2 47 cataulacus guineensis smith, 1853 0 0 1 crematogaster solenopsides emery, 1899 163 284 1558 crematogaster striatula emery, 1892 1 28 42 crematogaster africana mayr, 1895 38 371 0 crematogaster sp.9 23 0 1 crematogaster nigronitens santschi, 1917 18 1 0 crematogaster sp.14 66 23 115 crematogaster sp.22 0 0 25 crematogaster sp.17 0 0 692 crematogaster sp.21 0 0 1 monomorium dolatu bolton, 1987 1 0 5 monomorium floricola jerdon, 1851 9 97 10 monomorium inquietum santschi, 1926 37 57 287 monomorium pharaonis linnaeus, 1758 0 13 0 monomorium sp.2 5 0 63 monomorium sp. 3 0 0 1 pheidole megacephala fabricius, 1793 13 32 0 pheidole sp.2 0 10 0 pheidole sp.6 0 2 0 pheidole sp.7 0 0 11 pheidole sp.8 0 0 2 terataner velatus bolton, 1981 0 0 1 tetramorium lucayanum wheeler, 1905 1 4 0 tetramorium quadridentatum stitz, 1910 1 0 0 tetramorium sp.3 4 0 0 tetramorium sp.4 0 0 2 ponerinae platythyrea conradti emery, 1899 0 1 0 pseudomyrmicinae tetraponera mocquerysi andré, 1890 1 1 0 total 602 2800 3670 table 3. similarity index (jaccard) between ant assemblages collected by the three sampling methods. arboreal pitfall funnel trap beating arboreal pitfall 0.53 0.30 funnel trap 0.22 table 4. arboreal ant species composition between three sampling method. cd yodé, k dosso, lmm kouakou, k yeo, w dekoninck, s konate, pk kouassi – the “funnel trap”: a new sampling method for arboreal ants498 natural and disturbed habitats as the “funnel trap” can capture some larger ant species such as, camponotus puberulus emery, 1897, camponotus compressiscapus andré, 1889, platythyrea conradti and smaller ant species like pheidole sp.2, monomorium floricola jerdon, 1851, plagiolepis alluaudi emery, 1894 and tapinoma lugubre santschi, 1917. interestingly, 48 hours after bait placement in “funnel trap”, some ant species workers were still active and alive in the traps although the bait was totally consumed. thus, it is possible the “funnel trap” also offers unique possibility to observe the existence of competition and interactions between ant species. for example, in some trap, we have observed a high number of killed workers of both camponotus solon forel, 1886 and oecophylla longinoda latreille, 1802, suggesting a strong competition between these two ant species in canopy. the funnel trap is an efficient sampling technique to the study of arboreal canopy ant communities. in addition, it allows to capture several other orders of insects like blattodea, diptera, other hymenoptera, coleoptera, orthoptera and lepidoptera. funnel trap can also be used for sampling in the agroecosystem canopy or in other natural area besides savanna or forest. funnel traps collect also ant species that will not easily be collected with the usually used arboreal pitfall trap. acknowledgements the authors would like to express their sincere thanks to the african excellence center on climate change, biodiversity and sustainable agriculture of felix houphouët boigny university of côte d’ivoire for funding our research. we also thank kouassi dorgeles and, koffi kouame françois for their useful help during this work. this paper is a result of several ant-course training projects with financial support from the belgian directorate-general for development cooperation (dgd), global taxonomy initiative, within the framework of the cebios programme. authors’ contributions christine dakélé yodé: design of the work, methodology, data collection, data analysis, interpretation of data for the work and writing the original draft. wouter dekoninck, kolo yeo: contributed to revising draft, and final approval of the version to be published. lombart m. maurice kouakou, kanvaly dosso, souleymane konate, phillipe kouassi kouassi: contributed to conceptualization, revising draft critically for important intellectual content and final approval of the version to be published. references abbadie, l., gignoux j., lepage, m., & roux, x.l. 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(2018). ant mosaics in bornean primary rain forest high canopy depend on spatial scale, time of day, and sampling method. peer j, 6: e4231. doi: 10.7717/peerj.4231. doi: 10.13102/sociobiology.v67i2.4617sociobiology 67(2): 268-280 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the pollination exercised by stingless bees in the amazon is considered to be a great ecological benefit to this biome. the amazon region contains a rich fauna of these insects, guaranteeing the production of fruits and seeds through pollination, and promoting the conservation of plant species and the survival of their colonies (ferreira & absy 2017a, b; absy et al., 2018; rezende et al., 2019). meliponiculture is an activity that has been gaining prominence in the northern region of brazil, because it exploits the potential of native flora and helps to generate income through family farming, allowing for the commercialization abstract this study aimed to identify the pollen grains found in honeys of melipona (michmelia) seminigra pernigra moure & kerr and melipona (melikerria) interrupta latreille in two communities of the tapajós-arapiuns extractive reserve, lower amazon (pará, brazil) between december 2016 and november 2017. twenty-four samples of honey were processed, 12 samples from m. seminigra pernigra collected in the suruacá community and 12 samples from m. interrupta in the vila franca community. after acetolysis, 103 pollen types were identified, distributed across 22 families, plus eight indeterminate types. fifty-nine types were exclusive to m. seminigra pernigra, 29 types were exclusive to m. interrupta and 15 pollen types were shared between both species. anacardiaceae, burseraceae, melastomataceae, and myrtaceae were the most attractive pollen families, providing key resources for maintenance of these bee populations. the sharing of pollen types between both bee species revealed a high similarity in preference for certain resources. m. seminigra showed greater diversity (h’ = 1.928) than m. interrupta (h’ = 1.292). furthermore, the diversity (h’) and equitability (j’) indexes showed a more homogeneous pattern in the pollen spectrum of honeys from m. seminigra in most months studied. these data suggest that meliponiculturists should consider the diversity of plant species found in the two communities and keep them close to the meliponary, which will favor honey management and production, as well as the local biodiversity. sociobiology an international journal on social insects rr souza1, ada pimentel2, ll nogueira1, vhr abreu1, js novais3 article history edited by cândida aguiar, uefs, brazil received 24 july 2019 initial acceptance 13 november 2019 final acceptance 11 march 2020 publication date 30 june 2020 keywords stingless bees, trophic resources, melipona (michmelia) seminigra pernigra, melipona (melikerria) interrupta, meliponiculture, melissopalynology. corresponding author jaílson santos de novais universidade federal do sul da bahia rod. porto seguro-eunápolis, br 367, km 10, cep: 45810-000 porto seguro-ba, brasil. e-mail: jailson.novais@ufsb.edu.br of bee by-products, such as honey, pollen, and propolis, while also being in accordance with the precepts of sustainable natural resource use (absy et al., 2013). the genus melipona illiger is only found in neotropical america – south america, central america, and the caribbean islands –, being most diversified in the amazon basin (michener, 2007). in this group can be found the principal native stingless bees used in the practice of meliponiculture in the amazon region (brito et al., 2013; ferreira & absy 2017a). these bees are popularly known as jandaíra, jandaíra-preta, and jandaíra-da-amazônia, names that can be attributed to numerous different species of meliponines, depending on the region (nogueira-neto, 1997; silveira et al., 2002; oliveira et al., 1 universidade federal do oeste do pará, santarém-pa, brazil 2 instituto nacional de pesquisas da amazônia, manaus-am, brazil 3 universidade federal do sul da bahia, porto seguro-ba, brazil research article bees resources collected by two melipona illiger, 1806 (apidae: meliponini) species based on pollen spectrum of honeys from the amazon basin sociobiology 67(2): 268-280 (june, 2020) 269 2013). the trophic resources collected by melipona species in the brazilian amazon have been studied by various groups of researchers since the 1970s (absy & kerr, 1977; absy et al., 1980; marques-souza, 1996; marques-souza et al., 2002; oliveira et al., 2009; ferreira & absy, 2013, 2015, 2017a, b; rezende et al., 2019) using melissopalynological methods. through pollen analysis is possible to determine the botanical and geographical origin of the product, the flowering period of the plants, and their value as suppliers of nectar and pollen for bees (barth, 1989; jones & bryant, 2004; novais et al., 2009; vossler et al., 2014). in the amazon, several riverside communities have developed activities to raise native bees. in the tapajósarapiuns extractive reserve (resex), located in the lower amazon region, pará, souza et al. (2018) found a high diversity of meliponines being utilized: frieseomelitta longipes (smith), friseomellita silvestrii (friese), melipona (melikerria) interrupta latreille, melipona (michmelia) seminigra pernigra moure & kerr, scaptotrigona sp. and tetragona clavipes (fabricius). however, according to the local meliponiculturists, the main obstacles to developing meliponiculture in the region include the lack of: legislation regarding the activity, technical assistance and training for the proper management of products from bees, and melissopalynological studies focused on local stingless bees products. based on this, the current study aimed to identify, through analysis of pollen grains present in honey, the plants used by m. seminigra pernigra and m. interrupta in two communities in resex tapajós-arapiuns in the lower amazon region (pará, brazil). with this, it will be possible to subsidize stingless beekeeping strategies and increase regional meliponiculture activity, as these are the principal productive bees managed by meliponiculturists in resex. material and methods study area the study was carried out within the tapajós-arapiuns extractive reserve, in the communities of suruacá (2°54’00”s 55°09’52”w) and vila franca (2°21’21”s 55°00’24”w). the resex has a land area of 647,610 hectares and is distributed between the territories of santarém and aveiro, in the western part of the state of pará (icmbio, 2014). the reserve was created in 1998 (brasil, 1998) in the category of sustainable use within the conservation unit (uc) (law no. 9,985, 18 july 2000). the reserve contains 72 communities and lies within the basins of two principal rivers, tapajós and arapiuns. the community of vila franca is situated on the left bank of the arapiuns river, while the community of suruacá is on the right bank of the tapajós river, and access to both is exclusively by river. suruacá is one of the largest communities in the resex tapajós-arapiuns, with a total of 127 resident families, or about 500 inhabitants. the community economy is based on the cultivation of cassava and its derivatives. in addition to the cultivation of cassava, economic activities include fishing, hunting, raising small animals, handicrafts, small-scale logging, extraction, processing of oils (andiroba and copaiba), rubber cultivation, and meliponiculture. in the community of vila franca, there are 74 families totaling 298 people. the indigenous culture is still persistent and traditions of flour, taruba, cachaça, manicuéra, handicrafts, and cultural dances remain. agroextractivism is the principal economic activity. most vila franca families survive from the production of cassava flour, corn, or tapioca, and from the sale of handicrafts and products extracted from the forest. meliponiculture was introduced into the community 10 years ago and contributes as an extra economic activity for some meliponiculturists (projeto saúde e alegria, 2015). identification of stingless bees to identify the bees, five worker bees were collected from beehives located in the meliponaries of communities suruacá and vila franca, with the aid of killing jar for insects. next, the specimens were packed, assembled, and labeled according to the usual entomological standards and sent to be identified by a specialist in the laboratory of bionomics, biogeography, and insect systematics (biosis) at the federal university of bahia (ufba), where they remain deposited. collection of honey samples twenty-four honey samples were collected between december 2016 and november 2017, 12 from m. interrupta in the community of suruacá and 12 from m. seminigra in vila franca. the honey samples were always obtained from the same bee hive every month, using sterile plastic pipettes. about 10 ml of honey were conditioned in plastic pots, sealed with a lid and properly identified. the samples were kept in a refrigerator at a temperature of about 10 °c until the beginning of laboratory processing. chemical processing of honey samples the honey samples were treated according to louveaux et al. (1978) and acetolysed (erdtman, 1960). we also followed the recommendation of jones and bryant (2004) for honey dilution with 95% ethanol (etoh). following the acetolysis process, at least three slides of each sample were prepared using kisser’s glycerinated gelatin (salgado-labouriau, 1961) and sealed with paraffin (j. müller modified in erdtman 1952). after the completion of the analysis, they were deposited in the pollen library of the laboratory of botany and palynology (labpal) at ibef/ ufopa. analysis of pollen grains in the honey and statistical indexes the methodology used to count pollen grains followed moar (1985), with a minimum of 500 grains per sample. all of the different pollen types found were microphotographed at 100× magnification. in order to identify the pollen types, palynological bibliographies (roubik & moreno, 1991; carreira rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil270 fig 1. photomicrographs of the most frequent pollen types found in honey samples of melipona seminigra pernigra. anacardiaceae, spondias mombin (a) and tapirira guianensis (b); burseraceae, protium heptaphyllum (c); fabaceae, dialium (d) and mimosa pudica (e); melastomataceae, bellucia (f), miconia (g) and mouriri (h); myrtaceae, eugenia (i) and myrcia (j); sapindaceae, talisia (k); solanaceae, solanum (l). et al., 1996) were used, and the pollen library at the national institute of amazonian research (inpa) was consulted. to define the frequency classes, the pollen types were grouped according to louveaux et al. (1978): predominant pollen (pd = >45%), secondary pollen (pa = 16 to 45%), important pollen (pii= 3 to 15%), and important minor pollen (pio = <3%). the amplitude of the trophic niche in the honey samples was calculated using the diversity index (h’) of shannon-weaver (1949), which is based on the proportion of pollen types found in the monthly samples, according to the formula h’= − σ (pi x ln pi), where h’ is the diversity index, pi is the proportion of each pollen type found in the monthly samples, and ln is the natural logarithm. the equitability index (j‘) of pielou (1977) allows estimating the degree of uniformity of the plant species visited by the bees in a given month. it is calculated according to the formula j’ = h’/ h’max, where h’ is the diversity index and h’ max is the natural logarithm of the total number of pollen types present in the sample. the uniformity index can vary from 0 to 1, indicating an interval between completely heterogeneous use and completely homogeneous use of resources, respectively. precipitation data for the sampling period was obtained from the national institute of meteorology, pará meteorological station (inmet, 2018). the botanical nomenclature for the fabaceae family follows the new classification based on phylogenetic analyses (lpwg, 2017). results one hundred and three pollen types collected by m. seminigra pernigra and m. interrupta bees were identified, distributed across 22 families, with eight indeterminate pollen types. of this total, 59 pollen types were collected exclusively by m. seminigra pernigra, 29 were exclusive to m. interrupta and 15 were shared by both species (table 1). the pollen types found at rates above 50% in the honey samples and shared by both bee species were bellucia (melastomataceae), eugenia (myrtaceae), miconia (melastomataceae), myrcia (myrtaceae), protium heptaphyllum (burseraceae), and spondias mombin (anacardiaceae). pollen spectrum of m. seminigra pernigra the number of pollen types found per month in m. seminigra honeys ranged from five in october to 21 in february. mimosa pudica (fabaceae/caesalpinioideae), was the most representative pollen type found in honey from this species (august, 65.20%), being classified as a predominant pollen. secondary pollen types were identified as: indeterminate (type 3), in july (39.60%); miconia (melastomataceae), december (38.00%), march (37.60%), september (35.00%), and june (30.00%); mouriri (melastomataceae), in october (33.60%); myrcia (myrtaceae), in january (30.00%); tapirira guianensis (anacardiaceae), in october (28.20%); protium heptaphyllum (burseraceae), in november (28.20%); spondias mombin (anacardiaceae), in november (27.20%); and mimosa pigra (fabaceae/ caesalpinioideae), in november (17.40%) (fig 1). a total of 30 pollen types were classified as important pollen, distributed across nine families, with emphasis on bellucia (melastomataceae), in january (12.80%); eugenia (myrtaceae), in september (10.00%); and talisia (sapindaceae), in march (10.00%), as they occurred at a percentage of 10.00% or more. another 34 pollen types collected by m. seminigra were classified as important minor pollen, distributed across 15 families, with emphasis on fabaceae (eight types). considering the pollen types with a percentage greater than 10.00%, bellucia, eugenia, miconia, mimosa pigra, mouriri, myrcia, protium heptaphyllum, spondias mombin, talisia, and tapirira guianensis were considered attractive to m. seminigra. pollen spectrum of m. interrupta melipona interrupta honey samples contained fewer than 10 pollen types in nine of the 12 honey samples analyzed, with the highest number of pollen types found in may (13) sociobiology 67(2): 268-280 (june, 2020) 271 and the lowest number of pollen types in july, september, and november (5). the following pollen types were considered predominant: protium heptaphyllum (burseraceae), in february (76.20%), january (74.20%), march (61.00%), and august (59.60%); miconia (melastomataceae), in november (76.00%), april (70.00%), and september (64.00%); and spondias mombin (anacardiaceae), in january (56.40%). the pollen types grouped into the secondary pollen class were: bellucia (melastomataceae), in september (35.00%); mouriri (melastomataceae), in may (28.40%); eugenia (myrtaceae), in july (18.80%) and august (18.20%); and cassia (fabaceae/caesalpinioideae), in august (18.60%) (table 2, fig 2). for the important pollen frequency class, 18 types were found, distributed across eight families, with emphasis on myrtaceae, having five types (table 2). the bellucia, cassia, and eugenia pollen types were represented at over 10.00% in at least one sample, and were therefore considered attractive for m. interrupta. nineteen pollen types were found as important minor pollen, distributed across 11 botanical families. ecological indexes the diversity obtained for the pollen spectrum of the m. seminigra pernigra honeys exhibited the highest values in february (h’ = 2.635), april (h‘= 2.344) and march (h’ = 2.273), while the lowest values were found in july (h’ = 1.621), october (h’ = 1.311) and august (h’ = 1.301). with regard to equitability, the collections were most uniform in october (j’ = 0.945), november (j’ = 0.891) and february (j’ = 0.866). in contrast, the lowest uniformity values were recorded in march (j’ = 0.757), december (j’ = 0.712) and august (j’ = 0.592) (table 1). fig 2. photomicrographs of the most frequent pollen types found in honey samples of melipona seminigra. anacardiaceae, spondias mombin (a); araliaceae, schefflera morototoni (b); burseraceae, protium heptaphyllum (c); fabaceae, cassia and copaifera (d, e); melastomataceae, bellucia (f), miconia (g) and mouriri (h); myrtaceae, eugenia (i), myrcia (j) and psidium (k); solanaceae, solanum (l). rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil272 the diversity index registered for m. interrupta had its highest values in june (h’ = 2.128), may (h’ = 1.879), and august (h’ = 1.548), and its lowest values in july (h’ = 1.34), february (h’ = 0.952), and november (h’ = 0.929). the uniformity index for this bee species was highest in june (j’ = 0.856), and september (j’ = 0.806) and may (j’ = 0.733), with the lowest values being obtained in january (j’ = 0.505), december 2016 (j’ = 0.443), and february (j’ = 0.433) (table 2). climatic data the environmental variables recorded during the sampling period showed a high variation in rainfall volume, with the highest values measured in january (247 mm) and march (256 mm). in contrast, the lowest values were recorded in august/october (10 mm) and november (zero). for relative humidity, the highest values were recorded in january (91%) and february/march (92%), and the lowest values were measured in october (80%) and november (79%). with regard to temperature, the highest values were obtained in september/ october (28 °c) and november (29 °c), while the lowest values were recorded in january, february and march (26 °c), followed by april and june (25 °c) (inmet, 2018) (fig 3). the month of february was when the greatest number of pollen types were found in the samples from m. seminigra pernigra. for m. interrupta, the month with the greatest number of types was may. fig 3. diversity (h’) and uniformity (j’) records for pollen types found in samples of honey from melipona seminigra pernigra (msp) and melipona interrupta (min), as well as temperature (°c), relative humidity (%), and precipitation (mm) data for the communities of suruacá (su) and vila franca (vf), resex tapajós-arapiuns, pará, brazil, between december 2016 and november 2017. discussion among the stingless bees reared in apiaries in the amazon, the genus melipona stands out. a number of melissopalynological studies have been carried out in this region with this genus (absy & kerr, 1977; absy et al., 1984; oliveira et al., 2009; ferreira & absy, 2013; ueira-vieira et al., 2013; rezende et al., 2019). these studies emphasize the importance of polliniferous plants related to the miconia and bellucia (melastomataceae) pollen types as key species for the feeding of these pollinating insects (ferreira & absy, 2017a; rezende et al. 2019). these plant species have low seasonality, guaranteeing the availability of the resource for long periods in the year. these were also classified in this study as predominant pollen types for m. interrupta and secondary pollen grains for m. seminigra pernigra. this is due to the fact that some species of bees pollinate by vibration, grabbing the anthers (predominantly with poricidal dehiscence) and vibrating their thorax, shaking the anthers and releasing the pollen. this is common for some families, such as fabaceae/caesalpinioideae, melastomataceae, and solanaceae (buchmann, 1983; pinheiro & sazima, 2007; nunes-silva et al., 2010). in our study, the pollen types miconia (melastomataceae) and m. pudica (fabaceae/caesalpinioideae) refer to plant species that produce a lot of pollen and little or no nectar. similarly, barth (1989) noticed a high percentage of pollen grains from polliniferous plants in brazilian honey samples. on the other hand, we identified several pollen types botanically related to plants that are probably good sources of nectar sociobiology 67(2): 268-280 (june, 2020) 273 and produce little pollen: talisia (sapindaceae), tapirira guianensis (anacardiaceae), syzygium (myrtaceae), protium heptaphyllum (burseraceae) and eugenia (myrtaceae). the frequent occurrence of miconia (melastomataceae) and mimosa pudica (fabaceae/caesalpinioideae) in m. seminigra and m. interrupta honey samples in this study may be related to the size of pollen grains, which vary from small to very small and are always released in large quantities by plants (absy et al., 1980; ferreira & absy, 2017b; rezende et al., 2019). some pollen types from polliniferous plants, when found in the pollen spectrum of honeys, may indicate the geographical origin of the samples, as well as, suggest potential pollen sources for bees. according to roubik and moreno (2013), predominant pollen in honey is often no indication of nectar source, if flowers are nectarless. the principal pollen types responsible for high pollen frequencies in the samples were: for m. seminigra, mimosa pudica (65.20%), and for m. interrupta, protium heptaphyllum (76.20%), miconia (76.00%), and spondias mombin (56.40%). ferreira and absy (2017a), studying the honey pollen spectrum and trophic interactions between the colonies of melipona (michmelia) seminigra merrillae cockerell and m. interrupta, found a strong relation between the nutrition of these species and the mimosa pudica (fabaceae/ caesalpinioideae) pollen type, aligning with the results of this study for the community of suruacá. meanwhile, oliveira et al. (2009) observed pollen resources collected by stingless bees in a forest fragment in the manaus region and highlighted the importance of the burseraceae family, with pollen type protium heptaphyllum. this pollen type was used by all bees and was an important source for m. seminigra merrillae. in the same study, the family anacardiaceae was represented by the types t. guianensis and spondias mombin, collected by the foragers of m. seminigra merrillae and m. fulva. the reward gained by the bees, from most species of anacardiaceae, is its nectar. in these species, the flowers have ripe anthers and their pollen is fully exposed, favoring opportunistic collection. the flowers of this family are usually not very showy, being hermaphrodite or unisexual (ribeiro et al., 1999). in the current study, some pollen type families were found to be well represented, such as fabaceae, with mimosa pudica for m. seminigra; burseraceae, with protium heptaphyllum and melastomataceae, with miconia, for m. interrupta. according to judd et al. (2010), the melastomataceae, fabaceae, and myrtaceae families are predominantly pollinated by bees. pollen types with less than 10.00% representativity can act as complementary resources and become important in the maintenance of colonies for limited periods, when the supply of the principal resources is subject to seasonal variations (ramalho et al., 1985). the m. seminigra pernigra species had greater pollen richness (n = 74) in its honey samples. in contrast, m. interrupta had lower richness (n = 44). results similar to these were found in the study by ferreira and absy (2017b), who analyzed pollen types in honey and trophic relationships between colonies of m. seminigra merrillae and m. interrupta raised in meliponaries in the city of manaus, amazonas. this approach can show the general tendency of these bees, as discussed by ramalho et al. (2007). the diversity (h’) values for m. seminigra pernigra in the community of suruacá were found to be higher throughout the year in comparison to m. interrupta. the equitability analysis (j’) showed that m. seminigra pernigra utilized a larger pollen spectrum and that its use of different pollen types was more homogeneous, indicating that this species can be considered more generalist in its use of resources. meanwhile, m. interrupta presented a very similar pollen spectrum, with differences in the abundance of the pollen types utilized, being less generalist and showing greater preference for a few pollen types. the lowest diversity for m. seminigra, recorded in august, and for m. interrupta, in november, suggest the use of few sources that could have been more attractive due to a greater abundance of types, whereas, in the rainy season, pollen was collected from a greater number of sources at lower frequency levels. these results are those similar to those found by oliveira et al. (2009). they verified that rainfall is the most important factor influencing the extension of the pollen spectrum, promoting greater diversity in collection due to the low level of flowering. rainfall was verified to be an important factor that influences the extension of the pollen spectrum, as verified in the month of february for m. seminigra, and in the month of may for m. interrupta, when the greatest number of pollen types were obtained. the bees collected a total of 147 pollen types during the rainy season and 98 during the driest period, in the two communities. this can be explained by the supply of resources during certain periods. during the dry period, the most intense flowering of certain species, such as t. guianensis, occurs. the similarity between diversity indexes shows that these bees exhibit a similar collection potential, which may compromise the maintenance of more sensitive systems or populous species, depending on the diversity of flowers and the behavior of the bees with regard to these flowers (ferreira & absy, 2017a). in this study, pollen from the families anacardiaceae, melastomataceae, fabaceae/caesalpinioideae mimosoid clade, and myrtaceae was associated with both species of bees and these pollen types were found in abundance in the honey samples. although there have been several studies on the pollen types collected by stingless bees in the amazon region and the importance of these studies for the development of meliponiculture in local communities, few studies have prioritized pollen analyses of honey samples from these species, even though this is the most valuable resource for beekeepers (novais et al., 2015; ferreira & absy, 2017b; rezende et al., 2019). rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil274 conclusion of the 103 pollen types found to be present in honey samples from m. seminigra pernigra and m. interrupta, 15 pollen types were shared by both species. considering the proportion of pollen types found in the samples, the botanical families being explored by both bee species were, in descending order, melastomataceae, fabaceae/caesalpinioideae, burseraceae, myrtaceae, and anacardiaceae. the species m. seminigra pernigra collected different pollen types more homogeneously, indicating that this species can be considered more generalist in its use of resources. meanwhile, m. interrupta honeys had a pollen spectrum similar to m. seminigra, with differences regarding the abundance of pollen types used and being less generalist, with greater preference for a few pollen types. the predominant pollen types were mimosa pudica (fabaceae/ caesalpinioideae) for m. seminigra honeys, and miconia (melastomataceae), protium heptaphyllum (burseraceae) and spondias mombin (anacardiaceae) for m. interrupta. some of these pollen types are related to polliniferous plants, such as m. pudica and miconia. however, even though they do not contribute to the nectariferous composition of honey, they may indicate polliniferous preferences to be investigated in further studies. acknowledgments the authors gratefully acknowledge dr favízia freitas de oliveira (ufba) for identifying the bee species; the meliponiculturists from the resex tapajós-arapiuns for authorizing the research in their meliponaries; dr maria lúcia absy (inpa) for allowing the access to the palynotheca of the inpa’s laboratory of palynology; the coordenação de aperfeiçoamento de pessoal de nível superior (capes – brazil) for a master’s grant provided to r.r. souza; the programa de pós-graduação em ciências e tecnologias ambientais (ufsb) for financial support. authors’ contributions rrs, jsn and vhra conceived the main idea of the research; rrs, adap and lln collected, processed and analyzed the samples; rrs wrote the text; rrs, adap, lln, jsn and vhra revised the text. plant family/pollen type dec 16 jan 17 feb 17 mar 17 apr 17 may 17 jun 17 jul 17 aug 17 sep 17 oct 17 nov 17 anacardiaceae anacardium 5.00 spondias mombin 11.20 2.80 2.60 7.00 8.80 6.00 27.20 tapirira guianensis 5.80 3.60 8.20 10.00 28.20 10.40 type 1 6.00 type 2 1.00 araliaceae schefflera morototoni 5.00 type 1 1.60 type 2 6.20 type 3 9.60 arecaceae elaeis 1.00 2.00 maximiliana maripa 1.20 asteraceae ambrosia 1.00 1.00 0.60 bactris 3.40 burseraceae protium heptaphyllum 16.00 12.20 20.20 4.60 24.00 17.00 13.20 23.40 6.20 13.00 28.20 cyperaceae scleria 1.20 euphorbiaceae alchornea 2.00 1.20 5.40 type 1 1.20 table 1. frequency of pollen types found in honey samples of melipona seminigra pernigra (apidae: meliponini) collected from december 2016 (dec 16) to november 2017 (nov 17) in the community of suruacá, tapajós-arapiuns extractive reserve, santarém (pa), brazil. sociobiology 67(2): 268-280 (june, 2020) 275 fabaceae/ caesalpinioideae cassia mimosoides 3.40 delonix regia 1.00 inga 1.20 leucaena 4.00 mimosa 17.40 mimosa pigra 3.00 mimosa pudica 2.80 4.00 6.60 10.80 4.60 21.00 29.20 65.20 mimosa sensitiva 2,00 stryphnodendron 1.00 2.00 stryphnodendron guianense 2.00 2.00 type 1 3.40 type 2 3.60 type 3 2.20 fabaceae/ detarioideae copaifera langsdorffii 1.60 hymenaea 1.00 1.00 8.20 hymenaea parvifolia 1.00 1.00 1.40 fabaceae/ dialioideae dialium 7.40 fabaceae/ papilionoideae swartzia 1.60 loranthaceae phthirusa 1.00 malpighiaceae byrsonima 3.00 2.00 type 1 0.60 type 2 0.80 malvaceae rhodognaphalopsis minor 1.20 malvaceae type 2.00 melastomataceae bellucia 5.60 12.80 10.40 4.00 9.00 11.00 miconia 38.00 18.00 37.60 27.00 30.00 2.20 13.40 35.20 mouriri 6.00 34.40 33.60 type 1 7.20 type 2 2.60 table 1. frequency of pollen types found in honey samples of melipona seminigra pernigra (apidae: meliponini) collected from december 2016 (dec 16) to november 2017 (nov 17) in the community of suruacá, tapajós-arapiuns extractive reserve, santarém (pa), brazil. (continuation) plant family/pollen type dec 16 jan 17 feb 17 mar 17 apr 17 may 17 jun 17 jul 17 aug 17 sep 17 oct 17 nov 17 rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil276 myrtaceae eugenia 1.40 6.00 3.20 6.00 5.00 10.00 7.60 eugenia stipitata myrcia 22.00 30.00 15.80 7.00 psidium 1.00 1.20 2.00 psidium guajava 1.60 syzygium 1.60 4.40 3.00 type 1 4.00 type 2 6.80 type 3 3.00 type 4 3.20 type 5 3.00 passifloraceae passifloraceae type 1.20 2.00 polygonaceae triplaris 1.00 rubiaceae rubiaceae type 1.00 rutaceae zanthoxylum 1.00 sapindaceae serjania 1.00 talisia 0.80 8.40 10.00 1.00 1.00 sapotaceae pouteria 2.00 sapotaceae type 5.00 solanaceae solanum 2.00 1.40 7.40 5.00 urticaceae cecropia 3.00 4.60 indeterminate type 1 6.00 type 2 1.20 type 3 39.60 type 4 2.80 type 5 7.00 type 6 5.20 total % 100 100 100 100 100 100 100 100 100 100 100 100 number of pollen types 11 13 21 20 17 10 9 7 9 14 5 12 diversity (h’) 1.708 2.056 2.635 2.273 2.344 1.903 1.703 1.621 1.301 2.123 1.311 2.161 equitability (j’) 0.712 0.802 0.866 0.757 0.811 0.826 0.775 0.833 0.592 0.804 0.945 0.891 table 1. frequency of pollen types found in honey samples of melipona seminigra pernigra (apidae: meliponini) collected from december 2016 (dec 16) to november 2017 (nov 17) in the community of suruacá, tapajós-arapiuns extractive reserve, santarém (pa), brazil. (continuation) plant family/pollen type dec 16 jan 17 feb 17 mar 17 apr 17 may 17 jun 17 jul 17 aug 17 sep 17 oct 17 nov 17 sociobiology 67(2): 268-280 (june, 2020) 277 plant family/pollen type dec 16 jan 17 feb 17 mar 17 apr 17 may 17 jun 17 jul 17 aug 17 sep 17 oct 17 nov 17 anacardiaceae spondias mombin 2.00 56.40 0.80 2.00 araliaceae schefflera morototoni 5.00 5.60 1.60 1.20 3.00 araliaceae type 2.30 1.80 arecaceae arecaceae type 3.00 asteraceae ambrozia 1.00 burseraceae protium heptaphyllum 74.00 34.80 76.20 61.00 3.20 2.80 2.60 59.60 44.40 10.00 3.00 3.40 cunoniaceae cunoniaceae type 1.00 cyperaceae cyperus 3.40 fabaceae/ caesalpinioideae cassia type 1 4.00 18.60 cassia type 2 3.00 inga 3.00 type 1 2.00 type 2 1.00 type 3 1.60 type 4 1.20 type 5 1.00 fabaceae/ detarioideae copaifera 1.00 1.20 9.20 7.40 fabaceae/ papilionoideae aldina 1.00 diplotropis 1.00 loranthaceae phthirusa 1.00 malpighiaceae byrsonima 2.60 malpighiaceae type 1.00 1.00 malvaceae malvaceae type 0.80 melastomataceae bellucia 3.00 2.40 5.00 7.20 35.00 7.00 miconia 10.00 2.40 10.60 8.60 70.00 33.4 35.4 19.60 43.00 64.00 76.00 mouriri 28.4 type 1 10.00 8.00 type 2 8.00 7.00 table 2. frequency of pollen types found in honey samples of melipona interrupta (apidae: meliponini) collected from december 2016 (dec 16) to november 2017 (nov 17) in the community of vila franca, tapajós-arapiuns extractive reserve, santarém (pa), brazil. rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil278 myrtaceae eugenia 5.00 11.40 13.00 18.80 18.20 5.00 11.00 5.00 eugenia stipitata 1.00 myrcia 1.20 4.00 9.60 psidium 6.00 3.60 psidium guajava 5.60 type 1 2.60 type 2 11.40 type 3 3.80 type 4 8.00 polygonaceae polygonum 1.00 rubiaceae borreria 3.00 sapindaceae sapindaceae type 0.60 solanaceae solanum 7.00 8.00 5.00 type 1 1.00 indeterminate type 1 3.40 type 2 4.80 total % 100 100 100 100 100 100 100 100 100 100 100 100 number of pollen types 11 8 9 8 7 13 12 5 7 5 7 5 diversity (h’) 1.063 1.051 0.952 1.309 1.072 1.879 2.128 1.034 1.548 1.297 1.248 0.929 equitability (j’) 0.443 0.505 0.433 0.63 0.551 0.733 0.856 0.643 0.796 0.806 0.641 0.518 table 2. frequency of pollen types found in honey samples of melipona interrupta (apidae: meliponini) collected from december 2016 (dec 16) to november 2017 (nov 17) in the community of vila franca, tapajós-arapiuns extractive reserve, santarém (pa), brazil. 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(2009). recurso polínico coletado por abelhas sem ferrão (apidae, meliponinae) em um fragmento de floresta na região de manausamazonas. acta amazonica, 39: 505–518. doi: 10.1590/s0044-59672009000300004 pielou, e.c. (1977). mathematical ecology. new york: john wiley & sons. pinheiro, m.t. & sazima, m. (2007). visitantes florais e polinizadores de seis espécies arbóreas de leguminosae melitófilas na mata atlântica no sudeste do brasil. revista brasileira de biociências, 5: 447–449. projeto saúde e alegria (org.). (2015). almanaque da reserva extrativista tapajós-arapiuns: prazer em conhecer. santarém: ceaps projeto saúde e alegria. ramalho, m., imperatriz-fonseca, v.l. & kleinert-giovannini, a. (1985). exploitation of floral resources by plebeia remota holmberg (apidae-meliponinae). apidologie, 16: 307–330. rr souza, ada pimentel, ll nogueira, vhr abreu, js novais – pollen in melipona spp. honey from the lower amazon, brazil280 ramalho, m., silva, m.d. & carvalho, c.a.l. (2007). dinâmica de uso de fontes de pólen por melipona scutellaris latreille (hymenoptera, apidae): uma análise comparativa com apis mellifera l. (hymenoptera, apidae), no domínio tropical atlântico. neotropical entomology, 36: 38–45. rech, a.r. & absy, m.l. (2011). pollen sources used by species of meliponini (hymenoptera: apidae) along the rio negro channel in amazonas, brazil. grana, 50: 150–161. doi: 10.1080/00173134.2011.579621 rezende, a.c.c., absy, m.l., ferreira, m.g., marinho, h.a. & santos, a.o. (2019). pollen of honey from melipona seminigra merrillae cockerell, 1919, scaptotrigona nigrohirta moure, 1968 and scaptotrigona sp. moure, 1942 (apidae: meliponini) reared in sataré mawé indigenous communities, amazon, brazil, palynology, 43: 255–267. doi: 10.1080/01916122.2018.1458664 ribeiro, s.l.e., hopkins, g.j.n., vicentini, a., sothers, a.c., costa, s.a.m., bento, m.j., souza, d.a.m., martíns, p.h.l., lohmann, g.l., assunção, l.c.a.p., perreira, c.e., silva, s.c., mesquita, r.m. & procópio, c.l. (1999). flora da reserva ducke: guia de identificação das plantas vasculares de uma floresta de terra firme na amazônia central. manaus: inpa, 816 p roubik, d.w. & moreno, j.e. (1991). pollen and spores of barro colorado island. st. louis: missouri botanical garden. roubik, d.w. & moreno, j.e. (2013). how to be a beebotanist using pollen spectra. in p. vit, s.r.m. pedro & w.w. roubik (eds.), pot-honey: a legacy os stingless bees (pp. 295–314). new york: springer. salgado-labouriau, m.l. (1961). palinologia: fundamentos, técnicas e algumas perspectivas. revista brasileira de geografia, 23: 695–717. shannon, c.e. & weaver, w. (1949). the mathematical theory of communication. illinois: university of illinois press. silveira, f.s., melo, g.a.r. & almeida, e.a.b. (2002). abelhas brasileiras: sistemática e identificação. belo horizonte: fundação araucária, 253 p souza, r.r., abreu, v.h.r., novais, j.s., pimentel, a.d.a. & nogueira, l.l. (2018). a meliponicultura em comunidades da reserva extrativista tapajós-arapiuns, santarém, pará. cadernos agroecológicos, 13: 1–7. ueira-vieira, c., silva-nunes, c.g., absy, m.l., pinto, m.f.f.c., keer, w.e., bonetti, a.m. & carvalho-zilse, g.a. (2013). pollen diversity and pollen ingestion in an amazonian stingless bee, melipona seminigra (hymenoptera, apidae). journal of apicultural research, 52: 173–178. doi: 10.3896/ ibra.1.52.3.09 vossler, f.g., fagundez, g.a. & blettler, d.c. (2014). variability of food stores of tetragonisca fiebrigi (schwarz) (hymenoptera: apidae: meliponini) from the argentine chaco based on pollen analysis. sociobiology, 61: 449–460. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5792sociobiology 68(1): e-5792 (march, 2021) introduction epicharis klug, 1807 (hymenoptera, apidae, centridini) is an exclusively neotropical genus of bees representing about 35 species, which exhibit solitary behavior, the habit of digging their nests in the ground, usually in large aggregations (roubik & michener, 1980; hiller & wittmann, 1994; gaglianone, 2005; thiele & inouye, 2007; rocha-filho et al., 2008; rozen, 2016; dec & vivallo, 2019). epicharis belongs to a group of approximately 400 species known as oil-collecting bees, due to the female behavior of collecting floral oils, which are used for both cell construction and larval provisions (alves-dossantos et al., 2007; gaglianone et al., 2011). aspects of the nesting biology for different species of epicharis, including details on the nest architecture and female behavior during nest construction and cell provisioning, have been studied in seven out of nine subgenera belonging to abstract we investigated the nesting behavior of females of epicharis dejeanii and the architecture of their nests, in a large aggregation in a restinga area, on ilha do superagui, southern brazil. surveys were carried out intermittently through the warm-wet seasons from different years between 2013 and 2017. the nest aggregation occupied an area of approximately 2,000 m2 and was situated on a sand bank and on flat sandy soil. each nest consisted of a long unbranched tunnel, averaging 1.45 ± 0.35 m (n = 8), connected to a single brood cell with a mean length of 3.13 ± 0.2 cm (n = 13) and mean diameter of 1.2 ± 0.1 cm (n = 11). on average, females carried out 4.0 ± 2.4 foraging trips per day (n = 109) to collect floral resources for provisioning brood cells. similar times were spent by females in their foraging trips for: only pollen (15.8 ± 14.3 min, n = 72), oil (22.5 ± 15.7 min, n = 45), or both resources (17.0 ± 15.1, n = 63). our findings reveal that some variation in both nesting architecture and female behavior of e. dejeanii during nesting activities can occur in different locations from the same region. sociobiology an international journal on social insects natalia uemura1, andré luiz gobatto1, welber da costa pina1,2, rafael hideki ono1, silvia helena sofia1 article history edited by evandro nascimento silva, uefs, brazil received 22 august 2020 initial acceptance 10 december 2020 final acceptance 18 january 2021 publication date 03 march 2021 keywords solitary bees; nest aggregation; bee behavior; nesting biology; atlantic forest. corresponding author silvia helena sofia departamento de biologia geral, ccb universidade estadual de londrina rod. celso garcia cid, km 380 cep: 86057-970, londrina-pr, brasil. e-mail: shsofia@uel.br the genus (see gaglianone, 2005). in general, the different species and subgenera of epicharis studied so far construct the brood cells deep down in sandy soils (gaglianone, 2005; thiele & inouye, 2007; rocha-filho et al., 2008; rozen, 2016; dec & vivallo, 2019). detailed nest descriptions have also been used in assessments on the evolution of nest habitat and architecture in this genus (thiele & inouye, 2007). epicharis (anepicharis) dejeanii lepeletier, 1841, the only member of this subgenus (moure et al., 2012), is a univoltine species that constructs its nests in aggregations in sandy soil (hiller & wittmann, 1994; dec & vivallo, 2019). this species occurs in south america (moure et al., 2012). in brazil, e. dejeanii is widely distributed, occurring from the northern (amazonia) to the southern region (atlantic forest) (hiller & wittmann, 1994; steiner et al., 2010; moure et al., 2012). to date, although information on the nest architecture of e. dejeanii has already been published by hiller and 1 universidade estadual de londrina, londrina, paraná, brazil 2 universidade do estado da bahia, teixeira de freitas, bahia, brazil research article bees nest structure, seasonality and female behavior of epicharis (anepicharis) dejeanii lepeletier (hymenoptera, apidae, centridini) in a restinga ecosystem, in southern brazil n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii2 wittmann (1994) and, more recently, by dec and vivallo (2019), some aspects related to nest structure of this species, such as number of cells per nest, are divergent in both studies. concerning this subject, specifically in the preliminary study carried out by hiller and wittmann (1994), the authors inferred the number of brood cells per nest, reporting a number of 52 brood cells found in an area containing only five nests; suggesting thus, approximately 10 cells per nest (hiller & wittmann, 1994; reviewed by gaglianone, 2005). however, in a new publication on nesting biology of e. dejeanii a very distinctive number of brood cells (varying from 1 to 2) per nest was identified (dec & vivallo, 2019). since both studies were carried out in southern brazil, a provocative question that emerges at this point is: could the number of cells constructed by females per nest vary considerably, revealing a plasticity in nesting behavior for e. dejeanii? another relevant aspect related to studies on bee nest aggregations concerns the length of time of these aggregations. although michener (1974) states that nest aggregations are sometimes very long-lived, few reports on nest aggregations of epicharis (e.g. gaglianone, 2005; thiele & inouye, 2007; martins et al., 2019) monitored the aggregation for more than one or two years (roubik & michener, 1980; hiller & wittmann, 1994; rocha-filho et al., 2008; werneck, 2012; dec & vivallo, 2019). therefore, in the current study we bring more information on nesting biology of e. dejeanii, mainly concerning the number of cells constructed by females per nest, on the persistence of nest aggregation along the time and on behavior of females during the nest construction activity. to achieve our aims successfully, we have focused on the following questions: i) how long is the burrowing phase? ii) how many cells are constructed per nest? iii) how long is the provisioning phase? iv) how many pollen flights/pollen loads are necessary to provision one cell? v) does pollen collection occur throughout the day or mainly in the morning/evening? vi) how long is the activity phase of the aggregation? vii) how long does the aggregation last? material and methods study area the studied nest aggregation of e. dejeanii was first identified in december 2013, in a restinga area of the ilha do superagui (is), about 1 km from the study base of the chico mendes institute for biodiversity conservation (icmbio), 25°27.51.4”s 48°14.07.1’w. the area belongs to the parque nacional do superagui (pns), located in the environmental protection area (apa) of guaraqueçaba (icmbio, 2016), in the state of paraná, southern brazil. the landscape of the islands includes the atlantic forest (af), comprising the serra do mar corridor, one of the most diverse areas of the af (aguiar et al., 2005). the climate of the region is classified as subtropical humid (köppen classification cfa). the average annual temperature is 21°c. annual rainfall is higher than 2500 mm. mean temperature for the coldest month is lower than 18°c, and for the warmest, higher than 22°c (ipardes, 2001; giangarelli & sofia, 2018). the vegetation comprises a combination of dense ombrophilous forest and pioneer formations. among these is the restinga, which covers 30% of the total area of the island, being gradually replaced by forest as the distance from the sea increases (schmidlin et al., 2005). in the current study, the area where the aggregation was located showed herbaceous and grassy vegetation scattered among the nests. data collection data of this study correspond to a set of surveys conducted intermittently in the study area in different months from different years, since december 2013 until november 2018. however, in november 2014 and 2018 the study area was visited only for checking if the aggregation was in activity in the area. although desirable, unfortunately these limitations are due to some difficulties of accessing the study site (e.g., limited number of boat crossing times available, site isolation, many rainy days from november to january etc), samplings could not to be carried out through an entire warm season, when the studied species is active (see hiller & wittmann, 1994; dec & vivallo, 2019). thus, the findings presented here result from data sets gathered over different years. all dates of field studies in is, where samplings were conducted, are shown in table s1 (real time, not brazilian summertime). in total, we carried out 29 days and approximately 249 hours of field work. the behavior of e. dejeanii females was observed for approximately 160 h. observations on the nesting behavior of bees were based on gaglianone (2005) and rocha-filho et al. (2008), with some modifications. with exception of rainy days, the bee behavior observations were carried out mostly from 6:00 am to 11:00 am and from 1:00 pm to 6:00 pm; thus, with an interval of two hours between 11:00 am to 1:00 pm (table s1). this procedure was adopted to avoid the excessive tiredness of the collectors due to the strong collection effort usually under high temperature conditions. however, aiming to fill the gap on the information regarding the period between 11:00 am to 1:00 pm, we decided to conduct five samplings of 12 hours, from 6:00 am to 6:00 pm; these observations were conducted from 31 october 2017 to 04 november 2017 (table s1). in addition, complementary observations were conducted to identify possible twilight activity of the bees in december 2016 from 5:00 to 7:00 am and from 6:00 to 8:00 pm. nest excavation activities were conducted in december 2013, from 7:00 to 12:00 am, and in february and december 2016, from 7:00 am to 7:00 pm and from 8:00 am to 6:00 pm, respectively (table s1). during the period of study (from november to february), the sunrise in the region is at approximately 05:15 am and the sunset at 07:00 pm. sociobiology 68(1): e-5792 (march, 2021) 3 behaviors of female bees were observed by two observers, who simultaneously observed females from different nests, from a distance between 40 cm and 100 cm from the entrance of the nests marked with tape and styrofoam numbered plates, fixed on wooden rods (figure 1b). the following activities of the bees were recorded: i) the behavior of females during the nest building, including all the nest excavation process. in this latter case, after a few centimeters of tunnel depth, the behavior of the bee could only be deduced based on the removal of the sand; ii) type of floral resource present in the scopae (pollen, oil, oil + pollen), which was detected through visual observations when females returned to the nest; iii) time spent during the foraging trips for collection of the pollen and oil resources;; iv) time spent inside the nest after each foraging trip v) the number of fights performed by females during the period of observation. the duration of activities was recorded with a chronometer. all these parameters have also been evaluated and described in different studies on epicharis nesting biology (gaglianone, 2005; rocha-filho et al., 2008; martins et al., 2019). the density of the nests was calculated by random marking of four quadrants of 4 m × 4 m (i.e., 16 m2 each) along the aggregation. in these quadrants, the nests found with open and closed entrances were counted during the months of samplings, in order to compare the density of nests/m² during the months of activity of the species. for each month the number of nests in all quadrants was summed up, divided by 64 and expressed as nests/m2. to study the architecture of the nests, a mixture of plaster and water was poured into the nest entrances of 17 nests with a funnel, until the tunnel was filled (michener, 1964). after a few hours when the plaster was dry and hard, the soil around the entrance of the nest and along the burrow was excavated in search of the brood cells. with a tape or ruler, the length and depth of the tunnels and cells connected to them were measured. during excavations, brood cells not connected to the burrow (i.e., belonging to other nests) were found (n = 5), and their depth and size cells were also measured, totaling eight cells analyzed. throughout this stage, photographic records were made. some casts of tunnels and all cells found were taken to the laboratory of genetics and animal ecology (lagea) of the state university of londrina (uel). data analysis the kruskal-wallis rank sum test, followed by the post-hoc dunn test, when necessary, was used to test for possible statistical differences in the time spent by females on foraging trips for different types of feeding resources (i.e., pollen, oil or both pollen and oil). this comparison was performed on two sets of data: i) between the types of floral resources, considering all females together; ii) to check for possible differences among females, considering each floral resource. the statistical analysis was carried out on r software, version 3.2.3 (r development core team, 2015), and values were considered significantly different where p < 0.05. results features of the nest aggregation and nesting period the aggregation area was in sandy soil, typical of restinga, and had a total extension of approximately 2000 m². the nests were distributed in part along a gully and in part in flat ground. nests were easily detected due to the mound of sand, called tumulus, which forms outside the entrances fig 1. (a) general view of epicharis dejeanii nest aggregation in the study area. (b) a view of several tumuli around the nest entrances and styrofoam plates positioned next to the nest entrances for identification. (c) detail of the tumulus. n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii4 from the excavated sand. the tumulus has a lighter color than the surrounding soil (figure 1a-c). the nests were often established near the roots of the restinga vegetation (figure 1a-c). of the 27 nests studied for observing female behavior, 18 were found in this situation. individuals of e. dejeanii were active for a period of three months (november to january). in 2015, although we started to conduct our observation on 11 november, previous visits to the study area showed that females first appeared on 08 november, when they started to dig the first burrow. on november 8, we also observed the activity of males flying over the place where the nests would be built later. in 2017, the first two nests were constructed on 31 october, therefore one week earlier than in 2015. in both years, new females were observed burrowing tunnels on the following days. a nest density peak was observed in december 2016, showing 0.9 open nests per m2 (or 17.5 and 14.7 nests/ quadrant). although in january 2016, 1.1 nests built/m2 was registered, only 0.6 were in activity (open), (figure 2). it is also noticeable that the decline in nest construction started in january. nesting activities of e. dejeanii ended in february, since in this month the number of open nests dropped drastically (figure 2). in addition, during this month, no bee activity was observed at the aggregation site during the sampling days. fig 2. density (nests/m2) of open (in activity) and closed nests per month in an aggregation of epicharis dejeanii on ilha do superagui (is) during the warm-wet season, when the nests are constructed, showing data of november 2015 (n =29), january 2016 (n = 70), february 2016 (n = 28) and december 2016 (n = 59). the representation of density of the open nests in december 2016 is shown in different color (dark gray) to highlight that the nests from this month were surveyed in the following reproductive season. nest architecture the entrance of nests is circular, measuring approximately 1.5 cm in diameter. the nest is formed by a single narrow tunnel, which extends vertically (in some cases, making small curves) into the ground, ending in a single brood cell. no marks indicating branches or tunnel connections were visible on the plaster. the tunnels had the same diameter as the entrances of the nests, slightly wider than the body of the bee, which measures approximately 1 cm in diameter. the tumuli ranged from 3 to 8 cm in height (`x = 5.8 ± 1.7; n = 24), and 9.5 to 19 cm (`x = 15.5 ± 3.3; n = 24) in diameter at the base (figure 1a-c). from a total of 17 nests filled with the plaster and water mixture, we succeeded in excavating only eight thoroughly, with tunnel and cell (figure 3a). these eight nests were in depths ranging from 64 cm to 184 cm (`x = 145 ± 35 cm; n = 8). however, due to the fact that several excavated tunnels were not linear in their trajectories, from the nest entrance to the brood cell, showing some curves, the total length of nests ranged from 71 cm to 216 cm (`x = 160 ± 39 cm; n = 8). only one cell per nest was found. the total length of these varied from 2.90 to 3.5 cm (`x = 3.1 ± 0.19; n = 13). cells were oval and had a rough, hard, and resistant outer layer (figure 3b, c), composed of sand particles which were clearly visible and detectable by touch, but the sand was darker than that around the tunnel. the inner surface of the cells was smooth, dark and a bit shiny. the brood cells, in the end of each tunnel, were aligned in different directions in relation to the surface. nesting behaviors and female foraging activities the results on nesting behavior and foraging activities of e. dejeanii females reported herein are based on different periods of observation and, therefore, on behaviors and activities from different groups of females during our study. in november 2015, we observed the complete sequence of three nest foundations by distinctive females and, three phases have been clearly identifiable: i) nest site selection; ii) burrowing and cell construction and iii) brood cell provisioning. when choosing a nesting site, the females flew in circles over the ground at a height of approximately 10 cm over a large area and then in smaller circles, focused on a smaller region, until selecting the site to land and start construction (figure s1a, b). in those nests, the females excavated with their front legs, then transferred the sand to their middle and hind legs respectively, so that, as they dug, they pushed the sand to the surface. the removed sand was accumulated on the outside, forming a growing tumulus, which was arranged quickly with the hind legs every time they returned, coming up abdomen first. in addition, the distal tergal was used to compact the soil, probably to avoid collapse of the tunnel (vide-not included due to size). on rainy days, the tumulus collapsed. when the nest was closed for some reason, like rain for example, females returning from their trips dug and re-opened the entrance burrow (figure s2). the females carried out the nest excavation for periods ranging from 14 to 166 min (`x = 66.6 min ± 38.1; n = 12). the excavation was interrupted by a few brief flights, ranging from 13 to 27 min (`x = 19.7 min ± 5.1; n = 7; data not shown), after which they returned to the nests without any floral resources in their scopae and, re-started digging. the females started digging in the morning and, at the end of the day, at 6:00 pm, they were still building the nest (probably the tunnel). in two out of the three nests observed sociobiology 68(1): e-5792 (march, 2021) 5 for nesting construction, the females started the nest digging at 7:30 am (nest 1) and at 9:02 am (nest 2), while the nest 3 excavation began in the end of the morning (at 10:23 am). therefore, the nest excavation lasted all day for nest 1, totaling at least 630 min (> 10 h) of digging. as already mentioned, from a certain tunnel depth, we were only able to infer that females were still excavating, since they emerged from time to time, pushing sand with their abdomen, accumulating it in the tumulus. we also observed that although nest excavations occurred mostly in the morning, nests could be initiated any time during the day. for instance, in november 2015, we registered females from other two nests nearby starting the excavation late in the afternoon (after 5:00 pm). in the morning of the following day, at 6:00 am, no more excavation activities were observed for the three nests; at this time, the bees were already performing foraging trips, returning to their nests. therefore, the three females observed during the nest building phase took from 540 min to 630 min throughout the day and, at least, part of the night performing the excavation. however, it is not possible to state with certainty how many hours the females spent in the total phase of nest construction, since they remained inside their nests after the sunset and we do not have data on their nocturnal activity. after the nest burrowing and cell construction phase, the female starts the brood cell provisioning phase, carrying out foraging trips to collect oil and pollen. it is important to highlight that although the nests were frequently very close to each other, no antagonistic interaction was observed between the females in the aggregation during all period of observation. during the period of study, observations on foraging activities of females from 27 nests revealed that the they spent most of their time, from early morning to early evening, outside the nest on foraging trips for oil, pollen or both resources, with usually short intervals inside the nest for unloading (figure 4). they stayed most of the time, up to 10 minutes inside the nests after their trips and approximately one hour outside the nests foraging (figure 4). the average time spent inside the nests fig 3. (a) detail of an excavated nest, showing the main tunnel and the cell (arrow) connected to it. (b) the outer view of the cell shown laterally. (c) longitudinal view of an opened cell with larva inside it (arrow). n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii6 after arriving was: with pollen`x = 15.8 ± 14.3 min (n = 72); with pollen and oil `x = 1517.0 ± 15.1 min (n = 63), and with oil `x = 22.5 ± 15.7 min (n = 45), (figure 5); no significant differences were found among these values. in november 2017, for five days, from 6:00 am to 6:00 pm, we registered the period of foraging activity, mean number of foraging trips and number of trips for pollen, oil and for both pollen and oil carried out by females from three nests (table 1). in all these nests females had already initiated the foraging trips when the observations started, which means that the nests were in the cell provisioning phase. as shown in table 1, during the period of study, the mean number of foraging trips performed per day by the three females varied from 2.40 (± 1.52; n = 3) to 4.00 (± 1.83; n = 3). although the mean number of foraging trips for both pollen + oil (`x = 2.33 ± 1.15; n = 6) was below those registered for only pollen (`x = 5.33 ± 3.21; n = 16) or only oil (`x = 5.00 ± 2.00; n = 15). these measures did not differ statistically (h = 3.56, df = 2; n = 3). fig 4. time spent by epicharis dejeanii females inside the nest on cell provisioning (n = 215 records) and outside the nest on foraging trips (n = 174 records) during the observation period. results are shown in 23 frequency distribution classes (in intervals of 10 min). observations were carried out at the entrance of 27 nests in total, on november 15, january 16, december 16 and november 17. observations conducted from 08 to 14 january 2016 on foraging activities of females from 13 nests revealed that the average number of trips carried out was`x = 23.38 (± 4.41; n = 13), ranging from 23 to 35. distinctively, the number of foraging trips carried out by females in november 2015 was`x = 2.67 ± 1.23 (n = 5), ranging from 1 to 5 per day, while in november 2017 was`x = 3.14 ± 1.70 (n = 3), varying between 1 and 6. in january 2016, the females performed a statistically superior average number of foraging trips compared to those females from november of both years (h = 27.02, df = 5, p < 0.0001). fig 5. average time spent by epicharis dejeanii females inside the nest, after each foraging trip, and on foraging trips for collecting floral resources (only pollen, pollen + oil and only oil). sociobiology 68(1): e-5792 (march, 2021) 7 in the observations conducted during a week, in january 2016, estimates of the time spent by the 13 females during the collection of the resources showed that the pollen trips lasted from 8 to 114 min (`x = 64.34 ± 27.29; n = 38), oil trips between 30 to 103 min (`x = 60.18 ± 20.57; n = 34) and trips where females carried pollen and oil together in the scopae lasted 25 to 118 min (`x = 64.71 ± 21.42; n = 35). females of these nests foraged for the three types of resources along the day (figure 6). the number of foraging trips increased after 7:00 am and remained stable throughout the day, with a small peak in the period between 1:00 pm to 3:00 pm. however, since we only had a few samplings between 11:00 am and 1:00 pm, this period of the day was not considered. thus, we are not able to state what the female activity was like during this period. nest period of activity in days total number of foraging trips* mean number of trips per day (± ds) number of foraging trips with only pollen number of foraging trips with only oil number of foraging trips with pollen and oil 3a 4 16 4.00 (± 1.83) 9 5 1 4a 5 16 3.20 (± 1.60) 3 7 3 5a 5 12 2.40 (± 1.52) 4 3 3 all `x = 4.67 `x = 14.67 (± 2.31) `x = 5.33 (± 3.21) `x = 5.00 (± 2.00) `x = 2.33 (± 1.15) * the total number of trips registered was superior to the sum of the numbers of trips for the different resources, since for some foraging trips it was not possible to identify for sure the type of resource in the scopae of the females when they returned to the nest. table 1. period of activity (in days) by females of epicharis dejeanii in november 2017, during a survey conducted, from 6:00 am to 6:00 pm, at ilha do superagui, south of brazil, in a nest aggregation. it is also shown the total number of foraging trips along the study period, mean number of foraging trips per day (± sd) and number of foraging trips for different types of resources (oil, pollen and oil + pollen). the observations were carried out at the entrance three nests, by two observers. in all nests, when the observations started, the females had already initiated the cell provisioning activities. for these 13 nests, we also registered that the females performed for up to 6 days of foraging trips, varying from 4 to 6 days. moreover, when we interrupted our observations on 14 january, most females had not finished the foraging trips yet nor abandoned their nests. only in two out of the 13 nests under observation, females stopped the foraging trips before the sixth day. in this case, females left their nests on the fourth and the fifth days and did not return. in these 13 nests, the mean number of foraging trips per day ranged from 3.8 (± 2.0; n = 23) to 5.8 (± 3.5; n = 35). in additional observations to confirm the period females ceased their activities outside the nest, we registered that they ended near twilight, when the last bee was seen entering the nest at 7:18 pm. lastly, since no bee emerged from cells taken to our laboratory, this result is not shown in our study. fig 6. number and diurnal distribution of trips carried out by females of epicharis dejeanii, at ilha do superagui, brazil, to collect the three classes of resources: pollen, pollen + oil and oil. // = period without observations on bee behavior. n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii8 discussion nest architecture our results on e. dejeanii agree in great part with those of hiller and wittmann (1994) as well as dec and vivallo (2019), who also studied the nest architecture of this species in southern brazil. the aggregations studied by these authors, as in our study, were constructed in sandy soil and nests were similar in their general structure, including the characteristics of the brood cell and diameters of tunnel, nest entrance and tumulus surrounding the entrance. however, some differences can be pinpointed here. for example, in the aggregation at is we never detected the occurrence of more than one entrance giving access to the nest as reported in both studies (hiller & wittmann, 1994; dec & vivallo, 2019). in addition, all eight nests excavated in our study presented a descending tunnel, predominantly vertical, with only slight curves (or inclinations) going down in the soil, until it found the brood cell (as shown in figure 3). this finding differs from those reported by dec and vivallo (2019), who found a conspicuous heterogeneity in the tunnels from 15 dug nests. these authors frequently found nests presenting a vertical tunnel, which after a slope ended in a horizontal tunnel, a condition we never found. concerning the number of brood cells per nest, our results are more closely in line with the findings of dec and vivallo (2019), who reported one or two brood cells per nest. however, considering that the latter excavated a higher number of nests (n = 15) than the current study (n= 8), this divergence might be reflecting the difference in sampling efforts. despite this, both results by dec and vivallo (2019) and ours support the fact that the number of brood cells per nest of e. dejeanii is inferior to those estimated in hiller and wittmann (1994). in this case, either the higher number of cells per nest found by hiller and wittmann (1994) is a result of a plasticity in behavior of e. dejeanii, as already reported for e. nigrita (martins et al., 2019) or it could be due to the difference in the methodology employed in the preliminary study on e. dejeanii (hiller & wittmann, 1994). in fact, considering that digging deep nests seems to be an energetically expensive task to solitary bees, by producing several brood cells per nest rather than just one, a female could optimize its digging effort, having reproductive advantage with this behavior. (martins et al., 2014). it has also been suggested that, among species that show variation in number of cells built per nests, the low production is environmentally mediated (michener, 1974), and that it is possible that perturbation by other nesting females or parasites may cause the nesting female to close the nest and start another one (martins et al., 1994). however, in our study, the number of cells per nest was invariably one, despite most of nests surveyed are deep in the ground and independently of the number of days used for provisioning the nests by females. thus, other factors could be mediating the construction from one (this study) to more than one brood cell per nest (hiller & wittmann, 1994; dec & vivallo, 2019) in e. dejeanii, which we are not able to point out for sure here. the mean depth of nests excavated in the aggregation at is was higher than those already described for e. dejeanii (hiller & wittmann, 1994; dec & vivallo, 2019). in fact, our results revealed the deepest nests so far described for epicharis (e.g. gaglianone, 2005; rocha-filho et al., 2008; werneck, 2012; dec & vivallo, 2019). the construction of cells deep in sandy soils is common in epicharis, and probably represents an important feature to avoid parasites (gaglianone, 2005). it should be highlighted that although in our study the deepest cell was found at 185 cm deep, the total length of the tunnel excavated by the female, reached more than 210 cm, suggesting great effort of the bee in digging down the soil. many reports in the literature have shown that ground-nesting bee aggregations can persist for a long time, even decades, in the same area (michener, 1974; rozen & michener, 1988; cane, 2008; danforth et al., 2019; martins et al., 2019). in fact, the persistence of bee nest aggregations for years or even decades in a same area may be more common than we know (cane, 2008). in our study, we observed that the large aggregation of e. dejeanii at is persisted in activity for at least six years (from 2013 to 2018) in the same area. a combination of different appropriate factors, including soil texture and plenty and stable food resources have been pointed out as affecting the long-term persistence of such aggregations (danforth et al., 2019). in addition, the habit of build nests in aggregations shown by a number of species of bees seems to have some advantages, including, for example, both high chances of encounters between partners for mating (pina et al., 2020) and detections of cleptoparasites by the host bees (litman, 2019). nesting and foraging behavior our results also agree with findings that pointed out e. dejeanii as an univoltine and a seasonal species, active only during the warm and wet season (hiller & wittmann, 1994; dec & vivallo, 2019). since the three studies on nesting biology of e. dejeanii were carried out in the south of brazil, in areas located between the following geographical coordinates: 25°27.51.4”s 48°14.07.1’w (current study) and 29o22’33” 51o06’43”w (hiller & wittmann, 1994), under subtropical climate, we could expect a similarity in the period of activity of the studied species. whereas our results concerning the period of activity of e. dejeanii showed a complete overlapping with that described by dec & vivallo (2019), who also reported the activity of bees from november to january, the activity of this species started a little later in a region located farther south (hiller & wittmann, 1994). therefore, even within a limited range of latitude and longitude, some variation can be found in the period of activity of e. dejeanii. the seasonal activity and univoltine cycle detected for e. dejeanii seems to be almost a pattern in epicharis, having been reported for different species of this genus (roubik & michener, 1980; gaglianone, 2005; gaglianone et al., 2015; martins et al., 2019), although exception for univoltine life cycle was reported to the species epicharis bicolor smith, 1958 (rocha-filho et al., 2008). sociobiology 68(1): e-5792 (march, 2021) 9 concerning the period of diurnal activity of e. dejeanii at ilha do superagui (is), as reported by hiller and wittmann (1994), we also observed that this species began its activity very early in the morning, at about daybreak, particularly between 5:00 am and 5:30 am in is, and one hour earlier compared to the findings of dec and vivallo (2019) in the state of santa catarina. our results at the end of the diurnal activities of e. dejeanii corroborate that the females ceased their external activities at dusk as reported by hiller and wittmann (1994). the behavior of spending the night inside the nest herein described was also observed in the two previous study with e. dejeanii (hiller & wittmann, 1994; dec & vivallo, 2019). by spending more time away from the nest, the female bee leaves it more vulnerable to predators and parasites (kline & joshi, 2020). despite the activity of parasites and predators of bee nests is more intense during the day (kelber et al., 2005), the behavior of females in spending the night inside the nest could be related to a more effective strategy in protecting their nests. diversely from the brief breaks for nectar uptake, the foraging trips for collecting oil, pollen or both usually took longer, most of which between 30 and 90 min, averaging about 60 min. our results also revealed that the time of the foraging trips carried out by females of e. dejeanii at is were very superior than those reported by dec and vivallo (2019), for this species in ilha das flores in santa catarina state. thus, it is possible to infer that the higher time of foraging trips at ilha do superagui is probably a consequence of the longer distance to the food plants. in fact, at is, although we walked around the study area looking for females of e. dejeanii foraging on flowers nearby, we only registered this species in shrubs of tibouchina spp, suggesting that the females foraged for plants located far from the nest aggregation. surely, the availability of food resources in abundance close to the study area favors shorter foraging trips. gaglianone (2005) observed that e. nigrita performed shorter foraging trips (20 – 40 min), during the peak of the nesting activity, precisely when the main plant used as resource by this species was in the peak of flowering in the studied area. on the other hand, the foraging trips carried out by e. nigrita lasted up to two hours, probably because of the scarcity of food resources. the intense activity by e. dejeanii carrying out foraging trips all day, starting soon after dawn and only ceasing the trips at dusk, was reported to e. nigrita by gaglianone (2005). particularly for females of solitary and univoltine bees, this intense activity in nest tasks is expected, since they usually live for only a few weeks (willmer & stone, 2004; danforth et al., 2019) and have only a short time during the year to carry out the nesting activities to raise their offspring. as shown by gaglianone (2005) for e. nigrita, we also detected that, after returning to the nest from foraging trips, most of the times (@70%), females of e. dejeanii took less than 20 min to unload the different types of resources carried in their scopae. our findings are also in line with those from dec and vivallo (2019), who reported a mean time of 23 min (± 3.55; n = 20) for e. dejeanii inside the nest after the foraging trips. thus, at least for e. dejeanii and e. nigrita (gaglianone, 2005) the time for unloading and possibly handlining the plant resources collected was similar between these two species, as well as didn’t vary among different locations. a conspicuous difference between our findings and those reported by dec and vivallo (2019) is related to the period in days that females worked in their nests. according to these authors, the total period ranging from the nest excavation and the abandonment of the nests occurred in 3 to, rarely, 4 days. however, in our study, this period was usually longer, since in january 2016, for example, we observed 13 nests for a week, when all these nests were already in the provisioning phase. in addition, when observations were stopped after a week, 11 out of 13 females had not abandoned their nests and were carrying out foraging trips, indicating that they still had not finished their activity for brood cell provisioning. hiller and wittmann (1994), on the other hand, estimated a period of five days of female activity. a reasonable explanation to the longer period performing nesting tasks detected for e. dejeanii at ilha do superagui, could be the high rainfall rates (> 2,500 mm per year) in the region where this island is located, with the highest incidences of rain coinciding exactly with the period of activity of this species. in fact, we observed that e. dejeanii had to frequently interrupt its external activities due to unfavorable weather conditions. based on our results on behavior of e. dejeanii females during their nesting activities and comparing our findings with two previous studies (hiller & wittmann, 1994; dec & vivallo, 2019), we can conclude that in spite of a number of similarities found among these studies, a variation or plasticity in nesting behavior of this species can occur in different locations from the same region. together, these three studies contribute with complementary information to more detailed knowledge on the nesting biology of e. dejeanii, until date, the only species representing the anepicharis subgenus. acknowledgments we are grateful to the araucaria foundation and to the coordination for the improvement of higher education personnel (capes) for the scholarship awarded to n. uemura and a.l. gobatto; to the cnpq –pibic for the fellowship to r.h. ono; to dr. maria cristina gaglianone (uenf); to douglas c. giangarelli, robson rockembacher, diogo mazzaro, eliza tanaka, henrique zotarelli, thales f. lizarelli and wilson frantine-silva by helping during samplings; to ibama (brazilian institute of the environment and renewable natural resources)/icmbio-system (institute chico mendes – mma) for permission to collect samples and to the icmbio – superagui national park for logistic support; silvia h. sofia is research fellow at the cnpq (pq 305343/2018-1). we also would like to thank the reviewers for their suggestions, which substantially contributed to the improvement of this paper. we also thank the two anonymous reviewers for their valuable comments and help. n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii10 funding this work was supported by the coordenação de aperfeiçoamento de pessoal de nível superior [finance code 001]; araucaria research foundation of parana state and brazilian council for scientific and technological development (cnpq). disclosure statement no potential conflict of interest was reported by the authors. authors’ contributions n. uemura: conceptualization, methodology, investigation, formal analysis, writing s.h. sofia: conceptualization, methodology, formal anlysis, writing a.l. gobatto: investigation, data curation, formal analysis w.c. pina: investigation r.h. ono: investigation . references aguiar, a.p., chiarello, a.g., mendes, s.l. & matos, e.m. 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(2005). mapeamento e caracterização da vegetação da ilha de superagüi utilizando técnicas de geoprocessamento. floresta, 35: 303-315. doi: 10.5380/rf.v35i2.4618. steiner, j., zillikens a., kamke, r., feja, e.p. & falkenberg, d.b. (2010). bees and melittophilous plants of secondary atlantic forest habitats at santa catarina island, southern brazil. oecologia australis, 14: 16-39. doi: 10.4257/oeco.2010.1401.0. thiele, r. & inouye, b. (2007). nesting biology, seasonality, and mating behavior of epicharis metatarsalis (hymenoptera: apidae) in northeastern costa rica. annals of the entomological society of america, 100: 596-602. doi: 10.16 03/0013-8746. werneck, h.a. (2012). biologia de nidificação, sazonalidade e inimigos naturais de epicharis (epicharoides) picta (smith, 1874) (apidae: centridini) no município de viçosa, mg-brasil. (master dissertation in entomology) federal university of viçosa, minas gerais. willmer, p.g. & stone, g.n. (2004). behavioral, ecological, and physiological determinants of the activity patterns of bees. advances in the study of behavior, 34: 347-466. doi: 10.1016/s0065-3454(04)34009-x. n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii12 table s1. dates of samplings conducted at ilha do superagui, state of paraná, southern brazil, during some months in warm-wet season, from december 2013 to november 2017, types of activities carried out by study team in each date and total time spent in each sampling day. supplementary material date period of sampling total sampling time type of activity 22/12/2013 7:00 – 12:00 am 5 h nest excavation 23/12/2013 7:00 – 12:00 am 5 h nest excavation 11/11/2015 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 12/11/2015 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 13/11/2015 6:00 – 11:00 am 2:00 – 3:00 pm* 6 h bee behavior observation 14/11/2015 6:00 – 11:00 am 2:00 – 4:00 pm* 7 h bee behavior observation 15/11/2015 6:00 – 8:00 am* 2 h bee behavior observation 16/11/2015 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 17/11/2015 6:00 – 11:00 am 1:00 – 5:00 pm* 9 h bee behavior observation 18/11/2015 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 08/01/2016 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 09/01/2016 6:00 – 11:00 am 1:00 – 4:00 pm* 8 h bee behavior observation 11/01/2016 7:00 – 11:00 am* 1:00 – 6:00 pm 9 h bee behavior observation 12/01/2016 1:00 – 6:00 pm* 5 h bee behavior observation 13/01/2016 6:00 – 11:00 am 1:00 – 6:00 pm 10 h bee behavior observation 14/01/2016 6:00 – 11:00 am* 5 h bee behavior observation 13/02/2016 7:00 am – 7:00 pm 12 h nest excavation 14/02/2016 7:00 am – 7:00 pm 12 h nest excavation 15/02/2016 7:00 am – 7:00 pm 12 h nest excavation 16/02/2016 7:00 am – 7:00 pm 12 h nest excavation 13/12/2016 5:00 – 10:00 am 6:00 – 8:00 pm 5 h 2 h bee behavior observation 14/12/2016 5:00 – 6:00 am 6:00 – 8:00 pm 1 h 2 h bee behavior observation 15/12/2016 8:00 am – 6:00 pm 10 h nest excavation 31/10/2017 6:00 am – 6:00 pm 12 h bee behavior observation 01/11/2017 6:00 am – 6:00 pm 12 h bee behavior observation 02/11/2017 6:00 am – 6:00 pm 12 h bee behavior observation 03/11/2017 6:00 am – 6:00 pm 12 h bee behavior observation 04/11/2017 6:00 am – 6:00 pm 12 h bee behavior observation total 249 h * = samplings interrupted due to bad weather conditions (rain) sociobiology 68(1): e-5792 (march, 2021) 13 figure s1. (a) detail of a female of epicharis dejeanii flying over the ground at a height of approximately 2 cm; (b) on the ground, beginning the construction of the nest. n uemura, al gobatto, wc pina, rh ono, sh sofia – nesting biology of epicharis dejeanii14 figure s2. sequence of images (a-f) showing moments of a nest re-excavation by a female of epicharis dejeanii after a rainy period, which caused the obstruction of the nest entrance. doi: 10.13102/sociobiology.v67i3.5014sociobiology 67(3): 478-480 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 syllophopsis (hymenoptera: formicidae) is a genus of small, inconspicuous ants, with 21 valid species (bolton 2020). most syllophopsis species are known only from the afrotropical bioregion. syllophopsis sechellensis (emery), however, has achieved broad distribution in asia and australia, and on islands the indian, pacific, and atlantic oceans (wetterer & sharaf, 2017). in the new world, all published records of s. sechellensis come from west indian islands (wetterer & sharaf, 2017). here, i report the first records of s. sechellensis from north america. syllophopsis workers are small, monomorphic, yellow to light brown in color, and have 12-segmented antennae with 3-segmented terminal clubs (bolton, 1987). syllophopsis workers resemble small solenopsis thief ants. however, the two genera can be distinguished easily because solenopsis workers have 2-segmented terminal clubs. like most syllophopsis species, s. sechellensis workers have tiny eyes. one character, abstract syllophopsis sechellensis (emery) (formerly monomorium sechellense) (hymenoptera: formicidae) is a small, inconspicuous ant species native to the old-world tropics. syllophopsis sechellensis is widespread in asia and australia, and on islands the indian, pacific, and atlantic oceans. in the new world, all published records come from west indian islands. here, i report the first records of s. sechellensis from north america: from four sites in miami-dade and broward counties, florida, more than 1500 km from the closest records in the west indies. the ants of florida have been well-studied in the past, so s. sechellensis appears to be a recent arrival. sociobiology an international journal on social insects jk wetterer article history edited by evandro nascimento silva, uefs, brazil received 17 february 2020 initial acceptance 22 june 2020 final acceptance 22 june 2020 publication date 30 september 2020 keywords biogeography; biological invasion; nonnative species. corresponding author james k. wetterer wilkes honors college florida atlantic university 5353 parkside drive, jupiter, fl 33458, usa. e-mail: wetterer@fau.edu however, that distinguishes s. sechellensis from other members of the genus is that its entire mesopleuron is matte and reticulate punctate, whereas in all other described syllophopsis species the mesopleuron is glossy and smooth. this character easily separates s. sechellensis from the only other syllophopsis species known from the new world, syllophopsis subcoeca emery. materials and methods i surveyed ants at sites in peninsular florida primarily using two methods: vegetation beating for arboreal ants and leaf litter extraction for subterranean ants. for the litter samples, i sampled mostly under slash pine (pinus elliottii engelmann), where the leaf litter is typically 10-30 cm thick. in earlier research, wetterer et al. (2018) found a non-native trap jaw ant, anochetus mayri, was common in slash pine litter in southeastern florida. florida atlantic university, jupiter, florida, usa first north american records of the old-world tramp ant syllophopsis sechellensis (hymenoptera: formicidae) short note sociobiology 67(3): 478-480 (september, 2020) 479 results i collected syllophopsis sechellensis through litter extraction at four sites in southeast florida (fig 1), spanning 46 km from southwest to northeast (geo-coordinates and dates in parentheses): miami-dade co.; university park; fiu preserve, slash pine (25.755, -80.379, 23-sep-19). broward co.; fort lauderdale; 28th terrace, vacant lot, slash pine (26.093, -80.181, 25-sep-19). miami-dade co.; westview; nw 107th street, vacant lot, leaf litter (25.8721, -80.2267, 17-nov-19). broward co.; fort lauderdale; sailboat bend park, slash pine (26.1179, -80.1607, 19-nov-19). mostafa sharaf (king saud university, saudi arabia) confirmed my identification of these syllophopsis sechellensis specimens. pinned vouchers will be deposited in the united states national museum, the florida state collection of arthropods, archbold biological station, and the personal collections of mostafa sharaf and james k. wetterer. wetterer and sharaf (2017) reported s. sechellensis from 12 west indian islands (barbados, guadeloupe, grenada, martinique, mona, puerto rico, st croix, st lucia, st martin, st thomas, st vincent, and trinidad). subsequently, i reexamined specimens i collected at montpelier botanical gardens on nevis (17.123, -62.595, 14-may-07) and determined fig 1. records of syllophopsis sechellensis in florida (map made using carto.com). jk wetterer – first north american records of syllophopsis sechellensis480 them to be a mix of both s. sechellensis and s. subcoeca. in addition, although deyrup (2016) reported that he collected s. subcoeca on dominica, these specimens (cabrits national park; 15.587, -61.474; 2-may-2006) were actually misidentified s. sechellensis (m. deyrup, pers. comm.). discussion syllophopsis sechellensis has widespread records from the old-world tropics and subtropics, particularly from islands (wetterer & sharaf, 2017). syllophopsis sechellensis is certainly exotic to the new world, where it was first found in 2003 on the island of barbados (wetterer et al., 2016). syllophopsis sechellensis is now known from 14 west indian islands (see results). the first north american records of s. sechellensis, which i report here, are from four sites in miami-dade and broward counties, southeast florida, more than 1500 km from the closest records of this species in the west indies. the ant fauna of florida has been well-studied in the past (deyrup, 2016), so s. sechellensis appears to be a recent arrival. because it is small and subterranean, s. sechellensis can be easily overlooked and it may have a broader range in the new world than is currently known. although all known records of s. sechellensis in the new world are relatively recent, some earlier specimens of s. sechellensis from the new world may have been misidentified as s. subcoeca (see results). how far s. sechellensis will spread in florida remains to be seen. syllophopsis sechellensis is not known as a pest species and it seems unlikely that it will become one. it is just one more addition to the growing list of non-native ant species with established populations in florida. acknowledgments i thank m. wetterer for comments on this manuscript, m. deyrup for unpublished collection data, and florida atlantic university for financial support. references bolton b. (1987) a review of the solenopsis genus-group and revision of afrotropical monomorium mayr (hymenoptera: formicidae). bulletin of the british museum (natural history), entomology 54: 263–452. doi: 10.5281/zenodo.26850 bolton b. (2020) syllophopsis santschi, 1915. antcat: an online catalog of the ants of the world. http://www.antcat.org/ catalog/429732?qq=syllophopsis (accessed 16 february 2020). deyrup m. (2016) ants of florida. identification and natural history. crc press. boca raton, fl. 423 pp. wetterer jk, sharaf mr (2017) worldwide distribution of syllophopsis sechellensis (hymenoptera: formicidae). florida entomologist 100: 281-285. doi: 10.1653/024.100.0224 wetterer jk, deyrup ma, bryant a. (2018) spread of the nonnative trap-jaw ant anochetus mayri (hymenoptera: formicidae) in florida. transactions of the american entomological society 144: 437-441. doi: 10.3157/061.144.0201 wetterer jk, lubertazzi d, rana j, wilson eo. (2016) ants (hymenoptera: formicidae) of barbados. breviora, 548: 1-34. doi: 10.3099/brvo-548-00-1-34.1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i2.145-149sociobiology 60(2): 145-149 (2013) alternative control of the leaf-cutting ant atta bisphaerica forel (hymenoptera: formicidae) via homeopathic baits vm ramos, f cunha, kc khun, rgf leite, wf roma introduction the insects of the family formicidae are known for their highly organized colonies that consist of millions of individuals. in terrestrial ecosystems, ants may constitute 15 to 20% of the total animal biomass (schultz, 2000). the leafcutting ants belong to the tribe attini, are present throughout tropical portions of the americas and are the greatest consumers of vegetal mass in brazil when compared with other insects and even with mammals. when they carry the leaves under the soil, the large quantity of organic material that is made available becomes a source of carbon and other nutrients for other organisms. these insects play an important role in ecosystems, but, despite the benefits they confer, they become pests when in proximity to agriculture and need to be controlled (santos, 2010). the environmental impact of anthropogenic activity directly affects terrestrial ecosystems, such as, for example, abstract leaf-cutting ants are pests that afflict diverse crops, and are most efficiently controlled by chemical methods that are widely utilized. other methods have been investigated aiming to efficiently control these insects while reducing the environmental impact of applying such chemical products. therefore, an assay was conducted to evaluate the efficiency of baits, formulated homeopathically, in nests of the leaf-cutting ant atta bisphaerica, in the field. thirty (30) colonies were chosen and divided into 10 repetitions for each of the following treatments: control (without baits), standard (8 g/m2 of loose soil of baits based on sulfluramid 0.3%) and homeopathic (60 g/m2 of loose soil of homeopathic baits parceled into 20g/m2 doses applied on 3 consecutive days). at 24 hours after bait application on active foraging trails of colonies, the evaluation of of the following parameters was initiated: transport and devolution of the baits, foraging and mortality. the completed assay demonstrated that the transport of baits was greater in the standard (80%) than in the homeopathic treatment (50%). on the other hand, the devolution of baits was significantly higher in the homeopathic treatment (15%) versus the conventional, where devolution/rejection did not occur. colony mortality was 20% under the homeopathic treatment, differing statistically from the 80% value produced by the standard treatment. thus, the homeopathic treatment is not demonstrated to be efficient at controlling leaf-cutting ants, suggesting the need for new studies with different methodologies. sociobiology an international journal on social insects 1 universidade do oeste paulista, presidente prudente, sp, brazil research article ants article history edited by qiuying huang, hau china received 19 december 2012 initial acceptance 08 march 2013 final acceptance 11 april 2013 key words ant control, pests control, attini, grass-cutting ants corresponding author vânia maria ramos laboratório de entomologia agrícola faculdade de ciências agrárias universidade do oeste paulista rodovia raposo tavares km 572 presidente prudente, sp, brazil 19067-175 e-mail: vaniaramos@unoeste.br the application of agrochemicals. before the physicochemical dispersion or biotransformation of insecticides, they can act as an important element in the disruption of trophic interactions in terrestrial ecology (tiepo et al., 2010). large quantities of insecticides are used for combating pests, but the introduction of xenobiotic molecules in the environment can damage the equilibrium of the ecosystem because synthetic insecticides may present high toxicity and, generally, low biodegradability, which can lead to a persistent toxic action (tiepo et al., 2010). a large amount of chemical pesticides is utilized to meet commercial demands of industry and consumers, but given the growing public concern about the presence of their residues in water supplies or food, many producers are coming to adopt production systems that are purely organic or more ecologically integrated (boff et al., 2008). in order to reduce the harm caused by leaf-cutting ants, humans have pursued diverse forms of control, ranging from homemade methods to the use of advanced techniques (souza vm ramos, f cunha, kc khun, rgf leite, wf roma control of atta bisphaerica with homeopathic baits146 et al., 2011). the economic and environmental aspects have led businesses to improve the operational yield of the chemical control techniques employed (baits and thermal nebulization), and to experiment with new technologies and new toxic active ingredients (boaretto & forti, 1997). during the longterm evolution in the methods of controlling leaf-cutting ants, one of the great concerns has been to reconcile the conflict among the three principles of efficiency, economy and safety (cantarelli et al., 2005). as a consequence of the unfavorable aspects presented by granulated baits (deterioration of the environment, elimination of natural enemies and emergence of resistance), research lines have been generated to discover products with greater specificity and smaller environmental impact (hebling et al., 2000). in developed countries, intensive agriculture has not only augmented agricultural productivity, but also, due to its high dependence on great quantities of nonrenewable energy and raw materials, frequently has resulted in soil degradation, environmental pollution and damage to wildlife. for this reason, recent years have seen growing interest in agricultural methods that are both economically and environmentally sound (betti et al., 2009). among these, the agrohomeopathy has raised the interest of researchers. its potential benefits are significant because homeopathic preparations, on account of their extremely high dilution, are relatively inexpensive, have little or no ecological side effect and appear to be, when considered jointly, inoffensive (elmaz et al., 2004; lotter, 2003). all of these attributes make homeopathy optimally adjusted to the holistic approach of organic agriculture, and above all, biodynamic, in which the plants and their interactions with the environment are treated as a unified living organism (carpenter-boggs et al., 2000; heimler et al., 2009). furthermore, the new approach of applying homeopathic principles can also add to the improvement of nutritional properties (for example the levels of components that induce physiological benefits to human health) (fonseca et al., 2006) and physiological and qualitative characteristics of plants, in addition to their resistance to stress factors of both biotic (insects and pathogens) and abiotic in nature (physical and chemical damage) (betti et al., 2009). in recent years, increasing levels of resistance to insecticides and concerns about insecticide residues in agricultural products have stimulated a growing demand for products cultivated under new strategies of control, raising, in this context, the question of whether homeopathic preparations are able to control pest species (wiss et al., 2010). material and methods the assay was conducted on a cattle ranch located in presidente prudente, sao paulo state, brazil, in the period from august to october of 2011. the experimental area was constituted by a pasture composed of brachiaria decumbens and some spots of paspalum notatum, with the presence of colonies of the leaf-cutting ants atta bisphaerica and atta capiguara. from the experimental area 30 adult colonies of atta bisphaerica were selected and identified with numbered wooden stakes one meter in height. after this procedure, the area measurements of loose soil were estimated for each colony, with the aid of a measuring tape, namely, the greatest length and greatest width. the quantity of baits to be applied to each colony was calculated from its apparent external area. the following treatments were applied: t1 – control (0 g of baits/m2 of loose soil), t2 standard (8 g of commercial bait based on 0.3% of sulfluramid per m2 of loose soil) and t3 – homeopathic (baits formulated homeopathically and applied at the dose of 60g/m2 of loose soil). treatment t3 was formulated in a private commercial laboratory and the chosen dose had been recommended by the manufacturer, being that the total applied in each colony was parceled into 3 equal portions of 20g/m2 of loose soil, applied for the next 3 days. each treatment was composed of 10 repetitions, with each repetition constituting one colony. prior to application in the field, the baits were weighed in the laboratory of entomology at western paulista university, according to the measures obtained in the field for each colony, with the total quantity of each repetition being individualized in a plastic sack identified and duly stored until their use. in the field, before application of the treatments, for each repetition one active foraging trail near its area of loose soil was selected to ensure the immediate transport of granules and to avoid competition with workers of other colonies. the parameters evaluated were: transport and devolution of the baits, foraging, presence of recent loose soil, intoxication of individuals and mortality, according to established methodology as a protocol for experiments of this nature (zanuncio et al., 1997; nagamoto et al., 1999; ramos et al., 1999; zanuncio et al., 2002; forti et al., 2003; zanetti et al., 2004). the transport and devolution of baits were evaluated 24h, 48h and 72h after application, and attributed ratings of 0, 1, 2, 3 or 4, to represent the approximate respective equivalents of 0%, 25%, 50%, 75% and 100% of “pellets” transported and returned by the ants. the transport was evaluated by observing the trails were the baits had been applied, to verify the remaining quantity, while the devolution was evaluated by observing the mound of loose soil of each colony, in order to visualize and quantify the granules deposited on the ground, in relation to the quantity effectively applied. at 7, 15, 30, 60 and 90 days after installation of the experiment in the field, the remaining parameters were evaluated, verifying the presence or absence of the following: a) foraging – presence of individuals foraging on trails around the nests or on their loose-soil mound, b) loose soil – presence of granules of loose dirt on the mounds of nests, indicating recent activity of excavation, and c) intoxication: presence of young individuals (with clear tegument) on the loose-soil mound, signaling intoxication inside the nests, since under sociobiology 60(2): 145-149 (2013) 147 normal conditions, such workers are engaged only with tasks internal to the colonies. these parameters were used to supply data for evaluating the death (or absence thereof) among the colonies. in the final evaluation, at 90 days, as a function of the established criteria, the presence or absence of mortality was attributed to the colonies, considering dead those that did not show any signal of recent cutting or transporting of leaves and excavation of soil, with their mound of compacted loose soil and their foraging trails inactive. from the obtained data the mean percentages were calculated, and the results transformed into arcsine √x + 0.5/100, and then submitted to analysis of variance, comparing the means by the test of tukey at a 5% probability level, in an entirely randomized model of experimental design. results the transport of baits by the ants to the interior of the nests produced an 80% value under the standard treatment versus 50% via the homeopathic approach, differing statistically. the control treatment was represented by the value 0 on account of not having received baits (fig. 1-a). the devolution of the pellets transported by ants occurred only in homeopathic treatment, at the rate of 15%, differing from the control and standard treatments, which did not present devolution (fig. 1-b). the treatments differed as to efficiency, evaluated through the mortality of the colonies, being null in the control treatment, as expected, 20% in the homeopathic treatment and 80% in the conventional treatment (fig .1-c). discussion the 80% bait transport rate under the conventional treatment had been expected since prior experiments utilizing products formulated with the same active ingredient at the same concentration also obtained a high pellet transport rate in the field (zanuncio et al., 1997; zanetti et al., 2004). the transport of baits is one of the parameters that define treatment efficiency since the product can only act and express its potential if it is found attractive initially, accepted after investigation and then effectively transported by workers. the baits formulated based on homeopathy were not shown to be sufficiently attractive to the workers, as reflected in the unsatisfactory pellet transport value obtained (50%),which may have negatively influenced the efficiency of the treatment. the cause of the low rate of transport for homeopathic baits was not investigated, and thus it could not be affirmed whether it is a function of homeopathic formulation or of some other random factor such as the unknown quality of citric pulp utilized in the manufacturing of baits. the devolution of baits, as measured by the quantity of granules deposited on the mound of loose soil, was not observed in the standard treatment, a recurrent fact in field and laboratory assays that similarly evaluated sulfluramid-based baits (zanuncio et al., 1997; zanetti et al., 2004). in a contrary and significant manner, a portion (15%) of the homeopathic baits transported were returned by workers onto the loosesoil mound soon after transport, thus demonstrating that, for some reason, they were rejected during the process of postselecting foraged material, which occurs in the interior of the colonies. camargo et al. (2003) observed the occurrence of a post-selection process in colonies of acromyrmex subterraneus brunneus when the colonies were offered, in the laboratory, different inert materials such as plastic, polystyrene and clay, which were rapidly differentiated and not selected by the workers for cultivation of the symbiotic fungus. the authors reported that the post-selection of foraged material constitutes figure 1. transport of baits by workers (a), devolution of baits by workers (b) and, mortality of atta bisphaerica colonies (c), 90 days after application of treatments, in the field. presidente prudente, sp, brazil, 2011 (mean percentage values ± mean standard error, obs.: means followed by the same letter do not differ from each other for p < 0.05 by the test of tukey). a b c vm ramos, f cunha, kc khun, rgf leite, wf roma control of atta bisphaerica with homeopathic baits148 strong evidence of the cognitive abilities of workers and of the colony as a whole. devolution is an important parameter to be evaluated in experiments of this nature since it demonstrates the acceptance or rejection of a product by the ants. thus, it becomes evident that the lethality of the active ingredient to leaf-cutting ants is necessary but not sufficient since it may be prematurely perceived and rejected by the workers when formulated into baits, a scenario that would compromise the efficiency of the product, which may have occurred in the present assay. the efficiency of treatments, represented by the mortality of the colonies, was significantly superior in the standard treatment. the high mortality rate of colonies that received application of sulfluramid baits had been expected since there are reports in the literature of innumerable studies of the same nature that presented similar results (zanuncio et al., 1997; zanuncio et al., 2002; zanetti et al., 2004). sulfluramid is included among the active ingredients currently used for controlling leaf-cutting ants jointly with chlorpyrifos, given that chlorpyrifos is more toxic to mammals, aquatic organisms, fish and bees than sulfluramid (tiepo et al., 2010). the effect of the homeopathic treatment on the colonies was inconsistent, a coherent finding if considered a function of low transport of the baits presented and of the devolution rate, despite the low mortality observed. geisel et al. (2012) found that homeopathic preparations of adults and acromyrmex fungus reduced the activity of ant foraging trails in the field, from the sixth day of application (spraying), extending this effect until 20 days after last application. these results do not agree with those obtained in this test, but it is hard to compare them because the implementation and evaluation methodologies are completely different, with different goals, since in the geisel et al. (2012) study it was not evaluated the mortality of colonies, the main focus of this study. similarly, in an assay conducted on potato plants sprayed with diverse homeopathic preparations, in order to verify the incidence of diseases (phytophthora infestans and alternaria solani) and pests (diabrotica speciosa), boof et al. (2008) found no significant difference between the homeopathic products tested and the control (water), for all parameters evaluated. in another study, aiming to evaluate the effect of homeopathic preparations on the aphid dysaphis plantaginea in apple seedlings, wyss et al. (2010) reported no significant difference between homeopathic treatments and the control, by evaluating the quantity of individuals in the seedlings, the damage to the leaves and the fresh weight of the plants. in an assay performed by tiepo et al. (2010) to assess a natural formicide considered a “green pesticide,” the findings were promising. the product is a mixture of caffeine and common fatty acids, with citric pulp and apple as attractive agents. when its effects were observed on microbiological soil activity and the biomass of worms and plants, short-term toxicity was not detected. the authors reported that the formicide did not present toxic action on the experimental organisms on account of its natural composition, but that when the leaf-cutting ants ingested this product, despite not dying immediately, manifested behavioral disturbances. field observations, according to the authors, demonstrate a disruption in the social structure of the colony, which leads to the subsequent death of the individuals, considering that the communication constitutes the basis of the ant social structure. the term “green pesticide” should be understood as a natural or synthetic pesticide produced according to the principles of “green chemistry”, acting specifically and effectively on the precise target without deleterious or dangerous effects on non-target components of the ecosystem (tiepo et al., 2010). it is expected that the toxicity of this new and necessary class of “green pesticides” would be low or nonexistent (kahkonen & nordstrom, 2008). the data obtained herein do not justify a recommendation to control leaf-cutting ants by homeopathic baits. however, it is important to emphasize that, to the best of our knowledge, the literature contains no other scientific data on studies of this nature, a scenario that does not permit us to accept or reject definitively the hypothesis that homeopathy is a viable alternative for controlling these insects. in a review study on the use of homeopathic preparations in agriculture, to control pests and pathogens, betti et al. (2009) concluded that most of the works published and consulted do not provide sufficient information to enable a clear interpretation, in particular, due to the statistical analysis being inadequate or entirely absent, the number of repetitions being unspecified and, frequently, the experimental methodology being poor. even so, the authors concluded that the studies evaluated can serve as a starting point for future experiments that are more comprehensive and controlled. similarly, the results presented in the present assay do not permit us to resolve the question of whether homeopathy can control leaf-cutting ants, but rather call for initiation or expansion of this research line for greater investigation, conducted within scientific premises necessary to achieve a complete understanding of the subject. references betti l., trebbi g., majewsky v., scherr c., shah-rossi d., jäger t., baumgartner s. (2009). use of homeopathic preparations in phytopatological models and in field trials: a critical review. homeopathy, 98: 244-266. doi:10.1016/j. homp.2009.09.008. boaretto m.a.c., forti l.c. (1997). perspectivas no controle de formigas cortadeiras. série técnica ipef,30: 31-46. boff p., madruga e., zanelato m., boff m.i.c. (2008). pest and disease management of potato crops with homeopathic preparations and germplasm variability. ifoam organic world congress (http://orgprints.org/view/projects/conference.html). sociobiology 60(2): 145-149 (2013) 149 camargo r.s., forti l.c., matos c.a.o., lopes j.f., andrade a.p.p., ramos v.m. (2003). pos-selection and devolution of foraged material by acromyrmex subterraneus brunneus (hymenoptera: formicidae). sociobiology, 42: 93-102. cantarelli e.b., costa e.c., oliveira l.s., perrando e.r. 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(2011). efeitos de extratos naturais de folhas vegetais em leucoagaricus gongylophorus (möller) singer, (agaricales: agaricaceae). ambiencia, 7: 461-471. doi:10.5777/ambiencia. 2011.03.04. tiepo e.n., corrêa a.x.r., resgalla c., cotelle s., férard, j.f., radetski c.m. (2010). terrestrial short-term ecotoxicity of a green formicide. ecotoxicol. environ. saf., 73: 939-943. wiss e., tamm l., siebenwirth j., baumgartner s. (2010). homeophatic preparations to control the rosy apple aphid (dysaphis plantaginea pass.). the scient. world j., 10: 3848. zanetti r., days n., reis m., souza-silva a., moura m.a. (2004). efficiência de iscas granuladas (sulfluramida 0.3%) no controle de atta sexdens rubropilosa forel, 1908 (hymenoptera: formicidae). cienc. agrotec., 28: 878-882. zanuncio j.c., santos g.p., firme d.j., zanuncio t.v. (1997). uso da isca granulada com sulfluramida 0,3 % no controle de atta sexdens rubropilosa forel, 1908 (hymenoptera; formicidae). cerne, 3: 47-54. zanuncio j.c., sossai m.f., oliveira h.n. (2002). influência das iscas formicidas mirex-s max e blitz na paralisação de corte e no controle de atta sexdens rubropilosa (hymenoptera: formicidae). rev. arvore, 26: 237-242. 1335 survival rate, food consumption, and tunneling of the formosan subterranean termite (isoptera: rhinotermitidae) feeding on bt and non-bt maize by cai wang1, gregg henderson1*, fangneng huang1, bal k. gautam1 & chenguang zhu2 abstract although several termite species were reported to be susceptible to some bacillus thuringiensis (bt) subspecies, no research has been conduced to evaluate the possible non-target effect of genetically modified (gm) bt crops on termites. in this study, plant tissues of three commercial planted bt maize (yieldgard* corn borer, genuity* vt triple protm and genuity* smartstaxtm) and two non-bt maize hybrids were provided to formosan subterranean termite, coptotermes formosanus, as food. five food sources including wood blocks and filter paper treated with maize leaf extract as well as leaves, stalks, and roots of maize were tested in the laboratory. the experiment was maintained for two weeks and the survival rate of termites, food consumption, and tunneling behavior were recorded. the results revealed no significant differences in survival rate, food consumption and length of tunnels between termites feeding on bt and non-bt maize planting materials, indicating that bt proteins expressed in the three bt maize products did not negatively affect c. formosanus. however, compared to wood block and filter paper treatments, termites feeding on maize tissues showed different consumption pattern and tunneling behavior. our study also suggests that maize stalk is a good candidate for termite bait matrices. key words: coptotermes formosanus, gm bt maize, non-target effect, consumption behavior, tunneling 1department of entomolog y, louisiana state university agricultural center, baton rouge, la 70803, usa 2department of applied mathematics and statistics, state university of new york at stony brook, new york 11794, usa * corresponding author, e-mail: grhenderson@agcenter.lsu.edu 1336 sociobiolog y vol. 59, no. 4, 2012 introduction bacillus thuringiensis (bt) is a group of gram-positive, spore forming bacteria that have great agricultural importance. since it was first isolated in 1901 by japanese biologist, ishiwata shigetane, a considerable number of studies have been conducted for its application as a biological pesticide (schnepf et al. 1998, roh et al. 2007). based on their flagellar antigens, phage susceptibility and plasmid profiles, approximately 100 bt subspecies have been identified and have found targets to a variety of insect hosts and nematodes (mohan et al. 2009, sanahuja et al. 2011). although not considered as typical pathogens of termites, some bt subspecies were reported to be toxic to some termite species, such as reticulitermes flavipes, nasutitermes ehrhardti, heterotermes indicola, microcerotermes championi, bifiditermes beesoni, microcerotermes beesoni and microtermes obesi (smythe and coppel 1965, khan 1981, castilhos-fortes et al. 2002, khan et al. 1977, 1978, 1985, 2004, singha et al. 2010). the pathogenic mechanism of bt on its target insects depends on two types of crystal proteins, cry and cyt toxin (also known as δ-endotoxins), and other toxins such as vips (vegetative insecticidal proteins) (frankenhuyzen 2009, hernández-rodríguez et al. 2009, bravo et al. 2011). with the advance of modern molecular technolog y, some cry and vip genes have been cloned and transformed to maize and cotton against a variety of pests (koziel et al. 1993, vincent 2010). presently, gm bt crops are critically important for modern agriculture. by 2011, bt crops (maize and cotton) were planted on 65 million hectares worldwide ( james 2011). our interest in the relationship between gm bt maize and termites was based on two academic facts: (1) very few studies have focused on the nontarget effect of gm bt maize on termites; and (2) maize stalks and other agricultural waste have already been used as a termite bait matrix in china for years (zhang et al. 1995, li et al. 2001, henderson 2008, zhang et al. 2009). in this study, formosan subterranean termite, coptotermes formosanus, an important economic pest in the southern united states, was fed with materials of three bt maize hybrids, yieldgard® corn borer (bt yg), genuity* vt triple protm (bt vt 3pro) and genuity* smartstaxtm (bt smt) , and two non-bt maize hybrids (nbt-1 and nbt-2). yieldgard* corn borer maize expressing the cry1ab protein was the most commonly planted bt maize for 1337 cai wang, c. et al.— survival rate, food consumption, and tunneling controlling stalk borers in the world before 2010. genuity* vt triple protm and smartstaxtm are two new bt maize technologies which contain multiple bt genes. the objectives of this study were to determine if c. formosanus was susceptible to toxins expressed by bt maize hybrids and to study the consumption behavior of c. formosanus feeding on maize materials. materials and methods termites c. formosanus was collected from brechtel park, new orleans on march 17, 2011, using milk crate traps as described in gautam and henderson (2011a). termites were maintained in trash cans (140l) with wet wood under high relative humidity conditions for less than one month. bt and non-bt maize hybrids. plants of three bt maize and two non-bt maize (monsanto company, st. louis. mo) were collected from a green-house located at louisiana state university agricultural center, in baton rouge, la. the three bt corn hybrids were dkc 67-23 rr2 containing yieldgard* corn borer trait, dkc 67-88 expressing genuity* vt triple protm traits and dkc 61-21 possessing genuity* smartstaxtm traits. yieldgard* corn borer contains a single bt gene, cry1ab, which was the most commonly planted bt maize for controlling stalk borers worldwide before 2010. genuity® vt triple protm is a stacked/pyramided bt corn that expresses three bt genes including cry1a.105 and cry2ab2 for controlling above-ground lepidopteran species and cry3bb1 for managing underground rootworms, diabrotica spp. genuity® smartstaxtm corn contains all bt genes expressed in genuity® vt triple protm plus cry1f targeting lepidopteran species and cry34ab1/cry35ab1 targeting rootworms (monsanto, 2012). genuity® vt triple protm and smartstaxtm maize were among the first stacked/ pyramided bt maize technologies that were commercialized in 2010 in the united states and canada. the two non-bt maize hybrids were dkc 61-22 and dkc 67-86 . the hybrid, dkc 61-22, was genetically closely related to the bt maize hybrid, dkc 61-21, while dkc 67-86 was closely related to the bt corn hybrids dkc 67-23 and dkc 67-88. expression of cry proteins in the corn hybrids was confirmed using an elisa-based technique (envirologix, quantiplatetm kits, portland, me). leaves, stalks and roots of each maize hybrid were put in separate ziploc* bags with a small amount 1338 sociobiolog y vol. 59, no. 4, 2012 of water and stored in 4 °c for less than one week. before use, the plant tissues were carefully washed with distilled water to clean the pollen and dust off the surface. experimental design. a two-way completely random design was used in the study with corn hybrid and food source as the two main factors. the experiment contained five corn hybrids mentioned above. for each corn hybrid, tests were conducted in five different ways as food sources for the termite: (1) wood block containing maize leaf extract, (2) filter paper containing maize leaf extract, (3) maize leaf tissue, (4) maize stalks, and (5) maize root. in addition, wood block and filter paper treated with distilled water only were also included in the tests as blank controls. there were five replications in each treatment combination. therefore, a total of 27 treatment combinations and 135 experimental units were tested in this experiment. substrate and bioassay arena autoclaved (121°c 45min) sand was weighed and mixed uniformly with distilled water in a ziploc* bag to make the 15% moisture sand by weight. thirty grams wet sand was placed in each petri dish (100×15mm) and pressed by bottom side of a smaller petri dish (60×15mm) to form a thin layer as the substrate for termites. wood block and filter paper wood blocks (1.9×1.9×0.9cm southern yellow pine) were autoclaved (121°c, 15min) and dried in an oven dryer (45 °c, 1d). dry weight of wood blocks and filter paper (4.25 cm diameter, whatman*) was recorded. maize leaves (25 g ) were cut into small pieces and extracted with 20 ml distilled water. approximately 10 ml of extract was collected from each hybrid. one ml of extract was added to the surface of wood block and filter paper and air-dried at room temperature. wood blocks and filter paper treated with 1 ml of distilled water only were used as blank control. maize leaves, stalks and roots maize stalks were straight-cut to check infection of stalk bores, which could make tunnels inside the stalk. leaves and non-infected stalks were cross-cut into small segments (4-5 cm). roots of maize (5 g ) were weighted and cut 1339 cai wang, c. et al.— survival rate, food consumption, and tunneling into 3 cm segments and mixed with 30 g sand containing 15% moisture in each replicate of the root treatment. bioassays and data recording based on the colony structure, 50 termite workers and 2 nymphs (wing budded individuals) were introduced into each experimental unit. the bioassays were maintained at room temperature (23±1 ° c) for two weeks. dead termites were removed daily and distilled water was added when necessary. after two weeks, live termites of each experimental unit were counted. wood blocks, filter paper, leaves and stalks were carefully brushed clean of sand. the bottom side was scanned to observe the consumption areas and patterns. after completely drying in an oven dryer (45 °c, 1d), the weight of wood blocks and filter paper were recorded to determine consumption. because maize leaf, stalk and root used in this test were fresh, the consumption calculated by difference of dry weight was not available. the bottom side of each petri dish was scanned to record the tunneling behavior and length of tunnels. statistical analysis. the assumptions of independent and normal distribution were verified by the diagnostics plots in sas 9.3 (sas institute, 2011), a two-way analysis of variance (anova) was performed using proc mixed procedure to compare the survival rate, consumption, and tunnel length of termites feeding on different maize hybrids and different food sources. post anova comparisons were performed using tukey’s hsd test. significant levels were determined at α=0.05. results survival rate the mean survival rates of the two controls at 14 d were 89.6% (wood block) and 85.4% (filter paper) (table 1). the main effect of food source on survival rate of termites at 14 d was significant (f = 24.57; df = 4,99; p < 0.0001), but the effect of maize hybrid and the interaction of food source and maize hybrid was not significant (f = 1.41; df = 4,99; p = 0.2348 for maize hybrid and f = 0.91; df = 16,99; p = 0.5652 for interaction). an average of 36.9% of termites feeding on maize stalks survived after 14 days across the five maize hybrids (both bt and non-bt), which was significantly less (p < 0.05) than that observed for any other food source. survival rate of termites feeding on 1340 sociobiolog y vol. 59, no. 4, 2012 fig.1 (a) mean survival rate nd (b) mean tunnel length of coptotermes formosanus on diferemnt food sources across bt and nd non-bt maize hybrids. mean v alues followed by the same letter are notsignkficantly different (p>0.05) 1341 cai wang, c. et al.— survival rate, food consumption, and tunneling ta bl e 1: m ea n su rv iv al ra te (± se ), fo od c on su m pt io n (± se ), an d tu nn el le ng th (± se ) o f c . f or m os an us fe ed in g on d iff er en t f oo d so ur ce s c on ta in in g b t a nd n on -b t m ai ze p la nt ti ss ue o r e xt ra ct . nb t1 nb t2 b t y g b t v t 3 pr o b t s m t b la nk c on tr ol su rv iv al ra te (% ) w oo d bl oc k 78 .8 ± 3. 7 83 .1 ± 3. 5 76 .6 ± 7. 9 83 .1 ± 4. 7 84 .6 ± 3. 7 89 .6 ± 2. 1 fil te r p ap er 73 .8 ± 2. 9 81 .5 ± 4. 1 76 .1 ± 8. 9 80 .0 ± 2. 3 73 .8 ± 5. 3 85 .4 ± 2. 4 le af 71 .2 ± 5. 5 72 .7 ± 5. 2 72 .7 ± 5. 2 46 .9 ± 8. 2 58 .1 ± 10 .7 st al k 43 .5 ± 13 .1 52 .3 ± 9. 6 32 .3 ± 13 .8 27 .7 ± 10 .4 28 .8 ± 8. 8 ro ot 61 .8 ± 7. 9 59 .5 ± 14 .4 65 .1 ± 7. 8 67 .3 ± 4. 7 49 .8 ± 10 .2 c o n su m p ti o n (g ) w oo d bl oc k 0. 09 6± 0. 01 3 0. 09 2± 0. 01 1 0. 09 8± 0. 01 1 0. 10 0± 0. 00 3 0. 10 2± 0. 00 6 0. 12 0± 0. 01 3 fil te r p ap er 0. 04 6± 0. 00 9 0. 06 6± 0. 00 8 0. 05 8± 0. 00 9 0. 04 0± 0. 00 4 0. 04 6± 0. 00 7 0. 04 8± 0. 00 6 tu nn el le ng th (m m ) w oo d bl oc k 25 3. 1± 13 .7 22 6. 8± 36 .2 24 1. 2± 25 .7 24 1. 2± 7. 2 21 9. 3± 11 .3 25 8. 8± 29 .2 fil te r p ap er 23 6. 2± 23 .4 29 0. 1± 13 .2 27 8. 4± 18 .6 23 9. 5± 8. 5 26 1. 6± 24 .3 29 2. 1± 15 .8 le af 18 1. 6± 26 .3 21 1. 6± 18 .5 15 8. 8± 21 .4 14 5. 3± 30 .4 14 3. 6± 37 .0 st al k 12 1. 6± 17 .1 78 .4 ± 20 .5 12 0. 2± 17 .2 12 6. 5± 9. 4 13 8. 4± 17 .1 ro ot 28 7. 8± 26 .6 33 1. 4± 9. 9 27 7. 0± 24 .1 28 0. 8± 16 .5 31 6. 0± 20 .5 1342 sociobiolog y vol. 59, no. 4, 2012 wood bock was 81.2%, which was significantly greater (p < 0.05) than that observed for those feeding on maize leaf tissue (64.3%) or root (60.7%), but it was not significantly different compared to the survivorship (77.1%) of the fig. 2. consumption pattern of coptotermes formosanus feeding onnon-bt (on nonbt-1, and bt maize leaves. 1343 cai wang, c. et al.— survival rate, food consumption, and tunneling termites on filter paper. a difference was also significant (p < 0.05) between the filter paper and maize root, but not significant between filter paper and maize leaf tissue or between leaf tissue and root (fig. 1a). amount of food consumption. the mean consumption of the two controls at 14 d was 0.120 g (wood block) and 0.048 g (filter paper) (table 1). as observed in the survival rate, the main effect of food source on food consumption after 14 days was significant (f = 69.90; df = 1,40; p < 0.0001), but the effect of maize hybrid and the interaction of food source and maize hybrid was not significant (f = 0.42; fig. 3.(a) holes made by c, formosanus pn the srface of split stalks at day 2-3 and the consumption pattern of termites feeding of (b) n-bt-1, (c) nbt-2, (d) btyg, (e) btvt, (e) btvtproaand (f ) btsmt maize stalks. 1344 sociobiolog y vol. 59, no. 4, 2012 df = 4,40; p = 0.7908 for maize hybrid and f = 1.21; df = 4,40; p= 0.3204 for interaction). an average of 0.098 g wood block was consumed after 14 days across the five maize hybrids, which was significantly greater than that (0.051 g ) recorded for the termites feeding on filter paper. tunnel length the mean tunnel length of the two controls after 14 days was 258.8 mm (wood block) and 292.1 mm (filter paper) (table 1). similarly as observed in the termite survival and food consumption, the main effect of food source on survival rate of termites at 14 d was significant (f = 58.98; df = 4,100; p < 0.0001), but the effect of maize hybrid and the interaction of food source and maize hybrid was not significant (f = 0.62; df = 4,100; p = 0.6511for fig. 4. tunneling pattern of c, formosanus in the bottom sides of petrie dishes containing food sources withbt and non-bt plant tissue extracts. 1345 cai wang, c. et al.— survival rate, food consumption, and tunneling maize hybrid and f = 1.30; df = 16,100; p = 0.2092 for interaction). the length of tunnels among different food sources from the highest to the lowest was: root (298.6 mm) > filter paper (261.2 mm) = wood block (236.3 mm) > leaf (168.2 mm) > stalk (117.0 mm) (fig. 1b). consumption pattern and tunneling behavior of termites feeding on maize materials in the leaf treatments, both the daily observations and scanned pictures of leaf tissue (fig. 2) showed that termites prefer to eat primarily the vein. a tunnel inside the vein was regularly observed. observations also revealed that termites prefer to stay on the surface of sand and leaf tissue. in the stalk treatments, termites made tunnels inside the stalks (fig. 3b-f ). within the second or third day after release of termites, 1 to 3 holes were made on the surface of split stalks by termites (fig. 3a). observations and scanned pictures also showed that termites stay inside the split stalks, rather than making tunnels in the sand, resulting in fewest tunnels in sand substrate when compared to other food source treatments (fig. 4). in the root treatments, the termites consumed a large quantity of roots and broke them down into small pieces and pellets. extensive tunneling was found in the root treatments (fig. 4). discussion despite their great value in modern agriculture, non-target effects of gm bt crops have been of major concern. meta-analysis showed that, by 2008, more than 360 original papers focusing on the non-target effect of gm bt crops had been published (naranjo 2009). however, among those papers, few studies related to termite species were included. in nature, termites could interact with gm bt crops in various ways. for example, more than 10 termite species, such as ancistrotermes latinosus, macrotermes falciger, pseudacanthotermes spiniger, cornitermes cumulans, procornitermes triacifer, ancistrotermes latinosus, attack maize directly; some of them even cause 20 to 50% loss in corn yield (mill 1992, nkunika 1994, rouland-lefèvre 2011). in addition, bt toxins produced by gm bt crops can be released into soil by residue decomposition and root exudates (tapp and stttzky 1998, muchaonyerwa and waladde 2007, saxena 2010, helassa et al. 2011, das and chaudhary 2011). subterranean termite species such as c. formosanus are likely to be exposed to 1346 sociobiolog y vol. 59, no. 4, 2012 bt toxins remaining in soil (muchaonyerwa and waladde 2007). our results suggest that three gm bt maize involved in our study have no effect on c. formosanus. husseneder and grace (2005) developed a method to deliver foreign genes to termite colonies through genetically modified gut bacteria, which indicates a potential application of bt toxin(s) in termite control. however, despite studies on susceptibility of termites to bt subspecies, no termite-targeted toxin have been identified. our study showed that seven bt toxins expressed in three gm bt crops, including cry1ab, cry1a.105, cry2ab2, cry3bb1, cry1f, cry34ab1 and cry35ab1, do not negatively affect c. formosanus. this result will provide valuable information for the future screening work of termite-sensitive bt toxins. significant difference in survival rate of termites was found among different food sources (fig. 1a). the lowest survival rate observed in termites feeding on maize stalk could be caused by fungi growing on the surface of stalks observed from day 5 of the experiment. gautam and henderson (2011b) showed that, in laboratory conditions, attack of pathogenic fungi may lead to high mortality of termites. however, in nature, various strategies are used by termites to control fungi. for example, chouvenc and su (2012) reported that c. formosanus avoid the entomopathogenic fungus metarhizium anisopliae by employing several behavioral patterns. some anti-fungal chemicals associated with termites also inhibit the growth of fungi in natural conditions (chen et al. 1998, bulmer and crozier 2004, rosengaus et al. 2007). although c. formosanus is not considered an agricultural pest, some studies showed that they consume herbaceous crops such as sugarcane and bamboo (dai and luo 1980, su and scheffrahn 1986, chen and henderson 1996, hapukotuwa 2011). chen and henderson (1996) stated that the feeding preference of c. formosanus for sugarcane may be caused by glutamic acid and aspartic acid in sugarcane juice. li et al. (2000) reported that sugarcane powders were significantly preferred by c. formosanus over pine wood powders or starch. our study reveals that, leaves, stalks and roots of maize also can be alternative food sources for c. formosanus. moreover, termites showed special consumption and tunneling behaviors when feeding on maize tissues. one possible application of this maize consumption behavior is to develop a stalk bait for use against c. formosanus. in china, maize stalks have already 1347 cai wang, c. et al.— survival rate, food consumption, and tunneling been used as a termite bait matrix to control subterranean termites such as reticulitermes chinensis (zhang et al 1995, zhang et al. 2009). compared to traditional bait matrices such as pinewood and cardboard, stalk bait shows some obvious advantages. firstly, as an agricultural waste, maize stalk is a quite abundant resource for bait production, thus reducing the cost and the over utilization of forestry resources. moreover, since termites made tunnels inside the stalks, more contact area can be attained between termites and the stalk bait, which may enhance toxicant contact and transfer. acknowledgmments we thank david wangila for providing help in collecting maize plants. dr. yunlong yang provided valuable suggestions in experimental design. dr. binghao luo made editorial comments to the last draft, which is much appreciated. this publication was approved by the director of the louisiana state university agricultural center as 2012-234-7442. references bravo, a., s. likitvivatanavong, s. s. gill, m. soberón. 2011. bacillus thuringiensis: a story of a successful bioinsecticide. insect biochem. molec. biol. 41: 423-431. bulmer, m. s., and r. h. crozier. 2004. duplication and diversifying selection among termite antifungal peptides. mol. biol. evol. 21: 2256-2264. castilhos-fortes, r., t. s. matsumura, e. diehl, and l. m. fiuza. 2002. susceptibility of nasutitermes ehrhardti (isoptera: termitidae) to bacillus 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issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.5141sociobiology 67(3): 425-432 (september, 2020) introduction coptotermes wasmann (isoptera: rhinotermitidae), is a genus of subterranean termites that currently has 21 validated species (chouvenc et al., 2016), of which 16 are classified as pests of economic importance in various parts of the world (krishna et al., 2013). while only two of these species, c. formosanus shiraki, and c. gestroi (wasmann) have demonstrated remarkable invasive abilities, most coptotermes species still have an economic impact in their native range. coptotermes have the ability to infest live trees, create large, abstract coptotermes testaceus (l.) (rhinotermitidae) is a subterranean termite species that causes damage in urban and agricultural areas in the neotropics. despite its economic importance, there are no studies on its basic biological aspects for laboratory management and the development of strategies for its control. the objective of the present study was to evaluate the relative humidity, temperature, substrate moisture and preference to different wood substrates for the best c. testaceus survival under laboratory conditions. for this, a range of eight relative humidity (from 9 to 100%), three temperatures (20, 25 and 30 °c), six substrates (pinus sp, cedrela odorata (l.), cocos nucifera (l.), eucalyptus urophylla (s. t. blake), haematoxylum campechianum (l.) and tabebuia rosea [bertol.] dc) and four substrate moistures, (0 to 60%) were tested. the results of this study indicated a significant effect of all factors on termite survival or termite preference. when tested independently, the highest survival percentage of c. testaceus was obtained with humidity of 100%, temperature of 20 °c, substrate moisture of 60% and the eucalyptus urophylla substrate, reaching 83.33% survival at 21 days of observation. with these preliminary assays on small termite groups, it is concluded that with the appropriate percentages of humidity, temperature and substrate and the interaction of these three factors, further research can be conducted using larger termite groups in biologically relevant conditions, in order to study various aspects of c. testaceus biology. sociobiology an international journal on social insects co pozo-santiago1, m pérez-de la cruz2, m torres-de la cruz2, a de la cruz-pérez2, s capello-garcía2, ma hernández-gallegos3, jr velázquez-martínez4 article history edited by og desouza, ufv, brazil thomas chouvenc, ufl, usa received 10 april 2020 initial acceptance 29 june 2020 final acceptance 17 august 2020 publication date 30 september 2020 keywords termite, subterranean, woods, agricultural, control, eucalyptus urophylla. corresponding author j rodolfo velázquez martínez carretera villahermosa-teapa, km 25+2 ranchería la huasteca 2 da seccion, 86298 villahermosa, tabasco, méxico. e-mail: jrodolfovelazquez@gmail.com populous colonies, with extensive foraging abilities, which can reach more than 100 m of underground galleries (greaves, 1962; king & spink, 1969; su & scheffrahn, 1988). coptotermes testaceus (l.) is the only endemic coptotermes species in the new world and is established throughout most of the neotropics (scheffrahn et al., 2015; chouvenc et al., 2016). this species has been found affecting plantations of forest importance such as rubber (hevea brasiliensis [willd. ex a.juss.] müll.arg.) (apolinário & martius, 2004; krishna et al., 2013) and eucalyptus (eucalyptus urophylla s. t. blake) (santos et al., 1990; amaral-castro, 2004); agricultural crops 1 facultad maya de estudios agropecuarios-universidad autónoma de chiapas. catazajá, chiapas, méxico 2 división académica de ciencias biológicas-universidad juárez autónoma de tabasco. villahermosa, tabasco, méxico 3 división académica multidisciplinaria de jalpa de méndez-universidad juárez autónoma de tabasco. jalpa de méndez, tabasco, méxico 4 división académica de ciencias agropecuarias-universidad juárez autónoma de tabasco.villahermosa, tabasco, méxico research article termites survival of coptotermes testaceus (isoptera: rhinotermitidae) to environmental conditions (relative humidity and temperature) and preference to different substrates co pozo-santiago et al. – survival of coptotermes testaceus (isoptera: rhinotermitidae) to environmental conditions and preference to different 426 such as cassava (manihot esculenta c.), cocoa (theobroma cacao l.) and sugar cane (saccharum officinarum l.) (krishna et al., 2013); and wooden constructions in urban areas (bandeira et al., 1989). in mexico, the c. testaceus population represents the northern most distribution of the species (light, 1933; sheffrahn et al., 2015) and was reported to have an agricultural impact, damaging roots and stems of crops of economic, social and cultural importance (lópez-vera et al., 2018; capetillo-concepción et al., 2019). due to the economic importance of c. testaceus as a pest, it is necessary to find strategies for its control. however, to date, the environmental requirements to keep populations alive for prolonged periods of c. testaceus in the laboratory that allow the establishment of bioassays to evaluate their control, have not been reported. on the other hand, there is a continuous search for friendly alternatives to the environment and human health, for termite control. these studies generally start with laboratory bioassays, for example, the use of target-specific chemicals (baits and termiticides). while a lot of research efforts have been focusing on botanicals (essential oil, seed, bark, leaf, fruit, root, wood, resin) and entomopathogens (fungi, bacteria and nematodes), none has resulted in practical or commercial application (verma et al., 2009; chouvenc et al., 2011). however, achieving the establishment of live subterranean termite colonies in the laboratory is logistically complicated, time consuming and challenging (chouvenc, 2018), due to factors such as temperature (fei & henderson, 2002; nakayama et al., 2004; gautam & henderson, 2011; wiltz, 2012), humidity (nakayama et al., 2004; wong & lee, 2010; gautam & henderson, 2011; wiltz, 2012) and the food source (smythe & carter, 1969; su & tamashiro, 1986). particularly, desiccation is determining factor for termite survival, so it is important that in laboratory survival studies, not only relative humidity is considered, but also the substrate moisture and food (hu et al., 2012; zukowski & su, 2017). in order to provide preliminary information of the basic biological requirements for the survival and maintenance of c. testaceus in laboratory conditions, the aim of this study was to evaluate the survival of c. testaceus at various levels of relative humidity, temperature, substrate moisture, and preference for different substrates, to obtain the appropriate conditions that allow establishing live termite colonies in the laboratory, to ultimately carry out bioassays to study the control of c. testaceus. materials and methods obtaining c. testaceus the specimens were collected in the botanical garden of the academic division of biological sciences of the universidad juárez autónoma de tabasco. traps (modified from tamashiro et al., 1973) built with a metal cylinder (18.5 cm high x 15.5 cm in diameter) with exposed ends and internal walls lined with wood were used, a roll of corrugated paper 10 cm in diameter was placed in the center. the trap was placed in the basal part of trees that had damage caused by c. testaceus, buried in the ground 20 cm deep with the top of the trap covered. the corrugated paper cylinder with termites was removed after five days and transferred to the laboratory. in this study only workers were used of a single colony (a total of 1740). c. testaceus survival at different percentages of relative humidity (rh) and temperatures the methodology for this section was proposed by zukowski and su (2017), with modifications. environmental chambers (ec) were conditioned using plastic containers with lid (24.7 × 17 × 6.4 cm), with a 2.7 cm diameter hole in the central part of the lid to introduce a digital hydrometer. the rh inside the ec was stabilized using various stabilizing materials (rhsm) in different amounts (table 1). to achieve a high rh, the water was placed in plastic containers (6 cm diameter x 3.6 cm height) and cotton, covering the bottom of the ec; the salts and the silica gel were placed in the plastic containers described above and for the lowest rh, the cacl2 was spread at the bottom of the ec. the rh in the ec was assessed for 15 days with a digital hygrometer (vwr; traceable ™). the temperature was stabilized at 20, 25 and 30 °c in an incubator (novatech, mod. dbo-200). the bioassay was carried out using five petri dishes in each stabilized ec, the petri dishes (60 mm x 15 mm) contained a filter paper disk (60 mm) to facilitate locomotion of the termites and as a source of food was pine sawdust (200 mg), 10 termite workers were placed in each box. the daily record of live termites was monitored, reporting the survival percentage. dead termites were removed daily. the bioassay required five repetitions with ten workers in each repetition for the eight rh at the three temperatures tested (a total of 1,200 workers). rhsm formule amount rh obtained (%)a water, in cotton h2o 200 ml 100 ± 0.25 water h2o 100 ml 83.25 ± 0.55 undiluted salt mg(no3)2 100 g 75.28 ± 0.77 saturated saline solution nacl 100 ml 64.88 ± 0.59 silica gel silica gel 100 g 61.12 ± 0.83 saturated saline solution mgcl2 50 ml 42.98 ± 2.43 undiluted salt cacl2 100 g 23.88 ± 1.55 undiluted salt cacl2 300 g 9.89 ± 0.11 amean (m) ± standard error (se). n = 18. table 1. rhsm and amounts used to obtain different rh. sociobiology 67(3): 425-432 (september, 2020) 427 c. testaceus preference towards different substrates (woods) sawdust from pine (pinus sp.), cedar (cedrela odorata l.), coconut fiber (cocos nucifera l.), eucalyptus (eucalyptus urophylla s. t. blake), blackwood (haematoxylum campechianum l.) and pink poui (tabebuia rosea [ bertol.] dc) were used with a homogeneous particle size (sieve no. 16, 1.13 mm spacing). they were dehydrated in a drying oven (felisa® brand) at 50 °c for 24 h and stored in a desiccator with dehydrator until use for bioassays. a device was designed to allow a high rh and for termites to choose between different substrates (fig 1). small environmental chambers (sec) were used, which consisted of 8 x 5.5 cm (diameter x height) circular plastic containers, which inside contained plastic vials of 4 x 2 cm, suspended inside each sec. cotton was placed at the bottom of each radial sec with water to obtain the rh of 100% and the substrates were placed in the vials (fig 1a). using 6 x 1 cm plastic tubes, six radial sec were connected to a central chamber. the device was placed in the incubator at 20 ºc and the rh and temperature were stabilized for 4 h. once the rh and temperature had stabilized, 30 termites were deposited in the central chamber and at 24 h the number of termites within each sec with substrate was recorded, the termites found in the connection tubes were also included in the counts. the aggregation of termites to substrates was considered as preference. the rh and temperature were monitored during the bioassay and the percentage of preference was reported. the bioassay required ten repetitions with 30 workers in each repetition (a total of 300 workers). survival of c. testaceus at different substrate moisture the most preferred substrate previously conditioned was used. in petri dishes (60 mm x 15 mm) 3 g of the substrate were placed and sterile distilled water was used to obtain substrate moistures (sh) of 0, 20, 40 and 60%.the formula used to obtain the desired sh (nmx-aa-16-1984) was as follows: h = g – g1 x 100 g where: h = % of humidity g = wet sample weight in g g1 = dry sample weight in g six petri dishes were placed in environmental chambers (used in the survival test at different rh and temperature) with the stabilized rh of 100 ± 0.25% (fig 2a). groups of ten termites were placed in each petri dish with the substrate at the required humidity (fig 2b). this bioassay was incubated at 20 °c in total darkness. the daily count of living termites was recorded, and the survival (percentage of live termites) was reported. the bioassay required six repetitions with ten workers in each repetition for the four sh evaluated (in total 240 workers). fig 1. device for high rh and preference of termites towards different substrates. a) radial small environmental chamber, with suspended vial; b) central chamber for release of termites with vial connected to six radial small environmental chambers; c) device with the six substrates for multiple choice; d) device with modified caps for hr monitoring. fig 2. a) petri dishes in environmental chambers with the stabilized rh of 100 ± 0.25%; b) group of ten termites placed in the petri dish with the substrate at the required humidity. experimental design and statistical analysis for all bioassays, a completely randomized simple design was used. the statistical analysis used for the termite survival test at different rhs and temperatures was an analysis of variance (anova) for an 8 by 3 factorial experimental design (8 rhs and 3 temperatures), the response variable was the percentage of survival. statistical analysis for preference towards different substrates was performed with a simple analysis of variance (anova) and the percentage of preference as the response variable. a comparison of means with fisher’s lsd with an α = 0.05 was performed for these two trials. finally, for the survival of termites towards different sh, a kaplan-meier logrank analysis and the comparison of holm sidak means with an α = 0.05 were used, where the co pozo-santiago et al. – survival of coptotermes testaceus (isoptera: rhinotermitidae) to environmental conditions and preference to different 428 highest median lethal time (lt50) was considered to select the best treatment. because the results of the first two trials were expressed as a percentage, it was necessary to transform the data to the square root of the arcsine prior to anova. the statistical package used for all analyzes was sigmaplot 12.0. results c. testaceus survival at various rh levels and temperatures table 2 shows the survival rate of c. testaceus 24 hours after being exposed to eight rh and three temperatures. the rh of 100% presents the highest percentage of survival at 24 hours of observation for the three temperatures evaluated (f = 109,313; df = 7; p <0.001). there was a greater survival of termites at a temperature of 20 °c (f = 192,213; df = 2; p < 0.001). on the other hand, the interaction between the two studied factors, in the same way, generated a significant effect (f = 7,885; df = 14; p < 0.001), being the combination of 20 °c and 100% rh the treatment that provided the highest termite survival, with 88.4 ± 0.07%. likewise, it was observed that as the rh decreases, the termite survival also does it gradually, while at higher temperatures, it’s a lower survival. c. testaceus preference for various substrates (woods) termites showed a statistically different response in their aggregation toward a substrate after 24 hours of observation (f = 4,630; df = 5; p < 0.001, fisher’s lsd α = 0.05). the most preferentially aggregated on eucalyptus, with 68.75%, followed by blackwood with 12.5% and pine with 10%. the substrates that obtained the lowest preferences were coconut fiber, pink poui and cedar, with 4.38, 4.37 and 0.0% respectively (fig 3). survival of c. testaceus at different substrate moisture (sh) the four shs showed a significant difference (statistic = 385,312; df = 3; p < 0.001) with respect to c. testaceus survival during the bioassay. the sh of 60% obtained the highest survival, a lt50 value was not recorded, because after 21 days of the bioassay the percentage of live termites was 83% (lt50 > 21 days). the sh of 40%, 20% and 0% obtained a lt50 of 21, 13 and 1 days, respectively (fig 4). rha h2o h2o mg(no3)2 nacl síl gel mgcl2 cacl2 cacl2 t°c (100 ± 0.3%) (83.25 ± 0.5%) (75.28 ± 0.8%) (64.88 ± 2.5%) (61.12 ± 3.5%) (42.98 ± 2.4%) (23.88 ± 0.4%) (9.89 ± 0.1%) 20 88.4 ± 0.07aa 71.8 ± 0.07ab 70.5 ± 0.07ab 69.8 ± 0.07ab 39.6 ± 0.07ac 31.5 ± 0.07acd 31.5 ± 0.07acd 20.7 ± 0.07ad 25 74.7 ± 0.06ba 59.5 ± 0.06ab 9.3 ± 0.06bc 9.3 ± 0.06bc 6.7 ± 0.06bc 0.0 ± 0.0bc 0.0 ± 0.0bc 0.0 ± 0.0bc 30 74.7 ± 0.05ba 23.2 ± 0.05bb 13.9 ± 0.06bbc 9.26 ± 0.05bcd 4.63 ± 0.05bcd 0.0 ± 0.0bd 0.0 ± 0.0bd 0.0 ± 0.0bd a different lowercase letters within a row or different uppercase letters within a column indicate significant differences between means. table 2. percentage of survival (m ± se) of coptotermes testaceus 24 hours after being exposed to eight rhs and three temperatures. n = 5. p re fe re nc e (% ) pine cedar eucalyptus f.coconut blackwood pink poui 0 20 40 60 80 100 treatments a cdbc b d n/a fig 3. percentage of preference of coptotermes testaceus workers 24 hours after being exposed to six substrates (woods), at an rh of 100% and a temperature of 20 °c, n = 10. different letters indicate significant differences between treatments. sociobiology 67(3): 425-432 (september, 2020) 429 discussion the results of this study showed that the highest survival (88.4%) of c. testaceus was at 100% rh and temperature of 20 °c. this shows that both rh and temperature are important for the survival of c. testaceus, but they are even more so when the interaction between these factors occurs. this agrees with that reported by gautam and henderson (2011) as well as wiltz (2012), who found the highest survival percentages for c. formosanus at low temperatures (20 and 10 °c) and high rh (98 and 99%) respectively. the results of this study show an effect of rh and temperature, however, these two factors alone cannot maintain the in vitro survival for long periods of time (24 h for this study), which is necessary for studies focusing on control aspects of this pest. lenz (2005) points to substrate as another factor that must be considered to obtain successful results in bioassays. the substrate can be used for the construction of their galleries, food or as indicated by hu et al. (2012) and zukowski and su (2017), a resource for obtaining moisture. that is why, in this study, the preference that c. testaceus had towards six different woods that could function as a substrate was also evaluated. the results obtained in this work indicated that eucalyptus was the substrate with the greatest choice. this coincides with studies carried out in agricultural systems where it was reported that c. testaceus was the main responsible for infestations in eucalyptus plantations (amaral et al., 2004). this could be due to the fact that, as zabel and morrel (1992) points out, the different kinds of wood have the presence of extractable (chemicals). the part of the wood in which the extractables are produced is what determines if they play a role as attractants or repellents, i.e. in the sapwood, there is a higher concentration of starch and carbohydrates which makes this part of the wood more palatable to the biological agents that attack it. on the other hand, the heartwood has a series of components that make it less preferred to these agents (kollmann, 1959). this leads us to think that eucalyptus was the most attractive substrate due to its chemical composition, in addition to cellulose, which is the main food source of xylophagous insects, such as termites (bignell & eggleton, 2000; ramírez & lanfranco, 2001; shimada & maekawa, 2010). this coincides with that reported by scheffrahn (1991), who likewise attributes the preference of termites to this type of (extractable) substances. finally, and once the most preferred substrate was identified, different humidity levels were evaluated in this substrate. mcmanamy et al. (2008) pointed out that the humidity of the substrate is an important factor for the prolonged survival of species of subterranean termites, this due to the fact that after subjecting reticulitermes flavipes (kollar) (isoptera: rhinotermitidae) to ideal conditions of temperature and rh, but exposed to low moisture wood, time (days) 0 5 10 15 20 25 su rv iv al (% ) 0% 20% 40% 60% a b c d 100 80 60 40 20 0 fig 4. percentage of survival of coptotermes testaceus workers after being exposed to four substrate moistures (0%, 20%, 40% and 60%) at 20 °c and 100% rh, n = 6. different letters on substrate moisture indicate significant differences (holm sidak, α = 0.05). co pozo-santiago et al. – survival of coptotermes testaceus (isoptera: rhinotermitidae) to environmental conditions and preference to different 430 the termites did not survive. our results indicated that the humidity of the substrate of 60% (the highest tested), was the one with which the longest survival time of the termite (21 days) was reached, reaching 83.33% of live insects at the end of this time. this coincides with that reported by zukowski and su (2017) for the termite species c. formosanus, which, after three weeks of observation (21 days), reached 90% survival with wet food, but when the food source provided was dry, survival was 0%. these results lead us to agree with what was pointed out by gautam and henderson (2014), who pointed out that subterranean termite species are extremely susceptible to desiccation, therefore, they require not only high rh, but also other sources of humidity for its greatest survival. at a global level, although studies of this type were conducted on pest species of subterranean termites (rhinotermitidae), the current study provides preliminary information on optimal experimental and rearing conditions in the laboratory for c. testaceus. as previously shown, the importance of the biological relevancy of a bioassay when testing control method against subterranean termites in laboratory conditions is critical (su, 2005; chouvenc, 2018), to study the applicability of such approach in a field situation. the current study therefore provides initial guidelines for the manipulation of c. testaceus in the laboratory, thus being able to carry out future studies aimed at testing control approach for this species. acknowledgments cordial thanks to the editor and referees for their reviews and comments on our manuscript. to the universidad juárez autónoma de tabasco, for facilitating access to the laboratories, materials, and equipment of the academic division of biological sciences (dacbiol), the academic division of agricultural sciences (daca), the academic division of multidisciplinary of jalpa de mendez (damjm).to the national council of science and technology (conacyt) of mexico for the scholarship awarded to carry out this study. contribution of the authors co pozo-santiago, conception, design, data collection, analysis, interpretation of results and document writing; m pérez-de la cruz, conception, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; jr velázquezmartínez, conception, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; m torres-de la cruz, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; a de la cruz-pérez, design, critical review for important intellectual content, and final approval of the version to be published; s capello-garcía, design, critical review for important intellectual content, and final approval of the version to be published; ma hernández-gallegos, critical review for important intellectual content, and final approval of the version to be published. references amaral-castro, n.r., zanetti, r., moraes, j.c., zanuncio, j.c., freitas, g.d. & santos, m.s. 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(2017). survival of termites (isoptera) exposed to various levels of relative humidity (rh) and water availability, and their rh preferences. florida entomologist, 100: 532-539. doi: 10.1653/024.100.0307. 1167 management of social wasp colonies in eucalyptus plantations (hymenoptera:vespidae) by thiago elisei, cleber ribeiro junior, aluísio jose fernandes junior, juliana vaz e nunes, andré rodrigues de souza & fabio prezoto* abstract polistes paper wasps have shown potential for the biological control of agricultural plagues. twenty post-emergent colonies of p. versicolor were transferred from human constructions to artificial shelters installed on a eucalyptus plantation. we obtained 85% success in colony transference, as determined by the permanence of individuals in the colony after the colony transference. transferred colonies stayed active at the plantation for 64.05 ± 38.43 (8-123) days. we demonstrated that post-emergent colonies of p. versicolor can be easily transferred to areas where they can act as biological control agents. key words: polistes versicolor, artificial shelters, colony transference. introduction social wasps are known to prey upon caterpillars (de souza et al. 2008; bichara et al. 2009; da rocha et al. 2009; picanço et al. 2010). in fact, the presence of polistes paper wasps in different cultures is associated with decreased damage from lepidopterous pests of cotton (kirkton 1970), tobacco (lawson et al. 1961), cabbage (gould & jeanne 1984), coffee (gravena 1983) and maize (prezoto & machado 1999a). despite these previous works demonstrating the ability of social wasps to regulate pest populations in agroecosystems, we are not able to make general rules for social wasp management in biological control programs (bcps). this is because wasps vary greatly in their lifecyles, behavior and ecolog y (raveret-richter 2000), which reflects on the specific conditions of colony management. for example, ropalidia marginata accepts colony transference to artificial shelters, while a closely related speprograma de pós-graduação em ciências biológicas comportamento e biologia animal, universidade federal de juiz de fora, brazil. * correspondence author: fabio.prezoto@ufjf.edu.br 1168 sociobiolog y vol. 59, no. 4, 2012 cies, ropalidia cyathiformys, does not (gadagkar 2001). another aspect that illustrates this situation is the social wasps' homing ability, which predicts the adequate distribution of colonies in the culture. according to species, this ability varies from 20 to 650 meters from the colony (gobbi 1978; hibino 1981; giannotti 1994; santos et al. 2000; prezoto & gobbi 2005). so, the use of social wasps in bcps requires specific protocols for each species. during bcps with social wasps, colonies are managed according to the culture of interest in order to increase populations of pest enemies (raveretrichter 2000). so, the development of social wasp colony management techniques is an essential step for their use in bcps. for these purposes, a variety of artificial shelters composed by wood or plastic have been developed (kirkton 1970; gillaspy 1979; shang-shiu 1976; turillazzi 1980; prezoto & machado 1999b). another required step is to know how much time colonies remain active after transference. this allows inferences to be made about the necessity of colony reintroduction in the culture. despite its relevance, there is no information about this last topic. colonies of polistes versicolor (olivier, 1791) can be initiated by one or a few inseminated females, who produce one or more generations of workers, followed by males and reproductive females. these reproductive forms leave the original colonies (end of colony cycle), copulate and then, females start a new colony (gobbi 1977). in general, colonies are less than 100 individuals (west-eberhard 1969). nests are fixed on the substrate by a single peduncle and larger nests can reach around 11x10 cm (marques & carvalho 1993). these characteristics facilitate colony transference and allow the possibility of colony self-maintenance in the culture. previous studies have already investigated some essential aspects that will make possible the use of p. versicolor in bcps, such as the finding that (i) this species is widely found in south america (richards 1971), (ii) foragers prey upon caterpillars, when foraging in monocultures (elisei et al. 2010), (iii) they are able to forage 200 meters around the colony (gobbi 1978) and (iv) they are naturally found in eucalyptus monocultures (de souza et al. 2011). so, in this paper, we attempt to advance on two additional aspects. first, we describe a new technique of colony management and then we verify how much time colonies remain active after the transference to a monoculture. 1169 elisei, t. et al. — management of social wasp colonies in eucalyptus plantations materials and methods period of study and study area between january and june of 2007, we transferred 20 early post-emergent colonies of p. versicolor from human constructions to artificial shelters in a eucalyptus plantation, both of them located in the city of juiz de fora, minas gerais state, southwestern brazil (21º 47’s 43º 38’w, elevation 730 m). human constructions and the eucalyptus culture was around 25 km apart from each other. the eucalyptus plantation comprised approximately three ha of early regrowth eucalyptus urograndis (eucalyptus urophylla x eucalyptus grandis), nearly four years old following the last felling. eucalyptus trees varied in height from 2-10 m, interspersed with other plant species. no pesticide treatments were applied in the plantation during the study period. management technique colonies were collected at the end of the afternoon (5-8 p.m.), a period in which most of the wasps are in the colony (prezoto & machado 1999b). we collected colonies by wrapping them up with a transparent plastic sack (sack dimensions: 50x80 cm; 0.05 cm of thickness). to separate nest and substrate, we used a sharp tool. after colony collection, we separated wasps from the nest. to do this, we turned the sack’s open side to the ground. wasps show a very accurate positive phototropism (authors' personal observation), moving immediately to the higher and brightest part of the sack. as a result, they relocated close to the sack’s open side. at this time, with the use of an adhesive ribbon, wasps were isolated in a particular area allowing the nest removal from the sack. nests were attached to the internal central superior part of the shelter by taking advantage of the peduncle as a collage point (super-bonder®). the shelter consisted of a white plastic box, opened only in the inferior part, measuring 13x17x11 cm (fig. 1). the group shelter + nest were fixed to the eucalyptus plants with nails, around 1.80 meters from the ground. after the fixation, this group (shelter + nest) was wrapped by the plastic sack containing the wasps, allowing wasps to acclimate to the nests. this new group (shelter + nest + wasps) stayed wrapped by the plastic sack until the following morning, between 8 p.m.-6 a.m., and then, we removed the sack and individuals could forage freely. 1170 sociobiolog y vol. 59, no. 4, 2012 colony transference success and activity time of transferred colonies we conducted a daily census for 123 days in the monoculture to check transferred colonies. the success of the transference technique was considered by the individuals' presence in the colony at least five days after transference. from this time on, we verified the time in which colonies were actives, as determined by individuals' presence in the colonies on the following days after the colony success evaluation period. when no individual was found in the nest for three consecutive days, we considered this the end of the colony activity period. data analysis the relative frequency of active colonies after the transference to a eucalyptus plantation was submitted to a linear regression analysis with a 5% significance level and 95% confidence interval. results successful p. versicolor colonies in eucalyptus monocultures were obtained in 17 out of 20 colonies. three out of 20 transferred colonies didn't accept transference, being abandoned immediately after the transference processes (fewer than five days). the 17 remaining colonies stayed active for 64.05 ± 38.43 (9-123) days. during this period, three colonies were abandoned after a storm. in 10 colonies, activity lasted until the end of the colonial cycle and in four colonies activity was observed during the entire period of study, lasting more than 123 days. we identified three periods (fig. 2): the first (colony success evaluation period) was characterized by some failure in colony transference. in the secfig. 1. a colony of polistes versicolor in an artificial shelter. 1171 elisei, t. et al. — management of social wasp colonies in eucalyptus plantations ond period (stabilized period), colonies tended to stay active in the culture. in the last one (decline period) some colonies were abandoned after storm sor due to the end of the colony cycle. discussion the success of the p. versicolor colony management technique in this study (85%) was greater than that observed for previously utilized methodologies for the same species (butignol 1992), which had a success rate of 60% using wooden shelters. so, our technique enables a cheaper and more practical method of polistes colonies management. wasps are opportunistic insects who feed on abundant food resources (raveret-richter 2000), and are easily found in agrosystems (auad et al. 2010; de souza et al. 2011) and urban areas (lima et al. 2000, alvarenga et al. 2010). elisei et al. (2010), studying the foraging activity of p. versicolor in a eucalyptus plantation, verified that wasps foraged for nectar, water, vegetal fiber and prey. thus, the colonies’ survival after transfer to the culture was expected. however, we observed a decrease in the proportion of active colonies after transference. according to these dynamics of p. versicolor colony durability in eucalyptus culture, we can make some recommendations related to colony management. in the first period (1-5 days after transference) colonies fig. 2. relative frequency of active colonies of the social wasp polistes versicolor after transference to a eucalyptus plantation in minas gerais state, brazil (linear regression: r = 0.69; p = 0.04, n = 20 colonies). 1172 sociobiolog y vol. 59, no. 4, 2012 should be checked daily in order to detect abandonments. if so, a new colony can be established. in the second period (6-80 days after transference), visits could be less frequent because colonies are less likely to be abandoned. since storms were found to cause colony abandonments, it seems to be reasonable to propose personal inspections in the colonies after each storm, even in the stabilized period. visits should be intensified in the last period (after 80 days) because colonies can be abandoned due to the end of the colony cycle. these procedures could keep wasps in the culture continuously, maximizing their control of plagues and saving potential costs with pesticides. acknowledgments we would like to thank the brazilian agencies coordenação de aperfeiçoamento de pessoal de nivel superior, conselho nacional de desenvolvimento científico e tecnológico and fundação de amparo à pesquisa do estado de minas gerais for financial support. references alvarenga r.b., m.m.castro, h.h. santos-prezoto & f. prezoto 2010. nesting of social wasps (hymenoptera, vespidae) in urban gardens in southeastern brazil. sociobiolog y 55: 445-452. auad, a.m., c.a. carvalho, m.a. clemente & f. prezoto 2010. diversity 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j.f.s. lopes & f. prezoto 2008. foraging behavior and dominance hierarchy in colonies of the neotropical social wasp polistes ferreri saussure, 1853 (hymenoptera, vespidae) in different stages of development. sociobiolog y 52:293-303 1173 elisei, t. et al. — management of social wasp colonies in eucalyptus plantations elisei, t., j.v. nunes, c. ribeiro junior, a.j.f. junior & f. prezoto 2010. uso da vespa social polistes versicolor no controle de desfolhadores de eucalipto. pesq. agropec. bras. 45:958-964. gadagkar, r. 2001. the social biolog y of ropalidia marginata: toward understanding the evolution of eusociality. harvard university press, cambridge, massachusetts. giannotti, e. 1994. notes on the biology of polistes simillimus zikán (hymenoptera:vespidae). bioikos 8(1/2):201-206. gillaspy, j.e. 1979. management of polistes wasps for caterpillar predation. sowthwestern entomol. 4:334-352. gobbi, n. 1977. ecologia de polistes versicolor (hymenoptera: vespidae). phd. thesis. 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artificiais e sua manutenção em uma cultura de zea mays l.. rev. bras. entomol. 43:239-241. raveret-richter, m. 2000. social wasp (hymenoptera: vespidae) foraging behavior. annual review of entomolog y 45: 121-150. richards, o.w. 1971. the biolog y of the social wasps (hymenoptera: vespidae). biol. rev. 46:483-528. santos, g.m.m., v.p.g. santana-reis, j.j. resende, p.d. marco & c.c. bichara-filho 2000. flying capacity of swarm-founding wasp polybia occidentalis occidentalis oliver, 1791 (hymenoptera, vespidae). revista brasileira de zoociências (2): 33-39. shang-shiu. 1976. a preliminary study on the bionomics of hunting wasps and their utilization in cotton insect control. acta entomologica sinica 19:313:318. turillazzi, s. 1980. use of artificial nests for rearing and studying polistes wasps. psyche 87(1/2):131-140. west-eberhard, m.j. 1969. the social biolog y of polistine wasps. ann arbor: museum of zoolog y, university of michigan 140:1– 110. doi: 10.13102/sociobiology.v67i2.4834sociobiology 67(2): 308-311 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the brazilian fauna is composed of 22 genera of 304 species of registered social wasps, of which 104 occur exclusively in brazilian territory (silveira et al., 2016). three tribes occur in brazil: mischocyttarini (mischocyttarus), polistini (polistes) and epiponini (carpenter & marques 2001; ctfb, 2017). these wasps have high predation potential and are important in population regulation of pest insects (richards, 1978; richter, 2000; brügger et al., 2019a; prezoto et al., 2019). social insects forage for food resources (proteins, carbohydrates) material for building nests (plant fibers and clay), and water (richter, 2000; brügger et al., 2019b). environmental factors such as humidity, sunlight, and temperature regulate these activities (kovac et al., 2018). brazil has a large insect fauna and wasp diversity is poorly abstract social wasps play an important role in communities, whether in natural or agricultural ecosystems, performing pollination and/ or predation on other organisms, especially caterpillars, which reveals their potential for biological control. we register species of predatory wasps found in a eucalypt reforested area compared with a native rainforest. five species of social wasps were found: agelaia myrmecophila (ducke), mischocyttarus punctatus (ducke), polistes carnifex (fabricius), polybia liliacea (fabricius), and polybia striata (fabricius), with higher numbers in the eucalypt monoculture than in the atlantic rainforest, suggesting no negative impact of the monoculture on the population of that natural enemies. sociobiology an international journal on social insects r silva-filho1, bp brügger2, jc zanuncio2, pcr cassino3 article history edited by gilberto m. m. santos, uefs, brazil received 22 october 2019 initial acceptance 12 december 2019 final acceptance 07 april 2020 publication date 30 june 2020 keywords epiponini; mischocyttarini; occurrence; polistini; predator. corresponding author bruno pandelo brügger departamento de entomologia/bioagro universidade federal de viçosa, cep 36570-900, viçosa, minas gerais, brasil. e-mail: brunopb2002@yahoo.com.br known, so new records may contribute to the understanding of species diversity and richness in an area (de souza et al., 2017; brügger et al., 2019b; somavilla et al., 2019). eucalyptus trees play an important role in the brazilian economy. because they are locally abundant, this type of biomass is used as an energy source in agribusiness (lenz et al., 2019), but its monoculture can favor the occurrence of pests such as defoliator caterpillars, which must be controlled to reduce their damage (masson et al., 2017; munique et al., 2018; zanuncio et al., 2018). the collection and identification of social wasps in a given region provide information for studies on the ecology of these organisms and their interactions with the environment (silveira et al., 2002). therefore, the objective was to record the occurrence of social wasps in an area with eucalyptus and atlantic forest. 1 departamento de biologia animal, universidade federal de viçosa, viçosa-mg, brazil 2 departamento de entomologia e fitopatologia, universidade federal rural do rio de janeiro, seropédica-rj, brazil 3 departamento de entomologia/bioagro, universidade federal de viçosa, viçosa-mg, brazil research article wasps social wasps in exotic forest planting and atlantic rainforest in the neotropical region sociobiology 67(2): 308-311 (june, 2020) 309 materials and methods social wasps were collected from may 2008 to march 2009 in an area of 117.07 ha with four-year hybrid eucalyptus (eucalyptus grandis x eucalyptus urograndis) and in 35.05 ha of secondary atlantic forest in the municipality of dionísio, minas gerais; brazil (19º 48 ‘s 42º 45’ w and 315 m elevation). the distance between the areas was 953 meters. pet bottle traps with three 2.5 cm diameter side openings adapted to 200 ml plastic pots containing baits (sardines or honey) were used to capture wasps (fig 1). thirty traps were used in the eucalyptus plantation or atlantic forest, being 15 with fish and 15 with honey, spaced every 50 m, 1.60 m high and verified every 24 hours for 5 days, every month for 12 months. species frequency was calculated with the equation f = / σs1.100 / σs2, where s1 = total number of species and s2 = <0 .0001) and the areas of eucalyptus (f = 65.91, p <0.001). the average of all wasp species was higher in eucalyptus than in the atlantic forest, and those of a. myrmecophila higher and m. punctatus lower in both environments (fig 2). species atlantic rainforest eucalypt number frequency (%) number frequency (%) agelaia myrmecophila 224 63.8 559 67.6 polybia striata 54 15.4 111 13.4 polistes carnifex carnifex 43 12.2 99 12.0 polybia liliacea 22 6.3 42 5.1 mischoyttarus punctatus 8 2.3 16 1.9 total 351 827 table 1. social wasps sampled in eucalypt and atlantic rainforest from may 2008 to march 2009 in the municipality of dionísio, minas gerais state, brazil. total number of wasps collected. results five species, 827 and 351 individuals of social wasps, agelaia myrmecophila (ducke) (559 and 224), polybia striata (fabricius) (111 and 54), polistes carnifex carnifex (fabricius) (99 and 43), polybia liliacea (fabricius) (42 and 22) and, mischocyttarus punctatus (ducke) ( 16 and 08) were collected in the areas of eucalyptus or atlantic forest respectively (table 1). the number of individuals of a. myrmecophila (f = 59.68, p <0.001), m. punctatus (f = 6.32, p = 0.003), p. carnifex carnifex (f = 9.47, p = 0.001), p. liliacea (f = 7.34, p = 0.0038) and p. striata (f = 10.57, p <0.0001) were significantly different between atlantic forest (f = 35.87, p fig 1. trap used for wasp capture (silva-filho, 2008). discussion the largest number wasp individuals in the eucalyptus area in relation to the atlantic forest is due to the abundance of prey in this area, which may be present in trees or in the understory (kato, 1996; de souza et al., 2011). eucalyptus monoculture has defoliating caterpillars (zanuncio et al., 2018), fig 2. number of wasp individuals (average ± standard error) of the social wasps agelaia myrmecophila (a.m.), mischocyttarus punctatus (m.p.), polistes carnifex carnifex (p.c.c.), polybia liliacea (p.l.) and polybia striata (p.s.) (hymenoptera: vespidae: polistinae) captured with traps from may 2008 to march 2009 in eucalypts reforested and native atlantic rainforest areas. municipality of dionísio, minas gerais state, brazil. bars with the same letters (comparison within a species between two environments) and the same small letters (comparison within a species within an environment) tukey’s test (p> 0.05). r silva-filho, bp brügger, jc zanuncio, pcr cassino – social wasps in forest planting and atlantic rainforest310 favoring the wasps due to pest insect outbreaks (elisei et al., 2010). however, the use of only attractive traps, may have influenced the result, since in environments with less food availability (eucalyptus) (de souza et al., 2011) it may have captured more social wasps in relation to an environment rich in food (atlantic forest) (brügger et al.,2019b). in addition, the higher light intensity in eucalyptus monoculture than in the atlantic forest may have contributed to the number of wasp individuals, as these insects use marked clues or points for short or long-distance orientation in relation to their nests (steinmetz & schmolz, 2004; warrant et al., 2006; mandal, 2018; silva-filho et al., 2020). daytime insects can make navigation errors in low sunlight conditions (spiewok & schmolz, 2005). in social wasps it is common for the colonies’ foundations to be in forest areas due to their cryptic aspect, using monoculture only as a foraging site (jeanne, 1975). the maintenance of atlantic forest areas is important because among the management strategies that can favor the performance of biological control agents, there is the preservation of areas of refuge (van driesche & bellows, 1996; menezes et al., 2017). in brazil, some studies have evaluated the effects of fragments of native forest on natural enemies, in crops such as: eucalyptus (eucalyptus spp.) (murta et al., 2008), corn (zea mays l.) (sousa et al., 2011) and sugar cane (saccharum officinarum l.) (demite et al., 2015; duarte et al., 2015). the lower number of individuals of both a. multipicta and m. punctatus in both environments was expected because agelaia has the largest colony size among wasps (zuchi et al., 1995; noll et al., 1997; london & jeanne, 2000). the colonies of agelaia and polybia are founded by swarms, consequently with larger numbers of individuals than that of independent foundation such as polistes or mischocyttarus (wenzel, 1998). the higher number and frequency of social wasps in the eucalyptus compared to the atlantic forest demonstrates the importance of maintaining refugee areas for biological control, as it can positively influence the diversity of social wasps in monoculture. however, the use of only the attractive trap methodology, may underestimate the results, due to differences in food supply in the areas, so we recommend the consortium between the active search methodologies and the attractive traps. acknowledgments we thank the following brazilian agencies “conselho nacional de desenvolvimento cientifico e tecnológico, coordenação de aperfeiçoamento de pessoal de nível superior (finance code 001), “fundação de amparo à pesquisa do estado de minas gerais” and “programa cooperativo sobre proteção florestal do instituto de pesquisas e estudos florestais” for scholarships and financial support. references brügger, b.p., la cruz r.a., carvalho, a.g., soares, m.a., prezoto, f & zanuncio, j.c. 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(1995). agelaia vicina, a swarm–founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103(2): 129-137. 731 food preferences of workers of coptotermes formosanus (isoptera: rhinotermitidae) by jing li1, xiaodong yuan1, ruyin xu1 , xiaoqing nan1 & jianchu mo2* abstract coptotermes formosanus shiraki is an amphibious termite which severely damages wood components of housing construction, stored cellulose materials, living trees, and communication facilities. the food preference of this termite was investigated in laboratory conditions. our research results indicated that among 8 kinds of woods (teak tectona grandis, chinese fir cunninghamia lanceolata, oak quercus mongolica, taxis melia azedarach, birch betula allegansis, beech fagus syliatica, pine pinus massoniana, and poplar populus tremula), the consumption rate of pine wood by c. formosanus (30.46%) is higher than that of seven other species of wood. after pine was treated by eight different methods, the pine wood treated repeatedly (three times) by the method of dipping 24 hours in water and drying in the sun (sd method) was significantly preferred by c. formosanus. when adding 30% sugar into water for treatment of pine wood blocks in the sd method, the pine wood blocks attracted more termites to feed compared to other concentrations of sugar in water. moreover, adding 10% formosan subterranean termite nest material into 30% sugar solution increased the preference to the treated pine wood blocks by c. formosanus. this information shows that c. formosanus likes to feed on pine wood blocks treated repeatedly by the method of dipping in mixed solutions of formosan subterranean termite nest material and 30% sugar for 24 hours and drying in the sun. keywords: copototermes formosanus, pine wood block, consumption rate, efficacy, attraction. 1hangzhou institute of termite control, hangzhou, zhejiang 310016, p. r. china; 2ministry of agriculture key laboratory of molecular biolog y of crop pathogens and insects, institute of insect sciences, zhejiang university at zijingang, hangzhou, zhejiang 310012, p. r. china. * corresponding author. email: mojianchu@zju.edu.cn 732 sociobiolog y vol. 59, no. 3, 2012 introduction although the efficacy of traditional chemicals chlordane and mirex to control termites is good, these two chemicals have been banned because of their high toxicity, long persistent period and harm to environment and human health. use of bait to control termites has also been one of the most important and effective measures in china because of its simplicity, feasibility, minimal pollution, and high efficacy. however, many factors affect the feeding of termites on bait. some researchers (becker 1969; behr et al. 1972; carter et al. 1983; waller & lafage 1987; delaplane & lafage 1989; sornnuwat et al. 1995) have summarized some factors that affect the feeding preference in termites. of these, food components is one of the most important factors. in china, coptotermes formosanus shiraki is found in areas north of latitude 33.5° and is a amphibious termite which severely damages wood components of housing construction, stored cellulose materials, living trees, and communication facilities. it causes tremendous economic losses every year in china. therefore, people hope to improve the bait to better control the damage of c. formosanus and to reduce the economic losses. in this paper, we determined the feeding preference of c. formosanus to different wood materials in laboratory conditions. materials and methods source of termites a coptotermes formosanus nest was collected from the field and was maintained in laboratory conditions for two years. woods for experiment eight species of wood were used in this study. they were teak tectona grandis (a), chinese fir cunninghamia lanceolata (b), oak quercus mongolica (c), taxis melia azedarach (d), birch betula allegansis (e), beech fagus syliatica (f), pine pinus massoniana(g), and poplar populus tremula (h). they were purchased from the sanhe timber factory (deqing county, zhejiang province) and processed into wood blocks (5 cm×5 cm×2.5 cm) before experiments. 733 li, j. et al. — food preferences of c. formosanus experimental procedures experiments were divided into four steps. the first step was to determine the feeding preference of c. formosanus to different wood species. before experiments, the 8 kinds of wood blocks were put into an oven for 24 hours at 70°c, and then immediately weighed. when experiments started, one wood block of each wood species was randomly placed on the surface of the c. formosanus nest. two weeks later, the rest of the wood blocks were taken out and the mud on the wood was removed with water. finally, the blocks were put in a room to air dry. after that, they were baked for 24 hours at 70°c and weighed again. according to the weight variation of wood blocks, the wood species mostly preferred by c. formosanus was determined. the second step was to determine the feeding preference of c. formosanus to the pine wood blocks treated with different methods. before experiments, the pine wood blocks were treated according to the methods shown in table 1. when the experiment was done, the pine woods were tested as the method described on the first step. table 1. methods for pine wood treatment. treatment methods of treating pine wood blocks for experiments. drying in the sun (sa) dipping in water for 24 hours and then drying in the sun (sb) repeating sb method 2 times (sc) repeating sb method 3 times (sd) drying in oven at 70°c (oa) dipping in water for 24 hours and drying in oven at 70°c (ob) repeating ob method 2 times (oc) repeating ob method 3 times (od) the third step was to determine the feeding preferences of c. formosanus to wood blocks treated with different concentrations of sugar solution. before experiments, the pine wood blocks were treated by the best method screened out from the second experiment (that is, the pine wood blocks treated by this method were the ones most preferred by c. formosanus) but water solutions were replaced with 10%, 20%, 30%, 40%, 50% or sugar solution when wood blocks were treated. when the experiment was done, the consumption of the wood blocks was evaluated by the method described on the first step. the fourth step was to determine the feeding preference of c. formosanus to the pine wood blocks treated with 30% sugar solution and different concentrations of formosan subterranean termite nest material extract solutions. 734 sociobiolog y vol. 59, no. 3, 2012 before experiments, the pine wood blocks were treated by the best method screened out from the third step experiment (that is, the pine wood blocks treated by this method were the ones most preferred by c. formosanus) but the sugar solution contained 1%, 2%, 5%, 10%, and 20% of formosan subterranean termite nest material when pine wood blocks were treated. when the experiment was done, the consumption of the wood blocks was evaluated by the method described in the first step. statistical analysis dps data processing system was used to analyze the experimental data. one way anova was used to analyze difference of all data through duncan's new multiple range method for multiple comparisons (tang & feng 2010). results feeding preference of c. formosanus to eight species of wood among eight species of woods tested, c. formosanus most preferred the pine wood (fig.1). the consumption rate of pine wood (30.46%) was significantly higher than that of other seven species of wood (p <0.05). consumption rate of poplar wood (18.44%) and beech wood (14.63%) ranked second and fig.1. the consumption rate of different wood blocks by c. formosanus. the same letter means the difference between groups was not significant (p>0.05); each test was repeated eight times. 735 li, j. et al. — food preferences of c. formosanus third, respectively. there were no significant differences in the consumption rate among oak, birch or chinese fir, (p> 0.05). in addition, c. formosanus did not like teak wood or taxis wood. feeding preference of c. formosanus to pine woods treated with different methods among the eight treatment methods, the sd method most significantly enhanced the feeding of c. formosanus on pine wood blocks (fig.2). the consumption rate of pine wood blocks treated with sd method by c. formosanus (46.09%) was significantly higher than that treated with other seven methods (p <0.05). moreover, there were no significant difference among other seven methods (p>0.05), the consumption rate of pine wood blocks treated with other seven methods ranged from 22.54% to 29.42%. feeding preference of c. formosanus to pine woods treated with different concentrations of sugar solution among five concentrations of sugar solutions tested, 30% sugar solution most significantly enhanced the feeding of c. formosanus on pine wood blocks treated (fig.3). the consumption rate of pine wood blocks treated with 30% sugar solution by c. formosanus (46.77%) was significantly higher than that fig.2. the consumption rate of treated pine wood blocks treated with different methods by c. formosanus. the same letter means the difference between groups was not significant (p> 0.05); each test was repeated eight times. 736 sociobiolog y vol. 59, no. 3, 2012 fig.3. the consumption rate of pine wood blocks treated with different concentrations of sugar solution by c. formosanus. the same letter means the difference between groups was not significant (p> 0.05); each test was repeated eight times. fig.4. the consumption rate of pine wood blocks treated with different concentrations of formosan subterranean termite nest material by c. formosanus. the same letter means the difference between groups was not significant (p> 0.05); each test was repeated eight times. 737 li, j. et al. — food preferences of c. formosanus treated with other four sugar solutions (p<0.05). there were no significant differences among the other four glucose concentration (p> 0.05). feeding preference of c. formosanus to pine woods treated with different concentrations of formosan subterranean termite nest material among five concentrations of formosan subterranean termite nest material tested, 10% nest material solution most significantly enhanced the feeding of c. formosanus on pine wood blocks treated (fig.4). the consumption rate of pine wood blocks treated with 10% nest material solution (25.74%) was significantly higher than that treated with the other four concentrations of nest material solution (p<0.05). discussion among the 8 kinds of wood species studied, c. formosanus preferred to feed on the pine wood. our results were consistent with results of other researchers. song (1993) reported that pine wood mainly contains cellulose, lignin, little volatile oil (turpentine) and rosin (resin), etc. volatile oil includes α-pinene, β-pinene and less t-camphene. rosin contains rosin anhydride, hydrocarbon resins. preference of c. formosanus to pine wood may be due to the strongest temptation of turpentine or rosin. morales-ramos & rojas (2003) found formosan subterranean termites significantly preferred the bait matrix over southern yellow pine wood (pinus taeda l.) in laboratory and field evaluations. at the same time, the consumption rate of teak wood, taxis wood and other woods is low, which is probably related with large density and hardness of these woods. wood density is a physical property of wood, which is positively correlated with the hardness of wood. considering research on wood species attracting termites, many researchers found that termites have preference for low-density tissue wood types. peng-soon et al. (2004) indicated that rubber, jelutong and terentang were the most preferred species among the 29 wood species tested in the laboratory on four malaysian termite species (coptotermes gestroi wasmann, coptoterme curvignathus holmgren, globitermes sulphureus haviland and microcerotermes crassus snyder). delaplane & lafage (1989) indicated that c. formosanus preferred low density and moist wood or wood damaged by conspecifics. arango et al. (2006) found that wood density was 738 sociobiolog y vol. 59, no. 3, 2012 inversely correlated with wood consumption rate by reticulitermes flavipes kollar in tropical regions of the hard wood species in north america.on the contrary, the wood density was positively correlated with wood consumption rate by r. flavipes among the soft wood species. among the 8 treatment methods in our study, the consumption rate of the pine wood blocks treated with the sd method by c. formosanus was the largest. timber contents include fat, fatty acids, fatty alcohols, phenols, steroids, resin acid, resin, wax, and many other trace organic chemicals. most plants, especially the ones in tropical environments, use these substances as defensive components. therefore, the allelochemicals of wood such as terpenoids, quinones, phenols and flavonoids are generally considered as repellent and/or toxic for termites. yatagai et al. (2002) reported three kinds of wood acetic acid which were extracted from pseudotsgu menziesii, oak and pine could effectively kill reticulitermes speratus kollbe. these compounds increased the mechanical strength of woods and had anti-feeding repellent and toxic effects on termites. in addition, pine contains some poisonous compounds that are toxic to termites. in this study, use of repeated dipping-drying treatment methods can effectively remove these poisonous compounds, and therefore improve preference of c. formosanus to pine wood. many researchers have found high concentrations of carbohydrates could highly appeal to termites (waller & curtis 1996; reinhard & kaib 2001). the study of saran & rust (2008) found that adding carbohydrates can significantly increase intake of hexaflumuron baits by reticulitermes hesperus. choice tests by swoboda et al. (2004) determined that several types of sugars and uric acid would stimulate preferential feeding in the presence of a competing food resource. haifig et al. (2008) found heterotermes tenuis fed preferentially on filter paper treated with 0.03 g/ml trehalose and 0.015 g/ml urea solutions. in addition, zhang et al. (2004) also found that certain concentrations of sucrose can improve the attractive ability of woods to c. formosanus and reticulitermes flaviceps. lin et al. (2011) via bioassay showed that pine wood blocks with 20% glucose had the strongest attraction to c. formosanus. our study showed use of 30% sugar solution to treat the sd method-treated pine wood blocks could significantly enhance the feeding of c. formosanus on the wood. this means a high concentration of sugar solution is indeed a feeding stimulus for c. formosanus. 739 li, j. et al. — food preferences of c. formosanus adding 10% formosan subterranean termite nest material in to 30% sugar solution also increased feeding of c. formosanus on the sd method-treated pine wood blocks. this means the nest material also contains stimulant compounds to c. formosanus. li et al. (2001) found that adding homemade additive into pine sawdust significantly increased its attractiveness to c. formosanus, and the additive improved feeding addiction of termites on bait as well as food consumption. cornelius (2003) reported that ergosterol acted as a feeding stimulant at a concentration of 1 mg/g of filter paper for formosan subterranean termites. our study indicated that if pine wood blocks were treated repeatedly three times by the method of dipping in water solution with 30% sugar and 10% formosan subterranean termite nest material for 24 hours and drying in the sun, it would be preferred strongly by c. formosanus. therefore, pine wood blocks treated by this method treatment may be used in baiting systems for the monitoring of c. formosanus in the field. acknowledgments this study was supported financially by the ministry of housing, urban and rural development of china (project no. 2011-k6-32). references arango, r.a., iii.f. green & k. hintz 2006. natural durability of tropical and native woods against termite damage by reticulitermes flavipes (kollar). international biodeterioration and biodegradation 57: 146-156. becker, g. 1969. rearing of termites and testing methods used in the laboratory.in: k. krishna & f.m. weesner (eds.). biolog y of termites academic press, new york 1: 351-385. behr, e.a. 1972. termite resistance of northern white cedar. research report. agricultural experiment station, michigan state university 11. carter, f.l. & r.h. beal 1983. termite responses to susceptible pine wood treated with antitermitic wood extracts. international journal of wood preserve 2: 185-191. cornelius, m.l. 2003. evaluation of semiochemicals as feeding stimulants for the formosan subterranean termite (isoptera: rhinotermidae). sociobiolog y 41(3): 583-591. delaprane, k.s. & j.p. lafage 1989. preference of the formosan subterranean termite (isoptera: rhinotermitidae) for wood damaged by conspecifics. journal of economic entomolog y 82: 1363-1365. haifig, i., a.m. costa-leonardo & f.f. marchetti 2008. effects of nutrients on feeding activities of the pest termite heterotermes tenuis (isoptera: rhinotermitidae). journal of economic entomolog y 132: 497-501. 740 sociobiolog y vol. 59, no. 3, 2012 li, x.s., y.z. li, w.x. wang, j.c. mo & j zhang. 2001. development of high-efficiency bait for coptotermes formosanus and trapping experiments. journal of central south forestry university 20: 75-77 (in chinese). lin, a.s., l.h. feng, y.y. hu, a.j. yi & j.c. mo 2011. attractiveness of five kinds of sugar on coptotermes formosanus and reticulitermes flaviceps. forest pest and disease 30: 10-12 (in chinese). morales-ramos, j.a. & m.g. rojas 2003. formosan subterranean termite feeding preference as basis for bait matrix development (isoptera: rhinotermitidae). sociobiolog y 41: 71-79. peng-soon, n., t. ai, y. tsuyoshi, j. zairi & l. chow-yang 2004. wood preference of selected malysian subterranean termites (isoptera: rhinotermitidae, termitidae). sciobiolog y 43: 535-548. reinhard, j. & m. kaib 2001. thin layer chromatography assessing feeding stimulation by labial gland secretion compared to synthetic chemicals in the subteranean termite reticulitermes santonensis. chemical ecolog y 27: 175-187. saran, r.k. & m.k. rust 2008. phagostimulatory sugars enhance uptake and horizontal transfer of hexaflumuron in the western subterranean termite (isoptera: rhinotermitidae). journal of economic entomolog y 101: 873-879. song, x.g. 1993. screening of matrix for termite bait. science and technolog y of termites 10: 11-15 (in chinese). sornnuwat y.c., t. vongkaluang & t. yoshimura 1995. natural resistance of seven commercial timbers used in building construction in thailand to subterranean termite, coptotermes gestroi wasmann. journal of environment entomolog y 7: 146–150. swoboda, l.e., d.m. miller, r.j. fell & d.e. mullins 2004. the effect of nutrient compounds (sugars and amino acids) on bait consumption by reticulitermes spp. (isoptera: rhinotermitidae). sociobiolog y 44: 547-563. tang, q.y. & m.g. feng 2010. dps data processing system for practical statistics. beijing : science press (in chinese). waller, a.d. & j.f. lafage 1987. food quality and foraging response by the subterranean termite coptotermes formosanus shiraki (isoptera: rhinotermitidae). bulletin of entomological research 77: 417-424. waller, a.d. & a.d. curtis 1996. termite (rhinotermitidae: reticulitermes) preference for sugar-coated foods. in: presentation at the annual meeting of the entomological society of america. louisville, kentucky. yatagai, m., m. nishimoto & k.h. ohira 2002. termiticidal activity of wood vinegar, its components and their homologues. journal of wood science 48: 338–342. zhang, j.h., m.h. zhang, j.s. wang & j.h. huang 2004. the seduction of cane sugar, sodium cyclamate and white wine to termites. journal of human university of arts and science (natural science edition) 16: 56-58 (in chinese). 971 analysis of ant communities comparing two methods for sampling ants in an urban park in the city of são paulo, brazil by felipe m. ribeiro¹, neiva sibinel², giordano ciocheti³ & ana e. c. campos² abstract this study aimed to analyze the species composition and functional groups of the ant community and to assess the efficiency of two sampling methods, pitfall and leaf litter sampling, in an urban park. a total of 1,401 ants were collected, which belonged to six subfamilies and 36 species. the predominant species was wasmannia auropunctata (present in 45.36% of the samples), while the functional group of opportunistic ants were the most frequent (present in 83.75% of the samples) and abundant (95.29% of the total collected specimens) functional group. the jaccard similarity index showed a low similarity between the two sampling methods, as the difference of the number of individuals for each species between these two methods was not significant in only one case (linepithema sp. 1, p = 0.4561). the fungusgrowing and cryptic ants were more collected in leaf litter samples (p<0.0001; p = 0.0348 respectively). although there was no significant difference (p = 0.6397) between the two sampling methods for the total individuals of opportunistic ants, more species of this group were collected in pitfall traps. this difference was not significant because of the high presence of w. auropunctata, an opportunistic ant, in samples of leaf litter. due to the predominance of tramp ants in the studied area, this article illustrates the importance of green urban areas in ant control strategies, since these sites could be used as a source of new colonization for these ants. furthermore, the combination of the two sampling methods seems to be complementary for obtaining a more complete picture of the ant community. key words: ant community, urban areas, tramp ants, functional group, urban areas, pitfall trap, leaf litter sample 1 laboratório de ecologia espacial e conservação, depto de ecologia unesp rio claro, av. 24a, 1515, 13506-900 rio claro, sp, brasil. email: felipemartello@gmail.com 2 instituto biológico, avenida conselheiro rodrigues alves, 1.252, 04014-002, são paulo sp – brasil 3 laboratório de estudos subterrâneos, depto de biologia e ecologia evolutiva – ufscar – rod. washington luís, km 235, 13565-905, são carlos – sp brasil 972 sociobiolog y vol. 59, no. 1, 2012 introduction ants are one of the groups of animals with the greatest diversity in the planet. different species can be found in all regions except the poles (hölldobler & wilson 1990). among the 2,500 known species of ants in brazil, around 50 species are known as tramp ants due to their ability to survive in urban environments (bueno & campos-farinha 1999 campos-farinha 2002). this ability to survive in highly disturbed environments is related to certain characteristics that these ants present, such as unicolonialism, polyg yny, sociotomy, small size of workers, and migration and fragmentation of colonies in response to changes in environment (fowler et al. 1994, passera 1994, bueno & campos-farinha 1999, vega 2001). the damage that these ants cause in urban areas comes from their presence in residences, invasion and damage of electronic appliances and structures of buildings, like wood ceilings and door frames. they also cause public health problems due to their presence in food facilities, hospitals, health centers, as they can mechanically vector many pathogens that pose risks to human health. in addition, some species such as solenopsis invicta show aggressive behavior and some of their victims, who are allergic to their stings, may go into anaphylactic shock, which can lead to death (bueno & campos-farinha 1995, zarzuela et al. 2002, zarzuela et al. 2005). studies on ants in urban environments go beyond the simple understanding of their biolog y and control, focusing on how the whole ant community responds to the environment, especially in urban parks and squares (silva & loeck 1999, yamaguchi 2004, clarke et al. 2008, iop et al. 2009). these green areas are important for the conservation of plants and animals that are more sensitive to anthropization due to milder environmental conditions (rodrigues et al. 1993). the development of research on ant communities in green areas can provide important information on the environmental quality, since ants are essential to ecological processes such as decomposition, pollination, seed dispersal, nutrient cycling, etc. (hölldobler & wilson 1990, moutinho 1998, lobry de bruyn 1999). moreover, ants are considered good bioindicators due to the diversity of the group, the facility of sampling individuals, for being susceptible 973 ribeiro, f.m. et al. — comparison of ant sampling methods to environmental changes and also for being a well-known taxa (andersen 1997, silva & brandão 1999). in order to evaluate ant species as bioindicators, species are grouped into functional groups based on characteristics such as diet, nest location and response to habitat disturbance (andersen 1995, delabie et al. 2000, silvestre & silva 2001). with this grouping we can go beyond simply assessing the environment for species richness, but one can analyze how these groups react differently to environmental disturbance (philpott et al. 2010). the two main methods of sampling ants in urban environments are active collection and attractive baits (piva & campos-farinha 1999, yamaguchi 2004, zarzuela et al. 2005, clark et al. 2008, piva & campos 2012). this methodolog y is efficient for collecting urban species, particularly within households (alder & silverman 2005, vital 2007). in areas with greater vegetation covers where there is the presence of leaf litter, as some urban parks and squares, there is a possibility to use two other sample methods: pitfall traps and leaf litter samples. these two methods are part of the protocol for collection of leaf litter ants (agosti & alonso 2000) created to standardize the collection methodolog y and enable better comparisons among studies. although this protocol was widely used for sampling ants in forest physiognomies (fisher et al. 2000) and it has been also validated for other environments with very different physiognomies as brazilian savannah (lopes & vascocelos 2008), little is known about its efficiency in urban green areas. the objective of this study was to evaluate the species composition and functional groups of the ant community in an urban park, and compare two methods of sampling ants in this locality: pitfall traps and leaf litter sampling. the results of this research were also compared with two other studies conducted in the same neighborhood, but in households. material and methods study site the studied area was the 3 ha park of instituto biológico, a research institution located in one of the oldest areas in the city of são paulo, the vila mariana neighborhood, 5 km away from downtown. the site is next to a major avenue 974 sociobiolog y vol. 59, no. 1, 2012 that crosses the city from north to south and separates the studied area from the ibirapuera park, the most visited park in the city, which covers an area of 1,584 km² and receives about 220,000 visitors each week. ant sampling data collection was conducted in april, september and october 2005. two transects were designed to cover the largest area of the park. along each transect 36 pitfall traps were placed, spaced 10 meters apart (n = 72). each trap consisted of a 500 ml disposable plastic cup filled with 3% formalin and detergent. the traps were laid in the soil and collected 48 hours later. five transects of 40 m each were established in the main grassed areas, in order to collect leaf litter. in each transect one square meter of leaf litter was collected every10 meters (n = 25), later processed in a winkler extractor. data analysis frequency (number of traps where the species occurred relative to total number of traps) and relative abundance (total number of individuals collected of the species relative to total number of individuals of all species) were calculated for ant fauna sampled in pitfall traps and leaf litter. species richness was calculated through the chao 2 richness estimator and species accumulation curve using the mao tao estimator with estimates, version 8.2 (colwell 2004). chao 2 is an incidence-based estimator of species richness, which relies on the number of unique units and duplicates (species found in only one and two sample units) (chao, 2004) and species accumulation curve illustrates the rate at which new species are found (magurran 2004). species richness was analyzed for each sample methodolog y separately and also for both methods together. ant species were grouped into functional groups adapted from delabie et al. (2000) and silvestre & silva (2001). to compare the two sampling methodologies, the chi-square test was applied to the number of specimens (zar 1996). in this analysis those species or functional groups where the sum of individuals between the two methods was less than 11 were discarded. the jaccard similarity index was calculated to estimate the similarity between the species richness in both sampling methods. the list of ant species of two other studies conducted in the same neighborhood (piva & campos-farinha 1999, piva & campos 2012) was also 975 ribeiro, f.m. et al. — comparison of ant sampling methods grouped in order to compare data with the ant fauna collected at the instituto biológico park. results a total of 1,401 ant specimens were collected, distributed in 36 species and six subfamilies, myrmicinae was the richest (19 species), followed by formicinae (6 species), ponerinae and dolichoderinae (both with 4 species), ectatomminae (2 species) and pseudomyrmecinae (1 species) (table 1). the chao2 index estimated 43 species for both sampling methods, seven more species than the observed number. the species accumulation curve is represented in fig. 1 for each method and for both when analyzed together. wasmannia auropunctata was the predominant species in the samples with a total frequency (45.36%) two times greater than the second most frequent species (pheidole sp.1 22.68%) and accounting for over half of the collected specimens (abundance = 53.96%) (table 1). the genus pheidole was the richest (9 species) followed by solenopsis (3 species). these two genera after w. auropunctata represented the most frequent group of ants (pheidole spp. = 37.11% and solenopsis spp. = 34.02% from the 97 pitfall traps and leaf litter samples). the predominant functional group in the community was the opportunistic ants, since most species fitted in this group (25 species table 1), which were the most frequent in the 97 samples (f = 83.75%) and the most abundant (95.29% of the total collected specimens) (table 2). ants with aggressive behavior and omnivorous species were grouped as “opportunistic ants”, regardless of taxonomic group or if they show features such as massive recruitment and dominance of baits, since we did not use baits. the groups of cryptic species (p < 0.0001) and fungus-growing (p = 0.0348) were the only two that showed significant differences between the two sampling methodologies. the group of arboreal ants was discarded for not reaching the assumptions of the test (table 2). comparing the two sample methodologies, pitfall traps and leaf litter collection, only linepithema sp. did not show a significant difference between the two sampling methodologies according to the number of collected specimens (p = 0.4561). twenty four ant species were discarded from analysis 976 sociobiolog y vol. 59, no. 1, 2012 table1. relative abundance (ab) and frequency (f) of ant species collected with pitfall traps and leaf litter samples, the comparison between the two sample methodologies (chi-square test) and the classification of species into functional groups (fg), where op = opportunistic, fu = fungus-growing, cr = cryptic, lp = large predator and ar = arboreal. collections in the park of instituto biológico, são paulo, brazil. pitfall leaf litter total species fg ab (%) f (%) ab (%) f (%) p ab (%) f (%) dolichoderinae dorymyrmex sp.1 op 1.70 5.56 0.86 4.12 dorymyrmex sp.2 op 1.28 6.94 discarded 0.64 5.15 linepithema sp.1 op 2.84 16.67 3.59 40.00 0.4561 3.21 22.68 tapinoma melanocephalum op 0.28 2.78 0.29 4.00 discarded 0.29 3.09 formicinae brachymyrmex sp.1 op 0.57 4.17 0.14 4.00 discarded 0.36 4.12 brachymyrmex sp.2 op 1.28 9.72 0.14 4.00 discarded 0.71 8.25 camponotus sp.1 op 0.14 1.39 discarded 0.07 1.03 nylanderia fulva op 12.07 13.89 0.29 8.00 >0.0001 6.21 12.37 paratrechina longicornis op 8.10 6.94 >0.0001 4.07 5.15 paratrechina sp.1 op 9.80 8.33 3.01 24.00 >0.0001 6.42 12.37 myrmicinae cyphomyrmex sp.1 fu 0.57 4.17 0.29 8.00 discarded 0.43 5.15 monomorum floricola op 0.29 8.00 discarded 0.14 2.06 monomorium pharaonis op 0.29 8.00 discarded 0.14 2.06 mycocepurus goeldii fu 0.57 4.17 discarded 0.29 3.09 pheidole sp.1 op 9.23 30.56 >0.0001 4.64 22.68 pheidole sp.2 op 0.28 1.39 discarded 0.14 1.03 pheidole sp.3 op 0.28 1.39 discarded 0.14 1.03 pheidole sp.4 op 0.57 4.17 discarded 0.29 3.09 pheidole sp.5 op 1.99 9.72 0.0002 1.00 7.22 pheidole sp.6 op 0.14 1.39 discarded 0.07 1.03 pheidole sp.7 op 1.00 16.00 discarded 0.50 4.12 pheidole sp.8 op 1.15 8.00 discarded 0.57 2.06 pheidole sp.9 op 0.14 4.00 discarded 0.07 1.03 solenopsis sp.1 op 11.93 20.83 1.15 4.00 >0.0001 6.57 16.49 solenopsis sp.2 op 0.14 1.39 0.57 12.00 discarded 0.36 4.12 solenopsis sp.3 op 7.39 18.06 0.29 8.00 >0.0001 3.85 15.46 strumigenys sp.1 cr 0.28 2.78 3.01 40.00 >0.0001 1.64 12.37 trachymyrmex sp. fu 0.14 1.39 discarded 0.07 1.03 wasmannia auropunctata op 26.85 30.56 81.35 88.00 >0.0001 53.96 45.36 977 ribeiro, f.m. et al. — comparison of ant sampling methods as they represented less than 11 specimens in both sample methodologies (table 1). the chao2 index estimated 28 species for leaf litter sampling and 34 for pitfall traps, meaning that the difference between what was estimated and what was found in both methodologies was six species (fig. 1) and the similarity between the species collected in the two sampling methods was 0.388 ( jaccard similarility index). table1 (continued). relative abundance (ab) and frequency (f) of ant species collected with pitfall traps and leaf litter samples, the comparison between the two sample methodologies (chi-square test) and the classification of species into functional groups (fg), where op = opportunistic, fu = fungus-growing, cr = cryptic, lp = large predator and ar = arboreal. collections in the park of instituto biológico, são paulo, brazil. pitfall leaf litter total species fg ab (%) f (%) ab (%) f (%) p ab (%) f (%) ponerinae anochetus sp.1 cr 0.14 4.00 discarded 0.07 1.03 hypoponera sp.1 cr 1.72 24.00 0.0005 0.86 6.19 odontomachus sp.1 lp 0.14 4.00 discarded 0.07 1.03 pachycondyla sp.1 lp 0.28 1.39 discarded 0.14 1.03 ectatomminae ectatomma sp.1 lp 0.85 5.56 0.14 4.00 discarded 0.50 5.15 ectatomma sp.2 lp 0.14 1.39 0.86 4.00 discarded 0.50 2.06 pseudomyrmecinae pseudomyrmex sp.1 ar 0.28 2.78 discarded 0.14 2.06 table 2. relative abundance of functional groups for each sampling methodolog y and their sum, the comparison between the sampling methods (chi-square test). functional group pitfall (%) leaf litter (%) p total (%) opportunistic 96.875 93.687 0.640 95.289 arboreal 0.284 0.000 discarded 0.143 cryptic 0.284 4.878 <0.0001* 2.498 large predator 1.278 1.148 0.808 1.285 fungus-growing 1.278 0.287 0.0348* 0.785 *significance at 5% level 978 sociobiolog y vol. 59, no. 1, 2012 discussion composition of the ant community the high richness of ants found in the park contrasts with the results obtained in houses in the same neighborhood where 23 ant species were found in a 1999 study (piva & campos-farinha 1999) and 25 species were found between 2009 and 2011 (piva & campos 2012). certainly this difference is table 3. comparison of functional group species richness (number of species) among the two sampling methods to survey ants and data from piva &campos-farinha (1999) and piva & campos (2012) in vila mariana neighborhood, são paulo, brazil. functional group pitfall leaf litter total piva & camposfarinha 1999 piva & campos 2012 oportunistic 20.00 14.00 25.00 19.00 20.00 arboreal 1.00 0.00 1.00 0.00 1.00 cryptic 1.00 3.00 3.00 1.00 2.00 large predator 3.00 3.00 4.00 1.00 2.00 fungus-growing 3.00 1.00 3.00 0.00 0.00 fig.1. species accumulation curve for ants collected with pitfall traps and leaf litter samples in the park of instituto biológico, são paulo, brazil. 979 ribeiro, f.m. et al. — comparison of ant sampling methods due to the less disturbed environment of the park in relation to the residences and also because the sampling methods were not the same. the latter authors used baits for ant collection. although the accumulation curve (fig. 1) did not stabilize in the total sampling and also for each sampling methodolog y and the chao2 estimator showed a difference from what was expected and what was collected, this result is expected in tropical areas due to the large number of rare species in the samples (longino et al. 2002, leponce et al. 2004). therefore, it is likely a greater sampling effort would reduce this difference. w. auropunctata, the most frequent and abundant species in this study, is one of the most common tramp ants in southeastern brazil (campos-farinha et al. 2002), and it is an outstanding invasive species (orivel et al. 2009) despite being native to south america, its distribution currently extends to central america, tropical regions of north america and oceanic islands, and galapagos (robinson 2005). in contrast to this result, in residences and in their surroundings this species showed low frequency (f = 3.8%) (piva & campos 2012) or it was absent (piva & campos-farinha 1999), showing that despite being considered a tramp ant, the presence of w. auropunctata in urban areas is more common outside households, such as backyards and gardens (bueno & campos-farinha 1999), where it can nest in the soil under substrates such as leaf litter and stones (wetter & porter 2003). ants of the genus pheidole and solenopsis along with w. auropunctata represented 72.16% of the total abundance in both samples. these two genera are cosmopolitan ants with a high richness of species in both tropical and temperate regions (robinson 2005). functional groups the species mentioned above formed the functional group of opportunistic ants along with paratrechina spp., nylanderia fulva, brachymyrmex spp., monomorium spp., linepithema sp., and tapinoma melanocephalum (table 1). this group was the most frequent and abundant (table 2), and all these genera or species are recognized as tramp ants. some characteristics used to classify ants in the group of opportunistic ants, such as omnivory, are among those that identify tramp ants, besides others 980 sociobiolog y vol. 59, no. 1, 2012 such as polyg yny, sociotomy, migration and colony budding in response to environmental disturbances (passera 1994). the analysis of community composition by functional groups in our data compared to households and their surroundings in two studies conducted in the same neighborhood (piva & campos-farinha 1999; piva & campos 2012) suggests that, despite the similar dominance of opportunistic species, there is a greater number of species belonging to other functional groups in our research. the occurrence of such species in the studied area is related to the presence of leaf litter (table 3) and of course due to the different collection methodologies used in the different studies. vital (2007) considered the use of baits in consortium with pitfall traps and active search, as an efficient methodolog y to assess the diversity of ants in urban squares, where leaf litter is not always present, something that must be considered when urban ant communities are being assessed. therefore it is important to remember that generalist ants may be collected with more frequency and also if active search is performed, the results are more effective the higher the research efforts. thus, while in households and in certain urban areas such as squares, the use of baits, active search and pitfall traps combined may be a good approach, in green areas, in these same urban environments, the leaf litter must also be sampled, combined with pitfall traps, due to the great number of ant species present in this substrate as shown in this study. pitfall x leaf litter samplings the jaccard similarity index (0.388) indicates a low similarity between the two sampling methodologies, which points to the importance of combining the two types of sampling for a better evaluation of the ant fauna. the difference between these two methods was expressive mainly for the cryptic species hypoponera sp. (p = 0.0005) and strumigenys sp. (p < 0.0001), as well as for w. auropunctata (p < 0.0001), which were found more frequently in the samples of leaf litter, clearly because they use this substrate for nesting and foraging. the results from the two methodologies are not significantly different for the group of opportunistic ants (p = 0.6397), however, for dorymyrmex sp.1, pheidole sp.1, solenopsis sp.1, solenopsis sp.4, the two species of paratrechina 981 ribeiro, f.m. et al. — comparison of ant sampling methods and nylanderia fulva the difference was significant, indicating that pitfall traps are more efficient than leaf litter samplings in collecting opportunistic ants. moreover, the difference between the two methods for the group of opportunistic ants was not significant only because of the large number of individuals collected of w. auropunctata, a species that, despite being classified as opportunistic, nests in substrates such as leaf litter. excluding w. auropunctata from this group, the difference changes drastically to 493 specimens of opportunistic ants in pitfall traps against 130 in the leaf litter, a significant difference (p <0.0001). this finding is close to the results of lopes & vasconcelos (2008) who evaluated the effectiveness of these two methods and baits to assess the communities of ants in brazilian savannah. they found that the collection of leaf litter was more effective where there was greater abundance of this substrate, while in areas where it was scarce the use of pitfall traps was the best methodolog y. according to these authors, although a single methodolog y is enough to compare very different environments, they suggest a combination of methods to produce a more complete inventory, particularly pitfall traps and leaf litter sampling. the large number of opportunistic species present in the samples, particularly in pitfall traps (with the exception of w. auropunctata), suggests that baits, in urban environments with similar characteristics to the studied area, are not necessary, as they attract species that have omnivorous feeding habits, which also tend to be the species most collected in the pitfall traps. final considerations although at first glance the ant community of the studied area shows high species richness, approximately 90% were opportunistic ants, and some of them such as w. auropunctata and the genera solenopsis and pheidole, showed high frequency and abundance. these results raise a question about the control of tramp ants in urban areas: are squares and parks being used as a source for dispersion of tramp ant colonies instead of increasing the diversity of species? if this does happen the control of ants in urban areas, especially near parks and squares, should evaluate the potential of these sites as sources of new colonization of tramp ants. 982 sociobiolog y vol. 59, no. 1, 2012 the hypothesis of these areas serving as a source of dispersion for the opportunistic species is supported by the presence of other functional groups that apparently do not compete directly with the tramp ants. thus, the mere presence of other functional groups, in addition to opportunistic ants, does not seem to be the most important factor to assess the condition of the local community of ants. moreover, it is important to determine if in the opportunistic group there are only tramp ants. the presence of opportunistic species that are not tramp ants could be a sign that the ant community has better quality, since it supports species that compete directly with these urban species. this study also showed that the combination of pitfall traps to collect leaf litter is a valid methodolog y for sampling ants in urban areas with leaf litter. the large number of omnivorous species collected suggests that the use of baits may not be necessary in these areas, although more specific studies that focus on methodologies for sampling ants in green areas in urban environments are essential. references agosti, d. & l.e. alonso 2000. the 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river. 944pp. zarzuela, m.f.m., m.c.c. ribeiro & a.e.c. campos-farinha 2002. distribuição de formigas urbanas em um hospital da região sudeste do brasil. arq. inst. biol. 69(1): 85-87. zarzuela, m.f.m., a.e.c. campos-farinha & m.p. peçanha 2005 evaluation of urban ants (hymenoptera: formicidae) as carriers of pathogens in residential and industrial environments: i. bacteria. sociobiolog y 45(1): 9-14. 1037 the importance of bees for eggplant cultivations (hymenoptera: apidae, andrenidae, halictidae) by gleiciani bürger patricio1; bruno barufatti grisolia1; ivan cesar desuó2; paula carolina montagnana1; felipe gonçalves brocanelli1; elizandra goldoni gomig1 & maria josé de oliveira campos1 abstract the increasing demand for food and the “pollination crisis” have emphasized the importance of better understanding the potential of different wild bee species for pollinating crops. the aim of this study was to investigate how dependent solanum melongena l. is on bees for fruit production and if it is possible to observe any insufficiency of pollination in four (two organic and two conventional) eggplant cultivations. bee samplings were performed during the eggplant’s peak flowering. three pollination tests (t1= without insect visitation; t2= free insect visitation; t3= pollen complementation) were carried out in order to evaluate the importance of bees for fruit setting in s. melongena l. most of the bee species collected on eggplant flowers were buzz-pollinators – bombus, xylocopa, exomalopsis, centris, oxaea and many species of halictidae, and can promote the eggplant pollination. trigona sp. and apis mellifera were also collected on flowers, but they can’t vibrate their anthers, although apis presented a flying adaptation while visiting the flowers and eventually can pollinate the flowers. most of the unvisited flowers (t1) failed to form fruits and when it happened, those ones were much lighter and smaller than those formed from flowers of t2 and t3; demonstrating the importance of bees for eggplant pollination. no statistical differences were found between the weight of eggplants in t2 vs. t3 within each area, however, the weight of fruits from t2 tests varied and differed significantly between the four studied areas. our results indicated no pollen insufficiency in the studied areas, although the use of pesticides may disrupt crop-pollinator interactions, which may cause pollination insufficiency. furthermore, land 1departamento de ecologia, instituto de biociências, unesp, campus rio claro, sp, brazil, gleipatricio@gmail.com, cep:13506-900, (5519) 35269115. 2departamento de zoologia, instituto de biociências, unesp, campus rio claro, sp, brazil, cep:13506-900, (5519) 35264298. 1038 sociobiolog y vol. 59, no. 3, 2012 management seems to be a factor that determines efficiency of pollination in agricultural landscapes and ensures pollination services in cropped areas. introduction in tropical regions, about 25% of crops are considered dependent on bees for pollination and, consequently, for fruit setting and economically viable seeds (heard 1999; richards 2001). in a global scale, it is suggested that one third of the food consumed by humans depends directly or indirectly on pollinators (williams 1995; klein et al. 2007; ollerton et al. 2011). in the past decades, many species of pollinators have disappeared from agricultural areas (allen-wardell et al. 1998; corbet 1991; kearns et al. 1998; kevan & phillips 2001; steffen-dewenter et al. 2005; williams 1994), in particular the population of honeybees, which has declined all around the world. in north america, at the end of the 90s, this decline was so abrupt that the abundance of pollinators was significantly reduced when compared with any period over the past fifty years (allen-wardell et al. 1998). in fact, in the early 90s, the iucn/ssc (world conservation union/ species survival commission committee responsible for formalizing the extinction of species) estimated for the next decades, a worldwide loss of more than 20,000 species of plants; this loss was largely attributed to the decline of co-dependent pollinators (heywood 1993). the increasing demand for food and the “pollination crisis”, which could affect food production all around the world, have emphasized the importance of better understanding the potential of different wild bee species on pollinating crops (hein 2009). the knowledge about pollinator species related to a crop is the first step for the land management in order to preserve the services of pollination. many species of cultivated plants have particular traits, which demand different pollinators with different size and foraging behavior for a proper pollination, (e.g. eggplants) (solanum melongena l.). like most plant species of genus solanum, eggplants have poricidal anthers, which require specialized behavioral and morphological adaptations by potential pollinators (avanzi & campos 1997). in this species, the length of stamens is about one centimeter, with very short filaments and long bright yellow anthers; each one 1039 patricio, g.b. et al. — the importance of bees for eggplant cultivation presenting two apical pores. effective eggplant pollinators can acquire pollen by vibrating the anthers. s. melongena l. is native to the tropical regions of asia and has been cultivated for centuries by arabs and chinese. its consumption is increasing mainly in europe and the united states (vieira et al. 1996), where it represents an important crop. in brazil, the consumption of this vegetable has increased due to the medicinal properties attributed to the eggplant (gonçalves et al. 2006; guerra et al. 2007) as well as its nutritional content. according to the data of ceasa – campinas (2007), the production of eggplants in the state of são paulo (brazil) reaches 47,549 tons/year and is responsible for generating more than one thousand jobs. given the importance of eggplant cultivation, both in terms of nutrition and economic value, and considering the increasing need for alternatives that guarantee the sustainability of agricultural systems, more studies are required in order to obtain more information on the composition of pollination guilds, pollination efficiency, as well as the pollinators' responses to land management. in this study we aimed to answer two main questions: (1) how dependent is s. melongena on bees for fruit production? (2) is it possible to observe any insufficiency of pollination in eggplant plantations? material and methods study areas the studies on the importance of bees to fruit set and the effects of crop management to fruit quality were conducted in two organic and two conventional commercial plantation of solanum melongena l., napoli cultivar, located in são paulo state, brazil (table 1). the köppen climate classification of the region is “cwa” (humid subtropical climate with dry and mild winters from april to september and warm and wet summers, from october to march) (teixeira 2004). sampling of bees samplings were performed in sunny days, between 7:00 and 18:00 h, during the peak of flowering period, in a total of 48 hours in each area. the collector walked along a line through the plantation and observed groups of six flower1040 sociobiolog y vol. 59, no. 3, 2012 ing plants at a time, for about 5 to 10 minutes. visiting bees were observed and their behavior in flowers was recorded. they were then collected with an entomological net, killed in ethyl acetate, dry mounted and identified until genus. we used silveira et al. (2002) key to identify the bees. pollination tests in order to evaluate the importance of bees for fruit formation in s. melongena and to identify insufficiency of pollination in the crops, a series of tests (t1, t2 and t3) were carried out. in order to standardize the experimental design, the tests were conducted in plots positioned in the center of each cropped area and delimited with six hundred specimens of eggplants, thus using the smaller plantation as reference (vaissière et al. 2009). all tests were realized during the peak of eggplant’s flowering. for each test, 50 flower buds in pre-anthesis stage were used. only long-styled flowers were included. in t1 (without insect visitation), buds were bagged one day before anthesis and flowers remained covered for one week to avoid visitation by insects during the fertile period. flowers were then uncovered, marked on the pedicel and the fruit development was observed for one month after when they were harvested. in t2 (free insect visitation) flower buds were marked in the pedicel and left uncovered, free to insect visitation; fruit formation was observed for a month until ripening when the fruits were then harvested. in t3 (pollen complementation), the treatment was the same as in t2, except that extra pollen were deposited on the stigma of each flower around 48 hours post-anthesis, with the aid of a brush. table 1. crop management, localization, number of specimens cultivated and period of study in the four studied areas. area: localization (crop management) coordinates studied period number of plants on cropped area i: corumbataí (conventional) 22o 13’15.33’’ s 47o 37’18.95’’ w february to april, 2010 2000 ii: corumbataí (conventional) 22o 14’29.34’’ s 47o 36’40.04’’ w september to november, 2010 1000 iii: limeira (organic) 22o 34’57.00’’ s 47o 27’36.95’’ w may to july, 2010 600 iv: ajapí (organic) 22° 18’39.46’ s 47º 32’29.55” w september to november, 2011 600 1041 patricio, g.b. et al. — the importance of bees for eggplant cultivation all the fruits formed in these tests were harvested, weighed and had the largest diameter and length measured. data analysis since there is a positive correlation between the number of seeds and the weight in eggplants (gemmill-herren & ochieng 2008), the weight of the fruits was used to indicate pollination efficiency. data were log transformed to minimize the effects of variability. in order to verify if there was statistical difference among the mean weight of eggplants in the experiments, a two-way anova and a posteriori tukey hsd t-test were performed, in which the factors considered were the different treatments (free insect visitation [t2] vs. pollen complementation [t3]) and the different areas. this analysis allowed two different approaches: (1) to compare the mean weight of eggplants between the two treatments in each area separately and consequently to test the hypothesis of pollination insufficiency; (2) to compare the mean weight of t2 among areas in order to verify the effect of a specific land management on the weight of the eggplants and, consequently, on the pollination efficiency. results sampling of bees the species of bees collected in flowers of eggplants are presented in table 2. most of the visiting bees collected on eggplant flowers are species that can perform buzz pollination by vibrating the tubular anthers and releasing pollen through apical pores, such as those of bombus, xylocopa, exomalopsis, centris, oxaea and many species of halictidae. apis mellifera and trigona sp. are not able to vibrate their anthers they collect pollen fallen on petals. trigona sp. can also chew the peak of the anthers and apis mellifera, besides collecting the pollen fallen in flowers, presents a flying adaptation in which they release the pollen by grasping the tip of the anther's cone and flying up and down shaking the flower. pollination tests table 3 presents the percentage of fruit formed in the different treatments (t1, t2 and t3) and the mean weight, diameter and length of the fruits harvested in four study areas, while table 4 and fig. 2 present comparisons 1042 sociobiolog y vol. 59, no. 3, 2012 table 2. number of bee specimens collected on eggplants (s. melongena l.) flowers and their pollen gathering behavior. absence of bees. bees collected pollen gathering behavior number of individuals area i area ii area iii area iv andrenidae oxaea sp. vibrating 2 1 1 4 apidae apis mellifera theft 7 1 1 1 bombus vibrating 24 1 1 centris vibrating 1 1 centris sp.1 vibrating 3 epicharis vibrating 1 euglosisni vibrating 1 1 1 exomalopsis vibrating 2 10 23 4 thygater vibrating 5 trigona theft 6 10 10 12 xylocopa vibrating 4 15 1 3 halictidae vibrating 2 18 6 9 fig.1. top: fruit produced from t2 test (free insect visitation); bottom: fruits produced from bagged flowers (t1). 1043 patricio, g.b. et al. — the importance of bees for eggplant cultivation between the mean weight of formed fruits in the different tests (t2 vs. t3) within each study area and the comparisons between those values for fruits formed in the free insect visitation test (t2) in different studied areas. most of the bagged flowers (t1) failed to form fruits and when it happened, those fruits were much lighter and smaller than those formed from flowers left free for bee visitation or with pollen complementation (t2 and t3, respectively, table 3, fig. 1). the percentage of fruit set in t2 (free insect visitation) and t3 (pollen complementation) were quite expressive in table 3: percentage of formed fruits and mean weight, diameter and length of fruits formed in different pollination tests and in different studied areas. t1: without insect visitation test; t2 = free insect visitation test; t3 = pollen complementation test; % ff= percentage of fruit set; w = weight (g ) ; d = diameter and l = length (cm), -= none or only one fruit formed (no mean value). area t1 t2 t3 % ff w (g ) d (cm) l (cm) % ff w (g ) d (cm) l (cm) % ff w (g ) d (cm) l (cm) i 8 162.5 6.63 17.13 84 386.5 8.46 23.68 100 368.44 8.13 23.23 ii 2 98 242.41 7.07 22.57 94 218.51 6.7 20.93 iii 14 149.29 5.81 17.07 96 302.29 7.30 22.80 86 301.74 7.15 23.11 iv 0 82 492.14 8.02 25.26 72 535.39 8.53 26.46 table 4. comparisons of the mean weight of fruit set in different tests (t2 and t3) within each study area and comparisons of those values for fruit set in (t2) among those areas (two-way anova and a posteriori test tukey hsd t-test; t1 = without insect visitation test; t2 = free insect visitation test t3 = pollen complementation test). within each area t1 mean±sd(n) t2 mean±sd(n) tukey hsd p<0.05 i 386.50±85.14(40) 368.44±75.00(48) ns ii 242.40±59.33(49) 218.51±67.42(47) ns iii 302.29±119.13(48) 303.57±105.09(42) ns iv 466.92±108.47(26) 535.38±151.23(26) ns among areas (t2) i vs. ii * i vs. iii * i vs. iv * ii vs. iii * ii vs. iv * iii vs. iv * ns – no statistical significance *tukey hsd p<0.05 1044 sociobiolog y vol. 59, no. 3, 2012 all properties (table 3). however, when comparing t2 and t3 at area i, it is possible to observe an increase in the percentage of fruits formed from pollen complementation (t3). the results of statistical analyses are shown in table 4 and fig. 2. the two-way anova showed that at least one of the comparisons involving the two factors studied (different treatments and different areas) was statistically significant (f=43.78; p<0.001). tukey t-test for multiple comparisons was performed and the following results were found: (1) no statistical differences in the mean weight of eggplants were found between t2 and t3 within each area, suggesting no pollen insufficiency in the studied areas; (2) the mean weight of fruits formed from t2 differed significantly among areas, and the eggplants were heavier in the area iv (ajapí: organic). such results suggest fig. 2. boxplots indicating the distribution of weights of eggplants collected for analyses. it is possible to visualize that within the same area no significant differences were found between the free visitation test (0) and pollen complementation test (1). however, a significant variation in the weight of eggplants was found when (1) was compared among areas i, ii, iii and iv. 1045 patricio, g.b. et al. — the importance of bees for eggplant cultivation that land management affects significantly the weight of eggplants and the pollination efficiency. discussion many of the bee species collected in s. melongena flowers can be considered potential pollinators for this crop. in all of studied areas, we observed a predominance of bees capable of handling the anthers of eggplant flowers in a proper way, promoting the sonication of the anther's cone (king & buchmann 2003) and the deposition of pollen grains on the surface of the stigma. among them, large bees (romero et al. 2011) such as those of bombus, xylocopa, centris, epicharis and oxaea genus and small bees (romero et al. 2011) such as exomalopsis and many halictidae species are included (table 2). the importance of these bee species to pollination of other species of solanum was reported by different authors and is related to their ability to manipulate the poricid anthers, and also the large time spent in flower manipulation. halictidae and bees of genus exomalopsis are reported to vibrate each anther separately (avanzi & campos 1997; forni-martins et al. 1998; bezerra & machado 2003), but the simultaneous sonication of the entire anther cone, promoted by large bees, is considered to be more effective (carvalho et al. 2001; bezerra & machado 2003; gomig et al. 2007). despite a. mellifera and bees of genus trigona being considered thieves of pollen and not very effective in eggplant pollination, the described behavior of a. mellifera, which flies up and down, very close to the flower, waving the anthers, can be somewhat effective in eggplant pollination. amoako & yeboa-gyan (1991) reported that the pollination of tomatoes, peppers and eggplants by a. mellifera could increase the weight of fruits, if compared to those fruits formed without bee pollination. pollination tests emphasized the importance of bees for fruit setting in s. melongena. the exclusion of pollinator visits, by bagging flowers, induced flower drop and resulted in only a few fruits (table 3), many of them, small and malformed (fig. 1). rylski et al. (1984), pointed out that some of the fruits observed in pollinator exclusion experiments could be produced by parthenocarpy and, in these cases, small and malformed fruits should be expected. 1046 sociobiolog y vol. 59, no. 3, 2012 by contrast, the percentage of fruits formed from flowers, which received bee visitation (t2) or bee visitation plus pollen complementation (t3), was very high in all the four studied areas (table 3). gomig (2007) observed very similar results, but montemor & souza (2009) found a comparatively high percentage of fruit setting in flower bagging experiments (20%) and an unexpected low percentage of fruits formation in free visited flowers (40%). in this case, the authors didn’t specify the length of stigma flowering during the tests. s. melongena is a hetero-styled species (the flowers present short and long styles) and short-styled flowers rarely form fruits; on the other hand, about 90% of long-styled flowers result in well-formed fruits (rylski et al. 1984; kowalska, 2006). the use of short-styled flowers in the pollination tests could result in a low percentage of fruit set, even in free visitation or pollen complementation experiments. pollen complementation did not result in an increase in fruit setting success (table 3) or in heavier fruits (table 4, fig. 2), which suggests that probably, there is no pollination insufficiency in the studied sites. the only exception occurred in area i, which pollen complementation resulted in an increase in the percentage of fruit setting success. in the other areas it was possible to observe a decrease in the percentage of fruit setting when extra pollen was deposited on stigma (t3). stephenson (1981) reported that, in many plant species, a high level of pollination efficiency may cause an increase in fruit abortion rate, justified by the allocations of resources only to a few fruits, ensuring better fruit development and more viable seeds. in area i, the pollen complementation tests (t3) resulted in an increase in the percentage of fruit formation (100%), compared to the free insect visitation tests (t2, 84%), which could suggest that pollination services can be insufficient in this area. on the other hand, there was no significant difference in mean weight of the fruits formed in t2 vs.t3 (table 4, fig. 2), and the abundance of bumblebees in the area was very high bees of genus bombus are considered the most efficient pollinators of s. melongena (aback et al. 1995; kowalska 2006, 2008; gemmill-herren & ochieng 2008; montemor & souza 2009). although the presence of effective pollinators in area i suggests a higher level of pollination, pesticides, especially insecticides, were frequently used in 1047 patricio, g.b. et al. — the importance of bees for eggplant cultivation this area, even during eggplant flowering periods. pesticides can cause changes in foraging behavior of bees, by decreasing the visitation rate, the time spent in flowers or even the amount of transferred pollen grains. behavioral changes such as those were described for honeybees and bumblebees with sub lethal doses of insecticides (thompson & hunt 1999; bortolotti et al. 2003; yang et al. 2008). thompson (2003) found that the oral administration of insecticides induced errors in the wagtail dance in apis, with consequent failure in the communication of the position of food sources. according to this author, insecticides can also repel bees from flowers and disturb their orientation. brittain et al. (2010) found that the successive application of pesticides in cropped areas was followed by a decrease in richness and abundance of bee species. despite the intensive use of insecticides, area i presented the more diversified fauna of visiting bees. to better understand the relationship between pesticide use, fauna diversity and pollination effectiveness, an investigation of the history of cleaning and other disturbances in the area will be necessary. the mean weight of eggplant fruits formed from flowers that received free visitation of bees (t2) varied significantly among different areas (table 4, fig.2). since in this study we tried to understand the effects of land management on the relationship between cropped areas and pollinator services, we did not interfere with agricultural practices in any of studied areas. thus, many factors may be related to this high variability, such as the nutritional status of plants, pest control and soil irrigation. considering the importance of bees for fruit setting and the effects of the use of pesticides on bee foraging behavior, in this study, we emphasized the aspect of crop management. fruits formed in area ii presented the smaller mean weight, although no significant difference was observed when fruits formed in t2 were compared to those formed in t3 (table 4; fig. 2). during the period of this study, no pesticides were used; however, the use of insecticides in the crops, which occupied the area before eggplant cultivation, may have affected the bee fauna in that area. another aspect to be considered is the soil management. despite the fact that the needs of eggplant crops in terms of soil nutrition are well known (chen & li 1996), no soil nutrition or ph corrections were observed in that area. the same soil management was observed in area iii. if the amount of 1048 sociobiolog y vol. 59, no. 3, 2012 nutrients necessary to supplement growth of eggplants was applied to this area, it would be possible to observe an increase in the mean weight of the fruits. in area iv, on the other hand, where cropping follows organic techniques and management, nutrients are provided by organic fertilization. furthermore, the manager allows plants, which constitute pollen and nectar sources for bees, to grow around the planted area and provides nesting sites for bees, such as decayed wood, bare ground and bamboo clumps. these practices contribute to the maintenance of bees around cropped areas, even in periods when there is no flowering crop (campos et al. 2006; goulson et al. 2008). in this area, the percentage of the fruits formed in the t2 experiment was smaller (table 2 82%) when compared to the other areas, however, the mean weight of the fruits was the highest, indicating a higher pollination efficiency (table 4, fig.2). the relatively small percentage of fruit set in t2 in area iv (table 3) can be related to the presence of herbivores, such as diabrotica speciosa (coleoptera), which eats the stigmas and causes a great number of flower and fruit abortions. kessler (2011) reported that in solanum peruvianum (solanaceae), volatile organic compounds, released after attack by herbivores, could repel other insects and interfere in pollinator-plant interactions and consequently on fruit setting. another aspect to be considered is the presence of leaf cutting ants in the area; these ants cut off fruits in different development stages. since this is an organic farm, it is common to observe a greater diversity of insects associated with the crops. however, in area iv, the manager commonly reaps the fruits 15 days after the opening of flowers, in contrast to the other areas, where the fruit harvest is performed at about 30 days after flower opening. the faster fruit development indicates that the area and soil management, together, can promote a more effective harvest and contributes to pollinator conservation. conclusion the present study pointed out the importance of bees in promoting good yields in eggplant crops. land management seems to be a factor that determines a better efficiency of pollination in agricultural landscapes, ensuring the supply of pollination services in cropped areas. 1049 patricio, g.b. et al. — the importance of bees for eggplant cultivation good practices may enhance the establishment of viable populations of pollinators in the fields, but other practices, such as the continuous use of pesticides, can disrupt the crop–pollinator interactions by changing the composition of bee communities and the foraging behavior of bees, which may cause pollination insufficiency. although there is research supporting that these changes are possible, most studies were conducted in laboratory conditions. so, it is still necessary to investigate the effects of pesticides on the foraging behavior of bees (duration of visits and flower manipulation behavior) under field conditions. acknowledgments the authors gratefully acknowledge the financial support of capes and cnpq. we also give special thanks to the owners of the studied areas: mr. josué oliveira, mr. vanderlei canhone and the directory of apae (limeira) for their permission and cooperation in the studies. we finally acknowledge matheus m. roberto, iêda and benedito ap. patricio for field support. references aback, k., n. sari, m. paksoy, o. kaftanoglu & h. yeninar 1995. efficiency of bumble bees on the yield and quality of eggplant and tomato grown in unheated glasshouses. acta horticulturae 412:268-274. allen-wardell, a.g., p. bernhardt, r. bitner, a. burqez, s. bushmann, j. cane, p.a. cox, v. dalton, p. feinsinger, m. ingram, d. inouye, c.e. jones, k. kennedy, p. kevan & h. andrén 1998. effects of habitat fragmentation on birds and mammals in landscapes with different proportions of suitable habitat. oikos 71:355-366. amoako, j., k. & yeboa-gyan 1991. insecto pollination of three solanaceous vegetable crops in ghana with special reference to the hole of african honey bee (apis mellifera adansonii) for fruit set. acta horticulturae 288:255-259. avanzi, m.r. & m.j.o. campos 1997. estrutura de guildas de polinização de solanum aculeatissimum jacq. e s. variabile mart. 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[rio claro (sp)]: departamento de geografia, universidade estadual paulista “júlio de mesquita filho”. ciências agrárias e veterinárias, universidade estadual paulista p.86. 1052 sociobiolog y vol. 59, no. 3, 2012 thompson, h.m. 2003. behavioural effects of pesticides in bees their potential for use in risk assessment. ecotoxicolog y 12:317-330. thompson, h.m. & l.v. hunt 1999. extrapolating from honeybees to bumblebees in pesticide risk assessment. ecotoxicolog y 8(3):147-166. vaissière, b.e., b. freitas & b. gemmill-herren 2011. layout of experimental sites. in: vaissière, b.e., b. freitas & b. gemmill-herren (eds.) protocol to detect and asses pollination deficits in crops: a handbook for its use. food and agriculture organization of the united nation, rome, 29-30. vieira, a.r., l.r. angelocci & k. minami 1996. efeito do estresse hídrico no solo sobre a produção da berinjela (solanum melongena l.). revista brasileira de agrometeorologia 2(4):29-33. williams, i.h. 1994. the dependences of crop production within the european union on pollination by honey bees. agricultural zoolog y reviews 6:229-257. willians, c.s. 1995. conserving europe´s bees: why all the buzz? tree 10(8):309-310. yang, e.c., y.c. chuang, y.l. chen, & l.h. chang 2008. abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (hymenoptera: apidae). journal of economic entomolog y 101(6):1743-1748. doi: 10.13102/sociobiology.v62i3.655sociobiology 62(3): 364-373 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 two cleptoparasitic ant crickets (orthoptera: myrmecophilidae) that share similar host ant species differentiate their habitat use in areas of sympatry in japan introduction habitat segregation may enable potentially competing organisms to coexist in a given region (schoener, 1974). habitat segregation has been well-studied in species pairs of various taxa, including herbivorous mammals (e.g. main & coblents, 1990; conradt et al., 1999), birds (e.g. lynch et al., 1985), and aquatic organisms (e.g. hearn, 1987; leibold, 1991). however, such segregation can be interpreted as a result of either interspecific competition (e.g. chiba, 1996) or differences in habitat preference (e.g. steen et al., 2014). there are only a few cricket taxa that are myrmecophilous and myrmecophilus (orthoptera: myrmecophilidae) is one of them (kistner, 1982; ingrisch, 1995). these ant crickets live in ant nests and exploit debris, ant eggs and larvae, and other food resources in diverse ways (wheeler, 1900; wasmann, 1901; hölldobler, 1947; henderson & akre, 1986; sakai & terayama, 1995; akino et al., 1996; komatsu et al., 2009). some myrmecophilus species mimic the ant colony’s chemistry by acquiring cuticular hydrocarbons from the ants via physical contact, abstract myrmecophilus crickets (myrmecophilidae, orthoptera) are typical ant guests. in japan, about 10 species are recognized on the basis of morphological and molecular phylogenetic frameworks. we focused on two of these species, m. kinomurai and m. kubotai, and compared their host and habitat use. previous work based on a limited sampling effort suggested that these two species share some ant species as hosts, but that their habitat preferences (open versus shaded) differ. here, on the basis of exhaustive sampling across japan, we confirmed that m. kinomurai and m. kubotai do not differ in their host ant preferences: both prefer formicine ants as hosts. as for habitat preferences, m. kubotai occurred significantly more often in open habitats than in shaded ones (p < 0.05). in contrast, m. kinomurai showed no habitat preference in areas where m. kubotai did not occur. however, m. kinomurai showed an obvious preference for shaded environments in areas of sympatry with its potential competitor m. kubotai. this pattern suggests that interspecific competition between m. kinomurai and m. kubotai is a factor causing habitat differentiation in areas of sympatry. sociobiology an international journal on social insects t komatsu,1 m maruyama,2 t itino1 article history edited by gilberto m. m. santos, uefs, brazil received 10 october 2014 initial acceptance 21 january 2015 final acceptance 30 april 2015 keywords habitat segregation, host preference, competition. corresponding author t. komatsu institute of tropical agriculture kyushu university hakozaki 6-10-1, higashi-ku, fukuoka 812-8581, fukuoka, japan e-mail: corocoro1232000@yahoo.co.jp causing the ants to recognize them as nest mates (henderson & akre, 1986; sakai & terayama, 1995; akino et al., 1996). in japan, at least 10 species of myrmecophilus have been recognized on the basis of body surface structures, and each of these species has been collected from nests of specific ant species (maruyama, 2004). by using molecular phylogenetic methods, komatsu et al. (2008) detected seven well-supported mtdna lineages in japanese myrmecophilus; these lineages do not completely agree with the morphological taxonomy. komatsu et al. (2008) were able to group the lineages into at least two categories on the basis of their host specificity: specialists, which are commensally associated with a few ant species, and generalists, which are commensally associated with many ant species or genera. additionally, each mtdna lineage appeared to show a habitat use preference (komatsu et al., 2008). in this study, we investigated host and habitat use by the two most commonly occurring species, m. kinomurai (mtdna lineage d+g of komatsu et al., 2008) and m. kubotai (lineage e+f of komatsu et al., 2008), on the basis of exhaustive sampling across japan. research article ants 1 shinshu university, nagano, japan 2 the kyushu university museum, fukuoka, japan sociobiology 62(3): 364-373 (september, 2015) 365 myrmecophilus kubotai is found only in honshu and shikoku, whereas m. kinomurai is distributed widely from hokkaido to kyushu. thus, there is partial overlap in their distribution areas (maruyama, 2006). komatsu et al. (2008) investigated the host and habitat preferences of these two cricket species by random sampling in honshu, where they coexist, and reported that both m. kubotai and m. kinomurai most frequently use formicine ants as host, and one particular formicine ant species, lasius japonicus, was used by both cricket species (komatsu et al., 2008). moreover, komatsu et al. (2008) reported that the collected m. kubotai samples were from open habitats such as grassland or wasteland whereas the collected m. kinomurai samples were from shaded habitats such as forests. these results may indicate that m. kubotai and m. kinomurai are in a competitive relationship for adequate host ant species and that they differentiate their habitat to avoid encountering each other. however, because komatsu et al. (2008) sampled these two cricket species mainly in honshu, where they coexist, they could not determine whether their habitat preferences would differ between the areas of coexistence (honshu and shikoku) and areas inhabited by only one (m. kinomurai) of the two species (hokkaido and kyushu). in this study, we conducted exhaustive sampling, mainly in honshu, where these two cricket species coexist, but also in hokkaido and kyushu, where m. kubotai does not occur, and examined their habitat use and how it differed in different areas. we then compared host-ant and habitat use between the two cricket species. materials and methods collection of samples sampling was conducted from hokkaido to kyushu, japan, in 2004-2008 (appendix 1). adult or nymph myrmecophilus crickets were collected from randomlyselected ant nests. we collected as many crickets as possible by excavating the nest if it was subterranean or by spraying insect rejectant (repellent to keep mosquitoes out) into the nest if it was arboreal. the collected cricket samples were immediately preserved in absolute ethanol. when we collected samples, we recorded the habitat type (open versus shaded) where the host ant nests were located. if there were some sort of masking objects (e.g., trees) around the nest entrance (within 10m in radius), we determined the habitat as shaded. if not so, we determined the habitat as open. the collected cricket samples were identified by using field-emission scanning microscopy (jeol, jsm-6390) or mtdna sequencing (see below). the specimens were digitally micrographed without coating. voucher specimens are deposited in the institute of tropical agriculture, kyushu university, fukuoka, japan. some of the samples used in this study were also used by komatsu et al. (2008, 2010). dna analysis we used mitochondrial sequences of collected samples to distinguish the target lineages of m. kinomurai and m. kubotai (see below). komatsu et al. (2008, 2010) used the cytochrome b (cytb) gene for molecular phylogenetic analysis. however, the primers for that gene region did not successfully amplify mtdna in many of the m. kinomurai samples. therefore, we used the 16s ribosomal rna (16s rrna) gene, which has a lower substitution rate and has been used in studies of several orthopteran insects (e.g. allegrucci et al., 2005; lu & huang, 2006). dna was extracted from the hind legs of the crickets by using a dneasy blood & tissue kit (quiagen); the other body parts were preserved for morphological identification. a 511-bp fragment of the mitochondrial 16s ribosomal rna (16s rrna) gene corresponding to positions 12887–13398 in the drosophila yakuba mtdna genome was amplified by polymerase chain reaction (pcr) with the primers 16sbr (5’ ccg gtc tga act cag atc acg t -3’) and 16sar (5’cgc ctg ttt aac aaa aac at -3’) (simon et al., 1994) and using the following temperature profile: 35 cycles of 95 °c for 30 s, 55 °c for 30 s, and 72 °c for 90 s. after amplification, the pcr products were purified using a qiaquick pcr purification kit (quiagen). cycle sequencing reactions were performed with bigdye terminator ver 1.1 cycle sequencing kit on an abi 3100 automated sequencer. we used the obtained 16s rrna dataset and mega4 software (tamura et al., 2007) to reconstruct the phylogeny by the neighbor-joining (nj) method. our previous phylogenetic analysis based on the cytb gene revealed two cryptic lineages in both of the morphospecies m. kinomurai and m. kubotai (lineages d and g in m. kinomurai and lineages e and f in m. kubotai; komatsu et al., 2008, 2010), and we recognized a similar split lineage in our analysis of the 16s rrna gene. however, one 16s rrna lineage of m. kinomurai, corresponding to lineage d of komatsu et al. (2008), was quite rare and was collected from a fairly limited area. in addition, one 16s rrna lineage of m. kubotai, corresponding to lineage f of komatsu et al. (2008), almost exclusively used the myrmicine ant, tetramorium tsushimae, as host. therefore, we excluded samples of these lineages (corresponding to lineages d and f of komatsu et al., 2008) from the analysis. statistics details of the sampled ant crickets (host ant species, collected habitats, etc.) are given in appendix 1. we used one-way chi-squared test to compare the frequency of occurrence of m. kinomurai and m. kubotai in formicine ant nests relative to other ant subfamilies. we arbitrarily divided japan into six occurrence areas: hokkaido, northern honshu (tohoku), eastern honshu (kanto and chubu), western honshu (kansai and chugoku), shikoku, and kyushu. t komatsu, m maruyama, t itino two myrmecophilus species differentiate their habitat use366 we next sorted the areas into two categories: coexistence areas (eastern honshu, western honshu and shikoku) where both m. kinomurai and m. kubotai distributed and noncoexistence areas (hokkaido, northern honshu, and kyushu) where only m. kinomurai distributed. within each of the two categorized area, the preference of the two cricket species for open or shaded habitats was determined by using a oneway chi-squared test. for example, the proportion of m. kinomurai individuals collected from open habitat among all collected m. kinomurai individuals (observed proportion in open habitat) in a given area was compared to the proportion of investigated ant nests in open habitats among the total investigated ants nests (expected proportion in open habitat). if the crickets inhabited the open habitat significantly more often than expected, we judged that they “preferred” that habitat. the statistical analyses were performed with the r software package (ver. 2.3.1; r development core team, 2005). results and discussion we surveyed a total of 1250 ant colonies representing 69 species (appendix 2). these 69 species were distributed among ant subfamilies as follows: amblyoponinae (am), 1 species (2 colonies); formicinae (fo), 31 species (699 colonies); dolichoderinae (do), 2 species (17 colonies); myrmicinae (my), 24 species (459 colonies); ponerinae (po), 8 species (69 colonies); and proceratiinae (pr), 3 species (4 colonies). in addition, one m. kubotai cricket individual was collected from a termite nest, and six individuals (including one each of m. kinomurai and m. kubotai) were collected from outside ant nests (e.g. on the ground) (appendix 2). host specificity we collected a total of 880 myrmecophilus individuals (appendix 2) of the five species, m. gigas, m. kinomurai, m. kubotai, m. sapporensis, and m. tertamorii. for m. kinomurai and m. kubotai, most individuals were collected from formicine ant nests (m. kinomurai vs. m. kubotai, %, am: 0 vs. 0, fo: 93 vs. 88, do: 0 vs. 0, my: 5 vs. 8, po: 0 vs. 0, pr: 0 vs. 0). in addition, one m. kubotai cricket individual was collected from a termite nest, and six individuals (including one each of m. kinomurai and m. kubotai) were collected from outside ant nests (e.g. on the ground) (appendix 2). thus, we confirmed the previous finding of komatsu et al. (2008) that m. kinomurai and m. kubotai preferred formicine ants as hosts. we detected no significant difference in preference toward formicinae between m. kinomurai and m. kubotai (chi-squared test for fo and my, p = 0.54). further, the most-preferred host ant species was the same (lasius japonicus) between these two cricket species (m. kinomurai, 25 of 58 hosts; m. kubotai 24 of 52 hosts). myrmecophilus kinomurai was collected in all six occurrence areas of japan, but m. kubotai was collected only in eastern honshu, western honshu, and shikoku (not in hokkaido, northern honshu, or kyushu). phylogenetic analysis the dna lineages of the m. kinomurai and m. kubotai samples were determined by nj analysis of a 530-bp sequence of the mitochondrial 16s rrna gene. of the 110 analyzed samples, 58 belonged to m. kinomurai (corresponding to lineage g of komatsu et al., 2008) and 52 belonged to m. kubotai (corresponding to lineage e of komatsu et al., 2008). habitat preferences in areas of sympatry with the closely related m. kinomurai (i.e. coexistence areas), m. kubotai occurred significantly more often in open habitats, whereas m. kinomurai showed an obvious preference for shaded environments (table 1, fig 1). in contrast, where m. kubotai sampling area species (no. of samples) environment (no. of samples) expected proportion (no. of investigated ant nests in that habitat / total investigated ant nests) observed proportion (no. of crickets collected from that habitat / total collected crickets ) p non-coexistence area m. kinomurai (27) open (19) 250/393 19/27 ns shaded (8) 143/393 8/27 m. kubotai (0) open (0) shaded (0) coexistence area m. kinomurai (31) open (7) 487/857 7/31 + +(s) ++(o) shaded (24) 370/857 24/31 m. kubotai (52) open (43) 487/857 43/52 　 　 shaded (9) 370/857 9/52 table 1. habitat specificity of the ant crickets determined by chi-squared test for biased habitat preferences of the ant crickets in those areas where only m. kinomurai occurred and where both crickets were distributed. if the crickets inhabited open (or shaded) habitats in a significantly higher proportion than the expected proportion (i.e., the proportion of that habitat among total available ant nests), they were judged to “prefer” that habitat. see text for details. ++, p < 0.01; ns, not significant. o, open habitat; s: shaded habitat. sociobiology 62(3): 364-373 (september, 2015) 367 did not occur, m. kinomurai did not show an obvious preference for either habitat type (table 1, fig 1). these results suggest that interspecific competition between m. kinomurai and m. kubotai is a factor causing m. kinomurai to shift its habitat use in areas of sympatry. this observed habitat shift of m. kinomurai in areas of sympatry with its potential competitor m. kubotai can be interpreted in two ways. one possible interpretation is that the primary host ant species shows a habitat difference between coexistence areas (e.g. western honshu) and non-coexistence areas (e.g. hokkaido). in this study, many m. kinomurai individuals were collected from nests of lasius japonicus (appendix 2). the studied nests of l. japonicus were found mainly in open habitats both in coexistence areas (number of nests in open vs. shaded habitat, 99 vs. 35) and non-coexistence areas (99 vs. 10) although the habitat preference m. kinomurai was different between the areas (chi-squared test, p < 0.01). therefore, it is unlikely that the observed habitat difference of m. kinomurai between the two types of areas reflected a habitat difference of the primary host ant species between them. a second possible interpretation is that in the coexistence areas m. kinomurai shifts to a host ant species inhabiting open habitats rather than shaded habitats because of interspecific competition in those areas. in fact, the host ant species used by m. kinomurai differed significantly between the coexistence areas and non-coexistence areas (co-existence areas vs. noncoexistence areas, number of samples: aj, 1 vs. 0; cj, 1 vs. 2; co, 0 vs. 1; fj, 1 vs. 2; fs, 1 vs. 0; fy, 1 vs. 0; lc, 1 vs. 0; lf, 5 vs. 3; lj, 6 vs. 19; lni, 4 vs. 0; lsp, 5 vs. 0; lu, 1 vs. 0; pl, 1 vs. 0; ppu, 1 vs. 0; tt, 1 vs. 0; out, 1 vs. 0; chi-squared test; p = 0.03; see appendix 1 for host ant species codes). in coexistence areas, m. kinomurai tended not to use l. japonicus, an openland dweller (as mentioned above); instead, it used shaded habitat dwellers such as l. nipponensis and l. spathepus. moreover, most m. kinomurai were collected from shaded environments in coexistence areas (fig 1). therefore, in the coexistence areas m. kinomurai may shift to a host ant species preferring shaded environments. divergence of habitat use resulting from interspecific competition between sympatric species has been reported before (i.e., a form of ecological character displacement; chiba, 1996). because m. kinomurai and m. kubotai are dominant species and share the same host ant taxon (formicinae), it is plausible that there is competitive interaction between the two species. because myrmecophilus crickets use basically the same resource, the co-occurrence of more than one myrmecophilus species within the same ant nest may threaten the survival of one the cricket species. thus, m. kinomurai may shift its habitat by shifting its host ant species from an open habitat-dwelling ant species to a shaded habitat-dwelling species in areas in which it coexists with m. kubotai. such avoidance may have another advantage. at least in japanese myrmecophilus, the morphological differentiation of genital characters among species is too minor to be used for taxonomic differentiation (maruyama, 2006), which suggests that different species of japanese myrmecophilus can potentially mate with each other. therefore, differentiation of habitat or host use, or both, may function as a premating isolation mechanism in this genus (i.e., a form of reproductive character displacement). the nature of the ecological or reproductive competition between m. kinomurai and m. kubotai remains unknown. the adverse effects of interactions between these species on their fitness and survival need to be investigated by examining interspecific behavioral interactions and their effect on fertility and survival experimentally, for example, by artificially introducing both species into a single host ant colony. acknowledgments we thank s. ueda for his comments on the manuscript, and t. befu, y. hagiwara, k. harukawa, a. ichikawa, y. itino, s. nomura, k. kinomura, y. koshiyama, t. kurihara, k. maruyama, y. mori, t. ito, t. takagi, s. takaishi, and y. tsuneoka for sampling. this work was supported by jsps kakenhi grant numbers 19-6495, 26-932 to t. k., and by research and education funding for japanese alps inter-universities cooperative project, ministry of education, culture, sports, science and technology, japan to t. i. references akino, t., mochizuki, r., morimoto, m. & yamaoka, r. 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(1900). the habits of myrmecophila nebrascensis bruner. psyche, 9: 111-115. sociobiology 62(3): 364-373 (september, 2015) 369 sp ec ie s sa m pl e n o. h os t a nt l oc al ity ( ita lic s in di ca te no nco ex is te nc e ar ea s) h ab ita t d at e (y y. m m .d d) c ol le ct or a lti tu de ( m ) a cc es si on n o. m . k in om ur ai 19 8b fj a sa m us hi on se n, a om or i, a om or i s 05 .0 4. 29 k om at su t 11 2 a b 81 82 61 m . k in om ur ai h9 9a l j k ita , s ap po ro , h ok ka id o s 06 .0 9. 08 k om at su t 15 a b 81 82 57 m . k in om ur ai h9 9b l j k ita , s ap po ro , h ok ka id o s 06 .0 9. 08 k om at su t 15 a b 81 82 58 m . k in om ur ai h8 4 l j ta ka do m ar i, f uk ag aw a, h ok ka id o s 06 .0 9. 06 k om at su t 10 0 a b 81 82 59 m . k in om ur ai h8 5 l j ta ka do m ar i, f uk ag aw a, h ok ka id o s 06 .0 9. 06 k om at su t 10 0 a b 81 82 60 m . k in om ur ai h3 0a l j to yo ok a, h id ak a, h ok ka id o s 06 .0 8. 29 k om at su t 25 0 a b 81 82 62 m . k in om ur ai 10 27 l j k am ia ga ta , t su sh im a, n ag as ak i s 08 .0 4. 26 k om at su t 50 a b 81 82 91 m . k in om ur ai 10 54 l j k am ia ga ta , t su sh im a, n ag as ak i s 08 .0 4. 27 k om at su t 50 a b 81 82 92 m . k in om ur ai 20 0a c j sa nn ai , a om or i, a om or i o 05 .0 6. 19 k om at su t 21 .5 a b 81 82 45 m . k in om ur ai 20 1 c j sa nn ai , a om or i, a om or i o 05 .0 6. 19 k om at su t 21 .5 a b 81 82 46 m . k in om ur ai 25 6a c o h ita ch in ai , a ni , a ki ta o 05 .0 8. 26 k om at su t 25 0 a b 81 82 56 m . k in om ur ai h1 8 fj to ya -k o, s ob et su , h ok ka id o o 04 .0 8. 28 k om at su t 25 0 a b 81 82 54 m . k in om ur ai h9 7a l f k ita , s ap po ro , h ok ka id o o 06 .0 9. 08 k om at su t 15 a b 81 82 43 m . k in om ur ai h5 8a l f p in ne si ri , n ak at on be ts u, h ok ka id o o 06 .0 9. 04 k om at su t 63 a b 81 82 49 m . k in om ur ai h5 8c l f p in ne si ri , n ak at on be ts u, h ok ka id o o 06 .0 9. 04 k om at su t 63 a b 81 82 50 m . k in om ur ai 20 2 l j ta ka no za ki , i m ab et su , a om or i o 05 .0 6. 21 k om at su t 0 a b 81 82 39 m . k in om ur ai h1 7a l j m in am iu su , d at e, h ok ka id o o 06 .0 8. 28 k om at su t 5 a b 81 82 40 m . k in om ur ai h1 7b l j m in am iu su , d at e, h ok ka id o o 06 .0 8. 28 k om at su t 5 a b 81 82 41 m . k in om ur ai h1 7c l j m in am iu su , d at e, h ok ka id o o 06 .0 8. 28 k om at su t 5 a b 81 82 42 m . k in om ur ai h9 6 l j k ita , s ap po ro , h ok ka id o o 06 .0 9. 08 k om at su t 15 a b 81 82 44 m . k in om ur ai 17 5 l j sa nn ai , a om or i, a om or i o 04 .0 8. 12 k om at su t 22 a b 81 82 47 m . k in om ur ai 17 6 l j sa nn ai , a om or i, a om or i o 04 .0 8. 13 k om at su t 22 a b 81 82 48 m . k in om ur ai h5 9g l j p in ne si ri , n ak at on be ts u, h ok ka id o o 06 .0 9. 04 k om at su t 63 a b 81 82 51 m . k in om ur ai h5 9h l j p in ne si ri , n ak at on be ts u, h ok ka id o o 06 .0 9. 04 k om at su t 63 a b 81 82 52 m . k in om ur ai 18 0b l j ta pp i, m in m ay a, a om or i o 04 .0 8. 21 k om at su t 80 a b 81 82 53 m . k in om ur ai h2 0a l j to ya -k o, s ob et su , h ok ka id o o 04 .0 8. 28 k om at su t 25 0 a b 81 82 55 m . k in om ur ai 17 7a l j h ak ko ud a, a om or i, a om or i o 04 .0 8. 14 k om at su t 13 00 a b 81 82 63 m . k in om ur ai 18 2 a j sa to ya m ab e, m at su m ot o, n ag an o s 04 .0 9. 13 k om at su t 65 0 a b 81 82 77 a pp en di x 1 . o ve rv ie w o f th e sa m pl ed s pe ci m en s of m . k in om ur ai ( m td n a c yt b lin ea ge g ) an d m . k ub ot ai ( m td n a c yt b lin ea ge e ) (h ab ita t p re fe re nc es a re s um m ar iz ed in f ig . 1 ) an d th ei r 16 s g en b an k ac ce ss io n nu m be rs . h os t a nt s pe ci es c od es : c j, c am po no tu s ja po ni cu s; c o, c am po no tu s ob sc ur ip es ; f j, f or m ic a ja po ni ca ; f s, f or m ic a sa ng ui ne a; f y, f or m ic a ye ss en si s; l c, l as iu s ca pi ta tu s; l f, la si us fl av us ; l fu , l as iu s fu ji; l j, la si us ja po ni cu s; l ni , l as iu s ni pp on en si s; l sa , l as iu s sa ka ga m ii; l sp , l as iu s sp at he pu s; l u, l as iu s um br at us ; m j, m yr m ic a je ss en si s; m k, m yr m ic a ko to ku i; pl , p ol yr ha ch is la m el lid en s; p pu , p ri st om yr m ex p un ct at us ; p s, p ol ye rg us s am ur ai ; t rs , r et ic ul ite rm es s pe ra tu s; t t, te tr am or iu m ts us hi m ae ; a j, a ph ae no ga st er ja po ni ca ; o ut , c ol le ct ed fr om o ut si de a n an t n es t. o , o pe n ha bi ta t; s, s ha de d ha bi ta t; -, no d at a. * , m . k in om ur ai m td n a li ne ag e d ; * *, m . k ub ot ai m td n a li ne ag e f. t komatsu, m maruyama, t itino two myrmecophilus species differentiate their habitat use370 sp ec ie s sa m pl e n o. h os t a nt l oc al ity ( ita lic s in di ca te no nco ex is te nc e ar ea s) h ab ita t d at e (y y. m m .d d) c ol le ct or a lti tu de ( m ) a cc es si on n o. m . k in om ur ai 12 fj sa nd an i, ta ka ya m a, g if u s 04 .0 6. 27 m ar uy am a m 11 00 a b 81 82 88 m . k in om ur ai 10 6 fs sa to ya m ab e, m at su m ot o, n ag an o s 04 .0 5. 24 k om at su t 65 0 a b 81 82 76 m . k in om ur ai 27 1 l c sa to ya m ab e, m at su m ot o, n ag an o s 06 .0 7. 07 k om at su t 63 0 a b 81 82 74 m . k in om ur ai 44 c j sa to ya m ab e, m at su m ot o, n ag an o s 04 .0 4. 17 k om at su t 65 0 a b 81 82 75 m . k in om ur ai s5 0a l f k am ih an ba ra , o no , f uk ui s 08 .0 9. 16 k os hi ya m a y 90 0 a b 81 82 78 m . k in om ur ai s5 0b l f k am ih an ba ra , o no , f uk ui s 08 .0 9. 16 k os hi ya m a y 90 0 a b 81 82 79 m . k in om ur ai s5 0c l f k am ih an ba ra , o no , f uk ui s 08 .0 9. 16 k os hi ya m a y 90 0 a b 81 82 80 m . k in om ur ai 30 l j m iy am ot oh ig as hi ka ta , n ag ao ka , n iig at a s 04 .0 4. 11 k om at su t 60 a b 81 82 64 m . k in om ur ai 17 1b l j h ig as hi ka w ad e, a zu m in o, n ag an o s 04 .0 6. 30 k om at su t 62 0 a b 81 82 73 m . k in om ur ai 20 3 l j ir iy am ab e, m at su m ot o, n ag an o s 05 .0 7. 01 k om at su t 14 00 a b 81 82 90 m . k in om ur ai 13 6b l ni a ri ga sa ki , m at su m ot o, n ag an o s 04 .0 6. 03 k om at su t 58 5 a b 81 82 68 m . k in om ur ai 13 6d l ni a ri ga sa ki , m at su m ot o, n ag an o s 04 .0 6. 03 k om at su t 58 5 a b 81 82 69 m . k in om ur ai 16 7 l ni a ri ga sa ki , m at su m ot o, n ag an o s 04 .0 6. 24 k om at su t 58 5 a b 81 82 70 m . k in om ur ai 17 2 l ni a ri ga sa ki , m at su m ot o, n ag an o s 04 .0 7. 01 k om at su t 58 5 a b 81 82 71 m . k in om ur ai s2 1a l sp im am ur a, n eo , g if u s 04 .0 5. 08 m ar uy am a m 20 4 a b 81 82 66 m . k in om ur ai s2 1d l sp im am ur a, n eo , g if u s 04 .0 5. 08 m ar uy am a m 20 4 a b 81 82 67 m . k in om ur ai s1 2d l sp to go , o ki , s hi m an e s 04 .0 8. 02 k om at su t 62 a b 81 82 93 m . k in om ur ai s4 3a l sp te nn oza n, o ya m az ak i, k yo to s 08 .0 9. 05 it o t 27 0 a b 81 82 94 m . k in om ur ai s4 3b l sp te nn oza n, o ya m az ak i, k yo to s 08 .0 9. 05 it o t 27 0 a b 81 82 95 m . k in om ur ai 19 9b l u ir iy am ab e, m at su m ot o, n ag an o s 05 .0 6. 06 k om at su t 14 00 a b 81 82 89 m . k in om ur ai s3 b pl h at og ay a, s hi ra ka w a, g if u s 05 .0 5. 25 m ar uy am a m 60 0 a b 81 82 72 m . k in om ur ai s3 9 pp u k uc hi ka m og aw a, s hi m an to , k ou ch i s 08 .0 3. 16 b ef u t 30 0 a b 81 82 96 m . k in om ur ai s5 1 ou t n ak ao ku bo , t oy am a, t oy am a s 08 .1 0. 21 k os hi ya m a y 82 a b 81 82 65 m . k in om ur ai 45 5a fy n ag as ak a, s ak ae , n ag an o o 07 .0 5. 04 k om at su t 10 00 a b 81 82 81 m . k in om ur ai 83 2b l f o ku hi da on se n, t ak ay am a, g if u o 07 .0 8. 17 k om at su t 12 50 a b 81 82 83 m . k in om ur ai 15 1 l f ir iy am ab e, m at su m ot o, n ag an o o 04 .0 6. 13 k om at su t 14 00 a b 81 82 87 m . k in om ur ai 45 8 l j n ag as ak a, s ak ae , n ag an o o 07 .0 5. 04 k om at su t 10 00 a b 81 82 82 m . k in om ur ai 20 6a l j h iji ri ko ge n, o m im ur a, n ag an o o 05 .0 7. 13 k om at su t 12 60 a b 81 82 84 a pp en di x 1. o ve rv ie w o f th e sa m pl ed s pe ci m en s of m . k in om ur ai ( m td n a c yt b lin ea ge g ) an d m . k ub ot ai ( m td n a c yt b lin ea ge e ) (h ab ita t p re fe re nc es a re s um m ar iz ed in f ig . 1 ) an d th ei r 16 s g en b an k ac ce ss io n nu m be rs . h os t a nt s pe ci es c od es : c j, c am po no tu s ja po ni cu s; c o, c am po no tu s ob sc ur ip es ; f j, f or m ic a ja po ni ca ; f s, f or m ic a sa ng ui ne a; f y, f or m ic a ye ss en si s; l c, l as iu s ca pi ta tu s; l f, la si us fl av us ; l fu , l as iu s fu ji; l j, la si us ja po ni cu s; l ni , l as iu s ni pp on en si s; l sa , l as iu s sa ka ga m ii; l sp , l as iu s sp at he pu s; l u, l as iu s um br at us ; m j, m yr m ic a je ss en si s; m k, m yr m ic a ko to ku i; pl , p ol yr ha ch is la m el lid en s; p pu , p ri st om yr m ex p un ct at us ; p s, p ol ye rg us s am ur ai ; t rs , r et ic ul ite rm es s pe ra tu s; t t, te tr am or iu m ts us hi m ae ; a j, a ph ae no ga st er ja po ni ca ; o ut , c ol le ct ed fr om o ut si de a n an t n es t. o , o pe n ha bi ta t; s, s ha de d ha bi ta t; -, no d at a. * , m . k in om ur ai m td n a li ne ag e d ; * *, m . k ub ot ai m td n a li ne ag e f. (c on tin ua tio n) sociobiology 62(3): 364-373 (september, 2015) 371 sp ec ie s sa m pl e n o. h os t a nt l oc al ity ( ita lic s in di ca te no nco ex is te nc e ar ea s) h ab ita t d at e (y y. m m .d d) c ol le ct or a lti tu de ( m ) a cc es si on n o. m . k in om ur ai 11 5 l j ir iy am ab e, m at su m ot o, n ag an o o 04 .0 5. 29 k om at su t 14 00 a b 81 82 86 m . k in om ur ai 20 7 t t h iji ri ko ge n, o m im ur a, n ag an o o 05 .0 7. 13 k om at su t 12 60 a b 81 82 85 m . k ub ot ai 44 0 fj u en o, t ai to , t ok yo s 07 .0 4. 21 k om at su t 10 a b 81 83 27 m . k ub ot ai 12 63 c l fu y at a, m is hi m a, s hi zu ok a s 08 .0 8. 12 k om at su t 15 a b 81 83 28 m . k ub ot ai 12 63 d l fu y at a, m is hi m a, s hi zu ok a s 08 .0 8. 12 k om at su t 15 a b 81 83 29 m . k ub ot ai s4 0 l j h at to ri -r yo ku ch i, to yo na ka , o sa ka s 08 .0 5. 30 ic hi ka w a a 26 a b 81 83 44 m . k ub ot ai s4 2a l j a ka sh ik ou en , a ka sh i, h yo go s 08 .0 9. 06 ic hi ka w a a 28 a b 81 83 45 m . k ub ot ai s3 7a l j c hi sh im a, o sa ka , o sa ka s 08 .0 5. 11 ic hi ka w a a 33 a b 81 83 46 m . k ub ot ai s3 8 l j c hi sh im a, o sa ka , o sa ka s 08 .0 5. 11 ic hi ka w a a 33 a b 81 83 47 m . k ub ot ai 65 8 l j a id o, j in ze ki ko ge n, h ir os hi m a s 07 .0 6. 05 k om at su t 52 5 a b 81 83 48 m . k ub ot ai 18 3b l ni a ri ga sa ki , m at su m ot o, n ag an o s 04 .0 9. 22 k om at su t 58 5 a b 81 83 30 m . k ub ot ai 36 2a c j a w as hi m a, n um az u, s hi zu ok a o 05 .1 0. 20 k om at su t 0 a b 81 82 97 m . k ub ot ai 36 2b c j a w as hi m a, n um az u, s hi zu ok a o 05 .1 0. 20 k om at su t 0 a b 81 82 98 m . k ub ot ai 52 c j ta ki be , a zu m in o, n ag an o o 04 .0 4. 21 k om at su t 57 1 a b 81 83 13 m . k ub ot ai 16 8 c j a sa hi , m at su m ot o, n ag an o o 04 .0 6. 25 k om at su t 62 0 a b 81 83 18 m . k ub ot ai 91 4a c j n ad a, k ob e, h yo go o 07 .0 9. 16 k om at su t 25 0 a b 81 83 39 m . k ub ot ai 60 fj sh im ok an uk i, n um az u, s hi zu ok a o 04 .0 4. 23 k om at su t 0 a b 81 83 01 m . k ub ot ai s2 7 fj is hi ok a, i ba ra ki o ts un eo ka y 10 0 a b 81 83 06 m . k ub ot ai 24 1a fj a sa hi , m at su m ot o, n ag an o o 05 .0 9. 30 k om at su t 62 0 a b 81 83 14 m . k ub ot ai 24 1b fj a sa hi , m at su m ot o, n ag an o o 05 .0 9. 30 k om at su t 62 0 a b 81 83 15 m . k ub ot ai 97 b fj a sa hi , m at su m ot o, n ag an o o 04 .0 5. 18 k om at su t 62 0 a b 81 83 16 m . k ub ot ai 97 d fj a sa hi , m at su m ot o, n ag an o o 04 .0 5. 18 k om at su t 62 0 a b 81 83 17 a pp en di x 1. o ve rv ie w o f th e sa m pl ed s pe ci m en s of m . k in om ur ai ( m td n a c yt b lin ea ge g ) an d m . k ub ot ai ( m td n a c yt b lin ea ge e ) (h ab ita t p re fe re nc es a re s um m ar iz ed in f ig . 1 ) an d th ei r 16 s g en b an k ac ce ss io n nu m be rs . h os t a nt s pe ci es c od es : c j, c am po no tu s ja po ni cu s; c o, c am po no tu s ob sc ur ip es ; f j, f or m ic a ja po ni ca ; f s, f or m ic a sa ng ui ne a; f y, f or m ic a ye ss en si s; l c, l as iu s ca pi ta tu s; l f, la si us fl av us ; l fu , l as iu s fu ji; l j, la si us ja po ni cu s; l ni , l as iu s ni pp on en si s; l sa , l as iu s sa ka ga m ii; l sp , l as iu s sp at he pu s; l u, l as iu s um br at us ; m j, m yr m ic a je ss en si s; m k, m yr m ic a ko to ku i; pl , p ol yr ha ch is la m el lid en s; p pu , p ri st om yr m ex p un ct at us ; p s, p ol ye rg us s am ur ai ; t rs , r et ic ul ite rm es s pe ra tu s; t t, te tr am or iu m ts us hi m ae ; a j, a ph ae no ga st er ja po ni ca ; o ut , c ol le ct ed fr om o ut si de a n an t n es t. o , o pe n ha bi ta t; s, s ha de d ha bi ta t; -, no d at a. * , m . k in om ur ai m td n a li ne ag e d ; * *, m . k ub ot ai m td n a li ne ag e f. (c on tin ua tio n) t komatsu, m maruyama, t itino two myrmecophilus species differentiate their habitat use372 ant subfamily (no. of species) genus species no. of crickets found (total nests surveyed) no. of m. kinomurai found no. of m. kubotai found amblyoponinae (1) amblyopone a. silvestrii 0 (2) 0 0 formicinae (31) acropyga a. nipponensis 0 (3) 0 0 camponotus c. devestivus 0 (3) 0 0 c. japonicus 13 (43) 3 5 c. kiusiuensis 0 (2) 0 0 c. nawai 0 (1) 0 0 c. nipponicus 0 (1) 0 0 c. obscuripes 3 (4) 1 0 c. vitiosus 0 (4) 0 0 formica f. fukaii 0 (1) 0 0 f. hayashi 13 (40) 0 0 f. japonica 206 (115) 3 9 f. lemani 0 (15) 0 0 f. sanguinea 5 (2) 1 0 f. yessensis 4 (8) 1 0 lasius l. capitatus 3 (3) 1 1 l. flavus 56 (46) 8 0 　 　 l. fuji 6 (6) 0 2 lasius l. hayashi 1 (22) 0 0 l. japonicus 221 (243) 25 24 l. nipponensis 13 (5) 4 3 l. orientalis 5 (2) 0 0 l. productus 0 (1) 0 0 l. sakagamii 5 (15) 0 2 l. spathepus 18 (9) 5 0 l. talpa 0 (3) 0 0 l. umbratus 2 (7) 1 0 nylanderia n. flavipes 0 (87) 0 0 n. sakurae 0 (5) 0 0 plagiolepis p. flavescens 0 (1) 0 0 polyrhachis p. lamellidens 3 (1) 1 0 prenolepis p. sp. 0 (1) 0 0 dolichoderinae (2) ochetellus o. glaber 0 (15) 0 0 technomyrmex t. gibbosus 0 (2) 0 0 appendix 2. overview of ant nests surveyed and numbers of ant crickets found. sociobiology 62(3): 364-373 (september, 2015) 373 ant subfamily (no. of species) genus species no. of crickets found (total nests surveyed) no. of m. kinomurai found no. of m. kubotai found myrmicinae (24) aphaenogaster a. japonica 4 (23) 1 0 cardiocondyla c. kagutsuchi 0 (1) 0 0 crematogaster c. matsumurai 0 (4) 0 0 c. osakensis 0 (21) 0 0 c. vagula 0 (7) 0 0 myrmica m. jessensis 1 (1) 0 1 myrmica m. kotokui 6 (38) 0 1 m. taediosa 0 (2) 0 0 pheidole p. fervida 1 (51) 0 0 p. noda 0 (26) 0 0 p. pieli 0 (5) 0 0 pristomyrmex p. punctatus 2 (36) 1 0 pyramica p. benten 0 (4) 0 0 p. hexamera 0 (1) 0 0 p. membranifera 0 (1) 0 0 solenopsis s. japonica 0 (9) 0 0 　 monomorium m. intrudens 0 (4) 0 0 carebara c. yamatonis 0 (2) 0 0 strumigenys s. lewisi 0 (25) 0 0 temnothorax t. spinosior 0 (2) 0 0 tetramorium t. bicarinatum 0 (3) 0 0 t. tsushimae 281 (159) 1 2 vollenhovia v. emeryi 0 (10) 0 0 ponerinae (8) cryptopone c. sauteri 0 (8) 0 0 hypoponera h. nubatama 0 (6) 0 0 h. sauteri 0 (1) 0 0 pachycondyla p. chinensis 0 (35) 0 0 pachycondyla p. javana 0 (5) 0 0 p. pilosior 0 (11) 0 0 ponera p. japonica 0 (1) 0 0 p. scabra 0 (2) 0 0 proceratiinae (3) proceratium p. itoi 0 (1) 0 0 p. japonicum 0 (1) 0 0 　 　 p. watasei 0 (2) 0 0 termite (1) reticulitermes r. speratus 1 0 1 outside nest 6 1 1 total 　 　 880 (1250) 58 52 appendix 2. overview of ant nests surveyed and numbers of ant crickets found (continuation). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5227sociobiology 68(1): e-5227 (march, 2021) introduction between 15,000 and 20,000 plant species, including agricultural species, have flowers with poricidal dehiscence anthers (e.g. de luca & vallejo-marín, 2013) and foraging insects usually release the plant pollen through thorax vibration in a behavior known as buzzing (buchmann, 1983; thorp, 2000; portman et al., 2019). alternatively, some bee species “scrape” pollen (wille, 1963; thorp, 2000), using their mouthpieces in a “milking” movement to collect pollen from the poricidal flowers (portman et al., 2019). these behaviors under standard greenhouse conditions, the tomato fruits resulting from spontaneous self-pollination are expected to be of lower quality than those from bee pollination. in addition, the use of species with different behaviors is expected to change productivity differently. to test these hypotheses, we evaluated the pollination services from the use of three native stingless bee species with distinct foraging behaviors, melipona bicolor lepeletier, 1836, nannotrigona testaceicornis (lepeletier, 1836), and partamona helleri (friese, 1900) during the blooming period of cherry tomatoes in greenhouses. fruit quality parameters resulting from pollination experiments were measured and the acclimatization of the analyzed bee species was evaluated. visits of m. bicolor and n. testaceicornis to the tomato flowers contributed significantly to increases in the average weight, seed number, and thickness of the pericarp (only for n. testaceicornis) of the fruits, compared to the spontaneous selfpollination treatment. partamona helleri, however, did not show any pollen collection behavior in the experimental conditions. although n. testaceicornis did not perform the buzzing behavior, fruits from its pollination were equivalent to fruits from pollination by m. bicolor. the use of bee species with different flower-visiting behaviors can optimize tomato pollination in greenhouses and help to standardize fruit weights, contributing significantly to the quality of the fruits and increasing productivity, with consequent increases in commercial value. sociobiology an international journal on social insects maira coelho moura-moraes1,2, wilson frantine-silva1, maria cristina gaglianone1, lucio antonio o. campos2 article history edited by solange augusto, ufu, brazil received 27 april 2020 initial acceptance 05 october 2020 final acceptance 31 october 2020 publication date 22 february 2021 keywords buzzing, scraping, melipona, nannotrigona, protected cultivation, solanum lycopersicum. corresponding author maira c. moura-moraes https://orcid.org/0000-0002-0518-5193 universidade estadual do norte fluminense darcy ribeiro – uenf centro de biociências e biotecnologia, laboratório de ciências ambientais av. alberto lamego, 2000, parque califórnia cep 28013-602, campos dos goytacazes, rio de janeiro, brasil. e-mail: mcoelhobio@gmail.com performed by a high diversity of pollinators is a key factor for quality enhancement in crop production (westerkamp & gottsberger, 2000), including tomatoes (depra et al., 2014; gaglianone et al., 2018). the tomato (solanum lycopersicum l.) is an autogamous solanaceae with functionally poricidal anthers that must be vibrated to release the pollen grains through the apical pores (mcgregor, 1976). in open-field tomato crops, the wind action and bee visitation can promote the necessary vibration and pollen deposition on the flower stigma (free, 1993; depra et al., 2014; gaglianone et al., 2018). on the 1 laboratório de ciências ambientais – lca, programa de pós-graduação em ecologia e recursos naturais, universidade estadual do norte fluminense darcy ribeiro – uenf, campos dos goytacazes, rio de janeiro, brazil 2 departamento de entomologia, universidade federal de viçosa, viçosa, minas gerais, brazil research article bees the use of different stingless bee species to pollinate cherry tomatoes under protected cultivation abstract mc moura-moraes, w frantine-silva, mc gaglianone, lao campos – stingless bee pollination in cherry tomatoes2 other hand, greenhouse crops have a scarcity of wind and animal pollination conditions that are usually counterbalanced by mechanically induced vibration and airflow (bell et al., 2006). these pollination techniques increase production costs and represent losses to the commercial value (picken, 1984; westerkamp & gottsberger, 2000; cruz & campos, 2009). therefore, in recent years, the management of tomato pollinators in greenhouse crops has been tested through the introduction of bees including amegilla (notomegilla) chlorocyanea cockerell (hogendoorn et al., 2006); apis mellifera l. (bispo dos santos et al., 2009); xylocopa species (reviewed in velthuis & van doorn, 2006); and the stingless bees nannotrigona perilampoides (cresson) (cauich et al., 2004; palma et al., 2008) and melipona quadrifasciata lepeletier (del sarto et al., 2005; bispo dos santos et al., 2009; hikawa & miyanaga, 2009; bartelli & nogueira-ferreira, 2014; silva-neto et al., 2018). the stingless bees partamona helleri (friese), melipona bicolor lepeletier, and nannotrigona testaceicornis lepeletier display different behavior during visits to poricidal anthers, buzzing (p. helleri and m. bicolor) and scraping (n. testaceicornis), and there is no information about these species on greenhouse crops. although n. testaceicornis has been surveyed in some openfield crops (dos santos et al., 2008; cruz & campos, 2009) and p. helleri in wild plants in brazil (lopes de carvalho et al., 1999, ramalho, 2004, taura & laroca, 2004, azevedo et al., 2007, carvalho, 2007), we did not find any data about their interaction with plants in greenhouses. interest in managing stingless bees for pollination of agricultural crops has increased considerably in recent years. this is due to the high richness of species, their little aggressive behavior with easy and known handling and multiplication of colonies (nogueira-neto, 1997). in the current study, we evaluated the viability of the stingless bees melipona bicolor, nannotrigona testaceicornis, and partamona helleri for pollination of cherry tomatoes (solanum lycopersicum) in greenhouses, as well as their effectiveness in simultaneous use. our expectation was that pollination by more than one bee species would enhance the fruit set and quality of the fruits produced. material and methods crop implementation the study was conducted in two greenhouses at the federal university of viçosa (20°45’14”s e 42°52’53”w), municipality of viçosa, mg (brazil), implementing crops between april and july 2014. cherry tomato seedlings (hybrid “chipano®” red) were obtained by sowing and after 30 days, 200 seedlings were transplanted to 8 kg pots filled with commercial vegetable substrate, individually fertilized, and equally distributed in two vegetation greenhouses (100 in each). the tomato plants were carried with only one stem, eliminating all lateral shoots. the anthesis of the flowers started 30 days after transplanting the seedlings. we used arch-shaped greenhouses 3.2 m high at the sides and 5.2 m at the top (total area of 108 m2), closed at the sides with 50 mesh antivirus screen, covered with 150 micron light diffuser antiuv agricultural film. an automated irrigation system was programmed to irrigate plants twice a day with about 600 ml of water to each pot. the cover fertilization was applied to each pot every 20 days with 40 g of ammonium sulfate. temperature and relative humidity data were obtained over 24 hours using data logger (hobo® u10). the experiment lasted 120 days. beehives placement we selected strong nests (with large numbers of individuals) of melipona bicolor, nannotrigona testaceicornis, and partamona helleri, from the central apiary of the federal university of viçosa. the number of individuals was estimated indirectly from the observation of a large number of brood cells and food storage pots. we opted not to use colonies with reduced food reserves and few brood cells due to the risk of loss during the experiment, as it was conducted during a period considered as low flowering when colonies generally decrease the production and storage of resources. the nests were transported from the central apiary and installed in the greenhouses seven days before the anthesis of the first flowers. in greenhouse a, we installed one nest of m. bicolor and two nests of n. testaceicornis, and in greenhouse b we installed two nests of n. testaceicornis and one of p. helleri. the nests were kept closed for 07 days before being opened to provide access to the interior of the greenhouses. the access was opened at night. in order to minimize the effects of temperature on nest development and bee behavior, the nests were installed on 1.5 m high supports positioned outside the greenhouse and their entrances were connected to the insides through a pvc pipe. only p. helleri was kept inside the greenhouse in order to preserve the nest entrance architecture and facilitate the return of these bees to the nest. throughout the study, artificial feeders containing aqueous honey solution (70%) and water were placed inside the greenhouse to supply the nests with energetic food (tomato flowers do not produce nectar). we randomly distributed geometric shapes (square, triangle, and round) of different colors (yellow, green, and black) throughout the greenhouse in order to assist the flight orientation of the bees. bees learn and memorize visual features such as shapes, patterns, direction, depth, contrast, intensity, and movement of light that are important in flight control and use visual coordinates to explore places of interest such as nest and flower locations (srinivasan, 1994; giurfa & lehrer, 2001). pollination tests and behavior of the bees during the pollination experiments, we also recorded the pollination behavior and average time spent on each single visit by m. bicolor and n. testaceicornis workers between 08:00 and 11:00 on two non-consecutive days. sociobiology 68(1): e-5227 (march, 2021) 3 the flowers of the fourth branch of both plantations were subjected to the following treatments: (i) spontaneous self-pollination (sp): 20 branches with flowers in pre-anthesis phase were marked and protected with organza bags (16x30 cm) to avoid any eventual visit; (ii) induced self-pollination (isp): 20 branches were protected with organza bags and manually vibrated using cuttings during anthesis phase (higuti et al., 2010); (iii) hand cross-pollination (hcp): the pre-anthesis flowers in 20 branches were covered and then manually pollinated 24 hours after anthesis, touching the stigmatic surface with a pollen mixture extracted from anthers of 20 flowers from different plants; (iv) pollination by m. bicolor and n. testaceicornis (mbnt): open flowers from 20 branches were available for visits of the two bee species during three consecutive days; (v) pollination by m. bicolor (mb): same as in the mbnt treatment, but only visited by m. bicolor; (vi) pollination by n. testaceicornis (nt): performed as mb treatment, but preventing foraging of m. bicolor by closing its nest; (vii) pollination by any bee (bp): visits inferred through necrotic marks on the anthers, regardless of the visitor species; (viii) single visit by m. bicolor (smb): 60 flowers monitored and bagged after the single visit; (ix) single visit by n. testaceicornis (snt): 60 flowers monitored and bagged after the single visit; and (x) single visit by p. helleri (sph): 60 flowers monitored and bagged after the single visit. the flowers or inflorescences were protected with an organza bag immediately after the completion of each of the treatments mentioned above. to perform the hand cross-pollination test (hcp), stigma receptivity was tested on 10 pre-harvest flowers and 10 open flowers after 24 h using 3% hydrogen peroxide. the receptivity was confirmed by detecting the air bubble formation on the surface of the stigma due to the activity of the peroxidase enzyme, indicating that the stigma was receptive (kearns & inouye, 1993). in all treatments, the bags were removed after the beginning of fruit formation. the complementarity of the pollination services of the three bee species was analyzed through comparisons of the fruit quality parameters resulting from the experiments. to determine the influence of pollination tests on tomato production and fruit quality, the first three fruits of each branch were harvested, and five attributes were verified: (1) weight, (2) number of seeds, (3) transverse diameter, (4) longitudinal diameter, and (5) pericarp thickness. fruit weight was determined with the aid of a precision scale. transverse diameter, longitudinal diameter, and pericarp thickness were measured using a digital caliper. the number of seeds was manually counted by opening the fruit and removing its pulp to access the seeds. for the data analysis, we tested data normality using the kolmogorov-smirnov test. to evaluate the independence of the tested variables we used the pearson’s correlation coefficient. we assumed that width and height would be previously correlated, then we expressed this relationship resuming the values as volume in cm3 considering an irregular sphere: , where r is the radius for each sphere axis. we used the kruskal-wallis and the pairwise dunn post hoc tests with the sequential bonferroni correction (p < 0.05) to assess the differences among fruit parameters from the 4th branch in the different pollination treatments (sp, isp, hcp, mbnt, mb, nt, bp, smb, and snt) (zar, 1999). in addition, we evaluated the median differences from the sp treatment through the box-plot method, since sensitive median differences may be important when evaluating production increases. all tests were performed at a significance level of 5%, using past v 3.26 (hammer et al., 2001). results stingless bee behavior regarding the species behavior, after opening the entrances of the m. bicolor, n. testaceicornis, and p. helleri nests to access the greenhouse, foragers of the three species displayed the behavior of flying towards sunlight and colliding with the screen, where they remained and consequently died of exhaustion. flight behavior toward sunlight ceased after three days of confinement. after this period, outside activities were restricted to short flights to remove trash from the nests. sixteen days after the opening of the p. helleri nest entrance, some foragers were observed to be under the petals, branches, and leaves. however, the pollination tests for p. helleri (sph) were not possible, as this species did not show any pollen collection behavior under the experimental conditions. during the experiment period in which p. helleri was confined, the average temperature in the greenhouse was 19.28ºc (max 32.09°c and min. 10.05°c) and average relative air humidity was 85% (max. 98.53% and min 62.44%). twenty-four days after the opening of the nest entrances to the interior of greenhouse a, foragers of the species m. bicolor and n. testaceicornis began the visits to the flowers (fig 1). the average temperature during the period in which the bees were confined in this greenhouse was 19.83°c (max 35.04°c and min. 9.68°c) and average relative air humidity was 82.21% (max. 97.14% and min 47.86%). in greenhouse b, the foragers of n. testaceicornis began foraging in tomato flowers 27 days after the opening of the nest entrance to the greenhouse. foragers of n. testaceicornis and m. bicolor started foraging activities in both greenhouses at around 8:00 am and ceased at 11:00 am, with the highest activity recorded at 09:30 am. during the period in which both species had free access, foragers of n. testaceicornis always drove away m. bicolor foragers from the flowers and then remained collecting the pollen grains exposed after the m. bicolor visit, as well as the pollen grains inside the anthers. no more interactions were observed between the two species on flowers. it was very common to observe two foragers of n. testaceicornis collecting pollen from the same flower. mc moura-moraes, w frantine-silva, mc gaglianone, lao campos – stingless bee pollination in cherry tomatoes4 bees of n. testaceicornis showed a high floral constancy, randomly foraging flowers in either the same or distinct plants. when landing on the anthers, the foragers of n. testaceicornis went to the apical portion of the anthers and circled around it, introducing their glossa into the anther’s pore in a behavior of scraping the extremities. pollen grains accumulated in the ventral portion of the thorax during visits were transferred to the corbiculae. when foraging the flowers, m. bicolor vibrated the flowers of the tomatoes by buzzing. the individuals of m. bicolor explored several flowers of the same plant before moving to the next plant, almost always in the same line. flowers on the first day of anthesis were not visited by m. bicolor or n. testaceicornis foragers. on the 2nd day of anthesis, m. bicolor and n. testaceicornis performed longer visits on flowers not yet visited, compared to flowers that had already been visited by any bee. the visits of m. bicolor lasted on average 12 s (22 ± 7 s) on unvisited flowers and 3.8 s (6 ± 2) on flowers already visited on the same day. similarly, n. testaceicornis spent an average of 91 s (140 ± 50) on unvisited flowers and 29.5 s (42 ± 11) on already visited flowers. visited flowers could be recognized by necrosis marks on the anthers (fig 1). pollination experiments the results from the greenhouse a experiment showed significant differences in all pollination tests and attributes evaluated, except for pericarp thickness (fig 2a), when compared to fruits from spontaneous self-pollination treatment (sp) (table 1). fruits from bee pollination treatments were an average of 4.69 ± 0.71 g heavier and had 52.44 ± 5.3 more seeds than those from sp treatment. in addition, pericarp thickness was larger in the treatment with pollination by n. testaceicornis (nt) fig 1. visitation and necrotic lesions in flowers of cherry tomatoes (solanum lycopersicum l). (a) visitation by n. testaceicornis buzzing behaviour; (b) visitation by m. bicolor pollination scraping behavior; (c) necrotic lesions left by n. testaceicornis; (d) necrotic lesions left by m. bicolor visitation. black arrows indicate the exact point of the lesions in both flowers. sociobiology 68(1): e-5227 (march, 2021) 5 (3.41 ± 0.16 cm) compared to sp (2.93 ± 0.65) in greenhouse b. on the other hand, we did not find consistent significant differences between treatments involving induced selfpollination (isp) or hand cross-pollination (hcp) compared to the bee treatments. fruits from snt, mb, and mbnt showed significant differences (p < 0.05) only for weight (fig 2b) and number of seeds (fig 2c), compared to hcp in greenhouse a, and nt compared to hcp in greenhouse b. transverse and longitudinal diameters, and therefore the volume were highly correlated with the weight (person’s r > 0.96, p < 0.001), and the statistical tests followed the same pattern throughout the different comparisons. fig 2. box-plot and statistic differences among four parameters of cherry tomatoes measured in the present study in greenhouses a (blue) and b (yellow). (a) pericarp thickness; (b) weight; (c) number of seeds; (d) volume derived from transverse and longitudinal diameters. boxplot bars depicts quartile intervals, inner line the median position, out lines the boundaries of the distribution and circle the outliers. letters over the box-plots indicate the statistical differences (p < 0.05) according to the kruskal-wallis and dunn’s post hoc tests. boxplot bars depicts quartile intervals, spontaneous self-pollination (sp); induced self-pollination (isp); hand cross pollination (hcp); pollination by m. bicolor and n. testaceicornis (mbnt); pollination by m. bicolor (mb); pollination by n. testaceicornis (nt); pollination by bees (bp); single visit pollination by m. bicolor (smb); and single visit pollination by n. testaceicornis (snt). greenhouse experiment n weight (g) number of seeds transverse diameter (mm) longitudinal diameter (mm) pericarp thickness (mm) a sp 60 9.00 ± 0.53 a 18.41 ± 2.95 a 23.11 ± 0.50 a 24.83 ± 0.58 a 4.00 ± 0.56 a isp 64 12.70 ± 0.50 b,c 52.98 ± 3.43 b 26.62 ± 0.35 b,c 28.28 ± 0.41 b.d 3.71 ± 0.08 a hcp 39 11.07 ± 0.56 a,c 56.74 ± 2.71 b 25.32 ± 0.45 a.b 26.76 ± 0.50 a,b,c 3.50 ± 0.12 a bp 56 13.34 ± 0.57 b,c 66.71 ± 2.57 b,c 26.95 ± 0.43 b,c 29.11 ± 0.47 d 3.72 ± 0.07 a mb 122 13.39 ± 0.27 b 70.11 ± 1.37 c 27.27 ± 0.20 c 29.13 ± 0.23 d 3.57 ± 0.04 a mbnt 20 14.64 ± 0.79 b 73.65 ± 4.97 c 27.96 ± 0.62 c 29.98 ± 0.64 d 3.70 ± 0.12 a snt 17 14.37 ± 0.89 b 72.58 ± 3.87 b,c 27.70 ± 0.84 c 30.13 ± 0.64 d 3.84 ± 0.15 a smb 30 12.71 ± 0.62 b,c 71.20 ± 4.00 b,c 26.76 ± 0.44 b,c 28.46 ± 0.52 c,d 3.63 ± 0.10 a b sp 60 7.03 ± 0.45 a 20.45 ± 3.48 a 21.52 ± 0.08 a 22.43 ± 0.52 a 2.93 ± 3.48 a isp 63 11.21 ± 0.38 b 45.86 ± 3.21 b 25.68 ± 0.31 b 26.94 ± 0.34 b 3.48 ± 0.07 b hcp 31 8.74 ± 0.47 a,c 39.06 ± 3.56 b,c 23.92 ± 0.53 a,b 24.54 ± 0.48 a 3.00 ± 0.12 a,c nt 102 10.42 ± 0.34 b,c 52.48 ± 2.38 c 24.96 ± 0.31 b 26.43 ± 0.35 b 3.41 ± 0.16 b,c table 1. means followed by different letters (a-d) represent statistical differences (p <0.05) for the same attribute by the kruskal-wallis test and dunn’s posterior test. mc moura-moraes, w frantine-silva, mc gaglianone, lao campos – stingless bee pollination in cherry tomatoes6 discussion the behavior on the first days after the transfer of m. bicolor, n. testaceicornis, and p. helleri nests into the greenhouse was similar to that reported in studies with other stingless bee species (cauich et al., 2004; cruz et al., 2004; bomfim et al., 2014). the limited size of an artificial environment such as a greenhouse imposes physical barriers to flying activities, with collisions against the greenhouse cover being very common in the first days (slaa, 2006). older foragers already have certain established habits and experiences from the environment in which they were previously maintained, so they have greater difficulty acclimating to the protected environment (free, 1993). this explains the high mortality of foragers of the three species studied in the first days of introduction of the nests in the greenhouse. acclimatization of stingless bees to greenhouse conditions can vary greatly between species and between colonies of the same species (cauich et al., 2004; cruz et al., 2004; bartelli et al., 2014; bartelli & nogueira-ferreira, 2014; bomfim et al., 2014; da silva et al., 2016; silva-neto et al., 2018). for instance, foragers of nannotrigona perilampoides (cresson, 1878) may take from five to nine days to consistently start their activities in tomato greenhouse crops (cauich et al., 2004). in different crops, such as small watermelons, colonies of scaptotrigona sp. started foraging after the second day and m. subnitida started to explore resources on bell pepper flowers after seven days in the greenhouse (cruz et al., 2004; bomfim et al. 2014). the acclimatization time of melipona species seems to be variable; bartelli et al. (2014) observed that it took 22 days for m. quadrifasciata workers to start foraging tomato flowers. bumblebee species on the other hand might expend 9 to 10 days for acclimatation (morandin et al., 2001a). partamona helleri was not considered a pollinator of cherry tomatoes under the conditions of this study. several factors may be involved in this outcome, such as lack of interest in the floral resources or inability to acclimate to greenhouse conditions (bomfim et al., 2014). a determining factor for bees to use their functions is a temperature not exceeding 30 °c (brand, 2005; kiss, 2006). in the greenhouses, the maximum temperature recorded during the day was 32.09 °c and the relative maximum humidity was 98.53%. the temperature is the factor that most influences the internal and external activities of the colony; when very high, foraging decreases and ventilatory behavior increases in order to stabilize the internal temperature of the colony (michener, 2000; roubik, 1989; teixeira & campos, 2005). according to kleinert et al. (2009) stingless bees have excellent foraging activity when the temperature remains between 20 and 30°c, and the relative humidity values are between 30% and 70%. foraging activities were concentrated in the early hours of the day, when the temperature in the greenhouse was mild, which agrees with observations from other studies for different stingless bee species (cruz et al., 2004; teixeira & campos, 2005; nunes-silva et al., 2013; bomfim et al., 2014; silva-neto et al., 2018). however, one factor that may have caused the absence of foraging activity observed in the afternoon is the high number of visits per flower in the morning, leading to pollen depletion. some bee species are able to evaluate the amount of pollen available during the visit (buchmann & cane, 1989; shelly et al., 2000; nunes-silva et al., 2013), and the perception that flowers would have a low amount of pollen may lead to the bees no longer visiting these flowers. some studies reported that two visits per flower are enough for pollination of tomatoes in greenhouses and that higher levels of necrotic markings on the anthers lead to a decrease in return visits (morandin et al., 2001b). the foraging behavior of m. bicolor exploring several flowers on one plant is expected and often described for species from the genus melipona (camargo, 1972; cruz et al., 2004). fruits from flowers visited only by n. testaceicornis in both single and multiple visits, showed higher average values in all analyzed parameters when compared to fruits from flowers visited by m. bicolor. previous studies reported that bee species from the genus melipona were efficient pollinators of tomatoes (del sarto et al., 2005; bispo dos santos et al., 2009; hikawa & miyanaga, 2009; bartelli & nogueira-ferreira, 2014; silva-neto et al., 2018). although we did not measure the pollen grain removal rate, the fact that all evaluated attributes present values similar to the fruits of the induced self-pollination tests (isp) and higher than those found in self-pollinated fruits (sp) corroborates the effective pollination in the different types of foraging, scraping (n. testaceicornis) and buzzing (m. bicolor) (cauich et al., 2004; del sarto et al., 2005; dos santos et al., 2008; palma et al., 2008; bispo dos santos et al., 2009; hikawa & miyanaga, 2009; roselino et al., 2009; bartelli & nogueiraferreira, 2014; silva-neto et al., 2018). in addition to foraging behavior, the time spent visiting flowers seems to maximize the distribution of pollen grains on stigma, essential for fruit development (mann, 1943). the differences in visit time between m. bicolor and n. testaceicornis species may be related to different visitation behaviors. the buzzing behavior of m. bicolor can be considered more efficient for pollen extraction and lower energy expenditure, when compared to the time spent in each visit of n. testaceicornis. the time spent by n. testaceicornis may be the main factor in maintaining a greater floral constancy in flowers of the same and nearby plants, which could contribute to higher mean values of the analyzed tomato parameters. even without statistical significance, we can highlight the higher medians and quartiles distribution found in the weight and number of seeds of the fruits produced from the visit of the two bees, corroborating the expectations of the simultaneous use of these species for the pollination of cherry tomatoes. based on the observed results, we conclude that stingless bees m. bicolor and n. testaceicornis can be used simultaneously to pollinate cherry tomatoes grown in greenhouses. the fruit set resulting from non-buzzing n. sociobiology 68(1): e-5227 (march, 2021) 7 testaceicornis visits is similar to the fruit set after visits from m. bicolor, a buzzing species. in future work perspectives, we suggest testing the effectivity of simultaneous use of stingless bees in other tomato varieties. acknowledgements we thank gef/fao/pnuma/mma/funbio/cnpq (project on the conservation and management of pollinators for a sustainable agriculture through an ecossystem approach) and the federal university of viçosa, for the facility structure, and the coordenação de aperfeiçoamento de pessoal de nível superior (capes) financial code 001 and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the financial support provided. wfs thanks to capes and ppgern for the pnpd grant (id: 88882.314552/2019-01) and cnpq (303894/2018-0) and fundação de amparo à pesquisa do estado do rio de janeiro (faperj) (203.321/2017) for grants to mcg. the authors are also grateful to geraldo paiva, iris stanciole, geraldo meri, camila folly, cristiane marques, paula netto and riudo paiva for their help in the fieldwork. we also thank msc. bianca gonçalo meyrelles and dr. weyder cristiano santana for suggestions during the experiments. references azevedo, r.l., carvalho, c.a.l., pereira, l.l. & nascimento, a.s. 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(1999). biostatistical analysis. fourth edition. usa: prentice hall, 208-214 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.187-191sociobiology 62(2): 187-191 (june, 2015) a remarkable new dimorphic species of solenopsis from argentina introduction the ant genus solenopsis westwood (myrmicinae: solenopsidini) is a huge taxonomic challenge, not only for the great number of species that it contains, but also from phylogenetic and ecological points of view. at present, nearly 185 species of this genus have been recognized (bolton, 1987; pitts et al., 2005; tschinkel, 2006), with about 117 species in the new world, most of them neotropical (pacheco & mackay, 2013). until recently, there were no solid proposals regarding the systematics and phylogeny of solenopsidine ants. in 2003, bolton proposed the solenopsidini tribe group, including the tribes solenopsidini and stenammini. within the first tribe (solenopsidini), the same author distinguished two genus groups: that of solenopsis and that of carebara. recently, ward et al. (2015) published the first molecular phylogeny of the subfamily myrmicinae, reducing the 26 tribes to only six, finding the tribe solenopsidini (new sense) to be the sister group abstract solenopsis westwood (myrmicinae: solenopsidini) is an ant genus that represents a taxonomic challenge, including about 117 species in the new world, most of them neotropical. solenopsis can be divided into two artificial groups: “fire ants” and “thief ants”. the second group is represented by species often difficult to identify because of their small size and uniformity of color and sculpture. most of the thief ants are pale yellow, monomorphic, and lestobiotic, inhabiting small colonies often inside the nests of other ant species. in this paper we describe a new species of thief ant, solenopsis longicephala sp. n., characterized by extreme dimorphism and with a set of characters probably convergent with other genera of myrmicinae, such as carebara and pheidole. sociobiology an international journal on social insects f cuezzo1; f fernández2 article history edited by ted schultz, smithsonian institution national museum of natural history, usa received 30 november 2014 initial acceptance 04 march 2015 final acceptance 13 april 2015 keywords ants, formicidae, solenopsidini, south america, alpha taxonomy. corresponding author fabiana cuezzo instituto superior de entomología dr. a. willink facultad de ciencias naturales e iml miguel lillo 205, t4000jfe, san miguel de tucumán, tucumán, argentina e-mail: fcuezzo@csnat.unt.edu.ar of the combined tribes attini (new sense) + crematogastrini (new sense). according to ward et al. (2015), solenopsidini is expanded to include the tribes adelomyrmecini, myrmicariini, and stegomyrmecini. solenopsis (including carebarella) appears as a strongly monophyletic group, sister to the clade kempfidris + tropidomyrmex, both neotropical (ward et al., 2015). currently solenopsis comprises two informal groups. the first of these is the so-called “fire ants”, including about 20 species (trager, 1991; taber, 2000). this group contains the larger-sized species and is taxonomically better known. the name “fire ants” refers to the painful sting. it is this set of solenopsis species that has received more attention because some of them are invasive, having been introduced from their places of origin to different regions of the world, causing serious economic losses and damages in these zones (tschinkel, 2006). fire ant phylogeny has been explored in recent works (pitts et al., 2005; shoemaker et al., 2006). other studies focus on the genetic and population biological aspects of the research article ants 1 instituto superior de entomología dr. a. willink, facultad de ciencias naturales e iml, san miguel de tucumán, tucumán, argentina 2 instituto de ciencias naturales, universidad nacional de colombia, bogotá d.c., colombia f cuezzo; f fernández – new species of solenopsis188 invading species (e.g. ross & trager, 1990) or summarize general taxonomic information (wilson, 1952; ettershank, 1966; buren, 1972). the second group consists of species of thief ants formerly placed in the subgenus “diplorhoptrum”. most thief ant species are difficult to identify because of their small size and uniformity of color and sculpture. most species are pale yellow, monomorphic, and lestobiotic, inhabiting small colonies often inside the nests of other ant species. the combination of lestobiosis and subterranean nests may explain why many thief ant species have been collected at low frequencies and are underrepresented in biotic surveys at local or regional scales. the biology and life cycles of thief ants are poorly known. the only complete new world revision of the thief ant group is that of pacheco and mackay (2013), which proposes several species groups, many synonymies, and several new species. in argentina, a total of 72 species and subspecies have been documented and the only available regional key is that of kusnezov (1978), which is now out of date. in the present work a new remarkable species of solenopsis is described. this new species is dimorphic and possesses a set of characters unusual for the genus, probably convergent with traits of other myrmicinae genera, such as carebara (now member of crematogastrini, ward et al., 2015). material and methods photographs were taken with a leica dfc295 camera attached to a leica stereomicroscope and processed with automontage software ver. 4.4.0 and adobe photoshop ver. cs5. all scale bars are in mm. measurements were made using a stereomicroscope at 40x. all measurements are in mm. the following abbreviations are used: hl (head length). the length of the head capsule excluding the mandibles; measured in full-face view, as a straight line from the mid-point of the anterior clypeal margin to the mid-point of the posterior margin. hw head width. the maximum width of the head behind the eyes, measured in full-face view. el (eye length). maximum length of compound eye in lateral view. ew (eye width). maximum diameter of compound eye in lateral view. sl (scape length). the maximum length of the scape, excluding the basal radicle. sw (scape width). maximum width of the scape in full-face view. pw (pronotal width). the maximum width of the pronotum in dorsal view. wl (weber’s length of mesosoma). the diagonal length of the mesosoma in profile, from the anteriormost point of the pronotum to the posterior basal angle of the metapleuron. fl (femur length) length of the profemur. pl (petiole length), in dorsal view. ppl (postpetiole length), in dorsal view. ci (cephalic index). (hw/hl)*100. si (scape index). (sl/hw)* 100. ei (eye index). (ew/el)*100. examined material will be deposited in the following institutions: mzsp. museum of zoology, são paulo, brazil. icn. insect collection, instituto de ciencias naturales, universidad nacional de colombia, bogotá d.c., colombia. ifml. instituto fundación miguel lillo, san miguel de tucumán, tucumán, argentina. results and discussion systematic treatment solenopsis longicephala, cuezzo and fernández new species – figures 1 (a-b); 2 (a-b) major worker measurements: holotype (measurements of paratypes in parentheses, n=14): hw 0.675 (0.670-0.700), hl 0.975 (0.950-1.025), sl 0.425 (0.375-0.500), sw 0.1 (0.1), el0.050 (0.050-0.075), ew 0.050 (0.050), pw 0.450 (0.3000.425), wl 0.950 (0.950-1.025), fl 0.425 (0.450-0.525), pl 0.300 (0.300), ppl 0.375 (0.350-0.400), ci 69 (68-71), si 63 (56-71), ei 100 (67-100). body and appendages golden yellow. head: noticeably longer than wide (ci 68-71). posterior cephalic border sinuous, concave in middle, sides straight. mandibles conspicuous, smooth and shining with a few short setae scattered on dorsal face; masticatory margin with four stout teeth and denticle behind third tooth. mandibular teeth darker than rest of mandible. median part of clypeus bicarinate. clypeal carinae short, divergent, ending at posterior margin of antennal sockets. anterior clypeal margin with two teeth and two extralateral denticles (see fig.1a). one central seta in clypeal anterior margin, besides 6 short setae present on anterior margin of clypeus reaching to halfway point of closed mandibles. sides of clypeus with four to five extremely short setae. palpal formula 2-1. maxillary palp geniculate. second segment of labial palp distally swollen. both palps (labial and maxillary) with two long setae, distally attached. compound eye small (ei 67-100) with four to seven ommatidia, placed anterior to cephalic midline. median ocellus present (fig 1a). antennae 10-segmented with apical club of two articles. scape exceeding posterior margin of compound eye, but failing to reach vertexal border by more than its maximum width. scape stout and flattened, ventrally concave. all segments of funiculus wider than long, except last one. frontal lobes well developed, rounded, completely covering antennal sockets. mesosoma (fig 1b): straight in lateral view, with shallow notopropodeal suture. propodeum convex, low, and unarmed. propodeal spiracle circular, well developed, equidistant from propodeal border. dorsopropodeum twice as long as sociobiology 62(2): 187-191 (june, 2015) 189 posteropropodeum. from posterior view, posteropropodeum with central concavity, probably used to house anterior face of petiole. metasoma (fig 1b): petiole wider than long in dorsal view, with short peduncle and well-defined high node. sternopetiolar process well developed with short anterior tooth. postpetiole dorsally convex, lower than petiole. postpetiole, in dorsal view, rounded. gaster with five visible segments, the anterior segment the longest (fig 1b). sting well developed, but generally withdrawn inside gaster. body smooth and shiny, except for the gena, which has fine parallel grooves, and for the side of the propodeum, which is covered by fine transverse striation. cephalic dorsum with scattered punctures. entire body covered by erect to subdecumbent light yellow hairs, more abundant on all gastral tergal plates. body and appendages light brown. minor worker, measurements (n=15): hw 0.3000.400, hl 0.450-0.520, sl 0.250-0.300, sw 0.038-0.050, el 0.025, ew 0.012, pw 0.200-0.275, wl 0.400-0.525, pl 0.125, ppl 0.150, ci 60-77, si 75-83, ei 50. body and appendages light yellow. head: longer than wide (ci= 60-77) with sides slightly convex. cephalic dorsum with scattered punctures. posterior cephalic margin concave in middle (fig 2a). extremely small eyes with only one ommatidium, placed at lateral cephalic margin in full-face view. compound eyes difficult to see in dorsal view (fig 2a). mandibles with four well-developed teeth along masticatory margin, with two diastema, one between subapical tooth and third tooth and the other between 3rd and 4th teeth. dorsal face of mandible smooth and shining, covered with yellowish, subdecumbent hairs. anterior clypeal margin with one central short seta. central part of clypeus bicarinate, each carina ending in a well differentiated tooth. two extralateral teeth, visible only at more than 60x magnification, each with an apical long hair (see fig 2a). gena without striation observed in major worker. sides of clypeal margin with 5-6 short hairs. antennae 10-segmented with 2-segmented apical club with two articles. antennal sockets close to the posterior clypeal margin. funicular segments 2x-5x wider than long. palpal formula: 2-1. mesosoma, petiole and postpetiole (fig 2b): in profile similar to major worker. both sternopetiolar and sternopostpetiolar processes present, with antero-ventral denticle or projection. body smooth and shining, including gena. sides of propodeum covered by fine, transverse striation. mesosoma with more than 10 erect hairs. gaster with scattered subdecumbent setae, more abundant in last two segments. queen and male unknown. type data. holotype major worker: argentina, tucumán, dpto. burruyacú. las lajitas (26° 41.74’ s64° 94.47’ w) 10i-2011, f. cuezzo coll. (deposited in ifml). paratypes (same data): 10 major workers and 10 minor workers (deposited in icn), 5 major workers and 10 minor workers (deposited in mzsp), rest of the series (n= 33 major workers and 439 minor workers) deposited at ifml. fig 1a-e. habitus of solenopsis longicephala sp. n. major worker: a head in full-face view; b left side of body, in profile; c– dorsal view of body;d – petiole and postpetiole, lateral view; e – holotype labels. f cuezzo; f fernández – new species of solenopsis190 distribution. known only from the type locality. biology: one nest was found under a small rock. comments. this species can be differentiated from all other solenopsis by the combination of the following traits: dimorphic ants (see fig 3); major worker with long and abundant hairs on the dorsal face of mesosoma and gaster; head in major and minor workers extremely elongated; eye reduced to 1-5 ommatidia; and palpal formula 2-1. according to pacheco and mackay (2013) most species of thief ants are monomorphic or weakly polymorphic; only five species are strongly dimorphic (s. iheringi; s. johnsoni; s. thoracica, s. tetracantha, and s. vinsoni) and none of the polymorphic species have the particular combination of character states mentioned above. all of the species described as dimorphic belong to the fugax complex except s. iheringi, which belongs to the wasmanni complex. south american species with minor workers similar to those of s. longicephala sp. n. include s. leptanilloides and s. tetracantha, both also found in argentina. the first species is monomorphic and much smaller than s. longicephalasp. n. other characters of s. longicephala sp. n. useful for separating it from these two species include: clypeal carina well developed in major and minor workers (nearly absent in s. leptanilloides); anterior clypeal border with 4 teeth, two central and two extralateral (extralateral teeth are absent in s. leptanilloides). the elongated anterior clypeal margin of s. tetracantha and the lower process of the postpetiole (present in s. longicephala sp. n.) are enough to differentiate major workers of both species. minor workers of s. tetracantha and s. longicephala sp. n. are extremely similar, but differ in size; s. longicephala sp.n. is larger. the major workers of s. longicephala sp. n. are similar to those of s. wasmannii, but solenopsis longicephala sp. n. can be recognized by the extremely elongated head, whereas the head of s. wasmannii is only slightly longer than broad. most myrmicines possess heads not longer or slightly longer than wide. some species of this subfamily, however, have majors with elongated heads. this trait is known in adlerzia, carebara, and some pheidole (especially in the aberrans groups and p. dispar, formerly in machomyrma). because these are distantly related to solenopsis (ward et al., 2015), the presence of an elongated head in solenopsis longicephala sp. n. is here interpreted as a convergent trait. the biological functions of elongated heads are obscure because little is known of the biology of the mentioned ants. most carebara appear to be soil-inhabiting ants, perhaps living near or inside the nests of other ants or termites, with supposedly lestobiotic habits. however, some pheidole species in the aberrans group fig 2a-d. habitus of solenopsis longicephala sp. n. minor worker: a head in full-face view; b left side of body, in profile; c – dorsal view of body; d – paratype labels. fig 3. graph showing two quite separate groups of workers (major and minor) taking in account the relationship between pronotal width (pw) and head width (hw) in solenopsis longicephala sp. n. (n=30), measurements in mm. sociobiology 62(2): 187-191 (june, 2015) 191 appear to be surface foragers and no data are available for pheidole dispar and adlerzia. other subterranean myrmicines lack elongated heads, e.g., the “majors” of tranopelta. longer heads and cylindrical bodies could be associated with hard substrates or narrow passages in the soil or in twigs or may be associated with a specialization for defense in the soldier caste. solenopsis longicephala sp. n. is a member of the solenopsis pygmaea species complex of pacheco and mackay (2013). this complex comprises ants with elongated heads and strongly punctate integuments, often possessing a bicarinate clypeus; 4-5 well-developed clypeal teeth, with different degrees of development but always present, and extremely small minor workers with poorly developed eyes. also, the notopropodeal suture is only slightly impressed and the postpetiole nearly circular. all the characters above mentioned are present in s. longicephala. some members of the solenopsis wasmannii complex also have elongated heads, workers that are extremely small (total length up to 1.45 mm), and the head surface with very coarse punctures. however, those species possess a very particular form of the clypeal carinae different from that in s. longicephala sp. n. also, the compound eyes of their major workers have more ommatidia (10 or more) than those of s. longicephala sp. n. (4 7). acknowledgments this work was received financial support from ciunt 26/g413 and the national council of research (conicet, argentina). references bolton, b. (1987). a review of the solenopsis genus-group and revision of afrotropical monomorium mayr (hymenoptera: formicidae). bulletin of the british museum of natural history, entomological series, 54:263-452. bolton, b. (2003). synopsis and classification of formicidae. memoirs of the american entomological institute, 71: 1-370. buren, w.f. (1972). revisionary studies on the taxonmy of the imported fire ants. journal of the georgia entomological society, 7:1-26. ettershank, g. (1966). a generic revision of the world myrmicinae related to solenopsis and pheidologeton. australian journal of zoology, 14, 73-171. kusnezov, n. (1978). hormigas argentinas: clave para su identificación. miscelánea. instituto miguel lillo, 61, 1-14:1147+2. pacheco, j.a. & mackay, w.p. (2013).the systematics and biology of the new world thief ants of the genus solenopsis (hymenoptera: formicidae). edwin mellen press, lewiston, ny. pitts, j.p.; mchugh, j.v. & ross, k.g. (2005). cladistic analysis of the fire ants of the solenopsis saevissima speciesgroup (hymenoptera: formicidae). zoologica scripta, 34: 493-505. doi:10.1111/j.1463-6409.2005.00203.x. ross, k.g. &trager, j.c. (1990). systematics and population genetics of fire ants (solenopsissaevissima complex) from argentina. evolution, 44: 2114-2134. shoemaker, d.d.; ahrens, m.e. & ross, k.g. (2006). molecular phylogeny of fire ants of the solenopsis saevissima species group based on mtdna sequences. molecular phylogenetics and evolution, 38: 200-215. doi:10.1016/j.ympev.2005.07.014. taber, s.w. (2000). fire ants. texas a&m university press. college station. isbn 0-8096-945-0. thompson, c.r. (1989). the thief ants, solenopsis molesta group, of florida (hymenoptera: formicidae). florida entomologist, 72: 268-283. doi: 102307/3494907. trager, j.c. (1991). a revision of the fire ants, solenopsis geminata group (hymenoptera: formicidae: myrmicinae). journal of the new york entomological society, 99: 141-198. tschinkel, w.r. (2006). the fire ants. harvard university press, cambridge, ma. isbn: 0-674-02207-6. ward, p.s., brady, s.g., fisher, b.l. & schultz, t.r. (2015). the evolution of myrmicine ants: phylogeny and biogeography of a hyperdiverse ant clade (hymenoptera: formicidae). systematic entomology, 40:61-81. doi: 10.1111/syen.12090. wilson, e.o. (1952). o complexo solenopsis saevissima na america do sul (hymenoptera: formicidae). memórias do instituto oswaldo cruz, 50: 49-68. doi: 10.1590/s007402761952000100003. doi: 10.13102/sociobiology.v60i4.362-366sociobiology 60(4): 362-366 (2013) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 insecticidal activity of the whole grass extract of typha angustifolia and its active component against solenopsis invicta zx zhang1, 2, y zhou1, xn song1, hh xu1, 2, dm cheng1, 3 introduction typha angustifolia l. is a perennial marsh typhaceae plant considered as a rare medicinal herb with economical and environmental importance. this plant is used as food and ornament for landscape use. t. angustifolia l. is a wild plant with rich natural resources cultivated and widely distributed in china. dried pollens of t. angustifolia l. are commonly used as a chinese medicinal herb (li et al., 2011; liu & zhang, 2009; yan & xu, 1996) because these pollens have hemostatic and diuretic effects; t. angustifolia l. pollens can also remove blood stasis. whole grass of t. angustifolia l. is also used as good medicine and food (yu et al., 2007). furthermore, t. angustifolia l. is widely used in wastewater treatment, artificial wetland system, and purified water resource (yuan et al., 2012). different parts of t. angustifolia l. are used as food with refreshing taste and high nutritive value. dried grass of t. angustifolia l. is used as good bedding for coops or poultry cages because this plant part can repel abstract in this study, the toxicity of whole grass typha angustifolia l. extract was determined in vitro by a “water tube” method to investigate the bioactivity of t. angustifolia l. against micrergates of red imported fire ants. results indicated that the ethanol extract exhibited toxicity against the micrergates of red imported fire ants. mortality was 100% after the micrergates were treated with 2000 mg/ml of ethanol extract for 72 h. after 48 h of treatment, lc 50 values of ethanol extract and petroleum ether fraction were 956.85 and 398.73 mg/ml, respectively. after 120 h, lc 50 values of the same substances were 271.23 and 152.86 mg/ml, respectively. a bioactivity-guided fractionation and chemical investigation of petroleum ether fraction yielded an active component (compound 1). nmr spectra revealed that the structure of compound 1 corresponded to 3β-hydroxy-25-methylenecycloartan-24-ol. compound 1 also exhibited strong toxicity against the micrergates of red imported fire ants, thereby eradicating all of the tested ants treated with 240 mg/ml for 120 h. lc 50 values of compound 1 at 48 and 120 h were 316.50 and 28.52 mg/ml, respectively. sociobiology an international journal on social insects 1 key laboratory of natural pesticide and chemical biology, ministry of education south china agricultural university, guangzhou, china 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 department of plant protection, zhongkai university of agriculture and engineering, guangzhou, china research article ants article history edited by kleber del-claro, ufu, brazil received 15 april 2013 initial acceptance 11 june 2013 final acceptance 05 august 2013 keywords red imported fire ants, bioactivity, active component corresponding author dongmei cheng department of plant protection zhongkai university of agriculture and engineering guangzhou, china, 510225 e-mail: zdsys@scau.edu insects that parasitize warm-blooded animals, such as hens. however, reports about the bioactivity of t. angustifolia l. extracts or their constituents against insects have not been published yet. previous studies on the constituents of whole t. angustifolia l. grass focused on flavonoids and acidic components (gallardo-williams et al., 2002; jia et al., 1986; kong et al., 2011; zhang et al., 2008). in the present study, extracts of whole t. angustifolia l. grass were isolated and purified by column chromatography to identify their chemical compositions. the toxicities of the extracts and 3β-hydroxy-25methylenecycloartan-24-ol were determined to evaluate their effectiveness to control micrergates of red imported fire ants. solenopsis invicta is an aggressive invasive species. natural products for its environmental friendly characteristic are always regarded as important alternative for synthetic organic pesticides. thus, this study would focus on the possibility of t. angustifolia l. controlling red imported fire ants. sociobiology 60(4): 362-366 (2013) 363 material and methods plant material fresh whole t. angustifolia grass without flowers was collected from wangchang town, qianjiang city, hubei province, china in september 2009 and then dried at 40 °c. extraction and isolation the whole grass dried powder of t. angustifolia (17.51 kg) was extracted with ethanol (175 l ×3). the combined and concentrated ethanol extract was dissolved in a small amount of ethanol. the resulting mixture was resuspended in water and gradually partitioned with petroleum ether and ethyl acetate to produce 0.45 and 0.22 kg of dried organic extracts, respectively. these different solvent fractions were subjected to bioactivity assays against the micrergates of red imported fire ants. petroleum ether-soluble fraction elicited the highest potent activity. this petroleum ether-soluble fraction (200 g) was further fractionated in a silica gel column (200to 300-mesh column; 1 kg; 3.8 cm × 70 cm) by using a gradient mixture of petroleum ether-acetone at increasing polarities (20:1 to 1:1 and pure acetone). active constituent of t. angustifolia the active compound was isolated using various chromatographic methods because of different insecticidal activities of petroleum ether fraction. the isolated compound was subjected to structural determination by spectroscopic analyses (1h nmr and 13c nmr) and by direct comparison with an authentic reference compound. origin and rearing of micrergate of red imported fire ants solenopsis invicta colonies were collected from the suburbs of guangzhou and maintained in the laboratory for bioassays (lv et al., 2006; huang et al., 2007). the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor l.). a test tube (25 mm × 200 mm) partially filled with water and plugged with cotton was used as a water source. the ants were maintained in the laboratory at 25±2 °c. toxicity tests “water tube” method (huang et al., 2007) with slight modifications was used to determine the effectiveness of t. angustifolia in controlling the micrergates of red imported fire ants. the ants for the toxicity tests were transferred into a beaker with a bottom diameter of 10 cm whose vertical wall coated with fluon emulsion after drying for 24 h to prevent ants from escaping. the water source was a test tube filled with approximately two-thirds full of water and tightly fitted with a saturated cotton which was pushed into at least 3 to 5 cm from the open end of the tube. for the toxicity tests, all the test ants were from the same colony. a group of 30 ants of them were used for each toxicity test and each test was replicated thee times. the water source tubes containing the acetone solution, ethanol extract, petroleum ether fraction, or compound 1 were placed in the beakers for the toxicity tests. ethanol extract and petroleum ether fraction were tested at concentrations of 2000, 1000, 500, 250, and 125 µg/ml in acetone/water (1:99) mixture. compound 1 was tested at concentrations of 240, 120, 60, 30, and 15 µg/ml in acetone/water (1:99) mixture. acetone/ water (1:99) mixture was used as a control treatment. during the tests, mortality was recorded at an interval of 24 h for a total of 120 h, and no food was provided for the micrergates of red imported fire ants. statistical analyses percent of fire ant mortality was determined and transformed to arcsine square-root values for anova. treatment means were compared and separated using scheffe’s test at p = 0.05. means ± se of untransformed data were reported. results and discussion chemical compositions of t. angustifolia the following properties were obtained for compound 1: m. p. 155 °c to 165 °c analyzed for c30h50o2 (m+ at m/z 442). 1h nmr spectrum suggested that compound 1 comprised a mixture of c-24 epimers of 3β-hydroxy-25methylenecycloartan-24-ol (table 1) at a ratio of 60:40. 1h nmr spectrum of the major compound showed typical resonances of a cyclopropane methylene [δ 0.33 and 0.55 (ab, q, j = 4.2 hz)], six methyl groups (δ 0.81–1.72), and a terminal methylene [δ 4.93 (br, s), 4.83 (br, s) (2h-26)]. these two diastereomers could not be physically separated in the present study, but spectroscopic data (escobedo-martínez et al., 2012) have revealed the diastereomeric nature of the components of compound 1. c-24 epimers of 3β-hydroxy25-methylenecycloartan-24-ol in t. angustifolia l. have not been discovered yet in similar studies. toxicity of ethanol extract the toxicities of different concentrations of t. angustifolia l. ethanol extract against red imported fire ants were determined (fig. 1). fig. 1 shows that 2000 µg/ml of ethanol extract completely eradicated the micrergates of red imported fire ants treated for 72 h. the corrected mortality as concentrations and treatment time were increased. as concentration was decreased to 1000 µg/ml, the ethanol extract resulted in zx zhang, et al insecticidal activity of typha angustifolia against solenopsis invicta364 toxicity of petroleum ether fraction the toxicities of petroleum ether fraction against micrergates of red imported fire ants were evaluated (fig. 2). petroleum ether fraction was toxic against the micrergates of red imported fire ants. in particular, 2000 µg/ml of petroleum ether fraction achieved 100% mortality against the micrergates treated for 96 h. the mortality of the micrergates treated with 500 µg/ml petroleum ether fraction for 120 h was 98.67%. lc50 values of petroleum ether fraction administered for 48 and 120 h were 398.73 and 152.86 µg/ml, respectively (table 2). toxicity of 3β-hydroxy-25-methylenecycloartan-24ol (compound 1) 3β-hydroxy-25-methylenecycloartan-24-ol (compound 1) was isolated from t. angustifolia l. petroleum ether fraction and exhibited strong toxicity. the toxicity results of compound 1 are shown in fig. 3. approximately 240 µg/ml of compound 1 eradicated all of the tested ants treated for 120 h, indicating that compound 1 had strong toxicity against micrergates of red imported fire ants. compound 1 treated for 120 h exhibited stronger toxicity at lc50 of 28.52 µg/ml than lc50 values of compound 1 treated for 48 and 120 h (table 2). thus, compound 1 was responsible for an excellent toxicity of t. angustifolia l. against micrergates of s. invicta. natural products may be considered as important alternative insecticides to control the micrergates of red imported fire ants. the results from this study demonstrated that t. angustifolia extracts gradually exhibited an effective toxicity. the major component in ethanol extract accounting for its toxicity was petroleum ether fraction that has an important function in controlling the micrergates of red imported fire ants. compound 1 isolated from petroleum ether fraction also had strong toxicity. thus, t. angustifolia l. ethanol extract, petroleum ether fraction, and compound 1 were effective and environmentally friendly agents that could be used to control micrergates of red imported fire ants. 0 20 40 60 80 100 120 0 24 48 72 96 120 treatment time (h) m or ta lit y（ % ） 2000μg/ml 1000μg/ml 500μg/ml 250μg/ml 125μg/ml 0 20 40 60 80 100 120 0 24 48 72 96 120 treatment time (h) m or ta lit y (% ) 2000μg/ml 1000μg/ml 500μg/ml 250μg/ml 125μg/ml fig. 1 mortality of typha angustifolia ethanol extract against the micrergate of red imported fire ant. each data point represents mean ± se of three replicates. each replicate contains 30 tested ants. position 600 mhz, cdcl3 150 mhz, cdcl3 δh (j, hz) δc a (major) b (minor) a (major) b (minor) 1 32.11 2 30.60 3 3.28, m 79.06 4 40.70 5 47.32 6 21.33 7 26.23 8 48.20 9 20.20 10 26.29 11 26.68 12 33.10 13 45.50 14 49.02 15 35.77 16 1.88, m; 1.29, m 1.88, m; 1.29, m 28.35 28.30 17 52.39 18 0.96, s 18.25 19 0.55, d (4.2); 0.33, d (4.2) 30.11 20 36.13 36.16 21 0.88, d (6.0) 0.88, d (6.0) 18.54 18.53 22 32.11 23 1.41, m; 1.61, m 1.41, m; 1.62, m 31.87 31.71 24 4.02, t (6.5) 76.58 25 148.00 147.70 26 4.93, br, s; 4.83, br, s 4.92, br, s; 4.83, br, s 111.62 111.11 27 1.72, br, s 1.72, br, s 17.82 17.42 28 0.96, s 25.65 29 0.81, s 14.22 30 0.89, s 19.53 table 1. 1h and 13c nmr data of 3β-hydroxy-25 methylenecycloartan-24-ol. fig. 2. mortality of typha angustifolia petroleum ether fraction against micrergates of red imported fire ants. each data point represents mean ± se of three replicates. each replicate contains 30 tested ants. 92.45% corrected mortality of micrergates of red imported fire ants treated for 120 h. lc50 values of the ethanol extract treated for 48 and 120 h were 956.85 and 271.23 µg/ml, respectively (table 2). sociobiology 60(4): 362-366 (2013) 365 0 20 40 60 80 100 120 0 24 48 72 96 120 treatment time (h) m or ta li ty ( % ) 240μg/ml 120μg/ml 60μg/ml 30μg/ml 15μg/ml fig. 3. mortality of 3β-hydroxy-25-methylenecycloartan-24-ol against micrergates of red imported fire ants. each data point represents mean ± se of three replicates. each replicate contains 30 tested ants. acknowledgments the work was supported by grants from the national department public benefit research foundation of china (no. 30571235). references escobedo-martínez, c., concepción lozada, m., hernándezortega, s., villarreal, m. l., gnecco, d., enríquez, r. g., & reynolds, w. (2012). 1h and 13c nmr characterization of new cycloartane triterpenes from mangifera indica. magn. reson. chem., 50: 52-57. doi: 10.1002/mrc.2836 gallardo-williams m. t., geiger c. l., pidala j. a. & martin d. f. (2002). essential fatty acids and phenolic acids from extracts and leachates of southern cattail (typha domingensis p.). phytochemistry, 59: 305-308. doi: 10.1016/s0031-9422(01)00449-6 table 2. lc50 (µg/ml) of typha angustifolia ethanol extract, petroleum ether fraction, and 3β-hydroxy-25-methylenecycloartan-24-ol against micrergates of red imported fire ants. treatment 48 h 120 h lc50 (µg/ml) 95% fiducial limit lc50 (µg/ml) 95% fiducial limit ethanol extract 956.85 710.43–1288.75 271.23 201.44–365.21 petroleum ether fraction 398.73 297.60–534.23 152.86 118.41–197.34 3β-hydroxy-25-methylenecycloartan-24-ol 316.50 146.64–683.12 28.52 20.93–38.85 huang t. f., xiong z. h. & zeng x. n. (2007). studies on the contact toxicity of insecticides against the worker ants of solenopsis invicta. j. south china agric. univ., 28: 26-29. (in chinese) http://file.lw23.com/6/6a/6ab/6ab60791-9e014d0a-9d2c-8185732d3e22.pdf jia s. s., liu y. l., ma c. m. , yang s. l., zhou h. m., zhao d. c., liu d. & li s.y. (1986). studies on the constituents of the flavonoids from the pollen of typha angustfolia l. (puhuang). acta pharmac. sin., 21: 441-446. (in chinese) http://en.cnki.com.cn/article_en/cjfdtotalyxxb198606006.htm kong x. p., chen p. d., zhang l., shan m. q., cao y. d. & ding a. w. (2011). study on chemical constituents in pollen of typha angustifolia. jilin j. trad. chin. med., 31: 72-74. (in chinese) doi: 10.3969/j.issn.1003-5699.2011.01.038 li j. h., chen c. f. & li w. w. (2011). an overview of research progress on pharmacological activity and clinical application of pollen typhae. j. anhui agric. sci., 39: 9604-9606. (in chinese) doi: 10.3969/j.issn.0517-6611.2011.16.056 liu c. b., zhang s. c. (2009). pharmacology and clinical study on a traditional chinese drug pollen typhae. world j. integr. trad. western med., 4: 149-152. (in chinese). doi: 10.3969/j.issn.1673-6613.2009.02.034 lv l. h., feng x., cheng h. y., liu j., liu x. y. & he y. r. (2006). a technique for field collecting and laboratory rearing red imported fire ant, solenopsis invicta. chin. bul. entom., 43: 265-267. (in chinese) doi: 10.3969/j.issn.04528255.2006.02.034 yu h. f., du l. l. & li x. q. (2007). comprehensive utilization of typha. modern chin. med., 9: 31-34, 38. (in chinese) doi: 10.3969/j.issn.1673-4890.2007.09.013 yuan k. w., xu w. h. (1996). chemistry and pharmacological activity of pollen typhae. chin. trad. herbal drugs, 27: 693-696. (in chinese) http://www.cnki.com.cn/article/ cjfdtotal-zcyo199611024.htm zx zhang, et al insecticidal activity of typha angustifolia against solenopsis invicta366 yuan y. r., li x. y. & gao g. l. (2012). research on characteristic and medical value of typha angustifolia l.. modern agric. sci. technol., 9: 112-113, 115. (in chinese) doi: 10.3969/j.issn.1007-5739.2012.09.064 zhang s. m., qu g. w., xie f. x., qiu y. k. & wang p. p. (2008). study on chemical constituents in pollen of typha angustifolia. chin. trad. herbal drugs, 3:, 350-352. (in chinese) http://www.cnki.com.cn/article/cjfdtotalzcyo200803009.htm open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.441-448sociobiology 61(4): 441-448 (december, 2014) pollen and nectar foraging by melipona quadrifasciata anthidioides lepeletier (hymenoptera: apidae: meliponini) in natural habitat introduction flight activity in stingless bees have been studied for many species in brazil, such as: melipona quadrifasciata quadrifasciata lepeletier, melipona marginata marginata lepeletier, melipona obscurior moure, melipona bicolor bicolor lepeletier (review in hilário et al., 2000), trigona hyalinata lepeletier (contrera et al., 2004), tetragona clavipes fabricius (rodrigues et al., 2007), melipona bicolor schencki gribodo (ferreira-junior et al., 2010), melipona rufiventris lepeletier (fidalgo & kleinert, 2010), melipona eburnea friese (nates-parra & rodríguez, 2011), plebeia remota holmberg (hilário & imperatriz-fonseca, 2012), scaptotrigona depilis moure (figueiredo-mecca et al., 2013) and geotrigona mombuca smith (gobatto & knoll 2013). in the majority of abstract this study shows the influence of meteorological factors on the collection of nectar and pollen by melipona quadrifasciata anthidioides lepeletier foragers in their natural habitat. five m. quadrifasciata anthidioides colonies were studied in parque das neblinas, mogi das cruzes district (23º44’52”s/46º09’46”w), from october 2009 to september 2010. the foraging activity of the worker bees was observed monthly and the temperature and relative humidity were registered and the pollen grains types from the pollen loads were identified. the peaks of pollen collection occurred between 08:30-09:50 am, while nectar was gathered along the day. the relation between resources sampling and environmental temperature is best described with a polynomial function, while in relation to relative humidity the curves of foraging activity is slightly asymmetric to left. a total of 24 pollen types were identified and the most frequents were myrtaceae (eucalyptus, myrcia), melastomataceae and solanaceae. the tolerance to the environmental conditions is discussed, as well as the plants explored for pollen sources. sociobiology an international journal on social insects c oliveira-abreu1, sd hilário2, cfp luz3, i alves-dos-santos2 article history edited by cândida maria l. aguiar, uefs, brazil received 30 september 2014 initial acceptance 11 november 2014 final acceptance 05 december 2014 keywords stingless bees, foraging behavior, nectar, pollen. corresponding author sergio dias hilário departamento de ecologia, instituto de biociências, universidade de são paulo rua do matão, trav. 14, 321 cidade universitária 05508-090, são paulo, sp, brazil e-mail: sedilar@usp.br these studies the flight activity of the workers was associated with environmental conditions like temperature, barometric pressure, relative humidity, wind speed and even time of the day. however, any of these conditions can interact with biotic factors such as bees’ physiology or biological clocks (hilário et al., 2003; gouw & gimenes, 2013), influencing the performance of the individuals. in fact, the amplitude of tolerance to many ecological factors (with the minimum and maximum limits of tolerance) may have synergistic effects. besides the environmental conditions, the availability of food resources also play an important role on the limits where the species can live, grow and reproduce. in stingless bees, the essential food resources are pollen and nectar, which are stored in pots inside the nest (michener, 1974; roubik, 1989). the health and size of the colonies may reflect research article bees 1 universidade de são paulo, ribeirão preto, brazil 2 universidade de são paulo/ibusp, são paulo, brazil 3 instituto de botânica de são paulo, são paulo, brazil c oliveira-abreu, sd hilário, cfp luz, i alves-dos-santos running title: pollen and nectar foraging by melipona quadrifasciata anthidioides442 the availability of the floral resources in the field. thus, the number of individuals in the nest and the colony survival along the years are dependent, among other factors, on the amount of food collected and stored. on the other hand, the population size of each nest characterizes the colony as small (or weak), medium or large (or strong) and has a direct relationship with the foraging activities among other factors (kleinertgiovannini & imperatriz-fonseca, 1986; hilário et al., 2000; hilário et al., 2003; hilário & imperatriz-fonseca, 2009). melipona quadrifasciata lepeletier, has a relative broad geographic distribution along eastern brazil, reaching the misiones region in argentina and paraguay (camargo & pedro, 2008; batalha-filho et al., 2009). this species is commonly associated with the atlantic rainforest habitats in southern and southeastern brazil. the colony of this species is less numerous than other stingless bees, approximately 300-400 individuals (tóth et al., 2004). in nature the nest is constructed inside trees hollows, but it is easily kept and maintained into artificial hives. concerning the plants visited by m. quadrifasciata this species is considered generalist, but several species of myrtaceae, asteraceae, melastomataceae and solanaceae are among the preferred floral resources (ramalho et al., 1989; wilms & wiechers, 1997; antonini et al., 2006a; antonini et al., 2006b). the aim of this study was to evaluate the pattern of pollen and nectar collection by m. quadrifasciata and their relationship with environmental factors like the temperature and relative humidity under natural conditions. this species has an economic importance for the beekeeper and can be used as pollinator in greenhouse, in tomato production for example (del sarto et al., 2005; bispo dos santos et al., 2009; bartelli et al., 2014; bartelli & nogueira-ferreira, 2014). material and methods study area this study was conducted in the parque das neblinas, located in the mogi das cruzes district in sao paulo state, brazil (23º44’52”s/46º09’46”w; 700 to 1100m a.s.l.). the park has 2,788.15 hectares and the main vegetation is the atlantic rainforest. the climate of the region according to köppen is af, considered as tropical and always humid with the average temperature above 18°c during the summer. annual precipitation varies between 1600 and 2000 mm. there is no winter dry season and only a decrease of precipitation is registered. external activities of the bees five colonies of m. quadrifasciata were maintained in free-foraging wooden nest boxes at the park. during the first month the colonies were fed every 2 weeks with a sugar syrup (1:1 sugar and water). the flight activity of the bees was observed monthly between october 2009 and september 2010. for each month, the number of bees returning from the field to the nest was registered along two consecutive days, during five minutes per hour, between 05:30 am to 04:30 pm, which corresponded to 1430 observations. pollen was easily observed on the corbiculae of foragers, due to the color and texture of the load. on the other hand, foragers returning to the colonies, without load on the legs, were considered as nectar foragers considering that nectar is stored and transported inside the honey stomach (roubik, 1989; pierrot & schlindwein, 2003; souza et al., 2006). other materials, like resin and mud were not considered in the analysis. during the observations the temperature and relative humidity were registered every 30 minutes, using dataloggers (hobo pro rh/temp), which were installed in the area and later the data were transferred to the computer. pollen analysis one day after the field observations, samples of the honey collected from the pots and the pollen collected from the corbiculae of the foragers were taken from the five nests from december 2009 to september 2010. for that, the entrance of the colony was closed for five minutes and five arriving foragers were caught with entomological net. the pollen loads were extracted from their corbiculae and kept on separated plastic tubes (eppendorff) and the bees were released. for each colony samples of 10ml of honey from three new storage pots were sampled with syringes and kept separated into sterilized vials. in february honey and pollen were not sampled due to excessive rainfall. the honey samples and pollen pellets were prepared fresh according to the standard european protocol (maurizio & louveaux, 1965) for palynological studies. three microscopic slides with glycerine jelly were prepared for each sample and were sealed with paraffin. pollen grains identification was carried out using literature data (e.g., barth, 1989; roubik & moreno, 1991) and the reference of pollen grains previously identified and maintained in slides in the collection from the research center of palynology, botanical institute, department of environment of são paulo state. the pollen grains were identified using the term “pollen type” that means pollen from a single plant species as well as a group of species or higher taxa presenting similar pollen morphology. data analyses since our data had a non-normal distribution, we performed nonparametric correlation spearman’s rho (zar, 1999) to verify if there was relationship between the meteorological factors and the pattern of nectar and pollen collection. for statistical analysis the spss for windows release 8.0 was used. sociobiology 61(4): 441-448 (december, 2014) 443 results the temperature range during the experiment was between 5.8° and 37.0°c, and the relative humidity range was between 15% and 100%. foraging flights were registered within a temperature range of 13.7°-36.8°c, while relative humidity (rh) oscillated between 15.7% and 100%. the number of pollen and nectar foragers was positively correlated to the air temperature, and negatively with the relative humidity (table 1). these correlations were weak, since that there was a peak in intermediates temperature and relative humidity ranges (figs 1 and 2). pollen and nectar collection occurred at the relative humidity intervals of 60.1-70.0% (fig 2). table 1. spearman’s correlation coefficients (rho) for pollen and nectar collection by melipona quadrifasciata anthidioides foragers and meteorological factors. the relation of resources sampling and environmental temperature was best described with a polynomial function, showing an optimal interval between ca. 17.0°c -29.0°c, which corresponded to an intense foraging activity for nectar and pollen (fig 1). it is also possible to observe that there is a limit of tolerance in temperature below 13.7°c, and above 35.0°c. peaks of pollen and nectar collection occurred at the temperature intervals of 20.1°-23.0°c and 17.1°-20.0°c, respectively (fig 1). in relation to relative humidity the tendency to polynomial function is not clear and the curves of resources sampling are slightly asymmetric to left (fig 2). foraging activity increased with 40% relative humidity. both peaks of fig 1. pollen and nectar collection by melipona quadrifasciata anthidiodes related to the environmental temperature intervals. solid and dashed bars represent standard deviation of pollen and nectar collection, respectively. fig 2. pollen and nectar collection by melipona quadrifasciata anthidiodes related to the relative humidity intervals. solid and dashed bars represent standard deviation of pollen and nectar collection, respectively. fig 3. hourly variation of pollen and nectar collection related to the meteorological factors (temperature and relative humidity). solid and dashed bars represent standard deviation of pollen and nectar collection, respectively. the number of bees collecting resource increased until 09:50 am, with a peak of nectar sample between 08:30-08:50 am and pollen gather between 09:30-09:50 am. after this period the income of pollen decreased gradually, while the bees remain collecting nectar for the rest of the day (fig 3). rho p pollen temperature 0.174 0.001 (s) relative humidity -0.057 0.030 (s) nectar temperature 0.208 0.001 (s) relative humidity -0.063 0.017 (s) n=1430; s= significat, ns=non-significat; p=probability. c oliveira-abreu, sd hilário, cfp luz, i alves-dos-santos running title: pollen and nectar foraging by melipona quadrifasciata anthidioides444 the number of pollen types from honey pots and corbiculae was moderately correlated to the number of bees entering inside the nest with pollen and nectar (rho = 0.410; p = 0.009; fig 4). in samples of the honey pots and pollen of the corbiculae a total of 24 pollen types were recorded, recognizing 18 genera, 16 families. the most frequent pollen types in samples of honey and pollen of were myrtaceae (eucalyptus, myrcia), melastomataceae and solanaceae, which were also presented along all the year (table 2; fig 5). discussion the concept of niche proposed by hutchison (hutchinson, 1957) refers to the way that the tolerance and needs of the many conditions interact with the resources used by a species. the variables analyzed in this work are part of the ecological niche of m. quadrifasciata anthidioides in their natural environment, since we recorded the ideal temperatures and humidity for the foraging activity. in other words, environmental conditions influences the adaptative value in this species. in addition another important component of the niche, which are the plants explored for pollen sources, was also recorded. according to cobert et al., (1993) the temperature is the environmental factor that most affects the flight activity on bees. the extreme values of temperature affect the individual thermoregulation prerequisite to flight behavior (heinrich & esch, 1994). our results demonstrate that m. quadrifasciata anthidioides reduces the collection of resources with temperatures below 13.7°c and over 29.0°c when the foraging activity decreases considerably, in the studied area. the lowest value is very similar to temperatures found to this species in an urban table 2. pollen types present on the corbiculae of the workers (*) and honey pots (+) in 5 melipona quadrifasciata nests, sampled during 9 months (december 2009 to september 2010) in the parque das neblinas. fig 4. relationship between the number of pollen grain types (from corbiculae loads and honey from pots) with the total numbers of pollen and nectar foragers returning to the nest. pollen types/ months dec jan mar apr mai jun jul ago sep aegiphila (+) x alchornea (+) x baccharis (*) (+) x x x x x x begonia (*) x eucalyptus (*) (+) x x x x x x x x x euterpe/syagrus (+) x x hibiscus (+) x x inga (+) x machaerium (+) x x x maranthaceae (+) x melastomataceae (*) (+) x x x x x x x x x mimosa caesalpiniaefolia (+) x x x mimosa scabrella (*) x monocot (+) x x myrcia (*) (+) x x x x x x x x x schefflera (+) x serjania (+) x x x x solanaceae (*) (+) x x x x x x x x sorocea (+) x struthanthus (+) x stylosanthes (+) x triumfetta (+) x vernonia (*) (+) x x x not identified (+) x sociobiology 61(4): 441-448 (december, 2014) 445 fig 5. light microscopic (lm) pollen grain types micrographs observed in both honey and pollen pellets samples of melipona quadrifasciata in parque das neblinas, mogi das cruzes district, são paulo, brazil. figure a. aegiphila. b. baccharis. c. begonia. d. eucalyptus. e. euterpe/syagrus. f. hibiscus. g. inga. h. machaerium. i. melastomataceae. j. mimosa caesalpiniaefolia. k. mimosa scabrella. l. myrcia. m. serjania. n. solanaceae. o. vernonia. scales = 10µ. c oliveira-abreu, sd hilário, cfp luz, i alves-dos-santos running title: pollen and nectar foraging by melipona quadrifasciata anthidioides446 area (guibu & imperatriz-fonseca, 1984), but the highest temperature limits seems to be higher in our studied area, comparing also to other melipona. this may occurs because of the local shaded environment. the relation between gathering food and temperature fits better in a quadratic curve, demonstrating an optimum range and limits of tolerance. this seems to be truth for many other stingless bees, but the limits vary a lot among the species. sometimes the limits are tested, for instance when a sudden mass flowering provoke a copious collection of pollen outside the foraging pattern (pierrot & schlindwein, 2003). in the subtribe trigonina (tribe meliponini) the optimal range seems to be shorter, starting with temperature higher. for example mouga (1984) observed that for paratrigona subnuda moure the greatest flight activity occurs between temperatures of 24.0°c and 25.0°c; in plebeia saiqui friese in southern brazil, the collection of pollen had a higher intensity in the range of 18.0° to 19.0°c (pick & blochtein, 2002). some melipona species shows the optimal range wider and the lowest limit lower, like m. marginata (19.0°-30.0°c), m. bicolor bicolor (16.0°-26.0°c) and m. rufiventris (16.0°30.0°c) for the activity of related pollen and nectar collection (kleinert-giovannini & imperatriz-fonseca, 1986; hilário et al., 2000; fidalgo & kleinert, 2007). in cruz das almas, bahia, northeastern brazil, nascimento et al., (2012) recorded a significant negative correlation of temperature to the flight activity of m. quadrifasciata, and the peaks of bee flow occurred in temperature between 21.2° and 23.3°c. all these mentioned bee species are of different size, and probably the body dimension plays an important role on the tolerance of most suitable climatic factors for foraging. another important dimension of the niche for melipona bees is the environmental relative humidity. in this case we recorded that humidity higher than 30-40% is ideal for pollen gathering, while nectar collection occurred in a wide range of rh. but, it is important to consider that pollen is more available earlier in the day, when humidity is higher, while nectar in general is available the whole day. furthermore, the habitat of the coastal atlantic rainforest is moist almost all over the year, due to the dynamic of the trade winds producing precipitation or fog daily. more than 1/3 of our data were recorded with rh above 90%, in the studied area. silva et al., (2011) suggested that foragers from large species of melipona use rh as indicator of pollen availability in the environment. as it was expected for melipona bees the foraging activities are concentrated early in the morning and tend to decrease after midday. but in the studied area the peaks of pollen and nectar collection are slightly delay, probably due to the fogs formed at dawn and early in the morning. for many melipona species the peaks of pollen harvest occurs before 08:00 am (bruijn & sommeijer, 1997; fidalgo & kleinert, 2007) for example. the moderate correlation among the number of pollen types and bees entering the nest suggest that an increasing in flowering plants elicited an increasing in foraging intensity. but we need to consider that pollen type diversity not necessary correspond to floral abundance. it is know that meliponini species use massive flowering plants (roubik, 1989; wilms & wiechers, 1997). according to antonini et al., (2006a) m. quadrifasciata anthidioides is considered a generalist species, since it visited more than ten plant species. but, there is no doubt that myrtaceae (eucalyptus) pollen play an important role in the diet of m. quadrifasciata anthidioides. this was also verified in other areas (ramalho et al., 1989; wilms & wiechers, 1997; antonini et al., 2006b). similarly, more than 75% of myrtaceae pollen grains was found in the honey and in the larval food from five species of melipona in the amazon forest (cortopassi-laurino et al., 2007). luz et al., (2011) and serra et al., (2012) also found preference for myrtaceae pollen in melipona capixaba moure and camargo. according to roubik (1989) the foraging behavior of a bee is determined by the resources it has access, being influenced by the quality, dispersion, quantity and competition for these resources plus the environmental conditions. in the study area, eucalyptus is abundant, since it was used in the park reforestation program for a period, and so represents no competition with other foragers. hilgert-moreira et al., (2014) demonstrated that regardless of the landscape characteristics, m. obscurior foragers were able to collect pollen from eucalyptus throughout the year. according to these authors, this persistence is due to the capability of eucalyptus in supporting more than one foraging species, which results in low or inexistent competition for the resources. in samples of the honey pots many species were represented just once during the year. we believe that some of these species (like the first of the list: aegiphila, verbenaceae) are just nectar sources for m. quadrifasciata anthidioides, and probably the foragers were contaminated with the pollen on the body during nectar collection. an alternate explanation would be that their bloom just for short period. the environmental conditions and availability of particular resources define where the species can live, grow and reproduce. furthermore, we have to remember that the niche dimensions (temperature and relative humidity for example) act together. under natural conditions the ecological niche of m. quadrifasciata anthidioides is composed of multiple dimensions, i.e. tolerance to many other conditions and requirements that were not measured in this study, but it seems that in the studied area, m. quadrifasciata anthidioides are finding the good requirements since the five colonies kept the reproduction the whole year. acknowledgment we are grateful for the financial support of capes, fapesp, cnpq and the permission and help in the field of sociobiology 61(4): 441-448 (december, 2014) 447 the instituto ecofuturo. the authors express their gratitude to roberto shimizu for his assistance in data analysis, denise de araujo alves and carlos eduardo pinto that helped in the discussion of the data and the two anonymous referees for their valuable comments and suggestions. references antonini, y., costa, r.g. & martins, r.p. 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(1999). biostatistical analysis. fourth ed. upper saddle river, new jersey: prentice-hall, 664p. 241 structure-invading pest ants in healthcare facilities in singapore by lai-sum man1 & chow-yang lee2 abstract a survey of structure-invading pest ants was conducted at 17 healthcare facilities (hfs) in singapore using the index card method. a total of 18 species (anoplolepis gracilipes (fr. smith), meranoplus sp., monomorium destructor ( jerdon), monomorium floricola ( jerdon), monomorium pharaonis (l.), monomorium sp., odontomachus sp., oecophylla smaragdina (f.), pachycondyla sp., paratrechina bourbonica (forel), paratrechina longicornis (latreille), paratrechina pubens (forel), pheidole megacephala (f.), pheidole parva (mayr), pheidole sp., tapinoma indicum (forel), tapinoma melanocephalum (f.) andtetramorium bicarinatum (nylander)) were trapped. of these, the most common species were p. parva (25.9%), p. megacephala (25.2%), p. longicornis (14.1%), m. pharaonis (9.6%), and t. indicum (8.1%). most of these ant species were found in and around the premises. introduction singapore has a world-class healthcare system that was ranked sixth best in the world and the best in asia by the world health organization in 2000. as asia’s leading medical hub, healthcare facilities (hfs) in singapore emphasize excellence, safety, and trustworthiness (lim 2004). this means that the public’s expectations of quality service are high, and this includes low tolerance of pests within the premises. nevertheless, the majority of hfs through out the world are prone to pest infestation (burgess 1984). in singapore, hfs are vulnerable to pest intrusion and infestation through numerous entrances, exits, and food establishments within the premises, as well as through winurban entomolog y laboratory, vector control research unit, school of biological sciences, universiti sains malaysia, 11800 penang, malaysia 1present address: iss pest management pte ltd, 51 benoi road blk 8 #01-02 liang huat industrial complex singapore 629908. 2corresponding author. email: chowyang@usm.my 242 sociobiolog y vol. 59, no. 1, 2012 dows, ceilings, wall cracks, and gaps around plumbing and pipes. they also can hitchhike in commercial deliveries and patient’s clothing. in general, hfs provides an ideal environment in which pests can thrive (murphy & oldbury 1996). compared to other types of premises, hfs are sensitive environments because they house patients with compromised health. pests can carry diseases, and in the process of eliminating pests, patients may be exposed to unforeseen pesticide risks that may not be easily diagnosed (owens 2003). pest ants carry pathogens that may cause diseases that pose a threat to public health (lee 2002). for example, pharaoh ants collected from nine hospitals were shown to carry numerous pathogenic organisms (beatson 1972). pharaoh ants also can contaminate food and sterile instruments (beatson 1972, wilkinson 1988). however, according to who (2008), no cases of patients being affected by pharaoh ant-mediated mechanical contamination have been reported. fire ants pose a different risk; their stings are painful and cause a burning sensation (deshazo et al. 1990, who 2008). in addition, deaths due to an anaphylactic reaction to fire ant stings have been reported (deshazo et al. 1990, rhoades et al. 1989). until now, information about pest ant infestation in hfs in singapore has not been available. surveys carried out in hfs in this study showed that pest ants are the major pest group affecting these facilities. the goal of this study was to determine the species composition of structure-invading pest ants in hfs in singapore. the resulting data can be used to assist in the management efforts against pest ants in these facilities. materials and methods seventeen hfs in singapore were surveyed. for ethical reasons, the identity of these facilities cannot be revealed. of the 17 hfs, 3, 3, 1, 6, and 4 were located in the north, northeast, east, central, and west parts of singapore, respectively (fig. 1). they were located in concentrated urban areas surrounded by plants, plots of greenery, open car parks, roads, sidewalks, and tree-lined streets. ants were collected using the index card method (lee 2000, lee & lee 2002, lee et al. 2003). blank index cards (7.5 x 12 cm) baited with peanut butter (24.3% protein and 47.2% fat) and honey (83% carbohydrate) were used to attract the ants. baited index cards were placed at locations within the 243 man. l.-s. & c.-y. lee — pest ants in healthcare facilities in singapore hfs where staff members had seen ant trails or activities, and each location was listed on a checklist. these locations included cafeterias, staff pantries, patient wards, offices, the building perimeter, and loading/unloading bays. all baited and placed index cards were checked for the presence of ants after 40–50 min. the ants on the index card were photographed using a digital camera (nikon d90, nikon corp, bangkok, thailand). the ants then were collected, placed in plastic vials, and brought back to the laboratory for identification. identifications were performed using a stereoscopic zoom microscope (smz800, nikon corp., yokohama, japan) and using identification keys in antweb (2006), hedges (2010), and na and lee (2001). results and discussion eighteen pest ant species were collected and identified (table 1), and most were found both indoors and along the building perimeter of the hfs. pheidole parva mayr and pheidole megacephala (f.) were the two most common ant species encountered; together they accounted for more than 50% of the total collection (25.9% and 25.2%, respectively). earlier studies conducted in malaysia also reported that p. megacephala was the most commonly found ant species (lee et al. 2002, na & lee 2001). in a survey conducted in urban fig. 1: a map of singapore showing the locations of the healthcare facilities at which the pest ant surveys were conducted. 244 sociobiolog y vol. 59, no. 1, 2012 ta bl e 2: s pe ci es c om po si tio n of p es t a nt s s am pl ed a t e ac h he al th ca re fa ci lit y. sp ec ie s % o f a nt s t ra pp ed a t e ac h he al th ca re fa ci lit y to ta l n o. an ts tr ap pe d to ta l (% ) fr eq ue nc y of oc cu rr en ce n 1 n 2 n 3 n e1 n e2 n e3 e1 c 1 c 2 c 3 c 4 c 5 c 6 w 1 w 2 w 3 w 4 n o. o f a nt s t ra pp ed 20 47 12 82 66 2 40 0 17 64 64 7 98 7 13 3 66 7 81 8 17 08 63 6 31 5 14 46 42 9 83 9 29 2 15 07 2 10 0 su bf am ily m yr m ic in ae m on om or iu m fl or ico la 11 .3 4. 1 2. 2 23 6 1. 6 0. 2 m on om or iu m p ha ra on is 27 .0 13 .3 18 .7 0. 4 87 .3 10 .0 3. 0 0. 6 58 .9 14 60 9. 6 0. 6 m on om or iu m d es tr uc to r 44 .3 17 7 1. 2 0. 06 7 m on om or iu m sp . 12 .7 2. 2 23 8 1. 6 0. 13 ph ei do le m eg ac ep ha la 39 .5 32 .4 87 .2 75 .0 88 .1 16 .7 4. 1 13 .1 41 .1 37 92 25 .2 0. 6 ph ei do le p ar va 5. 9 22 .1 36 .7 30 .8 31 .6 13 .3 5. 3 60 .2 24 .0 26 .8 42 .3 4. 1 61 .8 49 .2 41 .8 38 99 25 .9 1 ph ei do le sp . 11 .2 23 0 1. 5 0. 06 7 te tr am or iu m b ic ar in at um 9. 5 4. 7 3. 1 2. 0 1. 6 4. 8 23 6 1. 6 0. 4 m er an op lu s s p. 0. 3 2 0. 01 0. 06 7 su bf am ily d ol ic ho de rin ae ta pi no m a m el an oc ep ha lu m 4. 1 20 .3 2. 6 0. 8 0. 3 36 .7 2. 7 86 5 5. 7 0. 47 ta pi no m a in di cu m 19 .6 2. 3 15 .5 0. 2 13 .5 7. 3 45 .1 0. 3 5. 3 7. 0 32 .3 -12 22 8. 1 0. 73 su bf am ily f or m ic in ae pa ra tr ec hi na lo ng ico rn is 10 .4 15 .1 33 .8 11 .7 61 .8 5. 5 24 .1 0. 3 1. 8 11 .1 0. 6 22 .8 27 .7 17 .4 21 26 14 .1 0. 93 pa ra tr ec hi na b ou rb on ic a 0. 7 4. 6 2. 3 0. 3 7. 8 1. 2 6. 9 0. 1 23 2 1. 5 0. 53 pa ra tr ec hi na p ub en s 5. 9 1. 8 10 .6 0. 4 27 2 1. 8 0. 27 o ec op hy lla sm ar ag di na 1. 2 25 0. 2 0. 06 7 a no pl ol ep is gr ac ili pe s 1. 8 7. 6 56 0. 4 0. 13 su bf am ily p on er in ae pa ch yc on dy la sp . 0. 1 1 0. 00 6 0. 06 7 o do nt om ac hu s s p. 0. 8 3 0. 02 0. 06 7 245 man. l.-s. & c.-y. lee — pest ants in healthcare facilities in singapore areas of singapore in 2004 (lee & tan 2004), there was no documentation of collection of p. parva. it is possible that p. parva was present but identified as pheidole sp. more studies in other urban areas in singapore are needed to determine whether the presence and dominance of p. parva is unique to areas around hfs in singapore. crazy ants, paratrechina longicornis (latreille) were the next most frequently encountered ant species (14.1%), followed by pharaoh ants, monomorium pharaonis (l.) at 9.6% and ghost ants, tapinoma indicum (forel) at 8.1%. in the past, it was reported the infestations of pharaoh ants only occurred indoors, but subsequent studies showed that they can occur in outdoor areas as well (klotz et al. 1995, knight & rust 1990). in our study, pharaoh ants were found both indoors and along outdoor perimeter areas. this may be due to singapore’s tropical climate, which allows pharaoh ants to establish nests or forage in outdoor areas due to the warm temperature (kohn and vlček 1986). five or more ant species were found within the vicinity of the majority of the hfs (> 75%) surveyed (table 2). the ant diversity within individual hfs might have been influenced by the surrounding landscape areas. ant populations often are affected by vegetation structure, with a decrease in vegetation diversity causing a reduction in ant diversity because vegetation plays a major role in regulating the microclimatic conditions that affect ant activity (perfecto & snelling 1995, perfecto & vandermeer 1996, retana & cerdá 2000, vasconcelos et al. 2008). of the 18 ant species found in this study, p. parva had the highest frequency of occurrence (fo): it was found at 15 of the 17 hfs surveyed (table 2). however, p. parva was not always the most abundant species found at those hfs (< 50%). p. longicornis had the highest fo (0.93) after p. parva. p. longicornis can thrive well in hfs because the facilities generally are surrounded by greenery, such as trees, planters, and lawns; landscape mulch, the undersides of potted plants and logs, and tree holes provide ideal nesting sites for crazy ants (hedges 2010). p. longicornis also can forage long distances from their nest ( jaffe 1993), and their quick random movements while foraging for food (lee 2002) help them locate food sources quickly. p. megacephala had the fourth highest fo (0.6) after t. indicum (fo = 0.73) although it was the most abundant ant species in five of the nine hfs 246 sociobiolog y vol. 59, no. 1, 2012 in which it was found. the high abundance of this species in some locations may occur because few other ant species are present in locations where p. megacephala occurs. invasion by p. megacephala reportedly reduced the native ant species and other invertebrate species (tryon 1912, vanderwoude et al. 2000), but its foraging activity declined when other tramp ant species (e.g., p. longicornis and m. pharaonis) were present (loke & lee 2004). only a small percentage of anoplolepis gracilipes (fr. smith) was found in two hfs (fo = 0.13). this is a highly invasive ant species, and chong and lee (2010) reported that it showed aggressive behavior towards other tramp ant species such as m. pharaonis, monomorium floricola ( jerdon), monomorium table 1: the pest ant species sampled and collection locations at the healthcare facilities. species common name location collected total number of ants trapped total (%) indoors outdoor perimeter subfamily myrmicinae monomorium floricola none x x 236 1.6 monomorium pharaonis pharaoh ant x x 1460 9.6 monomorium destructor singapore ant x 177 1.2 monomorium sp. none x 238 1.6 pheidole megacephala big-headed ant x x 3792 25.2 pheidole parva none x x 3899 25.9 pheidole sp. none x 230 1.5 tetramorium bicarinatum guinea ant x x 236 1.6 meranoplus sp. none x 2 0.01 subfamily dolichoderinae tapinoma melanocephalum ghost ant x x 865 5.7 tapinoma indicum ghost ant x x 1222 8.1 subfamily formicinae paratrechina longicornis crazy ant x x 2126 14.1 paratrechina bourbonica robust crazy ant x 232 1.5 paratrechina pubens hairy crazy ant x x 272 1.8 oecophylla smaragdina weaver ant x 25 0.2 anoplolepis gracilipes long-legged ant x x 56 0.4 subfamily ponerinae pachycondyla sp. none x 1 0.006 odontomachus sp. none x 3 0.02 247 man. l.-s. & c.-y. lee — pest ants in healthcare facilities in singapore destructor ( jerdon), p. parva, t. indicum, tapinoma melanocephalum (f.), p. longicornis, and oecophylla smaragdina (f.). our finding differs from results from malaysia, where an increase in a. gracilipes infestations has occurred in the past few years (chong & lee 2010). this difference could be due to the differences in survey locations. many of the ant species trapped in the survey were tramp ants. tramp ants are considered the most difficult group of ants to control, as they thrive successfully in environments where human activities provide them with shelter, moisture and food, which ensure their survival (hedges 2001, silverman 2005). this study had several limitations. not all of the indoor areas were surveyed, as some areas were no-entry zones for pest management professionals. other areas could not be sampled because hospital authorities wanted to avoid unnecessary questioning from the patients or the public and thus would not grant permission. in singapore, food manufacturing plants are among the few sectors that demand and implement the best possible pest management programs. these programs heavily emphasize exclusion methods to prevent pest intrusion and limit the use of chemical treatments to ensure food safety. hfs are another sensitive environment, as they house patients who have serious health conditions. more studies are needed in the hfs of singapore to develop a sustainable pest management program that focuses on integration of various methods to control pests, with use of insecticides only as the last option. acknowledgments the authors thank a.l.-c. seow, k.-h. teo, l.-y. lim and a. nasir of iss pest management pte ltd., singapore for support and technical assistance; dr y.-f. how and e.h.-p. lim for valuable comments on the manuscript. the work reported herein is a section of the graduate research project of the senior author (l.-s.m). references antweb, 2006. http://www.antweb.org/ (accessed 11/05/2011). beatson, s.h. 1972. pharaoh’s ants as pathogen vectors in hospitals. lancet 1: 425-427. burgess, n.r.h. 1984. hospital design and cockroach control. trans. roy. soc. trop. med. hyg. 78: 293-294. 248 sociobiolog y vol. 59, no. 1, 2012 chong, k.f. & c.y. lee 2010. interand intraspecific aggression in the invasive longlegged ant (hymenoptera: formicidae). j. econ. entomol. 103: 1776-1783. deshazo, r.d., b.t. butcher, & w.a. banks 1990. reactions to the stings of the imported fire ant. n.e. j. med. 323: 462-466. hedges, s.a. 2001. the best of stoy hedges. gie media inc publishers, 368 pp. hedges, s.a. 2010. field guide for the management of structure-infesting ants. 3rd edition. gie inc publishers, richfield, ohio. 325 pp. jaffe, k. 1993. surfing ants. flor. entomol. 76: 182-183. klotz, j.h., j.r. mangold, k.m. vail, l.r. davis, jr., & r.s. patterson 1995. a survey of the urban pest ants (hymenoptera: formicidae) of peninsular florida. flor. entomol. 78: 109-118. knight, r.l. & m.k. rust 1990. the urban ants of california with distribution notes of imported species. southwest entomol. 15: 167-178. kohn, m. & m. vlček 1986. outdoor persistence throughout the year of monomorium pharaonis (hymenoptera: formicidae). entomol. gener. 11:213-215. lee, c.y. 2000. performance of hydramethylnonand fipronil-based containerized baits against household ants in residential premises. trop. biomed. 17: 45-48. lee, c.y. 2002. tropical household ants: pest status, species diversity, foraging behavior and baiting studies. pp. 3-18. in: s.c. jones, j. zhai & w. h. robinson [eds.], proceedings of the 4th international conference on urban pests. pocahontas press, blacksburg, virginia. lee, c.y. & l.c. lee 2002. field and laboratory evaluation of a boron-based containerized dual-bait formulation against the pharaoh ant, monomorium pharaonis (hymenoptera: formicidae). sociobiolog y 40: 655-665. lee, c.y., c.y. lim, & i. darah 2002. survey on structure-infesting ants (hymenoptera: formicidae) in food preparative outlets. trop. biomed. 19: 21-26. lee, c.y., j. zairi, h.h. yap, & n.l. chong 2003. urban pest control – a malaysian perspective. second edition. universiti sains malaysia press, penang, malaysia. lee, c.y. & e.k. tan 2004. guide to urban pest ants of singapore. spma for pest management professionals, singapore. 40 pp. lim, m.k. 2004. quest for quality care and patient safety: the case of singapore. bmj quality safety 13: 71-75 loke, p.y & c.y. lee 2004. foraging behaviour of field populations of big-headed ant, pheidole megacephala (hymenoptera: formicidae). sociobiolog y 43: 211-219. murphy, r.g. & d.j. oldbury 1996. the role of environment health departments in ensuring pest free hospitals. pp. 579-586. in: k.b. wildey [eds], proceedings of the 2nd international conference on urban pests. na, j.p.s. & c.y. lee 2001. identification key to common urban pest ants in malaysia. trop. biomed. 18: 1-17. owens, k. 2003. healthy hospitals: controlling pests without harmful pesticides. washington dc: health care without harm & beyond pesticides. 57 pp. perfecto i. & r. snelling 1995. biodiversity and the transformation of a tropical agroecosystem: 249 man. l.-s. & c.-y. lee — pest ants in healthcare facilities in singapore ants in coffee plantations. ecol. appl. 5: 1084-1097. perfecto, i. & j. vandermeer 1996. microclimatic changes and the indirect loss of ant diversity in a tropical agroecosystem. oecologia 108: 577-582. retana j. & x. cerdá 2000. patterns of diversity and composition of mediterranean ground ant communities: tracking spatial and temporal variability in the thermal environment. oecologia 123: 436-444. rhoades, r.b., c.t. stafford, & f.k. james, jr. 1989. survey of fatal anaphylactic reactions to imported fire ants stings. j. allerg y clin. immunol. 84: 159-162. silverman, j. 2005. why do certain ants thrive in the urban environment? pp. 29-31. in: c.y. lee & w.h. robinson [eds], proceedings of the fifth international conference on urban pests, perniagaan ph’ng @ p&y design network, malaysia. tyron, h. 1912. the naturalization of an exotic ant (pheidole megacephala fab.). qld nat. 1: 224-229. vanderwoude, c., l.a. lobry de bruyn, & a.p.n. house 2000. response of an open-forest ant community to invasion by the introduced ant, pheidole megacephala. austral ecol. 25: 253-259. vasconcelos, h.l, m.f. leite, j.m.s. vilhena, a.p. lima, & w.e. magnusson 2008. ant diversity in an amazonian savanna: relationship with vegetation structure, disturbance by fire, and dominants ants. austral. ecol. 33: 221-231. who 2008. public health significance of urban pests. who regional office for europe. 569 pp. wilkinson, p.j. 1988. food hygiene in hospitals. j. hosp. infect. 11: 77-81. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7286sociobiology 68(4): e7286 (december, 2021) red wood ants (the formica rufa group) are polyphagous predators (laakso, 1999). although captured prey only constitutes about 20% of their diet, they strongly affect the abundance and behaviors of many arthropod taxa in the temporal and boreal forests of europe (skinner, 1980; laakso, 1999; reznikova & doroshea, 2004). in addition to direct predation, they may influence other animals through resource competition or non-consumptive interactions (hawes et al., 2002, maňák et al., 2013). several studies describe the predatory capacity of wood ants to reduce the population densities of forest pests, e.g., pristiphora abietina (christ.), bupalus piniarius (l.), panolis flammea (denis & schiffermüller), dendrolimus pini (l.), lymantria dispar (l.), operophtera brumata (l.), and oporinia autumnata (bkh.) (otto, 1967; laine & niemelä, 1980; weseloh, 1994, nielsen et al., 2018). in defoliated plantations, the effect of ant predation can be observed as “green islands” of undefoliated trees around their nests (karhu & neuvonen, 1998). adlung (1969) points out abstract red wood ants (the formica rufa group) are important predators which affect animal communities in their territory. therefore, they are useful in forest protection. on the other hand, they also prey on beneficial organisms. we have asked whether formica rufa l. affects the abundance of the parasitic fly ernestia rudis (fallén). ten anthills situated in about 40-yearold pine plantations were used for the study. the presence of e. rudis cocoons was assessed in eight soil samples excavated in the surrounding of each nest at a distance of 2–17 m. our results show a considerably lower abundance of e. rudis only to 4.5 m from the nests. the occurrence of formica rufa ants therefore had no significant effect on the beneficial e. rudis population in plantation forests, where ants populations are low. sociobiology an international journal on social insects adam véle1, jovan dobrosavljević2 article history edited by evando nascimento silva, uefs, brazil received 30 june 2021 initial acceptance 25 july 2021 final acceptance 14 august 2021 publication date 23 december 2021 keywords beneficial organisms, distance, pest, production forest, red wood ants. corresponding author adam véle forestry and game management research institute strnady 136, 252 02 jíloviště, czech republic. e-mail: adam.vele@centrum.cz that as a result of their foraging opportunism, ants also prey on nonharmful and beneficial species, thereby hindering the contribution of the latter to forest protection. in our study, we have asked whether formica rufa l. affects the abundance of ernestia rudis. this species of parasitic fly of the family tachinidae is an important population regulator of the moth panolis flammea, which causes extensive damage in pine plantations. one e. rudis larva can parasitize one panolis flammea caterpillar and subsequently crawl to the ground, where it pupates (křístek & urban, 2013). formica rufa is a typical representative of wood ants. in central europe, it is common from lowlands to mountains, where it inhabits mainly coniferous and mixed forests, preferring lighter areas (czechowski et al., 2002; bezděčka & bezděčková, 2011). the study area was a large (ca 35 km2) plantation forest of pinus sylvestris l. in south moravia, czech republic (48°57′27.516″ n, 17°12′46.611″ e). three hundred ha of 1 forestry and game management research institute. jíloviště, czech republic 2 faculty of forestry, university of belgrade, belgrade, serbia formica rufa ants have a limited effect on the abundance of the parasitic fly ernestia rudis in scots pine plantations short note mailto:adam.vele@centrum.cz adam véle, jovan dobrosavljević – wood ants do not affect ernestia rudis abundance2 this area were equally and severely damaged by p. flammea in 2018 and 2019. in line with this, the abundance of the parasitic e. rudis increased in 2019. for the study, ten anthills situated in about 40-year-old forests were chosen. all nests were isolated, i.e., their territories did not overlap. eight soil samples were dug up in the surrounding of each nest. the centers of the soil sampling patches (25 × 25 cm) were situated at a regular distance of 2.1, 4.3, 6.5, 8.7, 10.9, 13.1, 15.3, and 17.5 m from the nests. a distance of 17.5 m is roughly equivalent to an average wood ant territory (horstmann, 1974; skinner, 1980). all samples were collected on november 15, 2019, and transported to the laboratory, where cocoons were separated. to determine the predictors (mound volume, distance from nest) explaining the e. rudis cocoon number, a generalized linear model with log-normal distribution was used. analyses were done in statistica 13.0. the mean mound volume was 0.11 ± 0.08 m3. the number of e. rudis cocoons increased with distance from nests (p ≤ 0.01), independently of mound volume (p = 0.4). a considerably lower abundance of e. rudis was determined up to a distance of 4.5 m (fig 1). the short distance found in this study is probably a consequence of the low volume of mounds (sorvari, 2009). the mound volume is correlated with the size of the nest population (skinner, 1980). it follows that a larger colony consumes more prey (trainello, 1989; domisch et al., 2009). mound volumes measured in this study are lower than the size commonly reported for f. rufa and the closely related species f. polyctena först., hence the all examined anthills fall into the category of small nests (cezchowski, 2002; mabelis, 1979; frouz, 1996; kadochová & frouz, 2014; rybnikova & kuznetsov, 2015). this may be the reason why mound volume was found to be insignificant in this study and why other authors describe strong predatory abilities of ants up to a distance of 1735 m (koehler, 1976; laine & niemelä, 1980; oloffson, 1992). another explanation may be that the above studies primarily focus on prey on trees. however, only 25% of the foraging activity of wood ants takes place on the forest floor (sudd & lodhi, 1981). the caterpillars in the vicinity of the anthill may have been carried by the wind from more distant trees, as described by edland (1971) on the example of the larvae of o. brumata. wellenstein (1954), who counted cocoons of diprion pini (l.), found within 7 m of the nest only 32% of pupae occurring in the vicinity, which roughly confirms our results. also reznikova and dorosheva (2004) found a similar distance when studying the dynamic density of carabids around ant nests. this may be explained by the number of workers, which decreases with increasing distance from the nest (mabelis, 1979). our study provides the first data that suggest the marginal influence of wood ants on the occurrence of the cocoons of e. rudis. outbreaks of p. flammea (and subsequently e. rudis) are rare in central europe, as evidenced by the fact that the previous outbreak at the study site occurred more than 100 years ago. the onset of p. flammea outbreaks was related to a combination of sufficient temperature and precipitation (haynes et al., 2014; véle & liška, 2019). in the future, shorter gradation cycles can be expected due to climate change (haynes et al.. 2014). in light of our findings, future studies should be carried out to describe and evaluate the number of predated e. rudis larvae or compare it with the number of p. flammea caterpillars carried to the nest. fig 1. box plots despicts mean, standard error (box) and standard deviation (whiskers) of the number of e. rudis cocoons according to distance from wood ant nests. sociobiology 68(4): e7286 (december, 2021) 3 our results are roughly consistent with karhu, neuvonen (1998), who generally determined the border of the ecological importance of wood ants to a distance of 8 m and do not support adlung’s (1969) claim of a negative effect of ants on beneficial insects. this may be because plantation forests are not conducive to ants, so that their nest densities and population sizes are low here (sorvari & hakkarainen 2005, 2007). acknowledgment the study was supported by the national agency for agricultural research, ministry of agriculture, czech republic (project qk1920406). conflict of interest the authors declare no conflicts of interest. references adlung, k.g. (1966). a critical evaluation of the european research on use of red wood ants (formica rufa group) for the protection of forests against harmful insects. zeitschrift für angewandte entomologie, 57: 167-189. doi: 10.1111/j. 14390418.1966.tb03822.x bězděčka, p. & bezděčková, k. (2011). mravenci ve sbírkách českých, moravských a slezských muzeí. jihlava: muzeum vysočiny jihlava, 147p czechowski, w.; radchenko, a.; czechowska, w. (2002). the ants (hymenoptera, formicidae) of poland. warsaw: museum and institute of zoology, polish academy of sciences, 200 p domisch, t., finer, l., neuvonen, s., niemelä, p., risch, a.c., kilpeläinen, j., ohashi, m. & jurgensen, m.f. (2009). foraging activity and dietary spectrum of wood ants (formica rufa group) and their role in nutrient fluxes in boreal forests. ecological entomology, 34: 369-377. doi: 10.1111/j.13652311.2009.01086.x edland, t. (1971). wind dispersal of the winter moth larvae operophtera brumata l. (lep., geometridae) and its relevance to control measures. norwegian journal of entomology, 18: 103-105. frouz, j. (1996). the role of nest moisture in thermoregulation of ant (formica polyctena, hymenoptera, formicidae) nests. biologia, 51: 541-547. hawes, c., stewart, a.j.a. & evans, h.f. (2002). the impact of wood ants (formica rufa) on the distribution and abundance of ground beetles (coleoptera: carabidae) in a scots pine plantation. oecologia, 131: 612-619. doi: 10.1007/s00442002-0916-6 haynes, k.j., allstadt, a.j., klimetzek, d. (2014). forest defoliator outbreaks under climate change: effects on the frequency and severity of outbreaks of five pine insect pests. global change biology, 20: 2004-2018. doi: 10.1111/gcb.12506 horstmann, k. (1974). untersuchungen über den nahrungswerb der waldameisen (formica polyctena foerster) im eichenwald. iii. jahresbilanz. oecologia, 15: 187-204. kadochová, s. & frouz, j. (2014). red wood ants formica polyctena switch off active thermoregulation of the nest in autumn. insectes sociaux, 61: 297-306. doi: 10.1007/s00040014-0356-4 karhu, k. & neuvonen, s. (1998). wood ants and geometrid defoliators of birch: predation outweighs beneficial effects through the host plant. oecologia 113: 509-516. koehler, w. (1976). ksztaltowanie sie stosunkow trofobiotycznych przy sztucnej kolonizacii formica polyctena. prace instytutu badawczego lesnictva, 499 p. křístek, j. & urban, j. (2013). lesnická entomologie. praha: academia, 445 p laakso, j. (1999). short-term effects of wood ants (formica aquilonia yarr.) on soil animal community structure. soil biology and biochemistry, 31: 337-343. doi: 10.1016/s00380717(98)00131-x laine, k. & niemelä, p. (1980). the influence of ants on the survival of mountain birches during an oporinia autumnata (lep., geometridae) outbreak. oecologia, 47: 39-42. mabelis, a. a. (1979). wood ant wars. the relationship between aggression and predation in the red wood ant (formica polyctena foerst.). netherlands journal of zoology, 29: 451-620. maňák, v., nordenhem, h., björklund, n., lenoir, l., & nordlander, g. (2013). ants protect conifer seedlings from feeding damage by the pine weevil hylobius abietis. agricultural and forest entomology, 15: 98-105. doi: 10.1111/ j.1461-9563.2012.00597.x nielsen, j.s., nielsen m.g., damgaard, c. & offenberg, j. (2018). experiences in transplanting wood ants into plantations for integrated pest management. sociobiology, 65: 403-414. doi: 10.13102/sociobiology.v65i3.2872 olofsson, e. (1992). predation by formica polyctena förster (hym., formicidae) on newly emerged larvae of neodiprion sertifer (geoffroy) (hym., diprionidae). journal of applied entomology, 114: 315-319. doi: 10.1111/j.1439-0418.1992. tb01132.x otto, d. (1967). die bedeutung der formica-völker für die dezimierung der wichtigsten schadinsekten–ein literaturbericht. waldhygiene, 7: 65-90. reznikova, z. & dorosheva, h. (2004): impacts of red wood ants formica polyctena on the spatial distribution and behavioural patterns of ground beetles (carabidae). pedobiologia, 48: 15-21. doi: 10.1016/j.pedobi.2003.06.002 adam véle, jovan dobrosavljević – wood ants do not affect ernestia rudis abundance4 rybnikova, i.a. & kuznetsov, a.v. (2015). complexes of formica s. str. nests in the darwin nature reserve and causes of their degradation. entmological review, 95: 947-952. doi: 10.1134/s0013873815080023 skinner, g.j. (1980). territory, trail structure and activity patterns in the woodant, formica rufa (hymenoptera: formicidae) in limestone woodland in northwest england. journal of animal ecology, 49: 381-394. sorvari, j. & hakkarainen, h. (2005). deforestation reduces nest mound size and decreases the production of sexual offspring in the wood ant formica aquilonia, annales. zoological fennici, 42: 259-267 sorvari, j. & hakkarainen, h. (2007). wood ants are wood ants: deforestation causes population declines in the polydomous wood ant formica aquilonia. ecological entomology, 32: 707-71. doi: 10.1111/j.1365-2311.2007.00921.x sudd, j.h. & lodhi, q.k. (1981). the distribution of foraging workers of the wood-ant formica lugubris zetterstedt (hym.: formicidae) and their effect on the numbers and diversity of other arthropoda. biological conservation, 20: 133-145. traniello, j.f.a. (1989). foraging strategies of ants. annual review of entomology, 34: 191-210. véle, a. & liška, j. (2019). sosnokaz borový na jihovýchodní moravě v roce 2019. lesnická práce, 9: 46-47. wellenstein, g. (1954): what can we expect from the red wood ant for the forest protection? beiträge zur entomologie, 4: 117-138. weseloh, r.m. (1994). forest ant (hymenoptera: formicidae) effect on gypsy moth (lepidoptera: lymantriidae) larval numbers in a mature forest. environmental entomology, 23: 870-877. doi: 10.1093/ee/23.4.870 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.714sociobiology 62(3): 347-350 (september, 2015) foraging behavior of leaf cutting ants: how do workers search for their food? introduction leaf cutting ants present a notable division of labor during foraging and cultivation of the symbiontic fungus, generating food resources for the colony (hölldobler & wilson, 1990). for this purpose, workers need to select and forage a large number of plant species (rockwood, 1976). foraging depends on the size, density of quality of the available food source (traniello, 1989). selection, which is an important foraging step, is related to the content of nutrients and secondary compounds of plant species and to the physical resistance to cutting (cherrett & seaforth, 1970; rockwood, 1975; waller, 1982; cherrett, 1972; nichols-orians & schultz, 1990; roces & hölldobler, 1994; tautz et al., 1995). however, until finding an adequate plant species, the scout worker needs to search for it in the environment where it is located. once the scout worker has found a food source, the loaded forager returns to the nest, communicating the location and quality of the discovery to the nest mates (therien, 1988; abstract foraging behavior of leaf cutting ants: how do workers search for their food? forager ants search for adequate food sources in nature and, after their discovery, they decide whether the source is suitable or not for the colony. however, we asked “how do workers seek out the substrate for cultivation of the symbiontic fungus on which they feed? to answer this question, we evaluated the distance traveled by individual workers in the search of food and the distance traveled to return to the nest, as well as the time and velocity necessary for these activities. the results showed that the distance traveled by the leaf cutting ant, atta sexdens rubropilosa (linneus, 1758), in the search of food was greater than the distance traveled to return with the substrate to the colony. on the other hand, the mean time and velocity were similar for food search and return to the colony. these results support the hypothesis of information transfer, according to which the worker needs to return to the nest at the beginning of foraging to transfer information to other workers and thus to establish the process of worker ant foraging. it can be concluded that workers travel large distances in a random manner until finding their substrate, but the return to the nest is efficient considering the shorter distance traveled. sociobiology an international journal on social insects rv travaglini, lc forti, rs camargo article history edited by evandro do nascimento silva, uefs, brazil received 24 november 2014 initial acceptance 19 february 2015 final acceptance 11 march 2015 keywords leaf cutting ants, foraging, atta sexdens rubropilosa. corresponding author roberto da silva camargo laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas universidade estadual paulista (unesp) caixa postal 237, 18603-970 botucatusp, brazil e-mail: camargobotucatu@yahoo.com.br roces, 1994, 2002; lopes et al., 2004). the recruited workers are conditioned to the odor of the food fragment carried by the scout worker and the learned odor is then used as a clue at the time of decision-making about which plant to collect, as demonstrated experimentally in acromyrmex lundi (roces, 1990, 1994; lopes et al., 2004, bollazzi & roces, 2011). obviously, the establishment of mass recruitment requires an increase in the deposition of pheromone along the trail, leading workers to participate in the demarcation and maintenance of chemical trails (jaffé & deneubourg, 1992). this recruitment is influenced directly by the scout worker, but the decision making process is also affected by interactions between workers (de biseau & pasteels, 2000; roces, 2002). furthermore, the previous experience of workers also influences the selection of plants during foraging (fowler, 1982; howard et al., 1996; camargo et al., 2003). since scout workers are responsible for seeking out the adequate plant and for recruiting nest mates, we asked “how do workers seek out the substrate for cultivation of the symbiontic fungus on which they feed?” to answer this research article ants universidade estadual paulista unesp, botucatu, sp, brazil rv travaglini, lc forti, rs camargo food location by leaf cutting ants348 question, we evaluated the distance traveled by individual workers in the search of food and the distance traveled to return to the nest, as well as the time and velocity necessary for these activities. material and methods three atta sexdens rubropilosa colonies, maintained in the laboratory of social insects, lisp, unesp, botucatu, were used. each colony consisted of an acrylic box (500 ml) containing the fungus garden connected to two containers that served as a feeder and waste deposit by the ants. the colonies were fed acalypha spp. and ligustrum spp. and were maintained at a temperature of 24 ± 2 °c and relative humidity of 80% under a 12-h light period. an apparatus resembling a table was constructed to permit the observation of the behavior of foragers. the table, which is illustrated in figure 1, had stainless steel legs and a glass base (95 x 210 cm) delimited by glass borders containing fluon and was elevated 1.5 m above the floor. the total time necessary for the foraging of leaves was greater in the first release and decreased during subsequent releases. the longest time observed was 4 h and 23 min and the shortest time was 19 min. the mean time necessary for scout workers to find the leaves in the center of the table was 441 ± 313.10 seconds in the presence of leaves and 469 ± 322.84 in their absence, with no significant difference between times (t = -0.4147; d.f. = 88; p = 0.6794). the mean velocity was 0.585 ± 0.35 m/min during food search and 0.606 ± 0.23 m/min during leaf transport. this difference was not statistically significant (t = 1.6697; d.f. = 88; p = 0.0989). discussion forager ants search for adequate food sources in nature and, after their discovery, decide whether the source is suitable or not for the colony. this acceptance depends on the physical and chemical characteristics of the plants (howard, 1987) and on the previous experience of the workers (saverschek et al., 2010). if the food source is adequate, the workers return to the nest (with or without load), depositing a chemical trail and interacting with nest mates along the trail and/or inside the nest (roces, 1990; roces, 1993; jaffé & howse, 1979). however, it remains unknown how workers search for food sources in space and, once discovered, how they return to the nest. the present results showed that workers travel a large distance until finding a food source, but the distance is shorter when returning to the nest. this behavior of the first foragers agrees with the observation of bollazzi and roces (2011) that this faster return to the nest is related to the communication of information about food to workers residing inside the nest, which actively stimulates foraging. this foraging characteristic is supported by the hypothesis of information transfer (núñez, 1982; roces & núñez, 1993; roces & bollazzi, 2009), an adaptive advantage of leaf cutting ants which permits to rapidly monopolize a newly discovered source at an early stage (roces & hölldobler, 1994). the foraging period is limited by environmental variables (lewis et al., 1974) and by neighborhood competition for the same food source (whitehouse & jaffé, 1996). interestingly, the time of travel for food search and return to the colony by scout workers was similar despite the difference in the distance traveled. this finding is due to the fact that, at the time of return, workers deposit venom gland secretions to indicate the rapid transport of these leaves to the nest, establishing chemical trails so that the other workers can find the food source (vilela & della lucia, 1987). the mean velocity of substrate search and leaf transport to the nest was similar (0.585 and 0.606 m/min, respectively). these values agree with those reported by schlindwein (1996) who found a maximum velocity of 1.57 m/min and a minimum velocity of 0.571 m/min. usually, 68.8% of workers travel at a velocity of 0.5 to 1.0 m/min (gomides, 1997). in a. cephalotes, the velocities are higher, 1 m/min for loaded individuals and 2 m/ fig 1. schematic drawing of the observation apparatus. the observer is positioned beneath the apparatus. the colony was connected to one end of the table and remained without food on the day prior to the experiment. the food source (leaves of the preferred plant species such as acalypha wilkesiana, gmelina arborea and ligustrum japonicum) was placed on the opposite end. after release of the ants, the pathway of the individual until finding the food source and their return to the nest were observed. fifteen releases were performed per colony, for a total of 45 releases. the trajectory of this scout worker was recorded on the lower surface of the glass with a marker pen and measured with an analog curvimeter, which provided measures of the distance traveled. the time of displacement was recorded with a chronometer to calculate the mean velocity. the following parameters were evaluated: total foraging trip duration of the individuals, total distance traveled, and mean velocity. the data were analyzed by the paired t-test (α=0.05) using the bioestat 5.0 program. results the mean distance traveled in the search of food was greater than the distance traveled during return with the food to the colony (4.3 ± 2.2 versus 1.3 ± 0.5 m), with the difference being statistically significant (t = 8.9491, d.f. = 88, p < 0.0001). sociobiology 62(3): 347-350 (september, 2015) 349 min for unloaded individuals (lewis et al., 1974). however, this velocity is directly affected by environmental factors, such as the distance between the nest and food source, foraging trail gradient, and roughness of the substrate through which the workers pass (ribeiro, 2013). in summary, the distance traveled during substrate search is greater than the distance traveled during return with or without load. however, we observed a similar time and mean velocity for food search and return to the nest in the leaf cutting ant, atta sexdens rubropilosa. these results corroborate the hypothesis of information transfer (núñez, 1982; roces & núñez, 1993; roces & bollazzi, 2009), according to which the worker needs to return to the nest at the beginning of foraging to transfer information to other workers (bollazzi & roces, 2011). it can be concluded that workers travel large distances in a random manner until finding their substrate, but the return to the nest is efficient considering the shorter distance traveled. references bollazzi, m. & roces, f. 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(1996) ant wars: combat strategies, territory and nest defence in the leaf-cutting ant atta laevigata. animal behaviour, 51: 1207-1217. 81 screening of multiple potential control genes for use in caste and body region comparisons using rt-qpcr in coptotermes formosanus by matthew r. tarver1, christopher p. mattison2, christopher b. florane1, doug j. hinchliffe3, dunhua zhang1 & alan r. lax1 abstract formosan subterranean termites, coptotermes formosanus, are a significant worldwide pest. molecular gene expression is an important tool for understanding the physiolog y of organisms. the recent advancement of molecular tools for coptotermes formosanus is leading to the advancement of the understanding of termite physiolog y. one of the first steps in analyzing gene expression is the normalization to constant reference genes. stable reference genes that have constant expression across multiple treatments are important for accurately comparing target genes' expression. the objective of this investigation was to analyze and validate a set of potential reference genes including 18s rrna; glyceraldehyde 3-phosphate dehydrogenase (gadphd); ribosomal protein l7 (rpl); β-actin (ba1); α-tubulin (atube); α-actin (aactin); and elongation factor (elong) as standards for analysis of transcriptional changes in the termite coptotermes formosanus, across two phenotypic castes, body regions, and colonies. we also compared the expression of hexamerin-1 and 2 using stable and unstable reference genes to demonstrate the importance of consistent control genes. our results demonstrate that 18s and rpl can serve as reliable expression standards when comparing these different castes and body regions, and we show that c. formosanus hex-1 and hex-2 have expression patterns similar to that previously described in r. flavipes. introduction formosan subterranean termites (coptotermes formosanus) are an extremely destructive urban pest and are expanding their range throughout united states department of agriculture, agricultural research service, southern regional research center, 1100 robert e. lee blvd., new orleans, la 70124 1formosan subterranean termite research unit 2food processing sensory quality research unit 3cotton chemistry and utilization research unit 82 sociobiolog y vol. 59, no. 1, 2012 the world. following introduction of the species into the united states, they have successfully spread through the southern gulf coast states (su & scheffrahn 2000, lax & osbrink 2003). research focused on the control of this species has advanced through the years but there is an increased need for species-specific control methods. the implementation of molecular biolog y resources has added to the knowledge of the basic biolog y of this serious pest. understanding the molecular processes underlying termite growth, differentiation, and response to stress will lead to the identification of novel target sites for termite-specific control. gene expression profiles will be beneficial in focusing the search for termite control targets. one established method for measuring gene expression is through reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr). the rt-qpcr technique is an extremely sensitive method for nucleic acid detection, genotyping, snp analysis and the quantification of specific messenger rna (mrna) levels. several factors, including quality of template rna, choice of enzyme and other reagents, selection of control(s) (or reference) genes, and method of analysis can have a significant effect on results. slight variations in the transcript abundances of chosen reference gene(s) thought to be temporally and/or spatially static can artificially amplify or lessen apparent changes in target transcript quantification. application of insufficient reference standards can cause inaccurate normalization of experimental data leading to increased error or incorrect conclusions. rather than using a single reference gene, the use of several internal reference standards can be applied to more accurately measure changes in gene expression (vandersompele et al., 2002). careful consideration must be taken in the choice of these standards for each system and experimental condition analyzed to ensure adequate normalization for accurate analyses of results. in previous studies characterizing gene expression in termites, several different control genes were utilized (scharf et al. 2003, 2005a, 2005b, zhou et al. 2006a, 2006b, 2006c, 2007a, 2007b, 2008, wheeler et al. 2010, korb et al. 2009, wiel et al. 2009, hojo et al. 2009). however, it is unclear from these studies which control genes provide reliable high-quality standards for gene expression studies in c. formosanus termites. these types of analyses would benefit from a well characterized and validated set of control genes that provide a stable standard for analysis across various experimental conditions. 83 tarver, m.r. et al. —screening of multiple control genes in c. formosanus the objective of this investigation was to analyze and validate a set of potential reference genes including 18s rrna; glyceraldehyde 3-phosphate dehydrogenase (gadphd); ribosomal protein l7 (rpl); β-actin (ba1); α-tubulin (atube); α-actin (aactin); and elongation factor (elong) as standards for analysis of transcriptional changes in the termite coptotermes formosanus, across two phenotypic castes, body regions, and colonies. in addition we incorporated one exogenous (alien) rna control into our study. a wide range of ct (threshold cycle) variation was observed among this set of candidate control genes, and to test their utility we applied these control genes as standards for the analysis of two target genes hexamerin 1(hex-1) and hexamerin 2 (hex-2). when we applied the most stable set of reference genes, we found that hex-1 and hex-2 expression in c. formosanus was similar to reticulitermes flavipes, with an overall up-regulation in workers and increased expression in the carcass. predictably, target gene comparison between variable and non-variable control genes resulted in statistically different results, underscoring the idea that careful selection of control genes is essential for accurate gene expression analysis. materials and methods termites formosan subterranean termites were collected from cardboard rolls placed in bucket traps at various locations in new orleans city park (new orleans, la.). the termites were removed from the collection material, separated, and placed into plastic containers (17.1 x 12.2 x 6.0cm pioneer plastics) containing slats of moist spruce (picea sp.) wood. the containers were then placed into an incubator maintained at 25°c, 70 % relative humidity and constant darkness. individuals collected from two independent colonies were used in the experiments. 16s mitochondrial rdna sequencing was used to verify their identities as coptotermes formosanus (szalanski et al. 2003). dissection a total of four sets of 25 workers and soldiers from a colony designated 1732 were dissected to obtain the tissue from each individual’s head, gut and carcass for extraction of rna. termites were dissected using an olympus szx-12 dissecting microscope (olympus america inc., center valley, 84 sociobiolog y vol. 59, no. 1, 2012 pa). each individual was decapitated and the entire gut removed with fine forceps by gently squeezing the hind end and pulling until the entire gut was withdrawn from the carcass. the gut was placed into a 1.5 ml eppendorf tube containing 100ul phosphate buffered saline (pbs). the remaining head and carcass were placed into separate 1.5 ml eppendorf tubes. this procedure was repeated 25 times to provide isolated head, gut, and carcass samples for each individual termite in separate tubes. the tubes were then placed into a -80°c freezer prior to extraction of rna. this process was repeated to obtain 4 biological replicates of 25 heads, guts and carcasses from worker termites. the same procedures were followed to obtain 4 biological replicates of heads, guts, and carcasses from soldier termites as well. this was then repeated to obtain 4 biological replicates of 25 from workers and soldiers from a second colony designated 1314. extraction of rna total rna was isolated from each set of the 25 worker or soldier frozen body regions using the sv total rna isolation kit (promega corp., madison, wi) according to manufacturer’s instructions. rna was similarly extracted from the extirpated guts using the same kit. the extracted rna was quantitated using a nanodrop instrument (nanodrop technologies inc., wilmington, de). design of primers gene targets and primers were chosen based upon homolog y to past termite control genes and control genes from other species (hojo et al. 2009, tarver et al. 2010. zhou et al. 2006a,b,c; 2007a,b). specific gene sequences were identified from an in-house est library (d.z. usda-ars-srrc, unpublished results). eight control and two target genes were chosen. primers were designed using beacon designer 7.61 (premier biosoft, palo alto, ca) for sybr qrt-pcr. hexamerin-1 and -2 were chosen as test target genes based on previous research (zhou et al. 2006a, 2007b). all primer sequences are listed in table 1. primers were purchased from integrated dna technologies (idt www.idtdna.com). reverse transcription quantitative real-time pcr reverse transcription reactions for cdna synthesis were performed using a quantitect reverse transcription kit (qiagen inc., valencia, ca) and the 85 tarver, m.r. et al. —screening of multiple control genes in c. formosanus alien reference rna qrt-pcr detection kit (agilent technologies inc., santa clara, ca). a 1 μl aliquot of the alien rna transcript (3x1010 copies / μl) was diluted with 59 μl of rnase free water to a final concentration of 5x108 copies / μl. from the initial 1:60 dilution, four 10-fold serial dilutions were made to achieve a final concentration of 5x104 copies / μl. for the initial reaction, 2 μl of 7x gdna wipeout buffer (qiagen inc.) and a 2 μl aliquot of the 5x104 concentration alien rna template were added. the amount of template rna used from each sample was 130 ng and 240 ng for colonies 1732 and 1314 respectively. additional rnase free water was then added as needed until the final volume of each reaction was 14 μl. samples were then incubated at 42ºc for 2 minutes and immediately placed on ice. to each reaction, 1 μl of quantiscript reverse transcriptase, 4 μl of 5x quantiscript reverse transcriptase buffer and 1 μl reverse transcriptase primer mix was added to bring the total volume of each sample to 20 μl. samples were then incubated at 42ºc for 15 minutes followed by a 3 minute incubation at 95ºc to inactivate the quantiscript reverse transcriptase. all above reagents were from quiagen (valencia, ca) the qpcr reactions were performed in a bio-rad cfx96 real time pcr detection system (bio-rad laboratories, hercules, ca) using the fast protocol table 1. primer pair sequences designed for rt-qpcr. rt-qpcr primer pairs ( 5’ 3’ ) blastx sequence description forward reverse ribosomal protein l7 (rpl) attgacaacagagttcgtaggc cgttatgcaccagtaccttcc 18s ribosomal rrna (18s) cgagattgagcaataacaggtc acgtaatcaacgcgagcttatg β-actin (ba1) ctatgtcgccctggactt cgttgccaatggtgatga glyceraldehyde 3-phosphate dehydrogenase (gadphd) ggtagttaatggtcacaagatt gcagaagccttatcaatgg α-tubulin (atube) cttaccacgagcagttgagt cgaggatcacacttcaccat α-actin (aactin) tcttggatgattacgagtgtga tctcaatcctctcagcaattct elongation factor (elong) cttggatggtggaaaggagtaact atcttggattggaaggcgaagt hexamerin 1 (hex-1) gaatctgctgctacccaagg aatcccagtggcttgtcatc hexamerin 2 (hex-2) actctgctccgaaaggtgaa tcgaaatcatgttcgctctg 86 sociobiolog y vol. 59, no. 1, 2012 with itaq™ fast sybr® green supermix (bio-rad laboratories). all primer sets were first run at a temperature gradient (53-63°c) to obtain the optimal annealing temperature. optimal annealing temperature was determined to be 60°c. all samples (worker head, gut, carcass; soldier head, gut, carcass; colony 1 and colony 2) had four biological replicates per treatment (n=48). three technical replicates were run for each biological sample. a dissociation curve was generated and used to validate that a single amplicon was present for each rt-qpcr reaction. data analysis averaged biological c t values of the reference genes were analyzed using bestkeeper software (pfaffl et al., 2004) to obtain a ranking for control gene stability. two reference genes (18s, rpl) and the alien exogenous control gene were used to calculate the relative expression of the two target genes hex-1 and hex-2 according to the abi guide to performing relative quantitation of gene expression using real-time quantitative pcr (applied biosystems, foster city, ca) with the following modification: the geometric mean of the reference genes and alien exogenous control gene was used to calculate the ∆c t values for the individual target genes (vandersompele et al., 2002). the ∆c t values were normalized to the treatment that had the lowest overall expression to calculate the δδc t . the values were then graphed using the sigmaplot 11.0 software (systat software, inc., san jose, ca). statistical differences were calculated by analysis of variance (anova) and student’s t-test using jmp® 8.0 (sas institute inc., cary, nc, usa). statistical differences were not calculated using the δδc t values because normalization using the δδc t leads to incorporation of values from one of the treatments and violates the anova rules. to validate our findings and establish a set of stable control genes, the target gene hex-1 was analyzed using the two most consistent (18s and rpl) reference genes, and the least consistent (atube) reference gene independently for normalization. results / discussion the rt-qpcr technique provides a precise method for characterizing changes in gene transcription. inclusion of an appropriate normalization strateg y is essential to the integrity of the data and its analysis. to establish a set of reliable control genes for coptotermes formosanus we analyzed the expression pattern of several potential standards. 87 tarver, m.r. et al. —screening of multiple control genes in c. formosanus gene stability and ranking we first compared the ct values for each gene across all treatments to visualize the expression variability (fig. 1). data in fig. 1 indicated that the 18s and alien controls provided the most consistent c t values. both the rpl and gadph c t values varied slightly more, while the β-actin, α-actin, α-tubulin, elong, hexamerin-1, and hexamerin-2 c t values varied widely from the various body parts. c t values were also entered into the bestkeeper analysis program and the results summarized in table 2. this analysis provided a ranking of the expression of control genes based upon their standard deviation. the ranking based upon cv value was very similar with the alien and 18s genes providing the most stable reference standard. the α-actin and elong were not included in the bestkeeper analysis because of the increased number of reactions for which no product was detected (α-actin 26/48 rx, elong 41/48 rx ). undetectable reactions resulted in a lack of measurable product, which caused excessive variation in c t value. however, none of the samples lacked rna as evidenced by the expression levels of the other genes, indicating that transcript abundances of α-actin and elong were below the limit of detection in these samples. the transcript abundances of valid reference genes should be present at sufficient levels to reliably generate detectable amplification products, and so α-actin and elong may be invalid for use as reference genes due to their low expression levels. target gene analysis (single versus multiple gene normalization) to validate a set of reference genes that can provide accurate normalization, we chose to normalize hex-1 and hex-2 expression profiles using 18s and table 2. rt-qpcr c t values obtained from bestkeeper software rpl 18s ba1 gadpdh atube alien n 48 48 48 48 48 48 geo mean [c t ] 23.4 13.69 21.75 23.27 28.04 27.97 am mean [c t ] 23.47 13.73 21.88 23.34 28.72 27.99 min [c t ] 21.3 11.17 17.93 20.77 19.27 26.45 max [c t ] 29.07 17.33 27.44 28.45 37.56 32.68 std dev [± c t ] 1.45 0.79 1.85 1.38 5.21 1 cv [% c t ] 6.19 5.77 8.47 5.9 18.13 3.59 88 sociobiolog y vol. 59, no. 1, 2012 fig. 1. control and target gene ct values across all treatments. 89 tarver, m.r. et al. —screening of multiple control genes in c. formosanus rpl, two internal control genes, and one exogenous control gene “alien”. the results with this set of three reference standards indicate that both hex-1 and hex-2 were more highly expressed in workers compared to soldiers (fig. 2 a,b). hex-1 and 2 were also more expressed in the worker and soldier carcass compared to the head and gut. these findings are similar to results obtained with r. flavipes (zhou et al. 2006c) that demonstrated up-regulation of hex-1 and -2 in the termite fat body. our results clearly demonstrate an upregulation in the carcass which contained the fat body, therefore our results are consistent with the findings of zhou et al. 2006c. to demonstrate the potential pitfalls associated with application of a single non-validated reference gene for rt-qpcr normalization, we varied the application of our set of reference genes in the normalization of one target gene, hex-1. the expression levels of hex-1 across soldier body regions were compared using individual control genes (18s, rpl, alien, and αtube). our findings indicated a similar expression pattern between two consistent control genes (18s, rpl), and demonstrated a higher hex-1 expression in the carcass compared to the head and gut (fig. 3a and b). even between these two “stable” control genes there is a significant difference in hex-1 expression in the gut. we find that with the application of 18s alone as a reference, hex-1 expression in the gut is relatively higher compared to normalization with rpl alone (fig. 3a and b). analysis of hex-1 expression using the most variable control gene (atube) significantly alters the results. applying atube as a normalization control, hex-1 expression is lowest in the gut compared to the head and carcass (fig. 3c). these results are dramatically different from the results using the two previous normalizers (18s, rpl) and clearly demonstrate that using only atube as a control gene standard could result in misleading data analysis. this examination is consistent with similar types of analyses of multiple reference genes from other model systems and clearly supports the use of multiple standards (vandesomepele et al. 2002). when performing gene expression analysis it is important for researchers to characterize and validate a set of control genes specific for the type of study being performed. relatively few studies have been conducted on termite gene expression using rt-qpcr. the majority of the past experiments have used only one gene as a reference, and in most cases this was the β-actin gene (scharf et al. 90 sociobiolog y vol. 59, no. 1, 2012 fig. 2. hexamerin 1 (a) and hexamerin 2 (b) relative expression in worker and soldier termites in different body locations. 18s, rpl and alien were used as control genes with the “soldier head” being the control treatment. hex-2 relative expression in worker carcass (b) was too high to fit on to the graph (5663.5 ± 981 se). different letters represent significant differences between treatments (p<0.05). 91 tarver, m.r. et al. —screening of multiple control genes in c. formosanus fig. 3. relative hexamerin 1 expression in soldier termites after application of different reference standards (a) 18s, (b) rpl, and (c) atube. soldier head was used as the normalizing treatment. different letters represent significant differences (p<0.05). 92 sociobiolog y vol. 59, no. 1, 2012 2003, 2005a, 2005b, wheeler et al. 2010). zhou et al. (2006b) used only nadh-dh to compare different regions of the termite gut, while korb et al. (2009) used only 18s to compare gene expression in workers with different social situations. weil et al. (2009) also used only 18s and to compare gene expression in cryptotermes cynocephalus and cryptotermes scundus female neotenics. a number of researchers utilized the bestkeeper software to compare three control genes. zhou et al. (2006a; 2006b) used the bestkeeper software to compare β-actin, hsp-70 and nadh-dh and determined that β-actin was the most stable when measuring hexamerin expression levels in worker termites. in another set of experiments zhou et al. (2006b) used the bestkeeper software to compare β-actin, hsp-70 and nadh-dh and determined that nadh-dh was the most stable when comparing cytochrome p450 expression levels in termite workers and hexamerin body location. weil et al. (2007) used the bestkeeper software to compare 18s, β-actin, and hexamerin and determined that, similar to our findings, 18s was the most stable when comparing castes in c. secundus. hojo et al. (2009) also used the bestkeeper software and compared 18s, β-actin, eelong, and nadh-dh when measuring gene expression to compare soldier and minor workers in a higher termite. this study demonstrated that β-actin was the most stable and compared the stability of each potential control gene with each other. zhou et al. (2008) again used the bestkeeper software to compare β-actin, hsp-70 and nadh-dh, but this time normalized to all three control genes when documenting rnai expression knockdown of hexamerin expression. to date, the most extensive control gene identification experiment was performed in a study by tarver et al. (2010) that analyzed 49 different genes across multiple treatments, days and colonies in r flavipes. this study compared the standard deviations of the c t values across all genes and treatments and selected three genes with low variation to use as control genes (stero-1, lim and mev-1). taken together, these different rt-qpcr gene expression studies indicate a need for control genes capable of performing as reference standards independent of termite colony, body-region, and experimental condition. our results suggest that 18s and rpl may serve as stable control genes when comparing the various samples that we investigated (caste, body location, 93 tarver, m.r. et al. —screening of multiple control genes in c. formosanus and colony) in our termite species, c. formosanus. testing other treatments or species of termites may require a different set of control genes and a suite of control genes needs to be evaluated for variability. predefined suites of control genes that can be applied across multiple species, time points, and treatments are highly desirable and would provide ease of use if it is possible to define them. finally, the use of the alien reference rna (stratagene) has a number of advantages. by “spiking” in an exogenous “alien rna” before cdna synthesis and then measuring alien gene expression, a valid control is created to compensate for systematic error associated with pipetting and multiple reactions. increased use of the alien reference rna in future rt-qpcr studies will assist in the reduction of erroneous observations. care must be taken not to rely solely on the alien reference rna, but the addition of this reference with appropriate internal control genes should provide an additional reliable standard to more accurately quantify gene expression. conclusion the rt-qpcr technique provides a precise method for characterizing gene transcription changes. inclusion of an appropriate normalization strateg y is essential to the accuracy of the data and its analysis. while it is labor and reagent intensive, the use of several well characterized reference genes ensures that small variations in target genes are more likely to be detected and this translates into more accurate results. here we have characterized six potential control genes and applied the most stable (18s, rpl, alien) to measure target gene expression across body region (head, gut, and carcass) and between castes (worker and soldier). our results demonstrate that 18s and rpl can serve as reliable expression standards when comparing these different castes and body regions, and we show that c. formosanus hex-1 and hex-2 have expression patterns similar to that previously described in r.. flavipes. further use of these reference genes when comparing expression levels among various potential termite control treatments will allow more accurate assessment of effects on target gene expression. acknowledgments we would like to thank drs. maureen wright and michael santiago cintrón for manuscript review and helpful comments. 94 sociobiolog y vol. 59, no. 1, 2012 references hojo, m., k. toga, i. itai, & k. maekawa 2009. reference genes for real-time quantitative reverse transcriptase-pcr in the higher termite nasutitermes takasagoensis (isoptera: termitidae) comparing soldiers with minor workers. sociobiolog y 54: 509-520. korb, j., t. weil, k. hoffmann, k.r. foster & m. rehli 2009. a gene necessary for reproductive suppression in termites. science 324: 758. pfaffl, m.w., a. tichopád, c. prgomet & t.p. neuvians 2004. determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper – excel-based tool using pair-wise correlations. biotechnolog y letters 26:509-515. lax, a.r., & w. la. osbrink 2003. united states department of agriculture – agriculture research service research on targeted management of the formosan subterranean termite coptotermes formosanus shiraki (isoptera: rhinotermitidae). pest management science 59:788-800. scharf, m.e, d. wu-scharf, b.r. pittendrigh & g.w. bennett 2003. casteand development associated gene expression in a lower termite. genome biolog y 4, r62. scharf, m.e., c.r. ratliff d. wu-scharf, x. zhou & b.r. pittendrigh 2005a. effects of juvenile hormone iii on reticulitermes flavipes: changes in hemolymph protein composition and gene expression. insect biochemistry and molecular biolog y 35: 207-215. scharf, m.e., wu-scharf d., pittendrigh b.r. & g.w. bennett 2005b. gene expression profiles among immature and adult reproductive castes of the termite reticulitermes flavipes. insect molecular biolog y 14: 31-44. su, n., & r.h. scheffrahn 2000. termites as pests of buildings. termites: evolution, sociality, symbiosis, ecolog y (eds. t. abe, d.e. bigness & m. higashi), pp.437-454. dordrecht, kluwer acedemic publishers. szalanski, a.l., austin j.w. & c.b. owens 2003. identification of reticulitermes spp. (isoptera: reticulitermatidae) from south central united states by pcr-rflp. journal of economic entomolog y 96: 1514-1519. tarver, m.r., zhou x., & m.e. scharf 2010. socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite reticulitermes flavipes. bmc molecular biolog y 11: 28. vandesomepele, j., depreter k., pattyn f., poppe b., van roy n., depaepe a., & f. speleman 2002. accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes. genome biolog y 3: 34. weil, t., korb j. & m. rehli 2009. comparison of queen-specific gene expression in related lower termite species. molecular biolog y and evolution 26: 1841-1850. weil, t., rehli m. & j. korb 2007. molecular basis for the reproductive division of labour in a lower termite. bmc genomics 8: 198.1-198.9. wheeler, m.m., tarver m.r., coy m.r. & m.e. scharf 2010. characterization of four esterase genes and esterase activity from the gut of the termite reticulitermes flavipes. archives of insect biochemistry and physiolog y 73: 30-48. 95 tarver, m.r. et al. —screening of multiple control genes in c. formosanus zhou, x., oi f.m. & m.e. scharf 2006a. social exploitation of hexamerin: rnai reveals a major caste-regulatory factor in termites. proceedings of the national academy of sciences 103: 4499-4504. zhou, x., song c., grzymala t.l., oi f.m. & m.e. scharf 2006b. juvenile hormone and colony conditions differentially influence cytochrome p450 gene expression in the termite reticulitermes flavipes. insect molecular biolog y 15: 749-761. zhou, x., tarver m.r., bennett g.w., oi, f.m. & m.e. scharf 2006c. two hexamerin genes from the termite reticulitermes flavipes: sequence, expression, and proposed functions in caste regulation. gene 376: 47-58. zhou, x., smith, j.a., koehler, p.g., oi, f.m., bennett, g.w. & m.e. scharf 2007a. correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite r. flavipes. gene 395: 29-39. zhou, x, tarver m.r. & m.e. scharf 2007b. hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect. development 134: 601-610. zhou, x, wheeler m.m., oi f.m. & m.e. scharf 2008. rna interference in the termite reticulitermes flavipes through ingestion of double-stranded rna. insect biochemistry and molecular biolog y 38: 805-815. doi: 10.13102/sociobiology.v67i1.4598sociobiology 67(1): 106-111 (march, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction vespidae comprise species that are remarkably abundant, widely distributed (carpenter & marques, 2001), and actively participate in local food webs (santos et al., 2007). the most common subfamilies in brazil are polistinae, eumeninae and masarinae (carpenter & marques, 2001; pickett & carpenter, 2010). polistinae comprise the most diverse group, with more than 950 species described worldwide (pickett & carpenter, 2010), of which about 300 species occur in brazil (carpenter & marques, 2001; ctfb, 2017). the polistinae social wasps are important components of neotropical ecosystems due to their ubiquity and diversity, as well as their complex interactions with other organisms (silveira, 2002; somavilla et al., 2016). the growing interest in studies on the diversity of social wasps is due to the recognized ecological importance, mainly abstract this work aims to recognize and record the occurrence of species of social wasps (polistinae) in organic tobacco crops, as well as to point out possible species that may be used in future biological control programs. the research was conducted in virginia-type tobacco farming with organic management in two regions in south of brazil. the collections were carried out with malaise trap, during the harvests 2008/2009, 2009/2010 and 2010/2011. a total of 2.738 individual of wasps, from which 23 species distributed in six genera was collected. polistes was the most diverse genus (eight species), followed by polybia (7), mischocyttarus (4), agelaia (2) and bachygastra and protonectarina with one species. polybia scutellaris was the most abundant species, being considered eudominant. agelaia multipicta and polybia sericea with are also among the most abundant. this information is relevant for the insertion of social wasps with potential agents in the biological control of tobacco pests. sociobiology an international journal on social insects k schoeninger1, a somavilla2, a kohler1 article history edited by gilberto m. m. santos, uefs, brazil received 10 july 2019 initial acceptance 12 december 2019 final acceptance 23 january 2020 publication date 18 april 2020 keywords agroecosystem, diversity, natural enemies, polistinae, taxonomy. corresponding author karine schoeninger laboratório de entomologia departamento de biologia e farmácia universidade de santa cruz do sul – unisc santa cruz do sul, rio grande do sul, brasil. e-mail: karine.schoeninger@gmail.com as predators, in agricultural system environments (carvalho & souza, 2002; prezoto et al., 2008; prezoto et al., 2016). predatory wasps play a key role in pest control, preferring to prey on caterpillar (lepidoptera), which are economically important, such as spodoptera frugiperda (smith, 1797), alabama argillacea (hübner, 1823), anticarsia gemmatalis hübner , 1818 and heliothis virescens (fabricius, 1777), and others, which constitute the most common pests on small farms in tropical environments (picanço et al., 2012; prezoto et al., 2019; lourido et al., 2019). according to prezoto et al. (2008), social wasps capture adult and/or immature insects to feed their offspring. recently, lourido et al. (2019) reported seven species of social wasps as predators of larvae of diaphania hyalinata (linnaeus, 1767) (lepidoptera), considered cucumber pest in amazonas. according jacques et al. (2015) the presence of wasps of the genus polistes in different cultures is associated with reduced damage caused 1 laboratório de entomologia, departamento de biologia e farmácia, universidade de santa cruz do sul (unisc), santa cruz do sul, rio grande do sul, brazil 2 coordenação de biodiversidade (cobio), instituto nacional de pesquisas da amazônia (inpa), manaus, amazonas, brazil research article wasps occurrence of social wasps (hymenoptera: vespidae) in farming of organic tobacco (nicotiana tabacum l.) in south of brazil sociobiology 67(1): 106-111 (march, 2020) 107 by pests on cotton (kirkton, 1970), tobacco (lawson et al., 1961), cabbage (gould & jeanne, 1984), coffee (gravena, 1983) and corn (prezoto & machado, 2009), showing the importance of this genus for studies on the biological control of pests. thus, in addition to contributing to the regulation of insect pest populations, the presence of social wasps may also contribute to the reduction of insecticide use, with natural biological control being a significant contribution to this process (gallo et al., 2002). therefore, the knowledge of the richness and biology of social wasps present in crop systems can contribute to future studies aiming the use of these as a tool in the biological control in agroecosystems. tobacco (nicotiana tabacum l.) is a plant belonging to the family solanaceae, native to tropical and subtropical americas, with medium height of 90 to 150 cm, according to their variety (kuppert & thomas, 2010). it is a culture of recognized importance for brazil, especially for rio grande do sul, where it is widely cultivated, with all its leaves being used in the commercial production of cigarettes and cigars (kuepper & thomas, 2010). despite the great economic importance and capacity to generate jobs in various sectors, the issue of environmental sustainability should be the focus of attention. many problems are caused by the large area devoted to growing tobacco, as well as the great demand on use of synthetic products for their maintenance, causing environmental impacts and damages the health of farmers and the population in general (specht, 2006). considering this, more substantial researches, as well as effective actions that are able to reduce the environmental impacts of tobacco crop without a production affect, assume great importance and urgency. in order to obtain information on biodiversity that subsidizes ipm, research entomofauna surveys is of fundamental importance it allows the recognition of pest insects and their natural enemies. aiming at the current needs of cropping systems to the detriment of conservation and maintenance of the environment, this work aims to recognize and record the occurrence of species of social wasps (polistinae) in organic tobacco crops, as well as to point out possible species that may be used in future biological control programs. material and methods the research was conducted in virginia-type tobacco farming with organic management in two municipalities (santa cruz do sul and passa sete) located in south of brazil (fig 1). organic tobacco farming in santa cruz do sul is located at adet (worldwide center for agronomic development, extension and traning), a property of japan tobacco international (jti), and in passa sete, on a private property with the same conditions. the adopted management is annually inspected and certified by cee 2092/91 (european union) and usa nop – 7 cfr part 205 (usa national organic program). santa cruz do sul (29˚43’59”s, 52˚24’52”w) is a city that covers an area of 733.5 km2, located in the centro oriental riograndense mesoregion, as well as the geomorphological complex of periferic sul-riograndense depression, in a low altitudes belt, with no more than 150 m (leifheit, 1978). according to köppen’s classification, climate is subtropical humid. the medium temperature is 20.1°c, with the high of 26.4°c and minimum of 13.8°c. about the annual precipitations, occur in a period of 91-153 days, with variation between 858,8 and 2.325,4 mm (hoppe, 2005; diedrich et al., 2007; ruoso, 2007). the municipality of passa sete (29°27’12” s, 52°57’41” w), covers a total area of 303.58 km2, with an average elevation of 590 m. according to köppen’s classification, climate is subtropical humid. the medium temperature is 17.4°c, with the high of 28.0°c and minimum of 8.4°c. about the annual precipitations, the medium temperature is 1.565 mm (prefeitura municipal de passa sete, 2019). tobacco farming in santa cruz do sul has an area of 160 x 85 m, while the crop located in passa sete corresponds to an area of 60 x 30 m. in each crop, three malaise traps (townes, 1972) were arranged, distant from each other by approximately 30 m. the collections were carried out weekly during 2008/2009, 2009/2010 and 2010/2011 harvest in santa cruz do sul and only in the 2009/2010 harvest in passa sete. it should be noted that each harvest corresponds to the period from november to march in each year, and that only the 2009/2010 harvest in both locations was evaluated in this paper. the identification and quantification of the collected specimens of social wasps were realized at laboratory of entomology of university de santa cruz do sul, with stereoscope microscope and based on the taxonomic identification keys proposed by richards (1978) and carpenter and marques (2001), as well as by comparison with previously identified species from santa cruz do sul collection (cesc). the vouchers were deposited in the cesc’ collection. the relative frequency and dominance calculations were based on bodenheimer (1955). the dominance index expresses the relationship between the number of individuals of a given species and the total number of individuals of all species found. the classification adopted was eudominant (>10%), dominant (5-10%), subdominant (2-5%), recessive (1-2%) and rare (<1%). results throughout tobacco harvests, were collected 2.738 individual of wasps, from which 23 species distributed in six genera, 47% of occurred in rio grande do sul state (total of 49 species recorded until now for the state) by somavilla et al. (2010). polistes was the most diverse genera with eight species, followed by polybia (7), mischocyttarus (4), agelaia (2) and bachygastra and protonectarina with one species (table 1). k schoeninger, a somavilla, a kohler – social wasps in farming of organic tobacco108 fig 1. location of study areas at national, state and municipal levels. left images of tobacco crops under organic management. source: modified from laboratory of geoprocessing of the university of santa cruz do sul. polybia scutellaris was the most abundant species with 54% of the wasps collected and a relative frequency of 54,71%, being considered eudominant. agelaia multipicta with 281 individuals, polybia sericea with 225 individuals, polybia ignobilis (180), agelaia vicina (167), and brachygastra lecheguana (136) are also among the most abundant. polistes, the most specious genera among the social wasps, the present study a low abundance was verified, only 105 specimens were collected. discussion wasp surveys are lacking in agricultural ecosystems in brazil, making this a pioneer study for the tabacco crop. the polistinae richness and composition obtained in this study were similar than previous studies conducted in different crops. for example, in an area of silvipastoral culture of embrapa dairy cattle research center in minas gerais, a total of 205 social wasps specimens, distributed in 13 morphospecies sociobiology 67(1): 106-111 (march, 2020) 109 and four genera were captured (auad et al., 2010) and in eucalyptus plantation in minas gerais, a total of 1613 social wasp specimens, distributed in 12 species (de souza et al., 2011). in forest fragments with different surrounding matrices of sugarcane and citrus crops in são paulo, a total of 1460 social wasp specimens, distributed in 29 morphospecies and 10 genera were captured (tanaka junior & noll, 2011). most recent, somavilla et al. (2016), collected 977 specimens of social wasps, in 52 species in organic and conventional guarana cultivation in the north of manaus, amazonas. the superiority of both richness and abundance of social wasps in tobacco crops may be related to the period of cultivation and flowering, coinciding with the months of collection. wasps exhibit opportunistic behavior and are attracted to environments that provide greater amounts of resources such as water and food, which makes them explore cultivated environments, less complex in phytophysiognomy (santos et al., 2009; de souza et al., 2012; brugger et al., 2011). although polistes is one of the most diverse genera among the social wasps, in the present study a low abundance was verified, only 105 specimens. a similar result was observed in mischocyttarus, which, although having four species, had only 68 individuals collected. among the genera found nests, polistes and mischocyttarus nests by independent foundation represents only 6,3% of all wasps, with the rest of the genera having swarming behavior (epiponini genera), represented by 93.7% of the specimens collected. greater occurrences of swarming individuals were observed in studies by silveira (2002), souza et al. (2014), somavilla et al. (2016). this can be explained by the benefits resulting from this behavior in the process of founding colonies. the social wasp polybia scutellaris is very abundant species in rio grande do sul state (somavilla et al., 2010). furthermore, this species found colonies in open areas and close an urban environment, such as the edge of tobacco crop. this result may be related to the fact that these species construct larger nests, constituting colonies populous, what makes them frequent in the environments where the nests are located (richards, 1978; zucchi et al., 1995; hunt et al., 2001). the high abundance of polybia and agelaia registered in the current and other studies indicates that species of these genera find it easy to colonize several different types of microhabitats due to their protected nests, method of foundation and great number of individuals, which gives table 1. species of social wasps recorded for the farming of organic tobacco (nicotiana tabacum l.) at two different municipalities in rio grande do sul state. fr = relative frequency; dom = dominance, classification: eudominant (▲), dominant (►), subdominant (■), recessive (◊), rare (○). species adet santa cruz/rs passa sete/rs total fr (%) dom agelaia multipicta haliday, 1836 x x 281 10,26 ▲ agelaia vicina de saussure, 1854 x x 167 6,09 ► brachygastra lecheguana (latreille, 1824) x x 136 4,96 ■ mischocyttarus cassununga (von ihering, 1903) x 15 0,54 ○ mischocyttarus drewseni (de saussure, 1853) x x 46 1,68 ◊ mischocyttarus riograndensis richards, 1978 x 2 0,07 ○ mischocyttarus rotundicollis (cameron, 1912) x x 5 0,18 ○ polistes actaeon (haliday, 1836) x 1 0,03 ○ polistes billardieri fabricius, 1804 x 5 0,18 ○ polistes brevifissus richards, 1978 x 2 0,07 ○ polistes cavapytiformis richards, 1978 x x 53 1,93 ◊ polistes cavapyta de saussure, 1853 x 4 0,14 ○ polistes cinerascens de saussure, 1854 x 3 0,11 ○ polistes simillimus zikán, 1951 x 5 0,18 ○ polistes versicolor (olivier, 1791) x x 32 1,16 ◊ polybia fastidiosuscula de saussure, 1854 x x 34 1,24 ◊ polybia ignobilis (haliday, 1836) x x 180 6,57 ► polybia minarum ducke, 1906 x x 7 0,25 ○ polybia paulista h. von ihering, 1896 x 9 0,32 ○ polybia platycephala (richards,1978) x x 12 0,43 ○ polybia scutellaris (white, 1841) x x 1.498 54,71 ▲ polybia sericea (olivier,1791) x x 225 8,21 ► protonectarina sylveirae (de saussure, 1854) x x 16 0,58 ○ 2738 k schoeninger, a somavilla, a kohler – social wasps in farming of organic tobacco110 their colonies greater chances of success (hermes & köhler, 2004). in this study, their abundance was higher in the interior of the tobacco crop, showing a great capacity for dispersion, unlike the results of others studies where usually the uniform environments and interior of the crops lower their abundance, and suggests that these species encounter barriers to use resources outside the better-conserved environments (klein et al., 2015). interestingly, the six most abundant species of this study, namely agelaia multipicta, a. vicina, brachygastra lecheguana, polybia ignobilis, p. scutellaris, and p. sericea, are species with wide distribution in rio grande do sul, being registered in several localities in the state (somavilla et al., 2011), in this way, the great abundance in this study, together with the wide distribution and habit of predation, can be considered species with potential in the biological control of tobacco, especially caterpillars of lepidoptera, the most important pests in the culture. another important factor is that the presence of these social wasps may be contributing to the decrease of tobacco pests in the field, although this has not been evaluated, since the social wasps also have the habit of nesting in one place (probably in the adjacent vegetation) and feeding themselves in another place, in this case within the crop, and this has been proven with the large number of individuals and species at this location (silva-pereira & santos, 2006). thus, it is more advantageous for these wasps to nest near these sites, resulting in smaller non-foraging energy costs. this information is relevant since identifying and studying these predatory insects, especially in agricultural environments, are the first steps to identifying the best species to use in biological pest control. in this way, such information can support control strategies for tobacco pests, associated with integrated pest management. finally, we suggest some wasps species (like agelaia, polybia and polistes) should be considered for a potential use in a biological control program of tobacco pests and other agricultural pests in the rio grande do sul region, due to its high index of captured in malaise trap and intense foraging activity. references auad, a.m., carvalho, c.a., clemente, m.a. & prezoto, f. 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(1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). entomological society, 103: 129-137. 165 biodiversity management under limiting conditions: estimating effective population size using the molecular mark and recapture (mmr) method by kaori murase1 * & masaharu fukita1 abstract although many people have been paying attention to the decrease of biodiversity on earth in recent years, many local people, even staff of national parks, live under limiting conditions (such as a shortage of funds, specialists, literature, equipment for experiments and so on). to conserve biodiversity, it is important to be clear about which species decrease or increase. to find such information, it is quite important to know the dynamics of effective population size for each species. although a large number of papers have been written about how to improve the precision of the estimated effective population size, little has been studied on how to estimate the dynamics of the effective population sizes for many species together under limiting situations, very similar to the management methods of national parks in countries which have biological hot spots. in this paper, we are not concerned with the improvement of the precision of the estimates. we do, however, propose a simple method for the estimation of the effective population size. we named it the “mmr method.” it is not difficult to understand and is easily applied to many species. to show the usefulness of the mmr method we made simple virtual species, which included the first generation and the second generation, on a computer, and then we conducted simulations to estimate the effective population size of the first generation. we calculated three statistics to estimate whether the mmr method is useful or not. the three statistics showed that the mmr method is useful. key words: population size, biodiversity, methodolog y, population dynamics lab. of biometry and bioinformatics, graduate school of agriculture and life science, university of tokyo, yayoi1-1-1, bunkyo-ku, tokyo, 113-8657, japan * corresponding author. email: murasek@cc.tuat.ac.jp 166 sociobiolog y vol. 59, no. 1, 2012 introduction a large number of studies of the methods for conservation and management of biological resources have been made on the methods used on one specific or a few target species (e.g. nielsen et al. 2001; frankham et al. 2007). such studies needed measurements of prior information (such as reproduction patterns, mortality etc.) of target species, and constructions of specific models of the target species. as a result, these kinds of previous studies needed not only an expert, but also a lot of time, labor, and money. this kind of biological resource management is impractical when people have to manage a lot of species at the same time within limited budgets. although people have been emphasizing the need for biodiversity management for a long time, little is known about a method of biodiversity management that can be implemented under the constraints of a limited budget. we think using a simplified method of estimating population dynamics is one of the best ways when low cost is a key factor. however, little attention has been given to the point by bioinformaticians. in addition, no studies have ever tried to investigate the effectiveness of a simplified method to estimate population dynamics using bioinformatics. to solve this problem, we paid attention to the importance of the “order of magnitude” level information (for example, when we want to know the number of individuals). in fact, when people have to manage biodiversity within a limited budget, we want to know only “order of magnitude” numbers (like 100 or 1000); not too much precision (for example, 1011 individuals or 1014 individuals). we developed a simplified method to estimate population dynamics, which has the potential to be used for many species, and thus nullifying the need to construct species–specific models. so, what properties are needed in the simplified method to estimate population dynamics within limited budgets in order to maintain many species? (1) the method should be simple and easy. sometimes people, such as local people in the national parks in tropical areas, ask us: “how do we maintain the biodiversity in this area without a professional book on taxonomy, a university, a research institute, a specimen room and a specialist?” how many researchers can answer this question? not only in developing countries, but also in developed countries we have 167 murase, k. & m. fukita — simulation of the molecular mark and recapture method to depend on local people for the maintenance of biodiversity where there are no specialists or researchers stationed. examples of this can be found in mountain villages or isolated islands. thus, the method to estimate population dynamics must be simple and easy for local people. it will be more important for long-term biodiversity management that local people treat biodiversity management as their own problem or challenge. if local people can understand the simplified method to estimate population dynamics, the method will contribute to spreading the mindset amongst them that “we will conduct sampling by ourselves to maintain the biodiversity in this area.” moreover, if the method is simple, there are many advantages, such as applying the simple method to regionally specific assignments, and enhancing environmental education so that there can be a commonly held consciousness about the local nature and environment. (2) the method should be based on the assumption that molecular data analysis is used. the use of molecular data to estimate population dynamics is becoming an important tool in molecular ecolog y. however, to understand this study area, especially paternity, advanced statistical backgrounds are usually required (e.g. foltz & hogland 1981; amos et al. 1993; clapham & palsbøll 1997; nielsen et al. 2001). the simple traditional methods for estimating population dynamics is a mark-recapture method was first used for ecological study in 1896 by petersen; later, lincoln (1930) independently developed the method to estimate waterfowl populations (southwood & henderson 2000). these methods, however, cannot estimate the detailed ecological and lifecycle information. a new method should give us ecological and lifecycle information such as blood relations, sub-population structures, genetic diversity, reproductive patterns, and other structures using molecular data. our new method set out in this paper pays attention to the estimation of an effective population size only. however, once we get dna data, we can also estimate other ecological and lifecycle information from that data. moreover, it has the advantage of using molecular data for the simple estimation of population dynamics. other specialists can reanalyze the data in detail when someone finds some problem or trend using the new simple method. 168 sociobiolog y vol. 59, no. 1, 2012 we think that if local people know the importance of sampling on the assumption of molecular data analysis, many people will be able to grasp how important the samples are and what valuable information is included in them, which have been largely dismissed until recently. for example, how many local people know that if they only conduct random samplings once or twice (for example, before and after making a road), that they can know the extent of genetic diversity lost and the decrease in population size, etc.? in recent years, the cost of obtaining molecular data from samples has been decreasing remarkably. ordinary people can get the molecular data by offering a commission to a company to perform laboratory work. however, to date, there is little study on a sampling strateg y for local people based on the assumption of molecular data information. this study is an initiative to study sampling strateg y on the assumption of molecular data analysis. (3) the method should be used across generations. a long-term perspective is essential: it is important for biodiversity maintenance to know whether an effective population size decreases or increases across several generations. our method as set out in this paper can be used not only between two generations but also across many generations such as grandparents, parents, children and grandchildren generations by applying our simple method to each adjacent generation. thus, this method can also be used for estimating simple population dynamics as long as we prepare for more than two generations. (4) the method should allow use of samples that had been collected for other purposes. it is better that the new method is used to contribute to resource management not only for in the future but also for analyzing data from the past. the new method should estimate the effective population size with samples that had already been collected for other purposes (for example, to know the mating season of one insect species, researchers had collected the insect species by light traps monthly over several years). in most studies, such samples are thrown away after those samples are used only once for only one purpose. in the above example case, we lose information not only of a target species but also of other insect species, which had also been collected by light traps. even though studies that concentrate their efforts on one species have invested 169 murase, k. & m. fukita — simulation of the molecular mark and recapture method substantial amounts of money (for example, studies of deer), and there are many samples in museums or research institutes for ten years or more, the samples have not been used to estimate population dynamics effectively in the past. we think some samples, which had been collected for other purposes, can be used in our new method. (5) the method should be used not only in the biodiversity field but also the medical field. we add one more paragraph to focus on the medical field, because it is important to say that our new method is also useful in the medical field. especially in developing countries with biodiversity hotspot areas, it is not easy to survey the dynamics of many serious diseases under the constraints of a limited budget. basically, the new method is broadly applicable to many diseases such as insect-borne diseases, hereditary diseases and infectious diseases. (6) the method should be applicable to limited populations. in the study area of statistics, there are a lot of cases that tend to use an infinite population, as it is easier than using a limited population. however, most national parks, protected areas, and primary forests are found only in limited areas. we therefore make the qualification that a limited population be assumed. in conclusion, with this study we proposed a simple method (we named this method the mmr method) which combined the above six properties for estimating effective population size. moreover, we showed the usefulness of the mmr method. when showing the usefulness of the new method, gathering field data is not better because field samples include many kinds of environmental effects and species-specific life histories, and we cannot test repeatedly whether the mmr method is useful or not. thus, we used the bioinformatics method to test whether the mmr method is useful or not. first, we made two generations of simple virtual species in computer. next, we collected some samples from each generation using simulation programs that we had made. finally, we compiled three statistics that were calculated from results of the simulations to investigate whether the mmr method was useful or not. moreover, we discussed the cost and benefits of the sampling effort, especially the value of the contribution in improving estimation precision when we add one more sample. 170 sociobiolog y vol. 59, no. 1, 2012 materials and methods first, we describe a principle of the mmr method. mmr is the abbreviation of “molecular mark and recapture”. next, we describe estimation methods of the mmr method using the three statistics that we had made. third, we wrote our material which is simple virtual species in a computer. finally, we show the results of the three experiments (the three simulations) using the mmr method. the principle of the mmr method we want to estimate an effective population size of the first generation, but we can get only two sample sets from the first generation (f 1 ) and the second generation (f 2 ). thus, we estimated an effective population size from samples of two generations using formula (1), (1) where g1 is the estimated effective population size of the first generation, ng1 is the number of the sampled first generation, ng2 is the number of the sampled second generation and mg2 is the number of the second generation whose parent is found in the first generation (fig. 1). the probability distribution of the mmr method is hypergeometric distribution because this method is based on sampling without replacement (like ordinary mark-recapture method). in the case of the ordinary mark and recapture method, we must conduct sampling twice in field. in the case of the mmr method, if we can collect two generations together, we need collect samples only once because the mmr method uses dna information. in statistics, “point estimates” is a single value given as the estimate of a population parameter that is of interest, in our case an estimated effective population size of the first generation. “interval estimates” specifies instead a range within which the parameter is estimated to lie. the mmr method can give not only point estimates but also interval estimates. in this paper, however, we do not discuss the interval estimates because the most important aim in this paper is to show the principle of the mmr method simply. 2 2 1 1 ˆ g g g g nm n n = 171 murase, k. & m. fukita — simulation of the molecular mark and recapture method estimation methods of the mmr method using three statistics we calculated the three statistics to estimate whether the mmr method is useful or not. the first statistic is the mean value of the estimated effective population size of the first generation. the second statistic is the range of g1 (i.e. [g1]sd), which is obtained by substituting (2) into mg2of the equation (1), sd (2) where 2gm is the mean of the number of the sampled second generation and sd is the standard deviation. the third statistic is the quartile points (upper quartile and lower quartile) of the estimated effective population size of the first generation. a simple virtual species: two generations, 100 individuals in each generation we made a simple virtual species which had 100 adult individuals of the first generation (or parent generation). this establishment meant that the effective population size of the first generation was 100, and this number is an answer that we want from fewer samples. fig. 1. the concept of the mmr method. sdmg ±2 172 sociobiolog y vol. 59, no. 1, 2012 next, we established the 100 adult individuals of the first generation (or parent generation) made one child individual each of the second generation (or child generation). we also established that one adult individual of the first generation can be identified by one child individual of the second generation by the virtual dna (microsatellite) data. the simulation experiments in all experiments, at first, we collected some samples through random sampling from the first generation. next, we also collected some samples through random sampling from the second generation. finally, we estimated the effective population size of the first generation by the mmr method using virtual microsatellite data of the two sampling sets. we conducted the three steps 1000 times under the same condition. when we could not find any parents in samples of the second generation, we denoted the situation as ‘inf.’, which is the abbreviated form of infinite (i.e. unmeasurable). experiment 1 in experiment 1, we set a strict standard that the number of samples was not enough when a simulation set, which was 1000 repetitions, had even one inf. to know this strict standard sample number, we counted the number of inf in the 1000 times repetition. we collected the same number of samples (such as 10, 20, 30, 40, 50, 60, 70, 80, 90) from each generation. we estimated one effective population size of the first generation from one sample set of the first generation and one sample set of the second generation. we collected all samples randomly. experiment 2 from experiment 1, the number with no occurrence of inf. among 1000 repetitions was 30 individuals. thus, in experiment 2, we fixed the sample number of the first generation at 30 individuals. and we also collected 10, 20, 30, 40, 50, 60, 70, 80, 90 individuals from the second generations. we estimated one effective population size of the first generation from one sample set (30 individuals) of the first generation and one sample set (such as 10, 20, 30 … individuals) of the second generation. we collected all samples randomly. 173 murase, k. & m. fukita — simulation of the molecular mark and recapture method experiment 3 in experiment 3, we fixed the sample number of the second generation at 30 individuals. and we also collected 10, 20, 30, 40, 50, 60, 70, 80, 90 individuals from the first generations. we estimated one effective population size of the first generation from one sample set (such as 10, 20, 30 …individuals) of the first generation and one sample set (30 individuals) of the second generation. we collected all samples randomly. results experiment 1 the mean, which is the first statistic, showed around the expected value (100 individuals), independently of the number of samples (table 1). the second statistic showed when the sample number was 10, the range was wide (54.05125 1220.94065), whereas when the sample number was 20, the range reduced suddenly (70.52745 163.23536) (table 1). the third statistic showed when the sample number was 10, it was not possible to determine a specific range (50 inf.) because there were many inf, whereas when the sample number was 20, the range showed around an expected value (80.00 133.30) (table 1). table 1. the three statistics of experiment 1 of the virtual dna data by simulation. the number of sampling from first generation is ‘n. 1st g.’. the number of sampling from second generation is ‘n. 2nd g.’. the number of repetitions of the simulation is ‘n. repetition’. the mean value of the estimated effective population size of the first generation (the first statistic) is g1. the standard deviation of g1 (the second statistic) is [g1]sd. the upper quartile and lower quartile of the estimated effective population size of the first generation (the third statistic) is ‘upper – lower quartile’. if we could not measure g1 after 1000 times, we denoted the situation as ‘inf.’, which is the abbreviated form of infinite (i.e. unmeasurable). n. 1st g. n. 2nd g. n. repetition g1 [g1]sd upper -lower quartile inf. 10 10 1000 103.51967 (54.05125 1220.94065) 50 – inf. inf. 20 20 1000 98.49791 (70.52745 163.23536) 80.00 133.30 inf. 30 30 1000 100.7613 (81.59181 131.7045) 90.00 112.50 0 40 40 1000 100.38901 (87.34636 118.01045) 92.81 114.30 0 50 50 1000 99.60953 (90.72791 110.41874) 92.59 92.59 0 60 60 1000 99.71747 (93.23344 107.17079) 94.74 102.90 0 70 70 1000 99.97756 (95.80979 104.52441) 98.00 102.10 0 80 80 1000 99.97501 (97.53984 102.53488) 98.46 101.60 0 90 90 1000 99.9876 (98.89528 101.10443) 98.78 101.20 0 100 100 1000 100 (100 100) 100 – 100 0 174 sociobiolog y vol. 59, no. 1, 2012 when the sample number was 10, the number of inf. in the 1000 repetitions was 339. when the sample number was 20, the number of inf. was 10. when the sample number was more than 30, the number of inf. were 0 (table 1). we illustrated the same result of experiment 1 as histograms (fig. 2). the distributions were a discrete distribution. fig. 2 shows that as the sampling number increased, the tail of the distribution shortened. in the case of more than 50 samples, the distributions were like a normal distribution and the degree of concentration was extremely high around an expected value (100) (fig. 2). experiment 2 the first statistic showed around an expected value (100), independently of the number of samples (table 2). the second statistic also showed around an expected value (67.64291 176.16536) when the sample number of the second generation was 10 or higher. as the number of samples increased, the fig. 2 the histograms of the estimated effective population size using the same number of samples (10, 20, 30, 40, 50, 60, 70, 80, 90) from each generation in computer simulations. the upper right number of each graph is the number of samples. 175 murase, k. & m. fukita — simulation of the molecular mark and recapture method table 2. the three statistics of experiment 2 of the virtual dna data by simulation. the number of sampling from first generation is ‘n. 1st g.’. the number of sampling from second generation is ‘n. 2nd g.’. the number of repetitions of the simulation is ‘n. repetition’. the mean value of the estimated effective population size of the first generation (the first statistic) is g1. the standard deviation of g1 (the second statistic) is [g1]sd. the upper quartile and lower quartile of the estimated effective population size of the first generation (the third statistic) is ‘upper – lower quartile’. if we could not measure g1 after 1000 times, we denoted the situation as ‘inf.’, which is the abbreviated form of infinite (i.e. unmeasurable). n. 1st g. n. 2nd g. n. repetition g1 [g1]sd upper -lower quartile inf. 30 10 1000 97.75171 (67.64291 176.16536) 75.00 150.00 inf. 30 20 1000 101.18044 (77.32627 146.31754) 85.71 120.00 0 30 30 1000 99.14078 (80.46678 129.10149) 90.0 112.5 0 30 40 1000 100.64581 (85.59192 122.12515) 92.31 109.10 0 30 50 1000 100.2138 (86.7438 118.6362) 88.24 115.40 0 30 60 1000 100.22272 (88.81483 114.99309) 90.0 112.5 0 30 70 1000 100.21953 (90.91904 111.63962) 95.45 110.50 0 30 80 1000 100.2088 (92.9704 108.6694) 96.00 104.30 0 30 90 1000 100.3195 (95.4510 105.7114) 96.43 103.80 0 30 100 1000 100 (100 100) 100 – 100 0 table 3. the three statistics of experiment 3 of the virtual dna data by simulation. the number of sampling from first generation is ‘n. 1st g.’. the number of sampling from second generation is ‘n. 2nd g.’. the number of repetitions of the simulation is ‘n. repetition’. the mean value of the estimated effective population size of the first generation (the first statistic) is g1. the standard deviation of g1 (the second statistic) is [g1]sd. the upper quartile and lower quartile of the estimated effective population size of the first generation (the third statistic) is ‘upper – lower quartile’. if we could not measure g1 after 1000 times, we denoted the situation as ‘inf.’, which is the abbreviated form of infinite (i.e. unmeasurable). n. 1st g. n. 2nd g. n. repetition g1 [g1]sd upper -lower quartile inf. 10 30 1000 101.0101 (68.8818 189.3087) 75.00 150.00 inf. 20 30 1000 99.86684 (76.54911 143.61300) 85.71 120.00 0 30 30 1000 99.14078 (80.46678 129.10149) 90.0 112.5 0 40 30 1000 100.00833 (83.95997 123.64156) 85.71 120.00 0 50 30 1000 100.08006 (86.92943 117.91875) 88.24 115.40 0 60 30 1000 100.52496 (89.29582 114.98451) 94.74 112.50 0 70 30 1000 100.25302 (91.12135 111.41878) 95.45 105.00 0 80 30 1000 100.20877 (93.37477 108.12211) 96.00 104.30 0 90 30 1000 99.9889 (95.2401 105.2361) 96.43 103.80 0 100 30 1000 100 (100 100) 100 – 100 0 176 sociobiolog y vol. 59, no. 1, 2012 range reduced (table 2). the third statistic also showed around an expected value (75.00 150.00) when the sample number was 10 or more. as the number of samples increased, the range reduced (table 2). there was no inf. except in the case where the number of samples was 10. we illustrated the same results of experiment 2 as histograms (fig. 3). the distributions were a discrete distribution. fig. 3 shows that as the sampling number increased, the tail of the distribution shortened. in the case of more than 50 samples, the distributions were like a normal distribution and the degree of a concentration was extremely high around an expected value (100), however the degree of concentration was lower than experiment 1 (fig. 3). experiment 3 the first statistic showed around an expected value (100), independently of the number of samples (table 3). the second statistic also showed around fig. 3 the histograms of the estimated effective population size using two sample sets which are the 30 individuals from the first generation and 10, 20, 30, 40, 50, 60, 70, 80, 90 individuals from the second generations in computer simulations. the upper right number of each graph is the number of samples of the second generation. 177 murase, k. & m. fukita — simulation of the molecular mark and recapture method an expected value (68.8818 189.3087) from when the sample number of the first generation was 10. as the number of samples increased, the range reduced (table 3). the third statistic also showed around an expected value (75.00 150.00) from when the sample number was 10. as the number of samples increased, the range reduced (table 3). there was no inf. except the case of the number of samples was 10. we illustrated the same result of experiment 3 as histograms (fig. 4). the distributions were a discrete distribution. fig. 4 showed that as the sampling number increased, the tail of the distribution shortened. in the case of more than 50 samples, the distributions were like a normal distribution and the degree of a concentration was extremely high around an expected value (100), however the degree of concentration was lower than experiment 1 (fig.1, 4). fig. 4 the histograms of the estimated effective population size using two sample sets which are 10, 20, 30, 40, 50, 60, 70, 80, 90 individuals from the first generation and the 30 individuals from the second generation in computer simulations. the upper right number of each graph is the number of samples of the first generation. 178 sociobiolog y vol. 59, no. 1, 2012 discussion our results showed the estimated three statistics were stable around the correct answer (100 individuals) in all of the three simulations. thus, the mmr method gave unbiased estimated values. these results indicated the mmr method was one of the best methods to roughly estimate the effective population size, especially in cases of the management of biodiversity within the constraints of a limited budget. as the number of samples increased, the precision of the estimated effective population size of the first generation also increased in all three of the simulations. this result is consistent with the classical sampling theories (petersen 1896; lincoln 1930). our results also indicated that when we collect samples, we should collect samples impartially, i.e., equally, between the two generations (for example, samples of 20 individuals from each generation is better than samples of 10 individuals from one generation and 30 individuals from another generation). however, in some cases, it is difficult to collect exactly the same number of samples from each generation. which generation’s sample is more informative to estimate the effective population size in such cases? the results of experiment 2 and experiment 3 showed that the effect of increased samples was almost the same between the two generations. this result indicated that the value of samples of both generations is the same if we collect samples randomly. this result also indicated that when it is difficult to collect the same number of samples from each generation, it is preferable to collect more samples from one generation so as to increase the precision of the effective population size (provided that we collect the samples randomly), rather than halting the collection of samples for the sake of impartiality, i.e., numerically equal samplings across the two generations. regarding optimal sampling strateg y, our results showed that when the number of samples was increased extensively, the precision of the estimated effective population size of the first generation did not increase appreciably (fig. 2, 3, 4). for example, let’s see the width of the value of ([g1]sd) in experiment 1. when the number of samples was increased from 10 to 20, the precision of the estimates increased almost 10 times. however, when the number of samples was increased from 80 to 90, the precision was almost the 179 murase, k. & m. fukita — simulation of the molecular mark and recapture method same, even though the increase in the number of samples was the same as the previous increase (10 individuals). in field researches, many people collect excessive samples because they do not know an efficient number of samples with which to estimate some ecological information from their target species. because the mmr method can estimate the effective population size with a small number of pre-samples, less sampling is required. our results in experiment 1 showed that when the number of samples (the first generation and the second generation) was 20 individuals, there was not any order-level failure of estimation among 1000 replicates. this result indicated that 20% of the total sample is one of the objective percentages to collect in cases of rare species. our results in experiment 1 also showed that when the number of samples (the first generation and the second generation) was 30 individuals, there was not any inf. among 1000 replicates. this result indicated that 30% of the total sample is one of the objective percentages to collect in cases of management of biodiversity under a limited budget. our results in experiment 1 and 2 showed that when the number of samples of one generation was 20 and the number of samples of another generation was 30, there was not any inf. among 1000 replicates. this result indicates that 30% of the one generation and 20% of another generation is also one of the objective percentages to collect in the case of the management of biodiversity within the limits of a budget and/or time. (in the field, however, if you can, it is better to collect more specimens from adult generations to avoid genealogical problems; for example, some people may collect samples which are offspring generation from only one or few families.) we think we can use this mmr method to estimate the effective population size and the population dynamics from certain samples which were already collected for other research purposes. for example, many colonies of one social ant species were already collected over 10 years to study their life history including their social structure, mating strateg y and so on. these ant samples can be used to understand population dynamics across a 10 year period using the mmr method. another example: many samples of some harmful animals such as deer and bears were collected for several years to maintain low density. these samples of the harmful animals can also be used to understand their population dynamics and the level of effectiveness of the sampling. 180 sociobiolog y vol. 59, no. 1, 2012 it is also necessary to consider the relation between cost (the sampling effort, money spent and so on) and benefit. in conclusion, the mmr method is less expensive than other standard methods. in the case of the study of wild bears, researchers use dna information from hair traps and software “mark” to estimate the effective population size (e.g. boulanger 2002). the cost of this method is more than 10 times as high as the mmr method. because there were many hairs in a trap, and researchers need to continue collecting hair many times from each trap. it is difficult to raise the considerably high budgets needed for this kind of research every year. on the other hand, the mmr method needs only one sample set that is collected at one time (in the case of bears, we can collect two generations together). moreover, even though in cases of species from which it is dangerous to collect samples without special skills such as hunting and capture techniques, e.g., bears, we can estimate the effective population size of the species using the mmr method easily by collecting fresh dung from both adult and young bears in the target study site. thus all the costs, such as the field sampling effort, experimental effort in a laboratory, the cost of chemical reagents and so on, of the mmr method is very low. in cases of this type of species which attracts people’s attention, we can construct a new management scheme to combine the mmr method and the standard method (for example, we change the two estimate methods depending on the year). in this study, we used a virtual population to illustrate the principle of the mmr method and the results of the simulations simply. however, can we apply the mmr method to real life? our answer is, needless to say: “yes”. even though unique species have some specific life history, we can use the mmr method through small improvements or adjustments. for example, murase applied the mmr method to social insects (k. murase, unpublished data). finally, we want to emphasize the point that we hope that local people who live under limited conditions (such as a shortage of funds, specialists, literature, equipment for experiments, etc. ) will become aware that they can contribute to maintaining biodiversity (sometime the biodiversity is also a a part of local people’s living environment). maintenance of biodiversity requires only samples collected under random sampling. funds, specialists, literature and equipment are not necessarily needed. of course, people must record the 181 murase, k. & m. fukita — simulation of the molecular mark and recapture method environmental information (such as the date and time, the locality, whether the sample is from an adult or young specimen) for each sample; and they must know some basic techniques, such as how to preserve the sample for later use (for example, plant leaves must dry before being put into a plastic bag ). however, if local people all over the world conduct the management of their local biodiversity using the mmr method, we think it has great potential to enhance management methods of biodiversity on earth. acknowledgments this work was supported partly by japan society for the promotion of science ( jsps) research fellowships for young scientists (research project number: 05j10734). we thank tohru nakashizuka and tamiji inoue for providing opportunities to study in tropical areas. we also thank each and every one of the researchers of social insects in japan, especially yoshiaki ito, seiki yamane, tadao matumoto, keiichi masuko, kazuki tuji, keiko hamaguchi, toshiyuki sato, mamoru terayama, toru miura, kiyoto maekawa, tamito sakurai and kyoichi kinomura for helpful comments and encouragement. we also thank hirohisa kishino, yasuo ukai, yasushi takano, hiroyoshi iwata, hiroshi omori who are/were the faculty members of laboratory of biometry and bioinformatics, the university of tokyo for useful comments. we thank udo bartsch for the advice on english usage. references amos, b., c. schlötterer & d. tautz. 1993. breeding behavior of pilot whales revealed by dna fingerprinting. heredity 67: 49–55. boulanger, j., g.c. white, b.n. mclellan, j. woods, m. proctor & s. himmer 2002. a meta-analysis of grizzly bear dna mark-recapture projects in british columbia, canada. ursus. 13:137-152. clapham, p.j. & p.j. palsbøll. 1997. molecular analysis of paternity shows promiscuous mating in female humpback whales (megaptera novaeangliae, borowski). proceedings of the royal society lond. ser. b 264: 95–98. foltz, d. w. & d. w. hogland. 1981. analysis of the mating system in the black-tailed prairie dog (cynomys ludovicianus) by likelihood of paternity. journal of mammalog y. 62: 706-712. frankham, r., j.d. ballou & d.a. briscoe. 2007. introduction to conservation genetics. cambridge university press. cambridge. 182 sociobiolog y vol. 59, no. 1, 2012 lincoln, f.c. 1930. calculating waterfowl abundance on the basis of banding returns. united states department of agriculture circular. 118: 1-4. nielsen, r., d.k. mattila, p.j. clapham & p.j. palsbøll. 2001. statistical approaches to paternity analysis in natural populations and applications to the north atlantic humpback whale. genetics 157: 1673–1682. petersen, c.g.j. 1896. the yearly immigration of young plaice into the limfjord from the german sea, report of the danish biological station (1895), 6: 5-84. southwood, t.r.e. & p.a. henderson. 2000. absolute population estimates using capturerecapture experiments. pp. 73-140. in: ecological methods 3rd ed. blackwell science, oxford. 1223 a new synonymy of coptotermes formosanus (isoptera: rhinotermitidae) by zhi-qiang li, jun-hong zhong & wei-liang xiao1 abstract the taxonomy of the genus coptotermes is far from settled in the world, especially in china. coptotermes formosanus shiraki had been described or misidentified as several different species in china based on measurement characteristics. in this paper, the synonyms of c. formosanus from china are listed, and it is proposed that c. guizhouensis is a new junior synonym of c. formosanus. key words: termites, coptotermes formosanus, coptotermes guizhouensis. introduction the taxonomy of the genus coptotermes lacks some careful revisions (eggleton 1999; vargo & husseneder 2009). the formosan subterranean termite (fst), coptotermes formosanus shiraki, 1909, which originated from china, is so far the most important and destructive invasive termite species (evans 2011). the widespread and destructive species appears to be paraphyletic with respect to several other species in the coptotermes genus (gentz et al. 2008). the size of individuals of c. formosanus has been observed to increase with increasing age (huang et al. 2000). in the 1980s and 1990s, a substantial number of coptotermes species in china were described, and the genus is in serious need of revision in china (li et al. 2011). even worse, the original descriptions of those coptotermes species were described in chinese, and they are often not reviewed by foreign taxonomists. a high degree of variation exists in the measurements of c. formosanus among different ages and different populations in china, and seven synonymies have been reported, including c. hongkongensis oshima, 1914, c. eucalyptus ping, 1984, c. xiaoliangensis ping, 1984, c. heteromorphus ping, 1985, c. guangzhouensis ping, 1985, c. communis xia et he, 1986, guangdong entomological institute, guang zhou 510260, china 1corresponding author, email : xwl@gdei.gd.cn 1224 sociobiolog y vol. 59, no. 4, 2012 c. rectangularis ping et xu, 1986 (snyder 1949; huang et al. 2000). in this paper, a new junior synonym of c. formosanus is proposed. materials and methods based on the original description of the related species of c. formosanus regarding the chinese termite literature, the characteristics and measurements of soldiers and imagoes were carefully compared with c. formosanus reported by harris (1966), xia & he (1986), roonwal & chhotani (1989), and huang et al. (2000), especially the measurement characteristics of c. formosanus among different ages in a laboratory colony (huang et al. 2000). the following acronyms are used in the paper: guangdong entomological institute (gdei); shanghai institute of entomology, academia sinica (siec); dipartimento di entomologia, italy (ddei); naturhistorika riksmuseet, sweden (nrs). synonymy coptotermes guizhouensis he et qiu, 1992 resembles c. formosanus, and soldiers of both species have two pairs of setae at the base of the fontanelle. he & qiu (1992) originally described this species. he & qiu (1992) indicated that the diagnostic characteristics for c. guizhouensis were that the widths of pronotum of soldiers were narrower compared with those of c. formosanus, and the number of antennal segments of c. guizhouensis imagos was 19 segments. however, the range of measurements of c. formosanus has been extended, which showed that the measurement of c. guizhouensis fell within the overall range of the measurement characteristics of c. formosanus (table 1). therefore, the diagnostic characteristics of c. guizhouensis according to he & qiu (1992) are untenable, which implies that c. guizhouensis lacks obvious and reliable characteristics with which it can be distinguished from c. formosanus. c. guizhouensis is therefore a junior synonym for c. formosanus. coptotermes formosanus skiraki, 1909 coptotermes formosanus skiraki, 1909, japanses trans. entom. 2: 239. type: ?. type locality: taiwan. termes raffrayi matsumura, 1910, die schädlichen und nützlichen insekten vom zuckerrohr formosas : 1. 1225 li, z.-q. et al. — a new synonymy of coptotermes formosanus coptotermes formosae holmgre, 1911:192. syntypes: [nrs]. type locality: taiwan. coptotermes hongkongensis oshima, 1914, annotationes zoologicae japonenses 8:559. type: ?. type locality: hongkong. coptotermes intrudens oshima, 1920, proc. hawaiian entomol. soc. 4:262. type: ?. type locality: hawaii. cryptotermes hongkongensis campbell, 1922:82. coptotermes remotes silvestri, 1928, bollettino del laboratorio di zoologia general e agraria 21:93. snytypes: [ddei]. type locality: south africa. coptotermes communis xia et he, 1986, contr. shanghai inst. entomol. table 1. measurements (in millimeters) of soldiers and imagos of coptotermes formosanus and coptotermes guizhouensis. measurement coptotermes formosanus c. guizhouensis he & qiu (1992) harris (1966) xia & he (1986) roonwal & chhotani (1989) huang et al. (2000) soldier length of head to lateral base of mandibles 1.36-1.59 1.50-1.60 1.43-1.65 1.43-1.68 1.42–1.51 max width of head 1.14-1.28 1.13-1.15 1.05-1.30 1.07-1.25 1.20–1.27 left mandible length 0.95-1.00 0.95-1.08 0.95-1.03 0.90-1.05 0.91 max length of postmentum --0.90-0.93 0.88-1.08 0.93-1.00 0.96–1.03 max width of postmentum --0.40-0.43 0.40-0.43 0.37-0.43 0.41–0.43 min width of postmentum --0.23-0.28 --0.21-0.26 0.24–0.26 max width of pronotum 0.77-0.86 0.78-0.88 0.75-0.93 0.80-0.94 0.77–0.79 max length of pronotum --0.45-0.53 0.40-0.53 0.38-0.50 0.41–0.46 length of hind tibia 1.04-1.14 1.08-1.15 --0.90-1.05 1.01–1.08 imago max width of head with eyes 1.53-1.63 1.45-1.53 1.50-1.71 1.42-1.63 1.37–1.39 max diameter of eye 0.43-0.45 0.37-0.40 0.43-0.45 0.37-0.45 0.37–0.39 min diameter of eye --0.38-0.38 --0.32-0.37 0.32 min distance of eye-lower margin of head --0.12-0.18 --0.16-0.18 0.14 min eye-ocellus distance 0.03-0.05 0.02-0.04 0.03-0.05 0.03-0.07 0.02 max diameter of ocellus 0.16-0.20 0.20-0.23 0.16-0.20 0.16-0.22 0.17–0.18 min diameter of ocellus --0.15-0.18 --0.13-0.15 0.13 max width of pronotum 1.30-1.43 1.33-1.35 1.30-1.52 1.21-1.46 1.27 max length of pronotum --0.88-0.90 0.80-0.93 0.77-0.87 0.77 length of forewing sacle --1.13-1.15 1.00-1.28 1.10-1.16 0.96 length of hindwing sacle --0.75-0.75 0.60-0.80 0.68-0.74 0.60 length of hind tibia --1.38-1.50 --1.32-1.53 1.37–1.37 number of antennal segments --19-20 20-21 19-21 19 1226 sociobiolog y vol. 59, no. 4, 2012 6:166. holotype: soldier, imago, work [siec]. type locality: jinhua [china, zhejiang ]. coptotermes eucalyptus ping, 1984, ecol. syst. trop. subt. fore. res. 2:186. holotype: soldier [gdei]. type locality: xiaoling [china, guangdong ]. coptotermes guangzhouensis ping, 1985, entomotaxonomia 7:137. holotype: soldier [gdei]. type locality: guangzhou [china, guangdong ]. coptotermes heteromorphus ping, 1985, entomotaxonomia 7: 320. holotype: soldier [gdei]. type locality: guangzhou [china, guangdong ]. coptotermes rectangularis ping et xu, 1986, sci. silv. sin. 22: 157. holotype: soldier [gdei]. type locality: congjiang county [china, guizhou]. coptotermes xiaoliangensis ping, 1984, ecol. syst. trop. subt. fore. res. 2:184. holotype: soldier [gdei].type locality: xiaoling [china, guangdong ]. coptotermes guizhouensis he et qiu, 1992, science and technolog y of termites 9:1. holotype: soldier, [siec]. type locality: conjiang county [china, guizhou]. new synonymy. acknowledgements this work was supported by the national natural science foundation of china (31172163) and the guangdong academy of sciences foundation for excellent young scientists and technicians (200901). references eggleton, p. 1999. termite species description rates and the state of termite taxonomy. insectes sociaux 46:1–5. evans, t.a. 2011. chapter 19. invasive termites, pp. 519–562. in: d.e. bignell, et al. (eds.). biolog y of termites: a modern synthesis. springer science+business media, new york, 576 pp. harris, w.v. 1966. on the genus coptotermes in africa (isoptera: rhinotermitidae). proceedings of the royal entomological society of london (b) 35(11–12): 165166. he, x.s. & q.s. qiu 1992. a new species of the genus coptotermes from guizhou province, china (isoptera: rhinotermitidae). science and technolog y of termites 9(2): 1–3. huang, f.s., g.x. li, & s.m. zhu 1989. the taxonomy and biolog y of chinese termites (isoptera). tianze press, beijing, 605 pp. huang, f.s., s.m. zhu, z.m. ping, x.s. he, g.x. li, & d.r. gao 2000. fauna sinica, vol. 17, insecta, isoptera. science press, beijing, 961 pp. 1227 li, z.-q. et al. — a new synonymy of coptotermes formosanus li, z.q., b.r. liu, q.j. li, w.l. xiao & j.h. zhong 2011. two new synonyms of coptotermes gestroi (wasmann) (isoptera: rhinotermitidae) in china. sociobiolog y 58(2): 449456. roonwal, m.l. & o.b. chhotani 1989. the fauna of india and the adjacent countries. isoptera (termites). vol. i (introduction and families termopsidae, hodotermitidae, kalotermitidae, rhinotermitidae, stylotermitidae and indotermitidae). zoological survey of india, calcutta. pp. 434-441. snyder, t. 1949. catalog of the termites (isoptera) of the world. smithsonian miscellaneous collections, 112:78. vargo, e.l. & c. husseneder 2009. biolog y of subterranean termites: insights from molecular studies of reticulitermes and coptotermes. annual review of entomolog y 54:379–403. xia, k.l. & x.s. he 1986. study on the genus coptotermes from china (isoptera: rhinotermitidae). contr. shanghai inst. entomol. 6(6):157-182. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4938sociobiology 67(2): 223-231 (june, 2020) introduction the urbanization process is considered to be one of the main areas of concern in conservation biology. the creation and expansion of urban boundaries is responsible for the replacement of natural environments in various anthropogenic forms of land use (mckinney, 2002), resulting in a large loss of habitat and the fragmentation of natural environments in different shapes and sizes (pacheco & vasconcelos, 2007). the remnants of a natural landscape (e.g. forest fragments and urban parks) represent important reservoirs of native biodiversity (nielsen et al., 2013), since many organisms have niche requirements that do not allow them to survive in an urban matrix (viana & pinheiro, 1998). thus, it is necessary to urgently develop strategies for the conservation abstract restingas are lowland sandy ecosystems located between mountain ranges and the sea. for living organisms in this ecosystem, restingas can be seen as habitats formed by islands of vegetation separated by a sandy matrix. these organisms are highly influenced by the environmental conditions and physical characteristics of the landscape, including size, connectivity and environmental heterogeneity. given the recognized effectiveness of ants as bioindicators, this study uses these organisms as a model to assess how do the physical characteristics of a landscape influence the structures of ant communities. the study was carried out in the parque das dunas, salvador bahia, brazil. within this park, four categories of vegetation islands (small, medium, large and continuous) were delimited, where the ant fauna was sampled and the forms of vegetal life were analyzed, using the raunkiaer analysis. a total of 69 ant species were collected from 31 genera and six subfamilies. we found a positive relationship between ant richness and diversity of plant life forms (shannon index). in general, there was a significant difference in the composition of ant species between the areas of continuous vegetation and the different vegetation islands. we conclude that there is a bottom up effect mediating the ant community associated with dune vegetation, where the local richness of ant species responds to vegetation heterogeneity. sociobiology an international journal on social insects mva lopes¹,², gmm santos¹ article history edited by kleber del-claro, ufu, brazil received 24 december 2019 initial acceptance 03 february 2020 final acceptance 09 march 2020 publication date 30 june 2020 keywords conservation, habitat types, mirmecofauna, vegetation complexity. corresponding author marcos v. a. lopes universidade estadual de feira de santana laboratório de entomologia av. transnordestina s/nº, cep 44031-460, feira de santana-ba, brasil. e-mail: marcosvalopes@gmail.com of biological diversity in these types of environment, for example through the implementation of strategies such as the execution of inventories, in order to record the fauna and flora of a certain place (pearson, 1994), which can generate subsidies for conservation policies and biodiversity management programs. among the vegetal formations found in brazil, the rain forest and its associated ecosystems are one of the environments most affected by the urbanization process (myers et al., 2000). restingas are lowland sandy ecosystems located in coastal zones (mantovani, 2003). for living organisms in this ecosystem, restingas can be seen as habitats formed by islands of vegetation separated by a sandy matrix. these organisms are highly influenced by environmental conditions. according to macarthur and wilson (1967), the number of 1 universidade estadual de feira de santana, programa de pós-graduação em ecologia e evolução. feira de santana-ba, brazil 2 universidade católica do salvador, centro de ecologia e conservação animal. salvador-ba, brazil research article ants do bottom-up effects define the structuring of ant (hymenoptera: formicidae) communities in a restinga remnant? mva lopes, gmm santos – structuring of ant (hymenoptera: formicidae) communities224 species on an island is determined by a balance between species migration/colonization and extinction processes, with these factors being regulated by the physical characteristics of the landscape, such as size, connectivity and environmental heterogeneity. environmental heterogeneity can directly influence animal diversity, promoting conditions and resources for a greater coexistence of species (corrêa et al., 2006, resende et al., 2011, silva et al., 2014). in this sense, studies have demonstrated positive relationships between environmental heterogeneity and ant diversity over time, by comparing areas with different phytophysiognomies (kumar & mishra, 2008; resende et al., 2013) or relating vegetation structure to diversity of ants (santana-reis & santos, 2001; gomes et al., 2010; vasconcelos et al., 2019). ants are excellent bioindicators, abundant and highly diverse in most terrestrial ecosystems (holldobler & wilson, 1990), performing several functions (oliveira et al., 1999; fernández, 2003). these organisms are highly dependent on habitat vegetation structure, as it provides food resources and nesting sites (vasconcelos et al., 2019). thus, this study answers the following question: how do the physical characteristics of a landscape influence the structures of ant communities? our working hypotheses are: (i) ant species richness is positively correlated to environmental heterogeneity, because complex environments offer wider niches for species and (ii) ant species composition varies among different sized landscapes, since each island size presents a specific set of niches that can be realized for the species. material and methods study area the city of salvador is the fourth largest city in brazil founded in the year 1549 and its administrative headquarters is located at the geographical coordinates 12.97°s 38.51°w. located within the atlantic forest biome, its local climate is humid tropical, classified as af (köppen) and characterized by an annual average temperature of 17ºc and 81% annual mean humidity (bdmep, 2017). the study was realized in parque das dunas (12°55’6.54”s, 38°19’5.83”w) located in the city of salvador and inserted in the apa lagoas e dunas do abaeté (state decree nº 351 of september 22, 1987), comprising an area of approximately six million square meters. the park is located in the neighborhood of praia do flamengo, situated in a restinga ecosystem. it is formed by a heterogeneous set of vegetation, with flooded areas, lagoons and dune areas covered by shrub, arboreal and herbaceous vegetation, which play an important role in fixing the dunes and protecting the soil against erosion (britto et al., 1993). sample design the samples were collected during april and july 2018. four categories of vegetation islands were sampled along the parque das dunas: (1) small, (2) medium, (3) large and (4) continuous, which are treated as environments with different microhabitats in this study, since each island size presents a specific set of realizable niches. fig 1. map of parque das dunas and their respective sample points. yellow = small microhabitats; gray = medium microhabitats, blue = large microhabitats and red = continuous microhabitats. sociobiology 67(2): 223-231 (june, 2020) 225 we selected and collected eight sample points (sps) for the small (< 250m²), medium ( ≥ 250 m² and ≤ 1500 m²), large ( ≥ 1500 m²) microhabitats , and 12 sps for the continuous area (fig 1), with a minimum distance of 30 meters between them, totaling 36 sps. at each sampling point, a 10 m² quadrant was delimited where we analyzed the ant communities and vegetation complexity. in order to characterize the ant community, the soil and vegetation fauna were sampled at each sp for greater detection of the diversity of ants with different habits, using the following methods: winkler extractor: for sampling the edaphic ants that inhabit the litter. in each sp, 1m² of litter was sampled, where the collected material was placed in winklers extractors for a period of 24 hours. pitfall trap: for sampling epigeous ants. five pitfall traps (polystyrene cups 15cm high and 7cm in diameter, containing alcohol 70%) were installed in each sp and remained in the field for a period of 24 hours. entomological umbrella: for sampling understory ants. in each area, an umbrella sample was made, where each sample was composed of fauna from 10 shrubs (up to 3 meters high) shaken for 10 seconds on top of the umbrella. following the sampling procedure, the structural complexity of the vegetation was analyzed through the classification of the plant communities according to raunkiaer (1934). all plants in each quadrant were analyzed and classified in cactaceae (ca), creepers (ce), chamephytes (cm), cryptophytes (cr), epiphytes (ep), phanerophytes (ph), hemicryptophytes (hm) and therophytes (te). the plants were not collected. the collected ants were morphotyped and fixed. some of these were sent to the collection of the laboratory of mirmecology (acronym cpdc), itabuna, ba (curator: j. delabie), executive committee of the cocoa plan (ceplac), where they were identified and deposited. the specimen collections received authorization nº 61299-3 from mma/sisbio. data for species incidence in the collections were used to calculate the faunistic indexes of (1) frequency, (2) similarity and (3) richness, to characterize myrmecofauna (marques et al., 2009). statistical analysis ants and plant community parameters were analyzed for each sampled point through a frequency matrix. the shannon’s (h’) diversity index for plants and ants was analyzed using the vegan package in program r version 3.4.3. in order to compare the plant life form between each microhabitat we applied the multiple response permutation procedure (mrpp), using the sorensen (bray-curtis) distance (mccune & grace, 2002). in order to compare ant species composition between each microhabitat and for the peerto-peer comparisons, we applied the multiple response permutation procedure (mrpp), using the sorensen (braycurtis) distance (mccune & grace, 2002). a hierarchical clustering analysis was performed to evaluate how the environments are grouped according to ant species composition, using sorensen’s distance (bray-curtis) and the method of connection average method (mccune & grace, 2002). cluster analysis and mrpp were performed using the pc-ord © 6.0 program (mccune & mefford, 2011). a normality test was performed for species richness using the shapiro test and the data showed normal distribution (p > 0.05). the effect of plant structural diversity on the ant community was analyzed using a general linear model (glm) using the r program version 3.4.3. ant species richness was considered a dependent variable and the diversity of plant life forms (h’) was an independent variable. results the restinga phytophysiognomy showed a high heterogeneity of plant life forms (fig 2). this environment had areas with predominantly chamephytes plants (frequency fig 2. spectrum of plant life forms found in parque das dunas. ca = cactaceae, ce = creepers, cm = chamephytes, cr = cryptophytes, ep = epiphytes, ph = phanerophytes, hm = hemicryptophytes, te = therophytes. mva lopes, gmm santos – structuring of ant (hymenoptera: formicidae) communities226 19%), while the plants therophytes (11%), creepers (11%) and epiphytes (8%) were the least representative. among the studied microhabitats the small vegetation islands differed in the composition of life forms in relation to the other sampled habitats. the small vegetation island life forms were composed mainly of chamephytes (36%) and cactaceae (18%) with the absence of phanerophytes and epiphytes. the medium sized vegetation islands showed a higher formation of chamephytes plants (22%) and hemicryptophytes (17%). the large vegetation islands showed a higher formation of phanerophytes, chamephytes and cactaceae plants (14% both). on the other hand, the continuous area presented the greatest fairness in the distribution of plant life forms among the microhabitats sampled, with the chamephytes and phanerophytes plants (15% both) being predominant (fig 2). solenopsis saevissima, strumigenys alberti, strumigenys subedentata and thaumatomyrmex fraxini showed frequencies of less than 1% (table 2). subfamily habitat % species sma med lar con dolichoderinae dorymyrmex sp. 1 12 7 5 13 0.15 dorymyrmex sp. 2 13 8 10 10 0.16 dorymyrmex sp. 3 10 11 12 10 0.17 dorymyrmex sp. 4 1 0 0 0 0.01 dorymyrmex thoracicus gallardo, 1916 2 2 3 0 0.03 forelius brasiliensis (forel, 1908) 4 3 4 2 0.05 ectatomminae ectatomma muticum mayr, 1870 0 0 0 3 0.01 gnamptogenys moelleri (forel, 1912) 0 0 0 12 0.05 gnamptogenys striatula radoskowsky, 1884 1 3 7 13 0.10 formicinae brachymyrmex admotus mayr, 1887 20 16 10 15 0.24 brachymyrmex heeri forel, 1874 4 3 2 4 0.05 brachymyrmex sp. 1 1 0 0 5 0.02 camponotus arboreus (fr. smith, 1858) 1 7 7 10 0.10 camponotus atriceps (fr. smith, 1858) 1 2 6 0 0.04 camponotus crassus mayr, 1862 0 1 0 0 0.01 camponotus fastigatus roger, 1863 3 4 9 2 0.07 camponotus vittatus forel, 1904 1 2 4 2 0.04 paratrechina longicornis (latreille, 1802) 0 0 0 1 0.001 myrmicinae acromyrmex aspersus (smith, 1858) 0 1 0 3 0.02 acromyrmex sp. 1 0 1 0 0 0.001 atta opaciceps borgmeier, 1939 1 3 3 0 0.03 carebara sp. 1 0 0 0 1 0.001 cephalotes minutus (fabricius,1804) 1 5 6 6 0.07 cephalotes pinelii de andrade, 1999 1 1 2 0 0.02 table 1. pairwise comparison of plants life form community. t = variation between groups, a = intra-group variation, p = statistical significance. significant p-values are in bold. medium large continuous small t = -1.0862 t = -6.0993 t = -8.0052 a = 0.0445 a = 0.2704 a = 0.2584 p = 0.1350 p = 0.0003 p < 0.00001 medium t = -1.9452 t = -2.4028 a = 0.0694 a = 0.0718 p = 0.0434 p < 0.0235 large t = 0.9840 a = -0.0292 p = 0.8476 table 2 . ant frequency (0 100%), in small, medium large and continuous microhabitats in parque das dunas. sma = small, med = medium, lar = large, con = continuous area. in general, there was a significant difference in the plants life form between microhabitats (mrpp: p < 0.000009, a intra-group variation = 0.1856, t variation between groups = -6.3213). we found a significant difference between continuous vegetation and the small and medium microhabitats. however, we did not find significant differences in plants life form similarities between small/medium and large/continuous microhabitats (table 1). a total of 69 ant species were collected and distributed across 31 genera and six subfamilies. the most representative subfamilies were myrmicinae (39 species), formicinae (nine) and ponerinae (seven) representing 76.8% of the species collected. the most representative genera were pheidole (nine species), solenopsis (six), camponotus and pseudomyrmex (five species each) representing 36.2% of the species collected (table 2). in general, the species with the highest frequency of occurrence were: pheidole radoszkowskii (f = 42%), brachymyrmex admotus (f = 24%) and crematogaster erecta (f = 16%). on the other hand, the species camponotus crassus, cyphomyrmex costatus, ectatomma muticum, paratrechina longicornis, megalomyrmex drifti, monomorium floricola, mycetophylax morschi, nesomyrmex asper, odontomachus brunneus, pheidole midas, rogeria lirata, solenopsis geminata, sociobiology 67(2): 223-231 (june, 2020) 227 table 2 . ant frequency (0 100%), in small, medium large and continuous microhabitats in parque das dunas. sma = small, med = medium, lar = large, con = continuous area. (continuation) crematogaster erecta mayr, 1866 12 7 6 16 0.16 crematogaster victima fr. smith, 1858 0 1 3 2 0.02 cyphomyrmex costatus mann, 1922 0 0 0 1 0.001 cyphomyrmex rimosus (spinola, 1853) 1 2 0 3 0.02 cyphomyrmex transversus emery, 1894 6 4 6 2 0.07 megalomyrmex drifti kempf, 1961 0 0 0 2 0.01 monomorium floricola (jerdon, 1852) 0 0 0 2 0.01 mycetophylax morschi (emery, 1888) 1 0 0 0 0.001 myrmicocrypta sp. 1 2 0 0 2 0.02 nesomyrmex asper (mayr, 1887) 1 0 1 0 0.01 nylanderia fulva (mayr, 1862) 2 3 2 3 0.04 pheidole (group diligens) sp. 2 0 0 0 1 0.001 pheidole (group fallax) sp. 1 1 4 3 11 0.08 pheidole (group fallax) sp. 2 0 0 1 0 0.001 pheidole (group flavens) sp. 3 3 2 1 4 0.04 pheidole (group tnistis) sp. 4 0 1 0 1 0.01 pheidole (group tnistis) sp. 5 0 0 1 4 0.02 pheidole (group tnistis) sp. 6 0 0 1 2 0.01 pheidole midas wilson, 2003 0 0 0 1 0.001 pheidole radoszkowskii radoskowsky, 1884 29 23 21 32 0.42 rogeria lirata kugler, 1994 1 0 0 1 0.01 solenopsis geminata (fabricius, 1804) 0 0 0 1 0.001 solenopsis saevissima (smith, 1855) 0 0 1 0 0.001 solenopsis globularia (fr. smith, 1858) 6 3 3 0 0.05 solenopsis sp. 1 0 1 1 2 0.02 solenopsis sp. 2 3 3 3 8 0.07 solenopsis sp. 3 1 0 1 1 0.01 strumigenys alberti forel, 1893 0 0 0 1 0.001 strumigenys subedentata mayr, 1887 0 0 1 0 0.001 strumigenys sp. 1 1 0 0 0 0.001 strumigenys sp. 2 0 0 1 0 0.001 trachymyrmex sp. 1 1 0 4 10 0.06 wasmannia auropunctata (roger, 1863) 1 6 0 0 0.03 ponerinae anochetus diegensis forel, 1912 2 1 4 0 0.03 hypoponera sp. 1 0 0 0 2 0.01 hypoponera sp. 2 0 0 0 5 0.02 neoponera bactromica (fernandes, 2014) 2 1 4 4 0.04 odontomachus bauri emery, 1891 9 12 10 2 0.13 odontomachus brunneus (patton, 1894) 1 1 0 0 0.01 thaumatomyrmex fraxini d’esquivel & jahyny, 2017 0 0 0 1 0.001 pseudomyrmecinae psedomyrmex elongatus (mayr, 1870) 0 2 1 5 0.03 pseudomyrmex (group pallidus) sp. 1 1 1 1 0 0.01 pseudomyrmex (group pallidus) sp. 2 2 0 0 1 0.01 pseudomyrmex (group pallidus) sp. 3 3 1 1 0 0.02 pseudomyrmex (group pallidus) sp. 4 1 0 1 0 0.01 richness 42 38 41 48 subfamily habitat % subfamily habitat % the characterization of the ant communities showed that each sample point has specific ants richness. 48 species of ants were collected in the area of continuous restinga, with 13 species occurring exclusively in this environment;42 species were collected in the small vegetation islands; 38 species in medium sized vegetation islands and 41 species in large vegetation islands (table 3). the shannon’s diversity ranged between 3.12 +/3.37, with the continuous area showing the greatest ants species diversity among the studied microhabitats (table 3). the studied microhabitats presented high species similarity, with values varying between 0.55 +/0.77 (table 4). table 3. average number of species per sample point, species richness (s) of ants and ants shannon diversity (h’) in the four different microhabitats. microhabitats average number of ants species species richness (s) diversity (h’) small 12 42 3.12 medium 13 38 3.21 large 14 41 3.36 continuous 14 48 3.37 mva lopes, gmm santos – structuring of ant (hymenoptera: formicidae) communities228 in general, there was a significant difference in the composition of ant species between microhabitats (mrpp: p<0.0005, a intra-group variation = 0.0276, t variation between groups = -3.8002). we found a significant difference between continuous vegetation and the other three microhabitats. however, we did not find significant differences in ant species similarities between small, medium and large habitats (table 5). the one-way cluster analysis between microhabitats indicated the presence of three distinct groups, where the continuous area separated sharply from the others (70% similarity), and the large and medium areas were very similar to each other (100% similarity). finally, medium and large vegetations showed about 75% of similarity with small microhabitat. a positive relationship was found between ant richness and the diversity of plant life forms (h’) (fig 3). many ant species were common to several dunes, regardless of the type of vegetation, however, the diversity of plant life forms explains 12% of the variance in the species richness of ants (glm: df= 34, p< 0.03, r² = 0.123). discussion among brazilian habitats, the restinga is composed of a mosaic of vegetation types with great variations in their life forms. in this sense, this ecosystem is considered to be one of the most complex and heterogeneous ecosystems in brazil (mantovani, 2003), thus enabling a high number of realizable niches and the coexistence of a large number of species. the restingas are naturally fragmented with obvious differences between areas; however, they demonstrate high similarity in the composition of life forms. only small and medium dunes, as observed on oceanic islands, differ from dunes of other sizes due to a lack of space required to support a wider range of life forms. overall, studies have shown that restinga environments have a high diversity of ants. cardoso and schoereder (2014) collected a total of 71 ant species for a restinga environment in santa catarina; while vargas et al. (2007) collected 92 species in a restinga environment in rio de janeiro. the results of our study were not different. we found a high diversity of ants (69 species) for the restinga areas sampled, thus suggesting table 4. ants similarity index among the studied microhabitats. sma = small island, med = medium island, lar = large island, con = continuous area. fig 3. positive relationship between ant richness and diversity of plant life forms (h ‘). curve equation (9.9414+2.1449* x). analyses based on sample points. thirty-six sample points are not seen in the graph due to data overlap. sma med lar con sma 1 med 0.730 1 0.772 0.633 lar 0.678 0.772 1 0.616 con 0.557 0.633 0.616 1 table 5. pairwise comparison of ant communities similarity. t = variation between groups, a = intra-group variation, p = statistical significance. significant p-values are in bold. medium large continuous small t = 0.2251 t = -1.3906 t = -5.6366 a = -0.0021 a = 0.0146 a = 0.0423 p = 0.5645 p = 0.0936 p < 0.0001 medium t = 1.7167 t = -2.7170 a = -0.0163 a = 0.0201 p = 0.9670 p < 0.0100 large t = -4.2035 a = 0.0302 p < 0.0004 sociobiology 67(2): 223-231 (june, 2020) 229 the necessity of the expansion of studies on invertebrate biodiversity in these environments and reinforcing the importance of forest remnants as environments capable of maintaining a high diversity of organisms. the high relative ant richness of the myrmicinae and formicinae subfamilies was expected as they have the highest species richness among ant subfamilies around the world (bolton, 2019). in turn, the species belonging to these subfamilies make up communities of stressed environments, natural or anthropic. restingas are naturally fragmented environments and are subjected to high thermal amplitudes at ground level, which favor the presence of xerophilous and opportunistic species. this fact supports the highest frequency of occurrence found for the species pheidole radoszkowskii, brachymyrmex admotus and crematogaster erecta in this study. in view of this fact, the existence of 13 species of ants exclusive to the continuous area is explained by greater soil cover, a greater number of plant species, greater vertical structure and, consequently, the greater buffering of climatic factors such as soil temperature and incidence luminosity. despite the high richness found in the environment as a whole (gamma diversity), we observed small variations in ant richness among the studied microhabitats (beta diversity). these fluctuations may be due to physical differences present in each sampled environment, where structural differences are responsible for several changes in animal communities, directly influencing species diversity and consequently, modifying the distribution and richness of organisms (lach et al., 2010). in our study, a significant relationship was found between ant richness and plant life diversity, showing a bottom up effect. the use of the raunkiaer scale can be considered as an important substitute for assessing and characterizing the structural complexity of a habitat (santos et al., 2007). several studies have shown that vegetation structure is one of the main factors that can influence the structure of ant communities (corrêa et al., 2006; resende et al., 2011; silva et al., 2014; vasconcelos et al., 2019). thus, complex environments with high environmental heterogeneity are able to act directly on the realizable and fundamental niches of species, allowing for the coexistence of a large number of species by providing nesting structures, food resources or foraging areas. the composition of ant species was significantly different between the studied microhabitats and between the continuous area and the different vegetative islands. it is common and expected that ant communities from different habitats, although in close proximity or even bordering each other, present distinct compositions (guimarães et al., 2013; lutinski et al., 2013; achury & suarez, 2018). this disparity is mainly expressed through the presence of species unique to each habitat. however, ant communities from similar ecosystems are also expected to have similar compositions, at least at the generic level, even if they are geographically separate (resende et al., 2011), while distinct ecosystems (cerrado, caatinga, atlantic forest) have a set of species that characterize them. the existence of differences between types of dune vegetation phytophysiognomies reinforces the mosaic system structure of this environment. this difference is most exacerbated when comparing the different vegetation island sizes with the continuous area. thus, this suggests that the sampled environments present a great variety in habitat structure and that it is a completely heterogeneous environment, a fact proven by the presence of species exclusive to both habitats. however, although each island has unique species, we found no significant differences in species similarities between islands of different sizes. in island systems such as sandbanks, factors such as the shape, size and degree of isolation of a habitat can influence the composition of component species (mcarthur & wilson 1967). thus, variations in species composition should be related to the characteristics of the studied landscapes. among landscape metrics, connectivity acts directly on the migration and dispersal processes of biological communities (magle et al., 2009). these mobility rates influence the similarities of species assemblages between habitats (magura et al., 2001) and can be explained by the island biogeography theory (macarthur and wilson, 1967), which postulates the importance of the size of the species’ receiving areas and the distance of these areas from source areas, in explaining these similarities. ecological community theory highlights habitat isolation as one of the most significant explanations for differences and similarities in species richness and composition in a community (pacheco & vasconcelos, 2007; öckinger et al., 2009; lizée et al., 2012), since isolated habitats tend to be more easily colonized. conclusion there is a bottom up effect mediating the ant community associated with dune vegetation, where the local ant species richness responds to the heterogeneity of the vegetation. diversity of plant life affects ant richness. different plant life allows for the establishment of ants with different niches, directly influencing the richness of ant species across habitats. restinga has high ant richness and diversity and should be preserved as a refuge for ants and associated communities, and is therefore important for the preservation, maintenance and conservation of arthropod biodiversity in urban environments. acknowledgments this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. to many colleagues that contribut ed to this study, specially sara gomes, bruno coutinho, matheus lopes and carlos azevedo for helped with field sampling, and janete resende with morphospeciation. references achury, r., & suarez, a.v. 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(2011). the ecology of urban areas and their functions for species diversity. landscape and ecological engineering, 7: 231-240. doi: 10.1007/s11355-011-0153-4 1495 vombisidris tibeta, a new myrmicine ant species from tibet, china with a key to the known species of vombisidris bolton of the world (hymenoptera: formicidae) by zheng-hui xu1 & na-na yu1 abstract seventeen species of the myrmicine ant genus vombisidris bolton are recognized in the world. a new species collected from the south slope of himalaya mountain, v. tibeta sp. nov., is described. a key to the 17 known species of the genus is provided based on the worker caste. illustrations are provided for each species. key words: hymenoptera, formicidae, vombisidris, new species, key. introduction since the establishment of the myrmicine ant genus vombisidris bolton (1991), 16 species of the genus were recorded in the world (bolton, 1995, 2012). firstly, 9 species of the genus were described by bolton (1991) with the erection of the genus. secondly, 3 species described in other genus before 1991 were transferred to vombisidris by bolton (1991), i.e. leptothorax australis wheeler, 1934, l. bilongrudi taylor, 1989, and l. renateae taylor, 1989. thirdly, 1 species described in other genus, atopula jacobsoni forel, 1915, was transferred to vombisidris by bolton (1995, 2003) and sorger (2011). fourthly, 3 species of the genus were described after 1991, i.e. vombisidris humboldticola zacharias et rajan, 2004, v. umbrabdomina huang et zhou, 2006, and v. philippina zettel et sorger, 2010. it is difficult to have a complete grasp of the total 16 species of the genus due to the following shortcomings. firstly, the detailed information of atopula jacobsoni forel, 1915, was excluded from the papers of taylor (1989) and bolton (1991). fortunately, sorger (2011) had sufficiently improved the description and illustrations of the species. secondly, bolton (1991) 1key laboratory of forest disaster warning and control in yunnan province, college of forestry, southwest forestry university, kunming, yunnan province 650224, china, e-mail: xuzhenghui1962@163.com 1496 sociobiolog y vol. 59, no. 4, 2012 provided good illustrations and descriptions for the profile characters of the 9 species described, but without illustrations and good descriptions of the full face characters of the head, which are very useful for the identification of the species. thirdly, the other 3 species described after 1991 were published separately and without a key relating them to the formerly known ones, and the profile figure of v. humboldticola zacharias et rajan is not clear. so, it is necessary to produce a good key with sufficient illustrations for the known species of the genus, in order to facilitate identification of the species. recently, the high quality images of 9 species of the genus are presented on the antweb and antbase, which provides as good references for the preparation of the key. in the ant diversity investigation of southeastern tibet, a new species of vombisidris was found in the valley tropical rainforest of medog county, the south slope of the himalaya mountain. the new species is described. a key to the 17 known species of the genus of the world is provided based on the worker caste. however, illustrations of the worker caste of v. australis (wheeler), and illustrations of the full face head of v. harpeza bolton, v. lochme bolton, v. occidua bolton, and v. humboldticola zacharias et rajan are still absent from this paper. materials and methods the worker caste of v. tibeta sp. nov. was collected by the sample-plot method. descriptions and measurements were made under a xtb-1 stereo microscope with a micrometer. illustrations were made under a motic-700z stereo microscope with illustrative equipment. standard measurements and indices are as defined in bolton (1987), in addition, ed is supplemented: tl-total length: the total outstretched length of the ant from the mandibular apex to the gastral apex. hl-head length: the length of the head proper, excluding the mandibles, measured in a straight line from the mid-point of the anterior clypeal margin to the mid-point of the occipital margin, in full-face view. in species where the occipital margin or the clypeal margin is concave, the measurement is 1497 xu, z.h. & n-n. yu — ant species from tibet taken from the mid-point of a transverse line spanning the anteriormost or posteriormost projecting points, respectively. hw-head width: the maximum width of the head in full face view, excluding the eyes. ci-cephalic index = hw×100 / hl. sl-scape length: the maximum straight-line length of the antennal scape excluding the basal constriction or neck close to the condylar bulb. si-scape index = sl×100 / hw. ed-eye diameter: the maximum diameter of the eye. pw-pronotal width: the maximum width of the pronotum in dorsal view. al-alitrunk length: the diagonal length of the alitrunk in profile view from the point at which the pronotum meets the cervical shield to the posterior base of the metapleuron. all measurements are expressed in millimeters. the type specimens are deposited in the insect collection, southwest forestry university (swfu), kunming, yunnan province, china. illustrations of the worker caste of v. acherdos bolton, v. bilongrudi (taylor), v. nahet bolton, and v. philax bolton, and illustrations of the queen caste of v. australis (wheeler), were drawn from the antweb images. illustrations of the worker caste of v. dryas bolton, v. philippina zettel et sorger, v. regina bolton, and v. xylochos bolton were drawn from the antbase images. and illustration of the worker caste of v. humboldticola zacharias et rajan was drawn from the figure of zacharias & rajan (2004). key to known species of vombisidris of the world based on the worker caste (all figures cited are in the appendix) 1 subocular groove entirely absent (papua new guinea) (figs. 1-3)……… ...........................................................................................v. bilongrudi (taylor) subocular groove at least present from mandibular insertion to the eye… …………………................................................................................................…….2 1498 sociobiolog y vol. 59, no. 4, 2012 2 subocular groove incomplete, running from mandibular insertion to the anteroventral margin of the eye, never beyond the eye (papua new quinea) (figs. 4-6)……………............................................................v. acherdos bolton subocular groove complete, running from mandibular insertion to the anteroventral margin of the eye, then passing through a shallow angle and continuing along the side to the lateroccipital margin………......................3 3 in profile view, dorsum of mesonotum with a pair of short vertical sharp teeth at each side. anterior 2/3 of the first gastral tergite longitudinally rugulose (indonesia) (figs. 7-9) ......................................... v. philax bolton in profile view, dorsum of mesonotum without a pair of vertical sharp teeth, at most with a pair of blunt prominence. first gastral tergite smooth… ………………………………………….........................................................................4 4 in profile view, petiolar node dome-like or roughly triangular, dorsum roundly convex, without distinctly differentiated dorsal face………………… ……….....…………………............................................................................…………5 in profile view, petiolar node roughly trapezoidal, with distinctly differentiated dorsal face, anterodorsal and posterodorsal corners more or less distinct……..................................................................……………………………..10 5 in profile view, metanotal g roove shallow but obviously de pressed…….............................................................................……………………….6 in profile view, metanotal groove entirely absent .................................... 7 6 in full face view, occipital margin weakly concave. anterior clypeal margin strongly convex. in profile view, propodeal spines weakly curved down, longer than propodeal dorsum. postpetiolar node evenly convex (china: tibet) (figs. 10-13)…………………………..….......................…v. tibeta sp. nov. in full face view, occipital margin weakly convex. anterior clypeal margin weakly convex. in profile view, propodeal spines straight, as long as propodeal dorsum. postpetiolar node strongly convex (china: hunan province) (figs. 14-15)………………..........................v. umbrabdomina huang et zhou 7 in profile view, petiolar node roughly triangular, dorsal corner distinct……………….......................................................................................…………8 in profile view, petiolar node dome-like, dorsal corner indistinct……………..............................................................................…………………..9 8 in profile view, propodeal spines about as long as propodeal declivity. postpetiolar node evenly convex. in dorsal view, lateral sides of petiolar node 1499 xu, z.h. & n-n. yu — ant species from tibet convex. head and alitrunk blackish brown, gaster lighter brown (india) (fig. 16)……………............................…………………………….v. occidua bolton in profile view, propodeal spines distinctly longer than propodeal declivity. postpetiolar node strongly convex. in dorsal view, lateral sides of petiolar node almost straight. head and alitrunk yellowish brown, gaster blackish brown (india) (fig. 17)…....................v. humboldticola zacharias et rajan 9 in full face view, occipital corners roundly prominent. eyes evenly convex. anterior clypeal margin without a middle notch (indonesia) (figs. 1820)………................................................................……………..…v. nahet bolton in full face view, occipital corners rounded. eyes strongly extruding. anterior clypeal margin with a middle notch (malaysia) (figs. 21-23)…… ……............................................................…………………..……..v. regina bolton 10 in profile view, metanotal groove strongly depressed and trenchlike…….......................................................................................…...………...……11 -in profile view, metanotal groove vestigial to absent, without a trench-like notch………..................................................................................................…….13 11 in profile view, promesonotum nearly straight, anterodorsal corner angled. propodeal spines about two times as long as propodeal dorsum (brunei) (figs. 24-26......................................................................……v. xylochos bolton -in profile view, promesonotum weakly convex, anterodorsal corner rounded. propodeal spines about as long as propodeal dorsum………………………………………......................................………………..……12 12 in full face view, lateral sides of head divergent behind eyes. in profile view, anterior peduncle shorter than the dorsal face of petiolar node. first gastral tergite finely superficially reticulate (indonesia) (fig. 27)…………… ………………………………….........................................…………..v. lochme bolton -in full face view, lateral sides of head nearly parallel behind eyes. in profile view, anterior peduncle about as long as the dorsal face of petiolar node. first gastral tergite almost completely smooth (malaysia) (fig. 2830)…………..............................………………………………..………v. dryas bolton 13 in profile view, anterior peduncle of petiole relatively short, shorter than the dorsal face of petiolar node, the dorsal face nearly straight (malaysia) (fig.31)……….................................................................…..…v. harpeza bolton -in profile view, anterior peduncle of petiole relatively long , about 1500 sociobiolog y vol. 59, no. 4, 2012 as long as the dorsal face of petiolar node, the dorsal face weakly convex……………………………………….....................................................…………..14 14 in profile view, anterodorsal corner of petiolar node bluntly ang led, hig her than posterodorsal corner, the latter rounded……………………......................………………………………………………......…..15 in p r o f i l e v i e w, b o t h a n t e r o d o r s a l a n d p o s t e r o d o r s a l corners of p etiolar no de bluntly prominent, at the same le vel……………..................……………………………………………………………………16 15 in full face view, occipital margin straight. in profile view, propodeal spines relatively short, shorter than or about as long as propodeal declivity. ventral face of petiole nearly straight. dorsal surface of alitrunk with short blunt hairs (australia) (figs. 32-34 ..................v.australis (wheeler) -in full face view, occipital margin evenly convex. in profile view, propodeal spines relatively long, longer than propodeal declivity. ventral face of petiole concave. dorsal surface of alitrunk with moderate long tapered hairs (australia) (figs. 35-37)….................…………….…v. renateae (taylor) 16 in full face view, occipital margin nearly straight. anterior clypeal margin weakly convex. dorsal surface of alitrunk with short blunt hairs. color yellow to lighter brown (indonesia) (figs. 38-40)…........ v. jacobsoni (forel) -in full face view, occipital margin weakly convex. anterior clypeal margin strongly convex. dorsal surface of alitrunk with moderate long tapered hairs. color blackish brown (philippines) (figs. 41-43)……………………… …………………………......................................……v. philippina zettel et sorger description of new species vombisidris tibeta sp. nov. figs. 10-13 holotype worker: tl 3.9, hl 0.83, hw 0.68, ci 82, sl 0.65, si 96, ed 0.23, pw 0.53, al 1.13. in full face view, head roughly rectangular, longer than broad. occipital margin weakly concave in the middle, occipital corners rounded. lateral sides weakly convex. mandibles triangular, masticatory margin with 3 apical teeth, a long deastema, and 2 blunt basal teeth. anterior clypeal margin strongly 1501 xu, z.h. & n-n. yu — ant species from tibet convex, posteriorly extended portion very broad, about 2.5 times as broad as frontal lobes. antennae 12-segmented, apices of scapes just reached to occipital corners, antennal clubs 3-segmented. frontal carinae fine and long, extended backward and close to the occipital corners. eyes large, situated slightly before the midpoints of the lateral sides. in profile view, subocular groove complete, running from mandibular insertion to the lateroccipital margin. promesonotum weakly convex, promesonotal suture vestigial on the dorsum. metanotal groove shallowly notched. propodeal dorsum straight and weakly slope down backward. propodeal spines strong and long, slightly curved down backward, about 1.8 times as long as propodeal dorsum. declivity weakly concave, about as long as propodeal dorsum. propodeal spiracle small and circular, high up on the side. propodeal lobes moderately developed, rounded at apices. petiolar node elongate and dome-like, both anterior and posterior faces gently slope down, without distinct dorsal face; anterior peduncle very short, spiracle situated at about the mid-length of the peduncle; ventral face weakly convex about in the middle, and weakly concave afterwards; anteroventral corner acutely toothed. postpetiolar node evenly convex, ventral face nearly straight. in dorsal view, lateral sides of pronotum roundly convex. lateral sides of mesonotum without prominence. propodeal spines weakly curved inward. lateral sides of petiole nearly straight, slightly widened backward; petiolar node longer than broad, length : width = 2:1. postpetiole wider than petiole, lateral sides weakly convex; postpetiolar node wider than long, width : length = 1.2:1. mandibles smooth and shining, sparsely punctured. head and alitrunk coarsely reticulate. clypeus and lateral sides of metanotum longitudinally striate. propodeal declivity longitudinally striate and densely finely punctured. petiole and postpetiole finely reticulate and densely finely punctured. gaster smooth and shining, basal costulae distinct, about 1/2 length of the postpetiole. dorsal surfaces of head and body with sparse suberect to subdecumbent tapered hairs and abundant decumbent pubescence. scapes with abundant suberect to subdecumbent tapered hairs and dense decumbent pubescence. tibiae with sparse subdecumbent tapered hairs and abundant decumbent pubescence. color brownish yellow, middle portion of gaster black, legs yellow. 1502 sociobiolog y vol. 59, no. 4, 2012 paratype workers: tl 4.0-4.1, hl 0.85-0.88, hw 0.70-0.73, ci 82-83, sl 0.63-0.65, si 89-90, ed 0.24, pw 0.53-0.58, al 1.15-1.25 (2 individuals measured). as holotype worker, but metanotal groove even more shallow, sting extruding in one worker. holotype worker: china: tibet, medog county, damu town, damu village, 1200m, collected from a canopy sample in the valley tropical rainforest, 2011.vii.20, xia liu leg., no.a11-3928. paratypes: 2 workers, with the same data as holotype worker, and both collected from canopy samples respectively, but nos. a11-3876, a113943. comparative notes: this new species is close to v. umbrabdomina huang et zhou, but in full face view, occipital margin weakly concave in the middle; anterior clypeal margin strongly convex; in profile view, propodeal spines weakly curved down backward, and distinctly longer than propodeal dorsum; postpetiolar node evenly convex. etymology: the new species is named after the type locality “tibet”. biological notes: according to the collection data, all the 3 workers of the species were collected from canopy samples separately. it seems this species lives in the valley tropical rainforest and forages on the plants. acknowledgments this study is supported by the national natural science foundation of china (nos. 31260521, 30870333) and the key subject of forest protection of yunnan province. we thank the following persons or institution for their special helps in this study: xia liu (phd candidate of forest protection, beijing forestry university, beijing ) who collected the type specimens with us; daniela magdalena sorger (natural history museum, vienna) who supplied a valuable paper and permitted to use the figures in her paper; robert w. taylor (australian national insect collection, canberra), barry bolton (the natural history museum, london), merry zacharias & priyadarsanan dharma rajan (ashoka trust for research in ecolog y and the environment, bangalore), and jian-hua huang & shan-yi zhou (guangxi normal university, guilin) who granted permission to cite figures in their papers; 1503 xu, z.h. & n-n. yu — ant species from tibet california academy of sciences (san francisco) who permitted the use of the images of v. acherdos bolton, v. bilongrudi (taylor), v. nahet bolton, v. philax bolton, and v. australis (wheeler) on the antweb.org (http://www. antweb.org/); martin pfeiffer (national university of mongolia, ulaanbaatar) who permitted the use of the images of v. dryas bolton, v. philippina zettel et sorger, v. regina bolton, and v. xylochos bolton on the antbase.net (http:// www.antbase.net/). references bolton, b. 1987. a review of the solenopsis genus-group and revision of afrotropical monomorium mayr. bulletin of the british museum (natural history) (entomolog y) 54: 263-452. bolton, b. 1991. new myrmicine ant genera from the oriental region. systematic entomology 16: 1-13. bolton, b. 1995. a new general catalogue of the ants of the world. harvard university press, 504 pp. cambridge, massachusetts. bolton, b. 2003. synopsis and classification of formicidae. memoirs of the american entomological institute 71: 1-370. bolton, b. 2012. an online catalog of the ants of the world. http://www.antcat.org/. forel, a. 1915. fauna simalurensis. hymenoptera aculeata, fam. formicidae. tijdschrift voor entomologie 58: 22-43. huang, j. & zhou, s. 2006. vombisidris bolton, a new record genus in china, with description of a new species. acta zootaxonomica sinica 31: 206-207. sorger, d. m. 2011. redescription and history of vombisidris jacobsoni (forel, 1915). revue suisse de zoologie 118: 149-155. taylor, r. w. 1989. australian ants of the genus leptothorax mayr. memoirs of the queensland museum 27: 605-610. wheeler, w. m. 1934. an australian ant of the genus leptothorax mayr. psyche 41: 6062. zacharias, m. & rajan, p. d. 2004. vombisidris humboldticola (hymenoptera: formicidae): a new arboreal ant species from an indian ant plant. current science 87: 1337-1338. zettel, h. & sorger, d. m. 2010. on the ants (hymenoptera: formicidae) of the philippine islands: iv. the genus vombisidris bolton 1991. entomologica austriaca 17: 37-44. appendix starts on page 1504 1504 sociobiolog y vol. 59, no. 4, 2012 figs. 1-3: worker of vombisidris bilongrudi (taylor); 1. head and body in profile view; 2. head in full face view; 3. alitrunk, petiole, and postpetiole in dorsal view. (drawn from antweb images, pilosity and sculpture omitted) figs. 4-6: worker of vombisidris acherdos bolton; 4. head and body in profile view; 5. head in full face view; 6. alitrunk and petiole in dorsal view. (drawn from antweb images, pilosity and sculpture omitted) figs. 7-9: worker of vombisidris philax bolton; 7. head and body in profile view; 8. head in full face view; 9. alitrunk and petiole in dorsal view. (drawn from antweb images, pilosity and sculpture omitted) figs. 10-13: worker of vombisidris tibeta sp. nov.; 10. head and body in profile view; 11. head in full face view; 12. mandible in dorsal view; 13. body in dorsal view. (pilosity and sculpture omitted) 1505 xu, z.h. & n-n. yu — ant species from tibet figs. 24-26: worker of vombisidris xylochos bolton; 24. head and body in profile view; 25. head in full face view; 26. alitrunk and petiole in dorsal view. (drawn from antbase images, pilosity and sculpture omitted) figs. 18-20: worker of vombisidris nahet bolton; 18. head and body in profile view; 19. head in full face view ; 20. alitrunk, petiole, and postpetiole in dorsal view. (drawn from antweb images, pilosity and sculpture figs. 21-23: worker of vombisidris regina bolton; 21. head and body in profile view; 22. head in full face view; 23. alitrunk, petiole, and postpetiole in dorsal view. (drawn from antbase images, pilosity and sculpture omitted) figs. 14-17: workers of vombisidris bolton; 1415: worker of v. umbrabdomina huang et zhou; 16: worker of v. occidua bolton; 17: worker of v. humboldticola zacharias et rajan; 14, 16, 17. head and body in profile view; 15. head in full face view. 1506 sociobiolog y vol. 59, no. 4, 2012 figs. 35-37: worker of vombisidris renateae (taylor); 35. head and body in profile view; 36. head in full face view; 37. alitrunk, petiole and postpetiole in dorsal view. (drawn from figures of taylor, 1989, pilosity and sculpture omitted) figs. 27-30: workers of vombisidris bolton; 27: worker of v. lochme bolton; 28-30: worker of v. dryas bolton; 27, 28. head and body in profile view; 29. head in full face view; 30. alitrunk and petiole in dorsal view. (27. cited from bolton, 1991; 28-30. drawn from antbase images; pilosity and sculpture omitted) figs. 31-34: worker and queen of vombisidris bolton; 31: worker of v. harpeza bolton; 32-34: queen of v. australis (wheeler); 31, 32. head and body in profile view; 33. head in full face view; 34. alitrunk, petiole, and postpetiole in dorsal view. (31. cited from bolton, 1991; 3234. drawn from antweb images; pilosity and sculpture omitted) 1507 xu, z.h. & n-n. yu — ant species from tibet figs. 38-40: worker of vombisidris jacobsoni (forel); 38. head and body in profile view; 39. head in full face view; 40. body in dorsal view. (drawn from figures of sorger, 2011, pilosity and sculpture omitted) figs. 41-43: worker of vombisidris philippina zettel et sorger; 41. head and body in profile view; 42. head in full face view; 43. body in dorsal view. (drawn from antbase images, pilosity and sculpture omitted) 893 negative effects of azteca ants on the distribution of the termite neocapritermes braziliensis in central amazonia by pedro a. c. lima pequeno1* & pauline o. pantoja1 abstract termites play important roles in tropical ecosystem functioning, and their evolutionary success has been linked to their defense mechanisms. however, microhabitat overlap with potential aggressors may constrain their distribution and thus, their environmental impacts on an ecological timescale. we investigated a possible negative effect of abundant generalist ants (azteca sp.) on the termite neocapritermes braziliensis. both taxa frequently build their nests attached to trees. we determined the densities of their active nests in 10 plots (250 x 10 m) systematically distributed over 5 km2 in central amazonia, brazil, and recorded their co-occurrence in individual trees. using generalized nonlinear modeling in a bayesian framework, we found good support for a negative effect of azteca’s nest density on n. braziliensis’. this effect conformed to a power law, and accounted for more than half of the variation in the termite’s nest density (r2 = 0.56). additionally, of all counted n. braziliensis mounds, only 1.08 percent was attached to trees also hosting azteca. such patterns may have arisen due to n. braziliensis’ inability to establish new nests within azteca territories, or predation by ants on established colonies of the termite. we suggest that even non-strictly termitophagous ant species may have important impacts on termite populations and, consequently, on their roles in nutrient cycling and ecosystem engineering. key words: bayesian inference; generalized nonlinear modeling ; population limitation; power law; social insect; species distribution. introduction population size is subject to different constraints, which is the basis for spatial variation in species abundance (krebs 2002). there has been a long instituto nacional de pesquisas da amazônia (inpa), programa de pós-graduação em ecologia. av. andré araújo 2936, petrópolis, caixa postal: 478. manaus, am, brasil. cep: 69011-970. *corresponding author. email: pacolipe@gmail.com 894 sociobiolog y vol. 59, no. 3, 2012 debate on whether populations are mainly driven by bottom-up (resources) or top-down forces (predation) (hairston et al. 1960; hunter & price 1992; polis & strong 1996; borer et al. 2006). it has been recently argued that under most circumstances animal populations are limited by food quality or quantity (white 2008). however, while resource availability may set an upper limit to prey populations, predation may prevent these populations from reaching carrying capacity (mcintosh et al. 2005), especially under the presence of generalist predators ( jiang & morin 2005). this might be reinforced when a predator is both generalist and more abundant than a particular prey, so that the predator may limit this prey’s populations without being limited in turn. nonetheless, it is likely that any such effect depends on the spatial scale under consideration (levin 1992). ants (hymenoptera: formicidae) and termites (blattaria: isoptera) are well known for their convergent social evolution, but they are also closely connected by trophic interactions. in fact, ants are generally more abundant than termites and, although few ant species are strictly termitophagous, most ants prey upon termites if allowed to (hölldobler & wilson 1990). this, coupled with the diverse array of termite defense strategies, has been taken as evidence for an evolutionary arms race between both taxa (noirot & darlington 2000). nevertheless, current ant-termite interactions remain poorly explored, especially in tropical rainforests, where they are remarkably abundant and diverse (scholtz et al. 2008). given the acknowledged roles of social insects in the functioning of these ecosystems, understanding interactions between these groups can contribute to the conservation of the environmental services they provide (chapman & bourke 2001). in the amazon rainforest, nests of several ant and termite species are conspicuous. namely, arboreal ants of the genus azteca (formicidae: dolichoderinae) are dominant in some areas. they build large carton, stalactite-like nests on trees and attend coccid hemipterans for honeydew (blüthgen et al. 2000), although they can be predatory too (dejean et al. 2009). similarly, neocapritermes braziliensis (termitidae: termitinae) is a common, although poorly studied, mound-building termite from the same areas. it feeds on rotting wood, and its mounds are often attached to the base of tree trunks (constantino 1992). therefore, encounters between n. braziliensis and azteca are likely. 895 pequeno, p.a.c.l. & p.o. pantoja — effects of azteca ants on termites being a cryptic forager, n. braziliensis should be inaccessible to predators most of the time and the combination between cryptic behavior and the physical structure of the mound may allow the termite to avoid predation by ants (buczkowski & bennett 2008). however, termite nests are potential targets for predation because they represent concentrated food resources. also, neocapritermes species have rather low soldier/worker ratios (around 1:100; krishna & araujo 1968), a trait likely to affect termite susceptibility to predation by ants (wells & henderson 1993). because azteca and n. braziliensis overlap in their nest substrates and can be locally abundant, their point interactions may result in agonistic patterns at larger scales. here, we investigate whether n. braziliensis and azteca are negatively associated at the levels of individual trees and nest densities across the landscape. material and methods the study was undertaken in a lowland rainforest area of 10 km2 between the purus and madeira rivers, amazonas state, brazil (3°41'9.85"s, 60°19'9.65" w). local topography is weakly undulated, and much of the substrate is waterlogged in the wet season. fieldwork was carried out from may to june 2009. we surveyed ten plots of 250 x 10 m, distributed along two parallel 5-km trails 1 km apart from each other; both trails contained five sampling plots also 1 km apart from each other. plots were oriented along topographic contour lines so as to reduce soil variation (as well as its correlates) within sampling units, thus maximizing between-plot variation (costa & magnusson 2010). in each plot, we counted active nests of both taxa, which were recognized by their typical architecture and residents. we also recorded whether each nest co-occurred with a nest of other taxon in the same substrate. samples were taken for taxonomic confirmation and deposited in the entomological collection of the national institute for amazonia research (inpa), manaus, brazil. since all surveyed ants were equivalent in terms of their nesting and foraging modes, they were pooled for analysis. following graphical inspection of the data, we modeled n. braziliensis nest density (y) as a power function of azteca nest density (x), assuming a log link (i.e. log(y) = β 0 .xβ1) and poisson distributed errors given the count 896 sociobiolog y vol. 59, no. 3, 2012 response. the model was fit in a bayesian framework, which allows direct probabilistic statements about parameters and produces exact results even for small sample sizes (link & barker 2010). we used uninformative priors (normally distributed with zero mean and standard deviation of 1000) so that inference was mainly driven by the data themselves (i.e. likelihood). three parallel markov chain monte carlo simulations (mcmc) were iterated 300,000 times each using the gibbs sampler, starting from random values and with a thinning rate of 10. the first 1000 realizations of each chain were discarded as burn-in. chain convergence was checked with trace plots, autocorrelation plots and the brooks-rubin-gelman statistic (brg). once validated, chains were pooled for inference (n = 89,700), and the means of the resulting posterior distributions were used as point estimates for model parameters. we assessed model adequacy by plotting residuals against fitted values and by squaring the correlation between model predictions and the observed response (r2) (zheng & agresti 2000). we refrained from formal inference regarding nest substrate overlap since our sample was biased towards the presence of nests. modeling was carried out in r 2.10.1 (r core development team 2009) coupled to winbugs 1.4 (spigelhalter et al. 2003), with the r package “r2winbugs” (sturtz et al. 2005) as interface between the two programs. results we counted 30 nests of azteca and 54 of n. braziliensis. these values correspond to local densities of 12 nests/ha and 21.6 nests/ha, respectively. the model for the relation between nest densities was fit as log(y) = 2.35 . x-0.49 (fig. 1) and accounted for more than half the variation in the response (r2 = 0.56). mcmc diagnostics indicated chain convergence (brg = 1) and no autocorrelation, whereas residual analysis revealed no clear patterns, thus validating the model. the 95% credible interval (i.e. within which there is a 95% chance the true parameter value is found) for the constant β 0 ranged from 1.98 to 2.69, and from -0.25 to -0.76 for the exponent β 1 . also, only two nests of n. braziliensis (1.08% of this species’ total) were attached to trees hosting azteca nests. 897 pequeno, p.a.c.l. & p.o. pantoja — effects of azteca ants on termites discussion this study provides evidence that arboreal azteca ants act as limiting factors of n. braziliensis in the study area. not only nests appear to strongly segregate among substrate trees, but there is also a negative, nonlinear relationship between nest densities across the landscape. presumably, these patterns emerge from ant-termite behavioral interactions at the level of individuals. hence the observed patterns suggest a scalable negative effect. we did not observe aggression by azteca against undisturbed termites during the present study, though we did observe the ants attacking both n. braziliensis workers and soldiers that were removed from their nests. impacts of ants on termites have been recorded several times in the field. for instance, jutsum et al. (1981) detected a negative association between fig.1. mean response (solid line) of neocapritermes braziliensis’ nest density to azteca’s, estimated as log(y) = 2.35 . x-0.49. dotted lines are 95% credible limits. 898 sociobiolog y vol. 59, no. 3, 2012 the presence of azteca sp. and nasutitermes corniger (syn. n. costalis) on trees in trinidad; leponce et al. (1999) argued that the presence of the ant crematogaster irritabilis was a limiting factor for arboreal termites in new guinea, causing a two-fold decrease in their local abundance; gonçalves et al. (2005) recorded a strong negative effect of predatory ants on arboreal termite activity; and dejean et al. (2007a) found that the nest densities of arboreal nasutitermes were about three times lower in plots occupied by the invasive ant pheidole megacephala than in those ones without this species. we note, however, that azteca has neither the morphological nor behavioral traits known from specialized, termite-hunting species (e.g. leal & oliveira 1995). thus, it is improbable that it normally raids termite nests. nonetheless, most ant taxa are known to prey on exposed termites in central amazonia (bandeira 1979), and our results suggest that even ant species that do not specialize in termitophag y (such as azteca spp.) may have strong negative effects on natural termite populations. the fact that this observation involves a termite species presumably well protected by the mound structure such as n. braziliensis, somewhat contradicts previous suggestions (mill 1982; buczkowski & bennett 2008). we propose two hypotheses to account for the observed patterns. azteca could affect n. braziliensis populations by inhibiting newly mated reproductive pairs from founding new colonies within its territories. different colonies of the same termite species can liberate their alates in synchronized events, and these attract many opportunist predators – including ants (mill 1983). in this scenario, azteca would prey upon n. braziliensis only sporadically, due to random encounters during swarming. this would suffice to produce segregation between nesting substrates. if the probability of encounters were a function of nest density, then higher densities of azteca nests would also reduce n. braziliensis’ nest founding success and thus, the termite’s nest densities themselves. our second hypothesis is that azteca could shift its foraging at least partially towards termites if they become locally abundant. arboreal ants exhibit strong preference for protein-rich baits in comparison to litter-dwelling ones, suggesting that their colonies are limited by nitrogen availability (kaspari & yanoviac 2001). thus, azteca may take advantage of the protein supply represented by n. braziliensis colonies. termite mounds can be cracked by rainfall, 899 pequeno, p.a.c.l. & p.o. pantoja — effects of azteca ants on termites tree fall or specialized predators (e.g. anteaters, armadillos, nest-raiding ants), which may allow generalist ants to raid nests. these two hypotheses are not mutually exclusive, though. why would n. braziliensis nest density relationship with azteca’s follow a negative power function? ants are territorial insects and thus are expected to compete for space (parr & gibb 2010). for instance, experimental work has revealed that higher nest densities intensify intraspecific conflict in the fire ant solenopsis invicta (adams & tschinkel 2001). accordingly, a shift in the strength of interactions from interto intraspecific ones as ant nest density increases may dampen the predatory impact of ants on termites and thus, the rate of decline in termite abundance. dominant arboreal ants engage in the defense of well-marked territories against conspecifics as well as other ant species, which can lead to the establishment of unoccupied zones or “no ant’s lands” between adjacent, competing colonies (dejean et al. 2007b). hence it may also be that these areas provide termites with refuges from the ant “crossfire” (e.g. by going unnoticed during earlier, more vulnerable stages of colony development). in addition, dominant ant species only have absolute control of the immediate nest neighborhood; there is plenty of within-territory space for opportunistic foraging (andersen 2008) and, perhaps, termite nest founding and growth. the present study suggests a role for non-strictly termitophagous ants as effective limiting factors of natural termite populations at multiple scales. it has been observed that temporal gaps in termite activity can be a function of predation risk by ants (korb & linsenmair 2002; desouza et al. 2009). thus, the limiting effects of ants on termite populations may further impair termite roles in ecosystem functioning such as nutrient cycling and ecosystem engineering. however, further study of the mechanisms creating the observed patterns is necessary. we encourage the application of bayesian inference in future studies, as they could build on informative priors (i.e. posterior distributions as estimated here) to strengthen the robustness of their conclusions (mccarthy & masters 2005). acknowledgments we thank albertina lima and fabricio baccaro for their support during field work. f. baccaro also helped in identifying the ants and improving the 900 sociobiolog y vol. 59, no. 3, 2012 manuscript. william magnusson provided valuable suggestions on an earlier version of the manuscript. p. a. c. lima pequeno received a posgrad scholarhip from the fundação de amparo à pesquisa do estado do amazonas (fapeam) during this study, and p. o. pantoja received a scholarship from the coordenação de aperfeiçoamento de pessoal de 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henderson. 1993. fire ant predation on native and introduced subterranean termites in the laboratory: effects of high soldier number in coptotermes formosanus. ecological entomolog y 18(3):270–274. zheng, b. & a. agresti. 2000. summarizing the predictive power of a generalized linear model. statistics in medicine 19(13):1771–1781. 605 discovery of two aleocharine staphylinid species (coleoptera) associated with coptotermes formosanus (isoptera: rhinotermitidae) from central japan, with a review of the possible natural distribution of c. formosanus in japan and surrounding countries by munetoshi maruyama1, taisuke kanao2 & ryûtarô iwata3* abstract two termitophilous staphylinid beetles, japanophilus hojoi maruyama & iwata and sinophilus yukoae maruyama & iwata (staphylinidae: aleocharinae) are known to inhabit nests of a termite species, coptotermes formosanus shiraki (rhinotermitidae). previously, these beetles had only been collected from c. formosanus nests in the nansei archipelago, between kyushu and taiwan. here, were report their existence in nests in the coastal regions of wakayama prefecture, central japan. a review of published records strongly supports the hypothesis that nansei archipelago, kyushu and the coastal areas of kii peninsular of honshu, constitute, at least, part of the natural geographic range of c. formosanus. introduction the formosan subterranean termite, coptotermes formosanus shiraki (rhinotermitidae) is regarded as the world’s most economically damaging termite species (su & tamashiro, 1987). via accidental introductions, this species has expanded its range in warm temperate areas, and is currently inflicting significant destruction in urbanized parts of the southern united states (la fage,1987; woodson et al. 2001). for an effective control strateg y, precise determination of a pest species’ natural range is important, since it 1the kyushu university museum, hakozaki 6-10-1, fukuoka, 812-8581 japan 2entomological laboratory, graduate school of bioresource and bioenvironmental sciences, kyushu university, fukuoka, 812-8581 japan 3department of forest science and resources, college of bioresource sciences, nihon university, fujisawa, 252-0880 japan *corresponding author: iwata@brs.nihon-u.ac.jp 606 sociobiolog y vol. 59, no. 3, 2012 is here that natural enemies may exist that may be exploited for biological control measures (iwata, 2000). although c. formosanus is thought to have originated in east asia, its natural distribution has not been fully mapped. in particular, it is not known whether japanese populations from the southwest part of honshu, shikoku, kyushu, and nansei archipelago (islands between kyushu and taiwan) are natural or introduced. termitophiles are organisms that live in close association with termites; they are often “species-specific”, as natural enemies are (iwata, 2000). the presence of termitophiles within a termite colony may be a good indicator that the colony is within its natural geographic range: kistner, in an elaboration of emerson’s (1955) idea, argued that termite dispersal depends largely on short-range movement of winged reproductives; consequently, seas and oceans pose a barrier to the spread of termites, and also the movement of their termitophilous guests (kistner, 1969, p. 538). the presence at a certain locality of species-specific inquilines, e.g. termitophilous staphylinids, may thus help delineate the natural distribution of a given termite species. conversely, evidence that a colony has been introduced outside of its natural distribution may be the complete absence of termitophiles within nests at such localities. herein, we call this hypothesis the “emerson–kistner principle” (ekp). importantly, kistner (1985) described sinophilus xiai kistner (aleocharinae: termitohospitini), the first termitophilous staphylinid species specifically associated with c. formosanus. sinophilus xiai was recovered from nests in zhejiang and guangdong provinces, south china. based upon ekp, he designated this area of south china the putative origin (the center of the natural distribution) of c. formosanus. later, maruyama and iwata (2002a) described two further termitophilous staphylinids, again, specifically associated with c. formosanus (two species and one genus): sinophilus yukoae from iheya, ishigaki and iriomote islands (nansei archipelago), and japanophilus hojoi from yakushima, tokara-nakanoshima and tokara-suwanosejima islands. they proposed that the natural distribution area of c. formosanus should be expanded to include this japanese archipelago. furthermore, they noted the similarity of japanophilus with an unidentified staphylinid collected from a c. formosanus nest in fukuoka prefecture, kyushu, japan by nawa (1914, writing under the pseudonym “konchû-ô”). this implied the additional inclusion of kyushu in the natural range of c. formosanus, but until the present paper, 607 maruyama m. et al.— natural distribution of c. formosanus . there have been no bona fide records of termitophilous staphylinid species from c. formosanus colonies on mainland japan (excluding kyushu). other inquilinous insects associated with c. formosanus likely fall into the category of “termitariophiles”: species having a weaker, often facultative relationship with termites, in which the nest may be used simply as shelter and food. such species include madrasostes kazumai ochi, johki & nakata (coleoptera: ceratocanthidae), from tokara-nakanoshima is. and other islands (e.g., iwata et al., 1992; kôgata, 1999), and lorelus sasajii masumoto & akita (coleoptera: tenebrionidae), from iriomote, ishigaki and amamioshima islands (masumoto & akita, 2001). it is not clear if these termitariophiles are host-specific; for example, m. kazumai has also been found in a nest of hodotermopsis sjostedti holmgren (termopsidae) (maruyama & iwata, 2002b). hence, these taxa are less useful guides for judging the natural range of c. formosanus according to ekp. as yet, there have been no records of termitophiles from c. formosanus nests in southern parts of usa, hawaii and other regions clearly outside the natural geographic range. this observation would seem to further substantiate ekp. here, we present new records of two japanese termitophilous termitohospitini species (j. hojoi and s. yukoae) found in c. formosanus nests from the coastal areas of kii peninsula (wakayama prefecture), central mainland japan, a region located far from the nansei archipelago. we discuss the implications of these records for the biogeography and biological control of c. formosanus. records the collection data are as follows: japanophilus hojoi, 2 exs. (fig. 1): uragami, nachi-katsuura, wakayama pref., 1.vi.2009, leg. katsuo oya. j. hojoi, 2 exs.; sinophilus yukoae, 4 exs. (fig. 2): tahara, kushimoto, wakayama pref., 24.x.2009, leg. katsuo oya. j. hojoi, 2 exs.; s. yukoae, 8 exs.: urakatsu, nachi-katsuura, wakayama pref. x. 2009, leg. katsuo oya. figure 3 incorporates all the localities of the termitophiles and termitariophiles mentioned in the present paper. 608 sociobiolog y vol. 59, no. 3, 2012 figs. 1, 2. two termitophilous staphylinids associated with coptotermes formosanus. 1. japanophilus hojoi maruyama & iwata. 2. sinophilus yukoae maruyama & iwata. 609 maruyama m. et al.— natural distribution of c. formosanus . discussion validity of the ekp (emerson–kistner principle) estimating natural geographic ranges using the ekp comes with some caveats. for example, the ekp may be violated by cases in which an entire nest of termites, or soil containing live termites, has been transferred along with any resident termitophiles. one case that at first appears to contradict ekp comes from snyder (1934), who reported a human-aided introduction of a european termite species (reticulitermes lucifugus) to massachusetts, usa, and mentioned a nest in which “beetle termitophiles” were found. since snyder did not describe the termitophilous species, it is unclear whether the beetle was of european or north american origin. it is possible that a fig. 3. localities in japan of the termitophiles and termitariophiles associated with coptotermes formosanus. a, nachi-katsuura, wakayama pref.; b, kushimoto, wakayama pref.; c, sata-misaki, kagoshima pref.; d, yaku island; d, e: naka-no-shima island; f: suwanose island; g, amamiô-shims -shima : h, kakeroma island; i, iheya island, j, okinawa island; k, ishigaki island; l,iriomote island. circle, sinophilus yukoae; triangle, japanophilus hojoi; square, madrasostes kazumai kazumai; cross, m. kazumai hisamatsui, diamond, lorelus sasajii. 610 sociobiolog y vol. 59, no. 3, 2012 north american termitophile host-shifted from american reticulitermes to the introduced r. lucifugus. indeed, the establishment of a european termitophilous beetle has not been reported from massachusetts since then. there have been cases in japan where kistnerium japonicum naomi & iwata, a termitophilous staphylinid (aleocharinae: mesoporini) with rather a low host-species specificity, changes its host from the major host, reticulitermes speratus (kolbe) to c. formosanus and even to an ant species (iwata et al., unpublished). therefore, snyder’s (1934) case does not “disprove” ekp; however, until thorough phylogeographic studies are carried out, employing genetic markers, it remains a largely conceptual hypothesis, which, although useful, lacks solid empirical support. literature survey on reticulitermes spp. and coptotermes formosanus as endemic to japan amongst the japanese termite taxa, reticulitermes spp. (r. speratus and its allies; excluding r. kanmonensis, a putative introduced species: takematsu & morimoto, 1999) are, based on molecular evidence, thought to have all derived from an east china stock in the early pleistocene (park et al., 2006). notably, one north american representative [r. flavipes (kollar)] of this holarctic termite genus engages in a very distinct symbiosis with a highly derived termitophilous staphylinid trichopsenius (aleocharinae: trichopsenini), in which the beetle is provided with cuticular hydrocarbon compositions identical to those of the host (howard et al., 1980). such a reticulitermes– trichopsenius association also exists in japan (iwata & naomi, 1998), a fact that firmly verifies the resident reticulitermes spp. (excluding r. kanmonensis) as endemic to japan. as for the populations of c. formosanus in japan (ogasawara islands, south kanto district, south chubu district, south kinki district, south chugoku district, shikoku, kyushu, nansei archipelago), ikehara (1966), in discussing the termite biogeography and fauna of nansei archipelago, did not mention whether japanese c. formosanus is endemic or introduced, but suggested a recent expansion, due to the fact that it is not distributed in daito island, an island off the nansei archipelago. bess (1970) included japan in its geographic range, without including it in its artificial range, and there have been other statements that japan comprises part of its natural range (gay, 611 maruyama m. et al.— natural distribution of c. formosanus . 1969; coaton & sheasby, 1976). however, since su & tamashiro (1987) explicitly illustrated the expansion history of c. formosanus as an invasion from south china into japan in the 16th century (stating “300 years have elapsed since its introduction to japan”), almost all termitologists have followed this view (e.g. mori, 1987). ohmura & tokoro (2003) even mentioned “goshuin-sen bôeki” (shogunate-authorized ship trade with china) as the cause of its introduction to japan. yet, the introduction hypothesis has never been substantiated by any data. coptotermes formosanus: dna analyses more recently, vargo et al. (2003), under an assumption that japanese c. formosanus consists of introduced populations, investigated the microsatelite markers of populations from the mainland and gotô-rettô (nagasaki prefecture, kyushu district). they obtained no evidence of a “bottle-neck effect” due, they stated, to “the more than 300-year history of this species in japan”, but found that “the japanese populations contained 26% fewer alleles than did a native population from guangdong province, china, at the six microsatellite loci studied”. they ascribed this fact to the introduction history. these results seem somewhat equivocal, and the interpretation ad hoc. the genetic diversity of c. formosanus in hawaii (an introduced population) is known to be extremely low (broughton & grace, 1994), yet, that of populations in south china is also low, despite the fact that this species is endemic there. this may be due to human activity and disturbance (fang et al., 2008). on the other hand, a phylogenetic study using mitochondrial coii dna from the us populations also revealed an extremely low genetic diversity, giving the conclusion that at least two c. formosanus introductions to the mainland of usa had occurred (austin et al., 2006). such low genetic diversity is common in populations from taiwan (li et al., 2009) and japan (a. yamada, pers. comm.). coptotermes formosanus: colony reproduction analyses lenz & barrett (1982) and lenz et al. (1986) stated that in the genus coptotermes, as well as in the other rhinotermitid genera, alate swarming is generally followed by new colony foundation by the royal pair, and that this is the main reproduction method in the natural range of the species. brachypterous neotenic formation upon removal of the primary queen, leading 612 sociobiolog y vol. 59, no. 3, 2012 to reproduction and satellite nest formation, is an alternative method, and this latter process is thought to predominate in recently-introduced populations (lenz & barrett, 1982). in studying c. formosanus, husseneder et al. (2005) classified colonies into two categories, namely “simple family” (colony headed by a single pair of outbred reproductives) and “extended family” (colony headed by low numbers of multiple kings and/or queens that were likely the neotenic descendants of the original colony). however, no obvious relationship could be found between the mode of colony reproduction and the invasion success of this species. in investigating colony reproduction in c. formosanus from south china, hawaii and southern mainland of usa, husseneder et al. (2008) found that, surprisingly, in only south china did all colonies contained multiple inbreeding neotenics; in hawaii and southern mainland of usa, only about half to two thirds of colonies contained neotenics, the rest being founded by royal pairs. these results together suggest that the theory proposed by lenz & barrett (1982) cannot be applied to c. formosanus. however, it may be that it is valid for a limited time window after the introduction of c. formosanus. coptotermes formosanus in japan and adjacent areas: introduced or endemic? molecular biological studies over last 10 years have described the biogeographical and genetic relationships among c. formosanus populations from china, japan, hawaii and mainland usa (e.g. wang & grace, 2000; husseneder & grace, 2001; vargo et al., 2003). nevertheless, the origin and the natural distribution range of c. formosanus remain unclear. data on the distribution of japanophilus hojoi and sinophilus yukoae associated with c. formosanus (kii peninsula, kyushu and nansei archipelago), combined with ekp appear to support the idea that kyushu and kii peninsula of honshu comprise part of the natural geographic range. our conclusion thus differs from that of su & tamashiro (1987), who suggested that c. formosanus was introduced to japan from china relatively recently. what about the existence of c. formosanus in other regions of japan and surrounding countries? populations of c. formosanus in south kanto districts, including yokosuka (kanagawa pref.) and tateyama (chiba pref.), may indeed be the result of introduction, since there are no prewar distribution 613 maruyama m. et al.— natural distribution of c. formosanus . records (iwata, 2004). likewise, c. formosanus was not distributed in the ogasawara islands in prewar times, and the outbreak of swarming activities after 1976 clearly suggests the artificial introduction by the occupying us forces (minamiyama, 1978; mori, 1979). in taiwan, c. formosanus populations are considered endemic, forming the center of the species’ distribution, as the specific epithet indicates, and are genetically most allied to japanese populations (li et al., 2009). its presence in lanyu island, an accompanying island of taiwan, is thought to be via introduction from mainland taiwan (li et al., 2008). in the northern part of vietnam, as an extension from the putative distribution center, south china, c. formosanus was found exclusively in cultivated land and residential areas (vu et al., 2007), again suggesting the likelihood of artificial introduction. yaga (1997) stated that okinawa (the southern part of the nansei archipelago) has a history of termite control dating back more than 2500 years, although he did not describe this in detail. this strongly suggests that in this region, the threat of c. formosanus has been recognized since prehistory. due to global warming, c. formosanus may well expand its range in japan (iwata, 2004); indeed the species was recently recorded in the metropolitan area around tokyo (e.g. tomioka et al., 2009). in this case, host-specific termitophiles, like japanophilus and sinophilus, may be unable to expand their ranges as rapidly as their host, as the ekp predicts. in conclusion, we propose that the ekp is a viable method for determination of the natural distribution of c. formosanus. future detailed surveys of host-specific termitophiles in japan, china and surrounding countries will further resolve the natural geographic range of this species. such work may prove of great use in efforts to effectively control this notorious pest species in an ecologically-sound manner. acknowledgments the authors thank mr. katsuo oya, oya termite research institute, wakayama, japan, for providing us with the termitophilous staphylinids reported here, and also permitting us to the present paper. our sincere thanks are also due to dr. joseph parker, columbia university, and the late dr. kunio tsunoda, kyoto university, for their critical readings of the manuscript. this paper is dedicated to the memory of the latter, dr. k. tsunoda, who greatly contributed to the control technolog y of termites. 614 sociobiolog y vol. 59, no. 3, 2012 references austin, j.w., szalanski, a.l., scheffrahn, r.h., messenger, m.t., mckern, j.a. & gold, r.e. 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[in japanese.] doi: 10.13102/sociobiology.v62i1.18-22sociobiology 62(1): 18-22 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 moth flies (diptera: psychodidae) collected in colonies of the fire ant solenopsis virulens (smith) (hymenoptera: formicidae), with description of two new species introduction association between diptera and formicidae (hymenoptera) has been reported for many species of phoridae (borgmeier, 1938; brown, 2009), some microdontinae (syrphidae) (reemer, 2013) and in nematocerous diptera of the families culicidae, ceratopogonidae, cecidomyiidae and sciaridae (kistner, 1982, evenhuis et al., 2007). the association between psychodidae (diptera) and ants was only recorded in three psychodid species, each one of which in distinct subfamilies: adults of lutzomyia texana (dampf) (phlebotominae) in atta texana buckley nest, southern u.s.a. (young & perkins, 1984); pupa of nemapalpus mopani de león (bruchomyiinae) in eciton hamatum (fabricius) nest, guatemala and ecuador (kistner et al., 2001) and larvae of alepia longinoi quate & brown (psychodinae) in nests of two species of the genus azteca forel, costa rica (quate & brown, 2004). a recent diptera collection in ant nests in the atlantic rain forest biome in the state of bahia, brazil, has resulted in the abstract three species of psychodidae were collected in colonies of the fire ant solenopsis virulens (smith, 1858) (hymenoptera: formicidae), in an area of atlantic forest on the south of bahia state, brazil. two of them are new to science, herein described: trichomyia myrmecophila sp. nov. and quatiella truncata sp. nov., the last one also occuring in other states of northeastern brazil (paraíba and ceará) and in the amazonian region. a new record of trichomyia annae bravo 2001 is given. sociobiology an international journal on social insects tpl pereira1, f bravo1, mx araújo2, d cordeiro1, c chagas3, jhc delabie4 article history edited by edilberto giannotti, unesp, brazil received 17 september 2014 initial acceptance 12 february 2015 final acceptance 11 march 2015 keywords neotropical region, taxonomy, psychodid, myrmecophile. corresponding author thalles platiny lavinscky pereira universidade estadual de feira de santana laboratório de sistemática de insetos av. transnordestina, s/nº, 44036-900, feira de santana, bahia, brazil e-mail: thallesplp@gmail.com discovery of three species of psychodidae associated with colonies of solenopsis virulens (smith). despite of its wide distribution, being found in forest areas of northeastern south america, from bolivia to the state of bahia at atlantic coast (trager, 1991), there were already no report of myrmecophile interactions for this fire ant. herein we describe two new species of moth flies, and also record for the first time trichomyinae species associated with ant nests. material and methods the adults of psychodidae were collected from fragments of 12 nests of solenopsis virulens found on fallen tree trunks and at the base of trees. approximately 2000 cm³ of the nests were collected, carried to the laboratory in enclosed plastic container and accommodated in an emergency trap, during the period of 15 days. the moth flies were fixed in 70% ethanol, then cleared with 10% sodium hydroxide and mounted in canada balsam. the terminology of the morphological descriptions follows cumming research article ants 1 universidade estadual de feira de santana, feira de santana, ba, brazil 2 universidade federal do paraná, curitiba, pr, brazil 3 instituto nacional de pesquisas da amazônia, manaus, am, brazil 4 cepec-ceplac & universidade estadual de santa cruz, ilhéus, ba, brazil sociobiology 62(1): 18-22 (march, 2015) 19 and wood (2009). the moth flies specimens are deposited in the prof. johann becker entomology collection at the zoological museum of the universidade estadual de feira de santana, bahia, brazil (mzfs) and in the coleção de invertebrados do instituto nacional de pesquisas da amazônia, manaus, amazonas, brazil (inpa). specimens of the host ant were deposited in the myrmecology laboratory collection (cpdc) of the cocoa research center, at ilhéus, bahia, brazil results trichomyia annae bravo, 2001 material examined: brazil, bahia: igrapiúna, reserva ecológica da michelin, 13º50´s 39º10´w, 1 male, 22.v.2013, pereira, t.p.l. col. (mzfs). from a nest of s. virulens. distribution: brazil, bahia: serra da jibóia (bravo, 2001), igrapiúna (new record). trichomyia myrmecophila araújo & bravo, sp. nov. type material: brazil, bahia state, ituberá, reserva ecológica da michelin, 13º50´s 39º10´w, 22.v.2013, holotype male, pereira, t.p.l. col. (mzfs). from a nest of s. virulens. etymology: the new species is named after its discovery in the nest of the fire ant solenopsis virulens. diagnosis: base of r5 and r-m unsclerotized; gonocoxites fused with two internal expansions, the dorsal longer than ventral one, straight, narrow ending in a point and the dorsal ending in a bifurcated apex; gonocoxite with one pair of arms and with a strong setae in the interior margin of this structure; cercus pyriform, wider basally narrower apically with two distinct apical setae. description: male. head subcircular. antenna incomplete in the studied specimen; scape subcylindrical; pedicel subspherical (fig 1); basal flagellomeres pyriform and eccentric (fig 1); ascoids c-shaped with the same length of flagellomere (fig 4). palpus three segmented; palpus formula 1.0:0.5:0.6 (fig 2); first segment with sensilla in depressed pit on inner side (fig 2). wing: r5 with base unsclerotized; r-m unsclerotized, m-cu present and sc-c unsclerotized (fig 3). male terminalia: hypandrium and gonocoxites fused with two internal expansions, the dorsal longer than ventral one, straight, narrow ending in a point and the dorsal ending in a bifurcated apex; (fig 5). internal margin of gonocoxite with strong bristle (fig 6). dorsal arm of gonocoxite narrow with long setae along the margins and in the apex (fig 5). gonostylus slightly sclerotized, articulated ventrally to gonocoxite, bare, longer figs 1–6. trichomyia myrmecophila araújo & bravo sp. nov. 1. scape, pedicel and basal flagellomeres. 2. palpus. 3. left wing; 4. flagellomeres with ascoid. 5-6. male terminalia, dorsal 5. gonocoxites, gonostyli and ejaculatory apodeme 6. cerci, epandrium and hypropoct. abbreviations: agx= arm of gonocoxite, cerc= cercus, ej apod = ejaculatory apodeme, epand= epandrium, gst = gonostylus, hyprct = hypoproct. tpl pereira et al moth flies collected in colonies of the fire ant solenopsis virulens20 than gonocoxite (fig 5). aedeagus bifid fused apically (fig 5). ejaculatory apodeme, narrow, 1.5 times the length of gonostylus. epandrium subretangular (fig 6). cercus pyriform, wider basally narrower apically with two distinct apical setae (fig 6). hypoproct with apical micropilosity (fig 6). female: unknown. distribution: known only from the type locality, ituberá, bahia, brazil. remarks: no other neotropical species of trichomyia with one pair of arms have a pyriform cercus with two apical setae. quatiella truncata chagas & cordeiro sp. nov. type material: brazil. holotype male, bahia state, igrapiúna, reserva michelin, 13º50´s 39º10´w, 22.v.2013, pereira, t.p.l. col, (mzfs). from nest of s. virulens; paratypes: 42 males, pará state, santarém, chácara nossa senhora de nazaré, km 138, cdc – mata, 27.xi.1998, freitas, r., naiff, r.d., silva, f.l. cols. (21 males inpa, 21 males mzfs); 1 male, pará, santarém, estação do aeroporto, km 13, comunidade santa maria, chácara n. sr. nazaré, ponto 8, mata alterada, 25.xi.1998 (inpa); 2 males, amazonas state, manacapuru, cajatuba, ponto 4, cdc, 21.iv.1998, lote: 01 04, col. queiróz, r., nonato, r. cols (1 male inpa, 1 male mzfs); 2 males and 1 female paraíba state, areia, brejo paraibano, (1 male 06º 58’ s 39º 44” w), 25-29.ix.2011, figs 7-18. quatiella truncata chagas & cordeiro sp. nov. 7. head, anterior. 8. head, posterior. 9. scape, pedicel and basal flagellomeres. 10. flagellomeres. 11. palpus. 12. labellum. 13. wing. 14-17. male terminalia. 14. lateral view. 15. ventral view: epandrium, epiproct and hipoproct. 16. dorsal view: gonocoxites, gonostyli and aedeagal complex. 17. aedeagal complex in detail, lateral view. 18. female terminalia on ventral view showing subgenital plate and internal structures. sociobiology 62(1): 18-22 (march, 2015) 21 nascimento, e. & silva-neto, a. cols (mzfs); 1 male ce [ceará state], parque nacional ubajara (03º 50’ 21” s 40º 54’ 59” w), 2229.x.2011, nascimento, e & silva-neto, a. cols (mzfs). etymology: latin truncata, refers to the truncate apex of aedeagus. diagnosis: male cercus slightly curved, with one apical long tenaculum, around 0.7 length of cercus; gonocoxites almost contiguous; gonocoxal apodemes forming a ventral sheath to aedeagus; aedeagus straight, long and slender, with truncate apex; female subgenital plate wider than long, bilobed, with a pair of strongly sclerotized round structures. description: male: head semicircular in frontal view; vertex higher than width of eye bridge (fig 7); occipital foramen in upper position; frons hair patch extending to first row of eye bridge; eye bridge with 4 facet rows, separated by 0.2 x facet diameter in its closest point; vertex hair patch extending continuously to posterior margin of eyes (fig 8); 7 supra-ocular setae, nearly continuous with the 4 larger occipital alveoli; interocular suture with short medial spur; clypeus wider than long with 4–5 larger lateral alveoli; frontoclypeal suture present; antenna with cylindrical scape, little longer than subspherical pedicel (fig 9), and 14 flagellomeres, 12– 14 reduced, 11–13 without necks, all separated, 14 conically shaped (fig 10); ascoids y-shaped (fig 9); palpal formula 1.0:1.7:2.0:2.8 (fig 11); labellum compact, fleshy, with 3 spines on inner margin and 5 latero posterior setae (fig 12). wing (fig 13): wing membrane bare; sc vein short; r1 ending on the same of cua2; radial fork apical to medial fork, both complete, medial fork weakened. leg: distitarsi without apical projection. male terminalia: cercus entirely pilose (fig 14), slightly curved, about same length of epandrium, with one apical tenaculum, one or two subapical setae, three subapical papilla and a group of short setae (probably pilose papilla) on inner margin at base; tenaculum long, around 0.7 length of cercus; epandrium with one small foramen (fig 15); epiproct simple and short, pilose; hypoproct wider than long, pilose; hypadrium indistinguishable; gonostylus as long as gonocoxites, with several aparsely distributed short setae and one thin and long setae near apex (fig 16); gonocoxites almost contiguous; gonocoxal apodemes ventral to aedeagus, surrounding the aedeagus to form a dorsal sheath to aedeagus; aedeagus straight, long and slender, with truncate apex; aedeagal apodeme simple and short, 0.4 length of aedeagus (fig 17). female: head, antenna and wing same as male. genitalia as figured; subgenital plate slightly wider than long, pilose, bilobed with lobes well delimited (fig 18); a pair of round sclerotized structures interior to the subgenital plate. distribution: brazil (amazonas, pará, bahia). remarks: this species is very similar to quatiella cubana, but they can be differentiated by genitalic characters, as follows: in quatiella truncata sp. nov. males present a long dorsal sheath to aedeagus (which may be fused parameres) and females presents subgenital plate wider than long with well delimited apical lobes and round internal structures, while in q. cubana the males have no dorsal sheath to aedeagus and the subgenital plate of female is slightly longer than wide with a pair of saculiform structures internally. the genus quatiella was already considered a subgenus and a synonym of philosepedon eaton, 1904 by duckhouse (1973, 1974, respectively), but was accepted as a genus by others (vaillant, 1973, 1974, 1990, 1991; ježek, 1985; wagner & masteller, 1996; ibánez-bernal, 2004). this genus is similar to feuerborniella (ježek, 1985 even suggested they could be synonyms) by having eyes proximate, bulbous labellum with short spines on inner margin, 14 flagellomeres, 12-14 reduced, ascoids y-shaped, male genitalia with cercus bearing one tenaculum at apex. but they can be differentiated by male terminalia characters. in quatiella, the tenaculum is longer, at least half the length of cercus, the hipoproct is subretangular, much wider than long, and the gonocoxites are not separated by the hypandrium. in contrast, feuerborniella has a shorter tenaculum, not reaching half of cercus, hipoproct projecting posteriorly, and gonocoxites separated by the hypandrium. the paratypes of quatiella truncata were collected with malaise and cdc light traps and not found emerging from a fire ant nest, as the holotype. however, at least the specimens from the states of amazonas and pará were collected in a forest area, as the holotype, where s. virulens is known to occur (trager, 1991). discussion this is the first study about myrmecophilous interactions with fire ant solenopsis virulens. however, inside fire ants geminata group (trager, 1991) – in the first study to survey myrmecophilous species associated with nests of solenopsis saevissima richteri forel, collins & markin (1971) cataloged fifty-two species of arthropods, including crustaceans, millipedes, arachnids, and eight orders of hexapoda . to explain the coexistence and permanence of these insects within the nests vander meer et al. (1989) discovered that many of the insects myrmecophilous have similar or identical cuticular hydrocarbons to those of their host. although we can not affirm what kind of association the moth flies described here present with the ant nests, kistner et al. (2001) and quate and brown (2004) notes on psychodidae larvae inside ant nests show that moth flies may be capable of using this type of habitat for breeding. in a recent work (mendes et al, 2009), a new genus of nicoletiidae (zygentoma), allotrichotriura was discovered in colonies of solenopsis saevissima, demonstrating the need to further study the biodiversity of those insects that live in nests of ants. acknowledgments we convey our thanks to the graduate and post-graduate students who assisted in data collection of this study. to coordination for the training of higher education personal tpl pereira et al moth flies collected in colonies of the fire ant solenopsis virulens22 (capes) for the fellowship to thalles platiny lavinscky pereira. the michelin ecological reserve (rem) supported this study. the authors acknowledge the support of the secti/fapesb-cnpq pronex program pnx0011/2009. references borgmeier, t., (1938). alguns phorideos myrmecophilos de costa rica e do brasil e quatro espécies novas de melaloncha brues (dipt. phoridae). revista de entomologia (río de janeiro). 9(1/2), 39-53. bravo, f. 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(ed.). catalogue of the americas south of the united states. papeis avulsos do dep. zoologia, são paulo, são paulo, pp. 1–29. duckhouse, d. a. (1974). redescription of the neotropical psychodidae (diptera, nematocera) described by knab, dyar and coquillet. journal of entomology series b, 42(2): 141-152. evenhuis, n.l., sarnat, e. & tokota` a, m. (2007). new genus and species of sciarid ant guest from fiji (diptera: sciaridae) with an annotated checklist of fiji sciarids.. fiji arthropods ix. edited by neal l. evenhuis & daniel j. bickel. bishop museum occasional papers, 94: 3-10. ibañez-bernal, s. (2004). notes on the known species of trichomyia haliday of mexico, with the establishment of a synonymy and the description of a new species (diptera: psychodidae). zootaxa, 523: 1-14. ježek, j. (1985). contribution to the knowledge of a new subtribe trichopsychodina (diptera, psychodidae) from czechoslovakia. acta museu nationalis pragae, 40: 65-92. kistner, d.h. (1982). social insects bestiary, p. 1-244. in: h.r. hermann (ed.). social insects. new york, academic press, 437p. kistner, d. h., disney, r. h. l. & williams, p. (2001). larval and pupal bruchomyiinae (diptera, phlebotomidae) from army ant colonies (hymenoptera, formicidae). sociobiology, 37: 237-238. mendes, l. f., fox, e. g., solis, d. r., & bueno, o. c. (2009). new nicoletiidae (zygentoma: insecta) from brazil living in fire-ant (hymenoptera: insecta) nests. papéis avulsos de zoologia (são paulo), 49(34): 467–475. quate, l.w. & brown, b.v. (2004). revision of neotropical setomimini (diptera: psychodidae: psychodinae). contribution in science: natural history museum of los angeles county. (500):1-117. reemer, m. (2013). review and phylogenetic evaluation of associations between microdontinae (diptera: syrphidae) and ants (hymenoptera: formicidae). psyche 2013, article id 538316, 1-9. doi: 10.1155/2013/538316 trager, j. c. 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(1989). chemical mimicry in a parasitoid (hymenoptera: eucharitidae) of fire ants (hymenoptera: formicidae). journal of chemical ecology, 15: 2247-2261. wagner, r. & masteller, c. (1996). new moth flies (diptera: psychodidae) and a key to species from puerto rico. proceedings of the entomological society of washington, 98: 450–464. young, d.g. & perkins, p.v. (1984). phlebotomine sand flies of north america (diptera: psychodidae). mosquito news 44: 263-304.) doi: 10.13102/sociobiology.v61i4.502-509sociobiology 61(4): 502-509 (december, 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 adaptive and foraging behavior of two stingless bee species (apidae: meliponini) in greenhouse mini watermelon pollination iga bomfim¹, ad de m bezerra¹, ac nunes², fas de aragão², bm freitas¹ introduction the growth of protected cultivation in world agriculture has led to crops traditionally cultivated in open field to be grown in protected environments, but this condition creates a physical barrier which prevents the access of natural pollinators to flowers (guerra sanz, 2008). hand pollinating the flowers in a protected environment, as well as the application of hormones to induce fruit production, is laborious and increases production costs (cruz & campos, 2009). thus, a more economical alternative would be the introduction of native pollinators that are able to adapt to the conditions of closed environments and to meet the pollination requirements of crops under these conditions (banda & paxton, 1991; cruz & campos, 2009). the most promising pollinator in brazil seems to be the stingless bees (subtribe meliponina), since they occur abstract the growth of protected cultivation in world agriculture has driven crops traditionally cultivated in open field to greenhouse, which, in turn, prevent the access of pollinators to flowers. therefore, it is necessary to identify suitable pollinators for closed environments. stingless bees have been identified in brazil as the ideal pollinators for crop pollination under greenhouse conditions. colonies of two stingless bee species (melipona subnitida ducke) and scaptotrigona sp. nov. were evaluated in a greenhouse during the flowering of seeded (diploid) and seedless (triploid) mini watermelon (citrullus lanatus t.) varieties. the feasibility of using these bees in this production system was based on their adaptation and foraging behavior. m. subnitida did not show any interest to the crop under the experimental conditions; and this species stopped foraging trips and egg laying in a diapause-like stage. in comparison, scaptotrigona sp. nov. foragers were active and collected floral resources soon after their introduction. moreover, the foragers of scaptotrigona sp. nov. showed an essential behavior for mini watermelon pollination, because they visited staminate and pistillate flowers from both seeded and seedless genotypes for nectar collection. thus, we concluded that m. subnitida did not adapt to greenhouse mini watermelon cultivation under the experimental conditions and should not to be used for pollination purposes in this environment; however, scaptotrigona sp. nov. adapted well as a pollinator in the greenhouse and can be used for mini watermelon pollination in protected culture. sociobiology an international journal on social insects 1 universidade federal do ceará, fortaleza, ce, brazil 2 embrapa agroindústria tropical, fortaleza, ce, brazil article history edited by cândida maria l. aguiar, uefs, brazil received 30 september 2014 initial acceptance 06 november 2014 final acceptance 30 november 2014 keywords citrullus lanatus, meliponini, protected environment, pollinators. corresponding author isac gabriel abrahão bomfim faculdade de tecnologia centec (fatec) sertão central, eixo tec. de recursos naturais, quixeramobim, ce, brazil e-mail: isacbomfim@yahoo.com.br naturally throughout brazil and have a stunted vestigial sting that makes them safe for workers carrying out daily cultural practices in this confined environment. these insects also form perennial colonies that are usually active all over the year, and they can be managed in small hives which can be easily transported to the site requiring pollination services (heard, 1999; slaa et al., 2006). several stingless bees species have been determined to provide adequate pollination services to many different crops grown under protected cultivation, such as strawberry (fragaria x ananassa d.), eggplant (solanum melongena l.), sweet pepper (capsicum annuum l.), tomato (lycopersicon esculentum mill.) and cucumber (cucumis sativus l.) (malagodi-braga, 2002; cruz et al., 2005; del sarto et al., 2005; santos et al., 2008; nunes-silva et al., 2013; nicomedo et al., 2013). since the stingless bees form a very diverse group, c.a. 400 species, with very different physical and behavioral characteristics, it is possible to select research article bees sociobiology 61(4): 502-509 (december, 2014) 503 species with more suitable characteristics for certain plant species or type of protected cultivation (slaa et al., 2006). the use of stingless bees for pollination of cucurbit species inside greenhouse is restricted to cucumber (santos et al., 2008; nicomedo et al., 2013). however, the watermelon [citrullus lanatus (thunb.) matsum & nakai] is of great economic interest and its cultivation in protected environments is increasing in brazil and the world, especially with the use of genotypes that produce small fruits weighting up to 1.5 kg. these small, mini watermelon fruits are higher in price per kilogram than conventional large fruits, both in the domestic and international markets (bomfim et al., 2013). this crop has high pollination requirements, because flowers require multiple bee visits to deposit pollen on the stigma in order to set fruits (stanghelliniet al., 1997; walters & schultheis, 2009; winfree et al., 2007). regarding seedless watermelon varieties (triploid 3n), their pollination requirements is even greater (walters, 2005). the objective of this study was to evaluate the feasibility of using colonies of the stingless bees, melipona subnitida ducke and scaptotrigona sp. nov., to pollinate seeded and seedless mini watermelon varieties under protected culture. material and methods site and agricultural practices the experiment was conducted from august to october 2011 in a 160 m² arched-roof greenhouse (8 m wide x 20 m long x 3.5 m high), fitted with automated drip fertigation and temperature control systems situated at embrapa tropical agroindustry, fortaleza, ceará, brazil. the greenhouse was covered with a high density polyethylene film treated with anti-uv additives (material that filters ultraviolet radiation), having white colored roof and transparent light diffuser color on the sidewalls. it also had a 50% aluminet® type screen installed 2.5 m above the ground, which function was to lower the temperature inside the greenhouse. throughout the experiment, the plants were drip-fertigated with water and nutrients suitable for each stage of plant growth. seeds of two seeded mini watermelon varieties (2n) (minipol and polimore) and three seedless mini watermelon varieties (3n) (ha-5106, ha-5158 and ha-5161) were sown in plastic trays filled with a commercial substrate composed of dried coconut powder. twelve days later, 408 seedlings were transplanted to 5 liter-plastic pots, previously filled with raw coconut fiber and powdered coconut fiber (1:1). the pots were spaced 0.8 m between rows and 0.4 m between plants. a 3:1 ratio between triploid and diploid varieties was used in dedicated rows (fiacchino & walters, 2003; dittmar et al., 2009) for seedless watermelon cultivation. plants were trellised to facilitate management of this crop in a protected environment greenhouse. as suggested by mohr (1986) and campagnol et al. (2012), only one fruit was allowed to develop on each plant, so that later developing fruits would not influence the development of the first fruit. thus, additional fruits that set were removed during their early days of development. preparation of stingless bee colonies before being introduced in the greenhouse, four colonies of m. subnitida and two of scaptotrigona sp. nov. (oliveira & jesus, personal information) were selected in the meliponary of the federal university of ceará (ufc), and then, colonies of each species were standardized in respect to the quantity of brood and food stores. these colonies were introduced in the greenhouse as soon as the plants emitted their first staminate flowers. adaptive behavior to the protected environment bee flight activity, defined as foraging bees leaving their hives during the time that flowers were open (anthesis), was monitored throughout the experiment. foraging bees were enumerated by using a manual tally counter to determine the number of individuals that flew out their hives within a 10-minute period of each hour. therefore, it was possible to calculate the mean daily number of foragers leaving their hives within a 10-minute period of each hour. the activity performed by bee foragers after leaving their colonies and the amount of days that each species begin foraging on mini watermelon flowers after their introduction in the experimental greenhouse were also determined. bee colony development was monitored during the period in which they were used for pollination services. parameters such as changes in brood area, food store and queen oviposition (considering it active or not) were collected. additionally, data on luminosity (klux) were measured using a digital lux meter and data on temperature (°c) and relative humidity (%) were measured hourly by a data logger placed inside the greenhouse. foraging behavior in mini watermelon flowers the general foraging behavior of individuals belonging to both bee species were observed to determine the kind of resources collected from mini watermelon flowers, and if they touched the anthers (in staminate flowers) or the stigma (in the case of pistillate flowers) during visitation. it was also determined how many ways these bees approached the flowers and what part of their body contacted the anthers or stigma, depending on the flower approach. between 07:00 and 10:00 a.m. during two non-consecutive days, we recorded the frequency of bee visitation to flowers, measured by the number of visits received by a flower for five minutes. finally, we used a chronometer to record the mean time spent by bees per flower during a single visit. iga bomfim, ad de m bezerra, ac nunes; fas de aragão, bm freitas testing pollinators for greenhouse mini watermelon cultivation 504 data analysis data regarding the flux of foraging bees leaving their hives were submitted to regression analysis with analysis of variance of the curve by f-test using the program table curve 2d version 5.01. other data were analyzed by descriptive statistics, such as means with standard deviations (sd) or standard errors. results and discussion adaptive behavior to the protected environment during the first day of adaptation to greenhouse, both bee species showed a similar behavior. many foragers flew out their hives at first sunlight, flew towards the sunlight and collided against the plastic covering of the greenhouse. as the sun moved in the sky during the day, foragers collided against different parts of the greenhouse structure, as they followed the sun as a reference for navigation. most of these foragers became disoriented, unable to return to their hives, and continued to collide against the plastic structure until their death by exhaustion. according to several researches (free, 1993; malagodi-braga, 2002; cruz et al., 2004), apis mellifera l. and stingless bee foragers usually become disorientated, attempt to escape and eventually collide against the greenhouse covering in the early days after being introduced into this enclosed environment. in comparison, species belonging to the genus bombus do not become disorientated and begin pollination services as soon as they are introduced into a protected greenhouse environment, rarely colliding against the greenhouse covering (fisher & pomeroy, 1989; guerra sanz, 2008). after the death of several foragers and a few days of adaptation, there was a large decrease in the numbers that tried to escape, which resulted in less collision against greenhouse structures and disorientation behaviors. as soon as a colony is introduced in the greenhouse, the oldest and most experienced bees of the colony are those that are the first to go out foraging and fly high in the sky determining their usual flight paths. however, the artificial environment of a greenhouse has a limited size, which imposes physical barrier to the flight of foragers. therefore, it is understandable that bees initially collide against the transparent covering of the greenhouse in an attempt to continue the foraging activity they did before being introduced into this enclosed environment (free, 1993; slaa, 2003). after initial attempts to escape, m. subnitida foragers showed little movement at the hive entrances throughout the entire period they were in the greenhouse (fig 1a and c). furthermore, their activities were primarily restricted to brief flights of short distances to remove litter from inside their hives (pieces of dead larvae, pupae and adult bees). since foragers of this species did not visit the crop flowers, colony inspections showed a progressive reduction in food stores, brood area and queen laying. after 14 days inside the greenhouse, the colonies of m. subnitida had to be removed, because their populations were considerably reduced and colony internal development conditions were greatly reduced, resulting from the lack of food storage, high brood mortality, and disruption of egg laying by the queen (fig 2a, b, c and d). furthermore, some colonies had already completely sealed the entrance to the nest with resin to prevent them from being attacked by other colonies that were less affected. although stingless bees will generally adapt well in protected environments, some species will not adapt to the conditions imposed by certain greenhouse structures or to the crop being grown. malagodi-braga (2002) reported that the foragers of schwarziana quadripunctata (lepeletier) and scaptotrigona bipunctata (lepeletier) stingless bees did not visit the flowers of the strawberry grown under protected environment due to the lack of interest in the strawberry floral resources or they were not able to adapt to the conditions imposed by protected greenhouse structure. unlike the present study, cruz et al. (2005) were successful in using colonies of m. subnitida for pollination in protected cultivation. however, the crop was sweet pepper, and the greenhouse had different features, such as a glass roof and insect-proof screens covering the lateral sides and did not have an automated temperature control system. this non-adaptation of m. subnitida may have been caused by the coating material treated with anti-uv additives, and may not be related to the attractiveness of the mini watermelon crop for these bees. according to guerra sanz (2008) and bartelli et al. (2014), a preventive measure against insect pests in greenhouses is using coverings that have the ability to block or reduce the entry of ultraviolet (uv) light. these materials are able to transmit the band of light that the plant needs for photosynthesis, but diminish or block the entrance of uv light, which is the most important part of color spectrum visible to insects. although this type of plastic covering reduces the use of pesticides, which is desirable to consumers and also to the bees introduced for pollination purposes, it can also alter the activity of pollinators and may even prevent them from accomplishing what they were intended to do inside the greenhouse. another possible reason of the non-adaptation of m. subnitida colonies to the experimental greenhouse was the interaction between temperature (maximum average of 33.4 °c) and high humidity (maximum average of 97.4%) imposed by the experimental environment (fig 3). these of environmental conditions are not found in the caatinga (xeric shrub land and thorn forest), the region where the colonies used in this experiment were native. although the foragers of scaptotrigona sp. nov. during the first day in the protected environment showed a similar behavior to m. subnitida foragers, including high mortality by incessant collisions against the covering structures of the greenhouse, they began foraging in mini watermelon flowers in the second day after their introduction. this initial contact occurred about 10:00 a.m. of the second day, when a few foragers returned to their hives after visiting some flowers. then, sociobiology 61(4): 502-509 (december, 2014) 505 fig 1. outgoing flux of foragers during the anthesis of mini watermelon (citrullus lanatus) flowers under a greenhouse. a mean daily number with standard errors of melipona subnitida workers leaving their hives within a 10-minute period of each hour. b mean daily number with standard errors of scaptotrigona sp. nov. workers leaving their hives within a 10-minute period of each hour. c mean hourly number of melipona subnitida workers leaving their hives within a 10-minute period of each hour. d mean hourly number of scaptotrigona sp. nov. workers leaving their hives within a 10-minute period of each hour. suddenly, there was a great increase in the flight activity, with individuals of scaptotrigona sp. nov. flying directly to the flowers and gradually expanding their range within the greenhouse. from the third day until the end of the experiment, these bees visited mini watermelon flowers throughout their anthesis all over the entire area of the greenhouse. older bees usually have the task of collecting floral resources, and already have certain habits and experiences established with the previous environment, therefore, they have a greater difficulty in adapting to the protected greenhouse environment, which also limits their flight range (free, 1993). this explains the high mortality of foragers of both species on the first day after introduction. on the other hand, according to free (1993), cauich et al. (2004) and cruz et al. (2004), the sudden change in behavior on the second day can be explained by the young foraging bees, who did not perform foraging activities before the introduction of the colonies in the protected environment. for that reason, they had not yet established their flight paths, and consequently, did not have much trouble in adapting to the new conditions imposed by protected cultivation. after adapting to the greenhouse, which was when bees start of foraging on flowers, only a very small fraction of foragers continued to collide against the greenhouse covering and focused on removing debris from their colonies until the experiment terminated. from the third day after introduction of colonies into the protected environment, the vast majority of scaptotrigona sp. nov. foragers that left their hives flew directly to the flowers in search of food resources, and continued that behavior until the end of experiment (fig 1b). the period of adaptation for stingless bees to a greenhouse conditions can vary greatly between species as well as between colonies of the same species. some species, such as, nannotrigona perilampoides (cresson) require anywhere from five days to eight weeks to be consistent in their visitation to tomato flowers grown in greenhouse (macias et al, 2001; cauich et al., 2004). cruz et al. (2004) reported that m. subnitida foragers started collecting floral resources and providing pollination services on sweet pepper cultivation seven days after being introduced into greenhouse. malagodi-braga (2002) indicated other species of stingless bees that were consistent in their adaptation to pollinate strawberry grown in a greenhouse. for tetragonisca angustula (lepeletier) colonies, the adaptation period varied from one day to three weeks, while the adaptation period for nannotrigona testaceicornis (lepeletier) was about iga bomfim, ad de m bezerra, ac nunes; fas de aragão, bm freitas testing pollinators for greenhouse mini watermelon cultivation 506 three days. also in the same experiment, a wild nest of trigona spinipes (fabricius) was found to take only four hours to have its foragers consistently visiting the strawberry flowers. in this study, the foragers of scaptotrigona sp. nov. took only c.a. 30 hours to start foraging in mini watermelon flowers. moreover, the internal colony conditions and bee population levels were not considerably affected throughout the period of confinement under protected culture conditions (fig 2e and f), although they were faced with adverse envorinmental conditions, such as high temperatures, high humidity and lack of ultraviolet light (uv). also, after returning to the meliponary these colonies recovered to levels similar to those prior to their introduction in the greenhouse in less than 30 days. these results suggest that scaptotrigona sp. nov. has potential for commercial uses as a pollinator in greenhouse because it quickly adapts to the protected environment, initiates flower visitation in a few hours after introduction, internal colony development and bee population are not affected by the confinement, and populations recover fast after leaving the greenhouse. foraging behavior in mini watermelon flowers the foraging activity of scaptotrigona sp. nov. foragers usually began at 5:30 a.m. (temperature = 24.5°c; humidity = 97% and luminosity = 1.79 klux; fig 3), which was shortly after the first sunlight appeared in the greenhouse and the beginning of the anthesis of mini watermelon flowers. although only 2.46 ± 3.49 bees flew out of their hives at this time, numbers increased rapidly with a maximum peak occurring at 08:00 a.m. (27.71 ± 15.36 bees; fig 1d). after this time, the number of foragers flying out of their nests decreased slowly until they almost ceased by 2:00 p.m., when there was a mean of 3.82 ± 5.10 foragers (fig 1d). it appears that these bees remained active outside their nests only while the flowers were open, since flowers were almost completely closed by 2:00 p.m. also, the foragers of scaptotrigona sp. nov. had their maximum visitation peak within the optimal period of stigma receptivity and pollen viability for many cucurbit crops, which, according to free (1993), nepi and pacini (1993) and kwon et al. (2005), take place up to five to six hours after flower opening. unlike a. mellifera colonies that direct a certain amount of foragers to collect pollen from watermelon flowers early in the morning (azo’oela et al., 2010), scaptotrigona sp. nov. foragers did not appear to be collecting this resource. fig 3.temperature (°c), relative air humidity (%) and luminosity (klux) inside the greenhouse where the mini watermelon (citrullus lanatus) was cultivated and pollination services by stingless bees were required. these bees primarily seek nectar in both pistillate and staminate flowers, although during their visits to staminate flowers, they had their bodies dusted with a great amount of pollen grains (fig 4a). a portion of these pollen grains was transferred to the pollen basket and later taken to the hives (fig 4b), but a certain amount of pollen grains remained on their bodies, mainly in the head, legs, and thorax and abdomen area. when approaching the flower, foragers used the petals of both floral types (staminate or pistillate) as a landing platform and then proceeded to walk in the direction of the nectary at the bottom of corolla; unintentionally, the reproductive parts of the flower come in contact with the head and dorsal portion of the thorax (fig 4c). another way the flowers were approached by bees was to directly land on the reproductive fig 2. development stages of stingless bees colonies used for pollination of mini watermelon (citrullus lanatus) under a greenhouse. a brood combs in a melipona subnitida colony at the beginning of the pollination services. b food stores in a melipona subnitida colony at the beginning of the pollination services. c brood combs in a melipona subnitida colony at the end of the pollination services. d food stores in a melipona subnitida colony at the end of the pollination services. e brood combs and food stores in a scaptotrigona sp. nov. colony at the beginning of the pollination services. f brood combs and food stores in a scaptotrigona sp. nov. colony at the end of the pollination services. sociobiology 61(4): 502-509 (december, 2014) 507 the length of each time for nectar collection visit by a scaptotrigona sp. nov. forager varied between 2.27 and 43.95 seconds, with an average of 13.10 ± 8.86 s (n = 68). scaptotrigona sp. nov. foragers remained in flowers much longer than that reported by njoroge et al. (2004) and azo’oela et al. (2010) and araújo et al. (2014) for a. mellifera foragers visiting watermelon flowers in search of nectar, with means varying from 0.56 to 8.7 seconds. therefore, in addition to the forager behavior of often landing and walking on the reproductive parts of the flower, the long time that the scaptotrigona sp. nov. foragers spend visiting a flower seems to maximize the distribution of pollen grains on the three lobes of the stigma, which is essential for the development of fruit without deformation (mann, 1943). in contrast to this behavior, the foragers of a. mellifera rarely change their position after landing on the watermelon flower, typically restricting their contact to only one of the lobes of the stigma per visit (adlerz, 1966). therefore, the distribution of pollen grains by a. mellifera in the watermelon stigma is more dependent on multiple visits rather than the time each forager spends on a flower, or even the movement performed by a forager in the flower after landing. besides moving around in the flower, scaptotrigonasp. nov. foragers made multiple visits to the mini watermelon flower. each flower received an average of 6.0 ± 3.69 visits (n = 24) over a period of five minutes. after leaving a flower, scaptotrigona sp. nov. foragers, tended to move to the nearest flower, which were generally in the same plant or in the same row. however, foragers also moved between rows as well as only visiting one flower per plant before moving to another. probably, these variations of movements favored the transfer of pollen between diploid plants and, more importantly, from diploid to triploid plants. such behavior is essential for producing seedless watermelon fruits, because flowers from seedless varieties (3n) require pollen grains of seed varieties (2n) for setting fruits (walters, 2005). although indirect measurements are less informative than direct measurements (gross, 2005), they are an alternative way to measure pollinator effectiveness. the more types of information collected, the greater the likelihood that a visitor can be accredited as a pollinator. therefore, these results suggest that the introduction of colonies of scaptotrigona sp. nov. could be an alternative to pollinate seeded and seedless mini watermelon grown inside greenhouses, since these bees adapted quickly to confinement, maintained high population levels, and exhibited foraging behaviors compatible to provide pollination services required by this crop. however, in contrast, m. subnitida species did not adapt to the greenhouse mini watermelon cultivation under the experimental greenhouse conditions and should not be used for pollination purposes in such situation. further studies are needed to determine exactly what are the factors that affect the adaptation and visitation of m. subnitida species to flowers of crops grown inside greenhouses. fig 4. foraging behavior of scaptotrigona sp. nov.workers in mini watermelon (citrullus lanatus) flowers grown in a greenhouse. a worker covered with pollen grains and stopping on a petal of a staminate flower while brushing herself before taking off. b forager returning to the nest with pollen on her cobiculae. c worker collecting nectar while on a staminate flower and touching the anthers with her head and dorsal part of the thorax. d forager collects nectar while sitting on the anthers of a staminate flower, acquiring pollen on her ventral parts of the thorax and abdomen. e worker with pollen on her corbiculae, while moving over the stigma of a pistillate flower in a search for nectar. f forager moving from the petal to the stigma of a pistillate flower. parts of flowers (anthers and stigma in staminate pistillate flowers, respectively), where they proceeded towards the nectary (fig 4d and e). in this type of approach, bees usually contacted the reproductive parts of the flowers with practically the entire ventral portion of their body (thorax, abdomen and legs). regardless of the way that bees approached flowers, pollen was transferred to the body of the bees. however, a larger portion of the body of these individuals came in contact with the reproductive parts of the flowers as they approached the flowers and landed directly in their reproductive parts. some foragers were also observed performing both behaviors in the same flower during the same visit. in addition, some foragers of scaptotrigona sp. nov. walked on the petals and on the stigmatic surface of pistillate flowers (fig 4f) in order to reach the other side of the nectary, thus touching and transferring pollen grains in a single visit to more than one of the stigmatic lobes. according to mann (1943) and delaplane and mayer (2000), watermelon flowers require that pollen grains be distributed to all the three lobes of the stigma in order to develop fully formed fruits. iga bomfim, ad de m bezerra, ac nunes; fas de aragão, bm freitas testing pollinators for greenhouse mini watermelon cultivation 508 acknowledgements authors are grateful to hazera genetics for providing the seeds; top plant for cultivating the seedlings and funcap and cnpq, brasília-brazil, for a phd scholarship to isac gabriel a. bomfim, an undergraduate scholarship to antonio diego de m. bezerra and a research fellowship to breno m. freitas (proc. # 302934/2010-3). authors also thank the two anonymous referees for the valuable suggestions that improved this manuscript. references adlerz, w.c. 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(1997). the effects of honey bee and bumble bee pollination on fruit set and abortion of cucumber and watermelon. am. bee j., 137: 386-391. walters, s.a. (2005). honeybee pollination requirements for triploid watermelon. hortscience, 40: 1268–1270. walters, s.a. & schultheis, j.r. (2009). directionality of pollinator movements in watermelon plantings. hortscience, 44: 49-52. winfree, r., williams, n. m., dushoff, j. & kremen, c. (2007). native bees provide insurance against ongoing honey bee losses. ecol. lett., 10: 1105-1113. doi: 10.1111/j. 1461-0248.2007.01110.x. 1297 nest architecture, colony composition and feeding substrates of nasutitermes coxipoensis (isoptera, termitidae, nasutitermitinae) in subtropical biomes of northeastern argentina by enrique r. laffont1,2, juan m. coronel1, maría c. godoy1 & gladys j. torales1 abstract the moundbuilding termite nasutitermes coxipoensis is commonly found at subtropical environments of northeast argentina. this study gives new data about its nest architecture, biolog y and nutritional habits, comparing these results with other populations of the species. the volume of the analyzed nests varied between 0.91 dm3 y 207.33 dm3, a wider range than previously reported for n. coxipoensis. the external and internal characteristics of the nests were similar to descriptions from other sites although it was not possible to differentiate the royal cell. the societies were monog ynic and the queens laid eggs throughout the year. the body length and weight of primary reproductives were reported. the presence of alates within the mounds was higher in october and november. feeding substrates consumed by n. coxipoensis at these habitats are also reported. key words: isoptera, termitidae, nasutitermes, argentina. introduction nasutitermes coxipoensis (holmgren) is a moundbuilding termite widely distributed in the neotropical region, cited from french guyana, brazil and argentina (constantino 2012). previous field studies on its biology correspond to populations located in savannas of french guyana (lefeuve 1987), in the regions of brazilian campos and cerrado (mathews 1977, buschini 1996, garcia et al., 2006, buschini et al. 2008 ) and the province of corrientes, argentina (torales et al. 2006). 1 facultad de ciencias exactas y naturales y agrimensura (universidad nacional del nordeste). avenida libertad 5470. cp:3400. corrientes, argentina. 2 corresponding author, email: erl@exa.unne.edu.ar 1298 sociobiolog y vol. 59, no. 4, 2012 other authors have reviewed taxonomic (silvestri 1903, holmgren 1910, mathews 1977), morpho-anatomic (xavier soares & costa leonardo 2002, buschini & costa leonardo 2002) and histological aspects of the species (cunha et al. 2009 a y b), as well as its reproductive mechanisms (lefeuve 1987, buschini & costa leonardo 1999) and analyses of substrates for survival tests (albuquerque et al. 2008). in some widely distributed termite species, the nest characteristics sometimes show variations due to geographic, climatic or edaphic differences, reflecting intraspecific plasticity in the construction of these structures (korb & linsenmair 1998, 1999; noirot & darlington 2000, korb 2010). also, some species can vary in several life history traits, reproductive strategies or nutritional habits at different zones of its geographical distribution (leponce et al. 1995, jeyasingh & fuller 2004, arango 2007, lenz et al. 2009). in northeastern argentina, the populations of n. coxipoensis occupy several vegetation units of the paranaense (campos district) and chaco (chacoan eastern district) phytogeographical provinces (torales et al. 1997, 2005, figure 1. mound built by n. coxipoensis. note the irregular external surface. 1299 laffont, e.r. et al. — nest architecture of n. coxipoensis 2007, 2008, 2009, laffont et al. 2004). given the broad distribution of this species, inhabiting different climatic and landscape units in a wide latitudinal range, this study provides new data on the biolog y, nutritional habits and nest architecture of n. coxipoensis in subtropical environments from northeastern argentina and observations on its population dynamics. materials and methods the study areas were located in three sampling sites at mburucuyá (28º04’09”s, 58º 15’23” w, mburucuyá department) and two sites at villa olivari (27°38’01”s, 56º 54’19” w, ituzaingó department) of the province of corrientes, argentina. the climate is subtropical and the mean annual temperatures oscillate between 21.5ºc and 19.5°c. rainfalls are irregular, more frequent in autumn and spring but without a noticeable dry season, with annual average of about 1.5001.000 mm (carnevali 1994). fig. 2. internal view of a partially dissected n. coxipoensis nest. 1300 sociobiolog y vol. 59, no. 4, 2012 at mburucuyá, the populations of n. coxipoensis were located at andropogon lateralis nees and elyonurus muticus (spreng.) kuntze grasslands, as well as in paspalum notatum flügge pastures with cattle grazing. at villa olivari, the nests were found in plantations of exotic trees (pinus sp. and eucalyptus grandis hill ex maiden) and sandy dunes with several species of grasses, bordering the banks of the parana river. in both localities, the n. coxipoensis nests were found scattered among the epigeal nests constructed by other termitidae (cornitermes cumulans kollar, cortaritermes fulviceps silvestri or both). the nest architecture was described based on 41 n. coxipoensis termitaria selected from such populations. the arrangement of runways and the castes present in each of these nests were examined in the field and at the laborafig. 3. section of n. coxipoensis nest. arrow: lower galleries inside the soil. 1301 laffont, e.r. et al. — nest architecture of n. coxipoensis tory, applying a modification of the methodolog y described by torales et al. (2006) to study the qualitative composition of colonies. the nest volumes were calculated according to the formula used by buschini & costa leonardo (1999), for comparison, assuming the similarity of the nests to a hemisphere. however, since the nests also have a hypogeal sector, the calculated volume is considered only approximate. the depth to which cells spread within the soil profile were also measured. the total body length (tbl) and wet weight (w) of the reproductives was recorded according to the specimen availability, because some of them were damaged while dissecting the nests. the lengths were measured with a caliper mitutoyo 530-312, and weights were recorded with a precision balance uwe njw-150. photographs were taken with a digital camera kodak 913 (8.0 mpx). fig. 4. detail of the nest chambers (arrow indicates one of the “pillars”). 1302 sociobiolog y vol. 59, no. 4, 2012 results and discussion the field observations of n. coxipoensis epigeal nests showed that despite some unremarkable variations, the caps were approximately hemispherical, with mean values of 1.39 m (basal diameter), 0.24 m (height) and 0.25 m (depth) (table 1). a similar morpholog y is recognized for nests of this species in the brazilian cerrado (buschini et al. 2008). these mounds grew slightly deeper in the substrate and its maximum height (0.42 m) was lower than the record of lefeuve (1987), who reports nests up to 1.00 m in french guiana savannas. the volume of the analyzed n. coxipoensis nests ranged from 0.91 dm3 to 207.33 dm3, with an average of 38.46 dm3 (table 1). these values extend the range reported for the species by lefeuve (1987) and buschini & costa leonardo (1999). large differences in size between colonies were evident in these populations, such as those from sao paulo, brazil (buschini & costa leonardo 1999). of the nests analyzed in this study, 65.85% were smaller than the mean volume and in seven of them the volume was less than 9 dm3. in the province of corrientes, n. coxipoensis nests have only been detected so far in sandy soils. however, in the brazilian cerrado, although the number is higher in this type of soils, they are also located in lateritic surfaces (mathews 1977, buschini et al. 2008). the outer surface of the nests was irregular, with granular-like protuberances corresponding to protruding internal cells. this surface was constituted by a thin layer (34 mm thick) of fragile sandy consistency, which was easily removed and range in color from light or dark brown to reddish brown (fig. 1). unlike the records of mathews (1977), there were fig. 5. incipient nest of n. coxipoensis with covered runways departing from it. 1303 laffont, e.r. et al. — nest architecture of n. coxipoensis table 1. nest locations, collection date and dimensions, and colony composition of n. coxipoensis. d: depth, r: reproductives, e: eggs, l: larvae, ws: white soldiers, w: workers, s: soldiers, n: nymphs, a: alates, +: present, -: absent or not found. nest month locality volume (dm3) d r ♀ ♀ e l ws w s n a 1 feb mburucuyá 28.36 0.25 1 + + + + + + 2 feb mburucuyá 15.83 0.24 1 1 + + + + + + 3 feb mburucuyá 2.66 0.14 + + 4 feb mburucuyá 20.37 0.14 + + + + + 5 mar v. olivari 88.28 0.28 + + + 6 mar v. olivari 30.20 0.26 + + + + + 7 mar v. olivari 92.94 0.30 + + + + + 8 mar v. olivari 1.13 0.18 1 + + + + 9 apr c. laurel 33.65 0.53 + + + + + + 10 may s. antonio 38.78 0.30 + + + + + 11 jun mburucuyá 15.83 0.30 + + + + + + 12 jun mburucuyá 21.23 0.20 + + + + + + 13 jun mburucuyá 31.36 0.25 1 + + + + 14 jul v. olivari 0.91 0.06 + + + 15 jul v. olivari 2.20 0.20 1 1 + + + 16 ag c. laurel 46.79 0.35 + + + + + 17 ag c. laurel 23.72 0.21 + + + + + 18 ag v. olivari 54.82 0.26 + + + + + 19 ag v. olivari 35.57 0.28 + + + + + 20 sep ituzaingó 9.39 0.32 + + + + 21 sep mburucuyá 7.87 0.12 + + + + 22 sep mburucuyá 52.00 0.25 1 + + + + + + 23 sep mburucuyá 12.91 0.20 1 1 + + + + + + 24 sep mburucuyá 58.41 0.50 1 1 + + + + + 25 oct c. laurel 41.76 0.40 + + + + + + 26 oct c. laurel 33.29 0.35 + + + + + 27 nov mburucuyá 57.10 0.31 + + + + + + + 28 nov mburucuyá 18.79 0.26 1 + + + + + + 29 nov mburucuyá 38.61 0.20 + + + + + + 30 nov mburucuyá 39.78 0.25 + + + + + 31 nov mburucuyá 47.17 0.27 1 + + + + + + 32 nov mburucuyá 20.54 0.20 + + + + + + 33 nov mburucuyá 33.94 0.22 + + + + + 34 nov mburucuyá 52.25 0.24 + + + + + + 35 nov mburucuyá 12.90 0.17 + + + + + + 36 nov mburucuyá 13.20 0.15 + + + 37 dic v. olivari 207.33 0.30 + + 38 dic v. olivari 13.71 0.22 + + + + + 39 dic v. olivari 7.07 0.24 + + + + + 40 dic v. olivari 8.01 0.18 + + + 41 dic v. olivari 206.21 0.40 + + + + + 1304 sociobiolog y vol. 59, no. 4, 2012 no loose particles of soil covering the mounds. most nests showed a distinct color contrast between the inner carton (dark brown) and the covering layer (light brown). the interior of the mounds were made of cardboard-like material (carton), with an approximately homogeneous structure, without remarkable differences between the peripheral and central regions of the nest. the cells were arranged mostly irregularly although several horizontal layers could be recognized in some areas (fig. 2). the carton used as a building material included partially digested plant debris and sand particles coated with excreta of the workers. the nests had a soft consistency, and are more easily broken than other epigeal termite nests of this region. next to the hypogeal sector of the nests, the horizontal strata became discontinuous and the cells took a nearly concentric arrangement around a harder central area. the cells in this core area housed the reproductives and, sometimes, groups of eggs. it was not possible to differentiate the royal cell mentioned by lefeuve (1987). fig. 6. nest of n. coxipoensis and runways on the sandy dunes. 1305 laffont, e.r. et al. — nest architecture of n. coxipoensis some cells of different sectors of the nests contained plant debris and/or dry cattle dung possibly transported by workers to the nest, as in other foraging and litterfeeding species where the nests also work as a food reserve site (thorne et al. 1996, noirot & darlington 2000). tunnels of the hypogeal sector continued to the soil depth (fig. 3). the walls of all the examined n. coxipoensis nests included clumps of grass, as was observed by mathews (1977) and lefeuve (1987). therefore, the cells were distributed between stems and leaves. some thin stalks were cut into tiny fragments and coated with carton, to be used as reinforcements in the fig. 7. n. coxipoensis tunnels between grass tussocks. 1306 sociobiolog y vol. 59, no. 4, 2012 construction of internal chambers, acting as “pillars” in different layers of cells (fig. 4). in some nests, two or three layers were bound by the same piece of plant material acting as a column. in the populations of n. coxipoensis located on sandy dunes at villa olivari, environmental characteristics allowed us to distinguish clearly the arrangement of numerous covered runways departing from the nests (fig. 5). there a b fig. 8 a, b. physogastric queens of n. coxipoensis. bar= 3 mm. 1307 laffont, e.r. et al. — nest architecture of n. coxipoensis were very conspicuous dark tunnels arranged in different directions, on loose light colored sand. some completely tubular runways stretched to the tussocks of panicum sp., probably one of the main nutrient sources in the dune for n. coxipoensis (fig. 6). the maximum lengths of these runways were 3.20 m for those between two nests, 3.70 m from a nest to a panicum clump and 2.50 m between two clumps of these grasses. these runways were occupied by foraging groups of workers and soldiers. the connections through tunnels between nests and between mounds and grass tussocks, previously recorded for this species (mathews 1977, lefeuve 1987) were also observed in villa olivari. furthermore, tunnels linking together grass plants were registered, indicating movement of the groups between them during foraging activity (fig. 7). the analysis of the qualitative composition of n. coxipoensis societies (table 1) showed that the colonies in which reproductives were found (10) were monog ynous, with primary reproductives located in the core region or central area of the nests. these observations support the fact that n. coxipoensis societies have a single royal couple, as has been reported for other populations (lefeuve 1987, buschini & costa leonardo 1999, torales et al. 2006). these data do not exclude the possibility that, at least sometimes, the colonies are capable of producing multiple reproductives as in several species of nasutitermes (noirot 1956, thorne 1982, thorne & noirot 1982, thorne 1984, roisin & pastels 1986, atkinson & adams 1997, roisin 2000, hartke & baer 2011). experimentally, it has been shown that n. coxipoensis colonies are able to replace the primary reproductives through replacement individuals (adultoids) within relatively short time, involving a small number of third instar nymphs (lefeuve 1987). however, this author does not rule out the possibility that table 2: total body length (tbl) and wet weight (w) of primary reproductives of n. coxipoensis. tbl (mm) w (g ) n x r s n x r s females 10 17.4 11.023.0 3.59 6 0.159 0.2150.110 0.040 males 4 6.0 5.07.0 0.81 4 0.012 0.0100.015 0.003 1308 sociobiolog y vol. 59, no. 4, 2012 replacement sexuates could be originate from alates that remain in the nest after swarming. replacement reproductives were not found in this study. the total body length (tbl) and wet weight (w) of reproductives of both sexes are presented in table 2. all primary female reproductives detected showed varying degrees of fisogastry, with higher ltc values than males (fig. 8 a, b). comparatively, the queens of n. coxipoensis analyzed in this study showed similar weight to the specimens from french guyana ( x = 0.144 g, lefeuve 1987) but higher than those from the brazilian cerrado ( x = 0.071 g, buschini & costa leonardo 1999). for kings, the individual weight and the mean value were similar to those of brazil (buschini & costa leonardo 1999). under the environmental conditions of this survey, it was observed that queens of n. coxipoensis were capable of laying eggs during the four seasons although eggs were not detected in all nests analyzed at each month. in spring, 77% of the nests contained numerous eggs, so it would seem the most favorable period for oviposition. such observations are supported by the presence of larvae in 95% of the nests from february to december, with fig. 9. piece of dead wood partially excavated by n. coxipoensis. 1309 laffont, e.r. et al. — nest architecture of n. coxipoensis predominance of the first instars ones during spring and summer. the continuous presence of larvae throughout the year confirms our previous records (torales et al. 2006). white soldiers were detected more frequently in nests examined during the warmer months (september to february: 53.8% of nests) than in the cold season (march-august: 26.6% of nests). the nymphs were registered in 65% of nests in autumn and winter, declining sharply between september and december (22.7% of colonies). the simultaneous presence of nymphs and alates was only detected in two of the 41 examined nests. in october and november, 91.6% of the nests contained numerous alates, located in cells near the nest core. during the dissection of the mounds, most of them did not attempt to fly, despite the fact that their wing development seemed to be complete, but tried to hide into the nest. during december, no alates were found inside the mounds, which would confirm that spring is the season when swarming occurs in n. coxipoensis as well as in other termitidae in the province of corrientes (torales et al. 1999, 2006, coronel et al. 2001, fig. 10. longitudinal section of dry cow dung, invaded by n. coxipoensis. 1310 sociobiolog y vol. 59, no. 4, 2012 torales & coronel 2004,). alates are probably released once a year. in n. coxipoensis colonies from french guyana, the alates are in the nest between october and march and the flights take place from march to may (lefeuve 1987). with respect to the feeding substrates consumed by n. coxipoensis, stems and roots of grasses partially excavated and colonized by workers and soldiers were frequently observed at all sampling sites in both locations. it was also found that n. coxipoensis consumed dead wood from trunks and branches of eucalyptus grandis. the termites deeply excavated the lower surface of the pieces of wood, partially covering those areas (fig. 9). grass and dead wood seem to be the main nutrient sources of n. coxipoensis in the studied areas, as previously registered in brazil (mathews 1977) and this region (torales et al. 2009). another resource exploited by n. coxipoensis was dry cattle dung. the foraging groups of workers and soldiers were detected in cattle grazing areas, where the masses of manure were thoroughly excavated in the bottom surface contacting the soil, generating cells and galleries occupied by workers and soldiers (fig. 10). for the construction of these cells, the workers used their feces mixed with plant fibers included in the cattle manure or a mixture of sand and these fibers. the addition of dry cattle dung to their diet increases the exploitation of available food for the species within these habitats. this species was also recorded feeding on dung in the cerrado (freyman et al. 2008). in addition, the workers of n. coxipoensis are able to forage in the open at night (mathews 1977) and invade living trees in e. grandis forest plantations (laffont et al. 1998). in laboratory bioassays, the consumption of sugar cane and paper kraft by n. coxipoensis was also registered (albuquerque et al. 2008). the results reported here allow more detailed descriptions of n. coxipoensis nests and expand their recorded volumes. also, the periods of alate presence and oviposition of queens in the region were reported. the type and number of reproductives agreed with previous observations for this species. moreover, the substrates consumed in natural and anthropogenic environments of northeastern argentina revealed a wider feeding plasticity than previously known in this region for n. coxipoensis. 1311 laffont, e.r. et al. — nest architecture of n. coxipoensis acknowledgments this work was supported by the secretaria general de ciencia y técnica, universidad nacional del nordeste (sgcytunne). references albuquerque, a.c., f.m. cunha, m.a. oliveira, a.f. veiga & e.a. luna-alves lima. 2008. análise de sustratos para testes de sobrevivéncia com nasutitermes coxipoensis (holmgren) (isoptera: termitidae). arquivos do instituto biologico (sao paulo) 75: 529532. arango, r.a., f. green & g.r. esenther. 2007. seasonal response of feeding, differentiation and growth in the eastern subterranean termite reticulitermes flavipes (kollar) in wisconsin. the international research group on wood protection irg/wp 0710604. 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from nasutitermes coxipoensis nests (isoptera: termitidae, nasutitermitinae), from a locality of the province of corrientes (argentina). sociobiolog y 48 (1): 223236. torales, g.j., j.m. coronel, e.r. laffont, m.c. godoy & j.l. fontana. 2007. termite (insecta, isoptera) faunal composition in natural forests of the humid chaco (argentina). sociobiolog y 50 (2): 419433. torales, g.j., j.m. coronel, m.c. godoy, e.r. laffont & v. romero. 2008. additions to the taxonomy and distribution of isoptera from argentina. sociobiolog y 51 (1): 3147. torales, g.j., j.m. coronel, e.r. laffont, j.l. fontana & m.c. godoy. 2009. termite associations (insecta, isoptera) in natural or semi-natural plant communities from argentina. sociobiolog y 54(2): 383437. xavier soares, h. & a.m. costa leonardo. 2002. survey of the leg exocrine glands in termites (isoptera). revista brasileira de entomologia 46(1): 16. 1national termite control center of china, moganshan road 695, hangzhou, zhejiang 310011, p. r. china; 2ministry of agriculture key laboratory of molecular biolog y of crop pathogens and insects, institute of insect sciences, zhejiang university at zijingang, ruhangtang road 866, hangzhou, zhejiang 310058, p. r. china. * corresponding author. email: mojianchu@zju.edu.cn 1365 ivermectin dust for the control of coptotermes formosanus in residential areas by jing yang zhao1, ying dong2, baoting yu2, zhen zhang2 & jianchu mo2* abstract coptotermes formosanus shiraki is an important termite severely damage wood components of housing construction and old living trees in residential areas in south of china. the 70% mirex dust was one of important chemical products to control this termite damage. as the completely elimination of mirex in may 2009 in china, one of key works for chinese researchers on termite control is to find the alternatives of mirex product. in present study, we evaluated the effect of 3% ivermectin dust to eliminate the colonies of c. formosanus in residential areas of hangzhou city, zhejiang province, china from april 2010 to may 2012. the results indicated that when 20-30 grams of 3% ivermectin dust were applied onto the body of termites feeding in four monitor devices at 18-80 meter far from the c. formosanus nests, the termite colonies would be eliminated completely within three and half to eight months. this means 3% ivermectin dust was a good alternative of 70% mirex dust and could be used for subterranean termite control through the method of dusting in the monitor devices. keywords: copototermes formosanus, ivermectin dust, colony, dusting technolog y.introduction the formosan subterranean termite, coptotermes formosanus shiraki, is a native species of termites to china (kistner 1985), and causes worldwide over 2 billion u.s. dollars in damage and control costs each year (potter 1997). before may 2009, mirex dust and mirex bait are main products for control1366 sociobiolog y vol. 59, no. 4, 2012 ling the damage of c. formosanus in china. as the completely elimination of mirex in chinese termite control industry, ivermectin becomes a potential alternative of mirex. some researches found that ivermectin had good lethal and sublethal effects on termites (mo et al. 2005; mo et al. 2006; wang et al. 2007). when termites in monitor-controlling devices were treated with ivermectin dust and bait, damage of termites could be inhibited greatly (mo et al. 2006; wang et al. 2007; jiang et al. 2011). however, it is not clear whether ivermectin dust could eliminate completely the colony of c. formosanus when it was applied in the monitor devices far to termite nest until now. thus, the objective of this study is to evaluate the feasibility of 3% ivermectin dust to eliminate the c. formosanus colonies by means of monitor devices. materials & methods chemical the 3% ivermectin dust was provided by deqing institute of termite control, zhejiang province, china. monitor-controlling device the monitor-controlling devices for this study were provided by the hangzhou changlian pest control technolog y co., ltd. (hangzhou, china). this device was hollow cuboid and was 8 cm in length, 8 cm in width and 18 cm in height. there were eight pine wooden blocks in its inner wall and empty cavity. test sites the termite colonies tested was locating at the residential area of hangzhou city, zhejiang province and totally five coptotermes formosanus colonies were tested. test procedures firstly, some monitor devices were installed in the area near to the nest of c. formosanus in the reason of termite activity of 2010. when most of monitor devices were infested by termites, all termites in two monitor devices 50 cm far to the termite nest were collected and labeled with filter papers treated by 0.5% neutral red solution in laboratory. then labeled termites were released into original two monitor devices. after that, the monitor devices were checked every week within one month. during check, if dyed termites in 1367 zhao, j. et al. — vermectin dust for coptotermes termite control a monitor devices were found, and the position of the monitor device was marked on the map. after the activity territory of termite colony was determined, application of dust was done. for each treated colony, only foraging worker termites feeding in four monitor devices farthest to the termite nest were treated by dust (5-10 grams of dust was applied to each device and totally 20-30 grams of dust for one colony were applied) and other monitor device with termite infestation were left for the observation of termite activity. then the termite activity in untreated and treated monitor devices was checked every month. during check, if the woods were all consumed by termites, new woods would be added continuously into the monitor devices. when termite activities in untreated and treated monitor devices were not found within at least six continuing months in termite activity seasons, the test was ended. results colony one totally 40 monitor devices was installed in the place near to the formosan subterranean nest located in the zhijiang community of hangzhou city on april 5, 2010 (fig. 1). termite invasion was found in 10 monitor devices near to the nest on april 25. there was termite invasion in 22 monitor devices on june 9. termite individuals dye labeled test showed that the termites within these monitor devices belonged to the same colony. termites in four monitor devices were treated with about 25 grams of 3% ivermectin dust on july 11, 2010. the distance from the farthest devices treated to the termite nest was 24 m. when check on august 15, no termite was found in four devices treated but there were still some termites to be found in other untreated devices. when check on october 25, 2010, no termite activity was found in all devices with originally termite infestation. there was still no termite activity to be observed in these monitor devices from april 2011 to may 2012.( termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) colony two totally 25 monitor devices was installed in the place near to the formosa subterranean nest located in the huajiachi community of hangzhou city 1368 sociobiolog y vol. 59, no. 4, 2012 on june 11, 2010 (fig. 2). termite invasion was found in 22 monitor devices on july 9 and dye labeled test verified that the termites within these monitor devices were from the same colony. after using about 20 grams 3% ivermectin dust to treat the termites feeding in four monitor devices (the farthest one to the nest was 38 m) on august 12, 2010, no termite activity was found in all untreated and treated devices when check on december 16, 2010. moreover, no termite renewed to invade into these monitor devices during the period from april 2011 to may 2012. colony three totally 30 monitor devices was installed in the place near to the formosa subterranean nest located in the yuquan community of hangzhou city on july 14, 2010 (fig. 3). of which, 23 monitor devices was invaded by termites up to august 5 and dye labeled test showed that these termites were from the same colony. we used about 25 grams of 3% ivermectin dust to treat the termites feeding in four monitor devices (which the farthest one to the nest was 18 m) on september 10, 2010. only few termites were found in part of fig. 1. sketch map of termite monitor device distribution. termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) 1369 zhao, j. et al. — vermectin dust for coptotermes termite control monitor devices when check on december 2, 2010 and no termite activity was found in all untreated and treated devices when check again on may 10, 2011. meanwhile, we did not also find any termites in these monitor devices during the period from june 2011 to may 2012. colony four totally 30 monitor devices was installed in the place near to the formosa subterranean nest located in the xixi community of hangzhou city on june 18, 2010 (fig. 4). among these monitor devices, 19 monitor devices was infested by termites when check on july 6 and dye labeled test showed that these termites were from the same colony. about 30 grams of 3% ivermectin dust were dusted onto the body of termites feeding in four monitor devices (which the farthest one to the nest was 80 m) on august 18. we did not find any termites in all untreated and treated devices when check on december fig. 2. sketch map of termite monitor device distribution. termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust 1370 sociobiolog y vol. 59, no. 4, 2012 20, 2011. in addition, we did not also find any termites to enter into these monitor devices during the period from late april 2011 to late may 2012. (termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) colony five. totally 35 monitor devices was installed in the place near to the formosa subterranean nest located in the lianhua community of hangzhou city on june 21, 2010 (fig. 5). consequently, 22 monitor devices detected termite activity up to july 19 and we found the termites in these monitor devices came from the same colony through dye labeled test. about 30 grams of 3% ivermectin dust were applied onto the termites feeding in four monitor devices (which the farthest one to the nest was 41 m) on august 22. up to november 5, 2010, we did not find any termite activity in all untreated and treated devices. during the check from late april 2011 to late may 2012, no. fig. 3. sketch map of termite monitor device distribution termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) 1371 zhao, j. et al. — vermectin dust for coptotermes termite control fig. 4. sketch map of termite monitor device .with termite distribution.distribution. device distribution termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) ig. 4. sketch map of termite monitor device .with termite distribution.distribution. device distribution termite nest; monitor device with termite infestation; monitor device without termite infestation; monitor device treated with 3% ivermectin dust) 1372 sociobiolog y vol. 59, no. 4, 2012 discussion in china, c. formosanus locates at the area of north latitude 33.5° and is an amphibious termite which severely damage wood components of housing construction, stored cellulose materials, living trees, and communication facilities. it causes tremendous economic losses every year in china. therefore, people hope to have better method to control the damage of c. formosanus. at present, there are two potential approaches to the management of subterranean termite colony: exclusion and population suppression (su 1993). since the monitoring-baiting station was developed to detect and eliminate foraging population of subterranean termites (su 1995), this technolog y has been broadly used in subterranean termite management in china. however, the success of this technolog y relied greatly on the bait for termite control. unfortunately, there is no perfect bait in chinese market until now. in past decades, chinese termite control operators have been using dusting technolog y to control termite damage. under the condition without good baits, after the completely elimination of mirex in may 2009, they hope to have suitable alternatives to be used in the dusting technolog y. the 3% ivermectin dust was one of alternatives of 70% mirex dust. our test results indicated that 20~30 grams of 3% ivermectin dust could eliminate completely the formosan subterranean termite colonies within three and half to eight months when it was dusted onto the body of foraging workers feeding in four monitor devices at 18-80 meter far from the termite nests. clearly, 3% ivermectin dust could replace completely the 70% mirex dust and be used for subterranean termite control. lin et al. (2011) reported that when about 30 grams of 0.3% fipronil dust was applied onto termites feeding in a bait box, the whole colony of c. formosanus could be extinguished within one month. this means 3% ivermectin dust applied into four monitor devices needs longer time to eliminate completely a c. formosanus colony compared with 0.3% fipronil dust applied in a bait box. the reason caused this phenomenon could be that there are more termites in a bait box than in four monitor devices. in the near future, the effect of 3% ivermectin dust and 0.3% fipronil dust to control formosan subterranean termites feeding in monitor devices should be further evaluated. 1373 zhao, j. et al. — vermectin dust for coptotermes termite control acknowledgments this work was supported financially by the natural science foundation of china (project no.: 31170611) and the zhejiang provincial natural science foundation of china (project no.: z3100211). references jiang, z.y., w.f. wu, j.q. wei, l. chen & j.c. mo 2011. evaluation of ivermectin for the control of odontotermes formosanus shiraki (isoptera: termitidae) and macrotermes barneyi light (isoptera: termitidae) in seedling nursery. sociobiolog y 57(2):355-360. kistner, d.h. 1985. a new genus and species of termitophilous aleocharinae from mainland china associated with coptotermes formosanus and its zoogeographical signifcance (coleoptera: staphylinidae). sociobiolog y 10(1):93-104. lin, a.s., l.h. feng, y.y. hu, a.j. yi, d.f. wu, l. chen & j.c. mo 2011. application of fipronil dust to control coptotermes formosanus shiraki (isoptera: rhinotermitidae) in trees. sociobiolog y, 57(3):519-525. mo, j.c., h.y. he, x.g. song, c.r. chen & j.a. cheng 2005. efficacy of ivermectin against reticulitermes flaviceps (oshima) (isoptera: rhinotermitidae). sociobiolog y 46(3):603-613. mo, j.c., z.y. wang, x.g. song, j.q. guo, x.x. cao & j.a. cheng 2006. effects of sublethal concentrations of ivermectin on behaviors of coptotermes formosanus shiraki (isoptera: rhinotermitidae). sociobiolog y 47(3):687-696. mo, j.c., z.y. wang, x.g. song, j.q. guo, y.g. gong, w.l. lu & a.g. nei & j.a. cheng 2006. combination of ivermectin and monitoring device for control of coptotermes formosanus shiraki (isoptera: rhinotermitidae) in field. sociobiolog y 47(3):915-926. potter, m. 1997. termites. in: hedges, s.a. (ed.). handbook of pest control. mallis g..i.e., cleveland, oh: 232–332. su, n.y. 1993. managing subterranean termite populations. proceeding of the first international conference on insect pests in the urban environment. in: wildey, k.b. & robinson w.h. (eds.). cambridge (united kingdom): international conference on insect pests in the urban environment: 45-50. su, n.y., thomas e.m. & ban p.m. 1995. monitoring-baiting station to detect and eliminate foraging population of subterranean termites (isoptera: rhinotermitidae) near structures. journal of economic entomolog y, 88(4):932-936. wang, x.j., j.q. wei, j.c. mo, w.g. mao & t.j ye 2007. the susceptibility of reticulitermes chinensis synder (isoptera: rhinotermitidae) to sulfluramid, imidacloprid and ivermectin. sociobiolog y 50(2):561-572. wang, z.y., j.q. guo, y.g. gong, w.l. lu, a.g. lei, w.j. sun & j.c. mo 2007. control of dam termites with a monitor-controlling device. sociobiolog y 50(2):399-408. doi: 10.13102/sociobiology.v61i4.348-354sociobiology 61(4): 348-354 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 the stingless bee fauna in brazil (hymenoptera: apidae) introduction biological collections are essential for studying and understanding biodiversity because they store not only the specimens themselves but also important information associated with those organisms (e.g. zaher & young, 2003; wheeler et al. 2012). the specimens preserved in collections and museums can be an important source of reliable information about the biodiversity, present and past, as well as historical biogeography, if they are adequately studied by taxonomists. the insects or hexapoda are the most diverse group of organisms, corresponding to ca. 60% of the known species and their representation in entomological collections can reach thousands of specimens mainly due to their abundance in nature (rafael, 2012). its large territorial extension and variety of biomes make brazil one of the most diverse country in terms of insects, housing an estimated >400,000 species (rafael, 2012). these organisms are important not only in the socio-economic field with direct, both positive or negative impact on human population, but mainly through the role insects play in maintaining many ecosystem services, serving as food to vertebrates and invertebrates, acting as decomposers, recycling abstract the stingless bee fauna currently known from brazil is summarized, including geographic records by brazilian states. a total of 244 valid species and about 89 undescribed forms, placed in 29 genera, are recorded for the country. the survey is based mainly on the catalogue of bees (moure’s bee catalogue) and specimens housed in the camargo collection – rpsp. an evaluation of the current taxonomic status and some short comments on biology are also included. sociobiology an international journal on social insects srm pedro article history edited by denise araujo alves, esalq-usp, brazil received 06 october 2014 initial acceptance 03 november 2014 final acceptance 01 december 2014 keywords meliponini, taxonomy, nests, geographic distribution, behavior. corresponding author silvia regina de menezes pedro dept. biologia, faculdade de filosofia, ciências e letras de ribeirão preto, ffclrp universidade de são paulo, usp av. bandeirantes, 3900, cep: 14040-901 ribeirão preto são paulo, brazil e-mail: silviarmp@ffclrp.usp.br nutrients, or maintaining the plant community by dispersing seeds and especially pollen (gullan & cranston, 2008). the hymenoptera concentrate the most important groups of pollinators, and researchers, governments and ngos have increasingly given their attention in the last two decades especially to the native bees in order to preserve them (e.g. international pollinator initiative – ipi and iniciativa brasileira de polinizadores, mma, 2006). some actions have been proposed in order to establish strategies for monitoring and conservation of these organisms, considering their relevance to both wild and cultivated plants (e.g. kevan & imperatriz-fonseca, 2002; imperatriz-fonseca et al., 2006). the stingless bees, or meliponini, a pan-tropical and very ancient group (camargo, 2008, 2013), deserve special attention as a valuable socio-economic resource. they contribute directly to the pollination of important cultivated plants such as strawberry, “cupuaçu”, “camu-camu”, tomato, avocado, cucumber and others, by rising crop yields and improving fruits quality (e.g. roubik, 1995; slaa et al. 2006). also, these often mild-mannered bees are easily reared and have become an important source of income for beekeepers in the recent decades, by producing mainly honey and wax (e.g. nogueira-neto, 1970, 1997; cortopassi-laurino et review universidade de são paulo, ffclrp, ribeirão preto, são paulo, brazil sociobiology 61(4): 348-354 (december 2014) 349 al., 2006; contrera et al, 2011; alves, 2013). in addition to the importance of the “honey-pot” (term coined by p. vit in order to point out the differences between the honey produced by stingless bees and that produced in combs by apis, especially a. mellifera l.;vit, 2013; vit et al., 2013a; alves, 2013) as food, the historical and socio-cultural aspects of the traditional and medicinal uses of the by-products of the stingless bees are also worthy of notice (e.g. schwarz, 1948; camargo & posey, 1990; engels, 2013; jones, 2013; ocampo rosales, 2013; quintal & roubik, 2013; vit et al. 2013b; zamora et al., 2013). concerning the importance and diversity of the stingless bees – 417 species for the neotropical region up to the last update of the moure’s bee catalogue (camargo & pedro, 2007a, 2013), in addition to others still not described, a panorama of the stingless bee fauna currently living in brazil is presented, including geographic records by brazilian states. material and methods the survey of species that occur in the brazilian territory was based mainly on the material housed in the camargo collection – rpsp, department of biology, faculty of philosophy, sciences and letters of ribeirão preto, university of são paulo, with reliable determinations made by j.m.f. camargo, j.s. moure or by the author, in addition to material studied by the author more recently. most of information presented here was already compiled in the moure bee catalogue (camargo & pedro, 2007a, 2013) and recent taxonomic revisions (camargo & moure, 1994, 1996; camargo, 1996; camargo & pedro, 2003, 2004, 2005, 2008, 2009; pedro & camargo, 2003, 2009; marchi & melo, 2006; albuquerque & camargo, 2007). information from other articles with taxonomic notes such as melo (2013) was also considered. results and discussion a total of 244 valid species, and about 89 undescribed forms (species already recognized by the author, but which have not been published yet), affiliated to 29 genera, are recorded for brazil (table 1). about 87 are endemic to brazil, corresponding to ca. 20 % of the estimated neotropical stingless bee species. two genera are currently known only from brazil, friesella and the singular trichotrigona (camargo & pedro, 2007b). of course, the results presented here will be drastically changed with the taxonomic revision of some diversified genera that need reevaluation, mainly plebeia, trigona, melipona, scaptotrigona and trigonisca, which include many undescribed species. unfortunately, good taxonomists are vanishing and few students show interest for classical taxonomy which demands time and dedication, besides background in different areas of knowledge, mainly morphology. it is also worth noting that in addition to the taxonomic diversity, the stingless bees exhibit a large diversity of behaviors and ways of life (e.g. camargo, 2008, 2013), including cleptobiosis (nogueira-neto, 1970), necrophagy (camargo & roubik, 1991), mutualistic association with coccids (camargo & pedro, 2002) and yeast (camargo & pedro, 2004), as well as other associations yet poorly studied, such as for trichotrigona (camargo & pedro, 2007b). the diversity of architectural solutions and use of different substrates, even including gigantic nests of aggressive bees of the genus trigona, made from the bees’ own pollen feces (roubik & moreno, 2009), and association with nests of other organisms such as ants, wasps, termites and birds to construct their nests is impressive (e.g. camargo & pedro, 2003; rasmussen & camargo, 2008; and compilation by roubik, 2006). all this biological and taxonomic diversity exhibited by the stingless bees living in brazil make them amazing objects of study. table 1. stingless bees (meliponini) found in the brazilian territory. genus number of valid species and additional undescribed forms [indicated in square brackets] species found in the brazilian territory (†) total occurring in brazil aparatrigona 2 1 impunctata (ducke, 1916) (ac, ap am, mt, pa, ro, rr) camargoia 3 3 camargoi moure, 1989 (ap, am) nordestina camargo, 1996 $ (ba, ce, pi, to) pilicornis (ducke, 1910) $ (ma, pa) celetrigona 4 4 euclydiana camargo & pedro, 2009 (ac) hirsuticornis camargo & pedro, 2009 $ (ac, am, mt, ro) longicornis (friese, 1903) (am, go, ma, mt, pa, ro) manauara camargo & pedro, 2009 (ap, am, pa) cephalotrigona 5 2 [+1] capitata (smith, 1854) # (ap, ce, es, mt, mg, pa, pr, sc, sp) femorata (smith, 1854) (ap, am, ma, pa, ro) dolichotrigona 10 7 browni camargo & pedro, 2005 (ac, mt, ro) clavicornis camargo & pedro, 2005 $ (ac, am, ro) longitarsis ((ducke, 1916) (ac, am, ma, mt, pa, ro) mendersoni camargo & pedro, 2005 $ (ac, am, ro) moratoi camargo & pedro, 2005 $ (ac, am) rondoni camargo & pedro, 2005 $ (ro) tavaresi camargo & pedro, 2005 $ (ac, am) duckeola 2 2 ghiliani (spinola, 1853) (ap, am, mt, pa, ro) pavani (moure, 1963) (am) friesella 1 1[+1] schrottkyi (friese, 1900) $ (es, mg, pr, sp) frieseomelitta 16 13 [+5] dispar (moure, 1950) $ (ba, es, mg, pb) doederleini (friese, 1900) $ (ba, ce, ma, mt, mg, pb, pe, pi, rn) flavicornis (fabricius, 1798) (ap, am, pa, rr) francoi (moure, 1946) $ (ba, es, pb, pe, se) freiremaiai (moure, 1963) #(ba, es) languida moure, 1990 $ (ba, go, mg, sp) longipes (smith, 1854) # (pa) meadewaldoi (cockerell, 1915) # (*) paranigra (schwarz, 1940) (am) portoi (friese, 1900) (am, ma, pa) silvestrii (friese, 1902) (mt) trichocerata moure, 1990 # (ap, am, pa) varia (lepeletier, 1836) (ba, go, mt, mg, sp, to) srm pedro stingless bees in brazil350 geotrigona 22 10 [+1] aequinoctialis (ducke, 1925) $ (ce, ma, pa) fulvohirta (friese, 1900) (ac, am) kwyrakai camargo & moure, 1996 $ (pa, ro) mattogrossensis (ducke, 1925) (mt, pa, ro) mombuca (smith, 1863) (ba, go, ma, mt, ms, mg, pa, pi, sp, to) subfulva camargo & moure, 1996 $ (am) subgrisea (cockerell, 1920) (rr) subnigra (schwarz, 1940) (ap, am, pa) subterranea (friese, 1901) $ (ba, mg, pr, sp) xanthopoda camargo & moure, 1996 $ (pb, pe) lestrimelitta 23 14 ciliata marchi & melo, 2006 $ (pa) ehrhardti (friese, 1931) $ (al, es, mg, pr, rj, sc, sp) glaberrima oliveira & marchi, 2005 (ap) glabrata camargo & moure, 1996 (ac, am, mt, rr) limao (smith, 1863) $ (ba, ce, df, go, ma, mg, ro, sp) maracaia marchi & melo, 2006 $ (am, ro, rr) monodonta camargo & moure, 1989 $ (am, ma, pa, rr) nana melo, 2003 $ (ap) rufa (friese, 1903) (am, mt, pa, ro) rufipes (friese, 1903) (am, ba, ce, es, go, ma, mt, mg, pa, pr, pi, rs, ro, rr, sc, sp, to) similis marchi & melo, 2006 $ (pa) spinosa marchi & melo, 2006 (pa) sulina marchi & melo, 2006 (pr, rs, sc) tropica marchi & melo, 2006 $ (ba, ce, rj) leurotrigona 4 3 gracilis pedro & camargo, 2009 $ (ac, am, ro) muelleri (friese, 1900) #(ba, es, go, ma, mt, ms, mg, pr, pb, ro, sc, sp) pusilla moure & camargo, in moure et al., 1988 (ap, am, pa) melipona 74 40 m. (eomelipona) (15) 10 [+2] amazonica schulz, 1905 $ (ac, ap, am, pa, ro) asilvai moure, 1971 $ (al, ba, ce, mg, pb, pe, pi, rn, se) bicolor lepeletier, 1836 (ba, es, mg, pr, rj, rs, sc, sp) bradley schwarz, 1932 (ap, am, mt, pa, ro, rr) illustris schwarz, 1932 (pa, mt) marginata lepeletier, 1836 $ (ba, ce, es, go, mg, rj, sp) ogilviei schwarz, 1932 (ap, am, pa, to) puncticollis friese, 1902 (am*, ma, pa) torrida (= m. obscurior) friese, 1916 (mt*, pr, rs, sc, sp); see melo, 2013 tumupasae schwarz, 1932 (ac) m. (melikerria) (10) 5 [+1] compressipes (fabricius, 1804) (ap, am, rr) fasciculata smith, 1854 $ (ma, mt, pa, pi, to) grandis guérin, 1834 (ac, am, mt, ro) interrupta latreille, 1811 (ap, am, pa) quinquefasciata lepeletier, 1836 #(ce, df, es, go, mt, ms, mg, pr, rj, rs, ro, sc, sp) m. (melipona) (13) 5 [+1] favosa (fabricius, 1798) (rr) mandacaia smith, 1863 $ (al, ba, ce, pb, pe, pi, rn, se) orbignyi (guérin, 1844) (mt, ms) quadrifasciata lepeletier, 1836 (al, ba, es, go, ms, mg, pb, pr, pe, rj, rs, sc, sp, se) subnitida ducke, 1910 $ (al, ba, ce, ma, pb, pe, pi, rn, se) m. (michmelia) (36) 20 [+10] brachychaeta moure, 1950 (mt, ro) captiosa moure, 1962 (ap, am) capixaba moure & camargo, 1994 $ (es) cramptoni cockerell, 1920 (rr) crinita moure & kerr, 1950 (ac, am, ro) dubia moure & kerr, 1950 $ (ac, am, ro) eburnea friese, 1900 (ac, am) flavolineata friese, 1900 # $ (ce*, ma, pa, to) fuliginosa lepeletier, 1836 (ac, am, ba, es, mt, pa, pi*, sp) fulva lepeletier, 1836 (ap, am, pa, rr) fuscopilosa moure & kerr, 1950 (ac, am) lateralis erichson, 1848 (ap, am, pa, rr) melanoventer schwarz, 1932 $ (ac*, am, ma, mt, pa, ro) mondury smith, 1863 $ (ba, es, mg, pr, rj, rs*, sc, sp) nebulosa camargo, 1988 (ac, am, pa) paraensis ducke, 1916 (ap, am, pa) rufiventris lepeletier, 1836 # $ (ba, go, mt, ms, mg, pi, sp) scutellaris latreille, 1811 $ (al, ba, ce, pb, pe, rn, se) seminigra friese, 1903 # (ac, am, ma, mt, pa, ro, rr, to) titania (gribodo, 1893) (am) meliwillea 1 0 mourella 1 1 caerulea (friese, 1900) (pr, rs, sc) nannotrigona 10 7 [+1] chapadana (schwarz, 1938) (go, mt) dutrae (friese, 1901) $ (pa) melanocera (schwarz, 1938) (ac, am) minuta (lepeletier, 1836) # $ (pa*) punctata (smith, 1854) (ap, pa) schultzei (friese, 1901) (ap, am, pa) testaceicornis (lepeletier, 1836) (ba, es, go, ms, mg, pr, rj, rs, sc, sp) nogueirapis 4 2 [+1] butteli (friese, 1900) (am) minor (moure & camargo, 1982) (ap, am) oxytrigona 11 5 [+5] flaveola (friese, 1900) # (es) ignis camargo, 1984 $ (am, ma, pa) mulfordi (schwarz, 1948) (ac, ro) obscura (friese, 1900) (ap, am, mt, pa, ro) tataira (smith, 1863) # (ba, es, ms, mg, pr, rj, sc, sp) parapartamona 7 0 paratrigona 32 16 [+3] catabolonota camargo & moure, 1994 $ (am) compsa camargo & moure, 1994 $ (am) crassicornis camargo & moure, 1994 $ (pa) euxanthospila camargo & moure, 1994 $ (am) table 1. stingless bees (meliponini) found in the brazilian territory. (cont.) genus number of valid species and additional undescribed forms [indicated in square brackets] species found in the brazilian territory (†) total occurring in brazil sociobiology 61(4): 348-354 (december 2014) 351 femoralis camargo & moure, 1994 (ap)*** haeckeli (friese, 1900) (mt, pa, ro) incerta camargo & moure, 1994 $ (ba, mg, pb) lineata (lepeletier, 1836) # (ba, ce, go, ma, mt, mg, pa, pb, pe, pr, pi, sp, to) lineatifrons (schwarz, 1938) $ (am, pa) melanaspis camargo & moure, 1994 $ (am) nuda (schwarz, 1943) (ac) myrmecophila moure, 1989 $ (ro) pacifica (schwarz, 1943) (ac, ro) pannosa moure, 1989 (ap, am, pa) peltata (spinola, 1853) $ (ma, pa) prosopiformis (gribodo, 1893) (ac, am, pa, ro) subnuda moure, 1947 $ (ba, mg, pa, pr, rj, rs, sc, sp) paratrigonoides 1 0 partamona 32 23 [+3] ailyae camargo, 1980 (ac, am, ce, go, ma, mt, ms, mg, pa, pi, ro, sp, to) auripennis pedro & camargo, 2003 (ap, am, pa) batesi pedro & camargo, 2003 $ (ac, am) chapadicola pedro & camargo, 2003 $ (ma, pa, pe, pi, to) combinata pedro & camargo, 2003 (ac, df, go, ma, mt, ms, mg, pa, ro, sp, to) criptica pedro & camargo, 2003 $ (es, mg, rj, sp) cupira (smith, 1863) $ (df, go, ms, mg, sp) epiphytophila pedro & camargo, 2003 (ac, am) ferreirai pedro & camargo, 2003 (ap, am, pa, rr) gregaria pedro & camargo, 2003 $ (am, pa) helleri (friese, 1900) $ (ba, es, mg, pr, rj, sc, sp) littoralis pedro & camargo, 2003 $ (pb, rn) mourei camargo, 1980 (am, pa, rr) mulata moure, in camargo, 1980 (mt, ms) nhambiquara pedro & camargo, 2003 (go, mt, ms, pa, ro) nigrior cockerell, 1925) (rr) pearsoni pedro & camargo, 2003 (ap, am, ma, pa) rustica pedro & camargo, 2003 $ (ba, mg) seridoensis pedro & camargo, 2003 $ (ce, ma, pb, pe, rn) sooretamae pedro & camargo, 2003 $ (ba, es) subtilis pedro & camargo, 2003 (ac) testacea (klug, 1807) (ac, ap, am, ce, ma, pa, ro) vicina camargo, 1980 (ac, ap, am, mt, pa, ro, rr) plebeia 40 19 [+10] alvarengai moure, 1994 $ (am, mt, pa, ro) catamarcensis (holmberg, 1903) (ms, rs) droryana (friese, 1900) # (ba, es, mg, pa, pe, rj, rs, sc, sp) emerina (friese, 1900) (pr, rs, sc, sp) flavocincta (cockerell, 1912) $ (ba, pb, pe, pi) grapiuna melo & costa, 2009 $ (ba) julianii moure, 1962 # (pr) lucii moure, 2004 $ (es, mg) margaritae moure, 1962 (am, mt, ro) meridionalis (ducke, 1916) # (es, mg, pr, rj) minima (gribodo, 1893) # (ac, ap, am, ma, mt, pa) mosquito (smith, 1863) $ # (mg, rj) nigriceps (friese, 1901) (pr, rs, sc, sp) phrynostoma moure, 2004 $ (es, mg) poecilochroa moure & camargo, 1993 $ (es, mg) remota (holmberg, 1903) (es, mg, pr, rs, sc, sp) saiqui (friese, 1900) $ (mg, pr, rs, sc, sp) variicolor (ducke, 1916) (am, pa, ro) wittmanni moure & camargo, 1989 (rs) proplebeia 4 0 ptilotrigona 3 2 lurida (smith, 1854) (ac, ap, am, ma, mt, pa, ro, rr) pereneae (schwarz, 1943) (ac, am) scaptotrigona 22 9 [+10] affabra (moure, 1989) $ (pa, ro) bipunctata (lepeletier, 1836) (ac, ce, ma, mg, pa, pr, rj, rs, sc) depilis (moure, 1942) (ms, mg, pr, rs, sp) fulvicutis (moure, 1964) (ap, am) polysticta moure, 1950 (ac, go, ma, mt, mg, pa, pi, ro, sp, to) postica (latreille, 1807) # (pa)* tricolorata camargo, 1988 (am, mt, ro) tubiba (smith, 1863) # $ (es, mg, rj, sp)* xanthotricha moure, 1950 $ (ba, es, mg, pr, rj, sc, sp) scaura 5 4 [+2] atlantica melo, 2004 $ (ba, es, mg) latitarsis (friese, 1900) # (ac, ap, am, mg, pa, pr, rj, ro, sp) longula (lepeletier, 1836) # (ap, am, ba, go, ma, mt, mg, pa, sp) tenuis (ducke, 1916) (am, mt, pa) schwarziana 2 2 [+2] see melo, 2003 mourei melo, 2003 (go, ms, mg, to) quadripunctata (lepeletier, 1836) (ba, es, go, mg, pr, rj, rs, sc, sp) schwarzula 2 2 coccidophila camargo & pedro, 2002 (ac, am, ro) timida (silvestre, 1902) (ac, am, mt, ms, mg, pa, ro, sp) tetragona 13 10 [+5] beebei (schwarz, 1938) (am, pa) clavipes (fabricius, 1804) # (rr) dorsalis (smith, 1854)# (ap, am, ce, ma, pa, ro) elongata lepeletier & serville, 1828 # **(sp, mg) essequiboensis (schwarz, 1940) (am, ro) goettei (friese, 1900) (ac, am, mt, pa, ro) handlirschii (friese, 1900) (ap, am, pa, rr) kaieteurensis (schwarz, 1938) (am, pa) quadrangula $ (go, ma, mg, mt, pa, sp, to) truncata moure, 1971 (am, go, ma, mt, pa, ro) tetragonisca 4 3 [+2] angustula (latreille, 1811) # (brazil) fiebrigi (schwarz, 1938) (mt, ms, pr, rs, sp) weyrauchi (schwarz, 1943) (ac, mt, ro) trichotrigona 1 1 [+1] extranea camargo & moure, 1983 $ (am) trigona 32 21 [+12] albipennis almeida, 1995 # (ac, am, mt, pa, ro) amalthea (olivier, 1789) (am, mt, ro) amazonensis (ducke, 1916) (ac, am, mt, pa, ro, to) branneri cockerell, 1912 (am, go, ma, mt, pa, ro, to) table 1. stingless bees (meliponini) found in the brazilian territory. (cont.) genus number of valid species and additional undescribed forms [indicated in square brackets] species found in the brazilian territory (†) total occurring in brazil srm pedro stingless bees in brazil352 braueri friese, 1900 (ba, es, pr, rj, sp) chanchamayoensis (ac, am, mt, pa, ro) cilipes (fabricius, 1804) (ac, ap, am, go, mg, mt, pa, ro, rr, sp) crassipes (fabricius, 1793) (ap, am, mt, pa) dallatorreana friese, 1900 (ap, am, ma, mt, pa, ro, to) dimidiata smith, 1854, (am, mt, pa, ro) guianae cockerell, 1910 # (ac, ap, am, ce, mt, pa, pb, ro, to) hyalinata (lepeletier, 1836) (ba, df, go, ma, mt, ms, mg, pa, pi, sp, to) hypogea silvestri, 1902 # (am, ma, mt, pa, sp) lacteipennis friese, 1900 (ac, am, go, mt, pa, ro, rr) pallens (fabricius, 1798) # (ac, ap, am, go, ma, pa, ro, rr, to) pellucida cockerell, 1912 $ (mt, pa, ro) recursa smith, 1863 # (ac, am, ce, go, ma, mt, mg, pa, pi, ro, sp, to) sesquipedalis almeida, 1984 (ap) spinipes (fabricius, 1793) # (al, ba, ce, es, go, ma, mt, ms, mg, pa, pb, pr, pe, pi, rj, rn, rs, sc, sp, se, to) truculenta almeida, 1984 (ac, ap, am, ba, go, ma, mt, ms, mg, pa, ro, sp) williana friese, 1900 (ac, ap, am, ma, mt, pa, ro, rr) trigonisca 25 16 [+10] bidentata albuquerque & camargo, 2007 $ (ro) ceophloei (schwarz, 1938) (am) dobzhanskyi (moure, 1950) (am, pa) duckei (friese, 1900) (am, ce, ma, mt, pa, rr) extrema albuquerque & camargo, 2007 $ (am) flavicans (moure, 1950) $ (am) fraissei (friese, 1901) $ (am, mt, pa, ro) graeffei (friese, 1900) (am) hirticornis albuquerque & camargo, 2007 $ (ro) intermedia moure, 1900 $ (ba, es, mt, mg, sp) meridionalis albuquerque & camargo, 2007 $ (ma, mt, mg, pa, sp) nataliae (moure, 1950) $ (ma, mt, pa, ro) pediculana (fabricius, 1804) (ba, ce, ma, pb) unidentata albuquerque & camargo, 2007 $ (am) variegatifrons albuquerque & camargo, 2007 $ (mt, pa, ro) vitrifrons albuquerque & camargo, 2007 $ (am, pa) total 418 244 [+89] # taxonomic reevaluation is needed or it is currently under revision, in such a way that the geographic distribution is not well defined (only the type locality is reliable); $ species found only in the brazilian territory; * geographic record uncertain; ** tetragona elongata (sm.) was considered as a junior synonym of tetragona clavipes in moure’s bee catalogue, but the name is being validated here in order to include the form from southeastern brazil, previously determined as t. clavipes group or t. aff. clavipes or t. cf. clavipes or even as t. clavipes (fab.); *** gabriel a. r. melo, personal information. (†) brazilian states: acre (ac); alagoas (al); amapá (ap); amazonas (am); bahia (ba); ceará (ce); espírito santo (es); goiás (go); maranhão (ma); mato grosso (mt); mato grosso do sul (ms); minas gerais (mg); pará (pa); paraíba (pb); paraná (pr); pernambuco (pe); piauí (pi); rio de janeiro (rj); rio grande do norte (rn); rio grande do sul (rs); rondônia (ro); roraima (rr); santa catarina (sc); são paulo (sp); sergipe (se); tocantins (to). table 1. stingless bees (meliponini) found in the brazilian territory. (cont.) acknowledgments the author is grateful for the improvement of the manuscript through the many corrections and suggestions by the anonymous referees. references albuquerque, p.m.c. & camargo, j.m.f. (2007). espécies novas de trigonisca moure (hymenoptera, apidae, apinae). rev. bras. ent., 51: 160-175. alves, r.m.o. 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(2003). as coleções zoológicas brasileiras: panorama e desafios. ciênc. cult., 55: 24-26. zamora, g., arias, m.l., aguilar, i. & umaña, e., 2013. costa rican pot-honey: its medicinal use and antibacterial effect. in p. vit, s.r.m. pedro & d.w. roubik (eds.), pot honey: a legacy of stingless bees (pp. 507-512). new york: springer. doi: 10.1007/978-1-4614-4960-7_37 doi: 10.13102/sociobiology.v62i1.82-87sociobiology 62(1): 82-87 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 antifeedant and repellent effects of neotropical solanum extracts on drywood termites (cryptotermes brevis, isoptera: kalotermitidae) introduction solanaceae is a cosmopolitan plant family, being particularly varied and abundant in tropical and subtropical regions of central and south america (gutiérrez et al., 1999). they are known for producing a wide range of toxic molecules (pomilio et al., 2008). despite these secondary metabolites, some also have properties with low insecticidal activity, e.g., sugar esters (tingey & laubengayer, 1986; hori et al., 2011). particularly, species belonging to the genus solanum produce a vast array of chemically diverse bioactive secondary metabolites. solanum is heterogeneous and widely distributed in the new world (vázquez, 1997). most species of the genus have trichomes on stems, leaves, and inflorescences (mentz et al., 2000) which produce chemical substances with anti-insect activity (silva et al., 2003; silva et al., 2005; lovatto & thomé, 2004; leite, 2004; srivastava & gupta, 2007; szafranek et al., 2008). glandular trichomes from s. berthaultii have been used as morphological abstract antifeedant and repellent effects of two different extracts from native solanum species, s. bistellatum and s. sisymbrifolium on cryptotermes brevis were evaluated. the extracts obtained, particularly the dichloromethane extracts and the enriched fraction of sugar esters from both species, showed antifeedant and repellent activity against the termite. the antifeedant effect of dichloromethane extract from s. sisymbrifolium at the concentration of 25mg/ml reached 100%, while the repellent action of the dichloromethane extracts and the dichloromethane-acetone extract for sugar esters (enriched fraction of sugar esters) at 1mg/ml was greater than 90%. in the case of s. bistellatum, the antifeedant effect of the dichloromethane extract and dichloromethane-acetone extract for sugar esters at the concentration of 25.00mg/ml was 43% and 57%, respectively. the repellent action of the dichloromethane extracts and of the enriched fraction of sugar esters at a concentration level of 2.5mg/ml was greater than 92%. sociobiology an international journal on social insects v rech-cainelli1, nm barros1, sg giani1, ac sbeghen-loss1, h heinzen2, ar diaz2, i migues2, a specht3, mv cesio2 article history edited by rudolf h scheffrahn, ufl, usa received 23 july 2014 initial acceptance 02 september 2014 final acceptance 12 december 2014 keywords cryptotermes brevis; pest control; solanum species; bioactive compounds; sugar esters. corresponding author maría verónica cesio cátedra de farmacognosia y productos naturales, facultad de química universidad de la república av. gral. flores 2124, 11800 montevideo, uruguay e-mail: cs@fq.edu.uy markers to denote resistance to aphids in new potato cultivars. these trichomes produce sugar esters that are well known for their anti-insect properties (yencho et al., 1993). the bioactivity of a new family of sugar esters isolated from s. sisymbrifolium type iv trichomes leaves was reported by cesio et al. (2006). as s. sisymbrifolium is a widely distributed weed that produces relatively high amounts of such compounds, it was planned to evaluate the antitermitic activity of these compounds. the relationship between chemical family and biological activity deserves more in-depth investigation. therefore, we searched latin american solanum species for leaf morphology and chemical composition of the obtained extracts that might yield secondary compounds with similar structural features like those described in s. sisymbrifolium (cesio et al., 2006). termites are among the major pests of wood, and particularly the west indian drywood termite, cryptotermes brevis walker (isoptera: kalotermitidae), which is endemic to latin america (constantino, 2002), and has been disseminated research article termites 1 university of caxias do sul, caxias do sul, rs, brazil 2 universidad de la república, montevideo, uruguay 3 embrapa cerrados, planaltina, df, brazil sociobiology 62(1): 82-87 (march, 2015) 83 unintentionally into many regions of the world in boats and wooden packaging material (scheffrahn et al., 2009), damaging furniture, buildings, and even the wooden statues from ouro preto among other masterpieces in brazil. the most widely used method to control termites is based on the application of chemical insecticides which might possess negative effects on humans and the environment (cabrera et al., 2001; stephenson & solomon, 2007). alternative control strategies, like biological control or the use of less toxic plant extracts, (smith, 1989; harborne, 2001) should be considered. nevertheless, little bio-prospecting for active plant extracts against termites is reported in the literature despite the great potential that the unexplored flora of latin america might possess. using a chemotaxonomic approach to find bioactive compounds from plants against termites, it was hypothesed that the solanaceae family could be a source of new compounds due to its high chemodiversity. the objective of the present study was to assess the antifeedant and repellent effects of leaf extracts from s. bistellatum and s. sisymbrifolium on c. brevis, from the acyl sugars secreted by type iv trichomes. material and methods plant material solanum sisymbrifolium samples were collected in montevideo, uruguay, and s. bistellatum in rio grande do sul, brazil. the species were labeled as vouchers 3520 and 4327, respectively, and kept at the jose arechavaleta herbarium in the faculty of chemistry, udelar, uruguay. extracts three different extracts were obtained from each plant species, as follows: 1) dichloromethane extract: 300 g of fresh leaves were immersed portion wise in 1000 ml of dichloromethane for 30s. the dichloromethane solutions were evaporated under reduced pressure to dryness. 2) dichloromethaneacetone extract for sugar esters: 1g of the dichloromethane extract was dissolved in acetone (100 ml) and the solution was cooled to -20°c and kept overnight at this temperature. the resulting precipitate was discarded and the acetone extract was evaporated under reduced pressure to dryness to yield the extract containing the enriched fraction of sugar esters. 3) aqueous-ethanolic extract: the remaining plant material from the first procedure was immersed in an ethanol/water (70:30) solution for 24h. the ethanol solution was filtered and evaporated under reduced pressure to yield the final extract. crude extract composition ultrapure reagents (sigma aldrich) were used in the analysis. thin-layer chromatography (tlc) was conducted using polygram sil/uv 254 on 0.25-mm-layered plates (macherey-nagel). nmr spectra were measured using a bruker advance 400mhz spectrometer. standard pulse sequences were used for the different nmr experiments. samples were dissolved in cdcl3, (sigma-aldrich). gas chromatography-mass spectrometry (gcms) was performed with an hp 6890/6973 using an hp-5 25-m-long, 0.5 mm (i.d.) capillary column. the temperature program used was ti=60ºc for 10 min, 10 ºc/min to tf=280 and hold for 20min. the conditions of the ms quadruple were the standard. the compounds were identified using the nist library with a si > 90%. all solutions were evaporated under reduced pressure at ≤ 60°c. chemical analysis the phytochemical study was based on bioguided fractionation (hostettmann, 1999) aiming to characterize the most bioactive fractions but also taking into account our previous findings on the bioactivity of sugar esters and the extracts that contain them (cesio et al., 2006; dutra et al., 2008). thin-layer chromatography was performed with the different extracts obtained from s. bistellatum and s. sisymbrifolium, using ch2cl2/meoh (9:1) as the mobile phase. compounds were detected by spraying tlc plates with a universal dye reagent of 5% cuso4 in 10% aq. h3po4 and subsequent heating in oven at 20-140ºc (4ºc/min). specific dyeing reagent used for sugars was diphenylamine, aniline, and acetone phosphoric acid (anonymous, 1974). the major compounds of s. bistellatum were isolated from the enriched fraction of sugar esters by column chromatography. fifty mg of extract was seeded at the top of an open column packed with 10 g of silica gel (macherey-nagel mn, 60). elution was performed using a solvent gradient of increasing polarity: 30ml of dichloromethane; 50ml of dichloromethane/methanol (9.5:0.5); 50ml of dichloromethane/ methanol (9:1); dichloromethane/methanol (8.5:1.5). fractions of 2ml were collected and their composition was confirmed with tlc using the same conditions stated above. the fractions containing the same compound were combined, the solvent was evaporated under reduced pressure, and structural elucidation using nmr analysis was performed. the fatty acid composition of the acyl sugars was investigated following the procedure described by dutra et al. (2008). one to 10mg of the sugar ester was treated at room temp. with 0.5ml of 0.5n methanolic koh for 15min. one ml of 1n methanolic hcl was added and the solution left at room temp. overnight. the precipitated kcl was then filtered off and the methanol evaporated to dryness. the residue was redissolved in acoet and analyzed by gcms. insects cryptotermes brevis pseudergates were collected from wooden houses located in the state of rio grande do sul, brazil. the wood was collected and stored in plastic containers covered with cloth. the wood pieces were stored under appropriate conditions of temperature and humidity for termites (24 ± 1ºc and 70± 5% rh, respectively). v rech-cainelli et al. – antifeedant and repellent effects of solanum extracts on c.brevis84 bioactivity of solanum spp. extracts on c. brevis bioactivity was assessed following the methodology described by sharma and raina (1998), with a focus on antifeedant and repellent activities. to obtain the required concentrations, the dichloromethane extract and the enriched fraction of sugar esters were diluted in acetone, while the aqueous-ethanol extract was diluted in water. in all assays, 1ml of the extract solutions was applied to filter paper discs (9.0cm diam.). control groups consisted of filter paper discs treated with solvent only. to evaporate the solvent, discs were maintained under a laminar-flow hood for 48h. the discs were then placed in petri dishes to assess the behavioral response of 30 c. brevis pseudergates which were placed in the bioassay units. bioassays were run for 30 days in five replicates under appropriate conditions of temperature and humidity for termites (24 ±1ºc; 70 ±5% rh; 24h scotophase). antifeedant activity substrate consumption was determined by mass loss of filter paper exposed to the termites where: [(initial mass = post-impregnation with the product) – (final mass = postexposure to the termites)]/initial mass x 100. the difference in mass considered filter paper discs dried (60ºc) for 4h and weighed with an analytical balance. feeding inhibition (fi) of each extract concentration (2.5, 12.5, and 25 mg/ml) was determined by the equation (fi%) = (control disc consumption – treated disc consumption/control disc consumption + treated disc consumption) x 100 (simmonds et al., 1990). the concentration per unit (mg/cm2) area was calculated using the following formula: c(mg/cm2)=c(mg/ml)/πr2. in our case, values for the antifeedant assay were c= 2.5, 12.5, and 25 mg/ml/3.14159 x (4.5)2 and for the repellent assay c= 2.5, 12.5, and 25 mg/ ml/3.14159 x (4.5)2. repellency repellency was assessed by gravimetric measurement of termite feeding on the treated or untreated paper. filter paper discs were cut into two halves of which one-half was treated with an extract and the other half with solvent only. thirty termites were placed in the middle of the plate and their location counted every four days for 30 days. the following concentrations of the extract were used: 0.25, 1, and 2.5 mg/ml. repellency percentage (rp) was calculated daily using the following formula of measurement and finally the data pool was statistically calculated with: rp = (ca – ta) / (ca + ta) x 100, where ca = number of termites present on control area and ta = number of termites present on treated area. negative values for rp were considered as null (mcdonald et al. 1970). statistical analysis was performed using parametric tests and spss software version 18.0 for windows. data were analyzed using two-way anova followed by tukey’s test. results and discussion the antifeedant effect of extracts on c. brevis showed varying results. the aqueous-ethanol extract had little or no effect on filter paper consumption (table 1), but both organic extracts showed significant antifeedant effects. the acetone fraction from the dichloromethane extract from s. bistellatum had a higher antifeedant effect than the crude dichloromethane extract, whereas the opposite was observed for the extracts obtained from s. sisymbrifolium. the dichloromethane extract and the enriched fraction of sugar esters of s. bistellatum showed mild antifeedant activity (30-59%) on c. brevis. on the other hand, the aqueous-ethanol extract showed phagostimulant activity at concentrations of 12.5 and 25 mg/ml. the dichloromethane extract of s. sisymbrifolium inhibited feeding completely at 25 mg/ml concentration, but the enriched fraction of sugar esters at the highest concentration produced an antifeedant effect of 78% (table 1). plant speciesextract concentration extract dichloromethanedichlomethane-acetone*water-ethanol s. bistellatum 2.50 mg/ml31.67 % aa32.46 % ab9.92 % ba 12.50 mg/ml39.81 % aa52.80 % aa-6.9 % ba 25.00 mg/ml43.29 % aa57.46 % aa-1.4 % ba s. sisymbrifolium 2.50 mg/ml 10.79 % ab16.23 % ab-7.0 % bb 12.50 mg/ml 86.91 % aa63.85 % aa9.96 % ba 25.00 mg/ml 100 % aa78.20 % aa8.66 % ba table 1. antifeedant index (%) shown by c. brevis on filter paper treated with different solanum spp. extracts. * enriched fraction of sugar esters mean of five replicates (n=30 termites per replicate). values followed by the same letter,in lines(capital letters) for each plant extract and concentration in columns (lower case letters), are not different from each other (tukey’s test, p<0.05). sociobiology 62(1): 82-87 (march, 2015) 85 the effects of solanaceae plant extracts on brevicoryne brassicae (cabbage aphid) were assessed by lovatto et al. (2004), who reported s. sisymbrifolium as the only species with significant repellent activity with a 5% flower extractbeing the most effective. the glandular trichomes present in solanaceae are related to the plants resistance against herbivore attacks. investigations of the leaf surface of salpichroa origanifolia (solanaceae) identified type iv trichomes, while chemical investigations confirmed the presence of acyl sugars with antifungal activity levels similar to those of agrochemicals (dutra et al., 2008). the bioactivity of the assayed fractions from the obtained extract from the leaf surface of s. tuberosum on the beetle leptinotarsa decemlineata is deterrent or phagostimulant, as reported by szafranek et al. (2008). the repellency had similar results in both cases. dichloromethane extracts and their acetone soluble fraction of the two solanum species evaluated in the present study showed repellency against c. brevis, particularly at their highest evaluated concentration (table 2). solanum sisymbrifolium was the most active of the dichloromethane extracts at low concentration. the dichloromethane-acetone extract for sugars ester from s. sisymbrifolium showed an increased repellent effect on c. brevis. the same extracts of s. bistellatum and s. sisymbrifolium showed a repellent effect >90% at 2.5 mg/ml.the solanum bistellatum dichloromethane-acetone extract contained a 50% acyl sugar compounds and the s. sisymbrifolium extract contained up to 70% of acylsugars and showed high repellent activity. the dichloromethane extracts contained waxy compounds embedded in the cuticle at the plant surface where they form a hydrophobic barrier that, protects leaves against dehydration, attack by insects, and diseases (cesio, 2004; smith, 1989; garcia et al., 1995). nevertheless, the most non-polar hydrophobic compounds in the epicuticular wax like hydrocarbons and wax esters have only dehydration protection capacity and the compounds with antiplant speciesextract concentration extract dichloromethanedichlomethaneacetone* water-ethanol s. bistellatum 0.25 mg/ml50.70ab± 12.1028.73bc± 13.0743.81ac± 15.06 1.00 mg/ml42.70ab± 13.6941.90ab ± 17.2056.09ab± 7.28 2.50 mg/ml93.20aa± 4.1292.14aa± 5.4986.32aa± 7.28 s. sisymbrifolium 0.25 mg/ml79.91ab± 3.0984.53ab± 2.6832.74bb± 6.60 1.00 mg/ml92.21aa± 2.0890.06aa± 2.0137.31bb±6.68 2.50 mg/ml82.67ab± 3.1891.91ba± 1.9850.73ca± 7.02 *dichloromethane-acetone extract for sugar esters. mean of five replicates (n=30 termites per replicate). values followed by the same letter in each column, for each plant, are not different from each other (tukey’s test, p<0.05). table 2. mean repellency (%) of solanum spp. extracts on c. brevis. insect and antifungal properties are medium polarity compounds (garcia et al., 1995), which were obtained after partitioning the residue with acetone and freezing it overnight. the chromatographic analysis of the resulting acetone extracts confirmed the absence of the less polar hydrocarbons, wax esters, and n-alkanols and a higher proportion of sugar esters (50% for s. bistellatum and 75% for s. sisymbrifolium), which are compounds released by glandular type iv trichomes found in plants of the solanum genus. in order to isolate the bioactive compounds, the sugar ester fraction of both solanum species was fractionated by preparative column chromatography. the main compound in each of them was isolated, in order to characterize them spectroscopically. nmr analyses (13c, 1h and hsqc and tocsy-hsqc), performed on the purified fractions of s. sisymbrifolium, confirmed the structure reported by cesio et al. (2006): a glycoside of β-hydroxypalmitic with an arabinoxilane. for the compounds isolated from s. bistellatum, the same set of nmr experiments were performed allowing the identification of a similar structural pattern as those isolated from s. sisymbrifolium. the same glycoside of β-hydroxypalmitic acid and β-xylopyranosil(1-5)-α-furoarabinose template was esterified with two molecules of β-hydroxypalmitic acid and palmitic acid in s. bistellatum, as deduced from the gcms analysis and integration of the diasterostopic hydrogens attached to the α carbon to the carboxyls that appeared in the 1h nmr spectra between δ=2.0-3.0ppm (fig 1). fig 1. 1h nuclear magnetic resonance of the major sugar ester compound isolated from s. bistellatum. the hsqc-tocsy distinguished three spin systems, identifying the sugar template: one for the arabinose in furopyranose form, (δ(13c) = 109.3ppm, δ (1h)= 5.23 ppm,) ( 1, 2 and 3) the second one belonging to xylose in glycopyranose form (δ (13c)= 104.0ppm, δ (1h) = 4.40ppm) and the third one to the hidroxy acids (δ(13c)= 71.40, 78.40ppm, δ (1h) = 4.10ppm). v rech-cainelli et al. – antifeedant and repellent effects of solanum extracts on c.brevis86 fig 3. hsqc-tocsy nmr of the major sugar ester compound isolated from s. bistellatum. fig 2. 13c nuclear magnetic resonance of the major sugar ester compound isolated from s. bistellatum. fig 4. structure for the major active compound from s. bistellatum. the hmbc experiment was useful to identify the glycosidic unions that were the same reported previously. nevertheless, we were not able to identify the position where the fatty acids were attached. the proposed structure is depicted in fig 4. according to the structural data, the most important acyl sugar fraction in s. bistellatum has similar structural features as the acyl sugars from s. sisymbrifolium (cesio, 2004): a disaccharide glicosidated with a β-hydroxypalmitic acid. the disaccharide portion of the molecule is also diesterified by two fatty acid residues. to develop termite control strategies is not straightforward. once termites colonize the wood, they are not accessible to surface treatments. one alternative is to use preventive and repelling products applied to the wood surface before structural use. antifeedants are an example of possible surface treatments which could be used to inhibit the insects’ ability to penetrate wood. antifeedant treatments for termite control can be a useful tool for protecting wood as well (gutiérrez et al., 1999; sbeghen-loss et al., 2009). the products and extracts described in the present study show repellent activities that can possibly protect wood against termite attack. natural products are easily degraded in the environment by many microorganisms (zhou et al., 2013; you et al., 2014). particularly, sugar esters are very simple compounds consisting of simple sugars esterified by fatty acids. these properties should be tested in field trials in order to evaluate the persistence of the natural active compound in the environment or design more stable analogs. as stated above, a possible strategy to protect wood against termites is to prevent wood colonization by repelling termites from surface entry. in this context, the present work contributes with new tools in that regard by reporting the chemical composition of type iv trichomes exudates from little known solanaceae spp. which show signficant properties as antifeedants and repellents against the drywood termite, c. brevis. the extracts and sugar esters isolated and described in this study show very interesting repellent activities which may be used in pest management. acknowledgments the authors grateful acknowledge the financial support from capes, universidade de caxias do sul, facultad de química-udelar and dicyt-cnpq. references anonymous, 1974. dyeing reagents for thin layer and paper chromathography. darmstadt: merck. p, 6. cabrera, rr., lelis, at., berti filho, e. 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(2013). dissipation and residue of rotenone in cabbage and soil under field conditions. bulletin of environmental contamination and toxicology, 91: 251-255. 1075 structure of nests and colony sizes of the european hornet (vespa crabro) and saxon wasp (dolichovespula saxonica) (hymenoptera: vespinae) in urban conditions by jerzy nadolski1 abstract studies of different groups of insects in urban areas sometimes show large populations, greater than in non-urban areas. the reason for this is a presence in the cities some of dominating species which often occur almost en masse. this group includes, inter alia, hymenopterans and especially social wasps (vespinae). colonies and nests of two wasp species, the european hornet (vespa crabro) and saxon wasp (dolichovespula saxonica) in areas of the city of łódź in poland were studied. whole colonies positioned both in buildings as well in natural places, size of societies, parameters their nests, differentiation of cells in combs and their location were investigated. we also studied the correlation between the size of the nest and societies of these insects. it was found that nests established in the buildings are much larger, produced more individuals of reproductive castes and thus obtained a better reproductive success especially for the hornet colonies whose queens prefer the buildings as a place to nest. key words: urban fauna, social wasps, vespa crabro, dolichovespula saxonica, hymenoptera, vespinae, nests of wasps. introduction due to the diversity of habitats and wealth of species as well their small size, there is often a lack of information regarding insects existing in urban areas. individual insect species react differently to urbanization pressure and mosaics of environment within cities and only some of them are able to effectively inhabit these areas with very specific ecological niches. studies conducted so far on various groups of insects in urban areas sometimes showed large total populations which were higher than those known from 1 natural history museum, university of łódź, kilińskiego 101, 90–011 łódź, poland, e-mail: nadolski@biol.uni.lodz.pl 1076 sociobiolog y vol. 59, no. 4, 2012 non-urban areas. the reason for this is a presence in the cities of dominant species which often occur almost en masse. this group includes, inter alia, hymenopterans and especially ants (formicidae) and social wasps (vespinae) (chudzicka, skibińska 1994). the presence of wasps (vespidae) in urban areas is commonly known. (czechowski 1979; haeseler 1982; skibińska 1982; skibińska 1987; ahrné 2008), including the city of łódź in poland (kowalczyk 1991; nadolski 2004; kowalczyk & kurzac 2005; kowalczyk &szczepko 2008; nadolski et al. 2011). numerous and large-scale studies determined the species composition of this group of insects, their proportions as well as the dynamics of quantitative changes in the urbanized areas (pawlikowski 1998; nadolski 2000b; nadolski 2001; pawlikowski et al. 2005; hermes & köhler 2006; christie & hochuli 2008; langowska et al. 2010). the study of this group of insects is important because wasps in the city can be hazardous for people. they can inflict dangerous stings (mcgain et al. 2000; nadolski 2000a) and they can cause the spread of pathogens (nadolski et al. 2000). organization of insect societies for many years is of interest to many study centers. an excellent example of this is the study of honey bee (apis mellifera) societies. special scientific centers whose research interests are focused solely around this species of insect are known. moreover, there are many papers on the subjects of the organization of wasp societies, of which a classical model of these studies are paper wasps – species of the genus polistes. their nests, built without protective envelopes, are easy to observe and therefore have become a common source of information on the behavior of wasps (strassmann 1989; tibbetts 2004; starks & fefferman 2006). the observations of vespinae are definitely more difficult, although not impossible even in the case of hornets (yamane & makino 1977; bunn 1988; nakamura & sonthichai 2004). however, it is often difficult to accurately determine the quantitative parameters of colonies of these animals. the specificity of their nests, tightly surrounded by layers of envelopes, whose aim is to protect the colony and maintain an adequate microclimate, constant humidity and temperature (klingner et al. 2005; klingner et al. 2006), as well high number of individuals and aggressiveness of their societies may often pose serious impediments. hence sizes of colonies of these insects are often subject to speculation. parameters of vespinae nests are occasionally determined, during studies of these insects’ 1077 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica biolog y (brian 1965; haeseler 1997; leathwick & godfrey 1996), however, exceptionally little is known about the real size of societies of these wasps, especially in urban conditions. therefore, the author undertook this difficult task, whose aims were to obtain information about the structure and the real size of societies and nests of two species of vespinae in an urbanized area. the main purpose of these studies were therefore to determine the parameters and structures of their nests characterizing the size of their colonies and tracking the steps of the building of nests and development of societies in the aspect of adaptation of this group of insects to urban conditions. studies were conducted on two species of social wasps – the european hornet vespa crabro (linnaeus, 1758) and saxon wasp dolichovespula saxonica (fabricius, 1793). both species differ in size of individuals, as well as colonies, and their nests are convenient for research because they are built in places relatively easy to locate. the typical habitat both of these wasps is deciduous woodland. they usually build their nest in hollow trees, on branches of the trees, sometimes in natural burrow.s in the city, their colonies are established usually in different buildings and urban infrastructures, and also in bird nest-boxes (nadolski 2004; langowska et.al 2010). recent studies greatly increased the scope of various activities aimed at reducing the urban populations of social wasps (nadolski 2001). numerous media campaigns increase interest in this issue and heighten concerns about the city’s inhabitants caused by the presence of colonies of wasps. this fear, which is not always fully justified, causes that people massively inform municipal services about practically all of the observed colonies of wasps and a large part of the nests is removed. methods the studies were conducted in the area, located within the administrative boundaries of the city of łódź as well as within areas directly adjacent to it. łódź is a city in central poland covering 294.4 km2 (diehl 1997) of which green areas including parks and forests comprise a significant area 55.5 km2, which constitutes about 19% of the whole administrative area of the city. the climate is intermediate between the continental climate – eastern european and maritime climate – western european, with the average annual rainfall of 573 mm (dubaniewicz 1990). łódź as a city has a specific concentric and 1078 sociobiolog y vol. 59, no. 4, 2012 regular arrangement of streets and buildings, which is sometimes disturbed by traffic routes. this system of street, led to the creation of characteristic three zones each with a different character. the correlation between the area classified to the given zone, and the qualitative and quantitative composition of the studied different groups of organisms was found (kuziel & halicz 1979; markowski et al. 1998; nadolski 2000b; nadolski 2001, markowski et al. 2004). in recent years, the expansion of residential areas as well as conducted road investments gradually changed the original layout of the zones. however, their general character and separateness still exist. the first zone this is a center of the city with the rest of the former lodz at the turn of the 19th and 20th century. this zone, consisting of a old, historic areas, is cut by a chessboard streets running perpendicular to one another, where historic art nouveau buildings are located. this area is replenished by newly residential and office buildings. this zone is poorly differentiated biologically, and only to a small extent this area is enriched by the urban green. however, the presence the old city parks, forming a sort of enclave of greenery, enriched by the old, centuries-old plantings, create very specific living conditions for different animal groups, including insects. these parks are often a number of discrete and bounded through the streets and buildings and often form specific, partially isolated ecosystems. the second zone, formed by areas occupied by modern blocks including skyscrapers and their infrastructure as well as by industrial sites, despite the presence of large number of young trees and large green areas, makes an impression of rather poor and monotonous. by its specificity, in a very small degree, it allows for free migration of animals from the area of the city center to suburban and non-urban areas. therefore in this area observed, as in the city center, large qualitative impoverishment of many taxonomic groups of animals, including insects (markowski et al. 2004). the third zone runs along the administrative border and surrounds whole of the city and thus other zones also. this is an area of detached houses and old farms, which is in direct contact with agricultural lands and forests located outside the city. therefore, this area may be regularly replenished by migrating species of animals. green belts, which connect in some places all three of zones, as well as railway lines, which run deeply even into the dense urban development, also allow for limited migration of species to places, located within the very city centre (kowalczyk & nadolski 2007). 1079 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica information about the location of nests came, in most cases, from the municipal services, especially from the fire brigade department to which all such information are reported, forest services as well as from the telephone notification and other survey data obtained from citizens of łódź, as well as especially from author’s own observations. vespa crabro and dolichovespula saxonica colonies came from different places in all zones of the city. the european hornet is a large, easily distinguishable wasp and finding and identification of colonies of this insect are very easy. however, all incoming information was checked. being aware of possible defects of these methods of search of nests, but the only usable, it was necessary to estimate the possible their error. we must be aware of the fact that łódź has a surface of almost 300 km2, with a dense urban structure, with multiple industrial regions, agricultural areas, forests and parks. the author of this study has decided to carry out detailed inventory all of wasps nests in of randomly selected 10 squares, each with an area of 1km2. efforts were made in these areas to record all nests of vespinae, particularly colonies of vespa crabro and data obtained in this way compare from the number of colonies of the same squares noted as a result of notification, surveys, and data obtained from all of the municipal services. it turned out that in case of vespa crabro only a few nests established on the premises of abandoned factories and fallow lands were not included in the notifications. these studies showed very high over 90% effective registration all of colonies of hornet in the city. locate and recognize the nest of dolichovespula saxonica is much more difficult, and hence the above described method could not be used. therefore, the total number of nests of this species in the city of łódź was not specified. after verified, received data were entered into a specially created database in access (microsoft inc 1997 and 2000). only part of nests was included in further research which was dependent on the adopted procedure. some vibrant and active colonies were removed, along with all individuals of wasps who remained in it. to collect all individuals of societies of wasps, the liquidation of these nests was carried out at night. studies of social wasps’ nests are limited due to technical reasons. the choice of the nest is dependent on many factors which are essential to use it for further study. for most of these analyzes could be used only complete and undamaged nests. for this reason, underground nests of wasps, as well as placed high in the branches of trees, could not be used. 1080 sociobiolog y vol. 59, no. 4, 2012 for further detailed analysis were selected the appropriate number of nests collected from all study areas and zones in the city, that met the necessary requirements. for comparative analysis of some parameters of nests and castes were used all collected colonies at the final stage of their development, derived with two or three consecutive years. after freezing, whole nests were kept at a temperature -18 °c for further study. both active as well as the empty nests, were subjected to a thorough analysis. graphics and statistical analyses were made using statistica 9 (statsoft, inc. 2009). standard statistical methods were used. for better clarity, precise description of the used methods sometimes will complement the information about the results achieved that were obtained relating to specific topics included in this paper. results the place of establishment of the colony in spring, the young new queens of wasps, born and mated the previous autumn begin the building of nests, choosing for this places which are often characteristic for the given species. based on the material obtained within the years 2000-2008 which was recorded in the database, the preferences in this aspect were analyzed. every recorded place was qualified into two categories. to first category was classified ‘natural places‘, the typical and characteristic for particular species which were not places inhabited by humans or arising as a result of human activity. the only exception were the breeding boxes for birds, which because of their characteristic construction resemble hollows of trees, therefore they were classified to places in ‘natural’ groups (photo 1). to the other category “buildings” were included places directly related to the structure of the urbanization of the city residential buildings and outbuildings with the associated infrastructure. within years 2000-2008 in the area of łódź and its immediate neighborhood, based on the conducted research, 365 developed nests with workers of vespa crabro (table 1) were recorded. from the obtained results, the fact of a large number of nests established in residential or utility buildings, which as the ‘unnatural’ places included in the second category, really deserves attention. most often, wasps’ colonies were found in unused attics of buildings and in cellars (photo 2 and 3). they were often found in layered walls, in the insulation of roof or flat roof, as 1081 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica photo. 1. dolichovespula saxonica nest in a breeding box for birds. 1082 sociobiolog y vol. 59, no. 4, 2012 well in the chimney or ventilation flues. it happened that nests were formed directly on windowsills and window blinds, on balcony grilles and various objects left over in cellars, in clothes being on balconies, coca-cola containers kept at the back of shops, window curtains or even in bullet-proof firewall of military shooting range. study of the structures of nests. the catalogued and collected nests underwent a thorough quantitative and qualitative analysis. 34 nests of vespa crabro and 93 nests of dolichovespula saxonica, at the final stage of development, were accurately investigated both active colonies and empty nests. for the statistical analysis of parameters of cells and combs, only such nests were used which possessed fully developed photo. 2. vespa crabro nest in an attic table 1. the choice of place for nests of vespa crabro within the particular zones of łódź city centre housing estate suburban area including forests species natural places % buildings % natural places % buildings % natural places % buildings % nests total vespa crabro 3 0.8 33 9.0 24 6.6 130 35.6 11 3.0 164 45.0 365 1083 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica photo. 3. vespa crabro nest in a cellar during a rebuilding 1084 sociobiolog y vol. 59, no. 4, 2012 and not damaged combs. size of whole nests (diagonals), thickness of envelopes, size and shape of combs, number and size of individual cells (diagonals and depth of 10-50 randomly selected cells, depending on the size of combs, sequentially counted every fifth cell), the utilization of the cells in different stages of colonies development, location of meconium (fecal pellets in cells) and the number and proportions of castes. the surfaces of combs were calculated using the model of a geometric fig. most resembling the shape of the comb. these were most often the surfaces of a circle, ellipse, triangle or rectangle. the volume of the nest was calculated, depending on the shape, with the use of the formula for the volume of a sphere, ellipsoid, cylinder and sometimes cuboids. table 2 presents size of nests of vespa crabro and dolichovespula saxonica coming from both types of places (natural and buildings). values of parameters of cells (width and depth) in nests of studied wasp’s species were subjected to statistical analysis. as a result of these analyzes three types of cells, referred to herein as (small, medium and large) were distinguished. table 3 presents the results of the analysis of parameters of nests (combs and cells) of the studied wasps’ colonies. no statistically significant differences between parameters of combs and cells of the nests from natural places and buildings. for all nests of vespa crabro, analysis of the width of cells table 2. mean volumes of nests of vespa crabro and dolichovespula saxonica in the final stage of their development from the city of łódź in years 2000 2008. species nests in buildings volume (dm3) ± se nests in natural places volume (dm3) ± se vespa crabro 21.00±11.31 4.37±0.24 dolichovespula saxonica 1.32±0.29 0.78±0.12 table 3. values of parameters of nests of vespa crabro and dolichovespula saxonica within the area of łódź at the final stage of their development. surface of comb (cm2) dimensions of small cells (mm) dimensions of medium cells (mm) dimensions of large cells (mm) species max. min. n width.±se depth.±se n width.±se depth.±se n width.± se depth.±se vespa crabro 1963 9 100 7.65±0.038 17.69±0.88 140 8.7±0.032 20.34±1.63 170 10.15±0.029 28.41±5.78 dolichovespula saxonica 177 2 110 3.89±0.035 9.78±0.65 200 5.51±0.025 12.85±0.98 160 7.06±0.030 17.97± 2.65 1085 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica conducted using lévene’s test showed homogeneity of variances (f 2. 407 = 1.64, p = 0.196, α = 0.05), the differences between mean values of the width of cells are statistically significant (anova: f 2.407 =1448.6, p < 0.00001). the post-hoc analysis with fisher’s nir test demonstrated important statistical differences between the mean values of the width of small, medium and large cells (ms intergroup = 0.145, df = 407.00, for all variants p< 0.05). for the second studied species dolichovespula saxonica the diversification of these variables was also statistically significant (levene’s test (f 2,467 = 1.830, p = 0.162) (anova: f = 98655.13 p < 0.0001) fisher’s nir (ms intergroup = 0.135, df = 467, for all comparisons p<0.05). the statistical analysis of the depth of cells in the nests of both species demonstrated lack of homogeneity of variance. nonparametric test also confirmed the variation within the studied cells into three types ( anova kruskal-wallis test for v. crabro: h 2,410 = 233.52 p < 0.0001 and for d. saxonica: h 2,470 = 363.06 p < 0.0001). the distributions of mean values of width of cells, present in consecutive combs and different nests of vespa crabro are presented in graphs in figs. 1 and 2. we can clearly see the differences between cell types. parameters of fig. 1. the distribution of mean values of the width of different types cells, in several nests of vespa crabro, in the final development stage of colonies. 1086 sociobiolog y vol. 59, no. 4, 2012 a given type of cell are similar but not identical in different combs and also between different nests of hornets. diversity and location of particular type of cells both within a single nest and between various nests were studied. the parameters of one type of cell of the same species are subject to minor changes. the study showed little difference of cells size between nests, but much more noticeable are the differences between the combs, even within the same nest. to examine the distribution of mean values of parameters of different types of cells under the influence of two factors grouping : combs and nests (vespa crabro), statistical analysis was performed using a hierarchical anova. levene’s test demonstrated homogeneity of variance for statistics – the width and the depth of cells for particular their types, in hornet’s nests. statistical analysis univariate test of significance for the variables the width of small cells of two, two-stage grouping factors (nest and comb) are shown in fig. 3 (hierarchical anova). the obtained results acknowledge the statistically significant differences of the mean value of this parameter. small cells differ in width, both in particular combs in a nest and in combs between nests. graphical illustration of the distribution of mean values of cell width is shown in graphs (figs. 4 and 5). fig. 2. the distribution of mean values of the width cells in particular combs of nests of vespa crabro in the final development stage of colonies. 1087 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica fig. 3. distribution of the mean values of the width of small cells in different combs for the nests of vespa crabro at the final stage of development of colonies (hierarchical anova f 2,90 = 59.040, p< 0.00001) fig. 4. distribution of the mean values of the width of small cells in consecutive combs and separate nests of vespa crabro at the final stage of development of colonies (hierarchical two-factor anova, f 7,90 = 2.221, p = 0.04) 1088 sociobiolog y vol. 59, no. 4, 2012 these graphs clearly show the differences of mean values of analyzed variables, especially between successive combs. the distributions of mean values of depth of all the identified types of cells have also been analyzed. analyses have shown that the differences between the mean values of depth of small cells are not statistically significant, both between combs in one nest and combs of different nests of vespa crabro (hierarchical anova). for medium-sized cells, also were observed a similar variation, although cells in the last combs are smaller than in previous (fig. 6). the carried out analysis one-dimensional test of significance for the variable– the width of medium cells for two two-stage grouping factors (nest and comb) is presented in figs. 7 and 8 (hierarchic anova). the obtained results confirm the statistically significant differences of the mean value of this parameter. medium cells differ in width both between combs in a given nest and in combs of various nests. fig. 5. distribution of the mean values of the width of small cells in different nests and separate combs of vespa crabro at the final stage of development of colonies (hierarchical two-factor anova, f 7,90 = 2.221, p = 0.04) 1089 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica fig. 6. the distribution of mean values of the width of medium cells in different combs in all study nests of vespa crabro in the final stage of development of colonies (hierarchical anova f 3,126 = 14.899, p<0.00001) fig. 7. the distribution of mean values of width of medium cells in different nests and consecutive combs of vespa crabro in the final stage of development of colonies (hierarchical two-factor anova, f 10,126 = 2.501, p = 0.009) 1090 sociobiolog y vol. 59, no. 4, 2012 fig. 8. the distribution of mean values of width of medium cells in different combs and separate nests of vespa crabro in the final stage of development of colonies (hierarchical two-factor anova, f 10,126 = 2.501, p = 0.009) fig. 9. the distribution of mean values of the width of large cells in combs in nests of vespa crabro in the final stage of development of colonies (hierarchical anova f 6,153 = 1.272, p = 0.27) 1091 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica this graph shows the differences between mean values of the analyzed variable – the width of medium cells. the observed mean values of this parameter in fourth combs are smaller than in third. this is probably associated with the disorder of structure of these combs caused by appending to them the large cells, in later stages of developing of colony, and the necessity of small changes in the size of medium-sized cells adapting them to the further stages of extension of combs. the two last graphs demonstrate more precisely the complexity of structure of consecutive combs in nests of vespa crabro. one can see clearly, that differentiation of this parameter is greater for various nests than for different combs. the differences between the mean values of the depth of medium cells, as it is in the case of small cells, are not statistically significant, both between combs of a given nest and for various nests of vespa crabro. width of large cells proved to be a constant parameter, both between different nests of hornets, as well as the combs within the same nest. the results of conducted analysis one-dimensional test of significance for the variable – the width of large cells for two two-stage grouping factors (nest and comb) – is presented in figs. 9-11 (hierarchical anova). the obtained results do not confirm statistically significant differences between mean values of this parameter. width of large cells do not differ significantly both among combs in the nest as well as in combs between nests and the observed differences are not statistically significant (p>0.05). the obtained results of the statistical analysis of this parameter show its great conservatism clearly. it is understandable because these cells are built in the last phase of the development of a colony and they are used for development of castes which are the most precious for wasps’ societies – reproductive castes. because the size of the imago individuals depends on the size of the cell in which they grow (author’s own unpublished observations), therefore, the stability of this parameter, which determines the normal development of future queens (g ynes) and males (drones) is very important for the colony and one can expect that natural selection favors just such a solution. the observed higher, than in other cases, dispersion of the values defining the confidence intervals, is probably associated with differentiation of large cells into two types used for, future queens and drones, which have various sizes. 1092 sociobiolog y vol. 59, no. 4, 2012 fig. 10. the distribution of mean values of width of large cells in consecutive combs and different nests of vespa crabro in the final stage of development of colonies (hierarchical two-factor anova, f 10,153 = 0.805, p = 0.62) fig. 11. the distribution of mean values of width of large cells in different nests and consecutive combs of vespa crabro in the final stage of development of colonies (hierarchical two-factor anova, f 10,153 = 0.805, p = 0.62) 1093 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica the obtained results for depth of large hornet’s cells, not confirm statistically significant differences between the mean values of this parameter also . large cells no differ from one another significantly in depth both between consecutive combs in a given nest and in combs from various nests. similar statistical analyses were conducted for nests of dolichovespula saxonica. levene’s test demonstrated homogeneity of variance for statistics – the width of cells for particular their types, d. saxonica nests. the statistical analysis of this variable one-dimensional test of significance for two two-stage grouping factors (nests and combs) is presented in figs. 12-15 (hierarchical anova). the differences between the mean values of the width of small cells are statistically significant both between combs of the same nest and various nests of dolichovespula saxonica. the graph in fig. 13 shows the distribution of mean values of the width of small cells in the nests of distorted structure, which was caused by the presence of reproductive workers, which build additional cells from which fig. 12. the distribution of the mean values of the width of small cells in consecutive combs in nests of dolichovespula saxonica in the final stage of development of colonies (hierarchical anova f 2,90 = 129.92, p < 0.00001) 1094 sociobiolog y vol. 59, no. 4, 2012 drones of workers will develop. in the graph are clearly seen small cells also in 4th comb. in the nests where ‘worker policing or queen-controlled’ (foster & ratnieks 2001; foster et al. 2001) is effective, such cases are rare. for further analysis, small cells from the fourth comb were not included. the presented graphs demonstrate that the occurrence of small cells is limited mainly to the two first combs; rarely can they be found in the third one. their presence in the remaining indicates certain anomalies in the structure of social organization of a colony. for width of medium cells statistical analysis of this variable one-dimensional test of significance for two two-stage grouping factors (nest and comb) is presented in figs. 16-18 (hierarchical anova). the conducted analysis indicates that the differences between the mean values of the width of medium cells are statistically significant both between combs of the same nest and various nests of dolichovespula saxonica. the presented graphs indicate that medium cells, used for the growth of worker caste, are located mainly in combs 1 4, rarely in the last ones. fig. 13. the distribution of mean values of the width of small cells in successive combs in the nests dolichovespula saxonica in the final stage of the colony. the graph illustrates the presence of small cells built on the fourth comb probably by “reproductive” workers (limited ‘worker policing’) (foster et al. 2001; wenseleers et al. 2005a). 1095 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica fig. 14. the distribution of mean values of the width of small cells in particular nests in consecutive combs of dolichovespula saxonica in the final stage of development of colonies (hierarchical two-factor anova, f 7,90 = 26.857, p < 0.00001) fig. 15. the distribution of mean values of width of small cells in consecutive combs in different nests of dolichovespula saxonica in the final stage of development of colonies (hierarchical two-factor anova, f 7,90 = 26.857, p < 0.00001) 1096 sociobiolog y vol. 59, no. 4, 2012 fig. 16. the distribution of mean values of the width of medium cells in consecutive combs in nests of dolichovespula saxonica at the final stage of development of colonies (hierarchical anova f 5,180 = 56.541, p < 0.00001) fig. 17. the distribution of mean values of the width of medium cells in different nests in consecutive combs of dolichovespula saxonica at the final stage of development of colonies (hierarchical two-factor anova, f 14,180 = 74.737, p < 0.00001) 1097 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica moreover, their width is varied and differs among colonies of dolichovespula saxonica, but its parameters depend also on the location in a particular nest. the differences are statistically significant. for width of large cells, statistical analysis of this variable one-dimensional test of significance for two two-stage grouping factors (nest and comb) is presented in figs. 19-21 (hierarchical anova). the differences between the mean values of the width of large cells are not significant statistically, both between combs of a particular nest and various nests of dolichovespula saxonica. in nests of this species, widths of large cells are similar in all studied colonies. attention should be paid to the great similarity of the results of statistical analyses of cell parameters of nests of vespa crabro and dolichovespula saxonica. for both species one can observe significant statistical diversity of parameters of small and medium cells as well as their similar, though not identical, location within a nest. for both species one can observe stability of sizes of large cells for reproductive castes, but their function associated with the use of them for two types of castes (g ynes and drones), makes, that values fig. 18. the distribution of mean values of the width of medium cells in consecutive combs in different nests of dolichovespula saxonica in the final stage of development of colonies (hierarchical two-factor anova, f 14,180 = 74.737, p < 0.00001) 1098 sociobiolog y vol. 59, no. 4, 2012 fig. 19. the distribution of mean values of the width of large cells in particular combs in nests of dolichovespula saxonica in the final stage of development of colonies (hierarchical anova f 6,144 = 0.511, p < 0.8) fig. 20. the distribution of mean values of the width of large cells in consecutive combs of different nests of dolichovespula saxonica in the final stage of development of colonies (hierarchical two-factor anova, f 9,144 = 0.267, p < 0.99) 1099 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica of their mean and confidence intervals are relatively high. based on the analysis of location and size different forms of wasps development in colonies, so eggs, larvae, pupae, as well quantities of meconium in individual cells (yamane & makino 1977; star & jacobson 1990) attempt was made to reconstruct the sequence of formation and exploitation of the combs and cells by wasps. fig. 22 shows the results of these analyses for vespa crabro. the presented schematic drawings show the results these studies, demonstrating the sequence built of rings of cells in successive combs. the newly built ring is the basis of choice of area of combs, as places for eggs lying by the queen. the same color indicates areas of different combs formed more or less simultaneously. the schematically, real number and location of individual cells in consecutive combs in one of the studied nests of vespa crabro were shown in (fig. 23). one can clearly see here the ring system of distribution of individual forms of development of these insects. characteristic here is primarily creating and use and gradual increasing of the number of cells in few combs simultaneous (2-3), and after a certain stage of their formation (ring 4 6 cells), further continuing of their construction, with the growth of colonies. fig. 21. the distribution of mean values of the width of large cells in particular nests in consecutive combs of dolichovespula saxonica in the final stage of development of colonies (hierarchical two-factor anova, f 9,144 = 0.267, p < 0.99) 1100 sociobiolog y vol. 59, no. 4, 2012 at the maximum number of combs (6-8), wasps do not build each of them separately till the end, but they are gradually and annularly expanded (photo 4). the cells in first combs are used for the development of larvae and pupae usually two or three times, especially around the central point, which is the place of start construction of the primary comb, further cells are used usually twice, in last combs (6, 7 and 8), containing reproductive castes, only once. of course, there are often deviations from this typical pattern which result from disruptions in the working of insect’s societies related with the shape and volume of the place where the nest is located (construction obstacles, the place too small, narrow, etc.) (photo 5), presence of parasitoids (photo 6), including social usurpers or the death of the queen. fig. 22. vespa crabro nest analysis schema forming combs (different colors mean areas of cells built at the same time) 1101 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica fig. 23. detailed analysis of one of the vespa crabro nests in the final stage of development of the colony with the location of the individual cells 1102 sociobiolog y vol. 59, no. 4, 2012 photo. 4. vespa crabro comb showing successive rings of cells photo. 5. vespa crabro nest in the attic showing nest structure disorder caused by an obstacle in the roof structure 1103 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica colony size studies of colony size in the nests of the social wasps were based on data obtained by counting the individuals of each caste, the number of eggs, larvae and pupae as well as meconium in cells (yamane & makino 1977). based on the number of meconium, how many times used different groups of cells was studied. after analyzing the data, the potential number of all wasps of different castes presented in a given society over a full season was obtained. table 6 presents examples of exceptionally large societies of vespa crabro and dolichovespula saxonica in the area of the city of łódź. for these calculations 24 largest nests were used, in the final stage of development, both from ‘natural’ photo. 6. dolichovespula saxonica nest destroyed by aphomia sociella, showing combs built crosswise 1104 sociobiolog y vol. 59, no. 4, 2012 places and ‘buildings”. in table 7 there are presented mean values of volume of nests coming from both types of places as well as the mean values of the number of individuals of castes and number of cells in nests of vespa crabro. number of individuals in the colony should be dependent on the size of the socket, and above all on the number of cells. correlations between the number of individuals, and the quantitative parameters of the nest were examined. a statistical analysis of linear correlation – pearson r test between the size of the nest and various parameters determining the size of the studied societies of vespa crabro and dolichovespula saxonica was conducted. for these analyzes, only fully developed nests were used. there is a strong correlation (spearman test) between the total number of worker’s cells and the number of workers both in the nests of vespa crabro established in natural places (r = 0.69; p < 0.05) and in buildings ( r = 0.80; p < 0.05) (cohen 1988). there were very strong correlation between the total number large cells and counts of drones and g ynes together in the nests of vespa crabro established in natural places (r = 0.95; p < 0.05) and in buildings (r = 0.97; p < 0.05). these results reflect the effective use all of large cells. the determine count of meconium showed, that these cells, for the development of reproductive castes are used only once. the results of these studies are so interesting that require additional discussion. not surprisingly there is strong correlation between the number of various cells in the nest, and the number of individuals in the colony. but it table 6. abundance of castes in the example of two large colonies of vespa crabro and dolichovespula saxonica from the vicinity of łódź (total number of all individuals from different castes, which were born and lived in the nest) species workers queens males total number of individuals nest volume vespa crabro 1018 497 546 2061 23.552 dm3 dolichivespula saxonica 937 156 251 1344 3.203 dm3 table 7. the summary of mean values of the number of hornets from the castes, number of cells and the volume of vespa crabro nests in the final stage of development of colonies, which were established in natural places (nat.) and buildings (bld.) in the area of łódź (n = 24) workers ± se g ynes ± se males ± se number of cells ± se nest volume (dm3) ± se nat. bld. nat. bld. nat. bld. nat. bld. nat. bld. 767±62 915±138 56±8 238±59 80±10 270±63 708±56 1281±207 1.37±0.13 21.00±11.31 1105 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica is worth noting difference in r value between the nests from ‘natural’ places and from ‘buildings’. these differences are results from the limitations of the size of nests, connected with the volume of natural places in which colonies were established (nesting boxes and tree hollows) and the lack of such limitations in cases of nests in buildings. societies of wasps whose development of nests are limited by capacity of the place, are somehow forced to eliminate potential losses through additional use of the same cells, which is done more often than in the case of nests built without these restrictions, and located in the buildings (while maintaining the similar number of workers). the total number of individuals of all castes also should therefore be correlated with the overall size of the whole colony the volume of the nest both for vespa crabro as well as dolichovespula saxonica (figs. 24 and 25). the very strong correlation (cohen 1988) between the basic parameters of a nest its volume of a nest and the count of imago individuals is so understandable, although it must be noticed that the parameter nest volume cannot be the value directly proportional to the size of the society, as the volume of a sphere is not directly proportional to its radius (exponential function). moreover, the aforementioned fact that the same cells are used many times fig. 24. the correlation between the volume of a nest and the total count of individuals of vespa crabro (n = 27; r = 0.83; p<0.05) 1106 sociobiolog y vol. 59, no. 4, 2012 fig. 25. the correlation between the nest volume and the count of individuals of dolichovespula saxonica (n = 41; r = 0.84; p<0.05) fig. 26. the correlation between the count of combs and the count of individuals of vespa crabro (n = 27; r = 0.84; p<0.05) 1107 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica for the development of larvae has a fundamental importance here as well. the number of combs, though strongly correlated with the number of individuals (fig. 26) is not a parameter that can be used uncritically to assess the size of the colony. it is so because in some special cases, the volume of the place where the nest is located (low attic, flat roofs or narrow slits) necessitates the construction of only 1-2, a maximum of 3 combs, but with much larger surfaces on which all cell types are located the comparison between the success of vespa crabro nesting in natural sites and buildings ranks of the total number of large cells in studied nest, established in both types of places were compared. the differences were statistically significant (anova nonparametric mann-whitney u test) (fig. 27).to assess the effect of nest site for potential reproductive success of vespinae, hornet’s nests were used only. nests and colonies of dolichovespula saxonica are so small that their location in a ‘buildings’ or ‘natural places’ has no practical significance for future size of the colony. differences in the number of workers between colonies in both types of places are not statistically significant. fig. 27. the number of large cells of the nests of vespa crabro established in buildings and natural places in 2006-2008. anova nonparametric mann-whitney u test. u = 9.00; p = 0.002. 1108 sociobiolog y vol. 59, no. 4, 2012 fig. 28. the correlation between the total count of cells and the count of individuals of dolichovespula saxonica (n =41; r = 0.98; p<0.05) fig. 29. the correlation between the number of combs and the number of individuals of dolichovespula saxonica (n = 41; r = 0.92; p<0.05) 1109 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica for nests of dolichovespula saxonica analysis showed no differences between those parameters for colonies of this species of wasp established in natural places and buildings. however, the correlation between the number of cells and combs in the nest, and the number of individuals in the colony are very strong correlated (figs. 28 and 29). as can be inferred from these analyzes, the location of the slot is the primary determinant of colony size for large societies of wasps, including the european hornet. numbers of cells are especially strongly correlated (all r > 0.7) with the counts of individuals in a wasp colony. proportions of castes based on the data obtained from the quantitative assessment of nests and societies, including the abundance of individual castes, the comparative analysis of proportions between g ynes and young males (drones) of the studied species of wasps was conducted (fig. 30). the statistical analysis of proportions between reproductive castes g ynes / drones in the nests of the studied wasp species, showed homogeneity of variances (levene’s test). fig. 30. the comparison of proportions between reproductive castes: g ynes / drones in the nests of the studied species of wasps (vespa crabro and dolichovespula saxonica). anova single-factor ( f 1,19 = 9.22, p = 0.007) 1110 sociobiolog y vol. 59, no. 4, 2012 the differences between the mean values of these proportions are statistically significant (fig. 30). these proportions between the colonies of the same species, both of the european hornet and saxon wasp were not statistically significant. the obtained values of these proportions (about 0.8) for each colony of vespa crabro are proving that drones have a small quantitative advantage over the g ynes. in case of societies of dolichovespula saxonica these proportions are different (about 0.6) from hornet because the count of young males in the saxon wasps is increased. it should be assumed that this is one more proof of the limited ‘worker policing’ observed in this species (foster, ratnieks 2001; foster et al. 2001). the correlation between the total number of workers and the number of drones and g ynes together in the nests of vespa crabro, established in buildings were very strong (n = 11; r = 0.82; p < 0.05) but there is no correlation between the total number of workers and the number of drones and g ynes together in the nests of hornet, established in natural places. for the saxon wasp, correlations between the number of large cells and the number of workers both from “natural” and “building” places were strong (n = 27; r = 0.62; p < 0.05) (cohrn 1988). the presented results of the analysis between the number of large cells and the number of workers in nests of vespa crabro and dolichovespula saxonica indicate no correlation of these variables in nests of v. crabro located in natural places and strong correlation in nests in buildings and for all colonies of d. saxonica. this confirms the assumption of smaller structure stability of nests of hornet caused among other things by ‘technical problems’ occurring in colonies established in places too “cramped” for this species. in case of d. saxonica, the small sizes of nests of this wasp do not cause problems with locating the colony in a freely chosen place. discussion a large city is a specific ecosystem which, like no other, undergoes continuous changes resulting from constant, planned or accidental human intervention. as an environment, it is characterized by exceptional mosaics and diversity of microhabitats. the dynamic process accompanying these changes in result of which we may observe loss of some species and inflow of others may sometimes lead to the increase of species diversity within certain animal groups. according to wanat (1987), in suburban part of łódź, species diversity of 1111 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica weevils (coleoptera, curculionidae) can be comparable or even larger than that found in białowieski national park. previous studies on the presence of different groups of animals in łódź show a decrease in their diversity in the direction of town centre. however, the specific character of downtown habitats is tolerated relatively well by some groups of hymenopterans (hymenoptera) especially by forest species (markowski et al. 2004). because of large, located near each other houses, a specific microclimate is created, which stands out due to reduced amplitude of temperature and humidity, even when compared with directly neighboring areas devoid the urban infrastructure. these conditions are multiplied especially inside buildings due to the presence of heating and air conditioning, heating pipes, sewage system, culverts of various types, wells, tunnels and cellars. this situation makes it possible for thermophilic species to exist in a city (banaszak 1978), for whom survival during the winter in conditions outside the city would be difficult or impossible. the study of aculeata in the warsaw agglomeration has shown that social insects are a group that relatively easily, as it seems, overcomes the barrier of urban pressure (banaszak 1978; skibińska 1978). some representatives of aculeata have long been using human houses and homesteads to form colonies in them, and certainly many times all of us have found in attics and lofts of houses traces of their existence as active or already empty and abandoned nests. however, the only urban conditions have created a new quality that allowed for a much larger scale existence of many representatives of this group of insects in the immediate vicinity of people. the species, which ‘were able’ to adapt their biolog y to urban environment using the existing food resources and the infrastructure abundant with places for nesting, began their expansion at an unparalleled scale, as it seems (skibińska 1982, skibińska 1987; nadolski 2000b; nadolski 2001). both studied species the saxon wasp dolichovespula saxonica and european hornet vespa crabro are species which, as is clear from these research, often form colonies in the city, but the size of the d. saxonica nests and its societies is always rather small. besides the breeding boxes for birds, the presence of its colonies was recorded in different places in residential and utility buildings. the colonies of the european hornet, which because of its size, builds large nests and there is a species most often recorded in forests (dvořák 2007b; 1112 sociobiolog y vol. 59, no. 4, 2012 pawlikowski 2009), maybe not in large counts, but also were found inside residential and utility buildings, within the very centre of łódź. however, it builds its nests most often in suburban zone. furthermore, analysis of the spatial distribution of v. crabro nests showed no clear preference in the selection of łódź area by this species (nadolski unpublished). reproductive success of european hornet in urban conditions is achieved by ability of efficiently creating large colonies by this wasp and skills of efficient using of specific conditions of “housing”, which were made by man. in the city of łódź, even in places most similar to “natural”, vespa crabro prefers “buildings” as a place for the development of its colonies. interestingly, apart from isolated cases, not been demonstrated, that colonies of european hornet were created several times in the same places, even in cases of nests formed in breeding boxes for birds. so unlike in poznań studies have shown (langowska et al. 2010). perhaps this is due to surplus of potential nest sites in urban area. it seems that the key element conditioning reproductive success of vespinae is the choice of place to build a nest, made in the spring by the young queen. these studies demonstrated undoubtedly (table 1) that urban populations of both species prefer places in residential and utility buildings and other accompanying urban infrastructure. it may be understandable because the number of ‘natural’ places for nests is usually limited in urban area, while in ‘buildings’ is plenty of them. this seemingly obvious as it might seem cause, should fully explain the observed trends. however, there are situations that cannot be unambiguously explained this way. this applies especially as species as the saxon wasp which, according to their biolog y, usually can build their nests on the branches of trees and the european hornet as the ‘tree hollow’ species prefers attics of buildings and sheds than neighboring nest-boxes for birds. the cause of these observed trends is not probably only a limited number of available natural places on the nest, but rather their quality. places in ‘buildings’ provide relatively stable temperature and protection from precipitation, predators. wasp colonies ‘installed’ in buildings in majority of cases may reach much larger sizes (tables 2 and 7) than those from natural places and at the same time achieve definitely bigger reproductive success. the number of all individuals of a given society is closely correlated with the nest size (figs. 24-26 and 28-29), which is also confirmed by studies of jeanne and bouwma (2002), and size of the nest built depends on the volume 1113 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica of the place chosen for its establishment. limitations to the size of the nest resulting from the volume of the place of its location are compensated by the wasp society using the same cells many times. however, the beginning of final stage of colony development forces the society to build large cells which are used for the development of reproductive castes only once. in consequence, the number of large cells being built, limited by the volume of the place, is a few times smaller than in nests of colonies which do not have such problems, i.e. located in buildings (fig. 27). urban conditions make it possible to form exceptionally large societies of vespinae and the parameters of their nests recorded in the city of łódź were sometimes impressive (table 6). it is worth adding that the largest nest of vespa crabro found in łódź, which was located in an attic of a residential building, was 70 cm in diameter and its volume including envelopes was about 180 dm3. however, it is difficult to compare it with the size of colonies of other species of hornet (seeley & seeley 1980; star & jacobson 1990), especially perennial societies (pickett et al. 2001). based on the results of statistical analyses of nests of two species of social wasps, it can be considered, that the differentiation of cells in nests of vespinae into small, medium and large can be a universal feature (figs. 1 and 2). the conducted analysis of nest structure distinguished two types of workers’ cells, of which the smaller ones described as small are formed mainly in the period of initiation of the nest and in the first comb are built by the queen. later they are seen in the central areas of the first three combs (figs. 35) and as a result of the analysis of meconium, are often used twice for the development of larvae, rarely three times. small cells are sometimes built later as well, by ‘reproductive’ workers, which sometimes lay eggs in these cells and from which develop only small drones. thus, the described types of cells can not to be synonymous to the development stages of nests: qn, ls and lc (pawlikowski & pawlikowski 2003; nadolski 2004). their characteristics are based on cell size in combs, with respect to the period of their building at a given stage of development of insect societies and therefore not by their location in the comb and sizes, but since their time of formation. small cells are also observed in the areas of nest whose structure was disturbed by parasitoids. in the latter case, formed empty combs, sometimes are not positioned parallel to other combs but are set in relation to them almost at right angles (photo 6) (author’s own 1114 sociobiolog y vol. 59, no. 4, 2012 observations). this may testify to the disorder of the system of the assessment of spatial position of nest, which conditions its proper structure, including combs and envelopes, characteristic of individual species (wenzel 1991) and conditioned by various factors (karsai & pénzes 1993; karsai & pénzes 1996; karsai & pénzes 1998), including the phenomenon of gravity (ishay et al. 2008). disturbances of the nest structure can certainly have a negative impact on its internal homeostasis, which determines the normal development of larvae and pupae (ishay et al. 2002a; ishay et al. 2002b; klingner 2006). workers’ larger cells, described here as medium are located in the areas of combs which are added gradually. this is the largest group of cells occurring in a nest, some of which are used twice, very rarely, only in cases of nests of species with large societies (e.g. vespa crabro) formed in cramped with too low volume places, three times (e.g. in breeding boxes). the position of cells in the nests of both species of wasps exhibits some regularity. along with the formation of successive combs are increased the size of small cells (figs. 3-5 and 12-15), however, no significant differences in the width of these cells in the same type of combs (eg, the first or the second) between various nests (figs. 5 and 15). in colonies of dolichovespula saxonica differences in size of small cells between nests is bigger, especially when the structure of the nest is disturbed by “reproductive” workers (limited “worker policing”) (foster et al. 2001; wenseleers et al. 2005a) (fig. 13). parameters of medium cells are also variable depending on combs on which they occur (figs. 6-7 and 16-17), but these differences are greater between different nests (figs. 8 and 18). differences in depth between small cells and between medium cells in different combs and nests in both colonies of vespa crabro and dolichovespula saxonica are not statistically significant. cells large, regardless of their location have a high stability of these parameters for both species (figs. 9-11 as well 19-21). these cells are built mainly in the last combs or on the periphery of earlier built ones and this depend on the location place of the nest. if the spatial conditions make it impossible for the nest to develop along its axis, and in consequence forming new combs, the wasp society builds the nest in the transverse direction by forming new rings of cells in combs of existing ones. cells for g ynes and drones are not isolated from one another, although there are some homogeneous clusters of the same type of cells for reproductive of the same caste. presence of “reproductive” workers causes 1115 nadolski, j. — nest structure and colony sizes of v. crabro & d. saxonica produce of males also in other areas of the nest. however, in this case, the medium cells (sometimes small) are used and drones developed from them are always significantly smaller than those of large cells (author’s own observations). nests parameters, especially the number of cells, are highly correlated with the size of the wasp’s societies. this gives grounds for assessing the size of the society based on the volume of the nest (figs. 24 and 25), although this dependency may be fraught with error because of the possibility of using these same cells twice or three times in the case of societies, limited by small volume of the place where the colony is located. still, this estimate may be used effectively when analyzing most nests; especially those, that already have been deserted. sometimes observed deviations from these general principles of the construction of nests may be due to disturbances of its structure caused by various external factors. it may be the size of the place of the nest location, or technical obstacles (photo 5) as well as the presence of parasitoids (photo 6), and the death of the queen and the action of usurpers. the results of the analysis of number of particular castes are also worth thorough discussion. the differences in proportions between reproductive castes and workers between of the studied species did not prove to be statistically significant and this parameter exhibits relatively high changeability even between different colonies of the same species. thus, it may be assumed that the number of workers may have no influence on the number of ‘produced’ males and queens. the analysis of nest structure showed, that the number of large cells in nests of vespa crabro is correlated with the number of the rest of cells only for colonies established in buildings and for colonies of dolichovespula saxonica. this correlation, though it is not high occurs in both types of places for nests (natural and buildings). interestingly, the proportions between the number of g ynes and drones in societies vespa crabro (about 0.8) are constant for different colonies and nests places. reducing of the value of dolichovespula saxonica to about 0.6 is probably related to the more frequent presence of egg-laying workers in this species (foster et al. 2001) in connection with the limited control of male reproduction by the workers “queen or worker policing” (foster & ratnieks 2000; foster et al. 2000; foster & ratnieks 2001; foster et al. 2001; foster et al. 2002; takahashi et al. 2004; wenseleers et al. 2005a; wenseleers et al. 2005b) which is typical for wasps belonging to the genus dolichovespula 1116 sociobiolog y vol. 59, no. 4, 2012 (rohwer, 1916). the research of hornet nests (vespa sp. linnaeus 1785) in japan done by matsuura (yamane & makino 1977) demonstrated for species of large and numerous societies, similar proportions between drones and g ynes and twice bigger number of males than future queens in small nests of vespa analis insularis dalla. thus, it is difficult to assess the causes and adaptive nature of these proportions, which in the case of nests vespa crabro, give only the result of about o10-20% more drones than g ynes. briefly summarizing these considerations, it should be noted that the city is a special kind of environment that creates for wasp societies very good living 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[curculionidae (coleoptera) of łódź] acta universitatis. lodziensis, folia zoologica and anthropologica 5: 27-86. wenseleers, t., n.s. badcock, k. erven, a.tofilski, f.s. nascimento, a.g. hart, a.t. burke, m.e. archer & f.l.w. ratnieks. 2005a. a test of worker policing theory in an advanced eusocial wasp, vespula rufa. evolution, 59(6) 1306-1314. wenseleers, t., a.tofilski & f.l.w. ratnieks. 2005b. queen and worker policing in the tree wasp dolichovespula sylvestris. behavioral ecolog y and sociobiolog y 58: 80-86. yamane, s. & s. makino. 1977. bionomics of vespa analis insularis and v. mandarinia latilineata in hokkaido, northern japan, with notes on vespine embryo nests (hymenoptera: vespidae). insecta matsumurana (n.s.) 12: 1-33. doi: 10.13102/sociobiology.v67i3.5143sociobiology 67(3): 444-448 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction studies on social wasps are mainly focused on females, queens and workers, which are responsible for the reproduction and maintenance of colony respectively. males receive less attention, in part because of the brief period adult males remain in the nest in most species (west-eberhard, 1969; jeanne & castellón, 1980; gadagkar, 1991; starks & poe, 1997). males are frequently attacked by females (o’donnell, 1999) and are expelled from their natal nests (west-eberhard, 1969; kasuya, 1983). males contribute very little to the maintenance of the colony and alter the nutritional balance, being evicted to reduce additional costs (kasuya, 1983; suzuki, 1986). in colonies of neotropical swarmabstract in colonies of neotropical swarm-founding wasps (epiponini), males are thought to be produced only in periods when there are many queens present. little information is available regarding male behavior in and out of the nest, and male mating strategies are poorly understood. here, a behavioral study of males of synoeca surinama is provided and copulation behavior inside a nest is described for the first time. a description of an aggregation of non-natal males at a nest is also provided. the behavior of males was observed in three colonies subjected to removal of queens for another study. nest envelope was partially removed, and the observations were made directly (seen by “the naked eye”) and indirectly (through camcorder recordings), daily. production of males occurred in large colonies with number of workers being more relevant than the number of the queens. males of the aggregation were marked and filmed. the aggregation of non-natal males was observed for seven consecutive days next to the largest colony; some of the males attended the aggregation on consecutive days. the males were observed accessing the comb by the opening in the envelope and courting new queens. one successful copulation on the comb of the nest was observed. in the colony where the male aggregation was observed, 47.6% of the new queens were inseminated while in the other two colonies, no new queens were inseminated. these observations suggest males are attracted to, aggregate and seek mating opportunities at nonnatal colonies that are producing young adult queens. sociobiology an international journal on social insects ca dos santos, m da silva, fb noll article history edited by gilberto m. m. santos, uefs, brazil received 06 april 2020 initial acceptance 23 june 2020 final acceptance 20 july 2020 publication date 30 september 2020 keywords copulation, male aggregation, reproductive behavior, swarm founding wasps. corresponding author marjorie da silva universidade estadual paulista departamento de zoologia e botânica cristóvão colombo, 2265, jardim nazareth são josé do rio preto-sp, brasil. e-mail: marjoriebio@gmail.com founding wasps (tribe epiponini) males are rarely seen and seem to be produced only in periods when there are many queens present (queller et al., 1993, boomsma & grafen, 1991; pamilo, 1991). a great diversity of mating strategies is observed in hymenoptera. vespinae and polistinae, independent founders, are known to have sexual pheromones produced by females to attract males and stimulate mating behavior (downing, 1991), and pheromones produced by males to mark territory (landolt et al., 1998). in many species, males mark territory or other points rubbing the abdomen in the substrate to attract or intercept flying females to mate, suggesting that external glands produce pheromones (west-eberhard, 1982; post & jeanne, 1983; wenzel, 1987; beani & turillazzi, 1988; reed universidade estadual paulista, departamento de zoologia e botânica, são josé do rio preto, são paulo, brazil research article wasps on the production, mating and behavior of males in synoeca surinama (vespidae: polistinae: epiponini) sociobiology 67(3): 444-448 (september, 2020) 445 & landolt, 1991; beani et al., 1992; beani, 1996). in other social hymenoptera, such as apis, the males patrol or expect females in certain places (alcock et al., 1978). in stingless bees (meliponini), the males form aggregations near active nests that have young new queens (cortopassi-laurino, 2007). there is little information regarding male behavior inside and outside of the nest in epiponini and even less about mating strategies. male aggregations distant from nests were observed in polybia sericea (west-eberhard, 1982) and polybia occidentalis (jeanne, 1991). recently, chavarría and noll (2014) observed males of chartergellus communis trying to enter a nest with new females, possibly seeking to copulate. however, mating in situ has never been observed. in this work, we provide behavioral observations of males of a species of epiponini and describe, for the first time, copulation behavior inside the nest. a description of an observed aggregation of foreign males is also provided. material and methods the behavior of males was observed in three colonies (i, ii and iii) of synoeca surinama (linnaeus, 1767), located at farms in the municipality of indiaporã, são paulo state – brazil (19° 59’ s; 50° 18’ w). these colonies were subjected to removal of queens, to investigate the production of new reproductives for another study (table 1). the nest envelope was partially removed to stimulate construction and foraging activities. most workers were marked with quick-drying ink, according to the task performed. a sample of newly emerged females as well as four and 16 males, who emerged in the period, were marked in colonies i and ii, respectively. queens were captured when laying eggs and the newly emerged wasps at the time of emergence. the certainty of marking newly emerged was due to the fact that, in this period, they do not have flying ability. males were identified based on some morphological differences as follows: antennae little longer (13 segments in males and 12 in females) and thinner, the mandibular teeth are smaller; the gena is slightly narrower with its upper ends somewhat less protruding; and the last visible gastral sternite is flattened (richards, 1978). all resident males were marked to distinguish them from males coming from other colonies. after marking, wasps were observed directly and on video recordings, from daily bouts (2 to 3 hours) of filming (sony handycam dcr-sr 85 digital camcorder) for four days, to survey the behaviors and interactions between workers, queens and newly emerged adults. one week after the beginning of observations, all queens and unmarked individuals were removed. in the following days, males and newly emerged females were marked and their behavior monitored. at colony i, a few days after the start of oviposition, an aggregation of males formed. the aggregation was observed and filmed throughout the period in which it persisted. number of males was estimated based on the videos and counting individually. because males and females of s. surinama are very similar morphologically, a sample of 60 individuals from the aggregation was identified with the aid of a hand lens (using the morphological characters mentioned above) to confirm that the wasps observed were males. fifteen of the 60 identified males were marked with quick-drying ink. demographic, behavioral and physiological data from six more colonies were used for comparisons with the colonies studied here. spearman’s correlation tests were performed, using r (r core team, 2019), to compare frequencies and for correlation analysis respectively. results behavior, interactions, and permanence of young males in colonies synoeca surinama males were observed performing ventilation behavior by fanning their wings at the comb, usually before self-cleaning. also, they showed cell inspection, although they did not insert the whole head into the cells as the workers do. it is important to note that these behaviors were very sporadic in such a way that they did not contribute to the maintenance of the colony. males remained grouped to the nest peripheries (between the comb and the inner edge of the envelope) with queens and newly emerged females. sporadically they walked on the comb and interacted with the workers to obtain resources by trophallaxis. however, in colonies i and ii, many males were seen receiving aggression from workers, such as bites on the body and, on many colony period of observations nº of queens before removal nº of workers at the beginning of observations nº of new produced queens days between removal of the queens and the collection of the colony nº of nest-born marked males i 01/04/2012 to 02/05/2012 15 1374 84 25 57 ii 11/04/2012 to 02/05/2012 18 812 56 12 53 iii 10/09/2012 to 10/10/2012 28 571 38 23 69 table 1. period of observations and demographic characteristics of observed colonies of synoeca surinama. ca dos santos, m da silva, fb noll – on the male production in synoeca surinama (vespidae: epiponini)446 occasions, they were held and both male and workers in struggle, fell from the nest. as newly emerged males had little ability to fly, many were seen on the ground. the number of workers was positively related with the number of males produced by a colony (spearman’s correlation rho = 0,71; n = 9; p < 0.05). number of queens was not positively related with the number of males produced (spearman’s correlation rho = -0.51; n = 9; p = 0.16). the residence time of males in the nest (adult emergence to departure) was short (no more than seven days after emergence), even in colony iii, where attacks were observed. only three of the 179 marked males in the three colonies (1.7%) were collected at the end of the observations and these had emerged only five days before the collection. on the other hand, from the 733 newly emerged females marked, 408 (55%) were collected in their natal nests. these results suggest that most males are expelled by workers a few days after emergence and/or they leave the nest permanently by themselves, to probably join mating aggregations. aggregation of non-natal males and mating of new queens eleven days after the removal of the queens in the largest colony (i), new queens starting laying eggs. four days after the beginning of oviposition, the formation of a foreign male aggregation was observed in the tree canopy nest substrate. the aggregation was six meters high and three meters distant from the nest. we estimate more than 150 males flying nimbly and landing on the leaves of trees, where they remained for a few minutes and flew to other leaves again. males were observed alone or in small groups (up to three) in the leaves. as the vegetation in a radius of 50 meters was basically grasslands, males were observed only in the leaves of the tree where the nest was built. the aggregation was formed for seven consecutive days but, each day, the number of males apparently decreased. males arrived approximately one hour before sunset and flew away to a small fragment of forest (15.000 m2), located 300m from the nest, few minutes after the beginning of the sunrise. some of the 15 marked foreign males were seen in consecutive days, which suggests the same males form the aggregation during various days. we observed males accessing the comb by entering the opening we made in the envelope. fifteen landings were observed on the comb during four days of the period in which the aggregation lasted. in 10 of the 15 landings males were immediately recognized and attacked. nevertheless, not always the males were recognized. on one occasion a male landed and walked for about three minutes before finding a new queen and try to copulate. on five occasions they managed to mount the females but successful copulation was observed only one time since the new queens bend the gaster trying to prevent the union of the genitals. sometimes they were noticed by the workers, who immediately attacked, seized and bit the males. most males escaped and flew off. several males landed without apparently being noticed. in this case, they walked across the comb directly to a new queen, who was behaviorally identified, and then attempted copulation. the males mounted the female’s back, holding her with his legs, stretching their abdomen to expose the genitals and bending the end of the gaster in an attempt to unite his genitalia with that of the new queen. the observed mating behavior performed by males was similar to that observed and described by westeberhard (1969) for polistes fuscatus. no males were attacked when they were away from the nest, although they landed very close (within one meter) and, no sign of competition was observed among the males. copulation attempts by resident males (belonging to the observed colonies) were not observed. in colonies ii and iii, no new queens were inseminated, on the other hand, in the colony where the aggregation of males was present (i), 40 of the new queens (47,6%) were inseminated and oviposition of new queens was observed only in this colony. in addition, although the establishment of a new set of queens (females with developed ovaries) occurred in all colonies, oviposition by the news queens was observed only in the colony i, the colony where the aggregation of foreign males was observed. discussion in synoeca surinama males are produced in large colonies, with many workers, regardless of the number of queens. this same tendency was observed in asteloeca ujhelyii (nascimento et al., 2004) and metapolybia aztecoides, where the males are produced in large polygynous colonies (westeberhard, 1978). these three species belong to the same clade, being closely related (wenzel & carpenter, 1994; pickett & carpenter, 2010) and, it is possible that in this group the production of males in colonies with large numbers of workers (larger working force) is an adaptive strategy to minimize the risk of decline of the colonies for the costs males impose. concerning behaviors performed by males when they eclose, cell inspection is probably related to obtaining resources via trophallaxis with larvae (hunt et al., 1982; suryanarayanan & jeanne, 2008) and the ventilation behavior is possibly a form of training of the wings. males have weak flying ability in the first days after emergence and remain no more than seven days in the nest, being driven off by workers to avoid additional costs (o’donnell, 1999) or leaving themselves permanently. thus, they may need to be able to fly sooner than newly emerged females. in the social wasps of independent foundation, as polistes and mischocyttarus, males also leave their home nest in a few days after emergence (west-eberhard, 1969; gadagkar, 1991). in mischocyttarus drewseni, for example, they remained in the nest for 4.8 days on average (jeanne, 1972). in addition, newly emerged males of s. surinama do not accompany the females in swarming (santos, unpublished data), as parachartergus fraternus (mateus, 2011) and polybia occidentalis (bouwma et al., 2000) males do. sociobiology 67(3): 444-448 (september, 2020) 447 observations about the formation of aggregation of non-natal males near a nest suggest they were attracted chemically, although no sex pheromone has been chemically characterized in the subfamily polistinae (ayasse et al., 2001). it is highly likely the release of some volatile pheromone by new queens was perceived by foreign males at a great distance. the fact that an aggregation of males did not occur next to the colonies where oviposition was not observed corroborates this hypothesis. possible sources of attractive components would be the poison and dufour glands (ayasse et al., 2001), or even the venon gland, as observed by polistes fuscatus (post & jeanne, 1984; 1985). jeanne (1991) observed aggregation of males in polybia occidentalis in scrub vegetation far from a nest, suggesting that mating occurs away from nests. several young females were seen leaving and returning to the nest in this study, suggesting that mating would also occur in s. surinama. chavarría and noll (2014) observed males trying to enter a nest of chartergellus communis with new females, suggesting that mating would also occur near or inside the nest. mating in s. surinama may also occur within the nest, as well as outside, as we have seen males mounting and copulating with new queens in the comb. in this case, the nest envelope was partially opened, facilitating the access of males to comb, however, under natural conditions, with a closed envelope, access would have to be through the entrance at the top of the nest, which have workers watching. workers assault on foreign males was also documented for other species of epiponini like chartergellus communis (chavarría & noll, 2014) and polybia liliacea (jeanne, personal communication), suggesting that workers attack males, as if they do not recognize them as colony members, probably because they lack the same chemical signals (pfennig et al., 1983). conclusions this work contributes important information concerning production and behavioral aspects of males of synoeca surinama. copulation behavior was observed and described, for the first time, and the results suggest that mating may also occur at the comb, inside the nest. production of males occurs in large colonies, with a large number of workers, as observed for other epiponines. considering the lack of studies on the behavior and biology of males in epiponini, and the wide variation within the group, we need further research on different species of this tribe to understand strategies that allow the evolution of all the complexity observed for epiponini in terms of social organization. acknowledgments the authors would like thank capes and fapesp fundação de amparo à pesquisa do estado de são paulo (grant # 2011/06058-5) for financing this research. authors contribution all authors contributed to the study conception and design of the work. material preparation and data collection were performed by carlos alberto dos santos. analysis were performed by carlos alberto dos santos and fernando barbosa noll. the first draft of the manuscript was written by marjorie da silva and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. references alcock, j., barrow, e.m., gordh, g., hubbard, l.j., kirkedall, l., pyle, d.w., ponder, t.l. & zalon, f.g. 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(1982) the nature and evolution of swarming in tropical social wasps. in p. jaisson (ed.), social insects in the tropics (pp. 97-128). paris: université paris nord. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.6204sociobiology 68(2): e6204 (june, 2021) introduction competition is a major process related to community structuring, where the resource quality and the competitive ability of species determine the interaction outcomes (traniello et al., 1989; fonseca & robinson, 1990; dáttilo et al., 2013). it is known that competitively superior species can exclude their competitors, being able to dominate certain resources; however, it is not common to observe, in complex ecological systems, the total exclusion of a given population (brose, 2008; camarota et al., 2016). this occurs, partially, because of the great morphological and behavioral variation intrinsic to most species which grant them both competitive advantages and disadvantages. for instance, some animals are aggressive and large, but not so efficient when searching for food; others, abstract ecological interactions and environmental conditions are associated with community structuring, where the resource quality and availability, in addition to interspecific competition, determine potential local interactions. using grounddwelling ant species as a methodological tool model, we evaluated how three different daily periods affected species richness, recruitment, and resource dominance (baits) within two cerrado (brazilian savanna) phytophysiognomies. we found a total of 34 ant species and significant differences in species composition between the cerrado areas. camponotus and pheidole genera were the most abundant in both studied areas in which several species shared baits. ant richness did not vary between the cerrado areas, but ant recruitment was affected by observation periods: in both areas, the hottest period (afternoon) reflected the lowest recruitment, while the coolest period (morning) had the highest recruitment. we observed that the ant species that first found baits had a 29% chance of being the only species observed using the resource, while those that arrived after other species had only 7%. both areas promote the coexistence of ant species by shaping their interactions. we suggest that environmental structuring promotes niche partitioning in both spatial and temporal scales, providing possibilities of microhabitats that allow species to explore the environment and forage. sociobiology an international journal on social insects liegy resende dos santos1,2, renan fernandes moura2 article history edited by eduardo calixto, university of florida, usa received 29 january 2021 initial acceptance 17 february 2021 final acceptance 20 march 2021 publication date 14 may 2021 keywords abiotic factors, baits, competition, environmental conditions, interspecific interactions. corresponding author renan fernandes moura laboratório de ecologia comportamental e de interações (leci), instituto de biologia, universidade federal de uberlândia av. amazonas, bloco 6z sala 06, umuarama cep 38405-317uberlândia-mg, brasil. e-mail: renanmourabio@gmail.com despite being fragile, may have keen senses, being able to detect resources from great distances (bestelmeyer & wiens, 2001; parr & gibb, 2010). this high interspecific variation observed in nature is mediated by trade-off mechanisms and allows the coexistence of countless species by reducing the intensity of competitive interactions (fellers, 1987; tillman, 2000; miller & chesson, 2009). in addition to the competitive ability of species, it is known that the environmental conditions also interfere in the outcome of interspecific interactions, since they affect the resource distribution and microclimatic conditions (e.g., local temperature and moisture) (traniello, 1989; pol & casenave, 2004; dáttilo & izzo, 2012; mooney et al., 2016). species are delimited by a heterogeneous distribution of resources and environmental conditions (wang et al., 2001), which determine 1 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, ribeirão preto-sp, brazil 2 laboratório de ecologia comportamental e de interações, instituto de biologia, universidade federal de uberlândia, uberlândia-mg, brazil research article ants local and temporal effects on ant richness, recruitment, and resource dominance in two cerrado areas id https://orcid.org/0000-0001-6525-9177 lr santos, rf moura – local and temporal effects on ants from two cerrado areas2 potential local interactions (perfecto & vandermeer, 1996; camarota et al., 2016; queiroga & moura, 2017). for instance, some authors discuss how annual temperature and rainfall affect ants’ survival and how warmer and wetter conditions can increase the diversity and frequency of ant-plant interactions (costa et al., 2018; anjos et al., 2019; bujan et al., 2020). while the competitive capacity of species has relative stability with time, the environmental conditions are more unstable, where certain physical conditions such as temperature and rainfall may have large variation within a single day. in this sense, the resource domination exhibited by some species may only occur on specific periods of the day where the abiotic quality is within their physiological tolerance, occurring, thus, a turnover of species with time (pol & casenave, 2004; anjos et al., 2017). bestelmeyer (2000), for example, showed that dominating ants are not as heat tolerant as subordinate ant species. he observed that subordinate ants dominated food baits in periods where the temperature reached extreme values, while dominating ants were not able to forage during periods of extreme temperatures. the ants (hymenoptera: formicidae) are a good model for studying competition and dominance behaviors as they are omnipresent , with more than 15,000 species around the world (bolton, 2014). they have great morphological and behavioral variation, with many species presenting strong territorial behavior and high recruitment capacity allowing them to size critical food resources (levings & traniello, 1981). the cerrado (brazilian savanna) biome has one the greatest numbers of endemic species (myers et al., 2000). it is characterized by a well-delimited seasonality and a mosaic of plant phytophysiognomies with particular characteristics ranging from more open, heterogeneous, and dry areas such as the cerrado stricto sensu to dense and homogeneous forests such as the cerradão (oliveira-filho & ratter, 2002). this biome represents an adequate landscape for the study of ants since the wide availability and distribution of resources and the vegetal mosaic (klink & machado, 2005) allow ants to be the most successful insects in terms of biomass, abundance, and ecological impact by acting as bioindicators, cycling soil nutrients, and providing plant defense against herbivores (silva et al., 2004; silva et al., 2011; tibcherani et al., 2018; swanson et al., 2019; calixto et al., 2020). in this study, our goal was to assess the species richness, recruitment, and resource dominance of ant species within two cerrado areas (phytophysiognomies) by offering food baits during three periods of the day. we hypothesized that (i) ant composition, species richness (hereafter ant richness), and recruitment capacity will be distinct between the two cerrado areas; (ii) that ant dominance, richness, and recruitment will change during the observation periods; (iii) the discovery moment of food baits will affect their dominance by the ant species; (iv) the ant recruitment capacity will also affect the food resource dominance. we predicted that (i) ant dominance and richness will be higher in cerrado stricto sensu, due to its high habitat heterogeneity, than in cerradão, a more homogeneous phytophysiognomy; (ii) ant dominance, richness, and recruitment will be lower during the hottest observation periods; (iii) ants that discovered food baits before other species will have a higher chance of dominating it and (iv) species with increased recruitment will dominate more food baits than species with low recruitment ability. material and methods study area the study was conducted in november 2015 at the parque estadual serra de caldas novas (pescan) located in goiás state, brazil (17°46’28.88”s 48°40’46.07”w). the climate is seasonal with a dry (may to september) and a wet (october to april) season, with a mean temperature of 22.7 ºc (ferreira & torezan-silingardi, 2013). the region is characterized by a cerrado biome with 123 km2 of area and 1,043 m of altitude. the vegetation is a mosaic composed of fields, savanna, and forest formations (lima et al., 2010). the area combines several cerrado types: cerrado stricto sensu, campo sujo, campo limpo, cerradão, and gallery forests (lopes et al., 2009). for this study, we selected two cerrado areas according to the landscape description developed by ribeiro and walter (1998). the first area represents a cerrado stricto sensu located within a plateau region characterized by the presence of xeromorphic shrubs and grasses, that do not form a continuous canopy, and a latosol with medium-low organic deposition. the second area is characterized by a cerradão (xeromorphic forest) located at the plateau’s base, with latosol of medium fig 1. characterization of the study areas. cerrado stricto sensu (a) and cerradão (b) in pescan, caldas novas, goiás, brazil. sociobiology 68(2): e6204 (june, 2021) 3 organic deposition, bearing both forest and cerrado stricto sensu tree species that form a continuous canopy (fig 1). data sampling in each cerrado area, we established 10 sampling plots 10 m away from the main trail border and 20 m away from each other. in the center at each site, we sampled ants by offering, on a napkin sheet, 20 g of a protein-based bait made of sardine. the distance between sampling sites and the time of observation (see below) was established accordingly to the all protocol (fisher et al., 2000). we performed two trials at each cerrado area which were three kilometers apart from each other. in the first trial, we observed ant visitation on all 10 baits within two hours. we spent 12 minutes observing each bait until the arrival of the first ant, collecting it right away. for the second trial, we returned to the first bait to initiate a second round of observations, but we also collected at least two individuals of each ant species on each bait for identification and through a picture estimated the number of individuals (recruitment) (iphone 7 camera with 16 megapixels, ƒ/2.2 opening, 30 mm lens, field of vision of 73º). furthermore, during the second trial, we classified the observed ant species as resource dominating and non-dominating, where we considered as dominating those species that did not share the bait with others (i.e., only one species per bait). the method involving the first and second trials was repeated during three observation periods (period 1 = 8:00 to 10:00; period 2 = 12:00 to 14:00; period 3 = 16:00 to 18:00), at the same sampling sites so we could observe the arrival and dominance of ant species along the periods. all individuals collected were stored on microtubes with alcohol 70% and, afterward, they were identified with the aid of a taxonomic expert and deposited in the taxonomical collection of the behavioral ecology and interactions laboratory (leci). when performing bait trials during the three observation periods, we replaced each used sardine bait with a clean bait and removed all ants using the resource from the previous observation period. all new baits were positioned in the same place so we could observe potential changes of species over the three observation periods. all study was conducted during stable weather conditions (sunny days) to minimize data variability and avoid confounding effects caused by environmental factors such as rain. data analysis we performed all statistical procedures using the r software, version 3.5.2 (r development team, 2018). to compare species composition between the two cerrado areas, we calculated the jaccard similarity index (magurran, 2013) by using the following formula: j = c / (a + b c), where a is the number of ant species present at cerrado stricto sensu, b is the total number of species in cerradão, and c is the sum of ant species from both areas. this index varies from 0 to 1, where numbers around 1 indicate high species similarity between compared areas. we performed an analysis of similarity (anosim; see clarke, 1993) to analyze differences in species composition between the cerrado areas, using the vegan package of r (oksanen et al., 2013). we calculated the bray-curtis distance and performed 999 permutations using data on ant presence and recruitment (number of ants). after anosim, we performed a non-metric multidimensional scaling (nmds) in order to graphically expose the results. at last, we performed a similarity percentage breakdown (simper) to identify what species most contributed to the observed patterns of dissimilarity between areas (neves et al., 2013). furthermore, to evaluate the most common ant species in each cerrado area, we observed the number of baits occupied by distinct species. due to the high number of rare ant species, we selected only the species that occupied at least 10% of offered baits in each observation period. to understand whether the ant richness varied according to the cerrado areas and observation periods, we modeled the total richness per offered bait by using a linear mixed model with gaussian distribution. since we repeated bait offers per sampling site in three observation periods, we included it as a random variable represented as 1|bait (bates et al., 2015). we performed the same procedure to evaluate whether ant recruitment depended on the period of observation and cerrado area. we performed linear mixed models using the lmer function from the lme4 package of r (bates et al., 2015). we analyzed the effect of ant recruitment from each ant species on the number of dominated baits in each cerrado area by performing a generalized linear model (glm) with gaussian distribution. also, we performed a chi-square test to verify whether the arrival order of ant species on the resource (first or second trial) influenced their capacity of dominating baits. we performed three tests, one for each studied cerrado area and one using the data from both cerrados, independently of the period of observation. results we found 34 ant species, 24 from cerrado stricto sensu, 19 from cerradão, and 9 species shared by the two areas (table 1). the total number of observed individuals (recruitment) was 2,739, and the most abundant species was solenopsis geminata (849 individuals), found only in cerrado stricto sensu followed by camponotus and pheidole that were the most abundant genera in both studied areas. the jaccard similarity index revealed moderate similarity (j = 0.47) in species composition between the two sampled cerrados. this result means that the cerrado areas share 47% of all sampled ant species. the differences among species composition between the two areas were significant (anosim: r = 0.53, p = 0.001; fig 2). solenopsis geminata, camponotus sp. 1, camponotus sp. 2, c. novogranadensis, and c. senex strongly influenced the ant species composition, characterizing 32% of species dissimilarity between the two areas (simper: p = 0.001). lr santos, rf moura – local and temporal effects on ants from two cerrado areas4 we observed that the most frequent species, that is, those found on at least 10% of the offered baits (see data analysis, for details), differed between both the cerrado areas and observation periods. specifically, in cerrado stricto sensu, we found six abundant species during all three observation periods. in cerradão, we found four species, being camponotus sp.1 recorded on more than 50% of baits during period 2 and more than 40% during period 3 (fig 3). species cerrado stricto sensu cerradão observation periods 1 2 3 1 2 3 atta laevigata 15 atta sp.1 20 camponotus atriceps 120 8 6 camponotus blandus 180 23 1 camponotus novogranadensis 19 16 camponotus sp.1 2 76 50 97 camponotus sp.2 8 9 6 cephalotes depressus 1 cephalotes pusillus 5 3 1 1 crematogaster sp.1 131 5 117 10 80 dinoponera australis 1 ectatomma permagnum 7 ectatomma brunneum 3 ectatomma opaciventre 4 gracilidris pombero 2 forelius sp.1 5 linepithema sp.1 2 6 neoponera apicalis 7 nylanderia sp.1 2 odontomachus chelifer 1 pheidole diligens 51 24 3 14 pheidole gertrudae 60 pheidole oxyops 1 80 51 240 pheidole sp.1 3 58 23 pheidole sp.2 4 pheidole sp.3 40 20 pheidole sp.4 44 30 pheidole sp.5 1 pheidole sp.6 7 pheidole sp.7 33 32 pheidole sp.8 32 solenopsis geminata 197 37 615 total 613 104 966 341 302 422 table 1. the recruitment of ant species observed in each cerrado area and period of observation. fig 2. ant species composition present in two adjacent cerrado areas. each dot represents a sample spot (food bait). the ant species composition formed two distinct groups according to the cerrado area (anosim: r = 0.53, p = 0.001). in the cerrado stricto sensu, five species maintained resource dominance during the second observation period (table 2). furthermore, we observed that none of the species present in both cerrados were dominant in both areas. in cerrado stricto sensu, only camponotus sp.2 found and dominated baits during periods 2 and 3. in contrast, in cerradão, pheidole oxyops dominated baits during periods 1 and 3, while camponotus sp.1 was observed dominating baits during all periods. several species shared resources during the second trial with up to three other species. in cerrado stricto sensu, the camponotus genus was observed 34 times on the offered baits and shared them on six occasions: three times with camponotus sp.2 and three times with different species of pheidole. pheidole ants were observed 10 times on the offered baits and shared them three times with other species. in cerradão, the camponotus genus was observed 41 times, where camponotus sp.1 shared the resource 19 times with other species, while pheidole ants were observed on baits 35 times and shared them with other 23 species, pheidole diligens specifically, was observed sharing baits on 6 occasions. according to the linear mixed models, there was no effect of the cerrado area on ant recruitment (f = 0.17, p = 0.68), but there was a difference regarding the observation periods (f = 6.95, p = 0.002). in period 3, we observed higher recruitment on both cerrado areas, while in period 2 we noticed low recruitment in cerrado stricto sensu (tukey post-hoc: p < 0.05). we also verified an interaction effect between the observation periods and the cerrado areas, where ant recruitment was very low in cerrado stricto sensu (f = 4.12, p = 0.022; fig 4a). there were no differences regarding ant richness per bait between the cerrado areas (f = 2.53, p = 0.12), among the observation periods (f = 0.94, p = 0.37) and neither interaction effects between these two variables (f = 1.43, p = 0.25; fig 4b). sociobiology 68(2): e6204 (june, 2021) 5 species in cerrado stricto sensu a b atta laevigata 1 0 *camponotus atriceps 2 2 *camponotus blandus 8 4 camponotus novogranadensis 12 2 camponotus senex 2 0 *camponotus sp.1 2 0 camponotus sp.2 8 2 cephalotes depressus 2 0 *cephalotes pusillus 3 1 *crematogaster sp.1 3 4 dinoponera australis 1 0 dorymyrmex sp.1 1 0 ectatomma brunneum 1 0 ectatomma opaciventre 1 0 forelius sp.1 1 1 gracilidres pombero 1 0 odontomachus chelifer 1 0 *pheidole diligens 3 1 pheidole gertrudae 1 0 *pheidole oxyops 1 0 *pheidole sp.1 3 0 pheidole sp.2 1 0 *pheidole sp.3 1 0 solenopsis geminata 7 4 species in cerradão a b atta sp.1 1 0 *camponotus atriceps 2 1 *camponotus blandus 1 0 *camponotus sp.1 18 3 *cephalotes pusillus 1 0 *crematogaster sp.1 2 0 ectatomma permagnum 3 1 linepithema sp.1 2 0 neoponera apicalis 2 0 nylanderia sp.1 1 0 *pheidole diligens 5 0 *pheidole oxyops 8 2 *pheidole sp.1 6 1 *pheidole sp.3 1 0 pheidole sp.4 7 0 pheidole sp.5 1 0 pheidole sp.6 2 0 pheidole sp.7 4 1 pheidole sp.8 2 0 table 2. the number of food baits in which ant species shared the resource (a) and the number of baits in which they did not share it (dominating ants) (b) in each cerrado area. asterisks represent species found in both studied areas. fig 3. the proportion of ant species found on food baits according to the cerrado area and observation period (period 1 = 8:00 to 10:00; period 2 = 12:00 to 14:00; period 3 = 16:00 to 18:00). only the most frequent ant species – those that occupied at least 10% of all baits – were considered (see material and methods for details). in (a), camponotus novogranadensis was found during all three periods of observation, being predominant during period 1. in (b), there was a smaller number of ant species, with the predominant ant, camponotus sp. 1, being observed on more than 50% of baits during period 2 and 40% during period 3. species in light gray boxes have a lower proportion when compared to the species below (dark gray). species matching colors within the same bar have equal proportions. we found a significant, positive relationship between ant recruitment and the number of dominated baits for both cerrado stricto sensu (f1,22 = 51.75, r² = 0.70, p < 0.001) and cerradão (f1,18 = 11.24, r² = 0.38, p = 0.004). in cerradão, the chi-square tests revealed an association between the ant arrival moment (observation trials) and resource dominance (χ² = 8.40, p = 0.004); species that arrived during the first trial had a 29.73% chance of dominating the resource, while those arriving during the second trial had only a 7.14% chance of dominating it. we did not observe significant differences regarding the ant arrival moment and resource domination in the cerrado stricto sensu (χ² = 1.81, p = 0.18). lr santos, rf moura – local and temporal effects on ants from two cerrado areas6 discussion in this study, we evaluated how ground-dwelling ants varied in richness, recruitment, and resource dominance between two cerrado areas and distinct day periods (observation periods). we observed differences in species composition between the two areas, where ant richness and recruitment were greater in cerrado stricto sensu than in cerradão, as predicted by our first hypothesis. the camponotus and pheidole genera were highlighted in this study not only by their increased abundance but for being found in both cerrados. for both areas, there were no differences in richness among the observation periods, but cerrado stricto sensu had the lowest recruitment during the highest temperature period (period 2), while the highest recruitment occurred at the end of the day (period 3), partially corroborating our second hypothesis. for both areas, the recruitment capacity determined bait domination, where ants with increased recruitment were frequently observed seizing baits, which corroborates our fourth hypothesis. in cerradão, but not in cerrado stricto sensu, ant species that arrived early had increased chances of dominating the resource, partially corroborating our third hypothesis. the differences in ant richness and recruitment found between the cerrados are influenced by competitive interactions and environmental structuring and conditions (neves et al., 2013). we observed fewer ant species in cerradão than in cerrado stricto sensu possibly due to their distinct habitat heterogeneity. the environmental structuring in savannas is positively related to species richness, increasing in open habitats such as cerrado stricto sensu, and decreasing in shaded forests such as cerradão (andersen, 1992; oliveira filho & ratter, 2002; fisher, 2010). in addition to the environmental structuring, temperature is another abiotic factor able to shape the ant community throughout the day (dáttilo & izzo, 2012). ants often forage during daylight, but their activity levels drop during the hottest periods and increase during the coolest ones (wittman et al., 2010). we observed this pattern since the most intense ant activity (recruitment) occurred during the cooler period in both cerrado areas. in addition, we have to consider the effects of biotic factors such as interspecific competition as these interactions interfere and determine the coexistence, richness, and abundance of species (cavender-bares et al., 2009; gibb & parr, 2010). in fact, some of the main dominant ant species found in this study (e.g., camponotus genus) are usually very aggressive and tend to monopolize food resources (fagundes et al., 2017) the species dissimilarity found between the sampled areas reflects their structural differences, determining the distribution and coexistence of species (gibb & parr, 2010). the general composition of species was not very similar between the cerrado areas, showing species with distinct evolutionary histories and different capacities to find and explore resources. pheidole and camponotus genera are usually found in great abundance not only within the cerrado (andrade et al., 2007) but also within the atlantic forest (gomes et al., 2014) and crop areas of cerrado and caatinga (neves et al., 2013). in our study, these genera were not only the most abundant but also those that most often found and dominated the offered resources in both areas. pheidole and camponotus are highly abundant, aggressive, and generalist groups able to actively patrol the substrate in search of food (silvestre & silva, 2001; fonseca & dihel, 2004, queiroga & moura, 2017). given this, their outstanding capacity in finding and dominating food baits relies not only on their recruitment capacity but their high aggressiveness should play a major role in order to subdue weaker competitive species (andersen, 1992; parr et al., 2005). fig 4. the effects of cerrado area (stricto sensu and cerradão) and observation period (period 1 = 8:00 to 10:00; period 2 = 12:00 to 14:00; period 3 = 16:00 to 18:00) on ant recruitment (a) and richness (b). in (a), the linear mixed models revealed differences in ant recruitment considering the observation period, where recruitment was particularly low during period 2 of cerrado stricto sensu. ant richness has not varied significantly in any treatment (b). bars represent means, and all variability measures represent the standard error of the mean. https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2656.2008.01450.x#b3 https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2656.2008.01450.x#b38 sociobiology 68(2): e6204 (june, 2021) 7 in cerradão, we observed that species that found food baits in the first trial had higher chances of dominating it until the second trial. this challenges the idea that species with good abilities in finding resources are not effective in maintaining the resource; in other words, it does not indicate trade-off effects concerning the resource encounter and domination abilities (fellers, 1987; lebrun et al., 2007; wiescher et al., 2011). similar results were observed with invasive/alien species since their superior competitive capacity prevents or reduces the possibility of turnover with native species (holway, 1999; lessard et al., 2009; bestelsmeier & courchamp, 2014; bertelsmeier et al., 2015). according to parr and gibb (2012), trade-offs between encounter and dominance of resources represents an exception and not a requisite to the structuring and coexistence of ant species, meaning that the observed interactions here might be explained by other mechanisms such as the ant nest distance from resources (resource accessibility) and distribution (wagner, 1997; fagundes et al., 2015). our study demonstrated that the cerrado phytophysiognomy is an important factor that shapes the diversity and interactions of ants. environmental structuring is reflected by niche partitioning in both spatial and temporal scales (albercht & gotelli, 2001), providing several possibilities of microhabitats that allow species to explore the environment and forage (gibb, 2005; fagundes et al., 2005; neves, 2013). we emphasize the importance of studies comparing the richness, abundance, and behavior of ant species between different phytophysiognomies and biomes. such studies may allow us to understand the ecological role of many species, a fundamental knowledge useful for conservation practices involving not only ants but all kinds of organisms. acknowledgments we thank the parque estadual de caldas novas (pescan) and the graduation program in ecology and conservation of natural resources (universidade federal de uberlândia) for offering the field ecology course. we also thank our colleague dr. jonas maravalhas for helping us identifying the sampled ant species and our friend dr. gudryan jackson barônio for statistical advice. author's contribution lrs: conceptualization, investigation, methodology, writing rfm: conceptualization, methodology, formal analysis, writing. references albercht, m. & gotelli, n.j. 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(2010). species interactions and thermal constraints on ant community structure. oikos, 119: 551-559. doi: 10.11 11/j.1600-0706.2009.17792.x http://dx.doi.org/10.13102/sociobiology.v64i4.2107 doi: 10.13102/sociobiology.v67i4.5741sociobiology 67(4): 514-525 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction leaf-cutting ants are known in brazil as saúvas, the genus atta fabricius (tribe attini, subfamily myrmicinae). atta occurs throughout the neotropical region (mariconi, 1970) and can cause severe losses to the forest and agricultural sector of latin american countries (mariconi, 1970; fowler et al., 1989; jaffé, 1993). however, leaf-cutting ants are ecosystem engineers as they move massive amounts of soil during nest construction and remove foliage, which may change plant composition, acting as herbivores in natural systems (farji-brener & illes, 2000; urbas et al., 2007; costa et al., 2008; meyer et al., 2011; leal et al., 2014; stephan et al., 2015). in agricultural areas these alteration of the soil and abstract leaf-cutting ants are widely distributed in brazil, particularly species of the genus atta. we therefore described the occurrence of leaf-cutting and grass-cutting ant species of the genus atta. five routes comprising some of the main highways in the south, southeast, midwest, and north of brazil were sampled, in addition to ants received from other 82 municipalities, composing 300 municipalities sampled. this is the first comprehensive study of atta in brazil. the following species and subespecies were found: a. laevigata, a. capiguara, a. sexdens rubropilosa, a. sexdens piriventris, a. sexdens sexdens, and a. cephalotes. atta laevigata and a. capiguara were the species present in the largest number of the brazilian municipalities sampled. atta sexdens piriventris was only recorded in the southern region of brazil. atta bisphaerica presented lower expansion than a. capiguara. atta cephalotes and atta opaciceps are species of very restricted occurrence. southeastern region was characterized by the occurrence of a. capiguara and a. laevigata. atta laevigata exhibited a generalized pattern of occurrence in the midwest and north. our study contributes to a better understanding of the so far unknown occurrence of leaf-cutting and grass-cutting ants within brazil. sociobiology an international journal on social insects lc forti1, js rando2, rs camargo1, aa moreira3, ma castellani3, sa leite3, kka sousa1, n caldato1 article history edited by evandro nascimento silva, uefs, brazil received 30 july 2020 initial acceptance 07 september 2020 final acceptance 12 october 2020 publication date 28 december 2020 keywords attini, brazilian biomes, grass-cutting ants, leaf-cutting ants. corresponding author aldenise alves moreira https://orcid.org/0000-0002-2362-9624 departamento de fitotecnia e zootecnia universidade estadual do sudoeste da bahia 45083-300, vitória da conquista-ba, brasil. e-mail: aldenise.moreira@gmail.com the attack on plants led to the classification as pest insects (fowler et al., 1989). according to mariconi (1970), leaf-cutting ants show a wide geographic distribution, occurring from the south of the united states (latitude 33o n) to central argentina (latitude 44o s). the geographic distribution, frequency and density of attini ants in certain habitats is related to the environmental conditions such as type of vegetation, soil type, cultivation systems, climate change (fowler, 1983; farji-brener & ruggiero, 1994; gusmão & loeck, 1999; farji-brener et al., 2016), among others. according to brandão et al. (2011), the genus atta includes 19 species, of which nine occur in brazil, atta bisphaerica forel, 1908, atta capiguara gonçalves, 1944, 1 vegetal protection department, laboratory of social insects-pests, state university são paulo, school of agricultural sciences, botucatu, brazil 2 vegetal production department, state university of northern paraná, campus luiz meneghel, bandeirantes, brazil 3 departament crop science and animal science, state university southwestern bahia, vitória da conquista, brazil research article ants occurrence of leaf-cutting and grass-cutting ants of the genus atta (hymenoptera: formicidae) in geographic regions of brazil sociobiology 67(4): 514-525 (december, 2020) 515 atta cephalotes (linnaeus, 1758), atta goiana, gonçalves, 1942, atta laevigata (smith, f. 1858), atta opaciceps borgmeier, 1939, atta robusta borgmeier, 1939, atta sexdens linnaeus, 1758 and atta wollenweideri forel, 1893 (bolton et al., 2006; delabie et al., 2011). the species a. sexdens comprises three subspecies, a. sexdens sexdens, a. sexdens piriventris and a. sexdens rubropilosa (mariconi, 1970; della lucia et al., 1993; bacci et al., 2009). the available information on the distribution of leafcutting ants in brazil is found in a dispersed and unevenly updated form (delabie et al., 2011). for the brazilian regions, few studies on the subject have been conducted, highlighting the pioneering works of gonçalves (1942, 1945, 1951, 1955, 1960, 1961, 1967, 1971) and kempf (1972), who generated a broader knowledge on the distribution of species in the different regions of brazil, and paula (1956) for the state of paraná and mariconi (1966, 1970) for the state of são paulo. more recent are the studies developed by loeck and grützmacher (2001) and grürzmacher et al. (2002), for leafcutting ants in rio grande do sul and corrêa et al. (2005) and brito et al. (2012) for the northeastern region, states of alagoas and bahia, respectively. the contributions on timely reports of the occurrence of leaf-cutting ants, such as carvalho and tarragô (1982) for rio grande do sul, delabie (1989) for bahia and souza et al. (2009) for alagoas are also noteworthy. some authors have compiled the existing literature on the occurrence and/or geographic distribution of leaf-cutting ants in brazil, such as the work of della lucia et al. (1993), forti and boaretto (1997) and delabie et al. (2011). in general, a. sexdens presents the broadest geographical distribution, occurring in all regions of brazil, followed by a. laevigata, while the other species are restricted to certain brazilian regions or states, such as a. robusta, which only occurs in rio de janeiro (mariconi, 1970; della lucia et al., 1993) and espírito santo (teixeira et al., 2003). it is observed that studies on the distribution of leaf-cutting ants of the genus atta in brazil date from at least five decades. the objective of this study was to describe the current occurrence of leafcutting and grass-cutting ants of the genus atta in geographic regions of brazil. material and methods for the survey of leaf-cutting and grass-cutting ants of the genus atta, five routes comprising some of the main highways in brazil were sampled, beginning with the city of botucatu, são paulo (22°50’46”s and 48°26’02”w). the sampling was performed in 218 municipalities of the five itineraries established: 1) botucatu (são paulo state) to iepê (são paulo state), covering 48 municipalities; 2) ibiporã (paraná state) to quintana (são paulo state), with 30 municipalities; 3) oiapoque (amapa state) to santa isabel do pará (para state), covering four municipalities, 4) pirajuí (são paulo state) to avaré (são paulo state), with 83 municipalities; 5) aparecida do taboado (mato grosso sul state) to ivinhema (mato grosso sul state), comprising 53 municipalities (table 1). every 100 km, in a 500 m long by 50 m wide strip, marked at random, specimens of soldier ants were collected from the colonies found. the colonies were visually searched for and selected because of the huge size of their nests (large amount of loose soil removed). we selected the municipalities nearest to the main roads. at each sampling site, at least five exemplars of the ants found on the nests or on the foraging trails were collected. all material collected at each sampling site was stored in glass flasks containing 70% alcohol and labeled with the data obtained for subsequent analysis. latitude and longitude of the sampling municipalities were obtained with a global positioning system (sony gps – 360), whenever possible. after each trip, all material collected was sent to the laboratory of social insect pests (lisp) at fca/unesp, botucatu, sp. in addition to the collections made in routes, biological material from other 82 municipalities in different regions of brazil were duly collected and sent for identification, making up the total of 300 sampled municipalities (table 2). after screening, the specimens were mounted and identified under a stereo microscope based on the data published by gonçalves (1961) and using the identification keys of borgmeier (1959) and mariconi (1970). for atta sexdens, the division into subspecies according to bacci et al. (2009) was considered. all material obtained was compared with specimens stored in the museum of zoology, university of são paulo, and in the ângelo moreira da costa lima entomology collection of universidade federal rural do rio de janeiro. the material of this study was stored in museum of zoology, university of são paulo. results considering the 300 sampled municipalities (table 3), frequency of leaf-cutting ant species are: atta laevigata (32.6%), a. capiguara (20.0%), a. sexdens rubropilosa (10.0%), a. sexdens piriventris (9.7%), a. sexdens sexdens (2.7%), a. cephalotes (1.6%), a. opaciceps (0.3%), a. bisphaerica (0.3%), and a. vollenweideri (0.3%). our results showed that atta laevigata and a. capiguara were the species present in the largest number of the sampled municipalities (fig 1); atta sexdens piriventris was only recorded in the south region of brazil, with predominance in the states of santa catarina and rio grande do sul; atta bisphaerica presented restricted occurrence, only at the state of são paulo. atta cephalotes and a. opaciceps, were collected in the expected regions, north and northeast, respectively. the southeast region was characterized by the occurrence of a. capiguara and a. laevigata, in this order, with a. laevigata being the most frequent species in minas gerais and a. capiguara in paraná. the occurrence of a. laevigata was recorded for the first time in amapá and rio grande do sul. lc forti, js rando, rs camargo, aa moreira, ma castellani, sa leite, kka sousa, n caldato – leaf-cutting in geographic regions of brazil516 state county longitude latitude alagoas maceió -09o66’58” -35o73’52” amapá porto grande -00o71’33” -51o41’33” bahia barra do cacau -11o08’94” -43o14’16” bahia barra do rocha -14o21’05” -39o60’19” bahia mangue seco -11o13’42” -36o29’14” bahia maraú -14o10’30” -39o01’47” bahia salvador -12o97’11” -38o51’08” distrito federal brasília -15o77’97” -47o93’00” goiás caldas novas -17°74’16” -48o63’05” maranhão são luís -02o52’97” -44o30’27” minas gerais araxá -19o59’33” -46o04’05” minas gerais itaguara -20o30’22” -44o48’75” minas gerais jaíba -15o33’83” -43o67’44” minas gerais piumhi -20o46’52” -45o95’80” minas gerais são gonçalo do abaeté -18°33’83” -45°83’33” mato grosso campo novo dos parecis -13°67’52” -57°89’19” mato grosso sapezal -12o98’94” -58o76’41” pernambuco petrolina -09o39’86” -40o50’08” paraná arapoti -24°15’07” -49°82’66” paraná bandeirantes -23o11’00” -50o36’75” paraná cornélio procópio -23o18’11” -50o64’66” paraná curitiba -25o42’77” -49o27’30” paraná doutor camargo -23o55’58” -52o21’80” paraná guarapuava -25o39’52” -51o45’80” paraná matelândia -25o24’08” -53o99’63” paraná palotina -24o28’38” -53o84’00” paraná paranaguá -25°31’00” -48°31’00” paraná paula freitas -26o20’83” -50o93’80” paraná porto vitória -26°16’11” -51°23’16” paraná telêmaco borba -24°32’38” -50°61’55” rio de janeiro angra dos reis -23o06’61” -44o31’80” rio de janeiro parati -23o21’77” -44o71’30” rio grande do sul passo fundo -28o26’27” -52o40’66” rio grande do sul arroio dos ratos -30o07’72” -51o72’91” rio grande do sul butiá -30o11’97” -51o96’22” rio grande do sul charqueadas -30o73’27” -51o64’53” rio grande do sul gravataí -29o94’44” -50o99’19” rio grande do sul porto alegre -30o03’30” -51o23’00” rio grande do sul tapes -30o67’33” -51o39’58” santa catarina abelardo luz -26°56’47” -52°32’83” santa catarina caibí -27°07’16” -53°24’77” santa catarina cordilheira alta -26°95’88” -52°76’11” table 2. geographical location of the collect sites of material received in the laboratory of social insects-prague for identification. santa catarina coronel freitas -26°90’86” -52°70’30” santa catarina cunha porã -26°89’36” -53°16’80” santa catarina descanso -26°82’61” -53°50’16” santa catarina dionisio cerqueira -26°25’05” -53°63’97” santa catarina galvão -26°45’05” -52°68’58” santa catarina irani -27°02’47” -51°90’16” santa catarina ita -27°29’05” -52°32’30” santa catarina itapiranga -27°16’94” -53°73’22” santa catarina jupiá -26°39’83” -52°72’77” santa catarina maravilha -26°76’08” -53°17’25” santa catarina nova erexim -26°90’25” -52°90’58” santa catarina palmitos -27°06’75” -53°16’11” santa catarina pinhalzinho -26°84’80” -52°99’19” santa catarina piratuba -27°41’97” -51°77’19” santa catarina quilombo -26°72’61” -52°72’05” santa catarina são carlos -27°07’75” -53°00’38” santa catarina são josé cedro -26°45’05” -53°49’41” santa catarina são lourenço do oeste -26°35’91” -52°85’11” santa catarina são miguel do oeste -26°72’52” -53°51’80” santa catarina saudades -26°92’41” -53°00’30” santa catarina vargeão -26°86’36” -52°15’05” santa catarina xaxim -26°96’16” -52°53’47” são paulo agudos -22o46’91” -48o98’75” são paulo altinópolis -21°02’55” -47°37’38” são paulo assis -22°66’16” -50°41’22” são paulo cananéia -25°01’47” -47°92’66” são paulo eldorado -24o52’00” -48o10’80” são paulo franca -20o53’86” -47o43’03” são paulo igarapava -20o03’83” -47o74’69” são paulo itapetininga -23°59’16” -48°05’30” são paulo itatinga -23o10’16” -48o61’58” são paulo jaboticabal -21°25’47” -48°32’22” são paulo juquiá -24o32’08” -47o63’47” são paulo lençóis paulista -22o59’86” -48o80’02” são paulo mongaguá -24°09’16” -46°61’77” são paulo nazaré paulista -23o18’11” -46o39’05” são paulo peruíbe -24°32’00” -46°99’83” são paulo piracicaba -22°72’52” -47°64’91” são paulo santa rosa de viterbo -21°47’27” -47°36’30” são paulo ubatuba -23o43’38” -45o07’11” state county longitude latitude sociobiology 67(4): 514-525 (december, 2020) 517 species north northeast midwest southeast south brazil a. laevigata 6.0 0.3 20.3 5.0 1.0 32.6 a. capiguara -* 8.7 7.0 4.3 20.0 a. sexdens rubropilosa 0.3 2.7 2.0 5.0 10.0 a. sexdens piriventris 9.7 9.7 a. sexdens sexdens 2.7 2.7 a. cephalotes 1.3 0.3 1.6 a. bisphaerica 0.3 0.3 a. opaciceps 0.3 0.3 a. vollenweideri 0.3 0.3 *no collection discussion atta laevigata atta laevigata was the only species found in all regions of brazil (fig 1). the results are consistent with previous studies that already indicated the occurrence of this species in the five brazilian geographic regions (castro et al., 1961; della lucia et al., 1993). gonçalves (1967) reported the presence of a. laevigata in manaus, óbidos and são gabriel (northern region), where this species was found on roadsides and in crops, including under the shade of trees in covered areas. the cerrado region is an adequate habitat for a. laevigata, as observed by high-density nests recorded in cerrado areas also highlight the suitability of this vegetation type (costa & vieira-neto, 2016). although in the present study the occurrence of a. laevigata was not recorded in the northeast of brazil, this species was found by gonçalves (1951) in ceará, pernambuco and bahia in dicotyledons, wild grasses, eucalyptus and cassava. subsequently, brito et al. (2012), recorded the occurrence of a. laevigata in four municipalities in southwestern bahia, in areas of pasture, eucalyptus and forest. these municipalities are inserted in environments of caatinga, cerrado and deciduous seasonal forest. the southeastern region of brazil showed the third highest frequency of a. laevigata according to antunes (1996), part of the southeastern region is occupied by cerrado and a large area of minas gerais is covered with this type of vegetation. for the two states of the southeastern region (minas gerais and são paulo), the occurrence of a. laevigata had already been recorded (della lucia et al., 1993). the cerrado biome (brazilian savanna) covers 2 million km2 representing 23% of the area of the country. it is an ancient biome with rich biodiversity, estimated at 160,000 species of plants, fungi and animals (mma, 2020). the predominant vegetation of the woods is composed of small trees with twisted trunks with irregular ramifications; with shrubs and sub-shrubs that may have xylopodios, underground organs, which allow regrowth after burning or cutting, this being denominate cerrado senso stricto, one of the various components of the physiognomy mosaic of the cerrado biome. in the southern region, a. laevigata was rarely frequent (table 3). however, the record of its occurrence in rio grande do sul (southern region, municipality of tapes). in general, a. laevigata partially present in many municipalities (fig 1), mainly due to the opening of highways and expansion of livestock, which serve as means for the dispersion of the species (vieira-neto et al., 2016), as well as the opening of agricultural frontiers in cerrado areas, which involve the deforestation of natural forests and the plantation of monocultures and pastures. the first reference of this species in espírito santo was confirmed in the work of delabie (1998), whose author, after morphological studies of several samples of a. laevigata from different regions of brazil, concluded that atta silvai gonçalves, registered in that state, is a junior synonym of a. laevigata. these species occurred in a greater percentage on sampled municipalities in relation to a. sexdens. this result is probably related to the collection sites that covered areas close to the highways and that favor the nesting of a. laevigata (forti et al., 2011). a. laevigata is aggressive in the selection of nesting areas, since nests are built in both sunny and shady (mariconi, 1970; pereira da silva, 1975; moreira et al., 2004) and the forager workers select mono and dicotyledonous plants as growth substrate of the symbiont fungus (della lucia et al., 1993; forti et al., 2011). atta capiguara the species a. capiguara, commonly known as brown leaf-cutting ant, exploits monocotyledon plants, basically grasses, and is economically important in sugarcane fields and pastures (amante, 1967; mariconi, 1970; forti, 1985). atta capiguara was found in the southeast, mid-west and south regions in 7.0%, 8.7% and 4.3% of sampled municipalities more restricted in its brazilian distribution range. its occurrence had already been reported for the states of são paulo, mato grosso and minas gerais (della lucia et al., 1993) and also for paraná, mato grosso do sul and goiás (forti & boaretto, 1997). the highest concentration of a. capiguara was observed in the state of paraná (southern region) (fig 1). the common table 3. percentage of municipalities (n = 300) with the occurrence of species and subspecies of atta north, northeast, midwest, southeast and south regions of brazil. lc forti, js rando, rs camargo, aa moreira, ma castellani, sa leite, kka sousa, n caldato – leaf-cutting in geographic regions of brazil518 fig 1. atta species recorded in the municipalities of the north, northeast, midwest, southeast and southern regions of brazil. characteristics of municipalities where a. capiguara was found are sandy soil and plant nutrient deficiency, as well as the native vegetation classified as tropical forest (brazil, 1998). smaller size forests resembling secondary forests occur in these soils were naturally fertility is poor and used as pasture lands (wons, 1985). the characteristics of the cerrado are typical in most parts of this ecoregion, but gradual changes are observed due to climatic influences of neighboring regions (adámoli et al., 1986). two parameters should be highlighted since they define the characteristics of the seasonal regime of this region: the average annual rainfall of 1,200 to 1,800 mm and the duration of the dry season that ranges from 5 to 6 months. the average annual temperature is 22 °c in the southern part of this ecoregion and 27 °c in the northern part. its vegetation consists of isolated or groups of winding small trees on a continuous grass rug (brazil, 1998). the importance of a. capiguara has been known since the 1940s, with observations of nests in some localities with predominance of grasses in municipalities of the state of são paulo (gonçalves, 1945). surveys conducted in other municipalities increased the occurrence of this species to 17 municipalities (mariconi et al., 1961). mariconi (1966b) also recorded the occurrence of a. capiguara in 104 municipalities in the western region of the state of são paulo. amante (1967) cited the occurrence of a. capiguara in mato grosso do sul and minas gerais. although not mentioning the municipalities, thomas (1990) reported that a. capiguara had become a serious problem in two municipalities of the northwestern region of paraná after 1975 and, by 1990, this species had damaged pastures in more than 32 municipalities of that state. many factors could have contributed to the population explosion of this species, such as disturbances caused by humans when they replaced the natural vegetation with grass monocultures (forti & boaretto, 1997). according to thomas (1990), the reduction of areas with natural vegetation and the substitution of crops such as coffee, peanuts and other crops for pastures facilitated the rapid growth of a. capiguara infestation in paraná. the increased availability of monocotyledonous plants, especially exotic grasses, allows grass-cutting ants to expand their distribution and increase population density (cherrett, 1981). atta sexdens the subspecies a. sexdens rubropilosa was registered in all brazilian regions (fig 1), except in the north, while a. sexdens piriventris and a. sexdens sexdens were of more restricted occurrence, only in the south and north regions, respectively. this result reflects the adaptability de a. sexdens rubropilosa to the different environmental conditions of the country (farji-brener & ghermandi, 2008). a large number of studies on this leaf-cutting ant subspecies are available and its occurrence has been reported in several states, including minas gerais, espírito santo, goiás, mato grosso, são paulo, paraná, rio de janeiro and distrito federal (gonçalves, 1945; della lucia et al., 1993). atta sexdens piriventris was observed in the south region. the occurrence of this subspecies was sociobiology 67(4): 514-525 (december, 2020) 519 predominant in santa catarina. in the state of rio grande do sul, a. sexdens piriventris occurred in most of the sampled municipalities, corroborating the studies by loeck and grützmacher (2001) and grürzmacher et al. (2002). the main environmental characteristics that distinguish this region from other areas are lower average temperatures (16° c and 20° c) and uniform rainfall throughout the year, which are typical of subtropical climates (antunes, 1996). associated with this climate, the vegetation ranges from atlantic rainforest, tropical forest and araucaria forest to fields and coastal vegetation (carraro, 1994). supporting the hypothesis that this species tends to occupy climate environments such as those described above, in the state of paraná this species was only found in the municipality of paula freitas, located at the southern end of the state (fig 1). the north of paraná, where a significant number of the municipalities visited are located, is found in an intertropical transition zone characterized by high average temperatures and a rainy period concentrated in the summer months (antunes, 1996). this distribution in paraná resembles that observed in the southeast and, within this region, in the state of são paulo (fig 1). this fact is possibly associated with the similar environmental conditions in these border states, since the municipalities sampled in paraná are concentrated in the northern and northwestern regions of the state as mentioned above. the subspecie atta sexdens sexdens was exclusively recorded in the northern region (fig 1). however, the occurrence of this subspecies has already been reported by della lucia et al. (1993) for the mid-west, northeast and southeast regions. according to delabie (1989), this leaf-cutting ant occurs in the state of bahia (northeast region of brazil) in more open areas and is more generalist in its foraging, attacking several dicotyledonous plants of economic importance (cacao, cassava and citrus) and also forage grasses, being more abundant in pastures. the occurrence of this ant in the state of alagoas (northeast region of brazil) was recorded by souza et al. (2009). atta cephalotes atta cephalotes was found in the north region, in 1.3% of sampled municipalities. the state of amazonas (north region) was represented by only one locality (guajará) where a. cephalotes was collected. the occurrence of a. cephalotes was recorded during the travels in oiapoque (amapá) and in cruzeiro do sul and mâncio lima (acre) (fig 1), as well as in material sent for identification from maraú in bahia (north region) (fig 1), sites of occurrence of this species already cited in the literature. this result was expected, because a. cephalotes is a species easily found in the forests of amazonia and is the most demanding leaf-cutting ant in terms of soil moisture (gonçalves, 1960). in the northeast region, this ant had already been reported to the states of maranhão, pernambuco, bahia (mariconi, 1970; kempf, 1972) and alagoas (corrêa et al., 2005). atta bisphaerica in this work, a. bisphaerica occurred only in the southeast region (fig 1), in the municipality of botucatu, são paulo, which was not expected, considering that the possibilities of expansion of this ant, as well as in a. capiguara, are associated with the substitution of natural vegetation and agricultural crops by pasture and sugarcane, since this species preferentially cuts monocotyledonous plants, it was expected an increase of its occurrence. for example, in the state of são paulo, a. capiguara (amante, 1972) and a. bisphaerica (precetti et al., 1988) are of great economic importance in sugarcane and pasture, but only a. capiguara has high occurrence. nevertheless, a. bisphaerica has been more associated to sugarcane and a. capiguara to pasture areas. in sugarcane crops, the chemical control of leaf-cutting ants is quite intense, with the systematic use of thermonebulization, contributing to a significant reduction of nest density, which usually does not occur in pastures, which could explain the lower expansion of a. bisphaerica in relation to a. capiguara. the occurrence of a. bisphaerica had already been reported in other states of southeastern brazil (minas gerais and rio de janeiro), as well as in the mid-western region (mato grosso) (della lucia et al., 1993). atta opaciceps the occurrence of a. opaciceps was observed in the northeast region, state of alagoas (municipality of maceió) (fig 1), a finding also reported by souza et al. (2009), but was distributed in the caatinga region. the occurrence of this species was also reported for the states of sergipe (delabie et al., 1997) and bahia (delabie et al., 1997; brito et al., 2012) for the north and southweste regions of brazil. the hypothesis raised by fowler et al. (1990) that this leaf-cutting ant could be in extinction has not been confirmed. atta vollenweideri atta vollenweideri, which usually cuts the leaves of grasses and dicotyledons, was recorded only in the south region, in the municipality of arroio dos ratos, state of rio grande do sul (fig 1). this species is of very restricted occurrence, being recorded only for two brazilian states, rio grande do sul (gonçalves, 1960; 1971; jonkman, 1978; della lucia et al., 1993) and mato grosso (della lucia et al., 1993). in general, the results expand the knowledge about leafcutting ants of the atta genus in brazilian regions, since the work encompassed many municipalities until then not sampled. the first record of the occurrence of a. laevigata in amapá and rio grande do sul, as well as the presence of a. opaciceps in municipalities of alagoas, a quite what positive aspect once it were already raice possibility treat of being a species extinction. atta laevigata, a. sexdens (considering its subspecies) and a. capiguara are species of wide occurrence in the brazilian territory. atta cephalotes, a. bisphaerica, a. opaciceps and a. vollenweideri are species of very restricted occurrence. lc forti, js rando, rs camargo, aa moreira, ma castellani, sa leite, kka sousa, n caldato – leaf-cutting in geographic regions of brazil520 we consider that the data presented in this work can be integrated into the databases on leaf-cutting ants, the example of created and used by delabie et al. (2011), to expand the studies of regional clusters with identification of the main associated vectors and that allow the prediction of the occurrence of atta species. acknowledgements lcf was the recipient of a fellowship from conselho nacional de desenvolvimento cientifico e tecnológico (grant 301718/2013-0). state route county longitude latitude são paulo i botucatu -22°50’46” -48°26’02” são paulo i bauru -22o31’47” -49o06’05” são paulo i marília -22o21’38” -49o94’58” são paulo i rancharia -22°25’50.8” -50°59’33.7” são paulo i presidente prudente -22°21’42.4” -51°08’09.4” são paulo i ourinhos -23°00’16.2” -49°51’31.2” são paulo i santa cruz do rio pardo -22°89’88” -49°63’25” são paulo i iepê -22°66’05” -51°07’61” paraná i santo inácio -22°42’59.7” -51°46’09,8” paraná i colorado -22o83’75” -51o97’30” paraná i paranacity -22°55’00” -52°09’09.4” paraná i inajá -22o74’91” -52o19’80” paraná i são joão do caiuá -22°56’25.5” -52°22’32.4” paraná i santo antonio do caiuá -22o73’47” -52o 34’22” paraná i tamboara -23°13’29.2” -52°35’19.9” paraná i santa helena -24o86’02” -54o33’27” paraná i umuarama -23o76’63” -53°32’05” paraná i assis chateaubriand -24o42’00” -53°52’13” paraná i toledo -24o71’36” -53°74’30” paraná i vera cruz do oeste -25o05’77” -53°87’69” paraná i missal -25o09’19” -54o24’75” paraná i são miguel do iguaçú -25o34’80” -54o23’77” paraná i foz do iguaçú -25°54’77” -54°58’80” paraná i cascavel -24o95’58” -53o45’52” paraná i ampere -25o91’05” -53o47’27” paraná i capitão leônidas marques -25o47’91” -53o61’41” paraná i rio negro -26°06’00” -49°48’00” paraná i campo do tenente -25°97’80” -49°68’27” paraná i ponta grossa -25°06’00” -50°10’00” paraná i ortigueira -24o20’83” -50o94’94” paraná i imbaú -24°44’05” -50°76’08” paraná i astorga -23o23’25” -51°66’55” paraná i maringá -23o42’52” -51°93’86” paraná i londrina -23°31’02” -51°16’27” paraná i assaí -23°37’33” -50°84’13” table 1. geographical location of the collect sites visited by routes (i to v), during the trips. paraná i ibaiti -23°84’86” -50°18’77” paraná i santo antônio da platina -23°29’05” -50°07’72” santa catarina i chapecó -27°07’00” -52°37’00” santa catarina i cunhaporã -26°89’36” -53°16’80” santa catarina i xanxerê -26°56’02.2” -52°29’55.4” santa catarina i catanduvas -27°07’05” -51°66’16” santa catarina i campos novos -27°40’16” -51°22’05” santa catarina i curitibanos -27°18’10.7” -50°42’24.4” santa catarina i blumenau -26°55’00” -49°03’00” santa catarina i lages -27°81’61” -50°32’61” santa catarina i pouso redondo -27°15’29” -49°46’02” santa catarina i ibirama -27°05’69” -49°51’77” santa catarina i corupá -26°42’52” -49°24’30” paraná ii ibiporã -23o26’91” -51°04’80” paraná ii sertanópolis -23°05’86” -51°03’63” paraná ii bela vista do paraíso -22°99’66” -51°19’05” paraná ii florestópolis -22°86’33” -51°38’72” paraná ii jaguapitã -23°11’27” -51°53’19” paraná ii guaraci -22°97’30” -51°64’97” paraná ii santa fé -23°03’75” -51°80’52” paraná ii flórida -23°08’72” -51°95’36” paraná ii atalaia -23°10’50.6” -52°05’50.7” paraná ii nova esperança -23°12’41” -52°11’31” paraná ii paranavaí -23°07’30” -52°46’52” paraná ii presidente castelo branco -23°27’80” -52°15’16” paraná ii são jorge do ivaí -23°43’27” -52°29’30” paraná ii cianorte -23°66’33” -52°60’05” paraná ii campo mourão -24°04’55” -52°38’30” paraná ii jaguariaíva -24°79’11” -50°01’19” paraná ii guaíra -24°08’00” -54°25’58” state route county longitude latitude sociobiology 67(4): 514-525 (december, 2020) 521 table 1. geographical location of the collect sites visited by routes (i to v), during the trips. (continuation) paraná ii pérola -23°80’47” -53°68’36” paraná ii cafezal do sul -23°90’22” -53°51’25” paraná ii santa isabel do ivaí -23°00’27” -53°19’66” são paulo ii porto primavera -22°29’08” -52°35’43” são paulo ii teodoro sampaio -22°53’25” -52°16’75” são paulo ii mirante do paranapanema -22°29’19” -51°90’63” são paulo ii presidente epitácio -21°76’33” -52°11’55” são paulo ii presidente venceslau -21°54’24” -51°48’08” são paulo ii dracena -21°48’25” -51°53’27” são paulo ii flórida paulista -21°61’47” -51°17’36” são paulo ii osvaldo cruz -21°79’66” -50°87’86” são paulo ii universo -22°16’27” -50°68’15” são paulo ii quintana -22°07’25” -50°87’86” amapá iii oiapoque -03°84’30” -51°83’05” amapá iii clevelândia do norte -03°92’21” -51°84’10” amapá iii macapá -00°03’88” -51°06’64” pará iii santa isabel do pará -01°29’86” -48°16’05” são paulo iv pirajuí -21°99’86” -49°45’72” são paulo iv penápolis -21°41’97” -50°07’05” são paulo iv andradina -20°89’61” -51°37’94” mato grosso do sul iv três lagoas -20°75’11” -51°67’83” mato grosso do sul iv água clara -20°31’30” -52°35’43” mato grosso do sul iv mutum -20°17’23” -52°39’51” mato grosso do sul iv ribas do rio pardo -20°44’30” -53°75’91” mato grosso do sul iv campo grande -20°29’55” -54°36’39” mato grosso do sul iv anhanduí -20°37’65” -54°13’49” mato grosso do sul iv nova alvorada do sul -21°46’58” -54°38’38” mato grosso do sul iv jaraguari -19°46’27” -54°21’42” mato grosso do sul iv bandeirantes -19°36’35” -54°23’17” mato grosso do sul iv são gabriel do oeste -19°03’03” -54°47’58” mato grosso do sul iv rio verde de mato grosso -18°91’80” -54°84’41” mato grosso do sul iv coxim -18°50’66” -54°76’00” mato grosso do sul iv piúva -18°27’33” -54°15’02” mato grosso do sul iv sonora -17°57’69” -54°75’77” mato grosso iv anhumas -17°23’47” -54°45’27” mato grosso iv pedra preta -16°62’30” -54°47’38” mato grosso iv jaciara -16°42’13” -54°72’57” mato grosso iv juscimeira -16°05’05” -54°88’44” mato grosso iv são pedro da cipa -16°00’05” -54°92’13” mato grosso iv são vicente -15°49’20” -55°25’00” mato grosso iv cuiaba -15°23’32” -55°59’11” mato grosso iv jangada -15°23’55” -56°48’91” mato grosso iv rosário oeste -14°17’25” -55°33’68” mato grosso iv nobres -14°36’10” -55°14’36” mato grosso iv posto gil -14°26’42” -55°11’36” mato grosso iv nova mutum -13°37’57” -52°02’04” mato grosso iv piúva -13°18’45” -56°24’21” mato grosso iv lucas do rio verde -13°05’02” -55°91’11” mato grosso iv sorriso -12°54’52” -55°71’13” mato grosso iv sinop -11°86’41” -55°50’25” mato grosso iv chapada dos guimarães -15°26’04” -55°46’49” mato grosso iv campo verde -15°39’27” -55°12’29” mato grosso iv coronel ponce -15°18’45” -53°44’07” mato grosso iv primavera do leste -16°10’21” -56°28’54” mato grosso iv poxoréo -16°10’20” -56°28’54” mato grosso iv presidente murtinho -15°37’59” -53°57’49” mato grosso iv paredão grande -15°48’73” -53°22’81” mato grosso iv coronel meruri -15°44’23” -53°19’67” mato grosso iv alto araguaia -17°31’47” -53°21’52” mato grosso iv barra do garças -15°46’36” -52°33’44” goiás iv bom jardim de goiás -16°19’43” -51°56’35” goiás iv piranhas -16°26’52” -51°38’31” goiás iv arenópolis -16°38’61” -51°56’02” goiás iv iporá -16°44’19” -51°11’77” goiás iv israelândia -16°31’77” -50°90’80” goiás iv jussara -15°85’05” -50°86’80” goiás iv fazenda nova -16°04’58” -50°48’11” goiás iv santa fé de goiás -15°76’91” -51°10’55” goiás iv juscelândia -15°40’67” -51°43’58” goiás iv aruanã -15°02’40” -51°05’54” goiás iv araguapaz -15°09’08” -50°63’22” goiás iv faina -15°44’61” -50°36’05” goiás iv goiás -15°09’08” -50°63’22” goiás iv itaberaí -16°02’02” -49°81’02” goiás iv itauçú -16°11’20” -49°36’29” goiás iv inhumas -16°18’26” -49°31’30” state route county longitude latitude state route county longitude latitude lc forti, js rando, rs camargo, aa moreira, ma castellani, sa leite, kka sousa, n caldato – leaf-cutting in geographic regions of brazil522 goiás iv anápolis -16°32’66’’ -48°95’27” goiás iv leopoldo de bulhões -16°61’91” -48°74’36” goiás iv silvania -16°65’88” -48°60’80” goiás iv vianópolis -16°74’19” -48°51’63” goiás iv ourizona -17°10’23” -48°18’35” goiás iv urutaí -17°46’36” -48°20’16” goiás iv ipamerí -17°72’19” -48°15’97” goiás iv catalão -18°16’58” -47°94’63” minas gerais iv araguari -18°64’72” -48°18’72” minas gerais iv uberlândia -18°43’54” -48°13’22” minas gerais iv tupassiguara -18°53’36” -48°39’41” minas gerais iv monte alegre de minas -18°87’05” -48°88’08” minas gerais iv prata -19°23’42” -48°53’49” minas gerais iv frutal -20°02’47” -48°94’05” são paulo iv nova granada -20°53’38” -49°31’41” são paulo iv são josé do rio preto -20°81’97” -49°37’94” são paulo iv catanduva -21°13’77” -48°97’27” são paulo iv santa adélia -21°24’27” -48°80’41” são paulo iv ururaí -21°39’62” -48°29’34” são paulo iv matão -21°60’33” -48°36’58” são paulo iv boa esperança do sul -21°50’38” -49°34’52” são paulo iv barra bonita -22°49’47” -48°55’80” são paulo iv espírito santo do turvo -22°69’22” -49°43’02” são paulo iv avaré -23°89’06” -48°92’58” mato grosso do sul v aparecida do taboado -20°08’66” -51°09’36” mato grosso do sul v raimundo -19°25’22” -51°23’03” mato grosso do sul v mâncio lima -07°34’09” -72°49’42” mato grosso do sul v guajará -07°03’23” -72°34’24” mato grosso do sul v dourados -22°15’40” -54°69’18” mato grosso do sul v caarapó -22°63’41” -54°82’22” mato grosso do sul v amambai -23°04’58” -55°08’41” mato grosso do sul v sanga puitã -22°58’35” -55°87’29” mato grosso do sul v ponta porã -22°53’61” -55°72’55” mato grosso do sul v jateí -22°48’19” -54°30’25” mato grosso do sul v deodápolis -22°31’52” -54°12’30” mato grosso do sul v ivinhema -22°30’47” -53°81’52” goiás v paranaíba -19°47’06” -52°06’54” goiás v itajá -19°05’46” -51°37’50” goiás v cassilândia -19°04’58” -51°42’33” goiás v aporé -19°02’42” -51°48’51” goiás v serranópolis -18°30’61” -51°96’22” goiás v jataí -17°88’13” -51°71’44” goiás v mineiros -17°56’94” -52°55’11” goiás v perolândia -17°52’86” -52°06’41” mato grosso v portelândia -17°35’36” -52°67’86” mato grosso v santa rita do araguaia -17°32’55” -53°20’52” mato grosso v alto garças -16°94’38” -53°52’80” mato grosso v poconé -16°01’11” -56°39’06” mato grosso v cangas -16°23’47” -56°12’65” mato grosso v cáceres -16°07’05” -57°67’88” mato grosso v mirassol d’oeste -15°49’47” -58°11’03” mato grosso v porto esperidião -15°85’27” -58°46’02” mato grosso v pontes e lacerda -15°22’61” -59°33’52” rondônia v vila bela da santíssima trindade -15°00’80” -59°95’05” rondônia v comodoro -13°66’30” -39°78’58” rondônia v padronal -12°55’57” -60°02’35” rondônia v vilhena -12°74’05” -60°14’58” rondônia v marco rondon -12°11’32” -60°49’38” rondônia v cacoal -11°43’86” -61°44’72” rondônia v castanhal -01°29’38” -47°92’63” rondônia v ji-paraná -10°87’55” -61°94’91” rondônia v ouro preto do oeste -10°71’63” -62°24’77” rondônia v jaru -10°43’88” -62°46’63” rondônia v nova vida -10°27’30” -62°15’28” rondônia v ariquemes -09°49’47” -58°11’03” rondônia v porto velho -08°44’50” -63°53’08” rondônia v jamari -08°45’44” -63°44’41” rondônia v josé bonifácio -09°43’46” -64°08’37” rondônia v jaci paraná -09°18’05” -64°36’16” rondônia v mutum paraná -09°18’05” -64°36’16” rondônia v abunã -09°40’58” -65°05’27” pará v pimenta bueno -11°67’25” -61°19’36” acre v vista alegre do abunã -09°38’45” -65°09’32” acre v extrema de rondônia -09°44’56” -67°03’42” acre v plácido de castro -10°14’46” -67°40’43” acre v rio branco -10°61’07” -67°45’24” amazonas v cruzeiro do sul -07°36’07” -72°43’23” state route county longitude latitude table 1. geographical location of the collect sites visited by routes (i to v), during the trips. 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(hymenoptera: formicidae). neotropical entomology, 44: 123–128. doi: 10.1007/s13744-014-0267-0 teixeira, m.c., schoereder, j. h. & mayhé-nunes, a.j. (2003). geographic distribution of atta robusta borgmeier (hymenoptera: formicidae). neotropical entomology, 32: 719-721. doi: 10.1590/s1519-566x2003000400026 thomas, j.c. (1990). formigas cortadeiras: instruções básicas para o controle. curitiba: emater. 32 p. urbas, p., araújo, j.r.m.v., leal, i.r. & wirth, r. (2007). cutting more from cut forests: edge effects on foraging and herbivory of leaf-cutting ants in brazil. biotropica, 39: 489495. doi: 10.1111/j.1744-7429.2007.00285.x vieira-neto, e.h.m, vasconcelos, h.l. & bruna, e.m. (2016). roads increase population growth rates of a native leaf-cutter ant in neotropical savannahs. journal of applied ecology, 53: 983-992. doi: 10.1111/1365-2664.12651 wons, i. (1985). geografia do paraná: física-humana-econômica. curitiba: ensino renovado, 172 p. doi: 10.13102/sociobiology.v67i4.5513sociobiology 67(4): 526-534 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction in recent decades, efforts to increase knowledge about bees have been performed in response to the rapid decline and collapse of their populations (freitas et al., 2009; potts et al., 2016; nielsen et al., 2017; castilhos et al., 2019). the most probable causes for this worldwide phenomenon are the use of pesticides in the agriculture, infections by pathogens, and habitat fragmentation (cortopassi-laurino et al., 2006; del sarto et al., 2014; goulson et al., 2015; guimarães-cestaro et al., 2020). stingless bees (meliponini sensu michener, 2007) are the most numerous group of social bees widely distributed abstract stingless bees are social insects widely distributed in the neotropical region but their populations have declined due to loss of habitats and the increase use of pesticides in agriculture. thus, the knowledge of the biology of these insects, including reproductive biology is important for their conservation and management. this study describes the morphological changes in the male reproductive tract during the sexual maturation in the stingless bee scaptotrigona xanthotricha (moure, 1950). the gross morphology and histology of the reproductive tract was investigated from pupae to 22 days old adults. the male reproductive organs in s. xanthotricha are a pair of testes, each with four follicles, pair of vasa deferentia with enlarged seminal vesicle region that open in an ejaculatory duct. in the male reproductive tract of this bee there are no is accessory glands. from brown-eyed pupae to newly-emerged adults, the epithelia of the seminal vesicle and postvesicular vasa deferentia have prismatic cells, which release secretion to the organs lumen. in 5-days old adults the testes undergo degeneration, the seminal vesicle is filled with secretion and spermatozoa, and its epithelium has cubic cells rich in inclusions in the basal region suggesting that males reach the sexual maturation. structural modifications in the reproductive tract during sexual maturation are discussed concerning the reproductive biology of meliponini. sociobiology an international journal on social insects va araújo1, je serrão2, y antonini3, lg dias4, j lino-neto2 article history edited by evandro nascimento silva, uefs, brazil received 24 june 2020 initial acceptance 20 august 2020 final acceptance 13 october 2020 publication date 28 december 2020 keywords hymenoptera; metamorphosis; seminal vesicle; reproduction; vasa deferentia. corresponding author vinícius albano araújo https://orcid.org/0000-0001-9387-7378 instituto de biodiversidade e sustentabilidade (nupem) universidade federal do rio de janeiro av. são josé barreto, 764 são josé do barreto, cep 27965-045 macaé-rj, brasil. e-mail: vialbano@gmail.com vialbano@nupem.ufrj.br in tropics (michener, 2007, 2013; cortopassi-laurino & nogueira-neto, 2016). in the neotropics there are more than 400 species into 32 genera (camargo, 2013), which performing fundamental ecosystem services as pollinators (michener, 2007; freitas et al., 2009) pollinating 40-90% of native plants (brosi et al., 2009) as well as some crops (slaa et al., 2006). the morphology of the male reproductive tract has been described in some species of meliponini (cruz-landim, 2001; dallacqua & cruz-landim, 2003; ferreira et al., 2004; araújo et al., 2005; lima et al., 2006; lino-neto et al., 2008; brito et al., 2010; ferreira et al., 2019). although those studies have contributed in some extension to understand the reproduction 1 instituto de biodiversidade e sustentabilidade (nupem), universidade federal do rio de janeiro, macaé-rj, brazil 2 departamento de biologia celular, universidade federal de viçosa, viçosa-mg, brazil 3 departamento de biodiversidade, evolução e meio ambiente, instituto de ciências exatas e biológicas, universidade federal de ouro preto, ouro preto-mg, brazil 4 departamento de ciencias biológicas, facultad de ciencias exactas y naturales, grupo de investigación bionat, universidad de caldas, caldas, colombia research article bees structural changes in the male reproductive tract of the stingless bee scaptotrigona xanthotricha moure, 1950 (meliponini, apidae) during sexual maturation sociobiology 67(4): 526-534 (december, 2020) 527 in these insects, they are restricted to few species and gaps about the reproductive biology and evolutionary history of stingless bees needs to be fills. an intriguing finding in the male reproductive tract of stingless bees is the absence of accessory glands (kerr, 1948; ferreira et al., 2004, 2019). in insects, the accessory glands associated with the male reproductive tract produce compounds that are involved in functions such as spermatozoa maturation and activation, sperm and spermatophore formation, activation of oviposition in females, polygamy control including the formation of the mating plug (chen, 1984; gillot, 2003; boosmam et al., 2005; colonello & hartfelder, 2005; fuessl et al., 2014). because these glands are absent in stingless bees their function has been suggested to be replaced by secretory cells in other reproductive tract regions, mainly in the vas deferens and seminal vesicle (dallacqua & cruz-landim, 2003; araújo et al., 2005; brito et al., 2010; ferreira et al., 2019). in these organs, the epithelial cells seem to produce components of the semen fluid (dallacqua & cruz-landim, 2003; araújo et al., 2005; brito et al., 2010). in social bees the spermatogenesis begins in the pupal phase and is completed in adult males with different ages according to the species (happ, 1992; cruz-landim & dallacqua, 2002; dallacqua & cruz-landim, 2003; linoneto et al., 2008). males emerge sexually immature and continue to undergo structural changes in the reproductive tract until complete formation and migration of spermatozoa to the seminal vesicles, when they then are sexually mature to mate and fertilize the eggs (araújo et al., 2005; lima et al., 2006; brito et al., 2010; ferreira et al., 2019). among 33 genera of meliponini endemic of the neotropical region, scaptotrigona has 21 species with nine in brazil (pedro, 2014). scaptotrigona xanthotricha (moure, 1950) is widely distributed in the atlantic rainforest where they build nests in hollows of trunks (kerr, 2001). together with other bee species, s. xanthotricha is responsible for 90% of pollination of atlantic forest native trees (kerr, 2001; custodio et al., 2017) and has economic importance due to the honey and as pollinators of crops (slaa et al., 2006; nunessilva et al., 2013; witter et al., 2015), such as cucumber, strawberries, carrots and watermelon (dos santos et al., 2008; roselino et al., 2009; nascimento et al., 2012). considering ecological and economic importance as well as the need to expand the knowledge about the reproductive biology of stingless bees, this study describes the morphological changes in the male reproductive tract of s. xanthotricha during sexual maturation. materials and methods insects brood combs were obtained from colonies of s. xanthotricha kept in the central apiary of the federal university of viçosa, minas gerais state, brazil. the pupae were removed by opening the brood cells with aid of tweezers and males were identified by the presence of gonostylus. the age of pupae was classified according to the pattern of compound eyes color and body pigmentation, assigned into five different classes: 1) pink-eyed pupae; 2) brown-eyed pupae; 3) black-eyed and non-pigmented body pupae; 4) black-eyed pigmented body and non-pigmented wing pupae, and 5) black-eyed pigmented body and wing pupae. to obtain adults, brood combs were placed in petri dishes at 28 ± 2 ºc, and newly-emerged adult males were marked on the thorax with non-toxic ink to control age of each individual. these males were caged in wooden box together with workers of different ages (in order to feed young males), resin, and brood combs containing young larvae (to be handled by workers) at 28 ± 2 ºc. bees were fed on pollen grains stored in the colonies and 50% sucrose aqueous solution ad libitum. light microscopy seven males every pupal stages and adults 0-22 days old were cryo-anesthetized at -5 ºc for 2 min the reproductive tracts were dissected in 0.1 m sodium cacodylate buffer ph 7.2, and transferred to 2.5% gluteraldehyde in the same buffer for 4 h. then, samples were washed in the same buffer and post-fixed in 1% osmium tetroxide for 2 h following dehydration in a graded ethanol series (50, 70, 90 and 95%). the pieces were embedded in historesin (leica historesin) and 2 µm thick sections obtained with a glass knife in a rotary microtome leica rm 2255 were stained with 1% toluidine blue-sodium borate and analyzed with light microscope. for anatomical analysis, freshly fixed reproductive tracts were photographed with an olympus bx-60 microscope. some histological sections were submitted to the pas histochemical test (junqueira & junqueira, 1983) to detect polysaccharides and glycoconjugates. positive control was performed with bombus terrestris midgut (carneiro et al., 2018). results the male reproductive tract of s. xanthotricha had a pair of testes, a pair of vasa deferentia and seminal vesicles opening in an ejaculatory duct. each testis had four follicles opening into efferent ducts that fused to form a vas deferens. the proximal region of vas deferens, closely to the testis, had an enlarged region forming the seminal vesicle, followed for a long postvesicular region opening in a short ejaculatory duct (fig 1a). during post-embryonic development and adult lifespan there were morphological changes in male reproductive tracts. from pupae to 4-day-old adult, the testes were voluminous and seminal vesicles slightly dilated (fig 1b). in 5-day-old adults, the testes begun to degenerated, which was complete in the 10th day (fig 1a). in the young pink-eyed pupae the vasa deferentia and seminal vesicles had empties lumen, and their single layered epithelia had prismatic cells (65 µm high) with well-developed median nuclei rich in decondensed chromatin (fig 1c). va araújo, je serrão, y antonini, lg dias, j lino-neto – sexual maturation in scaptotrigona xanthotricha528 from brownto black-eyed pigmented body and nonpigmented wings pupae, the epithelia of the vasa deferentia and seminal vesicles were rich in basal large vesicle-like structures and apical granules which were released into the lumen, resulting in increased secretion accumulation according to the age (fig 1d, g). fig 1. the male reproductive tract of scaptotrigna xanthotricha. a. sexually mature male 10-day-old showing degenerated testes (t), enlarged seminal vesicles (sv), vasa deferentia (vd) and the ejaculatory duct (ed). b. black-eyed pupae showing well-developed testes (t) and dilated seminal vesicles (sv). c-d. cross section of a seminal vesicle in pink-eyed pupae (c) and brown-eyed pupae (d) showing empty lumen (l), simple prismatic epithelium (ep), vesicles (arrow), and a muscle layer (mu). e-f. cross section of vasa deferentia (e) and seminal vesicles (f) in black-eyed and non-pigmented body pupae showing the secretion in the lumen (arrows). g. longitudinal section of a vas deferens in black-eyed and pigmented body pupae, showing the different secretions in the lumen (arrows). bars: a-b = 0,7mm; c-g = 50µm; sociobiology 67(4): 526-534 (december, 2020) 529 from black-eyed pigmented body and wings pupae to 2-day-old adult, the vasa deferentia and seminal vesicles epithelia had prismatic cells with median and well-developed nuclei (fig 2a). in both organs, the epithelial apical region was dilated, rich in vesicles with granular content, which protruded and detached into the lumen (fig 2a). threeand 4-day-old adults, showed the epithelia of the vasa deferentia and seminal vesicles with tall cells with vesicle-like structures in the basal region and nuclei smaller (5 µm diameter) (fig 2b) than those from previous developmental stages (8 µm diameter). the lumens of these ducts were filled with vesicles of granular content (fig 2b, c). fig 2. light micrographs in transverse sections of the seminal vesicles and post-vesicular vasa deferentia of scaptotrigona xanthotricha. a. black-eyed and pigmented body pupae showing the epithelium of the seminal vesicle (ep) with prismatic cells rich in apical vesicles, median nuclei (nu) and vesicles inclusions in the base (in). b-c. 3-day-old adult male showing vesicular inclusions (in) at the base of the epithelial cells (ep) and secretions in the lumen (arrow) of the vasa deferentia. d. 5-day-old adult male showing cubic epithelium (ep), with basal cell nuclei (arrow) and sperm at the apical edge of the epithelium (arrow head). note the vesicular inclusions (in) at the base of the epithelium that is lined a muscle layer (mu). bars: a-c = 20µm; d = 10µm. va araújo, je serrão, y antonini, lg dias, j lino-neto – sexual maturation in scaptotrigona xanthotricha530 five-day-old adult revealed the seminal vesicles filled with spermatozoa with cubic epithelium (32 µm high) showing basal nucleus (fig 2d) and the apical region rich in vesicles with granular content (fig 3a). in the 6and 7-day-old adults, the seminal vesicles were filled with spermatozoa (fig 3b), the cubic epithelium had median nucleus and the basal region was rich in vesicles (fig 3b). in these males, the post-vesicular vas deferens was filled with secretion (fig 3c), whereas in the pre-vesicular region there was some spermatozoa in the lumen (fig 3d). fig 3. light micrographs of transverse sections of the seminal vesicles and post-vesicular vasa deferentia of scaptotrigona xanthotricha. a. seminal vesicle of a 5-day-old adult showing lumen with spermatozoa (spz), and vesicles (arrowhead) at the apex and base (in) of the epithelial cells (ep). nucleus (arrow). b. seminal vesicle with cubic epithelium showing sperm (spz) in the lumen (l), and cells with spherical nucleus (arrowhead) and vesicular inclusions in the basal region. c-d. vasa deferentia with secretions (arrowhead) and sperm (spz) in the lumen. e-f. seminal vesicle of 10-day-old males showing sperm (spz) in the lumen, and many vesicular inclusions (in) in epithelial cells (ep). nucleus (arrowheads). external muscle (mu). bars: a-b = 10µm; c = 15µm; d-f = 30µm. sociobiology 67(4): 526-534 (december, 2020) 531 males from 8to 10-day-old aged showed the nucleus of epithelial cells of the seminal vesicle with irregular shape and, sometimes, compressed by numerous and large vesicles (fig 3e, f). many spermatozoa were associated with the apical surface of the epithelium. the post-vesicular vas deferens showed a similar pattern with cubic epithelium and nucleus compressed by vesicles (fig 4a). after the 20th day of adulthood, there was an increase in size and quantity of vesicles in the epithelium of the seminal vesicle (fig 4a, d). in addition, some spermatozoa bundles were associated with deep folds of the epithelium (fig 4c). the post-vesicular vas deferens showed luminal secretion in male 22-day-old (fig 4b) and the epithelium with nuclei showing an increase in the amount of condensed chromatin (fig 4b, d). during the development and sexual maturation, the vesicles in the apical region of the epithelium in the seminal vesicle and vas deferens had some pas-positive granules (fig 4e). fig 4. light micrographs of transverse sections of the seminal vesicles and post-vesicular vasa deferentia of scaptotrigona xanthotricha. a. seminal vesicle in 10-day-old male showing sperm (spz) in the lumen, muscular cells lining externally (mu), and the cubic epithelium (ep) with cell nuclei (arrowhead) associated with vesicle-like inclusions (in). b-d. 22-day-old males showing (b) vas deferens secretion (arrow) in the lumen and nuclei with some clumps of condensed chromatin (arrowhead); (c) spermatozoa (spz) associated with deep epithelial fold (ep); and (d) basal region of the epithelium with many vesicle-like inclusions (in) and the nuclei with clumps of condensed chromatin (arrowhead). erepresentative epithelium of seminal vesicle in black-eyed pupae showing pas-positive reaction (arrowheads). bars: a and d = 20µm; b = 30µm; c and e = 10µm. va araújo, je serrão, y antonini, lg dias, j lino-neto – sexual maturation in scaptotrigona xanthotricha532 discussion the male reproductive tract of s. xanthotricha is similar to that described for other meliponini with a pair of testes, each with four encapsulated follicles, a pair of vasa deferentia and seminal vesicles opening in an ejaculatory duct without accessory glands (ferreira et al., 2004; brito et al., 2010). in s. xanthotricha, from young pink-eyed pupae to 5-day-old adults the testes are well-developed and the enlarged seminal vesicles empty, suggesting that the spermatogenesis is in progress such as reported for other bees (dumser, 1980; heinze & holldobler, 1993; cruz-landim & dalacqua ,2002; boomsma et al., 2005; lima et al., 2006; brito et al., 2010). however, in the stingless bees melipona mondury (lima et al., 2006) and m. quadrifasciata (ferreira et al., 2019), the lumen of seminal vesicle in the pupae is filled with amorphous content. when the seminal vesicles are filled with spermatozoa, the testes undergo degeneration and males can be considered sexually mature (cruz-landim & dallacqua, 2002; araújo et al., 2005; brito et al., 2010). thus, our results indicate that s. xanthotricha males reach sexual maturity from the 5th to the 10th day of adulthood because testes undergo degeneration and seminal vesicles are filled with sperm. in this way, they no longer produce spermatozoa after sexual maturation likely in other eussocial hymenoptera (boomsma et al., 2005). in s. xanthotricha, the spermatozoa begin to migrate from testes to the seminal vesicles in the 4th day of adulthood and testes are almost totally degenerate in 10-day-old male. the sexual maturation in bees varies according to the species with spermatozoa stored in the seminal vesicles of m. mondury on the 4th day of adult life (lima et al., 2006), 7th day in m. quadrifasciata (camargo, 1984), 9th day in friesella schrottkyi (brito et al., 2010), and 12th day in apis mellifera (snodgrass, 1978) and bombus terrestris (tasei et al., 1998). the absence of male accessory glands in the reproductive tract of s. xanthotricha and all studied meliponini is intriguing because these glands play important reproductive functions in other bees, such as the production of the mating plug, mating signals, changes in the female behavior, and maintenance of spermatozoa viability (colonello & hartfelder, 2005; den boer & boomsma, 2009). in sexually mature males of s. xanthotricha it was observed the storage of secretions in the seminal vesicles (araújo et al., 2005), but results here obtained show that during sexual maturation, substances are produced and stored by seminal vesicles and the post-vesicular vasa deferentia. in some stingless bees, secretion rich in glycoproteins are produced by the epithelium of the seminal vesicles in pupae and adults, although these compounds occur in small amounts in the lumen (dallacqua & cruz-landim, 2003; araújo et al., 2005; lima et al., 2006; ferreira et al., 2019). therefore, it is plausible to suggest that in s. xanthotricha, the vas deferens, including the region of the seminal vesicle, might have different functions during sexual maturation, such as synthesis of glycoproteins in early stages of post-embryonic development and storage after sexual maturation. the vesicles in the epithelial cells of vasa deferentia and seminal vesicles in sexually mature s. xanthotricha males have been reported as myelin figures under high resolution (araújo et al., 2005) and may indicate a function of spermophagy throughout the epithelium, as found in m. bicolor (dallacqua & cruz landim, 2003) and in the ant camponotus spp. (wheleer, 1992). however, myelin figures have also been suggested to be structures of storage and secretion, mainly of lipids with specialized functions in the extracellular environment (schmitz & muller, 1991). these vesicles occur during sexual maturation of s. xanthotricha, such as in f. schrottkyi (brito et al., 2010), which may release components of the sperm, including lipids to be transferred to female during mating. this work describes the relationship between age and spermatogenesis, testicular degeneration and sexual maturation in s. xanthotricha. these male bees reach sexual maturity between the 5th and 10th day of adulthood with morphological changes in the testes, epithelia of the vasa deferentia and seminal vesicles throughout the sexual maturation process. overall, these data contribute to a better understanding of reproductive biology contributing to the management and conservation of this important stingless bee. acknowledgements to professor lucio o. campos (ufv) for providing the insects and employees of the apiary of the federal university of viçosa. this research had the financial support of the brazilian agencies cnpq and fapemig. author contributions all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by vinícius albano araújo, josé eduardo serrão, yasmine antonini, lucimar gomes dias, josé linoneto. the first draft of the manuscript was written by vinícius albano araújo and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. references araújo, v.a., zama, u., neves, c.a., dolder, h., lino-neto, j. 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(2015). stingless bees as alternative pollinators of canola. journal of economic entomology, 108: 880-886. doi: 10.1093/jee/tov096. 653 molecular cloning and expression of an estrogen receptor-related receptor gene in apis mellifera (hymenoptera: apidae) by gengsi xi1*, xiaoming liu1 & xiahui ouyang2 abstract estrogen receptor-related receptors (errs) belong to a subfamily of orphan nuclear receptors where the proteins are closely related to the estrogen receptors (ers) in structure. err homologs have been found in many animals and play an important role in the regulation of physiologic processes. in our study, the estrogen receptor-related receptor homolog gene (referred to as amerr) was cloned from apis mellifera linnaeus. the full-length cdna of the amerr gene is 1779 bp, containing a 5’untranslated region (5’utr) of 197 bp and a 3’-utr of 277 bp. the open reading frame of 1,305 bp encodes a 434-amino acid protein. using real-time quantitative rt-pcr to study amerr mrna expression patterns indicated that this gene is differentially expressed during honeybee development. there is a remarkable increase of relative expression of amerr mrna from pupa to adult , which may indicate that the gene plays an important role in the bee’s differentiation of tissues and development. a different expression in castes and abundance expression in adults of amerr mrna may suggest it is relevant to functions in social communities. key words: molecular cloning, estrogen receptor-related receptors(err), apis mellifera , real-time quantitative rt-pcr introduction nuclear receptors (nr) constitute a large family of proteins that can be considered as ligand-inducible transcription factors, which bind directly to dna and regulate expression of down stream target genes (ouyang & xi 1 college of life sciences, shaanxi normal university, xi’an, 710065, people’s republic of china 2 college of life sciences and engineering,northwest university for nationalities,lanzhou,730030 ,people’s republic of china *corresponding author. email: xigengsi@snnu.edu.cn 654 sociobiolog y vol. 59, no. 3, 2012 2009). all members of this superfamily are composed of two main domains, the dna-binding domain (dbd) and the ligand-binding domain (lbd), which show varying degrees of similarity. however, putative receptor molecules for the ligands have not been found, and they are classified as orphan nuclear receptors (blumberg & evans 1998). the orphan receptors are distributed in all protein subfamilies (escriva et al. 2000). the estrogen-related receptors (errα, errβ, and errγ) belong to a subfamily of orphan nuclear receptors, share the similar structure with ers, bind to the steroid receptor coactivator family without any ligands, and drive transcription activity of the target genes (giguere et al.1988; giguere 2002; pettersson et al.1996). the results from various laboratories have suggested mammalian errs collaborate with estrogen signaling, and are involved in many physiological and developmental processes, as well as the proliferation and differentiation of cells (cheung et al. 2005; yang et al. 1996; zhang et al. 2000; vanacker et al.1999a; vanacker et al. 1999b ;kraus et al. 2002). in this study, for the first time, we have cloned the full-length cdna of an estrogen-related receptor homolog gene named amerr from the eusocial insect, apis mellifera. the expression pattern of amerr mrna was studied by using real-time rt-pcr. the results show that this gene is expressed differentially at distinct development stages and in different castes. material and methods experimental insects the colonies of apis mellifera were obtained at the apiary of huarun honeybee breeding base in shaanxi province, china. embryos, larvae, pupae and adults (workers, queens, and drones) were collected from the colonies, immediately immersed in liquid nitrogen, and stored at -80°c for rna extraction (lü et al. 2008; guo et al. 2010). rna preparation and cdna synthesis total rna was extracted from pooled samples of 2 frozen drones (selected randomly) with rnaiso plus® (takara bio inc.), and then immediately reversetranscribed for the generation of cdna using the first-strand cdna synthesis kit® with oligo(dt) primer (fermentas life sciences, burlington ontario). all procedures were performed according to the manufacturer’s instructions. 655 xi, g. et al. — molecular cloning in apis mellifera the cdna templates of different developmental stages and different castes for the real-time quantitative rt-pcr experiments were performed by the same methods described above. molecular cloning and sequencing of amerr the full-length cdna of amerr was cloned based on the scheme shown in fig.1. initially, partial cdna fragment of amerr was amplified by reverse-transcription chain reaction (rt-pcr) using degenerated primers a1and a2 (table 1), which were designed by primerpremier 5.0, oligo 6.0. the primers a1and a2 located at the conserved dna binding domain and ligand binding domain of the published amerr homologues from other insect species (drosophila melanogaster, culex quinquefasciatus ,nasonia vitripennis, tribolium castaneum ). pcr products were cloned into pmd19-t vector (takara, dalian, china), and individual clones were sequenced. based on the first obtained fragment, further specific and degenerated primers (b1 and b2; c1 and c2) (table 1) were designed and used to obtain most of the amerr gene coding sequence. full-length cdna of amerr was obtained by rapid amplication of cdna ends, using the 3'-full race core set® and 5'-full race kit® (takara bio inc), following the manufacturer’s instructions .the gene specific primers used for race are shown in table 1. structural and phylogenetic analysis of amerr fig.1. cloning strateg y and map of the primers used for amplifying the full sequence of the amerr gene: a1&a2 for seq.a of 385 bp, b1&b2 for seq.b of 934 bp, c1&c2 for seq.c of 509 bp, 5ro,5ri, 5i and 5r for seq.5of 783bp; 3r,3i,dt1 and dt2 for seq.3 of 385 bp. 656 sociobiolog y vol. 59, no. 3, 2012 the open reading frame (orf) of the amerr gene was searched using the ncbi orf finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html).the identities of amerr protein and known err protein sequences were analyzed using the megalign (dnastar package). sequence alignments based on the amino acid sequences of the known errs were performed by clustal x 1.81 ( jeanmougin et al.1998), followed by manual inspection. from these alignments, a phylogenetic tree was constructed by mega 4.0 (tamura et al. 2007), according to the neighbor-joining method with a bootstrap test calculated with 2000 replicates and a poisson correction model. real-time quantitative rt-pcr the expression of amerr was quantified by real-time quantitative rtpcr . total rnas were isolated from honeybees of different developmental periods and different castes. we used 4.5 micrograms of total rnas for reverse transcription using the method mentioned above. to prevent genomic dna table 1. oligonucleotide primers used for cdna cloning and real-time quantitative rtpcr. target name primer sequence 5'-3' a a1 caagghaayatcgagtaca a2 atctgyttcgcccavccrat b b1 aggtakccctsgaastcc b2 cgcttgtcctggtcttactgc c c1 catytcdacraasagcttgttcat c2 aacagattccaggattcagca 3’race 3r aaggcttcaaagattgggatt 3i ttacccagtctcaggcaggtc oligo(dt)1 oligo(dt)2 ggccacgcgtcgactagtac(t) 17 ggccacgcgtcgactagtac 5’race 5ro catggctacatgctgacagccta 5ri cgcggatccacagcctactgatgatcagtcgatg 5r cgtcaacgtcgtggtaggg 5i cggtggaccttctgtattt real-time f tccaggattcagcagtttagcttt r tctaccattatttggcttgcttctc β-actin s ccctcttccagccatcgttc a ccaccgatccagacggagta note: k: g or t; m: a or c; w: a or t; y: c or t; d: a, t or g; r: a or g 657 xi, g. et al. — molecular cloning in apis mellifera contamination, the rnas for real-time pcr were treated with rnase-free dnase (roche applied science, mannheim,germany;http://www.rocheapplied-science.com/index.jsp). reactions were performed using an iq5® apparatus (bio-rad laboratories, inc., hercules, california; http://www. bio-rad.com/) with a sybr premix ex taq kit® (takara bio inc). primers (f,r) of amerr and primers (s,a) of β-actin were used in real-time rt-pcr (table 1). the detailed protocol was as follows: 95°c for 1min, 40 cycles of 95°c for 10s and 58°c for 25s, followed by a dissociation-curve program from 55 to 95°c with a heating rate of 0.5°c every step and continuousfluorescence acquisition. all rt-pcr reactions were performed in triplicate. to obtain precise quantification,the specific pcr products and the absence of primer-dimers were confirmed by viewing the single peak in the melting curve of the tested genes. one of the cdna samples was used to construct standard curves for amerr and β-actin after serial dilution and the slopes of the curves were obtained. the relative quantification of amerr was determined using the formula f=2 -∆∆ct. in order to analyze the expression of amerr at different developmental stages, we chose the cdna of first instar as the calibrator, while analyzing amerr expression in the three castes, the drone was selected as the calibrator. amerr relative expression levels were analyzed using one-way analysis of variance (anova) with dunnett’s multiple comparison (spss inc. 2004). details of the procedures for measurement were same as those described previously. results cloning and characterization of amerr cdna the full-length cdna of amerr was amplified by rt-pcr and race. the full-length sequence is 1779 bp, contains a 1305 bp orf which encodes a 434 aminoacid protein (fig. 2). the length of the 5’utr is 197 bp ,and the 3’-utr is 277 bp. a putative polyadenylation signal is found upstream from the 19-nucleotide poly (a) tail, which coincides with the fact that the polyadenylation signal is most often present 11-30 nucleotides upstream from the poly (a) tail (fitzgerald & shenk 1981). the calculated molecular mass of deduced amerr protein is 49.131kda,and the isoelectric point is 7.43. 658 sociobiolog y vol. 59, no. 3, 2012 fig.2. thecdna and deduced amino acid sequence of amerr. the sequence is1779bp long encoding a protein of 424 amino acid residues. the initiating codon atg and the stop codon tga are underlined and in shadow, and a dbd domain is in shadow, a lbd domain is boxed. 659 xi, g. et al. — molecular cloning in apis mellifera the genbank accession number of the amerr cdna is ef506198. alignment analysis and phylogenetic-tree construction in insects a multiple alignment of the deduced animo acid sequence of amerr with other known err homologues was performed with clustal x 1.81 (fig.3) and modified by boxshade (european molecular biolog y network; http://www.ch.embnet.org/software/box_form.html). species and genbank accession numbers mentioned are as follows:apis mellifera (accession no.xp_392385); nasonia vitripennis(accession no.xp_1604033); drosophila melanogaster(accession no.np_729340); aedes aeg ypti (accession no.eat34188);culexquin quefasciatus (accession no.xp_1862743);polyrhachis vicina (accession no. ef474463);homo sapiens l. (accession no.np_004443); teleogryllus emma (accession no. acw84414); bombus terrestris (accession no. xp_003395673); tribolium castaneum (accession no.xp_001812322). the phylogenetic tree was constructed using the neighbor joining method with poisson correction, with fig.3. amino acid sequence alignments of amerr with other homologous sequences from other organisms. the highly conservative amino acid residues are highlighted in black and grey. 660 sociobiolog y vol. 59, no. 3, 2012 homo sapiens l. used as an outgroup. the results of phylogenetic analysis revealed that amerr shares high identity with polyrhachis vicina and bombus terrestris, as expected (fig.4). also, the phylogenetic relationship of amerr is closer to human err than to drosophila err in the evolutionary pathway. the phylogenetic relationship of err in insects shows the protein is very highly conserved in the evolutionary pathway. expression analysis of amerr mrna the relative level of expression of the amerr mrna in different developmental periods and castes was analyzed by means of real-time quantitative rt-pcr. previous studies clearly indicated that the analyzed β-actin mrna levels remain fairly constant in tissues of insects regardless of their developmental or physiological condition (claeys et al. 2003; simonet et al. 2004). the results showed that amerr is expressed in each tested sample. during the development stages of the three castes, the highest expression levels are all found in adults. during the development stages of worker bees (fig.5), the lowest expression level is found in the third instar, then it increased from pupae to adults. during the development stages of queen bees (fig.6), first, the expression level increased from the first instar to the second instar, then fig.4. the phylogenetic tree based on nj method, confidence values by 2000 repeats are shown in the nodes; the abbreviation of the species is shown in the text. 661 xi, g. et al. — molecular cloning in apis mellifera it declined significantly in the third instar, and finally, the expression of the pupa increased again. the expression level of amerr in drones (fig.7) is similar to that in queen bees. among three caste adults (fig.8), the highest expression of amerr was in queen bees, the second-highest was in worker bees, and the lowest was in drones. additionally, we analysed the amerr mrna expression among the developmental periods of the three castes (fig.9), the results showed that the expression level increases gradually durfig.5. relative expression of amerr in different developmental stages of worker bees (bars with different letters indicate means are significantly different, t-test for lsd, p<0.05). fig.6. relative expression of amerr in different developmental stages of queen bees (bars with different letters indicate means are significantly different, t-test for lsd, p<0.05). 662 sociobiolog y vol. 59, no. 3, 2012 ing the development stages. a remarkable increase of the amerr expression from pupa to adult may indicate the gene has important roles in regulating tissue differentiation. the differerent expression in castes and high mrna levels in adults may suggest amerr is related to various functions in social communities. discussion in this study, we have isolated and characterized a full-length cdna sequence of an estrogen receptor-related receptor homolog from the honeybee species apis mellifera. the cdna sequence of amerr and its deduced amino acid sequence reflect a high degree of homolog y with the err homologs identified from other animals, indicating that this newly isolated cdna encodes the apis mellifera estrogen receptor-related receptor protein. the results of multiple alignments showed that the dbd and the lbd of the amerr proteins are highly conserved, suggesting that these regions might include the main functional domains. real-time quantitative rt-pcr analysis indicated that amerr is expressed in each sample we tested, but at different levels. the highest expression level was found in adults. during the developmental stage, the expression level increased from the first instar to second instar, and declined during the third fig.7. relative expression of amerr in different developmental stages of drones (bars with different letters indicate means are significantly different, t-test for lsd, p<0.05). 663 xi, g. et al. — molecular cloning in apis mellifera instar, then in pupae the expression increased again. the high mrna levels in adults suggest the essential roles of err in honeybee development. in three different castes of adults, the amerr expression was highest in queen bees, second-highest in worker bees, and lowest in drones. the amerr mrna expression analysis among the development stages of the three castes fig.8. relative expression of amerr in different castes of apis melifera (bars with different letters indicate means are significantly different, t-test for lsd, p<0.05). fig.9. relative expression of amerr in different developmental stages of apis melifera g1-g5,x1x5,c1-c5 represent five developmental stages respectively of workers, drones and queens (bars with different letters indicate means are significantly different, t-test for lsd, p<0.05). 664 sociobiolog y vol. 59, no. 3, 2012 showed that, no matter which stage, the expression level of queen bees is always the highest. these results indicate that amerr may have an essential role of regulating development in honeybee social communities. however, more research is needed to determine the precise role of err in the different castes of apis melifera. in summary, this study describes an err homolog gene cloned from apis melifera, abbreviated as amerr. the results indicated that the evolutionary pathways of err genes in animals, especially insect err homolog genes, are similar to each other. amerr also is similar to mammalian errs in both sequence and structure, especially in the dbd and lbd, which implies that amerr may be evolutionarily related to the mammalian errs. the findings that amerr mrna is differentially expressed at distinct developmental stages and different castes suggest that the amerr protein may play a role in regulating honeybee development through its production. it will be interesting to extend the analysis on the precise role of err homolog gene in the different castes and anatomical parts of apis melifera. acknowledgements we thank prof. zhezhi wang for help in using rt-pcr. this work was supported by the national natural science foundation of china, nsfc (31171195 ) and the innovation funds of graduate programs, snu (2011cxb007). references cheung, c.p., s. yu, k.b. wong, l.w. chan, f.m. lai, x.h. wang, m. suetsugi, s. chen & f.l. chan 2005. expression and functional study of estrogen receptor-related receptors in human prostatic cells and tissues. the journal of clinical endocrinolog y and metabolism 90:1830-1844. claeys, k.g., g.a. orban, p. dupont, s. sunaert, p.v. hecke & e.d. schutter 2003. involvement of multiple functionally distinct cerebellar regions in visual discrimination: a human functional imaging study. neuroimage 20:840–854. escriva, h., f. delaunay & v. laudet 2000. ligand binding and nuclear receptor evolution. bioessays 22:717-727. fitzgerald, m. & t. shenk 1981.the sequence 5'-aauaaa-3'forms parts of the recognition site for polyadenylation of late sv40 mrnas. cell 24(1): 251-260 giguere, v. 2002. to err in the estrogen pathway. trends in endocrinolog y and metabolism 13: 220-225. 665 xi, g. et al. — molecular cloning in apis mellifera giguere, v., n. yang, p. segui & r.m. evans 1988. identification of a new class of steroid hormone receptors. nature 331: 91-94. guo, x.j. & g.s. xi 2010. molecular cloning and expression analysis of the gene encoding mef2 in the ant polyrhachis vicina (hymenoptera :formicidae). sociobiolog y 56(1):235-248 jeanmougin, f., j.d. thompson, m. gouy, d.g. higgins & t.j. gibson 1998. multiple sequence alignment with clustal x. trends in biochemical science 23: 403-405. kraus, r.j., e.a. ariazi, m.l. farrell & j.e. mertz 2002.estrogen-related receptor alpha 1 actively antagonizes estrogen receptor-regulated transcription in mcf-7 mammary cells. the journal of biological chemistry 277:24826-24834. lü, s. m., g.s. xi & x.h. wang 2008.molecular cloning, characterization, and expression analysis of a qm homologue in the ant polyrhachis vicina (hymenoptera:formicidae). the canadian entomologist 140:312-323. ouyang, x.h., g.s. xi & c.p. bu 2009. molecular cloning and expression of an estrogen receptor-related receptor gene in the ant polyrhachis vicina (hymenoptera: formicidae). annals of the entomological society of america 102(2):295-302. pettersson, k., k. svensson, r. mattsson, b. carlsson, r. ohlsson & a. berkenstam 1996. expression of a novel member of estrogen response element-binding nuclear receptors is restricted to the early stages of chorion formation during mouse embryogenesis. mechanisms of development 54:211-223. simonet, g., j. poels, i. claeys, t.v. loy, v. franssens, a.d. loof & j.v. broeck 2004. neuroendocrinological and molecular aspects of insect reproduction. journal of neuroendocrinolog y 16:649 -659. spss inc. 2004. spss version 13.0.spss inc., chicago. tamura, k., j. dudley, m. nei & s. kumar 2007. mega4: molecular evolutionary genetics analysis (mega) software version 4.0. molecular biolog y and evolution 24:15961599. vanacker, j.m., k. petterson, j.a. gustafsson & v. laudet 1999a. transcriptional activities of the orphan nuclear receptor err alpha (estrogen receptor-related receptor-alpha). molecular endocrinolog y 13: 764-773. vanacker, j.m., k. petterson, j.a. gustafsson & v. laudet 1999b. transcriptional targets shared by estrogen receptor-related receptors(errs) and estrogen receptor (er)α, but not by (er) β. the embo journal 18: 4270-4279. yang, n., h. shigeta, h. shi & c.t. teng 1996. estrogen-related receptor, herr1,modulates estrogen receptor-mediated response of human anlactoferrin gene promoter. the journal of biological chemistry 271:5795-5804. zhang, z. & c.t. teng 2000. estrogen receptor-related receptor alpha 1 interacts with coactivator and constitutively activates the estrogen response elements of the human lactoferrin gene. the journal of biological chemistry 275:20837-20846. 1053 diversity of social wasps in the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil by gabriel de castro jacques1, ancidériton antonio de castro1, gabriely köerich souza1, reinildes silva-filho1, marcos m. de souza1 & josé cola zanuncio1* abstract social wasps can be used as ecological indicators. however, the diversity of species of this group in minas gerais state, brazil, is poorly studied, especially in anthropized environments. the objective was to study diversity of these insects in the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil, from may to june 2011 with two methodologies (attractive traps and active search). eight hundred and eighty-nine individuals of social wasps of 10 genera and 25 species were collected. the high species richness found can be explained by the diverse environment of the area studied with greater availability of microhabitats, greater protection against predators, and high availability and diversity of food and substrates for nesting which may favor the coexistence of a greater number of species. in addition, combined use of two collecting methodologies provided a greater diversity of wasps captured. key words: vespidae, biodiversity, species richness. introduction hymenoptera species have different habits and levels of social complexity, including those of the family vespidae that are important as pollinators (hunt et al. 1991; brodmann et al. 2008; mello et al. 2011), predators (prezoto & machado 1999; prezoto et al. 2006; silveira et al. 2008; gomes & noll 2009) and bioindicators (souza et al. 2010). this family includes solitary wasps (euparigiinae, masarinae and eumeninae) and species with some degree of socialization (stenogastrinae, polistinae and vespinae) (elpino-campos et al. 2007). 1 departamento de entomologia, universidade federal de viçosa, 36570-000 viçosa, minas gerais state, brazil. e-mail: gabrieljacques@yahoo.com.br, anciagro@gmail.com, gaby.florestal@gmail.com, rennefilho@gmail.com and magalhaescajubi@bol.com.br * corresponding author: zanuncio@ufv.br 1054 sociobiolog y vol. 59, no. 3, 2012 eusocial wasps (polistinae) are cosmopolitan and diverse in the neotropics (auad et al. 2010). brazilian polistinae are among the most widespread wasp group in south america, with 22 genera and 316 species of three tribes: polistini, mischocyttarini and epiponini (prezoto et al. 2007). many species of this subfamily are predators of insects and other arthropods (richter 2000), but also an important food resource for insectivorous birds and ants (kumano & kasuya 2006). social wasps are viable for biological control programs, but the survey and identification of these insects are the first step for integrated pest management programs (prezoto et al. 2006). degradation of natural environments results in losses of biological diversity, with wasp species being endangered before the study of basic aspects of their biolog y (nascimento et al. 2004). this biodiversity must be known and evaluated in terms of number of species, distribution and interaction aspects to facilitate natural ecosystem preservation (del-claro 2004). social wasps, due to their distribution, abundance and richness of interactions, are considered a special group (prezoto et al. 2009), easily sampled because they forage and return to a central place (nest). moreover, they are active in all seasons in most tropical ecosystems and can be sampled in a relatively short period (kumar et al. 2009). sensitivity of these wasps to changes in abiotic conditions (light, temperature and humidity) shows that they can be used as indicators of environmental quality (souza et al. 2010). the surveying and identification of genera and species of animals and plants are important to know the natural resources available in a specific area and to understand ecological characteristics of an ecosystem (elpino-campos et al. 2007). global destruction of rainforests highlights the importance of these studies (buschini & woiski 2008). the diversity of social wasp species in brazil was studied in “campos rupestres” (silva-pereira & santos 2006), brazilian savanna (elpino-campos et al. 2007; souza & prezoto 2006), amazon rainforest (silveira 2002; silveira et al. 2008), semidecidual forest (gomes & noll 2009), riparian vegetation (souza et al. 2010; pereira & antonialli junior 2011) and mangrove, atlantic forest and restinga vegetation (santos et al. 2007). however, the wasp fauna of minas gerais state, mainly in anthropized environments, is poorly studied. the objective was to obtain preliminary data on wasp diversity 1055 jacques, g. c. et al. — diversity of social wasps in viçosa within the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil. materials and methods social wasps were collected in the campus of the “universidade federal de viçosa” (ufv) in viçosa, minas gerais state, brazil from may to june 2011 using two methodologies. this area has very diverse anthropized environments including buildings, agriculture areas, artificial lakes and atlantic forest fragments. attractive traps traps were prepared with two-liter plastic bottles with three triangular lateral openings (2 x 2 x 2 cm) located 10 cm from their base (souza & prezoto 2006). attractive substances were: 1natural passion fruit juice (passiflora edulis f. flavicarpa deg.; passifloraceae) with 1 kg of fruit mixed with 250 g of granulated sugar and two liters of water; 2250 g of sardine (sardinella brasiliensis steindachner 1789) with two liters of water, and 3pure honey. each bottle received 150 ml of attractive substance. twenty bottles were used per attractive substance. these traps were set up at 1.5m high in different places in the campus “universidade federal de viçosa”. wasps were collected from the traps every seven days and preserved in 70% alcohol for identification. active search active searches were performed in the campus of the “universidade federal de viçosa”. trunks and natural cavities (empty termite nests and rocks), broadleaf vegetation, flowers and buildings were inspected (souza & prezoto 2006; elpino-campos et al. 2007). the species collected were identified with keys (richards 1978; carpenter 2004); diversity and species dominance calculated using the shannon-wiener diversity index (h’) and the berger-parker dominance (d pb ), through the dives program (diversity of species v2.0) at the base 10 logarithm (rodrigues 2005). identifications were confirmed by dr. orlando tobias silveira from emílio goeldi museum of belém, pará state, brazil. 1056 sociobiolog y vol. 59, no. 3, 2012 results and discussion eight hundred and eighty-nine social wasps of 10 genera and 25 species were collected (table 1). the number of vespidae species was the sixth highest in collections of this group in brazil (table 2). heterogeneous substrates may favor the coexistence of a greater number of species due to greater availability of microhabitats, protection against predators, and a high availability and diversity of food resources and substrates for nesting (santos et al. 2007). in addition, combined use of collecting methods provides a greater diversity of wasps captured (silveira 2002; souza & prezoto 2006; elpino-campos table 1. frequency of social wasp species collected in the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil through active search (as) and attractive traps (at) species frequency as at total agelaia multipicta (haliday) 48 53 101 agelaia vicina saussure 0 295 295 apoica pallens (f.) 0 1 1 brachygastra lecheguana (latreille) 7 0 7 mischocyttarus atramentarius zikán 6 0 6 mischocyttarus sp.1 1 0 1 mischocyttarus cassununga (von. ihering ) 24 219 243 mischocyttarus drewseni saussure 1 1 2 mischocyttarus parallellogrammus zikán 2 2 4 mischocyttarus sp.2 2 0 2 parachartergus fraternus (gribodo) 1 0 1 polistes actaeon haliday 6 0 6 polistes simillimus zikán 16 0 16 polistes versicolor olivier 32 5 37 polybia bifasciata saussure 0 2 2 polybia fastidiosuscula saussure 38 8 46 polybia ignobilis (haliday) 0 6 6 polybia jurinei saussure 1 4 5 polybia platycephala (richards) 72 9 81 polybia paulista (von. ihering ) 0 1 1 polybia sericea (olivier) 5 13 18 polybia sp. 2 1 3 protonectarina sylveirae (saussure) 1 0 1 protopolybia exígua (saussure) 12 0 12 synoeca cyanea (fabricius) 1 0 1 279 620 899 1057 jacques, g. c. et al. — diversity of social wasps in viçosa et al. 2007; silva & silveira 2009; auad et al. 2010; souza et al. 2010). only sixteen of the species collected were present in a riparian area in the city of barroso, minas gerais state (souza et al. 2010), despite the distance of 160 km between the cities of viçosa and barroso. records of new species of the table 2. comparison between total number of species in this research (*) and other studies in the literature source number of species silveira 2002 (amazon rainforest) 79 silva & silveira 2009 (amazon rainforest) 65 souza & prezoto 2006 (cerrado and semidecidual forest) 38 souza et al. 2010 36 elpino-campos et al. 2007 (cerrado) 29 * this research 26 hermes & köhler 2004 (atlantic forest) 25 santos et al. 2009 (cerrado) 19 pereira & antonialli junior 2011 (riparian vegetation) 18 santos et al. 2007 (atlantic forest) 18 santos et al. 2007 (restinga vegetation) 16 auad et al. 2010 (silvipastoral system) 13 silva-pereira & santos 2006 (campos rupestres) 11 arab et al. 2010 (atlantic forest) 10 santos et al. 2007 (mangrove) 8 gomes & noll 2009 (semidecidual forest) 7 silveira et al. 2008 (amazon rainforest) 6 table 3. richness, diversity and dominance calculated with active search (as) and attractive traps (at) of social wasps in the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil as at total species richness (s’) 21 15 26 shannon-wiener diversity index (h’) 0.9814 0.5753 0.8584 berger-parker dominance index (d pb ) 0.2581 0.4758 0.3281 1058 sociobiolog y vol. 59, no. 3, 2012 genus mischocyttarus are due to a sum of factors such as small nests with few individuals. moreover, this genus is the largest group of social wasps, with nine subgenera and 245 species. for this reason, surveys of richness and diversity can increase the chance of obtaining new records for this genus (cooper 1998; silveira et al. 2008). the diversity index of social wasps in the ufv campus was h’= 0.8584 and richness s’= 25, which was lower than those obtained in atlantic forest area (h’= 2.61) and with lower number of species (s’= 18) (santos et al. 2007). this difference is explained by high dominance (d pb = 0.3281) of a few species in our study. in addition, the period of collections was cold and dry, which may interfere in the number of wasps collected and thus leading to dominance of a few species. the rainy season increases plant biomass and food resources such as nectar and prey (auad et al. 2010). a greater number of species and colonies of wasps were reported in periods with higher temperature and rainfall (souza & prezoto 2006). collection of social wasps in various locations at different times produced different insects even in areas with similar conditions. this is due to the fact that a number of species are represented by one or few individuals. thus, the number of individuals per species and a list of species in the sample are important, and may differ in the space and time (buschini & woiski 2008). agelaia vicina saussure and mischocyttarus cassununga (von. ihering ) were the most collected species with over 60% of the total individuals sampled. agelaia is the most common genus in brazil (arab et al. 2010). nests of this genus were not observed but, some of its species can establish colonies with an estimated population up to one million adults (zucchi et al. 1995), which increases the chances of capturing specimens of this group (hunt et al. 2001). the high abundance of this genus was also reported in different ecosystems in brazil (gomes & noll 2009; arab et al. 2010; pereira & antonialli junior 2011). mischocyttarus cassununga, a species with a high degree of synanthropism and easily found near buildings, was the second most abundant and the most commonly found around the campus buildings. the high degree of anthropization enables this species to establish many colonies (alvarenga et al. 2010). moreover, the presence of more than one inseminated female per colony, with well developed ovaries and the ability to oviposit viable 1059 jacques, g. c. et al. — diversity of social wasps in viçosa eggs enables a wider dispersal of m. cassununga (murakami & shima 2006; murakami et al. 2009). nests were found for 13 of the 25 species collected: mischocyttarus atramentarius zikán, mischocyttarus cassununga (von. ihering ), mischocyttarus sp.1, mischocyttarus sp.2, parachartergus fraternus (gribodo), polistes actaeon haliday, polistes simillimus zikán, polistes versicolor olivier, polybia fastidiosuscula saussure, polybia platycephala (richards), protonectarina sylveirae (saussure), protopolybia exigua and synoeca cyanea (fabricius). this is due to the fact that nests of mischocyttarus, protopolybia, polybia and polistes are commonly found on buildings which facilitate their location (alvarenga et al. 2010). the greater number of wasps collected with traps may be due to the cold and dry period of the survey where water availability became a survival factor, due to decreasing food resources (nectar and insects) (elpino-campos et al. 2007). traps represented a resource to be explored during this period of the year when the water is used by the wasps for colony thermoregulation, while the nectar and other sugary substances are the main food item for adult wasps (prezoto & gobbi 2003). the total number of species collected and the shannon-wiener diversity (’) showed that the active searching methodolog y was more effective than the traps (table 3). ten species were collected only with this method. this was also observed in other studies (silveira 2002; souza & prezoto 2006; elpinocampos et al. 2007; pereira & antonialli junior 2011). however, only five species were collected with traps which demonstrates the importance of active search. apoica pallens (f.) is generally captured with attractive traps because it forages mostly at night (hunt et al. 1995; pickett & wenzel 2007) which makes it less likely to be captured during the day by active search. these results show the importance of using combined sampling methods. conclusions social wasp fauna showed high diversity in the campus of the “universidade federal de viçosa” in viçosa, minas gerais state, brazil, and with high diversification and species richness. the number of species was higher than that observed by other authors, even with the shorter collection period of social wasps and unfavorable weather conditions. moreover, the active search 1060 sociobiolog y vol. 59, no. 3, 2012 and attractive traps showed the importance of using more than one method to register the largest possible number of species of these wasps. acknowledgments to the ‘‘conselho nacional de desenvolvimento científico e tecnológico (cnpq)’’, ‘‘coordenação de aperfeiçoamento de pessoal de nível superior (capes)’’ and ‘‘fundação de amparo à pesquisa do estado de minas gerais (fapemig)’’. references alvarenga, r.b., m.m. castro, e.h. santos-prezoto & f. prezoto 2010. nesting of social wasps (hymenoptera, vespidae) in urban gardens in southeastern brazil. sociobiolog y 55 : 445-452. arab, a., i. cabrini & c.f.s. andrade 2010. diversity of 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(2006) demonstrated that secondary compounds in nectar are effective visitor filters, which lead to a specialization in the pollination system. toxicity abstract spathodea campanulata is an african plant introduced into south america and other tropical and subtropical areas for ornamental purposes. this plant has been linked to insect mortality, bees included. however, its effects on the neotropical melipona are as yet unknown. thus, the aim of this study was to evaluate the effect of s. campanulata nectar and pollen on the survival of melipona fasciculata smith and melipona seminigra friese workers. a total of 120 newly emerged workers of each species were divided into groups of 10 individuals and confined in boxes. they were submitted to the following diet treatments: s. campanulata nectar or 11% sucrose solution (nectar control); 11% sucrose solution and s. campanulata pollen or 11% sucrose solution and the species’ original pollen (pollen control). a higher mortality of workers was detected in the groups fed with toxics nectar and pollen (m. fasciculata, p<0.01; m. seminigra, p<0.01) than on the respective controls. our results demonstrate that nectar and pollen from s. campanulata affected the survival of m. fasciculata and m. seminigra worker bees. we thus recommend that s. campanulata should not be provided as food source for stingless bees. sociobiology an international journal on social insects 1 embrapa amazônia oriental, belém, pa, brazil 2 universidade federal do pará (ufpa), belém, pa, brazil article history edited by denise araujo alves, esalq, usp, brazil received 04 september 2014 initial acceptance 14 october 2014 final acceptance 14 november 2014 keywords meliponine bees, plant toxicity, survival, meliponiculture. corresponding author ana carolina martins de queiroz embrapa amazônia oriental laboratório de botânica trav. dr. enéas pinheiro s/n, caixa postal 48, cep 66095-100, belém/pa, brazil e-mail: carolina.queiroz@embrapa.br for several animals is normally due to the secondary compounds found in all plant parts, especially on those most important for survival and reproduction (levin, 1976). these secondary compounds associated with resistance to herbivory have been frequently observed in floral nectar. adler (2000) detected nectar that was toxic or had secondary compounds in at least 21 different plant families. according to ott (1988), at least three psychoactive phytotoxin categories occurred in toxic honeys, and, consequently, in the nectar from which it was produced. toxins found amongst some plants are nicotine, rotenones, pyrethrins and tannins (bueno et al., 1990). dimorphandra mollis benth. (fabaceae; popularly called in brazil “fake barbatimão") and stryphnodendron adstringens (martius) coville (fabaceae; the “real barbatimão”), are rich in tannins and may cause serious losses to beekeepers due to larvae mortality and a reduction in adult apis mellifera linnaeus longevity. the toxicity of barbatimão is attributed to the pollen and nectar, and the pollen is research article bees sociobiology 61(4): 536-540 (december 2014) 537 considered more harmful (carvalho & message, 2004; santoro et al., 2004; cintra et al., 2005). spathodea campanulata beauv. (an exotic species of african origin introduced for ornamental purposes [nogueiraneto, 1997]) has been reported as toxic to stingless bees (tribe meliponini, sensu michener, 2007). portugal-araújo (1963) was one of the pioneers in reporting these effects. he recorded dead stingless bees on s. campanulata flowers in gabon, along with nogueira-neto (1997) and oliveira et al. (1991) in brazil. trigo and santos (2000) monitored dead insects in s. campanulata flowers for to up to five days after anthesis and stated that meliponine bees represented 97% of the dead insects. calligaris (2001) confirmed its nectar toxicity on scaptotrigona postica (latreille) and a. mellifera worker bees in laboratory bioassays, although pollen toxicity was not verified. thus, in this study we evaluated the effects of s. campanulata nectar and pollen consumption on the survival of two species of melipona illiger worker bees from the brazilian amazon used in meliponiculture, melipona fasciculata smith and melipona seminigra friese. material and methods studied species and study site we used m. seminigra and m. fasciculata workers to study the effect of s. campanulata nectar and pollen on the worker survival. m. seminigra occurs in the brazilian amazon states of acre, amazonas, maranhão, mato grosso, pará, rondônia, roraima, tocantins, and m. fasciculata in the brazilian states of maranhão, mato grosso, pará, piauí and tocantins (camargo & pedro, 2012). all experiments were carried out from january to may 2012 in the meliponary of embrapa amazônia oriental (1º26’11.52’’s, 48º26’35.50’’w). the area is composed of secondary forests patches of native plants (several hundred species, including trees and shrubs) and several agricultural crops such as assai trees (euterpe oleraceae, arecaceae) and other species. spathodea campanulata is a large tree (up to 20m) with numerous large flowers, externally red and internally yellow (francis, 1990). in its region of origin (africa), s. campanulata is pollinated by birds and bats, but is also visited by bees attracted by the abundant nectar and colorful flowers (ayensu, 1974; rangaiah et al., 2004; corlett, 2005). in the study site, there was only one tree of s. campanulata, situated 20 meters from the nests. experimental design effect of nectar and pollen on the survival of workers to analyze the effect of s. campanulata nectar and pollen on the survival of m. fasciculata and m. seminigra workers, we used newly emerged workers, obtained from eight different nests, four for each bee species. a total of 120 bees of each species were used (30 from each colony), from which 60 were destined for two control groups (30 for each species) and 60 for each experimental group: s. campanulata nectar and pollen. the workers were divided into groups of 10 individuals and confined in polyethylene boxes (8 x 8 x 4 cm) without the queen, and. the boxes were kept in a bod incubator (model dl-sedt 02) at 28 ± 1°c. every day the number of live bees was checked, any dead individuals were removed, the plastic box’s rubbish dump area was cleared and water was added to maintain humidity (method adapted from costa & venturieri, 2009). inflorescences with flower buds and newly opened flowers were gathered from trees located at the research campus – embrapa amazônia oriental – to prepare the s. campanulata nectar that was offered to m. fasciculata and m. seminigra workers. the nectar was removed with an automatic micropipette and the percentage of total sugars was measured with a field refractometer adapted to small volumes (bellinghanstanley™). for pollen sampling, anthers of the flower buds were removed and kept in 2 ml microtubes. a total of 1ml of water with 11% sucrose was added to the tubes with the anthers (the same concentration of sugar found in s. campanulata nectar) to wash and assist pollen extraction. the material was centrifuged for five minutes at 2000 rpm, the liquid part of the microtube was drained and the accumulated pollen on the bottom was collected and offered to the bees as protein source. a botanical sample of the plant’s reproductive structures was identified and stored at the ian herbarium (embrapa) under the number 187659. workers were submitted to the following diet, according to the treatment: nsc bees fed on s. campanulata nectar; nec bees fed on 11% sucrose solution; psc bees fed on an 11% sucrose solution and s. campanulata pollen; poc bees fed on 11% sucrose solution (nectar control) and its own pollen (m. fasciculata or m. seminigra pollen; pollen control). workers were daily fed with: (1) nsc: 240 µl of nectar and (2) nec: 240 µl of 11% sucrose solution. the workers from the psc treatments were offered 240 µl of 11% sucrose solution and 0.1 g of s. campanulata pollen daily. for the poc treatment, 240 µl of 11% sucrose solution and 0.1g of the species’ pollen was offered. the food was weighed daily in an analytical balance with a 10-³ g precision to check the consumption of each item. the carbohydrate (11% sucrose solution or s. campanulata nectar) and protein (pollen of s. campanulata and m. fasciculata m. seminigra colonies) foods were renewed whenever completely eaten (pollen), or daily (sucrose). analyses kaplan-meier survival curves were made for each species’ different treatments. the survival of control workers (pollen and nectar) was monitored until half or more of the treatment individuals died. a cox-mantel test was carried out, using the software statistica® 8.0, to compare the survival curves of the treatments used for each species (5% significance level). the data regarding workers that acm queiroz, fal contrera, gc venturieri effects of toxic pollen and nectar on bee survival538 were alive at the end of the experiment were treated as censured and from the workers monitored until death as complete data for the survival curve setting (crawley, 2007). results effect of s. campanulata nectar and pollen on the survival of workers there was food consumption in all the studied groups (table 1). the death rate was high for the 30 m. fasciculata bees that received s. campanulata nectar as a carbohydrate source (nsc). only nine bees from the group nsc remained alive at the end of the experiment. furthermore, from the 30 bees from group nec, only three died on the second day, so that 27 bees remained alive at the end of the experiment (fig 1a). a similar pattern of mortality due to s. campanulata nectar was detected for m. seminigra (fig 1b). mortality of m. fasciculata and m. seminigra workers was significantly higher in the experimental group (s. campanulata nectar) than in the control group (cox-mantel: m. fasciculata, p<0.01; m. seminigra, p<0.01). only nine of the 30 m. fasciculata workers submitted on the treatment with s. campanulata pollen (psc) remained alive at the end of the experiment. there was high mortality on the four experiment days. mortality was lower on the pollen control group (poc), with 24 live workers at the end of the experiment (fig 2a). again, the pattern was similar for m. seminigra (fig 2b). for both species the survival of the control group was significantly higher than for the experimental group (s. campanulata pollen) (cox-mantel: m. fasciculata, p<0.01; m. seminigra, p<0.01). there was no difference on m. fasciculata survival between s. campanulata nectar (nsc) and pollen (psc) treatments (cox-mantel, p=0.55). however, the intake of s. campanulata nectar by m. seminigra had a stronger impact on the survival of workers than pollen (cox-mantel, p<0.01). there were no significant differences between the longevity of bees that consumed 11% sucrose solution and pollen from their own boxes for the control groups (nsc and psc) (coxmantel: m. fasciculata: p=0.29; m. seminigra: p=0.45). discussion in this study we were able to prove the effect of s. campanulata pollen and nectar in reducing the survival of two meliponine species (m. fasciculata and m. seminigra) when fed on it. both pollen and nectar of s. campanulata reduced the survival of worker bees undergoing these treatments. since the food was consumed in the experimental cages, this indicates that the tested bee groups actually died due to s. campanulata nectar and pollen ingestion, and due to starvation. in general, s. campanulata nectar and flower bud secretion are referred to as toxic, although little has been studied about its pollen. calligaris (2001) found s. postica and a. mellifera survival reduction when s. campanulata nectar was added to the bees’ diet in bioassays. portugal-araújo (1963) attributed the death of insects in s. campanulata flowers, including stingless bees, to floral bud secretion toxicity. in a periodic survey, nogueira-neto (1997) also reported many meliponini bees in fallen flowers, highlighting plebeia droryana (friese), tetragonisca angustula (latreille), s. postica, trigona spinipes (fabricius) and friesella schrottkyi fig 1. survival curves for workers confined in groups of 10 individuals and submitted to dietary treatments. nscbees fed on s. campanulata nectar (filled circles); necbees fed on 11% sucrose solution (empty circles). a melipona fasciculata. b melipona seminigra. species/ treatment nsc (µl) nec (µl) psc (mg) poc mg) m. fasciculata 19.62 ± 1.91 29.73 ± 8.45 9.11 ± 11.40 5.20 ± 3.70 m. seminigra 27.01 ± 3.41 30.24 ± 6.81 5.00 ± 3.70 1.40 ± 0.70 table 1. daily consumption rate per worker (mean±s.d.) of melipona fasciculata and melipona seminigra, confined in groups of 10 individuals and submitted to dietary treatments. nscbees fed on spathodea campanulata nectar; nec bees fed on 11% sucrose solution; psc bees fed on an 11% sucrose solution and s. campanulata pollen; poc bees fed on 11% sucrose solution (nectar control) and its own pollen (pollen control). sociobiology 61(4): 536-540 (december 2014) 539 (friese). stingless bees represented up to 97% of the dead insects in the flowers, especially s. postica (trigo & santos, 2000). trigo and santos (2000) tested different flower mucilage concentrations on newly emerged s. postica workers, in laboratory bioassays. mucilage at a concentration of 25% reduced bee longevity by 52.9%, while pure mucilage reduced it by 95.2%. however, this last result is considered ambiguous, since there is no evidence that bee feed on pure mucilage. both pollen and nectar of s. campanulata are considered toxic. calligaris (2001) did not detect a reduction in the survival of s. postica and a. mellifera when fed on 5% pollen. however, oliveira et al. (1991) reported the death of t. spinipes bees due to the s. campanulata pollen found in its gizzard. in this study we found a marked reduction of m. fasciculata and m. seminigra survival rates when fed on pure pollen. trigo and santos (2000) suggested the existence of a defense mechanism in s. campanulata that protects flower buds from nectar and pollen thieves. otherwise these resources could be stolen by some meliponine bees, such as s. postica, or other efficient pillagers, before flower opening. in this case, vertebrate pollination would be reduced or even prevented. indeed, endress (1994) noted that some plants, including bignoniaceae, produce a mucilaginous or watery liquid to protect juvenile flower organs before anthesis. flower bud secretion would thus be a plant defense system, of chemical or physical nature, suffocating the bees (trigo & santos, 2000). considering these effects and the actual expansion of meliponiculture in brazil (contrera et al., 2011; venturieri et al., 2012), the use of s. campanulata trees is not recommended in areas foraged by stingless bees. such a recommendation has already been made regarding a. mellifera (modro et al., 2011). acknowledgements we thank the two anonymous referees for the comments that improved our manuscript. we also thank elisângela rêgo, lourival lucas and miguel nascimento for technical assistance. funding was obtained from the fundação amazônia paraense de amparo a pesquisa (fapespa) and the conselho nacional de desenvolvimento cientifico e tecnológico (cnpq). we also thank the dean of research and post-graduate studies of ufpa (próreitoria de pesquisa e pós graduação propesp/ufpa) and foundation for research support and development (fundação de amparo e desenvolvimento de pesquisa fadesp), for the support for through the program of qualified publication support (programa de apoio à publicação qualificada papq). references adler, l.s. (2000). the ecological significance of toxic nectar. oikos 91:409-420. doi:10.1034/j.1600-0706.2000.910301.x ayensu, e. s. (1974). plant and bat interactions in west africa. ann. miss. bot. gard. 61: 702-727. barker, r.j. (1990). poisoning by plants. in r. a. morse & r. nowogrodzki (eds.), honey bee pests, predators and diseases (pp. 309-315). new york: cornell university press. bueno, o.c., hebling-beraldo, m.j., aulino-silva, o., pagnocca, f., fernandez, j.b. & vieira, p.c. (1990). toxic effect of plants on leaf-cutting ants and their symbiotic fungus. in r.k. jaffé & k.a. cedeno (eds.), applied myrmecology: a world perspective (pp. 420-426). san francisco: westview press. calligaris, i.b. (2001). toxicidade do néctar e do pólen de spathodea campanulata (bignoneaceae) sobre operárias de apis mellifera (hymenoptera: apidae) e scaptotrigona postica (hymenoptera: apidae). dissertation, universidade estadual paulista júlio de mesquita filho. camargo, j.m.f. & pedro, s.r.m. (2012). meliponini lepeletier, 1836. in j.s. moure, d. urban & g.a.r. melo (orgs) catalogue of bees (hymenoptera, apoidea) in the neotropical region online version. http://www.moure.cria.org.br/catalogue. (accessed date 27 august, 2013). fig 2. survival curves for workers confined in groups of 10 individuals and submitted to dietary treatments. psc bees fed on 11% sucrose solution and s. campanulata pollen (filled circles); pocbees fed on 11% sucrose solution and its own pollen (empty circles). a melipona fasciculata. b melipona seminigra. acm queiroz, fal contrera, gc venturieri effects of toxic pollen and nectar on bee survival540 carvalho, a.c.p. & message, d. (2004). a scientific note on the toxic pollen of stryphnodendron polyphyllum (fabaceae, mimosoideae) which causes sacbrood-like symptoms. apidologie 35:89-90. doi: 10.1051/apido:2003059 cintra p., malaspina, o. & bueno, o.c. (2005). plantas tóxicas para abelhas. arq. inst. biol. 72:547-551. contrera, f.a.l., menezes, c. & venturieri, g.c. (2011). new horizons on stingless beekeeping (apidae, meliponini). rev. bras. zootec. 40(suppl. esp.): 48-51. corlett, r. t. (2005). interactions between birds, fruit bats and exotic plants in urban hong kong, south china. urban ecosyst. 8: 275-283. costa, l. & venturieri, g.c. (2009). diet impacts on melipona flavolineata workers (apidae, meliponini). j. apicult. res. 48(1): 38-45. doi: 10.3896/ibra.1.48.1.09. crawley, m.j. (2007). the r book. wiley & sons ltd, west sussex, 1051p. dafni, a. & kevan, p.g. (2003). field methods in pollination ecology. cambridge: ecoquest. endress, p.k. (1994). diversity and evolutionary biology of tropical flowers. cambridge: cambridge university press, 511p. erdtman, g. (1960). the acetolysis method. a revised description. sven. bot. tidskr. 54:561-564. francis, j.k. (1990). african tulip tree (spathodea campanulata beauv.) res. note so-itf-sm-32. http://www. fs.fed.us/global/iitf/spathodeacampanulata.pdf. (acessed date 1 february, 2013). johnson, s.d., hargreaves, a.l. & brown, m. (2006). dark, bitter-tasting nectar functions as a filter of flower visitors in a bird-pollinated plant. ecology 87: 2709–2716. levin, d.a. (1976). the chemical defenses of plants to pathogens and herbivores. annu. rev. ecol. syst. 7:121-159. michener, c.d. (2007). the bees of the world. 2nd ed. baltimore: the johns hopkins university press. modro, a.f.h., message, d., luz, c.f.p. & meira-neto, j.a.a. (2011). flora de importância polinífera para apis mellifera (l.) na região de viçosa, mg. rev. árvore 35: 1145-1153. doi:10.1590/s0100-67622011000600020. nogueira-neto, p. (1997). vida e criação de abelhas indígenas sem ferrão. são paulo: ed. nogueirapis, 446p. oliveira, r.m., giannotti, e., machado, v.l.l. (1991). visitantes florais de spathodea campanulata beauv. (bignoniaceae). bioikos 5:7-30. ott, j. (1988). the delphic bee: bees and toxic honeys as pointers to psychoactive and other medicinal plants. econ. bot., 52 (3): 260-266. portugal-araújo, v. (1963). o perigo de dispersão da tulipeira do gabão (spathodea campanulata beauv.). chácaras e quintais 107: 562. rangaiah, k., rao, p.s. & raju, a.j.s. (2004). birdpollination and fruiting phenology in spathodea campanulata beauv. (bignoniaceae). beitr. biol. pflanz. 73(3):395-408. roubik, d.w. (1989) ecology and natural history of tropical bees. new york: cambridge university press. santoro, k.r., vieira, m.e.q., queiroz, m.l., queiroz, m.c. & barbosa, s.b.p. (2004). efeito do tanino de stryphnodendron spp. sobre a longevidade de abelhas apis mellifera l. arch. zootec. 53:281-291. trigo, j.r. & santos, w.f. (2000). insect mortality in spathodea campanulata beauv. (bignoniaceae) flowers. rev. bras. biol. 60:537-8. venturieri, g.c., alves, d.a., villas-boas, j.k., carvalho, c.a.l., menezes, c., vollet-neto, a., contrera, f.a.l., cortopassi-laurino, m., nogueira-neto, p. & imperatrizfonseca, v.l. (2012). meliponicultura no brasil: situação atual e perspectivas futuras para uso na polinização agrícola. in: v.l. imperatriz-fonseca, d.a.l. canhos, d.a. alves & a.m. saraiva (orgs.), contribuição e perspectivas para a biodiversidade, uso sustentável, conservação e serviços ambientais (pp.213-236). são paulo: edusp. 183 urban termites of recife, northeast brazil (isoptera) by auristela c. albuquerque1,3, gicela r. r. s. matias1, alane a. v. o. couto1, marco a. p. oliveira1 & alexandre vasconcellos2 abstract this study surveyed the termite fauna in urban properties of the city of recife, state of pernambuco, northeastern brazil. six-hundred properties were inspected in search for termites or signs of their activity, such as abandoned nests, galleries on walls, and fecal pellets. seven termite species, belonging to seven genera and three families (kalotermitidae, rhinotermitidae, and termtidae) were found causing some type of damage. nasutitermes corniger and cryptotermes dudleyi were the most frequent species within buildings (82.4% and 9.5 %, respectively). such species, along with coptotermes gestroi, were responsible for the greatest damage recorded. with the exception of cryptotermes dudleyi and coptotermes gestroi, all of the species are native and relatively common in northeastern brazil’s atlantic forest. keywords: isoptera, urban entomolog y, pest status introduction termites are mainly distributed throughout tropical and subtropical regions. in south america there are approximately 400 termite species, of which 77 have already been recorded in environments altered by human activity; however, only about 5% of the south american species have real pest potential in agricultural or urban areas (constantino 2002). the urbanization process has caused a gradual substitution of natural habitats for urban spaces. this causes the local eradication of several termite species and favors the selection or installation of synanthropic species that may become pests (milano 1998, milano & fontes 2002, vasconcellos & bandeira 2006). 1departament of biolog y, federal rural university of pernambuco, recife, brazil. 2departament of botany, ecolog y and zoolog y, bioscience center, federal university of rio grande do norte, natal, brazil. 3corresponding author: auritermes@yahoo.com.br 184 sociobiolog y vol. 59, no. 1, 2012 termite infestations in urban areas have already been reported in several south american countries (e.g. mill 1991; aber & fontes 1993, fontes 1995, torales et al. 1997, bandeira 1998, constantino & dianese 2001, costa et al. 2009). in the northeastern region, eleven termite species have been recorded causing some type damage specifically within buildings (bandeira et al. 1998, vasconcellos et al. 2002). little is known about urban termites of the city of recife. serpa (1993) has listed three termite genera (coptotermes, cryptotermes, and nasutitermes), without citing the species, causing damage to buildings in several points of the city. milano & fontes (2002) have reported the occurrence of coptotermes gestroi (wasman) damaging the woodwork of recife’s warehouses. this study aimed to survey the fauna of urban termites of the city of recife (pernambuco, brazil) and to report the factors that favor these insects’ occurrence, their habits, and the damage they cause. materials and methods located on the northeastern brazilian coast, the city of recife (8º 04’ 03’’ s, 34º 55’ 00’’ w) is 219,493 km2 and has a population of approximately 1.422.905 inhabitants. its climate is hot and humid and the average temperature is of 25.2°c (prefeitura da cidade do recife 2011). this study was carried out between august 2006 and june 2008, in six neighborhoods of recife: ibura, casa amarela, várzea, água fria, afogados, and dois irmãos. twenty streets were chosen randomly from each neighborhood and five homes were chosen from each street, totalizing 600 sampled homes. inspections were carried out in each home, where we examined both the external area (trees, debris, fences, walls, and other structures that could serve as shelter to termites) and the internal area (furniture, wood from the roof, doors, steps, and cracks). specimens located were collected and later stored in labeled plastic containers along with pieces of nests or tracks to facilitate identification. the material collected was then identified and conserved in 80% alcohol. samples are deposited in the entomological collection of the biolog y department of the federal rural university of pernambuco and in the isoptera collection of the bioscience center of the federal university of rio grande do norte. 185 albuquerque, a.c. et al. — urban termites of northeast brazil results and discussion seven termite species belonging to seven genera and three families (kalotermitidae, rhinotermitidae, and termitidae) were found causing some kind of damage to recife’s urban environment (table 1). with the exception of cryptotermes dudleyi banks and coptotermes gestroi, all of the species are native and relatively common to the atlantic forest of northeastern brazil (vasconcellos 2010). table 1. termite species of the urban environment of recife, ne brazil. family/species occurrence of each species (%) pest status* kalotermitidae** cryptotermes dudleyi 9.46 ++ rhinotermidiae coptotermes gestroi 2.25 ++ heterotermes longiceps 1.80 + termitidae nasutitermes corniger 82.40 +++ neocapritermes opacus 0.45 + amitermes amifer 3.15 + microcerotermes sp. 0.45 + * +: minor; ++ intermediate; +++ major status. ** cryptotermes brevis has been recorded in recife’s urban environment, but was not collected in this study. nasutitermes corniger motschulsky was the most frequent species (82.4%) among the properties visited and was responsible for the greatest damage to the city of recife. this species was found in trees, furniture, and walls, but predominantly infecting the woodwork of roofs. a study carried out by bandeira (1998) indicated species of the nasutitermes genus (especially n. corniger) as responsible for over 50% of the damage caused by termites to the city of belém, northern brazil. vasconcellos et al. (2002) listed n. corniger as the most frequent species within properties of joão pessoa, northeast brazil; there it was responsible for 43% of the damage caused by termites in that city. there are also records of great damage caused by n. corniger in the city of corrientes, argentina (torales 2002). according to bandeira et al. (1998), the predominance of the nasutitermes genus in several cities of the brazilian northern and northeastern regions may be related to 186 sociobiolog y vol. 59, no. 1, 2012 these species’ low selectivity to the type of wood eaten, as they may attach dry or humid wood, hard or soft, wrought or not. additionally, the construction of compound nests is an important characteristic of n. corniger biolog y, which increases the complexity of the control measures needed to deal with this species in urban environments (vasconcellos & bandeira 2006). cryptotermes dudleyi was the second most frequent species (9.46%) and was found mainly attacking furniture. indicated by bandeira (1998) as a pest in the city of belém, c. dudleyi has also been observed by vasconcellos et al. (2002) causing damage to buildings of joão pessoa. although it was not collected from the buildings visited, there are samples of cryptotermes brevis (walker) deposited in the isoptera collection of the federal university of rio grande do norte that come from recife. cryptotermes brevis, along with n. corniger, has been considered a serious urban pest in the city of joão pessoa, where it was found in 61% of the properties analyzed (vasconcellos et al. 2002). the great success of c. brevis as a pest is possibly due to new colonies’ rapid adaptation to the large variety of urban conditions and to their high response level to orphanhood (mcmahan 1962, edwards & mill 1986). the species coptotermes gestroi, heterotermes longiceps (snyder), and amitermes amifer silvestri were found in only two of the six neighborhoods visited. coptotermes gestroi has been indicated by fontes (1998) and laera (1998) as one of the species responsible for the greatest damage to urban trees in the cities of são paulo and rio de janeiro, southeastern brazil. according to the same authors, this is a serious issue as these trees may spread their infestation to buildings and worsen the problem. fontes & milano (2002) have already reported this same species consuming wood in warehouses of recife. heterotermes longiceps and amitermes amifer were found infesting fences and eating decomposing wood in yards. heterotemes longiceps has also been found in the urban environments of the cities of brasília by constantino & dianese (2001), corrientes by torales (2002), belo horizonte by milano & fontes (2002), joão pessoa by vasconcellos et al. (2002), and goiânia by costa et al. (2009). amitermes amifer, in turn, has also been found in joão pessoa causing superficial damage to wood objects in gardens (vasconcelos et al. 2002). the remaining species collected in this study occurred less frequently and apparently cause insignificant losses to recife’s properties. 187 albuquerque, a.c. et al. — urban termites of northeast brazil the occurrence frequency and the intensity of the damage caused to the properties analyzed suggest that only n. corniger, c. dudley, and c. gestroi can be treated as pests in recife. the remaining species were found causing little damage to fences and other cellulose objects in gardens and can not be considered as true pests in the city. references aber, a. & l.r. fontes 1993. reticulitermes lucifugus (isoptera, rhinotermitidae), a pest of wooden structures, is introduced into the south american continent. sociobiolog y 21 (3):335-339. bandeira, a. g., c. s. miranda & a. vasconcellos 1998. danos causados por cupins em joão pessoa. pp. 75-85, in: fontes, l. r. & e. berti-filho (eds), cupins: o desafio do conhecimento. piracicaba: fealq. bandeira, a. g. danos causados por cupins na amazônia brasileira. 1998. pp. 87-98, in: fontes,l. r. & e. berti-filho (eds), cupins: o desafio do conhecimento. piracicaba: fealq. constantino, r. 1999. chave ilustrada para identificação dos gêneros de cupins (insecta: isoptera) que ocorrem no brasil. papéis avulsos de zoologia 40(25): 387-448. constantino, r. 2002. the pest termites of south america: taxonomy, distribution and status. journal of applied entomolog y. 126(7/8): 355-365. constantino, r. & e.c. dianese 2001. the urban termite fauna of brasília, brazil. sociobiology. 38: 323-326. costa, d. a; k. e. santo-filho & d. brandão 2009. padrão de distribuição de cupins na região urbana de goiânia. iheringia, sér. zoológica. 99(4): 364-367. costa-leonardo, a. m. 2000. cupins urbanos: conhecer para combater. ciência hoje.18(165): 74-77. edwards, r & a. e. mill 1986. termites in buildings: their biolog y and control, east grimstead, rentokil limited, 261p. fontes, l. r . 1998. considerações sobre a complexidade da interação entre o cupim subterrâneo, coptotermes havilandi, e a arborização no meio urbano. pp. 109-124, in: fontes, l. r. & e. berti-filho (eds), cupins: o desafio do conhecimento. piracicaba: fealq. fontes, l.r. & s. milano 2002. térmites as na urban problem in south américa. sociobiology. 40(1): 107-147. laera, l. h. n. 1998. cupins na arborização urbana do rio de janeiro, brasil. pp: 125-132, in: fontes, l. r. & e. berti-filho (eds), cupins: o desafio do conhecimento. piracicaba: fealq. mcmahan, e. 1962. laboratory studies of colony establishment and development in cryptotermes brevis (walker) (isoptera: kalotermitidae). proceedings of the hawaiian entomological society 18 (1):145-153. 188 sociobiolog y vol. 59, no. 1, 2012 milano, s. & l. r. fontes 2002. cupim e cidade: implicações ecológicas e controle. são paulo, brasil. 142p. milano, s. 1998. diagnóstico e controle de cupins em áreas urbanas. pp. 45-74, in: fontes, l.r & e.berti-filho (eds), cupins: o desafio do conhecimento. piracicaba: fealq. mill, a. 1991. termites as structural pests in amazonia, brazil.sociobiolog y19 (2):339349. prefeitura da cidade de recife. 2011. a cidade do recife. http://www.recife.pe.gov.br/pr/ secplanejamento/inforec. serpa, f. g. 1993. considerações sobre “cupins” na cidade de recife. boletim da associação brasileira de preservadores de madeira, 75: 1-7. torales, g. j., e.r. laffont, m.o. arbino & m.c. godoy 1997. primeira lista faunística de los isopteros de la argentina. revista de la sociedade entomologica argentina 56 (1-4):47-53. vasconcellos, a. 2010. biomass and abundance of termites in three remnant areas of atlantic forest in northeastern brazil. revista brasileira de entomologia, 54(3): 455–461. vasconcellos, a., a. g. bandeira, c. s. miranda & m. p. silva 2002. termites (isoptera) pests in buildings in joãopessoa, brazil. sociobiolog y 40(3): 639 644. vasconcellos, a. & a. g. bandeira 2006. populational and reproductive status of a polycalic colony of nasutitermes corniger (isoptera, termitidae) in the urban area of joão pessoa, ne brazil. sociobiolog y 47(1): 165 – 174. 985 worldwide spread of the moorish sneaking ant, cardiocondyla mauritanica (hymenoptera: formicidae) by james k. wetterer abstract cardiocondyla spp. are small, inconspicuous ants, native to the old world. until recently, cardiocondyla mauritanica forel, 1890 was a little known species recorded almost exclusively from the semi-arid subtropics of north africa, the middle east, and neighboring islands. in contrast, cardiocondyla nuda mayr, 1866 was considered a cosmopolitan tramp species, spread broadly around the world through human commerce. a recent taxonomic reanalysis by b. seifert, however, found genuine c. nuda restricted to australia, new guinea, and western oceania, and that published records of ‘c. nuda’ from outside this region were based on misidentifications of other species, notably c. mauritanica. in addition, cardiocondyla ectopia, known from north america, was found to be a junior synonym of c. mauritanica. here, i examine the worldwide spread of c. mauritanica. i compiled published and unpublished c. mauritanica specimen records from >250 sites, documenting the earliest known records for 47 geographic areas (countries, island groups, major islands, and us states), including several for which i found no previously published records: barbados, bonaire, curaçao, grenada, saba, and saudi arabia. cardiocondyla mauritanica is found primarily in semi-arid and urban environments. cardiocondyla mauritanica shows an apparently continuous distribution and geographic variation in morpholog y from northwest africa to india suggesting that c. mauritanica is native throughout this subtropical expanse. old world records of c. mauritanica far from this range come from ascension, zimbabwe, and several indo-pacific islands. the sole temperate record of c. mauritanica comes from ukraine. cardiocondyla mauritanica was first found in the new world in 1967, and has spread through the southwestern us, northern mexico, florida, and the west indies. part of the wilkes honors college, florida atlantic university, 5353 parkside drive, jupiter, fl 33458, usa. e-mail: wetterer@fau.edu 986 sociobiolog y vol. 59, no. 3, 2012 success of c. mauritanica in exotic locales may relate to its ability to co-exist with dominant invasive ants, such as the argentine ant, linepithema humile (mayr, 1868). key words: biogeography, biological invasion, exotic species, invasive species introduction cardiocondyla (hymenoptera: formicidae) ants are small, inconspicuous species, native to the old world. until recently, cardiocondyla mauritanica forel, 1890 was a little known species recorded almost exclusively from north africa, the middle east, and neighboring islands. in contrast, cardiocondyla nuda mayr, 1866 was long considered a cosmopolitan tramp species, spread broadly around the world through human commerce. seifert (2003), however, concluded that genuine c. nuda in fact has a fairly restricted range in australia, new guinea, and oceania. all specimens from outside this region previously identified as ‘c. nuda’ that he re-examined were actually less familiar species, notably c. mauritanica. seifert (2003) also recognized that cardiocondyla ectopia snelling, 1974, known from many sites in north america, was actually a junior synonym of c. mauritanica, and concluded “the cosmopolitan c. mauritanica is one of the most abundant and most widely distributed cardiocondyla species of the world and comprises about 12% of all investigated samples.” here, i examine the worldwide spread of c. mauritanica. taxonomy forel (1890) described cardiocondyla nuda mauritanica (= c. mauritanica) from tunisia. ortiz & tinaut (1987) raised c. mauritanica to a full species. junior synonyms include cardiocondyla emeryi nitida bernard, 1948 from libya, cardiocondyla ectopia snelling, 1974 from north america, and leptothorax caparica henin, paiva & collingwood, 2001 from portugal. seifert (2003) placed c. mauritanica in the “c. nuda species-group” along with c. nuda, cardiocondyla atalanta forel, 1915 from australia, cardiocondyla strigifrons viehmeyer, 1922 from indonesia, cardiocondyla kagutsuchi terayama, 1999 from east asia and oceania, and cardiocondyla paranuda seifert, 2003 from tunisia. cardiocondyla mauritanica appears to be most 987 wetterer, j.k. — worldwide spread of cardiocondyla mauritanica closely related to c. cf. kagutsuchi (seifert 2008, oettler et al. 2010). whereas c. mauritanica males are all wingless, c. cf. kagutsuchi has both winged and wingless males (oettler et al. 2010). cardiocondyla mauritanica was sometimes misidentified as c. nuda because c. mauritanica has a relatively indistinct metanotal groove (i.e., a dorsal furrow separating the propodeum from the promesonotum). this groove is only suggested in c. nuda, but it is well developed in several common cardiocondyla species, including three widespread “tramp” species, spread by human commerce: cardiocondyla emeryi forel, 1881, cardiocondyla obscurior wheeler, 1929, and cardiocondyla wroughtonii forel, 1890. nonetheless, it is fairly simple to distinguish c. mauritanica from c. nuda. in c. mauritanica, the lower surface of the postpetiole is nearly flat and the height of the postpetiole less than that of the petiole. in c. nuda, however, the lower surface of the postpetiole bulges out, giving it a convex profile and a taller postpetiole with a height equal to that of the petiole. another widespread cardiocondyla species lacking a distinct metanotal groove is cardiocondyla minutior forel. seifert (2003) presented a range of allometric measures that allow the identification of these and all other described cardiocondyla species. deyrup et al. (2000) coined the common name “sneaking ant” for cardiocondyla, apparently due to their inconspicuous nature. “mauritanica” is latin for “moorish.” the term “moor,” derived from the mauri people of northwestern africa, has long been used in europe as a colloquial term for all inhabitants of north africa. two other north african ants share this specific name: aphaenogaster mauritanica dalla torre, 1893 from “barbaria” (northern algeria) and cataglyphis mauritanicus emery, 1906 from tunisia and algeria. i have used the common name “moorish sneaking ant,” analogous to the common names for other north african mauritanica species, e.g., the moorish gecko, tarentola mauritanica (l., 1758) and the moorish viper, macrovipera mauritanica (duméril & bibron, 1848). methods using published and unpublished records, i documented the worldwide range of c. mauritanica. i obtained unpublished site records from museum specimens in the collections of the museum of comparative zoolog y (mcz, identified by s. cover) and the smithsonian institution (si, identified by 988 sociobiolog y vol. 59, no. 3, 2012 m. smith). in addition, i used on-line databases with collection information on specimens by antweb (www.antweb.org ), the asociación ibérica de mirmecología (www.formicidae.org ), the global biodiversity information facility (www.gbif.org ), myrmecolog y forum (antfarm.yuku.com), bugguide (bugguide.net), fauna europaea (radchenko 2004), and ants of eg ypt (taylor 2010). i received unpublished c. mauritanica records from j. heinze (bonaire) and b. seifert (malta, saudi arabia). finally, i collected c. mauritanica in california, florida, madeira, and on several west indian islands (all identified by s. cover). i obtained geographic coordinates for collection sites from published references, specimen labels, maps, or geography web sites (e.g., earth.google. com, www.tageo.com, www.geonames.org, and www.fallingrain.com). if a site record listed a geographic region rather than a “point locale,” and i had no other record for this region, i used the coordinates of the largest town within the region or, in the case of small islands and natural areas, the center of the region. in a number of cases, publications did not include the collection dates for specimens, but i was able to determine the date based on information from other museum specimens, on the collector’s travel dates, or limit the date by the collector’s date of death. results i compiled published and unpublished c. mauritanica specimen records from >250 sites worldwide (fig. 1). i documented the earliest known c. mauritanica records for 47 geographic areas (countries, island groups, major islands, and us states; tables 1-3), including saudi arabia plus several west indian islands for which i found no previously published records: barbados (one urban park site; leg. j.k. wetterer), bonaire (one garden site; leg. j. heinze), curaçao (five sites: four urban and one zoo; leg. j.k. wetterer), grenada (17 sites: seven disturbed forest, five urban, three beaches, one mangrove, and one sugarcane; leg. j.k. wetterer), and saba (one coastal site; leg. g.d. alpert). to date, c. mauritanica has never been collected in mauritania, a country whose name derives from the same source. cardiocondyla ants are notoriously difficult to identify to species. in the past, many authors have used the name ‘c. nuda’ as a catchall for several cardiocondyla species with a relatively indistinct metanotal groove. fortunately, 989 wetterer, j.k. — worldwide spread of cardiocondyla mauritanica c. mauritanica has not been used as a catchall name, and published records of c. mauritanica and its junior synonym in the new world, c. ectopia, appear to be reliable. authors reporting these relatively obscure taxa no doubt first determined the specimens were not c. nuda. i found no cases where a specimen that was identified as c. mauritanica was later re-identified as a different species, though seifert (2008) incorrectly listed c. mauritanica from kiribati through a typographical error (b. seifert, pers. comm.). except for this one record, i included all published records of c. mauritanica. still, the difficulties of separating closely related species of cardiocondyla should not be underestimated. as seifert (pers. comm.) wrote: “the clustering of kagutsuchi-mauritanica-nuda-atalanta is stable in multivariate analyses but even an experienced ‘cardiocondyla man’ like me cannot identify each specimen by simple eye inspection. you must measure.” fortunately, except for seifert’s (2003) three indo-pacific records, there have been no other reports of c. mauritanica from within the known geographic ranges of c. kagutsuchi, c. nuda, and c. atalanta. misidentifications emery (1884) identified specimens from tunisia as c. nuda, but emery (1891) re-identified them as c. mauritanica. seifert (2003) re-examined specimens reported as c. nuda and found many palearctic records of ‘c. fig. 1. worldwide distribution records of cardiocondyla mauritanica. 990 sociobiolog y vol. 59, no. 3, 2012 nuda’ were actually based on misidentification of c. mauritanica (e.g., pisarski 1967, bolton 1982, heinze et al. 1993). a number of other studies have also re-examined specimens first reported as c. nuda and found them to be c. mauritanica (e.g., barquín 1981 re-identified by espadaler & bernal 2003). cagniant (1962) listed c. nuda as the only cardiocondyla known from morocco, but cagniant & espadaler (1993) and cagniant (2006) listed cardiocondyla batesii forel, 1894, c. emeryi, and c. mauritanica. comín & furió (1986) reported c. batesii from majorca, later re-identified as c. mauritanica (see gómez & espadaler 2006). it seems likely that additional published records of c. nuda and c. batesii from the mediterranean region and the middle east are actually c. mauritanica, e.g., records of c. nuda from table 1. earliest known records for cardiocondyla mauritanica from the old world subtropics of southern europe, north africa, the middle east, south asia, and neighboring islands. si = smithsonian institution. + = no previously published records. location earliest record tunisia 1891 (a. forel, si): oasis by gabes algeria ≤1904 (forel 1904) libya 1906 (mayr 1908) cyprus ≤1909 (emery 1909) eg ypt ≤1911 (forel 1911, karavajev 1911) iraq 1918 (seifert 2003) israel 1922-1928 (menozzi 1933) canary islands 1949 (espadaler & bernal 2003) afghanistan 1953 (seifert 2003) iran 1974 (seifert 2003) pakistan 1974 (seifert 2003) balearic islands 1976 (gómez & espadaler 2006) india 1978 (seifert 2003) spain 1982 (ortiz & tinaut 1987) turkey 1984 (seifert 2003) malta 1984 (seifert 2003) pantelleria 1987 (mei 1995) crete 1990 (seifert 2003) portugal 1991 (seifert 2003) nepal 1991 (seifert 2003) morocco 1991 (seifert 2003) greece 1994 (seifert 2003) united arab emirates 1995 (seifert 2003) jordan 1996 (seifert 2003) madeira 2002 (wetterer et al. 2007a) sicily ≤2004 (radchenko 2004) gibraltar ≤2008 (guillem 2008) +saudi arabia 2009 (m.r. sharaf, b. seifert pers. comm.): riyadh 991 wetterer, j.k. — worldwide spread of cardiocondyla mauritanica morocco (delye & bonaric 1973), saudi arabia (collingwood 1985), yemen (collingwood & agosti 1996), eg ypt (mohamed et al. 2001), and southern europe (radchenko 2004). izhaki et al. (2009) listed c. nuda in a table of ants collected at mount carmel, israel, but instead listed c. nuda mauritanica in a figure, no doubt meaning c. mauritanica in both cases. cardiocondyla mauritanica is not the only species that has been misidentified as c. nuda. for example, seifert (2003) reported that many japanesepacific specimens reported as c. nuda were actually c. kagutsuchi. wilson & taylor (1967) designated c. minutior as a junior synonym of c. nuda, but heinze (1997, 1999) revived c. minutior as a full species. between 1967 and 1997, when c. minutior was considered a junior synonym of c. nuda, most authors reported records of c. minutior as c. nuda. based of web photos, b. table 2. earliest known records for cardiocondyla mauritanica from old world tropical and temperate areas. location earliest record sudan ≤1911 (karavajev 1911) oman 1989 (seifert 2003) ascension 1990 (ashmole & ashmole 1997) zimbabwe 1995 (seifert 2003) ukraine 1995 (seifert 2003) indonesia 1999 (seifert 2003) philippines 1999 (seifert 2003) papua new guinea ≤2003 (seifert 2003) table 3. earliest known records for cardiocondyla mauritanica from the new world. unpublished records include collector, museum source, and site. mcz = museum of comparative zoolog y. si = smithsonian institution. location earliest record california 1967 (snelling 1974 as c. ectopia) arizona 1975 (c. chandler, si): phoenix florida 1981 (seifert 2003) puerto rico 1982 (seifert 2003) mexico ≤1986 (rojas-fernández 2001 as c. ectopia) texas ≤1992 (o’keefe et al. 2000 as c. ectopia) +grenada 2003 ( j.k. wetterer, mcz): grenville +curaçao 2004 ( j.k. wetterer, mcz): piscadera +barbados 2006 ( j.k. wetterer, mcz): bridgetown +bonaire 2006 ( j. heinze, pers. comm.): kralendijk +saba 2008 (g.d. alpert, mcz): south coast 992 sociobiolog y vol. 59, no. 3, 2012 seifert (pers. comm.) determined that specimen casent 0102306 from diego garcia, identified as c. nuda on antweb.com, is almost certainly c. minutior. discussion the earliest records of c. mauritanica come from subtropical north africa, the middle east, and neighboring islands (table 1). the only c. mauritanica record older than 1967 from outside the old world subtropics comes from an urban site in neighboring sudan (fig. 1; table 2). seifert (2003) found that “c. mauritanica specimens from india (punjab, himachal pradesh) have slightly narrower postpetiole and slightly shorter spines. furthermore, there is a certain trend from nw africa east to india to have the petiole node lower and more rounded in profile (not quadrate as in the tunisian type population).” this cline of geographic variation suggests that c. mauritanica is native throughout this region, spanning much of the subtropics of north africa, the middle east, and south asia. espadaler & bernal (2003) considered c. mauritanica to be also native to the canary islands at the far western edge of this range. it is uncertain how far the native range of c. mauritanica extends. reyeslópez et al. (2008) considered c. mauritanica to be an exotic in southern spain. if this is true, it could explain why all known records of c. mauritanica from spain are recent (table 1). gómez & espadaler (2006) wrote that c. mauritanica “seems to be rapidly extending its range from north africa and southern spain to the north along the west mediterranean coast (k. gómez & x. espadaler, unpubl.) and the balearic islands.” wetterer et al. (2007a) found c. mauritanica only at urban sites in madeira and considered it exotic there. wetterer et al. (2007b) proposed that, given its widespread occurrence in isolated, uninhabited arid parts of ascension, it seemed possible that c. mauritanica was native to ascension. after mapping the worldwide distribution of c. mauritanica, however, i now realize that madeira is very close to the apparent native range of c. mauritanica, while ascension, in the middle of the south atlantic, is very distant from any populations of this species in north africa, making it very unlikely that ascension is part of its native range (fig. 1). other old world records of c. mauritanica far from its native range come 993 wetterer, j.k. — worldwide spread of cardiocondyla mauritanica from zimbabwe and several indo-pacific islands (fig. 1). the sole temperate record of c. mauritanica comes from ukraine (seifert 2003). cardiocondyla mauritanica was first found in the new world in 1967 (table 3), but it has since spread extensively through the southwestern us, northern mexico, florida, and the west indies (fig. 1). whereas in the old world, there are few records of c. mauritanica from the tropics, i have found this species at numerous tropical sites in the west indies. ecology most records of c. mauritanica come from semi-arid areas or urban sites, agreeing with seifert’s (2003) conclusion that “it is mainly a species of semideserts and other xerothermous habitats” and ward’s (2005) observation that c. mauritanica “occur in disturbed (mostly urban) habitats in california, where they nest in sidewalks and along roadways.” seifert (2003) noted that in tropical east asia and oceania, “c. kagutsuchi seems to replace c. mauritanica. the fact that c. mauritanica, a most widely distributed cosmopolitan tramp species, could not substantially penetrate the range of c. kagutsuchi is intriguing.” it may be that c. mauritanica is less well adapted for the humid tropical climate of this region. cardiocondyla mauritanica has habitat preferences that overlap broadly with those of the argentine ant, linepithema humile (mayr, 1868), an invasive ant from the subtropics of south america. wetterer et al. (2000) found that c. mauritanica (as c. ectopia) and l. humile were the only non-native ants on santa cruz island, off the coast of southern california. part of the success of c. mauritanica and other cardiocondyla species in exotic locales may be due to their ability of co-exist with dominant invasive ants (heinze et al. 2006). ward (2005) reported that both c. mauritanica and c. minutior “are able to survive in sites invaded by the argentine ant.” gulmahamad (1997) observed c. mauritanica (as c. ectopia) “co-existing with the argentine ant at four different geographical locations in southern california. at one site, it was surviving in a nest with the entrance located only 8 cm from the nest of the argentine ant and only 3 cm from an active trail of this species.” gulmahamad (1997) proposed several factors that promoting this co-existence, notably c. mauritanica’s use of chemical defenses against l. humile. gómez & espadaler (2006) reported c. mauritanica “on 994 sociobiolog y vol. 59, no. 3, 2012 several irrigated housing estates in ibiza, mallorca and menorca, usually in coexistence with the argentine ant, linepithema humile.” gómez & espadaler (2006) noted that c. mauritanica “seems to be the first case to join the argentine ant in its invasion, with no apparent problems in gardens infested by linepithema... both species are highly aggressive to each other. in encounters between foraging ants of the two species, the c. mauritanica worker initially crouches down to the floor and remains quiet while the argentine worker antennates it. if pulled, the cardiocondyla worker repeatedly fiercely stings the argentine ant until it retreats... thereafter, recruitment by the argentine ant is not triggered and its vast numeric prevalence has no local effect.” the ability to co-exist with dominant invasive ants extends to other cardiocondyla species as well. wilson & taylor (1967) reported that l. humile “excludes other larger ant species, including the formidable pheidole megacephala. one species found to be compatible with it on hawaii is the diminutive cardiocondyla nuda” (probably referring to c. minutior, which they had designated a junior synonym of c. nuda). wetterer (2012) noted that c. emeryi appears to be more common in areas dominated by african big-headed ant, pheidole megacephala (fabricius, 1793). at sites with high densities of p. megacephala on islands of the pacific, atlantic, and the west indies, i usually also found c. emeryi, but few other ants. it may be that dominant ants such as l. humile and p. megacephala benefit cardiocondyla species indirectly, through elimination of competing ant species. six species of cardiocondyla are now known to be cosmopolitan, having achieved broad distributions in both the old world and the new world (from largest to smallest in order of head size, from seifert 2003): c. venustula, c. mauritanica, c. obscurior, c. minutior, c. wroughtonii, and c. emeryi. by far, the most widespread and common is c. emeryi (wetterer 2012). none of these species are known to have significant ecological impacts, and it seems unlikely that any of these inconspicuous sneaking ants will ever become significant pests as they continue to spread, largely unnoticed, around the world. acknowledgments i thank m. wetterer, b. seifert, and j. heinze for comments on this manuscript; b. seifert, x. espadaler, and s. cover for help, encouragement, and ant identification; s. cover (mcz) and t. schultz (si) for help with their 995 wetterer, j.k. — worldwide spread of cardiocondyla mauritanica respective ant collections; b. seifert and j. heinze for providing unpublished records; w. o’brien for gis help; d.p. wojcik and s.d. porter for compiling their valuable formis bibliography; r. pasos and w. howerton of the fau library for processing so many interlibrary loans; florida atlantic university and the national science foundation (des-0515648) for financial support. references ashmole, n.p. & m.j. ashmole 1997. the land fauna of ascension island: new data from caves and lava flows, and a reconstruction of the prehistoric ecosystem. journal of biogeography 24:549-589. barquín, j. 1981. las hormigas de canarias. taxonomía, ecología y distribución de los formicidae. colección monograficas, secretariado de publicacíones de la universidad de la laguna 3:1-584. bolton b. 1982. afrotropical species of the myrmicine ant genera cardiocondyla, leptothorax, melissothorax, messor and catalaucus. bulletin of the british museum of natural history (entomolog y) 45/4:307-370. cagniant, h. 1962. etude de quelque fourmi marocaines. statistique provisoire des formicidae du maroc. bulletin de la societé d’histoire naturelle de l’afrique du nord 53:83-118. cagniant, h. 2006. liste actualisee des fourmis du maroc (hymenoptera: formicidae). myrmecologische nachrichten 8:193-200. cagniant, h. & x. espadaler 1993. liste des espèces de fourmis du maroc. actes des colloques insectes sociaux 8:89-93. collingwood, c.a. 1985. hymenoptera: fam. formicidae of saudi arabia. fauna of saudi arabia 7:230-302. collingwood, c.a. & d. agosti 1996. formicidae (insecta: hymenoptera) of saudi arabia (part 2). fauna of saudi arabia 15:300-385. comín, p. & v. furió 1986. distribución biogeográfica de las hormigas (hymenoptera, formicidae) en las islas del mediterráneo occidental. bolleti de la societat d’historia natural de les balears 30:67-79. delye, g. & j.c. bonaric 1973. les fourmis arenicoles du sud marocain (hym. formicidae). bulletin de la société entomologique de france 3:107-110. deyrup, m., l. davis, & s. cover 2000. exotic ants in florida. transactions of the american entomological society 126:293-326. emery, c. 1884. materiali per lo studio della fauna tunisina raccolti da g. e l. doria. iii. rassegna delle formiche della tunisia. annali del museo civico di storia naturale giacomo doria (genova) (2)1(21):373-386 emery, c. 1891. revision critique des fourmis de la tunisie. exploration scientifique de la tunisie. zoologie-hyménoptères. imprimerie nationale, paris. 21 pp. 996 sociobiolog y vol. 59, no. 3, 2012 emery, c. 1909. beiträge zur monographie der formiciden des paläarktischen faunengebietes. 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(hymenoptera: vespidae): the influence of the weather on the reproductive success of wasps societies in urban conditions j nadolski introduction the presence of different species of social wasps (vespidae) in urban habitats is commonly known (edwards, 1980; matsura & yamane, 1984; haesler, 1982; ahrnè 2008), also in the cities from poland (skibińska, 1987; kowalczyk, 1991; nadolski et al., 2011). previous studies determined the species composition of these insects, their proportions and structures of nests and sizes of their colonies (nadolski, 2012), including their parasitoids, parasities, pathogens and other microorganisms accompanying their nests (nadolski, 2013) as well as the dynamics of quantitative changes in the urbanized areas (christie & hochuli, 2008). however, it is still difficult to determine which factors have important impacts on the effective colonization of cities by many species of social wasps. the most characteristic features of these areas are a warmer and more stable microclimate than in other variable environments, and abundant food resources and a large variety of niches to be used (christie & hochuli, 2008). wasp colonies in cities abstract in subsequent years, changes in the period of colonies activity for both species of wasps were observed as well as the longer period of development of wasp societies in the city than in the forest. there was not found a clear influence of weather conditions in winter on date of colonies initiation. however, low temperatures and rainfall in may caused a delay in the initiation of nesting. the temperature in winter had a little impact on the number of queens that survived the winter and established their colonies in spring, but the humidity in winter had influence on the count of queens and thereby on the number of future nests especially in the forest area. sociobiology an international journal on social insects university of łódź, kilińskiego, łódź, poland. research article wasps article history edited by sulene n. shima, unesp, brazil received 08 may 2013 initial acceptance 12 july 2013 final acceptance 20 august 2013 keywords urban fauna; social wasps; phenology of wasps; vespa crabro, dolichovespula saxonica corresponding author jerzy nadolski faculty of biology and environmental protection, natural history museum university of łódź kilińskiego 101, 90–011 łódź, poland e-mail: nadolski@biol.uni.lodz.pl are mostly established in buildings such as roofs, attics, and within the walls of houses, and their nests are larger and societies are more numerous than those from rural or forested areas (nadolski, 2012). subjects concerning the biology of social wasps have been the basis of many studies (matsura, & yamane, 1984; starr & jacobson, 1990; leatwick & godfrey, 1996; haesler, 1997; nakamura & sonthichai, 2004), but only a few are directly related to the phenology of this group of insects in poland (pawlikowski et al., 2005; tryjanowski et al., 2010). however previous studies were based only on observed of wasp flight activity, which method does not always correctly characterizes the current stage of development of the colony.. the purpose of this study was to determine the phenology and the duration of reproductive periods of two wasp species the european hornet vespa crabro linnaeus, 1758 and saxon wasp dolichovespula saxonica (fabricius, 1793) based on the study of whole colonies and nests in the łódź city in poland and to determine whether meteorological factors may have an impact on their reproductive success. j nadolski phenology of european hornet in urban conditions478 materials and methods study sites the study was conducted in the city of łódź (51o45’n 19o28’e) in central poland. the climate of łódź is intermediate between the continental climate – eastern european and maritime climate – western european. the study of phenology of two species of social wasps (vespinae), the european hornet vespa crabro (linnaeus, 1758) and saxon wasp dolichovespula saxonica (fabricius, 1793) was carried out in two 10 km-distant areas in different habitat types. the first of these, the botanic and zoological gardens, was located at a parkland habitat (51o45n 19o24e) with the area about 84 ha. it is located in the western part of the city. the whole area, a remnant of an ancient deciduous forest, is dominated by hornbeam carpinus betulus and oaks querqus robur and q. petrea as well as scots pines pinus silvestris, birches betula sp. and different exotic tree and shrub species. south and south-east sides of these gardens directly border on big residential area with numerous blocks of flats and with population over one hundred thousand. the other study area, a woodland habitat, was the łagiewniki forest (51o50’n 19o29’e). it is a rich deciduous forest of considerable size about 1250 ha that is a remnant of the former of primeval forest and which was partly chopped down 70 years ago and reforested later. it is located in north-eastern part of łódź, a few kilometers from the city centre. because of its exceptional nature and landscape, part of this forest was turned into a nature reserve ‘las łagiewnicki’ with the area of 69.85 ha; whose main object of protection is a complex of natural phytocenoses with oak and hornbeam. collecting data this study was conducted between 2000 and 2009. the data concerning of the establishing of nests (the day of initiation nesting) were gathered based on the regular weekly recording of wooden nest boxes and other places (tree hollows, branches, outbuildings) where studied species could potentially form their nests by the young queen. wooden nesting boxes had dimensions of 13 x 13 x 36 cm and an entrance hole with 29 mm of diameter located 29 cm above the bottom of the box. the arising colonies were then regularly monitored, every two weeks, until the moment when reproductive castes (gynes and drones) left the nest. during each season the boxes occupied by wasps were observed and their colonies were studied for the presence of particular castes. the data obtained in this way, concerning both the date of initiation nesting and the appearing of castes, especially reproductive ones, gave ground to phenological analyses of biology of studied species. for the statistical analyses, the system of specification of dates was simplified by standardizing them. dates were coded as numbers, starting with april 1st as number 1of the given year (so that may 1st = 31, june 1st = 62 and so on). these activities were correlated with the studies on breeding of blue tits cyanistes caeruleus and great tits parus major in łódź (alabrudzińska et al., 2003; marciniak et al., 2007; bańbura et al., 2010). together 200 boxes during years 2000-2001 (110 located in the woodland site and 90 in the parkland site), 300 in years 20022003 (180 located in the woodland site and 120 in the parkland site) and 450 during the years 2004-2008 (250 located in the woodland site and 200 in the parkland site) were located on studied areas. after each season all the boxes were cleaned of all organic waste, including the remains of birds and nests of wasps as well as other impurities. weather data were obtained from historical weather records published on a website http://www.tutiempo.net/en/ climate/europe.htm. graphics and statistical analyses were conducted using statistica 9 package (statsoft, inc. 2010). results and discussion phenology of the establishment of a nest a total of 182 colonies of vespa crabro and 347 colonies of dolichovespula saxonica were investigated on studied areas in the city. the dynamic of development stages of colonies of v. crabro and d. saxonica are showed in the fig 1. for analysis, introduced designations (qn, and e) where qn (the queen) it is the nest of queen without workers yet, and e (the end) it is the nest ending the development, where been able to already observed the presence of first reproductive castes (gynes young queens and drones). analysis anova nonparametric, kruskal-wallis test: (h 3,529 =448.03 p = 0.00) and p-values for multiple comparisons showed statistical significance between ranks all of analyzed variables (p<0,05) except ranks of qn period for both studied species (p=1.0) (fig 1). figures 2 and 3 present the results of statistical analysis of initiation nesting dates (the period of qn without larvae and pupae yet) for d. saxonica (anova nonparametric, kruskal-wallis test: h 9,189 = 94.5 p =0.00) and v. crabro (anova nonparametric, kruskal-wallis test: h 9,84 = 56.0 p =0.00) in particular years. worthy of note are the minimum and maximum values which demonstrate the differences in dates of initiating nest-building in a given year although for some of multiple comparisons p-values were higher than 0.05. influence of urban conditions and weather on the reproductive success of social wasps (vespinae). means values of ranks of nest initiation dates, between two studied areas (parkland and woodland) were analyzed. fig 4 presents the mean value of standardized dates for establishing nests by vespa crabro in both studied areas habitats (the sociobiology 60(4): 477-483 (2013) 479 first residential areas with parkland and the second woodland). the analysis anova nonparametric kruskal-wallis test (h3,203 = 163.5 p = 0.00) demonstrated statistical significance between studied areas. multiple comparisons showed statistical significance (p-values ,<0.05) between means values of ranks of the time of appearing of reproductive castes for both habitats. for initiation dates, these differences are not statistically significant, although the mean of dates for the parkland habitat date is lower (earlier date). in the woodland habitat males and gynes appear in the nest about a week earlier than in the parkland habitat. for dolichvespula saxonica these differences were not statistically significant, probably due to the short period the full development cycle of colonies of this wasp. the assessment of the influence of weather conditions in the period of establishing of a nest by young queens of d. saxonica and v. crabro was conducted as well as the correlation between the number of nests in a given year and weather parameters in the winter (the average weather parameters of december, january, february and march) preceding the study season and in spring. in figures 5 and 6, correlations between the mean temperature in may and date of initiation of nests were demonstrated for nests of d. saxonica and v. crabro. temperature, precipitation and humidity in winter practically did not affect on the date of initiation of their nests and the precipitation and humidity in june had a weak effect. other weather factors for both species had a weak influence on date of nesting initiation by queens. the influence of weather conditions on the count of established nests was also studied. in table 1 are demonstrated r-values for correlations between count of established wasp nests and weather conditions in winter and spring in the studied period. fig 1. stages of development for dolichovespula saxonica and vespa crabro colonies in łódź in 2000-2009. d.sax.qn – the stage of queen for dolichovespula saxonica, v.crabro.qn – the stage of queen for vespa crabro, d.sax. e – the ending stage of development for dolichovespula saxonica, v.crabro e – the ending stage of development for vespa crabro fig 2. date of initiation for dolichovespula saxonica nests in łódź in 2000-2009. fig 4. date of initiation of nests by queens and time of appearing of reproductive castes for vespa crabro nests in two habitats in łódź, in 2000-2009. fig 3. date of initiation of vespa crabro nests in łódź in 20002009. j nadolski phenology of european hornet in urban conditions480 a large city is a specific ecosystem which, like no other, undergoes continuous changes resulting from constant, planned or accidental human intervention. the dynamic process accompanying these changes, in result of which we may observe loss of some species and inflow of others may sometimes leads to the increase of species diversity within certain animal groups. in urban habitats specific microclimate is created, which stands out due to reduced amplitude of temperature and humidity. these conditions are multiplied especially inside buildings due to the presence of heating and air conditioning, heating pipes, sewage system, culverts of various types, wells, tunnels and cellars. specific character of these habitats is tolerated by some groups of hymenoptera, including social wasps (banaszak,1978; skibińska,1978). the species, which ‘were able to” adapt their biology to urban environment, began their expansion at an unparalleled scale (skibińska, 1987). the european hornet vespa crabro and saxon wasp dolichovespula saxonica are species which often form colonies in the city and sizes of their nests and their societies are different and depend on the location of their colonies (nadolski, 2012). current knowledge of the phenology of vespinae in urban condition is clearly insufficient. one of the many reasons of this state of affairs are specific problems connected with the study of particular stages of the development of nests and societies, especially the time of produce particular forms and castes. a nest of vespinae is a tightly closed structure and some kind of external interference can irritate the society of these insects and leads to their attack in defense of the colony. monitoring of wasp societies based on observations of flight of particular forms of imago allows, as it seems, relatively accurate assessment of time of appearance and disappearance of particular forms and castes. special attention should be paid to the work of the research centre headed by tadeusz pawlikowski (pawlikowski et al., 2005; pawlikowski &pawlikowski, 2009; tryjanowski et al., 2010). however, studies of phenology based only on observations of wasps’ flight do not fully reflect the real phenomena occurring in their societies. many different factors can affect the time of departure from the nest of the particular individuals, especially the weather conditions and food availability. at the same time these factors may have a different effect on the wasps inside the nest. for example, adverse weather conditions, rain and low temperatures significantly reduce the penetration of the area by the wasps, however it does not mean they are not present inside r-values for winter conditions r-values for spring conditions habitat temperature humidity precipitation temperature humidity precipitation dolichovespula saxonica residential and parkland wn wn wn 0.30 wn wn woodland wn 0.62 0.32 0.35 -0.65 -0.57 vespa crabro residential and parkland wn 0.52 wn wn wn wn woodland wn 0.81 wn 0.37 0.35 -0.36 table 1. the correlation between the weather conditions and the count of dolichovespula saxonica and vespa crabro colonies. wn weak or none the correlation (r <0.3), numbers in bold indicate p<0.05 fig 5. the correlation between the mean temperature in may and date of initiation of dolichovespula saxonica nests in łódź, in 2000-2008 (n = 180; r=-0.54; p<0.05). fig 6. the correlation between the mean temperature in may and date of initiation of vespa crabro nests in łódź, in 2000-2008 (n = 53; r=-0.55; p<0.05). sociobiology 60(4): 477-483 (2013) 481 the nest. availability of food may be equally important factor in accelerating or delaying the flight of wasps from their nests. studies on vespa crabro (nadolski, unpubl.) in open breeding conditions demonstrated that when wasps have enough food (artificial feeding), neither gynes nor drones did not leave the nest even in late autumn. in autumn in natural conditions is running low on food because the number of workers that transported food are decreased both gynes and drones have to leave their nest. the effects of this phenomenon can be noticed most clearly in late autumn when reproductive castes leave nests suddenly and on mass scale. studies of hornet’s colonies conducted right before the abundant leaving of nests by wasps demonstrates large presence of gynes and drones (often few hundred) and only tens of workers. as earlier studies have shown (nadolski, 2012) the total number of gynes and drones leaving the nest in the autumn is close to the sum of all cells, previously occupied by larvae of reproductive castes. however, the emergence of particular individuals in nest is gradual as is apparent from the analysis of distribution of larvae and pupae in combs (nadolski, 2012) and it starts still in summer, not only in colonies of dolichvespula saxonica which development is completed in july as well as in colonies of vespa crabro which have active colonies in october yet (fig 1). this indicates that probably only very few individuals of reproductive castes probably leave their nest earlier. this situation is fully understandable because, there is necessity to synchronize the departures of these castes from various colonies. individuals which left the nest too early have little chance to find the partner. in studied years, changes in the period of activity of both species of wasps were observed (fig 2 and fig 3) but presence of hornets seems to be more stable. in the research of nests of both species of wasps based on the analysis of structure and the time of appearance of particular castes were demonstrated a statistically significant difference between dates of start and end of colony development. in fig 1 it can be seen that for hornet a stage qn sometimes lasted until the turn of june and july, and f stage was beginning in mid-august. thus, the presence or absence on the ground, the representative of reproductive castes is not equivalent to its presence or absence in the nest. it is worth paying attention to that on average, reproductive castes appear in nests for vespa crabro between the standardized value 160 and 170, i.e. the beginning of september, and the first males-drones and gynes can be observed in nests already in the middle of august. the comparison of the date of nesting initiation by young queens of vespa crabro and dolichovespula saxonica (fig 2 and fig 3) demonstrated differences in particular years. however, it is difficult to compare these data with the results of studies done for many years in kujawy region in poland (pawlikowski & pawlikowski, 2009; tryjanowski et al., 2010), because these studies were carried out on another area and with other methods. however, it can also be observed that in recent years the initiation of wasp nests is delayed and the period of time during which young queens begin to build nests is shortened also in łódź. the weather and urban conditions in particular years may have a significant impact on biology of wasps. the results concerning the nesting initiation for vespa crabro colonies based on the analysis of their nests showed nearly a week the difference between studied habitats residential-parkland and woodland (fig 4) although these differences were not statistically significant. it is interesting that similar results are obtained in studies of certain other groups of organisms. observations conducted over many years in the city of łódź showed similar phenological differentiation in vegetation of some plant species between these zones (bańbura et al., 2010). even more interesting is a significant difference between the periods of ending of colonies development of vespa crabro in both habitats (fig 4). in the forest a small but statistically significant accelerate the emergence of reproductive castes were observed. in conjunction with the later date of initiation of colonies this situation must result in shortening of the period of full development of society of hornets, and so consequently development of a smaller number of workers, gynes and drones and weaker reproductive success. demonstrated differences of presence of reproductive castes in the colony from zones of łódź, confirms assumptions that is a small but noticeable impact of urbanization degree on the time of wasps transformation. a longer period of colonies activity in urban conditions causes that wasp nests can be larger and achieve the better reproductive success. these results again confirm that urban conditions are more favorable for social wasps, than conditions outside the city (nadolski, 2012). in these studies a clear influence of weather conditions of the preceding winter season, on nest initiation date in the next year in both studied habitats there was not found. however the weather conditions preceding directly the time of establishing of nests by young mothers proved to be significant. the strong negative correlation (cohen, 1988) (r = -0.61) between the mean temperature in may and date of initiation of nests was demonstrated for nests of saxon wasp (dolichovespula saxonica) (fig 5). for the european hornet (vespa crabro), there was also found a strong negative correlation (r=-0.55) (fig 6) between mean temperature in may and date of initiation of nests. other weather factors for both species had no significant effect on date of nesting initiation. these results are partly consistent with the date for the “first queen” of vespula germanica (fabricius, 1793) obtained in kujawy region in poland (tryjanowski et al., 2010). winter weather conditions can have an effect on the number of wasp queens who have survived the winter and established colonies. winter temperatures and temperatures in spring do not have an influence on the number of colonies of wasps in the city (table 1). these relationships with spring temperatures are different for other social insects, such as the honey bee apis mellifera (gordo & sanz, 2006) and spiewok and schmolz (2006) showed that ambient temperature has an influence on the flight performance of wasps, especially hornets. this demonstrates that the phenology of social wasps j nadolski phenology of european hornet in urban conditions482 is difficult to investigate only based on an assessment the activity of flights, because wasps are active first of all in sunny and warm days and their absence on the ground does not mean, that they are not in the nest. in urban conditions wasps are not so strongly related to temperature for different reasons. first of all, the urban habitat can provide more comfortable and safer conditions for wintering for many groups of insects, protecting them from too low temperature during diapauses. it is different in the forest area where insects can survive the winter only in natural places in which temperature and humidity can be important. interestingly, this study indicates that no severe frosts, but rather mild and snowless winters may have a negative impact on the survival of young queens (table 1). in the light of current studies, it is understandable, because obtained ltemp50 values for gynes indicate that even a severe winter should not have influence on their survival (nadolski & bańbura, 2010). however, for both studied species were demonstrated a strong positive correlation between the level of humidity in the period preceding winters and the count of the established nests within the area of łagiewniki forest (r = 0.62 for dolichovespula saxonica and r = 0.81 for vespa crabro). this parameter is therefore important for the survival of gynes during diapauses in natural conditions. for vespa crabro, proper humidity in winter in urban conditions is also important (table 1). for vespa crabro, the humidity in winter in the residentialparkland habitat had also strong (r = 0.52) influence on the number of established colonies. it can be assumed that these differences between studied species may result from different places where young queens (gynes) overwinter. queens of hornet rarely overwinter inside in buildings (author observations) in contrast to queens of the saxon wasp. in natural areas intense precipitation and higher humidity also in spring have a negative influence on the very process of forming a nest and its further development especially for colonies of dolichovespula saxonica (table 1). especially nests on the branches of trees and bushes as well as colonies in breeding boxes and in tree hollows may be infected with fungi. for colonies in the urban area these factors are less important for initiation of the nest, because of the possibilities of alternative nest location in the buildings. the temperature in may had moderate influence on the count of nests of saxon wasps (moderate positive correlation r = 0.30-0.35) in both habitats and the weather condition in the spring had moderate influence on the count of colonies of vespa crabro in forest (table 1). the results of these studies demonstrate a clear influence of weather conditions on the phenology and reproductive success of social wasps. however, urban conditions can significantly reduce this impact in favor of insects. references ahrnè , k. 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(2010): does climate influence phenological trends in social wasps (hymenoptera: vespinae) in poland? europ. j. entomol., 107: 203-208. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4985sociobiology 67(2): 153-162 (june, 2020) introduction northern algeria is a forest-based country; despite the damage caused by humans and herds, beautiful stands are still in place (cagniant, 1968). the national forest inventory of northern algeria (established in 1983) confirms that the quercus suber forests cover an area of 2,000,000 ha totaling 34,000,000,000 trees (all strata combined). cork oak is strongly represented in the country’s east, rare and scattered in the west. the deciduous oak forests are represented by the algerian oak (q. canariensis) and the afares oak (q. afares) both of which thrive from 800 mm of rain. often in mixture with the corkoak they invade at some fresh stations. it should be noted that the afares oak is a species endemic to great kabylia and babor kabylia (louni, 1994). abstract in order to contribute to the myrmecological fauna knowledge of the guerrouche forest massif based in northeastern algeria. a study was conducted on three oak groves (quercussuber, q. canariensis and q. afares). a total of 60 plots were surveyed by using four sampling methods (manual capture, bait, pitfall and winkler). the inventory revealed 34 ants species belonging to 15 genus and 4 sub-families; dolichoderinae, formicinae, myrmicinae and ponerinae. dispatche as follow, 22 species for cork oak, 14 for algerian oak and 17 for afares oak. myrmicinae dominate in cork oakand in algerian oak (82.83 and 81.23% respectively) while in the afares oak, formicinae are largely in the lead (68.54%). this study revealed an endemic species to algeria (aphaenogaster testaceo-pilosa ssp canescens), three endemic’ species to algeria and morocco (camponotus laurenti, messor antennatus and aphaenogaster foreli) and one endemic to algeria and tunisia (bothriomyrmex decapitans). comparison of the four sampling methods effectiveness, used reveals that it’s the bait (80.7% of the species total number harvested in the all stations) and manual capture (49.69% of the species total number harvested in the all stations), which allowed the capture of the largest number of species followed by pitfalls traps (31.64% of the species total number harvested in the all stations). the winkler was much less effective (5.55% of the species total number harvested in the all stations). sociobiology an international journal on social insects a henine-maouche, a tahraoui, r moulaï article history edited by evandro nascimento silva, uefs, brazil received 23 january 2020 initial acceptance 24 january 2020 final acceptance 25 january 2020 publication date 30 june 2020 keywords formicidae, inventory, wet forests, oak forest, guerrouche, algeria. corresponding author anissa henine-maouche laboratoire de zoologie appliquée et d’écophysiologie animale faculté des sciences de la nature et de la vie université de bejaia, algérie. e-mail: anissa.maouche3@gmail.com among the most preserved and beautiful oak forests in algeria, the forest massif of guerrouche shelters to pure and mixed oak forests (algerian oak, afares oak and cork oak) of which algerian oak is the main species (a.s.a., 2006). this forest is home to the magot monkey (macaca sylvanus) and the algreian nuthatch (sitta ledanti) a strict endemic bird of algeria (bounar, 2014). the guerrouche forest fauna is much diversified but, unlike the flora, we know little about the subject, due to the lack of specific studies. faunal studies carried out in this forest are more concerned with mammals and birds. entomological studies, particularly myrmecological studies, are almost nonexistent (the best known being that of cagniant, (1968) on forest ants in algeria). in algeria, myrmecofaune is only known through the work of bernard (1958, 1968 and 1976) and cagniant (1968, laboratoire de zoologie appliquée et d’écophysiologie animale, faculté des sciences de la nature et de la vie, université de bejaia, algérie research article ants ants’ diversity (hymenoptera: formicidae) in the algeria’s humid forests, case of the gerrouche forest massif (taza national park) a henine-maouche, a tahraoui, r moulaï – ants’ diversity (hymenoptera-formicidae) in the algeria’s humid forests154 1970 et 1973). since then, the formicidae’s systematics and diversity in algeria has not been revised except recently with the work of barechet al. (2011, 2015, 2016 and 2017); chemala et al. (2017). the lack of knowledge of the guerrouche forest myrmecofauna creates an attraction that justifies the choice of this work, whose main objective is to carry out an analysis of the diversity of ants in guerrouche and as a second objective to compare the effectiveness of sampling methods. from a methodological point of view, the majority of authors who have worked on ants have used two sampling methods: quadrats, pifalls or both (cagniant, 1968; barech, 2016). in terms of sampling, this study presents a novelty by using four sampling methods: the winkler method (harvesting by extracting individuals from leaf litter), pitfall trap, bait and manual capture (agosti & alonso, 2000; bestelmeyer et al., 2000). a study using these four sampling methods has already been carried out in causses aveyronnais (france) (groc, 2006) and has given good results. methodology covering 10859.7 hectares and located 30km southwest of jijel in northeast algeria, the national forest of guerrouche is 19 km long from east to west and 13 km wide from north to south. this forest is characterized by some of the best preserved oak formations in algeria with an afforestation rate of 44%. the species distribution is as follows: corkoak: 4568 ha, algerian oak: 1026 ha (in pure stands up to 700 m altitude) and in association with cork oak at low altitude and afares oak, respectively: 1977 ha in pure stands (at high altitudes, above the algerian oak, from 900 m) (ziane, 1979). this study was conducted in three oak formations in the guerrouche forest more precisely in the taza national park and at different altitudes (fig 1): 1. the cork oak station is located in the jijelian cornice heart in the taza national park western limit (which is located inside the national forest of guerrouche) (36°42’26.46”n 5°32’57.264” e) at an altitude of 95m. the pure cork oak formation represents an open forest characterized by a shrub stratum as erica arborea, cytisus villosus, pistacia lentiscus, cistus monspelliensis, rubus ulmifolius, myrtus communis and calycotom espinosa. 2. the algerian oak station is located in the guerrouche forest heart at an altitude of 800 metres, the coordinates of which are: 36°41’45.62”n 5°38’27.36”e. this is a regular pure forest station (boudy, 1954) with a very dense canopy where grows only a much undeveloped undergrowth (bellatrèche, 1994). we can distinguish hedera helix which suffocates a certain amount of algerian oak. the shrub stratum consists of crataegus monogyna, laurus nobilis, cytisus villosus and rubus ulmifolius. the herbaceous stratum is dominated by vinca major and viola munbyana. 3. the afares oak station is located at the guerrouche forest top, at an altitude of 950 metres (36°41’34.10 “n 5°39’3.77 “e). it is a gardened forest of pure afares oak. we notice the rare algerian oak individual’s presence and the undergrowth are characterized by cytisus villosus, drimia maritima, ampelodesmos mauritanicus erica arborea and rubus ulmifolius. the myrmecological inventory carried out at these different stations took place from april to june 2018, twice a month. in this study, four sampling methods were applied to these environments: the winkler method (harvesting by extracting individuals from leaf litter), pitfall trap, bait and manual capture, as described by agosti and alonso (2000), bestelmeyer et al. (2000) and brinkman et al. (2001). 1. manual capure: during prospecting, manual capture is the appropriate. this is a random capture, pending 4 to 5 hours, using a mouth aspirator. all species seen with the unaided eye are sampled. fig 1. the three study stations location in taza national park (algeria). sociobiology 67(2): 153-162 (june, 2020) 155 2. bait: it is possible to actively attract ants with different baits placed in all areas. the bait used is pieces of oil’s sardines placed on an absorbent paper piece on the ground for three hours. 3. pitfall traps: the traps we used were 11 cm deep and 10 cm in diameter cans buried vertically, so that the opening was flush with the ground, to avoid the barrier effect for small species. each pitfall was filled to 5% with soapy water, which, in principle, is neither attractive nor repellent for ants. after 48 hours of continuous sampling, the traps were removed and the ants harvested. 4. winkler method: in each plot, the litter from a 1m² quadratand 15 cm of depth was collected in a numbered bag for later extraction in the laboratory. in the laboratory, a berlèse device was developed. the area total sampling for each environnent is 0.2 hectares. each area was divided into 5 transects to 40m long and 10m wide. each transect was then divided into four 10m square plots where each sampling method was applied. thus, for each environment, we obtained 20 samples of each method (fig 2). the standard protocol for myrmecofauna sampling proposed by agosti and alonso, (2000) and fisher et al. (2000) suggests a minimum 20 separate sampling points 10m apart to capture at least 70 ℅ of a site’s myrmecofauna. after display and drying, ants are identified after examining some each species systematic criteria specific. the determination always takes place under the binocular loupe. the most species identification is carried out by referring to various guides and identification keys such as those of bernard (1968) and cagniant (1968, 1970, 1997, 2005, 2006, 2009). professor henri cagniant has also contributed to the identification of some species. fig 2. experimental protocol for each sampled environment (groc, 2006). once the ants have been sorted, determined and counted, we measured them with a strip of graph paper in order to know their sizes. in order to exploit the results obtained from the study of the myrmecofauna of three humid forests, we used ecological indices such as specific richness, centesimal frequency, shannon-weaver diversity index, evenness index, sorensen coefficient and chao formula (ramade, 1984; chao, 1987). variance analysis (anova) is used to compare the diversity between stations. results our study identified 34 ant species representing 15 genus and 4 subfamilies; formicinae, myrmicinae, dolichoderinae and ponerinae. 22 species have been recorded in the cork oak, 14 species in the algerian oak and 17 species in the afares oak. (table 1). fig 3. formicidae centesimal frequencies of grouped by sub-family in the three study stations (guerrouche). a henine-maouche, a tahraoui, r moulaï – ants’ diversity (hymenoptera-formicidae) in the algeria’s humid forests156 in the cork oak and algerian oak stations, myrmicinae are largely in the lead (with respectively 82.83 and 81.23%) followed by formicinae with respectively 17.17 and 18.77%. in the afares oak station, the opposite is true, formicinae were the most captured (68.54%) followed by myrmicinae (18.04%) and dolichoderniae (12.69%). ponerinae are far behind with 0.73% (fig 3). among the cork oak’s ants found, the species; crematogaster scutellaris et aphaenogaster testaceopilosa are the most frequent with rates of 45.62 and 12.61% respectively followed by tetramorium forte and plagiolepis schmitzii (with 8.04 and 8.64%, respectively). the other species have frequencies ranging from 0.12 to 4.44% (table 1). in algerian oak, aphaenogaster crocea, crematogaster scutellaris and aphaenogaster testaceo-pilosa were the most captured (all methods combined) with 38.27; 28.15 and 12.46% respectively. the other species have frequencies that range from 0.29 to 7.04% (table 1). in afares oak, plagiolepis schmitzii is the most common with a rate of 59.88%. it is followed by the species and bothriomyrmex decapitans and crematogaster scutellaris (with 12.69 and 6.98%, respectively) (table 1). the species number per plot varies between 2 and 8 in cork oak, 1 and 6 in algerian oak and finally 4 and 9 in afares oak. the average richness (sm) per plot is higher in the cork oak and afares oak (respectively 5.05 and 5.7) (table 2). table 1. formicidae listed’s percentage and occurrence frequencies of at the three study stations in the guerrouche forest (algeria) (fc: percentage frequency, fo: occurrence frequency) (*endemic species). species cork oak algerian oak afares oak fc fo fc fo fc fo cataglyphis bicolor 0,36 5 cataglyphis viatica 0,12 5 0,29 10 0,18 10 camponotus spissinodis 1,92 25 camponotus cruentatus 1,32 10 *camponotus laurenti 0,36 10 0,29 5 0,59 15 camponotus lateralis 1,32 25 camponotus piceus 0,24 10 camponotus micans 2,88 55 0,88 25 2,36 60 camponotus gestroi 0,14 5 formica cunicularia 1,47 5 1,59 20 formica fusca 3,08 10 lasius niger 7,04 20 3,67 50 plagiolepis schmitzii 8,64 35 5,72 10 59,88 100 plagiolepis pallescens 0,14 10 aphaenogaster testaceopilosa 12,61 85 12,46 35 0,95 15 *aphaenogaster testaceo-pilosa ssp canescens 0,48 10 1,61 35 4,40 85 aphaenogaster gibbosa 4,32 25 *aphanogaster foreli 2,04 25 aphaenogaster depilis 0,12 5 aphaenogaster crocea 38,27 85 1,81 40 messor straticeps 0,24 10 *messor antennatus 0,12 5 crematogaster scutellaris 45,62 75 28,15 40 6,98 80 crematogaster auberti 0,12 5 crematogaster auberti laevithorax 2,88 10 crematogaster sordidula 1,80 15 pheidole pallidula 4,40 15 oxyopomyrmex emeryi 0,15 5 solenopsis occipitalis 3,54 5 tetramorium forte 8,04 15 0,44 5 0,09 10 temnothorax curtulus 0,15 10 temnothorax rottenbergii scabriosus 0,27 10 *bothriomyrmex decapitans 12,69 40 ponera coarctata 0,73 10 individuals number 833 682 2206 richness 22 14 17 sociobiology 67(2): 153-162 (june, 2020) 157 the calculated shannon-weaver diversity index indicates good specific diversity at all stations (table 2). the evenness values indicate that the species numbers in the cork and algerian oak stations tend to be in equilibrium (e = 0.64), while at the afares oak, species numbers are less balanced. to determine if the three studies are different in terms of richness, we used the analysis of variance (anova). the latter revealed significant differences between the three stations for a threshold-specific richness parameter (p < 0.05). to determine if the sampling of the three oak forests’ myrmecofauna of guerrouche forest was effective, we used chao’s formula. the latter estimates the total of cork oak’s specific richness at 26 species, which indicates that with our 80 samples (all methods combined), 84.61% of the area’ s specific richness was observed. for algerian oak, formula chao estimates the total specific richness at 15, indicating that 93.33% of the station was correctly sampled. for afares oak, the inventory can be considered complete because all species are represented by at least two individuals. the four sampling methods used allowed the capture variable-sizes workers (from 2 to 10 mm long) the respective distribution is shown in figure 4. the latter clearly shows us that the 2 mm class is the most represented in our inventory (fc=43.99%), followed by the 3, 4, 5 and 7 mm classes with frequencies respectively 14.81 ; 14.03 ; 12.98 and 8.22% . the species frequencies according to the workers size collected using the four sampling methods on guerrouche forest are shown in figure 5. pitfall mainly trapped medium sized ants (between 5 and 7mm) while the winkler has been used to catch mostly small ants (2 mm). the baits mainly caught small species (between 2 and 4mm) as well as medium-sized species of 5 to 7mm. as for manual capture, it allowed a complete species harvest the regardless of the workers’ size. stations cork oak algerian oak afares oak specific richness 22 14 17 average richness 5,05 3 5,7 shonnon-weather (h) 2,86 2,44 2,19 maximum diversity (hmax) 4,46 3,81 4,09 evenness 0,64 0,64 0,54 sorensen index cork oak algerian oak afares oak cork oak 100% 44,44% 41,02% algerian oak 100% 70,96% afares oak 100% table 3. sorensen similarity index values calculated for the two-to-one comparison of sampled environments in the guerrouche forest (algeria) table 2. specific and average richness, shannon-weaver diversity index and evenness distribution of ants recorded in three study stations in the guerrouche forest (algeria) environment sampling method species number species percentage (%) cork oak manual capture baits pitfall winkler 15 12 11 5 68,18 54,54 50,00 22,72 algerian oak manual capture baits pitfall winkler 12 5 3 3 85,71 35,71 21,42 21,42 afares oak manual capture baits pitfall winkler 15 10 4 1 88,23 58,82 23,52 5,88 table 4. total number of species and species percentage according to sampling methods in the guerrouche forest (algeria) fig 4. the species percentages’s representation according to the workers size harvested by all sampling methods used (winkler, pitfall, baits and manual capture) and in the three study stations of the guerrouche forest (algeria). according to sorensen’s index, the species communities in each environment are neither identical nor totally different. despite this, they share a fairly large common characters number. the greatest similarity was found between algerian oak and afares oak (70.96%) (table 3). the four sampling methods used allowed us to identify a significant species number, however, manual capture and bait were the most effective in this study (table 4). manual capture, baits and pitfalls harvested the most ant species in the oak; the winkler was relatively effective (22.72% ℅ of the total number of species) (table 4). regarding algerian oak, the manual capture allowed the largest species number catching (85.71 ℅ of the total number of species); followed by bait (35.71 ℅ of the total number of species). pitfalls and winkler were much less effective (21.42 for each method) (table 4). for afares oak, manual capture and bait made it possible to sample the largest species number (88.23 and 58.82 ℅ of the total species number); followed by pitfall and winkler method (table 4). a henine-maouche, a tahraoui, r moulaï – ants’ diversity (hymenoptera-formicidae) in the algeria’s humid forests158 discussion the myrmecological inventory carried out in three forests, of the guerrouche massif in algeria, allowed us to identify 34 species from 4 sub-families; formicinae, myrmicinae, dolichoderinae and ponerinae. at the cork and algerian oak stations, myrmicinae frequencies exceed 80%, followed by formicinae with frequencies ranging from 17.17 to 18.77%. while in afares oak, formicinae were the most captured (68.54%) followed by myrmicinae (18.04%) and dolichoderniae (12.69%). ponerinae are far behind with 0.73%. these last two sub-families have only been recorded in this last station. in a study on the the ant richness in the ermenonville forest in oise (france), formicinae slightly dominate myrmicinae in terms of species (16 and 14 taxa respectively). they are also the two richest subfamilies of the myrmecological fauna of these regions. the ponerinae and dolichoderinae represent only 1% for each one (colindre, 2017). among the 15 genus recorded, three genus were particularly well represented among the harvested ants: the genus camponotus (7 species), aphaenogaster (6 species) and crematogaster (4 species). commented list of harvested species the species’s classification is based on the systematic order adopted in the work of cagniant (2006) and taheri et al. (2014). for each species, its distribution in the algerian forests and its distribution in the guerrouche forest are presented, according to the three forest habitats surveyed. subfamily of dolichoderinae bothriomyrmex decapitans (santschi, 1911) was inventoried by cagniant (1968), in his preliminary list of forest ants in algeria, in the forest of guerrouche (jijel, algeria). according to borowiec (2014), this species is endemic to algeria and tunisia. in this study, it was sampled only in the afares oak. subfamily of formicinae genus plagiolepis mayr, 1861 plagiolepis schmitzii (forel, 1895) was captured in all study stations but it is better represented in afares oak because the heavy humus is very favourable to it (bernard, 1973). indeed, the largest workers numbers were found in the winkler. cagniant (1968) found that the species is extremely common when there is an onset of deforestation. it is a species common to all environments, from the coast to the saharan atlas, with a clear preference for open forests, brush and clearings (up to 2000m) (cagniant, 1970). genus lasius fabricius, 1804 lasius niger (linnaeus, 1758) is common in the open forests of pine, oak and humid areas and at all altitudes (cagniant, 1968, 1970). in this study, it was observed in algerian oak and afares oak. genus camponotus mayr, 1861 c. micans (nylander, 1856) is found throughout northern algeria, from coastal plains to mountains, up to 1200 m in the tell and 1700 m in the aurès (cagniant, 1970). he noted that this species is associated with the holm oak, kermesse and algerian oaks of the blida’s atlas, tala guilef, djurdjura and the beni-imbul forest (cagniant, 1968). c. laurenti (santschi, 1939) is a common underground species in the ouarsenis, in particular in a pine forest mixed with holm oak (900 m) and a cedar forest mixed with algerian oak (1500 m) from algiers (cagniant, 1970). fig 5. species frequencies’s representation according to the workers size harvested using the four sampling methods of the guerrouche forest (algeria). sociobiology 67(2): 153-162 (june, 2020) 159 c. micans and c. laurentiwere found in large numbers in the afares oak station and are not very abundant in the other stations. c. lateralis (olivier, 1791) is a fresh forests and mountain ant from 800 to 1900 m. the species has been observed in the hafir forest in oranie (a mixture of holm, cork and algerian oak), in algiers, in theniet-el-had forest (cedar grove mixed with algerian oak) and in blida holm oak groves and kabylia and numidia atlases (southern flank of djurdjura). but also, in a cork forest of the edough forest (between annaba and el-marsa) and at the calle (now el kala) (cagniant, 1970). c. laurenti is endemic to algeria and morocco, (borowiec, 2014). c. spissinodis (forel, 1909) is an open and clear forests species (cagniant, 1968). c. cruentatus (latreille, 1802) is found mainly in tellian atlas holm oak forests between 800 and 1200 m and along the edges of clearings (cagniant, 1968). c. gestroi (emery, 1878) is a tyrrhenian distribution species and rare one in algeria, it has been observed in algiers and kabylia in algerian oak stations. it nests are located at the oaks’s foot. c. lateralis, c. spissinodis, c. cruentatus and c. piceus were only caught in the cork oak while c. gestroi was only observed in the afares oak. it should be noted that the camponotus mentioned above were not sampled by cagniant (1968, 1970) in the guerrouche forest. genus cataglyphis foerster, 1850 cataglyphis bicolor (fabricius, 1793) is found in all open and sunny places from the seaside to the mountains tops (cagniant, 1968). it has been found in the diets of two insectivorous babors birds (henine-maouche et al., 2017; belkacem et al., 2019). in this study, the species is only included in the cork oak list. cataglyphis viatica (fabricius, 1787) is common to forest edges (cagniant, 1968). it does not appear in the preliminary list of ants in the algerian forests of cagniant in guerrouche (1968, 1970). in our study, it was sampled in all environments but with low densities. genus formica linnaeus, 1758 formica fusca (linnaeus, 1758) is an introduced ant from europe (cagniant, 1968). it is a well temperate forests species. it has been observed in akfadou (algerian oak and afares between 900 and 1300 m) (cagniant, 1970). in guerrouche, it was only captured in algerian oak. formica cunicularia (latreille, 1798) is an edges species of cedar forests. it was observed in an algerian oak in theniet-el-had forest and tala-guilef cedar forest (cagniant, 1968; 1970). in guerrouche forest, it was located in the algerian oak and afares oak. subfamily ponerinae ponera coarctata (latreille, 1802) is a species widespread throughout the mediterranean region and humid forests in good condition (cagniant, 1968). it was observed in a blida atlas holm oak forest, in yakouren ( cork, algerian and afares oak), in akfadou (algerian and afares oak) as well as a algerian of guerrouche forest and djebel el ghoufi (cagniant, 1970). in this study, the ant was only observed in the afares oak. subfamily myrmicinae solenopsis occipitalis (santschi, 1911) is common in cedar glades or pastures pseudo-alpine in djurdjura and aurès (cagniant, 1966). the latter did not record this ant at guerrouche. the species is fond of wetlands (bernard, 1968). in this study, it was collected at the afares oak. genus tetramorium mayr, 1855 tetramorium forte (forel, 1904) was sampled at all environments with greater abundance at the cork oak. genus aphaenogaster mayr, 1853 a. gibbosa (latreille, 1798) is a forest species in algeria (bernard, 1968) and is common to open forests near wadis (cagniant, 1968). a. foreli (cagniant, 1996) is an algerian-moroccan species of the middle and high atlas, between 1300 and 1800 m (cagniant, 1996; borowiec, 2014). it prefers the lawns, pastures, clearings, coppices and maquis of the holm oak floor. in this study, it appeared only in cork oak. a. depilis (santschi, 1911) is a species of high mountain on shallow ground. it was seen in a cedar grove in the blida atlas and telmet pass (belezma and aurès), in tikjda and jebel chelia as well as in a djurdjura pseudo alpine pasture (cagniant, 1968). a. gibbosa, a. depilis and a. foreli have only been found in guerrouche cork oak. a. foreli is endemic to algeria and morocco (cagniant, 1996; borowiec, 2014). a. testaceo-pilosa (lucas, 1849) was collected from all study stations but its number is declining in the algerianoak and afares oak. it has been identified in the western babors by henine-maouche et al. (2017) and belkacem et al. (2019). according to cagniant (1968), it is very common in algiers. a. testaceo-pilosa ssp canescens (lucas, 1849) is a kabylia and numidia subspecies on humus soil (cagniant, 1968). these taxa are limited to algeria while it is also found in europe (borowiec, 2014). in guerrouche, it was sampled in all stations with a higher abundance in the afares oak. a. crocea (andré, 1881) is commonplace in numidia and aurès but more uncommon elsewhere (cagniant, 1966). this autor found it in the cork, algerian oak and afares oak formations of kabylia and numidia as well as in the forests a henine-maouche, a tahraoui, r moulaï – ants’ diversity (hymenoptera-formicidae) in the algeria’s humid forests160 of mizrana, yakouren, akfadou and guerrouche, on the jebel babor and el ghoufi or even the melab pass (cagniant, 1968). genus messor forel, 1890 m. striaticeps (andré, 1883) is a mountain species. in algeria, cagniant (1968, 1970) observed it only in djurdjura pseudo-alpine pasture. m. antennatus (emery, 1908) is endemic to algeria and morocco (borowiec, 2014). it does not appear in the cagniant’s preliminary list of ants in algerian forests (1968 and 1970). in a study on the young picus vaillantii diet in western babors, henine-maouche et al. (2017) found this species in the young woodpeckers faecal bags. our investigations in guerrouche, allowed us to capture only one individual at the cork oak. genus oxyopomyrmex andré, 1881 oxyopomyrmex emeryi (santschi, 1908) is a new species in algeria (cagniant, 1968), it was observed in ghardaia. it is steppe and not forest (delye, 1971). according to bernard (1973), this species is rarely forested. in this study, only one individual was captured at the algerian oak. genus pheidole westwood, 1839 pheidole pallidula (nylander, 1849) is a common species, found everywhere (cagniant, 1968). in this study, it was only captured in the cork oak. genus crematogaster lund, 1831 c. scutellaris (olivier, 1791) is present in all study stations of guerrouche. this species is common especially in cork oak, it excavates its nests and galleries in the bark of trees. the latter are recognizable by the entry holes dug by these ants. c. scutellaris is a major factor in the deterioration of cork (bernard, 1968; mouro, 2011). it was identified by henine-maouche et al. (2017) and belkacem et al. (2019) in the algeria’s babors region. in this study, it was observed at all stations with a high presence in the suberaie. c. sordidula (nylander, 1848) prefers forests in lowlevel degradation (cagniant, 1968). c. auberti (emery, 1869) occurs in coastal forests and is less common in the interior (cagniant, 1968). the low density of the latter two species in the cork oak is probably due to the study station relief. indeed, the slopes are not very populated by these two species (bernard, 1973). c. auberti laevithorax (forel, 1902) is an ant from southwest algeria (cagniant, 2005). it was also sampled in the study of henine-maouche et al. (2017) at an altitude of 372 m. c. sordidula, c. auberti and c. aubertilae vithorax were only sampled in cork oak. genus temnothorax mayer, 1861 they are good ecological and biocenotic indicators, are sensitive to environmental disturbances and disappear when deforestation, erosion and overgrazing occur, facilitating the intrusion of anthropophilic ants (cagniant & espadaler, 1997). in guerrouche cork oak, the temnothorax are absent. temnothorax curtulus has only been recorded in the algerian oak and temnothorax rottenbergii scabriosus in the afares oak. in specific richness terms, the guerrouche cork oak formation is richer with 22 species compared to 17 species for afares oak and 14 species for algerian oak. in the cork oak forest mâamora (morocco), 43 species have been recorded (bernard, 1945). in talassemtane national park (morocco), cork oak shelters 25 species of formicidae (taheri et al. 2014). in ermenonville forest (oise-france), the ants species number inventoried is 32 (colindre, 2017). the shannon-weaver diversity index calculated for the all study stations, gave values of 2.86 bits for the cork oak, 2.44 bits for algerianoak and 2.19 bits for the afares oak. this indicates a good specific diversity of all stations. the evenness values for two stations (cork and algerian oak) are equivalent (e=0.64), it tells us that this stations’s individuals are tending to be balanced, the evennessin the afares oak (e=0.54) indicates that the individuals are fairly balanced. in the in talassemtane national park’s cork oak (pntls) at rif (morroco), the diversity index shows that this environment is quite diversified (1.49 bits). individuals of various ant species are far from equilibrium (e = 0.46) (taheri et al. 2014). despite their abundance and ease to collect in most ecosystems, several ants’ biology characteristics complicate their sampling (crist & wiens, 1996; wiernasz & cole, 1995). according to chao formula results; for an equal sampling effort and with identical sampling techniques, each environment appears to have been sampled overall with the same efficiency. in this study, manual capture and baiting were the most effective, while at the causses oak grove, it is the winkler extraction then the baits that allowed the the largest species number capture (81.8 and 63.6 ℅ of the species total number); pitfalls and manual capture were considerably less effective (45.5 and 40.9% of the total number of species) (groc, 2006). the use of several sampling methods simultaneously is the best way to assess biodiversity. all methods have their strengths and weaknesses and only a several combination of them will provide a representative sample for most research objectives (marshall et al. 1996). frequency distributions of workers size suggest that each sampling method traps preferentially some species category of given size ants and that the hypothetical completeness of sampling could only be achieved through a different collection methods combination (groc, 2006). this list does not claim to represent all the the guerrouche national forest myrmecofauna because our research was focused exclusively on oak forests. nevertheless, it results from this study that the inventoried environments have a good myrmecological diversity and that the main factors determining the formicidae’s distribution are altitude and the habitat’s type frequented. sociobiology 67(2): 153-162 (june, 2020) 161 conclusion our investigations revealed 34 species and subspecies. 32 species do not appear in the cagniant inventory (1968 and 1970) carried out in guerrouche. aphaenogaster testaceo-pilosa ssp canescens is an endemic species in algeria. three species are algeria and morocco’s endemic (camponotus laurenti, messor antennatus and aphaenogaster foreli). one species is endemic to algeria and tunisia (bothriomyrmex decapitans). some species have not reappeared in the scientific literature since their description especially in the genus: messor, bothriomyrmex, crematogaster and solenopsis. in the interest of a better knowledge of the forest ants’current status in algeria, it would be useful to extend investigations to other algerian wet forests, especially those found at high altitudes. acknowledgements we thank professor henri cagniant for his help with his many recommendations but also for his valuable contribution in some ants’ determination and identification. we also thank professor xavier espadaler for all the documentation he provided us. references a.s.a. 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(eds.), ants: standard methods for measuring and monitoring biodiversity. smithsonian press, washington. groc s., (2006). diversité de la myrmécofaune des causses aveyronnais-comparaison de différentes méthodes d’échantillonnage. mémoire de desups. univ. paul sabatier. 38p. henine-maouche a., bougaham a.f., moulaï r. & nicolauguillaumet p., (2017). première données sur le régime alimentaire des jeunes pics de levaillant picus vaillantii. alauda, 85: 152-154. marshall s.a., anderson r.s., roughley r.e., behan-pelletier v. & danks h.v., (1994). terrestrial arthropod biodiversity: planning a study and recommended sampling techniques. a brief. bulletin of the entomological society of canada, 26(1) supplement. 33 p. mouro c., (2001). inventaire de l’entomofaune du chêne liège dans la forêt domaniale de m’sila (wilaya d’oran). mémo. ing. univ. tlemcen. 82 p. louni d., (1994). les forêts algériennes. forêts méditerranéenne. vol. xv: 59-63. ramade f., (1984). eléments d’écologie – ecologie fondamentale. ed. mc graw-hill, paris. taheri a., reyes-lopez j., and bennas n., (2014). contribution à l’étude de la faune myrmécologique du parc national de talassemtane (nord du maroc): biodiversité, biogéographie et espèces indicatrices. boletin de la sociedad entomologica aragonesa (s.e.a), 54: 225-236. wiernasz d.c., & cole b.j., (1995). spatial distribution of pogonomyrmex occidentalis: recruitment, mortality and overdispersion. the journal of animal ecology, 64: 519-527. ziane b., 1979. etude phénologique de quercus suber l. et de quercus faginea l., dans la forêt de guerrouch (w. de jijel).thèse d'ingénieur agronome, i.n.a. algérie, 27 p. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5451sociobiology 68(1): e-5451 (march, 2021) introduction honeybees visiting flowers is such a common picture that it is hard to remember it is just a part of the pollination story. first, not all pollinators are honeybees. pollination stands as one of the most important classes of animal–plant interaction, as ~85% of angiosperms are pollinated by animals (ollerton et al., 2011). such interactions were key drivers of the evolutionary radiation of both angiosperms and insects across the world (crepet et al., 1991; grimaldi, 1999). within hymenoptera, one of the largest insect orders, honeybees and other bee species are frequently cited as major pollinators (cardinal et al., 2012; kevan & baker, 1983; potts et al., 2010), but other insects such as wasps are also important pollinators (heithaus, 1979; freitas & sazima, 2006; sühs et al., 2009; abstract basic research assessing environmental effects on entire pollinator communities are still uncommon, particularly for rare and commercially unattractive plantpollinator partners. we investigated the community of flower visitors of erythroxylum myrsinites to check for potential pollinators and to check the extent of weather influence of visitor behaviour, registered as the number of visitors attending flowers of e. myrsinites. we then calculated species’ dominance and constancy and assessed location of pollen attachment in each visitor’s body. we correlated weather variables with the composition and abundance of visits carried out by the entire community and by most constant and dominant species. the wasps polybia sericea, p. ignobilis and p. fastidiosuscula showed the highest values of constancy, dominance and attached pollen. there was a community-level effect of atmospheric pressure, solar radiation and wind speed on the number of visits. atmospheric pressure affected the number of visits of eudominant species p. fastidiosuscula and p. sericea, while solar radiation affected the number of visits of p. ignobilis. our results demonstrate the influence of weather variables on flower visiting insects and suggest the importance of native wasps in pollen transport and potential pollination. sociobiology an international journal on social insects rafael barbizan sühs1, alexandre somavilla2, eduardo luís hettwer giehl1 article history edited by solange augusto, ufu, brazil received 26 may 2020 initial acceptance 15 december 2020 final acceptance 29 december 2020 publication date 22 february 2021 keywords community ecology, insect-community, insect-plant interaction, polybia, wasps. corresponding author rafael barbizan sühs https://orcid.org/0000-0001-5921-7181 laboratory of human ecology and ethnobotany, universidade federal de santa catarina, campus florianópolis av. prof. henrique da silva fontes, 2754 trindade, santa catarina, brasil. e-mail: rbsuhs@gmail.com somavilla et al., 2010; rader et al., 2016). second, not all insects visiting flowers are pollinators. among other resources, flower visitors seek for pollen or nectar (faegri & van der pijl, 1971; proctor et al., 1996) and are only pollinators if visits are frequent and pollen is regularly transferred to another flower (stigma) of the same species (rech et al., 2014) or within the same individual. last, the study of unusual plant-pollinator systems and factors driving pollination success may still uncover gaps in the “story of pollination”. pollination success can be driven by factors such as flower and pollinator morphology and plant phenology or pollinator behaviour. over short temporal and spatial scales, successful reproduction of plant pollinated species can result from the activity and identity of pollinators, whereas the behaviour and activity of pollinators can be constrained by 1 universidade federal de santa catarina (ufsc), florianopolis, santa catarina, brazil 2 national institute for amazonian research (inpa), manaus, amazonas, brazil research article wasps weather variables affecting the behaviour of insect flower visitors and main pollinators of erythroxylum myrsinites martius (erythroxylaceae) rb sühs, a somavilla, elh giehl – drivers of visits to e. myrsinites flowers2 abiotic factors (case & barrett, 2004). air temperature and humidity, atmospheric pressure, solar radiation and wind intensity may influence insect visits, insect flight activity or even whether the insects leave their nests at all (abrol, 1988; burrill & dietz, 1981; fijen & kleijn, 2017; kearns & inouye, 1993; lundie, 1925; nielsen et al., 2017). however, studies usually focus on the effect of weather variables either on few or a single species (e.g. honeybees: burrill and dietz, 1981; lundie, 1925). this leaves a gap in the understanding of how weather affects the behaviour of entire pollinator communities (fijen & kleijn, 2017), particularly for rare and economically unattractive plant-pollinator partners. erythroxylum myrsinites mart. (erythroxylaceae) is a shrub native to south america, occurring in argentina, brazil, paraguay and uruguay (amaral jr., 1980). in brazil, e. myrsinites is found in southern and south-eastern regions, in the atlantic forest vegetation domain (loiola & costalima, 2015) and is considered endangered in parts of its range (são paulo 2016). even though pollination of e. myrsinites is still underexplored, bees, wasps, and flies are known to be the main flower visitors and pollinators of several species of erythroxylum (barros, 1998; freitas & sazima, 2006; rosas & domínguez, 2009). here we gathered information on the flower visitors of e. myrsinites and assessed what drives the activity and number of visitors on its flowers. by assuming pollination systems are normally evolutionary conserved (macior 1982; johnson et al. 1998; but see ollerton 1996) and based on previous studies for erythroxylum spp. (barros, 1998; da silva et al., 2007; freitas & sazima, 2006), we hypothesized that bees, wasps, and flies are the main flower visitors of e. myrsinites. regarding the drivers of visitors’ activity, we expected air temperature and relative humidity, atmospheric pressure, solar radiation and wind intensity (e.g. nielsen et al. 2017) to affect the community of visitors by influencing the number of visiting insects. by checking these hypotheses, we tried both to improve basic knowledge of a poorly known plant species and its interacting insect community and to apply communitylevel modelling techniques to assess environmental drivers of the behaviour of flower visitors. materials and methods study system the study was conducted in municipality of santa cruz do sul (29º41’s – 52º26’w), southern brazil. vegetation type is seasonal deciduous forest, being inside the atlantic forest domains. following koeppen’s classification (köppen, 1931), the regional climate is cfa (subtropical humid). erythroxylum myrsinites individuals were found growing in the edge of an isolated forest remnant of ca. 9 ha at 45 m above sea level. study plant erythroxylum myrsinites is a light-demanding shrub and grows on moist soils, reaching up to 3 m in height (amaral jr., 1980). flowers are solitary, small, light cream coloured and slightly perfumed (amaral jr., 1980). it is a distylous plant with brevistylous and longistylous flowers, very similar in size, produced from august to april (amaral jr., 1980). this kind of floral dimorphism, in which anthers and stigmas of the two floral morphs are in different positions, may promote pollen flux between the two flower types and increase cross-pollination (barros, 1998). the fruit is a small drupe (ca. 7 mm length and 3.5 mm wide), red, monospermic and probably bird-dispersed. fruiting spans from september to may (amaral jr., 1980). sampling methods and data collection flower visitors of seven individuals of e. myrsinites were collected during their complete flowering period between september and october 2008 (a different flowering period reported in literature). visitors were collected with entomological nets, between 9h00 and 17h00. this temporal sampling extent was determined after preliminary observations where the period with the highest visitor activity was identified. two additional observations were done at night to check if there were any nocturnal visitors, and no flower visitors were observed. each sampling event lasted 30 min. total time of observations and collections spanned over 32 h and was distributed along 16 days. insects were deposited in individual vials and stored in the entomological collection of university of santa cruz do sul (cesc). subsequently, all collected individuals were examined under a stereoscopic microscope (at 40x) for identification and to verify if there was pollen attached to their integument and in which body part pollen grains were located (corbicula, face, frontal legs, mesosome, metasome, propleuron and scopa). from a fraction of these individuals, pollen was removed and visualised in optical microscope (at 1000x) to confirm if pollen grains belonged to e. myrsinites (same procedure than in sühs et al. 2009). weather data (air temperature and relative humidity, solar radiation, wind intensity and atmospheric pressure) were collected by a weather station (davis®, model vantage pro plus), located ca. 500 m away from the study site. data was collected every 30 min and were provided by university of santa cruz do sul (unisc). data analysis to verify the first hypothesis of this study, we recorded the identity, dominance, and constancy of flower visitors. as a complementary information regarding flower visitor behaviour, we also verified the number of individuals with pollen attached and the location of pollen on visiting species. dominance (d) was calculated as: d = (i / t) × 100, where i = total number of individuals of i species; t = total number of collected individuals. dominance was used to classify species in the following scheme: d > 10% = eudominant; d > 5 ≤ 10% = dominant; d > 2 ≤ 5% = subdominant; d > 1 ≤ 2% = recessive; d ≤ 1% = rare (sühs et al., 2009). constancy (c) sociobiology 68(1): e-5451 (march, 2021) 3 was calculated as: c = (p / n) × 100, where p = number of collections (number of records containing the species) and n = total number of records (thomazini & thomazini, 2002). when c ≤ 25% the species was considered accidental, when c > 25 ≤ 50%, accessory; and when c > 50%, constant. we then listed species as potential pollinators when they showed high values of dominance and constancy, and when they showed e. myrsinites pollen on their bodies. we used generalized linear models for multivariate responses (glmmv) with negative binomial errors to test the effects of weather predictors (air temperature, atmospheric pressure, relative humidity, solar radiation and wind speed) on community composition and on species individual responses. following preliminary exploratory analyses, we decided to include also quadratic terms for all weather variables. correlation amongst explanatory variables was previously checked using variance inflation factor (vif) and redundant variables removed when vif > 4, so that all kept variables were uncorrelated. all weather predictor variables were standardized before the analysis to avoid collinearity. p-values were calculated through 999 pit-trap resamplings. even though we applied a high number of tests on univariate responses (each species against predictors), we reported unadjusted p-values, but interpret the results with caution. to reduce the risk of type-i errors, we inform and discuss only responses of most abundant species (eudominant). initial model contained all predictors, including quadratic terms. model simplification was performed through backward elimination. model selection was based on the sum of computed akaike information criterion (aic by selecting models with lowest sum-of-aic values) and validation was determined by graphical analysis of residuals. analyses were performed on the r interface (r development core team 3.0.1., 2013) using package mvabund (wang et al., 2013) for model building. results erythroxylum myrsinites flowering period (since the opening of buds) lasted 19 days. the number of eudominant and subdominant visitors followed a bimodal pattern, peaking from 11h00 to 12h00 and from 13h00 to 14h00 and being low from 9h00 to 10h00, 12h00 to 13h00, and 16h00 to17h00 (fig 1). we collected 376 visiting insects, 367 belonging to hymenoptera and nine to diptera. nineteen species were recorded, 16 belonged to hymenoptera and three to diptera. within hymenoptera, ten species belonged to vespidae, four to halictidae and two to apidae. within diptera, syrphidae had two species and stratiomyidae had one species (table 1). three visiting species were classified as eudominant and constant, all three belonging to the genus polybia (vespidae). these three species were: p. sericea (number of collected individuals, n = 182; constancy, c = 87.5%); p. ignobilis (n = 57; c = 57.8%); and, p. fastidiosuscula (n = 56; c = 56.3%). four species were classified as subdominant and accessories: apis mellifera (n = 16, c = 21.9%), mischocyttarus rotundicollis (n = 14, c = 18.8%), augochloropsis sp. (n = 13, c = 15.6%) and brachygastra lecheguana (n = 9, c = 14.1%). there were no species classified in the dominant and accidental classes. the remaining 12 species were classified as either recessive or rare regarding dominance (table 1). fig 1. number of individuals (ni) of eudominant and subdominant species collected per 0.5h periods over time of sampled days in erythroxylum myrsinites’ flowers, southern brazil. rb sühs, a somavilla, elh giehl – drivers of visits to e. myrsinites flowers4 taxon n do (%) do status co (%) co status pollen location o.m. (%) c/s fa fl ms mt pp hymenoptera vespidae agelaia multipicta (haliday, 1836) 3 0.8 rr 4.7 ac na 0 0 0 0 0 0 brachygastra lecheguana (latreille, 1824) 9 2.4 sd 14.1 ac na 2 2 0 0 0 0 mischocyttarus rotundicollis (cameron, 1912) 14 3.7 sd 18.8 ac na 3 4 1 0 2 0 pachodynerus guadulpensis (saussure, 1853) 2 0.5 rr 3.1 ac na 0 0 1 0 1 0 polistes cavapyta saussure, 1853 1 0.3 rr 1.6 ac na 0 0 0 0 0 0 polybia fastidiosuscula saussure, 1854 56 14.9 ed 56.3 co na 4 3 9 0 10 0 polybia ignobilis (haliday, 1836) 57 15.2 ed 57.8 co na 23 32 35 0 22 26.3 polybia platycephala richards, 1951 1 0.3 rr 1.6 ac na 0 0 0 0 0 0 polybia scutellaris (white, 1841) 1 0.3 rr 1.6 ac na 0 0 0 0 0 0 polybia sericea (olivier, 1792) 182 48.4 ed 87.5 co na 66 144 153 0 110 62.1 apidae apis mellifera l. 1759 16 4.3 sd 21.9 ac 10 6 5 0 1 5 62.5 plebeia sp. 6 1.6 rc 9.4 ac 2 0 0 0 1 0 33.3 halictidae augochlorella urania (smith, 1853) 1 0.3 rr 1.6 ac 0 0 0 0 0 0 0 augochloropsis sp. 13 3.2 sd 15.6 ac 2 1 3 3 1 1 33.3 dialictus sp. 2 0.5 rr 3.1 ac 1 0 1 0 0 0 50 neocorynura aenigma (gribodo, 1894) 3 0.8 rr 4.7 ac 1 0 0 0 1 0 66.6 diptera stratiomyidae chorisops sp. 7 1.9 rc 7.8 ac na 0 0 0 0 0 0 syrphidae taxomerus sp.1 1 0.3 rr 1.6 ac na 0 0 0 0 0 0 taxomerus sp.2 1 0.3 rr 1.6 ac na 0 0 0 0 0 0 table 1. recorded species in erythroxylum myrsinites’ flowers, southern brazil. n = number of individuals, do = dominance, do status = dominance status (ed = eudominant, sd = subdominant, rc = recessive and rr = rare). co = constancy, co status = constancy status (co = constant and ac = accessory). pollen location (number of records): c/s (corbicula or scopa); fa (face); fl (frontal legs); ms (mesosome/thorax); mt (metasoma/abdomen); pp (propleuron). o.m. (individuals with pollen analyzed in optical microscope). na (structure not available in taxon). we observed that 70% of the visiting insects had pollen attached to their integument. we analysed 229 wasps (vespidae) in detail (totalling 60.9% of all collected wasps) and the most common body structures showing attached pollen were mesosome (62% of inspected individuals), forelegs (56.7%), propleuron (44.5%) and face (29.4%) (table 1). of the collected wasps, 26.6% had no pollen attached. the highest number of records of pollen in body structures was recorded for polybia sericea, followed by p. ignobilis (fig 2). for bees (apidae and halictidae), 24 individuals (totalling 58.5% of all collected bees) had pollen attached to their bodies. we found pollen of e. myrsinites attached to all visiting insects inspected in optical microscope (n = 147). out of those insects, 77% belonged to the wasp species p. sericea (fig 3) and 10.1% to p. ignobilis (vespidae), and 12.9% belonged to bees (apidae and halictidae). at the community level, the number of visiting insects correlated with atmospheric pressure (secondorder parameter: lr = 42.5, p = 0.001), solar radiation (first-order: lr = 37.9, p = 0.001) and wind speed (firstorder: lr = 35.5, p = 0.001; all results from the selected most parsimonious model described in detail in table 2). graphical analysis showed that residuals of the model were adequate. more information on model selection can be found in supplementary file (table s1). sociobiology 68(1): e-5451 (march, 2021) 5 fig 2. number of records (nr) of pollen in body structures in individuals of eudominant and subdominant insect species in erythroxylum myrsinites’ flowers, southern brazil. fig 3. images of polybia sericea visiting erythroxylum myrsinites flowers, southern brazil. the two images (a and b) show the visiting pattern of the wasp as well as pollen grains attached to its body. at the species level, weather variables had an overall effect on the eudominant species polybia fastidiosuscula (lr = 8.21, p = 0.032) and polybia sericea (lr = 8.09, p = 0.039), both affected by atmospheric pressure (second order). p. gnobolis, although affected by solar radiation (lr = 3.57, p = 0.041), was not affected by the set of weather variables (lr = 5.15, p = 0.125; table 3). wind speed did not affect significantly any eudominant visitor species. graphical information about weather variables over the collection period is shown in figure 4. table 2. selected most parsimonious generalized linear model for multivariate response data (glmmv) built for abundance of visitors to erythroxylum myrsinites flowers, southern brazil. lr = likelihood ratio. significant p-values are in bold. variable lr p-value model aic model test statistics model p-value intercept 182.18 0.001 1141.2 101.5 0.0009 atmospheric pressure² 42.54 0.001 solar radiation 37.9 0.001 wind speed 35.46 0.001 rb sühs, a somavilla, elh giehl – drivers of visits to e. myrsinites flowers6 model parameters polybia fastidiosuscula polybia ignobilis polybia sericea lr p-value lr p-value lr p-value intercept 1.34 0.235 0 0.995 59.06 0.001 atmospheric pressure² 7.189 0.008 1.543 0.206 4.015 0.049 solar radiation 1.242 0.268 3.568 0.041 3.487 0.080 wind speed 2.747 0.097 1.331 0.233 0.858 0.368 univariate test statistics 8.211 0.032 5.152 0.125 8.086 0.039 table 3. univariate estimates of deviance and associated p-values of modelled variables for eudominant species recorded on erythroxylum myrsinites flowers, southern brazil. lr = likelihood ratio. significant p-values are in bold. fig 4. variation of weather variables during the flowering period of erythroxylum myrsinites, southern brazil. lines represent daily averages and shaded areas the standard deviations. discussion we found wasps are the most important flower visitors of e. myrsinites. the wasp polybia sericea (vespidae) was the most abundant species with high levels of constancy and pollen of the studied plant species attached to their integument, and thus can be considered the visitor with the highest potential to pollinate e. myrsinites. other two species of wasps of the sociobiology 68(1): e-5451 (march, 2021) 7 same genus (p. ignobilis and p. fastidiosuscula) also had high abundance and constancy. these eudominant species had their visiting activity associated with atmospheric pressure, solar radiation, and wind speed. the genus erythroxylum has a variety of potential pollinators, especially flies, bees and wasps. we recorded all those groups visiting flowers of e. myrsinites, a pattern also reported by da silva et al. (2007), for e. cf. macrophyllum. bees and wasps were considered the main flower visitors of e. campestre st. hil., e. suberosum st. hil. and e. tortuosum mart. (barros, 1998). wasps and flies were recognized as the main pollinators of e. microphyllum (freitas & sazima, 2006) and bees were considered the main pollinators for e. havanense (rosas & domínguez, 2009). in addition, our results suggest the wasp polybia sericea potentially pollinates e. myrsinites, an additional instance to the intrinsic relationship between other species of erythroxylum and wasps (barros, 1998). wind intensity, air temperature and solar radiation are important weather variables influencing insect visits and may directly affect the community of flower visitors (abrol, 1988; kearns & inouye, 1993; kjøhl et al., 2011; nielsen et al., 2017). our results showed that atmospheric pressure, solar radiation, and wind speed affected the number of visits of the entire community of insects found on flowers of e. myrsinites. except for wind speed, the same set of variables (either together or alone) affected eudominant species and, thus, can be important to regulate success of e. myrsinites pollination. moreover, models were of quadratic form, suggesting the existence of optimal values (peaks) in the number of visits. air temperature did not affect the community of visitors, which may be due to its low variation along the studied period. although weather conditions and abiotic stress are important factors in determining the success of insect visits (case & barrett, 2004; kearns & inouye, 1993), few studies directly evaluated their effects on the community level. our results showed that 87% of collected individuals were wasps (vespidae) while 11% were bees (apidae and halictidae). the same pattern was found for the species schinus terebinthifolius raddi, in the same forest fragment (sühs et al., 2009). visitation patterns found in both studies might be linked to local microclimatic conditions and visitor availability instead of a visitor preference for the flowers. however, the abundance and the seasonal pattern of common visitor species differ. in this study (during the spring), polybia sericea was the most abundant species, with 55% of vespidae individuals, followed by p. ignobilis and p. fastidiosuscula, both with 17% of collected vespidae individuals. conversely, these species individually accounted for less than 15% of collected vespidae individuals and had great part of individuals collected in winter (sühs et al., 2009). although these studies were carried out in different years, it seems that there is a preference of distinct wasp species for flowers of either e. myrsinites or s. terebinthifolius. there was a high production of fruits in all sampled individuals after the study, indicating that pollination event occurred. although not tested, based on our results, we can infer that pollination probably was mainly crossed and done by insects because floral dimorphism in erythroxylum species – considered a mechanism of self-incompatibility (pailler et al., 1998) – promotes the pollen flux between the two types of flowers, enhancing cross-pollination (barros, 1998), and a previously reported rejection of pollen tubes resulting from self-pollination and interbreeding for several species of erythroxylum (barros, 1998; ganders, 1979). nevertheless, we encourage future studies in evaluating the effectivity of different groups of flower visitors as pollinators, which besides depending on number and constancy of visits, also depends on the quantity of pollen grain still available for the pollination of the flowers transported on the body (e.g. garibaldi et al., 2013). although carried out over a short time, this study provides novel data on flower visitors and possible pollinators of a little known and threatened plant species – at least in part of its range. wasps were the main flower visitors, pollen carriers and potential pollinators of erythroxylum myrsinites. the wasp polybia sericea was considered the main potential pollinator of the species because of high values of dominance and constancy, and a high number of individuals with pollen attached to several of its body parts. on the community-level and for eudominant species, we found weather variables such as air humidity, atmospheric pressure, wind speed and solar radiation to affect the community of visitors. this study reinforces the role of wasps in pollen transport and potential pollination. acknowledgements we thank david warton for the suggestions with statistical analyses and the anonymous referees for their contributions. the authors declare no conflict of interest. author’s contributions rb sühs, elh giehl, a somavilla – conceptualization rb sühs, elh giehl, a somavilla – methodology rb sühs, a somavilla – formal analysis rb sühs, a somavilla – investigation rb sühs, a somavilla – data curation rb sühs, a somavilla – visualization rb sühs, elh giehl, a somavilla – writing and revising supplementary material doi: 10.13102/sociobiology.v68i1.5451.s3401 http://periodicos.uefs.br/index.php/sociobiology/rt/suppfiles/5451/0 references abrol, d.p. 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(2013). package “mvabund”: statistical methods for analysing multivariate abundance data. methods in ecology and evolution, 3: 471-474. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.181-186sociobiology 62(2): 181-186 (june, 2015) pheidole symbiotica wasmann, 1909, an enigmatic supposed social parasite, is a nematodeinfested form of pheidole pallidula (nylander, 1849) (hymenoptera: formicidae: myrmicinae) introduction pheidole symbiotica wasmann, 1909 is one of the most enigmatic european ants. it was described from material collected by p.w. deckelmeyer in a pheidole pallidula (nylander, 1849) nest in barrô [portugal, águeda county, 40° 32’ 11” n 8° 28’ 12” e]. in original description wasmann (1909) noted only morphology of a specimen (no information about the number of examined specimens), presented sketch figures of dorsal view and head and discussed biological status of the species. he suggested that the described specimen may be a worker or ergatoid gyne. in subsequent supplementary paper (wasmann, 1910) he provided information about four ergatoid gynes (two of them not fully sclerotized), one pupa with ergatoid gyne and six pupae with males collected by deckelmayer. the material contained also a specimens of host species including one major worker and 19 minor workers. unfortunately, present location of the type material is unknown. in supplementary description the putative male of abstract pheidole symbiotica wasmann, 1909 hitherto known only from type series collected in portugal is recorded from italy for the first time. dissection of abdomen of this supposed social parasite of pheidole pallidula (nylander, 1849) showed that it is infested by a large mermithid nematode. that allows us to conclude that pheidole symbiotica is only a teratological form of pheidole pallidula. in consequence, we propose synonymy of pheidole symbiotica wasmann, 1909 under pheidole pallidula (nylander, 1849). high quality photographs of this form are presented for the first time and an illustration of its parasite is also given. sociobiology an international journal on social insects l borowiec, s salata article history edited by rodrigo m. feitosa, ufpr, brazil received 27 november 2014 initial acceptance 31 january 2015 final acceptance 11 march 2015 keywords hymenoptera, formicinae, pheidole, myrmicinae, social parasite, italy, redescription. corresponding author lech borowiec department of biodiversity and evolutionary taxonomy university of wrocław przybyszewskiego 63/77, 51-148 wrocław, poland e-mail: cassidae@biol.uni.wroc.pl p. symbiotica is distinguished from male of p. pallidula only by the structure of antennae (basal segment slightly wider than subsequent segments and last segment twice longer than penultimate segment at p. symbiotica whereas male of p. pallidula has globular basal segment, twice wider than subsequent segments and last segment only 1.5 times as long as penultimate segment). according to these differences in male's body structure wasmann (1910) definitely stated that p. symbiotica represents a distinct species, a social parasite of p. pallidula. in 1913 forel published a note about a nest of p. pallidula (found by m. sahlberg on the corfu, greece) in which apart from the typical major and minor workers occurred a specimen of intermediate size. he suggested that it represents p. symbiotica and concluded that this form is only a simple intermediate worker of p. pallidula. however, there is no certainty that the specimen studied by forel (1913) represents the form described by wasmann (1909) as a p. symbiotica. emery (1915), in list of synonyms of p. pallidula, noted p. symbiotica as “var. ? symbiotica wasm.” with a short research article ants university of wrocław, wrocław, poland l borowiec, s salata – pheidole symbiotica, an enigmatic supposed social parasite 182 comment “an sp. distinguenda?”. collingwood (1978) in the checklist of iberian ants noted p. symbiotica as a good species but missed this taxon in the key to iberian species. in recent catalogues (bolton, 1995; bolton et al., 2006; borowiec, 2014) p. symbiotica is treated as a good species. although pheidole pallidula, a supposed host of p. symbiotica, is widely distributed and common species in the mediterranean area (borowiec, 2014) it is surprising that the form described by wasmann (1909) under the name p. symbiotica has never been found again. p. pallidula is a pioneering species, one of the first to inhabit areas transformed by man, such as roadsides of new roads, mines, gravels and excavations of sand, but also it is common on sun-exposed rock walls, in grasslands and meadows. during our entomological travels to several mediterranean countries we found several hundred nests of p. pallidula but we have never collected specimens distinguished from typical major and minor workers. such rarity of this enigmatic taxon suggested that perhaps pheidole symbiotica is not a good species, but only a form of p. palidula, teratological due to parasitic infections. some recent papers suggested that various ant parasites can affect the morphological changes of all castes. trabalon et al. (2000) described modification of morphological characters of leptothorax nylanderi (förster) induced by cestodes. csösz (2012), using noninvasive x-ray microtomography, proved that two myrmica species: m. myrmecophila wasmann, 1910 and m. symbiotica (menozzi, 1925) are teratological forms of myrmica sulcinodis nylander, 1846 and myrmica scabrinodis nylander, 1846 developing as a result of infection by mermithid nematodes. poinar (2012) gives an overview of examples of parasitic nematoda infecting ants and affecting the morphology of workers and queens. the paper presents a photo of the nest of pheidole pallidula with two atypical major workers infected by nematoda. unfortunately, lack of access to type material of p. symbiotica precluded verification of the hypothesis that this taxon is a teratological form of supposed host p. pallidula. unexpectedly, during senior author’s trip to southern italy (campania and calabria, august-september 2014) nine specimens with characters described for p. symbiotica were collected in two nests of p. pallidula. we dissected abdomen of two specimens and discovered that they are infested with large mermithid nematodes filling the entire abdomen (fig 6). below we present a detailed redescription of “symbiotica” form with high-quality photographs and propose synonymization of pheidole symbiotica wasmann with pheidole pallidula (nylander). materials and methods ants were collected in italy, campania, salerno prov., cilento national park, monte bulgheria n. poderia. description, measurements and photos were made under nikon smz 1500 stereomicroscope, nikon d5200 photo camera and helicon focus software. examined specimens are deposited in the collection of senior author. standard measurements and indices are as defined in bolton (1975): measurements hl – head length; measured in straight line from midpoint of anterior clypeal margin to mid-point of occipital margin; in full face view; hw – head width; width of head at anterior margin of eyes in full-face view; el – eye length; measured along the maximum diameter of eye; ew – eye width; measured along the maximum width of eye perpendicular to el; sh – scape width; measured in the widest point of the scape; sl – scape length; maximum straight-line length of scape; pnw – pronotum width; maximum width of pronotum in dorsal view; ml – mesosoma length; measured as diagonal length from the anterior end of the neck shield to the posterior margin of the propodeal lobe; ph – petiole height; maximum height of petiole in lateral view; pl – petiole length; maximum length of petiole in lateral view; pph – postpetiole height; maximum height of postpetiole in laterall view; ppl – postpetiole length; maximum length of postpetiole in lateral view; htl – hind tibia length; maximum length of hind tibia. example of measurements: 0.875 ± 0.032 (0.8380.927) = the average measurement ± standard deviation (range of variation). indices ci – cephalic index: hw/hl x 100; si1 – scape index 1; sl/hl x 100; si2 – scape index 2; sl/hw x 100; mi – mesosoma index; ml/pnw x 100; pi – petiole index 1; pl/ph x 100; ppi – postpetiole index; ppl/pph x 100; hti – hind tibia index; htl/hw x 100. all lengths are in mm. nomenclatorial notes pheidole pallidula (nylander, 1849) myrmica pallidula nylander, 1849: 42 (terra typica: sicily). oecophthora subdentata mayr, 1853: 145 (terra typica: austria); mayr, 1855: 456 (as synonym of pallidula). pheidole pallidula var. arenarum ruzsky, 1905: 647 (terra typica: kazakhstan); atanassov & dlussky, 1992: 123 (as synonym of pallidula). pheidole pallidula subsp. koshewnikovi ruzsky, 1905: 648 (terra typica: russia). sociobiology 62(2): 181-186 (june, 2015) 183 pheidole pallidula var. tristis forel, 1907: 204 (terra typica: tunisia), not pheidole tristis (smith, 1858: 132). pheidole symbiotica wasmann, 1909: 693 (terra typica: portugal), new synonymy. pheidole pallidula var. emeryi krausse, 1912: 169 (terra typica: sardinia), not pheidole tristis mayr, 1887: 589; pheidole pallidula var. obscura santschi, 1936: 200 (= pheidole pallidula var. emeryi krausse 1912: 169 not mayr, 1887: 589). pheidole pallidula var. cicatricosa stitz, 1917: 340 (terra typica: algeria); collingwood, 1978: 68 (as synonym of pallidula). pheidole pallidula var. inermis stitz, 1917: 340 (terra typica: algeria), not pheidole inermis mayr, 1870: 984; pheidole pallidula selenia özdikmen, 2010: 805 (= pheidole pallidula var. inermis stitz, 1917: 340 not pheidole inermis mayr, 1870: 984). pheidole pallidula subsp. orientalis müller, 1923: 69 (= pheidole pallidula ssp. arenarum var. orientalis emery, 1915: 230 unavailable name, terra typica: russia); baroni urbani, 1964a: 3 (as synonym of pallidula). pheidole pallidula var. recticeps menozzi, 1932: 452 (= pheidole pallidula ssp. tristis var. recticeps forel, 1909: 391 unavailable name, terra typica: egypt). xenoaphaenogaster inquilina baroni urbani, 1964b: 50 (terra typica: sicily), not inquilina wheeler, 1903: 664; bolton, 1987: 291 (as synonym of pallidula). redescription of “symbiotica” form (figs 1-5) measurements: infested workers (n = 9): hl: 0.875 ± 0.032 (0.838-0.927); hw: 0.869 ± 0.031 (0.816-0.916); sl: 0.721 ± 0.014 (0.704-0.739); sh: 0.085 ± 0.005 (0.0780.089), el: 0.151 ± 0.008 (0.134-0.156); ew: 0.108 ± 0.012 (0.089-0.123); ml: 1.04 ± 0.026 (0.993-1.079); htl: 0.748 ± 0.028 (0.691-0.782); pl: 0.421 ± 0.039 (0.352-0.464); ppl: 0.313 ± 0.021 (0.279-0.345); ph: 0.261 ± 0.03 (0.2120.302); pph: 0.257 ± 0.025 (0.223-0.291); pnw: 0.542 ± 0.019 (0.514-0.564); ci: 99.3 ± 1.7 (97.4-101.3); si1: 82.5 ± 2.9 (78.3-87.0); si2: 83.1 ± 2.5 (79.6-86.3); pi: 161.2 ± 18.7 (137.0-203.3); ppi: 122.7 ± 16.0 (107.6-154.7); hti: 85.9 ± 1.8 (83.0-88.4); mi: 192.0 ± 6.2 (184.3-202.7) redescription: forms were observed in two colors. in paler form the head is yellowish with brown frons and vertex, thorax, antennae and legs yellowish, abdomen brown, apical margins of abdominal segments yellowish (figs 1, 2). in darker form the head is mostly black with dark brown clypeus and yellowish brown underside, antennae and legs yellowish brown to partly brown, pronotum black, mesonotum brown, propodeum from brown to yellowish brown, petiole and postpetiole yellowish brown, abdomen brown, apical margins of abdominal segments yellowish (figs 3, 4). in full-face view head rectangular, almost as wide as long (fig 5). posterior margin shallowly concave, posterior corners rounded. mandibles elongately triangular, their surface with setose, partly elongate punctures but without figs 1-2: pale worker of teratological form “symbiotica” of pheidole pallidula. 1. dorsal; 2. lateral. scale bar = 1 mm. striation, space between punctures shiny. masticatory margin mostly straight, with large apical and slightly smaller preapical tooth, small tooth in the middle and two small teeth basally. anterior margin of clypeus straight or shallowly emarginate, surface shiny, without striation or ridges, on sides with few fine, setose punctures, basal clypeal lobe triangular, deeply impressed, sharply bordered from triangular frontal lobes. frons bicornute anterad, triangular anterior lobes with fine longitudinal striation, frontal lateral margins sharp, short, extending to 1/3 length of head, central part of frons shiny, figs 3-4. dark worker of teratological form “symbiotica” of pheidole pallidula. 3. dorsal; 4. lateral. scale bar = 1 mm. l borowiec, s salata – pheidole symbiotica, an enigmatic supposed social parasite 184 with sparse setose punctures. antennal scrobes deep, with semicircular striation surrounding scrobe dorsally and laterally. gena with longitudinal ridges, prolongated along inner margin of eyes up to 1/2-2/3 length of head, postocular area, top and venter of head without ridges or striation, with fine setose punctures. eyes small, almost round, approximately 0.4 times as long as tempora and 0.5 times as long as gena. whole surface of head shiny, covered with semi-erect and erect setae, the longest on clypeus, along frontal lateral ridges, on top of head and partly on ventral surface of head. the longest setae twice longer than eye length. antennae moderately elongate, scapes slightly shorter than head width, funicles approximately 1.4 times as long as scapes, with large 3-segmented club 1.3 times as long as eight basal funicle segments joined. scapus and funicles covered with long semierect to erect setae. mesosoma 1.9 times as long as wide, promesonotum subangulate in profile, pronotum separated from mesonotum by sharp margin. dorsal surface of mesonotum almost flat with small tubercle laterally, distinctly separated from propodeum by moderately deep transverse sulcus. dorsal surface of propodeum flat in profile, with small, triangular propodeal spines then with oblique, concave posterior surface (figs 2, 4). surface of pronotum shiny, with sparse, small setose punctures, top of mesonotum shiny, sides with distinct microreticulate sculpture, propodeum dorsally with transverse ridges and microreticulate sculpture, laterally with microreticulate sculpture and fine oblique to semicircular striation. petiole elongate with long peduncle, its anterior face straight to shallowly concave, node triangular to slightly obtuse in profile. posterior face of petiole straight, ventral margin straight, without spine or angulation. in dorsal view, petiole with concave sides and more or less angulate anterior and posterior corners. postpetiole in profile rounded to subangulate. in dorsal view postpetiole 1.7 times as long as wide, regularly widened posteriorly, apical half with gently rounded sides. material: nine infested workers from two nests of pheidole pallidula, italy, campania, salerno prov., cilento national park, monte bulgheria n. poderia (40,08709n / 15,39717e), 380 m, leg. l. borowiec. habitat and behavior notes: ants were collected in two nests of p. pallidula under limestone stones on the side of the road covered by a rock rubble which was scantily overgrown by xerothermophilic plants. after lifting the stone, the individuals of the “symbiotica” form immediately began to hide between the grains of gravel, while specimens of p. pallidula, in a manner typical for workers of this species, exhibited aggressive behavior. the area was placed on northern slope of monte bulgheria mountain which is exposed to sunlight in the middle of the day. the following ant species were recorded in the same area: camponotus aethiops (latreille), camponotus dalmaticus (nylander), camponotus lateralis (olivier), camponotus nylanderi emery, camponotus piceus (leach), camponotus vagus (scopoli), crematogaster scutellaris (olivier), crematogaster sordidula (nylander), formica gagates latreille, lasius emarginatus (olivier), lasius myops forel, lasius paralienus seifert, messor structor (latreille), myrmica scabrinodis nylander, plagiolepis pygmaea (latreille), solenopsis fugax latreille, solenopsis cf. lusitanica, temnothorax cf. exilis, temnothorax flavicornis (emery), temnothorax kraussei (emery), temnothorax leviceps (emery), temnothorax lichtensteini bondroit), temnothorax luteus (forel), temnothorax recedens (nylander), and tetramorium bicarinatum (nylander). finding of tetramorium bicarinatum in natural habitat is very interesting because this exotic species is usually collected in anthropogenic areas such as city parks, greenhouses, ruderal areas and roadsides but it was previously recorded from open habitats in italy near cropani marina, catanzaro province (jucker et al., 2008). comments: at first glance the “symbiotica” form looks intermediate between minor and major workers of p. pallidula. the most striking feature is the very large abdomen in this form, which is longer than the summed total length of the head and thorax, while in typical workers of pheidole fig 5. head of teratological form “symbiotica” of pheidole pallidula. scale bar = 0.5 mm. fig 6. mermithid nematode prepared from abdomen of teratological form “symbiotica” of pheidole pallidula. scale bar = 0.5 mm. sociobiology 62(2): 181-186 (june, 2015) 185 pallidula the abdomen is clearly shorter. the development of such a large abdomen is probably stimulated by the parasite, which is large and tightly fills the whole volume of the abdomen. form “symbiotica” does not have any features of the gyne, even ergatoid form, there are no wing buds and body proportions and construction of trunk are similar to the body shape of a big major worker. the parasite is believed to stimulate the development of workers to such a large size, so that it could accommodate in abdominal cavity such a large nematode specimen (csösz & majoros, 2009; csösz, 2012). discussion true social parasitism was observed in the genus pheidole westwood (buschinger, 2009). although wilson (1984) noted that this phenomenon is rare in tropical ants but pointed that neotropical pheidole microgyna wheeler, 1928 is a temporary social parasite of pheidole minutula mayr, 1878 and described two species from the oriental region: pheidole languinosa and pheidole parasitica, both parasitizing in common and widespread pheidole indica mayr, 1879. he listed another six pheidole species as potential social parasites including pheidole symbiotica (sic!) (kusnezov, 1951) described from argentina. this homonymic name was later replaced by pheidole kusnezovi by wilson (2003). our observations of the “symbiotica” form show that for the description of a new species of parasitic ant from a single specimen of caste is not enough. we confirm csösz’s (2012) conclusion that especially taxa reported only once or only few times should be verified by allowing the possibility of infection by internal parasites. thus far the most drastic changes in the morphology and development of ants was observed in the specimens infected by nematodes (csösz & majoros, 2009; poinar, 2012), but there are also examples of altered host morphology resulting from infections by other internal parasites (heinze et al., 1998; trabalon et al., 2000; miura et al., 2006). acknowledgements thanks to jolanta świętojańska (university of wrocław, poland) for her assistance during field trips of the senior author and marek l. borowiec (university of california, davis, usa) for language verification and other comments. the junior author would like to thank the university of wrocław for supporting grant no. 2127/m/ kbte/14. references atanassov, n. & dlussky, g. m. 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(2003). pheidole in the new world. a dominant, hyperdiverse ant genus. cambridge, mass.: harvard university press, [ix] + 794 pp. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.453-458sociobiology 60(4): 453-458 (2013) evaluation of formic acid toxicity to subterranean termite, reticulitermes chinensis snyder yj xie, q du, qy huang, cl lei introduction formic acid is the simplest organic acid, present in many organisms. it is found in most plant species, including polygonum hydropiper, urttca dioica, urtica urens (hutchens 1992, chopra & chopra 2006, rahmatullah et al. 2010). in animal, formic acid is the major component of ants from the subfamily formicinae. the produced formic acid concentration in such ant produced substances can reach up to 54% (hölldobler & wilson 1990). formic acid also occurs in carabid beetles (will et al. 2010, rossini et al. 1997, attygalle et al. 1992) and notodontid caterpillars (weatherston et al. 1979, attygalle et al. 1993). formic acid was reported to possess significant insecticidal activity. it has been used as a fumigant to control the varroa mite, varroa destructor anderson (sharma et al. 1983, underwood & currie 2004, 2005, 2007, vanengelsdorp et al. 2008, calderone 2010, giovenazzo & dubreuil 2011), and the tracheal mite, acarapis woodi rennie (hoppe et al. 1989, wilson et al. 1993, nelson et al. 1994), and red imported fire abstract this study examined formic acid contact and fumigation toxicity to reticulitermes chinensis in the laboratory. in the contact toxicity bioassay, the ld 50 values ranged from 267.86 to 287.68 μg adult-1 for workers, 279.09 μg adult-1 for alates (male and female) and 223.08 μg adult-1 for soldiers after 24 h, respectively. in the fumigation bioassays, the lc 50 values ranged from 0.84 to 1.08 μg ml-1 for workers, 1.19 μg ml-1 for alates (male and female) and 0.57 μg ml-1 for soldiers after 24 h, respectively. at the concentration of 2.50 μg ml-1, the kt 50 value of formic acid ranged from 25.38 to 34.75 min for workers, from 42.21 to 45.62 min for alates (male and female), from 32.18 to 36.37 min for soldiers. although formic acid was significantly less toxic to subterranean termite than bifenthrin, but higher toxic to many other pest insects. the findings of this study provide important confirmation of formic acid with fumigation toxicity against termite. it may be worth investigating the use of formic acid for managing subterranean termite. sociobiology an international journal on social insects huazhong agricultural university, wuhan, china. research article termites article history edited by alexandre vasconcellos, ufpb, brazil received 02 april 2013 initial acceptance 19 may 2013 final acceptance 18 june 2013 keywords: formic acid; contact toxicity; fumigation toxicity corresponding author chao liang lei hubei insect resources utilization and sust. pest manag. key laboratory huazhong agricultural university, wuhan 430070, china e-mail: cl_lei@yahoo.com.cn ioir@mail.hzau.edu.cn ants, solenopsis invicta buren (chen et al. 2012), and stored product insects, sitophilus oryzae and rhyzopertha dominica (chaskopoulou 2007), and the mosquito larvicidal, culex quinquefasciatus and aedes aegypti (welling & paterson 1985), and drosophila and houseflies (song & scharf 2008a, b, 2009). termites are world-wide pests threatening agriculture and the urban environment (verma et al. 2009) worldwide. they cause over 3 billion dollars worth of damage to wooden structures annually throughout the u.s. (su & scheffrahn 1998). control and repair costs due to termites in china, have been increasing annually. reticulitermes chinensis snyder (isoptera: rhinotermitidae) is a species of termites with broad distribution that damages the wooden structures of buildings and the xylems of living old trees in china (wei et al. 2007). over the past two decades, the control of termite was usually accomplished by using organochlorines and organophosphates, which have been banned owing to environmental and human health concerns (potter 1997). at the present time, several termiticides, including bifenthrin, chlorfenapyr, cyperyj xie, q du, qy huang, cl lei formic acid toxicity to reticulitermes chinensis454 methrin, fipronil, imidacloprid and permethrin, were registered for termite control around the world (unep report 2000). however, the persistent use of synthetic chemicals to control termites raises several concerns related to environment and human health. in order to reduce the negative impact of pesticides on the environment and to minimize the development of insecticide resistance in target pest species, as a result, the search for good efficacy and environmentally friendly insecticide is essential. as a safer or ‘green’ alternative, plant derived compounds have been used in termite management as contact insecticides or repellents, such as β-thujaplicin, thujopsene, t-muurolol, cinnamaldehyde and nootkatone (nakashima & shimizu 1972, yoshida et al. 1998, maistrello et al. 2001, chang & cheng 2002, cheng et al. 2004). another potential source of natural toxins against termite is the defensive/offensive chemicals in ants. some genus such as leptogenys, centromyrmex, termitopone, megaponera and pheidole are all obligate predators of termites (hölldobler & wilson 1990). in addition, kenne et al. (2000) observed that myrmicaria opaciventris can quickly subdued macrotermes bellicosus soldier with its pretarsus and abdominal thorn, thereby attacking the workers. nascimento (2001) reported that the pheidole pallidula preyed on reticulitermes lucifugus, which play an important role in suppressing the establishment of termite colonies. pachycondyla analis, living in the dry forests of the tanzanian coast and feeding on termites, can attack the termite nest (bayliss & fielding 2002). ke et al. (2008) found that diacamma rugosum, harpegnathos venator, pachycondyla astuta and polyrhachis dives had strong agonistic behavior and attacking capabilities to c. formosanus, and also observed the ant nest adjacent to the o. formosanus nest and the ant predation behavior to termite in the wild. the defensive chemicals of ants how to affect termite is not well understood, let alone the utilization of those chemicals in termite control. therefore, the aim of present work was to study the contact and fumigation toxicity of formic acid to r. chinensis in the laboratory. methods termite the termite samples of r. chinensis colonies were collected from huazhong agriculture university, wuhan, china. colonies has been reared on wood pieces in a glass container (70 cm × 40 cm × 30 cm) in dark at 26 ± 1ºc and 80 ± 5% relative humidity for more than six months. contact toxicity contact toxicity of formic acid (98%, sinopharm chemical reagent co., ltd. china) to r. chinensis workers, alates (male and female) and soldiers was determined. for the ease of handling and obtaining uniform body weight, large workers were selected in the bioassay. acetone was used as solvent, and the solution was applied with a 1.0 μl capillary tube. for workers, alates (male and female) and soldiers, doses of 100, 150, 200, 250, 300 and 350 μg adult-1 were applied. controls were treated with acetone. twenty adults of r. chinensis workers, alates (male and female) and soldiers were used for each concentration and control, and the experiment was replicated 3 times. four colonies were used for workers. for alates (male and female) and soldiers, only one colony was tested. the contact toxicity of bifenthrin (98%, huangma agrochemicals co., ltd, jiangsu, china) to r. chinensis workers was also estimated using 2 colonies. doses of 3.125, 6.25, 12.5, 25.0 and 50.0 ng adult-1 were used. bifenthrin is the most widely used commercial termiticide as positive control. each dose was replicated 3 times, and each replicate consisted of 20 workers. both treated and control insects were then transferred to glass petri dishes, and kept under the same environmental conditions described for the rearing. mortality percentages were recorded after treatment for 24 h and ld50 values were calculated according to finney (1971). fumigation toxicity to determine the fumigant toxicity of formic acid and the median effective time to cause mortality in 50 % of the workers from four different colonies (lt50 values), filter papers (whatman no 1, cut into 2 cm diameter pieces) were impregnated with an appropriate concentration of 1.25, 2.5, 5.0, 10.0 and 20.0 μg ml-1, respectively. the impregnated filter paper was then attached to the undersurface of the 1000 ml glass jar’s (10 cm in diameter × 12.5 cm in height) screw cap, respectively. the cap was tightly screwed onto the jar, which contained 20 termites. in addition, workers from two other colonies were tested only at 2.5 μg ml-1. alates (male and female) and soldiers were also tested at 2.5 μg ml-1. two colonies were used for alates (male and female) and soldiers. each concentration and control was replicated three times. mortality was counted every minute after the formic acid had been delivered into the tube until all termites were dead. when no leg or antennal movements were observed, insects were considered dead. another experiment was designed in order to determine the 50% lethal concentration. for the mortality bioassay, 20 workers, 20 alates (male and female) or 20 soldiers were placed in the bottle. the number of dead termites was counted after the formic acid had been delivered into the bottle for 24 h. six concentrations were tested, including 0.20, 0.40, 0.60, 0.80, 1.0 and 1.2 μg ml-1 for workers, alates (male and female) and soldiers, respectively. three colonies were used for workers, and only one colony was used for alates (male and female) and soldiers. for each concentration × colony combination, there were three replicates. all bioassays were conducted at 26 ± 1ºc and 80 ± 5% relative humidity. the fumigation toxicity of bifenthrin (huangma agrochemicals sociobiology 60(4): 453-458 (2013) 455 co., ltd, jiangsu, china) on workers was also measured. six concentrations were tested, including 0.125, 0.25, 0.625, 1.25 and 2.5 ng ml-1. two colonies were used, and there were three replicates for each colony. data analysis polo plus v.1.0 (leora software, petaluma, ca) was used to estimate ld50, kt50 and lc50 with 95% confidence intervals (cis). the relative toxicity ratio with their upper and lower 95% confidence limits was used to evaluate the significance of the difference between ld50, kt50 and lc50 values. the significance was set at the p = 0.05 probability level. if the 95% confidence interval of the ratio between two ld50, kt50 or lc50 values included 1, these were not considered to be significantly different (robertson et al. 2007). results toxicity of formic acid against r. chinensis was shown in table 1. the ld50 values of formic acid were ranging from 267.86 to 287.68 μg adult-1 for workers, which were larger than those of bifenthrin with ld50 values ranging from 7.16 to 10.39 ng adult-1, and no significant difference was also found in ld50 values of formic acid for workers between colony a and the other three colonies. alates and soldiers were more sensitive to formic acid than workers. the ld50 values for alates (male and female) and soldiers were at 279.09 and 223.08 μg adult-1, respectively. discussion when applied topically, formic acid was significantly less toxic to subterranean termite than bifenthrin, but higher toxic to many other pest insects. the lc50 value of formic acid for rice weevil, sitophilus oryzae, was 6.03-7.60 μg ml-1, and for the mosquito, culex quinquefasciatus, was 3.67 μg ml-1 (welling & paterson 1985). formic acid displayed low toxicity against yellow fever mosquito, aedes aegypti, at 6.00 μg ml-1 (chaskopoulou 2007). as demonstrated recently by chen et al. (2012), the lc50 value of formic acid was 0.260.70 μg ml-1 for red imported fire ants. this indicated that the toxicity of formic acid to subterranean termite, r. chinensis, was higher than that of the insects studied so far, but similar to red imported fire ants. a w: works; a: alates (male and female); s: soldiers. b positive control. as can be seen from table 2, the lc50 values ranged from 0.84 to 1.08 μg ml-1 for workers, 1.19 μg ml-1 for alates (male and female) and 0.57 μg ml-1 for soldiers after treatment for 24 h, respectively. the lc50 values of a well-known commercial pesticide used as a positive control in this study, bifenthrin, were 0.40 and 0.42 ng ml-1, respectively (table 2). at a concentration of 2.50 μg ml-1, the kt50 value of formic acid ranged from 25.38 to 34.75 min for workers, from 42.21 to 45.62 min for alates (male and female), from 32.18 to 36.37 min for soldiers (table 3). meanwhile, the higher the concentration, the smaller the kt50 value was. within a colony, the kt50 value of workers was always significantly smaller than that of alates (male and female), and soldiers. table 1ld50 values of formic acid by contact against workers, alates and soldiers of r. chinensis. table 2lc50 values of formic acid applied as fumigant for 24 h against workers, alates and soldiers of r. chinensis. a w: works; a: alates (male and female); s: soldiers. b positive control. table 3kt50 values of formic acid applied as fumigant against workers of r. chinensis. the action mode of formic acid was to inhibit the activity of mitochondrial cytochrome c oxidase (nicholls 1975, petersen 1977). recently, formic acid has been reported to have a significant excitatory effect on the nervous system of housefly larvae (song & scharf 2008a, b). song & scharf (2009) reported the formic acid have a significant excitatory effect on the nervous system of drosophila melanogaster. it may explain why formic acid has such a low contact toxicity a w: works; a: alates (male and female); s: soldiers. chemical colony caste a slope (±se) ld50 95% ci formic acid bifenthrin b a b c d e e f w w a w w s w w 2.36(±0.31) 2.09(±0.29) 2.91(±0.35) 2.54(±0.32) 2.21(±0.29) 2.93(±0.31) 2.52(±0.26) 2.46(±0.24) 287.68 (μg adult-1) 286.24 (μg adult-1) 279.09 (μg adult-1) 273.77 (μg adult-1) 267.86 (μg adult-1) 223.08 (μg adult-1) 7.16 (ng adult-1) 10.39 (ng adult-1) 250.02-347.80 245.04-322.94 248.91-250.48 241.01-322.79 232.15-322.98 200.83-250.48 5.99-8.41 8.82-12.20 chemical colony caste a slope (±se) lc50 95% ci formic acid bifenthrin b a c b c d w w w a s w w 6.71(±0.76) 4.56(±0.52) 4.39(±0.59) 4.72(±0.73) 4.77(±0.54) 3.06(±0.29) 2.80(±0.26) 0.95 μg ml-1 0.84 μg ml-1 1.08 μg ml-1 1.19 μg ml-1 0.57 μg ml-1 0.40 ng ml-1 0.42 ng ml-1 0.88-1.03 0.77-0.93 0.98-1.23 1.07-1.38 0.49-0.65 0.32-0.50 0.35-0.50 colony id caste a dosage (μg/ml) slope (±se) kt50 (min) 95% ci a b c d e f w w w w w w w w w w a s w w w w w w w w w w w a s w 1.25 2.5 5.0 10.0 20.0 1.25 2.5 5.0 10.0 20.0 2.5 2.5 1.25 2.5 5.0 10.0 20.0 1.25 2.5 5.0 10.0 20.0 2.5 2.5 2.5 2.5 4.32(±0.77) 7.87(±0.75) 9.06(±1.00) 9.63(±1.43) 5.10(±0.58) 2.32(±0.28) 8.45(±0.81) 9.47(±0.88) 6.24(±0.64) 7.15(±0.72) 7.77(±1.06) 4.19(±0.51) 9.84(±0.84) 7.95(±0.63) 11.32(±0.96) 14.18(±1.37) 15.62(±1.63) 14.05(±1.35) 12.03(±1.13) 6.02(±0.66) 8.56(±0.79) 5.72(±0.64) 12.94(±1.24) 9.40(±1.47) 8.30(±1.44) 6.95(±0.62) 91.67 26.71 16.79 8.74 5.14 107.45 32.85 19.70 9.55 4.74 45.62 36.37 84.12 25.38 19.63 7.74 5.25 79.53 29.74 17.90 6.31 3.79 34.75 42.21 32.18 28.25 82.27-109.12 25.47-28.01 15.80-17.82 7.78-9.34 3.87-5.48 94.26-123.01 31.61-34.25 18.97-20.49 9.05-10.05 4.42-5.03 43.34-48.79 33.21-41.01 81.25-86.94 24.21-26.51 19.05-20.20 7.44-8.04 5.08-5.42 76.53-82.67 28.49-31.07 16.54-19.72 5.94-6.68 3.01-4.54 33.52-36.02 40.14-45.72 30.30-35.30 26.44-29.90 yj xie, q du, qy huang, cl lei formic acid toxicity to reticulitermes chinensis456 compared with fumigation. fumigation has been extensively applied as building treatment in termite management programs. fumigants include methyl bromide, sulfuryl fluoride, dichloroethane and dibromoethane. however, compared to contact insecticides, fumigation has lower toxicity. therefore, the high efficacy of the conventional synthetic contact insecticides may have slowed down the development of any alternatives. using formic acid directly as fumigant against subterranean termite may not be suitable for its own acidity and corrosiveness. however, formate ester is much less corrosive than formic acid and should be much easier to handle. scharf et al. (2006) and nguyen et al. (2007) also found the low molecular weight formate esters are volatile compounds with fumigant insecticidal activity. similarly, chaskopoulou et al. (2009) reported the volatile low molecular weight compounds against aedes aegypti and culex quinquefasciatus. previous research reported some formate esters had neurological activity on drosophila melanogaster meig and musca domestica l, owning to their hydrolyzed metabolite, formic acid (song & scharf 2008a, b). previously, haritos & dojchinov (2003) reported some formate esters exert toxicity in the stored product beetle sitophilus oryzae l. for its hydrolyzed metabolite, formic acid, which causes mitochondrial impacts. therefore, formate esters may also be as good candidates for subterranean termite, particularly as fumigant in treatment. however, the prerequisite is that formic acid can be liberated from formate esters in termite bodies. this is definitely worth for further investigation. the formic acid fumigation bioassay to termite was carried out in an enclosed environment for termite control, such as house, storage spaces and timber. in order to make fumigation effectively, the enclosed environment must be intact during the application. simultaneously, other factors such as temperature, humidity, the concentration and time of formic acid applied all should be considered. these are the key of successful application of formic acid in the termite control. in addition to considering their potential fumigant effect, like other commercial fumigant, the more important concern of formic acid application is its toxicity to humans and the environment. the current occupational safety and health administration (osha) permissible exposure limit (pel) is 3 mg m-3 as the 8 h time-weighted average (twa) concentration for ddvp, 9 mg m-3 for formic acid, 80 mg m-3 for methyl bromide, 250 mg m-3 for methyl formate and ethyl formate is 300 mg m-3 (chen et al. 2012). therefore, the effect of formate esters against termite is more worth to investigate, as it seems that formate esters may be more practical to use than formic acid. acknowledgments this study was supported by the fundamental research funds for the central universities (2010py065). references attygalle a. b., j. meinwald & t. eisner 1992. defensive secretion of a carabid beetle, helluomorphoides clairvillei. j. chem. ecol., 18: 489-498. attygalle, a. b., s. r. smedley, j. meinwald & t. eisner 1993. defensive secretion of two notodontid caterpillars (schizura unicornis, s. badia). j. chem. ecol., 19: 2089-2104. bayliss, j. & a. fielding 2002. termitophagous foraging by pachycondyla analis (formicidae: ponerinae) in a tanzanian coastal dry forest. sociobiology, 39: 103-122. calderone, n. w. 2010. evaluation of mite-away-ii for fall control of varroa destructor (acari: varroidae) in colonies of the honey bee apis mellifera (hymenoptera: apidae) in the northeastern usa. exp. appl. acarol. 50: 123-132. chaskopoulou, a. 2007. testing vapor toxicity of formate, acetate, and heterocyclic compounds to aedes aegypti and musca domestica. ms thesis, university of florida, gainesville, fl. chaskopoulou, a. s. nguyen, r. m. pereira, m. e. scharf & p. g. koehler 2009. efficacy of 31 volatile low molecular weight compounds against aedes aegypti and culex quinquefasciatus. j. med. entomol., 46: 328-334. chang, s. t. & s. s. cheng 2002. antitermitic activity of leaf essential oils and components from cinnamomum osmophleum. j. agr. food chem., 50: 1389-1392. chen, j., t. rashid & g. l. feng 2012. toxicity of formic acid to red imported fire ants, solenopsis invicta buren. pest manag. sci. 68: 1393-1399. cheng, s. s., c. l. wu, h. t. chang, y. t. kao & s. t. chang 2004. antitermitic and antifungal activities of essential oil of calocedrus formosana leaf and its composition. j. chem. ecol., 30: 1957-1967. chopra, r. n. & i. c. chopra 2006. indigenous drugs of india. bimal kumar dhur of academic publishers, india. nascimento, f. s. 2001. behavioral responses of mediterranean termite reticulitermes lucifugus (rossi) (isoptera: rhinotermitidae) under presence effects of ant pheidole pallidula (nyl.) 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koen yoshishu, 40: 311-315. 1121 isolation and characterization of p450 gene from the formosan subterranean termite, coptotermes formosanus (isoptera: rhinotermitidae) by yue zhou1, jianchu mo2, keping chen1 & qinggang xu1* abstract a cytochrome p450 gene belonging to family9 was isolated from the midgut transcriptome of the termite coptotermes formosanus shiraki, for screening enzymes related to biomass degeneration. some studies show that insect p450 enzymes have ligninase activities for catalyzing lignin degradation. we employed the race method to clone this cytochrome p450 gene, named cyp9ax1 (genbank accession no.jn969113). to the best of our knowledge, cyp9ax1 is the first member of the cyp9 family cloned from this termite. the full-length cyp9ax1 cdna was 2242 bp long and included a 1599bp open-reading-frame (orf), a 61-bp 5’-untranslated region (utr) and a 592-bp 3’-utr (excluding the poly-a tail). the cyp9ax1 protein deduced from the orf contains 532 amino acids with a predicted signal peptide composed of 20 amino acid at its n-terminal and the classic heme-binding domain fxxgxxxcxg (residues 468-477). at position 473, residue arg (r) changes to gln (q), this suggests that cyp9ax1 is a new type of cyp subfamily 9a. the phylogenetic tree showed that c. formosanus has high genetic relationship with blattella germanica and diploptera punctata. quantitative rt-pcr assays demonstrated that cyp9ax1 was expressed most abundantly in malpighian tubules, and slightly lower in the head, foregut, midgut and hindgut. the results suggested that cyp9ax1 may be involved in enzymatic detoxification systems of the delignification process in c. formosanus. 1institute of life sciences, jiangsu university, zhenjiang 212013, china 2ministry of agriculture key laboratory of molecular biolog y of crop pathogens and insects, institute of insect sciences, zhejiang university at zijingang campus, yuhangtang road 388, hangzhou, zhejiang 310012, china *correspondence: qinggang xu, institute of life sciences, jiangsu university, zhenjiang, 212013, china. email: xuqg@ujs.edu.cn 1122 sociobiolog y vol. 59, no. 4, 2012 keywords: characterization, coptotermes formosanus shiraki, cytochrome p450, real-time pcr introduction the cytochrome p450 (cyp) monooxygenases have numerous functional roles in oxidative transformation of endogenous substrates and xenobiotics (nelson et al. 1996). in insects, cytochrome p450s metabolize hormones and pheromones but are best known for their roles in the metabolism of insecticides and host plant chemicals (feyereisen 1999; scott et al. 1998). many studies focus on the function of p450 on insect development and detoxification contributes to insecticide resistance (scott 1999, scott & wen 2001) . the first member of the cyp9 family, cyp9a1 from heliothis virescens, is constitutively over-expressed in resistant, thiodicarb-selected tobacco budworms (rose et al. 1997), and may play a role in pesticide metabolism (stevens et al. 2000). many studies showed that cyp9 genes such as cyp9a2,4,5, cyp9m10, cyp9a12,17, cyp9q, are mainly involved in detoxification of plant allelochemicals and pesticides (hardstone et al. 2010; itokawa et al. 2010; mao et al. 2011; stevens et al. 2000; zhou et al. 2010). interestingly, some insect p450 enzymes show ligninase activities for catalyzing lignin degradation (geib et al. 2008; scharf & boucias 2010). the termite is an insect can survive on highly lignocellulosic materials. the termite cytochrome p450 enzyme may be involved in lignin degradation. it is a candidate pre-treatment enzyme for delignification of lignocelluloses (scharf & boucias 2010). recent evidence suggested that lignin degradation is a key pre-treatment process in biomass transformation into biofuels (geib et al. 2008; ke et al. 2011). therefore, revealing the nature and roles of the p450 monooxoygenases of c. formosanus will facilitate the understanding of the mechanism of this enzyme in lignocellulose pre-treatment. in this paper, a new p450 gene belonging to the cyp9 family named cyp9ax1was isolated from the termite c. formosanus shiraki. our study shows that this cf p450 is mainly expressed in malpighian tubules. this newly identified cyp9ax1 appears to be important for c. formosanus to detoxifiy plant allelochemicals from digestion of lignocelluloses. 1123 zhou, y. et al. — isolation and characterization of p450 gene from termites materials and methods termites termites were collected from hangzhou city of zhejiang province. the termites were maintained in the laboratory in a dark container on wet wood at 27°c~30°c and 70% relative humidity. rna isolation and gene cloning ten worker specimens were washed in sterile phosphate-buffered saline (pbs: 5mm na 2 hpo 4 , 5mm nah 2 po 4 , 130mm nacl), then the digestive organs (consisting of the salivary gland, foregut, midgut and hindgut) were dissected and homogenized from the worker termites, and total rnas were extracted from the different homogenates using an rnapure isolation kit (bioteke, china). a reverse transcription reaction was carried out to synthesize the first strand cdna using reverse transcript kit2.0 (takara, china). briefly, reverse transcription was performed for 1 h at 42°c in 10μl reaction mixture, containing 1 μg of total rna, 25 pmol oligo dt primer, 0.5 mmol/l dntps, 10 units of recombinant rnasin ribonuclease inhibitor and 50 units of primerscripttm reverse transcriptase (takara, china). the cdna was diluted to 30μl. the pcr reactions were performed in 25μl reaction mixture containing 1μl of single strand cdna, 1.5 mmol/l mgcl2, 30 pmol of each primer, and 1 unit of la taq dna polymerase (takara, china) using eppendorf 2724 thermal cycler (usa). one pair of pcr primers was designed based on the p450 gene sequences from the transcriptome of termite midgut (unpublished). the primers for amplifying the c. formosanus p450 gene (cf p450) were as follows: p450f: 5’ -atggactggaactgggcgctgtcagac5-3’ and p450r: 5’ -agaacttctgagctccagccc-3’. the pcr reaction was performed as follows: 94°c for 1 min, 30 cycles of 94°c for 30 s, 60°c for 30 s, and 72°c for 1 min, and a final extension for 5 min at 72°c. the pcr product of c. formosanus p450 was about 1500 bp long. the amplified cdna fragments were separated by 1% (w/v) agarose gel and purified with a dna recovery kit (axygen, china), subcloned into pmd18-t vector (takara, china), and sequenced by sangon (shanghai, china). 1124 sociobiolog y vol. 59, no. 4, 2012 rapid-amplification of cdna ends of cf p450 a race (rapid-amplification of cdna ends) reaction was conducted to amplify the 5’ and 3’ utr of the cf p450 gene using a smarter™ race cdna amplification kit (clontech, usa). the gene-specific primer (gsp) (5’ race primer: p450-gsp1: 5’ -ggatcgcatgtccttccaacgctgacctgac-3’ and 3’ race primers: p450-gsp2: 5’ –gcttgaacctggggacagactcttc-3’) was designed to amply the full-size cf p450 gene. the pcr reaction was performed at 94°c for 5 min for dna predenaturation, then 35 cycles of 94°c for 1 min, 50°c for 1min and 72°c for 1 min 30 s, followed by one final extension at 68°c for 10 min. both fragments of 5’and 3’-race were analyzed by 1% agarose gel, then purified and cloned into pmd-18t vector (takara, china). positive clones were sequenced. bioinformatics analysis some online bioinformatics software was used to characterize the cf p450 gene. signalp 4.0 server was used to predict n-terminal signal peptide cleavage sites in amino acid sequences (http://www.cbs.dtu.dk/services/signalp/). the transmembrane domain was predicted with the tmhmm tool (http:// genome.cbs.dtu.dk/services/tmhmm/). the protein post-translational modification of glycosylation sites and phosphorylation sites were analyzed with dictyoglyc 1.1 server and netphos 2.0 server (http://www.cbs.dtu. dk/services/dictyoglyc/; http://www.cbs.dtu.dk/services/netphos/). the secondary structure was predicted by gor method (http://npsa-pbil.ibcp. fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html). phylogenetic analysis the relationship between cf p450 and other insect p450s was determined using phylogenetic analysis. amino acid sequences of insect p450 family 9 were aligned by the clustalw program (larkin et al. 2007). a maximum likelihood (ml) tree was constructed by mega5 with 1000 bootstrap replications (tamura et al. 2011). bayesian inference tree was also reconstructed using mrbayes v3.1.2 (ronquist & huelsenbeck 2003). four independent markov chain monte carlo (mcmc) chains were run for 1,000,000 generations with the default temperature of 0.1 and parameter sampling occurring every 500 generations. posterior probabilities for internal node were calculated 1125 zhou, y. et al. — isolation and characterization of p450 gene from termites from the posterior density of trees. mrna expression assay by real-time pcr mrna expression levels of cf p450 in different digestive organs were assessed by real-time pcr using a bio-rad cfx96 real-time pcr detection system and a sybr real-time premixture kit (bioteke, china). the reverse transcription reaction was carried out as previously described to synthesize the first strand cdna. cf p450 cdna fragments were amplified by pcr with forward primer p450ft 5’ aaagcccgacattatccagcacctcat -3’ and reverse primer p450rt 5'gtatccagtccggccacaagaaac -3'. the product of cdna fragments is 159 bp. serial 5×dilutions of cdna templates was performed in order to assess amplification efficiency of pcr. a pair of primer was designed to amplify termite actin as a reference gene to normalize the target gene expression levels. the primers were actinf, 5' aagcgcctctgaacccaaaagcaaa -3' and actinr, 5' tggcatggtgcacagcataaccttc3'. the product of termite actin is 204 bp. real-time pcr of each cdna sample and template free was performed in three independent replicates. the pcr reaction volume was 20μl. the pcr protocol consisted of an initial denaturation at 95°c for 2 min, followed by 40 cycles: 95°c for 10 s, 55°c for 30 s, and 72°c for 30 s. to verify the specificity of the pcr amplification, a melting curve analysis was carried out after amplification by taking continuous fluorescence readings while increasing the temperature from 65 to 95°c in steps of 0.5°c with 5 s at each step. biorad cfx manager 2.0 software was used to determine the threshold cycle (ct) value. the relative expression levels of target genes were calculated by the comparative ct method as described by livak and schmittgen (2001). results characterization of the cf p450 the full-length c. formosanus cyp gene revealed by race was 2242 bp long. it includes a 1599bp open-reading-frame (orf), a 36-bp 5’-untranslated region (utr) and a 592-bp 3’-utr (excluding the poly-a tail) which includes the canonical aataaa polyadenylation signal at nucleotides 2208-2213. the cf p450 protein deduced from the orf contains 532 amino 1126 sociobiolog y vol. 59, no. 4, 2012 fig.1. the full-length cdna sequence of cyp9ax1 and its deduced amino acid sequence. the start codon atg is underlined and the stop codon, tga is indicated in bold and by an asterisk. the five conserved motifs of insect p450 enzymes are indicated by the boxed amino acids. the amino acids thickly underlined are the positions of transmembrane region, which also belongs to the region of signal p prediction. 1127 zhou, y. et al. — isolation and characterization of p450 gene from termites acids with a 20 amino acid predicted signal peptide at its n-terminal. the comparative analysis of the cf p450 protein sequence with other insect cyp genes showed that cf p450 has the highest homolog y to the cyp9 family. the cf p450 protein sequence was named cyp9ax1 (genbank accession no. jn969113) by the p450 nomenclature committee (d. nelson, personal communication). the p450 amino acid sequences have some conserved signature motifs, though they have tremendous sequence diversity. in cyp9ax1, an fxxgxxxcxg (nelson 1998) motif is presented at amino acid residues 468-477, and the cysteine residue in this motif is known as the important ligand for heme binding (nebert & gonzalez 1987). this motif is also characterized as fxxgxrxcxg (feyereisen 1999), but in cyp9ax1, there is one amino acid residue change from arg (r) to gln (q) at position 473 (fig. 1). the helix c, wxxr (rewitz et al. 2006), present at amino acid residues 134-138, appears as wkdmr. the helix i motif believed to create an oxygen-binding pocket, a/ggxe/dtt/s (rewitz et al. 2006), present at amino acid residues 335-339, appears as agldtt. the helix k motif, exlr (rewitz et al. 2006), present at amino acid residues 385–395, appears as ylgmvvsetlr. the perf motif (rewitz et al. 2006) present at amino acid residues 441-452, appears as ypdperfdperf. bioinformatic analysis of cfcyp9ax1 as in other microsomal proteins, in cyp9ax1, a signal sequence of approximately 20 amino acids is predicted by the signal p analysis (fig. 2). this signal sequence is from amino acid residues 1 to 20, and the cleavage site is between position 20 and 21. in this signal sequence, 13 hydrophobic residues out of 20 are extremely hydrophobic. tmhmm software analysis showed that the cyp9ax1 protein has a highly hydrophobic n terminus transmembrane domain that functions as a membrane-anchor signal (sakaguchi et al. 1987), with an area number from 7 to 29 amino acids (fig. 3). the transmembrane domain of cyp9ax1 may also play a role as a membrane receptor or ion channel protein (zhang et al. 2011). a glycosylation site was predicted by dictyoglyc projections at pos.282 1128 sociobiolog y vol. 59, no. 4, 2012 fig. 2. signal sequence predication of cyp9ax1 by signalp server. fig. 3. transmembrane domain analysis of cyp9ax1 with tmhmm software 1129 zhou, y. et al. — isolation and characterization of p450 gene from termites g in cyp9ax1 (fig. 4). the phosphorylation site analysis of cyp9ax1 with netphos2.0 server showed that there were 9 serine (ser) phosphorylation sites, 9 threonine (thr) phosphorylation sites and 5 tyrosine (tyr) phosphorylation sites uniformly distributed throughout the polypeptide chain (fig. 5). gor secondary structure analysis of cyp9ax1 protein showed that 34.77% of α helix and 55.66% of random coil form the largest parts of the fig. 4. o-glycosylation site predication of cyp9ax1 by dictyoglyc projections fig. 5. phosphorylation site predication of cyp9ax1 by netphos2.0 server fig. 6. gor secondary structure analysis of cyp9ax1 1130 sociobiolog y vol. 59, no. 4, 2012 structural elements of cyp9ax1 (fig. 6). the extended strand accounting for 9.59% of cyp9ax1 was distributed throughout the protein (fig. 6). phylogenetic relationship of cf p450 with other insect cyp9 genes the cf p450 and sixteen full length cyp9 genes were analyzed using mega 5 and mrbayes v3.1.2 software. the cyp9ax1 encoded protein has 51% identity to the german cockroaches blattella germanica (aak69410 and q964t2) and diploptera punctata (aar97606). the phylogenetic trees showed that c. formosanus has high genetic relationship with b. germanica and d. punctata (fig. 7). the maximum likelihood tree based on the cyp9 fig. 7. maximum likelihood tree showing the relationship between c. formosanus and other insect cyp9 proteins. the ml tree and bayesian tree have identical topologies. branch confidence (bayesian posterior probability/ml bootstrap value) values are shown at the nodes. cf p450. the species abbreviations and the accession numbers are shown in table 1. 1131 zhou, y. et al. — isolation and characterization of p450 gene from termites proteins has the same topological structure as the bayesian tree. the cf p450 has a long genetic distance between the subfamily that is also present in the phylogenetic tree. mrna expression levels of cyp9ax1 we analysed c. formosanus cyp9ax1 transcription in the head, foregut, midgut, hindgut and malpighian tubules by real time-pcr. the relative expression levels of cyp9ax1 in different organs are illustrated in figure 8. the mrna levels in the foregut, midgut, hindgut and malpighian tubules were 1.6, 1.8, 2.5 fold and 10.6 fold higher than those in the head, respectively. this result indicated that cyp9ax1 was expressed most abundantly in malpighian tubules and expressed at roughly equal levels in the foregut, midgut and hindgut (fig. 8). discussion p450s are heme-thiolate proteins conducing oxidative attack of diverse fig. 8 relative expression levels of cyp9ax1 in different organs. real-time quantitative pcr data were evaluated using the formula 2-δct(-δct = ct of actin − ct of cyp9ax1) to calculate the relative expression levels. each column and bar represents the average of three amplification reactions. 1132 sociobiolog y vol. 59, no. 4, 2012 substrates through catalyzing activation of molecular oxygen. they are functionally involved in carbon source assimilation, biosynthesis of hormones and degradation of xenobiotics (werck-reichhart & feyereisen 2000). according to the identity at the amino acid level, p450s are grouped into different families (share >40% identity) and subfamilies (share >55% identity) (nelson et al. 1996). the members of cytochrome p450 family 9 (cyp9) were well known to be related in the oxidative metabolism of insecticides (feyereisen 1999). cyp9a1 is the first member of family 9 cloned from tobacco budworms, heliothis virescens (rose et al. 1997). to date, p450 variants belonging to the cyp9 family have been cloned from many insects such as cockroaches, ants, wasps and other insect species, and the majority of them are closely related to the metabolism of exogenous compounds such as plant toxins and insecticides (hardstone et al. 2010; itokawa et al. 2010; mao et al. 2011; stevens et al. 2000; zhou et al. 2010). cyp9a13 from mamestra brassicae is putatively involved in the metabolism of odorant compounds and xenobiotic clearance (maibeche-coisne et al. 2005). cyp9a20, a gene from b. mori, was presumably also related to insecticide resistance (yamamoto et al. 2010). insect cyp genes can be detected in a wide range of tissues. highest exprestable 1. the species name, abbreviations and accession numbers of p450 genes species name gene name abbreviation accession number blattella germanica cyp9e2 bgcyp9e2 aak69410 cyp9e2 bgcyp9e2 q964t2 bombyx mori cyp9a19 bmcyp9a19 abq08709 coptotermes formosanus cyp9ax1 cfcyp9ax1 jn969113 cnaphalocrocis medinalis cyp9a38 cmcyp9a38 az65619 culex quinquefasciatus cyp9b2 cqcyp9b2 xp_ 001855237 diploptera punctata cyp9e2 dpcyp9e2 aar97606 leptinotarsa decemlineata cyp9v1 ldcyp9v1 aaz94269 manduca sexta cyp9a5 mscyp9a5 aad51038 nasonia vitripennis cyp9p3 nvcyp9p3 np_001165945 cyp9p5 nvcyp9p5 np_001166017 cyp9ag4 nvcyp9ag4 np_001166010 plutella xylostella cyp9g4 pxcyp9g4 ach88357 tribolium castaneum cyp9d1 tccyp9d1 np_001034541 cyp9z4 tccyp9z4 np_001164248 cyp9f2 tccyp9f2 np_001127706 zygaena filipendulae cyp9a36 zfcyp9a36 acz97417 1133 zhou, y. et al. — isolation and characterization of p450 gene from termites sion levels usually appear in the midgut, fat bodies and malpighian tubules, but the expression level of individual cyp genes can vary (scott et al. 1998; scott & wen 2001) . for example, in bombyx mori, cyp9a19 was detectable in the brain and midgut, while cyp9a20 and cyp9a21 were not detectable in the midgut (ai et al. 2010). cyp9a12 mrna was detected in all the tested tissue of helicoverpa armigera larvae, whereas cyp9a17 was only detectable in the midgut and fat bodies (zhou et al. 2010). the expression level can be inducible by exogenous substances such as insecticides (stevens et al. 2000). in this study, cyp9ax1 was expressed most abundantly in the malpighian tubules, and less abundantly in the head, foregut, midgut and hindgut of c. formosanus. the digestion of lignocellulose in termites requires enzymes to cooperate with each other. the hindgut of termites is the most important digestive location for lignocellulose. before degeneration of the cellulose, delignification of biomass is very important. it is believed that many enzymes such as p450, laccase, catalase, and peroxidases are involved in the delignification process (geib et al. 2008; scharf & boucias 2010). ke et al. (2011) reported that the modifications of lignin began in termite foregut but happened mainly in the midgut, which indicated that the enzymes involved in lignin degradation or modification may be excreted in the foregut and midgut of the termite itself (ke et al. 2011). the role of p450s in the insect midgut has been well established in detoxification as well as possible pheromone synthesis (aw et al. 2010). high transcription levels of cyp9ax1 in the malpighian tubules could also correlate with detoxification given their role in metabolism and excretion of endogenous solutes and xenobiotics from biomass delignification processes (chung et al. 2009). however, these findings need further functional validation. the cloning and characterization of cyp9ax1 will contribute to our understanding of the biological roles of p450s in lignocelluloses digestion in termites. acknowledgments we thank dr david r. nelson for classifying the c. formosanus p450s before their publication. this work was supported by grants from the china 1134 sociobiolog y vol. 59, no. 4, 2012 postdoctoral science foundation funded project (no.20110491363), the research foundation for advanced talented scholars of jiangsu university (no.10jdg098), the national basic research program of china (no.2012cb114600), the natural science foundation of china (project no.: 31170611), and the zhejiang provincial natural science foundation of china (project no.: z3100211). references ai, j., q. yu, t. cheng, f. dai, x. zhang, y. zhu & z. xiang 2010. characterization of multiple cyp9a genes in the silkworm, bombyx mori. mol biol rep 37: 1657-1664. aw, t., k. schlauch, c.i. keeling, s. young, j.c. bearfield, g.j. blomquist & c. tittiger 2010. functional genomics of mountain pine beetle (dendroctonus ponderosae) midguts and fat bodies. bmc genomics 11: 215. chung, h., t. sztal, s. pasricha, m. sridhar, p. batterham & p.j. daborn 2009. characterization of drosophila melanogaster cytochrome p450 genes. proc natl acad 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j insect sci 11: 37. zhou, x., c. ma, m. li, c. sheng, h. liu & x. qiu 2010. cyp9a12 and cyp9a17 in the cotton bollworm, helicoverpa armigera: sequence similarity, expression profile and xenobiotic response. pest manag sci 66: 65-73. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7296sociobiology 69(2): e7296 (june, 2022) introduction wasps, as well as bees, demonstrate a behavioral spectrum that varies from solitary to eusocial. this is an important aspect for studies of social evolution in these groups (jeanne, 1980; carpenter, 1991; leadbeater et al., 2011; piekarski et al., 2018; manfredini et al., 2019; da-silva, 2021). wasps leave their nests daily to search for resources to maintain their colony. that foraging behavior is extremely important for understanding the social organization of wasps, their evolutive success, and the relationships between adults and young individuals in terms of the selection of resources to be gathered (cruz et al., 2006). abstract the study of foraging activity in wasps is important to understand the social organization and its evolutionary success. we examined aspects of the daily and seasonal foraging activities of mischocyttarus nomurae richards wasps, in terms of individual trip durationin the collection of different resources. the study was undertaken in two areas in the municipality of rio de contas, bahia state, brazil. observations were done for 10 colonies of m. nomurae in their post-emergence phase under natural conditions, five during the rainy period and five during the dry period. the amplitudes of the activity hours were similar between the two periods. the foraging efficiency index was higher (80.56%) during the rainy period than during the dry period (74.42%), with greater percentages of returns with all foraged items (with the exception of prey captures). temperature influenced positively and significantly the number of trips performed during the rainy period, while temperature and luminosity positively influenced the number of trips performed during the dry period. the mean duration of trips for different resources were greater during the dry period (with the exception of wood pulp), although those differences were not statistically significant. wasps spent the most part of their time nectar (83.60 min), followed by prey (21.06 min), and wood pulp (1.40 min). we observed that 52.56% of the foraging individuals of m. nomurae collected only a single resource type. sociobiology an international journal on social insects ramona s. da silva1, agda a. rocha1, edilberto giannotti2 article history edited by fabio s. nascimento, usp, brazil received 05 july 2021 initial acceptance 21 august 2021 final acceptance 18 april 2022 publication date 05 july 2022 keywords etology; polistinae; resources collected; social wasps. corresponding author ramona soares silva universidade federal da bahia campus anísio teixeira rua rio de contas, quadra 17, lote 58, bairro candeias, cep: 45029-094 vitória da conquista-ba, brasil. e-mail: ramonasoaress@yahoo.com.br foraging activity is generally influenced by external factors such as temperature, humidity, and luminosity (giannotti et al., 1995; andrade & prezoto, 2001; resende et al., 2001; paula et al., 2003; rocha & giannotti, 2007). however, depending on the necessities of the colony, there can be interactions between internal and external factors that influence foraging rhythms (detoni et al., 2015). the resources collected by social wasps include water to cool the nest, wood pulp to construct and enlarge the nest, carbohydrates from nectar, and the exudates of insects and proteins from prey that are used to feed larvae (jeanne, 1972; giannotti et al., 1995; raveret-richter, 2000; silva & noda, 2000; carpenter & marques, 2001). 1 universidade federal da bahia (ufba), campus anísio teixeira, vitória da conquista-ba , brazil 2 universidade estadual paulista júlio de mesquita filho (unesp), rio claro-sp, brazil research article wasps trip durations of daily and seasonal foraging activities in mischocyttarus nomurae (richards) (hymenoptera, vespidae) mailto:ramonasoaress@yahoo.com.br ramona s da silva, agda a rocha, edilberto giannotti – foraging activities in mischocyttarus nomurae (richards) (hymenoptera, vespidae)2 various studies have been undertaken focusing on the life hystory of social wasps and diverse aspects of their foraging behaviors, as well as analyzing the influences of environmental variations on them, as seen in the publications of giannotti et al. (1995), silva and noda (2000), andrade and prezoto (2001), resende et al. (2001), paula et al. (2003), cruz et al. (2006), rocha and giannotti (2007), hernández et al. (2009), castro et al. (2011), silva et al. (2012), elisei et al. (2013), and prezoto et al. (2016). mischocyttarus nomurae richards, 1978 is a littleknown species of social wasp. the only published materials available were the description of the species collected in ceará state, brazil (richards, 1978), the description of males from chapada diamantina, bahia state (silveira, 2004), and the study with the eggs and immatures this species (rocha & giannotti, 2016). souza et al. (2015) recorded the species for minas gerais state. considering the importance of foraging activities for wasps, and the fact that little is known about m. nomurae, we studied the foraging activities of m. nomurae to determine if: there were differences in the durations of trips to collect different types of resources; there were differences in the durations of those trips in relation to the rainy and dry periods; there existed any relationships between those activities and climatic and/or colony factors; each forager collects only a single type of resource. material and methods the present study was undertaken in two separate areas: cachoeira do fraga and raposo chalé, both within the municipality of rio de contas, bahia state, brazil. cachoeira do fraga is located 3 km from the city of rio de contas, while raposo chalé is 1.5 km distant. the municipality of rio de contas (13°34’44’’s; 41°48’41’’w) is situated in the centro sul baiano geographic mesoregion, in the southern extent of the chapada diamantina mountain range, at an altitude of 999 masl, and 612 km from the state capital of salvador. the region is included within the semiarid zone of brazil, with a subhumid to dry climate, a mean annual temperature of 19.1 °c, and a mean annual rainfall rate of 813.2 mm. the rainy period lasts from october to april. the vegetation is considered to be of the montane ecological refuge type with cerrado-caatinga contacts (sei, 2011). our observations were made under natural conditions during two periods of the year: in the rainy season (from october to april/2014) and in the dry season (from august to september/2014). five colonies in their post-emergence phase were accompanied during each period, totaling 10 colonies studied; the post-emergence phase is considered according to the criteria established by jeanne (1972): occurs from the emergence of the 1st adult to the beginning of irreversible reduction of the immature of the colony. one or two days before undertaking the observations of each colony, all of the adult wasps were marked with colored dots (using porcelain marking pens) on the dorsal region of their mesothorax. in this way each individual had a unique color identification code. to apply those markings, an insect net was extended near the nest to capture adult individuals. then, using a forceps to handle the insects, each individual was marked and subsequently released. additionally, the nests were mapped and the eggs, larva, and pupa in each nest were counted. each wasp colony of m. nomurae was monitored for 12 hours, beginning at 06:00h until 18:00h, totaling 120 hours of observations. temperature and relative humidity were recorded every 30 minutes (measured using a digital thermalhygrometer) as well as luminosity (using a digital light meter) (silva & noda, 2000). we recorded the time of each wasp leaving the nest, the time of its arrival back, as well as the resource it was carrying. the observations and identifications of the collected materials were based on the criteria of silva and noda (2000). the normality of the data was verified using the shapiro-wilk test, and homoscedasticity was verified using the levene test. the pearson correlation was used to analyze the relationship between the numbers of individuals in the colony and the numbers of foraging trips (for parametric data); the spearman correlation was used for data having nonparametric distributions. simple linear regression analysis was used to verify the influence of each environmental variable on foraging trips. the parametric student t-test was used to analyze the mean durations of the foraging trips in each season (for data demonstrating normal distributions); data demonstrating nonnormal distributions were analyzed using the mann-whitney nonparametric test. r program version 4.0.2 (r core team 2020) was used for those analyses, considering a significance level of p <0.05. the foraging efficiency index (giannotti et al., 1995) was calculated according to the formula: efficiency index = fr/fn x 100 where fr = nº of foragers that arrived with resource and fr = nº of foragers that arrived in the nest. results foraging activity patterns and colony factors the study focused on 10 colonies; their parameters are presented in table 1. the observations made during the rainy period identified the first foraging trips of m. nomurae occurring at 07:00 h, while the last return was recorded at 17:43 h, configuring amplitude of 10h and 43min of foraging per day (fig 1). that activity was less intense during the morning hours until 11:00 h, and after 17:00 h, when the numbers of returning foragers increased considerably. activity peaks during the rainy period occurred between 11:01 and 12:00 h, and again between 14:01 and 17:00 h. the foraging activities during the dry period did not differ from those of the rainy period. the first trips initiating at 07:05 h and the last returns at 17:41 h, with an amplitude sociobiology 69(2): e7296 (june, 2022) 3 of 10 h and 46 min (fig 2). the foraging activity increased as the day progressed, peaking at 13:01 to 14:00h, and then decreasing. comparing figures 1 and 2, it can be seen that foraging activities occurred more uniformly in the rainy period than in the dry period. the mean frequency of foraging trips remained the same during the two seasonal periods, with 2.87 ± 1.58 (0.20 – 4.80) trips during the rainy period and 2.85 ± 1.58 (0.00 – 5.60) trips in the dry period. the frequency of returns during the rainy period was slightly greater than in the dry period. period sampling date colony sub-phase of post-emergence (jeanne, 1972) nº de cells individuals e l p a rainy 04/18/2014 1 pre-male 44 13 16 9 8 04/17/2014 2 pre-male 44 13 14 7 7 04/20/2014 3 pre-male 81 14 29 16 7 04/20/2014 4 post-male 43 20 3 3 6 08/15/2014 5 post-male 33 7 20 4 10 dry 08/15/2014 6 pre-male 42 10 14 6 7 08/16/2014 7 pre-male 24 4 8 10 7 08/17/2014 8 pre-male 24 6 6 5 6 09/27/2014 9 post-male 47 18 17 9 14 09/28/2014 10 post-male 77 32 25 9 13 table 1. populational information concerning the 10 colonies of mischocyttarus nomurae wasps studied during the rainy and dry periods of 2014, in the municipality of rio de contas, bahia state, brazil. legend: e: eggs; l: larva; p: pupa; a: adults. fig 1. patterns of foraging activities of the five colonies of mischocyttarus nomurae wasps studied, and the mean values of the climatic factors of temperature (°c), relative humidity (%), and luminosity (lux) during the day, during the rainy period of 2014. ramona s da silva, agda a rocha, edilberto giannotti – foraging activities in mischocyttarus nomurae (richards) (hymenoptera, vespidae)4 fig 2. patterns of foraging activity of the five colonies of mischocyttarus nomurae wasps studied, and the mean values of the climatic factors of temperature (°c), relative humidity (%), and luminosity (lux) during the day, during the dry period of 2014. fig 3. correlation between the numbers of individuals in the nests and the foraging activities of mischocyttarus nomurae (r: pearson correlation for parametric data; ρ: spearman correlation for data having nonparametric distributions).*significant values sociobiology 69(2): e7296 (june, 2022) 5 during the rainy period the mean frequency of return flights was 3.07 ± 1.58 (0.20 – 4.80), and 2.78 ± 2.07 (0.00 – 6.00) during the dry period. all of the factors positively influenced the number of forage flights leaving the nest, especially the numbers of eggs and adults, which demonstrated correlation values above 50%; although only the number of eggs was statistically significant (fig 3). the foraging efficiency index varied between the two seasonal periods, being greater in the rainy period (80.56%) than during the dry period (74.42%). foraging activity and climatic factors temperature and luminosity values were lower at the beginning and end of the observation periods (fig 1 and 2). during the rainy period, the mean temperature was 27.6 °c (± 3.8); during the dry period the mean was 24.3 °c (± 3.5). luminosity varied greatly between the two periods, with a mean of 1001.6 lux (± 579.3) during the rainy period, and 513.9 lux (± 301.5) during the dry period. the means of relative humidity did not demonstrate variations between the two seasonal periods, being 51.4% during the rainy period and 51.9% during the dry period. in the rainy period, humidity negatively influenced foraging activity while luminosity and temperature hat a positive influence. although, only temperature demonstrated a statistically significant positive influence. during the dry period, temperature and luminosity both significantly positively influenced foraging activity (fig 4). fig 4. linear regression analysis among the environmental factors and foraging activities of mischocyttarus nomurae wasps during the rainy and dry periods. * significant values ramona s da silva, agda a rocha, edilberto giannotti – foraging activities in mischocyttarus nomurae (richards) (hymenoptera, vespidae)6 resource mean duration of the trips during the rainy period (min) mean duration of the trips during the dry period (min) results (mann-whitney ort test) nectar 67.91 (± 11.40) 83.60 (± 28.02) t = 1.1818 p = 0.2712 wood pulp 3.36 (± 7.51) 1.40 (± 1.95) u = 11.00 p = 0.7540 prey 11.15 (± 16.02) 21.06 (± 31.45) t = 0.6278, p = 0.5476 unfruitful 37.20 (± 16.43) 54.40 (± 45.18) t = 0.80173, p = 0.4459 table 2. mean duration of the foraging trips (min) to collect different resources during each period of the study, and the results of the tests used to compare the means between the rainy and dry periods of 2014, in the municipality of rio de contas, bahia state, brazil. resources collected and trip durations there were 168 returns during the dry period, and 184 during the rainy period, although the majority of both were fruitless; among successful foraging returns, the most frequent was with nectar, followed by prey, wood pulp, and unidentified materials (fig 5). water was not observed as a collection item in either of the seasonal periods. the greatest percentage of successful returns (considering all items) occurred during the rainy period, with the exception of returns with prey. fig 5. return frequencies of foraging individuals of mischocyttarus nomurae with items collected during the rainy and dry periods of 2014, in the municipality of rio de contas, bahia state, brazil. the mean duration of foraging trips made by m. nomurae to harvest different resources was greatest during the dry period, with the exception of wood pulp (table2). however, these differences were not significant statistically. based on this, the data showed that seasonality did not influence the mean duration of the foraging trips. of the 78 foraging individuals marked and accompanied, 52.56% (n = 41) were involved in the collection of only a single resource (nectar); the second most numerous group of individuals (24.36%; n = 19) collected two types of resources (nectar + wood pulp or nectar + prey); 21.79% (n = 17) collected nothing; 1.28% (n = 1) collected three resources (fig 6). prey, wood pulp, and nectar were observed being collected only in colony c10, with only a single individual collecting wood pulp. nectar was collected in colonies c3, c4, c6 and c8, with the majority of foragers performing those collections; on the other hand three of the seven foraging individuals in colony c6, collected nectar. wood pulp collection was only observed in three colonies (colonies c2, c9 and c10). in colony 2, three of the seven foraging individuals performed those collections, while in colonies c9 and c10, only one individual of each collected this resource. prey collection was observed in colonies c1, c5, c7, c9 and c10, with the foraging individuals collecting only that material (or nectar in addition to that material). sociobiology 69(2): e7296 (june, 2022) 7 discussion the foraging activities of m. nomurae were similar in terms of amplitude and the numbers of trips during both the dry and rainy periods. these results differ from were demonstrated in some species of wasps (giannotti et al., 1995 – polistes lanio lanio; silva & noda, 2000 – mischocyttarus cerberus; ribeiro-junior et al., 2005 – protopolybia exigua and castro et al., 2011 – mischocyttarus cassununga), in which the activity amplitude was higher in the warm and humid seasons (ranging between 9 to 12h in the total) than in cold and dry periods (ranging between 4 to 8h in the total). the foraging efficiency index of m. nomurae was greater during the rainy period. giannotti et al. (1995) likewise reported a greater foraging efficiency index of p. lanio lanio during the hot and humid season (89.3%) as opposed to the cold and dry season (68.8%). in addition to considerations of resource availability, the conditions of the colony, and the environment during any study, one must consider that foraging efficiency indices will reflect the unique characteristics of each species (silva & noda, 2000; ribeiro-junior et al., 2005; rocha & giannotti, 2007; castro et al., 2011). as such, and according to studies currently available, patterns of wasp activity tend to be different at different times of the year. foraging during the dry period is generally initiated in the second half of the morning and terminates relatively early, and is different from that observed in the rainy period, when foragers leave the nests in the first hours of the morning and continue foraging until the end of the day (silva & noda, 2000; ribeiro-junior et al., 2005). the pattern of foraging activity of m. nomurae in the dry period was similar to that of the rainy period – a result that differs from published accounts for other species. that result may reflect the fact that two of the five colonies observed during the dry period (c9 and c10), were monitored near the end of that season, and the climatic conditions at that time may not have been fully typical of the dry period. another factor that could have influenced the greater foraging activities of those two colonies (c9 and c10) when compared to the others (c6, c7 and c8), is the fact that c9 and c10 were larger than the latter three colonies, and consequently had more individuals available to undertake foraging activities. rocha et al. (2009) observed a positive correlation between the numbers of larva, eggs, pupa, and females of p. exigua and the numbers of trips from their nests. our results likewise evidenced that greater numbers of eggs in a nest positively influenced the numbers of foraging trips. however, the other members of the colony (larvae, pupae and adults) did not present correlation. in relation to foraging activity and climatic factors, it was found that only temperature positively and significantly influenced the number of foraging trips during the rainy period, while temperature and luminosity influenced that number during the dry period. studies have demonstrated that foraging activities are normally positively correlated with temperature and negatively correlated with relative humidity in different social wasp genera in the neotropical region (hernández et al., 2009; detoni & prezoto, 2021). studies undertaken with polistini and mischocyttarini wasps evidenced that temperature and luminosity were positively correlated with the numbers of foraging trips, and that relative humidity demonstrates a negative correlation with that behavior (silva & noda, 2000; montagna et al., 2009; elisei et al., 2013). fig 6. individual specializations of mischocyttarus nomurae wasps collecting different types of resources. r0 – no resource collected; r1– collection of a single resource; r2 – collection of two resources; r3 – collection of three resources. ramona s da silva, agda a rocha, edilberto giannotti – foraging activities in mischocyttarus nomurae (richards) (hymenoptera, vespidae)8 m. nomurae was never observed collecting water in either of the environmental periods, which indicates that temperatures had not risen sufficiently to require that resource for thermal regulation of the nests. among successful foraging trips, the resources harvested with the greatest frequencies corresponded to items used to feed the colony (nectar and prey) and wood pulp to expand the nest. that same result was reported in post-emergence phase colonies of polistes simillimus zikán by prezoto et al. (1994), of polistes ferreri by andrade and prezoto (2001), and of polistes versicolor olivier by elisei et al. (2010). studies with some species of wasps (andrade & prezoto, 2001 – p. ferreri; prezoto et al., 1994 – p. simillimus; rocha et al., 2009 – p. exigua) reported that the nectar was the most collected material, followed by prey and wood pulp. therefore, as in the present study, nectar was the most consistently collected resource. according to earlier publications (giannotti et al., 1995; giannotti, 1999; andrade & prezoto, 2001; rocha et al., 2009) that result can be explained by the fact that nectar serves as a food resource for larva as well as for adult individuals, and requires less energetic outlay than collecting live prey. the frequency of collecting wood pulp depends on the necessity of amplifying the numbers of cells in a nest (silva et al., 2012). the high percentage of prey resources captured by m. nomurae during the dry period was similar to that reported by giannotti et al. (1995) for p. lanio lanio, with 61.4% of the foragers returning with nectar and 5.3% with prey during the hot, rainy season, and 39.6% returning with nectar and 10.7% with prey during the dry, cold season. there was no significant influence of the season of the year on the mean duration of foraging flights by m. nomurae. in m. cerberus, however, the mean duration of foraging flights to collect different resources was greater during the cold season. resources are often scarce during cold periods, so that foragers will tend to spend more time on those trips (silva & noda, 2000). the time spent by m. nomurae collecting nectar (83.60 min) was considerably greater than that observed in other species of the same genus, such as m. cerberus (22.6 min) (giannotti, 1999) and mischocyttarus consimilis zikán (36 min) (montagna et al., 2009). foraging individuals of m. nomurae spent the most time collecting nectar resources, followed by prey and wood pulp, as it was similarly observed with m. consimilis (montagna et al., 2009), indicating that wasps encounter more difficulties searching for food resources. the fact that the mean duration of trips by m. nomurae for collecting wood pulp was shorter during the dry periodthan the rainy period can perhaps be explained by the fact that nest enlargement activity is lower during the dry period. in terms of individual foraging, m. nomurae demonstrated behaviors different from p. lanio lanio, with 56.3% of the latter wasps collecting two types of resources while 43.7% collected only a single resource type (giannotti et al., 1995). silva and noda (2000) likewise reported that most of the foragers of m. cerberus collected two different types of resources. giannotti et al. (1995) and silva and noda (2000) both observed that there was no individual specialization in p. lanio lanio or m. cerberus, as most foraging individuals collected two types of resources – which was different from the results of the present work, where most foraging individuals of m. nomurae collected only a single type of resource. although m. nomurae foragers collected all of the different resources, more than half of them collected only a single type. of the 78 foragers of m. nomurae accompanied, 21.5% left the nest but returned without any resource at all. that value was different from data for m. cerberus (silva & noda, 2000), in which of the 30 foraging trips only 6.6% returned without any collected resource. the elevated percentage of nonproductive trips observed in m. nomurae nests may be related to the fact that those individuals were only just initiating their foraging activities and were still in a learning phase. according to o’donnell and jeanne (1992) in their studies of p. occidentalis, as the ages of the wasps increased so did their tendency to collect only a single type of resource, together with the increased success of those trips. the largest percentages of foraging individuals of m. nomurae returning to their nests in both seasons studied were apparently unsuccessful. that result may be explained by the lack of resource availability. furthermore, recently emerged individuals that leave the nest for foraging, however, may still be in their learning phase and, therefore, will not always be successful (o’donnell & jeanne, 1992). detoni & prezoto (2021) hypothesized that the youngest wasps, still in their learning phase, will only make short flights around the nest to memorize visual cues and landmarks in the environment; as they grow older and more experienced their foraging flights will be more successful, as it was observed by o’donnell and jeanne (1992) with p. occidentalis. final considerations the present study provided new information about the poorly studied wasp species m. nomurae. its foraging activities were found to be influenced by temperature in both seasons, as well as by luminosity during the dry period. the presence of eggs in the nest was also found to significantly influence foraging activity. there were not observed collection patterns among the different colonies. the items were collected depending on the colonies necessities, resource availability, and the capacity of each individual to forage at any given time. it would therefore be interesting to examine the behavior ecology of that species and determine whether it demonstrates age polyethism. authors' contributions rss: conceptualization, methodology, investigation, formal analysis, resources, writing. sociobiology 69(2): e7296 (june, 2022) 9 aar: conceptualization, methodology, supervision, resources, formal analysis, writing. eg: supervision, formal analysis, writing. acknowledgments the authors thank adriele frança, luisa sodré and thatiana andrade for their help in collecting the data. to messrs. juraci and marcos for making their properties available for this work. references andrade, f.r. & prezoto, f. 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(2015). new occurrences of social wasps (hymenoptera: vespidae) in semideciduous seasonal montane forest and tropical dry forest in minas gerais and in the atlantic forest in the state of rio de janeiro. entomobrasilis, 8: 65-68. doi: 10.12741/ebrasilis.v8i1.359. about:blank about:blank about:blank about:blank about:blank _hlk69764781 _hlk69760443 _hlk69759884 _hlk69762117 _hlk64880666 _hlk69886778 _hlk66563066 107 population divergence of melipona scutellaris (hymenoptera: meliponina) in two restricted areas in bahia, brazil by cristovam alves de lima junior1, carlos alfredo lopes de carvalho1, lorena andrade nunes2 & tiago maurício francoy3 abstract melipona scutellaris latreille has great economic and ecological importance, especially because it is a pollinator of native plant species. despite the importance of this species, there is little information about the conservation status of their populations. the objective of this study was to assess the diversity in populations of m. scutellaris coming from a semideciduous forest fragment and an atlantic forest fragment in the northeast brazil, through geometric morphometric analysis of wings in worker bees. in each area, worker bees were collected from 10 colonies, 10 workers per colony. to assess the diversity on the right wings of worker bees, 15 landmarks were plotted and the measures were used in analysis of variance and multivariate analysis, principal component analysis, discriminant analysis and clustering analysis. there were significant differences in the shape of the wing venation patterns between colonies of two sites (wilk’s λ = 0.000006; p < 0.000001), which is probably due to the geographical distance between places of origin which impedes the gene flow between them. it indicates that inter and intrapopulation morphometric variability exists (p <0.000001) in m. scutellaris coming from two different biomes, revealing the existence of diversity in these populations, which is necessary for the conservation of this bee species. key words: geometric morphometrics, diversity, conservation. 1 insecta research group, centro de ciências agrárias, ambientais e biológicas, ufrb, c. postal: 118, 44380-000, cruz das almas-ba. brazil. e-mail: cristovamalves@yahoo.com.br; calfredo@ufrb. edu.br 2 escola superior de agricultura “luiz de queiroz”, universidade de são paulo, 13418-900, piracicabasp. brazil. e-mail; lorenunes1@yahoo.com.br 3 escola de artes ciências e humanidades, universidade de são paulo, 03828-000, são paulo-sp. brazil. e-mail: tfrancoy@usp.br 108 sociobiolog y vol. 59, no. 1, 2012 introduction the bees of the genus melipona illiger, 1806 occur throughout the neotropical region, and this genus that has the largest number of species in the subtribe meliponina (silveira et al. 2002). among the species of melipona occurring in bahia state, in northeast brazil, m. scutellaris latreille 1811, m. quadrifasciata lepeletier 1836 and m. mandacaia smith 1863, have an emphasis as honey producers (carvalho et al. 2003). despite its economic and ecological importance, there are few studies about the current state of the populations conservation of these species. according to nunes et al. (2007), this kind of information is important in the preparation of management plans for the preservation of m. scutellaris, since human actions, such as deforestation, use of insecticides and inadequate management of colonies, result in a decrease in populations of stingless bees and the consequential loss of biodiversity. in this context, the use of morphometric techniques have been used as a tool for morphological, taxonomic, phenotypic and geographic studies among and within bee populations, as in the works carried out by crewe et al. (1994), diniz-filho & malaspina (1996 ), araujo et al. (2004), mendes et al. (2007) and francoy et al. (2011). among the morphometric methods, geometric morphometrics provides powerful tools for the study of morphological variation among populations and therefore offers promising prospects for many problems such as the study of biodiversity patterns (albert et al. 2001). in this technique, the first step is the definition of a structure and landmarks that are homologous among organisms under study (monteiro & reis 1999). in bees, wings have been used in morphometric analysis because they are quite flat structures and due to the ease of measuring the size and shape (diniz-filho & bini 1994, nunes et al. 2007). the objective of this work was to study the population divergence through the shape of wing venation of worker bees in m. scutellaris, originating from colonies living in two restricted areas, a semideciduous forest fragment in the chapada diamantina and an atlantic forest fragment area in the coastal zone in the state of bahia. 109 lima, c.a. et al. — population divergence of m. scutellaris in brazil material and methods populations of m. scutellaris were sampled between january and february 2009 in two restricted areas, one in an atlantic forest fragment coastal zone in the city of vera cruz, bahia, itaparica island (12°57’37’’s; 38°36’31’’w; altitude: 13 m) and the other in a semideciduous forest fragment in the chapada diamantina region, in the municipality of novo mundo, bahia (12°02’59”s and 40°29’43”w, altitude 604 m). these areas have a distance of 230 km between them. traits of the right forewings of worker bees were used, with 10 workers of each colony and 10 colonies per area, totaling 200 subjects from 20 colonies. the colonies were named according to the place of origin, and to the semideciduous forest fragment – colonies sff 01 to 10, and the atlantic forest fragment – colonies aff 01 to 10. the wings were removed and placed between two plates to capture images with the motic 2.0 ml program using a digital camera coupled to a stereomicroscope, with 7.5 x magnification. the wings of each subject were identified and stored in eppendorf tubes of 1.5 ml and deposited at the núcleo de estudos dos insetos (experimental center for the studies of insects) (insecta), at the centro de ciências agrárias, ambientais e biológicas (center of agricultural, environmental and biological sciences) of the federal university of reconcavo of bahia. geometric morphometric analysis 15 landmarks on the forewing were defined and recorded using the tpsdig program, version 2.12 (rohlf 2008a) as shown in fig. 1. fig. 1. landmarks used in morphometric analysis of the forewing of melipona scutellaris. 110 sociobiolog y vol. 59, no. 1, 2012 with the tpsrelw program, version 1.46 (rohlf 2008b), using the archive of recorded images of landmarks, we extracted the x and y coordinates of each landmark of the aligned wings (alx and aly), the centroid size of the wings and the matrix w which consists of the deformed partial scores plus the estimate of the uniform components (rohlf 1996). this matrix w can be used as a data matrix in order to perform various multivariate analyzes (rohlf 2008b). centroid size is the square root of the sum of squared distances from each landmark to the centroid (center of mass of the configuration), and this measure is, in the absence of allometry (correlation of size and shape), the only variable size that is not correlated with the variables of form (rohlf 2008b). the partial deformation vectors are generated from the projection of the coordinates of each subject in the main deformation, in this way the partial distortions are the characteristics of each subject and are interpreted as the coordinates of each specimen (ferreira 2009). the uniform components are calculated from all the landmarks in relation to the average shape and express information on a global scale (uniform) of the shape variation (monteiro & reis 1999). the visualization of the displacement of landmarks and the deformation grids was performed using the morphoj software, version 1.02g (klingenberg 2011). the programs used for morphometric analysis are available on http://life. bio.sunysb.edu/morph. statistical analysis the coordinates of the landmarks of the aligned wings and the centroid size were used in principal component analysis (pca). the data matrix w were used in multivariate analysis of variance (manova) and cluster analysis by upgma (unweighted pair-group method using arithmetic average) performed with the statistica 9.0 (stat soft 2010) program and discriminant analysis performed using the xlstat software, version 2011.1.02. the cophenetic correlation coefficient was calculated by the statistical genes program (cruz 2006). the centroid size was also assessed using an analysis of variance in the statistical statistica 9.0 (stat soft 2010) program. 111 lima, c.a. et al. — population divergence of m. scutellaris in brazil results and discussion multivariate statistics principal component analysis and analysis of multivariate variance there were 27 principal components generated using the cartesian coordinates of landmarks of the aligned wings and the centroid size of the wings. it took the first nine components to explain over 76.0% of the variation of the data (table 1). the separation of the bees by area of origin was plotted graphically in a twodimensional space formed by the scores of the first two principal components that explained 16.95% and 13.57% of the variation, respectively (fig.2). fig. 2. dispersal of populations of melipona scutellaris of two restricted areas in the state of bahia with the principal component analysis. points with the same format in the graph represent the worker bees belonging to the respective areas as shown in the legend. sff = semideciduous forest fragment; aff = atlantic forest fragment. 112 sociobiolog y vol. 59, no. 1, 2012 variables aly4, aly9, aly11 and aly15 were the ones that contributed most to the first principal component, while for the second principal component, the variables that most influenced it were alx1, aly1, and alx2 aly13 (table 2). the multivariate analysis of variance (manova) showed the existence of a highly significant difference in the shape of wings between colonies of different areas (wilk’s λ = 0.000006; p < 0.000001). we also found difference among colonies from the same location (sff: wilk’s λ = 0.000045; table 1. eigenvalues and total and cumulative variation of the principal components obtained from the cartesian coordinates of the aligned wing landmarks and the centroid size of the wings of melipona scutellaris of two restricted areas in the state of bahia. principal component eigenvalue total variation (%) cumulative eigenvalue cumulative variation (%) 1 5.2537 16.9475 5.2537 16.95 2 4.2060 13.5677 9.4597 30.52 3 3.6318 11.7155 13.0915 42.23 4 2.6447 8.5313 15.7362 50.76 5 2.1901 7.0647 17.9263 57.83 6 2.0854 6.7270 20.0116 64.55 7 1.3303 4.2914 21.3420 68.85 8 1.2040 3.8840 22.5460 72.73 9 1.0800 3.4839 23.6260 76.21 10 0.9275 2.9920 24.5535 79.20 11 0.7939 2.5610 25.3474 81.77 12 0.6834 2.2044 26.0308 83.97 13 0.5996 1.9340 26.6304 85.90 14 0.5601 1.8069 27.1905 87.71 15 0.5082 1.6395 27.6987 89.35 16 0.4417 1.4249 28.1405 90.78 17 0.3966 1.2795 28.5371 92.06 18 0.3549 1.1448 28.8920 93.20 19 0.3355 1.0824 29.2275 94.28 20 0.2971 0.9584 29.5246 95.24 21 0.2918 0.9411 29.8164 96.18 22 0.2362 0.7620 30.0526 96.94 23 0.2296 0.7405 30.2822 97.68 24 0.2270 0.7322 30.5091 98.42 25 0.2107 0.6797 30.7198 99.10 26 0.1537 0.4959 30.8736 99.59 27 0.1264 0.4079 31.0000 100.00 113 lima, c.a. et al. — population divergence of m. scutellaris in brazil p < 0.000001 and aff: wilk’s λ = 0.000168; p < 0.000001), demonstrating the existence of morphometric variability between and within the areas of origin of these colonies. all 26 variables used of the matrix w used in manova contributed significantly (α = 0.05%) to the separation of the colony. in a similar context, francoy et al. (2009) found that the obtained partial deformation of the wings of both males and the worker bees have contributed significantly to the separation of five species of stingless bees. it indicates that the variables obtained in the geometric morphometric analysis can be used to assess the morphometric variability between and within species of bees. the change of the overall shape shows the greatest variations in the landmarks 04, 11 and 05 (fig. 3). since landmarks 04 and 11 are located in the extreme points of the wing, it indicates a variation in the total length of this structure among the sampled bees. the size variation of the wings from the two populations was confirmed whit the significant differences found in the variance analysis of the centroid size (f = 70.3579; p <0.001). the bees originating from the semideciduous forest fragment have the centroid size greater than the bees from the atlantic forest fragment, indicating that the wings of workers of m. scutellaris from the semideciduous forest table 2. contribution of variables to the princip a l c omp onents o bta ine d from cartesian coordinates of the aligned wing landmarks and the centroid size of melipona scutellaris. alx and aly = x and y coordinates of each landmark of the aligned wings. variable principal component cp01 cp02 centroid size -0.550453 -0.243003 alx1 -0.042075 0.776222 aly1 -0.306264 0.641001 alx2 -0.188666 -0.606984 aly2 0.202185 -0.121721 alx3 -0.177111 -0.518128 aly3 -0.250889 -0.136957 alx4 0.656952 0.434852 aly4 -0.787486 0.351908 alx5 -0.630051 -0.532158 aly5 -0.199672 -0.377015 alx6 0.343194 -0.241252 aly6 0.165304 0.175745 alx7 0.236879 0.066574 aly7 0.276928 -0.080970 alx8 0.207306 -0.229745 aly8 0.524310 -0.306929 alx9 0.194469 -0.113525 aly9 0.754889 -0.058321 alx10 0.148413 -0.162990 aly10 0.533427 -0.180559 alx11 -0.687989 0.309534 aly11 0.542941 0.173048 alx12 0.381650 0.135767 aly12 0.202381 -0.396282 alx13 -0.425072 -0.473367 aly13 -0.273201 -0.676573 alx14 0.002385 -0.037379 aly14 -0.014791 -0.282355 alx15 -0.073616 0.384956 aly15 -0.685634 0.435803 114 sociobiolog y vol. 59, no. 1, 2012 fig. 3. deformation grid showing the change in the overall shape of the wings of melipona scutellaris of two restricted areas in the state of bahia. fig. 4. wing centroid size of melipona scutellaris of two restricted areas in the state of bahia. sff = semideciduous forest fragment; aff = atlantic forest fragment. 115 lima, c.a. et al. — population divergence of m. scutellaris in brazil fragment are larger (fig. 4). araújo et al. (2004) in studies with six species of stingless bees, among which there were three species of melipona, revealed that the generalized wing size is strongly correlated to the flight distance, which suggests that the stingless bees occupy a maximum effective space proportional to body size, especially with wing dimensions, which might constitute strong constraints on local populations restricted to forest fragments. discriminant analysis in the discriminant analysis performed using the matrix w, it can be seen that 91.0% of the bees were correctly classified within their respective colonies (table 3). the semideciduous forest fragment colonies stood out with five colonies that had correct classification rates of 100.0% (colonies sff 03, 04, 06, 07 and 10). as for the atlantic forest fragment area, there were four colonies with 100.0% of correct classification (colonies aff 01, 03, 04 and 07). the colonies that had the lowest rates of correct classification (70.0%) were the colonies sff 01 and aff 10. mendes et al. (2007), noted that in stingless bees, geometric morphometrics can be used as a technique for the discriminant analysis even in small populations. by cross-validation (table 3), the average of correct identification of individuals to its correct colony of origin was 72.0%, with the colonies of the aff 01 and 03 and sff 06 standing out, which had 100.0% of subjects correctly identified. the discriminant analysis performed for the classification of subjects between areas revealed that in the atlantic forest fragment a correct classification rate of 100.0% was obtained, while the semideciduous forest fragment rate was 99.0%, and the overall average was 99.5%. in cross-validation tests the overall average was 97.5%, and for the semideciduous forest fragment the correct identification of its subjects was 98.0%, while for the atlantic forest fragment the rate was 97.0%. despite the absence of a record of discriminant analysis in populations of m. scutellaris, rates of correct classification and identification obtained in this study are considered high when compared to the works of tofilski (2008) and francoy et al. (2008), which had rates of 84.9 and 97.8% of correct identification by cross-validation for discrimination of subspecies of apis mellifera with geometric morphometric variables, but also with those of mendes et al. 116 sociobiolog y vol. 59, no. 1, 2012 (2007) and francoy et al. (2009) in studies of species of stingless bees that reached from 77.6 to 100.0% of ratings and from 69.79 to 100.0% of correct identifications of subjects in respective groups. comparing the rates of correct classification and identification considering the areas and also the colonies as a separate group, the highest correct rates found in the discrimination per area can be verified, since these higher rates of classifications and identifications are due to the greater variability in shape that are occurring between the areas of origins of the colonies than among the colonies themselves. table 3. classification and correct identification of subjects within their respective colonies and areas of origin by discriminant analysis and cross validation, respectively. sff = semideciduous forest fragment; aff = atlantic forest fragment. colony correct classification (%) correct identification (%) sff 01 70.00 60.00 sff 02 90.00 70.00 sff 03 100.00 80.00 sff 04 100.00 40.00 sff 05 90.00 80.00 sff 06 100.00 100.00 sff 07 100.00 80.00 sff 08 80.00 50.00 sff 09 90.00 90.00 sff 10 100.00 80.00 aff 01 100.00 100.00 aff 02 90.00 70.00 aff 03 100.00 100.00 aff 04 100.00 90.00 aff 05 90.00 80.00 aff 06 80.00 70.00 aff 07 80.00 40.00 aff 08 100.00 80.00 aff 09 90.00 40.00 aff 10 70.00 40.00 average 91.00 72.00 area correct classification (%) correct identification (%) semideciduous forest fragment 99.00 98.00 atlantic forest fragment 100.00 97.00 average 99.50 97.50 117 lima, c.a. et al. — population divergence of m. scutellaris in brazil cluster analysis the mahalanobis d2 distances between colonies were calculated for the cluster analysis (table 4). all distances were statistically significant (α = 0.05) indicating the existence of significant differences among the colonies. based on the mahalanobis distance the clustering of the colonies was performed by the method of upgma. in the dendrogram obtained (fig. 5) it can be observed the formation of nine groups consisting of: i – colonies sff 01 and 02; ii – colonies sff 04, 08 and 09; iii – colonies sff 10; iv – colonies sff 03 and 05; v – colonies sff 06 and 07; vi colony aff 01; vii – colony aff 02; viii colony aff 03, and ix – colonies aff 04, 05, 06, 07, 08, 09 and 10. the cophenetic correlation coefficient (ccc) for the dendrogram was 0.80 (p <0.01). the ccc shows a good fit between the graphical representation of the original distances and its original matrix according to bussab fig. 5. dendrogram generated by upgma through the mahalanobis distances between colonies of the semideciduous forest fragment (sff) and the atlantic forest fragment (aff). roman numbers represent the groups formed. 118 sociobiolog y vol. 59, no. 1, 2012 ta bl e 4 . d is si m ila ri ty b et w ee n co lo ni es o f m el ip on a s cu te lla ri s c om in g f ro m a se m id ec id uo us f or es t f ra gm en t a nd an a tla nt ic f or es t f ra gm en t i n th e s ta te of b ah ia . a t t he to p of th e ta bl e ar e th e m ah al an ob is d is ta nc es d 2 a nd a t t he b ot to m o f t he ta bl e is th e st at is tic al si gn ifi ca nc e (p ). sf f = s em id ec id uo us fo re st f ra gm en t; a ff = a tla nt ic f or es t f ra gm en t. c ol on y sf f0 1 sf f0 2 sf f0 3 sf f0 4 sf f0 5 sf f0 6 sf f0 7 sf f0 8 sf f0 9 sf f1 0 a ff 01 a ff 02 a ff 03 a ff 04 a ff 05 a ff 06 a ff 07 a ff 08 a ff 09 a ff 10 sf f0 1 9. 46 48 .8 7 24 .6 8 61 .3 6 45 .3 8 51 .9 9 33 .0 3 29 .0 1 28 .2 4 87 .5 4 83 .9 2 10 5. 16 57 .1 2 49 .3 5 83 .5 5 83 .5 6 58 .8 3 56 .5 1 66 .0 1 sf f0 2 0. 05 38 .9 7 23 .6 1 48 .6 0 41 .1 2 45 .8 2 26 .7 4 34 .0 4 30 .0 7 97 .9 3 89 .9 6 10 8. 78 60 .3 5 57 .3 6 85 .4 4 94 .2 3 68 .2 7 64 .6 3 76 .4 0 sf f0 3 0. 00 0. 00 27 .6 3 18 .3 1 38 .3 5 33 .2 2 25 .0 4 27 .6 4 35 .1 2 86 .1 4 63 .0 4 57 .4 0 54 .0 7 62 .9 8 50 .1 6 73 .1 3 55 .3 5 42 .9 4 43 .8 3 sf f0 4 0. 00 0. 00 0. 00 39 .1 3 39 .7 0 45 .3 6 13 .6 5 28 .0 3 32 .9 9 77 .9 5 56 .7 4 78 .6 2 38 .8 0 33 .9 8 58 .0 6 68 .6 0 45 .2 5 38 .5 4 41 .8 8 sf f0 5 0. 00 0. 00 0. 00 0. 00 45 .8 9 23 .2 6 38 .8 0 43 .2 3 51 .4 9 11 1. 66 67 .1 2 67 .3 9 63 .4 5 73 .4 7 68 .6 9 93 .7 9 58 .6 9 53 .5 0 66 .1 6 sf f0 6 0. 00 0. 00 0. 00 0. 00 0. 00 27 .0 2 32 .5 3 30 .1 8 54 .2 1 92 .2 8 71 .0 3 75 .9 3 58 .7 8 56 .1 9 53 .7 8 73 .0 0 67 .2 6 45 .7 0 58 .2 0 sf f0 7 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 30 .9 1 32 .4 2 43 .8 2 10 2. 32 75 .6 8 89 .3 4 62 .0 6 76 .9 5 70 .4 6 85 .4 5 66 .3 5 55 .5 4 76 .4 7 sf f0 8 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 23 .7 1 38 .7 1 87 .0 3 61 .5 0 83 .5 9 42 .2 6 50 .2 1 66 .6 3 73 .5 1 59 .9 7 45 .5 1 55 .5 4 sf f0 9 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 37 .0 4 63 .9 8 59 .8 7 78 .3 3 50 .6 6 43 .7 0 53 .9 5 67 .9 0 63 .5 1 35 .0 2 43 .8 5 sf f1 0 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 99 .1 5 84 .0 3 10 3. 57 54 .0 4 72 .8 5 84 .9 4 87 .9 1 64 .2 0 57 .3 5 75 .7 6 a ff 01 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 38 .6 0 56 .2 0 40 .1 9 32 .3 4 53 .7 0 49 .3 3 51 .0 7 36 .5 6 37 .8 8 a ff 02 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 30 .2 7 28 .1 0 34 .9 2 47 .7 9 51 .4 6 32 .8 0 27 .7 5 43 .5 9 a ff 03 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 39 .7 7 50 .6 6 32 .4 4 42 .7 7 33 .9 3 32 .3 7 32 .2 8 a ff 04 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 19 .8 1 33 .1 8 26 .1 4 21 .1 1 19 .5 6 32 .0 1 a ff 05 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 28 .7 8 26 .9 7 22 .5 3 17 .3 0 20 .5 7 a ff 06 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 12 .4 6 26 .3 1 18 .4 6 15 .8 1 a ff 07 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 20 .6 3 19 .8 9 22 .6 6 a ff 08 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 22 .8 1 21 .1 9 a ff 09 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 17 .0 9 a ff 10 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 119 lima, c.a. et al. — population divergence of m. scutellaris in brazil et al. 1990, thus enabling the realization of inferences by means of visual assessment of the figure. in this dendrogram the formation of groups composed of a variable number of colonies stands out, with the existence of clusters with up to seven colonies (group ix) and also colonies that are in isolation (groups iii and vi). this group formation reveals the similarity of the shape of wings among the colonies, and colonies grouped into a single set of wings are more similar to each other, while colonies that were isolated have wings more divergent in the populations studied. it was also noted that there is not any clustering of colonies from different origins at any time, which also shows the divergence between these colonies with respect to the location from which they were collected. according to francoy & imperatriz-fonseca (2010), the variables generated in geometric morphometrics are independent of the size of structures, which eliminate part of the environmental influence that affects the development of organisms, especially insects. thus, the shape variation found among the colonies, mainly coming from different areas may be due to genetic differences between these colonies. the subject’s phenotype (e.g. morpholog y, in this case the shape of the wing ) is the result of their interaction with genetic and environmental factors and when the effect of these environmental factors is eliminated there is a greater effect of genetics on the phenotype of the subject. as the distance of the stingless bee swarming is short (kerr et al. 1996), the geographical distance between these environments can be considered as a factor that prevents the exchange of genetic material between the colonies, as there is no reference that there has been any handling or transport of the colonies from one area to another. gonçalves (2010), when studying variability in frieseomelitta varies by geometric morphometrics and molecular analysis of wings, suggested that the genetic diversity found in the small population under study is the result of gene flow via males, which have greater ability to fly and thus may originate from further distant regions and may have fertilized queens of the isolated population, thus introducing new alleles in that population. this hypothesis can also be adopted as the source of the diversity found among colonies of the same geographical origin, because near the locations where the bees were collected from there is a forest area with colonies in their natural state. 120 sociobiolog y vol. 59, no. 1, 2012 the wing shape variations found in this work show that the use of geometric morphometrics of wings is a simple, practical, inexpensive and important tool in assessing diversity within populations of bees. conclusion the geometric morphometric analysis of wings indicates that inter and intrapopulation morphometric variability exists in melipona scutellaris coming from a semideciduous forest fragment and an atlantic forest fragment in the state of bahia, revealing the existence of diversity in these populations, which is necessary for the conservation of this bee species. acknowledgments the authors thank conselho nacional de desenvolvimento científico e tecnológico (cnpq) (proc. 303237/2010-4), coordenação de aperfeiçoamento de pessoal de nível superior (capes) (aux-pe-pnpd-1598/2008) for the scholarships and financial support and dr. wyratan da silva santos for his critiques and suggestions. references addinsoft sarl 2011. xlstat versão 2011.1.02. para windows. alibert p., b. moureau, j.l. dommergues & b. david 2001. differentiation at a microgeographical scale within two species of ground beetle, carabus auronitens and c. nemoralis (coleoptera: carabidae): a 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(http://life.bio.sunysb.edu/morph/index.html, acessado em 21/outubro/2008). 122 sociobiolog y vol. 59, no. 1, 2012 rohlf, f.j. 2008b. program tpsrelw version 1.46. department of ecolog y and evolution, state university of new york, stony book. (http://life.bio.sunysb.edu/morph/index. html, acessado em 21/outubro/2008). stat soft 2010. statistica for windows: versão 9.0. tulsa. tofilski, a. 2008. using geometric morphometrics and standard morphometry to discriminate three honeybee subspecies. apidologie 39:558-563. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.461-469sociobiology 61(4): 461-469 (december, 2014) pollen analysis of food pots stored by melipona subnitida ducke (hymenoptera: apidae) in a restinga area introduction stingless bees (apidae: meliponini) occur in most tropical or subtropical regions of the world and the melipona genus is exclusively found in the neotropical region (camargo & pedro, 2007). they have perennial colonies with hundreds to thousands of workers and require continuous foraging activity to meet their food requirements (roubik, 1989). faced with the need to forage several food resources, the stingless bees have a generalist behavior concerning the plants visited, but a small number of plant species are most exploited in local communities (ramalho et al., 1989). the study of the plant-pollinator interaction can be performed by sampling bees in flowers (imperatriz-fonseca et al., 2011) or indirectly by morphological identification of pollen loads transported on the workers’ corbiculae or stored in food pots inside their nests (barth, 2004). stingless bees usually store food resources in pots built with cerumen (the mixture of bees wax and resin) for the abstract the geographic distribution of melipona subnitida ducke covers the dry areas in the northeastern brazil, where it plays an important role as pollinator of many wild plant species. in the current study, the botanical species this bee uses as pollen and nectar sources in a restinga area of maranhão state, brazil, were identified by analyzing pollen grains present in their storage pots in the nests. samples were collected from five colonies bimonthly, from april 2010 to february 2011. in all the samples, 58 pollen types were identified; the families fabaceae (8) and myrtaceae (5) had the largest number of pollen types. in the pollen pots, 52 pollen types were identified; fabaceae, melastomataceae, myrtaceae and dilleniaceae species were dominant. in honey samples, 50 pollen types were found, with a predominance of nectariferous and polliniferous plant species. out of the total of pollen types from nectariferous plants identified in honey, 20 pollen types contributed to the honey composition. humiria balsamifera occurred in high frequency and was predominant in october. chrysobalanus icaco, coccoloba sp., cuphea tenella and borreria verticillata were also important for honey composition. the occurrence of a high number of minor pollen types indicated that m. subnitida visits many species in the locality; however, it was possible to observe that its floral preferences are very similar to those from other melipona species. sociobiology an international journal on social insects rs pinto, pmc albuquerque, mmc rêgo article history edited by denise araujo alves, esalq-usp, brazil received 30 september 2014 initial acceptance 06 november 2014 final acceptance 10 december 2014 keywords pollen, honey, nectariferous plants, floral resources, stingless bee, meliponini. corresponding author rafael sousa pinto universidade federal do maranhão departamento de biologia laboratório de estudos sobre abelhas av. dos portugueses, 1966, 65080-805 são luís, ma, brazil e-mail: rafael_spinto@hotmail.com colony’s future use. the pots that contain pollen or honey are irregularly distributed, using all the free spaces of the hollow or cavity where they are located, and have a completely random arrangement (camargo, 1970). however, in general, the pollen pots are located closer to the brood combs and opposite to the honey pots. several studies have identified the plants species collected by stingless bees by analyzing the contents of pollen pots (ramalho et al., 1989; wilms & wiechers, 1997; pick & blochtein, 2002) and pollen grains present in honey (iwama & melhem, 1979; carvalho et al., 2001; martins et al., 2011). using these analyses, it is possible to define the floral preferences of the visitors; the most abundant pollen types have greater relevance for the bees’ species. the knowledge of the floral resources are necessary for the maintenance of bee communities in their habitats is crucial to understanding the mutualistic relationship between plants and bees and to developing management programs for pollinators, reforestation and environmental restoration research article bees universidade federal do maranhão, são luís, ma, brazil rs pinto, pmc albuquerque, mmc rêgo pollen analysis of food pots stored by melipona subnitida462 (luz et al., 2007). for example, strategies for planting natural resources can be developed to supply food during periods of shortage that bees may face. the aim of the current study was to identify the botanical species that are sources of pollen and nectar for melipona subnitida ducke in a restinga (coastal sandy plain) environment. m. subnitida, locally known as jandaíra, occurs in brazil only in the northeast region and is very frequent in the caatinga biome (martins, 2002). in maranhão state, this species occurs in a restinga area of the lençóis maranhenses nacional park (rego & albuquerque, 2006) and in the parnaíba delta (silva et al., 2014). m. subnitida is of great importance for the pollination of the regional native flora (ferraz et al., 2008) and cultivated plants (cruz et al., 2004; silva et al., 2005) and is traditionally reared for honey production, which has a high economic value. materials and methods the current study was conducted on a m. subnitida meliponary located in the municipality of barreirinhas, in the lençóis maranhenses national park (parque nacional dos lençóis maranhenses; 2º58’12”s, 42º79’56”w), maranhão state, brazil. the climate in the study region is classified as tropical megathermal (aw’ type, according to the köppen classification). the average annual temperature is approximately 27°c, and the annual precipitation is approximately 2,000 mm. there are two well-defined seasons: a rainy season from january to july and a dry season from august to december (brazilian institute of environment and renewable natural resources ibama, 2002). the vegetation of the lençóis maranhenses national park covers an area of 453.28 km², of which 405.16 km² are predominately composed of restinga vegetation. the rest of the vegetation consists of mangroves and riparian forests. the restinga area has plant species that are specific to this type of vegetation and plants characteristic of cerrado (brazilian savanna), caatinga (semi-arid) and rainforest. shrub species are dominant, and herbaceous communities are also present in large areas surrounding lakes (brazilian institute of environment and renewable natural resources ibama, 2002). samples of the pollen and honey pots were collected in april, june, august, october and december 2010 and february 2011 from five randomly chosen nests. in each nest, 2-3 g of pollen and 10 ml of honey were extracted with a spatula and a disposable syringe, respectively. in total, 30 pollen samples and 30 honey samples were collected (5 nests x 6 months). pollen and honey samples were acetolysed by erdtman’s method (1960) to facilitate the observation of the outer pollen wall (exine). however, the honey samples (10 ml) were divided into two test tubes before they were subjected to the acetolysis process (erdtman, 1960). distilled water (10 ml) was added to each tube, and the tubes were centrifuged for 5 min at 2,000 rpm (louveaux et al., 1978). the supernatant was discarded, and the pellet was subjected to the acetolysis process. slides were then prepared using glycerin jelly for optical microscopy analysis. the pollen grains were separated into pollen types according to their morphology and were photographed under a zeiss primo star optical microscope. the identification of the pollen grains was performed by comparing them with both a pollen collection from the regional flora and the literature (roubik & moreno, 1991; carreira & barth, 2003; silva et al., 2010). the classification system adopted for the level of family was the apg iii (2009). in total, 2,000 and 1,000 pollen grains from each pollen and honey pot were counted, respectively. the monthly means of five pollen and honey samples were calculated. the quantitative results were classified as frequency classes (louveaux et al., 1978): predominant pollen ‘p’ (more than 45% of the grains counted), secondary pollen ‘s’ (15% to 45%), important minor pollen ‘i’ (3% to 15%) and minor pollen ‘m’ (less than 3%). based on the literature and floristic surveys performed in the study region, the pollen types identified in honey that were considered to be from nectariferous plants were separately analyzed to determine which species actually contributed to the honey composition. minitab®15 software was used to generate the dendrograms of percentage similarity of the two types of pots analyzed. results fifty-eight pollen morphospecies were observed in the m. subnitida food samples. in the qualitative analysis of the pollen pots, 52 pollen types belonging to 29 families, 40 genera and 29 species were recorded. only two pollen types were not identified (table 1). fifty pollen types were present in the honey samples, which were grouped into 29 families, 37 genera and 28 species. four pollen types were indeterminate (table 1). the botanical families with greatest species diversity in the samples’ pollen spectra were fabaceae (8), myrtaceae (5), malpighiaceae (3) and melastomataceae (3). most plants species occurred occasionally (minor pollen) in the samples. of the pollen types identified in the pollen pots, only 12 (23.07%) were identified with percentages higher than 3% in any given month. in the honey, 17 pollen types (34%) were considered at least once as important minor, secondary or predominant pollens. figure 1 shows the percentage frequencies of the main species identified in the pollen (fig 1a) and honey samples (fig 1b) throughout the study. in pollen pots, mimosa misera benth. (fabaceae) (fig 2a), chamaecrista ramosa (vogel) h.s. irwin & barneby (fabaceae) (fig 2b), mouriri guianensis aubl. (melastomataceae) (fig 2c), doliocarpus sp. (dilleniaceae) (fig 2d), myrcia obtusa schauer (myrtaceae) (fig 2e), comolia lythrarioides naudin (melastomataceae) (fig 2f), myrcia sp. 3 (myrtaceae), tibouchina sp. (melastomataceae), myrcia sylvatica (myrtaceae) and eugenia sp. (myrtaceae) were particularly abundant. in april, the presence of orbignya phalerata mart. (arecaceae) was also noteworthy. sociobiology 61(4): 461-469 (december, 2014) 463 pollen pots honey pots family pollen type apr jun aug oct dec feb apr jun aug oct dec feb amaranthaceae alternanthera sp. m m anacardiaceae anacardium microcarpum m m m m m m arecaceae arecaceae type m m m m m m m orbignya phalerata i m m m m m m i m m asteraceae wulffia baccata m m m m bignoniaceae arrabidaea dispar m m m bignoniaceae type m m boraginaceae heliotropium sp. m m burseraceae protium heptaphyllum m m m m m caryocaraceae caryocar brasiliense m m m chrysobalanaceae chrysobalanus icaco m m m m m m m m i i i i licania sp. m m clusiaceae clusia grandiflora m m m m m m dilleniaceae doliocarpus sp. m i m m m p i m i m m s euphorbiaceae croton sp. m m m mabea pohliana m m m m fabaceae abarema cochleata m m centrosema sp. m m m m m chamaecrista ramosa m s s s m m i i m m m m copaifera sp. m m m m m m mimosa caesalpiniifolia m m m m m mimosa misera s p s p m m s s i i m m senna sp. m m m m m stryphnodendron adstringens m m m m m m m m m gentianaceae irlbachia pratensis m humiriaceae humiria balsamifera m m m m m m i i i p i i loranthaceae phthirusa pyrifolia m m m m m m lythraceae cuphea tenella m m m m m i m m m m m malpighiaceae byrsonima chrysophylla m m m m m byrsonima crassifolia m m m m m m m m m m mascagnia sp. m m m malvaceae sida sp. m melastomataceae comolia lythrarioides i i i m m i i p m m i mouriri guianensis s m i i s p i i i i s i tibouchina sp. i m i m i i m m m i meliaceae meliaceae type m m menyanthaceae nymphoides indica m m m m m myrtaceae eugenia sp. i m m m i i m m m myrcia obtusa m m m m s m m m m m i i myrcia sylvatica i m i m i m i s myrcia sp.3 m m m m s m m i myrcia sp.4 m m m i ochnaceae ouratea racemiformis m m m m passifloraceae passiflora sp. m turnera ulmifolia m m m m m m poaceae poaceae type m m m m m m m polygonaceae coccoloba sp. i m m m m i m i i rubiaceae borreria latifolia m m m borreria verticillata m m m m m m i i m m m m rutaceae citrus sp. m sapindaceae paullinia pinnata m sapotaceae pouteria sp. m m m m m xyridaceae xyris paraensis m m m m not identified type 01 m type 02 m m m m type 03 m type 04 m type 05 m m m m table 1. occurrences and frequencies of pollen types in pollen and honey pots of melipona subnitida in a restinga area, maranhão state, brazil. classification: p – predominant pollen (more than 45% of the grains counted), s – secondary pollen (15 to 45%), i – important minor pollen (3 to 15%) and m – minor pollen (less than 3%). apr (april), jun (june), aug (august), oct (october), dec (december), feb (february). rs pinto, pmc albuquerque, mmc rêgo pollen analysis of food pots stored by melipona subnitida464 the major pollen types identified in the honey samples were m. misera, humiria balsamifera aubl. (humiriaceae) (fig 2g), m. guianensis, c. lythrarioides, doliocarpus sp., chrysobalanus icaco l. (chrysobalanaceae) (fig 2h) and coccoloba sp.. other species also stood out in a few of the months, such as o. phalerata, c. ramosa, cuphea tenella hook. & arn. (lythraceae), tibouchina sp., borreria verticillata (l.) g. mey. (rubiaceae) and all the species of the family myrtaceae. in the similarity dendrogram of the pollen pots, the samples from april, june, august and october were grouped together with 86.89% of similarity; the samples from june and october had 98.43% of similarity. conversely, february and december were the least similar months compared to the other months (fig 3a). in the similarity dendrogram of the honey samples, december and february had 75.70% of similarity. the samples collected in august had 73.55% of similarity with the samples collected in april and june. samples collected in april and june had the highest similarity (95.87%). the samples collected in october showed less similarity with those collected in the other months (fig 3b). fig 1. percentages of pollen types identified in the samples from (a) pollen pots and (b) honey pots of melipona subnitida in a restinga area, maranhão state, brazil. based on floristic surveys performed in the region and specialized literature, we separated the genera and species of nectariferous plants present in honey. of the 50 pollen types recorded in the honey samples, 20 were from plants that contributed to the production of m. subnitida honey (table 2). fig 2. photomicrographs of pollen types observed in food pots of melipona subnitida. (a) mimosa misera, (b) chamaecrista ramosa, (c) mouriri guianensis, (d) doliocarpus, (e) myrcia obtusa, (f) comolia lythrarioides, (g) humiria balsamifera, and (h) chrysobalanus icaco. bar scale – 10 µm. fig 3. similarity dendrogram based on the percentage data of (a) the 52 pollen types in pollen pots and (b) the 50 pollen types in honey pots of melipona subnitida. assessing the number of pollen quantified in nectariferous plants only, we calculated the percentage frequencies of those pollen types in each month. humiria balsamifera occurred as sociobiology 61(4): 461-469 (december, 2014) 465 al. (2012) in a botanical survey on the border of ceará and rio grande do norte states, also noted that the m. subnitida visited many fabaceae species. this seems to fit the pattern of phylogenetically close species that interact with a similar set of species in a wide geographic range, with highly conserved associations (thompson, 2005; rezende et al., 2007). fabaceae and myrtaceae families had the greatest number of observed pollen types, a finding that is closely related to the large richness of fabaceae and myrtaceae species and specimens in restinga areas of northeastern brazil (freire & monteiro, 1993; santos-filho et al., 2013). three melastomataceae species were very important for m. subnitida. this plant family is one of the most representative in south america (ramalho et al., 1990). given that many species of this family bloom sequentially throughout the year (wilms & wiechers, 1997), a high frequency of melastomataceae in the food samples would be expected because of the intrinsic preference of melipona for this plant family. the preference of m. subnitida for species of melastomataceae and chamaecrista ramosa must be attributed to the fact that the flowers of those plants have poricidal anthers, which restrict the number of floral visitors. this feature is important for the foraging activity of melipona, the only highly honey pots family pollen type apr jun aug oct dec feb anacardiaceae anacardium microcarpum 6.67 2.09 0.73 asteraceae wulffia baccata 0.12 0.75 0.09 boraginaceae heliotropium sp. 0.03 burseraceae protium heptaphyllum 7.72 1.95 3.40 1.95 caryocaraceae caryocar brasiliense 0.15 0.04 chrysobalanaceae chrysobalanus icaco 3.13 5.44 24.56 13.32 41.84 27.80 euphorbiaceae mabea pohliana 6.84 fabaceae centrosema sp. 0.09 copaifera sp. 0.36 0.52 0.09 mimosa caesalpiniifolia 0.72 2.93 0.42 1.08 stryphnodendron adstringens 0.39 2.72 0.05 1.04 1.03 gentianaceae irlbachia pratensis 0.09 humiriaceae humiria balsamifera 28.33 32.58 43.63 82.14 31.59 21.11 loranthaceae phtirusa pyrifolia 0.05 0.40 0.13 lythraceae cuphea tenella 20.74 0.81 4.23 0.14 3.39 0.16 menyanthaceae nymphoides indica 0.33 0.06 passifloraceae turnera ulmifolia 5.55 6.05 3.34 0.05 polygonaceae coccoloba sp. 11.75 12.47 13.37 20.62 41.97 rubiaceae borreria verticillata 21.94 22.40 1.15 0.14 1.08 7.71 sapotaceae pouteria sp. 0.37 0.07 table 2. frequencies (%) of pollen types of nectariferous species from honey samples produced by melipona subnitida in a restinga area, maranhão state, brazil. secondary pollen in five of the six samples; in october, it was the predominant nectariferous species, with 82.14% of the total grains counted. chrysobalanus icaco was identified as secondary pollen in august, december and february. coccoloba sp. was considered secondary pollen in december and february. two other species worth noting were borreria verticillata and cuphea tenella (table 2). discussion in the current study, we observed that m. subnitida depends on a variety of plant species to obtain pollen and nectar, but this specie prefers a particular plant spectrum. in the restinga region of the maranhão eastern coast, m. subnitida collected pollen preferably from fabaceae, melastomataceae, myrtaceae and dilleniaceae species, which are frequently observed in palynological studies of pollen of the genus melipona (kerr et al., 1986/1987; ramalho et al., 1989; marques-souza, 1996; wilms & wiechers, 1997; luz et al., 2011). in the brazilian semi-arid region, where m. subnitida is abundant, the floral preferences of this species also agree with our study. silva et al. (2006), in paraíba state, observed the dominance of mimosa and other fabaceae species in pollen stored by m. subnitida. in addition, maia-silva et rs pinto, pmc albuquerque, mmc rêgo pollen analysis of food pots stored by melipona subnitida466 eusocial genus that performs buzz pollination – the capacity to vibrate flowers with poricidal anthers (buchmann, 1983; nunes-silva et al., 2013). however, pollen collection is not restricted to this flower type, and the behavior of vibrating the thoracic flight muscles also occurs when visiting non-poricidal flowers, such as several myrtaceae species and mimosa (nunessilva et al., 2010), which were abundant in the study samples. the predominance of doliocarpus in the february pollen samples is likely due to the great supply of this resource in that month. all the plant species visited have flowering peaks – periods with the highest number of flowers available, which is crucial for attracting foraging bees. therefore, consecutive months tend to be more similar in regards to the plants visited, as observed by the similarity analyses of bimonthly samples. orbignya phalerata (the babassu, a native species of the transition zone between the cerrado and the south amazonian open forests (albiero et al., 2007)) was recorded in the pollen pots in april, and it was also a relevant species among the studied samples, although to a lower degree. several species of family arecaceae are visited by bees due to the plants prolonged flowering and pollen abundance in their inflorescences (oliveira et al., 2009). regarding the honey analysis, there was a high richness of pollen types in the samples, which usually hinders the determination of the nectariferous species. the polliniferous species tend to be greatly represented in honey and are considered contaminants because either the pollen grains were attached to the bee’s body or the flower of the nectariferous species was contaminated by pollen from anemophilous plants that attached to the nectaries (barth, 1989). the pollen types of plants that do not produce nectar but are present in honey are important because they help to broaden the knowledge of the flora where the colonies are located. mimosa misera was the most representative plant in the pollen pots and was abundant in the honey samples collected in april and june. mimosa misera is a polliniferous species (novais et al., 2009) and was therefore excluded from the honey analysis because it was considered a contaminant. nevertheless, some authors consider mimosa species to be nectar sources for m. subnitida (almeida-muradian et al., 2013; silva et al., 2013). however, in general, the mimosa genus consists of plants that produce high amounts of pollen and relatively little nectar, as they are over-represented in the pollen spectra. to be considered free of these contaminating plants, the honey samples need to have more than 98% of pollen from nectariferous species (barth, 1989). it is noteworthy that the polliniferous chamaecrista ramosa and melastomataceae and myrtaceae species were abundant in the honey samples but did not contribute to its production. according to buchmann (1983), plants adapted to buzz pollination, such as species of melastomataceae and c. ramosa, usually experience secondary loss of floral nectar. the true nectariferous importance of many myrtaceae species is also doubtful because their flowers supply high amounts of pollen grains as their main resource but produce small amounts of nectar (wilms & wiechers, 1997; gressler et al., 2006). in floristic surveys performed in the study region, myrtaceae species were only considered as pollen sources. of the more abundant nectariferous species in the honey samples, humiria balsamifera was present in all samples, since it blooms throughout the entire year (machado et al., 2007). on average, each specimen of this plant has 50,000 small flowers (viana & kleinert, 2006), and they produce a small amount of nectar (machado et al., 2007). thus, m. subnitida foragers may compensate for the small of amount of nectar offered by the plants by continually visiting the abundant flowers. humiria balsamifera was considered predominant in october; therefore, honey sampled in october can be characterized as unifloral honey, due to the pollen dominance of this species (barth, 2004). although h. balsamifera was less frequent in other months, ohe et al. (2004) noted that honey samples may be underrepresented in the pollen from plants that provided the nectar. for example, the occurrence of 2% to 42% of citrus pollen in a honey sample is enough to characterize it as unifloral. the nectariferous species chrysobalanus icaco, coccoloba sp. and borreria verticillata, which were greatly important in different samples, are examples of subrepresentations, not reflecting the real importance of those plants for m. subnitida. the possibility cannot be ruled out that at certain periods of the year, some honeys are produced solely from one of those plants. the only representative of lythraceae family (cuphea tenella) was observed throughout the entire study in honey samples, but the percentage was only high in april. this finding may be related to increased flowering in that period and the bees’ preference for certain food sources, depending on the ease of food gathering, the quantity and quality of trophic resources and interactions with competitors (cortopassi-laurino & ramalho, 1988). in the coastal dunes of abaeté, cuphea brachiata mart. ex koehne produces nectar constantly but with low sugar concentrations (viana & kleinert, 2006), which can also occur with c. tenella, thus making it less attractive to bees. in the pollen and honey samples, most of the pollen types was considered as minor pollen, indicating the polylectic nature of this bee. plants with low pollen frequencies may have reduced pollen production or may be present in the samples due to the bees’ indirect collection behavior. it is also known that different pollen types may be either stored for prolonged periods or be immediately offered to brood; therefore, minor pollens are poorly represented in the analyses of pollen pots (malagodi-braga & kleinert, 2009). it must be considered that less frequent plants complement the colony’s needs, ensuring nutritional balance in environments where the floral resource supply constantly changes due to seasonality. sociobiology 61(4): 461-469 (december, 2014) 467 the spectrum of floral sources visited by m. subnitida indicates its benefit for the pollination of several native species of the lençóis maranhenses restinga, as the bees clearly have a strong mutualistic relationship with both pollen-supplying and nectar-producing plants. in general, m. subnitida has botanical affinities that are very similar to those of melipona genus. acknowledgments the authors thank dr. léa carreira of the museu paraense emílio goeldi, msc. angela corrêa and dr. maria amélia cruz of the botanical institute of são paulo (instituto de botânica de são paulo) for their cooperation in identifying pollen types. the authors also thank irene aguiar, mr. emídio and mrs. maria, the owners of the jandaíra meliponary, and the research and scientific development foundation of maranhão (fundação de amparo à pesquisa e desenvolvimento científico do maranhão – fapema) for financial support. references albiero, d., maciel, a.j.s., lopes, a.c. & gamero, c.a. 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(1997). floral resource partitioning between native melipona bees and the introduced africanized honey bee in the brazilian atlantic rain forest. apidologie 28: 339-355. doi: 10.1051/apido:19970602 251 relationship between hygienic behavior and varroa destructor mites in colonies producing honey or royal jelly by priscila wielewski1, vagner de alencar arnaut de toledo2*, elias nunes martins2, fabiana martins costa-maia1, patrícia faquinello3, daniela andressa lino-lourenço2, maria claudia colla ruvolo-takasusuki2 & carlos antonio lopes de oliveira2 & maria josiane sereia4 abstract genetic and phenotypic parameters for hygienic behavior, invasion and infestation rates, total and effective reproduction of varroa destructor in africanized honeybee colonies producing honey (20 hives) or royal jelly (30 mini-hives) were analyzed. the significance of monthly fixed effects, type of product (honey and royal jelly) and their interactions were verified through generalized linear model procedures. software winbugs (bayesian inference using gibbs sampling ) with bayesian inference was employed for (co)variance estimates. the average values for colonies producing honey or royal jelly were 74.38 and 71.40% for hygienic behavior in 24 hours; infestation rates 8.30 and 11.40%; invasion rates 9.50 and 7.50%; total reproduction 1.02 and 0.55%; effective reproduction was 0.62 and 0.33%, respectively. the additive genetic variance for invasion (0.16), total reproduction (0.25) and effective reproduction (0.94) rates of the mite were higher than estimates for hygienic behavior in 24 hours (0.05) and infestation rate (0.04). mean heritability for hygienic behavior, infestation and invasion rates, total and effective reproduction of the mite were 1universidade tecnológica federal do paraná, campus dois vizinhos estrada para boa esperança, km 04 cep 85660-000, dois vizinhos – pr, brasil2 2universidade estadual de maringá – avenida colombo, 5790, cep 87020-900 maringá – paraná, brasil 3universidade federal do recôncavo da bahia – rua rui barbosa, 710, cep: 44380-000, cruz das almas –bahia, brasil. 4universidade tecnológica federal do paraná, campus campo mourão br 369 – km 0,5 cep 87301006 – caixa postal: 271 – campo mourão-pr, brasil *author for correspondence abelha.vagner@gmail.com 252 sociobiolog y vol. 59, no. 1, 2012 0.58, 0.54, 0.56, 0.63 and 0.61, respectively. the genetic correlation of -0.48 for hygienic behavior with total reproduction rate of varroa destructor shows that hygienic behavior may be the most interesting trait for selection. besides a heritability of high magnitude, when combined with the total reproduction rate of the mite, it has a high and antagonistic correlation. consequently, in cases of high infestation of varroa destructor, the selection for hygienic behavior would decrease the reproduction rate of the mite. key-words: bee breeding, heritability, genetic correlation, phenotypic correlation, bayesian inference resumo foram analisados os parâmetros genéticos e fenotípicos para o comportamento higiênico e as taxas de invasão, infestação e reprodução total e efetiva do ácaro varroa destructor em colônias de abelhas apis mellifera africanizadas produtoras de mel (20) ou geleia real (30). foi verificada a significância dos efeitos fixos de mês, tipo de produto e suas interações, utilizando o procedimento de modelos lineares generalizados. para a estimação das (co)variâncias foi utilizado o software winbugs por meio de inferência bayesiana. as médias para colônias produtoras de mel ou geleia real, respectivamente, foram: para o comportamento higiênico em 24h, 74,38 e 71,40%; taxas de infestação 8,30 e 11,40%; taxas de invasão 9,50 e 7,5%; taxas de reprodução total 1,02 e 0,55 e reprodução efetiva 0,62 e 0,33. as herdabilidades médias para comportamento higiênico, taxa de infestação, taxa de invasão, taxa de reprodução total e efetiva do ácaro foram 0,58, 0,54, 0,56, 0,63 e 0,61, respectivamente. a correlação genética encontrada de -0,48 para a característica comportamento higiênico com taxa de reprodução total do ácaro varroa destructor indicou que o comportamento higiênico pode ser considerado a característica mais interessante para seleção, pois além de apresentar herdabilidade de alta magnitude, quando associada à taxa de reprodução total do ácaro apresenta uma correlação alta e 253 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies antagônica, sendo assim, em casos de alta infestação do ácaro varroa destructor, selecionando-se para comportamento higiênico estaríamos diminuindo a taxa de reprodução do ácaro. palavras-chave: melhoramento de abelhas, herdabilidade, correlação genética, correlação fenotípica, inferência bayesiana introduction africanized honeybees are specifically significant owing to their positive adaptation in brazil and to their honey production. since the honey bee has high genetic variability between queens from the same geographical region (winston et al. 1983) with regard to economical features, the honeybees are interesting examples within a genetic improvement program. the peculiarity of honeybees makes genetic improvement difficult due to environmental influences and genetic differences in mating level (bienefeld et al. 2007). however, researchers in many countries are undertaking genetic improvement with some success. the best honeybee production selection programs in the usa necessarily include the selection of hygienic honeybees. hygienic behavior is an efficient resistance mechanism against brood diseases and has been well-documented since the 1940s when much research was undertaken in the area (message 2006). this hygienic behavior includes the colony capacity to inhibit infestation by mites of the genus varroa. the mite varroa destructor, which causes varroatosis in apis cerana and apis mellifera, was introduced in brazilian apiculture at the beginning of the 1970s. called varroa jacobsoni, the genus was introduced in brazil through paraguayan beekeepers (morse & gonçalves 1979). in turn, the queens bought from paraguay had been imported from japan. population dynamics of the mite varroa have varied by region (calderón et al. 2010). in brazil were greatly different from those reported in other varroatosis infested regions. over 20% rates, initially reported, 254 sociobiolog y vol. 59, no. 1, 2012 brought concern to researchers and beekeepers (moretto et al. 1991). however, as the mite spread itself throughout brazilian regions, it became clear that infestation rates, although initially high, declined some years after the first infestation. a balance seems to have been reached between the mite varroa destructor and africanized honeybees in climate conditions of brazil (moretto & mello 2001). in some regions of the country the varroatosis infestation rates reached the very low percentage of 2% (moretto et al. 1993, carneiro et al. 2007, calderón et al. 2010). this research was carried out to estimate phenotypic and genetic parameters which report hygienic behavior with the population dynamics of varroa destructor in africanized honeybee colonies which produce royal jelly or honey. the possible inclusion of these traits in genetic improvement programs in brazil may be achieved. material and methods the assay was undertaken at the central apiary of the iguatemi experimental farm of universidade estadual de maringá, maringá pr brazil, from february 2009 to november 2009. fifty colonies of africanized honeybees, 30 in mini-hives which produced royal jelly, and 20 in langstroth hives with supers, which produced honey which was used. production of queens queen production for the production of their daughters from honeyproducing colonies to replace the queens of royal jelly-producing colonies was undertaken in october 2008, april 2009 and august 2009. modified doolittle (1889) method for queen production, or rather, the grafting of worker larvae from their original cell to acrylic cups with royal jelly diluted in distilled water (1:1), was employed. whereas ten queens were randomly replaced in january 2009 in the 30 royal jelly-producing colonies, nine queens were once more randomly replaced in april and 12 in august. 255 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies production of royal jelly a modified doolittle (1889) method was employed for royal jelly production whilst its grafting and collection were undertaken twice a week. so that appropriate larvae for royal jelly production could be obtained, grafting was previously programmed with the introduction of an empty comb four days prior to grafting and placed in the center of the different colonies in the ten-frame nests or five-frame nuclei in langstroth hive model. after larvae grafting, the cup bars were carefully placed in their respective langstroth hive or mini-hive. hygienic behavior rothenbühler (1964) and newton et al. (1975), revised by spivak & downey (1998) reported that the hygienic behavior test establishes the time spent by honeybee colonies to detect, uncap and remove dead brood worker honeybees by freezing (-20ºc during 24h) from one section of the 5 x 6 cm comb (with approximately 100 capped brood pupae of the comb), separated from the colony nest for evaluation, and to determine whether the colony is or not hygienic. royal jellyand honey-producing colonies underwent hygienic behavior test (taber 1982, gramacho 1995). a comb with capped brood worker bees, aged 17-18 days, or rather, during the rose-colored pupa phase, placed on both sides, a and b, was removed from each colony. the central section with 5 x 10 cm had been removed from the comb, was photographed, and frozen at -20ºc for 24h. after that, the comb section was then conditioned in an incubator at 34ºc and 60% humidity during 4h to dry and establish the same internal temperature of the colonies. after restored to their respective colonies, the sections were again photographed 24h for counting of the uncapped cells. hygienic behavior percentages were obtained by calculating the number of alveoli of capped brood at zero hour as a function of the number of alveoli of the remaining capped brood in 24 h. further, 15, 11, 12, 14, 11 and 7 out of the 20 honey-producing matrix colonies were evaluated for hygienic behavior respectively in february, 256 sociobiolog y vol. 59, no. 1, 2012 march, april, may, june and july 2009. similarly, 14, 18, 11 and 13 out of 30 royal jelly-producing colonies were evaluated respectively in february, march, april and september 2009. estimates for hygienic behavior were obtained by calculating : ch x = (to zero hour – ao x )/ to zero hour ) where, chx is the relationship between the number of cleaned alveoli and the total number of capped brood cells in which x reaches 24h; tozero hour is the total number of capped brood cells at zero hour; aox is the total number of capped brood cells in which x reaches 24h. the number of cells with partially removed pupae (crp), pointed (cp) and uncapped (cd) which, as a rule, demonstrate the hygienic behavior corresponding to uncapping activities (pointing and destruction of the opercula or uncapping ). variables analyzed with regard to varroa destructor consisted of: infestation rate of adult worker honeybees the mite infestation rate, as proposed by stort et al. (1981), was undertaken to verify the number of mites in worker honeybees. the method retrieves approximately one hundred adult worker honeybees from a comb at the centre and placed in a dish with alcohol 70% and the contents stirred. total number of worker honeybees and mites was counted to evaluate each colony infestation percentage. invasion rate in worker pupae observation of the number of varroa mites, as proposed by de jong & gonçalves (1981), was performed to estimate the number of mites in the worker pupae. the method included the retrieval of a comb of capped brood from each colony. opercula were removed with insect pin from 100 (50 from each side of the comb) pupae. female mites and their offspring in pupae and alveoli were analyzed by attached light. tests were undertaken in the twenty honey-producing colonies: five colonies were tested during february 2009; six in march; 11 in april; eight in may; 15 in june and 10 in july 2009. 257 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies in the 30 royal jelly-producing colonies the test was undertaken in 11 colonies during february 2009; seven in march; nine in april and nine in september. reproduction rates of the mite varroa destructor mite total reproduction rate was calculated as follows: trt = total number of descendents/ (number of adult females) mite effective reproduction rate was calculated as follows: tre = number of deutonymphs + young adults / (number of female adults) whereas total reproduction rate (trt) is the total number of descendents produced by the mite, effective reproduction rate is the number of viable descendants. since worker pupae under analysis were approximately 18 – 19 days old, there was no sufficient time for the final development of the mite immature forms, in africanized honeybees. therefore, only deutonymphs, female adults and young females could parasitize the forthcoming worker honeybee one to two days after analysis. this fact actually contributed towards the mite reproduction. when toledo & nogueira-couto (1996) investigated africanized honeybees and hybrids from caucasian, italian and carniolian sister queens, they found no statistical difference for varroa total and effective reproduction rates. data analysis r development core team statistic program (2009) gave a previous analysis of data to verify the significance of fixed month effects, type of product (royal jelly or honey) and their interactions. since shapiro-wilk test showed non-normality of data, generalized linear models (glm) procedures were used, in which it was assumed the gama distribution and inverse link function, for variables. data were corrected when any significant effect occurred. since worker honeybees that performed the hygienic behavior are middle-aged, averaging 15.2 days old (arathi et al. 2000), worker groups 258 sociobiolog y vol. 59, no. 1, 2012 executing the same task in the colony are changed approximately at every 35.2 days. fig. 1 shows the relationship among colonies of queen bee mothers and those of the queen bee daughters amounted to 0.25 since fatherhood is unknown. corrected covariance among colonies of queen bee mothers and those of queen bee daughters amounts to 0.25 of additive genetic variance. the two colonies may be described as featuring aunt-niece relationships, or rather; the queen bee from which c1 derived is the queen mother that produced c2. analysis strateg y took the traits in pairs while considering that the same characteristic in the queen bee mother colonies was another characteristic when evaluated in the queen bee daughter colony. data were organized through the pairing of recording of the queen bee mother colonies and those of the queen bee daughter colonies. the archive in each analysis with two traits was formed by four columns: the first refers to recording of trait 1 in the queen bee mother colonies; the second refers to recording of trait 2 in queen bee mother colonies; the third refers to recording of trait 1 in the queen bee daughter colonies; the fourth refers to recording of trait 2 in the queen bee daughter colonies. employing the following multivariated structure, we have: fig.1. relationship among honey-producing (c1) and royal jelly producing colonies (c2). 259 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies in the case of matrix σk , the variance matrix and (co)variance in the reports, inverted wishart distribution, was taken up by σk ~ iw(rk, j) with r k =i j =i 4 , in which i is the identity matrix and the scale parameter is equal to four. marginal distribution for parameters was obtained a posteriori by software winbugs 1.4.2 (spiegelhalter et al. 1994) which precedes bayesian analysis. one million rates were generated for each parameter in a monte carlo markov chain (mcmc) process, declining initial discarding period and with all sampled rates inserted within a final chain. chain convergence was tested by criteria proposed by heidelberger & welch (1983) and geweke (1992), implemented in a coda library, available in the computer system r development core team (2009). based on estimated rates for σk and employing a program developed by computer system r development core team (2009), phenotypic (σ2yc1 and σ 2 yc2) and additive genetic (σ 2 ac1 and σ 2 ac2) variance for the two traits and phenotypic ( σ2yc1yc2)and additive genetic (σ 2 ac1ac2) covariance between the two was calculated as follows: yijk ~ nmv (μk; σk), or rather,                                  2 2 2 2 4342414 4332313 4232212 411211 ; 0 0 0 0 yyyyyyy yyyyyyy yyyyyyy yyyyyyy nmv σσσσ σσσσ σσσσ σσσσ 3             4 3 2 1 y y y y 2 22 2 1 31 yy yc σσ σ + =σ 2 yc1 2 22 2 2 42 yy yc σσ σ + =σ 2 yc2 260 sociobiolog y vol. 59, no. 1, 2012 so that heritability and correlation would be within the parameter space established for them, namely, from 0 to 1 and from -1 to 1, respectively, samples that failed to satisfy the above mentioned conditions were discarded from the markov chain. the new chain underwent convergence tests proposed by heidelberger & welch (1983) and geweke (1992). mean distribution rate, median, standard error, credibility interval and high density region were set at 95% for each variance component and genetic parameter. results and discussion percentage mean for hygienic behavior in 24h (ch24), adult infestation rate, invasion rate in the pupae, total and effective reproduction rate for honey and royal jelly-producing colonies were respectively 74.38 and 71.40%; 8.30 and 11.40%; 9.50 and 7.5%; 102.40 and 54.60%; 62.00 and 33.00%. previous data analysis did not show effects of production types for hygienic behavior in 24h (ch24). interaction existed for product and period in the case of mite infestation in adult honeybees. this fact shows that during the cold months there was an increase in infestation rates for honey-producing colonies. the number of colony individuals during this period decreased and an increase in mite concentrations occurred (nogueira-couto 1991). 2 3241 21 yyyy ycyc σσ σ + =σ2yc1yc2 42 42 2 yyac σσ =σ 2 ac2 31 42 1 yyac σσ =σ2ac1 2121 4 ycycacac σσ = σ2ac1ac2 yc1yc2 261 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies product type, honey and royal jelly, was significant for the pupa invasion rate, respectively with means of 9.5 and 7.5%. according to segndo lee et al. (2010) the mite prefers the nurse bees, which tend to stay in the brood frame. thus, the colonies submitted to royal jelly production, which need a greater amount of nurse bees responsible for feeding the queen larvae, are probably more affected by varroa mites. moretto et al. (1991) verified invasion rates in pupae in three regions and concluded that africanized honeybees were more resistant to varroa than italian honeybees bred with africanized honeybees. in brazil, africanized honeybees tolerate the mite, and high swarming rates contribute to this tolerance (boecking & ritter 1993). total and effective reproduction rates of varroa mites were not affected by period or product type. table 1 shows estimates of addictive genetic and phenotypic covariance in a two-trait analysis for variables under analysis. additive genetic variance for invasion (0.16), total (0.25) and effective (0.94) reproduction rates of the mite are higher than those estimated for hygienic behavior in 24h (0.05) and infestation (0.04). estimates for phenotypic variance had a similar behavior. higher additive genetic variance proportion for the mite reproduction rates may be due to higher precision of tye method employed when compared to infestation, invasion and hygienic behavior. table 2 shows estimates of heritability and the respective credibility intervals and high density regions in a two-trait analysis. mean heritability for hygienic behavior, infestation rate, invasion rate, total and effective reproduction rate of mites were respectively 0.58, 0.54, 0.56, 0.63 and 0.61. although spivak & gilliam (1993) said that this behavior is genetically determined, but not always expressed, it seems to be dependent on environmental factors of the colony. harbo & harris (1999) recorded heritability estimates of 0.65 for such behavior in 24h. in the following year, boecking et al. (2000) estimated heritability at 0.36 for the removal of capped brood perforated between 13 and 15h. 262 sociobiolog y vol. 59, no. 1, 2012 table 1. estimates of components of addictive genetic and phenotypic covariance with respective intervals of credibility and high density regions, at 95%, in a two-trait analysis for hygienic behavior in 24h infestation, invation and total and effective reproductive rates of varroa destructor in africanized honeybees. components* estimates credibility interval high density region σ2 a1 0.05 0.005 – 0.12 0.0004 – 0.10 σ a1 2 -0.001 -0.06 – 0.05 -0.06 – 0.05 σ a1a3 -0.001 -0.10 – 0.10 -0.10 – 0.10 σ a1a4 -0.21 -0.65 – 0.21 -0.63 – 0.23 σ a1a5 0.004 -0.35 – 0.35 -0.35 – 0.35 σ2 a2 0.04 0.004 – 0.10 0.0005 – 0.10 σ a2a3 0.001 -0.04 – 0.04 -0.04 – 0.04 σ a2a4 -0.07 -0.47 – 0.28 -0.45 – 0.30 σ a2a5 0.07 -0.29 – 0.41 -0.29 – 0.41 σ2 a3 0.16 0.02 – 0.38 0.002 – 0.33 σ a3a4 0.07 -0.08 – 0.23 -0.08 – 0.23 σ a3a5 0.01 -0.10 – 0.12 -0.10 – 0.12 σ2 a 4 0.25 0.05 – 0.52 0.02 – 0.47 σ a4a5 0.26 -0.36 – 0.82 -0.32 – 0.85 σ2 a5 0.94 0.18 – 2.31 0.06 – 1.98 σ2 y 1 0.09 0.05 – 0.15 0.05 – 0.15 σ y1y2 -0.0003 -0.01 – 0.01 -0.01 – 0.01 σ y1y 3 -0.0002 -0.02 – 0.02 -0.02 – 0.02 σ y1y 4 -0.05 -0.16 – 0.05 -0.16 – 0.05 σ y1y5 0.001 -0.08 – 0.08 -0.08 – 0.08 σ2 y2 0.08 0.05 – 0.13 0.05 – 0.12 σ y2y 3 0.0002 -0.01 – 0.01 -0.01 – 0.01 σ y2y 4 -0.02 -0.12 – 0.07 -0.11 – 0.08 σ y2y5 0.02 -0.07 – 0.10 -0.07 – 0.10 σ2 y3 0.29 0.16 – 0.52 0.14 – 0.47 σ y3y 4 0.02 -0.02 – 0.05 -0.02 – 0.05 σ y3y5 0.002 -0.02 – 0.02 -0.02 – 0.02 σ2 y4 0.37 0.21 – 0.66 0.18 – 0.59 σ y4y 5 0.07 -0.09 – 0.20 -0.08 – 0.21 σ2 y5 1.37 0.65 – 3.01 0.54 – 2.59 * a and y represent the addictive genetic and phenotypic effects, respectively, for all components; indexes 1, 2, 3, 4 and 5 correspond to the hygienic behavior in 24h, infestation, invasion, total and effective reproduction rate of the mite, respectively. 263 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies costa-maia (2009) estimated heritability of 0.28 for this component by bayesian inference. credibility intervals for heritability estimates were not only broad and positive but also coincided with high density regions. falconer (1987) reported on the importance of heritability due to the fact that it helps in the estimate of addictive genetic variance associated to a trait within a specific population. table 3 shows estimates of genetic and phenotypic correlation among the traits under analysis. the positive correlations are: adult infestation table 2. estimates of heritability (h2) in a two-trait analysis and respective credibility intervals and high density regions at 95%, and mean heritability for hygienic behavior in 24h, infestation, invasion and reproduction rates of mite varroa destructor in africanized honeybees. characteristic two-trait analysis estimates credibility interval high density region h2 a1a2 0.52 0.06 – 0.97 0.09 – 0.99 1 h2 a1a3 0.57 0.07 – 0.98 0.12 – 0.99 h2 a1a4 0.56 0.07 – 0.98 0.12 – 0.99 h2 a1a5 0.66 0.12 – 0.99 0.19 – 0.99 mean h2 0.58 – – h2 a2a1 0.55 0.07 – 0.98 0.11 – 0.99 2 h2 a2a3 0.52 0.06 – 0.97 0.09 – 0.99 h2 a2a4 0.47 0.05 – 0.96 0.03 – 0.93 h2 a2a5 0.64 0.13 – 0.98 0.19 – 0.99 mean h2 0.54 – – h2 a3a1 0.56 0.07 – 0.98 0.11 – 0.99 3 h2 a3a2 0.55 0.07 – 0.98 0.11 – 0.99 h2 a3a4 0.56 0.07 – 0.98 0.11 – 0.99 h2 a3a5 0.57 0.07 – 0.98 0.12 – 0.99 mean h2 0.56 – – h2 a4a1 0.67 0.17 – 0.99 0.23 – 0.99 4 h2 a4a2 0.59 0.09 – 0.98 0.15 – 0.99 h2 a4a3 0.58 0.08 – 0.98 0.13 – 0.99 h2 a4a5 0.68 0.16 – 0.99 0.23 – 0.99 mean h2 0.63 – – h2 a5a1 0.68 0.16 – 0.99 0.24 – 0.99 5 h2 a5a2 0.56 0.07 – 0.98 0.12 – 0.99 h2 a5a3 0.54 0.06 – 0.98 0.11 – 0.99 h2 a5a4 0.66 0.15 – 0.99 0.21 – 0.99 mean h2 0.61 – – 264 sociobiolog y vol. 59, no. 1, 2012 rates and the effective reproduction rate (0.21); invasion rate in pupae and the total reproduction rate (0.46); total reproduction rate and effective reproduction rate (0.43). the other correlations are only slightly genetically and phenotypically related owing to their low rates. the most important negative genetic correlation is hygienic behavior and the mite total reproduction rate (-0.48). the above antagonism, namely, the selection of africanized honeybees for hygienic behavior, decreases varroa destructor mite total reproduction rate. table 3. estimates of genetic (rg) and phenotypic (ry) correlation coupled to their respective credibility intervals and high density regions at 95%, within a two-trait analysis, for hygienic behavior in 24h, infestation, invasion and total and effective reproduction rates of the mite varroa destructor in africanized honeybees components* estimates credibility interval high density region r g1,2 -0.02 -0.95 – 0.94 -0.99 – 0.88 rg 1,3 -0.007 -0.95 – 0.94 -0.99 – 0.89 rg1,4 -0.48 -0.99 – 0.69 -0.99 – 0.50 rg 1,5 0.01 -0.93 – 0.94 -0.88 – 0.99 rg 2,3 0.02 -0.93 – 0.94 -0.88 – 0.99 rg 2,4 -0.19 -0.96 – 0.89 -0.99 – 0.80 rg 2,5 0.21 -0.87 – 0.97 -0.75 – 0.99 rg 3,4 0.46 -0.73 – 0.99 -0.52 – 0.99 rg 3,5 0.06 -0.93 – 0.95 -0.87 – 0.99 rg 4,5 0.43 -0.65 – 0.98 -0.44 – 0.99 ry 1,2 -0.003 -0.15 – 0.14 -0.15 – 0.15 ry 1,3 -0.001 -0.16 – 0.15 -0.16 – 0.16 ry 1,4 -0.08 -0.19 – 0.07 -0.21 – 0.07 ry 1,5 0.002 -0.17 – 0.17 -0.17 – 0.17 ry2,3 0.003 -0.14 – 0.15 -0.14 – 0.15 ry2,4 -0.03 -0.16 – 0.11 -0.17 – 0.11 ry2,5 0.03 -0.13 – 0.18 -0.12 – 0.18 ry3,4 0.07 -0.07 – 0.19 -0.06 – 0.20 ry 3,5 0.01 -0.14 – 0.16 -0.14 – 0.16 ry4,5 0.07 -0.09 – 0.19 -0.08 – 0.20 *indexes 1 , 2 , 3 , 4 and 5 represent hygienic behavior in 24h, infestation, invasion, and total and effective reproduction rate, respectively. 265 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies since credibility intervals for all broad estimates included a zero value in their intervals, no correlation lies among the analyzed traits. however, when data symmetry is investigated, their probability in fact exists with higher and lower rates than zero value. figs. 2 to 11 (see appendix) show estimates of phenotypic and genetic correlation to demonstrate probabilities and whether one trait affects or not another trait. figs. 2 (hygienic behavior with infestation), 3 (hygienic behavior with invasion), 4 (hygienic behavior with effective reproduction rate), 5 (infestation with invasion) and 6 (invasion with effective reproduction rate) show that phenotypic and genetic correlation have graphic symmetries and highest frequencies close to zero. this fact shows that correlation is very low and thus traits are genetically only slightly related. no correlation exists between hygienic behavior and varroa infestation rates, since these traits are not interdependent (fig. 2). when colonies with hygienic behavior are selected, there may not be any impact on the mite invasion rates (fig. 3). this fact is shown in table 3 by correlation -0.007 with a credibility interval ranging between -0.95 and 0.94. fig. 5 shows that infestation rate is not correlated with the varroa invasion rate. this may be explained by worker grooming, which may prevent the mites from invading the cells of the capped brood. grooming may have an important role in the maintenance of low infestation rates ( junkes et al. 2007). figs. 7 (hygienic behavior with total reproduction rate), 8 (infestation with total reproduction rate), 9 (infestation with total reproduction rate), 10 (invasion with total reproduction rate) and 11 (total reproduction rate with the mite effective reproduction rate) show that phenotypic and genetic correlation with a high probability. in fact, these are asymmetrical figures displaced from zero rates. figs. 4 and 7 show the highest frequencies of less than zero rates and represent great probability that genetic correlations exist and are negative. hygienic behavior may be antagonistic to total reproduction rate of the mites (fig. 7). this fact may be explained by the characteristics' 266 sociobiolog y vol. 59, no. 1, 2012 independence. in fact, the mite may reproduce itself regardless of the colonies' efficient hygienic behavior. conversely, the mite may be in the colonies without reproducing itself (fig. 8). however, if the infestation rate increases, the mite effective reproduction rate may increase too (fig. 9). this fact may occur since mite reproduction rates may increase owing to age with a possible introduction of a new recently occurring haplotype of the mite in brazil. the reproductive capacity of the mite may be thus increasing (carneiro et al. 2007). increasing the invasion rate might also increase the total reproduction rate of the mite (fig. 10). fig. 11 shows that there may be a correlation among reproduction rates of the mite when above zero rates occur. this is due to the fact that rates are mutually dependent. since criteria for selection should be chosen so that selected honeybees do not lose their adaptive characteristics, such as their relative resistance to diseases (toledo & mouro 2005), a higher negative correlation for hygienic behavior with total reproduction rate, as that found in the current research, should be taken into account and analyzed with great care. actually, hygienic behavior associated with all the other traits had low correlation and all traits associated with total reproduction rates had significant correlation. this may occur because hygienic behavior simulates problematic brood and total reproduction rate might be a highly precise measurement since the pupae analyzed for this rate (brown eyed pupae) indicate the precise phase of the mite reproduction. it should be also emphasized that the infestation rate of the mite varroa destructor in brazil is relatively low. the rate interferes with the mite reproduction rate since when no infestation occurs, no reproduction rate exists. however, at low infestation rates the hygienic behavior trait is still the most recommended selection criterion. selections for the above trait would solve the reproduction problem of the mite varroa destructor in the context of brood diseases. 267 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies when high infestation rates of the mite exist, the most important criterion is hygienic behavior. in fact, it presents high level heritability and negative correlation with total reproduction rate of the mite varroa destructor. acknowledgments the authors would like to thank the national research council (cnpq, process no. 301943/2005-2 and araucaria foundation 37/2005 4025 for financial support. references arathi, h.s., i. burns & m. spivak 2000. etholog y of hygienic behaviour in honey bee apis mellifera l. (hymenoptera: apidae): behavioural repertoire of hygienic bees. etholog y 106:365-379. bienefeld, k., k. ehrhardt & f. reinhardt 2007. genetic evaluation in the honey bee considering queen and worker effects – a blupanimal model approach. apidologie 38:77-85. boecking, o. & w. ritter 1993. grooming and removal behaviour of apis mellifera in tunisia against varroa jacobsoni. journal of apicultural research 32:127-134. boecking, o., k. bienefeld & w. drescher 2000. heritability of the varroa-specific hygienic behavior in honey bees (hymenoptera: apidae). journal animal breeding and genetics 117:417-424. calderón, r.a., j.w. van veen, m.j. sommeijer & l.a. sanchez 2010. reproductive biolog y of varroa destructor in africanized honey bees (apis mellifera). experimental and applied acarolog y 50:281-297. carneiro, f.e., r.r. torres, r. strapazzon, s.a. ramírez, j.c. jr. guerra, d.f. koling & g. moretto 2007. changes in the reproductive ability of the mite varroa destructor (anderson & trueman) in africanized honey bees (apis mellifera l.) colonies in southern brazil. neotropical entomolog y 36:949-952. costa-maia, f.m. aspectos genéticos da produção de mel e comportamento higiênico em abelhas apis mellifera africanizadas. 2009. 77f. thesis (phd in animal science) uem, universidade estadual de maringá, maringá. de jong, d. & l.s. gonçalves 1981. the varroa problem in brazil. american bee journal 121:186-189. doolittle, g.m. 1889. scientific queen-rearing as practically applied. chicago: illinois, 163p. falconer, d.s. 1987. introdução a genética quantitativa. viçosa, mg: ufv, 279p. geweke, j. 1992. evaluating the accuracy of sampling-based approaches to the calculation of posterior moments. bayesian stat 4:625-631. 268 sociobiolog y vol. 59, no. 1, 2012 gilliam, m., s. taber & g.v. richardson, 1983. hygienic behavior of honey bees in relation to chalkbrood disease. apidologie 14:29-39. gramacho, k.p. estudo do comportamento higiênico em apis mellifera, como subsídio a programas de seleção e melhoramento genético em abelhas. 1995. 103f. dissertation (master’s degree in entomolog y). faculdade de filosofia, ciências e letras de ribeirão preto, ribeirão preto. harbo, j.r. & j.w. harris 1999. heritability in honey bees (hymenoptera: apidae) of characteristics associated with resistance to varroa jacobsoni (mesostigmata: varroidae). journal of economic entomolog y 92:262-265. heidelberger, p. & p. welch 1983. simulation run length control in the presence of an initial transient. operations research 31:1109-1144. junkes, l., j.c.v. guerra jr. & g. moretto 2007. varroa destructor mite mortality rate according to the amount of worker broods in africanized honey bee (apis mellifera l.) colonies. acta scientiarum animal science 29:305-308. lee k.v., r.d. moon, e.c. burkness, w.d. hutchison & j. spivak 2010. practical sampling plans for varroa destructor (acari: varroidae) in apis mellifera (hymenoptera: apidae) colonies and apiaries. journal of economic entomolog y 103:1039-1050. message, d. 2006. devemos utilizar produtos quimioterápicos para controlar doenças e parasitoses das abelhas africanizadas? in: nogueira-couto, r.h. & l.a. couto. apicultura: manejo e produtos. jaboticabal: funep, p.147-156. momot, j.p. & w.c. rothenbuhler 1971. behaviour genetics of nest cleaning in honeybees vi. interactions of age and genotype of bees, and nectar flow. journal of apicultural research 10:11-21. moretto, g., l.s. gonçalves & d. de jong 1993. heritability of africanized and european honey bee defensive behavior against the mite varroa jacobsoni. revista brasileira de genética 16:71-76. moretto, g., l.s. gonçalves, d. de jong & m.z. bichuette 1991. the effects of climate and bee race on varroa jacobsoni oud. infestation in brazil, apidologie 22:197-203. moretto, g. & l.j. mello 2001. infestation and distribution of the mite varroa jacobsoni in africanized honey bee (apis mellifera) colonies. interciencia 26:394-396. morse, r.a. & l.s. gonçalves 1979. varroa disease, a threat to world beekeeping. bee culture 107:179-181. newton, d.c., g.c. cantowell & e.p. bourquin 1975. removal of freeze-killed brood as an index of nest cleaning behavior in honeybee colonies (apis mellifera l.). american bee journal 115:388-406. nogueira-couto, r.h. produção de alimento e cria de colméias de apis mellifera infestadas com varroa jacobsoni, em regiões canavieiras. 1991. 131f. thesis (livre docência em apicultura) universidade estadual paulista, faculdade de ciências agrárias e veterinárias, jaboticabal. r development core team. 2009. r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. isbn 3-900051-07-0, url http://www.r-project.org. 269 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies rinderer, t.e. 1977. a new approach to honey bee breeding at the baton rouge usda, laboratory. american bee journal 117:146-147. rothenbühler, w.c. 1964. behavior genetics of nest cleaning in honey bees iv. responses of f1 and backcross generations to disease-killed brood. american zoologist 4:111-123. spiegelhalter, d.j., a.p. dawid, s.l. lauritzen & r.g. cowell 1993. bayesian analysis in expert systems. statistical sciences 8:219-283. spivak, m. & d.l. downey 1998. field assays for hygienic behavior in honey bees (hymenoptera: apidae). journal of economic entomolog y 91(1):64-70. spivak, m. & m. gilliam 1993. facultative expression of hygienic behavior of honey bees in relation to disease resistance. journal of apicultural research 32:147-157. stort, a.c., l.s. gonçalves, o. malaspina & f.a. moura duarte 1981. study on sineacar effectiveness in controlling varroa jacobsoni. apidologie 12:289-297. taber, s. 1982. bee behavior: breeding for disease resistance. american bee journal 122:823825. terada, y., c.a. garofalo & s.f. sakagami 1975. age-survival curves for workers of two eusocial bees (apis mellifera and plebeia droryana) in a subtropical climate, with notes on worker polytheism in p. droryana. journal of apicultural research 14:161-170. toledo, v.a.a. & g.f. mouro 2005. produção de geléia real com abelhas africanizadas selecionadas cárnicas e híbridas. revista brasileira de zootecnia 34:2085-2092. toledo, v.a.a. & r.h. nogueira-couto 1996. infestação de colônias híbridas de abelhas apis mellifera pelo ácaro varroa jacobsoni. ars veterinária 12:104-112. winston, m.l. the biolog y of the honey bee. london: harvard university, 1987. 281p. winston, m.l. & s.j. katz 1981. longevity of cross-fostered honey bee workers (apis mellifera) of european and africanized races. canadian journal of zoolog y 59:1571-1574. winston, m.l., o.r. taylor & g.w. otis 1983. some differences between temperate and tropical african and south american honeybees. bee world 64:12-21. appendix figures 2-11 begin on page 19 270 sociobiolog y vol. 59, no. 1, 2012 fig. 2. phenotypic (a) and genetic (b) correlation of hygienic behavior with infestation rate of the mite varroa destructor fig. 3. phenotypic (a) and genetic (b) correlation of hygienic behavior with invasion rate of the mite varroa destructor. 271 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies fig. 4. phenotypic (a) and genetic (b) correlation of hygienic behavior with effective reproduction rate of the mite varroa destructor. fig. 5. phenotypic (a) and genetic (b) correlation of infestation rate and invasion rate of the mite varroa destructor. 272 sociobiolog y vol. 59, no. 1, 2012 fig. 6 . phenotypic (a) and genetic (b) correlation of invasion rate and effective reproduction rate of the mite varroa destructor. fig. 7 . phenotypic (a) and genetic (b) correlation of hygienic behavior with total reproduction rate of the mite varroa destructor. 273 wielewski, p. et al. —hygenic behavior and varroa destructor in honeybee colonies fig. 9. phenotypic (a) and genetic (b) correlation of infestation rate with effective reproduction rate of the mite varroa destructor. fig. 8. phenotypic (a) and genetic (b) correlation of infestation rate with total reproduction rate of mite varroa destructor. 274 sociobiolog y vol. 59, no. 1, 2012 fig. 10. phenotypic (a) and genetic (b) correlation of invasion rate with total reproduction rate of the mite varroa destructor. fig. 11. phenotypic (a) and genetic (b) correlation of total reproduction rate with effective reproduction rate of the mite varroa destructor. 1563 a new species of atheta (coleoptera: staphylinidae) from sri lanka found with termites (isptera: termitidae) by david h. kistner* abstract atheta dominguezae (coleoptera) is described from sri lanka where it is found with hospitalitermes monocerus (isoptera). behavioral observations indicate that the beetles are termitariophiles. what is novel about this ttermitariophile is that this species was found to live in this same nest over a period of 13 years . key words: atheta dominguezae, hospitalitermes monoceros, termitariophiles, sri lanka material and methods field methods: specimens of the new species were collected on two occasions' both from what appeared to be the same nest of hospitalitermes monoceros (koenig ) (det. a.e. emerson, 1963). since the first captures (1960) had no speacific termitophilous adaptations, we simply collected a small series although we did note that the beetles were active in the same trails as the termites. later (1973), i returned to sri lanka and found the identical tree in the botanic gasrden at kandy. i took about 4 hours to cut part of the nest off the tree and then studied the beetles interactions with their host termites in the exposed trails. laboratory methods: specimens were studied dried and mounted on microscope slides after being cleared in koh, washed in h 2 o, and mounted in hoyer's medium. multiple photographs were made through either a binocular dissecting microscope or a regular compound scope. the photographs were all augmented using the automontage program and photoshop. measureaments were made using an ocular micrometer. *department of biological sciences, california state university, chico, chico, ca 95929-0515, usa, email: dkistner@csuchico.edu, btaylor5@mail.csuchico.edu 1564 sociobiolog y vol. 59, no. 4, 2012 fig. 1. atheta dominguesae, dorsal view related to atheta graminincola gravenhorst =the generatype of atheta (blackwelder 1952) through its geral habitus aand color but distinguished there from by its smaller size and the shape of the spermatheca. see fig. 6 for a photo of the spermatheca of atheta graminincolaa. overall appearance slender as in fig. 1. head wider than long and about as wide as the pronotum, shaped as in fig. 2a. neck short. sides of gula bowing mesad; submentum separate from the mentum. mouthparts similar to other myrmedoniina, but with variations from the mouthparts of the other species. labrum wider at the apex than at the base, shaped as in fig. 2c. mandibles shaped as in figs. 2e and f, both with only traces of median teeth and so are practically mirror images of each other. maxillae shaped as in fig. 2b, with the lacinia about 20% shorter than the galea. maxillary palpi 4-segmented with the 3rd segment about equal in length to the 2nd, the 1st segment short, the 4th segment spindle shaped and about 4x longer than the 1st. acetabulae of the maxillae emarginate. labium and submentum shaped as inall athaaeta; labial palpi 3-segmented with segment 1 longer than segment 2 but with segment 3 longer than segment 2 but shorter than segment 1. . entire antenna shaped as in fig. 2d. pronotum wider than long and wider thsns the head, somewhat flattened dorsally, as in figs. 1 & 3a, with many setae presumed to results the specimens are a new species of atheta, described below. atheta (atheta) dominguezae new species figs. 1-5 1565 kistner, d.h. — new atheta from sri lanka vary by species. prosternum carinate (fig. 3a). elytra shaped as in fig. 3b, with sculpture and chaetotaxy which probably vary by species. metanotum and abdominal segment i shaped as in fig. 3c. metanotum about equal to 4x the length of abdominal segment i. mesonotum (scutellum) shaped as in most myrmedoniiina (fig. 3c). mesoand metasternum shaped as in fig. 3d, with the mesosternal intercoxal process wide, blunt, and carinate; and with the mesothoracic acetabulae completely margined. . pro-, meso-, and metathoracic legs shaped as in figs. 4a, b, & c,, respectively; tarsal formula 4-5-5. empodia of all legs shaped normally and of normal length. wings shaped as in fig 3e overall abdominal shape as in fig. 5.a segment ii represented by the tergite alone (fig.5a). segments iii-vi consisting of a tergite, a sternite, and 2 pairs of paratergites each (fig. 5a. segment vii (fig. 5a) consisting of a tergite, a sternite and 2 pairs of paratergites with the inner paratergite small and difficult to see. segment viii consisting a tergite and a sternite alone (fig. 5a). segment ix of male with long anterior apodemes and a sternite (fig. 5d); the long apodemes are lacking in females (5b). median lobe of the male genitalia shaped as in fig. 5e lateral lobe of the male genitalia shaped as in a e f a b c d fig. 2. atheta dominguesae,: a, head, dorsal; b, maxilla:c, labrum; d, antenna; e, left mandible; f, right mandible. 1566 sociobiolog y vol. 59, no. 4, 2012 fig. 5e. spermatheca shaped as in fif 5c. color dark reddish brown throughout with head, abdomen and legs a a b c d e fig. 3. atheta dominguesae,: a, pronotum, dorsal; b, left elytron; c, meonotum; metanotum, and abdominal segment 1';d, meso-and metasternum; e, wing. 1567 kistner, d.h. — new atheta from sri lanka lighter reddish brown sculpture of the dorsal surface of the head and abdomen deeply punctate with each puncture bearing a seta. the ventral surface of the body and to a lessor extent the dorsal surface of the abdomen with a a b c fig. 4. atheta dominguesae,: a, prothoracic leg ; b, mesothoracic leg ; c, metathoracic leg. 1568 sociobiolog y vol. 59, no. 4, 2012 fig. 5;. theta dominguesae,: a, abdominal segments ii-viii, dorsal; b, female abdominal segment ix; c, female spermatheca; d, male abdominal segment ix; e, male median lobe of male genitalia; f, lateral lobe of male genitalia. a b c d e f even vestiture of fine yellow setae. macrochaetotaxy of abdominal tergites ii-viii: 2,2,2,2,2,0. measurements: (in mm. ) pronotum length, 0.13-0.16; ; elytra length, 0.110.14. number measured, 10. holotype: male, no.21323, sri lanka, kandy, botanic garder , 50 may 1973, dex nest t-622, collected by d.h. kistner, no. 3153. in the collection of d.h. kistner to be eventually deposited in the field museum of natural history, chicago. 1569 kistner, d.h. — new atheta from sri lanka paratypes: 70, ( including 4 partially on slides), same data as the holotype (f.m.n.h., d.k.); 6, (including 3 partially on slides), sri lanka, kandy, botanic garden, 16 august 1960, ex nest t-25, collected by d.h. & a.c. kistner, no. 655, (f.m.n.h.). notes: at the time of captire of lot 655, sri lanka was called ceylon and those specimens are so labelled. this species is named for ms esmeralda dominguez who greatly assisted the author in reading pin labels and other materials that were difficult for his aged eyes. behavior of the species interaction between the termites and the atheta were watched in the columns and in petri dishes for over four hours. the termites were never aggressive to the athetta nor vice versa. soo far as i could see, the atheta fig, 6. spermatheca of atheta graminincola 1570 sociobiolog y vol. 59, no. 4, 2012 walked among the termites but were ignored. thus they fit in the category of termitariophiles as defined by araujo (1970). what this species adds to the concept is longevity as the species persisted in the same nest over a period of 13 years. acknowledgments thanks are given to kumar krishna, american museum of natural history, n.y. for identifying part of the termites. the field work was carried out under a grant from the national science foundation from many years ago. reference araujo, renato l. 1970. termites of the neotropical region. chapter 12 in biolog y of termites, edited by kumar krishna and frances m. weesner, academic press, new york & lon1951on, pp. 527-576. blackwelder,richard e.1952.the generic names of the beetle family staphykinidaee with an essay on genotype. smithsonian institution, united states national museum bulletin 200, pp 1-483. doi: 10.13102/sociobiology.v67i3.4837sociobiology 67(3): 388-400 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction in recent years, a growing number of studies have shown that the yields of many agricultural crops increase when pollinated by bees. the role of wild bees has garnered further attention due to the realization that human food security is directly dependent on this ecosystem service (potts et al., 2016; klein et al., 2018). according to garibaldi et al. (2013), pollination by wild insects is more efficient than that carried out by honey bees alone; mallinger et al. (2019) quantified the economic value of pollination by wild bees in sunflower crops in the united states at 56.7 million us dollars; two solitary bee species were identified as the most abstract many are the anthropogenic drivers of pollinator decline, but the loss of suitable habitats, among other effects caused by agricultural intensification, deserves special attention. reduction in the availability of floral resources negatively affects bee communities, compromising bee species composition, foraging behavior, corporal size, and fitness. our study aims to understand whether the presence of herbaceous plants, acting as foraging sites, next to crops contributes to bee species richness in smallholder rural properties. bee sampling was performed on smallholder rural properties in the municipality of guapiara, southern são paulo state. individuals who visited the flowers of ruderal plants and crops were collected, using an entomological net, for ten months. a total of 61 bee species were identified, with the highest species richness being sampled in ruderal plant flowers in the three properties studied. only in one property, ruderal plants hosted a more diverse bee assemblage (shannon-wiener and taxonomic diversity indices), but species composition differed from that sampled in crop plants (jaccard index) in all properties. thirty-two species were sampled exclusively in ruderal plants, versus 9 only in crops and 20 species in both types of plants. pollen analysis showed that of the 22 species of bees that were sampled only on flowers of ruderal plants, 9 species carried pollen of tomato and one species of bee carried pollen of kabocha squash. ruderal plants can provide an alternative food resource for pollinators, enabling these insects to remain in or be attracted to crop areas, where, in addition to visiting such plants, they also visit the cultivated plant flowers. allowing coexistence between crops and ruderal plants, provided that the issues of plant health are observed, is a simple and low-cost measure for farmers and will provide both economic and environmental benefits. sociobiology an international journal on social insects pc montagnana, mjo campos article history edited by celso martins, ufpb, brazil received 24 october 2019 initial acceptance 27 april 2020 final acceptance 21 july 2020 publication date 30 september 2020 keywords plant diversity, crops, smallholder rural properties, atlantic forest. corresponding author pc montagnana departamento de ecologia instituto de biociências universidade estadual paulista júlio de mesquita filho avenida 24 a, 1515, bela vista cep: 13.506-900, rio claro-sp, brasil. e-mail: paula-eco@hotmail.com efficient pollinators of this crop. crops such as strawberries (herrmann et al., 2019; macinnis & forrest, 2019), rapeseed oil (halinski et al., 2018; perrot et al., 2018) and blueberry (nicholson & ricketts, 2019), among others, are also better pollinated by wild bees. the anthropogenic drivers of pollinator decline are many (see potts et al., 2010), but the loss of suitable habitats, among other effects of agricultural intensification, deserves special attention. food and nesting resources are drastically reduced in intensively farmed landscapes because such areas have a high rate of land-use change and severe disturbances such as tillage, mowing, and grazing (potts et al., 2010). specialist species, whether habitat or dietary, experience the universidade estadual paulista júlio de mesquita filho (unesp), rio claro, são paulo, brazil research article bees ruderal plants providing bees diversity on rural properties sociobiology 67(3): 388-400 (september, 2020) 389 greatest population decline (biesmeijer et al., 2006). as the proportion of a landscape’s agricultural cover increases, bee abundance and richness decrease, and much phylogenetic diversity is lost, leaving bee communities in such landscapes composed of more closely related species (grab et al., 2019). reduction in the availability of floral resources, regardless of the responsible causes, affects bee communities negatively in terms of species composition, foraging behavior, corporal size, and fitness (jha & kremen, 2013; campbell et al., 2018; rollin et al., 2019). larval development and offspring size suffer when females provide brood cells with little pollen or pollen of poor nutritional quality (peterson & roitberg, 2006; vanderplanck et al., 2014; moerman et al., 2015; renauld et al., 2016). since the amount and type of nutrients present in pollen and nectar tend to vary from one plant species to another (london-shafir et al., 2003; mao et al., 2013; abbas et al., 2014) bees’ access to a diverse flora ensures better-nourished insects. sites within rural properties that harbor plant diversity can help maintain pollinator populations and thus provide pollination services in agricultural landscapes (albrecht et al., 2007), for both crops and native plants. ghazoul (2006) found that there were more pollinator visits to raphanus raphanistrum l. flowers when individuals of this species occurred close to individuals of three more herbaceous species. according to the author, his study confirmed the hypothesis that pollination facilitation exists among co-flowering plants whose floral displays differ, attracting greater pollinator diversity. thus, in addition to being an alternative source of food, ruderal plants also contribute to greater diversity in environments at the farm scale, which favors a greater abundance of pollinators (chateil & porcher, 2015). our study aims to understand whether the presence of herbaceous plants, acting as foraging sites, next to crops contributes to bee species richness in smallholder rural properties. we believe that the bee species richness and diversity sampled on ruderal plant flowers are greater than those sampled on crop plants; bee species composition also differs between these two types of plants. material and methods study area the study was carried out at three smallholding rural properties in guapiara municipality, southern são paulo state, brazil (fig 1). according to köppen typology, the climate in guapiara is cwa (humid temperate with dry winter and wet summer). the mean temperature is 15.8ºc in the coldest month (july) and 23.2ºc in the hottest month (february); the average rainfall is 53.3 mm in the driest month (august) and 217.3 mm in the wettest month (january) (cepagri, 2014). guapiara contains part of the intervales state park and is in the heart of the region that comprises the mosaic of conservation units of serra de paranapiacaba, which preserves the largest remaining area of atlantic forest in brazil, with 120,000 hectares of protected areas (fundação florestal, 2019). fig 1. smallholder rural properties overview. s1: property s1 showing cultivated and post-harvest areas. s2: property s2 showing a post-harvest area. s3: property s3 showing flat bean plantation. pc montagnana, mjo campos – ruderal plants as food sources390 property s1 is the largest in the area (8.67 ha), followed by property s2 (4.20 ha) and s3 (3.24 ha). the longest euclidean distance between the properties is 8.63 km (s2s3), with properties s1 and s2 being closer (4.61 km); s1 and s3 are 6.91 km apart (fig 2). in all, six crops were grown on the three rural properties over a year (zucchini, kabocha squash, tomato, flat bean, peach, and cucumber). however, on s1 property five crops were grown (s1 = kabocha squash, tomato, flat bean, peach, and cucumber), blooming from july/2012 to march/2013 (table 1). properties s2 and s3 had three crops each (s2 = flat bean, cucumber, and zucchini; s3 = flat bean, tomato, and kabocha squash), but with only one crop in common and different blooming periods (fig 3). blooming period refers to the time when there were crop flowers on the property, as there were plantations of the same crop with different ages within the properties. a common practice in the properties studied is to allow the free growth of ruderal plants in post-harvest areas and crop borders so that throughout the year there is a turnover of areas that concentrate a greater abundance of these plants. fig 2. study area location. south america, brazil. blue square indicating the study area in the são paulo state. fig 3. crop plants blooming period at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. data collection for a period of ten months, from may 2012 to march 2013 (except november 2012), we collected bees at three rural properties. bees were collected from 8 a.m. to 1 p.m., always by two collectors, with a change of collectors every sampling month. using an entomological net one collector sampled the bees walking the crop lines, and the other one collected the bees on ruderal flowers present on the crop edges and in transects open in post-harvest areas. when there were no crops in bloom on any of the three rural properties, the two collectors sampled the bees on ruderal flowers in the post-harvest and surrounding areas. each day of collection involved four to five 1-hour sampling cycles at 20-minute intervals, totaling 50 hours/property. at the time of insect collection were noted the plant type where the capture occurred (whether cultivated sociobiology 67(3): 388-400 (september, 2020) 391 crop bee species rural properties s1 s2 s3 tomato (lycopersicon esculentum mill.) anthrenoides meridionalis (schrottky, 1906) x augochloropsis cupreola (cockerell, 1900) x augochloropsis electra (smith, 1853) x augochloropsis sp. 2 x augochloropsis sp. 3 x x bombus (fervidobombus) morio (swederus, 1787) x x bombus (fervidobombus) pauloensis friese, 1913 x x exomalopsis (exomalopsis) analis (spinola, 1853) x x m. (melipona) quadrifasciata quadrifasciata lepeletier, 1836 x oxaea flavescens (klug, 1807) x oxaea sp. x paratrigona subnuda moure, 1947 x x psaenythia bergii (holmberg, 1884) x pseudaugochlora indistincta (almeida, 2008) x tetragonisca angustula (latreille, 1811) x trigona spinipes (fabricius, 1793) x xylocopa (neoxylocopa) suspecta (moure & camargo, 1988) x xylocopa (stenoxylocopa) artifex (smith, 1874) x zucchini (cucurbita pepo l.) augochlora (augochlora) amphitrite (schrottky, 1909) x augochlora (augochlora) foxiana (cockerell, 1900) x bombus (fervidobombus) morio (swederus, 1787) x bombus (fervidobombus) pauloensis friese, 1913 peponapis fervens (smith, 1875) x trigona hyalinata (lepeletier, 1836) x trigona spinipes (fabricius, 1793) x x kabocha squash (cucurbita maxima duchesne) apis mellifera linnaeus, 1758 x bombus (fervidobombus) pauloensis friese, 1913 x melissoptila thoracica (smith, 1854) x paratetrapedia fervida (smith, 1879) x peponapis fervens (smith, 1875) x x trigona hyalinata (lepeletier, 1836) x x trigona spinipes (fabricius, 1793) x x flat bean (phaseolus vulgaris l.) apis mellifera linnaeus, 1758 x x augochlora (augochlora) amphitrite (schrottky, 1909) x bombus (fervidobombus) morio (swederus, 1787) x x x bombus (fervidobombus) pauloensis friese, 1913 x x x paratrigona subnuda moure, 1947 x x peponapis fervens (smith, 1875) x schwarziana quadripunctata (lepeletier, 1836) x trigona spinipes (fabricius, 1793) x x x xylocopa (neoxylocopa) brasilianorum (linnaeus, 1767) x xylocopa (neoxylocopa) frontalis (olivier, 1789) x x xylocopa (stenoxylocopa) artifex (smith, 1874) x x cucumber (cucumis sativus l.) bombus (fervidobombus) morio (swederus, 1787) x paratrigona subnuda moure, 1947 x peach (prunus persica (l.) batsch) trigona hyalinata (lepeletier, 1836) x table 1. bee species sampled on crop flowers at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. pc montagnana, mjo campos – ruderal plants as food sources392 or ruderal), the identity of the rural property (if number 1, 2 or 3), the collector (identified by the first letter of the name) and the number of the insect (based on the amount that each collector sampled). bee specimens collected were euthanized in ethyl acetate and stored in 70% alcohol. in the laboratory, the specimens were pressed, appropriately tagged, and stored in entomological drawers. species identification was carried out by dr. silvia pedro from faculdade de filosofia, ciências e letras de ribeirão preto, brazil. the specimens are currently stored in the collection of the grupo de estudos em ecologia e conservação de abelhas silvestres laboratory, são paulo state university (unesp), rio claro, são paulo, brazil. the sampling of ruderal plants present in rural properties was also carried out monthly. each new ruderal plant that was identified in bloom was photographed, flower buds were collected for pollen analysis and a sample of the plant for the confection of exsiccates. the same procedure was adopted for crop plants in bloom. pollen analysis to complement the data of which plants were used as a food source for the bees sampled in the three rural properties, we carried out the analysis of pollen grains adhered to the specimens’ bodies, scopes, and corbicles. the pollen samples were obtained from the body of 115 individuals collected on crop flowers and 267 individuals collected on ruderal flowers and transferred to test tubes containing 50 ml of ethanol solution 70%. after 24 hours, the ethanol was discarded and the samples were placed in 4 ml of glacial acetic acid for 24 hours (silva et al., 2010). after that, the pollen material was submitted to the acetolysis process following the method described by erdtman (1960), and when the acetolysis process was finished the samples were then put in an aqueous glycerine solution of 50%. after another 24 hours, small amounts of acetolized pollen were removed with cubes of glycerine jelly for the preparation of three microscopic slides per individual. photomicrographs were taken and pollen types were identified by comparisons with the reference slides created with pollen samples collected from floral buds of the ruderal and crop plant species sampled in the three rural properties studied. pollen grains removed from anthers contained in flower buds were submitted to the same process described above, with three microscopic slides per plant species. the ruderal plants sampled were identified using the book “plantas daninhas do brasil” (lorenzi, 2008). data analysis we compared the bee assemblages from flowers of ruderal and crop plants for each of the three properties. to estimate species richness, we used the chao 1 index, which is based on abundance and is a function of the ratio of singletons to doubletons (magurran, 2013). average taxonomic breadth, (δ+; clarke & warwick, 1998; andersson et al., 2013) and shannon’s (h’) diversity index were calculated to estimate the sample diversity, while the dominance was calculated through the simpson dominance index (d). average taxonomic breadth is a diversity index that uses information from a linnaean classification tree of the taxonomic relations between all sampled bee species. high δ+ values indicate a taxonomically broad, therefore taxonomically diverse, bee community; low δ+ values indicate a community composed of closely related species. simpson dominance index ranges from 0, where all taxa are equally present, to 1, where one taxa dominates the community (magurran, 2013). finally, to verify whether bee species composition differed between the assemblage sampled from ruderal plants and that sampled from crop plants, we used the jaccard’s dissimilarity index (magurran, 2013). the analyses were performed using rstudio software version 3.6.3 (r core team, 2020) and the package vegan (oksanen et al., 2019). results a total of 61 bee species were identified (families apidae, halictidae, megachilidae, colletidae, and andrenidae) among the 666 specimens collected across the three properties (table 2). property s2 harbored the greatest bee species richness (n = 41 species), followed by properties s3 (n = 37) and s1 (n = 31). bombus pauloensis was the most abundant species in the three rural properties (s1 = 66 specimens; s2 = 65; s3 = 30), with trigona spinipes the second most abundant in two properties (s2 = 23 specimens; s3 = 28) and bombus morio the second most abundant in the s1 property (s1 = 40 specimens). twenty-nine bee species were collected on crop flowers, where the tomato was the crop with the greatest bee species richness visiting its flowers (n = 18 species) (table 1) and the bee species bombus morio that visited more varieties of crops. twenty-eight species of ruderal plants were identified overall, with 25 species occurring on property s1, 20 species on property s2, and 15 species on property s3 (table 3). pollen analysis showed that leonurus sibiricus and sonchus oleraceus were the ruderal species visited by the largest number of bee species, 22 species each. meanwhile, the rural property with the greatest ruderal plants species richness (s1 = 25 ruderal species) was not the place where there was the greatest bee species richness (s2 = 41 bee species), nor the place with the greatest bee species richness visiting ruderal plants (s2 = 39 bee species). there were nine bee species collected only on crop flowers and 32 bee species collected only on ruderal flowers (table 2); however, 20 bee species were collected on both types of plants. in the discussion section, we will pay special attention to species that have visited both ruderal and crop plants. pollen analysis also showed that of the 22 species of bees that were sampled only on flowers of ruderal plants, 9 species carried pollen of tomato and one species of bee carried pollen of kabocha squash, being augochloropsis sp 1, dialictus sp., exomalopsis (exomalopsis) planiceps, exomalopsis sp 2, megachile (chrysosarus) pseudanthidioides, melissodes (ecplectica) nigroaenea, neocorynura oiospermi, psaenythia sp, sociobiology 67(3): 388-400 (september, 2020) 393 thygater (thygater) analis, and exomalopsis sp 1, respectively. the same happened with the bee species that were collected only on crop flowers, of 9 species 5 carried pollen grains from ruderal plants: augochlora (augochlora) foxiana, augochloropsis electra, exomalopsis (exomalopsis) analis, oxaea sp., and paratetrapedia fervida. table 2. bee species, abundance and type of plant visited at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. species plants abundance/ rural property crop ruderal s1 s2 s3 andrenidae oxaeini oxaea flavescens klug, 1807 x 1 oxaea sp x 6 panurginae protandrenini anthrenoides meridionalis (schrottky, 1906) x x 9 1 anthrenoides jordanensis urban, 2007 x 1 cephalurgus anomalus moure & lucas de oliveira, 1962 x 3 psaenythia bergii holmberg, 1884 x x 1 1 2 psaenythia sp x 3 rophitulus sp x 1 apidae apinae apini apis mellifera linnaeus, 1758 x x 42 18 10 bombini bombus (fervidobombus) brasiliensis lepeletier, 1836 x 1 1 bombus (fervidobombus) morio (swederus, 1787) x x 40 10 5 bombus (fervidobombus) pauloensis friese, 1913 x x 66 65 30 emphorini melitoma segmentaria (fabricius, 1804) x 1 eucerini melissodes (ecplectica) nigroaenea (smith, 1854) x 3 4 2 melissoptila richardiae bertoni & schrottky, 1910 x 1 1 melissoptila thoracica (smith, 1854) x x 4 3 peponapis fervens (smith, 1875) x x 7 3 5 thygater (thygater) analis (lepeletier, 1841) x 2 trichocerapis mirabilis (smith, 1865) x 1 euglossini eulaema (apeulaema) nigrita lepeletier, 1841 x 1 exomalopsini exomalopsis (exomalopsis) analis spinola, 1853 x 9 11 exomalopsis (exomalopsis) planiceps smith, 1879 x 1 1 exomalopsis (exomalopsis) sp 1 x 3 exomalopsis (exomalopsis) sp 2 x 1 meliponini geotrigona subterranea (friese, 1901) x 4 3 m. (melipona) quadrifasciata quadrifasciata lepeletier, 1836 x x 2 3 paratrigona subnuda moure, 1947 x x 28 22 10 schwarziana quadripunctata (lepeletier, 1836) x x 10 6 1 tetragonisca angustula (latreille, 1811) x x 1 1 trigona hyalinata (lepeletier, 1836) x x 12 3 pc montagnana, mjo campos – ruderal plants as food sources394 trigona spinipes (fabricius, 1793) x x 23 23 28 tapinotaspidini paratetrapedia fervida (smith, 1879) x 1 tetrapediini tetrapedia aff. diversipes klug, 1810 x 1 xylocopinae ceratini ceratina (ceratinula) cf. oxalidis schrottky, 1907 x 1 xylocopini xylocopa (neoxylocopa) brasilianorum (linnaeus, 1767) x x 2 1 xylocopa (neoxylocopa) frontalis (olivier, 1789) x x 5 4 1 xylocopa (neoxylocopa) suspecta moure & camargo, 1988 x x 2 xylocopa (stenoxylocopa) artifex smith, 1874 x x 3 3 2 colletidae colletinae colletes rugicollis friese, 1900 x 1 halictidae halictinae augochlorini augochlora (augochlora) amphitrite (schrottky, 1909) x x 2 1 7 augochlora (augochlora) foxiana cockerell, 1900 x 1 augochlora (oxystoglossella) morrae strand, 1910 x 1 1 1 augochlora (oxystoglossella) sp x 1 augochloropsis cupreola (cockerrell, 1900) x x 3 2 2 augochloropsis electra (smith, 1853) x 2 augochloropsis nasuta moure, 1944 x 1 augochloropsis notophos (vachal, 1903) x 1 augochloropsis sp 1 x 2 augochloropsis sp 2 x x 7 3 2 augochloropsis sp 3 x 1 1 neocorynura oiospermi (schrottky, 1909) x 3 4 1 pseudaugochlora indistincta almeida, 2008 x 1 halictini dialictus creusa (schrottky, 1909) x 1 dialictus sp 1 x 1 dialictus sp x 1 2 3 pseudagapostemon (neagapostemon) cf. cyanomelas cure, 1989 x 1 megachilidae megachilinae anthidiini anthidium manicatum (linnaeus, 1758) x 1 1 megachilini megachile (chrysosarus) pseudanthidioides moure, 1943 x 1 1 megachile (melanosarus) sp x 2 megachile sp 1 x 2 megachile sp 2 x 1 table 2. bee species, abundance and type of plant visited at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. (continuation) species plants abundance/ rural property crop ruderal s1 s2 s3 sociobiology 67(3): 388-400 (september, 2020) 395 a comparison between the richness estimator chao 1 and the sampled species’ richness values indicates that there may be even more bee species in the study area (table 4) and there were greater bee species on ruderal flowers than on crops. the assemblages of bees collected on ruderal plants are not more diverse than those of crop plants, nor are they taxonomically more diverse (table 4). property s2 differs from these results, due to a smaller number of bee species collected on crop plants; this difference may be a consequence of not having planted tomatoes that year, a mass-flowering crop and prevalent in the guapiara municipality. property s3 was the only one that showed greater dominance in the bee assemblage collected on ruderal plants. however, the composition of bee species differs between crop and ruderal for the three properties (table 4). family species resource floral visiting rural properties bee species (n) s1 s2 s3 apocynaceae asclepias curassavica n/p x x x asteraceae ageratum conyzoides n/p 7 x x x bidens alba n/p 6 x x bidens pilosa n/p 11 x x x emilia fosbergii n/p 4 x x galinsoga paviflora n/p 1 x x senecio brasiliensis n/p 4 x x sonchus oleraceus n/p 22 x x synedrella nodiflora n/p 2 x x tilesia baccata n/p 2 x x tithonia diversifolia n/p x vernonia westiniana n/p x x bignoniaceae pyrostegia venusta n/p x boraginaceae cordia curassavica n x brassicaceae brassica rapa n/p 6 x convulvulaceae ipomea nill n/p x x x ipomea triloba n/p x euphorbiaceae ricinus communis p 1 x x lamiaceae leonurus sibiricus n 22 x x x malvaceae sida rhombifolia p 4 x x phytolaccaceae phytolacca americana n/p x x primulaceae anagallis arvensis p x x rosaceae rubus rosifolius n/p 1 x x x solanceae solanum americanum p x x solanum erianthum p 1 x x solanum paniculatum p 2 x x x verbenaceae lantana camara n x x x stachytarpheta cayennensis n 4 x x x total species 25 20 15 table 3. ruderal plant species, resource available for pollinators according to literature data (p: pollen; n; nectar), and number of floral visiting bee species (according to pollen analysis) at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. in bold are the ruderal plant species visited by the greatest number of bee species. discussion as we expected, species richness of bees visiting ruderal flowers was greater than the richness of bees visiting crop flowers, as well as differences in species composition. but we did not expect that there will be no differences concerning diversity (shannon index) and average taxonomic breadth (δ+). however, our results reinforce the importance of ruderal flora for increasing plant diversity on rural properties and thus contributing to bee diversity in agricultural landscapes. studies conducted mostly in europe and the united states have already evaluated the importance of plant diversity in agricultural areas, mainly in set-aside systems, and its positive effects on pollinator diversity (carreck & williams, 2002; potts et al., 2003; hopwood, 2008; kuussaari et al., 2011; tscharntke et al., 2011; kovács-hostyánszki et al., 2011; torné-noguera et al., 2014; venturini et al., 2017). pc montagnana, mjo campos – ruderal plants as food sources396 ruderal plants can provide an alternative food resource for pollinators, enabling these insects to remain in or be attracted to, crop areas where they visit the cultivated plant flowers as well as the ruderal plant flowers (nicholls & altieri, 2013). our pollen analysis shows that both bee species that were sampled only on crop or ruderal flowers carried pollen grains from both types of plants, together with the result that 20 bee species were sampled in both types of plants, it strengthens the argument that ruderal plants are an alternative food source in agricultural landscapes (luz et al., 2011; luz et al., 2018; santos et al., 2020). however, there is a lot of concern about ruderal plants competing with crop plants for pollinator visitation. for some crops, though, studies are showing that such competition does not occur (lundin et al., 2017; alomar et al., 2018; knapp et al., 2019); instead, the presence of a richer ruderal plant community at the field scale favored a more diverse bee community (norris et al., 2018; knapp et al., 2019). in sunflower crops, for example, the presence of a diverse ruderal plant community within the crop field was responsible for increasing the diversity of flower visitors while also mitigating the negative effects of isolation on crops that were far from native vegetation remnants (carvalheiro et al., 2011). of the 20 bee species in common between crop and ruderal plants (table 1), some are well-studied crop pollinators. as is the case of bee genera apis, bombus, melipona, trigona, and xylocopa, which are among effective pollinators of various crops, including those planted in our three rural properties studied (giannini et al., 2015). apis mellifera, bombus morio, bombus pauloensis, melipona quadrifasciata, trigona spinipes, and xylocopa frontalis are among the 14 most important bee species for the pollination of brazilian crops, considering the economic value of the pollination and the number of pollinated crops by them (giannini et al., 2020). for good agricultural production, a diverse pollinator fauna is necessary (garratt et al., 2014), which in turn is maintained by a diverse flora at crop scale and adjacent off-crop habitats (medeiros et al., 2017; thompson et al., 2019). tomato is the predominant crop in the guapiara municipality, the longest time flowering period (6 months) and also with the greatest bee species richness as floral visitors. due to its long flowering period and wide distribution in the municipality, it can be considered a mass-flowering crop and an important pollen source, mainly for native bee species since they are the only ones capable of performing buzz pollination releasing pollen from the poricidal anthers of its flowers (nunes-silva et al., 2010). although studies indicate positive and negative effects of mass-flowering crops on bee species richness and abundance (holzschuh, et al., 2011; holzschuh et al., 2013; diekotter et al., 2014; holzschuh et al., 2016), it is recognized that a diversified flora in the region provide food resources in periods when the crops are not blooming, favoring pollinator populations permanence. plant diversity at crop scale is crucial for providing bees with diverse food sources, especially in agricultural regions with few remnants of native vegetation. simplified landscapes, such as those dominated by monocultures, tend to harbor lower plant diversity and, therefore, lower quantity and quality of available food resources, which can compromise the fitness of bee individuals in the region. renauld et al. (2016) verified that the offspring from the solitary bee andrena nasonii robertson, 1895 in homogeneous landscapes had a smaller body size than those born in landscapes with lower percentages of agricultural occupation. the availability of food resources throughout the seasons is closely related to plant diversity, which positively influences the fitness and population growth of social bees as well (kaluza et al., 2018). at the rural properties sampled in the present study, it is common practice for farmers to allow the free growth of ruderal plants in the post-harvest areas (fig 4 c, e) and at the borders of the crop fields (fig 4 a). even though the practice is not intended to provide foraging places for bees, such sites are widely used by apicultural fauna (fig 4 d, f). measures such as those adopted by the farmers of guapiara municipality favor the permanence of wild bee populations in agricultural areas, and they can potentially be replicated in other localities. allowing ruderal plants to coexistence with crops, provided that the issues of plant health are observed, is a simple and low-cost measure for farmers and provides both economic and environmental benefits, as already presented by carvalheiro et al. (2011). our study has led us to conclude that ruderal plants help to enhance plant diversity at crop scale, which is crucial for providing bees with diverse food sources contributing to the permanence of these pollinators in agricultural landscapes. property s1 property s2 property s3 crop ruderal crop ruderal crop ruderal species richness 19 23 6 39 18 28 chao 1 26 44.25 7 73.33 29.66 48.6 shannon-wiener (h’) 2.35 2.4 1.76 2.69 2.3 2.95 simpson (d) 0.85 0.87 0.8 0.86 0.84 0.92 average taxonomic breadth (∆+) 80.46 80.15 58.83 87.66 80.63 80.86 jaccard (j) 0.7142 0.9052 0.7709 table 4. richness estimators, diversity and dominance indexes for bees sampled from crop and ruderal plants at three smallholder rural properties in guapiara, são paulo state, brazil, between may 2012 and march 2013. sociobiology 67(3): 388-400 (september, 2020) 397 acknowledgments we thank the owners of the sites, mr. lauro, paulo family, and mr. josé, who allowed us access to the plantations for the sampling of bee fauna. funding provided by cnpq (project “conservação e manejo de polinizadores para uma agricultura sustentável através da abordagem ecossistêmica”), global environmental facility (gef), the united nations environment programme (unep), the food and agriculture organization (fao), the brazilian biodiversity fund (funbio), and the brazilian ministry of the environment (mma). authors contribution p. c. m. carried out the fieldwork and performed the statistical analysis; m. j. o. c. participated in the design of the study and, p. c. m. and m. j. o. c. drafted the manuscript. all authors gave final approval for publication. references abbas, m., klein, a.m., ebeling, a. et al. 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(2017). pollination reservoirs for wild bee habitat enhancement in cropping systems: a review. agroecology and sustainable food systems, 41: 101-142. doi: 10.1080/21683565.2016. 1258377 839 load capacity of workers of atta robusta during foraging (hymenoptera: formicidae) by fabiola bonicenha endringer1, ana maria viana-bailez1, omar e. bailez1, marcos da cunha teixeira2, victor luiz de souza lima1 & josé hildefonso de souza1 abstract worker ants are highly polymorphic in the genus atta and they are usually classified into castes according to the specific functions they perform in a colony. minor workers (head width ≤ 2.0 mm) help to maintain and grow the symbiotic fungus whereas larger workers (head width > 2.0 mm) cut and transport plant fragments. this study investigated the roles in the cutting and transporting of different plant resources of different atta robusta worker classes that were classified based on the size of their head capsule. experiments were conducted in the restinga of grussaí/iquipari, são joão da barra, rio de janeiro state, brazil. in each month between october 2009 and september 2010, we collected 100 ants and their respective loads from the trails of four nests of atta robusta. the samples were individually transported to the laboratory, where the ants and their loads were weighed and the head capsules of the ants were measured. large ants transported heavier loads. these ants usually transported more fruit and seeds than smaller ants. keywords: leaf-cutting ants, atta robusta, restinga, load transport, polymorphism introduction in the genus atta, individual ants display a high degree of polymorphism and they are classified into castes according to the specific functions they perform within a colony (wilson 1971). in atta sexdens, the gardener ants have a head capsule of 0.8 to 1.0 mm and they perform tasks such as the care of the fungus and the offspring, as well as the final treatment of the fungal 1laboratório de entomologia e fitopatologia – universidade estadual do norte fluminense darcy ribeiro28013-602 – campos dos goytacazes, rj; brasil. email: amaria@uenf.br 2centro de ciências agrárias, ambientais e biológicas ccaabuniversidade federal do recôncavo da bahia 840 sociobiolog y vol. 59, no. 3, 2012 substrate. generalist ants have a head capsule that measures about 1.4 mm in width and they perform various tasks such as the processing of plant material, attending the offspring during ecdysis, queen care, waste disposal, and rebuilding the fungus. the foragers have a head capsule width that varies from 2.0 to 2.2 mm and they explore, cut, and transport vegetable material that is destined for the nest. the guards or soldier ants are the largest and they have head capsules that measure 3.0 mm on average. they defend the nest, but they also forage because they possess large mandibles that facilitate the task of cutting leaves (wilson 1980). this division of castes has also been described in the genus acromyrmex, although polymorphism is not as conspicuous in this genus as it is in atta (forti et al. 2004; moreira et al. 2010). the performance of a specific task that is a function of worker body size is referred to as alloethic (wilson 1980). this can be considered as a quantitative relationship for characterizing polyethism. foraging and fungus cultivation have a strong alloethic correlation because of the gradual change in the skills with the reduction in worker body size (forti et al. 2004). the ant atta robusta transports large amounts of fruit and seeds to the nest throughout the year, which belong to various plant species (teixeira 2007). in general, atta workers carry loads that are appropriate to their body capacity as observed in atta colombica (lighton et al. 1987); atta cephalotes (wetterer 1990) and atta vollenweideri (kleineidam et al. 2007). however, the seed freight capacity of atta robusta might possibly be increased due to the cooperation and participation of soldiers in the transport of larger seeds (teixeira 2007). this study aimed to clarify how distinct classes of atta robusta workers participate in the transport of resources. we tested whether the size of the ant influenced the size of the load carried and whether there was specialization in the transport of specific resources in different forager size classes. materials and methods study area the study was conducted at the restinga ecosystem from grussaí/iquipari complex, located in são joão da barra (21 44’s; 41 02’o), north of rio de janeiro state, brazil. 841 endringer, f.b. et al. — load capacity of atta robusta workers establishment of worker size classes the foragers were separated into classes based on the size of the head capsule, using a scale of 1 mm per class. the workers with a head width less than 1 mm were not considered in the tests because they were not abundant. we obtained five classes of foragers with the following head capsule sizes: class 1 (1 to 2 mm); class 2 (2.1 to 3 mm); class 3 (3.1 to 4 mm); class 4 (4.1 to 5 mm) and class 5 ( ≥ 5.1 mm). relationship of the transported resource with the workers size of the atta robusta each month, ants were collected from trails as they carried resources to four nests located in physiognomic units that were defined as beach grass and shrub formations. we collected 100 ants and their loads at 30 cm from the entrance of each nest. the ants and their loads were individually placed in glass vials. samples were collected after 5:00 pm, when the major foraging flow was established. the collected material was stored in a refrigerator to reduce any mass loss by the evaporation of water. the mass of the material collected was measured using a precision balance (marte, ay 220. 0,0001g ). measurements of the head capsules of ants were made using a digital caliper (digimess, resolution of 0,005mm/0,002”). the reliability of these measurements were assessed by comparing them with measurements made using a binocular stereoscopic microscope and an ocular micrometric (wilson 1980). there was no difference so we preferred to make the measurements using a caliper because it was faster and it made the handling easy. the loads carried by workers were classified as leaf, dry leaf, flower, cladode, fruit, seed, stem, bud, or other. data analysis to compare the load mass with the size of the head capsule of ants we used an analysis of variance (anova) to compare the means with a tukey test at a p < 0.01 significance level. to test whether there was specialization in the transportation of specific resources according to the class of workers, we made comparisons using the chi-square test at a p < 0.01 significance level. to determine whether there was any specialization in the worker in the transport of specific resources such as cladodes, fruit, and seeds, we analyzed samples from nests when 842 sociobiolog y vol. 59, no. 3, 2012 these resources were being foraged. we counted the total fruit and seeds that were foraged and the total of other resources and we compared them using a chi-square test for each class of foraged fruit, seeds, and other resources. the same comparison was made for the cladode resource. results atta robusta foragers were classified into five classes based on the size of their head capsule which ranged from 0.95 mm to 5.62 mm. it was verified that the average mass of load carried by the foragers was 0.0365g and the maximum load was 0.450 g. most foragers present on the trails were in classes 1 and 2, and these two classes combined constituted 95% of all ants collected from the trails (fig. 1). we showed that there was a significant difference in the mass of the loads transported by the different classes of workers (anova, f = 243.26, p < 0.01). fig. 1. percentage (± se) of workers of atta robusta of different size classes that foraged during one year in restinga ecosystem from grussaí/iquipari complex. class 1 (head capsule 1 to 2 mm), class 2 (head capsule 2.1 to 3 mm), class 3 (head capsule 3.1 to 4 mm), class 4 (head capsule 4.1 to 5 mm) and class 5 (head capsule ( ≥ 5.1 mm). 843 endringer, f.b. et al. — load capacity of atta robusta workers ants with a larger head capsule transported the plant fragments with the highest mass (p < 0.01). classes 3 and 4 did not differ in the mass of the load they transported (p = 0.69) (fig. 2). all classes transported all the different types of plant material, i.e., leaf, flower, fruit, seed, cladodes, and other. in nests (n = 4) that foraged for fruit and seeds, this corresponded to 8% of all the resources foraged. it was shown that workers in class 3 (χ2 = 75.13; p < 0.0001), class 4 (χ2 = 5.44; p < 0.0197), and class 5 (χ2= 44.78; p < 0.0001) transported more fruit and seeds than other resources. thus, it was shown that when a colony exploited a substrate source producing fruit or seeds, the larger workers (class 3, 4 and 5) transported these resources preferentially (fig. 3). in nests where the ants collected cladodes, these constituted approximately 30% of all the resources foraged. workers in class 4 transported significantly more cladodes than other resources (fig. 4). fig. 2. mass (g ) (mean ± se) of load transported by different classes of workers of atta robusta in restinga ecosystem from grussaí/iquipari complex. class 1 (head capsule 1 to 2 mm), class 2 (head capsule 2.1 to 3 mm), class 3 (head capsule 3.1 to 4 mm), class 4 (head capsule 4.1 to 5 mm) and class 5 (head capsule ( ≥ 5.1 mm5 to 6 mm). different letters indicate significant difference by tukey test, p < 0.01. 844 sociobiolog y vol. 59, no. 3, 2012 discussion atta robusta has foragers with the same pattern of caste distribution of the other attines. this was demonstrated by the large amount of workers in class 1 and 2 (head width from 1 to 3 mm) on trails sampled throughout a year of sampling. colonies of the genus atta contain a small number of larger foraging individuals, whereas most workers are of a medium size (wilson 1985; helanterä & ratnieks 2008). the division of tasks inside a colony may be related to age polyethism, where young workers execute tasks inside the nest whereas older workers come out to forage (hölldobler & wilson 1990). other factors, such as colony size and the worker size distribution, can also affect the division of tasks in the nest (roces & hölldobler 1994). larger a. robusta workers belonging to class 5 (head capsule width greater than or equal to 5 mm) transported loads that weighed significantly more than fig. 3. percentage of foragers of atta robusta that transported fruits and seeds compared with that transporting other resources. ns = no significant difference by test χ2, * significant difference, p ≤ 0.0197 and ** significant difference, p <0.0001. class 1 (χ2 = 21.13 and p <0.0001), class 2 (χ2 = 0.39 and p = 0.53), class 3 (χ2 = 75.13 and p <0.0001), class 4 (χ2 = 5.44 and p <0.0197) and class 5 (χ2 = 44.78 and p <0.0001). 845 endringer, f.b. et al. — load capacity of atta robusta workers those of other classes. according to kleineidam et al. (2007), larger workers of a. vollenweideri transport heavier loads. the high degree of polymorphism in the genus atta means that larger workers are proportionally more efficient in terms of energ y returns, which compensates for the increased expenditure on their production (hölldobler & wilson 1990). large atta cephalotes workers specialize on leaves with a different thickness and hardness when exploiting leaves from a great diversity of plants. however, the foragers of acromyrmex octospinosus are small in size so they cut a lower range of plants (wetterer 1992). the foraging of leaves by atta vollenweideri was divided into at least two phases, where the larger ants cut the leaves while the smaller ants carry the fragments to the nest (röschard & roces 2003). in atta, larger workers are responsible for the defense of the colony, but can also cut leaves. they have well-developed head capsules and mandibles (evison 2007). in atta robusta, the larger workers in class 4 participated in the cutting of cladodes from cereus fernambucensis lem. (cactaceae) and they fig. 4. percentage of foragers of atta robusta that transported cladodes compared with those that transported other resources. ns = no significant difference by test χ2, * significant difference, p <0.0171. class 1 (χ2 = 0.07 and p = 0.79), class 2 (χ2 = 0.60 and p = 0.43), class 3 (χ2 = 0.44 and p = 0.50), class 4 (χ2 = 5.69 and p <0.0171) and class 5 (χ2 = 0.76 and p = 0.38). 846 sociobiolog y vol. 59, no. 3, 2012 collected proportionally more cladodes than other resources. the cladodes have a hard exterior, so workers with larger mandibles are required to cut this type of resource. during the cutting of c. fernambucensis, it was shown that larger workers cut the exterior surface of the cladodes, which is the hardest, whereas medium foragers in classes 1 and 2 collected the internal plant material that was easier to remove. these results reinforce the concept that colony polymorphism is an important factor that allows colonies to exploit a greater diversity of resources. furthermore, it allows the colony to respond more efficiently to changes in the quality of available resources. furthermore, dussutour et al. 2009 also demonstrated the flexibility of foraging behavior in the leaf-cutting ant atta colombica. the size and shape of the fragments of foraging material brought back to the nest were significantly modified when a constraint was placed on the trail, independent of their size, forager ants cut smaller and rounder fragments in the presence of a height constraint than in its absence. in general, the atta carry loads that are below their body capacity (lighton et al. 1987; wetterer 1990; kleineidam et al. 2007). however, the seed load capacity of atta robusta is increased, which is possibly due to cooperation and the participation of larger workers in the transport of larger seeds (teixeira 2007). when larger workers were foraging, we found that classes 3, 4, and 5 preferentially transported heavier loads such as fruit and seeds rather than other resources. according to evison (2007), larger atta laevigata and atta sexdens workers also transport fruit. in atta laevigata colonies, only a small proportion of larger workers forage and they preferentially cut large pieces of fruit using their long mandibles (evison 2007; helanterä & ratnieks 2008). in the restinga of grussaí/iquipari, atta robusta workers with significantly larger head capsules carried fruit and seeds. the transport of fruit and seeds by workers in classes 3, 4, and 5 supports the hypothesis of teixeira (2007), which suggests that larger atta robusta workers are responsible for the transport of larger seeds from several species of plants in the restinga, in addition to their defensive role in the colony. thus, they have an important role in the ecosystem, because they may help the dispersal of seeds in the restinga. several studies have shown that most of the fallen fruit from plants was removed by species of leaf-cutting ants, which played a relatively important role in terms of the amount of dispersed seeds, especially for plants with small fruit (leal 847 endringer, f.b. et al. — load capacity of atta robusta workers & oliveira 1998; 2000; christianini & oliveira 2009). thus, leaf-cutting ants may disperse seeds found beneath plants or that were not dispersed by other animals (christianini & oliveira 2009). this study demonstrated that larger a. robusta workers preferentially transported fruit and seeds. this behavior indicates the importance of larger workers, although they are present in smaller quantities on the foraging trail, in the exploitation of heavier resources such as fruit and seeds and their potential role in seed dispersal. thus, this study contributes to the available evidence on the importance of a. robusta in the restinga ecosystem and this species should be considered in future management programs aimed at the conservation of these areas. acknowledgements we thank conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the scholarship awarded to fabíola bonicenha endringer. we also thank t. portal, j. mulinari, and a. almeida for their help in the field work. references christianini, a.v. & oliveira p.s. 2009. the relevance of ants as seed rescuers of a primarily bird-dispersed tree in the neotropical cerrado savanna. oecologia 160: 735-745. dussutour, a., deneubourg, j., beshers, s., fourcassié, v. 2009. individual and collective problem-solving in a foraging context in the leaf-cutting ant atta colombica. animal cognition. 12 (1): 21-30. evison, s.e.f. & f.l.w. ratnieks 2007. new role for largers in atta leafcutter ants. ecological entomolog y 32: 451–454. forti, l.c., r.s. camargo, c.a.o. matos, a.p.p. andrade & j.f. lopes 2004. aloetismo em acromyrmex subterraneus brunneus forel (hymenoptera, formicidae), durante o forrageamento, cultivo do jardim de fungo e devolução dos materiais forrageados. revista brasileira de entomologia 48: 59-63. helanterä, h. & f.l.w. ratnieks 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ant, atta colombica. guer. physiological zoolog y 60: 524-537. moreira, d.d.o., a.m. vianabailez, m. erthal jr., o. bailez, m.p. carrera & r.i. samuels 2010. resource allocation among worker castes of the leaf-cutting ants acromyrmex subterraneus subterraneus through trophallaxis. journal of insect physiolog y 56: 16651670. roces, f. & b. hölldobler 1994. leaf density and a trade-off between load-size selection and recruitment behavior in the ant atta cephalotes. oecologia 97: 1-8. röschard j. & f. roces 2003. fragment-size determination and size-matching in thegrasscutting ant atta vollenweideri depend on the distance from the nest. journal of tropical ecolog y 19: 647-653. teixeira, m.c. 2007. dispersão de sementes por atta robusta borgmeier 1939 (hymenoptera: formicidade) na restinga da ilha de guriri. tese (doutorado em entomologia) – viçosa – mg, universidade federal de viçosa ufv, 72p. wetterer, j.k. 1990. load size determination in the leaf-cutting ant, atta cephalotes. behavioral ecolog y 1: 95-101. wetterer, j.k. 1992. foraging ecolog y of the leaf-cutting ant acromyrmex octospinosus in a costa rican rain forest. psyche 98 :361-371. wilson, e. o. 1971. the insects societies. cambrige: harvard university press 1971, 548p. wilson, e.o. 1980. caste and division of labor in leaf-cutter ants (hymenoptera: formicidae: atta) i. the overall pattern in a. sexdens. behavioral ecolog y and sociobiolog y 7: 143-156. wilson, e.o. 1985. the sociogenesis of insect colonies. science 228: 1489-1495. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i4.5023sociobiology 67(4): 593-598 (december, 2020) pollinator insects in agricultural landscapes are dwindling over the years due to the use of agrochemicals, diseases, land fragmentation and rapid urbanization (biesmeijer et al., 2006; potts et al., 2010). the intensive agricultural practices make the pollinators and natural enemies devoid of nectar, pollen, shelter, and nesting sites (cane 2008, pywell et al., 2011). the easiest and feasible way for conservation of these pollinators could be the maintenance of flora attractive to pollinators such as small patches of pollinator gardens in urban and farmlands, which support them with food and shelter over time. the consequence of such efforts would be the resulting enhancement of pollination service to our food crops. these gardens not only serve as a reservoir of both pollinators and biological control agents like predators and parasitoids but also help in educating the public and enhancing the aesthetic value of the urban and farm ecosystems (kells et al., 2001; sheffield et al., 2008). planting abstract pollinators are important providers of ecosystem services through plant and crop pollination. however, pollinator population/colony decline has raised concern for their conservation in farm lands as well as in urban areas. given the need for conservation of these pollinators, we developed a pollinator garden at yelahanka campus of icar-national bureau of agricultural insect resources in an area of one acre by planting over 50 plant species. thirty-nine species of bees were documented from the flora of the pollinator garden. out of the thirty-nine species of bees, nineteen species of bees belong to non-apis families viz., megachilidae and halictidae. apart from foraging on the flowers, the solitary bees like megachile sp. were found nesting in the stems, fallen dried flowers in the pollinator garden. the bees were found year-round foraging upon the flora in the pollinator garden. pollinator garden is a way to provide in-situ conservation of native bees while sustaining the valuable pollination service in various crop plants. sociobiology an international journal on social insects tm shivalingaswamy, u amala, a gupta, a raghavendra article history edited by evandro nascimento silva, uefs, brazil received 24 february 2020 initial acceptance 15 october 2020 final acceptance 26 october 2020 publication date 28 december 2020 keywords bees, conservation, in-situ, non-apis bees, pollinator garden, pollen, shelter. corresponding author udayakumar amala https://orcid.org/0000-0002-2394-259x scientist (entomology), division of germplasm conservation and utilization, icar-national bureau of agricultural insect resources h a farm post, pb no 2491, bengaluru 560024, karnataka, india. e-mail: amala.uday@gmail.com diverse flowering plants in the pollinator garden supports both bee diversity and density in addition to the provision of food and nesting sources for the native bees (kremen et al., 2002). an effort has been made to develop two patches (approximately one-acre area) of pollinator gardens in icarnbair-yelahanka campus. the campus is spread over 8.5 ha area in the north of bengaluru city (13° 5’ 48.8724’’ n 77° 33’ 59.7168’’ e). over 50 species of plants belonging to diverse families (trees, shrubs, herbs and climbers) were brought from a local state recognized scientific nursery and planted during 2012 and nurtured and observed for their flowering and attractiveness to pollinator insects, especially bees. we studied forty-six species of plants in the established pollinator gardens. observations were made on the visitation of different species of bees at 15 days interval over a period of three years (2013-2015). the bees were collected using sweep nets and killed using ethyl acetate. the killed specimens division of germplasm conservation and utilization, icar-national bureau of agricultural insect resources, bengaluru, karnataka, india non-apis bee diversity in an experimental pollinator garden in bengaluru – a silicon valley of india short note tm shivalingaswamy, u amala, a gupta and a raghavendra – pollinator garden to conserve diversity of bees594 were relaxed and dry mounted for taxonomic identification. some of the bee specimens were identified using taxonomic keys and others were identified by experts whose names are acknowledged in this publication elsewhere. the plant species were ranked into different categories based on the number of bee species attracted (table 1, fig 2) the reward (nectar or pollen) and other nest building materials collected by each species of the bees were recorded. thirty-nine species of bees were documented from different species of plants maintained in the pollinator garden (table 2 & fig 1). the major plant families attracting the bees in the increasing order of attraction were lamiaceae (ocimum basilicum and o. gratissimum), convolvulaceae (argyreia cuneata and jacquemontia violacea), acanthaceae (asystasia gangetica), asteraceae (gaillardia pulchella), passifloraceae (passiflora edulis) and lythraceae (woodfordia fruticosa). fig 1. diverse species of pollinators observed in the pollinator garden. argyreia cuneata (convolvulaceae) was recorded to bloom between april to october, exhibiting attractive purple flowers. the bee activity on this plant was recorded from 0800 hrs to 1500 hrs, between may to october. there was a reduction in its population coinciding with the reduction in the blooming of a. cuneata. tetralonia (thygatina) macroceps (anthophorinae: apidae) was found to forage only on this species. another congener, argyreia nervosa, was found to be visited by xylocopa sp. (xylocopinae: apidae) and lithurgus sp. (lithurginae: megachilidae) but not by tetralonia. the presence and absence of target flora as a determining factor for bee frequencies was reported by frankie et al. (2009). this behavior shows the floral constancy of the bees. tetralonia (thygatina) sp. was reported to prefer the herbaceous plant a. populifolia (convolvulaceae) (inoka et al., 2002). bees belonging to the genus thyreus were recorded to frequently visit asystasia gangetica (acanthaceae). the tubular flowers present in this plant were found to attract long tongued bees as they have typical landing platform for the bees to rest and collect the reward. carpenter bees belonging to the genus xylocopa were found to forage on calotropis gigantea (apocynaceae), performing nectar robbing activity, a common behavior of carpenter bees according to zhang et al. (2007). woodfordia fruticosa (lyrthaceae) was found to attract leaf cutting species megachile anthracina, which was observed employing leaf bits as resources for nest construction. the nectar rich flowers of w. fruticosa were found to be actively foraged upon by the little bees apis florea and a. cerana. apis florea was found to build its nest in the branches of w. fruticosa with the ideal proximity of rich nectar source in the sociobiology 67(4): 593-598 (december, 2020) 595 fig 2. ranking of plants based on number of bees visited.   a  b c d e f  g  h  i j  k  l m n o p q r rank iv: a. scaveola taccada, b. adhatoda zeylanica, c. hedychium coronaria, d. asclepias curassavica, e. citharexylum substratum, f. aristolochia ringens, g. woodfordia fruticosa, h. sauropus androgynus, i. calotropis gigantea, j. budleja asiática, k. cestrum diurnum, l. quisqualis indica, m. crotolaria retusa, n. alpinia sp., o. chrysophyllum cainito, p. hamelia patens, q. mansoa alliacea, r. clerodendrum infortunatum fig 2. ranking of plants based on number of bees visited.  a  b  rank i : a. ocimum basilicum, b. argyreia cuneata a  rank ii : a. asystasia gangetica, b. adhatoda zeylanica b  b ca  rank iii : a. antigonon leptopus, b. gaillardia pulchella, c. ruta graveolans tm shivalingaswamy, u amala, a gupta and a raghavendra – pollinator garden to conserve diversity of bees596 table 1. different plant species in the pollinator garden along with ranking based on the number of bees visited. family plant species rank lamiaceae ocimum basilicum* ocimum gratissimum strobilanthus barbatus strobilanthus hamiltoniana i convolvulaceae argyreia nervosa argyreia cuneata* ipomoea pescaprae jacquemontia violacea i acanthaceae adhatoda zeylanica andrographis paniculata asystasia gangetica* ii polygonaceae antigonon leptopus* iii asteraceae gaillardia pulchella iii passifloraceae passiflora edulis iii rutaceae ruta graveolans iii verbenaceae citharexylum substratum clerodendrum viscosum* vitex negundo iv elaeocarpaceae elaeocarpus floribundus*elaeocarpus sphaericus iv bignoniaceae mansoa alliacea tecoma capensis* iv solanaceae cestrum diurnum*cestrum nocturnum iv zingiberaceae alpinia calcarata iv amaranthaceae alternanthera sessilis iv aristolochiaceae aristolochia ringens iv annonaceae artabotrys odoratissimus* cananga odorata iv apocynaceae asclepias curassavica iv plantaginaceae bacopa moniera iv scrophulariaceae budleja asiatica iv fabaceae butea monospermacrotolaria retusa* iv apocynaceae calotropis gigantea iv sapotaceae chrysophyllum cainito iv vitaceae cissus quadrangularis iv mimosaceae adenanthera pavonina iv rubiaceae hamelia patens iv zingiberaceae hedychium coronaria iv malpighiaceae hiptage benghalensis iv lythraceae lagerstromia indicawoodfordia fruticosa* iv oleaceae nyctanthes arbor-tristes iv phyllanthaceae sauropus androgynus iv goodeniceae scaevola taccada iv malpighiaceae tristellateia australasiae iv ranking: i15-20 species of bees attracted to the plant, ii 10-15, iii5-10 and iv-0-5 * the plant species which was more attractive compared with other species in the same families flowers of the plant. the flowers of w. fruticosa are a major source of nectar and pollen visited by apis cerana and a. mellifera in shiwalik hills (kaur & mattu, 2016). the pithy stems of clerodendrum viscosum (verbenaceae) was utilized by small carpenter bee, ceratina hieroglypica for nest building activity. the destructive sampling of c. viscosum revealed the brood nests of the small carpenter bee c. hieroglyphica harbouring its life stages of pollen food. pithy stems of caesalpinia pulcherrima after pruning the branches were reported to be natural nesting sites of small carpenter bee, c. binghami (amala & shivalingaswamy, 2019). continuous availability of flowers in the pollinator garden was found to sustain different species of bees from spring to summer. plants like asystasia sp. (acanthaceae) and hamelia patens (rubiaceae) were found to have long blooming periods supporting the bee fauna with pollen and nectar rewards. similar observations were recorded by wojcik et al. (2008) and reported that flowers with long blooming periods sustained different species of bees in a seasonal sequence. the plant tristellateia australasiae (malpighiaceae) was found to be foraged upon by little bee a. florea in large numbers. the composite flowers of gaillardia pulchella (asteraceae) was found to be foraged by different species of halictid bees viz., nomia curvipes, seladonia propinqua in search for pollen. blue banded bees amegilla zonata (anthphorinae: apidae) and sweat bees hoplonomia westwoodi (nominae: halictidae) were recorded as some of the buzz pollinators of tomato and eggplant present in the pollinator garden. six different aromatic plants belonging to the family lamiaceae were reported to attract and support many species of bees and hover flies (barbir et al., 2016). raju (2005) reported that three species of bees viz., apis cerana indica, trigona iridipennis and ceratina simillima visited the flowers of woodfordia floribunda salisb. (lythraceae) for the collection of pollen and nectar. plants belonging to the family convolvulaceae viz., argyreia populifolia, ipomoea cairica, i. mauritiana and i. pescaprae attracted five species of solitary bees lithurgus atratus, lasioglossum halictoides, l. serenum, systropha tropicalis and tetralonia sp.1 in sri lanka (karunaratne et al., 2005). the flowers of the family asteraceae with typical daisy like flower was reported to attract solitary bees, hoverflies, and ‘other’ flower-visiting insects (rollings & goulson, 2019). peters (2014) reported that trigona fulviventris, halictids, ceratina sp and bombus pullatus visited the flowers of hamelia patens (rubiaceae) for pollen and nectar collection. the flowers of plant, asystasia chelonoides (acanthaceae) were reported to be visited by four different species of bees viz., amegilla comberi, a. puttalama, a. scintillans and apis cerana (karunaratne et al., 2005). xylocopa latipes and x. pubescens as a floral visitor and pollinator of calotropis gigantea and c. procera was reported by zafar et al. (2018). holistically, the plants and the flora in the pollinator garden were found to attract a diverse assemblage of bee sociobiology 67(4): 593-598 (december, 2020) 597 table 2. non-apis bee and scolid wasp species recorded in the pollinator garden. s. no. bee species family 1 amegilla confusa (smith, 1854) apidae 2 amegilla violacea (lepeletier, 1841) apidae 3 amegilla sp. (zonata group): apidae 4 apis cerana fabricius, 1793 apidae 5 apis dorsata fabricius, 1793 apidae 6 apis florea fabricius, 1787 apidae 7 braunsapis sp. halictidae 8 ceratina binghami cockerell, 1908 apidae 9 ceratina hieroglyphica smith, 1854 apidae 10 ceratina smaragdula (fabricius, 1787) apidae 11 ceratina sp.1 apidae 12 ceratina sp.2 apidae 13 coelioxys basalis smith, 1875 megachilidae 14 coelioxys confusus smith, 1854 megachilidae 15 coelioxys sp. megachilidae 16 hoplonomia westwoodi (gribodo, 1894) halictidae 17 lasioglossum (ctenonomia) sp. 1 halictidae 18 lasioglossum sp. 2 halictidae 19 lithurgus atratus smith, 1853 megachilidae 20 megachile anthracina smith, 1853 megachilidae 21 megachile bicolor (fabricius, 1781) megachilidae 22 megachile cephalotes smith, 1853 megachilidae 23 megachile disjuncta (fabricius, 1781) megachilidae 24 megachile lanata (fabricius, 1775) megachilidae 25 megachile sp.1 megachilidae 26 megachile sp.2 megachilidae 27 nomia curvipes (fabricius, 1793) halictidae 28 pachynomia sp. halictidae 29 scolia affinis guérin-méneville, 1830 halictidae 30 seladonia propinqua (smith, 1853) halictidae 31 seladonia sp. halictidae 32 tetralonia (thygatina) macroceps (engel & baker,2006) apidae 33 thyreus histrio (fabricius, 1775) apidae 34 thyreus massuri (radoszkowski, 1893) apidae 35 thyreus sp. apidae 36 xylocopa aestuans (linnaeus, 1758) apidae 37 xylocopa amethystina (fabricius, 1793) apidae 38 xylocopa latipes (drury, 1773) apidae 39 xylocopa sp. apidae species belonging to the families apidae, megachilidae, and halictidae. plants belonging to the family lamiaceae and convolvulaceae could be ideally used to conserve native apis/ non-apis bees. the concept of pollinator garden is a vital tool to conserve the native pollinators by providing them food source (nectar and pollen) and habitat (nests construction). pollinator gardens could be encouraged in urban habitats to enhance the aesthetic value, educative tool for school children and finally to sustain the ecosystem services provided by the pollinators. acknowledgements the authors are thankful to dr. c. a. viraktamath, emeritus professor, principal investigator, icar network project on biosystematics, university of agricultural sciences, gkvk bangalore. dr. k. d. prathapan, professor, kau, thiruvananthapuram for confirmation of a specific bee encountered on argyreia cuneata as tetralonia (thygatina) macroceps. we thank dr. chandish r ballal, director, icarnbair for her constant support and encouragement in our efforts in conservation of pollinators. authors’ contribution tms conceptualized and coordinated the conduct of the study. ag identified the bee specimens. ar assisted in recording field observations. ua analyzed the data and drafted the manuscript. all authors have read and approved the manuscript. references barbir, j., azpiazu, f.r.b., fernandez-quintanilla, c. & dorado, j. (2016). functionality of selected aromatic lamiaceae in attracting pollinators in central spain. journal of economic entomology, 109: 529-536. biesmeijer, j., roberts, s.p.m., reemer, m., ohlemuller, r., edwards, t.m.j., peeters, m., schaffers, a.p., potts, s.g., kleukers, r.j.m.c., thomas, c., settele, j. & kunin, w. (2006). parallel declines in pollinators and insect-pollinated plants in britain and the netherlands. science, 313: 351-354. cane, j.h. (2008). a native ground-nesting bee (nomia melanderi) sustainably managed to pollinate alfalfa across an intensively agricultural landscape. apidologie, 39: 315-323. frankie, g.w., thorp, r.w., hernandez, j., rizzardi, m., ertter, b., pawelek, j.c., witt, s.l., schindler, m., coville, r. & wojcik. v.a. (2009). native bees are a rich natural resource in urban california gardens. california agriculture, 63: 113-120. frankie, g.w.m., rizzardi, m., vinson, s.b. & griswold, t.l. (2009). decline in bee diversity and abundance from 1972-2004 on a flowering leguminous tree, andira inermis in costa rica at the interface of disturbed dry forest and the urban environment. journal of the kansas entomological society, 82: 1-20. inoka, w.a., karunaratne, p. & edirisinghe, j.p. (2002). bee diversity and floral hosts in selected habitats of the peradeniya university park. ceylon journal of science, 30: 21-36. inoka, w.a., karunaratne, p., edirisinghe, j.p. & gunatillieke, c.v.s. (2005). floral relationships of bees in selected areas of sri lanka. ceylon journal of science 34: 27-45. tm shivalingaswamy, u amala, a gupta and a raghavendra – pollinator garden to conserve diversity of bees598 kells, a., holland, j. & goulson, d. (2001) the value of uncropped field margins for foraging bumblebees. journal of insect conservation, 5: 283-291. kremen, c., williams, n.m. & thorp, r.w. (2002). crop pollination from native bees at risk from agricultural intensification. proceedings of the national academy of sciences of the united states of america, 99: 16812-16816. peters, v.e. (2014). intercropping with shrub species that display a ‘steady state’ flowering phenology as a strategy for biodiversity conservation in tropical agroecosystems. plos one, 9: e90510. potts, s.g., biesmeijer, j.c., kremen, c., neumann, p., schweiger, o. & kunin, w.e. (2010). global pollinator declines: trends, impacts and drivers. trends in ecology and evolution, 25: 345-353. pywell, r.f., meek, w.r., hulmes, l., hulmes, s., james, k. l., nowakowski, m. & carvell, c. (2011). management to enhance pollen and nectar resources for bumblebees and butterflies within intensively farmed landscapes. journal of insect conservation, 15: 853-864. raju, a.j.s. (2005). passerine bird pollination and seed dispersal in woodfordia floribunda salisb. (lythraceae), a common low altitude woody shrub in the eastern ghats forests of india. ornithological science, 4: 103-108. rollings, r. & goulson, d. (2019). quantifying the attractiveness of garden flowers for pollinators. journal of insect conservation, 23:803-817. sheffield, c.s., westby, s.m., smith, r.f. & kevan, p.g. (2008). potential of bigleaf lupine for building and sustaining osmia lignaria populations for pollination of apple. the canadian entomologist, 140: 589-599. wojcik, v.a., frankie, g.w., thorp, r.w. & hernandez, j. (2008). seasonality in bees and their floral resource plants at a constructed urban bee habitat in berkeley, california. journal of the kansas entomological society, 81: 15-28. zafar, r., raju a.j.s. & kumar, b.d. (2018). floral biology and carpenter bee pollination in calotropis gigantea and calotropis procera (asclepiadeceae). journal of palynology, 54: 85-99. zhang, y.w., robert, g.w., wang, y. & guo, y. h. (2007). nectar robbing of a carpenter bee and its effects on the reproductive fitness of glechoma longituba (lamiaceae). plant ecology, 193: 1-13. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.5146sociobiology 67(3): 473-477 (september, 2020) social wasps are excellent predators (elisei et al., 2010; raveret richter, 2000), however, are prey of other insects too as considerable resources are stored in their nests. there are many parasitoids of polistes species, including lepidoptera (gelechiidae, crambidae, cosmopterigidae, tineidae, and pyralidae (strassmann, 1981; yamane, 1996)), strepsiptera (xenidae), diptera (phoridae, sarcophagidae, and tachinidae (somavilla et al., 2015; zeegers et al., 2014; benadé et al., 2014)), and hymenoptera (pteromalidae, chalcididae, eulophidae, torymidae, ichneumonidae, trigonalidae, mutillidae, and even vespidae itself (benadé et al., 2014; somavilla et al., 2015; hodges, 2003; whiteman & landwer, 2000; gumovsky 2007; silva-filho 2007; kozyra et al., 2014; kudo et al., 2014; madden et al., 2010; de souza tavares et al. 2013)). polistes myersi bequaert, 1934 is found from panamá to venezuela (richards, 1978). their nests, of up to 100 cells, are found in perturbed habitats (cubillos & sarmiento, 1996; london & jeanne, 2000), however, little is known about its biology and its relationships with predators and parasitoids. here we report several parasitoid species of this wasp. abstracts information about parasitoids of neotropical vespids is scarce. parasitoids collected from 43 colonies of polistes myersi bequaert, 1934 and one of polistes erythrocephalus latreille, 1813 are reported from an andean region of colombia. colony parasitism rates in p. myersi ranged from 35 % to 57 %, being higher in colonies with more cells; however, the number of parasitized colonies did not differ when considering the mean number of adult wasps (8.2 vs. 8.1 respectively). parasitoidism ranged from one up to four species per colony. p. myersi parasitoids were: seminota laeviceps (cresson, 1879) (trigonalidae); signiphora polistomyiella richards, 1935 (signiphoridae); elasmus polistis burks, 1971 (eulophidae, elasminae); and a new species of xenos (strepsiptera, xenidae). the latter three are first records for colombia. p. myersi and p. erythrocephalus are the first host reports for the trigonalid s. laeviceps. we also report an unknown tachinid fly species of the tribe blondeliini attacking p. myersi. sociobiology an international journal on social insects d mayorga-ch, ce sarmiento article history edited by marcel hermes, ufla, brazil received 07 april 2020 initial acceptance 20 may 2020 final acceptance 08 june 2020 publication date 30 september 2020 keywords neotropics; eulophidae; signiphoridae; trigonalidae; tachinidae; xenidae. corresponding author carlos sarmiento carrera 30, nº 45-03. edificio 425 oficina 303, bogotá, colombia. e-mail: cesarmientom@unal.edu.co a total of 43 colonies with either late instar wasp larvae or pupae were collected from fusagasugá, cundinamarca, colombia (4º18’996” n, 74º26’475” w, 1309 m) in may and october 2017, and in august 2019. each colony was established in transparent pvc cages (25 x 16 x 16 cm) and stored in a rearing chamber (28 °c, 44% rh, 12/12 h photoperiod). colonies had access to water and a mixture of water, honey, and pollen ad libitum. depending on the colony size either one or two late instar larvae of galleria mellonella linnaeus, 1758 (pyralidae) were offered every other day. colonies increased their size and their larvae reached adulthood or even started new colonies. colonies were checked daily and emergent parasitoids were preserved in ethanol 96%; these were identified using appropriate keys and help from specialists: for hymenoptera the following references: fernandez and sharkey (2006), burks (2019), subba rao (1974), carmean and kimsey (1998), and the following contacted specialists: d. carmean, r. burks, j. wooley, and a. dal molin. for strepsiptera the specialist jerry cook, (sam houston natural history collections). and instituto de ciencias naturales, universidad nacional de colombia, bogotá d.c., colombia parasitoids of polistes myersi bequaert, 1934 (vespidae, polistinae) short note d mayorga-ch, ce sarmiento – polistes myersi parasitoids474 for diptera the specialist juan manuel perilla (wright state university). parasitoids of a single incidental colony of polistes erythrocephalus latreille, 1813 are also reported. specimens were deposited in the instituto de ciencias naturales (icn), universidad nacional de colombia, bogotá (catalogue numbers table 1). a total of 19 colonies were infected by parasitoids; parasitism ranged from 35 % to 57 %. parasitized nests were larger than non-parasitized ones (average cell number 64 vs 38 respectively, table 1). however, the mean number of adult females did not differ between parasitized and non-parasitized colonies (8.2 vs. 8.1 respectively, table 1). the following parasitoid species of p. myersi colonies are reported: xenos n. sp. (strepsiptera, xenidae); elasmus polistis burks, 1971 (hymenoptera, eulophidae); signiphora polistomyiella richards, 1935 (hymenoptera, signiphoridae); seminota laeviceps (cresson, 1879) (hymenoptera, trigonalidae), and an unknown fly (diptera, tachinidae, blondeliini) (fig 1). except for xenos n. sp. all other parasitoids were collected during two or the three field trips. parasitoid species infected nests* nests characteristics # cells # females signiphora polistomyiella (icn 101081)^ 9 54.3 ± 57.8 7.6 ± 3.8 elasmus polistis (icn 101078) 8 71.3 ± 57.0 9.1 ± 9.2 blondeliini sp. (icn 101079) 7 45.1 ± 16.3 6.0 ± 3.7 seminota laeviceps (icn 101080) 3 60.3 ± 51.9 11.3 ± 16.3 xenos n. sp. (icn 101082) 1 72 13 non-parasitized 24 38.0 ± 23.7 8.2 ± 5.9 table 1. characteristics of the parasitized nest of polistes myersi where these parasitoids emerged (mean ± sd). *as some nests harbor more than one parasitoid species, the number of nests will not add up. ̂ museum collection number. fig 1. habitus of the parasioids reported in the colonies of polistes myersi and polistes erythrocephalus. (a) seminota laeviceps, (b) elasmus polistis, (c) signiphora polistomyella, (d) xenos n. sp, and (e) blondellini. sociobiology 67(3): 473-477 (september, 2020) 475 from most of the colonies a single parasitoid species was obtained, with the following exceptions: two colonies with s. polistomyiella and e. polistis, one colony with e. polistis and a blondeliini fly, and one colony with s. polistomyiella and a blondeliini fly, one colony with s. polistomyiella, e. polistis and a blondeliini fly, and one colony with s. polistomyiella, e. polistis, xenos n. sp. and a blondeliini fly. the number of parasitoids per colony could be higher as several of them are known hyperparasitoids. below we give an account of the taxa recorded: elasmus (eulophidae) is known as parasitoid of multiple taxa including diptera, lepidoptera and hymenoptera families such as braconidae, ichneumonidae, and vespidae (askew et al., 1997; gumovsky et al., 2007; krombein, et al., 1951). it is also hyperparasitoid of diptera (noyes, 2019). elasmus polistis is reported from brazil, india, germany, mexico, u.s.a. and the virgin islands (burks, 1971; dorfey & köhler, 2011; noyes, 2019), this is the first report for colombia. signiphora polistomyiella (signiphoridae) is parasitoid of blondeliini (tachinidae, diptera) (de santis, 1981) and hyperparasitoid of vespidae such as polistes pacificus and mischocyttarus surinamensis (vesey-fitzgerald 1938). nine colonies of this study had s. polistomyiella parasitoids. s. polistomyiella is reported from peru and trinidad and tobago (subba rao, 1974), this is the first report for colombia. seminota (trigonalidae) is a hyperparasitoid genus of several vespidae genera (carmean & kimsey, 1998; somavilla, 2015; weinstein & austin, 1991). however, santos and noll (2013) suggest that primary parasitoidism may also occur. the genus has been reported in several countries of the neotropics (carmean & kimsey, 1998; smith, 2012; santos & noll 2013) but this is the first report for colombia. to our knowledge, this is the first record of hosts for seminota laeviceps, wich includes p. myersi and polistes erytrocephalus. xenos, (xenidae) as indicated by cook’s (2019) catalogue, is a widely distributed and known parasitoid of vespids (kathirithamby, 2012; cook, 2019). it was reported from colombia as parasitoid of polistes erythrocephalus (girón, 2006). the specimen from our study belongs to a new species (cook pers. comm.) under description. the tachinidae fly recorded belongs to the tribe blondeliini. unfortunately, there are no thorough taxonomic studies on this tribe for the neotropics. consequently, no more accurate identification is possible. blondeliini is a tribe with global distribution (fuentes, unpub.). they are parasitoids of lepidoptera, coleoptera, and the hymenoptera families argidae tenthredinidae, braconidae and vespidae (zeegers, 2014; guimaraes, 1977). the number of parasitized colonies of p. myersi (average 44%) was very high compared to other polistes species. hodges et al. (2003) reported a parasitism rate of 23% out of 303 studied nests for polistes metricus in the usa. keeping and crewe (1983) reported five colonies of belonogaster juncea and b. petiolata parasitized out of 63 colonies sampled in south africa. the only higher record of parasitism rate than the present study was found in a small study with seven out of ten sampled colonies of polistes dorsalis; these were parasitized by elasmus polistis in the usa (macom & landolt, 1995). the location of p. myersi in a highly disturbed area could explain these numbers. acknowledgements the authors thank r. burks, d. carmean, j. perilla, d. benda, j. cook, j. wooley and a. dal molin for their help identifying the specimens. acknowledge to casa buenos aires, for their hospitality during the field trips, as well as the editor and the anonymous reviewers for their careful reading of the manuscript. this project was funded by the vicerrectoría of the universidad nacional de colombia grant number hermes 34846. authors contribution daniela mayorga-ch: field work, data collection and analysis, writing paper. carlos e. sarmiento: project design, field work, writing paper. references askew, r.r., segade, c., blasco-zumeta, j. & pujade, j. 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(2014). tachinidae (diptera) reared from ropalidia nests (hymenoptera: vespidae) from madagascar, with two new species of anacamptomyia. tijdschrift voor entomologie, 157 (2–3): 95-103. doi: 10.1163/22119434-00002041 903 parameters that influence the establishment and volume of microcerotermes exiguus and nasutitermes corniger nests in an atlantic forest fragment in northeastern brazil (isoptera: termitidae) by alane ayana vieira de oliveira couto¹, andré ribeiro de arruda¹, josivan soares da silva¹, eliana sofia fajardo vega1, carina carneiro de melo moura¹, sérgio luíz muniz¹ & auristela correia de albuquerque2 abstract this study aimed to evaluate the parameters that influence the establishment and volume of microcerotermes exiguus and nasutitermes corniger nests in an atlantic forest fragment in northeastern brazil. between september and october 2011, five 1000 m² parcels (100 x 10 m; 10 m apart) were examined and the volume was estimated for all m. exiguus and n. corniger nests. additionally, the following variables were measured: distance between nest and soil, height and diameter at breast height (dbh) of the supporting tree, and the minimum distance to the nearest nest. data was checked for normality with the shapiro-wilk test and the variation between the parcels was calculated using the kruskal-wallis nonparametric test. the analyses were carried out using bioestat 5.0. a total of 72 nests were sampled, of which 59 belonged to m. exiguus and 13 to n. corniger. species occurred in different strata, which prevented direct competition between the two. dbh had the greatest influence on colony volume. keywords: termites, nesting, rain forest introduction the atlantic forest is one of the richest biomes in the world in terms of biodiversity; nevertheless, due to the rapid process of fragmentation initiated by anthropic disturbances, it has become one of the areas with the highest 1departament of biolog y, post-graduation in ecolog y, federal rural university of pernambuco, 52171-900, recife-pe, brazil. 1 departament of biolog y, federal rural university of pernambuco, recife, brazil. corresponding author: alane ayana vieira de oliveira couto. departament of biolog y, federal rural university of pernambuco, 52171-900, recife-pe, brazil. e-mail: alane.couto@gmail.com 904 sociobiolog y vol. 59, no. 3, 2012 conservation priority (dário and almeida 2000; morellato and haddad 2000). in south america, approximately 35% of the termite species that occur in tropical rainforests such as the atlantic forest construct conspicuous nests (martius 1994), and a rapid analysis of these species can promptly provide information about their habitat (vasconcellos et al. 2008). however, in order to understand this process well, all of the factors that influence the establishment and distribution of termite colonies must be known. although such studies are still incipient, the diameter at breast height of the supporting tree seems to be an important parameter that influences the establishment of termite colonies (gonçalves et al. 2005; polatto and alvesjúnior 2009). similarly, other parameters such as exposure to wind and sunlight, the growth shape of the supporting tree and the biological characteristics of the termite species studied may strongly influence nest establishment in the environment (thorne 1984; gonçalves et al. 2005; leite et al. 2011). according to vasconcellos et al. (2007) and vasconcellos et al. (2008), nest volume is positively correlated with population size. thus, it is possible to say that larger colonies are better established in the environment. within this perspective, this study aimed to evaluate the parameters that influence the establishment and volume of microcerotermes exiguus and nasutitermes corniger nests in an atlantic forest remnant. materials and methods study area the study was carried out in an atlantic forest remnant (alto da buchada) located in the tapacurá ecological station (8°2’56.9”s, 35°13’14.5”o), são lourenço da mata municipality, state of pernambuco, brazil (azevedo-júnior 1990). the station is approximately 776 ha, of which only 382 ha are made up of atlantic forest; the rest is occupied by the lake created after the tapacurá river was dammed (coelho 1979). the station is classified as a semi-deciduous seasonal forest, which average yearly rainfall is of 1300 mm; the five drier months are september-january (condepe 2000). climate is type as’ and the vegetation is typical of dry, predominantly arboreal forest, which trees reach approximately 30 m in height (lyra-neves et al. 2007). 905 couto, a.a.v.o. et al. — microcerotermes exiguus and nasutitermes corniger nests sampling design & data collection data was collected between september and october 2011. five 1000 m² parcels (100 x 10m) were established in the study area, 10 m apart, going from the forest edge to its interior. all of the m. exiguus and n. corniger nests in these parcels had their volume estimated and parameters that could influence colony establishment were evaluated. in order to estimate nest volume, mathematical formulas (for cylinder or hemiellipsoid shapes) were used according to the format observed. to obtain more precise estimates, when surrounded by a nest the volume of the trunk was calculated and subtracted from the total value found for the colony. nests of the same species built in the same tree and connected by galleries were treated as a single colony, which volumes were added (adapted from vasconcellos et al. 2008). the following parameters were evaluated in this study: minimum distance between each colony and from the soil, height and diameter at breast height (dbh) of the supporting tree and minimum distance to the nearest colony. a malleable metric tape with a 0.1 cm precision was used to measure these parameters and to estimate nest volume. parcels were characterized regarding the type of predominant vegetation, tree size, luminosity, distance from the forest’s edge, and successional stage (table 1). data analysis volume data from microcerotermes exiguous nests were submitted to the shapiro-wilk normality test. the next step was to analyze the variation between parcels by applying the kruskal-wallis nonparametric test. all tests were carried out using bioestat 5.0. a descriptive analysis was not carried out for n. corniger as the amount of data obtained insufficient to undertake more robust statistical tests. in order to evaluate the correlation between the parameters measured, descriptive canonical correlation was applied with the aid of statistica 8.0. the parameters with the most correlations were then submitted to spearman’s correlation test using bioestat 5.0. 906 sociobiolog y vol. 59, no. 3, 2012 results and discussion a total of 72 nests were sampled – 59 of m. exiguus and 13 of n. corniger. the greatest occurrence of m. exiguus was recorded in parcel 2, the same with the lowest occurrence of n. corniger. conversely, the greatest occurrence of n. cornniger was recorded in parcel 1, the same with the lowest occurrence of m. exiguus. this result could indicate interspecific competition, as even when the two species were found using the same supporting tree, we observed that m. exiguus used the lower part of the trunk (below 1 m) while n. corniger used the higher portion (above 1.5 m). thus, trees are used in a specific manner, avoiding elimination by direct competition. according to levings and adams (1984) and adams and levings (1987), territoriality mediates the growth and expansion of neighboring colonies by making distinct species explore different areas or die after contact between colonies. therefore, niche specialization can reduce interspecific competition (bourguignon, leponce and roisin 2011) all of the parcels had a lower percentage of n. corniger. this species – which has a certain plasticity and adaptive capacity – is commonly found in urban table 1. characteristics of parcels studies in an atlantic forest fragment at the tapacurá ecological station, pernambuco, brazil. parcel characteristics 1 located parallel to the edge of the fragment studied; thus, it receives more sunlight and wind. presence of adult individuals of brazil wood (caesalpinia echinata), common to the area studied, and some representatives of family arecacea. vegetation with several development strata. 2 located 20 m from the fragment’s edge. few representatives of family arecacea, but a great number of creeping plants and individuals from family bromeliaceae. 3 located 40 m from the fragment’s edge. presence of small clearings, which promotes the incidence of sunlight. bamboos (poaceae: bambusoideae) and individuals of family melastomatacea in an initial developmental stage. a large amount of creeping plants. 4 located 60 m from the fragment’s edge. great occurrence of individuals from family bromeliaceae. presence of adult individuals of family melastomatacea. great occurrence of herbaceous plants. 5 located 80 m from the fragment’s border. great occurrence of adult and juvenile individuals of family arecaceae. presence of bromeliads (bromeliaceae). bamboos (poaceae: bambusoideae) and individuals of family melastomatacea in an initial developmental stage. 907 couto, a.a.v.o. et al. — microcerotermes exiguus and nasutitermes corniger nests or agricultural environments that have undergone some kind of disturbance, where may cause serious damage (constantino 2002; costa, espírito-santo filho and brandão 2009; novaretti and fontes 1998). such an adaptation to altered environments has also become clear in studies carried out in atlantic forest areas where this species was more frequent in environments which had undergone a shorter period of regeneration (reis and cancello 2007; vasconcellos et al. 2008). thus, the low occurrence of this species may indicate the conserved state of the studied area. for m. exiguus, the greatest nest volumes were observed in parcel 1, which was significantly different from parcels 3, 4 and 5 (fig. 1). parcels number 1 and 4, where the greatest nest volumes were found, were those with the lowest density of nests. similarly, while studying an assemblage of termites that build conspicuous nests in areas of atlantic forest in different stages of regeneration, vasconcellos et al. (2008) observed that m. exiguus colonies occurred less densely in the area which had undergone the longest regeneration process; the greatest nest volumes were also in this area. the same authors suggested that this might be related to the vegetation’s regeneration stage, lower climactic oscillation and greater resource availability. neverthefig. 1: variation in the volume of microcerotermes exiguus nests among the parcels sampled at the tapacurá ecological station, based on the kruskal-wallis test (p<0.05). 908 sociobiolog y vol. 59, no. 3, 2012 less, such an explanation does not apply to this study because parcel 1 is subjected to the greatest climactic oscillations among the parcels examined, such as sunlight and wind. this divergence in results might also be related to the scale being evaluated: while vasconcellos et al., (2008) examined two areas of atlantic forest that measured 1 ha each, this study compared five 1000 m2 parcels. the descriptive canonical correlation demonstrated that, among the parameters evaluated, dbh had the greatest influence on the volume of m. exiguus (table 2). although it was not possible to establish such a correlation for n. corniger due to the small sample obtained, spearman’s correlation coefficient showed that the dbh positively influences this species’ nest volume (r s = 0.4286; p= 0.1439) just as it positively influences the volume of m. exiguous nests (r s = 0.3538; p= 0.0059). a positive correlation between nasutitermes sp. nest volume and the circumference at breast height of the supporting trees has been verified by polatto and alves-júnior (2009). gonçalves et al. (2005) has also shown that the volume of nasutitermes spp. and m. exiguus nests is positively correlated with the circumference at breast height of the supporting trees. one of the explanations for such results is that trees with greater circumferences could represent more stable habitats (gonçalves et al. 2005). araújo et al. (2010) confirmed that the greater the tree diameter (and, consequently, the more the resource is available), the greater the number of trails, which would indicate an increase in colony activity. although the volume of the colonies of the two species studied was influenced by the supporting trees’ dbh, m. exiguus colonies established themselves in trees with an average dbh of 0.113 ± 0.05 m, while for n. corniger colonies that average was of 0.173 ± 0.18 m. considering that the m. exiguus nests’ average volume was of 0.022 ± 0.07 m3 and that of n. corniger was of 0.068 ± 0.08 m3, such a preference for greater or smaller dbhs reflects the need of table 2: descriptive canonical correlation between the volume of microcerotermes exiguus nests and supposedly influential parameters at the tapacurá ecological station, são lourenço da mata, pernambuco, brazil. parameters dbh 0.316 height of the supporting tree 0.225 nest height in relation to the soil 0.002 distance to the closest nest -0.011 909 couto, a.a.v.o. et al. — microcerotermes exiguus and nasutitermes corniger nests each species. greater nests need trees with larger dbh values, because large circumferences offer better adherence for nest fixation; this, in turn, would allow an increase in nest volume with a lower risk of the nest becoming loose from the supporting tree (polatto and alves-júnior 2009). although they were not analyzed in this study, some factors related to the biolog y of the species involved undoubtedly influence colony establishment and success. it is known, for instance, that n. corniger is a facultative polygamist and that the presence of multiple queens can directly influence the colony’s success, as the high growth rates of colonies with multiple queens increases the probability of survival and decreases the time needed to produce fertile winged individuals (thorne 1982; thorne 1984). we verified that the supporting tree’s dbh had the greatest influence on the establishment and volume of m. exiguous colonies. the dbh was also positively correlated with the volume of n. corniger colonies. the biological characteristics of each species certainly influenced colony implantation. thus, we recommend that new studies be carried out to investigate how these features might be linked to environmental characteristics, such as those evaluated here, for to influence colony success and establishment. references adams, e.s. & s.c. levings 1987. territory size and population limits in mangrove termites. journal of animal ecolog y. 56:1069-1081. araújo, f. s. et al. 2010. bottom-up effects on selection of trees by termites (insecta: isoptera). sociobiolog y. 55 (3). azevedo-jr., s. m. 1990. a estação ecológica do tapacurá e suas aves. in: anais do encontro nacional dos anilhadores de aves (enav). recife, 4:92-99. bourguignon, t., m. leponce & y. roisin, y. 2011. are the spatio-temporal dynamics of soil-feeding termite colonies shaped by intra-specific competition? ecological entomolog y. 36: 776–785. coelho, a.g.m. 1979. as aves da estação ecológica do tapacurá, pernambuco. natural biolog y. 2: 1-18. condepe. 2000. base de dados do estado – climatologia: descrição dos tipos. recife: governo do estado de pernambuco, instituto de planejamento de pernambuco. constantino, r. 2002. the pest termites of south america: taxonomy, distribution and status. journal of applied entomolog y. 126(7/8): 355-365. costa, d. a., k. espírito-santo filho & d. brandão. 2009. padrão de distribuição de cupins na região urbana de goiânia. iheringia, sér. zool. porto alegre. 99 (4):364-367. 910 sociobiolog y vol. 59, no. 3, 2012 dário, f.r. & a.f. almeida. 2000. influência de corredor florestal sobre a avifauna de mata atlântica. scientia forestalis, 58: 99-109. gonçalves, t.t., o. desouza, r. reis jr, & s.p. ribeiro= 2005. effect of tree size and growth form on the presence and activity of arboreal termites (insecta: isoptera) in the atlantic rain forest. sociobiolog y. 46 (2): 1-12. leite, g.l.d; r.v.s. veloso, j.c. zanuncio, s.m. alves, c.a.d. amorim & o. desouza. 2011. factors affecting constrictotermes cyphergaster (isoptera: termitidae) nesting on caryocar brasiliense trees in the brazilian savanna. sociobiolog y. 5(1):1-16. levings, s. c. & e.s. adams 1984. intra and interspecific territoriality in nasutitermes (isoptera: termitidae) in a panamanian mangrove forest. journal of animal ecolog y. 53 (3): 705-714. lyra-neves, r.m. m.a.b. oliveira, w.r. telino-júnior & e.m. santos. 2007. comportamentos interespecíficos entre callithryx jacchus (linnaeus) (primates: callitrichidae) e algumas aves de mata atlântica, pernambuco, brasil. revista brasileira de zoologia. 3(24): 709-716. martius, c. 1994. diversity and ecolog y of termites in amazonian forest. pedobiolog y, 38: 407-428. morellato, l. p. c. & c. f. b. haddad. 2000. introduction: the brazilian atlantic forest. biotropica. 32(4b):786-792. novaretti, w.r.t. & l.r. fontes. 1998. cupins: uma grave ameaça à cana-de-açúcar no nordeste do brasil, pp. 163-172. in: fontes, l.r. & berti-filho, e. cupins: o desafio do conhecimento. piracicaba: fealq. polatto, l. p. & alves-júnior, v. v. 2009. distribuição e densidade de nasutitermes sp. (isoptera: termitidae) em mata ribeirinha do rio miranda, pantanal sul-matogrossense, brasil. entomobrasilis. 2(1): 27-30. reis, y. t. & cancello, e. m. 2007. riqueza de cupins (insecta, isoptera) em áreas de mata atlântica primária e secundária do sudeste da bahia. iheringia. série zoologia. 97: 229-234. vasconcellos, a., araújo, v.f.p., moura, f.m.s. & bandeira, a.g. 2007. biomass and population structure of constrictotermes cyphergaster silvestri (isoptera: termitidae) in the dry forest of caatinga, northeastern brazil. neotropical entomolog y. 36: 693-698. vasconcellos, a., bandeira, a.g., almeida, w.o. & moura, f.m.s. 2008. térmitas construtores de ninhos conspícuos em duas áreas de mata atlântica com diferentes níveis de perturbação antrópica. neotropical entomolog y. 37: 15-19. thorne, b. e. 1984. behavioral ecolog y and sociobiolog y. polyg yny in the neotropical termite nasutitermes corniger: life history consequences of queen mutualism. 14 (2): 117-136. thorne, b. e. 1982. polyg yny in termites: multiple primary queens in colonies of nasutitermes corniger (motschuls) (isoptera: termitidae). insectes sociaux. 29 (1): 102-117. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.6247sociobiology 69(1): e6247 (march, 2022) introduction dolichoderus lund, 1831 is a large dolichoderine genus of the tribe dolichoderini (bolton, 2003). members of the genus are distributed worldwide except in the afrotropical and malagasy regions (bolton, 1995, 2003; shattuck & marsden, 2013; antweb, 2021). in general, workers of some species are active day and night, and regarding fedding behavior they are scavengers scavengers as well as tending aphids and other hemiptera for honeydew. they often forage in columns on the ground or lower vegetation and trees. most species are arboreal, sometimes using plant fibre to form coverings during nest construction (shattuck & marsden, 2013). in our field surveys, we observed herds of dolichoderus cuspidatus workers surrounding small tree branches and sometimes on ropes tied to wooden trunks in forest alleys to escape from wet during the rainy season. abstract dolichoderus lund, 1831 is one of the large ant genera in the world and belongs to the subfamily dolichoderinae. currently, 130 species and 19 subspecies are known in this genus. a new species of the dolichoderus thoracicus species group, dolichoderus bakhtiari sp. nov. is here described based on the worker caste. the type series of the new species was collected from shrub trees in a primary upland evergreen forest (ca. 800 m a.s.l.). a key to the thai d. thoracicus species group is provided. sociobiology an international journal on social insects z barabag1, w jaitrong2 article history edited by himender bharti, pup, india received 18 february 2021 initial acceptance 01 december 2021 final acceptance 07 january 2022 publication date 07 february 2022 keywords ant, dolichoderus bakhtiari, new species, species group, distribution, thailand. corresponding author zakaria barabag institute for tropical biology and conservation, university malaysia sabah kota kinabalu, sabah, malaysia 88400. e-mail: barabagg@gmail.com a total of 130 species and 19 subspecies are recognized in the genus (antcat, 2021). among them, about 32 species and 21 subspecies have been recorded from southeast asia. thirteen species are known from thailand in four species groups: d. cuspidatus, d. scabridus, d. sulcaticeps and d. thoracicus species groups (dill, 2002; khachonpisitsak et al., 2020). of these, five species belong to the d. thoracicus species group: dolichoderus affinis emery, 1889; dolichoderus butteli forel, 1913; dolichoderus brevis santschi, 1920; dolichoderus taprobanae smith, 1858 and dolichoderus thoracicus (smith, 1860). among these species, d. taprobanae was described based on the queen and we must recognize the worker of this species. based on the present study of southeast asian dolichoderus, we found a new species of the d. thoracicus species group from thailand, described based on the worker caste. a list of extant species and subspecies of the d. thoracicus species group is provided (table 1). 1 institute for tropical biology and conservation, university malaysia sabah, kota kinabalu, sabah, malaysia 2 office of natural science, national science museum, 39 moo 3, khlong 5, khlong luang, pathum thani, thailand research article ants a new species of the ant genus dolichoderus lund, 1831 (hymenoptera: formicidae) from thailand urn:lsid:zoobank.org:pub:ee659cb6-1d48-45b3-a026-d8f01a0e4120 mailto:barabagg@gmail.com z barabag, w jaitrong – a new species of the ant genus dolichoderus lund2 materials and methods holotype and paratypes of dolichoderus bakhtiari sp. nov. were point-mounted and gyne and male are not reported. we compared dealate and alate queens of dolichoderus taprobanae collected from thailand with the high-resolution images of the d. taprobanae holotype (alate queen) available on antweb (2021). the type specimens of the new species were compared with workers from the colonies from thailand to which the dealate and alate queens belonged. the highresolution images of the syntypes of dolichoderus semirufus (junior synonym of d. taprobanae), hypoclinea gracilipes (junior synonym of d. taprobanae), dolichoderus taprobanae tonkina (subspecies of d. taprobanae), and dolichoderus taprobanae siamensis (subspecies of d. taprobanae) which are available on antweb (2021) were also examined. taxonomic analysis was made with a zeiss discovery v12 stereoscope. multi-focused montage images were produced using nis-elements-d from a series of source images taken with the nikon digital sight-ri1 camera attached to a nikon az100m stereoscope. the holotype and ten paratypes were measured for the following parts using a micrometer (accurate to 0.01 mm). the abbreviations used for the measurements and indices are as follows: tl total length of the body, length of the body’s long axis from the anteriormost point of the anterior clypeal margin to the posteriormost point of the gaster (measurements of specimens that were not straightened out are composed of hl+msl+pl+length of gaster). hl maximum head length in full-face view, measured from the anterior clypeal margin to the midpoint of a line down across the posterior margin of head. hw maximum head width in full-face view. mdl mandible length. with head in full-face view, length of mandible measured along outer margin, the chord distance from lateral insertion to mandible apex. sl scape length excluding the basal of constriction and condylar bulb. species distributions (* = type localities) 1. d. affinis emery, 1889 india, china, myanmar*, vietnam, laos, thailand, malaysia (borneo), indonesia and philippines 2. d. affinis glabripes forel, 1895 india* 3. d. affinis mus santschi, 1920 vietnam* 4. d. brevis santschi, 1920 laos* and thailand 5. d. burmanicus bingham, 1903 myanmar* 6. d. butteli forel, 1913 thailand, malaysia, singapore and indonesia (sumatra*) 7. d. carbonarius emery, 1895 malaysia (malay peninsular*) 8. d. carbonarius latisquamis emery, 1900 indonesia (sumatra*) 9. d. gibbus (smith, 1861) indonesia (sulawesi*) 10. d. lactarius (smith, 1860) indonesia (maluku islands*) 11. d. moggridgei forel, 1886 afghanistan, china (yunnan) and india (assam*) 12. d. moggridgei bicolor santschi, 1920 india (sikkim*) 13. d. taprobanae (smith, 1858) pakistan, nepal, bhutan, india, sri lanka*, bangladesh, china, vietnam, laos, thailand, malaysia (borneo) and indonesia (sumatra) 14. d. taprobanae borneensis forel, 1911 malaysia (borneo*) and indonesia (sumatra) 15. d. taprobanae ceramensis stitz, 1912 indonesia (maluku islands*) 16. d. taprobanae friedrichsi forel, 1914 vietnam* 17. d. taprobanae gracilipes (mayr, 1879) india*, sri lank, china, myanmar, vietnam, malaysia (borneo), and indonesia (sumatra) 18. d. taprobanae siamensis forel, 1911 vietnam and thailand* 19. d. thoracicus (smith, 1860) india, sri lanka, china, vietnam, cambodia, laos, thailand, malaysia, singapore, indonesia* and philippines 20. d. thoracicus bilikanus santschi, 1925 indonesia (sumatra*) 21. d. thoracicus borneonensis (roger, 1863) malaysia (borneo*) and indonesia (sumatra) 22. d. thoracicus emarginatus santschi, 1920 vietnam* 23. d. thoracicus lacciperdus santschi, 1925 cambodia* 24. d. thoracicus levior karavaiev, 1926 indonesia (sumatra*) 25. d. thoracicus nasutus karavaiev, 1935 cambodia* 26. d. thoracicus rufescens stitz, 1925 malaysia (borneo*) and indonesia table 1. list of the southeast asian species and subspecies of the dolichoderus thoracicus species group. sociobiology 69(1): e6247 (march, 2022) 3 ml mesosomal length measurement from the point at which the pronotum meets the cervical shield to the posterior margin of metapleuron in profile. pl petiole length measurement from anterior margin to posterior-most point of petiolar in profile view. ph height of petiole in profile measurement as the perpendicular distance from the apex of the petiolar process to a line tangent to the dorsal-most point of a node. dpw dorsal petiole width. maximum width of the petiolar node in dorsal view. ci cephalic index. hw/hl x 100. mdi mandibulo-cephalic index. mdlx100/hl. oi ocular index. edx100/hw. si scape index. sl/hw x 100. abbreviations of the type depositories are as follows: thnhm thailand natural history museum of national science museum thailand skyc seiki yamane’s collection at kitakyushu museum of natural history and human history, japan results dolichoderus thoracicus species group dill (2002) defined the worker caste of this species group as follows: small to moderately sized species (hw 0.59–1.25 mm, tl 3.0–4.5 mm); variable coloration and sculpturing; head, mesosoma, and gaster sparsely to densely pilose, mostly pubescent; scape usually with short erect hairs, longest hairs shorter than the maximum width of scape; antenna moderately long (si 71–114); flagellomeres void of erect hairs; in full-face view, posterior margin of head concave emarginate; head about as long as broad; mesosoma unarmed; mesonotum round and only moderately vaulted; propodeum mostly distinctly narrower than pronotum; propodeum mostly somewhat rising, its declivitous face varying from not to distinctly concave, its rising dorsal face always with erect hairs; petiole scale in dorsal view mostly compressed, i.e. distinctly broader than long; its dorsal margin usually emarginate. dolichoderus bakhtiari barabag & jaitrong sp. nov. (fig 1) urn:lsid:zoobank.org:act:643ce703-f414-4d76-a8c8-ca86603a6154 types. holotype worker (thnhm-i-22732, thnhm), ne thailand, nakhon ratchasima province, pak chong district, moo sri subdistrict, 9. ii. 2020, w. khaikaew leg., from a colony (wk090220-01). paratypes: 32 workers (thnhm-i-22733 to thnhm-i-22764), same data as holotype (skyc, thnhm). measurements: holotype: tl 3.20, hl 0.79, hw 0.73, el 0.17, mdl 0.30, sl 0.76, ml 0.96, pl 0.23, ph 0.33, dpw 0.23, ci 92, oi 23, mdi 38, si 105. paratypes (n = 10): tl 3.14–3.63, hl 0.73–0.79, hw 0.69–0.76, el 0.13–0.17, mdl 0.26–0.33, sl 0.69–0.76, ml 0.89–0.99, pl 0.23–0.26, ph 0.26–0.33, dpw 0.20–0.26, ci 91–96, oi 19–23, mdi 35–43, si 100–105. worker diagnosis: body entirely yellowish to reddish; entire head and mesosoma covered with fine erect hairs; sparse dark spots present over dorsum of head; with mesosoma in lateral view mesonotum strongly convex with steep posterior slope, nearly as high as propodeum; propodeum dorsally weakly convex with rather a flat dorsum and blunt posterior angle; metanotal groove deeply and broadly incised. description of worker (holotype and paratypes): structure. head in full-face view, oval, slightly fig 1. dolichoderus bakhtiari sp. nov. holotype worker, thnhm-i-22732. a) head in full-face view, b) habitus in profile view, c) habitus in dorsal view. z barabag, w jaitrong – a new species of the ant genus dolichoderus lund4 longer than broad (ci 91–96); posterior margin weakly convex and shallowly emarginate medially, lateral margin convex; eye relatively large, located anterior to midlength of head laterally, with 14–15 ommatidia along the longest axis; antennal scape long (si 100–105), scape extending posteriorly slightly beyond the posterolateral corner of head; pedicel and flagellomeres longer than broad; antennal segment xii almost as long as segments x and xi combined; mandible subtriangular; masticatory margin with sharp apical tooth followed by 11–12 smaller teeth, and basal margin straight with 4–5 small denticles; frontal lobe narrow covering the antennal socket; frontal carina short, reaching half-length of head; clypeus broad, with anterior margin almost straight but feebly concave medially; mesosoma in profile pronotum dorsally flat, mesonotum convex and steeply sloping to metanotal groove; in dorsal view, mesonotum with shallow longitudinal groove; in profile view propodeum weakly convex; propodeal junction right-angled; mesopleuron demarcated from metapleuron by distinct groove, anterior margin of mesopleuron forming sharp triangle; metapleuron not clearly demarcated from lateral face of propodeum; petiole scale-like, convex dorsally and elevated anteriorly, anterior margin shorter than the posterior margin; legs relatively long (compared with other smaller species such as d. taprobanae). sculpture. head entirely superficially reticulate with slightly smooth and shiny interspaces; pronotum mostly punctate but slightly shiny; mesonotum, mesopleuron, and dorsal face of propodeum irregularly rugulose and punctate; metapleuron mostly smooth and shiny; petiole smooth and shiny; gaster superficially reticulated with smooth and shiny interspaces; coxae and femora superficially reticulated with smooth and shiny interspaces; tibiae are finely micropunctate. pilosity and coloration. dorsal surface of head, mesosoma, and gaster covered with dense short white decumbent hairs mixed with dense brown erect hairs; longest erect hair on pronotum about three times as long as erect hairs on gastral tergites; legs with short suberect hairs; entire body yellowish brown with sparse dark spots on the head dorsum. etymology. the specific name is dedicated to the late dr. bakhtiar effendi yahya, who worked on the taxonomy of malaysian ants between the years 2000-19. distribution. thailand (nakhon ratchasima province). bionomics. the type series foraged on leaves of shrub trees (fig 3) in a primary upland evergreen forest at about 800 m a.s.l. in nakhon ratchasima province, northeastern thailand. remarks. dolichoderus bakhtiari sp. nov. is most similar to dolichoderus taprobanae in general appearance as they share some common characters like reddish to yellowish body color; head microreticulate with smooth and shiny interspaces; relatively small body size; dorsally flat pronotum; nearly right-angled propodeal junction. however, d. bakhtiari sp.nov. can be easily separated from d. taprobanae by the following combination of characters: body larger in size (tl 3.14–3.63 mm, hl 0.73–0.79 mm, hw 0.6–0.76 mm); head slightly longer than broad; body entirely yellowishbrown; dorsal outline of propodeum weakly convex; lateral face of pronotum relatively smooth with more developed of frontal carinae. while in d. taprobanae body smaller in size (tl 2.51–2.93 mm, hl 0.66–0.69 mm, hw 0.49–0.52 mm); head slightly shorter than broad; vertex of head and gaster dark brown; dorsal outline of propodeum straight; lateral face of pronotum punctate with less developed of frontal carinae. here, the d. taprobanae images are attached for comparison (fig 2). fig 2. dolichoderus taprobanae worker (colony number wjt06-n6, 27.v.2006). a) head in full-face view, b) habitus in profile view, c) habitus in dorsal view. sociobiology 69(1): e6247 (march, 2022) 5 key to the thailand dolichoderus thoracicus species group 1 head densely and finely rugulose or punctured; dorsa of head, mesosoma, and gaster with dense pubescence and standing hairs; larger species (hw ˃ 0.90 mm).....….........…………..2 head superficially reticulate or smooth and shiny; dorsa of head, mesosoma, and gaster with sparse pubescence and standing hairs; smaller species (hw ˂ 0.80 mm)...………… 3 2 propodeal junction sharply angulated; dorsal outline of propodeum almost straight.....……….....dolichoderus affinis propodeal junction bluntly angulated; dorsal outline of propodeum convex....…….………..dolichoderus thoracicus 3 mesosoma foveolate............………………………..……. 4 mesosoma punctured, not foveolate...........….……….......5 4 propodeal junction sharply angulated; dorsal outline of propodeum straight; pronotum flat......... dolichoderus butteli propodeal junction bluntly angulated; dorsal outline of propodeum convex; pronotum convex......dolichoderus brevis 5 larger species (tl 3.14–3.63 mm, hl 0.73–0.79 mm, hw 0.6–0.76 mm); head slightly longer than broad; body entirely yellowish-brown; dorsal outline of propodeum weakly convex; lateral face of pronotum relatively smooth with more developed of frontal carinae...............................dolichoderus bakhtiari sp. nov. smaller in size (tl 2.51–2.93 mm, hl 0.66–0.69 mm, hw 0.49–0.52 mm); head slightly shorter than broad; vertex of head and gaster dark brown; dorsal outline of propodeum straight; lateral face of pronotum punctate with less developed of frontal carinae........…….………. dlichoderus taprobanae acknowledgments we thank the thailand museum of natural history of the thailand national science museum for allowing z. barabag to use facilities and to examine ant specimens. the authors also thank mr. w. khaikaew who donated us the type series. this project is supported by the university malaysia sabah postgraduate research grant (gug0268-2/2018), the malaysia ministry of education scholarship, and partly by thailand science research and innovation. we would like to thank seiki yamane and liew thor seng for their constructive comments on the manuscript. references antweb. 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(2013). australian species of the ant genus dolichoderus (hymenoptera: formicidae). zootaxa, 3716: 101-143. https://www.biotaxa.org/zootaxa/ article/view/zootaxa.3716.2.1/0 https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://antwiki.org/wiki/images/5/54/dill_2002.pdf https://zookeys.pensoft.net/article/54902/download/pdf/480278 https://zookeys.pensoft.net/article/54902/download/pdf/480278 https://www.biotaxa.org/zootaxa/article/view/zootaxa.3716.2.1/0 https://www.biotaxa.org/zootaxa/article/view/zootaxa.3716.2.1/0 _hlk62258148 _hlk91127985 _hlk53839511 _hlk90952965 _hlk54275641 _hlk62088609 _hlk64559727 _hlk91581640 doi: 10.13102/sociobiology.v62i3.708sociobiology 62(3): 450-456 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 use of flight interception traps of malaise type and attractive traps for social wasps record (vespidae: polistinae) introduction the scientific literature reports different colection methodologies applied on studies about social wasps biodiversity approaching several brazilian biomes, such as cerradothe brazilian savannah, rainforests, rupestrian fields, riparian forest, caatinga, as well as the areas with monoculture (diniz & kitayama, 1998; souza & prezoto, 2006; santo et al., 2007; silva & silveira, 2009; souza et al., 2010; melo et al., 2015). the most used methods for capturing polistinae are the active search. the use of attractive traps, flight interception traps of malaise type and collection close to floral resources. among these, the malaise traps are among the methodologies most commonly used (silveira, 2002; kumar et al., 2009; noll & gomes, 2009; somavilla & oliveira, 2013) and so are the attractive traps (souza & prezoto, 2006; elpino-campos et al., 2007), even though they are less efficient than the abstract the literature provides different methodologies for sampling social wasps, including, flight intercept trap type malaise and attractive trap, however, there is no consensus on its use. in this respect, the aim of this study was to evaluate the best use of malaise traps and attractive trap in biodiversity work of social wasps, and generate a collection protocol for the use of these traps. the study was conducted in the parque estadual do rio doce, located in the east of the state of minas gerais, in the years 2000, 2001, 2002 and 2004 and in the botanical garden of the federal university of juiz de fora, located in the southeastern state of minas gerais, in years 2011, 2012 and 2013. 15 species were collected using malaise traps, and, 26 species of social wasps were collected using attractive traps. although the negative aspects of both traps, complementary methodologies surveys varying social wasps are useful and it is recommended to choose for using in accordance with the logistical field. sociobiology an international journal on social insects mm souza1, ln perillo2, bc barbosa3, f prezoto3 article history edited by gilberto m. m. santos, uefs, brazil received 18 november 2014 initial acceptance 18 june 2015 final acceptance 11 august 2015 keywords atlantic rainforest, collection methodologies, diversity, inventory. corresponding author marcos magalhães de souza instituto federal de educação, ciência e tecnologia do sul de minas campus inconfidentes praça tiradentes nº 416, centro, 37576-000 inconfidentes-mg, brazil e-mail: marcos.souza@ifsuldeminas.edu.br active search for nests of social wasps (silveira, 2002; noll & gomes, 2009). authors have already presented data about the use and the efficiency of malaise and attractive trap for recording social wasps, but there is not a defined pattern for using this methodology (silveira, 2002; silva & silveira, 2009; locher et al., 2014; barbosa, 2015). the flight interception traps type malaise (townes, 1962; 1972) are applied for sampling different kinds of insects, besides social wasps, such as diptera (andrade-filho et al., 2008), coleoptera (linzmeier et al., 2006) and different families of hymenoptera with single habits (tanque & frieiro-costa, 2011; kumagaia, 2002). the malaise trap, due to the possibility of exposition for long continuous periods in autonomous way (andrade-filho et al., 2008), are commonly used individually or in small amounts. however, there is not much data on the efficiency to sample the community of species in a small number of traps or the necessary time for them to be left on the field (fraser et al., 2008). research article wasps 1 instituto federal de educação, ciência e tecnologia do sul de minas, minais gerais, brazil 2 bocaina ciências naturais e educação ambiental, minas gerais, brazil 3 universidade federal de juiz de fora, juiz de fora, minas gerais, brazil sociobiology 62(3): 450-456 (september, 2015) 451 the use of attractive traps to capture social wasps began in brazil with the pioneer work of lorenzato in 1985, which performed a study about the occurrence of such insects in apple and peach orchards in caçador, santa catarina, southern brazil. in this study the author used three types of attractive substances (wine vinegar at 25%, brown sugar at 5% and grape juice at 25%), that were displayed in bottles which are used to catch flies, model valenciano, with the result of 12 species of wasps – belonging to five genus – being captured. more than ten years later, santos (1996) has also made use of attractive traps to collect social wasps in citrus orchards in a farm in the surroundings of goiânia, goiás, central region of brazil. in this study, the author has used orange juice at 50% and sugar at 10% in units of 200ml in bottles that capture flies as attractive baits. after one year of collection, nine species of wasps, belonging to five genus, were reported. ten years later, there was a resumption on the studies about diversity of social wasps using attractive traps, with the studies of souza and prezoto (2006) and elpino-campos et al. (2007), which were inspired on the previously quoted studies and modified and adapted the collection methodology. the authors started to use pet bottles as containers, in which different kinds of attractive substances were tested (sardine broth, orange juice, passion fruit and water with sugar). the goal of this work was to evaluate the advantages and disadvantages of using malaise traps and attractive traps in studies of biodiversity of social wasps, as well as to generate a sampling protocol for the use of such traps. materials and methods malaise trap the study was conducted in rio doce state park, eastern minas gerais (420 38’w 480 28’w 190 45’s 190 30’s), conservation unit that hosts the biggest remainder of rainforest in the state, with 36,970 hectares. the climate is hot and wet, with two well-defined seasons: one of them is dry and cold (april to september), with temperatures ranging from 70 c to 200 c, and the other is wet and hot (october to march), with temperatures ranging from 280 c to 390 c. the average precipitation ranges between 1,350mm and 1,900mm and the heights vary from 236m to 515m (cetec, 1983; antunes, 1986). by using the malaise trap, it was possible to sample polistinae in three different successional stages, defined according to the gilhuis classification (1986): low and high second-growth, as well as primary kinds of forest. in each place three spots were selected and, in each one of them, three malaise traps were installed, coming up to nine traps by successional stage. they were simultaneously set in all the spots and each one was kept for three consecutive weeks in the field in each climate season. the collected materials were analyzed during the dry (july) and the wet (between october and november) seasons of 200, 2001, 2002 and 2004, coming to four years of samples with more than 12,000 hours of traps for each spot. attractive traps the study was performed at the botanical garden of federal university of juiz de fora (21° 43’ 28” s 43°16’ 47” o),a fragment of semideciduous seasonal montana forest (ibge, 2012), located in the urban area of juiz de fora, southeast region of minas gerais, 750m above the sea level, and presents subtropical hot climate with a dry winter and rainy summer (cwa), according köppen-geiger (sájúnior et al., 2012) classification. the area, with 84 hectares of extension, was recently classified by santiago et al. (2014) as complex of expressive richness, diversity and floristic heterogeneity of tree vegetation, with some endangered species and with predominance of pioneer plants, aside the remarkable existence of exotic species, it is considered an urban green area (marciel & barbosa, 2015) the records of social wasps occurred from september /2011 to august/2013. the collections happened monthly with duration of five days, 32 bottles used for each collection; the traps were divided in two height levels: 1.5m from the ground and 10m from the ground, with the possibility of variation according to the local vegetation characteristics. the traps were evenly distributed in two areas. for each one, the distance between the spots was 10m. the attractive substances were natural passion fruit juice and natural guava juice. the traps were kept in the field for five days (summing up to 120 days of collection with traps), the screening of the material was performed in loco, using a strainer to make the separation of liquids and insects easier. after this, the insects were stored in glass containers (5ml) with alcohol 70°gl that were properly labeled. results and discussion with malaise traps, 15 species of social wasps of five genus were collected, and the most frequent species were agelaia angulata (fabricius, 1804) and agelaia centralis (cameron, 1907). and for the attractive traps, 26 species of 10 different genus were collected, and the most frequent species were agelaia multipicta (haliday, 1836) e polybia platycephala (richards, 1951). species of genus agelaia are usually abundant in studies of diversity due to the great size of the colonies (zucchi et al., 1995; hunt et al., 2001). the greatest richness of species that was reported in this study for the polybia genus corroborates with other data collections performed in other brazilian regions (diniz & kitayama, 1994; elpino-campos et al., 2007; silva & silveira, 2009; grandinete & noll, 2013; barbosa et al., 2014). this fact may be related to the number of species that make the group (57, 44 of which are recorded mm souza, ln perillo, bc barbosa, f prezoto – standardizing two methods for social wasp sampling452 in brazil), the abundance of members in each colony, and its distribuction, that goes from united states to argentina, and it is considered the most frequent genre of social wasps in south america (richards, 1978; jeanne, 1991; carpenter et al., 2000; carpenter & marques, 2001). this number species collected by malaise trap in low, when compared with other studies in the same area usind active search as main sampling method for social wasps, as showed by souza et al. (2012), where 38 species were reported during one year of collection with 20 days of sampling. all the species that were collected using malaise were reported in the active search (souza et al., 2012). this lower efficiency may be explained by the scope of operation of the social wasps, which is generally around 150 meters (cruz et al., 2006), and restrain the efficiency of capture to the presence of colony close to the traps. another point to be considered is that the traps keep set up in the same spot during all the experiment, reducing thus the chance of sampling members of other colonies of different species. studies conducted in the brazilian amazon also showed this lower efficiency of malaise traps, compared to the active search (silveira, 2002; silva & silveira, 2009). aiming to make this methodology a pattern, a description is shown above of the standard used in most of the works: the traps are built with a fine, lightweight and durable fabric – organza, nylon or bias are preferable – making an open tent, placed close to the ground, with a septum in the central area. if possible, make it with dark walls and light roof (fig 1). this contrast between the colors and the bottom and top sections in essential to induce the insects to fly to the top, searching for the light while they are actually conducted the sampling container (almeida et al., 1998). they can be assembled during all the study or be replaced to other areas, according to the logistics and the size of the sampled area. in wide areas, there must be used the most traps possible, located from 0.5 to 1km away from each other. fig 1. malaise trap, assembled in the field. fig 2. scheme of correct assembling and dimensions of each part of conventional malaise trap. the shape is triangular, like a camping tent, with the measures showed on figures 2 (almeida et al., 1998). the dimensions may be changed according to the goal. an inclined cover, in a light color to lead the insects to the sampling container (which must be totally or partially transparent), located in the higher spot. the sampling container must contain a preservative liquid, alcohol is preferred (reminder: when exposed to the environment, the alcohol evaporates). in addition to monitoring the trap every seven or ten days (depending on the environmental condition), it is recommended the use of a more concentrated alcohol (80-90%). to maintain the trap standing, a pole is used to hold the front part (where the bottle is fixed) and the ends are tied with ropes attached to stakes or branches in the vegetation, making the fabric the most stretched it can be. the efficiency of the trap depends on the quality on the assembling time. when it is set in dense forests, it should be put in trails or open glades, to make the capture of the insects easier. regardless sociobiology 62(3): 450-456 (september, 2015) 453 of the environment where it is to be used, the place where the bottle is put should be directed to the region that receives the most light during the year or towards to the north, so not to influence on the collection rate of those insects guided by the sunlight and the moonlight. the containers should be replaced every 15 days, at maximum, and the biological material collected have to be taken to a laboratory. the best scenario is that in which the traps are kept during all the seasons of the year is the seasonality is an issue to be studied. the advantages of this methodology are : the collection of a great number of members of each specie; possibility of exposition for continuous periods and autonomously, dismissing the researcher in loco; increase in the chance of sampling species with big colonies, such as the genus agelaia and the possibility of sampling the same species of the active search, increasing the logistics on the field. on the other side, the disadvantages are: the time wasted on screening the material on the laboratory; the price of each trap; the time spent to change all the bottles; the relocation of these traps throughout the year or, if they stay in the same area, the risk of sampling the same species throughout the year (correlated to the presence of the colonies close to the trap); reduced chances of sampling species with small colonies with few members, such as the genus mischocyttarus. the attractive trap in the present study, as well as in the work of locher et al. (2014) (23 species), showed higher numbers, showing thus a better performance that the malaise traps. some advantages on using the attractive bait methodology can be highlighted. the advantages of this methodology are: practicality and low cost: the bottles used as containers are of type pet, with capacity for 2 liters, and can be easily obtained with zero cost. the attractive substances are made from fruit juice, which can be produced from in natura fruits (ribeiro júnior, 2008; barbosa, 2015) or from purchasing the industrialized juice (togni et al. 2014). fiing the bottler on the trail can be easily performed using sting or a nylon clamp to hold them together, both with low cost. optimization on the collection efforts: another advantage of this method is that it can be applied in consortium with other methodologies. the method consists on installing the traps in a day and its removal after a period (usually around 5 days). in the meanwhile, the researcher can perform another collection while the traps attract the wasps. the disadvantages: lower number of members collected of each specie; smaller life cycles in the field and the necessity of having the researcher in loco. aiming to make the use of this methodology for sampling social wasps a pattern, the following orientation is proposed: the attractive traps must be produced using two liters pet bottles, with three triangle-shaped openings on the side (2 x 2 x 2 cm) on the middle section (approximately 15 cm from the bottom) (fig 3). when it comes to the attractive substances, two preparations are recommended: in the first case it is possible to prepare the substance from the fruit itself, following the steps: 1kg of pulp, beat in blender with 250g of sugar and two liters of water. in the second case, the industrialized juice can be used. the choice of this juice should consider the specificities of each scope of study. for a work in the northern brazil, the recommended juices are cupuaçu, açaí, camu-camum buriti among others, as these fruits are typical from the region. for a study on the northeast part of brazil, the cashew juice is suggested. as a protein substance, sardine broth has been used, and it should be prepared using two cans of sardine (125 grams), beat with two liters of water. in each bottle, 200ml of the attractive substance should be inserted. this amount is the most commonly used in the studies published so far and it is enough to avoid the complete evaporation of the fluid during the sampling, even in periods of high temperatures. the distribution of the traps must throughout a transect, 1,5m above the ground and respecting the minimum distance of 10 meters between the bottles, alternating the sequence, as schemed on figure 4. that distance may vary according to fig 3. view of attractive trap in two-liter pet bottle, containing 200 ml of passion fruit juice and installed 1,5m above the ground on tree trunk. inside the red circle the triangle-shaped of 2x2x2cm can be seen. mm souza, ln perillo, bc barbosa, f prezoto – standardizing two methods for social wasp sampling454 the place where the study is conducted, with the number of traps and with the goal of the study. the traps must stay in the field for a period of five days, and installed in the beginning of the first day and taken on the fifth day, and next discarded or sanitized to avoid contamination. the screening of the material can be performed in the place itself, with a strainer to make the separation of the liquid and the insects easier. next, the insects should be stored in plastic containers containing alcohol 70°gl, and every container should be rightly labeled for further identification and analysis. the success of these collection techniques could be attested in the recent years, as many authors have been using the methodologies for studying diversity of social wasps in different biomes, usually in consortium with other methodologies, such as the active search. thus, despite the negative characteristics of both traps, they make complementary methodologies that are useful for data collection on diversity of social wasps, so their choice is recommended, according to the logistics of the field. aknowledgements we would like to thank professor jcr fontenelle and the researchers that took part on the program ecmvs/ufmg. to doctor orlando tobias silveira, emílio goeldi museum, for the identifications. this study was provided by cnpq and u.s. fish wildlife & services. to the biologist epifânio porfírio pires, for the cooperation on screening the biological material. to conselho nacional de desenvolvimento científico e tecnológico (cnpq) (f. prezoto 310713/2013-7) for providing financial support. references almeida, l.m., costa, c.s.r. & marinoni, l. 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(1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). entomology am-ny, 103: 129137. doi: 10.13102/sociobiology.v61i4.386-392sociobiology 61(4): 386-392 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 exocrine glands in the legs of the stingless bee frieseomelitta varia (lepeletier) (apidae: meliponini) introduction social insects display an enormous variety of exocrine glands, the secretions of which play multiple roles in their social organization (hölldobler & wilson, 1990; billen & morgan, 1998; billen, 2011). following their cellular organization, all glands can be classified in one of two major groups as first described in the pioneer paper on insect exocrine glands by noirot and quennedey (1974): class-1 glands are formed by simple epithelial secretory cells, while class-3 glands are formed by a number of bicellular units, each unit comprising a large spherical secretory cell and an accompanying slender duct cell. the ducts and the corresponding pores at their opening site have a characteristic diameter of 0.5-1 µm. glands of both groups can either occur underneath the external cuticle, and in this case release their secretion directly to the exterior, or they can be part of an invaginated reservoir sac, where secretion can be temporarily stored. this leads to five anatomical types (fig 1). the various glands are not only confined to the head, thorax and abdomen, but can also be found in the appendages such as the antennae, mouthparts and legs. in ants, an astonishing variety of 20 leg glands has been described (billen, 2009). bees, as well, have several glands in their legs, and especially stingless abstract social insects are known for their overwhelming diversity of exocrine glands. this study examines the glands in the legs of workers of frieseomelitta varia (lepeletier). a variety of 15 glands was found, with glands occurring in every leg segment, whereas previous studies only described 5 glands in stingless bee legs. six glands are novel exocrine structures for social insects. glands occurring in the articulation region between adjacent leg segments may occur in a repetitive pattern, and probably have a lubricant function. for most glands, however, the function is not yet known, and will require further experimental work. sociobiology an international journal on social insects j. billen1, l. vander plancken1 article history edited by denise araujo alves, esalq-usp, brazil received 30 september 2014 initial acceptance 25 october 2014 final acceptance 30 october 2014 keywords legs, exocrine glands, morphology, meliponini. corresponding author prof. johan billen k. u. leuven, zoological institute naamsestraat 59, box 2466, b-3000 leuven, belgium e-mail: johan.billen@bio.kuleuven.be bees have been studied the most (cruz-landim et al., 1998). the latter study presented data on leg glands in the three castes of 13 meliponine species belonging to 7 genera. its most important conclusions were the occurrence of epithelial glands only in the basitarsus and pretarsus (with a report that queens have sacculiform glands also in their femur), the widespread occurrence of class-3 glandular cells in the various leg segments, and that tarsomeres 2 to 4 did not contain any gland cells (cruz-landim et al., 1998, updated in cruz-landim, 2002). our present study deals with workers of frieseomelitta varia (lepeletier), a species that was not included in the survey study by cruz-landim et al. (1998), but which revealed the occurrence of several other leg glands, that therefore can be considered as novel exocrine structures. material and methods both callow and foraging workers of f. varia were collected from natural colonies at the apiary of the universidade de são paulo campus in ribeirão preto, sp, brazil. for the histological work, we used forager workers only (n=3), as they are relatively old. this is important to recognize epithelial glands, as callow workers may display a thick tegumental epithelium because of cuticle formation at their young age. as research article bees 1 zoological institute, university of leuven, leuven, belgium sociobiology 61(4): 386-392 (december 2014) 387 cuticle formation is completed in foragers, a thick epithelium in such older individuals can be considered as glandular tissue. in order to allow proper penetration of fixative and the various chemicals used for dehydration and embedding, fore-, midand hindlegs were cut into smaller pieces by making cross cuts in the femur, tibia and basitarsus. tissues were fixed in cold 2% glutaraldehyde, buffered with 50 mm na-cacodylate and 150 mm saccharose, followed by postfixation in 2% osmium tetroxide. after dehydration through a graded acetone series, tissues were embedded in araldite resin. serial semithin sections with a thickness of 1 µm were made with a leica em uc6 ultramicrotome, stained with methylene blue and thionin, and examined with an olympus bx-51 light microscope. for scanning microscopy, however, we only used callow workers (n=3), as they have clean legs whereas forager legs may be dirty and/or covered with resin material. legs were mounted on stubs, gold coated, and viewed in a jeol jsm6360 scanning microscope. results and discussion the various exocrine leg glands will be presented from most proximal (coxa) to most distal (pretarsus). figure 2 and table 1 give a survey of the occurrence of the various glands. all photographs are shown with the dorsal side up; all sections are longitudinal, the proximal side is always to the left. 1. coxal gland the distal region of the coxae of the three leg pairs contains a few rounded class-3 cells with a diameter around 30 µm. their ducts open through the articulation membrane with the trochanter (fig 3a). similar coxal glands have also been described in ants (schoeters & billen, 1993), and because of their opening through the articulation membrane, they possibly have a lubricant function. cruz-landim et al. (1998) and cruz-landim (2002) also report the existence of class-3 glandular cells in workers of several meliponine species, but in the proximal dorsal part of the coxae, and with unknown opening site of the ducts. 2. coxal epithelial gland besides the class-3 coxal gland cells, the coxae of the three leg pairs also contain a glandular epithelium with a thickness between 10 and 15 µm (fig 3a, b), that occurs as a kind of distal belt along the entire circumference of the coxae. the glandular epithelium is distinctly different from the much thinner proximal coxal epithelium. this gland was not mentioned in the review papers on meliponine leg glands by cruz-landim et al. (1998) and cruz-landim (2002), and it is also different from the epithelial basicoxal gland in ants, that occurs dorsally in the proximal region of the midand hindlegs of poneromorph ants (billen & ito, 2006). it therefore represents a novel exocrine structure for social insects with currently unknown function. 3. distal trochanter gland the distal part of the trochanter of the three leg pairs contains a ventral glandular epithelium with a thickness around 10 µm (fig 3c). an epithelial trochanter gland has also been described in several ant species, but occurs in the proximal ventral part only and it is presumed to have a lubricant function (billen, 2008). cruz-landim et al. (1998) and cruzlandim (2002) do not mention about an epithelial gland in the trochanter of meliponini, but instead report the occurrence of class-3 gland cells in the dorsodistal region near the articulation with the coxa, where their ducts open through the external cuticle (cruz-landim, 2002). our study of frieseomelitta varia, however, did not show any class-3 gland cells in the trochanter. 4. proximal femoral gland sections through the proximal part of the hindleg femur revealed the occurrence of a linearly arranged cluster of class-3 cells that are wedged in between the extensive femoral musculature (fig 3d). the cells appear rather square on sections and measure approx. 50x20 µm. we did not find similar cells in the foreand midlegs. the ducts open through the external dorsal cuticle (arrow in fig 3d), where they can also be seen with scanning microscopy (fig 3e). a similar proximal femoral gland was also reported, albeit without information in which leg pairs, in all meliponine species investigated by cruz-landim et al. (1998). the function of this gland remains unknown. 5. dorsodistal femoral gland the dorsodistal region of the femur in the three leg pairs contains a cluster of approx. 10 rounded class-3 glandular cells that have a diameter around 25 µm (fig 3f). their fig 1. general gland types, with types a and b formed by epithelial class-1 glandular cells, types c, d and e formed by bicellular secretory units containing class-3 gland cells. gland types a and c have no reservoir and release their secretion directly to the exterior, types b, d and e have a reservoir for temporary storage of secretion. this reservoir can be formed by an invagination of an intersegmental membrane, see type e (from billen, 2009a). j. billen, l. vander plancken exocrine glands in the legs of frieseomelitta varia388 ducts open through the external cuticle (arrows in fig 3f), and can also be recognized with scanning microscopy (fig 3g). as their opening site is not directly associated with the articulation membrane with the tibia, a lubricant function is less probable. cruz-landim et al. (1998) and cruz-landim (2002) do not mention about this gland in meliponini, while also ants do not have it (billen, 2009). scattered class-3 glandular cells and pores of the corresponding duct cells at the femur-tibia junction, and especially in the various tarsomeres, were found in the wasp polistes dominulus (christ, 1791), and are probably involved in pheromone production (beani & calloni, 1991). 6. distal femoral gland the distal region of the femur also contains, at least in the foreand midleg, a few ovoid class-3 glandular cells measuring approx. 30 by 20 µm (fig 4a). their ducts open through the articulation membrane with the tibia, which is probably an indication of a lubricant function. similar gland cells have been reported in ants (billen, 2009), but were not yet listed for meliponini (cruz-landim et al., 1998; cruz-landim, 2002). 7. ventrodistal femoral gland besides the dorsodistal femoral gland (which occurs dorsally and opens externally at the tip of the femur, see under 5) and the distal femoral gland (which occurs centrally and opens through the intersegmental membrane, see under 6), the distal femur of the hindleg contains a ventrally located cluster of numerous class-3 glandular cells. contrary to the dorsodistal femoral gland, however, this ‘ventrodistal femoral gland’ has long ducts that run posteriorly in an upward direction to open eventually at the dorsolateral femoral surface (fig 4c). the scattered ducts are clearly visible with scanning microscopy (fig 4b). as this gland was not observed in other stingless bees by cruz-landim et al. (1998) and cruz-landim (2002), and it is also lacking in ants (billen, 2009), it can be considered a novel exocrine gland for social insects. as the opening site of the ducts is quite far from the junction with the tibia, a lubricant function is not very probable. 8. dorsodistal epithelial femoral gland in all three leg pairs, the femur also contains underneath its dorsodistal region a glandular epithelium with a thickness around 20 µm (figs 4a, 4c). the occurrence of this gland was not noticed in other meliponini (cruz-landim et al., 1998; cruz-landim, 2002), nor does it occur in ants (billen, 2009). the function of this novel gland remains unknown. cruz-landim et al. (1998) found an internal sacculiform epithelial gland inside the femur of a few stingless bee species, but only in the queens, and without information of where this sac-like gland opens to the outside. 9. dorsodistal epithelial tibial gland the dorsodistal region of the tibia in the forelegs contains a glandular epithelium with a thickness around 10 µm (fig 4d). this gland was not found in other meliponini (cruzfig 2. schematical survey of the various exocrine glands in the legs of frieseomelitta varia workers. gland numbers correspond with table 1 and the main text. the anatomical organization in the distal tarsomeres is shown in the detail drawing. tc = undefined tibial cells in the hindlegs. sociobiology 61(4): 386-392 (december 2014) 389 landim et al., 1998; cruz-landim, 2002), nor in ants (billen, 2009), and thus forms another novel exocrine structure for social insects. its function remains as yet unknown. 10. dorsodistal tibial gland in the dorsodistal region of the foreand midlegs, we found some large rounded class-3 glandular cells with a diameter around 40 µm (fig 5b). their ducts open through the upper external cuticle (fig 4e), which make this gland a tibial equivalent of the dorsodistal femoral gland (see under 5). similar glandular cell clusters have also been described in other stingless bee species (cruz-landim et al., 1998; cruz-landim, 2002), as well as in ants (billen, 1997). as for the dorsodistal femoral gland, a lubricant function seems not to be very likely as the duct openings are situated on the external surface instead of opening through the articulation membrane. undefined tibial cells as in the majority of most other bees, the hindleg tibia of frieseomelitta varia workers is considerably enlarged as it forms part of the pollen collecting apparatus (fig 2). inside the distended part of the tibia, we found a massive number of large ovoid to polygonal cells of 40-50 µm with a rounded nucleus and highly vacuolar cytoplasm (fig 4f). their initial appearance reminds of duct cells, but we could not find any ducts, neither on sections nor under the scanning microscope. following the description and classification of the insect fat body as given in the review paper by roma et al. (2010), they may be oenocytes or trophocytes, although it remains unclear why they are so numerous in the hindleg tibia. 11. antenna cleaner gland a common characteristic for most hymenoptera is the occurrence of an antenna cleaning apparatus on both forelegs, that consists of a prominent tibial spur and a semicircular comb-like differentiation of the proximal basitarsus (fig 5a). the tegumental epithelium underneath the basitarsal comb is formed by cylindrical cells with a height around 40 µm (fig 5b). a major part of this complex structure is formed by nervous tissue, as sensillar structures extend into the comb cuticle (fig 5b). the other part probably forms part of the antenna cleaner gland that was first described in the ant messor rufitarsus (fabricius) by schönitzer et al. (1996). in spite of its name, however, no experimental evidence exists for a direct function of this gland in the process of antennal cleaning. no mention of this gland in meliponini was made in the review papers by cruz-landim et al. (1998) and cruz-landim (2002). 12. epithelial basitarsal gland the epithelium of the entire (foreand midleg: fig 5c) or dorsal part of the basitarsus (hindleg: fig 5d) is differentiated into a thickened glandular tissue. the existence of this epithelial basitarsal gland was first reported for a few meliponine species and for the honeybee (cruz landim & de moraes, 1994). its function as yet remains unknown. 13. epithelial tarsomere glands the entire dorsal epidermis of the three intermediary tarsomeres t2-t3-t4 in the three leg pairs appear as a clear glandular epithelium with a thickness of up to 15 µm, whereas the ventral side shows a squamous epithelium of hardly 1-2 µm (fig 5e). these conspicuous epithelial glands have never been described table 1. survey of the various leg glands in workers of frieseomelitta varia, and their occurrence in the three leg pairs. gland names shown in bold represent exocrine structures that have not been reported before for social insects. gland numbering is the same as in figure 2 and in the main text, gland type corresponds with figure 1. + indicates gland presence, indicates absence. the right column ‘cl’ indicates whether or not the occurrence of a gland was reported in the 1998 review article on leg glands in meliponini bees by cruz-landim et al. and the same author’s book chapter update in 2002. nr gland type foreleg midleg hindleg first reference cl 1 coxal gland e + + + schoeters & billen, 1993 yes 2 coxal epithelial gland a + + + this paper no 3 distal trochanter gland a + + + this paper no 4 proximal femoral gland c + cruz-landim et al., 1998 yes 5 dorsodistal femoral gland c + + + beani & calloni, 1991 no 6 distal femoral gland e + + billen, 2009 no 7 ventrodistal femoral gland c + this paper no 8 dorsodistal epithelial femoral gland a + + + this paper no 9 dorsodistal epithelial tibial gland a + this paper no 10 dorsodistal tibial gland c + + cruz-landim et al., 1998 yes 11 antenna cleaner gland a + schönitzer et al., 1996 no 12 epithelial basitarsal gland a + + + cruz landim & de moraes, 1994 yes 13 epithelial tarsomere glands a + + + this paper no 14 distal tarsomere glands a + + + billen et al., 2000 no 15 arolium gland b + + + arnhart, 1923 yes j. billen, l. vander plancken exocrine glands in the legs of frieseomelitta varia390 previously, also not in other meliponini (cruz-landim et al., 1998; cruz-landim, 2002). in ants, a glandular differentiation of the dorsal tarsomere epithelium only occurs in the proximal region, where it forms part of the articulation with the previous tarsomere, probably indicating a lubricant function (billen, 1997, 2009). the glandular nature of the entire tarsomere upper surface, as now described for f. varia, however, can hardly be linked with any lubricating role. 14. distal tarsomere glands in the three leg pairs, tarsomeres t1-t2-t3-t4 show a semicircular mediodistal glandular epithelium with a thickness of approx. 20 µm (fig 5e). their repetitive occurrence and their location in the articulation region with the adjacent tarsomere support a lubricant function. these distal tarsomere fig 4. a. junction between midleg femur and tibia, showing the dorsodistal epithelial femoral gland (defg) and distal femoral gland (dfg). b. scanning micrograph of hindleg femur, arrows indicate duct openings of the dorsodistal femoral gland (inset shows region where picture is taken). c. section through distal region of hindleg femur. arrows indicate long ducts of the ventrodistal femoral gland (vdfg; white arrows show ducts penetrating cuticle). d. distal part of foreleg tibia with dorsodistal epithelial tibial gland (detg). e. scanning micrograph of foreleg tip, arrows indicate duct openings of the dorsodistal tibial gland. f. distal region of hindleg tibia with numerous probably non-glandular tibial cells (tc). ddfg: dorsodistal femoral gland, ln: leg nerve, tb: tibia. glands have not been reported for other meliponini species (cruz-landim et al., 1998; cruz-landim, 2002), but do exist in ants (billen et al., 2000). 15. arolium gland the pretarsus (= tarsomere t5) in the three leg pairs contains the conspicuous arolium gland, which is formed by a glandular epithelium with a thickness of up to 30 µm (fig 5e). the lumen of the gland is continuous with the interior arolium. the reservoir sac is ventrally penetrated by the leg tendon, which in turn makes contact with the ventral wall of the arolium. although there is a transverse ventral groove in this region, there is no direct contact between the lumen of the arolium gland and the exterior (fig 5e). the arolium gland was first described almost a century ago by arnhart (1923) in the honeybee, and fig 3. a. distal region of midleg coxa with coxal epithelial gland (ceg) and coxal gland (cg). arrows indicate ducts of the coxal gland penetrating the articulation membrane with the trochanter. b. mediodistal region of midleg coxa and articulation with trochanter (trc), showing belt-like coxal epithelial gland (ceg). c. distal region of midleg trochanter with distal trochanter gland (dtrg). d. proximal region of hindleg femur with proximal femoral gland (pfg), arrow shows duct penetrating cuticle. e. scanning micrograph of proximal hindleg femur, arrows indicate opening of proximal femoral gland ducts. f. distal region of midleg tibia and articulation with basitarsus (bt). arrows indicate opening of ducts of the dorsodistal tibial gland (ddtg) through the cuticle. g. scanning micrograph of distal part of foreleg tibia, arrows indicate duct openings of dorsodistal tibial gland. cx: coxa. sociobiology 61(4): 386-392 (december 2014) 391 conclusion the legs of f. varia workers represent an impressive glandular environment with 15 different glands, of which 6 are novel exocrine structures for social insects in general. earlier work documenting the leg glands of 13 stingless bee species (cruzlandim et al., 1998; cruz-landim, 2002) listed only one third of the glands we have found in frieseomelitta. this may be due to a less systematic approach previously, as we studied semithin serial sections of 1 µm thickness, whereas cruz-landim et al. (1998) worked with 6 µm sections. we found glands in all leg segments, which often show a similar pattern in the three leg pairs. although difficult to prove experimentally, several glands probably have a function in the production of lubricant substances, especially when their opening site is situated at the junction between adjacent segments, while they may also occur in a repetitive pattern such as the distal tarsomere glands. also the arolium gland as part of a hydraulic system that regulates arolium shape and hence adhesion to the substrate (federle et al., 2001) and the antenna cleaner gland have mechanical functions, although empirical evidence for the latter is not yet clear. other leg glands may be involved in pheromone production. ants are substrate-bound and therefore often rely on trail pheromones, for which several hindleg glands have been shown as the anatomical source (billen, 2009). wasps have been reported to use leg glands for scent marking as well (beani & calloni, 1991), while lensky & slabezki (1981) already reported how honeybee queens inhibit the construction of royal cells with ‘foot-print pheromones’. the only known example in meliponine bees of leg glands having a pheromonal function is the food marking in m. seminigra with secretions from the femoral and tibial tendon glands (jarau et al., 2004). further behavioral and chemical work in future will hopefully reveal more insight in the role of the multiple glands that are present in the legs of stingless bees. acknowledgements we are very grateful to an vandoren for her help with section preparation, to alex vrijdaghs for his assistance with scanning microscopy, and to denise araujo alves for providing us with the bees. references arnhart, l. (1923). das krallenglied der honigbiene. arch. bienenkunde, 5: 37-86. beani, l. & calloni, c. (1991). leg tegumental glands and male rubbing behavior at leks in polistes dominulus (hymenoptera: vespidae). j. chem. ecol., 4: 449-462. billen, j. (1986). etude morphologique des glandes tarsales chez la guêpe polistes annularis (l.) (vespidae, polistinae). actes colloq. insectes soc., 3: 51-60. it is the most common leg gland, that is present in all social hymenoptera (bees: arnhart, 1923; cruz landim & staurengo, 1965; bumblebees: pouvreau, 1991; wasps: billen, 1986; ants: billen, 2009). initially, the gland secretion was thought to play a role in the adhesion to smooth surfaces (arnhart, 1923; pouvreau, 1991), which is very doubtful as there is no contact between the gland lumen and the exterior. instead, gland secretion can be pumped into the arolium and thus probably acts as a hydraulic system, with the resulting changes in arolium shape responsible for adhesion (federle et al., 2001). the eventual role in substrate marking (goulson et al., 2000) may not be attributed to the arolium gland for the same anatomical reason, but has been shown to be the result of secretions from femoral and tibial tendon glands, that are deposited at the pretarsus ventral surface through the leg tendon (jarau et al., 2004, 2005). these femoral and tibial tendon glands, elegantly described by jarau et al. (2004) in the three leg pairs of melipona seminigra friese, were not found in our study of f. varia. fig 5. a. scanning micrograph of the antenna cleaning apparatus at the junction between the foreleg tibia (tb) and basitarsus (bt), which is formed by the tibial spur (ts) and a basitarsal comb (bc). b. section through the foreleg tibia-basitarsus junction with indication of the dorsodistal tibial gland (ddtg), antenna cleaner gland (acg) and epithelial basitarsal gland (ebg). c. foreleg basitarsus with epithelial basitarsal gland (ebg). d. hindleg basitarsus and second tarsomere (t2), showing the epithelial basitarsal gland (ebg), epithelial tarsomere gland (etg) and distal tarsomere gland (dtg). e. section through foreleg tarsomeres with the arolium gland (ag), epithelial tarsomere glands (black arrows) and distal tarsomere glands (white arrows). ar: arolium, lt: leg tendon, ss: sensillar structures, t1-t5: tarsomeres 1-5. the arolium gland lumen is marked with asterisks (*). j. billen, l. vander plancken exocrine glands in the legs of frieseomelitta varia392 billen, j. 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(1993). glandes coxales de pachycondyla obscuricornis (formicidae, ponerinae). actes colloq. insectes soc., 8: 183-186. schönitzer, k., dott, h. & melzer, r.r. 1996. the antenna cleaner gland in messor rufitarsis (hymenoptera, formicidae). tissue cell, 28: 107-113. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.471-476sociobiology 60(4): 471-476 (2013) selectivity of old and new organophosphate insecticides and behaviour of vespidae predators in coffee crop fl fernandes, pr da silva, jer gorri, lf pucci, iw da silva introduction the social wasps collect water, plant fibers and carbohydrates, and hunt arthropod prey or scavenge animal protein (edwards, 1980). social wasps are generalist foragers, but individuals are able to learn and may specialize by hunting for prey or collecting other resources at specific locations (raveret, 1990). the foraging behavior of generalist social insects, which are able to associate the presence and quality of resources with colors, odors, shapes, and features of the environment, has greatly influenced the evolution of floral characteristics (faegri & van der pijl, 1979; barth, 1985). the lepidopteran are the more attractive prey for natural predators (osborn & jaffe, 1998; marques et al., 2005). in coffee crops, many predators act as biological control agents of leucoptera coffeella (guérin-mèneville) (lepidoptera: lyonetiidae). this pest causes severe damage to coffee crops, with losses that abstract organophosphates are old agrochemicals that are toxic for wasps (vespidae) that are predators of insect pests. however, chlorantraniliprole is the first insecticide produced from the new anthranilic diamide class, which binds to ryanodine receptor modulators. this study uses chlorpyrifos, triazophos, pyridaphenthion and new chlorantraniliprole insecticides that have not yet been extensively tested for effects on non-target organisms. adults of the predatory wasps were exposed to concentrations of organophosphate inseticides and the most toxic was used for a behavior test. the trial area, located in the rio paranaíba, minas gerais, received two treatments: with insecticide and without insecticide. chlorpyrifos, the most toxic insecticide, was used in the dose of 1.5l/ ha. in order to evaluate the predation, the number with predation action (injuries made by the wasps) in the leaves were accounted. behavior activities collect of nine colonies protonectarina sylveirae, brachigastra lecheguana, polybia sp. and polistes versicolor works were quantified. all colonies were in the adult phase. the colonies were observed by 12h, from 6:00 a.m. to 6:00 p.m. the number of wasps that leaved and returned to the nest, type of sources collected (glucidic foods, prey larvae, matherial for construction of the nest). the most toxic insecticide was chlorpyrifos and the most selective was chlorantraniliprole. the species and frequencies of wasps predator were: p. sylveirae (71.50%), b. lecheguana (12.00%), polybia sp. (7.00%) and p. versicolor (1.00%). chlorpyrifos presented not selective and changes in the behavior to the predatory wasps. sociobiology an international journal on social insects universidade federal de viçosa, rio paranaíba, minas gerais, brazil research articles wasps article history edited by: gilberto m m santos, uefs, brazil received 03 april 2013 initial acceptance 11 july 2013 final acceptance 05 august 2013 keywords chlorantraniliprole, chlorpyrifos, pyridaphenthion, social wasps, toxicity, triazophos corresponding author flávio lemes fernandes universidade federal de viçosa campus de rio paranaíba rio paranaíba, mg, brazil. 38810-000 e-mail: flaviofernandes@ufv.br may reach 50% of the total production. in coffee crops, the most common wasp species are the vespidae protonectarina sylveirae (saussure), polybia sp. and protopolybia exigua (saussure) (parra et al., 1977). tuelher et al. (2003) reported 90% predation by the action of vespidae predators on l. coffeella in coffee trees. nowadays, the organophosphate insecticides dissulfotom, etiom, metil parathion and chlorpyrifos are the most used to control this pest. however, the intensive use of them has caused negative impacts to humans, the environment and predatory wasps (guedes & fragoso, 1999; santos et al., 2003; fernandes et al., 2008a). among organophosphorus, chlorpyrifos [o,o-diethyl o-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is one of the leading products due to its worldwide use in agriculture and both outside and inside the home environment. chlorantraniliprole is the first insecticide produced from the new anthranilic diamide class, which binds to ryanodine fl fernandes et al. selectivity of organophosphorate insecticides and behaviour of vespidae472 receptor modulators (whalon et al., 2008). this insecticide was released for use in the control of pests in brazil in 2009 (mapa, 2013). in spite of the importance of l. coffeella and of the potential of predatory wasps for biological control, there are few works testing the ecological and physiological selectivity of this new insecticide to these natural enimies. furthermore, there is a lack of information on the effects of this new insecticide on the behavior of social wasps. this study had two objectives: 1) to evaluate the selectivity of the older organophosphates and of the new group of insecticides chlorantraniliprole (rynaxypyr) to predatory wasps; and 2) to evaluate the effect of the insecticides on the behavior of social wasps in coffee plantations. material and methods experiment 1: study of physiological selectivity the insecticides used in the bioassays were: triazophos (hostathion 400 br, containing 400g/l-1, bayer cropscience, são paulo, sp), chlorpyrifos (lorsban 480 br, containing 480g/l-1, dow agrosciences, são paulo, sp), pyridaphenthion (ofunack 400 ec, containing 400 g/l-1, sipcam upl, uberlândia, mg) and chlorantraniliprole (altacor 350 wg, containing 350g/ l-1, du pont, barueri, sp). these insecticides are the most frequently used for l. coffeella control in brazil. bioassays were conducted in the laboratory of integrated pest management of ufvrio paranaíba in four replications in a completely randomized design using adults of b. lecheguana, p. sylveirae and protonectarina spp. leaves of coffee (coffea arabica) were immersed in insecticide solution or water (control) for ten seconds. treated leaves were dried at room temperature for 2h and were placed on the bottom of petri dishes (90 mm × 20 mm). ten wasps were transferred to each petri dish using aspirators. the dish was then covered with organza and tied up with a rubber band. in each experimental unit a honey solution (10%) was added. the petri dishes with the treated leaves and insects (experimental units) were maintained at 25 ± 0.5ºc and 75 ± 5% of relative humidity. mortality was recorded 48h after treatment and the mortality was defined as insects that were not able to fly (sena et al., 2008). the mortality data was corrected for control using the method of abbott (1925). the results were transformed to arcscene (x/100)*0.5 in order to reach the assumptions of the analysis of variance (anova) and to compare the averages by the scott-knott clustering average test at 5% of significance (scott & knott, 1974). experiment 2. toxicity and behavior the experiment was carried out in rio paranaíba, minas gerais state (coordinates: 19.21s; 46.14w). the experiment was set up in an area with 5,000 coffee plants (c. arabica), variety “catuaí amarelo” in production phase. the plants were arranged in a spacing of 3.0m between lines and 1.5m between plants. the experimental site was constituted of a coffee field with a circular area measurung 22,500m² (fig. 1). fig. 1. experimental area in a coffee system irrigated by central pivot. the letter a is the side with insecticide spraying and the letter b is the side without insecticide spraying. the dashed line shows the division between the two areas. the solid line shows the rows of plants. treatments after the experimental area was delimited, the treatments were assigned. the experimental area was divided in 2 parts, which received two treatments: area a received treatment 1 (chlorpyrifos 1.5l/ha) and area b received treatment 2 (without insecticide) (fig. 1). both areas were separated by a distance of 60 meters, in order to avoid contamination of the treatment 2 area by insecticide drift. each treatment had five replicates, each one comprised of 65 contiguous coffee plants. within each coffee plant, two leaves were marked in the mid third part in the coffee plants, summing up 130 leaves/replicate or 650 leaves/treatment. each replicate (rows with 65 plants), in both treatments, were separated by a distance of 28 meters to assure independence. in order to simulate the real effect in the field, the insecticide chlorpyrifos 480 br was used in the recommended dose of 1.5 l/ha. insecticide spraying was carried out once, in august. we used a prior compression sprayer with 5l capacity, conic beak and manometer to apply the insecticides. the presence of active leaf mines (with larvae present) was an a priori criterion to include a leaf in the experimental replicate. predatory wasps species identification the monitoring of predatory wasps was carried out on a weekly basis, during the whole period of the experiment. the collection of specimens was made in points with 20m spacing. the observations were made for 3h at the hottest periods (periods specified in table 1) when the wasps were more present. the wasp adults found in the coffee crop were collected, stored in alcohool at 70% concentration and then, they were taken to the insect taxonomy laboratory at the federal university of viçosa in order to be identified. sociobiology 60(4): 471-476 (2013) 473 table 1. occurrence and frequency of vespidae predators in coffea arabica during the trial period. months of the year time of evaluation species of wasps f* may 09:30-12:30 polybia sp. protonectarina sylveirae 05 14 june 13:00-16:00 protonectarina sylveirae 10 july 09:34-12:34 protonectarina sylveirae polistes versicolor 21 01 august 10:25-13:25 protonectarina sylveirae polybia sp. brachigastra lecheguana 25 02 12 september 12:00-15:00 protonectarina sylveirae 01 october 11:23-14:23 protonectarina sylveirae 0.5 * frequency of visits (%) chlorpyrifos toxicity on l. coffeella and vespidae predators during the whole experimental period (before and after the insecticide application), the coffee leafminer attack intensity and its predation by wasps were weekly monitored. in order to assess the chlorpyrifos toxicity on this pest and on predators action, the mines were opened with metalic blade. so, in order to evaluate the coffee leafminer attack, the numbers of active mines (alive larvae), inactive mines (dead larvae by insecticide) and mines with predation evidence (injuries made by the wasps) in the leaves were recorded. behavior of predatory wasps behavior activities collect of four colonies p. sylveirae, b. lecheguana, polybia sp. and polistes versicolor works were quantified. all colonies were in the adult phase. the colonies were observed by 12h, from 6:00 a.m. to 6:00 p.m. the number of wasps that leaved and returned of the nest, as well as the type of food sources collected (glucidic foods, prey larvae, matherial for construction of the nest) were recorded. the identification of the material collected by wasps was evaluated based on their behavior and the collected material. the wasps that collected prey and/our material for the construction of the nest were identified by flight and source collected. trophallaxis was used to identify the wasps that collected glucidic resource. the behavior was evaluated before and after insecticide application on coffee plants. statistical analysis the data on the observation of adult predator wasps were used in order to calculate the frequency of the species presence at the experimental area. in order to determine the densities of l. coffeella and of vespidae predators, the number of alive larvae and dead larvae/100 leaves and the number of ripped mines per wasps/100 leaves were calculated and graphs were made with the monthly average of the populational densities. the results were corrected in relation to the mortality obtained in the outgroup using the formula (abbott, 1925). the results were transformed to arcscene (x/100)*0.5 in order to run the analysis of variance (anova) and to compare the average by the clustering average test by scott-knott 5% significance (scott & knott, 1974). based on the number of species that returned to the nest, we calculated the eficiency index in the search for resources used in the construction of the nest, glucidic foods and prey: eficiency index (ei) = number of wasps that returned with resources/number of wasps that left the nest x 100. results and discussion physiological selectivity the organophosphate insecticides showed no selectivity in favour of predatory wasps. chlorantraniliprole showed selectivity to all species of wasps (p. sylveirae, b. lecheguana and polybia sp.) with mortalities of 10.32, 7.01 and 9.15, respectively. in contrast, the insecticide chlorpyrifos was about 8.44, 10.55 and 10.56 times more toxic to p. sylveirae, b. lecheguana and polybia sp. than chlorantraniliprole. on the other hand, insecticides triazophos and pyridaphenthion were more toxic to polibia sp. than to other species of wasps, being 7.57 and 6.32 times more toxic than chlorantraniliprole (table 2). in general, no significant differences in the toxicity of insecticides between wasp species were found, except for the insecticide pyridaphenthion, which was more toxic to p. sylveirae (50.66%) and table 2. mortality (%) of adults of protonectarina sylveirae, brachygastra lecheguana and polybia sp. treated with old and new organophosphorate insecticides. insecticides mortality (%) protonectarina sylveirae brachygastra lecheguana polybia sp. chlorpyrifos 87.12±5.10aa 74.01±13.98aa 97.36±10.05aa triazophos 55.22±6.40ba 67.00±0.14ba 78.13±15.12aa pyridaphenthion 50.66±1.65ba 35.11±1.20cb 65.19±7.79aa chlorantraniliprole 10.32±5.10ca 7.01±0.72da 9.15±5.03ba ¹averages followed by the same capital letter in a column and lower case in a line belong to the same group according to the scott-knott test at p < 0.05. fl fernandes et al. selectivity of organophosphorate insecticides and behaviour of vespidae474 polibia sp. (65.19%) than to b. lecheguana (35.11%). the bioassays were carried out under extreme conditions of insect exposure to the insecticide. however, low mortality rates are expected to be found in field conditions due to problems such as insecticide drifting, degradation and loss during application. thus, an insecticide that showed some high selectivity could be used selectively (physiological selectivity), in order to reduce the exposure predatory wasps. the selectivity of clorantraniliprole to wasps may be related to an increased rate of metabolism of the compound by natural enemies than the pest, or to changes in the target of action of insecticides against the natural enemy (yu, 1987). the clorantraniliprole has shown high selectivity for parasitoids and predators such as predatory mites (dinter et al., 2008), parasitoid wasps (preetha et al., 2009) and predatory stinkbugs (lahm et al., 2009). possible mechanisms of physiological selectivity of these insecticides are not properly clarified due to few biochemical and physiological studies that may elucidate these mechanisms. moreover, the insecticide chlorpyrifos has showed greater toxicity than other insecticides (gusmão et al., 2000; fragoso et al., 2001; bacci et al., 2006). also, the insecticide triazophos is one of the organophosphate pesticides extensively used in agricultural practices throughout the world. triazophos is a broad spectrum systemic insecticide. lipophilic compounds have higher affinity to the insect cuticle and are more easily absorbed and translocated to the site of action. this hypothesis is based on the low water solubility of the insecticide chlorpyrifos (2 ppm) and triazophos (40 ppm) (kidd & james 1991; berg et al., 2003). the low toxicity of the insecticide pyridaphenthion to the species b. lecheguana has not yet been elucidated. however, it showed low toxicity to other species, which may be due to its low solubility (100ppm) (berg et al., 2003; kidd & james 1991). predatory wasps frequencies in coffee crops insecticide during the experimental period the species and frequencies of wasp predators found were: p. sylveirae (71.50%), b. lecheguana (12.00%), polybia sp. (7.00%) and p. versicolor (1.00%) (table 2). these found species were pointed out by several authors as the most important predator species of l. coffeella (parra et al., 1977; pereira et al., 2007). the biggest frequency of p. sylveirae was reported in works of pereira et al. (2007) who pointed out this species as a key predator of coffee leafminer in this crop. this species cut the tissue in the lesion upper part taking the l. coffeella larvae off through the superior part. on the other hand, b. lecheguana e p. paulista cut the tissue through the lesion lower part. the predation behavior of b. lecheguana e p. paulista was reported by fragoso et al. (2001). therefore, the nest preservation of this species would be really important in order to keep its predation potencial in high levels. the biggest species diversity in august can be related to the density dependent relation between coffee leafminer and predator vespidae species. fernandes et al. (2008b) verified that the spatial distribution of predatory wasps, as well as the dependent density relation between these species present positive correlation. but the low frequency can be a result of possible mortalities due to the use of organophosphate insecticide. chlorpyrifos selectivity to predatory wasps we detected a significant effect of the insecticides on the predatory activity of wasp species (f = 40.65, df = 2,58, p<0.001). furthermore, there was a significant difference of predatory activity of vespidae predators (f = 33.11, df = 5,53, p<0.001) over time. the larval mortality in the treatment without insecticide was low during all the experimental period. thus, the infestation of leaf miners reached levels of 35.23% in july, 30.34% in august, 25.09% in september and 39.12% in october, an infestation level that overpasses the economic damage level -edl (fig. 2a). on the othe hand, in the treatment with inseticide, populations of l. coffeella remained bellow the edl (fig. 2a). the predation by wasps increased up to 10% in september (fig. 2b), while in the area treated with insecticide thare was a reduction of predation by wasps that reached 0.5% in october (fig. 2c). these results showed that the insecticide chlorpyrifos did not present selectivity to the predatory wasps. the findings of fragoso et al. (2001) confirmed chlorpyrifos high toxicity on l. coffeella and low selectivity to the predatory wasps b. lecheguana, p. paulista and p. exigua. gusmão et al. (2000) observed that the insecticide chlorpyrifos caused mortality of 100% in the species apoica pallens, b. lecheguana and p. versicolor. fernandes et al. (2008a) when studying the seletivity of the organophosphate dimethoate 400 ec found mortality of 88% to p. sylveirae with the use of the recommended dose. the nonselectivity of this product to the predatory wasps can be related to the consume of poisoning larvae, bigger mobility, so being more exposed to the pesticides and higher percentage of product penetration in its cuticule. the high toxicity of organophosphate to predatory wasps can be related to the good perfomance of this insecticide group activity. in penetrating in the organism, these compounds suffer reactions and become more toxic. other factor possibly related to organophosphate toxicity is the lipofilic character of some insecticides associated to the thickness and the insect cuticule lipidic composition. this relation is responsible for the product penetration in the cuticule and the translocation up to the action target (leite et al., 1998). the lipofilic compounds present more afinity with the insect cuticule and are easily absorbed and translocated up to the action site. this hypothesis is based on the low chlorpyrifos insecticide solubility sociobiology 60(4): 471-476 (2013) 475 in water (2 ppm) (kidd & james, 1991; berg et al., 2003). behavior of predatory wasps before insecticide application: collection of glucidic food was the more important activity of p. sylveirae, b. lecheguana, polybia sp. and p. versicolor colonies. the colonies of these four species had eficiency indexes (ei) of 67.10, 55.78, 45.09 and 89.77 for the collection of glucidic food, 31.08, 44.22, 25.01 and 71.31 for prey search and 1.82, 0.00, 18.97 and 35.08 for the search for material to build the nest, respectively (fig. 2). after insecticide application: p. sylveirae, b. lecheguana, polybia. sp. and p. versicolor colonies, had eficiency indexes (ei) of 35.74, 40.18, 22.09 and 86.17, respectively, for the collection of glucidic food, 1.08, 4.33, 4.87 and 70.09 for the search of prey and 0.00, 0.00, 0.00 and 28.98 for the search of material to build the nest, respectively (fig. 2). we observed a reduction of activities in all behaviors after insecticide application in two colonies of predatory wasps (table 3). however, p. versicolor was the less affected species. in general, the effect of insecticides on behavior is a syndrome that affects motility, orientation, feeding, oviposition and learning. in many cases, insecticides act as repellents that are associated to the behavior of food searching. in some cases, repellence is the result of the contact with the host plant or prey treated with insecticides (desneux et al., 2007). in the present work, we have shown that chlorpyrifos clearly is not selective and interferes with the behavioral response of predatory wasps. because chlorpyrifos largely contributes to environmental pollution, and considering its broad spectrum of action, this insecticide can have important effects on nontarget insects and thus seriously modifies the equilibrium in natural populations. in addtion, the chlorantraniliprole belongs to the chemical group of anthranilic diamides and it has selectivity to the predatory wasp p. sylveirae. table 3. predation (%) of leucoptera coffeella according to the chlorpyrifos insecticide application in coffea arabica. year month 2010 insects2 predation (%) may 3.43±0.04 a june 5.90±0.06 a july 9.86±0.20 b august 11.97±0.10 b september1 2.60±0.20 a october 0.75±0.01 c 1 month of chlorpyrifos insecticide spraying; 2 means followed by the same letter in the column are not different according to the skott-knott test, p < 0.05. acknowledgements the authors would like to thank the brazilian agencies coordenação de aperfeiçoamento de pessoal de nível superior (capes), conselho nacional de desenvolvimento científico e tecnológico (cnpq) and fundação de amparo à pesquisa do estado de minas gerais (fapemig) for funding. we also thank two anonymous referees for helpful remarks in an early version of this manuscript. references abbott, w.s. 1925. a method of computing the effectiveness of an insecticide. j. econ. entomol., 18: 265-267. bacci, l., pereira, e.j.g., fernandes, f.l., picanço, m.c., crespo, a.l.b., campos, m.r. 2006. seletividade fisiológica de inseticidas a vespas predadoras (hymenoptera: vespidae) de leucoptera coffeella (lepidoptera: lyonetiidae) bioassay, 10: 1-7. barth, f.g. 1985. insects and flowers: the biology of a partnership. princeton, nj: princeton univ. press, 297 p. berg, g.l., sine, c., meister, r.t. & poplyk, j. 2003. farm chemicals handbook. willoughby, meister, usa, 1000p. dinter, a., brugger, k., bassi, a., frost, n.m. & woodward, m.d. 2008. clorantraniliprole (dpx-e2y45, duponttm rynaxypyr®, coragen® and altacor® insecticide)a novel fig. 2. average percentage ± standard error of alive vespidae predators in a central pivot irrigated coffe system without and with (a) insecticide spraying and presence of preadatory wasps (b and c). arrows pointing to the bottom indicate the moment of chlorpyrifos spraying; 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estacional e pela altitude. acta sci, 25: 119-124. doi: 10.4025/actasciagron.v25i1.2458. whalon, m.e., mota-sanchez d. & hollingworth r.m. 2008. analysis of global pesticide resistance in arthropods. in m.e. whalon (ed), global pesticide resistance in arthropods (pp.531). wallingford, cabi. yu, s.j. 1987. biochemical defense capacity in the spined soldier bug podisus maculiventris and its lepidopterous prey, pestic. biochem. physiol., 28: 216-223. doi: 10.1016/00483575(87)90020-4. 1 molecular cloning and expression analysis of th gene in the ant polyrhachis vicina (hymenoptera: formicidae) by wen-na zhang1 & geng-si xi1* abstract tyrosine hydroxylase (th) plays an important role in the synthesis of l-dopa. in this report, a th homologue was isolated from polyrhachis vicina roger (hymenoptera: formicidae). the full –length cdna of pvth is 2429bp, which contains an open reading frame of 1713 bp. the 5’-and 3’utrs are 357 and 359 bp long, respectively. the open reading frame encodes a deduced 570 amino-acid peptide with a calculated molecular mass of 65.09 kilodaltons and an isoelectric point of 5.26. to compare pvth mrna expression during p. vicina development and the heads of different castes, real-time quantitative reverse transcription polymerase chain reaction was used. the results show that pvth mrna is differentially expressed in the ant development, in whole bodies and in the heads of different castes. during the development, the highest expression level is in pupae. the levels also vary among castes, the highest level is in workers. the investigation revel that pvth is related with the developmental and caste-specifically at the level of transcription. key words: tyrosine hydroxylase (th), molecular cloning, polyrhachis vicina roger, real-time quantitative pcr introduction as a highly conserved enzyme, tyrosine hydroxylase (th) a pterindependent monooxygenase, has been found in vertebrates and invertebrates,, and contains ferrous iron at the active site (ulrich et al. 2007). as a first and rate limiting enzyme in the synthesis of catecholamine neurotransmitters (nagatsu et al. 1964), th is primarily expressed in the brain retina secreted area, sympathetic ganglia sympathetic noradrenergic neurons, sympathetic adrenal medulla norepinephrine epinephrine and norepinephrine cells college of life sciences, shaanxi normal university, xi’an, 710062, people’s republic of china *corresponding author. email: xigengsi@snnu.edu.cn 2 sociobiolog y vol. 59, no. 3, 2012 (peng 2002). in the presence of tetrahydrobiopterin (bh4) as a cofactor, th catalyzes the oxidation of l-tyrosine to l-dopa, h 2 o and quinoid dihydrobiopterin (haavik et al. 1987). in dopa decarboxylase activation, l-dopa is decarboxylated to dopamine (hopkins et al. 1992).th also catalyzes activity of other aromatic amino acids, such as hydroxylation of l-phenylalanine and l-tryptophan (meyer et al. 1992). in vertebrates, generally, the th gene encodes a single form. however, there are two isoforms in monkey (ichikawa et al. 1990) and four in human (kaneda et al. 1987). in insects, th gene also generally encodes a single form except drosophila melanogaster and pseudaletia separate which possesses two th isoforms encoded by a signle gene and generated by rna splicing (birman et al. 1994). th plays an important role in the stress response of insects (stathakis et al. 1999). the insect immune system is dependent on both humoral and cellular responses. adaptive immunity of insects is usually expressed by humoral response. melanin and the precursors which are necessary for many physiological processes such as wound healing, pathogen encapsulation, phagocytosis are cytotoxic and antimicrobial activity (gotz et al. 1985; söderhäll, et al. 1998; kazuhiko et al. 2008). studies have demonstrated that th participates in tanning and immune response, and is expressed in the epithelial cells underlying the darkly pigmented cuticle (futahashi et al. 2005; ninomiya et al. 2007; futahashi et al. 2009). th might be involved in neural activity in the larvae (wang. 2010). th and dopamine immunoreactivity appear to localize to the same neurons (budnik et al. 1988; nässel et al. 1992; lundell et al. 1994). in drosophila, dopamine plays a role in many complex neuronal processes such as sleep and arousal, stress response, learning, visual attention, and sexual behavior (andretic et al. 2005; ganguly-fitzgerald et al. 2006; kume et al. 2005; ye et al. 2004; neckameyer et al. 2005; schwaerzel et al. 2003; chang et al. 2006). so in the larval and adult central nervous system, th-immunoreactive neurons are commonly referred to as dopaminergic neurons (matthias et al. 2008). polyrhachis vicina roger is a typical social insect with extensive polymorphism and sophisticated behavior. we cloned the full-length cdna of th from the ant p. vicina for the first time and named pvth. the expression patterns of pvth mrna at distinct development stages and at the heads of different castes were studied by using real-time quantitative reverse 3 zhang, w. & g-s. xi — analysis of th gene in p. vicina transcription-polymerase chain reaction (rt-pcr). the results indicate that pvth mrna possesses a function to regulate the ant caste-specificity and development at the level of transcription. material and methods insects polyrhachis vicina colonies were purchased from hongfa edible ant research center in guangxi province, china. the ants were raised in a chamber supplied with fruit, fish food and honeydew under standard laboratory conditions at 28°c, 40% relative humidity and natural light-dark periods. embryos, larvae, pupae, workers, male and female ants were collected from the colonies, and immersed immediately in liquid nitrogen and stored at -80°c (lu et al. 2008; ouyang et al. 2009). rna preparation and cdna synthesis total rna was extracted from pooled samples of 15 frozen workers selected randomly with rnaiso plus (takara bio inc., shiga, japan; http://www. takara-bio.com/), and then immediately reverse-transcribed for generating cdna using the first-strand cdna synthesis kit with oligo(dt) primer (fermentas life sciences, burlington ontario; http://www.fermentas.com/). all the processes followed the manufacturer’s instructions. the full length cdna of th was cloned according to the scheme shown in fig. 1. a partial cdna fragment of th was obtained using degenerated primers (ts1 tx1) (table 1) that were designed on the basis of the conserved motifs of published th from other insect species (samia cynthia ricini, papilio xuthus, manduca sexta, drosophila melanogaster, nasonia vitripennis, fig.1. cloning strateg y and map of the primers for amplifying the full sequence of the pvth gene; ts1 and tx1 for seq 1 of 739bp; ts2 and tx2 for seq 2 of 860bp; 3r1 and 3r2 for seq 3 of 710bp; 5r1 and 5r2 for seq 4 of 676bp. 4 sociobiolog y vol. 59, no. 3, 2012 apis mellifera). based on the above fragment, primers ts2 and tx2 (table 1) were designed and used to obtain the seq2 fragment. pcr products were purified from agarose gel using a gel extraction kit (qiagen, germany http:// www1.qiagen.com/) and subcloned into the pmd19-t simple vector (takara, japan). pcr positive clones were obtained for sequencing. 3’-and 5’rapid amplification of cdna ends the full length cdna pvth was amplified by specific primers used in nested pcr of 3’and 5’rapid amplification of cdna ends (race) (3r1, 3r2 for 3’-race and 5r1,5r2 for 5’-race) (table 1) using 3’full race core set and 5’full race core set (takara bio inc.), following the manufacturer’s instructions. the pcr products of 3’-race and 5’-race were cloned into pmd19-t simple vector (takara) for sequencing. structural and phylogenetic analyses of pvth the open reading frame (orf) of pvth was searched using the national center for biotechnolog y information orf finder (http:// www.ncbi. nlm.nih.gov/gorf/gorf.html). the secondary structure of the pvth protein was predicted by sopm (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat. pl?page=/npsa/npsa_sopm.html), and the tertiary structure were predicted by swiss model first approach mode (http://swissmodel.expasy. org/). signal peptide prediction was performed using the singalp program target name primer sequence 5’-3’ expected tm size (bp) (°c) th fragment ts1 caaccayctbatgacbaart 739 56 tx1 accarcgdckgaayttbtc ts2 gcyatcaagaaatcytacag 860 48 tx2 gtccagcacggtgttgaga 3’race 3r1 tcaattctcacaagaaatcggtcggtctg 710 55 3r2 gcttatggtgctggtttgttgtc 5’race 5r1 cttccgctttcgcttcttctttc 676 62 5r2 ctacccaaagaaccaataccctc real time rts agctgattaggaacctgcgacag 81 55 rtx gatctttgatgctaacggaattg β-actin y1 ccctcttccagccatcgttc 250 55 y2 ccaccgatccagacggagta “y ” is c or t; “b” is c or g or t; “r” is a or g; “d” is a or g or t; “k” is g or t. table 1. oligonucleotide primers used for cdna cloning and real-time quantitative reverse transcription-polymerase chain reaction (rt-pcr). 5 zhang, w. & g-s. xi — analysis of th gene in p. vicina (centre for biological sequence analysis; http://www.cbs.dtu.dk/services/ signalp/; bendtsen et al. 2004). sequence alignments based on the amino acid sequences of known ths were performed with clustal x1.81 ( jeanmougin et al. 1998) followed by manual inspection. phylogenetic tree were generated using mega version 4.0 from these alignments (tamura et al. 2007) based on the neighbor-joining method with bootstrap test calculated with 2000 replicates and a poisson correction model. real-time quantitative pcr the mrna expression levels of pvth at different development stages and from the heads of ants of different castes was quantified by quantitative rt-pcr. reactions were performed using a iq5® apparatus (bio-rad laboratories, inc.) with a sybr premix ex taq kit (takara bio inc.). the β-actin gene was used as the endogenous control. primers (rts, rtx) of pvth and primers (y1, y2) of β-actin were used in real-time rt-pcr (table 1). the detailed protocol was as follows: 95°c for 1 min, 40 cycles of 95°c for 10 s and 55°c for 30 s, followed by a dissociation-curve program from 55 to 95°c with a heating rate of 0.5°c every step and continuous-fluorescence acquisition. expression levels were determined basing on the formula f=10δct,t/atδct,r/ar (zhang et al. 2005). the cdna of the forth instar was selected as the calibrator in analyzing pvth expression at different development stages in this study and the heads of workers was selected as the calibrator in analyzing pvth expression in the heads of the three castes. to analyze the relative expression levels, all the counted f values were put into spss 13.0 software using a one-way analysis of variance (anova) with dunnett’s multiple comparison (spss inc. 2004). results cloning and characterization of pvth cdna the full–length cdna of pvth, 2429bp, was obtained using rt-pcr and race. it contains a orf of 1713 bp that encodes a 570-amino-acid protein. the full-length cdna sequences are shown in fig.2. the 5’-and 3’ utrss are 357 and 359 bp long, respectively. a putative polyadenylation signal aataaa is found 14 bp upstream form the 25-nucleotide poly (a) tail, which coincided with the fact that the polyadenylation signal is most 6 sociobiolog y vol. 59, no. 3, 2012 7 zhang, w. & g-s. xi — analysis of th gene in p. vicina often present 11-30 nucleotides upstream from the poly (a) tail (fitzgerald et al. 1981). the deduced pvth protein consists of 570 amino-acid residues with a calculated molecular mass of 65.09 kilodaltons and an isoelectric point (pi) of 5.26. the genbank accession number is jf499654. analysis with the signalp program showed that there was no n-terminal signal sequence in this protein. it may suggest that pvth may be an unglycosylated protein. using prosite protein, a series of predicted function motifs were found in the pvth protein (table 2). alignment analysis and phylogenetic-tree construction the predicted secondary structure of the pvth shows that alpha helix is 55.44%, extended strand is 10.53%, beta turn is 6.32% and random coil is 27.22% (fig.3). the result of the tertiary structure prediction from the swiss model first apprpach shows that the protein model is 2xsnd (human tyrosine hydroxylase); catalytic domain; moedlled residue range, 231-568; sequence identify, 57.69%; x-ray resolution, 2.68; e value, 0.00e-1. a multiple alignment of the deduced animo acid sequence of pvth with other known th homologues was performed with clustalx 1.81 (fig. 5). megalign analysis of the pvth protein sequence with other th protein sequences revealed that the pvth protein shares 99% identity with a.mellifera. a phylogenetic tree was constructed using mega based on the neighbor-joining method (tamura et al. 2007; fig. 6). result show that pvth clustered with the ths of other hymenoptera and is most closely related to that of a.mellifera. fig.2 (facing page). the nucleotide and deduced amino acid sequence of pvth. the sequence is 2429bp long and encodes a protein of 570 amino acid residues. the initiating codon atg and stop codon taa are underlined and shaded; the olyadenylation signal aataaa is underlined. a series of important functional motifs in the pvth protein sequence are marked. two n-glycosylation sites at 60-63 (ndtn), 297-300 (netw); two campand cgmp-dependent protein kinase phosphorylation sites at 29-32 (rrrs), 348-351 (krnt); seven protein kinase c phosphorylation sites at 163-165 (skk), 228-230 (sik), 334-336 (shr), 354-356 (tlr), 363-365 (tar), 472-474 (sdk), and 531533 (tqr); eighteen casein kinase ii phosphorylation sites at 32-35 (slvd), 50-53 (svle), 62-65 (tnad), 85-88 (ssdd), 109-112 (sdsd), 125-128 (teee), 134-137 (tiae), 176-179 (thve), 183-186 (skke), 214-217 (sald), 228-231 (sikd), 239-242 (sdld), 249-252 (tkye), 293-296 (tete), 306-309 (tvld), 363-366 (tard), 427-430 (sdee), and 504-507 (sfed); three tyrosine kinase phosphorylation sites at 448-456 (kegvevkay), 489-495 (kyqdqey), and 521-527 (rpfevry); two n-myristoylation sites at 360-365 (glltar), 459-464 (gllsay); one biopterindependent aromatic amino acid hydroxylases signature at 394-405 (pdcihellghmp). 8 sociobiolog y vol. 59, no. 3, 2012 table 2. predicted function motifs in the pvth protein of polyrhachis vicina. domain name canonical sequence position in sequence n-glycosylation site n[^p][st][^p] 60-63(ndtn), 297-300(netw) campand cgmp-dependent [rk]{2}.[st] 29-32(rrrs), 348-351(krnt) protein kinase phosphorylation site protein kinase c phosphorylation site [st].[rk] 163-165(skk), 228-230 (sik), 334-336(shr), 354-356(tlr), 363-365(tar), 472-474(sdk), 531-533(tqr) casein kinase ii phosphorylation site [st].{2}[de] 32-35(slvd), 50-53(svle), 62-65(tnad), 85-88(ssdd), 109-112(sdsd), 125-128(teee), 134-137(tiae), 176-179(thve), 183-186(skke), 214-217(sald), 228-231(sikd), 239-242(sdld), 249-252(tkye), 293-296(tete), 306-309(tvld), 363-366(tard), 427-430(sdee), 504-507(sfed) tyrosine kinase phosphorylation site [rk].{2,3}[de].{2,3}y 448-456(kegvevkay), 489-495(kyqdqey), 521-527(rpfevry) n-myristoylation site g[^edrkhpfyw].{2} 360-365(glltar), [stagcn][^p 459-464(gllsay) fig.3. predicted secondary structure of the pvth protein, h is alpha helix; e is extended strand; t is beta turn; c is random coil. 9 zhang, w. & g-s. xi — analysis of th gene in p. vicina analysis of pvth mrna expression levels of expression of pvth mrna at different developmental stages and in different castes were detected by means of real-time quantitative rt-pcr. pvth relative expression levels were calculated using the formula f=10δct,t/ at-δct,r/ar for each replicate (table 3). pvth was expressed in all templates intable 3. pvth expression levels calculated for three replicates based on the formula f=10δct,t/at-δct,r/ar . th c t β-actin c t δct,t δct,r f=10δct,t/at-δct,r/ar embryo 21.65 18.65 0 0 1 21.48 18.44 0 0 1 21.73 18.11 0 0 1 first instar 17.24 14.80 -4.38 -3.60 1.82 16.69 14.49 -4.93 -3.91 2.16 16.90 14.68 -4.72 -3.72 2.12 second instar 22.45 20.07 0.83 1.67 1.74 22.61 19.02 0.99 0.62 0.77 22.15 19.82 0.53 1.42 1.81 third instar 22.37 21.28 0.75 2.88 4.17 22.86 20.73 1.24 2.33 2.05 22.60 20.92 0.98 2.52 2.79 fourth instar 19.52 14.65 -2.10 -3.75 0.34 19.37 14.36 -2.25 -4.04 0.31 19.73 14.22 -1.89 -4.18 0.22 pupa 21.99 21.79 0.37 3.39 7.66 21.89 21.73 0.27 3.33 7.88 22.06 21.58 0.44 3.18 6.33 worker 20.83 21.30 -0.79 2.90 10.02 20.69 21.28 -0.93 2.88 13.53 20.31 21.00 -1.31 2.60 17.36 female 22.90 17.39 1.28 -1.01 0.21 23.64 17.43 2.02 -0.97 0.13 24.48 17.25 2.86 -1.15 0.06 male 21.75 21.63 0.13 3.23 8.11 22.35 21.88 0.73 3.48 6.34 22.23 21.67 0.61 3.27 5.98 head worker 27.17 26.30 0 0 1 26.68 26.26 0 0 1 26.59 26.40 0 0 1 female 29.07 31.02 2.26 4.70 4.85 29.35 31.77 2.54 5.45 6.58 29.03 31.58 2.22 5.26 7.19 male 23.23 25.96 -3.58 -0.36 8.50 23.28 25.91 -3.53 -0.41 7.96 23.19 25.66 -3.62 -0.66 7.17 10 sociobiolog y vol. 59, no. 3, 2012 vestigated at different levels (figs.7, 8). during development, the level of expression of pvth transcripts was low in embryo and each instar, especially in fourth instar larvae. however, the level of expression greatly increased in pupae. in adults, the gene expression was highest in workers and lowest in females. in the heads of different castes, the expression was highest in the heads of males and lowest in the heads of workers. note: the three relicate samples yielded three c t values. δct,t is the difference in c t values for pvth between the samples and the calibrator. δct,r is in c t values for β-actin. in the analysis of development, the f value for embryos is designated 1 and the slope of the pvth curve and the β-actin curve was -3.325 and -3.406. in the analysis of the head, the f value for worker heads is designed 1 and the slope of the pvth curve and theβ-actin curve was -3.47 and -3.515 discussion a full-length cdna sequence of th from p.vicina was isolated and characterized in this study. the results of multiple aligments showed that pvth reflects a high degree of homolog y with the th genes identified from other animals. phylogenetic tree which shows the evolutionary relationship of pvth with other members of the th family constructed on the basis of alignment of the amino acid sequences of th homologues. numbers at branch nodes indicate percent bootstrap confidence values derived from 2000 replications. results showed that pvth clustered with the ths of other species and is most closely related to that of a.mellifera. it is consistent with the species' evolution. real-time quantitative rt-pcr indicated that pvth mrna is expressed at all stages of p.vicina development and the heads of different castes, but at different levels. in embryos and larvae, the expression was very low, espefig.4. predicted tertiary structure of catalytic domain in the pvth proteins by swissmodel first approach. 11 zhang, w. & g-s. xi — analysis of th gene in p. vicina fig.5. amino acid sequence alignments of pvth (polyrhachis vicinna (pvi)) with other species: apis mellifera (ame) np_001011633; tribolium castaneum (tca) np_001092299; mythimna separata (mse) baf32573; bombyx mori (bmo) np_001138794; manduca sexta(mas) abq95973; papilio xuthus (pxu) bae43824; papilio polytes (ppo) baj07593; papilio machaon (pma) baj07587; samia cynthia ricini (scr) baf64534; plutella xylostella (pxy) adk12633; drosophila melanogaster (dme) np_476897; homo sapiens(hsa) aai43615; xenopus laevis(xla) np_001091392. conserved amino acids in all ths are shown in black and residues that are similar with respect to side chains in gray; gaps are introduced to optimize the alignments. 12 sociobiolog y vol. 59, no. 3, 2012 cially in fourth larval stages. in pupa, the expression reached very high levels. it is widely known that th is the first, and rate limiting, enzyme in the synthesis of dopamine. dopamine, which is necessary in development and melanin synthesis in insects, is an important neurotransmitter of the central nervous system and also a material to resist pressure (goldstein et al. 1992; kobayashi et al. 1995; restifo et al. 1990; monastirioti. 1999; hopkins et al. 1992; stathakis et al. 1999). tissues, including nervous tissue, are gradually developed from the embryonic stage to the fourth instar stage. the low expression may reflect incomplete development of nervous tissue. in complete fig.6. phylogenetic tree which shows the evolutionary relationship of pvth with other members of the th family constructed on the basis of alignment of the animo acid sequences of th homologues. numbers at branch nodes indicate percent bootstrap confidence values derived from 2000 replications. see figure 5 for the explanation of species abbreviations 13 zhang, w. & g-s. xi — analysis of th gene in p. vicina metamorphosis, the central nervous system is formed in the pupa period (truman 1996). in adults, pvth mrna expression is high except in females. the obvious differences in different castes may reflect the different functions in social insects. as the females of the colony are important, they are more well-protected. in the brain, the lowest level was found in workers. the expression is high in females and males. it may play a role in sexual behavior (chang et al. 2006). the highest level expression in bodies is different than in brains. the result may be related to the complex fig.7. relative expression profiles (± standard error of the mean; n=3) for pvth in different developmental stages of p.vicina based on real-time pcr. all expression levels are shown relative to the expression level in embryos. means followed by the same letter are not significantly different (p<0.05). fig.8. relative expression profiles (± standard error of the mean; n=3) for pvth in the heads of different castes. all expression levels are shown relative to the expression level in the heads of workers. means followed by the same letter are not significantly different (p<0.05). 14 sociobiolog y vol. 59, no. 3, 2012 physiological function of 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tenebrio molitor (insecta coleoptera) ye y., w. xi, y.peng, y.wang & a. guo 2004. long-term but not short-term blockade of dopamine release in drosophila impairs orientation during flight in a visual attention paradigm. european journal of neuroscience, 20:1001–1007. zhang, c.y., s.g. xu &x.x. huang 2005. a novel and convenient relative quantitative method of fluorescence real time rt-pcr assay based on slope of standard curve. progress in biochemistry and biophysiolog y, 32:883-888. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.401-406sociobiology 61(4): 401-406 (december 2014) social information in the stingless bee, trigona corvina cockerell (hymenoptera: apidae): the use of visual and olfactory cues at the food site introduction when foragers of eusocial insects approach new food sources, their decision to land can be influenced by the presence or absence of visual or chemical information provided by other individuals (slaa et al., 2003; danchin et al., 2004; leadbeater & chittka, 2007; yokoi & fujisaki, 2011). for example, foragers can either be attracted (local enhancement) or repelled (local inhibition) by the presence of conspecifics (von frisch, 1914; d’adamo et al., 2000; slaa et al., 2003). social information in general is any information about behavior, physical presence or remnant provided by a sender that reduces uncertainty of a receiver. the information can be produced on purpose or inadvertently and may be of different modalities, as for example visual, olfactory or tactile (danchin et al., 2004; dall et al., 2005; kendal et al., 2005; gruter & leadbeater, 2014). such publicly accessible information provided by conspecific or heterospecific individuals allows the observer to adaptively change its behavior to gain fitness benefits, such as assessing abstract for social insects, colony performance is largely dependent on the quantity and quality of food intake and thus on the efficiency of its foragers. in addition to innate preferences and previous experience, foragers can use social information to decide when and where to forage. in some stingless bee (meliponini) species, individual foraging decisions are shown to be influenced by the presence of social information at resource sites. in dual choice tests, we studied whether visual and/or olfactory cues affect individual decision-making in trigona corvina cockerell and if this information is species-specific. we found that t. corvina foragers possess local enhancement: they are attracted by olfactory and visual cues released by conspecifics but avoid feeders associated with heterospecific individuals of the species tetragona ziegleri (friese). overall, olfactory cues seem to be more important than visual cues, but information by visual cues alone is sufficient for discrimination. sociobiology an international journal on social insects fmj sommerlandt1, w huber2, j spaethe1 article history edited by tom wenseleers, katholieke universiteit leuven belgium received 14 september 2014 initial acceptance 02 november 2014 final acceptance 01 december 2014 keywords social information, communication, odor marks, visual cues, recruitment. corresponding author frank m. j. sommerlandt department of behavioral physiology and sociobiology, biozentrum university of würzburg, am hubland, 97074 würzburg, germany e-mail: frank.sommerlandt@uni-wuerzburg.de the location and quality of a food source (chittka & leadbeater, 2005; goodale et al., 2010, yokoi & fujisaki, 2011). in bees, olfactory information at the food source can be provided by foragers through anal droplets, gland secretions or footprints originating from glandular epithelia of the claw retractor tendon (nieh et al., 2003; jarau et al., 2004; barth et al., 2008; wilms & eltz, 2008; jarau, 2009). in bumblebees and honeybees, for example, scent marks can be either short-lived and repellent, and used to avoid visitation of a recently depleted nectar source, or longer-lasting and attracting, and allows identification of particularly rewarding flowers (stout & goulson, 2001). the response to such marks depends also on the bee’s previous experience (hrncir et al., 2009; saleh & chittka, 2006) and can be modified through learning (reviewed in gruter & leadbeater, 2014). moreover, chemical information is also provided by the profile of epicuticular hydrocarbons, which is speciesand colony-specific (howard & blomquist, 1982; nunes et al., 2011). another potential modality depicts the physical presence of conspecifics or heterospecifics that serves as a visual stimulus. research article bees 1 university of würzburg, würzburg, germany 2 university vienna, vienna, austria fmj sommerlandt, w huber, j spaethe social information in trigona corvina402 in social wasps, for example, visual information about resident heterospecific wasps at the feeder site can appear attractive or repellent to foraging individuals, depending on characteristics of the involved species (size, aggressiveness, etc.; raveret richter & tisch, 1999). the use of conspecific and heterospecific visual information for decision-making in foragers is also reported for bumblebees (stout & goulson, 2001; dawson & chittka, 2012), which can learn to use this information to find profitable food sources (dawson & chittka, 2012). under natural conditions, signals and cues from different modalities are used to gain social information about the food source. findings in the stingless bee scaptotrigona mexicana (guérin) indicate a redundant function of visual and chemical cues in the bee’s recruitment system at the food site (sánchez et al., 2011). in general, in stingless bees the response to the presence or absence of nestmates or heterospecifics at the food site is usually species specific (slaa et al., 2003). the stingless bee trigona corvina cockerell plays an important role in plant pollination in the neotropics, since pollen from more than 70 different plant species has been found in a single colony (roubik & moreno patiño, 2009). known to be an aggressive mass-recruiter (roubik, 1981), the fitness of a t. corvina colony relies on an efficient recruitment system, where nestmates are guided to profitable food sources mainly by field-based mechanisms including pheromone trails and deposition of scent marks near the food source (aguilar et al., 2005; jarau et al., 2010). t. corvina foragers are known to be attracted to the food source by scent marks deposited by themselves (during previous visits), by nestmates or by conspecific non-nestmates, but no evidence was found for the use of heterospecific odor marks (boogert et al., 2006). whether t. corvina foragers also rely on visual cues provided by the presence of conspecifics and if there is a cross-effect (e.g. additive, redundant or hierarchical) of visual and chemical cues that affect the forager’s decision at the food site, needs to be investigated. here we aim to evaluate the significance of chemical and visual cues on the choice behavior of t. corvina foragers. we tested the significance of the presence of conspecific or heterospecific foragers, the effect of epicuticular hydrocarbons and the impact of conspecific forager-deposited odor marks. in particular, we addressed the following questions: i) do foragers of t. corvina possess local enhancement or local inhibition when making decisions at food sites? ii) which sensory modality (olfaction or vision) is used for decision-making? iii) can foragers use visual cues to discriminate between conspecifics and heterospecifics? material and methods experiments were performed in march 2013 (experiments 1, 5, 6) and march 2014 (experiments 1, 2, 3, 4, 5) at the tropical research station la gamba, golfito, costa rica (www.univie.ac.at/ lagamba/). workers of the stingless bee t. corvina were trained to a gravity feeder (spaethe et al., 2014) providing 0.1 m sucrose solution. a small sub-group of bees (up to 10-15 individuals) was then trained from the central feeder to a second feeder that was placed approx. 8-10 m away and that provided 0.3 m sucrose solution. this training procedure allowed us to work with a constant number of foragers during the experiments. the second feeder was made of a piece of foam material (85 mm x 85 mm x 10 mm) with a central hole (15 mm in diameter) covered with filter paper (fig. 1a). a snap-on cap with sucrose solution (or water during the test) was placed into the hole. this setup allowed a (olfactory) marking of the filter paper by visiting bees (see fig. 1b; comparable to sánchez et al., 2011). the experimental procedure consisted of two phases: a training phase and a test phase. during the training phase, the above mentioned second feeder (hereinafter referred to as training feeder) provided a sugar reward, and the bees were allowed to forage for 10 minutes to establish the food source. the first training phase of each day was prolonged to 60 minutes to obtain a stable number of foraging bees. afterwards, during the test phase, a choice test was performed where the bees had to choose between two new, unrewarded feeders (comparable to boogert et al., 2006) that provided different olfactory and/or visual cues according to the type of experiment (see results for the tested combinations). these feeders, consequently termed test feeders, were placed at 30 cm distance between each other. each bee was individually tested and immediately captured after landing on one of the test feeders to avoid multiple counting. after the decisions of six bees, the test feeders were removed and replaced by a fresh rewarding training feeder to start a new experiment. following a 10 minute training phase, a new randomly chosen combination of test feeders was presented and the next six bees were tested. during all experiments, the feeder positions were randomized to avoid position effects. we used different types of feeders: to test visual cues, we attached three hexane-washed conspecific individuals (fig. 1a) or individuals from the related species tetragona ziegleri (friese). t. ziegleri was chosen since it is of similar size as t. corvina (about 6 mm body length), but of different color (orange). thus, both species should be detectable by t. corvina foragers at a similar distance, but provide different chromatic information. the hexane-bath was applied for 24 hours and hexane was regularly exchanged to remove all species-specific odors. before the dummies were used in an experiment, they were sundried for 60 minutes. to assess the impact of epicuticular hydrocarbons, we attached three freshly killed (by freezing) bees. we used olfactory marked feeders with filter papers that were odormarked by foragers in previous visits to test for the relevance of odor markings of the feeder. the filter papers were marked by 10-15 foragers which completed multiple foraging bouts during the 10 minute training phases (see below). although we never observed active pheromone deposition by the bees through abdominal droplets, we cannot completely exclude pheromone marking. we chose randomly among a couple of marked filter papers for the ones that were used in the tests. overall, not more than 30 minutes passed between deposition of odor marks and the use in the experiments. for the control experiment, we used a new and empty control feeder covered with clean filter paper. sociobiology 61(4): 401-406 (december 2014) 403 experiments were performed in a randomized order to avoid temporal effects and with inter-test intervals of 10 minutes to obtain stable numbers of recruited foragers at the feeders. this time period corresponded to approximately four foraging flights per individual. we assume all tested bees belonged to a single colony, since no inter-individual aggression behavior was observed at the feeders and t. corvina foragers are known to defend food sources against competitors (roubik, 1981). all captured bees were released at the end of a day. as a consequence, we cannot exclude that some bees were counted multiple times during the following days. fig 1. training and test feeder. a, feeder with pinned bee dummies. b, bees feeding on a training feeder which allowed for the deposition of odor marks on the filter paper. fp, filter paper; sc, snap-on cap. results altogether, we counted 450 decisions in 6 different feeder combinations (fig. 2). to test if foragers of t. corvina possess local enhancement or local inhibition at the feeder site, bees had to choose between a feeder with three freshly killed conspecifics and a clean feeder (experiment 1). the majority of bees (83%) preferred the feeder with the (freshly killed) conspecifics compared to the plain feeder (p<0.001, chi²=24.000). to evaluate if odor cues, e.g. epicuticular hydrocarbons play a role in the recognition of nestmates at the feeding site, or if the visual presence of conspecifics is sufficient for the effect of local enhancement, we confronted the foraging bees with one feeder surrounded by three freshly killed conspecifics and a second feeder surrounded by the same number of hexane-washed dead conspecifics (experiment 2). significantly more foragers landed on the feeder occupied by freshly killed conspecifics (68%) compared to the feeder with odorless dummies (32%; p=0.034, chi²=4.513). in experiment 3, we tested if t. corvina foragers can distinguish between conspecifics and heterospecifics (both freshly killed). in this test, conspecifics were more attractive (70%) than heterospecifics of the fig 2. mean proportion of choices made by trigona corvina foragers towards each of the two feeders offered in each experiment. for each test combination, p-values are shown above the bars. for statistics see text. numbers in brackets indicate number of choices, dashed line indicates random choice level. the proportion of choices for all observed landings was calculated for each of the feeder types and shown as bar charts. to calculate the statistics, we used the observed number of landings on either feeder and compared it with the null hypothesis, which is a random choice (1:1 distribution on the two test feeders), using chi-square test. statistics were performed with ibm® spss® statistics software (version 20). species t. ziegleri (p=0.021, chi²=5.328). we then eliminated the epicuticular hydrocarbons by washing the dummies of both species with hexane, with the result that the tested foragers could only use visual cues to discriminate between conspecifics and heterospecifics (experiment 4). both species were similar in body size (approx. 6 mm body length) but differed in their chromatic appearance with t. corvina being almost entirely black and t. ziegleri possessing a mainly orange-colored body (jarau et al., 2009). foragers significantly preferred conspecifics over heterospecifics. 68% of the tested bees decided to land on the feeder occupied by three hexane-washed t. corvina, compared to 32% of the foragers that landed in the vicinity of t. ziegleri (p=0.034, chi²=4.513). we then tested the impact of conspecific odor marks (on filter paper) on recruited foragers (experiment 5). odor marks alone were significantly more attractive (72%) than clean feeders (p=0.002, chi²= 9.649). in experiment 6, we presented two attractive cues in a competition experiment: one feeder hosting hexane-washed conspecifics, whereas the second feeder provided a filter paper containing odor marks released by conspecifics. foragers possessed a preference towards the odor marks (76%), compared to the visual presence of conspecifics (p=0.032, chi²=4.593). to test for side-specific preferences of the bees, we also presented two clean feeders and found that bees showed no side-preference but chose randomly between both feeders (p=0.300, chi²=1.086, n=30; data not shown). discussion foragers of t. corvina use social information when deciding to land on an established food source and make use of olfactory and visual information. in accordance with findings by boogert et al. (2006), t. corvina foragers possess local enhancement, i.e. newly arriving foragers prefer fmj sommerlandt, w huber, j spaethe social information in trigona corvina404 to land close to other conspecifics (a possible distinction between local enhancement and stimulus enhancement and its relevance is reviewed in heyes, 1994 and was recently discussed in avarguès-weber & chittka, 2014). our species is described as relatively aggressive (johnson & hubbel, 1974; roubik, 1981); foragers often arrive at resources in groups and defend their foraging territories (roubik, 1981). following nestmates to a profitable food source could ensure the monopolization of the food source. t. corvina can make use of visual and chemical cues when orienting towards a food source that bears species-specific information, but odor marks seem to be more attractive than the visual presence of a nestmate, as already reported for another stingless bee species, s. mexicana (sánchez et al., 2011). as previously shown for olfactory cues (boogert et al., 2006), t. corvina foragers can discriminate conspecifics from heterospecifics by their visual appearance alone. in our experiments, feeders surrounded by freshly killed conspecifics – providing the visual information of the presence of the bees combined with the species specific profile of epicuticular hydrocarbons – were preferred by the recruited bees over clean feeders and hexane-washed conspecifics or heterospecifics (experiments 1-3). thus, the information of cuticular hydrocarbon profiles to recognize nestmates, as shown for several other stingless bee species (buchwald & breed, 2005; nunes et al., 2008; ferreira-caliman et al., 2010), plays a role in the recruitment behavior at the food site, as well. interestingly, conspecifics were also recognized and preferred over heterospecifics when the cuticular hydrocarbon profile and other species-specific odors were absent (due to hexane-washing), indicating that visual information alone is sufficient to identify and discriminate conspecifics from heterospecifics (experiment 4). both species used in our experiment were of approximately the same size (about 6 mm body length; jarau et al., 2009) but differed in color, with t. corvina being almost entirely black and t. ziegleri possessing a mainly orange-colored body. thus, aside from flower recognition (spaethe et al., 2014), color vision may also play an important role in species discrimination at resource sites in stingless bees. our results concur with findings in other species of the genus trigona (villa & weiss, 1990; spaethe et al., 2014), whereby the relevance of visual cues for decision-making at the food site was reported and the impact of visual cues for individual recognition could have been demonstrated for social wasps (raveret richter & tisch, 1999; sheehan & tibbetts, 2011). whether t. corvina foragers can identify heterospecifics that differ from conspecifics in size but not in color, needs to be investigated. when confronted with competing information (experiment 6), olfactory marks (on the filter paper) were preferred over visual cues of hexane-washed bees. we assume the olfactory marks in our experiment to be footprints secreted from the leg tips, as footprints were reported as a type of scent marks at the food source for other stingless bees (melipona seminigra friese: hrncir et al., 2004, melipona scutellaris latreille: hrncir et al., 2009). additionally, we did not observe any deposition of abdominal droplets. the efficacy of scent marks at the food source (experiment 5) was also shown in t. corvina (boogert et al., 2006) and other stingless bee species (sánchez et al., 2011) and emphasizes the importance of olfaction as the major sensory modality. this could be caused by either a bias in sensitivity for visual and olfactory stimuli, a difference in the stimuli’s action range or a true (innate) preference of the bees for scent marks over visual stimuli. except for the non-volatile longchained epicuticular hydrocarbons, odor marks can act over larger distances than visual cues. estimates of spatial visual resolution in the stingless bee tetragonisca angustula (latreille) which with 5-6 mm body length is about the size as t. corvina, suggest that objects of the size of conspecifics should be visually detectable at relatively short distances of approx. 10 to 20 cm (zeil & wittmann, 1993). in contrast, forager-deposited odor marks can be sensed by conspecific stingless bees over long distances up to 10 to 20 m (melipona panamica cockerell: nieh, 1998; scaptotrigona aff. depilis (moure): schmidt et al., 2003). here we report that foragers of the stingless bee t. corvina possess local enhancement at the food site and that they use visual cues in addition to, or in the absence of, olfactory marks, to identify conspecifics. whether these bees can also visually discriminate more similar heterospecifics, such as the similar-sized and colored trigona fuscipennis friese, needs to be investigated. acknowledgements we thank f. bötzl, j. kriegbaum, d. materna and a. weiglein for help with data collection, s. jarau for species identification, and the tropical research station la gamba (www.univie.ac.at/lagamba/), costa rica, for making available their laboratory facilities and tropical garden. the costa rican ministerio de ambiente y energía kindly granted research permits. we further thank two annonymous reviewers for their thoughtful comments on an earlier draft of this manuscript and j. plant for linguistic advice. this work was supported by a phd research scholarship offered by the free state of bavaria (elitenetzwerk bayern) to fmjs. references aguilar, i., fonseca, a. & biesmeijer, j. c. 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(1993). landmark orientation during the approach to the nest in the stingless bee trigona (tetragonisca) angustula (apidae, meliponinae). insectes soc., 40: 381-389 1 colony structure of the weaver ant, oecophylla smaragdina (fabricius) (hymenoptera: formicidae) by marcela, p. ª, abu hassan, a.ª, nurita, a.t.ª & kumara thevanb abstract the colony structure of oecophylla smaragdina within the compound of universiti sains malaysia, penang island, malaysia was determined. the current study involved a total of twelve nests which were taken from two different locations. each nest was dissected and the specimens were separated according to their caste. results show that the colony structure of o. smaragdina consisted of a dealate queen, eggs, larvae of female alates, pupae of major and minor workers, pupae of female alates, major workers, minor workers, female alates and male alates. our present study shows that o. smaragdina had a distinct caste system and the numbers of individuals in each caste reflect their respective functions which contributed to the success of their colony. keywords: oecophylla smaragdina; nests; caste; colony structure introduction weaver ants, or oecophylla, are eusocial insects of the family formicidae (order: hymenoptera). they are classified under the subfamily formicinae. weaver ants are obligate arboreal species and are known for their unique nest building behavior where workers construct nests by weaving together leaves using larval silk (hölldobler & wilson 1990). the asian weaver ant, o. smaragdina is well distributed throughout most of the oriental region from india to queensland and the solomon island (greenslade 1972). they are also common in the lowland of peninsular malaysia and have become an important ant in the tree canopies of the humid tropics of southeast malaysia, australia, and pacific islands ( jander & jander 1979; bluthgen & fiedler 2002). previous studies reported the existence of single or multiple queens in the colonies of o. smaragdina (greenslade 1971; peeters & andersen 1989; ªschool of biological sciences, universiti sains malaysia, 11800 penang, malaysia. bdepartment of forensic medicine, penang hospital, 10990 residensi road, penang, malaysia. corresponding author email: aahassan@usm.my 2 sociobiolog y vol. 59, no. 1, 2012 hölldobler & wilson 1983; 1990). however, little was known about the colony composition and caste system such as the existence of reproductive individuals and non-reproductive individuals of these weaver ants. despite being well-studied for other biological attributes, the colony structure of o. smaragdina has received less attention (schlüns et al. 2009). studies on colony structure and caste composition might be able to explain how the social structure inside the nest influence the behaviour of individuals outside the nest. therefore, the objective of the current study was to report the colony structure of o. smaragdina inside the nests of this species. materials and methods this study was conducted in november 2010 until february 2011 at the main campus of universiti sains malaysia, penang island, malaysia. two colonies of weaver ants, located 500 m from each other, were chosen for this study. the boundary of each colony was determined by following the ant trails. the first colony was located behind the “kelas al-quran dan fardu ain” building (kafa) and the second colony was located near the building of the school of industrial technolog y (ti). the kafa colony made nests on five mango trees (mangifera indica) whereas the ti colony made nests in a “mempari” tree (millettia pinnata). total nests sampled were twelve, where eight nests taken from the kafa colony and four nests from the ti colony. these nests were removed from the trees by cutting the base of the nest using a long cutter (sharpex) and placing it into plastic buckets (60 cm x 80 cm) containing cotton wads wetted with chloroform. all nests were collected in between 10 a.m. to 12 p.m., corresponding to the time of day when the weaver ants were least active which was determined in a preliminary study. each nest was kept separately in individual plastic buckets. these plastic buckets were brought back to the laboratory and kept inside a fume hood for 24 hours to ensure that the weaver ants were all dead. after 24 hours, the nests of o. smaragdina ants were dissected and sorted according to the following castes: dealate queens, egg, larvae, pupae, female alates, male alates, major workers and minor workers. the number of individuals of each caste for each nest were counted and recorded. the collected specimens were preserved in 95 % ethanol. the images of each specimen of o. smaragdina were 3 marcela, p. et al. — colony structure of oecophylla smaragdina captured and s measurements were determined using a stereomicroscope (olympus szx9) which was connected to image analyser cell a (dell inc. computer). the mean number and standard error of mean (s.e.m) for each caste was determined using statistical packages for the social sciences (spss 11.5). results table 1 shows the caste composition of o. smaragdina from the twelve nests sampled. one dealate queen was found in the kafa colony (fig. 1). the dealate queen of o. smaragdina found in the field was green to brown in color. in contrast, the male alates of o. smaragdina were black in color and smaller in size (fig. 2). the female alates of the kafa colony were green in color with unshed wings (fig. 3). however, the female alates of the ti colony were yellow in color with unshed wings (fig. 4). the major workers were bigger in size compared to minor workers (fig. 5; fig. 6). table 1 also shows that the highest number in any individual caste was male alates in the kafa colony (1384.38 ± 367.57), however no male alates were observed in the ti colony. in addition, the number of major workers was higher than the minor workers for both colonies (kafa= 553.38 ± 123.82; 441.88 ± 79.23) (ti= 390.00 ± 51.15; 193.25 ± 19.64). both eggs and larvae were white in color and similar in ta bl e 1. c as te c om po si tio n of o . s m ar ag di na fo r e ac h k a fa a nd t i c ol on y (m ea n ± s .e .m /n es t) . c ol on y c as te sy st em d ea la te q ue en eg gs la rv ae of fe m al e al at es pu pa e of w or ke rs pu pa e of fe m al e a la te s m aj or w or ke rs m in or w or ke rs fe m al e al at es m al e al at es k a fa m ea n ± s. e .m /n es t n= 8 ne st s 1 23 3. 38 ± 58 .7 9 35 .8 8 ± 1 0. 21 63 3. 50 ± 2 04 .8 5 83 .0 0 ± 2 0. 47 55 3. 38 ± 1 23 .8 2 44 1. 88 ± 7 9. 23 80 .0 0 ± 2 6. 09 13 84 .3 8 ± 3 67 .5 7 t i m ea n ± s. e .m /n es t n= 4 ne st s 0 8 4 .0 0 ± 25 .6 9 10 .7 5 ± 5 .6 5 91 .2 5 ± 3 2. 87 44 .7 5 ± 1 6. 91 39 0. 00 ± 5 1. 15 19 3. 25 ± 19 .6 4 11 .0 0 ± 5 .0 8 0 4 sociobiolog y vol. 59, no. 1, 2012 1 2 4 3 `` 4 5 5 6 figs. 1-6: fig. 1. dealate queen of o. smaragdina from kafa colony; fig. 2. male alate of o. smaragdina from kafa colony; fig. 3. female alate of o. smaragdina from kafa colony; fig. 4. female alate of o. smaragdina from ti colony; fig. 5. major worker of o. smaragdina from both colonies; fig. 6. minor worker of o. smaragdina from both colonies. shape but the larvae were bigger in size (fig. 7; fig. 8; fig. 9). the pupae had developed eyes, mouth, legs and wings that were very similar to the adults (fig. 10; fig. 11; fig. 12). 5 marcela, p. et al. — colony structure of oecophylla smaragdina discussion the current study showed the basic colony structure of o. smaragdina ants consisted of dealate queens, male alates, female alates, major workers, minor workers, eggs, larvae and pupae. this result corresponded to lee et al. (2003) figs. 7-12: fig. 7. eggs of minor worker of o. smaragdina from both colonies; fig. 8. eggs of major worker of o. smaragdina from both colonies; fig. 9. larvae of female alate of o. smaragdina from both colonies; fig. 10. pupae of minor workers of o. smaragdina from both colonies; fig. 11. pupae of major workers of o. smaragdina from both colonies; fig. 12. pupae of female alate of o. smaragdina from both colonies. 6 sociobiolog y vol. 59, no. 1, 2012 where the o. smaragdina ants had a complete metamorphosis consisting of immature stages (eggs, larvae, and pupae) and adults with reproductive stage (dealate queen) and non-reproductive stage (workers). the workers of o. smaragdina were all females and had a dimorphism or bimodal size-frequency distribution known as major and minor workers (wilson 1953; greenslade 1972; hölldobler & wilson 1990).the specific function of each individual adult in a colony is determined by the existence of the caste system and this division of labour is important for the continuous survival of the colony (lee et al. 2003). contrary to the current study, peng et al. (1998a; 1998b) reported that there were large numbers of major workers, eggs, early-instar larvae and some minor workers within each queen nest, but no pupae or medium-sized or large-sized larvae. way (1963) also reported that the brood (immature stages) of o. smaragdina is not raised in ‘pavilions’; the pavilions are constructed in the same way as nests where many homopterans are tended by ants, often several meters away from the nearest ant nest. only one dealate queen found in the kafa colony and no dealate queen was found in the ti colony. this may be due to the kafa colony was established much earlier than the ti colony. the nests which were taken from the ti colony were also fewer in numbers and smaller in size compared to the nests of the kafa colony. similar to the present study, peng et al. (1998b) reported that young mango trees which were closer to the matured mango trees with well-established o. smaragdina colonies were found to have more dealate queen nests. after being inseminated by a male ant during nuptial flight, the winged queen shed her wing (oster & wilson 1978; lokkers 1990). the queen would then begin to construct the first nest cell and rears its first brood of workers. soon, the workers would take over the tasks of foraging and brood care, allowing the queen to confine herself to egg-laying (oster and wilson 1978). therefore, the presence of a dealate queen in the kafa colony showed that the colony was well-maintained compared to the ti colony. the number of male alates in the kafa colony was higher than the female alates might be due to the female alates could mate with more than one male during swarming (1384.38 ± 367.57; 80.00 ± 26.09). this result was supported by schlüns et al. (2009) who reported that direct genotyping of the sperm carried by 77 queensland queens and worker genotypic arrays of 7 marcela, p. et al. — colony structure of oecophylla smaragdina established colonies of o. smaragdina showed that less than half of the queens mate only once, while some mate up to five times, which suggested that more males were involved during mating than the females. similar behavior was also observed in formica subpolita ants where the females controlled who they mated with and were observed to mate with up to 4 different males, before dispersing from swarm sites (o’neill 1994). female ants may mate just once, but multiple mating is apparently typical for many species (hölldobler & wilson 1990). lee et al. (2003) explained that male ants have only one task, which is to inseminate the queen. they usually possess wings and mating can occur within the nest or outside the nest (swarming ). shortly after mating, the males die and the queens settle and establish new colonies (schlüns et al. 2009; lee et al. 2003). for the female alates found inside the nests, the yellow color was also observed in the study of intercolony transplantation of o. smaragdina by krag et al. (2010). they reported that the o. smaragdina can be categorized into the following distinct groups: larvae, pupae, light yellow callow workers and red mature workers. lee et al. (2003) explained that a newly hatched adult which has not yet acquired its deep coloration is known as a callow. it will undergo coloration as it grows older. therefore, it could be that the yellow colored female alates of the ti colony were newly emerged adults that had not yet acquired its deep coloration (callow) (fig. 4). this result supported that the kafa colony was established much earlier than the ti colony due to the presence of the matured female alates with greenish color in the kafa colony. our results also show that the number of female alates in the kafa colony was higher than the ti colony (80.00 ± 26.09; 11.00 ± 5.08). the higher number of major workers compared to minor workers discovered in the current study could be due to task allocation. the major workers have more tasks than the minor workers do and therefore more major workers would be needed (lee et al. 2003). this result corresponds to previous studies which demonstrated that the major workers do virtually all of the foraging, defend the colony, care for the queen and assist in the care of the brood (chapuisat and keller, 2002), whereas the minor workers mostly remain inside the leaf nests and function as nurses of the brood studies (hölldobler & wilson 1978; 1990). this allocation of tasks to different castes of o. smaragdina creates an efficient society (saarinen 2006). 8 sociobiolog y vol. 59, no. 1, 2012 the eggs were small and ellipse-shaped with a size around 0.5 mm x 1.0 mm. the eyes and mouth of the larvae within the eggs could be seen when they were observed under a magnifying glass. the pupae could be differentiated from the mature adults because they were white in color (van mele & cuc 2007). the o. smaragdina colonies could produce a large number of “queen brood” every year. the “queen brood” refers to larvae and pupae, which are destined to become new queens as well as their last stage as imago virgin queens (cesard 2004; offenberg & wiwatwitaya 2010). the current study also showed that o. smaragdina ants built new nests within 14 days after their nests were taken. the new nests were located near to the nests which were taken for our study. regarding this condition, paimin and paimin (2001) reported that under normal conditions, weaver ants have a quick population recovery with regeneration time between 17 and 24 days. others reported it takes five days before new nests appear and around 20 days for the ants to produce new larvae (cesard 2004). a caste can be characterized as a set of individuals in a colony, morphologically distinct from other individuals and specialized in behavior which performs specialized task in the colony (oster & wilson 1978). the present study showed that different caste of o. smaragdina ants performed different but specific task to maintain their colony well. hölldobler & wilson (1990) stated a colony which is large enough to produce new, virgin queens known as “mature colony” and the efficiency of the mature colony is determined by the number of workers in each temporal caste at any given moment. this suggests that the kafa colony in the current study was a mature colony of o. smaragdina with the presence of a dealate queen and more workers compared to the ti colony. acknowledgments the authors thank the dean of the school of biological sciences, universiti sains malaysia for the facilities and funding provided for the research. references blüthgen, n. & k. fiedler. 2002. interactions between weaver ants, oecophylla smaragdina, homopterans, trees and lianas in an australian rainforest canopy. j. anim. ecol. 71: 793–801. 9 marcela, p. et al. — colony structure of oecophylla smaragdina cesard, n. 2004. harvesting and commercialization of kroto (oecophylla smaragdina) in the malingping area, west java, indonesia, pp. 61-77. in: 1-aisa., k. kusters & b. belcher (eds). forest products, livelihoods and conservation. case studies of non-timber forest product systems. center for international forestry research. 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(2nd edition). africa rice center (warda), cotonou, benin and cabi, egham, uk. 72 pp. way, m.j. 1963. mutualism between ants and honeydew-producing homoptera. annu. rev. entomol. 8: 307–344. wilson, e.o. 1953. the origin and evolution of polymorphism in ants. q. rev. biol. 28: 136-156. 667 aggregation and feeding behavior of the formosan subterranean termite (isoptera: rhinotermitidae) on wood decayed by three species of wood rot fungi by mary l cornelius*, kelley s. williams, mary p. lovisa & anthony j. de lucca ii abstract aggregation and feeding behavior of the formosan subterranean termite, coptotermes formosanus shiraki, was evaluated on wood decayed by three species of fungus, the brown rot fungus, gloeophyllum trabeum and two white rot fungi, phanerochaete chrysosporium and pycnoporus cinnibarinus. although termites aggregated on decayed sawdust from all three species in at least some of the tests, sawdust decayed by p. chrysosporium elicited aggregation behavior by termites over the greatest range of incubation periods. in some tests, termites avoided sawdust decayed by g. trabeum. termite feeding on blocks decayed for 90 d was significantly greater than on control blocks for all three species of fungi, despite the significantly lower decay rate of p. cinnibarinus. increasing our understanding of the interaction of termites with wood rot fungi could lead to the identification of chemicals that attract termites to bait stations. key words: coptotermes formosanus, fungus, aggregation, consumption, decay introduction lignocellulose is the major component of plant cell walls. the formosan subterranean termite, coptotermes formosanus shiraki, uses cellulases secreted from salivary glands and symbiotic flagellates to digest cellulose (nakashima et al. 2002, zhang et al. 2011). although there is evidence that termites produce cellulases, hemicellulases, and lignases, lignin is not metabolized by termites and acts as a physical barrier to cellulases (breznak & brune 1994, brune united states department of agriculture, agricultural research service, southern regional research center, 1100 robert e. lee blvd., new orleans, la 70124 *corresponding author: mary.cornelius@ars.usda.gov; this article presents the results of research only. mention of a commercial or proprietary product does not constitute endorsement or recommendation by the usda. 668 sociobiolog y vol. 59, no. 3, 2012 2006, geib et al. 2008, coy et al. 2010, ke et al. 2011, scharf et al. 2011). wood rot fungi may facilitate termite digestion of cellulose by modifying or metabolizing lignin. brown rot fungi circumvent the lignin barrier and metabolize cellulose and hemicellulose without removing the lignin. therefore, lignin remains the major component of plant cell walls degraded by brown rot fungi. however, brown rot fungi modify the lignin by demethylation and oxidation (green iii & highley 1997). in contrast, white rot fungi simultaneously degrade the three major components of the plant cell wall: lignin, cellulose, and hemicellulose (geib et al. 2008). two species of white rot fungi have different enzyme systems for lignin degradation. ligninolytic activity by, phanerochaete chrysosporium burdsall, is closely correlated with secretion of two specific peroxidases, lignin peroxidase (lip) and manganese peroxidase (mnp), whereas neither lip nor mnp was detected in the ligninolytic activity of the white rot fungus, pycnoporus cinnabarinus ( jacq.:fr.) karst. ligninolytic activity by p. cinnabarinus was characterized by a single dominant laccase (eggert et al. 1996, alves et al. 2004). many studies have documented the preference of subterranean termites for wood decayed by the brown rot fungus, gloeophyllum trabeum (persoon: fries) murrill over sound wood (amburgey 1979, lenz et al. 1991). studies have also demonstrated that chemicals in g. trabeum elicit trail following and directional tunneling behavior (esenther et al. 1961, matsumura et al. 1968, rust et al. 1996, su 2005). other researchers have found that the interaction between subterranean termites and g. trabeum is antagonistic because of the competition for cellulose ( jayasimha & henderson 2007a, 2007b). in previous research, the formosan subterranean termite has shown a significant preference for spruce, picea sp., sawdust inoculated with both the brown rot fungus, g. trabeum and the white rot fungus, p. chrysosporium, over control sawdust. however, termites strongly preferred sawdust inoculated with p. chrysosporium over sawdust inoculated with g. trabeum. also, there was significantly greater aggregation of termites on spruce sawdust that had only been decayed for three weeks by g. trabeum compared to sawdust decayed for 12 week (cornelius et al. 2002). the objective of this study was to examine the interaction of formosan subterranean termites with three species of wood rot fungi, the brown rot 669 cornelius, m.l. et al. — feeding behavior of termites on decayed wood fungus, g. trabeum and two species of white rot fungi, p. chyrsosporium and p. cinnabarinus at different stages of decay. because the two species of white rot fungi use different enzyme systems for lignin degradation, their effect on termite behavior may be different. this study examines the aggregation and feeding behavior of formosan subterranean termites on wood decayed by these three species of wood rot fungi. materials and methods termite collection and fungus cultures termites were collected from field colonies in city park, new orleans, la, using cylindrical irrigation valve boxes (22.5 by 14.8 cm; nds, lindsay, ca) buried in the ground so that the lids are level with the surface of the soil and filled with blocks of wood (spruce [picea sp.]). collections from different stations in the same section of city park were considered to be separate colonies based on a mark-release-recapture technique using the dye markers nile blue a and neutral red (sigmaaldrich, milwaukee, wi) to determine which stations were part of a single, interconnected tunneling system. any termites from stations containing dyed termites were considered to be part of the same colony as the termites in the station from which the dyed termites were released. termites were kept in the lab in 5.6-l covered plastic boxes containing moist sand and blocks of spruce until they were used in experiments. termites were used for experiments within two months of collection. the brown rot fungus, g. trabeum (madison 617), the white rot fungus, p. chrysosporium (46235) and the white rot fungus, p. cinnabarinus (10242) were obtained from the american type culture collection (atcc, manassas, va). fungal inoculation of sawdust northern red oak, quercus rubra l., sawdust was inoculated with the mycelium from one of the three species of fungus and decayed for different incubation periods (7, 14, 30, 60, 90, 120, or 150 d). there was no sporulation by any of the fungi in any of the tests. termites were only exposed to mycelium. nutrient plates were inoculated with one of the fungus species and placed in an incubator set at a temperature of 25° c with a photoperiod of 12 :12 (l:d) h for 3 d. for p. chrysosporium, potato dextrose agar (pda) 670 sociobiolog y vol. 59, no. 3, 2012 plates were used and for g. trabeum and p. cinnabarinus, potato dextrose yeast (pdy) plates were used. five liters of nutrient broth were inoculated with (1-cm square) plugs from the plates at a ratio of four plugs per liter of broth. the flasks were incubated at 25º c in an orbital shaker at 120 rpm. phanerochaete chrysosporium was cultured in sabouraud dextrose broth for 7 d, whereas g. trabeum and p. cinnabarinus were cultured in pdy broth for 14 d. afterwards, the fungal biomass was strained through a wire mesh strainer, and finally vacuum filtered through a buchner funnel using a sterile whatman #4 filter paper. sawdust was placed in an autoclavable polypropylene vent bags (32 by 40 cm) (unicorn imp. and mfg. corp., commerce, tx) with a single 0.2 micron filter (7 by 25 cm), 100 grams per bag. the bags were heatsealed and autoclaved vent side up, for 1 h on each of two consecutive days. an amount of mycelium equal to 43.4 g of mycelia per 100 g of sawdust was weighed and suspended in 18 ml of sterile water and blended in a stomacher 400 mark ii lab blender (spiral biotech, bethesda, md) on high for 60 s. after the autoclaved bags were cooled to room temperature, one corner of the bag was cut open, and the filtered mycelium was added to the bag using a pipette. after the sawdust was inoculated with the filtered mycelium, the opening in the vent bag was heat-sealed. the bags were kept in an incubator set at 25º c with 12 h photoperiod for different lengths of time (7, 14, 30, 60, 90, 120, or 150 d). aggregation behavior on decayed sawdust tests were conducted to determine if termites aggregated on sawdust inoculated with living fungal mycelium after different incubation periods. different batches of sawdust were inoculated at each incubation period to evaluate variability between different batches decayed for the same incubation period. three plastic screwtop containers (5cm diam and 4.7 cm height) were connected with 5 cm length pieces of pvc tubing. each distal container was filled to a height of 1 cm with either treated or control sawdust or with sawdust decayed by two different fungus species. a soil moisture meter (spectrum technologies, plainfield, il) was used to make sure that moisture levels were equivalent in treated and control sawdust. the center container was filled with 10 g of sand, moistened with 2 ml of distilled water. for each replicate, termites (190 workers and 10 soldiers) were released into the center 671 cornelius, m.l. et al. — feeding behavior of termites on decayed wood container. for each test, termites were collected from four different colonies, with three replicates of each colony. for 7 d, containers were kept in an unlit environmental chamber set at 28°c, 97% rh. at the end of the test, the number of termites in the tubing and in each container was counted. two tests were conducted with the first batch of sawdust decayed by g. trabeum for 30 d and 60 d because the behavior of termites toward the sawdust decayed for 30 d was different than toward the sawdust decayed for 60 d. tests were repeated with this batch to evaluate this change in termite behavior towards the decayed sawdust. fungus inoculation of wood blocks blocks of red oak (4 cm by 3.5 cm by 1 cm) were oven-dried at 90° c for 24 h, weighed, and numbered. blocks were then soaked in water for 3 d, wrapped in moist paper towels and two layers of aluminum foil, and then autoclaved for 60 minutes on two consecutive days. after cooling, each individual block was placed in a petri dish (100 mm by 20 mm) containing pda inoculated with one of the three fungus species. block numbers and fungus species were written on the lid of each petri dish. fungus-inoculated blocks were kept in an environmental chamber set at a temperature of 25° c with 12 h photoperiod for 30, 60, or 90 d. the fungal mycelium of each species gradually covered the block during the incubation period. at the end of each time period, blocks were cleaned, oven-dried at 90° c for 24 h, and re-weighed. decay rates were determined by calculating the weight loss of each block during the incubation period. feeding behavior on decayed blocks a no-choice test was conducted using blocks decayed by one of the three fungus species for 30 d, 60 d, or 90 d and undecayed control blocks. the assay was conducted using polystyrene, cylindrical screwtop containers (9 cm high by 7cm diam.) (consolidated plastics, twinsburg, ohio) filled with 50 g of sand (play sand, quikrete, atlanta, ga) and moistened with 10 ml of distilled water. after weight loss due to decay was determined, a block was placed on top of the sand in each container. for each replicate, termites (190 workers and 10 soldiers) were released into the container. for six weeks, containers were kept in an unlit environmental chamber set at 28°c, 97% rh. after six weeks, blocks were cleaned, oven-dried at 90° c for 24 h, and 672 sociobiolog y vol. 59, no. 3, 2012 re-weighed. wood consumption was determined by calculating the weight loss of each block. termites were collected from three different colonies, four replicates of each colony for each fungus species at each decay period, except that there were only two replicates of each colony for control blocks and for p. chrysosporium and g. trabeum at 30 and 60 d. statistical analysis in aggregation assays, the number of termites in treated and control containers was compared using a paired choice t-test. termites, located in the tubing and the center container, were not included in the analysis. in the nochoice feeding test, proportional weight loss due to decay and feeding, and proportional survival were compared for each treatment (fungus species + decay period) and for each fungus species at each decay period (30, 60, or 90 d) separately using a one-way kruskal-wallis anova. means were separated by dunn’s test with ranked sums due to unequal sample sizes. if the tests for both normality and equal variances passed, a one-way anova was used and means were separated with dunn’s test (systat software 2008). results and discussion aggregation behavior on decayed sawdust average survival of termites in the aggregation tests was 92.4 ± 2.3. sawdust decayed by p. chrysosporium had a highly significant effect on aggregation behavior for all batches and incubation periods from 7 d -120 d. when sawdust was decayed for 150 d, there was no significant difference in the number of termites on decayed and control sawdust (table 1). termite responses to sawdust decayed by g. trabeum were highly variable. in the first batch, termites aggregated on the control sawdust when sawdust was decayed for 30 d (p = 0.05). in contrast, termites aggregated on sawdust decayed for 60 d. when these tests were repeated with sawdust from the same bags, termites aggregated on control sawdust after an incubation period of 30 d (p =0.003) and aggregated on the decayed sawdust after an incubation period of 60 d (p = 0.001). in the second batch, termites aggregated on sawdust decayed for 30 d, and showed no response to sawdust decayed for 40, 50, or 60 d. in the third batch, they aggregated on control sawdust compared to sawdust decayed for 14 d. termites also appeared to aggregate on control sawdust after 40 d (p =0.05) (table 2). aggregation behavior on sawdust 673 cornelius, m.l. et al. — feeding behavior of termites on decayed wood table 1. mean (±se) number of termites in containers filled with red oak sawdust inoculated with phanerochaete chrysosporium and decayed for different lengths of time compared with containers filled with control sawdust after 7 d. mean (±se) number of termites in containers incubation time (days) fungus control p value1 batch 1 7 139.9 ± 11.6 4.7 ± 1.1 <0.0001 14 110.8 ± 6.2 30.6 ± 5.7 <0.0001 batch 2 14 116.7 ± 7.6 41.0 ± 8.4 <0.0001 30 135.8 ± 11.9 13.7 ± 3.9 <0.0001 40 110.9 ± 9.5 30.6 ± 7.9 <0.0001 50 99.5 ± 9.4 32.8 ± 5.2 <0.0001 60 125.6 ± 7.6 19.6 ± 4.6 <0.0001 batch 3 14 127.5 ± 8.7 12.2 ± 3.2 <0.0001 30 112.2 ± 12.1 19.6 ± 4.5 <0.0001 60 131.4 ± 9.4 14.2 ± 4.3 <0.0001 90 98.3 ± 9.9 28.7 ± 5.6 <0.001 120 123.2 ± 4.2 19.7 ± 3.0 < 0.001 150 87.4 ± 10.5 55.6 ± 11.4 0.12 1the number of termites in treated and control containers were compared using a paired choice t-test. table 2. mean (±se) number of termites in containers filled with red oak sawdust inoculated with gloeophylum trabeum and decayed for different lengths of time compared with containers filled with control sawdust after 7 d. mean (±se) number of termites in containers incubation time (days) fungus control p value1 batch 1 14 26.7 ± 12.3 67.4 ± 16.9 0.14 30 (test 1) 21.5 ± 6.7 63.3 ± 18.3 0.05 30 (test 2) 25.6 ± 7.5 81.3 ± 10.4 0.003 60 (test 1) 67.8 ± 14.2 23.8 ± 10.8 0.04 60 (test 2) 99.3 ± 13.6 31.8 ± 6.3 0.001 batch 2 30 68.0 ± 10.7 25.9 ± 5.1 0.01 40 58.2 ± 11.3 47.5 ± 10.8 0.63 50 46.9 ± 9.8 50.2 ± 9.9 0.84 60 37.1 ± 6.6 26.8 ± 6.8 0.30 batch 3 14 46.3 ± 9.7 79.8 ± 8.5 0.08 30 38.2 ± 5.0 53.4 ± 8.6 0.17 40 46.1 ± 8.2 82.2 ± 11.5 0.05 50 58.4 ± 7.2 58.1 ± 7.0 0.97 60 42.4 ± 8.3 61.2 ± 5.2 0.09 1the number of termites in treated and control containers were compared using a paired choice t-test. 674 sociobiolog y vol. 59, no. 3, 2012 decayed by g. trabeum was not consistent between batches and did not correspond with incubation period. in some cases, termites aggregated on control sawdust. because termites were exposed to living fungal mycelium in these tests, fungal growth rates were not measured. however, there may have been substantial differences in growth rates of g. trabeum between batches. because g. trabeum does not metabolize lignin, it can have a negative effect on termites by depleting the cellulose without removing the lignin. therefore, the range of conditions under which termites would aggregate on sawdust decayed by g. trabeum appears to be much narrower than for p. chrysosporium. termites aggregated on sawdust decayed by p. cinnabarinus for 14 d in both batches, but did not show a significant response to sawdust decayed for longer incubation periods in either batch (table 3). in choice tests of sawdust decayed by either p. chrysosporium or p. cinnabarinus for the same length of time, termites only displayed significant aggregation behavior on p. chrysosporium when sawdust was decayed for 120 d. termites were distributed equally between the two containers of sawdust decayed by the two fungi when sawdust was decayed for 14 or 60 d (table 4). these were the first tests conducted to evaluate the response of termites to p. cinnabarinus. although many factors, such as fungal growth rates and wood species, affect the interaction of subterranean termites with wood rot fungi, p. cinnabarinus did elicit aggregation behavior in termites when sawdust was only decayed for 14 d, but not when sawdust was decayed for a longer period. sawdust decayed by p. chrysosporium elicited aggregation behavior by formosan subterranean termites the most consistently and over the greatest range of incubation periods of the three fungus species tested. aggregation behavior of termites on a food source could increase the efficacy of baiting programs for termite control by increasing consumption of toxic baits. feeding behavior on decayed blocks in comparisons of rates of decay and feeding for the three fungi at each decay period, percent weight loss due to decay was significantly different at 30 d (h = 12.0, df = 2, p = 0.002), 60 d (h = 12.4, df = 2, p = 0.002), and 90 d (h = 19.7, df = 2, p < 0.001), but percent weight loss due to termite feeding was not significantly different at 30 d (f 2, 23 = 1.5, p = 0.24), 60 d (f 2, 23 = 1.4, p = 0.27), or 90 d (f 2, 35 = 3.1, p = 0.06). percent weight loss due to 675 cornelius, m.l. et al. — feeding behavior of termites on decayed wood decay by p. cinnabarinus was significantly less than percent weight loss due to decay by the other two species at all three decay periods (table 5). in comparisons of rates of survival, decay and feeding over all treatments, there were no significant differences in the survival of termites in containers with different treatments (h = 15.6, df = 9, p = 0.08). there were significant differences in the percent weight loss of blocks due to fungal decay (h = 56.5, df = 8, p < 0.001) and due to feeding damage (h = 51.2, df = 9, p < 0.001) for the different treatments. percent weight loss due to decay was significantly greater for p. chrysosporium and g. trabeum after 90 d decay compared to percent weight loss for p. cinnabarinus at 90 d. in addition, percent weight loss due to decay by p. chrysosporium was also significantly greater after 60 table 3. mean (±se) number of termites in containers filled with red oak sawdust inoculated with pycnoporus cinnabarinus and decayed for different lengths of time compared with containers filled with control sawdust after 7 d. mean (±se) number of termites in containers incubation time (days) fungus control p value1 batch 1 14 125.5 ± 11.4 62.3 ± 9.8 0.01 60 95.3 ± 16.4 48.2 ± 10.8 0.07 120 93.5 ± 15.3 80.0 ± 13.1 0.64 batch 2 14 131.3 ± 12.1 30.0 ± 7.1 0.0001 30 82.0 ± 12.5 73.1 ± 9.4 0.65 120 68.2 ± 9.9 55.8 ± 8.1 0.32 1the number of termites in treated and control containers were compared using a paired choice t-test. table 4. mean (±se) number of termites in containers filled with red oak sawdust inoculated with pycnoporus cinnabarinus compared with containers filled with phanerochaete chrysosporium when inoculated sawdust was decayed for different lengths of time after 7 d. mean (±se) number of termites in containers incubation time (days) pycnoporus phanerochaete p value1 14 68.3 ± 12.2 76.3 ± 18.1 0.79 60 80.5 ± 6.5 95.8 ± 5.6 0.22 120 31.0 ± 8.7 145.8 ± 8.5 <0.001 1the number of termites in treated and control containers were compared using a paired choice t-test. 676 sociobiolog y vol. 59, no. 3, 2012 d than percent weight loss for p. cinnabarinus at 60 d. termite feeding on decayed blocks was significantly greater than on control blocks for all three fungi after 90 d of decay (table 5). the effect of decay on feeding rates by termites is complex and affected by multiple factors. although termites avoided sawdust decayed by g. trabeum in some aggregation tests, termite consumption of blocks decayed by g. trabeum was significantly greater than consumption of control blocks after 90 d of decay. even though g. trabeum does not metabolize lignin, it seems to cause chemical changes to blocks that increase feeding rates by formosan subterranean termites. the chemical modification of lignin by g. trabeum appears to facilitate termite feeding under at least some conditions. by removing cellulose while leaving the lignin behind, g. trabeum gradually makes the wood less palatable to termites as the rate of decay increases (lenz et al. 2007, cornelius et al. 2002). the weight loss of blocks due to decay by p .cinnabarinus was much lower than the weight loss due to decay by the other two fungus species. even though weight loss of red oak blocks due to p. cinnabarinus was < 3% for all three incubation periods, the fungus may have been causing chemical changes to the blocks that facilitated termite feeding. after 90 d of incubation, wood consumption on decayed blocks was 12.6% compared to 4.8% on control blocks. other studies have also demonstrated that termite feeding rates on decayed blocks do not always correlate directly with amount of decay or length of incubation periods (amburgey & smythe 1977, lenz et al. 1980). although there are many factors that could influence the differences in termite behavior towards the two white rot fungi, p. chrysosporium and p. cinnabarinus, the difference in the enzymatic pathways utilized for lignin degradation may be important. further research is needed to elucidate the factors affecting termite aggregation and feeding behavior towards wood rot fungi. the interactions of termites with wood rot fungi range from obligate mutualism (darlington 1994, aanen et al. 2002) to antagonism (becker 1976, amburgey & beal 1977, jayasimha & henderson 2007a, 2007b). by breaking down or chemically modifying lignin, wood rot fungi can facilitate the ability of termites to consume lignocellulose. because of their symbiotic association with lignin-degrading fungi, fungus-growing termites are able to utilize virtually all of the lignocellulose they consume and they expel only a 677 cornelius, m.l. et al. — feeding behavior of termites on decayed wood small volume of feces (darlington 1994). in an analysis of feces of formosan subterranean termites fed southern pine, pinus australis f. michx, it was determined that lignin was modified, but not degraded, and that it retained its aromatic properties (ke et al. 2011). scharf et al. (2011) provide evidence that termites could gain a fitness advantage by utilizing gut symbionts to overcome end-product inhibition caused by the degradation of lignin. formosan subterranean termites may be able to utilize a greater percentage of lignocellulose from decayed wood due to the biochemical changes in the wood. therefore, feeding on decayed wood may provide a fitness advantage by allowing the wood rot fungi to partially degrade lignin prior to consumption. many researchers have investigated the potential of using attractants from wood rot fungi in termite baits (esenther & beal 1974, amburgey et al. 1981, rust et al. 1996, getty & haverty 1998, cornelius et al. 2009). however, optimal conditions for eliciting aggregation and feeding behavior by termites depends on many factors including, decay rate, wood species, and differences in fungal strains and species. elucidation of the biochemical changes in wood table 5. percent (±se) weight loss of red oak wood blocks decayed by three species of fungi for 30, 60, or 90 d. percent (±se) weight loss of blocks due to termite feeding and percent (±se) termite survival in a no-choice feeding test after six weeks. percent (±se) weight percent (±se) weight loss loss of blocks due to percent (±se) fungus species of blocks due to decay termite feeding termite survival undecayed control blocks ------------ 4.8 ± 0.3a 78.5 ± 1.5 30 d decay phanerochaete chrysosporium 5.4 ± 0.4aabc 10.9 ± 0.6a 88.7 ± 1.5 gloeophylum trabeum 5.3 ± 0.3aabc 9.8 ± 0.2a 86.0 ± 1.8 pycnoporus cinnabarinus 2.4 ± 0.5ba 10.7 ± 0.4a 86.9 ± 1.7 60 d decay phanerochaete chrysosporium 8.1 ± 0.8ac 9.5 ± 1.7a 85.3 ± 4.6 gloeophylum trabeum 7.9 ± 0.5abc 9.5 ± 1.1a 83.6 ± 7.7 pycnoporus cinnabarinus 2.9 ± 0.7bab 11.3 ± 0.4a 87.2 ± 1.3 90 d decay phanerochaete chrysosporium 11.11 ± 3.1ac 15.4 ± 1.1b 85.4 ± 1.7 gloeophylum trabeum 10.3± 0.4ac 14.5 ± 0.7b 87.8 ± 1.0 pycnoporus cinnabarinus 2.6 ± 0.1bab 12.6 ± 0.4b 88.0 ± 0.9 means followed by the same lowercase letter within an incubation period within a column were not significantly different (dunn’s test: p ≥ 0.05). means followed by the same uppercase letters within a column were not significantly different (dunn’s test: p ≥ 0.05) 678 sociobiolog y vol. 59, no. 3, 2012 due to decay by different fungus species may result in the identification of chemicals that could be used to improve baiting programs for termites. acknowledgements we thank erin lathrop, megan coyne, dominick maggio, and nick saybe for invaluable technical assistance on this project. we would also like to thank weste osbrink, matt tarver, and beverly wiltz for helpful comments on earlier drafts of this manuscript. references aanen, d. k., p. eggleton, c. rouland-lefevre, t. guldberg-froslev, s. rosendahl & j. j. boomsma. 2002 the evolution of fungus-growing termites and their mutualistic fungal symbionts. proceedings of the national academy of sciences 99 (23): 14887– 14892. alves, a. m. c. r., e. record, a.k 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nakashima, k., h. watanabe & j. i. azuma. 2002. cellulase genes from the parabasalian symbiont pseudotrichonympha grassii in the hindgut of the wood-feeding termite coptotermes formosanus. cellular and molecular life sciences 59: 1554-1560. rust, m. k., k. haagsma & j. nyug yen.1996. enhancing foraging of western subterranean termites (isoptera: rhinotermitidae) in arid environments. sociobiolog y 28 (3):275286. scharf, m. e., z. j. karl, a. sethi & d. g. boucias. 2011. multiple levels of synergistic collaboration in termite lignocellulose digestion. plos one 6 (7): e21709. su, n.-y. 2005. directional change in tunneling of subterranean termites (isoptera: rhinotermitidae) in response to decayed wood attractants. journal of economic entomolog y 98 (2): 471-475. systat software, inc. 2008.systat statistical package, version 12.0. systat software, inc., san jose, ca, usa. zhang, d., a. r. lax, j. m. bland & a. b. allen.2011. characterization of a new endogenous endo-b-1,4-glucanase of formosan subterranean termite (coptotermes formosanus). insect biochemistry and molecular biolog y 41: 211-218. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.459-465sociobiology 60(4): 459-465 (2013) multiple male and female reproductive strategies and the presence of a polyandric mating system in the termite reticulitermes labralis (isoptera: rhinotermitidae) j. wu, x. su, x. kong, m. liu, l. xing introduction high fertility of the reproductive caste is essential for maintaining and expanding a termite colony. the most important factors for fertility of termites are thought to be the number of individuals in the reproductive caste and the copulatory selectivity of individuals in the breeding system. life history and reproductive strategy often differ from one species to another (lainé & wright, 2003). the diverse and flexible breeding systems found in lower termites pre-adapt them to invade new environments or marginal habitats (dronnet et al., 2005). therefore, the study of the mating systems of termites has important significance to understand colony dispersal and establishment. in general, termite colonies are founded by a single primary king and a single primary queen that pair during nuptial flights, mate and subsequently produce the other colony members (thorne et al., 1999). since the reproductive system of termite is usually monogamous, the fecundity of the colony is abstract reproductive systems of termite colonies may involve the number of individuals in the reproductive caste and the copulatory selectivity of reproductive individuals (i.e., polyandry or polygamy), both of them impacting directly the fertility and genetic diversity of the colony. polygamy is widespread in the lower termites, whereas polyandry appears to be mostly absent in termites. in this paper, the differentiation of male and female neotenics was observed in orphaned experimental colonies of the subterranean termite reticulitermes labralis. the artificial orphaned colonies began to produce neotenics only a week after colony establishing, with more neotenics appearing in the same group as time went by. finally, each experimental group reserved multi-neotenics consisting of male and female neotenic individuals. our results demonstrated that these neotenic individuals retained in the colony participated in reproduction. a genetic analysis at four microsatellite loci showed that in addition to the conspicuous morphologically male reproductives, there were inconspicuous males or workers that had copulated with the females in the orphaned colony. multiple male and female reproductive individuals existed together in a single colony, and one female neotenic could mate with several male reproductives in a short time. thus, multiple male and female reproductive systems and a polyandric mating system are present in r. labralis. sociobiology an international journal on social insects college of life sciences, northwest university, xi’an, shaanxi province, china. research article termites article history edited by gilberto m m santos, uefs brazil received 26 april 2013 initial acceptance 17 june 2013 final acceptance 13 july 2013 keywords subterranean termite; neotenics; reproductive system; polyandry; microsatellite corresponding author xing lian-xi college of life sciences northwest university no. 229, north taibai rd., xi’an shaanxi province, 710069, p. r. china e-mail: lxxing@nwu.edu.cn relatively weak during the initial establishment of the colony (thorne, 1998; ishitani & maekawa, 2010; maekawa et al., 2010). in many termite species, abundant neotenics appear in colonies when the primary reproductives die, and take over the reproductive function to maintain the colonies (hayashi et al., 2003). some species even differentiate neotenics in the presence of primary reproductives and serve as supplementary reproductives (roisin, 2000; grube & forschler, 2004). in reticulitermes species, all castes are made up of individuals from both sexes (snyder, 1926; lainé & wright, 2003). field colonies of reticulitermes have often been found to contain a large number of neotenics, whose sex ratio is strongly female biased (howard & haverty, 1980; vargo et al., 2012). recently, a new breeding system named asexual queen succession (aqs) was confirmed in r. speratus and r. virginicus. these species undergo typical colony founding by a pair of primary reproductives. relatively early in the colony life cycle, the primary queen is replaced by numerous secondary queens (brachypterous female neotenics) that are produced asexually j. wu et al. reproductive strategies and mating system in the termite reticulitermes labralis460 by the primary queen. these neotenic queens mate with the primary king and produce workers, soldiers and new primary reproductives through sexual reproduction (matsuura et al., 2009; vargo et al., 2012). in other words, it is a multiple female and single male reproductive breeding system. although inconspicuous male reproductive was found in orphaned colonies in laboratory (fujita & watanabe, 2010), polyandry appears to be mostly absent in termites (hartke & baer, 2011). r. labralis is one of the most important economic termite species in central china, especially in urban areas. the main developmental pathway of this subterranean termite forks into two as other reticulitermes species (takematsu, 1992), following two larval instars (l1 and l2) (hereafter for terminology of termite castes we would stick to thorne, 1996). one pathway is the line of nymphs equipped with wing buds on the thorax, comprising six instars (n1-n6). the n6 nymphs can molt into alates (imagos). the other pathway is the line of apterous workers, where five successive instars (w1-w5) can be recognized morphologically. our early study presented three types of conspicuous male supplementary reproductives in r. labralis, ergatoids, nymphoids and adultoid form reproductives (floppy winged form and stay in the natal colony as supplementary reproductives after lost their wings) (xing lx unpubl. data), which were differentiated from w4-w5 workers, n4–n6 nymphs and n6 nymphs, respectively. the purpose of the present study is to investigate the inconspicuous male reproductives and the mating system of small orphaned groups of the r. labralis. material and methods termites a mature colony of r. labralis was collected in da xingshan temple, xi’an, shaanxi province, china, in april 2012, and experimental colonies were set up from this colony. ten experimental colonies were established containing all castes except larvae (1-2 instar juvenile) and neotenics. each experimental colony was composed of 100 individuals (20 n4-n6 nymphs, two soldiers and 78 w5 workers), being placed into a 120 ml transparent vial with moistened filter paper and pine wood at 20-26ºc in constant darkness. water was supplied regularly. we were carrying out observations once every two days in the first week, and recorded the number and time of reproductives differentiated in every experimental colony. during the second week until the period of larva appearance in the majority of colonies, we were observing every day and recorded the sex, number and time of differentiated reproductives. the experimental colonies were dissected after 60 days. all female and half of the male neotenics were fixed in bouin’s fluid at 4ºc for 24h, and the other half of male neotenics that had conspicuous morphological characteristics were selected for the subsequent experiment. neotenics (nymphoids, ergatoids and adultoid form neotenics) of r. labralis significantly differ in external morphology from those of workers or nymphs (i.e. a longer abdomen, darker pigmentation, slight sclerotisation and the presence of eyes). female and male neotenic reproductives can be distinguished by the seventh sternite: the female has an enlarged seventh sternite covering the eighth and ninth sternites, while the male has a seventh sternite similar to the previous sternites. in order to investigate whether the inconspicuous male neotenics existed or not, each conspicuous male neotenic (nymphoid or ergatoid) was placed into a separate container with 40 w5 workers (both male and female) and 10 n6 female nymphs that come from natal colony and reared for two months. we removed the nymphs from the container immediately after the first female neotenic (usually nymphoid) emerged in the colony. eight duplicates were established. two months later, the experimental colonies were dissected, and the abdomens of neotenics were soaked and fixed in bouin’s fluid for gonad histological observation, while the heads and thoraxes of neotenics and their offspring were preserved in 100% ethanol for genetic analysis. gonad histological observations the abdomens of neotenics (ten females and six males) were used for histological observation. paraffin-embedded sections were made and stained with hematoxylin and eosin. abdomens preserved in bouin’s fluid were dehydrated in increasing concentrations of ethanol, then transferred to xylene, and finally embedded in paraffin. serial parasagittal sections (7μm thick) were cut using a microtome (mrs80-074 ikemoto) and stained with hematoxylin and eosin. tissues on slides were observed using microscopes. genetic analysis whole genomic dna was extracted from parental head and thorax and offspring using tianamp genomic dna kit (tian gen biotcch co. ltd), respectively, pcr were performed with four pairs of microsatellite primers, rs68, rs78, rs76 (dronnet et al. 2004) and rf6-1 (vargo 2000). the primers were synthesized by invitrogen trading (shanghai) co., ltd. the pcr for the genotyping was performed in the 10μl reaction mixture consisting of 0.25 u of thermostable dna polymerase, 0.1 mm of each dntp, each 0.4 mm of primer pairs and genomic dna solution and 1×buffer for blend taq (containing 2μm mg2+). amplification conditions were 35 cycles of 30s denaturation at 94ºc, 30s annealing at 54ºc and 30s elongation at 72ºc. the pcr products were run on 7.5% polyacrylamide gels (25ma, 5h) and silver stained, then photographed by gel imaging system (bio-rad, beijing yuanye co., ltd.). microsatellite genotypes were determined in neotenics and their progeny for the above four microsatellite loci. pcr products amplified from each individual were sequenced by the commercial company (sangon biotech shanghai co., ltd) to verify subtle difference in the allele size. sociobiology 60(4): 459-465 (2013) 461 results number of neotenics per colony neotenics began to emerge shortly after the establishment of artificial orphaned colonies (6.6±1.3 day), and more neotenics produced by the same colony over time. the largest number of neotenics appeared in approximately 12 days, and the average number of neotenics were 14.9±3.1 in a colony. we could observe biting between neotenics, and the injured neotenics were intensively attacked by numerous workers to death. subsequently, the number of neotenics decreased with the appearance of physogastric neotenics in the colony. three weeks later, we could hardly see new neotenic differentiation, but the number of neotenics remained relatively stable in the colony (fig. 1). finally, each experimental colony contained multiple neotenics of both sex. after 60 days, 1.4±1.0 male and 3.1±1.1 female neotenics retained in one colony (table 1). the sex ratio of neotenics was significantly female biased (p<0.05), which was consistent with that of field investigations. gonad development of neotenics in orphaned colonies at the end of the experiments, only one colony contained one female neotenic among the 10 colonies; the other nine orphaned colonies had multi-neotenics (table 1). the ovaries of female neotenics were observed after 60 days. in female neotenics, each ovary was developed and contained plenty of vitellogenic oocytes in the end of the ovarioles, though the number of the vitellogenic oocytes was different among individual neotenics in the colony (fig. 2a). the observation of spermatogenesis in the testes of male neotenics showed that the testes consumed about half of the abdominal cavity and were full of sperms (fig. 2b). all the colonies had eggs and offspring at the end of the experiment, which suggested that all the female and male neotenics that eventually survived in the colony participated in reproduction. conspicuous and inconspicuous male neotenics in orphaned colonies in the orphaned colonies, in addition to the male reproductives that had conspicuous morphological characteristics, there were inconspicuous males or workers that participated in mating and reproduction. the eight duplication groups reared in the laboratory were used for genetic analysis. we found that in a 16-offspring colony of the eight duplications, 14 of the 16 offspring were not excluded from further analysis of paternity using the microsatellite assay (table 2). at the fig. 1. the fluctuation of reticulitermes labralis neotenics in the 10 orphaned colonies. the first neotenic began to appear after a week, and the number of neotenics became stable after 3 weeks of orphaning. the largest number of neotenic individuals appeared in colonies at approximately 12 days after the establishment of orphaned colonies. table 1 differentiation of conspicuous neotenics in different orphaned colonies. the neotenics refer to the total number of nymphiods, ergatiods and adultoids in the experiment. * asterisk denotes significant difference between the average female and male neotenics produced at the end of rearing (chi-square test, x2=5. 68 > x2df=1. p=0.05=3.84, p<0. 05). colony time of first neotenic a ppea ra nce (da y) pea k number of neotenics in the colony number of reta ined neotenics a t the end of rea ring ma le fema le 1 5 12 2 4 2 6 17 2 3 3 6 16 3 5 4 5 18 0 2 5 7 20 1 3 6 6 15 2 3 7 9 15 1 1 8 8 14 0 4 9 7 9 2 3 10 7 13 1 3 mea n±sd 6.6±1.3 14.9 ±3.1 1.4±1.0 3.1±1.1* j. wu et al. reproductive strategies and mating system in the termite reticulitermes labralis462 locus rs76, the allele sizes of the conspicuous male reproductive were 191/191, but the allele sizes in the offspring (no. 4 and 15) were 175/183 (fig. 3). therefore, we can exclude the conspicuous male reproductive from the paternity of these two offspring. in addition to that, offspring no. 4 and no. 15 were a combination of homozygote and heterozygote at the other four microsatellite loci, indicating that they were produced by a mere sexual reproduction. furthermore, two of the ten orphaned colonies did not emerge to be conspicuous male neotenics (table 1), but still they produced offspring in the colonies. these data suggested that besides the conspicuous male neotenics, there were inconspicuous males or workers that mated with female neotenics in the orphaned colony. discussion multiple female neotenics participated in reproduction in a single colony neotenics are found in many termite families (myles, 1999; roisin, 1999). these neotenic forms appear in termite colonies and take over the reproductive function to maintain the colonies when the primary reproductive individuals die. sometimes neotenics develop in the presence of primary reproductives as supplementary reproductive individuals (roisin, 2000; grube & forschler, 2004). in mature colonies, female neotenics lay eggs at a lower rate than that of the primary queens. however, they are usually present in large numbers and therefore collectively help produce larger communities even though their individual egg laying capacity is lower (thorne, 1996, 1998; thorne et al., 1999). in r. labralis, artificially orphaned colonies showed that many neotenics could coexist and laid eggs as secondary reproductives in a colony. polygamy is a common phenomenon in termites (neoh et al., 2010; hartke & baer, 2011), and multiple female neotenics are often found in field colonies (matsuura et al., 2009; vargo et al., 2012). although many females could lay eggs in the colony of r. labralis, the number of neotenics that retained as secondary reproductive in a colony was limited, which may be influenced by some queen-produced pheromones (keller & nonacs, 1993; yamamoto et al., 2012). the present study showed that r. labralis could not only differentiate multiple table. 2. allele sizes (bp) and their frequencies (n) of the conspicuous neotenics and offspring. two offspring (no. 4 and no. 15) were a combination of homozygotic and heterozygotic at the four microsatellite loci, indicating that the individuals were produced by sexual reproduction (*: allele sizes of no. 4. +: allele sizes of no. 15). rs78(n) rs76(n) rs68(n) rf24-2(n) male neotenic 178/182(1) 191/191 (1) 165/169 (1) 81/93 (1) female neotenic 178/182 (1) 175/183 (1) 165/169 (1) 81/81(1) offspring 178/178 (6)+ 183/191(6) 165/165 (3) 81/81(9)* 178/182(7)* 175/191 (8) 165/169 (9)*+ 81/93 (7)+ 182/182(3) 175/183 (2)*+ 169/169 (5) fig. 2. parasagittal sections of reticulitermes labralis neotenic gonads at the end of rearing. a: late vitellogenesis of ovary, the largest vitellogenic oocytes (vl) finished yolk accumulation, which were enveloped by degenerate follicle cells (fc) in the end of the ovarioles. b: the testis of male neotenic, the large number of mature sperm (sp) appeared in testis (t). fig. 3. genotypes of the conspicuous male neotenics, female neotenics and offspring at the microsatellite locus rs76. offspring of no. 4 and no. 15 did not have an allele from the conspicuous male reproductive in their colony. the conspicuous male reproductive was excluded from the paternity of these two offspring. sociobiology 60(4): 459-465 (2013) 463 female neotenics laying eggs in a colony, but also produce multiple male neotenics which had distinguishable morphological characteristics. the gonad histological observation suggested that these neotenic individuals all had ability of reproduction. our field investigation revealed also that multiple female and male neotenics coexisted in the field colonies of r. labralis (xing lx unpubl. data). polyandry was confirmed in r. labralis reproductive system of termites differ substantially from the one of hymenoptera insects, where polyandry is common and evolves independently (boomsma, 1996; strassmann, 2001; kronauer et al., 2004; baer, 2011), and polyandry appears to be mostly absent in termites (haetke & baer, 2011). in this study, genetic evidence provided by microsatellite locus rs76 revealed that two offspring (no. 4 and no. 15) were not paternally related to the conspicuous mature male reproductive of the rearing colony (fig. 3). three possible scenarios could explain this result: the female nymph had copulated in her parents’ colony before she was orphaned and her spermatheca stored residual spermatozoa, parthenogenic reproduction occurred in r. labralis, or there were other male individuals copulating with the female neotenic in addition to the conspicuous mature male. in contrast to the eusocial hymenopterans, termite copulations occur not during swarming and pair formation but after colony foundation and in the protection of the nest cavity (nutting, 1969). no sperm has been detected in the spermathecae of female swarming alates (dean & gold, 2004; ye et al., 2009), indicating that these individuals do not copulate within the parental nest during the development of the alates from the nymphs. our historical observation either found no sperms in the spermathecae of female nymphs and alates in the artificially orphaned colonies (xing lx unpubl. data). therefore, it is very likely that the nymphs used in this study did not copulate before the orphaned colonies were established. although parthenogenesis has been observed in some termite species (matsuura & nishida, 2001; matsuura et al. 2004, 2009; matsuura & kobayashi, 2007; vargo et al., 2012), our literature research found no evidence of the idea that r. labralis was capable of parthenogenesis. more importantly, parthenogenesis of termites is accomplished by automixis with terminal fusion, where two haploid pronuclei divide at meiosis fuse (matsuura et al., 2009). thus, offspring are homozygous for a single maternal allele at all loci (bignell et al., 2011; vargo et al., 2012; yamamoto & matsuura, 2012). however, individuals no. 4 and no. 15 in the present study were a combination of homozygote and heterozygote at the four microsatellite loci, thus excluding the possibility of parthenogenesis. the only reasonable explanation for the genotypes of individuals no. 4 and no. 15 is that there were inconspicuous males that copulated with the female neotenic in the experimental colony. in other words, polyandry was present in the subterranean termite of r. labralis. putting a focus on male neotenics in recent years, some researchers believed that the external morphology of some male neotenics did not differ significantly from those of workers or nymphs (pichon et al., 2007; fujita & watanabe, 2010). these male neotenics were designated as inconspicuous males, or reproductive males. as male reproductive ‘‘backups’’, the presence of inconspicuous males could insure against the loss of male reproductives, and be important for the continued functionality of the termite society. the majority of the reproductive males that are formed do not moult into the neotenic morph and are therefore inconspicuous in the overall population. indeed, inconspicuous male neotenics and workers have no significant differences in any measured aspect (fujita & watanabe, 2010). workers usually have three developmental pathways: remain workers, become soldiers, or evolve into ergatoid reproductives (haverty & howard, 1981). during the observations of r. labralis, we found that female neotenics laid eggs in the absence of conspicuous male neotenics in many orphaned colonies. as sperm can be observed both in w5 worker and the last instar of male nymph and male alate (nutting, 1969; su et al., 2010; hartke & baer, 2011), individuals no. 4 and no. 15 may be the offspring of inconspicuous mature males, but they could also be the offspring of workers, which requires further research. in the subsequent complementary tests, we dissected two replicate colonies at the end of 60-day experiment, one was a conspicuous male ergatoid colony with 28 remaining workers, the other was a conspicuous male ergatoid colony with 22 remaining workers. histology observation showed that there were 13 male workers in the 28-worker colony, with two workers having significantly bigger testes full of sperms, and nine male workers in the 22-worker colony, with one worker having significantly bigger testes and full of sperms. this confirms unconditionally the existence of sperms in the male worker of r. labralis. regardless of inconspicuous mature males or workers copulate with female neotentics, the present study has confirmed the existence of polyandry in the colony of r. labralis. reproduction system of r. labralis the life histories of lower termites are very complicated, and the establishment of a primitive colony requires the nuptial flight, nest building, sexual mating and reproduction (thorne et al., 1999). due to the fact that the impact on the reproductive system is usually monogamous, the copulatory selectivity of individuals in the reproductive caste is limited, and fecundity is relatively weak in the initial colony establishment (snyder, 1926; thorne, 1998; ishitani & maekawa, 2010; maekawa et al., 2010). when the colonies’ populations increase to a certain numj. wu et al. reproductive strategies and mating system in the termite reticulitermes labralis464 ber, numerous replacement reproductives appear in termite colonies, thus increasing the number of individuals in the reproductive caste and increasing the total colony reproductive output. increased reproductive output allows the colony to grow faster and larger because the combined egg production of dozens to hundreds of replacement reproductives far exceeds the egg-laying capacity of a single queen (thorne et al., 1999; grube & forschler, 2004; vargo et al., 2012). the japanese species r. speratus and the american species r. virginicus have been reported to undergo aqs to accelerate the development of the colony, that is, colonies are founded by monogamous pairs and the queens produce their replacements asexually but still continue using normal sexual reproduction processes to produce other colony members (matsuura et al., 2009; vargo et al., 2012). our results showed that in addition to the presence of numerous female neotenics in the reproductive caste, there were also numerous male reproductives in the r. labralis colony at the same time. moreover, there were inconspicuous males or workers that had the capacity for mating. one female neotenic could mate in a short time with several male reproductives, including inconspicuous males, in spite of the existence of conspicuous males. it is probable that the female neotenics mate with inconspicuous male neotenics to cover potential shortages in the spermatozoa of the conspicuous male neotenics. the current findings in r labralis have greatly expanded the known reproductive repertoire of termites. the individual copulatory options and the flexibility of the mating system are increased by multiple male and female reproductives participating in reproduction in the same colony. the diverse and flexible breeding systems of lower termites pre-adapt them to invade new or marginal habitats (dronnet et al., 2005). it is likely that multiple male and female reproductives and the polyandric mating system are present not only in r. labralis, but in more reticulitermes species as well. acknowledgments we would like to thank dr. kang huang for his help in genetic analyses. financial support for this work was provided by the national natural science foundation of china (31170363). we also thank two anonymous 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(2009). reproductive characteristics of imagos of reticulitermes flavipes (isoptera: rhinotermitidae). ann. entomol. soc. am., 102: 889-894. doi:10.1603/008.102.0515 doi: 10.13102/sociobiology.v67i3.4885sociobiology 67(3): 462-468 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction urbanization is increasing across the globe and it is recognized as a major factor affecting species, populations and assemblages (turner et al., 2004; grimm et al., 2008). although urbanization is recognized as a major threat to biodiversity, there is increasing evidence that urban habitats may play a role in conservation (evans et al., 2009). however, the general belief is that more natural environments, such as rural areas, provide a more suitable habitat for most species and thus for their conservation (turner et al., 2004; evans et al., 2009). indeed, human settlements in rural and urban areas differ in many structural and biotic components and even in human attitudes concerning wildlife (lepczyk et al., 2004). in urban landscapes, the presence of wildlife is limited by the availability of habitats, human disturbance, collisions with vehicles and behavioral shyness (ditchkoff et al., 2006). abstract urbanization is increasing across the globe and it is recognized as a major factor affecting species, populations and assemblages. although urbanization is recognized as a major threat to biodiversity, there is increasing evidence that urban habitats may play a role in conservation. the objective of this work was to verify the occurrence of polistes species (vespidae) and the substrates used for nesting in urban areas in south-western iberian peninsula. the study was carried out from march to august of 2018 in three small towns in the llanos de olivenza region (sw spain). active searching was conducted for colonies of social wasps along and for each colony that was found we identified the species, type of substrate used for nesting, height of the colony in relation to the ground level and orientation. 753 colonies of social wasps were found belonging to two species: polistes dominula and p. gallicus. the most used nesting substrate was clay roof tile followed by metals. the height of the nests was related to the height of the buildings on which they were built. in all the tree towns nests exposure were oriented to the se with mean angle values oscillating between 127.42° and 140.68°. in addition, our results confirm the prediction that wasps are more abundant in less or non-urbanized areas even if they are small urban areas such as those studied in our case. sociobiology an international journal on social insects jl pérez-bote, c mora-rubio article history edited by fabio nascimento, usp, brazil received 19 november 2019 initial acceptance 28 july 2020 final acceptance 13 august 2020 publication date 30 september 2020 keywords urbanization, nest abundance, nest substrates, nest orientation. corresponding author jl pérez-bote department of zoology faculty of sciences university of extremadura av. de elvas s/nº, 06006 badajoz, spain. e-mail: jlperez@unex.es previous studies on insects have demonstrated that urban habitats may not be as restrictive as expected (owen & owen, 1975; zapparoli, 1997). in addition, it has been observed that species richness and abundance can greatly vary within each city, indicating that different levels of urbanization have distinct effects on the local insect fauna (owen & owen, 1975; frankie & koehler, 1978; zapparoli, 1997; mcintyre, 2000; mcintyre et al., 2001). information about the responses of wasps to urbanization is important for a number of reasons. many wasp species, especially the eusocial ones, are key predators ecosystems (lasalle & gauld, 1993). wasps are sensitive to variations in abiotic conditions (e.g. temperature, luminosity and moisture), which may be related to changes in the urbanization level (morgan & jeanne, 1992). in addition, wasps are frequently found in urban environments, efficiently occupying different microhabitats (saure, 1996; raw, 1998). department of zoology, faculty of sciences, university of extremadura, 06006 badajoz, spain research article wasps nesting ecology of polistes species (hymenoptera, vespidae) in urban areas of southwestern iberian peninsula sociobiology 67(3): 462-468 (september, 2020) 463 in the south of the iberian peninsula, polistes species are widespread both in natural landscapes and in anthropogenic habitats, where the presence of the wasps usually alarm people living near the nests. numerous media campaigns have increased interest in this issue and have heightened the concerns of city inhabitants. this fear, which is not always fully justified, causes that people massively inform municipal services about practically all of the observed colonies of wasps. despite their ecological importance and their potential as indicators of environmental conditions (brown, 1991), few authors have analyzed the effects of urbanization on wasp assemblages in the iberian peninsula. in 1985 a study was conducted to analyze the influence of urban conditions on wasps and bees in the city of salamanca (gayubo & torres, 1990).these authors found that urbanization do not affect the populations of polistes dominula (christ, 1791) and polistes gallicus (l., 1767). in addition casamitjana (1989) studied some aspects related with the nesting of a small semiurban population of p. dominula located inside a brick-structure in an open space in the city of barcelona. in europe, the most complete study about nesting habits of p. dominula in urban areas was the conducted by höcherl and tautz (2015) in würzburg (germany). these authors concluded that p. dominula wasps are very flexible in their nesting behavior and that nesting itself is strongly linked to temperature conditions. outside its natural range only downing (2012) provides information about nest parameters of p. dominula, concluding that this species uses a wide range of nesting spots, some of which provided little shelter for the nest and were not locations that either of the native species would be found using. the objective of this work was to verify the occurrence of polistes species and the substrates used for nesting in urban areas in south-western iberian peninsula. we also took into account the height at which the nest was located and its orientation in relation to the north (azimuth). materials and methods from march to august of 2018, polistes nests were surveyed in three small towns in the llanos de olivenza region (sw spain): olivenza (ol; 12,008 inhabitants; urban area: 1,358,300 m2; 38° 41´8.03´´n, 7°6´3.24´´w; alt., 265 m a.s.l.), san francisco (sf; 466 inhabitants; urban area: 112,300 m2; 38°44´47.37´´n, 7°6´18.81´´w; 264 m a.s.l.) and san benito (sb, 575 inhabitants; urban area: 166,200 m2; 38°37´55.99´´n, 7°9´28.67´´w; 223 m a.s.l.). in the llanos de olivenza region the climate is typically continentalmediterranean with relatively cold wet winters and dry hot summers (mean temperature: 16.3 °c, rainfall: 432 mm yr-1). its flat to gently undulating landscape is dominated by a mosaic of dry winter cereal crops, olive groves, and vineyards. nest abundance was estimated along line transects of 100 m in each town. for each nest the species and the status: active, abandoned or old was recorded. we consider a nest as active when we detect the presence of wasps on the nest. in this case, the number of foundresses was recorded after detection of the respective nest and verified during the following weeks, until the first workers emerged. abandoned nests have the same structure and color as an occupied nest, they are usually gray-white nests with well-defined and undamaged edges. abandoned nests were visited for three consecutive days at different times to verify the absence of wasps. old nests are nests from previous years, they are generally darker in color than occupied nests, and their edges are irregular and their structure is damaged. in many occasions they appear broken or partially sagged. binoculars were used to observe and identify the species on high nests. active nests were left in place after species identification was noted for each nest. for species identification keys schmid-egger et al. (2017) were used. collected nests and wasps were deposited as voucher specimens in the zoology department insect collection (university of extremadura, badajoz, spain). the next features were estimated for each nest: height (using a laser distance meter), orientation (using a digital compass), and substrate (clay roof tile, crt; corrugated asbestos, ca; direct on wall, dw; metal tubes, mt; noncylindrical metallic structures, ncm; plant material, pm; others, ot). localization, height and orientation were estimated just above the nest, and in the case of the height from the soil to the nest. differences in height of the nests between species were tested with the mann-whitney u test. the preferences between substrates were tested by χ2 tests. we used program oriana (kovach, 2011) to calculate circular statistics for nest orientation: mean angle and circular standard deviation (c.s.d). rayleigh’s test of uniformity was used to test mean exposures for nonrandom orientations (batschelet, 1981). concentration (r) or length of the mean vector was calculated by circular methods, ranges from 0 to 1 and is affected by variation in circular data, sample size, and grouping. values of r near 1 indicate data points closely concentrated near the mean angle or, in our case, nest-sites with a preferred directional orientation. differences in mean direction between years were analyzed using the parametric watson–williams f-test in the cases where the assumptions underlying this test were fulfilled (batschelet, 1981). results a total of 744 wasp nests were found in the surveys, of which 583 were not active and 161 were active. of the active nests 106 were occupied by p. dominula and 55 by p. gallicus (table 1). the nests of p. dominula were significantly more abundant in olivenza (χ2 = 13.16, p < 0.001) and san benito (χ2 = 17.00, p < 0.001), while the nests of p. gallicus were more abundant in san francisco, but the differences were not significant (χ2 = 0.51, p = 0.5102). overall, nests of p. dominula were significantly more abundant than nests of p. gallicus (χ2 = 16.5, p < 0.001). in the case of p. dominula the number of foundresses per nest ranged between one and jl pérez-bote, c mora-rubio – nesting ecology of polistes in urban areas464 four (1.9230 ± 0.7344), whereas in the case of p. gallicus the number of foundresses per nest ranged between one and tree (1.1750 ± 04464). thus, we determined a ratio of 29.79% single-versus 70.51% multiplefoundress colonies for p. dominula, and a ratio of 84.62% single-versus 15.38% multiplefoundress colonies for p. gallicus. the multiple foundation was significantly higher in p. dominula than in p. gallicus (χ2 = 39.36, p < 0.001). the mean number of active nests per transect was higher in san francisco than in san benito and olivenza (table 2). the same occurred with the mean number of nonactive nests and the mean number of total nests. similarly, the number of active nests per 100 m was greater in san francisco (4.60) than in san benito (3.48) and olivenza (1.68, table 2). among the 753 colonies registered, the most used nesting substrate was clay roof tile in 60.02% (χ2 = 30.28, p<0.0001) of the records (fig 1). secondly, nets build directly on walls represented with 10.09%, followed by metals with 9.63% of nest found; the fourth position was occupied by plant material with 6.64%, followed by glass corrugated asbestos with 4.78%. the others categories include nests found on glass, plastic, and wood (in door, windows). in the case of the species, both p. dominula and p. gallicus preferred the clay roof tile as a material to build their nests. town active nests non active nests total p. dominula p. gallicus olivenza 52 21 382 455 san benito 32 7 95 134 san francisco 22 27 106 155 town active nests non active nests total active nest/100 m non active nest/100 m an active nest for each… an active nest by olivenza 0.55 ± 0.94 2.80 ± 3.28 3.40 ± 3.81 1.68 0.29 15344 m2 148 inhabitants san benito 2.38 ± 1.53 5.04 ± 2.29 7.42 ± 2.83 3.48 1.10 2825 m2 15 inhabitants san francisco 2.60 ± 2.06 6.33 ± 3.64 8.93 ± 3.91 4.60 1.48 1986 m2 9 inhabitants table 1. number of nests in each of the sampled localities. table 2. average abundance of active and non-active nests in the localities studied and relative abundance of nests with respect to the urban area and the number of inhabitants. the height of nests was 4.88 ± 1.59 m (range: 0.1514 m) in olivenza, 3.28 ± 1.14 m (range: 1.3-7.0 m) in san benito, and 3.35 ± 1.04 m (range: 1.6-7.0 m) in san francisco. mean overall height of nests was 4.23 ± 1.57 m (range: 0.1514 m). the height of the nests did differed between the three tows (kruskal-wallis test, h= 164.54, p < 0.001). fig 1. type of substrate selected by wasps in each city and by species (clay roof tile, crt; corrugated asbestos, ca; direct on wall, dw; metal tubes, mt; non-cylindrical metallic structures, ncm; plant material, pm; others, ot). in all the tree towns nests exposure were oriented to the southeast (se) with mean angle values oscillating between 127.42° and 140.68 ° (table 3). the length of the mean vector oscillated between 0.477 and 0.748. in all cases nests were significantly oriented on se direction (raleigh’s z test). differences in nest orientations were only significantly sociobiology 67(3): 462-468 (september, 2020) 465 between san francisco and san benito (watson-williams f-test, f = 4.3946, p = 0.036). overall, nests were oriented to the se with a mean vector of 131.94° and a length of mean vector of 0.474 (table 3). nests were significantly oriented to the se (raleigh’s z = 163.746, p < 0.0001).the analysis by species showed that nests of p. dominula and p. gallicus were significantly oriented to the se (table 3) and their orientations did not differed (watson-williams f-test, f = 0.8199, p = 0.368). town mean angle ± c.s.d length of the mean vector (r) raleigh´s z p value town olivenza 128.55˚ ± 69.76˚ 0.477 109.903 < 0.0001 san benito 140.68˚ ± 62.14˚ 0.555 41.326 < 0.0001 san francisco 127.42˚ ± 43.62˚ 0.748 91.847 < 0.0001 all towns 131.94˚ ± 70.01˚ 0.474 163.746 < 0.0001 species p. dominula 142.79˚ ± 36.99˚ 0.812 35.593 < 0.0001 p. gallicus 149.76˚ ± 51.61˚ 0.667 47.089 < 0.0001 table 3. nest orientations in the three sampled localities and by species. discussion it is generally assumed that urbanization reduces habitat complexity, mostly by reducing natural vegetation cover. consequently, it may be predicted that less urbanized areas are structurally more complex. for these reasons, it can also be predicted that less urbanized areas support a greater abundance and species richness of wasps and bees (zanette et al., 2005). previous works (torres & gayubo, 1989; zanette et al., 2005) have demonstrated that wasps were more abundant in the less or non-urbanized areas of cities. our results seem to confirm these predictions, as we have shown that in both species the number of nests decreases with increasing levels of urbanization (square meters built, number of inhabitants). according to zanette et al. (2005) the reduction in prey availability caused by the loss of vegetation may be one of the factors responsible for these results. in addition, the increase of pavements and buildings coupled with the reduction of vegetation cover increases the exposure of wasp colonies to possible predators and particularly to humans, which are the major cause of colony mortality in urban environments (fowler, 1983). one of the advantages of built nests in urban environments is the reduction of the competence for nesting sites (da silva et al., 2019). p. dominula and p. gallicus are the only synanthropic wasps species in the iberian peninsula (gayubo & torres, 1990) and the observed differences in nest abundance between these species in this study may be related with their biological traits. as a primitively eusocial wasp, the cooperation of queens in nest building and brood care of polistes species is common (reeve, 1991). we detected maximal four foundresses (in p. dominula) per nest during the study period. this number of foundresses is in contrast with observations made in other studies. for instance, zanette and field (2009) recorded nest buildings of p. dominula wasps by 1–10 foundresses in southern spain. the colonies of p. gallicus are founded by a single foundress in open locations (cervo & turillazzi, 1985). in our study it became apparent that p. gallicus preferred single over multiple foundation of colonies contrary to p. dominula. for this species turillazzi et al. (1982) determined a ratio of 36 % single-versus 64 % multiplefoundress colonies and zanette and field (2011) described a ratio of 7 % single versus 93 % multiple-founded nests. however, in germany (höcherl & tautz, 2015) the hibernating queens of p. dominula neither preferred multiple nor single founding (46 % and 54 %, respectively). one of the advantages of multiple founding is that the survival of nests increases in relation to single founding. another benefit of multiple-foundress nests is an increased productivity as measured by the number of cells per nest (queller et al., 2000; tibbetts & reeve, 2003). in addition, we have observed at our three study sites that p. dominula hibernating foundress emerges before and produces its first workers earlier than p. gallicus. according to gamboa et al. (2002) early worker production enhances productivity. polistes dominula may have other advantages over p. gallicus in addition to those we have considered. for example, p. dominula is less demanding than p. gallicus in relation to the selection of the nesting sites. downing (2012) demonstrated in the united states (where p. dominula is an invasive species), that p. dominula used a wide range of nesting spots, some of which provided little shelter for the nest and were not locations that either of the native species would be found using. in this way, gamboa et al. (2002) suggested that nesting sites may be a limiting resource, and its use of a wider diversity of nesting locations may provide some competitive advantages. the nesting sites chosen by wasps are strongly influenced by predation and the weather (jeanne, 1970). nesting on manmade substrates, in addition to natural ones, may be a useful strategy for wasp populations since their colonies may have higher developmental success as these locations may allow them to avoid their natural predators and take shelter from harsh weather conditions (alvarenga et al., 2010). polistes jl pérez-bote, c mora-rubio – nesting ecology of polistes in urban areas466 species may build their nest in man-made structures, such as eaves (cervo & turillazzi 1985) or attics (rusina et al., 2007), where metal, concrete, ceramic, stone, plastic, porcelain, asbestos, synthetic materials, and wood are highlighted as the substrates chosen by wasps in urban areas (lima et al., 2000; detoni et al., 2018). the apparent preference of the wasp for nesting under clay roof tiles (or arabian tile) might be related to the greater offer of this substrate in comparison to the others. probably in the iberian peninsula the tile is the material most used to cover the houses, especially in small towns and cities where buildings do not predominate. tiles are made of clay, are curved and protrude about 10-20 cm. with a thickness of 10-15 mm they provide excellent protection against cold and rain. the second category most used by wasps was metal tubes. some authors (lola et al., 2013; virgínio et al., 2016) have suggested that this preference was probably related with a phenomenon known as “heat islands”; the accumulated heat over the metallic superficies provides suitable conditions for the colonies to develop. we cannot rule out this fact, but in the south of the iberian peninsula very high temperatures are recorded in july and august, which can cause the death of the colony. in fact, most of the nests built on metallic tubes were located during the warmest months. according with da silva et al. (2019) the results might have been affected by each nesting substrate type availability rate in the area rather than preference. in this study nest height above ground (0.15 – 12 m) is greater than the reported for european species of polistes in natural habitats (15-25 cm, kozyra et al., 2016; 4-60 cm, cervo & turillazzi, 1985). this is no surprise, as wasps take advantage of the height of human structures to build their nests. in a similar study conducted in brazil (virgílio et al., 2016) nest were located at 2-4 m above ground. we are agreeing with kozyra et al. (2016) that nest height plays an important role in protecting the nest from predators. in this way we suppose, as occurs in birds (mainwaring et al., 2014), that wasps vary the height at which they build their nests in response to predators, as they build their nests higher from the ground in response to mammalian and ant predators and lower in response to avian predators. in many social insects, such as ants and termites, the geographic orientation of nests is not accidental and is of great significance for their thermoregulation (jones & oldroyd, 2006). nest orientation often influences the amount of solar radiation absorbed by a nest and the time of day that the highest radiation is received. many species orientate their nests so that it is warmed by solar radiation in the cool of the morning (jones & oldroyd, 2006). the orientation of nets of polistes in our study (131.94°) is very similar to those reported for p. nimpha in poland (110°, kozyra et al., 2016), and in italy (162°, cervo & turillazzi, 1985) and agree with orientations reports by yamane (1996) for p. snelli and p. biglumis (southern and southeastern slopes). although the above examples are for non-urban populations, the trend appears to be the same. in tropical areas the situation seems to be quite different, as occurs in brazil for example with other vespids. in the state of minas gerais most colonies of mischocyttarus cassununga (vespidae, mischocyttarini) were north-oriented, between northwest and northeast, 271° to 90º (de castro et al., 2014). in the lower amazon, colonies of m. drewseni were mainly oriented to the west (jeanne, 1972). in the state são paulo nests of m. cerberus styx were oriented to the east (giannotti, 1999), and in the state of mato grosso do sul montagna et al. (2010) did not find a preference for a particular nest orientation in mischocyttarus consimilli. however, for p. canadiensis virgílio et al. (2016) have pointed out that most of nests were oriented to the east. these results confirm that at least in temperate areas nest orientation is not accidental. according to sumner et al. (2018) scientists may provide robust evidence on the ecological and economic value of wasps to convince the public to view these insects favourably. an overall increase of scientific understanding could, in turn, help to improve the public’s perception of wasps. if this were achieved, we expect that the public would be more accommodating of wasps and more inclined to tolerate wasp nests in their local environment, rather than have them removed. increased tolerance will benefit the conservation of these important insects even in urban environments. acknowledgements we sincerely thank people for olivenza, san francisco de olivenza and san benito de la contienda for help and collaboration during the realization of this study. cmr was supported by a departmental collaboration grant ministry of education, spanish government. references alvarenga, r.b., castro, m.m., santos-prezoto, h.h. & prezoto, f. 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(1997). urban development and insect biodiversity of the rome area, italy. landscape and urban planning, 38: 77-86. doi: 10.1016/s0169-2046(97)00020-0 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.6232sociobiology 68(3): e6232 (september, 2021) introduction ants are social insects of the family formicidae in the order hymenoptera. they account for 15% of the world’s animal biomass (passera, 2016). more than 13,922 species are identified in the world (bolton, 2021). they are widely distributed over different latitudes and biotopes: underground, on the soil surface, in the air, and on plants and animals. throughout the ages, ants have been able to survive thanks to unique self-adapting and organizing mechanisms. as biological indicators of environmental quality, insects play abstract the current study deals with the diversity of ants (hymenoptera, formicidae) in kabylia of djurdjura. it has been carried out in two forest sites from the localities of azeffoun and assi-youcef, tizi ouzou (northern algeria). two sampling methods were adopted: manual capture and pitfall traps. various community metrics were used as key elements to assess ant biodiversity within the two study sites. they included the species richness, the relative abundance, the constancy, and the shannon-weaver and equitability indices. our inventory allowed identifying 24 species belonging to 12 genera and three subfamilies, which are the myrmicinae (58%), the formicinae (34%), and the dolichoderinae (08%). the highest species richness was registered for the two first subfamilies, while the subfamily dolichoderinae dominated numerically. the site of azeffoun, which is more submitted to human activities, recorded higher values in the number of individuals, the species richness, and the shannon-weaver diversity index. however, the difference between the two sites consisted mainly of the rare species, such as crematogaster laestrygon, goniomma sp. and palagiolepis sp., which were present in the azeffoun site. the local site conditions certainly have played a key role in ant species occurrence within the two study areas. azeffoun is more disturbed than assi-youcef, resulting in the recruitment of much more rare and accidental species in the first site. in contrast, the rate of accessory to omnipresent species is substantially higher in assi-youcef, which recorded a high species evenness. to the best of our knowledge, this is the first study to report the ant genus formica in kabylia of djurdjura. sociobiology an international journal on social insects sadou sid-ali1, sadoudi ali-hmed djamila1, metna ali-ahmed fatiha1, ourrad ouiza1, slimani said2 article history edited by evandro n. silva, uefs, brazil received 09 february 2021 initial acceptance 27 june 2021 final acceptance 23 july 2021 publication date 13 september 2021 keywords formicidae, mediterranean, species richness, taxonomy, algeria. corresponding author sadou sid-ali laboratoire protection, sauvegarde des espèces menacées et des récoltes, influence des variables climatiques. université mouloud mammeri de tiziouzou, algérie. e-mail: sidali.sadou@ummto.dz a key role in ecosystem functioning (alonso, 2000). however, their identification remains complex, which constitutes a major problem for their use in environmental studies (new, 1996). ants have attracted the attention of many researchers since the last century. the formicidae have been documented in several studies all over the world, with a focus on their biology and ecology. from the 1970, many myrmecofauna inventories were carried out through project aimed at strengthening our understanding of biodiversity (bernard, 1973, 1976; passera, 1984; jolivet, 1986; longino & colwell, 1997; delabie et al., 1 laboratoire protection, sauvegarde des espèces menacées et des récoltes, influence des variables climatiques. université mouloud mammeri de tizi-ouzou, algérie 2 laboratoire de production, amelioration et protection des végétaux et des denerées alimentaires. université mouloud mammeri de tizi-ouzou, algérie research article ants diversity of ants (hymenoptera, formicidae) in two forest sites from kabylia of djurdjura, northern algeria sadou sid-ali, sadoudi-ali ahmed djamila, metna-ali-ahmed fatiha, ourrad ouiza, slimani said – diversity of ants in two northern algerian sites2 2000; marinho et al., 2002; vasconcelos et al., 2003; hites et al., 2004; lepounce et al., 2004; lappola et al., 2006). the earliest studies on ants in north africa were those of emery (1914) in tunisia, dartigues (1988), doumandji et doumandji (1988), and barech et al. (2016, 2018) in algeria, and cagniant (2005) and taheri (2014) in morocco. it is noteworthy that since the first publications dedicated to the order hymenoptera in algeria, few studies have been dedicated to the family formicidae. many authors reported that data on ants in algeria in general and in the region of tizi ouzou, in particular, is scarce, old and fragmentary and that many sites remain unexplored and their fauna unknown (bernard, 1982, 1983; belkadi, 1990; cagniant, 1997, 2005; djioua & sadoudi-ali ahmed, 2015). the current study reports the first findings on ants (hymenoptera, formicidae) diversity in two forest sites from tizi ouzou (kabylia of djurdjura, northern algeria). these results provide relevant elements on the fauna in the study area and constitute vital information to support ongoing forest ecosystem management efforts in the region. material and methods study area the current study was carried out in two forest sites located at the extremities north and south of the wilaya of tizi ouzou (northern algeria) (fig 1). the two forested areas are situated some 50.5 km from one another. the site of azeffoun is situated on the northern slope, while assi-youcef is situated on the southern slope of the djurdjura mountain chain. the two study areas present important differences as regards their vegetation-soil-topography-climate association. the first site is located in the coastal zone of azeffoun (northern kabylia of djurdjura), at about 56 km north-east to tizi ouzou, at the geographical coordinates 36° 53’ 35” n and 4° 25’ 12” e. the sampled area is situated over an altitudinal gradient ranging from 50 to 300 m. it is characterized by a maritime mediterranean climate, wet and cool winter and a dry and warm summer. the total annual precipitation varies from 500 to 700 mm. the soil is derived from sandstone. this mediterranean coastal site is close to the urban area of azffoun, which makes it subjected to strong anthropic pressure, especially to grazing and forest fires. it consists of a reforested area of about 7,718 ha, dominated by a variety of introduced tree species of eucalyptus (e. globulus, e. sideroxylon, e. gomphocephala, e. cladocalyx, e. botryoides, e. occidentalis, e. maideni) and pinus (p. pinaster, p. pinea, p. canariensis). furthermore, there is a notable presence of the native and spontaneous oak species quercus suber. main understorey vegetation consists of pistacia lentiscus, daphne gnidium, erica arborea, cistus monspenliensis, and calycotome spinosa. the second site is located in a mountainous area belonging to the municipality of assi-youcef (southern kabylia of djurdjura), at about 50 km southwest to tizi ouzou, at the geographical coordinates 36° 27’ 54” n and 4° 05’ 47” e. fig 1. location of the study sites. sociobiology 68(3): e6232 (september, 2021) 3 the sampled campaign was performed over an altitudinal gradient ranging from 600 to 950 m. it is characterized by a mediterranean climate, wet and cold winter and dry and warm summer. the total annual precipitation exceeds 900 mm. the soil is derived from limestone. this site is situated within the national park of djurdjura. intensive grazing and forest fires are observed, but anthropic activities are much lower than in the first site. this study area consists of a mixed cedrus atlantica and quercus ilex forest with a very low density of the understory, mainly represented by juniperus oxycedrus, calycotome spinosa, erica arborea, cytisus triflorus, ampelodesma mauritanicum, and asphodelus microcarpus. sampling procedure myrmecofauna sampling campaigns were carried out within the two study sites during the years 2018 and 2019. the samples were collected from march to july, every ten days, three times a month. two sampling methods were applied in the current study: the barber pots and the manual capture. pitfall traps the first adopted method consisted of using pitfall traps. this technique allows capturing many walking arthropods and flying insects, which land on the surface of the trap or are driven to it by the wind (benkhelil, 1991). the traps are vertically buried, with the opening adjusted to the ground surface. to avoid any effect due to barriers towards the traps for tiny insects, the area around the placed barber pots was cleaned. a total of ten traps were placed in each site along a sampled transect determined by a twine (a trap every five meters). the traps were filled to 1/3 of their height with water, and a detergent is added to facilitate entrapment of attracted insects. the traps’ content was collected after 48 hours and stored in plastic tubes with a data tag. manual capture the manual capture method was performed from late april to late july, as described by mc gavin (2000). during this period of the year, the capture is easy, as ants are very active and occupy the superficial chambers of their nets. species identification the collected ants were preserved in 75% ethyl alcohol. their identification was performed using the taxonomic keys of bernard (1968) and cagniant (1968, 1970, 1997, 2005, 2006, 2009). statistical analyses a variety of statistical parameters were used to describe the biodiversity of ants in our study areas. the species richness (s) represents the total number of ant species collected at each site. the relative abundance (ra) is the percent composition of a given ant species relative to the total number of individuals collected in a given site. the species constancy (c), or the frequency of occurrence, is the number of samples in which a given species is found relative to the total number of samples. six constancy classes were considered: the species is rare if c ≤ 17%, accidental if 17% < c ≤ 34%, accessory if 34% < c ≤ 51%, regular is 51% < c ≤ 68%, constant if 68% < c ≤ 85% and omnipresent if c > 85%. the shannon–weaver diversity index (h’) was applied, using the following equation: h’ = -σpi log2 pi where pi = ni / n; ni being the total number of the species i and n is the total number of individuals. the equitability or evenness index (e) was estimated according to pielou as follows: e = h’/hmax where h’ is the shannon–weaver diversity index and hmax = log2 s, s being the species richness. in addition, sampling effort is assessed using the nonparametric species richness estimators of chao, jackknife 1, jackknife 2 and bootstrap, which calculate the number of cumulative observed and expected species in the study sites with 100 randomizations. fig 2. pitfall traps. sadou sid-ali, sadoudi-ali ahmed djamila, metna-ali-ahmed fatiha, ourrad ouiza, slimani said – diversity of ants in two northern algerian sites4 results our sampling campaigns allowed collecting 641 ant individuals: 454 in azeffoun and 187 in assi-youcef. ant species richness (s) within the two study sites is reported in table 1. a total of 24 species were identified. they belong to 12 genera and three subfamilies: myrmicinae (58%), the formicinae (34%) and the dolichoderinae (8%). all 24 species were present in azeffoun, while only 13 were found in assi-youcef. with seven genera and 13 species, the subfamily myrmicinae was the most diversified. each of the genera crematogaster, aphaenogaster, and messor were represented by three species. only one species represented the other genera (tetramorium, pheidole, goniomma and temmothorax). the subfamily formicinae registered ten species belonging to four genera. the genera cataglyphis and formica were represented by one single species, the genus plagiolepis recorded two species, and the camponotus was the richest genus, with six species. the least diversified subfamily was the dolichoderinae, which registered only one genus (tapinoma) and one species (tapinoma simrothi) (fig 3). from a quantitative perspective, the ant relative abundance (ra) varied between the two sites according to the species rank (fig 4 and 5). in azeffoun, tapinoma simorthi was the most abundant species (ra = 13.22%). it was followed by pheidole pallidula (ra = 12.56%), and cataglyphis viaticus, and crematogaster sticularis (ra = 11.01% for both). the most abundant species in assi-youcef was pheidole pallidula (ra = 22.32%). it was followed by tapinoma simorthi, palageolipis schmitzi, and cataglyphis viactus, with ra values of 16.04%, 11.76%, and 10.70%, respectively. all six considered constancy classes were observed while considering the two study areas (table 2). table 1. ant species richness in the two study sites. site subfamily species azeffoun assiyoucef camponotus toracicus + + camponotus spissinodis + camponotus ruber + camponotus latiralis + + formicinae camponotus alii + + camponotus barbarous + + cataglyphis viaticus + + formica sp. + palagiolepis schmitzi + + palagiolepis sp. + myrmicinae crematogaster sticularis + + crematogaster auberti + crematogaster laestrygon + aphaenogaster testaceopilosa + + aphaenogaster senilis + + aphaenogaster sardoa + messor barbarous + + messor lobicornis + messor capitus + pheidole pallidula + + goniomma sp. + temnothorax sp. + tetramorium bisekrens + + dolichoderinae tapinoma simorthi + + species azeffoun assi-youcef c category c category camponotus toracicus 25.00 ac 33.33 ac camponotus spissinodis 04.76 ra 00.00 absent camponotus ruber 04.76 ra 00.00 absent camponotus latiralis 06.66 ra 06.66 ra camponotus alii 28.33 ac 16.66 ra camponotus barbarous 08.33 ra 13.33 ra cataglyphis viaticus 66.66 reg 60.00 reg formica sp. 16.66 ra 0.00 absent palagiolepis schmitzi 66.66 reg 66.66 reg palagiolepis sp. 03.33 ra 00.00 absent crematogaster sticularis 75.00 con 40 acc crematogaster auberti 30.00 ac 00.00 absent crematogaster laestrygon 01.66 ra 00.00 absent aphaenogaster testaceopilosa 10.00 ra 06.66 ra aphaenogaster senilis 75.00 con 50.00 acc aphaenogaster sardoa 08.33 ra 00.00 absent messor barbarous 58.33 reg 50.00 acc messor lobicornis 05.00 ra 00.00 absent messor capitus 06.66 ra 00.00 absent pheidole pallidula 91.66 omn 93.33 omn goniomma sp. 01.66 ra 00.00 absent temnothorax sp. 05.00 ra 00.00 absent tetramorium bisekrens 08.33 ra 40.00 acc. tapinoma simorthi 93.33 omn 93.33 omn. table 2. constancy values (c) and their corresponding categories for the two study sites. five constancy classes were identified in azeffoun. accessory species were absent. a total of 14 species were rare. the rarest ones were goniomma sp. (c = 01.66%), crematogaster laestrygon (c = 01.66%) and palagiolepis sp. (c = 03.33%). three species were accidental: crematogaster auberti (c = 30.00%), camponotus alii (c = 28.33%) and camponotus toracicus (c = 25.00%). likewise the regular ones were represented by three species, which were palagiolepis schmitzi (c = 66.66%), cataglyphis viaticus (c = 66.66%) and messor barbarus (c = 58.33%). two species sociobiology 68(3): e6232 (september, 2021) 5 were constant: crematogaster sticularis (c = 75.00%) and aphaenogaster senilis (c = 75.00%). two other species were omnipresent: tapinoma simorthi (c = 93.33%) and pheidole pallidula (c = 93.33%). five constancy classes were also identified in assiyoucef. in this site, no ant species was identified as constant. four species were considered rare: camponotus alii (c = 16.66%), camponotus barbarus (c = 13.33%), camponotus latiralis (c = 06.66%) and aphaenogaster testaceopilosa (c = 06.66%). the class accidental was represented by only one species, camponotus toracicus (c = 33.33%). four accessory species were recorded: aphaenogaster senilis (c = 50.00%), messor barbarus (c = 50.00%), crematogaster sticularis (c = 40.00%) and tetramorium bisekrens (c = 40.00%). regular and omnipresent species were both represented by two species, which are palagiolepis schmitzi (c = 66.66%) and cataglyphis viaticus (c = 60.00%), and pheidole pallidula (c = 93.33%) and tapinoma simorthi (c = 93.33%) respectively. the majority of the species identified in azeffoun were rare (58.33%). they are followed by the accidental and the regular and species (12.50% for each category), and the least represented were the omnipresent (08.33%). in assi-youcef, the most present species belonged to the categories rare and accessory (30.77% for each class). they are followed by the regular and omnipresent species (15.38% for each of the classes), and the accidental ones came last (07.69%). 4 7 1 10 11 1 f o r m i c i n ae m y r m i c i n a e d o l i c h od er i n ae subfamillie number of genera number of species fig 3. number of genera and species for each subfamily in the two study sites. sp 1 sp 2 sp 3 sp 4 sp 5 sp 6 sp 7 sp 8 sp 9 sp 10 sp 11 sp 12 sp 13 sp 14 sp 15 sp 16 sp 17 sp 18 sp 19 sp 20 sp 21 sp 22 sp 23 sp 24 0,1 1 10 100 0 5 10 15 20 25 30r el at iv e ab un da nc e rank of species fig 4. rank-abundance curve for the relative abundance (log10ni/n) of ant species collected in azeffoun. sp 1 : tapinoma simorthi; sp 2: pheidole pallidula; sp 3: cataglyphis viaticus; sp 4: crematogaster sticularis; sp 5: aphaenogaster senilis; sp 6: palagiolepis schmitzi; sp 7: messor barbarus; sp 8: crematogaster auberti; sp 9: camponotus alii; sp 10: camponotus toracicus; sp 11: formica sp; sp 12: aphaenogaster testaceopilosa; sp 13: camponotus barbarus; sp 14: aphaenogaster sardoa; sp 15: temnothorax sp; sp 16: tetramorium bisekrens; sp 17: camponotus latiralis; sp 18: palagiolepis sp; sp 19: messor capitus; sp 20: messor lobicornis ; sp 21: camponotus spissinodis ; sp 22: camponotus ruber ; sp 23: crematogaster laestrygon; sp 24: goniomma sp. sadou sid-ali, sadoudi-ali ahmed djamila, metna-ali-ahmed fatiha, ourrad ouiza, slimani said – diversity of ants in two northern algerian sites6 it is worth mentioning that the absent species in assiyoucef registered a low presence in azeffoun: ten species out of 11 were rare, and one was accidental. moreover, the rare species in azeffoun were absent (ten species), rare (three species), or accessory (one species) in assi-youcef. on the contrary, the rare species in assi-youcef were rare (three species) or accessory (one species) in azeffoun. in addition, the two sites registered the same omnipresent species, which are tapinoma simorthi and pheidole pallidula. however, we notice that the rates of the rare and the accidental species were lower and that of the omnipresent species was higher in assiyoucef than in azeffoun. the results of the shannon-weaver (h’) and equitability (e) indices are presented in table 3. h’ recorded a higher value in azzefoun (2.61) than in assi-youcef (2.29). this also coincides with higher values in s and h’max. we notice that h’ is relatively close to h’max in assi-youcef, while in azeffoun, the difference between the two parameters is huge. on the contrary, assi-youcef registered a slightly higher value in e. sp 1 sp 2 sp 3 sp 4 sp 5 sp 6 sp 7 sp 8 sp 9 sp 10 sp 11 sp 12 sp 13 0,1 1 10 100 0 2 4 6 8 10 12 14r el at iv e ab un da nc e rank of spcies fig 5. rank-abundance curve for the relative abundance (log10ni/n) of ant species collected in assi-youcef. sp 1: pheidole pallidula; sp 2: tapinoma simorthi; sp 3: palagiolepis schmitzi; sp 4: cataglyphis viaticus; sp 5: aphaenogaster senilis; sp 6: messor barbarus; sp 7: crematogaster sticularis; sp 8: tetramorium bisekrens; sp 9: camponotus toracicus; sp 10: camponotus alii; sp 11: camponotus barbarus; sp 12: camponotus latiralis; sp 13: aphaenogaster testaceopilosa. parameter site azeffoun assi-youcef h’ 2.61 2.29 s 24 13 h’max 4.585 3.70 e 0.56 0.62 table 3. shannon-weaver (h’) and equitability (e) indices for the two study sites. sites azeffoun assi-youcef azeffoun assi-youcef estimators value completen-ess average completeness completeness average completeness observed richness 24 13 chao 44,63 ± 19.59 15,93 ± 3,63 53,77 % 67,09 % 81,60 % 81,60 % 81,90 % jackknife 1 33,72 ± 4,07 15,93 ± 1,37 71,17 % jackknife 2 40,36 16,90 59,46% 76,92 % bootstrap 28.58 ± 2,06 14,86 ± 0,77 83,97 % 87,48 % table 4. estimation of the total richness of the ants. in azeffoun, the estimated total species richness was higher than the observed value (sobs. = 24). according to the different used estimators, the number of new species to be found in this site is 16 to 20 with the chao 1 and jackknife 1 indices, 9 with the jackknife 2 index and four with the bootstrap index (table 4). in contrast, in assi-youcef, the estimated values are close to the observed, with a minimal estimate of one to three more species to be found in this site: sobs. = 13, chao = 15.93 ± 3.63, jackkniefe 1 = 15.93 ± 1.37, jackknife 2 = 16.90 and bootstrap = 14.86 ± 0.77). in addition, the inventory completeness rate reached a value of 67.09% in azeffoun and 81.90 in assi-youcef. moreover, the inadequacy of the sampling effort within the two sites is confirmed by the difference between the species accumulation curves (sobs.) and those obtained using the adopted estimators (fig 6 and 7). sociobiology 68(3): e6232 (september, 2021) 7 sample chao jackknife1 jackknife2 bootstrap3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 size 10 15 20 25 30 35 40 45 50 sp ec ifi c ric hn es s sample chao jackknife1 jackknife2 bootstrap 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 size 6 8 10 12 14 16 18 s pe ci fic r ic hn es s fig 6.curves of accumulation of the species, estimators of the richness in azeffoun. fig 7.curves of accumulation of the species, estimators of the richness in assi-youcef. sadou sid-ali, sadoudi-ali ahmed djamila, metna-ali-ahmed fatiha, ourrad ouiza, slimani said – diversity of ants in two northern algerian sites8 discussion the current investigation on the myrmecological fauna allowed identifying 24 species in the two studied sites. they belong to three subfamilies, which are: the myrmicinae, the formicinae, and the dolichoderinae. our findings showed that the species richness was much higher in azeffoun (24 species) than in assi-youcef (13 species). this difference may be due to the differences in ecological conditions within the two studied areas. the close relationship between ants and the type of habitats has been reported by agosti et al. (2000). indeed, the site of azeffoun is coastal and is located on the northern slope of the djurdjura mountain chain. on the contrary, assi-youcef is mountainous and is situated on the southern slope of the djurdjura massif. moreover, even if the two studied sites are forested areas, the site of azeffoun is a reforested one. it is close to the urban area, so it is more or less artificial and submitted to high anthropogenic pressure (overgrazing, forest fires, illegal loggers), while the site of assi-youcef consists of a spontaneous forest located in a protected area, which is the djurdjura national park. these differences suggest that the site of azeffoun is much more disturbed and open, which may play a key role in recruiting much more species than in assi-youcef. the soil type could also be a determining factor: in azeffoun, the soil is siliceous, while in assiyoucef, it is calcareous. a larger-scale study, carried out by djioua and sadoudi ali-ahmed (2015) in the region of tizi ouzou, involving four different sites (aghrib, azazga, ain el hammam, and tizi ouzou), allowed identifying 15 species. this richness is close to the one we found for assi-youcef. this result confirms the particular influence of the ecological conditions that prevail in our site of azeffoun, which recorded much higher species richness (24 species). the majority of the identified ant species in the two studied sites have already been reported by cagniant (1968, 1969, 1972, and 1973) and bouzekri et al. (2013) in their works on algerian ant populations. however, to the best of our knowledge, the current study is the first to report the genus formica in kabylia of djurdjura, occuring in the richest site, azeffoun. for both sites, the most abundant species were tapinoma simorthi and pheidole pallidula. similar results have been reported by belkadi (1990), djioua, and sadoudi ali-ahmed (2015) in the region of tizi ouzou and dehina (2009) in two stations in the algiers region. crematogaster scutellaris was also abundant. many authors have reported this species in the mediterranean forests; these results corroborate with those of janine (1972). our results are consistent with those reported by cagniant (2005) in morocco, who mentioned the genus crematogaster as one of the most abundant among the formicidae and corroborate those noted by djioua & sadoudi ali-ahmed (2015) in the region of tizi ouzou. the constancy index, reflecting the frequencies of occurrence, varies considerably among the identified ant species and confirms the difference mentioned between the two sites regarding their species richness. as mentioned above, the site of azeffoun recorded a higher number of identified species. however, the difference between the two sites was mainly represented by the category of rare species. consequently, more submitted to anthropogenic disturbances, the site of azeffoun tends to recruit more non-characteristic species. in contrast, located in a protected area, assi-youcef showed a higher rate of omnipresent species, which are characteristic of the forest environment. the values reported in table 2 were recorded from may to june. similar results have been reported in algeria by bakiri (2001) in the suburban environment near algiers and dehina (2009), who also listed tapinoma simrothi as an omnipresent species. however, different results have been reported concerning the omnipresent species, pheidole pallidula. hacini (1995) listed it among omnipresent species in staouali (northern algeria), while djioua & sadoudi-ali ahmed (2015) mentioned it as regular within four sites in the region of tizi ouzou. table 3 revealed that the shannon-weaver and equitability indices were high in both sites. however, the most disturbed site (azeffoun) recorded higher values of s, h’, and h’max. in contrast, the most stable one (assi-youcef) registered a higher value of e. in addition, this site showed close values of h’ and h’max, which suggests that many species have more or less the same abundance. on the contrary, the higher diversity in species of azeffoun is characterized by a high range of abundances among species. our findings stem from a sampling procedure associating manual capture and pitfall trap methods. other authors adopted this combined technique to assess ant biodiversity in algeria. their results showed lower h’ values: barech et al. (2016) noted values ranging from 1.35 bits in medbah to 1.47 bits in biskra. barech et al. (2018) reported an h’ value of 1.10 bits at the edges of the dam of el ksob (m’sila). this difference could be because our study areas are located in different climatic zones: our sites are situated in a humid area, facing the mediterranean sea, while the studies of barech et al. (2016, 2018) were carried out within sub-arid and arid zones, which are characterized by lower fauna biodiversity compared to the northern part of the country. according to the estimated total specifies richness, the adopted sampling methods allowed capturing two thirds (67.09%) of the ant species that would be present in azeffoun and over than three quarters (81.90%) of those expected to be found in assi-youcef. this justifies the differences exhibited between the accumulation curves of the captured and expected species within the two sites. the accumulation curve did not reach an asymptote for all samples. this suggests that the sampling effort is incomplete. hence, additional samples would be required in order to capture new species. sociobiology 68(3): e6232 (september, 2021) 9 conclusion a total of 641 ant individuals were collected. they belong to 24 species, 12 genera and three subfamilies, which are: the myrmicinae, the formicinae and the dolichoderinae. the local site conditions could have played a key role in ant species occurrence within the two study areas. the site of azeffoun is more submitted to human activities than assiyoucef, which is located in a protected area, the djurdjura national park. this promoted recruitment of much more rare and accidental species in that disturbed site, while the rate of accessory to omnipresent species is substantially higher in assi-youcef. to the best of our knowledge, the current study is the first to identify the genus formica in the region of tizi ouzou. compared to other works realized on ants since years. scientific research on this group of bio-indicator insects should be continued in order to better understand the biodiversity levels of these hotspots. acknowledgements the authors acknowledge the department of biology and the psemrvc laboratory, mouloud mammeri university of tizi-ouzou, for valuable support. we also thank doctor djaffer dib, slimani said and chafika ali-ahmed for their contribution. references alonso, l.e. 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(2003). influence of topography on the distribution of grounddwelling ants in an amazonian forest. studies on neotropical fauna and environment, 38: 115-124. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v66i4.4675sociobiology 66(4): 575-581 (december, 2019) morphometric variability among populations of euglossa cordata (hymenoptera: apidae: euglossini) from different phytophysiognomies introduction euglossa cordata (linnaeus, 1758) is a primitively social bee (garófalo, 1985; freiria et al., 2017), which pollinates plants of several botanical families, such as orchidaceae, apocynaceae, bignoniaceae and solanaceae (silva et al., 2007; lópez-uribe et al., 2008; coelho et al., 2017; ferreira-caliman et al., 2018). this species has wide distribution in the neotropical region (moure et al., 2012), occurring in several ecosystems in brazil (nemésio, 2009). furthermore, this bee is considered as indicator for dry, open and anthropic environments (aguiar & gaglianone, 2008; 2012; ferronato et al., 2017). abstract geometric morphometrics is a tool capable of measuring the response of organisms to different environmental pressures. we tested the hypothesis that e. cordata wing morphometry, as an indicator of response to environmental pressure, it would vary depending on habitat changes, in the atlantic forest, savanna and dry forest (caatinga). for analysis of wing shape and size, 18 landmarks were digitized at the intersections of the wing veins 348 individuals. except for the two populations sampled in chapada diamantina, the wing shape had significant statistical variations among the populations (p < 0.05). the wing size variation was also statistically significant among populations (p < 0.05). although e. cordata is a species tolerant to different environments, the observed morphometric variability may be related to population adaptations to the conditions of each phytophysiognomy. sociobiology an international journal on social insects ls carneiro1,2, cml aguiar1, wm aguiar3, es aniceto4, la nunes5, vs ferreira6 article history edited by celso martins, ufpb, brazil received 16 august 2018 initial acceptance 24 november 2019 final acceptance 28 november 2019 publication date 30 december 2019 keywords solitary bee, orchid bee, morphological variation, wing shape, wing size. corresponding author lázaro da s. carneiro laboratório de ciências ambientais-lca universidade estadual do norte fluminense av. alberto lamego, 2000, parque califórnia 280136002, campos dos goytacazes-rj, brasil. e-mail: lazarocarneiro16@gmail.com bee population responses to pressures in different environments can be observed through phenotypic variations (nunes et al., 2007; ribeiro et al., 2019). although it is easy to obtain samples of euglossini males, there is not enough knowledge about morphometric variability of these insects related to different environmental gradients (e.g. grassi-sella et al., 2018; ribeiro et al., 2019). this lack of studies may be related to the idea of variation absence in males of these bees, associated to haplodiploid characteristic, genetic determination of hymenoptera, in which most variation is conserved in females (zayed, 2009). furthermore, genetic studies on euglossa latreille, 1802 species have reported that environmental heterogeneity does not result in significant genetic variations 1 departamento de ciências biológicas, universidade estadual de feira de santana, feira de santana, brazil 2 centro de biociências e biotecnologia, universidade estadual do norte fluminense, campos dos goytacazes, brazil 3 instituto do meio ambiente e recursos hídricos, salvador, brazil 4 centro de ciências e tecnologias agropecuárias, universidade estadual do norte fluminense, campos dos goytacazes, brazil 5 faculdade de tecnologia e ciências da bahia, jequié, brazil 6 universidade federal do vale do rio são francisco, campus de ciências agrárias, petrolina, brazil research article bees ls carneiro et al. – morphometric variation of euglossa cordata576 among populations (dick et al., 2004; zimmermann et al., 2011; soro et al., 2017). this outcome seems to be related to the high dispersal capacity of these bees (pokorny et al., 2015), which results in gene flow between populations (cerântola et al., 2011). however, some studies verified the efficacy of morphological characters in males to identify morphotypes and cryptic species of the genus euglossa (francoy et al., 2012; quezada-euán et al., 2015), demonstrating the importance of using males in morphometric studies. geometric morphometrics, the study of shape and size associated with random factors (monteiro & reis, 1999; klingenberg, 2015), has been used as a tool for studies on intra and interpopulation variation in social bees species (mendes et al., 2007; prado-silva et al., 2016; combey et al., 2018) and solitary bees (ferreira et al., 2011; neves et al., 2012; dellicour et al., 2017; grassi-sella et al., 2018). such studies have shown the efficacy of that technique on understanding phenotypic expression of these insect populations associated to different environmental factors, such as altitude, geographic distribution, vegetation type and habitat fragmentation. considering that the interaction of organisms with the environment allows morphological variations, without necessarily causing genetic changes, which is a result of phenotypic plasticity (scheiner, 1993; schlichting & wund, 2014), we tested the hypothesis that e. cordata wing morphometry, as an indicator of response to environmental pressure, it would vary depending on habitat changes, in the atlantic forest, savanna and dry forest (caatinga). material and methods study area the sampling was carried out in five areas of bahia state, northeast of brazil, representing different phytophysiognomies: dense ombrophilous montane forest (dmf) and dense ombrophilous submontane forest (dsf) in the atlantic forest, semideciduous seasonal forest (ssf) and savanna (sav) on the highlands regions of chapada diamantina, and arboreal caatinga (arc), a dry phytophysiognomy typical in the northeast of brazil (fig 1). the distance between sampling sites ranges from 3 km (dmf x dsf) to 276 km (dsf x sav) (table 1). sampling the populations of the atlantic forest (dmf and dsf) and chapada diamantina (ssf and sav) were sampled between 2014-2015, while the population of caatinga (arc) was sampled between 2015-2016. three hundred and sixtytwo males of e. cordata were used in the analysis (table 2). sav dmf dsf ssf arc 222 216 219 209 sav 273 276 17 dmf 03 258 dsf 261 table 1. distance (km) among sampling phytophysiognomies. fig 1. phytosionomies sampled in the state of bahia, brazil. (dmf: dense ombrophilous montane forest; dsf: dense ombrophilous submontane forest; ssf: semideciduous seasonal forest; sav: savanna; arc: arboreal caatinga). sociobiology 66(4): 575-581 (december, 2019) 577 the bees were collected using bait traps (benzyl acetate, β-ionone, methyl cinnamate, eucalyptol, eugenol, methyl salicate and vanillin), based on the traps described by aguiar and gaglianone (2008). methods the left forewing of each individual was removed, fixed between two slides of microscopy, and photographed in leica stereoscopic microscope (96 dpi resolution). an image bank was created using the tpsutil 1.6 software (rolhf, 2013), that was opened in tpsdig 2.18 (rolhf, 2015), in which 18 landmarks were digitized at the intersections of the wing veins (fig 2). the wing size was estimated from the centroid size, which is the square root of the sum of of the landmarks distances relative to the center of gravity (klingenberg, 2015). subsequently, a variance analysis (anova one-away) and tukey comparison test were carried out. pearson’s test was used to verify if there was correlation between morphometric variables (wing shape and size) and environmental and geographic variables of each area (phytophysiognomy type, altitude, longitude and latitude). these tests were performed in the past 2.17c program (hammer et al., 2001). results in the principal components analysis (pca), 32 components were generated, of which the first nine components explained 72.4% of variation. in the canonical variable analysis (cva), the first two variables explained more than 80% of the data variation of wing shape. the first variable explained 48.5% and separated on the positive axis dmf and smf, and on the negative axis ssf and sav. the second variable explained 31.6% and separated on the positive axis sav and smf, and on the negative axis arc and sff (fig 3). the manova indicated low variation in wing shape between sff and sav (p = 0.05), and the difference in wing shape was statistically significant between the other e. cordata populations (wilk’s λ = 0.000; p < 0.05). the procrustes distance indicated higher wing shape similarity between arc and dmf populations, and higher dissimilarity between arc and sav (table 3). furthermore, from the test of permutation with 10.000 replicates associated to the procrustes distance, wing shape presented significant differences between populations (p < 0.05). table 2. number of individuals of euglossa cordata (l.) analyzed in each phytophysiognomy. (arc: arboreal caatinga; sav: savanna; ssf: semideciduous seasonal forest; dmf: dense ombrophilous montane forest; dsf: dense ombrophilous submontane forest). phytophysiognomy geographical coordinates altitude (m) number of individuals arc 11°57’s 39°32’w 235 46 sav 12°26’s 41°30’w 780 75 ssf 12°29’s 41°21’ w 516 51 dmf 13°53’s 39º27’w 660 93 dsf 13°54’s 39º27’w 500 97 total 362 fig 2. landmarks plotted at the intersections of the wing veins of euglossa cordata (l.). data analysis a procrustes superimposition was performed, eliminating any differences in size, position and orientation between configurations (klingenberg, 2015). the principal component analysis (pca), an exploratory analysis of data, was used to observe the variation level of the sample. in order to evaluate if there were morphometric differences in the wing shape between populations, it was used canonical variate analysis (cva) and a multivariate statistical analysis (manova). we have used the procrustes distance, i.e. values generated from the minimum sum of squares of the distances between landmarks after the procrustes superimposition (monteiro & reis, 1999), were used to evaluate the similarity of the wing shape between populations. these analyzes were made in morphoj 1.06d software (klingenberg, 2011). arc sav dmf dsf ssf arc 0.0001 0.0167 0.0001 0.0007 sav 0.0104 0.0001 0.0001 0.0006 dmf 0.0057 0.0092 0.0001 0.0001 dsf 0.0069 0.0077 0.0064 0.0001 ssf 0.0083 0.0074 0.0093 0.0083 table 3. procrustes distance generated from the canonical variate analysis (cva) between each phytophysiognomy (lower) and p value of the permutation test (10.000 permutations) (upper). ls carneiro et al. – morphometric variation of euglossa cordata578 the difference between means for wing size was statistically significant (anova one way, f = 10.58; p < 0.05). tukey’s multiple comparison test indicated that the wings of atlantic forest populations differed from the others (dsf x ssf, dmf x arc, dmf x sav, dmf x ssf). pearson’s test indicated positive correlation between wing shape and size with altitude and latitude, and negative correlation between wing shape and size with longitude and phytophysiognomy type, with high significance (p < 0.05) (table 4). environmental conditions (aguiar & gaglianone, 2008; 2012), the occurrence of interpopulation morphometric variations may reflect different environmental pressures in each phytophysiognomy, similar to what has been observed in social bees (nunes et al., 2007; combey et al., 2018). on the other hand, these variations observed in wing shape of e. cordata between the phytophysiognomies differed from those reported for populations of euglossa annectans dressler, 1982 and euglossa truncata rebêlo and moure, 1995 sampled in atlantic forest and savanna in brazil (grassi-sella et al., 2018). these authors reported the high morphological similarity between populations with the high dispersal potential of these bees. the wing shape variability may be related to factors such as genetic control, sexual selection and environmental heterogeneity (dellicour et al., 2017). euglossini bees present some ecological requirements that are scattered in the landscape (roubik & hanson, 2004). thus, it is possible that interpopulation wing shape variability in e. cordata reflects the adjustment of populations to fulfill their ecological requirements on different environments, since life history traits, such as foraging behavior, influence phenotypic adjustment (mendoza-cuenca & macías-ordóñez, 2005). the higher dissimilarity of e. cordata wing shape was observed between the populations sampled in caatinga (arc) and savanna (sav), although both are phytophysiognomies with open and shrub vegetation. the sav population comes from an area of 780 m of altitude in the chapada diamantina, north region of espinhaço moutain chain, and is separated by 222 km from the arc population, which is situated at lower altitude (235 m). furthermore, the geographic fig 3. scatter plot of the five populations of euglossa cordata (l.) analyzed, generated from the two canonical variables with higher variation of the canonical variate analysis (cva). table 4. result of pearson linear correlation (r) between the morphometrics variables (wing shape and size), with the geographic and environmental variables. morphometric variables geographic variables r p altitude 0.06 <0.05 wing shape latitude 0.24 <0.05 longitude -0.28 <0.05 phytophysiognomy -0.18 <0.05 altitude 0.23 0.21 wing size latitude 0.58 <0.05 longitude -0.46 <0.05 phytophysiognomy -0.31 <0.05 discussion based on wing shape and size analysis, populations of e. cordata showed morphometric variability. although this species has been indicated as tolerant to different sociobiology 66(4): 575-581 (december, 2019) 579 proximity between ssf (semideciduous seasonal forest) and sav areas could explain the low variation in wing shape between populations from these environments, as indicated in the manova test, because although they are distinct phytophysiognomies (forest and savanna), they are close to each other (~17 km), in the central region of the chapada diamantina. nunes et al. (2007) pointed out that the chapada diamantina mountains are an important barrier for other bees as melipona scutellaris latreille, 1811, precluding gene flow. the procrustes distance indicated that wing shape in the caatinga (arc) and atlantic forest (dmf and dsf) populations had higher similarity. it is possible that populations from caatinga still has some connectivity with populations in the atlantic forest, since these two biomes are neighbors and do not have geographic barriers separating them, which would facilitate gene flow, similar to what has been observed in other euglossa species (dick et al., 2004; zimmermann et al., 2011; soro et al., 2017). wing size showed significant variations between populations from the atlantic forest (dmf and dsf), with caatinga (arc), semideciduos seasonal forest (ssf) and savanna (sav) populations. in insects, size variations are related to quality and quantity of resources provided in larval stage (peruquetti, 2003; campos et al., 2018). the variations found in wing size may reflect the different ways in which floral resources are offered in each environment, since the dynamics of this supply may vary temporally in each phytophysiognomy, associated with variables such as pluviosity and temperature (morellato et al., 2000; conceição et al., 2007). campos et al. (2018) observed seasonal variation in the size of males and females of tetrapedia curvitarsis friese, 1899, which would be related to the amount of resources stored in brood cells in different seasons. the wing shape and size of e. cordata seems to be traits that vary between habitats, which may explain the presence of correlation found among these variables with phytophysiognomy type, latitude, longitude and altitude. these morphometric variations observed may be related to different populations responses to the local environmental characteristics of each phytophysiognomy, because insects may have plastic morphological adaptations in response to altitudinal and latitudinal geographical gradients (baranovská & knapp, 2018), or to local variations in landscape elements (ribeiro et al., 2019). grassi-sella et al. (2018) found a pattern of morphometric clustering in e. annectans and e. truncata related to the phytophysiognomy from which the populations were sampled, being such pattern attributed to phenotypic plasticity. thus, environmental variations resulted in morphological variability in e. cordata, despite its wide occurrence in different environments. genetic studies with these populations can indicate if the species presents a high interpopulation variability, which can subsidize conservation actions for these important pollinators. acknowledgements we are grateful to conselho nacional de desenvolvimento científico e tecnológico-cnpq (peld: 474065/2013-8) and organização de conservação da terra-oct for financial support. ls carneiro received scientific initiation fellowship from cnpq (process 114696/2016-0). ls carneiro and es aniceto receive master fellowship from coordenação de aperfeiçoamento de pessoal de nível superior – capes. references aguiar, w.m. & gaglianone, m.c. 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(2011). population genetic structure of orchid bees (euglossini) in anthropogenically altered landscapes. conservation genetics, 12: 1183-1194. doi: 10.1007/s10592-011-0221-1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.213-227sociobiology 62(2): 213-227 (june, 2015) check list of ground-dwelling ant diversity (hymenoptera: formicidae) of the iguazú national park with a comparison at regional scale introduction the atlantic forest (af) of south america houses 7% of the world’s species making it one of the most diverse ecosystems on the planet (quintela, 1990 in stevens & husband, 1998). in addition to its high biodiversity, the af is also characterized by high levels of endemism placing it among the world’s highest priorities for conservation (myers et al., 2000; galindo-leal & câmara, 2003). the af is considered one of the most endangered tropical rainforests because habitat loss and fragmentation due to agriculture and urbanization which has resulted in a drastic reduction in area (olson & dinesteirn, 2002; di bitetti et al., 2003; ribeiro et al., 2009). presently, only 6% of its original extent of 1,000,000 km2 remains along the east coast of brazil, in misiones province in northeastern argentina, and in eastern paraguay (galindo-leal & câmara, abstract we describe the ant fauna of iguazú national park (inp), a region of high biodiversity and endemism in northeastern argentina that includes the southernmost protected area of the atlantic forest (af). ants were sampled over seven periods from 1998 to 2011 using a variety of techniques. we also surveyed museum collections and the scientific literature to obtain additional records of ants from inp. in addition to providing a species list, we compare ant composition of inp to other sites in the upper paraná, serra do mar coastal forest and araucaria ecoregion of af. a total of 172 ant species belonging to 56 genera are reported; 56 species are new records for misiones province and 39 species are reported from argentina for the first time. alto paraná and canindeyú departments in paraguay present the most similar ant fauna to inp. serra da bodoquena in brazil and pilcomayo in argentina showed higher similarity with the upper paraná af ecoregion, despite that serra da bodoquena is composed of a mix of ecoregions. ant diversity was lower in upper paraná than in serra do mar coastal forest ecoregion. this difference may result from higher primary productivity and a greater altitudinal variation in the coastal region. sociobiology an international journal on social insects pe hanisch1; la calcaterra2; m leponce3; r achury4; av suarez4; rr silva5; c paris6 article history edited by john lattke, universidad nacional de loja, ecuador received 01 december 2014 initial acceptance 22 january 2015 final acceptance 02 march 2015 keywords ant biogeography, atlantic forest, faunistic surveys, neotropical ants. corresponding author priscila elena hanisch museo argentino de ciencias naturales ‘‘bernardino rivadavia’’ conicet buenos aires, argentina e-mail: hanisch.priscila@gmail.com 2003). however, this may be an underestimate; if secondary forest and small fragments are also included, approximaly 11.7% of original af still remains (ribeiro et al., 2009). the upper paraná atlantic forest ecoregion (upaf) is located at the southernmost extent of this forest. this region is an area of ecological transition among biomes including: the cerrado woodlands and savannas to the north, the pantanal and the humid chaco to the west, the campos and esteros ecoregion dominated by grasslands to the south, and finally to the east, it intermingles with araucaria forests, part of the af ecoregion complex (di bitetti et al., 2003). also, components of upaf extend south into argentina along the paraná and uruguay rivers. next to the araucaria forest is the serra do mar coastal forest ecoregion that includes one of the two largest remnants of the af. the second one is the protected area within the upaf (more than 10,000 km2) located across research article ants 1 museo argentino de ciencias naturales ‘‘bernardino rivadavia’’ conicet, buenos aires, argentina 2 fundación para el estudio de especies invasivas conicet, buenos aires, argentina 3 aquatic and terrestrial ecology unit, royal belgian institute of natural sciences, brussels, belgium 4 department of entomology, university of illinois, urbana, il, usa 5 museu paraense emílio goeldi, coordenação de ciências da terra e ecologia, belém, pa, brazil 6 universidad de buenos aires, buenos aires, argentina pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park214 the paraná state (brazil) and misiones province (argentina): the iguaҫu (br) and iguazú (ar) national parks (galindoleal & câmara, 2003). these parks are connected to the south with other areas of conservation and sustainable use given place to what is known as the misiones green corridor (garcía fernández, 2002). these two big remnants and the misiones province green corridor play an important role in the conservation of af flora and fauna at a regional scale (di bitetti et al., 2003; galindo-leal & câmara, 2003) because fragmented, poor connected areas or secondary forests cannot hold complete species assemblages sustained over time (e.g. chiarello, 2000; silva et al., 2007; bihn et al., 2010). given the unique nature of these areas, they are priorities for research for understanding the diversity and conservation value and the maintaining or restoring connectivity among them via land-use planning and restoration of other areas (ribeiro et al., 2009). insects, and particularly ants, are a valuable tool for conservation and restoration purposes as a result of the ecological services they provide (del-toro et al., 2012; small et al., 2013) and their use as environmental, ecological, or biodiversity indicators (see ribas et al., 2012). ants are considered as a key component of terrestrial ecosystems, directly or indirectly providing ecological services including seed dispersion control of herbivore populations, acceleration of forest recovery or plant succession, organic matter decomposition and nutrient cycling in the soil through their nest building and maintenance (folgarait, 1998; wilson & hölldobler, 2005; trager et al., 2010; del-toro et al., 2012). measures of ant species richness and functional composition are often used to understand community structure or as indicators of restoration success following great disturbance or environmental change (e.g. andersen & majer, 2004; underwood & fisher, 2006; bihn et al., 2010; pais & varanda, 2010; ribas et al., 2012). conversely, a few ant species that are native in af (e.g. linepithema humile, nylanderia fulva, solenopsis invicta, wasmannia auropunctata) have become invasive species around the world causing significant economic and ecological damage (holway et al., 2002; deltoro et al., 2012). documenting the distribution, ecology, and behavior of native populations of introduced ant species may provide insight for their control and guide monitoring programs for preventing invasions. studies on the biogeography and diversity of ants are of particular interest for effective conservation planning at multiple scales. in a global scale, due to ecological relevance and standardized samplings methods, ants are an ideal group to study diversity patterns along continents (dunn et al., 2007). at a regional scale, while vertebrates tend to persist only in larger forest remnants, invertebrates such as ants are valuable at identifying the conservation value of smaller remnants (kremen et al., 2008). in the af, butterflies and birds exhibit species richness peaks in coastal areas of brazil from 15º to 23°s (brown & freitas, 2000; galindo-leal & câmara, 2003). while some work has been done on the effects of forest loss on local ant assemblages in af (silva et al., 2007; bihn et al., 2008; bihn et al., 2010) we have little knowledge of ant diversity and its patterns across the af ecoregion complex. silva and brandão (2014) found a weak inverse latitudinal pattern in species richness with richer ant communities in higher than in lower latitudes. iguazú national park (inp) in northeastern argentina represents the southernmost protected area of the af, however, it remains poorly studied from the perspective of ants; only 5% of all studies conducted in the inp on biodiversity involved ants (http://www2.sib.gov.ar). the main objective of this work was to study the ant diversity in the inp in terms of species richness and species composition and compare these parameters with those from other protected areas located within and around the atlantic forest. materials and methods study site the study was carried out in semi-deciduous subtropical forest within inp, which is located in the northwestern of the misiones province, argentina (25°40’48.54’’s, 54°27’15.09’’w). the park was created in 1934 and covers more than 67,000 ha. the climate is subtropical humid without a defined dry season; annual rainfall ranges between 1800 and 2000 mm, and humidity between 70 and 90%. mean monthly temperatures range from 15°c (winter) to 26°c (summer). the topography of this region is undulating as a result of the erosion of a network of rivers with elevation running from 0 to 500 m (devoto & rothkugel, 1936; srur et al., 2007). variations in the local environment and soil properties allow a variety of plant communities to occur including: gallery forests, bamboo forests, palmito (euterpe edulis) forests, and araucaria forests (di bitetti et al., 2003; srur et al., 2007). we compared ant composition of inp to four previously studied areas at similar latitude within the af: alto paraná department (wild, 2007), rio cachoeira (bihn et al., 2008), parque estadual da ilha do cardoso (peic), and parque estadual do pau oco (pepo) (silva & brandão, 2014). we also compared our surveys to the canindeyú department where a special sampling effort was made in the mbaracayú forest reserve, a 65,000 ha fragment of af (wild, 2007), and seara municipality in santa catarina (south of brazil) as a represent of the araucaria forest ecoregion (silva & silvestre, 2004). finally, we included three other regions neighboring the af: serra da bodoquena national park (silvestre et al., 2012), pilcomayo national park (leponce et al., 2004), and iberá nature reserve (calcaterra et al., 2010). iberá nr located in corrientes province (argentina) has four main natural habitats (savanna, dry woodlands, gallery forest, and floating islands), but we only included the gallery forest habitat in this study, which share an important vegetation components with the af (fig 1). additional information about the study sites is provided in table 1. sociobiology 62(2): 213-227 (june, 2015) 215 fig 1. locations of sampled areas in paraguay, brazil and argentina used to compare myrmecological fauna against iguazú national park. atlantic forest is delimited by dotted areas meanwhile upper paraná ecoregion is shown with straight lines. within both regions remnants of forest are shown in grey. latitude longitude ecoregion sampling protocol (t = no. sampling sites; n = no. samples) iguazú np 25°40’48.54’’s 54°27’15.09’’w upper paraná af pitfall traps (t = 9; n=78); winkler sacs (t = 12; n=118); ground baits (t = 10; n=228); subterranean baits (t=6; n=57) and no systematic hand collecting. alto paraná 25°44’56.84’’s 54°44’34.60’’w upper paraná af no systematic: hand-collection, blacklighting, malaise traps, berlese funnels and ground baits. canindeyú 24° 9’20.86’’s 55°26’9.98’’w upper paraná af and cerrado no systematic: hand-collection, blacklighting, malaise traps, berlese funnels and ground baits. iberá nr 28°24’0.00’’s 57°14’0.00’’w gallery forest (upper paraná af) pitfall traps (t = 20; n=100), ground baits (t = 4; n=40), berlese funnels (t = 2; n=12) and winkler sacs (t = 2; n = 12) pilcomayo np 25°04’06’’s 58°05’36’’w wet chaco winkler sacs (t = 2; n=180) serra da bodoquena 21°07’16’’ s 56°28’55’’w cerrado, atlantic forest, amazonian forest and pantanal winkler sacs (t = 10; n=262) table 1. ant sampling sites used in this study, coordinates, habitat components and sampling information. more information in hanisch (2013), calcaterra et al. (2010), wild (2007), bihn et al. (2008), silva and brandão (2014), silva and silvestre (2004), silvestre et al. (2012) and leponce et al. (2004). pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park216 ant surveys ant surveys were conducted during 1998, 1999, 2003, 2005, 2008, 2009, and 2011 at 21 sites in inp (fig 2). surveys were performed mostly in spring and summer months and combined 72, 48, and 24 hours pitfalls traps (n=78), 48 and 24 hours winkler sacs of 1m2 plot (n=118), one hour ground (n=228) and 72 (n=48) and 92 hours (n=9) subterranean baits, and opportunist hand collecting. collections were primarily made along areas of relatively easy access and the distance between sampling stations varied from 0.2 to 28 km (fig 2). more detailed information about our collecting protocols can be found in hanisch (2013). collections by leponce in 1998 followed the a.l.l. protocol in term of spatial design and sampling techniques (agosti et al., 2000). vouchers of all specimens collected are deposited in the museo argentino de ciencias naturales (macn) bernardino rivadavia (numbers: macn_en17408 to macn_en17721), the royal belgian institute of natural sciences (rbins), the university of illinois (numbers: avs 705-798, 2344-2367, 2870-2912, 3053-3070, 4035-4066), the institute and museum miguel lillo (imml) and the bohart museum at uc davis (genus pseudomyrmyex). for specimens stored in rbins, sem images are available at: http://www.sciencesnaturelles.be/cb/ ants/collections/argentina%20ants/index.html. of each genus when possible. as with other studies conducted in the neotropics, many individuals from taxonomically difficult genera remain unidentified to species. these individuals were sorted to morphospecies, and when possible, compared across sample periods by peh and ra (samples from the peh, cip and avs collections). synonyms and trinomial names were updated following bolton (2014). data analysis species richness was compared between inp and those sites of the serra do mar coastal forest where a similar sampling methodology (e.g. winkler) was employed. we performed occurrence-based rarefaction curves for incidence data, this allows a standardization removing biases linked for example to ant activity (higher in good climatic conditions) (gotelli & colwell, 2001). an occurrence by species incidence matrix was produced for each sites and year of collection using estimates 9.0 (colwell, 2013). we only used the winkler data set (including morphospecies) from collections made in inp in 1998 and 2008 when the leaf-litter remained 24 and 48 hours in the winkler, respectively. winkler extraction is the best technique for capturing ants inhabiting leaf-litter (agosti et al., 2000; delabie et al., 2000; hanisch, 2013), the forest strata usually containing the most ant species. to evaluate the similarity in the species composition of known ant assemblages among our sites, we performed nonmetric multi dimensional scaling (nmds) based on a jaccard index in a matrix containing presence-absence data. nmds is an appropriated method for non-normal data and does not assume linear relationships among variables (mac cune & mefford, 1999). we used jaccard index as a measure of similarity, as this index gives the same weight to mismatches (0,1 or 1,0) and matches (1,1) of presence/absence of species between two sites. due to unresolved taxonomy, most studies included contain a high number of morphospecies in known diverse genera (e.g. pheidole, hypoponera, and solenopsis). we excluded these genera so our comparisons were not compromised by different estimates of undescribed species in diverse but unresolved groups. we also excluded exclusively arboreal genera (e.g. cephalotes) because nearly all studies focused on litter and soil ants. this allowed us to compare the ant community among sites based on common sampling techniques and taxa for which the taxonomy was best resolved, but without having to rely solely on common species. analyses were performed using pc-ord 4.0 software (mac cune & mefford, 1999). we also performed a cluster analysis in r (r core team, 2014) using jaccard index and upgma method with the vegan (oksanen et al., 2014) and cluster (maechler et al., 2014) packages. cutting level was defined as the maximum dissimilarity value between nodes height. to test if groups obtained with the cluster analyses were different of those expected by chance, we performed a nonparametric analysis of similarity (anosim) with past v.3.04 software (hammer et al., 2001). fig 2. area comprised by the national park of iguazú (inp) in grey within misiones province, argentina pointed by the arrow. survey points during years 1998, 1999, 2003, 2005, 2008, 2009, and 2011 are shown in black dots. our surveys were supplemented by examining previous collections and published records including: imml, macn, and antweb.org (california academy of sciences).we assessed if any ants could be qualified as new records for inp or the country of argentina based on the absence of the species in the main catalogs of neotropical and argentinean ant species listed in appendix. ant identification ants were sorted to species using available taxonomic keys. to confirm identifications, we sent samples to specialists sociobiology 62(2): 213-227 (june, 2015) 217 results ant diversity after pooling all collection events between 1998 and 2011, we recorded 172 ant species belonging to 56 genera and 10 subfamilies for inp: amblyoponinae (2 species), dolichoderinae (14), dorylinae (10), ectatomminae (7), formicinae (21), heteroponerinae (4), myrmicinae (85), ponerinae (21), proceratiinae (1), pseudomyrmecinae (7). among them, 56 were new to province and 39 new to country (appendix). including unidentified morphospecies not shown in appendix, the total number of species increased to 203 species: brachymyrmex (1), hypoponera (1), nesomyrmex (1), nylanderia (1), pheidole (8), solenopsis (18) and pseudomyrmex (1) of the 172 species recorded in inp, 141 species (82%) were captured during our surveys, while the remaining species were recorded from museum or personal collections. eighty seven of these 141 species (61%) were captured using winkler, 39 (28%) in pitfall traps, 26 (18%) were attracted to baits placed above the ground, and 17 (12%) were attracted to subterranean baits. concerning the strata, 66 species (47%) were collected on the soil surface using pitfall traps, baits and hand collecting, 86 (61%) in the leaf-litter, 22 (15%) in the soil (using subterranean bait and soil sieving), and 14 (10%) on the vegetation. the most diverse genera were pheidole with 19 species followed by camponotus with 15 species. species density and composition rarefaction curves based on winkler sampling suggested that the serra do mar coastal forests (pepo and peic) located at the same latitude of inp are richer in grounddwelling ant species than inp (fig 3). the estimated richness was clearly higher in pepo than in inp (both years), based conservatively on non-overlapping confidence intervals. pepo and 2008 inp sampling showed higher values over peic and 1998 inp respectively, however, these values are within the overlap in confidence intervals (fig 3). the nmds ordination and cluster analyses of the different regions show the same visual grouping among sites except within the upaf sites (table 1, fig 4a,b). the dendrogram obtained by hierarchical methods identified six groups (fig 4a) that were concordant in general with the belonged ecoregion of each site (table 1). however, these visual differences among groups were marginally not significant (anosim, r=0.90, p=0.06). visual grouping suggested that canindeyú and alto paraná departments were the most similar sites to inp. moreover, upaf was more similar to serra da bodoquena and pilcomayo than to serra do mar coastal forests and araucaria forest ecoregion (cachoeira, pepo, peic and seara municipality). however, the percent of species shared with inp was very similar among these fig 3. occurrence-based interpolation (rarefaction) based on species/morphospecies obtained with winkler litter samples from three sites on the atlantic forest with 95% unconditional confidence intervals (dashed lines). squares represents pe do pau oco (n= 50; sobs=105), diamons pe da ilha do cardoso (n= 50; sobs=94), circles inp 2008 (n=28, sobs=49) and triangles inp 1998 (n= 69; sobs=79) samplings. maximum species richness is found at pe do pau oco site, exceeding the species density at both inp collection years based in non overlapping confidence intervals. fig 4a. six clusters were defined according to the similarity found in the presence-absence matrix of 261 ant species among 10 sites. cluster analysis based on jaccard index and upgma as linkage method. cophenetic correlation=0.93. dashed line shows the level of cut used to group sites. we applied maximum dissimilarity value between node height as criteria for cutting level of dendrogram. supplementary material available at: http://periodicos.uefs. br/ojs/index.php/sociobiology/rt/suppfiles/724/0 doi: 10.13102/sociobiology.v62i2.219-233.s650 pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park218 regions (between 37-48% with serra do mar costal forest sites, 54 % with seara and 43% with serra da bodoquena, fig 5). despite the short geographic distance between seara and upaf ecoregion (fig 1), the ant community of seara was more similar to that from serra do mar coastal forests (fig 4a, b). finally, iberá showed to be the most different site according species incidence (fig 4a, b), despite that they shared near 66% of their species with inp (fig 5). pananá, peic, serra da bodoquena and seara), and the little fire ant, w. auropunctata (inp, iberá, canindeyú, alto paraná, peic, and serra da bodoquena). heteroponera mayri and heteroponera microps were only collected in the sites within the af (inp, cachoeira, pepo, alto paraná, and peic) with the exception of araucaria forest ecoregion for h. microps (supplementary material, appendix 1). a record of azteca instabilis from the imml collection was not included because of a lack of enough individuals to confirm this identification. our record for the rarely encountered anillindris bruchi is in agreement with schmidt et al.’s, (2013) assumption that rather than a locally uncommon species it is a broadly distributed but rarely collected species. several ant genera/species, such as eurhopalothrix (ketterl et al., 2004) and eciton quadriglume (watkins, 1976), that were expected to be collected given their known distribution in the af have not been found during this study. discussion we report the first compilation of ant species identified for iguazú national park (172 species), 141 species registered only from our samplings and the remained species were retrieved from museum collections and literature. more ant species will be added to our list and results could change once the status of some morphospecies will be resolved. fifty six species were new to province and 39 new to argentina. the majority of morphospecies that we could not identify belonged to diverse ant genera that include many similar species and also lack taxonomic resources for the region (e.g solenopsis, pheidole). as in other neotropical studies pheidole was the most diverse genus with 19 species (silva et al., 2007; donoso & ramón, 2009) followed by camponotus (15 species). the most efficient technique to capture ant species was winkler litter sampling, however, other methodologies including subterranean baits and hand collecting provided some unique records (e.g. linepithema neotropicum and heteroponera microps; appendix). because our methods concentrated on ground dwelling fauna, the list of canopy ant species is underestimated. due to the high heterogeneity of the af (galindo-leal & câmara, 2003) and the heterogeneous sampling efforts of the ant surveys, the estimated ant richness for the studied regions of the af is very variable among sites. in paraguay, wild (2007) found a total of 285 species in canindeyú department (mostly found in mbaracayú forest reserve), but only 89 species in alto paraná department, which is the closest study site to the inp. this is probably due to the lower sampling effort used and the poorer conservation state of this latter department. silva and brandão (2014) studying the leaf-litter ant fauna found 94 and 105 species in peic and pepo af sites, respectively. in our case, employing the same methodology and similar sampling effort, we found 79 species for inp (1998 sampling) using only winkler sacs (fig 3). in serra da bodoquena, silvestre et al. (2012) found 170 fig 4b. ordination of sites according to nmds analysis, based on jaccard index. stress = 0.11 presence-absence matrix of 261 species. fig 5. percent of ant species of each site exclusive and shared with the iguazú national park recorded in paraguay (alto paraná y canindeyú departments), brazil (cachoeira nr, pe do pau oco, pe da ilha do cardoso, serra da bodoquena), and argentina (iberá nr and pilcomayo np). the most widely distributed species were strumigenys denticulata (present in all sites but serra da bodoquena and iberá) and odontomachus meinerti (all sites but seara and iberá). followed by s. louisianae (inp, cachoeira, iberá, canindeyú, alto paraná, peic and seara), strumigenys crassicornis (inp, cachoeira, pepo, canindeyú, pilcomayo, alto paraná and seara) and octostruma rugifera (inp, pepo, canindeyú, pilcomayo, alto paraná and seara). other species that were present in at least six sites include: the fungus growing ant apterostigma pilosum (found in inp, iberá, canindeyú, pilcomayo, alto paraná, and serra da bodoquena), basiceros disciger (inp, pepo, alto sociobiology 62(2): 213-227 (june, 2015) 219 species over a four-year study. despite that sampling was conducted in multiple years, sampling efforts concentrated on hand collection and systematic winkler sampling and pitfall trapping in a sector of easy access of the inp close to the iguazú river. truly comprehensive faunal surveys take much more effort. for example, in a long term study (23 years) in the tropical forest of la selva biological station, costa rica, longino et al. (2002) found 437 ant species. la selva station is a small area (1,500 ha) when compared with the inp (~67,000 ha) or the cachoeira reserve (8,700 ha). our survey covered only approximately 15 ha of inp (0.02% of its surface) in seven sampling opportunities using several sampling methods, where most sampling sites were clumped in an easy-access area of the park (fig 2). as a consequence, other habitat types present in the park were not sampled, which potentially may have increased our ant species list. because canindeyú department represents so far the best studied area of upaf and shows a similar ant species composition with inp (fig 4a, b), ant richness in inp may be similar to that of canindeyú (~300 ground-dwelling ant species; wild 2007). the diversity of ground-dwelling ant species in the af seems to increase slightly with latitude along the brazilian coast (silva & brandão, 2014) and to decrease with the distance from the coast (fig 3). the longitudinal pattern found in this work is consistent with other taxa studied like birds and butterflies (brown & freitas, 2000; galindo-leal & câmara 2003). ribeiro et al. (2009) concluded that the northern af is presently in more dire need of conservation compared to the southern part, with the exception of the large mosaic in the south of the bahia region. this could mean that the observed latitudinal pattern in species richness could be a consequence of better conservation status in the southern af. also, a latitudinal gradient in primary productivity could be an alternative explanation for this pattern (silva & brandão, 2014; kaspari et al., 2000). in a large-scale leaf litter ant diversity study performed by ward (2000), altitude and latitude alone account for 47% of the variance in ant species richness; species richness peaked at mid-elevations in the tropics. in the upaf the elevation ranges from 0 to 500 m in contrast with those sites of serra do mar coastal ecoregion with montane and submontane forests (0 to 1000 m). larger elevation ranges could lead to a greater diversification and niche availability, resulting in higher species richness in ants as in other organisms (brown & freitas, 2000). also, sanders (2002) showed that other parameters, often confounded with area, can also play an important role in determining species richness patterns along elevation gradients. our survey of the incidence of ant species at a regional scale suggests that the sites studied in the af grouped according to each ecoregion (fig 4a, b; table 1). however, ant species composition was similar among all sites despite their strong environmental heterogeneity. many widespread species were shared among the different sites. however, it is important to consider that these results were based on tractable taxonomic species (i.e., excluding morphospecies of difficult genera), limiting our interpretation of this pattern. undoubtedly, these genera are characteristic components of the structure of local communities and the general pattern reported in this survey may change if more complete community compositions are estimated. despite this limitation, we still found differences in ant composition among sites. most habitats within upaf held a similar ant fauna (fig 4). canindeyú department, despite belonging also to the cerrado ecosystem, was similar in composition to inp (fig 4a). surprisingly, serra da bodoquena, a seasonal deciduous and semi deciduous forests with important influences of surrounding different biotas showed a closed relation with the upaf. cachoeira nr, peic, and pepo are characterized by lowland and submontane forests, with vegetation adapted to the sea when approaching to the coast (e.g. mangroves and restinga) (bihn et al. 2008). these vegetation differences together with the broader range of altitudes, probably increases the difference among ant communities of upaf and serra do mar coastal forest ecoregion, making upaf be more similar to serra da bodoquena. of the total ant species found in the gallery forest of iberá (15 species excluding morphospecies), a high proportion were shared with inp (fig 5), including the most widespread species (e.g. a. pilosum, camponotus rufipes, pachycondyla striata, s. louisianae). inp includes the native range of many ant species introduced in other regions of the world, including l. humile, n. fulva, pheidole obscurithorax, s. invicta and w. auropunctata (mcglynn, 1999; holway et al., 2002). however, most (if not all) source populations of these species are from further south along the corridor formed by the paraná and paraguay rivers from buenos aires to formosa province (calcaterra et al., 2015). some of these species are not common within inp. for example, l. humile was collected in 1995 by j. m. ramírez (imml collection) but was not found in our surveys (appendix). in fact, other species of linepithema are much more common within the forest. solenopsis invicta was often collected in open, disturbed environments in inp (e.g. near buildings and paths), but not collected in traps placed within the forest. further ecological studies on these species in their native ranges could enlighten biological control methods for invaded areas and also help identify how abiotic and biotic factors select for traits that confer invasion success. our work contributes to the general knowledge of ant diversity in the extreme south of the af (subtropical), one of their less studied regions, where we make a special effort identifying the majority of the capture species. our results suggest that study sites between upper paraná and serra do mar coastal atlantic forest ecoregion should be added to improve our knowledge of the longitudinal pattern and species turnover of ant species diversity in the af. for inp, more dispersed collection sites would potentially targeted different habitat types, which would include unknown ant species. regarding pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park220 the conservation implications, the study of ant richness in upaf is a valuable tool for the restoration and the sustainable development of non-protected areas of the atlantic forest in misiones province, a region that offers real opportunities to preserve extensive areas and reserves connected by corridors, maintaining genetic exchange among different populations (di bitteti et al., 2003; pais & varanda, 2010). supplementary material: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/724/0 doi: 10.13102/sociobiology.v62i2.213-227.s650 acknowledgements the authors thank to the administration of national parks in argentina, (specifically parque nacional iguazú), paula cichero, karina schiaffino, silvia fabri and cies for permissions to work in argentina and for logistic support. our appreciation to two anonymous reviewers for helpful comments on earlier versions of this manuscript. ants were collected under permit numbers: nea 200; nea 230; nea rev 1; dcm 272-rnv. 3; dcm 389;dcm 284 rnv. nea 1; dcm 284 rnv. nea 4; nea 286. we want to thank to p.s. ward, a. wild, c.m. ortiz, w. mackay, f. cuezzo, lina pedraza, l. theunis and j.h.c. delabie who contributed with the ant identifications. we thank to macn, imml and rbins institutions for the loan of specimens, with special thanks to dra. c. berta and lic. l. verde of imml for the help when reviewing the myrmecological collection. peh acknowledges financial support from the consejo nacional de investigaciones científicas y técnicas (conicet). reference agosti, d., majer, j.d., alonso, l., & schultz, t.r. 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(2000) broad-scale patterns of diversity in leaf litter ant communities. in d. agosti, j.d. majer, l.e. alonso, and t.r. schultz, eds. ants: standard methods for measuring and monitoring biodiversity, pp. 99-121. smithsonian institution press, washington, dc. watkins, j. f., ii 1976. the identification and distribution of new world army ants (dorylinae: formicidae). waco, texas: baylor university press, 102 pp. wild, a. (2007). a catalogue of the ants of paraguay (hymenoptera: formicidae). zootaxa, 1622, 1-55. wilson, e.o. & hölldobler b. (2005). the rise of the ants: a phylogenetic and ecological explanation. pnas, 102: 74117414. doi: 10.1073/pnas.0502264102. sociobiology 62(2): 213-227 (june, 2015) 223 source methodology collection amblyoponinae amblyopone prionopelta punctulata mayr kempf 1972 w imml; rbins stigmatomma elongatum santschi * kusnezov 1953 w avs dolichoderinae anillidris bruchi santschi kempf 1972 hc avs azteca alfari emery kempf 1972 hc avs;imml azteca adrepens forel ** b macna azteca lanuginosa emery kusnezov 1978 imml azteca luederwaldti forel kempf 1972 w imml; rbins dolichoderus bispinosus (olivier) kempf 1959 hc, b macna; avs dolichoderus lamellosus (mayr) shattuck 1994 dorymyrmex brunneus forel kempf 1972 hc macna linepithema humile (mayr) cuezzo 1998 imml linepithma iniquum (mayr) ** p, hc, b avs; macna linepithema micans (forel) kempf 1972 p,w,sb,hc,b avs; imml; lc; macna linepithema neotropicum wild ** hc avs linepithema pulex wild wild 2007 w, sb, b lc; macna tapinoma atriceps emery ** hc avs dorylinae acanthostichus brevicornis emery * kempf 1972 sb macna acanthostichus quadratus emery brandão 1991 s rbins eciton dulcium forel kempf 1972 imml eciton vagans (olivier) kusnezov 1978 hc avs; macna labidus coecus (latreille) kempf 1972 w, sb, hc avs, macna; rbins labidus praedator (smith) kempf 1972 p, w, hc lc, macna labidus spininodis (emery)* forel 1913 hc avs neivamyrmex punctaticeps (emery) bruch 1914 hc macn, macna neivamyrmex halidaii (shuckard) borgmeier 1955 nomamyrmex esenbeckii (westwood) kempf 1972 imml ectatomminae ectatomma edentatum roger kempf 1972 p, w, hc, b avs; imml; macna ectatomma brunneum smith kusnezov 1978 imml gnamptogenys caelata kempf ** w rbins gnamptogenys haenschi (emery) ** hc avs gnamptogenys rastrata (mayr) ** w, b lc gnamptogenys striatula mayr kempf 1972 p, w, hc, b, s avs; macna; rbins gnamptogenys sulcata (smith) kusnezov 1978 hc avs; imml appendix. ant species collected from iguazú national park. capture methodology is as follows: w (winkler sacs), sb (subterranean baits), s (soil samples), hc (hand collecting), b (baits), p (pitfalls). bibliography of species with previous records is provided. (*) new record only for misiones province, (**) new record for argentina. abbreviations of entomological collections used in this study: macn museo argentino de ciencias naturales, buenos aires, argentina; imml the institute and museum miguel lillo, tucumán argentina; rbins royal belgian institute of natural sciences; lc luis calcaterra personal collection, hurlingham, buenos aires, argentina; avs andrew v. suarez personal collection, university of illinois, urbana, usa; bmnh british museum of natural history, london, uk; casc california academy of sciences, san francisco, california, usa. records collected by the authors and stored in the macn are mark as macna to make a distinction from old records review in the museum collection. the abbreviation cf. before species name that were not included in published taxonomic list, means proximity to that species but require more taxonomic research and were considered as a new record, but subjective to be test against literature. pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park224 source methodology collection formicinae brachymyrmex aphidicola forel cuezzo 1998 p, w, sb, b macna brachymyrmex antennatus santschi ** sb macna brachymyrmex cordemoyi forel kempf 1972 w macna brachymyrmex cf. depilis emery ** w lc camponotus atriceps (smith) kusnezov 1978 p macna camponotus cingulatus mayr emery 1906 p, hc, b imml; macna; rbins camponotus crassus mayr kempf 1972 w, b imml; macna; rbins camponotus depressus mayr wp mackay record hc avs; macna camponotus cf. landolti forel wp mackay record p macna camponostus lespesii forel kempf 1972 p rbins camponotus mus roger kempf 1972 w lc camponotus punctulatus andigenus emery * kempf 1972 hc rbins camponotus renggeri emery kempf 1972 p, hc rbins camponotus rufipes (fabricius) kempf 1972 p, w, hc, b avs; imml; lc; macna; rbins camponotus scissus mayr kusnezov 1978 hc, p macna; rbins camponotus sericeiventris (guérin-méneville) kempf 1972 p, hc, b avs; imml; macna; rbins camponotus sexguttatus (fabricius) imml camponotus substitutus emery cuezzo 1998 hc avs camponotus trapezoideus mayr kusnezov 1978 w imml; lc; rbins myrmelachista cf.nodifera mayr kusnezov 1978 hc avs nylanderia fulva (mayr) bruch 1914 p, w, hc, b avs; macna; lc heteroponerinae heteroponera flava kempf ** w, hc, p avs; macna heteroponera dolo (roger) kempf 1972 w, hc imml; macna; rbins heteroponera mayri kempf ** p, w lc; rbins heteroponera microps borgmeier ** sb macna myrmicinae acromyrmex coronatus (fabricius) kusnezov 1978 hc avs acromyrmex hispidus fallax santschi kusnezov 1978 w, hc rbins acromyrmex laticeps (emery) kempf 1972 hc, b avs; imml; macna acromyrmex niger (smith) cuezzo 1998 p macna acromyrmex nigrosetosus (forel) ** hc macna apterostigma pilosum mayr kempf 1972 w avs; lc; rbins atta sexdens (linnaeus) kempf 1972 p, w, hc avs; imml; macna; rbins basiceros disciger (mayr) ** w rbins carebara cf. brasiliana fernández** w avs carebara brevipilosa fernández ** p, w, sb macna appendix. ant species collected from iguazú national park. capture methodology is as follows: w (winkler sacs), sb (subterranean baits), s (soil samples), hc (hand collecting), b (baits), p (pitfalls). bibliography of species with previous records is provided. (*) new record only for misiones province, (**) new record for argentina. abbreviations of entomological collections used in this study: macn museo argentino de ciencias naturales, buenos aires, argentina; imml the institute and museum miguel lillo, tucumán argentina; rbins royal belgian institute of natural sciences; lc luis calcaterra personal collection, hurlingham, buenos aires, argentina; avs andrew v. suarez personal collection, university of illinois, urbana, usa; bmnh british museum of natural history, london, uk; casc california academy of sciences, san francisco, california, usa. records collected by the authors and stored in the macn are mark as macna to make a distinction from old records review in the museum collection. the abbreviation cf. before species name that were not included in published taxonomic list, means proximity to that species but require more taxonomic research and were considered as a new record, but subjective to be test against literature (cont.). sociobiology 62(2): 213-227 (june, 2015) 225 source methodology collection myrmicinae (continuation) carebara urichi (wheeler) ** w avs; rbins cephalotes atratus (linnaeus) kempf 1972 imml; macn cephalotes borgmeieri (kempf) kempf 1951 cephalotes clypeatus (fabricius) kempf 1972 imml; macn cephalotes depressus (klug) kempf 1972 imml cephalotes minutus (fabricius) bruch 1914 w, hc avs; macna; rbins cephalotes pusillus (klug) kempf 1972 hc avs; imml macna crematogaster arata emery kempf 1972 hc avs crematogaster cisplatinalis mayr jtlc crematogaster corticicola mayr kusnezov 1978 imml crematogaster crinosa mayr hc avs;imml; jtlc crematogaster erecta mayr ** w, b macna crematogaster nigropilosa mayr ** p, w, b avs; macn crematogaster montezumia smith kempf 1972 imml cyphomyrmex minutus mayr ** p, w avs; macna cyphomyrmex olitor forel * kempf 1972 w lc cyphomyrmex rimosus (spinola) kempf 1972 w, hc avs; rbins hylomyrma balzani (emery) kempf 1972 w lc; macna hylomyrma reitteri (mayr) ** w lc; macna; rbins lachnomyrmex plaumanni borgmeier feitosa & brandão 2008 w rbins megalomyrmex drifti kempf ** w lc; macn megalomyrmex miri brandão ** w avs megalomyrmex silvestrii wheeler kempf 1972 w rbins monomorium pharaonis (linnaeus) cuezzo 1998 imml mycetarotes parallelus (emery) kusnezov 1978 & solomon et al. 2004 hc avs mycocepurus smithii (forel) kusnezov 1978 p, w macna myrmicocrypta foreli mann * leponce et al. 2004 w rbins nesomyrmex asper (mayr) kusnezov 1978 imml nesomyrmex argentinus (santschi) * kempf 1972 w rbins octostruma balzani (emery) cuezzo 1999 p, w avs; macna octostruma iheringi (emery) ** w, sb avs; macna octostruma rugifera (mayr) kempf 1972 w lc; rbins oxyepoecus reticulatus kempf * theunis et al. 2005 p, w macna pheidole aberrans mayr kempf 1972 macn pheidole bergi mayr * kempf 1972 w lc; macn pheidole bruchi forel kempf 1972 macn pheidole cf. diligens (smith) ** b macna appendix. ant species collected from iguazú national park. capture methodology is as follows: w (winkler sacs), sb (subterranean baits), s (soil samples), hc (hand collecting), b (baits), p (pitfalls). bibliography of species with previous records is provided. (*) new record only for misiones province, (**) new record for argentina. abbreviations of entomological collections used in this study: macn museo argentino de ciencias naturales, buenos aires, argentina; imml the institute and museum miguel lillo, tucumán argentina; rbins royal belgian institute of natural sciences; lc luis calcaterra personal collection, hurlingham, buenos aires, argentina; avs andrew v. suarez personal collection, university of illinois, urbana, usa; bmnh british museum of natural history, london, uk; casc california academy of sciences, san francisco, california, usa. records collected by the authors and stored in the macn are mark as macna to make a distinction from old records review in the museum collection. the abbreviation cf. before species name that were not included in published taxonomic list, means proximity to that species but require more taxonomic research and were considered as a new record, but subjective to be test against literature (cont.). pe hanisch; la calcaterra; m leponce et al. – ground-dwelling ants of the iguazú national park226 source methodology collection myrmicinae (continuation) pheidole fimbriata roger kempf 1972 p, w, sb imml; macna pheidole cf. flavens roger kusnezov 1952 p, s rbins pheidole gertrudae forel kempf 1972 w imml; rbins pheidole guilelmimuelleri forel kempf 1972 w rbins pheidole cf. inversa forel ** w lc pheidole jelskii mayr * bruch 1914 hc avs; macna pheidole mosenopsis wilson ** p, w, sb avs; macna pheidole nubila emery * kempf 1972 p, w rbins pheidole obscurithorax naves * kempf 1972 w, b lc pheidole radoszkowskii mayr vittar 2008 b lc pheidole subarmata mayr cuezzo 1998 w, sb, b avs; macna pheidole cf. subarmata sb, b macna pheidole rugatula santschi kempf 1972 macn pheidole rudigenis emery kempf 1972 w avs pheidole vafra santschi kusnezov 1952 imml pogonomyrmex naegelli emery kempf 1972 hc avs; macna procryptocerus hylaeus kempf ** hc avs; macna procryptocerus regularis emery kempf 1972 hc macna rogeria scobinata kugler * leponce et al. 2004 w, s macna; rbins solenopsis clytemnestra emery kusnezov 1978 w, s rbins solenopsis invicta buren * brandão 1991 hc avs;imml solenopsis solenopsidis (kusnezov) kusnezov 1962 solenopsis tridens forel kempf 1972 imml stegomyrmex vizottoi diniz casc strumigenys cf. siagodens (bolton) ** w macna strumigenys crassicornis mayr kempf 1972 w avs; macna strumigenys denticulata mayr kempf 1972 & cuezzo 1999 p, w, hc avs; imml; macna; rbins strumigenys elongata roger cuezzo 1999 w, sb avs; imml; macna; rbins strumigenys hindenburgi (forel) cuezzo 1999 imml strumigenys louisianae roger kempf 1972 p, w lc; macna; strumigenys ogloblini santschi kempf 1972 w rbins strumigenys silvestrii emery kempf 1972 & cuezzo 1999 w avs strumigenys splendens (borgmeier) bmnh strumigenys subedentata mayr ** w avs strumigenys tanymastax (brown) ** w avs appendix. ant species collected from iguazú national park. capture methodology is as follows: w (winkler sacs), sb (subterranean baits), s (soil samples), hc (hand collecting), b (baits), p (pitfalls). bibliography of species with previous records is provided. (*) new record only for misiones province, (**) new record for argentina. abbreviations of entomological collections used in this study: macn museo argentino de ciencias naturales, buenos aires, argentina; imml the institute and museum miguel lillo, tucumán argentina; rbins royal belgian institute of natural sciences; lc luis calcaterra personal collection, hurlingham, buenos aires, argentina; avs andrew v. suarez personal collection, university of illinois, urbana, usa; bmnh british museum of natural history, london, uk; casc california academy of sciences, san francisco, california, usa. records collected by the authors and stored in the macn are mark as macna to make a distinction from old records review in the museum collection. the abbreviation cf. before species name that were not included in published taxonomic list, means proximity to that species but require more taxonomic research and were considered as a new record, but subjective to be test against literature (cont.). sociobiology 62(2): 213-227 (june, 2015) 227 source methodology collection myrmicinae (continuation) wasmannia auropunctata (roger) kempf 1972 p, w, sb, b avs; lc; macna; wasmannia rochai forel ** w lc wasmannia sp. n. ** b, w lc ponerinae anochetus neglectus emery* brandão 1991 w macna dinoponera australis emery kempf 1972 p, w, hc, b avs; lc; macna; rbins hypoponera clavatula (emery) kempf 1972 w macna; rbins hypoponera distinguenda (emery) kusnezov 1978 p, w avs; macna; rbins hypoponera schmalzi (emery) ** w avs hypoponera foreli (mayr) ** w, sb macna; rbins hypoponera opaciceps (mayr) kempf 1972 imml hypoponera opacior (forel) kempf 1972 w imml hypoponera trigona (mayr) kempf 1972 w macna odontomachus chelifer (latreille) kempf 1972 p, hc, b avs; imml; lc, macna; rbins odontomachus meinerti forel * leponce et al. 2004 p, w, sb macna; rbins neoponera agilis (forel) kusnezov 1969 imml neoponera crenata (roger) kempf 1972 w, hc avs; imml; rbins neoponera marginata (roger) cuezzo 1998 hc macna neoponera moesta mayr mackay & mackay 2010 hc avs; macna neoponera villosa (fabricius) kempf 1972 hc imml; avs pachycondyla constricticeps mackay & mackay mackay & mackay 2010 p imml; macna; pachycondyla harpax (fabricius) kusnezov 1978; mackay & mackay 2010 p, w macna pachycondyla striata smith kempf 1972 p, w, hc, b, s avs; imml; lc; macna; rbins rasopone lunaris (emery) ** w macna thaumatomyrmex mutilatus mayr ** w rbins proceratiinae discothyrea neotropica bruch kempf 1972 w avs; rbins pseudomyrmecinae pseudomyrmex gracilis (fabricius) kempf 1972 hc avs; macna; pseudomyrmex kuenckeli (emery)* kempf 1972 hc rbins pseudomyrmex lizeri (santschi) ** hc avs pseudomyrmex phyllophilus (smith) kempf 1972 hc avs; imml pseudomyrmex rufiventris (forel) imml pseudomyrmex schuppi (forel) kempf 1972 hc imml pseudomyrmex simplex (smith) ** hc avs appendix. ant species collected from iguazú national park. capture methodology is as follows: w (winkler sacs), sb (subterranean baits), s (soil samples), hc (hand collecting), b (baits), p (pitfalls). bibliography of species with previous records is provided. (*) new record only for misiones province, (**) new record for argentina. abbreviations of entomological collections used in this study: macn museo argentino de ciencias naturales, buenos aires, argentina; imml the institute and museum miguel lillo, tucumán argentina; rbins royal belgian institute of natural sciences; lc luis calcaterra personal collection, hurlingham, buenos aires, argentina; avs andrew v. suarez personal collection, university of illinois, urbana, usa; bmnh british museum of natural history, london, uk; casc california academy of sciences, san francisco, california, usa. records collected by the authors and stored in the macn are mark as macna to make a distinction from old records review in the museum collection. the abbreviation cf. before species name that were not included in published taxonomic list, means proximity to that species but require more taxonomic research and were considered as a new record, but subjective to be test against literature (cont.). 189 do tunnel patterns of coptotermes formosanus and coptotermes gestroi (blattodea: rhinotermitidae) reflect different foraging strategies? by nirmala k. hapukotuwa1 & j. kenneth grace1* abstract tunnel network construction and time to food (wood) discovery by coptotermes formosanus shiraki and coptotermes gestroi (wasmann) (blattodea: rhinotermitidae) (formerly known as coptotermes vastator light in the pacific region) was examined when wood was present in a clumped distribution that mimics field conditions in the subtropical and temperate regions where c. formosanus naturally occurs. previous research has noted that the tropical species c. gestroi constructs a highly branched tunnel network, while the subtropical c. formosanus constructs longer tunnels with few branches. grace et al. (2004) hypothesized that this difference in tunneling behavior may relate to a more homogenous distribution of woody resources in the tropics vs. a disjunct and clumped distribution of fallen wood in the cooler subtropics. thus, c. gestroi may exhibit a thorough search of the immediate area where wood is initially located, while it may be more energetically efficient for c. formosanus to tunnel greater distances in search of scattered resources. to test this hypothesis, we placed two wood resources at the opposite ends of laboratory foraging arenas, and released 1500 termites (1350 workers: 150 soldiers) into each arena. arenas were observed every 24 hours for 14 days. we measured the total daily tunnel length, number of tunnels created in each quadrant of the arenas, and average time to discover food at both ends. total daily tunnel length was relatively longer with c. formosanus and average time to discover food at either end was longer for c. gestroi. although replication was limited in this study, these observations lend support to the hypothesis that c. formosanus is able to locate distant resources more efficiently than c. gestroi. 1college of tropical agriculture & human resources, university of hawaii at manoa, 3050 maile way, gilmore 202, honolulu hi 96822, usa *corresponding author. email: kennethg@hawaii.edu 190 sociobiolog y vol. 59, no. 1, 2012 introduction the tunneling behavior of subterranean termites is difficult to study due to their cryptic habitat (puche & su 2001b). however, laboratory studies with two-dimensional foraging arenas have provided insight into tunneling geometry (e.g., puche & su 2001a, campora & grace 2001, bardunias & su 2005, nobre et al. 2007). much research has been done in recent years on tunneling behavior in such arenas, including studies on the influence of the presence of wood and/or of wood discovery on tunnel orientation. campora & grace (2001) found that the basic pattern of tunneling by coptotermes formosanus shiraki was not influenced by the proximity of wood. changes in tunneling patterns appeared to reflect physical cues (as anomalies in the substratum) rather than the presence of edible wood. puche & su (2001a) and nobre et al. (2007) reported similar results in which the presence of wood in the soil did not influence termite tunnel orientation. however, other researchers have demonstrated that tunnel orientation can be influenced by the very close presence of sound or decayed wood (amburgey and smythe 1977 reinhard et al. 1997). hedlund et al. (1999) found that formosan subterranean termites exhibited changes in activity when available food size varied; when food size was increased, termite survival rates and food consumption rates also increased. similar findings were observed by akhtar & jabeen (1981) and waller (1988). reinhard & kaib (1995) found high feeding rates on large food resources, possibly due to the presence of chemical signals from labial gland secretions. laboratory studies of complex behavior are always subject to the possibility of artifacts occurring due to the artificial nature of the bioassay situation. with respect to the use of laboratory arenas to investigate termite tunneling behavior, however, campora & grace (2009) demonstrated by installing identical arenas in the field (connected directly to the soil) that the same tunneling patterns occur in both field and laboratory; although differences in rates of tunneling between different c. formosanus colonies were more apparent under field conditions, likely due to the much greater number of termites tunneling through the field arenas. there have been a limited number of comparative behavioral studies carried out on coptotermes formosanus and coptotermes gestroi (wasmann). 191 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies coptotermes gestroi was formally known as coptotermes vastator light in the pacific region, until synonymized by yeap et al (2007). shelton and grace (2003) noted higher mortality rates of c. gestroi than of c. formosanus under desiccating conditions. uchima & grace (2003), grace et al. (2004) and hapukotuwa & grace (2011b) reported that c. formosanus generally has a higher wood feeding rate than c. gestroi. uchima & grace (2009) reported that c. gestroi had higher mortality rates in interspecific agonism and foraging competition experiments with c. formosanus. grace et al. (2004) documented by visual observations that the tunnel systems of c. gestroi are distinctly different from those of c. formosanus. coptotermes gestroi makes long, thin, highly branched tunnels; whereas c. formosanus constructs longer, wider, and lessbranched tunnels. these visual observations were subsequently quantified by hapukotuwa & grace (2010, 2011a). the objectives of the present study were to evaluate the tunneling systems of c. formosanus and c. gestroi and the time to food location when food (wood) was present in the foraging arena as a discrete, clumped resource. as mentioned, grace et al. (2004) and hapukotuwa & grace (2010, 2011a) demonstrated that the tropical species c. gestroi constructs a highly branched tunnel network over a fairly limited area, while the subtropical c. formosanus constructs longer and wider tunnels with few branches. grace et al. (2004) hypothesized that this difference in foraging behavior may be related to foraging efficiency in the tropical vs. subtropical environs. woody resources would be expected to have a generally homogenous distribution in the tropics, as opposed to the more disparate and clumped distribution of such resources further from the equator in the subtropical and temperate regions. thus, an efficient foraging strateg y for the tropical c. gestroi would be to conduct an intensive local search of the immediate area where food (wood) is first found. in contrast, the subtropical c. formosanus may have to travel greater distances in search of discrete, scattered (or clumped) resources. the highly branched tunnel network of c. gestroi, and the longer, unbranched tunnels of c. formosanus appear to demonstrate these two different foraging strategies, each of which is most efficient for the particular environment inhabited by each termite species. although both species are distributed around the globe, they are only known to occur together in taiwan, florida, and hawaii, offering a unique opportunity in hawaii to directly compare their foraging strategies. 192 sociobiolog y vol. 59, no. 1, 2012 we chose to simulate the clumped distribution of wood in the subtropical and temperate regions by placing wood at opposite ends of a square foraging arena. the time taken to locate food at each end of the arena after release of either c. formosanus or c. gestroi in the center of each arena represents a measure of the efficiency of the foraging (tunneling ) strateg y of each species under these pseudo-subtropical conditions of clumped woody resources. support for the hypothesis offered by grace et al. (2004) would be demonstrated by more rapid food location, particularly of the second resource to be found, by c. formosanus, the subtropical species with longer and generally unbranched tunnels. under the conditions of this experiment, the tropical c. gestroi would be expected to spend more time in local search, constructing a densely branched network of fine tunnels, particularly once food was initially discovered; and would be expected to be slower to locate the separated resources, particularly the second resource to be found. materials and methods test arenas we used six test arenas, three for c. formosanus and three for c. gestroi. all test arenas consisted of two sheets of transparent acrylic. the upper half was 85x85x0.25 cm and the lower half was 85x85x0.50 cm. each half was fixed together using metal screws to create a space of 75x75x0.25 cm in the middle. on the inner space of the lower half, eight small squares with screw holes were affixed to the bottom of the lower half to keep the space uniform after assembly. the upper half of the arena had one chamber (termite release chamber, 8.5 cm in diameter) in the center. two pre-weighed douglas fir (pseudotsuga menziessii) wood blocks (2.5x2.0x0.5cm) were placed at each of the two corners of each arena (fig. 1), representing separated and clumped food resources for the termites. the space between the acrylic seats was filled with silica sand (40-100 mesh, 150–425 μm sieve, fisher scientific, fair lawn, nj) moistened with de-ionized water (approximately 18% by weight) (campora & grace 2004). sand was spread manually and as far as possible evenly across the arena with no space left between the lid and the sand surface (nobre et al. 2007). a wet filter paper was placed in the center termite release jar to provide initial food and moisture. all arenas were placed on a metal frame supported by glass jars 193 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies and kept in a dark room. fluorescent lights were placed under each arena to observe tunneling patterns and to take photographs. termites and bioassays coptotermes formosanus were collected from a site on the manoa campus of the university of hawaii (irradiator colony; 21o 17’ n; 157o 99’ w; 23.1m above sea level). c. gestroi was collected from kalaeloa , a field site on the west side of oahu, hawaii, 40 km from the university of hawaii at manoa campus (barber’s point horse stables; 21o 19’ n; 158o 02’ w; 9 m above sea level). termites were collected in wood within aggregation traps (tamashiro et al. 1973) at each site. termites were extracted from the wood and counted using techniques modified from those of tamashiro et al. (1973), su & la fage fig. 1. schematic drawing of the termite foraging arena. 194 sociobiolog y vol. 59, no. 1, 2012 (1984), and grace et al. (1995). we used 1500 termites comprised of 90% workers and 10% soldiers for each arena (1350 workers: 150 soldiers). a subset of 40 workers of each termite species was weighed before being added to the arenas. finally, termites were transferred to each arena’s central releasing chamber. arenas were obser ved daily (every 24 hours) over 14 days. temperature and humidity were recorded using an indoor data logger (hobo u10 temp/rh data logger). tunnel patterns were photographed daily using a nikon 12.3 mp digital camera. analyses the digital camera images were used to determine daily tunnel lengths (primary, secondary, tertiary and other) using adobe acrobat professional 8 software. in addition, to test if there was greater tunneling activity in the vicinity of the food sources once they were found by the termites, each digitized image was divided into four equal sectors (fig. 2) and the number of tunnels was counted in each sector.statistical analyses were carried out using sas 9.2 software. a general linear model (one-way anova) and ryan-einotgabriel-welsch multiple range test were used with total tunnel length per day (within each observation time) to test for differences in tunnel length over time between the two species c. formosanus and c. gestroi. student’s t-test was used to compare the frequency of tunnels in each sector to detect any differences in tunnel number with or without food present (for total length of the tunnels per sector relative to the total tunnel length per arena). mean wood consumption rates were calculated using initial and final dry weights of all wood blocks for each termite species. fig. 2. division of the foraging arena into four sectors. 195 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies fig.3. representative foraging arenas containing c. gestroi (left) and c. formosanus (right) on day 3 (top), day 4 (center), and day 14 (bottom). 196 sociobiolog y vol. 59, no. 1, 2012 results behavioral observations we observed both similarities and differences in the tunneling and other foraging activities of c. formosanus and c. gestroi. on the first day after release (after 12 hours) most of the termites (nearly 75%) remained in the central release chamber, with the remainder starting to make tunnels outward. from the 2nd to 8th day, both species were very active and constructed large numbers of tunnels throughout the arenas. as previously noted, c. formosanus made fewer, less branched, and longer tunnels; while c. gestroi constructed a larger number of highly branched tunnels (fig 3). the species demonstrated some common behaviors, such as moving back and forth, tunnel excavation, and resting in groups. during the last few days of the experiment (from day 9 to 14), activity of both termite species was greatly reduced. most gathered in small groups, while others moved very slowly. also, during the last few days the number of dead termites observed in the arenas was greater for both species than in the earlier days. during the experiment, temperature ranged from 230c to 24 0c and relative humidity ranged from 57% to 64%. both termite species were able to find the food (wood) on both sides of the arena over the course of the experimental period. however, they exhibited different time frames for discovery (fig. 4). for example, c .formosanus in arena no. 4 found wood in one side of the arena during the 2nd day and on other side on the 8th day; and in arena no. 5 found wood on one side on the 3rd day and on other side during the 4th day. coptotermes gestroi in arena no. 3 found wood on one side on the 3rd day and on the other side on the 8th day; and in arena no. 1 found wood on the first side during the 4th day and table 1: total number of tunnels in each section of foraging arenas (numbers in parentheses are ratio of number of tunnels in each section to total tunnel number of tunnels in the entire arena). termite species no. of tunnels in sec. 1 no. of tunnels in sec. 2 no. of tunnels in sec. 3 no. of tunnels in sec. 4 total no. of t unn e l s in arena s u r v i v i n g termites (w:soldiers) c. formosanus 23 (0.29) 20(0.25) 24(0.30) 13(0.16) 80 728:89 c. formosanus 25(0.19) 36(0.27) 48(0.36) 23(0.17) 132 743:101 c. gestroi 52(0.24) 60 (0.28) 80(0.37) 23(0.11) 215 690:90 c. gestroi 23(0.23) 19(0.19) 28(0.28) 29(0.29) 99 746:92 197 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies on the other side on the 9th day. two other arenas (no. 2 for c. gestroi and no. 6 for c. formosanus) had to be discarded discarded because of warped upper covers on the arenas. due to this bending, it was impossible to see any tunnels; and there was a space between the upper and lower layers through which termites could move. with the two arenas remaining for each termite species (total of four arenas), the average time to discover wood initially was 2.5 days for c. formosanus, and 3.5 days for c. gestroi. following this initial discovery, the average time to discover the wood on the opposite side of the arena was 6 days with c. formosanus, and 8.5 days with c. gestroi. thus, although replication is limited, the longer time taken for wood discovery by c. gestroi, and particularly for discovery of the second wood resource, supports the hypothesis presented by grace et al. (2004) that the tunneling pattern of c. formosanus is optimized for the more disjunct and clumped distribution of wood resources found further from the equator. statistical analyses overall, tunneling rates of both species did not differ significantly (df=01, f=0.34, p=0.5649). however, total tunnel length was relatively greater for c. formosanus than for c. gestroi (fig. 5). coptotermes gestroi constructed a larger number of secondary and tertiary tunnels compared to c. formosanus. fig. 4. average time to discover food at both ends of arenas for c. formosanus (cf ) and c. gestroi (cg ). 198 sociobiolog y vol. 59, no. 1, 2012 total tunnel numbers in each section of the arena (two sections with wood and two sections without wood) were not significantly different (t value=0.9, df=4, p=0.42). neither the total number of tunnels at the conclusion of the study in sections of the arena with wood present (df=1, f=0.55, p=0.536), nor the total tunnel number of tunnels in each section without wood differed significantly between the two species (df=1, f=0.8, p=0.465) (table 1). total wood consumption rates were also not significantly different between the two termite species in this study (df=1, f=1.24, p=0.2752), although mean mass loss values for c. formosanus in each arena were relatively higher than those for c. gestroi (fig. 6). mean percentage mortality values were also not significantly different between the two termite species (df=1, f=0.45, p=0.571), although c. gestroi suffered relatively greater mortality than c. formosanus, especially in arena no. 1 (fig. 7). discussion due to the unexpected loss of two of the six tunneling arenas used in this study, and the resulting limited replication, we were unable to statistically validate the hypothesis of grace et al. (2004) that the tunnel patterns of c. formosanus and c. gestroi reflect different foraging strategies that are tuned fig. 5. daily tunnel lengths for c. formosanus and c. gestroi over 14 days (termite species comparison by one-way anova and ryan-einot-gabriel-welsch multiple range test, p=0.565). 199 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies to the relative distribution of woody resources in subtropical/temperate vs. tropical environments. however, the more rapid discovery of both the first and second woody resources by c. formosanus (fig. 4) is consistent with this hypothesis. tunnel numbers at the conclusion of the study were not significantly different between the two termite species in arena quadrants where wood was present or quadrants where wood was absent. other authors have observed that the proximity of wood did not influence tunnel patterns for workers of fig. 6. mean mass loss values of wood in arenas with either c. formosanus or c. gestroi (one-way anova, p=0.275) fig. 7. percent mortality of c. formosanus and c. gestroi during the bioassay (one-way anova, p=0.571). 200 sociobiolog y vol. 59, no. 1, 2012 c. formosanus (campora & grace 2001), reticulitermes flavipes (puche& su 2001b), and r. grassei (nobre (2007). these findings are somewhat different from those of hedland & henderson (1999), akhtar & jaben (1981), and waller (1988) who noted that tunnel numbers increased as the size of the proximate food resource increased. we observed the same interspecific differences in tunneling patterns noted by grace et al. (2004) and hapukotuwa & grace (2010, 2011a). although we were not able to directly relate these tunnel patterns (in terms of tunnel numbers and lengths) to differences in resource usage, the differences in times to locate food noted in this study support the hypothesis of grace et al. (2004) that c. formosanus is able to locate distant resources more efficiently than c. gestroi. this difference is not yet fully resolved, as a result of the limited replication in the present study. however, the results obtained are consistent with the view that the tunneling behavior of c. formosanus is energetically more efficient when resources are distant and clumped, as in the subtropics and temperate regions, then the heavily branched, intensive local search strateg y employed by the tropical c. gestroi. future work might employ larger foraging arenas and/or incorporation of a wood distribution pattern to mimic the more homogenous tropical situation. acknowledgments we are grateful to r. oshiro and m. aihara-sasaki for technical assistance; and to j. r. yates iii, and m. g. wright for reviewing early drafts of the manuscript. thanks are also due to c. campora for providing us with assistance in arena construction, and to nh’s husband, chaminda wijesundara, for his help in various ways. funding for this research was partially provided by usda-ars specific cooperative agreements 58-6615-9-200 and 58-6435-8-294; and mcintire-stennis and hatch funds administered by the college of tropical agriculture and human resources, university of hawaii at manoa. references akhtar, m. s. & m. jabeen. 1981. influence of specimen size on the amount of wood consumed by termites. pakistan journal of zoolog y 13: 79-84. amburgey, t. l.& r. v. smythe. 1977. factors influencing termite feeding on brown-rotted wood. sociobiolog y 3: 3-12. 201 hapukotuwa, n.k. & j. k. grace — coptotermes foraging strategies bardunias, p. & n.-y. su. 2005. comparison of tunnel geometry of subterranean termites (isoptera: rhinotermitidae) in “two dimensional” and “three dimensional” arenas. sociobiolog y 45: 679-685 campora, c. e. & j. k. grace. 2001. tunnel orientation and search pattern sequence of the formosan subterranean termite (isoptera: rhinotermitidae). journal of economic entomolog y 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in foraging arenas. proceedings of the 2010 national conference on urban entomolog y. pp. 33-36. hapukotuwa, n. k. & j. k. grace. 2011a. comparative study of tunneling behavior of coptotermes formosanus shiraki and c. gestroi (wasmann) using foraging arenas. psyche. issn 1687-7438, doi:10.1155/psyche. in review. hapukotuwa, n. k. & j. k. grace. 2011b. preferences of coptotermes formosanus shiraki and coptotermes gestroi (wasmann) (blattodea: rhinotermitidae) among three commercial wood species. insects. issn 2075-4450, www.mdpi.com/journal/insects/. in press. hedlund, j. c. & g. henderson. 1999. effect of available food size on search tunnel formation by the formosan subterranean termite (isoptera: rhinotermitidae). journal of economic entomolog y 92: 610-616. nobre, t., l. nunes & d. e. bignell. 2007. tunnel geometry of the subterranean termite reticulitermes grassei (isoptera: rhinotermitidae) in response to sand bulk density and the presence of food. insect science 14: 511-518. doi: 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physiolog y part a: molecular and integrative physiolog y 134: 205-211. su, n.-y. & j. p. la fage. 1984. comparison of laboratory methods for estimating wood consumption rates by coptotermes formosanus isoptera rhinotermitidae. annals of the entomological society of america 77: 125-129. tamashiro, m., j. k. fujii & p. y. lai. 1973. a simple method to observe, trap and prepare large numbers of subterranean termites for labratory and field experiments. environmental entomolog y 2: 721-722. uchima, s. y. & j. k. grace. 2003. compartive feeding rates of coptotermes vastator and coptotermes formosanus (isoptera: rhinotermitidae). sociobiolog y 41: 289-294. uchima, s. y. & j. k. grace. 2009. interspecific agonism and foraging competition between coptotermes formosanus and coptotermes gestroi (blattodea : rhinotermitidae). sociobiolog y 54: 765-776. waller, d. a. 1988. host selection in subterranean termites: factors affecting choice (isoptera,rhinotermitidae). sociobiolog y 14: 5-13. yeap, b.-g., a. s. othman, v. s. lee & c.-y. lee. 2007. genetic relationship between coptotermes gestroi and coptotermes vastator (isoptera: rhinotermitidae). journal of economic entomolog y 100: 467-474. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.449-460sociobiology 61(4): 449-460 (december, 2014) variability of food stores of tetragonisca fiebrigi (schwarz) (hymenoptera: apidae: meliponini) from the argentine chaco based on pollen analysis fg vossler, ga fagúndez, dc blettler introduction honey and pollen stores from tetragonisca species have been reported as a culturally important and appreciated food as well as home medicine since ancient times (nogueira-neto, 1997; arenas, 2003; cortopassi-laurino et al., 2006; zamudio & hilgert, 2012; roig-alsina et al., 2013). several studies on diet have been done in tetragonisca angustula (latreille) such as imperatriz-fonseca et al. (1984), carvalho et al. (1999), novais et al. (2013, 2014) in brazil, sosa-nájera et al. (1994) and martínez-hernández et al. (1994) in mexico, obregón et al. (2013) in colombia, flores & sánchez (2010) in northwestern argentina, while only one for the aerial-nesting and aggressive tetragonisca weyrauchi (schwarz) (cortopassi-laurino & nogueira-neto 2003). no studies have been performed for the ground-nesting tetragonisca buchwaldi (friese) neither for tetragonisca fiebrigi (schwarz). it is important to study the botanical origin of food stores abstract honey and pollen mass samples of tetragonisca fiebrigi (schwarz) from the same and different nests, seasons, and forest types from the argentine chaco region were palynologically analyzed and multivariate techniques were applied. the samples from each forest type (palosantal and quebrachal) were grouped separately by cluster analysis but the phenological records detected that grouping was determined by the season when samples were taken. honeys and pollen masses were grouped together or fairly closed for all nests due to similar abundance of the different pollen types. furthermore, honeys were not clustered together with other honeys but with pollen masses. it can be assumed that both nectar and pollen were gathered from the same plant species, supporting the hypothesis that the dry chaco melittophilous vegetation is dominated by plants providers of both pollen and nectar, but not exclusively or predominately of one of them. results of principal component analysis revealed that the foraging behavior of t. fiebrigi was governed by random factors such as local differences of flower availability but not by preferences for some plant families. this idea can also be extended to other species of this genus as they concentrated their foraging on different families according to the local vegetation availability in the studied sites. sociobiology an international journal on social insects article history edited by cândida maria l. aguiar, uefs, brazil received 22 september 2014 initial acceptance 24 october 2014 final acceptance 13 november 2014 keywords honey, “rubiecita”, stingless bee, “yateí”. corresponding author favio gerardo vossler laboratorio de actuopalinología cicyttp-conicet/fcyt-uader dr. materi y españa, e3105bwa diamante, entre ríos, argentina e-mail: favossler@yahoo.com.ar of t. fiebrigi in the chaco region, a large plain of xerophylous forest of about 1,000,000 km2 in southern south america (prado, 1993). in this region, the “rubiecita/rubiecito” or “rubita/rubito” (t. fiebrigi) provides the most reputable honey by local people (arenas 2003) and it is the most intensively reared stingless bee, together with scaptotrigona jujuyensis (schrottky) (roig-alsina et al., 2013). furthermore, it is the most frequent meliponini species in the forest (vossler, 2012), being their colonies commonly harvested in the field (roig-alsina et al., 2013). due to the importance of stingless bees in good practices such as meliponiculture and crop pollination, it is desirable to assess the existence of seasonal and environmental foraging tendencies as well as differences in honey and pollen mass composition from the botanical origin of samples. the aim of this study was to analyze the variability degree in botanical composition of honey and pollen mass samples from the same and different nests, seasons, and forest types in the chaco region. research article bees laboratorio de actuopalinología, cicyttp-conicet/fcyt-uader, diamante, entre ríos, argentina fg vossler, ga fagúndez, dc blettler variability of food stores of tetragonisca fiebrigi450 materials and methods study area one sampling site (el sauzalito, argentina) was in palosantal forest while the other three (miraflores, juan josé castelli and villa río bermejito, argentina) were in quebrachal forest, which are located no more than 250 km away from each other. these sites have similar climate conditions, they are strongly seasonal with very hot summer (december to march) and low temperatures and frost during winter (july to september); there is a manifest yearly variation in rainfall, with a marked dry season in winter-spring and a rainy season from october to april (prado, 1993). the palosantal forest is characterized by the dominance of “palo santo” trees (bulnesia sarmientoi, zygophyllaceae) while the quebrachal forest by the dominance of “quebracho colorado chaqueño” (schinopsis balansae, anacardiaceae), “quebracho colorado santiagueño” (schinopsis lorentzii, anacardiaceae) and “quebracho blanco” trees (aspidosperma quebracho-blanco, apocynaceae). pollen analysis of samples honeys and pollen masses from the same nest were analyzed separately (i.e., they were taken into account as independent samples). a total of 11 nests were analyzed from the four seasons and two forest types (palosantal and quebrachal). as only honey was found in nest 14 while in nest 18 only the pollen mass, a total of 10 honey and 10 pollen mass samples were studied (tables 1 and 2). honey was sampled from different pots of a nest using a disposable plastic syringe and then homogenized. therefore, one representative sample of honey was kept per nest. closed pots were preferred for sampling honey. however, when only open pots were available, honey was kept from them. from 8 to 83.8 g of honey corresponding to between 6 and 130 honey pots per nest was studied (table 1). honey samples were pure in nests 1, 2, 10, 12, 14 and 15, while they were contaminated with pollen grains from masses during their sampling in nests 5, 6, 7 and 13 (table 1). the pollen masses (the content of pollen cerumen pots) of each nest were mixed and analyzed as an only sample. from 12 to 103 g of pollen corresponding to between 5 and 55 pollen pots per nest was studied (table 2). honey and pollen mass were weighed on an ohaus cs200 electronic balance with 0.1 gram of readibility. honey and pollen mass samples were dissolved with a glass rod in 200 ml of distilled water at 80-90 ºc and then with a magnetic stirrer for 1015 minutes. five milliliters of this mixture was centrifuged at 472 g (pendlenton, 2006) and the sediment was dehydrated using acetic acid and acetolyzed (erdtman, 1960), mounted in slides using a glicerine-gelatin mixture and identified using a nikon eclipse e200 light microscope at 400 and 1000 x magnification. pollen identification was carried out by comparing pollen provision slides with the pollen reference of plants grown in the sites sampled. the identification of the type gleditsia amorphoides was dubious, as it was absent in the sampled area. for this reason, its family was named as fabaceae?. the counting of 500 pollen grains per slide was made for honey samples, while a total of 300-500 grains for pollen masses. the reference pollen collection was made from flower buds of plant species collected in various localities from the chaco province of argentina (juan josé castelli (25°56’ s60°37’ w), villa río bermejito (25°37’ s-60°15’ w), miraflores (25°29’ s-61°01’ w) and el sauzalito (24°24’ s-61°40’ w)). these plant specimens were pressed, dried, identified by the author and deposited in the herbaria of the museo of la plata (lp) and the museo argentino de ciencias naturales “bernardino rivadavia” (ba), buenos aires, argentina. flowering phenology was recorded in these sites during most months except march, may and june (table 3). bees were identified by arturo roig-alsina and deposited in the entomology collection of the museo argentino de ciencias naturales “bernardino rivadavia”, buenos aires, argentina. multivariate analysis the cluster analysis and principal component analysis were the two multivariate techniques applied. the past statistic package (hammer et al., 2008) was used. for cluster analysis, the algorithm upgma (unweighted pair-group average) and bray-curtis distance were applied to the percentage values of the data matrix in q-mode. the highest similarity level is 1.00 and indicates 100% of similarity among pollen composition of samples. the cophenetic correlation coefficient was taken into account as a distortion measurement of the dendrogram (sokal & rohlf, 1962), being values higher than 0.8 indicators of well groupings in the dendrogram compared to the original similarity matrix (sneath & sokal, 1973). for principal component analysis (pca), a correlation matrix was applied to the data matrix in q-mode and r-mode. the scree plot (simple plot of eigenvalues) was used to cut-off the number of significant principal components. after this curve starts to flatten out, the corresponding components may be regarded as insignificant. the eigenvalues expected under a random model (broken stick) were also plotted and the ones under this curve represent non-significant components (jackson, 1993). the first three principal components (pc i, pc ii and pc iii) that made up the greatest part of the variability were graphed. results cluster analysis the dendrogram of samples of honeys and pollen masses of t. fiebrigi showed a high value of cophenetic correlation coefficient (0.879) (fig 1). two main groups can be seen within the dendrogram, diverging at 0.1 similarity level (samples of these groups are only 10% similar). group a (11 samples) is composed of four subgroups diverging at low similarity values (0.2-0.3) while group b of two subgroups diverging at a medium similarity value (0.4) (fig 1). sociobiology 61(4): 449-460 (december, 2014) 451 h on ey s am pl e co de tf 1h w in te r q tf 2h w in te r q tf 5h a ut um n q * tf 6h a ut um n q * tf 7h s um m er p * tf 10 h w in te r p tf 12 h s pr in g q tf 13 h s pr in g p* tf 14 h s pr in g p tf 15 h s pr in g p a m ou nt o f h on ey a na ly ze d (g ) n o da ta n o da ta 15 8 83 .8 30 n o da ta 71 30 20 n um be r of h on ey p ot s an al yz ed 6 19 10 10 55 60 7 ca . 1 30 ca . 5 0 ca . 6 0 d at e of s am pl in g a ug 2 00 6 a ug 2 00 6 a pr 2 00 8 a pr 2 00 8 fe b 20 08 se p 20 08 o ct 2 00 8 d ec 2 00 8 d ec 2 00 8 o ct 2 00 8 lo ca lit y of s am pl in g jj c jj c m ir m ir e ls e ls v rb el s e ls e ls pl an t f am ily a ca nt ha ce ae 0. 34 a ch at oc ar pa ce ae 3. 02 8. 17 6. 54 a na ca rd ia ce ae 63 .4 6 68 .6 0 52 .3 9 11 .3 0 30 .6 0 a po cy na ce ae 0. 34 a re ca ce ae 42 .8 2 85 .6 5 0. 48 a st er ac ea e 2. 05 2. 39 0. 60 1. 94 bi gn on ia ce ae 0. 60 2. 73 6. 05 ca pp ar id ac ea e 4. 83 2. 28 24 .1 1 3. 47 10 .4 1 ce la st ra ce ae 30 .7 7 25 .9 4 1. 60 18 .1 3 89 .3 5 1. 94 ce lti da ce ae 1. 92 0. 34 0. 65 0. 91 12 .9 8 51 .1 5 16 .7 2 2. 18 eu ph or bi ac ea e 0. 68 2. 40 fa ba ce ae , c ae sa lp in io id ea e 6. 34 0. 21 0. 32 3. 87 fa ba ce ae , m im os oi de ae 0. 64 0. 34 46 .8 3 8. 37 2. 88 8. 60 10 .4 1 13 .5 6 fa ba ce ae ? 2. 88 m al pi gh ia ce ae 2. 11 1. 69 n yc ta gi na ce ae 1. 02 po ly go na ce ae 0. 96 0. 63 7. 02 rh am na ce ae 3. 21 0. 34 3. 19 59 .6 2 0. 32 0. 97 sa po ta ce ae 8. 76 3. 37 37 .5 4 37 .5 3 zy go ph yl la ce ae 7. 85 13 .2 1 6. 30 u ni de nti fie d 2 3. 37 u ni de nti fie d 3 2. 88 ta bl e 1. r ef er en ce s o f t he t et ra go ni sc a fie br ig i h on ey sa m pl es st ud ie d an d ab un da nc e of p la nt fa m ili es p er n es t ( in p er ce nt ag e) . * in di ca te s c on ta m in at ed h on ey s; q = q ue br ac ha l f or es t; p = pa lo sa nt al fo re st . jj c (j .j .c as te lli ), (m ir ) m ir afl or es , ( e l s) e l s au za lit o, (v r b ) v ill a r ío b er m ej ito . fg vossler, ga fagúndez, dc blettler variability of food stores of tetragonisca fiebrigi452 c od e of p ol le n m as s sa m pl es t f1 p w in te r q t f2 p w in te r q t f5 p au tu m n q t f6 p au tu m n q t f7 p su m m er p t f1 0p w in te r p t f1 2p sp ri ng q t f1 3p sp ri ng p t f1 5p sp ri ng p t f1 8p w in te r q a m ou nt o f p ol le n an al yz ed (g ) 10 3 n o da ta 71 68 56 12 26 70 60 n o da ta n um be r o f p ol le n po ts a na ly ze d 40 5 45 55 23 7 20 15 20 n o da ta d at e of s am pl in g a ug 2 00 6 a ug 2 00 6 a pr 2 00 8 a pr 2 00 8 fe b 20 08 se p 20 08 o ct 2 00 8 d ec 2 00 8 o ct 2 00 8 ju l 2 00 9 l oc al ity o f s am pl in g jj c jj c m ir m ir e l s e l s v r b e l s e l s m ir pl an t f am ily a ca nt ha ce ae 0. 07 a ch at oc ar pa ce ae 3. 07 0. 81 2. 06 0. 03 a na ca rd ia ce ae 73 .1 9 10 0 40 .5 9 42 .6 7 42 .3 2 a re ca ce ae 0. 13 42 .9 0 56 .8 9 0. 95 0. 05 49 .1 3 a st er ac ea e 0. 05 6. 96 0. 15 1. 87 0. 07 0. 12 b ig no ni ac ea e 2. 47 9. 69 b ro m el ia ce ae 0. 51 c ap pa ri da ce ae 0. 82 1. 53 10 .7 9 12 .6 7 2. 20 39 .4 3 2. 37 0. 05 c el as tr ac ea e 30 .6 5 37 .6 0 0. 61 14 .7 9 0. 77 0. 34 c el tid ac ea e 0. 63 5. 52 0. 29 3. 42 3. 19 29 .4 3 3. 07 0. 47 c he no po di ac ea e 0. 09 e up ho rb ia ce ae 0. 01 1. 69 0. 14 fa ba ce ae , c ae sa lp in io id ea e 1. 72 3. 21 0. 47 0. 04 13 .3 3 fa ba ce ae , m im os oi de ae 11 .8 4 0. 30 25 .2 9 44 .3 4 30 .4 9 11 .8 5 24 .0 5 7. 08 fa ba ce ae ? 24 .6 7 3. 81 l or an th ac ea e 0. 18 0. 07 0. 14 m al pi gh ia ce ae 0. 34 6. 77 2. 10 o la ca ce ae 0. 02 po ly go na ce ae 0. 04 0. 09 1. 54 r ha m na ce ae 6. 31 28 .3 3 0. 67 0. 02 sa nt al ac ea e 2. 16 0. 44 sa po ta ce ae 8. 47 7. 34 4. 69 42 .3 5 si m ar ou ba ce ae 2. 70 u lm ac ea e 3. 19 0. 03 u ni de nt ifi ed 1 0. 19 ta bl e 2. r ef er en ce s of th e te tr ag on is ca fi eb ri gi p ol le n m as se s an al yz ed a nd a bu nd an ce o f pl an t f am ili es p er n es t ( in p er ce nt ag e) . q = q ue br ac ha l f or es t; p = pa lo sa nt al f or es t. jj c ( j. j. c as te lli ), (m ir ) m ir afl or es , ( e l s) e l s au za lit o, (v r b ) v ill a r ío b er m ej ito . sociobiology 61(4): 449-460 (december, 2014) 453 samples from group a are dominated by various plant families, while those from group b are dominated by one or codominated by two families. in each of the four subgroups of group a, different families are important. subgroup a has important percentages of rhamnaceae, b of celtidaceae and capparidaceae, c of celastraceae and fabaceae (mimosoideae), and d of sapotaceae. each subgroup has two sets differing according to the dominance and/or codominance of some of their families. subgroup a has one set (tf 12 h spring q) dominated by rhamnaceae and another codominated by fabaceae (mimosoideae), rhamnaceae and fabaceae? (tf 12 p spring q); subgroup b has one set (tf 13 h spring p) dominated by celtidaceae and in a lesser scale composed of capparidaceae and zygophyllaceae, while the other set (tf 13 p spring p) is codominated by capparidaceae and celtidaceae and in a lesser scale composed of celastraceae and fabaceae (mimosoideae); subgroup c has one set (tf 10 h winter p) dominated by celastraceae and another codominated and/or dominated either by celastraceae or fabaceae (mimosoideae); and subgroup d is composed of two sets, one of them codominated by sapotaceae, fabaceae (mimosoideae), capparidaceae (in honey) (tf 15 h spring p) or sapotaceae, fabaceae (mimosoideae), fig 1. dendrogram showing the two groups and six subgroups of honey and pollen mass samples of tetragonisca fiebrigi. fabaceae (caesalpinioideae) and bignoniaceae (in pollen mass) (tf 15 p spring p) while the other set is codominated by sapotaceae, anacardiaceae, celtidaceae and fabaceae (mimosoideae) (tf 14 h spring p). the subgroup a of group b is dominated by anacardiaceae, while the subgroup b is codominated by arecaceae and anacardiaceae. principal component analysis principal component analysis and cluster analysis partly agreed in the grouping of samples (figs 1, 2 and 3). in q-mode, the principal component i (figs 2 and 3) placed sample tf 2 h winter q on the top left corner (dominated by anacardiaceae and celastraceae), samples tf 12 h spring q and tf 12 p spring q (high percentages of rhamnaceae) on the extreme right and the remaining samples clustered together in the middle (figs 2 and 3). families rhamnaceae, fabaceae?, ulmaceae, unidentified 1 and chenopodiaceae are the major contributors to the principal component i (being 16.35% of the total variability) (supplementary material 1). the principal component ii (fig 2) separates samples tf 2 h winter q and tf 12 h spring q to the top of the graph and both fg vossler, ga fagúndez, dc blettler variability of food stores of tetragonisca fiebrigi454 of the same nest (codominated by rhamnaceae, fabaceae (mimosoideae) and fabaceae?) placing them to the bottom and top of the graph, respectively; and the rest in the middle (fig 3). the three unidentified families (unidentified 2, 3 and 1), chenopodiaceae and ulmaceae are the major contributors to the principal component iii (being 13.24% of the total variability) (supplementary material 1). the first three pc account for only 44.66% of the total variability. however, the greatest part of the variability of samples is made up by the first 8 principal components (83.91%) (supplementary material 2) as showed by the scree plot (supplementary material 3). variability of nest samples of tetragonisca fiebrigi the principal component i (r-mode) shows that certain families (anacardiaceae and arecaceae) are in the extremes of the axes, while principal component ii shows that fabaceae (mimosoideae), celastraceae and sapotaceae are the most distant (fig 4) the association of these families allowed for the differentiation of groups of samples, those from fall and winter (dominated by anacardiaceae and arecaceae) (pc i in supplementary material 1; group b of cluster analysis) samples of nest 15 (the honey sample codominated by sapotaceae, fabaceae (mimosoideae) and capparidaceae, and the pollen sample by sapotaceae, fabaceae (mimosoideae), fabaceae (caesalpinioideae) and bignoniaceae) to the bottom of the graph and the remaining samples to the middle. families bignoniaceae, fabaceae (caesalpinioideae), sapotaceae, fabaceae (mimosoideae) and euphorbiaceae are the major contributors to the principal component ii (being 15.02% of the total variability) (supplementary material 1). the principal component iii (fig 3) separates the honey sample of nest 12 (dominated by rhamnaceae) from the pollen sample from those from spring, summer and winter (in which anacardiaceae and arecaceae were absent) (pc ii in supplementary material 1; group a of cluster analysis). the families which were the greater contributors to each principal component (supplementary material 1) were those exclusive in the samples (such as rhamnaceae, fabaceae?, ulmaceae, unidentified 1 and chenopodiaceae for principal component i). due to the fact that only a low percentage of the variability (44.66%) was given by the first three principal components (supplementary material 2), the samples found in the extremes of the axes of each principal fig 2. two-dimensional graph of the principal components 1 and 2 showing the distribution of store samples. sociobiology 61(4): 449-460 (december, 2014) 455 plant family plant taxon j f m a m j j a s o n d lifeform acanthaceae acanthaceae (except ruellia) h achatocarpaceae achatocarpus praecox griseb. s-t anacardiaceae schinopsis balansae engl. t schinopsis lorentzii (griseb.) engl. t schinus fasciculatus (griseb.) i.m. johnst. s apocynaceae, apocynoideae aspidosperma quebracho-blanco schltdl. t arecaceae trithrinax schizophylla drude s-t asteraceae, astereae baccharis breviseta dc. h-s baccharis salicifolia (ruiz & pav.) pers. s baccharis trinervis pers. s solidago chilensis meyen h asteraceae, heliantheae ambrosia sp. h bidens spp. h melanthera latifolia (gardner) cabrera h parthenium hysterophorus l. h verbesina encelioides a. gray h xanthium spinosum l. h xanthium cavanillesii schouw h asteraceae, inuleae pterocaulon spp. h tessaria dodoneifolia (hook. & arn.) cabrera s bignoniaceae fridericia dichotoma (jacq.) l.g. lohmann c tabebuia impetiginosa (mart. ex dc.) standl. t tabebuia nodosa (griseb.) griseb. t bromeliaceae aechmea distichantha lem. h bromeliaceae (terrestrial species) h capparidaceae capparis atamisquea kuntze s capparis retusa griseb. s-t capparis salicifolia griseb. s capparis speciosa griseb. s-t capparis tweediana eichler s celastraceae maytenus vitis-idaea griseb. s-t moya spinosa griseb. s-t celtidaceae celtis spp. t chenopodiaceae chenopodiaceae spp. h-s euphorbiaceae cnidoscolus loasoides (pax) i.m. johnst. h croton argenteus l. h-s croton bonplandianus baill. h-s croton lachnostachyus baill. h-s jatropha spp. s sapium haematospermum müll. arg. t fabaceae, caesalpinioideae caesalpinia paraguariensis (d. parodi) burkart t cercidium praecox (ruiz & pav. ex hook.) harms t parkinsonia aculeata l. s-t table 3. flowering phenology of the plant taxa whose ascribed pollen types were found in the stores of tetragonisca fiebrigi in the dry chaco forest. during march, may and june, flowering was not recorded. plant life-forms: t = trees, s = shrubs (more than 1 m in height), c = climbers and lianas, h = herbs, semi-shrubs and shrubs less than 1 m in height, e = epiphytes. fg vossler, ga fagúndez, dc blettler variability of food stores of tetragonisca fiebrigi456 table 3. flowering phenology of the plant taxa whose ascribed pollen types were found in the stores of tetragonisca fiebrigi in the dry chaco forest. during march, may and june, flowering was not recorded. plant life-forms: t = trees, s = shrubs (more than 1 m in height), c = climbers and lianas, h = herbs, semi-shrubs and shrubs less than 1 m in height, e = epiphytes. (continuation). pterogyne nitens tul. t fabaceae, mimosoideae acacia aroma gillies ex hook. & arn. s-t acacia curvifructa burkart s albizia inundata (mart.) barneby & j.w. grimes s-t mimosa detinens benth. s-t prosopis alba griseb. t prosopis elata (burkart) burkart s-t prosopis kuntzei harms t prosopis nigra (griseb.) hieron. t prosopis ruscifolia griseb. t prosopis vinalillo stuck. t prosopis (hybrids) s-t loranthaceae struthanthus uraguensis (hook. & arn.) g. don e malpighiaceae mascagnia brevifolia griseb. c nyctaginaceae boerhavia diffusa l. var. leiocarpa (heimerl) adams h olacaceae ximenia americana l. s-t polygonaceae ruprechtia triflora griseb. s-t rhamnaceae ziziphus mistol griseb. t santalaceae acanthosyris falcata griseb. t sapotaceae sideroxylon obtusifolium (roem. & schult.) t.d. penn. t simaroubaceae castela coccinea griseb. s-t ulmaceae phyllostylon rhamnoides (j. poiss.) taub. t zygophyllaceae bulnesia sarmientoi lorentz ex griseb. t fig 3. two-dimensional graph of the principal components 1 and 3 showing the distribution of store samples. sociobiology 61(4): 449-460 (december, 2014) 457 component were different (but not very different) from the remaining samples although they shared many pollen types. discussion were samples clustered by type of forest or season? the samples from each forest type were clustered separately by cluster analysis with the exception of both provisions of nest 12 from quebrachal that were grouped together with palosantal samples. this could suggested that botanical composition of these two groups of samples (groups a and b) is an indicator of the strong differences existent in the floristic composition of these forest types. for instance, the abundance of schinopsis trees (anacardiaceae) in the palosantal is much scarcer than in the quebrachal (cabrera, 1971; prado, 1993). however, samples from group b (all from quebrachal) were sampled in the fall and winter, while those from group a (all palosantal plus two quebrachal samples) mostly in spring suggesting that grouping of samples might not have been achieved by the type of forest. moreover, the combination of phenological records and pollen analysis of stores detected the cause for these groupings. fall and winter provisions were composed mainly of floral resources bloomed during summer and fall such as arecaceae and anacardiaceae, while spring provisions were composed of spring flowerings alone but not summer-fall floral resources. for instance, the only two winter samples (both stores of nest 10) found in group a were not clustered in group b together with the remaining samples of winter because they were composed of pollen types from late winter flowerings but not of types foraged during summer-fall. therefore, it can be said that seasonality strongly influenced the grouping of these store samples. moreover, prado (1993) states that no differences in floristic composition exist between palosantal and quebrachal plant communities, but only in relative abundance of plant species, supporting the idea that seasonality was the cause of these groupings. are there differences in botanical composition among honeys and pollen masses? honeys and pollen masses were grouped together or fairly closed for all nests due to similar abundance of the different pollen types. sampling could be considered as a possible cause of honey contamination as the small cerumen pots of t. fiebrigi are densely packed and fragile. however, both pure (six samples) as well as contaminated honeys (four samples) were closely clustered with pollen masses from the same nests. although fig 4. two-dimensional graph of the principal components 1 and 2 showing the distribution of plant families composing the diet of tetragonisca fiebrigi. fg vossler, ga fagúndez, dc blettler variability of food stores of tetragonisca fiebrigi458 contamination can explain the clustering of honey and pollen mass from nests 5, 6, 7 and 13, it does not apply to the remaining six nests (not contaminated honeys). on the other hand, as honeys were not clustered together with other honeys but with pollen masses indicating a similar botanical composition, it can be assumed that both nectar and pollen were gathered from the same plant species. similar kind of clustering is also observed on the chaquenian stingless bees melipona orbignyi guérin and geotrigona argentina camargo & moure (vossler, unpublished data), supporting that the dry chaco melittophilous vegetation is dominated by plants providers of both pollen and nectar, but not exclusively or predominately of one of them. variability of plant family composition in food stored by tetragonisca bees the results of the present study would indicate that the botanical composition of samples of t. fiebrigi is governed by random factors such as local differences of flower availability but not by preferences for particular plant families. for this reason, bees of the genus tetragonisca have been associated to different plant families according to the vegetation of the study site where samples were taken. for instance, in two sites of chiapas (mexico) t. angustula mainly foraged on fabaceae (caesalpinioideae), celtidaceae, piperaceae, fig 5. acetolyzed pollen grains from nest stores, seen in light microscope at 40 x magnification. a–d pollen grains of type schinopsis (anacardiaceae) (1), celtis (celtidaceae) (2), trithrinax schizophylla (arecaceae) (3), sapium haematospermum (euphorbiaceae) (4), capparis retusa (capparidaceae) (5), castela coccinea (simaroubaceae) (6), prosopis (fabaceae, mimosoideae) (7), type maytenus vitis-idaea (celastraceae) (8), ziziphus mistol (rhamnaceae) (9). bars: 30 µm (a and c); 40 µm (b); 50 µm (d). sociobiology 61(4): 449-460 (december, 2014) 459 celastraceae, sapindaceae, amaranthaceae and clethraceae (sosa-nájera et al., 1994) and on asteraceae, euphorbiaceae, rubiaceae, rutaceae, celtidaceae, sapindaceae, anacardiaceae and phytolaccaceae (martínez-hernández et al., 1994). honeys from coffee agroecosystems from colombia were dominated by rubiaceae, rhamnaceae and malvaceae (obregón et al., 2013). in piracicaba (brazil), this bee species concentrated its foraging on liliaceae, myrtaceae, fabaceae (mimosoideae), fabaceae (papilionoideae), celtidaceae and fabaceae (caesalpinioideae) (carvalho & marchini, 1999; carvalho et al., 1999). in honeys from paraná (brazil), the most important families were apiaceae and fabaceae (caesalpinioideae) (cortopassi-laurino & gelli, 1991) while honeys from são paulo were dominated by euphorbiaceae, myrtaceae, apiaceae and anacardiaceae (iwama & melhem, 1979). pollen stores from são paulo were mainly composed of euphorbiaceae, cecropiaceae and celtidaceae (imperatriz-fonseca et al., 1989). honeys from different localities of são paulo state were dominated by either caricaceae, asteraceae, fabaceae (papilionoideae), myrtaceae or fabaceae (mimosoideae) (barth et al., 2013). in semi-arid areas from northeastern brazil covered by caatinga vegetation, the pollen types more common in honey were from malpighiaceae, asteraceae, myrtaceae, fabaceae (mimosoideae), solanaceae, anacardiaceae, arecaceae and fabaceae (caesalpinioideae) (novais et al., 2013). in this same region, pollen stores were dominated by fabaceae (mimosoideae), solanaceae, moraceae, fabaceae (caesalpinioideae) and malvaceae (novais et al., 2014). in yungas forest (northwestern argentina), honeys of t. angustula were mainly composed of fabaceae (mimosoideae), asteraceae, myrtaceae and rutaceae (flores & sánchez, 2010). in the rio negro channel (amazonas, brazil), pollen stores of t. gr. angustula were mainly composed of cecropiaceae and moraceae (rech & absy, 2011). in western amazonas (acre, brazil), honey samples of t. weyrauchi were dominated by myrtaceae (cortopassi-laurino & nogueira-neto, 2003). conclusion the use of multivariate methods from palynological data allowed the detection of clustering patterns in the food samples of t. fiebrigi. grouping of samples was determined by the season when samples were taken but not by type of forest. honeys and pollen masses were grouped together or fairly closed for all nests due to similar abundance of the different pollen types. furthermore, honeys were not clustered together with other honeys but with pollen masses. nectar and pollen were gathered from the same plant species, supporting the hypothesis that the dry chaco melittophilous vegetation is dominated by plants providers of both pollen and nectar, but not exclusively or predominately of one of them. the foraging behavior of t. fiebrigi was governed by random factors such as local differences of flower availability but not by preferences for some plant families. acknowledgments we wish to thank ricardo “nene” vossler, juan hiperdinger and césar albornoz for their warm hospitality and help during the field studies. we also thank nora brea for providing suggestions and comments on the manuscript. this study was supported by conicet (consejo nacional de investigaciones científicas y técnicas). references arenas, p. 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(2012). descriptive attributes used in the characterization of stingless bees (apidae: meliponini) in rural populations of the atlantic forest (misiones-argentina). j. ethnobiol. ethnomed., 8: 1-10. doi:10.1186/1746-4269-8-9. this article has online supplementary material that can be retrieved at: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/576/0 doi: 10.13102/sociobiology.v61i4..s713 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5188sociobiology 68(1): e5188 (march, 2021) introduction several experiments have shown that animals can use the geomagnetic field (gmf) for orientation and homing using a magnetoreception process (wiltschko & wiltschko, 2005). social insects, such as bees, ants and wasps, can use magnetoreception for magnetic orientation in guidance and navigation tasks (wajnberg et al., 2010; pereira-bomfim et al., 2015). unlike magnetic orientation, there are some reports of sensibility to magnetic fields (mf) by insects, where variations in mfs can change common behaviors. vowles (1954) presented a pioneer study with the ant myrmica laevinodis (nylander, 1846). he studied the orientation of ants to gravity and used tiny particles of soft iron cemented to abstract the present paper aims to study magnetosensibility and seek magnetic nanoparticles in ants. by living in colonies, the social insects developed very efficient methods of nestmate recognition, being less tolerant towards individuals from other colonies. therefore, any strange behavior between nestmates and/or conspecifics, besides those present in their behavioral repertoire, is not expected. the present paper analyzes whether changes in the intensity of applied magnetic fields on ectatomma brunneun (smith) ants can cause changes in the typical pattern of interaction between conspecifics. we used a pair of coils generating a non-homogeneous magnetic field to change the whole local geomagnetic field. magnetometry studies were done on abdomens and head + antennae using a squid magnetometer. the results show that changes in the geomagnetic field affect the usual pattern of interactions between workers from different colonies. the magnetometry results show that abdomens present superparamagnetic nanoparticles and heads present single-domain magnetic nanoparticles. behavior experiments show for the first time that ectatomma brunneun ants are magnetosensible. the change in nestmate recognition of ectatomma ants observed while a magnetic field is applied can be associated with disturbance in a magnetosensor presented in the body based on magnetic nanoparticles. sociobiology an international journal on social insects márlon césar pereira1,2, maria da graça cardoso pereira-bomfim2, ingrid de carvalho guimarães2, candida anitta pereira rodrigues1,2, jilder peña serna3, daniel acosta-avalos3, william fernando antonialli-junior2 article history edited by evandro n. silva, uefs, brazil received 12 april 2020 initial acceptance 26 january 2021 final acceptance 19 march 2021 publication date 31 march 2021 keywords aggression, ectatomminae, magnetometry, insect behavior. corresponding author marlon césar pereira universidade federal da grande dourados, cidade universitária rodovia dourados-itahum, km 12, cep 79804-970, dourados-ms, brasil. e-mail: marloncesarp@yahoo.com.br different parts of the ant body to generate a magnetic couple, in the presence of a magnetic field of about 300 μt, to be added to the gravitational couple. he aimed to identify where the gravitation sensor is located in the ant body. during the experiments, vowles observed that the magnetic couple does not alter the orientation to the gravitational force. still, he observed that ants changed their behavior when the magnetic field was on: they stopped their movement or often cleaning their antennae when the iron particles were located in the funiculi or the scape of both antennae (vowles, 1954). the study of kermarrec (1981) showed that acromyrmex octospinosus (reich, 1793) ants are sensitive to strong static mfs provided by magnets (of about 19100 μt and 27300 μt), by avoidance reactions including the repeatable movement 1 programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados, dourados-ms, brazil 2 laboratorio de ecologia comportamental, centro de estudos em recursos naturais, universidade estadual de mato grosso do sul, dourados-ms, brazil 3 centro brasileiro de pesquisas fisicas, cbpf, rio de janeiro-rj, brazil research article ants magnetosensibility and magnetic properties of ectatomma brunneun smith, f. 1858 ants mailto:marloncesarp@yahoo.com.br marlon césar pereira et al. – magnetosensibility of e. brunneum ants 2 of brood by workers. in the same study, no reaction was observed for magnetic fields of about 200 μt, and 500 μt. anderson and vandermeer (1993) showed that solenopsis invicta (buren, 1972) ants are sensitive to changes in the gmf direction analyzing the time of trail formation in an experimental arena. in those studies, with m. laevinodis, a. octospinosus, and s. invicta, the observed responses to mfs are examples of magnetosensibility, not necessarily related to orientation and homing. a magnetoreception mechanism essentially assumed in ants is the ferromagnetic hypothesis (johnsen & lohmann, 2005). it claims that the magnetic field transduction occurs through magnetic nanoparticles located inside some specialized cells associated with the nervous system. in pachycondyla marginata (roger, 1861) ants, the search for magnetic nanoparticles involved studies on their isolation (acosta-avalos et al., 1999), squid magnetometry (wajnberg et al., 2004), ferromagnetic resonance (wajnberg et al., 2000), and electron microscopy (oliveira et al., 2010). such studies allowed the identification of the antennae as the location of the magnetoreceptor. for other insects, such as atta colombica (guérin-méneville, 1844) and schwarziana quadripunctata (lepeletier, 1836) (lucano et al., 2006; alves et al., 2014), similar studies identify the antennae as the body part with a higher magnetic signal. like other social insects, ants show a high degree of cooperation among the individuals that interact in the colony (zinck et al., 2008). for instance, workers play an active role in nest construction, colony protection against predators, foraging, and brood care (ratnieks et al., 2006). the colony’s integrity depends on social interactions and communication among nestmates (crozier & pamilo, 1996). those abilities are well developed in ants, and individuals from a foreign nest are usually attacked and rejected from foreign colonies (crozier & pamilo, 1996; sturgis & gordon, 2012). however, the level of aggression towards conspecifics non-nestmates, i.e., during nestmate recognition, can vary among species (d’ettorre & lenoir, 2010). ants, in general, control and defend their territory, where they extract resources (newey et al., 2010). encounters among foragers from different colonies can generate conflicts that can turn into clashes, resulting even in individuals’ death (matthews & matthews, 2010). in some cases, ants can be tolerable to neighbors, implying that nestmate recognition and aggression can be associated with colony-recognition odors from environmentally derived cues, not only to the genetically derived ones (chen & nonacs, 2000; frizzi et al., 2015). as far as we know, magnetosensibility has not been applied to analyzing the aggressive behavior during nestmate recognition in the presence of different magnetic fields. the present study aimed to investigate magnetosensibility in ectatomma brunneum (smith). we hypothesized that the usual pattern of interaction among conspecifics could be affected by applied magnetic fields. we also looked for the presence of magnetic material in the ant’s body using squid magnetometry techniques. materials and methods behavior experiments we used a pair of coils connected to a digital power supply (skill tech, model skfa-05d) to test the effect of magnetic fields on ants’ intraspecific interactions, as described by pereira-bomfim et al. (2015). the coils had a 30 cm diameter with a space of 15 cm between them. they were built with 58 spirals of cu wire 14 awg (figs 1a and 1c), generating a non-uniform static magnetic field (fig 1b) applied to a plastic container called “arena of encounters” (fig 1c). the coils’ axis was kept in the south/north direction, oriented with a compass. when the power supply was switched on (15 v, 1.16 a), the horizontal component of the magnetic field (open circles in fig 1b) inside the coils presented its polarity inverted in the region from 4 cm to 12 cm (figs 1a and 1b). the intensity of the gmf in the lab was -16 μt horizontal, 21 μt vertical, and 16 μt perpendicular, meaning a total intensity of 31 μt. to measure the intensity of the mf, we used a sheet of graph paper. in this paper, we measured the intensity value in the vertical, horizontal and perpendicular directions at every centimeter. we took all measurements using a gaussmeter (globalmag, model tlmp-hall 050). the total magnetic field generated is shown in fig. 1b. the essential characteristics in the generated magnetic field, relative to the standard geomagnetic field, are the inversion in the vertical component (open squares in fig 1b), changing the sign of the inclination, the increasing of the magnetic field intensity from the center to the periphery, and the u-form of the field intensity. from 5 cm to 9 cm, the magnetic field gets its lower value of about 100 μt (about three times the local value in the lab, see the open triangles in fig 1b). the magnetic field generated by the pair of coils corresponds with a quadrupolar field generated by an antihelmholtz configuration. the electrical current circulates in the opposite direction in each coil (youk 2005). in contrast, in a helmholtz configuration, the current circulates in the same direction in each coil generating a dipolar field. this magnetic field configuration is interesting because it changes non-uniformly all the parameters of the local gmf (intensity, inclination and declination) and the increase in mf intensity is very low compared with that generated by magnets, as was done by vowles (1954) and kermarrec (1981). we collected a total of 56 foragers from six colonies of e. brunneum from their nests’ entrance. the colonies were in the campus of universidade estadual de mato grosso do sul, dourados, ms, brazil. after transfer to the laboratory, we kept the individuals in a 250 ml plastic container with holes in the sides and lid to allow air to circulate, with water and molasses ad libitum, wrapped in red cellophane to minimize the stress caused by collection and luminosity. for acclimatization to laboratory conditions (24ºc and 70% of humidity), a period of 24 hours was considered. then, we performed induced encounters among intraspecific workers. to this end, the sociobiology 68(1): e5188 (march, 2021) 3 ants were transferred to the “arena of encounters” (fig 1c), a container of 16cm x 10cm x 10cm, inside which there was a smaller container (6cm x 4cm x 3cm) in the central inner part, where we placed one ant. then, after one minute, a second worker from another colony was placed in the arena, outside the smaller container. this smaller container was only removed after 1 minute, thus enabling the encounter among the two workers. we induced 24 encounters, peer to peer, among 48 workers of six colonies of the species e. brunneum whose nests were at varying distances from each other (fig 2). as control of experimental conditions, we induced four nestmate encounters. we used each ant only once in the experiment to avoid pseudoreplication, and at each encounter, we used a new arena. we observed each pair interacting for 45 minutes in sessions of 15 minutes. in the first 15 minutes, the interactions occurred with the coils switched off, i.e., before the change in mf; for the next 15 minutes, the coils were switched on to generate a different mf in them; and in the last 15 minutes, the coils were switched off again. we kept the coils always in the same position to avoid their influence on the behavioral responses. to assess the level of aggression during the encounters, we scored behaviors from 0 to 2 as follows: 0 for ignoring (thomas et al., 2004), touching and avoiding (suarez et al., 1999); 1 for attempted seizure, seizure, antennal boxing, body lifting, gaster curling or aggression from one worker (monnin & peeters, 1999); and 2 for a fight, identified when both workers execute the aggressive behaviors (modified from suarez et al., 1999). we observed the evolution in the aggressive behavior during each encounter and recorded scores when the behavior changed. for each encounter, we calculated the arithmetic mean of the score regarding the observed levels of aggression. fig 1. pair of coils configuration and the magnetic field generated. a. representation of the orientation of the mf components measured among the coils. a* represents the arena of encounters. b. intensity of the magnetic field components as a function of the distance among the coils. the first coil is positioned at x = 1cm and the second coil is at x = 15cm. also is represented the total intensity of the magnetic field, whose variation corresponds with a quadrupolar field. c. photography showing the pair of coils, made of 58 spirals of cu wire 14 awg. in the middle is shown a plastic box that corresponds to the “arena of encounters”. while functioning, the coils can increase the temperature. to assess whether the increase in temperature could lead to behavioral response changes, we photographed all encounters with a thermal camera (testo® 870). we took thirty pictures at each step of the experiment from random encounters. with the aid of the software testo irsoft, we selected ten random points of thermal photos from both the arena and ants’ bodies to establish the mean temperature of the ants’ body during the encounters and assess whether the ant temperature varies when coils are switched on/off. squid magnetometry to assess whether there are magnetic nanoparticles on the ant’s bodies, we collected individuals of e. brunneum (eb). fig 2. distance in meters among the field colonies of ectatomma brunneum used in the experimental encounters. marlon césar pereira et al. – magnetosensibility of e. brunneum ants 4 afterward, we separated the heads + antennae (denominated only as head) and abdomens, kept these body parts in the fridge, and preserved them in a solution of 70% alcohol. we used a squid magnetometer (mpms quantum design) to search for magnetic material in these body parts. five abdomens and five heads of eb were dried and placed inside a gelatine capsule, which we placed in a plastic straw fixed on the magnetometer’s sample holder for magnetization measurements. we took two magnetization measurements as a function of temperature: zero field cooling (zfc), where the sample is cooled from room temperature in the presence of a null magnetic field; and field cooling (fc), where the sample is cooled in the presence of a magnetic field, which was 10000 µt (100 oe) in our measurements. after cooling the sample, a magnetic field was applied to it, and the magnetization was measured as the temperature increased. the temperature range was between 10 k and 330 k, under a magnetic field of 10000 µt (100 oe). we took hysteresis measurements at 300 k or 150 k, on a magnetic field range between -1t and +1t (1t = 106 μt). based on the hysteresis curves, we determined the saturation magnetization (ms), remanent magnetization (mr) and coercive field (hc). ms results from all contributions to magnetization, while mr is solely composed of the ferromagnetic component. as heads and abdomens have different sizes and masses, we calculated ms and mr, dividing the original values by analyzing the parts’ total mass. statistics we evaluated the difference in aggression levels among the three experimental conditions (coils off, on, and off again) using a kruskall-wallis test. to assess whether there were significant differences between the thermal photos’ mean temperature during the same encounters, we applied a kruskall-wallis test. we performed all statistical tests using the free software r 3.2.1 version. results aggressive behavior we recorded a total of 1571 behaviors during the encounters between workers of e. brunneum. from this total, 43.7% occurred before the change in mf, 27.6% during the change in mf, and 28.7% after the change in mf. the main behaviors performed by workers of e. brunneum were seizure (10.6%), attempted seizure (9.73%) and antennal boxing (9.35%). during controls, we did not observe aggressive behavior (touch and antennation represented 100% of behaviors). the mean level of aggression between workers from different colonies of e. brunneum was 1.5 ± 0.3 before the change in mf, 1.1 ± 0.1 during the change in mf, and 1.3 ± 0.2 after the change in mf (fig 3). the kruskal-wallis test showed significant differences between the groups (χ2 = 17.8 p < 0.05). fig 3. a. levels of aggression of ectatomma brunneum as a function of the distance among colonies. the intensity of the aggression displayed was registered during induced encounters among workers from different colonies, before the change in mf, during the change in mf and after the change in mf. the encounter number is related to the distance among the colonies as shown in fig 2. each point represents the mean value of the aggression level and the bar the standard error. b. boxplots for the levels of aggression displayed by workers of ectatomma brunneum. data acquired during the induced encounters, before, during and after changing the values of mf. the kruskal-wallis test shows that before, during, and after the coils switching on/off, there were no significant differences in the mean body temperature of e. brunneum workers (χ2 = 1.3 p > 0.052) (fig 4). magnetometry fig 5 shows the zfc and fc curves for the head and abdomen. all the curves are compatible with the presence of magnetic nanoparticles. for the abdomen, the nanoparticles show a strong dipolar interaction and a smaller size than the head. sociobiology 68(1): e5188 (march, 2021) 5 as for the head, in low temperatures, we observed a sudden increase in magnetization. this phenomenon is characteristic of paramagnetic contributions due to spins not compensated on the surface of the nanoparticles (peck et al., 2011). from fig 5 is observed that the blocking temperature for the abdomen is 282 k that can be associated with nanoparticles with an average diameter of about 24 nm (considering magnetite as the magnetic material). for heads, the blocking temperature must be higher than 340 k associated with the presence of magnetic nanoparticles whose sizes are bigger than 26 nm (considering magnetite as the magnetic material). fig 6 shows an example of magnetization curves for abdomens and heads after removing the diamagnetic + paramagnetic contributions. table 1 shows the results at 300 k for the coercive field hc, the remanent magnetization mr and the saturation magnetization ms. fig 7 shows ms and hc as a temperature function. the results show that the abdomen has higher ms values and lower hc values compared to the head. the value of mr/ms for the abdomen is compatible with the presence of superparamagnetic particles in the paramagnetic fig 4. boxplots for the distribution of ant body temperatures before the change in mf, during the change in mf and after the change in mf, for ectatomma brunneum. fig 5. zero field cooling (zfc) and field cooling (fc) curves from 10k to 330 k in the presence of a magnetic field of 10000 μt, for heads and abdomens of ectatomma brunneum. fig 6. example of hysteresis curve obtained for heads and abdomens of ectatomma brunneum. the graph shows the magnetization m as a function of the magnetic field b. the magnetic field increases from 1t to +1t (1 t = 10000 oe). the insert shows an amplification in the region from -200 oe to +200 oe, where can be observed the remanent magnetization mr and the coercive field hc. the magnetization is relative to the total mass. marlon césar pereira et al. – magnetosensibility of e. brunneum ants 6 state, producing higher ms values. on the other hand, those values for the head are compatible with single domain or blocked superparamagnetic nanoparticles, producing higher coercivities. the superparamagnetic fraction (fsp) was calculated as: fsp = [m(150k) m(300k)]/m(150k) (1) the values obtained for fsp are compatible with the values for mr/ms: the abdomen shows a higher amount of superparamagnetic particles than the head. the crude curves m(b) showed diamagnetic + paramagnetic contributions as a function of temperature. from these outcomes, it is possible to calculate the paramagnetic susceptibility χpm showed in table 1. the head shows a higher value of χpm compared to the value for the abdomen. that correlates with the low temperature behavior observed for heads in the zfc-fc curves (fig 5). pm’s higher value could mean that the nanoparticles in the head are single domains with defects in the surface producing an extra paramagnetic contribution (peck et al., 2011). nests. this recognition ability and consequent intolerance among themselves are related to competition for resources (temeless, 1994; sanada-morimura et al., 2003). this type of dispute has been described in the ants pogonomyrmex barbatus (smith, 1858) (gordon, 1989), pristomyrmex punctatus (smith, 1860) (sanada-morimura et al., 2003), linepithema humile (mayr, 1868) (thomas et al., 2004), and the termite nasutitermes corniger (motschulsky, 1855) (dunn & messier, 1999). we can explain the observed results through the change in the local temperature generated by the coils when they were turned on because of resistive losses. some studies have reported that temperature changes can affect the behavior of various animal groups (walther et al., 2002; deutsch et al., 2008; dell et al., 2011, 2014; huey et al., 2012; gilbert et al., 2014; sunday et al., 2014; vasseur et al., 2014; woods et al., 2015). however, our results demonstrate that the temperature variation in the surface of the ants’ body is not statistically significant (fig 4), so the temperature is not a stressing factor to explain the ant behavior observed. the fact of ants being intolerants to foreign ants is probably due to the ability to recognize non-nestmates through chemical signals, especially cuticular hydrocarbons already investigated in several studies (jutsum et al., 1979; liang & silverman, 2000; sorvari et al., 2008; van zweden et al., 2009; van zweden & d’ettorre, 2010; helanterä et al., 2011). when the coils were turned on, the change in mf decreased the aggression score (see scores for during in fig 3a). for e. brunneum, a significant decrease is observed only for further nests. when the coils were turned off, the aggression score increased, but it did not recover the same level as it had at the beginning of the encounter. therefore, when the coils were turned on, the change in mf seems to have caused disorientation on the ants, consequently changing the pattern of interaction displayed when the coils were turned off. a piece of evidence that supports the previous comments is that during the encounters with the mf turned on, ants showed “immobility”, an atypical behavior of them, remaining still without even moving the antennae and, after a period, they started to clean their antennae more often than usual. our results show for the first time that e. brunneum is magnetosensible, being perturbed by local changes in the mf. we cannot claim that mf changes affect the aggressive ant behavior but that changes in mf change the ant behavior. the magnetometry measurements show the presence of magnetic nanoparticles in the head and the abdomen of e. brunneum ants. in each case, the nanoparticles show different magnetic properties at room temperature: in the abdomen, fig 7. saturation magnetization (ms) and coercive field (hc) as a function of the temperature, for heads and abdomens of ectatomma brunneum. hc (oe) mr (emu/g) ms (emu/g) mr/ms fsp χpm abdomen 11 3.9x10-4 1.4x10-2 0.03 0.9 3.1x10-5 head 160 4.9x10-4 1.3x10-3 0.38 0.1 9.1x10-5 table 1. parameters obtained from the hysteresis curve at 300 k: coercive field (hc), remanent magnetization (mr) and saturation magnetization (ms). the superparamagnetic fraction fsp was calculated as indicated by eq. (1). χpm is the paramagnetic susceptibility. discussion in normal conditions, under the influence of the geomagnetic field, we found higher levels of aggression among e. brunneum ants (scores for before in fig 3a) whose nests were further apart in more than 100 m (fig 2). this finding means that these ants are tolerant to individuals from nests in a radius of 100 m. pereira et al. (2019) showed that e. brunneum colonies are more tolerant to ants from nearest sociobiology 68(1): e5188 (march, 2021) 7 the nanoparticles are mainly in the superparamagnetic state, and in the head, the nanoparticles are mainly single domains and superparamagnetic nanoparticles in the blocked state. magnetometry studies done in other ants have shown that all parts of the ant body are magnetic (wajnberg et al., 2010), but the abdomen and the head show different magnetic properties. those studies led to the proposal that the magnetosensor must be in the antennae for pachycondyla marginata, atta colombica, and schwarziana quadripunctata (wajnberg et al., 2004; lucano et al., 2006; alves et al., 2014). our results show that the abdomen and the head have different magnetic properties, and both can host the magnetosensor responsible for the observed magnetosensibility. in both cases, the type of magnetosensor must be different. on the one hand, the abdomen has an arrangement of superparamagnetic nanoparticles sensitive to the magnetic field intensity. on the other hand, the head has an arrangement of magnetic single domains sensitive to the magnetic field vector’s intensity and direction (johnsen & lohmann, 2005). our results and other studies (wajnberg et al., 2010) show that ants can detect mf intensity changes. oliveira et al. (2010) proposed that p. marginata ants can perceive the magnetic field through magnetic nanoparticles in the antennae. since the orientation of those magnetic nanoparticles and their magnetic moment can be rapidly altered when ants walk inside the “arena of encounters”, because the mf is displaced from the center of the arena (fig 1b), the increase in cleaning behavior suggests an attempt to adjust the magnetic sensor to the new mf configuration. another evidence that the mf is perceived by ants and causes changes in behavior is that soon after the coils were turned off, the ants stopped performing the abovelisted behaviors, and aggression levels increased again, but not to the same level of aggression shown before the coils were on. conclusion our results show that e. brunneum ants tolerate ants from other nests at distances lower than 100 m. for the first time, our results show that e. brunneum ants are magnetosensible, changing their behavior under the effect of applied mf. their body presents magnetic nanoparticles with different properties in the abdomen and the head. as the mf changes are about three times the local magnetic field, the change in behavior can be associated with disturbance and/or disorientation in some magnetosensor located in the ant’s body. our results do not discard the presence of a magnetoreceptor based on the radical pair mechanism. future experiments must address the impact of mf on several ants’ physiological and ethological traits, to establish the origin of the reported magnetosensiblity and understand its biophysical basis. acknowledgments the authors thank universidade federal da grande dourados, universidade estadual de mato grosso do sul, centro de estudos em recursos naturais and centro brasileiro de pesquisas físicas for technical support, coordenação de aperfeiçoamento de pessoal de nível superior (capes) and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for financial support, wf antoniallijunior acknowledges cnpq (nº 308182-/2019-7) and jp serna acknowledges cnpq by pci grant. authors’ contribution mc pereira: conceptualization, methodology, investigation, formal analysis, writing, review & editing mgc pereira-bomfim: conceptualization, methodology, writing, review & editing wf antonialli-junior: conceptualization, methodology, writing, review & editing ic guimarães: formal investigation, writing, review & editing jp serna: formal investigation, writing, review & editing d acosta-avalos: formal investigation, writing, review & editing cap rodrigues: writing, review & editing references acosta-avalos, d., wajnberg, e., oliveira, o.s., leal, i., farina, m. & esquivel, d.m.s. 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(2008). low intraspecific aggression level in the polydomous and facultative polygynous ant ectatomma tuberculatum. entomologia experimentalis et applicata, 126: 211-216. doi: 10.1111/j.1570-7458.2007.00654.x open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.5459sociobiology 69(2): e5459 (june, 2022) the behavior of forming sleeping aggregations on stems of bushes and trees or in the nesting site to sleep at night is characteristic of many species of bees and wasps (evans & linsley, 1960; linsley, 1962; alves-dos-santos et al., 2002). records of these insects forming sleeping aggregations have been made for more than a century, but there is no full understanding of the reasons for this phenomenon (banks, 1902; bradley, 1908; rau & rau, 1916). however, studies suggest that such behavior may be related to thermoregulation (evans & gillaspy, 1964; linsley & cazier, 1972), defense against predation (evans & linsley, 1960; alcock, 1998) or the evolution of social behavior (grassé, 1942). bees’ sleeping aggregations are usually composed of males because females usually spend the nights inside their nests (alcock, 1998). sleeping aggregations can last weeks, months (evans & linsley, 1960) or even years when shared by individuals of different generations (linsley, 1962). sleeping aggregations of male in the tribe eucerini (apidae) have already been reported for different species (table 1). here we describe the general aspects of the behavior of melissodes abstract bee males are sometimes found forming sleeping aggregations on stems of bushes or trees to sleep at night, but there is no complete understanding of the reasons for this behaviour. this note describes the behavior of melissodes (ecplectica) nigroaenea (smith, 1854) males forming temporary sleeping aggregations in dry inflorescences of bidens pilosa l. the sleeping aggregations of m. nigroaenea were observed for approximately 15 days in an area of cerrado, brasília, df. during the day m. nigroaenea males visit flowers of cosmos sulphureus cav. near the sleeping aggregations, where the females collect pollen. in the late afternoon, the males return to the sleeping aggregations, where they sleep at night. these data provide new information about the behavior of m. nigroaenea males. sociobiology an international journal on social insects wagner p. silva, rogério r. andrade article history edited by evandro nascimento silva, uefs, brazil received 16 december 2021 initial acceptance 19 march 2022 final acceptance 11 june 2022 publication date 24 june 2022 keywords bees, dormitory, night shelter, solitary bees. corresponding author wagner pereira silva laboratório de hymenoptera, instituto de biologia, universidade de brasília 70910-900, brasília, df, brazil e-mail: wagner.sillva@yahoo.com.br (ecplectica) nigroaenea (smith, 1854) males in two sleeping aggregation in central brazil. two sleeping aggregations of m. nigroaenea males were established for approximately 15 days, between march and april 2018, and observed (about 30 h) in an agroforest on the campus darcy ribeiro of the university of brasília (unb), federal district, brazil (15°45’51.1” s, 47°52’05.4” w), where native plant species are maintained and cultivated in a soil rich in exogenous organic matter and green manure. two species of asteraceae – bidens pilosa l. and cosmos sulphureus cav. – stand out in the place by the occurrence of several species of bees such as bombus (fervidobombus) pauloensis friese, 1913; epanthidium tigrinum (schrottky, 1905); megachile spp. latreille, 1802 using the reproductive structures of the plants to sleep. the sleeping aggregations of m. nigroaenea were recorded in inflorescences of b. pilosa, at about 40 and 60 cm above the ground, and approximately 15 cm between both. the highest number of males was recorded in march when five and ten males, respectively, in the 40 and 60 cm sleeping aggregations were recorded. laboratório de hymenoptera, instituto de biologia, universidade de brasília, brasilia-df, brazil male sleeping aggregation of melissodes (ecplectica) nigroaenea (smith, 1854) (hymenoptera, apidae, eucerini) in brazilian cerrado id short note mailto:wagner.sillva@yahoo.com.br https://orcid.org/0000-0002-5199-3124 running title: wagner p. silva, rogério r. andrade – male sleeping aggregation of melissodes (ecplectica) nigroaenea2 the collected specimens (n = 2) were assembled, identified and deposited in the entomological collection of unb (department of zoology). the first males arrived at the sleeping aggregation site around 16:00 h (figure 1). m. nigroaenea males used the mandibles to fix themselves to the dry inflorescences of b. pilosa. this way of attaching to the substrate has been observed in many bee species, f. ex. coelioxys deplanata cresson, 1878; melissoptila aff. bonaerensis holmberg, 1903; centris (paracentris) xanthomelaena moure & castro, 2001 (linsley, 1962; mahlmann et al., 2014; martins et al., 2018). after clinging to the inflorescences, the males used fore and middle legs to find the ideal position to establish in the sleeping aggregation and spend the night (figure 2). some males then frictioned the hind legs, concomitantly, sometimes scrubbing them against the sterna or terga. this behavior of scrubbing the legs and the sterna or terga may be related to chemical signaling, being observed also in tetrapedia species (alves-dos-santos et al., 2009). fig 1. male sleeping aggregation on dried inflorescence of bidens pilosa 60 cm above the ground (a) melissodes nigroaenea males arriving at the sleeping aggregation. (b) m. nigroaenea males trying to find a position in the sleeping aggregation. interactions between the m. nigroaenea males were registered in some situations, usually due to the arrival of a new individual at the sleeping aggregation, which collided with another male, establishing for a few seconds some contact. these interactions almost always occurred when the last males arrived at the sleeping aggregations, around 17:00 h, and other individuals were already resting. males left the sleeping aggregations early in the morning, resting individuals were not observed after 07:00 h. however, on two or three occasions m. nigroaenea males were recorded returning to their sleeping places and during the day when it was cloudy or raining. females of m. nigroaenea were observed (about 30 individuals) collecting pollen, mainly between 10:00 and 16:00 h, in flowers of c. sulphureus near the sleeping aggregations. m. nigroaenea males (n = 4) were often observed ingesting nectar from the flowers of c. sulphureus, but of copulations on flowers were not observed (figure 3). fig 2. melissodes nigroaenea males fixed in bidens pilosa through the mandibles at the sleeping aggregation 40 cm above the ground. sociobiology 69(2): e5459 (june, 2022) 3 according to chemsak & thorp (1962), melissodes robustior cockerell, 1915 males seem to present a preference for sleeping in cosmos sp. flowers, where females collect pollen. some authors suggest that establishing sleeping aggregations near flowers used by females as a source of floral resources may represent a strategy adopted by males in the search for mates on the next days (alves-dos-santos et al., 2009; pinheiro et al., 2017). mahlmann et al. (2014) also recorded a sleeping aggregation formed by m. nigroaenea males. the individuals of two eucerini species – m. nigroaenea and melissoptila aff. bonaerensis – formed an sleeping aggregation where the individuals remained fixed through the mandibles in dry inflorescences of hyptis sp. (lamiaceae). other studies report sleeping aggregations formed by individuals of both sexes (evans & linsley, 1960; starr & vélez, 2009; yokoi et al., fig 3. male of melissodes nigroaenea ingesting nectar in flower of cosmos sulphureus. table 1. sleeping aggregations records of eucerini available in the literature. species substrates sex reference florilegus (florilegus) condignus cresson 1878 on racemes of medicago sativa (fabaceae) male laberge & ribble (1966) gaesochira obscura (smith, 1879) stems of an unidentified species unknown rau & rau (1916) melissodes (ecplectica) nigroaenea (smith, 1854) dried inflorescence of hyptis sp. (lamiaceae) dried stems of bidens pilosa l. (asteraceae) male male mahlmann et al. (2014) present study melissodes (eumelissodes) agilis cress 1878 stems of an unidentified species in sunflowers (helianthus sp., asteraceae) unknown unknown bradley (1908) rau & rau (1916) melissodes (eumelissodes) denticulata smith, 1854 verbena stricta (verbenaceae) male mathewson & daly (1955) melissodes (eumelissodes) robustior cockerell, 1915 inside flower of cosmos sp. (asteraceae) male chemsak & thorp (1962) melissodes (eumelissodes) vernoniae robertson, 1902 verbena stricta (verbenaceae) male mathewson & daly (1955) melissodes (melissodes) bimaculata (lepeletier, 1825) stems of an unidentified species stems of an unidentified species melilotus sp. (fabaceae) unknown unknown unknown banks (1902) rau & rau (1916) rau (1938) melissodes verroniana robt. stems of an unidentified species unknown rau & rau (1916) melissoptila aff. bonaerensis holmberg, 1903 dried inflorescence of hyptis sp. (lamiaceae) males, females mahlmann et al. (2014) svastra (brachymelissodes) cressonii (dalla torre, 1896) petioles of leaves of unidentified species male cockerell (1915) svastra (epimelissodes) obliqua (say, 1837) stems of unidentified species male rau & rau (1916) svastra (idiomelissodes) duplocincta (cockerell, 1905) leaf or stem of encelia farinosa (asteraceae) leaf or stem of calliandra eriophylla (fabaceae) leaf or stem of ruellia peninsulares (acanthaceae) leaf or stem of justicia californica (acanthaceae) male male male male alcock (1998) alcock (1998) alcock (1998) alcock (1998) running title: wagner p. silva, rogério r. andrade – male sleeping aggregation of melissodes (ecplectica) nigroaenea4 2016, 2017). however, in the present study only m. nigroaenea males were observed in both sleeping aggregations. many explanations of the functions of aggregations have been proposed, but are still inconclusive. sleeping aggregations could represent a strategy to reduce the risk of nocturnal predation, although it could also represent a greater risk of predation as has already been described for stingless bees (evans & linsley, 1960; brown, 1997; alcock, 1998). another benefit that sleeping aggregations could provide would be the possibility of elevating the capacity of individuals to thermoregulate, since in the face of a possible predator, the bees could present a minimum ideal temperature to perform the flight activity and consequently escape (linsley & cazier, 1972). the hypothesis that males form sleeping aggregations near sources of pollen visited by possible reproductive partners cannot be discarded. thus, cosmos flowers could serve not only as a source of floral resource but also as a mating site for eucerini species (chemsak & thorp, 1962). besides sleeping in aggregations, some studies have reported eucerini males sleeping inside flowers mainly from cucurbitaceae and orchidaceae (hurd & linsley, 1964; dafni et al., 1981; willis & kevan, 1995; vereecken et al., 2012; watts et al., 2013). there is not yet full understanding of the factors that determine the establishment of sleeping aggregations. however, our observations add new information about the behavior of eucerini males. future studies, addressing how this behavior may be related to a possible strategy of defense or sexual selection in solitary bees are needed. acknowledgements the authors thank dr. antonio j.c. aguiar (department of zoology/unb) and dr. joão bernardo de azevedo bringel júnior (department of botany/unb), respectively, for the identification of the bees and plant species. wp silva thanks the coordenação de aperfeiçoamento de pessoal de nível superior (capes), under finance code 001, for granting a doctoral fellowship. rr andrade thanks the fundação de apoio à pesquisa do distrito federal (fapdf) for granting a undergraduate scholarship. references alcock, j. 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(2017). the choosing of sleeping position in the overnight aggregation by the solitary bees amegilla florea urens in iriomote island of japan. the science of nature, 104: 1-8. doi: 10.1007/s00114-017-1438-8 about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.4987sociobiology 67(3): 417-424 (september, 2020) introduction bees are considered the most important and effective pollinators for the reproduction of most angiosperms (roubik, 1989; rech et al., 2014). flowers benefit from pollination while providing nutritional essential resources to bees for the development and survival of individuals in the colony. bees have holometabolic development distributed into four stages: egg (embryo), larva, pupa, and adult (cranston & gullan, 2017). the first morphological and physiological transformations occur in the pupal stage, when externalization of anatomically distinct morphological structures appear and the larva passes to the adult stage (truman & riddiford, 2019). during this ontogenic process, developmental noises abstract transportation to long distances and handling of colonies can affect development and survival conditions of bees. our study investigated the stress intensity of individuals of melipona scutellaris latreille, 1811, due to transportation of colonies to long distances, within the natural range of the species. we used 746 bee workers. the right and left forewings were removed and measured using 15 landmarks in vein insertions. individuals were divided into four groups: (1) workers collected at the origin site, (2) workers emerged at the place of destination in pupal stage during transportation, (3) workers emerged at the destination site in the 3rd instar of larval stage during transportation, and (4) workers collected after three months of colony establishment at the destination site. the procrustes anova showed significant results as well as the presence of fluctuating asymmetry (fa) in all treatments for the shape of wings (p<0.01). however, in the comparison of groups using the oneway anova, only workers that emerged at the destination site in the 3rd instar of larval stage during transportation (group 3) significantly differentiated (p<0.05) from the others, with a higher fa index. the larval stage underwent more stress due to colony transportation. beekeepers should take good care of colonies during transportation in order to minimize damages to workers to prevent quality loss of services and products offered by bees. sociobiology an international journal on social insects br andrade1, eb santos2, la nunes3, as nascimento1, cal de carvalho1 article history edited by cândida aguiar, uefs, brazil received 27 january 2020 initial acceptance 03 july 2020 final acceptance 22 july 2020 publication date 30 september 2020 keywords stingless bee, uruçu, shape, forewing, migrating meliponiculture. corresponding author brunelle ramos andrade centro de ciências agrárias, ambientais e biológicas, universidade federal do recôncavo da bahia rua rui barbosa nº 710, centro, 44380-000, cruz das almas-ba, brasil. e-mail: insecta@ufrb.edu.br occur, which can affect asymmetry, including the wings, and can compromise the ability of individuals to collect food or escape from predators (møller, 1997). managed bees usually travel long distances from the hives in search of blooms, which is an alternative for colony survival during nectar scarcity. moreover, it benefits pollination of agricultural crops for commercial purposes and allows an exchange of colonies to ensure genetic variability of populations, as well as the implantation of meliponaries or apiaries (vanengelsdorp et al., 2012; tarpy et al., 2013; zhu et al., 2014). however, these management practices could stress bees, impairing their development, compromising their nutritional condition, and colony production (ahn et al., 2012; villas-bôas, 2012; simone-finstrom et al., 2016a; 2016b). 1 universidade federal do recôncavo da bahia – ufrb, cruz das almas-ba, brasil 2 universidade estadual de santa cruz – uesc, ilhéus-ba, brasil 3 universidade estadual do sudoeste da bahia – uesb, jequié-ba, brasil research article bees fluctuating asymmetry in melipona scutellaris (l.) 1811 (hymenoptera: apidae) associated to stress due to transportation of colonies br andrade, eb santos, la nunes, as nascimento, cal de carvalho – stress in melipona scutellaris due to colony transportation 418 the stress caused by the transportation process of colonies affects the bee health and causes oxidative stress (simone-finstrom et al., 2016a; glenny et al., 2017), influencing the ontogenetic development of individuals, as observed through the technique known as fluctuating asymmetry (fa) (lima et al., 2016; banaszak-cibicka et al., 2018). developmental noise is considered one of the causes that affect the individual bilaterality (clarke, 1998; tomkins & kotiaho, 2001). the fa allows identifying subtle differences in bilaterality in individuals and consequently their effects of genotypic or environmental instability. factors associated with stress result in organisms with higher asymmetry values and consequently greater difficulty to regulate their development (adaptive fitness) (lijteroff et al., 2008; klingenberg, 2015). the fa technique has been used to identify stress in bees during the ontogenic process and has been applied as a bioindicator associated to stress in the presence of pesticides during development in apis mellifera linnaeus, 1758 (nunes et al., 2015) and in melipona quadrifasciata anthidioides lepeletier, 1836 (prado-silva et al., 2018). in addition, the fa is also used to assess differences in stress related to handling in rational boxes and hives in m. subnitida ducke 1910 (lima et al., 2016). thus, fa is a useful and effective technique to infer ontogenetic changes in bees associated to genetic or environmental factors that lead to noise during development (del sarto et al., 2014). managed bees undergo stress during the handling and the transportation process associated to factors, such as food and temperature; however, the existence of morphological changes has not yet been observed during this transportation process and other associated factors. thus, this study used the fa technique to evaluate the effect of stress caused by the transportation of colonies over long distances, within the area of natural distribution of the species, based on the variation of size and shape of the forewings of individuals of melipona scutellaris. material and methods the bees used in the study were subjected to a displacement of 250 km for approximately 10 h, in the daytime, at the end of october. the place of origin was the municipality of mundo novo (11°51´00”s; 40°28´00”w), where the climate is classified as aw (according to köppen and geiger), with average annual temperature 22.1 °c and average annual rainfall 875 mm. the destination site was the municipality of cruz das almas (12°39´11”s; 39°7´19”w), which has af climate (according to köppen and geiger) with average annual temperature 23.0 °c and average annual rainfall 1136 mm. during the route, the trip was interrupted for 60 min, from 12h00 to 01h00, in the municipality of itaberaba in the state of bahia (fig 1). for the study, 400 adult workers of m. scutellaris were used, with 100 individuals per group. each group was represented by 10 colonies and 10 specimens were collected in each colony. the bees were divided into four groups: (g1) workers collected at the place of origin, (g2) workers emerged at the destination site in pupal stage during transportation, (g3) workers emerged at the destination site in 3rd instar of larval stage during transportation, and (g4) workers collected after three months of colony establishment at the destination site. to determine the age of immature insects and establish the exact time of collection of adults, cells containing pupae and larvae were identified by the wax color, also through a small opening in three brood cells chosen at random on the brood disc (set of brood cells) investigated. morphology of immature individuals was observed and the age of these individuals was determined. the disks (set of nest cells) of interest were marked with non-toxic water-based white paint. in these stages, the individuals were kept in the colony and were monitored in order to follow the emergence moment of bee workers. fig 1. route and distance of transportation of melipona scutellaris colonies. sociobiology 67(3): 417-424 (september, 2020) 419 the right and left forewings of the bees were removed with tweezers, placed between slides, and sealed for microscopy. photographic records were made with a leica® digital camera model dfc295 coupled to a leica® stereomicroscope model s8 apo. only the forewings were used because, as reported in previous works (nunes et al., 2015; lima et al., 2016; prado-silva et al., 2018), there is no need to use both wings, as they have evolved to operate together. in addition, all wings were photographed by the same equipment, at the same distance, without zoom, and with the structures centralized to avoid the distortion effects of the lenses, as suggested by klingenberg (2015). next, the images of left wings were mirrored to be similar to the right wings to minimize measurement errors. with the image database obtained, a tps file was generated from the images using the tpsutil software version 1.74 (rohlf, 2017a). we scanned 15 landmarks (fig 2) in the vein insertions of each wing using the software tpsdig version 2.30 (rohlf, 2017b). the wings were measured twice and by a single researcher to reduce errors associated with the measurer, as suggested by palmer (1994). the cartesian coordinates obtained were used as variables for the statistical analysis. after performing the procrustes superimposition, the procrustes anova test was applied to verify the fa level for the shape and size of the wings. the procrustes method provides an adjustment based on values related to least squares by comparing homologous points, while the procrustes anova allows quantifying the variation in shape at different levels. it has been used mainly in asymmetry studies and in the evaluation of the amount of measurement error associated to biological variation (palmer & strobeck, 2003; klingenberg, 2015). the fa method was applied to m. scutellaris individuals by varying the morphometric differences between the leftright sides between individuals. in the asymmetry analysis for the shape and size, the f value for the effects of sides and individuals, the side × individual interaction was used as the denominator. to check for significant differences in relation to fa, the side × individual interaction was used and the measurement of error as the denominator (palmer, 1994; klingenberg & mcintyre, 1998). the analyses were performed using the morphoj software version 1.06 (klingenberg, 2011). in addition, based on the procrustes coordinates (shape fa scores), the one-way anova was performed in the program r version 3.4.1. the tukey test was conducted to compare the means and verify differences in fa. subsequently, a principal component analysis (pca) was performed to analyze variation in the shape of the wings between the groups. fig 2. right forewing of melipona scutellaris with 15 landmarks in the vein insertion and used for the fluctuating asymmetry analysis. results no significant results were found for fa (p>0.05) for the analysis of wing size from the centroid size. however, all groups evaluated separately presented significant values for fa (p<0.01) for the wing shape, as well as pronounced values for directional asymmetry (p>0.01) (table 1). the one-way anova revealed significant differences between groups. specifically, bee workers in the 3rd instar of larval stage during transportation (g3) were statistically different from the others (p<0.05), presenting a higher fa index. in addition, the analysis of wing shape in the g4 stage showed no significant differences (p>0.05) after an adaptation period of three months at the destination site, when compared to individuals of the g1 stage (bees collected at the origin site) (fig 3). the pca analysis showed that individuals of g3 transported in the larval stage separated on the first axis of the main component (29%), especially from the g4 stage (fig 4). discussion all generations studied, represented by the groups g1, g2, g3, and g4, presented significant fa. fluctuating asymmetry is largely attributed to studies that assess the impact of developmental instability (palmer, 1994; klingenberg, 2015). br andrade, eb santos, la nunes, as nascimento, cal de carvalho – stress in melipona scutellaris due to colony transportation 420 change in the animal body symmetry caused by developmental instability can be attributed to the lack of ability of an organism to withstand disturbances during its development, hindering the production of a genetically predetermined phenomenon (clarke, 1995). environmental factors are causes of developmental instability, such as abrupt climatic conditions, nutritional changes, pesticides, parasitism, and genetics (parsons, 1990; møller & swaddle, 1997). the results for all groups that presented significant fa show that different factors contributed to the noise in the development of these insects. a comparative study between bees of the species melipona subnitida ducke, 1910 colonized in two types of nests showed that individuals showed significant asymmetry in both rearing systems. however, the comparison of nesting systems showed no significant difference between each other. in this study, lima et al. (2016) compared pre-established colonies without the influence of a stressor factor. another recent study showed that m. quadrifasciata anthidioides presented fa in the first pair of wings in both forage and newly emerged individuals (prado-silva et al., 2018). although fa occurs naturally in social bees of the genus melipona (lima et al., 2016; prado-silva et al., 2018), changes in these morphometric patterns have been reported effect ss ms df f p (param.) g1 centroid size individual 4462792.65 45078.713637 99 0.95 0.6080 side 31760.6803 31760.680285 1 0.67 0.4162 ind*side 4716286.20 47639.254564 99 1.00 0.4844 error 9495687.91 47478.439538 200 shape individual 0.04945959 0.0000192151 2574 3.79 <.0001 side 0.00126422 0.0000486239 26 9.58 <.0001 ind*side 0.01306128 0.0000050743 2574 2.70 <.0001 error 0.00976196 0.0000018773 5200 g2 centroid size individual 1572060.71 21244.063666 74 1.00 0.5000 side 43060.1051 43060.105050 1 2.03 0.1587 ind*side 1572085.12 21244.393563 74 0.99 0.5176 error 3229930.70 21532.871332 150 shape individual 0.04117770 0.0000214021 1924 3.37 <.0001 side 0.00099878 0.0000384144 26 6.04 <.0001 ind*side 0.01222677 0.0000063549 1924 3.37 <.0001 error 0.00735717 0.0000018865 3900 g3 centroid size individual 7278243.30 75033.436063 97 1.00 0.5029 side 1061372.25 1061372.2546 1 14.1 0.0003 ind*side 7288936.48 75143.675070 97 0.80 0.8840 error 16203668.0 93662.820942 173 shape individual 0.06466537 0.0000256405 2522 2.84 <.0001 side 0.00105218 0.0000404684 26 4.48 <.0001 ind*side 0.02279286 0.0000090376 2522 4.01 <.0001 error 0.01014514 0.0000022555 4498 g4 centroid size individual 2134202.14 21557.597355 99 0.97 0.5532 side 7393.77591 7393.775913 1 0.33 0.5647 ind*side 2192545.95 22146.928784 99 1.01 0.4772 error 4400425.44 22002.127161 200 shape individual 0.09048487 0.0000351534 2574 5.24 <.0001 side 0.00135201 0.0000520004 26 7.74 <.0001 ind*side 0.01728261 0.0000067143 2574 2.60 <.0001 error 0.01342102 0.0000025810 5200 (g1) workers collected at the place of origin, (g2) workers emerged at the place of destination, in pupal stage during transportation, (g3) workers emerged at the destination site, in 3rd instar of larval stage during the transportation, and (g4) workers collected after three months of colony establishment at the destination site. table 1. the procrustes anova of wing size and shape for worker bees of the groups (g1, g2, g3 and g4) and interaction between the effects between the body side and the individual. sociobiology 67(3): 417-424 (september, 2020) 421 for insects subjected to some type of stress (prado-silva et al., 2018). the one-way anova and pca tests conducted in our study revealed that, in addition to fa within each group, g3 stands out for presenting higher asymmetry values, indicating greater developmental instability in this group, which was in the larval stage at the time of transport. among the stress factors at the time of transport, temperature was possibly the key factor to trigger an increase in asymmetry of individuals in the g3 group, caused by stress. temperature can interfere with fecundity, population fluctuation, geographic distribution, and mainly with growth and development of insects (paul & keshan, 2016; lu et al., 2016; fig 3. the boxplot from one-way anova and the tukey test indicating the variation of fluctuating asymmetry among treatments. in the tukey test, equal letters do not differ statistically. (g1) workers collected at the place of origin, (g2) workers emerged at the place of destination, in pupal stage during transportation, (g3) workers emerged at the destination site, in 3rd instar of larval stage during the transportation, and (g4) workers collected after three months of colony establishment at the destination site. fig 4. principal component analysis for the groups (g1, g2, g3 and g4) of melipona scutellaris submitted to colony transport between two municipalities in the state of bahia. (g1) workers collected at the place of origin, (g2) workers emerged at the place of destination, in pupal stage during transportation, (g3) workers emerged at the destination site, in 3rd instar of larval stage during the transportation, and (g4) workers collected after three months of colony establishment at the destination site. br andrade, eb santos, la nunes, as nascimento, cal de carvalho – stress in melipona scutellaris due to colony transportation 422 li et al., 2017). another important factor is that an increase in temperature can be more harmful for tropical insects, as they live close to their maximum thermal limit, compared to insects in the temperate zone (evgen’ev et al., 2014; paul & keshan, 2016). a laboratory study showed that drosophila melanogaster meigen, 1830 (diptera: drosophilidae) that developed at a temperature of 30 °c showed an increase in fa of the sternopleural bristles, compared to individuals that developed at 25 °c (parsons, 1990). laboratory research on fa shows that insects undergo some type of stress in their development. welded termites of the species coptotermes formosanus shiraki, 1909 and c. gestroi wasmann, 1896 (isoptera: rhinotermitidae) from newly formed colonies showed significant asymmetry when compared to soldiers from colonies already established. a colony already established can ensure its homeostasis more easily; therefore, insects that grow in an established colony can overcome the developmental phase with much less stress (chouvenc et al., 2014). due to the consequences that stress causes to insects, it is crucial to understand and quantify the stress level that bees suffer, especially during a transport process, to assess the impacts of abiotic and biotic factors (simone-finstrom et al., 2016a; belsky & joshi, 2019). transportation and handling of colonies in the implementation of apiaries and meliponaries, pollinating cultures, or changing environments in search of resources increase the stress level, especially in individuals transported in the larval stage. when bees do not show an efficient response against the stress suffered during the physiological and behavioral processes, their activities inside the hive are compromised, as greater effort is applied in the control of homeostasis (domingos & gonçalves, 2014). in addition, the greater the stress factors in the younger stages, the greater the effects on the asymmetry analysis of adult individuals. as noted by nunes et al. (2015), individuals of apis mellifera that develop in areas under intense use of pesticides show greater deviations in symmetry compared to populations collected under other types of stress. although we did not measure the temperature values inside each colony and inside the vehicle, it is worth mentioning that the transportation was carried out throughout the day and during the hottest hours. in addition, the movement was interrupted for 60 min in the municipality of itaberaba. according to climate-data.org, the maximum average temperature in itaberaba, bahia, exceeds 30 °c, a value above those recorded for the municipality of origin of the evaluated colonies, mundo novo, bahia, in times with lower temperatures (approximately 17 °c). the loss of 30% of immature individuals in colonies transported compared to those who remain in fixed environments (pires et al., 2016) could be explained by the smaller capacity of these individuals to cushion the impacts caused by transport. several factors generate stress during bees ontogeny, such as feeding type (modro et al., 2012), homeothermal control (domingos & gonçalves, 2014), management practices (lima et al., 2016), and the use of pesticides (nunes et al., 2015; prado-silva et al., 2018). modro et al. (2012) showed that in summer, when temperatures are higher, individuals showed high fa levels when compared to other periods of the year. this shows deviations in the symmetry of individuals with bilateral symmetry, and fa may reflect the development of instability caused by stress due to high temperatures. it is known that each stingless larvae receive their liquid food integrally in their brood cell even before oviposition by the queen and the larvae have direct contact with this food upon hatching. the more initial the stage of insect formation, the greater the damage they can suffer from stress. our results of fa highlight the importance of taking great care when transporting colonies, regardless of the reason for transport, namely prioritizing days with lower temperatures, checking the resistance of colonies, and avoiding sudden temperature changes. the newly emerged bee workers analyzed (m. scutellaris) were not tested for their flight capacity; however, the stress level and variation in the symmetry pattern may also affect their flight performance, success of searches, and consequently performance of bee workers (hepburn et al., 1999). there are no studies that investigate developmental instability between different stages in insects. therefore, similar studies are needed to clarify issues related to the damping capacity that each stage has to overcome a stress. moreover, these studies could bring conclusive data regarding the damage that transport, acclimatization, food, and others can cause to social bee colonies. acknowledgements the authors thank the “coordenação de aperfeiçoamento de pessoal de nível superior” (capes), brazilian government (finance code 001) and “conselho nacional de desenvolvimento científico e tecnológico” (cnpq) (no. 305885/2017-0). authors’ contribution b r andrade: collected data and drafted the article. e b santos: collected data and drafted the article. l a nunes: designed the study, interpreted the results, and drafted the article. a s nascimento: designed the study, interpreted the results, and revised the article. c a l de carvalho: designed the study, interpreted the results, and revised the article. references ahn, k., xie, x., riddle, j., pettis, j. & huang, z.y. 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(2014). transportation and pollination service increase abundance and prevalence of nosema ceranae in honey bees (apis mellifera). journal of apicultural research, 53: 469-471. doi: 10.3896/ibra. 1.53.4.06 1151 development and isolation of 17 polymorphic microsatellite loci in coptoterms formosanus (isoptera: rhinotermitidae) by bing-rong liu1,3, jun-hong zhong2*, ming-fang guo2, zhi-qiang li2 & wen-hui zeng2 abstract seventeen polymorphic microsatellite dna loci for copototermes formosanus were isolated and characterized. polymorphism of these loci was assessed in a sample of 32 unrelated c. formosanus individuals. an average of 4.6 alleles per locus (3-8 alleles) was detected. observed and expected heterozygosities ranged from 0.2500 to 1.0000 and from 0.5591 to 0.8562, respectively. six loci were found to have deviated from hardy-weinberg equilibrium in the sampled population after bonferroni correction. no significant linkage disequilibrium was detected. these markers will be useful in population genetics, phylogenies and other relevant studies of c. formosanus. key words: coptotermes formosanus, microsatellite loci, polymorphism introduction the formosan subterranean termite (fst) coptotermes formosanus shiraki (isoptera: rhinotermitidae) is a major economic pest worldwide. it is thought to have originated in china, but has spread to many areas around the world where it is a highly destructive pest of wood structures (su & tamashiro 1987). recently, mitochondrial dna sequencing ( jenkins et al. 2002; austin et al. 2006; fang et al. 2008) has been used to study the genetic diversity of c. formosanus, mainly in china, america and japan, but this information is insufficient for investigating the genetic diversity and detailed spatial structure of these populations. microsatellite dna loci as polymorphic genetic markers are necessary for elucidating the details of colony organization, population structure, and relationships among introduced and native populations (ross 1south china botanical garden, the chinese academy of sciences, guangzhou 510650, china. 2guangdong entomological institute, guangzhou 510260, china. *corresponding author, e-mail: zhong@gdei.gd.cn. 3graduate school of the chinese academy of sciences, beijing 100049, china. 1152 sociobiolog y vol. 59, no. 4, 2012 2001, vargo et al. 2003). although vargo & henderson (2000) identified 12 microsatellite loci in c. formosanus, few subsequent studies have been performed. therefore we isolated and characterized 17 polymorphic microsatellite loci for c. formosanus following the protocol modified from yu et al. (2010), so as for further study of population genetics, phylogenies and other relevant study of c. formosanus. materials and methods total genomic dna was extracted from the heads of twenty c. formosanus individuals, using the regular phenol-chloroform method as described by zhang and hewitt (1998). approximately 4 ug high quality genomic dna was digested with restriction enzyme sau 3ai (promega), and the size-selected dna (400 900 bp) was isolated from an agarose gel using dna purification kit (takara). fragments were then ligated to a blunt-end adapter (saula: gcggtacccgggaagcttgg, saulb: gatcccaagcttcccgggtaccgc) with t4 dna ligase (takara) at 16°c for 14h. the ligation products were amplified by polymerase chain reaction using the adapter saula as primers. after denaturation at 95°c for 10 min, microsatellitebearing amplified fragments were selected with the biotin-labeled (ac) 12 and (agg) 8 (sangon) oligonucleotide probes in sodium phosphate buffer (0.5 m sodium phosphate, 0.5% sds, ph 7.4) at 50°c for 16h. the target fragments were amplified by pcr and the size-selected dna (400 900 bp) were excised from agarose gel and recovered. these recovered dna product were ligated with pmd19-t vector (takara) and transformed to e. coli dh5a competent cells (takara). eighty-two positive clones were identified from 196 recombinant colonies via pcr with the adapter saula as primers, and were sequenced with m13 primers in one direction. forty-eight sequences contained microsatellites, of which thirty-five possessed sufficient flanking sequences appropriate for primer design. the others had either insufficient or inappropriate flanking regions on one or both sides of their microsatellites. these thirty-five pairs of primers were designed by the premier 5.0 program (premier biosoft international, silicon valley, usa), and synthesized by sangon biotech (shanghai) co., ltd. a range of annealing temperatures (50 °c 68 °c) were tested and the 1153 liu, b-r. et al. —microsatellite loci in coptotermes formosanus temperature producing the cleanest and strongest pcr product when observed on an 1.5% agarose gel stained with goldview was selected for pcr. after these optimization procedures, seventeen of the thirty-five primer sets designed amplified successfully and were used to assess polymorphism based on 32 unrelated c. formosanus individuals. following genomic dna extraction, pcr was performed in a volume of 20 ul containing 50 100 ng of total dna, 0.25 0.5 units of taq polymerase (tiangen, china), 19 pcr buffer, 1.0 2.0 mm mgcl 2 , 0.2 mm dntps, and 0.2 1 um of each primer. the pcr profile was as follows: initial denaturation at 95 °c for 3 min, followed by 30 cycles of 94 °c for 30 s, a primer-specific annealing temperature for 30 s and 72 °c for 40 s, with a final extension at 72 °c for 10 min. the pcr products were checked by electrophoresis on 8% non-denaturing polyacrylamide gel, and visualized with silver staining. allele size was determined against a 50-bp dna ladder (tiangen, china) with software gel-pro analyzer 4.5. number of alleles, heterozygosity, test of hardy–weinberg equilibrium (hwe) and linkage disequilibrium (ld) were analyzed using genepop4.0.6 (raymond and rousset 1995). results conditions and characteristics of the 17 loci are shown in table 1. the number of observed alleles per locus ranged from 3 to 8. the expected and observed heterozygosity values ranged from 0.2500 to 1.0000 and 0.5591 to 0.8562, respectively. six loci (copf02, copf04, copf09, copf10, copf13 and copf17) significantly deviated from hardy-weinberg equilibrium after bonferroni correction (p<0.0029). no significant linkage disequilibrium was detected. we conclude that the markers obtained will be useful in population genetics, phylogenies and other relevant study of c. formosanus. acknowledgments we acknowledge the support of the national natural science foundation of china (31172163) and the guangdong academy of sciences foundation for excellent young scientists and technicians (200901). and we are grateful to dr. yuchun wu for revising the draft of the manuscript. 1154 sociobiolog y vol. 59, no. 4, 2012 ta bl e 1 c ha ra ct er iz at io n of 1 7 po ly m or ph ic c op to te rm es fo rm os an us m ic ro sa te lli te lo ci . m ot if, r ep ea t se qu en ce o f th e is ol at ed c lo ne ; t a, a nn ea lin g te m pe ra tu re ; a , t he n um be r o f a lle le s; h o, th e o bs er ve d he te ro zy go si ty ; h e, ex pe ct ed h et er oz yg os it y an d p, as so ci at ed p ro ba bi lit y va lu e o f c on fo rm at io n w ith h ar dy –w ei nb er g eq ui lib ri um (h w e) . l oc us a cc es si on n o. pr im er se qu en ce (5 ’– 3’ ) m ot if ta (° c ) a lle te si ze (b p) a h o h e p c op f0 1 jq 31 37 94 a t t c c t t c a c t t a c g c a c t t g t a c c c g a c a t c a t a c g c (c t )1 0t t (c t )8 65 29 7 7 0. 40 74 0. 79 32 0. 01 13 c op f0 2 jq 31 37 95 g g a a g a a g g a c c a a t c t g t a a c c a a g g a g c g t a a t g (g t )8 62 19 0 4 0. 96 88 0. 66 52 0. 00 00 c op f0 3 jq 31 37 96 a c c g a c t c c t c t g a t t g a c a c a t t a t g t t t c c a c g a c (g t )2 2（ g a ） 9 64 18 4 5 0. 58 06 0. 57 11 0. 00 69 c op f0 4 jq 31 37 97 t a c c g g a c t c t a a c a g a c a t c a g a g g a t t c t t a c c g a (a c )7 t c (a c )5 62 36 0 3 0. 31 25 0. 72 87 0. 00 00 c op f0 5 jq 31 37 98 g c a a t g a a g t g c c t c t g a a a c c t g g a c t c g a c c t t t (a c )1 9 65 26 1 8 0. 84 38 0. 69 35 0. 08 27 c op f0 6 jq 31 37 99 c a g t g g c a g c g a c g t a t a a t c c t g g a g t c c t a a g a a g c (a c )8 g c (a c )1 4 65 18 6 6 0. 25 00 0. 66 96 0. 01 96 c op f0 7 jq 31 38 00 c t c t t t g c t g c c a t a c g t c t c a g t t c c a t g c g g a c a (g t )1 8 65 24 2 5 1. 00 00 0. 55 91 0. 05 12 c op f0 8 jq 31 38 01 t c a a t g g c g t g c c t t c a c a g c t c a a c c a c t g c g t t t (c a c t )1 3( c a t t )1 6 62 26 3 6 0. 96 88 0. 72 77 0. 18 35 c op f0 9 jq 31 38 02 g t g c t g g c g t t c g g t a t t t t t g c t t g c c t a a a g t c g (a c )8 n (a c )6 n (a c )6 62 25 7 4 0. 87 50 0. 85 62 0. 00 01 c op f1 0 jq 31 38 03 a g g t g t t g a a t g g g c t g t t c c a a g c c t g c c a g a a a g t (a c )1 7 65 33 3 3 1. 00 00 0. 71 42 0. 00 00 c op f1 1 jq 31 38 04 c g a a g t t a t g c c t c t g t t t t t g g a t g c c t g g a t t a g (a c )8 62 28 6 4 0. 87 10 0. 67 85 0. 03 45 c op f1 2 jq 31 38 05 g t g c t g g a g t t t g g a t t t g a g g c g g t a g t a a c a a t a a g (g t )6 n (g t )6 62 33 4 3 0. 83 33 0. 68 70 1. 00 00 c op f1 3 jq 31 38 06 t a t t g t t g t t g c g g a a g c g t c g g c a g c a c t g a a g t a (g t )1 3 62 19 6 5 0. 39 29 0. 67 14 0. 00 00 c op f1 4 jq 31 38 07 c t a c a a g g c t a c c a t c a g g g g a a c a g c g a g a c g a g a t (c t )1 3 64 22 2 4 1. 00 00 0. 78 21 0. 52 83 c op f1 5 jq 31 38 08 t c t c c g t t c a t c a c a g c c c a g g g a a a g c a a c c a c a t c (g t )1 6 65 32 3 4 0. 70 37 0. 76 59 0. 10 70 c op f1 6 jq 31 38 09 c g t c a c g t t a t g g a g c a t a g c g g a c t t g a g g t t a g a (g t )1 6 64 26 7 4 0. 92 59 0. 69 81 0. 09 45 c op f1 7 jq 31 38 10 t g t t t c a c a g c c a t c a g a t g c t t g g t a a a t g g g t a g (a c )1 2 62 18 4 4 1. 00 00 0. 57 14 0. 00 00 1155 liu, b-r. et al. —microsatellite loci in coptotermes formosanus references austin, j.w., et al. 2006. genetic evidence for two introductions of the formosan subterranean termite, coptotermes formosanus (isoptera:rhinotermitidae), to the united states. florida entomologist 89:183-193. fang, r., l. huang & j.h. zhong 2008. surprising low levels of genetic diversity of formosanu subterranean termites in south china as revealed by the coii gene (isoptera: rhinotermitidae) sociobiolog y 51(1): 1-20. jenkins, t.m., r.e. dean & b.t. forschler 2002. dna technolog y, interstate commerce, and the likely origin of formosan subterranean termite (isoptera: rhinotermitidae) infestation in atlanta, georgia. j. econ. entomol. 95:381-389. raymond, m., f. rousset 1995. genepop (version 1.2): population genetics software for exact tests and ecumenicism. j hered 86:248-249. ross, k.g. 2001. molecular ecolog y of social behaviour: analyses of breeding systems and genetic structure. mol. ecol. 10: 265-284. su, n.y, m. tamashiro 1987. an overview of the formosan subterranean termite (isoptera: rhinotermitidae) in the world. in: biolog y and control of the formosan subterranean termite. college of agriculture and human resources, 3-15. vargo, e.l. & g. henderson 2000. identification of polymorphic microsatellite loci in the formosan subterranean termite coptotermes fomosanus shiraki. mol. ecol. 9:1935–1938. vargo, e.l., c. husseneder & j.k. grace 2003. colony and population genetic structure of the formosan subterranean termite, coptotermes formosanus, in japan. mol. ecol. 12: 2599-2608. varshney, r.k., a. graner & m.e. sorrells 2005. genetic microsatellite markers in plants: features and applications. trends biotechnol. 23:48–55. yu, d.m., b. ma, y.h. sun, & j.j. peng 2010. isolation and characterization of 16 microsatellite loci in an endangered fish ussuri cisco, coregonus usssruensis. conserv genet 11:11071109. zhang, d.x. & g.m. hewitt 1998. isolation of animal cellular total dna. molecular tools for screening biodiversity (eds. a. karp, p.g. isaac & d.s. ingram), chapman & hall, london, pp. 5-9. doi: 10.13102/sociobiology.v66i4.4601sociobiology 66(4): 602-605 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 urn:lsid:zoobank.org:pub:3a52127a-bb47-4c0b-bb0c-c581d3ec928e until 2010, chartergellus had only eight species described, the last one having been described by cooper (1993 – c. afoveatus). the genus was the subject of the phd thesis of andena (2007), who, at that time, had proposed the phylogeny of the group, which remains unpublished. however, since then, another four new species (c. golfitensis west-eberhard, 2010; c. jeannei andena & soleman, 2015; c. zucchii mateus & andena, 2015; and c. trinitatis carpenter & andena, 2019) were described. the genus seems more diverse than expected, based on it being uncommon, as pointed out by jeanne (1991) as well as chavarría and west-eberhard (2010). the genus is easily recognized by a curved bristle on the third labial palpomere; the maxillary and labial palpi five and three segmented, respectively; the metanotum rounded, lacking an occipital carina and a dorsal groove on mesepisternum (carpenter & marques, 2001; carpenter 2004). abstract a new species, chartergellus flavoscutellatus , collected in acre, northern region of brazil, is described and comparative remarks are given. sociobiology an international journal on social insects a somavilla1, sr andena2 article history edited by marcel gustavo hermes, ufla, brazil received 15 july 2019 initial acceptance 03 september 2019 final acceptance 19 september 2019 publication date 30 december 2019 keywords amazon forest; chartergellus; inpa collection; taxonomy; vespidae. corresponding author alexandre somavilla coordenação de biodiversidade instituto nacional de pesquisas da amazônia avenida andré araújo, 2936, petrópolis manaus, amazonas, brazil 69067-375 e-mail: alexandresomavilla@gmail.com the new species described herein was collected by malaise trap during a survey of the “biodiversity of insects of the amazon (rede bia)” project, in acre state, northern region of brazil. the species was deposited in the invertebrate collection of instituto nacional de pesquisas da amazônia – inpa. terminology employed follows richards (1978), west-eberhard et al. (2010), grandinete et al. (2015), mateus et al. (2015) and carpenter et al. (2019). the photos were taken with a digital camera leica dmc4500 attached to a stereomicroscope leica m205a and combined using the leica application suite software v4.10.0. chartergellus flavoscutellatus somavilla, new species (figure 1 a–d) urn:lsid:zoobank.org:act:b385d2b6-e35c-460a-af70-87086115dae0 1 instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus-am, brazil 2 universidade estadual de feira de santana, museu zoologia, divisão de entomologia, feira de santana-ba, brazil short note a new species of the swarming social wasp chartergellus bequaert, 1938 (vespidae: polistinae: epiponini) from acre, brazil sociobiology 66(4): 602-605 (december, 2019) 603 diagnosis: the species is easily diagnosed by the following combination of characters: scutellum and metanotum completely yellow; the clypeus touching the eyes for a distance equal to the width of antennal socket; mandible little raised, not forming a rim; mandibles and malar space, clypeus, inner orbits and supra-clypeal plate reddish, extending nearly up to median ocellus, base of antennal scape; tergum i–vi black; yellow apical band on tergum i and inconspicuous on tergum ii. description: female size: 9.0 mm. forewing length 7.0 mm. color: blackish species; mandibles and malar space reddish; apex of teeth black; clypeus, inner orbits and supraclypeal plate reddish, extending nearly up to median ocellus; base of antennal scape reddish, flagelomeres with dark brown appearance; gena with a wide yellow band; yellow band along the pronotal carina, posterior border of pronotum, in dorsal view; scutum blackish, anterior margin with a yellow band; anterior margin of mesopleura with a yellow band, in lateral view; scutellum and metanotum completely yellow (figure 1a); tegula black; legs black to dark-brown in tibiae and tarsi; tergum i–vi black; yellow apical band in tergum i and inconspicuous in tergum ii; sternum totally black without apical yellow bands; wings hyaline, venation dark-brown. head (figure 1c, d): (1) clypeus about 1.3 times wider than long, evenly convex, touching the eyes for a distance equal to the width of the antennal socket; lateral margins of the clypeus straight and upper margin sinuous; upper margin separated by antenna by less than the width of the antennal socket; surface of clypeus with gold pubescence and long bristles covering top half; punctures shallow, medium sized, separated by more than one diameter; (2) fig 1. chartergellus flavoscutellatus somavilla, new species. (a) dorsal view; (b) lateral view; (c) face, frontal view; (d) mandible in detail, the arrow indicates the mandible is little raised not forming a rim. a somavilla, sr andena – a new species of chartergellus604 frons and vertex with moderately long and spaced bristles and yellowish to white pubescence; punctures shallow, medium sized, separated by about one diameter; (3) eyes bare; (4) inner orbits and supra-clypeal plate with gold pubescence; (5) malar space shorter than second antennal flagellomere, shining; (6) mandible about 2.5 times longer than wide, little raised basally not forming a rim, with a band of long bristles on lower region; (7) gena about 0.75 width of eyes in profile; pubescence evident except on lower end, which is shining, reaching the malar space; punctures medium sized, separated by about little more than one diameter; (8) diameter of the medial ocellus 0,25 mm; (9) interocellar distance 0,23mm; (10) posterior region of head without occipital carina. mesosoma (figure 1a, b): (1) pronotum with short and dense pubescence, prominent on lateral part, some scattered short bristles on the anterior part of pronotum; punctation medium sized, separated by about 1.0 diameter; pronotal carina produced, slightly lamellate, extending to medial region; pronotal fovea in a shallow and oval concavity; punctures shallow, separated by less than one diameter; (2) mesopleura with same pattern of punctuation as pronotum, becoming sparser laterally, short and dense pubescence; scrobal furrow wide, shallow; (3) dorsal plate of metapleuron 1.5 times longer than wide at middle; lower plate with punctation very shallow and spaced, separated by more than two diameters, short and dense pubescence; (4) scutum as wide as long, with pubescence very spaced, present only on the borders, central area shining; punctation small, shallow, separated by one diameter or more, becoming sparser centrally; thin line in the anterior central region present; (5) scutellum with same pattern of punctuation as that of scutum, with a line in the anterior central region reaching a little more than half of the length of the scutellum; (6) metanotum with pubescence denser than that of scutellum, but not as that of pronotum; punctation very small and scattered; (7) propodeum with dense yellowish pubescence; long bristles centrally and laterally; (8) propodeal concavity shallow, wide; propodeal orifice large, rounded; (9) propodeal valvula narrow throughout and linear; (10) bristles on entire anterior and posterior wings. metasoma (figure1a, b): (1) tergum i cap-shaped, punctures very weak, spaced; (2) tergum ii wider than long, coriaceous, punctures very weak, spaced, pubescence present with a few bristles scattered; (3) posterior apical region of terga iii–vi with punctures very weak, spaced, pubescence present; (4) punctures on sternum ii–v very weak on posterior apical region, pubescence very weak. male: unknown. holotype: female ♀, brazil: acre, senador guiomard, fazenda experimental catuaba (coordinates: 10º04’28” s, 67º37’00” w). 14–31.i.2017. e.f. morato and j.a. rafael leg., [malaise trap]. instituto nacional de pesquisas da amazônia, inpa collection. paratype: 1♀: brazil: acre, bujari, fazenda experimental antimary (coordinates: 09º20’01” s, 68º19’17” w). 22.ix–06.x.2016. e.f. morato & j.a. rafael leg., [malaise trap]. instituto nacional de pesquisas da amazônia, inpa collection. measurements: total size 8.8 to 9.0 mm; forewing length 7.0 mm; clypeus width 1.3 mm; scutum width 2.4 mm, tergum ii width 3.5 mm. etymology: the name derives from latin, where flavus = yellow and scutellatus = scutellum. the name evokes the diagnosis, where the scutellum is completely yellow, resembling the yellow form of c. zonatus. distribution: brazil: acre. comments we compared chartergellus flavoscutellatus specimens with the holotype and paratypes of six chartergellus species: c. afoveatus, c. amazonicus, c. atectus, c. communis, c. nigerrimus, and c. punctatior, all of them deposited in the natural history museum (london). additionally, we also compared c. flavoscutellatus with the four recently described species: c. golfitensis, c. jeannei, c. trinitalis, and c. zucchii. we did not have access to type specimens of c. sanctus, described by richards (1978) and c. zonatus by spinola (1851), however we checked the original descriptions, and we compared it with a specimen of c. zonatus determined by james m. carpenter, and also specimens with the “yellow form” of c. zonatus, cited by richards (1978). chartergellus flavoscutellatus is similar to the yellow form of c. zonatus, which also has a yellow scutellum (fig 1a), the metanotum entirely yellow and yellow bands on terga i and ii. however, in c. zonatus the clypeus is separated from the eyes vs clypeus touching the eyes in c. flavoscutellatus (fig 1c), c. afoveatus, c. amazonicus, c. atectus, c. golfitensis, c. punctatior, and c. trinitatis, although, as pointed out by cooper (1993) and mateus et al. (2015), c. communis, and c. punctatior may have the clypeus narrowly separated from the eyes. chartergellus afoveatus and c. jeannei also present both states (clypeus narrowly separated from the eyes, sometimes touching). the mandible of c. flavoscutellatus is about 2.5 times longer than wide, a little raised not forming a rim. the rim of the mandible is a variable character, ranging from very feeble, as in c. sanctus (richards 1978), to strongly produced, as in c. communis (richards 1978). in c. flavoscutellatus the mandible is little raised not forming a rimas in c. afoveatus c. atectus, c. jeannei, c. nigerrimus, c. trinitatis, and c. zucchii. regarding this structure, we must emphasize that previous authors used the base of the mandible as the inferior region in frontal view (as in fig.1d). the pubescence covering the top half of the clypeus is another character of c. flavoscutellatus shared with c. communis, c. nigerrimus, c. punctatior, c. sanctus, c. zonatus, and c. zucchii. chartergellus afoveatus and c. trinitatis have the pubescence covering the entire clypeus, with only the apex bare. sociobiology 66(4): 602-605 (december, 2019) 605 moreover, the new species resembles, in some structures, the black form of c. punctatior and c. nigerrimus, but can be easily distinguished by the scutellum completely yellow. in addition, these two species have a smaller yellow band in the gena and the scutum black. moreover, the eyes of c. flavoscutellatus are bare, different from c. punctatior, which has hairy eyes. in c. afoveatus, the anterior margin of the scutellum and metanotum present yellow bands, differing from c. flavoscutellatus which is completely yellow. acknowledgements specimens were collected during a project coordinated by josé a. rafael and financed by the conselho nacional de desenvolvimento científico e tecnológico (cnpq, 407623/2013-2) through the program “rede bia biodiversidade de insetos na amazônia”. we sincerely thanks fundação de amparo à pesquisa do estado do amazonas for the post doctoral scholarship (fapeam – fixam, process number 062.01427/2018) to a. somavilla.we would like to thank dr. gavin broad and jaswinder boparai for support in natural history museum (london), james m. carpenter and marcel g. hermes for comments and to refauna program for financial support. reference andena, s.r. (2007). análise filogenética de alguns gêneros de vespas sociais neotropicais (hymenoptera, vespidae, epiponini). tese de doutorado, universidade de são paulo, ribeirão preto, são paulo. 161p. carpenter, j.m. (2004). sinonymy of the genus marimbonda richards, 1978, with leipomeles möbius, 1856 (hymenoptera: vespidae, polistinae), and a new key to the genera of paper wasps of the new world. american museum novitatis, 3465: 1-16. carpenter, j.m.& marques, o.m. (2001). contribuição ao estudo dos vespídeos do brasil. universidade federal da bahia, departamento de fitotecnia, bahia, cd-rom, 147p. carpenter, j.m., andena, s.r. & starr, c.k. (2019). a new species of the social wasp genus chartergellus bequaert from trinidad, (hymenoptera: vespidae, polistinae, epiponini). sociobiology, 66: 161-165. chavarria-pizarro, l. & west-eberhard, m.j. (2010). the behavior and natural history of chartergellus, a little-known genus of neotropical social wasps (vespidae polistinae epiponini). ethology, ecology and evolution, 22: 317-343. cooper, m. (1993). a new species of chartergellus (hym., vespidae, polistinae, polybiini) from bolivia. entomologist’s monthly magazine, 129: 165-166. grandinete, y.c., andena, s.r., soleman, s.a. & noll, f.b. (2015). chartergellus jeannei, a new species of the swarming social wasps from the amazon forest (hymenoptera: vespidae: epiponini). sociobiology, 62: 120-123. jeanne, r.l. (1991). the swarm-founding polistinae. in: ross, k.g. & matthews, r.w. (eds).the social biology of wasps. new york: cornell university press, p. 191-231. mateus, s., nascimento, f.s., aragão, m. & andena, s.r. (2015). a new species of the neotropical social swarmingwasp chartergellus bequaert (hymenoptera: vespidae: epiponini). sociobiology, 62: 105-108. richards, o.w. (1978). the social wasps of the americas excluding the vespidae. british museum (natural history), london. 580p. spinola, m. (1851). compterendu des hyménopteres inédits provenant du voyage entomologique de ghiliani dans le para. memorie della reale accademia della scienze di torino, 13: 19-94. west-eberhard, m.j., carpenter, j.m.,gelin, l.f.f. & noll, f.b. (2010). chartergellus golfitensis west-eberhard: a new species of neotropical swarm-founding wasp (hymenoptera: vespidae, polistinae) with notes on the taxonomy of chartergellus zonatus spinola. journal of hymenoptera research, 19: 84-93. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.5629sociobiology 68(2): e5629 (june, 2021) introduction the natural world varies across space with a mosaic of habitat fragments, and for any given species, some of these fragments are suitable and others are not, with the range of physical conditions where species can persist being called the fundamental niche (pianka, 1981). while the fundamental niche presents all the characteristics of the n-dimensional space essential for a species in the absence of other organisms, the realized niche is the portion of the fundamental niche abstract the ecological niche models can be important for biogeographic patterns and processes and geometric morphometrics involves identifying changes that have occurred and comparing them to other specimens from different places and/or environmental conditions, assessing whether the environment is influencing such change. the present work aimed to verify the potential model of distribution for apis mellifera and analyze if there is variation in the geometric morphometrics in wing venation in the pantanal. we followed the hypothesis that there is variation in the geometric morphometrics of wings and that the geographically closest groups are more similar. for niche modeling, 44 geographical points and 19 bioclimatic variables were used. for morphometrics, twenty-two anatomical landmarks were plotted at the intersection of the veins. the x and y coordinates were standardized through procrustes superimposition, and pca and manova tests were performed. the predictive model indicated that the center of the pantanal plain shows the greater probability of occurrence for the species. the most important bioclimatic variables were: average temperature in the rainiest quarter (84%) and average annual temperature (72%). morphometric analyzes indicate that there was variation between the most distant geographic points. the slight variation between some closely located points in the pantanal can be related to individual reflections of colonies from other points, since the species has great dispersion capacity. thus, the distribution of a. mellifera in the pantanal is possibly related to temperature also accompanied by human occupation and the geometric morphometrics of its wings reflecting aspects of dispersion and population dynamics in the brazilian pantanal. sociobiology an international journal on social insects alessandra c peil, rodrigo aranda article history edited by cândida aguiar, uefs, brazil received 20 july 2020 initial acceptance 03 december 2020 final acceptance 09 april 2021 publication date 19 may 2021 keywords africanized bees, beekeeper, predictive niche, wetland. corresponding author rodrigo aranda laboratório de ecologia de comunidade de insetos, universidade federal de rondonópolis av. dos estudantes, 5055, cidade universitária cep 78736-900 rondonópolis-mt, brasil. e-mail: rodrigoaranda.biologo@gmail.com actually occupied by that species, considering all interspecific interactions in such environment (hutchinson, 1978). the ecological niche is the last distributional unit for a species or subspecies (grinnel, 1917) that unites populational distributions with their environment, with species distributions reflecting the range of physical conditions that support individual survival (braunisch et al., 2008). in this context, the development of ecological niche models can help to investigate biogeographical patterns and processes, predicting the geographical distribution of species from sparse occurrence universidade federal de rondonópolis, instituto de ciências exatas e naturais, rondonópolis, mato grosso, brazil research article bees potential niche modeling distribution and wing geometric morphometrics of apis mellifera in the brazilian pantanal id mailto:rodrigoaranda.biologo@gmail.com https://orcid.org/0000-0002-4392-6423 alessandra c peil, rodrigo aranda – apis mellifera niche and morphometrics in pantanal2 data (guisan & thuiller, 2005). species distribution models are empirical precepts that relate field observations to environmental predictor variables, based on statistically or theoretically derived response surfaces (guisan & thuiller, 2005). modeling plays an important role, as it addresses several issues such as the analysis of an exotic/invasive species (peterson & vieglais, 2001; giovanelli et al., 2008) and implications for conservation (guisan & thuiller, 2005). animals are free to occupy different habitats, however, environments can change in time and space, and individuals must adjust by changing their behavior, physiology and even their structure (jungers et al., 1995). the measurements taken from structures where shape is studied contain information about both size and conformation (richtsmeier et al., 2002). geometric morphometrics aims to describe and represent the geometry of the conformations studied, clearly describing and locating regions where changes occur and graphically reconstituting such changes in shape (zelditch et al., 2012). geometric morphometrics involves identifying changes in homologous structures and comparing them to other specimens from different places and/or environmental conditions, making it possible to assess whether the environment is influencing such change (francoy et al., 2011). in the last decades, different applications in biology have used geometric morphometrics (adams et al., 2004). the studies quantify ontogeny (carvalho et al., 2011, komatsu et al., 2018), taxonomy, systematics or evolution of morphological characters (cardini et al., 2007, salas-lopez et al., 2017), sexual dimorphism (pretorius, 2005; olivier & aranda, 2018), ecomorphology, functional and biomechanical issues of biological forms (jungers et al., 1995) and intraspecific geographic variation (roggero & passerin d’entrèves, 2005; cardini et al., 2007). insect wings are very suitable structures for conducting morphometric studies, with many studies indicating geographic variation in wing structure (hoffmann & shirriffs, 2002; hoffmann et al., 2005). geometric morphometrics is precise enough to measure both greater variations between species and intraspecific variations, identifying colony origins in studies about bees as apis mellifera (francoy et al., 2008); an exotic species in brazil. african bees apis mellifera scutellata lepeletier, were introduced to brazil in 1956 (kerr, 1967). about one year later, 26 swarms of this bee with their respective queens, escaped and bred with the other subspecies of european honey bees that were introduced in the 19th century: the italian a. m. ligustica spinola, the german a. m. mellifera linnaeus, the austrian a. m. carnica pollmann and a. m. caucasica gorbachec. with this breeding process, polyhybrid populations called africanized bees arose, presenting predominantly african bee characteristics, such as great ability to swarm and rusticity, high capacity for defense, adaptation to inhospitable environments and ability to reproduce in a shorter life cycle than the other previously mentioned subspecies (kerr, 1967). such characteristics allowed the species to rapidly expand its biomass and significantly increase its population and geographic distribution (gonçalvez, 1994). apis mellifera is characterized as having very populous colonies with 5,000 to 180,000 individuals (lindauer & kerr, 1960), therefore, it has a high recruitment potential to forage and a high number of bees visiting flowers (kerr, 1967; sakagami et al., 1967). the africanized bee has a high swarm capacity and, due to the favorable conditions it is found in the neotropical territories, spread throughout the vegetation domains in brazil, including the pantanal floodplains. considering the characteristics of ecological niche and morphometric adjustments in relation to environmental variations, the present work aimed to verify the potential spatial distribution model of a. mellifera in the pantanal and to analyze if there is variation in the geometric morphometric of its wing venations, indicating where and how such variations occur and whether they are significant between different locations throughout the pantanal plain, since the region presents divergent characteristics. as hypotheses, we expected variation in the geometric morphometric of the wings and that the geographically closest groups would be more similar to each other within the pantanal area. material and methods study area the pantanal, one of the essential south american domains, is the largest continuous floodplain on the planet, with an area of approximately 150,000km2. the region’s climate is sub-humid tropical, with dry and rainy winters and average annual rainfall between 1,000 and 1,500 mm in the north and between 1,000 and 1,200 mm in the south (peel et al., 2007). the territory originates in the highlands, with predominance of cerrado, composed of brazilian savanna (cerrado stricto sensu), cerradão, natural fields, floodplains and environments such as freshwater or brackish ponds, rivers and streams (silva et al., 2000). furthermore, according to its hydrological, soil and vegetation characteristics, the pantanal can be divided into 11 distinct sub-regions. the altitude level varies from 80 m to 150 m and was formed by the subduction process of a large area, which occurred simultaneously with the appearance of the andes mountains (mercante et al., 2011). ecological niche modeling for niche modeling, 44 georeferenced a. mellifera occurrence points were used, 19 points where specimens were collected (aranda & aoki, 2018) and 25 points of occurrence records through bibliographic search (table 1). the following portuguese and english keywords were used for the search: “apis mellifera”, “pantanal”, “european bee”/“abelha europeia”, “bee production”/“produção apícola”. we used 19 bioclimatic variables from the worldclim database (http://worldclim.org) to build the niche distribution model. sociobiology 68(2): e5629 (june, 2021) 3 such data is correlated and allows for the visualization and understanding of the spatial pattern. for this model, the diva-gis georeferencing software (v. 7.5) was used to map and analyze spatial distribution data and elucidate geographic and ecological patterns (hijmans et al., 2012). we used the bioclim algorithm (hijmans et al., 2012) which calculates environmental similarity in multidimensional space with spatial resolution of 2.5 arch minutes (~5 km²) to create the predictive map. the result is a georeferenced distribution map that demonstrates the probability of species occurrence in a determined area presenting the basic conditions to maintain such species (hijmans et al., 2012). the model validation test was performed using the values of the area under the curve (auc), which is an accuracy measurement used for the prediction that measures the forecasting power based on real species distribution data. the auc ranges from 0 to 1, where a value of 0.5 indicates that the model is no different than random and a value of 1 perfectly discriminates between presence and absence records (hijmans et al., 2012). thus, auc values above 0.5 denote good accuracy and the closer to 1, the better the model. geometric morphometrics and statistical analyses sampling was carried out in 19 areas of the pantanal, from november 2015 to march 2016 during the hot and humid season (see aranda & aoki, 2018 for more sampling details). a total of 122 wings were measured. since some sampling points had few individuals collected from them and were located close together, the 19 collection points were grouped into eight spatial groups to facilitate data analysis. the groups correspond to the following sampling points: g1: points 1 and 2 (14 individuals); g2: points 3, 4 and 5 (23 individuals); g3: points 6, 7 and 8 (30 individuals); g4: points 9 and 10 (12 individuals); g5: points 11, 12 and 13 (15 individuals); g6: points 14 and 15 (6 individuals); g7: points 16, 17 and 18 (11 individuals) and g8: point 19 (11 individuals) (table 1, points 1 to 19). the specimens were screened, stored in 70% alcohol and mounted on an entomological pin to remove the wing, being established the removal of the right wing due to the firmness in the position of fixation of the specimens. the left anterior wing of each bee was removed near its insertion base on the thorax with an entomological scalpel, was mounted between blade and cover slip, and photographed with an opticam®opt 5.1mp camera coupled to a stereomicroscope (bel photonics series xtl). cartesian coordinates in the xy plane for 22 anatomical landmarks were manually plotted at the origin and intersection of the wing veins using opthd 3.7 software to construction of matrix data (fig 1). in order to observe divergence in wing shape within and between the populations of a. mellifera, the x and y coordinate data was standardized with procrustes superimposition to remove the effect of wing size, maintaining the shape in relation to the position of anatomical landmarks only. a principal component analysis (pca) was performed to identify where and how much was the magnitude of changes in landmarks wings. thin-plate splines were performed and warps of digitized x/y coordinates of reference points (landmarks) as a square grid associated deformations using the average shape as a reference, and shown expansion factor (or contraction) of the area around each reference point indicating the degree of local growth. the canculation uses the jacobian of the deformation, with the color-coded expansions being green for expansion and purple for contraction (dryden & mardia, 1998). to test the hypothesis that there is a separation of individuals in relation to their geographically closest groups, the scores of the main axes were tested using pca components with multivariate analysis of variance (manova) to analyze and test differences between the eight pre-established geographic groups (sadeghi et al., 2009). the analyses and graphics were performed using the free software past® 3.17 (hammer et al., 2001), with an alpha of 0.05 for the tests. fig 1. anatomical landmarks plotted at the intersections of veins on the left anterior wing of apis mellifera individuals collected in the brazilian pantanal. alessandra c peil, rodrigo aranda – apis mellifera niche and morphometrics in pantanal4 id region latitude longitude reference 1 pousada sinimbu 16°03’4.51”s 57°41’6.66”o aranda & aoki, 2018 2 estância seu preto 16°19’20.40”s 06°32’45.94”o aranda & aoki, 2018 3 sesc pantanal 16°30’52.51”s 06°22’45.36”o aranda & aoki, 2018 4 porto jofre 20°16’15.14”s 05°43’41.84”o aranda & aoki, 2018 5 parque nacional do pantanal 17°20’17.71”s 06°46’12.78”o aranda & aoki, 2018 6 rppn instituto homem pantaneiro 18°05’27.98”s 07°28’29.29”o aranda & aoki, 2018 7 assentamento pompeu 17°50’46.25”s 57°24’90.58”o aranda & aoki, 2018 8 base de estudo do pantanal-ufms 19°13’55.96”s 57°00’50.74”o aranda & aoki, 2018 9 fazenda santa clara 19°15’30.37”s 57°08’28.40”o aranda & aoki, 2018 10 fazenda arara azul 19°20’38.54”s 57°00’34.51”o aranda & aoki, 2018 11 porto da manga 20°11’42.18”s 56°30’26.74”o aranda & aoki, 2018 12 fazenda leque 19°24’51.95”s 57°31’0.99”o aranda & aoki, 2018 13 fazenda mutum 19°26’50.19”s 57°4’30.81”o aranda & aoki, 2018 14 fazenda bela vista 19°55’32.37”s 56°48’19.46”o aranda & aoki, 2018 15 povoado salobra 19°34’35.76”s 57°01’7.21”o aranda & aoki, 2018 16 fazenda aquapé 20°05’43.72”s 55°57’54.46”o aranda & aoki, 2018 17 fazenda taboco 20°04’60.91”s 55°38’38.69”o aranda & aoki, 2018 18 fazenda santa maria 19°44’20.05”s 57°06’16.31”o aranda & aoki, 2018 19 pousada camalote 21°43’09.93”s 57°53’47.25”o aranda & aoki, 2018 20 fazenda nossa senhora 16°00’02,0’’s 57°39’55’’o loureiro & galbiati, 2013 21 fazenda girau 16°04’55,0’’s 57°37’25’’ o loureiro & galbiati, 2013 22 poconé 16°13’15.60”s 56°39’51.30”o longo et al., 2019 23 poconé 16°20’43.60”s 56°32’10.10”o longo et al., 2019 24 poconé 16°15’51.80”s 56°34’53.20”o longo et al., 2019 25 poconé 16°15’34.90”s 56°36’51.50”o longo et al., 2019 26 poconé 16°15’17.30”s 56°33’12.70”o longo et al., 2019 27 poconé 16°21’03.40”s 56°39’06.50”o longo et al., 2019 28 poconé 15°48’11.00”s 56°32’22.40”o longo et al., 2019 29 poconé 16°22’20.70”s 56°37’07.80”o longo et al., 2019 30 barão de melgaço 16°20’51.70”s 55°49’43.20”o longo et al., 2019 31 barão de melgaço 16°34’13.50”s 56°12’6.19”o longo et al., 2019 32 barão de melgaço 16°8’50.50”s 55°56’11.40”o longo et al., 2019 33 barão de melgaço 16°8’34.10”s 55°55’42.40”o longo et al., 2019 34 retiro são bento 16°34’06.3”s 56°12’25.8”o longo et al., 2019 35 fazenda santana 18º06’56.08’’s 56º45’44.09’’o pott, 1986 36 fazenda ipanema 19º05’24.11’’s 56º50’40.78’’o pott, 1986 37 fazenda embrapa ládario 18°59’29.5”s 56°37’07.3”o marques et al., 2019 38 fazenda embrapa ládario 18º59’01.01’’s 57º30’15.13’’o marques et al., 2019 39 fazenda band’alta 19°08’34,6”s 57°35’12,1”o sambrana & reis, 2017 40 pantanal do rio negro 19º19’36,0’’s 57º03’13,0’’o carvalho et al., 2012 41 assentamento taquaral 19°06’02.3’’s 57°41’46.9”o almeida & reis 2016 42 assentamento tamarineiro ii 19º 09’ 00” s 57º 47’ 00” o almeida & reis 2016 43 aquidauana 20°28’15”s 55°47’13”o nunes, 2015 44 porto murtinho 21°42’04”s 57°53’06”o souza, 2016 table 1. georeferencing coordinates for analyzing occurrence sites of apis mellifera, in the pantanal. points 1 to 19 correspond to the locations where material was used for morphometric analysis. sociobiology 68(2): e5629 (june, 2021) 5 results niche modeling the potential geographic distribution of a. mellifera encompasses almost the entire region of the brazilian pantanal (fig 2). the predictive model indicates that the center of the pantanal plain, is the area with higher probability of a. mellifera presence. in the upper pantanal (mato grosso state), registration points are distributed in areas of low probability of occurrence such as poconé (dark green), medium probability of occurrence such as barão de melgaço, cáceres and poconé (light green), and high probability of occurrence as barão de melgaço (yellow). in the lower pantanal (mato grosso do sul state), the central area corresponding to paiaguás and nhecolândia presents areas with high probability of occurrence in the pantanal of aquidauana (yellow), very high probability of occurrence (orange) in the pantanal of miranda-abobral and nhecolândia, and excellent probability of occurrence in the pantanal of paiaguás (red). the main bioclimatic variables responsible for the a. mellifera model were: average temperature in the rainiest quarter (84%) and average annual temperature (72%) (table 2). the accuracy assessment (auc) of the prediction model indicates that the model has excellent accuracy (auc = 0.970). fig 2. occurrence report (black spots) and predictive model (bioclim) of potential geographical distribution of apis mellifera in the brazilian pantanal (blue area). fig 3. variation of the points on the x and y axes of thin-plate splines and warps from anatomical landmarks analyzed from the front wings of apis mellifera in the brazilian pantanal, representing locations with greater variations in the reference points. the color-coded expansions being green for expansion and purple for contraction. alessandra c peil, rodrigo aranda – apis mellifera niche and morphometrics in pantanal6 geometric morphometrics the wing landmarks of a. mellifera indivuduals showed differences in the measured positions according to the pca, where the points with the greatest variations and amplitude were the landmarks present in the x axis region (mark: 01, 04, 13, 14, 18, 19 20, 21 and 22) of the wing (fig 3; table 3). the central locations of vein intersections show differences on both the x and y axes, with the warmer colors representing the greatest deformations in the landmark positions (fig 3). landmarks number 1 and 22 showed high variation due to the cut at the base of the wing, which ended up making it difficult to mark the points (fig 3) and were generally not considered id variable unit cv 1 annual average temperature ºc 25.5 26.4 (72%) 2 average daytime temperature variation ºc 11.7 12.1 (36%) 3 isothermality ºc 65 66.7 (52%) 4 temperature seasonality ºc 213.7 243.2 (45%) 5 maximum temperature of the hottest month ºc 33.9 34.5 (34%) 6 minimum temperature of the coldest month ºc 16 16.7 (54%) 7 annual temperature variation ºc 17.4 18.1 (38%) 8 average temperature of the rainiest quarter ºc 27.1 28.2 (84%) 9 average temperature of the driest quarter ºc 23.8 24.5 (36%) 10 average temperature of the hottest quarter ºc 27.6 28 (50%) 11 average temperature of the coldest quarter ºc 23.2 23.7 (40%) 12 annual rainfall mm 1212.4 1291.6 (43%) 13 rainiest month precipitation mm 201 215 (50%) 14 precipitation in driest month mm 12 19.2 (36%) 15 precipitation seasonality mm 55.2 61.6 (38%) 16 rainier quarter rainfall mm 531.8 568.2 (29%) 17 precipitation of the driest quarter mm 43 63.4 (36%) 18 precipitation of the hottest quarter mm 490.4 556.2 (50%) 19 precipitation of the coldest quarter mm 86 108.2 (61%) table 2. bioclimatic variables and coefficient of variation (cv) corresponding to importance (%) in predicting the occurrence of apis mellifera in the brazilian pantanal. fig 4. multivariate analysis of variance test (manova). the colors represent the eight geographical groups: g1 = red; g2 = blue; g3 = green; g4 = purple; g5 = dark blue; g6 = light blue; g7 = mustard; g8 = yellow. sociobiology 68(2): e5629 (june, 2021) 7 in morphometric interpretation. we only used them to ensure accurate positioning of the areas for other measurements. variations in geometric morphometrics between groups were significant, with variation between geographical points (manova – pillai trace = 3.47; f = 1.78; p <0.05) (table 4). groups g3 and g8 showed the greatest difference, with g3 close to the serra do amolar region, which has a slightly higher altitude, and g8 porto murtinho, which is far from other points (axis 1 = 26.59; axis 2 = 15.74; fig 4). discussion the map shows a high percentage at high temperatures, with 25.5 ºc to 26.4 ºc and 27.1 ºc to 28.2 ºc. this is due to the topographic alignment arranged in the longitudinal direction, which exhibits well-defined morphological features: the plain to the west and the plateau to the east. this disposition influences the behavior of air masses. on one hand, the depression of the pantanal plain acts as a corridor for polar air masses during the winter in western-central brazil. on the other hand, seasons are responsible for the retention of hot and dry air causing its typical muggy weather conditions (alvare et al., 2014). temperatures are high throughout the year, with an annual average of 25 ºc, remaining above 26 ºc in autumn and winter, and never going below 30 ºc in spring and summer (alvare et al., 2014). vital et al. (2012) report few predictive models in the pantanal region, due to the ancestral subspecies of africanized bees and indicate that the best model for the neotropical region reflects colonization by a. mellifera scutellata, which has a low probability of occupation in the pantanal. the typical climatic conditions of the pantanal, with high temperatures and low rainfall in regions where there was better prediction of species occurrence, coincide with high temperatures. however, with a large number of extensive lakes and lagoons (pantanal region of nhecolândia and paiagúas), water availability would not be a limiting factor (gonçgalves et al., 2011; mercante et al., 2011). although high temperatures have been reported as limiting factors for the species (le conte & navajas, 2008), the availability of food resources throughout the year contributes to the species’ permanence in the area (salis et al., 2015). registration points are concentrated in transition areas with the best occurrence predictions (high, very high and excellent), being more concentrated in regions such as poconé in the northern and miranda-abobral in the southern pantanal, areas that are typically used for ecotourism and highly populated. human aspects associated with the use of exotic species, such as honey production with certification from the pantanal, can cause ecological losses in natural areas that have not yet been measured. understanding which areas are potentially susceptible to colonization can help mitigate future negative impacts. aspects of spatial distribution may not be continuous and populations may be isolated, and variations in morphometric characteristics could reflect genetic aspects within and between populations (roggero & passerind’entrèves, 2005; cardini et al., 2007; francoy et al., 2011), adaptations to natural (nunes et al., 2015) or anthropized environmental gradients (banaszak-cibicka et al., 2017), food stress (gumiel et al., 2003), or exposure to extreme physical factors in the environment (gumiel et al., 2003). the changes found in more distant geographic groups may reflect genetic aspects among the colonies in the pantanal (francoy et al., 2011; combey et al., 2018; henriques et al., 2020) or even in relation to resource use in case of some axis mean sd axis mean sd x1 118.66 6.10 y1 94.11 5.48 x2 521.25 16.92 y2 112.84 5.51 x3 594.53 17.65 y3 108.058 4.20 x4 938.00 30.55 y4 170.98 10.58 x5 135.86 13.30 y5 120.25 10.38 x6 484.13 15.64 y6 144.01 4.22 x7 566.55 17.48 y7 160.12 4.34 x8 61461 17.60 y8 127.39 4.21 x9 656.08 18.35 y9 140.98 4.53 x10 744.48 19.29 y10 151.41 6.05 x11 595.25 17.66 y11 183.46 4.56 x12 730.93 19.51 y12 207.83 10.33 x13 776.29 20.95 y13 241.14 7.05 x14 802.87 20.32 y14 242.23 7.82 x15 351.90 15.38 y15 180.73 7.88 x16 396.19 16.45 y16 179.43 4.61 x17 552.41 17.23 y17 216.32 4.57 x18 71235 25.33 y18 298.12 7.99 x19 343.75 36.34 y19 213.29 10.01 x20 539.12 36.70 y20 259.22 7.39 x21 521.67 18.39 y21 295.31 6.38 x22 119.39 11.05 y22 138.62 7.85 group 1 2 3 4 5 6 7 8 1 0.41 0.01* 0.41 0.15 0.43 0.45 0.49 2 0.03* 0.75 0.70 0.44 0.55 0.55 3 0.22 0.01* 0.47 0.06 0.01* 4 0.57 0.43 0.06 0.01* 5 0.46 0.01* 0.18 6 0.30 0.34 7 0.04* 8 table 3. mean values and standard deviation (sd) of the x and y axes for the anatomical landmarks of the left front wings of apis mellifera individuals collected in the brazilian pantanal. table 4. values of the multivariate analysis of variance (manova) test between geographic groups in the brazilian pantanal in relation to geometric morphometrics of left front wings of apis mellifera. (* corresponds to statistical significance = 0.05). alessandra c peil, rodrigo aranda – apis mellifera niche and morphometrics in pantanal8 bees (carneiro et al., 2019; ribeiro et al., 2019). the slight variation between some points in the pantanal could be related to the fact that some individuals collected were not from the same colony, that is, individual reflections of other distinct groups within the same collection point. a minimum distance of 20 km was established between the collection areas to avoid sampling individuals from one point that may be foraging at another point. however, large bees and those that forage massively can fly long distances in search of food resources (tornenoguera et al., 2014), which may have caused similarities in closer locations, highlighting differences in the most distant sampling points only and indicating good dispersion capacity. as expected, the closest geographic points showed greater morphometric similarity. geographically isolated points, such as the serra do amolar region (accessible only by water) with apiaries recently established due to the social actions of the ngo ecoa for income generation, or those with a smaller amount of bee records, such as the porto murtinho region, may still represent localities with possible genetic isolation another possibility could be related to the anthropic management of bees in the pantanal, which introduces different colonies to the same apiary or variations accumulated over time (francoy et al., 2009; francoy et al., 2011). additionally, migratory beekeeping that has been developed by a few beekeepers could have caused such divergence (reis & comastri-filho, 2003; francoy et al., 2009, nunes et al., 2012) due to fast development, adaptation and rusticity. furthermore, if a colony is removed from one place and installed in another place it could also lead to such effects, what happened when an a. mellifera colony was removed from a school in the barra do são lourenço community and relocated to the dois corações site, located at morro saquarema in the amolar region (almeida & reis, 2016). the removal of nests from natural areas to managed apiaries is a common practice adopted by riverside inhabitants in the pantanal. genetic analyzes of a. mellifera populations in the pantanal may reveal the history and routes of migration and colonization of the areas, since the morphology reflects the genetic aspects and we evidence significant differences in the populations (miguel et al., 2011). thus, the distribution of a. mellifera in the pantanal is related to temperature in the plain and the geometric morphometrics of wings show significant variation between the sampling points, reflecting aspects of dispersion and population dynamics in the brazilian pantanal, mainly in more isolated and distant areas. acknowledgments we thank the social service of commerce – sesc pantanal, instituto homem pantaneiro, and the national park of pantanal matogrossense headquarters for authorization to use the area and logistical support. we also thank the owners of the farms and private areas for support and access to their lands. the fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul – fundect for project financing no. 108/2015, siafem: 024501. author's contribution acp: conceptualization, methodology, investigation, writing. ra: conceptualization, methodology, investigation, formal analysis, writing. references adams, d.c., rohlf, f.j. & slice, d.e. 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(2012). geometric morphometrics for biologists: a primer. massachusetts: academic press. 488 p https://doi.org/10.1051/apido/2010048 http://dx.doi.org/10.13102/sociobiology.v66i2.3779 http://dx.doi.org/10.13102/sociobiology.v66i2.3779 https://doi.org/10.1002/ajpa.10174 https://doi.org/10.1371/journal.pone.0097255 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i1.4842sociobiology 67(1): 121-125 (march, 2020) introduction mischocyttarini is the most diverse tribe within the polistinae paper wasps, despite containing a single genus, mischocyttarus. these taxa encompass around 250 species and are widely distributed throughout south america and other tropical regions (silveira et al., 2008; barbosa et al., 2016a). new species are frequently recorded for the genus in brazil (cooper, 1998ab; silveira, 1998; silveira & felizardo, 2015; souza et al., 2015; borges & silveira, 2019), which holds the highest known species richness for mischocyttarus (carpenter & andena, 2013). studies on mischocyttarus have focused on the genus’ phylogeny, behavioural aspects, geographical distribution and abstract mischocyttarus saussurei nests show a curious architectural pattern which could be related to colony camouflage. since information on that species is scarce in literature, this study aimed to record ecological data on m. saussurei, as well as morphometric data on its nests. data was collected at the parque estadual do ibitipoca state park and at the municipalities of barroso and inconfidentes, minas gerais state, southeastern brazil. seven colonies were located, exclusively in conserved environments. five nests were dissected for morphometric analysis and for the assessment of the vegetal matter incorporated to comb walls. nests showed comb cells opening towards the substrate and covered by vegetal layers, in which three families of mosses and three of liverworts could be identified. we deduct that the nests’ morphometry and the incorporation of vegetal layers to the combs are related to the camouflaging of colonies amidst their substrate. sociobiology an international journal on social insects lr milani¹, f prezoto2, ma clemente3, pp gomes4, mm souza5 article history edited by evandro nascimento silva, uefs, brazil received 31 october 2019 initial acceptance 05 december 2019 final acceptance 05 december 2019 publication date 18 april 2020 keywords mischocyttarini, camouflage, nest, bryophytes, hepatics. corresponding author patricia p. gomes instituto federal de educação, ciência e tecnologia do paraná, campus umuarama rodovia pr 323, km 310 parque industrial cep: 87507-014 umuarama-pr, brasil. e-mail: patricia.gomes@ifpr.edu.br species ecology (raposo-filho & rodrigues, 1984; gianotti, 1998; o’donnell & joyce, 2001; silveira, 2008; barbosa et al., 2016b; souza et al., 2017; oliveira et al., 2017). despite the number of species sampled in diversity studies throughout brazil (barbosa et al., 2016), the ecological niches most of them fill are still poorly understood, which includes mischocyttarus saussurei zikán, 1949. for this species, the only data currently available on biology, geographical distribution, morphology and colony aspects are included in the works of zikán (1949) and richards (1978). mischocyttarus nests consist in a single comb attached to the substrate through a peduncle, without the presence of a protective envelope (jeanne, 1972; wenzel, 1991, 1998). some less-studied species show deviating architecture patterns; 1 biólogo colaborador do laboratório de zoologia do instituto federal de educação, ciência e tecnologia sul de minas, campus inconfidentes, minas gerais, brazil 2 universidade federal de juiz de fora, juiz de fora, minas gerais, brazil 3 instituto federal de educação, ciência e tecnologia sudeste de minas, campus barbacena, minas gerais, brazil 4 instituto federal de educação, ciência e tecnologia do paraná, campus umuarama, paraná, brazil 5 instituto federal de educação, ciência e tecnologia sul de minas, campus inconfidentes, minas gerais, brazil nesting behaviour of a neotropical social wasp mischocyttarus saussurei zikán, 1949 (hymenoptera, vespidae) short note lr milani, f prezoto, ma clemente, pp gomes, mm souza – nesting behaviour of a neotropical social wasp122 members of the artifex subgenus m. mirificus zikán, 1935, m. artifex ducke, 1914 and m. ypiranguensis da fonseca, 1926, for instance, build long, one cell-wide string-shaped combs. a similar pattern can be seen in species of the mischocyttarus iheringi zikán, 1935 group, which includes m. saussurei, with the additional deposition of vegetal matter on the comb’s external walls. this study aimed to present novel information on the nesting behaviour of m. saussurei, as well as ecological data for the species in different ecosystems in southeastern brazil. material and methods ecological data was collected during a study on social wasp species richness carried out between 2016 and 2017 in campos rupestres (“rupestrian grasslands”) environments within the of ibitipoca state park (21º43’ s 43º54’ w) and seasonal semideciduous montane atlantic forests in barroso (21º12’ s 43º55’ w) and inconfidentes (22º19’ s 46º19’ w) cities in the minas gerais state, southeastern brazil. when a colony was located, it was photographed and two wasp specimens were captured, killed, and stored for identification purposes. species confirmation was carried out on these individuals by the specialist dr. orlando tobias da silveira from the emílio goeldi museum, where samples were deposited. additionally, duplicates were deposited in the ifsulminas collection (inconfidentes campus) under voucher specimen numbers 03472-2017 034772017 (ifsulminas, 2018). after colonies were naturally abandoned, five nests were collected and analysed at the laboratório de ecologia comportamental e bioacústica (labec) and the botany department of the universidade federal de juiz de fora (ufjf). nest analysis was divided in two stages. the first stage consisted in the description of nest morphometry. for each nest we recorded the number of cells, cell width, cell length, peduncle width, and peduncle length. the second stage consisted in the identification of the vegetal matter found in each nest’s surface, carried out by specialist dr. andrea pereira luizi ponzo by comparing samples to items indexed in the herbarium collection of herbário prof. leopoldo krieger (cesj) of the universidade federal de juiz de fora. specialized literature was also used for reference during identification (gradstein et al., 2001; gradstein & costa, 2003). results and discussion six colonies were found nesting in rocky substrates and one in vegetal substrate. every site in which a colony was located was part of either a conservation unit or a highly conserved forest area (souza & prezoto, 2006; souza et al., 2017). this may indicate a relationship between the species and this ecological setting, since many species of social wasps are restricted to conserved environments or specific ecosystems (souza et al., 2017; detoni et al., 2018), as reported for the genus pseudopolybia (souza et al., 2010). the five dissected nests had, respectively, 15, 16, 26, 48 and 190 cells. the latter was atypically large, considering that mischocyttarus nests rarely present more than 100 cells (jeanne, 1975; castro et al., 2014). the finding of a nest with 190 cells may be evidence that the architecture pattern and nest camouflage are acting significantly to ensure colony success. by concealing themselves in the environment, nests may grow to reach high dimensions, which indicate a possible advantage of camouflage behaviour; however, further investigations are required to prove this hypothesis. cells had an average length of 1.16 mm ± 0.05 (1.1 – 1.2 n=5) and an average width of 3.2 mm ± 0.24 (3 – 3.5 n=5), which are comparable to those found in other studies on mischocyttarus (giannotti, 1999; montagna et al., 2010; downing, 2012). nest peduncles were always eccentrically fixed on the comb. they showed an average width of 1.46 mm ± 0.55 (0.9 – 2.2 n=5), which was negatively correlated to number of cells per nest according to a spearman’s correlation coefficient (p= 0.1 r = 0.73) (r development core team, 2017). conversely, a positive correlation has been found in other studies (montagna et al., 2010). wasp workers typically reinforce the peduncle as colonies increase in size and weight, but this was not verified in this study. peduncles had an average length of 4.48 mm ± 0.64 (3.7 – 5.3 n=5), which is comparable to studies on mischocyttarus drewseni (jeanne, 1972) and mischocyttarus cerberus styx (giannotti, 1999). every colony collected in this study was found nesting in moss-covered substrates, which made finding them challenging (figure 1) and reinforces the camouflage hypothesis, as discussed in barbosa et al. (2016b). the choice of nesting site is decisive for colony success and is an evolutionary response of social wasps to predation pressure (jeanne, 1975). colony camouflaging is more common in mischocyttarus than in other social wasp genera, acting as an indirect defensive behaviour (wenzel & carpenter, 1994; barbosa et al., 2016b). this is probably due to their morphological inability to use their stingers for defence, in conjunction to the absence of a protective envelope in their nests and their overall low aggressiveness (raposo-filho & rodrigues, 1984). another characteristic found in species of the mischocyttarus iheringi group is the presence of vegetal cover in the exterior of nests, avoiding contrast with the background and providing further visual camouflage. richards (1978) described colonies of the m. iheringi group with special emphasis to the substrate/colony relationship. richards cites the outward-facing comb’s bottom and sides being covered in moss, lichen and other vegetal materials as the group’s general feature. a curved peduncle, such as the ones found in this study, is necessary to ensure that cell openings are facing the substrate. this differs from the general architectural pattern in mischocyttarus nests. the sociobiology 67(1): 121-125 (march, 2020) 123 fig 1. (a) dorsal face of a mischocyttarus saussurei colony which was fixed on a vegetable substrate covered with moss. (b) detail of the curvature presented by the nest peduncle of mischocyttarus saussurei, so that the cell opening faces the nesting substrate, allowing the wasps to hide behind. (c) and (d) abandoned nests found on rocks covered by moss. nest’s vegetal cover could even be further added and kept by the wasps in the colony due to this feature being idiosyncratic to this species group. all collected colonies showed vegetal fragments. from these, we could identify three families of mosses (orthotrichaceae, sematophyllaceae and stereophyllaceae) and three of liverworts (frullaniaceae, metzgeriaceae and plagiochilaceae) (figure 2). three of these species were also found in nests of m. iheringi (barbosa et al., 2016b), whose nests are similar in architecture to m. saussurei’s, and whose vegetal cover was also discussed by authors as having a possible role in camouflage. m. saussurei nests show a morphometry particular to its group which, when associated to the incorporation of moss in the dorsal part of combs, make their visualisation extremely difficult amidst its substrate. this is likely to provide camouflage and have a positive effect on the colony’s success. future studies are necessary to prove this hypothesis. lr milani, f prezoto, ma clemente, pp gomes, mm souza – nesting behaviour of a neotropical social wasp124 acknowledgements the authors would like to thank dra. andrea pereira luizi ponzo for identifying the vegetal matter and the universidade federal de juiz de fora for infrastructural support. references barbosa, b.c., detoni, m., maciel, t.t. & prezoto, f. 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(2015). new occurrences of social wasps (hymenoptera: vespidae) in semideciduous seasonal montane forest and tropical dry forest in minas gerais and in the atlantic forest in the state of rio de janeiro. entomobrasilis, 8: 65-68. doi: 10.12741/ ebrasilis.v8i1.359 souza, m.m., brunismann, a.g. & clemente, m.a. (2017). species composition, relative abundance and distribution of social wasps fauna on different ecosystems. sociobiology, 64: 456-465. doi: 10.13102/sociobiology.v64i4.1839 wenzel, j.w. (1991). evolution of nest architecture. in: k.g. ross & r.w. matthews (eds.), the social biology of wasps (pp. 480-519.). ithaca: cornell university press, 678 p. wenzel, j.w.a. (1998). generic key to the nests of hornets, yellow-jackets, and paper wasps world wide (vespidae: vespinae, polistinae). american museum novitates, 3224: 1-39. wenzel, j.w. & carpenter, j.m. (1994). comparing methods, adaptative traits and tests of adaptation. in: eggleton, p. & vane-wright, r (eds.) phylogenetics and ecology (pp. 79101). london: academic press, 616 p. zikán, j.f. (1949). o gênero mischocyttarus saussure (hym., vespidae), com a descrição de 82 espécies novas. boletim de pesquisa do parque nacional do itatiaia, 1: 251. 11 the males of rhynchomicropteron (diptera: phoridae) by r. h. l. disney1 the genus rhynchomicropteron annandal (1912) has distinctive, myrmecophilous, flightless females. the females of the 17 oriental species are keyed by disney & kistner (1998), supplemented by disney (1999, 2010). until brown (1992) reared r. nudiventer papp in thailand, the males had been assigned to the genus gymnoselia schmitz (1927). this ‘genus’ contained the type species, r. curvescens (schmitz 1927) and r. nudicosta (brues 1907). neither of these have been linked to their females. recently leng yel (2011) has described both sexes of a species from israel, and reported the presence of this genus in africa and australia. the males remain poorly known. the purpose of this paper is to provide a key to the known males, even though most remain unknown and some are given code numbers only until they can be linked to their females. an initial impediment has been the recognition of schmitz’s species, r. curvescens. subsequent to his initial description, schmitz (1929) provided a more detailed description in which he included the statement that its palps are dark brown (‘dunkelbraun’). however, all the males i have examined, and r. nudicosta, have dusky yellow or yellow palps. in view of this i have borrowed its holotype of r. curvescens, kindly loaned by dr thomas pape (natural history museum of denmark, copenhagen). i have remounted it on a slide. its palps are clearly not dark brown, but are a slightly dusky yellow. the females of the r. nudiventer are largely recognised from negative characters. furthermore it is the most widely distributed species and is somewhat variable. it may prove to be a sibling species complex. the procurement of mating pairs, reared series or molecular barcodes will eventually provide an answer. however, i have compared its holotype female with a female from brown’s reared series (disney 2010). furthermore, chris von beeren recently sent me a reared series of r. nudiventer from malaysia that has allowed a detailed scrutiny of the details of its male. 1department of zoolog y, university of cambridge, downing street, cambridge cb2 3ej, u.k. email: rhld2@hermes.cam.ac.uk 12 sociobiolog y vol. 59, no. 1, 2012 notes on the known males rhynchomicropteron curvescens (schmitz) the holotype is from java. rhynchomicropteron nudicosta (brues) the holotype from india has been lost, but the original description indicates that its legs are reddish brown rather than the yellow of all other known males. rhynchomicropteron kuslitzkyi leng yel the type series is from israel. rhynchomicropteron nudiventer papp the holotype female was from n. e. india. the male was described by brown (1992) from thailand. females attributed to this species are also known from borneo and malaysia. rhynchomicropteron species 1 a male from malaysia collected with females of r. nudiventer at a colony of aenictus sp. was attributed to this species (disney 1992), but it was later found to differ from brown’s (1992) description of the male of this species (disney 2010). rhynchomicropteron species 2 a male from malaysia collected with females of r. necaphidiforme disney and r. nudiventer at a colony of leptogenys distinguenda (emery) (disney 1992) is probably r. necaphidiforme. rhynchomicropteron species 3 a male from malaysia was collected in a pitfall trap along with males of r. nudiventer and a female of r. beaveri (disney). rhynchomicropteron species 4 a male from a trap in indonesia , sulawesi. key to known males of rhynchomicropteron caution.the following key covers less than half the species known as females. 1. legs yellow ...........................................................................................................2 legs brown ..............................................................................nudicosta (brues) 2. costa with minute costal cilia along most of length (e.g. figs 9 & 13, see appendix) ..............................................................................................................3 13 disney, r.h.l— the males of rhynchomicropteron costa seemingly lacking costal cilia (as any are less than 0.02 mm long and are therefore not apparent at x100 or less) (figs 1, 5 and 17), but a few may be evident near base (fig. 6) .................................................................... 6 3. mesopleuron with fewer than 20 hairs. frons with fewer than 40 hairs ... .................................................................................................................................4 mesopleuron with more than 20 hairs. frons with more than 60 hairs. (hypopygium as figs. 8a & 8b in leng yel 2011) ......... kuslitzkyi leng yel 4. frons with less than 20 hairs (fig. 16). (hypopygium as figs 14 & 15) ... ...................................................................................................................species 2 frons with more than 20 hairs .........................................................................5 5 frons with a median furrow (fig. 12). mesopleuron with more than 8 hairs. hypopygium as figs. 10 and 11 ..............................................species 1 frons without a median furrow. mesopleuron with less than 8 hairs. hypopygium as figs 21 and 22 ................................................................species 4 6 anterolateral bristles present and almost as close to eye margins as mediolaterals (e.g. figs. 8 & 20) ......................................................................7 annterolaterals absent (fig. 4). (hypopygium as figs. 2 & 3) .................... .............................................................................................. curvescens (schmitz) 7. frons with more than 40 hairs (fig. 8). hypopygium as fig. 7 ................... ..................................................................................................... nudiventer papp frons with less than 40 hairs (fig. 20). hypopygium as figs 18 and 19 ... ...................................................................................................................species 3 acknowledgments i am grateful to dr thomas pape (natural history museum of denmark, copenhagen) for the loan of the holotype material of r. curvescens. my studies of phoridae are currently supported by grants from the balfour-browne trust fund (university of cambridge) and the systematics research fund of the linnean society and the systematics association (uk). references annandale, n. 1912. description of a micropterous fly of the family phoridae associated with ants. spolia zeylanica, colombo 8: 85-89. brown, b. v. 1992. life history, immature stages and undescribed male of rhynchomicropteron (diptera: phoridae). journal of natural history 26: 407-16. 14 sociobiolog y vol. 59, no. 1, 2012 brues, c. t. 1907. some new exotic phoridae. annales musei nationalis hungarici 5: 400413 + plate viii. disney, r . h. l., 1992. a further new species of rhynchomicropteron (dipt., phoridae) from malaysia. entomologist's monthly magazine 128: 219224. disney, r. h. l., 1999. two new species of rhynchomicropteron (diptera:phoridae) from borneo. sociobiolog y 34: 429-432. disney, r. h. l., 2010. newly recognised sibling species of rhynchomicropteron nudiventer (diptera: phoridae). sociobiolog y 56: 249-253. disney, r. h. l. & kistner, d. h., 1998. new species and new records of myrmecophilous phoridae (diptera). sociobiology 31: 291-349. leng yel, g. b., 2011. the first rhynchomicropteron annandale, 1912 (diptera, phoridae) species from the palearctic region with taxonomic and faunistic notes on the fauna of israel. zootaxa 2885: 23-32. schmitz, h. 1927. revision der phoridgattungen, mit beschreibung neuer gattungen und arten. natuurhistorisch maandblad 16, 30-40, 45-50, 59-68,72-79, 92-100, 110-116, 128-132, 142-148, 164, 176. schmitz, h. 1929. revision der phoriden. berlin: ferd. dummlers. 212 pp +tafel i and ii. appendix figures 1-22 fig. 1. rhynchomicropteron curvescens male, costal section 1. 15 disney, r.h.l— the males of rhynchomicropteron fig. 2. rhynchomicropteron curvescens male, left face of hypopygium. fig. 3. rhynchomicropteron curvescens male, right face of hypopygium. fig. 4. rhynchomicropteron curvescens male, frons. fig. 5. rhynchomicropteron nudiventer male, costal section 1. fig. 6. rhynchomicropteron nudiventer male, costal section 1, with a few costal cilia at base. 6 16 sociobiolog y vol. 59, no. 1, 2012 fig. 8. rhynchomicropteron nudiventer male, frons. fig. 10. rhynchomicropteron species 1 male, left face of hypopygium. (scale bar=0.1mm). fig. 11. rhynchomicropteron species 1 male, right face of hypopygium. (scale bar=0.1mm). fig. 9. rhynchomicropteron species 1 male, costal section 1. fig. 7. rhynchomicropteron nudiventer male, left face of hypopygium. 17 disney, r.h.l— the males of rhynchomicropteron fig. 12. rhynchomicropteron species 1 male, frons fig. 13. rhynchomicropteron species 2 male, costal section 1. figs 14-15. rhynchomicropteron species 2 male, hypopygium. fig. 14. right face; fig. 15. left face. (scale bar=0.1mm). fig. 16. rhynchomicropteron species 2 male, frons. fig. 17. rhynchomicropteron species 3 male, costal section 1. 18 sociobiolog y vol. 59, no. 1, 2012 fig. 18. rhynchomicropteron species 3 male, left face of hypopygium. fig. 19. rhynchomicropteron species 3 male, right face of hypopygium. fig. 20. rhynchomicropteron species 3 male, frons. fig. 21. rhynchomicropteron species 4 male, left face of hypopygium. fig. 22. rhynchomicropteron species 4 male, right face of hypopygium. 935 trichome removal by hitchhikers in two leaf-cutting ant species (hymenoptera: formicidae) by k. kitayama1*, leandro sousa-souto2, pedro de podestà uchôa de aquino1 & luiza xavier tenório1 abstract despite the known evidence that hitchhiker ants protect workers against attack by phorid parasitoids, several alternative hypotheses are suggested for the occurrence of hitchhikers on leaf-cutting ants. one hypothesis suggests that hitchhikers clean leaf fragments and remove pathogens. we hypothesized that hitchhikers can act in the removal of leaf trichomes. activities of hitchhikers (hh) are reported based on three laboratory and eight field colonies of leaf-cutting ants (atta spp.). we evaluated whether the presence of trichomes increases the frequency of hh in leaf fragments transported to the colony. furthermore, we evaluated if fragment size and the time that the fragment remains in the foraging arena could influence hh frequency. the removal of trichomes by hh of laboratory colonies was recorded on video. hitchhikers were more frequent in fragments with trichomes in both laboratory and field colonies. in the field, the distance from the foraging site did not influence the amount of hh. the proportion of hh in laboratory, however, was most frequent only during the first hour of foraging. the presence of hh is correlated to the size of fragments. we also observed removal of trichomes as an additional role of hitchhikers. key words: foraging behavior, atta sexdens, atta laevigata, social insects, trunk trails. resumo apesar das evidências que reforçam o papel de formigas caroneiras (hitchhikers) na proteção das operárias forrageadoras contra o ataque de forídeos, 1 departamento de zoologia, instituto de ciências biológicas, universidade de brasília, 70.910-900, brasília, df, brazil. 2 núcleo de ecologia, programa de pós-graduação em ecologia e conservação, universidade federal de sergipe, 49100-000, aracaju, se, brazil. e-mail: leandroufv@gmail.com *corresponding author: kiniti@terra.com.br 936 sociobiolog y vol. 59, no. 3, 2012 hipóteses alternativas tentam explicar a ocorrência desse comportamento em formigas cortadeiras. uma das hipóteses sugere que hitchhikers atuam limpando fragmentos de folhas contra patógenos. nossa hipótese é que hitchhikers podem atuar na remoção de tricomas foliares. atividades de caroneiras são relatadas com base em três colônias de atta spp. mantidas em laboratório e oito colônias de campo. nós avaliamos se a presença de tricomas aumenta a frequência de caroneiras em fragmentos de folhas transportados para a colônia. além disso, foi avaliado se o tamanho do fragmento e o tempo em que o fragmento permanece na arena de forrageamento poderiam influenciar na frequência de hitchhikers. a remoção de tricomas por caroneiras em laboratório foi gravado em vídeo. caroneiras foram mais frequentes em fragmentos com tricomas tanto em colônias de laboratório como nas de campo. no campo, a distância do local de coleta não influenciou a quantidade de caroneiras. a proporção de caroneiras em laboratório, no entanto, foi mais frequente apenas durante a primeira hora de forrageamento. a presença de caroneiras foi correlacionada com o tamanho do fragmento. a remoção de tricomas caracteriza-se como um papel adicional de caroneiras. palavras-chave: caroneiras, forrageamento, mata atlântica, trilhas introduction during foraging, the smallest workers (“minims”) of some leaf-cutting ant species are commonly seen “hitchhiking” on leaf fragments transported to the colony by larger foragers and the likely explanations for this behavior has been discussed in previous studies. several papers report the activity of hitchhikers (hh) in seven leaf-cutting ant species, most of which relates to forager defense against phorid parasitoids as the main function of these hitchhikers (lutz 1929; stahel 1943; eibl-eibesfeldt & eibl-eibesfeldt 1967; feener & moss 1990; erthal &tonhasca 2000; linksvayer et al. 2002; vieiraneto et al. 2006). linksvayer et al. (2002) reported hitchhiking behavior of atta cephalotes as a means of forager protection and, more recently, vieira-neto et al. (2006) tested, in addition to the aforementioned function, two other hypotheses concerning to hitchhiking behavior in a. sexdens and a. laevigata: defense against fungal contamination and leaf sap acquisition. these authors suggested that the hitchhiking behavior probably has multiple functions. 937 kitayama, k. et al. — trichome removal by hitchhikers in leaf-cutting ants for laboratory colonies, a previous study (kitayama et al. 2010) had shown that foragers of a. sexdens remove leaf trichomes prior to transport of leaf fragments to the colony. in field colonies, however, workers are seen carrying leaf fragments with trichomes (author’s personal observation). this suggests that trichome removal is performed during the journey to the colony or in the subterranean chambers of the nest. in this study, we test the hypothesis that there is a positive relationship between the presence of leaf trichomes and the number of hitchhikers, thus suggesting that hitchhikers may act in the removal of trichomes of the leaf fragments during the transport of them, to increase the efficiency of cleaning the substrate, an activity that has not been yet reported for this behavior. in addition, we tested whether larger foraging trails or large fragments have a higher proportion of hitchhikers and if this activity decreases with processing time of the plant material in the foraging area. material and methods laboratory colonies and experimental design experiments were carried out using three colonies of atta sexdens rubropilosa forel, 1908. the colonies were maintained in laboratory conditions at 25 ± 2 ºc under a 12 h photophase. colonies were daily supplied with leaves without trichomes of bauhinia variegata l. (fabaceae) and leaves with trichomes of ochroma piramidalis (bombacaceae). these plant species were chosen because they are commonly seen being cut by leaf-cutting ants in the field. the colonies (five years-old) were maintained in artificial nests, consisting of two interconnected plastic chambers with fungus and brood (volume: 40,000 cm 3 ) linked to a foraging arena (70 cm in length × 40 cm in width × 30 cm in height) by a transparent plastic tube (2.5 cm in diameter; 16 m in length). presence/absence of leaf trichomes and proportion of hitchhikers for each leaf type, small leaf discs (0.5 – 1.0 cm2) and large discs (1.1 – 1.8 cm2) were offered to ant colonies for 6 hours. the proportion of hitchhikers in the leaf discs was assessed by collecting all the workers carrying leaf fragments back to the colony during 30 minute intervals in two phases: (1) thirty minutes after the start of the experiment and (2) six hours after the first sampling. 938 sociobiolog y vol. 59, no. 3, 2012 the size of leaf discs collected as well as the head capsule of all collected ants (workers and hitchhikers) was measured using a digital caliper. trichome removal by hitchhikers the removal of trichomes by hitchhikers on the way back to the fungus garden from the foraging tray was videotaped inside the plastic tube and also in a transparent box of 20×10×5 cm in length, height and width connected to the plastic tubes. field colonies and sampling field study was conducted in an atlantic forest reserve of the parque estadual do rio doce (perd), state of minas gerais (19°30’21"s and 42°41'19" w). the park is considered the largest reserve of atlantic forest of minas gerais state with total area covering approximately 36,000 ha. colonies of leaf-cutters atta sexdens rubropilosa and a. laevigata are common on the edge of roads that cross the park. for this study we selected eight adult colonies (five a. sexdens rubropilosa and three a. laevigata) with nest area from 64 to 120 m2 and at least 100m apart from each other. the observations were made in three days (15-17) of july 2011, at the peak of foraging activity (18-23h). each colony had two to four well developed foraging trails, with an activity of approximately 68 ± 11 ants/minute. for each colony, a foraging trail was chosen arbitrarily for sampling. the total length of the trail (from the nest entrance to foraging site) was accessed and a fixed point was established 1 m away from the nest entrance. during the observation period (18 23h) we established a sampling period of 5 minutes at 60 minute intervals, totaling 5 samples per colony in total (25 minutes). in each sample we collected all loaded ants crossing a fixed point in the foraging trail and we determined the proportion of fragments with hitchhikers. also, the collected fragments were separated in relation to the presence/absence of trichomes. leaf fragments without trichomes were identified as being from trees of mabea fistulifera mart., while the plant species of fragments with trichomes could not be identified. data analysis to test whether the size of leaf discs may affect the proportion of hitchhikers, a wilcoxon rank test was performed. for laboratory colonies, a generalized 939 kitayama, k. et al. — trichome removal by hitchhikers in leaf-cutting ants linear model (glm; mccullagh & nelder 1989) with quasibinomial error distribution was performed to test the effects of presence of trichomes and foraging time (independent variables) on the proportion of hitchhikers in leaf fragments (dependent variable). for field colonies, the glm model was performed to test if the proportion of hh changes with leaf substrate (with or without trichomes) and length of the foraging trail. results laboratory colonies major leaf discs (1.55 ±0.76 cm²) had consistently higher number of hitchhikers than small leaf discs (1.02 ± 0.37 cm²) (w=0.85; p=0.04). as hh may climb up or down the leaf at anytime, it is likely that larger loads are more easily climbed, and this may explain the higher occurrence of hh in larger fragments. also, the presence of trichomes significantly affected the presence of hh. leaf fragments of ochroma sp. had more hh than leaf fragments of bauhinia sp. (f =38.61; df =1 p < 0.001) in the first hour of foraging (fig. 1). after a six hour interval, however, there was no significant difference in the proportion of leaf fragments with hh between leaf type (f = 1.87; p > 0.05). the decrease in the proportion of leaves with hh in ochroma treatment after six hours of exposure to leaves in the foraging arena may be due to the reduction of trichomes (kitayama et al. 2010). field colonies as in the laboratory, the field colonies showed a similar foraging behavior, with a higher proportion of hh on leaves with trichomes. in total, 3300 leaf fragments were collected, but only 132 (4%) had trichomes. the proportion of hh in these fragments, however, was three times higher than in fragments without trichomes (mean of 60% of leaves with hh versus 23% in fragments without trichomes) (f = fig. 1: hitchhiker presence (%) in response to trichomes in leaf discs in three laboratory colonies (mean ± se). 940 sociobiolog y vol. 59, no. 3, 2012 32.98 p < 0.001). the distance from the foraging site to nest entrance did not significantly influence the proportion of hh in the fragments (f = 1.58 p = 0.24) (table 1, fig. 2). discussion ants may hitch onto loads at anytime, anywhere along way back to the fungus garden (colony), for several different hypothetical reasons: (1) protection of leaf carriers from attack by phorid (diptera: phoridae) parasitoids; (2) to save energ y of walking themselves to the nest; (3) to feed on leaf sap from cut leaves; and (4) prior preparation of leaf fragments, removing microbial contaminants (eibl-eibesfeldt & eibl-eibesfeldt 1967; linksvayer et al. 2002; vieira-neto et al. 2006; gerstner et al. 2011). our results support the previous processing hypothesis since not only were there more frequent hitchhikers in leaf with trichomes but also because their frequencies were reduced after six hours of processing the leaf discs. in the absence of ant-parasitizing phorids, pathogens or trichomes, the function of hitchhikers is probably not much required. in addition, the energ y saving hypothesis (feener & moss 1990) was not supported, because the ratio of hh was not affected by the distance of the foraging site to the colony (fig. 2). table 1: glm analysis showing the effects of leaf type and distance of foraging site to the colony on proportion of hitchhikers of atta sexdens rubropilosa and a. laevigata colonies. response variable error distribution d.f. f-value p leaf type quasibinomial 2 32.98 0.001 distance to the nest quasibinomial 1 1.58 ns atta species quasibinomial 2 1.79 ns fig. 2: proportion of hitchhikers in response to distance of foraging site to the nest and trichomes in leaf fragments in eight field colonies (curves: y white circles = 0.0936+(0.0033*x) and y black circles = (0.401+(0.0033*x)). 941 kitayama, k. et al. — trichome removal by hitchhikers in leaf-cutting ants as the presence of phorid parasitoids may reduce foraging activity (bragança et al. 1998) parasitoid defence could be the primer function of hitchhiking. this fact is reinforced by the study of roces & holldobler (1995) that showed increased hitch behavior followed by sound signals caused by stridulatory vibrations during the cutting of leaf fragments before being loaded into the colony. so hitchhikers seem “to know” the right time to climb on the leaf fragments through this short-range recruitment signal. the occurrence of hitchhikers in periods that phorid parasitoids do not occur (night hours, for example), however, has raised several questions about alternative functions of hitchhikers on the trails of foraging. after observation of 14 field colonies of a. cephalotes, linksvayer et al. (2002) concluded that the prior preparation of leaves before entering the nest may be the most likely possibility for the occurrence of hitchhikers, as these authors found no evidence that hitchhikers were feeding on sap. this study reinforces the hypothesis that hh have multiple functions because hh were observed at night, with the highest proportion found in leaves with trichomes. moreover, as this behavior was observed in two different species in the field, cleaning leaves of trichomes can be performed by all species of leaf-cutting ants. similar functions performed by hh of different ant species were observed (vieira-neto et al. 2006). leaves without trichomes are preferred for cutting when there is a choice for ants (howard 1988; kitayama et al. 2010). in the present study, only 4% of the harvested leaves had trichomes. in fact, mabea fistulifera is one of the most abundant tree species of the study site (drumond & neto 1999; personal observation), being possibly the main plant resource cut by atta colonies throughout the year. other plant species, however, may be more suitable for workers and this may explain the harvesting of leaves with trichomes. it is possible that such leaves with trichomes were chosen by some either nutritional or stress factor of the host plant, since plants under stress (i.e. drought-sensitive individuals) suffer greater attacks on the edge of forest environments than healthy plants (meyer et al. 2006). in several brazilian ecosystems, however, climate seasonality leads to many plants developing defence mechanisms against desiccation and herbivores (saraiva et al. 1996; marques et al. 2000). thus, for several months, plants with leaves with abundant pubescence or trichomes are probably more resistant to drought and an 942 sociobiolog y vol. 59, no. 3, 2012 important resource to herbivores (woodman 1991). for ants in the cerrado and semi-arid habitats, leaves with trichomes are an important resource and the role of hitchhikers in these environments is crucial for the best utilization of this substrate for the colony. concerning the size of hh, we found that, according to our classification, 95.2% of ants are between 0.4 and 1.9 mm in head width and only 4.7% were larger than 2mm in head width. as nestmate (or load) defence is the main role of hitchhikers and small workers are more aggressive than larger ones, this size difference appears to correspond to this function. as workers smaller than 2mm in head width are more likely to remove trichomes (kitayama et al. 2010) and because hhs were also smaller than 2mm, a new function of hh was hypothesized and observed, that is, they could remove trichomes while moving back to the fungus garden. this new function was videotaped and is available for viewing at http://www.entomoufs.com.br/hitchhikers2012.wmv. acknowledgements we thank bianca ambrogi for suggestions and comments. we also thank conrado dos santos reis for his assistance with video editing. references bragança, m.a.l., a.jr. tonhasca & t.m.c. della lucia 1998. reduction in the foraging activity of the leaf-cutting ant atta sexdens caused by the phorid neodohrniphoa sp. entomologia experimentalis et applicata 89(3):305-311. drumond, m.a. & j.a.a.m. neto 1999. floristic and fitossociological compositions of a secondary forest in a site of the atlantic forest. ciência rural 29(4):657-661. eibl-eibesfeldt, i. & e. eibl-eibesfeldt 1967. das parasitenabwehren der minimaarbeiterinnen der blattschneider-ameise (atta cephalotes). zeitschrift für vergleichende physiologie 24:278-281. erthal, m.jr. & a.jr. tonhasca 2000. biolog y and oviposition behavior of the phorid apocephalus attophilus and the response of its host, the leaf-cutting ant atta laevigata. entomologia experimentalis et applicata 95(1):71-75. feener d.h.jr. & k.a.g. moss 1990. defense against parasites by hitchhikers in leaf-cutting ants: a quantitative assessment. behavioral ecolog y and sociobiolog y 26(1):17-29. gerstner a.t., m. poulsen & c.r. currie 2011. recruitment of minor workers for defense against a specialized parasite of atta leaf-cutting ant fungus gardens. etholog y ecolog y & evolution 23(1):61-75. 943 kitayama, k. et al. — trichome removal by hitchhikers in leaf-cutting ants howard, j.j. 1988. leaf-cutting ant diet selection: relative influence of leaf chemistry and physical features. ecolog y 69(1):250-260. kitayama, k., l. sousa-souto, j.d. hay, r.j.v. ottoni & p.p.u. aquino 2010. trichomes and atta sexdens (hymenoptera: formicidae): a study of foraging behavior in the laboratory. sociobiolog y 55(1b):107-116. linksvayer, t.a., a.c. mccall, r.m. jensen, c.m. marshall, j.m. miner & m.j. mckone 2002. the function of hitchhiking behavior in the leaf-cutting ant atta cephalotes. biotropica 34(1):93-100. lutz, f.e. 1929. observations on leaf-cutting ants. american museum novitates 388:121. marques, a.r., q.s. garcia, j.l.p. rezende & g.w. fernandes 2000. variations in leaf characteristics of two species of miconia in the brazilian cerrado under different light intensities. tropical ecolog y 41:47-60. mccullagh, p. & j. nelder 1989. generalized linear models. chapman & hall, london, 532p. meyer, s.t., f. roces & r. wirth 2006. selecting the drought stressed: effects of plant stress on intraspecific and within-plant herbivory patterns of the leaf-cutting ant atta colombica. functional ecolog y 20(6):973-981. roces, f. & b. hölldobler 1995. vibrational communcation between hitchhikers and foragers in leaf-cutting ants (atta cephalotes). behavioral ecolog y and sociobiolog y 37(5):297-302. saraiva, l.c., o. cesar & r. monteiro 1996. breeding systems of shrubs and trees of a brazilian savanna. arquivos de biologia e tecnologia 39(4):751-763. stahel, g. 1943. the fungus gardens of the leaf-cutting ants. journal of new york botanical garden 44(527):245-253. vieira-neto, e.h.m., f.m. mundim & h.l. vasconcelos 2006. hitchhiking behavior in leaf-cutting ants: an experimental evaluation of three hypotheses. insectes sociaux 53(3):326-332. wodman, r .l. & g.w. fernandes 1991. differential mechanical defence: herbivory, evapotranspiration, and leaf hairs. oikos 60:11-19. doi: 10.13102/sociobiology.v67i4.5081sociobiology 67(4): 508-513 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction eusociality is characterized by individuals of several generations living together and a non-reproductive caste caring for the offspring, thereby creating a labor division. social insect colonies are structured in a labor division system based on morphological and behavioral castes (wilson, 1971; wilson, 1980). the high density of related individuals in social insect colonies is expected to make them more susceptible to infections by increasing pathogen transmission rates (schmid-hempel, 1998). however, proper social insect organization may reduce the probability of pathogen transmission, thereby reducing pathogen establishment in the colonies (cremer et al., 2007). if workers can infect queens, e.g., workers can infect queens during their care behavior, their access to them should be abstract this study aimed to verify age polyethism occurrence in medium-sized (cephalic capsule = 2.3 ± 0.21 mm) and small-sized (cc = 1.4 ± 0.10 mm) workers from atta sexdens (linnaeus) colonies. four laboratory colonies were used, and they were maintained at 25 ± 2 °c, with 75 ± 3% relative humidity and a 12-hour photoperiod. workers from these colonies were marked after their emergence and observed throughout their lifetime to determine which tasks they performed. the number of ants performing each activity was analyzed using linear mixed-effect models (lme), considering the temporal effect and the block design (colonies). we found that fungal garden maintenance tasks were frequent for both sizes, but their occurrence decreased significantly from the ninth week. the foraging activity occurred gradually in both sizes, with stabilization in the number of workers from the fourth week onwards and declined in the last three weeks of lifespan. waste management tasks occurred throughout life but were more frequent during the first two weeks of life, in both medium and small workers. therefore, age polyethism may be related to all activities; however, foraging tasks presented a distinct pattern compared to tasks in the fungus garden and refuse dump, where younger ants were more frequently observed. sociobiology an international journal on social insects fg lacerda1; tmc della lucia2, l sousa-souto3, dj de souza4 article history edited by jacques delabie, uesc, brazil received 03 march 2020 initial acceptance 09 november 2020 final acceptance 02 december 2020 publication date 28 december 2020 keywords brood, leaf-cutting ant, foraging, fungus garden, waste. corresponding author danival josé de souza https://orcid.org/0000-0002-2839-1117 graduate program in plant production federal university of tocantins gurupi university campus cep 77410-530, gurupi, tocantins, brasil. e-mail: danival@uft.edu.br restricted since, and mainly when the colony depends on a single queen (monogyny) (wang & mofller, 1970). another pattern observed in several social insect species is labor division based on age. in this case, younger individuals assist the queen while older individuals forage (wilson, 1971). this labor division is called age polyethism and corresponds to task switching according to individual age. age polyethism is relatively common in social insects (kolmes, 1986), including bees (santos et al., 2010), termites (yanagihara et al., 2018), and ants (santana vieira et al., 2010). generally, younger individuals perform safer tasks inside the nest, while older workers perform riskier tasks outside, as the latter are “less valuable” to the colony (santana vieira et al., 2010). age polyethism in ants can be quite distinctive, as well as stereotypical or not. in pheidole hortensis (forel) colonies, older workers continue to perform characteristics of 1 department of biology (dbio), federal university of espírito santo, alegre-es,brazil 2 department of animal biology (dba), federal university of viçosa, viçosa-mg, brazil 3 department of ecology (deco), federal university of sergipe, são cristóvão-se, brazil 4 department of plant production, federal university of tocantins, gurupi-to, brazil research article ants age polyethism in atta sexdens (linnaeus) (hymenoptera: formicidae) sociobiology 67(4): 508-513 (december, 2020) 509 young workers (calabi et al., 1983). it has been shown that, in ectatomma opaciventre (roger) nests, outside activities such as defense and foraging are held by older workers, while offspring care is performed by the younger individuals (miguel & del-claro, 2005). age polyethism also occurs in dinoponera lucida (emery) workers (peixoto et al., 2008). in the leaf-cutting ants, some studies show that age polyethism is present in acromyrmex subterraneus brunneus (forel) colonies in which young workers remain inside the nest while older workers perform activities outside the colony (camargo et al., 2007). workers of atta cephalotes shift their behavior from leaf-cutting to leaf carrying when they become less effective at cutting leaves as they age and their mandibles become worn (schofield et al., 2011). in leaf-cutting ant colonies, workers perform various tasks, such as foraging and offspring, queen and fungus garden care, in addition to defending the colony. there are also hygienerelated tasks, which involve waste transport out of the fungus garden chamber and waste rearrangement in garbage piles. these tasks avoid the spread of waste that could promote disease propagation (lacerda et al., 2006). this study aimed to investigate age polyethism occurrence in nests of atta sexdens, commonly known as leafcutter ant. this species is widely distributed in all brazil regions and is economically significant because it damages agriculture and planted forests. material and methods we used four a. sexdens colonies maintained in the insectarium of the federal university of viçosa at 25 ± 2 °c, 75 ± 3% relative humidity, and a 12-hour photoperiod. the estimated population of each colony was 16,040; 17,620; 20,020 and 14,020 individuals, and colonies were designated as c1, c2, c3, and c4, respectively. population estimation was based on ant workers count in 50 ml of the fungus garden, and the result was extrapolated to the total volume of fungus garden in each nest. according to the available offspring, the highest possible number of medium (cephalic capsule width = 2.3 ± 0.21 mm) and small (cephalic capsule width = 1.4 ± 0.10 mm) pupae were withdrawn from each colony. thus, 308 medium and 250 small pupae were taken from c1 colony; 352 medium and 200 small pupae from c2 colony; 401 medium and 200 small pupae from c3 colony; and 138 medium and 80 small pupae from c4 colony. pupae were placed inside mini-colonies in the absence of a queen; each colony had its mini-colony, which consisted of a 200 ml plastic container with one-centimeter gypsum basal layer to maintain the humidity inside. seventy foraging workers and 200 gardening workers were placed in each mini-colony to provide offspring care. each container had an opening that allowed worker foraging and waste output and a perforated lid on top of which a cotton swab moistened with distilled water was placed. a 100 ml fungal garden fragment from each colony was placed in each container. this set was placed within a 4 l container whose borders were treated with a fluon® layer to prevent workers’ escape. acalypha wilkesiana müll. arg. and ligustrum japonicum thunb leaves were also placed within the larger containers to serve as the substrate for symbiotic fungi growth. after their emergence in the mini-colonies, adults were adequately marked and put back in their original colony. because adults did not emerge simultaneously, we marked them with different colors according to their emergence time to facilitate age identification. the activities of the newly emerged adults were observed 24 hours after they were returned to their original colonies. the fungus garden was placed in rectangular pots with a 192 cm2 area and 4 cm height to facilitate the marked colony workers’ observation. a one-centimeter gypsum layer was also placed at the base of each pot. for each colony, as many pots were used as necessary, according to fungus garden volume and its subsequent growth. fungus garden volume was approximately 1.5 l for c1 and c4 colonies and 2.0 l for c2 and c3 colonies. observation of workers’ behavior was performed daily for half an hour in each colony until no marked worker was present in each nest, i.e., when all were already dead or when marks had disappeared. the observed behaviors were fungus garden, offspring, queen care, foraging, and waste management. the first three behaviors were grouped into “fungus garden care” as offspring, and queen care activities were rarely seen. the number of daily occurrences of each behavior was grouped in weeks for each colony. because workers emerged at different times, resulting in three age classes for each ant size, the mean of the three ages about behavior occurrence each week and each colony was calculated. also, for improved interpretability of results, a mean of the four colonies relative to each behavior occurrence in each week was calculated. age polyethism observation methodology followed the example of camargo et al. (2007), with modifications, because these authors used only two colonies maintained in a closed foraging system and observed small, medium, and large workers. linear mixed effect models (lme) were used to compare differences in the number of ants marked in relation to their lifetime (weeks) and the cephalic capsule size. in these models, the number of ants (response variables) were tested with worker size, time (weeks), and the interaction worker size × time. complete models were fitted with all response variables as fixed effects, whereas colonies were used as random effects (crawley, 2012). results the behavior of fungus garden care in each week of the experiment was similar between small and medium workers (f1, 87 = 0.34; p > 0.05). the average number of workers performing the task remained stable until the eighth week and decreased from then until the end of the observations, fg lacerda; tmc della lucia, l sousa-souto, dj de souza – polyethism in atta sexdens510 showing two distinct periods in the activity (f12,87 = 48.97; p < 0.001; fig 1). there was no significant difference between the two size classes (f1,87 = 0.34; p = 0.55). the average number of medium and small-sized workers performing foraging activities gradually increased until the fourth week, remaining stable until the 11th week and declining after that until the end of the experiment (f13,94 = 5.35; p = 0.02; fig 2). there was significant difference between the two size classes, with the medium workers being slightly more frequent than the small ones (f1,94 = 4.54; p = 0.03) whereas the size x time interaction was not significant (f1,81 = 0.25; p = 0.9). fig 1. number of tasks occurrence within the fungus garden (garden, offspring and queen care) each week by medium and small-sized workers (mean ± se). fig 2. average number of medium and small-sized workers (± se) performing foraging tasks each week. sociobiology 67(4): 508-513 (december, 2020) 511 the occurrence of waste management activities performed by workers was more frequent in the first two weeks of post-emergence (f12,87 = 3.68; p < 0.001; fig 3) and was constant until the last two weeks of the experiment, where the lowest values were observed. in general, waste management activities were performed by workers of both sizes (f1,87 = 2.04; p > 0.05). fig 3. number of medium and small-sized workers (mean ± se) performing waste management each week. discussion in a. sexdens colonies, we observed that medium and small workers performed both the inside and outside tasks throughout their lives in varying degrees. inside work, such as fungus garden, offspring and queen care, was typical throughout their lives, although its occurrence decreased around the ninth and tenth weeks in medium and small workers, respectively. there may be a task specialization by those workers who remained caring for the garden until the end of their lives. because the queen mates multiple times, there is a certain degree of genetic diversity among the workers, and this factor could also partly explain the results obtained here. genetic-influenced behavioral specialization was found in acromyrmex versicolor (pergande) (julian & fewell, 2004) and acromyrmex echinatior (waddington et al., 2010). foraging activities also occurred throughout the lifetime of medium and small workers, becoming more common in the eleventh week, i.e., at the end of their lives. foraging may be a workers’ specialized task because it is performed by younger individuals, even if at low frequencies. the number of workers performing the fungus garden care behavior was very similar between medium and small workers, with a drastic reduction of this activity from the tenth week of the experiment. a similar number of workers of both sizes performing this activity is contrasting because one would expect this behavior to be more frequent among small workers. these medium-sized workers are likely part of a garden-care specialist caste. over the weeks, foraging was performed mainly by medium workers. their size is compatible with plant material cutting, loading, and transportation. waste compartment tasks were performed throughout the workers’ lives and were more frequent among the small workers during most of the experiment. it is noteworthy that the occurrence of waste management tasks was higher in the first two weeks. one would expect these tasks to be performed by older workers, considering that waste poses a greater risk of contamination with pathogens. one explanation is that not every microorganism in the nest refuse is hazardous to workers, or these workers have efficient defense mechanisms to inactivate potentially harmful microorganisms. some pathogenic fungus, like the garden parasite escovopsis (bot et al., 2001), the fungal garden antagonist trichoderma viride pers. ex gray (lacerda et al., 2006; ortiz & orduz, 2001), and the entomopathogenic aspergillus flavus link ex gray (lacerda et al., 2006) and metarhizium anisopliae (metsch.) sorokin (hughes et al., 2004), for example, has been found in waste material. however, several others microorganisms found in those studies are not considered pathogenic to ants or the mutualistic symbiotic fungus. leaf-cutting ants have fg lacerda; tmc della lucia, l sousa-souto, dj de souza – polyethism in atta sexdens512 many strategies to prevent colony contamination, such as the production of effective antibiotics by metapleural glands, including some against entomopathogens (poulsen et al., 2002), as well as actinomycetes associated with their bodies (currie et al., 1999) that inhibit escovopsis and entomopathogenic fungi (d’ângelo et al., 2016; mattoso et al., 2012; sen et al., 2009). in addition, there are behavioral strategies that help minimize contamination from waste, such as task sharing during waste transportation (ballari et al., 2007; hart & ratnieks, 2002; lacerda et al., 2006), allogrooming and self-grooming (lacerda et al., 2013). therefore, in our study, age polyethism was characterized by workers initially working in the garden and later in foraging activities; however, polyethism did not apply to waste-related tasks. acknowledgments the authors thank cnpq (project number 4037082013-3) and capes for funding this research. authors’ contribution fg lacerda: investigation and writing. tmc della lucia: conceptualization, methodology and writing. l souza-souto: formal analysis and writing. dj de souza: formal analysis and writing. references ballari, s., farji-brener, a.g. & tadey, m. 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(2018). age-based soldier polyethism: old termite soldiers take more risks than young soldiers. biology letters, 14: 20180025. doi: 10.1098/rsbl.2018.0025 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7194sociobiology 68(4): e7194 (december, 2021) introduction seeds of many plant species bear external appendages called elaiosomes (beattie & culver, 1981; handel et al., 1981; beattie, 1985; gaddy, 1986; servigne & detrain, 2008; lengyel et al., 2010). foraging ants attracted by elaiosomes bring back the seeds to their nests. although elaiosomes are consumed as food, the main parts of seeds are discarded inside or outside the nests (hughes & westoby, 1992a; gómez et al., 2005). consequently, mutualism between ants and plants is achieved. specifically, ants can obtain essential nutrition such as lipids and nitrogen-rich amino acids. in return, seeds are dispersed from the parent plants, which remove the seeds from the competition with other plants (higashi et al., 1989; abstract eggs of some stick insects bear external appendages called capitula. foraging worker ants attracted by capitula disperse eggs is similar to workers’ responses to elaiosomebearing seeds of many plants. for this study, we conducted rearing experiments in the laboratory to elucidate the interspecific relation between the queenless ant, pristomyrmex punctatus smith, and the stick insect, phraortes illepidus (brunner von wattenwyl), a species exhibiting eggs that bear capitula. eggs of p. illepidus were proposed to p. punctatus in the laboratory. capitula were removed from most of the eggs when ants were starved and when ants were well-fed. in large rearing containers, ants transported eggs from their origin, and many eggs were transferred horizontally on the surface. although some eggs were found in the artificial ant nests, it is likely that stick insects are not in active ant nests at hatching in nature because of p. punctatus nest-moving habits. the percentage of eggs buried in the sand was small. furthermore, most of the buried eggs were found at less than 3 cm depth. results show that many p. illepidus hatchlings can reach host plants safely without being attacked by ant workers. these results suggest that p. punctatus can be a good partner of p. illepidus. ants disperse eggs of slow-moving stink insects in exchange for some nutrition from capitula. sociobiology an international journal on social insects yoshiyuki toyama, izuru kuroki, keiji nakamura article history edited by evandro nascimento silva, uefs, brazil received 27 april 2021 initial acceptance 01 august 2021 final acceptance 08 october 2021 publication date 03 december 2021 keywords capitula, hatching, mutualism, rearing experiment, stick insect. corresponding author izuru kuroki department of biosphere – geosphere system science faculty of biosphere – geosphere system science okayama university of science ridai-cho 1-1, okayama 700-0005, japan. e-mail: pirukuru117@gmail.com lanza et al., 1992; gamman et al., 2005; boulay et al., 2006; fischer et al., 2008). stick insects are well-known for their twig-like forms of plant mimicry. the shapes of stick-insect eggs also resemble those of plant seeds. furthermore, in some stick insects, appendages, called capitula, are attached to the opercula of the eggs (clark, 1976). several studies have revealed that ants transfer stick-insect eggs with capitula (compton & ware, 1991; hughes & westoby, 1992b; windsor et al., 1996; stanton et al., 2015). ants are less attracted to eggs from which capitula are artificially removed or covered by paint (windsor et al., 1996; stanton et al., 2015). furthermore, when capitula removed from eurycnema goliath (gray) eggs are attached to polystyrene balls, workers of rhytidoponera department of biosphere – geosphere system science, faculty of biosphere – geosphere system science, okayama university of science, okayama, japan research article ants dispersal of phraortes illepidus (phasmida: phasmatidae) eggs by workers of the queenless ant, pristomyrmex punctatus (hymenoptera: formicidae) mailto:pirukuru117@gmail.com yoshiyuki toyama, izuru kuroki, keiji nakamura – dispersal of phasmida eggs by ant workers2 metallica (smith) intensively collect the balls (stanton et al., 2015). these studies show clearly that capitula of stickinsect eggs are attractive to ants, as are the elaiosomes of plant seeds. those results suggest that the transportation of eggs by ants is an adaptive strategy used by slow-moving stick insects to disperse their progeny (compton & ware, 1991; hughes & westoby, 1992b; windsor et al., 1996). ant nests can protect stick-insect eggs from egg parasitoids and fire. however, ant nests are not safe for stick insect hatchlings because of the predation risk by ants. compton and ware (1991) reported that workers of acantholepis capensis mayr ignored first instar nymphs of bacillus coccyx westwood emerged in their nests. nevertheless, it remains unclear whether or not workers ignore hatchlings in the other combinations of ants and stick insects. nest-moving habits of ants are important factors affecting stick insect survival. hughs and westoby (1992b) reported that the predation risk of stick-insect nymphs is mitigated by frequent nest moving in many ant species. specifically, even if eggs are stored in the nests, the long embryonic period of stick insects enables nymphs to hatch after the nests have already been moved elsewhere. even without ants, hatching success decreases when stick-insect eggs are buried deep into the soil. when eggs of podacanthus wilkinsoni macleay are buried at 6 cm or less in the soil, nymphs emerge successfully from 98% of the eggs (hughes & westoby, 1992b). however, nymphs reach the surface from only 11% of the eggs buried at 12 cm. consequently, ant species that transport eggs deep into the soil cannot be good partners for stick insects. the mortality of hatchlings can decrease if the eggs of stick insects are not stored in the ant nests. eggs of calynda bicuspis stål are not stored in the nests of ectatomma ruidum roger (windsor et al., 1996). specifically, many eggs are soon discarded outside of the ant nests. the risk of parasitism by egg parasitoids is decreased even though ants do not protect eggs in the nests. thus, we can infer that the horizontal transfer of eggs from the original places is effective in environments where forest fires are rare (windsor et al., 1996). pristomyrmex punctatus smith, widely distributed in southeast asia, is a widely dispersed ant species in western japan (japanese ant database group, 2003). this species transports seeds of plants such as thesium chinense turcz and sciaphila secundiflora thwaites ex bentham (suetsugu, 2015; suetsugu et al., 2017). pristomyrmex punctatus is queenless, and workers produce additional workers by thelytoky (mizutani, 1980, 1984; itow et al., 1984; tsuji, 1988a). a large colony can comprise several tens of thousands to several hundred thousand workers. nests are established under stones or inside decayed trees (japanese ant database group, 2003). however, permanent nests are not constructed: p. punctatus relocates its colony frequently (mizutani, 1982; tsuji, 1988b). phraortes illepidus (brunner von wattenwyl), a bisexual stick insect, is widely distributed in japan’s honshu, shikoku, and kyushu islands (okada, 1999). capitula are attached to the opercula of p. illepidus eggs (fig 1). it is expected that capitula attract ants and that they disperse stick-insect eggs. for this study, we conducted rearing experiments in the laboratory to examine 1) whether or not p. punctatus are attracted by p. illepidus eggs, and 2) where and how deep stick-insect eggs are transferred by ants. we first examined the response of p. punctatus to p. illepidus eggs. we presented stick-insect eggs to ants in small rearing containers and counted the capitula of eggs removed by ants. next, we examined the destination of p. illepidus eggs placed near the artificial nests of p. punctatus in large rearing containers. when eggs were buried in the sand, the burial depth of each egg was measured. the results demonstrated an interspecific relation between p. punctatus and p. illepidus. materials and methods collection and rearing of ants colonies of p. punctatus were collected in okayama city, japan (34.7 °n, 133.9 °e) during the spring-summer of 2011. small colonies were maintained in plastic containers with two levels. the bottom of each lower container (140 × 65 mm, 35 mm deep) was covered with plaster. dried fig 1. photograph of a phraortes illepidus egg, which bears a capitulum. sociobiology 68(4): e7194 (december, 2021) 3 bloodworm and insect jelly, an artificial diet for adult beetles (fujikon co. ltd., nose, osaka, japan), was placed on the upper containers (70 × 40 mm, 20 mm deep) once every week. a proper quantity of water was also provided through the plaster. large colonies were maintained in large plastic containers (31 × 17 cm, 22 cm in height). bottoms of the containers were filled with sand of approximately 2 mm maximum particle size, and the sand depth was 8-9 cm. a square plaster plate (approximately 10 × 10 cm, 2 cm in height), under which ants can construct a nest, was placed on the sandy surface of each container. in addition, a sufficient amount of dried bloodworm and insect jelly were provided once a week. water was provided through the plaster plate every day. the colonies were maintained under room conditions at approximately 20-25 °c. rearing of stick insects and collection of eggs first or second instar nymphs of p. illepidus were collected in okayama city, japan, in late april 2011. insects were reared in plastic containers (35 × 30 × 38 cm height) under room conditions at 20-25 °c. the rearing density of early instar nymphs was approximately 30 per container. after that, the respective densities of fourth and fifth instar nymphs and adults decreased to approximately 10 per container. insects were supplied with fresh leaves of cherry, prunus yedoensis matsumura, twice a week. eggs laid by adults were collected every other day and were used for the experiments. capitulum removal by ants experiments were performed with the two-level containers, which were used for the maintenance of the ants. in the first experiment, three colonies of p. punctatus were kept under room conditions at approximately 20-25 °c. ants were provided a sufficient food source in addition to stickinsect eggs. specifically, dried bloodworm and insect jelly were placed on the upper containers once a week. water was also provided once a week. in every ant colony, 50-100 eggs of p. illepidus within 48 h of oviposition were placed. one week later, all the eggs were collected; eggs without capitula and those moved to the lower containers were counted. for the next experiment, five colonies of p. punctatus were used. the experimental procedure was fundamentally identical to that of the experiment described above. no food source other than stick-insect eggs were provided to ants, and an adequate amount of water was provided once a week. in the upper part of the containers, several tens of p. illepidus eggs were placed within 48 h of oviposition. one week later, eggs without capitula and those on the lower containers were counted. then, all the eggs were removed from the containers. newly laid eggs were placed in the upper containers. the experimental treatment described above was applied repeatedly. the placing and removal of eggs were made twice: the total number of eggs was more than 100 for every colony. the fate of stick-insect eggs transported by ant workers five large colonies of p. punctatus were used in this study. experiments were performed in large plastic containers (31 × 17 cm, 22 cm in height), which were used to maintain ants. throughout the experimental period, dried bloodworm and insect jelly were provided once a week. water was provided through the plaster plates every day. three times a week, 40-80 eggs of p. illepidus were placed on a square aluminum foil of approximately 5 × 5 cm. they were placed on the sandy surface of each container. the aluminum foil square was located 5 cm away from the short wall of the container, on the opposite side of the plaster plate under which ants constructed a nest. the distance between the nearest edges of the plaster plate and aluminum foil was approximately 5 cm. one week later, new eggs were added to the aluminum foil square. all the eggs placed earlier were left as they were. egg-placing lasted until the cumulative egg number became more than 1000 per colony, except for one colony for which the egg number did not reach 1000 because of the death of stick insects. then, p. punctatus colonies were retained for an additional month. after that, all the eggs found in the containers were collected; the localities of the eggs were divided into five categories: sandy surface, under the plaster plate, less than 3 cm in the sand, 3-6 cm in the sand, and deeper than 6 cm in the sand. data analysis percentage data were evaluated using chi-square tests or fisher’s exact probability tests. results capitulum removal by ants stick-insect eggs were presented to p. punctatus that were kept in containers with two levels. when ants were provided diet, capitula were removed from more than 99% of the eggs (fig 2). when ants were not provided diet, capitula were removed from many eggs (81.0%). a significant difference was found in the percentages of eggs without capitula between the experimental treatments with and without food (p < 0.001, fisher’s exact probability test). the percentage of eggs found in the upper containers was 90.5% in the experimental treatment with dried bloodworm and insect jelly in addition to stick-insect eggs. as for the experimental treatment without food, we found 74.9% of eggs, which was significantly lower than in the experimental treatment with food (p < 0.001, fisher’s exact probability test). yoshiyuki toyama, izuru kuroki, keiji nakamura – dispersal of phasmida eggs by ant workers4 the fate of stick-insect eggs transported by ant workers we presented 462-1178 stick-insect eggs to large colonies of p. punctatus in this experiment. when workers carried the stick-insect eggs, they bit the capitula (fig 3). some eggs were transported into the nests under the plaster plates. however, we also observed that many eggs were not brought into the ant nests; capitula were removed from the eggs on the sandy surface. as a whole, only 50 of 4926 eggs were lost a month after the final egg placement (table 1). furthermore, the number of dead eggs (i.e., rotted eggs and debris of eaten eggs) was also small. the total percentage of eggs without a capitulum was 98.9% of the 4855 intact eggs. the results were almost identical among the five colonies. no eggs were found on the aluminum foil pieces where the eggs had been originally placed. many eggs were left on the sandy surface (table 2). under the plaster plates, a total of 629 eggs (13.0%) were found. only 246 eggs (5.1%) were buried into the sand. most of the buried eggs were found at depths of less than 3 cm from the surface. seventeen eggs were found at depths from 3 to 6 cm; only one egg was buried deeper than 6 cm in the sand. results were similar among colonies. discussion interspecific relation between ants and stick insects workers of ants are attracted by capitula of stickinsect eggs, and transfer the eggs to nest structures (compton & ware, 1991; windsor et al., 1996; stanton et al., 2015). the results obtained from this experiment reveal that workers of p. punctatus intensively remove capitula of p. illepidus eggs (table 1, fig 2). furthermore, ants transported stick-insect eggs from their place of origin (table 2). from these results, it can be inferred that p. punctatus responses to p. illepidus eggs are similar to those of many ant species to elaiosomebearing plant seeds and capitula of several other stick insects. adults of p. illepidus have no wings, similar to many other stick insects, and they do not move often. eggs are scattered on the ground by female adults on trees. consequently, egg transportation by ants can be an adaptive strategy for slow-moving p. illepidus to disperse progeny, as seems to be true also for other stick insects (compton & ware, 1991; hughes & westoby, 1992b; windsor et al., 1996). capitula as a diet for ants from elaiosomes of plant seeds, ants can obtain essential nutrition such as lipids and nitrogen-rich amino acids (higashi et al., 1989; lanza et al., 1992; gamman et al., 2005; boulay et al., 2006; fischer et al., 2008). capitula of stick-insect eggs also present nutrients such as several fatty acids. (stanton et al., 2015). the present study shows that ants removed capitula from p. illepidus eggs even when a sufficient food source other than eggs was provided (fig 2). this result suggests that p. punctatus can obtain necessary nutrition from the capitula of p. illepidus eggs. fig 2. percentage of phraortes illepidus eggs from which capitula were removed by pristomyrmex punctatus. closed and open columns respectively show percentages of intact eggs and eggs from which capitula were removed by ants. the percentages of eggs transferred to lower containers are also presented. table 1. capitula removal of phraortes illepidus eggs provided for pristomyrmex punctatus. colony number initial number of eggs number of lost eggs number of rotted eggs number of eggs eaten by ants number of intact eggs percentage of intact eggs without capitula colony 1 1085 25 4 1 1055 96,1 colony 2 1178 10 0 2 1166 99,7 colony 3 1099 7 0 3 1089 100,0 colony 4 1102 1 5 2 1094 99,4 colony 5 462 7 1 3 451 99,3 total 4926 50 10 11 4855 98,9 sociobiology 68(4): e7194 (december, 2021) 5 competition between establishing seedlings (higashi et al., 1989; hughes, 1991). by contrast, stick-insect nymphs that emerge in active nests might be in danger from the ants, and the danger is mitigated by the nest moving habits of many ant species (hughes & westoby, 1992b). specifically, stick insects are not in active ant nests at the time of hatching. this study revealed some eggs of p. illepidus in the nests of p. punctatus constructed under plaster plates (table 2). this result suggests that the survival rate of nymphs decreases if hatching occurs in the nests. however, p. punctatus has no permanent nest, and it moves its nest frequently (mizutani, 1982, 1984; tsuji, 1988b). furthermore, p. illepidus has a long embryonic period that may last from seven months to more than one year according to the timing of oviposition (s. matsumoto, personal communication, march 19, 2019). therefore, it is likely that the first-instar nymphs of p. illepidus from the eggs stored in the nests can safely reach the ground surface after p. punctatus has already moved its nest to another location. depth of p. illepidus eggs buried by p. punctatus if eggs are buried deep in ant nests, first instar nymphs of stick insects can have difficulty reaching the ground surface. hughes and westoby (1992b) showed that first instar nymphs of p. wilkinsoni could reach the surface from only 11% of eggs experimentally buried at 12 cm depth. consequently, ant species that do not bury eggs deep in the soil can be effective partners for stick insects. in the present study, the percentage of eggs buried in the sand was small (table 2). furthermore, most of the buried eggs were found near the surface, and only one of the 246 buried eggs was found at a depth greater than 6 cm from the surface. although we did not observe stick insect hatching, nymphs are likely to reach the host plants from p. punctatus eggs buried near the soil surface. horizontal transfer of stick-insect eggs on the ground surface germination of elaiosome-bearing seeds in the ant nets is regarded as adaptive for plants. specifically, nests can prevent predators from finding seeds and can protect seeds fig 3. photograph of pristomyrmex punctatus workers transferring phraortes illepidus eggs. table 2. distribution of phraortes illepidus eggs provided for pristomyrmex punctatus. colony number number of intact eggs number of eggs percentage of eggs on the sandy surface eggs on the sandy surface eggs under the plaster plates eggs buried less than 3 cm in the sand eggs buried 3-6 cm in the sand eggs buried deeper than 6 cm in the sand colony 1 1055 942 24 87 2 0 89,3 colony 2 1166 1050 93 23 0 0 90,1 colony 3 1089 751 255 71 11 1 69,0 colony 4 1094 909 176 10 0 0 83,0 colony 5 451 329 81 37 4 0 72,9 total 4855 3981 629 228 17 1 82,0 the percentage of eggs from which capitula were removed by ants was higher with supplemented than without supplemented food (fig 2). we expected the contrary, that starved ants would remove capitula more intensively. the average numbers of eggs per colony were, respectively, 77 and 170 in the experimental treatment with and without supplemented food. the colony sizes of the two experimental treatments were similar. the difference in egg numbers might affect the percentage of capitulum removal. a significant difference in the percentage of eggs transported to lower containers was found when ants were with and without food. specifically, ants not provided dried bloodworm and insect jelly intensively transported eggs to the lower containers. consequently, it might be possible that starved ants tend to store capitulum-bearing eggs of stick insects as a future diet. possible effects of p. punctatus nest-moving habits for plants, the nest-moving habits of ants affect the germination and survival of the progeny. the frequent relocation of ant nest entrances might result in the lack of nutritional enrichment, although it enables seeds to reduce yoshiyuki toyama, izuru kuroki, keiji nakamura – dispersal of phasmida eggs by ant workers6 from fire (o’dowd & hay, 1980; heithaus, 1981; beattie & culver 1981; bond & slingsby, 1984; bond & breytenbach, 1985; hughes & westoby, 1992a; ohkawara & higashi, 1994; espadaler & gómez, 1997). germination rates increase because of high available nutrients and aeration within the ant nests (horvitz, 1981; zettler et al., 2001; passos & oliveira, 2002). for stick insects, however, hatching in the ant nests is not adaptive, as discussed above. in c. bicuspis, many eggs are not stored in the nests but are instead transported horizontally on the ground surface (windsor et al., 1996). consequently, first instar nymphs that hatched out from eggs outside the ant nests can decrease the predation risk by ants. furthermore, the ground surface transfer of eggs decreases the risks of parasitism by egg parasitoids (windsor et al., 1996). most p. illepidus eggs were not found in ant nests under plaster plates in the study described herein. specifically, 82% of the intact eggs were found on the sandy surface (table 2). consequently, p. punctatus transfers eggs mainly horizontally on the surface, enabling first instar nymphs of p. illepidus to reach host plants safely without being attacked by ant workers. furthermore, eggs can escape from egg parasitoids by transfer from the original location. results suggest that aboveground transfer by p. punctatus is favorable also for p. illepidus eggs. conclusion the present study revealed that p. punctatus are attracted by capitula of p. illepidus eggs and transport the eggs from their place of origin. ants transport eggs mainly horizontally on the surface, which can decrease the predation risk of stick-insect hatchlings. although some eggs were stored in the ant nests or buried in the sand, first-instar nymphs can presumably reach the host trees safely because of the nestmoving habits of p. punctatus and the depth of buried eggs. based on the results, we infer that p. punctatus can be a good partner of p. illepidus: ants disperse stink-insect eggs safely, probably in exchange for some nutrition from the capitula of eggs. competing interests the authors declare no conflict of interest related to this report or the study it describes. authors’ contributions kn: conceptualization, methodology, writing yt: conceptualization, methodology, investigation, writing ik: investigation references beattie, a.j. 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(1988a). obligate parthenogenesis and reproductive division of labor in the japanese queenless ant pristomyrmex pungens. comparison of intranidal and extranidal workers. behavioral ecology and sociobiology, 23: 247-255. doi: 10.10 07/bf00302947. tsuji k. (1988b). nest relocations in the japanese queenless ant pristomyrmex pungens mayr (hymenoptera: formicidae). insectes sociaux, 35: 321-340. doi: 10.1007/bf02225809. windsor d.m., trapnell d.w. & amat g. (1996). the egg capitulum of a neotropical walkingstick, calynda biscuspis, induces aboveground egg dispersal by the ponerine ant, ectatomma ruidum. journal of insect behavior, 9: 353-367. doi: 10.1007/bf02214015. zettler, j.a., spira, t.p. & allen, c.r. (2001). yellow jackets (vespula spp.) disperse trillium (spp.) seeds in eastern north america. american midland naturalist, 146: 444-446. doi: 10.1674/0003-0031(2001)146[0444:yjvsdt]2.0.co;2. 595 contributions to the knowledge of the myrmecophilous pselaphines (coleoptera, staphylinidae, pselaphinae) from china. ix. a redefinition of the genus anaclasiger, with a description of a second species associated with prenolepis sphingthoraxa (hymenoptera, formicidae) by zi-wei yin1, bao-ping huang2 & li-zhen li1,3 abstract the genus anaclasiger raffray is redefined and its taxonomic placement briefly discussed, based on the discovery of a second species of the genus, a. zhudaiae sp. n., collected in association with the ant prenolepis sphingthoraxa from south china. the new species is described and illustrated. an identification key to species of anaclasiger is provided. introduction the monotypic genus anaclasiger raffray was established (raffray, 1890a) for a unique species, a. sinuaticollis raffray, from singapore. subsequently, the species was recorded from taiwan (raffray, 1914) and malaysia (nomura & idris, 2005). recently, a. sinuaticollis was redescribed (nomura et al., 2006) in detail with sem illustrations, and was reported from thailand and japan in the same paper. in 2010, the junior author, bao-ping huang, conducted several investigations on the local entomological fauna of shenzhen city, during which a small series of clavigerine beetles were collected from several colonies of the ant prenolepis sphingthoraxa zhou et zheng. among the collected specimens, some proved to represent an undescribed genus and species, recently named (yin et al. 2011) sinoclavigerodes yalianae, and a single individual belonged to an unknown species of the genus anaclasiger. 1lab of environmental entomolog y, department of biolog y, college of life and environmental sciences, shanghai normal university, 100 guilin road, shanghai, 200234, p. r. china. e-mail: yin_ziwei@yahoo.com 2 room 301, 88 bu-wei village, jingtian rd., futian district, shenzhen, 518034, guangdong province, p. r. china. 3corresponding author. e-mail: pselaphinae@gmail.com 596 sociobiolog y vol. 59, no. 3, 2012 the purpose of this paper is to redefine the genus anaclasiger and briefly discuss its taxonomic placement, to describe the new species with illustrations of its major diagnostic features, and to provide an identification key to species of the genus anaclasiger. material and methods a slash (/) is used to separate lines on the same label, a double slash (//) is used to separate different labels. the foveal terminolog y follows chandler (2001), except for using ‘ventrite’ instead of ‘sternite’. the following acronyms are used in the text: al–length of the abdomen; aw–maximum width of the abdomen, measured in dorsal view; bl–length of the body (= hl + pl + el + al); el–length of the elytra, measured along the sutural line; ew–maximum width of the elytra; hl–length of the head, measured from the anterior clypeal margin to the occipital constriction; hw–width of the head across eyes; pl–length of the pronotum along midline; pw–maximum width of the pronotum. the holotype is deposited in the insect collections of shanghai normal university. taxonomy genus anaclasiger raffray anaclasiger raffray, 1890a: 165 type species anaclasiger sinuaticollis raffray (subseq. mon. by raffray, 1890b: 216). diagnosis head cylindrical, frontal rostrum strongly projecting ; antennae formed by four antennomeres, antennomeres i–ii short, and antennomeres iii–iv long ; antennomeres iv rounded apically, lacking setose truncate cavity. pronotum lacking foveae; with distinct lateral antebasal constriction. elytra lacking basal foveae; discal striae present; lacking setal trichome at posterolateral margins. 597zi-wei yin et al.— the myrmecophilous genus anaclasiger redescription length 1.5–2.1 mm. head with prominent frontal rostrum, lacking antennal tubercles; nude vertexal foveae widely separated; antennae with four antennomeres, antennomeres i–ii short and subequal in length, iii–iv elongate; antennomeres iii straight, iv much longer than iii, curved laterally, with dense apical setae; gula broadly and shallowly swollen, with two gular foveae moderately separated. pronotum with thick discal setae in anterior half; mediobasal impression large and shallow; distinct lateral antebasal constriction covered with rows of long setae; lacking lateral antebasal foveae; lacking lateral procoxal foveae. elytra lacking basal fovea; discal striae extending from humeral angle and extending to at least elytral midpoint. mesoand metathorax lacking foveae; metathorax strongly convex medially; with broad posterior margin. legs short to elongate; tarsomeres i–ii very short, iii elongate; tarsomeres i often difficult to see. abdomen with tergites iv–vi fused into composite plate, with deep transverse basal impression, deep basolateral foveae in impression; thick setal trichome at lateral ends of impression; paratergites iv–vi well demarcated, iv with linear tufts of setae at base; sternites iv–v subequal in length, vi– vii short; sternite iv with large mediobasal foveae. aedeagus asymmetric, median lobe broad and enlongate; with sclerotized endophallus; large dorsal diaphragm opening oval. comparative notes. this is placed as a unique genus in the subtribe clavigerodina by the head being separated from the neck by an occipital carina, the elytra covered with short, thin and sparse setae arranged without order, and the composite tergite lacking a laterally well-defined furrow in the large transverse basal depression. anaclasiger shares only with microdiartiger sawada the short antennomeres i–ii and long antennomeres iii–iv. this genus also shares with sinoclavigerodes yin et al. the lack of the setal trichome at the elytral posterolateral margins. anaclasiger can be readily separated from all asian clavigerodine genera by the terminal antennomere being rounded apically (not truncate) and lacking an apical setose cavity, and the pronotum with a distinct lateral antebasal constriction. 598 sociobiolog y vol. 59, no. 3, 2012 fig. 1. dorsal habitus of anaclasiger sinuaticollis. scale: 0.3 mm. 599zi-wei yin et al.— the myrmecophilous genus anaclasiger dorsal habitus of anaclasiger zhudaiae, holotype. scale: 0.5 mm. 600 sociobiolog y vol. 59, no. 3, 2012 anaclasiger zhudaiae yin and huang, new species (figs. 2-9) type material. holotype female, labeled ‘china: guangdong prov. / shenzhen, yantian dist. / yantian station / 30.i.2010, b.p. huang leg. // [red] holotype / anaclasiger zhudaiae sp. n. / yin et huang det 2012, snuc'. description. female. body (fig. 2) length 2.06 mm; reddish brown, mouthparts and tarsi lighter. head (fig. 3) longer than wide, hl 0.37 mm, hw 0.27 mm; with sparse short setae dorsally; eyes each composed of about figs. 3-8. details of anaclasiger zhudaiae. 3. head and pronotum; 4. right antenna; 5. right elytron and tergal base; 6. tergite viii; 7. sternite viii; 8. female genitalia. scales: a–c = 0.3 mm; d–f = 0.1 mm. 601zi-wei yin et al.— the myrmecophilous genus anaclasiger 25 facets; length of antennomeres iii 0.22 mm, iv 0.61 mm, iii / iv = 1: 2.8 (fig. 4); antennomeres iv rounded apically, with dense long apical sensory setae. pronotum (fig. 3) slightly longer than wide, pl 0.42 mm, pw 0.32 mm; lacking punctation; lateral antebasal constriction formed by distinct marginal sulci; small mediobasal pit in large impression. elytra (fig. 5) wider than long, el 0.55 mm, ew 0.76 mm; sparsely with short setae dorsally, with row of long thick setae at posterolateral margins; discal striae extending from humeral angle to 2/3 of elytral length. composite plate strongly convex dorsally, with long discal setae and short setae around disc. tergite viii (fig. 6) transverse, roundly narrowed posteriorly, with thick setae. sternite viii (fig. 7) transverse, flat at posterior margin. female genitalia complex (fig. 8) well sclerotized. male. unknown. host ant prenolepis sphingthoraxa zhou and zheng, 1998 (det. by the junior author). this is the first host record for anaclasiger. biology. the single specimen was collected from the ant colony nesting under a rock near a stream (fig. 9). distribution guangdong province, south china. remarks the new species can be readily separated from a. sinuaticollis by the much longer antennae and legs, antennomeres iv slightly curving laterally at the apical half, elytra with sparse long and thick setae at the anterolateral margins, and the elytral discal striae extending past the elytral midpoint. anaclasiger sinuaticollis has relatively short antennae and legs, antennomeres iv are abruptly twisted laterally in the apical half, the elytra lack long thick setae at the anterolateral margins, and the discal striae end at the elytral midpoint. the habitat of the new species is shown in fig 9. etymology the specific name is dedicated to the junior author’s grandmother, zhudai zhang, to celebrate her coming 85th birthday. 602 sociobiolog y vol. 59, no. 3, 2012 key to species of anaclasiger raffray 1.antennomeres iii–iv relatively shorter, iv abruptly curved laterally at middle (nomura, 2006: 288, fig. 2-f). pronotum barely wider than long (nomura et al., 2006: 288, fig. 2-g). elytra lack row of long thick setae at anterolateral margins (nomura, 2006: 289, fig. 3-e). (singapore, thailand, malaysia, china: taiwan, japan) ............................. a. sinuaticollis raffray antennomeres iii–iv much longer, iv evenly curved laterally through entire length (fig. 4). pronotum slightly longer than wide (fig. 3). elytra with rows of long thick setae at posterolateral margins (fig. 5). (china: guangdong ) ..................................a. zhudaiae yin & huang, new species acknowledgments we thank shûhei nomura (tokyo, japan) for providing specimens of anaclasiger sinuaticollis for the comparative study. donald s. chandler (durham, u.s.a) critically read the manuscript and corrected the english. the present study is supported by the national natural science foundation of china (no. 31172134), and by shanghai normal university (no. sk01242). fig . 9. habitat of anaclasiger zhudaiae. 603zi-wei yin et al.— the myrmecophilous genus anaclasiger references chandler d.s. 2001. biolog y, morpholog y, and systematics of the ant-like litter beetles of australia (coleoptera: staphylinidae: pselaphinae). memoirs on entomolog y international 15: 1–560. nomura s. & idris a.g. 2005. faunistic notes on the pselaphine species of the supertribes giniaceritae, pselaphitae and clavigeritae from malaysia and singapore (coleoptera: staphylindae: pselaphinae). serangga 10(1-2): 1–36. nomura s., watana s. & idris a.g. 2006. a redescription and new distributional records of the clavigerine species anaclasiger sinuaticollis (coleoptera, staphylinidae, pselaphinae). elytra 34(2): 285–292. raffray a. 1890a. étude sur les psélaphides. v. tableaux synoptiques. notes et synonymie. revue d’entomologie 9: 81–172. raffray a. 1890b. étude sur les psélaphides. vi. diagnoses des espèces nouvelles sur lesquelles sont fondés des genres nouveaux. revue d’entomologie 9: 193–219, pls 2, 3. raffray a. 1914. h. sauter’s formosa-ausbeute. pselaphidae (col.) ii. supplementa entomologica 3: 1–5. yin z.w., hlaváč p. and zhao m.j. 2011. contributions to the knowledge of the myrmecophilous pselaphines (coleoptera, staphylinidae, pselaphinae) from china. v. sinoclavigerodes yalianae gen. et sp. nov. (clavigeritae) associated with anoplolepis gracilipes (formicidae). sociobiolog y 57(1): 1–9. erratum zi-wei yin et al. (2011) contributions to the knowledge of the myrmecophilous pselaphines (coleoptera, staphylinidae, pselaphinae) from china. v. sinoclavigerodes yalianae gen. et sp. nov. (clavigeritae) associated with anoplolepis gracilipes (formicidae). sociobiolog y 57(1): 1–9. the host ant of sinoclavigerodes yalianae was figured, but erroneously identified as anoplolepis gracilipes. the correct specific name should be prenolepis sphingthoraxa zhou & zheng. doi: 10.13102/sociobiology.v61i4.490-493sociobiology 61(4): 490-493 (2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 stingless bees (hymenoptera: apidae: meliponini) attracted to animal carcasses in the brazilian dry forest and implications for forensic entomology we santos1, lt carneiro2, acf alves1, aj creão-duarte1, cf martins1 introduction bees are known for foraging majorly on floral resources as pollen, nectar and oil (roubik, 1989; wcislo & cane, 1996), acting as pollinators of angiosperms. however, many bee species forage on other nutritional sources, as hemipteran’s honeydews (camargo & pedro, 2002), and on materials to be used in their nest building. mud, feces, plant parts, exudates and animal carcasses can be incorporated by bees to the material used in the construction and sealing of the nest (wille, 1983; baumgartner & roubik, 1989; noll et al., 1996). amongst the bees, the neotropical perennial social bees (meliponini) have been the most recorded on animal carcass or flesh bait (wille, 1962; baumgartner & roubik, 1989; silveira et al., 2005). stingless bees comprise over 500 species (michener, 2013), mainly generalist pollen and abstract the association of stingless bees with pig carcasses exposed in a brazilian dry forest area were examined. modified shannon traps were settled together to collect these insects during two seasons (dry and rainy). 564 bees were collected, belonging to three tribes and nine species. the majority of bees (75.5%) was collected during the dry season, and partamona seridoensis pedro & camargo (32.8%) and trigonisca sp. (20.9%) were the most abundant species. five decomposition stages were recognized, being the bloated, active and advanced decay the most attractive to the bees. considerations about seasonal foraging and use of bees in the forensic entomology scope are presented. sociobiology an international journal on social insects 1 universidade federal da paraíba (ufpb), joão pessoa, pb, brazil 2 universidade de são paulo (usp), são paulo, sp, brazil article history edited by cândida maria l. aguiar, uefs, brazil received 29 september 2014 initial acceptance 31 october 2014 final acceptance 13 november 2014 keywords caatinga, partamona seridoensis, trigonisca. corresponding author wellington emanuel dos santos programa de pós-graduação em ciências biológicas (zoologia), departamento de sistemática e ecologia, universidade federal da paraíba, ufpb, 58059-900 joão pessoa, pb, brazil e-mail: well-bio@hotmail.com nectar consumers. however three species are known by necrophagous obligate habits: trigona crassipes (fabricius), trigona hypogea silvestri and trigona necrophaga camargo & roubik (roubik, 1982; camargo & roubik, 1991). their corbiculae are not adjusted to transport pollen (camargo & roubik, 1991) and the workers gather animal flesh as protein resource which is transported, processed and stored in the food pots (noll et al., 1996). most species attracted to animal carcasses probably visit them only for nest materials, liquid exudates, or salts, rather than for protein source (baumgartner & roubik, 1989). the exploitation of carcasses by stingless bees appears to be common; however few studies have reported this behavior and their forensic use. several entomological surveys with baited traps and animal carcasses conducted across the andes (baumgartner & roubik, 1989; wolff et al., 2001), lowland tropical forests (cornaby, 1974; roubik, research article bees sociobiology 61(4): 490-493 (2014) 491 1982), amazon forest (silveira et al., 2005), atlantic forest (farias, 2012) and urban areas in the southeastern brazil (gomes et al., 2007), recorded many bee species foraging on these ephemeral sources. nevertheless, no information has been obtained from the brazilian seasonally dry tropical forest (caatinga), generally poorly investigated about the ecology of its insect fauna (aguiar & martins, 1997; vasconcellos et al., 2010; alves et al., 2014; santos et al., 2014). throughout the animal decomposition, a succession usually occurs as insect species exhibit associations to the decomposition stages providing better supply for their offspring (smith, 1986). this process can be useful in determining the postmortem interval (pmi) in a forensic approach (goff & flynn, 1991). furthermore, endemic species collected on corpses found in any environment can be the clues to know where the deaths took place (benecke, 1998). here we present a list of the carrion-foraging bees on pig carcasses in a seasonally dry tropical forest from the northeastern brazil. we asked: (1) do bees seasonally explore carrion in this environment? (2) how abundant were the bees throughout the carcass decomposition stages? we also briefly discuss about the concernment on bees as a tool in a forensic approach. material and methods the study was carried out at the private reserve for the environmental inheritance fazenda almas, são josé dos cordeiros, pb, brazil (07°28’19” s, 36°53’40” w). the reserve covers 3,505 ha (600-720 m a.s.l.) and the climate of the region is defined as warm semi-arid (bsh – köppen climate classification). the vegetation is highly deciduous during the dry season and ranges from open to dense arboreal. the soil is sandy and topographically irregular, with inselbergs and rocky outcrops (vasconcellos et al., 2010). we sampled the bees with a usual method for forensic entomology research (alves et al., 2014). two pig carcasses with ~15 kg in weight were placed nearly 50 m from each other in the dry season (october 2010) and in the rainy season (february 2011). the animals were slaughtered by a single gunshot to the head shot by a forensic examiner of the instituto de polícia científica da paraíba (ipc/pb). each carcass was exposed into an iron cage (3x10 cm mesh opening) with a modified shannon trap over it. a collecting tube containing 70% alcohol was connected to the top of each trap. the sampling was conducted daily until the end of the decomposition (15 days) and its stages were classified using the descriptions and terminology adopted by goff (2009). the dry stage (i.e. only the bones and hair retained) was not observed. the average temperature and relative humidity recorded along the dry and rainy period were 26.9±1.8°c / 63.8±19.4% and 24.5±1.3°c / 78.5±11.7%, respectively. voucher specimens were deposited in the entomological collection of the departamento de sistemática e ecologia, universidade federal da paraíba (dsec/ufpb). c.f. martins identified the collected bee specimens using keys and the reference collection of the same institution (dsec/ufpb). a license was granted by the comitê de ética no uso de animais (ceua/ufpb) for the study. results and discussion nine bee species in a total of 564 individuals attracted by the carcasses were collected in both seasons and the stingless bees (meliponini) were by far the most frequent (89.7%) among them. partamona seridoensis pedro & camargo was the most abundant species (n=185), followed by trigonisca sp. (n=118), plebeia flavocincta (cockerell) (n=83) and trigonisca pediculana (fabricius) (n=74) (table 1). the bees were more abundant in the dry season (75.5%) rather than in the rainy season. moreover, the four most frequent stingless bee species in the whole sampling were more recorded in the dry period indicating a remarkable seasonal tendency. on the other hand, the africanized honey bee apis mellifera linnaeus was more abundant in the rainy rather than in the dry period (table 1). five stages of decomposition were perceived: fresh, during 2.0±0 days; bloated, 2.5±0.5 days; active decay, 2.2±0.43 days; advanced decay, 3.2±1.3 days; and postdecay, 6.0±1.22 days. in general, the majority of the bees were collected in the bloated, active and advanced decay stages (table 1), when the liquid parts are available, the process of putrefaction increases, and a notable decomposition stink is emitted (goff, 2009). the exceptions were p. seridoensis, being more frequent in postdecay along the dry period, and p. flavocincta, the only species present in the fresh stage. in the caatinga ecosystem, where water is a real limiting resource, it is understandable that the bees are present in all stages of decomposition, even though showing differences in richness and diversity. payne and mason (1971) recorded bees on carrion only while fluids were present in the south caroline, usa. the authors observed a behavior of sucking up the foulsmelling juices. gomes et al. (2007) found bees mainly in the initial decomposition stages on pig carrions at southeastern brazil as well. surveys carried out in the brazilian seasonally tropical dry forest sampled low species richness for bees compared to other environments, e.g. cerrado (brazilian savanna) and tropical rain forest; however a high percentage of endemic species has been reported for the region (aguiar & martins, 1997; zanella, 2000). among the species we recorded, p. seridoensis is endemic to caatinga areas (zanella, 2000). thus, in the forensic entomology scope, whether these bees were found on a corpse in a different area, either hooked or dead inside the clothes, this would indicate a displacement from the place of death (benecke, 1998). farias (2012) collected 13 bee species on pig carcasses in an atlantic rainforest area localized in paraíba state as well; only p. flavocincta was found in common with the present study. this we santos, lt carneiro, acf alves, aj creão-duarte, cf martins stingless bees in animal carcasses 492 fact reinforces the application of theses insects in investigations of translocation of bodies. the stingless bee species are abundant in the caatinga (aguiar & martins, 1997). their perennial colonies are established under favorable conditions in the rainy season, with a short period of abundant floral resources and an extensive period of scarcity in the dry season, following the phenological dynamics of floral resources (machado et al., 1997). the increase in plant biomass during the rainy season in the caatinga represents an increase in resources for many insects, including hymenopterans (vasconcellos et al., 2010). on the other hand, a few plant species are able to produce flowers in the dry season (machado et al., 1997). thus, based on the seasonality, we suggest that bees use animal carcasses as a possible source of water, and salts during inhospitable periods. it is noteworthy the presence of small pots containing an aqueous acid substance, besides the typical honey and pollen pots, in a nest of p. seridoensis in the studied area, as observed in some other species of partamona (camargo & pedro, 2003). in another area of caatinga, close to a slaughterhouse, the honey of a colony of melipona asilvai moure presented a pronounced taste of leather in the dry season, and frieseomelitta dispar (moure) collected fat for nest building (c.f. martins, personal observation). as stated before, pasteurization of the honey of most stingless bee species is recommended for prevent contaminations (nogueira-neto, 1997). this is particularly relevant if honey is harvested in the dry season in the caatinga or if colonies are close to areas with animal feces or carcasses, as e.g. slaughterhouses. increasing frequencies of bees in carrions traps and carcasses during dry periods were also observed in amazon forest and atlantic forest areas (silveira et al., 2005; farias, 2012). however, carcasses are rare and unpredictable, and bees compete for them, but probably in a different way, with many other insects, mainly flies and beetles (alves et al., 2014; santos et al., 2014), which makes flesh an expensive resource (noll et al., 1996). table 1. absolute (n) and relative (%) frequencies of bees on pig carcasses along their decomposition stages in the dry season (oct/2010) and rainy (feb/2011) season in a dry tropical forest from the northeastern brazil. fr: fresh; bl: bloated; act: active decay; adv: advanced decay; pd: postdecay. acknowledgements we thank the reviewers for their comments on the manuscript, and the coordenação de aperfeiçoamento de pessoal de nível superior (capes) and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for their financial support. references aguiar, c.m.l. & martins, c.f. 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(1991). systematics and bionomics of the apoid obligate necrophages: the trigona hypogea group (hymenoptera: apidae; meliponinae). biol. j. linn. soc., 44: 13-39. bee species dry rainy total fr bl act adv pd n fr bl act adv pd n n % apidae apinae apini apis mellifera linnaeus 1 1 2 1 36 12 6 55 57 10.2 meliponini melipona asilvai moure 1 4 5 1 5 2 8 13 2.3 partamona seridoensis pedro & camargo 17 12 20 83 132 21 24 8 53 185 32.8 plebeia flavocincta (cockerell) 23 19 7 3 20 72 4 7 11 83 14.7 scaptotrigona aff. tubiba smith 1 1 1 0.2 trigona spinipes (fabricius) 6 1 22 29 1 1 1 3 32 5.7 trigonisca pediculana (fabricius) 27 11 17 12 67 6 1 7 74 13.1 trigonisca sp. 16 20 62 20 118 118 20.9 halictidae halictinae augochlorini augochlora sp. 1 1 1 0.2 total 23 85 51 104 163 426 6 72 43 17 138 564 100 sociobiology 61(4): 490-493 (2014) 493 roubik, d.w. (1989). ecology and natural history of tropical bees. cambridge: university press, x+514 p. santos, w.e., alves, a.c.f. & creão-duarte, a.j. 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(1962). a technique for collecting stingless bees under jungle conditions. insect. soc., 9: 291-293. doi: 10.1007/bf02329898. wille, a. (1983). biology of stingless bees. annu. rev. entomol., 28: 41-64. doi: 10.1146/annurev. en.28.010183.000353. wolff, m., uribe, a., ortiz a. & duque, p. (2001). a preliminary study of forensic entomology in medellín, colombia. forensic sci. int., 120: 53-59. doi: 10.1016/s03790738(01)00422-4. zanella, f.c.v. (2000). the bees of the caatinga (hymenoptera, apoidea, apiformes): a species list and comparative notes regarding their distribution. apidologie, 31: 579-592. doi: 10.1051/apido:2000148. cornaby, b.w. (1974). carrion reduction by animals in contrasting tropical habitats. biotropica, 6: 51-63. doi: 10.2307/2989697. farias, r.c.a.p. (2012). entomofauna associada a carcaças de sus scrofa l. expostas em remanescente de mata atlântica em joão pessoa, pb. tese de doutorado. joão pessoa: ufpb, 112 p. goff, m.l. & flynn, m.m. (1991). determination of postmortem interval by arthropod succession: a case study from the hawaiian islands. j. forensic sci., 36: 607-614. goff, m.l. (2009). early post-mortem changes and stages of decomposition in exposed cadavers. exp. appl. acarol., 49: 21-36. doi: 10.1007/s10493-009-9284-9. gomes, l., gomes, g., oliveira, h.g., junior, j.j.m., desuo i.c., queiroz, m.m.c., giannotti, e. & zuben, c.j.v. (2007). occurrence of hymenoptera on sus scrofa carcasses during summer and winter seasons in southeastern brazil. rev. bras. entomol., 51: 394-396. doi: 10.1590/s008556262007000300019. machado, i.c.s., barros, l.m. & sampaio, e.v.s.b. (1997). phenology of catinga species at serra talhada, pe, northeastern brazil. biotropica, 29: 57-68. doi: 10.1111/ j.1744-7429.1997.tb00006.x. michener, c.d. (2013). the meliponini. in: vit, p., pedro, s.r.m. & roubik, d.w. (eds.), pot-honey: a legacy of stingless bees (pp. 3-17). new york: springer, 175 p. nogueira-neto, p. (1997). vida e criação de abelhas indígenas sem ferrão. são paulo: editora nogueirapis, 446 p. noll, f.b., zucchi, r., jorge, j.a. & mateus, s. (1996). food collection and maturation in the necrophagous stingless bee, trigona hypogea (hymenoptera: meliponinae). j. kansas entomol. soc., 69: 287-293. payne, j.a. & mason, w.r.m. (1971). hymenoptera associated with pig carrion. p. entomol. soc. wash., 73: 132-141. roubik, d.w. (1982). obligate necrophagy in a social bee. science, 217: 1059-1060. doi: 10.1126/science.217.4564.1059. doi: 10.13102/sociobiology.v66i4.4892sociobiology 66(4): 606-609 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 with the growing need for food supplementation of swarms, especially in periods of reduced food supply in the wild, it is important to provide technological resources directly to producers, so that they can produce on their property the food that bees need. research reports the importance of nutritional supplementation, especially protein, when pollen sources in nature suffer significant reductions, colonies reduce their productivity due to their weakening (saffari et al., 2010; morais et al., 2013; paiva et al., 2019). however, the fermentation of this food becomes a fundamental factor in the animal’s acceptance and consumption, since this microbiological process makes the food closer to the beebread: more palatable and attractive (ellis & hayes jr, 2009; li et al., 2012). for the fermentation process to occur satisfactorily, the control of factors such as temperature guarantee the performance of the fermenting microorganisms (especially abstract considering the importance of offering food supplementation to the swarms during dearth periods, we developed in this project an artisanal incubator for fermentation of supplementary protein diets for apis mellifera bees, obtaining a fresh, nutritious and palatable product, made on the property, thus facilitating access to the beekeeper to this resource. sociobiology an international journal on social insects jplm paiva1, c gama1, pm drago2, c barbieri2, mm morais3 article history edited by evandro n. silva, uefs, brazil received 25 november 2019 initial acceptance 05 december 2019 final acceptance 05 december 2019 publication date 30 december 2019 keywords fermentative process, supplementary diet, honey bees, apiculture. corresponding author juliana pereira lisboa m. paiva instituto de ciência e tecnologia universidade federal de são paulo – unifesp rua talim, 330, vila nair cep: 12231-280 são josé dos campos, são paulo, brasil. e-mail: jplbiologia@gmail.com of the genus lactobacillus and pediococcus) that find in the range of 27º to 32°c the ideal point for the fermentation of sugars (sucrose) present in the food, converting them into lactic acid (kristensen et a.l, 2010; kung et al., 2011). this process reduces the ph of the food to 3.8, conserving its nutrients and preventing the attack of spoilage organisms, especially bacteria of the genus clostridium, capable of consuming the amino acids of the food, reducing its palatability and nutritional value (shao et al., 2002; schröder et al., 2013; strauber et al., 2016). thus, the food preserves its desirable characteristics, being consumed and used by the swarms, strengthening them and improving their productive performance, even in periods of reduced floral resources (paiva et al., 2016; paiva et al., 2019). the availability of a simple and inexpensive equipment such as incubator allows the producer to ferment the amount of food needed and store it in perfect condition until it is used by 1 universidade federal de são paulo, instituto de ciência e tecnologia, são josé dos campos-sp, brazil 2 universidade de são paulo, escola de artes, ciências e humanidades, são paulo-sp, brazil 3 universidade federal de são paulo, departamento de ecologia e evolução, diadema-sp, brazil a short note on an artisanal incubator for fermentation of apis mellifera artificial diets short note sociobiology 66(4): 606-609 (december, 2019) 607 fig 1. details of the constitution of the artisanal incubator: a. styrofoam and polyethylene lined box; b. cover with socket and lamp; c. thermostat and socket; d. inner face of box and lid; e. digital thermostat; f. ready incubator; g. diet before fermentation and h. fermented diet. a b c d e f g h jplm paiva, c gama, pm drago, c barbieri, mm morais – artisanal incubator for fermentation of artificial diets608 the swarms. it can prevent nutrient loss and deterioration and offer to the bees a beneficial, fresh, efficient food as it would avoid stocking the product for very long periods, causing diet rejection(carroll et al., 2017).according to this, the objective of this work was to create a facilitating artisanal incubator for beekeepers and stingless beekeepers interested in using fermented feed in their bee pasture, enabling the fermentation of bees feed and maintaining the temperature between 27º and 28º, reaching 30ºc. for this purpose, materials such as a styrofoam box (40x40x30cm) were used for the prototype. it was later replaced by a wooden box (35x35x35 cm) with a styrofoam coated lid and thermally insulated with carton packs lined with polyethylene and aluminum layers (used in milk or juice boxes), digital thermostat, porcelain socket; 15w light bulb; parallel wire; switch and socket (figure 1). system operation occurs through the digital thermostat, which has a minimum and maximum temperature setting display with a relay on/off system, as well as a thermal sensor, which reads the ambient temperature, showing it on the display. with set temperatures from 28°c minimum and 30°c maximum, the thermostat allows current to flow to the lamp when the sensor registers temperatures below the maximum. thus, the lamp lights generating heat inside the box. when the sensor records a maximum temperature of 30°c, the system will no longer allow current to pass, so turning off the lamp. after the incubator confection, four capped glass jars containing 100g of soybean meal, corn, sugarcane yeast, egg powder and vitamin-mineral premix feeds were inserted (paiva et al., 2019). 40 ml of 50% sucrose solution and 0.5 mg of microbial inoculum (lactobacillus plantarum and pediococcus sp) were added to each flask to form a paste. these flasks were kept for 7 days, 14 and 21 days at 30ºc, with daily process monitoring until completion. the ph of the rations was measured on the 1st, on the 7th day, on the 14th and 21st day of the experiment, for follow-up. after seven days, we observed that the rations fermented satisfactorily, presenting the formation of bubbles in the mass (due to the release of gases from microbial activity) and ph change (initial: 4.76; 7th day: 3.62; 14th day: 3.67 and 21st day, 3.77), the reduction of which is evidence of acid production by microorganisms from the fermentation of sugars. after the process was completed, 4g of feed were offered to 60 cage-confined worker bees, with 7 repetitions, for consumption of the fermented feed, which was well accepted by animals (paiva et al., 2019). considering the context, we can conclude that the use of affordable and low cost materials has enabled the production of an efficient artisanal incubator, allowing the beekeeper to ferment and conserve supplementary protein food for bees, especially during periods of greatest pollen deficiency in nature. with this resource, the beekeeper can have an effective food available, minimizing losses and helping the swarms to maintain the beekeeping production. acknowledgments the authors would like to thank ms. joanize paiva for comments on the use of the english language. references carroll, m.j., brown, n., goodall, c., downs, a.m., sheenan, t.h. & anderson, k.e. (2017). honey bees preferentially consume freshly-stored pollen. plos one, 12(4), e0175933. doi: 10.1371/journal.pone.0175933 ellis, a.m. & hayes jr, g.w. (2009). an evaluation of fresh versus fermented diets for honey bees (apis mellifera). journal of apicultural research, 48: 215-216. doi: 10.3896/ ibra.1.48.3.11 kung, l. & ranjit, n.k. (2011). the effect of lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage. journal of dairy science, 84: 1149-1155. doi: 10.3168/jds.s0022-0302(01)74575-4 kristensen, n.b., sloth, k.h., højberg, o., spliid, n.h., jensen, c. & thøgersen, r. (2010). effects of microbial inoculants on corn silage fermentation, microbial contents, aerobic stability, and milk production under field conditions. dairy science, 93: 3764-3774. doi: 10.3168/jds.2010-3136 li, c., xu, b., wang, y., feng, q. & yang, w. (2012). effects of dietary crude protein levels on development, antioxidant status, and total midgut protease activity of honey bee (apis mellifera ligustica). apidologie, 43: 576-586. doi: 10.1007/ s13592-012-0126-0 morais, m.m., turcatto, a.p., pereira, r.a., francoy, t.m., guidugli-lazzarini, k.r., gonçalves, l.s., de almeida, j.m. ellis, j.d. & de jong, d. (2013). protein levels and colony development of africanized and european honey bees fed natural and artificial diets. genetics and molecular research, 12: 6915-6922. paiva, j.p.lm., paiva, h.m., esposito, e. & morais, m.m. (2016). “on the effects of artificial feeding on bee colony dynamics: a mathematical model”. plos one, 11 (11): e0167054. doi: 10.1371/journal.pone.0167054 paiva, j.p.l.m., esposito, e., souza, g.i.h., francoy, t.m. & morais, m.m. (2019). effects of ensiling on the quality of protein supplements for honey bees apis mellifera. apidologie, 50: 414-424, doi: 10.1007/s13592-019-00661-4 saffari, a., kevan, p.g. & atkinson, j.l. (2010). palatability and consumption of patty-formulated pollen and pollen substitutes and their effects on honeybee colony performance. journal of apicultural science, 54: 63-69. shao, t., ohba, n., shimojo, m. & masuda, y. (2002). “dynamics of early fermentation of italian ryegrass (lolium multiflorum lam.) silage. asian-australian journal of animal science, 15: 1606-1610. doi: 10.5713/ajas.2002.1606 sociobiology 66(4): 606-609 (december, 2019) 609 schröder, j.j., visser, w., assinck, f.b.t. & velthof, g.l. (2013). effects of short-term nitrogen supply from livestock manures and cover crops on silage maize production and nitrate leaching. soil use and management, 29: 151-160. doi: 10.1111/sum.12027 strauber, h., lucas, r. & kleinsteuber, s. (2016). metabolic and microbial community dynamics during the anaerobic digestion of maize silage in a two-phase process. applied microbiology and biotechnology, 100: 479-491. doi: 10.1007/ s00253-015-6996-0 doi: 10.13102/sociobiology.v62i3.709sociobiology 62(3): 462-466 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nest biology and demography of the fungus-growing ant cyphomyrmex lectus forel (myrmicinae: attini) at a disturbed area located in rio claro-sp, brazil among the fungus-growing ant genera included in the tribe attini (myrmicinae), cyphomyrmex presents peculiar traits (e.g. nest structure and distribution, colony demography, two distinct forms of fungus cultivation: mycelium and “yeast” cultivation) and behavior that have called the attention of researchers (e.g., murakami & higashi, 1997; mueller & wcislo, 1998; schultz et al., 2002; klingenberg et al., 2007; leal et al., 2011; mehdiabadi et al., 2012). for example, along the atlantic seashore (called “restinga”), state of bahia, brazil, colonies of cyphomyrmex transversus were observed nesting under and inside dry coconuts (cocos nucifera l.) on the ground (ramos-lacau et al., 2012). coconuts not only may function as a physical barrier against natural predators of c. transversus, but also provide thermal insulation. according ramos-lacau et al. (2012), this behavior associated to the abstract cyphomyrmex ants are a basal group of small fungus-growers (myrmicinae:attini), which differ profoundly from their most studied relatives atta and acromyrmex. our objective was to investigate the nest biology and demography of the fungus-growing ant cyphomyrmex lectus in a transitional area (savanna-forest) disturbed by an annual fire regime, in southeast brazil. the colonies of c. lectus were located close to each other (mean distance between nests, 3.38 ± 2.75 m). its external nest structure consisted of a single circular nest-entrance hole but without a conspicuous nest mound. nests were relatively simple consisting of a single well-formed chamber and a single gallery connecting to the nest entrance. no relationships were found between ant number per colony and gallery length, as well as chamber volume. nonetheless, we detected an effect of the ant number per colony on chamber depth. in this sense, a deeper chamber could warrant a better protection against fire, natural enemies and offer favorable micro-conditions to yeast culture. the colonies of c. lectus are small (a mean of 70 ± 49.4 individuals/colony). alates, larvae and pupae were found only in one out of eight colonies. a single dealate queen per colony was observed strongly suggesting that this species is monogynous. further studies could evaluate whether (1) nests density differ before and after fire periods, (2) fire occurrence affects the survival and establishment rate of new colonies, as well as (3) the nest microclimatic conditions necessary to yeast culture. sociobiology an international journal on social insects ls ramos-lacau1, psd silva1, jhc delabie2, s lacau1,2, oc bueno3 article history edited by kleber del claro, ufu, brazil received 19 november 2014 initial acceptance 20 february 2015 final acceptance 05 may 2015 keywords fire, colony, establishment. corresponding author lucimeire s. ramos-lacau laboratório de biossistemática animal departamento de ciências exatas e naturais (dcen), universidade estadual do sudoeste da bahia br 415, km 03, s/nº, 45700-000 itapetinga, ba, brazil e-mail: fflormiga@hotmail.com simultaneous occurrence of multiple dealate queens per colony, conferring to the population a greater capacity for population expansion and maintenance of competitive nest densities to c. transversus in harsher environmental conditions, such as those seen in the restinga of brazilian coastal zones. however, little was published in particular on nest ecology and demography of cyphomyrmex lectus (e.g. kempf, 1964), as well as other aspects on its biology. our study provides new information about its nesting distribution, its internal and external nest architecture and the population structure of c. lectus colonies in a transitional zone between the brazilian cerrado and the atlantic forest affected by annual fire regime. fire is considered a major but normal disturbance in the brazilian savanna (cerrado) (miranda et al., 2002), and may influence ant ecology (frizzo et al., 2012) short note 1 laboratório de biossistemática animal, uesb/dcen, itapetinga, ba, brazil 2 laboratório de mirmecologia uesc/cepec-ceplac, centro de pesquisa do cacau, itabuna, ba, brazil 3 centro de estudos dos insetos sociais, ib-unesp, rio claro, sp, brazil sociobiology 62(3): 462-466 (september, 2015) 463 and ant colony establishment (araújo et al., 2003). in details, we investigated (1) the occurrence of other cyphomyrmex species around the nests of c. lectus and (2) described the respective nest internal and external architecture of c. lectus colonies. secondly, we evaluated whether colony size of c. lectus affects positively its nest internal architecture. this was done correlating ant number per colony with gallery length, nest chamber depth and chamber volume. therefore, we discuss about what factors can be favoring colony survival of c. lectus in an area on fire annual regimes based on nest structure. our study was carried out in an area influenced by an annual fire regime and located at the floresta estadual edmundo navarro de andrade (feena), campus of unesp (universidade estadual paulista), rio claro, sp, brazil (22°23’44”s 47°32’03”w). as cited above, feena is considered a transitional zone between the brazilian savanna (cerrado) and the atlantic forest (pirani et al., 2005). the vegetation is composed by several grassland species with scattered shrubs, typical vegetation of brazilian savanna (see oliveira-filho & ratter, 2002; formore information about vegetation physiognomies and woody flora of the cerrado bioma). mean annual precipitation is 1534 mm (dry season extends from april to september and rainy season from october to march) and mean annual temperature is 20.6°c (pirani et al., 2005). in june 2005, immediately after a bushfire, cyphomyrmex colonies were located and two workers per colony were collected to confirm the identification of the species. vouchers were deposited in the centro de estudos de insetos sociais (ceis), unesp, rio claro – sp, and also in the cpdc collection of the laboratório de mirmecologia (ceplac / cepec / secen, ilhéus-ba), brazil. eight nests of cyphomyrmex lectus were thus excavated (five in june and three in october) aiming to study the internal nest architecture in seven of them and all the ants were collected for the demography survey. in each colony studied for nest structure, five biological traits were measured, such as (1) nest distance between colonies, (2) diameter of nest role entrance, (3) principal gallery length (i.e., length from nest role entrance to next superficial gallery), (4) chamber depth, (5) chamber dimensions, i.e. maximum width and height in order to calculate chamber volume. single t tests were used to verify the differences between colonies regarding diameter of nest-entrance hole, gallery length, chamber depth, chamber volume and number of individuals. single regressions were performed to evaluate the effect of ant number per colony on gallery length, nest chamber volume and chamber depth. the analyses were developed using the software bioestat (ayres et al., 2007). all the average values are informed followed by standard deviation (sd). in the sampled locality, the colonies of c. lectus were aggregated and located relatively close to each other, with a mean distance between them of 3.38 ± 2.75 m. the external nest structure of the ant consisted of a single circular nestentrance hole with mean diameter of 0.21 ± 0.06 cm but without a well-formed nest mound on the ground surface. the diameter of nest-entrance hole varied significantly between colonies (t test = 8.22; p < 0.001). the internal structure was formed by a single oval chamber connected to the outside via a single gallery. a yeast-like fungi forming a spongy mass was observed and disposed at the bottom of the chamber. no dedicated garbage chamber was observed in all studied colonies. gallery length varied from 8.5 to 17 cm with mean length of 11 ± 3 cm. mean chamber depth varied from 2 to 3.8 cm reaching a mean of 3.05 ± 0.6 cm. chamber volume varied from 8.2 to 54.3 cm³ with average value of 27.5 ± 15.1 cm³. these measured nest attributes varied significantly between colonies (i.e. gallery length: t test = 9.7 and p < 0.001; chamber depth: t test = 13.4 and p < 0.001; and chamber volume: t test = 4.8 and p = 0.003). two other species of the cyphomyrmex genus (i.e. c. strigatus and c. rimosus) were simultaneously observed in the area with similar patterns of external nest architecture of c. lectus. for c. lectus colonies, the mean number of workers was of 70 ± 49.4 individuals per colony. no significant effect of total number of ant individuals (i.e., colony size) on both gallery length (f= 0.059, g.l. = 1; p = 0.83; fig 1a) and nest chamber volume (f= 0.22, g.l. = 1; p = 0.65; fig 1b) was observed but the nest chamber depth increased significantly with increasing the number of ants per colony (f= 7.88, g.l. = 1; p = 0.04; fig 1c). ant number per colony explained 54% of the observed variation regarding the chamber depth. as observed to all studied nest attributes, the number of ant individuals also varied significantly between colonies (t test = 4.83 and p = 0.003). alates (young reproductive females and males), larvae and pupae were found only in one colony (sampled in october). this colony contained eight females, two males, three larvae and nine pupae. none of the eight investigated colonies contained eggs. a single dealate queen was observed in each eight studied colonies strongly suggesting that this species is monogynous. the nest external and internal architecture in cyphomyrmex genus is considered geometrically simple (e.g. leal et al., 2011; ramos-lacau et al., 2012) when compared to atta and acromyrmex genera (wirth et al., 2003; silva junior et al., 2013). here, one single circular nest-entrance hole without a well-formed nest mound was observed in c. lectus. similar structure was also reported to cyphomyrmex morschi (klingenberg et al., 2007) and in other attine genera such as mycocepurus forel, mycetarotes emery, trachymyrmex forel and sericomyrmex mayr (mayhé-nunes, 1995; leal et al., 2011). in our study area, two other species of the cyphomyrmex genus (c. strigatus and c. rimosus) were also observed with similar patterns of external nest architecture as c. lectus. however, other cyphomyrmex species (e.g. c. transversus and c. cornutus) present a highly distinctive nesting biology compared to c. lectus, c. strigatus and c. rimosus (adams & longino, 2007; ramos-lacau et al., 2012). for example, in costa rica, on atlantic slopes, adams ls ramos-lacau et al nesting biology and demography of cyphomyrmex lectus464 contrasting with another cyphomyrmex species (rimosus group) described by leal et al. (2011), the fungus garden in c. lectus was well-defined (yeast-like fungi formed a sponge mass) and arranged at the bottom of the chambers. this pattern has been also illustrated for c. transversus (ramos-lacau et al., 2012). according to leal et al. (2011), the fungus garden in cyphomyrmex colonies was usually characterized by yeast nodules scattered among the superficial soil and leaf litter, in brazilian savanna (cerrado). in this biome, the same authors detected three basic types of fungus garden in six attines genera: (1) pieces of substrate enveloped by yeast (cyphomyrmex, rimosus group), (2) laminar fungus suspended in the chamber ceiling or plant roots (mycetarotes, mycocepurus, and myrmicocrypta), and (3) amorphous fungus on the floor of the chamber (sericomyrmex and trachymyrmex; see also solomon et al., 2004; rabeling et al., 2007; mehdiabadi et al., 2012). with respect to the demographic observations, the colonies of c. lectus had an average worker number per colony smaller compared to two other cyphomyrmex species studied, c. cornutus (adams & longino, 2007) and c. transversus (ramoslacau et al., 2012). colonies of c. lectus presented a single dealate queen. the reproductive and alates forms were not common in these colonies during our two sampling periods, since juvenile mates were observed only in a single colony sampled in october. the occurrence of colonies with multiple dealate queens in habitats on fire regimes could provide a significant competitive advantage and facilitates the persistence of colonies. in attines, studies have demonstrated that colonies with multiple dealate queens may have a higher number of workers, a better developed fungus garden and higher survival rates (e.g., mintzer, 1987; for atta texana). colonies with multiple dealate queens are frequently observed among attines (delabie, 1989; leal et al., 2011; ramos-lacau et al., 2012), where the colonies can be both haplometrotic and pleometrotic (fernández-marín et al., 2004). regarding the absence of alates in our study, it is possible that this occurred simply because the majority of colonies (five) were sampled in june, during the middle of the dry and cold season. this month may be unfavorable to the alate production. in our study, although there is no significant relationship between colony size with some nest attributes, such as gallery length and chamber volume, we detected a positive effect of the colony size over chamber depth possibly, colonies with deeper chambers could be better protected against fire and natural enemies, and offer more favorable microclimatic conditions to yeast culture. further studies on cyphomyrmex species, especially in brazilian savanna, could evaluate whether (1) the nests density differ before and after fire periods, (2) fire affect survival and establishment rate of new colonies and, finally, (3) fire affect the microclimatic conditions necessary to yeast culture. indeed, the vegetation cover reduction by fire can promote ant species composition by altering the species dominance status (see vasconcelos et al., 2008), given that the soil surface becomes exposed to the wind and solar radiation incidence (miranda et al., 2002). fig 1. ant number effect on (a) gallery length, (b) chamber volume and (c) chamber depth in colonies of c. lectus observed in rio claro-sp, brazil. and longino (2007) described nest of cyphomyrmex cornutus nests formed by large masses of accreted soil suspended in the low arboreal zones. in fact, as referred by schultz et al. (2002) to cyphomyrmex longiscapus¸ cyphomyrmex genus comprises species that may be considered as a model of organism for the study of behavior, ecology, mating frequency, cultivar specificity, etc. sociobiology 62(3): 462-466 (september, 2015) 465 acknowledgments we are indebted to the ceplac trainees and technicians at ilhéus, adriany leão, gilmar batista costa, josé crispim soares do carmo and lucileide de souza ramos nascimento. we thank ana gabriela bieber for helpful comments on the manuscript. to ‘conselho nacional de desenvolvimento científico e tecnológico – cnpq’ for the granting of several scholarship modalities. during manuscript preparation, p. s. d. silva was supported by ‘fundação de amparo à pesquisa do estado da bahia – fapesb’ (postdoctorate fellowship). references adams, r. m. m. & longino, j. t. (2007). nesting biology of the arboreal fungus-growing ant cyphomyrmex cornutus and behavioral interactions with the social-parasitic ant megalomyrmex mondabora. insectes sociaux, 54: 136-143. doi: 10.1007/s00040-007-0922-0 araújo, m. s., della llucia, t. m. c.,ribeiro, g. a. & kasuya, m. c. m. (2003). impact of sugar cane foliage burning on the nesting and establishment of atta bisphaerica forel colonies (hymenoptera: formicidae). neotropical entomology, 32: 685-691. doi: 10.1590/s1519-566x2003000400021 ayres, m., ayres, d. l. & santos. a. a. s. (2007). bioestat: aplicações estatísticas nas áreas das ciências bio-médicas. universidade federal do pará, belém, pará. 364 p delabie, j. h. c. (1989). observações sobre a ocorrência de poliginia em colônias de acromyrmex subterraneus brunneus forel 1893, em cacauais (formicidae, myrmicinae, attini). anais da sociedade entomológica do brasil, 18:193-197. fernández-marín, h., zimmerman, j. k. & wcislo, w. t. (2004). ecological traits and evolutionary sequence of nest establishment in fungus-growing ants (hymenoptera, formicidae, attini). biological journal of the linnean society, 81:39-48. doi: 10.1111/j.1095-8312.2004.00268.x frizzo, t. l. m., campos, r. i. &vasconcelos. h. l. (2012). contrasting effects of fire on arboreal and ground-dwelling ant communities of a neotropical savanna. biotropica, 44: 254-261. doi: 10.1111/j.1744-7429.2011.00797.x kempf, w. w. (1964). a revision of the neotropical fungusgrowing ants of the genus cyphomyrmex mayr. part i: group of strigatus mayr (hym., formicidae). studia entomologica, 7:144. klingenberg, c., brandão, c. r. f. & engels. w. (2007). primitive nest architecture and small monogynous colonies in basal attini inhabiting sandy beaches of southern brazil. studies on neotropical fauna and environment, 42: 121-126. doi:10.1080/01650520601065509 leal, i. r., silva, p. s. d. & oliveira. p. s.(2011). natural history and ecological correlates of fungus-growing ants (formicidae: attini) in the neotropical cerrado savanna. annals of the entomological society of america, 104: 901908. doi: 10.1603/an11067 mayhé-nunes, a. j. (1995). filogenia de los attini (hym., formicidae): un aporte al conocimiento de las hormigas fungívoras. universidad simón bolívar, caracas, venezuela. mehdiabadi, n. j., mueller, u. g., brady, s. g., himler, a. g. & schultz., t. r. (2012). symbiont fidelity and the origin of species in fungus-growing ants. nature communications, :1-7. doi:10.1038/ncomms1844 mintzer, a. c. (1987). primary polygyny in the ant atta texana: number and weight of females and colony foundation success in the laboratory. insectes sociaux, 34:108-117. doi: 10.1007/bf02223829 miranda, h. s., bustamante, m. m. c. & miranda a. c. (2002). the fire factor. in oliveira , p. s. & marquis, r. j. (eds.). the cerrados of brazil: ecology and natural history of a neotropical savanna (pp. 51-68). columbia university press, new york. mueller, u. g. & wcislo w. t. (1998). nesting biology of the fungus-growing ant cyphomyrmex longiscapus weber (attini, formicidae). insectes sociaux, 45: 181-189. doi: 10.1007/s000400050078 murakami, t. & higashi. s. (1997). social organization in two primitive attine ants, cyphomyrmex rimosus and myrmicocrypta ednaella, with reference to their fungus substrates and food sources. journal of ethology, 15:17-25. doi: 10.1007/bf02767322 oliveira-filho, a. t. & ratter j. a. (2002). vegetation physiognomies ans woody flora of the cerrado bioma. in p. s. oliveira & marquis, r. j. (eds.). the cerrados of brazil: ecology and natural history of a neotropical savanna (pp. 91120). columbia university press, new york. pirani, a. c., sartori filho, a., freitas, c. a. d., reis, c. m., zanchetta, d., silva, d. a. d., honda, f. a., pinheiro, g. s., fachin, h. c., mendes, i. a., ahmad, i. t., monteiro, j. b., pagani, m. i. & monteiro r. (2005). caracterização dos fatores abióticos da floresta estadual edmundo navarro de andrade – feena. in diniz, f. v., pinheiro, l. s. & pontalti, s. f. l. (eds.). plano de manejo da floresta estadual edmundo navarro de andrade (pp. 31-42). fundação florestal, governo do estado de são paulo, sistema ambiental paulista, são paulo. rabeling, c., verhaagh, m. & engels w. (2007). comparative study of nest architecture and colony structure of the fungusgrowing ants, mycocepurus goeldii and m. smithii. journal of insect science, 7:1-13. doi: 10.1673/031.007.4001 ramos-lacau, l. s., silva, p. s. d., lacau, s., delabie, j. h. c. & bueno o. c. (2012). nesting architecture and population structure of the fungus growing ant cyphomyrmex transversus ls ramos-lacau et al nesting biology and demography of cyphomyrmex lectus466 (formicidae: myrmicinae: attini) in the brazilian coastal zone of ilhéus, bahia. annales de la societe entomologique de france, 48: 439-445. doi: 10.1080/00379271.2012.10697789 schultz, t. r., solomons, s. a., mueller, u. g., villesen, p., boomsma, j. j., adams, r. m. m. & norden b. (2002). cryptic speciation in the fungus-growing ants cyphomyrmex longiscapus weber and cyphomyrmex muelleri schultz and solomon, new species (formicidae, attini). insectes sociaux, 49: 331-343. doi: 10.1007/pl00012657 silva junior, m. r., castellani, m. a., moreira, a. a., d’esquivel, m., forti, l. c. & lacau s. (2013). spatial distribution and architecture of acromyrmex landolti forel (hymenoptera, formicidae) nests in pastures of southwestern bahia, brazil. sociobiology, 60: 20-29. doi:10.13102/sociobiology. v60i1.20-29 solomon, s. e., mueller, u. g., schultz, t. r., currie, c. r., price, s. l., da silva-pinhati, a. c. o., bacci, m. & vasconcelos. h. l. (2004). nesting biology of the fungus growing ants mycetarotes emery (attini, formicidae). insectes sociaux, 51:333-338. doi: 10.1007/s00040-004-0742-4 vasconcelos, h. l., leite, m. f., vilhena, j. m. s., lima, a. p.& magnusson.,w. e. (2008). ant diversity in an amazonian savanna: relationship with vegetation structure, disturbance by fire, and dominant ants. austral ecology, 33:221-231. doi: 10.1111/j.1442-9993.2007.01811.x wirth, r., beyschlag, w., ryel, r., herz, h., & hölldobler b. (2003). the herbivory of leaf-cutting ants. a case study on atta colombica in the tropical rainforest of panama. ecological studies, springer verlag berlin, heidelberg, new york. 230 p. sociobiology 60(2): 210-213 (2013) doi: 10.13102/sociobiology.v60i2.210-213 observation of trigona recursa smith (hymenoptera: apidae) feeding on crotalaria micans link (fabaceae: faboideae) in a brazilian savanna fragment tmr santos1, jt shapiro1, ps shibuya1, c aoki2 introduction plants of the genus crotalaria (l.) contain pyrrolizidine alkaloids, which are known to be toxic to humans and animals, particularly livestock and poultry (rose et al., 1957; alfonso et al., 1993). monocrotaline, the primary toxin of this genus, has been shown to damage hepatocytes, astrocytes, and glial cells, interfere with cell growth, cytoskeleton protein expression, and atp production, damage dna and cause apoptosis (silva-neto et al., 2010;pitanga et al., 2011). trigona jurine 1807 is a neotropical stingless bee genus that occurs from mexico to northern argentina, paraguay and uruguay (camargo & pedro, 2012). the greatest diversity of the genus is found in the amazon and the central region of brazil and a total of 19 species can be found within brazilian territory (rebêloet al., 2003).relatively little is known of the feeding behavior of t. recursa. individuals mark their paths and food sources for their nestmates to follow, using pheromones produced in the labial glands (jarau et al., 2003). abstract in this paper we present observations of individuals of the bee species trigona recursa feeding on the fruits of crotalaria micans. this plant, which contains pyrrolizidine alkaloids, is known to be toxic to humans, mammals and poultry. over the course of three days, we observed a large number of bees feeding on many individual crotalaria micans plants in an urban fragment of brazilian savanna. the bees preferred greener fruits, which are the softest and most toxic. consumption of the plant had no immediately apparent fatal effect on the bees, since we did not find any dead individuals near the observation site.some insect species are known to use pyrrolizidine and alkaloids for defense by incorporating them into their body or using them as precursors to pheromones. trigona recursa and other bee species have not been previously recorded consuming crotalaria micans and it is unclear what their motivation may be. we present these observations as a novel finding of the feeding behavior of trigona recursa. sociobiology an international journal on social insects 1 universidade federal de mato grosso do sul, campo grande – ms, brazil 2 universidade federal de mato grosso do sul, aquidauana – ms, brazil short note article history edited by: celso f. martins, ufpb brazil received 17 january 2013 initial acceptance 26 february 2013 final acceptance 16 march 2013 keywords entomotoxicity, feeding behavior, geographic distribution, monocrotaline, stingless bee corresponding author julie teresa shapiro laboratório de zoologia universidade federal de mato grosso do sul, cidade universitária s/n, campo grande, ms, brazil 79090-900 julie.teresa.shapiro@post.harvard.edu they tend to exploit floral resources, although they have also been observed using non-floral sources and gathering sweat (lorenzon & matrangolo, 2005). other species of the genus are known to be generalists feeding on pollen from a variety of plants (oliveira et al., 2009). three neotropical trigona species are necrophagous (noll et al.,1996). previous studies of crotalaria retusa have found that few insects visit these plants, which contain toxins throughout all their parts, (kissmann & groth, 1999) with two carpenter bee species, xylocopa grisescens and x. frontalis making up for 90% of visits (jacobi et al., 2005). trigona spinipes has been observed visiting occasionally in order to obtain nectar by perforating the base of the flowers (jacobi et al., 2005). materials and methods during three consecutive days in february 2012, individuals of the bee species t. recursa were observed eating the fruits of c. micans in an urban cerrado fragment on the sociobiology 60(2): 210-213 (2013) 211 campus of the universidade federal de matogrosso do sul (federal university of mato grosso do sul), campo grande, mato grosso do sul, brazil (20º 30’ s, 54º 36’ w). the first observation was carried out in the afternoon before sunset at approximately 18:00. on the following days, the observations were repeated twice per day, the first at 12:00 and the second at 18:00. each observation period lasted 30 minutes. at the end of the second day of observation, seven individual bees, as well as a sample from the plant were collected. these specimens were processed and given to specialists for identification. the bees were deposited in the entomological collection pe. jesus s. moure (dzup) of the department of zoology at the universidade federal do paraná (federal university of paraná). results we observed the presence of a large number of individuals of t. recursa (n>100) consuming the fruit of several dozen individual c. micans plants during all observations. apparently, the number of t. recursa was lower during the observations at 12:00 than at 18:00. however, because of their large numbers and frequent, fast movements around and between the plants, it was not possible to quantify the exact number of individuals and therefore it is impossible to be certain of the quantity of individuals consuming the fruits during the different hours of observation. the fruits of c. micans are dry. the bees scraped the velvety-textured external layer of the fruits with their tongues, apparently marking them (fig. 1). the bees demonstrated a preference for the younger and softer fruits. older and drier fruits were discarded after partial consumption. each plant had over one hundred fruits and the ratio between consumed and not consumed fruits was approximately 1:1, although a few individual plants had almost 100% of their fruits consumed. all plants had at least some of their fruits eaten. we did not observe any indication of fatal intoxication in the bees. over the three consecutive days of observation, no dead bees were found in the area of the plants. possible intoxication later could not be analyzed. discussion our observations of t. recursa feeding on plants of c. micans are notable because it is not a known food source for this bee speciesand toxins are found throughout the entire plant, including the fruit (kissmann&groth, 1999). although t. spinipesis known to occasionally collect nectar from crotalaria species (jacobi et al., 2005), the individuals of t. recursa observed did not appear to collect nectar. furthermore, the scraping method that we observed them using has not been previously recorded. it is possible that the bees scraped the fruits in order to differentiate the consumed fruits from those that had not yet been consumed. they may have also been marking them to recruit nest-mates to this food source (jarau et al., 2003). studies have shown that secondary metabolites of plants can be toxic to bee species. for example, nicotine at high levels can reduce bees’ fitness, although at lower, naturally occurring levels, there are no apparently detrimental effects and the substance can even be beneficial (kohler et al., 2012). other toxins produced in flowers have also been shown to reduce the life span of bees in laboratory settings, depending on the dose (santoro et al., 2004; rother et al.,2009; rocha-neto et al.,2011). rother et al. (2009) tested the effects of ricinine (a toxic compound of ricinus communis euphorbiaceae) on bees of the species apis mellifera and scaptotrigona postica and observed that a. mellifera had a high mortality within 72 hours for two of three concentrations tested (0.05 and 0.1%), while s. postica presented high mortality rate only after 14 days. santoro et al. (2004) demonstrated that a. mellifera was also susceptible to tannins of stryphnodendron spp. with mortality rates increasing on day 3 at all concentrations tested (1.25, 2.5 and 3.75%). these two studies showed that a. mellifera seems to be sensitive and have a rapid response to the toxins of native plant species, while the same did not occur with s. postica native bee species. we note that for the above studies, bees were exposed to toxic treatments with extracts in laboratory experiments, whilehere we report that t. recursa spontaneously consumed c. micans. however, in more similar circumstances, del lama and peruquetti (2006) observed that the consumption of the toxic plant caesalpinia peltophoroides in a natural setting caused a large number of mortalities in bees, with 273 indifig. 1.bees feeding on the fruit ofcrotalariamicans. the arrow indicates the scraped portion of the fruit. tmr santos, jt shapiro, ps shibuia, c aoki observation of trigona recursa feeding on crotalaria micans212 viduals of 20 different species dying after visiting the plant. toxicity seemed to vary by time and individual tree. although the long term effects could not be observed, most bees dropped to the ground in narcosis and died immediately species of the orders lepidoptera, coleoptera, diptera, and orthoptera are known to sequester pyrrolizidine alkaloids, including those found in crotalaria, for purposes of defense against predators and as precursors to pheromones (boppré, 1990). notably, studies have shown that the moth species utetheisa ornatrix obtains the toxins from plants during its larval stage, retaining the compounds as an adult and then passing it on to their eggs (eisner & eisner, 1991). laboratory and field experiments have confirmed that the consumption of pyrolizidine alkaloids protects individuals from predation (dussourd et al., 1988; boppré, 1990; eisner & eisner, 1991). in this case, there was no immediately apparent negative effect on t. recursa from consuming c. micans. although we cannot be sure of the longer-term effects on the bees’ fitness, it is possible that t. recursa benefits from the protection from monocrotaline. the toxins could also have a longer-term negative effect or intoxicate the bees after several days, but we were unable to confirm or rule out any of these possibilities. we also note that this is the first record of t. recursa in the state of mato grosso do sul. the species has been previously recorded in the neighboring states of são paulo, goiás, and mato grosso (camargo & pedro, 2012). acknowledgements we would like to thank gabriel melo for assisting in insect identification, thales henrique dias leandro for plant identification, and the fulbright program for the support of julie shapiro. we thank two anonymous reviewers for valuable comments that improved this article. references alfonso, h.a., sanchez, l.m., figeurdo, m.a. & gomez, b.c. 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(2010). genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from crotalaria retusa, in a model of glial cells. toxicon, 55 (1): 105–117. doi: 10.1016/j. toxicon.2009.07.007. 1239 aggressive behavior and the role of antennal sensillae in the termite reticulitermes chinensis (isoptera: rhinotermitidae) by qiuying huang1*, chunsun guan1, qiang shen1, chengqiang hu1 & binbin zhu abstract this study examined aggressive behavior between the colonies in the termite reticulitermes chinensis from china. strong aggression was observed among workers and soldiers. intercolonial aggression was strong during the first 0.5 h and then reduced gradually in all the treatments. after cutting the five terminal antennal segments of workers and soldiers, there was still strong intercolonial aggression among workers and soldiers. however, after removal of the ten terminal antennal segments of workers and soldiers, almost no intercolonial aggression happened among workers and soldiers. sem results of antennae indicated that antennal sensillae mainly occurred on the ten terminal segments including four types of trichode sensilla, both in workers and soldiers of this species. few antennal sensilla occurred on the basal segments except for a few sensilla chaetica and basiconic capitate peg sensilla. the above findings suggest that antennal sensillae may play a role in nestmate recognition in r. chinensis. keywords: aggressive behavior, antennal sensilla, reticulitermes chinensis, nestmate recognition. introduction all social insects need to distinguish nestmates from non-nestmates (kaib et al. 2004). nestmate recognition ensures integration in a colony and prevents non-colony members from exploiting the colony’s resources (crozier & pamilo 1996, yusuf et al. 2010). the presence of non-nestmates (intruders) usually induces active aggressive behaviors (vander meer & morel 1998). in the eusocial termites, there is not only interspecific aggression 1hubei insect resources utilization and sustainable pest management key laboratory, huazhong agricultural university, wuhan 430070, china 2yichang center for disease control and prevention, yichang 443005, china *corresponding author, e-mail: qyhuang2006@mail.hzau.edu.cn 1240 sociobiolog y vol. 59, no. 4, 2012 but also intercolonial aggression in the same species (polizzi & forschler 1998). intercolonial aggression among subterranean termites is highly variable (cornelius & osbrink 2009). in the united states, some colony pairs of coptotermes formosanus showed high levels of aggression, while other colony pairs exhibited almost no aggression (su & haverty 1991, shelton & grace 1997, husseneder & grace 2001, cornelius & osbrink 2003). there was strong aggression among colonies of odontotermes formosanus in china (huang et al. 2007). italian reticulitermes lucifugus populations were moderately aggressive (uva et al. 2004). however, there was no aggression among colonies of r. santonensis in france and r. flavipes in massachusetts (bulmer & traniello 2002, dronnet et al. 2006). intercolonial aggression was correlated with cuticular hydrocarbon profiles in macrotermes subhyalinus (kaib et al. 2004), but there was no correlation between cuticular hydrocarbons and levels of intercolonial aggression in c. formosanus (su & haverty 1991). however, pairings of workers from different cuticular hydrocarbon phenotypes resulted in immediate aggression in reticulitermes (haverty et al. 1999). aggressive behavior among colonies of r. lucifugus was unrelated to intercolonial geographic distance ( jmhasly & leuthold 1999, uva et al. 2004). there was a positive correlation between genetic distance and levels of intercolonial agonism in c. formosanus (husseneder et al. 2005). in addition, group size, diet, gland secretions, bioassay design and length of time in the laboratory affected intercolonial aggression in termites (polizzi & forschler 1998, florane et al. 2004, zhang et al. 2006, huang et al. 2007, cornelius & osbrink 2009). the termite r. chinensis (isoptera: rhinotermitidae) is widely distributed in china, including beijing, tianjin, shanxi and the yangtze river drainage basin (wei et al. 2007). this termite species builds its nests in soil and wooden structures (liu 2003), and is an important pest of forest trees and urban buildings (li et al. 2010). however, knowledge about social behaviors of r. chinensis is currently limited, such as foraging behavior, trophallaxis and aggressive behavior. in this study, we combine results derived from aggression tests among colonies, effect of antennal sensillae on intercolonial aggression, and sem photomicrographs of antennal sensilla to better understand intercolonial aggression in r. chinensis. 1241 huang, q. et al. — role of antennal sensillae in r. chinensis methods and materials termites two colonies of r. chinensis were collected from pine stumps in shizi hill, huazhong agriculture university (30°29' n, 114°21' e), wuhan city, china at the same time. the two colonies were recorded as col. a and col. b respectively. the distances among them were more than 300 meters. one week later, termites were removed from stumps brought into laboratory. soldiers were used to identify species (huang et al. 2000). each colony was maintained in separate glass containers with lids. containers were lined with damp pines slats (1 mm×2 cm×8 cm). glass containers were kept in a dark laboratory at constant conditions (25±2°c, 85±5% rh). termites in glass containers were used in the following experiments during three months. aggressive behavior each group of 300 workers from one colony were placed in a 15.0 cm diameter glass petri dish lined with filter paper (15.0 cm) stained with 1.0 g/l solution of neutral red in distilled water (huang et al. 2007), and provided with 2 ml of distilled water for moisture. the dishes were then placed in a climate cabinet maintained in complete darkness at 25±2°c and 85±5% rh. the termites were forced to feed on the stained filter paper for 24 h, and then the marked individuals were selected to be used in the study. simultaneously, healthy workers without staining from the other colony were also selected to be used in this study. 60 workers from col. a and col. b were mixed together in a 9.0 cm diameter glass petri dish with filter paper (9.0 cm diameter) together in ratios of 50:10, 45:15, 30:30, 15:45 and 10:50. the aggressive encounters of workers between col. a and col. b were recorded in 5 min intervals at 0, 0.5, 1, 1.5 and 24 h after workers were combined. then numbers of dead termites were recorded at 24 h. there were 6 replicates for each combination in the experiment on aggression in workers between col. a and col. b. there were 3 replicates with marked workers for col. a and col. b respectively in each combination. for the experiment of aggression in soldiers between col. a and col. b, the abdomens of soldiers were painted by a black marking pen so that the soldiers from different colonies could be distinguished. the soldiers from col. a and col. b were placed in a 9.0 cm diameter glass petri dish with filter paper (9.0 cm diameter) together in ratios 1242 sociobiolog y vol. 59, no. 4, 2012 of 20:10, 15:10, 10:10, 10:15 and 10:20. the other test processes were the same as described above. for the experiment of aggression between soldiers from col. a and workers from col. b, because there were obvious morphological differences between soldiers and workers, the staining process was not needed. the soldiers from col. a and the workers from col. b were placed in a 9.0 cm diameter glass petri dish with filter paper (9.0 cm diameter) together in ratios of 20:10, 15:10, 10:10, 10:15 and 10:20. there were only 3 replicates for each combination in the experiment. the other test processes were the same as the above experiments. intracolonial aggression in col. a and col. b was respectively as control experiment, with the same caste combinations and experimental methods as described above. effect of antennal sensillae on aggression workers or soldiers in petri dishes (9.0 cm diameter) were immobilized on the ice, and then the five terminal or ten antennal segments were cut for workers and soldiers under the microscope. the treated workers and soldiers were left at room temperature for 30 min to recover and then used in the following aggression trials between col. a and col. b. there were four types of treatments: (1) aggression in workers without the five segments of antennae, (2) aggression in soldiers without the five segments of antennae, (3) aggression in workers without the ten segments of antennae, (4) aggression in soldiers without the ten segments of antennae. the 20 termites from col. a (10 termites) and col. b (10 termites) were placed in a 9.0 cm diameter glass petri dish with filter paper (9.0 cm diameter) together. the numbers of dead termites were recorded at 0.5 h, 1.0 h, 1.5h and 24 h. there were 5 replicates for each treatment. the other test processes were the same as the above experiments. sem of antennal sensillae two samples were observed by scanning electron microscope (sem), including a worker and a soldier from col. a. the heads were removed from the bodies and were rinsed in phosphate buffer. heads were twice dehydrated through a graded ethanol series of 30, 50, 70, 85, 95 and 100% (each for 15 min). the heads were placed in a sample basket which was then put in a sample chamber of a critical point dryer. after covering with a lid, liquid co2 was injected into the sample chamber up to submerge the sample. the 1243 huang, q. et al. — role of antennal sensillae in r. chinensis temperature of the sample chamber first increased to 15°c for 10 min and then increased to 35°c which made all the liquid co2 gasified. after all the co2 was discharged, the sample could be taken out. the samples were sputter-coated with gold for 5 min. finally the sample was observed under a jsm-6390lv sem at 20 kv. statistical analyses mortality of termites (%) = number of dead termites × 100 / initial number of termites. the 2-tailed paired-samples t test was used to analyze mortality of termites between the two colonies (spss inc., 1989-2002). results intercolonial aggression when the termites from col. a and col. b were placed in the same dish in the five ratios, they showed strong aggression (fig.1a, fig.2a and fig.3b). for example, one used its mandibles to clamp the head, maxilla or abdomen of the other one until it became immobile. for each combination in aggression in workers between col. a and col. b, the aggressive encounters were more than 9 in 5 min at 0 h after combining (fig.1a). when the testing time continued, the aggressive encounters between workers decreased. at 24 h after combining workers from the two colonies, the aggressive encounters between workers from the two colonies were less than 3 (fig.1b). for each combination in aggression in soldiers between col. a and col. b, the aggressive encounters fig.1. aggressive behavior in workers between col. a and col. b in five combinations; a, aggressive encounters of workers; b, worker mortality at 24 h. **, p < 0.01; *, p < 0.05; n.s., no significant difference. 1244 sociobiolog y vol. 59, no. 4, 2012 were more than 11 in 5 min at 0 h after combining (fig.2a). over time, the number of aggressive encounters between workers and soldiers decreased. at 24 h after combining, the aggressive encounters between soldiers from the two colonies were less than 3 (fig.2a). for each combination in aggression between soldiers from col. a and workers from col. b, the aggressive encounters were more than 13 in 5 min at 0 h after combining (fig.3a). when the testing time continued, the aggressive encounters between soldiers and workers presented the decreasing trend. at 24 h after combining, the aggressive encounters between soldiers from col. a and workers from col. b were less than 3 (fig.3a). there was no intracolonial aggression observed in all the control experiments in col. a and col. b. worker mortality of col. a was significantly less than that of col. b at 24 h when the ratios were 50:10 (t= -16.579, df=5, p=0.001) and 45:15 fig.2. aggressive behavior in soldiers between col. a and col. b in five combinations; a, aggressive encounters of soldiers; b, soldier mortality at 24 h. **, p < 0.01; *, p < 0.05. fig.3. aggressive behavior between soldiers from col. a and workers from col. b in five combinations; a, aggressive encounters of soldiers and workers; b, mortality of soldiers and workers at 24 h. **, p < 0.01; *, p < 0.05; n.s., no significant difference. 1245 huang, q. et al. — role of antennal sensillae in r. chinensis (t= -10.07, df=5, p=0.001) (fig.1b). however, worker mortality of col. a was significantly higher than that of col. b at 24 h in 10:50 (t=4.398, df=5, p=0.007) and 15:45 (t=3.045, df=5, p=0.029) (fig.1b). there was no significant difference in work mortality between col. a and col. b at 24 h in 30:30 (t= -0.002, df=5, p=0.999) (fig.1b). soldier mortality of col. a was significantly less than that of col. b at 24 h when ratios were 20:10 (t= -4.111, df=5, p=0.009), 15:10 (t= -11.087, df=5, p=0.001) and 10:10 (t= -2.739, df=5, p=0.041) (fig.2b). however, soldier mortality of col. a was significantly higher than that of col. b at 24 h in 10:15 (t=8.032, df=5, p=0.001) and 10:20 (t=10.0, df=5, p=0.001) (fig.2b). soldier mortality of col. a was significantly less than worker mortality of col. b at 24 h when the ratios were 20:10 (t= -6.518, df=2, p=0.023), 15:10 (t= -22.522, df=2, p=0.002) and 10:10 (t= -7.00, df=2, p=0.020) (fig.3b). however, there was no significant difference between soldier mortality of col. a and worker mortality of col. b at 24 h in 10:15 (t= -3.973, df=2, p=0.058) and 10:20 (t= -4.158, df=2, p=0.053) (fig.3b). effect of antennal sensillae on aggression after five terminal segments of the two antennae of workers were cut, strong aggression still occurred in workers between col. a and col. b. the aggressive encounters were 20 in 5 min at 0 h after combining (fig.4a). when the testing time continued, the aggressive encounters between workers presented the decreasing trend. at 24 h after combining, the aggressive encounters between workers from the two colonies were 3 (fig.4a). there fig.4. aggressive behavior in workers without the five segments of antennae between col. a and col. b when the combination was 10:10; a, aggressive encounters of workers; b, worker mortality. *, p < 0.05; n.s., no significant difference. 1246 sociobiolog y vol. 59, no. 4, 2012 was no significant difference between worker mortality of col. a and worker mortality of col. b at 0.5 h (t= -2.138, df=4, p=0.099), 1 h (t= -2.449, df=4, p=0.07) and 1.5 h (t= -2.236, df=4, p=0.089) (fig.4b). however, worker mortality of col. a was significantly less than that of col. b at 24 h (t= -3.773, df=4, p=0.02) (fig.4b). after cutting five terminal segments of the two antennae of soldiers, there was strong aggression in soldiers between col. a and col. b. the aggressive encounters were 21.8 in 5 min at 0 h after combining (fig.5a). when the testing time continued, the aggressive encounters between soldiers presented the decreasing trend. at 24 h after combining, the aggressive encounters between soldiers from the two colonies were 2.6 (fig.5a). soldier mortality of col. a was significantly less than that of col. b at 0.5 h (t= -4.00, df=4, p=0.016), 1.0 h (t= -3.207, df=4, p=0.033), 1.5 h (t= -5.715, df=4, p=0.005), 24 h (t= -3.207, df=4, p=0.033) (fig.5b). however, after cutting fig.6. aggressive behavior in workers and soldiers without the ten segments of antennae between col. a and col. b when the combination was 10:10; a, aggressive encounters of workers; b, aggressive encounters of soldiers. fig.5. aggressive behavior in soldiers without the five segments of antennae between col. a and col. b when the combination was 10:10; a, aggressive encounters of soldiers; b, soldier mortality. **, p < 0.01; *, p < 0.05. 1247 huang, q. et al. — role of antennal sensillae in r. chinensis ten terminal segments of the two antennae of workers, there was almost no aggression in workers between col. a and col. b (fig.6a). similarly, almost no aggression happened in soldiers without ten terminal segments between col. a and col. b (fig.6b). sem observation of antennal sensilla antennal sensillae of both workers and soldiers of r. chinensis occurred mainly on the ten terminal segments of antennae (fig.7a; fig.8a), which included four types of trichodea sensilla (ts) (fig.7b, d; fig.8b, d). there were few antennal sensillae on the basal segments of antennae of both workers and soldiers of r. chinensis (fig.7c; fig.8c), exception for a few sensilla chaetica (sch) and basiconic capitate peg sensilla (bcps) (fig.7e, f; fig.8e, f). discussion in this study, we observed strong intercolonial aggression in the termite r. chinensis. for the five combinations, all the aggressive encounters were almost more than 9 in 5 min at 0 h after termites from the two colonies were combined, but those dropped to less than 3 in 5min at 24 h. there are two fig.7. sem photomicrographs of antennal sensillae of r. chinensis worker; a, entire antenna; b, terminal segments of antenna; c, basal segments of antenna; d, trichodea sensilla (ts); e, sensilla chaetica (sch); f, basiconic capitate peg sensilla (bcps). 1248 sociobiolog y vol. 59, no. 4, 2012 potential explanations for this reducing trend: 1)some of the termites were already dead or injured due to their strong aggression, 2) remaining individuals were calm because of large amounts of energ y consumption (huang et al. 2007). the worker mortality of col. a was significantly less than that of col. b at 24 h when the combinations were 50:10 and 45:15 (col. a : col. b), and the worker mortality of col. a was significantly higher than that of col. b at 24 h in 10:50 and 15:45 (col. a : col. b). these results indicated that there was positive correlation between group size and levels of intercolonial aggression in workers of r. chinensis, unlike the aggressive behavior in workers between r. flavipes and r. virginicus (polizzi & forschler 1998). the soldier mortality of col. a was significantly less than that of col. b at 24 h when the combinations were 20:10 and 15:10 (col. a : col. b), and the soldier mortality of col. a was significantly higher than that of col. b at 24 h in 10:15 and 10:20 (col. a : col. b). these results also suggested that the advantage of individual numbers could produce an agonistic advantage in soldiers of r. chinensis (huang et al 2007). furthermore, the soldier mortality of col. a was significantly less than worker mortality of col. b at 24 h when the combinations were 10:10 (col. a : col. b), and there was no significant fig.8. sem photomicrographs of antennal sensillae of r. chinensis soldier; a, entire antenna; b, terminal segments of antenna; c, basal segments of antenna; d, trichodea sensilla (ts); e, sensilla chaetica (sch); f, basiconic capitate peg sensilla (bcps). 1249 huang, q. et al. — role of antennal sensillae in r. chinensis difference between soldier mortality of col. a and worker mortality of col. b at 24 h in 10:15and 10:20 (col. a : col. b). these results indicated that the soldier caste were more aggressive than the worker caste in r. chinensis (binder 1988), which is possibly correlated with soldiers’ strong mandibles and frontal gland secretions (he et al. 2006; zhang et al. 2006). after cutting five terminal segments of the two antennae of workers and soldiers, there was still strong aggression among workers and soldiers from col. a and col. b. however, after cutting ten terminal segments of the two antennae of workers and soldiers, almost no aggression happened among workers and soldiers between the two colonies. these results indicate that chemosensory sensilla with function of nestmate recognition mainly located in the ten terminal segments of antennae in r. chinensis (he 2006). as shown by scanning electron microscopy, antennal sensillae of both workers and soldiers of r. chinensis occurred mainly on ten terminal segments of antennae, including four types of trichodea sensilla, but there were few antennal sensilla on the basal segments of antennae of both workers and soldiers, except for a few sensilla chaetica and basiconic capitate peg sensilla (tarumingkeng et al. 1976; li et al. 2009). these findings suggest that antennal sensillae might be responsible for nestmate recognition in r. chinensis (ozaki et al. 2005). the above findings suggest that antennal sensillae may play a role in nestmate recognition in r. chinensis. however, the other factors involved in aggression in termites should also be studied for r. chinensis, such as diet, geographic distance, genetic distance, bioassay design and length of time in the laboratory (cornelius & osbrink 2009). currently, very little is known about molecular mechanisms of aggressive behavior in termites (dierick & greenspan 2006; alaux et al. 2009), so identification of genes affecting aggression in r. chinensis should also be initiated. acknowledgments we thank ganghua li for assisting with sem of antennal sensillae. we also thank dr. claudia husseneder, dr. xuguo zhou and dr. shichang zhang for revising the manuscript. this work was supported by the national natural science foundation of china (31000978), the international foundation for science (d/4768-1), and the national department public benefit (agriculture) research foundation (201003025). 1250 sociobiolog y vol. 59, no. 4, 2012 references alaux, c., s. sinha, l. hasadsri, g. j. hunt, e. guzmán-novoa, g. degrandi-hoffman, j. l. uriberubio, b. r. southey, s. rodriguez-zas & g. e. robinson 2009. honey bee aggression supports a link between gene regulation and behavioral evolution. proceedings of the national academy of 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variation in the composition of the frontal gland secretion of coptotermes formosanus (isoptera: rhinotermitidae). sociobiolog y 47: 553–561. 681 carrying and effect of granulated baits formulated with entomopathogenic fungi among atta sexdens rubropilosa colonies (hymenoptera: formicidae) by sandra r. s. cardoso1, nilson s. nagamoto1,*, luiz c. forti1 & efrain s. souza1 abstract the present study aimed to evaluate the carrying and effect of (dry) granulated baits containing conidia of entomopathogenic fungi among colonies of the leaf-cutting ant atta sexdens rubropilosa in the laboratory. this bait type was chosen to facilitate its eventual commercial use. two applications were performed: in the first, baits with 1x108 conidia/g were utilized while in the second employed concentrations 5 to 8.6 times greater. the baits were formulated with a citric pulp base, with 2 isolates of beauveria bassiana, 1 of paecilomyces lilacinus and 1 of isaria fumosorosea. the following controls were utilized: (i) baits with sulfluramid insecticide, (ii) without active ingredient, and (iii) acalypha spp. leaf discs. it was verified that the baits containing fungal conidia were rapidly carried to the nest interior in both applications and were rejected minimally. thus, the (dry) granulated bait formulation appears to be an adequate vehicle for entomopathogenic fungi. at the doses and concentrations utilized, the fungi provoked only limited worker mortality, not killing the colonies. given the rapid carrying and low rejection, a higher conidial dose per colony can, perhaps, kill them. thus, it is inferred that all the isolates tested present potential as an agent to control colonies of leaf-cutting ants. keywords: atta sexdens rubropilosa, beauveria, paecilomyces, isaria, bait, leaf-cutting ant. introduction the leaf-cutting ants, genera atta and acromyrmex (hymenoptera: formicidae: attini), utilize exotic and native plants as substrate for cultivating 1defesa fitossanitária, faculdade de ciências agronômicas (fca), univ estadual paulista (unesp); p.o.box 237, 18603-970, botucatu, sp, brazil. *correspondence author, e-mail: nsnagamoto@yahoo.com 682 sociobiolog y vol. 59, no. 3, 2012 symbiotic fungus (a basidiomycete, and therefore a macrofungus) (hölldobler & wilson 1990), and are considered pests that have a great impact on agriculture, ranching and silviculture (boaretto & forti 1997). to control these pests, in general, a chemical approach must be employed, principally via granulated toxic baits (dry), with a citric pulp base, given their practicality and effectiveness. relatively persistent synthetic insecticides are always utilized as the active ingredient (ai) in commercial granulated baits, in such a manner that most of them have been gradually prohibited or discontinued (boaretto & forti 1997; grosman et al. 2002; nagamoto et al. 2004; gunasekara et al. 2007). this context has prompted a search to develop alternatives including microbial control with entomopathogenic fungi (kermarrec et al. 1986; machado et al. 1988; lopez & orduz 2003). although the literature has already demonstrated the pathogenicity of these fungi to leaf-cutting ants, they remain far from being usable in the field on a large scale. furthermore, to the best of our knowledge, such fungi have never before been tested in a dry granulated formulation based on citric pulp, despite high attractiveness of this (nagamoto et al. 2004, 2011). in general, fungi are considered important agents for controlling insects (alves 1998); however, leaf-cutting ants possess a diverse array of defenses against these microorganisms: the use of specific and generic antibiotics, grooming, and removal of contaminated material (kermarrec et al. 1986; hölldobler & wilson 1990; bot et al. 2001; currie & stuart 2001; poulsen et al. 2002; caldera et al. 2009), represent a great challenge in the development of efficient products that are commercially viable. thus, the present study aimed to evaluate the carrying and the effect of (dry) granulated baits containing conidia of entomopathogenic fungi among colonies of atta sexdens rubropilosa forel, under laboratory conditions. material and methods the present work was carried out in the laboratory of social-pest insects (lisp), fca, unesp, botucatu, in 2010. the baits utilized were in the form of (dry) granulated pellets and had been manufactured artisanally with an attractive citric-pulp substrate and ais comprised of the following fungi isolates for testing : bbot11 and bbot12 (beauveria bassiana), pbot33 683 cardoso, s.r.s. et al. — baits with entomopathogenic fungi for a. s. rubropilosa (paecilomyces lilacinus) and ibot25 (isaria fumosorosea), which had been previously isolated from queens of atta spp. (cardoso 2010). the formulation process consisted of mixing the dried ground citric pulp, 5% cmc (carboxymethylcellulose) agglomerating agent, conidial suspension (1.0 ml of suspension/gram of final intended bait weight), refined soy oil at 5% w/w and distilled water, similar to the description of nagamoto et al. (2011), but utilizing some different ingredients and sterile conditions. on account of dehydration, and considering the conidial weight to be negligible, the final concentration was 1x108 conidia/gram of bait. the conidial suspensions were prepared by scraping mycelium from colonies of fungi (cultured in a appropriate culture medium, pda), adding distilled water, homogenizing in an orbital vortex shaker, and filtering the result through a double layer of cheesecloth; then, the conidia were quantified under a microscope utilizing a neubauer chamber. the bait fillets were produced according to the method of nagamoto et al. (2011), added onto petri dishes and dehydrated in a bod chamber at 5 oc for 48 hours; next, they were placed into a plastic bag and manually broken into pellets about ~0.5 cm in length, similarly to commercial baits (lima et al. 2003; nagamoto et al. 2011). finally, the pellets were conserved at 5 oc for subsequent use. the study consisted of two applications. at the first moment, the formulated baits were utilized with 1x108 conidia/gram of bait. due to the low mortality of worker ants after 144 hours, a second application was performed at the same dose, but utilizing higher concentrations (with the totality of conidia available): 6.0x108, 8.6x108, 5.0x108 and 5.0x108 (conidia/g ), respectively, for bbot11, bbot12, pbot33 and ibot25. the control baits tc1 (with standard ai, sulfluramid, from dinagro agropecuária ltd., at 0.3% w/w), and tc2 (neutral, without ai), were also manufactured under sterile conditions. to control against a possible effect of the bait components, a third control treatment (tc3) was utilized: fresh 8.0 mm (ø) leaf discs of acalypha spp, cut with the aid of a hole puncher. colonies of atta sexdens rubropilosa with about 500 ml of fungal sponge were used, maintained at 25oc. in each colony 1 g of bait or leaf discs was applied in the foraging arena. the delineation utilized in the first application was completely randomized, with 7 treatments and 4 repetitions. in the second application, each colony 684 sociobiolog y vol. 59, no. 3, 2012 received the same type of product, except the colonies treated with sulfluramid, which had died and were thus excluded from this new application. prior to the applications, the colonies stayed 24 hours without being supplied leaves and only 48 hours after the application this procedure was resumed. after application, the total carrying time was evaluated and, at 6 and 24 hours after application, the percentages of pellets and discs carried were determined. it was observed whether this material was incorporated into the fungus culture or rejected (foraging arena or waste chamber). the effect of baits was evaluated through the number of dead workers. for this, the numbers of dead ants in the foraging arena and waste chamber were counted, cumulatively, every 24 hours. the carrying time was assessed through descriptive analysis (mean, standard deviation), besides maximum and minimum carrying time observed by treatment. the percentage of pellets carried and the mortality data were submitted to anova and the means compared by the tukey test at 5% probability results and discussion in both applications, baits formulated with fungus were carried and incorporated into symbiotic culture (fungal sponge), with the isolates bbot12 (b. bassiana) and pbot33 (p. lilacinus) presenting the shortest mean carrying times, 0.52±0.37 h and 0.75±0.26 h, respectively in the first application, and 0.56±0.30 h and 0.91±0.53 h, in the second one (fig. 1). in the control treatments, at 6 and 24 hours after application, there were still pellets in the foraging chamber in the majority of colonies, while colonies treated with fungal baits presented carrying near or equal to 100% (fig. 2). the carrying percentage among treatments was not significative at 24h (p = 0.6996 and 0.2234, respectively, for applications i and ii). thus, the baits containing conidia of entomopathogenic fungi were carried rapidly, evidencing the favorability of these baits, since in field conditions, more rapid carrying of baits implies their lesser exposure to adverse climatic conditions, which may increase the efficiency of control (boaretto & forti 1997; lima et al. 2003). the rejection behavior was verified only in colonies under sulfluramid treatment and in one colony treated with bbot12 (b. bassiana), corresponding to the paucity of pellets found in the waste chamber. 685 cardoso, s.r.s. et al. — baits with entomopathogenic fungi for a. s. rubropilosa as to the effect of the baits on the ant population, it was verified, starting at 96h of application, that treatment tc1 (sulfluramid) provoked a greater number of ant deaths than all the others (table 1). in the second application, the worker mortality provoked by pbot33 (p. lilacinus) was greater than in the other treatments in all evaluation periods (table 2), although significant differences appeared only in the final evaluations (table 2). none of the colonies treated with fungal isolates died, but the dead ants found in the waste chamber presented extrusion of the pathogen applied (data not shown). the fact that the fungus-treated colonies presented almost no rejection of baits, and incorporated them into the fungus garden appears to indicate that the formulation utilized, dry granulated bait, can also be a good vehicle for entomopathogenic fungi, like the moist bait of lopez & orduz (2003), formulated with orange juice (50%), wheat bran, and spores of metarhizium anisopliae and/or trichoderma viride. the advantage of the formulation utilized in the present work is its practicality of application and eventual commercialization without requiring adaptation of the bait formulation for large-scale production, storage and distribution. the presence of dead ants fig. 1. mean bait carrying time (hours) by atta sexdens rubropilosa workers after application of baits with fungal isolates: bbot11 and bbot12 (beauveria bassiana), pbot33 (paecilomyces lilacinus) and ibot25 (isaria fumosorosea), after first and second application. 686 sociobiolog y vol. 59, no. 3, 2012 parasitized by the applied fungus and the increase in waste volume produced in the waste chamber, findings also observed by lopez & orduz (2003), indicate its deleterious effect on the colonies, and thus the possibility that fungal isolates would kill the colonies at higher concentrations or doses. there are still many important gaps in the research of leaf-cutting ants (nagamoto et al. 2004), including lack of knowledge about the manner and extent to which some microfungi such as trichoderma, in the form of spores, can sometimes be tolerated or not perceived (lopez & orduz 2003), and at fig. 2. percentage (%) of baits carried by atta sexdens rubropilosa workers, by treatment, at 6 and 24 hours after application of baits with fungal isolates: tf1 (isolate bbot11 of beauveria bassiana), tf2 (isolate bbot12 of beauveria bassiana), tf3 (isolate pbot33 of paecilomyces lilacinus), tf4 (isolate ibot25 of isaria fumosorosea), bait with the active ingredient sulfluramid (tc1), bait without active ingredient (tc2), and acalypha leaf discs (tc3), in (a) application i and (b) application ii. 687 cardoso, s.r.s. et al. — baits with entomopathogenic fungi for a. s. rubropilosa other times, intensely excluded (currie & stuart 2001). in fact, the most likely expectation is that the workers seek to evade and eliminate microfungi and other undesirable microorganisms (bot et al. 2001; currie & stuart 2001); however, the reality is that this does not always occur (lopez & orduz 2003, table 1. number of atta sexdens rubropilosa workers found dead after being supplied baits with conidia of beauveria bassiana, paecilomyces lilacinus and isaria fumosorosea at the concentration 1x108 conidia/g – application i. hours after application** treatment* 24 48 72 96 120 144 bait with ai 11.50 a 48.50 a 144.00 a 212.00 a 270.50 a 270.50 a neutral bait 0.50 a 0.00 b 1.00 b 1.00 b 1.00 b 1.00 b leaves 0.50 a 0.50 b 0.50 b 1.5 b 1.50 b 6.50 b bbot11 0.00 a 0.00 b 0.00 b 0.00 b 0.50 b 3.00 b bbot12 3.00 a 4.00 a 5.50 ab 10.50 b 6.50 b 14.00 b pbot33 3.00 a 1.00 ab 1.50 ab 2.50 b 1.50 b 5.00 b ibot25 0.00 a 1.00 ab 1.00 b 1.00 b 0.50 b 3.00 b p 0.061 0.033 0.035 0.029 0.035 0.033 * bait with ai: with ai sulfluramid (synthetic insecticide); neutral bait: without ai; leaves: leaf discs of acalypha; baits with fungal isolates: bbot11 (b. bassiana), bbot12 (b. bassiana), pbot33 (p. lilacinus), and ibot25 (i. lilacinus). ** means followed vertically by the same letter do not differ among the treatments by tukey’s test at 5% probability. table 2. number of atta sexdens rubropilosa workers found dead after being supplied baits with conidia of beauveria bassiana, paecilomyces lilacinus and isaria fumosorosea at the concentration 5x108 to 8x108 conidia/g – application ii. hours after application** treatment* 24 48 72 96 120 144 168 336 672 neutral bait 5.25 a 5.25 a 6.50 a 8.25 a 12.50 a 14.50 a 16.00 a 18.00 ab 71.00 ab leaves 2.50 a 3.25 a 4.75 a 6.75 a 8.75 a 9.75 a 10.00 a 15.00 b 24.50 b bbot11 1.50 a 4.50 a 3.25 a 5.75 a 7.25 a 9.25 a 10.00 a 14.50 b 45.50 ab bbot12 4.47 a 6.75 a 8.25 a 13.25 a 13.50 a 26.50 a 26.75 a 38.50 ab 125.25 ab pbot33 13.00 a 21.00 a 30.50 a 36.00 a 46.25 a 55.00 a 62.50 a 97.00 a 133.25 a ibot25 4.00 a 5.75 a 9.50 a 14.00 a 16.25 a 19.00 a 22.25 a 23.50 ab 103.25 ab p 0.3297 0.2982 0.1777 0.1691 0.2111 0.1774 0.1321 0.0281 0.0214 * neutral bait: without ai; leaves: leaf discs of acalypha; baits with fungal isolates: bbot11 (b. bassiana), bbot12 (b. bassiana), pbot33 (p. lilacinus), and ibot25 (i. lilacinus). ** means followed vertically by the same letter do not differ among the treatments by tukey’s test at 5% probability. 688 sociobiolog y vol. 59, no. 3, 2012 present study). a surprising case has already been observed in which most baits with abundant growth of some microfungi, including paecilomyces sp., were carried extensively (carlos et al. 2009). it is concluded that, although the (dry) granulated baits with fungi of the present study failed to overcome the defenses of the colonies (kermarrec et al. 1986; machado et al. 1988; hölldobler & wilson 1990; bot et al. 2001; currie & stuart 2001; poulsen et al. 2002; caldera et al. 2009), these isolates show potential as a control agent by virtue of all having presented optimum carrying and provoked deleterious effects. thus, new studies can be performed on these fungi, in this formulation, especially with an increase in conidial concentration in the baits, in order to better evaluate the control potential for leaf-cutting ants. acknowledgments we wish to thank fapema for the fellowship granted to the first author and fapesp for financial support (2008/07032-7). and a. rodrigues for help in fungi identification. l.c. forti acknowledges the support of cnpq (472671/2008-1). references alves, s.b. 1998. fungos entomopatogênicos, p. 289-381. in: alves, s.b. (ed.), controle microbiano de insetos. fealq, piracicaba. boaretto, m.a.c. & l.c. forti. 1997. perspectivas no controle de formigas cortadeiras. série técnica ipef 11(30): 31-46. bot, a.n.m., c.r. currie, a.g. hart & j.j. boomsma. 2001. waste management in leafcutting ants. etholog y, ecolog y & evolution 13(3): 225-237. caldera, e.j., m. poulsen, g. suen & c.r. currie. 2009. insect symbioses: a case study of past, present, and future fungus-growing ant research. environmental entomolog y 38(1): 78-92. carlos a.a., l.c. forti, a. rodrigues, s.s. verza, n.s. nagamoto & r.s. camargo. 2009. influence of fungal contamination on substrate carrying by the leafcutter ant atta sexdens rubropilosa (hymenoptera: formicidae). sociobiolog y 53(3): 785-794. cardoso, s.r.s. 2010. morfogênese de ninhos iniciais, mortalidade de atta spp. (hymenoptera, formicidae) em condições naturais e avaliação da ação de fungos entomopatogênicos ph.d. thesis, faculdade de ciências agronômicas univ estadual paulista, br (unpublished). currie, c.r., & a.e. stuart. 2001. weeding and grooming of pathogens in agriculture by ants. proceedings of the royal society b 268: 1033-1039. 689 cardoso, s.r.s. et al. — baits with entomopathogenic fungi for a. s. rubropilosa grosman, d.m., w.w. upton, mccook f.a. & r.f. billings. 2002. attractiveness and efficacy of fipronil and sulfluramid baits for control of the texas leaf cutting ant (atta texana (buckley) (hymenoptera; formicidae)). southwestern entomologist 27(3–4): 251–256. gunasekara, a.s., t. truong, k.s. goh, f. spurlock & r.s. tjeerdema. 2007. environmental fate and toxicolog y of fipronil. journal of pesticide science 32(3): 189–199. hölldobler, b. & e.o. wilson. 1990. the ants. harvard university press, harvard, 732 pp. kermarrec, a., g. febvay, & m. decharme. 1986. protection of leaf-cutting ants biohazards is there a future for microbiological control?, pp. 339-356. in c.s. lofgren, r.k. vander meer (eds.), fire ants and leaf-cutting ants biolog y and management. westview press, boulder. lima, c.a., t.m.c. della lucia, r.n.c. guedes & c.e. veiga. 2003. desenvolvimento de iscas granuladas com atraentes alternativos para atta bisphaerica forel (hymenoptera: formicidae) e sua aceitação pelas operárias. neotropical entomolog y 32(3): 497501. lopez, e., & s. orduz. 2003. metarhizium anisopliae and trichoderma viride for control of nest of the fungus-growing ant, atta cephalotes. biological control 27: 194-200. machado, v., e. diehl-fleig, m.e. silva & m.e.p. lucchese. 1988. reações observadas em colônias de algumas espécies de acromyrmex (hymenoptera-formicidae) quando inoculadas com fungos entomopatogênicos. ciência e cultura 40(11): 1106-1108. nagamoto, n.s., l.c. forti, a.p.p. andrade, m.a.c. boaretto & c.f. wilcken. 2004. method for the evaluation of insecticidal activity over time in atta sexdens rubropilosa workers (hymenoptera: formicidae). sociobiolog y 44(2): 413-431. nagamoto, n.s., r.f. barbieri, l.c. forti, s.r.s. cardoso, s.m. moreira & j.f.s. lopes. 2011. attractiveness of copperleaf-based bait to leaf-cutting ants. ciência rural 41(6): 931-934. poulsen, m., a.n.m. bot, m.g. nielsen & j.j. boomsma. 2002. experimental evidence for the costs and hygienic significance of the antibiotic metapleural gland secretion in leafcutting ants. behavioral ecolog y and sociobiolog y 52(2): 151-157. 849 impact of aqueous plant extracts on trigona spinipes (hymenoptera: apidae) by maria emilene correia-oliveira1*; júlio césar melo poderoso2; adailton freitas ferreira3; ricardo alves de olinda4 & genésio tâmara ribeiro5 abstract the stingless bees are an important component of the insect biomass in many tropical areas, due to their collection of nectar and pollen. trigona spinipes is a widely distributed species in south america, and described as a pollinator of many crops that can be used in a commercial pollinating system. the effects of plant extracts on insects are studied because of the demand for organic food and their selectivity to natural enemies. plant insecticides are reported as a potential agent for the control of insect pests, however little is known about their impact on beneficial insects. this study investigated the survival of trigona spinipes (hymenoptera: apidae, meliponini) fabricius, after exposure to the leaf extracts of azadiracha indica (meliaceae), lippia sidoides (verbenaceae), sapindus saponaria (sapindaceae), anonna squamosa (anonnaceae) cymbopogon winterianum (poaceae), corimbia citriodora (myrtaceae), jatropha curcas (euphorbiaceae) and ricinus communis (euphorbiaceae) and of seeds of azadiracha indica, ricinus communis nordestina and al guarany varieties and jatropha curcas. the extracts that had the greatest influence on the survival of the bees were a. indica at 3% and 7% of concentration, a. squamosa at a concentration of 10% with 68.89% survival and green leaf of r. communis at a concentration of 7%. the results show that 1programa de pós-graduação em entomologia da escola superior de agricultura luiz de queiroz, universidade de são paulo, 13418-900, piracicaba, são paulo, brasil. 2programa de pós-graduação em entomologia, universidade federal de viçosa, 36571-000, viçosa, minas gerais, brasil. 3programa de pós-graduação em ciências agrárias, universidade federal do recôncavo da bahia, cruz das almas, 44.380-000, bahia, brasil. 4departamento de estatística, universidade estadual da paraíba, 58101-001, campina grande, paraíba, brasil. 5departamento de engenharia florestal, universidade federal de sergipe, 49100-000, são cristóvão, sergipe, brasil. * corresponding author: emilenebio@hotmail.com 850 sociobiolog y vol. 59, no. 3, 2012 although the extracts were effective in controlling pests, they may also affect the pollinator trigona spinipes. key words: stingless bees, plant insecticides, trigonineos. introduction one third of human food comes from plants pollinated by bees, making the study on bees important to ensure food safety and security. this shows the need for production models integrating forest ecosystems and agroforestry to improve the performance of these insects (ashman et al. 2004, biesmeijer et al. 2006, williams 1994). stingless bees are an important component of the insect biomass in many tropical areas, due to their collection of nectar and pollen ( johnson and hubbell 1974). the genus trigona includes one of the most populous groups of stingless bees (wille 1983). this genus has highly efficient communication mechanisms for the location of food sources (kerr 1963). the trigona genus is described as pollinator of many crops and can be used in a commercial pollinating system (sanchez et al. 2001). trigona spinipes is a widespread species in south america (schorkopf et al. 2007). it has exposed nests, partially protected by a strong layer of bitumen, which maintains temperature regulation inside the nest, protecting the offspring (wille 1983). these bees usually leave the nest to pollinate after other pollinators have already made their visits to the flowers. typically, two individuals work together in the same flower; however, up to four bees may be simultaneously working in the same flower (koschnitzke 2011). in the last two decades, focus has been placed on the search for new environmentally friendly pesticides aiming to reduce the use of harmful synthetic pesticides (lobitz et al. 1997, pereira et al. 2002; isman 2006). the effects of plant extracts on insects are studied because of the demand for organic food and their selectivity to natural enemies ( junior jones et al. 1979; matos neto et al. 2004). one research line with plant extracts deals with the deleterious effect on organisms (wheeler et al. 2001; brahmachari 2004; gonzález-coloma et al. 2005; defagó et al. 2006). plant insecticides cause less impact and are relatively harmless to natural enemies, pollinators and other non-target organisms. in addition, they are 851 correia-oliviera, m. et al. — impact of plant extracts on t. spinipes rapidly degraded in the environment and present a low risk to users and consumers (isman 2006; schmutterer 1990, tsuzuki et al. 2000). plant extracts from species such as lippia sidoides, sapindus saponaria and azadirachta indica have bactericidal, fungicidal, molluscicidal, larvicidal insecticidal and acaricidal properties (choudhury 1994; girão et al. 2003; isman 2006; kunle et al. 2003; murgu et al. 2006, ross et al. 1995). however, the effects of plant extracts on pollinators are not well known. this study investigated the survival of trigona spinipes (hymenoptera: apidae, meliponini) fabricius exposed to aqueous solutions prepared from leaves and seeds of azadirachta indica a. juss (meliaceae), lippia sidoides cham. (verbenaceae), sapindus saponaria linnaeus (sapindaceae), anonna squamosa linnaeus (annonaceae), corymbia citriodora hill & johnson (myrtaceae), cymbopogon winterianum jowitt. (poaceae), ricinus communis linnaeus (euphorbiaceae) and jatropha curcas linnaeus (euphorbiaceae). materials and methods the test bioassays were conducted at the laboratory of agricultural and forestry pests of the department of forest engineering/federal university of sergipe – brazil – at 25ºc ± 2°c, relative humidity (rh) 60% ± 10% and photophase of 12 hours. we used leaf extracts of azadiracha indica, lippia sidoides, sapindus saponaria, anonna squamosa, cymbopogon winterianum, corimbia citriodora, and ricinus communis and seeds of ricinus communis varieties nordestina and al guarany, jatropha curcas and azadiracha indica. the leaves and seeds were dried in an oven at 40°c for two days, then, milled to obtain powder. the extract was prepared with 10g of the plant powder and 100ml of water. the mixture was stored in a hermetically sealed container for 48 hours. the suspensions were filtered through cotton fabric, thereby obtaining an aqueous extract at 10% that was preserved in amber flasks. the concentrations used in the bioassays were prepared by diluting 10% of the concentrated solution in distilled water to obtain concentrations of 3% and 7%. insects (10 individuals for each concentration and plant species tested) were inoculated in the chest with 1μl of the aqueous extract at different concentrations (3, 7 and 10%) and control (distilled water). the bees were 852 sociobiolog y vol. 59, no. 3, 2012 assessed at 24, 48 and 72 hours after inoculation, kept in petri dishes and fed with sugar solution. we used a completely randomized 4x3 factorial design, consisting of 12 treatments, and the factors were analyzed at three different concentration levels and the control, which were evaluated at three different times. the analyses were carried out in the anova through the family of optimal transformation box-cox (1964) and by applying the hartley test (1950) to check the homogeneity of variances. after using the anova, we applied the f test (p <0.05) to verify possible differences between the factors and the interaction, which, when present, were submitted to the tukey test (p <0.05). results the plant extracts made from leaves of l. sidoides, c. winterianum, j. curcas and seeds of the nordestina variety r. communis did not affect the survival of t. spinipes bees during the evaluation period. other extracts had a greater influence on the survival of the bees. the extracts of a. indica at concentrations of 3% and 7% produced 62.2 and 68.89% survival, respectively, a. squamosa at concentration of 10% produced 68.89% survival and green leaf of r. communis at concentration of 7% produced 63% survival (table 1). when the time factor was isolated for the extracts used in this experiment, we observed that time had a negative influence on the survival of t. spinipes. the mortality of the bees increased after 48 hours for the extracts of leaves of a. indica and r. communis and seeds of j. curcas and after 72 hours for the leaf extracts of c. citriodora, c. winterianum and s. saponaria (table 2). the combination of factors for concentration and time showed a negative effect on the survival of the bees for the leaf extracts of s. saponaria (table 3) and seeds of j. curcas (table 4). these results show that the factors influenced most negatively when analyzed together rather than separately. extracts of j. curcas and r. communis, although belonging to the same family (euphorbiaceae), have different insecticidal action according to the structure of the plant used. both extracts reduced the percentage of surviv853 correia-oliviera, m. et al. — impact of plant extracts on t. spinipes ing individual bees; however, only the leaf extract of r. communis and the seed extract of j.curcas showed significant effects on bee survival. this may be related to the presence of secondary compounds at higher concentration in leaves and seeds of the oil plant jatropha. table 1. survival of t. spinipes after inoculation with the plant extracts used at different concentrations. s. r. communis al = seeds of r. communis variety al guarany. s. r. communis n = seeds of r. communis variety nordestina. f.v. r. communis = green leaves of r. communis. s. j. curcas = seeds of j. curcas. f. j. curcas = leaves of j. curcas. extract 0% 3% 7% 10% l. sidoides 93.33a 96.66 a 95.55a 94.44a s. saponaria 93.33a 83.33b 88.75ab 90.00ab a. squamosa 93.33a 72.22ab 90.00ab 68.88b c. citriodora 93.33a 90.00a 75.55b 85.55ab a. indica 93.33a 62.22c 68.89bc 87.77ab c. winterianum 93.33a 88.89a 86.66a 84.44a s. r. communis al 93.33ab 100.00a 98.89ab 91.11b s. r. communis n 93.33a 94.44a 100.00a 97.78a f.v. r. communis 93.33a 73.33b 63.33b 76.66ab s. j. curcas* 1.97a 1.97a 1.99a 1.84b f. j. curcas 93.33b 100.00a 97.78ab 97.78ab means followed by the same lowercase letter in the rows do not differ (p<0.05) by the tukey test. *data transformed by box-cox (1964) and subjected to the hartley test (1950) corresponding to 93.33% (0 and 3%), 98.90 (7%) and 75.56 (10%). table 2. survival of t. spinipes in the inoculation time for the extract studied. extract 24h 48h 72h l. sidoides 84.17a 76.67 a 73.33a s. saponaria 92.50a 84.17ab 81.67b a. squamosa 87.50a 79.17a 76.67a c. citriodora 93.33a 90.00a 75.55b a. indica 93.33a 62.22c 68.89bc c. winterianum 95.83a 87.50ab 81.67b s. r. communis al 97.50a 95.00a 95.00a s. r. communis n 98.33a 95.83a 94.54a f.v. r. communis 86.67a 71.67b 71.67b f.j. curcas 99.17a 96.67a 95.83a s.j. curcas* 1.97a 1.94b 1.94b means followed by the same lowercase letter in the rows do not differ (p<0.05) by the tukey test. *data transformed by box-cox (1964) and subjected to the hartley test (1950) corresponding to 94.4%, 87.76% and 86.66% respectively, in the times studied. 854 sociobiolog y vol. 59, no. 3, 2012 discussion among the plant families with a potential for use as insecticides, the meliaceae and annonaceae stand out as the main sources of active insecticidal principles affecting the development, behavior and reproduction of insects (schmutterer 1988). this is corroborated by the data obtained in this work, where we observed a. indica with the lowest survival rate of a representative of the meliaceae family. the effect of these plant insecticides is attributed to terpenoids which are chemica ls found in plants that protect them from insect attack (bernays and chappman 1994). the impact of tetranortriterpenoids (secondary components) can vary and include behavioral, physiological or repellent manifestations (tedeschi et al. 2001; qiu et al. 1998). azadiracha indica contains a diverse group of bioactive substances with high biological effect, among which are azadirachtin, meliantrol, salanina and vilasinina (lee et al. 1991). the effect caused by extracts from the family anonnaceae may be related to alkaloids, acetogenins and diterpene, chemical compounds found in extracts of leaves, which are derivatives that exhibit toxic effects on a wide range of organisms (chen et al. 2004; mirela et al. 2007; oliveira et al. 2002). the combined action of all of these substances and their action separately produce different effects on the insects, such as repellency, sterility, egg-laying table 4. results of the interaction between concentration and time of the aqueous extract of seeds of j. curcas on t. spinipes. time (hours) concentration 24 48 72 0 1.98aa 1.95aa 1.96aa 3 1.98aa 1.96aa 1.96aa 7 2.00aa 2.00aa 1.96aa 10 1.93aa 1.77bb 1.77bb means followed by the same lowercase letter in the columns and uppercase letters in the rows do not differ (p<0.05) in the tukey test. table 3. results of the interaction between concentration and time of the aqueous extract of the leaf of s. saponaria on t. spinipes. time (hours) concentration 24 48 72 0 96.66aa 90.00aa 93.33aa 3 96.66aa 80.00aab 73.33abb 7 96.66aa 86.66aa 80.00aba 10 96.66aa 90.00aa 70.00bb means followed by the same lowercase letter in the columns and uppercase letters in the rows do not differ (p<0.05) in the tukey test. 855 correia-oliviera, m. et al. — impact of plant extracts on t. spinipes disorientation, lethal effects, and altered activity of growth regulators, among others (tedeschi et al. 2001; qiu et al. 1998). in this work, we can observe an effect on the survival of bees submitted to a. indica extract; however, this effect may go beyond the survival reduction, and may include factors such as disorientation, which could interfere with bees locating the colony and food source. ricinus communis was another extract in our experiment that caused effects on bees which may have been caused by secondary compounds known for their antimicrobial activity and also for inhibiting mitochondrial respiratory reactions (ferraz 1999). ricinine (secondary compound) is an alkaloid that can be found in all parts of r. communis. the ricinine content varies according to the plant part, and 1.3% of ricinine is found in leaf dry matter; 2.5% in etiolated seedlings; 0.03% in the endosperm of the seed and 0.15% in the fruit shell (holfelder 1998). in j. curcas, the toxic compound curcina is capable of inhibiting the formation of proteins in a fashion similar to ricinine, and can be found at higher concentrations in the seed (arruda et al. 2004; gandhi et al. 1995, kumar and sharma 2008; makkar et al. 1998). this fact is corroborated by our data where the extract of green leaves of r. communis caused more damage than the seed extracts. this may be related to the higher concentration of the compound in the leaves than in seeds. it is possible that the smaller damage caused by the seed extracts can be related to stronger action of glutathione s-transferase enzymes, which act more easily given that the ricinine concentration is lower in seeds. these enzymes belong to a group of multifunctional proteins that play an important role in the detoxification insects and may contribute to the development of resistance to insecticides, catalyzing the insecticide degradation (yu 1996). the induction of the detoxifying enzyme, glutathione s-transferase, usually occurs in the fatty body and midgut. the fatty body is more sensitive to ricinine. the fact that extracts from the leaves of l. sidoides, j. curcas, c. winterianum and seeds of a. communis variety nordestina did not affect the survival of t. spinipes may be attributed to the action of detoxifying enzymes; however, the production of these enzymes has a cost that may not be associated with survival 856 sociobiolog y vol. 59, no. 3, 2012 after 72 hours of evaluation. therefore, further studies should be conducted on the trade-off deriving from the production of these enzymes by bees. conclusion this study shows that although the extracts were effective in controlling pests, they may also affect the pollinator t. spinipes which warrants the need for further research involving the effect of plant insecticides and their effects on beneficial organisms in order to obtain greater safety, selectivity, economic viability and applicability of 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invasive red imported fire ants (rifas), solenopsis invicta buren, on native ant communities at three habitats in south china. by using paired control and treatment plots, the change in diversity and community structure of native ants due to the invasion of red imported fire ants could be observed. ant species richness was reduced by 46 and 33% at rifa-infested lawn and pasture habitats, respectively; however, the ant species richness in the lichee orchard was not affected by red imported fire ants. our results indicated that red imported fire ants became one of several dominant species or the only dominant species in all three habitats in south china. key words: solenopsis invicta buren, ant species, community structure introduction the red imported fire ant (rifa), solenopsis invicta buren, has been listed as one of the most serious invasive alien species in the world (lowe et al. 2004). s. invicta was first introduced into north america, at the seaport of mobile, alabama, usa from south america (buren 1972, vinson & sorensen 1986). after the introduction, its territory increased continually due to natural mating flights and human transportation. s. invicta then invaded australia, new zealand, mainland china and taiwan in 2001-2004 (henshaw et al. 2005, hoffmann & o’connor 2004, zeng et al. 2005a, zeng et al. 2005b, he et al. 2006). the first s. invicta specimen was collected from wuchuan, guangdong on september 23, 2004, and was identified on september 28, 2004 in mainland china (zeng et al. 2005a). later, s. invicta was found in three other prov1red imported fire ant research center, south china agricultural university, guangzhou 510642, p. r. china *corresponding author. email: insectlu@163.com 276 sociobiolog y vol. 59, no. 1, 2012 inces and two special administration districts, i.e. guangxi, hunan, fujian, hongkong and macau (zeng et al. 2005a; zeng et al. 2005b; zhang et al. 2007). s. invicta is a successful invader due to several reasons, including a wide range of climate tolerance, an ability to use a broad range of food resources, high fecundity and reproduction, and rapid colony establishment, especially in disturbed habitats (vinson, 1997, porter & savignano, 1990, callcott & collins, 1996, gotelli & arnett, 2000, korzukhin et al. 2001, porter et al. 1992). in texas, the invasion of s. invicta in the late 1980’s decimated the indigenous ant fauna, and the species richness of ants in rifa-infested areas dropped by 70%, while the total number of native individuals dropped by 90% (porter & savignano 1990). competitive replacement between s. invicta and local ants in the same or similar niches happened slowly (porter et al. 1988, vinson 1990). several studies have also reported that the overall diversity and richness of native ant communities declined after the invasion of fire ants (porter & savignano 1990, wojcik 1994, cook 2003). s. invicta has infested urban, suburban and disturbed habitats in mainland china (zeng et al. 2005, li et al. 2005). a prediction of potential distribution area of s. invicta in china showed that these ants could occupy wide areas in the southeastern part of china with a northern boundary of shandong and tianjin as well as the southern parts of hebei and shanxi provinces (xue et al. 2005, morrison et al. 2004). the objective of this study is to explore the effect of this pest invasion and infestation on pre-existing ant communities in mainland china. this study was initiated in the summer of 2005 and completed in the autumn of 2007. materials and methods study areas three areas, a lichee orchard, a pasture and lawn areas, infested with red imported fire ants in shenzhen of guangdong province in south china were chosen for sampling. the lichee orchard was located around a lake at longgang, shenzhen. the total acreage of the lichee orchard was approximately 100 ha. the percentage of weeds covering the ground surface was 60~80%. two blocks in the lichee orchard were chosen as the experimental sites. the infested block was 1.3 ha, and the density of active fire ant mounds was 18/ ha. the density of the uninfested (control) block was 1.2/ha. the pasture 277 lu, y.-y. et al —effects of solenopsis invicta on chinese ant communities and lawn areas were also in longgang, shenzhen. the percentage of the grass and weeds covering the ground surface was 80~95% at the pasture and lawn areas. the infested pasture area was 1.1 ha, and the control area was 1.3 ha. the density of active fire ant mounds reached 93/ha in the pasture area, but the average was approximately 34/ha. the infested lawn area was about 1.5/ha with an active fire ant mound density of 95/ha, and the control area was 0.8/ha. few weeds were found in the lawn area with the grass coverage of 100%. survey of ant community abundance and diversity ground-dwelling ant species in both treatment and control sites were sampled using pitfall traps, bait vials, and visual searching according to previous description (heyer et al. 1994). the combination of these methods is ideal for biodiversity monitoring programs and the comparison of ant communities among different habitats (greenslade 1973, heyer et al. 1994). the collection was done twice a month from january to december. in each plot, 20 pitfall traps were divided into 5 groups. the interval distance between groups was 10 m, and total area of 4 pitfall traps in each group was 1 m2. traps were 100 ml plastic vials (15 cm in length, and 3 cm in diameter) filled with 45% ethyl alcohol at 1/3 volume as a preservative. each trap was inserted into the ground and the upper rim of the vial was same level as the soil surface. after 24 h, each trap was removed from the soil, capped, and brought to the laboratory. bait vials were 50 ml plastic vials (7 cm in length, and 3 cm in diameter) containing a 5 × 25 mm circular hotdog slice (shuanghui™, guangdong shuanghui co., guangodng ) with several drops of honey (baoshengtm, baosheng yuan co., shanghai). in total, 30 bait vials were placed at a distance interval of 10 m in each plot. vials were set up at 08:00-10:00 during the warmer months and 12:00-14:00 during the colder months. vials remained exposed for 30 min after they were covered, sealed, and then transported to the laboratory. ants were collected and kept in 75% ethyl alcohol for identification and counting. if the bait was dominated by an individual ant species before the end of 30 min time period, it was covered and sealed to collect ants as soon as possible. the bait vial experiments were completed twice a month from january to december. 278 sociobiolog y vol. 59, no. 1, 2012 the third method for collecting ants was visual searching. each sampling plot was visually observed for two man-hours by three graduate students with previous survey experience of red imported fire ants. litter, bare ground, tree trunks, foliage, decaying wood, and other surfaces were searched. representative ants were collected and preserved. this method was also conducted twice a month from january to december. all ants collected by these methods were initially identified through the comparison with specimens housed in the south china agricultural university. identifications of ant species were confirmed by weiqiu zhang (department of entomolog y, south china agricultural university, guangdong ), shanyi zhou (school of biological science, guangxi normal university, guangxi) according to the books written by tang et al. (1995), wu et al. (1995), and zhou (2002). voucher specimens have been deposited in the red imported fire ant research center, south china agricultural university, guangdong, china. statistical analysis at each sampling site, diversity indices of ant communities in rifa-infested and uninfested areas were calculated using identified ant species from above three methods. the shannon-weaver species diversity index (h’) was used to assess community structure (keping ma et al. 1994). the shannon-weaver diversity index (h’) was calculated by following equation: h’ = -σ p i × ln (p i ), where p i is the proportion of individuals in the ith species relative to total species (keping ma et al. 1994). the diversity index h’ was compared between the infested and uninfested areas through t-test (keping ma et al. 1994). results ant species table 1 shows all species collected at each site through visual searching, pitfall traps and baits. the collected samples had 24 ant species belonging to 18 genera, 5 subfamilies, which included 5 genera and 5 species of ponerinae, 1 genus and 1 species of dorylinae, 7 genera and 10 species of myrmicinae, 2 genera and 2 species of dolichoderinae, and 3 genera and 5 species of formicinae. 279 lu, y.-y. et al —effects of solenopsis invicta on chinese ant communities only 7 ant species were observed in the lawn area infested by red imported fire ants when all sampling methods were combined, while 13 ant species were found in the uninfested area. in addition, 10 and 15 ant species were observed at the rifa-infested and uninfested pasture area respectively. 13 ant species were recorded at the rifa-infested lichee orchard sites, and 14 ant species were recorded at the uninfested sites. the reduction of ant species richness was up to 46, 33 and 7% at rifa-infested lawn, pasture, and lichee orchard sites respectively when compared with the uninfested area (table 1). generally, compared to the uninfested sites, the reduction of ant abundance was mainly due to the disappearance of some ant species in the infested sites of table 1. ant species present in both infected and control plots at three habitats. subfamily species lichee orchard pasture lawn s. invicta present s. invicta absent s. invicta present s. invicta absent s. invicta present s. invicta absent ponerinae diacamma rugosum √ √ odontoponera transversa √ √ √ √ leptogenys chinensis √ √ √ √ √ hypoponera confinis √ √ pachycondyla luteipes √ dorylinae dorylus orientalis √ myrmicinae pheidole pieli √ √ √ √ √ pheidole yeensis √ √ √ √ √ crematogaster biroi √ tetramorium smithi √ √ √ √ tetramorium bicarinatum √ pheidologeton diversus √ √ √ √ √ solenopsis invicta √ √ √ monomorium concolor √ √ √ √ √ monomorium orientale √ √ √ √ monomorium pharaonis √ √ meranoplus bicolor √ dolichoderinae tapinoma melanocephalum √ √ √ √ √ iridomyrmex anceps √ formicinae plagiolepis rothneyi √ √ √ paratrechina flavipes √ √ √ √ paratrechina longicornis √ √ √ √ √ paratrechina bourbonica √ √ camponotus dolendus √ *a √ designates the species as present. 280 sociobiolog y vol. 59, no. 1, 2012 two habitats. for example, 7 ant species (o. transversa, l. chinensis, h. confinis, p. luteipes, m. orientale, m. pharaonis and p. rothneyi) were not present at the pasture sites with red imported fire ants. while at the infested sites, several ant species (p. flavipes, p. longicornis, and p. bourbonica) were also recorded at the corresponding uninfested sites. dominant species in ant communities dominant species of ants in three habitats with and without red imported fire ants are listed in table 2. the compositions of ant community were obviously changed after the introduction of red imported fire ants in our test sites in southern china. the fire ants became one of the dominant species or the only dominant species. the dominant ant species in the lichee orchard without infestion of fire ants were t. melanocephalum, p. pieli, and p. yeensis (table 2). however, in the rifa-infested lichee orchard, t. melanocephalum, s. invicta and p. pieli were dominant (table 2). moreover, the most dramatic change in table 2. dominant species of ants in lichee orchard, pasture and lawn areas. habitat number of dominant species dominant species percentage (%) *of dominant species lichee orchard s. invicta present 3 tapinoma melanocephalum solenopsis invicta pheidole pieli 36.8 a 26.3 b 20.1 bc s. invicta absent 3 tapinoma melanocephalum pheidole pieli pheidole yeensis 66.7 a 18.1 b 11.3 bc pasture s. invicta present 2 solenopsis invicta tapinoma melanocephalum 74.6a 21.3 b s. invicta absent 3 pheidologeton diversus pheidole pielii tapinoma melanocephalum 42.7 a 16.8 b 15.4 b lawn s. invicta present 1 solenopsis invicta 98.9 s. invicta absent 3 tapinoma melanocephalum pheidologeton diversus pheidole pielii monomorium concolor 32.7 a 26.5 ab 20.1 b 11.4 c data of treatment and control in the same habitat followed by same letter represents no significance at 0.05 (ducan’s dmrt). 281 lu, y.-y. et al —effects of solenopsis invicta on chinese ant communities ant community structure was observed at the lawn site. before the invasion of red imported fire ants, 4 dominant ant species were t. melanocephalum, p. diversus, p. pieli and m. concolor. in contrast, once the red imported fire ants occurred at the lawn site, they changed into the only dominant ant species. diversity of ant community three parameters of ant communities in different habitats were calculated (table 3). the results revealed an obvious difference in ant species richness among three habitats. compared with the area without red imported fire ants, the total number of individuals in the infested sites exhibited a significant increase. in addition, species diversity indices revealed a decrease at the rifa-infested pasture and lawn sites. for example, the total ants captured at the rifa-infested lawn site were 22475, while only 5865 ants were captured at the uninfested lawn sites. species diversity index h’ dropped from 1.66 to 0.07. however, the ant species diversity index h’ in the lichee orchard without red imported fire ants exhibited an increase. discussion the invasion of red imported fire ants resulted in a decline in species richness and changed the community structure of native ants in two habitats of southern china. moreover, red imported fire ants replaced previously table 3. indices of ant communities in different habitats. habitat species number (s) total number of individuals (n) species diversity index (h’) lichee orchard s. invicta present 14 10145 1.61* s. invicta absent 14 6162 1.03 pasture s. invicta present 10 19466 0.70 s. invicta absent 15 6935 1.78** lawn s. invicta present 7 22475 0.07 s. invicta absent 13 5865 1.66* *and ** represent data where treatment and control in the same habitat were significantly different at 0.05 and 0.01, respectively. 282 sociobiolog y vol. 59, no. 1, 2012 dominant ant species and became one of the dominant species or the only dominant species. however, several problems in this study still need to be mentioned. the first one is that the density of active red imported fire ant mounds is relatively low at all experimental sites, which can result in a great variability in the population density of s. invicta. for example, the density of active mounds varies from 20-2100 colonies/ha at some places in wuhucan, and from 12-1600 colonies/ha at other places of shenzhen (zeng et al. 2005a). previous studies have revealed an overall negative correlation between s. invicta density and overall ant species richness as well as abundance (stein & thorvilson 1989, camilo & phillips 1990, gotelli & arnett 2000). in the present study, we have analyzed the impact of fire ants at a low population density on native ant communities in some habitats of southern china. therefore, ecological changes such as invasion of high-density fire ants are likely to cause much more dramatic impacts at some locations. the second problem is that there are many habitats in southern china. three common habitats such as lichee orchard, pasture, and lawn were selected as representative experimental sites to study the community structure and diversity of ants, but it is possible that high false positive results could have occurred due to the selection of limited types of ant habitats. given the environmental factors, differing impacts of s. invicta on ant diversity in three types of habitats probably showed that invader eco-function or eco-effect was related to the types of ecosystems and habitats in south china. the small impact of fire ants on the native ant community in lichee orchards may be due to high-population trees. in contrast, an obvious change in ant community structure at the lawn and pasture areas are likely due to the open community that is easily invaded by alien ant species. the ecological impacts of red imported fire ant invasion have been explored in the past decades in the us. the invasion of red imported fire ants has been proved to reduce the species richness and abundance of many native ants and other arthropods (nichols & sites 1989, camilo & phillips 1990, porter & savignano 1990, morris & steigman 1993, jusino-atresino & phillips 1994, kaspari 2000, gotelli & arnett 2000, summerlin et al.1984, vinson 1991, stoker et al. 1995). however, a positive correlation between s. invicta density and the diversity of ants and arthropods in stable post-invasion habitats has 283 lu, y.-y. et al —effects of solenopsis invicta on chinese ant communities also been established (morrison & porter 2003). although a possible reason is that good environmental factors are good for both ants and other species, the post-invasion correlation probably does not reflect the ecological impacts of the original invasion. after red imported fire ants were discovered at two locations in brisbane, australia in february, 2001, ecological effects of s. invicta were further investigated (nattrass & vanderwoude 2001), revealing much lower abundance in ant species and invertebrates at the rifa-infested sites when compared with uninfested sites (nattrass & vanderwoude 2001). these studies indicate that the invasion of s. invicta may have a substantial impact on species richness and diversity of ant communities in disturbed urban and rural areas of china. however, the overall impact of s. invicta on ecosystems and biodiversity in mainland china can only be determined by longer term and larger scale investigations in a wider variety of habitats. acknowledgments we would like to thank zhendong song and jun huang for sample collection and surveys as well as weiqiu zhang for the identification of ant specimens. we also thank sanford porter (usda-ars) for manuscript editing. this work is supported by the national natural science foundation of china (award# 305712427) and the national basic research program of china (award# 2009cb119206). references buren, w.f. 1972. revisionary studies on the taxonomy of the imported fire 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university press 123 molecular marker to identify two stingless bee species: tetragonisca angustula and tetragonisca fiebrigi (hymenoptera, meliponinae) by ana lúcia paz barateiro stuchi1, vagner de alencar arnaut de toledo2, denise alves lopes1, liriana belizário cantagalli1 & maria claudia colla ruvolo-takasusuki1* abstract tetragonisca angustula and t. fiebrigi esterases were biochemically characterized by their inhibition pattern and thermostability. workers of both species were collected from nests at the state university of maringá. in t. fiebrigi three esterases were observed: est-1 (β-esterase, cholinesterase i), est-2 (α-esterase, cholinesterase ii) and est-4 (αβ-esterase, carboxylesterase). in t. angustula two esterases were detected: est-3 (β-esterase, acetylesterase) e est-4 (αβ-esterase, carboxylesterase). t. angustula est-3 showed the highest thermostability, and it was not observed above 54°c, while in t. fiebrigi est1 and est-2 were not detected above 52°c. through this characterization, it was observed that est-4 of t. angustula and t. fiebrigi showed identical biochemical characteristics, and probably those esterases are encoded by the same gene in the two species. together, the biochemical characterization and molecular markers show that the two species are differentiated and secondary contact between the populations can still be occurring. key words: stingless bees, isoenzymes, inhibition, thermostability, taxonomy introdution the stingless bees evolved from a group of wasps which at some point in evolution no longer transmitted to its descendants the genetic characteristics universidade estadual de maringá, departamento de biologia celular e genética, av. colombo, 5790, maringá – paraná cep 87.020-900, (44) 3261-4678. 2universidade estadual de maringá, departamento de zootecnia, av. colombo, 5790, maringá – paraná cep 87.020-900. *corresponding author: mccrtakasusuki@uem.br 124 sociobiolog y vol. 59, no. 1, 2012 responsible for the sting formation (alonso 1998). the explanation for the sting loss in this group of bees is probably related to the fact that their colony is not exposed when the bees swarm, and they usually nest in concealed and well protected places (alonso & paim 2001). the stingless bees are among the most common pollinators of tropical environments, and in certain regions they are the dominant bee species, visiting various cultures (macías-macías et al. 2009). these insects are a diverse group, in which includes over 400 species which shows high variability in physiolog y, morpholog y and size, ranging from 0.2 mm in the genus trigonisca to over 20 mm in some melipona species (michener 2000; moure et al. 2007). the meliponines are though to have originated in the gondwana western continent; this hypothesis is based on the fossil findings and by biogeography (camargo & menezes-pedro 1992). they dwell mostly in regions of tropical and temperate subtropical weather of the world (nogueira-neto 1997). according to moure (1961), there are two tribes in the meliponinae subfamily: meliponini and trigonini. the meliponini are characterized for not building real cells, therefore, queens, workers and males born and develop to adulthood in cells of the same size. the trigonini consists of a highly diversified group, with dozens of genera and often build real cells, bigger than the other, where queens-to-be emerge (nogueira-neto 1997). according to castanheira & contel (2005), in tetragonisca angustula (trigonini), there are two known subspecies, which are differentiated through the coloring of the mesepisterna. t. angustula angustula presents a black mesepisterna, while t. angustula fiebrigi presents a yellow one. however, according to camargo & pedro (2007), they were considered as two taxonomically distinct species, t. angustula and t. fiebrigi. considering that there is still disagreement regarding the classification of these bees in species or subspecies, the development of new studies to detect the occurrence of markers which enable an accurate classification is essential. the geographical distribution for t. angustula fiebrigi was firstly described by schwarz (1938) and nogueira-neto (1970). this subspecies is present in brazil in mato grosso state, in the paraná river basin and also in paraguay and argentina. in 2005, castanheira & contel (2005) related the occurrence of t. angustula fiebrigi in north-western paraná, londrina and maringá counties. this subspecies was also collected in altônia county, paraná state (ruiz 2006; alves 2006). 125 stuchi, a.l.p.b. et al. —molecular markers to identify two tetragonisca species the subspecies t. angustula angustula is distributed in most states throughout brazil (iwama & melhem, 1979; camargo & posey, 1990), as well as in panama (roubik 1983), venezuela (vit et al. 1994) and costa rica (veen & sommeijer 2000a, b). regarding the population genetics studies and the differentiation between these two species, oliveira et al. (2004), by using the molecular marker rapd, identified a marker for the tetragonisca subspecies, the primer opl-11. later, baitala et al. (2006) showed that with the use of rapd markers, it is only possible to separate populations of tetragonisca, not being able to detect a marker for subspecies, even using the primer mentioned above. among the molecular markers, the esterase isoenzymes which present high multifunctional hydrolytic activity and catalyze the hydrolysis of a large number of esters can be highlighted (walker & mackeness 1983). based on the sensibility to the synthetic substrate which those enzymes hydrolyze in vitro, two groups can be distinguished in insects, the α-esterases which hydrolyze preferably α-naphthyl-acetate, and β-esterases which hydrolyze preferably β-naphthyl-acetate (oakeshott et al. 1993). also as a classificatory criterion, according to the sensibility to different inhibitors of the enzymatic activity and to the amino acid residues in its active site, there are four esterase classes, the acetylesterases (e.c. 3.1.1.6), the arylesterases (e.c. 3.1.1.2), the carboxylesterases (e.c. 3.1.1.1) and the cholinesterases which includes the acetylcholinesterases (e.c. 3.1.1.7) and the pseudocholinesterases (e.c. 3.1.1.8) (healy et al. 1991). little is known about the esterases in t. angustula and t. fiebrigi, known as the jataí stingless bee. ruvolo-takasusuki et al. (2006) showed the esterase activity regions in t. angustula, and these authors have found two regions, which were characterized as est-1 (β-esterase) and est-2 (αβ-esterase). the objective in the present study is to identify isoenzyme esterase as a biochemical marker which differentiates the two species of jataí stingless bee, t. angustula and t. fiebrigi. materials and methods material adult jataí stingless bee workers were collected from two natural nests located within the state university of maringá (universidade estadual de 126 sociobiolog y vol. 59, no. 1, 2012 maringá), paraná (23o24’40’’ s; 51o56’23’’ w), one nest was of t. angustula species and the other of t. fiebrigi. after collection, the bees were euthanized and stored in properly labelled and numbered containers, at –20ºc. preparing of the samples and electrophoresis page each bee worker had its head/thorax removed and homogenized individually in propylene tubes 1.5 ml containing 35 µl of 0.1% 2-mercaptoetanol solution plus glycerol at 10%. the samples were centrifuged at 56.000g for 10 minutes at 4ºc. vertical electrophoreses were performed, using page gels at 8% concentration and stacking gel at 5% concentration. tris-glicine at 0.1 m ph 8.3 was used as buffer. the gels were submitted to electrophoresis at 200 v for 5 hours. the gel was incubated for 30 minutes in 50 ml of sodium phosphate buffer solution (0.1 m ph 6.2), for staining. then the buffer was discarded and the staining solution was added 50 ml of sodium phosphate buffer at 0.1 m ph 6.2, 0.03 g of α-naphthyl acetate; 0.03 g of β-naphthyl acetate, 0.06 g of fast blue rr salt. the gel was incubated until the bands became visible. the gels remained in fixation solution (acetic acid at 75% and glycerol at 10%, dissolved in 1.000 ml of distilled water) for at least 24 hours. the gels were then soaked in gelatin at 5% and placed between two sheets of wet cellophane paper, stretched, pressed and kept in room temperature until completely dry (ceron et al. 1992). inhibition tests the head/thorax extracts of each worker were used twice in the same page gel, the first trial as the control, and the second as the inhibition test. for the staining, first, the gel was cut and separated in two parts: control and inhibition. each part, separately, was incubated for 30 minutes in 50 ml of sodium phosphate buffer solution (0.1 m ph 6.2). the inhibitor to be tested was the incubation buffer for the test gel (organophosphate – 60 µl, parachloromercuriobenzoate (ρ-cmb) – 0.01 g or eserine sulphate – 0.06 g ). after the incubation period, the buffer was discarded and the staining solution added as previously described. for the test, the inhibitor was added in the amounts described above. bands in the control gel were compared with the inhibitor gel, and an inhibition table was produced. 127 stuchi, a.l.p.b. et al. —molecular markers to identify two tetragonisca species thermostability the thermostability test for the esterases was done through the pre incubation of the samples for 5 minutes at a temperature that ranged from 52oc to 58oc. after the incubation, 10 µl of the supernatant was applied in the page gel and submitted to electrophoresis. as control, head/thorax extracts of t. angustula and t. fiebrigi which were not submitted to pre incubation were used. after the electrophoresis, the gels were stained for the esterase visualization as described above. results and discussion many differences were detected in the electrophoretic analysis, regarding the number of observed esterases, migration pattern, affinity to the substrate and thermostability, providing input that the tetragonisca genus has two species, t. fiebrigi and t. angustula, as suggested by camargo & pedro (2007). the number of regions with esterase activity varied according to the species; in t. fiebrigi, three esterases were observed, which were called est-1 (showing a most anodic migration), est-2 (showing a intermediary migration) and est-4 (showing a least anodic migration), while in t. angustula, two esterase activity regions were observed: est-3 (most anodic) and est-4 (least anodic) (fig. 1). ruvolo-takasusuki et al. (2006) and stuchi et al. (2008) classified the t. angustula esterases according to their migration pattern as follows: est-1 (most anodic) and est-2 (least anodic). however, due to the results obtained in the present study, it has been suggested that these esterases could be classified as est-3 (most anodic) and est-4 (least anodic), and est-3 is a specific region of t. angustula (fig. 1). according to the specificity to the substrates α-naphthyl-acetate and β-naphthyl-acetate in t. fiebrigi the est-1 has been classified as β-esterase, est-2 as α-esterase and est-4 as αβ-esterase (fig. 1). the biochemical characteristics described above identified the est-1 and est-2 as molecular markers for t. fiebrigi, and est-3 as molecular marker for t. angustula. insect esterases have been divided in four distinct classes based on their sensibility to three groups of inhibitors: organophosphates, eserine sulphate and sulphidril reagents (healy et al. 1991). in this work, after the electro128 sociobiolog y vol. 59, no. 1, 2012 fig. 1. esterase isoenzymes profile in the page system from head/thorax extracts of the tetragonisca fiebrigi (a) and tetragonisca angustula (b) workers. table 1. inhibition pattern for the characterization of esterases using the inhibitors malathion, ρ-cmb, and eserine sulfate in tetragonisca fiebrigi and tetragonisca angustula. (+) inhibition, (-) no inhibition. esterase malathion ρ-cmb eserine sulfate classification t. fiebrigi est-1 + + cholinesterase (i) est-2 + + + cholinesterase (ii) est-4 + + carboxylesterase t. angustula est-3 acetylesterase est-4 + + carboxylesterase 129 stuchi, a.l.p.b. et al. —molecular markers to identify two tetragonisca species phoresis, head/thorax extracts from the worker bees were submitted to the three groups of inhibitors, the results concerning the inhibition pattern can be observed in table 1. due to their inhibition pattern against the used inhibitors, est-1 and est2 in t. fiebrigi can be classified as cholinesterases, est-1 as a cholinesterase type i, while est-2 is a cholinesterase type ii. the est-4 from both species is a carboxylesterase (table 1). in t. angustula, est-3 is an acetylesterase. the analyzed esterase inhibition pattern shows that t. fiebrigi presents two cholinesterases and a carboxylesterase and that t. angustula presents a carboxylesterase (table 1). the carboxylesterases and the cholinesterases are common isoenzymes in insects, possibly due to their fundamental role in the process of detoxification of xenobiotic compounds, contributing to a resistance to insecticides. regarding the organophosphates, these mechanisms involve the increase in the metabolic detoxification by hydrolysis or the kidnapping of these compounds (hemingway 2000; lee & lees 2001; cui et al. 2007) or structural changes in the acetylcholinesterase, the primary target for this insecticide class (hsu et al. 2006). therefore, bees from the genus tetragonisca may be used in future studies as bio-indicators of the presence of pesticides in natural and cultivated areas. the thermostability tests have shown that there are differences in the esterases inhibition depending on the temperature in which they are submitted. in t. fiebrigi, when the head/thorax extracts were submitted to a temperature of 52°c, the est-4 presented a partial reduction of its activity, while the est-1 and est-2 presented total inhibition. regarding t. angustula, partial inhibition was observed for est-4, while est-3 was not inhibited at the same temperature. however, from 54°c, every esterase lost its activity (fig. 2 and table 2). concerning the thermostability it is possible to observe that the est-4 from t. fiebrigi and est-3 and est-4 from t. angustula are more thermostable than the est-1a from apis mellifera. ruvolo-takasusuki et al. (1997) have observed that there was no activity of the est-1a of a. mellifera when extracts from abdomen were previously incubated at 50°c or for over 4 minutes, while the t. angustula and t. fiebrigi ones were submitted to 52°c for 5 minutes and still remained active (table 2). 130 sociobiolog y vol. 59, no. 1, 2012 est-3 of t. angustula was the most thermostable esterase (fig. 2), losing its activity at 54°c. ruvolo-takasusuki et al. (1998) observed a high thermostability for a. mellifera est-2, which showed activity after pre-incubation at 60°c for 8 minutes. the esterase biochemical characterization showed that only est-4 is common in the two species. the est-1 (t. fiebrigi) and est-3 (t. angustula) probably have differentiated by mutations along the evolution of both fig. 2. thermostability profile of the esterase isoenzymes in the page system from head/thorax extracts of tetragonisca fiebrigi and tetragonisca angustula workers. samples 1-2, 5-8 and 13-16 are head/thorax extracts of tetragonisca angustula and samples 3-4, 9-12 and 17-20 are head/thorax extracts of tetragonisca fiebrigi. table 2. inhibition patterns of esterases in tetragonisca fiebrigi and tetragonisca angustula at 52 °c and 54 °c. (++) total inhibition, (+) partial inhibition, (-) no inhibition. temperature t. fiebrigi t. angustula est-1 est-2 est-4 est-3 est-4 52°c ++ ++ + + 54°c ++ ++ ++ ++ ++ 131 stuchi, a.l.p.b. et al. —molecular markers to identify two tetragonisca species species, and est-2 (t. fiebrigi) may have been originated by duplication and later mutations. the use of rapd markers has shown that t. a. angustula and t. a. fiebrigi may be separated in two groups considered subspecies (oliveira et al. 2004). on the other hand the use of rapd by baitala et al. (2006), in five t. a. angustula and t. a. fiebrigi populations from junqueirópolis (sp), maringá (pr) and cianorte (pr), have not permitted the separation of the two subspecies; only the populations from são paulo and paraná states were separated by rapd markers. alves (2006) has applied rapd for the analysis of three jataí populations in north-eastern paraná state, ivatuba, umuarama and altônia counties, and in ivatuba collected only t. a. angustula and in umuarama and altônia only t. a. fiebrigi. the results have shown that the two bee species are separated with genetic distance values which justify their taxonomy, as between t. a. angustula and t. a. fiebrigi the genetic distance value according to nei (1978) was 0.23 and between the two t. a. fiebrigi populations was 0. 0507. castanheira & contel (2005) have performed a morphometric analysis of the wings, mesepisterna staining and hexokinase polymorphism of t. a. angustula and t. a. fiebrigi bees from various regions within são paulo, paraná, mato grosso do sul and minas gerais states of brazil. the mesepisterna coloring and the allele hk88 frequency have enabled the authors to suggest that there is clinal distribution or race mixing between the two subspecies. a high correlation between the yellow coloring of the mesepisterna and the allele hk88 frequency has been observed. diniz-filho et al. (1998) have performed morphometric analysis of t. angustula from central and south-eastern brazil. the obtained variations could be explained by secondary contact among previously isolated races. the controversy regarding the two species or subspecies of tetragonisca seems to be nearly over, because the electrophoretic profile and the esterase biochemical characterization of these stingless bees have shown that they are two distinct species. the obtained results by the esterase biochemical characterization have shown that there are differences in these loci between the two species, which justifies their classification according camargo & pedro (2007). 132 sociobiolog y vol. 59, no. 1, 2012 the results show that the jataí stingless bees are an important group for speciation studies. the two species have probably originated from one that may have been differentiated due to geographical isolation. anthropic action and a relatively short period of geographical isolation may have put the two species in touch again which could be causing the hybridization detected in the study performed by castanheira & contel (2005). the observed similarities using the rapd marker could be due to the use of nonspecific primers and the short time that the two species have been apart. molecular markers such as pcr-rflp may contribute to new taxonomic analysis and the systematics of these native stingless bees. finally, it may be concluded that the results presented confirm that the jataí stingless bees are actually two species, t. fiebrigi and t. angustula, and that the est-1, est-2 and est-3 are molecular markers which identify them efficiently. references alonso, w. j. 1998. abelhas sem ferrão: centenas de espécies para polinização, produção de mel, lazer e educação. artigos técnicos. animais de criação – abelhas. 626. 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meliponinae). brazilian journal of morphological science 23: 431-434. stuchi, a. l. p. b., m. c. c. ruvolo-takasusuki & v. a. a. toledo. 2008. análise da genética de populações em abelhas jataí (tetragonisca angustula latreille) por meio de isoenzimas, magistra 20: 68-77. schwarz, h. f. 1938. the stingless bees (meliponidae) of british guiana and some related forms. bulletin of the american museum of natural history 74: 437-508. veen, j. w. van & m. j. sommeijer. 2000a. observation of g ynes and drones around nuptial flights in the stingless bees tetragonisca angustula and melipona beecheii (hymenoptera, apidae, meliponinae). apidologie 31: 47-54. veen, j.w. van & m. j. sommeijer. 2000b. colony reproduction in tetragonisca angustula (apidae, meliponini). insect sociolog y 47: 70-75. vit, p., s. bogdanov, v. kilchenmann. 1994. composition of venezuelan honeys from stingless bees (apidae: meliponinae) and apis mellifera l. apidologie 25: 278-288. walker, c. h. & m. i. mackness. 1983. esterases: problems of identification and classification. biochemical pharmacolog y 32: 3265-3269. 203 comparison of coptotermes formosanus and coptotermes gestroi (blattodea: rhinotermitidae) field sites and seasonal foraging activity in hawaii by nirmala k. hapukotuwa1 & j. kenneth grace1* abstract field surveys were carried out from january 2010 to june 2011 to record the environmental properties of coptotermes formosanus shiraki and c. gestroi (wasmann) (blattodea: rhinotermitidae) field sites on the island of oahu, hawaii; and to document seasonal patterns in c. formosanus and c. gestroi foraging activity. the two field sites selected differed in elevation, soil characteristics, vegetation, and mean temperature and humidity patterns. the c. formosanus colony was located on the manoa campus of the university of hawaii, near miller hall; while the c. gestroi colony was located 40 km away at the barber’s point horse stables in kalaeloa, oahu (formerly barber’s point naval housing ). mean temperature and humidity were recorded monthly at each field site using a hobo® data logger (1000-1100h), soil samples were taken from each site and analyzed for physical properties, and vegetation type/s were observed, photographed, and samples brought to laboratory for identification. during each site visit, the number of active termite collection traps (termites present) out of a total of 22 traps per site were counted. the c. gestroi field site was generally warmer than that of c. formosanus. both termite species exhibited irregular activity throughout the year, although c. formosanus was more active in general during cooler months (winter) than c. gestroi; while c. gestroi was generally more active during late spring and summer months. these results, as well as introduction histories, may help to explain c. gestroi distribution patterns in hawaii. 1college of tropical agriculture & human resources, university of hawaii at manoa, 3050 maile way, gilmore 202, honolulu hi 96822, usa *corresponding author. email: kennethg@hawaii.edu 204 sociobiolog y vol. 59, no. 1, 2012 introduction the hawaiian islands are an archipelago of eight major islands in the north pacific ocean, formed by volcanic activity over a hotspot in the earth’s mantle. the hawaiian island archipelago is the most isolated group of islands on earth. the islands have a climate typical of the edges of the tropical region, with temperatures ranging between 250c 320c. the islands receive most of their rainfall from the trade winds during the winter months (october to april) (ziegler 2002). eight introduced termite species have been recorded in hawaii (grace et al. 2002). among them, the formosan subterranean termite, coptotermes formosanus shiraki (blattodea: rhinotermitidae), was introduced over 100 years ago and is the most economically important and widely distributed insect pest in hawaii (tamashiro et al. 1987, grace et al. 2002). in contrast, the more recently introduced asian subterranean termite, coptotermes gestroi (wasmann), formally known as coptotermes vastator light in the pacific (yeap et al. 2007), has a very limited and localized distribution on the island of oahu (woodrow et al. 2001). of the drywood termites (kalotermitidae), incisitermes immigrans and neotermes connexus are long-term residents of hawaii that are only very rarely found in structural wood (woodrow et al. 1999), and cryptotermes brevis is the most severe drywood termite pest. cryptotermes cynocephalus and incisitermes minor have limited distributions on the island of oahu (grace et al. 2002). a rotten wood termite, zootermopsis angusticollis (termopsidae), is known to occur in the kula region, on the island of maui (grace et al. 2002). c. formosanus is primarily found in subtropical and temperate regions, whereas c. gestroi occurs largely in the tropics (su 2003, grace 2006). the distributions of these two species overlap in florida (scheffrahn & su 2005), taiwan (shiraki 1909, tsai & chen 2003, li et al. 2009), and hawaii (swezey 1914, weesner 1965, grace 2006). in hawaii, c. formosanus is distributed throughout the island of oahu (as well as other islands), while c. gestroi is currently limited to the southwest side of oahu. coptotermes gestroi appears to be expanding in distribution on oahu relatively slowly (uchima & grace 2009; grace, unpublished observations). 205 hapukotuwa, n.k. & j.k. grace — coptotermes foraging activity in hawaii uchima & grace (2003a) estimated the foraging distance and foraging population size of two c. gestroi colonies on oahu using the mark-releaserecapture method. the two c. gestroi colonies were present in a residence in kalaeloa, oahu (formerly barber’s point naval housing ) and in the barber’s point horse stables in kalaeloa. they estimated the foraging population in the horse stables as 679,193±120,065 individuals, foraging area as 287.2m2 and foraging termite biomass as 1.6±0.3 kg. the foraging population in the residence in kalaeloa was estimated as 186,593±51,910 individuals, the foraging area as 10.5m2 and the foraging termite biomass as 0.5±0.1kg. these results suggested that c. gestroi may have smaller colonies and foraging areas than c. formosanus in hawaii. the objectives of the present study were: (1) to record environmental properties of two field sites of c. formosanus and c. gestroi in order to obtain a basic idea of their comparative habitats; and (2) to document any seasonal variation in c. formosanus and c. gestroi foraging activity by checking field collection traps every month. materials and methods site descriptions the two field sites of the two different termite species on the island of oahu differ in elevation and temperature. for c. formosanus, we used a colony near miller hall on the manoa campus of the university of hawaii (210 17’ n, 1570 99’ w; 23.47m above sea level; average rainfall 267.21cm; annual mean temperature 18.610c). for c. gestroi, we used a colony located 40 km from the manoa campus, at the barber’s point horse stables (kalaeloa) (210 19’ n, 1580 02’ w; 9.14 m above sea level; annual rainfall 53.09 cm; annual mean temperature 240c). field surveys field surveys were carried out from january 2010 to june 2011. to document environmental properties and seasonal variations in termite activity, temperature and humidity at each site was measured monthly using a hobo® data logger (1000-1100h), soil samples were taken from each site and analyzed for physical properties, and vegetation type/s were observed, photographed, and samples brought to laboratory for identification. during each monthly 206 sociobiolog y vol. 59, no. 1, 2012 site visit, the number of active termite collection traps (termites present) was counted. each site contained 22 wooden box-type termite aggregation traps, after the design of tamashiro et al. (1973). results and discussion we observed some differences in temperature, humidity, vegetation, soil types and soil water content at the two field sites (table 1). at kalaeloa (c. gestroi field site), there were a few thorny shrubs (most of the time very dry), high silt content in the soil, low soil water content and relatively high temperature. the c. formosanus field site at the university of hawaii at manoa had some vegetation (most of the time wet), low silt content in the soil compared to kalaeloa, relatively high sand content, high soil water content and a relatively lower temperature than the kalaeloa field site. many studies have correlated climatic variables such as minimum and maximum temperatures and annual rainfall to the range limits of species ( jeffree & jeffree 1996, bullock et al. 2000). our study presents preliminary data regarding climatic variation between two termite habitats that may help to explain the differences in their distribution on oahu. coptotermes gestroi currently has a very limited distribution on the southwest side of oahu, a region with high temperatures year-round. in temperature, this region may be similar to their natural tropical environment. arntzen & themudo (2008) found that geographical variations in ecological parameters determine the range limit of species. subtropical c. formosanus is essentially found throughout oahu, except in high forested areas. according to the research of grace et al. (2004), c. formosanus colonies may extend large distances by making wider, relatively unbranched, and longer tunnels in the soil than c. gestroi. also the foraging area of c. formosanus colonies appears to be larger than that of c. gestroi in hawaii (uchima & grace 2003a) and c. gestroi colonies may be behaviorally constrained by the presence of c. formosanus in the immediate area (uchima & grace 2009). uchima & grace (2003b) also found that the feeding rate of c. formosanus was higher than that of c. gestroi. hence, for these various reasons, as well as the history of their introductions, c. formosanus exhibits a much broader distribution than c. gestroi on the island of oahu. we also observed differences in seasonal activity between c. formosanus and c. gestroi (fig. 1). we did not observe any regular pattern of foraging 207 hapukotuwa, n.k. & j.k. grace — coptotermes foraging activity in hawaii activity (number of active traps) with either species. in the case of c. formosanus, however, the greatest number of active traps was observed during winter (december 2010), and it was 13 out of 22 traps. the lowest number fig. 1. mean number of active collection traps (out of 22 traps per site) at c. formosanus and c. gestroi field sites on oahu, hawaii, plotted against month from january 2010 –june 2011. table 1: environmental properties of the two field sites (soil separation test; sand (0.05mm), silt (0.002-0.005mm) and clay (0.002 or less). species/ field site /area vegetation soil type/s soil water content mean annual temperature (oc) mean annual humidity (%) c. gestroi kalaeloa 246.75 m2 prosopis pallida (kiawe tree), asystasia gangetica (chinese violet) sand 4.7%, silt 52%, clay 14.1% 6.33% 29.81 55.23 c. formosanus miller hall 28.6 m2 asystasia gangetica (chinese violet), murraya paniculata (mock orange) sand 6.7%, silt 26.7%, clay 6.7% 23.66% 26.58 63.64 208 sociobiolog y vol. 59, no. 1, 2012 fig. 2. mean number of active collection traps (out of 22 total) at the c. formosanus field site in comparison to mean temperature from january 2010 –june 2011. fig. 3. mean number of active collection traps (out of 22 total) at the c. gestroi field site in comparison to mean temperature from january 2010 –june 2011. 209 hapukotuwa, n.k. & j.k. grace — coptotermes foraging activity in hawaii of active traps (5 out of 22) was observed during summer ( july 2010 and may 2011). from january to march 2010 and from october 2010 to march 2011, c. formosanus showed a peak activity from 9 13 active traps. on the other hand, c. gestroi had greatest activity during summer ( july 2010) and spring (february 2011) (all 22 traps active). the lowest active trap number recorded for c. gestroi was 7 in january and may 2010. the peak activity periods for c. gestroi were july 2010 and november 2011, with the number of active traps ranging from 21-22. fei & henderson (2004) reported that temperature and moisture were the most important factors in the distribution of subterranean termites. bouillon (1970) found that seasonal variations are directly correlated with termite foraging activities. buxton (1981) also noted that the number of termites in the colony, the production of new individuals and the amount of food already stored in the nest fluctuated according to the season. in contrast, however, dawes-gromadzki & spain (2003) found no direct relationships between rainfall and termite species richness, or frequency and intensity of attack on baits during a field investigation of seasonal patterns in the activity and species richness of surface-foraging termites at paper baits in a tropical australian savanna. throughout the experimental period (from january 2010 to june 2011) there were fluctuations of temperature and relative humidity at both field sites. temperature was relatively steady, however, in comparison to relative humidity. at the c. formosanus field site, temperatures ranged from 23 0c to 30 0c; however, for c. gestroi it ranged from 27 0c to 33 0c (figures 2 & 3). at the c. gestroi kalaeloa field site, relative humidity ranged from 46% to 70%; whereas at the c. formosanus miller hall site it ranged from 51% to 80% (figs. 4 & 5). in conclusion, the c. gestroi field site was generally warmer than that of c. formosanus. both termite species exhibited irregular activity patterns (as represented by the number of active termite collection traps) throughout the year, although c. formosanus was more active in general during cooler months (winter) than c. gestroi; while c. gestroi was generally more active during late spring and summer months. it is worth pointing out that both subterranean termite species distributions and the territory sizes exhibited by individual termite colonies may change in the future as a result of global climate change, 210 sociobiolog y vol. 59, no. 1, 2012 fig. 4. mean number of active collection traps (out of 22 total) at the c. formosanus field site in comparison to mean relative humidity from january 2010 –june 2011. fig. 5. mean number of active collection traps (out of 22 total) at the c. gestroi field site in comparison to mean relative humidity from january 2010 –june 2011. 211 hapukotuwa, n.k. & j.k. grace — coptotermes foraging activity in hawaii as recently modeled by lee & chong (2011). however, different temperature preferences as well as different histories of introduction may help to explain both the current limited distribution of c. gestroi in hawaii, and eventual limits to its future distribution in the islands. acknowledgments we are grateful to r. oshiro, m. aihara-sasaki, m. mason and r. tong for their help in various ways with the field survey; and to j. r. yates iii, and m. g. wright for reviewing early drafts of the manuscript. funding was partially provided by usda-ars specific cooperative agreements 58-6615-9-200 and 58-6435-8-294; and mcintire-stennis and hatch funds administered by the college of tropical agriculture & human resources, university of hawaii at manoa. references arntzen, j. w. & g. e. themudo. 2008. environmental parameters that determine species geographical range limits as a matter of time and space. journal of biogeography 35: 1177-1186. bouillon, a. 1970. termites of the ethiopian region. pages 153-280 in krishna and f.m.weesner, editors. biolog y of termites. academic press, new york london. bullock, j. m., r. j. edwards, p. d. carey & r. j. rose. 2000. geographical separation of two ulex species at three spatial scales: does competition limit species’ ranges? ecography 23: 257-271. buxton, r. d. 1981. changes in the composition and activities of termite communities in relation to changing rainfall. oecologia (berlin) 51: 371-378. dawes-gromadzki, t.& a. spain. 2003. seasonal patterns in the activity and species richness of surface-foraging termites (isoptera) at paper baits in a tropical australian savanna. journal of tropical ecolog y 19: 449-456. fei, h. & g. henderson. 2004. effects of temperature, directional aspects, light conditions, and termite species on subterranean termite activity (isoptera: rhinotermitidae). environmental entomolog y 33: 242-248. grace, j.k. 2006. when invasives meet: coptotermes formosanus and coptotermes vastator in the pacific. proceedings of the 2006 national conference on urban entomolog y. raleigh-durham, north carolina; 21-24 may, 2006. pp. 92-94. grace, j. k., m. aihara-sasaki & j. r. yates. 2004. differences in tunneling behavior of coptotermes vastator and coptotermes formosanus (isoptera: rhinotermitidae). sociobiolog y 43: 153-158. grace, j. k., r. j. woodrow & j. r. yates. 2002. distribution and management of termites in hawaii. sociobiolog y 14: 87-93. 212 sociobiolog y vol. 59, no. 1, 2012 jeffree, c. e. & e. p. jeffree. 1996. redistribution of the potential geographical ranges of mistletoe and colorado beetle in europe in response to the temperature component of climate change. functional ecolog y 10: 562-577. lee, s.-h. & t.-s. chong. 2011. effects of climate change on subterranean termite territory size: a simulation study. journal of insect science 11(80). available online: insectscience. org/11.80. li, h.-f., weimin ye, n.-y. su, & n. kanzaki. 2009. phylogeography of coptotermes gestroi and coptotermes formosanus (isoptera: rhinotermitidae) in taiwan. annals of the entomological society of america 102: 684-693. scheffrahn, r. h. & n. y. su. 2005. distribution of the termite genus coptotermes (isoptera: rhinotermitidae) in florida. florida entomologist 88: 201-203. shiraki, t. 1909. japanese termites. transactions of the entomological society of japan 2: 229-242. su, n. y. 2003. overview of the global distribution and control of the formosan subterranean termite. sociobiolog y 41: 7-16. swezey, o. h. 1914. notes and exhibitions. proceedings of the hawaiian entomological society 3: 27. tamashiro, m., j. k. fujii & p.-y. lai. 1973. a simple method to observe, trap and prepare large numbers of subterranean termites for laboratory and field experiments. environmental entomolog y 2: 721-722. tsai, c. c. & c. s. chen. 2003. first record of coptotermes gestroi (isoptera: rhinotermitidae) from taiwan. formosan entomolog y 23: 157-161. uchima, s. y. & j. k. grace. 2003a. characteristics of coptotermes vastator (isoptera: rhinotermitidae) colonies on oahu, hawaii. sociobiolog y 41: 281-288. uchima, s. y. & j. k. grace. 2003b. compartive feeding rates of coptotermes vastator and coptotermes formosanus (isoptera: rhinotermitidae). sociobiolog y 41: 289-294. uchima, s. y. & j. k. grace. 2009. interspecific agonism and foraging competition between coptotermes formosanus and coptotermes gestroi (blattodea : rhinotermitidae). sociobiolog y 54: 765-776. weesner, f. w. 1965. the termites of the united states. the national pest control association, elizabeth, nj. woodrow, r. j., j. k. grace & s. y. higa. 2001. occurrence of coptotermes vastator (isoptera: rhinotermitidae) on the island of oahu, hawaii. sociobiolog y 38: 667-673. woodrow, r. j., j. k. grace & j. r. yates. 1999. hawaii’s termites: an identification guide. college of tropical agriculture & human resources, university of hawaii at manoa. household and structural pests 1: 0-6. yeap, b.-g., a. s. othman, v. s. lee & c.-y. lee. 2007. genetic relationship between coptotermes gestroi and coptotermes vastator (isoptera: rhinotermitidae). journal of economic entomolog y 100: 467-474. ziegler, a. c. 2002. hawaiian natural history, ecolog y and evolution. university of hawaii press. 1315 eggs of a eusial aphid’s predator are protected against attacks by aphid soldiers by mitsuru hattori1* & takao itino2,3 abstract predators generally have traits that enable them to efficiently capture their prey and thus improve their survival. natural selection should also favor traits of predators that improve the survival rate of their eggs, which are immobile and incapable of active resistance. we hypothesized that eggs of atkinsonia ignipicta, a specialist predator of the eusocial aphid ceratovacuna japonica, exhibit a defensive trait against aphid soldiers. we found that the hatchability of a. ignipicta eggs did not differ significantly between the experimental treatments with and without soldiers, which suggests that the eggs have a defensive trait that protects them from soldier aphids. moreover, although the soldiers occasionally exhibited attack behavior when they encountered an egg, they did not continue the attack. we have observed a similar interruption of attack behavior by soldiers that attacked their aphid siblings by mistake, suggesting that the eggs may chemically mimic the soldiers' siblings. this study thus provides evidence for adaptation in a specialist predator of a eusocial aphid. key words: antagonistic adaptation, atkinsonia ignipicta, ceratovacuna japonica, eusocial aphid, predation, predator–prey interaction introduction interspecific interaction is one of the most important driving forces of organismal evolution (ehrlich & raven 1964; vermeij 1994; philipp et al. 1department of mountain and environmental science, interdisciplinary graduate school of science and technolog y, shinshu university, 3-1-1 asahi, matsumoto, nagano 390-8621, japan 2department of biolog y, faculty of science, shinshu university, 3-1-1 asahi, matsumoto, nagano 390-8621, japan 3institute of mountain science, shinshu university, 3-1-1 asahi, matsumoto, nagano 390-8621, japan *corresponding author: mtr.hattori@gmail.com 1316 sociobiolog y vol. 59, no. 4, 2012 2006). the interspecific interaction between predator and prey is antagonistic (slobodkin 1980; kishida et al. 2006), and numerous studies have investigated adaptation in interacting predators and prey. many have focused on the defensive traits of prey species (lively 1986; dodson 1989; petersson & brönmark 1997; agrawal 1998; kishida & nishimura 2004; toju & sota 2006; for a review see tollrian & harvell 1999), and others have examined how the attack traits of a predator enable it to efficiently capture its prey and thus improve its survival (abrams 2000; michimae & wakahara 2002; toju & sota 2006). investigations of reciprocal adaptation between prey and predator can not only deepen our understanding of adaptive evolution but also of the evolution of biodiversity (thompson 2005). the moth larva atkinsonia ignipicta (butler) (lepidoptera: stathmopodidae) is a specialist predator of the eusocial aphid ceratovacuna japonica (takahashi) (homoptera: hormaphidinae) (morimoto & shibao 1993). individuals of the defensive caste of c. japonica, the soldiers, have long horns and forelegs that they use to protect the aphid colony against predators (m. hattori & itino 2008). when the soldiers encounter a predator, they grasp it with their forelegs and then pierce it with their frontal horns, sometimes killing it (hattori et al. in prep). females of a. ignipicta lay eggs directly in aphid colonies (fig. 1; hattori personal observation). therefore, the fitness of eggs of a. ignipicta without any defensive trait against aphid soldiers would be decreased when soldier aphids were present. in fact, soldiers of pseudoregma bambucicola (takahashi) (homoptera: hormaphididae) (ohara 1985) and ceratovacuna lanigera (zehntner) (homoptera: hormaphidinae) (aoki et al. 1984) attack and crush the eggs of their generalist predators that are laid on or near their colonies. these observations suggest that natural selection might favor traits that improve the survival rate of the eggs, which are immobile and incapable of active resistance. indeed, some predators display an oviposition behavior that improves the survival rate of their eggs (ohara 1985; arakaki 1992). thus, we hypothesized that a. ignipicta eggs, which are laid directly in the center of a c. japonica colony, might have a defensive trait that protects them against the soldier aphids. therefore, we examined whether the hatchability of a. ignipicta eggs is influenced by the presence of soldiers. 1317 hattori , m.& t. itino— eggs of a eusial aphid’s predator materials & methods atkinsonia ignipicta the specialist predator of c. japonica, a. ignipicta, is widely distributed in japan from kyushu to hokkaido (moriuti 1982). adults of a. ignipicta are abundant during june and july, when they lay their yellow, barrel-shaped eggs in aphid colonies (fig. 1). immediately after they hatch out, a. ignipicta larvae attempt to prey on nearby aphids. if they are successful, they then spin silken nests in the aphid colony, where they cannot be attacked by the soldier aphids, occasionally coming out to prey on the aphids. ceratovacuna japonica the eusocial aphid c. japonica, which is widely distributed in japan (m. hattori personal observation), has a heteroecious (i.e., host alternating ) and cyclically parthenogenetic (i.e., an asexual phase and a sexual phase) life history. it has one primary host, styrax japonica (sieb. et zucc.) (ebenales, styracaceae), and several secondary hosts (in central japan, poaseae species, e.g., sasa senanensis [rehd.] [poales, poaceae]) (aoki & kurosu 1991, 2011). although this species has a complete life cycle and on its secondary hosts, it sometimes produces winged sexuparae, it has merely been observed on its primary host (carlin et al. 1994). here, we define a colony as an aggregation of aphid individuals on a single leaf of the secondary host s. senanensis. such a colony can persist for up to several months. each colony has three morphologically distinctive castes: young reproductive adult females, which produce nymphs or soldiers parthenogenetically; and sterile soldiers. soldiers are first fig. 1. photograph of the eggs of the predatory month a. ignipicta laid on a s. senanensis leaf (among the spiny trichomes) in the center of a colony of the eusocial aphid c. japonica. 1318 sociobiolog y vol. 59, no. 4, 2012 instar nymphs, and they do not develop into second instar nymphs (aoki et al. 1981). soldiers have longer horns and forelegs than non-soldier aphids of the same instar (hattori & itino 2008), and they protect their colony from predators (hattori et al. in review). the primary predators of c. japonica are larvae of taraka hamada (druce) (lepidoptera: lycaenidae) and of a. ignipicta, which prey exclusively on c. japonica (morimoto & shibao 1993). although other potential predators such as syrphidae and chrysopidae species are able to prey on c. japonica, they are seldom seen on c. japonica colony (hattori personal observation). the hatchability of a. ignipicta eggs in the presence of soldiers to test whether a. ignipicta eggs have any defensive trait that protects them against soldiers of c. japonica, we compared hatching rates of eggs placed in dishes with and without soldier aphids. we designed three treatments, each with different numbers of non-soldier and soldier aphids (fig. 2) we haphazardly collected a. ignipicta eggs (n = 50) and 30 aphid colonies from a wild aphid population on mt. nabekanmuri, nagano, central japan (36°16’56”n, 137°49’35”e) in july 2009. this aphid population inhabits the edges of a deciduous forest, where the secondary host plant s. senanensis is abundant. after collecting the eggs, we returned to the laboratory and immediately introduced the eggs and aphids into petri dishes, mixing the aphid individuals from the 30 aphid colonies. in the course of these manipulations, we observed fig. 2. the design of the egg hatching experiment. each petri dish was 3.5 cm in diameter. in treatments 1 and 2, the number of non-soldier first instar aphid nymphs was equal. in treatments 2 and 3, the total number of aphids (non-soldier first instar nymphs plus soldiers) was equal. 1319 hattori , m.& t. itino— eggs of a eusial aphid’s predator no confounding events such as soldiers attacking conspecifics. we observed the eggs at 24-h intervals for 1 week or until they hatched. also, every 24-h, we removed all aphid individuals from the petri dishes and introduced newly collected aphid individuals from the same population to the petri dishes according to treatment (fig. 2). statistical analysis we compared the hatching rate of the eggs among the treatments by using fisher’s exact test ( jmp v. 9.0.0 statistical package, sas institute). results in every treatment, the hatching rate was high (table 1), and hatchability was not significantly different among treatments (df = 2, χ2 = 0.26, p > 0.05). some eggs that did not hatch gradually turned black, and two individuals of a parasitic wasp, trichogramma sp., emerged from each of these eggs. other eggs gradually became moldy and distorted in shape (table 1). when the soldiers encountered an egg, they rarely showed attack behavior toward it. those that initially showed attack behavior did not continue in their attack, but immediately stopped displaying the behavior. table 1: numbers of hatched and dead a. ignipicta eggs among the three treatments. 1320 sociobiolog y vol. 59, no. 4, 2012 discussion the high hatching rate of the a. ignipicta eggs in the treatment with soldier aphids suggests that a. ignipicta has some defensive trait that protects it against soldiers of c. japonica. this result contrasts markedly with the findings of previous studies that soldiers of other social aphid species often crush the eggs of generalist predators (p. bambucicola, ohara 1985; c. lanigera, aoki et al. 1984). at present, we do not know whether c. japonica soldiers crush the eggs of their generalist predators. future studies should investigate this possibility. although we occasionally observed attack behavior of soldiers toward eggs of a. ignipicta, the soldiers did not persist but immediately stopped attacking the eggs. one of us has observed a similar interruption of attack behavior by soldiers that attacked one of their siblings by mistake (m. hattori personal observation). these observations suggest that the eggs of a. ignipicta may chemically mimic the aphid. for example, the cuticular hydrocarbons (chcs) of the eggs may be similar to those of the aphids, just as myrmecophilous insects produce chcs that mimic those of ants so that they can penetrate the ant colony (vander meer et al. 1989; akino et al. 1999; hojo et al. 2009). moreover, a similar chemical mimicry has been observed in predators of ant-tending aphids (lohman et al. 2006). a future study should compare the chcs of a. ignipicta with those of c. japonica nymphs. most studies of predator–prey interaction in social aphids have focused on the morpholog y and function of the soldier’s defensive traits (kutsukake et al. 2004; hattori & itino 2008; hattori et al. in review). in contrast, even though it has been suspected that predators of eusocial aphids have various anti-soldier traits (arakaki & yoshiyasu 1988; aoki & kurosu 1992; for a review see stern & foster 1996), whether a predator’s defensive traits can protect it from soldiers has not been investigated quantitatively. further study of the defensive traits of predators will deepen our understanding of not only reciprocal adaptation in predator–prey interactions but also of the evolutionary stability of eusociality in aphids. 1321 hattori , m.& t. itino— eggs of a eusial aphid’s predator acknowledgments the authors thank dr. osamu kishida (hokkaido universsity) for helpful chemical advice. this work was supported by grants from the japan society for the promotion of science to m. h. 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shelomi, 2020). while non-chemical therapies and pesticides certainly have applications in medicine and integrated pest management respectively, the demand for “natural” chemicals and rejection of anything seen as “artificial” can lead to people rejecting safe and effective products in favor of alternatives lacking in safety or proven effectiveness but often higher in price, and thus suffering needlessly (johnson et al., 2017). using chemophobia to market a product that is not cost-effective is abstract an accumulation of questionable scientific reports on the use of natural plant extracts to control household pest insects, using biologically irrelevant experimental designs and extremely high concentrations, has resulted in a publication bias: “promising” studies claiming readily available plants can repel various insects, including social insects, despite no usable data to judge cost-effectiveness or sustainability in a realistic situation. the internet provides a further torrent of untested claims, generating a background noise of misinformation. an example is the belief that cucumbers are “natural” ant repellent, widely reported in such informal literature, despite no direct evidence for or against this claim. we tested this popular assertion using peel extracts of cucumber and the related bitter melon as olfactory and gustatory repellents against ants. extracts of both fruit peels in water, methanol, or hexane were statistically significant but effectively weak gustatory repellents. aqueous cucumber peel extract has a significant but mild olfactory repellent effect: about half of the ants were repelled relative to none in a control. while the myth may have a grain of truth to it, as cucumber does have a mild but detectable effect on ants in an artificial setup, its potential impact on keeping ants out of a treated perimeter would be extremely short-lived and not cost-effective. superior ant management strategies are currently available. the promotion of “natural” products must be rooted in scientific evidence of a successful and cost-effective implementation prospect. sociobiology an international journal on social insects matan shelomi, bo-jun qiu, lin-ting huang article history edited by evandro nascimento silva, uefs, brazil received 26 august 2020 initial acceptance 11 february 2021 final acceptance 19 february 2021 publication date 15 april 2021 keywords cucurbitaceae; formicidae; repellency; cucumber; ant. corresponding author matan shelomi department of entomology national taiwan university no 27 lane 113 sec 4 roosevelt rd taipei 10617 taiwan. e-mail: mshelomi@ntu.edu.tw itself a negative, as it involves profiting from logical fallacies and misinformation, or at worst committing fraud by making false statements. insisting that all claims be supported by evidence is a solid ethical position, and one can apply the scientific method to confirm or reject “hypotheses” promoted by the natural products industry. this “mythbusting” (zavrel, 2016) can expose ineffective remedies as unethical placebos, but also can reveal genuine effects of compounds that can be further investigated, as has happened for several safe and effective pharmaceuticals derived from plant-based chemical remedies. a good example is the mosquito repellent paramenthane-3,8-diol (pmd), derived from lemon eucalyptus (corymbia citriodora (hook.) ssp. citriodora) and endorsed by the centers for disease control and prevention (cdc) as similarly effective as deet (cdc, 2019). [it is worth noting that “natural” oil of lemon eucalyptus is not an effective department of entomology, national taiwan university, taipei, taiwan opinion cucumber vs ants: a case against the myth of the uses of plant extracts in insect pest management mailto:mshelomi@ntu.edu.tw m shelomi, bj qiu, lt huang – a case against myths of plant extracts against ants that are pests2 repellent, and that pmd is considered less safe than deet, having more documented side effects and stronger restrictions on its use (cdc, 2019; shelomi, 2020).] the difference between science and pseudoscience lies in the rigor and replicability of the relevant work. demonstrating pmd’s effectiveness required years of research from multiple groups around the world using appropriate, standardized repellency testing protocols (carroll & loye, 2006). unfortunately, too frequently one sees papers published in legitimate or predatory scientific journals concluding that a product is “very promising,” but with methodological flaws such as impractically high concentrations or biologically irrelevant experiment designs that render the results meaningless. individuals involved in promoting a certain product (or denigrating a competitor) can thus misuse scientific publications to provide a veneer of academic respectability to what is otherwise pseudoscience (weigmann, 2018). in pest management this problem manifests as scientists taking any plant or food readily available to them and testing its effects against a pest, often in field-unrealistic concentrations with low sample sizes and confined laboratory experiments, and concluding that this “home remedy” formulation is useful, without any safety testing, any effort to identify the compounds responsible for the effect, and how to scale it in a cost-effective implementation. examples include a study on 41 essential oils that claimed eight of them were 100% effective, but only following “a peculiar formulation to fix them on the human skin” (amer & mehlhorn, 2006); and a study that claimed a common seaweed kills mosquitoes, but only when mixed with a lethal dose of insecticide (prasanna kumar et al., 2012). such research is abundant yet unhelpful and rarely leads to a costeffective product, meaning a product that is safe, effective, and long-lasting to the point that it is worth using. the target of study in this paper is the popular myth that cucumber (cucumis sativus l.) repels ants. a quick internet search reveals over a million hits promoting cucumber as a “natural” ant repellent. methods of control include leaving slices or peels of cucumber anywhere in the home one wants ants to avoid, using an extract of cucumber in water or another “natural” chemical solvent, or purchasing an expensive cucumber-based “natural” product. the same search also reveals youtube videos of ants devouring cucumbers without trouble, so clearly ants are not completely put off by cucumber. some online sources state “bitter cucumber” is a better repellent: we could not identify what plant “bitter cucumber” could exactly relate to, but suspect it refers to a cucurbitaceae plant, bitter melon (momordica charantia l.) like most such claims, the scientific evidence for cucumber as repellent is scant, though the possibility that certain cucurbitaceae contain a compound that, at a high concentration or in a purified form, repels ants is non-zero. a report from 1982 testing an “old wives’ tale” that cucumbers repel cockroaches found that, while whole cucumbers did nothing, sliced cucumber repelled roaches 80% of the time, and the active ingredient, trans-2-nonenal, repels roaches 100% of the time (maugh, 1982). the same researchers identified two more chemicals inside cucumbers, (e,z)-2,6nonadien-1-al and (e)-2-nonen-1-al, that repel cockroaches (scriven & meloan, 1984). the active moiety of these molecules can be applied to make even more potent synthetic compounds, like diisopropyl ether and 5,5-dimethyl-3-enebutyrolactone, that are much more effective repellents. the author noted, however, that these compounds are all highly volatile (maugh, 1982): it is likely that cucumber’s repellency effect wears off quickly, at which point it becomes a slice of rotting food that would only serve to attract more pests. the only evidence of cucumbers explicitly repelling ants in the “scientific” literature are two poor-quality studies from 2013 and 2014 by the same researcher published in predatory journals, which we are ethically dis-inclined to cite in order to combat the scourge of predatory publishing (clark & smith, 2015; kurt, 2018). in the spirit of mythbusting, we thus tested the hypotheses that cucumber and bitter melon can function as gustatory and/ or olfactory repellents for applicable solutions against ant infestations. material and methods our methods are all derived from published bioassay literature, albeit of varying quality and relevance to ants, yet nonetheless with sufficient citations to justify publication. whole, raw cucumbers and white bitter melon were purchased from a grocery store in taipei, washed thoroughly, and the peel grated off. to make aqueous solutions, 20 g of either grated peel were added to 100 ml of reverse-osmosis purified water and extracted for 24 hours at 4°c. these extracts were sterilized of any microbes by filtering through millex® gp filter units with 0.22 µm pore-size millipore express® pes membranes. to produce other extracts, 20 g of peel were sequentially extracted in 100 ml each of analytical grade hexane, isopropanol, and methanol for 24 hours each at -20°c. extracts were centrifuged to eliminate solid particles. ants were trapped from around the national taiwan university entomology museum building using a bait of canned tuna, fructose syrup, and rolled oats. the ants were identified morphologically as the invasive pheidole megacephala (fabricius) (wetterer, 2007). worker ants were collected into a 50 ml centrifuge tube just before use. each individual ant was used once, then killed by freezing. only the minor worker ants were used. major worker ants (soldiers) were not used. experiments were performed in 55 mm diameter plastic petri dishes, the sides of which had been painted with fluon® to keep the ants from escaping. the methods are modified from those used to test responses to chemicals in drosophila (monte et al., 1989). to test for gustatory repellency, a circle of cardstock that can fit inside the dish is sociobiology 68(2): e5813 (june, 2021) 3 cut in half. one half is dipped quickly in the extract, the other as a control dipped in the solvent. the two halves are placed in the dish and allowed to air dry, and twenty ants placed in the center of the dish with a paintbrush. to control for visual cues, a box is placed over the petri dishes to block out the light. after 15 minutes, the box was lifted and the number of ants standing on the control or extract paper was recorded. for each plant-solvent combination, a total of 10 replicates was run. the samples with the most significant gustatory response were tested for olfactory response. in those tests, each dish contained two hole-punches of cardstock placed equidistant from either end of the petri dish, one wetted with 20 µl of extract and the other with the control solvent. twenty ants were released in the center, and a box used to block visual cues. after 15 minutes, the number of ants in each half of the arena was recorded. for statistical analysis, a response index (ri) (monte et al., 1989; amer & mehlhorn, 2006) was calculated from the number of ants on the control (c) and extract (e) side using this equation: ri=(e-c)/(e+c). the mean ri over all replicates was recorded as the strength of the effect: a strongly attractant substance has an ri of 1, a strongly repellent substance has an ri of -1, and a completely neutral substance has an ri of 0. the number of insects on the e and c sides over all replicates for each solvent was compared using a two-tailed, two-sample, paired t-test, which estimates the statistical significance of the repellency (ie: how often or how reliably a repellent effect can be observed). two-tailed tests are more conservative but allow for the possibility of an attractant effect. the same test was also used to compare the effects of cucumber to bitter melon with the same solvent. results the results are summarized in table 1. t-tests comparing the results of gustatory tests for cucumber and bitter melon in each of the four extracts showed no significant difference in their effect (p > 0.1). the extracts in water and in hexane had weak (-0.4 < ri < -0.2) but significant (p < 0.01) repellent effects, and extracts in methanol had very weak (-0.2 < ri < -0.1) but significant (p < 0.01) repellent effects. extracts in isopropanol showed no effect (0 < ri < 0.2, p>0.1). for water and hexane only, we performed olfactory testing. bitter melon extracts and cucumber in hexane had no effect (p > 0.05). only aqueous extract of cucumber had a statistically significant effect (p < 0.001), with an ri of -0.544, meaning on average 77% of ants were found on the side of the petri dish with the control water disk, compared to the 50% expected in a negative control discussion the results suggest that cucumber may indeed repel some ants, slightly, sometimes. more accurately, the results show that a purified extract of taiwanese c. sativus peel in dihydrogen monoxide (“water”) repelled a slim majority of pheidole megacephala workers harvested from a single colony from one half of a confined container to another, with an effect lasting at least 15 minutes. we found no significant differences between the gustatory repellency of bitter melon and cucumber, and no olfactory repellency for bitter melon at all: whatever the online sources regarding “bitter cucumber” are talking about, it is not momordica charantia. the results suggest cucumber is not a particularly powerful way to repel ants, as some ants were always present on or near cucumber extract disks or papers. we also cannot tell from the data how long the effect lasts, how far the effect spreads, or what concentration of volatiles is needed for maximum effect. isolating the compound[s] responsible may provide interesting results: it is unlikely to be trans2-nonenol, (e)-2-nonen-1-al, or (e, z)-2, 6-nonadien-1-al (scriven & meloan, 1984), as those are insoluble in water. that said, because the observed effect is still weak, adding such an analytical chemistry component to this study would serve mostly to make it appear more publishable and appease chemistry-minded reviewers. fruit solvent gustatory olfactory mean ri p-value result mean ri p-value result cucumber water -0.284 0.007 weak repellent -0.544 <0.001 half repellent hexane -0.400 0.019 weak repellent 0.205 0.194 no effect isopropanol 0.207 0.421 no effect methanol -0.165 0.014 very weak repellent bitter melon water -0.356 0.001 weak repellent -0.21 0.100 no effect hexane -0.342 0.003 weak repellent -0.156 0.221 no effect isopropanol 0.003 0.501 no effect methanol -0.172 0.023 very weak repellent data is based on 10 replicates for each assay with 20 ants per replicate. no olfactory experiments were done for isopropanol or methanol extracts. p-values are based on a two-tailed, two-sample t-test. ri = response index. table 1. results of gustatory and olfactory repellency tests of cucumber and bitter melon extracts on pheidole megacephala (fabricius). m shelomi, bj qiu, lt huang – a case against myths of plant extracts against ants that are pests4 to summarize, while this research did find “statistically significant” effects of cucumber peel extract on ants, it does not at all suggest cucumber is a good or even promising repellent. the extract did not approach 100% repellency, even after only 15 minutes of time, while typically repellents are measured in terms of hours of absolute repellency (ri of -1.0). cucumber extract cannot be considered a cost-effective repellent, both because of its low efficacy and because cucumber provides much higher value as a food. cucumberbased products marketed as “natural” repellents [and likely priced accordingly] are almost certainly a waste of money, unless they have been adulterated with actual repellents. fresh cucumber as recommended by the internet would likely be even less effective: the bulk of the cucumber is not particularly aromatic but is rich in nutrients that worker ants would eagerly take back to their colonies. spreading ant food around places where ants congregate does not seem like an effective strategy for ant management. there are better uses for cucumbers and better solutions for ant control. indeed, the very idea of an “ant repellent,” natural or otherwise, was misguided from the start: it is a marketing gimmick for “natural” product pushers, but was never a costeffective pest management tool. repellents are valuable for temporary personal protection against pests that cannot be easily eradicated, such as against mosquitoes when hiking in a natural forest. however, pests that can infest households or in sensitive field setups (crops, recreational areas, pastures, etc.), repellency is simply impractical. repellents eventually wear off, and then the pests will return. for cost-effective control of household insects such as ants or cockroaches, the simple preventive action of keeping potential food items out of access can prevent or limit infestation levels, and insecticides that have demonstrated their efficacy and sustainability can be used thoughtfully to mitigate pest problems. one of the most common solutions used for ant control at home is a mixture of borates (borax or boric acid) with bait such as sugar, which the workers will take back to share with the colony, causing significant population reduction, while using a extremely small quantity of active ingredient. unlike cucumber, borates are both safe and effective, with plenty of peer-reviewed and rigorous publications supporting their use (klotz et al., 1998; gore & schal, 2004), with the marketing bonus of also being “natural,” for anyone who still values that term. acknowledgements special thanks to chi-man leong for ant identification, anonymous reviewers and the editors for their suggestions and revision. authors’ contributions ms: conceptualization, methodology, writing and revision bjq and lth: methodology, investigation. references amer, a. & mehlhorn, h. 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(1989). characterization of the larval olfactory response in drosophila and its genetic basis. behavior genetics, 19: 267-283. doi: 10.1007/bf01065910 prasanna kumar, k., murugan, k., kovendan, k., naresh kumar, a., hwang, j.-s. & barnard, d. r. (2012). combined effect of seaweed (sargassum wightii) and bacillus thuringiensis var. israelensis on the coastal mosquito, anopheles sundaicus, in tamil nadu, india. science asia, 38: 141-146. doi: 10.2306/scienceasia1513-1874.2012.38.141 scriven, r. & meloan, c.e. (1984). (e, z)-2, 6-nonadien1-al and (e)-2-nonen-1-al present in crushed cucumbers are natural repellents for the american cockroach (periplaneta americana). the ohio journal of science, 84: 82-85. shelomi, m. (2020). who’s afraid of deet? fearmongering sociobiology 68(2): e5813 (june, 2021) 5 in papers on botanical repellents. malaria journal, 19: 1-3. doi: 10.1186/s12936-020-03217-5 weigmann, k. 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(2016). pedagogical techniques employed by the television show “mythbusters”. the physics teacher, 54: 476-479. doi: 10.1119/1.4965268 1447 adaptive nesting tactics in a paper wasp, polistes riparius, inhabiting cold climatic regions by satoshi hozumi1 & kazuyuki kudô2 abstract thermal effects on the nest sizes constructed by polistes riparius foundresses, their nesting activities, and colony compositions were determined in laboratory conditions during the pre-emergence period. in this study, foundresses were placed in cold (20°c) and warm conditions (27°c), and ample food and nest materials were supplied. nest sizes were larger in the cold condition; particularly, the elongation of cells was remarkable. the number of cells between the 2 conditions was not significantly different. inter-condition colony composition was similar, while more honey drops were observed in the cold condition nests. a positive relation was observed when the relationships between body weight and developmental days were investigated, i.e., small offspring required shorter developmental days in both thermal conditions. in the cold condition, the first offspring were very small, whereas offspring size increased steeply after the second order, and was comparable to that of the foundresses; the size increase in the warm group was slight. this result indicates that the cold condition foundresses manipulated the sizes of their offspring. when inter-condition foundresses activities were compared , cold condition foundresses spent more time and energ y on flight activities. these results support the view that foundresses change nesting activities in relation to environmental temperatures, i.e., they can improve the thermal condition of nests by adding extra cells and manipulate the sizes of adult offspring. keywords: nesting tactics, polistis, nest building, body weight, cold climate, hymenoptera, vespidae. 1chigakukan secondary school, 310-0914, japan. e-mail: shoz@tokiwa.ac.jp, tel.: +81-29-2123311 2laboratory of insect ecolog y, department of biolog y, faculty of education, niigata university, 950-2181, japan. e-mail: kudok@ed.niigata-u.ac.jp 1448 sociobiolog y vol. 59, no. 4, 2012 introduction it has been revealed that social wasps vary their nesting habits flexibly in order to maximize their fitness in relation to their environments (wheeler 1986). among the activities of social wasps, the nest building is one of the most important and interesting aspects because the nest is where social activities take place, and it is used for nursing the brood, i.e., it is directly connected with reproduction ( jeanne 1991). many studies have shown that wasps build nests in response to the environments they inhabit (matsuura 1990; yamane 1996), since the development of the immature stages depends on the ambient temperature. in tropical and cold climatic ecozones, and nest thermoregulation is carried out by constructing nests with unique structures (hozumi et al. 2007, 2008ab, 2009). in tropical regions, wasps insulate nests to maintain a constant temperature against daily temperature fluctuations (15–40°c) (hozumi et al. 2010); in these regions, nests must be prevented from overheating. on the other hand, in cold climatic regions such as the arctic and high mountainous areas, the most pressing environmental problem is the low temperature, which shortens the development of the immature stages and activities of adult individuals (henrich 1993). due to the short nesting period, the strateg y in cold regions is that wasps should increase colony production by accelerating the development of the immature stages and produce g ynes as early as possible. in paper wasps belonging to the genus polistes, the colony cycle is divided into 3 periods: pre-emergence, worker, and reproduction. the foundresses (independent founding ) build exposed nests and manage all tasks before the worker individuals emerge (pre-emergence period). after the workers eclose (worker period), the adult individuals steeply develop colony production, and finally produce as many reproductive g ynes as possible; therefore, the wasps make an effort to shorten the colony cycle (yamane 1969; yamane & kawamichi 1975; miyano 1980). among these periods, shortening of the pre-emergence period is more important in the colony cycle, since this period is the longest of the 3 periods (1.8 months/3 months) and is when most colony failure occurs (kudô 1997). a major cause of colony failure is the loss of foundresses during extranidal activities such as foraging for prey and nest materials. in order to reduce the 1449 hozumi, s. & k. kudo — adaptive nesting tactics in polistes riparius risks during the pre-emergence period, foundresses hasten development of the immature stages by 2 methods, i.e., nest thermoregulation and control of larval feeding : incubation of brood (miyano 1981; jeanne & morgan 1992), feeding intensely on ample food (kudô et al. 1998), and manipulating worker sizes to be as small as possible (miyano, 1980). polistes wasps often improve the thermal conditions of their nests. for example, the foundresses of polistes riparius, which inhabit cold areas in northern japan, not only choose warm sites, but also construct extra cells around the brood cells (yamane & kawamichi 1975). such long and numerous cells further increase nest temperatures when the nest receives sunlight (hozumi et al. 2008b). generally, nest construction requires a very high cost in the form of extranidal activities and resources for nest material; foundresses have to collect nest materials and forage for prey to produce an oral secretion that is used for construction and maintenance of the nest (yamane et al. 1998). in nests built by lone p. riparius foundresses, both the amount of nest materials and oral secretion are twice of that in nests with no extra cells (kudô et al. 1998), i.e., p. riparius foundresses have to carry out more extranidal work despite the high cost and high risk of colony failure. however, whether the nest building activities of foundresses are influenced by cold ambient temperature has not been studied. regarding the control of larval feeding, it has been emphasized that very small first adults are produced to end the pre-emergence period as early as possible, even in temperate regions (“small worker” hypothesis; miyano 1981). recently, further manipulation by foundresses has been reported. fucini et al. (2009) reported that p. biglumis inhabit severely cold areas; the very short nesting period changes the nesting behavior in that foundresses produce g yne-like females instead of worker-like individuals, whereas worker-like individuals are produced in warm areas. this means that paper wasp foundresses can change nesting tactics in response to ambient temperature and shorten the colony cycle by omitting the worker period in order to produce g ynes immediately (“omission” hypothesis). however, it is still unknown whether p. riparius foundresses plastically change the caste of offsprings in cold conditions. this study aimed to determine whether the nesting activities of p. riparius foundresses are influenced by the environmental temperature. i hypothesized that foundress responses against a cold environment would be revealed in 1450 sociobiolog y vol. 59, no. 4, 2012 activities improving the thermal conditions of the nest, i.e., construction of extra cells and manipulation of the sizes of foundress-reared adult individuals to end the pre-emergence period immediately. to achieve this objective, foundresses work more, build a larger nest, and produce small first adult individuals (“small worker”); otherwise, they produce foundress-sized adults or g ynes (“omission”). i defined 2 goals for this study. the first goal was to confirm that larger nests are built in colder, rather than warmer conditions. the second goal was to compare the size of foundress-reared adult individuals reared in 2 different thermal conditions. i expected the first adults in the cold condition to be very small; in addition, foundress-sized adults or g ynes would be produced instead of workers. in order to examine this, the foundresses were placed in different thermal conditions, and the behaviors and the nest size constructed by lone foundresses were compared. furthermore, the dry weights of foundress-reared offspring were compared to determine whether foundress manipulation of body size occurred. materials and methods colonies used and experimental conditions ten p. riparius foundresses were captured together with their nests on 20 may 2000 in sapporo (43°03’n, 141°20’e), hokkaido, japan. at collection, all nests were virtually at the same development stage with only a few short cells. all of the eggs previously laid in the nests were removed prior to the experiment in order to equalize the initial conditions of the immature development. foundresses and their nests were divided into 2 groups: 5 colonies were kept at 20 ± 1.0°c (hereafter, cold group); the other 85 colonies were kept at 27 ± 1.5°c (hereafter, warm group). the dry weight of foundresses ranged 35.5–45.9 mg, and the mean weights (± sd) of foundresses in the cold and warm groups were 39.9 ± 1.4 mg (n = 5) and 41.1 ± 1.4 mg (n = 5), respectively, and there was no significant difference (mann–whitney u-test, n = 10, u = 7.0, p = 0.2506). hence, i regarded the influence of the mother’s body size on nesting activities as small or negligible in this study. the initial size of the nests did not differ between the conditions: cell lengths in the cold and warm groups were 8.2 ± 0.2 mm (n = 5) and 8.4 ± 0.3 mm (n = 5), respectively, with no significant difference determined (mann–whitney 1451 hozumi, s. & k. kudo — adaptive nesting tactics in polistes riparius u-test, n = 10, u = 9.5, p = 0.5309); numbers of cells in the cold and warm groups were 12.2 ± 3.0 (n = 5) and 12.6 ± 4.0 (n = 5), respectively, and there was no significant difference (mann–whitney u-test, n = 10, u = 12.0, p = 0.9161). each foundress and her nest was placed in a wire-screened cage measuring 30·30·40 cm (l·w·h), and was provided with ample water, and diluted honey and silkworms (bombyx mori) for food. in addition, filter paper was provided as nest material. the photoperiod for both groups was defined as follows: light, 16 h; dark, 8 h. all colonies were maintained under constant conditions until the first workers emerged, and the activities of the foundresses were observed throughout the experiment. on the day the first adult offspring emerged, the foundresses were removed and the experiment was stopped; eggs and larvae were carefully removed from the nests with tweezers. to determine the body size of adult offspring reared only by foundresses, the pupae were kept intact in the nests, and further incubated under each temperature condition to allow the remaining pupae emerge. foundresses and foundress-reared adult wasps were dried in an electric oven for more than 48 h at approximately 70°c, and the dry body weights were measured to the nearest 0.1 mg by an electronic scale. the pattern of cell arrangement and each cell occupant (egg, larva, pupa, honey, or empty) in each colony were recorded. the following nest parameters were measured just after emergence of the first adults: (1) maximum cell length (mm), (2) number of cells, and (3) nest volume (ml). cell lengths were measured by inserting a scaled stick (ø <1 mm) into each cell, and the length of the stick was measured with a vernier caliper to the nearest 0.1 mm; the longest value for each cell was recorded. the number of cells was counted right after the emergence of the first adults, and the value was standardized as follows; total number of cells minus initial number of cells. nest volume was measured as follows: cells were filled with minute and granular glass beads (ø <0.1 mm), and the volume of the beads was measured with a graduated cylinder to the nearest 0.1 ml. behaviors of foundresses observations of foundress behaviors in each colony began when the first larvae hatched. one observation episode spanned 3 h between 10:00 and 13:00 or 14:00 and 17:00. total observation time was 75 h (25 observation episodes) 1452 sociobiolog y vol. 59, no. 4, 2012 for 7 colonies in the cold group and 63 h (21 episodes) for 8 colonies in the warm group. foundress behaviors were categorized as follows: nest building, including elongation of cells, initiation of a new cell, reinforcement of petiole and licking of nest surfaces; collection of nest material; feeding the brood; hunting prey; collection of water or honey; seeking (seeking something or extranidal activities involving no visible tasks); oviposition; body cleaning ; resting ; and shivering. shivering activity often occurred just before flight in order to warm the body up by shivering the flight muscle of the thorax, and it required intense respiration, as it involved a sequence of rapid pumping motions with the abdomen. since most wasps and bees raise their body temperature up to 35°c, the duration of the pumping motion can be an index of pre-flight warm-up in hymenopteran insects (heinrich, 1993). in this study, direct measurements of body temperature by thermocouples was unfavorable, and the duration of pumping just before flight was measured as an index of environmental influence on the foundresses’ activities. statistics the mann–whitney u-test was employed to analyze the following features between the cold and warm groups: nest parameters, body weights of adult individuals, colony composition, and foundress activities. fisher’s exact test was used to determine the production rates for large females (see below). all analyses were made with statview ver. 5.0 (sas institute inc.) on a macintosh personal computer. results nest sizes and colony compositions in both groups, pupae were reared in center area of the nests, and larvae and eggs were located concentrically around the pupae. the elongation of the cells around the pupae was remarkable. honey dew and empty cells were found in the lower portions of the nests. more cells in the cold group were used to store honey than in the warm group (table 1) (mann–whitney u-test, n = 10, u = 2.0, p <0.05), whereas the numbers of immature stages and empty cells between groups were similar and no significant difference was detected. 1453 hozumi, s. & k. kudo — adaptive nesting tactics in polistes riparius table 1. mean (± sd) number of contents in the cells in cold (n=5) and warm (n=5) condtion observed at the end of pre-emergence period. eggs larvae pupae honey empty cool group 19.0 ± 15.6 15.1 ± 2.6 7.2 ± 2.8 15.7 ± 6.9 4.1 ± 3.1 warm group 22.1 ± 16.9 17.4 ± 5.5 7.0 ± 2.4 5.0 ± 4.0 6.8 ± 4.7 fig.1 body weights of foundress-reared adult wasps produced in cold and warm conditions; c1–c5, cold group colonies; w1–w5, warm group colonies. white bars indicate male offsprings. 1454 sociobiolog y vol. 59, no. 4, 2012 the nests in the cold group contained longer cells (37.7 ± 2.6 mm) than those in the warm group did (33.2 ± 2.5 mm) (mann–whitney u-test, n = 10, u = 2.0, p <0.05). cell numbers in the cold group (56.3 ± 13.9) were slightly smaller than in the warm group (56.4 ± 25.8), whereas no significant difference was found between the groups. the volume of nests in the cold group (19.1 ± 4.7 ml) was larger than that in the warm group (13.5 ± 2.1 ml) (mann–whitney u-test, n = 10, u = 3.0, p <0.05). figure 1 illustrates the sequent changes of body weights of foundress-reared adult wasps produced in cold and warm conditions. the mean weight of first offspring in the cold group (21.0 ± 3.8 mg ) was significantly lower than that of the warm group (26.0 ± 3.5 mg ) (mann–whitney u-test, n = 10, u = 3.0, p <0.05). the weight in the cool group steeply increased after the second offspring emerged, and the weight of some individuals was comparable to that of the foundresses (>35 mg ; the minimum weight observed in this study): 14 large female individuals were produced from 4 colonies, and 3 males were produced from 1 colony (fig. 1, from c1 to c5). however, the body weight of offspring in the warm group was relatively similar between individuals (about 30 mg ), and 4 large females (>35 mg ) were produced from 3 colonies(fig. 1, from w1 to w5); males were not produced. the number of large adults in the cold group was significantly greater than that in the warm group (fisher’s exact test, n = 10, p <0.05). foundress activities the mean times spent on each behavior during the observation period are listed in table 2. brood feeding activities were most frequently observed in both thermal conditions, followed by resting and nest building in the cold group or nest building and hunting prey in the warm group. comparison of activities between the groups determined that shivering in the cold group was significantly longer than in the warm group (mann–whitney u-test, n = 10, u = 3.0, p <0.05). the time distribution patterns for other activities were similar between both groups, and there was no significant difference. discussion it was confirmed that the foundresses in the cold group built larger nests, namely, by virtue of the length of the cells (“extra-cells”). cell elongation in 1455 hozumi, s. & k. kudo — adaptive nesting tactics in polistes riparius cells containing pupae was remarkable. the numbers of cells between groups were not different; hence, the volume of nests in the cold group was larger due to the elongation of cells containing pupae. the cell length, rather than the total number of cells, greatly affects cell temperature (hozumi et al. 2008b), and it is possible that the thermal effect of the cells accelerates development of the immature stages. in this study, the difference of cell length between the cold and warm groups was only 5 mm. however, even a 5-mm elongation may increase cell temperatures by 1–2°c when the nest receives solar radiation (hozumi et al. 2001, 2008b). in this study, environmental temperatures did not influence entire activity patterns of foundress. this means that the foundresses of cold group actually worked longer time because of the delay of the development of imamture stages. therefore, it cannot be denied that the larger nests built in the cold group were the result of prolongation of the pre-emergence period. however, such a buildinng manner, i.e., building extra-cells throughout the pre-emergence period, has not seen in other wasps inhabiting temperate ecozone. for example, p. chinensis antennalis perêz, a consubgeneric species of p. riparius dwelling in warm areas, the time for the construction behavior is short (less 2 min per hour) during the pre-emergence period, and the foundresses rarely leave nests at the end of founding phase, perhaps, in order to reduce the risks of predation and accident during the extranidal activites (kasuya 1981). table 2. mean time (min) of relative activities in an hour of observation. behavioral repertoire of the foundresses under cool (n=5) and warm (n=5) conditions was given. activity cool warm nest building* 9.2 ± 1.3 9.6 ± 2.6 collection of nest material 2.5 ± 0.6 3.0 ± 1.0 feeding the brood 20.3 ± 1.8 18.4 ± 1.2 hunting a prey 7.5 ± 0.4 8.8 ± 1.7 collection of honey-water and water 0.7 ± 0.3 1.0 ± 0.3 seeking 3.0 ± 1.5 4.8 ± 1.5 resting 11.8 ± 1.5 8.7 ± 2.0 cleaning 5.1 ± 0.5 5.5 ± 0.4 oviposition 0.2 ± 0.1 0.2 ± 0.1 shivering 1.7 ± 0.5 1.0 ± 0.4 *: including elongation of cells, initiation of a new cell and reinforcement of petiole and licking of nest surfaces 1456 sociobiolog y vol. 59, no. 4, 2012 commonly, the constrution of anew cell and elongation of cells is related to oviposition and development of larvae, respectively (delaeurance 1957), and it has been condidered that there would appear to be no advantage to be gained by constructing cells at a fast rate at a faster rate than the colonial development ( jeanne & bouwma 2004). the foundress’ overall effort probably represents an optimization of the trade-off between the benefit of increased colony reproduction and the risk of colony failure (wenzel 1996). according to yamane et al. (1998), p. riparius foundresses cost resources for nest building as much as rearing broods in terms of resource and time; i.e., foundresses enable to rear more immature stages during the pre-emergence period in spite of high risks during the extranidal activities. if so, foundresses should cost more for rearing immatures in order to produce more g ynes, otherwise should stay on the nest until the emergence of first worker in order to reduce the risks of colony failure. however, p. riparius foundresses worked throughout the observation hours, and the number of immature stages between the 2 groups was almost similar. it is suggested that with nest construction requiring more resources, insted of rearing more immature stages, the foundresses had attempted to improve the thermal condition of the cells in order to produce offspring immediately during the limited nesting period. among the nesting activities of foundresses, whereas the duration of shivering in cold group foundresses was remarkably longer than that of warm group foundresses. this means that cold group foundresses spent more time shivering just before extranidal activities, indicating that activities required a high cost in terms of time spent warming up and energ y consumption in cold conditions. many honey drops stored in the cold group may be used both as food for the colony and as an energ y source for warming up. this also supports the premise that extranidal activities require more resources under cold conditions. i confirmed that foundresses in the cold group manipulated the body sizes of the offspring they reared. as expected, the body size of the first adults in the cold group was very small, suggesting that the foundresses had tried to produce first workers as early as possible. in some colonies of cold condition, however, the body size of offspring subsequent to the second order increased steeply, and the body size of approximately half the newly emerged adults was comparable to that of the foundress. two hypotheses can be considered for the 1457 hozumi, s. & k. kudo — adaptive nesting tactics in polistes riparius altered body sizes: (1) foundresses produce large workers in cold conditions to increase working performance, or (2) produce g ynes instead of workers; i.e., omission of worker period. the performance hypothesis is well known in bumblebees inhabiting cold climates (heinrich & heinrich 1981). it is expected that large p. riparius workers perform nesting activities efficiently, and such large individuals will warm up easily before extranidal flight. regarding the omission hypothesis, a similar phenomenon has been reported in p. biglumis inhabiting high mountain regions (lorenzi and turrilazzi 1986); half of p. biglumis foundress-reared offspring were g ynes. despite the small number of samples, some male g ynes were also produced in this study, and 4 colonies produced very large females whose sizes were comparable to that of the foundresses. these results may support the hypothesis on worker period omission, and it is possible that p. riparius foundresses alternate nesting tactics in relation to environmental temperatures. references delaeurance, e.p. 1957. contribution à létude biologique des polistes (hyménoptères, vespidés). i. l’activité de construction. ann. sci. naturelles zool. biol. anim. 19: 91-222. fucini s, v. di bona, f. mola, c. piccaluga & m.c. lorenzi 2009. social wasps without workers: geographic variation of caste expression in the paper wasp polistes biglumis. insectes sociaux 56: 347-358. heinrich, b.1993. hot blooded insects. harvard university press, cambridge, massachusetts. p. 601. heinrich, b. and heinrich, m.j.e. 1983. size and caste in temperature regulation by bumblebees. physiol. zool. 56: 552-562. hozumi, s. & yamane, sô. 2001. incubation ability of the functional envelope in paper wasp nests hymenoptera, vespidae, polistes): i. field measurements of nest temperature using paper models. j. ethol. 19:39–46 hozumi, s., k. kudô & r. zucchi 2008a. promotion of thermoregulatory insulation in nests of neotropical wasps by building extra-combs with empty cells. neotropical entomol. 37: 159-166. hozumi, s., sô.yamane & h. katakura 2008b. building of extra cells in the nests of paper wasps (hymenoptera; vespidae; polistes) as an adaptive measure in severely cold regions. sociobolog y 51: 399-414. hozumi, s., k. kudô & s. mateus 2009. thermal characteristics of the mud nests of the social wasp polybia spinifex (hymenoptera; vespidae). sociobiolog y 53: 89-100. hozumi s, s. mateus, k. kudô, t. kuwahara, sô. yamane & r . zucchi 2010. nest thermoregulation in polybia scutellaris (white) (hymenoptera: vespidae). neotropical entomol. 39: 826-828. 1458 sociobiolog y vol. 59, no. 4, 2012 jeanne, r.l. 1991. the swarm-founding polistinae. in: the social biolog y of wasps (kg ross and rw matthews, eds), pp. 191–231. ithaca, ny: cornell university press. jeanne, r.l. & a.m. bouwma. 2004. divergent patterns of nest construction in eusocial wasps. j. kan. entomol. soc. 77: 429-447. jeanne, r. l. & r. c. morgan 1992. the influence of temperature on nest site choice and reproductive strateg y in a temperate zone polistes wasp. ecol. entomol. 17:135-141. kasuya e. 1981. polyg yny in the japanese paper wasp, polistes jadwigae. kontyû 49: 306313. kudô, k. 2000. variable investment in nests and worker production by the foundresses of polistes chinensis (hymenoptera: vespidae). j. ethol. 18: 35-39. kudô, k., yamane, sô. & yamamoto, h. 1998. physiological ecolog y of nest construction and protein flow in pre-emergence colonies of polistes chinensis (hymenoptera vespidae): effects of rainfall and microclimates. ethol. ecol. evol. 10: 171-183. lorenzi, m.c. & s. turillazzi 1986. behavioural and ecological adaptations to the high mountain environment of polistes biglumis bimaculatus. ecol. entomol. 11: 199204. matsuura, m. 1990. biolog y of three vespa species in central sumatra (hymenoptera, vespidae). in: sakagami, f. s., ohgushi, r. and roubik, d. w. (eds.), natural history of social wasps and bees in equatorial sumatra: pp. 113-124. hokkaido university press, sapporo. miyano s. 1980. life tables of colonies and workers in a paper wasp,polistes chinensis antennalis, in central japan (hymenoptera: vespidae). popul. ecol. 22: 69-88. miyano, s. 1981. brood development in polistes chinensis antennalis pérez i. seasonal variation of duration of immature stages and an experiment on thermal response of egg development. bull. gifu pref. mus. 2: 75-83. wenzel, j.w. 1996. learning, behavior programs, and higher-level rules in nest construction in polistes. in: turillazzi, s. & m.j. west-eberhard (eds.), the natural history and evolution of paper wasps. oxford university press, london, pp. 58-74. wheeler, d.e. 1986. developmental and physiological determinants of caste in social hymenoptera: evolutionary implications. am. nat. 128: 13-34. yamane, s. 1969. preliminary observations on the life history of two polistine wasps, polistes snelleni and p. biglumis in sapporo, northern japan. j facul. sci. hokkaido univ. ser. vi, zool. 17: 78-105. yamane, s. 1996. ecological factors influencing the colony cycle of polistes wasps. in: turillazzi, s. & west-eberhard, m. j. (eds.), the natural history and evolution of paper wasps. pp. 75-97, oxford university press, london. yamane, s. & kawamichi, t. 1975. bionomic comparison of polistes biglumis (hymenoptera, vespidae) at two different localities in hokkaido, northern japan, with reference to its probable adaptation to cold climate. kontyû 43: 214-232. yamane, sô., k. kudô, t. tajima, k. nihon’yanagi, m. shinoda, k. saito & h. yamamoto 1998. comparison of investment in nest construction by the foundresses of consubgeneric polistes wasps, p. (polistes) riparius and p. (p.) chinensis (hymenoptera, vespidae). j. ethol. 16: 97-104. doi: 10.13102/sociobiology.v61i4.494-501sociobiology 61(4): 494-501 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 foraging distance of melipona subnitida ducke (hymenoptera: apidae) introduction bees are primary pollinators in most regions of the world (bawa, 1990; silberbauer-gottsberger & gottsberger, 1988). their flight range strongly influences the sexual reproduction of most flowering plants and can further determine the genetic structure of plant populations (campbell, 1985; waser et al., 1996). the distance that bees travel in search of a resource can directly affect agricultural crops, given that bee pollination is necessary to generate 30% of the human food supply (slaa et al., 2006). to increase the efficiency of collection and exploitation of good resources (kerr, 1994; contrera & nieh, 2007), eusocial bees developed a sophisticated communication system that allows foragers to recruit other bees to profitable food sites (lindauer & kerr, 1960; kerr, 1969; wille, 1983; jarau et al., 2000; nieh, 2004; aguilar et al., 2005). to save time and energy, bees do not forage over long distances unnecessarily (frisch, 1967; seeley, 1994). for example, in abstract the current study aimed at estimating the maximum foraging distance of the stingless bee melipona subnitida ducke by comparing the efficacy of two methods: training of workers with an artificial feeding source and the capture-recapture technique, which consisted at marking bees that were released at different distances from the nest, after which the number of bees that returned to the colony was recorded. under the training method, the mean foraging distance of the three colonies studied was 1,120 m and maximum foraging distance of 1,160 m. yet the number of recruits and reactivated foragers for each colony were quantified, the average maximum distance until recruitment occurred was 886,66 m. in the capture-recapture method, the maximum flight distance of captured foragers ranged from 3,600 to 4,000 m, which was 2,700 m farther than the maximum flight distance recorded using the artificial feeding method. therefore, we verified that m. subnitida is a species that can travel long distances in search for food. our results also suggest that an abundance of resources near the nest can reduce its foraging area. sociobiology an international journal on social insects ag silva1, rs pinto1, fal contrera2, pmc albuquerque1, mmc rêgo1 article history edited by denise araujo alves, esalq-usp, brazil received 30 september 2014 initial acceptance 11 november 2014 final acceptance 09 december 2014 keywords artificial feeding, capture-recapture, distance, foragers, meliponini, stingless bees. corresponding author albeane guimarães silva universidade federal do maranhão ufma departamento de biologia, laboratório de estudos sobre abelhas. av. dos portugueses, 1966, 65080-805 são luís, ma, brazil e-mail: albeaneguimaraes@hotmail.com the summer, when there is a decrease in the food supply near the nest, bees of the genus apis may use an area 6 to 22-fold larger than the area used during spring or fall (couvillon et al., 2014). the methods that have been most commonly used for the study of the maximum flight range of honey bees in general, and stingless bees in particular, include training foragers to feed from artificial feeders, and the marked forager capture-recapture method (zurbuchen et al., 2010). the use of artificial feeders to train forager workers makes it possible to train them to the maximum distance that a species may travel in search of food. in this test, the workers that reach the feeder to collect a food sample are marked with a specific color or number combination, usually on the thorax or abdomen (seeley, 1995). measuring the maximum foraging distance for each stingless bee species provides information related to the communication and recruitment techniques used by stingless bees to obtain food in response to their environment (contrera & nieh, 2007; kuhn-neto et al., 2009). research article bees 1 universidade federal do maranhão (ufma), são luís, ma, brazil 2 universidade federal do pará (ufpa), belém, pa, brazil sociobiology 61(4): 494-501 (december 2014) 495 the capture-recapture method involves the release of previously marked workers at a given distance from the mother colony, and then counting the number of marked bees that return to the colony thereafter (roubik & aluja, 1983). according to kuhn-neto et al. (2009), this method may be less efficient when the workers are released in remote areas that they are not familiar with. despite a potential loss of foragers that may get lost in unfamiliar release spots, the number of bees that do return to the nest, it is possible to estimate the species’ flight radius, which could possibly be greater than obtained with others methods, and provide more reliable information about the distances that bees travel in the community away from their colony to search for resources (van nieuwstadt & iraheta, 1996). the current study aimed to estimate the maximum foraging distance of melipona subnitida ducke, a species distributed in the brazilian states of alagoas, bahia, ceará, maranhão, paraíba, pernambuco, piauí, rio grande do norte and sergipe (camargo & pedro, 2013), while comparing the efficacy of two experimental methods: (1) training workers to visit an artificial feeder; and (2) capture-recapture (adapted from roubik & aluja, 1983). foraging distance information is critical for understanding the scale at which bee populations respond to the landscape, assessing the role of bee pollinators in affecting plant population structure and planning conservation strategies for plants (greenleaf et al., 2007). materials and methods study area and colony selection this study was conducted at “ponta do mangue” village (2º58’12”s; 42º79’56”w), which is located in the municipality of barreirinhas along the eastern coast of the state of maranhão, brazil. the village is located within the national park of lençóis maranhenses. the park covers a total area of 155,000 ha, of which 453.28 km2 are covered by vegetation (brazilian institute of environment and renewable natural resources ibama, 2002). within the area covered by vegetation, 405.16 km2 are predominately composed of restinga, term that represents a set of physiognomically distinct plant communities under marine influence. the species that colonize these areas are mainly from other ecosystems, but with phenotypic variations that differ from those expressed in their original environments (freire, 1990). the restinga area has shrub species dominance, and herbaceous communities are also present in large areas surrounding lakes (brazilian institute of environment and renewable natural resources ibama, 2002). to test the efficacy of using artificial feeder and the capture-recapture methods to determine the flight range of m. subnitida, four colonies were selected from a meliponary consisting of natural nests and nest boxes kept at ponta do mangue village. two m. subnitida colonies were used for both methods (i.e., “colony 1” and “colony 2”), while the other two colonies were used individually for each method (i.e., “colony 3” artificial feeder and “colony 4” capturerecapture). strong colonies with a large number of workers (mean 890 foragers) and sufficient stored food were selected (mean of 115 pots food). artificial feeder method – foraging distance the experiments were performed in two different months: march 2013 and august 2013. in march, only the workers from colony 1 visited the artificial feeder. after the study with colony 1, which lasted five days, the same methodology was attempted with the other three colonies, though without success. the study continued in august with two other colonies (colony 2 and colony 3), thus totaling three colonies using the artificial feeder method. the weather in the days of study (march and august) did not differ (without rain), thus allowing for a comparison of the replicates. an artificial feeder consisting of a flat acrylic disc with grooves from the center to the edge was used to evaluate foraging distances. a jar containing food (2.5m, 60%: 40% sugar: water concentration), with the opening facing downwards so that the food would drain, was placed in the center of the disc. vanilla extract (9 μl/l) was added to the food to simulate floral odor and to increase the attractiveness of workers to the artificial food (fig 1). the feeder was placed atop a tripod, which allowed for the adjustment of the height and horizontal inclination of the food supply (method described by frisch, 1967; nieh et al., 2003). the first step of the experiment was to train the bees to visit the feeder. the feeder was placed right outside of the hive entrance and then moved a few cm away from the nest (thus, creating a food trail), so that when bees cleared the colony entrance, they tried the food and then followed the trail to reach the tripod with the artificial food source. each worker that visited the feeder was marked with a dab of acrylic paint on their thorax with one color or a combination of specific fig 1. feeder used to artificially train melipona subnitida to a food source. ag silva, rs pinto, fal contrera, pmc albuquerque, mmc rêgo foraging distance of melipona subnitida496 colors to differentiate them foragers from each other, as described in seeley (1995). foragers were classified as recruits or reactivated foragers. recruits were those foragers that had never visited the feeder before and found the feeder on their own, while reactivated foragers were those that had fed from the feeder when it was placed in a previous location, for some reason had stopped visiting the feeder temporarily, but found it again later (kuhn-neto et al., 2009). during the training process, when a minimum of 10 bees had visited the feeder and recruitment was in progress (i.e. when new foragers were recruited), the feeder was gradually moved away from the nest. to facilitate the forager’s memorization of the new location, the feeder was moved only when workers were feeding. the feeder was always moved only after the first bee recorded at a specific distance left the feeder probably to unload her food sample at the nest, and later returned to the feeder for more food. the number of workers that visited the feeder was recorded every 20 m. if the total number of foragers decreased by 50% from the maximum number recorded previously, the feeder was not moved throughout the rest of the day. the next morning, the feeder was placed at the final location reached on the previous day, and remained at this location until the same number of foragers arrived as was recorded the day before. if the same number of foragers did not arrive within two hours, the feeder was moved to the next distance. the experiments were performed daily during the period of natural foraging activity for m. subnitida (6 am – 5 pm) and ended when the workers stopped visiting the feeder. the experiments with colonies 1, 2, and 3 respectively lasted 56 h over 6 days, 33 h over 3 days, and 32 h over 3 days. trial duration was determined by the maximum distance to which the colony could be trained. the full path of the foraging range of each studied colony was classified as three distances: close foraging distance, average foraging distance and maximum foraging distance. the close foraging distance was defined as the distance from the colony’s nest to the point where the number of foragers remained above the overall mean of visiting workers along the path during the experiment ( vf = ), where tvw is the total number of visits by workers along the path, and ttp is the total number of points where bees were recorded along the path, dividing these values, we obtain xvf (the mean number of bees that visited the feeder). the average foraging distance was defined as the distance at which the number of workers fell below average to the last distance where workers were recruited. the maximum foraging distance was defined as the last point where occurred the foraging activities at the feeder. capture-recapture method – flight radius the capture-recapture experiment was performed in august 2013. three m. subnitida colonies were used, two of which were also used for the feeder test (2.2). workers (n = 150) were captured at the nest entrance and marked on the thorax with nontoxic paint of different colors, totaling 15 groups of 10 workers in each nest. initially, 10 linear points were marked every 200 m starting at the colony’s nest entrance, reaching a total distance of 2,000 m. from the 2,000-meter mark onwards, five more points were marked at a distance of 400 m apart from each other, reaching a total distance of 4,000 m. the distances at which the bees were released were measured from the colony entrance, and the points were located using a gps device. the bees were released between 9 and 10 am, and the number of bees that returned to the colony was recorded to calculate the percent of successful return. workers were recaptured at the colony entrance by observing the bees that arrived. to do this, one researcher released the bees at the established distances away from the nest, while another researcher stood at the nest entrance, observing and quantifying the bees that returned to the nest until 6 pm. the nest was kept closed during the experiments, and was only opened when a new worker approached the nest to prevent the erroneous recounting of bees that had already entered the nest. statistical analysis we performed linear regression between the total distribution of workers visiting the experimental feeders and the distance from the feeder to the colony to evaluate the effect of distance on foraging. we also made a linear regression on the number of recruits and reactivated foragers for each colony and distance (zar, 1999). in addition, for each colony, the distances at which we measured 75%, 95% and 100% of foraging activity were estimated to reflect the percentage of active workers relative to the total number of workers within a certain distance. to determine whether the number of workers that returned to the nest (as obtained through the capturerecapture method) differed between colonies, we used the non-parametric kruskal-wallis test. the relationship between the percentage of bees that successfully return to the nest and the distance at which they were released was assessed using a simple regression (zar, 1999). we also used the mann-whitney test to assess the significance of the difference between the two methods used in the current study (artificial feeder vs. capture-recapture). all statistical analyses were performed using the statistica 7.0 software with a critical p-value of 0.05 (statsoft inc., 2004). results foraging distance overall, m. subnitida foragers achieved a maximum foraging distance of 1,080 1,160 m and a maximum recruitment distance of 940 m. the mean foraging distances of sociobiology 61(4): 494-501 (december 2014) 497 the three colonies were as follows: close distance of 653 m, average distance of 863 m and maximum distance of 1,120 m (table 1). the mean values for 75%, 95% and 100% of foraging activity were 566 m, 833 m and 1,120 m, respectively (table 2). the number of workers found at each foraging distance is given in fig 2 for each of the three colonies studied. overall, the number of foragers trained and recruited to the artificial feeder decreased with distance away from the feeder. the linear regression analysis of the relationship between the number of bees that visited the feeder and distance showed a strong negative correlation for the three studied colonies (r = 0.67; p < 0.0001), i.e., the greater the distance, the lower the number of foragers that visited the artificial feeder table 1. foraging distances (m) calculated for the three colonies evaluated by the artificial feeder method. data shown as averages and ± sd. fig 2. total distribution of the number of melipona subnitida foragers (black diamonds), recruits (grey bars) and reactivated foragers (white bars) that were observed at artificial feeders to which the bees were trained over time. arrows indicate the distances defining the areas of 75%, 95% and 100% foraging activity (see methods for details). colony 1 colony 2 colony 3 average/ sd close distance 740 600 620 653.33 ± 75.71 average distance (maximum recruitment distance) 900 940 820 886.66 ± 61.10 maximum distance 1,160 1,120 1,080 1,120 ± 40.00 ag silva, rs pinto, fal contrera, pmc albuquerque, mmc rêgo foraging distance of melipona subnitida498 fig 3. percentage of returnees for each distance (m) at which the workers were released by the capture-recapture method: a – colony 1, b – colony 2, c – colony 3. (table 2). the correlation between the number of recruits and reactivated foragers and distance were also evaluated by linear regression for each colony. in all three colonies, both the number of recruits and the number of reactivated bees were negatively correlated with distance away from the nest (table 2). therefore, there was a decline in the number of recruits and reactivated foragers at more distant sites, compared to sites near the nest (fig 2). flight radius the number of workers released by the capturerecapture method and the number of workers that returned to the nest were significantly different in all three colonies studied (kruskal-wallis n = 350; p = 0.0175). thus, the data for the percentage of bees that returned to the nest were analyzed separately. the percentages of released bees that returned from different distances from the nest are shown in fig 3. the correlation between the percentage of bees that successfully returned to the nest and the distance traveled, as evaluated by linear regression, was highly negative for all three colonies. therefore, the number of bees that returned to the nest gradually decreased as the distance increased (table 3). the maximum flight distance of m. subnitida measured by the capture-recapture method was 3,600 m for colonies 2 and 3, and colony 1 reached 4,000 m (fig 3). table 2. linear relationships between distance (m) and the number of melipona subnitida workers that visited the feeder. in addition, distances that delimited the areas of 75%, 95% and 100% cumulative foraging activity. data shown as averages and ± sd. a – total number of bees, b – recruits, c – reactivated workers. a colony linear relationship between distance and the total number of bees that visited the feeder r 75% 95% 100% 1 y = 14.7203 0.0121x 0.70* 600 880 1,160 2 y = 24.3987 0.0193x 0.66* 540 820 1,120 3 y = 30.4169 0.023x 0.67* 560 800 1,080 avarege/ sd 0.67* ± 0.02 566,66 ± 30.55 833,33 ± 41.66 1,120 ± 40 b colony linear relationship between distance and the number of recruits r 1 y = 3.5068 0.0042x 0.48* 2 y = 6.9074 0.0074*x 0.80* 3 y = 7.0013 0.0076*x 0.76* c colony linear relationship between distance and the number of reactivated bees r 1 y = 1.3186 0.0013x 0.44* 2 y = 4.4289 0.0025x 0.27* 3 y = 4.3045 0.0021x 0.30* *p < 0.05 (significant) table 3. linear relationship between the distance (m) at which the melipona subnitida workers were released by the capture-recapture method and the percentage of return to the nest. data shown as averages and ± sd. colony linear relationship between distance and % of return r flight radius 1 y = 9.7306 0.0022x 0.95* 4,000 2 y = 9.9095 0.0024x 0.89* 3,600 3 y = 11.0524 0.0022x 0.83* 3,600 average/ sd 0.89 ± 0.06 373.33 ± 230.94 *p < 0.001 (significant) comparison of methods the results obtained for the foraging distance of m. subnitida workers were very different between the two methods used. there was a highly significant difference between the maximum foraging distances obtained by the two methods (u < 0.0001; p = 0.04; fig 4), with the maximum flight distance recorded in the capture-recapture method being approximately 2,700 m further than that observed in the artificial feeder method. sociobiology 61(4): 494-501 (december 2014) 499 discussion our results suggest that the mean foraging distance of m. subnitida, as obtained from the artificial feeder experiment, was approximately 1,120 m. this value was lower that reported by kerr (1987) for melipona fasciculata smith (approximately 2,470 m) and that reported by kuhnneto et al. (2009) for melipona mandacaia smith (1,800 m). interestingly, the three classifications of m. subnitida foraging distances revealed that the close foraging distance was approximately 653 m, indicating that at this distance, m. subnitida colonies have a greater ability to maintain a large number of foragers and a facilitated recruitment capacity. the close foraging distance is the distance within which the bees truly dominate a food resource and therefore have a higher probability of pollinating the plants where foragers (van nieuwstadt & iraheta, 1996). considering the distance at which it is possible to recruit workers, which in our study we found to be 863 m on average, it is still likely to establish an effective control of the food source. beyond this recruitment distance, it seems that it is possible for the foragers to find food, but they would probably have to explore it by themselves and potentially the exploitation of the food resource would be less efficient (kuhn-neto et al., 2009). according to van nieuwstadt and iraheta (1996), more than 75% of a stingless bee colony’s food-searching activity usually occurs within 40% of the maximum foraging distance. for m. mandacaia, 75% of the foraging activity (893.33 ± 11.54 m) occurred within corresponded to 50% of the maximum foraging distance (kuhn-neto et al., 2009). this is similar to what we observed for m. subnitida, in which 75% of the food-searching activity (566.66 ± 30.55 m) occurred within 51% of the maximum foraging distance in their restinga environment. therefore, it can be stated that the farther the colony is from the resource, the lower the number of interested or available foragers is, and consequently, the lower the recruitment of new workers to that food source. one factor that may have been crucial to the smaller flight radius of m. subnitida is the large amount of flowering plants near the colonies. m. subnitida workers visit flowers of humiria balsamifera (aubl.) a. st.-hil. and chrysobalanus icaco l. to collect nectar. these plant species bloom almost all year round in the studied region; they exhibited high flowering when colony 1 was trained but less flowering during the experiments with colonies 2 and 3. because other colonies did not follow the feeder in march, the experiment was resumed five months later. the results obtained in march and august were very similar. therefore, it can be inferred that an abundant supply of a profitable nectar source near the nest decreased foraging and recruitment to an artificial site located at more distant locations. the large food supply near the experimental colonies in march certainly influenced the number of bees of colony 1 that were recruited to the feeder. however, the foraging distance did not differ among the colonies. although it was not possible to verify such influence (abundant food sources), workers could potentially be “discouraged” in the search for a farther food source simply because there were other abundant food sources near their nest. a study looking at the foraging ecology of m. mandacaia, a native bee from the caatinga biome (kuhn-neto et al. 2009), found a larger foraging distance in that species than what we found in m. subnitida. the longer foraging distance of m. mandacaia may be in part because there is a marked reduction in the food supply in the caatinga biome during the prolonged dry season, compared to the constant nectar flow in the restinga region where our study was conducted. in the current study, the distances traveled by m. subnitida were measured without analyzing other factors. for instance, the distances traveled by bees responsible for foraging activities depend on several factors, including density and seasonality of the food supply, species (dornhaus et al., 2006), physiology, and body size (imperatriz-fonseca et al., 1985). furthermore, other factors, separately or combined, may also affect flight radius, such as the internal colony conditions and climatic factors (hilário et al., 2000). however, the days upon which the study was performed did not have any heavy rain and were thus favorable for the experiment because it is known that rain prevents or reduces bee activity (hilário et al., 2007). furthermore, in apoidea bees, body size may act as a limiting factor to the maximum flight capacity and therefore, maximum foraging distance (araújo et al., 2004; greenleaf et al., 2007; kuhn-neto et al., 2009; zurbuchen et al., 2010). however, it is likely that many species actually exercise a lower flight capacity (i.e., occupy a smaller space) depending on other variables such as foraging, specialization in resource searching, navigation, abundance of food resources, and availability of nesting sites. by using the capture-recapture method, the percentage of bees that returned to the nest was 80% if they were released within 1,000 m of their colonies. roubik and aluja (1983), released bees of the genus melipona at different distances from the nest, and found that the mean flight radius of these bees is fig 4. comparison of the efficacy of two methods to determine the foraging distance of melipona subnitida foragers: (a) training foragers to an artificial feeder, and (b) the capture-recapture method. ag silva, rs pinto, fal contrera, pmc albuquerque, mmc rêgo foraging distance of melipona subnitida500 2,100 m from the nest. the authors estimated the maximum flight radius for this genus using regression tests and concluded that it could be up to 2,400 m. taking into account that roubik and aluja (1983) used the same method as that used in our study our results in the capture-recapture experiment exceeded the maximum distance conjectured by roubik and aluja (1983). this difference could be attributable to the fact that, in the current study, we waited until the end of the day for the return of the workers, while roubik and aluja (1983) waited just a few hours. when comparing the two methodologies used in the current study, we observed that each technique has benefits and limitations. in the artificial feeder test, the foragers’ flight range could have been underestimated because some bees got lost in the course of the experiment, or because the artificial food is less attractive than a natural flower, which may discourage the search for a food supply at long distances (van nieuwstadt & iraheta, 1996). in the capture-recapture test, by contrast, an overestimation of the actual flight distance could be possible because the released bees only needed to fly back to the nest. alternatively, there may have been a smaller number of bees that returned to the nest after traveling greater distances, potentially because they became lost due to unfamiliarity with the environment where they were released and because the energy costs associated with orientation are high (van nieuwstadt & iraheta, 1996). the advantage of the artificial feeder method over the capture-recapture method is that it can assess the distance that workers can travel, the number of bees they recruit at each distance as well as the exact gradual reduction in the number of workers along the path, which would be closer to the distance travelled by the bees under natural conditions (kuhn-neto et al., 2009). nevertheless, even with the capturerecapture method, we can determine the distance at which the workers begin to return less successfully. when an environment is fragmented, numerous aspects of the landscape ecology are affected, as the reduction of dispersion and potential colonization of plant species (lovejoy et al., 1986; bierregaard et al., 1992). we can infer that such fragmentation of the landscape may have interfered with our results given that most of the vegetation in the study site is concentrated near the meliponary and the region is surrounded by dunes. according to roubik (1989), the behavior of bees is likely to be adapted to their environment and will also be determined by the “resource landscape,” which includes aspects such as resource quality. bees require a large area of vegetation to obtain food throughout the whole year and to nest in regions beyond the mother colony. as the lençóis maranhenses region is an area in constant flux due to the strong winds and the presence of dunes, the area covered by vegetation tends to decline (gonçalves et al., 2003), thus resulting in an increase in the distance that m. subnitida must fly in search of food. the information related to the foraging distance of m. subnitida reported in the current study can thus help to promote strategies for the conservation of this species. acknowledgments the authors thank the owners of the jandaíra meliponary, irene aguiar, mr. emídio and mrs. maria; 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(2010). maximum foraging ranges in solitary bees: only few individuals have the capability to cover long foraging distances. biol. conserv. 143: 669-676. doi: 10.1016/j.biocon.2009.12.003. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.7300sociobiology 69(1): e7300 (march, 2022) introduction bees (apoidea: hymenoptera) are typically considered the most important pollinators of cultivated plant species (delaplane et al., 2000). they use nectar and pollen for meeting their energy requirements and raising brood (bohart & nye, 1956; roswell et al., 2019; rodney & purdy, 2020). the pollination efficiency of bees is the function of their foraging behavior and body morphology (ohara & higashi, 1994; kudo, 2003). both the foraging behavior and body morphology not only vary among species but also within the sexes of a particular species (akram et al., 2019; tang et al., 2019). many studies have investigated the foraging behavior, life history and pollination efficiency of female solitary bees but how males forage and pollinate the crops largely remains abstract female and male solitary bees usually differ in their behavioral and morphological attributes and consequently in their pollination effectiveness. the current study was carried out at the research farm of the islamia university of bahawalpur, pakistan to compare the foraging behavior and pollination efficiency of female and male andrena savignyi spinola, 1838 on brassica campestris. the impact of different environmental factors (temperature, relative humidity, light intensity and wind speed) on foraging behavior was also studied. andrena savignyi was the most abundant floral visitor of b. campestris and comprised 52.17% of the floral visitors. female individuals fed on both nectar and pollen while male fed on nectar. visitation frequency, visitation rate, pollen harvest and pollen deposition of females were significantly higher than that of males because of their larger size and more dry weight. the maximum abundance of females was recorded at 12:00 pm followed by a sharp decline until 4:00 pm whereas males attained their maximum abundance at 2:00 pm. the female pollinated flowers resulted in greater pod weight, pod length, number of seeds per pod, seed weight per pod and germination percentage than the males. our results suggest that females of a. savignyi deliver better pollination of b. campestris than males in terms of its reproductive success and germination percentage. future studies should emphasis on exploring the biology and ecology of a. savignyi with special focus of its artificial nesting. sociobiology an international journal on social insects waseem akram, asif sajjad article history edited by alistair j. campbell, embrapa amazônia oriental, brazil received 08 july 2021 initial acceptance 05 august 2021 final acceptance 22 november 2021 publication date 07 february 2022 keywords andrena savignyi, brassica campestris, female, male, pollination effectiveness. corresponding author waseem akram department of entomology faculty of agriculture and environment the islamia university of bahawalpur bahawalpur, pakistan. e-mail: raowasiento@gmail.com unclear. females of solitary bees are considered as the better foragers than the males (o’toole & raw, 1991). female bees feed nectar from flowers to meet their energy requirements used for construction, maintenance and protection of their nests. besides this they gather abundant pollen grains for their young ones (ostevik et al., 2010; smith et al., 2019). they usually have high visitation frequency, better capability of utilizing anthesis timing, shorter foraging range and ability to deposit more viable pollen grains than the males (ne’eman et al., 2006, akram et al., 2019). female bees are sometimes ineffective pollinators of some plant species as they harvest more but deposit less pollen grains on the stigma. this is mainly due to deposition of heterospecific pollen grains as an outcome of intensive feeding on multiple plant species (thomson & thomson, 1992). department of entomology, faculty of agriculture and environment, the islamia university of bahawalpur, bahawalpur, pakistan research article bees pollination of brassica campestris (cruciferae) by andrena savignyi (andrenidae: hymenoptera): female vs. male pollination waseem akram, asif sajjad – pollination of brassica campestris by andrena savignyi2 moreover, pollen deposition on stigma depends on pollen carrying method as well as behavior on flower of a certain bee species. reason being females of all the bee species do not necessarily deposit more pollen grains on stigma (young et al., 2007). the males on the other hand, spend most of the time in searching for suitable mates to a longer distance (willmer & stone, 2004; roswell et al., 2019). although they fulfil their energy requirements from the nectar however, they can also carry some pollen grains. maybe because of this, they may transport pollen grains long distances (ne’eman, et al., 2006). some recent studies have shown that the females of andrena emeishanica, anthophora plumipes, habropoda tarsta, eucera nigrilabris and megachile cephalotes are better pollinators than the males in terms of foraging behavior (ne’eman et al., 2006; pascarella, 2010; cane et al., 2011; akram et al., 2019; smith et al., 2019; tang et al., 2019) and pollination effectiveness (akram et al., 2019; tang et al., 2019). keeping in view the inadequate information on foraging behavior and effectiveness of male and female counterparts especially under sub-tropical climatic conditions of indian subcontinent, we focused soil nesting andrena savignyi, the most abundant floral visitor of brassica campestris in the study area. both the sexes in genus andrena differ in their morphological features responsible for the pollination success i.e., females are larger than males and have some other pollen transport areas besides tibial scopa (tang et al., 2019). therefore, the present study was carried out to study whether females are better in (i) foraging behavior (visitation frequency, visitation rate and stay time), (ii) pollination facilitating morphological features (body length and proboscis length) and (iii) pollination effectiveness (pollen harvest, deposition and plant reproductive success) than the males of a. savignyi. the response of native bees to local environmental factors is unknown and as the environment at the study site is substantially different from the conditions elsewhere. the effect of environmental factors i.e., ambient temperature, relative humidity, light intensity and wind velocity -at the time of different observation hours across different observation dateson visitation frequency, visitation rate and stay time of both the sexes was also explored. material and methods study area and plant species the study was carried out at the research farm of the islamia university of bahawalpur (29.376830 n 71.762234 e; 181 meters above sea level), punjab, pakistan. the climate of the district bahawalpur is subtropical, blessed with cold winters and hot summers. the mean daily minimum and maximum temperatures are 18.8 °c and 33.5 °c in winter and summer, respectively and the average annual rainfall is 83218 mm (ahmad et al., 2019). the experimental plant species was field mustard, brassica campestris l. var. toria (cruciferae), sown in an area of half an acer. about 80% pollination of brassica spp. are usually performed by insects (chhuneja et al., 2007). the yellow flowers attract variety of insect orders i.e., hymenoptera, lepidoptera, diptera, neuroptera, thysanoptera and coleoptera (williams, 1978; ali et al., 2011). the study was lasted from last week of december 2020 to last week of march 2021 (i.e., from initiation of flowering until harvest). foraging behavior first, in order to assess the pollinators profile of b. campestris, we conducted a three days-long survey of floral visitors in district bahawalpur. for this purpose, three widely isolated (>10km) b. campestris fields of at least two acers each were selected. pollinators were collected for two hours (10:00 to 12:00) in each field and one field was surveyed in each day. a hand collection net was used for the collection of floral visitors. we regarded pollinator abundance as number of individuals in each species. the collected floral visitors – other than andrena savignyi – were identified to family level by using taxonomic keys proposed by borror et al. (1981). we focused on a. savignyi for further studies as both the male and female individuals were frequently available throughout flowering season. the sex of a. savignyi were distinguished on the wing i.e., females have scopa on hind legs while it is absent in males. the foraging behavior was recorded in terms of visitation frequency, visitation rate and stay time. we defined ‘visitation frequency’ as the number of individuals visited/ m2/120 seconds, ‘visitation rate’ as the number of flowers visited by an individual in 120 seconds and ‘stay time’ as the time spent by an individual on a flower during a single visit. only one foraging behavior was focused in a single day. the visitation frequency, visitation rate and stay time of a. savignyi was recorded at 09:00, 10:00, 12:00, 14:00, and 16:00 hours with three days interval throughout the flowering period. for measuring visitation frequency, visitation rate and stay time ten observations were made at each 09:00, 10:00, 12:00, 14:00, and 16:00 hours (total 50 observations/census). in case of visitation frequency, ten plants were randomly selected (at least 20 feet apart) from the field margins. a stopwatch was used to record these observations. a digital thermo-hygrometer, an anemometer and a lux meter were used before each observation to record temperature, relative humidity, wind speed and light intensity, respectively. morphological features ten individuals of each sex were caught and killed for measuring their body and proboscis length with the help of a digital vernier caliper. the dry weight of female and male a. savignyi specimens was measured by using an electronic weighing balance accurate to 0.001g (n = 10 for each sex) (gilbert, 2011). sociobiology 69(1): e7300 (march, 2022) 3 pollination effectiveness pollination effectiveness of both the sexes was measured in terms of pollen deposition on stigma in a single visit, pollen load carried by an individual bee during peak activity hours and reproductive success of b. campestris as a result of their single visits. to measure pollen deposition, 30 floral buds were caged with nylon mesh bags 24 hours before their opening. each bud was caged on a separate plant. the flowers were then uncaged during the peak activity timing of a. savignyi i.e., 11:00 to 2:00 hours. once a flower had been visited by a female or a male bee, the stigma was removed using a sharp blade. a stereoscopic microscope with 40× magnification was used to count pollen grains (ali et al., 2011). fifteen such observations each were made for female and male a. savignyi. to measure pollen load, fifteen specimens of each sex were collected and killed during peak activity timing. the pollen grains were removed from their body by placing them in a glass vial containing 70% ethanol. once the pollen grains were obtained within the solution, a 10,000µl sample was taken. ten subsamples of 10µl each were then taken and the pollen grains counted by using a stereoscopic microscope with 40× magnification. finally, the average number of pollen grains for the ten subsamples was multiplied by the total 10,000µl, and the result divided among the volume of each subsample i.e., 10µl (canto-aguilar & parra-tabla, 2000). to see the effectiveness of female and male a. savignyi in terms of plant reproductive success, some other floral buds were caged with nylon mesh bags 24 hours before they opened. each bud was caged on a separate plant. they were un-caged during peak activity timing of a. savignyi and recaged once a single visit had been made by a female or male bees. twenty-two such observations were made for each sex. the resultant pod weight (g), pod length (cm), number of seeds/pod and seed weight/pod (g) was recorded as the measures of pollination effectiveness. the seeds obtained from pods were subjected to germination tests, carried out in separate petri dishes containing filter paper. each petri dish was moisturized everyday with distilled water for three days. seeds were considered germinated when 2-3mm long radicles emerged. twenty-two open-pollinated (unrestricted insect visitation) and 22 caged (no insect visitation) flowers were also maintained on 44 different plants. data analysis to compare the foraging behavior of both the sexes, we used generalized linear mixed model (glmm). the dependent variables included visitation frequency, visitation rate and stay time whereas the predictor variables were the environmental factors (i.e., ambient temperature, relative humidity, light intensity and wind speed) and sex was the fixed factor. separate glmms were run for visitation frequency, visitation rate and stay time. we used poisson distributions with linear-log link functions. the means of pollen deposition, pollen harvest, body length, proboscis length and dry weight of female and male bees were compared using t-test as the data followed normal distribution (alpha 0.05). one-way anova was applied to see the significance in pod weight, pod length, number of seeds/pod, seed weight/pod and germination percentage among female, male and open-pollinated pods. means were compared by using tukey’s post hoc test at alpha 0.05. in order to know how much variation in foraging behavior (the dependent variables) of a. savignyi is explained by environment factors (the predictors) we performed multiple linear regression analysis separately for male and female. the foraging behavior included stay time, visitation rate and visitation frequency while environmental predictors included temperature, relative humidity, wind speed and light intensity. the linear regression’s f-test was applied with null hypothesis that model explains zero variance in dependent variables. in order to see the significant environmental predictors of foraging behavior, their regression coefficients were compared at alpha 0.05 by using t-statistics. all the analyses were performed in xlstat (xlstat, 2021). results a total of 991 individuals of 29 species i.e., 9 bees, 3 wasps, 1 ant (order, hymenoptera), 10 flies (order, diptera) and 6 butterflies (order, lepidoptera) visited the flowers of brassica campestris. hymenopterans were the most abundant (92.23%) followed by dipterans (6.36%) and lepidopterans (1.41%). among bees, andrena savignyi was the most frequent floral visitors of b. campestris and comprised 52.17% of the floral visitors followed by apis dorsata (23.01%), a. florea (13.97%) and lasioglossum albescens (1.01%). female to male population ratio of a. savignyi was 2:1 i.e., 349 and 168 individuals, respectively. the foraging activity of both female and male a. savignyi started somewhere between 9:00 to 10:00 am. the maximum abundance of females was recorded at 12:00 pm followed by a sharp decline until 4:00 pm. males attained their peak abundance at 2:00 pm (fig 1). 0.00 0.50 1.00 1.50 2.00 2.50 9:00 am 10:00 am 12:00 pm 2:00 pm 4:00 pm in di vi du al s/ pl an t/1 20 s ec on ds time female male fig 1. diurnal fluctuations in abundance of female and male a. savignyi. waseem akram, asif sajjad – pollination of brassica campestris by andrena savignyi4 females landed directly on the anthers and their thorax and legs came in contact with anthers while males landed on the petals and took up nectar by extending their proboscis. although males had dense hairs on the thorax but they rarely came in direct contact with anthers (fig 2). the comparison of foraging behavior of female and male a. savignyi is presented in table 1. the results of glmm showed that females had significantly higher visitation frequency and visitation rate while lower stay time than the males. the results of t-test showed that females harvested and deposited more pollen grains than the males, potentially due to the presence of scopa on the hind legs of females (fig 2). fig 2. hind legs of a. savignyi: (a) female with load of pollen grains, (b) male. visitation frequency1 visitation rate2 stay time3 female 2.44 ± 0.14 16.04 ± 0.32 3.06 ± 0.15 male 1.88 ± 0.14 12.58 ± 0.43 4.01 ± 0.26 wald chi-square 8.62 58.41 20.46 df 1 1 1 p 0.003 < 0.0001 < 0.0001 1individuals/m2/120 seconds, 2flowers/120 seconds and 3seconds/flower the comparison of morphological features of female and male a. savignyi is presented in table 2. there was a significant difference between female and male a. savignyi in terms of body length (p = <0.0001) and dry weight (p = <0.0001). female individuals have longer body and more dry weight than males. there was no significant difference between female and male a. savignyi in terms of proboscis length (p = 0.433). table 1. results of generalized linear mixed model. the results of multiple linear regression revealed that the model had very low r2 values therefore did not explain much of the variation in foraging behavior (visitation frequency, visitation rate and stay time) on account of environmental factors (temperature, relative humidity, wind speed and light intensity). however, the models were significant except for the stay time of female a. savignyi. the results of t-statistics revealed that visitation frequency of females significantly increased with increase in temperature, relative humidity and light intensity. visitation frequency of males on the other hand significantly increased with increase in temperature and light intensity. visitation rate of females increased with increase in wind speed and light intensity while the visitation rate of males increased with increase in temperature and wind speed. none of the environmental factors affected the stay time of females. stay time of males decreased with increase in light intensity (table 3). body length (mm) proboscis length (mm) dry weight (g) pollen deposition pollen harvest female 14.55 ± 0.024 3.10 ± 0.10 0.023 ± 0.001 351.33 ± 17.91 64853.33 ± 2371.78 male 11.15 ± 0.028 3.00 ± 0.075 0.015 ± 0.001 180.93 ± 9.62 3893.33 ± 78.20 results of t-test t-observed 9.216 0.802 9.258 8.381 25.688 t-critical 2.101 2.101 2.101 2.048 2.048 p-value <0.0001 0.433 <0.0001 <0.0001 <0.0001 df 18 18 18 28 28 table 2. comparison between female and male morphological features and pollination effectiveness. sociobiology 69(1): e7300 (march, 2022) 5 female bees produced higher pod weight (0.07g), pod length (4.84cm), number of seeds per pod (12.05), seed weight per pod (0.030g) and germination percentage (45.70%) than the males (table 4). the alternate floral resources of a. savignyi are presented in table 5. the maximum abundance was recorded on raphanus sativus and brassica napus. discussion in the present study, 29 insect species in three orders were observed foraging on brassica campestris in our experimental plot. pollinator composition and abundance vary with geographical area, time and latitude (ollerton & louise, 2002). due to generalized floral structure of brassica spp., a large group of floral visitors are attracted i.e., bees, flies, butterflies and wasps (kunin, 1993; mussury & fernandes, 2000). williams (1980; 1985) reported that the flowers of brassica spp. produce abundant pollen and nectar that makes it attractive to bees. among bees, andrena savignyi were the most frequent floral visitors of b. campestris. several factors determine the population abundance of a certain bee species in an ecosystem i.e., temperature fluctuations (bennett et al., 2015), agricultural intensification (connolly, 2013; woodcock et al., 2017), habitat loss (potts et al., 2010; belsky & joshi, 2019) and natural enemies (predators, parasites and diseases) (evison et al., 2012). table 5. pod weight, pod length, number of seeds/pod, seed weight/pod and germination percentage resulting from single visit by female and male a. savignyi. variables gender model f p-value r2 visitation frequency female y = -5.21+0.12 (t)* +0.03 (r.h)* -0.04 (w.s) +0.01 (l.i)* 16.202 <0.0001 0.180 male y = -2.82+0.07 (t)* +0.02 (r.h) -0.02 (w.s) +0.00 (l.i)* 9.367 <0.0001 0.113 visitation rate female y = -0.78+0.24 (t) +0.07 (r.h) +0.30 (w.s)* +0.01 (l.i)* 7.764 <0.0001 0.137 male y = -4.85+0.98 (t)* -0.08 (r.h) +0.35 (w.s)* -0.01 (l.i) 7.213 <0.0001 0.159 stay time female y = 7.80-0.11 (t) -0.02 (r.h) -0.06 (w.s) -0.00 (l.i) 1.831 0.125 0.037 male y = 11.80-0.14 (t) +0.02 (r.h) -0.04 (w.s) -0.01 (l.i)* 5.682 0.000 0.128 *t-statistics predicts significance at alpha 0.05 table 3. multiple regression analysis between foraging behavior of female and male a. savignyi and environmental factors (t = temperature, r.h = relative humidity, w.s = wind speed and l.i = light intensity). plant local name scientific name andrena savignyi individuals false flax camelina sativa 1 rapeseed brassica napus 12 cauliflower brassica oleracea 7 radish raphanus sativus 23 turnip brassica rapa 6 pod weight (g) pod length (cm) no. of seeds/pod seed weight/pod (g) germination percentage female 0.07 ± 0.01 b 4.84 ± 0.23 b 12.05 ± 1.19 b 0.030 ± 0.003 b 45.70 ± 9.82 b male 0.01 ± 0.00 c 0.75 ± 0.31 c 0.95 ± 0.89 c 0.001 ± 0.001 c 0.01 ± 0.00 c open 0.13 ± 0.01 a 6.18 ± 0.28 a 15.90 ± 0.93 a 0.045 ± 0.003 a 78.58 ± 7.37 a results of anova f 103 106 59 60 31 df 2 2 2 2 2 p value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 table 4. visitation of a. savignyi on alternate floral resources at bahawalpur, punjab, pakistan. female a. savignyi showed higher visitation frequency and visitation rate than the males. females have more energy requirements than the males as they have to build their nests, lay eggs, collect nectar and pollen for their immatures and defend and maintain their nests (eickwort, 1975; peterson & roitberg, 2006; danforth et al., 2019). consequently, they have to uptake more nectar than the males (roswell et al., 2019). tang et al. (2019) reported that the males of andrena emeishanica visited a smaller number of flowers than the females. in the present study, male a. savignyi showed higher stay time than the females. the female solitary bees spend less time on the flower because they have to visit several flowers for both nectar and pollen to fed their young ones. whereas the higher stay time in males is might be due to sole feeding on nectar to fulfil their own energy requirements (peterson & roitberg, 2006; roswell et al., 2019). bee species which are intensive foragers (having high foraging rates) usually work on rapid pace but stay for a shorter period of time on flowers waseem akram, asif sajjad – pollination of brassica campestris by andrena savignyi6 than species with less foraging rates (sajjad et al., 2008; ali et al., 2011; ali et al., 2014; ali et al., 2016; zameer et al., 2017, farooqi et al., 2021). female individuals of a. savignyi have longer body and heavier dry weight than males. large-bodied pollinators generally harvest and deposit more pollen grains per single visit than small-bodied ones (cruden & miller-ward, 1981; willmer & finlayson, 2014). the body size is positively related to pollen load capacity and heterospecific pollen deposition (ramalho et al., 1998; greenleaf et al., 2007). in our study, females of a. savignyi harvested and deposited more pollen grains than males. the andrena females have large hind tibiae with obvious scopa and about 90% of the pollen grains are collected on the hind legs (tang et al., 2019). in general, females collect both nectar and pollen grains whereas males prefer feeding on nectar and hardly collect pollen grains (thorp, 1979; ostevik et al., 2010). tang et al. (2019) found that females of andrena sp. usually collected ample quantity of pollen grains on their hind legs but contrarily, they deposited less on stigma whereas males harvested less but deposited more. the pollen transfer efficiency of males or females depends on how untidily the pollen grains are attached with the body. in the present study, female bees produced higher pod weight, pod length, number of seeds per pod, seed weight per pod and germination percentage than the males. female solitary bees are more efficient pollinators than males (michener, 2000; ne’eman et al., 2006; akram et al., 2019; tang et al., 2019). female bees move readily among flowers, carry abundant outcrossing pollen grains and therefore can have high single visit efficiency than the males (akram et al., 2019). in this study, the visitation frequency of females and males was significantly affected with temperature, relative humidity and light intensity. visitation rate of female was affected with wind speed and light intensity whereas in case of males, it was affected with temperature and wind speed. the comparison of female and male solitary bees in their foraging behavior towards different environmental factors is less understood. our results are in line with wang et al. (2009) who found that visitation rate of bees (andrena parvula, anthophora melanognatha, megachile abluta, m. spissula and xylocopa valga) increased with the increase in temperature and light intensity but decreased with increase in relative humidity. moreover, visitation frequency increased with increase in temperature, relative humidity and light intensity. while observing association between foraging behavior and environmental factors, biotic factors are often ignored. the environmental factors also influence the vegetative and reproductive growth of plants. during vegetative development of brassica spp. high mean temperature reduces the floral abundance (morrison & stewart, 2002) which ultimately affects pollinators’ abundance (nayak et al., 2015). conclusion it is concluded that females of a. savignyi exhibited better foraging behavior in terms of visitation frequency and visitation rate than males. they also proved to be the effective pollinators, as compared to males, in terms of pollen harvest, pollen deposition and plant reproductive success. therefore, in terms of conservation strategy, special focus should be given to females of ground nesting bees as they are effective pollinators than the males. acknowledgement this research was financed by the agriculture linkage program (alp) of pakistan agriculture research council (parc) under project “conservation of native bees through ecosystem approach for enhanced crop pollination”. we thank dr. john s. ascher for the identification of andrena savignyi. references ahmad, a., khan, m.r., shah, s.h.h., kamran, m.a., wajid, s.a., amin, m., khan a., arshad, m.n., cheema, m.j.m., saqib, z.a., ullah, r., ziaf, k., ul haq, a., ahmad, s., ahmad, i., fahad, m., waqas, m.m., abbas, a., iqbal, a., pervaiz, a. & khan, i.a. 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(2017). foraging behavior of pollinators leads to effective pollination in radish raphanus sativus l. asian journal of agriculture and biology, 5: 221-227. doi: 10.13102/sociobiology.v66i4.4679sociobiology 66(4): 560-567 (december, 2019) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction finding enough food is a central challenge for all the organisms. in social insects, the amount of collected food must counterbalance both individuals and collective requirements, following the ‘altruistic’ rules dictated by the inclusive fitness (abbot et al., 2011). escaping from starvation is, therefore, a collective concern that may deeply affect the foraging behavior of ants, since food shortage may be a serious threat for the whole colony. when resource supply is low, ant colonies may put in place collective behaviors to counterbalance food shortage, as it happens in lasius niger, in which the number of scouts that spontaneously leave the nest even in the absence of chemical or tactile signals increases in times of famine (mailleux et al., 2010). starvation may abstract in this study, we investigated the effect of starvation on the feeding behavior of the ant tapinoma nigerrimum. in particular, we tested the response of ants that had experienced different levels of starvation, toward sucrose solutions of increasing concentration. as expected, starved ants promptly reacted to the sugary food sources with a higher rate of acceptance as compared to satiated ones. acceptance increased both with sugar concentration and the length of the starvation period. however, a consistent fraction of the starved ants did not feed on the solutions, suggesting that starvation had different effects on different individuals, even though they all had food ad libitum before the beginning of the tests, had comparable body sizes, and were collected from the same trail. the different acceptance of sugary solutions may be, therefore, merely because ants fed on the experimental food at different times. interestingly, in all the experimental groups, ants appeared to satiate quickly, irrespective of the solution tested and fasting duration. this would suggest that the rate of ingestion was independent of these factors, a result partially at odds with previous studies. this study is one of the few ones dealing with the behavioral response of an ant species to a famine event. sociobiology an international journal on social insects f frizzi, k sudano, g santini article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 20 august 2019 initial acceptance 18 september 2019 final acceptance 01 october 2019 publication date 30 december 2019 keywords ants, feeding behavior, fasting period, famine, carbohydrates. corresponding author fillipo frizzi department of biology university of florence via madonna del piano nº 6, 50019 sesto fiorentino, florence, italy e-mail: filippo.frizzi@unifi.it negatively affect a colony’s fitness in many ways, which range from subtle metabolic impairment to direct foragers death. in aphaenogaster picea, for example, starvation reduces individual thermal tolerance (nguyen et al.,2017), while in formica exsecta, protracted food shortage affect the tissue-specific gene expression and impairs the efficiency of the tissues involved in the degradation of bacterial cell walls, melanization, and the encapsulation response (bos et al., 2016). all these effects may, in turn, elicit cascade effects as decreased brood and nest hygiene (dussutour et al., 2016), or unbalance interspecific interactions and facilitate the success of the species more resistant to the lack of food, as observed in linepithema humile (mcgrannachan & lester, 2013). although many details of the ecological and behavioral responses of ants to food shortage have been studied in the department of biology, university of florence, sesto fiorentino, florence, italy research article ants effect of starvation on the feeding activity toward sugary food in the ant tapinoma nigerrimum (nylander, 1856) sociobiology 66(4): 560-567 (december, 2019) 561 past, such as the inter-caste interactions (mailleux et al., 2011a), the spatial arrangement of the colony (mailleux et al., 2011b), the interspecific aggressiveness (liu et al., 2011), the exploration efficiency (mailleux et al., 2010), and even the communication (mailleux et al., 2006), the short-term behavioral responses to food resources received, surprisingly, little or no attention. tapinoma nigerrimum (nylander, 1856) is a dominant, polydomous, usually polygynous, and even potentially invasive ant species widespread along the mediterranean basin and a significant part of europe (blight et al., 2014; dekoninck et al., 2015). this species is widespread in anthropic habitats, and its nest holes can be easily found in gardens, or along the crevices of sidewalks. it has been assumed that similarly to t. sessile (buczkowski & bennett, 2008) this species can limit the expansion of the invasive argentine ant (l. humile), because of its supposed strong ecological competitiveness (blight et al., 2010, but see frizzi et al., 2017). moreover, this species is highly tolerant to high temperatures (frizzi et al., 2017) but seems to be sensitive to food shortage as six days without food appear sufficient to cause the death of the majority of workers (unpublished observation). as in many other ant species, carbohydrates are a fundamental resource for the survival of t. nigerrimum colonies, and they are usually obtained from mealybugs honeydew (cerdá et al., 1997; mansour et al., 2012). carbohydrate availability may affect individual behavior (e.g. aggressiveness, grangier & lester, 2014), colony size, and survival (wittman et al., 2018), and their importance is supported by the strong preference for this resource shown by many dominant species (e.g. grover et al., 2007; abril et al., 2007; frizzi et al., 2016). with this preliminary laboratory study, we aimed to assess the short-term behavioral effect of starvation on t. nigerrimum, in terms of acceptance of sugary solutions of increasing concentration. in particular, we observed the behavior of groups of ants in the first few minutes after the end of starvation and we expected that the acceptance of a solution changes with its sugar concentration and starvation duration. to our knowledge, this is one of the few studies dealing with the early effects of the starvation on the foraging behavior in ants. materials and methods the experiments were carried out from june to august 2014 within the sesto fiorentino university campus, central italy (43°49’05.05”n, 11°12’12.95”e). the sampling area is interspersed in an urban matrix, made up by university buildings surrounded by overgrown fields, public gardens with a few ornamental trees (acer sp.), and traversed by low-traffic roads. ants were collected from a paved lot (side 200m), where a total of 31 nest holes were counted. we randomly selected six nest holes, separated by at least 6 m from each other. as t. nigerrimum may form large super colonies (dekoninck et al., 2015), the likely non-independence of nest holes was taken into account in the statistical analyses. since t. nigerrimum shows considerable polymorphism and body size can affect both individual survival (kaspari & vargo, 1995; heinze et al., 2003) and the amount of food ingested, particularly carbohydrates (josens et al., 2018), we took care to collect only major workers having a similar size. at the end of each experiment, ants were measured under a stereomicroscope, to exclude the presence of outliers (total body length = 3.28 ± 0.21sd mm). in a single sampling event, we collected 300 ants from trails starting from a randomly selected nest hole. we repeated this procedure for each of the six nest holes. trails were used by the ants to exploit the honeydew of mealybugs infesting the trees bordering the lot, so we took care to capture only unfed individuals, i.e. those walking in the direction of trees. ants were housed in a plastic container (15 x 25 x 15 cm) with fluon® coated walls and acclimated to laboratory conditions for three days with 25 g/l sucrose solution and water available ad libitum to empty the gut of the ants (frizzi et al., 2017). after this acclimation period, we removed the sucrose solution only, and we collected five groups of 10 ants each (hereafter called sa, satiated ants), which were placed in a neutral arena (a 9 cm diameter petri dish with fluon® coated walls). after two minutes, a droplet of water or one of three different sucrose solutions (2, 4 and 16% weight/volume, expressed in g/l, following frizzi et al., 2016) was released in the center of each dish (fig 1). droplets were sufficiently large so that all the tenants could drink at the same time. two minutes later we started assessing the acceptance, measured as the proportion of drinking ants within a period of 15 seconds. we repeated this measure every two minutes for each group of ants, until a total time of 14 minutes and 15 seconds had elapsed. an ant touching the droplet with the mandibles was scored as drinking the solution only if contact lasted for more than 2 seconds. if the same ant drank more than one time during the 15 seconds of the observation, it was scored only once. to reduce the error probability, each trial was observed by at least two operators. at the end of each test, the ants were measured and released. the test was repeated after 48 and 96 hours (hereafter called ss, short starvation, and ls, long starvation, respectively). the length of the starvation period was chosen on the basis of previous observations, showing that t. nigerrimum ants well survived without access to food for at least 5 days. water was always available to all ants for the entire duration of the experiments. the 14 minutes duration of the observations was decided on the basis of preliminary tests, showing that after this time both in starved and non-starved ants ceased to feed. to do this, we performed individual tests with four groups of fed (10 ants per group) and four groups of starved ants (four days of fasting). to each group we supplied a 16% sucrose solution and we counted every two minutes the number of ants feeding (over 15 seconds), for a total period of 60 minutes. after 14 minutes, only one fed ant and two starved ants drank the solution at most. f frizzi, k sudano, g santini – effect of starvation on tapinoma nigerrimum562 to quantify how acceptance changes over the testing time for satiated (sa) and starved ants (ss and ls), we fitted, for each of the sucrose solutions tested, five generalized linear mixed models (glmm) of increasing complexity, with binomial distribution. these models were: the null model, two models including only one factor (level of starvation or testing time), a model including both factors, and finally a complete model including both factors and their interaction. we included the nest identity as a random factor to account for non-independence of nestmates. models were ranked using the akaike’s information criterion corrected for small size samples (aicc). to assess the differences in the acceptance between the differently starved groups in pairs, we performed tukey post-hoc multiple comparisons. to focus the acceptance change over time according to starvation, for all the observation times we compared the number of ants drinking among starvation levels using the kruskallwallis test, followed by dunn’s non-parametric post-hoc test. analyses were performed using the ‘lme4’ (bates e tal., 2015), ‘aiccmodavg’ (mazerolle, 2017), and ‘multcomp’ (hothorn et al., 2008) r 3.5.1 (r core team 2018) packages. results the main result of the experiments is shown in figure 2, which reports the temporal trends of acceptance of the four solutions tested, for the three groups of ants (sa, ss, and ls). results of model ranking and multiple comparisons are reported in table 1 and 2, respectively. water, was almost never accepted by ants, irrespective of starvation level, and no model performed better than the null one. when we analyzed the response to 2, 4 and 16% solutions, the best fitting model was always the full one, containing the length of starvation, time and their interaction as explanatory variables. in these cases, acceptance was always low or null for satiated (sa) ants, whereas for the 4 and 16% solutions both groups of starved ants showed a significantly higher acceptance. acceptance was also higher at the beginning but rapidly declined as time goes by. for the 2% solution, ls ants showed higher acceptance than either ss and sa (ss = sa, table 2). in table 3, all mean values of drinking ants and the results of kruskall-wallis and dunn’s tests are summarized. fig 1. experimental apparatus, which was used for all the three levels of starvation, satiated (sa), short starvation (ss), and long starvation (ls). table 1. aicc values of models ranked. in bold the significantly lowest aicc value. δaicc is the difference from the lowest value. percentages are weight/volume (g/l). starv = starvation level (sa, ss, and ls). time = time elapsed from the beginning of the test. water 2% 4% 16% model aicc δaicc aicc δaicc aicc δaicc aicc δaicc null 437.34 0 826.73 41.62 842.64 50.815 995.94 150.85 starv 438.95 1.61 817.92 32.81 825.50 33.672 963.46 118.37 time 438.92 1.58 798.24 13.13 813.32 21.496 891.49 46.40 starv + time 440.52 3.18 789.11 4.00 795.61 3.79 855.83 10.74 starv * time 439.23 1.89 785.11 0 791.82 0 845.09 0 table 2. results of multiple comparisons in the acceptance rate between differently starved groups in pairs. percentages are weight/ volume (g/l). sa = satiated ants; ss = short starved ants; ls = long starved ants. in bold significant p values. 2% 4% 16% contrast z p z p z p sa ss 1.672 0.212 3.332 0.002 4.385 <0.001 sa ls 4.448 <0.001 4.575 <0.001 4.821 <0.001 ss ls 3.356 0.002 1.592 0.244 1.439 0.297 sociobiology 66(4): 560-567 (december, 2019) 563 discussion as expected, the starved groups reacted quickly and started feeding, with concentration-dependent acceptance rates, while fed ants showed no interest for any of the test solutions. however, the results also showed unexpected behaviors, which are worth examining. first, the acceptance rate by starved ants was not as high as we expected, as they did not exceed 0.5 even in long starved ants on the most concentrated solutions. it is quite surprising if compared to a previous field study on the acceptance of different food sources for the same sucrose solutions concerning the mediterranean ant crematogaster scutellaris, which showed a level of acceptance ranging from 0.6 to 0.8 (frizzi et al., 2016). although this species belongs to a genus evolutionarily quite far from tapinoma and it likely has different nutritional needs, in absence of other previous studies regarding tapinoma these values can be taken as a general reference, because both species can be usually found in mild/ hot climates and urban contexts, thus climate conditions and, partially, feeding opportunities in sites they inhabit are similar. however, the possibility that the difference in the low level of acceptance of t. nigerrimum compared to c. scutellaris was completely due to the different biological features between these two species cannot be ruled out. nonetheless, other environmental factors may have a role. for example, the effect of water shortage and dehydration, which were effective in frizzi et al. (2016) study, but not here. the fact that not all ants promptly started consuming the sucrose solutions needs some further investigations, as its determinants are not entirely clear. this pattern could, in principle, be explained hypothesizing that not all ants in a group (and hence subjected to the same fasting period), shared the same level of hunger. in an ant colony, the inter-individual response to food shortage may be determined by several endogenous factors, such as the difference in morphology, the different allocation of fat bodies among nestmates, the physiological status, or age fig 2. proportion of drinking ants for water and all the three sucrose solutions tested over the experimental time, with their models fitted (black lines) and the standard errors of predicted values (grey lines). percentages are weight/volume (g/l). dotted lines, white circles = satiated ants (sa); dashed lines, grey circles = short starved ants (ss); solid lines, black circles = long starved ants (ls). in table 2 results of model comparisons are reported. f frizzi, k sudano, g santini – effect of starvation on tapinoma nigerrimum564 water kw contrasts time sa ss ls sa-ss sa-ls ss-ls 2 0.167 ± 0.167 0.333 ± 0.211 1.167 ± 0.477 ns 4 0.333 ± 0.211 0.500 ± 0.224 0.667 ± 0.333 ns 6 0.167 ± 0.167 0.667 ± 0.211 0.500 ± 0.227 ns 8 0.667 ± 0.494 0.667 ± 0.211 0.500 ± 0.227 ns 10 0.167 ± 0.167 0.167 ± 0.167 0.167 ± 0.167 ns 12 0.167 ± 0.167 0.667 ± 0.333 0.333 ± 0.211 ns 14 0.500 ± 0.224 0.667 ± 0.333 0 ns 2% kw contrasts time sa ss ls sa-ss sa-ls ss-ls 2 0.333 ± 0.211 1.167 ± 0.477 2.500 ± 0.671 * ns ** ns 4 0.333 ± 0.333 1.000 ± 0.365 3.333 ± 0.614 *** ns *** * 6 0.500 ± 0.341 0.500 ± 0.341 2.167 ± 0.601 * ns * * 8 1.167 ± 0.401 1.000 ± 0.365 1.167 ± 0.477 ns 10 0.167 ± 0.167 0.167 ± 0.307 0.500 ± 0.341 ns 12 0.500 ± 0.224 1.167 ± 0.401 0.833 ± 0.307 ns 14 0.500 ± 0.224 0.333 ± 0.333 0.167 ± 0.307 ns 4% kw contrasts time sa ss ls sa-ss sa-ls ss-ls 2 0 1.500 ± 0.563 2.833 ± 0.872 ** * ** ns 4 0.333 ± 0.333 1.667 ± 0.558 2.167 ± 0.601 ns 6 1.167 ± 0.308 1.500 ± 0.562 2.333 ± 0.803 ns 8 0.333 ± 0.333 0.833 ± 0.654 1.833 ± 0.307 * ns ** * 10 0.500 ± 0.341 0.500 ± 0.223 0.833 ± 0.307 ns 12 0.667 ± 0.422 1.000 ± 0.516 1.000 ± 0.258 ns 14 0.333 ± 0.333 0.167 ± 0.167 0.667 ± 0.333 ns 16% kw contrasts time sa ss ls sa-ss sa-ls ss-ls 2 0 3.000 ± 0.931 4.500 ± 0.341 *** ** *** ns 4 0 2.500 ± 0.428 3.000 ± 0.894 ** ** ** ns 6 0 2.167 ± 0.601 2.833 ± 0.601 ** ** *** ns 8 0.500 ± 0.341 1.333 ± 0.494 2.000 ± 0.817 ns 10 0.167 ± 0.167 1.167 ± 0.654 1.500 ± 0.341 ns 12 0.167 ± 0.167 0.833 ± 0.401 1.333 ± 0.421 ns 14 0.500 ± 0.341 0.833 ± 0.401 1.167 ± 0.401 ns table 3. mean number of ants drinking at solution droplets (± se), for each concentration and at each time (in minutes), and the results of kruskall-wallis and dunn’s tests. kw is the significance of the result of the kruskall-wallis global test. contrasts concern the dunn’s test. sa = satiated ants; ss = short starved ants; ls = long starved ants. significance levels: ‘ns’ not significant, ‘*’ < 0.05, ‘**’ <0.01, ‘***’ <0.001. (dussutour et al., 2016). in our experiments, we used only equally-sized ants collected from the same trail, which should imply similar morphological traits and size of fat bodies, and hence physiological status (couvillon et al., 2011). as for the effect of health state, when we measured the size of ants under the microscope, we also checked for the presence of ectoparasites or fungus infestations, and none of them was found positive. on the other hand, the effect of age cannot be excluded, although the majority of the ants carrying out the same taskforaging in this case should in principle have similar age (tripet & nonacs, 2004). overall, therefore, the contribution of all the quoted factors to the response variability should be rather low. the simplest explanation of these uneven food use is probably that during the three days when resources were fully available, ants ate different amounts of food and, therefore, some of them started the sociobiology 66(4): 560-567 (december, 2019) 565 satiation period with greater energy reserves. additionally, it is even possible that captured ants practiced an unbalanced trophallaxis among them (cassill & tschinkel, 1999), but, since we did not observe their behavior during the housing in the laboratory, we could not investigate the influence of this possible behavior. the second interesting result is that, despite starvation, ants continued to select the resources actively and consumed the richer solutions at a higher rate than the poorer ones. even though being selective is not surprising in well-fed animals, it seems unexpected for starved ones, particularly for those who underwent a long period of starvation that leads them close to death. this finding would imply that workers continue to evaluate, rank and accept the resources, even when starved. what we might have expected is that all the starved ants, particularly the ones which have experienced the most extended period of food shortage (four days), have fed on all the sucrose solutions, irrespective of the concentration, to avoid death from starvation. the effect of fasting is, therefore, not that to just switch on/off the acceptance of a solution, but that of increase or decrease the acceptance threshold. such behavior may arise because some of the ants, although they fasted for four days, were not enough starved to stop searching more nutritive resources, instead of filling their gut with a low energetic food, following the general principles of the contingency model (stephens & krebs, 1986). however, this point deserves further investigations. the last evidence concerns the variation of the acceptance rate with time, which decreased rapidly after a few minutes from the beginning of the experiments. for example, for the four-days starved ants, the acceptance rate rapidly declined over the six-eight minutes from the start, until the mean number of feeding ants become similar to that characterizing the less starved ones. this point is also underlined by the fact that significant differences in the acceptance among differently starved ants occurred particularly during the first minutes. interestingly, all ants seemed to be satiated approximately at the same time, irrespective of the fasting period, suggesting that the intake rate of feeding ants is similar in all the experimental conditions. this result seems to be partially in contrast with findings of previous studies, such as in camponotus mus, where the intake rate of the sugary food was found to be dependent on both the concentration (paul & roces, 2003) and the level of starvation (falibene et al., 2009). it should be noted that the concentrations of the solutions used in the tests did not significantly affect the viscosity of the liquid and hence a possible difference in the ingestion flow due to anatomic constraints should be excluded (josens et al., 1998). nonetheless, this point would suggest that when the feeding behavior is triggered, foragers eat until their gut is full, with no regulation due to the energetic value of the resource. this point should be surveyed more in-depth in future studies. finally, although carbohydrates are central resources for the ants, since the need for sugary sources may affect their behavior, from the interspecific interaction to the collective decision-making (e.g. grover et al., 2007; arganda et al., 2014; sola & josens, 2016; frizzi et al., 2018; wittman et al., 2018), an interesting improvement of the study would be to repeat the experiment with foods rich in fats or proteins, maybe including some different indicators in the analysis, such as hormone secretion, frequencies of trophallaxis and recruitment behavior. moreover, we selected these experimental solutions on the basis of previous studies, which showed a clear scalar response of workers. however, given that the acceptance was not full, it should be also useful to test more concentrated solutions in future. as a final issue, it must be stressed that t. nigerrimum can build large super colonies (seifert et al., 2017), and it is possible that most if not all the nests employed in the study belonged to the same large colony, which may be subjected to the same local environmental features. hence, the effect of the colony as analytical factor cannot be considered as fully investigated. elucidating this effect would require samples to be taken from distant and clearly independent nests. nonetheless, given the largeness of colonies and the relatively low number of individuals involved in the experiments, we believe that all trials can be considered as independent, and the experiment reliable in general. in conclusion, in this study, we assessed the behavioral responses of workers of t. nigerrimum subject to different levels of starvation toward different sugary solutions. we found a rapid reaction of the most starved ants, although the satiation appeared to occur at the same time, irrespective of the concentration supplied and level of starvation. moreover, it seems that the level of starvation tested was not sufficient to make the foragers entirely cease to evaluate and select the resources. to our knowledge, this is one of the few studies dealing with the effect of the starvation on the feeding behavior of the ants. references abbot, p., abe, j., alcock, j., alizon s., alpedrinha, j.a., andersson, m., et al. 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(2018). carbohydrate supply drives colony size aggression and impacts of an invasive ant. ecosphere, 9: e02403. doi: 10.1002/ecs2.2403 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5147sociobiology 68(1): e-5147 (march, 2021) introduction although life histories arise at the intersection of ecological and evolutionary dynamics (alonzo & kindsvater, 2008), attempts to link life history to habitat and ecological contexts remain diffuse and lack a comprehensive theory after decades since the synthesis of stearns (1997). stearns (1997) abstract stingless bees are abundant and generally numerically dominant in bee communities in the tropics. given that the collective foraging of their colonies is driven by communication systems, the preferential use and sharing of productive and spatio-temporally concentrated floral sources, are probably at the center of the foraging economy of the colonies of these bees. this study analyzed the influence of habitat anthropization on spatial variation in the survival of tropical stingless bee colonies. we investigated how three categories of habitats affect the survival of colonies within communities in a landscape of large fragments of disturbed natural forest and a matrix of rubber agroforestry. random sampling of replicates of each habitat type for two years located 118 nests of 14 species in the two natural forest habitats and 105 nests of six species in the anthropic habitat. monitoring revealed significant differences in colony survival between young and old-growth forests and between old-growth forests and anthropic habitats. the highest annual survival rates, ranging from 87% to 93%, were associated with three medium-large sized bee species that were most abundant and exclusive to the two forest habitats: melipona scutellaris latreille, 1811, scaptotrigona xanthotricha moure, 1950 and scaptotrigona bipunctata (lepeletier, 1836). these three species had similar survival rates in the two forest regeneration stages. another species abundant in the landscape was tetragonisca angustula (latreille, 1811), a small habitat generalist with the lowest survival rate among the three habitats in the anthropic (43%). euclidean distance analysis based on this habitat-generalist placed young forests closest to anthropic habitat and grouped the replicates of old-growth forest together. considering the observed spatial variation, we infer that the atlantic forest favors stingless bees with high colonial longevity. in contrast, habitat-generalists, such as t. angustula, with small body sizes, short colonial longevity and high reproduction rates are favored by deforestation and replacement of forest with anthropogenic habitats. sociobiology an international journal on social insects marilia dantas e silva1, mauro ramalho2, jaqueline figueirêdo rosa3 article history edited by kleber del-claro, ufu, brazil received 07 april 2020 initial acceptance 30 april 2020 final acceptance 04 december 2020 publication date 22 february 2021 keywords resilience, mass-effect, reproduction rate, habitat-generalist, tetragonisca angustula. corresponding author marilia dantas e silva instituto federal de educação, ciência e tecnologia baiano campus governador mangabeira rua waldemar mascarenhas, s/nº portão, cep 44350-000 – governador mangabeira, bahia, brasil. e-mail: marilia.silva@ifbaiano.edu.br concluded that it would be necessary to avoid approaches to life history as a whole (e.g. the r-k continuum) and focus on the impact of habitats on specific attributes, particularly ageand size-specific fecundity and mortality. similarly, the trade-off-centered approach among life history attributes proved unsatisfactory for understanding how the ecological context affects the overall fitness of individuals. in contrast, 1 instituto federal de educação, ciência e tecnologia baiano, campus governador mangabeira. governador mangabeira, bahia, brazil 2 laboratorio de ecologia da polinização-ecopol. instituto de biologia – departamento de botânica. universidade federal da bahia, campus universitário de ondina. salvador, bahia, brazil 3 instituto federal de educação, ciência e tecnologia baiano, campus serrinha. serrinha, bahia, brazil research article bees annual survival rate of tropical stingless bee colonies (meliponini): variation among habitats at the landscape scale in the brazilian atlantic forest md silva, m ramalho, jf rosa – survival rate of tropical stingless bee colonies (meliponini)2 stearns (1997) concluded that an approach based on specific attributes and defined lineages (or organisms of a given basic design) relative to habitats will contribute to expose “sets of patterns that could prove useful in future attempts to relate life history evolution to community ecology” (stearns, 1997). here, we adopt an exploratory approach to the ecological relationship between habitat and longevity for a lineage of tropical bees with perennial social colonies, known as stingless bees (meliponini). we are not aware of any studies on trade-offs between the life history attributes of these bees. slaa (2006) and silva et al. (2014) analyzed, respectively, variation in longevity and reproduction rates between natural forest habitats and anthropogenic habitats. however, this group has broad variability in characteristics related to life history, including interspecific variation in worker body size, colonial population size and reproduction rates (sakagami, 1982; kleinert et al., 2012, silva et al., 2013). assumptions about the role of these characteristics in diet breadth, food sharing, and competitive hierarchy are often implicit in studies of foraging behavior, local coexistence and species abundance in ecological communities (johnson, 1982; johnson et al., 1987; kleinert et al., 2012; biesmeijer & slaa, 2006; silva et al., 2013; lichtenberg et al., 2017). however, we are unaware of any ecological studies on the effects of these characteristics on individual or colonial fitness. stingless bees are abundant and generally numerically dominant in bee communities in the tropics (biesmeijer & slaa 2006; kleinert et al., 2012). in addition, these bee communities may be limited by food resources (e.g. roubik, 1983; biesmeijer & slaa, 2004, 2006; kleinert et al., 2012; silva et al., 2013; silva & ramalho, 2014; lichtenberg et al., 2017). due to their abundance, colonial perenniality and generalized foraging behavior (kleinert et al., 2012; silva et al., 2013), these bees also tend to control the structure of mutualistic interaction networks between plants and pollinators in some tropical communities (woitowiczgruchowski et al., 2020). given that the collective foraging of their colonies is driven by communication systems (nieh et al., 2004), the preferential use and sharing of productive and spatio-temporally concentrated floral sources, such as mass flowers, are probably at the center of the foraging economy of the colonies of these bees (ramalho, 2004; kleinert et al., 2012; woitowicz-gruchowski et al., 2020). this basic ‘colony foraging strategy’ should determine the ability to buffer food supply fluctuations in the restricted home range of a colony, and therefore affect colony longevity (e.g. annual survival rate) in the long run. in contrast, the level and temporal pattern of floral availability in tropical habitats, and the high and continuous demand for food (roubik, 1983; eltz et al., 2002), would be critical to the survival of the perennial and highly populated colonies of stingless bees. in this scenario, worker body size arises as a key phenotypic constraint because it determines flight range (araújo et al., 2004) and colony home range. in other words, viability of colonies of stingless bees (with hundreds to tens of thousands of workers of small body size) would depend fundamentally on the relationship between food demand (e.g. colonial population) and supply of floral resources in the restricted home ranges of colonies. therefore, the compromise between life history and local ecological conditions (stearns, 1997) must be reflected in spatial variation of key attributes of stingless bee colonies, such as annual survival or longevity, according to habitat heterogeneity or among habitats in a landscape. empirical studies that have compared the survival of colonies of stingless bees among habitats under natural conditions are rare. slaa (2006) detected similar survival rates between forest colonies and colonies in nearby cleared areas for most stingless bees in central america, with the exception being generalist species. the premise that the choice of resistant nesting cavities by stingless bee colonies was under selection (such as hollows in live hardwood trees that would be less susceptible to degradation or predator attack) due to potential effects on differential colony survival (roubik, 1989) was recently refuted by measures of non-selectivity of tree holes in a tropical forest of brazil (silva et al., 2014). in general, we assume that measured values of longevity of stingless bee colonies in local communities and their spatial distribution among habitats should simply reflect changing spatial ecological contexts in a landscape. the longevity of a colony is taken as an integrated response to local ecological contexts or habitat categories, which, in the focal landscape here, include natural tropical forests and rubber agroforestry. due to the perennial social lifestyle, and the aforementioned processes of structuring local communities, it is assumed that the expected corresponding variation in colony survival is inherent to “ecological fitting by phenotypically flexible genotypes” (sensu janzen, 1985; agosta & klemens, 2008). this would be a common ecological process to which these bees are subjected in landscapes with rapid changes in natural habitats and the expansion of anthropogenic habitats. thus, in this study, we tested the effects of habitats on colony survival rates in a landscape of native brazilian atlantic forest and deforested areas with anthropogenic habitats. we also explored the argument that the spatial variation in the community of these bees in the landscape are closely related to variation in colonial survival and reproduction rates among species and habitats. material and methods this natural field study was conducted in the northern portion of the central atlantic forest corridor of brazil, in the area of the ‘reserva ecológica michelin’ (rem) (13° 50’s, 39° 15’w). the rem consists of 992 ha of large remnants of legally protected tropical forest surrounded by extensive tropical forest to the north and numerous small fragments of dispersed forest and riparian forests to the south. the landscape sociobiology 68(1): e-5147 (march, 2021) 3 is dominated by extensive rubber agroforestry, with several small villages of rubber tappers. the sampled areas were distributed in a landscape of about 4,000 ha, of which 1,500 ha are covered by forest fragments of different extensions and in various stages of regeneration (including rem), and the rest by anthropogenic habitats, mainly due to rubber agroforestry. the natural forest is dense ombrophilous tropical forest at altitudes ranging from 150 to 330m (flesher, 2006). the regional climate is af (köppen, 1948), with temperatures between 18ºc and 30ºc, and high relative humidity (between 80% and 85%) and rainfall (about 2000 mm annually) (cei/ conder, 1993). sampling of nests in disturbed natural forests and anthropogenic habitats in the rem landscape disturbed natural forest was sampled within the largest forest fragments of rem. for comparative analyses, and according to regeneration stage, the forest plots were grouped into two categories: old-growth or advanced regeneration stage as; and young-growth or early regeneration stage es. the definition and field identification of forest regeneration stages in rem were based on historical data, plant physiognomy, stratification and the presence of key tree species (flesher, 2006). the two selected forest regeneration stages reflect different periods of forest restoration (secondary succession) after deforestation or selective logging. for example, in the young growth stage, fast-growing heliophitic trees are common and trees with diameters below 30 cm are very abundant. in the old growth stage, trees over 30 cm in diameter have the highest frequency and slow-growing centenary trees of over 1 m in diameter are common. the target anthropogenic habitat was of rubber agroforestry and associated with small villages of rubber tappers. the sampling of bee colonies in the anthropogenic habitat was concentrated on the walls of houses, other human structures and ‘urban trees’ widely dispersed over the total area of 1,500 ha of rubber agroforestry adjacent to the rem. this specific condition will here-on be referred to as ‘anthropic habitat’ (ah), while “rem landscape” will refer to the landscape formed by tropical natural forest and rubber agroforestry. bee colonies were sampled in 64 25x25-m randomlydistributed forest plots (32 plots per forest category) in three forest replicates (three large fragments, each > 300 ha) for a total of 32 ha sampled in the forest (16 ha / category of forest). few nests located along access trails near the forest plots were included in the analyses. replicates in the anthropic habitat were four villages of rubber tappers that were widely dispersed over an area of 1,000 ha of agroforestry. all sites with potential nesting cavities (walls, posts, sidewalks, scattered ‘urban’ trees, etc.) were surveyed, for a total sampled area of 29.9 ha. visual nest searches were performed between 07:00 and 15:00 h for five days per month in each habitat type (two forest categories and anthropic habitat), between july 2007 and january 2010. searches in the forest were more intensive in large trees (circumference > 60 cm), where most nests of species of meliponini are expected to be found (eltz et al., 2002; batista et al., 2003; silva et al., 2013). the nests found in the three habitats (young and old growth forests and anthropic habitat) were marked and georeferenced for monitoring. all found nests were monitored monthly for two years to check if the colonies were still alive. there was wide application of insecticides in the anthropic habitat in the second year of monitoring, and so survival rates there were calculated for only one year. recorded stingless bee species were grouped according to body length into the following arbitrary size categories: small (≤ 6mm), medium (> 6 ≤ 10mm) and large (> 10 mm). based on data from silva et al. (2013), bee species with more than 0.2 nests/ha in at least one habitat category were considered abundant. between five and 10 individuals per nest were collected to confirm species identification. specimens were separated by morphospecies at the pollination ecology laboratory of ibufba and identified by favizia freitas de oliveira from ibio-ufba. replicates of the collected material were deposited in the entomological collection of the museum of natural history of the federal university of bahia (mzufba). annual colony survival rate to estimate colony survival rate, all nests were monitored monthly for two years between july 2008 and january 2011. the presence or absence of a given species in the same nest each month was used as a measure of colonial survival. we were unable to monitor exceptional recolonizations of a nest by the same species following by colony mortality. annual survival rate is used as a synonym for colony longevity. data analysis multivariate analysis of variance (manova) was used to compare annual survival rates of colonies among the three habitat types (as, es and ah). annual survival rates were measured in each replicate of the three habitat categories: habitat type and species were the independent variables survival rates were considered as dependent variables and were based on measures of presence-absence of colonies at each nest site. each bee nest was taken as a sample unit and data on presence or absence of colonies after one or two years were used to construct the analysis matrix. the dependency between dependent variables is a premise of manova, which therefore covered the time dependence related to the fact that the nests are the same from year 1 to 2. tukey’s hsd test was used for multiple comparisons. nests of eight species with two or more occurrences were included in these analyses: lestrimellita sp., melipona scutellaris latreille, 1811, nannotrigona sp., paratrigona md silva, m ramalho, jf rosa – survival rate of tropical stingless bee colonies (meliponini)4 subnuda moure, 1947, plebeia sp., scaptotrigona bipunctata lepeletier, 1836, scaptotrigona xanthotricha moure, 1950 and tetragonisca angustula latreille, 1811. an exploratory analysis on the effect of bee size on colony survival was also conducted using manova, with bee sizes as independent variables and survival after one and two years as dependent variables. it was used three size categories based on bee body length: small (≤ 6 mm), medium (>6mm ≤ 10mm) and large (>10mm). this is a preliminary analysis, as there is an imbalance in the data in relation to the number of colonies/ body size category. the analysis of variance and tukey’s test were performed with ibm spss statistics software, version 25. the significance level adopted was 0.05. results and discussion annual colony survival rates were estimated based on monitoring 118 nests of 14 species in the two forest habitats (young and old-growth forest) and 105 nests of six species in the adjacent anthropic habitat in the landscape (table 1). all species with more than 0.2 nest/ha were considered abundant and with sufficient data for specific analyses. four species were in this category in the forest, and contributed 3.06 nest/ ha to the total of 3.68 nest/ha; only one species, t. angustula, was abundant in the anthropic habitat, which stood out with 2.77 nests/ha out of the total of 3.5 nests/ha. the highest survival rates were frequently associated with old-growth forest and the lowest rates with the anthropic habitat (table 1). the greatest longevities were associated with three medium-large-sized species that are abundant and exclusive to natural forest in the landscape – s. xanthotricha, m. scutellaris and s. bipunctata – which had survival rates (weighted averages) of 87.2%, 89.4% and 93.1%, respectively. lower rates were associated with a subset of non-abundant species (<0.2 nests/ha), which were more frequent in, or unique to, the anthropic habitat (e.g., lestrimelitta sp., nannotrigona sp., plebeia droryana (friese, 1900) and trigona hyalinata lepeletier, 1836). t.angustula was abundant in all three habitats with high variation in survival rate among them. during four years of natural nest monitoring, eltz et al. (2002) observed high annual survival (85.5% – 85% or = annual mortality rates of 14.5% – 15.0%) of stingless bee colonies in dipterocarp forests with different levels of disturbance (different intensities of selective logging), in northern borneo. in central america, slaa (2006) observed similar high survival rates (89% – 93%) in deforested and forested areas for colonies of the most abundant species, except for t. angustula. thus, the data presented here for the rem landscape also support slaa’s generalization that high annual survival rates (between 80% and 90%) are common for stingless bee colonies. there was no difference for the three most abundant bee species of the tropical forest of the rem landscape (s. xanthotricha, m. scutellaris and s. bipunctata), in colony survival rates between the two stages of forest regeneration during the two years of monitoring (table 1). these survival rates project low colony turnover with periods of over 10 years to completely replace colonies of the populations in these two forest habitats. this projection is very conservative considering estimates of about 20 years for total colony replacement (slaa, 2006) for some stingless bee populations residing in dry tropical forest of costa rica. the three most abundant species mentioned above also maintain similar abundances and very low reproductive rates in early and advanced stages of forest regeneration in the rem landscape (silva et al., 2013; 2014). high abundance and low turnover of colonies (high annual survival) suggest that the populations of this bee group are resistant to the long process of forest regeneration (e.g., from 20 years for young forest to more than a hundred years for old-growth forest with centenary trees). this subset of abundant stingless bees is likely playing a prominent role in the resilience of the rainforest bee community as a whole, especially when the forest disturbance was caused by selective logging. this can be explained, first, by these abundant stingless bees playing a central role in structuring mutualistic networks in the rainforest region (woitowicz-grushowski et al., 2020) and, second, they have low selectivity for tree cavities in these natural forests (silva & ramalho, 2014), and thus would not be affected by the slow replacement of tree species throughout the forest succession process. on the other hand, there is some evidence that colony survival or longevity depends on habitat type or ecological context, as well as the stingless bee species (table 1). the flexibility of this attribute and its involvement in ecological fitting (sensu janzen, 1985; agosta & klemens, 2008) is particularly apparent in t.angustula, with low to moderate survival rates in the two forest habitats (between 58% in es and 72% in ea) and very low survival in the anthropic habitat (43%) in the rem landscape. slaa (2006) also observed a lower survival rate (40%) for this species in dry forest compared to adjacent deforested areas in the landscape. therefore, the generalization that stingless bees invest more in colony survival (slaa, 2006) needs to be considered with reservations, since it is valid mainly for species that are restricted to the natural habitats of tropical forest. there were differences in the annual survival rate of colonies among species and between habitat types during the two years at rem (tables 2), but there was no interaction between habitat type and species (table 2). in any case, the spatial variation in longevity already described for t. angustula and the resulting similarities between habitats (fig 1, see below) indicate that this generalist is probably fitting colonial fitness to the ecological context, that is, to the heterogeneity of habitat types in the same landscape. in a comparative analysis of forested and deforested areas in costa rica, slaa (2006) also reported that the life history of stingless bees was affected by species or location. sociobiology 68(1): e-5147 (march, 2021) 5 bs as es ah tax. sobr. (%) year 1 tax. sobr. (%) year 2 tax. sobr. (%) year 1 tax. sobr. (%) year 2 tx. sobr. (%) year 1 annual survival variation **11±3,16 ***9,75±3,03 8,25±1,29 7,5±1,65 13±6 10±4,30 6±2,91 5,25±2,68 19,75±7,18 17,5±6,34 species lestrimelitta sp s * * * * 50% (n=4) melipona scutellaris latreille, 1811 l 83% (n=6) 100% (n=5) 100% (n=4) 75% (n=4) * nannotrigona sp s * * * * 66,6% (n=7) partamona sp1 m 66,6% (n=3) 100% (n=2) * * * partamona sp2 m 100% (n=1) 0% (n=1) * * * paratrigona subnuda moure, 1947 s 50% (n=2) 0% (n=2) 0% (n=4) * * plebeia droryana (friese, 1900) s * * 25% (n=4) 100% (n=1) 14,2% (n=6) scaptotrigona xanthotricha moure, 1950 m 92,8% (n=14) 100% (n=13) 81,8% (n=11) 66,6% (n=9) * scaptotrigona bipunctata lepeletier, 1836 m 100% (n=9) 88,8% (n=9) 83,3% (n=6) 100% (n=5) * scaura atlantica melo, 2004 s 0% (n=1) * * * * schwarziana quadripunctata lepeletier, 1836 m * * 0% (n=1) * * tetragonisca angustula latreille, 1811 s 85,7% (n=14) 58,3% (n=12) 59,3% (n=32) 57,8% (n=19) 42,6% (n=83) trigona braueri friese, 1900 m 100% (n=1) 100% (n=1) 100% (n=1) 100% (n=1) * trigona fuscipennis friese, 1900 m * * 100% (n=1) 100% (n=1) * trigona hyalinata lepeletier, 1836 m * * * * 75% (n=4) trigona spinipes fabricius, 1793 m * * 0% (n=1) * 100% (n=1) *species nest is absent. variation of annual survival rates among replicates of each habitat category: mean and standard deviation of total nests (**) and most abundant species (***). table 1. variation of the annual survival rate of stingless bee colonies in two forest categories and in the anthropic habitat, in the landscape of the ‘reserva ecologica michelin’ (rem) of the brazilian atlantic forest. asold-growth forest or advanced stage forest regeneration; es young forest or early stage forest regeneration; ah anthropic habitats. body size (bs): large (l), medium (m) and small (s). t. angustula was the only abundant species (> 0.2 nests / ha) in all the three habitat types in the rem landscape. the distance (similarity) analysis based on the survival of this habitat-generalist species places the young forest closer to the anthropic habitat than to the old forest (fig.1) and groups the old-growth forest replicates together. at the same time, the colonies of this species reproduce at higher rates in the anthropic habitat than in the adjacent old growth forest (silva et al., 2014; p.c.l. gouvêa personal inf. gouvêa & ramalho, n.p.). these patterns of survival and reproduction produce a spatially structured population, which is finding better conditions for growth in the anthropic habitat. therefore, t. angustula also has a mass effect on the bee community in the remnants of old-growth forest, which tends to increase with progressive loss of natural forest cover (personal information from pclgouvêa, gouvêa & ramalho, np). the expected trade-off between survival (longevity) and reproduction (stearns 1997) is apparent in all abundant stingless bee species residing in the atlantic forest: high survival rates (table 1) are related to low reproduction rates and, more specifically, to very low rates of swarming in trap nests (silva et al., 2014). again, the above detailed information about t. angustula supports phenotypic plasticity and opposing trends in spatial variation in survival and reproduction rates between anthropic habitat and the old growth forest. worker bee size had an apparent effect on colony survival. the multiple comparison tests revealed that small size differed from large (p < 0.0001) and medium (p < 0.0001) size but no difference between medium and large size (p = 0.9). these results, however, are preliminary and should be interpreted with some reserve as the number of colonies for the different sized classes differed, such as a much greater table 2. effect of habitat type and species on annual survival rate of stingless bee colonies. p = probability significance level. independent variables dependent variable (survival) after 1 year (p) after 2 year (p) species 0,084 0,001 habitat 0,000 0,002 interaction (species x habitat) 0,158 0,150 md silva, m ramalho, jf rosa – survival rate of tropical stingless bee colonies (meliponini)6 number of colonies of small species than of large species. the greatest longevities were associated with the three mediumlarge-sized species exclusive to natural forest. often, lowest rates were associated with the small-sized species more frequent in the anthropic habitat (see table 1) and the habitat generalist t. angustula. on the one hand, the relationship between worker size and colony survival was not expected, since perennial colonies of social stingless bees were presumed to persist as long as they were able to replace their long-standing laying queens (engels & imperatriz-fonseca, 1990). on the other hand, given a general relationship between body size and foraging area (e.g., araújo et al., 2004; kleinert et al., 2009), smaller bees are likely to be more exposed to seasonal or unpredictable variation in flower supply within the small home range of their colony. the implications of the central-place foraging and small colonial home range for the survival of stingless bee colonies become apparent considering that, first, these perennial colonies have very high rates of pollen consumption to maintain very high biomass replacement rates throughout the year (roubik, 1993), and, secondly, these bee communities are structured through the sharing of floral resources (kleinert et al. 2012). indeed, variation among colonies in stored food can lead to marked differences in the amount of sexuals produced by different colonies (moo-valle et al., 2001), which in turn causes variation in fitness. annual survival or longevity probably also depend on the specific resilience of colonies after seasonal population collapses, such as when they are exposed to critical unfavorable foraging conditions, as observed for species of melipona in brazilian dry forest (hrncir et al., 2019). in summary, it is likely that perennial colonies of stingless bees with smaller workers are more vulnerable to fluctuations in food supply that affect colony survival, due to implications of central-place foraging in very restricted colonial home range. an expected trade-off in such cases of unavoidable reduction in colony longevity would be an increase in reproduction rate, as the spatial pattern of t. angustula seems to confirm. based on independent empirical data on stingless bee species in various tropical vegetation types (brown & albrecht, 2001; ricketts, 2004; brosi et al., 2007; williams et al., 2010; silva et al., 2013; lichtenberg et al., 2017), we infer that deforestation and habitat anthropization contribute to increased species turnover among habitats, but reduce the number of stingless bee species at the landscape scale. the loss of species due to anthropic disturbances can not be compensated by the expansion of generalist species, fig 1. dissimilarities among the three habitat types (old-growth forest or advanced stage regeneration as; young forest or early stage regeneration es, and anthropic habitat – ah), as a function of variation in t. angustula annual survival rate. euclidian distances. sociobiology 68(1): e-5147 (march, 2021) 7 such as t. angustula, for example foraging traits and diet breadth are probably associated with interspecific variation in susceptibility to reduced forest cover due to anthropization (lichtenberg et al., 2017). the flexibility (phenotipic plasticity or reaction norm) of critical life history attributes (longevity and reproduction) can also contribute to the ‘ecological fitting’ (agosta & klemens, 2008) of habitat generalists to fast anthropogenic changes in the landscape. in this scenario, it is also expected that stingless bees, with high colonial longevity living in the forest, will be exposed to increased dispersal pressure from abundant generalists, and their mass effects, from adjacent anthropogenic habitats. aknowlegments we thank: michelin for logistical support; capes for granting a doctoral scholarship to the first author; and cnpq (processes no. 481113/2004-5 and 478271/2008) and fapesb (apr0114/2006, app0061/2016) for supporting the research. we also thank the pollination ecology laboratory team ecopol/ib-ufba for their help with the fieldwork. authors' contribution mds: conceptualization, methodology, investigation, data curation, project administration, visualization, writing. mr: supervision, conceptualization, methodology, project administration, resources, funding acquisition, writing jfr: methodology, formal, analisys, writing references agosta, s.j. & klemens, j.a. 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(2020). experimental field test of the influence of generalist stingless bees (meliponini) on the topology of a bee-flower mutualistic network in the tropics. ecological entomology, 45: 854-866. doi: 10.1111/een.12862. 755 community structure of social wasps (hymenoptera: vespidae) in riparian forest in batayporã, mato grosso do sul, brazil by maria da graça cardoso pereira bomfim1 & william fernando antonialli junior1,2 abstract reduction and destruction of riparian forests are harmful to the biota, especially the social wasps. this study analyzed the species constancy and the structure of the polistine wasp community associated with fragments of riparian forest in the municipality of batayporã, state of mato grosso do sul. eighteen species of social wasps were collected, by the methods of active searching and traps baited with honey and sardine. eight species were classified as infrequent, five as very frequent, and five as of intermediate frequency. the community structure, as represented by species richness, showed a significant negative correlation with the berger-parker dominance index, and no significant correlation with the width of the fragments and with the structural complexity of the vegetation, suggesting that the community, in this case, must be structured by the tolerance of the species and not by the vegetation characteristics, which did not limit the dispersal of the social wasps. keywords: polistinae, species richness, dominance index. introduction the term riparian forest (gallery forest; in portuguese “mata ciliar”) is synonymous with the official nomenclature (ibge 1992) of “seasonal semideciduous alluvial forest”. in spite of their importance and although they are legally preserved in brazil, as areas of permanent preservation, these forests are under anthropogenic pressures, with conflicting interests for use and occupation of the land, which have led to their destruction along entire watercourses for 1programa de pós-graduação em entomologia e conservação da biodiversidade, universidade federal da grande dourados. rodovia dourados/itahum, km 12, caixa postal 241, 79.804-970, douradosms, brazil airambio@yahoo.com.br; williamantonialli@yahoo.com.br. 2laboratório de ecologia, centro integrado de análise e monitoramento ambiental, universidade estadual de mato grosso do sul. rodovia dourados/itahum, km 12, caixa postal 351, 79.804-970, dourados-ms, brazil. 756 sociobiolog y vol. 59, no. 3, 2012 farming or logging (vestena & thomaz 2006). reduction and destruction of riparian forests are harmful to the biota, because these systems form ecological corridors, act to maintain the microclimate and biodiversity, and provide habitat, shelter, food and water to the fauna (kageyama et al. 2001). the family vespidae contains approximately 4,600 species, with one extinct subfamily and six current monophyletic subfamilies (carpenter & rasnitsyn 1990): euparagiinae, masarinae, eumeninae, stenogastrinae, vespinae and polistinae. social species occur in the last three subfamilies. these species share a series of characteristics, including cooperation in the care of the offspring until the emergence of the adult, gradual provision, reuse of the reproductive cells, nest sharing between different generations, trophallaxis between adults, and division of labor (carpenter 1991). the only eusocial subfamily that occurs in brazil is the polistinae, represented by the tribes polistini, mischocyttarini and epiponini (carpenter & marques 2001). the polistinae is a diverse group and it taxonomy is relatively well known, so that many species can be identified with precision through published keys (carpenter & marques 2001; garcete-barrett 1999; richards 1978). some species of wasps possess wide ecological valence; that is, they vary their habits of nest building as a function of the substrate and the environmental conditions (marques et al. 1993; santos & gobbi 1998). other species have relatively narrow ecological valence and only nest in sites with specific conditions (santos et al. 2007; santos et al. 2009; silva-pereira & santos 2006), selected for the type and density of the vegetation, the arrangement and shapes of leaves, and other plant structures (dejean et al. 1998; diniz & kitayama 1994; santos & gobbi 1998). a few recent studies have examined the community structure or the constancy of species of social wasps, in particular the work of santos et al. (2007) in atlantic forest, restinga, and mangrove ecosystems, and of silva-pereira & santos (2006) in montane savannah (portuguese “campo rupestre”); however, published studies on this subject are still few. the present study evaluated the constancy of the species and the relationship of the structure of the community of polistine wasps to the size and structural complexity of the vegetation, and to the dominance index associated with different fragments of riparian forest. 757 bomfim, m.g.c.p. & antonialli, w.f.j. — community structure of social wasps material and methods social wasps were collected in ten fragments of riparian forest near the municipality of batayporã, mato grosso do sul (22º18’00”s, 53º15’97”w). point 1: (22°15’80”s, 53°11’21”w); point 2: (22º14’82”s, 53º12’73”w); point 3: (22°20’98”s, 53°20’53”w); point 4: (22º13’86”s, 53º03’86”w); point 5: (22º14’58”s, 53º09’98”w); point 6: (22º18’78”s, 53º14’11”w); point 7: (22º18’59”s, 53º17’61”w); point 8: (22º11’93”s, 53º10’61”w); point 9: (22º20’89”s, 53º15’38”w); and point 10: (22º19’47”s, 53º19’22”w). in each fragment, 2,000 m² of riparian forest was selected, for a total study area of 20,000 m². the climate of mato grosso do sul is humid subtropical, with a warm rainy period from november through april, and a cooler dry period from may through october (zavatini 1992). we carried out ten collections in 30 days of fieldwork, in the period from october 2008 through march 2009. the collections were made by the methods of active searching and baited traps. active searching was carried out during the time of day when the wasps are most active, from 09:00 to 15:00 h (andrade & prezoto 2001; elisei et al. 2005; lima & prezoto 2003). we used a liquid bait, composed of a solution of sucrose (1:5, commercial sugar: water) and 2 cm3 of salt for each liter of solution, sprayed on the vegetation; and captured the wasps with an entomological net (noll & gomes 2009). in each forest fragment, we walked parallel to the watercourse at different distances from it, for six hours, totaling 60 hours of fieldwork for this method. in each fragment, we installed 40 baited traps, 20 containing a mixture of honey and water, and 20 containing a mixture of sardines and water, in 2 liter plastic bottles with side openings, as described by souza & prezoto (2006). in each forest fragment, the traps were attached 1.50 m above the ground, every 10 m along a 200 m transect, parallel to and about 5 m distant from the stream, and were removed after one week (elpino-campos et al. 2007; souza & prezoto 2006). this constituted a sampling effort of 60 baited traps. the specimens collected were identified by dichotomous keys proposed by carpenter & marques (2001), garcete-barrett (1999) and richards (1978) and by comparison with specimens of social wasps deposited in the comparative biolog y laboratory of paraná federal university. vouchers were deposited in the museu de entomologia of the universidade federal da grande dourados, ufgd. 758 sociobiolog y vol. 59, no. 3, 2012 constancy was calculated by the frequency of occurrence of each species. very frequent species were considered to be those that occurred in more than 50% of the fragments; of intermediate frequency, species that occurred in 25 to 50%; and as infrequent, species that were present in less than 25% of the fragments. to evaluate the existence of a correlation between dominance and the community structure, represented by the species richness, the berger-parker dominance index was calculated for each collection point. this index considers the highest ratio of the species with the largest number of individuals (rodrigues 2007), expressing the dominance of one or more species in a community, based on the importance value of each species. after the dominance index was calculated, an analysis of linear correlation was carried out, with a level of significance (α) of 0.05 between these parameters. the widths of the fragments of riparian forest, including both banks of the watercourse, were measured with the aid of a measuring tape. the structural complexity of the vegetation was evaluated by counting trees, saplings, shrubs, lianas, seedlings, and the thickness of the leaf litter. the counts were made in a sample plot of 4 m², at each collection point. to obtain the vectors associated with these variables, a principal components analysis was used (pca). to give the same importance to these variables, the value of each was divided by the square root of the total sum of squares (vieira et al. 2008). to evaluate if there was a significant correlation between the structure of the community and the width of the fragments, and between the structure of the community and the structural complexity of the vegetation, the linear correlation analysis was used, with a level of significance (α) of 0.05. results and discussion we collected 18 species of social wasps (table 1) belonging to six genera, with representatives of the tribes polistini, mischocyttarini and epiponini. tribe epiponini was most important, with 10 species (56%), followed by polistini with 7 (39%), and mischocyttarini with only 1 (5%). of the total of 529 individual wasps caught, 456 (86%) were members of the tribe epiponini, 46 (9%) polistini, and only 27 (5%) mischocyttarini. the predominance of epiponine wasps can be explained by the large numbers of individuals found in their colonies, in particular, the genus agelaia, which in 759 bomfim, m.g.c.p. & antonialli, w.f.j. — community structure of social wasps some species such as agelaia vicina (saussure 1854) may have up to a million adults in a single colony (zucchi et al. 1995). eight species (44%) were classified as infrequent, five (28%) as intermediate, and five (28%) as very frequent (table 1). the infrequent species were captured in less than 25% of the fragments. this may be related to the wide radius of action of the wasps during foraging. for example, polistes versicolor (olivier 1791) shows a 200 m effective radius of action (gobbi 1978), and polistes simillimus (zikán 1951) a 150 m radius of action (prezoto & gobbi 2005). this capacity of movement results in many captures of infrequent species in locations other than their permanent habitat. diniz & kitayama (1998) and silva-pereira & santos (2006) observed that some wasps can construct their nests in one environment and forage in locations distant from that environment. considering that the environment table 1. constancy of the species of social wasps, as a function of the relative frequency of occurrence in the fragments of riparian forest in batayporã, mato grosso do sul state, brazil. species frequency % constancy (c) 1 apoica pallens (fabricius) 30% intermediate frequency 2 agelaia pallipes (olivier) 70% very frequent 3 brachygastra augusti de saussure 30% intermediate frequency 4 polybia jurinei de saussure 10% infrequent 5 polybia ignobilis (haliday) 50% intermediate frequency 6 polybia sericea (olivier) 30% intermediate frequency 7 polybia chrysothorax (lichtenstein) 20% infrequent 8 polybia paulista (h. von ihering ) 70% very frequent 9 polybia ruficeps schrottky 20% infrequent 10 polybia occidentalis (olivier) 70% very frequent 11 polistes subsericeus de saussure 60% very frequent 12 polistes versicolor (olivier) 40% intermediate frequency 13 polistes simillimus zikán 10% infrequent 14 polistes brevifissus richards 20% infrequent 15 polistes billardieri (fabricius) 20% infrequent 16 polistes cinerascens de saussure 10% infrequent 17 polistes geminatus (fox) 10% infrequent 18 mischocyttarus drewseni de saussure 60% very frequent 760 sociobiolog y vol. 59, no. 3, 2012 that surrounds the fragments studied here consists of cerrado, the riparian forest may represent only part of the foraging area for those species that use the cerrado as a permanent habitat. supporting this hypothesis, polistes simillimus, for example, prefers to nest in dry habitats, and polistes geminatus (fox 1898), polistes billardieri (fabricius 1804) and polybia chrysothorax (lichtenstein 1796) prefer the cerrado (garcete-barrett 1999). therefore, riparian forest would be a transitory environment for many of these species. santos et al. (2007) also found the species polistes carnifex (fabricius 1775) and synoeca cyanea (fabricius 1775) foraging in mangrove areas, apparently a transitory environment for them, but their nests are restricted to the restinga and atlantic forest. santos et al. (2009) also observed the occurrence of protonectarrina sylveirae (de saussure 1854), parachartergus pseudoapicalis (willink 1859) and synoeca cyanea in agricultural systems other than their nest sites. the species with intermediate frequency can be considered as tolerant, changing their nest building habitats according to ambient conditions and the available substrata. apoica pallens (fabricius 1804), polybia ignobilis (haliday 1836) and polybia sericea (olivier 1791) are known to be tolerant (santos 2000) and both brachygastra augusti (de saussure 1834) and polistes versicolor (olivier 1791) can also be found in forests and the cerrado (garcete-barrett 1999). in contrast, agelaia pallipes (olivier 1791), polybia paulista (von ihering 1896), polybia occidentalis (olivier 1791), polistes subsericeus (saussure 1854) and mischocyttarus drewseni were very frequent in this study, probably because of their numerous colonies (zucchi et al. 1995), or because they found in the riparian forest fragments a more favorable environment than the cerrado. in particular, m. drewseni shows a clear preference for more humid environments (garcete-barrett 1999). the width of the fragments of riparian forest varied at the different collection points, from 26 to 650 m (table 2). no significant correlation between the width and the community structure in the fragments, represented by species richness, was found (r = 0.0476; t = 0.1347; p= 0.8962 and gl= 8). it can be suggested that the fragments of riparian forest do not represent islands for the polistines, and that the surrounding areas of cerrado do not represent a barrier to the dispersal of these species. 761 bomfim, m.g.c.p. & antonialli, w.f.j. — community structure of social wasps the variation in the structural complexity of the vegetation among the collection points can be represented by the first two axes of a principal components analysis (table 2). these axes accounted for 70.57% of the variance (eigenvalues 2.687 for axis 1 and 1.548 for axis 2) of the original data for the numbers of trees, shrubs, lianas, seedlings, and thickness of the leaf litter; the first axis (pca 1) explained 44.77% of this variance. for correlation analysis between the variable structural complexity of the vegetation (represented by pca axes 1 and 2) and the community structure, represented by the species richness, significant correlations between pca axes 1 (r = 0.1114; t = 0.3171; p = 0.7593 and gl = 8) and 2 (r = -0.0601; t = -0.1703; p = 0.8690 and gl = 8) with the community structure were also not found. therefore, the results demonstrate that the occurrence of species of polistine wasps in these fragments is not correlated with the vegetation complexity. this result differs from the data of santos et al. (2007), who found a direct correlation between the structural complexity of vegetation and the diversity of wasp species. table 2. dominance index, species richness, width of the fragments of riparian forest, and values of axes 1 and 2 of the principal components analysis (pca) representing the vegetation complexity of the sampling points. collection points berger-parker dominance dominant species species richness width (m) vegetation complexity pca 1 pca 2 1 0.63 polistes versicolor 6 300 -16.290 21.122 2 0.86 polybia paulista and polybia occidentalis 3 26 -0.7003 -17.453 3 0.57 apoica pallipes 8 650 42.221 0.4993 4 0.73 polybia occidentalis and agelaia pallipes 6 520 0.0288 0.4091 5 0.52 agelaia pallipes 8 30 0.1297 -0.7871 6 0.53 polybia paulista and polybia sericea 8 55 -0.6588 -18.211 7 0.98 agelaia pallipes 3 180 -0.2916 13.790 8 0.49 polybia paulista 11 50 -12.265 0.2321 9 0.75 mischocyttarus drewseni 5 100 -0.6985 0.0544 10 0.54 agelaia pallipes 5 150 0.8240 -0.3326 762 sociobiolog y vol. 59, no. 3, 2012 however, although the richness in a community can be influenced by the number of niches, which reflects the structural heterogeneity of the environment, it also can be partly determined by the tolerance of the species to the physical conditions (basic niche) and by interactions with other species (actual niche) (giller 1984; santos et al. 2007). tolerant species also possess wide ecological valence (marques & carvalho 1993; santos & gobbi 1998). the correlation analysis between the community structure, represented by species richness, and the berger-parker dominance index indicated a significant negative correlation (r = -0.8375; t = -4.3358; p = 0.0025 and gl = 8, fig. 1); the richness decreased as the dominance increased. in particular, the analysis indicated the dominance of polybia paulista (d= 0.49) at point 8, the co-dominance of polybia paulista (d= 0.57) and polybia occidentalis (d= figure 1. correlation between species richness and the berger-parker dominance index (α = 0.05; gl = 8). the numbers beside the points indicate the collection localities. 763 bomfim, m.g.c.p. & antonialli, w.f.j. — community structure of social wasps 0.29) at point 2, the co-dominance of polybia paulista (d= 0.29) and polybia sericea (d= 0.24) at point 6, the co-dominance of polybia occidentalis (d= 0.32) and agelaia pallipes (d= 0.41) at point 4, the dominance of agelaia pallipes at points 3, 5, 7 and 10, of polistes versicolor at point 1, and of mischocyttarus drewseni at point 9. acknowledgments we thank cnpq (133014/2009-6) for the master’s degree fellowship granted to the first author, and wfaj also acknowledges research support from this agency. we are grateful to bolivar r. garcete-barrett of the universidade federal do paraná, curitiba, brazil, for confirming species identifications. references andrade, f.r. & f. prezoto. 2001. horários de atividade forrageadora e material coletado por polistes ferreri saussure, 1853 (hymenoptera, vespidae), nas diferentes fases de seu ciclo biológico. revista brasileira de zoociências 3: 117-128. carpenter, j.m. 1991. phylogenetic relationships and the origin of social behavior in the vespidae. p. 7-32. in: k.g. ross & r.w. matthews. the social biolog y of wasps. ithaca, cornell university press. 678p. carpenter, j.m. & a.p. rasnitsyn. 1990. mesozoic vespidae. psyche 97: 1-20. carpenter, j.m. & o.m. marques. 2001. contribuição ao estudo dos vespídeos do brasil (insecta, hymenoptera, vespoidea, vespidae) [cd-rom]. cruz das almas – ba, brasil. universidade federal da bahia, escola de agronomia, departamento de fitotecnia / mestrado em ciências agrárias. série publicações digitais, 2. dejean, a.; b. corbara & j.m. carpenter. 1998. nesting site selection by wasps in the guaianese rain forest. insects sociaux 45: 33-41. diniz, i.r. & k. kitayama. 1994. colony densities and preferences for nest habitats of some wasps in mato grosso state, brazil (hymenoptera: vespidae). journal of hymenoptera research 3: 133-143. diniz, i.r. & k. kitayama. 1998. seasonality of vespid species (hymenoptera: vespidae) in a central brazilian cerrado. revista de biologia tropical. 46: 109-114. elisei, t.; c. ribeiro-júnior; d.l. guimarães & f. prezoto. 2005. foraging activity and nesting of swarm-founding wasp synoeca cyanea (fabricius, 1775) (hymenoptera, vespidae, epiponini). sociobiolog y 46: 317-327. elpino-campos, a.; k. del-claro & f. prezoto. 2007. diversity of social wasps (hymenoptera: vespidae) in cerrado fragments of uberlândia, minas gerais state, brazil. neotropical entomolog y 36: 685-692. ibge – fundação instituto brasileiro de geografia e estatística. 1992. manual técnico da vegetação brasileira. rio de janeiro, ibge, 92p. 764 sociobiolog y vol. 59, no. 3, 2012 garcete-barrett, b.r. 1999. guía ilustrada de las avispas sociales del paraguay (hymenoptera: vespidae: polistinae). london, the natural history museum, 56p. gobbi, n. 1978. determinação do raio de vôo de operárias de p. versicolor (hymenoptera, vespidae). ciência e cultura 30: 364-365. guiller, p.s. 1984. community structure and the niche. london, chapman and hall, 176p. lima, m.a.p. & f. prezoto. 2003. foraging activity rhythm in the neotropical swarm-founding wasp polybia platycephala sylvestris (hymenoptera: vespidae) in different seasons of the year. sociobiolog y 42: 745-752. kageyama, p.y.; 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(hymenoptera – vespidae) in a caatinga area, bahia state – brazil. journal of advanced zoolog y 19: 63-69. santos, g.m.m.; c.c.b. filho; j.j. resende; j.d. cruz & o.m. marques. 2007. diversity and community structures of social wasps (hymenoptera: vespidae) in three ecosystems in itaparica island, bahia state, brazil. neotropical entomolog y 36: 180-185. santos, g.m.m.; j.d. cruz; o.m. marques & n. gobbi. 2009. diversidade de vespas sociais (hymenoptera: vespidae) em áreas de cerrado na bahia. neotropical entomolog y 38: 317-320. silva-pereira, v. & g.m.m. santos. 2006. diversity in bee (hymenoptera: apoidea) and social wasp (hymenoptera: vespidae, polistinae) community in “campos rupestres”, bahia, brazil. neotropical entomolog y 35:163-174. souza, m.m & f. prezoto. 2006. diversity of social wasps (hymenoptera, vepidae) in semideciduous forest and cerrado (savanna) regions in brazil. sociobiolog y 47: 135 – 147. 765 bomfim, m.g.c.p. & antonialli, w.f.j. — community structure of social wasps vestena, l.r. & e.l. thomaz. 2006. avaliação de conflitos entre áreas de preservação permanente, associadas aos cursos fluviais e uso da terra na bacia do rio das pedras, guarapuava – pr. ambiência 2: 73-85. vieira, l.; f.s. lopes; w.d. fernandes & j. raizer. 2008. comunidade de carabidae (coleoptera) em manchas florestais no pantanal, mato grosso do sul, brasil. iheringia 98: 317-324. zavatini, j.a. 1992. dinâmica climática no mato grosso do sul. geografia 17: 65-91. zucchi, r.; s.f. sakagami; f.b. noll; m.r. mechi; s. mateus; m.v. baio & s.n. shima. 1995. agelaia vicina, a swarm-founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society 103: 129-1. doi: 10.13102/sociobiology.v61i4.378-385sociobiology 61(4): 378-385 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 environmental windows for foraging activity in stingless bees, melipona subnitida ducke and melipona quadrifasciata lepeletier (hymenoptera: apidae: meliponini) introduction due to their impact on many aspects of colony life, abiotic factors are considered key determinants for the geographical distribution of social bee species (michener, 1974). an environmentally influenced aspect of vital importance for colony functioning is food collection. the success of foragers, which is crucial for the maintenance and survival of the colonies, is affected both directly and indirectly by climatic factors. associated with the morphological and interrelated physiological peculiarities of a bee species – such as body size and colouration – abiotic factors determine the timing of food collection (daily onset and end) and the food patch choice (sunny versus shaded patches) (biesmeijer et al., 1999; abstract the foraging success of a bee species is limited to an environmental window, a combination of optimal ambient temperatures and resource availability. mismatches between flowering and optimal foraging temperature may lead to a reduction of a colony’s food intake and, eventually, of brood production. in the present study, we evaluated the pollen foraging activity of two native brazilian meliponine species melipona quadrifasciata lepeletier and melipona subnitida ducke at the campus of the university of são paulo at ribeirão preto (march, 2010 – january, 2011). whereas m. quadrifasciata naturally occurs in the study region (brazilian southeast), m. subnitida is restricted to the brazilian northeast. this difference in geographic distribution and concordant climatic specializations suggest differences concerning the environmental window between the two species. we investigated potential differences between the species concerning the thermal window within which foraging occurs, and associated differences in foraging activity, visited pollen sources, and colony survival. the lower temperature limit for m. subnitida (17 ° c) was 5 °c above the lower temperature limit found in m. quadrifasciata (12 °c). this difference resulted in a considerable time lag concerning the onset of foraging between the bee species (maximum: 120 minutes), mainly during the cold-dry season. due to this delay in foraging, m. subnitida could not benefit from highly profitable pollen sources (massflowering trees) that were in bloom during this time of the year. possibly because of this deficit in pollen intake, three of the six monitored colonies of m. subnitida did not survive the study period. sociobiology an international journal on social insects c maia-silva1,2, vl imperatriz-fonseca1,2, ci silva1,3, m hrncir2 article history edited by cândida maria l. aguiar, uefs, brazil received 05 october 2014 initial acceptance 04 november 2014 final acceptance 27 november 2014 keywords meliponini, pollen foraging, thermal window, resource overlap, colony survival . corresponding author camila maia-silva universidade federal rural do semi-árido departamento de ciências animais avenida francisco mota 572 mossoró, rn, 59625-900, brazil e-mail: maiasilvac@gmail.com pereboom & biesmeijer, 2003; hrncir & maia-silva, 2013). in addition to this direct influence of climatic factors, primarily of ambient temperature, on the foraging activity of a species, climatic factors affect the flowering phenology of plants and, consequently, the availability of floral resources for the bees. hence, the foraging success of a bee species is restricted to an environmental window (ew), a combination of optimal ambient temperatures and resource availability (stone et al., 1999; hilário et al., 2000). mismatches between flowering and optimal foraging temperature may lead to a dramatic reduction of a colony’s food intake and, eventually, of brood production, which depends on the availability of resources within the nest (ribeiro et al., 2003; ferreira-junior et al., 2010). research article bees 1 universidade de são paulo, ribeirão preto, sp, brazil 2 universidade federal rural do semi-árido, mossoró, rn, brazil 3 universidade federal do ceará, fortaleza, ce, brazil sociobiology 61(4): 378-385 (december 2014) 379 given the necessity of an accurate match between resource availability and foraging temperature (stone et al., 1999), the breadth of the environmental window of a bee species is determined by its capacity to acclimate to different abiotic conditions, the range of temperatures at which foragers may be active, and the dietary niche breadth. due to the bigger amplitude of foraging possibilities, the foraging success of species with broad ews, such as the honey bee apis mellifera linnaeus (apidae, apini), is bound to be less affected by variations in the abiotic and biotic environment than that of species with narrow ews. this, on the one hand, should result in a wider geographic distribution of broad ew-species compared to narrow ew-species. on the other hand, geographic ranges should be more dynamic for narrow ew-species than for broad ew-species. long-lasting climatic changes, such as global warming foreseen for the coming decades (marengo et al., 2009; nobre, 2011), may result in shifts in the geographic distributions particularly of narrow ew-species and, in consequence of the mutualistic interactions, of the plants they forage on (guisan & thuiller, 2005; hegland et al., 2009). an interesting group to study environmental windows are the stingless bees (apidae, meliponini), a group of highly eusocial bees with pantropical distribution (michener, 1974; michener, 2000; camargo & pedro, 2013). in contrast to a. mellifera, most meliponine species occur in rather narrow geographic ranges (camargo & pedro, 2013), which is typically attributed to physiological limitations and concomitant environmental specializations of the species. given their ecological importance as pollinators of many native plant species (imperatriz-fonseca et al., 2012), it has become a major concern to understand the possible impact of climatic changes on these bees (giannini et al., 2012). here, knowledge of the environmental windows of key species may form a solid background for the development of successful conservation plans. in the present study, we investigated the foraging activity of two meliponine species, melipona quadrifasciata anthidiodes lepeletier and m. subnitida ducke, at the campus of the university of são paulo at ribeirão preto-sp. of these, m. quadrifasciata naturally occurs in the study region in the brazilian southeast (camargo & pedro, 2013), whereas the occurrence of m. subnitida is restricted to the brazilian tropical dry forest, the caatinga, in the brazilian northeast (zanella, 2000). this difference in geographic distribution and concordant climatic specializations suggest that differences in the environmental window of these two species may exist. monitoring the pollen foraging activity of m. quadrifasciata and m. subnitida during 11 months, we tried to answer the following questions: (1) is there a difference between the species concerning the thermal window within which foraging occurs and, if so, does this difference result in differences in foraging activity? (2) is there a difference concerning the pollen resources collected by the species and, if so, may this be attributed to differences in foraging activity? (3) is there a difference concerning colony survival between m. quadrifasciata and m. subnitida and, if so, may this be explained by differences in resource access? material and methods study site and period the study was conducted from march, 2010 through january, 2011 at the experimental meliponary of the campus of the university of são paulo at ribeirão preto-sp (21◦10’30” s, 47◦48’38” w) in the brazilian southeast. the vegetation of the university campus is composed of both native species of seasonal semi-deciduous forest and exotic plants (vegetation cover of the campus: ~ 75 ha; pais & varanda, 2010). the area directly surrounding the meliponary presents a high diversity of potential food sources for bees (faria et al., 2012; aleixo et al., 2013). the local climate is characterized through two welldefined seasons, a hot-rainy season from september/october to april, and a cold-dry season from may to august/september. throughout our study, ambient temperature, relative humidity and precipitation were monitored by a weather station (wmr982, oregon scientific inc., u.s.a.) installed near the meliponary. bee species for our study, we monitored six colonies of m. quadrifasciata (mq) and six colonies of m. subnitida (ms). all bee colonies were housed in wooden observation hives that had been installed at the meliponary at least 3 months prior to the onset of the study. mq naturally occurs in the study area, characterized through seasonal semi-deciduous forest vegetation and, climatically, through two well-defined seasons (cold-dry season, hot-rainy season) (camargo & pedro, 2013; oliveira et al., 2013; aleixo et al., 2014). workers are 8 to 10.5 mm in length and have a thorax with of between 3.75 and 4.75 mm (schwarz, 1932). ms naturally occurs in the caatinga in the brazilian northeast, climatically classified as semi-arid with elevated annual temperatures and extended periods of drought (prado, 2003). this exclusively brazilian biome is characterized through tropical dry forest and scrub vegetation (sánches-azofeita et al. 2013). workers of this species are 7.5 to 8.5 mm in length and have a thorax with of 3.75 mm (schwarz, 1932). pollen foraging activity we evaluated the foraging activity of two of the monitored mq colonies and of at least three colonies of ms by counting the number of foragers returning to the nests with pollen loads between 5:30 am and 5:30 pm. during peak activity (usually, between 5:45 am and 10:00 am), the number of pollen foragers was registered for 5 minutes every 15 c maia-silva, vl imperatriz-fonseca, ci silva, m hrncir environmental foraging window of stingless bees380 minutes. when the colonies’ foraging activity decreased, bee counts were made for 5 minutes every 30 minutes (usually, between 10:00 am and 12:00 pm) and for 5 minutes every 1 hour (usually, between 12:00 pm and 5:30 pm). depending on the general activity of the colonies, bee counts were made on between 4 and 7 days per colony in each month. floral origin of pollen collected by bees in order to evaluate the floral origin of pollen collected by the bees, foragers with pollen loads were captured on their return to the nests. pollen sampling was performed twice or, when activity was high, three times between 6:00 am and 9:00 am on days on which we did not investigate the foraging activity. for the sampling, we blocked the nest entrances for a maximum of five minutes, and captured the returning pollen foragers individually in plastics vials when they tried to enter the closed colony. in order to avoid a significant reduction of the pollen foraging force on subsequent days, we caught a maximum of three bees during each collecting event (maximum of 9 bees per colony per day). the captured bees were chilled on ice for 5 minutes to reduce their mobility and facilitate manipulation. subsequently, the pollen pellets were removed from the bees’ corbiculae with alcohol-cleaned tweezers and stored in test tubes. thereafter, the foragers were released. after acetolysis of the individual pollen samples (method described by erdtman, 1960), the floral origin of the pollen loads was identified through comparison with reference material from university’s pollen collection. to evaluate the relative composition of potentially mixed pollen loads, we identified the floral origin of 400 pollen grains of the pellets of each forager (nagamitsu et al., 1999). samples containing between 95% and 100% of pollen grains of the same floral source were considered as pure samples (eltz et al., 2001). most of the samples consisted of only one type pollen (pure samples). in samples containing two or more pollen types, we regarded the most abundant type as the respective forager’s principal pollen source (nagamitsu et al., 1999). analysis of the thermal window of pollen foraging to assess the preferred temperature range for pollen collection, we evaluated the number of foragers returning to their nest at a given ambient temperature (to the nearest °c). this method slightly overestimated the actual foraging temperatures of the individuals because ambient temperatures steadily increased in the course of our observations and, thus, incoming pollen collectors, which forage for several minutes, were registered at the maximum temperature of their foraging trip. nonlinear regression analysis (gaussian peak model) with ambient temperature as predictor and the percentage of returning foragers as dependent variable was used to evaluate the preferred temperature range for pollen collection. from the regression model, we obtained the thermal window of both investigated species (temperature range within which 90% of activity occurs). the potential difference between the thermal windows of mq and ms was evaluated using a mann-whitney rank sum test. analysis of pollen foraging activity to evaluate the foraging activity of the investigated bee species, we calculated for each month the following parameters of foraging activity: fon, average time of foraging onset, fend, average end of pollen foraging activity, and fmax, average maximum number of foraging bees. fon, was the first 5 minute-interval in which we registered incoming pollen foragers. fend was the last 5 minute-interval in which we registered returning pollen foragers, followed by at least two 5 minuteintervals with zero-counts. for the statistical analyses, time data (hour:minute) were transformed into decimal numbers (hour + minutes/60, so that, e.g., 05:50 am became 5.83). potential differences in foraging activity parameters between mq and ms were evaluated using paired t-tests (monthly average value for mq paired with monthly average value for ms at a given month). monthly differences between the two bee species concerning the timing of foraging activity were described through the time lag between the foraging onsets (time lag = fon-ms fon-mq). analysis of pollen resource diversity and overlap for each month of our study, we assessed the plants visited by mq and ms to collect pollen. from these data, we calculated the monthly resource diversity of mq and ms through shannon’s diversity index, h’. potential differences concerning resource diversity between the bee species were evaluated using a paired t-test (monthly h’ for mq paired with monthly h’ for ms at a given month). additionally, we evaluated the monthly overlap in collected pollen sources between mq and ms using the morisita-horn overlap index, ch (ch = 0, no overlap; ch = 1, complete overlap). to determine whether and to which degree differences in resource use are associated with differences in foraging activity between the species, we evaluated the correlation between resource overlap (ch) and time lag of foraging onset using pearson product moment correlation. analysis of colony survival monthly, we evaluated the status of the monitored colonies (6 colonies mq, 6 colonies ms). a colony was considered as “dead”, in case no queen or worker bees were found in the colony. statistical analysis all statistical analyses were performed using the software packages sigmaplot 10.0/sigmastat 3.5 (systat software inc., u.s.a.) and statistica 8.0 (statsoft inc., u.s.a.). the α-level for significant differences was p ≤ 0.05. sociobiology 61(4): 378-385 (december 2014) 381 results climatic variations in the course of our study (march, 2010 through january, 2011), average temperatures varied by about 8 °c (16.8 °c – 24.5 °c). in the cold-dry season (may, 2010 through august, 2010), minimum temperatures were between 5.2 to 7.1 °c and maximum temperatures between 31.1 and 37.4°c. the total precipitation in this period was 38.0 mm. in the hot-rainy season (mach, 2010 through april, 2010, and september, 2010 through january, 2011), maximum temperatures (32.9 – 38.0 °c) did not differ much from maximum temperatures in the cold-dry season. minimum temperatures, by contrast, were considerably higher in the hot-rainy season (10.5 – 19.6 °c) than in the cold-dry season. the total precipitation during this period was 660.2 mm. during our study, average relative humidity remained above 60% (60.2 – 78.0 %) with exception of august (46.7%) and september (55.0%). in table 1, the monthly values of climatic variables obtained in the course of our study are presented in detail. thermal window of pollen foraging analysing the number of foragers returning to the colonies at a given ambient temperature, we observed a difference between m. quadrifasciata (mq) and m. subnitida (ms) concerning the temperature range at which foraging occurs (fig. 1). the thermal window for pollen foraging (90 % of returning foragers) of mq (n = 2 colonies; n = 3,295 foragers) was between 12 and 22 °c (maximum foraging activity calculated by gaussian peak model-analysis: 17.7 °c), and that of ms (n = 6; n = 2,772) was between 17 and 24 °c (maximum foraging activity: 19.3 °c). this difference was statistically significant (mann-whitney rank sum test: u = 2,312,623.5; p < 0.001). pollen foraging activity during our study, the pollen foraging activity of the investigated colonies of mq initiated between 5:30 am (december, table 1. variation of environmental variables (ev) in the course of our study. given are the respective values for each month: tavg, average temperature; tmax, maximum temperature; tmin, minimum temperature; rhavg, average relative humidity; rain, total precipitation. fig 1. thermal window of pollen foraging. scatterplot shows the amount of pollen foragers (proportional frequency relative to the total number of evaluated foragers) of melipona quadrifasciata (mq, filled circles and box) and m. subnitida (ms, open circles and box) returning to the colonies at a given ambient temperature (to the nearest ºc). dashed lines indicate the respective gaussian peak-models. horizontal boxplot indicates the median temperature (line within box), and the temperature ranges in which 50 % (box), 80 % (whiskers), or 90 % of the foragers (outliers) returned to to the colonies with pollen loads. month ev 03/10 04/10 05/10 06/10 07/10 08/10 09/10 10/10 11/10 12/10 01/11 tavg (°c) 24.0 21.2 18.1 16.8 19.1 20.2 23.2 22.2 23.2 24.5 24.0 tmax (°c) 33.6 32.9 31.4 31.1 31.9 37.4 38 37.7 35.4 35.2 35.6 tmin (°c) 16.5 11.3 5.3 5.2 7.6 7.1 10.5 10.5 12.3 17.2 19.6 rhavg (%) 67.2 73.3 72.7 67.9 60.2 46.7 55 60.2 67.2 76.2 78 rain (mm) 128.3 48.1 14.2 7.9 15.9 0.0 98.2 58.2 127 109.1 91.3 2010) and 7:15 am (july, 2010) and ended between 6:15 am (december, 2010) and 10:30 am (july, 2010). the colonies of m. subnitida started (6:00 am – 8:45 am) and ended (8:30 am – 11:15 am) their pollen collection significantly later than ms (paired t-test: fon, t = -3.94, df = 8, p = 0.004; fend, t = -4.73, df = 8, p = 0.001). the time lag between the foraging onsets of mq and ms was between 4 minutes (november, 2010) and 2 hours (august, 2010) (fig 2a). in two months of our study (may and june, 2010), both bee species showed almost no pollen collection activity (fig 2b). consequently, we could not evaluate the timing of foraging for these months. in most of the other months, the maximum number of pollen foragers (fmax) of mq was higher than that of ms. despite a significantly higher fmax of mq compared to ms considering the entire study period (paired t-test: t = 2.44, df = 10, p = 0.035), the maximum foraging force of both species was very similar in september and october, and in december, fmax of ms was even higher than that of mq (fig 2b). diversity and overlap of pollen resources we were not able to collect pollen from foragers in may and june, 2010 (virtually no pollen foragers) and in november, 2010 and january, 2011 due to heavy rainfall on collection days that impaired pollen foraging. in the course of the evaluable months, mq collected pollen at 23 plant species (between 3 and 11 species in each month) and ms at 18 plant species (between 3 and 8 species in each month) (fig 3a; table 2). the average diversity of collected resources did not differ significantly between mq and ms (h’mq = 1.39 ± 0.45; h’ms = 1.40 ± 0.36; paired t-test: t = -0.05, df = 6, p = 0.962). in march, september, and october, mq and ms collected pollen virtually at the same plant species (almost complete c maia-silva, vl imperatriz-fonseca, ci silva, m hrncir environmental foraging window of stingless bees382 resource overlap; ch = 0.91 – 0.94). on the other hand, resource overlap was low in july (ch = 0.28), august (ch = 0.00), and december (ch = 0.40). the average resource overlap was 65 % (ch = 0.65 ± 0.39) (table 2). resource overlap decreased with increasing time lag between the foraging activity of mq and ms (pearson product moment correlation: r = -0.88, p = 0.010, n = 7 months evaluated) (fig 3b). colony survival all six monitored colonies of mq survived until the end of our observations. by contrast, three of the six monitored colonies of ms died in the course of our study (one colony in july, 2 colonies in august). discussion in the present study, we evaluated the pollen foraging activity of m. quadrifasciata and m. subnitida at the campus of the university of são paulo at ribeirão preto. whereas fig 2. pollen foraging activity of melipona quadrifasciata (mq, filled symbols) and m. subnitida (ms, open symbols) in the course of our study. (a) monthly average onset (fon) and end (fend) of pollen foraging. (b) monthly average maximum of pollen foragers (fmax). nmq = 2 colonies; nms = 4 colonies (03-2010 to 06-2010) and 3 colonies (072010 to 01-2011). table 2. resource diversity and overlap. given are the numbers of pollen types collected by melipona quadrifasciata (mq) and m. subnitida (ms) in each month of our study (n indicates the number of analyzed foragers). from these, we calculated the resource diversity (shannon diversity index, h’) for each bee species and the resource overlap (morisita-horn overlap index, ch) between species. m. quadrifasciata naturally occurs in the study region in the brazilian southeast, the geographic distribution of m. subnitida is restricted to the brazilian tropical dry-forest in the northeast of the country (zanella, 2000; camargo & pedro, 2013), which is characterized through elevated annual temperatures and an extended hot-dry season. this limited occurrence of m. subnitida suggests strong environmental specialisations and survival strategies to cope with the long and often irregular periods of drought (maia-silva, 2013). in spite of the fact that this meliponine species has been repeatedly introduced into different environments (including são paulo state) for beekeeping or research purposes (nogueira-neto, 1997; koedam et al., 1999), these attempts often resulted in the loss of all established colonies (nogueira-neto, 1997). the results of our study now indicate that the possible reason for these and similar failures is the mismatch between optimal ambient temperature for foraging and resource availability mainly in the cold-dry season. the temperature range of m. subnitida within which pollen foraging occurred was between 17 and 24 °c. in its natural habitat, the brazilian tropical dry forest, the thermal window for pollen foraging of this bee was found to be between 21 and 29 °c (maia-silva, 2013). yet, despite the apparent capacity of acclimatization to the lower ambient temperatures in the brazilian southeast (minimum temperatures 5 to 20 °c) as compared to the brazilian northeast (minimum temperatures 18 to 21 °c; maia-silva, 2013), the lowtemperature threshold for m. subnitida was still 5 °c above the low-temperature threshold of m. quadrifasciata (12 °c). this difference concerning the thermal window between the two meliponine species is presumably related to the bees’ physiological adaptations to the climatic situation of their respective natural habitats. here, a critical factor is the absolute physiological limit of bees, determined by the temperature below which endothermic heating of the flight muscles becomes uneconomic (heinrich, 1993; stone, 1993; stone et al., 1999). number of collected pollen types shannon diversity index (h') morisita-horn overlap index (ch) month mq (n) ms (n) mq ms mq – ms 03-2010 07 (18) 08 (32) 1.72 1.80 0.91 04-2010 07 (19) 06 (41) 1.65 1.38 0.51 05-2010 (0) (0) 06-2010 (0) (0) 07-2010 11 (24) 08 (11) 2.09 1.97 0.28 08-2010 03 (20) 04 (06) 0.82 1.33 0.00 09-2010 05 (19) 04 (12) 1.16 1.20 0.92 10-2010 04 (25) 04 (24) 1.05 1.01 0.94 11-2010 (0) (0) 12-2010 04 (13) 03 (18) 1.23 1.07 0.98 01-2011 (0) (0) average ± sd 1.39 ± 0.45 1.40 ± 0.36 0.65 ± 0.39 total 23 (138) 18 (144) 2.68 2.28 0.74 sociobiology 61(4): 378-385 (december 2014) 383 (silva & pinheiro, 2007). in contrast to m. quadrifasciata, m. subnitida initiated foraging in july and august not before 8:30 am. thus, due to their elevated low-temperature threshold, the colonies of m. subnitida started pollen collection close to the time the mass-flowering pollen bonanzas became unprofitable. this observed mismatch between the thermal window of m. subnitida and the availability of mass-flowering food sources was presumably the main cause for the loss of 50% of the colonies. owing to extremely low ambient temperatures in the morning, both studied bee species did not collect pollen in may and june. consequently, the access to mass-flowering trees in the subsequent months was crucial for the bees to refill their pollen storage and resume their brood cell-construction, and, eventually, decisive for colony survival. the importance of highly profitable pollen plants, such as mass-flowering trees, for both m. quadrifasciata and m. subnitida became evident when evaluating the pollen collected by the colonies. the vast majority of the foragers’ pollen loads originated from mass-flowering plants belonging to the botanical families myrtaceae and fabaceae (m. quadrifasciata: 80% of pollen loads, 12 plant species; m. subnitida: 93% of pollen loads, 13 plant species). additionally, the bees collected pollen at plants with poricidal anthers belonging to the family solanaceae, which are highly attractive for bees owing to the high quantity of pollen present in the flowers (buchmann, 1983) (m. quadrifasciata: 12% of pollen loads, 4 species; m. subnitida: 2% of pollen loads, 1 species). thus, despite the availability of close to 300 plant species belonging to 73 botanical families at the university’s campus (aleixo fig 3. pollen source use and overlap. (a) bar-chart shows the amount of foragers (proportional frequency relative to the total number of evaluated foragers) of melipona quadrifasciata (mq, filled bars, n total = 138 analyzed pollen foragers) and m. subnitida (ms, open bars, n total = 144) returning to the colonies with pollen collected at the following plants: 1 anadenanthera peregrina; 2 mimosa sp.; 3 eugenia pyriformis; 4 eugenia involucrata; 5 eugenia uniflora; 6 eucalyptus moluccana; 7 leucaena leucocephala ; 8 eugenia brasiliensis; 9 eucalyptus grandis; 10 solanum sp.; 11 cecropia pachystachya; 12 pterocarpus violaceus; 13 solanum cernuum; 14 solanum paniculatum; 15 syzygium malaccense; 16 albizia lebbeck; 17 capsicum baccatum; 18 eucalyptus sp.; 19 solanum seaforthianum; 20 vernonia sp.; 21 indeterminate 1; 22 lagerstroemia indica; 23 serjania lethalis; 24 citrus limonia; 25 senegalia polyphylla; 26 eucalyptus citriodora; 27 handroantus sp.; 28 indeterminate 2; 29 psidium guajava. (b) scatterplot showing the correlation between the monthly average time lag concerning the onset of pollen foraging between m. subnitida and m. quadrifasciata and the monthly resource overlap (morisita-horn index, ch) between the two investigated bee species. see results for details on the correlation analysis. the observed differences concerning the low-temperature threshold between m. subnitida and m. quadrifasciata, and the consequent differences in foraging onset (fig 2), led to a segregation of the utilized resources among the species particularly in the coldest months of our study (fig 3). whereas both bee species forged at virtually the same plants in months with elevated ambient temperatures (high resource overlap in march, september, october, december), m. subnitida missed out on important pollen sources visited early in the morning by m. quadrifasciata in months with low morning temperatures. owing to a low-temperature threshold of 12 °c, m. quadrifasciata was able to initiate foraging before 7:00 am in july and august (minimum temperatures < 8 °c at around 5:00 am). in these months, m. quadrifasciata foragers collected pollen predominantly at the mass-flowering trees eugenia pyriformis and e. uniflora (july: e. pyriformis: 33.3% of the evaluated pollen; august: e. pyriformis: 35% of the evaluated pollen; e. uniflora: 60% of the evaluated pollen). massflowering plants, in general, produce an excessive number of flowers each day, thus providing large amounts of pollen and/or nectar to flower visitors (gentry, 1974; bawa, 1983). for stingless bees, mass-flowering plants offer an excellent opportunity to amass floral resources within their nests and are the predominant source of nectar and pollen, contributing by up to 90% to the annual nutritional input into the colonies (wilms et al., 1996; ramalho, 2004). the flowers of e. pyriformis and e. uniflora open early in the morning, around 5:00 am (proença & gibbs, 1994), and are rapidly exploited by bees before 8:00 am to 9:00 am c maia-silva, vl imperatriz-fonseca, ci silva, m hrncir environmental foraging window of stingless bees384 et al., 2014), the bees showed a high selectivity and floral preference for plants providing high amounts of pollen. the apparent dependency and specialisation of melipona bees on highly profitable pollen sources underlines the importance of an accurate match between the bees’ thermal window and the availability of these resources. here, species with broad environmental windows (broad ew-species) have considerable advantages over species with narrow thermal windows (narrow ew-species). owing to a wider temperature range within which foraging may occur, broad ew-species are less affected by thermal variations in the environment than are narrow ew-species (stone et al., 1999; hilário et al., 2000). this, on the one hand, results in a wider geographic distribution of broad ew-species, such as m. quadrifasciata (thermal foraging window: 12 – 22 °c; amplitude: 10 °), which occur from the brazilian southeast to northeast, compared to narrow ewspecies, such as m. subnitida (thermal foraging window: 17 – 24 °c; amplitude 7 °), which are restricted to parts of the brazilian northeast (zanella, 2000; camargo & pedro, 2013). on the other hand, broad ew-species may be less affected by long-lasting climatic changes, such as global warming predicted for the coming decades (marengo et al., 2009; nobre, 2011) than are narrow ew-species. increasing ambient temperatures presumably lead to shifts in the timing of both flowering and pollinator activity (memmott et al., 2007; hegland et al., 2009). these phenological responses to climate warming may occur at similar magnitudes in plants and bees, thereby maintaining existent mutualistic plant-pollinator relationships. however, broad ew-species presumably accompany phenological shifts of plants better than narrow ew-species that may suffer from any tiny temporal mismatch between the timing of flowering and foraging activity (memmott et al., 2007; hegland et al., 2009), as was the case in our study. so far, mismatches in pollination interactions have been poorly studied (hegland et al., 2009). here, the determination of the environmental window of a bee species may serve as important basis for understanding the potential decoupling of pollinator activity from the timing of flowering and its consequences for ecosystem functioning or species distribution. acknowledgments we would like to thank two anonymous reviewers for their critical comments that helped to improve the manuscript. this study complies with current brazilian laws and was financially supported by capes (cms) and cnpq (vlif: 482218/2010-0; mh: 304722/2010-3, 481256/2010-5). references aleixo, k.p., faria, l.b., garófalo, c.a., fonseca, v.l.i., & silva, c.i. 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(2000). the bees of the caatinga (hymenoptera, apoidea, apiformes): a species list and comparative notes regarding their distribution. apidologie, 31: 579-592. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.460sociobiology 62(3): 351-355 (september, 2015) bioactivity of cicuta virosa l. var. latisecta celak. (umbelliferae: cicutal) against red imported fire ant under laboratory and field conditions introduction the red imported fire ants, solenopsis invicta buren (hymenoptera: formicidae), is an exotic invasive pest ant species that has spread in many countries, including china. the imported fire ant workers sting and inject venom, which make them medically important insects (deshazo et al., 1999). less than 3% of the human population is sensitive to the venom of fire ants, but stinging incidences have resulted in serious medical problems or even death, particularly when the victim is young, old, or has a compromised immune system (drees et al., 2013). the fire ant has become a dominant pest in invaded areas, causing great economic loss and ecological problems. efforts have been made to control the red imported fire ants. insecticide use is an important control strategy. fipronil is a broad-spectrum pyrazole insecticide that could serve as a bait toxicant for control of ants. a 15 µg/mg granular bait abstract we have evaluated the bioactivities of compounds from cicuta virosa l. var. latisecta celak. against red imported fire ants (solenopsis invicta buren) under field and laboratory conditions. the compounds were as follows: methanol extract; petroleum ether, chloroform, and ethyl acetate fractions from the methanol extract; and the active compound isoimperatorin, which was isolated from the chloroform-fraction. the 7 d lc 50 values of the methanol extract, petroleum ether, chloroform, and ethyl acetate fractions and isoimperatorin toward micrergates were 111.20, 214.45, 40.90, 569.67, and 25.73 mg/kg, respectively. the corresponding lc 50 values toward macrergates were 155.78, 308.38, 75.01, 776.75, and 42.77 mg/kg, res pectively. under field conditions, baits containing 0.2% methanol extract, 0.1% chloroform fraction, and 0.05% isoimperatorin efficiently controlled s. invicta, with effectiveness percentages of 95.56%, 97.78%, and 95.56%, respectively on the 30th day after bait application. such effectiveness percentages were not significantly different from that obtained using the positive control fipronil. the present study showed that c. virosa l. var. latisecta has potential as a natural control agent for the red imported fire ants. sociobiology an international journal on social insects yq tian1, 2, dm cheng3, zx zhang1, 2 article history edited by evandro do nascimento silva, uefs, brazil received 16 july 2014 initial acceptance 14 may 2015 final acceptance 15 may 2015 keywords cicuta virosa l. var. latisecta, isoimperatorin, solenopsis invicta, insecticidal activity. corresponding author zhixiang zhang college of natural resources and environment, south china agricultural university wushan, tianhe, guangzhou 510640, people’s republic of china e-mail: zdsys@scau.edu.cn caused over 80% colony mortality at 6 and 12 weeks after broadcast application in non-grazed pastures (collins & callcott, 1998). the activities of other insecticides, such as bifenthrin, chlorfenapyr, and thiamethoxam were also evaluated against red imported fire ants (wiltz et al., 2010). biological control is also an important pest management method for this species. biological control strategies include the use of the following: pseudacteon spp. (diptera: phoridae) parasitoids; kneallhazia solenopsae (microsporidia: tubulinosematidae) (williams et al., 1999), which is the causative agent of fire ant disease; beauveria bassiana (balsamo) vuillem (bextine & thorvilson, 2002; oi et al., 1994); and insect viruses (valles & strong, 2005; hashimoto & valles, 2008). plants contain many compounds that have insecticidal activities. many of these compounds have been used as alternatives to synthetic chemical pesticides because of their desirable attributes, such as eco-friendly nature, availability, research article ants 1 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 2 south china agricultural university, guangzhou, china 3 zhongkai university of agriculture and engineering, guangzhou, china yq tian, dm cheng, zx zhang cicuta virosa l. var. latisecta celak. controls red imported fire ant352 safety, acceptability, minimal effect on beneficial organisms, low cost, good storage, and easy application. many plant-derived compounds have been widely used as commercial formulations, such as rotenone from the roots of derris trifoliata lour. and azadirachtin from the fruits of azadirachta indica a. juss. plantderived substances are globally known because they have few side effects and have insect control properties. however, information is lacking on the activities of plant-derived compounds against red imported fire ants. in our study on natural insecticides of plant origin, we obtained a methanol extract from cicuta virosa l. var. latisecta celak, which is a perennial herbaceous plant growing in northeast china; this plant is a chinese folk medicine used to treat abdominal pain (li et al., 2009) and has potency against the red imported fire ants. we analyzed the bioactivities of the methanol extract, fractions (petroleum ether, chloroform, and ethyl acetate fractions), and an isolated active compound, namely, isoimperatorin against fire ants under laboratory and field conditions. material and methods preparation of plant extract, fractions and isoimperatorin the whole plant of c. virosa l. var. latisecta celak was collected from the suburbs of changchun, jilin province, china in june 2013 and was air-dried at room temperature. methanol extract and fractions from the plant and isoimperatorin were prepared according to the author’s previously reported method (tian et al., 2013). the powdered dry plant material was extracted with methanol at room temperature, and the methanol solution was concentrated in vacuo. the residue was sequentially extracted with petroleum ether, chloroform (chcl3), and ethyl acetate (etoac). the resulting petroleum ether, chcl3, and etoac solutions yielded a deep brown syrup after concentration in vacuo. the active compound isoimperatorin was isolated from the chcl3-soluble extract using the same procedure described by the author in the abovementioned reference. origin and rearing of micrergate and macrergates of red imported fire ants s. invicta colonies were collected from the suburbs of guangzhou, china and maintained in the laboratory for bioassays (lv et al., 2006; huang et al., 2007). the collected ants were fed with a mixture of 10% honey and live insects (tenebrio molitor l.). a test tube (25 mm × 200 mm) partially filled with water and plugged with cotton was used as a water source. the ants were maintained in the laboratory at 25 ± 2 °c. preparation of fire ant bait c. virosa l. var. latisecta methanol extract, the three abovementioned fractions, and isoimperatorin were mixed with fire ant bait for bioassays under laboratory and field conditions. the bait was prepared as described by kafle et al. (2010, 2011) with some modifications. at first, the extract, fractions, and isoimperatorin were dissolved in acetone. subsequently, the acetone solutions were mixed with soybean oil, shrimp shell, and corn powders. finally, the prepared baits were placed in a ventilating cabinet for 1 h. the baits were maintained in a refrigerator (4 °c) for future use after the acetone had evaporated. laboratory bioassays the vertical wall of a beaker with a bottom diameter of 10 cm was coated with fluon emulsion after drying for 24 h to prevent the ants from escaping. this beaker was used as a foraging area. the artificial baits were placed 2 cm away from the inner wall of the beaker. a test tube was filled with water to approximately two-thirds full. the test tube was tightly plugged with water-saturated cotton, which was pushed 3 cm into the mouth of the test tube. the test tube was placed inside each beaker to serve as the water source for the insects. all ants used in the toxicity tests were from the same colony. a group of 30 micrergates or macrergates was transferred into the beaker for the toxicity tests, and each test was replicated thrice. the baits, which contained the abovementioned methanol extract, fractions, or isoimperatorin at serial concentrations were placed in the beakers for the bioassay. a blank bait mixed with only acetone was used as the negative control. the mortalities were corrected by abbott’s formula (abbott, 1925). the average corrected mortality of three replications at each concentration was calculated 7 d later. the lc50 value, which is the concentration that causes 50% insect mortality, was determined using probit analysis. during the tests, mortality was recorded at an interval of 48 h for 7 d. this study was conducted under ambient temperature and relative humidity, as follows: 27 ± 1 °c and 60 ± 5 % rh on average under 14 h : 10 h light : dark photoperiod. field trials the effectiveness of c. virosa l. var. latisecta baits containing methanol extract, chloroform fraction, and isoimperatorin on the inactivation of s. invicta mounds were evaluated under field conditions. the field studies were conducted in the zengcheng farm of south china agricultural university. the experiment was designed in a single factor random block. fifteen mounds were used for each bait and for control treatments. each treatment was replicated thrice. the baits were sprinkled around a 50 cm radius from the mounds’ center. the commercial insecticide fipronil (purity 96%, tuo qiu agricultural chemical ltd. co., jiangshu province, china) was used as the positive control. the blank bait mixed with only acetone was used as the negative control. the number of active mounds in the field was determined on the 1st, 5th, 10th, 20th, and 30th days after sociobiology 62(3): 351-355 (september, 2015) 353 treatment. a mound was designated as active when at least 20 adult workers exited the excavated soil when probed with a metal rod (5 mm in diameter) (kafle et al., 2011). the control effectiveness was calculated using the following equation: control effectiveness = [(scontrol – streatment)/(100 – scontrol)] × 100, where s = (number of active mounds before treatment number of active mounds after treatment)/number of active mounds before treatment × 100. statistical analyses the results were expressed as mean ± standard error. the treatment means were transformed into arcsine squareroot values for analysis of variance (anova). the means were compared and separated by scheffe’s test. p < 0.05 was the level of statistical significance. results and discussion laboratory bioassays c. virosa l. var. latisecta methanol extract and fractions (petroleum ether, chcl3, and etoac) and isoimperatorin showed insecticidal activities against micrergates of red imported fire ants (fig 1). the insecticidal activities were slow to show, but the corrected percentage mortalities of treated micrergates increased steadily with increasing treatment time. seven days after the treatment, the highest mortality (93.33%) occurred among the micrergates treated with 600 mg/kg methanol extract. the micrergate mortalities under the other treatments were in the following descending order: 200 mg/kg chloroform fraction (90.00%), 40 mg/kg isoimperatorin (70.00%), 200 mg/kg petroleum ether fraction (45.00%), 200 mg/kg ethyl acetate fraction (28.33%), and the control (3.33%). the 7 d lc50 values of the methanol extract and fractions (petroleum ether, chcl3, and etoac) and isoimperatorin for the micrergates were 111.20, 214.45, 40.90, 569.67, and 25.73 mg/kg, res pectively (table 1). fig 2. mortalities of cicuta virosa l. var. latisecta methanol extract, fractions and isoimperatorin against macrergates of solenopsis invicta under laboratory conditions. fig 1. mortalities of cicuta virosa l. var. latisecta methanol extract, fractions and isoimperatorin against micrergates of solenopsis invicta under laboratory conditions. treatment regression equation lc 50 (mg/kg) 95% fiducial limit correlation coefficients methanol extract y = 1.3499 + 1.7839 x 111.20 89.76 ~ 137.76 0.9917 petroleum ether fraction y = 0.8122 + 1.7963 x 214.45 175.07 ~ 262.69 0.9868 chloroform fraction y = 2.0932 + 1.8035 x 40.90 33.26 ~ 50.29 0.9907 ethyl acetate fraction y = -0.0535 + 1.8339 x 569.67 466.83 ~ 695.18 0.9966 isoimperatorin y = 2.4614 + 1.7999 x 25.73 21.03 ~ 31.48 0.9898 table 1. lc50 of cicuta virosa l. var. latisecta methanol extract, petroleum ether-, chloroformand ethyl acetate fractions, and isoimperatorin against micrergates of solenopsis invicta. for the macrergates, similar trends were observed (fig 2). the 600 mg/kg methanol extract resulted in the highest mortality (80.00%) at 7 d after treatment. the mortalities induced by the other treatments were in the following decreasing order: 200 mg/kg chloroform fraction (71.67%), 40 mg/kg isoimperatorin (53.33%), 200 mg/kg petroleum ether fraction (35.00%), 200 mg/kg ethyl acetate fraction (23.33%), and the control (1.67%). the 7 d lc50 values of the methanol extract and fractions (petroleum ether, chcl3, and etoac) and isoimperatorin for macrergates were 155.78, 308.38, 75.01, 776.75, and 42.77 mg/kg, res pectively (table 2). yq tian, dm cheng, zx zhang cicuta virosa l. var. latisecta celak. controls red imported fire ant354 field trials c. virosa l. var. latisecta methanol extract, chloroform fraction, and isoimperatorin could control s. invicta, but these botanical insecticides were slower to act than fipronil (table 3). on the 5th day after bait application, the control effectiveness of baits containing 0.2% methanol extract, 0.1% chloroform fraction, and 0.05% isoimperatorin were 33.33%, 31.11%, and 26.67%, respectively. these results were significantly lower than the positive control fipronil (66.67%) (f = 14.297, p = 0.0039). on the 10th day, the control effectiveness value of 0.01% fipronil-based bait was still significantly higher than the three baits (f = 11.407, p = 0.0068). however, on the 30th day, the control effectiveness of the baits containing methanol extract, chloroform fraction, and isoimperatorin increased to 95.56%, 97.78%, and 95.56%, respectively, and such percentages were not significantly different from the control effectiveness percentage of fipronil bait (100%) (f = 2.2, p = 0.1889). however, the control value of 0.01% fipronil bait treatment remained the highest throughout the 30 d period after bait application. in the present study, the bioactivity of the insecticidal plant (c. virosa var. latisecta) against the red imported fire ant was investigated. the crude methanol extract and fractions (petroleum ether, chcl3, and etoac) and the isolated compound isoimperatorin were verified to be active against micrergates and macrergates of the ants under both laboratory and field conditions. c. virosa l. var. latisecta compounds are safe for use as control agents of fire ants. c. virosa l. var. latisecta is used in northeast china to control aphids on vegetables. furthermore, the isolated active compound, isoimperatorin, is also a main active constituent in other safe-for-use traditional chinese medicinal plants, such as notopterygium forbesii boiss (zhou et al., 2007). c. virosa l. var. latisecta has potential as a source of natural insecticides for the control of the red imported fire ants. treatment regression equation lc 50 (mg/kg) 95% fiducial limit correlation coefficients methanol extract y = 1.2080 + 1.7295 x 155.78 126.60 ~ 191.67 0.9946 petroleum ether fraction y = 0.0045 + 2.0070 x 308.38 256.17 ~ 371.23 0.9899 chloroform fraction y = 1.9671 + 1.6174 x 75.01 58.85 ~ 95.61 0.9938 ethyl acetate fraction y = -0.5601 + 1.9237 x 776.75 636.97 ~ 947.18 0.9902 isoimperatorin y =1.9383 + 1.8771 x 42.77 35.08 ~ 52.13 0.9907 table 2. lc50 of cicuta virosa l. var. latisecta methanol extract, petroleum ether-, chloroformand ethyl acetate fractions, and isoimperatorin against macrergates of solenopsis invicta. treatment controlling effectiveness (%) 1d after treatment 5d after treatment 10d after treatment 20d after treatment 30d after treatment 0.2% methanol extract-based bait 0.00 ± 0.00 b 33.33 ± 3.85 b 57.78 ± 4.44 b 91.11 ± 2.22 ab 95.56 ± 2.22 a 0.1% chloroform fraction-based bait 0.00 ± 0.00 b 31.11 ± 5.88 b 66.67 ± 3.85 b 88.89 ± 2.22 ab 97.78 ± 2.22 a 0.05% isoimperatorin-based bait 0.00 ± 0.00 b 26.67 ± 3.85 b 62.22 ± 5.88 b 80.00 ± 3.85 b 95.56 ± 2.22 a 0.01% fipronil-based bait 15.56 ± 2.22 a 66.67 ± 3.85 a 86.67 ± 3.85 a 95.56 ± 2.22 a 100.00 ± 0.00 a table 3. controlling effectiveness of cicuta virosa l. var. latisecta methanol extract-, chloroform fraction-, isoimperatorinand fipronil-based baits on the inactivation of solenopsis invicta mounds under field conditions. values represent means from three replicates. means within the same row followed by the same letter are not significantly different (p < 0.05) using scheffe’s test of sas. acknowledgments the work was supported 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(in chinese) doi: 10.3321/j.issn:1001-5302.2007.07.003. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7259sociobiology 68(4): e7259 (december, 2021) introduction in tropical areas, 94% of plants require pollinators for reproduction and only a small number of species can reproduce by spontaneous self-pollination or agamospermy (ollerton et al., 2011). pollinators distribute pollen from one plant to another for successful cross-pollination; however, this process can often be affected by the presence of other floral visitors, like ants (torezan-silingardi et al., 2021). many plants are engaged in mutualisms with ants (kuriakose et al., 2018). the main role these mutualistic ants play on plants is the abstract the mutualism of ants and extrafloral nectary-bearing plants is known to reduce rates of herbivory. however, ants may have negative impacts on other mutualisms such as pollination, constituting an indirect cost of a facultative mutualism. for instance, when foraging on or close to reproductive plant parts ants might attack pollinators or inhibit their visits. we tested the hypothesis that ants on efn-bearing plants may negatively influence pollinator behavior, ultimately reducing plant fitness (fruit set). the study was done in a reserve at brazilian savannah using the efn-bearing plant banisteriopsis malifolia (malpighiaceae). the experimental manipulation was carried out with four groups: control (free visitation of ants), without ants (ant-free branches), artificial ants (isolated branches with artificial ants on flowers) and plastic circles (isolated branches with plastic circles on flowers). we made observations on flower visitors and their interactions, and measured fruit formation as a proxy for plant fitness. our results showed that pollinators hesitated to visit flowers with artificial ants, negatively affecting pollination, but did not hesitate to visit flowers with plastic circles, suggesting that they recognize the specific morphology of the ants. pollinators spent more time per flower on the ant-free branches, and the fruiting rate was lower in the group with artificial ants. our results confirm an indirect cost in this facultative mutualism, where the balance between these negative and positive effects of ants on efn-bearing plants are not well known. sociobiology an international journal on social insects rᴏᴅʀɪɢᴏ r. nᴏɢᴜᴇɪʀᴀ1, dᴀɴɪʟᴏ f. b. sᴀɴᴛᴏs2, eᴅᴜᴀʀᴅᴏ s. cᴀʟɪxᴛᴏ3, hᴇʟᴇɴᴀ m. tᴏʀᴇᴢᴀɴ-sɪʟɪɴɢᴀʀᴅɪ4, kʟᴇʙᴇʀ dᴇʟ-cʟᴀʀᴏ4 article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 01 june 2021 initial acceptance 31 july 2021 final acceptance 16 october 2021 publication date 15 december 2021 keywords malpighiaceae, interactions, tropical area, fruit set, brazil. corresponding author laboratório de ecologia comportamental e de interações (leci) instituto de biologia universidade federal de uberlândia caixa postal: 593, cep: 38400-902 uberlândia, minas gerais, brasil. e-mail: delclaro@ufu.br protection against herbivore attack; in turn plants provide different types of resources, such as places for nesting and food. one of the most common food resources offered by plants to ants is the extrafloral nectar produced by specialized glands known as extrafloral nectaries (efns) (calixto et al., 2018; del-claro et al., 2016; heil, 2015). indeed, some studies have shown that ants that hunt herbivores present in efn-bearing plants decrease leaf herbivory (rosumek et al., 2009; trager et al., 2010) and/or increase fruit production (del-claro and marquis, 2015; nascimento and del-claro, 2010). 1 postgraduation in entomology, faculdade de filosofia, ciências e letras, universidade de são paulo, ribeirão preto, sp, brazil 2 phd student (biology), the university of alabama, tuscaloosa, al, us 3 department of entomology and nematology, university of florida, gainesville, fl, usa 4 laboratório de ecologia comportamental e de interações, instituto de biologia, universidade federal de uberlândia, uberlândia, mg, brazil research article ants negative effects of ant-plant interaction on pollination: costs of a mutualism mailto:delclaro@ufu.br rodrigo r. nogueira et al. – tritrophic interaction between plant, pollinators and ants2 however, mutualistic ants attracted by plant-based resources may forage on entire plant structures, including inflorescences and flowers. although extrafloral nectar is a sugar-rich reward, ants also forage on plant reproductive parts looking for floral resources (such as nectar and pollen) and potential prey (such as floral herbivores) (blüthgen et al., 2003; blüthgen & fiedler, 2004; davidson et al., 2003; sinu et al., 2017; unni et al., 2021; hanna et al., 2015; fuster et al., 2020). the additional foraging behavior on these reproductive plant parts may negatively affect mutualistic plant-pollinator interactions. although many studies emphasize that ants are important mutualists for plants when defending against herbivores (calixto et al., 2018; del-claro et al., 2016; rosumek et al., 2009; trager et al., 2010), there are also cases where they have been shown to negatively impact plant pollination (assunção et al., 2014; melati & leal, 2018; ness, 2006; villamil et al., 2018). for example, byk and delclaro (2010) showed that the presence of cephalotes pusillus (formicidae: myrmicinae) negatively affects pollination of ouratea spectabilis (ochnaceae) by feeding on pollen and reducing viability of the pollen grains. assunção et al. (2014) demonstrated through experimental manipulation in heteropterys pteropetala (malpighiaceae) that ants, which feed on efns but also visit leaves and inflorescences, may have a negative impact on the plant fruit production by attacking or chasing pollinators. some studies have shown that shape and odor are important ant traits that can be detected by pollinators, who will avoid visiting these flowers (assunção et al., 2014; ballantyne & willmer, 2012; adam r. cembrowski et al., 2014). for example, using dummy ants on flowers, assunção et al. (2014) showed that pollinators easily recognize ant shape and color, which significantly reduced floral visitation rate and fruit production. when ants are present, floral visitors may approach a flower, but can also change their behavior by reducing the time spent on flowers (aguirre-jaimes et al., 2018; assunção et al., 2014; ness, 2006; sousa-lopes et al., 2020; villamil et al., 2018; sinu et al., 2017; unni et al., 2021; hanna et al., 2015; fuster et al., 2020), or they will not come into contact with the reproductive parts, becoming ineffective in pollination. in the brazilian tropical savannah, smaller bees tend to spend more time analyzing flowers with tending ants before approaching, since they are more affected by ant presence on flowers than larger bees (assunção et al., 2014; barônio and del-claro, 2017). thus, considering that pollinators can recognize ant shape and/or odor (ballantyne and willmer 2012; cembrowski et al. 2014), the ant presence on flowers can negatively affect the pollination, ultimately decreasing fruit production. banisteriopsis malifolia (malpighiaceae) is an abundant efn-bearing plant species in the brazilian savannah (henceforth “cerrado”), which is commonly visited by several ant species (torezan-silingardi, 2007). in addition, large bees represent the main pollinators of this species (torezan-silingardi, 2007). it is therefore an advantageous system to investigate indirect effects of ant-plant mutualisms on plant reproduction. the aim of this study was to investigate the impact of ants on floral visitor behavior and fruit production of an efn-bearing plant, b. malifolia. we tested the hypothesis that in ant-plant mutualisms mediated by efns, the ant presence on flowers will negatively interfere with floral visitor behavior and fruit production. thus, the two following hypotheses were evaluated: hypothesis 1 – the presence of ants on flowers decreases the total number of visits and the time spent on the flower by pollinators; hypothesis 2 – the presence of ants on flowers decreases the fruit set by influencing the pollinator behavior. material and methods study area the fieldwork was carried out from march to may 2018 in the brazilian cerrado, within the ecological reserve of the clube de caça e pesca itororó de uberlândia in the city of uberlândia, state of minas gerais, brazil (18º58’30”s, 48°17’27”w). this region has two well-defined seasons (awtype köppen climate): rainy, with a hot and humid climate from october to march, monthly rainfall of 270 ± 50 mm (average ± sd) and monthly temperature of 23 ± 5°c (average ± sd); and dry, with a cold and dry climate from april to september, precipitation of 22 ± 20 mm (average ± sd) and temperature of 19 ± 3 oc (average ± sd) (alvares et al., 2013; calixto et al., 2021b; vilela et al., 2017). the reserve’s cerrado vegetation ranges from open areas with grasses and small shrubs, to more closed-canopy areas, with trees reaching up to 12 meters in height (del-claro et al., 2019). pastures and properties with different levels of natural area conservation surround the ecological reserve. plant species banisteriopsis malifolia (nees & mart.) b. gates (malpighiaceae) is a common cerrado shrub, which blooms from march to june (calixto et al., 2021b; vilela et al., 2014). this species has efns (located on the underside of the leaves, at the junction with the petiole) that attract ants which feed on their secretion (barônio & del-claro, 2017). it has paniculate inflorescences with flowers that offer oil and pollen to floral visitors, which have five long-pedunculate petals and its reproduction depends on pollinators, with crosspollination (vogel, 1990). the flower can produce one to three schizocarpic fruits of the samara type (barroso et al., 2004) which are wind dispersed. leaves show alternating phyllotaxis. voucher specimens were deposited in the herbarium uberlandensis under number hufu 00013413. experimental set-up to test our hypothesis, 48 individuals of b. malifolia with similar phenological characteristics (height, number of branches, number of inflorescences, number of flower buds sociobiology 68(4): e7259 (december, 2021) 3 per inflorescence, active efns and reproductive stage) and 10 meters apart were marked along a 3 km transect. we did not use a block design, since the constant presence of plastic ants (see below) on the inflorescence could dissuade potential visitors of other treatments. individuals were randomly and equally divided into four treatments: control, artificial ants (hereafter “aa”), plastic circles (hereafter “pc”), and without ants (hereafter “wa”). in all treatments, in each individual plant, five flower buds were marked to ensure that the anthesis of the flower only occurred after the start of the experiment. flowers remain open about 2-3 days after the anthesis. preanthesis floral buds were bagged with voile bags and pollinator visitation was only allowed at the time of field observations, when the floral anthesis had already occurred, from 08:00 am to 12:00 pm (since bees’ activity is concentrated at this time (assunção et al., 2014), from which the flowers were bagged again. each morning one plant from each treatment was observed for four hours (08:00 to 12:00), totaling 192 hours of observation (4 hours per plant x 48 plants). in total, for each treatment we analyzed 60 flowers, resulting in a total of 240 flowers equally distributed across 48 plants. it was observed that the abundance of pollinators in the afternoon is much lower than in the morning. therefore, the collections were all concentrated in the morning period (an average of 1.3 visits in the afternoon and an average of 6.6 visits in the morning). in the control group, a tanglefoot resin (tree tanglefoot® pest barrier – rapids, michigan, usa) was applied 10 cm from the soil to half of the circumference of the stem base to control for a possible effect of the resin on the other treatments. in this way, access of ants and other arthropods was at least partly allowed. the treatment wa underwent the same procedure as the control group, however the application of tanglefoot was made around the entire circumference of the stem, to prevent any ants from accessing the plant. any ants present prior to the application of tanglefoot were removed manually from these plants and the surrounding vegetation to avoid contact bridges. the treatment aa received the same experimental manipulation as wa, but here artificial ants made of black plastic (based on the morphology of the most common ants that forage on this plant, ants of the genus camponotus, as shown in alves-silva, 2011; fagundes et al., 2017) (fig 1a and b) were glued onto the petals of all flowers open on a particular day of observation using white paper glue (fig 1c). all plastic ants and circles were glued in the same position on the flowers. to control for a possible effect of glue on the other treatments, a little drop was also applied to petals in the other treatments. five plastic ants were used on each plant to simulate an average ant colonization rate on each plant. artificial ants were removed at the end of each observation period. the last treatment, pc, underwent the same procedures as the aa group, but instead of artificial ants, black plastic circles made of the same material as the artificial ants were placed on the flower petals (fig 1d), and removed at the end of each observation. furthermore, 5 pieces of plastic were also used in each plant. the number of visits was quantified by counting the number of times that floral visitors, mostly bees, were observed collecting resources such as oil and pollen. the time spent visiting flowers was determined using a timer. the hesitation was scored by counting the number of floral visitors that approached a flower but did not actually visit it (it approaches a flower hovering over it and moves away without making the floral visit) (villamil et al., 2018). after all observation periods had finished, the flowers were bagged again, and after ca. 20 to 30 days, the fruits formed in each group were counted. fig 1. the black ant (camponotus crassus) frequently visitant of banisteriopsis malifolia foraging on the plant a); flower with a real ant on the petals of the flower b); flowers with examples of the treatments with artificial ants and plastic circles c) and d) respectively. statistical analysis all statistical analyses were performed in rstudio 4.0.0 (r core team, 2020) at 5% of significance level. model suitability, homoscedasticity and overdispersion (when applicable), were checked in all models. when overdispersion was detected for counting response variable, it was used the negative binomial distribution through package “mass” (venables & ripley, 2002). glm analyses were conducted with the package “stats” and “glmmtmb” (brooks et al., 2017) followed by likelihood-ratio test with the package “car” (fox & weisberg, 2011). pairwise comparisons were conducted using estimated marginal means with the package “emmeans” (lenth, 2020). to verify if there was no disturbance on floral display (number of open flowers per inflorescence) caused by opened flowers from different treatments, the total number of rodrigo r. nogueira et al. – tritrophic interaction between plant, pollinators and ants4 approaches (the sum of the number of visits and hesitations) per plant was compared between treatments. flower plant display did not influence the results since there was no significant difference in the number of approaches between treatments (glm: χ2 = 2.699, p = 0.440; fig s1). hypothesis 1 – the presence of ants on flowers negatively influences pollinator behavior to evaluate if pollinators present a different number of visits, and hesitations as well, among treatments, we used a glm with negative binomial error distribution controlling for overdispersion. treatments were fit as fixed factor and the total number of visits or hesitations per plant as count response variable. to evaluate the proportion of visited flowers per total flowers selected per plant, we used a glm with beta distribution and “logit” link function. treatments were considered as fixed factor and proportion of visits as the response variable. the results of this test (proportion of visited flowers per total flowers selected per plant) show the real impact that ants have on pollinators after a first visit. after landing on a flower with ant, the pollinator may not visit another flower in the same inflorescence due to ant presence in this first visited flower. to evaluate if pollinators differ in the time spent per flower per treatment, it was used a glm. treatment was considered as fixed factor and the average time spent visiting flowers per plant as the response variable. hypothesis 2 – the presence of ants on flowers decreases plant reproductive success to verify if fruit production differs between treatments, first the number of fruits produced was divided by the number of selected flowers (fruit proportion). then, it was used a glm with beta distribution and “logit” link function. treatments was used as fixed factors and the fruit proportion per plant as the response variable. since we found a significant difference between the number of visits and the number of fruits produced among groups (see results), we then fit a glm with poisson distribution, evaluating the influence of the number of visits (predictor variable) in the number of fruits produced (response variable). results hypothesis 1 – the presence of ants on flowers negatively influences pollinator behavior there was a significant difference in the number of visits between treatments (glm: χ2 = 29.553, p < 0.001, fig 2a). the aa group presented the lowest number of visits when compared to the other groups. the pc and wa groups had the highest number of visits, but did not differ statistically amongst themselves. the control group placed in the middle (table 1), presenting more visits than the aa group and fewer visits than the wa and pc groups, but with no statistical difference between wa and pc (fig 2a). mean ± se number of visits number of hesitations proportion of visits time (sec) proportion of fruits control 7.33 ± 1.31 1.25±0.44 0.74±0.10 4.29±0.83 0.70±0.05 artificial ants 1.08 ± 0.84 6.41±1.17 0.12±0.08 2.68±0.52 0.32±0.04 plastic circles 11.33 ± 3.38 0.00±0.00 0.99±0 5.55±0.76 0.64±0.09 without ants 9.91 ± 1.85 0.00±0.00 0.99±0 6.66±0.94 0.63±0.08 table 1. mean and standard error of each variables per treatment. the number of hesitations also showed a significant difference between treatments (glm: χ2 = 109.28, p < 0.001, fig 2b). the aa group had the highest number of hesitations, differing statistically from the other three treatments. on the other hand, the control, pc and wa groups did not present significant difference between them (fig 2b). the proportion of visits showed a significant difference between treatments (glm: χ2 = 38.522, p < 0.001, fig 2c). aa group was the least visited, statistically differing from the other three groups (fig 2c). the control, pc and wa did not statistically differ among themselves. the time spent visiting flowers statistically differed between treatments (glm: χ2 = 14.378, p < 0.01, fig 2d). the aa group differed from control, pc and wa, showing that pollinator spent less time when ants are constantly present than in control, pc and wa treatments (fig 2d). however, control, pc and wa groups did not differ from each other (fig 2d). hypothesis 2 – the presence of ants on flowers decreases plant reproductive success the proportion of fruits produced significantly differed between treatments (glm: χ2 = 13.329, p < 0.01, fig 3). the aa group showed the lowest production of fruits, which significantly differed from the other three groups. control, wa, and pc groups showed a similar proportion of fruits produced. (fig 3). we found a positive influence of the number of visits in the number of fruits produced (glm: χ2 = 11.753, p < 0.001, fig 4). sociobiology 68(4): e7259 (december, 2021) 5 discussion this study showed that artificial ants present on flowers have negative impacts on flower visitors’ behavior of b. malifolia, resulting in reduced fruit production; however, with no significant difference for the control group (natural conditions). in particular, the results demonstrated that artificial ants are responsible for a significant reduction in number and proportion of visits, time spent per flower by floral visitors, and a reduction in the number of fruits produced. although there was no significant difference in the variables analyzed between the control group and the isolation group, the considerable negative impact of artificial ants demonstrates that the ants’ visual cue alone is enough to drive away possible pollinators and negatively affect the fruiting of plants. the negative effects caused by artificial ants in this study is related to ants’ display, which is interpreted as threat to some visually oriented animals (aguirre-jaimes et al., 2018; dáttilo et al., 2016), such as bees. the fact that ants tend to attack all plant visitors and do not distinguish between herbivores and pollinators (tsuji et al., 2004; willmer et al., 2009) means that the presence of ants is an important factor for floral visitors (assunção et al., 2014; barônio & delclaro, 2017; galen, 1999; gonzálvez et al., 2013; ibarraisassi & oliveira, 2017; junker et al., 2007; tsuji et al., 2004; wagner, 2000; willmer et al., 2009). the first effect caused by ants on floral visitors is the reduction in their floral visit frequency and an increase in their number of hesitations (aguirre-jaimes et al., 2018; assunção et al., 2014; junker et al., 2007; sousa-lopes et al., 2020). a study developed by assunção et al. (2014) in the same study area showed negative effects of artificial ants on visitors’ behavior in heteropterys pteropetala, decreasing frequency of visits and fructification. fig 2. number of visits (a), number of hesitations (b), proportion of visits (c), and time spent per flower (d). the boxes represent mean (horizontal bars), maximum and minimum, raw data (points), and “violin plot” based on kernel density function. letters represent statistic difference among treatments by estimated marginal means. fig 3. proportion of fruits production among treatments. the boxes represent mean (horizontal bars), maximum and minimum, raw data (points), and “violin plot” based on kernel density function. letters represent statistic difference among treatments by estimated marginal means. rodrigo r. nogueira et al. – tritrophic interaction between plant, pollinators and ants6 similar results were also reported by barônio and del-claro (2017), who showed negative effects of natural ants on bee behavior in the same plant studied, b. malifolia, and in banisteriopsis campestris. however, in barônio and delclaro’ (2017) study, there was no guarantee of the presence of the image of an ant at the time that bees were visiting flowers. the authors used only two treatments manipulating the presence and absence of natural ants (control and isolation). in our study, as plastic ants were fixed, there was always an image of an ant at the time of visitation, even if it did not move. this shows something unprecedented in relation to the study by barônio and del-claro (2017), testing how the image of ants affects pollination. in addition, barônio and del-claro (2017) did not bag flowers before and after field observations, as our study did, guaranteeing the result of pollination after the visitation. finally, our study quantified the time of visitation in all groups used in the study, which was done only indirectly in the study by barônio and del-claro (2017). with all these improvements in the methodology of our study, we have significantly increased our understanding of how ant image cues can be important for pollinators behavior and plant pollination. on the other hand, ants can have no or positive effects on pollination of plants. for instance, almeida and figueiredo (2003) showed that in certain plant species, ants do not interfere with the activity of pollinators, and may even have a positive impact on pollination rates. in another study, santos and leal (2019) also found no negative impact of visiting ants of turnera subulataem on pollination rates or on plant reproductive success, suggesting that the ecological costs of the presence of ants may depend on the characteristics of the pollination system of the plant, being higher in plants that depend on pollinators more sensitive to ants (that demonstrate more hesitation behavior). finally, holland et al. (2011) showed that the presence of ants was beneficial to the pollination rates of pachycereus schottii, a species of sonoran desert cactus, when the abundance of ants in plants was higher. in this context, the results of these studies together with ours show how context-dependent the outcomes of this ant-plant-pollinator system can be. besides the number of visits and hesitations, the proportion of visits (flowers visited/total experimental flowers) can be an important variable for plant fitness (fig 4). studying the ant effects on flowers, altshuler (1999) showed that pollinators that were dissuaded by ants were discouraged to make successive visits to the next flowers on the same plant individual. as a result, even if floral visitors initially visit a flower on a plant, they may tend to concentrate their subsequent visits on other plant individuals without ants. in this way, the remaining flowers on the plant or inflorescences are unlikely to be visited, which can lead to a reduction to fruit set in individuals with ants on flowers (romero & koricheva, 2011). in our study, the aa group had the lowest proportion of visits, statistically different from the other three groups. fig 4. positive influence of the number of visits made by pollinators on the number of fruits produced in banisteriopsis malifolia. glm: χ2 = 11.753, p < 0.001. this result reinforces the impact that visual clues from plastic ants can have on plant pollination rates, causing pollinators look for plants without these visual clues. visitors spent in average less time per flower in aa than in the other three treatments. time is another important variable related to plant fitness, leading to a decrease in pollen removal and fruit set (calixto et al. under review) or an increase in cross-pollination, which can lead to an increase of fruit set (villamil et al., 2020). for instance, calixto et al. (under review) showed that more aggressive ants can significantly decrease the time spent by pollinators when visiting flowers, resulting in a significant decrease of fruits produced in qualea multiflora (vochysiaceae). on the other hand, aguirre-jaimes et al. (2018) showed that the effective pollinator of vigna luteola (fabaceae) spent less time visiting flowers in order to avoid ant predation, which might result in an increase of fruit set; and villamil et al. (2020) observed in turnera velutina (passifloraceae) that ants decreased pollinator foraging time and flower visit duration, but increased outcrossing rates. given that, it seems that the time spent by pollinators can have different effects depending on the system that they are involved, resulting in negative, neutral, or positive effects to plants (gonzálvez et al., 2013; sousa-lopes et al., 2020). artificial ants can negatively interfere with the proportion of fruits. this is likely a direct consequence derived from the other three effects (reduction of number of visits, proportion of visits, and time of visit) caused by the presence of artificial ants on flowers. similarly, as found by other studies, foraging ants on flowers are responsible to reduce fruit and seed set (assunção et al., 2014; barônio & del-claro, 2017; ibarra-isassi & oliveira, 2017). for instance, ness (2006) showed that ants interfered with pollinator behavior and not only reduced the number of fruits, but also the fruit and seeds weight. sociobiology 68(4): e7259 (december, 2021) 7 the ant species used as model for designing artificial ants (camponotus crassus) is often found foraging on b. malifolia, as well as on other efn-bearing plants (lange et al., 2019, 2017), protect them from herbivore attack (calixto et al., 2021a), resulting in a positive effect for plants. however, due to their presence and aggressive behavior towards pollinators, these ants can cause negative effects on pollinator behavior, leading to some cost of the mutualism. nonetheless, it is not clear to what extent or how the context can change the outcomes of these interactions. for instance, gonzálvez et al. (2013) showed that weaver ants deter less effective pollinators of melastoma malabathricum flowers, attracting more xylocopa bees, the more effective pollinators, ultimately resulting in a higher number of fruits produced. this result from gonzálvez et al. (2013) can be one of the explanations for the higher number of fruits produced in control compared to aa treatment. larger bees like bombus and xylocopa represent the main pollinators of b. malifolia and usually are not affected by c. crassus presence (barônio & del-claro, 2017; cembrowski et al., 2014; junker et al., 2007; calixto et al. under review). it was also observed the presence of trigona bees, smaller than bombus and xylocopa, which are considered the most effective pollinators of b. malifolia. the difference in behavior between bees of different sizes in relation to artificial ants was not analyzed in this study. however, barônio and del-claro (2017) verified the difference in the behavior of bees of different sizes in relation to natural ants. given that, we suggest that c. crassus might be deterring other smaller bees that could not pollinate the plant, as shown in our fig 2, but “allowing” visits by larger bees. however, it is also important to take into account that the fact that bees visit fewer flowers in the same individual and then move to another can favor the rate of cross-pollination. conclusion the presence of artificial ants on a flower could negatively affect plant pollination rates, reducing the frequency of visits, increasing the frequency of hesitations, reducing the time spent by the pollinators in the flower, and decreasing fruiting rates, affecting the competitive ability of plants, an essential factor in an environment that is rapidly being degraded, such as the cerrado. this demonstrates the importance that visual cues from a predatory ant on the plant can have on the behavior of pollinating bees, and ultimately on plant fitness. studies such as this, which investigate protective mutualisms and their costs, are needed to increase our understanding of multi-tritrophic interactions and their role in ecosystem biodiversity (bronstein, 2021). acknowledgments we thank the clube de caça e pesca itororó de uberlândia for providing the space where this work was done, the universidade federal de uberlândia (ufu) for the support and structure offered during the research, rosana for her support and assistance in transportation, and the coordenação de aperfeiçoamento de pessoal de nível superior (capes, finance code 001) for the financial support (rrn, dfbs, esc). funding sources this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) – finance code 001; and conselho nacional de desenvolvimento científico e tecnológico (cnpq pq kdc). references aguirre-jaimes, a., dáttilo, w., rodríguez-morales, d., canchola-orozco, s., cocoletzi, e., coates, r., angeles, g. 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(2009). floral volatiles controlling ant behaviour. functional ecology, 23: 888-900. doi: 10.1111/j. 1365-2435.2009.01632.x figure supplementary s1: number of approaches among treatments (glmm: χ2 = 1.300, p = 0.728). the boxes represent mean (horizontal bars), maximum and minimum, raw data (points), and “violin plot” based on kernel density function. letters represent statistic difference among treatments by estimated marginal means. _heading=h.1fob9te _heading=h.2et92p0 _heading=h.tyjcwt 19 a newly recorded genus and species, harpagoxenus sublaevis, from china with a key to the known species of harpagoxenus of the world (hymenoptera: formicidae) by zheng-hui xu1 abstract the newly recorded genus and species to china, harpagoxenus sublaevis (nylander), is reported from da hinggan ling, northeastern china. diagnosis of the genus and description of the species are provided based on a chinese specimen. a key to the 3 known species of the genus of the world is prepared based on the worker caste. key words: hymenoptera, formicidae, harpagoxenus, new record to china, taxonomy introduction the ant genus harpagoxenus forel, 1893, which is a very small genus in the subfamily myrmicinae (bolton 1994, 1995) is distributed in the palaearctic and nearctic regions . mayr (1861) established the genus tomognathus based on the type-species myrmica sublaevis nylander, 1849. later, tomognathus mayr, 1861, was recognized as a junior homonym of tomognathus agassiz, 1850, in the class pisces. therefore, forel (1893) proposed harpagoxenus as a replacement name for tomognathus mayr, 1861. currently, only 3 valid species of harpagoxenus forel are recognized in the world. myrmica sublaevis nylander was the first species moved into the genus. the second species, myrmica hirtula nylander, was also moved into the genus, but has been revised as a junior synonym of h. sublaevis (nylander) by mayr (1861) and radchenko (2007) respectively. the third species of the genus, h. canadensis, was described from canada by m.r. smith (1939) based on the queen, and the worker caste of the species was described by gregg (1945) afterwards. the forth species of the genus, h. zaisanicus, was described from 1 key laboratory of forest disaster warning and control in yunnan province, college of forestry, southwest forestry university, kunming, yunnan 650224, china, e-mail: xuzhenghui1962@163. com 20 sociobiolog y vol. 59, no. 1, 2012 mongolia by pisarski (1963). in addition, a subspecies of the genus, h. sublaevis caucasicus, was described from georgia by arnol’di (1968). in the ant species-diversity investigation of da hinggan ling, northeastern china, the palaearctic species, h. sublaevis (nylander), was found in mohe county, heilongjiang province. both the genus and species are newly recorded in china, the genus and species are reported in this paper based on a chinese specimen. a key to the 3 known species of the genus of the world is provided based on the worker caste. materials and methods the worker caste of h. sublaevis (nylander) was collected by the searchcollecting method. descriptions and measurements were made under a xtb-1 stereo microscope with a micrometer. illustrations were made under a motic-700z stereo microscope with illustrative equipment. standard measurements and indices are as defined in bolton (1987), in addition, ed is included: tl-total length: the total outstretched length of the ant from the mandibular apex to the gastral apex. hl-head length: the length of the head proper, excluding the mandibles, measured in a straight line from the mid-point of the anterior clypeal margin to the mid-point of the occipital margin, in full-face view. in species where the occipital margin or the clypeal margin is concave, the measurement is taken from the mid-point of a transverse line spanning the anteriormost or posteriormost projecting points, respectively. hw-head width: the maximum width of the head in full face view, excluding the eyes. ci-cephalic index = hw×100 / hl. sl-scape length: the maximum straight line length of the antennal scape excluding the basal constriction or neck close to the condylar bulb. si-scape index = sl×100 / hw. ed-eye diameter: the maximum diameter of the eye. pw-pronotal width: the maximum width of the pronotum in dorsal view. al-alitrunk length: the diagonal length of the alitrunk in profile view from the point at which the pronotum meets the cervical shield to the posterior base of the metapleuron. 21 xu, z-h. — a new record of harpagoxenus sublaevis in china all measurements are expressed in millimeters. the specimen is deposited in the insect collection, southwest forestry university (swfu), kunming, yunnan province, china. key to known species of harpagoxenus of the world based on the worker caste 1. in full face view, anterior margin of clypeus narrowly deeply concave in the middle. in profile view, propodeal spines long and slender, about as long as declivity (figs. 1-3) (canada) ................................... h. canadensis smith in full face view, anterior margin of clypeus widely shallowly concave along the margin. in profile view, propodeal spines short and acute, distinctly shorter than declivity .........................................................................................2 2. in full face view, occipital margin straight. in profile view, petiolar node narrow, anterodorsal corner prominent and rightly angled. subpetiolar process indistinct (figs. 4-7) (mongolia) ................. h. zaisanicus pisarski figs. 1-3: worker of harpagoxenus canadensis smith. 1. head and body in profile view; 2. head in full face view; 3. alitrunk, petiole, and postpetiole in dorsal view. (drawn from the images of antweb, california academy of sciences) figs. 4-7: worker of harpagoxenus zaisanicus pisarski; 4. alitrunk, petiole, and postpetiole in profile view; 5. head in full face view; 6. antenna in dorsal view; 7. alitrunk, petiole, and postpetiole in dorsal view. (cited from pisarski, 1963, slightly modified) 22 sociobiolog y vol. 59, no. 1, 2012 in full face view, occipital margin weakly concave. in profile view, petiolar node thick, anterodorsal corner bluntly prominent. subpetiolar process distinct, anteroventral corner toothed (figs. 8-11) (china: heilongjiang province; finland, slovak, georgia) ...................... h. sublaevis (nylander) genus harpagoxenus forel tomognathus mayr, 1861: 56. type-species: myrmica sublaevis, by monotypy. [ junior homonym of tomognathus agassiz, 1850: 376 (pisces).] harpagoxenus forel, 1893: 167. replacement name for tomognathus mayr, 1861: 56. diagnosis of worker: body small. head nearly rectangular. mandibles broad, triangular to trapezoidal, outer margin roundly convex, masticatory margin edentate. clypeus very short, without longitudinal carinae, the posteriorly extending portion broader the frontal lobes, anterior margin with paired hairs. frontal carinae long, well surpassed eyes, antennal scrobes distinct, antennal insertions completely concealed by frontal lobes. antennae short, 11-segmented, antennal clubs large, with 5 segments. eyes developed, located slightly before the mid-points of the lateral sides of the head. ocelli absent. palpal formula 5, 3. in profile view, promesonotum relatively high, promesonotal suture reduced, metanotal groove shallowly depressed. propodeum relatively low, with a pair of teeth or spines. propodeal spiracles small and circular, close to anterior middle of the lateral sides. propodeal lobes short, rounded apically. metapleural gland bullae large, orifices rounded. legs relatively short, ventral faces of femora longitudinally depressed, apices of hind tibiae without spurs. basal tarsi about as long as the other 4 tarsi together. claws simple. petiole with developed erect node, without anterior peduncle, subpetiolar process usually developed. postpetiolar node roundly convex, subpostpetiolar process tooth-like. pilosity abundant. taxonomic position: myrmicinae: formicoxenini. geographical distribution: palaearctic and nearctic regions. harpagoxenus sublaevis (nylander) (figs. 8-11) myrmica sublaevis nylander, 1849: 33 (w.) finland. adlerz, 1896: 62 (q.m.); viehmeyer, 1906: 58 (q.m.). 23 xu, z-h. — a new record of harpagoxenus sublaevis in china tomognathus sublaevis (nylander): mayr, 1861: 56. harpagoxenus sublaevis (nylander): forel, 1893: 167. worker: tl 4.4, hl 1.20, hw 1.00, ci 83, sl 0.60, si 60, ed 0.23, pw 0.65, al 1.23 (1 individual observed). in full face view, head rectangular, distinctly longer than broad. occipital margin weakly concave, occipital corners rounded. lateral sides nearly parallel, slightly convex behind eyes, and slightly concave before eyes. mandibles trapezoidal, masticatory margins edentate. clypeus without longitudinal carinae, anterior margin widely shallowly concave along the margin, anterolateral corners bluntly prominent. frontal carinae weakly divergent backward, about as long as antennal scapes. antennae 11-segmented, scapes concealable in the antennal scrobes; apices of scapes reach to 3/5 of the distance from antennal sockets to occipital corners; antennal clubs 5-segmented, decreased in segment length from apex to base. eyes relatively large, located slightly before the mid-points of the lateral sides of the head. in profile view, promesonotum relatively high, promesonotal suture slightly depressed. mesonotum weakly convex. metanotal groove moderately depressed. propodeum relatively low, dorsum evenly convex, posterodorsal corner with a pair of triangular teeth; declivity weakly depressed, about 1/2 length of dorsum; propodeal lobes short and small, rounded apically. petiolar node roughly trapezoidal, both anterior and posterior faces steeply sloped, dorsal face convex; anterodorsal corner bluntly prominent, posterodorsal corner rounded. subpetiolar process narrowly rectangular, anteroventral corner toothed. dorsum of postpetiole roundly convex, slightly inclined figs. 8-11 worker of harpagoxenus sublaevis (nylander); 8. head and body in profile view; 9. head in full face view; 10. mandible in dorsal view; 11. body in dorsal view. 24 sociobiolog y vol. 59, no. 1, 2012 forward, subpostpetiolar process toothed anteroventrally. in dorsal view, petiolar node transverse, length : width = 3:4, anterior margin weakly convex, lateral margins roundly convex, posterior margin straight; postpetiolar node nearly rhomboid, length : width = 1:2, lateral sides bluntly angled. mandibles relatively smooth, very weakly finely punctured. clypeus smooth and shining. dorsum of head densely longitudinally striate, lateral sides of head sparsely longitudinally striate and densely finely punctured. occipital margin and occipital corners smooth and shining. alitrunk, petiole, and postpetiole densely finely punctured, interfaces appear as fine reticulations; in addition, dorsum of alitrunk finely longitudinally striate, dorsa of petiolar node and postpetiolar node sparsely transversely striate. gaster smooth and shining. dorsa of head and body with abundant erect to suberect hairs and sparse decumbent pubescence. antennal scapes and hind tibiae with sparse decumbent hairs and dense depressed pubescence. color blackish brown. mandibles, scapes, and legs brownish yellow. gaster brownish black. specimen observed: 1 worker, china: heilongjiang province, mohe county, tuqiang town, san’erzhixian, 690m, forage on the ground in secondary conifer-broadleaf mixed forest, 2009.vii.30, zheng-hui xu leg., no. a09-2077. discussion: the worker specimen from china conforms well to the original description and images of worker of h. sublaevis (nylander) from slovak (antweb, casent 0178772, california academy of sciences). but with anterolateral corners of clypeus more prominent; in profile view, anterodorsal corner of pronotum more convex; propodeal lobes relatively narrower; anterodorsal corner of petiolar node bluntly prominent instead of roundly prominent; color comparatively darker. acknowledgments this study is supported by national natural science foundation of china (no. 30870333), rapid assessment program of biodiversity organized by peking university, and the key subject of forest protection of yunnan province. i thank the california academy of sciences (usa) for permission to use the images of harpagoxenus canadensis smith from the antweb. 25 xu, z-h. — a new record of harpagoxenus sublaevis in china references adlerz, g. 1896. myrmekologiska studier. 3. tomognathus sublaevis mayr. bihang till kongl. svenska vetenskaps-akademiens handlingar 21 (4): 1-76. arnol'di, k.v. 1968. vazhnye dopolneniya k mirmekofaune sssr i opisanie novykh form. zoologicheskii zhurnal 47: 1800-1822. bolton, b. 1987. a review of the solenopsis genus-group and revision of afrotropical monomorium mayr. bulletin of the british museum (natural history) (entomolog y) 54: 263-452. bolton, b. 1994. identification guide to the ant genera of the world. harvard university press, 222 pp. cambridge, massachusetts. bolton, b. 1995. a new general catalogue of the ants of the world. harvard university press, 504 pp. cambridge, massachusetts. forel, a. 1893. sur la classification de la famille des formicides, avec remarques synonymiques. annales de la société entomologique de belgique 37: 161-167. gregg , r .e. 1945. the worker caste of harpagoxenus canadensis smith. canadian entomologist 77: 74-76. mayr, g. 1861. die europäischen formiciden. 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fries et al., 2013) or to quantify nucleic acids of the species that are present (cilia et al., 2018). abstract nosemosis is an important disease that affects honey bees (apis mellifera lineu), caused by obligate intracellular parasites, nosema apis and/or nosema ceranae. since the initial detection of n. ceranae in a. mellifera coincided with recent large-scale losses of bee colonies worldwide, the impacts of this parasite under field conditions are of great interest. here we test two hypotheses, the first one, whether the climatic variables (temperature, air humidity and precipitation) influence the intensity of infection of the microsporidium nosema spp. in africanized honey bees (apis mellifera), and the second, whether the local of hive installation (outdoor or roofed) influences the intensity of infection of these spores in africanized honey bees. between august 2013 and august 2016, samples of africanized bees were collected weekly from 20 colonies, of which ten were located in an open area (outdoor apiary) and ten under a roof on a concrete floor (roofed apiary). n. ceranae was the only species present. the type of apiary did not influence (p > 0.05) the number of spores of n. ceranae in africanized bees. however, the infection intensities of the roofed apiary colonies were lower in the autumn. regarding the meteorological parameters, there was a negative correlation between the winter infection intensities and the minimum temperature in the roofed apiary and the humidity in the outdoor apiary. the highest infection intensities occurred in both apiaries in the spring and summer, which may be related to higher pollen production. on average, the infection intensity was 16.19 ± 15.81 x 105, ranging from zero to 100.5 x105, and there were no records of collapse during the three years. sociobiology an international journal on social insects l guimarães-cestaro1, ts maia2, r martins2, mltmf alves3, ip otsuk4, d message5 ew teixeira3 article history edited by evandro n. silva, uefs, brazil received 02 january 2020 initial acceptance 05 july 2020 final acceptance 08 august 2020 publication date 30 september 2020 keywords fungus, nosemosis, microsporidium. corresponding author lubiane guimarães cestaro av. prof. manoel césar ribeiro, 1920 santa cecília, cp: 07, cep: 12411010 pindamonhangaba, são paulo, brasil. e-mail: lubi.guimaraes@gmail.com initially, the only etiological agent of this pathology in apis mellifera bees was n. apis. however, the presence of n. ceranae (higes et al., 2006; huang et al., 2007) was also observed in the mid-2000s, initially in apis cerana (fries et al., 1996). it is believed, however, that n. ceranae began infecting a. mellifera bees a few decades ago, because analyses of historical samples have detected its presence in several places, such as uruguay (invernizzi et al., 2009), brazil (teixeira et al., 2013), italy (ferroglio et al., 2013), usa (fell et al., 2015) and méxico (guzman-novoa et al., 2011; guerreiro-molina et al., 2016) in decades prior to its first 1 postgraduate program in entomology, department of entomology, federal university of viçosa, minas gerais, brazil 2 cnpq scholarship (pibic): undergraduate student in biological sciences, unitau, taubaté-sp, brazil 3 laboratory specialized in honey bee health, biological institute/apta/saa, sp, brazil 4 institute of animal science/apta/saa, sp, brazil 5 pvns / capes fellow, graduate program in animal science, ufersa, mossoró-rn, brazil research article bees nosema ceranae (microsporidia: nosematidae) does not cause collapse of colonies of africanized apis mellifera (hymenoptera: apidae) in tropical climate sociobiology 67(3): 408-416 (september, 2020) 409 detection in bees of the genus apis in 1996 (fries et al., 1996). currently, the species n. ceranae is considered to be among the most prevalent infecting bees globally (fries, 2010; higes et al., 2010a; gisder et al., 2010; lecoq et al., 2016; paxton et al., 2007; higes et al., 2008; emsen et al., 2016). n. ceranae can infect all members of the colony, be they workers, drones or queens (alaux et al., 2011), including larvae (eiri et al., 2015). this microsporidium infects the bees through the ingestion of spores from contaminated sources (pollen and water) and is eliminated in the feces (fries et al., 1996). the activities of cleaning and feeding in the colony bring new sources of infection (fries, 2010; higes et al., 2010b; 2013; martin hernandez et al., 2018). the spores germinate in the middle intestine, where the epithelial cells are infected, among other consequences causing the suppression of the immune system (antúnez et al., 2009), energy stress (mayack & naug 2009; castelli et al., 2020), acceleration of age polyethism (lecoq et al., 2016), with a consequent decrease in longevity, as well as reduction of honey production and pollination (botías et al., 2013). infection with this microsporidium has been associated with colony collapse disorder (ccd) in parts of europe (higes et al., 2009; martín-hernández et al., 2007), but not in central europe (gisder et al., 2010), south america (invernizzi et al., 2009; pires et al., 2006), and united states (cox-foster et al., 2007; chen et al., 2008; guzman-novoa et al., 2016). according to fries (2010), the impact of this parasite is different depending on the environment, and research has found conflicting virulence data, either for individual bees and colonies, presenting distinct seasonal patterns between areas. in brazil, n. ceranae is widely distributed throughout the country, with no pattern of infection intensity observed during the year (teixeira et al., 2013; pires et al., 2016). their presence was first reported by klee et al. (2007), but later studies have demonstrated their presence since the 1970s (teixeira et al., 2013), although no studies have so far evaluated the consequences of this infection in the long term. in tropical countries, there is little information about infections and seasonal patterns of n. ceranae (guerrero-molina et al., 2016; fleites-ayil et al., 2018). since detection of n. ceranae in a. mellifera coincided with the recent large-scale loss of bee colonies worldwide, data on pathology and management are of significant interest (fries et al., 2013). epidemiological assessments and studies conducted in this sense, in brazil, may help to elucidate the causes of colony decline and sudden losses, since there is possible involvement of pathogens and parasites in this phenomenon (teixeira et al., 2008a; 2008b; 2012; 2013; santos et al., 2014; schwarz et al., 2014), including n. ceranae. here we test two hypotheses, the first one, whether the climatic variables (temperature, air humidity and precipitation) influence the intensity of infection of the microsporidium nosema spp. in africanized honey bees (apis mellifera), and the second, whether the local of hive installation (outdoor or roofed) influences the intensity of infection of these spores in africanized honey bees. material and methods from august 2013 to august 2016, samples containing about thirty africanized honey bees were weekly collected from 20 colonies in two apiaries of the honey bee health laboratory (lasa) of the biology institute of the são paulo state agribusiness technology agency (ib/apta), in pindamonhangaba, sp. langstroth hives were installed at easels (height of 50 cm), ten in the open air (here called “outdoor apiary”) and ten under roof (3.2 m high) (here called “roofed apiary”). the spore collections were performed according to teixeira and message (2010): the entrance to the hive´s was closed with a strip of common foam to allow the collection of honey bees arriving from the field. these honey bees were swept with a common brush (paint brush) 4 to 5 cm wide into a universal type plastic bottle, containing 70% alcohol. at least 30 bees were collected per hive. the counts of nosema spp. were performed according to cantwell (1970). for confirmation of the nosema spp., composite samples were prepared from each beehive containing bees belonging to each weekly collection. preparation of the samples for dna extraction was done according to teixeira et al. (2013) and for identification of the microsporidium species, the samples were submitted to duplex pcr reactions as described by guimarães-cestaro et al. (2016). data on temperature, air humidity and precipitation were obtained from the polo do vale do paraíba meteorological station in pindamonhangaba, sp, along with temperature and relative humidity obtained from a thermohygrometer installed in the covered apiary. in addition, pollen production data were obtained from september 2015 to august 2016 with pollen collectors of the intermediate type to evaluate the effect of this variable on the level of infection of the pathogen. although meteorological data and pollen quantity were collected daily, a weekly average was performed for the assessments (table 1 and 2). outdoor apiary spores air humidity precipitation max. temperature min. temperature autumn 360 30 33 36 36 spring 380 36 26 38 38 summer 390 26 27 39 39 winter 390 26 39 39 39 total 1520 118 125 152 152 table 1. sampling number (n value) for each studied variable on outdoor apiary: spores (honey bee samples), air humidity, precipitation, maximum temperature, and minimum temperature. l guimarães-cestaro et al. – n. ceranae does not cause collapse of honey bees in tropical climate in open or roofed apiaries410 a completely randomized factorial experimental design was used (2 apiary types x 4 seasons), with 10 replicates (represented by hives). the data were analyzed through the mixed procedure of the sas (statistical analysis system) program (sas institute, 2001), to determine the matrix structure of variance and covariance. the significance level adopted for the analysis of variance was 5%. the apiary averages were compared by the f-test and the seasonal differences by the tukey-kramer test. pearson and spearman correlations between spore fluctuation and climatic variables (maximum and minimum temperature, humidity, and precipitation) and pollen production in each season of the year were calculated. results all the analyzed samples presented positive results for the species nosema ceranae, while nosema apis was not detected (fig 1). table 2. sampling number (n value) for each studied variable on roofed apiary: spores (honey bee samples), air humidity, precipitation, maximum temperature, and minimum temperature. covered apiary spores air humidity precipitation max. temperature min. temperature autumn 337 22 33 22 22 spring 380 22 26 22 22 summer 382 26 27 26 26 winter 364 26 39 26 26 total 1463 96 125 96 96 season outdoor apiary covered apiary mean spring 16.86±17.40aa 21.51±16.39aa 19.19±3.29 summer 14.91±15.79aa 19.02±17.03aa 16.96±2.91 autumn 14.01±14.18aa 11.09±11.70ab 12.55±2.06 winter 14.34±15.99aa 18.21±15.03aa 16.38±2.74 mean 15.03±1.28 17.46±4.47 †means followed by different lowercase letters in the rows and upper case in the columns differ from each other by the tukey-kramer test (p ≤ 0.05). table 3. intensity of natural infection of nosema ceranae (average number of spores/bee x105) in hives located in open air and covered apiaries from august 2013 to august 2016 in relation to the seasons of the year. fig 1. agarose gel (2%) showing duplex pcr products (nosema apis/nosema ceranae). m: marker. 1-6: samples infected with nosema ceranae. c +: positive control of nosema apis (321bp) and nosema ceranae (218bp). c-: negative control. although outdoor hives had a lower mean number of spores than covered hives, n. ceranae infection intensities did not differ (p=0.0706) between the place of installation of the apiaries and the seasons, except for autumn in comparison with the other seasons in the colonies of the covered apiary (p<0.0001) (table 3). the meteorological data were correlated with the fluctuation of the number of spores per bee (p<0.0001) only when considering season. in winter, the minimum temperature had a negative correlation with the number of spores in the outdoor apiary (fig 2) and the humidity had a negative correlation with the number of spores in the covered apiary (fig 3). precipitation data had no influence on the intensity of infection (fig 4). the highest pollen production occurred in the summer and spring, when spore prevalence in bees was also high (fig 5), without, however, presenting a significant difference (table 4). season outdoor apiary r value (p value) covered apiary r value (p value) spring -0,4301 (0,1726) -0,4348 (0,1376) summer -0,4447 (0,1705) -0,2662 (0,4289) autumn 0,1043 (0,7345) -0,1505 (0,6235) winter -0,4215 (0,1724) -0,3537 (0,2593) table 4. pearson’s correlation coefficient between pollen production and spore fluctuation in each season evaluated. discussion the presence of nosema ceranae and absence of nosema apis corroborates the results obtained by teixeira et al. (2013), santos et al. (2014) and guimarães-cestaro et al. (2017a, 2017b), indicating possible supression of n. ceranae sociobiology 67(3): 408-416 (september, 2020) 411 fig 2. intensity of natural infection of nosema ceranae (mean number of spores/bee x105) and maximum and minimum temperatures in hives located in outdoor apiary and covered apiary from august 2013 to august 2016 in relation to seasons. fig 3. intensity of natural infection of nosema ceranae (mean number of spores/bee x105) and relative air humidity in hives located in open air apiary and covered apiary from august 2013 to august 2016 in relation to seasons. l guimarães-cestaro et al. – n. ceranae does not cause collapse of honey bees in tropical climate in open or roofed apiaries412 in the n. apis species in africanized apis mellifera bees in brazil, as observed in honey bees in europe (klee et al., 2007; paxton et al., 2007). however, it should be considered that reports of interactions between the two species of nosema in bees are contradictory. several authors have observed that in countries with hot summers and mild winters, there is predominance of n. ceranae (higes et al., 2006; klee et al., 2007; martín-hernández et al., 2007), but in countries with cooler and longer winters, n. apis is predominant (gisder et al., 2010; 2017). this can also be related to the presence of n. ceranae and the absence of n. apis in the samples analyzed in the present study and others performed in brazil (teixeira et al., 2013; santos et al., 2014; guimarães-cestaro et al., 2017a; 2017b). fig 4. intensity of natural infection of nosema ceranae (mean number of spores/bee x105) and precipitation in hives located in outdoor apiary and covered apiary from august 2013 to august 2016 in relation to the seasons. fig 5. intensity of natural infection of nosema ceranae (average number of spores/bee x105) and pollen production in hives located in open air apiary and covered apiary from august 2015 to august 2016 in relation to the seasons of the year. sociobiology 67(3): 408-416 (september, 2020) 413 it is believed that cold weather is one of the limiting factors of n. ceranae distribution (fries, 2010; gisder et al., 2010; ozkirim et al., 2019), although environmental variables and interspecies competition are important elements to explain the differential prevalence of nosema spp. in different climatic regions (martín-hernandez et al., 2018). in our study, the climatic parameters did not influence (p<0.0001) the number of spores per bee, but considering the seasons, the largest number of spores was observed in the spring and the smallest in the autumn. these results can be explained by the development of the swarm in two different periods of nectariferous secretion that occur at the study site during the year. the first and most important occurs from february to mid-may and the second from midjuly to september (silva et al., 1995). during the main flow, the colonies had a higher population of bees, and with the end of the flowering period (which coincides with autumn), the population decreased, with a substantial decline in the queen’s posture, but without completely ceasing. with the development of a secondary flow, the colonies resumed their growth, with increased posture and population. the lowest infection rate was observed when the colonies’ activity and population were reduced, due to lower temperatures and less availability of pollen, a product that is an important source of contamination by spores of nosema spp. and other bee pathogens (higes et al., 2008; zheng et al., 2014; guimarães-cestaro et al., 2016; teixeira et al., 2018). at the end of winter and early spring, with the increase of comb cleaning for posture and consequent feeding of the larvae, there was higher availability of pollen (fig 5), higher temperatures, in addition to greater wind intensity, which possibly facilitates the spread of spores through the air (sulborska et al, 2019), increasing prevalence of this pathogen. different results (p<0.05) were obtained only for colonies of the covered apiary, which presented negative correlation for minimum winter temperature (14.98 ± 1.58 °c) (fig 2), and for humidity in the open apiary (59.93 ± 10.91) (fig 3). similar data were obtained by chen et al., (2012) in taiwan, where infection by nosema spp. had negative correlation with temperature, and higher spore counts were observed at mean temperature of 15 ºc. these results were associated with an increase in the bee population in the spring (winston ,1987), and are also related to the fact that during this period, there is increased risk of the disease due to spore ingestion as the bees clean the combs and feed the larvae, while the presence of spores decreases in periods of low growth (guerreiro-molina et al., 2016). the type of apiary did not influence (p=0.0706) the number of spores per bee. except during the autumn, in all seasons the hives in the roofed apiary tended to have higher infestation of n. ceranae, but without statistical difference. the hives located in the open and roofed apiaries had, on average, 15.03 ± 1.28 x 105 and 17.46 ± 4.47x105 spores per bee, respectively, values close to those found by santos et al. (2014) (16.5 ± 114 x 105 spores per bee) at this same location, and higher than those obtained in other municipalities of the state of são paulo: 637 ± 36 x 103 (santos et al 2014) and 1070 x 103 (guimarães-cestaro et al 2017b). the highest intensity of infection may be related to the weeklyfrequency and kind of management adopted (equalization of the population, comb exchanges between colonies, supplementation, among others). in the current study, even considering the high infection rates in comparison to the other municipalities of the state of são paulo (santos et al., 2014; guimarães-cestaro et al., 2017a; 2017b), no negative effects were observed that could be associated directly to infection by n. ceranae, and no collapse of colonies occurred during the three years analyzed. this article presents the first data on the infection intensity of n. ceranae during three consecutive years in africanized bees in a tropical climate, allowing to infer that this pathogen does not cause a collapse in colonies frequently assisted of this honey bee hybrid in tropical areas. guerrero-molina et al. (2016), concluded that africanized bees infected with the pathogen suffer moderate effects when compared to european bees in temperate areas. santos et al. (2014) and guimarães-cestaro et al. (2017b) verified the highest prevalence of n. ceranae in the autumn, but it is important to note that in these studies, the collections occurred only once in the season, unlike the current study, where bees were sampled were performed weekly. according to teixeira et al. (2013) and pires et al. (2016), in brazil there is no pattern regarding the sporulation curve in different colonies and in geographically different places, as also mentioned by fries et al. (2010) in the northern hemisphere. the absence of colony collapses may be related with the constant and routinely management of apiaries, which made it possible to identify the early needs, for avoinding the substitution of queens, as consequence of absence or poor posture, as well as the needs of artificial suplementatation, timely offer of new wax foundations, adjustments in order to increase or decrease space according to population development, supplementation, among others. studies carried in cages, with artificial infection by nosema spp. have demonstrated the mortality of bees only a few days after infection (higes et al., 2006; paxton et al., 2007; martín-hernandez et al., 2011; dussaubat et al., 2012). the technical management routinely adopted may have been crucial to allow the colonies to survive and to avoid losses reported in studies under artificial or natural conditions but not as often supervised and kept watch over. acknowledgments we thank carmen l. monteiro and vilma c.v. takada for assistance in the analyses, and cnpq for specific funding (mapa/cnpq, process 2008-0 and apta/pibic program, e.w.t.). author contribution statement lgc did the molecular analysis and helped to wrote the manuscript, rm and tms executed sampling, helped with the l guimarães-cestaro et al. – n. ceranae does not cause collapse of honey bees in tropical climate in open or roofed apiaries414 experimental analysis and the initial drafts of the manuscript, ipo performed the statistical analyses, dm helped to plan the work, ewt and mltmfa were in charge of the assembly and maintenance of the hives and the monthly collection samples, ewt planed and coordinated the work, supervised all the steps of the investigation and wrote the manuscript, all authors collaborated in the revision of the final version of the manuscript. references alaux, c., folschweiller, m., mcdonnell, c., beslay, d., cousin, m., dussaubat, c., brunet, j.l. & le conte, y. 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(2017). long-term temporal trends of nosema spp. infection prevalence innortheast germany: continuousspread of nosema ceranae, an emerging pathogen of honey bees (apis mellifera), but no general replacement of nosema apis. frontiers in cellular and infection microbiology, 7: 1-14. doi:10.3389/ fcimb.2017.00301 guerrero-molina, c., correa-benítez, a., hamiduzzaman, m.m. & guzman-novoa, e. (2016). nosema ceranae is an old resident of honey bee (apis mellifera) colonies in mexico, causing infection levels of one million spores per bee or higher during summer and fall. journal of invertebrate pathology, 141: 38-40. doi: 10.1016/j.jip.2016.11.001 guimarães-cestaro, l., serrão, j.e., message, d., martins, m.f. & teixeira, e.w. (2016). simultaneous detection of nosema spp., ascosphaera apis and paenibacillus larvae in honey bee products. journal of hymenoptera research, 49: 43-50. doi: 10.3897/jhr.49.7061 guimarães-cestaro, l, alves, m.l.t.m.f., message, d, silva, m.v.g.b. & teixeira, e.w. (2017). a scientific note on occurrence of pathogens in colonies of honey bee apis mellifera in vale do ribeira, brazil. apidologie, 48: 384-386. doi: 10.1007/s13592-016-0481-3 guimarães-cestaro, l., alves, m.l.t.m.f., message, d. silva, m.v.g.b & teixeira, e.w. (2017). honey bee (apis mellifera) health in stationary and migratory apiaries. sociobiology, 64: 42-49. doi: 10.13102/sociobiology.v64i1.1183 guzman-novoa, e., hamiduzzaman, m.m., arechavaletavelasco, m.e., koleoglu, g., valizadeh, p. & correa-benítez, a. (2011). nosema ceranae has parasitized africanized honey bees in mexico since at least 2004. journal of apicultural research, 50: 167-169. doi: 10.3896/ibra.1.50.2.09 higes, m., martín, r. & meana, a. (2006). nosema ceranae, a new microsporidian parasite in honeybees in europe. journal of invertebrate pathology, 92: 93-95. doi: 10.1016/j.jip.2006.02.005 higes, m., martín-hernandez, r., botías, c., garrido-bail, o.e., gonzalez-porto, a.v. & barrios l (2008). how natural infection by nosema ceranae causes honeybee colony collapse. environmental microbiology, 10: 2659-2669. doi: 10.1111/j.1462-2920.2008.01687.x higes, m., martín-hernández, r., garrido-bailón, e., gonzálezporto, a.v., garcía-palencia, p., meana, a., del nozal, m j., mayo, r. & bernal, j.l. (2009). honeybee colony collapse due to nosema ceranae in professional apiaries. environmental microbiology reports, 1: 110-113. doi: 10.1111/j.1758-2229. 2009.00014.x higes, m., martín-hernández, r., garrido-bailón, e., gonzálezporto, a.v., meana & a, bernal, j.l. 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(2007). widespread dispersal of the microsporidian nosema ceranae, an emergent pathogen of the western honey bee, apis mellifera. journal of invertebrate pathology, 96: 1-10. doi: 10.1016/j.jip.2007.02.014 lecocq, a., jensen, a.b., kryger, p. & nieh, j.c. (2016). parasite infection accelerates age polyethism in young honey bees. scientific reports, 6: 22042. doi: 10.1038/srep22042 martín-hernández, r., meana, a., garcía-palencia, p., marrin, p., botías, c., garrido-bailón, e., barrios, l. & higes, m. (2009). temperature effect on biotic potencial of honey bee microsporidia. applied and environmental microbiology, 75: 2554-2557. doi: 10.1128/aem.02908-08 martín-hernandez, r., botías, c., barrios, l., martínezsalvador, a., meana, a., mayack, c. & higes, m. 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(2014). spore loads may not be used alone as a direct indicator of the severity of nosema ceranae infection in honey bees apis mellifera (hymenoptera: apidae). journal of economic entomology 107: 2037-2014. doi: 10.1603/ec13520 1175 four new species of the amblyoponine ant genus amblyopone (hymenoptera: formicidae) from southwestern china with a key to the known asian species by zheng-hui xu1 & jiao-jiao chu1 abstract four new species of the amblyoponine ant genus amblyopone erichson, 1842 collected from southwestern china are described, i.e. a. kangba sp. nov., a. zoma sp. nov., a. meiliana sp. nov., and a. awa sp. nov. the queen caste of a. awa sp. nov. is reported. measurements are supplemented to a. octodentata xu based on the newly collected specimens. a key to the 29 known asian species of the genus is provided based on the worker caste. illustrations are provided for each species except for a. quadrata (karavaiev). key words: hymenoptera: formicidae, amblyoponinae, amblyopone, new species, asian species. introduction the amblyoponine ant genus amblyopone erichson, 1842 is widely distributed in the world tropics and temperate zones (bolton 1995). before this study, 72 living species were recorded in the world (bolton 2012). the asian species of the genus were reported respectively by mayr (1879), emery (1895), forel (1900, 1912, 1913), bingham (1903), wheeler & chapman (1925), wheeler (1928), karavaiev (1935), arnol’di (1968), baroni urbani (1978), taylor (1965, 1979), terayama (1987, 1989, 2009), morisita et al. (1989), wu & wang (1992), onoyama (1999), xu (2001, 2006), zhou (2001), imai et al. (2003), and bharti & wachkoo (2011). before this study, 25 species of the genus were recorded in asia. in the ant diversity investigations of southwestern china, 4 new species of amblyopone were collected. the new species are described. the queen caste of a. awa sp. nov. is reported. measurements are supplemented to a. octodentata xu based on the newly collected specimens. 1 key laboratory of forest disaster warning and control in yunnan province, college of forestry, southwest forestry university, kunming, yunnan province 650224, china, e-mail: xuzhenghui1962@163.com 1176 sociobiolog y vol. 59, no. 4, 2012 in order to facilitate the identification of the species of amblyopone, a key to the 29 known asian species is provided based on the worker caste. illustrations are provided for each species except for a. quadrata (karavaiev). materials and methods the worker and queen castes of the new species and a. octodentata xu were collected by the sample-plot method. descriptions and measurements were made under a xtb-1 stereo microscope with a micrometer. illustrations of the new species were made under a motic-700z stereo microscope with illustrative equipment. figures of most species were drawn from the antweb images except for those cited from original descriptions. standard measurements and indices are as defined in bolton (1975), in addition, ml, ed, and al are supplemented: tl-total length: the total outstretched length of the individual, from the mandibular apex to the gastral apex. hl-head length: the straight-line length of the head in perfect full-face view, measured from the mid-point of the anterior clypeal margin to the midpoint of the occipital margin. in species where one or both of these margins is concave, the measurement is taken from the mid-point of a transverse line that spans the apices of the projecting portions. hw-head width: the maximum width of the head in full face view, excluding the eyes. ci-cephalic index = hw×100 / hl. sl-scape length: the straight-line length of the antennal scape, excluding the basal constriction or neck. si-scape index = sl×100 / hw. ml-mandible length: the straight-line length of the mandible measured from apex to the lateral base. ed-eye diameter: the maximum diameter of the eye. pw-pronotal width: the maximum width of the pronotum measured in dorsal view. al-alitrunk length: the diagonal length of the alitrunk in profile view, measured from the point at which the pronotum meets the cervical shield to the posterior basal angle of the metapleuron. 1177 xu, z.-h. & j.-j. chu — four new species of amblyopone from china pl-petiole length: the length of the petiole measured in profile from the anterior process to the posteriormost point of the tergite, where it surrounds the gastral articulation. ph-petiole height: the height of the petiole measured in profile from the apex of the ventral (subpetiolar) process vertically to a line intersecting the dorsalmost point of the node. dpw-dorsal petiole width: the maximum width of the petiole in dorsal view. lpi-lateral petiole index = ph×100/pl. dpi-dorsal petiole index = dpw×100/pl. all measurements are expressed in millimeters. the type specimens are deposited in the insect collection, southwest forestry university (swfu), kunming, yunnan province, china. key to known asian species of amblyopone based on the worker caste 1 antennae 10or 11-segmented .........................................................................2 antennae 12-segmented ......................................................................................5 2 antennae 10-segmented ( japan) (figs. 1-2) .................a. fulvida terayama antennae 11-segmented ......................................................................................3 3 in full-face view, head about as broad as long. mandibles deeply split at third tooth counting from apex. in profile view, subpetiolar process short and broad, roughly square (india) (figs. 3-4) .......... a. pertinax baroni urbani in full-face view, head distinctly longer than broad. mandibles not deeply split at third tooth counting from apex. in profile view, subpetiolar process long and narrow, slender ....................................................................................4 4 in full-face view, anterior clypeal margin with 5 teeth. in profile view, posterodorsal corner of propodeum and anterodorsal corner of petiolar node rounded. subpetiolar process oblique ( japan) (figs. 5-6) ............................ ......................................................................................... a. caliginosa onoyama in full-face view, anterior clypeal margin with 8 teeth. in profile view, posterodorsal corner of propodeum and anterodorsal corner of petiolar node bluntly angled. subpetiolar process horizontal (china: taiwan) (figs. 7-8) ......................................................................................... a. sakaii terayama 5 in full-face view, head nearly square, about as broad as long .......................6 1178 sociobiolog y vol. 59, no. 4, 2012 in full-face view, head elongate trapezoidal, distinctly longer than broad... .............................................................................................................................. 12 6 in full-face view, anterior clypeal margin with 4-6 minute rectangular denticles .......................................................................................................................7 in full-face view, anterior clypeal margin with 10-16 triangular or rectangular denticles .................................................................................................................9 figs. 1-8. 1-2: worker of amblyopone fulvida terayama. 1. head and body in profile view; 2. head in full-face view. (cited from terayama 1987, slightly modified) 3-4: worker of amblyopone pertinax baroni urbani; 3. head and body in profile view; 4. head in full-face view. (cited from baroni urbani 1978, slightly modified) 5-6: worker of amblyopone caliginosa onoyama. 5. head and body in profile view; 6. head in full-face view. (cited from imai et al. 2003, slightly modified) 7-8: worker of amblyopone sakaii terayama; 7. head and body in profile view; 8. head in full-face view. (cited from terayama 1989, slightly modified) 1179 xu, z.-h. & j.-j. chu — four new species of amblyopone from china 7 in profile view, anterodorsal corner of petiolar node bluntly angled; subpetiolar process roughly rectangular, anteroventral corner acutely angled (china: guangxi) (figs. 9-10) .............................................. a. eminia zhou in profile view, anterodorsal corner of petiolar node rounded, anterior face vertical to dorsal face; subpetiolar process triangular or square ................8 8 in full-face view, occipital margin narrowly deeply concave in the middle. in profile view, subpetiolar process triangular (china: hunan) (figs. 1112) ..............................................................................a. rubiginoa wu et wang in full-face view, occipital margin widely weakly concave. in profile view, subpetiolar process roughly square (china: tibet and yunnan) (figs. 1315) ........................................................................................... a. kangba sp. nov. 9 in full-face view, anterior clypeal margin with about 10 denticles, and a large protruding tooth on each side (india) (figs. 16-17) ....a. bellii forel in full-face view, anterior clypeal margin with 12-17 denticles, lateral sides without protruding large teeth ...................................................................... 10 10 in full-face view, anterior clypeal margin with about 17 denticles. eyes larger, with its diameter about as broad as the width of antennal scape (indonesia) (figs. 18-19) ......................................................a. reclinata mayr -in full-face view, anterior clypeal margin with about 12 denticles. eyes figs. 9-12. 9-10: worker of amblyopone eminia zhou; 9. head and body in profile view; 10. head in full-face view. (cited from zhou 2001, slightly modified). 11-12: worker of amblyopone rubiginoa wu et wang ; 11. head and body in profile view; 12. head in full-face view. (cited from wu & wang 1992, slightly modified) 1180 sociobiolog y vol. 59, no. 4, 2012 smaller, with its diameter narrower than the width of antennal scape .. 11 11 in full-face view, anterior clypeal margin with about 12 rectangular denticles. occipital corners rounded (china: taiwan) (figs. 20-21) .............. ...................................................................................................... a. bruni (forel) -in full-face view, anterior clypeal margin with about 12 triangular denticles. figs. 13-21. 13-15: worker of amblyopone kangba sp. nov.; 13. head and body in profile view; 14. head in full-face view; 15. body in dorsal view. 16-17: worker of amblyopone bellii forel; 16. head and body in profile view; 17. head in full-face view. (drawn from antweb images). 18-19: worker of amblyopone reclinata mayr; 18. head and body in profile view; 19. head in full-face view. (drawn from antweb images). 20-21: worker of amblyopone bruni (forel); 20. head and body in profile view; 21. head in full-face view. (drawn from antweb images). 1181 xu, z.-h. & j.-j. chu — four new species of amblyopone from china occipital corners blunt (vietnam) ....................... a. quadrata (karavaiev) 12 anterior clypeal margin with more than 10 denticles, the denticles not divided into lobes ............................................................................................. 13 -anterior clypeal margin with less than 10 teeth or lobes, some teeth maybe divided into lobes ............................................................................................. 15 13 anterior clypeal margin with 10 denticles, and a large triangular tooth on each side (myanmar) (figs. 22-23) ........................................a. feae (emery) -anterior clypeal margin with 12-15 denticles, lateral sides without large triangular teeth ................................................................................................. 14 14 in full-face view, occipital margin weakly concave. anterior clypeal margin figs. 22-30. 22-23: worker of amblyopone feae (emery); 22. head and body in profile view; 23. head in full-face view. (drawn from antweb images) 24-25: worker of amblyopone crenata xu; 24. head and body in profile view; 25. head in full-face view. (drawn from antweb images). 26-27: worker of amblyopone rothneyi forel; 26. head and body in profile view; 27. head in full-face view. (drawn from antweb images). 28-30: worker of amblyopone zoma sp. nov.; 28. head and body in profile view; 29. head in full-face view; 30. body in dorsal view. 1182 sociobiolog y vol. 59, no. 4, 2012 with about 12 denticles. in profile view, anterodorsal corner of petiolar node bluntly angled. posteroventral corner of subpetiolar process acutely toothed (china: yunnan) (figs. 24-25) .................................. a. crenata xu -in full-face view, occipital margin nearly straight. anterior clypeal margin with about 15 denticles. in profile view, anterodorsal corner of petiolar node rounded. posteroventral corner of subpetiolar process rightly angled (india) (figs. 26-27) ...............................................................a. rothneyi forel 15 anterior clypeal margin with a broad large middle lobe, each side with 1 or 2 simple teeth and a narrow small lateral lobe ...................................... 16 -anterior clypeal margin without a broad large middle lobe, teeth simple or pairly combined at base ............................................................................. 19 16 anterior clypeal margin with 2 simple teeth between the middle and lateral lobes. the middle lobe truncated at apex, and with a small denticle on each side (china: tibet) (figs. 28-30) ..........................a. zoma sp. nov. -anterior clypeal margin with 1 simple tooth between the middle and lateral lobes. the middle lobe with 4 denticles ....................................................... 17 17 in full-face view, occipital margin narrowly weakly concave in the middle. occipital corners rounded. eyes absent (china: yunnan) (figs. 31-32) .. ........................................................................................................... a. triloba xu -in full-face view, occipital margin widely weakly concave. occipital corners prominent. eyes present ................................................................................. 18 18 in full-face view, lateral sides of head nearly straight, anterolateral corner with a short tooth. lateral lobes of anterior clypeal margin bifid at apex. eyes each with 5 facets. in profile view, subpetiolar process roughly triangular (china: yunnan) (figs. 33-35) .............................a. meiliana sp. nov. -in full-face view, lateral sides of head weakly convex, anterolateral corner with a long tooth. lateral lobes of anterior clypeal margin simple. eyes each with about 18 facets. in profile view, subpetiolar process roughly rectangular (new guinea) (figs. 36-37) ...................... a. noonadan taylor 19 anterior clypeal margin with 6 teeth .......................................................... 20 -anterior clypeal margin with 7-8 teeth ........................................................ 22 20 anterior clypeal margin straight, with 6 isolated teeth, the lateral ones larger than the others (vietnam) (fig. 38) .......... a. amblyops (karavaiev) -anterior clypeal margin roundly convex, the median 2 teeth combined at 1183 xu, z.-h. & j.-j. chu — four new species of amblyopone from china base, the lateral ones not larger than the others ......................................... 21 21 the lateral 2 teeth of the anterior clypeal margin combined at base. mandibles relatively narrow (new guinea) (figs. 39-40) ...a. papuana taylor -the lateral 2 teeth of the anterior clypeal margin isolated. mandibles relatively broad (indonesia) (figs. 41-42) ..............................a. minuta (forel) 22 anterior clypeal margin with 7 teeth. anterolateral curve of clypeus with figs.31-38. 31-32: worker of amblyopone triloba xu; 31. head and body in profile view; 32. head in full-face view. (drawn from antweb images). 33-35: worker of amblyopone meiliana sp. nov.; 33. head and body in profile view; 34. head in full-face view; 35. body in dorsal view. 36-37: worker of amblyopone noonadan taylor; 36. head and body in profile view; 37. head in full-face view. (drawn from antweb images). 38: worker of amblyopone amblyops (karavaiev), head in full-face view. (cited from karavaiev, 1935, slightly modified) 1184 sociobiolog y vol. 59, no. 4, 2012 a tooth ................................................................................................................ 23 -anterior clypeal margin with 8 teeth. anterolateral curve of clypeus without a tooth ................................................................................................................ 24 23 in full-face view, head weakly longer than broad. eyes present. anterior clypeal teeth isolated, not pairly combined. in profile view, anterodorsal corner of petiolar node rounded, subpetiolar process roughly rectangular (israel) (figs. 43-44) ...................................... a. ophthalmica baroni urbani -in full-face view, head strongly longer than broad. eyes absent. anterior figs. 39-46. 39-40: worker of amblyopone papuana taylor; 39. head and body in profile view; 40. head in full-face view. (drawn from antweb images). figs. 41-42: worker of amblyopone minuta (forel); 41. head and body in profile view; 42. head in full-face view. (drawn from antweb images). 43-44: worker of amblyopone ophthalmica baroni urbani; 43. head and body in profile view; 44. head in full-face view. (drawn from antweb images). 45-46: worker of amblyopone boltoni bharti et wachkoo; 45. head and body in profile view; 46. head in full-face view. (drawn from antweb images) 1185 xu, z.-h. & j.-j. chu — four new species of amblyopone from china clypeal teeth pairly combined at base. in profile view, anterodorsal corner of petiolar node bluntly angled, subpetiolar process triangular (india) (figs. 45-46) ...................................................... a. boltoni bharti et wachkoo 24 anterior clypeal teeth pairly combined at base ......................................... 25 -anterior clypeal teeth isolated, not pairly combined at base ................... 27 25 dorsolateral sides of alitrunk and petiole strongly marginated (philipfigs. 47-56. 47-48: worker of amblyopone luzonica (wheeler et chapman); 47. head and body in profile view; 48. head in full-face view. (drawn from antweb images). 49-50: worker of amblyopone silvestrii (wheeler); 49. head and body in profile view; 50. head in full-face view. (drawn from antweb images). 51-53: worker of amblyopone awa sp. nov.; 51. head and body in profile view; 52. head in full-face view; 53. body in dorsal view. 54-56: queen of amblyopone awa sp. nov.; 54. head and body in profile view; 55. head in full-face view; 56. body in dorsal view. 1186 sociobiolog y vol. 59, no. 4, 2012 pines) (figs. 47-48) ...............................a. luzonica (wheeler et chapman) -dorsolateral sides of alitrunk and petiole rounded, not marginated ..... 26 26 in profile view, anterodorsal corner of petiolar node acutely angled, anterior face weakly concave, dorsal face straight (china: zhejiang and taiwan; korea peninsula, japan) (figs. 49-50) ......................a. silvestrii (wheeler) -in profile view, anterodorsal corner of petiolar node rightly angled, anterior face straight, dorsal face weakly convex (china: yunnan and tibet) (figs. 51-56) ........................................................................................... a. awa sp. nov. 27 anterior clypeal margin roundly convex, the clypeal teeth decreased in length from middle to lateral sides (turkmenia) (fig. 57) .......................... ...................................................................................................a. annae arnol’di -anterior clypeal margin straight, the clypeal teeth with the similar length .................................................................................................................. 28 28 in full-face view, occipital corners rounded, lateral sides of head nearly straight. in profile view, posterodorsal corner of propodeum rounded. anterior face of petiolar node straight (china: yunnan) (figs. 58-59) .... ................................................................................................... a. octodentata xu -in full-face view, occipital corners prominent, lateral sides of head evenly convex. in profile view, posterodorsal corner of propodeum bluntly angled. anterior face of petiolar node weakly concave (china: taiwan) (figs. 6061) ........................................................................................ a. zaojun terayama figs. 57-59. 57: worker of amblyopone annae arnol’di, head in full-face view. (cited from arnol’di, 1968, slightly modified). 58-59: worker of amblyopone octodentata xu; 58. head and body in profile view; 59. head in full-face view. (drawn from antweb images). 1187 xu, z.-h. & j.-j. chu — four new species of amblyopone from china descriptions of new species and supplementary measurements for a. octodentata amblyopone kangba sp. nov. (figs. 13-15) holotype worker: tl 6.7, hl 1.40, hw 1.40, ci 100, sl 0.80, si 57, ed 0.10, ml 1.17, pw 0.93, al 2.07, pl 0.70, ph 0.87, dpw 0.80, lpi 124, dpi 114. in full-face view, head square, as broad as long, slightly widened forward. occipital margin weakly widely concave, occipital corners bluntly angled. lateral sides nearly straight. anterolateral corners each with a reduced tiny tooth. mandibles elongate and linear, masticatory margin very short, about 1/4 length of the inner margin, with 3 simple teeth; inner margin with 2 rows of curved teeth, each row with 6 teeth, the basal tooth large and triangular. middle portion of anterior clypeal margin weakly protruding forward, slightly concave, with 4 tiny short rectangular denticles; anterolateral corners rightly angled. frontal lobes slightly surpassed anterior clypeal margin. antennae short, 12-segmented; apices of scapes reached to 2/3 of the distance from antennal sockets to occipital corners; funiculi incrassate toward apices. eyes small, located behind the midpoints of the lateral sides of head, each with about 9 facets. in profile view, dorsum of alitrunk weakly convex, promesonotal suture distinctly notched. mesonotum short and convex. metanotal groove absent. dorsum of propodeum nearly straight, about 1.5 times as long as declivity, posterodorsal corner rounded, declivity weakly convex. dorsal face of petiole weakly convex, anterior face straight, anterodorsal corner bluntly fig s. 60-61: worker of amblyopone zaojun terayama; 60. head and body in profile view; 61. head in full-face view. (cited from terayama, 2009, slightly modified) 1188 sociobiolog y vol. 59, no. 4, 2012 angled; ventral face oblique and weakly concave, subpetiolar process roughly square, with a circular sub-transparent fenestra. constriction between the two basal gastral segments distinct, sting strong and extruding. in dorsal view, mesothorax constricted, mesonotum very short. propodeal declivity weakly concave. petiole broader than long, width : length = 1.2:1. mandibles longitudinally striate. head with fine elongate reticulations. dorsal faces of alitrunk, petiole, and gaster sparsely punctured, the punctures decreased in diameter from alitrunk to gaster, interfaces smooth and shining. the longitudinal middle strip of pronotum without punctures. lateral sides of alitrunk, petiole, and gaster densely punctured; lateral sides of mesothorax and metathorax longitudinally striate. dorsal surfaces of head and body with abundant suberect short hairs and dense decumbent pubescence. scapes and hind tibiae with sparse suberect hairs and dense decumbent pubescence. color reddish brown. occiput blackish brown. antennae and legs yellowish brown. paratype workers: tl 6.5-7.0, hl 1.33-1.40, hw 1.33-1.40, ci 100102, sl 0.77-0.83, si 57-60, ed 0.07-0.08, ml 1.17-1.23, pw 0.87-0.97, al 1.93-2.07, pl 0.67-0.77, ph 0.87-0.90, dpw 0.78-0.83, lpi 117-130, dpi 109-118 (4 individual measured). as holotype, but middle portion of anterior clypeal margin with 4-6 tiny rectangular denticles; color yellowish brown to reddish brown. holotype: worker, china: tibet, zayu county, zhuwagen town, cibaqiao, 1750m, 2010.viii. 30, collected from a soil sample in the forest of pinus yunnanensis (pinaceae), xia liu leg., no.a10-3405. paratypes: 2 workers, with the same data as holotype; 2 workers, china: yunnan province, xichou county, xisa town, jiaokui village, 1480m, 2010. iii.31, collected from a soil sample in the forest of cyclobalanopsis glaucoides (fagaceae), zheng-hui xu leg., no.a10-1763. comparison notes: this new species is close to a. rubiginoa wu et wang, but in full-face view, occipital margin widely weakly concave, occipital corners bluntly angled; in profile view, subpetiolar process roughly square. etymology: the new species is named after a race of the tibetan people “kangba” who live in the southwestern tibet. 1189 xu, z.-h. & j.-j. chu — four new species of amblyopone from china amblyopone zoma sp. nov. (figs. 28-30) holotype worker: tl 4.5, hl 1.00, hw 0.85, ci 85, sl 0.48, si 56, ed 0.05, ml 0.68, pw 0.58, al 1.25, pl 0.48, ph 0.55, dpw 0.55, lpi 116, dpi 100. in full-face view, head roughly trapezoidal, widened forward and longer than broad. occipital margin widely weakly concave, occipital corners blunly angled. lateral sides weakly convex. anterolateral corners acutely toothed. mandibles elongate triangular, masticatory margin with a long apical tooth, a short subapical tooth, and 3 pairs of curved teeth; inner margin about as long as masticatory margin, with a pair of curved teeth, a short subbasal tooth, and a large basal tooth. anterior clypeal margin roughly triangularly protruding, with a large middle lobe, and 3 teeth on each side, the most lateral tooth large and lobe-like; the large middle lobe truncated at apex, with a small denticle on each side. antennae short, 12-segmented; apices of scapes reached to 4/7 of the distance from antennal sockets to occipital corners; funiculi incrassate toward apex. eyes small, each with about 6 facets, and located behind the midpoints of the lateral sides of head. in profile view, pronotum weakly convex. promesonotal suture deeply notched. mesonotum short and convex. metanotal groove absent. propodeal dorsum straight, about 1.5 times as long as declivity; posterodorsal corner very bluntly angled; declivity nearly straight. dorsal and anterior faces of petiole nearly straight, anterodorsal corner nearly rightly angled; ventral face oblique and weakly concave; subpetiolar process roughly rectangular, with a large elliptical sub-transparent fenestra, anteroventral corner prominent, ventral face straight, posteroventral corner toothed. sternite of the first gastral segment ill developed, anteroventral corner toothed, and the segment looks narrower. in dorsal view, mesothorax constricted. propodeum widened backward. petiole broader than long, width : length = 1.2:1, anterior and lateral sides weakly convex. mandibles longitudinally striate. head densely punctured, interfaces appear as micro-reticulations. pronotum densely punctured. mesonotum, propodeum, petiole, and first gastral segment abundantly punctured. the 1190 sociobiolog y vol. 59, no. 4, 2012 middle longitudinal narrow strip on alitrunk without punctures. lateral sides of mesothorax, metathorax, and propodeum longitudinally striate. gastral segments 2-5 finely sparsely punctured. dorsal surfaces of head and body with dense subdecumbent pubescence, gaster with sparse suberect hairs and dense subdecumbent pubescence. scapes with sparse suberect hairs and dense decumbent pubescence. tibiae with dense decumbent pubescence. head brown, eyes grey. body yellowish brown, legs yellow. holotype: worker, china : tibet, medog county, beibeng town, gangouhe, 740m, 2011.vii.19, collected from a soil sample in the valley tropical rainforest, zheng-hui xu leg., no. a11-3676. comparison notes: this new species is close to a. triloba xu, but in fullface view, occipital corners bluntly angled; eyes present, each with about 6 facets; anterior clypeal margin with 2 simple teeth between the middle and lateral lobes; the middle lobe truncated at apex, and with a small denticle on each side; the lateral lobes simple, not bifid at apex. etymology: the new species is named after a common female name “zoma” widely used in tibet. amblyopone meiliana sp. nov. (figs. 33-35) holotype worker: tl 4.9, hl 1.10, hw 0.95, ci 86, sl 0.63, si 66, ed 0.05, ml 0.78, pw 0.63, al 1.50, pl 0.53, ph 0.60, dpw 0.60, lpi 114, dpi 100. in full-face view, head roughly trapezoidal, widened forward and longer than broad. occipital margin widely weakly concave, occipital corners rounded. lateral sides nearly straight, anterolateral corners acutely toothed. mandibles elongate, masticatory margin about as long as inner margin, with a long apical tooth, a short subapical tooth, and 3 pairs of curved teeth; inner margin with a pair of curved teeth, a short subbasal tooth, and a large basal tooth. anterior clypeal margin roundly convex, with a broad middle lobe, a narrow lobe on each side, and a simple tooth between the middle and lateral lobes; the broad middle lobe with 4 denticles at apex, the lateral lobes slightly bifid at apices. antennae short, 12-segmented; apices of scapes reached to 2/3 of 1191 xu, z.-h. & j.-j. chu — four new species of amblyopone from china the distance from antennal sockets to occipital corners; funiculi incrassate toward apex. eyes small, each with 5 facets, and located well behind the midpoints of the lateral sides of head. in profile view, posterior 2/3 of pronotum nearly straight, mesonotum short and convex. promesonotal suture distinctly notched, metanotal groove weakly depressed. propodeal dorsum straight, about 1.5 times as long as declivity, posterodorsal corner rounded, declivity nearly straight. petiole roughly trapezoidal, dorsal and anterior faces nearly straight, anterodorsal corner rounded; ventral face oblique and weakly concave. subpetiolar process nearly triangular, with a large circular sub-transparent fenestra, anterior face rounded, ventral face straight, posteroventral corner rightly angled. in dorsal view, mesothorax constricted. propodeum widened backward. petiole slightly broader than long, width : length = 1.1:1, anterior and lateral sides weakly convex. mandibles and clypeus longitudinally striate. head densely punctured, interfaces appear as micro-reticulations. dorsal face of alitrunk densely punctured, the longitudinal middle strip on pronotum and propodeum smooth and shining. lateral sides of pronotum transversely striate. lateral sides of mesothorax, metathorax, and propodeum longitudinally striate. declivity nearly smooth. dorsal faces of petiole and gaster smooth, lateral sides of petiole finely reticulate, lateral sides of gaster finely punctured. dorsal surfaces of head and body with sparse suberect short hairs and dense decumbent pubescence. scapes and tibiae with sparse suberect hairs and dense decumbent pubescence. color reddish brown. eyes blackish. legs yellowish brown. holotype: worker, china: yunnan province, deqin county, yunling town, ming yong village, 3250m, 2004.x.10, collected from a ground sample in the conifer-broad leaf mixed forest on the east slope of the snow mt. meili, sheng-li shi leg., no. a04-536. comparison notes: this new species is close to a. triloba xu, but in fullface view, occipital margin widely weakly concave, occipital corners more prominent; lateral sides of head nearly straight; eyes present, each with 5 facets; in profile view, subpetiolar process roughly triangular. etymology: the new species is named after the type locality “snow mt. meili”, the highest mountain in yunnan province. 1192 sociobiolog y vol. 59, no. 4, 2012 amblyopone awa sp. nov. (figs. 51-56) holotype worker (figs. 51-53): tl 3.8, hl 0.80, hw 0.68, ci 84, sl 0.40, si 59, ed 0.03, ml 0.55, pw 0.45, al 1.05, pl 0.38, ph 0.43, dpw 0.43, lpi 113, dpi 113. in full-face view, head roughly trapezoidal, widened forward and longer than broad. occipital margin widely weakly concave, occipital corners bluntly angled. lateral sides weakly convex, anterolateral corners acutely toothed. mandibles elongate, masticatory margin with a long apical tooth, a short subapical tooth, and 3 pairs of curved teeth; inner margin about as long as masticatory margin, with a pair of curved teeth, a short subbasal tooth, and a large basal tooth. anterior clypeal margin with 8 teeth, which combined into 4 pairs. antennae short, 12-segmented; apices of scapes reached to about 2/3 of the distance from antennal sockets to occipital corners; funiculi incrassate toward apex. eyes very small, each with 3 facets, and located well behind the midpoints of the lateral sides of head. in profile view, pronotum weakly convex. promesonotal suture distinctly notched. mesonotum short and convex. metanotal groove absent. propodeal dorsum straight, about 2 times as long as declivity, posterodorsal corner rounded, declivity weakly convex. petiole trapezoidal, dorsal and anterior faces nearly straight, anterodorsal corner close to a rightly angle; ventral face oblique, nearly straight; subpetiolar process roughly rectangular, with a large elliptical sub-transparent fenestra, ventral face straight, posteroventral corner rightly angled. in dorsal view, mesothorax constricted. propodeum slightly widened backward. propodeal declivity longitudinally concave. petiole broader than long, width : length = 1.25:1, anterior and lateral sides weakly convex. mandibles longitudinally striate. head densely punctured, interfaces appear as micro-reticulations. pronotum densely punctured, the narrow longitudinal middle strip without punctures. dorsa of mesonotum and propodeum abundantly punctured. lateral sides of mesothorax and metathorax finely longitudinally striate. petiole and gaster finely sparsely punctured. dorsal surfaces of head and body with sparse suberect short hairs and dense decumbent pubescence. scapes with sparse suberect hairs and dense decumbent pubes1193 xu, z.-h. & j.-j. chu — four new species of amblyopone from china cence. tibiae with dense decumbent pubescence, but without suberect hairs. color reddish brown. eyes black. antennae and legs yellowish brown. paratype workers: tl 3.6-4.2, hl 0.78-0.90, hw 0.65-0.75, ci 81-90, sl 0.40-0.48, si 57-63, ed 0.03-0.04, ml 0.53-0.60, pw 0.43-0.50, al 1.03-1.20, pl 0.38-0.45, ph 0.43-0.48, dpw 0.41-0.48, lpi 106-113, dpi 103-113 (5 individuals measured). as holotype, but eyes with 3-6 facets; posteroventral corner of subpetiolar process toothed or bluntly angled; head and alitrunk reddish brown to blakish brown. paratype queens (figs. 54-56): tl 4.0-4.1, hl 0.80-0.85, hw 0.700.75, ci 88, sl 0.40-0.43, si 57, ed 0.13, ml 0.55-0.68, pw 0.53-0.55, al 1.18-1.25, pl 0.40-0.45, ph 0.45-0.48, dpw 0.45-0.48, lpi 106-113, dpi 106-113 (2 individuals measured). similar to holotype worker, but body feebly larger, vertex with 3 ocelli. eyes large, each with about 45 facets. mesonotum large in volume, tegulae present. in dorsal view, anterior margin of mesonotum roundly convex, scutum with a pair of posteriorly convergent longitudinal furrows, posterior margin roundly convex. scutellum rhombus, both anterior and posterior margins roundly convex. metascutum narrow and posteriorly arched. in profile view, posteroventral corner of subpetiolar process acutely toothed. holotype: worker, china: yunnan province, cang yuan county, banlao town, huguang village, 1720m, 2011.iii.17, collected from a soil sample in the monsoon evergreen broad-leaf forest, yong-qiang hao leg., no. a11855. paratypes: 1 worker, 1 queen, with the same data as holotype; 1 worker, china: yunnan province, cang yuan county, banhong town, nanban village, 1250m, 2011.iii.17, collected from a ground sample in the monsoon evergreen broad-leaf forest, yong-qiang hao leg,. no. a11-927; 1 worker, china: yunnan province, ximeng county, lisuo town, nankang village, 740m, 2011.iii.22, collected from a soil sample in the valley tropical rainforest, li zhang leg., no. a11-1671; 2 workers, 1 queen, china: tibet, linzhi county, lulang town, zhaqu village, 2380m, 2007.ix.25, collected inside decayed wood in the broad-leaf forest, zheng-hui xu leg., no. a07-432. comparison notes: this new species is close to a. silvestrii (wheeler), but in profile view, anterodorsal corner of petiolar node rightly angled, anterior face straight, dorsal face weakly convex; body smaller with tl 3.6-4.2 mm. 1194 sociobiolog y vol. 59, no. 4, 2012 etymology: the new species is named after an intimate call “awa” of the minority nationality “wa” people who commonly live in the area of the holotype locality. amblyopone octodentata xu (figs. 58-59) workers: tl 4.8-5.8, hl 1.03-1.38, hw 0.90-1.13, ci 82-91, sl 0.550.75, si 58-67, ed 0.04-0.08, ml 0.75-1.10, pw 0.60-0.75, al 1.38-1.70, pl 0.55-0.68, ph 0.58-0.68, dpw 0.53-0.68, lpi 100-114, dpi 95-109 (10 individuals measured). well conform to the original description. specimens observed: 9 workers, china: yunnan province, zhenxiong county, wufeng town, shangjie village, 1750m, 2009.iv.01, collected from a ground sample in the forest of cunninghamia lanceolata (taxodiaceae), zhifeng chen leg., no. a09-1322; 1 worker, china: yunnan province, yongshan county, xisha town, yangpu village, 1000m, 2009.iii.26, collected from a soil sample in the deciduous broad-leaf forest, li-mei li leg., no. a09-735. acknowledgments this study is supported by the national natural science foundation of china (nos. 30260016, 30870333), rapid assessment program of biodiversity organized by peking university, and the key subject of forest protection of yunnan province. we thank the following persons or institutions for their special help in this study: miss xia liu (phd candidate of forest protection, beijing forestry university, beijing ), mr. sheng-li shi (postgraduate of forest protection, southwest forestry university, kunming ), miss li-mei li & mr. zhi-feng chen (students of forest protection class 2005, southwest forestry university, kunming ), miss li zhang & mr. yong-qiang hao (students of forest protection class 2007, southwest forestry university, kunming ) who collected the type specimens with us; mr. himender bharti (punjabi university, patiala) who supplied a valuable paper; mr. konstantin vladamirovitsch arnol’di (anim. acad. sci. ussr, moscow), mr. cesare baroni urbani (naturhistorisches museum, basel), mr. vladimir aphanasjevich karavaiev (conservator of the zoological museum of the ukranian academy of science in kiew), mr. hirotami t. imai et al. (myrmecological society of japan, 1195 xu, z.-h. & j.-j. chu — four new species of amblyopone from china tokyo), mr. mamoru terayama (2-12-29-3, naka-cho, iwatsuki, 339-0054, japan), mrs. jiang wu & chang-lu wang (chinese academy of forestry, beijing ), and mr. shan-yi zhou (guangxi normal university, guilin) who granted permission to cite figures in their papers; california academy of sciences (san francisco) who permitted the use of the images on the antweb (http://www.antweb.org/). references arnol'di, k. v. 1968. vazhnye dopolneniya k mirmekofaune sssr i opisanie novykh form. zoologicheskii zhurnal 47: 1800-1822. baroni urbani, c. 1978. contributo alla conoscenza del genere amblyopone erichson. mitteilungen der schweizerischen entomologischen gesellschaft 51: 39-51. bharti, h. & a. a.wachkoo 2011. amblyopone boltoni, a new ant species (hymenoptera: formicidae) from india. sociobiolog y 58(3): 585-591. bingham, c. t. 1903. the fauna of british india, including ceylon and burma. hymenoptera 2. ants and cuckoo-wasps: 506 pp. taylor & francis, london. bolton, b. 1975. a revision of the ant genus leptogenys roger in the ethiopian region, with a review of the malagasy species. bulletin of the british museum (natural history) (entomolog y) 31: 235-305. bolton, b. 1995. a new general catalogue of the ants of the world: 504 pp. harvard university press, cambridge, mass. bolton, b. 2012. an online catalog of the ants of the world. http://www.antcat.org/. emery, c. 1895. viaggio di leonardo fea in birmania e regioni vicine. 63. formiche di birmania, del tenasserim e dei monti carin, raccolte da l. fea. annali del museo civico di storia naturale di genova (2) 14 [34] (1894): 450-483. erichson, w. f. 1842. beitrag zur insecten-fauna von vandiemensland, mit besonderer berücksichtig ung der geographischen verbreitung der insecten. archiv für naturgeschichte 8: 83-287. forel, a. 1900. les formicides de l’empire des indes et de ceylan. part 6. 3me sous famille ponerinae. journal of the bombay natural history society 13: 52-65. forel, a. 1912. h. sauter’s formosa-ausbeute: formicidae. entomologische mitteilungen 1: 45-81. forel, a. 1913. wissenschaftliche ergebnisse einer forschungsreise nach ostindien, ausgeführt im auftrage der kgl. preuss. akademie der wissenschaften zu berlin von h. v. buttelreepen. 2. ameisen aus sumatra, java, malacca und ceylon. gesammelt von herrn prof. dr. v. buttel-reepen in den jahren 1911-1912. zoologische jahrbücher. abteilung für systematik, geographie und biologie der tiere 36: 1-148. imai, h. t., a. kihara, m. kondoh, m. kubota, s. kuribayashi, k. ogata, k. onoyama, r. w. taylor, m. terayama, m. yoshimura & y. ugawa 2003. ants of japan: 224 pp. gakken, japan. 1196 sociobiolog y vol. 59, no. 4, 2012 karavaiev, v. 1935. neue ameisen aus dem indo-australischen gebiet, nebst revision einiger formen. treubia 15: 57-118. mayr, g. 1879. beiträge zur ameisen-fauna asiens. verhandlungen der k. k. zoologischbotanischen gesellschaft in wien 28 (1878): 645-686. morisita, m., m. kubota, k. onoyama, k. ogata, m. terayama, m. kondoh & h. t. imai 1989. a guide for the identification of japanese ants. 1. ponerinae, cerapachyinae, pseudomyrmecinae, dorylinae and leptanillinae: 42 pp. myrmecological society of japan, tokyo. onoyama, k. 1999. a new and a newly recorded species of the ant genus amblyopone from japan. entomological science 2: 157-161. taylor, r. w. 1965. new melanesian ants of the genera simopone and amblyopone of zoogeographical significance. breviora 221: 1-11. taylor, r. w. 1979. melanesian ants of the genus amblyopone. australian journal of zoolog y 26 (1978): 823-839. terayama, m. 1987. a new species of amblyopone from japan. edaphologia 36: 31-33. terayama, m. 1989. the ant tribe amblyoponini of taiwan, with description of a new species. japanese journal of entomolog y 57: 343-346. terayama, m. 2009. a synopsis of the family formicidae of taiwan. research bulletin of kanto gakuen university 17: 81-266. wheeler, w. m. 1928. ants collected by professor f. silvestri in japan and korea. bollettino del laboratorio di zoologia generale e agraria del r. istituto superiore agrario di portici 22: 96-125. wheeler, w. m. & j. w. chapman 1925. the ants of the philippine islands. part 1. dorylinae and ponerinae. philippine journal of science 28: 49-73. wu, j. & c. wang 1992. formicidae. in: peng, j. et al. (eds.). iconography of forest insects in hunan, china. forest bureau of hunan province. hunan scientific and technical publishing house, changsha: 1301-1320. xu, z. 2001. a systematic study on the ant genus amblyopone erichson from china. acta zootaxonomica sinica 26: 551-556. xu, z. 2006. three new species of the ant genera amblyopone erichson, 1842 and proceratium roger, 1863 from yunnan, china. myrmecologische nachrichten 8: 151-155. zhou, s. 2001. ants of guangxi. guangxi normal university press, guilin, china: 255 pp. 999 dominance and subordination interactions among nestmates in pre and post-emergence phases of the basal eusocial wasp mischocyttarus (monogynoecus) montei (hymenoptera, vespidae) by 1oliveira, vc, 1desuó, ic, 1murakami, asn & 2shima, sn abstract in basal eusocial wasps the social organization is based on a dominance hierarchy which is maintained through agonistic interactions. the dominant wasp is usually the most aggressive individual and assumes the reproductive function of the colony. these wasps lack morphological caste differences and the physiological conditions and behavioral repertoire define the role of each female in the nest. however, the position in the rank and its function in the colony are not definitive and tradeoffs in the social rank, at least in some species, are common. in this study 8 colonies of mischocyttarus (m.) montei in pre and post-emergence phases of colonial development were observed in field conditions in order to study the interactions of dominance and subordination among nestmates, thus allowing a better understanding of the establishment of the dominance hierarchy and consequently the social regulation of this species. our results showed that all colonies in pre-emergence were founded by an association of females which had established the hierarchy previously in their natal nest. such pattern may contribute to the success of association during pre-emergence, once it tends to reduce conflicts among cohorts during initial phases of colonial development, increasing the chances of colonial success. during post-emergence, the conflicts tend to be more intense, usually involving more physical contacts and in this phase, tradeoffs in the social rank are more frequent. during post-emergence, the number of females increases, more cells are available to lay eggs and the reproductive condition of the main egg-layer is probably reduced, aggravating the competition for reproductive dominance of the colony. 1. programa de pós-graduação, departamento de zoologia, instituto de biociências, unesp, av. 24-a, 1515, cep:13506-900, rio claro, sp, brazil. e-mail: ivan.desuo@yahoo.com.br. 2. departamento de zoologia, instituto de biociências, unesp, av. 24-a, 1515, cep:13506-900, rio claro, sp, brazil. 1000 sociobiolog y vol. 59, no. 3, 2012 key words: mischocyttarus, dominance hierarchy, polistinae, eussocial wasps. introduction in basal eusocial wasps, the social dominance represents the ability of a female to monopolize the reproductive control of the colony (west-eberhard 1969; wilson 1971; jeanne 1972). moreover, the maintenance of the reproductive control increases individual fitness, as the dominant wasp produces most of the colony brood. however, it also involves costs, since the main egglayer tries to avoid other females from laying eggs. in this group of wasps, the social roles of the nestmates are determined through aggressive interactions, leading to the establishment of a linear dominance/subordination hierarchy (pardi 1942; gadagkar 1991). the most aggressive female is usually coined as “the dominant wasp” and assumes the role of the main egg-layer, whereas the most subordinate individuals became typically foragers. in these wasps, the dominance hierarchy is not so static and tradeoffs in the dominance rank are commonly observed, especially in some species of polistes (reeve 1991). their nests are founded solitarily or by an association of few cohorts (pleometrosis), and the choice between nestling alone or in an association is direct related to environmental and biological restrictions, mainly the availability of nestling sites, survivorship insurance and nest usurpation (reeve 1991). the basal eusocial polistinae wasps are characterized by a lack of morphological caste differences resulting in flexibility in the social roles of adults; however, physiological conditions and behavioral repertoire differ among adults (gadagkar 1991; murakami & shima 2009, 2010). age, at least in some species of basal eusocial wasps, is important to determine dominance hierarchy and the most dominant female tends to be the oldest individual of the colony (murakami & shima 2009 2010 – mischocyttarus cassununga; giannotti & machado 1994 – polistes lanio; huges & strassmann 1988 – polistes instabilis; strassmann & meyer 1983 – polistes exclamans). the degree of aggressiveness is quite variable among basal eusocial wasps and depends on the phase of the colonial development (gadagkar 1991; reeve 1991; röseler 1991). in polistes the interactions are usually more intense, involving injuries among opponents (west-eberhard 1969; spradbery 1973; yamane 1985; röseler 1991; tindo & dejean 2000). this is especially true 1001 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei in the initial phases of colonial development in nests founded by association of other cohorts. in m. drewseni the rate of dominance encounters was low ( jeanne 1972) when compared with polistes gallicus (pardi 1948). in ropalidia species the dominance and subordination interactions seems to be generally less violent than those of polistes (gadagkar & joshi 1982; itô 1983, 1985). on the other hand, agonistic interactions in mischocyttarus are moderate and may not even involve physical contact. the genus mischocyttarus is the most diverse group of wasps, with 245 species and 9 subgenus (silveira 2008). however, it is almost restricted to the south america, with only two species occurring on the south and west sides of united states (gadagkar 1991). despite its great diversity, this genus is poorly studied and the mechanisms which regulate sociality are barely known. thus, the aim of this work was to study the interactions of dominance and subordination among the nestmates of the basal eusocial wasp mischocyttarus (monog ynoecus) montei in the pre and post-emergence phases of colonial development. this paper represents the first behavioral study in this species. materials and methods dominance interactions among nestmates were recorded under field conditions in 8 colonies of the basal eusocial wasp mischocyttarus (monog ynoecus) montei (fig. 1) in pre and post-emergence phases of colonial development. the observations were carried out at the campus of the universidade estadual fig.1. two colonies of mischocyttarus (m.) montei. it’s possible to see that the ventral portion of gaster (arrow) is whitish which differs from m. cerberus styx, where it is totally black. (photos by guilherme gomes) 1002 sociobiolog y vol. 59, no. 3, 2012 paulista (unesp), in rio claro, são paulo state, southeastern brazil. each colony was mapped weekly from march 2002 to june 2003 to establish their stages of development and observed during 164.4 hours (table 1). the colonial phases of development were identified and classified according to jeanne (1972) as follows: (1) pre-emergence (pe) – period from the colony foundation to the emergence of the first adult and (2) post-emergence (po) – period from the emergence of the first adult to the decline of the colony. a colony was considered to be in decline when it had more than 50% of empty cells and the presence of few nestmates. the individuals were marked with acrylic, non-toxic, fast-drying paint to identify their position in the dominance hierarchy. ethological data were collected based on the studies of several authors ( jeanne; 1972; itô 1985; west-eberhard 1986; noda et al. 2001) and a brief description of the behaviors used to discriminate the hierarchical position of the females is provided as follows: trophallaxis: exchange of fluids among nestmates. the dominant female usually receives a larger share of the load brought by incoming foragers (o’donnell 1998; prezoto et al. 2004); approach: the simple presence or approach of the dominant female, without any body contact. it may act as a signal of aggressiveness, leading the other nestmates to move away or change their position in the nest. commonly, the subordinate females assume a submissive posture, lowering the antennas, wrinkling the legs and wings; table1. colonies of mischocyttarus (m.) montei observed in this study. colony period of observation time of observation (hours) colony phase i* jan/03 to mar/03 16.3 pre-emergence ii* mar/03 to jun/03 12.0 pre-emergence iii* mar/03 to jun/03 12.3 pre-emergence iv* mar/03 to jun/03 10.0 pre-emergence v# apr/02 to feb/03 37.3 post-emergence vi# mar/02 to mar/03 35.3 post-emergence vii may/02 to dec/02 29.6 post-emergence viii nov/02 to jan/03 11.6 post-emergence * these colonies were found from known colonies in post-emergence stage and then the dominance hierarchy was pre-established; # in these two colonies tradeoffs in the hierarchical rank were observed, data was taken after the stabilization of the new social rank. 1003 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei attack type i: it occurs when a dominant female, initially in resting position, suddenly attacks a subordinate female in movement; attack type ii: while moving in the nest, a dominant female detects a subordinate resting and immediately attacks her. this behavior acts as a signal for the subordinate female to start an in-nest task or to forage; evade: once in the presence of the main egg-layer or during an attack attempt, the subordinate female tries to avoid physical contact with the dominant to preserve its body integrity. all statistics were conducted using statsoft statistica 8.0. the absolute frequency of dominance behaviors were corrected by the time of observation and the number of individuals which directly participated of the social hierarchy in each colony. since data did not follow a normal distribution nor the variances were homogenous, we performed non-parametric statistics. the comparisons were tested using mann-whitney u test. results and discussion in the four pre-emergence colonies of mischocyttarus (m.) montei (i, ii, iii, and iv), which were founded from previously known colonies (table 1), the first ranked female was the most dominant individual. these females performed the majority of interactions observed in this phase being responsible for 95.92%, 84.85%, 63.27% and 56.06% of the total number of interactions recorded in their colonies, respectively (tables, 2, 3, 4 and 5). these results are different from those reported by noda et al. (2001), which have demonstrated that during pre-emergence of m.cerberus styx, the totality of dominance acts were performed exclusively by the first ranked females. similar results were obtained in m. cassununga in the pre-emergence stage (prezoto et al. 2004). according to prezoto et al. (2004) the dominant females in colonies of pre-emergence of m. cassununga were by far the most aggressive individuals of the colonies, since they were engaged in ensuring their role as main egglayers in the nest. possibly, the differences found between m. cerberus styx, m. cassununga and m. montei are due to the fact that the colonies of m. montei observed in pre-emergence were founded by females whose hierarchy was defined in the natal nest, and consequently, the level of genetic relatedness among those females was high (full-sisters). such characteristics explain the low level of aggressiveness found in the pre-emergence phase of m. montei. 1004 sociobiolog y vol. 59, no. 3, 2012 table 3. absolute frequency of dominance acts registered in colony ii (pre-emergence) of mischocyttarus (m.) montei. rows represent dominance and columns subordination. rank 1 2 3 total 1 19 9 28 2 0 2 2 3 1 2 3 total 1 21 11 33 table 4. absolute frequency of dominance acts registered in colony iii (preemergence) of mischocyttarus (m.) montei. lines represent dominance and columns subordination. rank 1 2 3 4 5 total 1 11 10 4 6 31 2 0 6 2 5 13 3 0 0 0 2 2 4 0 0 1 1 2 5 0 0 1 0 1 total 0 11 18 6 14 49 table 5. absolute frequency of dominance acts registered in colony vi (preemergence) of mischocyttarus (m.) montei. lines represent dominance and columns subordination. rank 1 2 3 4 5 total 1 19 13 2 3 37 2 0 11 5 5 21 3 2 0 4 2 8 4 0 0 0 0 0 5 0 0 0 0 0 total 2 19 24 11 10 66 table 2. absolute frequency of dominance acts registered in colony i (pre-emergence) of mischocyttarus (m.) montei. rows represent dominance and columns subordination. rank 1 2 3 total 1 40 7 47 2 0 2 2 3 0 0 0 total 0 40 9 49 1005 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei it is interesting to note that despite the fact that these species belong to the same genera, they have unique characteristics that may affect the pattern of social dominance in each species. in m. cassununga, it is rare to find a foundation by pleometrosis and the hierarchy is remarkably stable (murakami & shima 2006, 2009, 2010). on the other hand, in m. montei, most of the colonies are founded by association of few cohorts and new colonies are commonly founded by females whose dominance hierarchy was pre-established during post-emergence in their natal nest. in fact, this occurred in 100% (n=4) of the colonies studied in pre-emergence (table 1). according to itô (1987), in species that found their nest mostly by association, such as m. montei, the agonistic interactions during pre-emergence tend to be softer. this would be adaptive, since females may tolerate each other in the same nest, the odds of mortality of the main egg-layer, as well as other individuals also essential to the maintenance of colonial functions, would be smaller and the chance of colony success would be higher. however, this proposal was never properly fig. 2. relative frequency of dominance acts performed by the females of mischocyttarus (monog ynoecus) montei in the different phases of colony development (t – trophallaxis; wc – dominance acts involving direct physical contact; nc – dominance acts without physical contact). 1006 sociobiolog y vol. 59, no. 3, 2012 tested. it is clear that the females of m. montei try to avoid direct contact, and the interactions without physical contact are much more common than those involving direct contact (fig. 2). another fact that may explain the low level of aggressiveness in pre-emergence colonies of mischocyttarus (m.) montei is the characteristics of the foundations observed in the colonies studied; every colony was founded by an association of females from a pre-existent colony in post-emergence, so the hierarchy was already established. this type of foundation is common in this species (oliveira 2003, 2007) and must be an important feature to guarantee the success of pre-emergence colonies. itô (1985) reported that aggressive interactions in pre-emergence colonies of ropalidia fasciata, polistes versicolor, mischocyttarus angularis and m. basimacula were significantly less intense than in p. canadensis. in m. angulatus, no agonistic interactions were recorded during the pre-emergence stage and p. versicolor sustained a low level of aggressiveness even during post-emergence stage. in m. angularis and m. basimacula, the intensity and frequency of dominance acts increased during post-emergence. noda et al. (2001) found the same pattern for m. cerberus styx; the absolute frequency of dominance acts was greater in post-emergence. in m. drewseni, as the first foragers emerge, the frequency of dominance acts increases and the dominant females establish its reproductive superiority during the first 10-15 days after the emergence of foragers. after the establishment of dominance, the frequency of acts decreased significantly once the hierarchy was already established ( jeanne 1972). tables 6, 7, 8 and 9 show the absolute frequency of dominance and subordination interactions during the post-emergence phase of m. montei. it is clear that the absolute frequency of agonistic interactions is greater than in the pre-emergence stage; this fact was also reported by other authors (noda et al. 2001; prezoto et al. 2004 and murakami & shima 2006). in post-emergence, the first ranked female performed a considerable share of the dominance behaviors recorded, represented by 60.6%, 45%, 35% and 61.7%, respectively. these values, however, were consistently smaller than in pre-emergence. the only exception occurred in colony vii, where the 2nd ranked female performed slightly more dominance acts than the first one; however, she was heavily dominated by the dominant female (table 8). in post-emergence, the number of individuals increases, more cells are available to lay eggs and consequently the competition for reproductive domi1007 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei nance is more intense. however, when comparing the absolute frequencies/ hour/individual between the two phases, no statistical differences were found (mann-whitney z=0.28, p=0.77) (fig. 3). such results may be explained by the fact that in post-emergence, more individuals participate of dominance and subordination interactions, including typical foragers (tables 6-9). this may have diluted the number of interactions in post-emergence, explaining the finding of no statistical difference between the two phases. in fact, 92% (646/701) of the total dominance acts displayed in post-emergence were performed by the three higher ranked females of each colony. it is also worth mentioning that, although the relative frequency of dominance acts were not table 6. absolute frequency of dominance acts registered in colony v (post-emergence) of mischocyttarus (m.) montei. lines represent dominance and columns subordination. rank 1 2 3 4 5 6 7 8 total 1 65 34 15 8 6 6 3 137 2 1 29 16 8 2 5 1 61 3 0 1 2 3 4 3 0 13 4 0 0 0 0 0 5 0 5 5 1 0 0 2 0 0 2 5 6 0 0 0 0 1 0 0 1 7 0 0 0 1 0 0 1 2 8 0 0 0 0 1 0 1 2 total 2 66 63 36 21 12 20 7 226 *the 1st ranked female disappeared from the colony, the 2nd assumed the post of dominant, and after that the 2nd lost its post to the 3rd. after the stabilization of the new rank, the data was collected. table 7. absolute frequency of dominance acts registered in colony vi (postemergence) of mischocyttarus (m.) montei. rows represent dominance and columns subordination. rank 1 2 3 4 5 6 total 1 36 27 6 7 8 84 2 1 46 11 11 4 73 3 1 1 9 5 2 18 4 3 1 0 1 3 8 5 0 0 0 0 0 0 6 0 0 0 0 0 0 total 5 38 73 26 24 17 183 *the 1st ranked female disappeared from the colony and its post was occupied by the 2nd ranked female. after the stabilization of the new rank, the data was collected. 1008 sociobiolog y vol. 59, no. 3, 2012 statistically different between the two phases, our data showed that the median frequency (%) of dominance interaction involving physical contact was statistically higher in the post-emergence (freq pe =13.66%; freq po =24.80%; z=2.02, p=0.04) (fig. 2). these results showed that the competition for reproductive dominance in m. montei during post-emergence stage is more intense than in pre-emergence. the exchanges in social rank occur more frequently during post-emergence phase. the dominant supersedure occurred in 50% (n=2, colonies i and ii, tables 6 and 7) of the colonies studied in this phase. queen supersedure was reported by some authors, especially in polistes, during the late post-emergence, when the competition for reproductive dominance is stronger (strassamann 1981; miyano 1986). recently, murakami and shima (2009) reported a case of dominant supersedure in m. cassununga, however, the authors classified this as a very rare event. in table 8. absolute frequency of dominance acts registered in colony vii (post-emergence) of mischocyttarus (m.) montei. rows represent dominance and columns subordination (f indicates foragers). rank 1 2 3 4 5 6 f(n=4) total 1 30 12 9 2 2 4 59 2 2 20 13 4 5 20 62 3 0 0 6 5 1 9 21 4 0 0 0 1 2 5 8 5 0 0 0 0 0 1 1 6 0 0 0 0 0 0 0 f(n=4) 0 3 0 0 5 1 2 11 total 2 33 32 28 17 11 41 162 table 9. absolute frequency of dominance acts registered in colony viii (post-emergence) of mischocyttarus (m.) montei. rows represent dominance and columns subordination. (f indicates foragers) rank 1 2 3 4 f(n=5) total 1 32 17 10 15 79 2 0 12 7 11 30 3 1 0 0 8 9 4 0 0 0 5 5 f(n=5) 0 1 0 1 3 5 total 1 33 29 19 42 128 1009 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei m. montei, exchanges are common (oliveira 2003; 2007), indicating a less stable hierarchy when compared to m. cassununga. this fact may explain the reason why a group of females leaves the natal nest and founds a new nest in the neighborhood. this is caused by a conflict which affects the productivity of the colony through cannibalism of eggs and larvae (in prep.). when this occurs, the only way to maintain the development of the natal nest in postemergence is the colonial fission, in which the most dominant female and the typical foragers remain in the natal nest, while the higher ranked females (usually involved in the colonial conflict) leave the nest. this colonial fission, which is different from a natural dispersion, once reproductive individuals are not being produced yet, is adaptive, since it allows the natal nest to keep developing to the natural decline. additionally, fig. 3. box-plots indicating the median, 25th and 75th percentiles and non-outlier range of the frequency/individuals/hour of dominance interactions in the two phases of colonial development. 1 – pre-emergence, 2 – post-emergence. the results of the mann-whitney test were not significant (z=0.28; p=0.77). 1010 sociobiolog y vol. 59, no. 3, 2012 this pattern allows the foundation of new colonies by highly related females, ensuring a low level of conflict during pre-emergence stage. if such fission, related to the dispersion of the higher ranked females, did not occur, the colony would decline prematurely. therefore, such a mechanism is adaptive, as it allows the species to maximize its dispersion without the cost of derailing the natal nest. there are few studies in the literature regarding this pattern of fission in social wasps. gadagkar and joshi (1985) reported that the high level of aggression found in ropalidia cyathiformes before the colony fission could be a consequence of the reproductive competition among nestmates. the authors suggested that the high level of aggression decreased the level of brood production. these results were similar to those found in m. montei (in prep). most of the studies available in the literature are related to the natural dispersion of the colonies; litte (1977) showed that the females of three nests of mischocyttarus mexicanus dispersed and regrouped in small groups during the fall (proper time for the dispersion of reproductive females). the authors also reported that all wasps within each group were derived from the same natal nest. finally, we conclude that: (1) the colonial fission caused by a conflict during post-emergence phase, but not by natural dispersion, may promote the foundation of new colonies by females with defined social dominance hierarchy, which may explain the low level of dominance interactions found in pre-emergence; (2) the fact that no statistical difference was found in the relative frequency of dominance interactions between the phases of development indicate that, in post-emergence, more females participated in these interactions, including typical foragers, which diluted the frequency of interactions in post-emergence. however, the median relative frequency of interactions involving physical contacts was statistically higher in postemergence, and these interactions were displayed mostly by the three higher ranked females of each colony; (3) m. montei have two types of colonial foundation: (a) by a fission caused by a conflict, such characteristics are adaptive since it prevents that the natal nest declines prematurely; (b) by natural dispersion, after production of reproductive individuals. in both cases, the colonies are maintained by females with high level of relatedness (this was inferred as all females were marked and observed continuously) increasing the odds of colonial success. 1011 oliveira, v.c. et al. — dominance interactions in mischocyttarus montei acknowledgment the author acknowledges capes for financial support. references gadagkar, r. & n.v. joshi. 1982. behaviour of the indian social wasp ropalidia cyathiformis on a nest of separate combs (hymenopetra, vespidae). journal of zoolog y 198:27-37. gadagkar, r. & n.v. joshi. 1985. colony fission in a social wasp. current science 54(2):5762. gadagkar, r. 1991. belonogaster, mischocyttarus, parapolybia and independent-founding ropalidia. in: ross k.g. & r.w. matthews (eds.). the social biolog y of wasps, ithaca(ny): cornell university press. p. 149-190. giannotti, e. & v.l.l machado. 1997. queen replacement in postemergent colonies of the social wasp, polistes lanio (hymenoptera, vespidae). revista brasileira de entomologia 41(1):911. hughes, c.r. & j.e. strassmann. 1988. age is more important than size in determining dominance among workers in the primitively eusocial wasp, polistes instabilis. behaviour 107:1-14. itô, y. 1985. a comparison of frequency of intra-colony agressive behaviours among five species of polistine wasps (hymenoptera, vespidae). journal of comparative etholog y (zeitschrift für tierpsychologie) 68:152-167. itô, y. 1987. roles of pleometrosis in the evolution of eusociality in wasps. in: y. itô, j. l. brown & j. kikkawa (eds.). animal societies: theories and facts. tokyo: japan society press. p.17-34. itô, y. 1983. social behaviour of a subtropical papar wasp, ropalidia fasciata (f.): field observations during founding stage. journal of etholog y 1:1-14. jeanne, r.l. 1972. social biolog y of the neotropical wasp mischocyttarus drewseni. bulletin of the museum of comparative zoolog y 144(3):63-150. litte, m. 1977. behavioural ecology of the social wasp mischocyttarus mexicanus (hymenoptera, vespidae). behavioral ecololog y and sociobiolog y 2:229-246. miyano, s. 1986. colony development, worker behavior and male production in orphan colonies of a japanese paper wasp, polistes chinensis antennalis (hymenoptera, vespidae, polistinae). researches on population ecolog y 28:347-361. murakami, a. s. n. & s.n. shima. 2006. nutritional and social hierarchy establishment of the primitively eusocial wasp mischocyttarus cassununga (hymenoptera, vespidae, mischocyttarini) and related aspects. sociobiolog y 48:183–207. murakami, a.s.n. & s.n. shima. 2009. queen replacement in mischocyttarus (monocyttarus) cassununga (hymenoptera, vespidae, mischocyttarini): a particular case. sociobiolog y 53(1):1-11. 1012 sociobiolog y vol. 59, no. 3, 2012 murakami, a.s.n. & s.n. shima. 2010. regulation of social hierarchy over time in colonies of the primitive eusocial wasp mischocyttarus (monocyttarus) cassununga. von ihering 1903 (hymenoptera, vespidae). journal of kansas entomological society 82(2):163-171. noda, s. c. m., e.r. silva & e. giannotti. 2001. dominance hierarchy in different stages of development in colonies of the primitively eusocial wasp mischocyttarus cerberus styx (hymenoptera, vespidae). sociobiolog y 38(3): 603–614. o’donnell, s. 1998. dominance and polyethism in the social wasp miachocyttarus mastigophorus (hymenoptera, vespidae). behaviral ecololog y and sociobiolog y 43:327-331. oliveira, v. c. 2003. comportamentos de dominância e subordinação da vespa eussocial primitiva mischocyttarus (monogynoecus) montei zikán 1949 (hymenoptera, vespidae, mischocyttarini). scientific initiation, unesp—campus de rio claro, rio claro. 71 pp. oliveira, v. c. 2007. diferenciação etológica e morfofisiológica das castas de mischocyttarus (monog ynoecus) montei, zikán 1903 (hymenoptera, vespidae, mischocyttarini), com especial referência à regulação social das colônias. master thesis, unesp—campus de rio claro; 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(hanson & gauld, 1995; carpenter & marques, 2001; west-eberhard et al., 2010). hitherto, six species were distributed in brazil, two being endemic (carpenter & marques, 2001). from acre state only c. nigerrimus had been recorded. the genus is diagnosed by a prominent curved bristle on the third labial palpomere, the maxillary palp five-segmented and labial palpi three-segmented, lacking an occipital carina, the mesepisternum lacking a dorsal groove, and the metanotum rounded (carpenter, 2004 apud west-eberhard et al., 2010). abstract a new species of chartergellus, collected in acre state, is described and comparative remarks are given. sociobiology an international journal on social insects s mateus1, fs nascimento1, m aragão2, sr andena2 article history edited by marcel gustavo hermes, ufpr, brazil received 14 august 2014 initial acceptance 04 september 2014 final acceptance 02 december 2014 keywords epiponini, new species, chartergellus zucchii. corresponding author sidnei mateus ffclrp universidade de são paulo av. bandeirantes, 3900 ribeirão preto-sp, brazil 14040-901 e-mail: sidneim@ffclrp.usp.br in this paper, the female, nest and males of a new species are described. comparative notes with c. atectus. c. punctatior and c. communis are given. chartergellus zucchii sp. n. mateus and andena (fig 1a and b) diagnosis the new species is similar to c. atectus and c. punctatior, in general coloration; however it is similar to c. communis in terms of the size. differently of c. atectus and c. communis, c. zucchii and c. punctatior have the propodeal valve narrow throughout. the mandible rises more abruptly than in c. communis, c. punctatior and in c. atectus, recalling that of c. afoveatus; the basal rim is absent in c. zucchii and in c. atectus. chartergellus zucchii is less hairy than c. punctatior, sharing it with c. atectus and c. communis. research article wasps 1 universidade de são paulo, ribeirão preto, sp, brazil 2 universidade estadual de feira de santana, feira de santana, ba, brazil s mateus et al a new species of the neotropical social wasp genus chartergellus 106 description size: 8.0 – 9.0 mm color: black species with reddish marks on frons, clypeus, mandible, gena and malar space; yellowish bands on upper part of gena, extending to middle region; scape blackish above, reddish beneath, the rest of antennomeres are blackish; yellow band on pronotal carina and dorsal margin of the pronotum; metanotum with yellow band on anterior region; metasoma blackish, without yellow bands. variation: some specimens have the frons, clypeus and mandible yellowish; pubescence on clypeus covering little less than a half; the ventral corner of the pronotum with a yellow spot; upper region of the gena blackish, becoming reddish toward inferior region; tarsomeres yellowish. head (fig 2a): (1) clypeus as long as wide, convex in profile, laterally sinuous; separated from the eyes; pubescence covering top half, bristles longer and denser on lower half than on top half; punctation deep, spaced, separated by more than 2.0 diameters; (2) eyes with short and spaced hairs; (3) frons and vertex with medial size punctation, deep, spaced, separated by more than 2.0 diameters; very short and spaced hairs; (4) malar space longer than 4th antennal article; (5) medial region of gena equal to eyes, narrowing toward upper region; punctation very spaced and shallow; pubescence present on top ¾, lower ¼ shinning, without pubescence; (6) mandible with very spaced and shallow punctation; raising abruptly, not forming a rim (fig 2b). mesosoma: (1) pronotum with short and dense pubescence, concentrated on upper part; punctation medium sized, separated by about 1.0 diameter; pronotal carina produced, slightly lamellated, extending to medial region; pronotal fovea oval, deep; ventral corner bulging; (2) mesopleura with very short and spaced pubescence; punctation medium sized, separated by about 1.0 diameter; dorsal groove shallow and wide; (3) upper plate of metasoma longer than wide, with large punctation separated by about 1.0 diameter; pubescence very short and spaced; lower plate with punctation very shallow and spaced, separated by more than 2.0 diameter; (4) scutum with very short and spaced pubescence, coriaceus; punctation medium sized, dense, separated by less than 1.0 diameter, becoming more spaced on central region; (5) scutellum with the same pattern of punctation and pubescence as that of scutum; (6) metanotum flat, with very spaced pubescence, very small, shallow and spaced punctures, separated by much more than 2.0 diameters; (7) propodeum with dense pubescence; spaced long hairs centrally, laterally more spaced; propodeal valve narrow throughout; (8) propodeal concavity shallow, wide. metasoma: (1) tergum i as long as wide, cap-shape; fig 1. chartergellus zucchii. a= lateral view; b= dorsal view. scale bars = 1.0 mm. fig 2. chartergellus zucchii. a= head, frontal view; b= mandible, frontal view. scale bars = 1.0 mm. sociobiology 62(1): 105-108 (march, 2015) 107 (2) tergum ii wider than long, coriaceous (3) sternum with the same pattern than tergum. fore wings: 7.5-8.0 mm, infuscated, costal region darker, venation brown; prestigma as long as wide. male: like female except that the gena and clypeus are narrower and entirely yellow; the mandible is blackish; the clypeus is covered with a silver pubescence; the frons is also yellowish with a central area becoming reddish; yellow marks are present beneath of the coxae and femur; yellow spot on upper region of mesopleuron. male genitalia: (1) paramere longer than wide, basal angle obtuse, apical angle truncate, long and curved spine, without bristles (fig 3a); (2) aedeagus with small triangular and irregular serration beneath, ventral process forming a ̈ u¨, area near serration darker (fig 3 b and c); (3) cuspis rounded apically, with small and spaced hairs (fig 3d); (4) digitus pointed apically, with small and spaced hairs (fig 3e). nest: we found two similar nests into the catuaba reserve, one populated and the other not. the description and photos refer to the populated nest. the nest is spherical, about 10 cm of diameter and 6 cm high (fig 4a), built on twigs with leaves incorporated on the top (fig 4b); the envelope is predominantly yellowish/grayish; long vegetable fibers; the construction lines are parallel giving a grooved aspect to the surface of the envelope; the entrance is short, (about 1,2 cm in diameter) located downward spout, forming a ring externally, measuring 10mm height (fig 4c); the combs are horizontal suspended from center to margins by multiple pedicels fused or buttressed sheet in the substrate, the second comb are fixed in the first one by the same structures, multiple pedicel fused or buttressed sheet (fig 4d). nest composition: two combs were found, one with 256 cells and the other with 226 cells, totaling 482 cells. one hundred seventy-two adults were collected, being 141 females and 31 males. due the great number of males, we classified the colony phase as “male producing”. etymology: the name zucchii is named in honor to prof. dr. ronaldo zucchi, in gratitude for his advice for many students’ in behavioral works over many years, a wasp worker and friend. distribution: a single populated nests was collected in fig 3. male genitalia. a= paramere, lateral view; b= aedeagus, ventral view; c= aedeagus, lateral view; d= cuspis, lateral view; e= digitus, lateral view. scale bars = 0.5 mm. fig 4. nest of chartergellus zucchii. a= spherical nest with envelope opened; b= detail of the nest attached on twigs with leaves incorporated; c= entrance with dark vegetal resin around the ring; d= combs connected by multiple pedicel fused or buttressed sheet. 27/xi/2002 by nascimento, f.s., in catuaba experimental farm (ufac), rio branco municipality, acre state, northern brazil (10° 04’ 36.’’s 67° 37’ 40.2’’w). it was found in a small orchard, hanging on a twig of a citrus sp., about 1.9 meters from the ground. holotype: 1 female catuaba experimental farm (ufac), rio branco municipality, acre state (10° 04’ 36.’’s 67° 37’ 40.2’’w); nascimento, f.s. col. [museu de zoologia da universidade de são paulo – são paulo, brazil (mzusp)] paratype: the paratypes belongs to the same nest of holoype and are deposited in the following institutions: 3 females and 3 males [museu de zoologia da universidade de são paulo, são paulo, brazil (mzusp)]; 3 females and 3 males [american museum of natural history, new york, usa (amnh)]; 3 females and 3 males [museu de zoologia da universidade estadual de feira de santana, feira de santana, brazil (mzfs)]; 3 females and 3 males [faculdade de filosofia, ciências e letras de ribeirão preto da universidade de são paulo, ribeirão preto, brazil (ffclrp-usp)]; 3 females and 3 males [natural history museum, london, uk (nhm)]. remarks chartergellus zucchii is grouped within those species in which the clypeus is separated from the eyes, however some aspects deserve consideration. chartergellus zonatus, c. communis, c. nigerrimus and c. zucchii have the eyes narrowly separated from the clypeus, while c. sanctus has the eyes widely separted from the clypeus. within the genus, c. amazonicus, c. atectus, c. golfitensis and c. punctatior have the clypeus touching the eyes, although, as pointed out by carpenter (pers. comm.) some specimens of c. puctatior have the clypeus narrowly separated from the eyes. such variation s mateus et al a new species of the neotropical social wasp genus chartergellus 108 is not uncommon in epiponini as cited by cely and sarmiento (2011) and carpenter et al. (2013) for the genus synoeca. the clypeus of c. zucchii is convex, like in c. punctatior and c. sanctus, and the pubescence cover the top half as all species within the genus, except c. amazonicus and c. atectus, which have the pubescence covering only the top third. the new species chartergellus zucchii, would be placed within couplet 3 of the richards’s key sharing the face reddish and the malar space longer with c. punctatior, c. communis and c. atectus, although, as cited above, c. zucchii may have the face yellowish. chartergellus communis and c. punctatior have the mandible forming a basal rim, a feature absent in c. zucchii and c. atectus. except c. communis, which has the eyes bare, the other three species have eyes with short and spaced hairs. the propodeal valves are narrow throughout in c. zucchii, c. punctatior and c. communis, and little wider in c. atectus. chartergellus punctatior has the scutum and pronotum with long and dense hairs, an exclusive feature of this species within the genus. we do not observe camouflage in the nest color, which differs from c. communis (richards, 1978; wenzel, 1998; mateus et al., 1999), c. golfitensis (chavarría-pizarro & westeberhard, 2010) and c. punctatior (wenzel, 1998). the grooves are not evident like in c. communis (richards, 1978; wenzel 1998; mateus et al., 1999). the entrance is similar to that of c. atectus (richards, 1978), different from others chartergellus species studied, the external ring of the entrance was impregnated with dark vegetal resin measuring 2 mm large, probably related to ant defense, a strategy also recorded in leipomeles dorsata, which uses ant guards on the leaf petiole (wenzel, 1998). such a strategy of ant defense was also seen in two nests of this species collected by sra and sm in presidente figueiredo, amazon state in 2005. both nests have dozens of small balls of resinous material attached to the leaf petiole. a similar strategy was observed in the related genus nectarinella: on the nest entrance and around the envelope of nectarinella xavantinensis a resinous material was impregnated (personal observation s.m.). cells may be lengthened after adult emergence due to accumulation of meconium layers. acknowledgment we thank fapesp (# 10/10027-5), and ffclrp-usp, departamento de biologia. references carpenter j.m., marques, o.m. (2001). contribuição ao estudo dos vespídeos do brasil. universidade federal da bahia, departamento de fitotecnia, bahia, cd-rom, 147p. carpenter, j.m., andena, s.r., noll, f.b., wenzel, j.w. (2013). well, what about intraspecific variation? taxonomic and phylogenetic characters in the genus synoeca de saussure (hymenoptera, vespidae). zootaxa, 3682: 421-431. doi: 10.11646/zootaxa.3682.3.3h cely, c.c., sarmiento, c.e. (2011). what about intraspecific variation? reassessment of taxonomic and phylogenetic characters in the genus synoeca de saussure (hymenoptera: vespidae: polistinae). zootaxa, 2899: 43-59. chavarria-pizarro, l., west-eberhard, m.j. (2010). the behavior and natural history of chartergellus a little-known genus of neotropical social wasps (vespidae polistinae epiponini). ethology, ecology and evolution, 22: 317-343. doi: 10.1080/03949370.2010.510035 hanson, p. e. & gauld, i.d. (1995): the hymenoptera of costa rica. oxford university press. 893 pages. mateus, s., noll, f.b., zucchi, r. (1999). caste differences and related bionomic aspects of chartergellus communis, a neotropical swarm-fouding polistinae wasps (hymenoptera: vespidae: polistinae: epiponini). journal of the new york entomological society, 107: 390-405. wenzel, j.w. (1998). a generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae, vespinae, polistinae). american museum novitates, 3224: 1-39. richards, o.w. (1978). the social wasps of the americas, excluding the vespinae. british museum (natural history), 580 p. west-eberhard, m.j., carpenter, j.m., gelin, l.f.f., noll, f.b. (2010). chartergellus golfitensis west-eberhard: a new species of neotropical swarm-founding wasp (hymenoptera: vespidae, polistinae) with notes on the taxonomy of chartergellus zonatus spinola. journal of hymenoptera research, 19: 84-93. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.6235sociobiology 69(1): e6235 (march, 2022) introduction intraspecific recognition in social insects involves the ability of the individuals to distinguish among their nestmates. this mechanism ensures colonial cohesion, as well as protection from intruders (oliveira & hölldobler, 1989). the recognition is mediated by chemical compounds called surface pheromones, which are volatile compounds of relatively low molecular mass or with longer chains (lenoir et al., 2001; sainz-borgo et al., 2011). the primary function abstract intracolonial recognition among social insects is performed mainly by means of cuticular hydrocarbons (chcs) that provide chemical communication, although their primary function is the avoidance of desiccation. therefore, the ability to adjust to climatic variation may be related to the composition of chcs. the hypothesis adopted in this work was that workers of the ant odontomachus brunneus, when exposed to higher or lower average temperatures, change the chcs composition, as a readjustment to the new conditions, and that this, in turn, leads to a change in intraspecific recognition capacity. to test this hypothesis, colonies of o. brunneus reared in the laboratory were subdivided into four groups. two groups were kept at the same temperature, in order to assess the effect of isolation itself, while one group was kept at high temperature and another was kept at low temperature. two groups were maintained at 25 °c, with no further conditions imposed. subsequently, encounters were induced between individuals from these groups and from the high and low temperature groups, followed by the extraction of chcs from each individual. the results indicated significant differences in recognition time and chc composition between the high/low temperature groups and those kept at 25 °c. antennation time during nestmate encounters was significantly longer for the groups submitted to temperature treatments (high and low), compared to those kept at 25 °c, suggesting recognition difficulty. in order to adjust to changing temperature conditions, o. brunneus undergoes changes in the composition of chcs and in intraspecific recognition capacity. sociobiology an international journal on social insects luiz c santos jr1, kamilla b michelutti1, rafaella c bernardi1, emerson p silva2, claudia al cardoso1, william f antonialli jr1 article history edited by kleber del-claro, ufu, brazil received 11 february 2021 initial acceptance 09 april 2021 final acceptance 29 november 2021 publication date 07 february 2022 keywords cuticular hydrocarbons; temperature; chain length; intraspecific recognition. corresponding author luiz carlos santos junior ecology laboratory center for integrated environmental monitoring and analysis universidade estadual de mato grosso do sul (uems) cidade universitária de dourados caixa postal 351 cep: 79804-97 dourados-ms, brasil. e-mail: lc.santosjunior@yahoo.com.br of these compounds is to protect against water loss, but by virtue of their structural complexity, they have evolved to form recognition pheromones that transmit the identity of an individual to other members of the insect society (blomquist & bagnères, 2010). in insect society, cuticular hydrocarbons (chcs, surface pheromones) encode chemical recognition signals that are signatures characteristic of each species, colony, and even caste (antonialli-junior et al., 2007; blomquist & bagnères, 2010). hence, chcs are important mediators of intraspecific 1 ecology laboratory, center for integrated environmental monitoring and analysis, universidade estadual de mato grosso do sul-ms, brazil 2 ecology laboratory, center for integrated environmental monitoring and analysis, universidade federal da grande dourados, dourados-ms, brazil research article ants you smell different! temperature interferes with intracolonial recognition in odontomachus brunneus mailto:lc.santosjunior@yahoo.com.br luiz c santos jr et al. – temperature interferes with intracolonial recognition 2 recognition, especially in ants (vander-meer & morel, 1998). these nonpolar hydrocarbons, which contain only carbon and hydrogen in their structures, may be either saturated (alkanes) or unsaturated (alkenes and alkynes, which are very common in insects) (blomquist & bagnères, 2010). the formation of a colonial chemical signature arises from a complex range of cuticular chemical compounds that are homogenized among individuals by behaviors such as allogrooming, where the postpharyngeal gland (ppg) acts as a mixing organ (boulay et al., 2000a; lenoir et al., 2001; soroker et al., 1994, 1998; soroker et al., 2003). this social interaction among nestmates is extremely important for the integration of workers in the colony, forming a characteristic odor of each colony. for example, in camponotus fellah, when workers were isolated individually from the mother colony for up to 20 days, their ppg and chcs presented different characteristics, compared to the non-isolated nestmates, and they were generally attacked when they were reintroduced into the mother colony, due to their different cuticular chemical profile. however, when the period of separation was reduced (to between 3 and 10 days), the isolated workers tended to perform the allogrooming behavior with greater intensity, as a way to rapidly regain the odor of the colony (boulay et al., 2000b). despite its relative uniformity, the colony odor is dynamic and there may be quantitative changes in the relative proportions of the different cuticular hydrocarbon classes (vander-meer et al., 1989; provost et al., 1993; dahbi & lenoir 1998b; nielsen et al., 1999). the chemical profiles of insects are strongly influenced by genetic and environmental factors (blomquist & bagnères, 2010). therefore, since one of the functions of chcs is to create a barrier against desiccation, it may be expected that the variation of physical factors, such as temperature and relative humidity, is likely to affect their composition (gibbs et al., 1997; boulay et al., 2017; menzel et al., 2017; michelutti et al., 2018). for example, species in humid climates tend to have more alkenes and fewer linear alkanes, compared to species in drier habitats, which can be explained by the different waterproofing capabilities of these compounds in the cuticle (menzel et al., 2017). on the other hand, particular chc compositions may also affect signaling properties during intraspecific interactions (chung & carroll 2015). several studies have discussed the dual function of chcs (gibbs, 2007; chung & carroll, 2015; boulay et al., 2017). one mechanism that ants use to adjust to new environmental conditions involves modification of the cuticular composition (menzel et al., 2017). hence, the need of the insect to protect itself against desiccation may negatively influence the capacity to transmit information, and vice versa (gibbs, 2007; chown et al., 2011; menzel et al., 2017). for example, there is strong evidence to suggest that compounds with intermediate melting points and volatility, such as alkenes and branched alkanes, are involved in both desiccation resistance and chemical communication (chung & carrol, 2015). some species produce other compounds responsible for communication, such as unsaturated compounds or branched alkanes, which can increase the risk of dehydration. therefore, increased permeability of the surface lipids at high ambient temperatures may also be a consequence of the need to interact with nestmates (chown et al., 2011). so far, however, no study has investigated the interdependence of these two chc functions. therefore, in the present work, our hypothesis was that exposure of o. brunneus worker ants to high or low temperature would lead to intraspecific non-recognition, due to changes in the chcs composition during the attempt to adjust to new environmental conditions. materials and methods three colonies of o. brunneus were collected in the urban perimeter of the city of dourados, mato grosso do sul state, brazil (22º13’16’’ s; 54º48’20’’ w), in september of 2016. all three colonies were collected from hollow trunks of caesalpinia pluviosa (fabaceae) using tweezers and plastic containers, and were transferred to the laboratory in artificial nests. these nests were constructed using plastic trays and plaster molds that simulated the nest chambers, and were connected to a foraging arena where food was offered to the colonies. the colonies were kept at a constant temperature of 25 ºc during an acclimation period of seven days, and were fed ad libitum with water and honey on dampened cotton inside an eppendorf tube. as a source of protein, 4 last instar larvae of tenebrio molitor were offered every 4 days. this was the standard diet used during the experiment. after the acclimation period, four groups were separated from each of the three mother colonies, according to the procedure described by sorvari et al. (2008), with adaptations. the groups were subjected to different temperatures: group “a”, composed of workers and queen, was kept at a constant temperature of 25 ± 2 ºc; group “b”, composed of 50 workers, was kept at a constant temperature of 25 ± 2 ºc; group “c”, composed of 50 workers, was kept at a constant temperature of 35 ± 2 ºc; and group “d”, composed of 50 workers, was kept at a constant temperature of 15 ± 2 ºc (fig 1). all the groups were maintained for 30 days, with the same standard diet, in biochemical oxygen demand (bod) chambers (model 347 cd, fanem, são paulo, brazil), where it was possible to control the temperature of each group, at constant humidity of 65 ± 5% and 12 h photoperiod. in this way, it was possible to assess the effect of isolation of the nestmates (when exposed to the same temperature condition, in this case), as well as the effect of exposure of the nestmates to high and low temperatures. the temperatures chosen were based on the averages for the two main seasons in the city of dourados in the previous sociobiology 69(1): e6235 (march, 2022) 3 year (zavatini, 1992). hence, it was possible to test the effect of temperature using values above the average temperature of the hot and humid season, which was 31.2 ± 3.2 ºc, and below the average temperature of the cold and dry season, which was 20.1 ± 5.0 ºc, according to data from the weather station of the brazilian agricultural research agency (embrapa). analysis of the tolerance levels of different treatment groups during ant encounters soon after the colonies were collected, the cuticular compositions of 10 individuals from each colony were analyzed. this enabled identification of any changes in the odors of the colonies without any type of treatment, during the course of the experiment. in order to evaluate whether low or high temperature, and/or isolation itself, might affect the level of intraspecific recognition and the antennation time, encounters were induced between one ant from the 25 ºc group (group “a”) and another ant from the other groups, resulting in four sets of induced behavioral encounters (fig 1). these encounters were conducted in a petri dish (100 mm x 20 mm), where a worker was kept trapped inside a glass container (50 mm x 20 mm) turned upside down in the center of the petri dish, in order to minimize the stress caused by manipulation, which might influence the level of recognition. a second worker was then placed outside the glass container (fig 2). after allowing one minute for the workers to become accustomed to the environment, the glass container was removed from the center of the petri dish, enabling the encounter between the two workers, which were then monitored for 15 minutes without interruption. the sets of induced behavioral encounters between the groups were organized as follows: 30 encounters between workers of group “a” itself, used as a parameter for evaluation of the other behavioral encounters; 30 encounters between fig 1. sample design showing how the groups of ants were formed from the mother colony, together with the corresponding temperatures at which the workers were maintained prior to the induced encounters. luiz c santos jr et al. – temperature interferes with intracolonial recognition 4 workers from groups “a” and “b”, in order to assess whether isolation could lead to increased antennation time; 30 encounters between groups “a” and “c”, in order to assess whether higher temperature, relative to group “a”, could lead to longer antennation time between workers; and 30 encounters between workers in groups “a” and “d”, in order to assess whether low temperature, relative to group “a”, could lead to longer antennation time (fig 1). the same ant was not used more than once, and at the end of the observation period, the ants were used for subsequent chemical analyses. before each induced encounter, the arena was sterilized with a towel soaked in alcohol. the same ant was not used in other behavioral tests, and as soon as the tests were completed, the ants were immediately used for extraction and analysis of the chcs. during the 15 minutes of blinded observations, the observer did not know which group the workers belonged to, hence minimizing any possible influence of the observer in evaluation of the ant behavior. the following behavioral parameters were considered: “ignore” (tomas et al., 2004), “touch the body of another worker”, “escape” (suarez et al., 2004), “attempt to apprehend”, “apprehend” (mercier et al., 1997), “antennal boxing”, “body lift, abdomen showing” (monnin, 1999), and “fight” (mercier et al., 1997). the execution time of each of these behaviors was counted from the first contact between the ants. in order to evaluate the level of aggression (if present) during the encounters between ants from the different treatments, the behaviors exhibited were assigned a score from 0 to 2, based on suarez et al. (2004), with modifications: 0 for “touch”, “escape”, and “ignore”; 1 for “attempt to apprehend”, “apprehend”, “antennal boxing”, “elevation of the body”, and “elevation of the abdomen”; and 2 for “fight”. for each encounter, an arithmetic average of the scores related to the levels of aggression presented was compiled. for the “touch the body of another worker” behavior, the time spent in antennation between the workers was measured, enabling calculation of the average time spent in intraspecific recognition during the induced encounters. the antennation time was used here as a parameter reflecting the degree of difficulty in the recognition of nestmates (starks et al., 1998). analysis of the effect of temperature on cuticular chemical composition for evaluation of the effects of the different treatments on the cuticular composition, the ants were sacrificed by freezing, followed by extraction of the cuticular chemical compounds from the 30 workers of each experimental set used in the behavioral encounters. each worker was immersed for 3 min in a glass vessel containing 2 ml of hexane (hplc grade, tedia). the resulting extracts were dried under a fume hood and stored in a freezer (at −20 °c) for a maximum of 30 days. for chromatographic analysis, each extract was solubilized in 400 μl of hexane. the analyses employed a gas chromatograph (gc-2010 plus, shimadzu, kyoto, japan) equipped with a mass detector (ultra 2010, shimadzu, kyoto, japan). the compounds were separated using a db-5 fused silica capillary column (j & w scientific, folsom, california, usa) coated with 5% phenyl-dimethylpolysiloxane (30 m length × 0.25 mm diameter × 0.25 μm film thickness). the analysis conditions were as follows: 1 μl injection, in splitless mode; heating program with initial temperature of 150 °c, ramp to 300 °c at 3 °c min−1, and maintaining the final temperature for 10 min; and injector temperature of 250 °c. the temperatures of the detector and transfer line were 300 and 280 °c, respectively. the mass spectrometry parameters included electron impact ionization voltage of 70 ev, 0.3 s scan interval, and scanning in the range from m/z 45 to 600. fig 2. scheme showing the procedure for the induced encounters between nestmate ants that had previously been maintained under different temperature conditions for 30 days. sociobiology 69(1): e6235 (march, 2022) 5 the compounds were identified from the retention index (bernier et al., 1998; howard, 2001; smith et al., 2012; moore et al., 2014; weiss et al., 2014; soares et al., 2017), comparison with databases (nist 21 and wiley 229), and interpretation of the mass spectra. the retention index was calculated employing a mixture of linear alkanes (c7-c40, purity ≥ 95%, sigma-aldrich) as an external reference. all the identified compounds were used in the statistical analysis. the major compounds for the different groups of ants were considered to be those that represented at least 10% of the total relative area. statistical analysis for all the behavioral encounters, the normality of the data was evaluated using the average time of interaction between the workers of each set. after identification of nonnormality of the data distribution, the kruskal-wallis test was applied to identify any significant differences between the median values for the time spent in antennation behavior during the encounters. for evaluation of whether the different sets of induced behavioral encounters caused significant differences in the cuticular chemical compositions of the ants, relative to group “a”, a discriminant analysis was applied, using the relative abundances (areas) of all the compounds from the cuticles of the different ant groups. for evaluation of the effects of low and high temperatures on the chain lengths of the chcs, discriminant analyses were applied separately for the different groups of compounds (short, intermediate, and long chains) from different ant groups, relative to group “a”. the compounds were separated into short, intermediate, and long chains based on the methodology of menzel et al. (2017), with adaptations. the distance analysis of mahalanobis was applied to evaluate the distance between the different groups, relative to the control, in a pairwise manner. results observations were made for a total of 30 h of interactions between the workers in the different sets of induced behavioral encounters. during all the encounters, the only behaviors observed were “touching the body of the other worker” and “ignoring”, representing “0” aggression. however, as shown in fig 3, significant differences were observed between the average antennation times of workers during “touching the body of the other worker” behavior, when the workers were kept at different temperatures (sets 3 and 4). no significant differences in antennation times were noted between the ants of set 1 and of set 2 (fig 3). a total of 17 chromatographic peaks were detected for the cuticle extract from the group “a” workers (table 1). of these, 8 were linear alkanes, 7 were branched alkanes, and 2 were alkenes. linear alkanes were the most representative, corresponding to 68.55% of the total area, followed by branched alkanes (18.09%) and alkenes (13.36%) (fig 4). three compounds were considered as major components: the linear alkanes octadecane (39.25%) and heptacosane (12.09%), and the alkene eicosene (11.39%), as shown in table 1. the cuticular compounds varied from c18 to c32. compounds with short, intermediate, and long chains were considered to be those with chain lengths in the ranges c18 to c21, c22 to c27, and c28 to c32, respectively. a total of 16 compounds were identified in the extract from the group “b” workers (table 1), including 7 linear alkanes, 7 branched alkanes, and 2 alkenes. the linear alkanes represented 67.27% of the area, the branched alkanes 22.87%, and the alkenes 9.86% (fig 4). the major compounds were octadecane (37.90%) and heptacosane (17.24%). for the extract from the group “c” workers, 19 compounds were detected (table 1), including 10 linear alkanes, 7 branched alkanes, and 2 alkenes (fig 4). the linear alkanes represented 65.71% of the area, the branched alkanes 26.68%, and the alkenes 7.61% (fig 4). the major compounds were octadecane (25.50%) and heptacosane (25.32%). for the group “d” workers, 19 compounds were detected in the extract, including 10 linear alkanes, 7 branched alkanes, and 2 alkenes (table 1). the linear alkanes represented 57.81% of the area, the branched alkanes 33.19%, and the alkenes 9.00% (fig 4). the major compounds were octadecane (14.57%), heptacosane (23.75%), and 12, 16-dimethyldotriacontane (11.85%). fig 3. median and standard deviation values for the antennation times measured during the behaviour “touching the body of the other worker”, for encounters between odontomachus brunneus workers previously maintained under different temperature conditions (n = 30). *significant difference (p < 0.05, kruskal-wallis = 85.69), compared to set 1. luiz c santos jr et al. – temperature interferes with intracolonial recognition 6 the cuticles of groups “c” and “d” (exposed to 35 ºc and 15 ºc, respectively) presented increases in the number of linear alkanes, relative to group “a”. on the other hand, the cuticles of the workers in groups “c” and “d” showed reductions in the abundances of linear alkanes and alkenes, but increases of branched alkanes, relative to group “a” (fig 4). the compounds hexacosane and octacosane only occurred in the cuticles of the ants exposed to low and high temperature, while 3-methyldotriacontane occurred in these groups and in group “b”. on the other hand, 5-methylheptacosane only occurred in the cuticles of the ants in group “a”. pentacosane was only observed for the ants in group “b” (table 1, fig 4). time cri lri compound group “a” (25 ºc) group “b” (25 ºc) group “c” (35 ºc) group “d” (15 ºc) abundance (% ± standard deviation) 10.77 1800 1800 octadecane * 39.25 ± 11.32 37.90 ± 8.77 25.50 ± 14.58 14.57 ± 6.39 13.25 1900 1900 nonadecane 3.79 ± 5.24 1.71 ± 0.48 1.40 ± 0.73 1.50 ± 0.75 15.71 1990 1990 eicosene * 11.39 ± 1.54 8.78 ± 1.85 6.04 ± 3.64 7.75 ± 4.62 15.88 2000 2000 eicosane 4.27 ± 0.76 2.99 ± 0.68 2.21 ± 1.24 2.82 ± 1.62 19.01 2119 x-methylheneicosane 1.67 ± 2.10 1.01 ± 0.23 0.71 ± 0.39 0.91 ± 0.50 21.12 2200 2200 docosane 5.79 ± 1.03 4.24 ± 1.12 3.03 ± 1.84 4.54 ± 2.78 24.53 2318 x-methyltricosane 1.14 ± 0.37 0.92 ± 0.18 0.53 ± 0.37 0.78 ± 0.51 26.39 2400 2400 tetracosane 1.43 ± 0.31 0.98 ± 0.33 0.76 ± 0.58 1.35 ± 0.89 29.07 2500 2500 pentacosane 0.50 ± 0.58 1.79 ± 1.97 1.19 ± 1.66 31.51 2600 2600 hexacosane 0.73 ± 1.52 0.47 ± 0.37 33.91 2700 2700 heptacosane * 12.09 ± 5.08 17.24 ± 5.83 23.75 ± 14.40 25.32 ± 12.72 35.05 2750 2751 5-methylheptacosane 0.61 ± 0.38 36.21 2800 2800 octacosane 0.44 ± 0.32 0.53 ± 0.26 38.45 2900 2900 nonacosane 1.42 ± 0.57 2.21 ± 0.97 6.10 ± 4.72 5.53 ± 4.80 42.25 3070 3070 3-methyltriacontane 3.24 ± 2.51 4.48 ± 3.37 6.43 ± 1.37 8.25 ± 2.72 42.33 3078 3078 13,17-dimethyltriacontane 2.53 ± 2.18 2.22 ± 1.73 1.40 ± 1.63 2.02 ± 1.96 45.85 3253 3254 12,16-dimethyldotriacontane 5.81 ± 4.98 7.56 ± 5.98 7.82 ± 3.60 11.85 ± 6.08 45.94 3259 3260 2-methyldotriacontane 3.08 ± 2.81 3.37 ± 3.06 1.62 ± 1.18 2.88 ± 1.74 46.28 3274 3275 3-methyldotriacontane 3.31 ± 3.01 8.17 ± 3.62 6.50 ± 3.41 46.31 3271 3271 tritriacontene 1.97 ± 1.93 1.08 ± 1.18 1.57 ± 1.60 1.25 ± 1.62 * major compounds. cri = calculated retention index. lri = literature retention index. table 1. relative abundances (peak areas) of the compounds detected in the cuticles of odontomachus brunneus workers submitted to different treatments. qualitative and quantitative differences were observed between the results for group “a” and those for the different treatments. for example, the ants in groups “c” and “d” (submitted to high and low temperatures, respectively) showed lower abundance of short-chain compounds and increase of long-chain compounds, compared to group “a” (table 1). discriminant analysis revealed significant differences in cuticular chemical composition, considering all the compounds as well as when the compounds were separated into the three chain length categories (fig 5, table 2). the discriminant analysis f-values showed that the cuticles of the ants in groups “c” and “d” presented significantly greater differences, compared to the ants in group “b”, relative to group “a” (table 2). fig 4. abundance of compounds of each class present in the cuticles of odontomachus brunneus workers submitted to different treatments. sociobiology 69(1): e6235 (march, 2022) 7 discussion the results demonstrated that the effects of exposure at different temperatures increased the difficulty in nestmates recognizing each other, probably due to the modification of cuticle chemical composition, as a function of readjustment to different temperatures. on the other hand, the ants of set 2, which were subjected to the same temperatures, but were isolated, did not show any change in intraspecific recognition. aggression was absent during all the induced behavioral encounters, although longer antennation times were observed during “touch the body of another worker” behavior for sets 3 and 4 (35 ºc and 15 ºc, respectively), compared to set 1 (25 ºc). antenna behavior allows these insects to capture chemical signals that identify them as members (or not) of the same colony (hefetz, 2007). hence, it can be inferred that a longer antennation time is related to increasing difficulty of recognition as nestmates (starks et al., 1998), which, on the other hand, may be related to any change in what howard and blomquist (2005) called a chemical signature of the colony. this recognition is fundamental for social insects to ensure protection of the colony against invasion by other insects, especially members of other colonies (hefetz, 2007). the results of the induced behavioral encounters between workers in set 1 and set 2, submitted to the same temperature condition, reinforced the hypothesis outlined above, since the time of contact between them did not suggest any difficulty in recognition (fig 3). therefore, the results demonstrated that temperature may, in fact, be related to alteration in the level of recognition among nestmates. in addition, it is important to note that the treatment processes may have caused some stress additional to that associated with increase or decrease of the temperature, since the handling of the ants may also have led to discomfort, which could also have influenced the levels of recognition. fig 5. scatter diagram obtained using the two canonical roots for differentiation of the compounds present in the cuticles of odontomachus brunneus workers submitted to different treatments, based on the relative abundances (peak areas) of the cuticular compounds. all compounds short-chain intermediate-chain long-chain f p f p f p f p treatments group “a” – group “b” 18.655 <0.001 5.869 <0.001 26.472 <0.001 4.272 <0.001 group “a” – group “c” 38.537 <0.001 12.830 <0.001 34.136 <0.001 35.600 <0.001 group “a” – group “d” 52.150 <0.001 30.782 <0.001 41.891 <0.001 49.258 <0.001 discriminant 24.121 <0.001 13.547 <0.001 17.948 <0.001 19.101 <0.001 wilks’ lambda 0.004 0.248 0.105 0.074 table 2. values of p and f for the mahalanobis distance analysis applied to the cuticle hydrocarbon data for odontomachus brunneus workers submitted to different treatments. luiz c santos jr et al. – temperature interferes with intracolonial recognition 8 however, chemical analysis of the cuticles of ants subjected to temperatures of 15 ºc and 35 ºc (groups “c” and “d”) revealed significant differences, compared to the ants subjected to 25 ºc (group “a”). therefore, it is possible that this alteration of cuticular composition could act as a mechanism that enables ants to readjust to new temperature conditions (menzel et al., 2017, 2018; michelutti et al., 2018; sprenger et al., 2018; duarte et al., 2019). the standard deviations of the means for the relative abundances (peak areas) of the compounds obtained from o. brunneus were relatively high (table 1), which could be explained by the fact that the workers originated from different colonies. previous studies have shown differences among the individual members of a colony (cuvillier-hot et al., 2001) and between members of different colonies (wagner et al., 2001). these differences may be due to genetic factors (wagner et al., 2001; howard & blomquist, 2005), as well as environmental factors (buczkowski et al., 2005; howard & blomquist, 2005). as shown in fig 5, the effects of temperature led to significant changes in cuticular chemical composition, with both qualitative and quantitative changes being observed in this study. qualitatively, there were increases in the numbers of linear alkanes in the ants submitted to both low and high temperatures, compared to group “a”. on the other hand, there was increased abundance of branched alkanes, but decreases of linear alkanes and alkenes. differently, in earlier work, wagner et al. (2001) found variation only in the relative abundances of the compounds present in the cuticle of pogonomyrmex barbatus, when the ants were submitted to heat and low humidity. the compounds hexacosane and octacosane only occurred in the cuticles of ants subjected to 35 ºc and 15 ºc (groups “c” and “d”), while 3-methyldotriacontane was present in groups “b”, “c”, and “d”, and 5-methylheptacosane only occurred in group “a”. these findings suggested that the ant cuticle has the ability to respond to temperature variation, sometimes synthesizing new compounds (or failing to do so). similarly, in other work, michelutti et al. (2018) identified qualitative and quantitative changes in the chcs of three species of wasps submitted to different temperatures, compared to the controls. the literature includes studies describing the ability of the cuticle to respond to the environmental conditions in which the animal is found (wagner et al., 2001; menzel et al., 2017; michelutti et al., 2018; duarte et al., 2019). it is likely that the occurrence (or not) of certain compounds may be related to the response to changes in the environment in which the animal is located. in insects, this ability to adjust the cuticular composition involves making the chc layer more viscous, to avoid water loss, when average temperatures are higher and relative humidity is low. conversely, the layer can be made more fluid, in order to facilitate the exchange of information between individuals (menzel et al., 2017; sprenger et al., 2018). under low temperature conditions, the cuticle tends to become less fluid, so to compensate for this, an increase in the abundance of branched alkanes would be expected, since these compounds increase the fluidity of the layer. the resulting increase of compound mobility makes the transfer of information by the cuticle more efficient (menzel et al., 2017). on the other hand, at high temperatures, a reduction in the abundance of alkenes would be expected, since these compounds liquefy at lower temperatures, compared to the corresponding linear alkanes (gibbs & pomonis, 1995). importantly, the trade-off between the requirements for waterproofing and communication makes the evolution and plasticity of chc profiles an intriguing field of research, with many open questions (sprenger et al., 2018). when o. brunneus responds to changes in temperature by changing the amounts of different classes of compounds, this makes recognition between nestmates more difficult. hence, it is necessary to perform tests with specific compounds, in order to detect key compounds responsible for these changes. although the number of linear alkanes increased at the highest temperature, there was a reduction of their abundance, while there was increased abundance of branched alkanes. linear alkanes are the main compounds required to avoid desiccation in social insects (gibbs, 1998; hefetz, 2007; wagner et al., 2001), but the branched alkanes (mono or dimethyl) are the main compounds required to mediate communication between nestmates (howard & blomquist, 2005). it is well known that methyl alkanes, and notably alkenes, liquefy at lower temperatures, compared to linear alkanes (gibbs, 1998). that is, methyl alkanes and alkenes have lower melting points than linear alkanes (gibbs & pomonis, 1995). hence, according to menzel et al. (2017), an increasing need for waterproofing should increase the abundance of linear alkanes and reduce the abundances of branched alkanes and alkenes. the ant groups subjected to 35 oc and 15 oc (groups “c” and “d”) both showed a reduction in the abundance of compounds with shorter chain length, together with increased abundance of compounds with longer chain length (table 1). in addition, analyses considering compounds of short, intermediate, or long chain lengths indicated a significant difference between ants of both group “c” and group “d”, compared to group “a” (table 2). these results were in agreement with gibbs et al. (1997), who found that under desiccation conditions, drosophila melanogaster presented chcs with longer chain lengths. gibbs (1998) reported that the longer chains of arthropod chcs tended to liquefy at higher temperatures. furthermore, gibbs and pomonis (1995), in experiments with synthetic hydrocarbons, found that increase of the chain length caused a corresponding increase in the melting temperature, and that the position of the alkane branching could also influence the melting temperatures of compounds present in social insects. essentially, the presence sociobiology 69(1): e6235 (march, 2022) 9 of compounds with longer chains may indicate a greater ability of the cuticle to withstand high temperatures. however, menzel et al. (2017) found no differences in the chain lengths of chcs present in ant species from different populations of the genera crematogaster and camponotus. it should be noted that insect surfaces are composed of more than a single pure compound, and not only hydrocarbons (gibbs & pomonis, 1995). the fusion of natural lipid mixtures occurs at the melting temperature of the cuticular compounds, with interactions between saturated and unsaturated chcs resulting in nonlinear effects (gibbs, 1998). furthermore, compounds with intermediate melting and volatility temperatures, such as alkenes and methyl alkanes, may have important effects in desiccation resistance and chemical communication (chung & carroll, 2015). conclusions the findings of this work showed that the cuticles of worker ants could undergo temperature-dependent compositional changes, with some categories of compounds decreasing in abundance, while others could increase as a means of compensating for the imposed conditions. on the other hand, although isolation itself caused alteration of the cuticular chemical composition, most likely due to the absence of homogenization by means of social interactions, the alteration was not sufficient (during the time tested) to lead to significant changes in the ability to recognize nestmates. the results validated the hypothesis adopted in this work, namely that o. brunneus undergoes changes in the composition of chcs, in order to adjust to changing temperature conditions, while such changes in chc composition also have consequences in terms of alteration of intraspecific recognition capacity. acknowledgements the authors thank coordenação de aperfeiçoamento de pessoal de nível superior (capes) for doctoral scholarships awarded to the first and second authors. fundação de apoio ao desenvolvimento do ensino ciência e tecnologia do estado de mato grosso do sul (fundect) awarded a doctoral scholarship to the third author (fundect n° 03/ 2014). conselho nacional de desenvolvimento científico e tecnológico (cnpq) provided productivity scholarships (wfaj: grant number 307998/2014-2; calc: grant number 310801/2015-0). author contributions conceived and designed the experiments: lc santosjunior and wf antonialli-junior. performed the experiments: lc santos-junior, ep silva, kb michelutti, and rc bernardi. analyzed the data: lc santos-junior, kb michelutti, cal cardoso, and wf antonialli-junior. wrote the paper: lc santos-junior and wf antonialli-junior. references antonialli-junior wf, lima sm, andrade lhc, súarez yr (2007). comparative study of the cuticular hydrocarbon in queens, workers and males of ectatomma vizottoi (hymenoptera, formicidae) by fourier transform infrared photoacoustic spectroscopy. genetics and molecular research, 6: 492-499. antonialli-junior wf, súarez yr, izida t, andrade lhc, lima sm (2008). intraand interspecific variation of cuticular hydrocarbon composition in two ectatomma species (hymenoptera: formicidae) based on 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(1998). pheromone communication in social insects: ants, wasps, bees, and termites. colorado, co: westview press. wagner d, tissot m, gordon d (20001). task-related environment alters the cuticular hydrocarbon composition of harvester ants. journal of chemical ecology, 27: 1805-1819. doi: 10.10 23/a:1010408725464 weiss k, parzefall c, herzner g (2014). multifaceted defense against antagonistic microbes in developing offspring of the parasitoid wasp ampulex compressa (hymenoptera, ampulicidae). plos one, 9: 1-14. doi: 10.1371/journal.pone. 0098784 zavatini ja (1992). dinâmica atmosférica no mato grosso do sul. geografia (rio claro). ageteo, 17: 65-91. 945 cloning and characterization of phospholipases a2 and hyaluronidase genes from the venom of the honeybee apis mellifera carnica (hymenoptera: apidae) by chunsheng hou1,2,3, liqiong guo1,2, jianrong wang1,2, linfeng you1,2, junfang lin1,2,﹡, wuhua wu1,2, chengshun wu1,2 &teng wang 1,2 abstract bee venom contains the allergic enzymes phospholipases a2 (pla2) and hyaluronidase. these enzymes have been extensively studied as therapeutic modalities because of their proven effects in pharmaceutical and clinical applications. the cdna cloning of pla2 and hyaluronidase was amplified by rt-pcr from the total rna of the venom gland of a honeybee (apis mellifera carnica). the lengths of the pla2 and hyaluronidase of apis mellifera ligustica were 504 and 1146bp, respectively. the genes of pla2 and hyaluronidase shared 90.94% and 96.65% homologies with a. mellifera ligustica and apis cerana cerana, respectively. some similar pla2 and hyaluronidase were also found in the venom of other bee species, we analyzed their sequences and compared them with those of other sources. a notable finding was that the two genes differed from those of a. mellifera ligustica and a. cerana cerana. the positions of the disulfide bonds of pla2 and hyaluronidase were also completely different from those previously reported. we used the available sequences to construct a phylogenetic tree and discovered that these two genes of a. mellifera carnica belonged to the western honeybee, and was more closely related to that of a. mellifera ligustica than to any other insect. keywords: phospholipase a 2 ; hyaluronidase; honeybee venom; cdna clone; sequence analysis; apis mellifera carnica 1 college of food science, south china agricultural university, guangzhou city, guangdong 510640, people’s republic of china 2 institute of biomass energ y, south china agricultural university, guangzhou city, guangdong 510640, people’s republic of china 3 institute of sci-tech information, guangdong academy of agricultural science, guangdong 510640,people’s republic of china ﹡corresponding author. e-mail address: junfanglin2003@yahoo.com.cn( jf.lin). 946 sociobiolog y vol. 59, no. 3, 2012 introduction bee venom is a natural toxin that contains numerous enzymes, peptides and other active substances. these substances are widely used in medical practice due to their antibacterial, anti-inflammatory, hypotensive, and immunity-promoting activities, among others (habermann 1972; gauldie et al.1976; zhang et al.2003). a number of phospholipase a2 (pla2) and hyaluronidase enzymes has already been cloned from different sources (luo et al.2010; sylvia et al.2010). some of these enzymes have improved cognitive ability in alzheimer’s disease patients (evelin et al.2009),demonstrated anti-inflammatory (bonfim et al.2009) as well as antibacterial activities (emmanuel & gérard 2000a), and inhibited the migration effects of cancerous cells (raoudha et al.2009). thus, several studies have recently focused on isolating the active components of bee venom to determine the underlying action mechanism (murat et al.2009; timothy et al.2009). the enzymes in bee venom are mainly pla2 and hyaluronidase, which account for 11% to 15% of the bee venom dry weight. pla2 (habermann 1972; kuchler et al.1989; shen et al.2002a), and hyaluronidase (soldatova and mueller 1998; gmachl and kreil 1993; shen et al. 2002b) in the venom glands of a. mellifera ligustica and a. cerana cerana worker bees have been cloned and their nucleotide sequences have been reported. the structure and catalytic mechanism of bee venom pla2 and hyaluronidase have also been studied (robert et al.1996; housley et al.2000). the molecular characterization of the pla2 gene from the bumblebee bombus ignites has been performed as well, and its activity has been verified (yu et al.2009). hyaluronidase from rhynchium brunneum has been cloned (xu and han 2008), and shown to be highly similar with that from vepula vulgaris. similarly studies on other species such as scorpions and snake have been conducted (valdez-cruz et al. 2007; frey francisco et al.2010). a. mellifera carnica is one of the four most superior bee species. it has a strong resistance against mites as well as excellent foraging and feed-saving abilities (wang 2005). pla2 and hyaluronidase are believed to be effective bee venom spreading factors. however, the absence of protein sequences data on these enzymes hinders the verification of their functions. there is no available report on the pla2 and hyaluronidase genes of a. mellifera 947 hou, c. et al. — characterization of venom of apis mellifera carnica carnica, although those of other bee species have been reported. whether a. mellifera carnica also contains these two genes in its venom is not yet clear. the molecular structures of the two genes are also not known. in the current paper, we report the nucleotide sequences of the pla2 and hyaluronidase genes of a. mellifera carnica (the genbank accession numbers are jq900376 and jq900377, respectively). we also compare them with those of a. mellifera ligustica and a. cerana cerana, and perform a phylogenetic analysis. materials and methods experimental material honeybee (a. mellifera carnica) workers were obtained from the apiary of the institute of jiangxi apiculture in china. the stingers were collected from the venom gland of the bees and immediately placed in liquid nitrogen for storage until use. the bacterial strain escherichia coli dh5α was from the laboratory of biomass energ y of the college of food science of the south china agricultural university. restriction endonucleases (bamh i and xho i), taq polymerase, x-gal, isopropyl-β-d-thio-galactoside(iptg), and dldna 4500 marker were purchased from the takara company. a first-strand cdna synthesis kit was obtained from the shanghai gereray company, and a pgem-t vector kit was from the promega company. all other chemical reagents used were available from our laboratory. general experimental procedures a pair of pcr primers was designed based on the sequences of pla2 and hyaluronidase from a. mellifera ligustica (nico et al.2005; scott et al.1990). the forward and reverse primers of the sequences are listed in table 1. total rna was extracted from the venom glands, frozen in liquid nitrogen, and table 1. primer sets used for the rt-pcr analysis of bee venom gene transcription. target gene primer sequence (5’-3’) predicted product size (bp) pla 2 forward tgtaacctccgcttccctt 504 reverse tccgccccgtgaatttatc hyaluronidase forward ggtgcgatcgtcgattcat 1149 reverse gtcacacttggtccacgct 948 sociobiolog y vol. 59, no. 3, 2012 ground into fine powder using a mortar and pestle. total rna was isolated using an rna kit (omega) according to the manufacturer’s instructions. the two cdna genes were synthesized from total rna by a reverse transcriptase kit following the manufacturer’s the protocol. pcr amplification was performed in a 50 ul reaction flask containing 5 μl of 10×taq buffer (mg2+ plus), 0.2 mm deoxyribonucleotide triphosphate, 10 μm each primer, 2.5 units of taq dna polymerase, and 100 ng of the template genomic rna of a. mellifera carnica. the pcr of pla2 was carried out as follows: 1.5 min at 94 °c; 33 successive cycles of 40 s at 94 °c, 40 s at 53 °c, and 40 s at 72 °c; and a final extension of 6 min at 72 °c. the pcr of the hyaluronidase was carried out as follows: 1.5 min at 94 °c, 33 cycles of 40 s at 94 °c, 40 s at 55 °c, and 1.5 min at 72 °c; and a final extension of 8 min at 72 °c. the rt-pcr products were examined by electrophoresis in 1.5% (w/v) agarose gels with ethidium bromide staining. the pcr products were purified by pcr purification kits and ligated into the pgem t-easy vector. component tg1 cells were transformed with the ligation products, and then grown on luria-bertani(lb-agar) plates containing 100 μg/ml ampicillin, 80 μg/ml x-gal, and 80 μg/ml iptg. white colonies were cultured in a 3 ml medium. plasmid dna was extracted and identified by bamh i and xho i digestion as well as pcr amplification. sequence analysis the positive recombinant plasmid dna was sequenced by the bgi company (shenzhen). the amino acid sequence was deduced from the cdna data using the ncbi database. sequence analysis was performed using the dnaman (version 5.0) program. two genes homologues of some vertebrates and invertebrates were obtained from ncbi. the full-length amino acid sequences were aligned using the clustal x1.83 program, and the phylogenetic tree was generated using the mega 4.0 software based on the neighbor-joining method. the protein sequences of the representative species, a. mellifera ligustica and a. cerana cerana, were obtained from published reports. the sequences were analyzed for identity using dnaman version 5.0. their biological characters were analyzed using online tools (http:// www.cbs.dtu.dk/services/netphos/, and http://web.expasy.org/cgi-bin/ protscale/protscale.pl?1). 949 hou, c. et al. — characterization of venom of apis mellifera carnica data processing the amino acid sequence was deduced from the cdna data using dnaman 5.0. sequence analysis was performed using the bioedit program, and the phylogenetic tree was generated using the mega 4.0 software based on the neighbor-jointing method. results the result of the agarose electrophoresis of the rt-pcr products amplified from the venom cdna of a. mellifera carnica is shown in fig. 1. the fragment size of the pla2 of a. mellifera carnica was consistent with that of a. mellifera ligustica in the corresponding region reported by (kuchler et al.1989), but differed from that of a. cerana cerana. the fragment size of the hyaluronidase of a. mellifera carnica was not consistent with those of a. mellifera ligustica and a. cerana cerana in the corresponding region reported by (gmachl and kreil 1993). the pcr products were purified and cloned into the pgem t-easy vector. the recombinant plasmids were identified by bamhi i and xho i digestion (fig. 2) and the pcr amplifications were sequenced. the two recombinant plasmids were named pgempla2 and pgemhyaluronidase. fig. 1. pcr products of the two genes from apis mellifera carnica by agarose electrophoresis. m :molecular weight marker(dl4500); lanes 1 and 2: phospholipase a 2 ; lanes 3 and 4: hyaluronidase. fig . 2. enzyme cut identification of the recombinant plasmids containing the target gene from apis mellifera carnica. m: molecular weight marker ( dl4500); lanes 1 and 2: pgemphospholipase a 2 ; lanes 3 and 4: pgem hyaluronidase. 950 sociobiolog y vol. 59, no. 3, 2012 sequencing results showed that the amplified fragments were 504 and 1146bp long, respectively. the multiple alignments showed that the pla2 and hyaluronidase genes shared more than 90.9% and 96.6% homologies with those of a. mellifera ligustica and a. cerana cerana in terms of nucleotide sequences. fig. 3. alignment of the deduced amino acid sequences of the phospholipase a2. diamond represents ca2+-binding site; underline represents active site; square represents n-myristoylation site; triangle represents n-glycosylation site; black dot represents casein kinase ii phosphorylation site; asterisk represents protein kinase c phosphorylation site. fig. 4. alignment of the deduced amino acid sequences of hyaluronidase. underline represents n-myristoylation site; broken line represents casein kinase ii phosphorylation site; dotted line represents n-glycosylation site; square represents tyrosine kinase phosphorylation site; arrowheads represents protein kinase c phosphorylation site; asterisk represents camp-and cgmp-dependent protein kinase phosphorylation site; black dot represents the b-cell epitope. 951 hou, c. et al. — characterization of venom of apis mellifera carnica using the bioedit program, the deduced amino acid sequences of the two genes were determined to be 168 and 382 amino acid residues in length, and predicted molecular weights of 41.2 and 92.257kda, respectively. multiple alignment (fig. 3) showed that the amino acid sequence of venom pla2 from a. mellifera carnica shared a high degree of homolog y with a. mellifera ligustica (the genbank accession number: np_001011607.1) and a. cerana cerana (the genbank accession number: q8lw54.1). on the other hand, fig. 5. phylogenetic tree constructed based on the alignment of the amino acid sequence of phospholipase a2 homologues. black dot represents the apis mellifera carnica. 952 sociobiolog y vol. 59, no. 3, 2012 the hyaluronidase gene had differences in many positions with those from a. mellifera ligustica and a. cerana cerana (fig. 4). phylogenetic analysis was performed based on the neighbor-joining method using the amino acids of these two gene sequences. the two genes from a. mellifera carnica were found to be most closely related to those of a. mellifer ligustica, and the trees illustrated the evolutionary relationship of the different bee species studied (figs. 5 and 6). discussion due to the abundance of pla2 and hyaluronidase in bee venom and their various toxic activities, they are two of the most commonly studied components of honeybee venom. in this study, the genes encoding these two proteins were fig. 6. phylogenetic tree constructed based on the alignment of the amino acid sequence of hyaluronidase homologs. black dot represents apis mellifera carnica. 953 hou, c. et al. — characterization of venom of apis mellifera carnica obtained from the venom of the honeybee a. mellifera carnica. the genes were cloned as well as identified, and their sequences were analyzed. there are two main interesting findings. first, the fragment size of hyaluronidase (1146bp) had a difference with those from a. mellifera ligustica (1149bp) and a. cerana cerana (1164bp). on the other hand, the fragment size of pla2 is the same as that of a. mellifera ligustica, although their sequences differ. second, the amino acid residues of the signal peptides of genes of the a. mellifera carnica genes are significantly different from that of a. cerana cerana (figs. 3 and 4). the phylogenetic tree shows that the relationship between the honeybee and other species, including some vertebrates, involves the conservation and variation of these genes during long-term evolution. the isoelectric points (pis) of pla2 and hyaluronidase are 6.36 and 8.82, respectively, and both are hydropathic. the secondary structure prediction of pla2 is α-helix (26.35%), extended strand (18.56%), beta turn (5.39%), and random coil (49.7%). the hyaluronidase secondary structure is α-helix (35.96%), extended strand (19.69%), beta turn (4.72%), and random coil (39.63%). the pis of pla2 and hyaluronidase from a. mellifera carnica differ from those (7.05 and 8.67) of a previous report (nico et al. 2005), although they all belong to the alkaline amino acid family. their structures of the two enzymes could explain their properties. pla2 contains the same his and asp amino acid residues as previously reported, which are involved in enzyme activity and the proton transfer system. pla2 also possesses trp, gly and asp amino acid residues with ca2+-binding site and active sites (scott et al. 1990). pla2 contains the same gly amino acid at position 143 as a. cerana cerana, although they belong to different families with different biological characterizations. using online anaylsis tools (http://clavius.bc.edu/~clotelab/dianna/), we find that the amino acid sequence of pla2 has nine cys amino acid residues, which can be formed into four disulfide bonds. the pla2 sequence also differs from those of a. mellifera ligustica and a. cerana cerana, its positions (42-64, 63-128, 94-138 and 96-103) also differed from that reported (9-31, 30-70, 37-63, 61-95 and 105-113)(sylvia et al. 2010). the hyaluronidase has only one disulfide bond in position 232 to 344, which also differs from a previous report wherein only two disulfides were observed (gmachl & kreil 1993). this result also differs from that of previous studies indicating that the mature peptide of the bee 954 sociobiolog y vol. 59, no. 3, 2012 enzyme is a single polypeptide chain, given that pla2 belongs to group iii phospholipases (valdez-cruz et al. 2007), which contain ten cys residues and are composed of one or two subunits (emmanuel & gérard 2000b). this discrepancy may have a relationship with allergens. hyaluronidase has the same structure as that reported, with a catalytic active center at d141 and e143 (housley et al. 2000). however, positions 141 and 143 are occupied by val and asp, and a conserved region with a hydrophilic ring, dpngnv, is present. moreover, only three out of nine b-cell epitopes have been reported (paclavattan et al. 2007). the amino acid sequences of pla2 and hyaluronidase, share four sites, namely, n-myristoylation, n-glycosylation, casein kinase ii phosphorylation and protein kinase c phosphorylation. there is a tyr kinase phosphorylation site in hyaluronidase, but none in pla2. pla2 and hyaluronidase are the main components of bee venom. they play important roles in anti-inflammatory and hemolytic actions, among others (habermann 1972; gauldie et al. 1976). we predict that their different functions could be related with their different structures, because they both cause hemolysis. however, pla2 causes indirect hemolysis, meaning that it dissolves phospholipids after melittin dissolves the lipoprotein layer of the erythrocyte surface. at the same time, both have activities related to allergic reactions (histamine release), which affects nearly 20% of the population (zhang et al. 2003; sutton and gould 1993). we do not know which the strongest allerg y cause is. the severest allerg y is induced by pla2 in some people and by hyaluronidase in others (wang et al. 1997). an allergic reaction results from the lack of aromatic amino acids, which can increase the rigidity of protein molecules, although this has not been proved by experiments. bee venom requires a posttranslational route independent of a signal identification particle and a docking protein, such as pla2 (boman et al. 1989). whether the two gene precursors have the same route when they slip into the signal peptide and proregion or the natured peptide is unknown. a. mellifera carnica and a. cerana cerana have different signal peptides (figs. 3 and 4), which could be useful in more in-depth studies of the mechanism of related proteins. this study is the first to describe a. mellifera carnica venom pla2 and hyaluronidase sequences, and provides a significant contribution to the honeybee 955 hou, c. et al. — characterization of venom of apis mellifera carnica database. the main proteins of bee venom have been widely studied and used in many fields. further studies are required to clarify the structure, biological characteristics, and expression mechanisms of the two genes. detailed studies on the effects of the two enzymes are important, considering that the complexity of the mechanisms involved hinder therapeutic approaches. therefore, studies on the molecular biolog y of bee venom proteins are necessary for further applications. our further studies shall focus on the expression of the recombinant forms of these two genes and their biological activities. acknowledgments we gratefully acknowledge fanggui xi (apicultural institute of jiangxi province,china) for providing the honeybee samples. this work was supported by the national natural science foundation of china (nos. 31071837 and 30371000). references boman, h.g., boman, a. i., andreu, d., zong, l., merrifield, r.b., schlenst, g., zimmermann, r., 1989. chemical synthesis and enzymic processing of precursor forms of cecropins a and b. j. biol. chem. 264:5852-5860. bonfim, v.l., carvalho, d.d., ponce-soto, l.a., kassab, b.h., marangoni, s., 2009. toxicity of phospholipases a2 d49 (6-1 and 6-2) and k49 (bj-vii) from bothrops jararacussu venom. cell biol. toxicol. 25:523-532. emmanuel, v., gérard, l., 2000a. what can venom phospholipases a2 tell us about the functional diversity of mammalian secreted phospholipases a2? biochimie 82: 815831. emmanuel, v., gérard, l., 2000b. increasing molecular diversity of secreted phospholipases a2 and their receptors and binding proteins. bba-proteins. proteom. 1: 59-70. evelin, l.s.; orestes, v.f.; 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http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i1.5772sociobiology 68(1): e5772 (march, 2021) introduction the subterranean termite globitermes sulphureus (haviland) (blattodea: termitidae) is a secondary pest species that has been introduced from its natural habitat to many parts of the metropolitan areas in peninsular malaysia after the elimination of primary termite pests such as coptotermes sp. (ab majid et al., 2007). in this study, we found infestations of globitermes sulphureus species around suburban or metropolitan environments at several locations in northern peninsular malaysia. the successful migration and establishment of g. sulphureus populations in metropolitan environments have created considerable concerns regarding biological invasions. invasive social insects such as termites often interrupt ecological communities and trigger serious economic destruction in their newly invaded locations (vargo & carson, 2006, ab majid & ahmad, 2011). abstract the subterranean termite globitermes sulphureus (blattodea: termitidae) can be found in tropical regions. we chose seven novel species-specific microsatellite markers to infer the breeding pattern of g. sulphureus based on its colony and population genetic structure in eight selected populations (natural-n = 4 and metropolitan-n = 4) in kedah and penang, malaysia. a strong correlation with their geographical location is shown by the acquired genetic gap for all studied populations from this study. the breeding pattern of family structure and comparisons of estimated f-statistics among g. sulphureus workers suggests 60% of all colonies are mixed families, whereas the remaining are simple families. average relatedness values within simple and mixed family colonies are similar (r = 0.121). positive fixation index f st values (f st = 0.086) indicate all eight populations (>500 m apart) have a significantly moderate genetic differentiation and low levels of inbreeding based on the low overall inbreeding coefficient f it value of 0.391. furthermore, four populations; palapes usm (pu), tmn astana (ta), kg teluk (kt), and penang national park (np), deviate from hardy–weinberg equilibrium (hwe, all p = 0.000) and five studied polymorphic loci (gs1, gs10, gs15, gs27 and gs29) are possibly under selection. the findings also reveal signs of a bottleneck effect in two populations: tikam batu (tb) and penang national park (np), indicating genetic drift. sociobiology an international journal on social insects nur aizatul nathasha khizam, abdul hafiz ab majid article history edited by qiuying huang, hau, china received 05 august 2020 initial acceptance 13 february 2021 final acceptance 11 march 2021 publication date 31 march 2021 keywords globitermes sulphureus; higher group termite; population genetics; polymorphism; natural regions; metropolitan regions. corresponding author abdul hafiz ab majid household & structural urban entomology laboratory, vector control research unit, school of biological sciences, universiti sains malaysia 11800 minden, penang, malaysia. e-mail: abdhafiz@usm.my in particular, g. sulphureus termites are considered a unique model system for analysing and distinguishing ecological elements that play a prominent role in the evolution of the introduced population (luchetti et al., 2013). furthermore, most comparative studies to date, including measuring the genetic variation level, genetic structure, and breeding pattern of g. sulphureus species, have failed to reveal any meaningful information. additionally, most termite species’ population studies have shown some genetic structure levels, and these population patterns are critical points for evolutionary biology research. thus, knowledge of the g. sulphureus breeding system and dispersal behavior is crucial to understand the observed patterns of the population genetic structure. molecular data such as microsatellite marker analysis enables researchers to infer the breeding system of the species and its dispersal behavior responsible for producing the pattern household & structural urban entomology laboratory, vector control research unit, school of biological sciences, universiti sains malaysia research article termites population genetic structure and breeding pattern of higher group termite globitermes sulphureus (haviland) (blattodea: termitidae) nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus2 structure and characterize the population’s subdivisions (thompson et al., 2007; fougeyrollas et al., 2018). khizam and ab majid (2019) developed highly polymorphic species-specific microsatellite markers for g. sulphureus to investigate the breeding pattern and population genetic structure. microsatellite markers are suitable instruments for understanding a colony’s breeding structure in social insects (ross, 2001). besides, there are a growing number of genetic research studies observing the social organization of termites colonies, especially among subterranean termites such as g. sulphureus (goodisman & crozier, 2002; bulmer et al., 2001; clement et al., 2001; dronnet et al., 2005; deheer et al., 2005). in general, the knowledge gained from species-specific microsatellite analysis helps assess the genetic structure, genetic diversity, and population structure, which can be applied in breeding system strategies and termite management. this study’s main objectives are to analyze and understand the breeding system and organization of the population genetic structure of g. sulphureus between natural and metropolitan populations in penang and kedah, malaysia. materials and methods termites collection we collected termite workers of g. sulphureus from eight different assigned sampling sites in natural and metropolitan locations in kedah and pulau pinang between september 2017 and february 2018. we collected a total of 10 worker termites from each nesting site and identified termite specimens based on tho (1974). figure 1 shows the sampling locations marked on the map using the pinmap web application. we recorded the addresses and geographic coordinates for the sampling sites (table 1) using a handheld garmin® gps 72h unit (garmin ltd, inc. usa). after collection, all worker termites from each sampling site were preserved in vials containing 90% ethanol. the sample was kept at -20°c before dna isolation. sampling site location geographical areas and environments determine the types of research sites, either natural or metropolitan regions. natural sites such as forest and agricultural areas were selected if the sites are in rural areas with a low-density population. consequently, metropolitan sites consist of many infrastructures with many human settlements such as cities, towns, urban and suburban areas. genomic dna isolation dna was extracted from the head of 10 termite workers individually from each sampling site using the real biotech corporation (rbc) dna extraction kit with modified protocols (seri masran & ab majid, 2018). all purified genomic dna was then quantified and tested for quality using a spectrophotometer nanodrop 2000c (thermoscientific, usa). the purified dna samples were observed under 2.0% agarose gel electrophoresis. microsatellite genotyping each termite worker was genotyped based on speciesspecific microsatellite loci obtained from khizam and ab majid (2019) (gs 1, gs 3, gs 4, gs 10, gs 15, gs 27, and gs 29) (table 2). we deposited all sequences in the national centre for biotechnology information (ncbi) and sequence read archive (sra) databases under accession number isolated code location (gps) state sources nesting sites collection_date tj n 5°38’ 51”; e 100°29’3” kedah tmn jubli dead log 28 oct 2017 pu n 5°21. 525”; e 100°17.596” penang palapes usm mound 31 oct 2017 ta n 5°35. 367”; e 100°31.134” kedah tmn astana mound 9 nov 2017 au n 5°21. 648”; e 100°18.365” penang arkeologi usm living tree 14 nov 2017 *tb n 5°35. 042”; e 100°25.915” kedah *tikam batu dead log 26 nov 2017 *sl n 5°39’23”; e 100°28’14” kedah *sg. layar tengah mound 13 jan 2018 *kt n 5°39’1”; e 100°27’44” kedah *kg. teluk mound 19 jan 2018 *np n 5°27’43”; e 100°12’14” penang *penang national park mound 20 jan 2018 tikam batu (tb), sg. layar tengah (sl), kg. teluk (kt), penang national park (np), tmn. jubli (tj), tmn. astana (ta), palapes usm (pu), arkeologi usm (au) *natural/rural regions was labelled. table 1. detailed of eight collection points of g. sulphureus worker individuals. sociobiology 68(1): e5772 (march, 2021) 3 srp132022 associated with bioproject prjna432461. the gdna was amplified using a pcr thermocycler machine in volumes of 50 μl containing 25 μl master mix (qiagen, valencia, ca), 15 μl of double-distilled water, 2.5 μl of each primer (0.05 μm), and 5 μl gdna. the pcr touchdown reaction was then subjected to the following settings; initial denaturation at 94 °c (30 seconds), 30 denaturation cycles at 94 °c (30 seconds), annealing at 60 °c (30 seconds), and extension at 72 °c (1 minute), followed by another 30 cycles at 94 °c for 30 seconds, 45 °c for 30 seconds, and 72 °c for 1 minute. the reaction was terminated at 72 °c for 10 minutes and held at 4 °c (seri masran & ab majid, 2018; khizam & ab majid, 2019). we measured exact fragment sizes for all pcr products after electrophoretic separation during fragment analysis. to separate the fragments according to their respective size, fig 1. eight sampling locations of g. sulphureus termite species between natural and metropolitan region abbreviations refer to table 1. we used the fragment analyzertm automated ce system (advanced analytical technologies, ankeny, iowa, usa) with an internal size standard of 35-1500 bp. microsatellite data were analyzed and hand-scored using the prosize 2.0 software package (advanced analytical technologies, ankeny, iowa, usa). microsatellites genotyping and tests for hw equilibrium to determine the definite tests of genotypic differentiation, we used genepop version 4.2 (available at http://wbiomed.curtin.edu.au/genepop) (rousset, 2008). the significant result obtained from the test exhibits different genotype frequencies among different groups of g. sulphureus workers, which indicates they were drawn from different colonies. using the hardy–weinberg equilibrium (hwe) nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus4 concept, the observation of allele frequency patterns within and between population(s) could also be carried out. unbalanced distribution of homozygote and heterozygote allele frequencies within the population indicate a deviation from the hwe principle (vargo & husseneder, 2009; perdereau et al., 2010; ab majid et al., 2018). colony breeding pattern a subterranean termite’s colony breeding structure is classified into three categories: simple, mixed, or extended family structure (vargo & husseneder, 2009; perdereau et al., 2010; ab majid et al., 2018). the simple family structure consists of a single pair of primary (winged) reproductive members in the subterranean termite colony. colonies are characterized as simple families if worker genotype patterns are consistent with a direct offspring expected from a single pair of reproductive parents. primary pair of reproductive parents can disperse and build a new colony, later producing secondary reproductive parents or neotenic (perdereau et al., 2010). the neotenic replaces the primary reproductive parents, fly, and initiate inbreeding. meanwhile, multiple neotenic forms an extended family structure having fewer than four alleles at any locus (vargo & husseneder, 2009; perdereau et al., 2010). on the other hand, colonies form mixed families if more than four or five alleles are observed at one or more loci and headed by more than a single pair of primary reproductive parents. colony genetic structure and relatedness coefficient additional understanding of the genetic structure of colonies is possible by analyzing relatedness coefficients. firstly, we used fit to measure the homozygosity of individuals relative to their population. secondly, fis indicates the level of inbreeding in individuals relative to the population. thirdly, fst refers to the inbreeding coefficient of individuals relative to their colony. 95% confidence intervals (cis) and standard errors (ses) were obtained by jackknifing over loci. we used the program fstat to compute the f-statistics (goudet, 2001) and to calculate the expected heterozygosity (he), the observed heterozygosity (ho), and the allelic diversity in the population. on the other hand, the f-statistic output can estimate the coefficient of relatedness (r) among individual workers from each population. thus, the colony data and population genetic structure of g. sulphureus within populations can be successfully assessed to extrapolate the number of alleles per loci and population. genetic isolation by distance genaiex v6.5 (peakall & smouse, 2012) a crossplatform tool was used to perform population genetic analysis, which runs within microsoft excel. data were received in the form of co-dominant genotypic microsatellite data with two columns per locus. the loci scored as fragment sizes were obtained from the previous fragment analysis. we performed an analysis of molecular variance (amova) (meirmans, 2012) to determine the extent of population differentiation and the distribution of genetic variation within and among the eight selected population sites of g. sulphureus. estimations of pairwise fst and significance were evaluated by a probability distribution from permutation tests (n = 1000). by comparing different populations, pairwise fst values were quantified for linear correlation with gene diversity values (he). besides, we used principal coordinates analysis (pcoa) (peakall & smouse, 2012) to characterize the population structure among the eight different population sites. this analysis generates the number of genetic clusters among the population sites based on each termite’s coordinate along the variation axes. bottleneck effect test the effect of a mutation on allele frequency in a population can be computed using two models of mutation– loci primer3 calculated value of possible allele size (bp) primer (5’-3’) type of repeat motif gs1 236 f: agcgatcggatgagcaagg r: acacgtctgtgtaaaggcag dinucleotide gs3 186 f: gtgccattccaccttcgtg r: cgtctcactagcagcaattatg dinucleotide gs4 316 f: tggtgtgagatggtgaaccc r: tcagttagcaaatgggaagcc dinucleotide gs10 303 f: tccagtaggtgtcctgttgc r: aaggctagcttccagttcag dinucleotide gs15 180 f: tgttgctgaaactaaatggctg r: ctgcacgtaaggagaagtctg dinucleotide gs27 294 f: acaatgaagggcacgtttgg r: gcaatggagtctaggtgtcg tetranucleotide gs29 431 f: ggacgactgcttaaagttgc r: actatgcctgggtttgatcc tetranucleotide table 2. information for seven chosen loci used in genotyping of microsatellite. sociobiology 68(1): e5772 (march, 2021) 5 drift equilibrium. we reviewed the results using the bottleneck v1.2.02 program (piry et al., 1999). the models used were the infinite allele model (i.a.m.) and stepwise mutation model (s.m.m.). as a newly formed population in metropolitan areas, the g. sulphureus termite population is expected to exhibit a recent genetic bottleneck. thus, we tested worker genotypes for excessive heterozygosity to obtain evidence of a bottleneck effect in each population. the first test aimed to detect any existence of significantly excessive heterozygosity with a relatively larger proportion of loci for a population at mutation-drift equilibrium. whereas the second test – known as the wilcoxon sign-rank test – aimed to detect significant average excessive heterozygosity across loci. results allelic diversity we detected a total of 363 alleles at seven appointed microsatellite loci assessed in 560 g. sulphureus genotypes. within the eight populations, allelic diversity is between 3 to 9 alleles per locus, with an average of 2.685. the mean percentage of the pic value is 94.9%. observed heterozygosity (ho) is 0.601, lower than the expected heterozygosity (he) of 0.967. from these findings, all of the studied populations in natural and metropolitan areas demonstrate a considerable variance in their genetic variability. as shown in table 3, 7 of the 30 loci examined in natural and metropolitan populations varies between 40 to 74 alleles per locus. locus k number of individuals (n) observed heterozygosity (ho) expected heterozygosity (he) pic f gs1 74 80 0.73 0.98 0.963 0.1481 gs3 40 80 0.44 0.95 0.946 0.3692 gs4 49 80 0.79 0.97 0.961 0.1004 gs10 46 80 0.45 0.96 0.959 0.3634 gs15 49 80 0.44 0.97 0.913 0.3768 gs27 43 80 0.53 0.96 0.942 0.2957 gs29 62 80 0.83 0.98 0.961 0.0837 k (number of alleles at each locus); f (null allele); pic (polymorphic information content). table 3. variability of seven polymorphic species-specific microsatellite loci in peninsular malaysia (kedah and penang) for natural and metropolitan populations. colony breeding pattern the subterranean termites collected from eight population sites were morphologically identified as g. sulphureus termite species according to hussin and ab majid (2017) and hussin et al. (2018). there is a diverse variation in terms of the distribution among simple and mixed family colonies presented in the eight assigned populations in peninsular malaysia. as shown in table 4, most colonies are derived from mixed families (60%) in both natural and metropolitan areas. mixed family colonies have more genotypes produce by a single pair of reproductive parents. each natural and metropolitan region also includes simple family colonies of g. sulphureus. population sites number of colonies number of simple family colonies number of mixed family colonies natural (tb, sl, kt, np) 10 subtotal = 80 1 (20%) 3 (60%) metropolitan (tj, ta, pu, au) 10 subtotal = 80 1 (20%) 3 (60%) all population sites combined = 160. abbreviations refer to table 5.1. table 4. breeding structure of natural and metropolitan populations of the globitermes sulphureus subterranean termite. genetic differentiation, colony genetic structure, and relatedness table 5 exhibits the f-values obtained from the computer simulations. for simple family colonies, all workers in two populations (pu and tb) are the most significantly inbred (fit = 0.483). the inbreeding (fit) measure has positive values, which indicates a general deficit of heterozygosity compared to the expected genotypic hwe. furthermore, both are relatively connected due to the moderate value of the relatedness coefficient (r = 0.121). simple families show a moderate genetic differentiation (fst = 0.090) since the fst ranges between 0.05 and 0.25. fst of 0 indicates the populations are not genetically differentiated. therefore, the populations show identical allele frequencies. meanwhile, fst of 1 indicates populations are fixed for different alleles. nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus6 compared to simple family colonies, mixed family colonies show less substantial inbreeding (fit = 0.358) in all six populations. average relatedness values within simple and mixed family colonies are similar (r = 0.121), as shown in table 5.5. fst of 0.082 is recorded for mixed families, showing a moderate genetic level differentiation among colonies. fst values in the range of 0 0.05 indicate the populations have little genetic differentiation. fst values between 0.05 0.25 represent moderate genetic differentiation, while fst values higher than 0.25 manifest a high genetic differentiation level. this study found a positive fis value (0.301) for mixed family colonies, indicating an absence of excessive heterozygosity, similar to the simple family colonies (fis = 0.432) of g. sulphureus. fst values of eight g. sulphureus populations are significantly greater than zero (positive), according to the permutation test (p < 0.005), which shows a significant genetic differentiation among colonies. thus, all eight populations of g. sulphureus have a moderate genetic differentiation (fst = 0.086) and a low inbreeding level based on the low fit value of 0.391. table 6 summarizes the f-statistics for g. sulphureus workers from natural and metropolitan populations, particularly in peninsular malaysia. the total inbreeding (fit) level for metropolitan populations (fit = 0.415) is higher than natural population sites (fit = 0.371). these values indicate metropolitan populations have a greater inbreeding level variation among individuals of g. sulphureus compared to natural populations. although both populations experience a moderate genetic differentiation, g. sulphureus individuals in metropolitan populations show slightly less differentiation, as evident from a relatively lower fst (0.082) than natural populations (fst = 0.097). moreover, both natural (fis = 0.303) colonies fit (s.e) fst (s.e) fis (s.e) r (s.e) simple families (n=2) 0.483 (0.128) 0.090 (0.020) 0.432 (0.130) 0.121 (0.019) mixed families (n=6) 0.358(0.055) 0.082 (0.010) 0.301 (0.056) 0.121 (0.013) all populations (n=8) 0.391(0.066) 0.086 (0.011) 0.333 (0.067) 0.124 (0.013) table 5. summary of f-statistics (fit, fis, and fst) and relatedness coefficient (r) for workers of globitermes sulphureus subterranean termite from simple, mixed family colonies and all populations focused in the northern part of peninsular malaysia. population sites classification fit fst fis natural (tb, sl, kt, np) n=4 forests paddy field oil palm trees or plantations 0.371 0.097 0.303 metropolitan (tj, ta, pu, au) n=4 street trees housing areas business premises 0.415 0.082 0.363 *abbreviations refer to table 1. table 6. summary of f-statistics (fit, fst, and fis) for workers of globitermes sulphureus subterranean termite from natural and metropolitan populations focused in the northern part of peninsular malaysia. population r fis (95% ci) palapes usm (pu), penang 0.087 0.204 tikam batu (tb), kedah 0.154 0.678 table 7. relatedness coefficient (r) between nestmate reproductive and inbreeding (fis) reproductive in simple family colonies of g. sulphureus. and metropolitan (fis = 0.363) populations experience a low level of inbreeding indicated by fis < 1. thus, the populations have a high level of heterozygosity in all seven loci. the finding suggests the wahlund effect may not affect the seven loci allele distribution within the eight populations. inbreeding coefficients (fis) for simple families’ reproductive and relatedness values between reproductive nestmates in the colonies are based on genotypes, as inferred from the worker offspring (table 7). both populations in penang (palapes usm) and kedah (tikam batu) have a high relatedness reproduction coefficient of r = 0.087 and r = 0.154, respectively. however, neither show a significance greater than zero (both p > 0.1, t-test). in palapes usm (penang), simple family colonies’ functional reproduction shows slightly less inbreeding than the workers in tikam batu (kedah). however, the differences are insignificant based on the overlapping 95% confidence intervals. table 8 shows the measured genetic diversity for g. sulphureus workers. mean of alleles per loci, observed (ho) and expected (he) heterozygosities, and estimations of fis within eight g. sulphureus termite populations (natural and metropolitan regions). the mean of alleles across loci is higher than 9 in most of the populations. two populations (mu and np) show the lowest mean of alleles per loci. interestingly, population mu is located in a metropolitan area, while population np is in a natural area. also, the heterozygosity deficit measured by fis is positive in most populations when averaged across loci, ranging from 0.015 to 0.066. the average fis across loci and populations ranging from 0.190 (population au) to 0.640 (population mu). among eight populations of g. sulphureus, pair-wise fst values show an overall genetic differentiation of 0.086 and pair-wise fst values ranging from 0.047 to 0.134. significant (α = 0.05) genetic differentiation is found after sequential bonferroni correction is performed to evaluate population pairs’ significance. sociobiology 68(1): e5772 (march, 2021) 7 genetic distance by pcoa and amova analysis based on the pcoa analysis (nm values), samples from four populations failed to prove different due to overlapping in a particular location site (figure 2). nm values are based on fst values, which indicate the genetic distance between populations. this study shows that pu and au populations are the most genetically related populations with an fst of 0.088. the sl population is related to the tj population with an fst of 0.045. although pu, au, sl, and tj populations might be closely related to each other, pu and au populations have the highest genetic correlation. the acquired genetic distance for g. sulphureus populations in this study shows a positive relation with geographical location for eight populations in peninsular malaysia (figure 1). tj, ta, kt, tb, and sl populations are located in northeast peninsular malaysia (near each other), while au, pu, and np populations are in southwest peninsular malaysia. the moderate level of individual relatedness within aggregated populations suggests g. sulphureus individuals are moderately related. the observed moderate values are due to the moderate genetic differentiation among populations and the moderate gene flow among them. these results demonstrate individuals of g. sulphureus could interbreed because of active diffusion, with a percentage of amova within the population of 94% and among populations of 6% (table 9). population q observed heterozygosity (ho) expected heterozygosity (he) fis (ic 95%) tj 9.57 0.671 0.887 0.253 pu 8.29 0.314 0.844 0.640 ta 11.29 0.586 0.923 0.378 au 10.71 0.743 0.908 0.190 *tb 9.14 0.686 0.877 0.228 *sl 9.71 0.657 0.893 0.275 *kt 9.71 0.700 0.881 0.214 *np 7.71 0.429 0.836 0.501 q (mean number of alleles per loci); * indicate natural populations. abbreviations refer to table 5.1. ** 10000 bootstrap over fis by population, ic 95% = confidence interval at 95%. table 8. gene diversity measures for workers of globitermes sulphureus subterranean termite from both natural and metropolitan populations. pu; au sl; tj kt tb ta np co or d. 2 coord. 1 principal coordinates (pcoa) populations fig 2. principal coordinates analysis (pcoa) among the eight population sites based on g. sulphureus colony coordinates along axes of variation. abbreviations refer to table 1. source df est. var. % among populations 7 0.219 6% within populations 72 3.260 94% within individual 80 0.000 0% total 159 3.479 100% *est. var. (estimation variance) table 9. summary of amova analysis. hardy–weinberg equilibrium (hwe) tests hwe results across loci and populations are shown in table 10 (a and b). following fisher’s statistical definite test for hwe, highly significant p-values (p < 0.001) for multilocus departures from hwe proportions are found in most populations. significant p-values (p < 0.05) are observed in tj, au, and tb populations. however, the single locus test across populations to access the deviation from hwe shows nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus8 no significant p-values other than for the gs4 microsatellite marker. other loci mostly show highly significant p-values. regarding the linkage disequilibrium for seven microsatellite loci pairs, the definite test results show seven of eight population combinations with significant linkage disequilibrium. however, inconsistent patterns are found across loci or populations. thus, the seven polymorphic microsatellite loci used in the analysis are independently assorted markers conforming to hwe and suitable for the colony and population genetic analysis. population p-value s.e. tj *0.0282 0.0091 pu 0.0000 0.0000 ta 0.0000 0.0000 au *0.0174 0.0045 tb *0.0300 0.0045 sl 0.0016 0.0016 kt 0.0000 0.0000 np 0.0000 0.0000 markov chain parameters for all tests: demorization: 1000; batches: 100; iterations per batch: 1000. s.e. (standard error). abbreviations refer to table 5.1. *no significant departure from hardy-weinberg equilibrium. table 10 (a). hardy-weinberg equilibrium (hwe) exact test in the subterranean termite g. sulphureus populations based on populations. locus p-values s.e. gs1 0.0000 0.0000 gs3 0.0003 0.0002 gs4 *0.0422 0.0122 gs10 0.0000 0.0000 gs15 0.0000 0.0000 gs27 0.0000 0.0000 gs29 0.0000 0.0000 markov chain parameters for all tests: demorization: 1000; batches: 100; iterations per batch: 1000. s.e. (standard error). * no significant departure from hardy-weinberg equilibrium. table 10 (b). hardy-weinberg equilibrium (hwe) exact test in the subterranean termite g. sulphureus populations based on loci. population i.a.m t.p.m s.m.m mode shift p(he) tj 0.464 0.592 0.575 normal 0.886 pu 0.025 0.174 0.641 normal 0.844 ta 0.020 0.034 0.652 normal 0.923 au 0.373 0.637 0.602 normal 0.908 tb 0.027 0.032 0.158 shifted 0.877 sl 0.162 0.444 0.305 normal 0.893 kt 0.394 0.552 0.110 normal 0.394 np 0.403 0.384 0.419 shifted 0.836 i.a.m (infinite allele model); t.p.m (two-phase model); s.m.m (stepwise mutational model) population i.a.m s.m.m tj 0.375 0.812 pu 0.007 0.578 ta 0.007 0.468 au 0.468 1.000 tb 0.007 0.015 sl 0.023 1.000 kt 0.375 0.039 np 0.109 0.812 i.a.m (infinite allele model); s.m.m (stepwise mutational model) table 11. bottleneck test analysis that showed p-value within eight populations of g. sulphureus in peninsular malaysia. bottleneck tests table 11 shows evidence of a bottleneck effect in two populations (tb and np). both populations undergo a heterozygosity deficit with the shift-mode estimation of allele frequencies, thus suggesting the occurrence of genetic drift (piry et al. 1999). also, the probabilities of mutation–drift equilibrium using both i.a.m. and s.m.m. are high for the au population at 0.46 and 1.00, respectively. kt population shows a moderate probability of mutation–drift equilibrium using i.a.m. (0.375) and s.m.m. (0.039) (table 12). discussion microsatellite genotyping is a tool to amplify and analyze individual loci. this method provides robust markers for target alleles containing simple repetitive sequences with a high mutation rate in the genome’s non-coding regions (guichoux et al., 2011). for instance, several studies on termites have used microsatellite marker accessibility to study the wooddwelling termite kalotermes flavicollis (luchetti et al., 2013), the subterranean termite reticulitermes grassei (dronnet et al., 2015), and the soil-feeding termites embiratermes neotenicus and silvestritermes minutus (fougeyrollas et al., 2018). table 12. summary of probability value for mutation-drift equilibrium from wilcoxon test. sociobiology 68(1): e5772 (march, 2021) 9 additionally, a genotyping assay from microsatellite markers reveals information on ancestries and relationships of individual termites, colonies, and populations, as discussed in the mendelian inheritance rules (glass, 2017). alleles with high variability in repetitive numbers and co-dominant characteristics have possible variations in length (keller & waller, 2002). therefore, the proportion of termite individuals carrying different alleles at gene loci on corresponding chromosomes (heterozygotes) in g. sulphureus colonies and populations can be detected and analyzed using f-statistics procedures (goodisman & crozier, 2002). from eight colonies of g. sulphureus screened for variability using seven primer pairs, all loci show variation within all population sites with 3 to 9 alleles per locus. we conclude that a large amount of genetic variation is detected. from the results shown in table 2, observed heterozygosity (ho) is less than expected heterozygosity (he) in seven polymorphic loci (gs1, gs3, gs4, gs10, gs15, gs27, and gs29) with significant deviation from hwe (p < 0.05). this might be due to the sample collection strategy or unique only to the tested populations. genetic diversity within the population varies as all seven tested loci are observed in all eight populations of g. sulphureus (table 7) due to the various populations’ heterozygosity levels. we propose that these populations are genetically diverse and have excellent survival prospects. genetically diverse populations with high heterozygosity levels have greater survival prospects during regular environmental changes. the opposite occurs with genetically homogenous populations with high homozygosity levels (frankham, 2005). although palapes usm (pu) and penang national park (np) are island populations, a constant phenomenon was not observed. an island population usually experiences low genetic diversity due to high genetic drift caused by its small population size. the outcome of genetic drift is severe in small and isolated populations (keller & waller, 2002). however, this population did not suffer low genetic diversity (he for pu = 0.844; he for np = 0.836), probably due to selection. kaeuffer et al. (2007) reported selection is the most likely mechanism responsible for heterozygosity to increase in a small population over time. selection may affect the changes in allele frequency. however, the genetic drift effect caused by selection is sometimes hidden. thus, selection may reduce the impact of genetic drift on the loss of genetic diversity. genetic drift can cause alterations in allele frequencies of a population from time to time due to variability of the reproductive success rate within a population. some individuals produce more offspring than others. the impact of genetic drift can be seen frequently in small populations such as an island population. however, all eight populations of g. sulphureus show no homozygosity (ho = 0). this finding suggests all of the studied populations have not experienced a high level of genetic drift and natural selection. therefore, some alleles are fixed in the populations and low genetic diversity. under natural selection, individuals tend to adapt to their local environmental conditions, leading to local adaptation (lenormand, 2002). there are two possibilities: (i) if an environmental condition prefers an allele, the selection direction skews towards it in the population, and (ii) upon a negative effect of mutation or the allele is less preferred, the selection process removes the allele from the population (kawecki et al., 1997), decreasing the genetic diversity level within a population. meanwhile, under genetic drift, alleles are randomly fixed or lost, leading to a low genetic diversity level (lande, 2015). genetic diversity is essential for the survival and adaptation of a population (frankham, 2005). reducing genetic diversity lowers alleles fitness and thereby decreases the evolutionary potential of species to adapt to a changing environment. this study establishes two types of colonies: simple family colonies (40 %) and mixed family colonies (60 % ). previous research on the colony’s breeding structure suggested most subterranean termite populations are composed of different proportions of simple and extended colonies, while mixed colonies are generally less common (vargo & husseneder, 2009). however, current data show mixed family colonies are predominant in six populations (sl, kt, np, tj, ta, and au), followed by simple family colonies (tb and pu). simple family colonies in palapes usm (pu) and tikam batu (tb) are headed by inbred, related, monogamous reproductive pairs, which suggests that dispersal by primary reproductive parents is limited in those populations. these findings support the notion that simple family colonies are classified as being headed by the original colony-founding reproductive pairs. on the contrary, colonies headed by more than a pair of primary reproductive parents having more than five alleles per locus are identified as mixed family colonies. mixed colonies form when two different individuals from two different colonies fuse and breed together (ab majid et al., 2013). the fusion of colonies, invasion of mature colonies by other alates, or sharing of foraging galleries by neighboring colonies is other factors that may lead to the formation of a greater mixed colonies proportion (deheer & vargo, 2004; aldrich & kambhampati, 2007). for instance, mixed family colonies result from colony fusions in natural and metropolitan populations of g. sulphureus termites. other than that, this study uses a large sample size, which might contribute to the greater percentage of mixed family colony formation in g. sulphureus populations (ross & carpenter, 1991). besides, high mixed families proportion in this study is commonly found in nature (kt, np, and sl) as supported by studies on zootermopsis nevadensis termite (howard et al., 2013) and wood-dwelling termite kalotermes flavicollis (luchetti et al. 2013). the breeding structure could not be resolved in colonies that did not fit the expected genotype frequencies for the progeny of a simple family containing less than four alleles at all loci. populations of g. sulphureus termites focus nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus10 in northern peninsular malaysia reveal a substantial variation in colony breeding structures, probably due to the mixed colonies’ inbreeding levels in the tb population. proportions of termite colonies with different breeding systems vary across populations depending on the age of colony structure, dynamics of the colony–colony interactions, food quantity and quality, soil characteristics, and disturbance or treatments (bulmer et al., 2001; aluko & husseneder, 2007). it has been suggested that fst between 0.05 to 0.15 indicates a moderate genetic differentiation, while values in the range of 0.15 to 0.25 indicate a great genetic differentiation. fst above 0.25 indicates an exceptionally great genetic differentiation. based on this rule, the fst of this study is in the range of 0.05 to 0.15 (fst = 0.082 to 0.097), indicating a moderate genetic differentiation. furthermore, the gst range is equivalent to fst obtained between natural and metropolitan populations of g. sulphureus (gst = 0.069), which also show a moderate genetic differentiation. the moderate genetic differentiation between both populations might indicate incomplete isolation (pamilo et al., 2016;). the dispersal or migration of g. sulphureus termites occurs in several ways. for instance, julio et al. (2002) claimed rafting of wood pieces containing reproductive pairs could be an effective means of dispersal for some termite species. in a tropical country such as malaysia, the rainy season occurs several times a year, facilitating the transportation of infested wood pieces to a new infestation area via rainwater flow. besides, blowing out alates from their nest by strong winds could be considered another possible dispersal mode. winged termites can disperse over 800 m. most studies on termites reveal that the emergence of alates usually occurs during the middle of the year, which corresponds to malaysia’s annual rainy season between july and november (tong et al., 2017). therefore, an alate flight phenomenon might occur since the collection of g. sulphureus termites was performed from october 2017 to february 2018. additionally, frequent migration among populations is necessary to achieve a moderate genetic differentiation between natural and metropolitan populations. the frequencies observed rarely occur by wood rafting due to a very stochastic process, but most probably is caused by the most frequent migration, the alates’ flight. furthermore, relatively low gst values between natural and metropolitan populations demonstrate a gene flow phenomenon between both populations. the current study demonstrates a considerable variation in the g. sulphureus colony structure over a small spatial scale, including colonies headed by monogamous outbred primary reproductives and colonies containing multiple inbred neotenic reproductives. this result reflects the number of reproductives and nestmate relatedness. polymorphic speciesspecific microsatellite markers are employed to determine the social organization of g. sulphureus colonies at two sites in peninsular malaysia. the level of nestmate relatedness and inbreeding coefficient within and among colonies are estimated. this information helps to infer the nature of colony founding and reproductive structure by comparing the empirical results with computer simulations for different breeding schemes. the results indicate a remarkable variation in g. sulphureus colony organization. thorne et al. (1999) stated the inbreeding phenomenon is common in subterranean termites. fis is the measure of inbreeding in populations under random mating. the positive value for all eight populations, as shown in table 7, indicates there are more related individuals in the population under random mating (wright, 1965). in a previous study on odontotermes termite, the high level of inbreeding suggests a shorter mating flight range; thus, they are more likely to pair with relatives during colony founding (cheng et al., 2013). inbreeding reduces heterozygosity, which has also called the wahlund effect. inbreeding also contributes to a high level of genetic relatedness among workers in colonies (dronnet et al. 2005). the wahlund effect shows excessive homozygosity in a population is due to the existence of subdivision fragmentation. this effect may have occurred in the current study since the seven tested loci had an overlapped distribution for all eight tested populations with unknown stratification. in summary, the high and significant fit and fst close to zero indicates lots of related breeders in each colony, leading to a significant genetic differentiation among colonies and inbreeding (painter et al., 2000). nm values are based on fst, which is equivalent to genetic distances between populations. in this study, several samples from four populations did not reach a clear distinction due to overlapping at a particular location site (figure 2). this study shows pu and au populations are the most genetically closely related populations with an fst of 0.088. in contrast, sl populations are close to tj populations with an fst of 0.045, a coefficient possibly resulting from the short distances between pu, au, sl, and tj sampling sites. comparison between island populations (np, pu, and au) and mainland populations (sl, tj, kt, tb, and tl) generally shows a high genetic distance. this outcome may be due to the small sample size of island populations which yields a high value of nei’s genetic distance (nei, 1987. as julio et al. (2002) described, the dispersal or migration of termites in islands such as pu and au populations may occur in several ways. for instance, the rafting of wood pieces containing reproductive parents could be an effective means of dispersal for g. sulphureus species (gathorne-hardy et al., 2000). strong winds hit penang island several times a year, facilitating the transportation of infested wood pieces to the sea via inland temporal and permanent rivers, carrying them to other islands. a similar study on nasutitermes takasagoensis termite in japan showed that strong typhoon winds blow out alates, possibly establishing a mode of overseas dispersal. nevertheless, anthropogenic dispersal is another possible means of putative dispersion for n. takasagoensis termites (julio et al. 2002). sociobiology 68(1): e5772 (march, 2021) 11 in particular, the obtained genetic distance for all eight populations in this study is positively related to the populations’ geographical locations in peninsular malaysia (figure 2) (pironon et al., 2015; schwalm et al., 2016). tj, ta, kt, tb, and sl populations are in northeast peninsular malaysia (near each other), while au, pu, and np populations are in southwest peninsular malaysia. several factors influence the relationship between allele frequencies and genotype frequencies, e.g., mutation, population size, mating strategy, natural selection, and gene flow. in the absence of these factors, allele and genotype frequencies conform to a simple relationship known as the hardy–weinberg equilibrium (hwe) (harrison et al., 2018. the concept of hwe states in non-evolving populations, allele and genotype frequencies remain constant from generation to generation. the populations are then regarded as being in hwe. therefore, at a single locus, any deviation from hwe is eradicated after one generation of random mating (allendorf, 2017). from table 10, four populations (pu, ta, kt, and np) deviate from hwe (all p = 0.000), indicating they are under selection, causing rapid changes in allele frequencies since many alleles are lost except for the favorable alleles (miller et al., 2001; williams, 2018). five polymorphic loci (gs1, gs10, gs15, gs27, and gs29) are probably under selection. besides, allele frequencies may also deviate from hwe if the sample size is small (salanti et al., 2005). however, the effect of sample size is very modest (ioannidis al., 2001). the sample size used in this study is sufficient to obtain a good hwe analysis (n = 10 per population). another factor contributing to hwe deviation is non-random mating or inbreeding, which commonly occurs in isolated populations (reddy, 2017). since pu and np are island populations, the effect of inbreeding may be high; thus, these populations deviate from hwe. when a population deviates from hwe, genotype frequency shifts. for example, as inbreeding occurs, the population stops undergoing random mating, and the homozygote genotypes frequency increases with the decreasing frequency of heterozygote genotypes (hamilton, 2011). this is due to relatives, by definition, are more likely to inherit the same ancestral alleles from a common ancestor; this is known as being identical by descent (ibd) (powell et al., 2010). a population consisting of inbred individuals is expected to show excessive homozygosity over hwe expectations, which can be detrimental as recessive mutations continue to segregate within the population, resulting in inbreeding depression (charlesworth & charlesworth, 1987). s.m.m signifies that an allele only mutates by losing or gaining single tandem repetitive alleles, possibly among alleles already present in the population. on the contrary, under i.a.m, a mutation involving any number of tandem repeats always produce alleles not commonly encountered in the population (hardy et al., 2003; roussel et al., 2004). since the estimation of effective population size and mutation rates depend on the mutation model, computational assays are performed to test the adequacy of each model with the observed data. the results (table 10) show five populations (tj, au, sl, kt, and np) might have a low probability of undergoing the mutation and genetic drift for the seven tested loci using both i.a.m. and s.m.m (p > 0.05). in contrast, mu, ta, and tb populations have high chances of mutation and genetic drift (p < 0.05) for i.a.m but not for s.m.m (p-value mu = 0.641; p-value ta = 0.652; p-value tb = 0.158). the cause of these outcomes may be these populations’ geographical location (wlasiuk et al., 2003). the nesting sites of g. sulphureus in these populations are surrounded by suboptimal local environments such as being placed far from any food source (taylor & hellberg, 2003). in particular, the probability of mutation-drift equilibrium using both i.a.m. and s.m.m. is high for the au population (0.46; 1.00) and moderate for the kt population (table 5.11). the latter shows a moderate (0.37) and low (0.03) probability of mutation-drift equilibrium using i.a.m. and s.m.m., respectively. this result can be evidence that the kt population may be suffering from a mutation effect. the mutation–drift equilibrium test can also be applied to study inbreeding depression by measuring the correlation between individual fitness and inbreeding. the result shows the heterozygosity level of individuals or the population. this test also provides insights into evolutionary interpretation using the information on relative values of mutation rates compared to the migration rate or population divergence (hardy et al. 2003). conclusion this study is the first one to focus on the population, colony genetic structure, and dispersal pattern of g. sulphureus termites within natural and metropolitan regions. in conclusion, geographic variation and urbanization affect the genetic structure of g. sulphureus colonies. a suitable and flexible environmental habitat plays a vital role in driving genetic differentiation. the current study suggests g. sulphureus in northern peninsular malaysia can be characterized by moderately related and inbred individuals between populations. the high level of genetic diversity among and within the populations and the moderate genetic differentiation found out in this study show that g. sulphureus is most likely to mate with moderately related mates. with increasing globalization due to trade, human-mediated transportation, seasonal weather, and tourism, subterranean termites of g. sulphureus spread rapidly to infest new regions such as housing and business areas. a comprehensive understanding of the population dynamics of g. sulphureus colonies provides an improvement in response to higher group termite colonies and population management tactics, especially for higher termite group baiting. acknowledgments the universiti sains malaysia funded this research work (usm) bridging research grant: 304/pbiologi/6316010. nur aizatul nathasha khizam, abdul hafiz ab majid – population genetics of globitremes sulphureus12 references ab majid, a.h. & ahmad, a.h. 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(hymenoptera: formicidae) in colombia the colonies of atta cephalotes (l.) are monogynous: i.e. has a single queen, fed and protected by thousands of workers. it is estimated that a queen lives for 15 years and is irreplaceable, that is, when it dies, the colony disappears (hölldobler & wilson, 2011). the workers, especially the soldiers, are territorial and defend the colony from the presence and or attack of intruders, even those of the same species (whitehouse & jaffe, 1995). in the same way, the workers reject winged queens from other colonies that approach their territory. they are attacked, and in most cases, dismembered (c. giraldo, foundation center for research on sustainable farming systems (cipav) personal communication, august 21, 2013). at present, it is unknown whether the foundation of nests of a. cephalotes with more than one queen occurs, or if a. cephalotes workers have the ability to adopt new queens into their nest without altering the work within the colony and/or affecting the surrogate queen. recently, the queen of a colony of a. cephalotes with eight years of establishment under abstract in nature, atta cephalotes (l.) is a monogynous species. each colony has a single, permanent queen fed and protected by thousands of sterile workers. at death the queen colony practically disappears. recently a colony established eight years in the international center for tropical agriculture (ciat), colombia, lost the queen being orphaned by three months. starting from the idea that whether this colony could take a surrogate queen, we collect a field young nest of a. cephalotes, which donated its queen to the orphan colony. overall, there was a slight aggression among workers without attacking the surrogate queen, which was adopted by the orphan colony. five months later, the surrogate queen was still alive and there was presence of larvae and pupae. the results show that the artificial colony of a. cephalotes, after a period of orphanhood, accepts surrogate queen and remains stable and active. sociobiology an international journal on social insects g sotelo1, ds ortiz-giraldo2, j rodríguez2, j montoya-lerma2 article history edited by inge armbrecht, universidad del valle, colombia received 20 january 2015 initial acceptance 06 august 2015 final acceptance 17 august 2015 keywords leaf-cutter ants, orphans, intruder. corresponding author diana sofía ortiz giraldo. facultad de ciencias naturales y exactas, departamento de biología, universidad del valle calle 13 nº 100-00, sede meléndez cali, colombia e-mail: dianasofiaog@hotmail.com laboratory conditions at the international center for tropical agriculture (ciat) in palmira, valle del cauca, died and the colony spent three months in orphanhood. this allowed us to question whether an artificial colony of a. cephalotes, after a period of orphanhood (i.e. without a queen), could accommodate an “intruder” queen and her progeny. starting from this question, a laboratory test was conducted in three stages; step a: removal and transfer of queen from donor ant colony. a small nest, obtained in the field, was deposited in a transparent plastic container with an airtight lid. this consisted of the queen, workers, and a quantity of symbiotic fungus. step b: conditioning, recognition, and acceptance. migration of small quantities of workers from the orphaned nest (from ciat) every 30 minutes for a period of four hours into the nest obtained in the field until these workers exceeded the number of workers found in the nest from the field. step c: assembly between nests. after four hours of testing, the nest from the field with its new population short note 1 forage laboratory ciat, palmira, colombia. 2 universidad del valle, cali, colombia. g sotelo et al. – adoption of a surrogate artificial queen in a colony of leafcutter ant614 queen in their colonies; however, the absence of the queen in this nest supplied evidence of the ability of workers to replace it. it is worth noting that there have been isolated cases of two queens within atta nests: in atta texana (b.) (moser & lewis, 1981) and atta laevigata (s.) (mintzer, 1990). in synthesis, this event shows how a leafcutter ant colony, a. cephalotes, can adopt a new queen, thus guaranteeing the continuity of the colony by restoring the production of new individuals who will become a future workforce. the adoption of a queen or even an event of secondary polygyny in nests of a. cephalotes is significant because they imply a challenge to the central dogma of nest removal by the extermination of queens. acknowledgements the authors thank the international center for tropical agriculture (ciat) and the leafcutter ant research group of the universidad del valle. references de souza, d., della lucia, t.m.c., errard, c., d’ettorre, p. & mercier, j.l. (2003). reconhecimento da prole por operárias companheiras e não companheiras de ninho em acromyrmex laticeps nigrosetosus forel, 1908 (hymenoptera, formicidae). ciência rural., 33: 91-95. doi: 10.1590/s010384782003000100014 holldöbler, b. & wilson, e. o. (2011). the leafcutter ants. first edition. new york: w. w. norton & company, inc., 160p mintzer, a.c. (1990). colony foundation in leafcutting ants: the perils of polygyny in atta laevigata (hymenoptera: formicidae). psyche, 98(1): 1-5. doi:10.1155/1991/71624 moser j.c. & lewis, j.r. (1981). multiple nest queens of atta texana (buckley 1860), hymenoptera: formicidae) leafcutting ant. turrialba, 31: 256-257. whitehouse, m.e.a. & jaffe, k. (1995). nestmate recognition in the leaf-cutting ant atta laevigata. insectes sociaux, 42: 157-166. doi: 10.1007/bf01242452 fig 1. artificial colony of atta cephalotes (ciat). a five months after the adoption of the surrogate queen. b live surrogate queen integrated into the orphan colony. (its own workers and orphan workers) joined the largest nest (orphan) through a communication channel and colony was observed during five months. in the first step, there was a slowmovement between colonies. this was accelerated in the step b, and at the outset of the migration, confusion was observed by orphan workers, and contact was observed between the workers, but without aggression. it is noteworthy that orphaned workers, upon detecting the surrogate queen, tried to remove her from the symbiotic fungus (field colony) and from the donor workers. after five months of adoption, this colony is stable (fig 1) and shows offspring production. this represents the first record, on the behavior of artificial colonies of a. cephalotes, aimed at understanding their social organization and the role of different castes within it, especially under a condition of catastrophe. de souza et al. (2003) indicated that atta queens, in order to maintain monogynous colonies, do not tolerate another 1389 natural biological control of lepidopteran pests by ants by rodrigo soares ramos1, marcelo coutinho picanço1*, paulo antônio santana júnior1, ézio marques da silva2, leandro bacci3, alfredo henrique rocha gonring4 & gerson adriano silva5 abstract the predatory ants (hymenoptera: formicidade) are social insects and important natural enemies of pests in agroecosystems. despite the importance of these predators, little is known about their role, especially in tropical regions. among the major lepidopteran pests of vegetables are ascia monuste (pieridae), diaphania nitidalis (crambidae), neoleucinodes elegantalis (crambidae) and tuta absoluta (gelechiidae). thus, this work aimed to study the natural biological control of a. monuste, d. nitidalis, n. elegantalis and t. absoluta by ants. for this, we evaluated the natural biological control of a. monuste on kale and d. nitidalis on cucumber both species in the dry season. whilst the natural biological control of n. elegantalis and t. absoluta on tomato plants were evaluated in the rainy and dry seasons. ants preyed on lepidoptera in the pupa stadium. they also preyed on eggs of d. nitidalis. the activity of predatory ants occurred mainly during the night. the ants were the main causes of pupae mortality of a. monuste, d. nitidalis and t. absoluta. beyond the ants, the physiological disturbances and birds were also important factors of pupae mortality of n. elegantalis. the ants labidus coecus and solenopsis sp. were observed preying on pupae whereas the paratrechina sp. was observed preying eggs of d. nitidalis. the pupae mortality of d. nitidalis and t. absoluta by ants were higher than the pupae mortality of n. elegantalis and a. monuste. the rate of pupae predation of n. elegantalis by ants was similar in rainy and dry seasons and the same occurred with t. absoluta. keywords: social insects, formicidae, ascia monuste, diaphania nitidalis, neoleucinodes elegantalis, tuta absoluta. 1 federal university of viçosa (ufv), department of entomolog y (dde), 36570-000, viçosa, mg, brazil, 2 ufv, rio paranaíba campus, 3 federal university of sergipe, 4dupont do brasil s.a, 5ufv, plant science department (dft), *e-mail: picanco@ufv.br 1390 sociobiolog y vol. 59, no. 4, 2012 introduction the ants (hymenoptera: formicidae) are social insects and they constitute the largest group among the insects. their colonies usually live in nests that have tunnels and chambers. the ant societies are organized into castes (groups where there is division of labor). the caste system enables execution of multiple tasks. in the society of ants sterile castes (workers and soldiers) are responsible for maintaining the colony (obtaining food, protection and care of offspring ). the fertile castes (queens and drones) have reproductive function (hölldobler & wilson 1990; ross & keller 1995). in agroecosystems there are species of ants which are pest and others which are predators and they act as natural enemies. ants are generalist predators and they usually have others insects as their main prey. ants prey on insects of various orders such as coleoptera, diptera, hemiptera, lepidoptera and orthoptera. ants are important predators of insect pests in annual and perennial crops such as fruits, vegetables, ornamental plants, grain crops, coffee, sugar cane and cotton. the story of ants as biological control agents of agricultural pests is from 300 b.c. however there are many aspects about the role of ants as predators of agricultural pests that need to be studied, especially in tropical regions, to enable the preservation of these agents of biological control (way & heong 2009, fernandes et al. 2010, choate & drummond 2011). among the main lepidopteran pests of vegetables are ascia monuste (godart) (pieridae), diaphania nitidalis cramer (crambidae) neoleucinodes elegantalis guenée (crambidae) and tuta absoluta (meyrick) (gelechiidae). these four lepidoptera species occur in the americas (gonring et al., 2003a; picanço et al., 2007; picanço et al. 2011). the t. absoluta in 2006 was introduced in europe and is currently distributed by practically this entire continent. also, t. absoluta invaded the mediterranean region of africa and asia (garcia-marí & vercher 2010, eppo 2012). a. monuste is a pest of brassica and d. nitidalis is a pest of cucurbitaceae family (picanco et al. 2000, picanco et al. 2010, picanco et al. 2011) whilst n. elegantalis and t. absoluta are pests of solanaceae, especially tomatoes (picanco et al. 2007; galdino et al. 2011, moreno et al. 2012). thus, this work aimed to study the natural biological control of a. monuste, d. nitidalis, n. elegantalis and t. absoluta by predatory ants. 1391 ramos , r.s.— natural biological control of lepidopteran pests by ants materials and methods this work was conducted in crops of kale, cucumber and tomato located in viçosa (20°48’45”s, 42°56’15”w, altitude of 660m), minas gerais state, brazil. the tomato crop was carried out in the rainy season ( january and february) and dry season (may and june) 2007. the crops of kale and cucumber were only conducted in the dry season. the cultivars used were portuguese clone (kale), hybrid sprint 440 ii (cucumber) and santa clara variety (tomato). the experimental design was randomized blocks with five replications. the repetition had six rows of nine plants. the spacing used was 1 x 0.5 m. therefore, each repetition had 54 plants. no pesticides were applied to the crop, and conventional cultivation practices were employed, according to filgueira (2000). the pest insects used in experiments were obtained from laboratory rearing conducted as liu (2005) for a. monuste, pratissoli et al. (2008) for d. nitidalis, frança et al. (2009) for n. elegantalis and galdino et al. (2011) for t. absoluta. kale plants were used for a. monuste, cucumber plants were used for d. nitidalis and tomato plants were used for n. elegantalis and t. absoluta. at the beginning of the experiment, 10 plants in each repetition were infested with 10 eggs and 10 larvae of each instar of lepidoptera species. the plants were about 60 days old when they were infested. in the stadia of eggs and larvae were monitored mortalities caused by ants in each lepidoptera species. as soon as the insects moved to the pupal stadium the mortalities caused by each factor for each lepidoptera species were monitored. mortality factors of the lepidoptera pupae were identified (number and causes). natural enemies observed causing mortality of lepidoptera pupae were noted according to their morphospecies. in plants unmarked, individuals of each morphospecies of these natural enemies were collected and assembled. at the end of the pupal stadium we collected those alive pupae in plastic pots of 500 ml. the pots were taken to the laboratory for evaluation of the pupae parasitism. the emerged parasitoids were collected. the natural enemies collected were identified in the museum of entomolog y at ufv. the morphospecies of ants were identified by dr. ivan cardoso nascimento (state university of southwest of bahia). from the experimental data the averages of lepidoptera mortality by ants in the eggs and larvae stadia were 1392 sociobiolog y vol. 59, no. 4, 2012 calculated. in the pupal stadium of each lepidoptera species the mortalities caused by each factor were calculated. we also calculated the 95% confidence intervals of mortality rates. results and discussion the ants preyed a. monuste, n. elegantalis and t. absoluta only in the pupal stadium. the d. nitidalis was preyed by ants in both eggs and pupae (fig. 1). pupae predation of these four species, probably occurred due to the main site of pupation of lepidoptera and nesting of ants to be at the same spot, ie the soil. however, the eggs and larvae of a. monuste, n. elegantalis and t. absoluta occur in kale and tomatoes leaves that do not have contact with the soil thus hindering predation by ants. the eggs of d. nitidalis are in cucumber leaves that often come in contact with the soil thus enabling their predation by ants. moreover, the larvae of d. nitidalis are located inside the fruit making it difficult to predation by ants (picanco et al. 2000). the report of ants as predators of a. monuste, n. elegantalis and t. absoluta is unprecfig. 1. mortality of diaphania nitidalis in each stadium by ants. 1393 ramos , r.s.— natural biological control of lepidopteran pests by ants edented. miranda et al. (2005) reported the occurrence of predatory ants in the tomato crop attacked by n. elegantalis and t. absoluta but these authors did not observe which prey those ants were preying. whilst gonring et al. (2003a) reported ants as predators of d. nitidalis. the predatory activity of ants occurred in the early morning, late afternoon and mainly during the night. we did not observe predation rate of ants in periods of high temperature and low relative humidity. the high air temperatures might elevate body temperature excessively of ants causing death or reducing their performance since they are biological poikilotherm organisms ( jensen 1978). the foraging activity of predatory ants in periods of mild temperatures and low or no light might be related to the fact that these periods is low activity of predators of ants as the birds (necr 2009, narendra et al. 2010). the absence of foraging activity of ants during periods of low relative humidity is possibly due to this condition cause dehydration to these insects. the ants were the main causes of pupae mortality of a. monuste, d. nitidalis and t. absoluta. beyond the ants, the physiological disturbances and birds were also important factors of pupae mortality of n. elegantalis. besides these factors, we observed mortality of pupae by rain (n. elegantalis and t. absoluta), hymenoptera: vespidae (n. elegantalis), hymenoptera parasitoids (t. absoluta and n. elegantalis), fungi (n. elegantalis), spiders (d. nitidalis) and heteroptera, pentatomidae (a. monuste) (fig. 2). the increased mortality of lepidoptera pupae by ants than the other natural enemies should be, possibly, their social organization. the social organization of ants allows the division of labor between castes, efficient localization of prey and more competitive than other natural enemies. also knutson & campos (2008) observed the ants were the most important predators of lepidoptera helicoverpa zea (bod.) (noctuidae). given the importance of ants as predators of lepidoptera pests they should have preserved their populations in crops. among the practices that enable the preservation of predators ants populations are: the reduction of pesticide applications, the application of products with physiological selectivity and selective use of pesticides. the decreased use of pesticides on crops may be obtained by adopting sampling and level of pest control (moura et al. 2003, bacci et al. 2006, gusmao et al. 2006). the use of insecticides with physiologi1394 sociobiolog y vol. 59, no. 4, 2012 fig. 2. factors of pupae mortality (a) ascia monuste, (b) diaphania nitidalis, (c) neoleucinodes elegantalis and (d) tuta absoluta. 1395 ramos , r.s.— natural biological control of lepidopteran pests by ants cal selectivity is important because these products are effective in controlling pests and they are somewhat toxic to natural enemies. in this context, moreno et al. (2012) found that the plant extracts acmella oleracea and azadirachta indica were selective in favor of the predator ant solenopsis saevissima (smith) as the insecticide permethrin was not selective. in the selective use of pesticides the aim is to reduce exposure of natural enemies with the products. for ants this must be achieved avoiding products to reach their site of the foraging, ie the soil. for this goal is reached we should avoid the application of pesticides in soil and spraying must be made using the smallest possible volume of solution to prevent much of the pesticide reaches the soil. also, we should select pesticides that degrade rapidly when they come in contact with the ground. ants observed preying on pupae of a. monuste, d. nitidalis, n. elegantalis and t. absoluta were labidus coecus (latr.) and solenopsis sp. also, we observed the ant paratrechina sp. preying on eggs of d. nitidalis. way & heong (2009) observed solenopsis species as an important predator of insect pests fig. 3. pupae mortality of ascia monuste, diaphania nitidalis, neoleucinodes elegantalis and tuta absoluta by ants. 1396 sociobiolog y vol. 59, no. 4, 2012 in rice whereas gonring et al. (2003ab) found l. coecus and paratrechina sp. preying diaphania spp. the pupae mortality of d. nitidalis and t. absoluta by ants were higher than the pupae mortality of n. elegantalis and a. monuste (fig. 3). this may be due to factors such as pupae size and pupation site of the pests. as ants fig. 4. pupae mortality of (a) neoleucinodes elegantalis and (b) tuta absoluta in rainy and dry seasons. 1397 ramos , r.s.— natural biological control of lepidopteran pests by ants forage mainly on soils (hölldobler & wilson 1990, way & heong 2009, fernandes et al. 2010) they must prey more intensely on those preys which has exclusive pupation site. in this context, we found that the ants preyed more lepidoptera pupate in the soil (d. nitidalis and t. absoluta) than those pupate in the plant and in the soil (n. elegantalis and a. monuste). another factor that may have influenced the change in predation rate of prey species by ants is the size of the pupae. we observed increased predation of lepidoptera pupae with smaller size (t. absoluta and d. nitidalis) than species with larger size pupae (n. elegantalis and a. monuste). the rate of pupae predation of n. elegantalis by ants was similar between the rainy and dry season. the same occurred with t. absoluta (fig. 4). therefore; the rainfall did not affect predation of lepidoptera by the ants. we did not observe predation rate of ants during rain events. in the rainy season the ants performed predation when the rains ceased. in conclusion, our results demonstrate the importance of ants as predators of a. monuste, d. nitidalis, n. elegantalis and t. absoluta. thus, the planning programs of pest management of vegetables it should preserve the predatory ants to maximize biological control of pests. acknowledgments we wish to thank cnpq, capes, fapemig, fapitec and bnb by scholarships and resources granted. dr. ivan cardoso nascimento from the state university of southwest of bahia for identifying the ant species. references bacci, l., m.c. picanço, m.f. moura, t.m.c. della lucia & a.a. semeão 2006. sampling plan for diaphania spp. (lepidoptera: pyralidae) and for hymenoptera parasitoids on cucumber. journal of economic entomolog y 99, 1980-1988. choate, b. & f. drummond 2011. ants as biological control agents in agricultural cropping systems. terrestrial arthropod reviews 4, 157-180. eppo 2012. european and mediterranean plant protection organization. reporting service. n.1. disponivel em: http://archives.eppo.org/epporeporting/2012/rse-1201.pdf. fernandes, f.l., m.c. picanço, m.e.s. fernandes, v.m. xavier, j.c. martins & v.f. silva 2010. controle biológico natural de pragas e interações ecológicas com predadores e parasitóides em feijoeiro. bioscience journal 26, 6-14. filgueira, f.a.r. 2000. novo manual de olericultura. viçosa: ufv. 1398 sociobiolog y vol. 59, no. 4, 2012 frança, s.m., j.v. oliveira, c.m. oliveira, m.c. picanço & a.p. lôbo 2009. efeitos ovicida e repelente de inseticidas botânicos e sintéticos em neoleucinodes elegantalis (guenée) (lepidoptera: crambidae). boletín de sanidad vegetal. plagas 35, 649-655. galdino, t.v.s., m.c. picanço, e.g.f. morais, n.r. silva, g.a.r. silva & m.c. lopes 2011. bioassay method for toxicity studies of insecticide formulations to tuta absoluta (meyrick, 1917). ciência e agrotecnologia 35, 869-877. garcia-marí, f. & r. vercher 2010. descripción, origin y expansión de tuta absoluta (lepidoptera; gelechiidae). phytoma-españa 20, 16-20. gonring, a.h.r, m.c. picanço, j.c. zanuncio, m. puiatti & a.a. semeão 2003a. natural biological control and key mortality factors of the pickleworm, diaphania nitidalis stoll (lepidoptera: pyralidae), in cucumber. biological agriculture & horticulture 20, 365-380. gonring, a.h.r., m.c. picanco, r.n.c. guedes & e.m. silva 2003b. natural biological control and key mortality factors of diaphania hyalinata (lepidoptera: pyralidae) in cucumber. biocontrol science and technolog y 13, 361-366. gusmão, m.r., m.c. picanço, r.n.c. guedes, t.l. galvan & e.j.g. pereira 2006. economic injury level and sequential sampling plan for bemisia tabaci in outdoor tomato. journal of applied entomolog y 130, 160-166. hölldobler, b. & e.o. wilson 1990. the ants. cambridge: harvard univiversity. jensen, t.f. 1978. annual production and respiration in ant populatios. oikos 31, 207213. knutson, a.e. & m. campos 2008. effect of red imported fire ant, solenopsis invicta on abundance of corn earworm, helicoverpa zea, on maize in texas. southwestern entomologist 33, 1-13. liu, t.x. 2005. biolog y and life history of ascia monuste monuste (lepidoptera: pieridae), a potential pest of cruciferous vegetables. annals of the entomological society of america 98, 726-731. miranda, m.m.m, m.c. picanço, j.c. zanuncio, l. bacci & e.m. silva 2005. impact of integrated pest management on the population of leafminers, fruit borers, and natural enemies in tomato. ciência rural 35, 204-208. moreno, s.c., g.a. carvalho, m.c. picanço, e.g.f. morais & r.m. pereira 2012. bioactivity of compounds from acmella oleracea against tuta absoluta (meyrick) (lepidoptera: gelechiidae) and selectivity to two non-target species. pest management science 68, 386-393. moura, m.f., m.c. picanço, e.m. silva, r.n.c. guedes & j.l. pereira 2003. plano de amostragem do biótipo b de bemisia tabaci na cultura do pepino. pesquisa agropecuária brasileira 38, 1357-1363. narendra, a., s.f. reid & j.m. hemmi 2010. the twilight zone: ambient light levels trigger activity in primitive ants. proceedings of the royal society of london. series b, biological sciences 277, 1531-1538. 1399 ramos , r.s.— natural biological control of lepidopteran pests by ants necr (natural england commissioned report) 2009. comparison of the abundance and distribution of birds along the northern shore of poole harbour by day and by night. sheffield: natural england (necr017). picanço, m.c., m.r. gusmão & t.l. galvan 2000. manejo integrado de pragas de hortaliças. in: zambolim, l. manejo integrado de doenças, pragas e ervas daninhas. viçosa: ufv, p.275-324. picanço, m.c., l. bacci, a.l.b. crespo, m.m.m. miranda & j.c. martins 2007. effect of integrated pest management practices on tomato production and conservation of natural enemies. agricultural and forest entomolog y 9, 327–335. picanço, m.c., i.r. oliveira, j.f. rosado, f.m. silva, p.c. gontijo & r.s. silva 2010. natural biological control of ascia monuste by the social wasp polybia ignobilis (hymenoptera: vespidae). sociobiolog y 56, 67-76. picanço, m.c., l. bacci, r.b. queiroz, g.a. silva, m.m.m. miranda, g.l.d. leite & f.a. suinaga 2011. social wasp predators of tuta absoluta. sociobiolog y 58, 621-634. pratissoli, d., r.a. polanczyk, a.m. holtz, t. tamanhoni, f.n. celestino & r.c. borges filho 2008. influência do substrato alimentar sobre o desenvolvimento de diaphania hyalinata l. (lepidoptera: crambidae). neotropical entomolog y 37, 361-364. ross, k.g. & l. keller 1995. ecolog y and evolution of social organization: insights from fire ants and other highly eusocial insects. annual review of ecolog y and systematics 26, 631-656. way, m.j. & k.l. heong 2009. significance of the tropical fire ant solenopsis geminata (hymenoptera : formicidae) as part of the natural enemy complex responsible for successful biological control of many tropical irrigated rice pests. bulletin of entomological research 99, 503-512. 875 queens and workers of the primitively eusocial wasp ropalidia marginata do not differ in their dufour’s gland morphology by aniruddha mitra1* & raghavendra gadagkar1, 2 abstract ropalidia marginata is a primitively eusocial paper wasp found in peninsular india, where recent work suggests the role of the dufour’s gland hydrocarbons in queen signaling. it appears that the queen signals her presence to workers by rubbing the tip of her abdomen on the nest surface, thereby presumably applying her dufour’s gland secretion to the nest. since the queen alone produces pheromone from the dufour’s gland and also applies it on the nest surface, the activity level of queen gland should be higher than that of worker gland, as the gland contents would have to get replenished periodically for queens but not for workers. the difference in activity level can be manifested in difference in dufour’s gland morpholog y, larger glands implying higher activity levels and smaller glands implying lower activity levels, as positive correlation between gland size and gland activity has been reported in exocrine glands of various taxa (including hymenopteran insects). hence we investigated whether there is any size difference between dufour’s glands of queens and workers in r. marginata. we found that there was no difference between queens and workers in their dufour’s gland size, implying that dufour’s gland activity and dufour’s gland size are likely to be uncorrelated in this species. keywords: dufour’s gland; morphometry; ropalidia marginata; queens; workers 1centre for ecological sciences, indian institute of science, bangalore 560012, india. 2evolutionary and organismal biology unit, jawaharlal nehru centre for advanced scientific research, jakkur, bangalore 560064, india. *corresponding author, email: mitra.aniruddha@gmail.com 876 sociobiolog y vol. 59, no. 3, 2012 introduction monopolization of reproduction by the queen is an important distinguishing feature of eusocial insect colonies. ropalidia marginata (hymenoptera: vespidae) is a primitively eusocial (queens and workers morphologically similar) paper wasp found in peninsular india. r. marginata queens are remarkably docile and thereby cannot be using dominance behavior to maintain reproductive monopoly (gadagkar 2001). therefore the queen must use other means, possibly pheromone, to suppress worker reproduction (sumana & gadagkar 2003). recent work has shown the dufour’s gland, a small exocrine gland located near the tip of the abdomen, to be at least one source of the queen pheromone. in a bioassay it was found that a macerate of the queen’s dufour’s gland can act as a proxy for the queen herself, thereby elucidating the role of this gland in queen signaling (bhadra et al. 2010). chemical analysis of dufour’s glands revealed long chain hydrocarbons that were found to be correlated with the state of ovarian activation of queens (bhadra et al. 2010; mitra et al. 2011; mitra & gadagkar 2011a, b), and it was also shown that the dufour’s gland hydrocarbon composition may act as an honest signal of fertility (mitra & gadagkar 2011a, b). these evidences unequivocally suggest the role of the dufour’s gland in queen signaling, and it appears that the queen signals her presence to workers by rubbing the tip of her abdomen on the nest surface, thereby presumably applying her dufour’s gland secretion to the nest (bhadra et al. 2007). workers appear to perceive the presence of their queen through her dufour’s gland compounds and react accordingly (bhadra et al. 2010). the activity of a gland is often correlated with the size of the gland. it has been found in both vertebrates as well as invertebrates that increase in activity of an exocrine gland can be reflected in increase of its size, and positive correlations between quantity of secretions and rates of biosynthesis with sizes of glands have been reported (herrera 1992; elmèr & ohlin 1969; ravi ram & ramesh 2002; hassani et al. 2010; tobe & stay 1985; roseler et al. 1980). with reference to the dufour’s gland, ali et al. (1988) have found that gland size corresponds with amount of secretion in workers of the ant formica sanguinea. other studies have also suggested a connection between gland activity, gland size and reproductive status. workers with well-developed 877 mitra, a. & r. gadagkar — dufour's gland morpholog y of ropalidia marginata ovaries and egg-laying workers had larger dufour’s gland diameters than workers with poorly developed ovaries in the bumble bee bombus terrestris, and gland size of queens also were found to increase with reproductive activity (abdalla et al. 1999a, b). in r. marginata, since the queen alone produces pheromone from the dufour’s gland and also applies it on the nest surface, possibly by rub abdomen behavior, the activity level of queen gland should be higher than that of worker gland, as the gland contents would have to get replenished periodically for queens but not for workers. the difference in activity level can be manifested in difference in gland size, larger gland size implying higher activity levels and smaller gland size implying lower activity levels. hence the objective of our study was to see whether there is any size difference between dufour’s glands of queens and workers. materials and methods: six post emergence nests of r. marginata were used in this study. r. marginata forms aseasonal, perennial colonies and nests are founded and abandoned throughout the year. the study was conducted from september 2008 to september 2009. nests were collected from various localities in bangalore (13° 00’ n and 77° 32’ e), and transplanted to the vespiary at the centre for ecological sciences, indian institute of science, bangalore. the nests were maintained in closed cages made of wood and fine mesh, and provided with ad libitum food, water and building material. all wasps were uniquely colorcoded with small spots of testors® enamel paints (gadagkar 2001) and, the queen was identified by egg-laying behaviour prior to beginning the study. the queen along with six randomly chosen workers were collected from each nest and the dufour’s gland of each wasp was dissected out along with the sting in ringer’s solution under a stereomicroscope (wild m3z). each gland was photographed using a camera attached to a stereomicroscope (leica s8ap0), using the software leica im 50, version 4.0. the following measurements were made from the image of each gland using the same software (fig 1): total length of the gland (breaking up the length into several short straightline segments and then summing them up) (µm) maximum width of the gland (µm) 878 sociobiolog y vol. 59, no. 3, 2012 fig 1: diagrammatic representation of the measurements made from the image of dufour’s gland of ropalidia marginata; (a): length of the gland (breaking up the length into several short straight-line segments and summing them up), (b): maximum width of the gland, (c): perimeter of the gland’s image, and (d): area covered by the gland’s image. (diagram made by tracing from photograph, drawing : aniruddha mitra.) 879 mitra, a. & r. gadagkar — dufour's gland morpholog y of ropalidia marginata perimeter of the gland’s image (µm) area covered by the gland’s image (µm2) analysis of variance (anova) was done to look at morphological difference between glands of queens and workers with respect to the four measurements made. dufour’s glands measurements were subjected to a principal components analysis and dufour’s gland size index calculated by taking scores of each individual on the first principal component (which accounted for 83.33% of total variance). dufour’s gland size indices of queens and workers were compared by a mann whitney u test. following morphometry, the dufour’s glands were subjected to gas chromatography for another study (mitra & gadagkar 2011a). body measurements were done for each wasp by making 24 measurements (in mm) of different body parts (table 1) using a stereomicroscope (wild m3z), and the measurements subjected to a principal components analysis. the score of each individual on the first principal component (which accounted for 55.6% of total variance) was used as an index of body size. correlation between body size index and dufour’s gland size index was checked by spearman rank correlation, and since a positive correlation was found, dufour’s gland size parameters were corrected by dividing with transformed body size index (made positive to get rid of negative values by linear transformation, done by adding 10 to all the values) and the resulting corrected values were subjected to anova. statistical analyses were done using statistixl 1.7. table 1: list of 24 measurements made from different body parts of ropalidia marginata for calculating body size index. # measurement 1 intra-ocellar distance 2 ocello-optic distance (right) 3 ocello-optic distance (left) 4 head width 5 head length 6 mesoscutum width 7 mesoscutum length 8 alitrunk length 9 wing length (left) 10 length of 1st marginal cell (left wing ) 11 number of hamuli (left wing ) 12 length of 1st gastral segment 13 width of 1st gastral segment 14 height of 1st gastral segment 15 length of 2nd gastral segment 16 width of 2nd gastral segment 17 height of 2nd gastral segment 18 clypeus length 19 clypeus width 20 length of 1st antennal segment (right) 21 length of 1st antennal segment (left) 22 width of 1st antennal segment (right) 23 width of 1st antennal segment (left) 24 inter-antennal socket distance 880 sociobiolog y vol. 59, no. 3, 2012 results overall morpholog y of the dufour’s glands of queens and workers appeared to be similar. we did not find any difference between queens and workers with respect to any of the four parameters of their dufour’s gland size (fig. 2). multivariate anova: f = 1.358, p = 0.267 univariate anova: length of gland: f = 0.007, p = 0.936 maximum width of gland: f = 0.250, p = 0.620 perimeter of gland’s image: f = 0.001, p = 0.973 area covered by gland’s image: f = 0.795, p = 0.378 in the dufour’s gland size index, it was found that workers had a greater range of gland size (having both undeveloped as well as large glands). queens had a smaller range of gland size, but were not different from workers (fig. 3) (mann whitney u test: u = 124, p = 0.586). fig 2: mean and standard deviation of (a) length of the dufour’s gland, (b) maximum width of the gland, (c) perimeter of the gland’s image and (d) area covered by the gland’s image in queens (q) and workers (w) of ropalidia marginata. bars are carrying the same letter signifying that there is no difference between queens and workers for any of the four variables (anova, p > 0.05, n = 6 queens and 36 workers). 881 mitra, a. & r. gadagkar — dufour's gland morpholog y of ropalidia marginata body size index and dufour’s gland size index were positively correlated implying that wasps with larger body size tend to have a larger dufour’s gland as well (spearman rank correlation: r s = 0.311, p = 0.046, n = 84). because body size was positively correlated with dufour’s gland size, all the dufour’s gland size parameters were corrected for body size and the resulting corrected values were subjected to anova. there was no difference between queens and workers even after correcting for body size. multivariate anova (corrected for body size): f = 1.163, p = 0.344 univariate anova (corrected for body size): length of gland: f = 1.631, p = 0.209 maximum width of gland: f = 1.165, p = 0.287 perimeter of gland’s image: f = 1.610, p = 0.212 area covered by gland’s image: f = 2.193, p = 0.147 fig 3: dufour’s gland size indices of queens (q) and workers (w) of ropalidia marginata. distributions carry the same alphabet indicating that they are not significantly different (mann whitney u test, p > 0.05, n = 6 queens and 36 workers). 882 sociobiolog y vol. 59, no. 3, 2012 discussion we found that there was no difference between queens and workers in their dufour’s gland size. in polistine wasps, fratini et al. (1996) found in polistes dominulus that dufour’s glands were larger in foundresses than in workers. in r. marginata however, the dufour’s gland of the queen is not different in size from that of the workers. since the queen is the sole reproductive in a colony and she alone produces pheromone from the dufour’s gland, and also applies it on the nest surface, possibly by rub abdomen behaviour (gadagkar 2001; bhadra et al. 2007; bhadra et al. 2010), we expected that the activity level of queen glands should be higher than worker glands, which can be reflected in size difference between dufour’s glands of queens and workers. our results suggest that even if there is activity difference between dufour’s glands of queens and workers, this does not translate to size difference. our results are reminiscent of what was found in polistes fuscatus, where egg laying foundresses do not necessarily have larger dufour’s glands than subordinates (downing & jeanne 1983). it may be possible that increase in secretory activity of a gland can be manifested in other ways like increase in abundance of cell organelles likely to be involved in secretion, rather than increase in gland size. some studies suggest that actual synthesis of compounds in hymenopteran exocrine glands might be low and such glands might be involved in uptake of compounds from the haemolymph and transporting them to the lumen of the gland, while the compounds themselves are synthesized elsewhere (abdalla & cruz-landim 2004; strohm et al. 2010). since the dufour’s gland in r. marginata has been found to contain long chain linear and branched alkanes, which are typically found on the cuticle of insects (bhadra et al. 2010; mitra et al. 2011), it is possible that the dufour’s gland compounds are synthesized in oenocytes, which are usually involved in synthesis of insect hydrocarbons, and are then transported through the haemolymph to the dufour’s gland, and get sequestered there, as has been suggested for other hymenopteran exocrine glands (strohm et al. 2010). hence difference in dufour’s gland activity need not necessarily translate to difference in gland size, as has been suggested in other endocrine and exocrine glands as well (roseler et al. 1980; zouboulis & boschnakow 2001). 883 mitra, a. & r. gadagkar — dufour's gland morpholog y of ropalidia marginata we conclude that dufour’s gland activity and dufour’s gland size are uncorrelated in r. marginata, and thereby any activity difference that exists between dufour’s glands of queens and workers does not translate to difference in dufour’s gland size. acknowledgments we thank the department of science and technolog y, the department of biotechnolog y, the council for scientific and industrial research and the ministry of environment and forests, government of india for financial assistance. dissections, morphometry and statistical analyses were done by am. the paper was co-written by am and rg, and rg supervised the overall work. all experiments reported here comply with the current laws of the country in which they were performed. references abdalla, f.c. & c. da cruz-landim 2004. occurrence, morpholog y and ultrastructure of the dufour gland in melipona bicolor lepleiter (hymenoptera, meliponini). revista brasileria de entomologia 48(1):9-19. abdalla, f.c., h. velthius, m.j. duchateau & c. da cruz-landim 1999. secretory cycle of the dufour’s gland in workers of the bumble bee bombus terrestris l. 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(ed.). advances in insect physiolog y, volume 18. london, academic press: 305-341. zouboulis, c.c. & a. boschnakow 2001. chronological ageing and photoageing of the human sebaceous gland. clinical and experimental dermatolog y 26:600-607. 287 decayed wood affecting the attraction of the pest arboretum termite nasutitermes corniger (isoptera: termitidae) to resource foods by vinícius gazal1,2, omar bailez2, ana maria viana-bailez2, elen de lima aguiar-menezes1 & eurípedes barsanulfo menezes1 abstract nasutitermes corniger shows preferential feeding for the wood of different tree species, but it is not known whether attractiveness is a function of the state of decay. this study examined the foraging behavior of n. corniger towards wood in different stages of decay. wood was exposed to weather for durations of 0, 3, 6 or 9 months. then the wood was placed in a standard foraging arena with termites. exploration and recruitment behavior were recorded for 1 h. separate bioassays were conducted for three species: pinus elliottii, eucalyptus grandis and manilkara huberi. in the tests with p. elliottii and e. grandis, more individuals were recruited to wood decayed for 6 months (191 and 185, respectively) than to undecayed wood (12 and 69, respectively). similarly, more individuals were recruited to decayed m. huberi wood than undecayed, but only after 9 months (249 and 7, respectively). decayed wood has therefore been demonstrated to be more attractive to n. corniger than undecayed wood. the different decomposition rates necessary to increase attractiveness may be explained by differences in wood density. key words: decaying wood, foraging behavior, nasutitermes, recruitment, termites, wood-decomposition. introduction the termite nasutitermes corniger (motschulsky) is one of the most harmful arboreal species in brazil, where it causes damage to wood on buildings and 1universidade federal rural do rio de janeiro-ufrrjvgazal@uenf.br departamento de entomologia e fitopatologia, rod. antiga rio-são paulo km 47-seropédica, rio de janeiro-brazil 2universidade estadual do norte fluminense darcy ribeiro-uenf, laboratório de entomologia e fitopatologia, setor de semioquímicos, av. alberto lamego 2000 – parque california 28013 602 campos dos goytacazes, rio de janeiro-brazil 288 sociobiolog y vol. 59, no. 1, 2012 indoor structures. control is difficult owing to the construction of polycalic nests, which are often some distance from the point of infestation (mill 1991; costa-leonardo 2002; vasconcellos & bandeira 2006). existing methods to reduce damage in urban areas are based on backfill barriers of potent residual insecticides as well as chemical wood treatment (su & scheffrahn 1998). alternative methods based on baits impregnated with small amounts of insecticide are preferred to reduce the risk of accidental poisoning and environmental contamination (su & scheffrahn 1993; su & scheffrahn 1998; rojas & morales-ramos 2001). these have been used successfully in the control of subterranean termites of the genera coptotermes and reticulitermes, but have so far been ineffective with nasutitermes termites (su 1994; costa-leonardo 2002). originally, n. corniger was assumed not to discriminate between the wood of different plant species (bustamante 1998). however, gazal et al. (2010) showed that eucalyptus grandis was more heavily attacked than pinus elliottii and manilkara huberi wood, when offered simultaneously. another factor that could influence the choice of n. corniger is the degree of wood decomposition. a positive effect of wood decay on attractiveness has been demonstrated in other termite species (lenz et al. 1980; lenz et al. 1991; bustamante 1998). this study aimed to investigate the attractiveness of wood altered by decomposition processes to n. corniger, with the intention of developing more suitable baits. material and methods maintenance of termites in the laboratory twenty mature colonies of n. corniger (with alates termites present) were collected from trees located in different non-urban areas. the colonies were established in glass containers 50 × 40 × 60 cm and maintained under laboratory conditions of 25±5ºc, 85±5% relative humidity (r.h.) and 10:14 h light:dark cycle (gazal et al. 2010). each colony was connected by a silicone tube (8 mm in diameter) to an exclusive foraging arena consisting of a 50 × 50 cm glass plate with a 5 cm high perimeter wall. to avoid pre-conditioning to the wood types used in the experiments, prior to the experiments the termites were fed with cedar wood, cedrela odorata l. (meliaceae) and hymenolobium elatum ducke (fabaceae). 289 gazal, v. et al. —attraction of termites wood in the experiments, wood from the following trees was used: pine, pinus elliottii engelm. (pinaceae); eucalypt, eucalyptus grandis hill (ex maiden) (myrtaceae); and massaranduba, manilkara huberi ducke (ex chevalier) (sapotaceae). these woods have very different densities (0.55, 0.81 and 1.10 g/cm3, respectively) and geographic origin: pinus elliottii originated from the united states, where it is considered a forest species of great importance (drescher et al. 2001); e. grandis is native to the east coast of australia; and m. huberi originated from brazil’s amazon region (azevedo et al. 2007). the wood densities were calculated after determining the dry mass of a piece of freshly fallen wood of known volume. the three wood species were cut into pieces of 5 × 2 × 2 cm and a part of these wood was stored in a room at 15±5ºc and 50±5% r.h. for a duration of 9 months to prevent decomposition (undecayed wood or control). the other part of the wood was exposed to weathering for 3, 6 or 9 months (decayed wood), respectively. the wood was exposed to weather as a monolayer on the terrace of the laboratory. the 9-month decay period began in june 2006, the 6-month period in september and the 3-month period in december of the same year, so they ended on the same date in march 2007 when the tests began. rainfall, temperature, relative humidity and radiation data were recorded. bioassays the bioassays were carried out separately for each of the three wood species. all the tests were carried out between 1:00 and 6:00 pm since a preliminary test in laboratory conditions demonstrated that n. corniger does not have a daily activity rhythm. before beginning the tests, the linking tube from the nest to the foraging arena was obstructed with mineral cotton for 15 minutes to prevent the entrance of insects into the arena. immediately, the maintenance food was removed and the termites within it were transferred to the nest. then, the treatments (decayed pieces of wood of the same species by 3,6 and 9 months) and the control (one undecayed piece of wood) were placed in the arena on separate glass plates of 5 × 2.5 cm, 10 cm apart from each other, equidistant (20 cm) from the point of access. for each test, the position of the treatment and control wood in the arena was randomized. the tests began when the access of the termites to the arena was reopened. each experiment was carried out on twenty colonies of termites (n = 20). 290 sociobiolog y vol. 59, no. 1, 2012 the tests started when the first termite contacted one of the wood samples. we then recorded the occurrence of: a) initial exploration, b) initial recruitment and c) massive recruitment behavior. initial exploration was considered to be when the first soldier made contact with a piece of wood, pressed its abdomen against it and then returned to the nest. initial recruitment was considered to be when the first workers arrived at the wood source. massive recruitment was considered to be when a large number of workers arrived at the wood source via a trail (gazal et al. 2010). after 1 h of observation, the plates containing wood and the termites present on them were removed and the termites present on each plate were counted. the proportions in which the behaviors initial exploration, initial recruitment and mass recruitment occurred in the tests were compared between the decayed wood treatments and the undecayed wood (control) with chi-square tests. the number of termites recruited at the end of the tests was compared by anova and the means compared by tukey’s test (p<0.05). due to the lack of normal distribution these data were log-transformed (ln). results the climatic conditions to which the wood types were exposed are presented in table 1. only one of the climatic variables differed between treatments. solar radiation was highest in the 3-month treatment (kruskal–wallis, h 1, 350 = 11.97, p < 0.001). this treatment was performed in the summer months only, when radiation is invariably higher. the mean of other variables (rainfall, relative humidity and temperature) did not differ significantly between treatments (kruskal–wallis test). therefore, the time of exposure to weather was assumed to be the main factor causing differences between the treatments. the first contact of the termites with one of the wood pieces generally occurred a few minutes after opening the access to the arena. initial exploration, initial recruitment and mass recruitment were observed to undecayed wood as well as to decayed wood, regardless of the treatment and species wood type (table 2). the proportion of initial exploration and initial recruitment did not differ between undecayed and decayed wood. however, the proportion of 291 gazal, v. et al. —attraction of termites massive recruitment of termites to decayed wood was always greater than to undecayed wood. in the tests with p. elliottii, recruitment was highest to wood exposed to weather for 6 months (12/20 vs 2/20 to undecayed wood, χ2=8.90, p < 0.01). a similar result was observed in tests with e. grandis (13/20 vs 3/20 table 1. mean daily rainfall (frequency and volume), temperatures (minimum and maximum), relative humidity and solar radiation during weather exposure of wood decomposition treatments (3, 6 and 9 months). treatment (months) rainfall temperature relative humidity solar radiationfrequency volume min. max. (n) (mm) (ºc) (ºc) (%) (wm-2) 0 (control) 0.0±0.0 0.0 10.0±0.3 20.0±0.2 50.0±5.0 0.0 3 0.5±0.1 6.3±1.5 21.4±0.1 30.2±0.3 84.0±0.7 237.0±8.0 6 0.5±0.1 5.2±1.0 20.2±0.2 28.6±0.3 83.0±0.6 218.0±6.0 9 0.4±0.1 3.8±0.6 19.0±0.2 28.3±0.2 81.2±0.5 204.4±4.7 *significant difference in kruskal–wallis tests, p < 0.01. table 2. percentage of tests with occurrence of the three phases of foraging behavior (exploration, initial recruitment and massive recruitment) of nasutitermes corniger on wood exposed to ambient conditions for 0 (control), 3, 6 and 9 months (treatments). the tests were replicated five times on four termite colonies (n = 20). experiments were performed separately with wood of pinus elliottii, eucalyptus grandis and manilkara huberi. wood treatment behavioral event species (months) initial exploration initial recruitment massive recruitment (%) (%) (%) p. elliottii 0 (control) 95 80 10 3 90 65 30ns 6 85 85 60* 9 90 75 40ns e. grandis 0 95 80 15 3 90 75 25ns 6 95 90 65* 9 95 70 45ns m. huberi 0 80 65 10 3 85 65 20ns 6 80 75 30ns 9 95 80 50* *significant difference from the control in χ2 tests, p < 0.05. 292 sociobiolog y vol. 59, no. 1, 2012 undecayed wood, χ2 = 8.44, p < 0.01). the total number of termites recruited to decayed wood (6 months) at the end of the tests was also higher than to undecayed wood, both for p. elliottii (191.4±51.9 vs 12.4±4.1, f 3,76 = 3.44, p < 0.05) and for e. grandis (185.2±44.6 vs 69.0±42.8, f 3,76 = 3.82, p < 0.05). this behavior was similar among the different replications (f 19,220 = 0.97, p = 0.41). in tests with m. huberi mass recruitment happened more times on decayed wood than on undecayed wood (10/20 tests vs 2/20 tests, χ2 =5.83, p < 0.05), but in this case to the wood exposed to weather for 9 months (table 2). similarly, more termites were also recruited to decayed wood exposed for 9 months than to undecayed wood (249.4±62.9 vs 7.3±2.1, f 3,76 = 2.91, p < 0.05, fig. 1). discussion attacks by n. corniger to the undecayed wood of pinus elliottii, eucalyptus grandis and manilkara huberi were recently described by gazal et al. (2010). given that the termites attacked the undecayed wood of all three species, even in situations in which decayed wood was also available, decay may not be a figure 1. number of nasutitermes corniger termites recruited [ln] (mean ± se) after 60 min to undecayed and decayed wood (3, 6 and 9 months) in a foraging choice arena. separate experiments were conducted with the wood of pinus elliottii, eucalyptus grandis and manilkara huberi. different letters indicate significant differences between wood treatments in tukey tests, p < 0.05. 293 gazal, v. et al. —attraction of termites prerequisite for termite attacks (esenther et al. 1961). on the other hand, the occurrence of mass recruitment to decayed wood of the three species indicates that the decay process was not restrictive to exploitation of wood as a resource. in our experiments, pine and eucalypt wood decayed for 6 months and massaranduba wood decayed for 9 months recruited more termites than the undecayed wood. increase attractiveness of decomposed wood to termites may be a consequence of changes in nutritional value due to the action of fungi. the wood decomposition process can release toxic compounds, but can also increase nutrient availability (waller & la fage 1987). further, changes in the physical structure caused by depolymerization of cellulose, including cell wall thinning, bore hole formation and rounded pit erosion, may ease fiber removal (erwin et al. 2008). in addition, allelochemicals that influence the foraging behavior of termites may also be released (cornelius et al. 2002), some of which, for example, may trigger trail following (esenther & beal 1979; rust et al. 1996). extracts from decayed wood have been used to direct subterranean termite foraging toward a bait station (su 2005). in this study, p. elliottii and e. grandis wood was most attractive to termites after a decay period of 6 months, but m. huberi wood was most appealing after a decay period of 9 months. this difference may be due to lower rates of water retention of dense wood resulting in slower decomposition (bultman & southwell 1976). wood with higher density, such as m. huberi, may need to be exposed to the weather for longer to undergo chemical or physical changes equivalent to that occurring in eucalypt or pine wood. contrary to expectation, e. grandis and p. elliottii decayed for 9 months were not preferred to undecayed wood. this indicates a drop in attractiveness at a certain point in the decomposition process. a similar response was found in studies with the termites coptotermes formosanus shirak and reticulitermes flavipes kollar, when wood decayed for 8 weeks was preferred to the undecayed wood, but wood decayed for 12 weeks was not (cornelius et al. 2003). perhaps the ease of fiber removal or gains in nutritional quality in the short term do not compensate indefinitely for nutrient losses associated with the decomposition process longer. alternatively, increased attractiveness may be offset by the simultaneous production of toxic or repellent chemicals. wood is often used as a bait substrate to facilitate the distribution of insecticide within termite colonies. in this study, we found that undecayed 294 sociobiolog y vol. 59, no. 1, 2012 and decayed wood offered together are attractive food sources for n. corniger. however, differences in the mass recruitment of foragers suggested that this termite species is more attracted to decayed wood. therefore, it may be beneficial to use decayed wood when preparing insecticide-impregnated baits for n. corniger termites. future research is needed to determine whether increased recruitment to decayed wood is a result of physical or chemical changes in the wood, and also to investigate the role of different decomposing agents in this process. acknowledgements we are indebted to veronica figueiredo, pamella rosa and arli de fatima, for helping in the laboratory. v.s.s. gazal was financially supported by capes. references 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nutritional ecolog y of termites. in: nutritional ecolog y of insects, mites, and spiders. eds. slansky f, rodriguez jg, wiley, new york, ny, 487-532. doi: 10.13102/sociobiology.v62i1.52-55sociobiology 62(1): 52-55 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 period and time of harvest affects the apitoxin production in apis mellifera lineu (hymenoptera: apidae) bees and expression of defensin stress related gene introduction apitoxin comes from a latin word and means: apis bee and toxikon – venom, being produced by bees as a way of defending and protecting the hive. the apis mellifera apitoxin composition varies according to the subspecies development stage and habits. the primary findings are variations in the concentrations of apitoxin proteins throughout the seasons of the year, showing an influence of the environment (abreu, 2010). the apitoxin injected into a sting contains about 50 mg of dry matter. the major proteins present are melittin; 50% of the dry weight of apitoxin (dwv), phospholipase a2 (dwv 12%), mast cell factor degranulation (3% dwv), hyaluronidase (3% dwv) and apamin (2% dwv). moreover, abstract the aims of this study were to determine the period effects (morning and afternoon) and harvest time (30 and 60 minutes) in the apitoxin production as well as the management effects in the expression of a stress related gene. five apis mellifera l. beehives were used. the harvest of apitoxin and honeybees occurred three times a week (morning and afternoon at 09h00 and 14h00), according to the following treatments (period and apitoxin harvest time): t1: morning/30 minutes, t2: morning/60 minutes; t3: afternoon/30 minutes; t4: afternoon/60 minutes. the apitoxin was collected by electric collectors. the stress level was monitored by defensin gene expression, using actin gene as control. the results were evaluated by anova followed by tukey-kramer test (p ≤ 0.05). it can be concluded that the better period and time to apitoxin harvest is in the morning for 60 minutes, associated to minor stress for honeybees. sociobiology an international journal on social insects ms modanesi, sm kadri, pem ribolla, dp alonso, ro orsi article history edited by celso f martins, ufpb, brazil received 29 july 2014 initial acceptance 06 october 2014 final acceptance 28 october 2014 keywords honeybees, collection, venom, welfare corresponding author dr. ricardo de oliveira orsi center of education, science and technology in rational beekeeping (nectar) college of veterinary medicine and animal sciences, unesp university distrito de rubião jr., s/n, 18618-000, botucatu, são paulo, brazil e-mail: orsi@fmvz.unesp.br biogenic amines are present, including histamine (1% dwv), dopamine (0.5% dwv) and norepinephrine (0.5% dwv) (cruz-landim et al., 2002). many volatile acetates which presumably stimulate the defensive behavior of other bees are also found in apitoxin (abreu et al., 2010). the apitoxin has beneficial effects to the humans, apitherapy (the use of bee products for the benefit of humans) has been used for many health problems, such as, eczema, tropical ulcers, infections such as laryngitis and mastitis, rheumatologic problems, cardiovascular, pulmonary and orthopedic, and help in the inhibition of ovarian cancer, multiple sclerosis and have an antibiotic effect (leite et al., 2005). the apitoxin harvest can be performed by electrical collectors placed at the entrance of the hive containing filaments research article bees universidade estadual paulista, botucatu, sp, brazil sociobiology 62(1): 52-55 (march, 2015) 53 conducting an electric current, thereby promoting accessory muscle contraction release of the apitoxina, without killing the bees (leite & rocha, 2005). however, even when not promoting the death of bees, there is still no knowledge of the influence of management on the hive. it is assumed that it can promote behavioral changes, interfering with routine activities of the colony, causing an acute or chronic stress. the main goal of this study was to investigate the effects of period (morning and afternoon) and harvest time (30 and 60 minutes) in apitoxin production by africanized honeybees and the influence of this management on the defensin gene expression. material and methods the experiment was conducted by the center for education, science and technology in rational beekeeping nectar, the apicultural sector, from college of veterinary medicine and animal science, experimental farm lageado, unesp, botucatu, brazil; with the following coordinates: 22o 49’’ s, 48°24’’ w and 623 meters high. in order to perform the experiment 10 africanized beehives in wooden langstroth hives model (five as control and five per treatment), externally oil painted light green, kept in 50cm racks numbered for easy identification were used. the selected colonies were standardized to the number of frames of brood and food. during the experimental period, each beehive received sugar syrup (50% water + 50% sugar) weekly through bordman feeder. to measure the defensive behavior of the colonies, we performed the defensiveness test, according to the methodology described by stort (1975) and brandenburg and gonçalves (1990). the test consisted of a black suede ball swinging in front of the entrance, attached to a string, for a minute. the first sting time (fst) were recorded and the number of stings left in the black suede ball (snb). the tests were repeated in triplicate. values above 36 stings designate defensive colonies. the bee and apitoxin harvesting to evaluate expression of genes related to stress occurred three times a week, from november 2011 to january 2012 (twelve weeks), and five beehives per treatment were assessed in the same day using the same treatment according to: t1: harvesting apitoxin in the morning, lasting 30 minutes; t2: harvest apitoxin in the morning, lasting 60 minutes; treatment 3: harvest apitoxin in the afternoon, lasting 30 minutes; treatment 4: harvesting apitoxin in the afternoon, lasting 60 minutes. thus, each treatment was realized in nine replicates the harvest took place in the morning, always starting at 09:00 and in the afternoon at 14:00, by electric collectors consisted of wire filaments with six volts of electric current, and a glass plate for the fall of the released apitoxin (leite & rocha, 2007). at the end of each harvest, the collectors and the glass plates were removed and sent to the laboratory of apiculture sector, kept at room temperature and protected from light, until the evaporation of the volatile phase. after, the apitoxin was scraped with a spatula, mixed (pool of five beehives), weighed and stored at -20oc. climatic variables (mean temperature, wind speed, rainfall and relative humidity) were provided by the department of environmental sciences, college of agricultural sciences, unesp, botucatu. the climatic data for the period of the study were as follows: minimum temperature of 17.2 ± 1.9 ºc, maximum temperature of 30.6 ± 2.2, precipitation of 5.0 ± 6.6 mm, relative humidity 60.13 ± 10.4%, solar radiation 477.1 ± 80.0 cal / cm², insolation 8.1 ± 3.4 hours decimal and wind speed 99.7 ± 39.8 km/day. the data for the analysis of stress-related genes were processed at laboratory for genetics research and analysis, parasitology department, biosciences institute, são paulo state university – unesp, botucatu campus. for the experiments of defensin expression, a stress related gene, the harvest of the after the placement of apitoxin collectors. ten internal worker bees and ten foragers were used. after harvest, the bees were immediately stored at 80°c for rna extraction. bees were collected also of five beehives control, without apitoxin collect, in the different treatments. for rna extraction, the head of each worker bee was separated from the body with the aid of a disposable scalpel (scharlaken et al., 2008). each sample consisted of a “pool” of five heads and rna extraction was performed by using the trizol method, using for each sample 500 ul of trizol (gibco brl) to disrupt the cells and release their contents. the extraction product was visualized on a 1% agarose gel and quantified using the nanodrop instrument (nd-1000 spectrophotometer). then all samples were stored at -80°c until ready to use. after, samples were treated with dnase, and cdna synthesis reaction was set as follows: a mix of oligodt solution (n = 18) 0.75 mm; random oligonucleotides (n = 8) 0.15 mm; 0.75 mm dntp and 11 µl of rna treated with dnase in the previous step , was prepared and incubated at 65°c for 5 minutes and then placed on ice for 1 minute. to this preparation, 0.5mm dtt, 40u of rnase out and 100u of super script iii were added. the reaction was then incubated at 50°c for 1 hour and then at 70°c for 15 minutes. the stress level of the bees was monitored by changes in defensin gene expression (scharlaken et al., 2008). as internal control for quantitative pcr reactions, we used the actin gene (scharlaken et al., 2008). the determination of gene expression was performed by real time polymerase chain reaction in triplicate, on a real time abi 7300 instrument (applied byosystems) using the sybr green pcr master mix kit (applied biosystems, foster city, ca) under the following conditions: one cycle at 50°c for 2 minutes, a cycle at 94°c for 10 minutes, followed by 40 cycles of 94°c for 15 seconds and 60°c for 1 minute. the dissociation curve was obtained as follows: 95°c for 15 seconds, 60°c for 30 seconds and 95°c for 15 seconds. the oligonucleotides sequences used and details are shown in the table 1: ms modanesi et al – apitoxin production and defensin gene expression 54 to calculate the efficiency for the oligonucleotides used, four dilutions of cdna samples were made: 1:5, 1:25, 1:125 and 1:625. the efficiency (e) was calculated using the formula e = 10 (-1/slope). relative quantification (r) was determined according to pfaffl (2001). data were calculated using bees taken before placing electric collectors as controls in relation to the other five harvests after the electric collectors were removed. the analysis of the data obtained was compared by anova followed by tukey test to check for differences between means. results were considered statistically different when p<0.05 (zar, 1996). results and discussion do not observed difference in the first sting time between the beehives of treatments and control (1.3±0.7 to 1.4±0.8 seconds) and number of stings left in the black suede ball (50.3±17.2 to 51.8±14.4). there was a higher apitoxin production in the treatment using 60 minutes in the morning (t2), which differed significantly from the other treatments (table 2). relative quantification of the defensin gene in foragers bees showed no significant difference between treatments and treatments versus control. in control worker bees was observed that treatment using 60 minutes in the afternoon (t4) showed significant difference between others treatments. internal workers showed significant difference between treatments 2 and 3 throughout the experimental period. the control beehives do not observed differences. it was observed significant difference between internal works to treatment 4 and the control. no difference was observed between the foragers and internal workers for all treatments and was observed difference between foragers and internal worker bees from t4 and control. the colonies used in the experiment can be classified as defensive, regardless the period of the day, during apitoxin production, according to the stort (1975) classification, which the bees are classified with high defensiveness when they sting 36 more times the black suede ball. we could observe that there was no influence of climatic variables on colonies defensiveness. previous work, in the same place, found no significant correlations between time and number of first sting in the black suede ball with climatic variables (lomele et al., 2010). gene gene bank number accession oligonucleotides sequences 5’-3’ amplification (pb) ta (oc)a ee (%)b actina ab023025 tgccaacact gtcctttctg agaattgac ccaccaatcca 155 61 99,11 defensina u15955.1 gtcggccttct cttcatggt gacctccagct ttacccaaa 200 61 91,70 ata optimal annealing temperature specific to each oligonucleotide be measuring the efficiency of the reaction real time pcr (calculated trough the standard curve) production period (min) morning (minutes) afternoon (minutes) 30 33.7±13.0 aa 24.6±10.0 aa 60 49.1±13.0 bb 24.9±6.0 aa table 2. mean values and standard deviation for apitoxin production (milligrams) of africanized honeybees according the day time and time of production. different small letters in the same column indicate statistical differences between averages (p<0.05). different capital letters in the same line indicate statistical differences between averages (p<0.05). the relative quantification (r) results from defensin gene, using actin as the endogenous gene in the samples from the different treatments and control are shows in table 3. treatment fw fw control iw iw control 1 2.02±1.6aaα 0.007±0.0abα 1.84±1.7abaα 0.005±0.0abα 2 1.23±0.1aaα 0.003±0.0abα 0.16±0.2aaα 0.06±0.01aaα 3 1.35±1.4aaα 0.002±0.0abα 2.97±1.6baα 0.07±0.0abα 4 0.59±0.4aaα 0.025±0.0bbα 0.56±0.6abaα 0.003±0.0abβ table 3. mean and standard deviation of defensin gene relative quantification of foragers workers (fw) and internal workers (iw), in different treatments with afr canized honeybees. different small letters in the same column indicate statistical differences between averages (p<0.05). different capital letters in the same line, to group of foraging or internal bees (treatment vs control), indicate statistical differences between averages (p<0.05). different greek letters in the same line, to foraging vs internal, indicate statistical differences between averages (p<0.05) treatment 1: apitoxin production in the morning, during 30 minutes; treatment 2: apitoxin production in the morning, during 60 minutes; treatment 3: apitoxin production in the afternoon, during 30 minutes , treatment 4: apitoxin production in the afternoon, during 60 minutes. table 1. genes and oligonucleotide sequences useoto avaluate the defensin expression. sociobiology 62(1): 52-55 (march, 2015) 55 the higher apitoxin production occurred in the morning period during 60 minutes. a probable cause for this result would be the foraging behavior of bees. malerbo and souza (2011) observed in the end of spring and in the beginning of summer, that nectar were collected by the bees throughout the day, with preference for the hottest hours of the day, between 10 and 14h, when the temperature was between 15 and 30°c. thus, a greater flow of bees results in greater amount of foragers through the collector and consequently a significant increase on the release of apitoxin through electrical stimulation. however, the apitoxin harvest could promote alterations in all beehives, affecting the grooming, brood care and hygienic behavior, besides could affect the immunity sistem. as the honeybees could be exposed to field pathogens, defensin gene related to stressor stimulus like changes in immune system was quantified (ursic-bedoya & löwenberger, 2007). there was significant increase in the defensin gene expression in the internal worker bees in the treatment 2 (morning/60 minutes) compared to the treatment 3 (late/30 minutes). this higher defensin gene expression in the internal worker bees could be related to the increase of alarm pheromones released at the time of apitoxin release by the electrical stimulation (isopentilacetate and 2 heptanone) promoting alertness in other worker bees to protect the hive, increasing discomfort and possibly higher stress in the afternoon period than morning. it was observed that the apitoxin harvest promote a highest defesin expression in treatments when compared as control, with exception for internal worker bees from treatment 2 that do not differ as control. its result could be explained by the worker bees alarm pheromone adaptation in the collector. this is important because apitoxin harvest would not promote stress in the bees, associated with highest production, and can be used by beekeepers. it can conclude that the better period and time to apitoxin harvest is in the morning for 60 minutes, associated the minor stress for honeybees. acknowledgments we would like to thank (capes), for the master’s degree scholarship and fapesp by financial support (process number 2012/23466-2). references abreu, r.m., moraes, r.l.m.s. & mathias, m.i.c. (2010) biochemical and cytochemical studies of the enzymatic activity of the venom glands of workers of honey bee apis mellifera l. (hymenoptera, apidae). micron, 41: 172-175. doi: 10.1016/j. micron.2009.09.003. brandeburgo, m.a.m. & gonçalves, l.s. (1990) environmental influence on the aggressive (defense) behaviour and colony development of africanized bees (apis mellifera). ciência e cultura 42: 759-771. http://www.cabdirect.org/ abstracts/19920510324.html (accessed date: 28 june, 2014). lomele, r.l., evangelista, a., ito, m.m., ito, e.h., gomes, s.m.a. & orsi r.o. (2010) natural products on the defensive behavior of apis mellifera l. acta scientarium animal science, 32: 285-291. doi: 10.4025/actascianimsci. v32i3.8486. funari, s.r.c., rocha, h.c., sforcin, j.m. & orsi, r.o. (2004) influence of smoke and lemon grass (cymbopogon citratus) on the defensive behavior of africanized bees and their hybrid european (apis mellifera l). boletim de indústria animal, 61: 121-125. http://revistas.bvs-vet.org.br/bia/article/ view/8704. (accessed date: 28 june, 2014). leite, g.l.d. & rocha, s.l. (2005) apitoxin. revista unimontes científica, 7: 115-125. http://www.ruc.unimontes. br/index.php/unicientifica/article/viewarticle/145. (accessed date: 28 june, 2014). malerbo-souza, d.t. & silva, f.a.s. (2011) comportamento forrageiro da abelha africanizada apis mellifera l. no decorrer do ano. acta scientarium animal sciences, 33: 183-190. doi: 10.4025/actascianimsci.v33i2.9252. pfaffl, m. (2001) a new mathematical model for relative quantification in real-time rt–pcr. nucleic acids research, 29: e45. scharlaken, b., graaf, d.c., goossens, k., peelman, l.j. & jacobs f.j. (2008) differential gene expression in the honeybee head after a bacterial challenge. developmental and comparative immunology, 32: 883-889. doi: 10.1016/j. dci.2008.01.010. stort, a.c. (1975) genetic study of the aggressiveness of two subspecies of apis mellifera in brazil. behavior genetics, 3: 269-274. http://link.springer.com/article/10.1007/bf01066178. (accessed date: 28 june, 2014). ursic-bedoya, r.j. & lowenberger, c.a. (2007) rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization. developmental and comparative immunology, 31: 109-120. doi: 10.1016/j.dci.2006.05.008. zar, j. h. (1996) biostatistical analisys. 4th ed. new jersey: prentice hall 663 p. 213 thermal tolerances of three tramp ant species (hymenoptera: formicidae) by daniel russ solis & odair correa bueno abstract tramp ant species present a set of adaptations to their urban habitats, and there is a paucity of knowledge about how they interact with abiotic factors, like temperature. temperature is well known to interfere with insect activity. the present study evaluated the temperature tolerance of three important tramp ant species: monomorium floricola ( jerdon), monomorium pharaonis (linnaeus) and tetramorium bicarinatum (nylander). tested temperatures were 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50ºc. ten repetitions with 20 workers each were done with each temperature and analyzed species. the number of dead workers was recorded every hour over a total of 8 hours. all procedures were done using thermal incubators at relative humidity within 50-95%. workers of m. pharaonis proved more tolerant to high temperatures (30-50ºc) than workers of m. floricola and t. bicarinatum. the higher the temperatures tested, greater was the recorded ant mortality, with temperature 50ºc being fatal to all species after 1h of exposition. the least tolerant species to temperatures below 20ºc was t. bicarinatum. low temperatures tested were not fatal to any of the tested species. key words: myrmicinae, monomorium floricola, monomorium pharaonis, tetramorium bicarinatum. introduction both biotic and abiotic factors exert great influence over the functionality of organisms. changes in these factors can be recurrent or intermittent, predictable or unpredictable, and each species differ in its ability to handle and adapt to a change of conditions (linksvayer & janssen 2008). according with the same authors, examples of abiotic factors would include range of centro de estudos de insetos sociais, instituto de biociências, universidade estadual paulista “júlio de mesquita filho”, unesp, rio claro, são paulo, brazil; e-mail: entomo75@yahoo.com 214 sociobiolog y vol. 59, no. 1, 2012 temperature and humidity, fires, droughts, hurricanes and climate changes. temperature is an important factor driving insect activity; the body temperature of insects reflect surrounding environmental conditions, and most of their internal heat is metabolically produced, particularly by wing muscles (chapman 1998). the urban ant fauna presents considerable medical and economic importance, as many species are pests of domiciliary, industrial, and commercial establishments, including some which can cause relevant structural damage; many ant species are serious pests of crops and natural habitats, and some are important mechanical vectors of pathogenic microorganisms in hospital areas, and can cause severe allergic reactions (vega & rust 2001; campos-farinha et al. 2002; klotz et al. 2008). among urban ants, there are those species baptized ‘tramp species’, which feature a set of advantageous characteristics that enabled them to adapt to humanized environments, as described in passera (1994). tramp ant species typically have a global geographical distribution, being only absent in the coldest world parts (e.g., antarctica) (wetterer 2008, 2009a, 2009b, 2009c, 2009d, 2010a, 2010b). based on the classification of ants into functional groups proposed by andersen (2000), it can be seen that the most common tramp ant species would include representative species from dominant dolichoderinae (e.g., linepithema), opportunists (e.g., tetramorium and paratrechina) and generalized myrmicinae (e.g., monomorium and pheidole). these functional groups vary in the way they respond to environmental stress and disturbances: generalized myrmicinae are better adapted to hot climates and shaded areas, while being only moderately sensitive to low temperature; opportunistic species are well adapted to cool, shaded, and open, warm habitats (andersen 1995). in spite of the importance of urban ants, little is known about their adaptability to abiotic factors at species level. for instance, a recent study with atta sexdens (linnaeus) (angilletta et al. 2007) compared the temperature tolerance of workers from urban and rural regions, and found that workers from colonies in urban environments can tolerate higher temperatures (42°c) for a longer time period than workers deriving from colonies in the countryside. however, the authors could not determine whether the differences in thermal tolerance derived of some genetic component or was the result of a more flexible response to the environmental conditions. 215 solis, d.r. & o.c. bueno — thermal tolerances of tramp ants the present study aimed at assessing the thermal tolerance of three tramp ant species: monomorium floricola ( jerdon), monomorium pharaonis (linnaeus), and tetramorium bicarinatum (nylander). we expect the finds to have applicable implications to managing these pest species in the urban environment, as knowledge about such basic biolog y parameters of pest species can elucidate how they adapted to the urban environment. material and methods maintenance of the ant colonies in the laboratory laboratory colonies of the ants m. floricola, m. pharaonis, and t. bicarinatum were held in a climatized room with controlled temperature (23-27ºc) and humidity (50-80%). the colonies were brought up inside wooden boxes (10.5 cm x 8.5 cm x 1.0 cm), or test tubes (25.0 cm x 2.5 cm), the tubes containing a piece of cotton soaked in water at the distal end and were lined with red cellophane. ants were fed with insects, larvae of tenebrio molitor (linnaeus) (coleoptera: tenebrionidae) and zophobas sp. (coleoptera: tenebrionidae), adults of gryllus sp. (orthoptera: gryllidae), and sugary liquids (water solution of honey and inverted sucrose), and pieces of sausage and sardine. food items were presented three times a week, tap water was offered ‘ad libitum’. assembly and analysis of the experiment randomly-picked workers from several colonies were exposed to the following temperatures: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50ºc. ten repetitions with 20 workers each were done with each temperature and analyzed species. these experiments were made inside a thermal incubator (eletrolab, model el 101/3) containing plastic trays with water to keep interior relative humidity within 50-95%; this being the range considered adequate for rearing these ants in the laboratory, as previously mentioned. temperature variation inside the incubator was set to a minimum of ±0.5ºc. in all cases, ant workers were kept in test tubes (25 cm x 2.5 cm) closed with perforated tissue as a screen. with the only exception of the temperature 25°c, the tested workers were acclimatized to the exposure temperature tests: they were brought from the rearing room (23-27ºc) to a thermal incubator at 25°c, and the temperature was slowly adjusted about 0.5°c/min to the experiment temperature. 216 sociobiolog y vol. 59, no. 1, 2012 the number of dead workers was recorded every hour over a total of 8 hours. according with hebling-beraldo (1978), ants are immobilized at lower temperatures due to some cold narcosis, thus when dealing with temperatures below 8°c we waited for 30 min at room temperature until living ants recovered at ambient temperature (18-21°c). statistical analysis resulting percentage of surviving workers from each temperature were transformed by arcsine to yield normal distribution. transformed data were then submitted to analysis of variance (one-way anova) followed by dunnett’s test, in order to check from which temperature mortality of workers became significant over the analyzed time periods tested. control temperature set for dunnett’s test was 25ºc. moreover, we calculated lt 50 (lethal temperature for 50% of workers) and tl 90 (lethal temperature for 90% of workers) for each species and period of exposure using probit analysis. obtained lethal temperature values were subjected to analysis of variance (two-way anova) followed by tukey’s test, comparing among different species and periods of exposure. results from analyzing the results of the 23 tested temperatures, it can be seen that total mortality was shared by the three species at the temperature of 50ºc upon the first hour of exposure. for the other exposure periods at temperatures over 25ºc, complete mortality is shown in fig. 2. however at temperatures below 25ºc, total mortality never occurred (fig. 1). from analyzing tables 1, 2, and 3, it can be observed that from temperatures above 25ºc at longer periods of exposure there is a decrease in the temperature value at which worker mortality becomes significant, demonstrating how workers are less tolerant to high temperatures at longer periods of exposure. most tolerant species to high temperatures was m. pharaonis, with the other two presenting the same amount of tolerance. from analyzing table 1, which presents results for temperatures below 25ºc, it can be seen that only t. bicarinatum exhibited significant mortality at the longer periods of exposure of 6, 7, and 8 hours. however, mortality at these temperatures with m. floricola and m. pharaonis was not significant, 217 solis, d.r. & o.c. bueno — thermal tolerances of tramp ants fig. 1. worker mortality of the tramp ant species monomorium floricola (○), monomorium pharaonis (☐), and tetramorium bicarinatum (∆) when exposed to different temperatures below 25ºc over eight hours of exposure. error bars display standard error of the mean. 218 sociobiolog y vol. 59, no. 1, 2012 fig. 2. worker mortality of the tramp ant species monomorium floricola (○), monomorium pharaonis (☐), and tetramorium bicarinatum (∆) when exposed to different temperatures above 25ºc over eight hours of exposure. error bars display standard error of the mean. 219 solis, d.r. & o.c. bueno — thermal tolerances of tramp ants suggesting that these species are more tolerant to lower temperatures than t. bicarinatum. discussion hebling-beraldo (1978) determined the lt 50 over six hours of exposure of the brazilian ant’s a. sexdens and atta laevigata (smith), and found their respective tolerance limits to be 2.8ºc and 36.3ºc, and 1.5ºc and 37.5ºc. from comparing their obtained tolerance limits with our results, it becomes clear that our tested species have a broader temperature tolerance. we think this might be correlated with the fact that they are exotic invasive species, which are expected to be more tolerant to altered abiotic conditions. as an table 1. temperatures from which worker mortality in three tramp ant species was significant when exposed to different experimental temperatures, according with dunnett’s test (p < 0.01) employing 25ºc as control temperature. species time (h) / temperature (ºc) 1 2 3 4 5 6 7 8 monomorium floricola 46 44 44 44 42 42 40 38 monomorium pharaonis 48 46 46 44 44 44 40 40 tetramorium bicarinatum 44 42 42 42 42 0;42 0;42 0;42 table 2. lt 50 (lethal temperature to 50% of sample) obtained for the tramp ants species over eight hours of continuous exposition to different temperatures above 25ºc. species1 time (h) / temperature (ºc)2 1a 2ac 3acd 4bc 5bd 6 bd 7 bd 8 bd monomorium floricola a 45 44.8 44.1 43 42.7 42.5 42.3 41.5 monomorium pharaonis b 48.8 46.9 45.2 44.6 43.7 43.1 42.7 42 tetramorium bicarinatum a 44.6 44.3 43.3 42.8 42.2 41.6 41.1 40.9 notes. 1figures on the same column followed by same letter did not differ by tukey’s test (p < 0.01). 2figures on the same line followed by same letter did not differ by tukey’s test (p < 0.01). table 3. lt 90 (lethal temperature to 90% of sample) obtained for the tramp ants species over eight hours of continuous exposition to different temperatures above 25ºc. species1 time (h) / temperature (ºc)2 1a 2ac 3ac 4ac 5ac 6bc 7bc 8bc monomorium floricola a 45.9 45.6 45.4 44 44 44 44.3 44 monomorium pharaonis b 49.6 47.8 46.1 45.8 45.2 44.5 44.2 44.5 tetramorium bicarinatum a 45.3 45.7 44.7 44.3 44.2 43.7 43.2 43.2 notes. 1figures on the same column followed by same letter did not differ by tukey’s test (p < 0.01). 2figures on the same line followed by same letter did not differ by tukey’s test (p < 0.01). 220 sociobiolog y vol. 59, no. 1, 2012 example of a brazilian ant invasive to other countries, xu et al. (2009) obtained the lt 50 for the fire ant solenopsis invicta (buren) of 43.5ºc after one hour of exposure, which is still lower than the herein recorded with other three species. tramp ant species have worldwide distribution and are typical of humanized areas, thus probably display physiological and behavioral adaptations that allow their survival in stressful environments wherein the native species cannot thrive (passera 1994). for instance, some species do not build their nests in an organized fashion into the soil (herein including tramp ant species), and living under rocks and fallen branches, there are physiologically less vulnerable to lower humidity or higher temperatures (hölldobler & wilson 1990). however, linksvayer & janssen (2008) stated opportunistic ant species are more tolerant to stressful and disturbed environments, but cannot out compete species adapted to extreme temperatures. for instance, walters & mackay (2004) compared heat tolerance of linepithema humile (mayr) (an invasive species) in australia in comparison with two native species: iridomyrmex rufoniger (lown) and rhytidoponera convex (mayr). these authors found all l. humile died after one hour of exposure to 50ºc, while others were more tolerant: total annihilation with r. convex only occurred after 2h of exposure, while i. rufoniger still presented a 50% survival after 3h of exposure to 50ºc. in a similar study, holway et al. (2002) compared l. humile with five species native to california and also found that l. humile was less tolerant to higher temperatures (all killed after 1h at 46°c) than native species (others were only annihilated at temperatures above 50°c). on the other hand, walters & mackay (2004) advised caution when interpreting such results as apparently ants can modulate behavior in response to environmental changes to avoid exposure to extreme conditions, and temperature should not be regarded isolated from other important abiotic factors. for instance, ant species living in deserts and other dry habitats can adapt by building their nests deeper into the soil, and diurnal species will usually forage in the morning and at nightfall (hölldobler & wilson 1990). in a study of diurnal foraging in three tramp ant species from the galapagos islands, meier (1994) found that foraging workers of m. floricola have a pattern of activity over the day, with peak activity during the day (from 06:00h 221 solis, d.r. & o.c. bueno — thermal tolerances of tramp ants to 18:00h). from analyzing the foraging pattern for m. floricola presented by meier (1994) on page 56, two significant decreases in the foraging at the peaks of activity can be perceived: between 11:00h and 13:00h and from 16:00h to 18:00h. upon comparing meier’s graph with another illustration on page 56 of meier (1994) presenting temperature variation over the day, it can be seen that the two reductions in foraging coincided with temperature variations: the first foraging reduction can be related with the peak temperatures of the day 30-32ºc while the second reduction can be related with temperature dropping below 26ºc. foraging ceased completely between 00:00h and 05:00h, period at which mean temperature remained around 22ºc. interestingly, worker mortality was null in our experiments within the temperature range 18-30°c, illustrating that m. floricola is well adapted to this temperature range. it should be emphasized, however, that even if a given ant species is tolerant to higher temperatures, this does not mean it will forage at such temperatures (cerda et al. 1998). cerda et al. (1998) evaluated the thermal tolerance of a spanish ant community of 11 species. one of their conclusions revealed a direct correlation between worker size and maximum critical temperature (i.e., a 10-min exposure to a given temperature in which 50% of worker wither died or were severely impaired): species with larger workers were more tolerant to higher temperatures. in our experiments, t. bicarinatum was the largest species and proved the least tolerant. francke et al. (1985) hypothesized that tolerance to different temperatures in ants can be influenced by worker age, ambient humidity, previous thermal regime, and colony diet, apart from colony location (angilletta et al. 2007). as all three species were reared under the same room conditions and the tests employed only workers found outside the colonies which are regarded as the oldest (hölldobler & wilson 1990) we think the observed differences between species directly reflect their intrinsic temperature tolerances. based on the results of this study we can state that workers of m. pharaonis are the most tolerant to higher temperatures among the tested species. regarding temperatures below 25ºc, t. bicarinatum proved also the least tolerant. other temperature studies with these species ought to evaluate their regulatory capacity at colony level, thus assessing general survival develop222 sociobiolog y vol. 59, no. 1, 2012 ment of colony members, and the correlation of temperature with other environmental variables (like humidity), and the existing behavioral and physiological mechanisms to cope with extreme conditions. acknowledgments we would like to thank capes institution for the financial support provided and to eduardo gonçalves paterson fox for kindly translating and revising the manuscript. references andersen, a.n. 1995. a classification of australian ant communities, based on functional groups which parallel plant life-forms in relation to stress and disturbance. journal of biogeography 22: 15-29. andersen, a.n. 2000. a global ecolog y of rainforest ants: functional groups in relation to environmental stress and disturbance. in: agosti, d., j.d. majer, l.e. alonso & t.r. schultz (eds). ants: standard methods measuring and monitoring diversity. smithsonian institution press, washington: 25-34. angilletta, m.j., r.s. wilson, a.c. niehaus, m.w. sears, c.a. navas & p.l. ribeiro. 2007. urban physiolog y: city ants possess high heat tolerance. plos one 2: e258. campos-farinha, a.e.c., o.c. bueno, m.c.g. campos & l.m. kato. 2002. as formigas urbanas no brasil: retrospecto. o biológico 64: 129-133. cerda, x., j. retana & s. cros. 1998. critical thermal limits in mediterranean ant species: trade-off between mortality risk and foraging performance. functional ecolog y 12: 45-55. chapman, r.f. 1998. the insects: structure and function. cambridge university press, cambridge. francke, o.f., l.r. potts & j.c. cokendolpher. 1985. heat tolerances of four species of fire ants (hymenoptera: formicidae: solenopsis). southwestern naturalist 30: 59-68. hebling-beraldo, m.j.a. 1978. tolerância às variações de temperatura, em operárias de saúvas. revista brasileira de biologia 38: 195-199. hölldobler, b. & e.o. wilson. 1990. the ants. harvard university press, cambridge. holway, d.a., a.v. suarez & t.j. case. 2002. role of abiotic factors in governing susceptibility to invasion: a test with argentine ants. ecolog y 83: 1610-1619. klotz, j., l. hansen, r. pospischil & m. rust. 2008. urban ants of north america and europe: identification, biolog y, and management. cornell university press, ithaca. linksvayer, t.a. & m.a. janssen. 2008. traits underlying the capacity of ant colonies to adapt to disturbance and stress regimes. systems research and behavioral science 26: 315-329. 223 solis, d.r. & o.c. bueno — thermal tolerances of tramp ants meier, r.e. 1994. coexisting patterns and foraging behavior of introduced and native ants (hymenoptera, formicidae) in the galapagos islands (ecuador). in: williams, d.f. (ed). exotic ants: biolog y, impact and control of introduced species. wetsview press, san francisco: 44-62. passera, l. 1994. characteristic of tramp species. in: williams d.f. (ed). exotic ants: biolog y, impact and control of introduced species. wetsview press, san francisco: 23-43. vega, s.j.v. & m.k. rust. 2001. the argentine ant – a significant invasive species in agricultural, urban and natural environments. sociobiolog y 37: 3-25. walters, a.c. & d.a. mackay. 2004. comparisons of upper thermal tolerances between the invasive argentine ant (hymenoptera: formicidae) and two native australian ant species. annals of the entomological society of america 97: 971-975. wetterer, j.k. 2008. worldwide spread of the longhorn crazy ant, paratrechina longicornis (hymenoptera: formicidae). myrmecological news 11: 137-149. wetterer, j.k. 2009a. worldwide spread of the ghost ant, tapinoma melanocephalum (hymenoptera: formicidae). myrmecological news 12: 23-33. wetterer, j.k. 2009b. worldwide spread of the destroyer ant, monomorium destructor (hymenoptera: formicidae). myrmecological news 12: 97-108. wetterer, j.k. 2009c. worldwide spread of the argentine ant, linepithema humile (hymenoptera: formicidae). myrmecological news 12: 187-194. wetterer, j.k. 2009d. worldwide spread of the penny ant, tetramorium bicarinatum (hymenoptera: formicidae). sociobiolog y 54: 811-830. wetterer, j.k. 2010a. worldwide spread of the flower ant, monomorium floricola (hymenoptera: formicidae). myrmecological news 13: 19-27. wetterer, j.k. 2010b. worldwide spread of the pharaoh ant, monomorium pharaonis (hymenoptera: formicidae). myrmecological news 13: 115-129. xu, y.j., y.y. lu, z.p. pan, l. zeng & g. liang. 2009. heat tolerance of the red imported fire ant, solenopsis invicta (hymenoptera: formicidae) in mainland china. sociobiolog y 54: 115-126. 1351 seasonal trends in honeydew-foraging strategies in the red wood ant, formica yessensis (hymenoptera: formicidae) by izumi yao1 abstract the red wood ants formica yessensis are known to support super colonies comprising thousands of nests, contain approximately 360 million workers, and over one million queens along the ishikari coast, hokkaido, northern japan. previous studies revealed the abundance of prey insects in ishikari is very limited; suggesting that honeydew collected from aphids is a critical resource for f. yessensis. furthermore, several reports suggested f. yessensis performs a generation change between late july and early august at the study site. the present study examined seasonal changes in f. yessensis honeydewforaging workers and specifically addressed the following : information transfer to aphid trees; fidelity to aphid trees; and changes in f. yessensis body size. observation of marked ants revealed that information transfer to aphid trees occurred by direct guidance from older to younger foragers. seasonal sampling indicated that honeydew-foraging ant body size decreased with progressive seasons. large gaster coefficient of variation (cv) values showed two honeydew-foraging ant worker types were present in the super colony. the results revealed older foragers exhibited a large body size, which decreased in number towards autumn. younger workers exhibited a smaller body size, and initiated honeydew foraging after emergence. honeydew is a critical resource, therefore information transfer to aphid tree location, and honeydew-foraging were the first priority tasks observed in f. yessensis at the study site. keywords honeydew-foraging , formica yessensis, generation change, fidelity, body size, gaster introduction consistent with many large colonial organisms specialised life histories and social system, formica yessensis worker ants are sterile. consequently workers 1department of ecolog y and systematics, graduate school of agriculture, hokkaido university, sapporo 060-8589, japan author's email: iyao@res.agr.hokudai.ac.jp. 1352 sociobiolog y vol. 59, no. 4, 2012 do not engage in mating practices, but maximize efforts on identifying and collecting food sources (traniello 1989). compared to prey insects, honeydew is a suitable and effective feeding resource; and foraging locations are stable. therefore, an optimal strateg y in f. yessensis is the transfer of geographic information regarding aphid tree locations from older to younger foraging generations. however, little is known about how information transfer occurs in ants. f. yessensis support super colonies comprising thousands of nests, approximately 360 million workers, and over a million queens distributed along the ishikari coast, hokkaido, northern japan (43˚n, 141˚e). cherix (1987) compared the diets of f. yessensis super colonies distributed at ishikari (the study site) and hakkenzan (adjacent forest sites). the number of prey insects in ishikari was approximately 1/28 that of hakkenzan, suggesting honeydew is a critical resource for f. yessensis in ishikari. the annual activity cycle, and development of nest structure for f. yessensis fig. 1 annual cycle of colony activities and nest development of nest in f. yessensis. figure 1 in ito (1973) was rearranged. arrows indicate ant collection (c1-3) and marking (m1-2 periods. 1353 i yao, i. — seasonal trends in honeydew-foraging strategies , i. in ishikari, including some environmental phenolog y was characterised by ito (1973). extra-nest activities by post-hibernating workers initiate in midapril, following thaw. full-scale activity starts in late may, when tuberculatus quercicola aphid honeydew becomes available on quercus dentata. budding is generally practiced generally from may through july, and new workers emerge in late july to mid-september (fig. 1). corresponding to ito’s (1973) description, the worker number in attending aphid colonies increases until late june, followed by a decline that continues until late july. in late july, early august, worker number sharply increases again. this period of emergence of new ant nest workers coincides with an increase in aphid colony workers in late july. therefore, it is reasonable to hypothesis that new workers correspond to a younger generation of ant workers. genotypic analyses using microsatellite markers revealed distinct genetic differentiation among samples of f. yessensis individuals representing seasonal populations collected in spring and summer (yao & akimoto 2009). these results suggested summer honeydew foragers were not members of the spring population. however, principle coordinate analysis for spring and summer population samples indicated a genetic relationship among populations. in f. yessensis, the daughter queen has been observed returning to natal nests (higashi 1983). consequently, information transfer regarding aphid tree locations is likely inherited, and occurs between late july and early august. this raises the question of whether or not morphological differences exist between older and younger generations of workers. if morphological differences are favoured in younger generations, body size variation in ants attending aphid colonies is expected to change with the progression of seasons. in addition to information transfer, ant-aphid tree fidelity is a critical strateg y to maintain stable resources. fidelity to honeydew has been examined in camponotus pennsylvanicus (fowler & roberts 1980), ectatomma ruidum (schatz et al. 1995), lasius fuliginosus (quinet & pasteels 1996), and formica obscuripes (mciver & yandell 1998). wilson (1971) demonstrated in highly polyg ynous populations, colony boundaries frequently appear in individuals that move freely among nests. therefore, in mutualisms between f. yessensis and t. quercicola, an aphid tree can have foragers composed of workers that belong to different nests. moreover, as insects and their larvae 1354 sociobiolog y vol. 59, no. 4, 2012 are low in abundance in spring but high in summer, aphid tree fidelity may diminish based on insect prey availability. the aims of the present study is to examine the mutualistic relationships between f. yessensis ants and t. quercicola aphids using the following approaches: (1) fidelity to honeydew by means of a marking methodolog y; and (2) examine the morphological differences between older and younger foragers by comparing workers collected in june, august, and september. fidelity to honeydew, and seasonal body size variation were discussed in terms of resource availability, and environmental factors. materials and methods marking fig. 2 tree distribution sampled in the present study. distances among trees (m) are given as solid line. the number in each circle is an assigned sample number. broken and solid curved lines indicate ant movement from the original ant marking tree to other trees in spring and summer. 1355 i yao, i. — seasonal trends in honeydew-foraging strategies , i. ten q. dentata trees were chosen in the field, and used to monitor marked workers (fig. 2). workers foraging honeydew, or moving on q. dentate were collected with tweezers from each tree, and marked by a paint marker (uni mitsubishi pencil). workers were marked on the dorsal surface of the gaster, and released to the trunk of the tree from which the worker was collected. from each tree, 1000 workers were marked. different colours were applied on each of ten trees. marking was performed twice on each tree from 6-12 june and 29-31 july 2011(table 1). weekly observations of marked individuals were made from 9:00 to 14:00. for each tree, the ant number on aphid colonies per q. dentata shoot, where seven-eight leaves gathered, was counted. the counting did not exceed 20 shoots supporting aphid colonies. if the number of aphid colonies was fewer than 20, the number of ants climbing up the trunk was counted for 20min. observations were conducted from 25 june to 4 october 2011. the fidelity coefficient defined by quinet & pasteels (1996) was employed: 100× (co/ co+de), where co (conservants) refers to the number of marked ants observed on the site where they were marked; and de (deviants), the number of marked ants observed at other sites. ant collection and measurement morphological differences between older and younger foragers were investigated by collecting a total of 50 f. yessensis workers per tree from t. quercicola aphid colonies. if the number of t. quercicola aphid colonies did not exceed 20, f. yessensis ants observed on the tree trunk were collected. ant collection was performed three times in 2011; 18-25 june, 3 august, and 23-26 september. ant samples were preserved in a collection tube with 100% ethanol and stored in -20℃ freezer. worker ant head width was measured as a body size index. in addition to measuring of head width, three body parts (head, thorax, and gaster) were weighed to evaluate each average weight and the coefficient of variation (cv) between seasons. the head was removed from each worker to measure head width. all legs were removed from each worker to extract genomic dna for another experiment. the remaining body i.e., the thorax and gaster, was divided into two parts. subsequently the head, thorax, and gaster were dried at 56℃ for a minimum 3 hr. each body part was weighed with an ultramicro 1356 sociobiolog y vol. 59, no. 4, 2012 fig. 3 seasonal changes in (a) the number of non-marked, spring-marked, and summer-marked ants observed on 10 sampled trees. the number of springand summer-marked ants was extracted from (a) and given again in (b). (kaleida balance se-2 (sartorius). the head, thorax, and gaster weights were summed as body weight. the head width length measurement, and body parts weight were ordered based on each q. dentata tree (trees 1-10), and the medians were used for statistical analyses. each cv value was converted to an arcsine1357 i yao, i. — seasonal trends in honeydew-foraging strategies , i. square root. because few ants were found on trees 6 and 9 in autumn, the total number of scores used in statistical tests resulted in 28. statistical analyses the effects of season and tree on body size were examined applying a twoway anova. the anova model analysed the effects of the independent variables ‘tree’ and ‘month’ (factors), on the dependent variable ‘body size’. a three-way anova was applied to investigate body parts with large cv values. the anova model contained three factors, ‘body parts’, ‘tree’, and ‘month’, and the interaction term ‘body parts×month’. results fidelity to aphid trees was revealed in younger and older generations. a high fidelity to aphid trees was detected in marked workers during early june and late august; 96-100% in the younger generation, and 95-100% in the table 1. fidelity to aphid trees in june and august 1358 sociobiolog y vol. 59, no. 4, 2012 older generation (table 1). mixed older and younger foragers in aphid trees were observed between 4 and 31 august (fig. 3). a significant seasonal effect was found in head width (f 2,16 = 17.92, p < 0.0001), and body weight (f 2,16 = 21.84, p < 0.0001). head width length and body weight were high in june, and low in august and september (head width, 1.31 ± 0.1 mm for june, 1.19 ± 0.06 mm for august, 1.12 ± 0.04 mm for september; body weight, 1.14 ± 0.22 mg for june, 0.89 ± 0.12 mg for august, 0.7 ± 0.08 mg for september) (fig. 4). the gaster exhibited the largest weight among body parts (f 2,66 = 33.07, p < 0.0001; 0.35 ± 0.06 mg for head, 0.35 ± 0.06 mg for thorax, 0.46 ± 0.08 mg for gaster). cv for all body parts decreased seasonally, with the exception of the gaster. the collective cv was high in june and august and low in september (f 2,66 = 14.57, p < 0.0001; 0.4 ± 0.08 for june, 0.42 ± 0.08 for august, 0.33 ± 0.07 for september) (fig. 5). discussion the present study showed that high fidelity to aphid trees (q. dentata) was transferred from older to younger foragers by means of older foragers guidance. i observed many workers emerging from nests during the summer, ig. 4 seasonal changes in (a) head width (mm), and (b) body weight (mg ) of f. yessensis. means ± sd. data points with different letters indicate a significant difference between seasons was detected using a multiple comparisons test (tukey’s hsd test). (kaleida graph 4.1.3 was used to produce tif fail and 1359 i yao, i. — seasonal trends in honeydew-foraging strategies , i. fi g. 5 ( a) b od y pa rt c om pa ri so ns ( he ad , t ho ra x, a nd g as te r) . ( b) s ea so na l c ha ng es in th e c v in ea ch b od y pa rt . d at a p oi nt s w ith d iff er en t l et te rs in di ca te a si gn ifi ca nt d iff er en ce b et w ee n se as on s w as d et ec te d us in g a m ul tip le c om pa ri so ns te st ( tu ke y’s h sd te st ). 1360 sociobiolog y vol. 59, no. 4, 2012 however fidelity to aphid trees was maintained. in formica species, it has been reported that daughter queens return to natal nests after mating. therefore, geographical information may be transferred from generation to generation. more importantly, direct guidance from older to younger workers assures the information of aphid tree location. honeydew is a more stable food resource relative to insect prey in terms of search cost. however, not all q. dentata trees maintain aphid colonies until autumn. indeed, no aphid colony was found on two of the ten trees in late september, and attending ants were not observed on those trees. in ant nests experiencing difficulty foraging new protein resources, the capacity to pass on knowledge of honeydew resource locations (i.e., nest sites) would be advantageous, and favoured by natural selection. f. yessensis workers actively entering and exiting the nest sites have been observed at the mounds near the study trees for 11 consecutive years; and new nests appear to be established close to natal nests adjacent to trees where honeydew might be available throughout the seasons. experimental studies have shown that in f. paralugubris b, new queens can mate and remain in their natal nest or seek adoption in a foreign nest following a mating flight (cherix et al. 1991). if f. yessensis queens possess similar philopatric behavior, genetic similarities may be maintained in a nest that exploits an aphid population on a host tree as a honeydew resource. it is suggested that the susceptible and narrowly distribution q. dentata has enabled aphid colonies to persist on a limited number of trees for a long period of time, resulting in a long-term mutualistic interaction between t. quercicola aphids, and attending f. yessensis ants. the present study showed that the body size of honeydew-foraging ant workers was not uniform throughout the summer through fall seasons, but ranged from 1.31-1.12 mm. it has been reported in some ant species that division of labour is related to body size (herbers 1979; mciver & loomis 1993), and age (seid & traniello 2006; vieira et al.2010; amador-vargas 2012). however, honeydew-foraging tasks in f. yessensis workers showed no relationship to body size or age. this may be responsible for differences in energ y requirements between collecting honeydew, and hunting and carrying prey. when ants demand honeydew by tapping their antennae, aphids quickly respond to the requirement. therefore, compared to hunting and carrying insects, collecting honeydew likely requires less energ y. consequently, large body size may not be required for honeydew collection. 1361 i yao, i. — seasonal trends in honeydew-foraging strategies , i. larger body size was observed in workers emerging in spring. this is due to selection for larger body size in ants to over-winter; during the long hibernation (approximately six months from november to late april) larger workers have lipid stores to survive the winter months (blanchard et al. 2000). in the period before new workers emerged, larger workers were observed undergoing multiple tasks, including hunting, foraging honeydew, and carrying insect prey. spring workers were observed on oak trees seeking prey in mid-may, even when aphids were absent on trees. furthermore, marked workers, which had collected honeydew in june, were recorded carrying pupae to new nests for nest budding in mid-july. it has been reported that new emergence workers care for eggs and pupae, in f. yessensis younger workers emerged from nests and foraged for honeydew. this pattern does not follow normal polyethism patterns in ants, where new emergence workers begin tasks inside the nests. however, f. yessensis exhibits highly aggressive behavior, and its mortality risks caused by predators is negligible even considering its small body size. in addition, assigning many small workers to aphids is reasonable in terms of worker production costs. gasters exhibited the highest cv in weight, indicating that gasters are the most flexible body part. this large cv may be partly explained by the existence of several honeydew collecting worker types. mciver & yandell (1998) reported that honeydew was often transferred mouth to mouth to other ants from honeydew-foraging workers called transporters. the two types of workers may differ in physical carrying capacity for honeydew, resulting in large variation in the extent of the distended gaster. cv for all body parts but the gaster decreased with progressive seasons, indicating that honeydew-foraging workers gradually became uniform in body size. these results indicated that in autumn, honeydew foragers were replaced with new emergence workers, and older foraging ants no longer took part in honeydew foraging. acknowledgments i thank s. ikeda for helping marking ants. this study was supported by a grant-in-aid for scientific research (c) (no. 21570012 to i.y.) financed by the japan society for the promotion of science ( jsps). 1362 sociobiolog y vol. 59, no. 4, 2012 references amador-vargas, s. 2012. behavioral responses of acacia ants correlate with age and location on the host plant. insectes sociaux 59:341-350. blanchard, g.b., g.m. orledge, s.e. reynolds & n.r. franks 2000. division of labour and seasonality in the ant leptothorax albipennis: worker corpulence and its influence on behaviour. animal behavior 59:723-738. cherix, d. 1987. relation between diet and polyethism in formica colonies. experientia supplementum 54:93-115. cherix, d., d. chautems, d.j.c. fletcher, w. fortelius, g. gris, l. keller, l. passera, r. rosengren, e.l. vargo & f. walter 1991. alternative reproductive strategies in formica lugubris zett. (hymenoptera formicidae). etholog y ecolog y & evolution, special issue 1:61-66. fowler, h.g. & r.b. roberts 1980. foraging behavior of the carpenter ant, camponotus pennsylvanicus, (hymenoptera: formicidae) in new jersey. journal of the kansas entomological society 53:295-304. herbers, j.m. 1979. caste-biased polyethism in a mound-building ant species. american midland naturalist 101:69-75. higashi, s. 1983. polyg yny and nuptial flight of formica (formica) yessensis forel at ishikari coast, hokkaido, japan. insectes sociaux 30: 287-297. ito, m. 1973. seasonal population trends and nest structure in a polydomous ant formica (formica) yessensis forel. journal of the faculty of science, hokkaido university. ser. 6, zoolog y 19:270-293. mciver, j.d. & c. loomis 1993. a size-distance relation in homoptera-tending thatch ants (formica obscuripes, formica planipilis). insectes sociaux 40:207-218. mciver, j.d. & k. yandell 1998. honeydew harvest in the western thatching ant (hymenoptera: formicidae). american entomologist 44:30-35. quinet, y. & j.m. pasteels 1996. spatial specialization of the foragers and foraging strateg y in lasius fuliginosus (latreille) (hymenoptera, formicidae). insectes sociaux 43: 333-346. schatz, b., j-p. lachaud & g. beugnon 1995. spatial fidelity and individual foraging specializations in the neotropical ponerine ant, ectatomma ruidum roger (hymenoptera, formicidae). sociobiolog y 26:269-282. seid, m.a. & j.f.a. traniello 2006. age-related repertoire expansion and division of labor in pheidole dentata (hymenoptera: formicidae): a new perspective on temporal polyethism and behavioral plasticity in ants. behavioral ecolog y and sociobiolg y 60:631-644. traniello, j.f.a. 1989. foraging strategies of ants. annual review of entomolog y 34:191210. vieira, a.s., w.d. fernandes & w.f. antonialli-junior 2010. temporal polyethism, life expectancy, and entropy of workers of the ant ectatomma vizottoi almeida, 1987 1363 i yao, i. — seasonal trends in honeydew-foraging strategies , i. (formicidae: ectatomminae). acta ethologica 13:23-31. wilson, e.o. 1971. the insect societies. harvard university press, cambridge. yao, i. & s. akimoto 2009. seasonal changes in the genetic structure of an aphid-ant mutualism as revealed using microsatellite analysis of the aphid tuberculatus quercicola and the ant formica yessensis. journal of insect science 9:1-9. available online: insectscience. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i3.217-221sociobiology 60(3): 217-221 (2013) ants visiting the post-floral secretions of pericarpial nectaries in palicourea rigida (rubiaceae) provide protection against leaf herbivores but not against seed parasites k del-claro1, r guillermo-ferreira2, em almeida3, h zardini1, hm torezan-silingardi1 introduction in some plant species, floral nectaries may remain active after pollination, attracting ants that defend the plants against herbivores. thus, these nectaries act as extrafloral nectaries (efns) and are sometimes called extra-nuptial nectaries (enns; delpino, 1886), postfloral nectaries (daumann, 1932), or more recently, pericarpial nectaries (pns; schmid, 1988). these glands have frequently been treated in the literature as efns. this treatment is partly justified by their morphological and functional similarities (see paiva, 2009 for a review). extrafloral nectaries are secretory glands that are not directly involved in pollination (fiala & machwistz, 1991) and may occur in any vegetative or reproductive plant part (keeler, 1989). efns have been reported in 3941 species in 21% of vascular-plant families, representing a widespread abstract pericarpial nectaries (pns) have frequently been treated in the literature as extrafloral nectaries (efns). this treatment is partly justified by their morphological and functional similarities in attracting bodyguard ants to protect the plant against herbivores. palicourea rigida is a common neotropical savanna treelet with tubular yellow flowers that are pollinated by hummingbirds. after pollination, the corolla falls, but the sepal ring remains and keeps the nectaries active over the ovarium throughout fruit development stages. using a standard ant-exclusion experiment, we tested whether these pns attract ants to protect the developing fruits against seed parasites and the leaves against chewing herbivores. we analyzed the differences between the initial and final leaf area. before full fruits maturity, they were collected and taken to the laboratory for weighing and to observe wasp emergence. the number of wasps per fruit and per plant was recorded. the results showed that after pollination, the floral nectaries of p. rigida act as efns, attracting visiting ants. ant-tended plants lost significantly less leaf area and had heavier fruits than untended plants. however, the ants did not protect the fruits against seed-parasitic wasps. in p. rigida, the post-floral secretions of pns play the same role as efns, and the ant-plant mutualism is context-dependent based on the type of herbivore and the plant tissue consumed. sociobiology an international journal on social insects 1 universidade federal de uberlândia, uberlândia, minas gerais, brazil. 2 universidade de são paulo, ribeirão preto, são paulo, brazil. 3 empresa de pesquisa energética, superintendência de meio ambiente, rio de janeiro, rio de janeiro, brazil. research article ants article history edited by gilberto m m santos, uefs, brazil received 02 may 2013 initial acceptance 30 may 2013 final acceptance 06 july 2013 key words mutualism, ant-plant interactions, parasitism, seed predation, herbivory corresponding author kleber del-claro instituto de biologia, l.e.c.i. universidade federal de uberlândia uberlândia, mg, brazil, 38400-902 e-mail: delclaro@ufu.br evolutionary trait that has evolved several times (weber & keeler, 2012). recent evidence suggests that efns are homologous within eudicots, sharing common genetic controls and homology with floral nectaries (lee et al., 2005; weber & keeler, 2012). a wide variety of predatory taxa exhibit mutualistic interactions with plants, providing protection against herbivores in exchange for nectar and thus complementing their diets by visiting plants with efns for their sugary secretions (byk & del-claro, 2011) while enhancing plant fitness (rosumek et al., 2009; nascimento & del-claro, 2010; romero & koricheva, 2011). for example, ants (oliveira & brandão, 1991), spiders (ruhren & handel, 1999; nahas et al., 2012) and reduviidae (guillermo-ferreira et al., 2012) have been observed feeding on efns and attacking approaching herbivores. k del-claro, r guillermo-ferreira, em almeida, h zardini, hm torezan-silingardi ant-plant mutualism in palicourea rigida218 such arthropod-plant interactions may represent a strong force driving the evolution of efns (e.g. del-claro & oliveira, 1993). however, because herbivory may affect all parts of the plant, the effects of carnivores on plant fitness may be context-dependent based on the type of tissue consumed by the herbivores (marquis, 1992). for example, ants and jumping spiders protect the vegetative parts of chamaecrista nictitans (caesalpiniaceae), thus increasing fruit and seed production, but these insects do not deter seed predation (ruhren & handel, 1999; ruhren, 2003; see also nahas et al., 2012). the continuous post-floral secretion (following pollination) by pns and the adaptive significance of this trait remain underexplored; only one study has suggested that pns function as true efns in attracting ants to protect the seeds against herbivores (keeler, 1981). however, it is not clear whether such protection extends to the vegetative components of the plant because ants generally visit the nectaries on fruits. the treelet palicourea rigida kunth. (rubiaceae) is a common shrub in the brazilian savanna (cerrado). the genus palicourea is neotropical and encompasses 200 species of small trees and shrubs (taylor, 1993). these species are distinguished by certain characteristics of their yellow-red tubeshaped corollas, which exhibit adaptations for hummingbird pollination (taylor, 1996). the fruits are fleshy, purple and ornithochorous (wütherich et al., 2001). in p. rigida, after the corolla falls, the sepal ring remains active and produces nectar over the fruits throughout their development (figure 1). the fruits commonly show marks of the exit holes made by seed-parasitic wasps that develop inside the growing seeds, as occurs in p. salicifolia (wesselingh et al., 1999). the aim of this study was to test the hypothesis that after pollination, the floral nectaries of p. rigida can be considered true efns. if so, ants visiting these nectaries should reduce plant herbivory (leaf-area loss) and parasitic-wasp attacks on the fruits. materials and methods this study was conducted in the cerrado area of the “clube de caça e pesca itororó de uberlândia” ecological reserve (ccpiu; 15º57’s, 48º12’w; 640 ha) (for additional details about the study area, see réu & del-claro, 2005). field experiments were performed during the reproductive season of p. rigida in 1996, 2001 and 2005. by the flip of a coin, we designated tagged shrubs as control plants (with unrestricted ant access; n=20 in 1996, n=25 in 2001, n=22 in 2005) or treatment plants (with ants excluded by applying the tanglefoot® resin on the main plant stem; n=20 in 1996, n=24 in 2001, n=22 in 2005). between october 1996 and february 1997, we collected and weighed the fruits of the control and treatment groups. between november 2001 and april 2002, we collected data on leaf herbivory by measuring the leaf-area loss (damage by chewing insects the three most apical and full expanded leaves of each plant) at the beginning and end of the experiment (sensu moreira & del-claro, 2005). between november 2005 and june 2006, we investigated the presence of seed-parasitic wasps by collecting the developing fruits and keeping them in plastic containers for 30 days until the wasps emerged from the pupae. the fruits were collected before full maturity to avoid interference by frugivorous birds. at the end of the study in 2005, 21 control and 18 treatment plants remained because 5 plants died during the experiment. to determine whether the ants protected the fruits against seed parasites, we calculated the infestation rates as follows: (i) the number of infested plants divided by the total number of plants and (ii) the number of wasps per fruit and per plant in the treatment and control groups. to assess the correlation between fruit production and infestation level, we used the spearman coefficient. the data on wasp-infestation rates, leaf herbivory and fruit weight were compared between groups using the mannwhitney (u test). the numbers of infested plants in the control and treatment groups were compared using fisher’s exact test. all tests were performed using the software statistica 10®. data under parenthesis represent median ± standard deviation. results the results showed that leaf damage increased significantly in ant-excluded plants (initial herbivory = 16.79 ± 14.10 %; final herbivory = 25.73 ± 16.52 %; u = 47.50, figure 1. inflorescence of the treelet palicourea rigida. (a) longitudinal section (in detail) of a flower and developing fruits on the same stem. (b) after pollination, the corolla falls, and the pericarpial nectaries remain active. (c) ants (camponotus crassus mayr, 1962) feed on the post-floral secretions. sociobiology 60(3): 217-221 (2013) 219 p < 0.05), while the initial (14.65 ± 12.17 %) and final (23.54 ± 20.62 %) leaf-area loss were not significantly different in plants with unrestricted ant access (u = 143.00 p > 0.05; figure 2). furthermore, the fruits were heavier in the control group (0.203 ± 0.114 g, n = 360 fruits) than in the treatment group (0.144 ± 0.092 g, n = 340 fruits) (u = 52.50, p < 0.05). however, there was no difference in fruit formation between plants with (0.32 ± 0.17) and without ants (0.36 ± 0.17). in the three years of experiments the main ant species observed on plants were: camponotus crassus mayr, 1862; cephalotes pusillus klug, 1824 and pseudomyrmex gracilis frabicius, 1804. figure 2. leaf-area loss in palicourea rigida (rubiaceae) in antexclusion experiments and with pericarpial nectaries visited by ants. *, p < 0.05 (mann–whitney u test). parasitic wasps, two braconidae, one pteromalidae and one eulophidae of galeopsomyia genus (tetrastichinae) were found in 44% (8/18) of the plants in the treatment group and 62% (13/21) of the plants in the control group. wasp infestation did not differ significantly between groups (fisher’s exact test, p = 0.13). the mean number of wasps per fruit did not differ between the control (0.14 ± 0.17) and treatment (0.07 ± 0.11) groups (u=145.00, p = 0.19), and the mean number of wasps per plant did not differ between plants with (12.87 ± 9.31) and without ants (8.50 ± 8.36) (u = 141.00, p = 0.18). in addition, the number of wasps recovered per plant increased with the number of fruits per plant (r = 0.54, n = 21, p < 0.02; figure 3). discussion the pericarpial nectaries of p. rigida attract ants that feed on the post-floral secretions and prey on or chase away chewing herbivores, significantly reducing leaf-area loss. thus, the pns act as true efns, confirming our primary hypothesis. however, our results also show that the ants do not provide any protection against seed-parasitic wasps. these wasps may affect plant fitness by reducing the number of viable seeds (huffman 2002), resulting in losses greater than 80% in some plant species (andersen 1989). the relationship between the number of fruits and the number of wasps per plant may explain these results because a large infructescence may represent a more heterogeneous and complex structure to be explored and defended by ants, enabling the small wasps to parasitize the fruits without notice (e.g., torezansilingardi, 2011; alves-silva et al., 2012). hence, contrary to prior assumptions that a nectary on the fruit must attract ants to protect the fruit, this study suggests that the pns of p. rigida most likely did not evolve to play a role in fruit or seed protection but to reward ants with nectar in exchange for protecting the vegetative parts of the plant. our results support this hypothesis because the ants protected the leaves against herbivory, thus indirectly increasing fruit weight. leaf-area reduction due to herbivory may negatively affect fruit and seed weight (thalmann et al., 2003) by reducing the resources available for fruit production (stephenson, 1980). by decreasing fruit weight, herbivory has a crucial negative impact on future generations because seed weight influences seedling growth, germination and mortality (fenner, 2006; rees & venable, 2007). figure 3. fruit-parasitoid wasp infestation level is positively correlated with fruit abundance in palicourea rigida (rubiaceae) (spearman correlation: r=0.54, n=21, p < 0.02). numerous studies have shown that ants protect efnbearing plants against herbivores, thus increasing their reproductive success, in various parts of the world (e.g. keeler, 1989; koptur, 1992). however, the majority of published data refer to plant species that bear efns on their vegetak del-claro, r guillermo-ferreira, em almeida, h zardini, hm torezan-silingardi ant-plant mutualism in palicourea rigida220 tive parts (see rico-gray & oliveira, 2007 for review). the present study provides data for a species that bears efns on its fruits. despite being borne on the fruits, these efns are responsible for the plant’s anti-herbivory strategy, attracting ants that prevent leaf herbivory. similar to other studies, our data show that the defensive function (or lack thereof) of nectaries in this type of ant-plant mutualism depends upon the context, especially upon the type of herbivore, the tissue consumed, the visiting predators and phylogenetic inertia (o’dowd & catchpole, 1983; marquis, 1992; rashbrook et al., 1992; cuautle & rico-gray, 2003; jones & callaway, 2007; byk & del-claro 2010; nogueira et al., 2011; weber & keeler, 2012). in conclusion, our data show that in p. rigida, the nectaries located on the fruits attract ants, which protect the plant against leaf-chewing herbivores. however, the prediction that the pns play a role in fruit and seed defense was not supported. along with keeler (1981), this study provides information about the adaptive significance and evolution of pns and efns. considering the few species of rubiaceae that are known to possess efns and the benefits that ants may provide to fruit production, we suggest that the pns in this group can be considered true efns selected as a defensive strategy through ant attraction. acknowledgements we thank cnpq for providing financial support (kdc 301248; kdc and hmts 476074, 472046, 473055). we thank christer hansson and john la salle for identifying the wasps. we also thank two anonymous reviewers for valuable comments that improved this article. references alves-silva, e., barônio, g.j., torezan-silingardi, h.m. & del-claro, k. 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(2001). seed dispersal in palicourea rigida, a common treelet species from neotropical savannas. j. trop. ecol., 17: 449–458. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i2.6725sociobiology 68(2): e6725 (june, 2021) introduction many factors have shaped the biota of west indian islands. each island has a different balance of these forces related to the island’s origin, age, topography, current and past size, and degree of access by potential biotic colonists. the result is a varied collection of natural experiments in island biogeography (wilson, 1988). barbados, grenada, and saint vincent are the three southernmost major islands of the lesser antilles, a chain of oceanic island bordered to the south by the continental shelf islands of trinidad and tobago. in earlier papers, wetterer et al. (2016, 2019) examined the ants of barbados and grenada. in the present study, i consider the ant fauna of st. vincent. st. vincent is the largest island in the nation of st. vincent and the grenadines. the ant fauna of st. vincent has long been one of the most thoroughly documented of any caribbean island and as such has played a central role in defining the taxonomy of many neotropical ant abstract the ants of saint vincent have long been one of the most thoroughly documented ant faunas of any caribbean island. ant specimens collected more than 100 years ago on st. vincent include 76 valid taxa. in ten days surveying ants on saint vincent, i found eleven species not found by previous researchers. eight are widespread neotropical species (anochetus inermis, camponotus claviscapus, cyphomyrmex minutus, odontomachus ruginodis, pheidole exigua, pheidole moerens, rogeria curvipubens, solenopsis corticalis) and three are old world exotics (cardiocondyla minutior, syllophopsis sechellensis, trichomyrmex destructor). ant records from st. vincent include more neotropical species (72) and fewer old world exotic species (15) than the neighboring caribbean islands of similar size: barbados and grenada. factors that may contribute to this pattern are that, compared to barbados and grenada, saint vincent has more mountainous terrain, more intact forest, lower human population density, and fewer international tourist visits. sociobiology an international journal on social insects james kelly wetterer article history edited by jacques delabie, uesc, brazil received 12 march 2021 initial acceptance 12 april 2021 final acceptance 22 april 2021 publication date 17 june 2021 keywords ants; biogeography; exotic species; island biogeography; west indies. corresponding author james kelly wetterer florida atlantic university jupiter, florida, usa. e-mail: wetterer@fau.edu species, largely thanks to the fieldwork of herbert huntington smith (1851–1919), an american naturalist who made extensive ant collections on st. vincent between may 1889 and january 1890. in total, 29 valid ant taxa (marked below with*) were first described from st. vincent. before smith’s work, only one ant species had been reported from st. vincent: neivamyrmex klugii* (shuckard 1840). smith’s ant specimens from st. vincent represent a total of 75 additional species (forel, 1893, 1901, 1909, 1912; emery 1894; snelling, 2005). these include 63 neotropical ant species (using current taxonomy): acropyga smithii*, anochetus mayri, anochetus testaceus*, brachymyrmex minutus*, brachymyrmex obscurior*, camponotus auricomus vincentensis*, camponotus conspicuus sharpi*, camponotus sexguttatus, cephalotes pallens, crematogaster crinosa, crematogaster curvispinosa, cyphomyrmex rimosus, dolichoderus lutosus, dorymyrmex antillanus*, hypoponera foeda*, hypoponera opaciceps, hypoponera opacior*, leptogenys arcuata, leptogenys mucronata*, leptogenys florida atlantic university, jupiter, florida, usa research article ants ants (hymenoptera, formicidae) of saint vincent, west indies mailto:wetterer@fau.edu james kelly wetterer – ants of saint vincent2 pubiceps vincentensis*, linepithema iniquum, monomorium ebeninum*, mycetomoellerius jamaicensis, mycocepurus smithii*, myrmelachista ambigua*, nylanderia guatemalensis, nylanderia pubens*, nylanderia steinheili*, odontomachus haematodus (= misidentified odontomachus bauri), pheidole antillana*, pheidole flavens, pheidole godmani*, pheidole jelskii, pheidole orbica*, pheidole radoszkowskii, pheidole sculptior*, pheidole subarmata, pheidole susannae, platythyrea punctata, prionopelta antillana*, pseudomyrmex curacaensis, pseudomyrmex elongatus, pseudomyrmex flavidulus, pseudomyrmex gracilis, pseudomyrmex simplex, pseudomyrmex termitarius, pseudoponera stigma, rogeria foreli*, solenopsis azteca*, solenopsis basalis, solenopsis castor*, solenopsis geminata, solenopsis globularia, solenopsis pollux*, solenopsis pygmaea, solenopsis succinea, strumigenys alberti*, strumigenys elongata, strumigenys gundlachi, strumigenys margaritae*, strumigenys smithii, wasmannia auropunctata, and wasmannia sigmoidea. in addition, smith’s specimens included twelve cosmopolitan old world tramp ant species, spread around the world by human commerce: cardiocondyla emeryi, hypoponera punctatissima, leptogenys maxillosa, monomorium floricola, monomorium pharaonis, paratrechina longicornis, pheidole megacephala, strumigenys emmae, strumigenys rogeri, tapinoma melanocephalum, tetramorium bicarinatum, and tetramorium simillimum. wheeler (1913) published collection of ants from the island of st. vincent, listing five ant species that f.o. hovey collected on st. vincent, none of which were new records: c. sexguttatus, d. antillanus, o. haematodus, p. stigma, and s. geminata. here, i present site records from st. vincent for ant species not collected by earlier researchers, based on my own collecting efforts. the presence of these species in st. vincent listed in wetterer et al. (2016, 2019) are based on the collection records reported here. material & methods i twice collected ants on st. vincent for a total of ten days: 8–10 june 2004 (12 sites) and 30 june – 6 july 2006 (30 sites). i collected primarily in heavily disturbed environment following standard methods used in numerous earlier ant faunal surveys (e.g., wetterer & wetterer, 2004; referred to as “direct sampling” by bestelmeyer et al., 2000). the primary goal was to collect the maximum number of different ant species in the time allotted. to do this, i collected at numerous sites in the widest range of habitats accessible and permitted, spending more time at sites where the new or rare species accumulation rate was greater, and adjusting collecting techniques to best sample different habitats (longino, 2000). the vial numbers for the 42 sites were as follows (with geo-coordinates; date): 134. arnos vale, riverside apartments (13.148, -61.201, 8-jun-04) 135–138. arnos vale, next to airport (13.142, -61.212, 8-jun-04) 139–141. kingstown, botanical garden (13.168, -61.228, 8-jun-04) 142. kingstown, waterfront (13.157, -61.231, 9-jun-04) 143. reiland, side of road (13.191, -61.242, 9-jun-04) 147–152. vermont, nature trail (13.210, -61.224, 9-jun-04) 144–146. montreal garden, under path stones (13.209, -61.189, 9-jun-04) 153. biabou, playing field (13.198, -61.137, 9-jun-04) 154. stubbs bay, in litter on cliff face (13.147, -61.162, 9-jun-04) 155–156. calliaqua, w side of road (13.129, -61.191, 10-jun-04) 157–162. yambou head, beachfront (13.165, -61.144, 10-jun-04) 163–164. calliaqua, by house (13.129, -61.184, 10-jun-04) 581. rose cottage, by inn (13.136, -61.205, 30-jun-06) 582–584. spring, by beach (13.185, -61.142, 30-jun-06) 585. henry’s vale, bananas (13.319, -61.145, 30-jun-06) 586–593. ratho hill, mango (13.127, -61.189, 30-jun-06) 594–596. belmont, by bar above school (13.165, -61.180, 30jun-06) 597–607. wallilabou river, 0.3 km up river (13.313, -61.232, 1-jul-06) 608–611. richmond beach, sw end of beach (13.310, -61.235, 1-jul-06) 612–619. trinity falls, trailhead forest (13.305, -61.214, 1-jul-06) 620–622. dark view falls, trailhead forest (13.292, -61.224, 1-jul-06) 623–624. coulls corner, cliff (13.277, -61.256, 1-jul-06) 625. arnos vale, in room (13.135, -61.202, 1-jul-06) 626–643. porter point, by roadside shack (13.380, -61.166, 2-jul-06) 644. chateau, stream forest (13.377, -61.155, 2-jul-06) 645–648. sandy bay, beach (13.364, -61.136, 2-jul-06) 649–659. orange hill, forest patch (13.322, -61.124, 2-jul-06) 660. belle vue, beach (13.234, -61.122, 2-jul-06) 664. kingstown, government area (13.155, -61.223, 3-jul-06) 665–670. wallilabou falls, forest patch (13.246, -61.259, 3-jul-06) 671–676. hermitage, above reservoir (13.247, -61.212, 3-jul-06) 677–678. hermitage, by reservoir (13.247, -61.216, 4-jul-06) 679–681. grove, up n fork (13.245, -61.227, 4-jul-06) 682–685. peter’s hope, forest fragment (13.221, -61.273, 4-jul-06) 686–687. prospect, beach (13.126, -61.182, 4-jul-06) 688–690. georgetown, waterfront (13.279, -61.117, 5-jul-06) 691–698. la soufriere, 3.5 km w rabacca (13.311, -61.146, 5-jul-06) 699–705. la soufriere trail, w of trailhead (13.318, -61.160, 5-jul-06) 706–720. la soufriere trail, trailhead parking (13.318, -61.156, 5-jul-06) 721–724. sans souci, grass by beach (13.228, -61.126, 6-jul-06) 725–729. rabacca, s of dry river (13.299, -61.117, 6-jul-06) 730–737. south rivers, by dam above town (13.246, -61.152, 6-jul-06) sociobiology 68(2): e6725 (june, 2021) 3 i originally estimated site geo-coordinates using a printed map. i later made small corrections to some records using google earth. unless otherwise stated, i made species identifications. identification of some specimens was hampered by taxonomic problems in several genera, most notably brachymyrmex, hypoponera, nylanderia, and solenopsis. i therefore only present new species records that have been identified with confidence. i deposited voucher specimens at the museum of comparative zoology and the us national museum of natural history. results i collected eleven ant species on the island of st. vincent not found by earlier researchers, bringing the total number to of ant species known from the island to 87. this count does not include five ant species with records from smaller islands in the grenadines, but not from the main island of st. vincent: discothyrea humilis on petit saint vincent (antweb.org specimen casent0318490), neivamyrmex adnepos on isle à quatre (casent0318513), neivamyrmex antillanus on union island (casent0729238), tetramorium lanuginosum on canouan (wetterer 2010) and petit canouan (casent0767676–9), and thaumatomyrmex zeteki on bequia (casent0318451). new species records numbers in parentheses refer to vial numbers listed above, with underlines grouping multiple vials from the same site. 1. anochetus inermis: seven sites (2004: 139, 154; 2006: 585, 608, 664, 665, 730, 734). forel (1897) reported that all ant species that h.h. smith collected in grenada had also been found in st. vincent, except one: anochetus inermis. i found that this widespread neotropical trap-jaw ant, know from the west indies and south america, is relatively common on st. vincent. 2. camponotus claviscapus: one site (2006: 718). this yellow-brown carpenter ant is known from south and central america, as well as numerous west indian islands (wetterer et al., 2019). in the lesser antilles. i have also collected this species in aruba, bonaire, curaçao, grenada, martinique, st. lucia, and trinidad. this species commonly nests in hollow twigs. camponotus claviscapus occultus is known from haiti and the dominican republic (lubertazzi, 2019). 3. cardiocondyla minutior: ten sites (2004: 153; 2006: 582, 585, 612, 626, 645, 649, 660, 691, 699). cardiocondyla minutior is an indo-malayan native that has become a cosmopolitan tramp species (wetterer, 2014). forel (1893) published a single record of cardiocondyla from st. vincent: cardiocondyla emeryi from one site. i also collected c. emeryi from just one site. all my other cardiocondyla records were c. minutior. 4. cyphomyrmex minutus: 24 sites (2004: 137, 138, 139, 141, 148, 154, 155, 164; 2006: 582, 594, 597, 598, 608, 612, 623, 644, 664, 665, 674, 677, 679, 682, 684, 686, 691, 695, 696, 701, 706, 707, 730). cyphomyrmex minutus is common across the west indies, though it has often been misidentified as cyphomyrmex rimosus (snelling & longino, 1992; wetterer et al., 2016). deyrup (2016) explain one way to distinguish the two species: “the most easily seen difference is the much greater development of the dorsal thoracic tubercles in rimosus. if one views the mesosoma from the front and imagines that the ant is large enough to ride, minutus would be an acceptable mount, while rimosus would be too uncomfortable to ride.” 5. odontomachus ruginodis: seven sites (2004: 138, 155; 2006: 608, 644, 645, 665, 725). odontomachus ruginodis is a widespread neotropical species (wetterer et al., 2019). i collected this brown-headed trap-jaw ant much less often on st. vincent than i collected its larger all-black congener odontomachus bauri (see below). exotic populations of this species are now spreading in florida (wetterer, 2020a). 6. pheidole exigua: eight sites (2004: 139; 2006: 597, 599, 608–609, 612, 626, 707, 709, 712, 725, 730, 734). identified by stefan p. cover. this is a widespread neotropical big-headed ant is known from south america, central america, and the west indies (wilson, 2003). 7. pheidole moerens: two sites (2006: 704, 706). identified by stefan p. cover. this neotropical big-headed ant has been reported from many west indian islands and from north america (wetterer et al., 2019). sarnat et al. (2015), however, found that records of pheidole moerens from outside of the west indies may all be based on misidentifications of pheidole navigans. 8. rogeria curvipubens: two sites (2004: 139; 2006: 586). identification confirmed by david lubertazzi. rogeria curvipubens is a widespread neotropical species known from northern south america, central america, and the west indies (lapolla & sosacalvo, 2006; wetterer et al., 2019). 9. solenopsis corticalis: six sites (2006: 597, 673, 677, 682, 691, 696, 734). identified by josé pacheco and william p. mackay. concerning this tiny neotropical thief ant, pacheco and mackay (2013) wrote: “care must be taken in the caribbean region where s. pollux and s. corticalis are common, as confusion could result between them, especially on the island of st. vincent.” james kelly wetterer – ants of saint vincent4 10. syllophopsis sechellensis: two sites (2006: 665, 721). identification confirmed by mostafa sharaf. syllophopsis sechellensis (formerly monomorium sechellense) is an old-world ant species that has recently been found for the first time on many west indian islands and in florida (wetterer & sharaf, 2017; wetterer, 2020b). 11. trichomyrmex destructor: four sites (2004: 142, 163– 164; 2006: 664, 686). the destroyer ant, trichomyrmex destructor (formerly monomorium destructor), is a widespread old-world pest in many tropical and subtropical areas, where it is notorious for chewing through the insulation of electrical wires, living in and destroying electrical equipment, and attacking people (wetterer, 2009). re-identification odontomachus bauri (misidentified as o. haematodus). in the past, the name odontomachus haematodus was applied to what we now know to be several distinct species, including odontomachus bauri (brown, 1976). non-native populations of o. haematodus have been found in florida. odontomachus bauri is widespread in the west indies (wetterer et al., 2016, 2019). discussion although the islands of st. vincent, grenada, and barbados are not far apart geographically and have similar climates, they differ in many ways, including geologic history, topography, geographic isolation, and human impacts (table 1). the present study indicates that, compared with grenada and barbados, st. vincent has more neotropical ant species and fewer old-world ant species records (table 1). these differences may be due to several possible factors. the higher neotropical ant species richness may be related to more mountainous topography and more remaining forest cover (table 1). some neotropical ant species that smith collected on st. vincent in 1889–1890, however, may now be extinct on the island. fewer exotic old-world ant species on st. vincent may be related to lower historical and on-going human impacts as indicated by lower human population density (table 1). in addition, st. vincent may have a lower rate of exotic ant arrivals, in part related to lower international tourism levels (table 1). forel (1897) wrote that the ant faunas of st. vincent and grenada were almost identical. in fact, wetterer et al. (2019) found that 54 of the 65 neotropical ant species known from grenada are also known from st. vincent, including all 35 neotropical ant species that wetterer et al. (2019) collected at six or more sites in grenada. area elev. % pop/ tourists/ # known ant species (km2) (m) forest km2 year neo ow total st. vincent 381 1234 69.2 285 80k 72 15 87 grenada 344 840 50.0 331 185k 65 17 82 barbados 462 336 14.7 668 680k 46 24 70 table 1. ant species richness known from st. vincent and the neighboring islands of barbados (wetterer et al., 2016) and grenada (wetterer et al., 2019). elev. = maximum elevation. neo = neotropical species, ow = old world species. forest cover, population density, and tourism data from united nations (2020). i have conducted ant surveys on more than 30 major islands of the eastern caribbean. as i write up the results from more islands, i expect to be able to evaluate larger scale trends in the biogeography of west indian ants. acknowledgments i thank the st. vincent and the grenadines forestry department for logistic help; m. wetterer for comments on this manuscript; s. cover, d. lubertazzi, j. pacheco, w. mackay, and m. sharaf for ant identifications; the national science foundation (deb-0515648) and florida atlantic university for financial support. references bestelmeyer, b.t., l.e. alonso & r.r. snelling (2000). the ants (hymenoptera: formicidae) of laguna del tigre national park, petén, guatemala. rap bulletin of biological assessment, 16: 75-83. brown, w. l., jr. 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(1988). the biogeography of the west indian ants (hymenoptera: formicidae). in: j.k. liebherr, ed., zoogeography of caribbean insects, pp. 214-230. cornell university press, ithaca, ny. wilson, e.o. (2003). pheidole in the new world: a dominant, hyperdiverse ant genus. harvard university press, cambridge, ma. 767 morphometric characterization of a population of tetrapedia diversipes in restricted areas in bahia, brazil (hymenoptera: apidae) by cynthia maria de lyra neves1; carlos alfredo lopes de carvalho1; adriane vieira souza1 & cristovam alves de lima junior1 abstract tetrapedia species are solitary bees which collect floral oils, being restricted to tropical regions of the americas. information on forms of nesting has been little researched in the literature, requiring studies on the diversity and variability of species to obtain better management and conservation strategies for their populations. morphometry is a efficient technique and has been used to detect variation and for identification of species of bees in order to detect changes in quantitative traits within and among populations of bees. this study aimed to compare the variability of the population of tetrapedia diversipes in artificial nests located in orchards and their surroundings (other fruit) of acerola in a restricted area of the reconcavo region of bahia, brazil. right wings were extracted from 155 individuals of the t. diversipes species, to perform the morphometric analysis. in conventional morphometry, 9 variables contributed significantly to the sexual dimorphism in the study areas (α = 0.05). the geometric morphometric analysis revealed low gene flow in populations of t. diversipes demonstrating loss of genetic diversity, requiring proper management of this bee for its conservation and maintenance of the associated flora. key words: solitary bees, tetrapediini, morphometry. introduction solitary bees represent approximately 330 species in the neotropical region (michener 2000) including the genus tetrapedia (tetrapediini) with 18 1 insecta research group; center of agricultural, environmental and biological sciences, federal university of bahia reconcavo (ufrb), 44380-000, cruz das almas-ba, brazil. e-mail: cmlneves@ msn.com; insecta@ufrb.edu.br 768 sociobiolog y vol. 59, no. 3, 2012 described species in brazil (silveira et al. 2002), presenting different morphological and behavioral characters in their nesting (cordeiro et al. 2010). the tetrapedia species use preexisting cavities as nesting sites which allows the sampling of these bees by means of trap-nests or artificial nests (camillo 2005), and this solitary behavior is another hallmark of the tetrapediini tribe characterized by independence of the female in the construction and supply their nest (alves-dos-santos et al. 2007). the study of oil-collecting bees and their importance in the pollination of many plant groups is an important tool to contribute to the knowledge of their ecolog y, economic importance (imperatriz-fonseca 2010) and morphological variation of bee populations in their natural environment (francoy & imperatriz-fonseca 2010). research for the evaluation of morphological diversity is important in ecological and genetic studies, and one of the techniques used in these studies are morphometric tools (rohlf 1990). in morphometric analysis variation is studied through the covariation between pairs of linear measurements, and geometric morphometry is able to more clearly describe and locate the regions of changes and rebuild and reconstruct these differences graphically (francoy et al. 2011). morphometry has been widely applied in many studies with social bees using tools to detect or describe genetic patterns in colonies, species identification, geographical variations and phylogenetic relationships among and within populations of bees (peruquetti 2003, mendes et al. 2007; nunes et al. 2008, souza et al. 2009, carvalho et al. 2011; ferreira et al. 2011; francoy et al. 2011, souza et al. 2010), and variability in shape and size of wings of social bees (nunes et al. 2007; quezada-euán et al. 2007). however, there are few morphometric studies on solitary bees (bosch 2008), with the exception of a study on the patterns of wing venation and geographical differences between populations of the centris genus (ferreira et al. 2011) and one study on the morpholog y of tetrapedia diversipes applying linear morphometric analysis to length and width of the wings, head width and cephalic and mouth appendages (smith et al. 2011). in this context, considering the importance of morphometric studies in populations of bees, it is necessary to collect data on solitary bees and offer suggestions for future research aimed at the morphological variability within and among populations, diversity and biolog y of nesting. the study aimed to compare the degree the morphological variability of tetrapedia diversipes 769 neves, c. et al. — morphometric characterization of tetrapedia diversipes residing in artificial nests located in orchards and their surroundings (other fruit) of acerola in a restricted area in the bahia reconcavo region, brazil. material and methods the experiment was conducted in embrapa (brazilian agricultural research corporation) cassava and tropical fruits in the period between march/2008 and august/2009 in the municipality of cruz das almas (12 ° 40’12 “s, 39 º 06’07” w, 220 m), located in the reconcavo of bahia, brazil. the nests were installed in four areas: area i active germplasm bank of malpighia emarginata dc; area ii other orchards (citrus spp., spondias sp., musa spp. and mangifera indica); area iii transition area (the area between the forest and other orchards) and area iv forest fragment. the artificial nests were made of colored paper timber of 5 mm in diameter with a length of 15 cm (machado 2011), and the species collected from the tetrapediini tribe was tetrapedia diversipes. the sealed nests were transferred to the laboratory of the center for the study of insects (insecta) of the federal university of bahia reconcavo (ufrb), and placed in bod (biologic oxygen demand) chambers at a temperature of 25 ± 1 ° c, humidity 80% ± 1% with a photoperiod of 12 hours. right wings of t. diversipes were removed and placed between two plates to capture images using the program motic 2.0 ml with a digital camera coupled to a stereomicroscope, with 7.5 x magnification (carvalho et al. 2011). the veins and cells wings were classified according to the methodolog y described by silveira et al. (2002), and biological material has been identified, and stored in eppendorf tubes of 1.5 ml and deposited in the collection of the laboratory of the center for the study of insects (núcleo de estudos dos insetos-insecta), at the center of agricultural, environmental and biological sciences (centro de ciências agrárias, ambientais e biológicas) of the federal university of bahia reconcavo (ufrb). geometric morphometric analysis 18 landmarks were defined and recorded using intersections of the veins of the forewings of tetrapedia diversipes specimens (fig. 1) using software tpsdig2 version 2.12 (rohlf 2008a). the alignment of the x and y coordinates of each line of the wings (alx aly) and the centroid size of the wings can be 770 sociobiolog y vol. 59, no. 3, 2012 used as a data matrix (matrix w) in order to perform multivariate analysis (rohlf 2008b). the coordinates of the landmarks of the wings and centroid size were used in principal component analysis (pca), the matrix w was used in multivariate analysis of variance (manova) and cluster analysis by upgma (unweighted pair-group method using arithmetic average). conventional morphometric analysis in this analysis we used 24 linear measurements in the right wings (figs. 2, 3) of 155 individuals measured by motic software 2.0 ml. measurements of the veins and cells were applied according to the methodolog y of de souza et al. (2010), which used 17 measures on the forewing (length c and width l of the wing, width and length of 1st, 2nd and 3rd submarginal cells, and 1st and 2nd medial cells (m), length of the subcostal veins + radial (sc + r) and anal-1 vein; length of the cells: marginal (m) and the 2nd cubital (cu); distance from the intersection of the anal-1 vein with cubital-anal (cu-a) until the intersection of the cubital vein with 1st middle-cubital (m-cu) and the hindwings with 7 measurements (length and width of the wing ; length of the ribs sector radial (r), anal-1 and cubital-anal veins; distance from the intersection of the rib anal -1 with cubital-anal until the intersection of the median-radial sector (rs-m) with the radial sector (rs) and radial (r) cell width. fig. 1 – landmarks used for geometric morphometric analysis of the forewing tetrapedia diversipes. 771 neves, c. et al. — morphometric characterization of tetrapedia diversipes the differences between the sexes of the individuals of the species t. diversipes were analyzed by multivariate variance (manova), canonical variables (avc), principal components (pca) and cluster analysis by the upgma method. results and discussion conventional morphometrics for the multivariate analysis of variance (manova), there was a significant difference between groups of individuals (wilk’s λ = 0.65109, p <0.00001), fig. 2 – linear measurements of morphological characters of the forewing (a) and hindwing (b) female tetrapedia diversipes by conventional morphometric. legend: length (l), width (w), radial + subcostal veins (sc + r), cubital vein (cu), cubital-anal (cu-a), middle-cubital or median-cubital (m-cu), radial (r), radial sector (rs), median-radial sector (m-rs). 772 sociobiolog y vol. 59, no. 3, 2012 with only 10 variables contributing significantly to the separation of individuals (α = 0.05), confirming the existence of sexual dimorphism of this species in relation to the size of the wings. this separation is represented graphically by principal component analysis (fig. 4). in principal component analysis (pca) performed to test the existence of sexual dimorphism of t. diversipes the first three variables explained 74.40% of the variation, with the first component explained 63.28%, the second and the third 6.70%, and 4.42% (table 1), and the variables that contributed most to the first component were length and width of forewings (table 2). fig. 3 – linear measurements of morphological characters of the forewing (a) and hindwing (b) male tetrapedia diversipes by conventional morphometric. legend: length (l), width (w), radial + subcostal veins (sc + r), cubital vein (cu), cubital-anal (cu-a), middle-cubital or mediancubital (m-cu), radial (r), radial sector (rs), median-radial sector (m-rs). 773 neves, c. et al. — morphometric characterization of tetrapedia diversipes to evaluate the differences between the areas another pca was performed (fig. 5), where an overposition between the three areas of the population occurred, demonstrating the existence of low morphometric divergence between individuals of the three areas. this can be explained by the vicinity of the sampled areas, which contributes to gene flow between the population and therefore, maintains a similar phenotypic pattern. the analysis of canonical variables showed morphometric differences between individuals in area i and ii, and area iii formed a group with characteristics that are similar to areas i and ii (fig. 6). multivariate analysis of variance (manova) for the three areas showed a significant difference between individuals collected in areas i, ii and iii (wilk’s λ = 0.57864, p <0.00001), where only 9 variables corresponding to the forewings contributed significantly to the separation of individuals in each area (α = 0.05). considering the mahalanobis d2 distances (table 3) between the areas, it appears that individuals of the areas i (active germplasm bank of m. emarfig. 4 graphic dispersion between males and females tetrapedia diversipes in relation to cartesian axes established by the first two principal components (pc1, pc2). 774 sociobiolog y vol. 59, no. 3, 2012 ginata) and ii (other orchards) showed highly significant morphological differences (p <0.00001). however, comparing areas i and iii (transition area: between the forest and other orchards), and ii and iii there was no significant difference between the total sample, demonstrating morphometric similarity, which can be attributed to the similarity of available resources between these areas. table 1 eigenvalues, total variation (%), proportion (%) and cumulative proportion of the 24 variables obtained with principal component analysis of tetrapedia diversipes wings in the areas studied, bahia of reconcavo region, brazil. principal component eigenvalue total variation (%) proportion (%) cumulative proportion 1 15.1888 63.2868 15.1888 63.2868 2 1.6056 6.6901 16.7944 69.9769 3 1.0622 4.4257 17.8566 74.4026 4 0.8999 3.7494 18.7565 78.1520 5 0.6954 2.8976 19.4519 81.0497 6 0.6015 2.5064 20.0535 83.5560 7 0.5423 2.2597 20.5958 85.8158 8 0.4739 1.9747 21.0697 87.7905 9 0.4454 1.8560 21.5152 89.6465 10 0.4061 1.6922 21.9213 91.3387 11 0.3427 1.4279 22.2640 92.7666 12 0.2890 1.2041 22.5530 93.9706 13 0.2375 0.9898 22.7905 94.9604 14 0.2114 0.8809 23.0019 95.8413 15 0.1979 0.8245 23.1998 96.6659 16 0.1609 0.6702 23.3607 97.3361 17 0.1494 0.6224 23.5101 97.9586 18 0.1162 0.4840 23.6262 98.4425 19 0.0985 0.4103 23.7247 98.8528 20 0.0695 0.2896 23.7942 99.1424 21 0.0636 0.2651 23.8578 99.4075 22 0.0609 0.2537 23.9187 99.6612 23 0.0464 0.1935 23.9651 99.8547 24 0.0349 0.1453 24.0000 100.00 775 neves, c. et al. — morphometric characterization of tetrapedia diversipes table 2. principal component analysis between males and females of tetrapedia diversipes, bahia of reconcavo region, brazil. variable principal component pc01 pc02 l1 0.6028 0.0439 l2 0.7141 0.0823 l3 0.8419 0.0173 l4 0.8128 0.1221 l5 0.7525 0.3258 l6 0.7385 0.3392 l7 0.7719 0.3525 l8 0.7364 0.2207 l9 0.9338 0.1445 l10 0.8580 0.2261 l11 0.7232 0.1663 l12 0.8485 0.3841 l13 0.8553 0.3358 l14 0.7541 0.1905 l15 0.7733 0.3139 l16 0.9335 0.1209 l17 0.9340 0.1748 l18 0.8054 0.1504 l19 0.7467 0.3711 l20 0.8668 0.1418 l21 0.4813 0.6517 l22 0.8023 0.0295 l23 0.7515 0.2033 l24 0.8975 0.0174 legend: l1: width of 1st submarginal cell; l2: length of the 1st submarginal cell; l3: length of marginal cell; l4: width of the 2nd submarginal cell: l5: length of the 2nd submarginal cell; l6: width of the 3rd submarginal cell; l7: length of the 3rd submarginal cell, l8: width of the 1st medial cell; l9: length of the 1st medial cell; l10: width of the 2nd medial cell; l11: length of the 2nd medial cell; l12: intersection of anal 1 vein with cu-a (anal 1-cubital) until intersection of cu (cubital vein) with 1st m-cu (1st median-cubital); ll3: length of the 2nd cubital cell; l14: length of sc + r (subcostal + radial); l15: length of anal 1; l16: length of forewing ; l17: width of the forewing ; l18: length of the radial sector (rs); l19: radial cell width; l20: width of the hindwing ; l21-length of the cu-a; l22: intersection of anal 1 with cu-a until intersection of m-rs (median-radial sector) with radial sector (rs); l23: the length of the anal 1 vein; l24: length of hindwing. 776 sociobiolog y vol. 59, no. 3, 2012 fig. 5 graphic dispersion of the population tetrapedia diversipes in relation to cartesian axes established by the first two principal components (pc1, pc2). table 3 mahalanobis d2 distances (superior part) and statistical significance (p) for each distance (inferior part) between areas, by conventional morphometry analysis: i active germplasm bank of malpighia emarginata; ii other orchards (citrus spp., spondias sp., musa spp. and mangifera indica) and iii transition area (the area between the forest and other orchards). area i ii iii i 2.646967 2.137111 ii 0.00001 5.670577 iii 0.758967 0.086292 777 neves, c. et al. — morphometric characterization of tetrapedia diversipes fig. 6 graphic dispersion of the population tetrapedia diversipes in relation to cartesian axes established by canonical variables (cv1, cv2) obtained from morphological characteristics of wings. table 4 mahalanobis d2 distances (superior part) and statistical significance (p) for each distance (inferior part) between areas, by geometric morphometry analysis: i active germplasm bank of malpighia emarginata; ii other orchards (citrus spp., spondias sp., musa spp. and mangifera indica) and iii transition area (the area between the forest and other orchards). area i ii iii i 3.83154 6.76865 ii 0.00001 10.84870 iii 0.239981 0.017491 778 sociobiolog y vol. 59, no. 3, 2012 ferreira et al. (2011) studied the degree of differentiation of populations in areas of centris aenea in acerola orchards, where they observed significant morphometric differences between individuals, corroborating the present study. it is very important to consider that there are fluctuations of trophic resources over time, and the phenotypic expression of the bees may vary according to availability of resources. geometric morphometrics multivariate analysis of variance (manova) with the matrix w to observe the difference between the areas, also found significant differences (wilk’s λ = 0.459, p <0.0001) between individuals of t. diversipes collected in the three areas, and all variables of the matrix w contributed significantly (α = 0.05) to discriminate between the groups in the areas. with the triangular matrix of mahalanobis d2 distances from the data coming from the matrix w, we found again significant differences between fig. 7 graphic dispersion between the bees t. diversipes in each area, in relation to cartesian axes established by canonical variables (cv1, cv2). 779 neves, c. et al. — morphometric characterization of tetrapedia diversipes area i and areas i and ii. however, among areas i and iii, there was no significant difference (table 4). ghaderi et al. (1984) found that when several characteristics are analyzed at once, the squared mahalanobis distances can be used as estimates of genetic diversity among the groups or areas where this variability is the result of morpho-physiological characteristics and ecological differences. the analysis of canonical variables produced a difference between individuals in areas i, ii and iii (fig. 7). the individuals of t. diversipes in areas i and iii showed similar morphometrics, so there is probably a greater genetic interaction between the bees in these two areas. this similarity can be attributed to a low level of gene flow, with the assumption of the occurrence of inbreeding. according to breda et al. (2004) inbreeding occurs among individuals related by descent, or is the union of individuals more closely related than the average population, increasing the homozygosity and phenotypic effect of expressing the recessive genes. it is possible that the fragmentation of the environment caused by anthropogenic activities and is mainly favoring the isolation of the population of t. diversipes, increasing the rate of inbreeding, weakening the genetic diversity and therefore favoring the disappearance of bees in these areas. similar data were found by mendes et al. (2007). analyzing the population nannotrigona testaceicornis collected in two urban areas and an area of natural vegetation, they found that in urban areas there were more similarities when compared with areas of natural vegetation, affecting gene flow of these bees. however, gonçalves (2010) noted that the geometric morphometry of wings was not sensitive enough to separate samples of frisieomelita varies in natural and disturbed areas and diverged from the present work. the conservation of native flora and reducing human disturbance around natural and agricultural areas can contribute to the maintenance of intrapopulation genetic material of t. diversipes, increasing their genetic variability and ensuring survival, demonstrating the need for further studies on the evolutionary variations of this species. conclusion a conventional morphometric analysis revealed the existence of sexual dimorphism of the species tetrapedia diversipes. however, geometric mor780 sociobiolog y vol. 59, no. 3, 2012 phometrics indicated the existence of low levels of gene flow, facilitating the isolation and weakening of the genetic diversity in the population. the study of morphometric characterization has provided important data on the degree of intrapopulational differentiation of t. diversipes, facilitating future research on other species of tetrapedia, supporting strategies for the conservation and maintenance of this bee. acknowledgments the authors thank conselho nacional de desenvolvimento científico e tecnológico (cnpq) (proc. 303237/2010-4 and proc. 506290/2010-7), coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarships, embrapa mandioca e fruticultura tropical for providing the study areas and phd. joão paulo morselli for the wing study design. references alves-dos-santos, i., i.c. machado & m.c. gaglianone. 2007. história natural das abelhas coletoras de óleo. oecologia brasiliensis 11: p. 544-557. bosch, j. 2008. production of undersized offspring in a solitary bee. applied animal behavior science 75: p. 809-816. breda, f.c.; r.f. euclydes, s.p. carmen, a.t. robledo, l.s.c. paulo, r.s. josé lindenberg, a.t.f. rodolpho & k.f.m. antonia. 2004. endogamia e limite de seleção em populações selecionadas obtidas por simulação. revista brasileira de zootecnia 33: p. 2017-2025. camillo, e. 2005. nesting biolog y of four tetrapedia species in trap-nests (hymenoptera, apidae, tetrapedini). revista de biologia tropical 53: p. 175-186. carvalho, c.a.l., w.s. santos, l.a. nunes, b.a. souza, g.a.c. zilse & r.m.o. alves. 2011. offspring analysis in a polyg yne colony of melipona scutellaris (hymenoptera: apidae) by means of morphometric analyses. sociobiolog y 57: 347-354. cordeiro, g.d., m. taniguchi, c.h.w. flechtmann, & i. alves-dos-santos. 2010. phoretic mites (acari: chaetodactylidae) associated with the solitary bee tetrapedia diversipes (apidae: tetrapediini). apidologie 42: p. 128-139. ferreira, v.s., c.m.l. aguiar, m.a. costa & j.g. silva. 2011. morphometric analysis of populations of centris aenea lepeletie (hymenoptera: apidae) from northeastern brazil. neotropical entomolog y 40: p. 97-102. francoy, t.m. & v.l. imperatriz-fonseca 2010. a morfometria geométrica de asas e a identificação automática de espécies de abelhas. oecologia australis 14: p. 317-321. 781 neves, c. et al. — morphometric characterization of tetrapedia diversipes francoy, t.m., m.l. grassi, v.l. imperatriz-fonseca, w.j. may-itzá & j.j.g. quezada-euán. 2011. geometric morphometrics of the wing as a tool for assigning genetic lineages and geographic origin to melipona beecheii (hymenoptera: meliponini). apidologie, 42: p. 499507. ghaderi, a., m.w. adams & a.m. nassib. 1984. relationship between genetic distance and heterosis for yield and morphological traits in dry edible bean and faha bean. crop science 24: p. 37-42. gonçalves, p.h.p. 2010. análise da variabilidade genética de uma pequena população de frieseomelitta varia (hymenoptera, apidae, meliponini) por meio de análise do dna mitocondrial, microssatélites e morfometria geométrica das asas. dissertação de mestrado, universidade de são paulo-usp. são paulo-sp. 140p. imperatriz-fonseca, v.l. 2010. conservação de polinizadores no ano internacional da biodiversidade. oecologia australis 14: p. 14-15. machado, c.s. 2011. biologia de nidificação e dieta das larvas dos polinizadores efetivos de malpighia emarginata d.c. em uma área restrita do recôncavo da bahia. tese doutorado em ciências agrárias, universidade federal do recôncavo da bahia-ufrb. cruz das almas-ba. 91p. mendes, m.f.m., t.m. francoy, p. nunes-silva, c. menezes & v.l. imperatriz-fonseca 2007. intra-populational variability of nannotrigona testaceicornis lepeletier 1836 (hymenoptera, meliponini) using relative warp analysis. bioscience journal 23: p. 147-152. michener, c.d. 2000. the bees of the world. baltimore. the johns hopkins university press. 913p. nunes, l.a., m.f.f.c. pinto, p. carneiro, d.g. pereira & a.m. waldschmidt 2007. divergência genética em melipona scutellaris latreille (hymenoptera: apidae) com base em caracteres morfológicos. bioscience journal 23: p. 1-9. nunes, l.a., e.d. araujo, c.a.l. carvalho & a.m. waldschmidt. 2008. population divergence of melipona quadrifasciata anthidioides (hymenoptera: apidae) endemic to the semi-arid region of the state of bahia, brazil. sociobiolog y 52: p. 81-93. peruquetti, r.c. 2003. variação do tamanho corporal de machos de eulaema nigrita lepeletier (hymenoptera, apidae, euglossini). resposta materna à flutuação de recursos? revista brasileira de zoologia 20: p. 207-212. quezada-euán, j.j.g., r.j. paxton, k.a. palmer, w.j.m. itzá, w.t. tay & b.p. oldroyd 2007. morphological and molecular caracters reveal differentiation in a neotropical social bee, melipona beechii (apidae: meliponini). apidologie 38: p. 247-258. rohlf, f.j. 1990. morphometrics. annual review of ecolog y and systematics 21: p. 299316. rohlf, f.j. 2008a. program tpsdig2 for windows version 2.12. departament of ecolog y and evolution, state university of new york, stony book. (http://life.bio.sunysb.edu/ morph/index.html, accessed 21/october/2008). 782 sociobiolog y vol. 59, no. 3, 2012 rohlf, f.j. 2008b. program tpsrelw version 1.46. department of ecolog y and evolution, state university of new york, stony book. (http://life.bio.sunysb.edu/morph/index. html, acess in 21/october/2008). silveira, f.a., g.a.r. melo & e.a.b. almeida. 2002. abelhas brasileiras: sistemática e identificação. 1 edição, belo horizonte mg. 253p. souza, d.l., a. evangelista-rodrigues, m.n. ribeiro, f.a. padilha, e.s.l. farias & w.e. pereira. 2009. análises morfométricas entre apis mellifera da mesoregião do sertão paraibano. archivos de zootecnia 58: p. 65-7. souza, a.v., c.a.l. carvalho, c.s. machado, w.s. santos, r. ritzinger & c.a. lima-júnior. 2010. análises morfométricas de centris tarsata (hymenoptera: apidae) no recôncavo da bahia. in: congresso brasileiro de fruticultura. natal-rn. anais. p. 1-4. 617 morphological characterization and molecular mediated genetic variation of thief ants (hymenoptera: formicidae) by ralph b. narain1, shripat t. kamble1, thomas o. powers2 abstract the morphological characterization and molecular genetic variation were determined in populations of thief ants, solenopsis molesta (say). the genetic variations were elucidated using mitochondrial deoxyribonucleic acid (mdna) sequences of cytochrome oxidase i. dna from thief ants was extracted with qiagen’s gentra puregene® dna isolation kit using their solid tissue protocol. polymerase chain reactions (pcr) were run on the extracted dna using primers lep-f1 (forward) and lep-r1 (reverse). the dna products were concentrated and purified by microcon centrifugal filter unit ym-100. purified dna samples were sequenced at the university of arkansas medical sciences (uams) dna sequencing core facility. the sequences were edited and aligned using codon code aligner. the contigs wee uploaded to www.phylogeny.fr and phylogenetic trees were produced (neighbor joining, maximum likelihood and bayesian). the trees displayed variation in genetic makeup of the thief ants from various geographic regions and genetic variation corresponded to the morphologic identification. the thief ants collected from different states were separated into three groups. ants collected from new york, indiana and one location in nebraska formed one group identified as s. molesta validiuscula, a second group formed with ants from louisiana identified as s. carolinensis and the third group consisted of ants from south dakota, washington, new jersey tennessee, kansas and two other locations in nebraska identified as s. molesta molesta. key words: thief ants, solenopsis sp., morpholog y, molecular genetics. introduction of the 40 species of common ants in urban environments, 10 species are consiconsidered economic pests. according to a survey of structural pest 1department of entomolog y, university of nebraska, lincoln, ne 68583 2department of plant patholog y, university of nebraska, lincoln, ne 68583 618 sociobiolog y vol. 59, no. 3, 2012 control operators by field et al. (2007), ants are considered the number one urban pest in the united states generating approximately $1.7 billion annually. the most important species include: carpenter ants, camponotus spp. (mayr), argentine ants, linepithema humile (mayr), odorous house ants, tapinoma sessile (say), pavement ants, tetramorium caespitum l., red imported fire ants, solenopsis invicta (buren), thief ants, s. molesta (say) plus others (bennett et al. 2005, klotz et al. 2008). most of these are considered nuisance pests, except the red imported fire ants due to their ability to sting and inject venom into the skin which causes welts or allergic reactions (rhoades et al. 1975). s. molesta is commonly called a “thief ” because they lives near the nests of other ants and “steals” the larvae and food from the other ant colonies to feed its own. hays (1920) determined the development time from egg to adult for thief ants ranges 52-64 days. colonies are generally composed of a few hundred to several thousand workers and several queens. the thief ants infest homes, contaminate food products and their presence is unsightly causing distress, especially in elderly residents. although thief ants do not cause physical harm to occupants of the structures, they are genetically related to red imported fire ants. therefore, precise separation of thief ants from imported fire ants or possibly from hybrid populations is critical. numerous molecular studies have been conducted on the red imported fire ant, s. invicta, (krieger & ross 2003, 2005) and the argentine ant, l. humile, (rosset et al. 2005), but very limited data exist on morpholog y of s. molesta and none on molecular genetic variations. materials and methods ant collection thief ant workers (s. molesta) were collected from various locations in lancaster county, nebraska and other states including : indiana, kansas, louisiana, new york, new jersey, south dakota, tennessee and washington (fig. 1). thief ant workers in nebraska were collected using traps made of cylindrical, plastic culture tubes (17 x 100 mm) (vwr, chicago, il) with 16 or 17 entrance holes in their bottom halves (husen et al. 2008). peanut butter was used as the food source within each trap. approximately 2-3 grams of peanut butter was placed on a small piece of paper; the paper was rolled and inserted into the collection tube, and collection tubes were placed 619 narain, r.b. et al. —morphological and genetic characterization of thief ants around the perimeter of a structure from 5:00 to 7:00 pm and were picked up the following morning from 8:00 to 10:00 am. ants were separated to species in the laboratory using a bausch and lomb dissecting microscope and specimens were preserved in 95% ethanol and stored at -20oc in vwr freezer (vwr, west chester, pa) for dna extraction, coi amplification and sequencing. morphological characterization thief ants were identified to genus using numerous keys (hayes 1920, creighton 1950, thompson 1989, bolton 1994, pacheco 2007). identifications were based on 10 specimens from each collection and enumerated with mean lengths (mm) of antennae and antennal club formed with apical and preapical segments (fig. 2), and two-segmented petiole connecting gaster and thorax (fig. 3). measurements were denoted in mm at 250x magnification, using a micrometer in the ocular lens of a wild dissection microscope. other morphological characters supplementing identification were body color, head shape, density of hairs on head and body, and prominence of eyes. morpholog y was further illustrated by the following measurements: i) total fig. 1. geographic collection points for thief ants. 620 sociobiolog y vol. 59, no. 3, 2012 fig. 2. morphological characters and various measurements of solenopsis spp. head in frontal view. (image: r. narain). fig. 3. morphological characters and various measurements of solenopsis spp. petiole dorsal view. (image: r. narain). 621 narain, r.b. et al. —morphological and genetic characterization of thief ants length (tl) from head to tip of gaster; ii) head length (hl) posterior border (top of head) to anterior margin of clypeus (just before mandibles) (fig. 2); iii) head width (hw) maximum width excluding eyes (fig. 2); iv) scape length (sl) excluding basal condyle (fig. 2); v) petiole length (pl) maximum length of nodes measured in dorsal view, starting at posterior edge of thorax and ending at anterior edge of gaster (fig. 3); and vi) petiole width (pw) maximum width of node measured in dorsal view (fig. 3). the indices used in further illustrations were calculated as follows: a) cephalic index (ci), hw/hl x 100; b) scape index (si), sl/hl x 100; and c) petiolar index (pi), pl/pw x 100. the measurements were recorded as number of units on the micrometer in the left ocular lens then converted to mm using a calibration scale. one unit viewed through the ocular lens at 250x magnification was equivalent to 0.4 mm on the calibration scale, so a recording of 4.68 units is equivalent to 1.87 mm. dna extraction and isolation ants were stored at -20oc in 95% ethanol were removed and the ethanol was allowed to evaporate. ten thief ants were used per extraction with a minimum of three extractions per location. dna from thief ants was extracted using a puregene® dna isolation kit (invitrogen 2010) using a modified tissue protocol from their manual. standard primers (smith et al. 2007) (forward primer >lepf1 attcaaccaatcataaagatattgg: reverse primer >lepr1 taaacttctggatgtccaaaaaatca) (invitrogen, carlsbad, ca) were used to amplify and sequence the mitochondrial cytochrome oxidase-i (coi) from thief ants (s. molesta). the extracted dna, once rehydrated was stored at 4.0oc until pcr amplification was completed. the concentration of the extracted dna was determined with the aid of a nanodrop 1000 spectrophotometer (thermo scientific, waltham, ma) and an equivalent of 80-100 ng/µl was used as template for the pcr reaction. polymerase chain reaction (pcr) amplification protocol the pcr reactions were performed in a total volume of 25.0 µl: 23.0 µl master mix (mm) and 2.0 µl dna template, and run on applied biosystem veriti 96 well thermocycler (applied biosystem, foster city, ca) the mm was prepared by combining the equivalent volume of each reagent into a 1.5 ml micro centrifuge tube. the equivalent volume was determined by the 622 sociobiolog y vol. 59, no. 3, 2012 number of pcr reactions to be run at that time. all solutions were allowed to thaw to room temperature and mixed, by uptake and release solution in pipette tip for at least 50 times, before extracting aliquot for mm. the mm was comprised a final concentration of 3.0 mm magnesium chloride, 400.0 µm dntp mix, 0.2 µm forward primer, 0.2 µm reverse primer and 0.05 units/µl jumpstarttm redtaq® (sigma, st. louis, mo). the mm was made homogenous by an uptake and release solution in pipette tip 100+ times before aliquot volume needed for pcr reaction was transferred into 0.2 ml thin walled dna free micro-centrifuge tube and dna template was added. before amplification, dna template and mm were also mixed by pipettor 20 times and pulse centrifugation. after each pcr reaction, tubes were prepared (mm + template). during sample preparation, dna template, pcr master mix and prepared samples were kept on ice to reduce template dna breakdown until all samples were ready and thermocycler program check completed. pcr program and sequencing the pcr program used was optimized from smith et al. (2007). the program consisted of an initial denaturation step at 94.0oc for 1min, then 5 cycles of denaturation for 40 seconds (sec.) at 94.0oc, an annealing step at 45.0oc for 40 sec., with an elongation step which lasted for 1min at 72.0oc. this was followed by the main pcr cycles which consisted of 35 cycles of denaturing at 94.0oc for 40 sec.; annealing at 51.0oc for 40 sec., and extension for 1min at 72.0oc. the pcr program was completed with a final extension/ fig. 4. images (a and c) of 1% agarose gel showing pcr amplified coi from thief ants and (b) invitrogen 100bp dna ladder used to estimate number of base pairs (invitrogen 2010) 623 narain, r.b. et al. —morphological and genetic characterization of thief ants elongation soak at 72.0oc for 5 min. the samples stayed in the thermocycler at 4.0oc (max 12 hr) until they were removed for the next step. after completion of the amplification process, 5.0 µl pcr product was loaded into 1.0% agarose gel in 0.5x tbe, stained with 0.1% ethidium bromide, electrophoresed in 0.5x tbe buffer solution at 100 volts for approximately 1 hr. the gel was viewed and photographed (fig. 4) on a bio-rad gel doc system (bio-rad, hercules, ca). successfully amplified products were concentrated and purified using microcon centrifugal filter unit ym-100 (fisher scientific, pittsburgh, pa). another 1.0% agarose gel was run to determine the approximate concentration of the clean pcr product by comparing the distance travelled by the cleaned pcr product and intensity of the bands to that of a low mass dna ladder (invitrogen carlsbad, ca). purified samples were diluted to 20 ng/ml and sent to the university of arkansas medical sciences (uams) dna sequencing core facility for sequencing. sequences received from uams were edited and aligned using codon code aligner (dedham, ma). the contigs were uploaded to phylogeny.fr (dereeper et al. 2008) where phylogenetic trees were produced. the trees obtained (figs 6, 7 and 8) from the sequences submitted indicated the relationship and variation in thief ant genetic make up for the location sampled. results and discussion morphological and genetic variation thief ant specimens were identified with morphological characters (table 1) as solenopsis molesta validiuscula, s. carolinensis and s. molesta molesta. solenopsis molesta validiuscula was identified from one collection site from each of three states: new york, indiana and nebraska. solenopsis molesta molesta was identified from two collection sites in nebraska, two collection sites in tennessee, and one collection site from each of the following states: kansas, south dakota, washington and new jersey. solenopsis carolinensis was collected from two locations in louisiana (table 2). the thief ants species identified from these nine states are not an exhaustive list, but were based on specimens collected. there could be additional thief ant species or subspecies found in those states. the latest revision of the thief ants by pacheco (2007) listed a total of 83 species. from a previous 149 available taxa, the author recognizes 72 valid 624 sociobiolog y vol. 59, no. 3, 2012 species and identified an additional 11 new species. one possible reason as to why there were 149 taxa listed previously was the use of synonymy, for example, listed below are several of the synonyms of s. carolinensis: s. texana carolinensis (forel 1901); s. molesta var. castanea (wheeler 1908); s. texana r. truncorum (creighton 1950). similar situations of synonymy are found in other thief ant populations. another reason for taxa overestimation could be because of the wide range of color and size of thief ants within a species. it is still possible that the number of species pacheco (2007) identified in his dissertation may be changed, either by addition of new species or review and re-identification of the species. one way to counter this problem is the use of morphological and molecular genetic identification simultaneously. solenopsis molesta species complex workers of most species in this complex are around 1.0-2.5 mm long and yellow to light brown ants. the head is somewhat elongated, two-node petiole table 1: morphometric measurements of thief ants. location total length head length head width scape length petiolar length petiolar width cephalic indexa scape indexb petiolar indexc 1 la71051 1.677 0.466 0.321 0.279 0.298 0.124 68.979 59.928 243.170 2 la70714 1.690 0.458 0.342 0.248 0.251 0.122 74.672 54.148 205.229 3 sd57701 1.791 0.474 0.327 0.289 0.271 0.123 68.987 60.886 220.292 4 tn37721 1.808 0.518 0.403 0.276 0.268 0.134 77.838 53.282 200.599 5 tn37996 1.853 0.511 0.384 0.282 0.264 0.131 75.294 55.278 202.917 6 ne68521 1.820 0.512 0.402 0.264 0.268 0.132 78.516 51.563 203.030 7 nj08901 1.822 0.524 0.336 0.270 0.256 0.126 74.122 51.527 202.532 8 ks66503 1.828 0.518 0.420 0.285 0.278 0.132 81.208 55.042 210.399 9 wa99224 1.866 0.524 0.405 0.338 0.299 0.132 77.328 64.504 225.982 10 ne68505 1.872 0.496 0.400 0.290 0.288 0.131 80.645 58.468 219.817 11 ny11741 1.949 0.576 0.436 0.291 0.293 0.157 75.676 50.579 186.441 12 ne68583 1.988 0.516 0.380 0.268 0.250 0.128 73.751 51.986 195.751 13 in47907 2.028 0.512 0.426 0.278 0.268 0.135 83.203 54.297 198.813 acephalic index = head width/head length x 100 bscape index = scape length/ head length x 100 cpetiolar index = petiole length/petiole width x 100 625 narain, r.b. et al. —morphological and genetic characterization of thief ants connecting the gaster to the thorax and 10 segmented antennae with apical two segments forming a large club. thief ants have small stingers and generally have small eyes (say 1836, hayes 1920, creighton 1950, thompson 1989, pacheco 2007). some species in s. molesta complex are difficult to conclusively identify with morphological features without the species’ queen and male. this limitation could be resolved with the use of dna barcoding. solenopsis carolinensis forel workers are small, yellow with the hairs on the posterior tibia are usually semi-erect, scape length 0.23-0.26 mm, head length 0.30-0.35 mm, a relatively narrower petiole (petiolar 0.12-0.13 mm, and the post petiolar width 0.250.26 mm. the lateral clypeal teeth are well developed, and the extra-lateral processes are developed at least into an angle. eyes are nearly circular, normal size between 0.03-0.04 mm, mostly brown to black (say 1836, hayes 1920, creighton 1950, thompson 1982, 1989) (fig. 5). the s. carolinensis species identified based on morphometric data in this study had been previously identified as the same species by hopper-bui (2010). table 2: identification of thief ant specimens collected from different states. site location and specimen identification state zip code specimen identification 1 louisiana 71051 solenopsis carolinensis 2 louisiana 70714 solenopsis carolinensis 3 south dakota 57701 solenopsis molesta molesta 4 tennessee 37721 solenopsis molesta molesta 5 tennessee 37996 solenopsis molesta molesta 6 nebraska 68521 solenopsis molesta molesta 7 new jersey 08901 solenopsis molesta molesta 8 kansas 66503 solenopsis molesta molesta 9 washington 99224 solenopsis molesta molesta 10 nebraska 68505 solenopsis molesta molesta 11 new york 11741 solenopsis molesta validiuscula 12 nebraska 68583 solenopsis molesta validiuscula 13 indiana 47907 solenopsis molesta validiuscula 626 sociobiolog y vol. 59, no. 3, 2012 solenopsis molesta molesta say and solenopsis molesta validiuscula emery the workers are small, yellow or light brown species, with two well-developed clypeal teeth and underdeveloped extra-lateral teeth (only small bumps). the smaller segments of the funiculus are about 0.12 mm long. this species of ants can be separated from s. carolinensis by the longer length of the smallest segments of the funiculus. it is difficult to separate subspecies of s. molesta validiuscula from s. molesta molesta. the workers of s. molesta molesta are often smaller (1.7-1.8 mm long ) than s. molesta validiuscula (1.92.0 mm long ), although the sizes of the workers overlap. the cephalic punctures of s. molesta validiuscula are nearly always moderately coarse, much larger than the hairs which arise from them, the punctures are often finer in s. molesta molesta, often difficult to see, and not much larger in diameter than the hairs which arise from them. the pedicel of s. molesta validiuscula is about 2/3 the length of the scape. the two species are nearly identical, but based on these characters; it appears that they are both valid subspecies and do not appear to be a synonym of s. molesta (say 1836, hayes 1920, creighton 1950, thompson 1989, bolton 1994). morphometric data generated with this research agreed with previously published work by the authors mentioned above. fig. 5. thief ants collected from louisiana (la 70714), identified as solenopsis carolinensis (image: r. narain). 627 narain, r.b. et al. —morphological and genetic characterization of thief ants solenopsis molesta validiuscula is relatively larger than s. molesta molesta, which is relatively larger than s. carolinensis. color ranges from yellow in s. carolinensis to yellow or light brown in s. molesta molesta and to a darker brown in s. molesta validiuscula. molecular based species genetic variation phylogenetic analysis using programs on www.phylogeny.fr separates the coi sequences collected into three groups, which correspond to the morphological identification using the measurements of key ant features and calculations of cephalic, antennae scape and petiole ratios. tetramorium caespitum and myrmica spp. were used as out-groups for the phylogenetic trees. the phylogenetic tree produced by maximum likelihood analysis (fig. 6) illustrates that the sequences analyzed fall into identifiable groups. one group consists of s. carolinensis from the two locations in louisiana. the second group consists of s. molesta validiuscula from new york, indiana and nebraska. the final group was s. molesta molesta from two collection sites in nebraska, two collection sites in tennessee, and one collection site from kansas, south dakota, washington and new jersey. bayesian analysis (fig. 7) and neighbor-joining (fig. 8) trees showed similar groupings with fig. 6. maximum likelihood phylogenetic tree branch supporting values of thief ants collected from nine states (13 locations) across its distribution range, rooted with tetramorium caespitum and myrmica spp. 628 sociobiolog y vol. 59, no. 3, 2012 the only differences being the supporting values displayed on the branches of each tree. since the morphological data generated in this research agrees with published data, these phylogenetic trees confirm the morphological identifications and are descriptive of the genetic variation of these thief ant species. the sequences generated could be used for identification of these species in future research. fig. 7: bayesian phylogenetic tree with branch supporting values of thief ants collected from nine states (13 locations). analysis done on phylogeny.fr program mrbayes, rooted with tetramorium caespitum and myrmica spp. fig. 8: neighbor joining profile with branch supporting values of thief ants collected from nine states (13 locations) across their geographic distribution range. 629 narain, r.b. et al. —morphological and genetic characterization of thief ants summary and conclusions this research shows that the use of coi sequences to identify thief ant species/subspecies that are difficult to key out via the dichotomous keys is a feasible process. specimens that are very minute, such as thief ants, or disintegrated due to age could be identified once coi sequences from previously identified specimen have been sequenced and the sequences deposited in gene banks, or via comparison of related species using phylogenetic trees. these coi sequences would be used to identify unknown or undetermined species. thief ants were found in all states sampled, distribution of species in the complex could vary within each state. the geographic distribution of thief ants varies; some species are likely to be localized, such as s. carolinensis, which was collected only from louisiana. other species were more universally distributed. solenopsis molesta molesta was identified within seven states and s. molesta validiuscula was identified from specimens collected in three states. the morphologic identification corresponded to genetic variation found within the samples analyzed. the thief ants collected from louisiana were morphologically identified as s. carolinensis and genetic variability supported this distinction. coi sequences generated and the protocol used in this research could be reproduced on thief ant specimens collected in other locations. this could aid in identification of the species, reducing the hassle and aggravation associated with morphologic identification of such tiny ants. significance of research based on literature reviewed, it was determined that this is the first attempt to correlate morphologic and genetic variation of thief ant species, and also the first submission of coi sequences from thief ants to genbank. a search for nucleotide sequences from solenopsis molesta in genbank on may 11, 2010 (http://www.ncbi.nlm.nih.gov/sites/entrez) returned seven entries. none of these entries were coi sequences of thief ants. this study also sought to update the geographic distribution map of the s. molesta species complex. previous geographic distribution data suggested that s. molesta validiuscula were more common in the western states. this research determined this to not be the case. solenopsis molesta validiuscula 630 sociobiolog y vol. 59, no. 3, 2012 was found in nebraska, indiana and new york suggesting that this species was always present in these states or its distribution range has increased. acknowledgments the authors would like to acknowledge the assistance of the supporting staff and fellow graduate students at unl entomolog y department, especially tim husen, neil spomer and abdul hafiz ab-majid. special thank you to drs: karen vail (tsu), changlu wang (rutgers), laurel hensen (wsu), linda hooper-bui (lsu), grzegorz buczkowski (purdue) and mrs. sharon dobesh (ksu) for sending me specimens their states. without these specimens, this research would have not been possible. references bennett, g.w., j.m. owens & r.m. corrigan. 2005. truman’s scientific guide to pest management operations. 6th ed. purdue university/questex media. west lafayette, in. bolton, b. 1994. identification guide to the ant genera of the world. harvard university press, cambridge, ma. creighton, w.s. 1950. the ants of north america. bulletin of the museum of comparative zoolog y of harvard college, 104: 1-585. dereeper, a., v. guignon, g. blanc, s. audic, s. buffet, f. chevenet, j.f. dufayard, s. guindon, v. lefort, m. lescot, j.m. claverie & o. gascuel. 2008. phylogeny.fr: robust phylogenetic analysis for the non-specialist. nucleic acids res. 2008 jul 1;36 (web server issue):w465-9. epub 2008 apr 19. field, h.c., w.e. evans sr., r. hartley, l.d. hensen & j.h. klotz. 2007. a survey of structural ant pests in the southwestern u.s.a. (hymenoptera: formicidae). sociobiolog y. 49: 151-164. forel, a. 1901. variétés myrmécologiques. annales de la societe entomologique de belgique. 45: 334-382. hayes, w.p. 1920. solenopis molesta say (hym.) a biological study. kansas agric. expt. stat. tech. bull. no. 7: 1–50. hooper-bui, l. 2010. identification of thief ants from louisiana. department of entomolog y, louisiana state university, baton rouge, la. personal communication may 17, 2010. husen, t.j., n.a. spomer, r. narain, s.t. kamble, d. branscome, e. roper & b. cartwright. 2008. efficacy of optigard® flex, termidor® sc, and i maxxpro® 2f against selected nuisance ants (odorous house, pavement, and thief ) when applied as perimeter treatments. arthropod management tests. 34: 6. invitrogen. 2010. invetrogen 100bp dna ladder (cat. no 15628-19) technical support guide http://tools.invitrogen.com/content/sfs/manuals/15628019.pdf 631 narain, r.b. et al. —morphological and genetic characterization of thief ants klotz, j., l. hansen, r. pospischil & m. rust. 2008. urban ants of north america and europe: identification, biolog y and management. cornell university press. ithaca, ny. krieger, m.j.b. & k.g. ross. 2003. molecular evolutionary analyses of mariners and other transposable elements in fire ants (hymenoptera, formicidae). insect mol. biol. 12: 155-165. krieger, m.j.b. & k.g. ross. 2005. molecular evolutionary analyses of the odorant-binding protein gene gp-9 in fire ants and other solenopsis species. mol. biol. evol. 22: 2090–2103. pacheco, j.a. 2007. the new world thief ants of the genus solenopsis (hymenoptera: formicidae). ph. d. dissertation, the university of texas at el paso, el paso, tx. rhoades, r.b., w.l. schafer, w.h. schmid, p.f. wubbena, r.m. dozier, a.w. townes & h.j. wittig. 1975. hypersensitivity to the imported fire ant: a report of 49 cases. j. allerg y and clinical immunolog y. 56: 84-93. rosset, h., l. keller & m. chapuisat. 2005. experimental manipulation of colony genetic diversity had no effect on short-term task efficiency in the argentine ant linepithema humile. behav ecol sociobiol. 58: 87–98. say, t. 1836. descriptions of new species of north american hymenoptera, and observations on some already described. boston journal of natural history. 1: 209-416. smith, m.a., d.m. wood, d.h. janzen, w. hallwachs & p.d.n. herbert. 2007. dna barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (diptera, tachinidae) are not all generalists. proc. natl. acad. sci. 104: 4967–4972. thompson, c.r. 1982. a new solenopsis (diplorhoptrum) species from florida (hym.: formicidae). j. kansas entomol. soc. 55: 485-488. thompson, c.r. 1989. the thief ants solenopsis molesta group of florida (hymenoptera: formicidae). fla. entomol. 72: 268-283. wheeler, w.m. 1908. the ants of texas, new mexico and arizona. (part 1.). bull. am. museum of natural history. 24: 399-485. 1015 comparison of foraging ability between solenopsis invicta and tapinoma melanocephalum (hymenoptera: formicidae) by lu yong-yue*, wu bi-qiu, zeng ling & xu yi-juan abstract in this study, we investigated the foraging ability of the invasive ant solenopsis invicta and native ant tapinoma melanocephalum (hymenoptera: formicidae) by measuring their searching and recruitment time for 5 types of food (sausage, sausage & honey, honey, mealworm and peanut oil) in infested wasteland and litchi orchards in south china. the searching time was determined by measuring the time required for the first ant to find the food. the recruitment time was determined by measuring the time to recruit 10 ants to the food which was placed on petri dish 30 cm away from nest entrances. 30 colonies each of t. melanocephalum and s. invicta were tested. in the infested wasteland, the searching time of s. invicta for sausage & honey, sausage, mealworm and honey and the recruitment time of s. invicta for sausage & honey, mealworm and honey were significantly longer than those of t. melanocephalum, but the searching time of these two species of ants for peanut oil was not significantly different. in the infested litchi orchard, the recruitment time of s. invicta for sausage was significantly longer than that of t. melanocephalum, while the recruitment time for the other four types of food was not significantly different between the two species of ants. the searching time for all the five types of food was not significantly different between the two species of ants in the infested litchi orchard. key words: solenopsis invicta, tapinoma melanocephalum, searching time, recruitment time introduction interspecific competition refers to interference or suppression between two or more species. the consequences of interspecific competition are often red imported fire ant research center, south china agricultural university, guangzhou 510642, p.r.china *corresponding author, email:luyong yue@scau.edu.cn, insectlu@163.com 1016 sociobiolog y vol. 59, no. 3, 2012 a reduction of efficiency in reproduction, growth and survival of one species due to the utilization or interference of common resources by another species. competition for scarce resources by two species leads to an adverse effect on both species. resource competition can be divided into exploitation competition and interference competition (reitz & trumble 2002). in exploitation competition, the ability of acquiring resources in one species is greater than that in another species. the mechanisms of exploitation competition include differential resource acquisition, differential female fecundity, differential searching ability, resource preemption, differential strategies and reactions to bad resources (reitz & trumble 2002, xu & cheng 2005). solenopsis invicta buren is a dangerous pest originally discovered in south america, brazil, argentina, paraguay and the panama canal (vinson 1997). it is currently distributed all over the 19 southern states and regions of the us, an area of 128 million hectares (callott & collin 1996, callott 2002, alfredo & jim 2004). on sep., 28, 2004, s. invicta was first identified in wuchuan, guangdong in china, and subsequently it was found in other areas of guangdong, guangxi, fujian, hunan, and hongkong (zeng et al. 2005a). this alien ant can reduce the diversity of the native ants and cause effects on other organisms directly or indirectly (holway et al. 2002). during the competition with native ants, the invasive ants can extend their searching and recruitment time due to their large popualtions, which enhances the exploitation competition ( johnson et al. 1987). in invaded areas, compared to the native ants, s. invicta normally has stronger exploitation competition, which allows them to rapidly find food, discover more food resources, recruit more ants and extend the recruitment time ( jones & phillips 1990, porter & saignano 1990, morrison 1999, calcaterra et al. 2008). previous studies have shown that invasion by s. invicta has already reduced the diversity and abundance of native ants in south china (sheng et al. 2007, wu et al. 2008, lu et al. 2012). however, further studies on the exploitation competition between s. invicta and native ants in south china are needed. while investigating the ant diversity in the areas with s. invicta in south china, we found that a small and fast-moving native ant (tapinoma melanocephalum fabricius) can co-exist with s. invicta, and is still the one of dominant species in the areas infected by s. invicta (lu et al. 2012). t. melanocephalum and s. invicta have similar reactions to the soil surface temperature: with 1017 yu, y-l. et al. —comparison of foraging ability of two ant species the increases of soil surface temperature, the percentage of ants appearing and controlling the food was decreased (zheng et al. 2007). however, the heat resistance of t. melanocephalum is slightly higher than that of s. invicta (zheng et al. 2007). how about the competition between these two species of ants? which is stronger? these questions need to be answered. to avoid the interference of other species of ants and reveal the foraging ability of t. melanocephalum and s. invicta, we investigated the foraging capabilities and recruitment dynamics of both ants at a short distance (30 cm to the ant nest) in different habitats. by comparing the exploitation competition between t. melanocephalum and s. invicta, we expected to provide potential mechanisms by which t. melanocephalum co-exists with s. invicta and remains a dominant species in the areas with s. invicta in south china. materials and methods experimental environment this study was conducted in longgang, shenzhen in 2007. s. invicta colonies in this district were polyg yne. based on the density and distribution of active s. invicta colonies (zeng et al. 2005b), s. invicta was considered to be introduced for more than two years at the infested wasteland and litchi orchard. the general information of the two regions is summarized in table 1. table.1 general information of infested wasteland and litchi orchard areas area type infested wasteland infested litchi orchard altitude(m) 46.5 40.7 longitude and latitude n22°44".260" e114°22'.340" n22°44'.227" e114°22'.303" area (m2) 1826 2241 agrotype yellow-red soil yellow lato soil grass coverage degree(%) 90 60 defoliation thickness (cm) 2-3cm shade density(%) 92 active s. invicta nests/100m2 0.93 0.18 1018 sociobiolog y vol. 59, no. 3, 2012 experimental design three fan-shaped side wall with equal areas (≈ 0.023 cm2) were removed from the bottom side of plastic petri dishes (diameter = 9 cm) and then wet filter paper (diameter = 9 cm) was placed on the bottom of the petri dishes. subsequently, five types of food including sausage (representing artificial food with abundant protein), sausage & three drops of honey (representing artificial food with abundant protein and carbohydrates), mealworms (representing natural food with abundant protein), peanut oil (representing artificial food with abundant oils) and honey (representing artificial food with abundant carbohydrates) were placed on the center of the petri dishes as baits. peanut oil and honey were dropped in the cotton wool at the center of the petri dishes with a plastic head dropper. five types of baits were placed (at a distance of 30 cm) around the nest of t. melanocephalum and s. invicta. the time from food placement in petri dishes to the first ant appearing on the food was the searching time, and the time required to recruit 10 ants to the food was recruitment time, and both of them were recorded by stopwatch. a total of 30 nests for t. melanocephalum and s. invicta respectively were tested in the wasteland and litchi orchard. because of the hot weather in august in shenzhen, we performed the experiments at the time of the relatively low temperature, from 8 am to 10 am in the morning or from3 pm to 6 pm. in the afternoon. during the experiment, the soil surface temperature in the wasteland was between 30.0°c and 42.4°c, and the relative atmospheric humidity was between 49% and 81%. the soil surface temperature in the litchi orchard was between 30.8°c and 36.9°c, and the relative atmospheric humidity was between 61% and 92%. statistical analysis each nest in wasteland and litchi orchard was regarded as one replicate and duncan's new multiple range method was used to compare the foraging ability between different types of food. non-paired student t test was used to compare the foraging ability between t. melanocephalum and s. invicta. all statistical analyses were conducted using spss, version 14.0 (spss inc., chicago, il, usa). 1019 yu, y-l. et al. —comparison of foraging ability of two ant species results foraging ability of s. invicta for different types of food in the wasteland, the shortest searching time (2.98 min) for s. invicta was observed for peanut oil, which was significantly shorter than the longest searching time (7.84 min) for sausage (n=30, p<0.05). searching time of s. invicta for sausage & honey, honey and mealworms was not significantly different. the longest recruitment time for s. invicta was 9.87 min, recorded for honey, which was significantly longer than that for sausage & honey, sausage and mealworms (n=30, p<0.05). s. invicta’s recruitment time for honey and peanut oil was not significantly different. in the litchi orchard, s. invicta’s shortest searching time was observed for peanut oil (2.1 min), while the longest searching time was observed for sausage (3.5 min) (n=30, p<0.05). the longest recruitment time was observed for honey (12 min), which was significantly longer than that for sausage & honey, sausage and peanut oil (n=30, p<0.05). the recruitment time for honey and mealworms was not significantly different (table 2). foraging ability of t. melanocephalum for different types of food in the wasteland, the longest searching time of t. melanocephalum was observed for peanut oil (3.02 min), while the shortest searching time was observed for honey (1.07 min). the searching time for peanut oil was significantly longer than that for sausage & honey (1.37 min) and honey (1.07 min) (n=30, p<0.05), but was not significantly different from that for sausage and mealworms. the longest recruitment time was observed for peanut oil (12.55 min), which was significantly longer than that for the other four types of food (n=30, p<0.05). in the litchi orchard, the longest searching time of t. melanocephalum was observed for mealworms (2.84 min), while the shortest searching time was observed for peanut oil (1.54 min). the searching time for mealworms was significantly longer than that for sausage & honey (1.85 min), honey (1.82 min) and peanut oil (2.84 min) (n=30, p<0.05), but was not significantly different from that for sausage. the longest recruitment time for peanut oil was observed for peanut oil (8.42 min), which was significantly longer than that for the other four types of food (n=30, p<0.05, table 2) . 1020 sociobiolog y vol. 59, no. 3, 2012 comparison of foraging ability between t. melanocephalum and s. invicta the results for the comparison of the foraging ability between t. melanocephalum and s. invicta are summarized in table 2. in the wasteland, the searching time of s. invicta for sausage & honey, sausage, honey and mealworms was significantly longer than that of t. melanocephalum (n=30, p<0.05). the searching time of s. invicta for peanut oil was not significantly different from that of t. melanocephalum. the recruitment time of s. invicta for sausage & honey, sausage, honey and mealworms was longer than that of t. melanocephalum (n=30, p<0.05). in the litchi orchard, all searching timse for five types of food between s. invicta and t. melanocephalum were not significantly different. the recruitment time of s. invicta for sausage, honey and mealworms was significantly longer than that of t. melanocephalum. discussion in the wasteland and litchi orchard, the shortest searching time of s. invicta was observed for peanut oil, while the longest searching time was observed for sausage. this is possibly due to the rapid attraction of s. invicta to the food by the evaporated peanut smell. when they discover food, s. invicta can recruit their companions rapidly to the sausage or sausage & honey, then cut and move the sausage back to the nests. the longest recruitment time of s. invicta was observed for the honey. these results suggest that s. invicta favors high-protein food, while high-carbohydrate food is least attractive for s. invicta, which is in agreement with previous studies (xu et al. 2006). t. melanocephalum prefers honey to the other food sources according to the shortest searching and recruitment time both in the wasteland and lithci orchard. in contrast, in the litchi orchard, the smell of the peanut oil attracts t. melanocephalum early, but the recruitment time of sausage & honey, sausage, honey and mealworms was significantly shorter than that of the peanut oil, suggesting that t. melanocephalum favors artificial or natural food with high levels of carbohydrate and protein. the litchi orchard has higher diversity of vegetation and provides more honey than wasteland. this may explain why t. melanocephalum in the litchi orchard does not search for honey or recruit as rapidly as in the wasteland. the results suggest that t. melanocephalum 1021 yu, y-l. et al. —comparison of foraging ability of two ant species ta bl e 2. s ea rc hi ng ti m e an d re cr ui tm en t t im e of s . i nv ic ta a nd t . m el an oc ep ha lu m fo r 5 fo od s i n in fe st ed a re as (m in ). fo od ty pe se ar ch in g tim e r ec ru itm en t t im e w as te la nd l it ch i o rc ha rd w as te la nd l ic hi o rc ha rd s. in vi ct a t. m el an oc ep ha lu m s. in vi ct a t. m el an oc ep ha lu m s. in vi ct a t. m el an oc ep ha lu m s. in vi ct a t. m el an oc ep ha lu m sa us ag e & h on ey 4. 61 ± 0. 68 bc 1. 37 ± 0. 23 bc ** 3. 13 ± 0. 66 ab 1. 85 ± 0. 37 b 4. 86 ± 0. 96 b 1. 87 ± 0. 40 b* * 3. 69 ± 0. 54 b 2. 42 ± 0. 24 b sa us ag e 7. 84 ± 1. 10 a 2. 44 ± 0. 83 ab ** 3. 50 ± 0. 57 a 2. 18 ± 0. 49 ab 4. 88 ± 0. 70 b 2. 76 ± 0. 85 b 4. 86 ± 0. 70 b 2. 07 ± 0. 33 b* * h on ey 5. 09 ± 1. 27 bc 1. 07 ± 0. 09 c* * 2. 41 ± 0. 33 ab 1. 82 ± 0. 37 b 9. 87 ± 2. 82 a 1. 85 ± 0. 57 b* * 12 .0 0± 3. 17 a 4. 09 ± 0. 82 b* * m ea lw or m 5. 99 ± 1. 01 ab 2. 11 ± 0. 45 ab c* * 2. 48 ± 0. 40 ab 2. 84 ± 0. 57 a 5. 02 ± 1. 02 b 1. 92 ± 0. 33 b* * 9. 24 ± 3. 30 ab 3. 21 ± 1. 11 b* * pe an ut o il 2. 98 ± 0. 34 c 3. 02 ± 0. 72 a 2. 10 ± 0. 28 b 1. 54 ± 0. 33 b 8. 09 ± 2. 11 ab 12 .5 5± 2. 74 a 5. 75 ± 1. 92 b 8. 42 ± 2. 34 a m ea ns w ith in a co lu m n fo llo w ed b y t he sa m e l et te r a re n ot si gn ifi ca nt ly d iff er en t ( p＞ 0. 05 ) u si ng th e d un ca n m et ho d of m ul tip le co m pa ri so ns , f ol lo w in g a on ew ay a n o va . “ *”, “ ** ” re pr es en t s ig ni fic an t d iff er en ce s a t p ＜ 0. 05 a nd p ＜ 0. 01 re sp ec tiv el y, be tw ee n m ea ns u si ng th e un pa ir ed sm al l s w at ch es tte st . 1022 sociobiolog y vol. 59, no. 3, 2012 searches for high-carbohydrate or high-protein food more rapidly than s. invicta. in addition, the recruitment of t. melanocephalum is faster than that of s. invicta. ants belonging to dolichoderinae and formicinae favor sweet foods (li et al. 2000). the honeydew secreted by aphids, scale insects, and angle cicadas, etc. is the favorite food of ants belonging to formicinae (wu & wang 1995). in this study, we found that t. melanocephalum favors high-carbohydrate foods, e.g., honey. we also found that food proficient in protein is the favorite food of both t. melanocephalum and s. invicta, which is one of the reasons leading to the competition between these two species of ants. however, t. melanocephalum also favors high-carbohydrate foods, but s. invicta does not, which provides a good condition for the survival and proliferation of t. melanocephalum when it lives in the areas with s. invicta. furthermore, for the high-carbohydrate food and high-protein food, the searching and recruitment ability of t. melanocephalum is stronger than that of s. invicta, indicating that the exploitation competition of t. melanocephalum is stronger than that of s. invicta. in addition, t. melanocephalum has a smaller size and moves more rapidly than s. invicta (li et al. 2008). therefore, t. melanocephalum can find and transport sufficient food before s. invicta arrives. we also found that t. melanocephalum lived in many s. invicta-abandoned nests (naturally abandoned or abandoned due to human activities), and sometimes the nests of these two speices were very close. the exploitative nesting habit and wide range of favorite food allows t. melanocephalum to survive in various areas (smith 1965). rapid food searching and recruitment of t. melanocephalum can explain why t. melanocephalum can co-exist with the alien ant s. invicta and continue to be one of the dominant species in areas invaded by s. invicta. this is also in agreement with the hypothesis that resource division (e.g., food, time and space) can result in ecological segregation between species. the areas with long-term (several years) invasion of s. invicta were chosen as experimental sites in this study. therefore, the effect of short-term invasion of s. invicta on the foraging ability of t. melanocephalum needs to be further studied. acknowledgments we would like to thank zhendong song and haiquan wu for their observations and records. our study was supported by the national basic research 1023 yu, y-l. et al. —comparison of foraging ability of two ant species program of china (award# 2009cb119200) and national natural science foundation of china (award# 305712427). references alfredo, f., c. jim. 2004. putting out the fire. agri. res. 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(47): 435-465. schoener, t.w. 1974. resource portioning in ecological communities. science,185:27-39. shen, p., x.l. zhao, d.f. cheng, y.q. zheng & f.r . lin. impacts of the imported fire ant solenopsis invicta invasion on the diversity of native ants.j. southwest nor.uni. 32 (4):93-97. smith, m.r. 1965. house-infesting ants of the eastern united states: their recognition biolog y and economic importance. u.s.d.a. tech. bull. 1326. vinson, s.b. 1997. invasion of the red imported fire ant (hymenoptera: formicidae): spread, biolog y and impact. am. entomol. 43(1):23-29. 1024 sociobiolog y vol. 59, no. 3, 2012 wu, b.q., y.y. lu, l. zeng, & g.w liang. 2008. influences of solenopsis invicta buren invasion on the native ant communities in different habitats in guangdong. chin. j. appl. ecol. 19(1):151-156. wu, j., c.l. wang.1995. chinese ants.beijing :chin. forestry pr. xu, r.m., x.y. cheng. 2005. insect population ecolog y-foundation and frontier. beijing : sci. and tch. press, 326. xu, y.j., y.y. lu, l. zeng, & n.d. li.2006.attraction of several baits to workers of red imported fire ant, solenopsis invicta.chin. b. entomol.43(6):856-857. zeng, l., y.y. lu & z.n. chen 2005. management and surveillance of red imported fire ant. guangdong science & technique press, guangzhou, china, 106pp. zeng , l., y.y. lu, x.f. he, w.q. zhang & g.w. liang. 2005. identification of red imported fire ant solenopsis invicta to invade mainland china and infestation in wuchuan，guangdong. chin. bul. entomol. 42:144-148. zheng,j.h., mao r.q., & r.j. zhang. 2007. comparisons of foraging activities and competitive interactions between the red imported fire ant (hymenoptera: formicidae) and two native ants under high soil-surface temperatures. sociobio. 50(3):1165-1175. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7343sociobiology 69(2): e7343 (june, 2022) introduction forel (1911) listed 15 tramp ant species, spread by human commerce, which had achieved or were in the process of achieving broad cosmopolitan distributions. over the past 100 years, many additional ant species, not on forel’s (1911) original list, have attained broad distributions in both the old world and new world (wetterer, 2015). here, i examine the geographic distribution of one of these new cosmopolitan species, nylanderia bourbonica (forel) (formerly paratrechina bourbonica). this species, native to the old-world tropics and subtropics, is now spreading through parts of north america and the west indies. abstract the robust crazy ant, nylanderia bourbonica (forel) (formerly paratrechina bourbonica), is native to the old-world tropics and subtropics. its earliest known record in the new world was collected in 1924 in miami, florida. here, i examine the subsequent spread of this species to other parts of north america and the west indies. i compiled published and unpublished new world n. bourbonica specimen records from 446 sites, documenting the earliest known records for 24 geographic areas (countries, island groups, major islands, and us states), including nine for which i found no previously published records: anguilla, antigua, barbuda, british virgin islands, jamaica, turks and caicos islands, missouri, new york, and washington. the vast majorityof new world site records for n. bourbonica (89%) come from florida, where this species is now known from 37 counties. most, if not all, of the 14 site records of n. bourbonica in north american north of 30.5°n come from indoors. although the earliest record of n. bourbonica from cuba dates to 1933, the spread of n. bourbonica to many west indian islands appears to be much more recent. in florida, n. bourbonica is a widespread, though relatively minor household and agricultural pest, and also is common in some more natural environments. it remains to be seen whether n. bourbonica will become a significant pest in the west indies or elsewhere in the new world. sociobiology an international journal on social insects james k. wetterer article history edited by jacques h.c. delabie, uesc, brazil received 12 august 2021 initial acceptance 17 august 2021 final acceptance 28 march 2022 publication date 17 june 2022 keywords ants; biogeography; exotic species; pest species. corresponding author james k. wetterer wilkes honors college florida atlantic university 5353 parkside dr. jupiter, florida, usa. e-mail: wetterer@fau.edu in 1924, alexander e. wight collected the earliest known n. bourbonica specimens in the new world in miami, florida (wheeler, 1932). smith (1930) reported that n. bourbonica “workers were observed running over sand and the pavement of sidewalks at miami. it would appear that this species is not only well established at miami but that it is capable of living outdoors. whether the species will prove to be a house infesting form is not known, but it would appear that there might be a strong possibility.” nylanderia bourbonica has since become a common household and agricultural pest throughout much of peninsular florida (klotz et al., 1995). trager (1984) reported that n. bourbonica was “established outdoors in the us in peninsular florida from gainesville florida atlantic university, jupiter, florida, usa research article ants new-world spread of the old-world robust crazy ant, nylanderia bourbonica (forel) (hymenoptera: formicidae) mailto:wetterer@fau.edu jk wetterer – new-world spread of nylanderia bourbonica2 south. i have also seen specimens from mobile, alabama which were probably collected outdoors”. in addition to florida and alabama, trager (1984) plotted n. bourbonica site records from kansas, south carolina, texas, cuba, and mexico on a distribution map. taxonomy nylanderia bourbonica is a large (total length = 2.6-3.2mm; trager, 1984), uniformly dark species, with thick straight hair on the mesosoma. forel (1886) described prenolepis nodifera bourbonica (= n. bourbonica) from réunion, an island in the indian ocean. some published records of n. bourbonica from the old world appear to be misidentifications of nylanderia vaga (forel), nylanderia amia (forel), and possibly other nylanderia species (williams et al., 2020). in the new world, however, n. bourbonica is easy to distinguish from all other species (see deyrup, 2016), and published n. bourbonica specimen records appear to be correctly identified (j.s. lapolla, pers. comm.). the genus is named in honor of william nylander (1822-1899), a finnish naturalist. the species is named after île bourbon (now réunion island), where the type specimens were collected. one common name for n. bourbonica is the robust crazy ant. robust refers to its relatively large and “husky” size compared to other species in the genus; crazy refers to the fast, erratic movements typical of nylanderia species. materials and methods using published and unpublished records, i documented the worldwide range of n. bourbonica. i obtained unpublished site records from museum specimens in the collections of archbold biological station (abs; identified by m.a. deyrup), the museum of comparative zoology (mcz; identified by s.p. cover) and the us national museum of natural history (usnm; identified by j.s. lapolla). in addition, i obtained unpublished site records from on-line databases by antweb (antweb.org) and idigbio (idigbio.org), which included collection information for n. bourbonica specimens in the mississippi entomological museum (mem), texas a & m university (tamu), and j.t. longino’s personal collection (jtlc). i received unpublished records from j.a. macgown, j.c.trager, and s.d. porter. i also included site records of n. bourbonica based on photographs posted on inaturalist from grand cayman (2), new york (1), and florida (15). starting in 2000, i collected n. bourbonica at numerous sites as part of various ant surveys in the west indies and florida (e.g., wetterer and o’hara, 2002; wetterer et al., 2007;wetterer & lombard, 2010). in 2018-2021, i made ant surveys at several hundred sites in peninsular florida primarily to map out the spread of non-native ants, including anochetus mayri emery (wetterer et al., 2018), camponotus novogranadensis mayr (wetterer, 2019), camponotus sexguttatus fig 1. new world site records of nylanderia bourbonica (mapped using carto.com). sociobiology 69(2): e7343 (june, 2022) 3 (fabricius) (wetterer, 2020a), camponotus planatus roger (wetterer, 2020b), odontomachus ruginodis smith (wetterer, 2020c), syllophopsis sechellensis (emery) (wetterer, 2020d), and syllophopsis subcoeca (emery) (wetterer & sharaf, 2021). i collected ants primarily through vegetation beating and leaflitter extractions, mostly in weedy areas along roads, hiking paths, and adjacent to commercial parking lots. i deposited voucher specimens at the mcz and usnm. geographic coordinates for collection sites came from published references, specimen labels, maps, or geography web sites (e.g., earth.google.com, www.tageo.com, and www. fallingrain.com). if a site record listed a geographic region rather than a “point locale,” and i had no other record for this region, i used the coordinates of the largest town within the region or, in the case of small islands and natural areas, the center of the region. i mapped site records using carto.com. results i compiled published and unpublished new world n. bourbonica specimen records from 446 sites (including 396 sites in florida; fig 1), documenting the earliest known records for 24 geographic areas (countries, island groups, major islands, and us states), including nine for which i found no previously published records: anguilla, antigua, barbuda, british virgin islands, jamaica, turks and caicos islands, missouri, and washington (table 1). i compiled n. bourbonica from 32 sites on 16 west indian islands, including published records from barbados (wetterer et al., 2016), new providence (deyrup et al., 1998), south bimini (smith, 1954), north andros (deyrup et al., 1998), san salvador (deyrup et al., 1998), cuba (trager, 1984; loddovega et al., 2001), and st croix (wetterer & lombard, 2010). of these, i collected n. bourbonica at 21 sites on eleven west indian islands (°n, °w; date): anguilla, katouche valley, lower forest (18.217, -63.075; 29may-2006) anguilla, long bay, golf course garden (18.190, -63.122; 21may-2006) antigua, long bay, waterfront (17.099, -61.689; 12-jul2007) barbuda, dulcina, by old hotel (17.594, -61.826; 8-jul-2007) barbuda, landing river, by wharf (17.591, -61.820; 8-jul-2007) grand cayman, 0.5 km up mastic trail (19.316, -81.196; 8-mar-2008) grand cayman, breezy castle, by building (19.302, -81.36; 9-mar-2008) grand cayman, midland acres, beach (19.297, -81.214; 8-mar-2008) great stirrup key, beach (25.8215, -77.919; 24-dec-2016) jamaica, montego bay, by burger king (18.483, -77.930; 17dec-2010) new providence, coral heights, shopping plaza (24.987, -77.456; 2-jul-2010) new providence, nassau, zoo (25.073, -77.363; 5-jul-2010) new providence, nw adelaide, scrub (25.004, -77.513; 2-jul-2010) new providence, s airport entrance, garden (25.027, -77.454; 2-jul-2010) new providence, w adelaide, beach (25.002, -77.504; 2-jul-2010) new providence, w of carmichael, roadside (25.005, -77.433; 2-jul-2010) north caicos, sandy point, by port (21.9377, -72.0452; 15jan-2016) st croix, krause lagune, boat pier (17.695, -64.7627; 5-nov-2005) st croix, sandy point, beach (17.68, -64.90; 3-mar-2006; wetterer & lombard, 2010) st vincent, georgetown, waterfront (13.279, -61.117; 5-jul2006; wetterer, 2021) tortola, boway, waterfront (18.382, -64.672; 15-nov-2005) table 1. earliest known records for nylanderia bourbonica from the new world. unpublished specimen records include collector, museum source, and site. + = no previously published records. * = indoor populations. mcz = museum of comparative zoology. usnm = us national museum of natural history. north america earliest record florida 1924 (wheeler, 1932) south carolina 1932 (d.e. read, usnm): summerville texas 1933 (p.j. darlington jr, mcz): brownsville kansas* 1974 (j.c. trager, pers. comm.): salina alabama ≤1984 (trager, 1984) mexico ≤1984 (trager, 1984) +missouri* late 1980s (j.c. trager, pers. comm.) arizona* 1990 (wetterer et al., 1999) massachusetts* 1994 (m. kelley, mcz): cambridge +washington* 1998 (j.t. longino, antweb): seattle center québec* ≤1999 (francoeur, 2000) +new york* 2019 (c. seltzer; inaturalist): ny botanical garden west indies earliest record cuba 1933 (n.a. weber, mcz): cienfuegos bahamas 1951 (smith, 1954) barbados 1998 (wetterer et al., 2016) us virgin islands 2005 (j.k. wetterer, mcz): krause lagune, st croix +british virgin islands 2005 (j. endeman, mcz): road town, tortola st vincent 2006 (wetterer, 2021) +anguilla 2006 (j.k. wetterer, mcz): long bay +antigua 2007 (j.k. wetterer, mcz): long bay +barbuda 2007 (j.k. wetterer, mcz): landing river +cayman islands 2008 (j.k. wetterer, usnm): midland acres, grand cayman +jamaica 2010 (j.k. wetterer, usnm): montego bay +turks & caicos islands 2016 (j.k. wetterer, usnm): sandy point, north caicos jk wetterer – new-world spread of nylanderia bourbonica4 outside of florida, i found only 18 north american site records of n. bourbonica, including three from mexico (trager, 1984; hernández-ruiz et al., 2009; coronado-blanco et al., 2013) and one from canada (biodôme greenhouse in montréal, québec; 45.5°n; s. johnson, 1999 pers. comm.; francoeur, 2000). of the 14 us sites outside florida, at least nine are indoor records (from north to south): washington, seattle, seattle center (45.5°n; 27-dec-98; j.t. longino; jtlc) massachusetts, cambridge, apartment (42.4°n; m. kelley, mcz) new york, ny botanical garden, conservatory (40.9°n; 12apr-2019; c. seltzer; inaturalist.org/observations/22398498) kansas, salina, hotel (38.8°n; jc trager pers. comm.) missouri, missouri botanical garden (38.6°n; j.c. trager, pers. comm.) missouri, st. louis zoo (38.6°n;j.c. trager pers. comm.) alabama, brent, motel (32.9°n; j.a. macgown, 6-jun-2003; mem) arizona, biosphere 2 greenhouse (32.6°n; wetterer et al., 1999) texas, houston, cockrell butterfly center (29.7°n; 2-feb1995; tamu) i do not know whether records of n. bourbonica from five remaining us sites are from indoors or outdoors: south carolina, summerville (33.0°n; smith, 1934) south carolina, charleston (32.8°n; smith, 1934) alabama, mobile (30.7°n; trager, 1984) texas, harris co.; houston, (29.8°n, 30-jun-1973; tamu) texas, brownsville (25.9°n; 1933; p.j. darlington jr; mcz) i compiled 396 n. bourbonica site records in florida (including 159 of my own records), from 37 counties: alachua, brevard, broward, charlotte, citrus, collier, desoto, duval, flagler, glades, hardee, hendry, hernando, highlands, hillsborough, indian river, lake, lee, levy, manatee, martin, miami-dade, monroe, okeechobee, orange, osceola, palm beach, pasco, pinellas, polk, putnam, sarasota, seminole, st johns, st lucie, sumter, and volusia (fig 2). marion county is the southernmost florida county without any n. bourbonica site records. the northernmost record in florida, collected at florida community college north (now called florida state college at jacksonville north campus) in jacksonville, florida (30.4°n; 20-apr-1991; m.a. deyrup; abs) was apparently collected outdoors. between 1972 to 2017, s.d. porter and d.p. wojcik collected n. bourbonica at a total of 17 sites along transects in gainesville, florida (29.6°n-29.7°n; s.d. porter; pers. comm.) using baits (fig 2: cluster of points in north florida). fig 2. site records of nylanderia bourbonica in the southeastern us and the western bahamas (mapped using carto.com). sociobiology 69(2): e7343 (june, 2022) 5 in addition to the above records from jacksonville and gainesville, i recorded only seven site records of n. bourbonica north of 29.5°n in florida, all of which i collected outdoors (°n, °w; date): st johns co.; st. augustine; murabella; sr-16 & 9 mile rd (29.9636, -81.4906; 16-may-2020) st. johns co.; vermont heights; by hikingtrail parking (29.804, -81.410; 21-sep-2019) st johns co.; elkton; church rd (29.7839, -81.4717; 22-apr2020) putnam co.; east palatka; by post office (29.6453, -81.6055; 12-jun-2020) alachua co.; gainesville; by campus museums (29.6365, -82.3697; 11-jun-2019) flagler co.; palm coast; old kings rd (29.5558, -81.2131; 27-mar-2020) putnam co.; rodman; rodman dam rd (29.5273, -81.7547; 1-apr-2020) kallal and lapolla (2012) presented a map (figure 205) that purported to show the distribution in north america of n. bourbonica and five other nylanderia species. oddly, this map included just three site records for n. bourbonica: one in the florida keys, one in palm beach county, florida, and one in eastern massachusetts. i interpret this last point as the first published record of n. bourbonica in massachusetts, perhaps based on the mcz specimen from cambridge (see above). nylanderia bourbonica was once included on an online list of the ants of georgia, but was removed when no voucher specimens could be found (j.a. macgown, pers. comm.). antweb once listed n. bourbonica as in illinois, but this list has been removed. boer (2019) listed n. bourbonica from curaçao, but more recently boer (2021a) did not include it, and boer (2021b) wrote “n. bourbonica unknown from the abc islands” (i.e., aruba, bonaire, and curaçao). discussion most n. bourbonica site records in new world come from florida (89%; figs 1 and 2). given how widespread n. bourbonica is in peninsular florida, it is surprising that it has been so rarely collected elsewhere in the new world (figs 1 and 2). although the first record of n. bourbonica from cuba was less than a decade after the earliest records in florida, the spread of n. bourbonica to many other west indian islands appears to be recent (table 1). while this may be, in part, an artifact of limited collecting on some islands, it is striking that n. bourbonica has not yet been recorded on puerto rico and hispaniola, where there has been much ant research in the past few decades (e.g., see torres & snelling, 1997; lubertazzi, 2019). since 1972, when n. bourbonica was first collected in gainesville (s.d. porter, pers. comm.), there have been only four florida records of this species north of gainesville (>29.7°n; fig 1; see results). this suggests that n. bourbonica populations have shown little northward spread in florida over the past 48+ years, and that the northern most outdoor populations in florida may be near their climatic limits in terms of cold weather. deyrup (2016), in fact, noted that n. bourbonica “records from mobile, alabama (trager, 1984), and from jacksonville, florida, might represent populations in urban sites protected from cold weather.” in the new world, there are only 12 records of n. bourbonica from sites north of 30.5°n, and at least nine of these were collected indoors (see results). in florida, n. bourbonica is now a widespread household and agricultural pest (klotz et al., 1995). wilson (1964) reported that n. bourbonica “workers were commonly seen foraging during the day at several locations on the streets of residential sections of key west.” although vail et al. (1994) reported that n. bourbonica is a major structure-invading pest in south florida, deyrup (2016) wrote “its chief crime seems to be that of traipsing about on the patio, where it seems to be a regular but not strikingly abundant visitor.” deyrup (2016) wrote: “in natural habitats in florida, bourbonica is most often found in wet areas that are naturally disturbed, such as the edges of marshes and upper zones of beaches.” in fact, wetterer et al. (2007) found n. bourbonica was the second most common ant (after solenopsis invicta buren) collected at tuna bait on sea turtle nests on beaches in palm beach county, florida. several other nylanderia species have been spread around the world through human commerce (wetterer, 2011; williams et al., 2020). one species, nylanderia fulva (mayr), originally from south america, has become a significant pest in parts of the southeastern us and in the west indies, where it can have enormous, localized population explosions followed by population crashes (wetterer et al., 2014). it remains to be seen whether n. bourbonica will become a significant pest in florida, the west indies, or elsewhere in the new world. acknowledgments i thank m.k. wetterer and j.c. trager for comments on this manuscript; sp cover for help, encouragement, and ant identification; j.a. macgown, j.c. trager, s.d. porter, and m. kubo for providing unpublished records; e. lebrun, k. wright, and a. wild for help concerning texas specimen records; j. endeman for sending me specimens; s.p. cover (mcz) and m.a. deyrup (abs) for help with their respective ant collections; j.s. lapolla for confirming identifications; florida atlantic university and the national science foundation (deb-0515648) for financial support. references boer, p. 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(2011). worldwide spread of the yellow-footed ant, nylanderia flavipes (hymenoptera: formicidae). florida entomologist, 94: 582-587. doi: 10.1653/024.094.0323 wetterer, j.k. (2015). geographic origin and spread of cosmopolitan ants (hymenoptera: formicidae). halteres, 6: 66-78. wetterer, j.k. (2019). spread of camponotus novogranadensis (hymenoptera: formicidae), a non-native carpenter ant in florida. transactions of the american entomological society, 145: 86-90. doi: 10.3157/061.145.0109 wetterer, j.k. (2020a). geographic distribution of camponotus sexguttatus (hymenoptera, formicidae), a neotropical carpenter ant spreading in florida. transactions of the american entomological society, 146: 239-250. doi: 10.3157/061.146.0107 wetterer, j.k. (2020b). geographic distribution of the compact carpenter ant camponotus planatus (hymenoptera: formicidae), a neotropical species spreading in florida. transactions of the american entomological society, 146: 409-420. doi: 10.3157/061.146.0207 wetterer, j.k. (2020c). spread of the neotropical trap-jaw ant odontomachus ruginodis (hymenoptera: formicidae) in florida. transactions of the american entomological society, 146: 591-600. doi: 10.3157/061.146.0309 wetterer, j.k. (2020d). first north american records of syllophopsis sechellensis (hymenoptera: formicidae). sociobiology, 67: 478-480. doi: 10.1653/024.100.0224 wetterer, j.k. (2021). ants (hymenoptera: formicidae) of st. vincent, west indies. sociobiology, 68: e6725; doi: 10.13102/ sociobiology.v68i2.6725 wetterer, j.k., o. davis & j.r. williamson (2014) boom and bust of the tawny crazy ant, nylanderia fulva, on st croix, us virgin islands. florida entomologist, 97: 1099-1103. wetterer, j.k., m.a. deyrup & a. bryant (2018). spread of the non-native trap-jaw ant anochetus mayri (hymenoptera: sociobiology 69(2): e7343 (june, 2022) 7 formicidae) in florida. transactions of the american entomological society, 144: 437-441. doi: 10.3157/061. 144.0201 wetterer, j.k. & c.d. lombard (2010). fire ants on an important sea turtle nesting beach in st. croix, us virgin islands. florida entomologist, 93: 449-450. doi: 10.1653/ 024.093.0321 wetterer, j.k., d. lubertazzi, j. rana & e.o. wilson (2016). ants of barbados (hymenoptera: formicidae). breviora, 548: 1-34. wetterer, j.k., s.e.miller, d.e. wheeler, c.a. olson, d.a. polhemus, m. pitts, i.w. ashton, a.g. himler, m. yospin, k.r. helms, e.l. harken, j. gallaher, c.e. dunning, m. nelson, j. litsinger, a. southern & t.l. burgess (1999). ecological dominance by paratrechina longicornis (hymenoptera: formicidae), an invasive tramp ant, in biosphere 2. florida entomologist, 82: 381-388. wetterer, j.k. & b.c. o’hara (2002). ants (hymenoptera: formicidae) of the dry tortugas, the outermost florida keys. florida entomologist, 85: 303-307. doi: 10.1653/0015 4040(2002)085[0303:ahfotd]2.0.co;2 wetterer, j.k., l.d. wood, c. johnson, h. krahe & s. fitchett (2007). predaceous ants, beach replenishment, and nest placement by sea turtles. environmental entomology, 36: 10841091. doi: 10.1603/0046-225x(2007)36[1084:pabran]2.0.co;2 wetterer, j.k. & m. sharaf (2021). worldwide distribution of syllophopsis subcoeca (hymenoptera: formicidae), an old-world species long known only from the west indies. journal of natural history, 55: 1465-1476. doi: 10.1080/ 00222933.2021.1948129 wheeler, w.m. (1932). a list of the ants of florida with descriptions of new forms. journal of the new york entomological society, 40: 1-17. williams, j.l., y.m. zhang, m.w. lloyd, j.s. lapolla, t.r. schultz & a. lucky (2020). global domination by crazy ants: phylogenomics reveals biogeographical history and invasive species relationships in the genus nylanderia (hymenoptera: formicidae). systematic entomology, 45: 730-744. doi: 10.11 11/syen.12423 wilson, e.o. (1964). the ants of the florida keys. breviora, 210: 1-14. 959 ant–aphid relations in costa rica, central america (hymenoptera: formicidae; hemiptera: aphididae) by xavier espadaler1, nicolás pérez hidalgo2 & william villalobos muller3 abstract we present the first catalogue of ant-aphid associations (hymenoptera: formicidae / hemiptera: aphididae) of costa rica. 29 species of ants and 18 species of aphids establish 48 relationships. those interactions seem not to be rare in costa rica. key words: formicidae. ants, aphididae. aphids, costa rica, central america resumen se establece el primer catálogo homiga-pulgón (hymenoptera: formicidae / hemiptera: aphididae) de costa rica, que consta de 29 especies de hormigas y 18 de áfidos que establecen 48 relaciones. se ofrecen datos sobre las especies citadas y se comenta la composición faunística. palabras clave: formicidae. hormigas. aphididae. áfidos. costa rica. centroamérica. introduction the relationships of ants with hemiptera in general, and with aphids in particular, have been treated in several aspects in many works since the 1950s (way 1963; hölldobler & wilson 1990; stadler & dixon 2008). in the context of the study of the aphid fauna of costa rica, sixty-four aphid species (hemiptera, sternorrhyncha: aphididae) were caught during 2008. the aphid catalogue consists now of 89 species (villalobos et al. 2010; pérez hidalgo et al. 2012) and the list of “host plant species − aphid species” 1 unidad de ecología y creaf, universidad autónoma de barcelona, e-08193 bellaterra, barcelona, spain. e-mail: xavier.espadaler@uab.es 2 departamento de biodiversidad y gestión ambiental, universidad de león, e-24071, león, spain. e-mail: nperh@unileon.es 3 centro de investigación en biología celular y molecular, universidad de costa rica, 11501-2060, san josé (costa rica). e-mail: william.villalobos@ucr.ac.cr 960 sociobiolog y vol. 59, no. 3, 2012 relationships was compiled by voegtlin et al. (2003) and complemented by villalobos et al. (2010), sánchez-monge et al. (2010) and pérez hidalgo et al. (2012). the aphid parasitoid fauna of costa rica has been recently reviewed by zamora mejías et al. (2010, 2011). nevertheless, the association with others arthropods, including ants, has not been suitably studied in costa rica. here we attempt to provoke further field research by noting the scarcity of published accounts of aphid-ant associations in the neotropics. as this short –but focusedfield sampling study shows, aphid-ant associations are indeed frequent and deserve enhanced attention. methods we carried out two intensive aphid collection campaigns in february and december 2008, covering almost the entire territory, as well as other sporadic expeditions that year and in 2009. the aphids were mainly collected by prospecting the plants and they were studied in alcohol or in microscopic slides. they are now in the university of leon zoological collection and in the university of costa rica (centro de biología celular y molecular) collection. the list of aphid species and plants is published in villalobos et al. (2010). the ants were identified using the excellent available keys, images and information at http://academic.evergreen.edu/projects/ants/ and the recent revision of linepithema by wild (2007). vouchers are in the museum of zoolog y of barcelona and the personal collection of x.e. we did an extensive search at formis (2011) using as keywords: colombia, costa rica, guatemala, honduras, nicaragua, panamá, or venezuela and aphids, áfidos, hemiptera, coccidae, mealybugs, pulgones, whiteflies, or leaf-, plant-, or tree-hoppers. in addition, general textbooks (beattie 1985; bristow 1991; dixon 1998; hölldobler & wilson 1990; rico-gray & oliveira 2007; stadler & dixon 2008 and reviews by way (1963), buckley (1987a, b), and delabie (2001) were also specifically searched for neotropical cases of aphid-ant interactions. results a total of 597 samples of aphids with colonies were studied, and only in 48 of them there were relations with ants (see appendix). 961 espadaler, x. et al. — ant-aphid interactions in costa rica neotropical aphid-ant literature reports seem to be very scarce homoptera-ant relationships are highly diverse in the neotropics but they concern mainly auchenorrhyncha (treehoppers, mealybugs) and non-aphididae sternorrhyncha (scales). aphid-ant relationships reports seem to be exceedingly scarce in costa rica and, by extension, in central america. kleinfeldt (1978) noted crematogaster longispina tending unidentified homoptera on pentaclethra macroloba (willd.) ktze. (leguminosae) and on pejibaye (bactris utilis benth. and hook; palmae) and ectatomma ruidum tending unidentified aphids on codonanthe crassifolia (focke) morton (gesneriaceae) in costa rica. vergara et al. (2007) mention solenopsis geminata and paratrechina sp. tending unidentified aphididae at the arboretum of medellín university (colombia). ramírez et al. (2001) in an extensive survey of deciduous forest fragments in colombia, mention a single association, that of cerataphis sp. with dolichoderus bispinosus. delabie (2001) and delabie & fernández (2003), in a couple of very useful reviews of the biological intricacies of ant-homoptera relationships, note the commonness of ant-attendance in temperate regions of the northern hemisphere albeit fail to mention but a case of tropical (asian) aphid-ant relationship, that of pseudoregma sundanica (van der goot, 1917) with several ant species (schütze & maschwitz 1991). here we have detected 48 aphid-ant associations involving 19 aphid species and 29 ant species. therefore, the image we obtained seems to be in contrast with what is currently known. on the specialist vs. generalist aphid-ant interactions as a mere exploration of the interactions, the numbers of partners in each group (ants, aphids) was analyzed. the frequency distribution of cases in which the number of partners is 1, 2, etc. is marginally significant (table 1; collapsing columns 4-9, chi square = 7.4; 3 d.f.; p = 0.0598) between both insect groups. there is a highly biased general distribution towards one-toone or few interactions. thus, we detected 20 ant species interacting with a single aphid species, and 6 aphid species attended only by one ant species. some species, though, are able to interact with many partners (8 or 9). thus, solenopsis geminata (black form) was detected attending eight aphid species and the polyphagous, world-wide distributed aphids aphis gossypii and aphis 962 sociobiolog y vol. 59, no. 3, 2012 spiraecola were attended by nine and eight ant species respectively. we refrained from performing any nestedness analysis because of the small data base. discussion scarcity of aphid-ant literature reports ants are ubiquitous everywhere in the neotropics (wilson 1990). thus, the scarcity of aphid-ant interactions in this region is not for shortage of ants. instead, this is attributable to the aphid component, either directly by the low number of aphids in the tropics or indirectly by the low density of their plant hosts (bristow 1991; dixon et al. 1987). notwithstanding, as this note shows, there may be geographical areas in which aphid-ant relationships are not rare, thus calling for regular, and more focused, field surveys of antaphid interactions in the neotropical region. a useful example to start from would be the revision of aphid-plant associations in peru by delfino (2005), where he notes 66 aphid species and 238 plant species. nine of those aphid species were found in our survey as well. peronti & sousa-silva (2002) listed 25 aphid species –mostly exoticsfrom 49 ornamental plant species in são carlos, brazil. the cosmopolite, polyphagous aphis gossypii and a. spiraecola were the most abundant in their list. in our aphid study the predominant aphid species (those most frequently captured) were a. gossypii and a. spiraecola, which likely have a stable extensive presence on a considerable number of plants all over the country. the nearctic species, aphis coreopsidis, always on bidens pilosa, present a strong relation with ants and aphis craccivora, hysteroneura setariae, myzus persicae and toxoptera aurantii, were also frequently captured, and, like the previously mentioned species, are alochthonous in central america and distributed worldwide, feeding on a large range of plant species (blackman & eastop 2006). most of the remaining aphid species were captured only once or very rarely. nevtable 1. frequency distribution of the number of aphid or ant species with different partners in costa rica (greenidea not included). number of partners 1 2 3 4 5 6 7 8 9 aphid species with number of partners 6 3 4 2 1 1 1 ant species with number of partners 20 5 1 2 1 1 963 espadaler, x. et al. — ant-aphid interactions in costa rica ertheless, species that do not establish relations with ants, as myzus ornatus, brachycaudus helichrysi, neomyzus circumflexus and aulacorthum solani have been captured often in our study, which confirms the scanty relation that aphids, as a group, establish with the ants in this territory. specialist vs. generalist aphid-ant interactions the results reported here contrast with the general non-specificity of aphidant interactions that is theoretically expected (law & koptur 1986), and usually accepted (bristow 1991; stadler & dixon 2008). the current concept of ant-homoptera interactions is one of facultative mutualism (letourneau & choe 1987; delabie & fernández 2003) depending on scale of analysis, viz. at the level of individual trees (gove & rico-gray 2006) or in the context of the presence/absence of extrafloral nectaries (oliveira & freitas 2004). of course, the most parsimonious hypothesis to explain the high abundance here detected of one-to-one species aphid-ant association, and vice versa, is one of defective sampling : our sample might be too small to show the expected trend towards a lack of specificity of the ant-aphid interactions. an alternative explanation would be that the pattern is genuine in costa rica but has been unattested for several reasons (scarcity of focused visits, or poor attention of aphid taxonomists visiting the neotropics, or lack of expectancy to find those associations). the test of both hypotheses is straightforward: more aphid-ant determined field sampling, looking specifically for aphid-ant relations. time will tell. acknowledgments to john t. longino for the splendid, and useful, work he is doing on the ants of costa rica. the authors thank the agencia española de cooperación internacional para el desarrollo (aecid) [joint project of the universities of león and costa rica d/010523/07] for supporting this research. xe is currently supported by micinn-feder grant cgl2010-18182. references beattie, a.j. 1985. the evolutionary ecolog y of ant-plant mutualisms. cambridge university press, new york. blackman, r.l.; v.f. eastop 2006. aphids on the world’s herbaceous plants and shrubs. john wiley & sons; london. 964 sociobiolog y vol. 59, no. 3, 2012 bristow, c.m. 1991. why are so few aphids ant-tended? in: ant-plant interactions, c. r. huxley and d. f. cutler (eds.). oxford university press, oxford: 104-119. buckley, r.c. 1987a. interactions involving plants, homoptera, and ants. annual review of ecolog y and systematics 18: 111-135. buckley, r.c. 1987b. ant-plant-homopteran interactions. advances in ecological research 16: 53-85. delabie, j. & f. fernández 2003. capítulo 11. relaciones entre hormigas y “homópteros” (hemiptera: sternorrhyncha, auchenorrhyncha). en: fernández, f. (ed.). introducción a las hormigas de la región neotropical. iirb alexander von humboldt, bogotá, colombia: 181-197. delabie, j. h.c. 2001. trophobiosis between formicidae and hemiptera (sternorrhyncha and auchenorrhyncha): an overview. neotropical entomolog y 30: 501516. delfino, m.a. 2005. inventario de las asociaciones áfido-planta en el perú. checklist of aphid-plant associations in peru. ecología aplicada 4(1,2): 143-158. dixon, a. f. g., p. kindlmann, j. leps, & j. holman 1987. why there are so few species of aphids, especially in the tropics? american naturalist 129: 580–592. dixon, a.f.g. 1998. aphid ecolog y, 2nd. edition. chapman & hall. formis 2011. formis: a master bibliography of ant literature. (at http://www.ars. usda.gov/saa/cmave/ifahi/formis; last accessed 3 february 2012. gove, a.d. & v. rico-gray 2006. what determines conditionality in ant – hemiptera interactions? hemiptera habitat preference and the role of local ant activity. ecological entomolog y 31: 568-574. hölldobler, b. & e.o. wilson 1990. the ants. belknapp press; harvard university press. kleinfeldt, s.e. 1978. ant-gardens: the interaction of codonanthe crassifolia (gesneriaceae) and crematogaster longispina (formicidae). ecolog y 59: 449-456. law, r. & s. koptur 1986. on the evolution of non-specific mutualisms. biological journal of the linnean society 27: 251-267. letourneau, d.k. & j.c. choe 1987. homopteran attendance by wasps and ants: the stochastic nature of interactions. psyche 94: 81-91. oliveira, p.s. & a.v.l. freitas 2004. ant–plant–herbivore interactions in the neotropical cerrado savanna. naturwissenschaften 91: 557-570. pérez hidalgo, n., d. martínez-torres, j.m. collantes-alegre, w. villalobos muller & j.m. nieto nafría 2012. a new species of rhopalosiphum (hemiptera: aphididae) on chusquea tomentosa (poaceae: bambusoideae) from costa rica. zookeys, 166: 59-73. doi: 10.3897/zookeys.166.2387. peronti, a.l.b. & c.r. sousa-silva 2002. aphids (hemiptera: aphidoidea) of ornamental plants from são carlos, são paulo state, brazil. revista de biologia tropical 50(1): 137-144. ramírez, m., p. chacón de ulloa, i. armbrecht & z. calle 2001. contribución al conocimiento de las interacciones entre plantas, hormigas y homopteros en bosques secos de colombia. caldasia 23(2): 523-536. 965 espadaler, x. et al. — ant-aphid interactions in costa rica rico-gray, v. & p.s. oliveira 2007. the ecolog y and evolution of ant-plant mutualisms. the university of chicago press. schütze, m. & u. maschwitz 1991. enemy recognition and defense within trophobiotic associations with ants by the soldier caste of pseudoregma sundanica (homoptera: aphidoidea). entomologia generalis 16: 1-12. stadler, b. & a.f.g. dixon 2008. mutualism. ants and their insect partners. cambridge university press, cambridge. vergara, e.v., h. echavarría & f.j. serna 2007. hormigas (hymenoptera formicidae) asociadas al arboretum de la universidad nacional de colombia, sede medellín. boletín sociedad entomológica aragonesa 40: 497−505. sánchez-monge, a., a. retana-salazar, s. brenes & r. agüero 2010. new records of aphid-plant associations (hemiptera: aphididae) from eastern costa rica. florida entomologist 93(3): 489-492. villalobos muller w, n. pérez hidalgo, m.p. mier durante, & j.m. nieto nafría 2010. aphididae (hemiptera: sternorhyncha) from costa rica, with new records for central america. boletín de la asociación española de entomología 34 (1-2): 145–182. voegtlin, d., w. villalobos, m.v. sánchez, g. saborío & c. rivera 2003. guía de los áfidos alados de costa rica / a guide to the winged aphids of costa rica. revista de biología tropical, 51, suplemento 2, 1−214. way, m.j. 1963. mutualism between ants and honeydew-producing homoptera. annual review of entomolog y 8: 307-344. wild, a.l. 2007. taxonomic revision of the ant genus linepithema (hymenoptera: formicidae). university of california publications in entomolog y 126: 1-159. wilson, e.o. 1990. success and dominance in ecosystems: the case of the social insects. excellence in ecolog y, 2. ecolog y institute. oldendorf/luhe. zamora mejías, d., p.e. hanson, & p. starý 2010. survey of the aphid parasitoids (hymenoptera: braconidae: aphidiinae) of costa rica, with information on their aphid (hemiptera: aphidoidea)-plant associations. psyche 2010: article id 278643, 7 pp. zamora mejías, d., p.e. hanson, p. starý, & e. rakhshani 2011. parasitoid (hym., braconidae, aphidiinae) complex of the black citrus aphid, toxoptera citricidus (kirkaldy) (hem., aphididae) in costa rica and its relationships to nearby areas. journal of the entomological research society 13(3): 107-115. the appendix begins on page 5 966 sociobiolog y vol. 59, no. 3, 2012 appendix ant-aphid-host plant catalogue brachymyrmex santschii menozzi, 1927 myzocallis pepperi on quercus sapotifolia, frailes, 27-12-2008. camponotus atriceps (f. smith, 1858) cerataphis brasiliensis on chamaedorea costaricana, san pedro montes de oca, 27-12-2008. camponotus novogranadensis mayr, 1870 aphis coreopsidis on bidens pilosa, cervantes, 5-12-2008. greenidea psidii on psidium sp., san pedro montes de oca, 21-12-2008. this relation must be considered to be doubtful because in all the samples of this species recorded by us in costa rica (pérez hidalgo et al. 2009) we have never observed relations with ants. probably the ants were attending to coccidae who were present also in the same host plant. camponotus planatus roger, 1863 aphis gossypii on thunbergia grandiflora, san pedro montes de oca, 7-122008. camponotus rectangularis emery 1890 aphis craccivora on pha seolus v ulgaris , filadelfia , 23-2-2008. myzocallis discolor (monell, 1879) on quercus oleoides, liberia, 24-122008. camponotus striatus (f. smith, 1862) sitobion avenae on penisetum purpureum, nuevo arenal, 24-12-2008. camponotus textor forel, 1899 toxoptera aurantii on unidentifiable plant, upala, 21-2-2008. crematogaster carinata mayr, 1862 aphis gossypii on unidentifiable plant, la fortuna , 21-2-2008. ap h i s s p i ra e c o l a o n s e n e c i o s p . , l a fo r t u n a , 2 0 2 2 0 0 8 . toxoptera aurantii on unidentifiable plant, sarapiqui, 25-12-2008. crematogaster limata f. smith, 1858 aphis spiraecola on unidentifiable plant, agua buena, cotobrus, 17-122008. ectatomma ruidum (roger, 1861) aphis gossypii on unidentifiable plant, tronadora , 24-12-2008. 967 espadaler, x. et al. — ant-aphid interactions in costa rica hysteroneura setariae on unidentifiable gramineae, liberia, 2212-2008. myzocallis discolor on quercus oleoides, liberia, 22-122008. myzus persicae, without data of host-plant, locality and date. rhopalosiphum maidis on panicum sp., liberia, 24-12-2008. ectatomma tuberculatum (olivier, 1792) aphis spiraecola on unidentifiable plant, monterrey, 21-2-2008; on iresine diffusa, la fortuna, 24-12-2008. pseudoregma panicola on panicum sp., la fortuna, 24-12-2008. linepithema angulatum (emery, 1849) sarucallis kahawaluokalani on lagerstroemia indica, san francisco de dos rios, 27-12-2008. linepithema iniquum (mayr, 1870) aphis spiraecola on conyza canadensis, braulio carrillo, 22-12-2008; on phenax rugosus, san pedro montes de oca, 5-12-2008. linepithema neotropicum wild, 2007 aphis gossypii on commelina sp., san pedro montes de oca, 5-12-2008. monomorium floricola ( jerdon, 1851) aph i s c ra c c iv o ra o n d e sm o di um sp . , pl ata n er a , 2 5 1 2 2 0 0 8 . myzocallis discolor on quercus oleoides, liberia, 25-12-2008. myrmelachista zeledoni emery, 1896 aphis spiraecola on phenax rugosus, san pedro montes de oca, 5-122008. nylanderia steinheili (forel, 1893) toxoptera citricidus on crescentia cujete, sarapiqui, 25-12-2008. paratrechina longicornis (latreille, 1802) myzus persicae on catharanthus roseus, san francisco de dos rios, 27-12-2008. sarucallis kahawaluokalani on lagerstroemia indica, san francisco de dos rios, 27-12-2008. pheidole bilimecki mayr, 1870 aphis coreopsidis on bidens pilosa, san pedro montes de oca, 7-12-2008. aphis gossypii on thunbergia grandiflora, san pedro montes de oca, 7-12-2008. aphis spiraecola on bidens pilosa, agua buena, cotobrus, 18-12-2008. hysteroneura setariae (thomas, 1878) on eleusine indica, san pedro montes de oca, 24-2-2008 and 7-12-2008. 968 sociobiolog y vol. 59, no. 3, 2012 pheidole fallax mayr, 1870 aphis gossypii on unidentifiable plant, santo domingo de heredia, 14-122008. pheidole flavens roger, 1863 aphis gossypii on blechum pyramidatum, la fortuna, 20-2-2008. pheidole megacephala (fabricius, 1793) cerataphis brasiliensis on chrysalidocarpus lutescens, alajuela, 6-122008. pheidole pugnax dalla torre, 1892 hysteroneura setariae on oplismenus burmannia, parque nacional santa rosa, 23-12-2008. pheidole punctatissima mayr, 1870 toxoptera aurantii on citrus sp., coronado, 28-12-2008. pheidole variegata emery, 1896 aphis coreopsidis on bidens pilosa, frailes, 27-12-2008. solenopsis geminata fabricius 1804 (black form) aphis gossypii on unidentifiable plant, tronadora, 24-12-2008. aphis spiraecola on ixora sp., aguas zarcas, san carlos, 25-12-2008; on priva lappulacea, cahuita, 25-12-2008. hysteroneura setariae on axonopus sp., estación experimental 28 millas, 26-12-2008. pentalonia nigronervosa on musa sp., cahuita, 25-12-2008. rhopalosiphum maidis on unidentifiable gramineae, upala, 21-2-2008; on penisetum purpureum, cahuita, 25-12-2008. schizaphis rotundiventris cyperus sp., upala, 21-2-2008. tetraneura fusiformis on eleusine indica, la fortuna, 21-2-2008. toxoptera citricidus on citrus limetoides, sarapiqui, 25-12-2008. solenopsis geminata (yellow form) ap h i s c o r e o p s i d i s o n b i d e n s p i l o s a , c e r v a n t e s , 5 1 2 2 0 0 8 . aphis craccivora koch, 1854 on phaseolus vulgaris, filadelfia, 23-2-2008. tetramorium bicarinatum (nylander, 1846) rhopalosiphum maidis on penisetum purpureum, cahuita, 25-12-2008. wasmannia auropunctata (roger, 1863) aphis gossypii on commelina sp., sarapiqui, 25-12-2008. aphis spiraecola on priva lappulacea, cahuita, 25-12-2008. cerataphis brasiliensis on alpinia purpurata, san josé, 24-2-2008. hysteroneura setariae on eleusine indica, sarapiqui, 25-12-2008. 969 espadaler, x. et al. — ant-aphid interactions in costa rica wasmannia sigmoidea (mayr, 1884) toxoptera aurantii, without data of host-plant, locality and date. aphid-ant catalogue aphis coreopsidis (thomas, 1878) camponotus novogranadensis, pheidole bilimecki, pheidole variegata, solenopsis geminata (yellow form) aphis craccivora koch, 1854 camponotus rectangularis, monomorium floricola, solenopsis geminata (yellow form) aphis gossypii glover, 1877 camponotus planatus, crematogaster carinata, ectatomma ruidum, linepithema neotropicum, pheidole bilimecki, pheidole fallax, pheidole flavens, solenopsis geminata (black form), wasmannia auropunctata aphis spiraecola patch, 1914 crematogaster carinata, crematogaster limata, ectatomma tuberculatum, linepithema iniquum, myrmelachista zeledoni, pheidole bilimecki, solenopsis geminata (black form), wasmannia auropunctata cerataphis brasiliensis (hempel, 1901) camponotus atriceps, pheidole megacephala, wasmannia auropunctata greenidea psidii van der goot, 1916 camponotus novogranadensis (see comment on this ant species). hysteroneura setariae (thomas, 1878) ectatomma ruidum, pheidole bilimecki, pheidole pugnax, solenopsis geminata (black form), wasmannia auropunctata myzocallis discolor (monell, 1879) camponotus rectangularis, ectatomma ruidum, monomorium floricola myzocallis pepperi boudreaux & tissot, 1962 brachymyrmex santschii myzus persicae (sulzer, 1776) ectatomma ruidum, paratrechina longicornis pentalonia nigronervosa coquerel, 1859 solenopsis geminata (black form) pseudoregma panicola (takahashi, 1921) 970 sociobiolog y vol. 59, no. 3, 2012 ectatomma tuberculatum rhopalosiphum maidis (fitch, 1856) ectatomma ruidum, solenopsis geminata (black form), tetramorium bicarinatum sarucallis kahawaluokalani (kirkaldy, 1907) linepithema angulatum, paratrechina longicornis schizaphis rotundiventris (signoret, 1860) solenopsis geminata (black form) sitobion avenae (fabricius, 1775) camponotus striatus tetraneura fusiformis matsumura, 1917 solenopsis geminata (black form) toxoptera aurantii (boyer de fonscolombe, 1841) camponotus textor, crematogaster carinata, pheidole punctatissima, wasmannia sigmoidea toxoptera citricidus (kirkaldy, 1907) nylanderia steinheili, solenopsis geminata (black form) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.517-522sociobiology 61(4): 517-522 (december, 2014) assessing sperm quality in stingless bees (hymenoptera: apidae) introduction in order to exploit the great potential of stingless bees as commercial pollinators, it is of great importance to improve management and optimize practices to produce colonies on a large scale. therefore, it is essential to develop accurate diagnostic tools that enable a better understanding of the reproductive biology of stingless bees. semen quality is known to determine male reproductive success across taxa (simmons, 2001). in turn, semen quality can be affected by factors such as the number, morphology and viability of sperm (garófalo, 1980; simmons, 2001; den boer et al., 2009). few studies have looked at sperm characteristics of bees in general, and even less in stingless bees. cortopassi-laurino (1979) counted the number of sperm in males of plebeia droryana (friese) and found 313,000 ± 102,050 sperm per male. garófalo (1980) evaluated the amount of semen produced by the males and stored in the female’s spermathecae in different species of bees, including eight stingless bees, and found that there is no relationship between social levels and the amount of sperm found in abstract although stingless bees have a great potential as commercial pollinators, their exploitation depends on the successful reproduction of colonies on a large scale. to do so, it is essential to develop accurate diagnostic tools that enable a better understanding of the reproductive biology of stingless bees. sperm counts, sperm morphology and sperm viability (the relative proportion of live to dead sperm), are key parameters assessing semen quality and potential fertilization success. here we present standardized protocols to assess these three parameters. we used scaptotrigona aff. depilis (moure) as a study model. semen extractions from the seminal vesicles were found to yield better results when performed in mature rather than in younger males. for morphology and viability analyses, the best semen dilution on hayes solution was adding 120 µl to the contents of the two seminal vesicles. for sperm counts, however, we recommend a higher dilution (1,000 µl). sperm viability values were higher when hayes solution was adjusted to ph 8.7, and when samples were analyzed before 24 hours from collection. based on these results we present standard protocols, hoping they will be useful to future researchers assessing sperm quality in other stingless bee species. sociobiology an international journal on social insects hm meneses1, s koffler2, bm freitas1, vl imperatriz-fonseca2, r jaffé2 article history edited by denise araujo alves, esalq-usp, brazil received 14 november 2014 initial acceptance 01 december 2014 final acceptance 01 december 2014 keywords sperm viability, sperm morphology, sperm counts, semen, meliponini. corresponding author hiara marques meneses av. mister hull 2977, campus do picí, bloco 808, 60021-970, fortaleza-ce, brazil e-mail: hiarameneses@gmail.com females and produced by males. conte et al. (2005) found that the small spermatids are lost during the early stages of spermiogenesis of melipona quadrifasciata anthidioides lepeletier. also addressing the spermatogenesis process, lino-neto et al. (2008) found that half of the spermatids formed during the spermatogenesis are not transformed into viable sperm cells in scaptotrigona xanthotricha moure. pech-may et al. (2012) found a strong positive relationship between the size of male and semen production in melipona beecheii bennett. camillo (1971) noted an increase of 2.35 times in the number of sperm of giant friesella schrottkyi (friese) males (produced occasionally when an unfertilized egg is raised on a royal cell) compared to what is found in common males. table 1 summarizes previous contributions to the study of stingless bee sperm. the viability of spermatozoa, or relative proportion of live to dead sperm, is an important parameter to appreciate reproductive success of males (simmons, 2005), and it is used to analyze semen quality in honeybees (collins & donoghue, 1999; collins, 2004; den boer et al., 2009; gençer et al., 2014). it is also commonly used in other organisms to study various aspects of their reproductive biology (garner et al., 1994; ball et al., 2001; paulenz et al., 2002). research article bees 1 universidade federal de ceará,ufc, fortaleza, ce, brazil 2 universidade de são paulo, usp, são paulo, sp, brazil hm meneses, s koffler, bm freitas, vl imperatriz-fonseca, r jaffé assessing sperm quality in stingless bees518 recent advances in the use of flow cytometry to assess sperm traits in social insects allowed significant improvements, decreasing processing times and increasing accuracy (cornault & aron, 2008; paynter et al., 2014). however, flow cytometry equipment is not common across the tropics (where stingless bees occur), and the costs involved in their use and maintenance are substantially higher than those involving fluorescence-based methods. we thus provide standard protocols for fluorescence-based methods, which are cheap and ready available across the tropics, aiming at facilitating further research on stingless bees. although less accurate than flow cytometry methods, fluorescence-based methods have been widely used and they are well accepted as standard methods (thomas & simmons., 2007; den boer et al., 2008, 2010; cobey et al., 2013). moreover, sperm viability obtained with fluorescence microscopy and flow cytometry has been found indistinguishable in some cases (paynter et al., 2014). to date, no standard protocol is available to assess sperm quality in stingless bees. here we fill this gap by presenting appropriate protocols for assessing sperm counts, sperm morphology and sperm viability in stingless bees. table 1. works addressing sperm biology in stingless bees. materials for details). males were collected from male aggregation and from the interior of colonies. those found in aggregations (fig 1) were considered mature and originated from different colonies (paxton et al., 2000). males collected inside the colonies were only distinguished as freshly emerged males (usually of a lighter color) or adult (darker) males. however, during dissections, we found marked differences between males in testis size and migration of sperm to the seminal vesicles. we thus used this information as a proxy for male age, where males with bigger testis and incomplete semen migration were characterized as immature males. fig 1. aggregation of scaptotrigona aff. depilis males. different semen dilutions were tested (40, 100 and 200 µl) to facilitate the counting of sperm cells (5 males per treatment). as ph was found to have a profound effect on sperm viability during our initial trials, we experimentally manipulated ph to identify the ph yielding maximal sperm viability (ph tested was 8.4, 8.7 and 9.0, with 3 males per treatment). likewise, because sperm cells start to die once the ejaculate has been collected, we also tested the effect of time on sperm viability (fresh, 1, 3, 5 and 24 hours after dissection, with 5 males per treatment). for the sperm counts and sperm morphology analyses we stained sperm using dapi (fig 2, see supplementary material for details). in order to assess the morphology of sperm cells, black and white images of the dapi-stained cells were taken (20x magnification). images were analyzed with the software image j, adjusting brightness and contrast for better visualization. for the measurement of the sperm head area, each image was adjusted by changing the threshold parameter, which enhances binary contrast. this modification was done until all head area was selected, excluding the sperm tail and background. after this procedure, head area was selected with the automatic selection tool (wand) and measured. because threshold adjustments may be subjective, training and standardizing the procedure is suggested before the real measurements are taken. illustrative results are presented for species topic reference friesella schrottkyi (friese) giant males camilo et al. (1971) plebeia droryana (friese) sperm number cortopassi-laurino (1979) friesella schrottkyi (friese) lestrimelitta limao (smith) melipona marginata lepeletier m. quadrifasciata lepeletier m. rufiventris lepeletier scaptotrigona postica (latreille) tetragonisca angustula (latreille) trigona hyalinata (lepeletier) sperm produced by males and stored in queen’s spermathecae garófalo (1980) melipona quadrifasciata lepeletier spermatogenesis conte et al. (2005) scaptotrigona xanthotricha moure spermatogenesis lino-neto et al. (2008) melipona beecheii bennett sperm number pech-may et al. (2012) material and methods we used established protocols for sperm counts and sperm viability in honeybees (apis mellifera linnaeus) (collins & donoghue, 1999; cobey et al., 2013), as a guideline to develop protocols to analyze stingless bee sperm. all tests were conducted in the bee laboratory, department of ecology, university of são paulo. our study model was scaptotrigona aff. depilis (moure), a common stingless bee of southeastern brazil. specimens of each colony were collected and identified with the aid of a specialist (dr. silvia r. m. pedro). voucher specimens can be found in the coleção entomológica paulo nogueira-neto (cepann), located in the university of são paulo, são paulo, brazil. adjustments were implemented to improve the collection of semen from the seminal vesicles (see supplementary sociobiology 61(4): 517-522 (december, 2014) 519 10 sperm cells from one male (mean ± se). sperm counts were estimated by diluting the total amount of sperm of each male 10,000 times and counting cells in three samples of 1 µl (air dried in microscope slides). the samples were also dapistained. counting began at the right edge of each sample, and continued until de left side of the sample was reached. the mean value obtained from the three samples was multiplied by 10,000, to estimate the total number of sperm per male. results for sperm counts are presented for six males (mean ± se). statistical analyses consisted in binomial generalized linear models, as they are the standard method to analyze proportion data (crawley, 2013), such as sperm viability (dead/alive). similar analyses are often implemented in studies of sperm viability (den boer et al., 2009; stürup et al., 2011). all statistical analyses were implemented in r. results semen was more easily collected from the seminal vesicles of mature males (fig 5) rather than younger ones (fig 6), as semen had already migrated from the testicles to the seminal vesicles in mature males only. sperm counts in s. aff. depilis resulted in an average (± se) of 1,487,778 ± 36,044 sperm cells per male. when assessing sperm morphology, we found a mean total length of 85.58 ± 1.06 µm, a mean head length of 9.50 ± 0.24 µm, a mean tail length 76.08 ± 0.98 µm and a mean head area of 23.42 ± 0.58 µm2. finally, sperm viability ranged from 52 to 87% (figs 7 and 8). we found that the best semen dilution for sperm morphology and viability assays was 100 µl hayes solution to the contents of the two seminal vesicles, in addition to the initial 20 µl hayes for emptying the vesicles. this was due to the greater ease during the identification and counting of sperm cells. for sperm counts, however, we recommend a higher dilution (10,000 x) in order to identify single isolated sperm cells. sperm viability was significantly affected by ph, being highest when hayes solution was adjusted to a ph of 8.7 (fig 6; table 2). we also found a significant effect of incubation time (table 3). no difference was found in sperm viability for the initial time intervals tested (fresh, 1, 3 and 5 hours after collection), but a marked decrease in viability was found after 24 hours (fig 7). fig 2. honeybee (apis mellifera) sperm cell marked with dapi. fig 3. live (green) and dead (red) honeybee (apis mellifera) sperm. fig 4 a. diagram summarizing the workflow of the semen extraction protocol; b. diagram summarizing the workflow of the sperm count and morphology protocol; c. diagram summarizing the workflow of the sperm viability protocol. we used the live / dead ® invitrogen sperm viability kit to assess sperm viability. employing a fluorescence microscope and a cell counter, we proceeded to count the first 400 cells found from the center of the cover slip. sperm cells were classified as green (live), red (dead) and green / red (dying) (fig 3). the workflow protocols are summarized in figs 4 a, b, and c. hm meneses, s koffler, bm freitas, vl imperatriz-fonseca, r jaffé assessing sperm quality in stingless bees520 fig 7. sperm viability of sperm from scaptotrigona aff. depilis diluted in hayes solution with different ph values. fig 8. sperm viability of sperm from scaptotrigona aff. depilis assessed on fresh semen, 1, 5 and 24 hours after collection. table 2. summary statistics for the effect of ph on sperm viability. table 3. summary statistics for the generalized linear mixed effect model for sperm viability in different intervals between semen collection and viability analysis. discussion semen extraction in stingless bees is different than in honeybees. first, stingless bees lack the enormous mucus glands found in honeybees, so the dissection of the reproductive tract is more delicate. second, the manual collection of the ejaculate from the end phallus is not possible, because the manual eversion of the endophallus fails to stimulate ejaculation. finally, dissection is more difficult in smaller species, requiring the use of specialized equipment. different protocols are available for counting sperm cells. counting cells using a hemocytometer was described for a. mellifera sperm (cobey et al., 2013; human et al., 2013), in which a known volume containing unstained cells is analyzed. a drawback of this method is that the counting procedure must be done immediately after dissection. in our protocol, microscope slides are prepared with samples of sperm in a known dilution, and only three samples are counted to estimate the total number of sperm cells. since samples are dried, the slides can be stored for later analyses, which is an advantage when a high number of males need to be analyzed. other studies implemented similar techniques successfully (baer et al., 2006; stürup et al., 2011). our results show that sperm counts, sperm morphology and sperm viability can be effectively assessed fig 6. reproductive system of immature male of scaptotrigona aff. depilis. fig 5. reproductive system of mature male of scaptotrigona aff. depilis. response predictor estimate se p-value sperm viability fresh semen 0.15 0.18 0.41 1h after collection 0.08 0.26 0.76 5h after collection 0.12 0.26 0.64 24h after collection -0.76 0.26 0.003 sociobiology 61(4): 517-522 (december, 2014) 521 in a stingless bee. for sperm morphology and viability, the best semen dilution was found to be 120 µl hayes solution to the total semen content of a male. for sperm counts, the best dilution factor was 10,000 x. while the ph yielding the highest sperm viability was at 8.7, we found that the best time to assess viability was before 24 h of semen collection. given the high susceptibility of sperm to manipulation, desiccation, ph and temperature extremes, we recommend that great care must be taken to ensure sperm are analyzed shortly after collection and under the best possible conditions. based on these results we present standard protocols, hoping they will be useful to future researchers assessing sperm quality in other stingless bee species. such studies could contribute advance basic knowledge on the reproductive biology of stingless bees, and facilitate their commercial use. acknowledgments we thank paulo cesar fernandes for technical assistance in the lab, prof. dr. ana castrucci for kindly providing access to her fluorescence microscope and fapesp for funding (rj, process 2012/13200-5). references baer, b., armitage, s.a.o. & boomsma. j.j. 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(1980). reproductive aspect and evolution of social behavior in bees (hymenoptera, apoidea). braz. j. genet. 3:139-152. gençer, h.v., kahya, y. & woyke j. (2014). why the viability of spermatozoa diminishes in the honeybee (apis mellifera) within short time during natural mating and preparation for instrumental insemination. apidologie 45: 757-770. doi:10.1007/s13592-014-0295-0. human, h., brodschneider, r., dietemann, v., dively, g., ellis, j., forsgren, e., fries, i., hatjina, f. hu, f., jaffé, r., jensen, a.b., köhler, a., magyar, j.p., özkýrým, a., pirk, c.w.w., rose, r., strauss, u., tanner, g., tarpy, d.r., van der steen, j.j.m., vaudo, a., vejsnæs, f., wilde, j., williams, g.r. & zheng, h.q. (2013). miscellaneous standard methods for apis mellifera research. j. apicult. res. 52. lino-neto, j., araújo, v.a. & dolder, h. (2008). inviability of the spermatids with little cytoplasm in bees (hymenoptera; apidae). sociobiology 51: 163-172. paulenz, h., soderquist, l., pérez-pé, r. & berg, k.a. (2002). effect of different extenders and storage temperatures on sperm viability of liquid ram semen. theriognology 57: 823-836. paynter, e., baer-imhoof, b., linden, m., lee-pullen,t., heel, k., rigby, p. & baer, b. (2014). flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee apis mellifera. cytometry part a 85: 463-472. paxton, r.j. (2000). genetic structure of colonies and a male aggregation in the stingless bee scaptotrigona postica, as revealed by microsatellite analysis. insect. soc. 47: 63-69 pech-may, f.g., medina-medina, l., may-itzá, w.j., paxton, r.j. & quezada-euán, j.j.g. (2012). colony pollen reserves affect body size, sperm production and sexual development in males of the stingless bee melipona beecheii. insect. soc. 59: 417-424. hm meneses, s koffler, bm freitas, vl imperatriz-fonseca, r jaffé assessing sperm quality in stingless bees522 simmons, l.w. (2001). sperm competition and its evolutionary consequences in the insects. priceton: princeton univ. press. simmons l.w. (2005) sperm viability matters in insect sperm competition. curr. biol. 15: 271-275. doi: 10.1016/j. cub.2005.01.032 stürup, m., den boer, s., nash, d., boomsma, j. & baer, b. (2011). variation in male body size and reproductive allocation in the leafcutter ant atta colombica: estimating variance components and possible trade-offs. insectes soc. 58: 47-55. thomas, m.l. & simmons, l.w. (2007). male crickets adjust the viability of their sperm in response to female mating status. am. nat. 170: 190-195. doi:10.1086/519404 this article has online supplementary at: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/628/0 doi: 10.13102/sociobiology.v61i4..s760 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.766sociobiology 62(4): 593-597 (december, 2015) the role of salivary enzymes in the detection of polysaccharides in the termite reticulitermes flavipes kollar (isoptera: rhinotermitidae) introduction termites are selective in which types of wood they feed on (smythe & carter, 1970; waller, 1988; waller et al., 1990; morales-ramos & rojas, 2001, 2003; ngee et al., 2004; aihetasham & iqbal, 2012). the mechanisms termites use to select certain wood products over others have been of interest for ecological and economic reasons. wood species vary in chemical make-up and structure providing a number of possible cues termites could use in food selection. natural compounds such as sugars (abushama & kamal, 1977; waller & curtis, 2003; saran & rust, 2005; swoboda & miller, 2005; haifig et al., 2008; haifig et al., 2010; wallace & judd, 2010; castillo et al., 2013), amino acids (swoboda & miller, 2005; castillo et al., 2013), urea (swoboda & miller, 2005; haifig et al., 2008; castillo et al., 2013), and phosphates (botch et al., 2010) were previously found to increase termite feeding. it has also been suggested that wood fiber density also plays an important role in food selection. waller et al. (1990) found that an equal or greater biomass was consumed by coptotermes formosanus when they were exposed to a wood that was abstract this study tested the ability of the termite reticulitermes flavipes to detect the presence of large polysaccharides when salivary enzymes catabolize them. previous work has found that the saliva of reticulitermes contains cellulase and amylase but not xylanase. in several experiments, lab colonies were given choices between glucose and starch in the presence and absence of an amylase inhibitor or a choice between xylan and xylose. the results found that there was no preference between artificial food that contained equal amounts of starch and glucose but termites did prefer food containing xylose over food containing xylan. in addition, the presence of an amylase inhibitor in an artificial food source reduced the termite’s preference for food containing starch. the results confirm that the enzymes are necessary for termites to detect the presence of polysaccharides. termites were previously found to prefer denser wood and higher concentrations of cellulose. the mechanism found here provides an explanation of how wood density is determined by termites. sociobiology an international journal on social insects ja cypret, tm judd article history edited by qiuying huang, huazhong agricultural university, china received 12 february 2015 initial acceptance 13 april 2015 final acceptance 07 august 2015 keywords termites, amaylase, food selection, sugars, reticulitermes. corresponding author timothy m. judd department of biology southeast missouri state university cape girardeau, mo 63048, usa e-mail: tjudd@semo.edu compressed beyond its normal density. denser woods have a higher amount of cellulose per unit volume. judd and corbin (2009) found that reticulitermes flavipes prefers food with higher concentrations of cellulose. however, it is unlikely that termites have a receptor for large polysaccharides such as cellulose or starch. if termites could catabolize these molecules in their saliva, then denser woods would produce higher concentrations of glucose (judd & corbin, 2009) and thus digestion and absorption through salivary fluid breakdown of polysaccharides may be a potential avenue for investigating how termites assess wood density. the breakdown of polysaccharides occurs in multiple points in the termite digestive tract. most termites have cellulose and amylase in their saliva (hewitt et al., 1974; la fage & nutting, 1978; inoue et al., 1997; tokuda et al., 2002; tokuda et al., 2005) but their saliva lacks hemicellulases such as xylanase (la fage & nutting, 1978; inoue et al., 1997; brune, 2014). in the hindgut, microorganisms produce multiple enzymes to digest polysaccharides including cellulase, amylase and hemicellulases such as xylanase (brune, 2014). thus, the saliva of termites is research article termites southeast missouri state university, cape girardeau, united states ja cypret, tm judd – polysaccharide detection in termites594 more limited in its digestive ability than the hindgut. this has implications in the ability of termites to respond to various polysaccharides. the feeding responses of the genus reticulitermes in response to sugars has been well studied. both glucose and xylose were found to increase the feeding response in species of this genus (waller & curtis, 2003; saran & rust, 2005; swoboda & miller, 2005; wallace & judd, 2010). thus, termites are able to detect both sugars. inoue et al. (1997) found that r. serratus has both cellulase in their saliva whereas there is virtually no xylanase activity. park et al. (2014) found that r. serratus produces α-amylase which is commonly found in insect salivary glands (cohen, 2004) and has been found in the saliva of other termite genera (hewitt et al., 1974; la fage & nutting, 1978). thus, if reticulitermes foragers are detecting polysaccharides salivary fluid then starch should be detectable to the termites whereas xylan would not be detectable. in this study, we determined if the termite reticulitermes flavipes could directly detect large polysaccharides or if they needed to break the polysaccharides down prior to detection. we used artificial food sources to compare the termites’ consumption of artificial foods containing potato starch or glucose in the presence or absence of an amylase inhibitor. we also tested if xylan has a similar palatability as xylose to termites. if the termites are determining density of polysaccharides in the food by breaking them down, then 1) they should prefer xylose over xylan but should not prefer glucose to starch, and 2) the amylase inhibitor should reduce the termites’ feeding rate on starch enriched foods. methods and materials collections: colonies were sampled from cape girardeau county using termite traps as described by judd and fasnacht (2007). traps were placed in known location in order to collect ten colonies. in the case of experiments 1 and 4, only eight of the ten traps had enough termites to conduct the experiment. experiment 1: in this experiment we tested whether r. flavipes showed a preference for food enriched with glucose or potato starch. r. flavipes has amylase in their salvia that breaks down starch (la fage & nutting, 1978) and thus the prediction would be that there should be no feeding preference between food enriched with starch and food enriched with glucose. food: two different artificial food sources were created in a similar manner as judd and corbin (2009). both foods contained 1.5 g agar heated in 100 ml of distilled water. in the starch-enriched food, 1.0 g of potato starch was added and in the glucose-enriched food 1.0 g of glucose was added (table 1). after the potato starch or glucose was added, 7.5 g of α-cellulose was added to both foods and the mixture was stirred and poured into petri dishes. because cellulose doesn’t dissolve in water, each dish was placed on a shaker to keep the cellulose suspended until the solution solidified. trials: from each of 8 colonies, 100 worker termites were extracted and housed in 17.8 x 17.8 x 5 cm sealed plastic containers filled with approximately 200 g of topsoil. only workers were used because soldiers do not directly feed on food sources (la fage & nutting, 1978). once each container was prepared, a 3.0 g piece of each food item (starch-enriched food and glucose-enriched food) was weighed and placed on a 3.5 cm x 3.5 cm note card. the food was then placed within each container in opposite corners and each corner was labeled to indicate what food item was present. three additional containers were created without termites as controls to measure water loss in the food (judd & corbin 2009). the containers were housed in a dark area at room temperature. because the bins were sealed the moisture levels were kept constant. the control bins were housed with the other bins. each trial period lasted 14 days and each food square was weighed every two days to determine how much food was eaten by the termites. during the weighing process, termites and soil were brushed away from the food before the mass was determined. throughout the experiment the food sources remained intact and thus there was no issue of overestimation of weight-loss due to crumbs lost in the soil. experiment 2: reticulitermes lacks xylanase in their saliva (inoue et al., 1997) therefore r. flavipes should not be able to break down xylan with their saliva. reticulitermes was previously shown to prefer food enriched with xylose over unenriched food (saran & rust, 2005; wallace & judd, 2010). the prediction of this experiment was that termites should preferentially feed on xylose-enriched food because they lack the salivary enzymes to free the xylose from xylan. the methods for this experiment were identical to experiment 1 except foods were enriched with xylan or xylose, respectively, rather than potato starch or glucose. 1.0 g of xylan was added to the xylan-enriched food and 1.0 g xylose was added to the xylose-enriched food (table 1). a total of 10 colonies were used in this experiment. experiment 3: if the amylase is necessary for termites to detect the presence of starch in food items, inhibiting the experiment food 1 food 2 number of colonies 1 potato starch glucose 8 2 xylan xylose 10 3 potato starch + amylase inhibitor potato starch 10 4 glucose + amylase inhibitor glucose 8* * one colony died table 1. summary of the molecules added to the two food items (see methods for full description of food) and the number of colonies tested in each experiment. sociobiology 62(4): 593-597 (december, 2015) 595 function of amylase should limit their ability to detect starch in foods. this experiment was identical to experiment 1 except the two food items contained potato starch (instead of potato starch and glucose) and an amylase inhibitor was added to one of the food items (table 1). if amylase increases the termites’ response to starch, then the termites should preferentially feed on the starch-enriched food that lacks the inhibitor. during the food preparation, 1.0 g of potato starch was added to each food item. the food was allowed to cool to room temperature before 0.5 mg of amylase inhibitor was added to one of the food solutions. this was done to prevent the amylase inhibitor from denaturing. a total of 10 colonies were used in this experiment. experiment 4: this experiment insured that the presence of the inhibitor itself did not negatively affect the feeding behavior of the termites. to control for this effect, two identical glucose enriched foods were used except one contained the amylase inhibiter (table 1). the amylase inhibiter should not affect the termites’ ability to detect the glucose; therefore, both food sources were predicted to be equally palatable. this experiment had the same methodology as experiment 1 except 1.0 g of glucose was added to both food types. the amylase inhibitor (0.5 mg) was added to one of the foods in the same manner as in experiment 3. a total of 8 colonies were used in this experiment; however, one colony died part way through the experiment and was eliminated from the analysis. data analysis the data were analyzed in the same manner as judd and corbin (2009). in each experiment, the final mass of the food was subtracted from the average final mass of the same type of food from three control bins. this controlled for change in mass loss due to water loss. the difference between the change in mass of the food and the average change in mass for the controls was the amount eaten by the termites. negative numbers were converted to zeros, signifying that no significant amount of food was consumed by the termites. this only happened in three cases in experiment 1 and three case in experiment 2, and in all cases the result was within 0.025 g of 0.0 g. for each experiment, the amount of each food type eaten by the termites (adjusted weight loss) was compared using the wilcoxon signed-rank test. results experiment 1: there was no significant difference between the adjusted weight loss of glucose or starch enriched foods (t=13, p=0.2734; fig. 1). thus, the termites showed no preference for glucose or starch enriched food sources. experiment 2: the adjusted weight loss for the xylose enriched food was significantly higher than the adjusted weight loss of the xylan enriched food (t=6, p=0.027; fig 1). thus, the termites preferred the xylose-enriched food source. experiment 3: there was significantly higher adjusted weight loss in the food that lacked the amylase inhibitor compared to the adjusted weight loss in the food with the inhibitor (t=6 p=0.014; fig. 1). these results suggest that the starch-enriched food item was preferred over the food enriched with starch and the amylase inhibitor. experiment 4: there was no significant difference in the adjusted weight loss between the food enriched with glucose and food enriched with glucose that included the amylase inhibitor (t=11.5, p=0.34; fig 1). this result confirms that the amylase inhibitor did not affect the feeding behavior of the termites. thus, the amylase inhibitor did affect the termites’ ability to detect starch but not glucose. discussion in this study we demonstrated that termites can detect larger polysaccharides only if the enzyme that hydrolyzes the specific polysaccharide is found in their saliva. in the experiments of this study, we provided termites food in which we controlled for the levels of a polysaccharide and its corresponding monosaccharide. based on the fact that reticulitermes has amylase in its saliva but not xylanase, we predicted that the termites should be able to detect the presence of starch but not xylan. as expected, there was no significant preference for food enriched with glucose compared to food enriched with starch but there was a significant preference for food enriched with xylose over food enriched with xylan (experiments 1 and 2). we also predicted that the addition of an amylase inhibitor would lower the phagostimulatory response of r. flavipes to starch but not glucose. the results of experiments 3 and 4 support this prediction. thus, the results of this study suggest that termites are able to detect starch because the amylase in their saliva hydrolyzes the starch into its monomer components. fig 1. median and quartiles of the percent weight loss of artificial food over a 14 day period due to termite feeding (adjusted for natural water loss from each food square) for all four experiments in this study. the four experiments were analyzed independently. an asterisk indicates a significant difference between percent weight loss (p <0.03) of the two food types in the experiment. ja cypret, tm judd – polysaccharide detection in termites596 enzymes that digest polysaccharides are produced in two major areas of the termite gut, the salivary glands and the hindgut (brune, 2014). the latter has a larger suit of enzymes that attach large polysaccharides due to the presence of symbiotic bacteria (könig et al., 2013; li et al., 2013; ni & tokuda, 2013; raychoudhury et al., 2013; brune, 2014). the enzymes produced in the salivary glands are generated by the termites themselves (brune, 2014). the role of the enzymes of the salivary glands is twofold: 1) they initiate the digestion of cellulose and starch from the food the termites consume (saadeddin, 2014); free up smaller, detectable molecules (demonstrated herein). reticulitermes saliva contains both amylases and cellulases (inoue et al., 1997) and thus based on our results, termites should be able to detect the presence of cellulose. indeed r. flavipes does prefer foods with higher concentrations of cellulose (judd & corbin, 2009). interestingly, the saliva of the little soldier of macrotermes subhyalinus has an endo-beta-d-glycosidase that is able to act on cellulose and xylan (bedel et al., 2012). it would be interesting to see how this termite responds to xylan. assessing food quality is one of the many critical roles of taste. for social insect colonies, the ability to assess the quality of food allows for the colony as a whole to optimize the distribution of its foraging force. this phenomenon was previously well documented in social hymenoptera such as honey bees and ants (sudd & sudd, 1985; stein et al., 1990; seeley, 1995; judd, 2006; cook et al., 2010, 2011). subterranean termites burrow to and into food sources and thus assessing the quality of a potential food source is critical to colony success. this is especially true if one considers the energetic investment of burrowing in soil and wood (lee et al., 2007; bardunias & su, 2009). indeed, many studies have shown that termites are selective in the types of wood they feed (morales-ramos & rojas, 2001; ngee et al., 2004; aihetasham & iqbal, 2012). the role of small molecules in food palatability has been well studied and much like social hymenoptera, termites are sensitive to different concentrations of sugars and regulate their feeding accordingly (waller & curtis, 2003; saran & rust, 2005; swoboda & miller, 2005). it was proposed by judd and corbin (2009) that the preference of denser wood and higher concentrations of cellulose was a result of termite enzymes freeing up detectable sugars from polysaccharides. higher concentrations of cellulose would produce higher levels of glucose per unit area when broken down by cellulase. thus, the termites are detecting higher concentrations of glucose in denser wood (judd & corbin, 2009). although cellulose was not directly tested in this study, the mechanism of polysaccharide detection found in this study confirms this mechanism is the likely explanation of how the concentration of cellulose is determined by the termites. the role of saliva in the regulation and enhancement of taste has been well studied in vertebrates (pedersen et al., 2002). this study demonstrates a case for a similar role for insect saliva. acknowledgements we thank dustin siegel and two anonymous reviewers for their comments on the manuscript. this project was funded by southeast missouri state university. references abushama ft, kamal ma (1977) the role of sugars in the food-selection of termite microtermes traegardhi (sjostedt). zeitschrift für angewandte entomologie, 84: 250-255. aihetasham a, iqbal s (2012) feeding preferences of microcerotermes championi (snyder) for different wooden blocks dried at different temperatures under forced and choice feeding conditions in laboratory and field. pakistan journal of zoology, 44: 1137-1144. bardunias p, su n-y 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doi: 10.13102/sociobiology.v61i4.560-565sociobiology 61(4): 560-565 (december 2014) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 chemical composition, antinociceptive and free radical-scavenging activities of geopropolis from melipona subnitida ducke (hymenoptera: apidae: meliponini) introduction many stingless bee species (meliponini) store in their nests a large amount of geopropolis, a mixture of wax, plant resins, pollen grains and mud (nogueira-neto, 1997). the bees use this material for sealing small crevices in their nest cavities, in order to avoid the entry of air, and for defense against pathogenic microorganisms (simone-finstrom & spivak, 2010). however, despite its popular use in folk medicine, very little is known about its chemical composition and biological activity. recently, studies investigating geopropolis from native bees have indicated a potential for bioactive compounds and biological activities. velikova et al. (2000) analyzed 21 samples of brazilian geopropolis from 12 different species of stingless bees and observed the presence of compounds such as diand triterpenes and gallic acid. the same samples showed activity against staphylococcus aureus rosenbach and cytotoxic activity. abstract like many stingless bee species, melipona subnitida ducke uses geopropolis (a mixture of wax, plant resins, pollen grains and mud) for sealing small crevices in their nest cavities, in order to avoid the entry of air, and for defense against pathogenic microorganisms. the aim of this study was to evaluate the antinociceptive and free radical-scavenging activities of ethanolic extracts of six geopropolis samples from m. subnitida and the phenolic fractions obtained by c18-spe extraction. the in vivo antinociceptive activity was analyzed on abdominal constriction induced by acetic acid in mice and in vitro free radical-scavenging activities by dpph and abts assays. additionally we analyzed the chemical composition of the phenolic fractions by hplc-dad. the six samples of geopropolis showed variations in the total phenolic content over the period, but not in the chemical profile observed by hplc-dad. geopropolis is a rich source of bioactive compounds as phenolics 6-o-p-coumaroyl-d-galactopyranose, 6-o-cinnamoyl-1-o-pcoumaroyl-β-d-glucopyranose, 7-o-methyl naringenin, 7-o-methyl aromadendrin, 7,4’-di-o-methyl aromadendrin, 4’-o-methyl kaempferol, 3-o-methyl quercetin, 5-o-methyl aromadendrin and 5-o-methyl kaempferol with potential antioxidant and antinociceptive activities. the antioxidant activity is related to the total phenolic content. sociobiology an international journal on social insects sa souza1, tlmf dias2, tmg silva1, ra falcão1, ms alexandre-moreira2, ems silva3, ca camara1, tms silva1 article history edited by denise araujo alves, esalq-usp, brazil received 23 september 2014 initial acceptance 01 november 2014 final acceptance 08 december 2014 keywords stingless bees, phenolic, antioxidant. corresponding author tania maria sarmento silva biofito depto. de ciências moleculares universidade federal rural de pernambuco rua dom manoel de medeiros, s/n, dois irmãos, recife, 52171-900, pe, brazil e-mail: sarmentosilva@gmail.com samples of melipona fasciculata smith geopropolis showed activity against streptococcus mutans clarke (liberio et al., 2011) and antioxidant capacity (dutra et al., 2014) and eleven compounds were tentatively identified as belonging to the classes of phenolic acids and hydrolysable tannins (gallotannins and ellagitannins). these compounds were responsible for the antioxidant activity and high phenolic content of the geopropolis produced by m. fasciculata (dutra et al., 2014). geopropolis produced by melipona scutellaris latreille has been shown to exhibit antimicrobial and antioxidant activities and has anti-inflammatory, antinociceptive and antiproliferative properties (franchin et al., 2012; cunha et al., 2013), and benzophenones have been identified as the major compounds (cunha et al., 2013). previous investigations in our laboratory have found that the geopropolis from melipona subnitida ducke has antioxidant activity. this study led to the isolation and characterization of two phenylpropanoids, one of which was a new compound, and research article bees 1universidade federal rural de pernambuco, recife, pe, brazil 2universidade federal de alagoas, maceió, al, brazil 3universidade federal do vale de são francisco, petrolina, pe, brazil sociobiology 61(4): 560-565 (december 2014) 561 seven flavonoids (souza et al., 2013). these findings suggested that m. subnitida geopropolis is highly bioactive and deserved further study to identify other potential biological activities. thus, the aim of this study was to evaluate the antinociceptive and free radical-scavenging activities of ethanolic extracts of six geopropolis samples from m. subnitida and its phenolic fractions. additionally, we analyzed the chemical composition of the phenolic fractions obtained by c18-spe extraction by hplc-dad. materials and methods geopropolis samples and fractionation for this study, six samples of geopropolis from four m. subnitida nests were collected in march 2010 (1), july 2011 (2), january 2012 (3), april 2012 (4), june 2012 (5) and july 2012 (6) at sítio riacho vieirópolis (a semi-arid region), paraíba state, brazil. each sample (200 g) was extracted with 100 ml of ethanol (etoh) in an ultrasonic water bath. the combined ethanolic extracts were completely evaporated under reduced pressure to a brown residue (2.7 g to 18.4 g). the etoh extract (100 mg) was dissolved in 2 ml of distilled water, and the solution was adjusted to ph 2.0 by adding concentrated hcl while stirring with a magnetic stirrer at room temperature for 10 min. a c18 cartridge (spe strata 1 g, phenomenex) was sequentially conditioned with 3 ml of meoh and 6 ml of distilled deionized water without allowing the cartridge to dry. the samples of geopropolis were passed through the cartridge and rinsed with 6 ml of water and the phenolic compounds were eluted with 8 ml of hplc-grade methanol. the eluate was dried under reduced pressure in a rotatory evaporator at 40 °c to yield 32 to 57 mg of phenolic fraction. these fractions were dissolved in methanol, filtered through a 0.45-µm nylon syringe filter (whatman) and injected into the hplc system. the phenolic samples were reconstituted with tween® 80 and carboxycellulose and also to evaluation for their antinociceptive and antioxidant activities. reagents and standards all reagents used were of analytical grade. folin-ciocalteu’s phenol reagent, dpph (1,1-diphenyl-2-picryl hydrazyl), potassium persulfate and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid) were supplied by acros organics (belgium). abts (2,2 azinobis 3-ethylbenzothiazoline-6-sulfonic acid) was purchased from fluka chemie gmbh (switzerland). ascorbic acid was from vetec (brazil). formic acid (merck) and methanol (tedia) were of analytical grade. dipirone, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium (mtt), gallic acid, carboxymethylcellulosecmc (sigma); tween® 80 (sigma-aldrich, usa) and dimethyl sulfoxide (dmso) were purchased from sigma-aldrich (usa). the compounds 6-o-p-coumaroyl-d-galactopyranose (1), 6-o-cinnamoyl1-o-p-coumaroyl-β-d-glucopyranose (2), 7-o-methyl naringenin (3), 7-o-methyl aromadendrin (4), 7,4’-di-o-methyl aromadendrin (5), 4’-o-methyl kaempferol (6), 3-o-methyl quercetin (7), 5-o-methyl aromadendrin (8) and 5-o-methyl kaempferol (9) had been previously isolated and identified from m. subnitida geopropolis (souza et al., 2013). hplc analysis of the phenolic all chromatographic analyses were performed using a shimadzu prominence lc-20at equipped with a spd-m20a diode array detector (shimadzu corp. kyoto, japan). the samples (20 µl) were injected into a rheodyne 7125i injector with a 20 μl loop. the column heater was set at 40 °c. the chromatographic separation was performed with a luna phenomenex c-18 column (250 mm x 4.6 mm x 5 μm). the compounds were separated using a mobile phase consisting of 1% aqueous formic acid (a) and methanol (b) at a flow rate of 1 ml/min. the mobile phase was delivered using the following solvent gradient: 0-10 min, 2025% b; 10-20 min, 25-60% b; 20-30 min, 60-70% b; 30-35 min, 70-100% b. the injection volume was 20 µl. chromatograms were recorded at 290 nm and 340 nm. the identification of the compounds was based on their retention times and uv spectra with authentic markers. animals male and female swiss mice weighing 20-25 g were used and given access to water and food ad libitum. we used six mice per experimental group. the animals were housed at a temperature of 25-28°c with a 12 h light/12 h dark cycle. the procedures described were reviewed and approved by the local animal ethics committee (ceua ufal process number 23065.004873/2011-01). determination of the total phenolic content the total phenolic content of the samples was determined with the folin ciocalteu reagent, according to the method of slinkard and singleton (1977), modified by using gallic acid as a standard phenolic compound. etoh extracts (100 µl) and phenolic fractions (1 mg/ml) were transferred to an eppendorf tube with 1 ml. folin ciocalteu reagent (20 µl), 820 µl of distilled water were added and the contents of the flask were mixed thoroughly. after 1 min, 60 µl of sodium carbonate (15%) was added and then the mixture was allowed to stand for 2 h. the absorbance was measured at 760 nm with an automatic biochrom asys uvm 340 microplate reader (cambridge, uk). the amount of total phenolic compounds was determined in micrograms of gallic acid equivalents using the equation obtained from the standard gallic acid graph. dpph● radical scavenging assay the free radical-scavenging activity was determined using the dpph assay, as described previously (silva et al., 2006) with sa souza, tlmf dias, tmg silva, ra falcão, ms alexandre-moreira, ems silva, ca camara, tms silvageopropolis from melipona subnitida562 modifications. the antiradical activity was evaluated using a dilution series to obtain five concentrations (1.0 to 80.0 µg/ml). this process involved mixing the dpph solution (23.6 µg/ml in etoh) with the appropriate etoh extracts and phenolic fractions followed by homogenization. after 30 min, the remaining dpph radicals were quantified by measuring the absorption at 517 nm with an automatic biochrom asys uvm 340 microplate reader (cambridge, uk).the percentage of inhibition was given by the formula: percent inhibition (%) = [(a0 a1)/a0] x 100, where a0 was the absorbance of the control solution and a1 was the absorbance in the presence of the sample and standards. abts●+ radical cation decolorization assay the radical cation decolorization assay was based on the method described by re et al. (1999) with modifications. abts was dissolved in water to yield a final concentration of 7 mm. the abts radical cation (abts●+) was produced by reacting the abts stock solution with 2.45 mm of potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 h before use. the abts●+ solution was diluted to give an absorbance of 0.70 ± 0.05 at 734 nm with ethanol before use. then, appropriate amounts of the abts●+ solution were added into 0.5 ml of the sample solutions in ethanol at five concentrations (140 µg/ml). after 10 min, the percentage inhibition of the absorbance at 734 nm was calculated for each concentration with an automatic biochrom asys uvm 340 microplate reader (cambridge, uk), relative to the blank absorbance (etoh). the capability to scavenge the abts●+ radical was calculated using the following equation: abts●+ scavenging effect (%) = [(a0-a1/a0) x100], where a0 was the initial concentration of the abts●+ and a1 was absorbance of the remaining concentration of abts●+ in the presence of sample. evaluation of activity ethanol extracts and fractions of geopropolis on abdominal constriction responses caused by acetic acid abdominal constrictions (writhes) were induced by the i.p. injection of acetic acid (1.2%) and carried out according to the procedure described previously (koster et al., 1959; collier et al., 1968; fontenele et al., 1996). mice were treated with etoh extracts and phenolic fractions (100 mg/ kg, i.p.) or dypirone (10 mg/kg, i.p.) 40 minutes before initiating nociceptive stimulus. dypirone was used as a positive control and the vehicle (cmc/tween® 80) (10 ml/kg, i.p.) was used as the negative control (the animals without treatment). the total numbers of writhes, which consisted of constriction of the flank muscles associated with inward movements of the hind limb or with whole body stretching, were counted cumulatively over a 20 min period. the antinociceptive activity was determined as the difference in number of writhes between the control group and the treated group. statistical analysis all analyses were performed in triplicate. the results were expressed as the standard error of the mean (mean ± s.e.m.) and were analyzed using graphpad prism 5.0 program (demo). comparisons between groups were made using analyses of variance (anova) followed by tukey’s test. significance was indicated by a p value ≤0.05. pearson’s correlation test was used to evaluate the correlations. results and discussion the aim of this study was to evaluate the antinociceptive activity of six samples of m. subnitida geopropolis collected over three years. etoh extracts and the phenolic fractions were evaluated in a model of nociception, and the free radicalscavenging activity was evaluated using the dpph and abts assays. the total phenolic content was determined by the folin ciocalteu reagent. in addition, chromatographic profiles were analyzed by hplc-dad, and the principal phenolics present in the geopropolis samples were identified. this study was conducted by an extraction of phenolics using a c18-spe cartridge as a simpler, less expensive and faster technique compared with the use of liquid-liquid solvent extraction. this technique has been used to determine flavonoid markers in honey (hadjmohammadi et al., 2009). interestingly, there is a correlation (r=0.85, p<0.05) between the total phenolic content present in the ethanolic extract and the amount of phenolics extracted by c18-spe. these samples showed a total phenolic content two times higher when compared with the etoac fraction (which is rich in phenolic compounds) obtained by the liquid-liquid extraction of a sample of m. scutellaris geopropolis collected in january 2010 (souza et al., 2013). the phenolic profiles of samples 1-6 also were analyzed by hplc-dad. the characterization of these compounds is important because they are associated with a variety of health benefits. the comparative analysis of the chromatograms (fig 1) shows a similar profile between the six samples obtained by the spe and the etoac fraction (souza et al., 2013) of geopropolis, again demonstrating that spe extraction is effective for extraction of phenolics. all phenols (phenylpropanoids and flavonoids) previously identified from etoac fraction (souza et al., 2013) were verified in the samples of this study; the 6-o-p-coumaroyl-d-galactopyranose compounds (1), 6-o-cinnamoyl-1-o-p-coumaroyl-β-d-glucopyranose (2), 7-o-methyl naringenin (3), 7-o-methyl aromadendrin (4), 7,4’-di-o-methyl aromadendrin (5), 4’-o-methyl kaempferol (6), 3-o-methyl quercetin (7), 5-o-methyl aromadendrin (8) and 5-o-methyl kaempferol (9) were identified (fig 1). further studies are necessary to quantify the compounds identified. the following plant species occur in the region and are resin-producing sources possibly collected by the bees for propolis production: myracrodruon urundeuva allemão (anacardiaceae), handroanthus impetiginosus (mart. & dc.) mattos (bignoniaceae), jatropha mollissima (pohl) baill. sociobiology 61(4): 560-565 (december 2014) 563 fig 1. chromatograms (hplc-dad 320 nm) of the phenolic fractions of melipona subnitida geopropolis (1-6) and of etoac fraction geopropolis collected in january 2010. compounds identified were: 6-o-p-coumaroyl-dgalactopyranose (1), 6-o-cinnamoyl-1-o-p-coumaroyl-β-d-glucopyranose (2), 7-o-methyl naringenin (3), 7-o-methyl aromadendrin (4), 7,4’-dio-methyl aromadendrin (5), 4’-o-methyl kaempferol (6), 3-o-methyl quercetin (7), 5-o-methyl aromadendrin (8) and 5-o-methyl kaempferol (9) (euphorbiaceae) and anadenanthera colubrina (vell.) brenan (fabaceae) (maia-silva et al., 2012). other studies to verify the presence of pollen in m. subnitida geopropolis are required, because pollen analysis in addition to chemical analysis is a method used to characterize regionally different propolis samples. pollen types that occur in low frequency in propolis samples can be regarded as an indicator of the botanical species supplying the resin (matos et al., 2014). it is a good tool for defining the phytogeographical origin of resins and quality of the propolis (barth et al., 2003). barth et al. (1999) and barth and luz (2003) showed that there is a fairly equal number of pollen grains between the samples of propolis from apis and geopropolis produced by meliponini, but a wider richness of pollen types is characteristic of geopropolis. in this regard, the meliponini visits more plant species than the apis bees. nevertheless, the occurrence of dominant and accessory pollen grains is more frequent in propolis samples, which reflects a higher generalization of honeybees. evaluating abdominal constrictions induced by acetic acid was initially used to evaluate the antinociceptive activity of the etoh extracts (100 mg/kg) of geopropolis and their phenolics fractions (100 mg/kg). the results showed in fig. 2a and table 1 demonstrate that the etoh extract (100 mg/kg), produced inhibition of abdominal constrictions induced by acetic acid in mice (p<0.05), with inhibitions of 96.9% (sample 5) to 100% (sample 1). phenolic fractions at the same concentration also inhibited the number of writhes (p<0.05) from 71.4% (sample 3) to 93.5% (sample 5), fig 2b and table 1. the inhibitory properties of the etoh extracts and the phenolic fractions versus the abdominal constrictions induced by acetic acid in mice is first suggestion of the antinociceptive potential of these materials. the acetic acid induced constrictions test is a typical model for inflammatory pain that has long been used as a screening tool for the assessment of analgesic properties. the fact that the etoh extracts showed slightly greater antinociceptive activities than the phenolic fractions suggests that geopropolis contains other compounds responsible for this activity and should be chemically investigated. the phenolic fraction is probably principally responsible for this activity. no reports on antinociceptive activity have been found in the literature for the identified constituents of m. subnitida geopropolis. fig 2. effects of injections of ethanolic extract of geopropolis and phenolic fractions on abdominal constriction induced by acetic acid in mice. control groups included the mice treated with only vehicle (negative control) or dypirone (positive control) 40 min before initiating nociceptive stimulus. data are expressed as the mean ± sem, n=6. symbols indicate significant differences (*p<0.05 and ***p<0.001, one way anova followed by dunnett’s test) compared to the control group. table 1. effects of injections of ethanolic extracts and phenolic fractions of geopropolis on abdominal constrictions induced by acetic acid in mice. a data are expressed as the mean ± sem, n=6. b symbols indicate significant difference ((*p<0.05 and ***p<0.001, one way anova followed by dunnett’s test) compared to control group. control was treated with vehicle (cmc/tween® 80) (10 ml/kg, i.p.), dypirone 100 mg/kg, i.p. 40 minutes before initiating nociceptive stimulus. samples numbers of writhers etoh extracts phenolic fractions media ± s.e.m.a % inhibition b media ± s.e.m.a % de inhibition control 38.4 ± 2.7 dipirone 18.8 ± 2.7 29.9 * 1 0.0 ± 0.0 100.0 *** 4.5 ± 1.0 85.4 *** 2 0.2 ± 0.2 99.4 *** 8.2 ± 1.6 73.5 *** 3 0.2 ± 0.2 99.4 *** 8.8 ± 2.5 71.4 *** 4 0.7 ± 0.3 97.5 *** 3.0 ± 1.9 90.3 *** 5 0.8 ± 0.6 96.9 *** 2.0 ± 0.7 93.5 *** 6 0.2 ± 0.2 99.4 *** 5.2 ± 3.1 83.2 *** sa souza, tlmf dias, tmg silva, ra falcão, ms alexandre-moreira, ems silva, ca camara, tms silvageopropolis from melipona subnitida564 conclusion the present results from six samples of m. subnitida geopropolis collected over three years showed that there is a variation in the total phenolic content over the years but not in the chemical profile. geopropolis is a rich source of bioactive compounds with potential antioxidant and antinociceptive activities. the antioxidant activity is related to the total phenolic content. the spe extraction was effective for the extraction of phenolic from m. subnitida geopropolis. acknowledgments this work was financially supported by grants from cnpq (cnpq-ppbio 503285/2009-9), facepe (grant no. pronem apq-1232.1.06/10) and capes. references almeida-da-silva, i.a., silva, t.m. s., camara, c.a., queiroz, n., magnani, m., novais, j.s., soledade, l.e.b., lima, e.o., de souza, a.l. & de souza, a.g. (2013). phenolic profile, antioxidant activity and palynological analysis of stingless bee honey from amazonas, northern brazil. food chem. 141: 3552-3558. doi: 10.1016/j.foodchem.2013.06.072 barth, o.m. (1998). pollen analysis of brazilian propolis, grana 37: 97-101. doi: 10.1080/00173130310012512. barth, o.m. & luz, c.f.p. (2003). palynological analysis of brazilian geopropolis samples, grana 42: 121-127. doi: 10.1080/00173130310012512 collier, h.o.j., dinneen, j.c., johnson, c.a. & schneider, c. (1968). the abdominal constriction response and its suppression by analgesic drugs in the mouse. brit. j. pharmacol. 32: 295table 2. total phenolic and free radical-scavenging activity of m. subnitida geopropolis samples. a mean value ± standard deviation: n=3, concentration of antioxidant required to reduce the original amount of the radicals by 50%. table 3. pearson correlation coefficients between the total phenolic content and the antiradical activity dpph and abts. geopropolis sample total phenolic content (mg gae/g ± sd) abtsa ce50 (µg/ml) dppha ce50 (µg/ml) etoh extract phenolic fraction etoh extract phenolic fraction etoh extract phenolic fraction 1 97.6 ± 5.7 273.9 ± 6.8 15.2± 0.8 4.3 ± 0.1 39.2 ± 0.9 8.4 ± 0.1 2 92.6 ± 8.1 204.5 ± 7.4 13.4 ± 0.7 8.9 ± 0.7 31.7 ± 0.5 17.7 ± 0.2 3 172.6 ± 4.5 305.3 ± 5.0 7.7 ± 0.1 3.4 ± 0.1 15.9 ± 0.4 7.6 ± 0.1 4 150.7 ± 5.1 282.4 ± 1.5 10.3 ± 0.2 4.5 ± 0.2 16.1 ± 0.4 9.8 ± 0.1 5 201.6 ± 4.2 322.4 ± 6.4 6.9 ± 0.3 3.1 ± 0.1 13.3 ± 0.4 7.5 ± 0.1 6 139.3 ± 6.9 261.3 ± 5.8 15.2 ± 0.5 6.0± 0.1 28.9 ± 1.2 10.5 ± 0.1 ascorbic acid 2.8 ± 0.0 2.8 ± 0.4 trolox 3.21 ± 0.0 3.21 ± 0.0 dpph abts etoh extracts phenolic fractions etoh extracts phenolic fractions total phenolic contentetoh extracts -0.90 -0.85 dpphetoh extracts 0.91 abtsetoh extracts 0.91 total phenolic content phenolic fractions -0.94 -0.97 dpph phenolic fractions 0.97 abts phenolic fractions 0.97 the free radical-scavenging activities of the etoh extracts and phenolic fractions from geopropolis are shown in table 2. the ce50 ranged from to 6.99-15.2 µg/ml (abts) and 13.3-39.2 µg/ml (dpph) for the etoh extracts and 3.2-8.9 µg/ml (abts) and 7.5-17.1 µg/ml (dpph) for the phenolic fractions. the lower ec50 value indicates a higher antioxidant activity. the etoh extracts and phenolic fractions showed a correlation between free radicalscavenging activity and the total phenolic content. the phenolic content ranged from 92.6-201.6 to etoh extract and 205.5 to 305.3 to phenolic fractions. a correlation between dpph-abts results for the etoh extracts (r=0.91) and the dpph-abts results for the phenolic fraction (r=0.97) was observed (table 3). these results suggest that total phenols, particularly the phenylpropanoids and flavonoids identified in m. subnitida geopropolis were responsible for the free radicalscavenging activity. geopropolis obtained from the other stingless bees showed important antioxidant activities (silva et al, 2013; dutra et al, 2014). in early studies other m. subnitida products such as the pollen (silva et al, 2006) and honey showed (silva et al, 2013) free radical-scavenging activity. the pollen collected by the stingless bees melipona rufiventris lepeletier (silva et al, 2009) and honey produced by melipona seminigra merrillae cockerell (almeida da silva et al., 2013) also were reported as having important antioxidant activities. sociobiology 61(4): 560-565 (december 2014) 565 310. retrieved from: http://www.ncbi.nlm.nih.gov/pmc/ articles/pmc1570212/ cunha, m.g., franchin, m., galvão, l.c.c., ruiz, a.l.t.g., carvalho, j.e., ikegaki, m., alencar, s.m., koo, h. & rosalen, p.l. 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(2013). phenolic constituents and antioxidant activity of geopropolis from two species of amazonian stingless bees. quim. nova. 36:628-633. doi: 10.1590/s0100-40422013000500003 silva, t.m.s., camara, c.a., lins, a.c.s., agra, f.m., silva, e.m.s., reis, i.t. & freitas b.m. (2009). chemical composition, botanical evaluation and screening of radical scavenging activity of collected pollen by the stingless bees melipona rufiventris (uruçu amarela). an. acad. bras. cienc. 81: 173-178. doi: 10.1590/s0001-37652009000200003 silva, t.m.s., camara, c.a., lins, a.c.s., barbosa-filho, j.m., silva, e.m.s., freitas, b.m. & santos, f.a.r. (2006). chemical composition and free radical scavenging activity of pollen loads from stingless bee melipona subnitida ducke. j. food composit. anal. 19:507-511. doi: 10.1016/j. jfca.2005.12.011 silva, t.m.s., santos, f.p., rodrigues, a.e., silva, e.m.s., silva, g.s., novais, j.s., santos, francisco, a.r. & camara, c.a. 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(2000). antibacterial ent-kaurene from brazilian propolis of native stingless bees. fitoterapia 71: 693-696. doi: 10.1016/s0367-326x(00)00213-6 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.109-115sociobiology 62(1): 109-115 (march, 2015) the cuticular hydrocarbons profiles in the colonial recognition of the neotropical eusocial wasp, mischocyttarus cassununga (hymenoptera: vespidae) introduction cuticular hydrocarbons (chcs) are present on the surface of all insects and play an important role in the life of insects. these cuticular lipids have evolved for prevention of desiccation, as a barrier to microorganisms and as a important signals in the recognition system of social insects (hadley, 1981; lockey, 1988; singer, 1998; howard & blomquist, 2005). each individual of a colony presents a blend of compounds on its cuticle that may carry information regarding its sex, age, caste, group task as well as colony (howard & blomquist, 2005; provost et al., 2008). these chemical messengers are determined by genetic (ratnieks, 1991; page et al., 1991; arnold et al., 1996) and environmental components (ratnieks, 1991; gamboa, 1996). based on differences of cuticular compounds, individuals from the same colony are able to detect specific chemical abstract cuticular hydrocarbons are chemical messengers with fundamental role in information transfer on the nestmate recognition, physical or behavioral caste, age, task specialization and reproductive status among individuals of colony. in basal (primitively) eusocial wasps, the role of cuticular hydrocarbons in the colonial recognition has been extensively studied in genus polistes (polistinae) and stenogastrinae. although the genus mischocyttarus (polistinae) is a large group of neotropical eusocial wasps and its cuticular hydrocarbons were only investigated in a few species. this study aimed to verify whether the cuticular hydrocarbons can discriminate the intercolonial identity in mischocyttarus cassununga. our results showed that the cuticular hydrocarbons potentially can play a role in intercolony recognition. sociobiology an international journal on social insects asn murakami1, tm nunes2, ic desuó1, sn shima1, s mateus2 article history edited by fabio s. do nascimento, usp, brazil received 01 september 2014 initial acceptance 23 october 2014 final acceptance 10 january 2015 keywords recognition nestmate, chemical signal, colony, mischocyttarini. corresponding author andré sunao nishiuchi murakami dep. de zoologia, instituto de biociências, universidade estadual paulista campus de rio claro av. 24-a, n. 1515, bela vista cep: 13506-900, rio clarosp, brazil e-mail: sunamigobio@yahoo.com.br signals in order to recognize the caste to which they belong as well as identify individually the nestmates, and differentiate the invaders (gamboa, 1996; lorenzi et al., 1997; singer & espelie, 1997; singer, 1998; howard & blomquist, 2005; richard & hunt, 2013). studies on chemical signaling have shown that the profiles of cuticular hydrocarbons can be used in the recognition among nestmates due to its colonyspecific composition in ants (bonavita-cougourdan et al., 1987; vander meer et al., 1989; wagner et al., 2000; sturgis & gordon, 2011; wilgenburg et al., 2011), bees (breed & stiller, 1992; breed et al., 1995; breed et al., 1998; dani et al., 2005; nunes et al, 2008; nunes et al, 2009; nunes et al., 2011), wasps (pfenning et al., 1983; bonavitacourgoudan et al., 1991; gamboa et al., 1996; dani et al., 2001; tannure-nascimento et al., 2007; bruschini et al., 2011; mitra et al., 2014) and termites (haverty & thorne, 1989; bagnères et al., 1991; kaib et al., 2002; kaib et al., 2004. research article wasps 1 universidade estadual paulista, rio claro, sp, brazil 2 universidade de são paulo, ribeirão preto, sp, brazil asn murakami et al cuticular hydrocarbons profiles and colonial recognition in mischocyttarus cassununga 110 in independent founding wasps, cuticular hydrocarbons have been mainly studied in the subfamilies polistinae (in polistes) and stenogastrinae. on the other hand, few studies have investigated the chemical communication in genus ropalidia and mischocyttarus. studies show that the mechanism of recognition of conspecifics depends mainly on the amount of relative concentration of cuticular compounds in a colony. the colony specific composition, where females of a given colony are able to recognize their conspecifics by odour, was demonstrated in polistes annularis (espelie & hermann, 1990) , in p. metricus (espelie et al., 1990; layton et al., 1994) , in p. dominulus (bonavita-courgoudan et al., 1991; dani et al., 2001; pickett et al., 2000; lorenzi et al., 2004; sumana et al., 2005) , in p. fuscatus (gamboa et al., 1996; panek et al., 2001; pfenning et al., 1983) in p. biglumis bimaculatus (lorenzi et al., 1997) , and p. satan (tannure-nascimento et al., 2007). in subfamily stenogastrinae, individual recognition through a similar or homogeneous colony odour was also demonstrated in liostenogaster flavolineata (cervo et al., 1996; cervo et al., 2002), in l. vechti (cervo et al., 1996) parischnogaster jacobsoni (cervo et al., 1996) and parischnogaster striatula (zanetti et al., 2001). and more recently, in ropalidia marginata has also demonstrated the importance of colonial profile of cuticular hydrocarbons (mitra et al., 2014). the tribe mischocyttarini is a large group of eusocial wasps with about 245 described species (carpenter, 1993; silveira, 2008), however the cuticular hydrocarbons and their role in chemical communication of this group of social insects were investigated in a few species (ferreira et al., 2012; neves et al., 2012). mischocyttarus cassununga is a species of neotropical wasp characterized by a linear and well stable dominance hierarchy, high longevity and rare replacement of the reproductive dominant female (“queen”) in the colony (murakami & shima, 2009; murakami & shima, 2010). the aims of the present study were to verify whether intercolonial identity could be discriminated using the cuticular hydrocarbons. material and methods the research was realized at universidade estadual paulista unesp, campus of rio claro sp, using eight (8) colonies of m. cassununga, during the post-emergence phase. all colonies were collected from the same population. chemical analysis of the colonies after the collection of colonies, all female wasps were placed separately in glass vial (1.5 ml) in freezer (temp. ca. -20oc) until the moment of the extraction of the cuticular compounds. the cuticular compounds of each sample were extracted by hexane (labsynth) at 500 ml for 1 minute. each sample extract was analysed by gas chromatography coupled with mass spectrometry (shimadzu, model qp2010, tokyo, japan), equipped with a zb-5ms column; length: 30 m; id: 0.25 mm; film thickness: 0.25 μm. the column was initially set at 150 oc for 3 min, then programmed at a rate of 3 oc/min to 280 oc, and then held at 280 oc for 15 min. the carrier gas was helium at a flow rate of 1 ml/min. the injection mode was splitless and the injection temperature was 250 oc. for the identification of the cuticular compounds it was performed comparisons of mass spectra with the wiley library and with the data of the diagnostic ions present in the literature. all the process of the hydrocarbons identification was carried out with the aid of the program gcms solutions for windows (shimadzu corporation). statistical analysis statistical analyses were conducted in statistica 7.0 for windows (statsoft inc., tulsa, ok, u.s.a.). the concentration of all compounds was transformed according to reyment’s formula (z = ln (ap / g (ap) )), where ap is the peak area, g (ap) is the geometric mean of the peak in each group of females and z is the transformed area of the peak (aitchison, 1986). compounds that, on average, contributed less than 1% to the overall chemical profile (i.e. linear alkanes, monomethyl and dimethyl alkanes) were excluded from the analysis, as made by kather et al. (2011). the areas of these peaks were recalculated, taking into account the reduced data set, and then reanalysed by statistical tests. first, a one-way anova was carried out to test for group differences (colonies) in total compound quantity for each chemical compound. at the same time, a two-way manova with pillai test and posthoc tukey test was used to test for group differences for each chemical compound separately. results and discussion the analysis of cuticular compounds of mischocyttarus cassununga resulted in a total of 88 peaks detected, divided into linear alkanes, methyl-branched alkanes and dimethylbranched alkanes, with chains between 16 and 36 carbon atoms. the selected peaks for chemical analysis are indicated in figure 1. the overall composition of hydrocarbons on an insect’s cuticle, termed as the profile, can range from highly complex to relatively simple (dani et al., 2001). for example, in the genus polistes, the most studied group of basal eusocial wasp, the species polistes biglumis bimaculatus (lorenzi et al., 1997) and p. dominulus (dapporto et al., 2004) have respectively 63 and 65 cuticular compounds, while p. metricus (layton et al., 1994) and p. satan (tannure-nascimento et al., 2007) have cuticular compounds 25 and 28, respectively. the objective of determining whether the entire mixture of cuticular compounds or some specific compounds are important for individual discrimination has generated discussion. however, there are evidences that linear alkanes are not involved in recognition processes. studies suggest that linear alkanes are relatively less colony-specific than the branched alkanes and other compounds present in the cuticle (gamboa et al., sociobiology 62(1): 109-115 (march, 2015) 111 1996; dani et al., 2001; dani et al., 2005). in honeybees, the alkenes or unsaturated fatty acids seem to influence more in the colonial recognition than the alkanes and unsaturated fatty acids (breed, 1998; breed et al., 1998). dani et al. (2005) similarly showed that linear alkanes have no effect on recognition in honeybee, in which only branched alkanes and alkenes are found. according to a comparative study of cuticular hydrocarbons of 78 species of 5 subfamilies of ants, the wide diversity of dimethylalkanes can indicate them as excellent compounds discriminatory recognition inter and intra-specific species (martin & drijfhout, 2009). dani et al. (2001) tested the effect of the concentration of some cuticular substances during the process of recognition between conspecific of polistes dominulus (vespidae), and found that linear alkanes did not cause any effect on the recognition response, unlike methylalkanes and alkenes. in the same species (p. dominulus), lorenzi et al. (2004) found that total quantities of chcs increased after emergence, with branched alkanes increasing drastically when compared with other classes of hydrocarbons. older wasps did not incorporate hydrocarbons, suggesting that the chemical profiles of mature wasps are less prone to chemical shifts than those of newly emerged wasps. in stingless bees, newly emerged workers have a very simple hydrocarbon pattern on their cuticle consisting almost entirely of linear alkanes (mainly c23; c25; c27 and c29) and guards were not able to discriminate between newly emerged nestmates and non-nestmates (nunes et al., 2011). in m. cassununga, all colonies significantly differed according to total quantity of fourteen cuticular hydrocarbons (=eight peaks) (table 1 and figure 2). the branched alkanes were the predominant compounds in the chemical differentiation of individuals. differently, in p. satan (vespidae), the linear alkanes were the main compounds responsibles by the differentiation of individuals through the colonial profile (tannure-nascimento et al., 2007; 2008). fig 1. total ion mass chromatograms of a mischocyttarus cassununga female wasp. peak numbers from 1 to 18 correspond to the selected compounds that contributed more than 1% to the overall chemical profile. 1= c27, 2= 7,16-dimec27, 3= 3-mec27, 4= c29, 5= 15-,13-,11-,9-mec29, 6= 7-mec29, 7= 11,15-dimec29, 8= 9,15 + 9,16-dimec29, 9= 7,15 + 7,18-dimec29, 10= 3-mec29, 11= 13,15-dimec29, 12= 15-,14-,13-,11mec30, 13= 11,15-dimec30, 14= 15-,13-,11-,9-mec31, 15= 7-mec31, 16= 11,16-dimec31, 17= 9,21-dimec31 and 18= 7,19 + 7,16-dimec31. asterisks represent identified contaminants in some of the samples. n. cuticular compound df ss f statistic p-value 1 c27 7 58.57 3.97 < 0.01 2 7,16-dimec27 7 29.82 0.92 0.49 3 3-mec27 7 29.22 6.54 < 0.001 4 c29 7 33.82 3.55 < 0.01 5 15-,13-,11-,9-mec29 7 6.39 4.30 < 0.001 6 7-mec29 7 17.20 2.85 0.01 7 11,15-dimec29 7 5.99 0.49 0.83 8 9,15 + 9,16-dimec29 7 14.11 0.88 0.52 9 7,15 + 7,18-dimec29 7 93.09 2.46 0.02 10 3-mec29 7 21.03 1.23 0.30 11 13,15-dimec29 7 75.53 15.07 < 0.001 12 15-,14-,13-,11-mec30 7 33.11 5.42 < 0.001 13 11,15-dimec30 7 2.63 1.34 0.25 14 15-,13-,11-,9-mec31 7 19.33 2.39 0.03 15 7-mec31 7 15.72 3.80 < 0.01 16 11,16-dimec31 7 13.09 10.42 < 0.001 17 9,21-dimec31 7 101.24 2.82 0.01 18 7,19 + 7,16-dimec31 7 15.07 0.94 0.32 table 1. group differences (colony) in total compound quantity for each chemical compound during the post-emergence phase in m. cassununga. n= number. (anova: p was significant for < 0.01 and < 0.001). manova test also showed that the overall chemical profiles of cuticular hydrocarbons are significantly different among colonies (manova: f= 2.61, d.f.= 315, 105, p < 0.001, pillai). a further analysis using tukey multiple comparison test (p < 0.001) shows the significant differences for each chemical compound separately (table 2). the peaks of the cuticular hydrocarbons 13,15-dimec29 and 11,16-dimec31 were the chemical compounds with significant differences in relative abundance among the higher number of colony groups (in eleven comparisons for both). colonies also differed significantly in the peaks 3-mec27 (in five comparisons), 15-,14-,13-,11-mec30 (in four comparisons), c29 (in two comparisons), 15-,13-,11-,9-mec29 (in two comparisons), 7-mec31 (in two comparisons) and c27 (in one comparisons). these results probably indicate that colonial recognition also occurs through the cuticular hydrocarbon profile in this neotropical species of social wasp (subfamily polistinae genus mischocyttarus). several studies on cuticular hydrocarbons show that differences on chemical profiles can be used as cues for nestmate recognition in polistinae (polistes and ropalidia) and stenogastrinae (pfenning et al., 1983; cervo et al., 1996; lorenzi et al., 1997; dani et al., 2001; zanetti et al., 2001; cervo et al., 2002; mitra et al., 2014). asn murakami et al cuticular hydrocarbons profiles and colonial recognition in mischocyttarus cassununga 112 fig 2. relative abundance of the chemical compounds in the colonies of m. cassununga. c27; 3-mec27; c29; 15-, 13, 11, 9-mec29; 13, 15-dimec29; 15-, 14-, 13-, 11-mec30; 7-mec31 and 11, 16-dimec31. error bars are standard errors. (anova: p < 0.01*; p < 0.001**). sociobiology 62(1): 109-115 (march, 2015) 113 despite the ability of colonial recognition has been studied for a long time, this aspect was not much investigated in the endemic species from the south america. in the neotropical species polistes satan, a variation in hydrocarbon profile was sufficiently strong to discriminate individuals according to their colony membership. according to this study, it seems that small differences in the proportion of these compounds can be detected and used as a chemical-based cue by nestmates to detect invaders and avoid usurpation (tannure-nascimento et al., 2007). the hydrocarbon analysis in m. cassununga shows that the differentiation of the cuticular profile among colonies is related to differences in abundance of a mixture of compounds, especially of branched alkanes. although we did not perform bioassays in this study, field observations show that female wasps are aggressive toward non conspecific individuals. this fact also may indicate that these wasps are capable to use these chemical differences for recognition. in conclusion, this work in m. cassununga represents a initial investigation of the potential role of cuticular hydrocarbons as mediators of nestmate recognition in the mischocyttarini tribe. however, more researches are necessary toward a better understanding of the importance of cuticular hydrocarbons in neotropical social wasps. acknowledgments thanks to partnership with faculdade de ciências farmacêuticas (universidade de são paulo usp, ribeirão preto sp). funding was provided by the capes (coordenação de aperfeiçoamento de pessoal de nível superior). references aitchison, j. 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(2001). nestmate recognition in parischnogaster striatula (hymenoptera, stenogastrinae), visual and olfactory recognition cues. journal of insect physiology, 47: 1013-1020. doi: 10.1016/s0022-1910(01)00077-4 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.393-400sociobiology 61(4): 393-400 (december 2014) the influence of the loss of brazilian savanna vegetation on the occurrence of stingless bees nests (hymenoptera: apidae: meliponini) introduction stingless bees (apidae: meliponini) are highly social bees considered by several authors as generalists, both in relation to floral resources and in relation to their choice of nesting substrate. their nests are found primarily in tree cavities, but, as apis mellifera linnaeus, they may also occupy different types of natural cavities (roubik, 1989), and some species may also use artificial cavities (e.g. zanette et al., 2005). unlike a. mellifera, however, their physiogastric queens are unable to fly, which has two major implications: first, they are unable to move the colony to another cavity if the nest site suffers a disturbance; second, the reproduction of the colony is made after the workers from the mother nest find a new cavity and prepare it to receive the new queen. after the new queen is established in the new nest, workers from the mother nest look after it up to six months (roubik, 2006). abstract the vegetation of the cerrado, also known as the brazilian savanna, is rapidly being replaced by agricultural and urban areas. the scope of this study was to evaluate the relationship between the reduction in savanna vegetation and the occurrence of nests of stingless bees (apidae: meliponini), particularly those of the genus melipona illiger. we mapped 33 surveys of stingless bees nests located in cerrado areas (25 sampled by us and 8 from literature) on landsat images and assessed the proportions of vegetated, urban and rural areas within a radius of 3 km from each nest. we performed two-step cluster analyses separately for common and rare meliponini species and melipona species and assessed the proportion of vegetation in the resulting clusters. common species such as trigona spinipes fabricius and tetragonisca angustula latreille were frequent in areas surrounded by vegetation as well as in degraded surround areas. some species, such as oxytrigona tataira smith, occurred predominantly in degraded areas. most melipona species were uncommon or absent in the degraded areas. it is possible that isolated trees in rural landscapes provide functional connectivity for opportunistic species of stingless bees but not for susceptible species such as species of melipona. this study is one of the first to attempt to understand the effects of the loss of cerrado vegetation on the occurrence of stingless bees’ nests. sociobiology an international journal on social insects fc pioker-hara¹, ms drummond², amp kleinert¹ article history edited by cândida maria l. aguiar, uefs, brazil received 30 september 2014 initial acceptance 11 november 2014 final acceptance 28 november 2014 keywords meliponini, cerrado, habitat loss, fragmentation. corresponding author fabiana curtopassi pioker-hara depto. de ecologia, instituto de biociências rua do matão, tr. 14 nº 101, 05508-090 cidade universitária, são paulo, sp, brazil e-mail: fpioker@usp.br thus, as stingless bees are unable to migrate their colonies and to establish a new nest too far from the mother nest, they are potentially sensitive to habitat degradation. to ensure the viability of stingless bees populations, the area around the nests must provide appropriate resources, as food and nesting sites. despite some species are able to live and to find resources in urban places, the environmental anthropization may have a deleterious effect on the community (zanette et al., 2005). besides the effects of deforestation, which is not yet known how it can affect bee populations (e.g. edge effect, increase of temperature and decrease of relative humidity), a direct and obvious problem is the reduction of available nesting sites, since most species nest in tree cavities. for many species, the availability of nesting sites may be a crucial factor limiting colonies reproduction (inoue et al., 1993). for sensitive species, however, not only quantity but mainly research article bees 1 universidade de são paulo, são paulo, sp, brazil 2 universidade federal do maranhão, são luís, ma, brazil fc pioker-hara, ms drummond, amp kleinert – conservation of stingless bees in a brazilian savanna394 quality of potential nesting sites is of greater importance. in some brazilian dry forests and savanna, most of the stingless bees nests were found in a few species of trees, despite the presence of many tree species with available cavities (antonini & martins, 2003; martins et al., 2004). among brazilian biomes, the cerrado is one of the most affected by deforestation. the biome is one of the largest savanna formations in the world and occupied an original area of approximately two million square kilometres (ab’saber, 1981). approximately half of the original area of the cerrado, however, has been converted to pastures and crops, primarily large monocultures of soybean, sugarcane and eucalyptus. a large portion has also been converted to urban land uses. only 33,000 km² is protected by a series of discontinuous protected areas (mittermeier et al., 1999; klink & machado, 2005). even so disturbed, the cerrado is considered a biodiversity hotspot. from the 33 neotropical genus of stingless bees, 23 are represented in the cerrado (camargo & pedro, 2013). bees also represent the most important group of cerrado pollinators (biesmeijer et al., 2005; gottsberger & silberbauer-gottsberger, 2006), and stingless bees may represent the highest biomass of insects that visit flowers in areas where they occur due to their large colonies, consisting of many workers that can be recruited to gather resources (michener, 1979). thus, stingless bees are very important to maintain genetic variability of the remnant flora of the cerrado. despite the high rate of devastation of brazilian environments, especially the cerrado, the effects of human landscape disturbances on the populations of most species of stingless bees are still poorly understood. most empirical studies of bees have generally focused on floral visitations, comparing the fauna in environments with different levels of anthropogenic disturbance within the same locality (e.g. antonini & martins, 2003; araujo et al., 2006; carvalho et al., 2007). surveys of flower visitors are important for characterising the current fauna but may mask the true status of stingless bee populations. first, the foraging area of stingless bees varies according to the size of the workers, and workers of some species, such as melipona compressipes smith, may forage more than 2 km from the colony (araujo et al., 2004). this maximum distance results in a foraging area of more than 2,500 ha, whereas the area sampled in surveys is usually not more than 100 ha (one hectare: 10,000 m²). thus, transient foraging visitors may be sampled in an area even though the foragers’ colony actually resides in an area with an entirely different vegetation composition. therefore, a study focused on the effects of human disturbances on the occurrence of stingless bee nests in a given area can provide a more precise understanding of the effects of these disturbances on bee populations. among stingless bees species, those of melipona genus have been considered particularly sensitive to habitat loss. one of the reasons for this susceptibility is because many species of melipona require large cavities for nesting, usually in large trees (roubik, 2006), which are preferred targets for commercial exploitation and what make these bees susceptible to deforestation. brown & albercht (2001), for example, found that amazonian melipona bees are sensitive to deforestation, showing a population decrease even where some forest remnants were kept in the surroundings. melipona species are on the list of endangered species in several brazilian states, including populations located in cerrado environment (machado et al., 1998), despite the studies have only included data from floral visitors surveys. given the great importance of stingless bees for pollination across the cerrado, understanding the effects of human disturbances on stingless bees nests is essential for ensuring the conservation of these bees and, consequently, the remnants of native cerrado vegetation. to evaluate the influence of the loss of cerrado vegetation on the occurrence of stingless bees nests, we surveyed bee nests in two areas of cerrado in the state of são paulo and 23 areas of cerrado in the state of maranhão and evaluated the degree of anthropogenic change in the landscape, analysing the data together with those from other nest surveys in the cerrado. the goal of this study was to understand if there was a relationship between loss of cerrado vegetation and the occurrence of the stingless bees nests found in the nest surveys, to answer the following questions: (1) does the presence of stingless bees nests decrease with loss of cerrado vegetation? (2) how does the frequency of nests of different species changes with the loss of cerrado vegetation? and (3) how does the frequency of melipona nests responds to the loss of cerrado vegetation? material and methods study areas and nest surveys meliponini nest surveys were conducted in 25 natural, vegetated areas of brazilian savanna: 23 areas of 4 ha in northeastern brazil and two areas of 5 ha in southeastern brazil. nests were actively searched for until each area was completely examined. all trees and abandoned termite and ant nests were inspected. location for each nest found was recorded using gps. bee workers were collected at nest entrances for further identification and sent to specialists when necessary. bees collected in the southeastern areas are deposited in the paulo nogueira-neto entomological collection, bee laboratory, institute of biosciences of the university of são paulo – usp (cepann) and those collected in the northeastern areas are deposited in the entomological collection of the federal university of maranhão. the data collected were analysed together with data from previous nest surveys reported on literature, selected based on the following criteria: (1) the surveys had been conducted in an area of brazilian cerrado; (2) they contained data from surveys of nests of different species of stingless bees; (3) the surveys were conducted following the same sociobiology 61(4): 393-400 (december 2014) 395 method used in this study; and (4) they included geographic coordinates or information that could be used to determine the exact location of the study area. an initial selection of 31 studies was identified, including two theses, a work of graduate course completion, and 28 articles, including two popular science articles (supplementary material available on line). based on the selection criteria, only eight of the 31 studies were retained (table 1). samples were collected in five geographic regions: são paulo, minas gerais, mato grosso, southwestern and northeastern maranhão (fig 1). characterisation of environmental variables sampled areas of each survey were located on georeferenced landsat satellite images, obtained through the website of the brazilian environmental institute (http:// siscom.ibama.gov.br/monitorabiomas/cerrado/ last accessed 10/25/2012). to verify the amount of remnant savanna vegetation surround the sampled areas, we drawn a circle with a 3 km radius around each one using the buffer function of the program arcgis 9.2 ®. this area size was selected based on the average maximum foraging radius of the stingless bees (araújo et al., 2004) inhabiting a nest located at the edge of the largest area sampled in the selected surveys. we thus tried to assess the scale of the landscape likely to be used by most of the stingless bees inhabiting the nests found in the studies. the images were manually classified by drawing polygons that delineated cover types: brazilian savanna vegetation (regardless of size or condition of the vegetation), rural areas (identified based on regular designs with a predominance of crops or bare soil), and urban areas. the area of open water visible at a resolution of 1:30000 was subtracted from the total. when in doubt, the classification of the landsat images was verified using images from google earth®. the areas of the polygons were calculated and the areas of polygons of the same cover type were summed and converted into a percentage to obtain the proportion of remnant savanna area in the landscape. to evaluate the effect of landscape on the distribution of species, the number of nests of each species in each survey was transformed into presence-absence and subjected to a principal coordinate analysis (pco). these analyses were performed in past 2.07 (hammer et al., 2001). data analysis due to the variation in the sizes of the areas sampled during the literature surveys and the few number of nests found in most of our surveys, the number of nests of each species in each study was transformed into presence or absence data. a two-step cluster analysis without a predefined number of clusters was used to investigate the relationship between species composition and the proportions of surround remnant savanna vegetation. we performed three separate analyses: one considering the species occurring in five or more surveys (“frequent species”); the second considering species with occurrences in four or fewer surveys (“rare species”); and the third considering only species of the genus melipona, regardless of the number of surveys in which they occurred. we performed these separate analysis to evaluate the effect of loss of cerrado table 1. locations of the included nest surveys. landsat: point-orbit of the landsat satellite where the surveys were located. % vegetation: amount of remnant cerrado vegetation in each survey. coordinates in utm – sad69. references in supplementary material available online. fig 1. overview of survey locations. stars: this study. circles: surveys from literature. surveys: 1: são paulo state surveys (eei, iab, santa teresa and usp ribeirão); 2: minas gerais state surveys (araguari and panga); 3: mato grosso survey (bacaba); 4 and 5: maranhão state surveys, being 4: arizona, bacabinha, balsas, bom lugar, cajueiro winnits, extrema, fazenda são pedro, fazenda walter, feio, formosa, gleba cajueiro, and são francisco; 5: arizona, buriti, carmo, castiça, cocalinho, jatoba, macaco, normasa, são lourenço, são luizinho, sipauba, tabocal, urbano santos, and urubu. the light delimited area corresponds to cerrado. survey name landsat longitude latitude % vegetation reference araguari 221-073 155049 7923805 69.8 siqueira et al. 2007 arizona 221-065 346637 9223868 55.4 this work bacaba 224-070 354877 8375344 41.2 pereira 2004 bacabinha 222-065 230705 9253082 73.5 this work balsas 221-065 368933 9146931 95.7 rego et al. 2008 bom lugar 222-064 239513 9415091 61.4 this work buriti 219-063 712945 9494380 94.5 this work caj. winnits 222-064 326864 9334741 84.7 this work carmo 219-063 713144 9537956 93.7 this work castiça 219-064 698053 9392620 95.0 this work chapadinha 220-063 666549 9548548 86.2 rego et al. 1998 cocalinho 220-064 676565 9377561 79.4 this work eei 220-075 199103 7541617 38.4 this work extrema 222-065 276484 9169159 99.3 this work faz. são pedro 221-065 312959 9153858 98.4 this work faz. walter 222-065 236598 9205941 95.4 this work feio 222-064 236312 9330425 48.7 this work formosa 221-065 367645 9273900 89.5 this work gleba cajueiro 222-065 299478 9237677 91.7 this work iab 220-075 203483 7545030 29.9 this work jatoba 220-064 585754 9357013 75.0 this work macaco 220-064 576991 9334800 77.7 this work normasa 220-064 668536 9380055 55.0 this work panga 221-073 141662 7875233 39.3 nogueira-ferreira & siqueira 2008 são lourenço 219-064 710963 9426135 98.1 this work são luizinho 220-064 582344 9404812 79.3 this work são francisco 221-064 328443 9302114 88.0 this work sipauba 220-063 639155 9478107 91.9 this work santa teresa 220-075 204430 7650779 11.6 alvarenga 2008 tabocal 219-064 678591 9312946 97.7 this work urb. santos 220-062 677454 9645344 75.3 serra et al. 2009 urubu 219-064 712108 9418736 90.8 this work usp ribeirão 220-075 203224 7657088 11.1 freitas et al. 2009 fc pioker-hara, ms drummond, amp kleinert – conservation of stingless bees in a brazilian savanna396 areas to the occurrence of each kind of species (common species, usually found in antropic environments; rare species, detected in few surveys; and melipona bees, reported in literature as sensitive to deforestation). to evaluate the effect of the variable order on the cluster arrangement, the position of common species was randomised using a monte-carlo method with 10,000 iterations, and its significance within the clusters was tested by a chi-square test. the significance of the environmental variables in the species composition of the clusters was assessed using a mann-whitney test. analyses were performed using the program spss 13.0. results the surveys compiled for the present study comprised a total of 1135 nests belonging to 19 genera and 63 species, of which 10 were unidentified (supplementary material available on line). the first axis of the pco of surveys from the species presence-absence data explained only 20% of the variation. the spread of the ordination coefficients along the first two axes showed that geographically closer surveys tended to have more similar species compositions, as expected for a large landscape scale (fig 2). frequency of occurrence of stingless bees species versus the reduction in cerrado vegetation fourteen species occurred in at least five sites (table 2). these “frequent species” formed tree natural clusters (a, b and c). cluster a had significantly less vegetation (43% ± 31%) than cluster b (82% ± 17%; z=-2.419, α=0,014) and than cluster c (80% ± 17%; z=-2.538, α=0,008), but cluster b and c are similar with respect to the amount of vegetation (z=-0.539, α= 0,614). cluster a showed 39% of the occurrences of the “frequent species”, and clusters b and c, together, showed 61% of the occurrences. of the 14 species, eight had significant results regarding the frequency in each cluster: frieseomelitta longipes smith, lestrimelitta limao smith, oxytrigona tataira smith, tetragona clavipes fabricius, trigona fulviventris guérin, trigona pallens fabricius, and trigona spinipes fabricius. the other six species exhibited random frequencies between clusters. excluding these random species, cluster a showed 51% of the occurrences and clusters b and c, together, showed 49% of the occurrences (fig 3). fig 2. pco analysis (1st coordinate x 2nd coordinate) of the ordination of sites by the presence-absence species data. circle: são paulo; triangle: minas gerais; x: mato grosso; square: sw of maranhão; cross: ne of maranhão. table 2. species that were found in five or more surveys. nº of nests: total of nests found in all surveys; occurrences: number of surveys where species are found; state: brazilian state where species were found. ma: maranhão state; mg: minas gerais state; mt: mato grosso state; sp: são paulo state. fig 3. percentage and standard deviation of natural cerrado vegetation in cluster a, b, c and b+c (dark gray) and percentage of occurrence of non-random clustered common species (light gray). from the eight non-random species clustered, four occurred mainly in cluster a (most degraded): o. tataira (100% of the species occurrence), l. limao (80% of the species occurrence), tetragonisca angustula latreille (62%) and t. clavipes (58%). t. pallens showed 50% of occurrence in cluster a and in cluster b+c. the other three species were less frequent or absent in most degraded cluster a (t. fulviventris, 43%; t. spinipes, 42%; and f. longipes, 0%). the 38 species with a frequency of four or fewer sites (supplementary material available on line) were grouped into two clusters (d and e) that not differed with respect to the amount of vegetation (z=-1.542, α= 0.13); 69% of the occurrence of the “rare species” was found in cluster c, against 31% of occurrences in cluster d. the significance of the position of each species in each cluster could not be determined because of their low frequencies of occurrence. species nº of nests occurrences state scaptotrigona postica moure 38 17 ma, mt, sp tetragona clavipes fabricius 75 13 ma, mg, mt, sp tetragonisca angustula latreille 149 12 ma, mg, mt, sp trigona pallens fabricius 22 12 ma, mt trigona spinipes fabricius 82 12 ma, mg, mt, sp melipona fasciculata smith 16 10 ma frieseomelitta flavicornis fabricius 51 9 ma frieseomelitta longipes smith 12 9 ma melipona flavolineata friese 13 9 ma trigona branneri cockerell 10 7 ma trigona fulviventris guérin 8 7 ma, mg oxytrigona tataira smith 26 6 ma, mg, mt lestrimelitta limao smith 12 5 ma, mg, mt, sp trigona truculenta almeida 5 5 ma, mg sociobiology 61(4): 393-400 (december 2014) 397 frequency of occurrence of melipona species versus the reduction in cerrado vegetation melipona species were grouped into two clusters (f and g), and none of the species occurred in more than one cluster. cluster f showed significantly less vegetation (63% ± 19%) than cluster g (vegetation: 85% ± 13%; z= -2.261, α=0.022). cluster f showed only 17% of melipona occurrences, against 83% of occurrences for cluster g (fig 4). of the six species of melipona identified in the surveys, three showed non-random position in clustering: melipona fasciculata smith, melipona flavolineata friese and melipona seminigra friese. 100% of the occurrence of these species was in cluster g. the 14 more frequent species found in the surveys showed a spread occurrence among the clusters, despite the amount of vegetation in them. in fact, from the eight frequent, non-random clustered species, the most degraded cluster showed the higher frequencies of four species and at least 40% of occurrence for three species. f. longipes was the unique of the frequent species not found in the more degraded cluster. the apparent insensitivity of these common species to the reduction in native vegetation may be related to the behavioural plasticity of each species. while many meliponini species are selective about their choice of nesting site (antonini & martins, 2003; macías-macías et al., 2014), most of the species frequent in degraded cluster are opportunistic and are commonly reported in surveys made in anthropical and urban environments. bees such as t. angustula, for example, are extreme generalists in their choice of nesting site, and can occupy both natural and artificial cavities and may even be more numerous in artificial cavities (sousa et al., 2002), being also able to take over nests of other species. l. limao is also a known parasitic bee, attacking other stingless bees nests. other, as o. tataira and trigona bees are aggressive foragers, being efficient in acquiring resources, including resource monopolization (e.g. breed et al., 1999, nieh et al., 2004; 2005). thus, the loss of cerrado vegetation and its replacement by a matrix of cropland and pastures is not enough to represent a loss of habitat connectivity to these common species. connectivity indicates the aggregation of similar landscape elements. connectivity at landscape level is considered structural, but there is a second type of connectivity, functional connectivity. in this case, the structure of the matrix and the landscape corridors may allow the movement of a particular taxon but be prohibitive to another, i.e. a given fragment can be isolated for an animal but connected by matrix elements that function as corridors (goodwin, 2003). thereby, structural fragmentation does not always reflect the loss of functional connectivity because this loss will depend on the mobility of each organism and the extent to which the fragmentation of the landscape represents a real loss of habitat (d’eon et al., 2002; goodwin, 2003; manning et al., 2004). thus, the loss of natural areas may have a greater effect on some species of stingless bees that are more sensitive to the reduction in these areas than on the most frequent species reported in this study. another factor to consider is the type of environment considered in this study, which was predominantly savanna. previous studies have shown that the richness of stingless bees decreases with increasing distance of forest fragments from native forest (brosi et al., 2007; 2008), as well as the sensitivity of many species to deforestation (brosi, 2009). these studies, however, were conducted in tropical forest areas, where there is a stark contrast between vegetation and agricultural areas. for the present study, we considered environments whose phytophysiognomies are characteristically more open, featuring mostly shrubby vegetation with a few isolated trees. in the works selected for this study, although the proportion of rural areas was fig 4. percentage and standard deviation of natural cerrado vegetation in cluster f and g (dark gray) and percentage of occurrence of melipona species (light gray). discussion from the results obtained, we found that the loss of cerrado vegetation may have a negative effect on the occurrence of nests of most species of stingless bees, mainly those from melipona genus. this effect, however, is not the same for all species, which makes it difficult to establish a general pattern of response for meliponini in relation to the degradation of vegetation. although not all stingless bees species are capable of nesting in the areas with most degraded surroundings, as observed in the cluster analysis, some species are more frequent in these areas. moreover, species composition may differ among surveys surrounded by degraded or preserved areas, as seen in the cluster analysis. frequency of occurrence of meliponini species versus the reduction in cerrado vegetation we could not establish a relationship between the frequency of rare species and the amount of vegetation. their clustering should be more related to geographic proximity, as shown in the pco (fig 2), than by the amount of vegetation. this was expected, since most of them occurred in the maranhão surveys (supplementary material available on line), that also concentrate the most vegetated areas. fc pioker-hara, ms drummond, amp kleinert – conservation of stingless bees in a brazilian savanna398 high in some sites, none featured a fragment of native vegetation completely isolated by agricultural fields. thus, the agricultural matrix interspersed with fragments of field type phytophysiognomies may not differ significantly from the natural phytophysiognomies from the perspective of habitat use by stingless bees as a whole. that is, the degradation of native vegetation may represent a loss of structural, but not functional, connectivity, especially for species that are less restrictive in their choice of nesting site. the species most frequently recorded in surveys are common species of stingless bees, some reared by man and others common in anthropogenic environments. moreover, for many species, a single isolated tree can support more than five nests of stingless bees, including multiple species (martins et al., 2004). thus, even if the proportion of native vegetation decreases, the matrix between fragments may provide functional connectivity to the most opportunistic species. the frequency of occurrence of melipona species versus the reduction in cerrado vegetation unlike found for the frequent species, the decrease of native cerrado vegetation may imply in a loss of habitat connectivity to melipona bees. the absence of the non-random clustered species of melipona from the cluster with the greatest reduction in native vegetation confirms the sensitivity of these bees to habitat reduction. one of the reasons may be the loss of available nest sites in areas with low cerrado vegetation. except for melipona quinquefasciata lepeletier, which builds nests in cavities in the soil (martins et al., 2004) and was not found in any of the selected works, bees of the genus melipona require large cavities for nesting, which are found in large trees. therefore, the absence of large trees may cause the absence of this type of bee from a given region. furthermore, the occurrence of some species at low frequencies in locations with a lower proportion of native vegetation may be a result of nests that have persisted from a pre-degradation stage because high longevity and low fecundity are characteristic of stingless bees (eltz et al., 2003) and colonies may remain active for more than a decade. in contrast, the replacement of natural areas and agricultural areas can be rapid (csribama, 2009). thus, species that are uncommon today in degraded areas may disappear from areas where they are unable to establish new nests in the long term. despite many melipona species are kept in artificial nests by beekeepers, there are no reports on spontaneous nesting in artificial cavities, as for t. angustula, for example. surveys on urban or anthropogenic environments usually do not report melipona bees, even where large trees are still remaining. thus, not only the loss of nest sites as the quality of the environment may represent a problem to melipona bees. therefore, the conservation of natural environments may represent an important step towards the viability of melipona species populations, either in forest environments (brown & albrecht, 2001) or in the brazilian savanna. study limitations in most of our surveys there was a few number of nests (one to five), and each survey presented a different size of the sampled area. these issues have become a problem for some analyses as direct relationships between the amount of vegetation and parameters as nest density, species diversity or richness, even for those based on rarefaction techniques. thus, we chose to run a robust analysis through the exploratory technique of two-step cluster analysis between species frequency of occurrence and the amount of vegetation. interpretations of the results of the present study should take into account three major limitations. first, there is a lack of peer-reviewed scientific journal articles that present data from nest surveys, especially for the cerrado. this deficiency is worrisome because the advancement of human disturbances across the remaining areas of cerrado is outpacing scientific studies. second, the absence of nest surveys in the central areas of the cerrado leaves a large gap in the present study. thirdly, the sampled areas of different sizes needed retrospective standardisation, limiting the interpretation of the results. conclusions even with the appropriate caveats, the present study is one of the first attempts to understand the effects of human disturbances of the cerrado on the nests of stingless bees, showing that the loss of native vegetation of the cerrado may have a deleterious effect on melipona species. our results produced the following answers to our initial questions regarding the reduction in the native cerrado: (1) does the presence of stingless bees nests decrease with the loss of cerrado vegetation? yes to melipona bees, but not to common, opportunistic stingless bees species; (2) how does the frequency of nests of different species changes with the loss of cerrado vegetation? common, opportunistic bee species are frequent either in degraded as in preserved areas, being sometimes more frequent in more degraded areas; frequency of rare species may not be related to the amount of vegetation in their area of occurrence, being their distribution in this study probably a reflection of geographical location; (3) how does the frequency of melipona nests respond to the loss of cerrado vegetation? nests of melipona bees are significantly less frequent in lesser vegetated areas, being absent of them when it is considered only non-random clustered species. acknowledgements we thank paulo c. fernandes, marcos r. hara, maria r. vianna, sandra m. c. pioker and samanta o. jorge for field help; favizia f. freitas and gabriel a. r. melo for help with identification of some bee species; mitti a. h. koyama for statistical support; paulo ruffino, instituto florestal, estação ecológica de itirapina and instituto arruda botelho for logistic support and leandro trambosi for help with arcgis software. financial support: são paulo research foundation fapesp (process no 06/60188-0). sociobiology 61(4): 393-400 (december 2014) 399 references ab’saber, a. n. 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(2006). stingless bee nesting biology. apidologie, 37: 124-143. doi: 10.1051/apido:2006026. sousa, l. m., pereira, t. o., prezoto, f., faria-mucci, f. m. (2002). nest foundation and diversity of meliponini (hymenoptera: apidae) in an urban area of the municipality of juiz de fora, mg, brazil. biosci. j., 18(2): 59-65. zanette, l. r. s., martins, r. p. & ribeiro, s. p. (2005). effects of urbanization on neotropical wasp and bee assemblages in a brazilian metropolis. landsc. urban plan., 71: 105-121. doi: 10.1016/j.landurbplan.2004.02.003. doi: 10.13102/sociobiology.v67i3.5773sociobiology 67(3): 358-363 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction typical and simple ant societies consist of an inseminated reproductive queen and multiple infertile workers in one nest (hölldobler & wilson, 1990). the subsequent social transformation steps in ant societies have evolved polydomy (when a colony has multiple nests), polygyny (when multiple inseminated queens are in one nest), polyandry (when a queen mates with multiple males), parasitism (i.e., of another ant species), production of intermediate reproductive females (intercastes, gamergates, and ergatoid queens), and worker reproduction (peeters & ito, 2001). generally, newly emerged queens conduct nuptial flights or attract males using sexual abstract in eusocial hymenoptera such as ants, bees, and wasps, the queen numbers are fundamentally important to maintain the social systems. in texas, usa, a fungus growing ant, mycetomoellerius turrifex wheeler (1903) (the genus name was changed from trachymyrmex to mycetomoellerius in 2019 (solomon et al., 2019)) was observed to have several non-inseminated queens that wandered outside the nest long after the mating season. however, the evolutionary and ecological factors causing the occurrence of such non-inseminated queens are still unclear. thus, i examined the worker-like behaviors of non-inseminated queens of m. turrifex in texas. fifteen ant colonies were collected over three years, between 1999 and 2001. the frequencies of non-inseminated queens, workers, and broods, as well as the depths of nest chambers, were observed in each collecting year. in nov. 1999 and may 2000, multiple nests contained queens that did not mate with males within their nests. these nests had a relatively larger colony size than those collected in oct. 2001. conversely, the colonies collected in oct. 2001 were completely monogynous, i.e., there were no non-inseminated queens in the nests. behavioral observations of each female revealed that the non-inseminated queens behaved significantly differently from the workers and the inseminated queens. the behaviors that distinguished different female castes were mutualistic fungus garden care, digging of nest floors, guarding, and resting in the nest. these data suggest that queens failed to mate due to severe environmental fluctuations in southern texas, but were accepted by the colony as a temporary labor force. sociobiology an international journal on social insects t murakami article history edited by evandro nascimento silva, uefs, brazil received 05 august 2020 initial acceptance 08 august 2020 final acceptance 20 august 2020 publication date 30 september, 2020 keywords mating failure, monogyny, polygyny. corresponding author takahiro murakami institution of decision science for a sustainable society kyushu university motooka 744, fukuoka, 819-0395, japan e-mail: taka_bci2000@yahoo.co.jp murakami.takahiro.023@m.kyushu-u.ac.jp pheromones and then mate with them. if a queen is unable to mate with males at that point, it will die within a short period of time. there are a few species in which non-inseminated queens survive and co-exist in their natal nest. for example, the presence of non-inseminated queens within colonies has been reported in myrmecina nipponica (ohkawara et al., 1993; murakami et al., 2002) and probolomyrmex longinodus (kikuchi & tsuji, 2005), but their behavioral ecology remains poorly studied and the adaptive factors to evolve the existence of non-inseminated queens in their natal nest are still controversial. fungus-growing ants (tribe attini), distributed only in the new world, have a specialized ability to cultivate institution of decision science for a sustainable society, kyushu university, fukuoka, japan research article ants non-inseminated queens have worker-like behaviors in colonies of fungus-growing ants, mycetomoellerius turrifex wheeler (attini, hymenoptera) sociobiology 67(3): 358-363 (september, 2020) 359 mutualistic fungi in the nest and feed them to queens and larvae (weber, 1972; hölldober & wilson, 1990; murakami & higashi, 1997; mueller & gerardo, 2002). attini consists of over 250 species and 17 genera, some of which have a simple colony structure (small colony sizes, monogyny, and no worker polymorphism), such as mycetosorites, myrmicocrypta, and cyphomyrmex, while others have a very complex society (huge colony sizes, multiple fungus cavities deep underground, and highly divided worker polymorphism), such as leaf-cutting ants, atta, and acromyrmex. the genus mycetomoellerius is the “higher” group that has relatively derived ecological characters and molecular phylogentic positions in attini (solomon et al., 2019). they have small to medium colony sizes (around 50–1000 workers in one nest), and their workers are slightly physically differentiated in subcastes. mycetomoellerius turrifex wheeler is distributed in texas, northeastern mexico, western louisiana, and oklahoma. the ant is abundant in the open desert habitats of west texas and dense populations have been observed in southern texas. nests consist of 1–5 chambers connected by vertical tunnels. the colonies are monogynous and have up to 300 workers (rabeling et al., 2007). in this paper, i report that this ant is basically functionally monogynous, but they have multiple noninseminated queens in their nests. colonies of m. turrifex in southern texas were sampled over three years, and individual behaviors of female castes were observed. the roles and functions of non-inseminated queens are discussed, and verified five hypotheses that will explain the existence of noninseminated queens in a natal nest. the hypotheses are related to environmental changes, social parasite strategy, mating strategy, trophic egg production, and labor force in a workerlike manner (kikuchi & tsuji, 2005). materials and methods sample collection i collected 15 m. turrifex colonies from 1999-2001 at the brackenridge field laboratory of the university of texas, u.s.a. (30°17´ n, 97°46´ w). additionally, in 2000 and 2001, the depths of each fungus garden chamber were measured. at each sampling time, the numbers of workers, queens, pupae, larvae, eggs, and males were counted. colonies were kept in the laboratory at room temperature with milled corn and oats as their fungus substrates. individual behavioral observations the behaviors of 34 queens from five colonies and 12 workers from four colonies were observed using a microscope (olympus sz-40) and recorded. i observed in detail the queens and the workers after individually marking them with oil-based pen dyes. their behaviors were checked every 10 min, for five h in each colony, and the total observation times were 25 h for queen ants and 20 h for worker ants. the recorded behavioral repertoires were divided into 24 types according to murakami (1998). after the observation period, all queens were dissected under a microscope and their egg laying abilities, oviposition experiences, and existing sperms into spermathecae, were assessed. statistical analysis in order to analyze the behavioral differences among each caste, canonical discriminant analysis, a type of multivariate analysis, was used to discriminate among three categories: inseminated queens, non-inseminated queens, and workers. the analysis used 24 behavioral repertoires as explanatory variables. this analysis allowed us to identify the discrimination rate for each category and the behavioral repertoire that significantly contributed to discrimination. the analysis was conducted using r (3.6.1) software. results as shown in table 1, the frequencies of inseminated queens, non-inseminated queens, workers, and broods changed every collecting year. in november 1999, there were significantly more workers (average: 71.8 individuals ± 60.9, n = 4) than that in other collecting years [one-way anova, tukey-kramer test; t = 1.93, p = 0.17 for may 2000 (average: 30.5 ± 18.5, n = 6); t = 2.77, p = 0.04 for oct. 2001 (average: 10.4 ± 8.6, n = 5)]. in nov. 1999 and may 2000, multiple non-inseminated queens were observed in 80% of collected colonies (12.8 ± 14.7 in 1999, 12.3 ± 10.9 in 2000). in contrast there were no non-inseminated queens in the colonies in oct. 2001 (one-way anova, tukey-kramer test; t = 2.18, p = 0.07, between 1999 and 2001; t = 2.88, p = 0.01, between 2000 and 2001). the number of broods (larvae, pupae, and eggs) in may 2000 was significantly larger than that in nov. 2001 (one-way anova, tukey-kramer test; t = 2.93, p = 0.03). there was no male production except for one colony in 1999, which reared 13 males. m. turrifex had on average three nest chambers per colony. the depths of the first chamber ranged from 3–10 cm, that of the second chamber ranged from 19–56 cm, and the last chamber was over 35 cm deep. the depths of first chamber were shallower, and that of the second and third chambers were deeper in 2001 compared with those in 2000. following detailed behavioral observations, individuals were identified as either queens or workers (table 2). after the observations, all queens were dissected, and four inseminated queens were identified. thirty non-inseminated queens did not produce even trophic eggs in the ovarioles. the inseminated queens had never performed any task outside the nests, and their top three behaviors were fungus licking (25%), resting inside the nests (23.1%), and antennations (22.5%). the non-inseminated queens performed 87.5% repertoires of all observed behaviors including those of worker ants. oviposition was never observed in these females. the top three behaviors of the non-inseminated queens were resting t murakami – non-inseminated queens having worker-like behaviors360 sa m pl in g d at e (y ym m dd ) q ue en s (t ot al ) in se m in at ed qu ee ns n on -i ns em in at ed qu ee ns w or ke rs l ar va e pu pa e n es t c ha m be r d ep th (c m ) st ag e i ii ii i q ue en s w or ke rs m al es e gg s m al es i ii ii i 99 11 01 2 1 1 43 1 1 3 0 3 0 1 0 nd nd nd 99 11 01 21 1 20 10 4 1 2 1 0 6 0 4 0 nd nd nd 99 11 01 31 1 30 13 8 0 0 0 0 0 0 0 13 nd nd nd 99 11 21 1 1 0 2 0 0 3 0 0 0 0 0 nd nd nd av er ag e 13 .8 1. 0 12 .8 71 .8 0. 5 0. 8 1. 8 0. 0 2. 3 0. 0 1. 3 3. 3 　 　 　 00 05 19 4 1 3 11 4 0 1 0 1 0 2 0 8 10 00 05 19 8 1 7 9 0 0 4 0 0 0 0 0 23 25 00 05 24 26 1 25 31 4 0 0 0 2 0 2 0 15 35 45 00 05 24 10 1 9 56 7 5 11 0 28 0 4 0 10 00 06 13 4 1 3 31 2 10 34 0 0 0 2 0 7 15 20 00 06 13 28 1 27 45 5 2 6 15 1 0 7 0 2 12 40 a ve ra ge 13 .3 1. 0 12 .3 30 .5 3. 7 2. 8 9. 3 2. 5 5. 3 0. 0 2. 8 0. 0 10 .8 19 .4 35 .0 01 10 05 1 1 0 10 0 0 0 0 0 0 0 0 2 60 >1 00 01 10 05 1 1 0 8 0 0 0 0 0 0 0 0 5 55 >1 00 01 10 05 1 1 0 6 0 0 0 0 0 0 0 0 7 45 >1 00 01 10 05 1 1 0 3 0 0 0 0 0 0 0 0 2 60 >1 00 01 10 05 1 1 0 25 0 0 0 0 0 0 0 0 1 60 >1 00 a ve ra ge 1. 0 1. 0 0. 0 10 .4 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 3. 4 56 .0 >1 00 　 inseminated queens non-inseminated queens workers observed individual numbers 4 30 12 collecting substrate 0 (0.0) 8 (0.3) 0 (0.0) licking substrate 0 (0.0) 70 (2.8) 7 (0.6) substrate weeding 0 (0.0) 46 (1.8) 0 (0.0) substrate care 0 (0.0) 14 (0.55) 21 (1.9) walking outside 0 (0.0) 215 (8.5) 255 (23.1) self-grooming (outside) 0 (0.0) 33 (1.3) 14 (1.3) antennation (outside) 0 (0.0) 8 (0.3) 0 (0.0) resting (outside) 0 (0.0) 3 (0.1) 7 (0.6) dumping 0 (0.0) 26 (1.0) 7 (0.6) guarding 0 (0.0) 274 (10.8) 18 (1.6) allo-grooming 16 (4.4) 4 (1.6) 52 (4.7) walking inside 55 (15.3) 478 (18.8) 168 (15.2) antennation 81 (22.5) 229 (9.0) 52 (4.7) digging 0 (0.0) 0 (0.0) 112 (10.1) resting (inside) 83 (23.1) 575 (22.6) 21 (1.9) self-grooming (inside) 26 (7.2) 206 (8.1) 63 (5.7) cleaning floor 0 (0.0) 69 (2.7) 4 (0.4) trophallaxis 1 (0.3) 20 (0.8) 18 (1.6) fungus care 0 (0.0) 14 (0.55) 283 (25.6) fungus licking 90 (25.0) 202 (8.0) 4 (0.4) brood care 0 (0.0) 6 (0.2) 0 (0.0) carcass care 0 (0.0) 4 (0.2) 0 (0.0) laval eating 7 (1.9) 0 (0.0) 0 (0.0) oviposition 1 (0.3) 0 (0.0) 0 (0.0) total observations 360 (100%) 2504 (100%) 1106 (100%) t ab le 1 . c ol on y co m po si tio ns o f m yc et om oe lle ri us tu rr ife x ov er th re e ye ar s. table 2. the frequencies of behavioral observations in each female caste in mycetomoellerius turrifex. inside the nests (22.6%), walking inside the nests (18.8%), and guarding behavior near the nest entrance (10.8%). the most frequent worker behaviors were fungus care (25.6%), walking outside the nests (23.1%), and walking inside the nests (15.2%). the twelve workers observed never performed substrate collection, substrate weeding, and brood care. the canonical discriminant analysis using the frequencies of 24 behavioral repertoires and each female category revealed that each category (inseminated queens, non-inseminated queens, and workers) could be divided with a discrimination rate of 100% (fig 1; axis 1: wilks lambda = 0.031, chi square = 116.04, df = 40, p < 0.0001; axis 2: wilks lambda = 0.301, chi square = 40.20, df = 19, p < 0.01). the behaviors that strongly contributed to axis 1 were fungus garden care and nest floor digging behaviors, and those strongly contributing to axis 2 were defense behaviors and resting in the nest. sociobiology 67(3): 358-363 (september, 2020) 361 discussion the number of queen ants is an important factor in social structure of ants. the numbers have had variable intra and interspecific, but its patterns of variation are adaptive to their environment and rarely change dramatically in a short period (eriksson et al., 2019). exceptions have been reported in invasive ant species, such as fire ants (solenopsis invicta) and argentine ants (linepithema humile), that have been introduced to new areas as a result of human activities. these ants are known to become polygynous in invaded areas even though they are monogynous in their native areas, forming huge supercolonies in invaded areas (helanterä et al., 2009). in my research, the continuous sampling of m. turrifex in texas grasslands for three years revealed a change in the social structure from functional monogyny with a high frequency of non-inseminated queens to simple monogyny. such social structural changes are rare under natural conditions, and might be due to the following factors: (1) environmental changes: in the summer of 1999 there was a record-breaking drought in texas, which resulted in no successful nuptial flights, nesting, or breeding of the newly emerged alate queens. this severe drought resulted in the temporary presence of multiple noninseminated queens in the colonies. (2) synchronization of colony dynamics: colony sizes, numbers of non-inseminated queens, brood numbers, and nest chamber depths tended to follow similar dynamics during the three years of sampling. it is likely that the dynamics of the ant populations in the region would be synchronized (teseo et al., 2013). (3) human impacts: the brackenridge field laboratory in austin, texas has a limited area of 0.33 km2 and it is estimated that the population of m. turrifex within the area is small. four to six colonies were sampled per year. while this is a low number, it is possible that it resulted in sampling pressure on the colonies in 2001, which may have been a factor in increasing the initial stage nests. it is difficult to determine from this study which of the three hypotheses above is the main contributor to the social structural changes in m. turrifex. it is likely that all three factors influenced the ant society. non-inseminated queens are very rarely found in ant colonies. previous studies have reported a few examples of non-inseminated queens in polygynous colonies of formica and solenopsis (bourke & franks, 1995). only myrmecina nipponica (ohkawara et al., 1993; murakami et al., 2002), pyramica hexamera (t. kikuchi, associate professor, chiba university, personal communication, april, 2020), probolomyrmex longinodus (kikuchi & tsuji, 2005), and acromyrmex echinatior (nehring et al., 2012) have been found to have monogynous colonies with extra noninseminated queens. mycetomoellerius turrifex is now the fifth ant species observed to have non-inseminated queens in a monogynous society. fig 1. canonical discriminant analysis using 24 behavioral repertoires in three female castes (inseminated queens, non-inseminated queens, and workers) of mycetomoellerius turrifex. each female caste could be completely identified at a discriminant rate of 100%. white squares represent inseminated queens, black squares represent non-inseminated queens, and white triangles represent workers. t murakami – non-inseminated queens having worker-like behaviors362 this study also shows for the first time that the noninseminated queens have a unique division of labor in the colony, and their labor repertoires are significantly different from that of the reproductive queens and the workers. canonical discriminant analysis revealed that the factor that divided the queens into inseminated and non-inseminated ones was fungus garden care, and the factors that separated the workers from the non-inseminated queens were nest entrance defense, nest floor digging, and resting in the nest. non-inseminated queens did not lay eggs at all and were less active in licking the symbiont fungus, which is the main food resource for queens (quinlan & cherrett, 1979). although the non-inseminated queens were more likely to engage in worker-like behaviors, their behaviors were not entirely consistent with those of the workers that defended the nest entrance by taking advantage of their large body size. these data suggest that the noninseminated queens remained in the colonies and secured their positions by undertaking a certain amount of labor. kikuchi and tsuji (2005) proposed five hypotheses for the existence of non-inseminated queens: (1) mating failure due to a severe environment, (2) social parasitism to produce unfertilized male-destined eggs, (3) strategies to shift the timing of mating to the next season, (4) egg laying of trophic eggs, and (5) staying in the colony as a labor force in a worker-like manner. three of these hypotheses are unlikely to explain the presence of non-inseminated queens in m. turrifex: (2) the social parasite hypothesis, (3) the strategy of shifting the timing of mating, and (4) the trophic egg producing hypothesis. hypotheses (2) and (4) do not hold because the non-inseminated queens did not lay eggs during the observation period and dissections revealed that not even trophic eggs were produced in the ovarioles. also, hypothesis (3) does not hold because the queens in the myrmicinae have wings and mate during their nuptial flights, then they shed the wings. in other words, they are not able to conduct nuptial flight afterwards and it would be impossible to mate with males. our data on the presence of non-inseminated queens in these ant colonies supports two hypotheses: (1) the severe environment hypothesis (due to the 1999 drought in texas) and (5) the labor force hypothesis. in areas at high risk of climate change, such as texas (summer droughts, heat waves, and hurricanes), it is estimated that alate queen ants often fail to mate during nuptial flights and fail to establish new colonies. in such cases, rather than the non-inseminated queens dying, it is beneficial to the colony that they return to their mother’s nest and take on labor. in particular, the relatively larger bodysize of queens would be suited to defend the nest. this is supported by the results of the present study. in contrast, the summer of 2000 had a stable climate in austin, texas, with a sufficiently high chance of nuptial flight and nesting. in such an environment, m. turrifex would be more likely to nest alone and avoid competition for resources. therefore, the temporarily acceptance of non-inseminated queens in m. turrifex is dependent on 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(1993). production and reproductive function of intercastes in myrmecina graminicola nipponica colonies (hymenoptera: formicidae). insectes sociaux, 40: 1-10. doi: 10.1007/bf01338828. sociobiology 67(3): 358-363 (september, 2020) 363 peeters, c. & ito, f. (2001). colony dispersal and the evolution of queen morphology in hymenoptera. annual review of entomology, 46: 601-630. doi: 10.1146/annurev.ento.46.1.601. quinlan, r.j. & cherrett, j.m. (1979). the role of fungus in diet of the leaf-cutting ant atta cephalotes (l.). ecological entomology, 4: 151-160. doi: 10.1111/j.1365-2311.1979. tb00570.x. rabeling, c., cover, s.p., johnson, r.a. & mueller, u.g. (2007). a review of the north american species of the fungus-gardening ant genus trachymyrmex (hymenoptera: formicidae). zootaxa, 1664: 1-53. doi: 10.5281/zenodo.180014. solomon, s.e., rabeling, c., sosa-calvo, j., lopes, c.t., rodrigues, a., vasconcelos, h.l., bacci jr., m. mueller, u.g. & schultz, t.r. (2019). the molecular phylogenetics of trachymyrmex forel ants and their fungal cultivars provide insights into the origin and coevolutionary history of ‘higherattine’ ant agriculture. systematic entomology, 44: 939-956. doi: 10.1111/syen.12370. teseo s., kronauer d.j.c, jaisson p. & châline n. (2013). enforcement of reproductive synchrony via policing in a clonal ant. current biology, 23: 328-332. doi: 10.1016/j.cub.2013.01.011. weber, n. (1972). gardening ants, the attines. philadelphia, american philosophical society, 146 pp. 27 the origin and in situ identification of uncultured gut bacteria in fourth-instar larvae of the red imported fire ant, solenopsis invicta (hymenoptera: formicidae) by albert h. lee1 & linda hooper-bui2 abstract this study corroborated the findings of lee et al. (2008) and relied on culture-independent techniques, particularly 16s rdna sequencing. results from the study demonstrated that ants in captivity completely change their gut bacterial diversity strongly supports the conclusion that fire ant gut bacteria are environmental in origin. bacteria in the fourth-instar larvae were present because they were consumed with food sources. from molecular data, a differing list of bacteria from each colony with different branching and clustering among bacterial clades in the phylogenetic trees were found. different bacterial diversity and evenness was found in all colonies, which provided evidence against the idea of the presence of obligate bactrial endosymbionts in solenopis invicta guts. obligate endosymbiont elimination through the use of broad-spectrum antibiotics did not significant affect ant mortality when compared to the untreated controls. this study did not find bacteriocytes in the midgut epithelium of fire ants. microscopic observations did not locate any bacteriocytes in fourth-instar s. invicta larvae. all identified bacteria appeared to have been derived from the food or the environment. though coadapted bacteria and specialized bacteriocytes were not found in the guts of fourth-instar larvae, such specialized organisms may be present elsewhere in the ant. coadapted symbionts may exist in other parts of the larvae’s body or in other stages of development. key words: bacteria, symbiosis, solenopsis invicta louisiana state university agricultural center 404 life science building, lsu. baton rouge, la 70808 1leeahy@gmail.com 2lhooper@agcenter.lsu.edu 28 sociobiolog y vol. 59, no. 1, 2012 introduction lee et al. (2008) recently described culture-independent identification of gut bacteria in fourth-instar larvae of the red imported fire ant, solenopsis invicta buren. they identified bacteria communities in three locations across louisiana with culture-independent methods in an attempt to identify coadapted mutualistic microorganisms. the data from the earlier study suggest that such organisms are not present in the guts of s. invicta larvae, and that the origin of the bacteria in the guts is environmental (lee et al. 2008). culture-independent molecular techniques, which they used, are a valuable tool in identifying unknown bacteria because they do not rely on selective conditions such as media (lee et al. 2008). in an effort to verify the observation made by lee et al. (2008), three studies were conducted to further explore the possibility of coadapted mutualistic bacteria in s. invicta larvae guts, as well as identifying potential origins. symbiotic associations between organisms are common, and these associations are described as an interaction between multiple organisms living together in intimate association (inside or on the body), or even the merging of two dissimilar organisms (sapp 1994). one type of symbiosis is endosymbiosis. this term refers to an organism associated internally within another organism (e.g. intracellular, inside guts). peloquin & greenberg (2003) investigated bacteria from fire ant guts by culturing gut contents on nutrient and blood agar. data from lee et al. (2008) suggested that their bacterial inventory may be incomplete due to the selective nature of growth media. additionally, li et al. (2005), used selective methods to grow bacteria from fire ants and used molecular techniques to identify what they grew. more than 85% of known naturally occurring bacteria cannot be cultured with currently available techniques (liu et al. 1997). lee et al. (2008) found 65 species of bacteria. as demonstrated by lee et al. (2008), culture-independent molecular techniques provide an opportunity to describe microbial diversity without the need to culture live bacteria (urakawa et al. 1999). these techniques remove biases associated with culturing and provide a comparatively unbiased inventory. the most common molecular approach to identifying yet-to-be cultured bacteria is to sequence the 16s rrna gene (liu et al. 1997). gene sequences can then be 29 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae compared to the public database genbank through blast (altschul et al. 1990) to identify the bacteria. microbial communities in s. invicta were investigated since this invasive ant has the capacity to greatly affect the environment, wildlife, and other invertebrates. the red imported fire ant is a medical, urban, and agricultural pest from south america. it has invaded the southern states of the us, california, puerto rico, australia, taiwan, and china. these invasive ants are successful because of their preference for disturbed habitats, high reproductive rates, and ability to feed on a wide variety of food items (vinson & greenberg 1986). fire ants have painful stings that are a serious health concern for people living in their invaded range. any new biological or physiological information may provide insight into their biolog y and could lead to research on control. the first steps in these investigations are to determine which microbes are important to solenopsis invicta and whether or not they are obligate or transient. the microbes in fire ants may provide an opportunity to control these invasive ants. most fire ant management techniques capitalize on fire ant foraging behavior in which the adult worker ants leave the nest in search of food. workers will find many food types but only consume liquids or minute particles due to their “waist.” they bring solid items to the fourth-instar larvae to be liquefied by digestive enzymes secreted externally (cassill & tschinkel 1995). investigation on this stage of larvae was emphasized to determine if their guts harbored symbiotic bacteria involved in nutrient distribution. the goal of the experiment was to determine if a laboratory diet, which differed from that on which the insects fed in the field, affected the bacterial community in the s. invicta larvae. that question was addressed with a study which focused on an ant colony that was observed in the published study (lee et al. 2008) and was maintained in the laboratory for two and a half months. the three colonies tested by lee et al. (2008) harbored different sets of bacteria in the guts of their fourth-instar larvae; this suggested that the bacteria communities can be changed based on what is fed to the ants. colonies from each unique location in louisiana were exposed to unique environmental conditions. since the bacterial communities appeared to be determined by foods and environment, it would be logical to examine the foods fed to the ants to see if the bacteria were transferred. a logical conclusion would be that bacteria found in ants in captivity would be different 30 sociobiolog y vol. 59, no. 1, 2012 from the time when they were first brought in to the lab due to differences in the environment. despite the great power of molecular techniques, they do have limitations in that abundant dna templates can mask the presence of less abundant ones during amplification. if a bacterium was not represented by a clone in the library that does not necessarily mean that it is not present in the gut microbial community. this leaves the possibility that the presence of symbionts may be masked by the environmentally-derived bacteria. due to this possibility, the problem was investigated with different tactics. ant colony fragments were treated with several broad-spectrum antibiotics and their mortality was studied versus an untreated control. coadapted obligate mutualistic microorganisms, if eliminated, should increase the mortality of s. invicta workers. sauer et al. (2002) found that the “candidatus blochmannia floridanus endosymbiont” in the ant camponotus floridanus is most important during embryogenesis and larval development. elimination of these endosymbionts from the oocytes had a negative effect on embryogenesis and larval development. this is reflected in reduced brood and an increase in mortality of adult ants. ants in the tribe cephalotini have specialized structures in their digestive tract that harbor bacteria (caetano 1989). these bacteria are facultative anaerobic bacteria with digestive functions (yurman & dominiquez-bello 1993). like the carpenter ants, these ants have bacteriocytes with obligate endosymbionts. jaffe (2001) fed antibiotics to cephalotes pusillus and found an increase in mortality. the researchers concluded that some of the bacteria in the digestive tract of c. pusillus were essential to the ant, probably forming a symbiotic relationship with their host. elimination of such bacteria caused an increase in mortality. similarly, if s. invicta are treated with antibiotics and mortality is increased, then a similar set of obligate endosymbionts may be in s. invicta . if mortality is no different from controls then the presented results will be more evidence rejecting the idea of obligate endosymbionts in s. invicta . camponotus floridanus was found to have bacteriocytes containing obligate endosymbionts through the use of transmission microscopy (sauer et al. 2002). obligate symbionts have been found in several ant species to supplement specialized diets. bacteriocytes in the carpenter ant midgut epithelium were full of endosymbiotic bacteria. sauer et al. (2002) believed that the “candidatus 31 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae blochmannia floridanus endosymbiont” in the ant of the species c. floridanus is most important during embryogenesis and larval development. adult ants are able to live without their bacterial endosymbionts under laboratory conditions. these symbionts seem to degenerate naturally over time, as observed in older queens, suggesting that the symbiosis may be of relevance during the early stages of the ants’ development (sauer et al. 2002). the omnivorous c. floridanus is also capable of feeding almost exclusively on sugary fluids due to their symbionts. candidatus blochmannia floridanus is able to supply nitrogen and sulfur compounds to the host through the host’s metabolic machinery (gil et al. 2003). members of the genus candidatus blochmannia are closely related to other endosymbiotic bacteria of insects such as buchnera of aphids, wigglesworthia of tsetse flies, and, more distantly, carsonella of psyllids. these bacteria form a clade contained within the family enterobacteriaceae within the phylum proteobacteria (sauer et al. 2002). the presence of bacteriocytes and the visibility of the bacteria provided unquestionable proof of the presence of endosymbiotic bacteria in other ants. fourth-instar larvae of red imported fire ants, the stage that can process solid foods (vinson & sorenson 1986) was the focus of this study. after experimenting with culture-independent methods and antibiotic mortality tests, the next step was to physically examine the guts of larvae and search for bacteriocytes similar to those found in c. floridanus. materials and methods antibiotic efficacy at the beginning of the experiment, larvae were collected and their guts aseptically removed and plated on bhi and bhi-kanamycin media. this was done to check for the antibiotics’ effectiveness at killing bacteria. the same was done on day 13 and day 25 to check for antibiotic resistance. effect of laboratory diet of the three colonies used by lee et al. (2008), the colony from bogalusa was the only one that still had queens and brood after two and a half months in captivity. as the sole-survivor of the three colonies initially studied, it could be accessed again for a follow-up analysis. fire ant colonies were reared in plastic boxes with teflon as described in lee et al. (2008). food and water 32 sociobiolog y vol. 59, no. 1, 2012 were provided in the form of dead crickets, tap water, and 50% sugar water. rearing methods were based on the protocol described by banks et al. (1981). all food sources and water were replenished as needed. all the available fourth-instar larvae (32) were taken out of the colony followed by aseptic isolation of their guts. larval guts were then homogenized in a common reaction tube and bacteria dna was extracted with the qiagen dneasy tissue kit. the dna was used as a template to perform 16s rdna pcr utilizing the methods described in lee et al. (2008). amplification was done with the universal 16s rrna primers 27f and 1492r (lane 1991). pcr products were purified and a clone library was established utilizing e. coli. clones with the 16s rdna were selected and m13 pcr was performed to amplify the dna of interest. the li-cor 4300 dna analyzer used to sequence the colonies described in lee et al. (2008) was malfunctioning at this point in the experiment so amplified ribosomal dna restriction analysis (ardra) was used in conjunction with a commercial sequencing service. detailed description of methods used for dna extraction, pcr, cloning, phylogenetic tree construction, and species accumulation curve construction can be found in lee et al. (2008). ardra required the 16s rdna fragments to be restricted to create unique banding patterns. purified 16s rdna products were digested with haeiii and rsai restriction enzymes to generate banding patterns for each clone. each digestion reaction contained 15 μl of pcr product, 2 μl of 10x nebuffer 1 (new england biolabs, beverly, massachusetts), 3 μl pcr quality water, 0.25 μl of rsai (2.5 units), and 0.25 μl of haeiii (2.5 units). each reaction was incubated in an mj research ptc-200 peltier thermal cycler. the incubation program consisted of 2 h at 37˚c, 20 min at 80˚c, and finally a storage temperature of 4˚c. incubated products were then loaded onto a 1% agarose gel and banding patterns were examined visually. clones with unique patterns were isolated and sequenced at the university of california, riverside’s core instrumentation facility. once the sequences were returned, vector nti version 7 was used to form contiguous sequences. sequences were then compared to entries in genbank through blast. once clone identities were established, a phylogenetic tree and species accumulation curves were created using mega 3.1 (kumar et al. 2004) and estimates (colwell 1994-2004), respectively. 33 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae antibiotic trials an ant colony fragment from an established laboratory colony was used. this colony was field-collected on february 23, 2005 (469 days). multiple queens and a large number of brood were available in the colony. larvae were numerous enough for the three treatments and controls, each with ten replicate colony fragments. three treatment sets represent the three antibiotics tested. each replicate had colony fragments of ~50-100 workers (counted at the end of the study), and five fourth-instar larvae and about ten larvae with an approximate age of first to third instars. control colony fragments consisted of one set with ten boxes (replicates) and were set up similar to treatment boxes. each treated colony fragment received water and antibiotics suspended in 50% sugar water, and freshly killed crickets. all food and water resources were replenished every day except for crickets. crickets were replaced when consumed or after one week. control colonies were not provided antibiotic solutions but were given all of the other food and water resources. plastic boxes (17 x 8 x 4 cm) were coated with teflon to prevent the ants from escaping the experimental enclosure. for treatment colony fragments, each coated box was furnished with two crushed crickets, 900 µl of water and 900 µl of 5% antibiotic solution (in treatments only) mixed with 50% sugar water in 6 x 50 mm borosilicate culture tubes plugged with moist cotton. harborage sites were provided for all colony fragments in the form of plaster of paris nests. each nest was constructed from a petri dish (3.5 cm in diameter) filled half-way with plaster of paris. a cotton shoestring (~ 6cm) was partially embedded in the plaster to facilitate moistening of the plate without disturbing the ants. once dry, the sides of the dishes were drilled to provide entry holes for the ants. three antibiotics were tested in this study; these included kanamycin, tetracycline, and rifampicin. the antibiotic was found to be effective against the endosymbionts of those ants. antibiotics were mixed with 50% sugar water to produce a 5% antibiotic solution. worker mortality was counted every day until the last larva pupated. the last larva pupated after 28 days of observation. dead ants were removed from each box after they were counted. at the end of the experiment, the live ants were killed and counted to determine the total number of ants in each repli34 sociobiolog y vol. 59, no. 1, 2012 cate for mortality percentages. sas version 9.1 (sas institute inc. 2002) was used to determine statistical differences between control and treated colonies. proc-glm was used to generate the residuals and univariate procedure was used to determine normality. the data passed the test for normality (sas institute 2002). mortality of antibiotic-treated adult ants for each day was compared to each other and the control using proc-mixed, which included an analysis of variance (anova) and the tukey-kramer analysis. larvae were examined and counted every day; once they all eclosed as adults the replicate was terminated. microscopy the ants used were from an established laboratory colony from february 23, 2005 and the experiment began on april 16, 2007 (782 days post collection). twenty fourth-instar larvae were taken from the colony and dissected for microscopy. the louisiana state university’s socolofsky microscopy center prepared the samples and processed the larval guts for microscopy after aseptic removal of the guts. multiple guts (five) were submitted to the facility to compensate for any errors that may occur during microscopy preparation. four gut samples were used to take images. light and electron microscopy were used to examine gut structures. three ant guts provided the best images with the clearest structures. entire guts of s. invicta fourth-instar larvae guts were pulled out and fixed in 2.5% glutaraldehyde and 1% paraformadehyde in 0.2 m cacodylate buffer (ph 7.2) and incubated at room temperature for 2 hours. after the incubation period, the mixture was post-fixed in 1% osmium tetroxide (oso 4 ) buffer for one hour, followed by a 0.1 m cacodylate buffer wash. upon completion of the wash, the mixture was placed in 1% uranyl acetate for block staining. the materials were dehydrated in an ethanol series and embedded in medium grade lr white resin (london resin company ltd., england). once in place, the resin was dried in carbon dioxide using a denton dcp-1 critical point drying apparatus. blocks of resin were cut with a microtome to produce 90 nm thin sections. thin sections were stained with lead citrate and then examined with a jeol jem 100cx transmission electron microscope (tem). the same preparations were also used for standard light microscopy. two ant guts were successfully prepared for scanning electron microscopy (sem). they were fixed and dehydrated with the same methods described 35 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae above. the guts were mounted on stubs, dissected, and coated with 25 nm of gold palladium using a hummer ii sputter coater, and examined on a cambridge s-260 scanning electron microscope. results effect of the laboratory diet bacteria initially found in the ants when they were collected in the field were no longer present after being reared in the laboratory (tables 1 and 2). the species accumulation chao1 curve of the lab-reared colony (fig. 1) leveled off at sample 111, which suggested that increased sampling should not reveal more bacteria species. chao1 indicated that between 47-96% of the actual bacterial richness was identified. the percentage range was obtained by dividing the number of unique species by the lower and upper bounds of the chao1 estimator 95% confidence interval. rare-species based estimator jack1 indicated that between 56-77% of the bacterial richness was identified. both the ace and ice estimators indicated that about 47-57% of the bacterial richness was identified. fig. 1. species accumulation analysis of the 16s rdna clone library from the lab-reared bogalusa s. invicta colony 36 sociobiolog y vol. 59, no. 1, 2012 table 1. ncbi blast results for the 16s rrna gene from 130 clones from the colony collected from the lab-reared bogalusa colony. closest match in genbank accession number % identity to closest match number of clones enterobacter cloacae aj251469 >99% 86 lactococcus garvieae isolate 36 ay946285 >99% 29 citrobacter freundii strain brn1 dq517285 >99% 3 enterococcus faecalis atcc 19433 dq411814 >99% 2 enterobacter cloacae subspecies dissolvens dq988253 >99% 2 uncultured bacterium p2d1-740 ef510442 >99% 2 lactococcus garvieae nric 0612 ab267905 >99% 1 enterococcus faecalis tua 2495l ab098122 >99% 1 bacterium spbl1 type3-1 dq321639 >99% 1 klebsiella sp. gr9 dq100465 >99% 1 enterobacter aerogenes taxon 548 af395913 >99% 1 uncultured bacterium rrna238 ay959011 >99% 1 the name, accession numbers, closest matches in genbank, and the number of clones identified to the species (category) are displayed. table 2. ncbi blast results for the 16s rrna gene from 104 clones from the field-fresh colony collected from bogalusa. closest match in genbank accession number % identity to closest match number of clones uncultured bacterium f4s2 dq068792 >99% 94 bacterium 2-4 dq163943 >99% 3 kluyvera cryocrescens 27 ay946284 >99% 1 pseudomonas fluorescens ost-5 dq439976 >99% 1 acinetobacter sp. dsm 590 x81659 >99% 1 serratia marcescens kred ab061685 >99% 1 klebsiella pneumoniae 573 ay830396 >99% 1 serratia marcescens cpo1(4) aj296308 >99% 1 uncultured bacterium f6s10 dq068816 >99% 1 the name, accession numbers, closest matches in genbank, and the number of clones identified to the species (category) are displayed. (data from lee et al. 2008) 37 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae bacteria from this colony were not closely related to the symbionts of carpenter ants, aphids, and tsetse flies, which formed a unique and well-supported branch in the phylogenetic tree (fig. 2). tree ant (tetraponera spp.) symbionts were found in neighboring clades. antibiotic trials mortality occurred evenly throughout the experiment (fig. 3), and survivorship curves represented type ii mortality survivorship (fig. 4). larvae were not attacked or eaten by the adult ants. larval remains were not found in any of the boxes. the controls had a worker mortality range between 30-93% after 28 days; mortality values averaged 78% when the experiment ended. ants that were given tetracycline exhibited a mortality range between 68-98% with an average of 83% at the end of the data collection period. ants treated with kanamycin experienced a mortality range of 25-98% with an avfig. 2. phylogenetic affiliation of the bacteria found in the bogalusa colony after 82 days. after 82 days. dotted species represent bacteria found in the lab-reared colony. all other entries are type strains or endosymbionts found in other insects. 38 sociobiolog y vol. 59, no. 1, 2012 fig. 3. mortality curve of untreated and treated s. invicta. fig. 4. survivorship curve of untreated and treated s. invicta. 39 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae erage of 74% at the end of the experiment. whereas ants received rifampicin demonstrated a mortality range of 54-92% with an average of 78% at the end of the data collection period. the analysis indicated that in the 28 days of antibiotic treatment, significant differences occurred on six days (days: p < 0.0001, df = 27, 983, f stat = 75.59, treatments: p < 0.0001, df = 3, 938, f stat = 24.81[sas 1999]). significant differences occurred between the control and rifampicin sets on days 8, 9, 10, and 15 (table 3). the control differed from tetracycline on days 17 and 18. aside from these six instances, there were no significant differences between the treatments and the controls. the treatments themselves were not significantly different from each other on any days. adult ant mortality was plotted against time (days) in fig. 3. the confidence intervals from each day overlapped each other in every treatment and control. microscopy hindgut images (fig. 5) revealed similar structures (fig. 5a) with the addition of circular muscles (fig. 5b) and bacteria (fig. 5c) near the gut lining. cross-sections of the hindgut (fig. 5) and foregut/midgut (fig. 6) did not contain any bacteriocytes. visceral muscles in the gut epithelium were clearly visible. scanning electron micrographs of the foregut/midgut (fig. 6) revealed that the structure was filled with bolus and fluids fed to the larvae by the workers. portions of the epithelium can also be seen. bacteriocytes were not observed in the scanning or transmission electron micrographs. hindgut scanning micrographs showed that the hindgut had structures similar to the foregut/ midgut (figs. 7 and 8) and no evidence of bacteriocytes. transmission electron micrographs of the hindgut also did not contain bacteriocytes (fig. 8). discussion effect of laboratory diet the study successfully inventoried bacteria in fourth-instar s. invicta larvae table 3. significant differences detected with the tukey-kramer analysis between treatments. treatment day p-value control vs. rifampicin 8 0.0497 control vs. rifampicin 9 0.0401 control vs. rifampicin 10 0.0426 control vs. rifampicin 15 0.0403 control vs. tetracycline 17 0.0318 control vs. tetracycline 18 0.0473 40 sociobiolog y vol. 59, no. 1, 2012 guts that have been lab-reared for 82 days. with the lab diet, the inventory of bacteria found in the ant guts completely differed from the one obtained when the colony was first brought in from the field (lee et al. 2008). such results meant that the laboratory diet contained a different bacterial inventory than the field diet. bacteria from the diet could completely replace those in the ant guts. comparisons between tables 1 and 2 indicated that no common species were found among the larvae in the colonies, despite using the same colony during both studies. the bacterial inventory was strongly affected fig. 5. light microscopy cross-section images of a fourth-instar larva’s hindgut. letters represent magnified areas in the hindgut epithelium and lumen. a. epithelial cell in the gut wall. b. circular muscles in the gut wall. c. bacteria in the lumen near the gut wall. 41 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae by laboratory rearing. the results suggested that bacteria associated with the laboratory diet of crickets and sugar water or the environment may have transferred to the ants. enterobacter cloacae and lactococcus garvieae may have been in high abundance in the laboratory diet. the presence of bacteria in the ant diet was not checked during this experiment. the species accumulation chao1 curve (fig. 1) leveled off at sample 111, which suggested that increased sampling should not reveal more bacteria species. chao1 indicated that between 47-96% of the actual bacterial richness was identified. the percentage range was obtained by dividing the number of unique species by the lower and upper bounds of the chao1 estimator 95% confidence inter val. rarespecies based estimator jack1 indicated that between 56-77% of the bacterial richness was identified. both the ace and ice estimators indicated that about 47-57% of the bacterial richness was identified. bacteria from this colony were not closely related to the symbionts of carpenter ants, aphids, and tsetse flies, which formed a unique and wellsupported branch in the phylogenetic tree (fig. 2). tree ants (tetraponera spp.) symbionts were found in neighboring clades. the results suggested that bacteria associated with the laboratory diet of crickets and sugar water or the environment may have transferred to the ants. this conclusion is supported by the work of jouvenaz et al. (1996). they fig. 6. scanning electron micrograph of the foregut/ midgut of a fourth-instar lar va longitudinal section. fig. 7. scanning election micrograph of the hindgut of a fourth-instar larva. 42 sociobiolog y vol. 59, no. 1, 2012 contaminated food with serratia marcescens and fed it to fourth-instar larvae. the s. marcescens were isolated from all tested larval midguts. data from lee et al. (2008) and that of jouvenaz et al. (1996) support the conclusion that the bacteria in the guts of fourth-instar fire ant larvae are composed of environmental bacteria. enterobacter cloacae are commonly found in human and animal feces and are not an enteric pathogen (bergey 1984). however, e. cloacae are an fig. 8. cross-section of the hindgut of a fourth-instar larva. 43 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae opportunistic pathogen capable of infecting the urinary tract, sputum, and respiratory tract of animals. as a fecal bacterium it is very common in water, sewage, and soil (bergey 1984). enterobacter cloacae’s presence in the inventory was not surprising since crickets could have easily picked them up as they foraged for food or simply by interacting with the environment. attempts were made to establish how the crickets were fed and reared by the supplier (fluker farms, port allen, la) but the company never responded. other identified bacteria in the inventory fell into the same niche as e. cloacae. these include enterococcus faecalis, klebsiella spp., uncultured bacterium rrna238, and uncultured bacterium p2d1-740. both uncultured bacteria identified in this study had similar dna sequences to fecal bacteria. lactococcus garvieae was the second most abundant bacteria in the inventory. identified as a major pathogen of fish (eldar et al. 1999) and a causative agent for bovine mastitis (bergey 1984), this bacterium may be present in soil and contaminated water. all three field-fresh colonies examined did not have a single clone representing l. garvieae. the bacterium’s presence clearly came from the laboratory food or laboratory environment. preliminary studies conducted while examining the bacterial community of whole larvae that have been reared in the lab for two weeks also had large numbers of l. garvieae in their systems. this study was not the first one to identify l. garvieae in s. invicta . peloquin & greenberg (2004) and li et al. (2005) found this bacterium in their culture-dependent methods. studies done by these researchers had one thing in common with our lab-reared colony. all of the colonies do not appear to be fresh from the field. antibiotic trials results from the antibiotic trials indicated no significant difference in overall mortality between control and treated colony fragments. worker ant mortality was not visibly affected by clearing most of the bacteria in the guts of fourth-instar larvae. likewise, larvae mortality was not visibly affected either. there were no larval skins found in the boxes, indicating that cannibalism did not occur. both larvae and workers appeared healthy during the duration of the study, which suggests that dosage of the drugs used had no adverse affects on the ants. all of the ants were both active and quick and did not appear sluggish or slow. bacteria with drug resistance genes were the only factor that may have affected the results. 44 sociobiolog y vol. 59, no. 1, 2012 when similar antibiotic experiments were done on cephalotes pusillus, adult mortality effects were observed after 24 hrs. jaffe et al. (2001) concluded that ant mortality was probably caused by the antibiotic killing one, some, or all of the associated bacteria that are susceptible to kanamycin. some of the bacteria in the digestive tract of c. pisillus are essential to the ant, probably forming a symbiotic relationship with the host ( jaffe et al. 2001). our study not only included kanamycin, but also tetracycline and rifampicin and found no significant differences in daily mortality in the ants except in six cases. time periods before and after these days were no different from each other and the control. differences found were determined to be outliers and the treatments and control were not significant different from each other. even though the presence of antibiotic-resistant bacteria may be available in this fire ant population, it is assumed that the majority of the bacteria in the larvae were not antibiotic resistant or if they were, they would not be resistant to all three antibiotics tested. from the data it would appear as though there are no obligate endosymbiotic bacteria in the guts of fourth-instar fire ant larvae that are important for survival. however, there are many species that are transient in nature. molecular data suggests that all bacteria inventoried from larvae was environmental in origin and were present in the guts because they were consumed by the ants (lee et al. 2008). a broad spectrum antibiotic that has been shown to have an effect on c. pusillus mortality had no significant effect on s. invicta . additionally, two other antibiotics demonstrated no adverse affects on s. invicta workers and larvae. however, li et al. (2005) found that certain bacteria (all but an uncultured bacterium and bacillus punilus) isolated from s. invicta were resistant to antibiotics (ampicillin, chloramphenicol, doxycycline, erythromycin, gentamycin, kanamycin, kalidixic acid, neomycin, spectinomycin, streptomycin, tetracycline, or zeocin). they found one bacteria that was resistant to all the drugs tested. in their study, most were susceptible to various drugs and only two were resistant to both kanamycin and tetracycline (li et al. 2005). these data suggest that the larval guts in this study may not have been rendered completely bacteria free. in the future, an antibiotic cocktail may be necessary to test on the ants since the ants would be closer to being bacteria-free. 45 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae microscopy the absence of specialized structures with obligate endosymbionts is evidence that bacteria in fourth-instar larvae are present because they were brought by the workers and fed to the young. only epithelial and muscle cells were found in the lining of guts of s. invicta larvae. gut lumen contained bacteria, fluids, and bolus. fourth-instar larvae are believed to be the only members in a s. invicta colony that can digest solid food. such abilities may be due to glands in the gut that secrete digestive compounds instead of specialized gut organisms that facilitate the process like protozoans in termites. experimental data from the study provided evidence of environmental sources of bacteria in s. invicta . microscopy did not find bacteria in bacteriocytes or even bacteriocytes themselves; bacteria were found in the lumen of the hindgut. carpenter ants may be capable of functioning as herbivores who subsist on a diet of sugary water causing the ants to rely on their obligate endosymbionts. candidatus blochmannia floridanus, the obligate endosymbiont in carpenter ants, is able to supply nitrogen and sulfur compounds to the host through the host’s metabolic machinery (gil et al. 2003). the endosymbiont makes it possible for the carpenter ant to feed exclusively on sugars and still be able to survive and multiply by providing nitrogen and sulfur. an alternative feeding strateg y may not be present or necessary in s. invicta . though coadapted bacteria and bacteriocytes were not found in the guts of fourth-instar larvae, such specialized organisms may be present elsewhere in the ant. coadapted endosymbionts may be present in the hemolymph, fat body, or other organs in fourth-instar larvae. adult ants may contain coadapted endosymbionts that help the colony survive by providing nutrients or other useful compounds. the reason for the lack of obligate symbiosis may be due to diet. s. invicta’s diet consists of diverse food items and rarely focuses on a narrow nutrient source such as honeydew. workers also collect honeydew and will forage for sweets, proteins, and fats (vinson & sorenson 1986). fire ants acquire all their essential nutrients from their diet. unlike termites, tetraponera spp., and camponotus floridanus, s. invicta may not need specialized obligate symbionts to supply it with supplemental nutrients. s. invicta gut bacteria communities are contributed by substances ingested from the environment. 46 sociobiolog y vol. 59, no. 1, 2012 conclusion observations from this study corroborated the findings of (lee et al. 2008) and relied on culture-independent techniques, particularly 16s rdna sequencing. ants in captivity completely change their gut bacterial diversity strongly supports the conclusion that gut bacteria are environmental in origin. studies performed with antibiotics and microscopy support the conclusion drawn from the 16s rdna sequencing work. the null hypothesis that fourth-instar larvae do not contain coadapted bacteria and that all the bacteria identified were environmentally derived was supported in every case. bacteria in the fourth-instar larvae were present because they were consumed with food sources. from molecular data, a different list of bacteria from each colony with different branching and clustering among bacterial clades in the phylogenetic trees was found. different bacterial diversity and evenness was found in all colonies, which provided evidence against the idea of the presence of obligate endosymbionts in s. invicta guts. direct methods described above did not indicate the presence of endosymbionts. likewise, indirect approaches came up with the same results. if the fire ants had obligate endosymbionts then clearing (or nearly clearing ) the guts with antibiotics should have a detrimental effect on them similar to that observed by jaffe et al. (2001) in c. pusillus. clearing out endosymbionts with antibiotics increased mortality in c. pusillus. the three broad-spectrum antibiotics that were tested provided no significant differences in ant mortality when compared to the untreated controls. following this method of study was light and electron microscopy. unlike sauer et al. (2002), who found bacteriocytes in carpenter ants, our study did not find bacteriocytes in the midgut epithelium of fire ants. microscopic observations did not locate any bacteriocytes in fourth-instar s. invicta larvae. all identified bacteria appeared to have been derived from the food or the environment. though coadapted bacteria and specialized bacteriocytes were not found in the guts of fourth-instar larvae, such specialized organisms may be present elsewhere in the ant. coadapted symbionts may exist in other parts of the larvae’s body or in other stages of development. however, the fourth-instar larvae remains the primary stage responsible for the digestion of solid food and the source of nutrients for the queen and adult workers through the production 47 lee, a.h. & l. hooper-bui — identification of gut bacteria in fire ant larvae of chymotrypsin-like and elastase-like proteinases (whitworth et al. 1998). in termite and carpenter ant examples, nutrients and enzymes that facilitate food digestion can be from obligate endosymbionts. solenopsis invicta may have other types of obligate endosymbionts in the form of protozoans and fungi in their systems however, based on the results, these proteinases are likely from the gut glands of s. invicta . acknowledgments we thank cindy henk and ying xiao from louisiana state university’s socolofsky microscopy center for her help in preparation of the ants for microscopy. the authors are grateful for helpful comments on this manuscript from drs. fred enright, claudia husseneder, christopher carlton, gregg henderson, and lane foil. references altschul, s.f., w.gish, w. miller, e.w. myers & d.j. lipman 1990. basic local alignment search tool. journal of molecular biolog y 215: 403-410. banks, w.a., c.s. lofgren, d.p. jouvenaz, c.e. stringer, p.m. bishop, d.f. williams, p.d. wojcik & b.m. glancey 1981. techniques for collecting, rearing and handling imported fire ants. u.s. department of agriculture science and education administration advances in agricultural technolog y aat-s-21. bergey, d.h. 1984. bergey’s manual of systematic bacteriolog y, volume 1. lippincott williams and wilkins. caetano, f.h. 1989. endosymbiosis of ants with intestinal and salivary gland bacteria. in schemmler w, editor. insect endocytobiosis, morpholog y, physiolog y, genetics, and evolution, pp. 58-76. crc press. cassill, d.l & w.r . tschinkel 1995. allocation of liquid food to larvae via trophallaxis in colonies of the fire ant, solenopsis invicta. animal behavior 50: 801-813. colwell, r.k., c.x. mao & j. chang 2004. interpolating, extrapolating, and comparing incidence-based species accumulation curves. ecolog y 85: 2717-2727. colwell, r.k. 1994-2004. estimates: statistical estimation of species richness and shared species from samples. available online: http://viceroy.eeb.uconn.edu/estimates. 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both sexes are diploid and hemimetabolous (gallo et al., 2002). the genus nasutitermes is the largest in the world in terms of number of species and occurs in bolivia, venezuela, paraguay, uruguay and brazil. in brazil, its species can be found in regions of tropical forest, the cerrado and caatinga. given its adaptability to urban settings, n .corniger is considered the primary pest in the genus nasutitermes abstract lippia is one of the main genera in the family verbenaceae, with 200 species described. despite its richness in bioactive molecules, with several scientifically proven applications, there is little information on the insecticidal potential of its species. this study aimed to assess the insecticidal potential of essential oils from the species lippia thymoides (martius & schauer); lippia lasiocalycina (schauer) and lippia insignis (moldenk) against nasutitermes corniger (motschulsky) (isopteratermitidae). insecticidal activity was evaluated by exposure to a contaminated surface, whereby plastic pots were lined with filter paper and imbibed in 1.5 ml of solution containing essential oils (10 µl/ml), with 10 n. corniger specimens per pot. the mortality count was performed at 24 and 48 h. the lc 50 was determined by diluting the essential oils to concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 µl/ml, which were chemically analyzed by gc-fid and gc-ms. the data indicated high toxicity for the essential oils for the lippia species tested. the lowest lc 50 (0.46 µl/ml) was recorded for l. lasiocalycina. the most common constituents were β-myrcene and (e)-ocimenone in essential oil of l. lasiocalycina, β-myrcene and limonene for l. insignis, and (e)-caryophyllene and caryophyllene oxide for l. thymoides. the results demonstrate the viability of developing biopesticides for n. corniger control. sociobiology an international journal on social insects dr santos1, lm oliveira2, am lucchese3, af espeleta3, jd cruz2 , ms lordêlo3 article history edited by evandro nascimento silva, uefs, brazil received 30 january 2020 initial acceptance 09 february 2020 final acceptance 10 february 2020 publication date 30 june 2020 keywords verbenaceae, lippia lasiocalycina, lippia insignis, lippia thymoides, termites, biopesticide. corresponding author daiane rodrigues dos santos graduate program in plant genetic resources feira de santana state university (uefs) av. transnordestina s/nº, novo horizonte feira de santana, bahia, brasil. e-mail: daibio@hotmail.com.br and causes significant economic losses, particularly in the construction and furniture industries (constantino, 1999; constantino, 2002). organo-synthetic insecticides such as bifenthrin, cypermethrin, chlorfenapyr, fipronil, imidacloprid and permethrin are the most widely used worldwide to control nasutitermes, but their harmful effects have prompted a growing search for new alternatives to combat these pests (lima et al , 2013; specht et al., 2014). in this respect, natural plant-based products have proved to be highly effective at controlling termites, as well as being economically viable and having less impact on the environment and human health (verma et al., 2009; cruz et al., 2012). the most prominent of these are essential oils, whose proven biological functions in the plant kingdom include attracting pollinating agents, 1 graduate program in plant genetic resources, feira de santana state university (uefs), bahia, brazil 2 departament of biological sciences, feira de santana state university (uefs), bahia, brazil 3 departament of exact sciences, feira de santana state university (uefs), bahia, brazil research article termites insecticidal activity of essential oils of species from the genus lippia against nasutitermes corniger (motschulsky) (isoptera: termitidae) sociobiology 67(2): 292-300 (june, 2020) 293 repelling insects and protecting against certain plant pathogens (rozwalka et al., 2008; silva et al., 2015; miranda et al., 2016), in addition to being widely used in the chemical, pharmaceutical, food and cosmetics industries (pascual et al., 2001). lippia is one of the most important genera of the family verbenaceae, with 200 species described and widely distributed in the neotropics, brazil being the largest endemic region (81 species described) (flora do brasil, 2017). some lippia species have proven biological activity, such as the antimicrobial and antioxidant properties of lippia origanoides (pinto et al, 2013; teles et al., 2014) or the spasmolytic, anti-diarrheal and antimicrobial activity of lippia thymoides (silva et al, 2015; menezes et al, 2018). the insecticidal efficiency of this genus has been reported for the species lippia gracilis and lippia sidoides against sitophilus zeamais (castro coitinho et al., 2006), aedes aegypti and culex quinquefasciatus (costa et al, 2005), respectively. although these studies reinforce the insecticidal properties of lippia species against other classes of arthropods, research on the insecticidal potential of other representatives of this genus remains scarce, particularly against termites (evans & iqbal, 2015; santos et al., 2017). thus, the present study aimed to investigate the chemical composition and insecticidal potential of essential oils of the species lippia lasiocalycina, lippia insignis and lippia thymoides to control nasutitermes corniger motsch (isoptera: termitidae). materials and methods plant material the plant species used in the experiments were obtained from the medicinal and aromatic plant collection of the forest garden (horto florestal) experimental unit belonging to feira de santana state university (uefs). the following were identified through exsiccates of the species deposited in the institutional herbarium (huefs): lippia thymoides mart. and schauer 115371; lippia lasiocalycina cham. – 197676 and l. insignis moldenke – 197674. leaves were collected during flowering, between 07:00 and 08:00 a.m., and dried out in the dark at room temperature for 10 days. essential oil (eo) extraction the eos were extracted by hydrodistillation, as described by teles et al. (2014), with some modifications. the dried leaves (200 g) were ground in a blender and extracted for 3h in a modified clevenger-type apparatus, using enough distilled water to completely cover the plant material. the oils extracted were dried out with anhydrous sodium sulfate. the eo content was calculated on a dry weight basis using the equation described by santos et al. (2004): u the moisture content or water present in the biomass, and 100 the percentage conversion factor. determining chemical composition in order to determine the chemical composition of the eos, 20 mg of oil was previously diluted in 1 ml of dichloromethane. quantification was performed by gas chromatography with a flame ionization detector (gc-fid) andthe components were identified by gas chromatographymass spectrometry (gc-ms). a varian® cp–3380 chromatograph equipped with an fid and chrompack cp-sil 5 capillary column (30 m x 0.5 mm) was used for cg-fid analyses, with a film thickness of 0.25 μm, injector and detector temperatures of 220 °c and 240 °c, respectively, and helium as carrier gas (1 ml.min-1). the oven temperature program was60 to 240 °c (3 °c.min-1), with a 240 °c isothermal run for 20 min.the gc-ms analyses were carried out in a shimadzu® gc-2010 chromatograph coupled to a shimadzu® gc/ms-qp 2010 mass spectrometer, equipped with a db-5ms capillary column (30 m x 0.25 mm, film thickness of 0.25 μm), using an injector temperature of 220 °c, helium as carrier gas (1 ml.min-1), 240 °c interface temperature and ionization source, 70 v electron ionization, 0.7 kv voltage, and a similar temperature program to that described above. the components were identified by calculating kovats retention indices and comparing these and the mass spectra to reference standards and literature data (adams, 2007). the relative percentages of the constituents were calculated by peak area normalization. insecticidal activity analysis the insects were transported to the laboratory and placed in transparent 500 ml plastic containers, sealed with voile held in place by an elastic band. to prevent contamination, the containers were kept in plastic trays with water. a sample of the termites was identified and deposited in the uefs zoology museum (mzuefs). insecticidal activity was evaluated by exposure to a contaminated surface, using the eos of lippia species at 1% concentration, diluted in water, with tween 20 as emulsifier. ten n. corniger individuals were placed in 140 ml plastic containers (7.5 x 6.5 cm) lined with filter paper imbibed in 1.5 ml of solution containing the essential oils. to better accommodate the insects, wood shavings from the tree where they were collected and fragments of their own nest were placed in the containers. the containers were sealed with voile held in place by an elastic band, kept in a room at ambient temperature throughout the experiment, and covered with black fabric to protect them from the light. insect mortality was assessed 24 and 48 hours after treatment application. insects that moved any part of their body, even when stimulated, were considered alive (santos et al., 2007). where: to is oil content in %, vo the volume of oil in ml, bm the plant biomass (g), dr santos, lm oliveira, am lucchese, af espeleta, jd cruz, ms lordêlo – lippia (verbenaceae) essential oils as potential termiticides294 a completely randomized design, consisting of 6 treatments (concentrations) for each lippia species and 4 repetitions, was used, with each repetition represented by one container housing 10 insects. determining the median lethal concentration (lc50) to determine the lowest lethal concentration capable of killing 50% of the individuals (lc50), the eos were diluted in water to obtain six different concentrations (0; 0.625; 1.25; 2.5; 5; 10 μl/ml), using tween 20 as dispersing agent. four repetitions were used per treatment, each consisting of one container holding 10 insects. the tween solution was used as control, in the same proportion. insecticidal activity analysis insecticidal activity was assessed using adult nasutitermes corniger motschulsky termites collected from nests in brazil wood trees (caesalpiniae echinata lam). the insects were transported to the laboratory and placed in transparent 500 ml plastic containers, sealed with voile held in place by an elastic band. to prevent contamination, the containers were kept in plastic trays with water. a sample of the termites was identified and deposited in the uefs zoology museum (mzuefs). insecticidal activity was evaluated by exposure to a contaminated surface, using the eos of lippia species at 1% concentration, diluted in water, with tween 20 as emulsifier. ten n. corniger individuals were placed in 140 ml plastic containers (7.5 x 6.5 cm) lined with filter paper imbibed in 1.5 ml of solution containing the essential oils. to better accommodate the insects, wood shavings from the tree where they were collected and fragments of their own nest were placed in the containers. the containers were sealed with voile held in place by an elastic band, kept in a room at ambient temperature throughout the experiment, and covered with black fabric to protect them from the light. insect mortality was assessed 24 and 48 hours after treatment application. insects that moved any part of their body, even when stimulated, were considered alive (santos et al., 2007). a completely randomized design, consisting of 6 treatments (concentrations) for each lippia species and 4 repetitions, was used, with each repetition represented by one container housing 10 insects. statistical analysis given the lack of variability in the results of some treatments, at both 24 and 48h, the initial insecticidal activity data were submitted to nonparametric analysis via the kruskal wallis test for multiple comparisons of the treatments (doses administered). significance was set at 5% probability. tests in which different doses or concentrations of a drug are administered to individuals are known as doseresponse analyses (demétrio, 2001), in which the status of these individuals changes within a certain period after administration (a dead insect indicates success and survival, failure). the aim of these experiments is generally to model the chances of success and determine effective or lethal concentrations (lcs). nonlinear logistical regression with two parameters (β1 and β2) is used to estimate this probability. in order to estimate the lowest lethal concentration capable of killing 50% of the individuals (lc50), the logistical model is linearized using the logit function. thus, lc50 is determined by dividing the two parameters in the model: lc50 = β1 / β2 all the results were obtained using r software (r core team, 2017). results eo content and chemical composition the highest essential oil content was obtained in l. thymoides (2.9%), followed by l. lasiocalycina (1.8%) and l. insignis (1.7%). analyses of the eo chromatograms of l. thymoides, l. lasiocalycina and l. insignis identified 83.80, 96.30 and 95.40 (%) of the chemical compounds, respectively. the major compounds observed in l. thymoides eos were sesquiterpenes, namely (e)-caryophyllene (29.55%), caryophyllene oxide (8.17%), germacrene d (6.59%) and cis-calamenene (5.59%). monoterpenes predominated in l. lasiocalycina and l. insignis, whereas the primary constituents in l. lasiocalycina were β-myrcene (31.17%), (e)-ocimenone (24.10%), p-cymene (7.17 %) and (z)-ocimenone (6.51%). the major compounds in the eos of l. insignis were (e)ocimenone (26.11 %), limonene (14.73%), β-myrcene (12. 48 %) andp-cymene (7.24%) (table 1). insecticidal activity the in vitro results indicated that the eos of the three species were highly efficient at controlling n. corniger, promoting up to 60% mortality after 48 hours.significant differences in insect mortality were observed in all the treatments when compared to distilled water (35.0%) and distilled water and tween (40.0%). however, insecticidal activity was still inferior to that of the commercial agrochemical, which exhibited 100% mortality. l. thymoides and l. lasiocalycina stood out for causing 100% termite mortality in the shortest time period (24 h) (table 2 fig 1). determining the median lethal concentration the median lethal concentration (lc50) of the essential oils (1%) was estimated by individually adjusting the models. l. lasiocalycina eo caused 50% n. corniger mortality at the lowest concentration (0.46 μl/ml), followed by l insignis (0.88 μl/ml) and l. thymoides (3.64 μl/ml) (table 3 fig 2). sociobiology 67(2): 292-300 (june, 2020) 295 compound kilit kicalc ll (%) ± sd li (%) ± sd lt (%) ± sd α-thujene 930 927-28 t 0.24±0.02 t α-pinene 939 939 t t 1.62±0.04 camphene 954 951 0.19±0.00 sabinene 975 973-75 1.23±0.07 0.16±0.00 1.83±0.55 β-pinene 977 977-79 t 0.88±0.04 β-myrcene 990 989-91 31.17±1.16 12.43±0.02 0.38±0.06 α-phellandrene 1002 1004 t α-terpinene 1017 1016-17 1.17±0.03 t p-cymene 1026 1024-26 7.17±0.62 7.24±0.84 0.78±0.51 limonene 1029 1029-31 0.31±0.00 14.73±0.60 2.75±0.16 eucalyptol 1031 1034 5.17±0.47 z-β-ocimene 1037 1038 t e-β-ocimene 1050 1049 1.67±0.06 e-β-ocimene 1050 1048 1.78±0.04 t γ-terpinene 1059 1059-61 2.29±0.02 6.99±0.14 0.48±0.14 cis-sabinene hydrate 1070 1068 t terpinolene 1088 1089 0.19±0.01 linalool 1096 1096-98 1.29±0.09 2.80±0.79 chrisanthenone 1127 1125 t trans-pinocarveol 1139 1140 0.18±0.00 trans-verbenol 1144 1142 t ipsdienol 1145 1144-46 0.43±0.05 0.85±0.12 0.20±0.00 myrcenone 1149 1151-53 4.05±0.35 6.37±1.14 borneol 1169 1163-66 1.34±0.05 0.43±0.02 terpinen-4-ol 1177 1177-79 t 0.56±0.16 α-terpineol 1188 1188-90 t 0.55±0.06 α-terpineol 1188 1189 t myrtenol 1195 1195 t (z)-ocimenone 1229 1231 6.51±0.13 5.29±0.49 thymol methyl ether 1235 1237 t (e)-ocimenone 1238 1240-41 24.10±0.80 26.11±1.05 geraniol 1252 1255 0.34±0.02 geranial 1267 1271 0.66±0.08 thymol 1290 1290-92 t t carvacrol 1298 1297-98 0.21±0.01 t δ-elemene 1338 1339 0.25±0.10 α-cubebene 1348 1352 0.79±0.09 piperitenone oxide 1368 1368 α-copaene 1377 1379 3.49±0.60 β-bourbonene 1388 1387 0.42±0.11 β-cubebene 1388 1391 0.31±0.07 β-elemene 1388 1392 0.39±0.05 β-elemene 1390 1393 0.50±0.05 α-gurjunene 1409 1411 0.39±0.02 e-caryophyllene 1419 1420-26 4.00±0.35 1.90±0.43 29.55±0.47 γ-elemene 1436 1435 0.22±0.01 α-guaiene 1439 1442 2.51±0.27 table 1. chemical composition of essential oils extracted from the leaves of lippia insignis (moldenk), lippia thymoides (martius & schauer) and lippia lasiocalycina (schauer). dr santos, lm oliveira, am lucchese, af espeleta, jd cruz, ms lordêlo – lippia (verbenaceae) essential oils as potential termiticides296 trans-muurola-3,5-diene 1453 1453 0.55±0.15 α-humulene 1454 1455-57 1.43±0.14 0.52±0.12 2.65±0.12 alloaromadendrene 1460 1464 0.79±0.11 germacrene d 1485 1481-85 0.41±0.04 1.97±0.60 6.59±2.98 trans-muurola-4(14),5-diene 1493 1493 0.99±0.16 bicyclogermacrene 1500 1496-98 0.41±0.05 2.55±0.90 α-muurolene 1500 1499 0.63±0.70 β-bisabolene 1505 1504-09 t 0.54±0.09 cuparene 1505 1507 2.18±0.57 α-bulnesene 1509 1508 1.22±0.20 cubebol 1515 1516 0.43±0.10 cis-calamenene 1529 1526 5.59±0.99 trans-cadina-1,4-diene 1534 1535 0.42±0.04 germacrene b 1561 1561 2.53±0.72 spathulenol 1578 1577-80 1.70±0.19 1.32±0.17 caryophyllene oxide 1583 1583-88 1.77±0.40 0.29±0.04 8.17±3.40 α-muurolol 1646 1648 0.70±0.05 total number of compounds identified 96.30±0.20 95.49±1.55 83.80±1.02 content 1.8% 1.7% 2.9% *kilit = kovats retention index from the literature; kicalc = kovats retention index calculated; (-) compound absent from the sample.lt: lippia thymoides, ll: lippia lasiocalycina, li: lippia insignis. table 1. chemical composition of essential oils extracted from the leaves of lippia insignis (moldenk), lippia thymoides (martius & schauer) and lippia lasiocalycina (schauer). (continuation) fig 1. mortality of nasutitermes corniger (motschulsky) 24 and 48 hours after exposure to a surface contaminated with essential oils of lippia. insignis (moldenk), lippia. lasiocalycina (schauer) and lippia thymoides (martius & schauer). disw: distilled water; disw+ tween: distilled water and tween (1%); fipronil: commercial insecticide; l lasio: lippia lasiocalycina. discussion the results obtained demonstrate the insecticidal properties of eos from the species l. lasiocalycina, l. insignis and l. thymoides in the biocontrol of n. corniger. despite their inferior insecticidal activity when compared to the commercial agrochemical, essential oils are less harmful to the environment and do not promote the development of resistance in insects (campos & andrade, 2002). this study is pioneering in that it assesses insecticidal activity against termites of lippia species, which are still underexplored. the significant insecticidal results of the eos studied can be justified by the presence of terpenes such as thymol, carvacrol, geranial, linalool, p-cymene, carvone, neral, limonene, (e)-caryophyllene, caryophyllene oxide, mircene. and γ-terpinene, whose insecticidal activity has been widely studied and confirmed (gomes et al., 2011 ; soares & tavaresdias., 2013; kamanula, j. f., 2017; blank, a. f., 2019 ). compound kilit kicalc ll (%) ± sd li (%) ± sd lt (%) ± sd sociobiology 67(2): 292-300 (june, 2020) 297 the authors attributed this activity to their high thymol content and synergistic effects of p-cymene and thymol methyl ether. tests using different chemotypes of l. gracillis against diaphania hyalinata (lepidoptera: pyralidae) demonstrated that chemical races consisting primarily of the phenolic terpenes thymol and carvacrol were more toxic (melo et al., 2018). essential oil of l. origanoides kunth, rich in carvacrol and p-cymene, was also efficient at controlling aedes aegypt linn, tetranychus urticae koch. and cerathapis lataneae boisd (mar et al., 2018). terpenes, the predominant compounds in lippia essential oils, encompass a series of substances whose defensive properties in plants are well-known. these metabolites protect the plants that produce them through neurotoxic action by inhibiting acetylcholinesterase, the main neurotransmitter responsible for acetylcholine reuptake in the synaptic cleft (tak & isman, 2017). another effect of eos is their inhibition of the neuromodulator octopamine, leading to hyperpolarization of the calcium channels modulated by gaba (gamma-aminobutyric acid) (priestley et al., 2003). octopamine acts as a neurohormone and neurotransmitter, responsible for regulating the heart rate, behavior and metabolism of insects (castro coitinho et al., 2011; enan et al., 200101). the toxicity of lippia eos causes delayed growth and appetite suppression in insects, as well as hampering maturation and reducing their reproductive capacity, leading to death (viegas júnior, 2003). although the phenolic terpene content in the species studied here was low, the presence of other compounds with proven insecticidal activity, such as limonene, p-cymene and ß-myrcene, may have contributed to this finding. limonene is one of the most common components of pesticides such as insecticides and insect repellent, and its presence has been essential oils treatments mortality 24 hrs 48 hrs lippia thymoides 100.0a 100.0a lippia lasiocalycina 100.0a 100.0a lippia insignis 95.0a 100.0a distilled water and tween 20 (1%) 20.0b 40.0b distilled water 25.0b 35.0b fipronil (c+) 100.0a 100.0a *data were submitted to nonparametric analysis via the kruskal wallis test and subsequently compared by rank. medians followed by the same lowercase letter in the column do not differ significantly. species cl50 (μl/ml) logit r2 l. insignis 0.88 y = -1.926 + 2.179 ci 0.74 l. thymoides 3.63 y = -1.610 + 0.443 ci 0.719 l. lasiocalycina 0.46 y = -0.841 + 1.800 ci 0.787 table 2. mortality of nasutitermes corniger (motschulsky) 24 and 48 h after treatment with essential oils of lippia thymoides (martius & schauer), lippia lasiocalycina (schauer) and lippia insignis (moldenk) at a concentration of 1%. fig 2. median lethal concentrations (lc50) of essential oils from lippia insignis (moldenk), lippia thymoides (martius & schauer) and lippia lasiocalycina (schauer) against nasutitermes corniger (motschulsky). the insecticidal properties of essential oils of l. lasiocalycina, l. insignis and l. thymoides corroborate the findings of lima et al. (2013), who studied the action of oes of l. alba, l. gracilis and l. sidoides against n. corniger, with l sidoides proving to be particularly effective. table 3. equations used to determine the medial lethal concentration (cl50) of eos of lippia thymoides (martius & schauer); lippia lasiocalycina (schauer) and lippia insignis ((moldenk) against nasutitermes corniger (motschulsky). dr santos, lm oliveira, am lucchese, af espeleta, jd cruz, ms lordêlo – lippia (verbenaceae) essential oils as potential termiticides298 reported in lippia alba eo, a species proven to be effective against spodoptera frugiperda (j. e. smith) (niculau et al., 2013). p-cymene, found in lippia sidoides and lippia lasiocalycina, exhibits proven insecticidal action against aedes aegypti (diptera: culicidae) and heterotermes sulcatus mathews (isoptera: rhinotermitidae) (costa et al., 2005). however, in termites, pinenes have been identified as the most volatile components in defending against n. corniger, enhancing the chemical response of these insects to pinene-rich products (lima et al., 2013). β-myrcene was active against adult sitophilus zeamais with a dose-dependent relationship to exposure time (yildirim et al., 2013). the median lethal concentrations (lc50) recorded in our study demonstrated that oes of l. lasiocalycina (0.46 μl/ ml) and l. insignis (0.88 μl/ ml) were more effective after 48 hours, indicating that very little oil is needed from these species to obtain a toxic response in n. corniger, using the contact method. another study investigated the median toxicity of lippia sidoides cham and pogostemon cablin benth eos against the termites microcerotermes indistinctus and amitermes a. cf. amifer. the authors found that pogostemon cablin eo was the most effective (lc50) at 0.32 μl/ ml and 0.29 μl/ ml, concentrations lower than those observed here (bacci et al.,2015), while l. sidoides was less effective, with a median effect on the termites tested at 2.41 and 3.47 μl/ ml, respectively. the insecticidal activity of l sidoides against cryptotermes brevis (walker, 1853) (kalotermitidae) has also been reported using the fumigation test, with cl50 estimated at 9.10 and 23.6 μl/ l of beans (santos et al., 2017). it is important to note that individual characteristics such as cuticle thickness, associated with different application methods, can influence inter or intragroup tolerance and resistance (haddi et al., 2015; pinto-zevallos & zarbin, 2013). given that their social behavior is one of the main barriers to successful termite control, in most cases the topical use of synthetic insecticides leads to recurrence (lima et al., 2013). as such follow-up studies should be conducted in the field to chemically control this insect pest, in conjunction with the partition and isolation of the chemical constituents of the oils tested, as well as preparing stable essential oil solutions from the lippia species studied. conclusions the essential oils of l. lasiocalycina, l. insignis and l. thymoides showed in vitro insecticidal potential to control n. corniger termites. l. lasiocalycina eo exhibited the lowest median lethal concentration, followed by l. insignis and l. thymoides. in conclusion, this study demonstrates the potential of developing bioinsecticides from the eos studied to control termite populations, particularly considering the substantial toxicity to humans of the synthetic products currently used, their significant environmental impact and the high recurrence of pests after topical treatment with these products due to their social behavior. acknowledgments the authors are grateful to the research support foundation for bahia state (fapesb) and the national council for scientific and technological development (cnpq) for the funding provided to carry out this project. we would also like to thank feira de santana state university (uefs) and its graduate program in plant genetic resources (ppgrgv/uefs. references adams, r.p. et al. 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(2013). insecticidal effects of monoterpenes on sitophilus zeamais motschulsky (coleoptera: curculionidae). journal of applied botany and food quality, 86: 198-204. doi: 10.5073/jabfq.2013.086.027. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7322sociobiology 69(2): e7322 (june, 2022) introduction stingless bees play a key role in various ecosystems, and they have been standing out as the primary agent for pollinating many native and cultivated plant species. they also ensure the maintenance of genetic variability, productivity, and the quality of many fruits (bartelli & nogueira-ferreira, 2014; roubik, 2018a). these bees can be found in urban environments as long as there are favorable conditions for their survival. however, their ability to succeed in urban environments will depend on several factors, such as food availability (e.g., nectar, pollen, and water) and nesting resources (e.g., mud, oil, resin), as abstract meliponiculture, the rational breeding of native stingless bees, is considered an excellent sustainable alternative to assist in the pollination process and is an economically viable activity. in the cerrado of tocantins, the meliponine species that stand out most due to their wide distribution are: scaptotrigona tubiba, melipona fasciculata,m. rufiventris and tetragonisca angustula. the bibliographic collection about these species is still little explored, hence there is a need for research to deepen the existing knowledge in the area. for this reason, the aim of this study was: a) to quantify the honey production of four meliponine species: t. angustula, m. fasciculata, m. rufiventris, and s. tubiba; b) to determine the physicochemical characteristics of the product; c) measure the biological parameters of the colony and d) evaluate the profile and sensory acceptance of honey in the municipalities, palmas and miracema, in the tocantins. the study evaluated the biological parameters of the colony, honey production, and physicochemical analysis. the highest honey production came from the species t. angustula in the two collections for the municipality of palmas. for miracema, the species s. tubiba and m. fasciculata were evaluated, respectively. the physicochemical parameters evaluated fit the norms assigned to honey quality control. results showed that honey from m. fasciculata was the sensory profile that obtained the best average among the characteristics observed in the study. there was a positive and negative correlation between the biological parameters, with a significant difference only between the characters’ height and diameter of the honey pot. sociobiology an international journal on social insects maisa f ribeiro¹, roberta z da silva², rosilene n domingos2 article history edited by evandro n. silva, uefs, brazil received 28 july 2021 initial acceptance 15 november 2021 final acceptance 21 april 2022 publication date 05 july 2022 keywords native bees, honey production, physicochemical analysis. corresponding author roberta z. da silva universidade estadual do tocantins 108 sul alameda 11, lote 03 caixa postal: 173, cep: 77020-122 palmas, tocantins, brasil. e-mail: robertazani@gmail.com well as other intrinsic factors related to the geographical distribution of each species (souza et al., 2005; antonini et al., 2012; singh, 2016). although almost three hundred species of native bees are known in brazil, only a few are properly studied. stingless bees of the genus meliponini stand out for their honey production (pereira et al., 2017). however, some species, for example, meliponini rufiventris, despite being endemic to the cerrado biome, are in danger due to devastating anthropic practices (silveira et al., 2018). stingless bees have a limited honey production compared to apis melífera (zuccato et al., 2017; shadan et al., 2018). the limitation is due to various factors, such as the 1 universidade federal do tocantins, gurupi, brazil 2 universidade estadual do tocantins, palmas, brazil research article bees quality of honey produced by four species of stingless bees in the central region of the state of tocantins maisa f ribeiro, roberta z da silva, rosilene n domingos – analyzing honey quality of four stingless bee species2 absence of standards of quality control, limited knowledge in the field, and low levels of industrial production. however, despite this limitation in the industry, stingless bee honey is available in traditional markets, and it has a significantly higher price than the honey of a. melífera (sekuan wei & ghoshalb et al., 2018). the composition of bee honey depends on the nectar and the physiology of the species that produces it, providing its physical, chemical, biochemical, and sensory characteristics. in addition to climatic and soil conditions, handling practices in processing also interfere with physicalchemical properties, product quality, and consumer preference (araújo et al., 2017). therefore, achieving the honey physicochemical parameters becomes essential not only for the characterization of the diversity of meliponini species but also to ensure the quality of the product on the market. (zielinski et al., 2014; nascimento et al., 2015). in addition, to the knowledge we already have on the types of honey, it is also necessary to understand the relevant characteristics of each species of bee to meet their needs. for example, characteristics of harvesting, food storage by bees, the nest structure, and the sanity of colonies (alves & imperatriz-fonseca, 2010; pereira et al., 2011). therefore, the objective of this study was: a) to quantify the honey production of four species of meliponines: t. angustula, m. fasciculata, m. rufiventris, and s. tubiba; b) to determine the physical and chemical characteristics of the product; c) to assess the biological parameters of the colony, and d) to evaluate the profile and sensory acceptance of the honey from stingless bees in two municipalities in the tocantins, palmas and miracema, as previously described. material and methods study area and stingless bee species the hives used in this study were situated in two municipalities of tocantins: palmas and miracema, 90.5 km away from each other. thus, we performed the procedures for honey harvesting in the respective cities. the physicalchemical and entomological evaluations were conducted at the laboratory of production, food technology, and entomology, in the agrarian sciences complex of the state university of tocantins, in the municipality of palmas. we used four species of stingless bees in the study: melipona rufiventris, scapotrigona tubiba, melípona fasciculata and tetragonisca angustula, popularly known as: uruçu amarela, tubi, tiúba and jataí, these are the species found most frequently in tocantins cerrado (a savanna like vegetation). these species were populated by processes of divisions and transfers of colonies. monitoring of the bee colonies for the monitoring of the colonies, four in each municipality – in the same locality, referring to the species mentioned above a review form was used with the following observation parameters: water supply, hive productivity, batum, nest development, and general needs. during the study, the colonies of both municipalities were monitored weekly. we made the water supply using disposable cups (50 ml) with stones (previously cleaned) placed in the bottom to prevent the melyphs from drowning. we observed the productivity of the hives by removing the lid from the boxes and visually diagnosing any products left by the melyphs. in addition, the batum was monitored by its presence. fig 1. bee colonies of the species: tetragonisca angustula (a and e), melipona fasciculata (b and f), scaptotrigona tubiba (c and g) and melipona rufiventris (d and h) in the municipalities of palmas and miracema, respectively. sociobiology 69(2): e7322 (june, 2022) 3 the development of the nest was carried out visually by observing the following characteristics: the presence of the queen and its sanity; the space occupied by the chicks in the tenements; the number of worker bees and the presence of drones in the hive. finally, we allocated space for observations not previously accounted for, which could interfere with each species’ honey production. images of the colonies of each species were taken in the same monitoring week in both locations, palmas and miracema (fig 1). experimental design we used a completely randomized factorial (2x2x4) experimental design. the first factor corresponded to the municipalities: palmas and miracema, the second to harvests (first and second), and the third corresponded to stingless bees: m. rufiventris, m. fasciculata, t. angustula, and s. tubiba. each treatment had three boxes with nest and over nest considering a repetition. the harvest interval was thirty-six days with the first one in september. the cities were chosen for their proximity and environment similarity, and the modular boxes were selected for the ease of handling and observation of the sanity parameters of the colonies exemplified in the study. evaluation of honey production the period defined for this study was around 7a.m. and 5p.m. these times were chosen so that the honey was first and second harvested with less interference regarding avoiding wear of the colonies. thus, avoiding the hottest times of the day. the boxes were handled as little as possible, using the syringe method with a capacity of 20ml. the honey was collected following hygiene protocols, avoiding possible contaminants, and using appropriate materials at the time of harvest and storage. after this process, the samples were kept at a temperature of 10°c to maintain the physicochemical characteristics, until the moment they were taken to the food technology laboratory of the agricultural sciences complex of the state university of tocantins, where the evaluations recommended in this study. evaluation of biometric parameters the colony biological parameters evaluated in the four species were: diameter and height of brood cells, height and diameter of honey pots, the weight of pollen mass deposited in closed pots, and height of pollen pots. both assessments were carried out in the same weekly period and in the morning, according to brito et al. (2013), adapted. for the evaluations of space occupied by the honey pots, height and diameter, and height of the pollen pot, the measurement was carried out directly using a manual caliper, with a measurement range of 0.01mm − 150mm and an error of 0.03mm. as for the other parameters, a number 60 sewing thread was used so that these cells would not be damaged. finally, to quantify the mass of the pollen pots, an analytical balance was used. evaluation of physical-chemical parameters we determined the physicochemical parameters following the brazilian legislation, which includes the physicochemical parameters of honey from apis melífera (azevedo & beser, 2000) all physicochemical parameters were determined in triplicate according to the standards of the international honey commission (ihc, 1997) and the official analysis methods of the association of official analytical chemists (with some modifications). the determination of moisture and total soluble solids was performed by the refractometric method, using a portable manual refractometer, model rt90 atc. the equipment was calibrated with distilled water. the samples had a mathematical correction, an addition of 0.00023, for each degree celsius higher than the established, 20°c ambient, according to brazilian legislation (brasil, 2000). we determined the ph according to the methodology adopted by moraes and teixeira (1998). the method is based on simple titration, made by measuring the potential difference between two suitable electrodes, immersed in the solution analyzed and using a benchtop ph metro, model sp 3611, with 15 ml of honey in becker, to monitor the ph measurement. the hach dr model spectrophotometer equipment measured the parameter “honey color” following the methodology by larana (1981). the wavelength worked at was 540 nanometers and the reagent chosen for equipment calibration was pure glycerin. the values obtained were in absorbance, and after the conversion of the wavelength into honey color, using the formula mm pfund = −38.70 + 371.39x abs, the honey color classification proceeded (ferreira et al., 2009) we performed a sensory analysis with the honey of the four stingless bee species in this study. the parameters used for the analysis were: appearance, color, odor, flavor, texture, and overall evaluation, through a free profile spreadsheet, with scoring amplitude from 1 (disliked very much) to 9 (i liked it very much). for this process, five tasters who were already consumers of this product, but had no experience with free profile sensory analysis, were invited. the samples were coded so that the compositions of notes were in accordance only with the preferences of each one. palate cleaning was also performed between the samples (grosso, 2006). statistical analyses statistical analysis of variance (anova) was used to evaluate the honey production of the four species of stingless bees in the two municipalities. when applicable, tukey multiple comparison tests at 5% probability were used to identify differences between treatments. the correlation between biological parameters and honey production of the four species was performed through maisa f ribeiro, roberta z da silva, rosilene n domingos – analyzing honey quality of four stingless bee species4 the construction of graphs that provided this interaction. according to the first and second collections, the analyses were carried out for the physicochemical characters, individualizing them by town. variance analyses and tukey tests were performed using the software sisvar (sisvar for windows v. 5.6, ufla, lavras, mg, br). we calculated the means and standard errors with the software ms excel (excel 2010, microsoft, redmond, wa, usa). then we produced the graphs with the minitab statistical software and minitab workspace. all analyses were performed using a significance level of 0.05. results and discussion not all melipona species produced honey during the harvest period in their respective municipalities. for example, there was no production in the first collection, in miracema, for the species m. rufiventris and s. tubiba. however, t. angustula produced 115.95 ml of honey in the same period. also, the species s. tubiba (91.56 ml) stood out, in the first collection, in palmas, followed by m. rufiventris (68.4 ml) and m. fasciculata (47.53 ml), according to fig 2. in the second collection, in miracema, the highest honey production was verified with the melipona species fig 2. honey production from the first and second harvest in the municipalities of miracema and palmas (to), brazil. t. angustula with the summation of 191.33 ml, followed by m. fasciculata and rufiventris (171.62 and 96.63 ml), respectively. s. tubiba did not produce honey during this period. in the municipality of palmas, honey production was not observed for s. tubiba and t. angustula bees, only for m. fasciculata and rufiventris, with averages of 159.81 and 135.96 ml, respectively (fig 2). honeybee production is represented by the number of pots and the volume of honey contained within (evangelistarodrigues et al., 2008). thus, colonies with higher values of these variables have higher production. furthermore, variations in the number of honey pots can easily occur, given the flowering time in each region. table 1 shows the significant differences in honey production, performed by the average of the three repetitions of each species of the four species of meliponines in the two municipalities, individually, in each harvest. it is possible to notice the range of quantification of production, both among bees and in the cities of the study. the municipality of miracema had higher rates of honey production except for t. angustula, which did not show any production. table 1 average production (ml) of honey from four species of stingless bees in the municipalities of palmas and miracema (to), brazil, in two harvest seasons. 1st harvest species municipality miracema palmas melipona fasciculata 57.2 aa 2.02 bb scaptotrigona tubiba 63.77 aa tetragonisca angustula 59.97aa melipona rufiventris 32.21 aa 2nd harvest species municipality miracema palmas melipona fasciculata 53.27 aa 15.84 aa scaptotrigona tubiba 30.52 a tetragonisca angustula melipona rufiventris 45.32 aa 21.6 aa *means followed by the same lowercase letter in the row and uppercase in the column do not differ statistically from each other by the tukey test at the 5% probability level. data were transformed into log (x+1). sociobiology 69(2): e7322 (june, 2022) 5 the current apicultural bloom in the city of palmas, visually observed was mainly weeds (undergrowth), for example, vernonia difusa less., sida rhombifolia l., and sida spinosa l., with potential for the production of pollen and nectar. these plant species have flowering times ranging from september to december in the region, given the favorable environmental conditions. in addition to these, the tree species syzygium cumini (l.) skeels is also easily found in the landscape composition of the studied municipality, with its flowering season extended from april to september (migliato, 2005). the plant composition in the municipality of miracema was slightly different from palmas, as there were a higher number of native trees, visually observed. the species most frequently found were: caryocar brasiliense camb, anacardium occidentale l., parkia pendula willd., cabralea canjerana (vell) and curatella americana l. according to migliato (2005), these species have distinct flowering seasons ranging from the second half of august to the end of november, given the appropriate edaphoclimatic conditions. the state of the colonies is a factor that maintains a close relationship with honey production. generally, when weak, they are more susceptible to temperature variations. they have activity shifted to later hours of the day compared to other colonies of the same species, drastically reducing their honey production (hilário et al., 2001). according to venturieri (2008) knowledge of the biometric parameters is necessary to understand the productive indexes, identify similarities or differences between individuals, and evaluate each species’ potential. another relevant point is the behavior and characteristics of production and reproduction in the boxes used in meliponaries. for ribeiro et al. (2014), the construction of brood discs can be influenced by several factors. for instance, the number of bees in the colony, food (mainly pollen), and a queen’s presence. concerning honey pots, the larger the store, the greater volume of honey. consequently, there will be more outstanding production and less waste of wax. however, population size can directly interfere with honey production at a proportional rate (alves et al., 2007; alves, 2010). the importance of the number of individuals refers to the fact that, typically, in populous colonies, many foragers collect more resources during flowering times, enabling defense against enemies, and maintaining adequate temperature for the development of the offspring (faquinello et al., 2013). there was a significant difference in the correlation between honey production and the diameter and height of honey pots (-0.28 and -0.33), see table 2. the other measured variables had a positive correlation, however, with no significant difference. table 2 correlation of production and biometric variables of colonies of four species of stingless bees in the municipalities of palmas-to, brazil. variables pm app mp ac dc dpm app -0,31ns mp -0.3 ns 0.366 ns ac -0.309 ns 0.917 ns 0.522 ns dc -0.264 ns 0.94 ns 0.489 ns 0.983 ns dpm -0.287** 0.804 ns 0.241 ns 0.732 ns 0.749 ns apm -0.334** 0.85 ns 0.249 ns 0.773 ns 0.791 ns 0.989 ns app = height of pollen pots; pm = honey production; mp = pollen pot mass; ac = height of brood cells; dc = diameter of brood cells; dpm = diameter of honey pots and; apm = height of honey pots, of the meliponous species: m. fasciculata, m. rufiventris, s. tubiba, and t. angustula. **significant at 1%; *significant at 5%; ns = not significant. the formation of honey pots with greater diameters and depths reduces the space occupied. it reduces honey consumption by workers bee in wax production, which leads to a more significant amount of honey produced, revealing the statistical difference shown in table 3 (alves et al., 2012). table 3 correlation of production and biometric variables of colonies of four species of stingless bees in the municipality of miracema-to, brazil. variables pm app mp ac dc dpm app 0.025 ns mp -0.266 ns 0.221 ns ac 0.202 ns 0.662 ns 0.657 ns dc 0.064 ns 0.613 ns 0.662 ns 0.954 ns dpm 0.176** 0.528 ns 0.741 ns 0.907 ns 0.87ns apm 0.279** 0.645 ns 0.57 ns 0.962 ns 0.9 ns 0.892 ns app = height of pollen pots; pm = honey production; mp = pollen pot mass; ac = height of brood cells; dc = diameter of brood cells; dpm = diameter of honey pots and; apm = height of honey pots, of the meliponous species: m. fasciculata, m. rufiventris, s. tubiba, and t. angustula. ** significant at 1%; * significant at 5%; ns = not significant. maisa f ribeiro, roberta z da silva, rosilene n domingos – analyzing honey quality of four stingless bee species6 these bees separate the pollen and honey. they are stored in two types of pots, differentiated by size. the pollen pots are cylindrical or conical with about 3 cm high, and the ovoid honey pots, with approximately 1.5 cm in height, vary according to the meliponous species (carvalho-zilse et al., 2012; pereira et al., 2012). however, rodrigues et al., (2018) state that empty pots, regardless of the final product, derives from the preparation of food stored in wax pots. thus, they identified that the biometric characteristics of pollen and honey pots are related to the colony’s size and vary according to their purpose. the table below presents the results of humidity, ph, color, and brix of the kinds of honey produced by the stingless bee species, s. tubiba, m. rufiventris, m. fasciculata, and t. angustula, in both harvests, first and second, in the municipalities of miracema and palmas. the moisture contents found ranged from 24% 31% (m/m) and 23% – 32% (m/m) for melipona fasciculata and melipona rufiventris, in the first and second collections, respectively. it was possible to observe that the species differ from each other and harvest periods (table 4). for example, the species t. angustula obtained 24.02% (m/m) in the first collection and 28% (m/m) in the second. samples humidity (%) ph color °brix 1m. fasciculata (a) 25.2 4.18 clear amber 73.48 1m. fasciculata (a) 27.3 3.79 amber extra clear 70.48 1m. fasciculata (a) 25.5 4.00 amber extra clear 73.48 1 s. tubiba (a) 28.2 4.19 white 70.48 1 t. angustula (a) 26.3 4.48 âmbar 72.48 1 t. angustula (a) 28.1 4.56 âmbar 70.48 1m. rufiventris (a) 31 3.99 âmbar 67.48 1m. rufiventris (a) 30.3 3.87 amber extra clear 68.48 1m. fasciculata (b) 24 nd clear amber 74.48 1m. fasciculata (b) 29.4 nd clear amber 71.48 1 t. angustula (b) 24.2 4.39 amber extra clear 74.48 2 s. tubiba (b) 31.3 4.0 white 67.98 2 s. tubiba (b) 29 4.03 white 69.48 2 m. rufiventris (b) 32.1 3.9 white 66.48 2 m. fasciculata (b) 23 4.09 white 75.48 2 m. rufiventris (a) 31.2 4.22 amber extra clear 67.98 2 m. rufiventris (b) 30 3.97 white 68.48 2 m. rufiventris (b) 31.5 3.82 clear amber 67.48 2 m. fasciculata (b) 28.9 4.06 amber extra clear 70.28 2 m. fasciculata (b) 32 3.58 white 66.48 2 m. fasciculata (b) 33 3.58 white 65.48 note: nd = undetected values; 1 and 2 = order of collection; (a) and (b) = miracema and palmas. table 4 analysis of moisture, ph, color, and total soluble solids (°brix) for honey collected by the four melipona species in the municipalities of palmas and miracema during the two collections periods. current brazilian and international legislation specific to honey from apis mellifera maintains that honey must contain a maximum of 20% moisture (brasil, 2000; codex stan 12, 2001). however, several studies that characterize honey from stingless bees show that higher humidity levels are a characteristic of this type of honey, such as the study by almeida-muradian (2013), silva et al. (2013), and sousa et al. (2016), when analyzing honey from t. angustula, m. asciculate, and m. scutellaris, they found values ranging from 23.4 to 25.6% (m/m), from 22.2 to 24.4% (m/m) from 23.9 and 28.9% (m/m), respectively. high levels of moisture can come from the low dehydration of the nectar during the natural transformation process into honey. and it may also be related to several factors such as the humid tropical environment; the collection of nectar from lower flowers, and ripe fruits with higher water content; harvest time; degree of maturity reached in the hive; edaphoclimatic conditions; or even from the different species of bees (guerrini et al., 2009; vit et al., 2013). high levels of moisture can also accelerate the fermentation process, resulting in the formation of undesirable organic compounds, which can give a bitter taste, and unwanted color, interfering with the product’s shelf life and, consequently, with its commercialization (da silva et al., 2016). for the soluble solids’ concentration (°brix), the values found ranged from 67.98 – 72.98° brix and 65.48 – 75.48° sociobiology 69(2): e7322 (june, 2022) 7 brix for m. asciculate and m. rufiventris and m. asciculate in both, in the first and second collection, respectively. it is possible to observe that the species differ from each other and harvest seasons (see table 4). for example, the species m. asciculate had a greater range of variation (10.00 °brix) in the same collection between cities. honey from apis ascicula bees generally has higher values of °brix. thus, lower values, obtained for honey from stingless bees, may be related to the high-water content found in this product and lower sugar content (de sousa et al., 2016). the ph values found ranged from 3.87 (melipona rufiventris) to 4.56 (tetragonisca angustula) and 3.58 (melipona asciculate) to 4.22 (melipona rufiventris), in the first and second collection, respectively (table 04), defining an acidic character to the samples, similar to the ph observed for apis ascicula kinds of honey (3.2 – 4.5), according to brazilian and international legislation (brasil, 2000; codex stan 12, 2001). according to crane (1985), ph values can be influenced by the ph of the nectar, soil, or association of nectars for its composition. in addition, even if honey is produced in the same town, there are mandibular substances added to the nectar during its transport to the hive (vit et al., 2013). currently, ph is not a mandatory parameter in the quality control of brazilian honey, nor is it recommended by international honey legislation. however, its evaluation is of great importance in defining the form of handling and storage of honey. itconsiders that ph and acidity are important antimicrobial factors, capable of providing greater stability and shelf life to the product, as well as influencing the speed of formation of 5-hmf (guerrini et al., 2009). the color of honey is considered a parameter of quality, preference, and acceptance among consumers. in our study, the color of the kinds of honey ranged from white to amber (fig 3). these colors fit the current regulations, brasil (2000) and codex alimentarius (2001), ranging from colorless to dark amber, for honey quality control that is based on the characteristics of honey produced specifically by a. mellifera bees. fig 3. color parameter of honey produced by stingless bees in the municipalities of palmas and miracema in two harvest seasons. maisa f ribeiro, roberta z da silva, rosilene n domingos – analyzing honey quality of four stingless bee species8 in our study, the predominance of the white color, with honey from the stingless bee species, agrees with the results obtained by souza et al. (2006) and anacleto et al. (2009). on the other hand, lacerda et al. (2010) found lighter hues for stingless bees, adhering to the variations in landscape composition, temporal, and intrinsic characteristics of each species. it is also noteworthy that several other factors namely, influence the color of honey: floral origin, honey maturation, climatic variations along the period of nectar flow,time, and temperature at which the honey remains stored in hives, presence of chemical elements, vitamins b and c, caramelization process, maillard reaction products, and the content of flavonoids and phenolic acids. the greater the concentration of these components, the more intense the color of the honey (lacerda et al., 2010; lira et al., 2014; braghini, 2016; da silva et al., 2016). the development and approval of regulation for stingless bee honey, which meets standards regarding identity and consumer safety, is important and establishes a reference basis for regulatory agencies, thus allowing official marketing. but, unfortunately, the brazilian legislation for honey does not behold the stingless bees. figure 4 shows the sensory profile of each species, performed by the average between the two municipalities. the average value attributed by the tasters to each attribute (appearance, color, texture, odor, flavor, and global evaluation) is marked on the corresponding axis. the center of the figure represents the zero point of the scale used in the assessment, while the intensity increases from the center to the periphery. the sensory profile is revealed when the points are connected (spider chart). fig 4. sensory analysis of honey produced by stingless bees in the municipalities of palmas and miracema in two harvest seasons. sensory analysis is a technique used for evaluating the attributes perceived by the sense organs (organoleptic attributes) applied in many fields. it allows for establishing the organoleptic profile of different products, indicating consumer preference (piana et al., 2004). sensory characteristics stimulate the senses and provoke various degrees of reactions of desire parameters especies of meliponines m. fasciculata s. tubiba t. angustula m. rufiventris appearence 8.2 a 7.2 b 5.4 c 5.4 c colour 8 a 7.2 b 4.4 d 6.2 c odour 7.2 a 7 b 5.6 c 4.4 d texture 8 a 7 b 6.4 c 6 d flavour 7.6 a 5 c 7.6 a 5.2 b global evaluation 8 a 6 c 7.2 b 5.8 d *means followed by the same lowercase letter on the line do not differ statistically from each other by tukey’s test at the 5% probability level. table 5 sensory analysis of honey produced by stingless bees in the municipalities of palmas and miracema in the two harvest seasons. or rejection, in which the consumer chooses a food based on its level of sensory quality (carvalho, et al., 2006). sensory analysis is essentialfor assessing consumer preferences according to honey processing, storage, and its impacts on botanical, geographic, and volatile identity (sodré et al., 2008; ribeiro et al., 2014; costa et al., 2017). sociobiology 69(2): e7322 (june, 2022) 9 table 5 shows the marks given by the tasters concerning the sensory attributes related to the species m. asciculate, s. tubiba, t. angustula, and m. rufiventris, where the first one presented higher marks for all the evaluated characters, differing statistically from each other at 5% probability by tukey test. conclusion the results of this study demonstrate that the highest production of honey (inthe first and second collections), came from the species t. angustula in the municipality of palmas. as for miracema, the highest production came from the species s. tubiba and m. fasciculata. considering the physicochemical analyses carried out, we verified that all the evaluated parameters fit the norms designated to the quality control of honey, even if specifically, of a. melífera. the specie m. fasciculata obtained the best honey sensory profile by average among the characteristics observed in this study. as indicated by the data analyses there was a positive and negative correlation between the biological parameters evaluated namely, height and diameter of honey pots and brood, and height and mass of pollen pots, with a significant difference only between the height and diameter of the character of the honey pot. in light of these results and beyond the scientific purpose, this study supports the creation of a standard identity and specific quality control for the honey of stingless bees, contributing to the production and marketing of thisproduct, like that, already used for apis mellífera honey. acknowledgments the authors acknowledge mr. jacinto fernandes (in memorian) for his contribution. authors’ contributions mr: conceptualization, methodology, formal analysis, investigation, resources, and writing. rs: conceptualization, methodology, investigation, resources, project administration, review & editing, and project administration. rd: methodology and formal analysis. references almeida-muradian, l.b., stramm, k.m., horita, a., barth, o.m., freitas, a.s., estevinho, l.m. 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(2017). entomological authentication of stingless bee honey by 1 h nmr-based metabolomics approach. food control, 82: 145-153. doi: 10.1016/j.foodcont.2017.06.024 doi: 10.13102/sociobiology.v62i2.296-299sociobiology 62(2): 296-299 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 first record of the dolichoderine ant genus gracilidris wild & cuezzo (hymenoptera: formicidae) from southern brazil introduction the ant genus gracilidris wild and cuezzo, 2006 (formicidae: dolichoderinae) includes a single extant species, g. pombero wild & cuezzo, 2006 and the fossil g. humilioides (wilson, 1985). morphologically, the genus is characterized by the unique combination of slender body, anterior margin of clypeus broadly convex, eyes located near the longitudinal midpoint of head and touching lateral margins of head in frontal view, antennal scapes longer than head length, petiole with a tall dorsal scale with apex curved posteriorly, and body dorsum devoid of erect pilosity. the few observations on the species biology suggest that the colonies are established in the soil with the nest entrance consisting of a small earthen turret. the small number of workers collected in nest excavations suggests a small colony size (wild & cuezzo, 2006). workers are often attracted to sardine baits in the soil which indicates that these ants may have generalist feeding habits (costamilanez et al., 2014) as also observed for other soil-nesting abstract the dolichoderine ant species gracilidris pombero, sole representative of the genus, is recorded for the first time in southern brazil. until now, the species was known only for the open fields of the south american dry diagonal and for a single locality in the colombian amazon. the specimens reported here were collected with pitfall traps in grasslands of the pampa biome, state of rio grande do sul, brazil. this record represents the southernmost occurrence for the genus, extending its distribution in approximately 450 km to the south in the neotropics and in almost 1,150 km to the south in brazil. sociobiology an international journal on social insects rm feitosa1, w dröse2, lr podgaiski2, ms mendonça jr. 2 article history edited by gilberto m. m. santos, uefs, brazil received 29 january 2015 initial acceptance 26 may 2015 final acceptance 28 may 2015 keywords dolichoderinae, pampa biome, grasslands, biogeography, dominican amber. corresponding author rodrigo machado feitosa departamento de zoologia universidade federal do paraná curitiba, pr, brazil 81531-980 e-mail: rsmfeitosa@gmail.com dolichoderine ants including dorymyrmex, forelius, and iridomyrmex, for example. according to wild and cuezzo (2006) gracilidris ants are probably nocturnal, which may explain their relative rarity in myrmecological collections. these authors mention that at the type locality (paraguay), foraging workers were captured at dusk and after dark, while visual searches and baiting during the day at the same locality did not reveal any workers. nothing is known about the reproductive biology of the genus. gracilidris was originally described based on specimens collected primarily along the so called south american dry diagonal, which includes the savannah lands of northeastern and central brazil (caatinga and cerrado, respectively), and the chaco areas of paraguay and argentina (prado & gibbs, 1993). until recently, it was thought that the extant lineage of gracilidris was restricted to the low scrub forests and open fields of this dry diagonal. however, guerrero and sanabria (2011) provided the first record of g. pombero for the northern portion of south america, more precisely for the foothills of the colombian amazon basin. research article ants 1 universidade federal do paraná, curitiba, pr, brazil 2 universidade federal do rio grande do sul, porto alegre, rs, brazil sociobiology 62(2): 296-299 (june, 2015) 297 the record of g. pombero in colombia and the presence of the fossil species g. humilioides in dominican amber, with age estimated in approximately 43 million years (ward et al., 2010), suggest an ancient and widespread presence of the genus in the neotropics. in this paper we report the first record of gracilidris pombero for south brazil, considerably extending the distribution limits for the genus to the south. we also update the geographic information on gracilidris and discuss the morphological variation found among populations of g. pombero. materials and methods gracilidris pombero specimens were identified among the ants collected in a long term ecological research (lter) network (peld campos sulinos – cnpq) at southern brazilian grasslands. these grasslands occupy a transitional zone between tropical and temperate climates, with hot summers and cool winters, and are composed of several phytophysiognomies and extremely high plant species richness. vegetation is characterized by an association of c4 and c3 grasses and a variety of forbs and shrub species, and its structure responds directly to factors such as climate, soil and disturbance history. cattle grazing is the traditional land use, which besides the economic importance also ensures the maintenance of typical grassland vegetation (overbeck et al., 2007; pillar et al., 2009). in the lter, an experimental setup was distributed over several natural grassland sites from pampa (in the southern and western regions of the state of rio grande do sul) and atlantic forest biome (on the highland plateau of rio grande do sul) to investigate the effects of grazing management (e.g. exclusion, traditional and sustainable grazing) on biological communities, ecosystems processes and services. in brazil, the pampa biome is restricted to and occupies 63% of the territory of the state of rio grande do sul, representing only 2.07% of the brazilian biomes (instituto brasileiro de geografia e estatística [ibge], 2004). ant specimens were collected with pitfall traps filled with formalin (formaldehyde 3%), set at the soil level, and exposed for seven days. after sorting, the ants were preserved in alcohol 80%. vouchers are deposited in the laboratório de ecologia de interações of the universidade federal do rio grande do sul, rs, brazil, and in the entomological collection padre jesus santiago moure of the universidade federal do paraná (dzup), pr, curitiba, brazil. we compared the rio grande do sul workers with those (including types) deposited at the myrmecological collections of the museu de zoologia da usp (mzsp), são paulo, and of the comissão executiva do plano da lavoura cacaueira (ceplac), bahia. high resolution images were obtained under a leica m125 stereomicroscope attached to a leica dfc 295 video camera. photos were combined using zerene stacker software at the dzup. images were then processed as tiff files in adobe photoshop cs5 to enhance parameters of brightness and contrast. the distribution map was generated by the software quantum gis 1.5.0 (tethys) with coordinates imported from google earth after consulting the records for gracilidris pombero in the literature (wild & cuezzo, 2006; guerrero & sanabria, 2011; neves et al., 2013; costa-milanez et al., 2014). results and discussion gracilidris pombero individuals were sampled in experimental sites distributed solely over the pampa biome. eighteen workers were recovered from pitfall traps. the specimens studied here came specifically from the traps installed in the experimental plots of lavras do sul (30º42’02”s, 53º58’53”w) and aceguá (31°38’55”s, 54°09’26”w), near the border with uruguay. both sites are placed in private farms that use traditional grazing management. workers were captured in 16th december, 2011 (one individual in lavras do sul, and eight individuals in aceguá), 8th december, 2012 (one individual in lavras do sul, and seven in aceguá), and 4th december, 2013 (one individual in aceguá). the records of gracilidris for rio grande do sul represent the southernmost register of the genus in the neotropics and the first for the pampa biome, extending its distribution in approximately 450 km to the south. the most meridional record for the genus until now was represented by specimens collected in santiago del estero, argentina (wild & cuezzo 2006). in brazil, the southernmost record so far consisted of two workers collected in agudos, state of são paulo, in 1957 (father w. w. kempf collection at the mzsp). thus, the specimens of rio grande do sul (aceguá county) extend the genus distribution in approximately 1,150 km to the south within brazil. workers examined here can be securely identified as g. pombero (fig 1), since they match most of the diagnostic characters of the species. when compared to the type specimens, the rio grande do sul workers present a slightly shorter head (head length range 0.75-0.76 mm in rio grande do sul workers and 0.80-0.93 mm in type specimens) and a more angulated propodeal dorsum (not gently convex as in the types). however, these discrete differences fall within the variation observed in northern populations of the species. the specimens registered in rio grande do sul were captured at the environment typically associated with the genus, grasslands and open fields. despite the few observations on g. pombero natural history (wild & cuezzo 2006), the present record reinforces the theory that these ants can tolerate some degree of anthropic disturbance (guerrero & sanabria, 2011), since they were recorded in natural grasslands under traditional grazing management. it is interesting to notice that despite the several ant inventories carried out in the states of paraná (pr) and santa catarina (sc), the first occurrence of gracilidris in south rm feitosa, w dröse, lr podgaiski, ms mendonça jr. – first record of gracilidris from south brazil298 brazil was reported from the southernmost state of this region, rio grande do sul. this fact suggests that (1) g. pombero probably occurs in pr and sc, but (2) the atlantic forest that predominantly cover these states has received the bulk of the sampling effort, while the natural open fields and grasslands of south brazil remain relatively undersampled. the presence of gracilidris in the fossil record of the dominican amber (wilson, 1985; wild and cuezzo, 2006) and the present register of the most meridional population known for the genus reinforces the hypothesis that gracilidris pombero is a remaining lineage of a previously widely distributed ant genus which predominantly occurred in open fields and/or arid zones (fig 2). the occurrence of the species in the colombian amazon is an enigmatic biogeographical issue that remains to be explained. guerrero and sanabria (2011) argue that the amazon basin could be a potential biogeographic barrier for the soil-nesting dolichoderine. since gracilidris occurs above and below the amazon basin (considering the fossil record), primarily in open environments, this could suggest that in the past gracilidris could have been found in almost all open fields of the neotropics, including the amazon region during a drier climate period. these authors also suggest that the increasing humidity and the expansion of the amazonian wet forest in the recent past could have resulted in local extinctions, with subsequent isolation of gracilidris in the extreme north and south of the amazon basin, and the subsequent extinction of the northernmost populations. a second hypothesis raised by guerrero and sanabria (2011) considers the effect of the rise of the andes on the amazon basin. in this case, the lifting of the eastern slope of the northern andes during the late miocene changed the course of the rivers in the amazon basin from the west to the east of the continent, resulting in the current amazonian river system, a new natural barrier that separated the populations of the different species in the genus. in both scenarios, the colombian populations of g. pombero were separated from those of southern south america, but the biogeographical mechanism could have been different. the presence of the fossil g. humilioides in the dominican amber and the peculiar distribution of the extant g. pombero reveal an interesting biogeographic scenario to be investigated. new collections of the genus in the open environments of northern south america and a detailed phylogeographic study of its single extant species would be extremely important to clarify the evolution patterns of the genus and even to confirm the precise identity of the different populations of g. pombero. fig 1. gracilidris pombero worker (aceguá, rs, brazil). a: head in frontal view. b: body in profile. fig 2. distribution map of gracilidris pombero in the neotropics. known distribution: red circles. new records from rio grande do sul, brazil: yellow triangles. acknowledgements thanks to brunno bueno and gabriel melo (dzup) for kindly helping with the preparation of the high-resolution images of g. pombero. arthropod sampling in lavras do sul and aceguá (peld campos sulinos) was supported by grants from cnpq to valério pillar (558282/2009-1 and 403750/2012-1). mmj was supported by a cnpq research productivity grant (309348/2012-9). sociobiology 62(2): 296-299 (june, 2015) 299 references costa-milanez, c.b., lourenço-silva, g., castro, p.t.a., majer, j.d. & ribeiro, s.p. (2014). are ant assemblages of brazilian veredas characterised by location or habitat type? brazilian journal of biology, 74: 89-99. doi: 10.1590/15196984.17612 guerrero, r.j. & sanabria, c. (2011). the first record of the genus gracilidris (hymenoptera: formicidae: dolichoderinae) from colombia. revista colombiana de entomologia, 37: 159-161. instituto brasileiro de geografia e estatística [ibge]. 2004. mapa de biomas e de vegetação. retrieved from: http://www. ibge.gov.br/home/presidencia/noticias/21052004biomashtml. shtm#sub_pesquisas. (accessed date: 23 january, 2015). neves, f.s., queiroz-dantas, k.s., rocha, w.d., delabie, j.h.c. (2013). ants of three adjacent habitats of a transition region between the cerrado and caatinga biomes: the effects of heterogeneity and variation in canopy cover. neotropical entomology, 42: 258-26. overbeck, g.e., müller, s.c., fidelis, a., pfadenhauer, j., pillar, v.d., blanco, c.c. boldrini, i.i., both, r. & forneck, e.d. (2007). brazil’s neglected biome: the south brazilian campos. perspectives in plant ecology, evolution and systematics, 9: 101-116. pillar, v.p., müller, s.c., castilhos, z.m.s. & jaques, a.v.a. (2009). campos sulinos conservação e uso sustentável da biodiversidade. brasília: ministério do meio ambiente, 403 p. prado, d.e. & gibbs, p.e. (1993). patterns of species distributions in the dry seasonal forests of south america. annals of the missouri botanical garden, 80: 902-927. ward, p.s., brady, s.g., fisher, b.l. & schultz, t.r. (2010). phylogeny and biogeography of dolichoderine ants: effects of data partitioning and relict taxa on historical inference. systematic biology, 59: 342-362. wild, a.l. & cuezzo, f. (2006). rediscovery of a fossil dolichoderine ant lineage (hymenoptera: formicidae: dolichoderinae) and a description of a new genus from south america. zootaxa, 1142: 57-68. wilson, e.o. (1985). ants of the dominican amber. 3. the subfamily dolichoderinae. psyche, 92: 17-37. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.281-295sociobiology 62(2): 281-295 (june, 2015) ants of the panga ecological station, a cerrado reserve in central brazil introduction one of the first steps for the establishment of effective conservation actions and monitoring programs is the generation and maintenance of species distribution records (agosti & alonso, 2000). in this sense, species lists play an important role because they provide essential data for a more comprehensive analysis of diversity patterns, including biogeographical patterns (fisher, 2010). the compilation of species lists can also determine the presence of rare, threatened, or ecologically important species (agosti & alonso, 2000) that otherwise could be missed in a single survey. in addition, recording the distribution of species is essential for the creation of a data network (agosti & alonso, 2000) that can be very important for basic research, allowing taxonomists to access new records of poorly known species. comprehensive invertebrate surveys are essential for biodiversity studies as invertebrates are highly abundant, diverse, and play key ecological roles in most terrestrial ecosystems abstract species lists are an invaluable tool for a more comprehensive analysis of diversity patterns. such lists, when derived from a comprehensive sampling effort, can indicate the presence of rare, threatened, or ecologically important species. this study aimed to generate a species list of the ants of the panga ecological station, a protected cerrado reserve in southeastern brazil. this list was generated through taxonomic identification or through unification of the morphospecies codes of all specimens collected at the reserve in ten different studies since 2003. information about the types of habitat and strata of occurrence of each species or morphospecies was also compiled. the data presented here represents one of the most intensive ant inventories conducted in the brazilian cerrado. we recorded 277 ant species belonging to 58 genera and nine subfamilies. this number is 1.63 to 3.69 times higher than the number of species recorded in other cerrado localities surveyed so far. more species were collected in the savanna (249 species) than in the forest habitats (108 species), and more species were collected on ground (226 species) than in arboreal vegetation (117 species). taxonomic identification was possible for 171 of the 277 species collected. three of the named species are recorded for the first time in brazil. among the 106 unidentified species, at least six of them represent new, undescribed species. together, these results highlight the conservation potential of this cerrado reserve. sociobiology an international journal on social insects gp camacho1, hl vasconcelos2 article history edited by gilberto m. m. santos, uefs, brazil received 26 december 2014 initial acceptance 16 march 2015 final acceptance 08 april 2015 keywords ant inventories, ant diversity, formicidae, species distribution, neotropical savannas. corresponding author gabriela p. camacho prog. de pós-graduação em entomologia universidade federal do paraná 81531-980 curitiba, pr, brazil e-mail: gabipcamacho@ymail.com (fittkau & klinge, 1973; alonso & agosti, 2000). ants are a particularly important group of terrestrial invertebrates, as they establish mutualistic associations with many organisms (ness et al., 2010), are predators of other invertebrates (philpott & armbrecht, 2006), secondary seed dispersers, and contribute to soil aeration (lee & foster, 1991) and nutrient cycling (hudson et al., 2009). however, as is also the case for many other invertebrate groups, one of the difficulties in generating a list of ant species from a given locality or region is the taxonomic identification. this identification is usually performed at the “morphospecies” level, what makes the compilation of data originated from different researchers very difficult or even not possible. more than 12,900 ant species, distributed in 324 genera, are known worldwide (bolton, 2014). this fauna has strong biogeographic patterns, with different regions containing endemic and strongly related taxa that show rapid species turnover within their borders (fisher, 2010). their abundance and diversity reach research article ants 1 universidade federal do paraná, curitiba, pr, brazil 2 universidade federal de uberlândia, uberlândia, mg, brazil gp camacho, hl vasconcelos ants of the panga ecological station282 a peak in tropical regions and decline in temperate latitudes (fernandez & sendoya, 2004). the neotropical region has the greatest ant diversity and the highest level of endemism among the different biogeographic regions in the globe (fisher, 2010), harboring 30% of all ant species currently described (fernandez & sendoya, 2004). nevertheless, there are few comprehensive inventories of the neotropical ant fauna (but see longino & colwell, 1997), particularly in non-forest ecosystems. the cerrado, one of the biodiversity hotspots worldwide (myers et al., 2000), comprises a mosaic of vegetation types, which includes grasslands, forests, and especially savannas of variable structure that originally covered over two million square kilometers of central brazil. about 10,000 plant species occurs in the cerrado region, of which approximately 40% are endemic (ratter et al., 1997). however, this biodiversity is highly threatened by the expansion of modern agriculture. the creation of conservation units in this biome is urgent, since only less than 3% of the original cerrado area is currently preserved (klink & machado, 2005). one of the best known ant faunas of cerrado is the fauna of the “reserva ecológica do panga” (hereafter panga ecological station), a reserve of 409 ha located in uberlândia, minas gerais, brazil. since 2003 several studies involving the ant fauna were conducted in this reserve (vasconcelos et al., 2008; campos et al., 2011; lopes & vasconcelos, 2008; lopes & vasconcelos, 2011; pacheco & vasconcelos, 2007; pacheco & vasconcelos, 2012; powell et al., 2011; vasconcelos et al., 2009; camarota et al., 2015). however, a complete list of the species collected in the reserve was never compiled, since each study generated its own list of species and morphospecies and the codes used to separate the morphospecies were unique for each study. the objective of this study was to integrate information derived from the various ant surveys performed at the panga ecological station, in order to provide a comprehensive list of species. for this, we examined the specimens collected in each study, identifying to species level whenever possible and, when this was not possible, assigning a standardized code for each recognized morphospecies. material and methods all ants listed in this study were collected at the panga ecological station (pes), a 409 ha reserved owned by the university of uberlândia (ufu) and located 30 km south of uberlândia, minas gerais state, brazil (19° 10’ s, 48° 24’ w). the region is characterized by a tropical climate with two distinct seasons: a dry winter (from may to september) and a rainy summer (from october to april). average annual temperature in this locality is 22.8° c and average annual rainfall is 1482 mm. soils at the site are primarily red latosoils. most of the reserve is covered by savannas of which cerrado sensu stricto is the predominant and in addition there are patches of forests and seasonally water-logged swamps. for a more detailed description of the vegetation of the study area see cardoso et al. (2009). the species list provided here results from a compilation of data from 10 different ant surveys performed at pes since 2003 (table 1). all specimens collected during these studies are deposited at the zoological collection of the federal university of uberlândia (ufu), in uberlândia, brazil. we examined all specimens deposited at ufu´s zoological collection. all material identified at the morphospecies level was revised in order to unify morphospecies codes across the studies. once this was done, we attempted to identify the specimens using taxonomic keys (longino, 2003; wild, 2005; mayhé-nunes & brandão, 2006; klingenberg & brandão, 2009; mackay & mackay, 2010) or by sending specimens to ant taxonomists (see acknowledgments). table 1. list of studies involving the ant fauna of the panga ecological station, including sampling effort, sampling method and habitats studied. source number of samples sampling method habitats sampled pacheco & vasconcelos (2007) 60 baited pitfall traps savanna (cerrado sensu stricto) lopes & vasconcelos (2008) 120 baits pitfall traps winkler extractor savanna (cerrado sensu stricto, cerrado ralo and cerrado denso), forest vasconcelos et al. (2008) 80 corn flour baits orange peel baits savanna (campo cerrado, cerrado sensu stricto, cerradão), semideciduous forest vasconcelos et al. (2009) 160 mini-winkler extractors savanna (cerrado denso) campos et al. (2011) 320 pitfall traps sardine and honey baits cerrado sensu stricto lopes & vasconcelos (2011) 90 vegetation beating active search savanna (cerrado denso) powell et al. (2011) 60 baited pitfall traps savanna (cerrado sensu stricto, cerrado ralo, cerradão) pacheco & vasconcelos (2012) 100 pitfall traps (epigeic and hypogeic) savanna (cerrado sensu stricto) koch (2014) 81 baited pitfall traps vegetation beating savanna (cerrado sensu stricto, cerrado ralo) camarota et al. (2015) 240 baited pitfall traps savanna (cerrado sensu stricto, cerrado ralo) sociobiology 62(2): 281-295 (june, 2015) 283 sampling completeness (across all studies) was determined using the chao 2 species richness estimator, which was calculated using the default options of estimates 9.1.0 (colwell et al, 2012). based on information existing on the specimens labels and/or through information in the original data sets of the various studies listed in table 1, we determined the habitats (savanna or forest) and the foraging strata (soil or woody vegetation) of occurrence (number of times the species was recorded in all studies) for each species and morphospecies. results we recorded a total of 277 ant species from 58 genera and nine subfamilies (appendix). using taxonomic keys or with the aid of experts we were able to name a total of 177 species (63.9% of the total). in the previous studies conducted at pes only 24 species had been formally named. therefore, the present study adds 153 species names for the ant fauna of the reserve.the total number of species recorded so far at the reserve represents 89.2% of the expected species number to be found there according to the species richness estimator chao 2 (310.4 species). the diversity-dominance curve (fig 1) shows a high proportion of rare species (24.9% of the total), i.e. of species that were collected only once or twice. the subfamily myrmicinae was the most diverse, with 153 species, followed by formicinae with 43 species and dolichoderinae and ponerinae, with 23 species each. the most diverse genus was pheidole with 39 species, followed by camponotus with 35 and cephalotes, with 17 species. the most frequent species was camponotus crassus mayr, 1862, with 442 records of occurrence, followed by cephalotes pusillus (klug, 1824), pheidole oxyops forel, 1908 and pheidoleradoszkowskii mayr, 1884 with 348, 337 and 306 records, respectively. fig 1. diversity-abundance curve of all ant species collected at the panga ecological station. more species were recorded in the savanna (249 species) than in the forest habitats (108 species), and more species were collected on ground (226 species) than in the arboreal vegetation (117 species). a total of 160 species from 56 genera were only found on ground, whereas 51 species from 23 genera were only found in the woody vegetation. regarding species occurrences in different vegetation types, 169 species were collected exclusively in the savanna habitats, 28 only in the forest habitats, and 141 in both. discussion the data presented here represents one of the most intensive ant inventories conducted in the brazilian cerrado. we found that at least 277 ant species coexist within the 409 hectares of panga ecological station. this number of species is nearly as high as the number of species recorded by silvestre et al. (2004) in seven different cerrado localities (333 species in total) and higher than the number of species recorded in savannas of africa and australia (table 2). table 2. number of ant species recorded in different tropical savannas areas of the world. locality sampled ant species richness number of samples sampling methodology type of habitat source brazil (sp e go) 56 120 baits cerrado silva et al. (2004) brazil, niquelândia (go) 158 50 baits and additional methods* cerrado silvestre et al. (2004) brazil, colinas do sul (go) 167 50 baits and additional methods* cerrado silvestre et al. (2004) brazil, campinaçu (go) 154 50 baits and additional methods* cerrado silvestre et al. (2004) brazil, uruaçu (go) 170 50 baits and additional methods* cerrado silvestre et al. (2004) brazil, luiz antônio (sp) 128 50 baits and additional methods* cerrado silvestre et al. (2004) brazil, cajuru (sp) 85 150 baits and additional methods* cerrado silvestre et al. (2004) brazil, aguas emendadas (df) 75 150 baits and additional methods* cerrado silvestre et al. (2004) brazil (mg) 61 51 baited pitfall traps cerrado campos et al. (2008) australia 27 100 sweep nets arboreal savanna andersen et al. (2007) africa 34 105 mini-winkler extractor and pitfall traps arboreal savanna parr&chown (2001) africa 160 120 pitfall traps arboreal savanna parr et al. (2004) africa 72 200 pitfall traps arboreal savanna sithole et al. (2010) *including manual collection, pitfall traps, subterranean pitfall traps, color trays, light traps, malaise traps and winkler extractors. gp camacho, hl vasconcelos ants of the panga ecological station284 however, it is important to mention that the elevated number of species recorded at pes may only reflect the higher diversity of sampling methods and/or the higher intensity of sampling at this reserve compared to the other savannas areas sampled so far. only 66 species were recorded both on ground and in the woody vegetation, indicating that, as also observed in tropical forests (bruhl et al., 1998; vasconcelos & vilhena, 2006; wilkie et al., 2010) and in other cerrado areas (campos et al., 2008), there is a vertical stratification of the ant fauna. camponotus, pseudomyrmex and pheidole were the most frequent genera in the vegetation, whereas pheidole, camponotus and solenopsis were the most frequent ones on ground. differences in species composition were also clear. the most abundant species in the vegetation were camponotus crassus and cephalotes pusilus, whereas on ground pheidole oxyops and pheidole radoszkowskii were the most abundant ones (fig 2). therefore, the high diversity of ants in the cerrado reserve we studied can be explained, at least in part, by the specialization of some species to forage and/or nest either on ground or in the vegetation (campos et al., 2008). habitat specialization is probably also involved since many of the species inhabiting the savannas were not recorded in the forest habitats and vice-versa (fig 3). fig 2. the most abundant ant genera (a) and most abundant species (b) collected on ground or in the woody vegetation. fig 3. the most abundant ant genera (a) and most the abundant species (b) collected in the savanna or forest habitats. taxonomic identification was possible for 171 of the 277 species collected at pes. although many of the named species are widely distributed in neotropics (fernandez & sendoya, 2004) some are poorly known. for instance, pheidole superba wilson, 2003, was previously known only from its type locality in colombiaand from panamá (wilson, 2003). similarly, crematogaster crucis forel, 1912 and myrmicocripta urichi weber, 1937 were recorded for the first time in brazil (ant wiki, 2014). cyatta abscondita sosacalvo, schultz, brandão, klingenberg, feitosa, rabeling, bacci, lopes & vasconcelos, 2014, the only representative of the newly described genus cyatta, is also present at pes. the same is true for cephalotes specularis brandão, feitosa, powell & del-claro, 2014, a newly described species that presents a remarkable social parasitism behavior (powell et al., 2014). in addition, pseudomyrmex curacaensis (forel, 1912) and pseudomyrmex euryblemma (forel, 1899) are recorded for the first time in the cerrado region (rodrigo m. feitosa, departamento de zoologia da universidade federal do paraná, personal communication, december 12, 2014). among the 106 unidentified species, at least six of them represent new, undescribed species, including five species of trachymyrmex (antônio mayhé-nunes, departamento de sociobiology 62(2): 281-295 (june, 2015) 285 biologia animal, universidade federal rural do rio de janeiro, personal communication, july 2010) and one species of xenomyrmex (lívia p. prado, museu de zoologia da universidade de são paulo, personal communication, february 14, 2014). together, these results highlight the conservational potential of this cerrado reserve. the panga ecological station harbors a surprisingly high number of ant species indicating that, although poorly studied, the cerrado has a highly diverse ant fauna. moreover, the large number of species that remain unnamed shows that the ant fauna of this region is taxonomically poorly known, emphasizing the need for a greater effort in the collection and description of new species. acknowledgments we are thankful to dr. rodrigo m. feitosa, dr. fernando c. fernandez, dr. phillip s. ward, dr. john t. longino, dr. ted r. schultz, dr. jeffrey sosa-calvo, dr. antônio mayhénunes, lívia p. prado and alexandre c. ferreira for their help with the identification of the ant species, and to brunno bueno rosa for help with the preparation of the images. we deeply thank renata pacheco, cauê t. lopes, bruna b. araújo, scott powell, elmo koch, flávio camarota and ricardo campos for providing their original data sets, without which some of the analysis presented here would be impossible.we also thank rodrigo feitosa, ricardo solar and an anonymous reviewer for reading and commenting on a previous version of the manuscript. financial support was provided by the brazilian council of research and scientific development (cnpq). references alonso, l. e. & agosti, d. 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(2003). pheidole in the new world: a dominant, hyperdiverse genus. cambridge: harvard university press. 794 p. gp camacho, hl vasconcelos ants of the panga ecological station288 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata. savanna forest ground woody vegetation total amblyoponinae prionopelta prionopelta cf. punctulata mayr, 1866 5 4 9 0 9 dolichoderinae azteca azteca sp. 01 32 0 0 32 32 azteca sp. 02 19 0 2 17 19 azteca sp. 03 10 0 5 5 10 azteca sp. 04 1 0 0 1 1 dolichoderus dolichoderus bispinosus (olivier, 1792) 6 0 0 6 6 dolichoderus diversus emery, 1894 1 0 1 0 1 dolichoderus imitator (emery, 1894) 2 0 2 0 2 dolichoderus lamellosus (mayr, 1870) 16 0 0 16 16 dolichoderus lutosus (f. smith, 1858) 32 0 0 32 32 dorymyrmex dorymyrmex prox. jheringi forel, 1912 8 0 8 0 8 dorymyrmex pyramicus forel, 1912 47 7 44 10 54 dorymyrmex sp. 01 1 0 1 0 1 dorymyrmex sp. 02 0 1 0 1 1 forelius forelius brasiliensis (forel, 1908) 6 0 3 0 6 forelius maranhaoensis cuezzo, 2000 2 0 2 0 2 gracilidris gracilidris pombero wild & cuezzo, 2006 1 0 1 0 1 linepithema linepithema aztecoides wild, 2007 98 0 73 25 98 linepithema cerradense wild, 2007 1 0 1 0 1 linepithema micans forel, 1908 12 0 11 1 12 linepithema sp. 01 13 6 19 0 19 tapinoma tapinoma sp. 01 7 0 7 0 7 tapinoma sp. 02 5 0 5 0 5 tapinoma sp. 03 13 0 0 13 13 dorylinae cerapachys cerapachys splendens borgmeier, 1957 2 2 4 0 4 eciton eciton vagans borgmeier, 1955 2 1 3 0 3 labidus labidus coecus (latreille, 1802) 11 0 10 1 11 neivamyrmex neivamyrmex sp. 01 6 0 6 0 6 nomamyrmex nomamyrmex esenbeckii (westwood, 1842) 2 0 2 0 2 sociobiology 62(2): 281-295 (june, 2015) 289 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). savanna forest ground woody vegetation total ectatomminae ectatomma ectatomma brunneum f. smith, 1858 10 0 8 2 10 ectatomma edentatum roger, 1863 12 1 13 0 13 ectatomma lugens emery, 1894 41 17 58 0 58 ectatomma opaciventre roger, 1861 27 6 33 0 33 ectatomma permagnum forel, 1908 104 7 111 0 111 ectatomma planidens borgmeier, 1939 16 0 16 0 16 ectatomma suzanae almeida, 1986 35 8 43 0 43 ectatomma tuberculatum olivier, 1792 14 5 4 15 19 gnamptogenys gnamptogenys acuminata (emery, 1896) 4 0 4 0 4 gnamptogenys horni (santschi, 1929) 2 0 2 0 2 gnamptogenys striatula mayr, 1884 5 2 10 0 10 typhlomyrmex typhlomyrmex sp. 01 1 0 1 0 1 formicinae brachymyrmex brachymyrmex sp. 01 40 3 39 4 43 brachymyrmex sp. 02 184 31 165 50 215 brachymyrmex sp. 03 2 0 2 0 2 camponotus camponotus arboreus (f. smith, 1858) 25 0 7 18 25 camponotus atriceps (f. smith, 1858) 163 7 35 135 170 camponotus blandus (f. smith, 1858) 74 47 68 53 121 camponotus cameranoi emery, 1894 11 0 0 11 11 camponotus cingulatus mayr, 1862 5 19 20 4 24 camponotus crassus mayr, 1862 442 0 125 317 442 camponotus dimorphus emery, 1894 0 3 0 3 3 camponotus fastigatus roger, 1863 4 0 0 4 4 camponotus genatus santschi, 1922 7 0 0 7 7 camponotus latangulus roger, 1863 1 15 9 7 16 camponotus lespesii forel, 1866 90 20 89 21 110 camponotus leydigi forel, 1866 5 2 3 4 7 camponotus melanoticus santschi, 1839 84 1 20 62 85 camponotus novogranadensis mayr, 1870 6 0 5 1 6 camponotus renggeri emery, 1894 64 0 41 19 64 camponotus rufipes fabricius, 1775 42 2 19 25 44 camponotus senex f. smith, 1858 21 0 0 21 21 camponotus sericeiventris guerin-meneville, 1838 111 16 46 79 127 camponotus tenuiscapus roger, 1893 21 0 10 11 21 camponotus textor forel, 1899 80 0 0 80 80 camponotus trapeziceps santschi, 1922 0 1 0 1 1 camponotus vittatus forel, 1904 1 0 1 0 1 camponotus sp. 01 62 0 6 56 62 gp camacho, hl vasconcelos ants of the panga ecological station290 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). savanna forest ground woody vegetation total camponotus (continuation) camponotus sp. 02 24 0 0 24 24 camponotus sp. 03 0 1 0 1 1 camponotus sp. 04 6 0 2 4 6 camponotus sp. 05 4 0 3 0 4 camponotus sp. 06 4 0 4 0 4 camponotus sp. 07 10 0 5 5 10 camponotus sp. 08 20 0 20 0 20 camponotus sp. 09 3 5 6 2 8 camponotus sp. 10 0 12 11 1 12 camponotus sp. 11 5 0 5 0 5 camponotus sp. 12 5 0 5 0 5 camponotus sp. 13 6 0 6 0 6 myrmelachista myrmelachista sp. 01 15 3 18 0 18 nylanderia nylanderia sp. 01 137 10 147 0 147 nylanderia sp. 02 2 0 2 0 2 nylanderia sp. 03 9 9 18 0 18 nylanderia sp. 04 14 0 14 0 14 heteroponerinae acanthoponera acanthoponera mucronata roger, 1860 1 0 0 1 1 myrmicinae acanthognathus acanthognathus rudis brown & kempf, 1969 1 1 2 0 2 acromyrmex acromyrmex ambiguus emery, 1888 1 0 1 0 1 acromyrmex balzani emery, 1890 9 0 9 0 9 acromyrmex subterraneus (forel, 1893) 0 2 2 0 2 apterostigma apterostigma sp.01 6 1 6 1 7 apterostigma sp.02 35 0 35 0 35 apterostigma gr. auriculatum sp.03 0 6 6 0 6 atta atta laevigata smith, f., 1858 18 2 13 7 20 atta sexdens linnaeus, 1758 1 5 6 0 6 cardiocondyla cardiocondyla emeryi forel, 1881 4 0 4 0 4 carebara carebara brevipilosa (fernandéz, 2004) 8 5 13 0 13 carebara urichi (w.m. wheeler, 1922) 1 0 1 0 1 carebara gr. lignata sp. 01 2 0 2 0 2 cephalotes cephalotes adolphi emery, 1906 1 0 0 1 1 cephalotes angustus mayr, 1862 7 0 0 7 7 sociobiology 62(2): 281-295 (june, 2015) 291 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). savanna forest ground woody vegetation total cephalotes (continuation) cephalotes atratus (linnaeus, 1758) 21 0 0 21 21 cephalotes clypeatus fabricius, 1804 3 0 0 3 3 cephalotes cordatus smith, f., 1853 4 0 0 4 4 cephalotes depressus klug, 1824 1 0 0 1 1 cephalotes eduarduli forel, 1921 5 0 2 3 5 cephalotes grandinosus (f. smith, 1860) 21 0 2 19 21 cephalotes liepini de andrade, 1999 2 0 0 2 2 cephalotes maculatus (f. smith, 1876) 7 0 0 7 7 cephalotes minutus (fabricius, 1804) 9 0 2 7 9 cephalotes pallidoides de andrade, 1999 7 0 0 7 7 cephalotes pellans de andrade, 1999 10 0 0 10 10 cephalotes persimilis de andrade, 1999 9 0 1 8 9 cephalotes pusillus (klug, 1824) 331 17 71 277 348 cephalotes simillimus kempf, 1951 1 1 1 2 3 cephalotes specularis brandão, feitosa, powell & del-claro, 2014 1 0 0 1 1 crematogaster crematogaster cf. crucis forel, 1912 0 1 0 1 1 crematogaster curvispinosa (mayr, 1862) 0 5 3 2 5 crematogaster distans (mayr, 1870) 2 2 4 0 4 crematogaster erecta (mayr, 1866) 0 3 3 0 3 crematogaster limata (smith, f., 1858) 2 1 2 1 3 crematogaster nitidiceps emery, 1895 5 17 9 9 22 crematogaster victima forel, 1901 1 0 1 0 1 crematogaster sp. 01 4 0 0 4 4 crematogaster sp. 02 3 0 0 3 3 crematogaster sp. 03 8 0 4 4 8 crematogaster sp. 04 0 3 2 1 3 crematogaster sp. 05 1 0 1 0 1 crematogaster sp. 06 1 0 1 0 1 cyatta cyatta abscondita sosa-calvo et al., 2014 1 0 1 0 1 cyphomyrmex cyphomyrmex olitor forel, 1903 34 11 45 0 45 cyphomyrmex rimosus (spinola, 1851) 15 14 30 0 30 cyphomyrmex gr. rimosus sp. 01 2 0 2 0 2 cyphomyrmex gr. rimosus sp. 02 4 0 4 0 4 cyphomyrmex gr. strigatus sp. 01 3 0 3 0 3 hylomyrma hylomyrma balzani emery, 1894 5 4 9 0 9 hylomyrma reitteri (mayr, 1887) 9 0 9 0 9 gp camacho, hl vasconcelos ants of the panga ecological station292 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). savanna forest ground woody vegetation total megalomyrmex megalomyrmex drifti kempf, 1961 0 2 2 0 2 megalomyrmex goeldii forel, 1912 0 3 3 0 3 megalomyrmex silvestrii (w.m. wheeler, 1909) 2 0 2 0 2 monomorium monomorium floricola jerdon, 1851 (introduced) 4 0 2 2 4 mycetagroicus mycetagroicus cerradensis brandão & mayhé-nunes, 2001 7 0 7 0 7 mycetarotes mycetarotes parallelus emery, 1906 5 0 5 0 5 mycocepurus mycocepurus goeldii (forel, 1893) 85 19 104 0 104 mycocepurus obsoletus emery, 1913 2 0 2 0 2 mycocepurus smithii (forel, 1893) 42 1 43 0 43 myrmicocripta myrmicocripta cf. urichi weber, 1937 0 3 3 0 3 myrmicocrypta cf. squamosa smith, f., 1860 10 10 20 0 20 myrmicocrypta sp. 01 3 0 3 0 3 nesomyrmex nesomyrmex cf. brasiliensis kempf, 1958 3 0 0 3 3 nesomyrmex spininodis mayr, 1887 13 2 5 10 15 ochetomyrmex ochetomyrmex semipolitus mayr, 1878 1 0 1 0 1 octostruma octostruma balzani (emery, 1894) 0 18 18 0 18 octostruma iheringi (emery, 1888) 1 0 1 0 1 oxyepoecus oxyepoecus longicephalus de albuquerque, lavor & brandão, 2004 1 0 1 0 1 pheidole pheidole fallax 4 0 4 0 4 pheidole fimbriata (roger, 1863) 0 8 8 0 8 pheidole flavens roger, 1863 70 37 107 0 107 pheidole gertrudae forel, 1886 2 0 2 0 2 pheidole nr. lucretii santschi, 1923 0 27 27 0 27 pheidole oxyops forel, 1908 253 83 330 7 337 pheidole pubiventris borgmeier, 1928 15 6 15 6 21 pheidole radoszkowskii (mayr, 1884) 262 44 212 94 306 pheidole nr. rugiceps wilson, 2003 11 6 17 0 17 pheidole nr. schmalzi emery, 1894 70 0 32 38 70 pheidole nr. susannae forel, 1886 26 0 24 2 26 pheidole superba wilson, 2003 2 0 2 0 2 pheidole triconstricta forel, 1886 58 28 86 0 86 pheidole sp. 01 122 12 134 0 134 sociobiology 62(2): 281-295 (june, 2015) 293 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata. (continuation). savanna forest ground woody vegetation total pheidole (continuation) pheidole sp. 02 35 0 35 0 35 pheidole sp. 03 2 5 7 0 7 pheidole sp. 04 2 0 2 0 2 pheidole sp. 05 9 0 9 0 9 pheidole sp. 06 3 16 19 0 19 pheidole sp. 07 8 0 8 0 8 pheidole sp. 08 1 0 1 0 1 pheidole sp. 09 29 5 33 1 34 pheidole sp. 10 1 0 0 1 1 pheidole sp. 11 9 0 9 0 9 pheidole sp. 12 20 0 10 10 20 pheidole sp. 13 1 0 1 0 1 pheidole sp. 14 1 0 1 0 1 pheidole sp. 15 1 0 1 0 1 pheidole sp. 16 6 0 6 0 6 pheidole sp. 17 0 3 1 2 3 pheidole sp. 18 12 0 12 0 12 pheidole sp. 19 11 11 22 0 22 pheidole sp. 20 5 0 5 0 5 pheidole sp. 21 9 0 7 2 9 pheidole sp. 22 0 5 5 0 5 pheidole sp. 23 12 11 22 1 23 pheidole sp. 24 50 0 46 4 50 pheidole sp. 25 1 1 0 2 2 pheidole sp. 26 3 0 3 0 3 pogonomyrmex pogonomyrmex naegelli emery, 1878 19 0 19 0 19 rogeria rogeria sp. 01 1 0 1 0 1 rogeria sp. 02 1 1 2 0 2 sericomyrmex sericomyrmex luederwaldti santschi, 1925 69 41 110 0 110 sericomyrmex scrobifer forel, 1911 24 0 24 0 24 solenopsis solenopsis substituta santschi, 1925 91 16 90 17 107 solenopsis tridens forel, 1911 97 66 97 66 163 solenopsis sp. 01 49 0 30 19 49 solenopsis sp. 02 126 0 104 22 126 solenopsis sp. 03 20 0 20 0 20 solenopsis sp. 04 56 30 40 46 86 solenopsis sp. 05 0 1 0 1 1 gp camacho, hl vasconcelos ants of the panga ecological station294 savanna forest ground woody vegetation total solenopsis (continuation) solenopsis sp. 06 3 0 3 0 3 solenopsis sp. 07 1 0 1 0 1 solenopsis sp. 08 0 1 1 0 1 solenopsis sp. 09 2 0 2 0 2 solenopsis sp. 10 0 1 1 0 1 solenopsis sp. 11 2 1 3 0 3 solenopsis sp. 12 1 0 1 0 1 strumigenys strumigenys denticulata mayr, 1887 15 24 39 0 39 strumigenys eggersi (emery, 1890) 34 6 40 0 40 strumigenys sp. 01 1 0 1 0 1 strumigenys grytava bolton, 2000 17 0 17 0 17 strumigenys zeteki (brown, 1959) 0 2 2 0 2 strumigenys nr. elongata roger, 1863 3 0 3 0 3 strumigenys louisianae roger, 1863 6 0 6 0 6 strumigenys perparva brown, 1958 12 8 20 0 20 strumigenys nr. trinidadensis (w.m. wheeler, 1922) 0 5 5 0 5 trachymyrmex trachymyrmex bugnioni (forel, 1912) 13 4 17 0 17 trachymyrmex cirratus mayhé-nunes & brandão, 2005 3 0 3 0 3 trachymyrmex cornetzi (forel, 1912) 3 0 3 0 3 trachymyrmex dichrous kempf, 1967 16 0 16 0 16 trachymyrmex farinosus emery, 1894 9 0 9 0 9 trachymyrmex holmgreni w.m. wheeler, 1925 4 0 4 0 4 trachymyrmex gr. urichi sp. 01 1 0 1 0 1 trachymyrmex gr. urichi sp. n. 02 19 0 19 0 19 trachymyrmex sp. n. 03 8 0 8 0 8 trachymyrmex sp. n. 04 3 0 3 0 3 trachymyrmex sp. n. 05 16 2 18 0 12 trachymyrmex sp. n. 06 24 0 24 0 24 tranopelta tranopelta gilva mayr, 1866 1 0 1 0 1 wasmannia wasmannia affinis santschi, 1929 12 0 6 6 12 wasmannia auropunctata (roger, 1863) 71 20 79 12 91 wasmannia rochai (forel, 1912) 9 0 0 9 9 xenomyrmex xenomyrmex sp. n. 1 0 0 1 1 ponerinae anochetus anochetus bispinosus (f. smith, 1858) 24 14 38 0 38 hypoponera hypoponera foreli mayr, 1887 2 3 5 0 5 hypoponera sp. 01 7 0 7 0 7 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). sociobiology 62(2): 281-295 (june, 2015) 295 savanna forest ground woody vegetation total hypoponera (continuation) hypoponera sp. 02 2 0 2 0 2 hypoponera sp. 03 0 4 4 0 4 hypoponera sp. 04 0 1 1 0 1 hypoponera sp. 05 10 11 21 0 21 hypoponera sp. 06 0 3 3 0 3 hypoponera sp. 07 3 2 5 0 5 hypoponera sp. 08 1 0 1 0 1 neoponera neoponera agilis (forel, 1901) 2 0 0 2 2 neoponera apicalis latreille, 1802 2 0 2 0 2 neoponera crenata (roger, 1861) 0 2 0 2 2 neoponera inversa f. smith, 1858 19 0 1 18 19 neoponera laevigata (f. smith, 1858) 1 0 1 0 1 neoponera verenae (forel, 1922) 95 14 109 0 109 neoponera villosa fabricius, 1804 41 0 10 31 41 odontomachus odontomachus bauri emery, 1892 5 0 5 0 5 odontomachus chelifer (latreille, 1802) 20 13 33 0 33 odontomachus laticeps roger, 1861 3 0 3 0 3 odontomachus meinerti forel, 1905 10 4 14 0 14 pachycondyla pachycondyla harpax fabricius, 1804 52 10 62 0 62 pachycondyla striata f. smith, 1858 1 7 8 0 8 pseudomyrmecinae pseudomyrmex pseudomyrmex curacaensis (forel, 1912) 29 0 0 29 29 pseudomyrmex elongatus mayr, 1870 59 0 0 59 59 pseudomyrmex euryblemma (forel, 1899) 2 0 0 2 2 pseudomyrmex gracilis (fabricius, 1804) 255 0 5 250 255 pseudomyrmex kuenckeli emery, 1890 9 0 0 9 5 pseudomyrmex maculatus f. smith, 1855 6 0 0 6 6 pseudomyrmex pallidus f. smith, 1855 13 6 12 7 19 pseudomyrmex sericeus stitz, 1913 4 0 0 4 4 pseudomyrmex simplex (f. smith, 1877) 36 0 0 36 36 pseudomyrmex tenuis fabricius, 1804 3 0 3 0 3 pseudomyrmex tenuissimus emery, 1906 14 0 0 14 14 pseudomyrmex termitarius f. smith, 1855 21 2 23 0 23 pseudomyrmex unicolor f. smith, 1855 9 0 0 9 9 pseudomyrmex urbanus f. smith, 1877 165 0 0 165 165 pseudomyrmex sp. 01 3 0 0 3 3 pseudomyrmex sp. 02 1 0 0 1 1 appendix. list of species recorded at the panga ecological station. numbers represent the total number of occurrences in different habitats or strata (continuation). 703 diversity of epigeal ants (hymenoptera: formicidae) in urban areas of alto tietê by d. r. de souza1; s. g. dos santos1; c. de b. munhae2 & m. s. de c. morini1 abstract the objective of this study was to conduct an inventory of the ant fauna, evaluating indicators of diversity, litter community composition and similarity among seven areas located around the city of mogi das cruzes (sp). in each area 20 pitfalls were distributed, which remained in the field for seven days. four samplings were performed, two in the rainy season and two during dry season. in total there were 92 recorded species, 36 genera, 19 tribes and seven subfamilies. the most frequent species belong to the genera pheidole and camponotus, both common in the neotropics. differences were observed in species composition with the formation of two groups, one under the influence of the atlantic forest and other in the urban region. only one exotic species, paratrechina longicornis, was recorded. the results indicate the importance of the forest surrounding the city to maintain the biological diversity of ant communities. keywords: anthropization, communities, atlantic forest, exotic ants, serra do itapeti introduction urban ecosystems are characterized as spatially heterogeneous and temporally dynamic sites, being usually recognized as areas under deep and constant human activities (mcintyre et al. 2001). these sites are effectively synonymous of disorder and reduction of biological diversity (murphy 1988; mcintyre et al. 2001; yamaguchi 2004; 2005). they are very similar all over the world in relation to the structure and function and differ only on the geographic location and size (savard et al. 2000). 1universidade de mogi das cruzes, núcleo de ciências ambientais, laboratório de mirmecologia. av. dr. cândido xavier de almeida souza n. 200. 08780-911. mogi das cruzes, sp, brasil. 2universidade estadual paulista, instituto de biologia, centro de estudos de insetos sociais. avenida 24 n. 1515. 13506-725. rio claro, sp, brasil. corresponding author. e-mail: morini@umc.br 704 sociobiolog y vol. 59, no. 3, 2012 some cities still have areas of native vegetation that represent important refuges for plants and animals (rodrigues et al. 1993). this is the case with the city of mogi das cruzes, located in the alto tietê river basin and protected by the watershed law (law n0 9.866). the region is composed of the main atlantic forest remnants in the southeastern brazil and at the same time it has an expressive population and industrial development (candido et al. 2010). ants are among the most abundant insects in urban environments (mcintyre et al. 2001; holway et al. 2002; lópez-moreno et al. 2003). the high diversity, numerical dominance and biomass in almost all habitats, ease of sampling and species identification, the presence of stationary nests, which allows the re-sampling over time (alonso & agosti 2000) make ants good indicators of biological diversity in urban areas (arcila et al. 2003). these insects also play critical functions for ecosystems, such as cycling of nutrients (hölldobler & wilson 1990; folgarait 1998; sanders & van veen 2011) and interaction with other organisms (schultz & mcglynn 2000). whereas the first step towards the conservation of certain areas or their use in a sustainable way are studies on biological diversity (scott et al. 1987) and the importance of formicidae ecosystems, this paper aims at presenting a list of species and descriptively assessing richness, diversity and similarity of communities around the city of mogi das cruzes. the urban area in this city grows around the serra do itapeti, which is a remnant of atlantic forest with high biodiversity (morini & miranda 2011) and its conservation is of paramount importance. materials and methods samplings were made around the city of mogi das cruzes (s 23º52’ 22”; w 46°18’ 55” / 742 m), which is located in the eastern region of são paulo city with 721 km2. seven sampling areas were delimited along a transect, whose ends are located in an urban park (parque leon feffer) and a conservation unit (parque natural municipal francisco affonso de mello) (table 1). in all areas, samplings were performed at sites under anthropogenic influence. four samplings were carried out in each area, two in the rainy season and two in the dry season (minuzzi et al. 2007). ants were collected with pitfall 705 de souza, d.r. — diversity of epigeal ants in alto tietê (n = 20), distributed every 20 m (baccaro et al. 2011). traps remained in field for seven days. the material was initially classified into subfamilies according to the proposal from bolton (2003), identified at the level of genus and named according to bolton (1994), baroni-urbani & de andrade (2007) and lapolla et al. (2010) and subsequently named into morphospecies comparing specimens with those from the formicidae collection of alto tietê. the taxa numbering sequence is according to this collection. the species were identified by comparison with specimens deposited in the zoolog y museum, university of são paulo (mzusp). vouchers were deposited at the university of mogi das cruzes (sp). richness was defined as the number of species and the abundance as the number of individuals of each species collected. the relative frequency of occurrence was based on the presence and absence data. we calculated the estimated richness (chao 2) and compared between different sampling areas using the kruskal-wallis test; the diversity indices of shannon-wiener (h ') and evenness (e). patterns of species composition and community structure were compared between sampling sites through ordination analysis (non-metric multidimensional scaling nmds). a similarity dendrogram was constructed table 1. characterization of sampling sites in the city of mogi das cruzes (sp). area location sampling site characteristics plf-parque leon feffer s 23°31’37.56”; w 46°13’45.28” it is an urban park, whose vegetation is found in different degrees of regeneration cec centro esportivo colégio joana d’arc s 23°31’19.36”; w 46°13’14” presence of grasses and a few tree species pnj parque nagib najar s 23°31’17”; w 46°13’01” it is an urban park, whose vegetation is at various stages of regeneration aui – urbanized area i s 23°30’49.24”; w 46’12’52.10” it is a country club, near a forest area; presence of grasses auii – urbanized area ii s 23°30’37.22”; w 46°12’32.22” area near a highway; presence of grasses ap – private area s 23°30’10.97”; w 46°11’55.55” little native vegetation, presence of grasses and exotic species pnmfam parque natural municipal francisco affonso de mello s 23°29’17”; w 46°11’43” it is a conservation unit, consisting of secondary vegetation of atlantic forest at advanced stages of regeneration 706 sociobiolog y vol. 59, no. 3, 2012 using bray-curtis as dissimilarity measure for the analysis of clusters formed by the complete connection method. the sotfware r (oksanen et al. 2009), estimates version 8.2 (colwell 2009), biostat (ayres et al. 2007) and dives (rodrigues 2005) were used for analyses. results and discussion the extensive inventory of epigaeic ant communities in areas surrounding the city of mogi das cruzes showed similar diversity in fauna and composed of 92 species, 36 genera, 19 tribes and seven subfamilies (table 2). myrmicinae was the richest subfamily. the total richness recorded has been possibly influenced by the atlantic forest surrounding the city (oliveira & camposfarinha 2005). the richest genera were pheidole and camponotus with total of 24 and 14 species, respectively. these genera are characterized by high species richness and abundance in different environments, being common in the neotropical region (ward 2000). species such as linepithema neotropicum wild and gnamptogenys striatula mayr, common in the study sites, are found in the litter of the atlantic forest in region (morini et al. 2012). the species accumulation curves indicated a plateau, indicating that the sampling effort was enough to sample the species from localities (figure 1). among all sampling areas, the estimated richness is significantly different (kruskal-wallis = 57.8806, df = 6, p < 0.01), and average richness ranged from 12 to 15 species (table 3). the lowest number of species was recorded in locations near the urban area or areas with high human interference, such as urban parks; the urbanized area i is an exception in this case. the urbanized area around i is composed of atlantic forest vegetation that may be providing increased flow of species among locations. the communities’ analysis confirms this observation once there was the formation of two groups (figures 2 and 3). one composed of areas that are closer to the urban area of the city of mogi das cruzes, being directly susceptible to the effects of urbanization, such as parque leon feffer, sports center joana d’arc, parque nagib najar and urbanized area ii. the other consists of areas that still have atlantic forest vegetation or are close to or within the conservation unit, ie. the urbanized area i, and parque municipal francisco affonso de mello. the areas forming the first group share 21 species while those of the 707 de souza, d.r. — diversity of epigeal ants in alto tietê ta bl e 2. r el at iv e fr eq ue nc y of o cc ur re nc e (f r o ) an d ab un da nc e (f r a ) ac co rd in g to th e ta xa a nd s am pl in g si te s lo ca te d in th e ci ty o f m og i d as c ru ze s ( sp ). su bf am ili es /s pe ci es sa m pl in g si te s pl f c ec pn j a u i a u ii a p pn m fa m fr o fr a fr o fr a fr o fr a fr o fr a fr o fr a fr o fr a fr o fr a d o l ic h o d e r in a e d or ym yr m ex sp .1 1. 32 0. 35 2. 75 1. 93 1. 72 0. 06 0. 36 0. 56 5. 33 3. 26 0. 35 0. 45 0. 38 0. 01 l in ep ith em a ne ot ro pi cu m w ild , 2 00 7 5. 28 4. 13 3. 44 1. 32 6. 01 1. 84 5. 73 9. 61 5. 33 3. 57 6. 71 18 .2 8 5. 32 3. 94 ec it o n in a e l ab id us p ra ed ad or ( sm ith f ., 18 58 ) 0. 33 0. 01 3. 44 25 .4 2 3. 22 1. 84 2. 12 10 .1 5 2. 66 25 .4 6 l ab id us co ec us ( la tr ei lle , 1 80 2) 0. 33 0. 01 0. 38 0. 01 ec t a t o m m in a e e ct at om m a br un ne um ( sm ith f ., 18 58 ) 2. 75 0. 12 2. 51 0. 67 1. 00 0. 31 0. 35 0. 04 e ct at om m a ed en da tu m r og er , 1 86 3 0. 33 0. 01 1. 37 0. 10 0. 43 0. 05 1. 07 0. 24 0. 66 0. 03 g na m pt og en ys st ri at ul a (m ay r, 18 84 ) 5. 61 3. 49 1. 03 0. 03 2. 15 0. 44 6. 09 9. 90 1. 66 0. 45 4. 24 3. 99 7. 60 12 .2 1 g na m pt og en ys sp .4 0. 69 0. 02 0. 33 0. 01 th yp ho lo m yr m ex ro ge nh of er i (m aj er , 1 86 2) 0. 36 0. 02 fo r m ic in a e b ra ch ym yr m ex h ee ri f or el , 1 87 4 0. 66 0. 04 0. 43 0. 01 1. 80 0. 13 0. 33 0. 01 0. 35 0. 01 2. 28 0. 76 b ra ch ym yr m ex p ic tu s m ay r, 18 87 3. 63 0. 82 1. 29 0. 14 0. 72 0. 38 1. 77 0. 16 1. 90 0. 12 b ra ch ym yr m ex in ci su s fo re l, 19 12 6. 60 7. 07 6. 87 15 .1 6 8. 15 8. 76 5. 01 2. 60 4. 66 3. 35 4. 95 1. 98 3. 04 4. 74 c am po no tu s r ufi pe s ( fa br ic iu s, 17 75 ) 6. 60 7. 15 6. 18 1. 69 7. 73 2. 97 6. 10 8. 61 4. 33 1. 04 5. 65 3. 08 4. 56 3. 77 708 sociobiolog y vol. 59, no. 3, 2012 c am po no tu s ( m yr m ap ha en us ) s p. 2 0. 99 0. 03 2. 06 0. 08 1. 29 0. 08 2. 87 0. 40 2. 00 0. 45 1. 06 0. 10 1. 14 0. 37 c am po no tu s s er ice iv en tr is (g ué ri nm én ev ill e, 1 83 8) 2. 06 0. 49 c am po no tu s (t ae na m yr m ex ) 0. 66 0. 02 0. 69 0. 10 2. 51 0. 40 0. 66 0. 03 0. 38 0. 03 c am po no tu s ( m yr m ap ha en us ) n ov og ra na de ns is 1. 32 0. 15 0. 69 0. 02 3. 00 0. 20 0. 36 0. 02 2. 00 0. 12 0. 70 0. 04 0. 76 0. 01 c am po no tu s s p. 7 0. 33 0. 01 0. 34 0. 02 0. 36 0. 04 1. 66 0. 18 c am po no tu s s p. 8 5. 94 1. 53 5. 50 1. 32 3. 43 0. 66 3. 59 0. 69 5. 00 0. 61 2. 83 0. 34 1. 90 0. 31 c am po no tu s s p. 9 0. 33 0. 01 1. 33 0. 15 0. 38 0. 03 c am po no tu s s p. 10 0. 33 0. 01 0. 76 0. 03 c am po no tu s s p. 11 0. 34 0. 02 0. 72 0. 11 2. 66 0. 45 2. 47 0. 21 1. 52 0. 15 c am po no tu s s p. 12 0. 36 0. 02 c am po nt ot us sp .1 3 0. 76 0. 08 c am po nt ot us sp .1 6 0. 86 0. 03 0. 36 0. 02 c am po nt ot us sp .1 8 0. 33 0. 01 m yr m el ac hi sta ca th ar in ae m ay r, 18 87 0. 38 0. 01 m yr m el ac hi sta ru sz ki i f or el , 1 90 3 0. 36 0. 02 1. 06 0. 05 m yr m el ac hi sta sp .4 0. 36 0. 04 n yl an de ri a fu lv a (m ay r, 18 62 ) 4. 95 4. 27 5. 50 4. 52 8. 58 42 .9 3 0. 72 0. 04 4. 66 7. 98 1. 41 0. 14 2. 66 1. 32 pa ra tr ec hi na lo ng ico rn is (l at re ill e, 1 80 2) 1. 33 0. 11 2. 28 0. 88 m y r m ic in a e a cr om yr m ex cr as sis pi nu s ( fo re l, 19 09 ) 5. 28 0. 58 0. 69 0. 02 2. 00 0. 20 0. 71 1. 54 a cr om yr m ex d isc ig er m ay r, 18 87 0. 34 0. 01 0. 33 0. 01 0. 35 0. 01 a pt er os tig m a sp .1 0. 69 0. 02 0. 36 0. 02 0. 33 0. 01 709 de souza, d.r. — diversity of epigeal ants in alto tietê a tta se xd en s (f or el , 1 90 8) 3. 09 0. 72 3. 43 0. 97 5. 38 4. 51 2. 12 0. 44 1. 52 0. 39 c ep ha lo te s p us sil us v. b re vi sp in os us 1. 72 0. 53 0. 72 0. 71 0. 38 0. 01 c re m at og as te r ( o rt ho cr em a) sp .1 0. 36 0. 13 1. 90 0. 39 c re m at og as te r s p. 2 0. 33 0. 05 0. 34 0. 01 0. 86 0. 02 2. 15 1. 22 0. 76 0. 12 c re m at og as te r s p. 3 0. 66 0. 01 0. 34 0. 10 0. 43 0. 03 2. 66 1. 25 c re m at og as te r s p. 7 0. 99 1. 01 0. 34 0. 16 1. 29 0. 49 1. 43 0. 44 0. 35 0. 01 c yp ho m yr m ex s p. 7 0. 33 0. 01 h yl om yr m a re itt er i ( m ay r, 18 87 ) 0. 33 0. 03 0. 38 0. 01 l ep to th or ax sp .1 0. 36 0. 04 m eg al om yr m ex sp .4 1. 06 0. 05 0. 76 0. 07 m yc et ar ot es p ar al le lu s em er y, 19 05 0. 69 0. 03 0. 33 0. 01 m yc et os or iti s s p. 1 2. 64 0. 15 0. 34 0. 01 2. 15 0. 11 0. 36 0. 02 1. 00 0. 08 1. 06 0. 12 o xy po ec us sp .2 0. 66 0. 07 5. 15 7. 92 0. 72 0. 58 4. 59 11 .8 7 3. 42 6. 33 ph ei do le s p. 1 2. 75 1. 35 1. 29 0. 02 0. 66 1. 30 2. 47 0. 33 ph ei do le sp .3 1. 98 0. 13 6. 18 3. 40 0. 43 0. 05 3. 22 0. 93 1. 66 0. 52 2. 83 6. 25 0. 76 0. 26 ph ei do le sp .4 5. 94 23 .1 0 2. 06 1. 97 4. 72 1. 88 2. 87 7. 28 6. 33 29 .1 3 2. 83 3. 08 2. 28 6. 58 ph ei do le sp .6 0. 66 1. 80 1. 29 0. 09 1. 43 0. 24 3. 66 2. 82 2. 28 1. 14 ph ei do le s p. 7 0. 33 0. 07 0. 86 0. 87 ph ei do le s p. 9 0. 43 0. 31 0. 36 0. 04 0. 38 0. 03 ph ei do le sp .1 3 0. 71 0. 09 1. 14 0. 17 ph ei do le sp .1 4 0. 35 0. 08 1. 52 0. 64 ph ei do le sp .1 5 0. 33 0. 05 0. 38 0. 03 710 sociobiolog y vol. 59, no. 3, 2012 ph ei do le s p. 16 3. 43 2. 76 0. 33 0. 01 1. 41 0. 07 0. 38 0. 03 ph ei do le s p. 17 1. 98 1. 49 1. 72 0. 49 3. 00 0. 50 2. 15 6. 19 3. 33 4. 36 0. 35 0. 03 1. 14 0. 65 ph ei do le s p. 18 0. 18 0. 03 ph ei do le s p. 20 0. 66 0. 48 0. 36 0. 04 0. 35 0. 11 0. 38 0. 31 ph ei do le s p. 22 0. 34 0. 08 1. 07 0. 07 0. 22 0. 05 0. 71 0. 27 ph ei do le s p. 23 1. 03 0. 15 0. 35 0. 40 ph ei do le sp .2 4 1. 98 0. 56 1. 03 0. 18 1. 07 0. 33 1. 00 1. 51 1. 41 2. 85 1. 14 0. 11 ph ei do le s p. 26 0. 38 0. 25 ph ei do le sp .2 8 0. 99 0. 46 0. 34 0. 02 2. 47 2. 19 1. 90 4. 11 ph ei do le sp .3 0 1. 32 0. 66 2. 75 2. 06 0. 72 0. 31 3. 89 6. 00 1. 14 4. 30 ph ei do le sp .3 6 3. 96 4. 88 3. 78 8. 47 2. 15 4. 76 0. 72 0. 84 2. 33 2. 16 2. 83 8. 18 ph ei do le sp .3 8 3. 63 8. 37 2. 75 0. 80 2. 15 0. 92 3. 22 3. 37 3. 66 2. 16 0. 71 0. 14 0. 38 0. 14 ph ei do le s p. 39 0. 38 1. 10 ph ei do le s p. 43 0. 66 0. 99 ph ei do le s p. 46 0. 33 0. 04 po go no m yr m ex a bd om in al is sa nt sc hi , 1 92 9 2. 66 0. 69 pr oc ry pt oc er us g r. pr . s ch m al zi 0. 36 0. 02 so le no ps is sa ev iss im a (s m ith , 1 85 5) 5. 61 19 .6 4 5. 15 12 .9 5 8. 58 18 .8 6 7. 17 18 .5 2 4. 33 7. 47 4. 95 6. 76 4. 56 8. 23 so le no ps is sp .2 2. 31 0. 66 2. 75 3. 63 4. 29 1. 43 5. 02 5. 26 3. 00 1. 73 3. 53 3. 42 6. 08 5. 10 so le no ps is sp .3 1. 32 2. 15 5. 58 4. 94 0. 72 10 .3 2 1. 00 0. 77 1. 41 2. 05 3. 04 0. 24 st ru m yg en ys sc hm al zi e m er y, 19 06 1. 32 0. 04 0. 43 0. 01 0. 72 0. 04 0. 66 0. 03 tr ac hy m yr m ex g r. se pt en tr io na le s 0. 35 0. 01 711 de souza, d.r. — diversity of epigeal ants in alto tietê w as m an ni a sp .3 2. 31 0. 13 1. 03 0. 08 4. 31 4. 63 0. 72 0. 20 3. 00 18 .7 2 2. 12 1. 36 1. 52 0. 39 p o n e r in a e a no ch et us a lti sq ua m is (m ay r, 18 87 ) 0. 36 0. 02 0. 71 0. 01 0. 38 0. 01 h yp op on er a sp .1 0. 69 0. 02 0. 72 0. 04 0. 35 0. 01 h yp op on er a sp .8 0. 33 0. 01 0. 36 0. 09 0. 33 0. 06 1. 52 0. 04 h yp op on er a sp .9 0. 38 0. 03 l ep to ge ny s s p. 3 0. 76 0. 03 o do nt om ac hu s a ffi ni s g ué ri nm én ev ill e, 1 84 4 0. 34 0. 01 1. 07 0. 13 1. 06 0. 22 1. 52 0. 15 o do nt om ac hu s m ei ne rt i f or el , 1 90 5 0. 71 0. 04 0. 76 0. 03 o do nt om ac hu s c he lif er (l at re ill e, 1 80 2) 1. 37 0. 04 0. 43 0. 01 1. 07 0. 11 7. 07 1. 87 6. 08 2. 27 pa ch yc on dy la st ri at a sm ith , 1 85 8 6. 60 4. 39 2. 75 0. 15 4. 72 0. 41 6. 09 1. 38 4. 66 1. 11 6. 01 0. 91 6. 84 1. 86 pa ch yc on dy la h ar pa x (f ab ri ci us , 1 80 4) 0. 43 0. 01 ps eu d o m y r m ec in a e ps eu do m yr m ex g ra cil is (f ab ri ci us , 1 80 4) 0. 72 0. 09 1. 33 0. 11 1. 77 0. 10 0. 38 0. 07 ps eu do m yr m ex p al lid us (s m ith , 1 85 5) 0. 36 0. 02 ps eu do m yr m ex p hy llo ph ilu s ( sm ith , 1 85 8) 2. 00 0. 40 pa rt ia l a bu nd an ce 10 .3 05 9. 21 7 8. 36 0 4. 50 4 6. 46 7 7. 25 9 7. 18 8 pa rt ia l r ic hn es s 44 46 36 56 49 49 56 to ta l a bu nd an ce 53 .3 00 to ta l r ic hn es s 92 sh an no n d iv er si ty 3. 31 3. 48 3. 21 3. 54 3. 58 3. 50 3. 62 ev en ne ss 0. 88 0. 91 0. 89 0. 88 0. 92 0. 90 0. 90 712 sociobiolog y vol. 59, no. 3, 2012 second group share 32 species. the high exchange rate of species between areas near the atlantic forest suggests the importance of preserving these sites for conservation of the regional ant fauna in the serra do itapeti and at the same time, they can act as buffer areas for the conservation unit. taxa with specialized morpholog y and biolog y (brandão et al. 2009) were observed in the areas under the influence of the atlantic forest, such table 3. average number of species per sample and richness estimates of epigaeic ants collected around the city of mogi das cruzes (sp). area average number of species (± sd) observed number of species richness estimate plf 15 (±1.84) 44 51.46 cec 15 (±3.78) 46 51.34 pnj 12 (±2.94) 36 42.65 aui 14 (±3.05) 56 67.28 auii 15 (±2.25) 49 59.45 ap 14 (±3.30) 49 55.53 pnmfam 13 (±4.51) 56 70.36 fig. 1. accumulation curves of epigaeic ant species sampled around the city of mogi das cruzes (sp). ( ) plf; ( ) cec; ( ) aui; ( ) pnj; ( ) auii; ( ) ap; ( ) pnmfam. 713 de souza, d.r. — diversity of epigeal ants in alto tietê as the urbanized area i, private area and the parque natural municipal francisco affonso de mello; especially ectatomminae and ponerinae species. myrmelachista, which is exclusively arboreal (longino 2006) since most of its species nests in cavities of tree trunks and branches of living trees (stout 1979; brown 2000; longino 2006; edwards et al. 2009), was found only in sites in contact with forest areas. only one exotic species, (paratrechina longicornis latreille) was recorded, including in the conservation unit. this species and p. megacephala fabricius are present in the central region of mogi das fig. 2. non-metric multidimensional scaling (nmds) comparing epigaeic ant communities sampled around the city of mogi das cruzes (sp). (stress = 27.71) ( ) plf; ( ) cec; ( ) pnj ( ) aui; ( ) auii; ( ) ap; ( ) pnmfam. 714 sociobiolog y vol. 59, no. 3, 2012 cruzes and neighborhoods built in the vicinity of the serra do itapeti (kamura et al. 2007; munhae et al. 2009; souza et al. 2012). however, exhaustive inventories of litter in places sheltered from the serra do itapeti have registered exotic species (morini et al. 2012; suguituru et al. in preparation). the absence of these species is important for maintaining the biodiversity of serra do itapeti since they have potential to reduce native fauna (nafus 1993; harris & barker 2007; vanderwoude et al. 2000; hoffmann 2010). however, it is necessary to monitor the populations of p. longicornis, as the species is already in areas of the parque natural municipal francisco affonso de mello where population’s visit is constant. this conservation area is the largest one of serra do itapeti under legal protection (law no. 6220 of 29.12.2008), being very rich in fauna and flora (morini & miranda, 2012), although located in the metropolitan area of são paulo city. acknowledgements the authors would like to thank the cnpq for the scholarships granted to the first two authors (grants 432.484/2008 and 100479/2009-5) and mscmorini (grant 301151/2009-1) and fapesp for for financial support (grant 06/52409-6). references alonso, l.e. & d. agosti 2000. biodiversity studies, monitoring and ants: a overview. p. 1-8. in: agosti, d., majer, j.d., alonso, l.e. & t.r. shultz (eds.). ants – standard methods for measuring and monitoring biodiversity. washington, dc: smithsonian institution press, washington. arcila, a.m.c. & f.h. lozanozambrano 2003. hormigas como herramienta para la bioindicación y el monitoreo. p. 159166. in: fernández, f. 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(eds.). ants: standard methods for measuring and monitoring biodiversity. smithsonian institution press, washington. yamaguchi, t. 2004. influence of urbanization on ant distribution in parks of tokyo and chiba city, japan, i. analysis of ant species richness. entomol. sci. 19:209-216. yamaguchi, t. 2005. influence of urbanization on ant distribution in parks of tokyo and chiba city, japan, ii. analysis of species. entomol. sci. 8:17-25. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4895sociobiology 67(2): 201-212 (june, 2020) introduction the ‘dehesas’ are agro-silvo-pastoral systems typical of central-western and south-western iberian peninsula. they consist of vast extensions of pastures with scattered adult acorn-producing trees that provide shade, shelter and food to livestock (duque-lazo & navarro-cerrillo, 2017). in general, the ecosystems of ‘dehesas’ are similar to savannas, due to the low level of soil moisture, little or none litter cover under trees and shrubs, which are usually evergreen, and the high temperatures reached (leiva & fernández-alés, 2003). the ‘dehesas’ have an anthropogenic origin, deriving from the pre-existing mediterranean forest through the elimination of the scrub and part of the tree cover, and thus promoting the growth of grass for livestock use (san miguel, 1994). although these landscapes come from earlier times, abstract the ‘dehesas’ are important and vast agro-silvo-pastoral systems typical of the iberian peninsula that are undergoing a crisis due to their low economic profitability and environmental degradation. thus, it is necessary to identify effective tools that provide a reliable idea of the status of these ecosystems as a starting point for future measures of conservation. in this study we analyse the possible role of ants as surrogates for epigeic arthropods, a common biodiversity indicator group. a total of 15 farms were sampled throughout sierra morena (andalusia, spain) with pitfall traps, both for the ‘dehesa’ habitats themselves and for different microhabitats within the study sites. first, we achieve a complete list of the species of ants of the area. the results indicate that the ‘dehesa’ habitats were very homogenous for all farms, while microhabitats showed differences in species richness and ant communities’ composition compared to the ‘dehesas’. to evaluate the role of ants as surrogates, the number of traps occupied by each order of arthropod and by each ant species was compared. we found a high correlation between them what confirm the surrogate character of ants for the rest of arthropods in these ecosystems. sociobiology an international journal on social insects f jiménez-carmona, s carpintero, jl reyes-lópez article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 28 november 2019 initial acceptance 12 january 2020 final acceptance 18 february 2020 publication date 30 june 2020 keywords bioindicators; formicidae; pastureland; holm oak; pitfall traps. corresponding author francisco jiménez-carmona university of córdoba, department of botany, ecology and plant physiology, edificio celestino mutis, 1ª planta campus de rabanales, 14071 córdoba, spain. e-mail: francisco.jimenez@uco.es there is evidence of the use of the word ‘dehesa’ from the early middle ages (álvarez-guzmán, 2016). thenceforth, traditionally they have been dedicated to different activities, mainly extensive livestock rearing (cattle, sheep, goats and/ or pigs), but also pasture and grain production, usually for livestock, or hunting use (klein, 1920; san miguel, 1994; martin, 1996). in spain the ‘dehesas’ comprise a total of 2,360,700 has (martin, 1996), principally in the communities of, extremadura, castilla la mancha, castilla y león and the largest extension in andalusia with 1,263,143 ha (costa pérez et al., 2006). not only do the ‘dehesas’ fulfill important roles in the spanish agriculture but also in the environmental protection. in this respect, they are included in the natura 2000 network as ecosystems of community interest (díaz esteban & pulido díaz, 2009; european directive 92/43/ecc; marañón et al., 2012), because they are emblematic examples university of córdoba, department of botany, ecology and plant physiology. campus de rabanales, córdoba, spain research article ants ants (hymenoptera: formicidae) as surrogates for epigeic arthropods in northern andalusian ‘dehesas’ (spain) f jiménez-carmona, s carpintero, jl reyes-lópez – ants as surrogates for arthropods in ‘dehesas’202 of mediterranean managed landscapes with high biodiversity, including threatened species such as the spanish lynx (lynx pardinus (temminck, 1827)) (díaz esteban & pulido díaz, 2009; álvarez-guzmán, 2016). they are also considered key cultural landscapes for their aesthetic, historic and ecotourism roles (marañón et al., 2012; maldonado et al., 2019). despite all their values, nowadays the ‘dehesas’ are undergoing a crisis as result of their low economic profitability and of the environmental degradation derived from the loss of traditional management and from other multiple factors, such as the decay of tree canopy or the fragmentation of habitat (díaz et al., 1997; plieninger et al., 2004; escribano et al., 2018). this forces the competent institutions to adopt the necessary measures to keep or restore their status of conservation (pulido & picardo, 2010). as a starting point to set future plans of protection, it is very important to consider what are the best measures to evaluate the current status of the ecosystems. one of the first tasks required when designing conservation strategies is to estimate the biodiversity (caro, 2010; laurila-pant et al., 2015). this knowledge is essential for a proper environmental management. when facing a biodiversity inventory, one of the most outstanding group are the arthropods, because they represent a high percentage in terms of biomass (bar-on et al., 2018) and they participate in essential functions of the ecosystems (yang & gratton, 2014; schowalter, 2017). but the main problem for the knowledge of this phylum is their megadiversity (smith et al., 2005). the experts are usually specialized in specific groups, such as orders, and even families or genders; this implies that to study all the arthropod taxa would be an extensive work and the need to involve numerous researchers. the estimation of species richness is one of the most common ways to measure the biodiversity of an ecosystem (noss, 1990), but many times this is a complex duty. in recent times, efforts have been made to simplify this task by searching for shortcuts (moreno et al., 2007). one of the possible methods is the search for groups whose diversity represents other taxa of the community whose study is more complex for any reason, these are the surrogate groups (moreno et al., 2007; lewandowski et al., 2010; lindenmayer & likens, 2011). the use of surrogates is an important option to mitigate the shortage of biodiversity data and by sampling only one group, instead of the entire community, time and money are saved (heino et al., 2005). there is no standard agreement to estimate the suitability of a taxon as a surrogate indicator, but efforts have been made for searching certain taxa as surrogates according to their representativeness of the diversity of other groups (see for example leal et al. (2010)). ants (hymenoptera: formicidae) display a series of characteristics that make them suitable as a good group of bioindicators (andersen, 1997; crist, 2009; nakamura et al., 2007; ribas et al., 2012; underwood & fisher, 2006; verdinelli et al., 2017). they present high ecological fidelity and are functionally important in the ecosystems, participating in multiple relevant functions, such as: the decomposition of organic matter, soil turning, pollination, zoochory, predation of other arthropods or being prey for many other groups of animals (folgarait, 1998; philpott & armbrecht, 2006; crist, 2009; diamé et al., 2017). moreover, they respond to disturbances in their habitats, regardless of their origin, in a predictable, quick and generally in a linear way (philpott et al., 2009). they are abundant and well distributed throughout the planet, being in all continents and ecosystems except in antarctica (hölldobler & wilson, 1990). finally, there is a good knowledge of their taxonomy and they are easily found in the field and sampled (agosti et al., 2000). all these factors may lead to conclude that ants are a group susceptible of being surrogate for other taxa. at this respect, some studies have shown that ants, either by their own or together with other groups of fauna or flora, can be considered as surrogates for plants (gadagkar et al., 1993; pfeiffer et al., 2003) or for a set of taxa, either just of invertebrates (sauberer et al., 2004; nakamura et al., 2007), or also including vertebrates and/or plants (sauberer et al., 2004; majer et al., 2007; leal et al., 2010). however, other studies reported negative results (osborn et al., 1999; allen et al., 2001; dauber et al., 2003; sackmann et al., 2006; bennett et al., 2009; uys et al., 2010; landeiro et al., 2012; pérez-fuertes et al., 2016; gibb et al., 2017; hanford et al., 2017; barton et al., 2019). these contradictory results are not rare. the accuracy of these shortcuts depend on different factors, which are: the studied the studied taxa, the scale of the study and, being the case, the environmental information used (moreno et al., 2007). indeed, everything indicates that ants may act as surrogates or not, depending on the group with which they are being related, as well as on the type of habitat. for example, tropical zones are megadiverse, which probably make difficult the task of describing completely their communities. therefore, individualized studies for every zone and taxon are required if we propose the search of a shortcut for assessing species diversity (moreno et al., 2007). within this context, given the importance of the ‘dehesas’ in the iberian peninsula and the scarcity of information on their conservation status, we wished to test the hypothesis of whether ants could be a good subrogated group for arthropods in these ecosystems. for this purpose, we developed this work with the following specific objectives: first, to carry out an exhaustive work for the knowledge of the ‘dehesa’ ants communities, then to evaluate the role of ants as a subrogated group for the rest of arthropods. material and methods study site this study was carried out in 15 ‘dehesas’ along andalusia (southern spain), in the years 2016 and 2017 (table 1). the distances between more remote farms is 300 sociobiology 67(2): 201-212 (june, 2020) 203 km (figure 1a and table 1). the study ‘dehesa’ ecosystems are dedicated to livestock, hunting and agriculture. the predominant arboreal species is quercus ilex l. subsp. ballota (desf.) samp. in some ‘dehesas’ olea europaea var. sylvestris brot. and quercus suber l. may appear occasionally, and with less frequency and scattered distribution: ceratonia siliqua l., pyrus bourgaeana decne., pinus pinea l., prunus dulcis (mill.) d.a. webb, quercus faginea lam. and quercus pyrenaica willd. depending on the ‘dehesa’, the herbaceous layer may include either natural or improved pastures, or monospecific crops (such as wheat, oats, barley, vetch or pea). regarding the livestock species, swines and bovines predominate. ovines, caprines, equines and even beehives are also present. the shrub layer, when present, forms patches in steep zones and low exploitation value areas, either for livestock or agriculture. the main species are cistus sp., quercus coccifera l., thymus spp. nerium oleander l., pistacia lentiscus l., retama sphaerocarpa (l.) boiss. fig 1. a) location map of the study ‘dehesas’ (layer of distribution of ‘dehesas’ from rediam: andalusian environmental information network). b) pitfall traps position according to both sampling types (‘dehesas’ and microhabitats) for the farm as05, as an example. most of the surface of the study farms shows the typical ‘dehesa’ landscape, but there are also small unmanaged areas (microhabitats) with different characteristics. they include: the dense scrub patches describe above; ponds; streams’ riparian forests; and vegetation zones adjacent to traditional stone walls (table 1). moreno et al. (2007) found that despite occupying a low proportion of the farm area, these microhabitats (called by them marginal habitats) contribute largely to the biodiversity of these ecosystems. therefore, for the study we differentiated two types of sampling areas: the landscape of the ‘dehesa’ itself (de) and the microhabitats (mh). climatology the predominant climate is the mediterranean, specifically mediterranean mountain climate (junta de andalucía, 2019), with hot summers and cold winters (annual average range between 11 and 18 ºc). the accumulated rains range between 600 and 1,200 mm, with maximums in autumn and winter and minimums in summer (gómez-zotano et al., 2015). experimental design sampling was performed with pitfall traps. these constitute an easy and effective sampling system for communities of epigeic arthropods and they are recommended as part of a standard protocol for measuring biodiversity (agosti et al., 2000; gotelli & colwell, 2001; prasifka et al., 2007; sheikh et al., 2018).traps consisted of 150-ml translucent plastic cups (upper ø 5.7 cm, base ø 5 cm, depth 7.3 cm. ref 409702, deltalab sl). they were set at ground level, flushed with the soil surface, and placed in the field for 48 h. traps were filled with a killing agent consisting of 30-35 ml of water with 1% of detergent, to break the surface tension of water and prevent the escape of little individuals. we did not employ any bait so the traps were suitable to calculate both presence of species and their relative abundance (wang et al., 2001). collected specimens were separated into two groups: formicidae and other arthropods. ants were identified to species level. their abundance was quantified by counting the number of workers per each trap, as well as by the number of traps occupied by each species (gotelli et al., 2011). in the case of the rest of the arthropods, they were identified to order level (except for subclass acari) and the number of traps occupied by each one was recorded. numerous studies have shown that the estimation of the diversity of arthropods at the taxonomic level of order can be a very useful tool for the f jiménez-carmona, s carpintero, jl reyes-lópez – ants as surrogates for arthropods in ‘dehesas’204 evaluation of the conservation status of different ecosystems (wettstein & schmid, 1999; cecil et al., 2019; holmquist & schmidt-gengenbach, 2019). according to crist and wiens (1996), ants may display different distribution patterns even at small scales. to avoid this possible effect and to cover the largest possible area of the study sites, we placed a single linear transect of 1600 m that crossed the larger axis of every farm (figure 1b). these transects consisted of 40 pitfall traps, separated one trap from the next by 40 m. if there were fences with pigs (sus scrofa domestica l.) they were avoided, since they often unearth and destroy the traps. as regards microhabitats (mh), we sampled two of them per farm. depending on the study site the mh type could vary (see in table 1). as the area of the mh is much smaller than the one of the de, the method to set the pitfall traps necessarily had to be different. accordingly, we set one transect per mh, with 10 pitfall traps separated each trap from the next 2 m. this methodology has been used by our research group in numerous occasions with proven effectiveness. therefore, in total per farm and for both sampling modalities, 60 pitfall traps were placed (40 in de and 10+10 in mh). the sampling was conducted during the springs and summers (may-june) of 2016 and 2017, being this the peak period of activity for most ant species of these latitudes (cros et al., 1997; carpintero et al., 2007). in table 1 it is specified the timing of sampling for every farm. statistical analysis adequation of the sampling systems ants the adequation of sampling effort and methodology was tested with ants’ rarefaction curves (mao’s tau) based on abundance of workers per species and trap. the sample coverage was calculated for each sampling area (chao & chiu, 2016; function “diversity”; package “spader”), this index measures “[…] the proportion of the total individuals in a community that belong to the species represented in the sample”, this is a measure of sample completeness (chao & jost, 2012). comparison of the fauna of de and mh and evaluation of farms homogeneity ants in order to compare the richness and composition of species (relative abundance of each species) of ants of the ‘dehesas’ versus microhabitats, the traps were divided into three categories: first comprised the twenty traps of the code farm location province coordinates surface (ha) years mean t. (°c) mean acu. rain. (mm) microhábitats ap05 la juanita alosno huelva 37.555913°, -7.082250° 191.14 17 17.81 639.01 sp, rf ap06 paymoguillo paymogo huelva 37.751957°, -7.330534° 109.4 16-17 17.00 671.00 vw, sp as02 el palomar de la morra pozoblanco córdoba 38.348277°, -4.819241° 96.69 17 15.70 544.00 sw, vw as05 lote de los pérez cazalla de la sierra sevilla 37.896797°, -5.875586° 107.7 16-17 16.27 661.59 sw, sp as06 las morrillas pozoblanco córdoba 38.360550°, -4.771079° 157.42 17 15.62 538.96 sw, p co01 las ánimas aroche huelva 37.962325°, -7.012390° 77.62 16-17 15.96 779.81 sw, vw co05 monterrey y carretero aroche huelva 37.905465°, -7.049219° 119.59 17 16.34 773.34 sw, vw co08 quebradahonda castillo de las guardas sevilla 37.653961°, -6.421098° 114.14 16-17 17.08 782.91 sw, vw co12 majada del indio el viso córdoba 38.545339°, -4.986985° 123.94 16-17 16.05 527.79 vw, sp en04 encinarejo alosno huelva 37.551441°, -7.148217° 281.33 16-17 17.65 617.79 sw, vw fa01 las hazas villanueva de córdoba córdoba 38.403720°, -4.603515° 459.09 16-17 15.35 594.48 sw, vw fa05 la panadera pozoblanco córdoba 38.381964°, -4.758971° 83.6 17 15.70 553.00 sw, sw fa11 santa clotilde cardeña córdoba 38.202356°, -4.286978° 292 16-17 15.48 887.26 vw, sp up23 oropesa fuente ovejuna córdoba 38.299421°, -5.463488° 110.69 16-17 15.73 564.71 sw, vw up24 las caras vilches jaén 38.229856°, -3.544986° 592.61 16-17 16.76 574.51 sw, sp table 1. codes for the study farms, their location, surface and years of sampling. five farms were full sampled in 2017 (‘dehesas’ and microhabitats). in the rest of the farms the ‘dehesas’ were sampled in 2016 and the microhabitats in 2017. means of annual temperatures (mean t.) and means of accumulated rainfall (mean acu. rain.) are included. microhabitats column shows the different types of microhabitats sampled per farm: zones adjacent to traditional stone walls (sw); scrub patches (sp); vegetation of temporary water courses (vw); ponds (p); riparian forest (rf). sociobiology 67(2): 201-212 (june, 2020) 205 microhabitats (mh); for the second and third categories we selected the first and second twenty traps of the ‘dehesas’ (d1 and d2 respectively), this is to have a balance design (20 traps per category). besides, comparing the first and second twenty traps of de it was also possible to test the homogeneity of these ecosystems. with these groups of traps (d1, d2 and mh) we performed two statistical analyzes: a factorial oneway anova analysis was performed with the number of ant species per trap to compare the richness between sampling categories (d1, d2 and mh); to compare the structure of ant assemblages (relative abundance of the species) per sampling category (d1, d2 and mh), a one-way permanova analysis was performed with the matrix of the number of traps occupied by each species. bray-curtis distances were applied, with 9999 permutations for the calculation of the similarity matrix. surrogacy study the role of ants as surrogates for arthropods was analysed by means of a plsr (partial least squares regression) analysis. this allows to establish a linear regression between two matrices of variables (predicted vs. observed variables) of unequal size (ants vs. arthropods), which are projected to a new space (abdi, 2010). the analysis was performed with the number of traps occupied by each species of ants versus number of traps occupied by the different groups of arthropods. the use of the covariance matrix and of the correlation matrix were evaluated to perform this analysis, using the system that explained more variance. spatial autocorrelation in order to account for spatial autocorrelation we carried out a mantel test (mantel, 1967), which calculated the conditional correlation of two matrices of the same rank (diversity of ants and of arthropods) eliminating the effect of a third one (geographical location of the study sites) (smouse et al., 1986). these analyses were performed with the statistical packages past 3.20 (hammer et al., 2001), r v 3.6.2 (r core team, 2019) and statistica v 8.0 (statsoft inc., 2001). results formicidae study ants species the species rarefaction curves (figure 2) for each site reached or approached their asymptote. these results are reinforced by the sample coverage analysis (table 3), that shows values close to 1, what confirms that all or most of the species of the sampling areas have been recorded. fig 2. rarefaction curves of ants for each farm, with the abundance of workers per species and trap. the curves are alternatively drawn in solid and dotted lines for a better differentiation. f jiménez-carmona, s carpintero, jl reyes-lópez – ants as surrogates for arthropods in ‘dehesas’206 species author, year abbrev nf nt nw aphaenogaster dulciniae emery, 1924 aphdul 11 45 79 aphaenogaster gibbosa (latreille, 1798) aphgib 15 139 322 aphaenogaster iberica emery, 1908 aphibe 14 288 1246 aphaenogaster senilis mayr, 1853 aphsen 9 170 865 camponotus cruentatus (latreille, 1802) camcru 11 186 1119 camponotus fallax (nylander, 1856) camfal 4 7 7 camponotus figaro collingwood & yarrow, 1969 camfig 1 2 2 camponotus foreli emery, 1881 camfor 8 16 76 camponotus lateralis (olivier, 1792) camlat 5 10 37 camponotus micans (nylander, 1856) cammic 4 8 9 camponotus pilicornis (roger, 1859) campil 14 50 94 camponotus sylvaticus (olivier, 1792) camsyl 7 12 22 cataglyphis hispanica (emery, 1906) cathis 15 612 2704 cataglyphis iberica (emery, 1906) catibe 9 60 234 cataglyphis rosenhaueri santschi, 1925 catros 7 34 107 colobopsis truncata (spinola, 1808) coltru 2 4 4 crematogaster auberti emery, 1869 creaub 11 49 191 crematogaster scutellaris (olivier, 1792) crescu 14 77 511 crematogaster sordidula (nylander, 1849) cresor 3 4 44 formica cunicularia latreille, 1798 forcun 3 5 15 formica gerardi (bondroit, 1917) forger 2 2 5 goniomma baeticum reyes & rodriguez, 1987 gonbae 8 18 34 goniomma hispanicum (andré, 1883) gonhis 13 46 139 goniomma kugleri espadaler, 1986 gonkug 3 5 5 iberoformica subrufa (roger, 1859) ibesub 15 448 6858 lasius grandis forel, 1909 lasgra 4 20 478 lasius lasioides (emery, 1869) laslas 13 45 97 messor barbarus (linnaeus, 1767) mesbar 15 484 6010 messor bouvieri bondroit, 1918 mesbou 5 20 120 messor celiae reyes, 1985 mescel 3 7 19 messor hispanicus santschi, 1919 meshis 8 23 65 messor lusitanicus tinaut, 1985 meslus 1 2 2 myrmica aloba forel, 1909 myralo 1 2 8 oxyopomyrmex saulcyi emery, 1889 oxysau 13 36 181 pheidole pallidula (nylander, 1849) phepal 9 38 627 plagiolepis pygmaea (latreille, 1798) plapyg 15 192 667 plagiolepis schimitzii (latreille, 1798) plasch 11 56 135 proformica ferreri bondroit, 1918 profer 1 5 25 solenopsis spp. solspp 14 56 105 tapinoma madeirense forel, 1895 tapmad 1 1 2 tapinoma nigerrimun cf. (nylander, 1856) tapnig 15 340 5227 temnothorax alfacarensis tinaut, in littere. temalf 1 2 4 temnothorax angustulus (nylander, 1856) temang 2 3 3 temnothorax racovitzai (bondroit, 1918) temrac 11 39 141 temnothorax recedens (nylander, 1856) temrec 3 8 12 temnothorax tyndalei (nylander, 1856) temtyn 2 5 8 tetramorium caespitum cf. (linnaeus, 1758) tetcae 5 11 34 tetramorium forte forel, 1904 tetfor 15 262 2364 tetramorium semilaeve andré, 1883 tetsem 15 310 1904 table 2. list of the species of ants and their abbreviations. number of farms where each species was located (nf); total number of traps occupied by each species (nt); total abundance of workers-individuals (nw). sociobiology 67(2): 201-212 (june, 2020) 207 a total of 32,820 workers from 49 different species, belonging to 19 genera, were captured. there was an average of 26 species per farm (21-33 species) (sm1). the following species were in every farm and with high abundance: aphaenogaster gibbosa (latreille, 1798), cataglyphis hispanica (emery, 1906), iberoformica subrufa (roger, 1859), messor barbarus (linnaeus, 1767), plagiolepis pygmaea (latreille, 1798), tapinoma nigerrimum cf. (nylander, 1856), tetramorium forte forel, 1904 and tetramorium semilaeve andré, 1883 (table 2 and 3, and sm1). farm spp. to sc spp. de spp. mh spp. co spp. ex. mh % spp. co % spp. ex. mh ap05 27 0.996 26 18 17 1 62.96 3.70 ap06 23 0.998 20 18 15 3 65.22 13.04 as02 27 1 23 20 16 4 59.26 14.81 as05 26 0.999 22 16 12 4 46.15 15.38 as06 24 0.999 23 12 11 1 45.83 4.17 co01 27 0.999 26 20 19 1 70.37 3.70 co05 22 0.999 18 16 12 4 54.55 18.18 co08 26 0.999 24 16 14 2 53.85 7.69 co12 25 0.998 22 21 18 3 72.00 12.00 en04 25 0.997 25 14 14 0 53.85 0.00 fa01 24 0.998 22 14 12 2 50.00 8.33 fa05 31 0.999 28 18 15 3 48.39 9.68 fa11 33 0.997 26 24 17 7 51.52 21.21 up23 30 0.999 27 22 19 3 63.33 10.00 up24 21 0.998 21 11 11 0 52.38 0.00 minimum 21 0.996 18 11 11 0 45.83 0.00 averrage 26.1 100 23.5 17.3 14.8 2.5 56.79 9.46 maximum 33 1.000 28 24 19 7 72.00 21.21 table 3. species of ants per farm: total number of species (spp.to); sample coverage (sc); species in ‘dehesas’ (spp.de); species in microhabitats (spp.mh); common species in both types of habitats (spp.co); species exclusive to microhabitats (spp.ex.mh). comparison of the fauna of de and mh and evaluation of farms homogeneity ants the results of the anova showed that there were significant differences in the number of ant species per trap according to the different category of traps: first or second twenty traps of the ‘dehesas’ transects (d1 and d2), and twenty traps of microhabitats transects (mh) (f=8.990, p<0.0001). the post-hoc tukey test hsd group to group delved into these results and revealed that d1 and d2 did not show significant differences (p=0.2978), while mh registered a significant higher capture rate than the other groups (d1 vs. mh p=0.0188 y d2 vs. mh p<0.0001). the sampling in microhabitats added 0-7 more species of ants to the list per farm, which accounted for 9.46 % of the species (table 3). moreover, there are three species that were only found in microhabitats: messor lusitanicus tinaut, 1985, myrmica aloba forel, 1909 and tapinoma madeirense forel, 1895. a one-way permanova showed that there were significant differences between d1, d2 and mh ant assemblages’ structure, according to the matrices of the number of traps occupied by each species (permanova one-way f = 5.1230, p = 0.0001). again, a comparison by pairs revealed that the differences were due to mh (d1 vs. d2 p = 0.5803, d1 vs. mh p = 0.0001, d2 vs. mh p = 0.0001) other arthropods non-formicid arthropods comprised 34 groups, with an average of 21 per farm (17-25 groups). all the groups and the abbreviations used in the figures are shown in sm2. the most abundant groups, and found in every farm, were: acari, araneae, coleoptera, diptera, entomobriomorpha, hemiptera, hymenoptera, orthoptera and symphypleona. comparison ants vs. other arthropods surrogacy study a plsr analysis performed with the matrix of covariance (first axis explained 66.38% of the variance) showed that the diversity of ants vs. other arthropods was highly and significantly correlated (r2=0.8134; p < 0.0001) (figure 3). study of the spatial autocorrelation: mantel test. the results of the test with the matrices of the number of traps occupied by ant species and by the arthropod groups was of r= 0.45 (p=0.0012; for 9999 permutations). then, the test analysed their correlation eliminating the possible effect of farms distance, with similar results (r = 0.44 p=0.0011, for 9999 permutations). therefore, the location of farms did not have any effect on ants and arthropods correlation. f jiménez-carmona, s carpintero, jl reyes-lópez – ants as surrogates for arthropods in ‘dehesas’208 discussion the present study supports the hypothesis that ants can act as a surrogate group for general epigeic arthropods diversity in ‘dehesa’ ecosystems. according to the ant fauna, the studied ‘dehesas’ constitutes a homogeneous habitat, with a group of common and very abundant species in all the sites sampled throughout sierra morena. these are mainly species adapted to open and warm ecosystems, such as m. barbarus and c. hispanica, or to open areas with the presence of a dispersed tree stratum (i. subrufa). some species are adapted to live in the litter layer, for example p. pygmaea. finally, there are a large group of generalist species, such as a. gibbosa, t. nigerrimun cf., t. forte and t. semilaeve (roig & espadaler, 2010). the fact that not only did we study the typical ‘dehesa’ habitat, but also the different microhabitats of the farms completed the information about the sites. these samplings increased the number of species, even with the appearance of species exclusively found in microhabitats. actually, the composition of ant assemblages in microhabitats showed significant differences with those of the typical habitat of ‘dehesas’. these results confirm what has already been said by numerous authors that small variations in the structure of habitats will bring modifications in the composition of ant species (menke & vachter, 2014; vasconcelos et al., 2014). thus, in the microhabitats there were found some species typical of shaded and/or humid areas, such as m. aloba or m. lusitanicus. regarding the use of ants as a surrogate group for epigeic arthropods in ‘dehesas’ of northern andalusia, in our work we have verified how both groups have a high correlation. at this respect, leal et al. (2010) proposed a benchmark for assessing if a surrogate group provides a reliable prediction of other groups. they consider a surrogate “reasonable” if it explains > 60% of total species richness, “good” if it explains > 70% and “excellent” if it explains > 80%. in our case, the value of correlation of ants’ diversity and arthropods is of r2 = 0.8134, therefore we may consider that the ants of the ‘dehesas’ of andalusia reflects to a large extent the community of epigeic arthropods. we found similar results in nakamura et al. (2007), where a strong relationship between ant species and orders of insects from forests and subtropical grasslands of eastern australia is found. other authors also obtain positive values of surrogacy using different taxa. for example, biaggini et al. (2007) studied in an area of similar climate to ours the possible role of the diversity of species of the family carabidae (coleoptera) as surrogate for other insect orders obtaining a significant correlation of more than 90% (p << 0.05). guan et al. (2018), analysed the status of the species of gastropods as surrogates for the invertebrate orders of the lakes of china and obtained also a high correspondence (r = 0.66, p << 0.05). these studies contribute to highlight the use of surrogate groups, at least in particular circumstances; we already established in the introduction the need to be wary and consider that there are multiple factors that may influence the validity of the surrogate groups (moreno et al., 2007). and fig 3. correlation pls of scores for axis 1 for the block of arthropod groups versus ants. sociobiology 67(2): 201-212 (june, 2020) 209 also, it would depend on one’s objectives, considering always the balance between the level of accuracy desired and the need to reduce the burden of addressing the study. in other words, as wiens et al. (2008) suggest, we need to take into account how good is good enough. in the case of studies related to conservation and management of ecosystems or species, many times we need to achieve results in an effective, quick way. the use of surrogate groups may be especially helpful in this context. with our study we conclude that just with the study of ants, a single group with a good taxonomic resolution in the iberian peninsula, the situation of the epigeic arthropod community can be extrapolated, and therefore they could be used as a tool that help to evaluate the state of conservation of the ‘dehesa’ ecosystem. acknowledgements this work has been financed by life biodehesa project (life11 / bio / es / 000726) ‘dehesa ecosystems: development of policies and tools for the management and conservation of biodiversity’, european union. we are very grateful to jose emilio guerrero-ginel (university of cordoba) for the coordination of this project. thank you to sergio andicoberry de los reyes (university of cordoba) and alma mª garcía moreno, belen caño vergara, pedro j. gómez giráldez (ifapa córdoba, junta de andalucía) for their participation in the field work. the revision of the manuscript by alba jiménez-guirval is gratefully acknowledged. references abdi, h. 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(2014). insects as drivers of ecosystem processes. current opinion in insect science 2: 26–32. doi: 10.1016/j.cois.2014.06.004 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.7202sociobiology 69(1): e7202 (march, 2022) introduction most solitary bees named megachilidae construct their nests in pre-existing natural cavities like hollow stems, dead woods, and manmade holes using materials like leaf, mud, resin, and chewed plant tissues (o’neill, 2001; cane et al., 2007). leafcutter bees use leaf bits or plant resin to line their brood cells (litman et al., 2011; maclvor, 2016). these bees possess an unusual nesting habitat preference, such as nesting in the fallen flowers of markhamia lutea (amala et al., 2017) and petioles of papaya (amala et al., 2019). usually, they cut the leaves in different shapes to line the brood cells and abstract solitary bees of the family megachilidae are the key pollinators of pigeon pea. artificial trap nests were used to study nesting parameters, such as occupancy rate, nest establishment time, and building pattern during the vegetative and flowering stages of the cajanus cajan (fabaceae). the installed traps were occupied by three different leafcutter bees (megachile lanata, m. laticeps, m. disjuncta) and one parasitic bee coelioxys sp. bees occupied the nest tubes for 16.01 ± 2.82 and 10.23 ± 2.30 days in the vegetative and flowering stages. we recorded 38.33 and 72.50% trap occupancy rates during crop vegetative and flowering stages, respectively. the percent parasitization by melittobia sp was significantly higher during the vegetative stage (53.67%). brood cells were parasitized by coelioxys sp (6.67%) during the flowering stage. many female bees tend to occupy trap nests with active nest construction during the flowering stage (7.21 ± 2.28 bees). in contrast, male bees merely took shelter inside the traps during the vegetative stage. thus, it is concluded that female bees possess more wing wear during the vegetative stage of the crop when compared to the flowering stage. we also observed a significant increase in the percent pod set, the number of seeds per pod, and 100 seed weight in the experimental plot compared to the control plot, proving the importance of leafcutter bees for the pollination of cajanus cajan crop. the present study results confirmed the role of artificial trap nests in providing habitats for the solitary leafcutter bees, thereby increasing their activity and nest abundance, which favors the pollination and better yield in pigeon pea. sociobiology an international journal on social insects amala udayakumar, timalapur m shivalingaswamy article history edited by evandro n. silva, uefs, brazil received 30 april 2021 initial acceptance 15 november 2021 final acceptance 24 january 2022 publication date 22 april 2022 keywords conservation, megachile, pod set, trap nests, pollination. corresponding author amala udayakumar icar – national bureau of agricultural insect resources h a farm post, post box nº 2491, hebbal bengaluru 560024. e-mail: amala.uday@gmail.com lay their eggs inside the leaf cell pre-provisioned with pollen (raw, 2004; buschini, 2006; michener, 2007). trap nests were widely used to manage other megachilidae wild bees like osmia cornifrons (maeta & kitamura, 1974), o. bicornis (gruber et al., 2011), o. lignaria (bohart, 1972; philips & klostermeyer, 1978), o. cornuta (bosch & kemp, 2002) and m. rotundata (bohart, 1972; fairey et al., 1989; pitts-singer & cane, 2011). trap nests were also utilized to monitor the nesting behavior of bees, their pollen/prey preference, their natural enemies in agro-ecosystem (roubik & villanuevagutierrez, 2009; ercit, 2014). an additional study conducted by junqueira et al. (2012) reported that erecting bee shelters indian council of agricultural research – national bureau of agricultural insect resources, bengaluru, india research article bees leafcutter bees (hymenoptera: megachilidae) as pollinators of pigeon pea (cajanus cajan (l.) millsp., fabaceae): artificial trap nests as a strategy for their conservation mailto:amala.uday@gmail.com amala udayakumar, timalapur m shivalingaswamy – farm conservation of solitary bees using trap nests in pigeon pea2 with suitably sized bamboo stalks can enhance the population of nesting carpenter bees by >200% over 23 months with the active emergence of new broods. pigeon pea (cajanus cajan (l.) millsp., fabaceae) is an often-cross pollinated crop. it attracts several bee pollinators, leading to potential yield enhancement (abrol & shankar, 2015). interestingly, leafcutter bees of the genus megachile were reported to be the key pollinators of pigeon pea as they are capable of ‘flower tripping’ behavior to access pollen of leguminous crops. hence, providing nesting structures amidst the cropped area would lead to suitable foraging and shelter, with the consequent conservation of solitary bees populations (gathmann et al., 1994; peterson & roitberg, 2006; joshi et al., 2020). due to the increasing loss of biodiversity of pollinators, there is a growing need to modify agricultural landscapes to restore the biodiversity of bee pollinators (villemey et al., 2018). the present study was conducted to test the hypotheses that nesting activity of leafcutter bees, pollination, and yield of pigeon pea during the vegetative and flowering stages can be influenced by providing artificial nesting structures. materials and methods study site the present study was carried out in the experimental farm of icar-national bureau of agricultural insect resources (nbair) yelahanka campus, bengaluru (13.096792n, 77.565976e), karnataka, india, during the year 2019 (july to december). the study area of 22 acres comprising of cultivated croplands with various annual crops like cereals and pulses, orchard blocks of mango (mangifera indica), sapota (manilkara zapota), and cherimoya (annona cherimola). also, two patches of pollinator gardens of about 1.5 acres with over 100 plant species of diverse plant families were part of this study location, which is right in the heart of a rapidly growing high-tech-city and capital of the southern indian state of karnataka. pigeon pea, cajanus cajan crop (var. brg-1) was cultivated on 0.5 acres. we adopted a standard package of practices recommended for pigeon pea cultivation in the region by the state agricultural university. an inter-row spacing of 60cm and plant to plant spacing of 15cm was adopted during seeding. care was taken not to apply pesticides to manage any insect pests as it may affect the activity of the bees in this experimental plot. the observations were recorded from july to september (vegetative stage), mid-october to november (flowering stage), coinciding with the peak flowering of the crop. trap nests two plots of pigeon pea were maintained, one installed with trap nests and another without trap nests. bamboo trap nests were installed during the vegetative and flowering stages of the crop. the artificial trap nests consist of bamboo culms of 15 mm in diameter, measuring 200 mm in length. a total of 120 trap nests made of bamboo were organized into ten different nest bundles. each bundle of a nest with 12 bamboo culms was installed using a wooden pole in the study site during the pigeon pea’s active vegetative and flowering stage. a total of 10 nest bundles were installed in the study area (fig 1). before installing the trap nests, the culms were split longitudinally into two halves to facilitate the easier examination of nests once they get occupied by the insects. the split halves were joined firmly using sticky tape. the trap nests were closed at one end with cotton wool and installed with the open end projecting out. fig 1. a. trap nest installation in a cajanus cajan (l.) millsp. (fabaceae) plantation of the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. b. leaf cutter bee, megachile latreille, 1982 entering trap nest with a leaf bit. sociobiology 69(1): e7202 (march, 2022) 3 nesting parameters of solitary bees the trap nests were monitored daily for occupancy by the insects for 30 days. the inhabited nest tubes were recorded and removed to be studied, and new ones replaced the removed nest tubes after being occupied by the insects in a bundle. the plug material used by the insect to cap its nest and plug appearance was also recorded. the occupied nests were checked for the presence of bees sheltering inside and the presence of active nests. the occupied nests were gently opened by cutting the sticky tape to examine the inner contents. the percent occupancy rate of trap nests at the vegetative and flowering stages was recorded. the material used to construct nests and partition the cells was observed and recorded. the number of male and female bees that occupied the trap nests during the vegetative and flowering stage was also recorded. the number of days the bees took to occupy the nest tubes after installation and the time to complete the nest construction were recorded. the trap occupancy rate was calculated by taking the ratio of the number of occupied tubes per bundle of the nest and the total number of tubes per bundle of the nest. the occupied nest tubes were collected and brought to the laboratory and placed inside polyethylene bags fastened with a rubber band to prevent the escape of emerging adult bees. the occupied nest tubes were observed daily, and the emerged adults were collected and dry preserved for taxonomic identification. the number of days taken from the end of nest closure to the emergence of adult insects from the trap nests was also recorded. the number of cells constructed by female bees inside the nest and the number of emerged bees per nest from the trap nests were recorded. non-emerged bees were examined for signs of brood parasitization and kept separately in aerated containers for the emergence of parasitoids. estimation of wing wear of foraging female bees the activity of female bees in the flowers was recorded during the vegetative and flowering stages of the crop. the foraging bees were collected using entomological nets at weekly intervals at six different time points viz., 8.00 am, 10.00 am, 12.00 pm, 2.00 pm, 4.00 pm, 6.00 pm. the wing wear of the foraging females was evaluated according to mueller & wolf-mueller (1993). a scale of 0 to 5 was attributed to wing wear, where 0 = a completely intact apical margin; 1 = 1–2 nicks on the apical margin; 2 = 3–10 nicks on the margin; 3= some wing margin intact, though heavily serrated, with >10 nicks; 4 = completely serrated with no apical wing margin intact, but with excisions less than half the width of the distal submarginal cell; 5 = wing as described in 4, but with excisions more than half, but less than the entire width of the distal submarginal cell. diversity of megachile bees the diversity indices of the genus megachile latreille, 1802 bees occupying the trap nests during the vegetative and flowering stage were calculated. the total number of solitary bees occupying the nests and emerging was recorded for 30 days at weekly intervals. the diversity indices were calculated using the past software. shannon wiener diversity index (shannon & wiener, 1949), taking into account the number of individuals as well as the number of taxa indicated by h =-sum((ni/n) ln (ni/n)) where, ni, is the number of individuals of taxon ‘i’. simpson’s index is also a measure of diversity that considers the number of species present and the relative abundance of each species. margalef’s richness index: (s-1)/ ln(n), was also calculated where s represents the number of taxa and n is the number of individuals. evenness index measures the distribution of a species in an ecosystem and was calculated using pielou’s (1966) formula eh/s, where h indicates the shannon wiener index and s indicates the number of taxa. effect of the visitation by megachile spp. on the yield of pigeon pea the effect of the installation of trap nests to increase the abundance of leafcutter bees over the pollination and yield of pigeon pea was studied. we compared the yield and pod set parameters in two plots, one with trap nests installed (treatment plot) and the other without trap nests (control plot). the pollination efficiency of megachile sp in pigeon pea was assessed exclusively by bagging 100 mature flower buds (5 mature buds per 20 different plants) a day before the experiment. the bagged flower buds were opened the next morning during the bright sunshine hours and allowed for single visitation by the leafcutter bees foraging near the artificial traps. the flowers were observed, and those visited by the leafcutter bees were labeled and bagged after the visitation. the fruiting rate due to the visitation by leafcutter bees was assessed using the formula, percent pod set = pl-pb/pl x 100 where pl denoted pods set in flowers visited by leafcutter bees, pb denoted pods set in bagged flowers. in both the plots, the pods were collected from the 20 marked plants at maturity. the number of seeds per pod, the percent pod set, number of seeds set per pod, and 100 seed weight (g) were recorded by bagging the flowers (fb) and by allowing flower visitation by the leafcutter bees (fl). data analysis analysis of variance (glm in sas 9.3; sas institute, cary, nc) was used to compare the effect of the leafcutter bee artificial nests (trap nests) on the pollination and yield of pigeon pea. mean percent acceptance of trap nests, the emergence of bees, and the number of male and female bees that emerged were also analyzed. when a significant difference was detected, treatment means were separated using tukey’s hsd test (0.5%). results trap occupancy by the leafcutter bees (megachile spp.) the installed artificial trap nests were occupied by insects belonging to different taxa viz: megachilinae (bees), amala udayakumar, timalapur m shivalingaswamy – farm conservation of solitary bees using trap nests in pigeon pea4 eumeninae (wasps), and araneae (spiders). three species of leafcutter bees, megachile lanata (fabricius, 1775); m. laticeps smith, 1853; m. disjuncta (fabricius, 1781 and one parasitic bee, coelioxys sp. were observed to occupy the installed trap nests (fig 2). megachile disjuncta used resin, resin + mud, and cellophane-like membrane to plug its nests. m. laticeps used leaf bits to construct and plug its nest entrance. m. lanata used leaves and sand to construct and plug its nest entry. fig 3. number of trap nests by leaf cutter bees megachile latreille, 1802 occupied at two different crop stages of pigeon pea (cajanus cajan (l.) millsp. fabaceae) plantation of the experimental farm of icarnational bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. a b c d fig 2. different types of nesting materials used by bees megachile latreille, 1982 in a cajanus cajan (l.) millsp. (fabaceae) plantation of the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india; a. and b. leaf bits c. resin d. mud. trap nests had a significant influence on trap rate occupancy by bees during the crop vegetative and flowering stages (f value = 84.03; p < 0.0001) (table 1). the trap occupancy rate was higher during the flowering (72.50%) than in the vegetative stage (38.33%). the mean number of trap nests occupied, the number of active nests recovered, and the number of nests half-filled by bees during the vegetative stage was 17, 12, and 17, respectively (fig 3). on the contrary, 43 trap nests were occupied during the flowering period, with 31 active nests and 13 half-filled nests. during the vegetative stage, the male bees were homing inside the tubes more than the female bees. such males occupied nest tubes lacked built-in cells, and these male bees were observed to take mere shelter inside the nest tubes. while no active nest construction was observed in the traps during the vegetative stage, the active nests were recovered from the tubes during the flowering stage. the collected nests were built using leaves, resin, and soil material. the female bee used the leaves of pigeon pea specifically for their nest construction. there was a significant difference in the trap occupancy rate by non-bee insects, viz., spiders (f value = 13.50; p < 0.0001) and the wasp (f value = 14.23; p < 0.0001) sociobiology 69(1): e7202 (march, 2022) 5 during the vegetative as well as flowering period. spiders’ percent trap nest occupancy was 3.33 and 13.33 during the vegetative and flowering stages, respectively. the eumenid wasp trap occupancy rate was 2.22 and 14.47 during the vegetative and flowering stages. the number of days taken by the bees to occupy the nest differed significantly between the vegetative and flowering stage (f value = 16.38; p < 0.0001) (table 2). during the vegetative stage, the traps were occupied for 16.01 ± 2.82 days, whereas, during the flowering stage, the bees occupied the nests for 10.23 ± 2.30 days. the nest completion time significantly varied between the vegetative and flowering stages (f value = 36.10; p < 0.0001). after occupying the nest tubes, the nests were completed in 6.20 days during the vegetative stage and 2.40 days during the flowering stage. there was a significant difference in the number of cells formed (f value = 15.62; p < 0.0001) and the number of emerged bees per nest (f value = 13.23; p < 0.0001) in the trap nests during the vegetative and flowering stage of the crop. the mean number of cells formed by the bees per nest was 1.60 ± 1.14 and 5.60 ± 0.54 during the vegetative and flowering stages, respectively. the number of bees that emerged per nest during the vegetative and flowering stage was 0.4 ± 0.54 and 5.00 ± 0.71, respectively. the average number of male bees observed to occupy and take shelter inside the nest tubes during the vegetative and flowering stages was 6.23 ± 0.5 and 1.21 ± 0.8, respectively (fig 4). the mean number of female bees observed to occupy and found involved in constructing the brood cells in the nest tubes during the vegetative and flowering stages was 1.01 ± 0.71 and 7.21 ± 2.28, respectively. table 1. trap occupancy details of leafcutter bees megachile latreille, 1802 at two different crop stages of pigeon pea (cajanus cajan (l.) millsp., fabaceae) in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. crop stages trap occupancy rate by megachile bees (%) trap occupancy rate by spiders (%) trap occupancy rate by wasps (%) vegetative stage 38.33b 3.33b 2.22b flowering stage 72.50a 13.33a 14.47a f value 84.03 13.50 14.23 p value p < 0.0001 p < 0.0001 p < 0.0001 fig 4. number of traps occupied by male and female bee megachile latreille, 1802 at two different crop stages of pigeon pea (cajanus cajan (l.) millsp. fabaceae) plantation of the experimental farm of icarnational bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. table 2. nest development parameters of leafcutter bees megachile latreille, 1802 at two different crop stages of pigeon pea (cajanus cajan (l.) millsp., fabaceae) in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. crop stages number of days to occupy the nest number of days to complete the nest number of cells formed per nest number of bees emerged per nest percent parasitization of brood cells vegetative stage 16.01 ± 2.82a 6.20 ± 0.84a 1.62 ± 1.14b 0.41 ± 0.12b 53.67a flowering stage 10.23 ± 2.30b 2.40 ± 0.55b 5.61 ± 0.55a 5.00 ± 0.71a 6.67b f value 16.38 36.10 15.62 13.23 3.27 p value p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 amala udayakumar, timalapur m shivalingaswamy – farm conservation of solitary bees using trap nests in pigeon pea6 natural parasitization by melittobia sp (53.67%) occurred in nest tubes during the vegetative crop stage. parasitization by coelioxys sp (6.67%) in the brood cells in the occupied nest tubes collected during the flowering stage was recorded (table 2). the adult parasitoids emerged from the brood cells collected from the nest tubes. the age categorization during the vegetative and flowering stage was recorded and presented in fig 5. the sampling of the female bees during the vegetative stage belonged to the wing wear category of 3 to 5. on the contrary, during the flowering stage, the foraged females belonged to the wing wear category of 0 to 2. fig 5. classification of foraging female of leaf cutter bees megachile latreille, 1802 as per wing wear in studied specimens from the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. diversity of leafcutter bees (megachile spp.) four species of solitary bees were observed occupying the trap nests. the data revealed that a higher diversity of solitary bees was recorded during the flowering stage than the vegetative stage (table 3). in the present study, the simpson and shannon wiener diversity indices recorded during the vegetative stage were 0.68 and 0.47, respectively. the simpson (1.33) and shannon wiener diversity indexes (0.72) were higher during the flowering stage. the evenness index recorded during the flowering stage (1.01) was higher than during the vegetative stage (0.98). the richness index of the trap nesting bees recorded during the vegetative and flowering stages was 0.68 and 0.98, respectively. effect of megachile spp. visits over the pollination and yield of pigeon pea the contribution of leafcutter bees to the pollination and yield of pigeon pea was recorded (table 4). it was observed that there was a significant difference in the percent of pod set due to the pollination by leafcutter bees in the plots installed with and without trap nests (f value = 17.75; p value < 0.0001). the percent pod set in the plots installed with and without trap nests were 62.70 and 38.06, respectively. the number of seeds per pod (f value = 26.75; p value < 0.0001) and test weight (f value = 90.85; p value < 0.0001) of harvested seeds differed significantly between the plots with and without trap nests. the mean number of seed set per pod from the plants in the plot with and without trap nests were 5.76 ± 0.43 and 3.24 ± 0.78, respectively. simultaneously, the seed weight from plots with trap nests recorded a significantly greater 100 seed weight (11.37 ± 0.87 g) than the control plot (8.31 ± 1.75 g). discussion four different species of solitary bees occupied the artificial trap nests installed in pigeon pea cultivation. other insects like spiders and eumenid wasp were also observed to crop stages simpson’s index shannon-wiener diversity index evenness index margalef richness index vegetative stage 0.68 0.47 0.98 0.68 flowering stage 1.37 0.72 1.01 0.98 table 3. diversity indices of leafcutter bees megachile latreille, 1802 in different crop stages of pigeon pea (cajanus cajan (l.) millsp., fabaceae) in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. sociobiology 69(1): e7202 (march, 2022) 7 occupy the trap nests. gathmann et al. (1994) reported fourteen species of apoidea, four species of sphecidae, one species of eumenidae, and four species of parasitoids reared from the reed internodes of the grass phragmites australis (cav.) trin. ex steud. (poaceae). solitary bees like megachile sp., xylocopa tenuiscapa westwood, amegilla zonata linnaeus, and nomia sp. were the major pollinators of pigeon pea in nagaland, india (singh, 2016). chaudhary & jain (1978) reported megachile lanata as the major pollinator of pigeon pea. the number of days taken by the bees to occupy the nest and complete the nest was significantly lesser during the flowering stage compared to the vegetative stage. the number of emerged bees per nest was significantly higher during the flowering stage compared to the vegetative stage. this can be attributed to the greater amount of food available in the area, which allowed more females to actively build their nests in the area during the flowering stage. the foraging patch of female leafcutter bee, m. lapponica, closer to its trap nests was reported by gathmann & tscharntke (2002). conversely, more male bees were observed occupying per nest during the vegetative stage. availability of lush green pigeon pea leaves serving as a nesting material with copious pollen-filled flowers might be the reason for more female bees constructing their nests during the flowering stage. it was observed in three solitary bee species (anthophora plumipes (pallas), habropoda tarsta (anthophoridae), and eucera nigrilabris lep. (eucerinidae), the proportion of ‘near’ visits was twice higher for females indicating that males performed more ‘far’ flights than females (neeman et al., 2006). consequently, males flew longer inter-floral distances than females. therefore, the energy expended by males during foraging activity must have been higher than that of females, indicating a lower foraging efficiency of males. the availability of specific materials for their nest construction and pollen for larval food are the pre-requisites for successful reproduction in solitary bees (westrich, 1996). regarding megachile’s natural enemies, the broods were parasitized by melittobia sp (hymenoptera: eulophidae) in the vegetative stage and coelioxys sp (hymenoptera: megachilidae) in the flowering stage of the crop. a significantly higher rate of parasitization of the brood cells was recorded during the vegetative stage than the flowering stage. as the proportion of female bees was significantly lesser in the trap nests during the vegetative stage of the crop, the brood care was significantly lower, which would have resulted in easier invasion by the brood parasites. the brood care by female leafcutter bees by capping its nest with layers of mud/leaf bits to evade attack by predators/parasites was reported by peterson et al. (2016). a parasitism rate of 85.55% by the parasitoid, melittobia hawaiiensis perkins, 1907 on broods of leafcutter bees in the trap nest of ipomea reeds was reported by veeresh kumar et al. (2015). sabino & antonini (2017) reported 15% parasitization of brood cells of leafcutter bee, megachile (moureapis) maculata inside the trap nests in a montane forest by cuckoo bee coelioxys (acrocoelioxys) sp. therefore, providing the trap nests in the agricultural cropped area helps in the dual advantage of conserving the solitary bees by providing habitats and attracting solitary predatory wasps for nesting resulting in biological control of pest insects infesting pigeon pea. wing wear of female leafcutter bees was reported to have a negative impact on the foraging ability and reproductive performance of the bees (foster & carter, 2010; rehan & richards, 2010). in the present study, wing wear occurred more during the vegetative period, and the female bees were observed to make more foraging flights in the crop from the leaf material. due to the lack of pigeon pea flowers, the bees have to engage in pollen foraging trips far away from the crop, resulting in wing wear during the vegetative period. this increased wing wear could be correlated with the reduction in the nesting success of the female bees, as evident from the low number of nests recovered in the artificial traps during the vegetative period. greater wing wear was reported to be linked with the reduced foraging success and sustainability of nesting performance of alfalfa leafcutting bee, megachile rotundata (fabricius, 1787) (neill et al., 2015). hence, the reason for the lowest wing wear during the flowering period might be due to the instant availability of patches of flowers of pigeon pea for pollen foraging. the diversity indices of the solitary bees that occupied the trap nests during the vegetative and flowering stage were presented in table 3. shannon’s index indicates both abundance and evenness of the species occurring in a community. the abundance of solitary bees was higher during the flowering stage due to the abundant pollen source from the blooming flowers of pigeon pea. the evenness index measures the distribution of a species in an ecosystem. the higher evenness index recorded during the flowering stage indicated the uniform distribution of solitary bees compared to the vegetative stage. treatments percent pod set number of seeds per pod 100 seed weight (g) plots installed with trap nests 62.70a 5.76 ± 0.43a 11.37 ± 0.87a control plot 38.06b 3.24 ± 0.78b 8.31 ± 1.75b f value 17.75 26.75 90.85 p value p < 0.0001 p < 0.0001 p < 0.0001 table 4. pollination efficiency of leafcutter bees megachile latreille, 1802 and yield parameters of pigeon pea (cajanus cajan (l.) millsp., fabaceae) in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus, india. amala udayakumar, timalapur m shivalingaswamy – farm conservation of solitary bees using trap nests in pigeon pea8 a higher richness index was recorded during the flowering stage, which indicated an increased number of different species of trap nesting bees compared to the vegetative stage. trap nests as an effective tool to investigate the species assemblage and community structure of cavity-nesting solitary bees were reported by buschini (2006). smaller solitary bees prefer to construct their nests near the floral resources rather than foraging for nectar and pollen at far distances, unlike large-sized bees (wcislo & cane, 1996; greenleaf et al., 2007; franceschinelli et al., 2017). in the present study, the percent pod set, number of seeds per pod, and 100 seed weight by the pollination of leafcutter bees were significantly higher in the plots where the trap nests were exposed compared to plots without trap nests. the females actively foraged over the opened flowers and returned to the nest in the plots installed with the trap nests. the examination of trap nests during the flowering stage revealed the presence of active broods inside the nests. the foraging bees were loaded with pollen in their abdominal scopa while entering the trap nests. the number of pollinating visits performed by leafcutter bees touching the stigmal surface of the flowers was significantly higher compared to the control plot. junqueira et al. (2013) reported that the installation of trap nests significantly increased the population and flower visitation of carpenter bees in passion fruit at minas gerais, southeastern brazil. the introduction of trap nests was reported to increase the densities of solitary bees belonging to the family megachilidae in apples, almonds, and alfalfa (bosch & kemp, 2002). usually, successful habitat management measures depend on factors like elevation, land fragmentation, source habitats, and the prevalence of nesting resources like dead woods to conserve solitary bees (murray et al., 2009; scheper et al., 2013). thus, providing nesting sites with rich pollen sources by the presence of a varied melitophyllous flora near the nesting site allows the solitary female bees to save time and energy budgets in foraging and enables them to spend more time in their nests thereby evading the attack by predators and parasitoids (batra, 1984). westrich (1989) reported that the availability of pollen sources plays a major role in the prevalence and structuring of bee communities rather than nectar source plants. conclusion our study shows the leafcutter bees being the key pollinators of pigeon pea. the installation of trap nests helped to increase their population, nesting activity, thereby increasing the flower visitation and pollination in pigeon pea. the trap nest installation in farmlands is an easiest way and also helps in the in-situ conservation of the leafcutter bees. acknowledgements the authors are thankful to former directors of icar-nbair, bengaluru, dr. chandish r ballal and dr n. bakthavatsalam for their constant support and encouragement in our efforts in the conservation of pollinators. authors contribution au: conceptualized, conducted, and recorded field observations in the study. tms: assisted in drafting the manuscript. both the authors have read and approved the manuscript. references abrol, d.p. & shankar, u. 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(2015). parasitisation of leafcutter bees (megachilidae: apoidea) by melittobia species. entomon, 40: 103-110. villemey, a., jeusset, a., vargac, m. bertheau, y., coulon, a., touroult, j., vanpeene, s. castagneyrol, b., jactel, h. & witte, i. (2018). can linear transportation infrastructure verges constitute a habitat and/or a corridor for insects in temperate landscapes? a systematic review. environmental evidence, 7: 5. doi: 10.1186/s13750-018-0117-3. wcislo, w.t. & cane, j.h. (1996). floral resource utilization by solitary bees (hymenoptera: apoidea) and exploitation of their stored foods by natural enemies. annual review of entomology, 41: 257-286. doi: 10.1146/annurev.en.41.0101 96.001353. westrich, p. (1996). habitat requirements of central european bees and problems of partial habitats. the conservation of bees (eds a. matheson, s.l. buchmann, c. o’toole, p. westrich & i.h. williams), pp. 1-16. academic press, london. _hlk79493030 _hlk79493097 _hlk79493597 _hlk53498348 _hlk79498044 doi: 10.13102/sociobiology.v67i3.5148sociobiology 67(3): 364-375 (september, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the expansion of cities is a conspicuous cause of the conversion of land into landscapes modified by man (mckinney, 2002; santos, 2016). these urban landscapes are usually spatially and temporally dynamic (mcintyre et al., 2001), and often associated with low biodiversity (vitousek et al., 1997; mcintyre et al., 2001; yamaguchi, 2004; 2005). all over the world, these urban landscapes are characterized by high heterogeneity, which can provide useful information on the management of biodiversity in other ecosystems (savard et al., 2000). abstract around the world, urban landscapes are environments modified by man, generally related to low diversity. the efficiency of a biodiversity inventory is linked to the choice of the sampling technique since the taxonomic and ecological responses of the taxons vary between methods. here we investigate differences in the ant’s composition sampled using three different techniques in two fragments of the urban forest in the brazilian amazon. we also assessed whether the different techniques maintained the same ecological responses. we sampled 12 collection points at each fragment, at vegetation, and manual collection and winkler extractor on the ground. at the same points, soil samples were collected to determine their granulometry, ph, and concentrations of organic matter, sodium, phosphorus, and potassium. we collected 115 taxa and 4720 ants. the cururu was the richest site with more species in general, as well as in the techniques of manual collection and winkler. we detected a complementary effect on sampling techniques, which collected different ants’ assemblages. the potassium concentration positively influenced the assemblage’s composition, but its effect varies according to the sampling techniques used. the studied fragments revealed diversity very similar to those registered in continuous amazonian forests. the use of sampling techniques together improves the representation of the diversity of ants in the studied fragments. edaphic environmental variables seem to have a predominant effect on ants, affecting their distribution in the landscape even in urban fragments. this highlights the importance of urban forest fragments and their inherent ecological processes. sociobiology an international journal on social insects jlp souza1, js araújo2 article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 09 april 2020 initial acceptance 06 july 2020 final acceptance 28 july 2020 publication date 30 september, 2020 keywords biodiversty surveys, formicidae, manaus, petroleum, tropical forest. corresponding author jorge lp souza instituto nacional da mata atlântica (inma) avenida josé ruschi nº 04, cep: 29650-000 santa teresa, espírito santo, brasil. e-mail: souza.jorge@gmail.com environments that ants inhabit are modified by natural disturbances, which vary in extent, magnitude, and frequency. also, many terrestrial ecosystems have been altered due to various human activities, including urbanization (philpott et al., 2010). urbanization is a driving force behind habitat destruction and has powerful effects on ants’ richness and composition. ecological surveys of urban ants usually address changes in species richness and composition (gibb & hochuli 2003; lessard & buddle 2005; pacheco & vasconcelos 2007; yamaguchi 2004), but little has been done regarding the choice or efficiency of sampling techniques. 1 instituto nacional da mata atlântica (inma), santa teresa, espírito santo 2 departamento de biologia, universidade federal do amazonas (ufam), manaus, amazonas, brazil research article ants evaluation of sampling techniques and influence of environmental variables on ants in forest fragments in an oil extraction area in the amazon sociobiology 67(3): 364-375 (september, 2020) 365 there is no suggestion of an ant sampling protocol for urban environments (munhae, 2017), which makes comparisons between works difficult. in urban green areas (i.e. parks or forest fragments) among the most used techniques, we have a manual collection, winkler extractors (if there is litter), and beating, which is on a smaller scale (munhae, 2017). the manual collection has a very low cost, which facilitates its use. however, the effectiveness of this technique depends on the researcher’s experience and the sampling effort (bestelmeyer et al., 2000). the winkler has a high initial cost and depends on the existence of a litter, however, it has the advantage of sampling a large portion of ants that are not detected with other methods (olson, 1991; bestelmeyer et al., 2000). beating on vegetation is often used to complement other techniques, as it underestimates species by not accessing the canopy (majer & delabie 1994; munhae, 2017). no sampling technique is effective for capturing all invertebrates present in a given area. some studies with several sampling techniques to capture ants have revealed that the information obtained by these techniques may be complementary to obtain a more complete survey of the richness of genera and species in the area (olson, 1991; delabie et al., 2000; antoniazzi et al. 2020). but may have some degree of redundancy of information (parr & chown, 2001; lopes & vasconcelos, 2008; souza et al., 2012). studies of a minimum number of sampling techniques usually seek to know which technique or combination of techniques is most efficient for an inventory with an emphasis on records of the diversity and abundance of taxa and are usually conducted in natural landscapes (e.g. garden et al., 2007; roy et al., 2007; ribeiro-junior et al., 2008). however, little is known about the choice of sampling techniques in urban environments (munhae, 2017). in aquatic systems, invertebrates have been widely used for biological monitoring (hawkins et al., 2000), however, in terrestrial environments they have been used less frequently (andersen & majer, 2004). while providing valuable indications of changes in biological integrity and ecosystem functions, terrestrial invertebrates are usually not considered by management agencies (andersen & majer, 2004) due to the high costs, especially those resulting from the long time needed to identify this megadiverse group, together with the lack of taxonomists and parataxonomists. however, some change has occurred in recent times and longterm inventories on biodiversity have included ants in their focus groups (e.g. fernandes & souza, 2018). among the most commonly used environmental characteristics for modeling the distribution of organisms are soil, topography, and vegetation structure (zuquim et al., 2007). for invertebrates, environmental factors, such as topography, texture, and soil chemistry, generate microhabitat variability that can affect the spatial patterns of invertebrate assemblages at local scales (vasconcelos et al., 2003; oliveira et al., 2009; mezger & pfeiffer, 2011). in the amazon, some studies already find effects of these variables on oribatid-mites (moraes et al., 2011), pseudoscorpions (aguiar et al., 2006), ants (oliveira et al., 2009; souza et al., 2009; gomes et al., 2018; torres et al., 2020), cockroaches (tarli et al., 2014) and termites (dambros et al., 2016). to be considered efficient, the sampling protocol must satisfy the taxonomic, ecological and financial aspects of the research and some studies have used the responses of taxa to the environment to calibrate sampling protocols in the amazon (souza et al., 2012; 2016). both the forest fragments areas around the isaac sabbá refinery (reman) and the surroundings of the cururu stream are now fragmented forest fragments with high vulnerability and constant anthropic action. although they are located within the city of manaus, there is no study report on surveys to characterize the terrestrial invertebrate fauna of these forest fragments, neither now nor in the past. this is the first biodiversity survey of the insects in both sites. the majority of the studies on the fauna of manaus invertebrates were carried out in the ducke reserve, a forest reserve belonging to the national research institute of amazonia inpa (magnusson et al., 2014). there is only one study evaluating the impact of the oil spill that occurred in 1985, on the water and edaphic insects of the riverbank, in cururu igarapé (couceiro et al., 2006). biodiversity surveys are one of the first steps to determine the conservation or sustainable use of a location or ecosystem (scott et al., 1987; national research council, 1992). for the characterization of ant´s assemblages in the two study areas, different sampling techniques were used, covering the available habitats, such as soil and vegetation of the understory. information on environmental variables that possibly affect the distribution of ant’s assemblages was collected in areas located around the isaac sabbá refinery reman and the marine command base at igarapé cururu. our goals are: 1) evaluate if sampling techniques have a complementary effect on ant’s assemblages on urban forest fragments and 2) test if environmental variables affect the distribution of the ant´s assemblages in the forest fragments studied. for this, we tested two hypotheses. the first is that the collection techniques would have a null or reduced complementary effect on biodiversity survey, due to the constant anthropic effect on the fragments. also, the ecological response of the ants collected with the techniques together will remain with the use of them separately. material and methods study area the study was conducted in two urban forest fragments, the first around the isaac sabbá refinery – reman (03° 08’13.47 “s – 59° 57’24.7” w). the second area sampled was the forest on the slope of the cururu igarapé, owned by the marine operations battalion of the brazilian navy (03° 07’47.72 “s – 59° 56’34.20” w), hereafter reman and cururu respectively (fig 1). the minimum distance between the sampling points and the fragment’s edge was 150m in cururu and 200m in reman. two sampling collections were carried jlp souza, js araújo – ants in an urban oil extraction area in the amazon366 out in two areas, the first from september 14 to october 27, 2006, and the second from february 14 to april 02, 2007. each sampling technique was used on different days, in a single collection event per year. the manual collection and winkler extractor were used for sampling ground-dwelling ants. in the manual collection, the litter samples were removed from a bounded area with 0.50 x 0.50 m (0.25 m2), placed in a white tray for the sorting of ants (bestelmeyer et al., 2000). the litter samples for winkler extraction were taken from a bounded area of 1 m2. the whole litter was collected manually and placed in a 1 cm2 mesh sieve. the sieve was stirred for about one minute and the sieved material was transported to the laboratory. in the laboratory, the samples were placed in the winkler extractor for 48 hours for extraction (bestelmeyer et al., 2000; souza et al., 2012). the manual collection and winkler extractor were collected 24 samples for each method in both areas. ants were identified at the genus level using the keys provided in baccaro et al. (2015). later, this material was identified by morphospecies or species whenever possible, using available taxonomic keys, comparison with material previously identified by specialists and deposited in the national institute of amazon research (inpa) entomological collections. environmental predictors the environmental variables related to soil factors (clay content, ph, organic matter, sodium, phosphorus, and potassium concentration) were selected due to their influence in previous research in the amazon basin (vasconcelos et al., 2003; oliveira et al., 2009; gomes et al., 2018; torres et al., 2020). along each of the plots, 12 soil samples were collected using an auger, at the same sampling points as the ants. the soil samples were collected up to 10 cm deep after removal of the leaf litter and large roots. after collecting the soil, the material was sent for analysis of texture and chemical characteristics, following the protocols, established by embrapa (silva, 2009). data analysis we used frequence data (presence/absence) to avoid the overestimation of species with larger nests (hölldobler & wilson, 1990; gotelli et al., 2011). some ant species have mass foraging strategies and their nests, or foraging/displacement trails (e.g. dorylinae) may be close to the sampling point (hölldobler & wilson, 1990; brühl et al., 2003). the frequency was calculated for each sampling point (trap). the data from the two sampling events (2006 and 2007) were grouped and analyzed jointly. thus, we have 12 winkler extractors, 12 manual collections and 10 palm samples for each fragment. we calculated the number of species in relation to the number of individuals collected using hill numbers (chao et al., 2014; hsieh et al., 2016). observed samples were used to calculate diversity estimates for rarefied and extrapolated samples and the confidence intervals associated with 95%. the generated curves plot the diversity estimates in relation to the sample size (chao & jost, 2012; colwell et al., 2012). fig 1. location of the two urban forest fragments near an oil and gas refinery in the city of manaus, amazonas, brazil. sampling techniques and identification to sampling ants, three different techniques were used: beating (entomological umbrella), manual collecting, and winkler extractor. in both areas, 12 sampling points approximately 15 m apart were allocated in plots, one in each fragment, installed on the ground, in the flattest location possible. each sampling technique was performed on the same plot in each fragment but on different days. the length of the plots was 180m for the techniques of manual collection and winkler extractor. for the beating, the length of the plots was variable to accommodate 10 palm trees, with a minimum distance of 10 m between sampled plants. the beating (entomological umbrella-hunting) technique was used to collect ants in the palms of the genera astrocarium, bactris and geonoma, which are found in the understory. these palms accumulate organic plant material in decay between the leaves and suspended from the ground between 0.5 m to 2 m in height. ten palm trees were selected for this study in each area. the palms were hit to dislodge ants from a tray underneath. this technique sampled the fauna associated with palm trees (bestelmeyer et al., 2000). sociobiology 67(3): 364-375 (september, 2020) 367 to evaluate the complementary between the sampling techniques, we used the permutational multivariate analysis of variance (permanova, anderson, 2001). we also evaluated the effect of the sites and their interaction with sampling techniques on the composition of the ant assemblage. we created a stratified permutation procedure to keep the nested structure of the data (plots nested in sites) in the permanova to control for possible spatial autocorrelation of the data. the permanova probability values were based on 999 permutations. the ground-dwelling ants collected in the winkler extractor and manual collection techniques were correlated with environmental variables related to soil factors (clay content, ph, organic matter and, potassium concentration). we used redundancy analysis (rda) to estimate how much of the variance in the response variable (ant’s species composition matrix) can be explained by the environment. we carry out these analyzes with the sampling techniques together and separately. we also include as variables in the rda, the collection technique and the sampling site. rda is a direct extension of multiple regression analysis to model multivariate response data (borcard et al., 2011). the statistical significance of rda models was tested by 1000 permutations per test. all analyses were performed with the r environment version 4.0.2 (r core team, 2020) using the vegan 2.5-6 (oksanen et al., 2019), car 3.0-3 (fox & weisberg, 2019), inext 2.0.19 (hsieh et al., 2019) packages. fig 2. species accumulation curves based on 1,000 randomization (rarefied and extrapolated values) for ant species sampled with three techniques in the cururu (a) and reman (b) urban forest fragments, in the city of manaus, amazonas, brazil. confidence intervals were calculated to 95%. results we collected 4720 ants, belonging to 8 subfamilies and 115 species. at cururu, we observed 98 species of ants and 74 species in reman. of these, 41 species occurred only in the cururu area, 17 exclusively in reman and 57 species were recorded in both areas. the rarefaction and interpolation curves indicate that the number of species tends to increase with the addition of individuals. winkler extractor was the technique that sampled the highest ant´s richness in both forest fragments. at the cururu site, it registered 59 species of ants and at reman 41. with the extrapolation to 2000 individuals, the estimate for cururu was 88 species and 65 for reman. in the cururu fragment, manual collection registered 51 species (88 estimated) followed by the beating that registered 32 (44 estimated). in the reman fragment, there was an inversion and the beating surpassed the manual collection in the number of species, collecting 36 (57 estimated) and 29 (49 estimated) species respectively (fig 2, table 1). in cururu, the greatest abundance of ants was recorded with winkler, followed by manual collection and beating. at reman, the greatest abundance of ants was detected at the beating; winkler and the manual collection had similar abundances (table 1). sampling techniques have an effect on the ant species composition (permanova: f5,53 = 6.811; r 2 = 0.19; p < 0.001). the assemblage sampled with the winkler (blue symbols) is concentrated in the center of the figure. around it, is the points of the manual collection samples (green symbols), and a more distinct group on the right (red symbols) are the points of the beating (fig 3). the effect of forest fragments on the composition of ants is small (permanova: f5,53 = 2.490; r2 = 0.03; p <0.01), so there is no evident separation between them (fig 3). the interaction term between sites and sampling techniques was not significant (permanova: f5,53 = 1.332; r2 = 0.04; p = 0.120). considering the winkler and manual collection techniques together, the ant assemblage composition was significantly correlated with tested variables (rda = 0.3975; p > 0.001). but, the environmental variables have a small effect on the ant’s assemblages sampled in the two forest fragments. jlp souza, js araújo – ants in an urban oil extraction area in the amazon368 the greatest detected effect on the assemblage is the sampling techniques (f = 12.4165; p > 0.001), followed by the effect of potassium concentration (f = 2.1492; p > 0.05; fig 4-a). analyzing winkler separately, the analysis is not significant (rda = 0.2726; p = 0.093), but the partial effect of potassium concentration is still detected (f = 2.1155; p > 0.05; fig 4-b). similarly, the manual collection also has a non-significant result (rda = 0.2577; p = 0.113), this time, without partial effects of environmental variables. disscusion although urban environments are usually associated with low biodiversity, we detected high biodiversity of ants in the studied urban forest fragments. we also verified the complementary effect of the sampling techniques used. besides, we detect that ants’ responses to environmental variables are subtle, and their detection will depend on the sampling technique used. fig 3. an nmds ordination (stress = 0.122) plot indicating the congruence in ant assemblages’ associations among two urban forest fragments and the three sampling techniques in the city of manaus, amazonas, brazil. circular symbols indicate sampling points of the cururu fragment and triangular ones from renan. different colored symbols indicate different sampling techniques. ants form a mega-diverse group with several species and high abundance. in the gradient along the amazon river on the amazon basin were recorded 42 genera and 166 species (vasconcelos et al., 2010). a non-urban fragment of dense ombrophilous forest near the city of manaus record 31 genera and 151 ant species (vasconcelos et al., 2003). in sites of dense ombrophilous forests near manaus, 51 genera and 219 species (torres et al., 2020) and 54 genera and 237 species were recorded (oliveira et al., 2009; souza et al., 2012). contrary to our hypothesis, the number of genera and ant species (42; 117 respectively) found in this work conducted in urban forest fragments, was very similar to the numbers found in studies carried out in ombrophilous forests in the amazon basin with greater sampling effort. ants are an example of successful arthropods in urban or fragmented landscapes (menke et al., 2011). the effects of urbanization are usually characterized as damaging to biodiversity (vitousek et al., 1997; mcintyre et al., 2001; yamaguchi, 2004; 2005), however, ecologically they can have positive effects, with the creation of new environments that do not occur anywhere else (niemelä, 1999; marshall & shortle, 2005). this indicates that the ant´s assemblages of the studied sites, although located in a forest fragment near to the oil refinery, and disconnected from larger areas of ombrophilous forest, harbor a relevant part of the genera and species already registered in the amazon region. the rarefaction curves of sampling techniques in both sites have similar trends and do not reach an asymptote. this is a recurrent fact in studies with ant’s assemblages where most of them usually do not reach the asymptote regardless of the sampled environment or collection technique employed sociobiology 67(3): 364-375 (september, 2020) 369 (e.g. king & porter, 2005, vasconcelos & vilhena, 2006; lopes & vasconcelos, 2008; souza et al., 2012). although they have similar tendencies, the techniques have different efficiencies between and within each fragment. in cururu, manual collection and winkler have similar efficiencies, with winkler collecting slightly more species and individuals. the beating was much less efficient both in species and in ant’s abundance. at reman, winkler was distinctly the most efficient, collecting the most species. manual collection and beating had similar efficiencies, sampling a resembling number of species, but differing in the total of individuals. the winkler is an efficient method in forest environments, as it collects species neglected by other techniques (bestelmeyer et al., 2000) and seems to maintain this efficiency in forest fragments. manual collection can be as efficient as other techniques and is widely used in work in urban areas (bestelmeyer et al., 2000; munhae, 2017). however, it seems that this technique is more sensitive to environmental differences and that is why it had different efficacy between the fragments, which may prove to be a limiting factor in its use. the beating added a few species to the total, and this fact had already been reported in the literature (majer & delabie, 1994). perhaps because of the three techniques used here, this is the least frequent in studies in the urban area (munhae, 2017). depending on the question to be answered and the financial and time cost of the survey, this increase in the sampling effort may render the survey unviable (souza et al., 2012). some studies have already indicated the complementary effect of sampling methods on the study of ants (olson, 1991; delabie et al., 2000, fisher, 1999) and this effect were also confirmed by some studies in the amazon region (souza et al., 2007; souza et al.; 2009). the successful and long-term conservation of a site depends on knowledge of its biological diversity, associated with efforts to protect and manage this biodiversity (national research council, 1992). the initial way of knowing the biodiversity of a given location or an ecosystem is to conduct a biodiversity survey (scott et al., 1987; gardner et al., 2008). in this study, the ant assemblages sampling through edaphic techniques (winkler extractor and manual collecting) are more similar, forming a separate group from the group of species sampling by the beating. this shows that the sampling techniques are quite particular, and the use of combined techniques is indicated to better portray the diversity of the fragments area. however, it is possible to find some degree of redundancy using more than one sampling method (parr & chown, 2001; lopes & vasconcelos, 2008), and depending on the research objective, the use of a single sampling technique can be efficient and more economical (souza et al., 2012). some studies have used ecological responses, based on environmental variables that determine the distribution of organisms, to evaluate the decision to reduce the number of sampling techniques (souza et al., 2012) or the selection of higher-taxon (souza et al., 2016). contrary to our hypothesis, the ecological response has changed between the sampling techniques used. only the winkler was able to maintain an ecological response similar to that detected with the two sampling techniques together. this efficiency of winkler in maintaining the ecological responses had already reported in other areas in the amazon (souza et al., 2012). investigating ecological relations, in addition to the usual metrics of diversity (richness and composition), increase more information when fig 4. a-an nmds ordination (stress = 0.167) plot indicating the congruence in ant assemblages’ associations among two urban forest fragments and the two edaphic sampling techniques in the city of manaus, amazonas, brazil. ban nmds ordination (stress = 0.229) plot indicating the congruence in ant assemblages’ associations among two urban forest fragments and the winkler extractor technique in the city of manaus, amazonas, brazil. circular symbols indicate sampling points of the cururu fragment and triangular ones from renan. the size of the symbols indicates the magnitude of their value. jlp souza, js araújo – ants in an urban oil extraction area in the amazon370 defining sampling protocols. depending on the research objective, the choice of a sampling technique that not only collects more ant species, but that is also efficient in detecting environmental responses can prove to be useful, especially in the situation of environments where the creation of a protocol is still eminent. anthropically impacted environments, such as forest fragments near the oil refinery and navy barracks, still maintain a high diversity of ants. the use of various sampling techniques provided a better overview of the ant diversity in the study sites and indicated that the tree fauna is quite different from that associated with the litter-soil interface. the effect of environmental variables on the distribution of ants were small, but they suggest that there are some complex processes in the structuring of assemblages, even in places considered environmentally simpler. its reinforces the importance of these forest fragments to maintain biodiversity in urban areas. table 1. abundance and richness of ant species by each sampling technique in both studied sites in the city of manaus, amazonas, brazil. subfamily / taxon cururu total reman total grand totalbeating manual collecting winkler extractor beating manual collecting winkler extractor dolichoderinae azteca sp.01 1 1 2 2 3 azteca sp.02 1 1 1 1 2 dolichoderus attelaboides (fabricius, 1775) 5 5 5 dolichoderus sp.01 1 1 1 1 2 tapinoma sp.01 2 4 6 10 10 16 tapinoma sp.02 2 2 2 dorylinae neocerapachys splendens (borgmeier, 1957) 3 3 3 ectatomminae ectatomma brunneum smith, 1858 2 1 8 11 1 1 12 ectatomma edentatum roger, 1863 1 4 5 5 ectatomma lugens emery, 1894 1 1 2 1 1 3 ectatomma tuberculatum (olivier, 1792) 2 2 2 gnamptogenys tortuolosa (smith, f., 1858) 69 69 1 1 70 gnamptogenys sp.02 8 16 24 24 gnamptogenys sp.03 1 1 1 formicinae brachymyrmex sp.01 105 3 1 109 165 15 180 289 brachymyrmex sp.03 1 7 8 3 3 1 7 15 camponotus atriceps (smith, 1858) 3 3 1 1 4 camponotus bidens mayr, 1870 4 4 1 1 5 camponotus latangulus roger, 1863 1 1 1 1 2 camponotus trapezoideus mayr, 1870 22 22 6 6 28 camponotus sp.02 3 3 3 camponotus sp.03 1 1 1 gigantiops destructor (fabricius, 1804) 3 3 1 1 4 nylanderia sp.01 1 1 1 1 2 nylanderia sp.02 1 1 3 5 8 9 nylanderia sp.03 5 17 22 22 nylanderia sp.04 31 7 38 167 167 205 myrmicinae acanthognathus sp.01 1 1 1 acromyrmex sp 01 1 1 1 atta sexdens (linnaeus, 1758) 1 3 4 4 blepharidatta brasiliensis wheeler, 1915 196 193 389 1 19 20 409 sociobiology 67(3): 364-375 (september, 2020) 371 myrmicinae carebara sp.01 4 4 1 1 5 cephalotes atratus (linnaeus, 1758) 2 2 2 cephalotes minutus (fabricius, 1804) 1 1 1 3 3 cephalotes opacus santschi, 1920 1 1 1 1 2 3 cephalotes pallens (klug, 1824) 1 1 1 1 2 cephalotes pinelii (guérin-méneville, 1844) 1 1 1 crematogaster arcuata forel, 1899 6 6 6 crematogaster brasiliensis mayr, 1878 2 2 463 6 469 471 crematogaster curvispinosa mayr, 1862 1 1 16 16 17 crematogaster erecta mayr, 1866 2 5 7 25 1 26 33 crematogaster flavosensitiva longino, 2003 35 35 35 crematogaster foliocrypta longino, 2003 11 11 11 crematogaster limata smith, 1858 4 67 6 77 25 2 27 104 crematogaster sotobosque longino, 2003 6 4 10 11 84 69 164 174 crematogaster tenuicula forel, 1904 7 1 8 8 crematogaster sp.01 1 1 1 cyphomyrmex sp.01 67 13 80 2 15 17 97 megalomyrmex sp.01 3 3 1 1 4 megalomyrmex sp.02 2 2 1 1 3 mycocepurus smithii (forel, 1893) 1 1 1 myrmicocrypta sp.01 2 2 2 ochetomyrmex semipolitus mayr, 1878 55 2 24 81 25 4 29 110 octostruma betschi perrault, 1988 3 3 6 2 2 8 octostruma iheringi (emery, 1888) 2 2 2 pheidole sp.01 12 12 2 2 14 pheidole sp.02 1 1 1 pheidole sp.03 93 165 258 20 79 54 153 411 pheidole sp.04 10 1 11 11 pheidole sp.05 5 5 5 pheidole sp.06 7 7 7 pheidole sp.08 1 3 4 2 1 3 7 pheidole sp.09 1 103 104 2 136 138 242 pheidole sp.10 1 1 1 procryptocerus attenuatus (smith, 1876) 1 1 1 strumigenys appretiata (borgmeier, 1954) 6 6 6 strumigenys beebei (wheeler, 1915) 1 1 1 strumigenys cincinnata (kempf, 1975) 1 1 1 strumigenys denticulata mayr, 1887 7 117 124 1 30 31 155 strumigenys elongata roger, 1863 4 2 6 6 strumigenys infidelis santschi, 1919 1 2 3 2 2 5 strumigenys perparva brown, 1958 3 3 3 strumigenys stenotes (bolton, 2000) 1 3 4 3 3 7 strumigenys trinidadensis wheeler, 1922 1 1 1 strumigenys trudifera kempf & brown, 1969 22 22 5 5 27 table 1. abundance and richness of ant species by each sampling technique in both studied sites in the city of manaus, amazonas, brazil. (continuation) subfamily / taxon cururu total reman total grand totalbeating manual collecting winkler extractor beating manual collecting winkler extractor jlp souza, js araújo – ants in an urban oil extraction area in the amazon372 table 1. abundance and richness of ant species by each sampling technique in both studied sites in the city of manaus, amazonas, brazil. (continuation) myrmicinae rhopalothrix sp.01 1 1 1 rogeria sp.01 1 1 2 1 1 3 rogeria sp.02 1 1 1 rogeria sp.03 1 1 2 2 3 sericomyrmex sp.01 1 1 1 1 2 solenopsis sp.01 117 134 251 82 19 23 124 375 solenopsis sp.02 13 52 65 9 2 12 23 88 solenopsis sp.03 13 15 28 13 25 38 66 solenopsis sp.04 2 2 9 13 1 1 2 15 solenopsis sp.05 4 4 4 solenopsis sp.06 5 5 5 solenopsis sp.07 122 21 143 1 1 144 paratrachymyrmex sp.01 1 2 3 1 1 4 paratrachymyrmex sp.02 1 1 1 paratrachymyrmex sp.03 1 1 3 1 4 5 paratrachymyrmex sp.04 1 1 1 wasmannia auropunctata (roger, 1863) 162 53 163 378 11 74 85 463 wasmannia rochai forel, 1912 1 1 1 ponerinae anochetus diegenis forel, 1912 2 2 1 1 3 anochetus emarginatus (fabricius, 1804) 63 63 63 anochetus horridus kempf, 1964 7 19 26 5 1 6 32 hypoponera sp.01 71 80 151 1 5 16 22 173 hypoponera sp.02 31 31 1 1 32 hypoponera sp.03 1 1 1 leptogenys linearis (smith, 1858) 1 1 1 mayaponera constricta (mayr, 1884) 6 6 12 12 neoponera apicalis (latreille, 1802) 1 1 1 neoponera crenata (roger, 1861) 6 6 6 odontomachus haematodus (linnaeus, 1758) 3 3 3 1 4 7 odontomachus sp.01 1 1 1 pachycondyla crassinoda (latreille, 1802) 1 1 1 pachycondyla harpax (fabricius, 1804) 1 1 1 pachycondyla sp.01 12 12 8 8 20 thaumatomyrmex atrox weber, 1939 1 1 1 proceratiinae discothyrea neotropica bruch, 1919 2 3 5 2 2 7 pseudomyrmicinae pseudomyrmex flavidulus (smith, 1858) 2 2 2 pseudomyrmex sp.02 1 1 1 pseudomyrmex sp.03 1 1 1 pseudomyrmex sp.04 2 2 2 pseudomyrmex sp.05 1 1 1 abundance 563 844 1334 2741 1127 427 425 1979 4720 species richness 32 51 59 36 29 41 subfamily / taxon cururu total reman total grand totalbeating manual collecting winkler extractor beating manual collecting winkler extractor sociobiology 67(3): 364-375 (september, 2020) 373 acknowledgements we thank nair aguiar for the invitation to participate in this biological survey and for his suggestions in the preliminary versions of this manuscript. to the reviewer, who provided useful suggestions for improving the quality of the manuscript. j.l.p.s. was supported by cnpq post-doctoral scholarship # 302301/2019-4. references aguiar, n.o., gualberto, t.l. & franklin, e. 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(2007). redução de esforço amostral vs. retenção de informação em inventários de pteridófitas na amazônia central. biota neotropica, 7: 217-223. doi: 10.1590/s1676-06032007000300023 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i3.5083sociobiology 67(3): 343-357 (september, 2020) introduction the brazilian atlantic forest is considered a global conservation hotspot (myers, 2000; mittermeier et al., 2005), as it is one of the richest biomes in biodiversity as well as one of the most threatened on the planet, since only 12.4% of forest remnants remain compared to the original coverage of 1,315,460 km2 in the 16th century (sos mata atlântica, 2018; inpe, 2018). with this significant reduction of its original coverage, the current landscape of the atlantic forest is composed by a mosaic of native vegetation fragments and crop areas, including forest remnants of over 3 hectares of different shapes and sizes, of these two main forest formations: abstract composed of two main forest formations, ombrophilous forest and seasonal forest, the brazilian atlantic forest biome is constituted currently by a mosaic of forest remnants and secondary vegetation. representatives of the ponerinae ant genus neoponera are observed mainly in both wet and seasonally dry forests. the aim of this study was to approach the diversity of the genus neoponera in the north of the atlantic forest of brazil (from the extreme north of its distribution to the doce river hydrographic basin in the south), associating the occurrence of ant species with the types of vegetation. we have compiled occurrence data from the collection of the myrmecology laboratory of the cocoa research center, on internet, or available in literature. we found information on 23 species of neoponera, including a new record for the atlantic forest, neoponera globularia (mackay & mackay, 2010), and a new record for brazil, neoponera fiebrigi forel, 1912. the relative composition of the neoponera assemblages was evaluated according to the types of vegetation. we found that the occurrence of the genus neoponera is mainly related to the types of vegetation of the focus region, principally dense forests where a higher diversity was observed. sociobiology an international journal on social insects ps silva1,2, eba koch2, a arnhold2,3, es araujo4, jhc delabie2,5, csf mariano4 article history edited by gilberto m. m. santos, uefs, brazil received 06 march 2020 initial acceptance 15 april 2020 final acceptance 23 june 2020 publication date 30 september 2020 keywords geographic distribution, species richness, forest fragmentation, forest formations, vegetation types, doce river. corresponding author priscila santos silva laboratory of myrmecology cocoa research center – cepec/ceplac caixa postal 7, cep: 45.600-970 itabuna, bahia, brasil. e-mail: priscilapitth@hotmail.com ombrophilous forest and seasonal forest (ribeiro et al., 2009; tabarelli et al., 2010; sos mata atlântica, 2018; inpe, 2018; mma, 2018). in perennial ombrophilous forests, the incidence of sunlight is low at the lower strata and the trees are tall; on the other hand, in semideciduous or deciduous seasonal forests, a considerable part of the foliage is lost during the dry season, which favors the penetration of sunlight until the floor, contributing to the formation of a more open vegetation structure with few epiphytes (pereira, 2009; colombo & joly, 2010). studies on the structure and composition of animal and plant communities are essential for the recovery and preservation of forest remnants (ferreira et al., 2018), as well as for the conservation of biological diversity. however, we 1 graduate program in ecology and biodiversity conservation, santa cruz state university (uesc), ilhéus, bahia, brazil 2 laboratory of myrmecology, cocoa research center, ceplac, itabuna, bahia, brazil 3 federal university of southern bahia (ufsb), training center in agroforestry sciences, ceplac, ilhéus, bahia, brazil 4 laboratory of social arthropods dcb/uesc, ilhéus, bahia, brazil 5 dcaa/uesc, ilhéus, bahia, brazil research article ants diversity of the ant genus neoponera emery, 1901 (formicidae: ponerinae) in the north of the brazilian atlantic forest, with new records of occurrence ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest344 must understand first the main patterns of their geographic distribution to carry out these studies (papes & gaubert, 2007; sigrist & carvalho, 2008). in view of their high diversity and sensitivity to changes in the physical and biological environment, the insects, especially the ants (hymenoptera: formicidae), are useful for such studies (santos et al., 2006; ribas et al., 2012; schmidt et al., 2013). some ants directly or indirectly control the availability of resources, changing the state of biotic or abiotic conditions for other organisms (jones et al., 1997). their role as ecosystem engineers, coupled with their abundance in terrestrial ecosystems, reveals the ecological importance of this group (folgarait, 1998). these ants contribute to structure the environment through soil ventilation and nutrient cycling, seed dispersal, mutualistic associations with plants and animals, on evolutionary as well as ecological scales (moreau et al., 2006; klimes et al., 2012; dejean et al., 2014). ponerinae is one of the most diverse groups within formicidae with regard to morphology and behavior. in this subfamily, neoponera is the second more genus in the neotropical region (after leptogenys) with 57 species (mackay & mackay, 2010; schmidt & shattuck, 2014), occurring from southern texas and northern mexico to northern argentina and southern brazil (mackay & mackay, 2010). to date, 35 species of neoponera have been recorded in brazil (feitosa, 2015), with representatives found throughout the country, preferably in humid forests, at ground level or in trees, but also in dry forests with seasonal rainfall (lattke, 2003; 2015). ants of the genus neoponera generally are much more frequent in conserved areas than in anthropized lands (campiolo et al., 2015). this fact strongly suggests that a set of information on the distribution of this group can help in monitoring environment quality and, furthermore, will provide valuable arguments to justify policies aiming at the implantation of conservation units. although the pattern of global ant diversity is similar to that of other taxa (for example, vascular plants), many regions have much less recorded diversity than expected. this can be related to several factors, including climate change and migration, emphasizing that regions with still unknown diversity are often the regions where deforestation is occurring most rapidly (guénard et al., 2012), such like the atlantic forest. the present study aimed to analyze the current diversity for the neoponera genus in the northern part of the brazilian atlantic forest biome, inserting new records, evaluating the richness and the association of neoponera species with different forest formations (types of vegetation) within the biome. material and methods study area the atlantic forest covers an area of approximately 1,110,182 km² (ibge, 2004a), being composed of the two main formations: coastal forest (perennials ombrophilous forests) and tropical seasonal forest (morellato & haddad, 2000), subdivided into different vegetation types according to the classification of the brazilian institute of geography and statistics (ibge) (fig 1/table1), such as the: pioneer formation areas under marine and fluvial influence (pf), pioneer formation under marine influence (pm), pioneer formations with agricultural activities (aa.p), savannah/seasonal forest with agricultural activities (aa.sn), savannah/ombrophilous forest with agricultural activities (aa.so), dense ombrophilous forest of lowlands (db), savannah/ombrophilous forest (so), open ombrophilous vegetation secondary vegetation and agrarian activities (vs-aa.a), seasonal deciduous forest secondary vegetation and agrarian activities (vsaa.c), dense ombrophilous forest secondary vegetation and agrarian activities (vs-aa.d) and semideciduous seasonal forest (secondary vegetation and agrarian activities) (vs.aa.f). in addition to associated ecosystems such as mangroves, sea shore vegetation, highland fields, inland swamps and forest entrances in the northeast, housing thousands of species of plants and animals (ribeiro et al., 2009; tabarelli et al., 2010; sos mata atlântica, 2018; mma, 2018). we defined the doce river as the southern geographic boundary to outline the studied area, which is the northern part of the brazilian atlantic forest (fig 1). the doce river fig 1. map of the study area. sociobiology 67(3): 343-357 (september, 2020) 345 is one of the larger brazilian rivers and its hydrographic basin presents a worrying picture of environmental degradation, since it is within the limits of two global biodiversity hotspots, 98% of its area is in the atlantic forest and the remaining 2% in the cerrado (mittermeier et al., 2005; azevedo-santos et al., 2016; pires et al., 2017). the doce river flows 888 km and its basin has an area of about 84,000 km2, of which 86% are in the state of minas gerais and 14% in that of espírito santo. it is considered as an effective biogeographical barrier at least since the pleistocene (carnaval & moritz, 2008). thus, the analyzed area covers from the southeast of brazil, from the north of espírito santo and part of minas gerais, to the northeast of the country, and includes the states of bahia, sergipe, alagoas, pernambuco, paraíba and rio grande do norte. in the northeast, the biome is limited to the caatinga and in the southeast, to the cerrado (pereira, 2009). in general, it presents precipitation index above 1,000 mm3 per year and several climatic types, such as humid tropical, hot and super humid, tropical altitudinal and mesothermal climates according to the köppen-geiger classification (pereira, 2009). data collection neoponera species data were set up from the myrmecology laboratory collection of the cocoa research center (cpdc), at ilhéus-ba, brazil, looking for information on specimen collection locations and geographic coordinates. occurrence data were also compiled from the online data networks antweb.org (accessed on 2018/2019) and antmaps. org (accessed on 2018/2019). another search was performed based on literature, using the species names as keywords. we consider the possibility that the species was registered with an outdated name, especially when included in the genus pachychondyla before the revision of this genus by schmidt and shattuck (2014). we reviewed information about these species across brazil and, subsequently, analyze their distribution in the north of the brazilian atlantic forest biome to compile the list of species and data (reference codes are available in table 5). statistical analysis we built a spreadsheet with data compiled for the occurrence of species recorded in different locations in the north of the atlantic forest biome. we assessed the structure of neoponera assemblages according to the types of vegetation (table 1; fig 1). for that purpose, we evaluated the similarity of neoponera assemblages (species presence/absence) according the vegetation types using the jaccard’s index. in addition, the association between species and vegetation types was illustrated using a multidimensional non-metric ordering (nmds) using the bray-curtis index. these analyzes were performed using the software r v. 3.6.1(r development core team 2019). description of classified vegetation classification legend pioneer formations under marine and fluvial influence pf pioneer formations under marine influence pm pioneer formations with agriculture aa.p savanna/seasonal forest with agriculture aa.sn savanna/ombrophilous forest with agriculture aa.so dense ombrophilous forest of lowlands db savanna/ombrophilous forest so open ombrophilous vegetation (secondary vegetation and agriculture) vs-aa.a seasonal deciduous forest (secondary vegetation and agriculture) vs-aa.c dense ombrophilous forest (secondary vegetation and agriculture) vs-aa.d semideciduous seasonal forest (secondary vegetation and agriculture) vs-aa.f source: vegetation map of brazil (ibge, 2004b available online at http:// www.ibge. gov.br). table 1. description and classification of vegetation types currently found in the atlantic forest in the studied region, brazil. results the total number of records of the survey comprises 23 species (table 2) of neoponera, distributed in the atlantic forest above the geographical boundary of doce river (tables 3 and 4). between these, neoponera globularia (mackay & mackay, 2010) is a new record for the atlantic forest, and neoponera fiebrigi forel, 1912 a new record for brazil. we also found records of neoponera goeldii forel, 1912 (species previously known from the amazonian biome only) in the state of bahia at least at 1,500 km further east from the nearest record. the species recorded in the higher number of vegetation types (table 2) were neoponera apicalis (latreille, 1802) (recorded in seven vegetation types, 63.6%), neoponera villosa (fabricius, 1804) and neoponera curvinodis (forel, 1899) (five vegetation types, 45.4% each). among the other species, seven were recorded in a single type of vegetation. the species with the highest number of occurrences by type of vegetation were n. apicalis (48 records), neoponera concava (mackay & mackay, 2010) (52), neoponera bucki (borgmeier, 1927) (18), and neoponera inversa (smith, 1858) (14), all for the vegetation vs-aa.d (table 2). the types of vegetation that presented a higher number of species of neoponera were: vs-aa.d (19 spp.), vs-aa.c (10) vs-aa.f (7) and so (6). the number of species in the other types of vegetation varied from one to three (fig 2). three vegetation types presented only a single species: vsaa.a and db (n. apicalis) and aa-sn (n. villosa) (fig 2). ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest346 when we test the similarity between vegetation types according to the presence/absence of neoponera species, we observed that db and vs-aa.a share the same assemblages (fig 3). in general, the other types of vegetation present low similarity between them (less than 50%), especially vsaa.d and vs-aa.c (about 40% of similarity). it is worth noting that the assemblages found in vegetation types pf and aa.p (38% similarity between them) have low similarity with the other vegetation types (fig 3). the relative superposition of certain vegetation types with some neoponera is observed on nmds graph (fig 4). the grouping with the species found in the same vegetation types is evident, such as n. globularia, n. goeldii, neoponera laevigata (smith, 1858), neoponera schultzi (mackay & mackay, 2010), neoponera venusta forel, 1912 occurring in vs-aa.d. the same occurs for n. fiebrigi, neoponera marginata (roger, 1861) and neoponera moesta (mayr, 1870) in vs-aa.c, as well as for neoponera carinulata (roger, 1861) in pm. the greatest distance was observed for neoponera species vegetation types aa.p aa.sn aa.so db pf pm so vs-aa.a vs-aa.c vs-aa.d vs-aa.f neoponera apicalis (latreille, 1802) 1 1 1 1 2 38 4 neoponera bactronica (fernandes, de oliveira & delabie, 2014) 1 1 5 neoponera bucki (borgmeier, 1927) 1 16 1 neoponera carinulata (roger, 1861) 1 4 neoponera cavinodis mann, 1916 1 1 neoponera concava (mackay, w.p. & mackay, e.e., 2010) 5 42 5 neoponera crenata (roger, 1861) 1 2 neoponera curvinodis (forel, 1899) 1 1 2 2 3 neoponera fiebrigi forel, 1912 2 neoponera globularia (mackay, w.p. & mackay, e.e., 2010) 2 neoponera goeldii forel, 1912 1 neoponera inversa (smith, f., 1858) 2 3 9 neoponera laevigata (smith, f., 1858) 2 neoponera magnifica (borgmeier, 1929) 1 1 1 neoponera marginata (roger, 1861) 1 neoponera moesta mayr, 1870 2 neoponera obscuricornis (emery, 1890) 1 neoponera schultzi (mackay, w.p. & mackay, e.e., 2010) 3 neoponera striatinodis emery, 1890 1 1 neoponera unidentata mayr, 1862 1 1 2 4 neoponera venusta forel, 1912 3 neoponera verenae (forel, 1922) 1 5 neoponera villosa (fabricius, 1804) 1 1 2 4 1 table 2. list of neoponera species recorded in different vegetation types in the brazilian atlantic forest biome. the values represent the number of records. fig 2. neoponera diversity according to types of vegetation of the brazilian atlantic forest biome. the codes on the x axis correspond to the types of vegetation listed in table 1. sociobiology 67(3): 343-357 (september, 2020) 347 obscuricornis (emery, 1890), an exclusive species of aa.so (fig 4). some species, although found in more than a single type of vegetation, were more frequent with one or two certain types, for example, neoponera cavinodis mann, 1916 with n. villosa, neoponera magnifica (borgmeier, 1929) and neoponera bactronica (fernandes, oliveira & delabie, 2014) with vs-aa.c and aa.sn. in turn, neoponera verenae forel, 1922, neoponera striatinodis (emery, 1890) and neoponera crenata (roger, 1861) group together more with vs-aa.f and aa.p (fig 4). fig 3. dendrogram of similarity (jaccard’s distance) comparing the vegetation types of the atlantic forest biome, according to the assemblages of species of the genus neoponera. fig 4. non-metric multidimensional scaling (nmds) of vegetation types in the brazilian atlantic forest based on neoponera frequency of occurrence. circles filled with the same color = exclusive occurrence in the type of vegetation with equivalent color. ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest348 species code neoponera apicalis 2, 7, 10, 11, 12, 14, 15, 19, 24, 25, 28, 29, 35, 38, 39, 42, 43, 46, 47, 48, 57, 59, 64, 66, 67, 72, 73, 74, 76, 77, 78, 79, 82, 88 neoponera bactronica 5, 24, 32, 41, 61, 63, 92 neoponera bucki 1, 3, 7, 10, 22, 26, 27, 28, 37, 50, 55, 74, 76, 80, 83 neoponera carinulata 10, 29, 64, 78, 83 neoponera cavinodis 13, 29 neoponera concava 1, 2, 3, 9, 10, 12, 13, 15, 23, 25, 26, 29, 33, 37, 40, 46, 55, 56, 66, 68, 71, 75, 77, 78 neoponera crenata 4, 31, 85 neoponera curvinodis 18, 24, 29, 34, 44, 49, 81, 90, 91 neoponera fiebrigi* 8, 81 neoponera globularia** 6, 32 neoponera goeldii 33 neoponera inversa 9, 13, 21, 24, 26, 29, 37, 53, 55, 68, 77, 78 neoponera laevigata 29, 78 neoponera magnifica 4, 17, 81 neoponera marginata 62 neoponera moesta 4, 81 neoponera obscuricornis 7 neoponera schultzi 43, 51 neoponera striatinodis 45, 86 neoponera unidentata 7, 20, 23, 54, 65, 69, 70, 77 neoponera venusta 29, 77, 83 neoponera verenae 16, 24, 46, 52, 80, 90 neoponera villosa 30, 36, 46, 50, 58, 83, 84, 87, 89 table 4. list of neoponera species reported from the study area. the codes of localities follow table 3. (*) new record for brazil, (**) new record for atlantic forest. discussion so far 35 species were recorded for the neoponera genus in brazil (feitosa, 2015). with this survey and insertion of new records and occurrences, this number can be updated to 36 species, 23 of which are found in the northern part of the atlantic forest biome. thus, n. fiebrigi, previously registered only in paraguay, argentina (mackay & mackay 2010) and panama (schmidt & schattuck, 2014), constitutes a new record for brazil and the atlantic forest. we observed that the occurrence of many species of the genus neoponera in the brazilian atlantic forest is related to dense vegetation, such like secondary forests or vegetation at climax. these environments offer a higher number of records and species richness than opened environments. the same was observed by campiolo et al. (2015) in a study that points out the ponerinae preferences for forest-type habitats with dense vegetation cover. these characteristics results from the particular habitat requirements of each species of the genus. for example, n. apicalis, n. villosa, n. carinulata, n. crenata, n. curvinodis and n. inversa are arboreal (mackay & mackay, 2010). some species can also nest in undergrowth, trunks and hollow branches fallen on the ground, where moisture and shade conditions are suitable, such as for example, n. verenae (delabie et al., 2008, araujo et al., 2019). the species recorded in a higher number of vegetation types, n. apicalis, is known to occur in both primary and secondary moist forests (mackay & mackay, 2010). however, delabie et al. (2008) suggest that the apicalis group of neoponera (sensu wild, 2005) is in fact a complex of at least nine cryptic species, including n. apicalis, n. obscuricornis and n. verenae (ferreira et al., 2010), which could explain the range of habitats chosen by the ants. the type of vegetation with the higher diversity of neoponera was the ombrophilous dense forest. this kind of forests presents moist soil covered by a thick layer of leaf-litter (pereira, 2009). because it uses to be more heterogeneous in diversified environments, the leaf-litter offers certainly a range of niches and, as a result, a well-diversified community of soil fauna; in addition, we can expect that the greater the amount of leaf-litter, the greater the availability of resources, such as food and nesting sites (santos et al., 2006; ferreira et al., 2018). however, the seasonal deciduous and semideciduous forests, which occur in places with 2 to 5 months of dry season (colombo & joly, 2010), also showed a relatively high species richness. lattke (2003) had already pointed out that some neoponera species are able to colonize dry forest areas with a marked rainfall regime. our observations point out some particularities such as the occurrence of n. goeldii in the atlantic forest biome, with an extremely reduced population living isolated in the península de maraú, bahia (j.h.c. delabie, personal communication, december, 2019), while this species is common in amazon (mackay & mackay, 2010). in fact, there are numerous physiographic, floristic, fauna and climatic similarities between the atlantic forest biome, in the coastal strip which runs from the south of the state of bahia to the north of the state of espírito santo (“central corridor of the mata atlântica”), and the amazon forest (pereira, 2009). according to joly et al. (1999), the occurrence of species typical of the amazon region in the atlantic forest of southern bahia confirms that these biomes have undergone expansion and retraction processes during the pleistocene climatic fluctuations. in other words, at one or several intervals in this period, transition bridges occurred allowing the arrival of typically amazonian species in the atlantic forest biome (ab’saber, 2003; costa, 2003). the ant species neoponera globularia, n. goeldii, n. laevigata, n. schultzi and n. venusta follow the same patterns of geographical distribution in the atlantic forest, occupying exclusively areas of ombrophilous dense forest. the same occurs with n fiebrigi, n. marginata and n. moesta, in the seasonal deciduous forest. neoponera schultzi and n. venusta, sociobiology 67(3): 343-357 (september, 2020) 349 state county coordinate vegetation type code alagoas quebrângulo 9.3167s, 36.4667w vs-aa.f 1 9.322s, 36.476w vs-aa.f bahia arataca 15.2195s, 39.4243w vs-aa.d 2 15.2803s, 39.3919w vs-aa.d aurelino leal 14.3311s, 39.3589w vs-aa.d 3 14.3679s, 39.4657w vs-aa.d 14.3828s, 39.4156w vs-aa.d barra do choça 14.8081s, 40.5897w vs-aa.d 4 14.8333s, 40.5536w vs-aa.d 14.8659s, 40.5779w vs-aa.d barra do rocha 14.2068s, 39.6031w vs-aa.c 5 barro preto 14.8097s, 39.4233w vs-aa.d 6 belmonte 16.0951s, 39.2745w vs-aa.d 7 16.1333s, 39.25w aa.so 16.1s, 39.2833w aa.so boa nova 14.3656s, 40.2075w aa.so 8 buerarema 14.6333s, 39.8833w vs-aa.d 9 14.7583s, 39.2411w vs-aa.c 15.0144s, 39.2999w vs-aa.f camacan 15.3833s, 39.55w vs-aa.d 10 15.4011s, 39.5664w vs-aa.d 15.4167s, 39.4833w vs-aa.d 15.4201s, 39.4964w vs-aa.d 15.4573s, 39.4516w vs-aa.d 15.5006s, 39.2206w vs-aa.d 15.5036s, 39.5156w vs-aa.d 15.6011s, 39.5211w vs-aa.d camaçari 12.6972s, 38.3332w vs-aa.d 11 camamu 13.9443s, 39.1046w vs-aa.d 12 14.0142s, 39.1667w vs-aa.d 14.1369s, 39.2775w vs-aa.d canavieiras 14.4094s, 39.0337w vs-aa.c 13 15.6752s, 38.9969w vs-aa.d 15.6775s, 39.9783w vs-aa.d caravelas 17.6794s, 39.6105w pm 14 cruz das almas 12.6736s, 39.1017w vs-aa.f 15 12.6799s, 39.0891w vs-aa.d ecoporanga 18.3709s, 40.8329w vs-aa.f 16 esplanada 12.1144s, 37.6969w vs-aa.d 17 estância 11.2687s, 37.4385w so 18 eunápolis 16.372s, 39.5825w vs-aa.f 19 firmino alves 14.9247s, 39.9196w vs-aa.d 20 floresta azul 14.8761s, 39.6931w vs-aa.c 21 gongogi 14.2742s, 39.4842w vs-aa.d 22 15.2742s, 39.4842w vs-aa.d governador lomanto junior 14.8158s, 39.4839w vs-aa.d 23 guaratinga 16.5625s, 39.899w vs-aa.d 24 16.5867s, 39.7753w vs-aa.d 16.5867s, 39.7808w vs-aa.d 16.6286s, 39.7983w vs-aa.d ibicaraí 14.8583s, 39.5918w vs-aa.d 25 14.9042s, 39.4836w vs-aa.f ibirapitanga 14.0709s, 39.4243w vs-aa.d 26 table 3. information from the sampled sites. the abbreviations vegetation type follows those reported in table 1. ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest350 14.1942s, 39.4231w vs-aa.f igrapiúna 13.8448s, 39.1127w vs-aa.d 27 iguaí 14.6416s, 39.198w vs-aa.d 28 14.6439s, 40.1533w vs-aa.d ilhéus 14.255s, 39.2314w vs-aa.d 29 14.4274s, 39.5705w vs-aa.d 14.5006s, 39.0676w vs-aa.d 14.5294s, 39.0661w vs-aa.d 14.5533s, 39.4275w vs-aa.d 14.6207s, 39.1397w vs-aa.d 14.6808s, 39.2567w vs-aa.d 14.6935s, 39.0966w vs-aa.d 14.7422s, 39.1056w vs-aa.d 14.755s, 39.2314w vs-aa.d 14.7561s, 39.2314w vs-aa.d 14.7813s, 39.0795w vs-aa.d 14.7935s, 39.0774w vs-aa.d 14.7961s, 39.211w vs-aa.d 14.7972s, 39.0798w vs-aa.d 14.798s, 39.1722w vs-aa.d 14.8239s, 39.1w vs-aa.d 14.9197s, 39.1997w vs-aa.d 14.9504s, 39.0631w vs-aa.f 14.9903s, 39.0583w vs-aa.f ipiaú 14.1349s, 39.7386w vs-aa.d 30 itabira 19.7501s, 43.2323w vs-aa.d 31 itabuna 14.7772s, 39.3674w vs-aa.d 32 14.78s, 39.2784w vs-aa.d itacaré 14.2794s, 39.4852w vs-aa.f 33 14.3092s, 39.0194w vs-aa.d 14.3568s, 39.1752w vs-aa.f itagi 14.2329s, 39.8579w vs-aa.c 34 itagibá 14.233s, 39.8579w vs-aa.d 35 itaju do colônia 15.1652s, 39.7756w vs-aa.d 36 itajuípe 14.6757s, 39.3725w vs-aa.d 37 14.7033s, 39.4981w vs-aa.d itamaraju 16.8675s, 39.9175w vs-aa.d 38 16.9167s, 39.2667w vs-aa.d 16.9917s, 39.4553w vs-aa.d 17.0382s, 39.5389w vs-aa.d itamari 13.7273s, 39.6312w vs-aa.d 39 itambé 14.6519s, 40.3397w vs-aa.f 40 itapebi 15.9693s, 39.5321w vs-aa.d 41 itapetinga 15.2461s, 39.9403w vs-aa.d 42 itapitanga 14.4228s, 39.565w vs-aa.d 43 14.4319s, 39.565w vs-aa.f 14.5108s, 39.6105w vs-aa.d itaquara 13.4537s, 39.8785w vs-aa.d 44 itati 13.9572s, 40.0308w vs-aa.c 45 itororó 14.9586s, 39.0643w vs-aa.c 46 14.9586s, 40.0425w vs-aa.f 14.9614s, 40.0425w vs-aa.d 14.9778s, 40.0364w vs-aa.d table 3. information from the sampled sites. the abbreviations vegetation type follows those reported in table 1. (continuation) state county coordinate vegetation type code sociobiology 67(3): 343-357 (september, 2020) 351 15.1185s, 40.0661w vs-aa.d 15.4744s, 40.0503w vs-aa.d ituberá 13.736s, 39.1466w vs-aa.c 47 jaguaripe 13.1956s, 39.0239w vs-aa.d 48 jequié 13.8591s, 40.0838w aa.sn 49 jiquiriçá 13.3193s, 39.5899w vs-aa.d 50 jussari 15.1406s, 39.5247w vs-aa.d 51 15.1535s, 39.5167w aa.p laje 13.1761s, 39.3414w pm 52 lauro de freitas 12.8642s, 38.2697w vs-aa.d 53 macarani 15.5303s, 40.3905w vs-aa.d 54 maraú 14.1503s, 39.1127w so 55 mascote 13.7189s, 39.41w vs-aa.d 56 15.5636s, 39.3094w vs-aa.d 15.5772s, 39.41w so 15.6969s, 39.445w vs-aa.d 15.7344s, 39.3844w vs-aa.d mucuri 18.0825s, 39.8909w vs-aa.d 57 mutuípe 13.2288s, 39.5047w vs-aa.d 58 nazaré 13.0399s, 39.0034w vs-aa.d 59 nilo peçanha 13.6494s, 39.2103w vs-aa.d 60 pau brasil 15.4897s, 39.6931w vs-aa.d 61 planalto 14.6991s, 40.4772w vs-aa.c 62 poções 14.6147s, 40.3411w vs-aa.c 63 porto seguro 15.6694s, 38.9942w pm 64 16.3883s, 39.1814w db 16.4444s, 39.0984w vs-aa.d pratas 15.1956s, 39.4453w vs-aa.d 65 presidente tancredo neves 13.3911s, 39.3183w vs-aa.d 66 salvador 12.9292s, 38.5014w vs-aa.d 67 12.9722s, 38.5014w vs-aa.d santa luzia 15.3897s, 39.305w vs-aa.d 68 15.4233s, 39.2791w vs-aa.d santo amaro 12.5465s, 38.7111w vs-aa.d 69 são francisco do conde 12.6655s, 38.59w vs-aa.d 70 são josé da vitória 15.0517s, 39.3133w vs-aa.d 71 15.0617s, 39.3442w vs-aa.d simões filho 12.7701s, 38.4219w vs-aa.d 72 12.7717s, 39.5219w vs-aa.c teixeira de freitas 17.54s, 39.7422w vs-aa.d 73 ubaíra 13.1192s, 39.6594w vs-aa.d 74 ubaitaba 14.2503s, 39.3214w vs-aa.d 75 14.2503s, 39.3242w vs-aa.d 14.3089s, 39.3226w vs-aa.d 14.4247s, 39.3233w vs-aa.d ubatã 14.0699s, 39.5278w vs-aa.d 76 14.2256s, 39.4656w vs-aa.d una 15.0892s, 39.295w vs-aa.d 77 15.177s, 39.1055w vs-aa.d 15.1844s, 39.0546w so 15.2028s, 39.0531w so 15.2091s, 39.196w vs-aa.d 15.2336s, 39.1844w vs-aa.d table 3. information from the sampled sites. the abbreviations vegetation type follows those reported in table 1. (continuation) state county coordinate vegetation type code ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest352 15.2617s, 39.1533w so 15.2792s, 39.0414w so 15.2792s, 39.0914w so 15.2795s, 39.0769w so 15.2808s, 39.089w so 15.2858s, 39.1175w so 15.3897s, 39.1975w so uruçuca 14.4514s, 39.0478w vs-aa.d 78 14.4649s, 39.0532w vs-aa.d 14.465s, 39.0567w vs-aa.d 14.5125s, 39.2003w vs-aa.d 14.5155s, 39.2999w vs-aa.d 14.5653s, 39.2739w vs-aa.d 14.6s, 39.2667w vs-aa.d valença 13.3422s, 39.1953w vs-aa.d 79 13.3709s, 39.0759w vs-aa.d vera cruz 13.0229s, 38.7159w vs-aa.d 80 vitória da conquista 14.7936s, 40.7231w vs-aa.c 81 14.8411s, 40.8389w vs-aa.c 14.8619s, 40.8445w vs-aa.c 14.8892s, 40.8034w vs-aa.d 15.0392s, 40.9097w vs-aa.c wenceslau guimarães 13.5539s, 39.7019w vs-aa.d 82 13.5832s, 39.6931w vs-aa.d espírito santo linhares 19.1514s, 40.0708w vs-aa.c 83 19.15s, 40.05w vs-aa.d 19.3947s, 40.0653w vs-aa.d 19.6455s, 39.855w vs-aa.d são mateus 18.7002s, 40.0633w vs-aa.d 84 minas gerais guanhães 18.7667s, 42.9167w vs-aa.d 85 timóteo 19.5816s, 42.6475w vs-aa.f 86 paraíba joão pessoa 7.1195s, 34.855w vs-aa.d 87 pernambuco são lourenço da mata 7.9s, 35.0833w vs-aa.a 88 rio grande do norte parnamirim 5.9072s, 35.1984w pm 89 sergipe laranjeiras 10.8122s, 37.1711w vs-aa.d 90 santa luzia do itanhy 11.419s, 37.4284w aa.p 91 são cristóvão 10.9238s, 37.1019w pf 92 table 3. information from the sampled sites. the abbreviations vegetation type follows those reported in table 1. (continuation) together with n. concava, which likewise predominates in dense forest, are endemic from brazil (mackay & mackay, 2010). in the atlantic forest, four areas of endemism are reported (silva et al., 2004; sigrist & carvalho, 2008; peres et al., 2020), and three of which are considered in our area of study: i) central bahia, ii) central corridor of the atlantic forest, and iii) pernambuco. these are biogeographic regions supported by several taxonomic groups, being a valid representation of regionalization for studies and conservation of biodiversity (peres et al., 2020). the neoponera data obtained in our study basically comprise records for the central corridor of the atlantic forest, an area of endemism that extends from the south of the state of bahia to the north of espírito santo, with n. schultzi being an endemic species of that region. thus, we understand that the occurrences of ants reinforce the importance of the conservation of these ants in the central corridor of the atlantic forest. some studies carried out in the atlantic forest (santos et al., 2006; silva et al., 2007) have shown that ant communities use to be affected by anthropogenic disturbances. it is expected that in areas where landscape fragmentation is dominant and land use is excessive and disordered, many species will not be able to face climatic changes and migrate at a sufficient rate to maintain their population (pearson & dawson, 2003). state county coordinate vegetation type code sociobiology 67(3): 343-357 (september, 2020) 353 table 5. references of the data sources used in the study. species state references neoponera apicalis bahia 1, 3, 9, 11, 14, 19, 22, 23, 25, 26, 31, 34 pernambuco 33 neoponera bactronica bahia 1, 5, 12, 13, 15, 35 sergipe 1, 13 neoponera bucki alagoas 10, 36 bahia 1, 34 espírito santo 2, 36 neoponera carinulata bahia 1, 23, 25, 34 espírito santo 1 neoponera cavinodis bahia 1, 6, 34 neoponera concava alagoas 1 bahia 1, 6, 26, 28, 32, 34 espírito santo 2 sergipe 1 neoponera crenata alagoas 10 bahia 1, 2, 5, 7, 15, 23, 24, 25, 27, 34 minas gerais 1 neoponera curvinodis bahia 1, 3, 7, 13, 25, 27, 34, 35 sergipe 1, 13 neoponera fiebrigi bahia 1 neoponera globularia bahia 1 neoponera goeldii bahia 1, 18, 21 neoponera inversa alagoas 10 bahia 1, 3, 5, 7, 8, 10, 13, 15, 17, 20, 25, 35, 37 espírito santo 13 neoponera laevigata bahia 1, 28 neoponera magnifica bahia 1, 15, 26 neoponera marginata bahia 1, 34 neoponera moesta bahia 1, 7, 8, 15, 24, 25, 34 neoponera obscuricornis bahia 1, 31 neoponera schultzi bahia 1, 34 neoponera striatinodis bahia 1, 19, 34 minas gerais 1 neoponera unidentata bahia 1, 5, 7, 11, 14, 15, 21, 22, 23, 25 neoponera venusta alagoas 30 bahia 1, 4, 9, 11, 14, 21, 25, 28, 30, 31 espírito santo 1, 2, 11, 36 neoponera verenae bahia 1, 3, 11, 19, 25, 26, 30, 34 espírito santo 1, 2 sergipe 1, 16 neoponera villosa bahia 1, 5, 7, 8, 9, 12, 13, 14, 20, 23, 25, 29, 31, 34, 35 espírito santo 1, 13, 35 paraíba 1, 13 rio grande do norte 1 references corresponding to the codes are available in the annex. nevertheless, we observed here that the vegetation types with the higher diversity of neoponera correspond to landscapes with secondary forests in association with agriculture. this has important implications for the future of the biome conservation, since it suggests that secondary forests conserve an important pool of species that, face to the climatic changes, will contribute to the genus resilience in the region. since ants are organisms faithful for a specific type of habitat, and may allow inferences about the rehabilitation of an area (schmidt et al., 2013), the occurrence of some neoponera in more than a single type of vegetation, like n. apicalis, n. villosa, n. bucki, n. curvinodis, neoponera unidentata (mayr, 1862) and n. inversa (fig 4), strongly suggest the connectivity between vegetation types. thus, the conservation of forest ps silva, eba koch , a arnhold, es araujo, jhc delabie, csf mariano – diversity of the neoponera in the north of the brazilian atlantic forest354 remnants and even some kinds of agriculture which maintains a forest structure (cocoa agroforestry, for example) can be decisive for the conservation of this group of ants (delabie et al., 2007; campiolo et al., 2015). further studies should evaluate the relationships between the occurrence and distribution of the species of neoponera and the habitat conservation including also the other areas covered by the biome. acknowledgments this study is part of the requirements for the obtention of the first author’s master degree. thanks are due to the programa de pós-graduação em zoologia of state university of santa cruz (uesc). this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) – finance code 001. pss acknowledges her study grant from capes; jhcd and csfm thank cnpq (conselho nacional de desenvolvimento científico e tecnológico) for their research grants. author contributions all authors conceived this study, pss and ea conducted performed research/acquisition of data. e.b.a.k performed the data analyses. all authors contributed to the writing, discussed the results and commented on the manuscript. references ab’sáber, a. n. 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(ascomycota: laboulbeniales) in the carpathian basin fungi of the laboulbeniales order are obligate ectoparasites of different arthropod taxa, mainly of coleoptera (santamaria, 2001; henk et al., 2003). six described laboulbeniales species infect ants, but only four of these have been found in europe (haelewaters et al., 2015a, santamaria & espadaler, 2015, d. haelewaters, pers. comm.): laboulbenia formicarum thaxt. is found in france, portugal and spain on two lasius species; laboulbenia camponoti s.w.t. batra in bulgaria and spain on five camponotus species; rickia wasmannii cavara in 17 countries on nine myrmica species; and rickia lenoirii santam. in greece on messor wasmanni krausse, 1910 and in france on messor structor (latreille, 1798). at the moment, only r. wasmannii and l. camponoti have been reported among these four laboulbeniales species in the carpathian basin (tartally et al., 2007; csata et al., 2013; báthori et al., 2014; tartally & báthori, 2015). as m. structor is widely distributed in the carpathian basin (schlick-steiner et al., 2006), the potential presence of r. lenoirii on this host ant species in this region was plausible, abstract rickia lenoirii has been reported in seven localities in the carpathian basin, six in hungary and one in romania, on messor structor (hymenoptera: formicidae) host specimens. this is the first occurrence of this fungus in two new (pannonian and continental) biogeographic regions. according to our findings, the northernmost (47°31’33.01”n) known occurrence of r. lenoirii is ferenc-hegy (ferenc hill) in budapest. sociobiology an international journal on social insects f báthori1, wp pfliegler2,3, a tartally1 article history edited by evandro do nascimento silva, uefs, brazil received: 17 august, 2015 initial acceptance 05 november, 2015 final acceptance 23 december, 2015 keywords formicidae, harvester ant, hungary, messor structor, romania corresponding author andrás tartally department of evolutionary zoology and human biology, university of debrecen egyetem tér 1, h-4032 debrecen, hungary e-mail: tartally.andras@science.unideb.hu especially as r. lenoirii is a recently described and extremely small species (santamaria & espadaler, 2015) that could easily escape notice. it should be noted that only m. structor and no other messor harvester ant species are distributed in the carpathian basin (schlick-steiner et al., 2006). thus, our aim was to investigate the presence of r. lenoirii within the carpathian basin by checking museum specimens of m. structor collected from several parts of this region. in order to discover the presence of r. lenoirii, all the m. structor specimens collected in the carpathian basin and deposited in the hymenoptera collection of the hungarian natural history museum were checked under an olympus szx9 stereomicroscope between 12.6-114x magnifications. in total, 499 m. structor specimens (428 workers, 28 males, and 43 queens) were examined, originating in 44 localities of the carpathian basin (35 sites in hungary, 6 in romania and 3 in slovakia; see supplementary file for details, available at http://periodicos. uefs.br/ojs/index.php/sociobiology/rt/suppfiles/901/0 and doi: 10.13102/sociobiology.v62i4.901.s1079). short note 1department of evolutionary zoology and human biology, university of debrecen, debrecen, hungary 2department of biotechnology and microbiology, university of debrecen, debrecen, hungary 3postdoctoral fellowship programme of the hungarian academy of sciences (mta), hungary sociobiology 62(4): 620-622 (december, 2015) 621 pinned host specimens with r. lenoirii thalli we resoaked in 70% ethanol overnight. thalli were removed with an insect pin and prepared onto slides in heinz-pva. microscopy images were taken with an olympus bd40 microscope equipped with a 100x lens and focus-stacked. specimens were compared with the original description and diagnostic characters (santamaria & espadaler, 2015) and determined on the basis of thallus form, size and cell number, and shapes of antheridia and perithecium. thirty specimens (6.0% of the investigated individuals) of m structor specimens were found to be parasitized by r. lenoirii (fig 1) at six hungarian sites (28 workers) and one romanian one (2 workers) (see details in the supplementary file). thus, r. lenoirii infection was recorded in 15.9% of the investigated sites in total. fungi were recorded on the legs and antennae of the hosts. as noted in the original description of r. lenoirii (santamaria & espadaler, 2015), deterioration and reduction of antheridia to amorphous secondary appendagelike structures was observable. brown trichogyne scars were visible on all photographed specimens. the tip of perithecia were in some cases less markedly truncated than in the original description (fig 1). no other messor species, as potential hosts, were recorded in the carpathian basin in the course of our research. rickia lenoirii specimens are deposited in the fungi collection of the hungarian natural history museum on permanent slides (inventory numbers: 107653-107659). our findings represent the first records of r. lenoirii since its recent description (santamaria & espadaler, 2015). this is the third ant-parasitic laboulbeniales species recorded in the carpathian basin (see section 1). the number of countries in which this fungus has been found thus has doubled, as at the time of its description it was known only from greece and france (santamaria & espadaler, 2015). now, r. lenoirii has been recorded in hungary and romania. furthermore, ferenchegy (ferenc hill), in budapest, is now the northernmost (47°31’33.01”n) known occurrence of r. lenoirii. the discovery of r. lenoirii on m. structor host specimens does not constitute the discovery of a new host ant species, but it should be noted that m. structor has cryptic species/lineages (seifert, 2007; schlick-steiner et al., 2006). it would be interesting to know whether each lineage is susceptible to r. lenoirii. similarly, it would be worth checking the presence of this fungus on the numerous other known (sub) species of messor harvester ants (see details in santamaria & espadaler, 2015) and comparing different populations of the fungus. as noted in the original description, thalli from the two known host specimens seemed to be slightly different in size, and the specimens recorded in the course of our work showed some variation in perithecium shape. rickia lenoirii has been found in the mediterranean biogeographic region (santamaria & espadaler, 2015), and it has now also been discovered in the pannonian and the continental regions (compare supplementary file with eea, 2012), where the host messor structor lives in xerothermic grasslands with rich seed vegetation (seifert, 2007). however, as discussed by santamaria and espadaler (2015), a relatively high level of humidity could promote r. lenoirii infection, as with other laboulbeniales fungi. our records seem to confirm this suggestion on a broad scale, as all the sites where this fungus has been found are close to big bodies of water (lake balaton or the danube river; see supplementary file). the danube river could even be a corridor of r. lenoirii, connecting the carpathian basin with the black sea. new research focusing on this recently described parasitic fungus would probably uncover new occurrences across europe. recent research has shown that ant parasitic r. wasmannii and l. formicarum have effects on their ant hosts (csata et al., 2014; báthori et al., 2015; konrad et al., 2015; pech & heneberg, 2015). similar experiments on r. lenoirii could contribute to a deeper understanding of the interactions of laboulbeniales fungi with their hosts. however, the main aim of this inquiry is to call the attention of mycologists and myrmecologists to these small but interesting fungi and to the importance of museum collections (see suarez and tsutsui, 2004, haelewaters et al., 2015b). fig 1. rickia lenoirii thalli (a: farkasrét, b: ferenc-hegy, c: balatonfüred, d: badacsony, e: révfülöp, f: herkulesfürdő) and a part of an infected leg of a messor structor host ant (g: budapest), recorded in the carpathian basin (see localities and their names in the supplementary file) f báthori, wp pfliegler and a tartally rickia lenoirii is reported from the carpathian basin622 acknowledgments to m. sipiczki and z. vas; 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(2014). effects of the ectoparasitic fungus rickia wasmannii on its ant host myrmica scabrinodis: changes in host mortality and behavior. insectes sociaux, 61: 247-252. doi: 10.1007/s00040-014-0349-3 eea (2012). biogeographic regions in europe. http://www.eea. europa.eu/data-and-maps/figures/ds_resolveuid/9afe2a4dadf9-45cd-a5a9-26e34640d494 (accessed date: 06 november, 2015). haelewaters, d., boer, p. & noordijk, j. (2015a). studies of laboulbeniales (fungi, ascomycota) on myrmica ants: rickia wasmannii in the netherlands. journal of hymenoptera research, 44: 39-47. doi: 10.3897/jhr.44.4951 haelewaters, d., zhao, s.y., de kesel, a., royer, i.r., handlin, r.e., farrell, b.d. & pfister, d.h. (2015b). laboulbeniales (ascomycota) of the boston harbor islands i: species parasitizing coccinellidae and staphylinidae. northeastern naturalist, 22: 459-477. doi: 10.1656/045.022.0304 henk, d., weir, a. & blackwell, m. (2003). laboulbeniopsis termitarius, an ectoparasite of termites newly recognized as a member of the laboulbeniomycetes. mycologia, 95: 561564. doi: 10.2307/3761931 konrad, m., grasse, a. v, tragust, s. & cremer, s. (2015). anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host. proceedings of biological sciences, 282: 20141976. doi: 10.1098/rspb.2014.1976 pech, p. & heneberg, p. (2015). benomyl treatment decreases fecundity of ant queens. journal of invertebrate pathology, 130: 61-63. doi: 10.1016/j.jip.2015.06.012 santamaria, s. (2001). los laboulbeniales, un grupo enigmático de hongos parásitos de insectos. lazaroa, 22: 3-19. http://revistas.ucm.es/index.php/laza/article/view/ laza0101110003a santamaria, s. & espadaler, x. 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(2015). does laboulbenia formicarum (ascomycota : laboulbeniales) fungus infect the invasive garden ant, lasius neglectus (hymenoptera: formicidae), in hungary? e-acta natualia pannononica 8: 117-123. http://epa.oszk.hu/01900/01957/00011/pdf/ epa01957_eactanat_2015_8_117-123.pdf tartally, a., szűcs, b. & ebsen, j.r. (2007). the first records of rickia wasmannii cavara, 1899, a myrmecophilous fungus, and its myrmica latreille, 1804 host ants in hungary and romania (ascomycetes: laboulbeniales; hymenoptera: formicidae). myrmecological news, 10: 123. doi: 10.13102/sociobiology.v62i4.792sociobiology 62(4): 578-582 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 determination of acute lethal doses (ld 50 and lc 50 ) of imidacloprid for the native bee melipona scutellaris latreille, 1811 (hymenoptera: apidae) introduction bees belong to the class insecta, order hymenoptera, as well as wasps and ants. these insects are the most important ones for biodiversity conservation since they have the biggest number of pollinators which use pollen and nectar as food and energy sources (nogueira-neto, 1997). during the collection process of these resources on flowers, they transfer pollen grains from the male reproductive organs to the female ones, ensuring pollination success. in brazil, the apidae family is the biggest one, with solitary and social bee species. among representatives of this family we find bees from the melipona illiger, 1806 genus (which belongs to the meliponini tribe), with more than 50 species. in this genus, the stingless and eusocial bee, melipona scutellaris latreille, 1811 (hymenoptera: apidae), also commonly known as “uruçu” or “uruçu do abstract the bee species melipona scutellaris latreille, 1811 (hymenoptera: apidae) is native to brazil and, stingless. in brazil, stingless bees are responsible for 40% to 90% of tree species pollination, depending on the considered ecosystem. however, their survival has been threatened since the country has been standing out as a big consumer of pesticides. many of the pesticides used are considered toxic to bees, including imidacloprid. although the bees are not the target of these substances, they are highly vulnerable to contamination. thereby, the objective of this study was to establish the mean lethal dose (ld 50 ) and the mean lethal concentration (lc 50 ) of imidacloprid for the m. scutellaris. in order to carry out this experiment, bees were collected and the test was performed according to oecd’s protocol (1998a, 1998b), developed for a. mellifera. for the determination of ld 50 and lc 50 , data was analyzed through the probit method. the topical ld 50 established in this study was 2.41 ng/ bee for 24 hours and 1.29 ng/bee for 48 hours. the oral lc 50 was 2.01 ng i.a./μl for 24 hours and 0.81 ng i.a./μl for 48 hours. thus, it is important to establish management methods which take this higher susceptibility into account to protect native species. sociobiology an international journal on social insects lm costa1, tc grella1, ra barbosa2, o malaspina2, rcf nocelli1 article history edited by celso f. martins, ufpb, brazil received 13 april 2015 initial acceptance 16 july 2015 final acceptance 14 august 2015 keywords stingless bee, neonicotinoid, toxicity. corresponding author leticia mariano da costa center of agricultural sciences federal university of são carlos (ufscar) anhanguera road, km 174, 13600-970 araras, são paulo, brazil e-mail: costa.l.mariano@gmail.com nordeste”, stands out (nogueira-neto, 1997; kerr et al., 2001; imperatriz-fonseca & santos, 2014). this species is endemic among brazilian northeastern region, inhabiting hot and humid forest regions of the bahia coast and chapada diamantina, and it is also adapted to the ecological and climatic conditions of the state of são paulo (nogueira-neto, 1997). in the atlantic rainforest this bee is found only in places which present a low level of disturbance, fact that can be considered as an indication of environmental quality (ramalho & batista, 2005). in addition to the pollination services, melipona provides products and by-products with high economic value, such as: honey, pollen, and propolis. however, the importance of these bees goes far beyond the economic benefits, they also help in the rebuilding process of tropical forests and in the conservation of the existing ones, being able to act as bioindicators of environmental quality and having a key role in the ecosystem processes in which they are involved (ballivián, 2008). research article bees 1 federal university of são carlos (ufscar), campus araras-sp, brazil 2 são paulo state university (unesp), campus rio claro-sp, brazil sociobiology 62(4): 578-582 (december, 2015) 579 unfortunately, little is known about brazilian bees and, for this reason, there are difficulties in establishing conservation initiatives for this insect. at the same time there is also a reduction of food sources and nesting areas, an intensive occupation of lands and, most importantly, an excessive and/or incorrect use of pesticides, contributing to the reduction of populations (kerr et al., 2001). neonicotinoids acts on post-synaptic nicotinic acetylcholine receptors in the central nervous system. after binding, nerve impulses are discharged at first, followed by failure of the neuron to propagate any signal. acetylcholinesterases are not able to break down the neonicotinoid. this binding process is irreversible. the symptoms resulting from intoxication are tremors, seizures and death (national pesticide information center [npic], 2010). although bees are not the target of these toxic compounds, they may do their foraging in contaminated fields, being highly vulnerable to contamination (instituto brasileiro do meio ambiente e dos recursos naturais renováveis [ibama], 2012). thereby, the objective of this study was to establish the mean lethal dose (ld50) and the mean lethal concentration (lc50) of imidacloprid for the m. scutellaris. material and methods bee collection foragers bees of m. scutellaris were collected in the apiary of the biosciences institute at são paulo state university “júlio de mesquita filho”, rio claro campus. for all the performed tests, 30 bees were used with three repetitions of 10 bees, being each one from three different colonies. the chosen colonies were free from diseases and presented a queen in good health. the tests were performed at the center for the study of social insects in the bioscience institute at unesp, rio claro. determination of mean lethal topical dose the procedures for the determination of the topical ld50 were based on the oecd (1998a) developed for a. mellifera (the temperature was changed to 28 °c). the imidacloprid pesticide (degree of purity 95%) was initially diluted in acetone and by means of successive dilutions were prepared the desired doses for application (2; 4; 8; 16; 32; and 64 ng a.i./μl to establish ld50 for 24 hours and 0.3; 0.6; 1.2; 2.4; 4.8; and 9.6 ng a.i./μl to establish ld50 for 48 hours). the collected bees were transferred to plastic containers with volume of 250 ml with perforations on the lid for air circulation. in each container there was a plastic microvial with food (50% sucrose solution) ad libitum. bees in the experimental group received a topical application of 1 μl of one respective the solution containing the tested substance in the dorsal area of the thorax. bees in the control group received only 1 μl of acetone. the bees were held in climatic room at 28 ± 1°c, the relative humidity of 70 ± 5% and darkness. determination of mean lethal oral concentration the procedures for the determination of the oral lc50 were based on the oecd (1998b) developed for a. mellifera (the temperature was changed to 28 °c). to obtain the desired concentrations of imidacloprid, from the stock solution were prepared some others by dilutions on cascade using as solvent a mixture of 85% water + 15% acetone. the concentrations used for the bee’s contamination were 0.3; 0.6; 1.2; 2.4; 4.8; and 9.6 ng/μl. for the control group, food was supplied without imidacloprid. the tests were conducted in bod chamber with a temperature of 28 ºc ± 1.0 and with 70% ± 5 of relative humidity. statistical analysis mortality data obtained from the assays were subjected to statistical analysis using the probit method (finney, 1952) using the r® software. ld50 and lc50 values were determined, as well as their respective 95% confidence intervals values. results and discussion the values for the topical ld50 of the imidacloprid obtained for m. scutellaris were: 2.41 and 1.29 ng a.i./bee (table 1 and figs 1 and 2), for 24 and 48 hours, respectively. the oral lc50 obtained were of 2.01 and 0.81 ng a.i./µl (table 1 and figs 3 and 4), for 24 and 48 hours, respectively. exposure mode time (hours) ld50 lc50 c.i.95% χ 2 d.f. topic ng a.i./ bee 24 2.41 _ 1.63 – 3.27 0.753 4 48 1.29 _ 0.813 – 1.903 2.642 4 ingestion ng a.i./ diet μl 24 _ 2.01 1.551 – 2.618 2.534 4 48 _ 0.81 0.264 – 1.538 4.001 4 (ld50) mean lethal dose; (lc50) mean lethal concentration; (c.i.95%) confidence interval 95%; (χ2) chi-square, and (d.f.) degree of freedom. table 1. acute toxicity values of imidacloprid for melipona scutellaris. according to johansen and mayer classification (1990), which consider insecticides with a ld50 < 2.000 ng/ bee as highly toxic to bees, imidacloprid is considered highly toxic for m. scutellaris. for a. mellifera there are several reports for the topical ld50 of imidacloprid, among them: 17.9 lm costa et al. – acute lethal doses of imidacloprid for melipona scutellaris 580 ng a.i./bee (24 hours) (iwasa et al., 2004); 24 ng a.i./bee (24 and 48 hours) (suchail et al., 2003); 42 – 1041 ng a.i./bee (48 hours) (schmuck et al., 2003); 49 – 1022 ng a.i./bee (48 hours) (nauen et al., 2001). the values of lc50 for a. mellifera are: 81 ng a.i./μl diet (48 hours) (nauen et al., 2001) and 40.9 ng a.i./μl diet (48 hours) (schmuck et al., 2001). these values show that the m. scutellaris bees are more sensitive to imidacloprid than a. mellifera. the fact that m. scutellaris bee is more sensitive than a. mellifera bee was also proved by lourenço et al. (2012a, 2012b) who did a toxicity study of fipronil with native bee species and verified that the pesticide is highly toxic, showing a topical ld50 (48 hours) of 0.41 ng a.i./bee and oral lc50 (48 hours) of 0.011 ng a.i./ μl diet. jacob et al. (2013) also demonstrated the higher sensitivity of native bee s. postica to fipronil. in this study, the topical ld50 (24 hours) determined was 0.54 ng a.i./bee and the oral lc50 (24 hours) was 0.24 ng a.i./μl diet. for a. mellifera the topical ld50 of fipronil is 4 ng a.i./bee and the oral lc50 is 1.27 ng/μl diet (tingle et al., 2003; decourtye et al., 2005). 1 this range is because one of the objects of study schmuck et al. (2003) it was to verify if there was difference between the sensitivity of bees from different european apiaries to imidacloprid. the toxicity tests were carried out in different european laboratories. the calculated ld 50 values did not indicate significant differences in sensitivity between honeybees of different apiaries. 2 this range is because one of the objects of study nauen et al. (2001) it was to verify if there was difference between the sensitivity of bees from different european apiaries to imidacloprid. the toxicity tests were carried out in different european laboratories. the calculated ld 50 values did not indicate significant differences in sensitivity between honeybees of different apiaries. fig 1. mortality of melipona scutellaris (24 hours) after the intoxication with imidacloprid by contact. our results corroborate with the work of soares et al. (2015), which determined the topical ld50 and oral lc50 of imidacloprid for native bee scaptotrigona postica latreille, 1807 (hymenoptera: apidae). the obtained values were: topical ld50 of 25.20 (24 hours) and 24.46 ng a.i./bee (48 hours) and oral lc50 of 42.5 (24 hours) and 14.3 ng a.i./ μl diet (48 hours), indicating that this species is also more susceptible to the neonicotinoid pesticide than a. mellifera. fig 2. mortality of melipona scutellaris (48 hours) after the intoxication with imidacloprid by contact. comparing values of ld50 and lc50 (48 hours) of imidacloprid for s. postica and m. scutellaris, it was noted that this bee is 19 times more sensitive when compared to the other bee. fig 3. mortality of melipona scutellaris (24 hours) after the intoxication with imidacloprid by ingestion. when we compare the lc50 and ld50 found in this study with values obtained by other studies presented here, it is possible to infer that m. scutellaris species is more sensitive to the fipronil than to imidacloprid. others studies that compare the tolerance between stingless and africanized honey bee species showed that the former are usually more sensitive to pesticides (moraes et al., 2000; valdovinos-núñez et al., 2009; del sarto et al., 2014). the toxicity of neonicotinoid pesticides for bees can be classified in two groups based on the presence of nitro or cyan grouping. the pesticides with nitro grouping are the most toxic ones, such as imidacloprid, because the presence of this functional group grants to the pesticide great affinity with the nicotinic receptor of acethylcholine and, therefore, its high toxicity (tomizawa & casida, 2003). the bees exposed to imidacloprid, either topically or orally, presented signs of paralysis, tremors and some of them were even dead, which are common symptoms of intoxication by neonicotinoid pesticides observed by suchail et al. (2001), since the target organ of this substance is the nervous system. sociobiology 62(4): 578-582 (december, 2015) 581 a study performed by suchail et al. (2003) indicated that the high oral toxicity of imidacloprid for a. mellifera might be caused by the fact that this molecule is rapidly metabolized in olefin and 5-hydroxyimidacloprid. such metabolites are toxic with acute exposure, highly suggesting that 5-hydroxyimidacloprid and/or olefin contribute for an increased action of imidacloprid in bees. depending on the cultivation and application method, imidacloprid can show an application concentration of 70 μg/ml, exceeding in 54 times the value capable of causing bee mortality (ld50 for 48 hours). therefore, the use of imidacloprid should be avoided during the bloom period (suchail et al., 2000). in conclusion, our study showed that m. scutellaris is highly sensitive to the action of the insecticide imidacloprid after topical and oral intoxication. because of this and of the economic and ecological importance, native species of stingless bees should be more studied, especially in relation to pesticide impact. acknowledgments the authors thank to fundação de amparo à pesquisa do estado de são paulo (fapesp) for the scholarship (process no. 2013/20666-3) and financial support (process no. 2012/50197-2). references ballivián, j.m.p.p. 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(2003). metabolism of imidacloprid in apis mellifera. pest management science, 3: 291-296. doi: 10.1002/ps.772 suchail, s., guez, d. & belzunces, l.p. (2000). characteristics of imidacloprid toxicity in two apis mellifera subspecies. environmental toxicology and chemistry, 7: 1901-1905. doi: 10.1002/etc.5620190726. suchail, s., guez, d. & belzunces, l.p. (2001). discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in apis mellifera. environmental toxicology and chemistry, 20: 2482-2486. tingle, c.c., rother, j.a., dewhurst, c.f., lauer, s. & king, w.j. (2003). fipronil environmental fate, ecotoxicology and human health concerns. rev. environ. contam. toxicol., 176: 1-66. doi: 10.1007/978-1-4899-7283-5_1 tomizawa, m. & casida, j.e. (2003). selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. annual review of entomology, 48: 339-364. doi: 10.1146/annurev. ento.48.091801.112731. valdovinos-núñez, g.r., quezada-euán, j.j.g., anconaxiu, p., moo-valle, h., carmona, a. & sánchez, e.r. (2009). comparative toxicity of pesticides to stingless bees (hymenoptera: apidae: meliponini). journal of economic entomology, 102: 1737-1742. doi: http://dx.doi. org/10.1603/029.102.0502. doi: 10.13102/sociobiology.v62i4.736sociobiology 62(4): 610-612 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 foraging behavior of fire ant solenopsis saevissima (smith) (hymenoptera: formicidae) in felis catus linnaeus (carnivora: felidae) carcass insects are the first organisms to detect and colonize dead animals, being present in every stage of decomposition (carvalho et al., 2000). carcass colonizers are classified as necrophagous, which feed on the corpse and are the first to arrive; natural enemies (predators and parasitoids), which feed on necrophagous in the corpse; omnivores, which feed both on the corpse and the associated fauna; opportunists, which use the corpse as refuge, warming and other purposes; adventives, which are found in the corpse with no apparent purpose (almeida et al., 2010). ecological and fauna studies aiming to assess the decomposition-related entomofauna in vertebrate corpses are essential to understand the cycling dynamics of nutrients in ecosystems, as to build knowledge on the diversity related to these processes (gomes, 2010). furthermore, insects associated to murder cases and corpse decaying processes may participate in crime scenes and serve as evidence to the solving of such cases (oliveira-costa & zuben, 2011). regarding activity, abundance and number of species, formicidae family is an important group of insects related to animals in decomposition (zara & caetano, 2010). the abstract solenopsis saevissima fire ants were found foraging in a felis catus carcass over tissues an secretions present in holes and mucosa. the ants built a dirt-made physical structure around the carcass, which prevented necrophagous flies from laying eggs or larvae in the body. these observations are relevant to increasing knowledge on the role of this ant genus in the decaying process of other animal corpses, including humans. sociobiology an international journal on social insects tt maciel, mm castro, bc barbosa, ef fernandes, hh santos-prezoto, f prezoto article history edited by gilberto m. m. santos, uefs, brazil received 08 december 2014 initial acceptance 02 june 2015 final acceptance 08 september 2015 keywords carcass decomposition, domestic cat, forensic entomology, necrophagous ants. corresponding author tatiane tagliatti maciel laboratório de ecologia comportamental e bioacústica – labec universidade federal de juiz de fora campus universitário, martelos, juiz de fora 36036-900, minas gerais, brazil e-mail: tatitagliatti@hotmail.com ecological role of ants in this process varies from predator to necrophagous (moretti & ribeiro, 2006). a relevant ant group for forensic entomology is the genus solenopsis westwood, 1840, which includes individuals popularly known as fire-ants. these ants play the role of necrophagous and predators, and are present in various phases of the decomposition process (oliveira-costa et al., 2008). the objective of this study was to register the presence and behavior of solenopsis saevissima in a domestic cat carcass (felis catus linnaeus, 1758). observation took place during daytime in august 30 and 31 of 2014, in a public road of a residential condominium (21º46’02,72’’s; 43º22’34,9’’w) located in the urban area of the municipality of juiz de fora, minas gerais state, brazil. the regional climate is characterized as warm subtropical – cwa, according to köppen (1970), and the weather conditions during the experiment were typical from winter (mean temperature of 14.1 ºc), with short precipitation periods (0,2mm). a domestic cat carcass (adult female) was found with unknown causa mortis. the corpse was in the first stages of the short note universidade federal de juiz de fora, minas gerais, brazil sociobiology 62(4): 610-612 (december, 2015) 611 decomposition process, transitioning through the fresh stage to the bloated stage, according to the classification suggested by scaglia (2014). it is believed that death happened about 24 hours before the observations started. an association between direct observation registers and spontaneous observation through ad libitum method (altmann, 1974) was used for data sampling on the behavior of s. saevissima ants in the carcass. ant individuals were collected and fixed in alcohol at 70% in eppendorf tubes for genus confirmation in laboratory. the fernández (2003) dichotomous key was used for identification, as well as comparisons with reference individuals properly identified by specialists and deposited in the entomological collection of the laboratório de ecologia comportamental e bioacústica (labec) of universidade federal de juiz de fora. observations ended in august 31 of 2014 due to removal of the carcass by the condominium maintenance crew. the s. saevissima ants (fig 1a) were found exploring the whole carcass; the tissues and secretions present in holes and mucosa were being used as food source. intense foraging activity by the ants was observed upon the carcass, as well as the construction of a physical structure made of dirt around the body (figs 1b and 1c). dirt accumulation around the corpse, associated with aggressive behavior of fire ants, may have prevented necrophagous flies from ovipositing (fig 1d), once these individuals could not find the appropriate place (body cavities) for oviposition. the ants’ action on the carcass also inhibited the presence of other decomposition-related insect species. celino et al. (2009) observed the same soil-disturbing phenomenon and suggested that this behavior is related to ant workers searching for larvae buried in the soil. however, in a very particular way, it stands out that the dirt accumulated by the ants for construction of the structure around the carcass was brought by the workers from another place (probably from the sidewalk), as the carcass was located in the interface of the asphalt and the sidewalk borders, thus offering no nearby resources to the construction of the structure. observations showed that ants have accumulated most of the dirt around the head of the corpse, which was partially covered (fig 1 b). such behavior shown by ants has limited the action of necrophagous insects (mainly flies), which had restricted access to the natural cavities of this region (mouth, ears, eyes and nostrils). according to oliveira-costa and zuben (2011), the head part is the necrophagous` main access way to oviposition, as it is the first body part to start decomposing. solenopsis ants` behavior of covering this part with dirt shows the dominance that this genus is able to place in a carcass, limiting the actions of other insects and changing the process of decomposition. wells and greenberg (1994) also inferred that the presence of ant solenopsis in carcasses cause failures in succession of other insects in the decomposition process. thus, such presence must be considered when using sequence data (succession pattern) to estimate postmortem intervals (pmis). zara and caetano (2010) relate that solenopsis ants may indeed use a carcass on purpose, becoming dominant over the resources and avoiding competitors by the mass recruitment shown by ants. this group can be found in urban and forest areas (moura et al., 1997) and its colonization fig 1. a ants of the solenopsis genus photographed under stereomicroscope. b and c dirt-made physical structure around the cat carcass. d necrophagous flies prevented from ovipositing in a hole (anal cavity) due to the presence of ants in the body and the structure around the carcass. tt maciel et al. – fire ant foraging in cat carcass612 changes the succession pattern and prevents other insect species from laying eggs in the carcasses (moura et al., 2005). the occurrence of solenopsis saevissima and the behavior of burying in a medium sized vertebrate carcass are proof of the workers` capacity to modify the decomposition process. these observations are relevant to increase knowledge on this genus` role in the decaying process of other animal corpses, including humans. aknowledgements the authors thank mateus f. de freitas salviato detoni, of laboratório de ecologia comportamental e bioacústica at universidade federal de juiz de fora for its contribution to this study. references almeida, e.c. et al. (2010). noções de perícia criminal e o vestígio entomológico na estimativa do ipm. in gomes l. (ed.), entomologia forense: novas tendências e tecnologias nas ciências criminais (pp. 133-167). rio de janeiro: technical books. altmann, j. (1974). observation study of behavior: sampling methods. behaviour, 49: 223-265. carvalho, l.m.l.; thyssen, p.j.; linhares, a.x. & palhares, f.a.b. (2000). a checklist of arthropods associated with pig carrion and human corpses in southeastern brazil. memórias do instituto oswaldo cruz, 95: 135-138. doi: 10.1590/s007402762000000100023 celino, t.b.; oliveira-costa, j.; mahye-nunes, a.; rosa, d.o.; costa, c.e.s.; fontoura, p. & mateini, n. (2009). papel ecológico de formigas do gênero solenopsis westwood relacionado à decomposição de carcaça de porco sus scrofa (l.). in ix congresso de ecologia do brasil, são lourenço. anais. são lourenço: ecologia, p. 1-3. fernández, f.(2003). introducción a las hormigas de la región neotropical. bogotá, instituto de investigación de recursos biológicos alexander von humboldt. 398p. gomes, l. (2010). entomologia forense: novas tendências e tecnologias nas ciências criminais. rio de janeiro: technical books. 523p. köppen, w. (1970). roteiro para classificação climática. [mimeo]. moretti, t.c. & ribeiro, o.b. (2006). cephalotes clypeatus fabricius (hymenoptera: formicidae): hábitos de nidificação e ocorrência em carcaça animal. neotropical entomology, 35: 412:415. doi: 10.1590/s1519-566x2006000300019 moura, m.o.; carvalho,c.j.b. & monteiro-filho, e.l.a. (1997). a preliminary analysis of insects of medico-legal importance in curitiba, state of paraná. memórias do instituto oswaldo cruz, 92: 269-274. doi: 10.1590/s007402761997000200023 moura, m; monteiro-filho, e.l.a. & carvalho, c.j.b. (2005). heterotrophic succession in carrion arthropod assemblages. brazilian archives of biology and technology, 48: 473-482. doi: 10.1590/s1516-89132005000300018 oliveira-costa, j.; celino, t.b. & santana, d.o. (2008). himenópteros de interesse forense no brasil. in oliveiracosta, j. (ed.), entomologia forense: quando os insetos são vestígios (pp. 241-252). campinas: millenium. oliveira-costa, j. & zuben, c.j.v. (2011). entomologia aplicada a procedimentos legais no brasil: considerações e estudo de casos. vetores e pragas, 27: 33-37. scaglia, j.a.p. (2014). manual de entomologia forense. leme: j.h.mizuno, 406 p. wells, j.d. & greenberg, b. (1994). effect of the red imported fire ant hymenoptera: formicidae) and carcass type on the daily occurrence of postfeeding carrion-fly larvae (diptera: calliphoridae, sarcophagidae). journal of medical entomology, 31: 171-174. zara, f.j. & caetano, f.h. (2010). mirmecologia e formigas que ocorrem em carcaças. in gomes, l. (ed.), entomologia forense: novas tendências e tecnologias nas ciências criminais. (pp. 237-269). rio de janeiro: technical books. 795 fire anthemipteran mutualisms: comparison of ant preference for honeydew excreted by an invasive mealybug and a native aphid by aiming zhou, yong yue lu, ling zeng, yijuan xu* & guangwen liang* abstract interaction between ants and honeydew-producing hemipterans is defined as mutualism which is beneficial for both species. red imported fire ants, solenopsis invicta, who tend the honeydew-producing hemipteran insects, can help reduce their predators and parasites. in return, ants receive honeydew as an important food resource. in this study, we tested the foraging intensity (fi), weight change and honeydew consumption (hc) of s. invicta on phenacoccus solenopsis, myzus persicae and infested plants by mixed-colony (both p. solenopsis and m. persicae) . our results showed that fi of s. invicta was gradually increasing with time on the plants infested by aphids and the mixed-colony, while inverse situation was found on mealybuginfested plants. within 10 and 15 days, fi on aphid and the mixed-species infested plant was significantly more than that on the mealybug infested plant. we compared the ant weight between the two moving directions, and the result showed that the weight of downward ants was significantly heavier than upward ants except that on the mealybug infested plant after 15 days. the study also indicated that there was no observable difference of hc among the three kinds of honeydew resource in one day and five days, while hc on aphid and the mixed colony infested plant in 10 and 15 days was significantly more than that on mealybug-infested plants. key words: solenopsis invicta, myzus persicae, phenacoccus solenopsis, foraging intensity, honeydew consumption. red imported fire ant research center, south china agricultural university, guangzhou 510642, china *corresponding authors: e-mail: xuyijuan@yahoo.com, gwliang@scau.edu.cn 796 sociobiolog y vol. 59, no. 3, 2012 introduction mutualistic interactions between ant species and honeydew-producing hemipteran insects, such as aphids, scales, mealybugs, membracids and lepidopteran larvae, have been described extensively in various ecosystems (nixon 1951, buckley 1987b, buckley 1987a, holway et al. 2002, ness & bronstein 2004). ants tend honey-producing hemipteran insects by reducing not only the predation and parasitism by natural enemies but also the risk of fungal infection. in return, the ants receive abundant honeydew from hemipteran insects as food (banks & macaulay 1967, tilles & wood 1982, yao et al. 2000, standler & dixon 1998). honeydew excreted by hemipterans is considered to be an important food resource for ants because it contains sugars mixed with various amino acids and energ y-rich materials (hölldobler & wilson 1990, douglas 1993, tobin 1994, davidson et al. 2004). previous studies showed that s. invicta colonies grew substantially larger when supplied with insect prey and honeydew produced by the invasive mealybug antonina graminis (maskell) (helms & vinson 2008). the intensity of mutualism between ants and honeydew-producing hemipterans is involved with various factors, such as host density, host plant quality, species and density of hemipterans and ants (addicott 1978, addicott 1979, auclair 1963, cushman 1991, breton & addicott 1992, bristow 1984). hibiscus rosa-sinensis is commonly infested by ant-tending aphids and mealybugs such as myzus persicae and phenacoccus solenopsis in south china. m. persicae is a native species which has abundant population density in the field. the mealybug p. solenopsis is native to the us and has spread throughout the world (fuchs et al. 1991). it has a wide geographic distribution and can be found in central america, south america and africa (williams & willink 1992, culik & gullan 2005). recently, p. solenopsis was reported to be an important invasive species in southern china (lu et al. 2008). the red imported fire ant, solenopsis invicta, is a new invasive pest in south china. negative effects of s. invicta on agriculture and forestry production, human health and poultry production have been reported in south china (zeng et al. 2005). like the aphid m. persicae, we found that s. invicta were also attracted by the honeydew-producing p. solenopsis in the field. in this study, we compare the fi and hc of s. invicta on the plant infested by m. persicae, p. 797 zhou, a. et al. — ant preference for honeydew solenopsis and the mixed-colony respectively, as well as to test the hypothesis that s. invicta had higher fi and hc on a plant infested by mixed-colony than that on a plant which was infested by a single colony of m. persicae or p. solenopsis. materials and methods host plants h. rosa-sinensis was purchased from a commercial horticultural farm. all plants had 25-30 uninoculated leaves and were approximately 25-30 cm in height. each plant was cultivated in plastic flowerpots (the diameters of the upper and lower edges were 18 cm and 14 cm, respectively, with a height of 17 cm) in greenhouses. mealybugs and aphids colonies of p. solenopsis and myzus persicae were fed on h. rosa-sinensis. the 1st instar p. solenopsis and m. persicae nymphs were inoculated on each plant and raised for several generations. all colonies were reared in the laboratory with the temperature maintained at 27 ±2°c and a relative humidity of 60-70%. fire ants colonies of s. invicta were collected from the suburb of guangzhou and reared in plastic boxes (116 l). all colonies were separated from the soil by dripping water into plastic boxes until the colonies floated ( jouvenaz et al. 1977). the ants were removed and reared in plastic boxes with tubes filled with distilled water. colonies were divided into several small colonies (approximately 1.0 g workers and one queen) measured with a microbalance (sartorius, bs, 224s). the ants were placed in a 9-cm plastic petri dish with moist plaster, which served as an artificial nest. the ants were given fresh live tenebrio molitor worms, and a 10% solution of honey mixed with water (50 ml) weekly. the colony was assigned randomly to each experimental treatment. h. rosa-sinensis seedling leaves were inoculated with 60 3rd instar p. solenopsis and m. persicae. artificial nests of s. invicta were transferred to plastic cases (40 cm × 28 cm × 22 cm). after 24 h, mealybug and aphid-infected plants were placed into each plastic case. a plastic hose (1.5 cm diameter) 798 sociobiolog y vol. 59, no. 3, 2012 was used to build a bridge between the ants’ nest and the basic stem of the plant to allow worker foraging. we randomly collected 30 workers from the bottom stalk as they were moving toward the hemipteran colony since the beginning of the experiment, and 30 more were collected after 1, 5, 10 and 15 days as they were returning from the colony. weights of the ants before and after foraging were measured with a microbalance (sartorius, bs, 224s). workers’ weight change of s. invicta was viewed as an indirect measure of p. solenopsis and m. persicae honeydew consumption. meanwhile, in order to measure the ant foraging intensity, we also counted all the ants present on each plant. in addition, honeydew consumption and foraging intensity of s. invicta were studied as was described in the preceding experiments when both mealybugs and aphids were present on the same plant (mixed-colony) (60 3rd instar nymph each hemipteran species). the studies were conducted in an enemy-free laboratory. all treatments were replicated ten times. statistical analysis difference of fi and hc of s. invicta between mealybug, aphid and mixed-colony inoculated plants and in four different experimental times were analyzed using a one-way anova followed by lsd tests for multiple comparisons. changes in ant weight between traveling up and down were analyzed with paired-sample t-tests. all statistical analyses were conducted using spss, version 14.0 (spss inc., chicago, il, usa). results foraging intensity of s. invicta we found that fi on mealybug-infested plants was gradually decreasing, the number of foraging ants in 15 days was significantly smaller than that after one day (f 3,36 =3.406, p=0.028, fig.1.a). fi on the plants infested by aphids or mixed-colony gradually increased, and the number of foraging ants on aphid-infested plants after 10 and 15 days was larger than that after one day (f 3,36 =3.749, p=0.019, fig.1.a). there was no marked difference of fi on the plant infested by mixed-colony among the four tests (f 3,36 =1.323, p=0.282, fig.1.a). in addition, there was no significant difference of fi in one day among the three kinds of honeydew resources (f 2,27 =1.967, p=0.159, fig.1.b), while the number of foraging workers on the plant infested by aphids 799 zhou, a. et al. — ant preference for honeydew and the mixed-colony in 10 and 15 days was significantly larger than that on mealybug-infested plant after one day (f 2,27 =9.701, p=0.001; f 2,27 =15.042, p=0.000, fig.1.b). honeydew consumption of s. invicta we recorded the difference of the weights of the workers moving in two directions. our results indicated that the weights of all downward workers were significantly heavier than upward ones on plants inoculated with the three kinds of heipteran species in the four tests, while only the result on mealybug-infested plants in 15 days was significant (fig.2.a, b and c). we also calculated the hc of s. invicta under different honeydew resources for four fig.1.comparision of the average number (m±se) of foraging ants per plant (a): the same honeydew resource and different testing time; (b): the same testing time and different honeydew resource. the same letter on bars indicates no significant difference (p≥0.05). 800 sociobiolog y vol. 59, no. 3, 2012 fig.2. mean (±se) weight of workers traveling up (❒) and traveling down (■) (a): h. rosa-sinensis infested with p. solenopsis only (b): h. rosa-sinensis infested with m. persicae only (c): h. rosa-sinensis infested with both p. solenopsis and m. persicae, (* indicate p<0.05, ** indicate p≥0.01). 801 zhou, a. et al. — ant preference for honeydew different testing times. the results showed that when plants were inoculated with the mixed-colony, there was no significant difference in hc of s. invicta among all the four testing times (f 3,36 =1.157, p=0.339 , fig.3.a). compared with hc in one day on aphid-infested plants, hc of s. invicta increased after 15 days (f 3,36 =2.398, p=0.084 , fig.3.a). but the trend on mealybug-infested plants was reversed (f 3,36 =3.688, p=0.021 , fig.3.a). in addition, there was no observable difference of hc among the three kinds of hemipteran species after one day and five days (f 2,27 =0.361, p=0.700; f 2,27 =0.373, p=0.692, fig.3.b), while hc on the plants infested by aphids and the mixed-colony after 10 and 15 days was significantly larger than that on mealybug-infested plants (f 2,27 =6.023, p=0.007; f 2,27 =9.292, p=0.001, fig.3.b). fig.3.comparision among the average weight (m±se) of honeydew consumption (a): the same honeydew resource and different testing time; (b): the same testing time and different honeydew resource. the same letter on bars indicates no significant difference (p≥0.05). 802 sociobiolog y vol. 59, no. 3, 2012 disscussion our results demonstrated that fi and hc of s. invicta decreased on mealybug-infested plants and increased on the plants infested by aphids or the mixed-colony (fig.1.a, fig.3.a). compared with the cases on the plant infested by single-colony, there was no significant increase of fi and hc of s. invicta when ants had access to the plants infested by the mixed-colony (fig.1.b, fig.3.b). we conclude that fi and hc of s. invicta were involved with the quantity of honeydew produced, the more honeydew produced by hemipterans, the more ants attracted to the infested plants. fi and hc of s. invicta on the plant infested by aphids and mixed-colony after 10 and 15 days was significantly more than that on mealybug-infested plants after one day (fig.1.b, fig.3.b). those results indicated that the m. persicae colony could produce more honeydew than p. solenopsis in 10 and 15 days. in addition, the quantities of honeydew produced between m. persicae and the mixedcolony showed no significant difference. different reproductive rates and inter-specific competition between m. persicae and p. solenopsis could be responsible for the above results. the developmental period of p. solenopsis from immature crawler to adult stage (females) was approximately 9-16 days with 23.3-30.2 and 40.5-92.5% rh (vennila et al. 2010). the developmental period of m. persicae from nymph to adult was approximately 5-6 days (liu 1991). a higher reproductive rate of m. persicae may lead to decreasing population and fitness of p. solenopsis when m. persicae and p. solenopsis feed on the same plant. this may explain why ants had stronger fi and more hc on the plants infested by mixed colony than on those only infested by m. persicae. our results accorded with the report that ants usually visited a rich hydrocarbon source with higher intensity rather than a less rich one, and would exploit much closer and more worthy sugar resources within their foraging range (hölldobler & wilson 1990, bonser et al. 1998, mailleux et al. 2000). previous studies showed that ants prefer honeydew which contained trisaccharides such as raffinose and melezitose (vökl et al. 1999). lasius niger showed marked preferences when collecting honeydew from three aphid species living on tansy (fischer et al. 2001). 803 zhou, a. et al. — ant preference for honeydew therefore, difference of honeydew quality between p. solenopsis and m. persicae may be another reason for the results seen here. however, the composition of honeydew excreted by p. solenopsis and m. persicae should be further studied. acknowledgment this study was supported by the national natural science foundation of china (no. 31101498). references addicott, j. h. 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and family vespidae, distributed in the subfamilies: stenogastrinae, vespinae, and polistinae (carpenter, 1993). the subfamily polistinae has high diversity in the neotropical region and is the only one having eusocial wasps occur in the brazilian territory, where we find 21 genera and 361 species (hermes et al., 2021). the wasps of this subfamily belong to three tribes: polistini, mischocytarini, and epiponini (carpenter & marques, 2001). these insects act as pollinators and floral visitors, collecting nectar for adult nutrition (hunt et al., 1991; brodmann et al., 2008; mello et al., 2011). social wasps, especially from the genus polistes, also act as predators of various agricultural pests, playing an important role in food chains and biological control, mainly attacking lepidoptera caterpillars (prezoto & machado, 1999; prezoto et al., 2006; abstract the social wasps of the order hymenoptera, family vespidae and subfamily polistinae perform important ecological functions such as pollination and predation, including pest insects, and can be an important resource in biological control. some species of parasitoids attack nests of social wasps causing mortality in the early stages of development, thus impairing the biological control by these insects. this study aimed to verify the occurrence and identify parasitoids in nests of polistes versicolor olivier, 1971, at the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg) – campus bambuí, brazil. in total, 14 nests of p. versicolor were collected. the parasitism rate was 28.57%, and parasitoids were recorded in four of 14 nests collected. the presence of parasitized nests may be due to the absence of a protective envelope, typical of the nest of p. versicolor. the emerged parasitoids belong to the species elasmus polistis burks, 1971 (hymenoptera: eulophidae), a species identified in nests of social wasps only in the state of rio grande do sul, southern brazil. to the best of our knowledge, this is the first study showing the occurrence of e. polistis parasitizing social wasps in minas gerais, southeastern brazil. sociobiology an international journal on social insects gabriel c jacques¹, sheliane cc francisco², luis cláudio p silveira³ article history edited by evandro n. silva, uefs, brazil received 14 december 2021 initial acceptance 02 february 2022 final acceptance 03 march 2022 publication date 22 april 2022 keywords parasitism; polistinae; social wasp. corresponding author gabriel de castro jacques instituto de educação, ciência e tecnologia de minas gerais campus bambuí, varginha farm bambuí-medeiros roadway km 05, minas gerais, brasil. e-mail: gabriel.jacques@ifmg.edu.br elisei et al., 2010; jacques et al., 2019). these caterpillars are torn, macerated, and given as food to larvae, being the main nutrition of wasps in their early stages of development (evans & west-eberhard, 1970) the subfamily polistinae is divided into two groups, according to the nesting behavior and architecture: 1 independent foundation form small colonies and build unprotected combs; 2 swarm: wasps build larger nests, protected by an envelope, with a well-defined social organization (carpenter & marques, 2001). the nests of social wasps are often invaded by parasitoids of larvae, pupae, and adults, and species of the genus polistes, which behave as an independent foundation, are more vulnerable to these natural enemies (burks, 1971; somavilla et al., 2015). the incidence of parasitism is higher in these nests due to the shorter time that wasps spend 1 instituto de educação, ciência e tecnologia de minas gerais, bambuí, brazil 2 instituto de educação, ciência e tecnologia de minas gerais, bambuí, brazil 3 universidade federal de lavras, lavras, brazil short note first record of elasmus polistis (hymenoptera: eulophidae), a parasitoid of polistes versicolor (hymenoptera: vespidae), in minas gerais, brazil mailto:gabriel.jacques@ifmg.edu.br gabriel c jacques, sheliane cc francisco, luis cláudio p silveira – first record of a parasitoid of polistes versicolor2 protecting the colony to forage, besides the absence of a nest protection envelope (soares et al., 2006; somavilla et al., 2015). the parasitism can compromise the colony, being the main cause of mortality among social wasps in the early stages of development (soares et al., 2006). this study aimed to verify the occurrence and identify parasitoids in nests of polistes versicolor olivier, 1971 (vespidae: polistinae), at the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg) campus bambuí, brazil. the experiment was performed at the bambuí campus of the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg), an anthropography environment characterized by the presence of several buildings and several agricultural crops. we collected 14 nests of p. versicolor on the campus. the species p. versicolor was chosen since it is dominant on the campus, facilitating the location of their nests (jacques et al., 2015). the nests, without the presence of adults, were placed in plastic containers covered by a fabric structure, with ventilation, kept in an incubator type b.o.d. for approximately 40 days, at 25 °c and relative humidity of 70% (somavilla et al., 2015). the number of cells and pupae of each nest was recorded. daily monitoring of nests was performed, recording the emergence of parasitoids. those who emerged were fixed in 70% alcohol and sent to prof. luís cláudio paterno silveira from the federal university of lavras for identification based on the key of brown (2010). the parasitism rate was estimated using the formula %tp = (nnp / nnt) × 100, where nnp represents the number of parasitized nests, and nnt, the number of total nests. two pearson correlation analyses were performed. one is between nest cell numbers and parasitoid numbers, and another is between pupae numbers and parasitoid numbers. we used the past program (hammer, 2005) to run the analyses. the nests of p. versicolor had an average of 110.64 ± 34.5 cells and 11.29 ± 7.39 pupae (table 1). we recorded a total of 301 parasitoids in four of the 14 collected nests, with an average of 75.25 ± 20.14 parasitoids/parasitized nests. the parasitism rate was 28.57% (table 1), similar to that found in polistes metricus say, 1831, in the usa, of 23% (hodges et al., 2003). nests of species of the genus polistes are severely parasitized because they do not have a specific caste of offspring caregivers (clouse, 1997) and consequently spend more time foraging and less time protecting the colony. moreover, in this genus, the nest has no protection envelope (somavilla et al., 2015). the emerged parasitoids belonged to elasmus polistis burks, 1971 (figure 1). the genus elasmus is the only member of the elasmini tribe (hymenoptera: eulophidae), having 248 described species (noyes, 2019). some species of elasmus are secondary parasitoids (hyperparasitoids) of braconidae and icheneumonidae, but most are primary parasitoids of lepidoptera, coleoptera, and hymenoptera (gibson, 1993; coote, 1997; dorfey & köhler, 2011). the species of this genus are characterized by the small size (1-3 mm) and the yellow body (dorfey & köhler, 2011). nest (n°) cell (n°) pupa (n°) parasitoid (n°) 1 31 2 2 20 3 163 7 4 31 7 5 297 14 188 6 36 4 7 33 8 19 1 9 390 106 32 10 20 1 11 300 12 44 12 155 13 26 14 28 4 37 mean ± standard error 110.64 ± 34.50 11.29 ± 7.39 75.25 ± 20.14 parasitism rate (%) 28.57 table 1. number of cells and pupae of the nests of polistes versicolor collected, number of elasmus polistis parasitoids emerged and percentage of parasitized nests. sociobiology 69(1): e7678 (march, 2022) 3 the analysis showed a high correlation between the number of cells in the nests and the number of parasitoids (person coefficient = 0.728; p < 0.05). three parasitized nests were the largest collected, with 390, 300, and 297 cells. this higher rate of parasitism found in larger nests was also reported in polistes myersi bequaert, 1934 (mayorga & sarmiento, 2020) and p. versicolor (somavilla et al., 2015). furthermore, these nests had the highest number of pupae and showed a correlation between the number of pupae and the number of parasitoids (person coefficient = 0.539; p < 0.05). e. polistis is a gregarious ectoparasite that feeds on the genus polistes’ pupae (nelson, 1976). thus, the larger nests with the highest number of pupae were more likely to be parasitized because the parasitoids obtained more food for their larvae. females of e. polistis usually oviposit in wasp pupae in their early stage and with cells already covered. in this site, the parasitoids enter the pupa state and are protected from attacks by host wasps (reed & vinson, 1979; lutz et al., 1984). as soon as the parasitoid larvae hatch, they feed on the entire content of the pupae. then, the larvae migrate to the bottom of the cell, build a protection and stay apart from the host remains. males usually appear first and wait for the females to mate (reed & vinson, 1979; macom & landolt, 1995). females, after mating, can stay in the nests they emerged from and oviposit in a pupa. (macom & landolt, 1995). e. polistis has already been identified as a parasitoid in nests of other polistes species, such as polistes exclamans (viereck, 1906), polistes annularis (linnaeus, 1763), polistes fuscatus (fabricius, 1793), polistes major palisot de beauvois, 1818, polistes metricus (reed & vinson, 1979), polistes dorsalis (fabricius, 1775) (macom & landolt, 1995), polistes myersi (mayorga & sarmiento, 2020), which are found in north and central america. in brazil, e. polistis has been recorded as a parasitoid of polistes cinerascens (saussure, 1854) and p. versicolor in rio grande do sul, southern brazil (dorfey & köhler, 2011; somavilla et al., 2015). there is a great distance between central america and rio grande do sul in brazil without any record of this parasitoid in wasp nests. our first record of e. polistis in minas gerais, southeastern brazil, is an important remark for the biogeographic comprehension of this ecological interaction. the species of the genus polistes have already been elucidated as an important tool in biological control in different crops, such as corn (prezoto & machado, 1999), cotton (kirkton, 1970), tobacco (rabb & lawson, 1957), cabbage (gould & jeanne, 1984), coffee (gravena, 1983) and kale (jacques et al., 2018). the species p. versicolor stands out, for it has already been tested in different studies with biological control (prezoto et al., 2006; elisei et al., 2010; jacques et al., 2019). the attack of parasitoids can cause high mortality among host social wasps, thus impairing the biological control by these insects (hanson & gauld, 2006). since p. versicolor is the dominant wasp in ifmg – campus bambuí (jacques et al., 2015), these parasitoids may impair the control of agricultural pests by this species in the crops of the campus, and further studies are necessary to test this hypothesis. acknowledgments the authors would like to thank the instituto de educação, ciência e tecnologia de minas gerais – campus bambuí for their support in the translation and publication of this article. author’s contribution gcj: conceptualization; formal analysis; resources; writing -review & editing. sccf: investigation; writing-original draft. lcps: supervision; methodology; writing-review & editing. references brodmann, j.r., francke, g., hölzler, q., twele, w. & zhang, m. 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(2015). record of parasitoids in nests of social wasps (hymenoptera: vespidae: polistinae). sociobiology, 62: 92-98. doi: 10.13102/ sociobiology.v62i1.92-98 225 genetic divergence of turkish apis mellifera subspecies based on sequencing of nd5 mitochondrial segment by fulya özdil1*& fatma i̇lhan1 abstract mitochondrial dna sequence variation can be used to infer honey bee evolutionary relationships. in this study, dna sequence diversity in the nd5 region of the mitochondrial genome was investigated in 93 samples of apis mellifera from 15 different populations in turkey. five novel haplotypes were revealed for the nd5 gene segment of turkish honeybees. the number of variable sites found was 6 for this region while 2 were parsimony informative sites. the average pairwise genetic distances were 0.3% for nd5 gene. in this study, the nj tree of nd5 gene segment were constructed with the published sequences of apis mellifera haplotypes. this study expands the knowledge about the mitochondrial nd5 region in apis mellifera and it is also the first comprehensive sequencing analysis of nd5 region in turkish honeybees. key words: apis mellifera l., dna sequence diversity, nd5, turkey introduction the honey bee, apis mellifera l., occurs naturally in europe, the middle east and africa (ruttner 1988). from morphometric and molecular studies, the 29 subspecies of the honey bee, apis mellifera l., are grouped into five evolutionary lineages: m from northern and western europe and northern africa, a from southern and central africa, c from the northern mediterranean region and eastern europe, o from the eastern mediterranean and the near and middle east region, and y from the east african country of ethiopia (ruttner 1988; hall and smith 1991; garnery et al. 1992; arias and sheppard 1996; kauhausen-keller et al. 1997; franck et al. 2000, 2001). 1department of animal science, faculty of agriculture, selçuk university, konya, turkey *corresponding author: fulyaozdil@selcuk.edu.tr the department where the study was conducted: selçuk university, faculty of agriculture, major of biometry & genetics, 42075 konya-turkey 226 sociobiolog y vol. 59, no. 1, 2012 based on morphometrics, the near eastern subspecies, anatolian (a. m. anatoliaca), caucasian (a. m. caucasica) and iranian (a. m. meda), had been grouped within the o branch (ruttner 1988; kauhausen-keller et al. 1997), however mtdna analysis showed that they belonged to the c lineage (smith et al. 1997; palmer et al. 2000; franck et al. 2000, 2001; kandemir et al. 2006; özdil et al. 2009a, 2009b; bouga et al. 2011). ruttner’s (1988) morphometric analyses concluded that a. m. anatoliaca, a. m. caucasica and a. m. meda exist in turkey. nearly all of turkey is occupied by a. m. anatoliaca. a. m. caucasica is found in the northeastern part of turkey and a. m. meda is found in the southeastern part of turkey. recently, the mitochondrial studies of turkish honeybees had also shown that a. m. carnica is found in the european part of turkey called thrace (palmer et al. 2000) and a. m. syriaca is found in the south part of the country near the hatay region (kandemir et al. 2006). the mitochondrial genome has been a useful molecule for population genetic studies of apis mellifera l. length and sequence variations within the mitochondrial genome of honeybees have been particularly useful in differentiating evolutionary lineages and groups of subspecies (hall and smith 1991; garnery et al. 1992, 1993; franck et al. 2000; palmer et al. 2000). apis mellifera ligustica was the first hymenopteran subspecies for which the whole mitochondrial genome has been sequenced (crozier and crozier 1993). to date, many mitochondrial regions, of which the trnaleu-coii region (formerly coi-coii intergenic region) (garnery et al. 1992, 1993; franck et al. 2000; de la rua et al.2001, 2004; palmer et al. 2000, sušnik et al. 2004; kozmus et al. 2007; muňoz et al. 2009; nedić et al. 2009; özdil et al. 2009a; magnus and szalanski 2010; szalanski and magnus 2010) was the first, have been sequenced and phylogenetic relationships among apis mellfera subspecies has been described. but we still have little knowledge about the other mitochondrial regions. the nd5 gene segment was first sequenced in greek honeybees and this is the only study of the phylogenetic relationships among east european subspecies (martimianakis et al. 2011). the objective of this research was to determine the genetic diversity and phylogenetic relationships of apis mellifera subspecies of turkey as determined by sequencing of nd5 gene segment. length variations and nucleotide substitutions found in this gene segment were compared with the other mitochondrial surveys and such results could be also useful in determining the genetic structure of honey bees. 227 özdil, f. & i̇lhan, f. genetic divergence of turkish honeybees materials and methods sampling and dna extraction a total of 93 honey bees each representing a different colony was collected from 15 widespread locations in turkey (fig. 1, table 1). workers were collected in 95% ethanol and subsequently air-dried. total dna was extracted from each bee’s thorax according to hall (1990). the concentration and purification of genomic dna was quantified with a nanodrop nd-1000 spectrophotometer, and 20 ng of genomic dna was used for the pcr. sequence analysis the nd5 mitochondrial gene segment was amplified according to bouga et al. (2005). the pcr products were purified using a gel purification kit (qiagen) and sequenced in both directions on an abi prism 3130 automated sequencer (applied biosystems) using standard protocols. sequences were aligned with the computer program clustal x (thompson et al. 1997). for estimates of the similarity index and evolutionary divergence between dna sequences mega5 software was used. resulting in a consensus of the phylogenetic tree, the methods of maximum parsi mony (mp) and neighbor-joining (nj) analysis were performed using the same software (tamura et al. 2011). fig. 1. sampling locations in turkey. number in parenthesis show the number of colonies sequenced at each site. 228 sociobiolog y vol. 59, no. 1, 2012 for the construction of the phylogenetic trees, we used apis cerena (accession number: nc_014295) sequences retrieved from genbank, as outgroup in order to root the trees. the sequences obtained in this study have been deposited to genbank with accession numbers jn410833 to jn410837. the resulting sequences were compared to published sequences available in genbank. results the sizes of the pcr-amplified nd5 segment for all populations studied were found to be 782 bp (primers excluded). five novel haplotypes were revealed for the nd5 gene segment in turkish honeybees. the number of variable sites was found to be 6 for this region 2 of which were parsimony informative sites. the average pairwise genetic distances were 0.3% for nd5 gene (kimura 1980). table 2 lists the different haplotypes of the nd5 gene found in this study and in martimianakis et al. 2011. the sequence information, genbank accession numbers and variable sites of these haplotypes and additional new table 1. sampling localities, geographical positions and number of colonies used for sequencing. locations abbreviation of the locations geographical position # colonies analyzed for sequence analysis adiyaman adi 37°46'n 38°16'e 7 ardahan ard 41°03'n 42°42'e 6 ankara / kazan kaz 39°58'n 32°52'e 6 ankara /auz* auz 40°12'n 32°41'e 6 antalya / elmali elm 36°44'n 29°56'e 8 balikesir bal 39°39'n 27°53'e 5 bingöl bin 39°00'n 40°41'e 6 bolu / yiğilca bol 40°58'n 31°27'e 6 hakkari hak 37°35'n 43°34'e 6 konya / akören ako 37°27'n 32°22'e 5 konya / selçuklu sel 37°57'n 32°26'e 6 konya / sizma siz 38°05'n 32°24'e 8 konya / suz ** suz 38°02'n 32°30'e 6 muş / varto mus 39°17'n 41°12'e 6 van / gevaş van 38°18'n 43°06'e 6 *auz: the apiary of the ankara university **suz: the apiary of the selçuk university 229 özdil, f. & i̇lhan, f. genetic divergence of turkish honeybees haplotypes that are found in this study are summarized in table 2. the mtdna nucleotide positions are taken from crozier and crozier (1993). the trees drawn by maximum parsimony and neighbor-joining analysis exhibited nearly the same topolog y for nd5 mtdna region so only the nj tree is presented here. at population level, sequencing of nd5 mitochondrial region has been studied less than the other mitochondrial regions. only martimianakis et al., (2011) gives information about sequencing of this region in samples from greece and some east european countries. the phylogenetic tree of greek and turkish honey bee haplotypes constructed by the nj method is shown in fig. 2. discussion several mitochondrial studies of turkish honey bee populations have been conducted (smith et al. 1997; palmer et al. 2000; kandemir et al. 2006, özdil et al. 2009a). with restriction digests, nearly all turkish colonies analyzed previously by the other surveys were found to belong to the c mediterranean fig. 2. neighbour-joining dendogram based on nd5 sequences of apis mellifera haplotypes. sequences obtained in this study are written in capital letters. 230 sociobiolog y vol. 59, no. 1, 2012 ta bl e 2. h ap lo ty pe s, g en ba nk a cc es si on n um be rs a nd v ar ia bl e si te s o f t he n d 5 re gi on o f a pi s m el lif er a l . th e va ri ab le si te s t ha t a re fo un d in th is st ud y ar e in di ca te d in b ol d nu m be rs . m td n a n uc le ot id e po si tio ns 7425** 7636 7697** 7777 7830 7968 7991** 8006 8011 8019 8026 8088 8089 8095** h ap lo ty pe s/ r ef er en ce g en b an k a cc es si on n um be rs c ro zi er a nd c ro zi er 1 99 3 n c _0 01 56 61 t c a t c a g g c t c c c t m ar tim ia na ki s et a l. 20 11 g u 06 04 66 2 * · · · a · · · · · t · t · g u 06 04 67 2 * · · · a g · · · · · · t · g u 06 04 68 2 * · · · a · · · · · · · t · g u 06 04 69 3 * · · · a · · a · · · · t · g u 06 04 70 3 * · · · · · · · · · · · · · g u 06 04 71 3 * · · · · · · · · · · · t · g u 06 04 72 3 * · · a a · · · · · · · t · g u 06 04 73 3 * t · · a · · · t c t t t · n ew – a n a t o 1 jn 41 08 33 4 a · · · a · · · · · · · t · n ew – a n a t o 2 jn 41 08 34 4 · · t · a · · · · · · · t · n ew – a n a t o 3 jn 41 08 35 4 · · · · a c · · · · · t · n ew – c a u c a 1 jn 41 08 36 4 · · · · a · · · · · · · t g n ew – m e d a 1 jn 41 08 37 4 · · · · a · · · · · · · t · 1 n c _0 01 56 6: 1 63 43 b p of a pi s m el lif er a lig us tic a co m pl et e m it oc ho nd ri al g en om e. 2 g u 06 04 66 -g u 06 04 68 : 5 53 b p of n d 5 ge ne ( ba se s fr om 7 59 5 to 8 14 7) ; 3 g u 06 04 69 -g u 06 04 73 : 6 87 b p of n d 5 ge ne (b as es fr om 7 46 1 to 8 14 7) ; 4 7 82 b p of n d 5 ge ne (b as es fr om 7 41 6 to 8 19 7) o f t ur ki sh h on ey b ee s f ou nd in th is st ud y. · i nd ic at es id en tic al n uc le ot id es a t t ha t s it e. * m ea ns th er e is n o se qu en ce in fo rm at io n at th at si te o f t he h ap lo ty pe . * * v ar ia bl e si te s t ha t a re n ew ly fo un d in th is st ud y ar e in di ca te d in b ol d nu m be rs . 231 özdil, f. & i̇lhan, f. genetic divergence of turkish honeybees lineage. this finding was expected because the turkish populations consist mainly of a. m. anatoliaca, a. m. caucasica and a. m. meda. previously, we had shown the phylogenetic relationships and pcr-rflp profile of turkish honey bees using several restriction enzymes in both the mitochondrial and nuclear genome of apis mellifera (özdil et al. 2009, 2011). in this study we presented a comprehensive sequencing analysis of the nd5 mitochondrial gene segment to verify genetic divergence in turkish honey bee populations. we found five different haplotypes for the nd5 gene segment. sequencing of the nd5 mitochondrial region has been studied less than the other mitochondrial regions. only eight different haplotypes of apis mellifera were reported in this region. here we added five new haplotypes in this study (table 2). three came from the anatolian geographical locations, one from each iranian and caucasian geographical location. while anato1 ( jn410833) haplotype was only obtained in some of the honeybees from siz population, anato2 ( jn410834) was found in both siz and elm populations. these two haplotypes were found to be the rarest anatolian haplotypes whereas anato3 ( jn410835) haplotype was found in a wide geographical area such as bal, bol, ako, sel and suz. caucasian haplotype, cauca1 ( jn410836), was obtained from ard, kaz and auz where ard was the center of caucasian honey bees. and meda1 ( jn410837) haplotype was found in honey bees from the south-east part of turkey where mostly a. m. meda is found. the base substitutions at position 7830 (c→a) and 8089 (c→t) which were reported in martimianakis et al. (2011), were observed in all of the turkish honeybees in this study. the nj dendogram (fig. 2) based on nd5 sequences of the two surveys showed that that these races cannot be discriminated from each other since all of them belong to east european (c) lineage. but it is seen that haplotype 1-4 (genbank records: gu060466-gu060469) in martimianakis et al. (2011) and the haplotypes that are found in turkish honeybees are much closer than the other haplotypes. in addition to previous findings of the nd5 gene, here we reported sequencing of this mitochondrial dna gene segment in turkish honey bees and compared these results with other east european races. our data showed 232 sociobiolog y vol. 59, no. 1, 2012 that samples from siz (konya/sızma) and ard (ardahan), having unique haplotypes, maintain their native origin and they might be pure a. m. anatoliaca and a. m. caucasica, respectively. high migratory beekeeping activity and the use of caucasian queens for queen rearing in turkey have resulted in the loss of genetic diversity. so it is highly important to identify the genetic structure of local honey bee races and improve strategies to conserve them in their areas. acknowledgments the authors would like to thank prof. dr. saim boztepe, (selçuk university, faculty of agriculture) and prof. dr. mehmet ali yildiz (ankara university, faculty of agriculture) for providing insight and guidance during various phases of this project. the authors also would like to thank hüseyin bayir for help in collecting samples from central region of turkey. references arias, m.c. & w.s. sheppard 1996. molecular phylogenetics of honey bee subspecies (apis mellifera l.) inferred from mitochondrial dna sequence. molecular phylogenetics and evolution 5: 557–566. bouga, m., p.c. harizanis, g. kilias & s. alahiotis 2005. genetic divergence and phylogenetic relationships of honey bee apis mellifera (hymenoptera: apidae) populations from greece and cyprus using pcr-rflp analysis of three mtdna segments. apidologie 36: 335–344. bouga, m., c. alaux, m. bienkowska, r. büchler, n.l. carreck, e. cauia, r. chlebo, b. dahle, r. dall’olio, p. de la rúa, a. gregorc, e. ivanova, a. kence, m. kence, n. kezic, h. kiprijanovska, p. kozmus, p. kryger, y. le conte, m. lodesani, a. m. murilhas, a. siceanu, g. soland, a. uzunov & j. wilde 2011. a review of methods for discrimination of honey bee populations as applied to european beekeeping. journal of apicultural research 50(1): 51-84. crozier, r.h. & y.c. crozier 1993. the mitochondrial genome of the honeybee apis mellifera: complete sequence and genome organization. genetics 133: 97–117. de la rúa, p., j. galián, j. serrano & r.f.a. moritz 2001. genetic structure and distinctness of apis mellifera l. populations from the canary islands. molecular ecolog y 10: 1733–1742. de la rúa, p., r. hernández-garcia, b.v. pedersen, j. galián & j. serrano 2004. molecular diversity of honeybee apis mellifera iberica l. 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apicultural research and bee world 49(2): 177–185. tamura, k., d. peterson, n. peterson, g. stecher, m. nei & s. kumar 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biolog y and evolution 28(10): 2731–2739. thompson, j.d., t.j. gibson, f. plewniak, f. jeanmougin & d.g. higgins 1997. the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids research 25: 4876–4882. doi: 10.13102/sociobiology.v62i3.650sociobiology 62(3): 396-400 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 mating behavior of the african weaver ant, oecophylla longinoda (latreille) (hymenoptera: formicidae) introduction the african weaver ant (awa), oecophylla longinoda latreille (hymenoptera: formicidae) is a predatory species which controls a wide range of insect pests in multiple crops (way and khoo, 1992; van mele, 2008). the specie is increasingly being used in biological control programs in tropical plantations (van mele, 2008). a stable and high population level is usually required for effective control of insect pests by o. longinoda (sporleder & rapp, 1998). introduction of o. longinoda into crop fields requires mapping of colonies in natural habitat, subsequent harvesting and transplanting. however, the transplantation of colonies into new plantations is hampered by the difficulties in finding the single maternal queen in colonies comprising up to several hundreds of nests (ouagoussounon et al., 2013; rwegasira et al., 2015). abstract mating in most species of ants occur during nuptial flights. in the african weaver ant, oecophylla longinoda latreille, mating has previously been hypothesized to take place within the nest before the nuptial flight but no research data has ever been presented to support this. understanding the mating strategy of o. longinoda is important for its successful application in biological control programs. here we report on the findings from studies conducted in tanzania to determine whether mating occur prior to dispersal flight. winged o. longinoda queens collected at four steps; before taking flight, immediately after leaving the nest, up to 12h after leaving the nest and after settling naturally following the dispersal flights were examined. mating in captivity with varied number of males and queens was also assessed. results showed that no eggs hatched from any of the 527 winged queens that were collected prior to their dispersal flights and no mating attempts in captivity lead to viable offspring. only eggs produced by queens collected after settling naturally (n=65) hatched into larvae. high percentages (88.73) of eggs that hatched were laid by queens that shed wings and laid their eggs within 3 days after nuptial flights. findings from the current study suggest that mating of o. longinoda queens take place during a nuptial flight and does not take place within the nest, as previously suggested. time from nuptial flights to shedding of wings and egg laying translates to hatchability of the eggs. sociobiology an international journal on social insects wa nene1, gm rwegasira1, j offenberg2, m mwatawala1, mg nielsen2 article history edited by gilberto m. m. santos, uefs, brazil received 02 december 2014 initial acceptance 21 may 2015 final acceptance 11 august 2015 keywords african weaver ant, mating behavior, nuptial flight, oecophylla longinoda. corresponding author wilson angelo nene sokoine university of agriculture department of crop science and production box 3005, morogoro, tanzania e-mail: wilsoninene@gmail.com oecophylla spp. queens disperse from their natal colonies at the onset and/or during the rainy season (vanderplank, 1960; offenberg & wiwatwitaya, 2010; peng et al., 2013). the copulation of unrelated sexual forms during nuptial flights (depa, 2006), provides an opportunity for genetic mixing and plays an important role in the life cycle of many ant species (dhami & booth, 2008). there are mainly two mating systems which have been reported in ants namely; male aggregation and female calling. male aggregation (ma) is a system that winged males and females meet during nuptial flights. females enter a swarm, which is dominated by males and subsequently mate with one or more of these males and return from the flight as fertile queens (kaspariet al., 2001). female calling (fc) is a system that winged females position themselves near their natal nests and emit pheromones to attract winged males with whom they mate, before they disperse or re-enter their research article ants 1 sokoine university of agriculture, morogoro, tanzania 2 aarhus university, aarhus, denmark sociobiology 62(3): 396-400 (september, 2015) 397 natal colony as fertile queens (kaspari et al., 2001). in rare cases, mating occur within the nest, for instance in formica aquilonia (yarrow) (forterius, 2005) and linepithema humile (myr) (aron, 2001). in independent founding species, including oecophylla spp., fertilized queens search for nesting places and shed their wings after the mating (hölldobler & wilson, 1978; dhami & booth, 2008). queens then rear their first brood by using food reserves stored in the fat body and their wing musculature (keller & passera, 1989). mating strategy of o. longinoda has never been established. understanding the mating strategy of o. longinoda is an important prerequisite for their successful use in integrated pest management (ipm), particularly in collection of mated queens to stock ant nurseries (ouagoussounon et al., 2013; peng et al., 2013). way (1954) collected alate and dealated queens of o. longinoda in the field, during the ants’ mating seasons in tanzania which laid eggs that subsequently hatched into viable offspring. however, it was not known how and when mating occurred. vanderplank (1960) hypothesized that mating by o. longinoda takes place within the nest, before dispersal of the queens. in a latter case, males liberated from their natal nests, disperse and enter into the nests of other colonies, where mating with alate queens takes place. reported facts about sexuals of the closely related asian green tree ant, oecophylla smaragdina, unveiled that mating occurs during nuptial flights (peng et al., 2013). similar case would be true to o. lonignoda but evidence based on data was required to disprove vanderplank’s hypothesis. the current study aimed at examining the mating strategies of o. longinoda by testing the fertility of queens at different dispersal stages (pre-dispersing, dispersing, postdispersing and natural settling). materials and methods the study area field experiments were conducted from 2012 to 2014 at naliendele agricultural research institute (nari) in mtwara region, southern tanzania (40º09´ 57.05” e, 10º 21´ 22.49” s). the region has a unimodal rainfall lasting from november/december to april/may and the mean annual rainfall ranges from 810 to 1090 mm. the mean maximum and minimum temperatures are 27 ºc and 23 ºc respectively. collection of queens at different stages of dispersal winged queens were caught during different stages of their dispersal from their natal nests. four different stages of nuptial flights were considered; i) pre-dispersal, ii) dispersal, iii) post-dispersal and iv) after naturally settling. in the first stage, alate queens were caught before dispersal, either from surface of the nests or from vegetation in the immediate neighborhood of nests. in the second stage, queens were collected immediately after they took off from their nests. the queens were trapped with mosquito nets that covered cashew trees in the field as well as plastic sheets that covered potted mango and cashew seedlings. in the third stage, queens that attempted to disperse from four colonies established on cashew seedlings were collected from the roof of the screen house, twelve hours after each dispersal flight. in the fourth stage, queens were collected by using artificial nests, after settling naturally as described by peng et al. (2013). in this technique, a total of 1000 artificial nests were constructed on 10 citrus trees. testing for fertility of queens collected at different stages of dispersal each collected queen was kept in a match box (53x36x17mm) placed on tables in a screen house and protected from other ants as described by way (1954). queens were provided with distilled water soaked in a cotton wool every day. a 20% sucrose solution was also provided after the first workers emerged. rearing continued for 60 days after the queens were collected. number of queens that shed wings, numbers of queens that laid eggs, and queens whose eggs hatched and developed into larvae, pupa and imago were recorded. the mortality of queens after 60 days of development was calculated and compared between the tested groups. testing for fertility of queens in captivity queens and males were collected from nest surfaces and nearby vegetation just before mating. a queen was paired with 0, 1, 2, 3, 4, or 5 males in a matchbox during the first mating season (december 2012 to april 2013). the same combinations of males were paired with two queens in a matchbox, except in the second season (december 2013 to april 2014). each combination was made of two different kinship relations; i.e with queens and males originating from the same or different colonies and replicated three times. queens and males were maintained for 60 days as in the queen fertility tests at varied stages of dispersal described above. the number of queens that shed their wings, numbers of queens that laid eggs, and queens whose eggs hatched and developed into larvae, pupa, imago workers or workers were recorded. developmental times of initial stages of weaver ants queens were collected using artificial nests placed on citrus trees at moma (040018´ 25.1” e, 100 39´ 71.7” s, 34 m asl) and sogea (040014´ 71.4” e, 100 37´ 79.1” s, 142 m asl) sites in mtwara from january to april 2015. founding queens were reared in matchboxes placed on cages in the shade house. number of queens that shed wings (n=103) as well as number of queens that laid eggs (n=103) within 3, 6 and above six days from nuptial flights were recorded. also recording wa nene, gm rwegasira, j offenberg, m mwatawala, mg nielsen – african weaver ant mating behavior398 was done on number of queens with hatched eggs from 10th to 14th and beyond 14th days after nuptial flights (n=69). the temperature during rearing period ranged between 28 and 29 0c. queens were provided with distilled water ad libitum. data analysis collected data were analysed using jmp 10.0 software (sas, 1995). chi square was used to test dependency of wing shedding, egg laying, egg hatching and queens’ survival, on different stage of queen dispersal. the dependency of wing shedding, egg laying and egg hatching, on days from nuptial flights were also analysed by chi square test. results fertility status and survival of collected queens all collected queens produced eggs, regardless of stage of collection. egg production was not dependent on stage of collection in both the first (x2=7.418, p=0.06) and second (x2=5.88, p=0.12) season. only queens that settled naturally after dispersal flights produced viable eggs (fig 1). the proportion of queens that produced eggs was higher than the proportion that shed their wings (fig 1). the proportion of queens that shed their wings varied between 60.6 and 100%. shedding of wings was significantly dependent on stage of queen collection in both first (χ2=24.83; p<0.0001) and second season (χ2= 28.60; p<0.0001). the highest proportion of queens that shed wings were those collected after settling naturally. results on survival of queens collected at the different stages of dispersal are presented in fig 2. in all cases, survival was above 88 %, except for queens that settled naturally after dispersal (73.7% to 94.1). survival of queens was significantly dependent on stage of collection in the first season (χ2=8.2, p=0.043) but not in the second season (χ2=0.80, p=0.85). queens collected pre-dispersal to 12 hours after dispersal had higher survival rates while those collected after settling naturally had lowest survival rate. mating in the captivity none of the males that were introduced to alate queens in match boxes survived, regardless of the combination. males died within approximately two days after they were placed in the boxes of alate queens. dead males were removed from the rearing chamber (match boxes). many of the test queens (n=83) kept in captivity shed off their wings and initiated egg laying but none of them (n=90) produced viable eggs. developmental times of eggs and wing shedding all the collected queens (experiment conducted from january to april 2015) laid eggs and shed their wings. however, only eggs from 71 queens were hatched out of 103, making eggs hatching successful by 68. 93 %. wing shedding was significantly dependent on time from nuptial flights (table 1). more queens shed their wings within 3 days after nuptial flights. similarly, egg laying was significantly dependent on time from nuptial flights (table 1). more queens (68.93%) laid eggs within 3 days after flights. a high percentage of eggs that hatched (88.73) were laid by queens that shed their wings and laid eggs within 3 days after nuptial flights (χ2=21, p=<0.0001, n=71). only 11.27% of eggs that hatched were laid by queens between 4th and 6 days after nuptial flights. egg hatching was also dependent on days from nuptial flights (table 2). a significantly high number of queens had their eggs hatched from day 11 to 13 after nuptial flights. no hatching was recorded in eggs laid 6 days after nuptial flights. fig 2. survival rates of queens caught at different stages of dispersal from their natal nests in 60 days of rearing. fig 1. reproductive success of queens collected at different dispersal stages. sociobiology 62(3): 396-400 (september, 2015) 399 discussion in this study only the queens that dispersed and subsequently settled naturally were able to produce viable offspring. these queens had an opportunity to fly to heights beyond 10 meters (the size of a shade house). this observation suggests that mating took place after their dispersal from the nest and that a certain flying height was needed before mating occurred. this correlates well with the australian species, o. smaragdina that mate at some greater heights in the air and not inside their nests (m.g. nielsen et al., unpublished data). mating at great heights is not a unique character to oecophylla species. other ant species are known to fly high before mating. for instance, solenopsis invicta has been reported to reach at height of 60-100 m during their mating flights (dhamb & booth, 2008). the current study has shed light on the myth that surrounded the collected alate oecophylla queens that produced viable offspring (way, 1954; lokkers, 1990) without clear understanding of the dispersal stage at which the queens were collected. it also brings to an end the speculation (vanderplank, 1960) that mating by o. longinoda on zanzibar took place inside their nests before dispersal. since vanderplank’s alate queens were reportedly collected from the nest surface and that the queens subsequently produced viable offsprings with imago workers. queens were collected after nuptial flights, and were most probably founding queens. centrally to that, the difference between vanderplank’s and the present study might be hard to explain unless behavioral variation among populations exists. if the behavior observed by vanderplank (1960) holds true, it would be a rare strategy only used by few species and not even by the sister species, o. smaragdina (peeters & molet, 2010). in the present study, many of the infertile queens shed their wings and laid eggs. thus, wing shedding and egglaying cannot be used as indicators of fertility or viability among o. longinoda queens. it is surprising that queens shed their wings while still unmated as they would need them in subsequent attempts to locate potential mating partners. furthers studies would be needed to unveil triggers for wing shedding in queens which occurred regardless of mating or fertilization of the queen’s eggs. queens that were allowed to settle naturally had lower survival rates exhaustion due to higher energy expenditure. as these queens had been on a mating flight and subsequently fed their developing larvae with food derived from their body reserves. they used more energy than queens that did not participate in mating flights. this higher energy consumption led to a slightly lower survival (king’ori, 2012). all the founding queens laid eggs and shed their wings but only eggs from 71 queens (68.93%) were hatched. thus, mating for viable offsprings may sometimes be unsuccessful. we hypothesized that hatching of the laid eggs is affected by the environmental conditions that prevailed in rearing units particularly the temperature. further research might be needed to confirm this supposition. time from nuptial flights to egg laying and shedding wing was associated with eggs hatching. early laid eggs hatched successfully unlike late laid ones. no eggs hatched from any queen that started laying eggs beyond 6th day after the flight. high percentage (88.73%) of queens with hatched eggs laid eggs within three days following nuptial flights. this means that, eggs hatching can be predicted by more than 88 % within three days rearing. variation in time taken to egg hatching was also noted. more egg hatches were observed in the 11th, 12 and 13th days, with the highest on the 13th day. these results contrast with vanderplank (1960) who reported that, time from nuptial flights to egg hatching is the same for all new queens reared at the same temperature. he reported that eggs from 24 queens reared at 28 ºc took 6 and half days to hatch where as at 33 ºc took only 4 days. we therefore conclude that mating is most likely to take place after queens disperse. this is further supported by the fact that attempts to mate queens in artificial nests was unsuccessful, and that several hundred attempts to mate australian and thai o. smaragdina sexuals in artificial enclosures have been, equally, unsuccessful (morgens g. nielsen, personal communication). eggs hatch day number of queens hatched % queens hatched statistics day 10 2 2.9 day 11 11 15.94 day 12 16 23.18 χ 2=60.07, df, 5, p=<0.0001 day 13 32 46.38 day 14 4 5.8 above 14 4 5.8 total 69 100 table 2: time taken to hatching of eggs into larvae. duration (days) 1-3 days 4-6 days >6 days statistic n % shedding wings 78.64 (81) 8.74 (9) 12.62 (13) χ2=52.12, df, 2, p=<0.0001 103 % laying eggs 68.93 (71) 13.59 (14) 17.48 (18) χ2=67.79, df, 2, p=<0.0001 103 table 1. time taken for initiation of wing shedding and egg laying in founding o. longinoda queens. wa nene, gm rwegasira, j offenberg, m mwatawala, mg nielsen – african weaver ant mating behavior400 acknowledgements this study was funded by danida through project dfc 10-025 au. we thank prof. renkang peng, for his technical guidance during experimental design. messrs. hasan libubulu and frank thadeo are acknowledged for their assistance in data collection. we thank naliendele agricultural research institute for providing the fields and screen houses for the experiments as well as working facilities. references aron, s. 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(1992). role of ants in pest management. annual review of entomology, 37: 479-503. way, m.j. (1954). studies of the life history and ecology of the ant oecophylla longinoda latreille. bulletin of entomological research, 45: 93-112. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.866sociobiology 62(4): 533-537 (december, 2015) pheidole protaxi sp. nov. (hymenoptera: formicidae), new species from tabuleiro forests of the atlantic forest biome introduction with more than 1,000 valid species in the world, and many others awaiting description, pheidole westwood, 1839 (formicidae: myrmicinae: attini) is one of the largest ant genera (longino, 2009). this genus has a worldwide distribution, and in the new world where more than 600 species were described, it is very abundant in all ecosystem (wilson, 2003). its species exhibit a wide range of foraging behaviors, nesting habits, and colony structure (wilson, 2003). here we describe a new species of pheidole after the morphology of its minor and major worker from southern bahia, brazil. this was discovered in a remnant fragment of forest belonging to the ecological system known as “floresta ombrófila densa das terras baixas” (ibge, 2012) or “floresta pluvial dos tabuleiros” (thomas, 2003). this ecosystem is a variety of the lowland ombrophilous forests from the central corridor of atlantic forest biome (thomas, 2003). locally, it covers large sedimentary plains of the barreiras formation, being located primarily south of the rio jequitinhonha south to the rio doce in espírito abstract hereafter a new species of pheidole westwood, 1839 (hymenoptera: formicidae) is described from southern bahia in brazil, after the morphology of its minor and major workers. this taxon is easily distinguished from any other congeneric species by a unique combination of characters. sociobiology an international journal on social insects ml oliveira1,2,3, csf mariano1,3, ma costa1, jhc delabie1,3, s lacau1,2 article history edited by kleber del-claro, ufu, brazil received 07 july 2015 initial acceptance 21 october 2015 final acceptance 07 december 2015 keywords taxonomy, pheidole westwood, 1839, protax. corresponding author muriel lima de oliveira universidade estadual do sudoeste da bahia laboratório de biossistemática animal itapetinga-ba, brazil e-mail: mlopheidole@gmail.com santo (thomas, 2003). they are flat formations with deep soils, bearing forests relatively tall (canopy of 40 m), but with some very large trees on flat terrain (thomas, 2003) and a few vegetal density in undergrowth, facilitating locomotion (ibge, 2012). the flora is relatively well characterized by ecotypes of the genera ficus, alchornea, handroanthus and ochlospecies tapirira guianensis aubl. (ibge, 2012), and the jussara palm euterpe edulis (thomas, 2003). while the floristical components of the tabuleiro forests are relatively well known, knowledge on the fauna is comparatively very poor, especially about arthropods. like this, there are still very few published studies on the diversity of ants in these forests. however, several standardized collects made in some remnant fragments in southern bahia, revealed a very high taxonomic diversity for the ants of leaf-litter, both at specific and generic level (lacau et al., in prep.). in this context, the present description of a new pheidole species contributes to increase the knowledge on the diversity of ants in this ecosystem. this paper is a partial integrative result of the research projects “contribution to the study of the ants fauna in lowland ombrophilous forests from the research article ants 1 universidade estadual de santa cruz (uesc), ilhéus-ba, brazil 2 universidade estadual do sudoeste da bahia (uesb), itapetinga-ba, brazil 3 u.p.a. laboratório de mirmecologia, convênio ceplac, ilhéus-ba, brazil ml oliveira, csf mariano, ma costa, jhc delabie, s lacau – pheidole protaxi sp. nov., new species from tabuleiro forests534 atlantic forest biome” and “hyperdiversity of the genus pheidole westwood (hymenoptera: formicidae) in bahia: emphasis on the atlantic forest” (mct/cnpq/dgp/geban), and “a biossistemática aplicada ao estudo das formigas (hymenoptera: formicidae) como instrumento de formação e capacitação em taxonomia integrativa” (uesc/ppg-zoo & mct/cnpq and mec/capes, edital nº 52/2010 – protax). material and methods field work was performed in november 2011, collecting manually the specimens using a pooter, featherweight forceps and a fine brush. all specimens were caught in a single nest. morphological examination of specimens was completed at various magnifications using a light stereomicroscope olympus szx7. morphological data were compared between species by using the bioinformatics software xper3 ©lis (http://www. xper3.fr/). morphometric measures were made with a carl zeiss measuring microscope and recorded to the nearest 0.01 mm. all measurements are given in millimeters, using the following definitions and abbreviations: el eye length: the maximum diameter of the eye. gl gaster length: the length of the gaster in lateral view from the anteriormost point of first gastral segment (third abdominal segment) to the posterior most point. hl head length: maximum distance from the midpoint of the anterior clypeal margin to the mid-point of the posterior margin of the head, measured in full-face view. hla1 anterior head length: in full-face view, perpendicular distance between two horizontal lines, one tangent to anteriormost projection of clypeus, one tangent to lowermost margin of compound eye. hla2 anterior head length: in full-face view, perpendicular distance between two horizontal lines, one tangent to anteriormost projection of cephalic capsule (excluding the anterior margin of clypeus), one tangent to lowermost margin of compound eye. hlp depth concavity vertexal median: in full-face view, perpendicular distance between two horizontal lines, one tangent to dorsalmost of vertexal lobes projection, and other passing by line from vertexal to bottom of groove. hw head width: the maximum width of the head in full face view. hwt width of head in height of torulus: measured in full-face view, the distance between the side edges of the head, the height of an imaginary line that passes through the torulus. mdl mandible length: length of a mandible measured in ventral view from its basal articulation to its apex. mfl metafemur length: maximum length of metafemur, measured from the junction with the trochanter to the junction with the tibia. pph postpetiole height: maximum height of postpetiole, measured in lateral view. ppl postpetiole length: the maximum length of postpetiole, in lateral view. ppw postpetiole width: maximum width of postpetiole, measured in dorsal view. psl propodeal spine length: maximum length of propodeal spines, measured in lateral view of the same pth petiolar node height: maximum height of petiolar node measured in lateral view. ptl petiole length: the maximum length of the petiole in lateral view. ptw petiolar node width: maximum petiolar node width, measured in dorsal view. pw pronotal width: maximum width of pronotum measured in dorsal view. sl scape length: maximum scape length, excluding basal condyle and neck. spl propodeal spiracle width: measured from the outer edge of ring that surrounds orifice. wh mesosoma height: distance between the inner base pro-thigh and the top edge of the dorsal prothorax. wl weber’s length: diagonal length, measured in lateral view, from the anterior margin of the pronotum (excluding the collar) to the posterior extremity of the metapleural lobe. the data for holotype are given in [brackets]; for the paratypes, the means with standard deviations are given in (parenthesis) and the maximum range observed are in { }. when available, direct comparisons with the morphology of other pheidole species were made through study of high-resolution microphotographs of type and non-type specimens in the following websites: ants of costa rica (http://academic.evergreen.edu/ projects/ants/genera/pheidole/specieslist.html), antweb http:// www.antweb.org/description.do?name=pheidole&rank=genus), mcz (http://140.247.119.225/mcz/recordlist.php), smithsonian (http://ripley.si.edu/ent/nmnhtypedb/public/search.cfm). standard microphotographs were made using the following sequential process: the specimen was first filmed using a video camera (sony fullhd1080 avchd, 10. 2mp) mounted on a light microscope (zeiss jena), while the resolution was continuously scanned from the top to the bottom of the holotype specimen; the videos (in format “.mts”) were processed using the free software imagegrab 5.0 (available at http://paul.glagla. free.fr/imagegrab.htm) in order to extract the sharpest images referable to differing focal points, and composite pictures were then assembled using the free software combine zm (available at http://www.hadleyweb.pwp. blueyonder.co.uk/index.htm). finally, each optimum microphotograph was improved using adobe element photoshop software (version 6. 0). depending of the morphological structure considered, morphological concepts and terminology in this paper follow richards (1956), eady (1968), harris (1979), gauld and bolton (1988), goulet and huber (1993), kugler (1994), bolton (1994), eguchi (2008), wilson (2003) and keller (2011). taxonomical nomenclature follows bolton et al. (2014). because of the messy state of the internal taxonomy for sociobiology 62(4): 533-537 (december, 2015) 535 the neotropical species of pheidole after that moreau et al. (2008) showed that nine of the 19 species groups suggested by wilson (2003) are not monophyletic, no attempt will be done in this paper for classifying the new species here described in any species-group. taxonomic information and specimens were managed by using the bioinformatics software mantis® version 2.0 (http://140.247.119.138/mantis/) (naskrecki, 2008). holotype and all paratypes associated with the new species described here have unique specimen-level identifiers affixed to each pin, being written in the text as: “[lbsa_sa_specimen codes]”. depository collections are referred to by the following acronyms: cpdc, centro de pesquisas do cacau, comissão executiva do plano de lavoura cacaueira (ceplac), itabunaba, brazil; inpa, instituto nacional de pesquisas da amazonia, manaus-am, brazil; mcz, museum of comparative zoology, harvard university, cambridge, massachusetts, usa; mnhn, muséum national d’histoire naturelle, paris, france; mpeg, museu paraense emilio goeldi, belém, pará, brazil; mzsp, museu de zoologia da universidade de são paulo, brazil. results class: insecta order: hymenoptera family: formicidae latreille, 1809 subfamily: myrmicinae lepeletier de saint-fargeau, 1835 tribe: attini smith, 1858 genus pheidole westwood, 1839 pheidole protaxi oliveira and lacau, new species (figs 1-3) http://zoobank.org/nomenclaturalacts/72a068980caf-4211-8228-39e918b888ca fig 3. pheidole protaxi sp. nov., habitus: dorsal view. e: holotype major worker, [lbsa_sa_14013667]. f: paratype minor worker, [lbsa_sa_14012040]. fig 1. pheidole protaxi sp. nov., head: full-face view. a: holotype major worker, [lbsa_sa_14013667]. b: paratype minor worker, [lbsa_sa_14012040]. fig 2. pheidole protaxi sp. nov., habitus: left lateral view. c: holotype major worker, [lbsa_sa_14013667]. d: paratype minor worker, [lbsa_sa_14012040]. type material: holotype (major worker, cpdc: [lbsa_ sa_14013667]): brasil, bahia, porto seguro, ceplac/cepec/ estação ecológica do pau brasil (espab) (16°23’50’’s, 39°10’28’’w), elev. 101 m, 5-6.11.2011, col. m.l. oliveira, y.a.m. velasco, & b.j.b. jahyny. paratypes: 6 major workers and 7 minor workers with the same data as holotype (one major worker and two minor workers deposited in cpdc: ml oliveira, csf mariano, ma costa, jhc delabie, s lacau – pheidole protaxi sp. nov., new species from tabuleiro forests536 [lbsa_sa_14014469; lbsa_sa_14012040; lbsa_sa_ 14014463], one major worker and one minor worker in inpa: [lbsa_sa_14014470; lbsa_sa_14014464], mcz: [lbsa_sa_14014471; lbsa_sa_14014465], mnhn: [lbsa_sa_14014472; lbsa_sa_14014466], mpeg: [lbsa_sa_14014473; lbsa_sa_14014467] and mzsp: [lbsa_sa_14014474; lbsa_sa_14014468]. etymology. the name of this new taxon, “protaxi”, is a latinization of the “protax” name, brazilian acronym for “programa de capacitação em taxonomia”, meaning: program of capacitation in taxonomy”. diagnosis. the minor and major workers of this new species exhibit all the diagnostic characters of the genus pheidole. it differs from all other known species by the unique following combination of characters. major worker – head in dorsal view: with a quadrate shape, as long as wide; vertexal margin deeply and widely concave. frontal carinae short and straight, posteriorly diverging, their tips reaching the imaginary transversal line crossing anterior edge of eyes. head in ventral view: submedian teeth of hypostomal carina with triangular shape, slightly blunt and strongly divergent. head in lateral view: median part of vertex with sub-rectilinear outline, slightly convex. antennae with 12 segments; scape relatively short, its tip not reaching the mid-point between posterior edge of eye and vertexal corner. mesosoma in dorsal view: with pronotum and mesonotum completely fused, the lateral margins with outline drawing a losange. mesosoma in lateral view: pronotum and mesonotum completely fused, the dorsal face with outline forming a single convexity. pronotum in posterior view: dorsal face with outline slightly convex; lateral faces with outline strongly concave. propodeum in lateral view: spines relatively short, postero-laterally directed. postpetiole (3rd abdominal segment) in dorsal view: slightly wider than petiole (2nd abdominal segment). first gastral segment (fourth abdominal segment) in dorsal view: anterior margin with outline convex. vertex almost entirely smooth and shining, except the median longitudinal sulcus with areolate microrugulae (no other sculpturing types). clypeus with a short longitudinal carina between midpoint of posterior edge and its anterior third; median and lateral parts separated by two symmetrical pairs of straight carinae, diverging anteriorly. mesosoma almost entirely sculptured with areolate microrugulae, except median part of its dorsal face, posterior part of the lateral faces of pronotum, and median part of katepisternum that are smooth and shining. antero-dorsal part of pronotum with fine transversal rugulae. petiole wholly sculptured with areolate microrugulae. postpetiole nearly all sculptured with areolate microrugulae, except its dorsal face. minor worker head in dorsal view: frontal carinae short and straight, posteriorly diverging, their tips reaching the imaginary transversal line crossing anterior edge of eyes. head in postero-lateral view: occipital carina present but discrete. antennae with 12 segments; scape reaching, but not exceeding, posterior margin of head. mesosoma in dorsal view: pronotal humerus developed as blunt tubercles. mesosoma in lateral view: pronotum and mesonotum completely fused, the dorsal face with outline forming a single convexity. propodeum in lateral view: spines relatively short, postero-laterally directed. postpetiole (3rd abdominal segment) in dorsal view: same width as petiole (2nd abdominal segment). first gastral segment (fourth abdominal segment) in dorsal view: anterior margin with subrectilinear outline, slightly concave. head almost entirely smooth and shining, except some rugae and areolate microrugulae on the dorsal part of preocular genae. mesosoma almost entirely sculptured with areolate microrugulae, except a small circular area smooth and shining on lateral faces of pronotum. petiole wholly sculptured with areolate microrugulae. postpetiole entirely smooth and shining. measurements. major workers (n= 7): el: [0,11] (0,12±0,01) {0,11-0,14}, gl: [0,78] (0,78±0,13) {0,54-0,92}, hl: [0,80] (0,88±0,04) {0,8-0,93}, hla1: [0,22] (0,25±0,02) {0,22-0,28} hla2: [0,16] (0,18±0,02) {0,15-0,21}, hlp: [0,07] (0,10±0,02) {0,07-0,12}, hw: [0,79] (0,85±0,03) {0,79– 0,90}, hwt: [0,64] (0,69±0,03) {0,64-0,72}, mdl: [0,38] (0,39±0,02) {0,35-0,41}, mfl: [0,54] (0,55±0,02) {0,52-0,58}, pph: [0,13] (0,15±0,01) {0,13-0,17}, ppl: [0,13] (0,13±0,01) {0,11-0,15}, ppw: [0,13] (0,14±0,01) {0,13-0,15}, psl: [0,08] (0,09±0,02) {0,06-0,10}, pth: [0,17] (0,18±0,01) {0,17-0,20}, ptl: [0,22] (0,23±0,01) {0,21-0,25}, ptw: [0,10] (0,11±0,01) {0,09-0,12}, pw: [0,37] (0,40±0,02) {0,37-0,43}, sl: [0,42] (0,43±0,03) {0,38-0,47}, spl: [0,05] (0,04±0,01) {0,03-0,05}, wh: [0,36] (0,39±0,03) {0,36-0,44}, wl: [0,73] (0,73±0,02) {0,70-0,77}. minor workers (n= 7): el: (0,09±0,01) {0,090,10}, gl: (0,48±0,06) {0,40-0,58}, hl: (0,48±0,02) {0,440,50}, hla1: (0,18±0,01) {0,16-0,19} hla2: (0,11±0,02) {0,08-0,13}, hw: (0,46±0,02) {0,44–0,48}, hwt: (0,40±0,02) {0,37-0,43}, mdl: (0,28±0,01) {0,26-0,30}, mfl: (0,43±0,02) {0,40-0,45}, pph: (0,10±0,01) {0,08-0,12}, ppl: (0,09±0,01) {0,08-0,10}, ppw: (0,10±0,01) {0,09-0,11}, psl: (0,07±0,01) {0,05-0,08}, pth: (0,14±0,01) {0,12-0,15}, ptl: (0,19±0,02) {0,16-0,20}, ptw: (0,07±0,01) {0,06-0,08}, pw: (0,30±0,01) {0,30-0,31}, sl: (0,39±0,02) {0,35-0,42}, spl: (0,03±0,00) {0,03-0,04}, wh: (0,31±0,02) {0,28-0,34}, wl: (0,59±0,02) {0,56-0,62}. geographic range. brazil: southern bahia state. biology. this species is only known from the type series which specimens were collected in a leaf litter sample of a great primary remnant fragment of tabuleiro forest (more than 7.000 ha). the colony was nesting between leaves, under a small rotten wood. discussion the recent revisionary works of wilson (2003) and longino (2009), and the availability on-line of a large number of microphotographs of the type-material for nearly all valid neotropical pheidole species, have turned easier their identification. thus, based on all available morphological sociobiology 62(4): 533-537 (december, 2015) 537 information, a comparative study with others congeneric species showed that pheidole protaxi sp. nov. is unique by the whole combination of characters offered in its diagnosis, and, for this, cannot be confused with any other species of the genus. acknowledgements we thank to y.a.m. velasco and b.j.b. jahyny for their help in the field. also, we are very grateful to capes (mec agency, brazilian government) and to cnpq (mct agency, brazilian government) for a scholarship granted to the first author and a financial support to the research project in which the new species is here described (mct/cnpq-mec/ capes edital nº 52/2010protax). by this, we wished dedicate this new species here described to all team members and creators of the protax, program which represents a very important politic action of the brazilian government, in order to combat the dramatic state of taxonomic impediment that suffers this country. this program is of extreme importance for training new taxonomists in brazil, aiming to increase the national ability of biologists to identify and catalog the biodiversity, thus making possible the elaboration of an efficient and rational policy to protect all ecosystems strongly endangered by the sixth mass extinction. mac and jhcd acknowledge their research grants fromcnpq. references bolton, b. (1994). identification guide to the ant genera of the world. harvard university press, cambridge, mass, usa, 222p. bolton, b. (2014). an online catalog of the ants of the world. http://antcat.org. (accessed date: 6 june ,2015). eady, r. d. (1968). some illustrations of microsculpture in the hymenoptera. proceedings of the royal entomological society of london, 43: 66-72. eguchi, k. (2008). a revision of north vietnamese species of the ant genus pheidole. zootaxa, 1902:1-118. gauld, i. & bolton, b. (1988). the hymenoptera. oxford university press, oxford .12 + 322 pp. goulet, h. & huber, j. t. (eds.) (1993). hymenoptera of the world: an identification guide to families. research branch, agriculture canada publication 1894/e. ottawa: canada communications group, vii + 668 pp. harris, r. a. (1979). a glossary of surface sculpturing. occasional papers on systematic entomology, 28: 1-31. ibge (2012). manual técnico da vegetação brasileira: sistema fitogeográfico, inventário das formações florestais e campestres, técnicas e manejo de coleções botânicas, procedimentos para mapeamentos. instituto brasileiro de geografia e estatística, rio de janeiro, 2a ed, ibge 275p. keller, r. a. (2011). a phylogenetic analysis of ant morphology (hymenoptera: formicidae) with special reference to the poneromorph subfamilies. bulletin of the american museum of natural history, 355: 1-90. kugler, c. (1994). a revision of the ant genus rogeria with description of the sting apparatus (hymenoptera: formicidae). journal of hymenoptera research, 3: 17-89. longino, j. t. (2009). additions to the taxonomy of new world pheidole (hymenoptera: formicidae). zootaxa, 2181: 1-90. moreau, c. s. (2008). unraveling the evolutionary history of the “hyperdiverse” ant genus pheidole (hymenoptera: formicidae). molecular phylogenetics and evolution, 48: 224-239. doi:10.1016/j.ympev.2008.02.020 naskrecki, p. (2008). mantis v. 2.0 a manager of taxonomic information and specimens. url: http://insects.oeb.harvard. edu/mantis. richards, o. w. (1956). hymenoptera – introduction and keys to families. handbooks for the identification of british insects. proceedings of the royal entomological society of london, 6: 1-94. thomas, w. w. (2003). natural vegetation types in southern bahia. in: prado p.i., landau e.c., moura, r.t., pinto, l.p.s., fonseca, g.a.b., alger, k. (orgs.). corredor de biodiversidade da mata atlântica do sul da bahia. publicação em cd-rom, ilhéus, iesb/ci/cabs/ufmg/unicamp. wilson, e. o. (2003). pheidole in the new world. a dominant, hyperdiverse ant genus. harvard university press, cambridge, mass, 794 p. doi: 10.13102/sociobiology.v67i2.4851sociobiology 67(2): 186-200 (june, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the soil harbors a large diversity of invertebrates that coexist in small areas, utilizing resources and occupying microhabitats above and below the surface (lavelle, 1996; ettema & wardle, 2002; nielsen et al., 2010). most soil invertebrates have relatively short life spans and respond to slight changes in edaphic environments, such as soil humidity and physical properties (stork & eggleton, 1992; moldenke et al., 2000; bardgett, 2002). in addition, the soil fauna can modify the surrounding environment and increase its value to other organisms (stork & eggleton, 1992; lavelle et al., 1997). among invertebrates, ants are considered good indicators of changes in terrestrial environments (folgarait, 1998; andersen & majer, 2004; underwood & fisher, 2006), abstract in the amazon basin, ants are often associated with environmental or edaphic factors. however, these associations may vary between the epigeic and hypogeic strata. here, we investigated differences in richness and composition of epigeic and hypogeic ant assemblages along an environmental gradient in the brazilian amazon. the four studied sites cover different topographic and soil characteristics. we sampled 25 plots of 250 m2 using 10 samples of epigeic pitfalls and 10 samples of hypogeic pitfalls installed at two depths (10 and 30 cm). the pitfalls remained in the field for 48 hours. in the same plots, soil clay content and terrain altitude were also measured. we collected 219 species or morphospecies, of which 14 were exclusively hypogeics. we found higher local richness in the epigeic compared to hypogeic assemblages. we also found an interaction between clay content and strata for ant species composition. overall, the species turnover was related to clay content, but the effect depended on the strata, with hypogeic fauna being more heterogeneous, compared with epigeic fauna. despite the relationship between clay content and ant´s assemblage’s composition, we did not find strong environment predictors for both strata, which suggests that other factors may structure ant assemblages in these sites. this reinforces the need for studies to define which environmental gradient determines the distribution of amazonian epigeic and hypogeic ants. sociobiology an international journal on social insects mt torres1, jlp souza2, fb baccaro3 article history edited by gilberto m. m. santos, uefs, brazil received 31 october 2019 initial acceptance 05 januarty 2020 final acceptance 09 march 2020 publication date 30 june 2020 keywords amazonia, community ecology, environment gradient, stratification, subterranean. corresponding author jorge luiz pereira souza instituto nacional da mata atlântica av. josé ruschi nº 04, cep: 29650-000 santa teresa, espírito santo, brasil. e-mail: souza.jorge@gmail.com and are relatively easy to collect and identify (majer, 1983; alonso & agosti, 2000). ant diversity and abundance can be affected locally by food resource availability and microhabitat specificities for colony establishment, which, with few exceptions, are stationary (kaspari & weiser, 2000; armbrecht et al., 2004). the distribution of nests in the tropical forest leaflitter is often gregarious, where grounddwelling ants concentrate their activities on resource patches and may respond to variation in edaphic factors, litter cover, humidity, vegetation cover and composition (levings & franks, 1982; kaspari & weiser, 2000; soares & schoereder 2004; vasconcelos et al., 2003). however, these environmental factors may not affect all ant species in a similar manner. for instance, hypogeic ant species usually nest, forage and migrate in below ground (silva & silvestre; 2004; rabeling et al., 2008), 1 secretaria de estado da saúde do tocantins (ses-to), palmas, tocantins, brazil 2 instituto nacional da mata atlântica (inma), santa teresa, espírito santo, brazil 3 programa de pós-graduação em entomologia, instituto nacional de pesquisas da amazônia (inpa), manaus, amazonas, brazil research article ants distribution of epigeic and hypogeic ants (hymenoptera: formicidae) in ombrophilous forests in the brazilian amazon sociobiology 67(2): 186-200 (june, 2020) 187 and have a number of characteristics related to low light environments and restricted space for movement (fowler & delabie, 1995; weiser & kaspari, 2006). these features include small body size, relatively short legs and narrowed body, smaller or absent eyes (often present only in the form of eyespots), and cuticle pallor (rabeling et al., 2008; andersen & brault, 2010; silva & brandão, 2010). hypogeic ants may also respond to variation in soil properties (oliveira et al., 2009) or soil-related variables, such as water-table fluctuation (baccaro et al., 2013). however, some studies have not detected any effect of soil properties on underground ants (e.g. jacquemin et al., 2012). in central amazonia the topography is closely-related to soil properties, where the bottomlands (valleys) harbor sandy soils and higher areas (plateaus) have soil with higher clay content (chauvel et al., 1987). topographic variation also influences soil moisture and forest cover (ranzani, 1980; luizão et al., 2004), which are good predictors of the distribution of epigeic ant assemblages in the amazon (vasconcelos et al., 2003; oliveira et al., 2009) and how ants interact with other organisms (dáttilo et al., 2013). these edaphic variables are related to primary production and the availability of nesting sites (vasconcelos et al., 2003; luizão et al., 2004; kaspari & yanoviak, 2008). soil type, texture and moisture can structure epigeic and hypogeic myrmecofauna, these factors are linked with soil compaction and gas exchange in soil organisms (ettema & wardle, 2002; decaëns, 2010). however, these factors may have different effects depending on the guild or microhabitat studied (austin, 1985). several papers have already indicated strong differences between epigeic and hypogeic ant fauna (fowler et al., 2000; andersen and brault, 2010; ryder wilkie et al., 2007; martins et al., 2020), which could lead to different responses even when subjected to similar environmental gradients (wilkie et al., 2010). however, most of the information on the effect of environmental gradients comes from grounddwelling ants (e.g. vasconcelos et al., 2003; oliveira et al., 2009; gomes et al., 2018), so the knowledge regarding how hypogaeic ants are distributed along environmental gradients is still incipient (jacquemin et al., 2012). here, we investigated the responses of epigeic and hypogeic ant assemblages (richness and composition) associated with edaphic gradients in four sites in the brazilian amazon. we expected a strong difference between hypogeic and epigeic ant species richness and composition, which could be correlated with different environmental predictors. specifically, we hypothesized interaction between edaphic factors (clay content and elevation) with strata (epigeic and hypogeic) for ant species richness and composition. material and methods study sites we investigated the distribution of epigeic and hypogeic ant richness and assemblage composition in four sites associated with the brazilian biodiversity research program (ppbio). these four sites are distributed along ~ 450 km, with a minimum distance of 50 km from one another, covering different topographies, soil characteristics and phytophysiognomies. the rio amazonas, separate the ufam from the other three sites, which are located along the br-319 highway (figure 1). all sites have seasonal rainfall, with rain concentrated from october to march (de marques-filho et al. 1981). coordinates, vegetation type, elevation range, rainfall and spatial layout of the study sites are summarized in table 1. fig 1. location of the four sampling sites in the brazilian amazon. mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution188 sampling design ants were sampled in permanent plots with ten samples each using the two sampling techniques. in total, we took 500 samples from 25 plots (table 1). we used the rapeld sampling design, which is based on a system of trails and permanent plots where a diverse range of taxa can be sampled (magnusson et al. 2005, 2013; costa and magnusson 2010). the permanent plots are 250 m2 (250 m x 1 m) and positioned to follow terrain contours to minimize the effects of topographical variation within plots. in each site, plots were 1 km distant from each other. the number of 250-m long sampled plots varied between areas (table 1), but all had the same standardized spatial design and were subject to the same sampling effort. table 1. vegetation, rainfall, elevation range, number of samples and sampling techniques in four sites in the brazilian amazon. sites ufam orquestra capanã jari coordinates 02°38’26.51”s 04°59’2.39”s 05°36’36.00”s 05°58’11.99”s 60°5’44.55”w 61°34’30.00”w 62°12’0.00”w 62°29’24.00”w vegetation type dense ombrophilous dense ombrophilous dense ombrophilous dense ombrophilous (number of 250-m long plots) forest (27) forest (5) forest (5) forest (5) elevation range (m.a.s.l.) 42-130 36-61 70-72 70-72 mean rainfall (mm) 2362 2200 2200 2200 sampling-grid area (km2) 24 5 5 5 number of plots with pitfall traps 10 5 5 5 number of plots with hypogeic traps 10 5 5 5 total of samples 200 100 100 100 sampling date september/2011 november/2010 november/2010 november/2010 ant sampling and identification epigeic ants were sampled with pitfall traps, consisting of plastic containers (6.5 cm diameter, 8 cm high, 500 ml volume) installed at ground level. the pitfall traps contained 70% alcohol to preserve the collected material and a few drops of detergent to decrease surface tension. the traps remained in the plots for 48 hours (souza et al., 2012). one pitfall-trap was installed in the ten sampling points in each plot. hypogeic ants were sampled using underground traps with canned commercial sardine oil as bait. these traps shared the basic features of the traps used by andersen and brault (2010) and schmidt and solar (2010). the underground traps used were composed of a plastic tube, 12 cm long, 3 cm in diameter and 50 ml volume (figure 2a). all tubes had 12 holes in their sides randomly positioned in their upper portion. each underground trap had an attractive bait inside. the traps were filled with 15 ml of 70% alcohol mixed with detergent. using an auger, two underground traps were installed, one above the other. one trap was installed at approximately 10 cm and the other at 30 cm below the soil surface (figure 2b). thus, a total of 20 traps were installed, distributed at two different depths, at the 10 sampling points in each plot (figure 2c). in the data analysis, the underground traps arranged on top of each other were combined and treated as a single sample. in order to standardize the sampling time, these traps remained buried for 48 hours (the same time as the pitfall-traps), according to the technique used by most studies with hypogeic ants (wong & guénard, 2017). ants were identified at genus level using the keys provided in baccaro et al. (2015). later, this material was identified by morphospecies or species whenever possible, using available taxonomic keys, comparison with material previously identified by specialists and deposited in national institute of amazon research (inpa) entomological collections. sociobiology 67(2): 186-200 (june, 2020) 189 environmental predictors we used clay content and elevation as environmental predictors due to their influence on the distribution of grounddwelling ants in previous studies in the amazon basin (vasconcelos et al., 2003; oliveira et al. 2009; gomes et al., 2018). along each of the permanent plots, soil samples were collected at six points 50 m apart. the samples were collected up to 20 cm deep after removal of the leaf litter and large roots. the six individual samples were mixed, yielding a sample representing the plot, of which 500 g was separated for particle size analysis. after dispersion with sodium pyrophosphate, the clay percentage was determined by drying 20 μm of the suspension in an oven at 105 ºc (http://ppbio.inpa.gov.br/knb/ metacat). the altitude above sea level (m.a.s.l.) of the plots was obtained from gps (global positioning system) data taken at the start of each plot. these data are available on the ppbio website (http://ppbio.inpa.gov.br/repositorio/dados). data analysis information about ant diversity and environmental factors were summarized to plot level in the analysis. ant species richness was evaluated in two forms, first considering the observed richness and then by comparing the species accumulation curves (colwell & coddington, 1994) of the epigeic and hypogeic strata at each site. the accumulation curves (interpolation and extrapolation) were based on 1000 randomizations and order zero of hill number (q = 0). a matrix of occurrence of species per plot was generated to minimize the possible effects of ants’ colonial habitats. several ant species have mass foraging strategies and their nests, or foraging/displacement trails (e.g. dorylinae) may be close to the sampling point (hölldobler & wilson, 1990; brühl et al., 2003). thus, the number of each species occurrence varied between zero and ten at each plot. we used a linear mixed model (lmm; laird & ware, 1982) to investigate the interaction effect of strata (epigeic and hypogeic) with clay content and strata with elevation on observed ant species richness per plot. the study sites were included as a random factor in the model to control for possible spatial autocorrelation of the data. in cases when the interaction term was nonsignificant, a simpler model without interaction was fitted. both clay content and elevation were scaled to zero mean and unit variance to increase model convergence. we also calculated the conditional and marginal r2 for the lmm model to measure the importance of the random variable (site). conditional r2 gives the variation explained by fixed and random effects, while marginal r2 indicates the variation explained by the fixed effects only (nakagawa & schielzeth, 2013). we used permutational multivariate analysis of variance (permanova, anderson, 2001) to investigate fig 2. representation of the collection trap and sampling unit for underground ants collection. (a) underground trap with lateral holes; (b) trap positioning below the surface; (c) plot with 250 m contour line with 10 sub-sampling units installed at ombrophilous forests in the brazilian amazon. a b c 11 c m 5 cm 40 cm 25 m} }{{ mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution190 the interaction effect of strata (epigeic and hypogeic) with clay content and the interaction of strata with elevation on ant assemblage composition. we created a stratified permutation procedure to keep the nested structure of the data (plots nested in sites) in the permanova to control for possible spatial autocorrelation of the data. we also built simpler models when the interaction terms were non-significant. the permanova probability values were based on 999 permutations. to further understand the composition patterns, we used the homogeneity of multivariate group dispersions analysis (permdisp, anderson 2006), to test the homogeneity of species composition between strata. permdisp is a multivariate analog of levene’s test for homogeneity of variances, based on permutations. the permdisp probability values were based on 999 permutations. all analyses were run in the r environment for statistical computing (r core team, 2019, version 3.6.1), using the packages vegan 2.5-5 (oksanen et al., 2019), nlme 3.1-142 (pinheiro et al., 2019) and inext 2.0.19 (hsieh et al., 2019). results we sampled 219 species/morphospecies of ants belonging to 52 genera and eight subfamilies. ectatomma lugens was more frequent in epigeic pitfalls, occurring in 20 (80%) plots sampled. solenopsis sp. 04 was the most frequent species sampled in the hypogeic traps, being detected in 10 (40%) plots of the total plots sampled. at the other extreme, 49.3% of the taxa were rare, as they occurred only once (singleton: 84 taxa), or only twice (doubleton: 36 taxa) in the studied sites (table 2). at ufam, 106 taxa (65.7% of the total) were sampled only from the epigeic stratum, five (4.3%) were restricted to the hypogeic stratum and eight taxa (6.8%) were recorded in both epigeic and hypogeic strata. in br-319 modules, at orquestra (km 300) 85 taxa (84.5%) occurred exclusively in the epigeic stratum, ten (9.7%) only in the hypogeic stratum and eight (7.8%) were recorded from both strata. at capanã (km 400) 90 taxa (84.1%) occurred exclusively in the epigeic stratum, nine (8.4%) only in the hypogeic stratum and eight (7.5%) were recorded from both strata, and in jari (km 450) 85 taxa (78%) occurred exclusively in the epigeic stratum, 15 (13.8%) were found only in the hypogeic stratum and nine (8.3%) were recorded from both strata (table 2). species accumulation curves did not reach the asymptote at the four sites studied for either strata (figure 3). the epigeic ant richness (112 taxa in the ufam, 93 taxa in the orquestra, 98 taxa in the capanã and 94 in the jari) was higher than the hypogeic richness (13 taxa in the ufam, 18 taxa in the orquestra, 17 taxa in the capanã and 24 in the jari). ant species richness differed between the epigeic and hypogeic strata (lmm: p < 0.001), but was not related to clay content (p = 0.725) or elevation (p = 0.261). as both interaction terms (strata x clay and strata x elevation) were not significant, both interactions were removed from the analysis. the strata explained a large amount of ant species richness variation (marginal r2= 0.85), with relatively little variation explained by fixed and random factors, sites in our case (conditional r2 = 0.91). fig 3. species accumulation curves based on 1,000 randomization of fex-ufam and br-319 modules samples, representing the epigeic and hypoggeic strata. shaded area represent the standard deviation of the richness average. sociobiology 67(2): 186-200 (june, 2020) 191 fig 4. an nmds ordination (stress = 0.151) plot indicating the congruence in ant assemblages associations among sudied sites and the sampling strata in 47, 250-m long plots in ombrophilous forests in the brazilian amazon. polygons delimit the epigeic and hypogeic strata and the color variation represents the clay gradient. the interaction between strata and clay content explained most of ant species composition variation (permanova: f3,46 = 6.471; r 2 = 0.22; p < 0.001, figure 4), compared with elevation alone (f3,46 = 6.471; r 2 < 0.01; p = 0.995). the interaction term between strata and elevation was not significant and was removed from the analysis. the homogeneity of group dispersion was also different between strata, with epigeic fauna being more heterogeneous, compared with pigaeic fauna (perdisp: f1,45 = 26.936, p < 0.001; figure 4). discussion even with a minimum distance greater than 50 km and possible geographic barriers (amazon and negro rivers) between the sampling sites, the ant richness and composition of the epigeic strata were more similar when compared to those collected in different strata (epigeic and hypogeic) separated by only 20 to 30 cm. due to the particularities of each stratum, differences in ant richness were expected. previous studies in an ombrophilous forest near manaus (vasconcelos & delabie, 2000), in the ecuadorian amazon (wilkie et al., 2010) and in southern region of brazil (martins et al., 2020) found higher number of ant species on the soil surface. however, this pattern is not always present. studies in a tropical forest in borneo (berghoff et al., 2003) and in araucaria forest fragments in southern brazil (silva & silvestre, 2004) did not detect differences in number of species between the strata. this may indicate that other factors such as soil type and vegetation may affect ant richness. the rarefaction curves show the low contribution of the hypogeic ants to total species richness. thus, hypogeic ants need more samples to reach the expected number of species when compared to epigeic ants. in addition, the large variance around the mean and the low average richness values per plot indicate that hypogeic ants were relatively more under sampled compared to epigeic ants. more plots or more subsamples per plot could increase the record of more cryptic species, which would lead to an increase in the average richness of hypogeic species. in addition, the lower local hypogeic richness of each site may be related to the bait (sardine oil) used in the underground traps. this bait generally mobilizes dominant ants, which exclude other species of subordinate ants, thus limiting a more accurate richness estimation (baccaro et al., 2012). in this context, solenopsis geminata and solenopsis sp. 04 dominated four of the five plots in the capanã site (km 450 of the br-319 highway), preventing another subordinate species from reaching the underground traps. in addition, the bait may not have mobilized ants with more restricted and/or distinct foraging habits (delabie & fowler, 1995; berghoff et al., 2003). an example of restricted behavior is the acropyga species, which was recorded only once (at orquestra); species from these genera are associated with specialized root hemiptera and are not attracted to sardine mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution192 bait (delabie & fowler, 1995; lapolla et al., 2002). thus, the species assemblage captured underground in our samples were probably the most abundant, opportunistic, generalist and active in this stratum (fowler et al., 2000; andersen & brault, 2010). the difference in species composition between the epigeic and hypogeic strata of the four sampling sites indicates a strong stratification of the ant assemblage. partitioning of ant assemblages into epigeic and hypogeic strata is often reported (fowler et al., 2000; vasconcelos & delabie, 2000; silva & silvestre, 2004; wilkie et al., 2010; wong & guénard, 2017). as well as stratification between leaf litter and canopy assemblages, this difference in epigeic-hypogeic composition may be one of the causes of the high local diversity of ants in tropical forests (benson & harada, 1988; brühl et al., 2003). assemblage stratification is one dimension of resource partitioning among species in the environment (schoener, 1974). for ants, stratification is related to nesting and foraging sites (vasconcelos & vilhena, 2006; delabie & fowler, 1995). however, there is an overlap in the exploitation of these resources, where ants that are usually associated with the epigeic stratum can fetch below-ground resources, and vice versa. the small fraction of species that occurs in both strata, 4.3% at ufam, 9.7% at orquestra (km 300), 8.4% at capanã (km 400) and 13.8% at jari (km 450), indicates that the overlap between epigeic and hypogeic assemblages is small. even with a small overlap between these strata, some records are at least curious. azteca sp. 01; ectatomma lugens and crematogaster flavosensitiva have characteristics of specimens that live on the surface and were recorded in both strata. prionopelta punctulata, is a typically hypogeic species and was recorded only in the surface pitfall. conversely, crematogaster sp. 07 is a species with epigeic characteristics and was recorded only in the hypogeic stratum. these records suggest some type of contamination when installing the traps in the field. differences between assemblages collected in the epigeic and hypogeic strata were greater than dissimilarities caused by distance or geographical location. in our study, the sampling sites are separated by at least 50 km. in addition, one of the study sites (ufam) is separated from the others (along the br-319 highway) by the amazon and negro rivers at a distance greater than 300 km. a similar result was reported between secondary and primary forests in the amazon and atlantic forest in bahia (fowler et al., 2000; wilkie et al., 2007). this reinforces the role of soil as a dominant vertical barrier in the separation of ant assemblages between strata. large geographical barriers such as sizeable rivers in the amazon basin are often cited to support hypotheses about limiting dispersion for birds, primates, amphibians and squamates (dias-terceiro et al., 2015; moraes et al., 2016). however, a recent study refutes this hypothesis for a wide variety of taxa in the amazon region, suggesting that other processes are also acting on dispersion processes (santorelli jr et al., 2018). our results are in line with the latter, suggesting that large rivers are not strong barriers to ant species. however, further studies using molecular data are needed to unravel the evolution of ant assemblages between riverbanks. the altitude of the terrain was not able to explain the variation in the richness and species composition of the ants in the studied sites and strata. however, the clay content associated with the strata explained some variation (22%) in the ant assemblage composition. clay content in the central amazon, is often related to other environmental properties, such as soil moisture retention, vegetation composition and leaf cover, which may support different numbers of ant species in both the epigeic and hypogeic strata (ranzani, 1980; luizão et al., 2004, oliveira et al., 2009). the influence of clay content and altitude on the assemblage composition of epigeic ant fauna is relatively well documented for the amazon region (vasconcelos et al., 2003; oliveira et al., 2009; gomes et al., 2018). the lack of effect of altitude on species richness and composition or the finding that an effect of clay content is only detected when associated with the strata at these study sites may be the result of the low variation in these predictors in our study. studies of the amazon basin that have detected an effect of clay content on the richness and composition of ants record greater amplitudes in this environmental variable (e.g. vasconcelos et al., 2003; souza et al., 2012; gomes et al., 2018). even though the four sites were distributed across ~ 450 km, we found low variation in clay content and altitude that may have attenuated the changes in ant’s species richness and composition between plots. the various soil types of the amazonian forests are directly correlated with forest architecture and altitude gradient (guillaumet, 1987; costa & magnusson, 2010). these environmental predictors are often associated with changes in ant assemblage structure (vasconcelos et al., 2003; oliveira et al., 2009, gomes et al., 2018), and may have great practical importance, going beyond showing correlations between the numbers of taxonomic entities (souza et al., 2016). environmental predictors can be used to evaluate management decisions about sampling techniques employed (souza et al., 2012, 2018) or the use of surrogates as a shortcut to assessing species diversity (souza et al., 2016) or to optimize ant research protocols. however, their lack of effect on ant fauna indicates that there is still no universal distribution pattern. as the distribution of ants is quite different between strata (andersen & brault, 2010; ryder wilkie et al., 2010; martins et al., 2020), it is probable that the factors determining the distribution of ants in the epigeic stratum are distinct from those that do so in the hypogeic stratum. this reinforces the relevance of future studies to clarify if and what environmental or biological factors affect the epigeic and hypogeic ant fauna, thus facilitating better prediction of the distribution of ants. sociobiology 67(2): 186-200 (june, 2020) 193 subfamilies / taxon fex-ufam orquestra (km 300) capanã (km 400) jari (km 450) frequence (%)epigeic hypogeic epigeic hypogeic epigeic hypogeic epigeic hypogeic amblyoponinae prionopelta punctulata mayr, 1866 2 4 dolichoderinae azteca sp. 01 1 1 1 1 2 1 14 azteca sp. 02 1 2 dolichoderus bidens (linnaeus, 1758) 1 2 dolichoderus bispinosus (olivier, 1792) 2 2 2 12 dolichoderus decollatus smith, 1858 1 2 dolichoderus sp. 02 1 2 dolichoderus sp. 03 4 2 12 dolichoderus sp. 05 1 2 dolichoderus sp. 07 1 2 6 dolichoderus sp. 10 1 2 linepithema sp. 01 1 2 dorylinae acanthostichus sp. 02 1 2 eciton burchellii (westwood, 1842) 1 2 eciton dulcium forel, 1912 1 2 labidus coecus (latreille, 1802) 3 5 1 18 labidus praedator (smith, 1858) 4 2 1 1 1 3 2 28 labidus spininodis (emery, 1890) 1 1 4 neivamyrmex sp. 03 1 1 4 neivamyrmex sp. 07 2 2 2 12 neivamyrmex sp. 08 1 1 4 neivamyrmex sp. 09 1 2 nomamyrmex esenbeckii (westwood, 1842) 1 2 ectatomminae ectatomma edentatum roger, 1863 8 1 18 ectatomma lugens emery, 1894 8 1 4 3 5 1 44 gnamptogenys acuminata (emery, 1896) 1 1 2 1 10 gnamptogenys haenschi (emery, 1902) 1 2 gnamptogenys horni (santschi, 1929) 6 1 1 16 gnamptogenys moelleri (forel, 1912) 1 1 4 gnamptogenys tortuolosa (smith, f., 1858) 4 1 10 gnamptogenys sp. 06 1 3 8 gnamptogenys sp. 11 1 2 formicinae acropyga sp. 02 1 2 brachymyrmex longicornis forel, 1907 1 2 brachymyrmex sp. 02 2 1 2 10 camponotus atriceps (smith, 1858) 1 2 camponotus balzani emery, 1894 4 3 4 22 camponotus femoratus (fabricius, 1804) 2 2 2 2 16 camponotus novogranadensis mayr, 1870 1 1 3 2 14 camponotus rapax (fabricius, 1804) 1 1 4 camponotus renggeri emery, 1894 2 1 3 12 table 2. occurrence and frequency of ant species and morphospecies sampled in the hypogeic (10 plots) and epigeic (7 plots) strata at ufam and on br 319 modules orquestra, capanã and jari (5 plots in both strata for each site). mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution194 camponotus sp. 04 1 2 camponotus sp. 06 2 1 1 8 camponotus sp. 10 1 2 camponotus sp. 16 3 2 3 16 gigantiops destructor (fabricius, 1804) 2 4 nylanderia sp. 01 2 1 3 3 18 nylanderia sp. 02 4 2 3 1 20 nylanderia sp. 03 9 4 2 2 34 nylanderia sp. 04 1 1 1 6 nylanderia sp. 05 1 1 4 myrmicinae acromyrmex sp. 01 2 1 6 acromyrmex sp. 03 4 5 5 28 adelomyrmex sp. 01 1 2 allomerus septemarticulatus mayr, 1878 1 2 apterostigma sp. 01 1 1 4 atta sp. 01 2 4 basiceros sp. 01 2 4 blepharidatta brasiliensis wheeler, 1915 8 16 carebara urichi (wheeler, 1922) 3 2 2 2 18 carebara sp. 01 2 1 2 1 2 2 20 carebara sp. 04 1 2 cephalotes atratus (linnaeus, 1758) 1 1 1 6 cephalotes marginatus (fabricius, 1804) 1 2 cephalotes pusillus (klug, 1824) 1 2 cephalotes sp. 08 1 2 crematogaster brasiliensis mayr, 1878 9 2 1 1 1 28 crematogaster flavomicrops longino, 2003 1 1 4 crematogaster flavosensitiva longino, 2003 1 1 1 4 14 crematogaster limata smith, 1858 7 4 4 2 34 crematogaster rochai forel, 1903 1 2 crematogaster sotobosque longino, 2003 5 3 2 2 24 crematogaster stollii forel, 1885 1 1 1 1 8 crematogaster tenuicula forel, 1904 2 1 6 crematogaster sp. 07 1 2 cyphomyrmex sp. 01 1 2 6 hylomyrma immanis kempf, 1973 1 2 megalomyrmex balzani emery, 1894 2 1 6 megalomyrmex cuatiara brandão, 1990 1 2 megalomyrmex goeldii forel, 1912 3 6 megalomyrmex leoninus forel, 1885 1 2 megalomyrmex sp. 01 2 3 1 12 megalomyrmex sp. 02 1 3 8 megalomyrmex sp. 03 1 2 monomorium floricola (jerdon, 1851) 1 2 table 2. occurrence and frequency of ant species and morphospecies sampled in the hypogeic (10 plots) and epigeic (7 plots) strata at ufam and on br 319 modules orquestra, capanã and jari (5 plots in both strata for each site). (continuation) subfamilies / taxon fex-ufam orquestra (km 300) capanã (km 400) jari (km 450) frequence (%)epigeic hypogeic epigeic hypogeic epigeic hypogeic epigeic hypogeic formicinae sociobiology 67(2): 186-200 (june, 2020) 195 myrmicocrypta sp. 01 2 1 1 8 ochetomyrmex semipolitus mayr, 1878 6 3 3 1 2 30 octostruma balzani (emery, 1894) 1 1 1 6 octostruma iheringi (emery, 1888) 1 2 pheidole vorax (fabricius, 1804) 1 1 1 1 8 pheidole sp. 01 2 4 pheidole sp. 02 2 4 pheidole sp. 03 1 2 pheidole sp. 04 1 2 pheidole sp. 05 1 1 4 pheidole sp. 06 3 4 1 3 4 30 pheidole sp. 07 1 1 4 pheidole sp. 08 2 2 3 1 16 pheidole sp. 09 1 2 pheidole sp. 10 7 3 2 2 28 pheidole sp. 11 4 2 1 14 pheidole sp. 12 1 2 1 8 pheidole sp. 13 2 4 pheidole sp. 14 1 3 3 2 18 pheidole sp. 15 1 2 1 1 1 12 pheidole sp. 16 1 1 2 1 10 pheidole sp. 17 1 2 pheidole sp. 18 1 2 pheidole sp. 19 2 2 8 pheidole sp. 20 6 4 3 3 32 pheidole sp. 21 2 4 pheidole sp. 22 1 2 pheidole sp. 23 1 1 4 pheidole sp. 26 1 4 3 16 pheidole sp. 27 3 3 1 2 18 pheidole sp. 28 3 1 8 pheidole sp. 29 3 6 pheidole sp. 30 2 4 pheidole sp. 31 1 1 4 pheidole sp. 32 2 1 1 1 10 pheidole sp. 33 2 1 1 8 pheidole sp. 35 2 1 6 pheidole sp. 36 1 2 pheidole sp. 37 1 2 2 2 14 pheidole sp. 37a 1 2 pheidole sp. 41 1 2 pheidole sp. 42 7 1 3 3 4 1 38 pheidole sp. 44 1 2 pheidole sp. 46 1 2 pheidole sp. 47 2 1 6 pheidole sp. 48 1 2 table 2. occurrence and frequency of ant species and morphospecies sampled in the hypogeic (10 plots) and epigeic (7 plots) strata at ufam and on br 319 modules orquestra, capanã and jari (5 plots in both strata for each site). (continuation) subfamilies / taxon fex-ufam orquestra (km 300) capanã (km 400) jari (km 450) frequence (%)epigeic hypogeic epigeic hypogeic epigeic hypogeic epigeic hypogeic myrmicinae mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution196 pheidole sp. 49 1 1 4 pheidole sp. 51 1 2 1 1 10 pheidole sp. 52 1 2 pheidole sp. 54 1 2 1 1 10 pheidole sp. 56 1 2 pheidole sp. 57 1 2 pheidole sp. 59 1 2 pheidole sp. 60 3 1 1 10 pheidole sp. 71 1 2 pheidole sp. 75 1 1 4 pheidole sp. 76 2 2 1 10 pheidole sp. 83 1 2 pheidole sp. 87 1 2 1 3 14 pheidole sp. 90 1 2 pheidole sp. 91 2 1 1 8 pheidole sp. 93 1 2 pheidole sp. 94 2 4 pheidole sp. 95 2 4 pheidole sp. 96 1 2 pheidole sp. 97 1 1 1 6 pheidole sp. 98 2 4 pheidole sp. 99 1 2 pheidole sp. 100 1 2 pheidole sp. 102 1 2 pheidole sp. 105 1 2 pheidole sp. 107 1 2 pheidole sp. 120 1 2 pheidole sp. 121 1 2 pogonomyrmex sp. 02 1 2 rogeria sp. 01 1 2 rogeria sp. 02 2 4 sericomyrmex sp. 01 1 3 2 12 solenopsis geminata (fabricius, 1804) 2 3 1 4 2 2 28 solenopsis sp. 01 1 1 2 8 solenopsis sp. 02 1 2 solenopsis sp. 03 4 1 2 1 16 solenopsis sp. 04 2 3 4 2 3 28 solenopsis sp. 05 6 1 3 1 3 1 30 solenopsis sp. 06 1 1 3 10 solenopsis sp. 07 1 2 solenopsis sp. 08 3 2 10 solenopsis sp. 09 8 16 solenopsis sp. 11 1 1 4 solenopsis sp. 14 4 2 2 16 solenopsis sp. 15 1 1 1 6 table 2. occurrence and frequency of ant species and morphospecies sampled in the hypogeic (10 plots) and epigeic (7 plots) strata at ufam and on br 319 modules orquestra, capanã and jari (5 plots in both strata for each site). (continuation) subfamilies / taxon fex-ufam orquestra (km 300) capanã (km 400) jari (km 450) frequence (%)epigeic hypogeic epigeic hypogeic epigeic hypogeic epigeic hypogeic myrmicinae sociobiology 67(2): 186-200 (june, 2020) 197 strumigenys beebei (wheeler, 1915) 1 2 strumigenys denticulata mayr, 1887 7 1 1 2 22 strumigenys elongata roger, 1863 1 2 strumigenys trudifera kempf & brown, 1969 1 2 strumigenys villiersi (perrault, 1986) 2 4 strumigenys zeteki (brown, 1959) 2 1 2 10 strumigenys sp. 01 3 5 2 20 strumigenys sp. 05 1 1 4 strumigenys sp. 11 1 2 paratrachymyrmex sp. 01 4 1 2 1 1 18 paratrachymyrmex sp. 02 1 2 paratrachymyrmex sp. 03 2 4 paratrachymyrmex sp. 05 2 4 paratrachymyrmex sp. 06 4 8 paratrachymyrmex sp. 07 1 2 tranopelta sp. 01 1 2 wasmannia auropunctata (roger, 1863) 6 1 1 16 wasmannia scrobifera kempf, 1961 1 2 ponerinae anochetus diegenis forel, 1912 2 1 6 anochetus horridus kempf, 1964 1 2 centromyrmex sp. 01 1 2 hypoponera sp. 01 1 2 hypoponera sp. 07 1 2 hypoponera sp. 08 1 1 4 hypoponera sp. 09 3 3 12 hypoponera sp. 11 1 2 leptogenys wheeleri forel, 1901 1 2 mayaponera constricta (mayr, 1884) 1 2 3 4 1 22 neoponera apicalis (latreille, 1802) 5 3 3 1 24 neoponera commutata (roger, 1860) 2 4 neoponera obscuricornis (emery, 1890) 3 2 10 neoponera unidentata (mayr, 1862) 1 2 odontomachus caelatus brown, 1976 1 2 odontomachus haematodus (linnaeus, 1758) 1 1 4 odontomachus laticeps roger, 1861 1 1 3 10 odontomachus opaciventris forel, 1899 1 2 odontomachus scalptus brown, 1978 1 2 pachycondyla crassinoda (latreille, 1802) 4 4 4 4 32 pachycondyla harpax (fabricius, 1804) 7 1 16 pachycondyla impressa (roger, 1861) 2 1 6 mayaponera arhuaca (forel, 1901) 1 1 4 pseudomyrmicinae pseudomyrmex sp. 02 1 2 pseudomyrmex sp. 10 2 4 1 14 table 2. occurrence and frequency of ant species and morphospecies sampled in the hypogeic (10 plots) and epigeic (7 plots) strata at ufam and on br 319 modules orquestra, capanã and jari (5 plots in both strata for each site). (continuation) subfamilies / taxon fex-ufam orquestra (km 300) capanã (km 400) jari (km 450) frequence (%)epigeic hypogeic epigeic hypogeic epigeic hypogeic epigeic hypogeic myrmicinae mt torres; jlp souza; fb baccaro – amazonian epigeic and hypogeic ants’ distribution198 acknowledgements we thank mael, pedro, claudio r. santos-neto and carlos a. nogueira, for their help in sampling ants. itanna o. fernandes confirmed the species identifications for this study. we thank marcos a. l. bragança for his logistical support to m.t.t., which resulted in the first draft of this manuscript. m.t.t. was supported by a cnpq master scholarship, j.l.p.s. was supported by cnpq post-doctoral scholarship and fbb is continuous supported by cnpq productivity grant 309600/ 2017-0. financial support was provided by projects pnpd/ capes 2180/2009, cnpq via pronex 16/2006, program for biodiversity research (ppbio) 558318/2009-6, and the national institute for amazonian biodiversity (cenbam). data are maintained by ppbio and cenbam. we also thank the reviewers of previous versions of this manuscript, who provided useful suggestions. references alonso, l.e. e agosti, d. 2000. biodiversity studies, monitoring, and ants: an overview. in d. agosti, j.d. majer, l.e. alonso e t.r. schultz (eds.), ants: standard methods for measuring and monitoring biodiversity (pp. 1–8). washington, d.c.: smithsonian institution press. andersen, a.n. & majer, j.d. 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(2017). subterranean ants: summary and perspectives on field sampling methods, with notes on diversity and ecology (hymenoptera: formicidae). myrmecological news, 25: 1–16. 297 techniques for the in vitro production of queens in stingless bees (apidae, meliponini) by ana rita baptistella¹, camila c. m. souza², weyder cristiano santana³* & ademilson espencer egea soares² abstract considering the ecological importance of stingless bees as caretakers and pollinators of a variety of native plants makes it necessary to improve techniques which increase of colonies’ number in order to preserve these species and the biodiversity associated with them. thus, our aim was to develop a methodolog y of in vitro production of stingless bee queens by offering a large quantity of food to the larvae. our methodolog y consisted of determining the amount of larval food needed for the development of the queens, collecting and storing the larval food, and feeding the food to the larvae in acrylic plates. we found that the total average amount of larval food in a worker bee cell of f. varia is approximately 26.70 ± 3.55 µl. we observed that after the consumption of extra amounts of food (25, 30, 35 and 40 µl) the larvae differentiate into queens (n = 98). therefore, the average total volume of food needed for the differentiation of a young larva of f. varia queen is approximately 61.70 ± 5.00 µl. in other words; the larvae destined to become queens eat 2.31 times more food than the ones destined to become workers. we used the species frieseomelitta varia as a model, however the methodolog y can be reproduced for all species of stingless bees whose mechanism of caste differentiation depends on the amount of food ingested by the larvae. our results demonstrate the effectiveness of the in vitro technique developed herein, pointing to the possibility of its use as a tool to assist the production of ¹biolog y department, laboratory of bee biolog y and genetics, faculty of philosophy, sciences and letters of ribeirão preto, university of são paulo, brazil. ² genetic department, laboratory of bee biolog y and genetics, medicine faculty of ribeirão preto, university of são paulo, brazil. ³ entomolog y department, federal university of viçosa, brazil. *correspondence author: weyder cristiano santana, entomolog y department, federal university of viçosa, ph rolfs avenue, s/n, zip code: 36571-000, viçosa, minas gerais, brazil. e-mail: weyder.santana@ufv.br 298 sociobiolog y vol. 59, no. 1, 2012 queens on a large scale. this would allow for the artificial splitting of colonies and contribute to conservation efforts in native bees. key words: stingless bees; production of queens; caste determination; frieseomelitta varia; larval food; colony splitting. introduction stingless bees (apidae, meliponini) are very common in the tropics and are the main pollinators of tropical ecosystems (roubik 1984; corlett 2004). it is believed that about 40 to 90% of native trees in brazil are pollinated by these bees (kerr et al. 1996). thus, the need to preserve the species of native bees lies mainly in the broad action of these agents as pollinators of our flora, ensuring the conservation of the fauna and all its associated biodiversity. one of the most striking features of the biolog y of stingless bees, besides that of being highly eusocial and a sister group of apis mellifera (apidae, apini) (michener 2000), is related to the larvae’s feeding process, which presents a strateg y of mass food supply in their brood cells (kerr 1948; nogueira-neto 1997; michener 2000). the mechanism of caste determination differs between the bees of the genus melipona and the bees from other genres of this tribe (kerr 1950; kerr & laidlaw 1956; maciel-silva & kerr 1991; hartfelder et al. 2006). the caste induction in most of the stingless bees is related to the amount of food ingested by the larvae during their development (wheeler 1986; hartfelder et al. 2006). the larvae destined to become queens eat an increased amount of food compared to those who will develop into workers (kerr 1948; hartfelder & engels 1989; engels & imperatriz-fonseca 1990; hartfelder et al. 2006) with the exception of bees from the genus melipona, where the process is also associated with genetic factors (kerr 1950; kerr & laidlaw 1956; kerr & nielsen 1966), and few species that produce miniature queens such as schwarziana quadripunctata (camargo 1974; imperatriz-fonseca & darakjian 1993; wenseleers et al. 2005), plebeia julianii ( juliani 1962), plebeia remota (ribeiro et al. 2006), nannotrigona testaceicornis (imperatriz-fonseca et al. 1997) and plebeia lucii (teixeira & campos 2005). among the species of the genera leurotrigona and frieseomelitta, as well as in plebeia lucii, the brood cells are not grouped and there is no construction 299 baptistella, a.r. et al. — in vitro production of stingless bee queens of typical royal cells (which are notably larger); the queens develop in cells formed from the junction of two equally sized brood cells, and the adjacent cell contains only the larval food (terada 1974; faustino et al. 2002; teixeira & campos 2005). with the advance of meliponiculture (cortopassi-laurino et al. 2006), the growing interest in using these bees as agricultural pollinators, as well as the commercialization of stingless bee products, such as pollen, honey, and propolis, the demand for colonies has considerably increased. with this in mind, we attempt to develop a technique for queen production in the laboratory as a first step to enable the production of the colonies on a large scale, allowing for their use both ecologically and economically, and to enable further study of the biolog y and caste determination of the stingless bees. methods the technique for “in vitro” production of stingless bee queens consists of three steps: determination of the amount of larval food needed for the development of queens the biological material was taken from three colonies of frieseomelitta varia (lepeletier, 1836), kept at the meliponary of the genetic department of the university of são paulo, ribeirão preto, brazil. from may 2007 to april 2008, recently capped brood cells containing eggs (n= 522) were collected. the eggs were removed in the laboratory and the food in each cell was collected and volumetrically measured with a micropipette (10µl). collection and storage of larval food the larval food was collected from the cells of frieseomelitta varia by vacuum suction with the aid of a vacuum pump (fig. 1) and rapidly cooled to 4°c. after collection, the material was stored in a freezer at -20°c for later use in larvae transference tests. transference of the larvae elisa-type 96 well 500 μl round bottom acrylic plates were used in this step. different volumes of stored larval food (25, 30, 35, 40 e 50 µl) were homogenized and transferred to the culture plate wells with a 50µl micropipette. 300 sociobiolog y vol. 59, no. 1, 2012 fig. 1. instrument used to collect larval food by suction: (i) vacuum suction pump; (ii) receptacle for the collection of waste; (iii) food collector tube kept in ice; (iv) food suction hose and (v) petri dish to accommodate the brood cells. fig. 2. (i) larvae of frieseomelitta varia transferred to artificial cells (elisa-type acrylic plate). immature developmental stages in vitro: (ii) feeding larva; (iii) final feeding phase; (iv) defecation phase, (v) queen pupa phase and (iv) queen. 301 baptistella, a.r. et al. — in vitro production of stingless bee queens larvae (immature) in the final stage of larval feeding (predefecant) with milky white coloration and a full gut were selected for this step. using round tip tweezers, the larvae were transferred to the cells and deposited in the same position onto the larval food. at that stage of development, the frieseomelitta varia larvae are easily identified because of their milky-white coloration, their food-filled gut and their larger size, which facilitated handling them during the transfer process. afterwards, the plates with the larvae were placed inside plastic containers with saturated nacl solution in order to keep the humidity between 70-80% and sealed with pvc film. they were kept in bod greenhouse at 28 ±½°c and the bees' development was observed until birth (fig. 2). when the virgin queens emerged, they were weighed with an electronic analytical balance metter® (ae50) and then compared with the recently emerged virgin queens produced naturally in the colony. in order to verify the dominant behavior of the in vitro produced queens, small orphaned colonies (without queen) were formed with brood combs containing imagines, 40 newly emerged workers, pollen reserves from the species itself and a 50% (p/v) sucrose solution. the queens were individually introduced to those nests, which were then covered with a transparent glass to allow for observation. the data was analyzed using the statistical test oneway anova and the tukey test a posteriori. results quantifying the amount of larval food in the brood cells of frieseomelitta varia we observed seasonal environmental variations with significant effect on the amount of food deposited in the brood cells (table 1, fig. 3, anova test p<0.001), during the months of larval food collection in frieseomelitta varia. the amount of larval food in the brood cells of f. varia ranged from 20 to 38 µl (fig. 3). analyzing the total number of uncapped cells and the total volume of food collected during this time (table 1), we observed that the average amount of larval food in a worker cell of f. varia is 26.7± 3.55 µl (n=522), which varied during the year according either to the higher precipitation periods 302 sociobiolog y vol. 59, no. 1, 2012 (february, april, may, november) or to the lower precipitation periods ( june and august) (fig. 3). in vitro production of queens a total of 98 queens of frieseomelitta varia were reared using the methodolog y developed for in vitro production of stingless bee queens (table 2). after analysis of the weight of individuals produced in vitro (n=98) and the naturally produces queens in the colonies of f. varia (n= 6), we observed that the queens produced in vitro weighed less than those produced naturally (p< 0.001) (fig. 4). table 1: average amount (μl) of larval food from frieseomelitta varia deposited in the brood cells during the collection months (2007-2008). sd= standard deviation, sem = standard error of the mean. amount of larval food in brood cells of frieseomelitta varia month of data collection total no. of cells total volume (µl) mean/cell sd sem february 115 2751 23.92 2.53 0.24 april 64 1476 23.06 3.11 0.39 may 112 2713 24.22 4.78 0.45 june 92 2564 27.87 4.37 0.46 august 63 2024 32.13 3.42 0.43 november 76 2202 28.97 2.72 0.31 total 522 13730 26.70 3.49 fig. 3. variation of the amount of larval food (µl) in brood cells of frieseomelitta varia (n=522 cells), during the collection months (2007-2008) and the relation with precipitation (mm). tukey’s test (p< 0,050). different letters indicate statistically significant differences among months. 303 baptistella, a.r. et al. — in vitro production of stingless bee queens the in vitro queens that were accepted by the workers in the new colonies, therefore assuming their role as dominant females (n=11), had a similar weight compared to the natural queens (n=6), without significant differences amoung them (p=0.083). the same did not occur in the other in vitro queens table 2: transference of frieseomelitta varia larvae to the in vitro production of queens with different amounts of larval food (µl) and their respective survival rates (%). test volume of larval food (µl) transferred larvae birth rate queens workers number % number % number % 1 25 100 82 82 13 16 69 84 2 30 100 20 20 15 75 5 25 3 35 100 31 31 30 97 1 3 4 40 100 50 50 40 80 10 20 5 50 100 0 0 0 0 0 0 total 500 183 37 98 54 85 46 fig. 4. comparison of the weight (mg ) of the virgin queens of frieseomelitta varia produced under natural conditions (n=6) and in vitro (n=98). 304 sociobiolog y vol. 59, no. 1, 2012 (n=87) that were rejected and therefore eliminated by the workers. they had a significantly lower weight than the others (p<0.001) (fig.5). discussion amount of larval food in worker cells an average total volume of 26.7 ± 3.55 μl (n= 522) (table 1) of larval food was found in the brood cells of f. varia. considering that the larvae which become queens feed on the contents of their cell and on the larval food of the auxiliary cell, the larvae that become queens ingest more food than the worker larvae (faustino et al. 2002). this results in an approximate 2:1 proportion of food, for queen larvae compared to worker larvae. compared with other species of meliponini, such as scaptotrigona aff. depilis, where the proportion of food is 4:1 (130 μl in royal cells and 31 μl in worker cells) (menezes 2010), we noted that there is not a great difference in the amount of food offered to the larvae in frieseomelitta varia; there is a fig. 5. comparison between the weight (mg ) of queens of frieseomelitta varia produced under natural conditions (n=6) and in vitro that were accepted by the workers (n=11), and queens produced in vitro that were rejected and eliminated by the workers (n= 87). a there is no significant difference between the queens’ weight (p=0,083), b there is a significant difference between the queens’ weight (p<0,001). 305 baptistella, a.r. et al. — in vitro production of stingless bee queens threshold for the amount of larval food responsible for differentiating a worker larva into a queen. this shows up as differences in weight, size of adults and phenotipic characteristics of queens, which are not much larger than their workers, when compared to the other species. we verified the occurrence of a variation of the amount of larval food inside the brood cells during the collection months (february, april, may, june, august, november) coinciding with the dry and the rainy seasons of the year when there is little or much food respectively available in the environment. this occurrence was also described by castilho-hyodo (2001) in schwarziana quadripunctata brood cells, where the amount of larval food was not the same year-round, but varied depending on the environmental conditions. we attribute the variation in the amount of food in the brood cells to the availability of food resources in the environment, to the food stored in the colony, and also to the number of individuals in the colonies which is directly related to the number of active foragers. thus, the higher the number of workers collecting food in the field, the higher the amount of food being brought into the colonies, therefore promoting an increase in the number of cells built and eventually increasing the number of individuals in the colony. on the other hand, when there are fewer resources available, the production of brood in the colony also decreases (fewer cells are built). consequently, the nurse workers of frieseomelitta varia feed the larvae and deposit larger amounts of larval food per cell in order to meet the needs of the colony. the phenomenon of a decrease in the number of brood cells and amount of larval food in each cell during times of “environmental stress” has been shown in four species of stingless bees: melipona quadrifasciata (ramalho et al. 1998), s. quadripunctata (castilho-hyodo 2001), plebeia remota (ribeiro et al. 2003), scaptotrigona aff. depilis (menezes 2010) and now in frieseomellita varia. this data indicates that this phenomenon is common in meliponini species, strengthening the importance and the relation of the larval food with the mechanisms of caste determination in stingless bees, with the environmental conditions as a promoting factor of intra and extra-nesting cycles, with the production of sexed individuals (queens and males) and with the ecological expansion of those bees. 306 sociobiolog y vol. 59, no. 1, 2012 transfer of the larvae to the production of in vitro queens assuming that the larvae used in these experiments had ingested the average amount of 25 µl of larval food prior to the transfer, we verified that after the consumption of extra food (25, 30, 35 e 40 µl), the larvae differentiated into queens. therefore, the total amount of food needed for the differentiation of a young larva of frieseomelitta varia into a queen is approximately 61.70 ± 5.00 µl, that is, the larva destined to become a queen consumes 2.31 times more food than a larva destined to become a worker. this threshold is the amount of food capable of inducing the differentiation and the morphogenetic changes in these larvae to become queens. we found that 100% of the larvae that received 50µl of extra larval food were not able to consume it and drowned inside the food. in this way, an excess amount of larval food, around 75 µl, exceeds the capacity of ingestion/ digestion by the developing larvae. these results confirm that in frieseomelitta varia the mechanism of caste determination depends exclusively on the amount of food provided to the developing larvae and that the larvae at the end of the feeding phase (predefecant) are able to keep feeding and to develop into queens; they are still conditioned to the availability of larval food and to the ability of ingestion. these findings agree with other studies related to the effect of larval nutrition on the caste determination in stingless bees (hartfelder et al. 2006). the variation in the amount of food provided to the larvae which become queens also varies by species of meliponini. in trigona spinipes, for instance, the queens consume 10 times more food (360 µl) than the workers (36 µl) (buschini & campos 1995; lisboa et al. 2005); in scaptotrigona postica, camargo (1972) demonstrated that extra supply of food to the worker larvae caused them to develop into queens. another discussed factor related to the amount of food in the brood cells of meliponini is the existence of dwarf queens that can emerge from worker cells if they receive an increased amount of food than that received by normal workers, as was noted by campos & costa (1989) and castilho-hyodo (2001) in schwarziana quadripunctata. the weight of emerged individuals does not seem to influence the final acceptance or rejection, once the produced queens were accepted as dominant 307 baptistella, a.r. et al. — in vitro production of stingless bee queens females in the new nests (n= 11) they had a similar weight compared to the queens produced in vitro that were eliminated by workers (n=87). most likely, this suggests that the workers do not interpret queen weight as an indication of fitness, a fact that is also evident in other behavioral and physiological features (hartfelder et al. 2006). on the other hand, in species of stingless bees in which the phenomena of miniature queens does occur such as nannotrigona testaceicornis, imperatrizfonseca et al. (1997) showed that recently emerged miniature queens weighed 7.67 mg (sd 0.87) on average, and workers weighed 7.13 mg (sd 0.7). there may be other intrinsic factors related to the dominant behavior (imperatriz-fonseca & zucchi 1995; jarau et al. 2009; jarau et al. 2010) such as patterns of cuticular hydrocarbons present in these bees (nunes et al. 2008) which interfere with the recognition of the individuals and are related to the process of acceptance of a new queen into the nest (nunes et al. 2009) or even group decision making regarding the choice of a new queen (tarpy & gilley 2004). this method developed for the in vitro production of stingless bees was shown to be efficient, with a success rate of 45.75% (n= 183, excluding the values of test 5). we believe that one of the factors responsible for the larvae’s high mortality of 54.25% (n=217) is related to the variation of the relative humidity during the first experiments. additionally, in some cases there was an excessive amount of microorganisms in the larval food that was responsible for larval death. another important factor regarding the transferring of larvae is the positioning of the larva inside the cell which can be responsible for larval mortality. one must carefully place the larva on top of the food in the same position it was found so that the larva can perform the gas exchange through the tegument when it is in contact with the air, thus avoiding its suffocation. eleven new colonies were obtained from the queens produced in vitro, allowing a fast increase of the number of colonies over a short period of time, demonstrating the efficiency of this technique. in this way, our methodolog y can be successfully applied as a tool in studies on the mechanism of caste determination, especially those involving the large-scale production of queens from many species of stingless bees. this will allow for the production of colonies which can be used for the economic 308 sociobiolog y vol. 59, no. 1, 2012 benefits of pollination and will also help in the conservation and reintroduction of stingless bees in their natural ecosystems. acknowledgements the authors would like to thank jairo de souza for helping with the colony management, ivan de castro for the preparation of the figures, juliana ramos martins for valuable comments and collaboration in the final work, michael a. t. freiberg (the pennsylvania state university) for his helpful review of the text and “centro integrado de informações agrometeorológicas” of são paulo state for the climatic data. we also thank fapesp (nº 04/15801-0), cnpq and capes for supporting this work. references buschini, m.l.t. & l.a.o. campos 1995. caste determination in trigona spinipes (hymenoptera, apidae): influence of the available food and the juvenile hormone. revista brasileira de biologia 55: 121 129. camargo, c.a. 1972. determinação de castas em scaptotrigona postica latreille (hymenoptera, apidae). revista brasileira de biologia 32 (1): 133-138. camargo, j.m.f. 1974. notas sobre a morfologia e biologia de plebeia (schwarziana) quadripunctata (hymenoptera, apidae: meliponinae). studia entomologica 17 (1): 433-470. campos, l.a.o. & m.a. costa 1989. determinação do sexo em abelhas: xxviii determinação das castas em schwarziana quadripunctata (hymenoptera, apidae). revista brasileira de biologia 49: 999-1001. castilho-hyodo, v.c.c. 2001. rainha ou operária? um ensaio sobre a determinação de castas em schwarziana quadripunctata (lepeletier, 1836) (hymenoptera, apidae, meliponini). phd thesis university of são paulo, são paulo, brazil: 134 p. corlett, r.t. 2004. flower visitors and pollination in the oriental (indomalayan) region. biological reviews 79: 497–532. cortopassi-laurino, m., v.l. imperatriz-fonseca, d. roubik, a. dollin, t. heard, i. aguilar, g. venturieri, c. eardley & p. nogueira-neto 2006. global meliponiculture: challenges and opportunities. apidologie 37: 275-292. engels, w. & v.l. imperatriz-fonseca 1990. caste development, reproductive strategies and control of fertility in honey bees and stingless bees. in: engels, w. 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(termitidae). the results showed that the total cellulase activity of the higher termite a. sichuanensis was markedly higher than that of the lower termites. co. formosanus had the highest activity of endo-β-1,4-glucanase and cellobiohydrolase. the activity of β-glucosidase was not significantly different among the three species. the proportion of cellobiohydrolase was higher in the higher termite. in terms of distributions of cellulolytic activity in the gut, the primary site of endo-β-1,4-glucanase activity was presented in the hindgut of both the lower termites but in the midgut of the higher termite, and the primary site of β-glucosidase activity was restricted to the midgut in the lower termites and the head/foregut in the higher termite. the functions of the gut segments were apparently differentiated between the lower and higher termites, with the role of the midgut becoming more important in the higher termite. for the endogenous cellulases, the main site of endo-β-1,4-glucanase activity was concentrated in the midgut in both the lower termite and the higher termite, but the main site of β-glucosidase activity was in the head/foregut in the higher termite. the results suggest that characters of cellulase activity could reflect the phylogeny of wood-feeding termites to a certain extent. key words: crypototermes domesticus, coptotermes formosanus, ahmaditermes sichuanensis, endo-β-1,4-glucanase, exoglucanase, β-glucosidase, cellobiohydrolase guangdong entomological institute, 105 xingang west road, guangzhou 510260, china, 1corresponding author, email: xwl@gdei.gd.cn 1158 sociobiolog y vol. 59, no. 4, 2012 introduction cellulose is the most abundant, widespread and renewable biomass on the earth. biotic fuels generated from cellulose could replace petroleum, but economic production of cellulosic biotic fuels is dependent on the growth of more efficient and cheaper cellulases that can be applied into both pretreatment and cellulolytic technologies (mosier et al. 2005; emsley 2008). in nature, xylophagous insects have evolved strategies to derive their energ y needs from celluloses with cellulases, of which termites are the most efficient decomposers of celluloses (sugimoto et al. 2000; watanabe & tokuda 2010). termites are important decomposers in tropical ecosystems (noble et al. 2009), and cellulase enzymes and cellulase genes in the digestive systems of termites may have potential for cellulosic ethanol production by biological processes (ni 2007; warnecke et al. 2007; li et al. 2009). termites have specialized cellulose-digesting systems (nakashima et al. 2002; tokuda et al. 2007; zhou et al. 2007). various cellulases are involved in degradation of celluloses in termites and their symbionts. the related investigation of cellulase activity in termites is an important research field. the three main types of cellulases are endo-β-1,4-glucanases (egs; ec 3.2.1.4), cellobiohydrolases (cbhs; ec 3.2.1.91), and β-glucosidases (bgs; ec 3.2.1.21), and cellulose degradation requires the synergistic action of the three types of glycoside hydrolases (lo et al. 2011). patterns and characters of cellulases in termites and their symbionts have been described extensively (watanabe and tokuda 2010; willis et al. 2010), and cellulase activity and their distributions in the digestive systems were different among diverse termites. recently, distributions of diverse cellulase activity in each gut segments of termites were mostly studied (willis et al. 2010), and tokuda et al. (2004, 2005) found that the expression of the endogenous cellulase genes has shifted from the salivary glands of lower termites to the midgut of higher termites. in addition, differences in total cellulase activity were compared among lower termites (mo et al. 2004; lu et al. 2010), and eg expression among different castes of hodotermopsis sjostedti (family termopsidae) and nasutitermes takasagoensis (family termitidae) have been reported (fujita et al. 2008). recent research suggests that the distributions of cellulase activity in termites are related to their evolutional levels (tokuda et al. 2004, 2005). 1159 li, z.-q. et al. — cellulase activity in higher and lower termites at present, there are approximately 2,600 described termite species around the world (kambhampati & eggleton 2000), and comprehensive studies of phylogenetic and taxonomic relationships among termite groups have been published (donovan et al. 2000; legendre et al. 2008). however, the cellulase activities of only around 16 termite species have been reported (lu et al. 2010; lo et al. 2011). additional research can expand the knowledge of the cellulose-digesting mechanisms of termites, and provide a reference basis for the development of cellulosic ethanol (rubin 2008) and environmentallyfriendly biotermicides (zhang et al. 2011). termites feed on dead plant material at all stages of decomposition. besides wood, they feed on soil, leaf litter, fungi, grass, or lichen (donovan et al. 2000). on the whole, cellulase activity levels in wood-feeding termites are far higher than those found in the fungus-growers and the soil-feeders (tokuda et al. 2004; lo et al. 2011). to provide further information about the relationship between cellulolytic activity and evolutionary status in woodfeeding termites from phylogenetically different lineages, the cellulase activity and distribution of workers were studied in three wood-feeding termites from phylogenetically different lineages; the lower termites, crypototermes domesticus (haviland) (isoptera: kalotermitidae) and coptotermes formosanus skiraki (rhinotermitidae), and the higher termite, ahmaditermes sichuanensis xia et al. (termitidae). materials and methods termites cr. domesticus were collected from two laboratory-maintained colonies. three co. formosanus colonies and two a. sichuanensis colonies were collected on luofu mountain, guangdong province in china. the caste of termites used for experiments were healthy adult workers, but pseudoworkers for cr. domesticus. the workers or pseudoworkers were directly put into liquid nitrogen before enzyme extraction. preparation of crude enzyme to prepare enzyme extracts, the workers or pseudoworkers were washed with precooling 0.09% normal saline. fifteen sets per termite colony of heads (including salivary glands) and whole guts were dissected from termites, and 1160 sociobiolog y vol. 59, no. 4, 2012 each set was divided into head/foregut, midgut, and hindgut. the three sections and other five bodies of worker per colony were collected in tubes and homogenized in 500 μl of 0.1 m sodium acetate buffer (sab) ( ph 5.6) on ice. the tubes were centrifuged at 12,000 rpm for 15 min at 4 °c, and the supernatants were brought to volume of 500 μl by adding 0.1 m sab and used as the enzyme extract. the same volume of 0.1 m sab was used as the control. assay of cellulase activity filter paper degrading activity circular filter paper (3.5 mg per piece) after high temperature sterilization was put into the microtubes with 120μl sab (ph 5.6), and the crude enzyme (12 μl) was incubated with the filter paper at 37 °c for 60 min. based on dinitrosalicylic acid method (eveleigh et al. 2009), 120 μl dinitrosalicylic acid solution was added and boiled for 5min. then, the tubes were cooled to room temperature rapidly. the glucose production was detected colorimetrically with victor 3 multi-label microplate reader (perkin elmer, us) at 540nm, using glucose as a standard. the protein content of the sample was determined spectrophotomerically at 660nm according to the coomassie brilliant blue g-250 method (lott et al. 1983), using bovine serum as a standard. one unit (u) of enzyme activity was defined as the amount of enzyme capable of releasing one μmol reducing sugar per minute. specific activity was expressed as units per mg protein. the enzyme assays were repeated three times. endo-β-1,4-glucanase and β-glucosidase activity the activity of both eg and bg were determined using 120 μl of 1% sodium carboxymethylcellulose and 120 μl of 1% salicin as the substrates, respectively. other steps and definitions were the same as the assay of filter paper degrading activity mentioned above. cellobiohydrolase activity cellobiohydrolase was assayed using p-nitrophenyl-β-d-cellobioside (pnpc) as the substrate. 120 μl of 0.1 mm pnpc was mixed with 12 μl crude enzyme at 37 °c for 60 min. the incubation was stopped by adding 120 μl of 1 m sodium carbonate, and diluted with sab to 500 μl. the amount of p-nitrophenyl was measured at 405 nm. 1161 li, z.-q. et al. — cellulase activity in higher and lower termites data analysis the data were analyzed by one-way analysis of variance with least significant difference post-hoc tests (lsd) with spss 16.0 for windows software. results differences in cellulase activity among termites the data (table 1) of cellulase activity of whole bodies of termite workers (or pseudoworkers) showed that the bg activities were not significantly different among the three termites, but activity of both eg and cbh was high in co. formosanus. regarding the proportion of different cellulose types within species, eg activity was the principal component in all three termites. however, cbh activity of a. sichuanensis was not significantly different from eg or bg. filter paper assay filter paper degradation is directly related to digestibility of naturally occurring cellulose. according to the data of filter paper degrading activity (table 2), the most evolved species, a. sichuanensis, had the highest activity of filter paper degrading activity in the whole body of three wood-feeding table 1 complete cellulase activity of whole bodies in the termite workers tested. species whole body activity endo-β-1,4-glucanase β-glucosidase cellobiohydrolase crypototermes domesticus 0.347±0.028 ba 0.116±0.007 ab 0.016±0.000 bc coptotermes formosanus 0.780±0.047 aa 0.426±0.057 ab 0.243±0.079 ab ahmaditermes sichuanensis 0.525±0.122 ba 0.747±0.470 aa 0.015±0.000 ba mean±s.e. with different small letter means significant difference in the same column, while different capital letter means significant difference in the same line at the 0.05 probability level. the same as below. table 2 filter paper degrading activity of different segments and whole body in the termite workers. species filter paper degrading activity whole body head/foregut midgut hindgut crypototermes domesticus 0.167±0.036 b 0.139±0.055aa 0.130±0.057aa 0.155±0.072aa coptotermes formosanus 0.241±0.031ab 0.131±0.027ab 0.107±0.017ab 0.229±0.022aa ahmaditermes sichuanensis 0.417±0.102a 0.213±0.087ac 0.326±0.157aa 0.288±0.051ab 1162 sociobiolog y vol. 59, no. 4, 2012 termites, but there were no significant differences among filter paper degrading activity of the three termites in each gut segment. regarding distributions of filter paper degrading activity in guts of the termites, a. sichuanensis showed significantly different filter paper degrading activity among three gut segments, and the midgut was the main segment of filter paper degrading activity in a. sichuanensis. distribution of eg and bg activities in the gut according to the data from the hindgut (table 3), it is suggested that flagellate-harbouring termites possess a higher percentage of intestinal microbial eg and bg than a. sichuanensis. in addition, activities of both eg and bg were not significantly different in the midgut among the three wood-feeding termites, and neither was bg activity in the hindgut. co. formosanus had the highest activity of eg in head/foregut and hindgut, and a. sichuanensis had the highest activity of bg in head/foregut. for endogenous endoglucanase and β-glucosidase, eg activities were not significantly different between the head/foregut and midgut of cr. domesticus, or co. formosanus. however, the higher termite a. sichuanensis had higher eg activity in the midgut. in contrast, the higher bg activity in a. sichuanensis was in the head/foregut. discussion termites play an important role in degradation of cellulosic materials in nature, and special attention is paid to the activity and expression of termite cellulases (watanabe & tokuda 2010). efficient cellulose digestion in termites requires both endogenous and intestinal microbial cellulases (tokuda 2007; table 3 endo-β-1,4-glucanase and β-glucosidase activity of different segments in the termite workers. cellulases speices cellulase acitivity in each gut segment head/foregut midgut hindgut endo-β-1,4-glucanase crypototermes domesticus 0.152±0.016bb 0.185±0.073ab 0.499±0.020ba coptotermes formosanus 0.363±0.016ab 0.295±0.120ab 0.818±0.126aa ahmaditermes sichuanensis 0.214±0.075bc 0.399±0.085aa 0.289±0.079bb β-glucosidase crypototermes domesticus 0.080±0.005bc 0.160±0.006aa 0.116±0.007ab coptotermes formosanus 0.153±0.068ba 0.314±0.107aa 0.200±0.033aa ahmaditermes sichuanensis 0.363±0.033aa 0.305±0.162ab 0.289±0.103ac 1163 li, z.-q. et al. — cellulase activity in higher and lower termites zhou 2007). the present study showed that the hindgut was the primary site of cellulose digestion in lower termites, which was consistent with the report of tokuda et al. (2005). as for endogenous termite cellulases, tokuda et al. (2004) proposed that the expression of the endogenous cellulase genes has shifted from the salivary glands of lower termites to the midgut of higher termites. in terms of the dynamic shift in termites, our results showed that activity of eg and filter paper degrading activity were most highly concentrated on the midgut of the higher termite studied than that of the lower termites, which supported the previous studies on eg (mo et al. 2004; tokuda et al. 2004, 2005; fujita et al. 2008; tokuda et al. 2009; lo et al. 2011), but the dynamic change of bg was contrary to the related report of fujita et al. (2008) and tokuda et al. (2009). they suggested that the main position of bg activity tended to the head/foregut with evolution of wood-feeding termites, and the evolved wood-feeding termites such as the termites of the rhinotermitidae and termitidae may have higher bg activity in the head/foregut. as for the comparison of cellulase activities in whole bodies of termites, it was suggested that the degrading activity of filter paper and the percentage value of cbh in complete cellulases were increased with a rise in evolutionary status. the higher termite a. sichuanensis had the highest degrading activity of filter paper and the highest percentage value in complete cellulases. however, a previous study showed that activity in the higher termite nasutitermes takasagoensis was markedly lower using microcrystalline cellulose as substrate than that of flagellate-harbouring termites (tokuda et al. 2005).because of this, tokuda et al. (2005) considered the cellulase activity of n. takasagoensis was likely to be similar to its requirement for energ y metabolism. in addition, filter paper, microcrystalline cellulose and cotton have been used as cellulase substrates to determine the existence of complete cellulases (willis et al. 2010), but there is evidence for the degradation of microcrystalline cellulose in insects lacking cbh activity (scrivener and slaytor 1994). co. formosanus is one of the most destructive and invasive pests around the world, and its cellulase activity has been intensively studied on different substrates (willis et al. 2010). co. formosanus had the highest activity of eg and bg among termites (tokuda et al. 2005; lu et al. 2010). the present study showed that co. formosanus had the highest degrading activity of not 1164 sociobiolog y vol. 59, no. 4, 2012 only eg but also cbh in the three termites studied, but its bg activity was not significantly different from species of the family kalotermitidae which was consistent with the report of mo et al. (2004). in addition, in terms of the synthetic cellulase activity in wood-feeding termites, a. sichuanensis and co. formosanus showed higher activities of eg and cbh. use of diverse cellulase substrates can help differentiate the specific contribution of specific insect gut areas to the digestion of plant material (tokuda et al. 2005). for cellulase activity assays, the data are often difficult to compare among studies. multinomial factors can influence the cellulase activity, such as temperature, substrate, detection method and so on (tokuda et al. 2005; willis et al. 2010). furthermore, phylogenetic analysis of cellulase genes from the termite lineages has received special attention (todaka et al. 2010). the relative expression levels of cellulase genes do not correspond with their activity, which could be affected by different regulators (fujita et al. 2008). in addition, for the workers of higher termites, the cellulose-digesting division of labor might indirectly influence the determination results (fujita et al. 2008). acknowledgments we thank professor li-ying li for english language revision. this work was supported by the guangdong academy of sciences foundation for excellent young scientists and technicians (200901) and the national natural science foundation of china (31172163). references donovan s., d. jones, w. sands, p. eggleton 2000. morphological phylogenetics of termites (isoptera). biological journal of the linnean society 70(3): 467-513. emsley a.m. 2008. cellulosic ethanol reignites the fire of cellulose degradation. cellulose 15(2): 187-192. eveleigh d.e., m. mandels, r. andreotti, c. roche 2009. measurement of saccharifying cellulase. biotechnolog y for biofuels 2(1): 1-8. fujita a., t. miura, t. matsumoto 2008. differences in cellulose digestive systems among castes in two termite lineages. physiological entomolog y 33(1): 73-82. gusakov a.v., t.n. salanovich, a.i. antonov, b.b. ustinov, o.n. okunev, r. burlingame, m. emalfarb, m. baez, a.p. sinitsyn 2007. design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. biotechnolog y and bioengineering 97(5): 10281038. 1165 li, z.-q. et al. — cellulase activity in higher and lower termites kambharnpati s. & p. eggleton 2000. taxonomy and phylogeny of termites. in: abe t, bignell de, higashi m (eds) termites: evolution, sociality, symbioses, ecolog y, pp.1-23. kluwer academic publishers, dordrecht. legendre f., m.f. whiting, c. bordereau, e.m. cancello, t.a. evans, p. grandcolas 2008. the phylogeny of termites (dictyoptera: isoptera) based on mitochondrial and nuclear markers: implications for the evolution of the worker and pseudergate castes, and foraging behaviors. molecular phylogenetics and evolution 48(2): 615-627. li x., h. yang, b. roy, d. wang, w. yue, l. jiang, e.y. park, y. miao 2009. the most stirring technolog y in future: cellulase enzyme and biomass utilization. african journal of biotechnolog y 8(11): 2418-2422. lo n., g. tokuda, h. watanabe 2011. evolution and function of endogenous termite cellulases. in: d.e. bignell et al. (eds.), biolog y of termites: a modern synthesis, pp.5167. springer science+business media, london new york. lott j., v.a. stephan, jr k. pritchard 1983. evaluation of the coomassie brilliant blue g-250 method for urinary protein. clinical chemistry, 29(11): 1946-1950. lu j., t. deng, j. li, j. mo 2010. activities of some lignocelluloses-degrading enzymes in workers of five common termites (isoptera). sociobiolog y 55(3): 749-762. mo j., t. yang, x. song, j. cheng 2004. cellulase activity in five species of important termites in china. applied entomolog y and zoolog y, 39, 635–641. mosier n., c. wyman, b. dale, r. elander, y.y. lee, m. holtzapple, m. ladisch 2005. features of promising technologies for pretreatment of lignocellulosic biomass. bioresource technolog y 96(6): 673-686. nakashima k., h. watanabe, h. saitoh, g. tokuda, j.i. azuma 2002. dual cellulosedigesting system of the wood-feeding termite, coptotermes formosanus shiraki. insect biochemistry and molecular biolog y 32(7): 777-784. ni j., g. tokuda, m. takehara, h. watanabe 2007. heterologous expression and enzymatic characterization of β-glucosidase from the drywood-eating termite, neotermes koshunensis. applied entomolog y and zoolog y 42: 457–463. noble j.c., w.j. miller, w.g. whitford, g.h. pfitzner 2009. the significance of termites as decomposers in contrasting grassland communities of semi-arid eastern australia. journal of arid environments 73(1): 113-119. rubin e.m. 2008. genomics of cellulosic biofuels. nature 454(7206): 841-845. scrivener a. & m. slaytor 1994. properties of the endogenous cellulase from panesthia cribrata saussure and purification of major endo-β-1, 4-glucanase components. insect biochemistry and molecular biolog y 24(3): 223-231. sugimoto a., d.e. bignell, j.a. macdonald 2000. global impact of termites on the carbon cycle and atmospheric trace gases. in: abe t, bignell de, higashi m. (eds) termites: evolution, sociality, symbioses, ecolog y, pp.409–435. kluwer academic publishers, dordrecht. todaka n., t. inoue, k. saita, m. ohkuma, c.a.nalepa, m. lenz, t. kudo, s. moriya 2010. phylogenetic analysis of cellulolytic enzyme genes from representative lineages of termites and a related cockroach. plos one 5(1): e8636. 1166 sociobiolog y vol. 59, no. 4, 2012 tokuda g., n. lo, h. watanabe 2005. marked variations in patterns of cellulase activity against crystallinevs. carboxymethyl-cellulose in the digestive systems of diverse, woodfeeding termites. physiological entomolog y 30(4): 372-380. tokuda g., lo n., watanabe h., arakawa g., matsumoto t., noda h. 2004. major alteration of the expression site of endogenous cellulases in members of an apical termite lineage. molecular ecolog y 13(10): 3219-3228. warnecke f., p. luginbühl, n. ivanova, m. ghassemian, t.h. richardson, j.t. stege, m. cayouette, a.c. mchardy, g. djordjevic, n. aboushadi 2007. metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. nature 450(7169): 560-565. watanabe h. & g. tokuda 2001. animal cellulases. cell. mol. life sci. 58(9): 11671178. watanabe h. & g. tokuda 2010. cellulolytic systems in insects. annual review of entomology 55: 609-632. willis j.d., c. oppert, j.l. jurat-fuentes 2010. methods for discovery and characterization of cellulolytic enzymes from insects. insect science 17(3): 184-198. zhang d., a.r lax., j.m. bland, a.b. allen 2011. characterization of a new endogenous endo-β-1,4-glucanase of formosan subterranean termite (coptotermes formosanus). insect biochemistry and molecular biolog y 41: 211-218. 135 distribution of resources collected among individuals from colonies of mischocyttarus drewseni (hymenoptera, vespidae) by eliani rodrigues da silva1, 3, olga coutinho togni2, 4 , gabriela de almeida locher 2,5 & edilberto giannotti2, 6 abstract the aim of this study was to analyze the distribution pattern of the food collected among groups of individuals in mischocyttarus drewseni colonies. this behavior is one of the first actions exhibited by the foragers when they arrive in the colonies. regarding nectar and prey collection, 95.90% of the collected nectar was given to larvae, whereas 3.57% to dominant individuals and 95.94% of the collected prey were given to the larvae, 2.54% to the dominant members, while the remainder of both was given to the workers. despite not being significant, it was possible to observe a difference in food distribution among larvae, with larger larvae receiving more food than others. when the forager returns to the nest with pulp, it adds this material to cells in 64.29% of the times. males showed agitated behavior with the arrival of the foragers, and sometimes took the foraged material from them. introduction mischocyttarus de saussure (1853) is the largest group of social wasps, consisting of 245 species, distributed in nine subgenera, being mainly neotropical (richards 1978; carpenter & marques 2001; silveira 2008). these wasps are considered primitive eusocial species, with independent foundation and without morphological caste differentiation. the dominant and subordinate females may assume any role, according to their colony's need, due to the small number of individuals and the fact they are all potentially reproductive (litte 1981; jeanne 1986; murakami 2007). 1 centro universitário de votuporanga, caixa postal 81, 15500006, votuporanga, sp, brazil 2 departamento de zoologia instituto de biociências, universidade estadual paulista, caixa postal 199; 13506-900, rio claro, sp, brazil. 3 eliani@fev.edu.br; 4 olgatogni@yahoo.com.br; 5 gabriela.locher@gmail.com; 6edilgian@rc.unesp.br 136 sociobiolog y vol. 59, no. 1, 2012 although these wasps have high behavioral plasticity, the dominance hierarchy creates a social dynamic based on the nutritional cost and benefits from the behaviors performed. the dominant members avoid activities with high energ y cost, such as foraging, by creating a nutritional reserve that improves their reproductive capacity in the colony; thus, increasing their dominance over other females (markiewicz & o’donnell 2001). therefore, the foraging activity, which consists of leaving the nest to collect resources for the maintenance of the colony, is a crucial aspect to elucidate issues related to the evolution of sociality (smith 2005), since the main behavioral interactions conducted by social wasps are related to the capture and division of materials collected among the members of the colony (rocha & giannotti 2007). the distribution of the resources collected among individuals from the colony is one of the first behavioral acts exhibited by foragers after the arrival in the field. this behavior is the main goal of the foraging process, and often the trophallaxis among adults is held immediately after the arrival of foragers, even before entering the nest (rocha & giannotti 2007; sugden & mcallen 1994; cruz et al. 2006). despite its importance, studies related to foraging activity in species of the genus mischocyttarus are scarce and have only been conducted for m. drewseni ( jeanne 1972), m. labiatus (litte 1981), m. flavitarsis (cornelius 1993), m. mastigophorus (o'donnell 1998), m. cerberus styx (silva & noda 2000) and m. consimilis (montagna et al. 2009). the objective of this study was to analyze the distribution of resources collected by foragers of mischocyttarus drewseni de saussure, 1857 in the following aspects: (1) the distribution of food among larvae (small, medium and large), (2) what proportion of the food is divided between the immature and adult females (dominant and subordinate), (3) if the forager shares wood pulp collected with other individuals in the colony and what proportion of pulp is shared among dominant and subordinate indivduals, and (4) in what proportion males receive the resources that arrive in the colony. material and methods the field work was carried out at the unesp – universidade estadual paulista, rio claro, são paulo state, southeastern brazil (22o24'36"s; 47o33'36"w 137 da silva, e.r. et al. — distribution of resources by m. drewseni and an average altitude of 612 meters), in a population of m. drewseni. data were obtained from 21 colonies in the pre-and post-emergence stages for 189 hours of observation. at the onset of the experiment, the colonies were found, identified and mapped to verify the number of cells, immature (eggs, larvae and pupae) and adults, and find out the colony stage of development. next, in order to identify the behavioral roles of each individual (dominant, subordinate and male) in the colony and their foraging habits, the wasps were collected, marked and then returned to the nest. the mark was made on the mesosoma with a ceramic paint pen. regarding the size of the larvae, the terminolog y described by giannotti & trevisoli (1993) was adapted for this study: sl small larvae (l1 and l2 in giannotti & trevisoli, 1993): the ventral lobes of the first abdominal segment are not evident; ml medium larvae (l3 and l4 in giannotti & trevisoli, 1993): ventral lobes of the first abdominal segment are small; ll large larvae (l5 in giannotti & trevisoli, 1993): ventral lobes of the first abdominal segment are fully developed. the identification of the collected material was made by observing the behavior of individuals in the colony (silva & noda 2000), as follows: nectar foraging: was considered to be when the wasp arrived at the nest with liquid food stored in its crop and performed adult-adult or adult-larva trophallaxis. prey foraging: was noted when the wasp arrived with a solid mass held in its mouthparts. in this case, the wasp might chew the food itself or share it with another wasp, and then offer this macerated protein to the larvae. pulp foraging: the collection of construction material was characterized by the arrival at the nest with a solid dark mass. this material was chewed and incorporated into the cells of the nest. water foraging: was noted when the liquid was deposited directly on the walls of the nest cells, without contact with another wasp. unfruitful foraging: collections were considered unfruitful, when the wasp returned without any apparent material, not performing trophallaxis or depositing any substance in the nest. after the foragers' arrival, the distribution of the collected material between the individuals of the nest was observed and more than one distribu138 sociobiolog y vol. 59, no. 1, 2012 tion type was recorded if it occurred in a single collection. during nectar or prey distribution among larvae, the frequency of each larva (small, medium or large) receiving such food was quantified. the sum of all data collected from the colonies and the results were compiled by dividing the number of receipts for each size of larvae by the total number of larvae of the same size. this method enabled comparisons between colonies with different amounts and sizes of larvae, as follows: average distribution to sl = times that each sl received food/total number of sl. average distribution to ml = times that each ml received food/total number of ml. average distribution to ll = times that each ll received food/ total number of ll. the kruskal-wallis test (p ≤ 0.05) was used to verify the significance of these differences, if samples represented only casual variations, and whether the distribution of nectar and prey is different for each size of larva. in order to quantify the food distribution between larvae, dominant females and subordinate females, it was checked how much each group received for each of the journeys made by the forager. next, an average per hour of observation and the proportion of each group of individuals receiving food was calculated. when the forager returned to the nest with wood pulp, it was observed whether it was directly added to cells or shared with other adults, and the average time the forager stayed with the material and shared with dominant and subordinates was calculated. the food sharing between females and males was also quantified by calculating the relative frequency of times that males received nectar or prey. results food distribution among small, medium and large larvae according to fig. 1, the mean values show that large larvae received more nectar (fig. 1a) (average of 2.25 times for each trip) and prey (fig. 1b) (mean 2.13 times for each trip) than the others the medium larvae received nectar 1.76 times for each trip, and the small larvae 1.03 times; while, the small larvae received prey more often (average of 1.68 times) than the medium larvae 139 da silva, e.r. et al. — distribution of resources by m. drewseni fig. 1. pattern of division of nectar (a) and prey (b) among small larvae (sl), medium larvae (ml) and large larvae (ll). 140 sociobiolog y vol. 59, no. 1, 2012 (average of 1.34 times). nevertheless, statistical results (kruskal-wallis test) showed that these differences are not significant, nor was the distribution of nectar (h = 3.6778, p = 0.1590, n = 195) or for distribution of prey (h = 5.3493, p = 0.0689, n = 83). food distribution among immature (larvae) and adult females (dominant and subordinate) as shown in tables 1 and 2, the distribution of food between immature and adults females of the colonies was differentiated. of the arrivals with nectar, 95.90% were distributed to larvae, 3.57% to dominant individuals and 0.54% to subordinates. on the instances where prey was brought to the colonies, 95.94% were delivered to the larvae, regardless of the size, 2.54% were given to the dominant adults and 1.42% to the subordinates the larvae received nectar 15.58 times per hour, the dominants 0.58 and subordinates 0.09. regarding the prey collection, larvae received the macerate an average of 28.63 times per hour, the dominant adults 0.76 times and subordinates 0.42. wood pulp distribution among adult females (foragers, dominant and subordinate) out of 42 arrivals with wood pulp (table 3), the dominant individuals received 33.33%, representing a value clearly higher than that for the subordinates, which received only 2.38% of the total, while 64.29% stayed with the foragers. from an average of 1.79 collections of pulp per hour, 1.17 were directly deposited in the cell by the foragers, while 0.58 were delivered to the dominant adults and 0.04 to the subordinates. food distribution among males and females males were restless with the arrivals of the foragers in the colony. on occasion one to three males investigated the female who had just arrived from the field and often took the foraged material, either nectar or prey. from a total of 85 arrivals of nectar, which was divided only among adults, 47 (55.3%) were given to female adults, subordinate or dominant, and 38 (44.7%) were divided between males. males received 11.5% of all prey that were brought to the colonies, and of seven total instances where males received the prey, three (42.8%) were partially delivered and four (57.2%) were given whole (table 4). 141 da silva, e.r. et al. — distribution of resources by m. drewseni discussion foragers of m. drewseni appear to perform an unequal distribution of food in the act of feeding the larvae, providing greater amounts of nectar and prey for larger larvae, although this difference is not statistically significant. this result is clear from fig. 1, and corroborates with the data observed by kudô table 1. distribution of nectar among larvae and adult females (dominant and subordinate). nectar larvae dominant subordinate total 1075 40 6 average/hour 15.58 0.58 0.09 standard deviation 12.47 0.79 0.28 percentage 95.90% 3.57% 0.54% table 2. distribution of prey among larvae and adult females (dominant and subordinate). prey larvae dominant subordinate total 945 25 14 average /hour 28.63 0.76 0.42 standard deviation 60.32 0.67 1.15 percentage 95.94% 2.54% 1.42% table 3. distribution of wood pulp among foragers (dominant and subordinate). wood pulp foragers dominant subordinate total 27 14 1 average/hour 1.17 0.58 0.04 standard deviation 1.00 1.06 0.20 percentage 64.29% 33.33% 2.38% table 4. distribution of nectar and prey among males and females of m. drewseni, specifying the number of occurrences (n) and the relative frequency (%). adults nectar prey n % n % females 47 55.3 54 88.5 males 38 44.7 7 11.5 total 85 100 61 100 142 sociobiolog y vol. 59, no. 1, 2012 (1998) in colonies of polistes chinensis where this difference was attributed to the fact that large larvae require more food than small larvae. in m. labiatus adult prefer to feed large larvae and have a tendency to concentrate their feeding efforts on one larva at a time (litte 1981). larval development time depends on feeding rates in social insects (wilson 1971). therefore, both in colonies with lone-foundresses and colonies with multifoundress, the older larvae are always given priority over the younger larvae and these older larvae pupate considerably earlier than the others present in the nests (litte 1981). this conclusion has been demonstrated during the pre-emergence phases of colonies of m. drewseni when the mean duration of the first larvae appearance is less than that of later-appearing larvae ( jeanne 1972), suggesting that in this species, feeding efforts are also concentrated on the first emerging larvae. moreover, the better fed larva has more chance to be the reproductive female of the colony, so the differential distribution of food among larvae may be related to caste determination (rossi & hunt 1988; hunt 1991; gadagkar et al. 1988; gadagkar et al. 1991). regarding the distribution of collected resources among individuals of the colony, it was found that most of the food is directed to larvae and distribution of supply among adults follows a hierarchy, with the dominants receiving more than the subordinates. the high rate of larva-adult trophallaxis in this study may be related to the attraction of adults by larval salivary secretions, which may have been the reason for the evolution of social wasp species in a context of trophallaxis as mutual exchange of food (roubaud 1916). reproductive maturation and capacity of oviposition appear to be influenced by larval trophallaxis, mainly in those species of primitive social wasps, in which morphological differences between reproductive and worker castes do not exist (gadagkar 1991). thus, food offered by the foragers to the larvae does not appear to be a purely altruistic act, since the females who collected and distributed the food to immatures will also be favored. it was found that the foragers of m. drewseni obey a hierarchy in the distribution of resources among females that remain in the colony, and the dominant adults have advantages over the subordinates at the time of division of nectar, pulp and prey, supporting the hypothesis of jeanne (1972). foloni 143 da silva, e.r. et al. — distribution of resources by m. drewseni & giannotti (1998) quantified the number of transfers of regurgitated liquid from an adult female of m. drewseni to another, and noted that there is a significant difference between dominants and subordinates, so that queens spend 1.60% of their time getting food, while the workers 0.34%. studies on polistes metricus confirm this hypothesis because they claim there is a division of labor among individuals, finding that the queens stay in the nest most of the time and their journeys are of short duration (gamboa et al. 1978). noda et al. (2001) found that the material collected by foragers of m. cerberus styx delivered to another female is an act of submission and stated that the species has a well-defined hierarchy. similarly, costa-filho et al. (2011) observed that queens of m. cerberus styx spends most of their time in the comb while intermediate females perform high frequencies of cell inspection and gaster rubbing ; however, they also forage and the workers or foragers spend most of their time in the field collecting. knowing that queens need food, both for survival and for development of the ovarioles, and present no foraging behavior, it becomes clear why they receive more food than subordinates (gamboa et al. 1978) the distribution of pulp was also distinguished, and probably beyond the reproductive function. the dominant individuals of the colonies observed in this work are likely responsible for building and increase of cells in the nest, since the dominant females received a higher percentage of pulp when compared with the subordinates. in polistes, the dominant females initiate the construction of most cells and perform oviposition soon after (westeberhard 1969; pratte 1989), as observed in colonies of p. lanio in the preemergence stage, when the only activity performed by the dominant females was collecting wood pulp to start new cells and then laying eggs (giannotti & machado 1999). in the same way the dominants of m. cerberus styx forage for pulp at a higher rate than the workers (3.1% and 0.3% respectively, giannotti 1999), and in mischocyttarus mastigophorus the wood pulp loads were never shared with nest mates, while food loads, especially insect prey, were often partitioned with other wasps (o’donnell 1998). this behavior may explain why a large percentage of pulp is shared with females of the highest hierarchical position. males received food from foragers, corroborating the findings by jeanne (1972), who also worked with m. drewseni and noted that these individuals 144 sociobiolog y vol. 59, no. 1, 2012 requested prey as soon as foragers arrive at the nest. often the request of the food is aggressive, as was observed in males of m. mastigophorus, which do not forage, but through aggressive behavior, solicit and consume part of the food brought by foragers, representing a considerable energ y cost to the colony (o’donnell 1999). jeanne (1972) suggested that larval feeding by males of m. drewseni is associated with the removal of liquid protein, since the macerate of prey becomes smaller when chewed by a male and in one case the portion was totally dropped and was not distributed among the immatures after macerating. makino (1993), observing polistes jadwigae, stated that from the total of 27 prey, that males received four which were completely discarded after chewing. according to the author these observations indicate that larval feeding by polistinae males is minimal, because they ingest a significant portion of the food themselves during this process. unlike the cases noted above, males of m. latior display participation in the colony activities and, in contrast to other studies, males have been observed foraging nectar, prey and water (cecílio 1999). in general, it may be concluded that the fact that foragers of m. drewseni leave the nest and go out to the field in search of resources for individuals who are not their offspring is certainly an altruistic behavior, because it involves high energ y cost and risk of predation. nevertheless, considering the theory of mutualism, when adult-larva trophallaxis occurs there is reciprocity on the part of the larvae in relation to foragers, featuring reciprocal altruism, which is reflected in the preference to feed larger larvae that solicit more food and probably produce more salivary secretion. also, the distribution of resources among adults of m. drewseni has an important role in determining and maintaining the dominance hierarchy of the colony. as in m. mastigophorus, dominant individuals foraged for food (nectar and prey) at lower rates than subordinate individuals. in contrast, dominant wasps performed most of the foraging for the wood pulp used in nest construction (o’donnell 1998). dominant individuals on the nest were more likely to take food from arriving foragers than subordinate individuals. nestmates can compete for access to loads of food provided by foragers for personal consumption. control of food materials influences individual physiolog y (hunt 1991), as well as that of the developing brood (markiewicz & o’donnell 2001). wasps may also 145 da silva, e.r. et al. — distribution of resources by m. drewseni compete to control the processing of building material loads. the ability to monopolize building materials allows individuals to regulate nest growth. in addition, it is possible to note that males can participate passively in the activities of the colony, just receiving the food brought by foragers, or actively, distributing food to the larvae or possibly foraging. acknowledgments we thank the fundação de amparo à pesquisa do estado de são paulo (fapesp) for financial support. references carpenter, j.m. & o.m. marques. 2001. contribuição ao estudo dos 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(80 workers, 27 major workers, 27 queens, 27 virgin queens and 27 males). results showed that cuticular hydrocarbons varied quantitatively and qualitatively among the groups and this variation was sufficiently strong to allow separation of castes and genders. we discuss the specificity of some compounds as possible regulatory compounds of worker tasks and reproduction in c. textor. key-words: cuticular hydrocarbons, camponotus textor, castes and sex introduction the integrity of social insect colonies is possible due to the ability of individuals to recognize and to discriminate nestmates, non-nestmates and heterospecific individuals (wilson 1971; hölldobler & michener 1980; breed & bennett 1987; nunes et al. 2008). in a nestmate recognition system, individuals detect and perceive the odor present in the cuticle of another individual and compare it with an internal template, thus distinguishing nestmate cues from non-nestmate cues (sturgis & gordon 2012; leonhardt et al. 2007). several studies have shown that cuticular hydrocarbons are the main compounds playing roles as chemical signatures of individuals, castes, 1departamento de biologia, faculdade de filosofia ciências e letras de ribeirão preto universidade de são paulo, av. bandeirantes 3900, 14040-901, ribeirão preto, são paulo, brazil 2 departamento de física e química, faculdade de ciências farmacêuticas de ribeirão preto, universidade de são paulo * author for correspondence: fsnascim@usp.br 1026 sociobiolog y vol. 59, no. 3, 2012 brood and colonies (vander meer & morel 1998; tannure-nascimento et al. 2009; reviewed in blomquist & bagneres 2010). in ants, for example, workers that perform distinct tasks present variable hydrocarbon profiles (wagner et al. 1998). patrollers and foragers of pogonomyrmex barbatus yield a higher amount of more complex compounds than nest maintenance workers (wagner et al. 2001). in this same species, greene and gordon (2003) showed that a worker can access the task status of a nestmate from its cuticular hydrocarbon profile. in addition, kaib et al. (2000) showed that, irrespective to their age, myrmicaria eumenoides workers performing nest tasks retain their hydrocarbon profiles unaltered. they stated that the variation of the cuticular blend is not only relatively correlated with age, but also strongly correlated with task allocation of each individual. moreover, cuticular hydrocarbons can mediate the reproduction and regulation of egg laying in ants (peeters et al. 1999; liebig et al. 2000) and the reproductive status is related to the cuticular hydrocarbons of queens, mated workers and gamergates (monnin et al. 1998; liebig et al. 2000; cuvillier-hot et al. 2001). reproductive queens have differences in their cuticular profiles when compared with infertile workers (heinze et al. 2002). in camponotus floridanus the presence and the fertility of queen may be indicated by presence of hydrocarbons directly from the queen or her eggs (endler et al. 2004). camponotus textor is a weaver ant that inhabits forests of central and south america (schremmer 1979a, 1979b). their nests are built with larval silk and present a round shape with several galleries around the branches and dead leaves (santos 2002; longino 2006). behavioral observations and natural history of this species has previously been studied (cited as c. senex: santos et al. 2005a, 2005b; santos & del-claro 2009). in the present study we evaluated and compared the chemical profile of cuticles of c. textor workers, queens, virgin queens and males. material and methods a mature colony (27 queens, approximately 24,600 workers and major workers, approximately 500 virgin queens and 500 males) of c. textor obtained from the university of são paulo (usp-ribeirão preto, são paulo, brazil), was collected and freezer-killed (-20°c). the cuticular hydrocarbons of 80 1027 campos, m.c.g et al. — cuticular hydrocarbon profiles of camponotus textor workers, 27 major workers, 27 queens, 27 virgin queens and 27 males were extracted. cuticular compounds were extracted from individuals in 0.5 ml hexane for 2 min. after evaporating the solvent, the apolar extract was resuspended in 160 µl of hexane. the samples were analyzed in a gc-ms (shimadzu, model qp2010 plus) equipped with silica capillary column of 30m and helium as carrier gas at 1 ml/min. the oven temperature was initially set to 150°c, and ramped up 3°c/min until it reached 280°c. analyses were performed in splitless mode. data were analyzed to characterize the mass spectrum. equivalent chain lengths were determined using n-alkane standards (sigma chemical co.) and quantification was based on the peak areas obtained from the chromatograms. the data were analyzed with gcms solutions for windows (shimadzu corporation) and the chemical compounds were identified based on their mass spectra by comparison with wiley library data and with a standard solution of different synthetic hydrocarbons. principal components analysis (pca) was used to define the main compound peaks to be compared. compounds missing in the most individuals of an analyzed group as well as compounds contributing less than 5% to the first two factors, as indicated by the pca, were excluded from the statistical analysis. following this, a stepwise discriminant function analysis was used to observe if combinations of variables could be useful in the predicting group. in this method, variables are successively added to the model based on the higher f to enter values, adding no more variables when the f ratio is no longer significant. wilks’ λ values were used to verify the individual contribution of each variable to the model. the wilks’ λ statistic for the overall discrimination is computed as the ratio of the determinant of the within-groups variance/ covariance matrix to the determinant of the total variance/covariance matrix. mahalanobis distance (d) was used as parameter to evaluate chemical distance between groups. to avoid errors in compositional sample data, the area under each peak was transformed according to the following formula: z = ln[ap/g(ap)], where ap is the area under the peak, g(ap) is the geometric mean for each individual compound group and z is the transformed peak area (aitchison 1028 sociobiolog y vol. 59, no. 3, 2012 1986). the statistical analyses were performed using the software statistica 7.0 (statsoft, inc.). results analysis of the worker cuticular waxes identified 90 hydrocarbons (table 1). these compounds were classified as linear alkanes, monomethylalkanes, and dimethylalkanes. the groups presented many qualitative differences in their respective cuticular profiles (table 1). of ninety compounds found, eighty-nine compounds were present in queens. apart from these, eight compounds were exclusively found in the queens and six compounds were only found in queens and virgin queens. workers, major workers and males had no exclusive compounds, but major workers and queens had four compounds in common, and one compound was only found in workers and queens. queens, workers and major workers presented nine compounds and these compounds were not present in virgin queens and males. one compound was present only in major workers. some compounds were more representative in the relative proportion of cuticular hydrocarbon profile of studied groups: 17-, 15and 13-me c32 (>10%), n-c 28 and n-c 30 (> 5%), 9-, 11and 13-, 15-dime c 32 (>6%), and 13-, 15-dime c 29 (>4%) (table 2). stepwise discriminant analyses of these compounds significantly separated castes and sex (table 2: global model: wilks’ λ= 0.001; f = 52,668; p< 0.001) and the most important hydrocarbons for this separation (f to enter >1) were 9-, 11-dime c 32 , 4-me c 30 , n-c 32 , 13-, 15-dime c 30 , n-c 30 , n-c 28 and n-c 29 . chemical distances (d) between castes and sex were extremely high (fig. 1): queens vs workers: 102.57, queens vs majors: 72.92, queens vs virgin queens: 127.87, queens vs males 184.58, workers vs majors: 41.95, workers vs virgin queens: 54.16, workers vs males: 58.20, majors vs virgin queens: 98.91, majors vs males: 121.06 and virgin queens vs males: 44.85 (all values were highly significant p<0.001). discussion in social insects, physiological variation of individuals is well reflected in their cuticular hydrocarbons (reviewed in blomquist & bagneres 2010). in 1029 campos, m.c.g et al. — cuticular hydrocarbon profiles of camponotus textor table 1: cuticular hydrocarbons present in the camponotus textor workers (w), major workers (lw), queens (q), virgin queens (vq) and males (m). rt = retention time in minutes. different letters indicate significant differences (p<0.001). compound rt w lw q vq m mean sd mean sd mean sd mean sd mean sd n-c 26 28.841 0.54 ±0.36a 0.28 ±0.17 a 0.16 ±0.06 b 0.03 ±0.03 b 0.10 ±0.09 b 12-me c 26 10-me c 26 29.669 0.12 ±0.09 0.09 ±0.04 3-me c 26 30.661 0.40 ±0.21 a 0.44 ±0.16 b 0.35 ±0.17 a 0.31 ±0.05 a 0.14 ±0.08 c n-c 27 31.359 1.32 ±1.56 d 0.45 ±0.25 c 0.24 ±0.11ac 0.05 ±0.02 b 0.09 ±0.05 ab 13-me c 27 11-me c 27 9-me c 27 32.124 0.15 ±0.15 0.18 ±0.05 0.15 ±0.06 5-me c 27 32.725 0.12 ±0.08 0.07 ±0.02 0.18 ±0.48 0.05 ±0.02 11-.13-dime c 27 13-.15-dime c 27 32.879 0.09 ±0.03 0.16 ±0.08 0.05 ±0.02 0.01 ±0.01 3-me c 27 33.122 0.21 ±0.09 0.11 ±0.05 n-c 28 33.828 9.63 ±4.28 d 4.60 ±2.68 a 2.75 ±1.28 ab 0.80 ±0.47 c 1.89 ±0.75bc 14-me c 28 13-me c 28 12-me c 28 34.561 4.13 ±1.72ac 3.62 ±0.98 ab 2.46 ±1.20 c 4.26 ±0.39 b 3.16 ±0.91 c 7-me c 28 34.745 0.43 ±0.53 b 1.33 ±0.34 a 0.28 ±0.13 b 1.47 ±0.17 a 1.02 ±0.22 a 6-me c 28 34.950 0.21 ±0.07 0.27 ±0.30 4-me c 28 35.162 0.28 ±0.26 a 0.22 ±0.06 a 0.19 ±0.13 a 0.23 ±0.07 a 0.04 ±0.03 b 2-me c 28 35.582 5.91 ±1.81 b 5.54 ±1.35 a 6.05 ±2.76 abc 4.15 ±0.86 c 8.79 ±1.75 d 3-me c 28 35.660 0.15 ±0.06 0.06 ±0.09 0.09 ±0.03 n-c 29 36.175 3.24 ±1.68 d 1.16 ±0.84ac 1.78 ±0.70 c 0.22 ±0.15 b 0.46 ±0.10 ab 15-me c 29 13-me c 29 11-me c 29 36.845 0.32 ±0.35 d 0.81 ±0.41bc 0.30 ±0.16 ad 0.92 ±0.15 c 0.54 ±0.23 ab 9-me c 29 36.970 0.11 ±0.05 0.16 ±0.11 11-.15-dime c 29 37.454 0.09 ±0.12 0.43 ±0.85 9-.13-dime c 29 37.593 0.21 ±0.12 0.23 ±0.08 0.37 ±0.18 3-me c 29 37.824 0.13 ±0.11 a 0.17 ±0.07 b 0.38 ±0.62 b 0.08 ±0.03 a 0.07 ±0.03 a n-c 30 38.491 7.22 ±3.11 b 3.91 ±2.40 a 6.56 ±2.73 b 1.00 ±0.68 a 2.77 ±0.95 a 13-. 15-dime c 29 38.941 5.12 ±1.63 1.74 ±2.34 9.29 ±1.28 11.15 ±2.87 16-me c 30 14-me c 30 12-me c 30 39.111 0.58 ±0.42 ab 7.71 ±1.89 d 1.00 ±0.86 a 0.30 ±0.09bc 0.19 ±0.09 c 1030 sociobiolog y vol. 59, no. 3, 2012 10-me c 30 39.205 1.11 ±0.57 d 0.26 ±0.10 c 1.61 ±0.78 a 1.96 ±0.25 b 1.89 ±0.40 ab ? 39.321 0.76 ±0.63 1.86 ±0.42 1.11 ±0.39 17-me c 30 15-me c 30 13-me c 30 39.556 1.24 ±0.52 0.82 ±0.84 1.24 ±0.13 0.21 ±0.40 11-.15-dime c 30 39.683 1.17 ±0.56 ab 0.79 ±0.30 c 0.65 ±0.37bd 0.46 ±0.08acd 1.36 ±1.21 d 9-. 17-di me c 30 39.756 1.29 ±0.69 a 0.65 ±0.48 ab 0.46 ±0.28 b 2.72 ±1.42a 8.86 ±2.84 c 13-.15 dime c 30 39.939 2.61 ±1.53 a 1.46 ±0.53 a 0.45 ±0.33 b 1.71 ±0.80 a 0.65 ±0.38 b 4-me c 30 40.151 4.04 ±1.23 b 3.92 ±1.77 b 7.70 ±3.24 c 2.05 ±0.75 a 1.04 ±0.19 a n-c 31 40.680 1.41 ±0.59 d 0.71 ±0.39 ab 3.82 ±3.26 c 0.73 ±0.28 b 0.47 ±0.13 a 14-me c 31 15-me c 31 41.279 1.19 ±0.45 b 2.15 ±0.68 a 1.17 ±0.58 b 2.27 ±0.60 a 1.59 ±0.28 c 9-me c 31 41.435 0.14 ±0.07 5-me c 31 41.579 0.45 ±1.00 11-.15-dime c 31 9-.13-dime c 31 41.898 0.52 ±0.21 b 0.96 ±0.30 a 1.50 ±0.90 a 0.43 ±0.19 b 0.25 ±0.13 c 7-. 15-dime c 31 7-. 17-dime c 31 41.998 0.59 ±0.41 5-. 13-dime c 31. 5-. 15-dime c 31 5-. 17-dime c 31 42.282 0.36 ±0.14 0.52 ±0.24 0.58 ±0.20 3-me c 31 42.465 0.39 ±0.41 0.12 ±0.05 n-c 32 42.843 1.47 ±0.79 a 0.74 ±0.45 a 5.36 ±2.06 c 0.16 ±0.13 b 0.33 ±0.11 b 13-me c 32 15-me c 32 17-me c 32 43.415 13.76 ±3.67 d 12.73 ±2.99 a 7.14 ±3.72 b 15.21 ±1.02 c 15.12 ±2.48 a 9-me c 32 43.533 1.95 ±0.50 a 0.37 ±0.33 c 2.84 ±1.10 b 2.04 ±0.19 b 2.24 ±0.74 ab 7-me c 32 43.645 2.41 ±0.56 2.02 ±0.71 10-. 14-dime c 32 5me c 32 43.859 5.00 ±1.55 c 2.00 ±0.66 a 4.46 ±2.61bc 2.87 ±0.32 ab 4.53 ±2.35bc 1216-dime c 32 43.957 1.12 ±0.60 c 1.98 ±0.60 a 2.00 ±2.24bc 1.27 ±0.18 ab 3.17 ±1.99 a 9-. 11-dime c 32 13-.15-dime c 32 44.055 4.15 ±1.12 c 0.95 ±0.29 b 1.54 ±1.11 b 9.45 ±0.99 a 7.27 ±1.42 a 6-. 10-dime c 32 6-. 14-dime c 32 6-. 16-dime c 32 44.222 2.52 ±1.40 7.66 ±2.08 1.50 ±0.79 table 1 (continued): cuticular hydrocarbons present in the camponotus textor workers (w), major workers (lw), queens (q), virgin queens (vq) and males (m). rt = retention time in minutes. different letters indicate significant differences (p<0.001). compound rt w lw q vq m mean sd mean sd mean sd mean sd mean sd 1031 campos, m.c.g et al. — cuticular hydrocarbon profiles of camponotus textor nc 33 44.519 2.28 ±0.61 c 1.30 ±0.34 ab 10.91 ±4.25 d 1.62 ±0.23bc 0.91 ±0.28 a 13-me c 33. 15-me c 33 17-me c 33 45.015 1.23 ±0.54 1.54 ±0.22 9-me c 33 45.153 0.06 ±0.10 0.78 ±0.49 0.78 ±0.19 7-me c 33 45.684 0.64 ±0.51 0.31 ±0.12 0.93 ±0.48 9-. 11-di me c 33 9-.13-dime c 33 45.759 0.59 ±0.38 6-me c 33 46.070 0.10 ±0.24 0.29 ±0.19 3.93 ±0.76 5-me c 33 46.300 0.21 ±0.26 1.22 ±0.41 0.53 ±0.74 3-me c 33 46.426 0.93 ±1.62 n-c 34 47.648 0.96 ±0.90 3.70 ±0.42 13-me c 34 48.531 2.41 ±2.23 ad 3.04 ±0.97 d 2.62 ±2.36 b 3.97 ±0.48 abc 2.86 ±0.55 c 11-me c 34 48.620 1.62 ±1.55 c 2.51 ±0.74 b 1.09 ±1.13 c 3.53 ±0.73 a 3.11 ±0.93 ab 5-me c 34 49.174 1.76 ±0.64 0.89 ±0.70 2.95 ±0.54 1.95 ±0.43 7-. 11-dime c 34 49.340 1.76 ±0.67 9-. 11-dime c 34 49.413 2.00 ±0.74 b 0.84 ±0.27 a 1.28 ±0.65 a 1.54 ±0.46 b 1.11 ±0.34 a 5-. 7-dime c 34 49.635 1.19 ±0.42 1.12 ±0.57 n-c 35 49.971 0.12 ±0.17 3.41 ±1.68 8-me c 35 51.046 1.29 ±0.76 table 1 (continued): cuticular hydrocarbons present in the camponotus textor workers (w), major workers (lw), queens (q), virgin queens (vq) and males (m). rt = retention time in minutes. different letters indicate significant differences (p<0.001). compound rt w lw q vq m mean sd mean sd mean sd mean sd mean sd table 2: classification matrix for group comparison of predicted functional group using discriminant analysis. % correct w lw q vq m w 98.76 80 1 0 0 0 lw 100 0 27 0 0 0 q 100 0 0 27 0 0 vq 88.88 0 0 0 24 3 m 100 0 0 0 0 27 total 97.88 80 28 27 24 30 1032 sociobiolog y vol. 59, no. 3, 2012 this study, castes and sex had qualitative differences in their respective cuticular profiles, mainly related to monomethylalkanes, and dimethylalkanes. the compounds that have distinct and variable isomers (double bonds or methyl groups) are those associated with colonial recognition. on the other hand, n-alkanes are considered important for physical protection of the individual (howard & blomquist 2005). in c. textor, some compounds were only found in queens and virgin queens, which suggest that these hydrocarbons are characteristics of reproductive females. in camponotus floridanus, the prime distinction of cuticular hydrocarbon profiles is observed between queens and workers, in which eighteen compounds are only found in fertile queens (endler et al. 2004, 2006). in harpegnathos saltator the hydrocarbon profiles were similar between gamergates and queens, but they were different between workers and young queens (peeters & hölldobler 1995), and in pachycondyla inversa many compounds were different in profiles of queens and workers (heinze et al. 2002). in the polyg ynous ant linepithema humile the cuticular hydrocarbon profiles of queens, young queens and workers were different (de biseau et al. 2004). dietemann et al. (2003) showed that myrmecia gulosa had differences between queen and worker hydrocarbon profiles. dahbi & lenoir (1998) showed the similarity of chemical compound profiles in queens and their fig. 1: discriminant analysis of cuticular hydrocarbons present in the castes. w (workers), lw (major workers), q (queens), vq (virgin queens) and m (males). 1033 campos, m.c.g et al. — cuticular hydrocarbon profiles of camponotus textor respective daughter-workers, indicating a significant correlation between cuticular profiles and fertility. in c. textor, no alkenes were present in the cuticular profiles. the same results were obtained for c. floridanus (endler et al. 2004) and c. fellah (lalzar et al. 2010).this pattern is different from that found in pogonomyrmex barbatus (wagner et al. 1998), harpegnathos saltator (liebig et al. 2000) and other 37 ant species (van wilgenburg et al. 2011). discriminant analysis of compounds of all studied groups showed that of 90 total compounds, 42 were common to all castes (queens and workers) and genders (females and males). 4-me c 30 , n-c 32 and n-c 33 were found in larger quantities in queens than in other groups, major workers had the compounds 16-,14and 12-me c 30 in more representative quantities than any other group. males had the compound 9-, 17-dime c 30 in larger quantities than other groups and virgin queens and males presented the compound 9-, 11and 13-, 15-dime c 32 in larger quantities than other groups. boulay et al. (2007) observed quantitative differences in the profile of aphaenogaster senile, where reproductive queens had five compounds in larger quantities and workers had five other compounds in larger quantities. moreover, endler et al. (2006) showed that queens had a higher proportion of compounds compared with workers. also in crematogaster smithii, workers, virgin queens, intermorphs and queens had qualitative differences in their compounds (oettler et al. 2008). colonial blend or gestalt colony odor seems to be more important for individual recognition than a specific group of hydrocarbons. however, recent revision of cuticular hydrocarbon profiles in ants suggests that dimethylalkanes are good candidates for species and nestmate discrimination (reviewed in martin & drijfhout 2009a). in c. textor, ten of ninety compounds were dimethylalkanes, and discriminant analysis showed that variation between groups was mostly yielded by these hydrocarbons. however, further behavioral bioassays are needed to understand the role of these compounds within the colonies. in summary, our study demonstrates that cuticular hydrocarbons varied sufficiently among castes and sex to separate individuals according to their functional roles. by using this variation, individuals may discriminate among workers, major workers, fertile queens, virgin queens and males like in other 1034 sociobiolog y vol. 59, no. 3, 2012 ant species. variation in cuticular hydrocarbons might therefore play an important role in the organization of colonies of polyg ynous ant species. acknowledgments the authors thank cnpq for a graduate research grant to m.c.g.c. and fapesp (proc. 10/10027-5 to f.s.n.). references aitchison, j. 1986. the statistical analysis of compositional data, monographs on statistics and applied probability. chapman and hall, london. blomquist, g.j. & a.g bagnères 2010. insect hydrocarbons: biolog y, biochemistry and chemical ecolog y. cambridge university press: 492. boulay, r., x. cerdá, t. simon, m. roldan, & a. hefetz 2007. intraspecific competition in the ant camponotus cruentatus: should we expect the ‘dear enemy’ effect? animal behaviour 1: 1-9. breed, m.d. & b. bennett 1987. kin recognition in highly eusocial insects. in: fletcher, d j.c. & michener, c.d. 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(eds) pheromone communication in social insects: ants, wasps, bees and termites. westview, boulder: 79–103. wagner d., m.j.f. brown, p. broun, w. cuevas, l.e. moses, d.l. chao & d.m. gordon 1998. task-related differences in the cuticular hydrocarbon composition of harvester ants, pogonomyrmex barbatus. journal of chemical ecolog y 24:2021-2037. wagner, d., m. tissot & d.m. gordon 2001. task-related environment alters the cuticular hydrocarbon composition of harvester ants. journal of chemical ecolog y 27: 1805– 1819. wilson, e.o. 1971. social insects. science, new series 172: 406. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.7104sociobiology 68(3): e7104 (september, 2021) introduction ants and plants interact in a variety of ways, from parasitism to mutualism (beattie, 1985), including interactions with diaspores (i.e.: dispersal unit) that can result in seed dispersal (anjos et al., 2020; luna et al., 2021). seed dispersal is a fundamental process for plant fitness because it determines the location in which seeds arrive and whether they will be able to develop and reach future stages in that location (wenny, 2001). ants can interact with non-myrmecochorous diaspores (without elaiosomes), in which the pulp and aryl abstract ants are able to interact with fruits and seeds that are not adapted for ant seed dispersal. in brazil, several studies show interactions of ants with non-myrmecochorous diaspores; however, few of them have studied the structure of ant-fruit networks. the use of the network approach allows visualising multiple interactions between partners and how they are shaped by the community context. our study aims to investigate ant-fruit networks as well as quantitative and qualitative dispersal components in a fragment of the brazilian atlantic forest. we investigated the structure of interaction networks, diaspore removal rates, diaspore destination and dispersal distance over two years of observation. we constructed three interaction networks: dry season, rainy season and total, with the latter comprising the two formers. the diaspore removal rate, dispersal distance and diaspore destination experiments were performed for the plant species miconia calvescens, miconia prasina, psychotria leiocarpa and inga edulis. we recorded a large number of interactions, with diaspore cleaning being more frequent than removal. ant-diaspore networks were nested, non-modular and little specialized. m. calvescens, m. prasina and i. edulis showed higher diaspore removal rates. diaspore removal distances were the same among m. calvescens, m. prasina and i. edulis. in m. calvescens and i. edulis, the main diaspore destination was the ant’s nest. our study shows that diaspore cleaning is the most common behavior in antdiaspore interactions and there are no differences in the organization of interaction networks over the seasons. these results have implications for the future structure of plant communities, considering that a small part of the diaspores is removed, and that most of them are cleaned, favouring germination at the deposition site. sociobiology an international journal on social insects bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 29 march 2021 initial acceptance 16 april 2021 final acceptance 03 july 2021 publication date 13 august 2021 keywords ant-fruit interactions, secondary dispersal, seed cleaning, seed removal, mutualistic networks. corresponding author bianca ferreira da silva laviski universidade federal rural do rio de janeiro – ufrrj km 07, zona rural, br-465 seropédica cep: 23890-000 seropédica, rio de janeiro, brasil. e-mail: biancalaviski@gmail.com work as an attraction for them (rico-gray & oliveira, 2007). several ant species have already been reported to disperse non-myrmecochorous diaspores across the globe (anjos et al., 2020). pizo and oliveira (2000), for instance, observed more than 800 interactions between 56 species of nonmyrmecochorous plants and 36 species of ants from monthly samplings in the atlantic forest over two years. the interactions between several ant and plant species can be represented by complex ecological networks at the community level, in which species are represented as nodes, and interactions, as links. the use of the network approach universidade federal rural do rio de janeiro, seropédica-rj, brazil research article ants structure of ant-diaspore networks and their functional outcomes in a brazilian atlantic forest mailto:biancalaviski@gmail.com bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas – ant-diaspore networks and their functional outcomes2 allows visualizing multiple interactions between partners and how they are shaped by the community context, in addition to joining different research fields (bascompte, 2007). most studies on ant-plant networks have focused on the interactions between ants and plants with extrafloral nectaries (efn), and only 6% have studied ant-seed networks (del-claro et al., 2018). furthermore, the majority of such studies have been performed in a few regions, such as the amazon and neotropical savanna, both in brazil, and on the coast of the gulf of mexico (del-claro et al., 2018). in interactions between ants and efn-bearing plants, studies show nested networks (del-claro et al., 2018), in which interactions are organised around a central core, and the interactions of less central species are a sub-set of the most generalist species (bascompte et al., 2003). studies on mutualistic and predation networks between diaspores and ants also have networks with a nested pattern (guimarães et al., 2007; anjos et al., 2018, 2019; luna et al., 2018), showing that species do not interact randomly. in addition, anjos et al. (2018) showed that removal and consumption networks are not modular in the brazilian savanna. network specialization, vulnerability and robustness were network metrics that were not affected by habitat and exclusion of the main disperser under study in the mediterranean landscape (timóteo et al., 2016). several factors have been pointed out to explain the origin and maintenance of structural patterns of ant-plant networks, such as temperature and precipitation (rico-gray et al., 2012), soil characteristics (dáttilo et al., 2013b) and plant phenology (lange et al., 2013; anjos et al., 2018). regarding the latter, as fruiting is seasonal, dry and rainy periods show differences in fruiting plant species, and this can affect the organization of interaction networks. in addition to a network approach, in order to understand whether plants gain advantages in interactions with ants – such as escape from areas with a high mortality rate, colonization of new areas, and deposition in suitable areas for development (wenny, 2001; rico-gray et al., 2007) –, approaches at the plant-population level are necessary. these approaches allow learning about the effective dispersers, which maximize the number of new adult plants by their dispersal activity, considering the quantitative and qualitative components (schupp et al., 2010). the number of seeds dispersed represents the quantitative component, and the distance and removal destination represent the qualitative component. diaspore removal rates by ants – quantitative component – may differ among different plant species, due to diaspore mass and chemical content (pizo & oliveira, 2000; pizo & oliveira, 2001). the lipid content of diaspores plays an important role in attracting ants for interaction. small, lipid-rich diaspores (>60%) are highly attended by ants, quickly removed and moved over long distances (>10m) (rico-gray et al., 2007). however, ants can also consume diaspore resources locally or remove parts and take them to the nest, which promotes diaspore cleaning (christianini et al., 2007; christianini & oliveira, 2010; gallegos et al., 2014). diaspore cleaning ensures germination rates approximately 20 to 60% higher than control tests (leal & oliveira, 1998; pizo & oliveira, 1998; silva et al., 2019), but not for all plant species (christianini et al., 2007). in addition, diaspore cleaning can increase the germination speed of some species (pizo & oliveira, 1998; silva et al., 2019). therefore, even without removal, diaspores have an advantage in interacting with ants. apart from the number of diaspores removed, the destination and removal distance are important to ensure that seeds are reaching places with improved conditions for seedling establishment and growth. thus, destination and dispersal distance are part of the qualitative dispersal component (schupp et al., 2010). the destination of antremoved diaspores is frequently the ants’ nest (rico-gray et al., 2007), where they are cleaned and deposited in ant dumps, which are located in subterranean chambers or on the surface (farji-brener & medina, 2000; giladi, 2006; luna et al., 2018). outer dumps are potentially important for plants, as the soil around the nest has different edaphic and microclimatic conditions from those in regular soil, as well as higher nutrient content (farji-brener & medina, 2000). these conditions favour germination in ants’ nesting soils for some plant species (passos & oliveira et al., 2002; leal et al., 2007). diaspore removal generally occurs at short distances, with a global mean dispersal distance of 2.39 m for nonmyrmecochorous diaspores (anjos et al., 2020). removal distance depends on ants’ nest density and on diaspore disposition in relation to the nests. furthermore, rainforest ecosystems have shorter removal distances than do savanna ecosystems (anjos et al., 2020). longer distances and the nest as a destination are more advantageous results for plants (anjos et al., 2020; ortiz et al., 2021). considering the different behaviors of ant species when interacting with diaspores, the aim of our study was to describe the network structure of ant-diaspore interactions and their temporal variation in the atlantic forest in south-eastern brazil, seeking to understand several aspects of ants’ role in the interactions. in order to do so, we attempted to answer the following questions: (1) what is the structure of the antdiaspore network in the study area? and does this network structure vary between dry and rainy seasons? we expected nested and non-modular networks to be observed, since they seem to be a pattern in mutualistic networks and a variation in this structure according to the season, due to the direct and indirect effect via plant phenology; (2) which ant behavior (removal or cleaning) is more common with diaspores in the community? as cleaning is a behavior with lower energy cost and performed by a wide variety of ant species, we expected it to be more common than removal; (3) what are the removal rates, distances and destinations of the diaspores carried by ants? finally, we used a series of experiments to study various aspects of the ant-diaspore interaction in four species of plants. our goal was to learn about the food preference of ants that remove diaspores in the study area, thus revealing their diaspore preferences and the role played by ants in the sociobiology 68(3): e7104 (september, 2021) 3 quantitative and qualitative components of dispersal. we expected to find different removal rates, removal distances and destinations for different plant species. methods study area we carried out the present study in a secondary forest located on marambaia island (23º 02’ s; 43º 35’ w), on a part of the area known as restinga de marambaia and located within the municipalities of rio de janeiro, itaguaí, and mangaratiba, in the sepetiba bay, south-eastern brazil. although it is called an island, the area corresponds to an enlarged portion of land (ca. 6 km) connected to the continent by a narrow sand strip. the northern part of the island faces the sepetiba bay and the southern part faces the atlantic ocean (conde et al., 2005). marambaia island shelters different vegetation types. this diversity of vegetation types is related to the geological processes that formed the island and originated soils with different levels of water saturation (menezes & araújo, 2005). among the most common vegetation types are mangrove, restinga (coastal shrub land on sandy soils), and the sloped atlantic forest (conde et al., 2005). the area was farmed from the 17th to the 19th centuries and had most of its forest removed, which is now under secondary succession (goés et al., 2005). the soil has high leaf deposition, slow decomposition and higher nutrient levels on the surface layer (pereira et al., 2008). the regional climate is classified as aw (tropical rainy climate), according to the köppen system, with average monthly temperatures above 20 ºc. the coldest period occurs from june to august (average minimum temperature around 18 ºc), and the warmest period occurs from december to march (average maximum temperature around 30 ºc; (mattos, 2005). rainfall has an annual average above 1,000 mm. the rainy season consists of november to march, when rainfall indices are above 100 mm (mattos, 2005). ant-plant interactions this study was conducted in a transect with 0.5 km in length, in a secondary rainforest, and the procedures were performed monthly, from january 2012 to december 2013. we established 50 observation stations every 10 m. this distance is sufficient to preserve independence between ant colonies (byrne & levey, 1993; kaspari, 1993). each observation station consisted of diaspores on filter paper (10 × 10cm) to facilitate ant visualizing. we collected mature diaspores from trees or which just fallen on the ground. we provided diaspores monthly according to their availability and abundance, ranging from one to 10 diaspores of a single plant species per station. the diaspores made available each month, as well as the total number and stations are presented in supplementary material (table s1). the diaspores were intercalated among stations in cases of availability of more than one species on the same day. we set up the stations at 8 a.m. and observed the interactions between diaspores and ants from 9 a.m. to 5 p.m., with two-hour intervals. the lack of nocturnal observations limits this experiment’s results, because there are species with nocturnal activity, such as odontomachus chelifer, which are known to remove diaspores (raimundo et al., 2009). thus, some species may appear less important in the networks when the interactions actually occur at another time. we recorded the date, time, ant behavior (removal or cleaning) and plant species, as well as collected worker ants for identification. we considered the visualization of any ant species in contact with the surface of the diaspore to be an interaction, as long as the ant was not only walking on the diaspore or touching it with its antennae. ants of the same species found in the same station at different times of the same day were considered to be the same interaction. the ant specimens were deposited in the costa lima entomological collection, of universidade federal rural do rio de janeiro (ufrrj). the plant exsiccates were included in the herbarium collection of the botany department of ufrrj (herbarium rbr). network analysis considering all observed interactions (removal and cleaning), we constructed interaction matrices a, where aij = 1 when ant species i was observed interacting with diaspore species j, and using aij = 0 where there was no interaction. we built three matrices: one for the whole observation time (referred to here as total network); one for only the dry months (april to october), and one for only the rainy months (november to march). we analyzed the connectance, nestedness, modularity and specialization network metrics. connectance is the proportion of links made by the total number of possible links in the network (jordano, 1987). connectance was obtained by the number of observed connections divided by the number of possible connections, calculated by the formula: c = i / ap, where c is connectance; i is the number of observed interactions; a is the number of ant species, and p is the number of plant species (mello et al., 2011). nestedness evaluates whether species with few interactions tend to interact with highly interactive species (i.e., generalist species) (bascompte et al., 2003; almeidaneto et al., 2007). we estimated the nestedness value by the nodf index in the aninhado software (guimarães-jr & guimarães, 2006). nodf values range from 0 (non-nested) to 100 (perfectly nested). we tested nestedness with 1,000 networks generated by the null model ii (bascompte et al., 2003). such null model assumes that the probability of an interaction to occur is proportional to the observed number of interactions of both ant and plant species (bascompte et al., 2003). in addition, we also evaluated modularity. modular networks are those in which species interact more frequently with a group than with species outside that group. we estimated modularity in the network by the m index in the modular bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas – ant-diaspore networks and their functional outcomes4 software (marquitti et al., 2013), based on a simulated annealing algorithm (guimerá & amaral, 2005). the m index ranges from 0 (no modules) to 1 (totally separated modules). we used 1,000 randomized networks by the null model ii with fixed total margins (bascompte et al., 2003). aiming at a more conservative approach (dáttilo et al., 2016), and counting on the availability of more data in the literature for comparison, we used binary data. also, it was considered that quantitative and binary data, with the latter defining the fundamental niche of species (fründ et al., 2016), can answer different questions. however, in order to measure specialization at the network level, we used quantitative data, where each cell in matrix a was filled with the frequency of interaction between the ant species i and the diaspore species j. we used the h2’ index, which ranged from 0 (completely generalist network, total overlap of interactions) to 1 (completely specialized network, without overlap of interactions). this index is robust to changes in sampling effort and network size (blüthgen et al., 2006). we simulated 1,000 null networks from each network, using patefield’s algorithm (patefield, 1981) to evaluate the significance of the h2’ index. we estimated 1,000 h2’values from the null networks, and then we compared if the observed h2’ value differed from those of the null model. to test the differences between the dry and rainy periods, we calculated the absolute difference of the h2’ index between the dry and rainy networks, and then compared it with the absolute difference of the same metric for the networks generated by the null models. for nodf and m, which are metrics affected by the network size, we used the methodology adopted by carvalho et al. (2021), where the observed and randomized values were standardized using z-scores. the transformed z-score is defined: z = [x μ] / σ, where x is the observed index value, μ is the mean of the values from the null networks, and σ is the standard deviation of the values from the null networks (almeida-neto et al., 2008). we used the calculated values of nodf and m for the null networks obtained on aninhado and modular, respectively. we estimated the significance of the difference in the metrics between the dry and rainy networks using z-scores with values greater than 2 (dormann & strauss, 2014). moreover, we defined whether the species were central or peripheral in the networks by the formula: gc = (ki – kmean) / σk, where ki is the mean number of connections for a given ant or diaspore species; kmean is the mean number of connections for all ant or diaspore species (connectance), and σk is the standard deviation of the number of connections for ant or diaspore species (dáttilo et al., 2013a). gc > 1 values indicated central species in the network, with a large number of connections. gc < 1 values indicated peripheral species in the network, with few connections. with the exception of nestedness and modularity, all other analyses cited were performed using the ‘bipartite’ package (dormman et al., 2019) implemented in r v.4.0.2 (r development core team, 2020). diaspore removal rate we selected four species of plants with high abundance of individuals and a large number of fruits in the study area: inga edulis mart., miconia calvescens dc., m. prasina (sw.) dc. and psychotria leiocarpa cham. & schltdl. (table 1). from june to october 2013, we picked 15 stations at random where we placed diaspores protected by screen fences (20 × 20 × 12 cm; 2-cm gap) fixed on the ground by 2-cm wires, so as to allow ant access and prevent disturbance by vertebrates. we then placed four diaspores of a single species in each station. we marked the diaspores with a small dot using a marker pen to identify the fruits utilized in the experiment. we set up the experiment at 07:00 a.m. and then checked it after 24 h, when we recounted the number of diaspores. we considered a diaspore removed if we did not find it in a radius of 30 cm around the cage (passos & oliveira, 2002). we repeated the experiment during the fruiting period of each evaluated diaspore species and while there were enough diaspores to perform the experiment. we used glmm with the poisson family to test for differences in the number of removals between diaspore species. we considered the number of removals to be the dependent variable; diaspore species, the fixed factor; and stations, a random factor. the analyses were performed using the ‘lme4’ (bates et al., 2019) and ‘multcomp’ (hothorn et al., 2008) packages implemented in r v.4.0.2 (r development core team, 2020). destination and distance of diaspore removal from june to october 2013, we selected one fruiting individual of each plant species selected in the previous experiment (table 1), and established three radial stations underneath it at approximately 1 m from each other, in which we assessed the diaspore destination and distance. each station was composed of filter paper (10 × 12 cm) used as substrate, and received two diaspores. thus, each observed plant had a total of six diaspores. we monitored the stations for ant removal from 8:00 a.m. to 5:00 p.m. with one-hour pauses between observations, for a total of 5 observation hours per day. observations were suspended during rain. the total hours observed in each species are shown in table 1. when a removal event took place, we followed the ants to their nests or until they disappeared in the leaf litter, and then measured the removal distance with a measuring tape. we replaced each diaspore after removal. we used glmm with the binomial family to test for differences in destination between diaspore species. we considered the destination the dependent variable; diaspore species, the fixed factor; and data and time, random factors. to test differences in removal distance between diaspore species, we used glmm with the gaussian family. we considered the removal distance the dependent variable; diaspore species, the fixed factor; and data and time, random factors. the analyses were performed using the ‘lme4’ (bates et al., 2019) and ‘multcomp’ (hothorn et al., 2008) packages implemented in r v.4.0.2 (r development core team, 2020). sociobiology 68(3): e7104 (september, 2021) 5 results ant-plant interactions we recorded a total of 1,032 interactions among 49 ant species (22 genera belonging to six subfamilies) (table 2) and 25 plant species belonging to 17 families (table 3). myrmicinae was the subfamily of ants with the largest number of species (s = 36 spp.; 73.47%), followed by ponerinae (s = 4 spp.; table 1. plant species used in the removal-rate, destination and removal-distance experiments. observation hours refer to the total time of removal-distance experiments. plant species diaspore size (cm) observation hours inga edulis mart. 1.20 ± 0.17 × 0.77 ± 0.13 5 miconia calvescens dc. 0.42 ± 0.05 × 0.37 ± 0.04 25 miconia prasina (sw.) dc. 0.51 ± 0.04 × 0.42 ± 0.04 25 psychotria leiocarpa cham. & schltdl. 0.57 ± 0.09 × 0.49 ± 0.06 26.5 sub-family / species code dolichoderinae 1. linepithema sp. 1 linep1 ectatomminae 2. ectatomma edentatum roger, 1863 e_ede 3. ectatomma permagnum forel, 1908 e_per formicinae 4. brachymyrmex sp. 1 brach1 5. brachymyrmex sp. 2 brach2 6. camponotus sp. 1 campo1 7. myrmelachista sp. 1 myrme1 myrmicinae 8. acromyrmex subterraneus forel, 1893 a_sub 9. atta sexdens rubropilosa forel, 1908 a_sex 10. carebara urichi (wheeler, 1922) c_uri 11. carebarella bicolor emery, 1906 careb1 12. crematogaster sp. 1 crema1 13. cyphomyrmex sp. 1 cypho1 14. mycocepurus sp. 1 mycoc1 15. octostruma rugifera (mayr, 1887) o_rug 16. pheidole diligens (smith, 1858) ph_dil 17. pheidole sigillata wilson, 2003 ph_sig 18. pheidole sp. 3 pheid3 19. pheidole transversostriata mayr, 1887 ph_tra 20. pheidole pedana wilson, 2003 ph_ped 21. pheidole subarmata mayr, 1884 ph_sub 22. pheidole sp. 7 pheid7 23. pheidole tijucana borgmeier, 1927 ph_tij 24. pheidole sp. 9 pheid9 table 2. ant species recorded in this study on marambaia island (rj) interacting with diaspore species. 25. pheidole sp. 10 pheid10 26. pheidole puttemansi forel, 1911 ph_put 27. pheidole lucaris wilson, 2003 ph_luc 28. pheidole sp. 13 pheid13 29. pheidole sp. 14 pheid14 30. pheidole sensitiva borgmeier, 1959 ph_sen 31. pheidole sp. 16 pheid16 32. pheidole sp. 17 pheid17 33. sericomyrmex sp. 1 seric1 34. solenopsis sp. 1 solen1 35. solenopsis sp. 2 solen2 36. solenopsis sp. 3 solen3 37. solenopsis sp. 4 solen4 38. solenopsis sp. 5 solen5 39. solenopsis sp. 6 solen6 40. trachymyrmex sp. 1 trach1 41. trachymyrmex sp. 2 trach2 42. wasmannia auropunctata (roger, 1863) w_aur 43. wasmannia sp. 2 wasm2 ponerinae 44. odontomachus chelifer (latreille, 1802) o_che 45. odontomachus meinerti forel, 1905 o_mei 46. neoponera apicalis (latreille, 1802) n_api 47. pachycondyla striata fr. smith, 1858 p_str pseudomyrmecinae 48. pseudomyrmex sp. 1 pseud1 49. pseudomyrmex sp. 2 pseud2 sub-family / species code myrmicinae (continuation) 8.16%) and formicinae (s = 4 spp.; 8.16%). for plants, the melastomataceae, meliaceae and rubiaceae families equated to over 50% of the interactions. we observed 1,016 diaspore cleaning interactions (98.45%) versus 16 diaspore removal interactions (1.55%). however, 275 diaspores disappeared from the observation area and may have been removed by the ants. we have included these diaspores as removals in table 3, which, therefore, has a different total number of removals from that shown here. considering these disappearances to be removals, the total number of removals was 291 (22.26% of interactions). the inclusion of such data overestimates removal by ants; however, the specific removal experiments (results in the topic below) showed that the removal was greater than 1.55%. the true percentage of diaspore removal by ants in the community must be between these two values (1.55% and 22.26%). ants that removed diaspores were acromyrmex subterraneus, atta sexdens rubropilosa, cyphomyrmex sp. 1, ectatomma edentatum, e. permagnum, neoponera apicalis, pachycondyla striata, pheidole sigillata and sericomyrmex sp. 1. bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas – ant-diaspore networks and their functional outcomes6 table 3. diaspore species explored by ants in this study on marambaia island (rj) with the total number of interactions recorded and ant species with which they interacted (see code in table 2). family / species unit of dispersal number of interactions number of removals ant species araceae 50. monstera adansonii var klotzschiana (schott) madison fruit 4 1 18-19; 34; 38 burseraceae 51. protium brasiliense engl. seed 61 41 3; 16-21; 25-26; 33-38; 42; 44; 47 erythroxylaceae 52. erythroxylum pulchrum a. st.-hil. fruit 74 19 12; 16-21; 25-27; 29-30; 34-36; 38-42; 47 fabaceae 53. inga edulis mart. seed 23 2 1; 3; 12; 16-19; 25; 40-41; 46-47 lauraceae 54. ocotea schottii (meisn.) mez fruit 89 1 1-2; 4-5; 14; 16-22; 25; 33-35; 38-40; 42; 48 malpighiaceae 55. niedenzuella acutifolia (cav.) w.r. anderson fruit 1 1 42 melastomataceae 56. clidemia hirta (l.) d. don fruit 26 17 16-21; 35; 40; 42-43; 47 57. miconia calvescens dc. fruit 70 30 1; 4; 8; 12; 16-21; 23; 25; 34; 38; 42 58. miconia prasina (sw.) dc. fruit 193 93 1; 3-4; 12; 16-21; 23; 25; 28; 33; 35; 38; 40-41; 43; 46; 49 meliaceae 59. guarea guidonia (l.) sleumer seed 132 35 2-3; 10; 12; 16-27; 34; 36-38; 42-43; 45-47 moraceae 60. ficus insipida willd. fruit 78 0 1; 3; 7; 10; 12; 16-21; 25; 33-38; 40; 42-44; 47 nyctaginaceae 61. guapira opposita (vell.) reitz fruit 7 0 16; 18-19; 25; 36; 47 passifloraceae 62. passiflora edulis sims seed 18 1 14; 16-20; 35-36; 42 piperaceae 63. piper amplum kunth fruit 2 0 6; 19 64. piper anisum (spreng.) angely fruit 3 0 16; 40; 42 65. piper caldense c. dc. fruit 5 0 9; 13; 19; 43 rubiaceae 66. coccocypselum cordifolium nees & mart. fruit 17 0 16-22; 36; 42 67. psychotria cf. hoffmannseggiana (schult.) müll. arg. fruit 14 1 1; 16-22; 31 68. psychotria deflexa dc. fruit 37 10 11-12; 16-20; 36; 40; 42-43; 47 69. psychotria leiocarpa cham. & schltdl. fruit 58 14 16-22; 28; 32; 34; 36; 40-43 sapindaceae 70. paulinia micrantha cambess. seed 33 6 9; 11; 16-21; 25; 33; 36; 40; 42 71. urvillea sp. fruit 3 0 18; 20 siparunaceae 72. siparuna guianensis aubl. seed 17 17 16-19; 29; 42 solanaceae 73. solanum pseudochina spreng. fruit 21 0 10; 16-20; 22; 36; 40; 47 verbenaceae 74. citharexylum myrianthum cham. fruit 46 2 1; 15-21; 25; 34-36; 40; 42-43; 47 sociobiology 68(3): e7104 (september, 2021) 7 fig 1. network of interactions between diaspores and ant species. plants are represented by triangles, and ants by circles. lines represent ant-diaspore interactions. (a) dry-season network. (b) rainy-season network. (c) total network. the ant codes are in table 2. cmyr = citharexylum myrianthum; chir = clidemia hirta; ccor = coccocypselum cordifolium; epul = erythroxylum pulchrum; fins = ficus insipida; gopp = guapira opposita; ggui = guarea guidonia; iedu = inga edulis; mcal = miconia calvescens; mpra = miconia prasina; mada = monstera adansonii; nacu = niedenzuella acutifolia; osch = ocotea schottii; pedu = passiflora edulis; pmic = paulinia micrantha; pamp = piper amplum; pani = piper anisum; pcal = piper caldense; pbra = protium brasiliense; phof = psychotria cf. hoffmannseggiana; pdef = psychotria deflexa; plei = psychotria leiocarpa; sgui = siparuna guianensis; spse = solanum pseudochina; urv1 = urvillea sp. network analysis the complete network between ants and diaspores (removal and cleaning interactions) showed a connectance of 0.237; it was significantly nested (nodf = 33.74; p < 0.001), not significantly modular (m = 0.23; p = 1.00; fig 1), and it had a higher level of specialization than the null models (h2’ = 0.099; p < 0.001). pheidole species (species 16-21 – table 2), solenopsis sp. 3, trachymyrmex sp. 1 and wasmannia auropunctata species were present in the core ant species. in plants, the species observed in the central core were erythroxylum pulchrum (erythroxylaceae), ocotea schottii (lauraceae), miconia prasina (melastomataceae), guarea guidonia (meliaceae) and ficus insipida (moraceae). bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas – ant-diaspore networks and their functional outcomes8 fig 2. (a) diaspores removal frequency of the plant species studied. (b) destination of diaspore removals (nest = black hatched bar, litter = grey bar). the network of only dry months showed a connectance of 0.271; it was significantly nested (nodf = 34.34; p < 0.001), not significantly modular (m = 0.25; p = 0.99), and it had a higher level of specialization than the null models (h2’ = 0.123; p < 0.001). the ant species in the central core were pheidole species (species 16-20 – table 2) and w. auropunctata. the diaspores in the central core were o. schottii (lauraceae), m. prasina (melastomataceae) and g. guidonia (meliaceae). the network of only rainy months showed a connectance of 0.279; it was significantly nested (nodf = 36.03; p < 0.001), not significantly modular (m = 0.25; p = 0.99), and it had no higher level of specialization than the null models (h2’ = 0.118; p = 0.086). the ant species in the central core were pheidole species (species 16-21 – table 2), solenopsis sp. 3, w. auropunctata and p. striata. the diaspores in the central core were protium guidonia brasiliense (burseraceae), e. pulchrum (erythroxylaceae) and g. guidonia (meliaceae). diaspore removal rate, destination and distance of diaspore removal miconia calvescens, m. prasina and inga edulis showed the highest removal rates (65.8%, 58.3% and 40.0%, respectively), whereas p. leiocarpa showed a low removal rate (2.1%; deviance = 310.03; d.f = 3; p < 0.001, fig 2a). in the experiments on destination and distance of diaspore removal, we observed 137 removals for m. calvescens, 14 for m. prasina and 17 for i. edulis. no removals were observed for p. leiocarpa. the ants that removed diaspores were a. sexdens rubropilosa and poneromorph species (pachycondyla and ectatomma species). the a. sexdens rubropilosa species removed the most diaspores of m. calvescens (95.62%; n = 131), whereas the poneromorph species removed the most diaspores of m. prasina (92.86%; n = 13). the a. sexdens rubropilosa species removed all the diaspores of i. edulis. the removal distance varied between 5 and 473 cm. the average removal was 107.50 cm for m. calvescens, 104.57 cm for m. prasina and 300.55 cm for i. edulis. the diaspores of i. edulis were removed farther than miconia diaspores (deviance = 1945.3; d.f = 3; p < 0.001). ants carried most of the diaspores of m. calvescens and i. edulis to their nests (83.94% and 94.12%, respectively; fig 2b). in most m. prasina removals, ants did not reach the nests and abandoned the diaspores in the leaf litter (92.86%; deviance = 134.02; d.f = 2; p < 0.001; fig 2b). we observed pieces of m. calvescens fall to the ground along the path of a. sexdens. we did not observe any subsequent discards by a. sexdens for any plant species after they entered the nest. discussion this study recorded a large number of interactions in an atlantic forest area on marambaia island, with diaspore cleaning being the main interaction. the networks analysed cleaning and removal interactions together, and they were nested and without modules for the total, dry and rainy seasons networks. the total and dry season networks were more specialized than the null models, but with low specialization values. the rainy season network did not show higher specialization than the null models. the central core ant species were virtually the same in the dry and rainy seasons. removal rates were high and equal for the miconia and inga edulis species, but low for psychotria leiocarpa. the removal distance was the same for the miconia species and i. edulis. however, the destination of m. calvescens and i. edulis was mostly the nest, while for m. prasina the destination was the leaf litter. ant-plant interactions diaspore cleaning was the most common ant behavior in the recorded interactions, being 3.5 times more frequent than diaspore removal. this result opposes to that found in ant-fruit interaction networks in the brazilian cerrado, where diaspore removal was more common than diaspore cleaning (anjos et al., 2018). despite not presenting values for comparison, pizo and oliveira (2000) report that the behavior sociobiology 68(3): e7104 (september, 2021) 9 of removing pieces and collecting liquids is more common than removing diaspores in the atlantic forest. in addition, passos and oliveira (2003) justify their systematic sampling in a restinga area by pointing the high speed of diaspore removal by large ponerines, which makes it difficult for them to be seen during active search. moreover, ants remove diaspores at longer distances in savanna areas than in rainforests (anjos et al., 2020). in addition to the distance, the removal rate may also be lower in rainforests. moreover, impacted areas of the atlantic forest and in secondary succession had lower removal rates when compared to undisturbed areas (zwiener et al., 2012; almeida et al., 2013; bieber et al., 2014). poneromorph species are the ant species that most remove in atlantic forest areas, and they are less often observed in impacted areas than in undisturbed areas (almeida et al., 2013; bieber et al., 2014). the fact that the study area was in the process of secondary succession explains the low removal rate. in addition, most interacting ant species were small and did not remove diaspores. an ant’s body size is also a key trait for removal (camargo et al., 2019). diaspore cleaning plays an important role in plant recruitment, since it increases germination rates in most plant species whose diaspores are cleaned by ants (christianini et al., 2007; camargo et al., 2016), and it may also decrease germination time (lima et al., 2013). in addition, diaspore cleaning decreases the chances of attacks by pathogens such as fungi, providing conditions for germination and seedling development (pizo & oliveira, 1998; passos & oliveira, 2002). for example, guarea guidonia, whose diaspores are cleaned by ants, is benefited by this interaction (silva et al., 2019). therefore, although there is no removal, diaspore cleaning brings benefits to the plants. the m. prasina species was the most frequent in the interaction records, followed by g. guidonia. miconia species are classified as ornitochoric and usually have high water and sugar content (silveira et al., 2012). the compounds present in these fruits serve as a resource for the ants and should promote the high number of interactions found for that species. guarea guidonia is an ornitochoric species whose seeds are covered with a red sarcotesta, a similar compound to aryl, usually rich in lipids (van der pijl, 1972). the presence of resources such as pulp is important for ant attraction (ricogray & oliveira, 2007). network analysis the networks showed low connectance, a nested pattern, absence of significant modules and low specialization. these results indicate low interaction between diaspores and ants and with some species of plants and ants dominating most interactions. other studies have shown that mutualistic networks between ants and diaspores were also nested (guimarães et al., 2007; anjos et al., 2018). in our study, we observed that some species of ants (e.g., pheidole species) and plants (e.g., g. guidonia, m. prasina) concentrated most of the interactions. this result agrees with those by palacio et al. (2016), which showed that generalist species play a central role in highly diverse plant-frugivorous networks. in addition, the specialization values of the networks were very low (although the total and dry season networks were more specialized than the null models). this indicates that ants that explore diaspores are generalists and interacting with any diaspore type, just as plant species (diaspores) interact with several ant species. this is probably the result of opportunistic interactions (anjos et al., 2018), and it is common in relationships with frugivorous insects (passos & oliveira, 2003), where most frugivorous species have generalist and opportunistic behavior and whose spectrum of fruits visited by different ant species commonly overlaps (blüthgen, 2011). in the case of frugivorous vertebrates, specialization varies among dispersal groups and it is influenced by fruit characteristics (donatti et al., 2011; garcía et al., 2018). the absence of differences in the values of nestedness, modularity and specialization between the rainy and dry networks indicates that ant-diaspore interactions remain stable despite differences in climate in our study area. thus, species of ants and plants interact throughout the year, regardless of seasonal variation. in line with our results, ruzi et al. (2017) found that the removal rate by ants for 12 species of neotropical pioneer trees was not affected by seasonality. the core of generalist species was stable throughout the years. the pheidole transversostriata, pheidole sp3, p. diligens, p. sigillata, p. pedana and wasmannia auropunctata species are almost always present in the generalist core, which makes them important species in the general structure of the network and within the community because they promote diaspore cleaning and removal. core species are known to be competitively superior and to monopolize resources in interactions between ants and efn-bearing plants (dáttilo et al., 2013a; dáttilo et al., 2014a, 2014c). a stable core appears to be robust to annual fluctuations, and core species tend to belong to lineages that are less volatile and/or generate multiple species in a short time span (burin et al., 2021). regarding the ant species in the core species in this study, pheidole species are extremely competitive, and they monopolize the resources that they explore, being considered dominant omnivores (silvestre et al., 2003), and w. auropunctata shows massive recruitment, which facilitates the control of the diaspore stations (delabie et al., 2003; silvestre et al., 2003). species of the pheidole and wasmannia genera can remove seeds, although that is not frequent (christianini et al., 2010). thus, a core of interactions composed by such species may indicate high diaspore cleaning rates, which corroborates our results, as well as potential for removing diaspores. the presence of p. striata in the core of central species in the rainy season network may indicate an increase in removal by that species during rainy period, since it removes seeds (christianini et al., 2010; christianini et al., 2012). this idea should be evaluated in future studies. bianca f s laviski, antonio j mayhé-nunes, andré f nunes-freitas – ant-diaspore networks and their functional outcomes10 diaspore removal rate, destination and distance of diaspore removal we observed high removal rates for two miconia species and i. edulis, but not for p. leiocarpa. differences in secondary removal rates occur among plant species (christianini et al., 2012; ruzi et al., 2017; ortiz et al., 2021). the presence of several attractive resources for ants is one of the causes for such differences (christianini et al., 2012; ruzi et al., 2017; clemente & whitehead, 2019; ortiz et al., 2021). the presence of secondary compounds, as occurs in psychotria fruits, can reduce the ant recruitment and result in low removal rates (cazetta et al., 2008; santana et al., 2013). as a result, diaspores ‘preferred’ by ants have greater removal (e.g., m. calvescens = 65.8% of removal), and ‘non-preferred’ diaspores are rarely or not removed (e.g., p. leiocarpa = 2.1% of removal). this had already been observed in granivory by ants (willot et al., 2000). the studied ants removed diaspores to short distances. removal distances are in accordance with data reported by anjos et al. (2020), in which rainforests ecosystems show shorter removal distances by ants than savanna ecosystems. hence, even at short distances diaspore removal decreases diaspore aggregation and helps the local plant population (gorb & gorb, 2000), as it may promote recruitment through a reduction in competition among seeds and a decrease in attacks by predators (guimarães-jr & cogni, 2002). despite their larger sizes (table 1), i. edulis diaspores were removed for longer distances than those of miconia. diaspore size is also a key trait influencing removal rates (pizo & oliveira, 2001). therefore, the high rates of removal of a large diaspore must have occurred due to the chemical composition of the fleshy portion. it is known that ants look for more fleshy fruits (passos & oliveira, 2003; rico-gray & oliveira, 2007), and the composition of these fruits can also be an important factor (pizo & oliveira, 2001; christianini et al., 2012). most of the removals of i. edulis and m. calvescens were to the nest, which is advantageous for seedling development (farji-brener & medina, 2000). however, the arrival of many seeds in the nest can increase competition (spiegel & nathan, 2012). therefore, behaviors in which ant species take some diaspores to the nest and abandons others can be advantageous for plants (ortiz et al., 2021). in removals by atta sexdens, nest deposition without subsequent seed discard may indicate that the diaspores serve as a substrate for the fungus and, therefore, dispersal does not occur. in the case of m. calvescens, in which the dispersal unit is the fruit with multiple seeds, some of them are abandoned along the way and can benefit from the removal distance. however, in i. edulis, in which the dispersal unit is the seed, a. sexdens may be acting as a predator. general conclusions in the atlantic forest, interactions between ants and diaspores are frequent and generalised, with ants playing an important role in dispersal stages. in general, ants interact with diaspores by cleaning them, and some species also remove them. ant-diaspore networks are generalist, nested and remain stable throughout the seasons. thus, we can conclude that diaspore cleaning and removal occur continuously. the removal distance and final destination of diaspores depend on the diaspore species and on the ant species that remove them. ants are good secondary dispersers for only some plant species, depending on their behavior and the identity of the ant species (christianini et al., 2012; clemente & whitehead, 2019). for most plants, ants would play a more important role in cleaning and promoting germination (pizo & oliveira, 1998). acknowledgements we thank d. azevedo and i. azevedo for helping us in the field. we also thank the assessment centre of marambaia island (cadim), which provided us with logistic support during our study, as well as capes-br for granting the scholarship. references almeida, f.s., mayhé-nunes, a.j. & queiroz, j.m. 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(2012). antdiaspore interactions during secondary succession in the atlantic forest of brazil. revista de biología tropical, 60: 933-942. doi: 10.15517/rbt.v60i2.4028 https://doi.org/10.5091/plecevo.2012.706 https://doi.org/10.1016/j.cub.2016.01.046 _hlk21444713 doi: 10.13102/sociobiology.v63i1.779sociobiology 63(1): 628-636 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant community in burned and unburned sites in campos rupestres ecosystem introduction fire represents an important evolutionary factor in forest distributions and tree cover (bond & keeley, 2005; hirota et al., 2011; veldman et al., 2015). ants play important interactions in ecosystem functioning and are good indicators of ecosystem response to fire disturbances (york, 2000; hoffmann, 2003; parr et al., 2004; andersen et al., 2006). ground dwelling ants have been reported to be resilient to burning in many tropical savanna ecosystems (e.g., frizzo et al., 2011; maravalhas & vasconcelos, 2014; anjos et al., 2015). fire events in this ecosystem promote a high biodiversity of ants mediated by fire-induced changes in the vegetation structure (maravalhas & vasconcelos, 2014). in other savannas, the fire effect on ant assemblages also depends on vegetation structure (farji-brener et al., 2002; parr et al., 2004). the increase in fire frequency regimes leads to ant community simplification and loss of biodiversity, while sensitive forest ant species are generally eliminated from the environment (bond & keeley, 2005; hirota et al., 2011; abstract ground-dwelling ants have shown consistent resilience to fire in savanna environments. we carried out a study to investigate how ant community structure responds to fire in campos rupestres sites. we studied the ant communities’ response to fire in two different elevations (800 m above sea level and 1300 m a.s.l.) in southeastern brazil. pitfall trap samples were set at three different periods: one, four, and ten months after a fire event. overall, one hundred and fifteen ant species were collected. at a local scale, ant species richness and composition did not differ between burned and unburned plots in the lower elevation (800 m). at the higher altitude (1300 m) the burned sites presented higher richness and different ant composition in comparison to unburned sites. moreover, ten months later no difference between the richness of ant species in burned and unburned plots was found at 1300 m, even though the plots showed distinct species composition. these data support the hypothesis that fire represents a structuring disturbance factor for ant assemblages and indicates that the recovering of the campos rupestres vegetation influences ant community structure at higher elevations. sociobiology an international journal on social insects fs neves1 , tc lana¹, mc anjos², ac reis1, gw fernandes¹ article history edited by gilberto m. m. santos, uefs, brazil received 11 march 2015 initial acceptance 13 october 2015 final acceptance 11 february 2016 publication date 29 april 2016 keywords disturbance, diversity, resilience, serra do cipó, conservation. corresponding author frederico s. neves laboratório de ecologia evolutiva e biodiversidade/dbg/icb/ufmg 31270-901, belo horizonte, brazil e-mail: fredneves@ufmg.br dantas et al., 2015). as a result, ant species more adapted to open habitats are selected (andersen et al., 2006) and then the effect of fire on ant assemblages depends both on habitat traits and on the regional context of the ant fauna (farji-brener et al., 2002; parr et al., 2004; arnan et al., 2006). in campos rupestres fire is a common phenomenon (alves & silva, 2011; conceição et al., 2013; fernandes et al., 2014; veldman et al., 2015; silveira et al., 2016). this ecosystem occurs mainly in espinhaço mountain range and presents a rich and endemic flora and fauna (lara & fernandes, 1996; giulietti et al., 1997). it is characterized by a variety of soil types, a mosaic of vegetation types, and many life forms, mostly adapted to fire (e.g., kolbek & alves, 2008; neves et al., 2011; alves et al., 2014; silveira et al., 2016). the pyrophilic characteristic is specially due to the high abundance of a herbaceous stratum of poaceae and cyperaceae (vitta, 2002; ribeiro & figueira, 2011; neves et al., 2011). the herbaceous layer generally possess high postfire resilience, great capacity of rapid reestablishment and almost complete recovery after months since the fire event research article ants 1 universidade federal de minas gerais (ufmg), belo horizonte-mg, brazil 2 universidade federal de viçosa (ufv), viçosa-mg, brazil sociobiology 63(1): 628-636 (march, 2016) 629 (neves & conceição, 2010). many graminoid species mainly found at higher altitudes sprout and blossom shortly after fire disturbances (kolbek & alves, 2008; neves et al., 2011; ribeiro & figueira, 2011; conceição et al., 2013; alves et al., 2014). in contrast to the herbaceous stratum, shrubs and trees exhibit thick, corky, or exfoliating bark that protect the gems and guarantees their survival after fire. on the other hand, recovery time is greater when compared to the herbaceous stratum (warming, 1892; mistry, 1998; miranda et al., 2002; maurin et al., 2014). the vegetation in characteristics exert a fundamental role in determining the ant community structure (warming, 1892; miranda et al., 2002; ribas et al., 2003; pacheco & vascocelos, 2012). in campos rupestres, the biota quickly re-colonize burned sites, benefiting from vegetation products induced by fire (conceição et al., 2013). this ecosystem presents a high diversity of ants (see costa et al., 2015), which has been argued to be highly resistant and resilient to fire disturbance (anjos et al., 2015). nevertheless, human induced impact on the campos rupestres is increasing at fast rates (fernandes et al., 2014). fire has now been known to promote biological invasion in campos rupestres by exotic plant species as well as to have a deep effect on the depletion of the seed bank (miranda et al., 2002; medina & fernandes, 2007). hence, considering the complexity and dynamics of campos rupestres environment, the presence of frequent fire events may change this structural complexity affecting the availability of nesting sites and food resources for ants (see fagundes et al., 2015). an extensive fire event occurred at serra do cipó in september 2011 offering an important opportunity to evaluate the effects of fire on ant community. our goal is to determine the response of ant species richness and composition after a fire event on a local scale in campos rupestres. moreover, we aimed to compare such effects at two different altitudes (800 and 1300 m a.s.l.) in this ecosystem. we postulated that ant species richness and composition would change after fire, and this variation would differ according to the altitude. materials and methods study site this study was conducted in serra do cipó, minas gerais, brazil. a study conducted by madeira and fernandes (1999) in the site reported mean annual precipitation of 1374 mm, with four distinguishable seasons: the rainy season from november to february; the ‘post-rain’ transition season from march to april; the dry season from may to september; and the ‘post-dry’ transition in october. the soil is acidic, poor in phosphorus, calcium, potassium and magnesium and rich in aluminum (medina & fernandes, 2007; negreiros et al., 2009). our study sites comprised two distinct campos rupestres sites: the low elevation located at the parque nacional da serra do cipó (19°17’49.6” s 19°17’49.6”w) at 800 m (a.s.l.) characterized by several shrub species belonging to the vellozia (velloziaceae) and lychnophora (asteraceae) genera (de carvalho et al., 2012) (fig 1). at this altitude, the fig 1. burned sites immediately after fire in september 2011 in campos rupestres at two altitudes at a) 800 and b) 1300 m. c and d: the same sites four months after fire, in the mid rainy season. tc lana et al. – ants and fire in campos rupestres630 of the dry season (in july), ten months after the fire event. ant specimens were identified to the species level whenever possible (baccaro et al., 2015) and deposited in the collection at the laboratório de ecologia de insetos, ufmg, belo horizonte, mg, brazil. statistical analysis analyses were performed separately for each elevation. to test the influence of fire through time on ant species richness, we compared burned and unburned plots through a linear mixed effects model for each altitude. this analysis fits linear mixed-effects model with specified mixtures of fixed effects and random effects, which exclude temporal pseudo-replication of data (crawley, 2013). data were grouped by sample points (pitfalls) as random effect, whereas the burning and time effects were added to the model as a fixed effect. sampling and burning dates were also incorporated as fixed units in order to test the effects of time and fire on ant species richness. we conducted all mixed effects models analyses with the package lmer in the r language and environment for statistical computing (r development core team, 2014). moreover, for each elevation we performed separate analysis of similarity on a monthly basis (one way anosim) to test changes in ant species composition in burned and unburned sites at each sample month. we used an absencepresence matrix, performing the similarity index of jaccard and each r-value with a corresponding p-value. in addition, non metric multidimensional scale (nmds) analysis was carried out to plot the significant differentiation in ant community composition. anosim and nmds analysis were performed with the program past (hammer et al., 2001). table 1. values of linear mixed effects model of burning and time since fire effects on ant species richness in campos rupestres at two altitudes (800 and 1300 m) and nmds’s values of dissimilarity between burned and unburned plots at each site (jaccard index), separated by time since fire (in months). elevation response variable parameters chisq d.f. aic p 800 m ant richness time*burn 5 143.44 time + burn 0.168 4 141.61 0.681 burn 1.134 3 140.75 0.287 time 8.532 3 148.15 0.003 1300 m ant richness time*burn 7 125.03 time + burn 2.788 5 123.82 0.249 burn 4.304 4 126.12 0.038 time 8.918 3 128.74 0.011 elevation response variable parameters time (moth) r p 800 m ant composition burn x unburned 1 0.04 0.180 4 0.01 0.391 10 0.08 0.065 1300 m ant composition burn x unburned 1 0.07 0.043 4 0.60 <0.001 10 0.59 <0.001 greater amount of available water, higher temperatures, deeper and fertile soils promote the presence of woody vegetation (ribeiro & figueira, 2011). the higher elevation site was at reserva natural vellozia (19°16’45”s, 43°35’27.8”w), at 1300 m (a.s.l.). this was a more typical campos rupestres environment, presenting rocky grasslands, formations of contiguous graminoid cover, some rock outcrop islands, as well as spots of sandy grassland habitats (see de carvalho et al., 2012) (fig 1). the graminoid taxa have been argued to be responsible for the spread of fire in fire-prone ecosystems (castro & kauffman, 1998; ribeiro & figueira, 2011). ant sample design a burned and an adjacent unburned site (control) were randomly chosen at 800 m and 1300 m elevations (fig 1). both elevations had the same experimental design: the burned and unburned sites were at least 500 m apart from each other and in each of them the sample design consisted of three parallel transects of 200 m long, spaced 200 m apart. this experimental design allowed the minimization of the effects of variation in soil structure, slope, topography, and other factors that could influence the diversity of soil organisms. along each transect, five pitfall traps were installed with intervals of 50 m among them, totalizing 15 pitfalls in the burned site and 15 in the unburned site. pitfall traps were 1 l volume plastic container, 15 cm of diameter, filled with 300 ml of soapy water, and left open for 48h in the field. the first sample was taken in october 2011, just one month after the fire event; at the beginning of the wet season. the second sample was collected after the rainy period, in the middle of the wet season (january), four months after the fire event. finally, the last sample was collected in the middle sociobiology 63(1): 628-636 (march, 2016) 631 exhibited significantly more species per pitfall compared to the unburned plot, as well as a dissimilarity of 60% in the community composition between them (table 1, fig 2 and fig 3). while the mean number of species per pitfall was 4.62 + 0.55 (n=15) in unburned plots in this period, in burned sites it was equal to 7.13 + 0.93 (n=15). ten months after the fire disturbance (july), the number of species per pitfall decreased and both plots exhibited again a non-significant difference in ant richness, although was presented distinct species composition. the ant species found more frequently in burned sites four and ten months after fire at 1300 m were dorymyrmex goeldii (f.), camponotus rufipes (f.) and ectatomma edentatum (r.), while brachymyrmex. sp 4. solenopsis sp. 6 was found exclusively in burned sites and completely absent from samples in unburned plots. we also observed the presence of the harvester ant pogonomyrmex naegelii (f.) in the burned plots, and its absence at the unburned site in these months. linepithema sp.1 and the argentine ant l. humile were by far the most frequent species in both unburned and burned plots at higher elevation of campos rupestres, in all studied months. in january, at this altitude, l. humile was more frequent in burned site than in the adjacent unburned site. discussion we found a resilient ground-dwelling ant community on a local scale at the altitude of 800 m (a.s.l.) considering that there was not a significant alteration in ant community structure after fire in this altitude. similar results were obtained in burned sites of the cerrado stricto sensu (see frizzo et al., 2011; vasconcelos et al., 2009; maravalhas & vasconcelos, 2014). therefore, ground-dwelling ants that nest and forage underground seem not to suffer from the negative effects of fire (frizzo et al., 2011; vasconcelos et al., 2009; maravalhas & vasconcelos, 2014; anjos et al., 2015). on the other hand, arboreal ant species may hide and protect themselves inside thick trunks of fire-adapted plant species in the cerrado (fagundes et al., 2015). the variation in ant richness at 800 m correlates with the climatic and phenological traits of vegetation (e.g., belo et al., 2013). we observed an increase in ant species richness in january, the warmer and wetter period, followed by a significant decrease in july, the colder and dryer period. many plant species bloom and disperse in the rainy season in serra do cipó (madeira & fernandes, 1999; ranieri et al., 2012; belo et al., 2013). the availability of food resources, mediated by plant phenology, influences positively the ant–plant interactions (see rico-gray et al., 2011; lange et al., 2013), which could reflect in greater ant species richness. in the same way, during the winter the number of ant species decreased, probably due to a combination of lower availability of plant-resources produced and lower ant activity due to cooler temperatures (rico-gray et al., 2011; lange et al., 2013). therefore, cold temperatures during winter may act as another environmental factor filtering ant species colonization and success. results we recorded 115 morphospecies of ground-dwelling ants foraging in campos rupestres of serra do cipó (appendix). these species were distributed into seven different subfamilies with myrmicinae being the most diverse in terms of species number (appendix). pheidole was the most diverse genus with 21 species, followed by camponotus with 20 ant species. as expected, the two elevations exhibited contrasting ant community composition and showed distinct patterns of ant community organization after the fire disturbance (table 1). ant species richness per pitfall at 800 m (a.s.l.) changed throughout time (p <0.01, table 1) but did not differ between burned and unburned plots in all sampling periods (p=0.681, table 1, fig 2). in january, incoming species that were absent in october such as trachymyrmex sp.2, cyphomyrmex lectus (f.), strumigenys schulzi (e.), pheidole sp.2, and tapinoma sp.1 were equally found in both burned and unburned plots. ant species composition at this elevation did not differ statistically between burned and unburned plots, regardless of time (table 1, fig 3). fig 2. mean number (+ se) of species per pitfall in burned and unburned plots a) site at 1300 and b) site at 800 m, one (october), four (january) and ten (july) months after fire. * indicates significant difference (p<0.05) in mean number of species per pitfall between burned and unburned plots. there was a temporal variation in species richness per pitfall in burned and unburned plots at 1300 m (a.s.l.), and this variation differed among plots in january, but did not differ in october and july (table 1, fig 2). one month after the fire (october) burned and unburned plots at 1300 m presented similar number of ant species and similar species composition. on the other hand, in january, burned plot tc lana et al. – ants and fire in campos rupestres632 at higher altitude (1300 m a.s.l.), ant community showed a distinct organization pattern from the one detected at lower altitude. fire apparently caused a positive effect on ant species richness and triggered a distinct organization on the community in burned plots from higher elevation. burned plots exhibited an increase in ant richness in january and different species composition from unburned plots. the new post fire ant community organization in january could have been related to the sharp increase in the graminoid resprouting biomass and to the blooming of some species plant induced by fire (see maurin et al., 2014; maravalhas & vasconcelos, 2014). the high plant regeneration capacity after fire (coutinho, 1990; maurin et al., 2014) provides quick recovery of ants in campos rupestres (see anjos et al., 2015). in this study, the reestablishment of grasslands of the burned sites was almost complete four months after the fire event (lana personal observation). moreover, the typical superficial layer of the campos rupestres, which is composed by rocky and sandy soils, offers protection against fire heat (coutinho, 1990; kolbek & alves, 2008; alves et al., 2014). fig 3. ant species spatial distribution in non metric dimensional scale (nmds) in campos rupestres at two altitudes (800 and 1300 m); filled symbols correspond to burned plots. the polygons represent significant differences between the plots (p <0.05). in addition, a massive flowering in burned site at 1300 m took place in january. this phenomenon followed by fruit dehiscence and germination of seeds has already been reported for campos rupestres (see mistry,1998; kolbek and alves, 2008; neves et al., 2011; conceição et al., 2013). this boom of resources are attractive for many insects (seyffarth et al., 1996; vieira et al., 1996), representing higher quality resources offered by the herbaceous stratum for herbivores and pollinators (vieira et al.,1996; conceição et al., 2013). some ant species are seeds’ harvesters and such resource represents a high-nutritional food for those ants (passos & oliveira, 2004). ectatomma edentatum, acromyrmex subterraneus (f.) and c. rufipes were found abundantly in burned site at 1300 m. species of those genera were already reported to remove and transport miconia seeds in campos rupestres of serra do cipó (lima et al., 2012), and also to collect seeds from feces in brazilian forest formations (pizo et al., 2005). moreover, ants were already reported to benefit from the seeds fallen after the fire event in other fire prone savannas worldwide (andersen, 1988; arnan et al., 2006). on the other hand, we are unaware sociobiology 63(1): 628-636 (march, 2016) 633 of any detailed experimental or even field study focusing on the resilience of the campos rupestres productivity and phenological shifts influencing the fauna. the presence of the harvester ant p. naegelii in burned site at 1300 m in january supported our hypothesis of greater availability of food. in cerrado, p. naegelii has a generalist and season-dependent diet comprised of many seed species and arthropod preys, as well as pieces of plant and animal matter (belchior et al., 2012). in california, abundant seeds of forbs appeared immediately after fire (underwood & christian, 2009). also, an increase of seed harvester ants was reported one year after burning and related to greater food availability represented by plant re-sprouting (underwood & christian, 2009; maravalhas & vasconcelos, 2014). also, harvester ants remove carcasses of insects killed by fire (zimmer & parmenter, 1998), and are associated with seed dispersion and establishment of “islands of fertility” in soil (macmahon et al., 2000). during the sampling we observed e. edentatum and c. rufipes widely interacting with trophobiont insects and extrafloral nectaries (efns) in the burned site. in the cerrado, higher sugar concentration in efns was found in fire induced re-sprouting plants when compared to unburned ones, and this fact affected positively abundance of ants on those plants (alves-silva & del-claro, 2013). previous studies also revealed that frequency of visiting efns by ants was higher in sites with more frequent fire events compared to rarely burned ones (knoechelmann & morais, 2008; vasconcelos et al., 2009). in summary, the increase of ant species only at higher elevations may have been influenced by fire-induced sprouting, blooming, and seed dispersion which occurred rapidly in the wet season resulting in a flush of resources highly palatable to ants (vieira et al., 1996; alves-silva & del-claro, 2013; conceição et al., 2013; maravalhas & vasconcelos, 2014; anjos et al., 2015). thus, we argue that fire adaptive traits of herbaceous stratum and rocky island plants at higher altitudes in campos rupestres may offer ants attractive and ephemeral resources after burning events (frizzo et al., 2011; alvessilva & del-claro, 2013; conceição et al., 2013), reflecting in more ant species foraging. regardless the shift from the original and indistinct situation of ant fauna richness between burned and unburned sites, of both altitudes, ten months after fire, burned sites at higher elevation still presented a distinct ant species composition in comparison to unburned plots. similar results were found in other study with campos rupestres (anjos et al., 2015). in this way, frequent fire events may provide a new community of ants in this system, and may also change the relationship with plants (del-claro & marquis, 2015) extending their effects on other trophic levels. this study reinforces that ants respond quickly to fire disturbance and the changes persists for at least some months after burning, which could promote a dynamic and diverse ecosystem. at the same time, rapid colonization of burned sites by ants may be positive for plants since they are potential seed dispersers in these environments (lima et al., 2012, anjos et al., 2015). however, long-term studies are needed to verify these hypotheses. even though, ant nests are known to be an important source of nutrients that contributes to accelerate the vegetation recovery after burning (sousa-souto et al., 2007). in campos rupestres the interaction among ants, plants and fire represent new avenues for future studies on the role of ants in this threatened fire-prone ecosystem. acknowledgments we thank j. h. delabie and w. rocha for identification of ant species and m. beirão and f. costa for their comments in this manuscript. we thank the parque nacional da serra do cipó/icmbio and reserva vellozia for logistical support. we thank the icmbio for the research permits (sisbio no. 23234-7). this work was in partial fulfilment for the phd dissertation of tcl, and was financially supported by peld/ cnpq, comcerrado/cnpq and fapemig. references alves, r. j. v., & silva, n. g. 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(1998). harvester ants and fire in a desert grassland: ecological responses of pogonomyrmex rugosus (hymenoptera: formicidae) to experimental wildfires in central new mexico. environmental entomology, 27: 282287. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.407-414sociobiology 61(4): 407-414 (december, 2014) distance and habitat drive fine scale stingless bee (hymenoptera: apidae) community turnover across naturally heterogeneous forests in the western amazon introduction the lowland amazon basin is notable for its exceptionally high levels of species diversity across a wide range of taxa (erwin, 1988; kress et al., 1998; pitman et al., 2001). high levels of species richness have been attributed to extreme habitat heterogeneity with particular emphasis on the mosaic of soil types found across the western amazon in peru (terborgh, 1985; whitney & alvarez, 1989). these variable soils create a patchwork of forest types differentiated by plant communities that have strong edaphic associations with nutrient poor white-sand soil patches and the more fertile brown-sand and clay soils which surround them (tuomisto & ruokolainen, abstract high tree species richness in the western amazon has been attributed to heterogeneous soils, which harbor edaphic specialist trees. while rapid transitions in tree communities are well documented across these variable soils, few studies have investigated the role of habitat heterogeneity in structuring animal communities. stingless bees are taxonomically diverse and important natural pollinators in neotropical forests. however, little is known about their community structuring at local scales in naturally heterogeneous environments. we systematically sampled stingless bee communities found across three paired sites that included adjacent patches of white-sand and non-white-sand forest in the lowland amazonian region of loreto, peru. we sought to understand: (1) how stingless bee species richness and abundance differ among white-sand and non-white-sand habitats and (2) the relative influence of fine scale geographic distance and habitat type in structuring stingless bee communities. we found that species richness did not differ between habitats and that species abundances were highest in white-sand habitats. community analyses for sampling sites pooled across all months demonstrated that location and soil type played a significant role in structuring bee communities and that community turnover may be more strongly influenced by distance in white-sand habitats than non-white sand habitats. our results suggest that distance and habitat play an important role in driving stingless bee community turnover at fine scales and that the interaction between habitat and geographic distance may promote higher stingless bee community turnover in white-sand habitats than non-white sand habitats. sociobiology an international journal on social insects tm misiewicz1,2, e kraichak2,3 , c rasmussen4 article history edited by denise araujo alves, esalq usp, brazil received 14 september 2014 initial acceptance 03 november 2014 final acceptance 26 november 2014 keywords meliponini, beta-diversity, distance-based redundancy analysis, peru, white-sand forest. corresponding author tracy m. misiewicz university of california, berkeley department of integrative biology 3060 valley life sciences building #3140, berkeley, california 94720 e-mail: tracymisiewicz@gmail.com 1994; fine et al., 2010). white-sand forests differ from surrounding forests in that they harbor significantly less species richness, are shorter in stature, and experience higher temperatures on average below the canopy due to increased light penetration than forests found on surrounding soils (medina & cuevas, 1989; fine et al., 2010). in the western amazon white-sand forests exist as small habitat islands, usually no larger than a few square hectares (fine et al., 2005). while the majority of research examining the role of edaphic variation in species turnover in the amazon has been centered on tree communities (tuomisto & ruokolainen, 1994; fine et al., 2010; but see alvarez alonso et al., 2013) similar habitat specialization may also be present in aniresearch article bees 1 university of california, berkeley, berkeley, california 2 the field museum, chicago, il, usa 3 kasetsart university, bangkok, thailand 4 aarhus university, aarhus, denmark tm misiewicz, e kraichak, c rasmussen stingless bee community turnover 408 mals. because forests constitute the primary habitat and food source for many forest dwelling animals we expect that abrupt changes in floristic composition across habitats may in turn drive turnover in animal communities. animals that provide pollination services play a particularly important role in tropical ecosystems where the majority of trees are reliant on animal interactions for pollen transfer (bawa, 1990). while turnover in bee communities across forest fragments and agriculturally modified landscapes has been well studied in relation to crop production (tylianakis et al., 2005; jha & vandameer, 2010) the role of naturally heterogeneous habitats in driving species turnover in the lowland amazon has largely been neglected (but see abrahamczyk et al., 2010). stingless bees (hymenoptera: apidae: meliponini) are an important taxon for studies of biodiversity and species turnover in the lowland amazon because they are highly diverse (ca 500 species) with their center of diversity found in the neotropics (ca 400 species) (michener, 2013). additionally, they are the most important native providers of pollination services in the amazon, making them essential for ecosystem functioning (engel & dingemans-bakels, 1980; roubik, 1995). most stingless bee species are considered to be generalist pollinators and they exhibit a wide range of variation in nesting habits across species. nests are usually arboreal or subterranean and are constructed using diverse construction materials including mud, wood pulp, feces, and plant exudates (schwarz, 1948; roubik, 1989). foraging distance away from nest site is dependent on the size of the bee with distances ranging from less than 500 m to 2 km (kuhn-neto et al., 2009). given the variation in nest site preferences between lineages relatively little attention has been paid to the fine scale distribution and ecology of neotropical stingless bees and no studies have investigated species turnover across naturally occurring environmental gradients in undisturbed forest sites. furthermore, because the movement of animal pollinators directly influences the distance, direction and degree of pollen dispersal, they ultimately determine the spatial pattern of gene movement within and among plant populations (garcia et al., 2007). if pollinators are restricted in their foraging area due to habitat preference (dieckmann, 2004) then the question of ecological specialization in bee communities may be of particular interest to plant ecologists as well. in this study we simultaneously examined the effect of habitat and distance in structuring stingless bee communities at a local scale. we systematically sampled native bee communities found across three paired sites that included adjacent patches of white-sand and non-white-sand forest across more than 100 km in the lowland amazon in the region of loreto, peru in order to answer two questions (1) how does stingless bee species richness and abundance differ among white-sand and non-white-sand habitats? (2) what is the relative influence of fine scale geographic distance and habitat type in structuring stingless bee communities? if stingless bees are generalist pollinators with relatively large foraging ranges, we expect that geographic distance will play a greater role in structuring bee communities than habitat type providing that trees exhibit similar flowering phenology across habitat types. alternatively, if bees prefer floral resources provided by soil specialist trees, nesting sites that are more common in one particular habitat (i.e. large vs. small stems or clay vs. sandy soil in the case of subterranean nesters) or environmental differences such as temperature or predation risk then we may find that habitat type plays a stronger role than distance in structuring stingless bee communities. materials and methods study sites three primary study areas, each containing adjacent white-sand and non-white-sand forest patches, were established in the region of loreto, peru (fig 1; table 1). area one and two are located within the allpahuayo mishana national reserve in the nanay river watershed and area three is located approximately 100km to the south in the ucayali river watershed. we consider each forest patch a sampling site. sampling design all trapping was conducted using bee pan traps. these traps are easily standardized and avoid collector bias (westphal et al., 2008). traps were created using 12-oz clear plastic soup bowls painted fluorescent blue, fluorescent yellow, or white in order to account for variation in color preference among bee species. four trapping stations consisting of six bee pan traps were established in white-sand and non-white-sand forest sites at each of the three areas for a total of 24 trapping stations across six collecting sites (fig 1). within each collecting site each trapping station was established 200-250 m distant from any other trapping station. fig 1. sample sites and soil types where stingless bees were sampled in the region of loreto, peru. numbered points represent the three areas where adjacent white-sand and non-white-sand forests were found. each individual sampling site is displayed in the inset. grey circles represent non-white-sand forests and white circles represent white-sand forests. sociobiology 61(4): 407-414 (december, 2014) 409 each trapping station contained two sets of yellow, blue and white traps. one set was suspended one meter above the ground with each individual bowl spaced at a distance of five meters to avoid bowl competition (droege, 2010). the second set of bowls was suspended at a height of 15–20 m in the canopy directly above the ground traps. bowls were filled with six ounces of soapy water solution (one tsp blue dawn brand soap per two liters of water). pan traps were set out at each site once per month for 24 h between march-july 2010. while loreto, peru exhibits very little seasonality our sampling period extended from the high water season, when rivers rise substantially, through the low water season. all trapped specimens were collected in the field and transferred to 96% ethanol. specimens were separated, pinned and identified and have been deposited at the essig museum of entomology at university of california, berkeley. identification of all specimens was done by c.r. by direct comparison with a large synoptic collection of peruvian stingless bees previously identified by j.m.f. camargo. general diversity we assessed the effectiveness of our sampling method using species accumulation curves and the chao estimator (chao, 1987). rarefaction curves were calculated using the individual-based species matrix and the species accumulation curve and chao estimates were calculated using the ‘specacum’ and ‘specpool’ functions respectively within the package ‘vegan’ (oksanen et al., 2013). we assessed the dominance structure within our dataset by ranking the relative abundance of each species using a regular base plot (magurran, 2004). fine scale community structure we investigated the influence of trap height, trap color, month collected, soil type and trapping site on species richness and abundance using generalized linear models (dobson, 1990). the full model was constructed using trap height, trap color, month collected, soil type and trap site as a fixed effect. poisson distribution and logarithmic link function were selected as an error distribution and link function, as it has shown to work well with count data (bolker et al., 2009). the significance of each explanatory variable was then tested using chi-square test to compare the reduction of deviance from the residual deviance (hastie & pregibon, 1992). we then identified the best-fitting model using backward step-wise model selection with aic criterion, using function “stepaic” in package mass. broad scale community structure in order to assess the broad scale effects of soil type and location as defined by the three sampling areas (fig 1), we combined all monthly collection data for each of the four trap stations within each of the six collecting sites for a total of 24 sample units. we transformed the data by first applying square root to the abundance data followed by standardization using the wisconsin method (bray & curtis, 1957), which reduces the effect of overly dominant species in the data set and c o n t r o l s f o r s a m p l i n g e f f o r t a t e a c h t r a p s i t e (legendre & gallagher, 2001). we first quantified relative contributions of soil type and location to the community structure by performing variance partitioning with the function ‘varpart’. the analysis partitions the explained variation in community structure into different components based on the studied environmental factors (borcard et al., 1992). then, in order to visualize results and specifically test the significance of effects of soil and location, as well as their interaction in driving community structure, we used a distance-based redundancy analysis (db-rda) with soil and location as constraints. this method allowed us to carry out constrained ordinations using non-euclidean distance measures (gower, 1966; gower, 1985; legendre & anderson, 1999; legendre & legendre, 2012). our distance matrix was created using bray-curtis distance, which only accounts for shared presences between two sites (anderson et al., 2011), and the redundancy analysis was carried out using the ‘capscale’ function (anderson & willis, 2003). the significance of constrained ordination was assessed using a permutation test for constrained correspondence analysis (legendre et al., 2011; legendre & legendre, 2012) using the function ‘anova.cca’. the p-value is calculated by comparing the observed f-value with the values from 999 permutations of community data. table 1. geographic coordinates, number of individuals, number of genera, and number of species collected at each trapping site as well as total abundance (a) and richness (s) for each habitat type and collection site. white-sand non-white-sand totals per site site lat. long. ind. (n) gen. (n) spp. (n) site lat. long. ind. (n) gen. (n) spp. (n) a s 1-ws -3.91 -73.55 215 8 16 1-nws -3.90 -73.55 92 9 15 307 20 2-ws -3.95 -73.40 112 5 6 2-nws -3.97 -73.42 236 9 12 348 14 3-ws -4.86 -73.61 415 9 16 3-nws -4.88 -73.64 31 6 9 446 20 a 742 a 359 s 27 s 24 tm misiewicz, e kraichak, c rasmussen stingless bee community turnover 410 we also used a permutational multivariate analysis of variance (permanova) to further test the effects of soil, location, and their interaction using the function ‘adonis’. this analysis is analogous to parametric multivariate analysis of variance (manova), but has been shown to be more robust for community data, as the p-value is derived from permutation, as opposed to the comparison against a known distribution (anderson, 2001). finally, using the function ‘mantel’ we implemented mantel tests (legendre & legendre, 2012) individually on whitesand and non-white-sand populations to determine if geographic distance was more important in structuring bee communities in one habitat or the other. all functions for community analysis are available the r package ‘vegan.’ all statistical analyses were carried out in r 3.0.3 (r development core team 2013). results general diversity we trapped a total of 1109 bees representing three families, 17 genera and 39 species. all but three taxa were apidae (appendix s1). thirty-one species (79%) were identified to the species level and the remaining eight were sorted to morphospecies. eight of the collected specimens representing six species were identified as solitary bees and were therefore discarded from the dataset for further analysis. all other specimens for the remaining 33 species were stingless bees and were included in all analyses (table 1). the species accumulation curve approached, but did not reach an asymptote (fig 2) suggesting that our sampling was adequate but not exhaustive. the estimated species richness across all habitats and sites was 38.6 species (standard error: ±3.85) meaning we captured approximately 77 percent to 95 percent of the estimated total number of stingless bee species. plebeia minima (gribodo) and plebeia sp. a were particularly abundant in our data set (n=584, n=235). 29 species were represented by medium to low abundances. six species were represented by singletons (table 2; fig 3). preliminary analyses showed little to no effect when singletons were omitted from the data. as a result, all further analyses were carried out with the inclusion of singletons. a total of 19 species (n=1,048) were found in both white-sand and nonwhite-sand habitats, five species (n=13) were found only in non-white-sand habitats and nine species (n=40) were found only in white-sand habitats (table 2). total abundance (a) and species richness (s) for each habitat type and sampling location are reported in table 1. fine scale community structure soil type and trapping site had a significant effect on species richness and abundance, while trap height, and month fig 2. species accumulation curves for stingless bees. a. species accumulation curves for white sand habitat, terrace habitat and total across habitats. b. species accumulation curves by sampling sites 1, 2 and 3 and total across all sampling sites. fig 3. species abundance distribution for all stingless bee species for all sites sampled. sociobiology 61(4): 407-414 (december, 2014) 411 table 2. bee species collected by soil type table 3. results of the generalized linear models for the effects of soil type, location, month, trap height and trap color on stingless bee richness and abundance at the smallest scale. fig 4. results from distance-based redundancy analysis. gray ellipses correspond to white-sand habitat and black ellipses correspond to nonwhite-sand habitat. 1, 2 and 3 refer to sampling sites. cap refers to ‘constrained analysis of principal coordinates’. species n ind. collected on ws n ind. collected on non-ws leurotrigona pusilla 1 1 leurotrigona muelleri 0 1 melipona bradleyi 2 0 melipona crinite 5 0 melipona gr. rufiventris 1 0 nannotrigona melanocera 73 5 nannotrigona schultzei 4 1 nogueirapis butteli 3 5 partamona epiphytophila 3 2 partamona testacea 0 1 plebeia minima 342 242 plebeia sp. a 200 35 plebeia sp. b 18 0 plebeia sp. c 3 1 plebeia sp. d 13 3 plebeia sp. e 1 0 ptilotrigona lurida 7 2 ptilotrigona pereneae 6 0 scaura latitarsis 1 0 scaura tenuis 0 6 schwarzula coccidophila 4 11 schwarzula timida 33 1 tetragona clavipes 6 3 tetragona dissecta 2 0 tetragona gr. dorsalis 1 8 tetragona handirschii 0 3 tetragonisca angustula 3 0 trigona amalthea 0 1 trigona cilipes 0 7 trigona guianae 3 2 trigona williana 2 1 trigonisca bidentata 4 15 trigonisca gr. ceophloei 1 1 collected only significantly affected on abundance (table 3). the multiple linear model that best explained species richness included soil type and trap site (δaic=6.28). the model that best explained species abundance included soil type, month collected, trapping site and height, but it was only marginally better than the full model with the trap color (δaic=0.96). mantel tests suggests that geographic distance is correlated with species turnover in both habitat types (white-sand, r=0.4, p<0.001; non-white-sand, r=0.3, p=0.02). discussion to our knowledge this is the first study to examine the role of geographic distance and forest type in structuring stingless bee communities at local scales across a naturally heterogeneous landscape in the western amazon. we found that both location and habitat were important in structuring stingless bee communities even over extremely small spatial scales. our results were consistent with other tropical bee studies, which demonstrate changes in bee communities across a variety of spatial scales and environmental gradients (tylianakis et al., 2005; abrahamczyk et al., 2011; batista matos et al., 2013). while the total amount of variance explained by these two factors seems relatively small these results are in line with other studies of community turnover particularly those using natural gradients. white-sand forests exist as small patches or habitat ‘islands’ surrounded by a matrix of non-white-sand forest. accordingly, factors such as migration, colonization and local extinction may play a stronger role in structuring white-sand bee communities than non-white-sand bee communities. if non-white-sand habitat is less favorable for bee species found in white-sand forests then these communities may experience higher levels of isolation due to the compounded effect of habitat and distance. in this case metacommunity dynamics could play an important role in bee species richness bee abundance df deviance p df deviance p null model 252 108.92 252 1326.7 soil type 1 6.37 0.010 1 62.55 <0.001 location 2 12.45 0.002 2 64.01 <0.001 month 1 1.59 0.200 1 49.38 <0.001 trap height 1 0.17 0.670 1 21.75 <0.001 pan color 2 0.27 0.870 2 2.72 0.25 tm misiewicz, e kraichak, c rasmussen stingless bee community turnover 412 references abrahamczyk, s., steudel, b. & kessler, m. 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(1998) a new herpsilochmus antwren (aves: thamnophilidae) from northern amazonian peru and adjacent ecuador: the role of edaphic heterogeneity of tierra firme forest. auk. 115: 559–576. doi: 10.13102/sociobiology.v62i4.772sociobiology 62(4): 484-493 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 do atta sexdens rubropilosa workers prepare leaves and bait pellets in similar ways to their symbiotic fungus? introduction the genus atta is characterized by colonies that contain a large number of workers and shows one of the broadest polymorphism among leaf-cutting ants (fowler et al., 1986). among ants of this genus, the lemon ant atta sexdens rubropilosa forel, 1908, is widely distributed in several brazilian states (mariconi, 1970; della lucia, 2011) and attacks different crops of agricultural importance, causing significant losses particularly in forest crops (amante, 1967). species of the genus atta forage on a variety of plants which serve as a substrate for culture of the symbiotic fungus. the latter is the primary food source for larvae and abstract leaf-cutting ants rely on the obligate symbiosis with a fungus, leucoagaricus gongylophorus, on which they feed. this fungus garden is maintained preferentially with green leaves in combination with other tissues of plant origin. the nutritional composition of this symbiotic relationship is extremely beneficial for the colony, representing the main food source for larvae and an important food component of adult workers. the objective of this study was to observe the behavioral repertoire of atta sexdens rubropilosa during the preparation and incorporation of two different substrates, leaves and bait pellets, as well as the participation of each caste in these processes this process. the first substrate offered to the colonies were citrus sp. leaves cut into disks and the second were pellets of citrus pulp bait formulated without the active ingredient. the observations were made under a stereomicroscope and were started when the first portion of the substrate offered was carried to the fungus garden. the behaviors executed during preparation and incorporation of the leaf disks, and subsequently of the bait pellets, were recorded using three colonies, with six repetitions of 5 and 7 hours per colony, respectively. eight behavioral acts were recorded for the three castes found, irrespective of the substrate processed by the workers. licking was the most frequently executed behavioral act, which occurred throughout the observation period, regardless of the substrate offered. gardeners and generalists were the castes most engaged in the execution of this behavior. considering the large number of individuals involved in the execution of this task, it can be concluded that this behavior is important for dispersion of the active ingredient and contamination of workers inside the colony. sociobiology an international journal on social insects lc silva1; rs camargo1; lc forti1; cao matos2; rv travaglini1 article history edited by evandro do nascimento silva, uefs, brazil received 04 march 2015 initial acceptance 09 august 2015 final acceptance 09 august 2015 keywords social insects, worker contamination, bait processing, leaf-cutting ants, atta. . corresponding author laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas/unesp caixa postal 237, 18603-970 botucatu-sp, brazil e-mail: camargobotucatu@yahoo.com.br is also part of the diet of adults, which is complemented by the ingestion of exudates during the activity of leaf-cutting and manipulation (littledyke & cherret, 1976; hubbell & wiemer, 1983; forti & andrade, 1999; mueller, 2002; silva et al., 2003; schneider, 2003). according to quinlan and cherret (1979), the ingestion of exudates accounts for about 95% of the dietary requirements of workers. however, silva et al. (2003) suggested the fungus to play a much more important role in the worker diet since they observed a decrease in the survival of a. s. rubropilosa ants in the absence of the fungus when workers were maintained only on fresh leaves or fungal mycelium. when supplied with pieces of the fungus garden, the ants survived for a longer period of time. the same was research article ants 1 faculdade de ciências agronômicas/unesp, botucatu-sp, brazil 2 campus experimental de itapeva, unesp, itapeva-sp, brazil sociobiology 62(4): 484-493 (december, 2015) 485 reported by benevides (2004) for acromyrmex subterraneus workers. nevertheless, the fungus is fundamental for feeding of the colony, regardless of whether it is consumed directly or through its exudates (moreira et al., 2011). the function of the symbiotic fungus includes the degradation of secondary plant compounds and their transformation into palatable substances. there is evidence that the fungus is able to degrade cellulose (martin et al., 1969; bacci et al., 1995; nagamoto et al., 2011). however, abril and bucher (2002) suggested that the fungus leucoagaricus gongylophorus müller (singer), whose basic energy reserve is glycogen, which are present in a form that can be readily assimilated by ants (quinlan & cherrett, 1979), is unable to degrade cellulose. the processing and incorporation of plant material are a complex activity and involve a series of different tasks which are performed according to the body size of workers. as a consequence, a high degree of polymorphism may be observed (wilson, 1980a, 1983). substrate processing and incorporation can be divided into four different phases. the first consists of the collection of plant material, the second and third of the physical and chemical treatment of the foraged fragments, respectively, and the fourth of incorporation of the substrate itself (diniz & bueno, 2009). after foraging, generalist workers with a head width of 1.3 to 1.6 mm lick and cut the leaves into fragments measuring 1 to 2 mm in diameter. next, minima workers (ants with a head width of 0.8 to 1.2 mm) chew the leaves along their borders until they are reduced to a moist pulp and incorporate these fragments into the fungus garden (wilson, 1980a; fowler, 1983; andrade et al., 2002; lopes et al., 2004; camargo, 2007; diniz & bueno, 2009, 2010). finally, hyphal tufts are inoculated onto the surface of the fragments (weber, 1956; lopes et al., 2004). it is important to note that these behaviors are specialized for culture of the symbiotic fungus and that, somehow, a large number of workers are involved in this process. this high demand may play an important role in the contamination of workers with insecticides, thus demonstrating that plant processing can be a major route of colony contamination. however, the role of oral trophallaxis among adults of leafcutting ants in the contamination of workers still remains debatable (andrade et al., 2002). therefore, the objective of the present study was to describe and to analyze the behavioral repertoire executed by a. s. rubropilosa workers during the preparation and incorporation of substrates into the fungus garden by comparing the processing of leaves and bait pellets material and methods the colonies used were collected in the municipality of botucatu, state of são paulo, brazil, and kept in the laboratory of social pest insects (laboratório de insetos sociais-praga lisp), school of agricultural sciences, unesp, botucatu. for observation, three colonies containing approximately 500 ml of the fungus were transferred to glass boxes (length: 20 cm, width: 10 cm, height: 2.5 cm). the boxes contained a layer of plaster for the maintenance of humidity and at their opposite ends connections to containers destined for foraging and disposal of exhausted waste (lopes, 2004). the ambient temperature was maintained at approximately 22 ± 2°c and humidity at about 70± 20%. prior to the observations, a sample of individuals of variable sizes were removed for the measurement of greatest head width under a stereomicroscope equipped with an ocular micrometer. the sample was classified and the castes were defined as proposed by wilson (1980a). since no soldiers were detected, the observations were made based on the behavior of gardening workers (head with of 0.8 to 1.2 mm), generalists (head width of 1.3 to 1.6 mm), and foraging workers (head width of 1.7 to 2.2 mm). the substrates offered to the colonies consisted of citrus sp. leaves and bait pellets made of citrus pulp and 4% soybean oil and formulated without the active ingredient. the surface of the two substrates was standardized to 63.58 and 62.86 mm², respectively. standardization of the substrate surface is important for the quantification of behavioral acts, avoiding duplication of the results. the observations were started when the first of the 15 leaf disks or pellets offered was carried to the fungus garden. the behavioral acts executed during preparation and incorporation of the leaf disks were recorded using three colonies with six repetitions of 5 hours each, for a total of 90 hours of observation. in view of the physical nature of the bait pellets, six repetitions of 7 hours each were performed per colony to record the behavioral acts executed during bait preparation and incorporation, for a total of 105 hours of observation. the ants were observed ad libitum, i.e., there was no time interval between recordings. the main behaviors executed by a. s. rubropilosa workers during preparation and incorporation of the different substrates into the fungus garden were recorded and the percentage of individuals performing the different behavioral acts was calculated according to size class of the workers. the results were compared by analysis of residuals standardized in contingency tables (z) (pereira, 2010). the wilcoxon-mannwhitney test was used to compare the behaviors between bait versus leaves (p=0.05). results processing and incorporation of leaf disks at the time when the leaf disks were carried by foraging workers into the fungal chamber, other workers of different size classes touched the leaf surface and borders with their antennae and mouthparts. these ants then started to lick the leaves continuously, while other workers transported and held the disks on the surface of the fungus culture. the disks were reduced to lc silva et al. – process of substrate preparation for symbiotic fungus culture486 increasingly smaller fragments. these fragments, in turn, had their borders pleated and were then chewed by the workers, which occasionally deposited fecal fluid on the fragments to induce their softening and moistening. after this sequence of behaviors, the substrate was finally incorporated into the outer region of the fungal sponge and inoculated with a small quantity of fungus. the most commonly observed behavior was licking the surface and borders of the leaf disk, which accounted for 38.88% of all behavioral acts executed (fig 1). basically, all castes contributed to the execution of this activity, although at different proportions (table 1). foraging workers performed 13.33% of this activity (zld1=5.311994;zld2= -0.14412; zld3 = 7.43419), generalists 25.05% (zld1=28.65059;zld2=19.27417; zld3 =21.34883), and gardeners 61.62% (zld1=78.58434;zld2=84.38611; zld3 =83.21173). the second least frequent behavior was transporting the disk to the surface of the fungus garden, accounting for 3.39% of all acts observed. however, the percentage of this behavior compared to the previously cited acts was low, corresponding to only 2.87% of all tasks. processing and incorporation of bait pellets the pellets were processed in a similar manner as the citrus sp. disks, with each size category contributing to at different proportions to the execution of these acts. however, the time spent on the complete incorporation of the bait into the fungus garden was longer than the time spent by workers to perform the whole process using leaves as substrate. once the bait pellets had been offered, the workers were immediately attracted and started touching the pellets with their antennae and palps. the pellets were then carried into the fungal chamber where workers started to lick them continuously for hydration. while some workers licked the pellets, others carried and held them on the surface of the fungus. once the pellets were well hydrated, they were fractionated into very small portions and incorporated into the fungus garden. as observed in the previous experiment, the most common behavior was licking the surface of the bait pellet, accounting for 38.68% of all behavioral acts executed (fig. 2). gardening workers were the main size class executing this behavior, which were responsible for 51.73% of this activity (zb1= 97.2501; zb2= 72.1057; zb3= 51.4991), followed by generalists performing 24.54% of this activity (zb1=36.6324; zb2= 14.7516; zb3=22.4027) and foraging workers performing 23.73% (zb1=23.8329; zb2= 18.6163; zb3= 4.95512) (table 2). observed as the second most frequent behavior, pressing the pellet fragment corresponded to 14.37% of all acts executed by workers. gardening workers were fig 1. percentage of each behavioral act performed by atta sexdens rubropilosa workers during preparation of the leaf disk substrate and its incorporation into the fungus garden. 1: transporting the disk to the surface of the fungus garden; 2: holding the disk on the surface of the fungus culture; 3: licking the surface and borders of the leaf disk; 4: cutting the leaf disk into smaller fragments; 5: pleating the borders; 6: deposition of fecal fluid; 7: incorporation into the fungus garden; 8: deposition of hyphae on the incorporated fragments. the second most observed behavior was pleating the borders of the leaf disk, which accounted for 17.7% of the acts executed by workers (fig. 1 and table 1), with the observation of excess of significant events in the groups of gardeners and generalists. gardening workers were effectively responsible for 54.94% of this activity (zld1= 22.68025;zld2= 25.2817;zld3= 25.56538), followed by generalists responsible for 28.57% (zld1=0.185945;zld2=5.377955;zld3=5.506622) and foragers responsible for 14.49% (zld1= -9.282169; zld2= -4.69528; zld3 = 5.93832). the behaviors of cutting the disks into smaller fragments and incorporating them into the fungus garden corresponded to 12.47% and 12.14% of the acts executed, respectively, and were performed mainly by gardening workers (table 1). holding the disks and depositing hyphae on the incorporated fragments corresponded to 6.58% and 5.98% of all behavioral acts executed by workers, respectively. the first activity was performed equally by generalists and foragers (33.98%) and the second by gardening workers (91.58%). fig 2. percentage of each behavioral act performed by atta sexdens rubropilosa during preparation of the bait substrate and its incorporation into the fungus garden. 1: transporting the pellet to the surface of the fungus garden; 2: holding the pellet; 3: licking the surface; 4: pellet fragmentation; 5: pressing the pellet fragment; 6: deposition of fecal fluid; 7: incorporation into the fungus garden; 8: deposition of hyphae on the incorporated fragments. sociobiology 62(4): 484-493 (december, 2015) 487 behavior head width (mm) observed frequency % behavior leaf disk substrate (zld) zld1 zld2 zld3 transporting the disk to the surface of the fungus garden 0.8 1.2 43 6.17 -16.94109 -15.8001 -16.4797 1.3 1.6 289 41.46 -12.77994 -10.096 -11.4801 1.7 2.2 365 52.37 -8.679104 -10.5815 -10.5163 total 697 100.00 holding the disk 0.8 1.2 465 32.05 -5.121023 -10.3994 -8.2273 1.3 1.6 493 33.98 -8.618798 -7.30461 -6.11903 1.7 2.2 274 33.98 -11.21198 -14.5258 -9.55251 total 1,232 100.00 licking the surface and borders 0.8 1.2 4,933 61.62 78.58434 84.38611 83.21173 1.3 1.6 2,005 25.05 28.65059 19.27417 21.34883 1.7 2.2 1,067 13.33 5.311994 -0.14412 7.43419 total 8,005 100.00 cutting leaf pieces into smaller fragments 0.8 1.2 980 38.16 4.648623 0.766112 1.952667 1.3 1.6 903 35.16 4.829542 -0.44753 -1.66152 1.7 2.2 685 26.67 -6.447765 -1.53981 -2.44460 total 2,568 100.00 pleating the borders 0.8 1.2 2,075 56.94 22.68025 25.2817 25.56538 1.3 1.6 1,041 28.57 0.185945 5.377955 5.506622 1.7 2.2 528 14.49 -9.282169 -4.69528 -5.93832 total 3,644 100.00 deposition of fecal fluid 0.8 1.2 512 86.78 -3.975201 -8.39689 -8.52849 1.3 1.6 64 10.85 -15.91588 -15.8001 -16.2388 1.7 2.2 14 2.37 -16.94109 -16.8924 -17.1423 total 590 100.00 incorporation into the fungus garden 0.8 1.2 1,926 77.07 24.36883 20.30576 19.84291 1.3 1.6 469 18.77 -7.412669 -7.48666 -8.58872 1.7 2.2 104 4.16 -14.89067 -14.5865 -16.0581 total 2,499 100.00 depositionof hyphae on the incorporated fragments 0.8 1.2 1,240 91.58 1.693606 14.29823 7.133007 1.3 1.6 93 6.87 -15.91588 -14.101 -16.1785 1.7 2.2 21 1.55 -16.82048 -16.8924 -16.8411 total 1,354 100.00 overall total 0.8 1.2 12,174 59.13 1.3 1.6 5,357 26.02 1.7 2.2 3,058 14.85 total 20,589 100.00 z: residuals standardized in contingency tables. (*) z >1.96 excess of significant events (alpha = 0.05); z < -1.96 scarcity of significant events (alpha = 0.05). table 1.standardized residuals in contingency table analysis of the behaviors executed by atta sexdens rubropilosa during the preparation and incorporation of leaf disksas substrate. lc silva et al. – process of substrate preparation for symbiotic fungus culture488 behavior head width (mm) observed frequency % behavior bait substrate (zb) zb1 zb2 zb3 transporting the pellet to the surface of the fungus garden 0.8 1.2 4 1.07 -18.721 -18.518 -20.155 1.3 1.6 69 18.4 -18.001 -16.614 -19.223 1.7 2.2 302 80.53 -13.347 -12.973 -14.874 total 375 100 holding the pellet 0.8 1.2 986 40.44 8.09667 -1.3232 -10.266 1.3 1.6 723 29.66 2.88821 -12.077 -9.0754 1.7 2.2 729 29.9 -2.5419 -12.301 -3.4839 total 2,438 100 licking the surface 0.8 1.2 5,099 51.73 97.2501 72.1057 51.4991 1.3 1.6 2,419 24.54 36.6324 14.7516 22.4027 1.7 2.2 2,339 23.73 23.8329 18.6163 4.95512 total 9,857 100 pellet fragmentation 0.8 1.2 1,504 45.37 8.2629 11.559 4.48916 1.3 1.6 873 26.34 -5.257 -3.6756 -1.4647 1.7 2.2 938 28.3 -10.909 -1.2672 4.95512 total 3,315 100.01 pressing the pellet fragment 0.8 1.2 2,087 56.98 8.53995 19.9605 26.648 1.3 1.6 959 26.18 -7.0855 -3.3956 4.43739 1.7 2.2 617 16.84 -13.845 -7.7084 -2.9662 total 3,663 100 deposition of fecal fluid 0.8 1.2 1,596 83.3 9.4265 14.6956 5.26576 1.3 1.6 179 9.34 -15.674 -14.934 -17.256 1.7 2.2 141 7.36 -17.281 -16.278 -16.479 total 1,916 100 incorporation into the fungus garden 0.8 1.2 1,871 74.87 4.99376 15.5917 22.8168 1.3 1.6 423 16.93 -13.5129 -11.181 -10.1109 1.7 2.2 205 8.2 -17.5024 -12.5812 -16.3754 total 2,499 100 deposition of hyphae on the incorporated fragments 0.8 1.2 1,372 96.62 -8.6369 13.7434 11.4268 1.3 1.6 41 2.89 -18.777 -17.678 -18.964 1.7 2.2 7 0.49 -18.832 -18.518 -19.896 total 1,420 100 overall total 0.8 1.2 14,519 56.98 1.3 1.6 5,686 22.31 1.7 2.2 5,278 20.71 total 25,483 100.00 z: residuals standardized in contingency tables. (*) z > 1.96 excess of significant events (alpha = 0.05); z < -1.96 scarcity of significant events (alpha = 0.05). table 2.standardized residuals in contingency table analysis of the behaviors executed by atta sexdens rubropilosa during the preparation and incorporation of bait pellets as substrate. sociobiology 62(4): 484-493 (december, 2015) 489 effectively responsible for 56.98% of this activity (zb1= 8.53995; zb2= 19.9605; zb3= 26.648), generalists for26.18% (zb1= -7.0855; zb2= -3.3956; zb3= 4.43739), and foraging workers for 16.84% (zb1= -13.845; zb2= -7.7084; zb3= -2.9662). the behaviors of fragmenting the pellet, incorporating the pellet into the fungus garden and holding the pellets were recorded at the following frequencies, respectively: 13.01%, 9.81% and 9.57% (fig. 2).the significant participation of gardening workers in the activities of pellet fragmentation, responsible for 45.37% of this activity (zb1 = 8.2629; zb2 = 11.559; zb3 = 4.48916) and pellet incorporation into the fungus garden, responsible for 74.87% (zb1 = 4.99376; zb2 = 15.5917; zb3 = 22.8168), should be highlighted. holding the pellets on the fungus garden was a behavior shared by the different size classes, with gardening workers performing 40.44% of this activity (zb1= 8.09667; zb2= -1.3232; zb3= -10.266), generalists 29.66% (zb1= 2.88821; zb2= -12.077; zb3= -3.4839),and foraging workers29.9% (zb1= -2.5419; zb2= -12.301; zb3= -3.4839). deposition of fecal fluid and deposition of hyphae on the incorporated fragments accounted for 7.52% and 5.57% of all behavioral acts recorded. gardening workers were the main size class executing these two acts, being responsible for 83.3% (zb1=9.4265; zb2= 14.6956; zb3= 26.648)and 96.62% (zb1= -8.6369; zb2= 13.7434; zb3= 11.4268) of these activities, respectively. the least common behavioral act was transporting the pellet to the surface of the fungus garden, which accounted for only 1.57% of all acts observed. foraging workers were the main size class responsible for the execution of this activity (80.53%) (zb1= -13.347; zb2= -12.973; zb3= -14.874). comparison of behaviors between bait versus leaves in general, the workers performed more behavioral acts during processing of the bait pellets compared to leaf processing (fig. 3). the frequency of the behavior of leaf disk/pellet transportation was higher for leaves (39.22±7.38) compared to bait (20.78±5.50), with the difference being statistically significant (u=9.00; p-value (bilateral)< 0.0001). on the other hand, the behavior of holding the leaf disk/pellet was significantly more frequent for bait (133.50±69.32) than leaves (67.94±22.83) (u =59.50; p-value (bilateral) =0.0012). the frequency of the licking behavior was higher for bait (546.22±87.76) than leaves (444.72±145.76), with the difference being significant (u =91.50; p-value (bilateral) =0.0257). the same was observed for cutting the leaf pieces into smaller fragments/ pellet fragmentation, with a significantly higher frequency for bait (184.22±60.58) compared to leaves (142.67±36.77) (u =94.00; p-value (bilateral) =0.0314). in contrast, the frequency of pleating the leaf borders/pressing the pellet fragments was similar for leaves (202.44±66.00) and bait (194.61±102.01) (u =157.50; p-value (bilateral)=0.8868). the behavior of fecal fluid deposition was more frequent for bait (106.44±30.53) than leaves (32.78±12.53), with the difference being significant (u =0.00; p-value (bilateral)< 0.0001). in contrast, the frequency of the behavior of incorporation into the fungus garden did not differ significantly between bait (138.83±57.85)and leaves (138.83±54.79) (u =160.00; p-value (bilateral)=0.9495). there was also no significant difference in the frequency of hyphal deposition (bait: 78.67±41.39 and leaves: 75.22±29.27) (u =148.50; p-value (bilateral)=0.9495). fig 3. mean number and standard deviation of the frequency of behaviors executed by atta sexdens rubropilosa workers during culture of the symbiotic fungus on bait pellets (gray bars) and leaf disks (white bars). c1: transporting the disk/pellet to the surface of the fungus garden; c2: holding the disk/pellet; c3: licking the surface and borders of the disk/pellet; c4: cutting the leaf disk into smaller fragments/pellet fragmentation; c5: pleating the borders/ pressing the pellet fragment; c6: deposition of fecal fluid; c7: incorporation into the fungus garden; c8: deposition of hyphae on the incorporated fragments. discussion processing and incorporation of leaf disks the present results show that the act of substrate licking wasin general the most frequently executed behavior, regardless of size class. by licking the surface of the substrate, workers remove deleterious impurities and thus guarantee the integrity of the fungus garden (stahel, 1943; andrade et al., 2002; andrade, 2002; mueller, 2002; diniz & bueno, 2009, 2010). furthermore, licking the leaves permits removal of the existing layer of wax, thus facilitating the growth of hyphae of the symbiotic fungus and increasing their inoculation potential. similar observations have been made for acromyrmex octospinosus (quinlan & cherret, 1977). the activity of gardening workers and generalists is unquestionably essential during execution of this behaviorand the demand of individuals of these size classes for the lc silva et al. – process of substrate preparation for symbiotic fungus culture490 execution of this behavior is high. studying the behaviors of a. s. rubropilosa during substrate preparation, andrade et al. (2002) reported that gardening workers were the main caste involved in the behavior of leaf licking, which were responsible for 52% of this task, followed by generalists (38%). in a similar study, diniz and bueno (2009) observed that in the grass-cutting ant species atta bisphaerica, gardening workers and generalists were responsible for 49% and 48% of this task, respectively. in the same study, but using atta laevigata, the same authors offered leaves of monocot and dicot plants and observed that gardening workers were the main caste involved in this task, irrespective of the substrate offered. in addition to being the most frequent, the act of licking also required most of the time of the workers involved, as demonstrated by the fact that they started to execute the task as soon as the disks were transported to the fungus chamber. similar results have been reported by andrade et al. (2002). the smaller size classes were found to execute this behavior throughout the observation period. the smaller castes, gardening workers and generalists, are the main castes responsible for preparation of the substrate and represent the majority of ants in a colony (weber, 1972). according to this author, gardening workers correspond to 60-67% of ants present in the fungus gardens, generalists to 30-33%, foraging workers to 1-2%, and soldiers (limited to the genus atta) to less than 1% of a colony. hence, the symbiotic balance is disturbed when workers directly involved in substrate processing are affected to an extent that compromises the care and maintenance of the main energy source of the colony. it is known that workers ingest exudates during substrate processing (peregrine & mudd, 1975; littledike & cherret, 1976; forti & andrade, 1999). workers are therefore able to simultaneously feed and eliminate microorganisms that are harmful to them and to the symbiotic fungus. the mechanism of substance exchange in leaf-cutting ants is widely debated. in addition to permitting food exchange, trophallaxis is an important and complex mechanism of communication among nest mates (hölldobler & wilson, 1990). abdominal or proctodeal trophallaxis is the consumption of anal excretion by the larvae (jaffé, 1993; schneider, 2003; lopes et al., 2005) and oral trophallaxis, which occurs among adult workers, consists of the transfer of liquid food stored in the crop to nestmates. however, some authors argue that oral trophallaxis is rare or even absent in this group (andrade et al., 2002; paul & roces, 2003), although subsequent studies have shown oral trophallaxis in leaf-cutting ants (moreira et al., 2006, 2010; da silva et al., 2009; richard & errard, 2009; moreira et al., 2014). one may therefore speculate that the transfer of substances between castes in the colony occurs during mutual cleaning and by oral trophallaxis. processing and incorporation of bait pellets during processing of the pellets, as observed in the previous experiment, licking the substrate was the most frequently executed task throughout the observation period. as observed for the leaf substrate, gardening workers and generalists more effectively participated in this behavior, although a larger number of ants exerted this function during pellet processing (table 2). in agreement with the present study, andrade (2002) also found that the size classes of acromyrmex workers involved in the process were the smallest of the colony. the larger number of workers performing this task may be attributed to the fact that ants are most attracted to the citrus pulp present in the pellet formulation (carlos et al., 2009). as a consequence, a large number of workers are attracted and come in direct contact with the active ingredient. these ants then become contaminated and die and no longer exert the functions attributed to them within the colony, causing an imbalance in the symbiotic relationship. baits are one of the most important control methods of leaf-cutting ants because of their efficiency. the bait formulated as pellets consists of a mixture of dehydrated citrus pulp meal (vehicle) and active ingredient dissolved in soybean oil (robinson, 1979; forti et al., 1998). the citrus pulp used as vehicle and attractant in toxic baits is appropriate for growth of the fungus cultured by leaf-cutting ants since it is slightly acid, has a high carbohydrate content and contains nitrogen, vitamins and microelements that provide good conditions for development of the symbiotic fungus (boaretto & forti, 1997). other factors such as contamination and humidity also play an important role in this control method, but attractiveness is the key factor for the efficiency of bait pellets, guaranteeing that the baits are carried by workers to the nest (etheridge & phillips, 1976, forti et al., 1998, nagamoto et al., 2004; verza et al., 2006). comparison of behaviors between bait versus leaves comparison of the behavioral acts executed during preparation of the two different substrates offered showed that physical factors influenced both the time spent on this process and the number of workers involved. physical factors of the substrate are known to influence the selection of plants and post-selection for incorporation of forage material into the fungus garden by leaf-cutting ants (camargo et al., 2004). for example, when the acceptance of new and old leaves by leaf-cutting ants are compared, a higher frequency of workers showing a preference for young leaves is noted, probably as a result of physical and chemical factors (cherrett & seaforth, 1970; barrer & cherrett, 1972; cherrett, 1972; rockwood, 1976; littledyke & cherrett, 1978; waller, 1982). the procedures involved in the preparation of the substrate for culture of the symbiotic fungus imply the manipulation of this substrate by workers (licking, cutting, ingesting exudates) and ants are able to perceive the composition of the materialduring the execution of these behavioral tasks (camargo et al., 2004). bait pellets are obviously more sociobiology 62(4): 484-493 (december, 2015) 491 resistant than leaf disks and their processing by workers is therefore more time consuming. this is likely due to the fact that the bait needs to be hydrated for incorporation into the fungus garden considering the absorption of water from the medium (fungal chamber). in the case of pellets, in addition to asepsis, hydration can be performed during licking and can be complemented subsequently by the deposition of fecal fluid. even considering the attempt to standardize the area of the substrates offered for foraging (see material and methods), an increase in the size of the pellets was visually observed after hydration, a fact that may explain the large number of ants involved in their processing. however, further investigation is necessary to test this hypothesis. on the basis of the behavioral repertoire obtained, it can be concluded that the behavioral acts executed during processing of the plant material and bait pellets were similar (e.g., licking and reducing the substrates tested to minute fractions) despite differences in worker demand and frequency. the present study described and analyzed the behavioral repertoire executed by a. s. rubropilosa workers during culture of the symbiotic fungus with leaves and bait pellets. the results showed that baits are more laboriously processed than leaves and can therefore act as a contaminating agent of workers in the colony during processing, with the consequent contamination of a large number of gardening workers and generalists by the active ingredient. references abril, a.b. & bucher, e. h. 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(2003). eficiência de isca formicida aplicada sobre o monte de terra solta de ninhos de atta sexdens rubropilosa (hymenoptera: formicidae). revista arvore, 27: 410-407. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7430sociobiology 68(4): e7430 (december, 2021) introduction ants are among the most ubiquitous organisms in terrestrial habitats, being highly diverse and comprising a significant part of the terrestrial arthropods’ biomass (holldöbler & wilson, 1990; lach et al., 2010). ants are terrestrial and cursorial organisms adapted for living on different solid surfaces, from the soil underground to the canopies of tropical rain forests. although water is present in 70% of the planet, interactions with water represent a formidable obstacle for such terrestrial organisms. successfully overcoming this obstacle can be essential to determine their persistence in some specific habitats. among these habitats are those constantly flooded, such as the intertidal zone and seasonally flooded areas, like floodplains and riparian forests. while some species avoid the water by constraining their foraging activities to drier habitats or climbing taller objects (adis, 1997), others developed ways to overcome this obstacle by swimming. abstract ants are among the most abundant organisms on earth, being adapted for living on different solid surfaces. however, in some habitats, like riparian forests and flooded plains, water can be a constant obstacle, and overcoming this obstacle can be essential to determine the persistence of ants in such habitats. while most ant species avoid the water during a flood by foraging at higher elevations or climbing on trees, a few species developed ways to overcome this obstacle by swimming. here, we report, for the first time, ants of the species linepthema micans (forel 1908) performing rafts. we observed 14 rafts in three consecutive days at approximately 1400 meters a.s.l. in serra do cipó, brazil. notably, this is the first record of ant rafting in tropical mountaintop grasslands, extreme habitats with shallow and sandy soils, where small temporary water pools are extremely common in the wet season. sociobiology an international journal on social insects geraldo w. fernandes, flávio s. de castro, flávio camarota, jessica c blum, renata maia article history edited by evandro nascimento silva, uefs, brazil received 24 september 2021 initial acceptance 27 september 2021 final acceptance 06 october 2021 publication date 19 november 2021 keywords campo rupestre, linepthema micans, mountaintop grassland, rupestrian grassland, serra do cipó. corresponding author flávio camarota universidade federal de minas gerais cep: 31270-901, belo horizonte, brasil. e-mail: camarotaflavio@gmail.com while ant swimming is not usual, it has been known for over a century that ants have some swimming ability (field, 1903; wheeler, 1910). the swimming behavior occurs in multiple ant genera distributed in different clades, and there is often considerable variation across different ant groups and even among species within genera (yanoviak & frederick, 2014). usually, larger ant species, like pachycondyla spp. and odontomachus bauri, are efficient swimmers, using their long legs to provide propulsion (yanoviak & frederick, 2014). perhaps, the most extreme cases involve ant species inhabiting mangroves (nielsen, 1997; robson, 2010). the australian ant polyhachis sokolova can have their colonies submerged for 3.5 hours during a normal tide cycle and covered by two meters of water under extreme tides (shuetrim, 2001). another interesting adaptation is related to ant species associated with pitcher plants and other epiphytes, such as camponotus schmitzi ants that live in symbiosis with the pitcher plant nepenthes bicalcarata, universidade federal de minas gerais, departamento de genética, ecologia e evolução, belo horizonte, brazil short note ant rafting in an extreme ecosystem geraldo w. fernandes, flávio s. de castro, flávio camarota, jessica c blum, renata maia – ant rafting in an extreme ecosystem2 in bornean rain forests (clarke & kitching, 1995; merbach et al., 2007). this interaction is unique because the ants actively forage on the water to prey on the insects captured by the pitcher plant (bohn et al., 2012). in the ground of tropical forests, some large-bodied ants, such as ectatomma ruidum and acromyrmex lundii, can walk to overcome small pools of water (adis, 1982; yanoviak & frederick, 2014). while most of the mentioned taxa are from tropical habitats, some cases were also reported for temperate forest ants, including the genera camponotus and formica (dubois & jander, 1985; yanoviak & frederick, 2014). some ant species, usually small-bodied, can overcome the lack of morphological adaptations for swimming by linking their body together in a kind of self-assemblage, the ant rafting, consisting of floating conglomerations of ants (purcell et al., 2014; avril et al., 2016). rafting is extremely important in habitats where colonies are often exposed to floods, and these floating masses can persist from some hours to even a few weeks (adams et al., 2011). the rafts comprise interconnected workers connected mainly by their tarsi and curling their appendages, forming velcro-like connections in the structure (adams et al., 2011). the rafting behavior gained much attention since it is used by one of the most prominent invasive ant species, solenopsis invicta, which can form rafts of thousands of individuals (mlot et al., 2011; 2012). the native habitats of s. invicta are mostly comprised of seasonally flooded plains, like the brazilian pantanal. this adaptation could have been extremely important in defining s. invicta as one of the numerically dominant ant species (adams et al., 2011). while rafting appears to be a formidable adaptation for those species living in constantly flooding habitats, only a few species have been recorded performing rafting. one common feature is that they were all found in habitats prone to inundation. on three consecutive days in april 2021, we observed the formation of 14 rafts at a small first order wash at approximately 1400 meters a.s.l. in serra do cipó, brazil (19•10’19,7” s 043• 60’ 49,2” w) (fig 1). on the first day, nine rafts were observed. these varied from 3 to 10 cm in diameter and were composed of a single ant species, linepithema micans (fig 2 a, b). the form of the rafts was mostly a circle, with a very large number of ants. only workers were observed in the rafting. curiously, ants swam actively towards the rafting, even against the wash’s weak but constant water flow. yet another interesting behavior was the jumping of many individuals into the water from some grass stems. this jumping behavior was observed in the morning (9:00 – 11:00) and lasted for ca. 15 minutes. on the following day, six rafts were observed; they had similar sizes. after the raft observations, we collected ant individuals to assure their species-level identification. the ants were stored in vials with 70% ethanol and brought to the laboratory. the ant identification was performed by flávio siqueira de castro, by comparisons with the extensive collection of formicidae from campo rupestre of the laboratory of insect ecology at the universidade federal de minas gerais, brazil. all ants collected are held at this referred collection of formicidae, which consists mostly of specimens from the peld/crsc project (silveira et al., 2019). here, we show, for the first time, ants of the species linepthema micans (forel, 1908) performing rafts. while this is not the first record for rafting on the genus linepithema (see nondillo et al., 2013), it is the first record of such complex social behavior in a tropical mountaintop grassland. l. micans is very common in the study area (costa et al., 2016; monteiro et al., 2020; calazans et al., 2020; castro et al., 2020) and generally exhibit high recruitment rates with many individuals fig 1. ant raft in a small water pool in serra do cipó, minas gerais, brazil. sociobiology 68(4): e7430 (december, 2021) 3 foraging on the ground (nondillo et al., 2013). the formation of rafts by l. micans has purposes similar to other species with this behavior. that essentially means the ants gather a significant part of the colony to find drier places to forage. the soils in the harsh and nutritionally poor habitats at high elevation in the espinhaço mountains are mostly shallow and sandy. during the wet season, the formation of small temporary water pools is extremely common (see reviews in fernandes, 2016). thus, while rafting is relatively uncommon among ant species, our observation suggests that this behavior may be more widespread than previously thought. although no specific swimming adaptation has been reported for this species, this is the first record of such a phenomenon. the region where the observations were taken is known for its high precipitation and rapid flood events due to the orographic rains and strong winds. during the winter, the moisture-laden clouds may bring important water and humidity to the region, representing another stressing variable among the others known for this mountaintop area (see fernandes, 2016). while we do not know the relevance of this behavior to colony success, it may represent an important observation on the plasticity of ants to deal with abrupt changes in extreme tropical environments. acknowledgements we thank the support provided by the brazilian national research council – cnpq, minas gerais foundation for science (fapemig), anglo american, and the project peldcrsc-17 (cnpq-mcti). author contributions all authors contributed to the study’s conception and design. all authors performed data collection and analysis. conflicts of interest/competing interests all authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript. references anstett, d.n., naujokaitis-lewis, i. & johnson, m.t. 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(2014). water surface locomotion in tropical canopy ants. journal of experimental biology, 217: 2163-2170. doi: 10.1242/jeb.101600 https://doi.org/10.1111/aec.12839 https://doi.org/10.1603/ec13392 https://doi.org/10.1371/journal.pone.0089211 https://doi.org/10.1242/jeb.101600 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i3.5778sociobiology 68(3): e5778 (september, 2021) introduction a significant important number of cases of phenotypic anomalies have been reported among bees. specimens with intersex phenotypes are the majority of these cases. these anomalies consist of the manifestation of primary and secondary characteristics of both sexes in the same individual, a phenomenon called gynandromorphism (michez et al., 2009). the origin of gynandromorphy has been attributed to genetic anomalies. though different hypotheses have been raised to explain it, the mechanisms that generate these abnormal individuals remain unclear. the phenomenon is presented in many groups within hymenoptera (see wcislo et al., 2004; michez et al., 2009). the gynandromorphs have been categorized in three accepted patterns: transversal or anteroposterior, mixed or mosaic, and bilateral, of which mixed abstract the discovery of the first case of gynandromorphism in eulaema meriana (olivier) is described and illustrated. a specimen from los ríos, western ecuador, discovered in the instituto nacional de biodiversidad (mecn) collection, exhibits mostly male features on the left and female features on the right, except for the jugal comb of the hind wing and terminalia. this finding is compared to other reported cases in orchid bees. sociobiology an international journal on social insects alex pazmiño-palomino1, marcio l. oliveira2 article history edited by solange augusto, ufu, brazil received 07 august 2020 initial acceptance 28 october 2020 final acceptance 27 may 2021 publication date 13 august 2021 keywords gynander, deviant phenotypes, euglossini, ecuador. corresponding author alex pazmiño-palomino división de entomología instituto nacional de biodiversidad rumipamba 341 y av. de los shyris, quito, ecuador. e-mail: alex.pazmino@biodiversidad.gob.ec and transverse appear to be the most common (dalla torre & friese, 1899; wcislo et al., 2004; hinojosa-díaz et al., 2012). gynandromorphy has been reported in all families of anthophila and all biogeographical regions. however, most cases have been documented from species of the holarctic region (hinojosa-díaz et al., 2012). fewer cases have been reported in the neotropics, despite containing one of the most diverse bee faunas, with many endemic taxa. neotropical orchid-bees (tribe euglossini) are conspicuous members of the corbiculate bees due to their long labiomaxillary complex, metallic coloration, and the male behavior of collecting fragrances from orchid flowers (hence their name). some cases of gynandromorphism in orchid bees have been reported in the last decade. the first was in euglossa iopoecila dressler (giangarelli & sofia, 2011), and several cases have been reported since: euglossa tridentata moure 1 división de entomología, instituto nacional de biodiversidad, quito, ecuador 2 laboratório de hymenoptera, coordenação de biodiversidade, instituto nacional de pesquisas da amazônia, manaus, brazil research article bees first case of gynandromorphism in the orchid-bee eulaema meriana (olivier) (hymenoptera: apidae) alex pazmiño-palomino, marcio l. oliveira – first case of gynandromorphism in eulaema meriana2 (hinojosa-díaz et al., 2012), eulaema atleticana nemésio (silveira et al., 2012), euglossa pleosticta dressler (camargo & gonçalves, 2013), euglossa gorgonensis cheesman (gonzález, 2014) and euglossa melanotricha moure (suzuki et al., 2015). this article describes the first recorded gynandromorph of the orchid-bee species eulaema meriana (olivier) coming from western ecuador. this is the seventh case within the euglossini and the first gynander bee from ecuador. material and methods dr. marcio de oliveira visited the entomological collection of the instituto nacional de biodiversidad (quito, ecuador) in july 2019, intending to review and identify bee specimens. along with the work, we found an individual of el. meriana exhibiting male and female-specific features on its body. we described the specimen according to morphological terminology used in michener (2007); description of the genitalia follows ospina-torres (1998). morphological measures and photographic records of characters were made using a stereomicroscope (olympus sz61r) adapted with a digital camera (the imaging source dfk23ux236) installed with the ic measure v. 2.0.0.161 software. all measures were performed three times with ic measure; the means of these measures are presented here and are given in millimeters (mm). images were then focus stacked in helicon focus and edited in adobe photoshop cs5. the genitalia of the specimen was dissected. before dissection, the individual was placed in a sealed plastic container with water-soaked tissue paper for about 72 hours. next, the terminalia were excised using an insect pin (size 0) and fine forceps to punch holes and pull apart the conjunctival membrane, separating the fifth and sixth metasomal terga and sterna. excised terminalia were placed in a solution containing 10% potassium hydroxide (koh) and water, boiled for about 5 minutes, and then put in acetic acid to neutralize the koh. finally, the genitals were transferred to a glass vial containing a small amount of glycerin for indefinite storage. the vial containing the genitals remains stored together with the specimen, pinned under it through the rubber cap. the gynander genitals were compared with those of a congeneric female, eulaema cingulata (fabricius), and the descriptions of male genitals were taken from the literature (ospina-torres, 1998). results gynandromorph description. the specimen is in good condition, except that most of the left antenna flagellomeres are missing. general measurements (mm): total body length 25.01, head width 7.64, upper interocular distance 3.07, lower interocular distance 4.43, intertegular distance 6.41, mesoscutellum length 3.06, right forewing length 21.06, left forewing length 21.39, right hind wing 14.01, left hind wing 13.99, maximum metasomal width 10.3, metasomal length 13.45. overall coloration black in head, mesosoma, and legs, metasoma with integument of the first tergum with a green metallic luster, or blue to golden green. marginal stripes of yellow hairs in the distal section of each tergum. terga 4-7 covered with ferruginous hairs. integument densely punctuate in head and legs. long face, malar area clearly longer than the diameter of the third flagellomere; distance between the lower corner of the clypeus and the orbit greater than the diameter of the middle ocellus. tongue elongate, nearly as long as the body. these general features match those of normal specimens of both sexes and the descriptions of the species (moure, 2000; oliveira, 2007). head. bilaterally asymmetric, right side with mainly female features, left side with only male features, as follows (fig 1a): right section of the labrum more developed with hairs twice as large as on the left side. right mandible (3.98), noticeably larger and broader, with three well-developed teeth; external part of the mandible with long hairs uniformly distributed along its surface. left mandible smaller (2.64) with no differentiable teeth and only a row of long hairs on the external margin, directed ventrally. smooth and shiny spot on the right malar area (at the intersection of the clypeus, mandible, and compound eye) are more developed and with a patch of yellow hairs longer than those on the left side. the ventral part of the mandibular insertion of the left side with a conspicuous bifurcated lobe, stouter on the external side and broader but weaker on the internal side, with a medial concavity. right side uniformly descending to the mandibular articulation, without lobes (fig 1b). right antenna with 12 segments with 10 flagellomeres, left antenna with the last flagellomeres missing. the dimensions of the head show little difference between right and left, the right side being slightly bulkier than the left one. mesosoma. the central thoracic axis presents slight differences in symmetry, the right side being more voluminous than the left. in addition, mesoscutelum with a small tuft is found, a character showed only by eulaema females. the most notable differences are those of the legs. the right legs largely correspond to those of a normal female: foreleg without chemical gathering tufts (fig 1c), without velvety area or tufts in mesotibia (fig 1e), metatibia with a well-developed concave corbicula (fig 1g). the left legs were very similar to those of a normal male: foreleg with well-developed chemical gathering tufts on second through fourth protarsomeres (fig 1d), an incipient velvety area, and tufts on the outer surface of mesotibia (fig 1f). in the hind leg, a flattened metatibia with the appearance of a deformed corbicula can be observed. in addition, there is a slit with a very irregular shape in its lower portion, widened in its initial and final parts, while in the middle portion it becomes a thin line. in normal male specimens, the slit has a regular width throughout (fig 1h). wings without notable difference in size. both hind wings with a jugal comb (typical of male euglossine bees), sociobiology 68(3): e5778 (september, 2021) 3 left wing with 20 blades, the right with only 15. both wings differ in the number of hamuli, the left having 37, and the right 35. however, this is not a sex-related character. based on our observations in specimens of both sexes and several species of eulaema, the number of hamuli can vary in the wings of the same individual. metasoma. abdomen with five visible terga and six sterna (tergite six is naturally lacking), which would be the number of visible terga for a normal female. it also appears to be bilaterally asymmetric, with slight differentiation in size between the right and left sides of the terga and sterna. the right side is slightly coarser than normal females, the left one being less bulky (fig 1i). a well-developed stinger (7.15), as in normal females, emerges from the final segments (fig 1j). terminalia. the genital parts, although slightly modified, match those of a female of the same species (fig 2a). fig 1. eulaema meriana gynander: a. front view of the head, b. ventral part of the mandibular insertion (left side with a conspicuous bifurcated lobe), c. right foreleg, d. left foreleg, e. right middle leg, f. left middle leg, g. right hind leg, h. left hind leg (with malformation), i. ventral view of the abdomen (bilaterally asymmetric), j. final tergites of the abdomen with a well-developed sting. scale bars = 2 mm. alex pazmiño-palomino, marcio l. oliveira – first case of gynandromorphism in eulaema meriana4 a normal female has two rami of valvulae that converge to form the stinger; however, one of the rami of valvulae (the lower) is not connected to the sting in the gynander specimen and is ending freely in the genital capsule. the longer one emerged from the upper part of the capsule, appearing as a longer and thinner ramus. sternite 6 similar to that of a normal female (fig 2b). material examined. ecuador, los ríos, buena fé, patricia pilar, centro científico río palenque, 140m, 00°34’55” s, 79°22’01” w; 12 feb 1987; calaway dodson leg. (collection method is not mentioned). deposited in instituto nacional de biodiversidad – inabio [mecn]. catalog number: mecn-en-hym-1626. m. l. oliveira det. fig 2. terminalia of el. meriana gynander: a. genitalia of gynander (in lateral view), sting emerges from the upper part of the genital capsule and not the lower one, b. sternum 6 in dorsal view. scale bars = 1 mm. discussion eulaema meriana (olivier) is one of the most widely distributed orchid bee species in the neotropical region, from guatemala to bolivia (oliveira, 2007). despite being a relatively common species, this is the first record of a specimen with morphological characteristics of both sexes and the seventh documented case in the tribe euglossini. in this specimen, most of the secondary sexual characters are distributed bilaterally, half male left and half female right. however, the hind wing jugal comb on both sides (male feature) and female terminalia would classify this case as mixed or mosaic gynandromorphy. the other known case within the genus, eulaema atleticana nemésio (silveira et al., 2012), is also classified as mixed. still, it differs from the case presented here because it does not present bilateral sexual secondary characteristics. on the contrary, each tagma has characteristics of one sex or another. furthermore, the head and mesosoma have characteristics of male and the metasoma of female, while there is an alternation of characters on the legs. the mixed or mosaic pattern of this case in el. meriana corresponds with all reports of orchid-bee gynanders, where male and female structures were distributed in patches throughout the body. however, the distribution of characters was irregular among body sections in prior cases (giangarelli & sofia, 2011; hinojosa-díaz et al., 2012; silveira et al., 2012; camargo & gonçalves, 2013; gonzález, 2014; suzuki et al., 2015) (see table 1), establishing a pattern of phenotypic intersexual anomalies in euglossini. this aligns with the trend of most known cases of gynandromorph bees worldwide that present this gynandromorph type (wcislo et al., 2004; michez et al., 2009; hinojosa-díaz et al., 2012). detection of gynandromorphism in orchid bees should be facilitated by the marked sexual dimorphism between males and females, a situation that does not occur in other groups of bees (hinojosa-díaz et al., 2012). males have specialized morphological structures to collect fragrances for reproductive purposes, while females are morphologically homogeneous, to the point that many are not possible to identify species without an associated male (roubik & hanson, 2004; rasmussen et al., 2015). sociobiology 68(3): e5778 (september, 2021) 5 on the other hand, the methodology with which the specimen described here was collected has not been written on the original label. but we presume that it could be collected with attractants for a male orchid bee. since our observations, most of the orchid bee specimens donated by c. dodson to inabio were collected with attractive fragrances such as eucalyptus oil, methyl salicylate, among others that only the males of this group collect for reproductive purposes. despite the notable morphological differences between the sexes and the anomalies that this specimen presents, it has taken more than 33 years since it was collected for a specialist to determine it as a case of gynandromorphism. this is due to the notable lack of knowledge of the ecuadorian bee fauna. although, in recent years, euglossines have been the relatively most studied group of this fauna, to the point that they are the only group that currently has a species checklist and several recent revisions (botsch et al., 2017; padrón et al., 2018; lópez, 2018; yanouch et al., 2018; suárez-torres, 2019; roubik, 2019). the case published here follows this trend of increasing melittological research and a positive outlook for greater knowledge of this fantastic pollinator group in ecuador and the neotropical region. species gynandromorph type deviant phenotypic characters reference euglossa iopoecila dressler mixed head right half ♀, left half ♂; mesosoma: left half ♂, right half ♀, (mesoscutellum, ♀-like); metasoma ♀, except left half of the second sternum ♂ giangarelli & sofia, 2011 euglossa tridentata moure mixed head and mesosoma: right half ♀, left half ♂, but labrum with reversed sides, mesoscutellum ♀-like, legs mixed; metasoma: right half ♀, left half ♂ basally (distal segment lost) hinojosa-díaz et al., 2012 eulaema atleticana nemésio mixed head and mesosoma ♂, metasoma ♀, legs mixed silveira et al., 2012 euglossa pleosticta dressler mixed head right half ♂, left half ♀; legs mixed; mesosoma ♀; metasoma ♀ camargo & gonçalves, 2013 euglossa melanotricha moure mixed head, mesosoma, and metasoma ♀; right legs ♀, left legs ♂ suzuki et al., 2015 euglossa gorgonensis cheesman mixed head and mesosoma ♂; legs mixed. gonzález, 2014 eulaema meriana (olivier) mixed head and mesosoma left half ♂; right half ♀ (mesoscutellum ♀-like); genitalia ♀. this work table 1. comparison of characters among orchid bee gynanders recorded until today. acknowledgments we thank the trilateral cooperation program germanybrazil-ecuador, which made collaboration between inabio (ec) and inpa (br) possible. thanks to roberto andreocci for help with the photographic material, terminalia dissections, and important corrections in the text. special thanks to john christopher brown and mario e. cohn-haft for the english revision. mlo thanks to productivity grant no. 305150/20200 awarded by cnpq. author’s contribution: mlo: conceptualization, writing-original draft, writingreview and editing, supervision. app: conceptualization, methodology, writing-original draft, writing-review and editing, supervision. references botsch, j.c., walter, s.t., karubian, j., gonzález, n., dobbs, e.k. & brosi, b.j. 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(2018). las abejas de las orquídeas: la tribu euglossini (hymenoptera, apidae): primeros registros en el valle de los manduriacus, cantón cotacachi, provincia de lmbabura. recinatur international journal of applied sciences, nature and tourism, 1: 44-52. retrived from: http://www.recinatur. periodikos.com.br/article/5c6edf7c0e88258a6f8e6fd6 doi: 10.13102/sociobiology.v62i3.760sociobiology 62(3): 412-416 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 quantitative analysis of the genes affecting development of the hypopharyngeal gland in honey bees (apis mellifera l.) introduction age-dependent division of labor is one of the most typical features of the social insect honeybees (robinson et al., 2005). some tissues such as the hypopharyngeal gland (hg), which is located in the head of bees and proven to produce and secrete the protein components of royal jelly, will change in parallel with the role change of the bees. young bees, before 6 days after eclosion, perform tasks inside the hive, including brooding, cleaning cells and feeding larvae within 3 days after eclosion in hives, and their hgs are small and not fully developed during this period. nurse bees (6-12 days) are mainly responsible for feeding queen and larvae within 3 days, in the meanwhile, the secretory activity of hgs reach a peak, facilitating the synthesis and secretion of royal jelly. after that, most of the worker bees generally mature into foragers to collect nectar outside the hive (michener, 1974; pankiw & page jr ,2000; brillet et al., 2002; seeley, 2009). the hgs gradually degrades during this stage, and the secretory protein components changed into digestive enzymes such as alphaabstract royal jelly has many important biological functions, however the potential molecular mechanisms of royal jelly secretion in hypopharyngeal gland (hg) are still not well understood. in our previous study, six genes (sv2c, eif-4e, pdk1, imp, cgnp and tgf-βr1) have been shown to be associated with royal jelly secretion. in this study, the relative expression levels of these genes were further determined in the hypopharyngeal gland of worker bees (apis mellifera l.) at different developmental stages (nurse, forager and reversed nurse stages) with quantitative real-time pcr (q-pcr) assay. our results indicated that the relative expression levels of sv2c, eif-4e, imp, cgnp and tgf-βr1 were decreased at forager stage relative to nurse stage, while were reversed at reversed nurse stage compared to forager stage. for the pdk1, it reduced continuously with the developmental stages. we concluded that sv2c, eif-4e, imp, cgnp and tgf-βr1, but not pdk1, were possibly potential candidate genes related to hg development and/or royal jelly secretion. sociobiology an international journal on social insects h liu, zl wang, lb zhou, zj zeng article history edited by evandro donascimento silva, uefs, brazil received 01 february 2015 initial acceptance 27 april 2015 final acceptance 04 june 2015 keywords honeybee, hypopharyngeal gland (hg), gene expression, quantitative real-time pcr (q-pcr). corresponding author zhi jiang zeng honeybee research institute jiangxi agricultural university nanchang, jiangxi, 330045, china e-mail: bees1965@sina.com glucosidase, amylase and glucose oxidase for brewing honey (kubo et al., 1996; ohashi et al., 1997, 1999). in addition, a large number of researches has demonstrated that the degeneration and secretory activity of hgs are quite flexible. a prior study has found that the hgs are well-developed but no royal jelly secretion occurs in most of the workers when a few brood need to be nursed in the winter (fluri et al., 1982). moreover, its growth degree depends on the needs of feeding larvae (free, 1961) and consumption rate of protein diets (al-ghamdi et al., 2011). besides that, huang and robinson (1996) proved that foragers would reverse to nurses (reversed nurses) and their hgs could also redevelop if there are no young bees or queen in the colony. previously, we performed a digital gene expression (dge) analysis of the hgs at three developmental stages (newly emerged worker, nurse and forager) between apis mellifera and apis cerana, the two sibling honeybee species representing the most typical subspecies for western and eastern bee, respectively, with the aim of searching for the degs affecting the development of hg and/or the research article bees jiangxi agricultural university, nanchang, jiangxi, china sociobiology 62(3): 412-416 (september, 2015) 413 secretion of royal jelly (liu et al., 2014). as a result, synaptic vesicle glycoprotein 2c (sv2c) was found to be expressed significantly high amounts in nurses among three developmental stages in both species and it was significantly higher in hgs of apis mellifera than that of apis cerana at the nurse stage. furthermore, there was no significant difference at newly emerged worker or forager stage between two species. additionally, there were another 5 genes displaying significantly abundant expressions at the newly emerged worker and nurse stages while reduced rapidly at forager stage, including eukaryotic translation initiation factor 4e (eif-4e), 3-phosphoinositide-dependent protein kinase 1 (pdk1), igf-ii mrna-binding protein (imp), cell growth-regulating nucleolar protein-like (cgnp) and tgf-β receptor 1 gene (tgf-βr1). furthermore, the five genes exhibited higher expressions in the hgs of apis mellifera than that of apis cerana at both the newly emerged worker and nurse stages. combined with the observations of expression patterns of these genes and the corresponding development stages in worker bees, we hypothesized that these genes might be associated with royal jelly secretion and/or hypopharyngeal gland development. in this study, quantitative real-time pcr (q-pcr) was used to further analyze the differential expression levels of these genes among the hgs of nurses, foragers and reversed nurses. materials and methods sampling collection the honeybees (apis mellifera ligustica) were bred in the honeybee research institute, jiangxi agricultural university, china (28.46 °n, 115.49 °e) using standard beekeeping techniques. three categories of workers (nurses, foragers and reversed nurses) were gathered alive from two colonies as independent biological replicates, immediately frozen in liquid nitrogen and then stored at -80 ºc until further processing. the nurses were caught at the time when they entered the cells and were nursing the larvae; the foragers could be easily identified by the pollen loads on their hind legs; and the reversed nurses were collected according to the method of huang and robinson (1996). a total of 360 workers were sampled randomly. the hgs were dissected in normal saline (137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 2 mm kh2po4) under a binocular stereo microscope. after rinsed with depc-treated water, the hgs from 20 individuals per colony were pooled together in a tube for subsequent processes. total rna extraction and cdna synthesis total rna was extracted using 1 ml of trizol reagent (invitrogen, usa) according to the manufacturer’s instructions. after subjected to quality inspection (absorption ratio 260 nm/280 nm) and concentration quantification by nanophotometertm p 300 (implen, germany), cdna was synthesized with 1 μg rna with 3 μl (50 μm) oligo-dt 18 primer (invitrogen, usa) in 23 μl depc-treated water and incubated at 70 ºc for 5 min and then immediately placed on ice for 5 min. afterwards it was added 10 μl rt m-mlv buffer (5×, takara), 10μl dntp mixture (2.5 mm each, takara), 2 μl m-mlv revertase (50 u/μl, takara), 2 μl rnase inhibitor (50 u/μl, transgen) into the rna mixture and incubated at 42 ºc for 1 h, and then at 75 ºc for 5 min. finally, the synthesized cdna was diluted with 50 μl depctreated water. the products were kept at -80 ºc for subsequent q-pcr reactions. primer design and quantitative real-time pcr assays the primers used for amplifying the 6 specific genes and two house keeping genes gpdh (huang et al., 2012) and arp1 (lourenço et al., 2008) were designed based on mrna using primer 5.0 software, the primers were listed in table 1. the q-pcr reactions were cycled in a thermal cycler iq5 (bio-rad, usa) using the following conditions: 5 μl sybr® premix ex taqtm (takara), 3.2 μl sterile water, 0.4 μl (10 μm /l, invitrogen) forward primer, 0.4 μl (10 μm /l, invitrogen) reverse primer and 1 μl cdna template. the q-pcr assay was performed using the following program: initial denaturation 95 ºc for 30 s, 40 cycles including 95 ºc for 10 s and 60 ºc for 1 min. finally, melting curves were recorded by increasing the temperature from 55 ºc to 95 ºc (increased 0.5 ºc per 10 s) subsequently to 95 ºc for 1 min and 55 ºc for 1 min. each sample had three technical replicates and biological replicates, respectively. the specificity of the pcr reaction was verified by melting curve analysis. gene name accession number sense and antisense sequences (5′–3′) sv2c xm_394660.4 actcggcttgtgctcggtatt / ctgctttgatttattcttttgcg eif-4e xm_624287.3 atcaaacatccacttcaacataca / tacatttacaacagccccacaa pdk1 xm_394208.4 tcaccaccatcaacccg / gtaggacaggacgaaatagagt imp xm_393878.4 agacgcaggaacaagcacaac / cgagaatacgaagcgggaagt cgnp xm_623800.3 gtgaaagaaagcaacaagaatg/ ga actgctgatggtattttgtgac tgf-βr1 xm_003251608.1 gaggtgttacgataaggtgcg / cgtgaggaggaataaagggc gpdh nm_001014994.1 gctggtttcatcgatggttt / acgatttcgaccaccgtaac arp1 nm_001185146.1 tcctggaatcgcagatagaatg / ggaaggtggacaaagaagcaag table 1. the q-pcr primer sequences used in this study. h liu, zl wang, lb zhou, zj zeng – analizing genes of hypopharyngeal gland in apis mellifera414 statistical analysis the threshold values (ct) were analyzed by using biorad iq5 2.1 standard edition optical system software. and the amplification efficiency (e) of each gene was obtained using the q-pcr package (spiess & ritz, 2010; hornik, 2011). relative quantification analysis with two internal control genes was performed using the formula according to huang (2012), the computational formula as follows: where e is the pcr amplification efficiency; ct is cycle threshold; i is i-th reference gene; n is number of reference gene; r is the relative gene expression. the significance of the differential expressions was analyzed by variance analysis (anova) using statview (v 5.01, usa). p-value less than 0.05 was regarded as statistically significant. results our experiments were carried out to examine whether the 6 genes were associated with the secretory activity of hgs, and the results are showed in figure 1. the relative expression level of sv2c significantly reduced at forager stage compared to nurse stage (p< 0.05), and then significantly recovered at reversed nurse stage (p< 0.05). contrary to our expectation, the expression of pdk1 decreased at forager stage and then further decreased at reversed nurse stage (p> 0.05). furthermore, eif-4e and cgnp showed much reduced at forager stage (p< 0.05) and then slightly recovered at reversed nurse stage (p> 0.05). in addition, the expression patterns of tgf-βr1 and imp indicated they reduced at forager stage (p> 0.05) and then recovered at reversed nurse stage (p> 0.05). discussion our results revealed that the relative expression levels of the sv2c, eif-4e, pdk1, imp, cgnp and tgf-βr1 genes were reduced from nurse to forager, which is consistent with our previous dge analysis (liu et al., 2014), suggesting that our previous dge results are reliable. sv2c is a member of transmembrane glycoproteins almost existing in all of the neuronal and neuroendocrine cells, which are closely related to the normal secretion of neurotransmitters. besides that, sv2 could be also found to be expressed in the neuroendocrine cells (feany et al., 1993). considering the expression pattern of sv2c in hgs of the three development stages, we speculated that sv2c might be associated with the secretion activity of royal jelly, and it could be considered as a marker for genetic improvement programs. however, this speculation should be treated cautiously as no supportive evidence has been reported in prior studies. it needs to be verified through subsequent. the imp, tgf, pdk1 and eif-4e belong to igf, tgf, pkb/akt and mtor signaling pathway respectively, which has the main role involved in the regulation of cell growth, proliferation, differentiation, cell death and motility (hafen 2004; ikushima & miyazono, 2011). the imp and tgf-βr1 could activate igf-ii and tgf-β respectively, the latters would activate phosphatidyl inositol 3-kinase (pi3k) signaling and finally lead to pkb/akt signaling, via pdk1dependent or -independent manner. upon the activation of pkb/akt regulates, a large number of genes with multifarious physiological functions would be expressed and exert their corresponding functions. in addition, the nucleolar proteins and cgnp are respectively involved in the biosynthesis of ribosomes and cell proliferation and death. in brief, these genes co-regulate multiple cellular and physiological processes including the metabolisms of carbohydrate, lipid and protein, cell differentiation, proliferation, apoptosis, lifespan, and so on (sarbassov et al., 2005; fanayan et al., 2000). both the present study and our prior observation (liu et al., 2014) suggested that the expression levels of eif4e, cgnp, imp and tgf-βr1 expressing higher at newly emerged worker, nurse and reversed nurse stages compared to forager stage which is in parallel with the physiological conditions of hgs: the hgs grew rapidly at newly emerged worker stage and synthesized a large amount of royal jelly fig 1. relative expression levels of the sv2c, pdk1, eif-4e, cgnp, tgf-βr1 and imp genes in hgs of honey. n: nurse stage; f: forager stage; rn: reversed nurse stage; sv2c: synaptic vesicle glycoprotein 2c; pdk1: 3-phosphoinositide-dependent protein kinase 1; eif-4e: eukaryotic translation initiation factor 4e; crnp: cell growth-regulating nucleolar protein-like; tgf-βr1: tgf-β receptor 1; imp: igf-ii mrna-binding protein. different letters on top of bars indicate significant difference (p< 0.05) between the groups. values are expressed as mean ± sem. sociobiology 62(3): 412-416 (september, 2015) 415 at the nurse stage, so many genes related to the biological metabolism and cell growth over expressed at these two stages and then reduced their expression quickly at forager stage in view of the degeneration of hgs and the cell apoptosis. however, the hgs re-growed and speeded up biological metabolism at reversed nurse stage lead to increased expression of many relevant genes. therefore, according to their biological functions in other organisms and expression patterns detected in this study, we speculated that these four genes might participate in the development of hgs. contrary to our expectations, pdk1 reduced continuously (p> 0.05) with the developmental stages. actually in our previous study, pdk1 declined continuously all the time (liu et al., 2014). hence we speculated that pdk1 has the function of controlling the lifespan of hgs. in fact, reddy et al. (2009) found pdk1 in mice could control oocytes lifespan by pdk1 pkb/akt p70 s6 kinase 1 (s6k1) ribosomal protein s6 (rps6) signaling pathway. in summary, we used q-pcr to investigate the differential expression levels of sv2c, eif-4e, pdk1, imp, cgnp and tgf-βr1 genes among the hgs of nurses, foragers and reversed nurses. our results indicated that sv2c might be a potentially strong candidate gene associated with the secretory activity of royal jelly in hgs, in addition, eif4e, tgf-β, cgnp and imp might be also associated with the development of hgs, while pdk1 might be not involved in this process. in addition, we generalized three signal pathways in figure 2 for better understanding. acknowledgements we thank li-zheng zhang, an yuan, and you li for their help in conducting experiments. this work was supported by the earmarked fund for china agriculture research system (no.cars-45-kxj12), the 555 talents project of ganpo of jiangxi province and the natural science foundation of jiangxi province (no. 20114bab214001). references al-ghamdi, a. a., al-khaibari, a. m., & omar, m. o. 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(2010). qpcr: modelling and analysis of real-time pcr data. r package version, 1-3. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.8308sociobiology 69(3): e8308 (september, 2022) introduction myrmecochory is an ecosystem function in which ants disperse approximately 11,000 species of myrmecochorous plants (lengyel et al., 2010). within this function, diaspore removal is the process in which ants transport diaspores, which is part of the seed dispersal. this is an asymmetric mutualism in which seeds of many angiosperm species are dispersed by a few ant species (warren & giladi, 2014). in general, ants are attracted by the high lipid content of seed elaiosomes for primary dispersal (bas et al., 2007; servigne & detrain, 2008; leal et al., 2014) and to the arils of diaspores on the ground for secondary dispersal (pizo & oliveira, 2000). ants, along with birds, are the major secondary removers of fleshy diaspores falling on the grounds of brazilian tropical abstract secondary diaspore removal on the ground is an important ecosystem process. in this process, solitary foraging ants with larger body sizes are more efficient because they may remove more diaspores, faster and carry them at greater distances. therefore, we sought to test the effects of the sizes of the morphological traits of ants, removal strategy, and nest distance on secondary diaspore removal, testing hypotheses related to the efficiency of this process. we evaluated the removal of artificial diaspores by ants in 15 areas of cerrado sensu stricto (tropical savanna), collecting data on diaspore removal strategy (solitary or group), nest distance, diaspore discovery time, diaspore removal time, and the number of diaspores removed. larger ants tended to remove diaspores alone and remove diaspores faster than smaller ones. ants that removed diaspores alone removed more diaspores than ants that removed diaspores in groups. however, we did not find a linear relationship between ant size and diaspore removal. this is likely due to a limitation on, or a preference by larger ants for removing larger diaspores, while the smaller diaspores may have hindered manipulation or been less attractive to larger ants. thus, the removal strategy was the best predictor of efficient diaspore removal performance, where the solitary foraging ants discover and remove diaspores quickly and remove more diaspores, mainly from the closest nests to the sampling point. however, the benefits (or not) of removing more diaspores still need to be evaluated. sociobiology an international journal on social insects icaro wilker¹, mariana a. rabelo¹, marina a. angotti¹,², carla r. ribas¹ article history edited by kleber del-claro, ufu, brazil received 23 june 2022 initial acceptance 29 june 2022 final acceptance 11 august 2022 publication date 12 september 2022 keywords functional traits, myrmecochory, seed removal, morphology, strategy foraging, animal-plant interactions. corresponding author icaro wilker programa de pós-graduação em ecologia aplicada, departamento de ecologia e conservação instituto de ciências naturais, universidade federal de lavras aquenta sol, s/nº, cep: 37200-900 lavras, minas gerais, brasil. e-mail: gonzagaicaro@gmail.com savannas (campagnoli & christianini, 2021; christianini & oliveira, 2010). these ants are typically large ants that forage solitarily and small ants that forage in groups, which are considered good and poor seed dispersers, respectively, due to their foraging characteristics and morphological traits (giladi, 2006). large species of ants that forage solitarily, which are characterized by omnivorous or scavenging guilds, are considered to be high-quality dispersers because they disperse diaspores in greater quantity and at greater distances from parent plants (giladi, 2006; leal et al., 2014). species that recruit many workers (generally small ants), such as omnivorous or granivorous ants, are considered to be poorquality dispersers because they tend to disperse fewer diaspores over shorter distances (hughes & westoby, 1992; 1 programa de pós-graduação em ecologia aplicada, departamento de ecologia e conservação, instituto de ciências naturais, universidade federal de lavras, lavras-mg, brazil 2 institudo federal de educação, ciência e tecnologia do mato grosso do sul (ifms), campus ponta porã, mato grosso do sul, brazil research article ants ant species that remove diaspores alone are more efficient removers icaro wilker, mariana a. rabelo, marina a. angotti, carla r. ribas – solitary remover ants are better diaspore removers2 giladi, 2006; leal et al., 2014). in addition, these ants often consume the aril, elaiosome, or even the seed at the site, performing few removals, making difficult the germination or even killing the seed embryo (fernandes et al., 2018; pizo & oliveira, 2000). conversely, large species are more attracted by elaiosomes with a greater content of some lipids because these components are also found in dead insects and in the prey of these ants (hughes et al., 1994). so, this leads large ants that forage solitarily to discover and remove diaspores before the granivorous ants consume them (hughes et al., 1994). moreover, larger ants probably transport diaspores to the nest faster than small ants, because long-legged ants show the best performance in carrying loads (nielsen et al., 1982; espadaler & gomes, 1996). conversely, the group-foraging ants, after finding resources and recruiting workers, continue to remove resources (dornhaus & powell, 2010). this behaviour can lead to faster removal of resources, due to the larger number of workers performing this function. on the other hand, although the solitary foraging ants do not recruit other workers, some ant groups, like some ectatomminae ants, can show path fidelity, i.e., the ants specialized in particular foraging zones around the colony (pie, 2004). in this foraging type, the ants find the resources and return to the nest quickly, which can also increase the speed of removal of resources. some morphological traits of ants are considered useful to evaluate the effectiveness of ant performance in ecosystem processes and functions. ants with larger bodies tend to carry more diaspores for greater distances (ness et al., 2004), and probably discover and remove lipid-rich diaspores faster (espadaler & gomes, 1996; hughes et al., 1994; nielsen et al., 1982). this apparently is beneficial for myrmecochory, allowing increased plant dispersal and conquest of new territories (bestelmeyer & wiens, 2003; ness et al., 2004). in addition, these ants often have larger legs, which indicate higher foraging speed and ease of movement on the ground (feener jr et al., 1988; hurlbert et al., 2008; silva & brandão, 2010; pearce-duvet et al., 2011). similarly, ants with larger mandibles can hold or carry larger food items (weiser & kaspari, 2006; silva & brandão, 2010; gibb et al., 2015), which may be beneficial for diaspore dispersal because it allows these large ants to remove a wider variety of diaspores. however, larger ants tend to prefer to handle larger diaspores due to possible limitations in the manipulation of small diaspores and the lower attractiveness of them (davidson, 1977; takahashi & itino, 2012; anjos et al., 2018). regarding orientation and search for resources, larger eyes may help in orientation (hölldobler, 1980) and identification of food (weiser & kaspari, 2006; gibb et al., 2015). larger antennae scapes indicate greater chemosensory perception in foraging (weiser & kaspari, 2006; silva & brandão, 2010; gibb et al., 2015). due to the apparent advantages already presented of larger ants in the removal of diaspores (feener et al., 1988; bestelmeyer & wiens, 2003; ness et al., 2004; hurlbert et al., 2008; pearce-duvet et al., 2011), if plants can attract omnivorous or scavenging ants (larger ants), this can be advantageous for diaspore dispersal. however, the opposite has been observed, with smaller ants participating in most of the interactions with diaspores (christianini & oliveira, 2010; anjos et al., 2020). in brazilian savannas, the larger interactions between ants and fleshy diaspores falling on the ground happen with small ants (christianini & oliveira, 2010). however, large ants remove myrmechorous diaspores at a higher rate because they are rich in lipids (hughes et al., 1994; giladi, 2006). it is important to evaluate the effects of ants of different sizes and with different foraging strategies on diaspore removal from the savanna ground, as this is a key part of myrmecochory. to evaluate these characteristics, we compared ant species to evaluate how morphological traits and solitarily or group foraging strategy affected secondary diaspore removal. we addressed the following questions and predictions: i) can the diaspore removal foraging strategy be defined by the ant size? we predicted that larger species of ants would remove diaspores alone, while smaller species would remove diaspores in groups; ii) do removal strategy, ant size and removal distance influence the time taken for diaspore discovery? we predicted that solitarily foraging ant species, larger ants with removal to greater distances, would discover diaspores faster; iii) do removal strategy, ant size, and removal distance influence the speed of diaspore removal? we predicted that larger ants (solitary foraging ants with removal to greater distances) would remove diaspores faster; iv) do ants remove diaspores increasingly faster? we predicted that after the first diaspore removal, the ants will remove diaspores quickly; v) do removal strategy, ant size, and removal distance influence the number of diaspores removed? we predicted that larger ant species (solitary foraging ants with removal to greater distances) would remove more diaspores than smaller ants (group-foraging ants). materials and methods study site we collected the data on cerrado sensu stricto vegetation (tropical savanna) within the área de proteção ambiental do rio pandeiros (apa rio pandeiros). this protected area encompasses 393,866 hectares and is considered the largest sustainable use conservation unit in the state of minas gerais, brazil. it is located in the north of the state (-15° 50′ s, -44° 76′ w) in a transitional region between the cerrado and caatinga biomes. the climate is semiarid, with well-defined seasonality. the temperature ranges from 9 °c in the cold (june and july) to 45 °c in the hot (october to january) season. the rainfall varies from 900 to 1250 mm throughout the year (nunes et al., 2009). field collections were performed in march 2016 during the hot and rainy season, the period of greatest ant activity in the cerrado (marques & del-claro, 2010). sociobiology 69(3): e8308 (september, 2022) 3 sampling design and diaspore removal we sampled 15 areas of cerrado spaced at least 500 m apart. in each area, 50 artificial diaspores were deposited at a sampling point 70 m from the edge of the cerrado to avoid edge effects. the artificial diaspores were made of beads weighing 0.03 g and 2 mm in diameter, considered small, usually as myrmecochory diaspores (anjos et al., 2020; pizo & oliveira, 2000), and an attractive paste composed of 75% hydrogenated vegetable fat, 7% casein, 5% maltodextrin, 4.8% fructose, 4.7% glucose, 3% calcium carbonate, and 0.5% sucrose. the beads simulated the solid part of the seed, while the attractive paste simulated the lipid-rich aril, a portion of the diaspore that is attractive to ants (see raimundo et al., 2004). we used artificial diaspores to standardize sampling, since natural diaspores may vary in shape, size, and ripening stage, which could influence their interactions with ants. the use of artificial diaspores is a method employed to avoid problems with not finding enough natural diaspores at the time of the experiment and to standardize samplings, since natural diaspores may vary in shape, size, and ripening stage, which can influence their interactions with ants (raimundo et al., 2004; bieber et al., 2014; rabello et al., 2015; angotti et al., 2018; rabelo et al., 2020, 2021). we defined diaspore removal as the event in which the ants transported the artificial diaspores from the sampling point to the nest. the artificial diaspores were left exposed on the ground from 7 am to 10 am (period of high activity for most ant species). during this period, we recorded the time of diaspore discovery by the ants, speed of diaspore removal from the sampling point to the nest, distance from the sampling point to the nest, diaspore removal strategy (solitary or group), and number of diaspores removed. we marked the nest with diaspore removal and collected one ant per nest after experiments. we also collected individuals from the nests that performed the removals for subsequent species identification and measurement of morphological traits. we assessed the removal strategy as whether ants transported the diaspores alone or in groups of workers. solitary foraging occurred when only one ant carried the diaspore from the sampling point to the nest. group foraging occurred when two or more workers jointly carried the diaspore to the nest. the groups of ants can vary in the number of individuals involved, both between different removal events and within the same removal event (during a removal, workers may join or leave the group, increasing or decreasing the number of ants). diaspore discovery occurred when one or more ants found and began to remove the diaspore. we defined discovery time as the number of seconds (s) from the beginning of the experiment (7:00 am) until ants began removal of an artificial diaspore. for each ant species, we calculated the mean discovery time in each of the 15 experimental areas. the removal time was the time that the ants (with solitary behaviour or in groups) took to transport the artificial diaspore from the sampling point to the nest. for each ant species, we calculated the mean removal time in each of the 15 experimental areas. to assess whether the removal time changed when the same nest performed more removals, we chose the nests where the ants performed at least three diaspore removals, and considered the removal times of first, second and last diaspore removal, regardless of how many removals the nest performed overall. we chose to perform the analyses only with species that had at least three replicates (species collected in at least three out of the 15 areas). for the analyses, we calculated the mean removal time of each ant species per area. for each of the 15 areas, we calculated the arithmetic mean diaspore removals per species. in all cases, the sampling unit was the diaspore-removing ant species collected in each area. we only consider diaspore removal when the ants load the diaspores out of the sampling point to the nest. other types of interactions between ants and diaspores, like ants that only interacted with diaspores on sampling point, which do not load diaspores until the nest, or that we do not achieve follow, were not considered in our study. we marked the nest with diaspore removal and collected one ant per nest after experiments. species identification and morphological measurements the collected individuals were identified according to palacio and fernández (2003), wilson and hölldobler (2005), and baccaro et al. (2015). the specimens were deposited in the reference collection of the laboratório de ecologia de formigas at universidade federal de lavras (ufla). one individual from each nest was taken for morphological measurements. in species for which we sampled only one nest, only one individual was measured, whereas in species for which we sampled more than one nest, the mean of all individuals measured was calculated. we measured the following morphological traits, which are considered important for diaspore removal: body size (weber’s length), which is the length from the anterior edge of the pronotum to the posterior edge of the propodeum (weiser & kaspari, 2006; gibb et al., 2015); mandible size, which is the length from the mandibular insertion to the most external point (weiser & kaspari, 2006; silva & brandão, 2010; gibb et al., 2015); eye width (weiser & kaspari, 2006; gibb et al., 2015); scape size, which was measured by its length (weiser & kaspari, 2006; silva & brandão, 2010; gibb et al., 2015); and leg size, which was the length of the femur added to the length of the tibia of the posterior metathorax (weiser & kaspari, 2006). all measurements were obtained in millimetres under a zeiss axio zoom v16 microscope. the data is available in supplementary material. icaro wilker, mariana a. rabelo, marina a. angotti, carla r. ribas – solitary remover ants are better diaspore removers4 data analysis first, we performed a correlation test on the morphological traits (weber’s length, mandible size, eye width, scape size, and leg size). the spearman method was used due to the non-normality of the data. all morphological measurements (weber’s length, mandible size, eye width, scape size, and leg size) were correlated with each other (table 1). to avoid problems of multicollinearity in the analyses, we chose only weber’s length as a predictor variable, considering that it synthesizes the other morphological traits in our data. to test whether larger ants removed diaspores alone and smaller ants removed diaspores in groups, we performed a linear mixed-effects model (lmm) where the explanatory variable was the removal strategy (solitary or group), and the response variable was the body size (weber’s length in mm) and the fixed variable was species. to test whether larger ants, solitary removal and larger distances removal is linked to discovery of diaspores and quickly diaspore removal, we constructed an lmm and a glmm (generalized linear mixed model), respectively: in lmm we are relating body size, removal strategy and removal distance (explanatory variables) with the discovery time (response variable), and the glmm we relating them to removal time (response variable). in glmm, we used the poisson distribution family, but due overdispersion, we used quasi-poisson distribution. in both models the fixed variable was species. to test whether, after the first removal, the ants removed the diaspores faster, we constructed a glmm to evaluate the effect of being the first, second, or last removal (explanatory variable) on removal time (response variable) and the fixed variable was species nest. we used the poisson distribution family, but due overdispersion, we used quasipoisson distribution. to test whether body size, foraging strategy and removal distance influenced the number of diaspores removed, we constructed a glmm relating body size, removal strategy and removal distance (explanatory variables) with the proportion of diaspores removed (response variable)and the fixed variable was species. we used a quasi-binomial distribution family because the values ranged from 0 to 1. weber’s length mandible length eye width scape length leg length weber’s length 0.003 <0.001 <0.001 <0.001 mandible length 0.61 0.007 <0.001 <0.001 eye width 0.93 0.56 <0.001 <0.001 scape length 0.88 0.72 0.89 <0.001 leg length 0.86 0.69 0.91 0.97 p value above the dash and spearman correlation below the dash. table 1. correlation coefficients in ants morphological traits. all analyses were performed using the software r version 4.1.2 (2022). for the correlation analysis, we set p < 0.05 and spearman’s correlation coefficient > 0.6 as statistically significant; the correlations were tested with the hmisc package (harrell & harrell jr, 2019) and plotted with the corrplot package (wei et al., 2017). for the lmms and glmms with poisson distribution family we used lme4 package (bates et al., 2015). for the glmms with quasipoisson and quasi-binomial we used mass package (venables & ripley, 2002). to calculate pseudo-r² we used jtools (long, 2022) mumin package (barton, 2013). we detected multicollinearity through vif (variance inflation factor) and excluded variables that had vif > 4.0. all graphs were generated using the ggplot2 package of r (wickham, 2011). results a total of nine species of diaspore-removing ants were identified. the subfamily ectatomminae was the most frequent, removing artificial diaspores in 13 of the 15 sampled areas. five ant species were classified as displaying a solitary foraging strategy in the 15 sampled areas: ectatomma opaciventre (roger, 1861), ectatomma edentatum roger, 1863, ectatomma planidens borgmeier, 1939, ectatomma brunneum smith, 1858, and odontomachus haematodus (linnaeus, 1758). four species displayed a group-foraging strategy in six sampled areas: blepharidatta conops kempf, 1967, pheidole jelskii mayr, 1884, pheidole capillata emery, 1906, and solenopsis tridens forel, 1911. we indeed found that (i) larger ant species removed diaspores alone and smaller ant species remove in groups (df = 19; χ² > 33.566; p < 0.001; pseudo-r² = 0.98; fig 1). on question (ii), we excluded ant size of model because detected multicollinearity through vif (vif = 6.004) and found that solitary remover ants (df = 18; χ² = 15.974; p < 0.001; pseudo-r² = 0.70; fig 2) and ants which remove for shorter distances discovered diaspores faster (df = 18; χ² = 5.384; p = 0.020; pseudo-r² = 0.70; fig 2). on question (iii), we excluded ant size of model (vif = 8.747) and found that solitary remover ants removed diaspores faster (df = 19; χ² = 52.274; p < 0.001; pseudo-r² = 0.92; fig 3), but the nest distance did not affect removal time (df = 18; χ² = 0.345; p = 0.556; pseudo-r² = 0.92). on question (iv), only one ant species was collected in more than three areas (replicate) and performed the three diaspore removals. in this case, we only made data analysis with ectatomma edentatum sociobiology 69(3): e8308 (september, 2022) 5 (larger species with solitary strategy). we found that the removal time decreases from the first to the last removal (df = 33; χ² = 13.127; p = 0.001; pseudo-r² = 0.76; fig 4). fig 3. solitary remover ants remove diaspores faster than group removal ants. the x-axis indicates the removal strategy, and the y-axis indicates the removal time in seconds. the centreline of each boxplot indicates the median of all values, and the boxes indicate the first quartile (median of the values above the central median) and third quartile (median of the values below the central median). the vertical lines of each boxplot are the tukey-style whiskers (1.5 × interquartile range). fig 1. smaller ants remove diaspores in groups, and larger ants remove diaspores alone. the x-axis indicates the removal strategy, and the y-axis indicates the ant body size in millimetres. the centreline of each boxplot indicates the median of all values, and the boxes indicate the first quartile (median of the values above the central median) and third quartile (median of the values below the central median). the vertical lines of each boxplot are the tukey-style whiskers (1.5 × interquartile range). the circles above each boxplot are outliers. fig 2. ants with solitarily removal and ants with nests less distance discover diaspores faster. the x-axis indicates nest distance in meters (m) the removal strategy (solitary and group), and the y-axis indicates the diaspore discovery time in seconds (s). the circles and the line indicate group removal ants, and the triangles and dashed line indicate solitary remover ants. fig 4. after the first diaspore removal, ectatomma edentatumremove diaspores more quickly. the x-axis indicates which diaspore removal (first, second and last) and the y-axis indicate removal time in seconds (s). the centreline of each boxplot indicates the median of all values, and the boxes indicate the first quartile (median of the values above the central median) and third quartile (median of the values below the central median). the vertical lines of each boxplot are the tukey-style whiskers (1.5 × interquartile range). the circle above the boxplot is an outlier. icaro wilker, mariana a. rabelo, marina a. angotti, carla r. ribas – solitary remover ants are better diaspore removers6 last, regarding objective (v), we found that solitary foraging ants removed more diaspores than group-foraging ants (df = 19; χ² = 11.793; p < 0.001; pseudo-r² = 0.39; fig 5) and larger distance of nests decreases diaspore removal (df = 18; χ² = 7.969; p = 0.004; pseudo-r² = 0.39; fig 5), but the ant size did not affect removal time (df = 17; χ² = 1.888; p = 0.169; pseudo-r² = 0.42). the search and removal of seeds alone (gomes et al., 2009; ostwald et al., 2018). in contrast, smaller ants are rarely able to hold and remove diaspores on their own, needing to recruit other workers to transport the resource. our results corroborate giladi (2006) and leal et al. (2014), who separate ants into “good dispersers”, composed of guilds of omnivorous or scavenging ants with solitary foraging, and “poor dispersers”, which are guilds of granivorous ants that recruit many workers. we can draw a parallel between this classification and the finding that group removal ants (genera: blepharidatta, pheidole and solenopsis) perform few removals and are often seen to consume the aril at the site (pizo & oliveira, 2000). these ants may in our study be considered “poor removers” since we only evaluated the characteristics of the diaspore removal process as part of the dispersal function. however, the small ants clean the seeds, decreasing the fungal attack and increasing the germination rate (oliveira et al., 1995). conversely, solitary remover ants (omnivorous or scavenging genera: ectatomma and odontomachus) removed more diaspores and were not observed to prey at the site, so they can be considered “good removers”. in a complementary manner, ectatomma edentatum was the species that most participated in interactions with diaspores in our study (≈60%). in addition to being a species with solitary removal, which discovers and removes resources faster, it is also considered a good disperser by leal et al. (2014). however, it is important to notice that removal is part of seed dispersal process, and goodquality removal does not mean dispersal, as dispersal also depends on where the seed is deposited, how the ants manipulate the seed, and whether the seed germinates. still regarding the effect of guilds on diaspore removal, we find many interaction between omnivorous and scavenging ants which are attracted to rich lipid diaspores (pizo & oliveira, 2000; bronstein et al., 2006), since our attractive paste contained 75% lipids, simulating fleshy diaspores, as done in other studies (raimundo et al., 2004; rabello et al., 2015; angotti et al., 2018). more specifically, some lipids are even more attractive to scavenging ants (hughes et al., 1994). this relationship between lipids and scavenging ants (large ants with solitary removal) occurs because these diaspore contents are the same as those found in insects that are prey to these ants (hughes et al., 1994). these factors make the diaspores more attractive to scavenging ants, which may make it easier for them to find the diaspores faster than other guild or functional groups of group foraging ants. moreover, the solitary foraging ants with nests closer to the diaspore, discovered diaspores faster than that with far nests, probably because they forage more around nests than in far areas. in addition to discovering diaspores faster, solitary remover ants also removed the diaspores faster, probably because they had greater ease in removing the resources than smaller ants. their larger legs provide greater ease and speed of locomotion (silva & brandão, 2010; feener et al., 1988). regarding the diaspore removal time to ectatomma edentatum (solitary foraging ant), we found a decrease in removal time after the first transport, which suggests that a given nest does become more efficient at removal. ectatomma fig 5. ants with solitarily removal and ants with nests less distance (x-axis) remove more diaspores (y-axis). the number of diaspores removed is expressed as a percentage (number of diaspores removed by the species/total diaspores available at the sampling point*100). the circles and the line indicate group removal ants, and the triangles and dashed line indicate solitary remover ants. discussion overall, we found that solitary foraging ants were more effective in removing artificial diaspores. when considering the removal strategy, solitary remover ants (larger ants) discovered and transported diasporas quickly and removed more diasporas than ants with group foraging removal (small ants), showing a greater efficiency in this process, which can generate benefits for the myrmecochory function. ectatomma edentatum, a solitary ant, removed the highest number of diaspores and with increased speeds, being more efficient. moreover, ants with nests closer to diaspores, discovered and removed more diaspores, which can affect the dispersal distance. larger ants removed diaspores alone, and smaller ants removed them in groups. this probably occurred because larger mandibles allow ants to hold diaspores of different sizes more easily, and they have larger bodies and legs that afford them better locomotion on the ground (silva & brandão, 2010; gibb et al., 2015; feener et al., 1988), important characteristics for performing more efficient diaspore removal. moreover, in addition to the morphological characteristics that can help these larger ants to remove alone, they have solitary foraging behaviour, providing sociobiology 69(3): e8308 (september, 2022) 7 edentatum are ectatomminae ants and can show path fidelity in particular foraging zones around the nest (pie, 2004). so, the ants find the resources and return to the nest quickly and remove diaspores in a growing speed, increasing removal efficiency. in addition, in our study some nests with group foraging ants performed only one or few removals. this may be related to body limitations among smaller ants, preference for other resources, or interactions at the sampling point (e.g., eat the diaspore at the sampling point). we observed that solitary remover ants removed more diaspores than ants in groups, but there was no direct relationship between ant size and the number of diaspores removed. this finding suggests that solitary remover ants may have a size limit in the morphological traits that would let them perform such processes more efficiently. as we observed, larger ants with solitary removal remove more diaspores, probably because they have more easily to execute this function than smaller ants with group removal. however, ants are limited in body size and diaspore size. as observed by anjos et al. (2018), there is a relationship between mandible size and diaspore size, and larger ants prefer to handle larger diaspores. in addition, takahashi and itino (2012) observed that large ants have difficulty handling small diaspores. in this sense, the standardization of the size of the diaspores we used, considered small, may have generated a limitation and a difficulty for some larger ants, such as ectatomma brunneum and odontomachus haematodus, which removed few diaspores. however, despite this limitation for larger size ants, when comparing solitary removers with group removers, ants that remove diaspores alone still have advantages and perform the removal process more efficiently due to the aforementioned characteristics that confer greater ease in finding and removing diaspores. moreover, ants with nests closer to diaspores, removed more diaspores. in the same way for diaspore discovery, foraging more around nests than in far areas, facilitates to remove more diaspores. however, this can lead to a limitation on the distance of seed dispersal, where more diaspores are removed only for short distances, and this should be investigated. we conclude that solitary remover ants are better diaspore removers than group removal ants. solitary remover ants are ants with larger body sizes that discover and remove diaspores faster and in larger quantities. these ants are probably more efficient at secondary seed dispersal, as we observed that they remove more diaspores, probably decreasing the competition under the parent plant, but remove more for short distances, which can be a problem for dispersal, and we suggest this evaluation in future research. in addition, solitary remover ants find and remove diaspores quickly, and ectatomma edentatum, the greater remover in our study, remove each time faster than the before one, decreasing the diaspore exposure time to fungi, which are likely to make the diaspores unfeasible (oliveira et al., 1995), or to vertebrate predators and granivorous ants (christianini & oliveira, 2010). smaller ants with group removal discover and remove diaspores slowly and in smaller quantities. they perform fewer dispersals (giladi, 2006) for shorter distances (ness et al., 2004) and are still often seen eating the attractant at the site, decreasing dispersal (christianini & oliveira, 2010), or eating the seed, killing the embryo. thus, although it has been an assumption that ant morphological traits are an indication of an efficient diaspore removal performance, this is not always true, since the morphological trait evaluated by us and by most studies on this topic – ant body size – were not the best predictor of diaspore removal, but rather the removal strategy was the best predictor. moreover, these results can change due to the chemical composition of diaspores. the larger ants in our study were probably attracted by lipid composition of diaspores, but the chemical composition influences the identity and groups of ants that interact with diaspores (campagnoli & christianini, 2021; pizo & oliveira, 2001). acknowledgements we thank msc ariel reis and dr graziele santiago silva for their help with data collection; dr antônio queiroz for his help with statistical analysis; dr rafael zenni, dr lívia audino, and msc laís da glória for their suggestions about preliminary versions of the manuscript; dr rodrigo feitosa and dr alexandre ferreira for confirming the species identification; we thank the anonymous referees and the associate editor dr. kleber delclaro of the sociobiology for their revision of the manuscript; we thank the centro de estudos em biologia subterrânea (cebs) for providing the microscope. this manuscript was partially produced during the course pec 533 – publicação científica em ecologia, programa de pós-graduação em ecologia aplicada, universidade federal de lavras. the funding agencies that supported this study were coordenação de aperfeiçoamento de pessoal de nível superior (capes, finance code: 001), companhia energética de minas gerais s.a. (cemig), conselho nacional de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado de minas gerais (fapemig), and programa institucional de bolsas de iniciação científica (pibic-ufla). this manuscript was edited for proper english language, grammar, punctuation, spelling, and overall style by one or more of the highly qualified native english-speaking editors at aje (american journal experts) due programa de apoio à publicação científica at universidade federal de lavras. code: 1259-5091-2f1b-f232-81fp. authors’ contribution iw: conceptualization, methodology, validation, formal analysis, data curation, writing-original draft, writing-review & editing, visualization. mar: conceptualization, methodology, validation, investigation, resources, data curation, writing-review & editing, supervision, project administration. maa: conceptualization, methodology, validation, investigation, writing-review & editing. crr: conceptualization, methodology, validation, resources, writing-review & editing, supervision, project administration, funding acquisition. icaro wilker, mariana a. rabelo, marina a. angotti, carla r. ribas – solitary remover ants are better diaspore removers8 references angotti, m.a., rabello, a.m., santiago, g.s. & ribas, c.r. 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(2005). the rise of the ants: a phylogenetic and ecological explanation. proceedings of the national academy of sciences of the united states of america, 102: 7411-7414. doi: 10.1073/pnas.0502264102 icaro wilker, mariana a. rabelo, marina a. angotti, carla r. ribas – solitary remover ants are better diaspore removers10 supplementary material table s1. sampling areas, species sampled, and variables collected: sample (sampled areas 1 to 15); specie (ants species hymenoptera: formicidae); ml (mesosomal or weber’s length millimeters mm); mandl (mandible length millimeters mm); el (eye length millimeters mm); sl (scape length millimeters mm); fl (leg length summing the femur and tibia length of posterior metathorax leg millimeters mm); strategy (foraging strategy solitary or group); discovery (discovery time of diaspores seconds s); time_r (removal time of diaspores seconds s); diaspore_removal (artificial diaspores removal by ants hymenoptera: formicidae). sample species ml mandl el sl fl strategy discovery distance time_r diaspore_ removal 1 ectatomma edentatum 2.549 0.909 0.291 1.481 4.075 solitary 660 4.56 289 1 1 pheidole jelskii 0.892 0.360 0.108 0.721 1.650 group 3240 0.35 1960 1 2 ectatomma opaciventre 4.506 7.782 0.475 2.661 7.260 solitary 4.11 4.50 198 4 2 pheidole jelskii 1.146 0.442 0.126 0.808 1.560 group 7320 5.42 960 2 3 ectatomma planidens 2.433 0.509 0.224 0.759 1.276 solitary 2160 1.09 128 20 4 ectatomma edentatum 2.744 0.941 0.267 1.579 4.023 solitary 880 2.44 177 15 5 ectatomma edentatum 2.993 1.268 0.392 1.515 4.700 solitary 960 1.86 101 35 6 ectatomma edentatum 3.064 1.311 0.435 1.578 3.995 solitary 1320 1.08 65 34 7 ectatomma edentatum 2.875 1.130 0.348 1.616 4.303 solitary 1120 1.99 116 15 8 ectatomma edentatum 2.489 1.182 0.364 1.637 4.194 solitary 1020 1.45 131 6 9 ectatomma edentatum 2.858 1.086 0.313 1.519 3.820 solitary 1900 4.52 146 9 9 pheidole jelskii 1.024 0.391 0.122 1.044 2.260 group 2340 2.13 960 1 10 ectatomma edentatum 2.818 1.050 0.323 1.557 4.095 solitary 330 2.55 156 13 11 ectatomma edentatum 2.758 1.048 0.302 1.612 3.950 solitary 480 3.60 280 4 12 odontomachus haematodus 3.221 1.017 0.262 1.527 2.924 solitary 1800 3.70 123 6 12 ectatomma opaciventre 4.471 1.828 0.537 2.568 7.759 solitary 5000 8.42 307 3 13 odontomachus haematodus 3.144 0.968 0.275 1.557 3.069 solitary 3240 2.42 200 1 13 ectatomma brunneum 3.581 0.905 0.359 1.137 3.510 solitary 4020 3.02 123 24 14 pheidole capillata 0.807 0.451 0.099 0.702 1.670 group 5640 2.14 1601 3 15 blepharidatta conopsi 0.994 0.330 0.073 0.639 1.450 group 5640 0.34 493 1 15 solenopsis tridens 0.930 0.276 0.072 0.656 1.390 group 6180 0.67 2100 1 sociobiology 69(3): e8308 (september, 2022) 11 table s2. nests sampled in each area, species sampled (only ectatomma edentatum), removal performed (first, second, and last), and removal time (s). nest species time_r n_removal a4n1 ectatomma edentatum 373 first a4n1 ectatomma edentatum 178 second a4n1 ectatomma edentatum 149 last a4n2 ectatomma edentatum 63 first a4n2 ectatomma edentatum 40 second a4n2 ectatomma edentatum 41 last a5n1 ectatomma edentatum 161 first a5n1 ectatomma edentatum 187 second a5n1 ectatomma edentatum 94 last a6n1 ectatomma edentatum 87 first a6n1 ectatomma edentatum 80 second a6n1 ectatomma edentatum 58 last a7n1 ectatomma edentatum 66 first a7n1 ectatomma edentatum 57 second a7n1 ectatomma edentatum 71 last a7n2 ectatomma edentatum 196 first a7n2 ectatomma edentatum 140 second a7n2 ectatomma edentatum 97 last a7n4 ectatomma edentatum 180 first a7n4 ectatomma edentatum 162 second a7n4 ectatomma edentatum 112 last a8n1 ectatomma edentatum 106 first a8n1 ectatomma edentatum 69 second a8n1 ectatomma edentatum 77 last a9n1 ectatomma edentatum 242 first a9n1 ectatomma edentatum 296 second a9n1 ectatomma edentatum 240 last a9n2 ectatomma edentatum 206 first a9n2 ectatomma edentatum 470 second a9n2 ectatomma edentatum 190 last a10n1 ectatomma edentatum 120 first a10n1 ectatomma edentatum 195 second a10n1 ectatomma edentatum 130 last a10n2 ectatomma edentatum 442 first a10n2 ectatomma edentatum 230 second a10n2 ectatomma edentatum 138 last _hlk104388684 doi: 10.13102/sociobiology.v63i2.976sociobiology 63(2): 792-799 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 is the initial nest depth adapted to favorable conditions for the incipient colony in leafcutting ants? introduction nest foundation in atta sexdens is claustral, where the queen uses body reserves for self-preservation and to cultivate the symbiotic fungus and its brood until the emergence of the first workers (autuori, 1940). moreover, the nest excavation by the queen requires significant energy investment due to the excavation hours and excavated soil volume (camargo et al., 2011). however, the ideal depth of the initial chamber and how natural selection acts on the queens are still unknown issues. one can hypothesize that a shallow initial chamber requires the queen’s less time and energy, but the queen, the symbiotic fungus and her brood would be exposed to environmental variables such as temperature and humidity abstract the nest dug by the leaf-cutter ant queen of the genus atta is a vertical tunnel interconnected to a small chamber which holds its offspring and the symbiotic fungus. the depth of the initial chamber of the atta sexdens rubropilosa ranges from 10 to 30 cm below the soil surface. with this information, we asked whether the ideal initial nest depth is adapted to favorable conditions for the initial colony. we hypothesized this depth can provide a minimum temperature range with almost constant temperature, leading to the development of symbiotic fungus and brood yet to emerge. to test this hypothesis, laboratory experiments were carried out and the soil temperature was measured at different depths. the colony development at different temperatures was studied in the laboratory and the brood production (number of eggs, larvae, pupae and adults) was measured until the first workers emergency. additionally, lipid content and the survival of queens at different temperatures were determined. our results show a suitable temperature range (ranging from 24.82±3.14°c to 24.11±1.30°c) at a depth of 5 to 25 cm from the ground, an ideal brood development at temperatures of 24 and 28 °c, and consequently a reduction in lipid content of the queens at high temperatures, without affecting their survival in the trial period. these results indicate that the depth of the initial chamber excavated by the queen is suitable for the success of the incipient colony. sociobiology an international journal on social insects rs camargo1, lc forti1, cao matos2, ncaldato1, os fonseca1 article history edited by inge armbrecht, university del valle, colombia received 05 january 2016 initial acceptance 07 april 2016 final acceptance 06 june 2016 publication date 15 july 2016 keywords atta sexdens, nest, leaf-cutting ants, social insects, claustral foundation. corresponding author roberto da silva camargo laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas/unesp caixa postal 237, 18603-970, botucatu-sp, brazil email: camargobotucatu@yahoo.com.br (bollazzi et al., 2008; lapointe et al., 1998). in contrast, a deep initial chamber could provide a more stable microclimate, but would require more time and energy spent with additional exposure to predators during its construction (autuori, 1940). in addition, the survival queen and colony development can be affected in several ways: first, predation is intense at the nuptial flight and nest excavation, the main agents is birds, frogs, lizards , armadillos and insects, such as canthon spp. (coleoptera, scarabaeidae) and ants (solenopsis, paratrechina and nonamyrmex) (autuori, 1950; erthal & tonhasca, 2001; forti et al., 2012); second, excessive nest digging and greater depths require the queen’s substantial digging effort, which directly affects her survival at this initial stage of the colony (camargo et al., 2011); third, entomopathogenic or parasitic 1 faculdade de ciências agronômicas/unesp, botucatu-sp, brazil 2 campus experimental de itapeva/unesp, itapeva-sp, brazil research article ants sociobiology 63(2): 792-799 (june, 2016) 793 fungi of symbiotic fungus may infect the founding colony (bento et al., 1991; rodrigues et al., 2010; marti et al., 2015; currie et al., 1999). probably, there is an adequate depth for the initial chamber, as there are various nest depths among the atta species. for example, initial chamber depths range from 7.5 to 12 cm in a. colombica (weber, 1972), 6.5 to 13 cm in a. cephalotes (weber, 1937), 15 to 25 cm in a. texana (moser, 1967), 10 to 30 cm in a. sexdens rubropilosa (camargo & forti, 2013), 10 to 15 cm in a bisphaerica (cardoso et al., 2014), 11 to 34 cm in a capiguara (mariconi, 1974) and 9 to 15 cm in a. insulares (bruner & valdes barry, 1949). it is known that digging behavior is affected by temperature and soil moisture, reflecting the nesting habits of species. the initial chamber depth significantly differed between the ant species, with a. bisphaerica showing deeper chambers than a. sexdens rubropilosa (cardoso et al., 2014). atta bisphaerica presents nests in full sun and forages predominantly grasses, while atta sexdens rubropilosa presents nests in shaded places and forages dicots (mariconi, 1970; fowler et al., 1986; nagamoto et al., 2009), therefore both species differ in nesting habits and foraging strategies. probably, the differences in nest depth between species are correlated to soil temperature, because shading alters soil temperature regimes by locally diminishing soil temperature (rosenberg et al., 1983). in this perspective, it is reasonable that nest exposed in grassland should have deeper fungus chamber than nest under shade of trees or inside the woods, given that soil temperature is negatively correlated with soil depth (rosenberg et al., 1983). for leaf cutting ants, soil moisture and temperature act together: (i) bollazzi et al. (2008) verified that workers’ thermopreferences lead to the construction of superficial nests in cold soils, and subterranean ones in hot soils; and (ii) pielström and roces (2014) verified that soil moisture also varies according to soil depth, and demonstrably affects the digging behavior of leaf cutting ants. so, is the ideal depth of the initial nest adapted to favorable conditions for the incipient colony? we hypothesized that this depth would provide a minimum temperature range with almost constant temperatures, conducive to the development of the symbiotic fungus and the brood yet to emerge. to test this hypothesis, we used brood production as a measure of the colony development at different artificially controlled temperatures in the laboratory. material and methods collection of queens of atta sexdens rubropilosa the collection was carried out during the nuptial flight that occurred on october 5, 2014, by capturing queens that were founding their colonies at the lageado experimental farm, faculdade de ciências agronômicas – universidade estadual paulista–botucatu, são paulo state, brazil. the newly collected queens were stored in plastic containers, 11 cm in diameter and 8 cm in height, containing 1 cm of plaster at the bottom to maintain humidity. the queens were transported to the laboratory of social-pest insects fca/ unesp – botucatu, where all experiments were performed. . the queens were allocated in laboratory (bod incubators) at different artificially controlled temperatures. field experiment soil temperature and initial nest depths the temperature was measured at different soil depths (dark red latosol): 0 cm (ground surface) 5 cm, 25 cm, 45 cm, 150 cm. the temperatures were measured using a thermal sensor with data logger testo (model 175-t2), with daily readings every five minutes using the thermal sensor in october (n=7801), november (n=8001), december (n=8001), january (n=6311), february (n=2000) and march (n=2000). the sensors were buried at different soil depths, as mentioned before. the readings were discontinued due to some days of excessive rain which could compromise the measurement accuracy. at the end of march, 89 early nests were excavated in order to measure the depth (cm) of the initial chamber (fig 2). additionally, for better view of its structure some were cement molded (two nest), according to the methodology used by moreira et al. 2004. for molding, a mixture of cement and water at a proportion of 2:10 (kg/l) was pumped into the holes of each nest. the nests were excavated one week after they had been filled with cement using small and manual tools to avoid destruction of the nests. after excavation, the nests were photographed (fig 2). laboratory experiment: temperature effect on the establishment of initial colonies we tested the following hypotheses: the observed depth would provide a small temperature range with almost constant temperature which leads to successful development of brood. the rationale of the experimental approach was to compare the influence of temperature in colony development, measured by offspring production over time (numbers of eggs, larvae, pupae and adults raised until the first workers emerge) of queens at different temperatures. to test this hypothesis, we used brood production as a measure of the colony development at different artificially controlled temperatures in the laboratory (bod incubators, eletrolab). the brood production (number of eggs, larvae, pupae and adult) was carefully examined and recorded weekly using a stereoscopic microscope (nikon, smz 1000) according to camargo et al. (2011). the experimental groups were divided as follows: 1development at 15°c: 25 queens remained under the effect of low temperature (15°c) until the emergence of the first workers. 2development at 20°c: 25 queens remained under the effect of medium temperature (20°c) until the emergence of the first workers. rs camargo et al. – favorable conditions for the incipient colony in leaf-cutting ants794 3development at 24°c: queens remained under the effect optimal temperature (24°c) until the emergence of the first workers. 4development at 28°c: 25 queens remained under the effect of high temperature (28°c) until the emergence of the first workers. temperatures of 15°c, 20°c, 24°c and 28°c were chosen due to temperature preference for the allocation of brood and fungus by worker, according to bollazzi and roces (2002), powell and stradling (1986). the mortality of queens was also measured weekly. lipid determination the queens were immersed in organic solvent (pentane) until they reached a constant weight (cook et al., 2010). the procedure was as follows: fresh weight was individually determined; queens were dried for 24 hours at 50°c and their dry weight was determined; lipids were extracted with pentane for 24 hours and then dried and weighed on an analytical balance. the procedure was repeated for 72 hours of extraction. values are expressed as a percentage (%) of the dry mass of the queen. statistical analysis the external and internal temperature (soil) was compared by the f test at each depth, assuming there is an equal temperature variation between the internal (soil inside) and external temperature (soil surface) (h0), with 5% significance. the total production (egg, larva, pupa and adult) was subjected to multiple comparison test of variances (ryan, 1960) between the temperatures 15°c, 20°c, 24°c and 28°c. after that, a generalized estimating equations model (generalized linear model for dependent data) was used in the calculation using an iterative process proposed by liang and zeger (1986) that was fitted using a poison probability model that has the same mean-variance relationship. a correlation structure that minimized pan’s qic was chosen. qic is a statistic that generalizes aic to generalized estimating equations, according to pan (2001), and comes in two versions. one version is used for selecting a correlation structure and the second version is used for choosing models, all of which were fitted with the same correlation structure. a correlation structure ar-1 that includes time dependence and reflects the manner in which the data were collected was selected for eggs and larvae. an independent correlation structure was selected for pupae. the model considering the variables time and temperature as discrete was selected from three models. the cox proportional hazards regression model was fitted to the queens’ survival at different temperatures. in addition, a linear regression was fitted between variations in lipid content of queens and temperature. statistical analyses and graphs were processed by r 2.9.0 for windows. results the temperature variance of the measured temperatures at different soil depths did not change (table 1). this result corroborates the hypothesis that the temperature is relatively constant at different soil depths when compared to the external temperature (table 1). the depth of the initial chamber was 17.5±5.5 (fig 2), in a temperature range varying from 24.82±3.14°c (5 cm) to 24.11±1.30°c (25 cm) considering the averages of every month (fig 1). as for the months, october had the lowest temperature at all depths, but over the following months it tended to increase and remain constant (fig 1). fig 1. mean temperature (°c) of soil and nest depth (cm± sd) of initial nest of atta sexdens for six months after nuptial flight. in the laboratory, the queens showed differences in offspring production under different temperatures. at 15°c, no queen produced offspring, there was only underdeveloped symbiotic fungus cultivation. at 20°c, the queens produced eggs (396±98.73) and larvae (15.85±17.51), adults during the six weeks of observation. at 24°c the queens produced eggs (309±67.07), larvae (173.12±78.38), pupae (51.44±31.88) and adults (1.40±0.55). and at 28°c the queens produced eggs (238.40±80.47), larvae (184.00±79.30), pupae (81.24±51.67) and adults (36.92±29.92). the variance of the total production (egg, larva, pupa and adult) was 0.00029for 15°c, 0.00096 for 20°c, 0.00313 for 24°c and 0.00892 for 28°c. the multiple variance comparisons showed no differences between the 15°c and 20°c groups (p-value f7,23 = 0.1117), but there were differences ranging between 20°c and 24°c (p-value f23,21 = 0.006973), 20°c and 28°c (p-value f23,19 = 1.863 10-6) and finally between 24°c and 28°c (p-value f21,19 = 0.02244) (table 2). the glm showed significant effects regarding the number of eggs produced during the weeks, in the 2nd week the average was 0.41 times higher than the average of the 1st week (p>|z| = 4.767 10-18). in larvae, in the 6th week the average was 0.48 times lower than the average of the 3rd week (p>|z| = 2,361 10-6). there was no significant effect for pupae and was not estimated for adults. sociobiology 63(2): 792-799 (june, 2016) 795 the average lipid content was 32.58±2.98% for queens at 15°c, 30.90%±2.94 at 20°c, 28.84±3.41% at 24°c and 26.78±5.70% at 28°c. linear regression analysis of the lipid content according to temperature was significant (p-value f1,74 < 0.0001), but due to the high variation, the quality of the model fit is low (r2 adjusted = 0.1836) (fig 3). depth thermal sensor october november december january february march 5 cm in 21.95±3.11 σ2=9.67 24.98±0.95 σ2=9.04 24.80±2.15 σ2=4.63 26.77±2.19 σ2=4.80 23.65±2.29 σ2=5.90 24.44±1.47 σ2=2.15 out 20.01±3.90 σ2=15.21 24.55±1.29 σ2=16.61 22.95±2.95 σ2=16.61 24.91±3.40 σ2=11.59 21.15±3.91 σ2=15.26 23.29±4.26 σ2=4.26 f test f=1.572 p≥0.05 f=1.8386 p≥0.05 f=1.88 p≥0.05 f=2.42 p≥0.05 f=2.88 p≥0.05 f=8,4323 p≥0.05 25 cm in 21.51±1.37 σ2=18.88 25.38±1.19 σ2=14.13 24.54±1.06 σ2=11.22 24.21±1.56 σ2=2.43 24.16±0.82 σ2=0.67 25.11±0.65 σ2=0.42 out 20.74±1.50 σ2=22.40 24.68±1.25 σ2=15.59 23.70±1.20 σ2=14.47 22.04±3.64 σ2=13.26 24.45±3.18 σ2=10.12 23.91±3.22 σ2=10.33 f test f=1.1867 p≥0.05 f=1.1031 p≥0.05 f=1.29 p≥0.05 f=5.46 p≥0.05 f=15.08 p≥0.05 f=24.67 p≥0.05 45 cm in 21.29±0.81 σ2=6.70 24.98±0.95 σ2=9.03 24.25±0.76 σ2=5.81 26.22±0.72 σ2=5.14 24.88±1.21 σ2=1.47 24.81±1.06 σ2=1.13 out 20.64±1.55 σ2=24.0 24.55±1.28 σ2=16.61 23.75±1.14 σ2=12.92 25.37±2.08 σ2=43.57 20.96±3.98 σ2=15.87 24.47±3.72 σ2=13.80 f test f=3.58 p≥0.05 f=1.8386 p≥0.05 f=2.22 p≥0.05 f=8.48 p≥0.05 f=10.77 p≥0.05 f=12.19 p≥0.05 150 cm in 21.29±0.21 σ2=0.05 23.36±0.77 σ2=0.59 24.21±0.24 σ2=0.05 25.53±0.10 σ2=0.01 24.93±0.14 σ2=0.018 25.16±0.05 σ2=0.002 out 20.61±1.67 σ2=2.80 24.59±1.30 σ2=1.71 23.84±1.11 σ2=1.23 21.65±3.74 σ2=14.01 23.02±3.16 σ2=10.01 23.78±3.13 σ2=9,85 f test f=6.179 p≥0.05 f=2.8692 p≥0.05 f=2.11 p≥0.05 f=1389.8 p≥0.05 f=548.19 p≥0.05 f=4311.59 p≥0.05 in – thermal sensor in the soil out – thermal sensor at soil surface table 1. summary statistics for soil temperature (mean ±sd) during 6 months. fig 2. initial nest of atta sexdens: a) excavated at 4 months after nest foundation; b) initial nest molded with cement at four months after nest foundation. group k number of groups n nominal level nominal level/2 significance 15-28 4 4 0,00833 0,00417 ** 15-24 3 4 0,0125 0,00625 ** 20-28 3 4 0,0125 0,00625 ** 15-20 2 4 0,025 0,0125 n.s. 20-24 2 4 0,025 0,0125 ** 24-28 2 4 0,025 0,0125 ** n.s. no significance table 2. summary of multiple comparisons of variances at 5% level experimentwise. in relation to queen survival, 32% of queens survived (17 deaths) at 15°c, 92% of queens survived (2 deaths) at 20°c, 88% of queens survived (3 deaths) at 24°c and 80% of queens survived (5 deaths) at 28°c. the cox proportional hazards regression model fitted to the data shows no difference for queens’ survival in relation to the different temperatures (p>|z|=0.729). discussion the depth that the queen of atta sexdens rubrupilosa digs her initial chamber promotes a minimum temperature range, with a temperature that is favorable to the development rs camargo et al. – favorable conditions for the incipient colony in leaf-cutting ants796 of symbiotic fungus and her brood. this hypothesis was confirmed by the results obtained which showed that the depth of the initial chamber was 17.5±5.5 in a temperature range varying from 24.82±3.14°c (5 cm) to 24.11±1.30°c (25 cm) (table 1, fig 1). combined with the laboratory results, the temperatures ranging from 24 to 28°c allowed the queens to produce in large numbers and rapidly in the 6th and in the 4th week there were adults (fig 3). the queen determines the initial chamber depth, this is a regulated process involving the use of internal references: queens excavated their tunnels either until a particular depth was reached or for some predetermined length of time according to fröhle and roces 2012. for example, atta sexdens queens dug around 11 to 30 cm in the soil to build their chamber (camargo & forti, 2013) to a depth for suitable temperature for fungus garden and brood. some studies have recorded fungus temperatures in field colonies: 25–28°c for atta sexdens (stahel and geijskes1940; eidmann 1935), 27.5 °c for atta vollenweideri (kleineidam & roces, 2000), and around 27°c for a. heyeri (bollazzi & roces, 2002). for brood, bollazzi and roces (2002) found that a. lundi workers avoid, while digging, soil temperatures that are unsuitable for brood development, and prefer those temperatures that are known to maximize fungal growth (bollazzi & roces, 2002). thus, our results show that soil temperature at depths of 5 to 25 cm in the months of october to march provide ideal conditions for the fungus garden and brood (fig 1). the number of eggs produced during the 2nd week was 0.41 times greater than the average of the 1st week, probably due to the cumulative effect on the number of eggs (incubation period greater than seven days) and the absence of larval oophagy (autuori, 1942, camargo et al., 2007). in larvae, in the 6th week the average was 0.48 times lower than the average of the 3rd week. this result is related to the larvae becoming pupae during the observation period, thus the number decreased. furthermore, there was no significant effect on pupae and adults, probably due to the low number found in the treatments. in general, comparing the development of initial colonies of atta with data from the literature (autuori, 1942; pereira-da-silva, 1979), differences were found in the fig 3. eggs, larvae, pupae and adults produced by the queens in different temperatures (15°c, 20°c, 24°c e 28°c) during six weeks. sociobiology 63(2): 792-799 (june, 2016) 797 incubation, larval and pupal periods. for atta sexdens, the incubation period lasted 25 days, the larval period 22 days and pupal period 10 days (autuori, 1942). camargo et al. (2011) obtained similar results from the same leaf-cutting ant species, incubation period lasted 30 days, larval period 25 days and pupal period 15 days. in atta capiguara, in the sampled periods, the eggs were most prevalent at 1 to 18 days, larvae at 21–38 days, pupae at 39–55 and adults at 58–67 days (pereira-da-silva, 1979). when comparing the quantity of brood produced with the findings of autuori (1942) and pereira-da-silva (1979), a pattern is verified in the production of eggs, larvae, pupae and adults, in other words, generally showing high variability (fig 3). the lipid content decreased in relation to increasing temperature (fig 4), or high temperatures (24 °c and 28°c) producing more offspring than at low temperatures (15°c and 20°c), thereby consuming more lipids. the high lipid content is extremely important to the queens of atta sexdens, responsible for maintaining a high brood production rate for several weeks (camargo et al., 2011). according to fowler et al. (1986), egg production by the queen during the 3 or 4 months of the colony lifespan is correlated with the activity cycle of the corpora allata. the corpora allata are responsible 3 months of atta colony life have many obstacles for queens and their incipient colonies (fowler et al., 1986; marti et al., 2015). the survival of the queen can be reduced in several ways: predation (erthal and tonhasca, 2001; forti et al., 2012), nest excavation effort (camargo et al., 2011) and entomopathogenic or parasitic fungi of symbiotic fungus (bento et al., 1991; rodrigues et al., 2010; marti et al., 2015; currie et al., 1999). in summary, our results show an appropriate temperature range ( from 24.82 ±3.14°c to 24.11±1.30°c) at a depth of 5 to 25 cm from the ground, good brood development at temperatures of 24 and 28 °c, thus reducing the lipid content of the queens at high temperatures without affecting their survival in the trial period. these results indicate that the depth of the initial chamber excavated by the queen is suitable for the success of the incipient colony. acknowledgements we are grateful to fundação de amparo à pesquisa do estado de são paulo (fapesp) for the financial support and stipends to the authors [grant numbers 2007/04010-0 and 2007/07091-0]. r.s. camargo thanks coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the postdoctoral fellowship. lcf thanks conselho nacional de desenvolvimento cientifico e tecnológico for the research assistance (301917/2009-4). references autuori m (1940). algumas observações sobre formigas cultivadoras de fungo (hymenoptera: formicidae). revista de entomologia, 11 (1-2): 215-226. autuori m (1942). contribuição para o conhecimento da saúva (hymenoptera: formicidae) ii o sauveiro inicial (atta sexdens rubropilosa forel, 1908). arquivos do instituto biológico, 13: 67-86. autuori m (1950). contribuição para o conhecimento da saúva (atta spp.). v. número de formas aladas e redução dos sauveiros iniciais. arquivos do instituto biológico, 19: 325-331. barker jf (1978). neuroendocrine regulation of oocyte maturation in the imported fire ant solenopsis invicta. general and comparative endocrinology, 35: 234–237. bento, j.m.s., della lucia, t.m.c., muchovej, r.m.c. & vilela, e.f. 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(1983). microclimate-the biological environment. new york: wiley. ryan ta (1960). significance tests for multiple comparison of proportion, variance, and other statistics. psychological bulletin, 57: 318-328 stahel g, geijskes dc (1940). observations about temperature and moisture in atta nests. revista de entomologia, 11: 766-775. weber na (1937). the biology of the fungus-growing ants – part ii. nesting habits of the bachac (atta cephalotes l.). australian tropical, 14: 223-226. weber na (1972). gardening ants – the attines. pa: the american philosophical society, philadelphia. doi: 10.13102/sociobiology.v63i2.936sociobiology 63(2): 770-776 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 toxicity of plant extracts from bahia, brazil, to atta sexdens sexdens (hymenoptera: formicidae) workers introduction ants of the atta and acromyrmex genera (formicidae: myrmicinae: attini) are the true leaf cutting-ants with their species utilizing fresh plant parts, mainly leaves, to cultivating fungus of the leucoagaricus and leucocoprinus genera (agaricaceae: leucocoprinae) (della lucia et al., 2014). the leaf cutting-ants are one of the most significant pests in neotropical america damaging almost all cultivated plants and causing losses to agriculture, forest and pasture plants (zanetti et al., 2000a; zanuncio et al., 2002). leaf cutting-ants use green leaves what justifies their importance by reducing survival, growth and reproduction of plant of economic value (zanetti et al., 2000b; zanetti et al., 2014). abstract ants of the atta and acromyrmex genera (formicidae: myrmicinae: attini) are the true leaf cutting-ants with species of economic importance in america neotropical and mainly controlled by toxic baits. there are few active ingredients for use in baits, being necessary studies to indicate molecules with insecticide potential. the aim of this study was to evaluate the toxicity of aspidosperma spruceanum benth ex. mull arg. (leaf and bark), casearia arborea (rich.) urb. (leaf and branch), casearia sylvestris sw. (leaf and bark), erythroxylum affine a.st.-hil. (leaf and branch), esenbeckia grandiflora mart. (leaf and bark), ocotea brasiliensis coe-teix (bark and branch), simarouba amara aubl. (bark), tabernaemontana bracteolaris mart. ex müll.arg. (leaf, bark and branch) and zanthoxylum rhoifolium lam. (leaf and branch) extracts to workers of atta sexdens sexdens l. (hymenoptera formicidae). the contact and ingestion toxicity of all extracts to this ant by was evaluated by topical application and addition in their diet, respectively. data of contact application were submitted to analysis of variance and tukey test while those from ingestion were compared by survival curves using the statistical test ¨log rank¨. through contact, the leaf and branch extracts of z. rhoifolium and of that of bark of s. amara were the most toxic ones. through ingestion, four extracts were toxic and showed delayed action. the extract of z. rhoifolium branches presented the slowest action (s 50 = 10 days). this characteristic is crucial for toxic baits. the z. rhoifolium leaf and branch extracts were the only ones with contact and ingestion toxicity to a. sexdens sexdens workers. sociobiology an international journal on social insects mcar gomes1, vf paula1, aa moreira2, ma castellani2, gel macedo1 article history edited by evandro nascimento silva, uefs, brazil received 16 october 2015 initial acceptance 21 february 2016 final acceptance 11 june 2016 publication date 15 july 2016 keywords atta sexdens sexdens, bioassays, insecticidal activity, zanthoxylum rhoifolium. corresponding author maria aparecida castellani department of plant and animal science state university of southwest bahia box 95, 45.083-900 vitória da conquista-ba, brazil e-mail: castellani@uesb.edu.br leaf cutting-ants are the main pests in cultivated forests of the pinus and eucalyptus genera (zanetti et al., 2014). damages by leaf cutting-ants is common in forest commercial cultivation in 2005 in 3.4 million of hectares planted with eucalyptus, 1.8 million with pinus and 326 thousand with other plant species (pereira & santos, 2008). the leaf cutting-ants can be controlled with different methods, but toxic baits are the most practical and efficient one (laranjeiro et al., 1995; zanuncio et al., 2000). on the other hand, thermal fogging with plant extracts could be an alternative to these baits, but the costs of equipments with this method is much higher compared to toxic bait application (zanetti et al., 2014). the active ingredient must be attractive to ants even distant from the nest to assure the efficiency of toxic baits. 1 universidade estadual do sudoeste da bahia (uesb), jequié-ba, brazil 2 universidade estadual do sudoeste da bahia (uesb), vitória da conquista-ba, brazil research article ants sociobiology 63(2): 770-776 (june, 2016) 771 initial rejection of these baits cannot occur and its toxic compound should have delayed action at the right time to completely contaminated the ant colony besides having low toxicity to nontarget organisms (nagamoto et al., 2004; verza et al., 2006). the sulfuramids (n-ethyl perfluoroctane sulfonamide) and fipronil are the most used active ingredients in ant baits (zanuncio et al., 2003; della lucia et al., 2014). the sulfuramid, categorized in annex b of the stockholm convention on persistent organic pollutants (pops) as an organic pollutant (united nations treaty collection, 2009), is the most used active ingredient in baits to control leaf cutting-ants, what makes urgent searching for active ingredients to replace it. the leaf-cutting ant [(atta sexdens forel) (hymenoptera: formicidae)] has attracted the attention of researchers (zanetti et al., 2003) and studies have been developed to searching for plants with toxic substances to this pest (peñaflor et al., 2009; gouvêa et al., 2010). the toxicity is due to secondary metabolites in plant that can be toxic to ants, to its fungus (morais et al., 2015) or to both. oil of ricinus communis l. and jatropha curcas l. seeds with different concentrations were toxic, through ingestion and topical application to a. sexdens, in laboratory tests, thus, suggesting the toxicity of these oils to this ant (alonso & santos, 2013). the search for alternatives that are effective to control leaf cutting-ants is important. the objective of this study was to evaluate the toxicity of extracts of several plant species collected in the bahia state, brazil, to a. sexdens sexdens workers, through topical application and ingestion, in laboratory conditions. material and methods plant species were collected in june 28th of 2012, in a forest fragment in brejo novo farm (13º56’41”s and 40º06’33.9”w) between 617 m and 755 m of altitude at 9 km from jequié, bahia sate, brazil. the plant material was dried in tecnal drying oven (te 394\2 model) at 40 °c for 48 h and submitted to cold maceration with methanol. extracts from different plant parts of the following species represented by their respective abbreviations were prepared: leaf (efas) and branch (egas) of aspidosperma spruceanum benth. ex müll. arg.; leaf (efca) and branch (egca) of casearia arborea (rich.) urb.; leaf (efcs) and bark (eccs) of casearia sylvestris sw.; leaf (efea) and branch (egea) of erythroxylum affine a. st.-hil.; leaf (efeg) and bark (eceg) of esenbeckia grandiflora mart.; bark (ecob) and branch (egob) of ocotea brasiliensis coe-teix; bark (ecsa) of simarouba amara aubl.; leaf (eftb), bark (ectb) and branch (egtb) of tabernaemontana bracteolaris mart. ex müll. arg.; and leaf (efzr), branch (egzr) and root (erzr) of zanthoxylum rhoifolium lam. the extract solution was filtered and concentrated under vacuum, in rotary evaporator (fisatom, 801 model), at 50 °c. the masses of dry plant materials and their respective extracts and their yields were obtained (table 1). the biological tests were conducted using a. sexdens sexdens workers collected from several nests in the myrmecology laboratory of the southwestern bahia state university. foraging workers of the similar size and width of head capsule between 1.7 and 3.0 mm were selected. family species plant abbr. mat. (g) ext. (g) yield (%) salicaceae casearia arborea leaf efca 91.45 29.91 33 branch egca 135.79 11.58 9 casearia sylvestris leaf efcs 91.08 34.77 38 bark eccs 147.4 19.37 13 erythroxylaceae erythroxylum affine leaf efea 56.50 13.23 23 lauraceae ocotea brasiliensis branch egob 704.75 30.41 4 bark ecob 388.48 55.14 14 apocynaceae tabernaemontana bracteolaris leaf eftb 81.38 26.54 33 bark ectb 281.07 32.99 12 aspidosperma spruceanum leaf efas 54.76 19.16 35 branch egas 152.57 8.35 5 simaroubaceae simarouba amara bark ecsa 362.27 13.07 4 rutaceae esenbeckia grandiflora leaf efeg 79.57 23.45 29 bark eceg 138.78 18.98 14 zanthoxylum rhoifolium leaf efzr 117.04 22.16 19 branch egzr 179.78 19.75 11 root erzr 122.23 14.58 12 table 1. family, species, plant parts (plant), abbreviation (abbr.), mass of dry material (mat.), mass of extract (ext.) and yield of plant species and extracts used in bioassays with atta sexdens sexdens (hymenoptera: formicidae) workers. mcar gomes – toxicity of plant extracts to atta sexdens sexdens772 the contact toxicity test (araújo et al., 2008) was conducted in a completely randomized design with 21 treatments (19 extracts and two controls) with three replications and each parcel having 10 ants. the extracts (table 1) were diluted in ethanol at the concentration of 1.0 mg.ml-1. each leaf cutting-ant worker was treated topically with 1.0 µl of this solution on its pronotum using a dosing micro syringe of 10 µl (hamilton, 701n model). two controls were prepared: one without application and another with the application of 1.0 µl of solvent (ethanol). the ants were conditioned in petri dishes per treatment with a cotton ball soaked in distilled water. the ant mortality was evaluated after 24, 48 and 72 of treatment application. in the ingestion toxicity test the ants were fed on a solid diet composed of (g.l-1): glucose (50), bacteriological peptone (10), yeast extract (1.0) and agar (10) dissolved in distilled water and autoclaved at 120 °c for 15 minutes (bueno et al., 1997). the experimental diets, except the controls, were obtained by adding the extracts efeg, eceg, efea, efzr, egzr, efca, eccs, ecsa, efas (concentration of 0.2 mg.ml -1) to the diet still hot, immediately after its removal from autoclave. the liquid was poured in petri dishes previously identified and kept under refrigeration. the ant mortality was evaluated in a daily basis during 25 days. the petri dishes were covered with paper filter, previously damped with distilled water. the diet was replaced daily when 0.4 g of the artificial diet was put per petri dish. the experimental design was completely randomized with 10 treatments, nine with the extracts and one control (diet without extract) with five replications using 10 ant workers each one. in both tests, the petri dishes (90 mm diameter) were kept at a temperature of 25 ± 1 °c and relative humidity from 70 to 80%. the data from the topical application test were submitted to analysis of variance and the averages compared by the tukey test (p< 0.05) using the sas institute program (2002). the data from ingestion tests were graphically analyzed comparing the survival curves of each treatment with that of the control by the statistical test “log-rank” using the prisma 6.0 program (graphpad software). for each treatment, the average survival period of ants (s50) was determined considering the day that 50% of them were still alive. results and discussion the topical application of the extracts efzr, egzr, ecsa, egas and efca caused higher mortality of ants than the controls. ants treated with ecsa, efca, egzr, efzr and egas presented cumulative mortality between 33% and 37% (table 2). this may be due to secondary metabolites toxics to treatment mortality (%) 1º day 2º day 2ºday (cumulative mortality) 3º day 3ºday (cumulative mortality) control with solvent 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00a control without solvent 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00a eftb 0.00±0.00 6.67±9.43 6.67±9.43 15.00±10.80 21.67±16.41a efeg 3.33±4.71 0.00±0.00 3.33±4.71 3.33±4.71 6.67±4.71 a egtb 0.00±0.00 3.33±4.71 3.33±4.71 3.33±4.71 6.67±4.71a egzr 16.67±9.73 24.34±9.55 41.01±15.38 0.00±0.00 41.01±13.21 b erzr 0.00±0.00 6.67±4.71 6.67±4.71 0.00±0.00 6.67±4.71 a eceg 6.67±9.43 4.17±5.89 10.83±15.32 3.33±4.71 14.17±12.47 a eccs 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±4.71 a efca 10.00±8.16 8.33±11.79 18.33±19.29 19.26±7.93 37.59±13.18 b ecsa 6.67±4.71 14.44±5.52 21.11±9.07 16.93±12.25 38.04±13.33 b efzr 6.67±9.43 21.67±8.50 28.33±14.34 11.11±15.71 39.44±5.47 b egca 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 a efcs 0.00±0.00 0.00±0.00 0.00±0.00 3.33±4.71 3.00±4.71 a ectb 0.00±0.00 0.00±0.00 0.00±0.00 3.33±4.71 3.00±4.71 a ecob 0.00±0.00 3.33±4.71 3.33±4.71 7.04±5.00 10.37±8.71 a egob 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±.4.71 a egea 0.00±0.00 0.00±0.00 0.00±0.00 6.67±4.71 6.67±4.71 a efea 6.67±4.71 3.70±5.24 10.37±8.62 0.00±0.00 10.74±8.17 a efas 3.33±4.71 7.41±10.48 10.74±15.19 0.00±0.00 10.74±14.16 a egas 3.33±4.71 14.44±13.97 17.78±18.53 21.48±18.17 39.26±12.74 b table 2. mortality (%) of atta sexdens sexdens (hymenoptera: formicidae) workers (mean and standard deviation) treated with plant extracts at concentration of 1 mg.ml-1 through topical application. different letters after the mortality value on the 3rd day, show significant difference in relation to the control (tukey at 5%). sociobiology 63(2): 770-776 (june, 2016) 773 a. sexdens sexdens and that these plants need further studies in field with thermal fogging. besides, secondary metabolites are usually found at low concentrations in extracts and their isolation is necessary to prove their respective activities. the quassinoids, the most active substances used in traditional medicine, is the main chemical components of s. amara and present inhibitory effect (fiaschetti et al., 2011). simarouba versicolor was toxic to cutting-ants at concentrations of 2.0, 1.6 and 0.3 mg.ml1 (peñaflor et al., 2009). species of casearia genus present therapeutic properties and active substances as diterpenes, called casearins (bento et al., 2013). aspidosperma has species, including a. spruceanum, important sources of indole alkaloids with therapeutic properties (oliveira et al., 2009). insecticidal activity for c. arborea, s. amara and a. spruceanum has not been found, but plant extracts may be active against leaf cutting-ants. for instance, ruta graveolens l. and ageratum conyzoides l. extracts employed in traditional medicine, induced mortality of a. sexdens workers through topical application, at concentration of 1 mg.ml-1 (araújo et al., 2008). in tests through ingestion, the extracts of leaves and branches of z. rhoifolium (efzr and egzr), leaves of e. grandiflora (efeg) and of bark of c. sylvestris (eccs) caused higher mortality of ants than the control (figure 1). results for the efca, eceg, efas, ecsa and efea extracts were similar to those of the controls. the average survival period (s50) of a. sexdens sexdens workers per treatment varied from 8 to15 days (table 3). the average survival period (s50) of ants in the efeg, eccs, efzr and egzr treatments was seven, eight, eight and 10 days, respectively, evidencing their mortality after ingesting them. the values of s50 showed that these extracts had delayed action and therefore, they may be considered for future use in the management of cutting-ants. the delayed toxic action occurs when the mortality of ant workers is lower or equal to 15% up to the first day of evaluation, and higher or equal to 90% after the twentieth first day of evaluation (nagamoto et al., 2004). the accumulated mortality showed that extracts with difference from control had this characteristic. further tests need to be develop to implement these extracts in field. an extract, to be used in field in toxic bait, must present action preferably through ingestion, delayed toxic action, lethality at low concentrations, environmental safe odorless and non repellent (boaretto & forti, 1997). in ingestion tests, the toxicity of plants studied to leaf cutting-ants are not found in literature, but other plants such as ricinus communis l. (bigi et al., 2004), sesamum indicum l. (morini et al., 2005), cedrela fissilis vell (bueno et al., 2005), helietta puberalla re fr. (almeida et al., 2007), simarouba versicolor st. hil. (peñaflor et al., 2009), jatropha curcas l. and ricinus communis l. (alonso & santos, 2013) treatment accumulated mortality (%)/day s50 1º 2º 3º 6º 8º 10º 14º 17º 21º 25º control 2 4 8 14 16 23 42 56 75 87 15 a efzr 2 2 4 30 56 72 86 92 94 94 8 b efeg 4 6 22 51 57 71 87 89 94 96 7 b efca 2 4 12 24 30 38 68 78 85 85 12 a eccs 4 10 28 48 60 86 92 92 94 98 8 b eceg 2 2 8 23 31 42 66 83 85 87 11 a efas 6 6 8 14 22 51 69 78 82 90 11 a ecsa 0 2 4 14 22 32 50 62 84 94 15 a efea 10 12 12 16 22 52 80 90 92 92 11 a egzr 6 16 26 32 34 58 80 86 98 98 10 b table 3. mortality (%) and average survival period (s50) in days of atta sexdens sexdens (hymenoptera: formicidae) workers fed on diet containing several plant extracts at the concentration of 0.2 mg.ml-1 fig 1. survival curves for atta sexdens sexdens (hymenoptera: formicidae) workers in ingestion test with plant extracts. average survival period (s50) is displayed besides the names of extracts, in brackets. different lower case letters indicate difference in relation to control (data obtained with the application of “log-rank” test). s50= average survival. letters after the value of s50 showed significant differences according to the “log-rank test” (b = 0.01<p<0.05). mcar gomes – toxicity of plant extracts to atta sexdens sexdens774 also presented toxicity to leaf cutting-ants. the toxicity of c. sylvestris extracts to leaf cutting ants may be due to its diterpenes, class of substances compounds with biological activities, including antifungal effect (bento et al., 2013). the alkaloids (januário et al., 2009) and coumarins from e. grandiflora were toxic to aedes aegypti larvae (oliveira et al., 2005). leaf and branch extracts from z. rhoifolium presented contact and ingestion toxicity to leaf cutting ants. the biological activities of this plant were described (negi et al., 2011; medhi et al., 2013), but insecticidal activity was observed only for its fruit essential oil (prieto et al., 2011). alkaloids are the main active pharmacological constituents of this plant (jullian et al., 2006), with promising molecules for the management of leaf cutting-ants (bigi et al., 2004; almeida et al., 2007). the activity of the egzr extract through topical application and delayed toxic action through ingestion to leaf cutting-ant deserve studies to define possibilities of its use in toxic baits, due to its mode of action preferably through ingestion (boaretto & forti, 1997). however, as this extracts showed both contact toxicity and delayed toxic action by ingestion, future studies are needed to elucidate this issue. zanthoxylum rhoifolium and e. grandiflora, are from rutaceae family, which has other species toxic to leaf cuttingants as citrus sp. (fernandes et al., 2002), raulinoa echinata r.s. cowen (biavatti et al., 2005) and helietta puberula r. e. fr. (almeida et al., 2007). conclusions 1the leaves and branches of zanthoxylum rhoifolium, bark of simarouba amara, branches of aspidosperma spruceanum and leaves of casearia arborea extracts are toxic by contact to a. sexdens sexdens, justifying studies to elucidate its active ingredient. 2the branches and leaves of z. rhoifolium, leaves of esebenckia grandiflora and bark of casearia sylvestris extracts were toxic through ingestion to a. sexdens sexdens. 3 the leaves and branches extracts of z. rhoifolium are the only ones with contact and ingestion toxicity to a. sexdens sexdens. 4the extracts of rutaceae plants are highlighted with toxicity to a. sexdens sexdens and, therefore, toxicity of other species of this family to leaf-cutting should be studied. acknowledgements the authors want to thank the petrobras program of human resources training – pfrh-pb211 (fomentation to training of human resources in monitoring, bio-production and recovery of degraded areas in the state of bahia) and to the coordination for the improvement of higher education personnel (capes), for the financial support and by granting master’s scholarship to the author m.c.a.r. gomes. references almeida, r.n.a., peñaflor, m.f.g.v., simote, s.y., bueno, o.c., hebling, m.j.a., pagnocca, f.c., fernandes, j.b., vieira, p.c., silva, m.f.g.f. 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(2000). utilización del cebo mirex-s (sulfluramida 0.3%) para el control de atta sexdens rubropilosa forel (hymenoptera: formicidae) en área estratificada de hormigueros. revista colombiana de entomologia 26: 157-160. zanuncio, r., zanuncio, j.c., mayhe-nubes, a.j., medeiros, a.g.b., souza-silva, a. combate sistemático de formigascortadeiras, com iscas granuladas, em eucaliptais com cultivo mínimo. revista árvore, 27:387-392. 2003. zanuncio, j.c.; lopes, e.f.; zanetti, r.; pratissoli, d.; couto, l. (2002). spatial distribution of nests of the leaf cutting ant atta sexdens rubropilosa (hymenoptera: formicidae) in plantations of eucalyptus urophylla in brazil. sociobiology, 39: 231-242. 97 botanical preferences of africanized bees (apis mellifera) on the coast and in the atlantic forest of sergipe, brazil by júlio césar melo poderoso1; maria emilene correia-oliveira2*; luan carlos paz3; tácito moreira da silva e souza3; franz zirena vilca4; priscylla costa dantas5 & genésio tâmara ribeiro3 abstract pollen analysis in honey can be used as an alternative method to research into flowers visited by bees in an area. this study aimed to indentify the main floral families in honey from apiaries in the atlantic forest and sergipe state coast. honey samples from these apiaries were studied, as well as plants that grow around them, which can be used as a source of foraging for bees. the palynological technique was used to compare the pollen content of honey samples with the pollen grains from leaves of plants found in the vicinity of the apiaries to assess whether they had been visited by bees. the results of studies in both sites were similar in terms of incompatibility of families found in the apiary vicinity and honey. thus, it was possible to observe that in honey samples from the coast and in the remaining atlantic forest, the number of families was greater than the number of families found in the apiary vicinity, which highlights the diversity of plants visited by bees and a possible expansion of the visited area for food search. this diversity suggests an adaptive foraging behavior to plant resources available in the environment, which may facilitate the pollination of these botanical families and consequently improve their genetic quality. 1programa de pós-graduação em entomologia da universidade federal de viçosa, 36571-000, viçosa, minas gerais, brasil 2programa de pós-graduação em entomologia da escola superior de agricultura luiz de queiroz, universidade de são paulo, 13418-900, piracicaba, são paulo, brasil 3departamento de engenharia florestal da universidade federal de sergipe, 49100-000, são cristóvão, sergipe, brasil 4programa de pós-graduação em ecologia aplicada da escola superior de agricultura luiz de queiroz e centro de energia nuclear na agricultura, universidade de são paulo, piracicaba, são paulo, brasil 5programa de pós-graduação em agronomia/entomologia da universidade federal de lavras, 3037, 37200-000, lavras, minas gerais, brasil * corresponding author: emilenebio@hotmail.com 98 sociobiology vol. 59, no. 1, 2012 key-words: palynolog y, food preferences, honey, pollen introduction bees visit plants searching for pollen, nectar and resins to maintain their colonies. the workers of apis mellifera l. move from flower to flower collecting food for the larvae and adults of the colony (wcislo & cane 1996). the pollen is the only protein source in the bees' diet, and it contains lipids, vitamins and minerals necessary for their development (day 1990). the nectar, a substance secreted by glands called nectaries, is a sugary compound that attracts animals (raven et al. 1992), which may contain varying amounts of sugars, according to the species and other environmental factors (crane 1999). there are other substances such as organic acids, ethereal oils, polysaccharides – mainly dextrin, proteins, enzymes, boron and alkaloids – provided by the nectar (percival 1965). the visited plants are an essential resource for the bees and knowing them constitutes a basic tool for the development of apiculture (pearson & braiden 1990). the composition of the bee flora of a region is the main factor for the installation and development of an apiary, as it influences the number of hives to be installed and the honey production of the apiary (ashman et al. 2004; biesmeijer et al. 2006; williams 1994; westerkamp & gottsberger 2000). pollen analysis in honey can be used as an alternative to identify the flowers visited by bees in an area (ramírez-arriaga & martínez-hernández 1998). one way to identify species visited by bees is through palynolog y, as the pollen spectrum obtained through this analysis shows the distribution of pollen sources and honey plants where the apiary is installed (durkee 1971). the flora of a site is composed of different species at distinct levels of importance; therefore, studying them is crucial as it provides information that enables the adoption of an optimal apiary management. this study aimed to identify the main floral families present in honey and apiaries located in biomes in the atlantic forest and on the coast of the state of sergipe – brazil. material and methods experimental area the study was conducted in the municipality of pacatuba (11º36’15” s 99 poderoso, j.c.m. et al. — pollen preference of africanized bees in brazil and 37º26’34 w) at the “tigre” apiary, representing the coastal area and in japaratuba (10° 35’ 34” s 36° 56’ 24” o) at the “treze de maio” apiary, representing the atlantic forest area. honey samples from these apiaries were studied, as well as plants that grow in their vicinity, which can be used as a source for bee foraging. collection of samples the samples were collected during the harvest season of honey, which corresponded to the period from november 2009 to january 2010. a honey sample was obtained from each apiary, and at the same time, plants in full bloom were collected from a radius of one kilometer around the apiaries. considering the four quadrants, a sample in each 100 meters was collected to represent the tree, shrub and/or herbaceous extracts of plants in full flower (fig. 1). the plant material collected was dried in an oven at 60±2ºc for 48 hours to assemble the exsiccates and sampling of flower buds to make a comparative slide collection, using the acetolysis technique, by removing anthers and breaking pollen sacs, which were placed in test tubes and combined with 2ml of acetic acid. fig , 1: representation of the method of collection of botanical samples selected in apiaries. 100 sociobiology vol. 59, no. 1, 2012 pollen analysis this analysis consists of fossilizing the pollen grains for comparison with images on the database and botanical identification of plants that bees visited for nectar and pollen collection. slides with samples of honey and anthers were made using the acetolysis technique and mounted in glycerol gelatin (erdtman 1960; barth & luz 1998; terrab et al. 2003). the pollen analysis was carried out in the laboratory of agricultural and forest pests of the federal university of sergipe and the slides were sent to the laboratory of useful insects of the college of agriculture luiz de queiroz (esalq\usp) where they were micrographed by a digital system of photo documentation coupled to an optical microscope with 40x objective. the qualitative assessment was carried out with the identification of the botanical families and botanical genera. the quantitative analysis was performed by counting 900 pollen grains per sample. the pollen species classified as dominant accounted for 46 %, accessory between 15 to 45% and occasional lower than 15%. the pollen type identification was carried out following image database and specialized bibliography. after, the pollen content of the honey samples were compared with the pollen grains of plants collected in the vicinity of the apiary to assess whether they had been visited by bees. results despite the representation of the mimosaceae family as dominant, it had four different types of pollen grains, in other words, the pollen contained four different botanical species, mimosa caesalpiniifolia 41%, an unidentified species with 6%, mimosa scrabella with 5% and m. pudica with 3% altogether amounting 55% of the pollen in the honey. although the mimosaceae pollen grains are dominant, the honey is considered wild, once no species showed isolated dominance, i.e., presence greater than 45% of pollen grains (figure 7). discussion both the coastal area and in the remaining atlantic forest showed that in the honey samples, the number of flower families was greater than the number of families found in the apiary vicinity, which highlights the diversity 101 poderoso, j.c.m. et al. — pollen preference of africanized bees in brazil of plants visited by bees and their possible expansion of the visited area to search for food. the expansion of the area to collect pollen is related to the preference for a specific botanical family and for the cost-benetif to search for available sources to maintain the hive, a behavior that is transmitted to the workers. by finding the ideal source, of forage, bees may recruit nestmates and indicate the direction to these sources, increasing, therefore, the number of recruited bees (queller and strassmann 2002; brockmann et al. 2003; breed et al. 2004; billen 2006; pianaro et al. 2007). the botanical families found in this study corroborate reports that africanized bees in the neotropical region use primarily asteraceae, anacardiaceae, euphorbiaceae, lamiaceae, fabaceae, melastomataceae, moraceae, myrtaceae, arecaceae, rubiaceae and solanaceae plant families (almeida-muradian et al. 2005; barth & luz 1998; ramalho et al, 1990). the families visited by apis mellifera are important sources of nectar and pollen. moreover, the presfig. 2. accessory pollen grains found in a sample of honey from the apiary tigre, pacatuba, se; a. fabaceae type macropitilia; b. leguminosae, papilionoidae; c. mimosaceae; d. arecaceae, 40x magnification. fig. 3. isolated pollen grains found in a sample of honey from the apiar y tigre pacatuba, se; a. anacardiaceae; b. araceae; c.rubiaceae; d. sapindaceae; e. verbenaceae, 40x magnification. 102 sociobiology vol. 59, no. 1, 2012 fig. 7. pollen types belonging to the family mimosaceae found in honey samples. a. mimosa caesalpiniifolia; b. mimosa pudica; c. mimosa scrabella; d. mimosa sp. (unidentified species), with 40x magnification. fig. 5. pollen grains in a sample of pacatuba honey, se. a. pollen grains of the family mimosaceae; b. pollen grains of the family myrtaceae, 40x magnification. fig. 6. pollen grains in a sample of pacatuba honey, se. a. anacardiaceae; b. melastomataceae; c. fabaceae; d. rubiaceae, 40x magnification. fig. 4. pollen of the poaceae family (higher grain) and bottom right, the pollen of the family cecropiaceae, 40x magnification. 103 poderoso, j.c.m. et al. — pollen preference of africanized bees in brazil ence of pollen of these families in the honey may be related to the abundance of available species in the flora (quiroz-garcía et al. 2001). the presence of pollen of the family arecaceae can be used as an indicator of geographical origin, since some species such as cocos nucifera are easily found in the coastal or in cultivated areas. the number of families visited by apis mellifera is linked to their general characteristics, because they visit a large number of spices of plants, mainly small flowers, with dense and longer-lasting inflorescences and flowers with corollas long and wide or enough to allow access to the nectar (heard 1999). the pollen diversity found in the samples can be used as an indicator of wild honey, which can favor honey producers because of the diversity of aromas and flavors that they may have, due to a mixture of various nectars. this diversity suggests an adaptive behavior of plants to resources available in the environment, which may facilitate the pollination of these botanical families and consequently improve their genetic quality. acknowledgment we are grateful to the “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, coordenação de aperfeiçoamento de pessoal de nível superior (capes). references almeida-muradian, l.b., l.c. pamplona, s. coimbra, & o.m. barth 2005. chemical composition and botanical evaluation of dried bee pollen pellets. journal of food composition 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centro de méxico. se estudiaron tres sitios que han estado bajo diferentes actividades humanas: un campo de cultivo abandonado (cca), una terraza con evidencia de actividades humanas actuales y una zona con pocas actividades dentro de un jardín botánico ( jb). la hipótesis fue que el sitio que ha sido más transformado (cca) tendría las tasas de remoción de semillas más bajas por presentar condiciones más adversas para la actividad de forrajeo de las hormigas. los resultados mostraron que la transformación del hábitat afecta las tasas de remoción de las cinco especies de cactáceas estudiadas. las tasas más bajas de remoción de semillas se encontraron en cca y las más altas en tah. este resultado podría deberse a que tah es un sitio muy heterogéneo que aún tiene algunas características que permiten una actividad de forrajeo alta por parte de las hormigas. también se encontró que la tasa de remoción de semillas varió ente las diferentes especies de cactáceas. las semillas de escontria chiotilla tuvieron la remoción más alta y las de opuntia decumbens la más baja. las diferencias en la remoción de semillas podrían estar asociadas con los cambios que las actividades humanas han ocasionado en la vegetación y en la superficie del suelo. sin embargo la morfología externa de las semillas también debe ser considerada ya que en este estudio se encontró que las semillas más pequeñas fueron las más removidas. ubipro, fes-iztacala, universidad nacional autónoma de méxico. av. de los barrios 1, los reyes iztacala, tlalnepantla 54090, estado de méxico, méxico. leticiarc@campus.iztacala.unam.mx * autor para correspondencia 50 sociobiolog y vol. 59, no. 1, 2012 palabras clave: hormigas granívoras, escontria chiotilla, méxico, opuntia decumbens, pachycereus hollianus, stenocereus stellatus, stenocereus pruinosus, tamaño de semillas, valle de tehuacán abstract one of the main consequences of human activities in semiarid zones is the transformation of habitats. in this work we studied the effect of this transformation on seed removal of five cacti species by the harvester ant pogonomyrmex barbatus in the tehuacán-cuicatlán valley, a semiarid zone in central mexico. seed removal was quantified at three sites which have been under the effect of human activities: an abandoned crop field (cca), a site with evidence of current human activities (tah), and a site inside a botanic garden ( jb). we hypothesized that sites which have been under intense human activities would have low rates of seed removal because they offer harsh conditions for harvester ants, reducing their foraging activity. results showed that vegetation and surface soil characteristics of the sites studied are affecting the rates of seed removal of the five cacti species studied. the lowest seed removal rate was found at cca, the most transformed site. in contrast with our hypothesis the highest seed removal was found at tah, the site which represents the intermediate transformation condition, because this site still conserves some characteristics which permit intense foraging activity by harvester ants. we also found that the seed removal rate varied among the different cacti species studied. seed of e. chiotilla had the highest removal rate, whereas o. decumbens had the lowest. differences in seed removal rate could be associated with the high heterogeneity found in sites with intermediate levels of transformation. another factor that must be considered is the external morpholog y of seeds since smaller seeds presented highest removal rates. key words: cacti seeds, harvester ants, escontria, méxico, opuntia, pachycereus, stenocereus, seed size, tehuacan valley introducción en las regiones áridas y semiáridas, la remoción de semillas por hormigas puede afectar la diversidad de la comunidad de plantas, por lo que esta interacción es importante para la estructura y funcionamiento de estos ecosistemas 51 rios-casanova, l. et al. — seed removal in transformed habitats (brown et al. 1986, whitford 2002). las hormigas consumen las semillas de una amplia variedad de especies, llegando a remover hasta 14 g de semillas en 12 h (whitford 2002). sin embargo, la remoción puede variar dependiendo de las características de los sitios en donde ésta ocurre (whitford 2002). en este sentido, fenómenos como la fragmentación, la transformación del hábitat y el disturbio pueden estar afectando dicha interacción (sassi et al. 1996; kerley & whitford 2000; nicolai et al. 2007). la importancia de la remoción de semillas por hormigas ha sido relativamente bien documentada en desiertos extra-tropicales de argentina, australia, chile, estados unidos, israel y sudáfrica (marone et al. 2000; pirk & lópez de casenave 2011). por el contrario, la información sobre la importancia relativa de esta interacción en desiertos tropicales es prácticamente inexistente (ibáñez & soriano 2004). los estudios previamente realizados en las zonas semiáridas del centro de méxico (valle de tehuacán) (guzmán 2004; ríos-casanova et al. 2004; ríos-casanova 2005; ríos-casanova et al. 2006) sugieren que las hormigas podrían tener un papel importante en la remoción de semillas en la región. sin embargo, hasta el momento no existen estudios sobre esta interacción ni sobre la respuesta de las hormigas a la transformación del hábitat y sus consecuencias sobre la remoción de semillas. la transformación del hábitat en las zonas semiáridas del centro de méxico ocurre principalmente debido a las actividades humanas que se realizan en estos lugares como la agricultura, la ganadería, la extracción de leña y la construcción de salinas. dichas actividades han originado una disminución en la estructura de la vegetación, y un aumento de las áreas de suelo desnudo, por lo que los sitios más transformados presentan fuertes evidencias de erosión (lópez-galindo et al. 2003; muñoz-iniestra 2008, gaytán 2011). debido a estos cambios, es posible encontrar sitios con una vegetación muy poco estructurada que reciben una alta insolación y tienen una baja disponibilidad de semillas, en los que se esperaría registrar tasas muy bajas de remoción de semillas. por el contrario, existen sitios con vegetación más estructurada y que por lo tanto se encuentran menos expuestos a la radiación solar y que ofrecen una mayor disponibilidad de recursos, en los cuales se esperaría que las tasas de remoción de semillas fueran más altas. el presente trabajo se llevó a cabo en una porción del valle de tehuacáncuicatlán, una zona semidesértica del centro de méxico en la que se ha 52 sociobiolog y vol. 59, no. 1, 2012 reportado a la hormiga pogonoyrmex barbatus como uno de los elementos granívoros más importantes (ríos-casanova et al. 2004, ríos-casanova 2005; ríos-casanova et al. 2006; guzmán-mendoza et al. 2010). se analizó la remoción de semillas de cactáceas que son las plantas dominantes en el sitio de estudio, el cual es considerado un centro de diversidad de esta familia de plantas muchas de las cuáles son endémicas (godínez-alvarez & valiente-banuet 1998). la remoción de las semillas de estas plantas se evaluó en tres sitios que tienen distintas características fisonómicas de la vegetación y del suelo, principalmente como producto de la realización de actividades humanas. se esperaba que las tasas de remoción de semillas por la hormiga p. barbatus fueran bajas en los sitios que como consecuencia de la realización intensa de actividades humanas tienen vegetación poco estructurada, mientras que los sitios que han recibido menos impacto por las actividades humanas y que tienen una vegetación más estructurada deberían tener tasas de remoción de semillas mayores debido a la mayor disponibilidad de recursos y a que las condiciones ambientales permiten a las hormigas periodos de actividad más amplios. materiales y métodos sitios de estudio el estudio se realizó en zapotitlán de las salinas, puebla (17º 48’-18º 58’ n, 97º 03’-97º 43’ w), que se ubica dentro de la reserva de la biosfera del valle de tehuacán-cuicatlán, el cual abarca la porción noreste del estado de oaxaca y el sureste del estado de puebla en el centro de méxico. el clima es seco con una estación de lluvias que ocurre entre mayo y septiembre. la precipitación media anual es de 412.4 mm y la temperatura media anual varía entre 17.6 y 23.7 ºc (valiente 1991). se sabe que en este sitio existen aproximádamente 290 especies de angiospermas de las cuales 81 son cactáceas (dávila et al. 1993). los principales tipos de vegetación son el “matorral espinoso” con mimosa luisana (mimosaceae) y prosopis laevigata (mimosaceae) y las “tetecheras” dominadas por el cactus columnar neobuxbaumia tetetzo (cactaceae) (osorio et al. 1996). para probar la hipótesis se realizaron experimentos de remoción de semillas por p. barbatus en tres sitios en los que los efectos de las actividades humanas son 53 rios-casanova, l. et al. — seed removal in transformed habitats notables: 1) campo de cultivo abandonado (cca), 2) jardín botánico ( jb) y 3) terraza con evidencias de la práctica de varias actividades humanas (tah). el cca se caracteriza por ser un área con varios campos de cultivo que no han sido labrados en los últimos cinco años (fig. 1a). no hay presencia de costras biológicas en la superficie del suelo y, alrededor de lo que fueron los campos, crecen algunos árboles como pirul (schinus mole) y mezquite (prosopis laevigata), algunas cactáceas del género opuntia y columnares como pachycereus hollianus, además de diferentes herbáceas anuales como viguiera dentata, ipomea violacea y algunas gramíneas (oliveros 2000). el jb es un sitio que pertenece al jardín botánico “helia bravo hollis”. predomina el cactus columnar neobuxbaumia tetetzo y crecen una gran variedad de plantas asociadas a esta tetechera como la cactácea p. hollianus, y las leguminosas mimosa luisana y parkinsonia praecox (osorio et al. 1996). la actividad humana en este sitio es principalmente el ecoturismo (fig. 1b). la tah presenta zonas con vegetación abundante y algunas áreas con escasa o ninguna cobertura vegetal, también se encuentran zonas cubiertas con costra biológica. presenta especies como p. laevigata, pachycereus hollianus, opuntia decumbens y o pilifera. es evidente la presencia de actividades humanas como la extracción de leña y el cultivo de pitahaya (hylocereus undatus) (oliveros 2000; fig. 1c). en el cuadro 1 se presentan las características de cada uno de los sitios en términos de algunos indicadores ecológicos como porcentaje de cobertura vegetal, porcentaje de claros en la superficie del suelo, porcentaje de pedregosidad, etcétera. los datos para cca y tah se obtuvieron de gaytán (2011) mientras que los datos para jb se obtuvieron siguiendo la metodología de herrick et al. (2005). figura 1. sitios de estudio. a) campo de cultivo abandonado, b) terraza con actividades humanas, c) jardín botánico. 54 sociobiolog y vol. 59, no. 1, 2012 semillas las semillas que se emplearon pertenecen a cinco especies de cactáceas que tienen una amplia distribución en el área de estudio además de ser dominantes: pachycereus hollianus, stenocereus pruinosus, s. stellatus, escontria chiotilla y opuntia decumbens. estas especies se escogieron debido a que fructificaron durante el año de estudio (2009) y por lo tanto representaron un gran número de semillas disponibles para las hormigas. todas estas cactáceas son endémicas de méxico, excepto s. pruinosus que también se encuentra en centroamérica. la mayoría son cactáceas columnares a excepción de o. decumbens que es un arbusto cuyos cladodios crecen de forma rastrera. las semillas de las cinco especies de cactáceas se obtuvieron de al menos 25 frutos maduros elegidos al azar en diferentes sitios del valle de zapotitlán. posteriormente se extrajo la pulpa la cual se lavó con agua corriente con ayuda de una malla que permitió el paso de la pulpa pero no de las semillas una vez lavadas, se colocaron sobre papel absorbente a temperatura ambiente, y ya secas se guardaron en bolsas de papel para ser utilizadas en los experimentos de remoción. experimentos de remoción para cuantificar la remoción de semillas, en cada uno de los tres sitios se localizaron 5 nidos de la hormiga pogonomyrmex barbatus. alrededor de cada nido se colocaron cuatro cajas de petri a 3 m de la entrada del nido y separadas 90° una de la otra. en la superficie de dichas cajas se pegó un cuadro de papel lija en cuyo centro se dibujo un cuadro de 1 cm2, el cual sirvió para determinar el número de semillas removidas por las hormigas (kaspari 1996). cuadro 1. características de los sitios estudiados. cca = campo de cultivo abandonado, tah = terraza con actividades humanas, jb = jardín botánico. promedios ± un error estándar. cca tah jb cobertura vegetal (%) 21.6 ± 5.8 39 ± 13.4 63.33 ± 5.81 suelo desnudo (%) 72.5 ± 6.7 13.33 ±8.0 6.66 ± 2.40 rocas en superficie del suelo (%) 1.66 ± 1.66 0 ±0 44.66 ± 2.66 hojarasca (%) 4 ±2.5 36.6 ±3.6 10 ± 4.16 obstrucción vertical por la vegetación (%) 20 ±6.5 33 ± 4.6 57.88 ± 6.83 estabilidad del suelo 3.2 ± 0.24 5.8 ± 0.18 4.83 ± 0.55 espacios en el dosel > 10 m (%) 23 ±13.9 42.6 ± 6.0 28.58 ± 5.6 espacios en la superficie del suelo > 10 m (%) 59.3± 8.7 62.65±11.5 40.9 ± 5.9 55 rios-casanova, l. et al. — seed removal in transformed habitats dentro de cada cuadro se colocó una capa de semillas de una de las especies de cactáceas y se revisaron las cajas cada media hora para contar el número de semillas removidas y remplazar con más semillas hasta llenar el cuadro de 1 cm2 (fig. 2). figura 2. disposición de las semillas ofrecidas alrededor de la entrada del nido de p. barbatus. cuadro 2. tamaños y características principales de las 5 especies de cactáceas utilizadas en los experimentos de remoción por la hormiga granivora p. barbatus. especies largo ancho mayor ancho menor grosor largo del hilio color e. chiotilla 1.53 ± 0.02 1.15 ± 0.02 0.88± 0.02 0.83± 0.06 0.62± 0.02 marrón brillante p.hollianus 2.66 ± 0.04 2.13 ± 0.03 1.82 ± 0.04 1.51 ± 0.02 1.54 ± 0.03 café obscuro s.pruinosus 2.28 ± 0.07 1.77 ± 0.03 1.44 ± 0.02 1.14 ± 0.02 0.89 ± 0.02 marrón s. stellatus 1.92 ± 0.03 1.41 ± 0.03 1.2 ± 0.02 0.93 ± 0.02 0.66 ± 0.02 marrón mate o. decumbens 3.1 ± 0.03 2.75 ± 0.03 1.89 ± 0.06 1.82 ± 0.04 0.38 ± 0.02 blanco opaco 56 sociobiolog y vol. 59, no. 1, 2012 debido a las dificultades que presenta el conteo de semillas pequeñas en el campo, en cada revisión solamente se anotaba si ¼, ½, ¾, o todas las semillas del cuadro de 1 cm2 habían sido removidas. para conocer el número de semillas de cada especie de cactácea que fueron removidas, en el laboratorio se rellenó el cuadro de 1 cm2 con las semillas de cada una de las especies ofrecidas a las hormigas para contar el número de semillas que caben en el cuadro. esta medición se hizo 20 veces para cada especie de semilla y se calculó un promedio. el número de semillas removidas de cada especie de cactácea se calculó multiplicando la fracción de semillas removidas en el campo por el número promedio de semillas que caben en el cuadro de 1 cm2. los experimentos de remoción se realizaron entre las 10 y las 15 horas (horario de verano) por dos días, haciendo un total de 10 horas de observación para cada especie. la remoción se midió como el número de semillas totales removidas por las hormigas, entre el número de horas de observación por especie (número de semillas removidas/hora). relación tamaño-tasa de remoción. para explorar si algunas de las características morfométricas de las semillas están relacionadas con las tasas de remoción encontradas, se midieron el largo, ancho mayor, ancho menor y grosor de las semillas, así como el ancho del hilio de 20 semillas de cada especie de cactácea y estas medidas se correlacionaron con la tasa de remoción de cada especie. para este caso se calculó una tasa de remoción general, es decir, sin tomar en cuenta el sitio, ya que las semillas provienen de sitios con diferentes características y no fue posible diferenciar su origen. los tamaños de las semillas se obtuvieron utilizando el programa motic mc images plus 2.0 ml., el cuál permite realizar mediciones digitales de objetos observados en el microscopio estereoscópico. análisis estadístico las tasas de remoción para cada especie de semillas fueron comparadas entre sitios utilizando una prueba de kruskal wallis siendo la tasa de remoción la variable de respuesta y los sitios la fuente de variación. las medias se compararon entre si utilizando la prueba de q (zar 1996). estos análisis estadísticos se realizaron utilizando el programa spss 9.0 para windows. para conocer la relación entre las medidas de las semillas y su tasa de remoción se hicieron correlaciones lineales para cada parámetro utilizando el programa jmp 3.1.2 (sas institute). 57 rios-casanova, l. et al. — seed removal in transformed habitats resultados los resultados mostraron que las características fisonómicas de los sitios debidas principalmente al efecto de las actividades humanas, afectan la tasa de remoción de las semillas por la hormiga p. barbatus. la menor y mayor tasas de remoción se registraron en cca y tah, respectivamente. la tasa de remoción en jb tendió a presentar valores intermedios (fig. 3). la tasa de remoción también varió dependiendo de la especie de cactácea (fig. 3). la remoción de las semillas de s. pruinosus y e.chiotilla fue significativamente diferente entre sitios (chi 2 = 7.02, p = 0.03 para ambas especies) siendo mayor en tah aunque no difirió de la tasa encontrada para jb, mientras que en cca no se registró remoción. este patrón fue similar para las especies s. stellatus y o. decumbens, aunque no se encontraron diferencias significativas entre los sitios (chi 2 = 3.37, p = 0.18; chi 2 = 4.61, p = 0.09 respectivamente). las semillas de p. hollianus fueron removidas en los tres sitios, sin embargo no se encontró efecto significativo del sitio (chi 2 = 0.43, p = 0.8) fig. 3. tasa de remoción de semillas por p. barbatus para cinco especies de cactáceas en tres sitios (cca = campo de cultivo abandonado, jb = jardín botánico, tah = terraza con actividades humanas) de zapotitlán de las salinas, puebla. medias marcadas con letras diferentes son estadísticamente diferentes (prueba de q, p < 0.05). 58 sociobiolog y vol. 59, no. 1, 2012 únicamente la longitud del ancho menor de las semillas se correlacionó con su tasa de remoción (r2 = 0.75, f = 9.23, p = 0.05; fig. 4). para las otras medidas de la semilla las correlaciones no fueron significativas (largo: r2 = 0.7, f = 7.0, p = 0.07; ancho mayor: r2 = 0.61, f = 4.6, p = 0.12; grosor; r2 = 0.56, f = 3.8, p = 0.15; largo del hilio: r2 = 0.04, f = 0.15, p = 0.73; p = 0.85). discusión los resultados obtenidos indican que la remoción de semillas llevada a cabo por la hormiga pogonomyrmex barbatus puede estar afectada por las transformaciones que han ocurrido en su hábitat como resultado de factores biológicos y de las actividades humanas que se llevan a cabo en la zona de estudio. las características propias de cada sitio, medidas por medio de indicadores, mostraron que los tres sitios se han transformado de manera diferente. en el sitio cca se registró la menor cobertura vegetal, menor porcentaje de obstrucción visual ocasionada por la vegetación, menor estabilidad del suelo y mayor porcentaje de espacios con suelo desnudo. todas estas características indican que este es el sitio con mayor grado de transformación, lo que puede ser resultado de la intensa actividad agrícola realizada en el pasado. de acuerdo con la hipótesis planteada, en este sitio se registraron las figura 4. regresión lineal entre el tamaño del ancho menor de las semillas y su tasa de remoción por la hormiga p. barbatus en zapotilán de las salinas, puebla. ecuación de la recta: y = -144.01x + 271.7. 59 rios-casanova, l. et al. — seed removal in transformed habitats menores tasas de remoción de semillas para todas las especies de cactáceas estudiadas. únicamente se observó la remoción de semillas de s. stellatus y p. hollianus y sus tasas de remoción fueron tan bajas que no sobrepasaron las 0.3 y 17 semillas por hora respectivamente. los sitios con poca cobertura vegetal como el cca, se encuentran expuestos a la radiación directa del sol por lo que son sitios en los que las hormigas están expuestas a las altas temperaturas y a la desecación (lópez et al. 1992). se ha demostrado que la temperatura es uno de los factores más importantes en la regulación de las actividades de las hormigas (whitford & ettershank 1975; mackay & mackay,1989; hölldobler & wilson 1990). particularmente para las hormigas del género pogonomyrmex se ha documentado que tienen su mayor actividad entre los 20 y 45 °c y no realizan ninguna actividad por arriba de los 60°c (mackay y mackay 1989; hölldobler & wilson 1990; ríos-casanova 2005). sitios como el cca pueden alcanzar temperaturas por arriba de los 50 °c en la superficie del suelo (observación personal) por lo que representa un lugar poco propicio para la búsqueda y remoción de las semillas ofrecidas durante los experimentos ya que predominan los sitios desprovistos de vegetación. por la misma razón, estas hormigas son vulnerables al ataque de depredadores. uno de los principales depredadores de p. barbatus son las lagartijas del género sceloporus. en el sitio cca, se observaron lagartijas de este género y se ha documentado que una gran proporción de la dieta de estas lagartijas en el valle de zapotitlán son las hormigas p. barbatus (serranocardozo et al. 2008). a pesar de lo anterior, en este sitio se observó forrajear a las hormigas en sitios cubiertos por los arbustos anuales que se encuentran alrededor de los campos de cultivo, entre ellas algunos pastos y asteráceas cuyas semillas eran ocasionalmente removidas por las hormigas. el sitio jb fue considerado como el menos deteriorado debido a que presentó mayor cobertura de la vegetación y por lo tanto mayor obstrucción visual provocada por la misma. igualmente, presentó pocos espacios en el dosel y el menor porcentaje de suelo desnudo; además este sitio presentó poca evidencia de la práctica de actividades humanas a excepción del ecoturismo. los resultados obtenidos difieren de la hipótesis planteada, ya que en este sitio se esperaba encontrar las mayores tasas de remoción. sin embargo, los valores de remoción encontrados, aunque fueron mayores que en el cca, 60 sociobiolog y vol. 59, no. 1, 2012 no fueron los más altos. en este sitio hubo remoción de semillas de todas las especies siendo e. chiotilla la especie con la mayor tasa de remoción (149 semillas por hora). se esperaba que en jb ocurriera la mayor remoción ya que por sus características, tiene el mayor porcentaje de sitios protegidos de la incidencia solar directa, lo que permitiría a las hormigas forrajear durante rangos mayores de tiempo provocando tasas de remoción altas (hensen 2002). sin embargo las tasas de remoción encontradas podrían deberse a que las hormigas forrajearon otras semillas que estaban disponibles en ese momento como por ejemplo semillas de gramíneas. también se ha sugerido que la dirección en la que las hormigas forrajean está orientada por marcadores químicos que las hormigas exploradoras de la colonia dejan en la superficie del suelo al detectar un recurso (gordon 2002). el sitio jb fue el sitio con el mayor porcentaje de rocas en la superficie, lo cual podría haber dificultado la búsqueda y el transporte de las semillas de las cactáceas ofrecidas, así como la comunicación química entre forrajeras. un estudio previo realizado en el jb señaló que en este sitio se observó la presencia de depredadores arácnidos cerca de los nidos, sugiriendo que habría una fuerte presión por depredación sobre las hormigas p. barbatus (guzmán 2004). lo anterior podría estar disminuyendo los periodos de actividad de las hormigas en este sitio y por lo tanto sus tasas de remoción de semillas. el sitio denominado terraza con actividades humanas (tah), fue llamado así por presentar evidencias de que ahí se realizan actividades como extracción de leña y cultivo de la cactácea hylocereus undatus, entre otras. la transformación de este sitio debida a la realización de dichas actividades le ha conferido una alta heterogeneidad, ya que existen zonas con mucha insolación, pero también zonas sombreados por arbustos y árboles, zonas cubiertos de costra biológica y zonas con suelo desnudo. aquí encontramos la mayor tasa de remoción para todas las especies de cactáceas estudiadas, desde 32 semillas por hora para o. decumbens, hasta 400 semillas por hora para e. chiotilla.. este patrón podría deberse a que es un sitio muy heterogéneo en el que hay sitios sombreados con temperaturas que pueden ser toleradas por las hormigas, sitios con diferentes texturas en el suelo, lo cual puede reducir las temperaturas del mismo, y una gran posibilidad de encontrar semillas de diferentes recursos en diferentes momentos. 61 rios-casanova, l. et al. — seed removal in transformed habitats igualmente, la alta heterogeneidad podría estar proveyendo a las forrajeras de sitios en los que pueden escapar de la depredación. se ha sugerido que la granivoría es resultado de cambios en la estructura y composición de los ensamblajes tanto de plantas como de la comunidad de granívoros en general y que los factores que ocasionan disturbio son una fuente de heterogeneidad lo cual repercute en las tasas de remoción llevadas a cabo por los granívoros (wilby & shachak 2000; sassi et al. 2006; lomov et al. 2009). en este sitio (tah) la semilla más removida fue e. chiotilla ya que se encontró que p. barbatus removió cerca de 400 semillas por hora. en otros estudios se ha encontrado que las hormigas del género pogonomyrmex pueden remover hasta 30 semillas en un día (mull & macmahon 1997), por lo que las tasas encontradas en este estudio indican una remoción sumamente elevada. en lo que respecta a las características de las semillas y sus tasas de remoción los resultados indicaron que las semillas más pequeñas tienen las tasas de remoción más altas. entre las características de las semillas sobre las cuales se ha sugerido que opera la selectividad de las hormigas se encuentran el tamaño, la morfología, el contenido de agua y la composición nutricional, así como las habilidades de las hormigas para reconocer el alimento (ibáñez & soriano 2004). en este estudio solo se consideraron aspectos relacionados con el tamaño de las semillas y se encontró que las semillas menos removidas fueron las de o. decumbens. estas semillas, además de ser las más grandes no presentan ornamentaciones evidentes en la testa, lo que podría estar dificultando su manipulación y traslado por parte de p. barbatus. se ha documentado que el transporte de semillas con superficies lisas es más difícil (pirk, 2002). también se ha documentado que las obreras de p. barbatus prefieren las semillas con prolongaciones y ornamentaciones que facilitan su manejo (pulliam & brand 1975). las semillas de e. chiotilla fueron las semillas más removidas en este estudio y, aunque no tienen prolongaciones, tienen una constricción que corresponde al ancho menor, que podría estar facilitando el manejo por parte de las hormigas. además estas semillas son las más pequeñas, lo que puede facilitar su transporte. las semillas de las otras especies, p. hollianus, s. stellatus y s. pruinosus, son semillas más grandes que, aunque no presen62 sociobiolog y vol. 59, no. 1, 2012 tan prolongaciones, si fueron removidas por p. barbatus aunque con menor frecuencia que las de e. chiotilla. otro factor que podría estar afectando directamente la preferencia de las semillas por las hormigas es su contenido nutricional (kelrick et al. 1986) y la presencia de compuestos tóxicos (carroll & janzen 1973). aunque en este estudio no se midió ninguna variable relacionada con la química de las semillas, datos obtenidos en la literatura indican que las semillas de s. stellatus , s. pruinosus y p. hollianus contienen alcaloides que podrían resultar tóxicos para las hormigas (ortega-nieblas et al., 2001). desafortunadamente no se cuenta con datos para las semillas del género escontria y por el momento no es posible conocer si las preferencias de p. barbatus podrían relacionarse con el contenido de nutrientes o de compuestos tóxicos de las semillas de esta especie. muchos de los estudios sobre remoción de semillas por hormigas han utilizado semillas exóticas y ya se ha demostrado que las tasas de remoción de esas semillas son generalmente más altas que las de semillas nativas (parmenter et al. 1984; folgarait & sala 2004). en este trabajo utilizamos semillas de cactáceas nativas del valle de zapotitlán de las salinas lo cual nos permite acercarnos al conocimiento de cómo las actividades humanas pueden estar afectando la interacción de la hormiga p. barbatus con los recursos disponibles en el sitio. para un futuro se podrían realizar experimentos con otras semillas además de las de cactáceas, puesto que a pesar de que son especies abundantes en la zona, hay muchas otras plantas como las asteráceas viguiera dentata y verbesina, leguminosas como el mezquite prosopis laevigata y el palo verde parkinsonia praecox, pastos y muchas otras anuales. el estudio de la remoción de semillas nativas en las zonas semiáridas intertropicales es un aspecto que aún requiere ser estudiado y el presente trabajo es un primer intento por abordar este tema. agradecimientos gracias a silvano santiago geraldo “chimino”, guía del jardín botánico helia bravo hollis, por acompañarnos a los sitios y por su entusiasmo durante los experimentos de remoción de semillas y al proyecto papca 2009-2010 otorgado por la facultad de estudios 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10.13102/sociobiology.v62i2.228-245sociobiology 62(2): 228-245 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 ant fauna in megadiverse mountains: a checklist for the rocky grasslands introduction the rocky grasslands, locally known as campo rupestre, are an ecosystem characterized by a montane, fire-prone vegetation mosaic, with rocky outcrops on quartzite, sandstone, or ironstone soils. they are inserted in a matrix of sandy and stony grasslands, and other vegetation types, such as cerrado (brazilian savanna), atlantic forest, and caatinga (giulietti & pirani, 1997; alves et al., 2014; fernandes et al., 2014). rocky grasslands occur mainly along the espinhaço mountains, a vast mountain range that has its southern limit in the state of minas gerais, southeastern brazil, encompass important smaller ranges, such as serra do caraça and serra do cipó, and ends in chapada diamantina, state of bahia, northeastern brazil (giulietti & pirani, 1997). rocky grasslands are also found in the mountains of central (e.g., serra da canastra) and southeastern brazil (e.g., serra da mantiqueira), whose geology and flora resemble those of the espinhaço mountains (giulietti & pirani, 1997; rapini et al., 2008; vasconcelos, abstract the rocky grasslands, environments locally known as campo rupestre, occur mainly along the espinhaço mountains and are considered local centers of biodiversity and endemism in brazil. however, knowledge of ant species richness (hymenoptera: formicidae) in this kind of environment is still poor. aiming at filling this gap, we compiled information from empirical studies and literature records. we found a total of 288 species of 53 genera and eight subfamilies recorded in rocky grasslands. myrmicinae and formicinae were the most representative subfamilies, with 53% and 18% of the total species richness, respectively. the genera with the largest number of species were pheidole (41) and camponotus (40). this large number of ant species recorded for the rocky grasslands surpasses those found in other studies conducted in several different places. ant species richness decreased with altitude; most species occur below 800 m a.s.l. (171), and only a few species occur above1600 m a.s.l. (17). some genera occur only at a specific altitude (e.g., azteca and dolichoderus at 800/900 m a.s.l.; leptogenys and labidus at 1400 m a.s.l.), which points out to the potential use of ants as biological indicators. our results suggest that the rocky grasslands favor high ant diversity. the patterns of ant richness associated with the altitudinal gradient reinforce the idea of considering the rocky grasslands as priority areas for biological conservation. moreover, we observed a lack of records on the occurrence of most ant species considered in the present study (93%), which shows that brazilian myrmecologists need to invest more in taxonomy, management, and data sharing. sociobiology an international journal on social insects fv costa, r mello, lana tc, fs neves article history edited by john lattke, universidad nacional de loja, ecuador received 01 december 2014 initial acceptance 18 january 2015 final acceptance 01 april 2015 keywords ant checklist, altitudinal gradient, campo rupestre, espinhaço mountains. corresponding author fernanda vieira da costa federal university of minas gerais institute for biological sciences insect ecology lab (e3257) av. antônio carlos, 6627 31270-901, belo horizonte-mg, brazil e-mail: fecostabio@gmail.com 2011). this complex geographic mosaic associated with a long evolutionary time turned this environment into a local biodiversity center, with high endemism (approximately onethird of its plant species are endemic) and several endangered species (giulietti & pirani 1997; rapini et al., 2008). tropical grasslands have been under severe threat and are consistently overlooked by conservation policies (parr et al., 2014). these ecosystems are subjected to several human pressures, such as mining, livestock raising, agriculture, road construction, tourism, and frequent fires (barbosa et al., 2010; fernandes et al., 2014). in addition to their large number of endangered species and human threats, montane ecosystems are also subjected to global changes (ipcc, 2013). climatic models predict a catastrophic future in which, by the end of this century, the rocky grasslands may lose up to 95% of their current area (fernandes et al., 2014). in this scenario, the development of effective conservation strategies is crucial. fauna and flora inventories are of fundamental importance, as describing the biodiversity of rocky grasslands is the first step towards their conservation (pearson, 1994). research article ants federal university of minas gerais, belo horizonte, mg, brazil sociobiology 62(2): 228-245 (june, 2015) 229 despite the information on the flora and fauna of rocky grasslands (giulietti et al., 1987; lessa et al., 2008; rapini et al., 2008; rodrigues et al., 2011), sampling in those environments has been heterogeneous and large areas remain unexplored (madeira et al., 2008). in addition, most of the literature on the biodiversity of rocky grasslands focus on plants and vertebrates (silveira et al., unpublished data). therefore other groups, such as invertebrates, remain unknown. some of the challenges to invertebrate conservation are a scarce and underfunded basic research, and the overlooking of invertebrates in most conservation policies (cardoso et al., 2011). conversely, invertebrates dominate most terrestrial environments and deliver several ecosystem services (cardoso et al., 2011). among invertebrates, ants (hymenoptera: formicidae) represent one of the most important and abundant terrestrial groups (hölldobler & wilson, 1990). well known for their functional roles, ants have been used as bioindicators due to their sensitivity to environmental and climate changes (lach et al., 2010). previous studies reported changes in ant diversity along altitudinal gradients (fisher, 1996; bharti & sharma, 2013; bishop et al., 2014). altitudinal gradients are excellent to model species distribution, due to differences in abiotic conditions. however, as most literature records came from temperate mountains, the lack of information on tropical mountains makes it difficult to elaborate conservation plans. what we know about ants from rocky grasslands comes from case studies on ant species associated with a particular plant or area (e.g., guerra et al., 2011; viana-silva & jacobi, 2012; fagundes et al., 2013). however, a complete record that comprises the whole diversity and distribution of ants is still missing. in order to fill this gap of knowledge and support invertebrate conservation in the rocky grasslands, we need a more thorough biodiversity survey. in the present study, we compiled a checklist of ant species and their occurrence from original data and published information. material and methods data sampling and database we searched for studies carried out in areas of rocky grasslands that informed the geographic coordinates of their sampling sites and identified ants to the species. we compiled records from a total of eight datasets, most of which original and collected in serra do cipó (lana, 2015). seven other datasets were found through an online survey in the web of knowledge, other academic search engines such as google scholar, and brazilian academic libraries. among those sources is one unpublished dataset from serra do cipó (hereafter “cipó”) (soares, 2003) and six published datasets from cipó (n = 1), serra do rola moça state park (hereafter “rola moça”) (n = 1), itacolomi state park (hereafter “itacolomi”) (n = 3), and ibitipoca state park (hereafter “ibitipoca”) (n = 1). all studied sites are located in the espinhaço mountains, except for ibitipoca. as we aimed at providing a broad inventory of the ant fauna, we used studies carried out with different sampling efforts and methods. table 1 describes the samples collected from the literature, including information on sampling method, environment, and location. details on species identification are given for each study. sampling sites reference sampling method environment altitudinal range (m) geographic coordinates provided in the study serra do cipó region lana, 20151 pitfalls, winkler, beating and sweep net cerrado ecotones, rocky grasslands 800 – 1400 19°21’36.2’’ s, 43°36’25.2’’ w 19°16’17.8’’ s, 43°36’18.1’’ w 19°15’50.6’’ s, 43°35’10.3’’ w 19°13’56.5’’ s, 43°34’34.8’’ w 19°17’43.0’’ s, 43°33’17.4’’ w 19°17’49.6” s, 43°35’28.2” w 19°16’59.3’’ s, 43°32’08.9’’ w itacolomi state park region almeida et al., 2014*2 active capture rocky grasslands, canga outcrops 1200 – 1500 20°22’30” s, 43°32’30” w 20°27’55.4” s, 43°35’59” w 20°21’47” s, 43°30’10” w 20º22’27” s, 43º32’22” w itacolomi state park fagundes et al., 2013*3 observations and active capture rocky grasslands 1400 20°26’26’’ s, 43°30’52’’ w serra do rola moça state park viana-silva & jacobi, 2012*4 ground baits canga outcrops 1400 – 1500 20°03’35.19’’ s, 44°00’41.9’’ w 20°03’33.57’’s, 44°01’52.01’’ w itacolomi state park rosumek, 2009*5 baits, observations and active capture canga outcrops 1320 1400 20º26’18” s, 43º30’35” w serra do cipó region soares, 2003*6 baited pitfalls rocky grasslands, cerrado ecotone 800 1600 19º10’00’’ to 19º40’00’’ s, 43º30’00’’ to 43º 55’00’’ w serra do cipó region araújo & fernandes, 2003*7 baits and active capture rocky grasslands, cerrado ecotone 800 – 1400 19º10’00’’ to 19º40’00’’ s, 43º30’00’’ to 43º55’00’’ w ibitipoca state park sales et al., 2014*8 active capture rocky grasslands 1400 21º42’00’’ s, 43º53’00’’ w *studies found through an online survey; superscript numbers provide reference for sources in the full list of species (table 2). table 1. sites where ants were sampled in the brazilian rocky grasslands. fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands230 in the original dataset (lana, 2015) ants were sampled in seven sites during the long term ecological research of the rocky grasslands of serra do cipó (peld-crsc, in the portuguese acronym). those seven sites were chosen along an altitudinal gradient in cipó, from 800 to 1,400 m a.s.l. in each site, three transects of 200 m were set up and five sampling points were established at 50 m from each other. in 2011 and 2012, ants were sampled quarterly, mainly with pitfalls traps, but also with beating, sweep nets, and winkler traps. ants were identified using a taxonomic key (fernández, 2003), by comparison with specimens deposited in the laboratório de mirmecologia do centro de pesquisas do cacau (cepec/ceplac), and by consulting specialists (jacques h. c. delabie). nomenclature followed bolton et al. (2005), with posterior improvements made available on the online catalog of the ants of the world (antcat). we built a complete species checklist (appendix) with information on several ant species organized by study, sampling locality, and altitude. as it was not possible to match the morphospecies hosted in different institutions and collections, we included in the analysis only one record for each morphospecies, regardless of its potential presence in more than one study, area, or altitude. each morphospecies (e.g., pheidole sp.1) probably represents more than one species, as the same nomenclature was established by different authors. however, excluding those records or trying to tell them apart could interfere with the estimation of the real diversity. although we are aware of this taxonomic limitation, in face of the difficulty of assigning names to several neotropical ant species (e.g., camponotus and pheidole) and the lack of current taxonomic revisions for many species-rich genera (e.g., brachymyrmex, cyphomyrmex, and solenopsis) (lach et al., 2010), this is the most parsimonious option for a study that aimed at estimating ant species diversity on a broad scale. study sites the espinhaço mountains are 50-100 km wide and 1,200 km long, and encompass several mountains (up to approximately 2,000 m a.s.l.) (giulietti & pirani, 1997). rocky grasslands occur mostly from 900 to 2,033 m a.s.l. in the basal part of the range, at altitudes between 800-1,000 m a.s.l., we found ecotones between savanna and rocky grassland. trees and shrubs are the most common life forms at lower altitudes, but their predominance decreases with altitude, as the soil profile also changes, and they gradually give way to outcrops and grasslands (giulietti & pirani, 1997; alves et al., 2014). similarly, together with the altitudinal gradient there is also a climate gradient, in which the mountaintop is colder and moister than the base (giulietti & pirani, 1988). serra do cipó is located in the southeastern part of the espinhaço mountains, state of minas gerais, southeastern brazil (fig 1). this region has a diversified mosaic of vegetations, which varies with soil type and altitude (from 800 to 1.600 m a.s.l.). this environment is covered by a low vegetation composed of shrubs and small trees and abundant grasses and sedges. there are also several watercourses, along which gallery evergreen forests grow (giulietti et al., 1987). itacolomi state park and serra do rola moça state park are characterized by rocky grasslands that grow on ironstone, locally known as canga. both areas are located in the iron quadrangle of minas gerais, a 7,200 km2 region in the southern part of the espinhaço mountains (fig 1). the iron quadrangle is geologically dominated by ironstone and represents one of the world’s main mineral provinces (jacobi et al., 2011). itacolomi comprises an altitudinal range varying from 700 to 1,772 m a.s.l. and a mosaic of rocky grasslands, canga, semi-deciduous montane forest, and associated vegetation types (gastauer et al., 2012). rola fig 1. the location of rocky grasslands in brazil (a). rocky grasslands along the espinhaço mountains and other brazilian mountain ranges (circumscribed) (b). sampling sites in the southern part of the espinhaço mountains and the southern mountains of minas gerais (c). sociobiology 62(2): 228-245 (june, 2015) 231 moça is located in an ecotone between the cerrado and atlantic forest biomes, which comprises several vegetation types, such as cerrado, semi-deciduous forest, riparian forest, and prominent rocky grasslands developing on canga on the mountaintops (1,200 –1,500 m a.s.l.) (jacobi et al., 2008). ibitipoca state park is a protected area located in serra da mantiqueira, southern minas gerais (fig 1). this site is characterized by a vegetation type composed of grasses, herbs, and shrubs on outcrops of quartzite rocks associated with shallow soils and high sun incidence (dias et al., 2002). results and discussion we recorded 288 ant species of 53 genera and eight subfamilies (appendix). myrmicinae was the most speciose subfamily, with 53% of the recorded species, followed by formicinae (18%), dolichoderinae (11%), ponerinae (6%), and ectatomminae (5%). the richest genus was pheidole (41 species), followed by camponotus (40), crematogaster (22), dorymyrmex (14), and solenopsis (13). the largest number of ant species was found in cipó (n = 265), followed by itacolomi (48), ibitipoca (20), and rola moça (14). similarly, cipó was the locality with the largest proportion of exclusive species (83%), which indicates that this site was the best sampled and the faunas of other sites are nested within it. the proportion of exclusive ant species in each site and ant species shared between at least two sites is shown in fig 2. among the identified species, only camponotus crassus (mayr 1862) occurred in all sites. only morphospecies of pheidole exhibited similar distribution. thus, camponotus and pheidole emerged as the most widespread genera currently recorded for rocky grasslands. the dominance of those genera is consistent with the patterns suggested for other neotropical ants (fernández & sendoya, 2004) and similar ecosystems, such as open cerrado (ribas et al., 2003; campos et al., 2011; pacheco & vasconcelos, 2012). likewise, myrmicinae and formicinae were also the most prominent subfamilies in ant inventories conducted in different environments, such as cerrado (ribas et al., 2003; campos et al., 2011), amazon (miranda et al., 2012), and caatinga (ulysséa & brandão, 2013). the large number of ant species recorded for rocky grasslands (288) deserves attention, as other studies carried out in wider geographical ranges found a smaller or similar number. for example, checklists made for the caatinga (ulysséa & brandão, 2013) and amazon (miranda et al., 2012) found 173 and 276 species, respectively. although we did not find a comprehensive inventory for the cerrado that could be used for comparison, studies carried out over large areas revealed about 150 species (ribas et al., 2003; campos et al., 2011; pacheco & vasconcelos, 2012). actually, it is hard to compare number of species among studies or environments, as different studies used different sampling efforts and methods. nevertheless, by analyzing the map in fig 1, we infer that the rocky grasslands have several sampling gaps (e.g., chapada diamantina, serra da canastra, northern and southern minas gerais). considering this gap of knowledge, high endemism, and complex environmental mosaic found on those mountains, we expect ant diversity in the rocky grasslands to be even higher than observed in the present study (288). moreover, the large number of unidentified species together with the inclusion of only one record per morphospecies point out to an underestimation of the number of ant species in the rocky grasslands. we observed a decrease in ant richness along the altitudinal range (appendix). the lowest altitudes, 800 and 900 m a.s.l., contributed with 171 and 127 ant species, respectively, whereas the highest altitude (1,600 m a.s.l.) had a smaller number of species (17). only solenopis occurred at all altitudes. those findings corroborate the general diversity pattern of ants that live on mountains, in which the number of species decreases with altitude (fisher, 1996; brühl et al., 1999; longino & colwell, 2011; bharti & sharma, 2013). nonetheless, very few studies have documented the altitudinal trends of ant biodiversity in brazilian montane ecosystems (but see araújo & fernandes, 2003). at 800 m a.s.l., there were 18 exclusive species, whereas at 1,400, 1,500, and 1,600 m a.s.l. there were 21, five, and two, respectively. we found some genera of dominant arboreal ants (azteca and dolichoderus) restricted to the mountain base (800/900 m a.s.l.), which indicates that altitude may restrict ant occurrences. similarly, at 1,400 m a.s.l., we recorded some unique genera, such as specialized predators (leptogenys) and legionary ants (labidus) (brandão et al., 2012). those findings corroborate the potential of ants as bioindicators, especially of climate change. similar patterns of restriction of functional groups to particular altitudes have already been observed for tropical (brühl et al., 1999) and temperate regions (bharti & sharma, 2013). however, those findings may have been biased by the sampling effort used in each site. ant responses to altitude, associated with the high richness found in a small geographic area, point to the importance of conserving the rocky grasslands. this conclusion is consistent with the strategies recommended for ant conservation, which state that efforts should be targeted to high biodiversity, high endemism, and extremely threatened areas (alonso, 2010). fig 2. proportion of ant species exclusive to each site and shared between at least two sites. sampling site abbreviations: serra do cipó (sc), itacolomi state park (it), ibitipoca state park (ib), and serra do rola moça state park (rm). dark bars represent exclusive proportion and light bars correspond to the shared proportion of species. fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands232 we also bring to light the need for investing in ant taxonomy, database management, and data sharing, which are essential tools for biodiversity conservation, though they are neglected in most brazilian research institutions. those gaps of knowledge became clear when we searched for information on ant species occurrence and distribution (only those with complete taxonomic identification) in online databases (antwiki, antweb, cria specieslink) and specialized catalogues (kempf, 1972; brandão, 1991). we noticed that most ant records (94%) neither were followed by a formal record for the rocky grasslands nor were hosted in databases (appendix, symbol*). approximately 5% of the records contained no information on geographic distribution and only 1% accounted this kind of information for the rocky grasslands. therefore, myrmecology in brazil needs to invest strongly in taxonomy and species inventories. despite some recent advances, rocky grasslands still have many sampling gaps for ants, and, therefore, they need more efforts in conservation. acknowledgments we thank j. h. delabie and w. rocha for their help in ant species identification; h. brant, m. c. anjos, and a. c. reis for their help in the field and laboratory work; and g. duarte for elaborating the map. we also thank parque nacional da serra do cipó/icmbio and reserva vellozia for the logistic support. this study was partially funded by peld/ cnpq/ fapemig and comcerrado/cnpq. references almeida, m.f., santos, b.l.r. & carneiro, m.a.a. 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(2012) myrmecofauna of ironstone outcrops : composition and diversity. neotropical entomology, 41: 263-271. doi: 10.1007/s13744-012-0045-9. sociobiology 62(2): 228-245 (june, 2015) 235 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 fo rm ic id ae a m bl yo po ni na e pr io no pe lta s p x x d ol ic ho de ri na e a zt ec a sp x x x d ol ic ho de ru s di ve rs us (e m er y, 1 89 4) * x d ol ic ho de ru s lu to su s (s m ith , 1 85 8) * x d or ym yr m ex b ru nn eu s (f or el , 1 90 8) * x x x x x d or ym yr m ex g oe ld ii (f or el , 1 90 4) * x x x x x x d or ym yr m ex jh er in gi (f or el , 1 91 2) * x d or ym yr m ex p yr am ic us (r og er , 1 86 3) * x x x x x x d or ym yr m ex s p x d or ym yr m ex s p1 x x x x d or ym yr m ex s p2 x x x x x d or ym yr m ex s p3 x x d or ym yr m ex s p4 x x d or ym yr m ex s p5 x d or ym yr m ex s p6 x d or ym yr m ex s p7 x d or ym yr m ex s p8 x d or ym yr m ex s p9 x fo re liu s br as ili en si s (f or el , 1 90 8) * x fo re liu s m ar an ha oe ns is (c ue zz o, 2 00 0) * x x x x fo re liu s sp x x x li ne pi th em a ce rr ad en se (w ild , 2 00 7) * x li ne pi th em a hu m ile (m ay r, 18 68 )* x x x x x x li ne pi th em a m ic an s (f or el , 1 90 8) † x li ne pi th em a pr ox . h um ile s p x fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands236 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 d ol ic ho de ri na e (c on tin ua tio n) li ne pi th em a sp 1 x x x x x x x x x x x x x x x x li ne pi th em a sp 2 x x x x x x x x x x li ne pi th em a sp 3 x x x x x x x x x x x x x x x x li ne pi th em a sp 4 x x x x x x x li ne pi th em a sp 5 x x x x x li ne pi th em a sp 6 x x x x x x ta pi no m a sp 1 x x x x x x x x x x x ta pi no m a sp 2 x x x x x x d or yl in ae la bi du s co ec us (l at re ill e, 1 80 2) * x la bi du s pr ae da to r ( sm ith , 1 85 8) * x le pt og en ys p ro x. li nd a sp x n ei va m yr m ex d ia na (f or el , 1 91 2) * x n ei va m yr m ex p se ud op s (f or el , 1 90 9) * x n ei va m yr m ex s p1 x x x n ei va m yr m ex s p2 x x x ec ta to m m in ae ec ta to m m a br un ne um (s m ith , 1 85 8) * x x x ec ta to m m a ed en ta tu m (r og er , 1 86 3) * x x x x x x x x x x x x x ec ta to m m a m uti cu m ( m ay r, 18 70 )* x x ec ta to m m a op ac iv en tr e (r og er , 1 86 1) * x x x x x ec ta to m m a pe rm ag nu m (f or el , 1 90 8) * x x x ec ta to m m a pl an id en s (b or gm ei er , 1 93 9) * x x x x ec ta to m m a sp 1 x x x x x ec ta to m m a sp 2 x x x x ec ta to m m a su za na e (a lm ei da , 1 98 6) ‡ x x ec ta to m m a tu be rc ul at um (o liv ie r, 17 92 )* x x x x sociobiology 62(2): 228-245 (june, 2015) 237 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 ec ta to m m in ae (c on tin ua tio n) g na m pt og en ys s p1 x g na m pt og en ys s tr ia tu la (m ay r, 18 84 )* x g na m pt og en ys s ul ca ta (s m ith , 1 85 8) * x x x x fo rm ic in ae br ac hy m yr m ex p at ag on ic us (m ay r, 18 68 )* x x x x x x br ac hy m yr m ex s p x x x x x br ac hy m yr m ex s p1 x x x x x x x x x x x x x x br ac hy m yr m ex s p2 x x x x x x x br ac hy m yr m ex s p3 x x x ca m po no tu s (h yp er co lo bo ps is ) s p1 x x x x x ca m po no tu s (h yp er co lo bo ps is ) s p2 x ca m po no tu s (h yp er co lo bo ps is ) s p3 x x ca m po no tu s (m yr m ap ha en us ) s p1 x x x x x x x x ca m po no tu s (m yr m ap ha en us ) s p2 x x x x x x x x x x ca m po no tu s (m yr m ap ha en us ) s p3 x x x x x x x x x x ca m po no tu s (m yr m ap ha en us ) s p4 x x x x x ca m po no tu s (m yr m ap ha en us ) s p5 x x x ca m po no tu s (m yr m ap ha en us ) s p6 x x x x ca m po no tu s (m yr m ap ha en us ) s p7 x x ca m po no tu s (m yr m ap ha en us ) s p8 x ca m po no tu s (m yr m ap ha en us ) s p9 x ca m po no tu s (m yr m ap ha en us ) s p1 0 x x x ca m po no tu s (m yr m ap ha en us ) s p1 1 x x x x x ca m po no tu s (t an ae m yr m ex ) s p1 x x x ca m po no tu s (t an ae m yr m ex ) s p2 x x x x x x x x ca m po no tu s (t an ae m yr m ex ) s p3 x x x x x x x x x ca m po no tu s (t an ae m yr m ex ) s p4 x x x x x x x x x x fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands238 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 fo rm ic in ae (c on tin ua tio n) ca m po no tu s (t an ae m yr m ex ) s p5 x ca m po no tu s at ri ce ps (s m ith , 1 85 8) * x ca m po no tu s bl an du s (s m ith , 1 85 8) * x x x x x x ca m po no tu s ci ng ul at us (m ay r, 18 62 )* x ca m po no tu s cr as su s (m ay r, 18 62 )* x x x x x x x x x x x x x x x x x x ca m po no tu s fa sti ga tu s (r og er , 1 86 3) * x x x ca m po no tu s ge na tu s (s an ts ch i, 19 22 )* x x ca m po no tu s le yd ig i ( fo re l, 18 86 )* x x x ca m po no tu s m el an oti cu s (e m er y, 1 89 4) * x x x x x x x ca m po no tu s no vo gr an ad en si s (m ay r, 18 70 )* x x x x x x x x ca m po no tu s pu nc tu la tu s (m ay r, 18 67 )* x x x x ca m po no tu s re ng ge ri (e m er y, 1 89 4) * x x x x x x x x x x x x ca m po no tu s ru fip es (f ab ri ci us , 1 77 5) * x x x x x x x x x x x x x x x x x ca m po no tu s se ne x (s m ith , 1 85 8) * x x x x x x x x ca m po no tu s se ri ce iv en tr is (g ué ri nm én ev ill e, 1 83 8) * x x ca m po no tu s sp 1 x x ca m po no tu s sp 2 x x x ca m po no tu s sp 3 x x x x x x x x x ca m po no tu s sp 4 x x x x x ca m po no tu s sp 5 x x x x x x ca m po no tu s sp 6 x ca m po no tu s vi tt at us (f or el , 1 90 4) ‡ x x x x m yr m el ac hi st a no di ge ra (m ay r, 18 87 )* x x x m yr m el ac hi st a sp x x m yr m el ac hi st a sp 1 x x x x x m yr m el ac hi st a sp 2 x x x m yr m el ac hi st a sp 3 x x x n yl an de ri a sp x pa ra tr ec hi na s p x x x sociobiology 62(2): 228-245 (june, 2015) 239 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae a cr om yr m ex b al za ni (e m er y, 1 89 0) * x x x x x x x a cr om yr m ex c ra ss is pi nu s (f or el , 1 90 9) * x x a cr om yr m ex fr ac tic or ni s (f or el , 1 90 9) * x a cr om yr m ex s p1 x x x x x x a cr om yr m ex s p2 x x x x x x x a cr om yr m ex s p3 x x x x x a cr om yr m ex s p4 x a cr om yr m ex s ub te rr an eu s su bt er ra ne us (f or el , 1 89 3) * x x x x x a pt er os tig m a (g r.p ill os um ) s p1 x x x a pt er os tig m a sp 1 x x a pt er os tig m a sp 2 x att a se xd en s (l in na eu s, 1 75 8) * x x x att a se xd en s ru br op ilo sa (f or el , 1 90 8) * x x o ct os tr um a ih er in gi (e m er y, 1 88 8) ‡ x ca re ba ra s p x x x x x ce ph al ot es a tr at us (l in na eu s, 1 75 8) * x ce ph al ot es m ac ul at us (s m ith , 1 87 6) * x x x ce ph al ot es m in ut us (f ab ri ci us , 1 80 4) * x x x ce ph al ot es p av on ii (l at re ill e, 1 80 9) * x ce ph al ot es p ro x. p al lid oi de s sp x x x x x x x x x x x x x x x x x x ce ph al ot es p us ill us (k lu g, 1 82 4) * x x x x ce ph al ot es s p1 x x x x ce ph al ot es s p2 x ce ph al ot es s p3 x x x cr em at og as te r a cu ta (f ab ri ci us , 1 80 4) * x x x x x x cr em at og as te r a rc ua ta (f or el , 1 89 9) * x cr em at og as te r b ra si lie ns is (m ay r, 18 78 )* x x x fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands240 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae (c on tin ua tio n) cr em at og as te r c om pl ex . c ri no sa s p1 x cr em at og as te r c om pl ex . c ri no sa s p2 x cr em at og as te r e re ct a (m ay r, 18 66 )* x x cr em at og as te r g oe ld ii (f or el , 1 90 3) * x cr em at og as te r m oe lle ri (f or el , 1 91 2) * x x x cr em at og as te r p ro x. e re ct a sp 1 x x x cr em at og as te r p ro x. o bs cu ra ta s p1 x cr em at og as te r s er ic ea (f or el , 1 91 2) * x cr em at og as te r s p x x x x x x x cr em at og as te r s p1 x x x x x x cr em at og as te r s p2 x x x cr em at og as te r s p3 x cr em at og as te r s p4 x cr em at og as te r s p5 x x x cr em at og as te r s p6 x cr em at og as te r s p7 x x cr em at og as te r s p8 x cr em at og as te r s p9 x x cr em at og as te r s p1 0 x x cy ph om yr m ex (g r.r im os us ) s p1 x x x x x x cy ph om yr m ex (g r.r im os us ) s p2 x x cy ph om yr m ex (g r.s tr ig at us ) s p1 x x cy ph om yr m ex (g r.s tr ig at us ) s p2 x cy ph om yr m ex (g r.s tr ig at us ) s p3 x x x x x cy ph om yr m ex le ct us (f or el , 1 91 1) * x cy ph om yr m ex p el ta tu s (k em pf , 1 96 6) * x cy ph om yr m ex s p1 x sociobiology 62(2): 228-245 (june, 2015) 241 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae (c on tin ua tio n) cy ph om yr m ex s p2 x x x x x cy ph om yr m ex tr an sv er su s (e m er y, 1 89 4) * x x x h yl om yr m a ba lz an i ( em er y, 1 89 4) * x x x h yl om yr m a sp x x ka la th om yr m ex e m er yi (f or el , 1 90 7) * x x le pt ot ho ra x sp 1 x x x x le pt ot ho ra x sp 2 x x x le pt ot ho ra x sp 3 x m eg al om yr m ex s p x x x m on om or iu m p ha ra on is (l in na eu s, 1 75 8) * x m yc et ar ot es s p x x m yc et op hy la x em er yi (f or el , 1 90 7) * x x x x x x m yc oc ep ur us g oe ld ii (f or el , 1 89 3) * x x x m yc oc ep ur us s m ith i ( fo re l, 18 93 )* x x m yr m ic oc ry pt a sp x n es om yr m ex p ro x. e ch in ati no di s (f or el , 1 88 6) x n es om yr m ex s pi ni no di s (m ay r, 18 87 )* x x o ch et om yr m ex s em ip ol itu s (m ay r, 18 78 )* x x x x o ch et om yr m ex s p x o xy ep oe cu s pr ox . b ru sc hi s p1 x x o xy ep oe cu s pr ox . b ru sc hi s p2 x x x o xy ep oe cu s sp 1 x x x x x x o xy ep oe cu s sp 2 x o xy ep oe cu s sp 3 x ph ei do le g er tr ud ae (f or el , 1 88 6) * x x x x ph ei do le o bs cu ri th or ax (n av es , 1 98 5) * x x ph ei do le o xy op s (f or el , 1 90 8) * x x x x x x x x fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands242 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae (c on tin ua tio n) ph ei do le ra do sz ko w sk ii (m ay r, 18 84 )* x ph ei do le s p x ph ei do le s p1 x x x x x x x x x x x x x x x x ph ei do le s p2 x x x x x x x x x x x x x x x ph ei do le s p3 x x x x x x x x x x x x x x ph ei do le s p4 x x x x x x x x x x x ph ei do le s p5 x x x x x x x x x x x x x ph ei do le s p6 x x x x x x x x x ph ei do le s p7 x x x x x ph ei do le s p8 x x x x x ph ei do le s p9 x x x x x x x x x x x ph ei do le s p1 0 x x x x x x x x x x x x x ph ei do le s p1 1 x x x x x x x x ph ei do le s p1 2 x x x x x x x x ph ei do le s p1 3 x x x x x x ph ei do le s p1 4 x x x x x ph ei do le s p1 5 x x x x x x ph ei do le s p1 6 x x x x x ph ei do le s p1 7 x x x x ph ei do le s p1 8 x x x x ph ei do le s p1 9 x x x x x x x x ph ei do le s p2 0 x x x x x ph ei do le s p2 1 x x x ph ei do le s p2 2 x x ph ei do le s p2 3 x x ph ei do le s p2 4 x x ph ei do le s p2 5 x x x x sociobiology 62(2): 228-245 (june, 2015) 243 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae (c on tin ua tio n) ph ei do le s p2 6 x x x x ph ei do le s p2 7 x x ph ei do le s p2 8 x x x x x x ph ei do le s p2 9 x x ph ei do le s p3 0 x x x x ph ei do le s p3 1 x x ph ei do le s p3 2 x x ph ei do le s p3 3 x x ph ei do le s p3 4 x ph ei do le s p3 5 x x ph ei do le s p3 6 x x po go no m yr m ex a bd om in al is (s an ts ch i, 19 29 )* x x x x x po go no m yr m ex n ae ge lii (e m er y, 1 87 8) * x x x x x x x po go no m yr m ex s p1 x pr oc ry pt oc er us s p x se ri co m yr m ex s p x x x x x so le no ps is (d ip lo rh op tr um ) s p x x x so le no ps is b on da ri (s an ts ch i, 19 25 )‡ x x x x x so le no ps is g lo bu la ri a (s m ith , 1 85 8) * x x x x x x x so le no ps is s ae vi ss im a (s m ith , 1 85 5) * x x x x x x x x x x x x so le no ps is s p1 x x x x x x x x x x x x x x so le no ps is s p2 x x x x x x x x x x x so le no ps is s p3 x x x x x x x x x x x so le no ps is s p4 x x x x x so le no ps is s p5 x x x x x x x x so le no ps is s p6 x x x x x x so le no ps is s p7 x fv costa, r mello, lana tc, fs neves – ant fauna of rocky grasslands244 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 m yr m ic in ae (c on tin ua tio n) so le no ps is s p8 x so le no ps is s p9 x x st ru m ig en ys c ra ss ic or ni s (m ay r, 18 87 )* x st ru m ig en ys e gg er si (e m er y, 1 89 0) * x st ru m ig en ys e lo ng at a (e m er y, 1 89 0) * x st ru m ig en ys lo ui si an ae (r og er , 1 86 3) * x st ru m ig en ys s ch ul zi (e m er y, 1 89 4) * x x st ru m ig en ys s p x tr ac hy m yr m ex s p1 x x x x x x x x x x x tr ac hy m yr m ex s p2 x x x x x x x x x tr ac hy m yr m ex s p3 x x x tr ac hy m yr m ex s p4 x x x x tr ac hy m yr m ex s p5 x tr ac hy m yr m ex s p6 x tr ac hy m yr m ex s p7 x x x tr ac hy m yr m ex s p8 x x x x tr ac hy m yr m ex s p9 x x x tr an op el ta g ilv a (m ay r, 18 66 )* x x x w as m an ni a au ro pu nc ta ta (r og er , 1 86 3) * x x x x x x x x x x x w as m an ni a sp x po ne ri na e a no ch et us d ie ge ns is (f or el , 1 91 2) * x a no ch et us in er m is (a nd ré , 1 88 9) * x x x h yp op on er a sp x x h yp op on er a sp 1 x x x x x x h yp op on er a sp 2 x x x x x n eo po ne ra b uc ki (b or gm ei er , 1 92 7) ‡ x x sociobiology 62(2): 228-245 (june, 2015) 245 a pp en di x. o cc ur re nc e an d di st ri bu tio n of a nt s pe ci es a lo ng th e al tit ud in al g ra di en t, lo ca lit ie s, a nd s tu di es c ar ri ed o ut in th e b ra zi lia n ro ck y gr as sl an ds . s am pl in g si te s: s er ra d o c ip ó (s c ), it ac ol om i st at e pa rk (i t ), ib iti po ca s ta te p ar k (i b ), an d se rr a do r ol a m oç a st at e pa rk (r m ). n um be rs in c ol um ns in di ca te th e lit er at ur e so ur ce s de sc ri be d in t ab le 1 (c on tin ua tio n) . * sp ec ie s w ith n o oc cu rr en ce in th e sa m pl in g ar ea ; ‡ s pe ci es w ith n o in fo rm at io n on d is tr ib ut io n; † sp ec ie s w ith o cc ur re nc e re co rd ed f or th e sa m pl in g si te . tá xo n a lt it u d es (m ) 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 sc sc sc sc it sc it sc ib it sc rm it sc sc 1 6 7 1 6 7 1 7 7 1 6 7 2 1 7 8 2 1 6 7 8 3 5 1 6 7 4 2 6 7 6 po ne ri na e (c on tin ua tio n) n eo po ne ra v ill os a (f ab ri ci us , 1 80 4) * x x x o do nt om ac hu s ba ur i ( em er y, 1 89 2) * x x o do nt om ac hu s br un ne us (p att on , 1 89 4) * x x o do nt om ac hu s ch el ife r ( la tr ei lle , 1 80 2) * x x x o do nt om ac hu s ha em at od us (l in na eu s, 1 75 8) * x o do nt om ac hu s in su la ri s (g ué ri nm én ev ill e, 1 84 4) * x o do nt om ac hu s m ei ne rti (f or el , 1 90 5) * x o do nt om ac hu s sp 1 x pa ch yc on dy la s p1 x x x pa ch yc on dy la s p2 x x pa ch yc on dy la s tr ia ta (s m ith , 1 85 8) * x x x x x x x x po ne ra s p1 x ps eu do m yr m ic in ae ps eu do m yr m ex (g r. pa lli du s) s p2 x ps eu do m yr m ex (g r.p al lid us ) s p x x x x x x x ps eu do m yr m ex c f. fla vi du lu s x x ps eu do m yr m ex e lo ng at us (m ay r, 18 70 )* x ps eu do m yr m ex g ra ci lis (f ab ri ci us , 1 80 4) * x x x x x ps eu do m yr m ex p up a (f or el , 1 91 1) * x ps eu do m yr m ex s p1 x x x x x x ps eu do m yr m ex s p2 x x x x x x x ps eu do m yr m ex te rm ita ri us (s m ith , 1 85 5) * x x x x x x x x x x x x to ta l n um be r of s pe ci es p er a lti tu de 17 1 12 7 11 1 11 6 11 7 12 1 11 2 45 17 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v67i2.4901sociobiology 67(2): 239-246 (june, 2020) introduction one of the main questions explored in invasion ecology is that of how exotic species become successful in their exotic ranges. the most used explanations to determine the success of invasion are competitive superiority, disturbance, biotic resistance, mutualistic facilitation and enemy release (lowry et al., 2013). another potential explanation is that successful exotic species have some sort of phenotypic plasticity (davidson et al., 2011) that helps them to shift their niches during the colonization of new areas. on the other hand, some studies have shown support for the niche conservatism hypothesis (wiens et al., 2010) in the context of species’ invasions, which predicts that exotic species retain traits related to their niches during the colonization of new areas (petitpierre et al., 2012; aguirre-gutiérrez et al., 2014; guisan et al., 2014; faleiro et al., 2015; but see: atwater et al., 2018). abstract the understanding of how niche-related traits change during species invasion has prompted what is now known as the niche conservatism principle. niche conservatism predicts that species are prone to not change during invasion of new areas and most studies that have tested the niche conservatism principle have focused on the extent to which the species’ climatic niches remain stable in their exotic distribution. however, it is equally important to address how biotic specialization, i.e. resource use, changes during exotic species invasions. here, we use the widespread european honeybee (apis mellifera) to understand whether its grinnelian and eltonian niches changed in its exotic distribution using tests of abiotic and biotic niche conservatism. we found that both niche domains of the european honeybee remained stable in its exotic distribution, which means that neither the climatic niche nor the biotic specialization showed significant differences between the native andthe exotic ranges. our findings that climatic and resource use are coupled can be explained by a. mellifera’s long history of domestication and the possibility that life history traits (e.g., polyandry) may have shaped this species’ large niche over the course of evolution and therefore facilitated exotic ranges colonization. sociobiology an international journal on social insects am medina, m almeida-neto article history edited by evandro nascimento silva, uefs, brazil received 11 december 2019 initial acceptance 31 december 2019 final acceptance 21 january 2020 publication date 30 june 2020 keywords apis mellifera; invasive species; climatic niche; niche breadth; niche overlap; resource use. corresponding author anderson matos medina departamento de ecologia, laboratório de interações ecológicas e biodiversidade universidade federal de goiás goiânia, goiás, brasil. e-mail: bojaum@gmail.com a species’ niche can be divided into two broad components: the grinnellian domain, which corresponds to how a species depends on certain environmental conditions and tolerates variations in such conditions; and the eltonian domain, which corresponds to how a species interacts with other species and uses its biotic resources (soberón & nakamura, 2009; soberón, 2007). both domains play a role in how an exotic species colonizes new areas. for example, an exotic species needs to tolerate the climatic conditions ofits exotic range and interact with different sets of antagonistic and mutualistic species (guisan et al., 2014; richardson et al., 2000). however, there is a bias in the literature toward the grinnelian domain, specifically in those studies that have evaluated the niche conservatism hypothesis (aguirre-gutiérrez et al., 2014; parravicini et al., 2015), wherein much less attention was paid to the eltonian domain (but see olallatárraga et al., 2017). departamento de ecologia, laboratório de interações ecológicas e biodiversidade, universidade federal de goiás, goiânia, goiás, brazil research article bees grinnelian and eltonian niche conservatism of the european honeybee (apis mellifera) in its exotic distribution am medina, m almeida-neto – grinnelian and eltonian niche conservatism: the case of a widespread exotic bee240 a more comprehensive understanding of niche conservatism can be achieved if both the grinnellian and the eltonian domains were evaluated at the same time (larson et al., 2010) rather than assuming they are congruent (soberón & nakamura, 2009). under niche conservatism, the importance of environmental conditions in limiting the spread of exotic species will depend on the extent to which environmental conditions are different between native and exotic ranges. the higher the difference in environmental conditions between native and exotic ranges, the higher the chance of a difference between the native and exotic ranges of a species’ realized climatic niche; if this difference is high, then the species is under niche shift (hill et al., 2017). furthermore, if the same rationale of niche conservatism applies to the eltonian domain, then the resource specialization of a species in its exotic range should not be higher than its resource specialization in its native range (emer et al., 2016). on the other hand, an increase in the set of resources used indicates biotic niche expansion; actually, this is analogous to the interaction release hypothesis (traveset et al., 2015), whereby a colonizing species acquires new interactions due to the absence of competitors or predators. in the present study, we used the european honeybee apis mellifera linnaeus, 1758 (hymenoptera, apidae) to evaluate whether limits in the exotic distribution of a widespread species can be explained by the grinnelian and eltonian domains of niche conservatism. if the grinnelian niche is conserved, then we could expect that the honeybee would not expand its climatic niche in exotic ranges in comparison to native ranges. alternatively, if the eltonian niche is conserved, then we could expect to find that the honeybee would be restricted in terms of the number of plants that it could visit in exotic ranges in comparison to native ranges. therefore, on the biogeographic scale, it is expected that the honeybee would interact with fewer plant lineages in its exotic range than in its native range. additionally, at the local scale, the honeybee would have a smaller niche breadth in its exotic distribution in comparison to its local niche breadth in its native distribution. a. mellifera is a particularly good model organism because of its widespread distribution, super generalist behavior (norfolk et al., 2018), and importance to the pollination of plants in natural habitats (hung et al., 2018). methods grinnelian niche we gathered a. mellifera’ soccurrences using the global biodiversity information facility database (gbif.org 2017) and retrieved a total of 88.653 points. afterward, we removed occurrences without geographic coordinates and duplicated points, ultimately keeping 19.416 points (fig 1a). we separated these occurrence data for the analysis of climatic niche on a native set (africa, europe, and middle east) and an exotic set (americas, asia, and oceania) because a. mellifera is native to africa, europe, and the middle east, but has exotic populations in americas, asia and oceania (han et al., 2012; moritz et al., 2005). we considered island populations in the seychelles and mauritius as native because there is genetic evidence of a. mellifera subspecies colonization before human settlements in these regions (techer et al., 2017). we opted to neglect a. mellifera subspecies differences because subspecies are known to interbreed (schneider et al., 2004), and no data was available to incorporate subspecies information in the climatic and biotic analyses. we downloaded bioclim variables from worldclim (fick & hijmans, 2017) with a spatial resolution of five minutes (ca. 10 km2 at the equator line) and selected variables with correlations less than 0.8 to avoid multicollinearity. we selected the following variables: annual mean temperature (bio 1), mean diurnal range (bio 2), temperature seasonality (bio 4), annual precipitation (bio 12) and precipitation seasonality (bio 15). variations in temperature are predictors of visitation frequency by a. mellifera (hung et al. 2018), and should, therefore, be good predictors of the climatic niche of the species. we used the framework developed by broennimann et al. (2012) to analyze climatic niche; this approach uses both the climatic space occupied (occurrences) and the climatic space available (background) to create an environmental principal component analysis (pca-env). the first axis of the pca-env accounted for 48.55% of the variation of the climatic variables while the second axis accounted for 32.16% of the variation. contributions of each environmental variable to the axis of the pca-env are shown in figure s1. this pca-env allows for the comparison of the climatic niches of a species in two regions, taking three components into consideration: niche stability, niche expansion, and niche unfilling (guisan et al. 2014). niche stability is the portion of the analogue climatic space available in both ranges used by a species. niche expansion is the portion of climatic space that a species uses in a new region but does not use in the other. lastly, niche unfilling is the portion of climatic space that is present in both regions but is not used by the species. in order to evaluate the grinnelian niche conservatism we used niche similarity and equivalency tests (warren et al., 2008). niche similarity randomizes the distribution of the exotic range, keeping the native range fixed and calculates the probability that niche overlap is caused by a random distribution in the exotic range. niche equivalency randomizes the distribution in both the native and exotic ranges and calculates the probability that niche overlap is caused randomly. we performed the pca-env, niche similarity test, and niche equivalency test using the r package ecospat (broennimann et al., 2015; di cola et al., 2017). eltonian niche we gathered 108 plant-pollinator networks that included a. mellifera from the web of life: ecological networks database (www.web-of-life.es). also, we complemented these networks with a non-systematic search that yielded 16 plant-pollinator networks and plant-pollinators inventories. the combination sociobiology 67(1): 239-246 (june, 2020) 241 of both data sets resulted in a total of 124 plant-pollinator networks (fig 1b; see table s1 for references). using these networks, we measured a. mellifera’s niche breadth using the standardized degree, a measure of dietary specialization which takes into account how many plants were visited by a. mellifera divided by the number of plants used by all pollinators (including a. mellifera). the standardized degree had the benefit of reducing the effects of networks with different sampling efforts because sampling effort can affect the richness of plants and pollinators found in networks. to measure the phylogenetic diversity of plants consumed by a. mellifera, we used the phylomatic tool (webb & donoghue, 2005) to build a phylogenetic tree for the 1843 plant species found in the plant-pollinator networks based on the megatree of the phylogenetic hypothesis of the angiosperm phylogeny group (r20120829). plants identified only at the genus (n=121) or family (n=32) levels were inserted as polytomies. we used the phylogenetic tree to calculate the mean phylogenetic distance of plants used by a. mellifera and the total mean phylogenetic distance of the network using the picante r package (kembel et al., 2010). for each plant-pollinator network, we calculated a standardized phylogenetic distance of plants used by a. mellifera by dividing the mean phylogenetic distance used by a. mellifera by the total mean plant phylogenetic distance. mean phylogenetic distance was not measured in four exotic networks and 30 native networks because a. mellifera used only one plant in each of these networks. at the biogeographic level, we compared the similarity between plant lineages used by a. mellifera in its native range and plant lineages used by a. mellifera in its exotic range by pooling all plants used in the local networks for both ranges. we measured the species phylogenetic similarity using the phylosor index in the betapart r package (baselga et al., 2013). we partitioned the phylogenetic similarity into two components: the turnover component, corresponding to the eltonian niche shift of the plant lineages visited by a. mellifera; and the richness component, corresponding to the eltonian niche expansion or unfilling of these plant lineages. fig 1. world map with the geographic distribution of the apis mellifera linnaeus, 1758 dataset. areas in grey denote exotic distribution, and white areas denote native distribution. exotic occurrences are indicated by white squares, and native occurrences are indicated by grey circles (a). white squares indicate networks in which a. mellifera is exotic, while grey circles indicate networks in which a. mellifera is native (b). am medina, m almeida-neto – grinnelian and eltonian niche conservatism: the case of a widespread exotic bee242 we tested the eltonian niche conservatism of a. mellifera using two different scales. on the biogeographic scale, a permutation test was used to determine the similarity (three components) between plant speciesused by a. mellifera in its native distribution and plant species used by a. mellifera in its exotic distribution. this permutation was akin to the niche similarity test sensu warren et al. (2008), which tests if the plant species used in the exotic range was different from that which occurred in a random invasion. therefore, the use of plants by a. mellifera was randomized in the exotic rangeand kept fixed, in the native range. furthermore, the richness of plant species used by a. mellifera in the exotic range was kept fixed and plant species were kept in their original distributions (native or exotic). on the local scale, the effect of a. mellifera’s range on the standardized degree and standardized mean phylogenetic distance was evaluated using two anovas. all analyzes were performed in the r programming environment (r core team 2017). results grinnelian niche the climatic niche of a. mellifera in its native range was more similar to its climatic niche in its exotic range than expected through random invasion (schoener overlap d = 0.395; p < 0.01; fig 2). the same trend was found in the results of the niche equivalency test, in which both climatic niches were randomized (p < 0.01; fig 2). the climatic niche was highly stable between both distributions (niche stability = 91.94%; fig 3), with a low niche expansion in the exotic range (8.06%) and almost no niche unfilling (0.04%). fig 2. niche similarity test and niche equivalency test comparing apis mellifera linnaeus, 1758 climatic niche overlap between its native and exotic ranges using 1000 randomizations of the exotic range. in the niche similarity, only the native range was randomized while in the niche equivalency test both ranges are randomized. fig 3. climatic environment based on a principal component analysis of the environment (pca-env) of apis mellifera linnaeus, 1758. the blue polygon corresponds to niche overlap, the red polygon corresponds to niche expansion, and the green polygon corresponds to niche unfilling. solid lines represents the total native (red) and total exotic (green) ranges and dashed lines represents 75% of the ocurrences of native (red) and exotic ranges (green). eltonian niche on the biogeographic scale, we found that a.mellifera used 50.39% of the available plants in its native range; whereas it only used 25.36% of the available plants in its exotic range. furthermore, phylogenetic similarity was lower than that expected from a random invasion (phylosor = 0.499; p < 0.001). specifically, the turnover component of phylogenetic similarity was higher than expected from a random invasion (phylosor = 0.341; p < 0.001), while the richness component of phylogenetic similarity was lower than expected from a random invasion (phylosor=0.159; p<0.001). on the local scale, we found that the standardized degree did not differ between native and exotic distributions (f1, 167 = 0.0036; p = 0.952; fig 4a). furthermore, we found that the mean phylogenetic distance of plant resources used by a. mellifera did not differ between distributions (f1, 133 = 0.369; p = 0.545; fig 4b). sociobiology 67(1): 239-246 (june, 2020) 243 discussion the climatic niche of a.melliferain its native range was conserved during its invasion of new ranges. this finding is in line with results of previous studies that showed evidence of niche conservatism in a. mellifera subspecies (vital et al., 2012). this honeybee species has a long history of domestication by human populations (bloch et al., 2010), which has helped the species achieve a global distribution and increased genetic diversity due to admixture (harpur et al., 2012; wallberg et al., 2014). in parallel, a. mellifera is among the most polyandrous of social insects, and this pattern increases population genetic diversity. an increase in genetic diversity buffers colonies from diseases (tarpy, 2003) and may also help a. mellifera occupy different climatic conditions. indeed, polyandrous colonies accumulate resources faster and have higher rates of winter survival than their monoandrous counterparts (mattila & seeley, 2007). furthermore, introductions of multiple subspecies can boost the spread of honeybees and the success of colonization (schneider et al., 2004), because phenotypic differences may help in the colonization of regions with different environmental conditions. we also found that a. mellifera conserved its eltonian niche on the regional and local scales. because a. mellifera uses a broad range of plant species in its native and exotic distributions, a. mellifera can interact with a similar number of plants found in a particular climatic region after the climatic barriers are overridden (emer et al., 2016). indeed, one study has shown that plants with two different modes of pollination (hummingbirds and bats) had similar climatic niches because their pollinators’ climatic niches did not differ (alexandre et al., 2017). therefore, if the climatic niche of a. mellifera overlaps with that of most of other native pollinators, then the possibility of interactions with other native pollinators’ host plants will increase. changes in community species richness may lead to a niche shift in pollinators due to mutual competition (fründ et al., 2013). additionally, if a. mellifera visits plant species with different floral traits from multiple lineages in its native range, then the possibility of interaction with native plants outside its native range will increase. indeed, previous studies have shown that pollinator overlap was higher for exotic plants with traits more similar to those of native plants (gibson et al., 2012; but see: montero-castaño & vilà, 2017) and that a. mellifera can incorporate exotic plants in its diet (monterocastaño & vilà, 2017).the ability of a. mellifera to use pollen from various plants to produce honey is demonstrated by the global increasing trend in honey yield (aizen & harder, 2009), notwithstanding crops pollination and fruit set are higher with pollination driven by wild insects than compared to honeybee visitations (garibaldi et al., 2013). moreover, even with the ongoing scenario of a. mellifera importance to agriculture is important to carefully understand possible impacts to native species, especially considering that honeybees can consume pollen much faster than native species (cane & tepedino, 2017). besides competing for resources, invasive pollinators can transmit pathogens, cause reproductive disruptions of native bees and modification of native plants communities (stout & morales, 2009). for example, honeybee spillovers from cultivated areas to native woodlands can have negative effects in the structure of plant-pollinator networks and seed set of native plants (magrach et al., 2017). our finding that the niche expansion of a. mellifera was below 10% differs from the results of a recent review (hill et al., 2017) that showed that most invasive insects often undergo fig 4. effect of distribution on apis mellifera’s linnaeus, 1758 resource use in plant-pollinator networks. standardized degree of plants used by a. mellifera (a). mean phylogenetic distance (mpd) of plants used by a. mellifera (b). square indicate means and vertical bars indicate standard errors. am medina, m almeida-neto – grinnelian and eltonian niche conservatism: the case of a widespread exotic bee244 niche shifts during the invasion process. indeed, a. mellifera differs from other insects in that it is actively spread through human beekeeping activities but can also spread by accidental releases in the wild, as was the case in the american continent (moritz et al., 2005). furthermore, we found major support for niche conservatism congruence between the grinnelian and eltonian domains. however, this finding should not encourage disregard for the eltonian domain. previous studies have shown that interactions are not irrelevant as predicted by the eltonian niche hypothesis (araújo et al., 2014) and that domains can be decoupled (larson et al., 2010). the next step for future research is to gain an understanding of how the anthropogenic domestication of a. mellifera has shaped the species’ niche over the course of evolutionary time and whether this domestication played a role in the coupling of the eltonian and grinnelian domains. authors contribution am medina conceived the idea; 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(2010). niche conservatism as an emerging principle in ecology and conservation biology. ecology letters, 13(10): 1310–24. doi: 10.1111/j.1461-0248. 2010.01515.x open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.738sociobiology 62(3): 439-445 (september, 2015) diversity of social wasps (hymenoptera: vespidae: polistinae) in an agricultural environment in bambuí, minas gerais, brazil introduction social wasps (vespidae) are insects belonging to the order hymenoptera, and may play an important ecological role in the environment, acting as pollinators (hunt, 1991; brodmann, 2008; mello et al., 2011) and predators (prezoto & machado, 1999; prezoto et al., 2006; silveira et al., 2008; gomes & noll, 2009). this family includes solitary species (euparigiinae, masarinae and eumeninae), and others with different degree of sociality (stenogastrinae, polistinae and vespinae) (carpenter, 1993; carpenter & marques, 2001). in this family, those wasps belonging to the subfamily polistinae are eusocial, cosmopolitan and highly diverse in the neotropical region (carpenter et al., 1996; carpenter & marques, 2001; silveira, 2002). brazilian species of polistinae belong to three tribes, namely polistini, mischocyttarini and epiponini, with 23 genus e 319 species (carpenter & marques, 2001). abstract studies on the diversity of social wasps in agricultural environments represent an important step to identify ideal species to be used in biological pest control programs. there is a growing effort to recognize the diversity of these insects, but information on anthropized environments is still rare. this study focused on obtaining data on the diversity of social wasps in a predominantly agricultural area in bambuí, minas gerais, brazil, and identifying, through dominance, species with potential use in biological control studies. sampling was conducted from july 2012 to july 2014 with two type of wasps’ capture methods: attractive traps and active search. this research confirms that a well diversified environment, even if anthropized, is rich in social wasp species. in addition, the great number of collected species shows the importance of a long-term survey and the use of more than one method of capture. the high rate of collections of polistes versicolor in a predominantly agricultural environment, coupled with other studies on this species as a predator of lepidopteran caterpillars, suggests the use of this species as a tool in biological control of pests. sociobiology an international journal on social insects gc jacques1,2, mm souza3, hj coelho1, lo vicente1, lcp silveira2 article history edited by fernando barbosa noll, unesp, brazil received 29 december 2014 initial acceptance 08 march 2015 final acceptance 26 may 2015 keywords biodiversity, polistes versicolor, polistinae. corresponding author gabriel de castro jacques departamento de entomologia universidade federal de lavras 37200-000, lavras-mg, brazil e-mail: gabriel.jacques@ifmg.edu.br the survival of these social wasps depends on their success in creating new colonies (dejean et al, 1998; hunt, 2007). the choice for a nesting site depends on certain morphological characteristics, such as protection from rain and predators (e.g., ants and vertebrates), besides proximity to food resources and material for nest construction (andena et al., 2009; santos et al., 2010; souza et al., 2010; souza et al. 2014a). disturbances caused by humans in natural environments are the main factors which may reduce their biodiversity (samways, 2007). social wasps act as pollinators (suhs et al., 2009) and the extinction of a pollinator can cause loss of plant species and trigger a “cascade of linked extinctions” (myers, 1986). some hymenoptera species are sensitive to variations of abiotic conditions (light, temperature and humidity), which may be related to changes in the level of environment degradation, thus representing potential environmental indicators (souza et al., 2010). research article wasps 1 instituto federal de educação, ciência e tecnologia de minas gerais, bambuí, mg, brazil 2 universidade federal de lavras, lavras, mg, brazil 3 instituto federal de educação, ciência e tecnologia do sul de minas, inconfidentes, mg, brazil gc jacques et al diversity of social wasps in agricultural environments440 biological inventories are the first step for the development of preservation studies, since it is essential to know first of all which resources are available in a particular area (elpino-campos et al., 2007). social wasps are easily sampled because they forage and return to a core area (nest). the survey and identification of these insects, especially in predominantly agricultural environments, are the first steps to identify ideal species to use in biological control of pests programs (prezoto et al., 2006). several studies have been developed on the diversity of species of social wasps in brazil, however, most of them focused only natural environments (silveira, 2002; silva-pereira & santos, 2006; souza & prezoto, 2006; elpino-campos et al., 2007; santos et al. 2007; silveira et al., 2008; gomes & noll, 2009; silva & silveira, 2009; santos et al., 2009; souza et al., 2010; bonfim & antonialli junior, 2012; simões et al., 2012; souza et al., 2012; grandinete & noll, 2013; locher et al. 2014; souza et al., 2014a, 2014b). therefore, the fauna of wasps in anthropized environments is still poorly known (jacques et al., 2012). the objective of this study was to obtain data on the diversity of social wasps in a predominantly agricultural area, instituto federal de educação, ciências e tecnologia de minas gerais (ifmg), campus bambuí, minas gerais, brazil, and to identify, through dominance, species with potential for use in biological control studies. material and methods this research was conducted at the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg), campus bambuí, minas gerais, brazil. the campus has a total area of 328 ha, with one anthropic site, but very diverse, with a predominance of buildings and agricultural areas. an area with 175 hectares is used for agricultural crops (corn, beans, sugar cane, orange, banana, coffee and vegetables) and pastures, and 34 acres of buildings, most being close to the cultivated areas. sampling was conducted in agricultural environments from july 2012 to july 2014 with two types of sampling techniques: attractive traps and active search. the traps were made with two-liter plastic soft-drink bottles, with three triangular lateral openings (2 x 2 x 2 cm) at a 10 cm distance from the base (souza & prezoto, 2006). substances used to attract insects were: 1natural passion fruit juice (passiflora edulis f. flavicarpa deg. passifloraceae) prepared with 1 kg of fruit blended with 250g granulated sugar plus two liters of water; 2sardine broth (sardinella brasiliensis steindachner 1789), which included two cans of sardines plus two liters of water; 3pure honey; 4 sugarcane molasses diluted to 50%. five bottles with 150 ml attractive substance for each type of bait, were assembled in eight dates, with total for forty bottles per attractive substance type (160 traps in total). these traps were set up next to the cultivars at the ifmg campus, at a 1.5 m height above the ground. the traps were active for seven days. after that time, the wasps collected were removed and preserved in 70% alcohol for later identification. active searches were conducted throughout the agricultural area of the campus bambuí at the ifmg. trunks of trees and other natural cavities, broad-leaved vegetation, flowers and edifications were surveyed and the wasps were collected with the aid of entomological nets (souza & prezoto, 2006; elpino-campos et al., 2007). species collected were identified using entomological keys (richards, 1978; carpenter, 2004). diversity was calculated by the shannon-wiener (h‘) index, and the dominance by the berger-parker index (dpb), through the dives program diversity of species v3.0.3, in the base 10 logarithmic (rodrigues, 2014). results and discussion five hundred and twenty-seven social wasps of 8 genus and 29 species were collected, with a total diversity index of 1.7406 (table 1). this great richness of species, compared with that observed by other authors (table 2), is explained by the fact that many species of social wasps present high level of synanthropism (fowler, 1983; michulleti et al, 2013). the study area has a very diverse environment, which may help explain the large number of species collected, since environments that are structurally more heterogeneous and complex may favor the coexistence of a larger number of species due to the greater availability of microhabitats, greater protection against predators and greater disposal and diversity of food resources and substrate for nidification (santos et al, 2007; souza et al, 2012). there was high dominance (dpb= 0.2789) of few species. polistes versicolor (olivier, 1791) presented the greater rate of total frequency (36.81%), it has been collected 194 times. it also had the higher frequency in capture by attractive traps (34.35%) and active search (47.47%). such occurrence may be explained by the fact that there are p. versicolor nests in both urban buildings and vegetation (oliveira et al., 2010; torres et al., 2014a), which makes it easy to be found. wasps of polistes are excellent predators of agricultural pests, especially caterpillars of lepidoptera (prezoto et al., 2006; elisei et al., 2010; souza et al., 2013). thus, the agrarian environment of the campus may have provided a favorable environment for p. versicolor, because the caterpillars present in different agricultural areas are the main feed of immature insects that develop in the colony (raveret-richter, 2000). in 120 hours of observation, 315 returns of p. versicolor foragers included preys, mostly lepidopteran caterpillars (elisei et al., 2010). eighty-nine preys (95% of lepidoptera) were captured by this species in juiz de fora, mg (prezoto et al., 2006). the predatory activity of this wasp was also studied on chlosyne lacinia saundersii (doubleday & hewitson, 1849) (lepidoptera: nymphalidae) (campos-farinha & sociobiology 62(3): 439-445 (september, 2015) 441 active search traps total species abundance frequency relative (%) abundance frequency relative (%) abundance frequency relative (%) 1 agelaia centralis (cameron, 1907) 1 1.01% 127 29.67% 128 24.29% 2 agelaia multipicta (haliday, 1836) 1 1.01% 47 10.98% 48 9.11% 3 apoica gelida van der vecht, 1973 0 0.00% 2 0.47% 2 0.38% 4 brachygastra lecheguana (latreille, 1824) 1 1.01% 2 0.47% 3 0.57% 5 mischocyttarus bahiae richards, 1949 1 1.01% 0 0.00% 1 0.19% 6 mischocyttarus cassununga (r. von. ihering, 1903) 2 2.02% 3 0.70% 5 0.95% 7 mischocyttarus cerberus (richards, 1940) 1 1.01% 1 0.23% 2 0.38% 8 mischocyttarus drewseni sausurre, 1857 2 2.02% 2 0.47% 4 0.76% 9 mischocyttarus ignotus zikán, 1949 3 3.03% 0 0.00% 3 0.57% 10 mischocyttarus latior (fox, 1898) 1 1.01% 0 0.00% 1 0.19% 11 mischocyttarus matogrossensis zikán, 1935 1 1.01% 0 0.00% 1 0.19% 12 mischocyttarus nomurae richards, 1978 1 1.01% 0 0.00% 1 0.19% 13 mischocyttarus paraguayensis zikán, 1935 6 6.06% 0 0.00% 6 1.14% 14 mischocyttarus rotundicolis (cameron, 1912) 12 12.12% 1 0.23% 13 2.47% 15 polistes actaeon haliday, 1836 1 1.01% 0 0.00% 1 0.19% 16 polistes satan bequaert, 1940 3 3.03% 8 1.87% 11 2.09% 17 polistes simillimus zikán, 1951 2 2.02% 21 4.91% 23 4.36% 18 polistes versicolor (olivier, 1971) 47 47.47% 147 34.35% 194 36.81% 19 polybia bifasciata saussure, 1854 1 1.01% 0 0.00% 1 0.19% 20 polybia chrysothorax (lichtenstein, 1796) 1 1.01% 12 2.80% 13 2.47% 21 polybia erythrothorax (richards, 1978) 2 2.02% 0 0.00% 2 0.38% 22 polybia ignobilis (haliday, 1836) 0 0.00% 25 5.84% 25 4.74% 23 polybia jurinei saussure, 1854 0 0.00% 18 4.21% 18 3.42% 24 polybia occidentalis (olivier, 1971) 2 2.02% 2 0.47% 4 0.76% 25 polybia paulista (r. von. ihering, 1896) 4 4.04% 0 0.00% 4 0.76% 26 polybia rejecta (fabricius, 1978) 1 1.01% 0 0.00% 1 0.19% 27 polybia sericea (olivier, 1971) 0 0.00% 10 2.34% 10 1.90% 28 protopobybia sedula (saussure, 1854) 1 1.01% 0 0.00% 1 0.19% 29 synoeca cyanea (fabricius, 1775) 1 1.01% 0 0.00% 1 0.19% total of individuals 99 428 527 richness of species (s`) 25 16 29 shannon-wiener (h`) index 0,9402 0,8004 1,7406 berger-parker (dpb) index 0,4747 0,3435 0,2789 table 1. richness, diversity and dominance of social wasp species collected at the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg), campus bambuí, minas gerais, brazil. september, 2014. gc jacques et al diversity of social wasps in agricultural environments442 pinto, 1996) and on heraclides anchysiades capys (hübner, 1809) (lepidoptera: papilionidae) (marques, 1996, 2005). moreover, the presence of wasps of the genus polistes in different cultures is associated with reduced damage caused by pests on cotton (kirkton, 1970), tobacco (lawson et al., 1961), cabbage (gould & jeanne, 1984), coffee (gravena, 1983) and corn (prezoto & machado, 1999), showing the importance of this genus for studies on the biological control of pests. the most collected genus was mischocyttarus de saussure, 1853, with 10 species, and mischocyttarus bahiae richards, 1949 was first recorded for the state of minas gerais, which shows the importance of surveys on social wasp richness and diversity. nests of mischocyttarus are easily found in edifications, making it easy to locate it (alvarenga et al., 2010). moreover, this genus consists of the largest group of social wasps, with 245 species of eleven subgenera, which added to fact that only a few have been studied for richness and diversity, increases the chance of unpublished records (cooper, 1998; silveira, 2008). of the 28 species found, 25 were located in their colonies. this was due to the long period of data collection, which is important, because collection must be carried out in the warmer and wetter periods of the year, considering that there is a positive correlation between these abiotic factors and the number of species and colonies (souza et al., 2014a). this is due to a greater availability of nesting sites, protection from predators and increased availability and diversity of food resources on those periods (prezoto et al., 2006; elpinocampos et al., 2007). the methodology of active search, considering the richness of the collected species, was more effective than the use of traps (table 1), whereas sixteen species were collected solely through this method. this has also been observed in other works (silveira, 2002; souza & prezoto, 2006; elpinocampos et al., 2007; jacques et al., 2012; loucher et al., 2014; souza et al., 2014b). four species, apoica gelida van der vecht, 1973, polybia ignobilis (haliday, 1836), polybia jurinei saussure, 1854 and polybia sericea (olivier, 1971), were collected solely through traps. this may be related to the fact that social species have the habit of nesting in one place and feeding in another (pereira & santos, 2006). furthermore, the genus apoica lepeletier, 1836 are generally captured by traps, because they mainly forage at night (hunt et al., 1995; pickett & wenzel, 2007), making it less likely to be captured by active search during the day. these results demonstrate the importance of using more than one method to record the highest possible number of wasp species (jacques et al., 2012). this work confirms that a well-diversified environment, even if anthropized, such as the campus bambuí of the instituto federal de educação, ciência e tecnologia de minas gerais (ifmg), brazil, includes a rich amount of species of social wasps. in addition, the great number of collected species, shows the importance of a long-term survey and the use of more than one method of collection. the high rate of collections of polistes versicolor in a predominantly agricultural environment, coupled with other studies on this species as a predator of lepidopteran caterpillars, suggests the use of this species as a tool in the biological control of pests. researches number of species silveira, 2002 (amazon rainforest) 79 silva & silveira, 2009 (amazon rainforest) 65 souza et al., 2014a (cerrado) 38 souza et al., 2012 (atlantic forest) 38 souza & prezoto, 2006 (cerrado and semidecidual forest) 38 souza et al., 2010 (riparian) 36 simões et al., 2012 (cerrado) 32 locher et al., 2014 (riparian vegetation) 31 *present work (agricultural environment) 29 elpino-campos et al., 2007 (cerrado) 29 souza et al., 2014b (riparian) 28 jacques et al., 2012 (anthropized environment) 26 grandinete & noll, 2013 (cerrado) 22 santos et al., 2009 (cerrado) 19 bonfim & antonialli junior, 2012 (riparian vegetation) 18 santos et al., 2007 (atlantic forest) 18 santos et al., 2007 (restinga vegetation) 16 silva-pereira & santos, 2006 (campos rupestres) 11 arab et al., 2010 (atlantic forest) 10 santos et al., 2007 (mangrove) 8 gomes & noll, 2009 (semidecidual forest) 7 table 2. comparison between the total number of species in this research work (*) and other surveys in the literature. agelaia centralis (cameron, 1907) and agelaia multipicta (haliday, 1836) also showed high frequency in the community in collections using attractive traps (29.67% and 10.98%, respectively). some species of genus agelaia lepeletier, 1836 can build colonies with a population estimated in up to a million of adults (zucchi et al., 1995), which means a greater ability of foraging by a greater amount of wasps and increased chances of finding specimen of this group (hunt et al., 2001). furthermore, these wasps have a scavenger habit, especially being collected by the attractive sardine broth, which is used as an additional source of protein for their larvae (prezoto & souza, 2006). the high prevalence of this genus was also reported in different ecossystems in brazil (gomes & noll, 2009; 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(1995). agelaia vicina, a swarm-founding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103:129-137. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.732sociobiology 62(3): 417-425 (september, 2015) comparative study of resistance and feeding preference of 24 wood species to attack by heterotermes indicola (wasmann) and coptotermes heimi (isoptera: rhinotermitidae) in pakistan introduction termites are the major constituents of the forest ecosystem in the tropical and subtropical areas and they are well known for their capacity to damage and destroy wood and wood products of all kinds (rashmi & sundararaj, 2013). the interaction of soil type and moisture availability influences the distribution of foraging termites in microhabitats (mary & weste, 2010). a lot of research has been done on wood resistance against termites by using various timbers but very little on bamboos (nirmala & kenneth, 2011). abstract the present study was conducted to evaluate the laboratory and field preference of 24 pakistani wood species by termite species heterotermes indicola and coptotermes heimi. the wood species evaluated, regarding attack and damage were: azadirachta indica (neem), pinus roxberghii (chir), dalbergia sissoo (sheesham), populus deltoides (popular), albizzia lebbeck (shirin), abies pindrow(fir), alstonia scholaris (alstonia), erythrina suberosa (gul-e-nister), eucalyptus citriodora (safaeda), ficus religiosa (bohar), heterophragma adenophyllum (beeri patta), melia azedarach (derek), pinus wallichiana (chir), terminalia arjuna (arjun), acacia arabica (kikar), betula utilis (birch) cedrus deodara (deodar), cordia oblique (lasura), mangifera indica (aam),ehretia serrata moringa oleifera (sohanjana), putranjiva roxburghii (lucky bean), syzygium cumini (jaman)and zizyphus jujube (berry). two weeks laboratory and four weeks field feeding trials were performed as described in standards of the american wood protection association. samples of each of the 24 wood species were individually exposed to 100 termites (10% soldiers); and termite mortality, wood mass loss and visual appearance of the samples (on a scale of 0-10) were recorded. results indicated that by no choice feeding assay, populus deltoides was the most preferred and dalbergia sissoo was the least preferred among the 24 wood species tested in laboratory against heterotermes indicola and coptotermes heimi. field studies by no choice feeding test against mixed termites and c. heimi indicated d. sissoo the least palatable and mangifera indica the most palatable wood. so it is recommended that though all 24 species evaluated in the present study differ in their susceptibility to termite attack, they would require additional protection to avoid termite attack. sociobiology an international journal on social insects f manzoor1, muneeza abbas1, mu latif2 article history edited by alexandre vasconcellos, ufpb, brazil received 03 december 2014 initial acceptance 25 may 2015 final acceptance 29 june 2015 keywords h. indicola; c. heimi; wood mass loss; laboratory test; no choice. corresponding author farkhanda manzoor department of zoology, lahore college for women university, lahore, pakistan e-mail: doc_farkhanda@yahoo.com in pakistan, the most common termite species causing damage to wood and wooden structures are coptotermes heimi (wasmann) and heteroterme indicola (wasmann) and microtermes obesi (holmgren) and odontotermes obesus (rambur) heterotermes indicola has become major structural pest of wood and wooden structures inside houses in pakistan and has been ranked as the most destructive termite species of the lahore. it not only destroys wood but has been found damaging paper, clothes and any cellulosic material (manzoor, 2010) coptotermes heimi (wasmann) is a widely distributed termite in pakistan and causes damaging effects in standing trees (khalid & hina, 2014). termites research article termites 1 lahore college for women university, lahore, pakistan 2 government degree college (boys), raiwind, lahore, pakistan f manzoor, mu latif – feeding preference of termites: a comparison of 24 wood species in pakistan418 are often regarded as decomposers of lignocellulosic waste (lenz, m. et al., 2011; shaomei et al., 2013). the ability of termites to degrade lignocellulose gives them an important place in the carbon cycle (brune, 2014). inside houses,the infestation of h. indicola is judged by galleries running on walls and ceilings. during survey of houses, it was also observed that it only eats softer part of wood and after attacking wood it always has connection with ground soil (its breeding place). its tunnels can be observed as hanging food tunnels made of mud tubes (manzoor, 2010). many wood species in pakistan are utilized as food by termites and certain wood species are preferred by others. a study by sheikh et al. (2010) showed that workers of o. obesus prefer fagus sp. (beech) and pinus wallichiana (kail) more whereas they least prefer abies pindrow (pertal) and cedrus deodara (diar). the importance of termite resistant (durable) timbers is environment friendly and cheap source to protect timber-in-service from attack and damage in pakistan. termite attack is related to the presence of resistant components which are not equally distributed to all part of plant. the occurrence of organic chemicals such as phenol, quinones, terpenoids and high concentration of lignin may also affect the areas where feeding takes place (ayesha & sumbal, 2012). heartwood and sapwood contains maximum concentration of components and top of stem contains minimum (henderson et al. 2001). sometimes the presence of essential oils also protects plants from termite attack. elahe et al. (2014) in their study found that eucalyptus essential oil may be an effective toxicant with suitable contact and digestive toxicity on microcerotermes diversus. some of the tropical forest plantation species having natural resistance to termites may offer an alternative for the use of chemicals products (peralta et al., 2004). factors affecting wood consumption by termites are numerous and complexly related (peralta et al., 2004). most important factors are wood species, hardness, presence of toxic substances, feeding inhibitors or deterrents, presence or absence of fungi, degree of fungal decay, moisture content of wood and soil (carter & smythe, 1974; nagnan and clement, 1990). qureshi et al. (2012) found in their study that the death of the termites is due to the mortality of their protozoan population during the period of experimentation which appears to be due to the toxic effect of corresponding wood and not because of non-feeding of the woods. it was also studied by morales-ramos and rojas (2001) that different wood combinations offered to termites in laboratory trials are important in choice feeding tests. keeping in view the significance of the above mentioned studies, the purpose of present study was to test the feeding preference of different wood species against two subterranean termite species in laboratory and field. the current study will also be effective in preliminary screening of naturally resistant timber species for chemical analysis in order to isolate the termite resistance components for commercial use as wood preservative and to determine which commercial species will require preservative treatment before use in regions with high termite hazard. materials and methods laboratory bioassays termite source: orphaned termite workers and third instar larvae of workers and soldiers were collected using bucket traps. the traps were brought to the laboratory, all the debris was removed and termites were kept in plastic boxes with moist filter paper. before exposure to different wood species, termites were kept in the laboratory at (26 ± 2oc, 80% r.h) to eliminate injured and inactive termites and kept in constant darkness. only active and healthy termite workers were used for the experiments. termites were kept in petriplates (90mm×15mm) containing moist filter papers until the experiments were conducted. termite species h. indicola and c. heimi were tested for feeding preference using no-choice feeding test in laboratory and in field conditions. all bioassay termites were collected from three colonies i.e. lcwu, jallo park and changamanga. test wood specimens: 24 important collected timber species (that were not decomposed and were dried naturally) were: azadirachta indica (neem), pinus roxberghii (chir), dalbergia sissoo (sheesham), populus deltoides (popular), albizzia lebbeck (shirin), abies pindrow (fir), alstonia scholaris (alstonia), erythrina suberosa (gulenister), eucalyptus citriodora (safaeda), ficus religiosa (bohar), heterophragma adenophyllum (beeri patta), melia azedarach (derek), pinus wallichiana (chir), terminalia arjuna (arjun), acacia arabica (kikar), betula utilis (birch) cedrus deodara (deodar), cordia oblique (lasura), elaeis guineensis (african palm oil), mangifera indica (aam), moringa oleifera (sohanjana), putranjiva roxburghii (lucky bean), syzygium cumini (jaman) and zizyphus jujube (berry), most of these were commonly used in wood work in pakistan. all pakistani woods used in this study were from heartwood. laboratory preference test no choice laboratory tests were executed to study wood preference of two termite species h. indicola and c. heimi in the laboratory. wooden blocks (20 x 20 x 20 mm) of each wood species were prepared, cleaned and pre weighed. wooden blocks were dried at 100°c for 24 hours. only one type of test or wooden block was placed in a glass petri dish and 100 termite workers (with 10% soldiers, as naturally given in colonies) were released and kept in darkness at 26°c and 60 + 5% relative humidity for 14 days in controlled chamber. filter paper treated with distilled water was used as control. the wooden blocks were kept suitably moist. three replicates of wooden blocks were used for each wood. after test period the wooden blocks were recovered, dried at the same temperature at which they were dried before exposure to termite workers and the amount of wood consumed was calculated by weighing. sociobiology 62(3): 417-425 (september, 2015) 419 field wood preference test against h. indicola (wasmann) and c. heimi (wasmann) the field trials were conducted at sites heavily infested with termites as evidenced from previous studies and where many active nests of h. indicola, m. obesi, odontotermes obesi and c. heimi were located at the workshop area of lahore college women university, lahore and jallo forest park, lahore. three replicates of each wood were prepared and wooden blocks were tied by copper wire into a bundle. each bundle was buried at each site, 15-20 cm deep into the soil (so that they may reach the nest) for a period of one month. each replicate was set 2 m apart from each other. at the end of experiment, wood samples were brought back to laboratory, cleaned, oven dried and weighed to determine the amount consumed. upon termination of experiment, wood samples were brought back to laboratory, cleaned, oven dried and weighed to determine the amount of wood consumed. other recorded parameters were total termite-contact, which was based on either one of the three criteria: termite feeding, deposited faecal material or mud gallery built on the wood. in both laboratory and field trials, tested wood specimens was assessed according to the standard method for laboratory evaluation to determine resistance to subterranean termites (awpa,1997). visual rating of the test blocks using the scale of 10 (sound, surface nibbles permitted), 9 (light attack), 7 (moderate attack), 4 (heavy attack), or 0 (failure). data analyses data in mass loss (g) were subjected to mean, standard error and analysis of variance, and difference in mass loss for each pair of wooden block was calculated by paired comparison t-test. means were separated using tukey’s hsd test. results and discussion results of the no-choice laboratory evaluations of the resistance and feeding preference of 24 wood species to the subterranean termites h. indicola and c. heimi are shown in table 1. interpreting the objectives of the awpa (1997) termite test relative to the present study, in no choice feeding bioassays, the wooden blocks of d. sissoo, p. wallichiana, e. deodara, p. roxberghii, c. deodara, a. indica, a. arabica and c. oblique scored 10 for the mean visual rating, indicating that these woods are sound and very resistant (vr) to termite attack with only surface nibbles were permitted. pinus roxburghii, t. arjuna, a. scholaris, a. pindrow, m. azedarach, scored 9 for the mean visual rating, showing light attack and the woods were identified as resistant (r), a. lebbeck, z. jujube, m. oleifera scored 7 for the mean visual rating, indicating moderate attacks and were identified as moderate resistant (mr). f. religiosa, b. utilis, e. suberosa, p. deltoides and m. indica scored 4 for the mean visual rating, indicating susceptible (s) to termite attack (table 1). in control, no termite mortality was recorded and tunnelling pattern was observed for both species. inside petri dish, tunnels of h. indicola were thin and were highly branched while tunnels of c. heimi were wider and less branched. h. indicola makes tunnel to top but c. heimi makes few tunnels to top. for h. indicola, the woods were arranged in the following descending order of preference: populus deltoides > mangifera indica > betula utilis > erythrina suberosa > moringa oleifera > eucalyptus citriodora > syzygium cumini > elaeis guineensis > ficus religiosa > zizyphus jujube > abies pindrow > melia azerdarach > heterophragma adenophyllum > terminalia arjuna > putranjiva roxburghii > acacia arabica > cordia obliqua > pinus wallichian > albizia lebbeck > alstonia scholaris > cedrus deodara > pinus roxburghii > azadirachta indica > dalbergia sissoo. while for no choice feeding test against c. heimi, in laboratory woods were arranged in the following descending order of preference: populus deltoides > mangifera indica > erythrina suberosa > betula utilis > elaeis guineensis > ficus religiosa > heterophragma adenophyllum > terminalia arjuna > moringa oleifera > putranjiva roxburghii > syzygium cumini > zizyphus jujube > melia azerdarach > abies pindrow > acacia arabica > eucalyptus citriodora > azadirachta indica > alstonia scholaris > cordia obliqua > albizia lebbeck > pinus roxburghii > cedrus deodara > pinus wallichian > dalbergia sissoo. mcmahan (1966) studied wood feeding preference of different woods and observed that poplar and maple both ranked as “more preferred” woods, with maple being perhaps slightly above poplar, yet incipient colonies reared on poplar strongly preferred it over maple. results of no choice feeding bioassay in the field for c. heimi confined to wooden blocks of different species of wood, the wooden blocks of the wooden blocks of d. sissoo, p. wallichiana, e. deodara, pu. roxberghii, c. deodara, a. arabica and c. oblique scored 10 for the mean visual rating, indicating that woods were sound and very resistant (vr) to termite attack with only surface nibbles were permitted. p. roxberghii, t. arjuna, a. scholaris, a.pindrow, m.azedarach, scored nine for the mean visual rating showing light attack and were identified as resistant (r), a. lebbeck, z. jujube, m. oleifera scored 7 for the mean visual rating, indicating moderate attacks and were identified as moderate resistant (mr). f. religiosa, b. utilis, e. suberosa, p. deltoides and m. indica scored 4 for the mean visual rating, indicating susceptible (s) to termite attack (table 2). so, it was observed that inside laboratory and in field studies the termites species c. heimi had the same mean visual rating. regarding mean wood mass loss, the lowest mean wood mass loss was for d. sisso which was the least preferred while p. deltoides was the most preferred wood by the species c. heimi (wasmann). the results of no choice field feeding test against h. indicola indicating that damage to wooden blocks was noted at the end of the four-week test. the loss in weight of wooden f manzoor, mu latif – feeding preference of termites: a comparison of 24 wood species in pakistan420 serial no. wood species mean visual rating* h.indicola c.heimi mean wood mass loss (g) mean % mortality mean visual rating mean wood mass loss (g) mean % mortality 1 populus deltoides (p.d) 4(s) 0.42±0.00057 18.3+0.0057 4(s) 0.48±0.006 11.66±0.577 2 azadirachta indica (a.i) 10(vr) 0.0213+0.0005 55.0+0.0057 9(r) 0.078±0.006 47.49±0.577 3 pinus roxberghii (pi.r) 9(r) 0.023+0.00057 57.0+0.0005 10(vr) 0.060±0.006 57.3±0.577 4 dalbergia sissoo (d.s) 10(vr) 0.019+0.00057 44.0+0.00057 10 (vr) 0.04±0.006 67.5±0.577 5 heterophragma adenophyllum (h.a) 7(mr) 0.059+0.00057 29.9±0.5774 7 (mr) 0.17±0.006 26.9±0.577 6 ficus religiosa (f.r) 4(s) 0.069+0.00057 29.4±0.5774 4(s) 0.23±0.006 16.88±0.577 7 terminalia arjuna (t.a) 9(r) 0.055+0.00057 34.7±0.5774 7(mr) 0.15±0.006 21.77±0.577 8 albizia lebbeck (a.l) 7(mr) 0.040+0.00057 27.7±0.5774 9(r) 0.065±0.006 39.7±0.577 9 pinus wallichiana (p.w) 10(vr) 0.040+0.00057 29.6±0.5774 10(vr) 0.050±0.006 57.5±0.577 10 alstonia scholaris (a.s) 9(r) 0.039+0.00057 29.1±0.5774 9(r) 0.074±0.006 41.50±0.577 11 erythrina suberosa (e.s) 4(s) 0.037+0.00057 27.9±0.5774 4(s) 0.32±0.006 14.58±0.577 12 eucalyptus citriodora (e.c) 10(vr) 0.082+0.00057 47.0±0.5774 7(mr) 0.083±0.006 40.55±0.577 13 abies pindrow (a.p) 9(r) 0.071+0.00057 32.3±0.5774 9(r) 0.09±0.006 40.4±0.577 14 melia azedarach (m.a) 9(r) 0.062+0.05774 47.2±0.5774 9(r) 0.10±0.006 38.9±0.577 15 putranjiva roxburghii (p.r) 10(vr) 0.045±0.020 44.44±0.5774 7(mr) 0.13±0.006 56.8±0.577 16 cedrus deodara (c.d) 10(vr) 0.048±0.024 38.44±0.5774 10(vr) 0.053±0.006 73.88±0.577 17 acacia arabica (a.a) 10(vr) 0.050±0.017 33.55±0.5774 9(r) 0.087±0.026 38.8±0.577 18 cordia oblique (c.o) 10(vr) 0.053±0.023 33.32±0.5774 9(r) 0.070±0.026 43.7±0.577 19 syzygium cumini (s.c) 4(s) 0.076±0.023 32.88±0.5774 7(mr) 0.12±0.043 26.9±0.577 20 zizyphus jujube (z.j) 7(mr) 0.072±0.028 32.44±0.5774 7(mr) 0.11±0.037 37.8±0.577 21 betula utilis (b.u) 4(s) 0.070±0.023 28.55±0.5774 7(mr) 0.28±0.029 30.7±0.577 22 ehretia serrata (e.s) 4(s) 0.074±0.032 27.77±0.5774 4(s) 0.24±0.043 24.6±0.577 23 moringa oleifera (m.o) 7(mr) 0.086±0.032 29.33±0.5774 7(mr) 0.14±0.0.20 24.21±0.577 24 mangifera indica (m.i) 4(s) 0.30±0.028 29.88±0.5774 4(s) 0.34±0.046 16.36±0.577 25 control 0.45±0.070 0.00 0.16±0.075 0.00 results expressed as mean±s.e (standard error). *visual rating according to awpa scale 1997 of 10(sound, surface nibbles permitted), nine (light attack), seven (moderate attack), four (heavy attack), or zero (failure). difference in mass loss for each pair of wooden block indicated by *=0.05 and **=0.01 are significantly different (paired comparison t-test). table 1. mean visual rating, mean wood mass loss and mean percentage mortality of different wood species during (no choice feeding) bioassay against h. indicola (wasmann) and c. heimi (wasmann) workers exposed under laboratory conditions. blocks served as a measure of termite attack. each block was then graded by the amount of termite damage by using an awpa scale (1997). analysis of variance also revealed mean difference were significantly different from one another (f, 7.859; d.f. 3:8:11; p<0.001). regarding the mean wood mass, results also indicate that the highest mean mass loss was for h. indicola was for p. deltoides (0.42±0.0005) and lowest mean mass loss was for d. sissoo (0.019±0.00057). similarly, the mean wood mass, results also indicate that the highest mean mass loss was for c. heimi was for p. deltoides (0.48±0.006) and lowest mean mass loss was for d. sissoo (0.04±0.006). the results indicated that the wood of d. sissoo was the least preferred and m. indica was the most preferred wood used for c. heimi. analysis of variance also revealed that means difference were significantly different from one another (f, 7.617; d.f., 3:8, p<0.001). regarding feeding preferences of h.indicola and c. heimi, the basic purpose was to know which species of local timbers possesses natural resistance against termites and which timber species are palatable. various researchers in pakistan had studied the feeding preference of c. heimi, o.obesus and b.beesoni (akhtar & jabeen, 1981). akhtar and ali (1979) studied feeding preference of o. obesus and arranged the woods in the following descending order of preference: populus eur-americana (s.w), abies pindrow (h.w), acacia arabica (h.w), cedrus deodara (h.w), mangifera indica, morus alba (h.w), pinus roxburghii (h.w) and dalbergia sissoo (h.w). however, no comprehensive data on the feeding preferences of different wood species was available. mcmahan (1966) studied wood feeding preference of different woods and observed that poplar and maple both ranked as “more preferred” woods, with maple being perhaps sociobiology 62(3): 417-425 (september, 2015) 421 slightly above poplar, yet incipient colonies reared on poplar strongly preferred it over maple. the present study was carried out to compare the feeding preference of two important species of termites. regarding mean percentage mortality in laboratory trials, the highest mean percentage mortality (57.0±0.0005) for h.indicola was for the wood pinus roxberghii and lowest mean percentage (0.045± 0.020) mortality was for putranjiva roxburghii. for c.heimi, the mean percentage mortality in laboratory trials was highest for cedrus deodara (73.88± 0.577) and lowest mean percentage (11.66±0.577) mortality was for populus deltoids (table 1). according to the results of field no choice feeding test for c.heimi, it was revealed from results that wood of dalbergia sissoo, azadirachta indica, eucalyptus citriodora, pinus wallichiana, acacia arabica were very resistant (vr) and had 10 mean visual rating showing only nibbles of termite attack, the wood of pinus roxberghi, a. pindrow, ficus religiosa, alstonia scholaris, melia azerdarach, and terminalia arjuna were resistant (r), to termite attack showing minimum wood mass loss and no significant portion of wood was eaten by c. heimi. the woods of albizia lebbeck, h. adenophyllum, m. olifera and z. cuminiwere moderately resistant (mr) indicating that some portion of wood is eaten by them. populus deltoids, erythrina suberosa, betula utilis and mangifera indica were susceptible (s) to termite attack and some portion of wood was significantly eaten by c. heimi (table 2). so 24 tested woods were arranged in following descending order of preferences: populus deltoides > mangifera indica > moringa oleifera > elaeis guineensis > erythrina suberosa > syzygium cumini > heterophragma adenophyllum > cordia oblique > ficusreligiosa > pinus roxburghii > alstonia scholaris > terminalia arjuna > albizia lebbeck > pinus wallichiana > melia azerdarach >betula utilis > abies pindrow > zizyphus jujube > eucalyptus citriodora > putranjiva roxburghii > acacia arabica > azadirachta indica > cedrus deodara > dalbergia sissoo. as far as mean wood mass loss is concerned, the maximum mean wood mass loss (0.40± 0.0152) is for populus deltoids wood and minimus wood mass loss is for dalbergia sisso(0.013±0.0057). if we compare laboratory and field wood preference for c.heimi results are almost similar, under field conditions three woods i.e. populus deltoids, elaeis guineensis and mangifera are susceptible to termite attack and maximum mean wood mass loss is observed for them. it is quite obvious that d. sisso is having high specific gravity as compared to populus and is hard in nature, one factor may be it is less eaten by both termite species in the laboratory and in field also (table 3-appendix). chaudhry et al., (1978) also studied natural resistance of twelve timbers to the attack of c. heimi and found that cedrus deodara and tectona grandis were resistant to termite attack, while salmalia malabarica, abies pindrow and picea smithiana were the most susceptible. akhtar (1981) reported that a. pindrow is resistant to c. heimi. in this study, our main question, to know which species of local timber was palatable but which possesses natural resistance against termites, was answered, and there is a need to further investigate the nature of compounds having anti-termitic properties within these woods. manzoor et al. (2009) studied the comparative studies on two pakistani subterranean termitespecies i.e. coptotermes heimi and microcerotermes championi (rhinotermitidae, termitidae) for natural resistance and feeding preferences in laboratory and field trials and the results of present study are in conformity with the previous ones so there is dire need to develop wood rating scales in pakistan also. similarly rasib and hina (2014) studied the feeding preferences of coptotermes heimi and the feeding preference of c. heimiin descending order based on wood consumption as a quantitative parameter were as follows: p. euramericana > ailanthus excelsa > azadirachta indica > p. roxburghii > butea monosperma > morus alba > bauhinia variegata > albizia lebbeck > dalbergia sissoo > heterophragma adenophyllum > erythrina suberosa > cassia fistula > t. grandis > mangifera indica > eucalyptus camaldulensis > jacaranda mimosifolia > bambusa bamboo > s. cumini. s. no. wood species mean visual rating mean wood mass loss (g) (mean+s.e) 1. dalbergia sisso 10 (vr) 0.013a±0.0057 2. pinus roxburghii 9(r) 0.026b±0.0115 3. azadirachta indica 10 (vr) 0.053c±0.0378 4. populus deltoides 4 (s) 0.40d±0.0152 5. albizia lebbeck 7(mr) 0.03+0.00632 6. abies pindrow 9(r) 0.08+0.00632 7. pinus wallichiana 10(vr) 0.12+0.00632 8. alstonia scholaris 9(r) 0.13+0.00632 9. melia azerdarach 9(r) 0.14+0.00632 10. terminalia arjuna 9(r) 0.17+0.00632 11. eucalyptus citriodora 10(vr) 0.18+0.00632 12. heterophragma adenophyllum 7(mr) 0.20+0.0632 13. ficus religiosa 4(mr) 0.21+0.00632 14. erythrina suberosa 4(s) 0.28+0.00632 15. moringa oleifera 7(mr) 0.19±0.003 16. cedrus deodara 10(vr) 0.014±0.026 17. cordia oblique 10(vr) 0.23±0.026 18. acacia arabica 109(vr) 0.11±0.033 19. betula utilis 4(s) 0.16±0.025 20. zizyphus jujube 7(mr) 0.16±0.026 21. syzygium cumini 4(s) 0.19±0.069 22. putranjiva roxburghii 10(vr) 0.28±0.075 23. elaeis guineensis 4(s) 0.41±0.120 24. mangifera indica 4(s) 0.42±0.153 table 2. mean visual rating and mean wood mass loss of various wood species during no choice feeding bioassay against c. heimi (wasmann) in the field. f manzoor, mu latif – feeding preference of termites: a comparison of 24 wood species in pakistan422 conclusion the present study was performed to determine the natural wood preferences of two subterranean species of termites c. heimi and h. indicola. subterranean termites cause extensive damage to wood and cellulose products in temperate and tropical climates. some wood species are preferred by termites over other species due to many factors e.g. palatability and digestibility of wood, essential oils and chemicals. twenty four commonly used woods were tested in laboratory and field by choice and no choice feeding tests. so it was concluded that populus deltoids and mangifera indica were most preferred along with highest mass loss ratio, whereas dalbergia sisso and cedrus deodara were least preferred by the both termite species in laboratory and field tests. references aihetasham, a., & iqbal, s. (2012). feeding preferences of microcerotermes championi (snyder)for different wooden blocks dried at different temperatures under forced and choice feeding conditions in laboratory and field. pakistan journal of zoology, 44: 1137-1144. akhtar, m. s. (1981). feeding responses to wood and wood extracts by bifiditermes beesoni (gardner) (isoptera: kalotermitidae). international biodeterioration bulletin, 17: 21-24. akhtar, m. s., and ali, s. (1979). wood preferences and survival of coptotermes heimi (wasmann) and odontotermes obesus (rambur) (isoptera). pakistan journal of zoology, 11: 303-314. akhtar, m. s., and jabeen, m. (1981). influence of specimen size on the amount of wood consumed by termites. pakistan journal of zoology. 13: 79-84. alavijeh, e. s., habibpour, b., moharramipour, s., & rasekh a. (2014). bioactivity of eucalyptus camaldulensis essential oil against microcerotermes diversus (isoptera: termitidae). journal of crop protection, 3: 1-11 awpa. 1997. standard method for laboratory evaluation to determine resistance to subterranean termites. standard e197. american wood preservers’ association, 279-282. brune, a. (2014). symbiotic digestion of lignocellulose in termite guts. nature reviews microbiology, 12: 168-180 carter, f. l., and smythe, r. v. (1974). feeding and survival responses of reticulitermes flavipes (kollar) to extractives of woods from 11 coniferous genera. holzforschung, 28: 41-45. chaudhry, i. m., and ahmad, m. (1972). termites of pakistan, identify, distribution and ecological relationships. final technical report.pakistan forest institute, peshawar, pakistan,13-19. cornelius, m. l. and w. osbrink (2010). effect of soil type and moisture availability on the foraging behavior of the formosan subterranean termite (isoptera: rhinotermitidae). journal of economic entomology, 103: 799-807. hapukotuwa, n. k., & grace, j. k. (2011). comparative study of the resistance of six hawaii-grown bamboo species to attack by the subterranean termites coptotermes formosanus shiraki and coptotermes gestroi (wasmann)(blattodea: rhinotermitidae). insects, 2(4): 475-485. he, s., ivanova, n., kirton, e., allgaier, m., bergin, c., scheffrahn, r. h., & hugenholtz, p. (2013). comparative metagenomic and metatranscriptomic analysis of hindgut paunch microbiota in wood and dung-feeding higher termites. plos one, 8(4): e61126. henderson, g., heumann, d.o., laine, r.a., maistrello, l., zhu, b. c. r., and chen, f. (2001). extracts of vetiver oil as a repellent andtoxicant to ant, ticks and cockroaches. us patent disclosure,serial number 09/932,555. lenz, m., lee, c. y., lacey, m. j., yoshimura, t., & tsunoda, k. (2011). the potential and limits of termites (isoptera) as decomposers of waste paper products. journal of economic entomology, 104: 232-242. mahmood iqbal sheikh (1993). trees of pakistan. pn abw250, 95254. manzoor, f., zameer, k., and malik, s. a. (2009). comparative studies on two pakistani subterranean termites (isoptera: rhinotermitidae, termitidae) for natural resistance and feeding preferences in laboratory and field trials. sociobiology, 53: 259-274. manzoor, f., and mir, n. (2010). survey of termite infested houses, indigenous building materials and construction techniques in pakistan. pakistan journal of zoology, 42: 693-696. mcmahan, a. e. 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(2012). protozoidal activities of eucalyptus cammeldulensis, dalbergia sissoo and acacia arabica woods and their different parts on the entozoic flagellates of heterotermes indicola and coptotermes heimi. african journal of biotechnology,11: 12094-12102. ngee, p. s., tashiro, a., yoshimura, t., jaal, z., and lee, sociobiology 62(3): 417-425 (september, 2015) 423 c. y. (2004). wood preference of selected malaysian subterranean termites (isoptera: rhinotermitidae, termitidae). sociobiology, 43: 535-550. peralta, r. c. g., menezes, b., carvalho, a. g., and aguiar, m. (2004).wood consumption rates of forest species by subterranean termites under field conditions. sociedade de investigações florestais, 28: 283-289. rasib, k. z.,& ashraf, h. (2014). feeding preferences of coptotermes heimi (isoptera: termitidae) under laboratory and field conditions for different commercial and noncommercial woods. international journal of tropical insect science, 34: 115-126. shanbhag, r. r., & sundararaj, r. (2012). host range, pest status and distribution of wood destroying termites of india. the journal of tropical asian entomology, 02: 12-27 sheikh n., a. mahmood qureshi, m. umair latif, and f. manzoor (2010). study of temperature treated woods for the preference and first food choice by odontotermes obesus (isoptera: termitidae). sociobiology, 56: 363-373. f manzoor, mu latif – feeding preference of termites: a comparison of 24 wood species in pakistan424 s. n o. w oo d sp ec ie s d en si ty st re ng th u se 1 a bi es p in dr ow (a .p ) sp ec ifi c gr av ity o f 0. 48 w ith a c al or ifi c va lu e of 4 50 0 kc al /k g l ig ht , s of t c on st ru ct io n, f ue l, fo dd er , w at er sh ed p ro te ct io n, p ac ki ng c as es , an d pl yw oo d. 2 a za di ra ch ta in di ca (a .i ) sp ec ifi c gr av ity o f 0. 68 a nd a c al or ifi c va lu e of 4 99 0 kc al /k g. h ea vy , h ar d, r es ili en t. fu rn itu re , f od de r, w oo d ca rv in g, t im be r, ag ri cu ltu re im pl em en ts an d ta nn in . 3 p in us r ox be rg hi i ( pi .r ) sp ec ifi c gr av ity o f 6. 48 a nd a c al or ifi c va lu e of 4 99 5 kc al /k g. m od er at el y ha rd , m od er at el y he av y c on st ru ct io n, a nd v ar io us w oo d pr od uc ts ( fu rn itu re , m at ch s tic ks , w in do w f ra m es , e tc . 4 d al be rg ia s is so o (d .s ) w oo d is h ea vy w ith a s pe ci fic g ra vi ty o f 0. 85 a nd a c al or ifi c va lu e of 5 00 0 kc a1 /k g. h ar d an d st ro ng , r es ili en t fo dd er , f ur ni tu re , f ue l a nd c ha rc ca l, m ed ic in al ( ro ot s an d ba rk ), ra ilw ay c ar ri ag es , s po rt in g go od s, f ar m im pl em en ts , a nd s ha de . 5 h et er op hr ag m a ad en op hy llu m ( h .a ) c al or ifi c va lu e of 4 80 0 kc al /k g. h ar d, s tr on g re si lie nt fu rn itu re , f ue l, an d or na m en ta l 6 f ic us r el ig io sa ( f. r ) m ed iu m so ft o rn am en ta l, fo dd er , f oo d (fi gs ), s m al l t im be r, an d m ed ic in al . 7 te rm in al ia a rj un a (t .a ) sp ec ifi c gr av ity o f 0. 9 an d a ca lo ri fic v al ue of 5 00 0 kc al /k g. h ar d, h ea vy , r es ili en t. fu el , i m pl em en ts , e ro si on c on tr ol , w he el s, s po ke s an d ax le s, fo dd er , m ed ic in al ( ba rk is a a st ri ng en t a nd c ar di ac s tim ul an t) , tim be r an d or na m en ta l. 8 a lb iz ia le bb ec k (a .l ) d en se w ith a s pe ci fic g ra vi ty b et w ee n 0. 55 an d 0. 64 , a nd a c al or ifi c va lu e of 5 10 0 kc al /k g. v er y st ro ng , r es ili en t fo dd er , f ue l, la nd s ta bi liz at io n, n itr og en fi xi ng , p ol es , a gr ic ul tu ra l im pl em en ts , s ha de , a nd a pi cu ltu re 9 p in us w al lic hi an a (p .w ) sp ec ifi c gr av ity o f 6. 48 a nd a c al or ifi c va lu e of 4 99 5 kc al /k g. m od er at el y ha rd , m od er at el y he av y c on st ru ct io n, f ue l, sl ee pe rs , a nd v ar io us w oo d pr od uc ts ( fu rn itu re , m at ch s tic ks , w in do w f ra m es , e tc .) 10 a ls to ni a sc ho la ri s (a .s ) h ea vy h ar d, b ri ttl e o rn am en ta l a nd m ed ic in al 11 e ry th ri na s ub er os a (e .s ) w oo d is li gh t a nd h as a c al or ifi c va lu e of 48 00 k ca l/k g. so ft a nd n ot d ur ab le , b ut fi br ou s an d to ug h fu el , n itr og en fi xi ng , o rn am en ta l a nd m ed ic in al b ar k as a fa br if ug e) . 12 e uc al yp tu s ci tr io do ra (e .c ) sp ec ifi c gr av ity o f 0. 78 a nd a c al or ifi c va lu e of 4 80 0 kc al /k g. h ar d, e la st ic a nd r es ili en t fu el , c ha rc oa l, fu rn itu re , p er fu m e (l ea ve s) , s he lte rb el t, ap ic ul tu re , pu lp , fi be r bo ar d an d to ol h an dl es 13 p op ul us d el to id es ( pd ) sp ec ifi c gr av ity o f 0. 46 a nd a c al or ifi c va lu e of 5 90 0 kc al /k g. m od er at el y lig ht , s of t fu el , p ac ki ng c as es a nd c ra te s, m at ch es , e ro si on c on tr ol a nd re fo re st at io n, p ly w oo d, p ul p, f od de r, an d ro ad si de tr ee 14 m el ia a ze da ra ch (m .a ) sp ec ifi c gr av ity o f 0. 56 a nd a c al or ifi c va lu e of 5 10 0 kc al /k g. l ig ht , m od er at el y ha rd , r es ili en t fu rn itu re , f od de r, or na m en ta l, tim be r, co ns tr uc tio n, a gr ic ul tu ra l i m pl em en ts , b ox es a nd p ac ki ng c ra te s, s po rt s eq ui pm en t, ve ne er a nd pl yw oo d an d m ed ic in al ( flo w er s an d le av es )p ou lti ce f or h ea da ch es 15 p ut ra nj iv a ro xb ur gh ii (p .r ) sp ec ifi c gr av ity o f 0. 47 a nd a c al or ifi c va lu e of 5 80 0 kc al /k g. l ig ht , s of t c on st ru ct io n, f ue l, ra ilw ay s le ep er s, w at er sh ed p ro te ct io n, p ac ki ng ca se s an d m ed ic in al ( ar om at ic w oo d ju ic e ia a c ar m in at iv e, di ur et ic ). 16 c ed ru s de od ar a (c .d ) sp ec ifi c gr av ity o f 0. 57 w ith a c al or ifi c va lu e of 5 20 0 kc al /k g w oo d is li gh t a nd n ot s tr on g fu el a nd ti m be r (c on st ru ct io n) 17 a ca ci a a ra bi ca (a .a ) w oo d is s of t, w ith a s pe ci fic g ra vi ty o f 0. 59 an d a ca lo ri fic v al ue o f 49 10 k ca l/k g h ar d, m od er at el y st ro ng fu el , f ru it, im pl em en ts , e ro si on c on tr ol a nd m ed ic in al ( fr ui t f or co ug h, c he st d is ea se s) a pp en di x. t ab le s ho w in g d en si ty , s tr en gt h an d us es o f di ff er en t w oo d sp ec ie s us ed in th e st ud y. sociobiology 62(3): 417-425 (september, 2015) 425 s. n o. w oo d sp ec ie s d en si ty st re ng th u se 18 c or di a ob liq ue (c .o ) a c al or ifi c va lu e of 4 90 0 kc al /k g h ar d, h ea vy a nd r es ili en t c on st ru ct io n, f ue l, fr ui t, m ed ic in al ( fr ui t i s a ca rm in at iv e, s ee d fo r tr ea tm en t o f di ab et es ), ta nn in , s he lte rb el ts , a pi cu ltu re , p ap er p ul p, sh ad e, f od de r an d ro ad si de p la nt in g 19 sy zy gi um c um in i ( s. c ) sp ec ifi c gr av ity o f 0. 70 a nd a c al or ifi c va lu e of 4 80 0 kc al /k g. h ar d, h ea vy a nd r es ili en t c on st ru ct io n, f ue l, fr ui t, m ed ic in al ( fr ui t i s a ca rm in at iv e, s ee d fo r tr ea tm en t o f di ab et es ), ta nn in , s he lte rb el ts , a pi cu ltu re , p ap er p ul p, sh ad e, f od de r an d ro ad si de p la nt in g 20 zi zy ph us ju ju be (z .j ) sp ec ifi c gr av ity o f 0. 93 a nd a c al or ifi c va lu e of 5 90 0 kc al /k g. h ar d, h ea vy , s tr on g fu el , c ha rc oa l, ag ri cu ltu ra l i m pl em en ts a nd f ru it 21 b et ul a ut ili s (b .u ) h ea vy st ro ng b ut lo w a s co m pa re d to ye llo w b ir ch fu el , f od de r an d fu rn itu re 22 e hr et ia s er ra ta r ox b sp ec ifi c gr av ity o f 0. 59 . h ar d, s tr on g fu el , f ru it, im pl em en ts , e ro si on c on tr ol , f ur ni tu re , 23 m or in ga o le ife ra (m .o ) m ed iu m so ft , s po ng y, w ea k o rn am en ta l, fo dd er , f oo d (l ea ve s, fl ow er s an d fr ui ts ), s ee d oi l (l ub ri ca tio n an d pe rf um e) , a nd g um ( ba rk ). 24 m an gi fe ra in di ca (m .i ) d en se , w ith a s pe ci fic g ra vi ty o f 0. 55 a nd a ca lo ri fic v al ue o f 46 00 k ca l/k g. st ro ng a nd d ur ab le fr ui t, lu m be r an d co ns tr uc tio n, c hi pb oa rd , o rn am en ta l, m ed ic in al (r ip e fr ui t is a l ax at iv e, s ee ds a re a st ri ng en t an d ve rm if ug e) a nd fo od p ic ke ls a pp en di x. t ab le s ho w in g d en si ty , s tr en gt h an d us es o f di ff er en t w oo d sp ec ie s us ed in th e st ud y ( c on tin ua tio n) . open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.369-377sociobiology 61(4): 369-377 (december 2014) diapause in stingless bees (hymenoptera: apidae) diapause a strategy for surviving unfavourable environmental conditions extreme environmental conditions may negatively affect the development of certain animals and many have evolved behavioral strategies in order survive. insects present a wide range of adaptive behaviors that have allowed them to respond successfully to adverse climatic conditions by temporarily interrupting some of their activities or development in the face of, for example, extreme temperatures (cold or warm) (tauber & tauber, 1976; denlinger, 1986; kort, 1990; tatar & yin, 2001; koštál, 2006). such behavioral responses have variously been termed dormancy, quiescence, hibernation, aestivation and diapause (tauber & tauber, 1976; denlinger, 1986). however, conceptual differences exist among these terms relating to the kind of environmental variable and whether the response is merely immediate (nonprogrammed) or rhythmical (endogenously and genetically abstract extreme environmental conditions may negatively affect the development of animals. insects show a wide range of adaptive behaviors that have allowed them to respond successfully to adverse climatic conditions by temporarily interrupting some of their activities or development. one such behavior is diapause. diapause can be defined as a gradual and progressive interruption in development or ontogeny of an organism in any phase of its lifecycle in order to survive cyclic unfavorable environmental conditions. this review presents an overview of the current knowledge of diapause in stingless bees and describes the various studies on this subject. it focuses on plebeia species, the most studied genus in this regard. in this group of bees, provisioning and oviposition behavior ceases in autumn/winter, a so-called reproductive diapause. besides the cessation of brood-rearing activity, other behaviors, such as foraging, are also modified. the mechanisms that induce the reproductive diapause are still unclear, but evidence points to temperature and photoperiodism as the main drivers of this behavior. sociobiology an international journal on social insects cf dos santos, p nunes-silva, r halinski, b blochtein article history edited by denise araujo alves, esalq usp, brazil received 30 september 2014 initial acceptance 02 november 2014 final acceptance 01 december 2014 keywords plebeia, provisioning and oviposition process, reproductive diapause, temperature, photoperiodism. corresponding author charles fernando dos santos depto. de biodiversidade e ecologia, faculdade de biociências, pontifícia universidade católica do rio grande do sul av. ipiranga, 6681, partenon porto alegre, rs, brazil e-mail: chasanto@gmail.com programmed) (tauber & tauber, 1976; denlinger, 1986, 2002; koštál, 2006). in a broader sense, dormancy is a term embracing several others (e.g., quiescence, hibernation, aestivation and diapause) and refers to any temporary arrest in behavior performed by an insect in response to any unfavorable climatic condition (tauber & tauber, 1976; denlinger, 1986). however, the terms quiescence and diapause have also frequently been used as synonyms of dormancy, although both are actually different kinds of dormancy (tauber & tauber, 1976; denlinger, 1986). basically, both quiescence and diapause are temporary interruptions in the developmental cycle or activity of an organism in response to environmental adversity. quiescence can be defined as any sudden, non-cyclic response, whereas diapause is a strictly cyclic or rhythmical response, i.e., genetically pre-programmed (tauber & tauber, 1976; denlinger, 1986). a concise and clear definition of diapause is a gradual and review pontifícia universidade católica do rio grande do sul, porto alegre, rs, brazil. cf dos santos, p nunes-silva, r halinski, b blochtein diapause in stingless bees370 progressive interruption in development or ontogeny of any organism in some phase of its life cycle in order to survive unfavorable environmental conditions that occur cyclically (tauber & tauber, 1976; denlinger, 1986; kort, 1990; tatar & yin, 2001; koštál, 2006). among the major insect orders, including hemimetabolous and holometabolous ones, diapause behavior has been best observed in orthoptera, diptera, coleoptera, lepidoptera, hemiptera and hymenoptera (tauber & tauber, 1976; herman, 1981; denlinger, 1986; lefevere et al., 1989; kort, 1990; greenfield & pener, 1992; van benthem et al., 1995; kipyatkov et al., 1997; pick & blochtein, 2002b). diapause behavior may be divided into at least three stages: (1) pre-diapause, (2) diapause, and (3) post-diapause, during which insects exhibit a series of behavioral, biochemical and morphological changes (denlinger, 2002; koštál, 2006). furthermore, diapause behavior may occur at any stage of the insect life cycle: egg, larval, pupal (or nymph) or adult (tauber & tauber, 1976; herman, 1981; denlinger, 1986; lefevere et al., 1989; kort, 1990; greenfield & pener, 1992; van benthem et al., 1995; kipyatkov et al., 1997; pick & blochtein, 2002b). when diapause occurs in sexually active insects, breeding is usually compromised due to the interruption of certain physiological processes related to: (1) female oogenesis, (2) the activity of male accessory glands, or (3) the reproductive behavior of both sexes (kimura, 1988; tatar & yin, 2001). diapause in sexually active insects has, therefore, been termed reproductive diapause, a period during which insects may temporarily cease egg-laying and searching for sexual partners (kimura, 1988; tatar & yin, 2001). predicting the arrival of adverse environmental conditions since diapause is a rhythmical biological strategy to survive unfavorable environmental conditions, it requires mechanisms that anticipate the arrival and ending of such conditions. indeed, many insects have been found to effectively decode certain environmental cues in order to enter and/or terminate diapause behavior (tauber & tauber, 1976; denlinger, 1986; kort, 1990; tatar & yin, 2001; koštál, 2006). such cues include the amount of food available in the environment, the relative humidity, the parasite load, and the physiological state of host plants (e.g., solute concentration, age, limb senescence) (tauber & tauber, 1976; derr, 1980; denlinger, 1986; greenfield & pener, 1992; takagi & miyashita, 2008; togashi, 2014). however, the most important environmental cues used by insects to prepare for the arrival or ending of adverse environmental conditions are photoperiodism and temperature (tauber & tauber, 1976; tauber & kyriacou, 2001; saunders, 2012, 2014). photoperiod, i.e., daylength, is a pivotal cue because of its seasonality and invariability (tauber & tauber, 1976; tauber & kyriacou, 2001; saunders, 2012, 2014). temperature is also a significant environmental cue, although it may fluctuate seasonally from year to year (tauber & tauber, 1976; tauber & kyriacou, 2001; saunders, 2012, 2014). nevertheless, both photoperiod and temperature often exert significant synergistic power influencing an insect’s response (kimura, 1988; vaz nunes & saunders, 1989; chen et al., 2014; saunders, 2014). why study diapause in bees? diapause has been observed amongst insects which play diverse ecological roles, e.g., herbivorous agricultural pests (adedokun & denlinger, 1985) and pollinators (e.g., van benthem et al., 1995; goulson, 2010). these insects may exert social and economic impacts in many regions of the world. however, beneficial insects acting as pollinators currently add high economic value to agriculture globally (€153 billion; gallai et al., 2009), in addition to their contribution to the reproduction of wild plants (gallai et al. 2009; ollerton et al. 2011). it is known that many bee species undergo diapause at some point in their life cycle, including solitary bee species, such as megachile rotundata (fabricius), nomia melanderi (cockerell), osmia rufa (linnaeus), and osmia lignaria say (hsiao & hsiao, 1969; bosch et al., 2010; fliszkiewicz et al., 2012; wasielewski et al., 2013). amongst the social bees, honey bees (apis mellifera linnaeus) show certain behavioral adaptations for surviving low temperatures, e.g., the storage of honey, the decrease or complete absence of broods during autumn and winter, increased longevity, and reduced metabolism of the workers (seeley, 1985). bumblebees (bombus latreille) have queens that survive the winter in diapause, and are certainly the most commonly investigated in this respect (plowright & laverty, 1984; beekman et al., 1998; goulson, 2010). while there are many aspects of diapause in bees that could be dealt with here, our focus is to review diapause in stingless bees, a subject that has still been little studied. stingless bees and the diapause stingless bees are found in tropical and subtropical regions all around the world (sakagami, 1982). they usually nest in holes in trees, in the soil, and even in cavities in human constructions, but some also construct aerial nests (nogueira-neto, 1997). their nests are perennial and they are active throughout the year (sakagami, 1982; nogueira-neto, 1997). however, some species may show quite a remarkable change in activities at the colony as a diapausal response to adverse climatic conditions. most descriptions in the literature of diapause in stingless bees have concerned the genus plebeia schwarz, e.g., plebeia droryana (friese), plebeia emerina (friese), plebeia wittmanni moure & camargo (friese), plebeia nigriceps (friese), plebeia remota (holmberg), plebeia julianii moure, and plebeia saiqui (friese) (juliani, 1967; terada et al., 1975; imperatriz-fonseca & oliveira, 1976; kleinert-giovannini, 1982; wittmann, 1989; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; witter et al. 2007; alves et al., 2009; nunes-silva sociobiology 61(4): 369-377 (december 2014) 371 ta bl e 1. b io lo gi ca l a nd e co lo gi ca l f ea tu re s pr es en te d by s tin gl es s be es ( h ym en op te ra : a pi da e: m el ip on in i) d ur in g th e re pr od uc tiv e di ap au se in a ut um n an d/ or w in te r in s ou th er n so ut h a m er ic a. l oc al ity : p r =p ar an á, b ra zi l. (1 )e as t a si an ( m ay r ea ch m ou nt ai no us r eg io ns u p to 2 ,5 00 m a bo ve s ea le ve l) ; (2 ) r ep ro du ct iv e di ap au se n ot d et ec te d, b ut m ar ke d de cr ea se in e gg -l ay in g in w in te r; (3 )m ea su re s (i n m m ) m ad e in a r ef er en ce b ee f ro m th e co lle ct io n of th e sc ie nc e an d te ch no lo gy m us eu m o f po nt if íc ia u ni ve rs id ad e c at ól ic a do r io g ra nd e do s ul ( pu c r s) ; (4 )d ru m on d et a l. (1 99 8) ; ( 5) w itt er 2 00 7; (6 )v an b en th em e t a l., 1 99 5; (7 )a ve ra ge m in im um te m pe ra tu re ( in st itu to n ac io na l d e m et er eo lo gi a [i n m e t ], 2 01 4) ; (8 ) m in im um te m pe ra tu re r eg is te re d du ri ng d ia pa us e ac co rd in g to m en tio ne d re fe re nc e. sp ec ie s o ve rw in te ri ng st ra te gy n es t ar ch ite ct ur e w or ke r si ze (3 ) n um be r of w or ke rs p er n es t m on th s in di ap au se m ax im al w or ke r ag e (d ay s) te m pe ra tu re (° c ) l oc al ity r ef er en ce s p le be ia d ro ry an a o bl ig at or y in vo lu cr um 3. 5 4. 7 2, 00 0 – 3, 00 0 3 – 11 .7 ( 7) ; 9. 8 (7 ) sã o pa ul o r io g ra nd e do s ul te ra da e t a l. (1 97 5) ; b lo ch te in ( pe rs . ob s. ) p le be ia e m er in a o bl ig at or y in vo lu cr um 4. 0 4. 5 – 3 10 7 11 .7 ( 7) ; 12 .1 6 (8 ) sã o pa ul o r io g ra nd e do s ul k le in er tg io va nn in i, (1 98 2) ; sa nt os e t a l. (2 00 9) p le be ia ju lia ni i o bl ig at or y pi lla rs 3. 0 30 0 (4 ) 3 – 4 – 11 .1 (7 ) ; 9. 8 (7 ) pa ra ná r io g ra nd e do s ul ju lia ni ( 19 67 ); w itt er e t a l. (2 00 7) p le be ia w itm an ni o bl ig at or y pi lla rs 4. 5 – 3 27 4 9. 8 (7 ) r io g ra nd e do s ul w itt m an n (1 98 9) p le be ia s ai qu i o bl ig at or y in vo lu cr um 4. 0 5. 0 70 00 (5 ) 2. 5 – 6 17 4 11 .0 ( 8) r io g ra nd e do s ul pi ck a nd b lo ch te in ( 20 02 a ) pi ck a nd b lo ch te in ( 20 02 b ) p le be ia n ig ri ce ps o bl ig at or y pi lla rs 3. 5 20 0 (4 ) 3 – 4 – 9. 8 (7 ) r io g ra nd e do s ul w itt er e t a l. (2 00 7) p le be ia r em ot a o bl ig at or y pi lla rs 2. 75 4 .0 2, 00 0 – 5, 00 0 (6 ) 2 – 5 – 11 .3 ( 8) sã o pa ul o va n b en th em e t a l. (1 99 5) ; r ib ei ro e t a l. (2 00 3) ; n un es -s ilv a et a l. (2 01 0) m el ip on a ob sc ur io r fa cu lta tiv e in vo lu cr um 7. 1 – 2 – 6 12 0 9. 8 (7 ) r io g ra nd e do s ul b or ge s & b lo ch te in ( 20 06 ); b lo ch te in e t a l. (2 00 8) m el ip on a qu ad ri fa sc ia ta fa cu lta tiv e in vo lu cr um 8. 6 9. 6 30 0 – 40 0 2 – 3 (? ) – 11 .7 ( 7) sã o pa ul o n og ue ir an et o (1 99 7) m el ip on a bi co lo r sh en ck i – (2 ) in vo lu cr um 8. 9 9. 1 – 3 – 9. 8 (7 ) r io g ra nd e do s ul b lo ch te in e t a l. (2 00 8) tr ig on a ve nt ra lis h oo za na (1 ) fa cu lta tiv e (? ) in vo lu cr um – 10 ,0 00 – – 5. 0 c hh ia yi c ou nt y, ta iw an su ng e t a l. ( 20 08 ) cf dos santos, p nunes-silva, r halinski, b blochtein diapause in stingless bees372 et al., 2010). however, diapause has also occasionally been observed or suspected in melipona quadrifasciata lepeletier, melipona obscurior moure and trigona ventralis hoozana strand (kleinert-giovannini & imperatriz-fonseca, 1986; nogueira-neto, 1997; borges & blochtein, 2006; sung et al., 2008) (table 1). due to the more frequent occurrence of diapause in plebeia species compared to the other genera of stingless bees, this paper mainly describes the diapause of plebeia species in southern brazil, with reference to diapause in other stingless bees whenever possible. in this sense, we know little about whether other stingless bee species in subtropical regions of south america also show any facultative or obligatory reproductive diapause. many stingless bee species occur in this region, such as melipona quinquefasciata lepeletier, melipona quadrifasciata lepeletier, mourella caerulea friese, paratrigona subnuda moure, scaptotrigona bipunctata (lepeletier), scaptotrigona depilis (moure), schwarziana quadripunctata (lepeletier), tetragonisca fiebrigi (schwarz) and trigona spinipes (fabricius) (camargo & pedro, 2013). not all of these species are managed in these areas due to their aggressiveness, rarity or fragility when kept in hives, making observations of the provisioning and oviposition process (pop) difficult. the colony reproductive phase plebeia spp. occurs from the northernmost parts of mexico to the southern parts of south america (camargo & pedro, 2013). in brazil, this genus is represented by at least 19 species (camargo & pedro, 2013), although many species remain to be identified and described (silveira et al., 2002; camargo & pedro, 2013). nine of these 19 plebeia species occur in the southern parts of brazil (silveira et al., 2002; camargo & pedro, 2013), and seven of them are known to undergo diapause (see above). within the tribe meliponini, plebeia is considered a phylogenetically basal group among the neotropical stingless bees (camargo & pedro, 1992). a remarkable feature of this group is the aggressive and physically intense queen-worker interactions (sakagami, 1982; zucchi, 1993). these interactions occur predominantly during the pop, which is highly ritualized and complex in plebeia spp. (sakagami, 1982; zucchi, 1993). briefly, the pop can be defined as the construction of brood cells and provisioning of larval food by nurse workers, followed by queen oviposition and the subsequent sealing of the brood cells by nurse workers (sakagami, 1982; zucchi, 1993). in plebeia, pop is characterized by a large number of brood cells being built simultaneously until they reach the collar stage, a phase that may last from 3.5 to 7 hours (occasionally less than 30 minutes) (van benthem et al., 1995; drumond et al., 1996, 1997, 1998, 2000). during this stage, some workers may lay trophic and/or reproductive eggs on the comb or in the periphery of a brood cell, and such eggs are often eaten by the queen (van benthem et al., 1995; drumond et al., 1996, 1997, 1998, 2000). while some brood cells are still in the collar stage, workers on the comb display intense agitation, apparently stimulated by the presence of the queen (van benthem et al., 1995; drumond et al., 1996, 1997, 1998, 2000). subsequently, many workers start to load larval food into the new brood cells, and after a certain volume has been reached, the queen quickly lays her eggs onto the food and the cells are sealed by workers (van benthem et al., 1995; drumond et al., 1996, 1997, 2000, 1998). hence, in the colony reproductive phase (outside of diapause) there is a clear sequence of brood cell building, provisioning and egg-laying, which differs according to species. furthermore, there is usually very little, if any, involucrum cover over the brood cell area (van benthem et al., 1995; drumond et al., 1996, 1997, 1998, 2000). it is usually thought that stingless bees have a rather low ability to effectively thermoregulate their nests. in general, these bees build lamellae of wax and propolis, often called cerumen involucra, which partially or entirely cover the brood combs during the cold season (nogueira-neto, 1997). such nest architecture provides thermal insulation that passively protects brood combs and the adult bee population during unfavorable climatic conditions, such as unduly low temperatures (jones & oldroyd, 2006). however, such passive thermoregulation in stingless bees is not nearly as effective as the active thermoregulation provided by honey bees, for example, which use contractions of their thoracic muscles to produce heat inside their nests (jones & oldroyd, 2006). the addition of wax and propolis lamellae by stingless bees may be directly related to their need for facultative or obligatory reproductive diapause. it has also been suggested that their reduced ability to effectively thermoregulate their nests and their reproductive diapause may be related to body size (blochtein et al., 2008). in stingless bee inhabiting southern brazil, there is a progressive increase in worker size from plebeia spp. (with obligatory diapause), through m. obscurior (with facultative diapause) to melipona bicolor schencki gribodo (with no diafig. 1. colony reproductive phase: plebeia emerina (hymenoptera: apidae: meliponini) nest showing many workers over brood combs, some brood cells being built in the periphery. involucrum layers can already be seen around combs. sociobiology 61(4): 369-377 (december 2014) 373 pause) (blochtein at al., 2008) (table 1). it is thought that in cold regions, such as southern brazil, there could be a relationship between bee body size and their ability to withstand low temperatures (blochtein et al., 2008). the diapause phase many populations of plebeia spp. (p. droryana, p. emerina, p. julianii, p. nigriceps, p. remota, p. saiqui and p. wittmanni) in southern south america (table 1) may be exposed to quite rigorous winters that may reach temperatures at or below zero in june to july (instituto nacional de metereologia [inmet], 2014). in preparation for these cold winters, the construction rate of brood cells falls gradually until it stops entirely. consequently, the queen’s egg-laying is interrupted in many plebeia colonies during the milder autumn months of march to may (juliani, 1967; terada et al., 1975; imperatriz-fonseca & oliveira, 1976; kleinert-giovannini, 1982; wittmann, 1989; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010). this kind of diapause in stingless bees has been called reproductive diapause (juliani, 1967; terada et al., 1975; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010). one of the more conspicuous signs that a given colony of plebeia spp. is preparing for reproductive diapause is the progressive construction of a multi-layered involucrum covering the brood comb area, as observed in p. droryana and p. emerina (drumond et al., 1996) and in p. remota (ribeiro et al., 2003) (figs 1 and 2). in other cases, a substantial increase in the number of cerumen pillars may occur, also over the brood combs, as in p. remota (ribeiro et al., 2003) (fig 3a, b; table 1). at the peak of reproductive diapause, when all of the brood has already emerged (“winter workers”), brood cells or combs are no longer constructed and no combs are found in the nest (ribeiro et al., 2003). many workers and the queen can be seen clustered in specific regions of the nest, under or over food pots or among the pillars (or lamellae), where they exhibit lethargic movements or remain completely immobile (ribeiro et al., 2003). these “winter workers” are longer lived than the workers emerging at other times (table 1) (blochtein et al., 2008; nunes-silva et al., 2010). during reproductive diapause, queens of p. remota may lose weight, as evidenced by their reduced physogastry (ribeiro et al., 2003). this lower queen physogastry observed during diapause has been ascribed to a regression in ovarian development due to the cessation of egg-laying (ribeiro et al., 2003). however, little is known about this phenomenon in stingless bees, although it is possible that glycogen and body fat reserves also significantly decrease during this period. reproductive diapause in stingless bees has been recorded as lasting from 2 up to 6 months (juliani, 1967; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010) (table 1). however, colonies inhabiting the same locality may not always commence their reproductive diapause in synchrony (juliani, 1967; van benfig. 2. diapause phase: plebeia emerina (hymenoptera: apidae: meliponini) nest showing brood combs entirely covered by involucrum layers. below right: some honey pots. fig. 3. post-diapause phase: plebeia remota (hymenoptera: apidae: meliponini) nest shortly after terminating diapause. a note that there is a large number of food pots, several wax pillars and an incipient brood comb being built by some workers; b – detail from a with some removed pillars. cf dos santos, p nunes-silva, r halinski, b blochtein diapause in stingless bees374 them et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010). while some colonies may start diapause early, during the milder months (march, april), others may not start until the winter months (may, june) (juliani, 1967; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010). in contrast to this variability in preparation for diapause, which occurs over a prolonged period and independently among colonies at the same site, termination of diapause among plebeia colonies seems to be faster and synchronous during the first weeks of spring (in august, september or eventually in october) (juliani, 1967; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; alves et al., 2009; nunes-silva et al., 2010). despite the arrest of pop throughout reproductive diapause in stingless bees, the nest population is not entirely lethargic or immobile. other tasks, e.g., manipulating waste, storing food (nectar, pollen), or foraging still occur (juliani, 1967; van benthem et al., 1995; pick & blochtein, 2002b; ribeiro et al., 2003; nunes-silva et al., 2010). regarding foraging, the activity of workers outside of the nest is more intense when ambient temperatures are milder and the wind is light or absent (pick & blochtein, 2002b; ribeiro et al., 2003; nunes-silva et al., 2010). the foraging pattern and food sources harvested by foragers also change from the reproductive phase to reproductive diapause, at least in p. saiqui (pick & blochtein, 2002a, 2002b) and p. remota (nunes-silva et al., 2010). during the reproductive phase, p. remota forage constantly throughout the day and both nectar and pollen are collected (nunes-silva et al., 2010). however, during diapause foraging does not commence until noon, when temperatures are higher, and the collection of pollen is greatly reduced compared to the reproductive phase (nunes-silva et al., 2010). a decrease in pollen collection and a shift in the time of peak foraging activity between phases has also been observed for p. saiqui (reproductive period: peak 11:00-13:00; diapause: peak 13:00-14:00) (pick & blochtein, 2002a, 2002b). it has therefore been suggested that, at least for p. remota, the higher nectar input during diapause affects the potential energy storage by bees, whereas reduced collection of pollen (a protein source) causes no harm because there is no egg-laying or production of new individuals during this period (nunes-silva et al., 2010). nevertheless, comparative observations should also be made for other plebeia and stingless bee species that show diapause. an overview of the process of reproductive diapause in stingless bees is presented in table 2. factors determining diapause in stingless bees our knowledge regarding which environmental cues (e.g., photoperiod and temperature) are indeed significant in determining the onset, permanence and termination of diapause in stingless bees is almost absent. these social insects live inside cavities in trees or dead wood, and also nest in soil or other cavities, so how could the population in the nest, especially nurse workers and the queen, foresee the onset of adverse climatic conditions? furthermore, how could they (or foragers) communicate and/or induce reproductive diapause in their nestmates? unfortunately, there are few studies addressing these questions. for example, an experiment with p. remota showed that workers have a significant role in promoting diapause behavior (ribeiro, 2002). by introducing p. remota queens from colonies in diapause into colonies that were in the reproductive phase (and vice versa), it was possible to observe that diapausal queens did not reinitiate egg-laying (ribeiro, 2002). moreover, it remains unknown just how individuals that have never left their nests, and thus table 2. overall behavioral characteristics concerning to the reproductive diapause process in stingless bees (hymenoptera: apidae: meliponini). note: diapause phases following koštál (2006). pre-diapause diapause post-diapause induction preparation initiation/maintenance termination environmental stimuli (e.g. photoperiod and temperature) achieve critical levels according to physiological sensitivity from bees. progressive reduction in brood cell building with a decrease in provisioning and oviposition process; increased presence of cerumen involucrum layers or pillars over brood cells; shift in foraging pattern (1): larger input nectar and less pollen; shift in foraging pattern (2): flight activity concentrated around noon. no brood cell actively built; emergence of the last brood, winter workers. some brood cells start to be built; eventually this can occur over food pots or cerumen layers. full removal or significant reduction in involucrum layers or pillars; usual provisioning and oviposition process (pop) is implemented. autumn / winter spring / summer sociobiology 61(4): 369-377 (december 2014) 375 have not been exposed to external environmental cues, are induced to enter, continue and terminate diapause. studies with other social insects might provide some clues. for example, it has been suggested that foraging honeybees may somehow synchronize their biological rhythms and inform nestmates about external environmental conditions through their body movements (bloch et al., 2013). another possibility could be some sort of chemical signaling that would induce entire nest populations to enter or terminate diapause. evidence for this has been obtained from myrmica rubra (linnaeus) ants in which larvae remain in diapause and do not pupate, while simultaneously queens do not lay eggs (kipyatkov, 2001). in this study, it was observed that when diapausal nests were interconnected to non-diapausal nests, and odors from the latter could enter the former, diapause was terminated in the diapausal colonies, most probably due to worker primer pheromones. however, the reverse effect was not observed (kipyatkov, 2001). unfortunately we do not yet know if this system could also apply in stingless bees, even though chemical communication is known to also play a pivotal role within their colonies. conclusions and perspectives possibly because nest thermoregulation in stingless bees is somewhat passive and not highly efficient, species exposed to winters that are more rigorous in southern brazil have apparently evolved reproductive diapause behavior as a strategy for overwintering. this strategy interrupts egg laying for up to 6 months, even though this is a critical activity promoting the growth and maintenance of their nests. stingless bees are subject to several environmental stresses (freitas et al., 2009). therefore, it is reasonable to assume that stingless bee colonies could be more susceptible to strong environmental pressures during diapause, due to the lack of population resilience. furthermore, very little is known about how climate change may affect the distribution of stingless bees which show diapause behavior. the future of stingless bee populations which show reproductive diapause will depend on how the scientific community, policy makers and society act to effectively preserve the natural populations of these pollinators. in conclusion, we have much to learn about reproductive diapause in stingless bee species. the use of a combination of methods involving molecular, biochemical, physiological and ecological techniques could allow new investigations on the mechanisms underlying the regulation of diapause in stingless bees. we hope that this review will stimulate further studies that address these questions and that it will lead to better conservaion practices to preserve these bee species. acknowledgements we thank conselho nacional do desenvolvimento científico e tecnológico (cnpq) [process number, 500458/20138], fundação de amparo à pesquisa do estado do rio grande do sul (fapergs) and coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarships (cfs, pns and rh). references adedokun, t.a. & denlinger, d.l. 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(1993). ritualized dominance, evolution of queen-worker interactions and related aspects in stingless bees (hymenoptera: apidae). in t. inoue & s. yamane (eds.), evolution of insect societies (pp. 207-249). tokyo: hakuhinsha. this article has online supplementary material at: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/641/0 doi: 10.13102/sociobiology.v61i4..s556 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.902sociobiology 63(1): 645-650 (march, 2016) daily foraging activity of acromyrmex (hymenoptera: formicidae) leaf-cutting ants introduction leaf-cutting ants (atta and acromyrmex, formicidae) are possibly the most important herbivores of terrestrial environments throughout the neotropics, leading to considerable economic losses to a variety of crops (hölldobler & wilson, 2011). foragers of these ants cut fragments of leaves and other plant parts and carry them to their nests to be used as substrate for the cultivation of a mutualistic fungus (weber, 1972; hölldobler & wilson, 1990). traditionally, attempts to control leaf-cutting ants involve the use of insecticides from diverse chemical groups and different formulations (liquid concentrates such as nebulizers, fumigants, but mainly granulated baits) (araújo et al., 2003). temperature and relative humidity have marked effects on the activities of all animal species (begon et al., 2006) and the behavior of leaf-cutting ants is strongly influenced by those parameters (roces & kleineidam, 2000; bollazzi & roces, 2002). temperature primarily affects both worker activities abstract leaf-cutting ants are well-known insects due to their remarkable activity as herbivores and the considerable economic damage they cause to many crops. the identification of season and time of day when leaf-cutting ants are most active is an important tool, not just to understand the foraging ecology of these ants, but also to optimize their control in plantation areas where they are pests. thus, the aims of this study are to evaluate the daily foraging activity of leafcutting ant species of the genus acromyrmex, which occur in forest plantations in southern brazil. foraging activity of acromyrmex crassispinus (forel) and acromyrmex subterraneus subterraneus (forel) were correlated with weather conditions, and it was more intense during spring and summer. workers that forage at night are significantly heavier than workers that forage during the day. this study showed that a. crassispinus and a. subterraneus subterraneus did not forage at temperatures below 10-11°c. then, the use of granulated baits to control these leaf-cutting ants species where they are pests should be done just under favorable conditions of temperature for acromyrmex foraging activity (over 12°c), to ensure maximum collection of baits by ants and the least left-over baits. sociobiology an international journal on social insects ma nickele1, w reis filho2, mr pie1, src penteado3 article history edited by evandro nascimento silva, uefs, brazil received 25august 2015 initial acceptance 06 march 2016 final acceptance 15 march 2016 publication date 29 april 2016 keywords attini, ecology behavior, forest pests, pinus. corresponding author mariane aparecida nickele universidade federal do paraná departamento de zoologia caixa postal 19020, cep 81531-980 curitiba-pr, brazil e-mail: manickele@ufpr.br by inhibiting or reducing foraging, as well as the speed of brood development, conditions that directly determine the growth rate ofa colony (gamboa, 1976; porter, 1988; porter & tschinkel, 1993). also, the fungus gardens have strict demands of high humidity and temperatures between 20 and 30ºc for proper growth (powell & stradling, 1986). foraging activity of atta sexdens (l.) and atta cephalotes (l.) is mainly daytime during the winter and nighttime during the summer in paraguay (fowler & robinson, 1979) and in costa rica (wetterer, 1990). foraging activity of atta colombica guérin-méneville is more frequent during the day in the republic of panama (wirth et al., 1997). atta capiguara gonçalves shows two daily foraging journeys, one in daytime and another one at night, a variation that is a function of dry and wet seasons (caldato, 2014). for acromyrmex species, studies showed that acromyrmex versicolor versicolor (pergande) is primarily nocturnal during the hot summer months, but shifted toward diurnal activity as fall progressed in tempe, arizona (gamboa, 1976). foraging research article ants 1 universidade federal do paraná, departamento de zoologia, curitiba-pr, brazil 2 epagri/embrapa florestas, centro nacional de pesquisa florestal, colombo-pr, brazil 3 embrapa florestas, centro nacional de pesquisa florestal, colombo-pr, brazil ma nickele et al. – daily foraging activity of acromyrmex646 activity of acromyrmex crassispinus (forel) occurred throughout the day during april in paraguay (fowler, 1979). the annual foraging activity of a. subterraneus subterraneus (forel) was exclusively nocturnal in viçosa, mg, brazil (maciel et al., 1995), while in acromyrmex balzani (emery) (mendes et al., 1992) and acromyrmex laticeps nigrosetosus forel (araújo et al., 1998) is mainly nocturnal in minas gerais, brazil. there are suggestions that the micro-weather near each nest, or even peculiar to each trail or nest-entrance may affect their foraging rhythms. nocturnal foraging may be encouraged by warm nights, by cool or very hot dry days, and by full clear moonlight; conversely, diurnal foraging is supposedly encouraged by cool nights, warm days and moonless or dull nights (lewis et al., 1974a). leaf-cutting ants’ workers are highly polymorphic (wilson, 1980; endringer et al., 2012). in some leaf-cutting ant species, there are dial changes in worker weight. workers that foraged at night were heavier than workers that foraged during the day. heavier leaf cutters do not forage during the day in order to avoid diurnal parasitoids that preferentially attack larger ants or they might avoid activity outside the nest during the day due to greater risks with desiccation or heat stress (wetterer, 1990). acromyrmex crassispinus and a. subterraneus subterraneus (forel) were the most commonleaf-cutting ant species in pine plantations in southern brazil (nickele et al., 2009). the identification of season and time of day when leaf-cutting ants are most active is an important tool, not just to understand the foraging ecology of these ants, but also to optimize their control in plantation areas where they were pests. thus, the aims of this study are to evaluate the daily foraging activity of leaf-cutting ant species of the genus acromyrmex, which occur in forest plantations in southern brazil. material and methods observations were conducted on one adult colony of a. crassispinus (nest size = 1 m²) and one adult colonyof a. subterraneus subterraneus (nest size = 6.25 m²) at the same time, during all four seasons of 2010 (february, 10, may, 10, july, 10, and october, 10). colonies were located in a pinus taeda (l.) plantationin rio negrinho city (26º15’16” s; 49º31’06” w; alt. 790 m), state of santa catarina, brazil. the colonies were monitored during 24 hours in all seasons. surveys began at 18:00h and ended at 17:00h the following day. foraging rates were measured at one-hour intervals by counting all ants passing a fixed point in the active foraging trail close to the nest entrance. workers leaving the nest and workers returning (either laden or unladen) were counted for 3 minutes at each sampling period (24 samples per colony per season). in order to investigate the correlation between ant activity and climate parameters, temperature and relative humidity of theair were measured usinga thermohygrometer. measurements were made near the monitored nests during the first minute of each sampling interval. in order to study the ant size variation during the activity period (diurnal or nocturnal workers), 20 workers were collected at each interval (10 ants per colony per hour) following the measurement of foraging rates. workers were stored in individual eppendorf® vials and they were weighed to the nearest 0.1 mg on a mettler balance. differences in body weight of ants that foraged during the day (ants collected in the period from 7:00h until 18:00h) or during the night (ants collected in the period from 19:00h until 6:00h) were tested using student’s t tests. results foraging activity of a. crassispinus started at 4:00h and stopped at 14:00h, on summer. there was significant correlation between foraging activity and humidity, in this season. on fall, a. crassispinus workers did not foraged from 2:00h until 08:00h. in this case, there was significant correlation in foraging activity and both climatic parameters (temperature and humidity). on winter, a. crassispinus workers did not foraged from 3:00h until 07:00h, and there was significant correlation in foraging activity and temperature. on spring, a. crassispinus workers showed foraging activity all day long and in this season there was not significant correlation in foraging activity and bothclimatic parameters. acromyrmex crassispinus workers did not forage at temperatures below 10°c (table 1, fig 1). season climate parameters species r p temperature a. subterraneus subterraneus -0.44 0.020 summer a. crassispinus -0.22 0.300 humidity a. subterraneus subterraneus 0.32 0.124 a. crassispinus 0.61 0.001 temperature a. subterraneus subterraneus 0.52 0.007 fall a. crassispinus 0.87 0.000 humidity a. subterraneus subterraneus -0.42 0.038 a. crassispinus -0.90 <0.001 temperature a. subterraneus subterraneus -0.08 0.695 winter a. crassispinus 0.40 0.050 humidity a. subterraneus subterraneus 0.31 0.134 a. crassispinus -0.14 0.503 temperature a. subterraneus subterraneus 0.17 0.430 spring a. crassispinus 0.23 0.269 humidity a. subterraneus subterraneus -0.02 0.939 a. crassispinus -0.30 0.151 table 1: correlation between ant activity and climate parameters. sociobiology 63(1): 645-650 (march, 2016) 647 foraging activity of a. subterraneus subterraneus started at 2:00h and continued all day long, on summer. there was significant correlation between foraging activity and humidity, in this season. on fall, workers did not foraged from 22:00h until 09:00h. in this case, there was significant correlation in foraging activity and bothclimatic parameters. on winter, workers did not foraged from 2:00h until 09:00h, fig 1. daily foraging activity of the leaf-cutting ant acromyrmex crassispinus in rio negrinho, sc, brazil, 2010. fig 2. daily foraging activity of the leaf-cutting ant acromyrmex subterraneus subterraneus in rio negrinho, sc, brazil, 2010. and there was significant correlation in foraging activity and temperature. on spring, workers did not foraged from 2:00h until 07:00h, and there was significant correlation in foraging activity and both climatic parameters. acromyrmex subterraneus subterraneus workers did not forage at temperatures below11°c (table 1, fig 2). 0 20 40 60 80 100 0 50 100 150 200 250 300 350 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs summer 0 20 40 60 80 100 0 50 100 150 200 250 300 350 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs fall 0 20 40 60 80 100 0 50 100 150 200 250 300 350 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs winter 0 20 40 60 80 100 0 50 100 150 200 250 300 350 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs spring leaving coming back unladen coming back laden temperature humidity 0 20 40 60 80 100 0 40 80 120 160 200 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs summer 0 20 40 60 80 100 0 40 80 120 160 200 te m pe ra tu re ( °c ) / h um id ity ( % ) n um be r of w or ke rs fall 0 20 40 60 80 100 0 40 80 120 160 200 te m pe ra tu re ( °c ) / h um id ity ( % ) n um be r of w or ke rs winter 0 20 40 60 80 100 0 40 80 120 160 200 te m pe ra tu re ( °c ) / h u m id ity (% ) n u m be r o f w or ke rs spring leaving coming back unladen coming back laden temperature humidity ma nickele et al. – daily foraging activity of acromyrmex648 foraging activity of a. crassispinus was more intense during the spring and foraging activity of a. subterraneus subterraneus was more intense during the summer. a considerable number of workers were observed returning unladen to the nest, for both acromyrmex species in all seasons (fig 1 and 2). there was variation in body weight during the activity period. workers that foraged at night were significantly heavier than workers that foraged during the day. the weight of a. crassispinus workers that foraged during the day was 5.32 ± 0.172 mg (n=219) )( xsx ± and 6.07 ± 0.264 mg (n=154) at night (t = -2.35, p = 0.0193). the weight of a. subterraneus subterraneus workers that foraged during the day was 7.73 ± 0.246 mg (n=247) and 9.72 ± 0.294 mg (n=178) at night (t = -5.46, p <0.001). discussion foraging activities of a. crassispinus and a. subterraneus subterraneus leaf-cutting ants occurred at night and during the day, however it was correlated with weather conditions, such as temperature and humidity. these acromyrmex species were most active during spring and summer, but they have also foraged during fall and winter. however, foraging intensity was lower in these two last seasons. similar results were observed for acromyrmex species in the high lands of santa catarina state, brazil, where ants were most active during the warmest months (geisel et al., 2008). temperature and humidity fluctuations might cause changes in ant respiration and water loss, as well as affect the water balance of the plants being cut, which indirectly affect handling time and nutritional quality of the plants as fungal substrate (fowler, 1979). acromyrmex crassispinus and a. subterraneus subterraneus did not forage at temperatures below 10°c and 11°c, respectively. in viçosa, mg, brazil, it was observed that a. subterraneus subterraneus cease their foraging activity at temperatures below 14°c (maciel et al., 1995). atta sexdens (l.) and atta mexicana (f. smith) do not forage at temperatures below 10°c and 12°c, respectively (fowler & robinson, 1979; mintzer, 1979). foraging activities of the leaf-cutting ant atta sexdens piriventris santschi were concentrated during the night in the summer, in the afternoon and in the winter, allowing ants to forage under favorable temperature conditions (geisel et al., 2013). a considerable number of workers were observed returning unladen to the nest in both studied acromyrmex species. a similar pattern was reported for acromyrmex heyeri forel, in which a large proportion of workers returned to the nest unladen at the initial foraging phase, which could represent a response to the high needs for information at the beginning of a daily foraging process (bollazzi & roces, 2011). however, in this study we observed a great number of unladen workers returning to the nest not only at the initial foraging phase. these workers could be scouts (lewis et al., 1974b), workers involved in trail clearing (lewis et al., 1974b; howard, 2001), transport of plant sap (stradling, 1978), reinforcement of the chemical trail (evison et al., 2008), or in a combination of recruiting and food transport, where these workers cut no fragments, but return to the nest displaying recruiting behavior (jaffé & howse, 1979). workers that foraged at night were significantly heavier than workers that foraged during the day. the same pattern was observed in atta cephalotes (l.) (wetterer, 1990; feener & brown, 1993). however, daytime a. sexdens foragers were not significantly smaller than nighttime foragers (tonhasca, 1996; tonhasca & bragança, 2000). one explanation is that heavier leaf cutters do not forage during the day in order to avoid diurnal parasites that preferentially attack larger ants (wetterer, 1990). atta cephalotes colonies shifted the size distribution of workers on foraging trails as a second line of defense against parasitism by neodohrniphora curvinervis (malloch) (feener & brown, 1993). however, it seems not to be an effective defense against phorids in acromyrmex, because a study reported that acromyrmex phorids selected ants from all sizes available outside the nests. phorids did not have preference to attack larger acromyrmex workers as in atta (elizalde & folgarait, 2011). another possible explanation for the diel changes in forager size is that larger ants might avoid activity outside the nest during the day due to greater risks with desiccation or heat stress (wetterer, 1990). this study showed that a. crassispinus and a. subterraneus subterraneus were most active during spring and summer and they did not forage at temperatures below 1011°c. this result is an important tool, not just to understand the foraging ecology of these ants, but also to infer in leaf cutting ants control. the use of granulated baits to control these leafcutting ants species where they were pests should be done just under favorable conditions of temperature for acromyrmex foraging activity (over 12°c), to ensure maximum collection of baits by ants and the least left-over baits. acknowledgements we thank elisiane de castro queiroz, mila ferraz de oliveira martins and priscila strapasson for their assistance during field work. we also thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for granting fellowship to m.a.n and battistella florestal for financial support and granting the study area. references araújo, m. da s., della-lucia, t. m. c., picanço, m. c., anjos, n. dos & vilela, e. f. 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(1997). annual foraging of the leaf-cutting ant atta colombica in a semideciduous rain forest in panama. journal of tropical ecology, 13: 741-757. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7204sociobiology 68(4): e7204 (december, 2021) introduction weaver ants (oecophylla sp.) are arboreal, eusocial insects (family: formicidae; order: hymenoptera). they play a significant role in pest management, food, and medicines (itterbeeck et al., 2014). oecophylla sp., also known as the red weaver ants and green tree ants, comprises only two extant species, o. smaragdina (fabricius), distributed in tropical asia to northern australia and o. longinoda (latreille) distributed in africa (cole & jones, 1948). weaver ants exhibit a unique strategy of nest building on the tree using the leaves for nest construction. the mated o. smaragdina queen starts its colony by laying the first batch of about 35 eggs underneath tree leaves and taking care of the eggs until they hatch into worker abstract weaver ants are known for their unique nest-building skills using leaves and larval-silk as a binding agent. the weaver ants, oecophylla smaragdina, are present in large numbers in the ri-bhoi district, meghalaya. this study is the first from this region on the nesting behavior and the population dynamics of these ants. it was noted that oecophylla smaragdina build nests more abundantly in needlewood trees (schima wallichi), locally known as ‘diengngan’. the nests of o. smaragdina are somewhat round-oval using the leaves of different sizes ranging from 8-32 cm2, and the average nest size is about 9,483 cm3. the nearest distance of the nest from the central tree trunk of s. wallichi is about 0.7 m, and the farthest is up to 3.4 m. the nests are made at a height ranging from 4-25m. they utilize about twenty leaves with a specific number of chambers to keep their broods, the queen, and food. the number of worker ants, pupae, and larvae are variable in nests of different sizes. the study shows that the active period for foraging and nest building of o. smaragdina is at 20-25 °c and significantly declines with the rise in temperature at 30 °c and above. this finding on the presence of an enormous population and familiarity with the nesting behavior of weaver ants o. smaragdina could be useful as an alternative source of nutrition and traditional medicine for the people in this region. sociobiology an international journal on social insects jonata sa sangma, surya b prasad article history edited by evandro nascimento silva, uefs, brazil received 30 april 2021 initial acceptance 01 august 2021 final acceptance 18 october 2021 publication date 23 december 2021 keywords oecophylla smaragdina, nest building, nest size, population. corresponding author surya bali prasad department of zoology school of life sciences north-eastern hill university shillong, india. e-mail: sbnehu@gmail.com ants (lokkers, 1990). the worker ants exhibit a bimodal size distribution in which the major workers are approximately 8-10 mm in length, and the minors are roughly half the length of the majors (weber, 1946; wilson & taylor, 1964). the function of the minor workers is mostly to feed the queen and primary workers in attending the broods, rearing, and milking honeydew from the scale insects. the significant workers function as soldiers, hunters, and nest builders (holldobler, 1983). while building the nest, the worker ants pull the leaves and glue them using their larvae silk. in the case of leaves that are far, a group of worker ants move towards the target leaves and form the ants chain to pull towards the nest site by shortening the chain by one ant at a time until the leaves are department of zoology, school of life sciences, north-eastern hill university, shillong, india research article ants population and nesting behaviour of weaver ants, oecophylla smaragdina from meghalaya, india jonata sa sangma, surya b prasad – population and nesting behavior of weaver ants2 close enough to be glued together. when the nest leaves die, these weaver ants abandon the nest and construct a new one (bharti & silla, 2011). they also clean the nest by applying venom gland secretions (formic acid) to avoid bacterial and fungal infections (tragust, 2016). in southeast asia, australia, and africa, o. smaragdina is consumed as food, used in traditional medicines, and pest management (offenberg et al., 2013; wetterer, 2017). they are used as natural bio-control agents against agricultural pests by indigenous farmers in southeast asia (peng & christian, 2005; crozier et al., 2010). in australia, o. smaragdina is used to treat cold, flu, and headache (crozier et al., 2010; bharti & silla, 2011). o. smaragdina has also been used for treating severe cough, cold, and flu in myanmar, africa, and india (rastogi, 2011). north-eastern india constitutes eight states with various ethnic groups showing unique cultural diversity (teron, 2019). different ethnic groups in assam have unique traditions and cultures distinct from each other and utilize weaver ants for food, medicine, and livelihood. (langthasa et al., 2017). the nyishi and the galo tribes of arunachal pradesh, india, use weaver ants to treat fever (chakravorty et al., 2011). a prominent state in northeast india, meghalaya comprises thirteen districts and three indigenous ethnic groups, the khasis, garos, and the jaintias. it has a rich diversity of flora and fauna, which are endemic to the region. in the garo tradition of meghalaya, o. smaragdina, locally known as ‘ketchra,’ is consumed as food (dey, 2013). there is no population dynamics study of these weaver ants from meghalaya. our preliminary survey showed that these weaver ants are present in large numbers in the ri-bhoi district, meghalaya. therefore, the present study was undertaken to examine the population dynamics of the weaver ants concerning the variation in leaves utilized for nest construction and the nest size. further, the study also evaluated the pattern of the weaver ants at different time intervals concerning the temperature for foraging and building a nest. considering that nesting biology of this species is poorly understood, the present study was carried out to test the hypothesis that: 1) the size of weaver ant nests is shaped by the multidimensionality of habitat hypervolume and population traits and 2) the foraging and nest building activity is influenced by temperature materials and methods study area the study was carried out during april 2019-february 2020 in umran-dairy village (25.78° n latitude and 91.88° e longitude) of the ri-bhoi district of meghalaya, india (fig 1). fig 1. maps showing the location of ri-bhoi district (collection/study site) of meghalaya, india (maps not at scale). ri-bhoi district is on the national highway-40 in between shillong and guwahati of assam. it is a hot and humid place with an ranging from 18 – 32 °c tand an average annual rainfall of 1267 mm. o. smaragdina nests were located in the study area and examined for the type and height of trees, the nests from the ground, and the nests’ distance from the central trunk (santos & del-claro, 2009). a behavioral observation like nest building, foraging, protecting the nest, and rearing scale insects was done in the field. the interaction between the ants and other organisms present in the tree was also studied. the study plan has been approved by the institutional ethics committee (animal models), northeastern hill university, shillong, india vide reference no. iec/ms/misc./21, 3-12-20. https://en.wikipedia.org/wiki/biological_pest_control sociobiology 68(4): e7204 (december, 2021) 3 nest structure and population study a total of 15 nests of these weaver ants were collected for further study. the nest size was calculated, taking the average length and breadth of the nests from three locations, as shown in fig 2. the height was also measured, and the area/size (cm3) of the nest was calculated as length x breadth x height (l x b x h) (fig 2). the number of queens, worker ants, larvae, and pupae were recorded to know the population in a nest. the number of chambers in the nest and leaves utilized for nest construction was also recorded. fig 2. diagrammatic representation of a nest and the location of the measurement taken for nest area. nesting and foraging activity nest building behavior was observed four times a day, i.e., morning at 8.0 a.m., afternoon at 1.0 p.m., evening at 6.0 p.m., and at night at 9.0 p.m.in the field for ten days. related activity such as hunting or foraging and interactions with scale insects was also observed during that time. statistical analysis pearson correlation, analysis of variance (anova), and principal component analysis (pca) were used as statistical tools to analyse the data and establish a correlation between different variables. fig 3. scree plot showing all components. the first principal component explains about 71% of nest size, the second and the third principal components explain about 16% and 8% respectively, accounting for 95% of the cumulative variation. jonata sa sangma, surya b prasad – population and nesting behavior of weaver ants4 results nesting trees and nest spatial location the o. smaragdina nests were found on mango trees (mangifera indica), litchi tree (litchi chinensis), jackfruit tree (artocarpus heterophyllus), needlewood trees (schima wallichi), and a few in other trees. compared to other trees, the nests of these weaver ants were found abundantly in schima wallichi or the needlewood trees locally known as ‘diengngan’. the approximate height of the trees where the nests were found ranged from 4-25 m. regarding the location of nests in the multidimensional spatial hypervolume, we found that the average nest height from the ground was about 6 m. we found the distance of the nest from the needlewood trees central trunk ranging from 0.7-3.4 m (table 1). sample nest nest size dimensions (cm) (l × b × h) area/size of the nest (cm3) height of the nest from the ground (m) distance of the nest from the central trunk (m) no. of leaves utilized for nest construction no. of chambers population of oecophylla smaragdina 1 25×15×15 5625 4.6 2.5 19 17 2850 2 10×5×8 400 4.5 0.7 7 5 979 3 30×15×17 7650 5.5 3 25 21 3321 4 15×14×10 2,100 3.7 2.3 10 6 1866 5 15×20×10 3,000 4.3 1 13 7 2031 6 20×17×11 3,740 4.9 2.6 15 13 4429 7 10×12×6 720 4.3 1.7 6 16 1896 8 20×18×16 5,760 4.3 2.5 14 15 4164 9 60×31×30 55800 4.4 3.1 53 45 8563 10 50×25×19 23750 10.7 3.4 48 38 9211 11 40×25×15 15000 8.5 2.9 41 32 8712 12 18×15×14 3780 9.1 2.6 12 12 3742 13 20×15×16 4800 3.6 1.1 16 13 6348 14 21×16×18 6048 9.4 1.8 18 15 4006 15 17×15×16 4080 9.8 1.6 15 11 2627 mean 9483.53 6.11 2.19 20.80 17.73 4316.33 table 1. demographic details of o. smaragdina nests on needlewood trees in ri-bhoi district of meghalaya. nest structure and its population the nest size was statistically analysed, showing a wide variation, with the smallest of 400 cm3 and the largest being 55,800 cm3 (table 1). pearson correlation coefficient analysis established that the total population, number of leaves utilized for nest construction, and the number of chambers to be positively correlated with the nest size (r2 = 0.731, 0.878, and 0.885, p ≤ 0.01, respectively) (table 2). data analysis using pca (fig 3) showed that three variables explain 95% of the cumulative variation in nest size. thus, o. smaragdina population cum nest size in the first principal component, explains about 71% of the total variance. the second and third components include the number of leaves utilized and the number of chambers explaining 16% and 8% of the total variance respectively. the analysis shows that the population of o. smaragdina, the number of leaves utilized, and the number of chambers contribute as essential factors for nest building (fig 4). the nests were divided into three groups, i.e., small nests ranging from 400-4,000 cm3, medium nests from 4,001– 10,000 cm3, and large nests ranging from 10,000 cm3 and above. the average number of leaves used for the construction of small, medium, and large nests was 10.5 ± 3.50, 17.83 ± 3.9, and 47.33 ± 6.02 having 9.83 ± 4.44, 15.33 ± 3.44, and 38.33 ± 6.5 chambers, respectively. similarly, the average population of the ants in these nests was 2491± 1,308.7, 386 ± 1,351.3, and 8,829 ± 339.4, respectively (table 3). the number of different castes of o. smaragdina varied in different nests (table 4). one or more queens may be present in a single nest, and the nest without the queen is probably the satellite nest, while more queens were observed in nest 9. the highest number of worker ants, pupae, and larvae were recorded in nest 10, nest 11 and nest 9, respectively and the least number of all the castes were recorded in nest 2. pearson correlation analysis was done to establish a relationship between the presence of a queen and the presence sociobiology 68(4): e7204 (december, 2021) 5 approximate nest area height of the nest from the ground distance of the nest from the central trunk no. of leaves utilized for nest construction no. of chambers population of oecophylla smaragdina approximate nest area pearson correlation 1 .086 .545* .878** .885** .731** sig. (1-tailed) .380 .018 .000 .000 .001 n 15 15 15 15 15 15 height of the nest from the ground pearson correlation 1 .316 .346 .292 .359 sig. (1-tailed) .126 .103 .146 .094 n 15 15 15 15 15 distance of the nest from the central trunk pearson correlation 1 .689** .724** .612** sig. (1-tailed) .002 .001 .008 n 15 15 15 15 no. of leaves utilized for nest construction pearson correlation 1 .959** .891** sig. (1-tailed) .000 .000 n 15 15 15 no. of chambers pearson correlation 1 .869** sig. (1-tailed) .000 n 15 15 population of oecophylla smaragdina pearson correlation 1 sig. (1-tailed) n 15 table 2. pearson correlation (one-tailed), n = 15, showing significance at **p ≤ 0.01 and *p ≤ 0.05 comparing the multidimensionality of different variables. fig 4. principal component plot showing important factors contributing to variations in o. smaragdina nest size. component 1 explaining about 71% variation and component 2 about 16%. jonata sa sangma, surya b prasad – population and nesting behavior of weaver ants6 of different castes in the nest. pearson correlation (table 5) shows a positive correlation between the number of queens and the number of workers (r2 = 0.607), pupae (r2 = 0.570), and larvae (r2 = 0.735). the number of worker ants shows a positive correlation with the number of pupae (r2 = 0.805) and the number of larvae (r2 = 0.794). also, the number of pupae is positively correlated with the number of larvae (r2 = 0.751). however, a comparison between nests with the queen and without the queen revealed that only the number of pupae shows significant differences between the groups (table 6). table 3. nest size range showing the average number of leaves used, chambers and population of o. smaragdina. mean ± sd, n = 6 for small and medium nest, n = 3 for large nest. nest category average nest size (cm3) average number of leaves for nest construction average number of chambers average population of oecophylla smaragdina small 400 4,000 11 ± 3.5 10 ± 4.4 2,491 ± 1,308.7 medium 4,001 10,000 18 ± 4.0 15 ± 3.4 3,886 ± 1,351.3 large 10,000 ≤ above 47 ± 6.0 38 ± 6.5 8,829 ± 339.4 sample nest no. of queen no. of worker ants no. of pupae no. of larvae nest 1 1 1375 963 511 nest 2 0 545 327 107 nest 3 2 1506 1498 315 nest 4 1 788 560 517 nest 5 0 896 763 372 nest 6 1 1675 1250 1503 nest 7 1 678 645 572 nest 8 1 1540 1298 1325 nest 9 3 3796 2589 2175 nest 10 2 4572 2518 2119 nest 11 1 3563 3716 1432 nest 12 0 1855 1430 457 nest 13 2 2536 2049 1761 nest 14 1 2742 576 687 nest 15 0 1796 574 257 mean ± sd 1.07 ± 0.88 1990.87 ± 1215.59** 1383.73 ± 958.91* 940.67± 705.38** no. of queen no. of workers no. of pupae no. of larvae no. of queen pearson correlation 1 .607** .570* .735** sig. (1-tailed) .008 .013 .001 n 15 15 15 15 no. of workers pearson correlation .607** 1 .805** .794** sig. (1-tailed) .008 .000 .000 n 15 15 15 15 no. of pupae pearson correlation .570* .805** 1 .751** sig. (1-tailed) .013 .000 .001 n 15 15 15 15 no. of larvae pearson correlation .735** .794** .751** 1 sig. (1-tailed) .001 .000 .001 n 15 15 15 15 table 4. details of different castes of o. smaragdina present per nest. the result is expressed as mean ± sd, n = 15, *p ≤ 0.05 and **p ≤ 0.01 compared to the presence or absence of the queen. (the significance value was taken from pearson correlation analysis shown in table 5). table 5. pearson correlation analysis (one-tailed) showing the correlation between the different castes. **p ≤ 0.01 and *p ≤ 0.05, n = 15. sociobiology 68(4): e7204 (december, 2021) 7 nesting behavior and foraging activity temperature plays a significant role in the nesting and foraging behavior of weaver ants. high activity for nesting with about 116 of these ants was observed at 9:00 pm, where the temperature was about 21 °c. moderate activity with about 55 and 71 ants at 8:00 am and 6 pm with an average temperature of 21 °c and 25 °c respectively was noted. the least activity was observed with only eight ants at 1.00 pm, recording an average temperature at about 30 °c and above (fig 5). the worker ants scout and locate the best spot for the nest building. few worker ants start to bite the leaves, pull them closer, and hold them until another worker ant comes along with the larva in its mandible to glue the leaves (fig 7). the last instar larvae are used for gluing in which the worker ant squeezes the larvae gently, and the silk is produced (fig 7c). the nests of o. smaragdina in this locality are somewhat irregular round-oval/ellipse in shape (fig 6a, b). the nest comprises many chambers depending on different castes of the ants, food, and environmental factors. the worker ants add another layer of leaves around the nest to increase the nest size and population. as the nest ages, the leaves turn from green to pale yellow to brown (fig 6c). once the nest turns brown, the worker ants constructed new nests, and side by side, the broods (larvae and pupae) were shifted to the new nest. the queen moves first, followed by the broods and, lastly, the eggs. with the increase in the nest population, the worker ants also construct another nest called the satellite nest in another location of the host tree not far from the mother nest, which is a nest without a queen. this satellite nest was constructed to ease the mother nest from over-population and shift some of its broods. major workers functioned as soldiers, few foragers, and minor workers to attend the broods. fig 5. relationship between temperature and the number of weaver ants performing foraging and nest building activities. sum of squares df mean square f sig. no. of workers between groups 2810904.824 1 2810904.824 2.044 .176 within groups 17876468.909 13 1375112.993 total 20687373.733 14 no. of pupae between groups 2031189.388 1 2031189.388 2.436 .143 within groups 10841895.545 13 833991.965 total 12873084.933 14 no. of larvae between groups 2251086.402 1 2251086.402 6.207 .027* within groups 4714790.932 13 362676.226 total 6965877.333 14 table 6. variation in the number of the different caste with respect to the presence or absence of the queen. anova, *p ≤ 0.05, n = 15. jonata sa sangma, surya b prasad – population and nesting behavior of weaver ants8 apart from nest building, foraging is a critical factor of o. smaragdina. their foraging action becomes very active at around 20 °c, with about 116 ants involved in foraging. as the temperature increases to about 30 °c and above, the number of foraging ants declines as only 32 ants were observed until late afternoon (fig 5). it was observed that the ants feed on different types of insects like cricket, grasshoppers, beetles, butterflies, flies, fly larvae, beetle grubs, earthworms, and sometimes dead rodents or birds. foraging activity for these weaver ants is not restricted only to their host trees, but sometimes they climb down from the tree to find potential prey to feed their colony. fig 6. representative photographs of an irregular oval-ellipse-shaped nests of o. smaragdina on needlewood tree (schima wallichi). smallest nest (a), large nest (b), and a nest containing pale yellow to brown leaves (c). fig 7. representative photographs of worker ants and nest construction by o. smaragdina in needlewood tree. (a) individual worker ant, (b) worker ants pulling the leaves together, (c) worker ants squeezing the larva during nest construction, and (d) weaver ants tending the scale insects (hemipterans). sociobiology 68(4): e7204 (december, 2021) 9 discussion the elucidation of the population dynamics and nesting details of o. smaragdina in a particular region should have a direct beneficial impact as these ants have been used as bio-control agents in agricultural pest management (peng & christian, 2005; offenberg et al., 2013), food and traditional medicines (jena et al., 2020). weaver ants generally thrive in warm and humid climatic conditions with an average temperature of 20-30 °c (crozier, 2010; bharti & silla, 2011). they are found in almost all kinds of trees with moderate to large-sized leaves, which are easier to pull and provide good shelter for nesting. in the ri-bhoi district of meghalaya, the temperature range of 18-32 °c and an annual rainfall of 1267 mm may be a perfect condition for these weaver ants to flourish. the o. smaragdina nests were found on mango trees (mangifera indica), litchi tree (litchi chinensis), jackfruit trees (artocarpus heterophyllus), and needlewood trees (schima wallichi). they are more abundant in s. wallichi trees due to the leaves that can be easily drawn for nest expansion. their large number of leaves may provide good camouflage from predators such as birds and an excellent cover from adverse environmental factors like heavy rain, hailstones. the height of these trees should also be suitable to receive accessible sunlight. the suitability for the activity and population of o. smaragdina may depend on the geographic location and vegetation. the high abundance of o. smaragdina was documented on pongamia pinnatta, zizyphys mauritiana, eucalyptus platyphylla, and canarium australianum in northern australia. it was suggested that the vegetation depending on the seasonality of rainfall, dry tropical climate, and average temperature, play a significant role in the distribution, colony structure, and activity of the green ant o. smaragdina (lokkers, 1990). population dynamics of o. smaragdina on two trees, prunus dulcis and morinda citrifolia, in annamalai university, tamil nadu, india, showed that in prunus dulcis, the highest number of the green nest, dry nest and leaf pavilion were found in april, may and march, respectively. in morinda citrifolia, most green and dry nests were found in july and october (nalini & ambika, 2019). in a study on oecophylla longinoda in tanzania, more weaver ants were recorded during cashew vegetative and reproductive phases than dormancy. rainfall and temperature negatively affected the number of nests, while relative humidity was negatively related to shoots with weaver ants (nene, 2016). the average nest height from the ground was about 6 m and, from the central trunk, the nearest distance of the nest is about 0.7 m and the farthest up to 3.4 m (table 1). the approximate height of the tree where the nests were found ranged from 4-25 m. this height and distance of the nests should be suitable for better living of the weaver ants in the climatic condition of the ri-bhoi district. the study on the ecology and behavior of the other weaver ant camponotus (myrmobrachys) senex in brazil showed that the weaver larvae have a fundamental function in nest building. the nests were always arboreal (one nest/plant), with a round form, beige in color, and leaves and shoots adhered to the silk nest. the average size and weight of the nests was 34.24 cm and 163.87g respectively. the nests contained up to 50,000 individuals and several queens (santos & del-claro, 2009). like other social ants, o. smaragdina comprises different castes such as the queen, workers, pupae, larvae, and the eggs (table 4). these weaver ants are known for their unique ability to construct nests above the ground on the trees. it was noted that the first nest is built with only one or two leaves by the queen by folding the leaves together then glued them using their first batch of larvae, and later when the first batch emerges as adult workers, they take over all the roles from nest building to feeding and hunting (crozier, 2010). the worker ants are differentiated into major and minor workers based on their sizes (bharti & silla, 2011). we also observed that the leaves of the host trees are used for building their nests, and the leaves are pulled by the worker ants and glued together using their larval silk (fig 6, 7). while gluing, the worker ants hold the larva at a specific place and squeeze the larva to secrete silk (fig 7). in the case of another weaver ants, camponotus (myrmobrachys) sensex, santos and del-claro (2009) categorized nests into two groups, small and medium, while bharti & silla (2011) categorized these nests into three groups, small, medium, and large. in the present study, based on the nest size, the nests were categorized into three groups to examine the variation in the number of leaves utilized and the number of chambers built in different groups (table 3). as the population of the weaver ants and nest size increase with time, the leaves die to turn their color from green to pale yellow and finally to brown (fig 6c). these weaver ants form long chains to pull the leaves far towards the nest for its expansion (bharti & silla, 2011). the number of chambers present in the nest also plays a vital role in increasing the ants’ population. it was observed that the average number of chambers built in the nests is about 17 (table 1). it was noted that these weaver ants utilize a certain number of leaves for nests of different sizes (table 1). the weaver ants population and number of leaves utilized in nest building are positively correlated since the increase in population is followed by an increase in the number of leaves and chambers in a nest. the study shows that the average number of leaves utilized for nest construction is about 20 (table 1). the scree plot (fig 3) indicates three components whereby the first principal component explains about 71% variance in nest size. the second and the third principal components explain about 16% and 8%, respectively, leading to a cumulative variance of 95%. these three components are the population of o. smaragdina, the number of leaves utilized for nest building and the number of chambers. the study also showed that the approximate area of the nest is influenced by the number of leaves utilized for nest construction, the number of chambers, and the total population (table 2). jonata sa sangma, surya b prasad – population and nesting behavior of weaver ants10 pearson correlation analysis showed a positive correlation between the number of queens and the number of larvae. in contrast, the number of queens in the nest showed less significance with the number of worker ants and pupae. moreover, the number of queens influences the number of larvae, which affects the number of pupae and worker ants (table 5). however, a comparison between nests with and without the queen showed that only the number of larvae has significant differences between the groups (table 6), confirming the positive correlation between the number of queens with the number of larvae. the nest-building behavior of these weaver ants is greatly affected by the temperature. the present study shows that the number of ants and nestbuilding activity increases when temperatures are between 20-25 °c (fig 5). along with nest building, foraging activity is an important factor in the living o. smaragdina. their foraging activity becomes elevated at around 20 °c, with about 116 ants involved in foraging. as the temperature increases to about 30 °c and above, the number of foraging ants declines since only 32 ants were observed until late afternoon (fig 5). it was observed that the weaver ants feed on different types of insects, fly larvae, beetle grubs, earthworms, and sometimes dead birds. the study on o. smaragdina in malaysia revealed that food higher in protein content was highly preferred by o. smaragdina than food with lipid and carbohydrate contents. the foraging activity of the o. smaragdina was significantly influenced by both temperature and relative humidity. thus, the weaver ants respond to different food and indirectly, forming a strategic foraging activity to maximize the food supplies for their colony (pimid et al., 2019). a large number of sensilla supports the workers’ role in foraging activity compared to other castes (barsagade et al., 2020). when the nest turns brown, the worker ants start building a new nest to transfer the host colony and the queen. it was also observed that these weaver ants build multiple nests when the population increased, expanding their territory and reducing the stress of only one nest (holldobler, 1983). these nests are called satellite nests, in which weaver ants look after brood till they hatch. only worker ants live in some of these satellite nests, and when threatened, these worker ants come out in large numbers and defend their colony. the findings from the present study documenting the immense population and specific nesting behavior of the weaver ants o. smaragdina in this region could be very useful as an alternative source of food and medicine for human society. the findings from the present study documented that o. smaragdina nest structure is more explained by the population size and caste structure than by their location on the spatial hypervolume (height from the ground and distance from main trunk) on trees. regardless of nest size, the study allowed to verify that o. smaragdina can spread their nests over almost all the vertical and horizontal axis of a tree. this is an important information when thinking of using this species as biocontrol agent in tree orchards. there was a direct influence of temperature on the foraging activity of o. smaragdina. the ants forage on the coolest hours of the day. this is also an important information for the conservation of this species in agricultural/orchard fields because technical recommendations for spraying insecticides indicate sprayings in the coolest hours. this hazardous recommendation should be carefully employed in cultivated areas with the presence of o. smaragdina nests. acknowledgment we are grateful to the local people of umran-dairy village, especially mrs. k. mukhim and her family, for allowing us to conduct experimental observations on their property. thanks to university grants commission, new delhi, for nonnet fellowship to jonata s. a. sangma. references barsagade, d.d., masram, p.d. & nagarkar, d.a. 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(1964). a fossil ant colony: new evidence of social antiquity. psyche, 71: 93-103. http://dx.doi.org/10.22214/ijraset.2020.5148 https://researchonline.jcu.edu.au/24114/ https://researchonline.jcu.edu.au/24114/ http://dx.doi.org/10.1080/09670870500131749 https://doi.org/10.1080/00222930902903236 https://doi.org/10.1080/00222930902903236 https://doi.org/10.25849/myrmecol.news_023:119 https://doi.org/10.1093/aesa/39.1.7 https://doi.org/10.1093/aesa/39.1.7 http://dx.doi.org/10.3157/061.143.0215 http://dx.doi.org/10.3157/061.143.0215 _hlk81135531 doi: 10.13102/sociobiology.v63i1.868sociobiology 63(1): 720-723 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 initial development and production of co 2 in colonies of the leaf-cutting ant atta sexdens during the claustral foundation annually, mature colonies of atta produce male and female winged ants that leave the colony to form new colonies. nuptial flights always occur after rains, which provide favorable conditions for the initial survival of initial colonies, facilitating the initial excavation of a vertical tunnel by the queen (autuori 1942). after the nuptial flight, a female of the atta sexdens (linneus, 1758) digs a vertical tunnel of about 15 cm and a chamber within which she is enclosed, caring for fungus culture and for brood (autuori, 1942; ribeiro, 1995, camargo et al., 2011; fröhle & roces, 2012). the process of constructing a new nest is the most crucial period in the life of an ant colony, whereas the mechanisms that optimize survival during this phase are of great value (wilson, 1971). the claustral foundation of atta species always involves great effort by the queen, who is solely responsible for cultivation of fungus and caring for herself and her offspring. in this period consumption of large portion of the queen’s reserves takes place (della lucia et al., 1995). mintzer (1990) abstract queens in the genus atta are solely responsible for fungus cultivation and for care of herself and her offspring. only few studies have investigated their nests in the claustral foundation and it is unknown the production rate of expelled carbon dioxide and/or of oxygen consumption in the initial colonies of leaf-cutting ants. thus, we have studied the development of 50 initial colonies of atta sexdens, and production of expelled carbon dioxide under laboratory conditions. the number of eggs was counted one week after nest foundation on the seventh day, the larvae counted on day 28, and the pupae between days 42 and 49. the workers emerged on the 63rd and 70th day. the co 2 concentration increased steeply in the 42nd days (20.60 ± 8.36%) and 49th days (15.37 ± 13.11 %), at 42nd days, and subsequently returned to lower values, for example, 3.35 ± 2.84% at week seven. it is the first record of co 2 emission data in initial nests, in their claustral foundation under laboratory conditions. sociobiology an international journal on social insects rs camargo1, ej silva1, lc forti1, cao de matos2 article history edited by evandro nascimento silva, uefs, brazil received 22 july 2015 initial acceptance 27 january 2016 final acceptance 21 february 2015 publication date 29 april 2016 keywords egg, larvae, nests, pupae, social insects. corresponding author roberto da silva camargo laboratório de insetos sociais-praga departamento de produção vegetal faculdade de ciências agronômicas/unesp caixa postal 237, cep: 18603-970 botucatu-sp, brazil e-mail: camargobotucatu@yahoo.com.br stated that the foundation of these nests demands the highest investment already observed among the queens of several ant species. thus, we can explain their large body size, due to the accumulation of energy reserves prior to the nuptial flight, so they can spend them during the formation of their colonies (fujihara et al., 2012; camargo & forti, 2013). considering that there are few studies investigating nests of atta in the claustral foundation, we have studied the development of 50 initial colonies of atta sexdens, as well as the production of carbon dioxide expelled under laboratory conditions. the study was conducted in the city of botucatu, são paulo state, brazil with a. sexdens rubropilosa nests being selected in an area of eucalyptus plantations belonging to fca/unesp, botucatu, sp (22o 50.833’ s and 48o 26.476’ w). the area presents the oxisol soil type (dark red latosol). the 50 queens were randomly selected at the moment of nest foundation on the 5th of october, 2013.the newly collected queens were stored in plastic containers, 11 cm in diameter short note 1 universidade estadual paulista (unesp), botucatu-sp, brazil 2 unesp, campus experimental de itapeva, são paulo, brazil sociobiology 63(1): 720-723 (march, 2016) 721 and 8 cm in height, containing 1 cm of plaster at the bottom to maintain humidity. the queens were transported to the laboratory of social-pest insects fca/unesp – botucatu. the initial development was measured through the production of brood (number of eggs, larvae, pupae and adults) until the first workers emerged. the brood of 50 colonies was counted weekly, by careful examination under a stereoscopic microscope. the mortality of colonies was measured for a period of 70 days after nest foundation. production of co2 by the colonies the colonies were enclosed in hermetic chambers for one week, simulating a claustral foundation found in a natural environment. co2 production was measured by respirometry. bacharach co2 sensor was employed, with a fixed probe (bacharach model 2800; more details at http://www. bacharach-inc.com), maintained at 25°c in the laboratory. co2 concentration in the surrounding environment was monitored during 10 weeks, resulting in a mean of 285.7 ± 63.9 ppm (n=70). consecutive readings (n=7) of the co2 concentration from a respirometric receptacle were carried out to estimate a mean value. the results were expressed in ppm and subsequently converted to ml or mg, through the known volume of the acrylic b respirometric receptacle. each colony had its own respirometric receptacle (mean volume = 455.9 ml). this environment was retro-fed by a peristaltic pump, thereby ensuring no loss of modified chamber atmosphere of the colonies. the number of eggs, larvae, pupae and adults was submitted to linear mixed-effects model. the simplified model was used: % co2 = eggs + larvae + pupae + adults + days / colonies + error. the variable number of eggs, larvae, pupae and adults during the period were categorized into three stages based on population growth (lack of population, population growth and population decrease), and it was submitted to linear mixed-effects model. the co2 percentage was the response variable, random effects were days given colonies, and fixed effects were eggs, larvae, pupae and adults. statistical analyses and graphs were processed by r 2.9.0 for windows. the queens of atta sexdens began to oviposit on the second and third day after the nuptial flight, but the eggs were here counted on the seventh day. the larvae were counted on the evaluation at 28th day, and the pupae between 42nd and 49th days. the workers emerged between the 63rd and 70th day (fig 1). the linear mixed-effects model showed a remarkable correlation among eggs, larvae, pupae, adults and concentration of co2 ( fig 1).. first, the eggs phase was verified two stages, increasing and decreasing stage. the co2 production was 4.56 times lower in decreasing than increasing stage (t=-3.12, d.f =378; p=0.0019). in the larvae phase was verified three stages: no larvae, increasing and decreasing stage of larvae number. the co2 production was 6.08 times bigger in increasing stage than without larvae (t=4.59, d.f.=378; p<0.001). although 3.80 times bigger in decreasing stage than no larvae (t=1.88, d.f.=378; p=0.0607). in the pupae phase was verified 3 stage: no pupae, increasing and decreasing stage of pupae number. the co2 production was 13.10 times lower in increasing stage than without pupae (t=-9.72, d.f.=378; p<0.001) and 14.51times lower in decreasing stage than without pupae (t=-8.44, d.f.=378; p<0.001).finally, adult phase was verified 2 stage: without and increasing of adults number. the co2 production was 11.36 times lower in increasing stage than without adults (t=-3.17, d.f.=378; p=0.0016). the mortality of colonies was high, with 24 (48 %) dead colonies at 70 days. the co2 concentration within the nest increased with time, with a peak between the 42nd and 49th days (fig 1), but decreased at following weeks. the linear mixed-effects model showed a remarkable correlation between brood and concentration of co2. it may be concluded the larvae were responsible for increasing of co2 concentration inside the colony, because they have a greater food consumption, and consequently, greater body mass (chapman, 1998). all this growth causes greater aerobic respiration, and of course, the production of co2 increases. in our study, the co2 volume emitted weekly varied from 20.60 ± 8.36% at 42nd days to15,37 ± 13.11 % at 49 th days (fig 1). fig 1 .number of eggs (black line), larvae (red line), pupae (green line), adults (dark blue line) and concentration of co2(%) (blue line), during a period of 10 weeks, within of a claustral foundation of atta sexdens. although this value of co2 concentration is high, it has been shown that in adult nests the values are much higher and that leaf-cutting ant workers are able to tolerate elevated co2 levels, especially the genus atta, which can withstand severe hypoxia (hebling et al., 1992), and also are capable of behaving normally at elevated co2concentrations, such as those of about 1.5 to 4.5% found in nests of a. capiguara and a. laevigata (bollazzi et al., 2012). another interesting example is found in ants that inhabit mangroves, whose nest interiors present carbon dioxide levels that become elevated rs camargo et al. – co2 production in colonies of atta sexdens722 during nest closure, reaching 11% (nielsen et al., 2003). under these hypercapnic and hypoxic conditions, the ants are able to sustain aerobic respiration even at co2 concentrations of up to 15% (nielsen et al., 2006; nielsen & christian, 2007). comparing the development of initial colonies of atta with data from the literature (autuori, 1942; pereira-da-silva, 1979), similarity was verified in the incubation, larval and pupal periods. for atta sexdens, the incubation period lasted 25 days, the larval period, 22 days and pupal period, 10 days (autuori 1942). camargo et al. (2011) obtained similar results from the same leaf-cutting ant species, even when measuring the excavation effort. in atta capiguara gonçalves, 1944, in the sampled periods, the eggs were most prevalent in the interval of 1-18 days, larvae at 21-38 days, pupae at 39-55 and adults at 58-67 days (pereira-da-silva, 1979). the incubation period is related to suitable temperature, and consequently, larva is fully formed and hatched by specific environmental stimuli (chapman, 1998). our study was carried out at 25°c in the laboratory, similar to temperature found on initial chamber in natural condition (25-28°c for atta sexdens (stahel & geijskes, 1940; eidmann, 1935), 27.5 °c for atta vollenweideri (kleineidam & roces, 2000), and around 27°c for a. heyeri (bollazzi & roces, 2002)). when comparing the quantity of brood produced with the findings of autuori (1942) and pereira-da-silva (1979), a pattern is verified in the production of eggs, larvae, pupae and adults, in other words, generally concise numbers with high variability (figure 1). this is due to the fact that the production of eggs is controlled by their endocrine system. according to fowler et al. (1986), egg production by the queen during the 3 or 4 months of the colony lifespan is correlated with the activity cycle of the corpora allata. the corpora allata are responsible for the synthesis of juvenile hormone, which acts during oviposition of founder queens, as verified in females that suffer allatectomy (barker, 1978). the juvenile hormone acts on the fatty body, initiating the synthesis of vitellogenins. thus, the production of brood depends on body reserves (lipids from the fatty body and protein from the wing muscles), since the queen does not feed during the claustral foundation. the queens with a claustral foundation present a greater quantity of lipids in their bodily reserves (keller & passera, 1989). in smaller queens of lower attini, it was verified that trachymyrmex septentrionalis mccook (1881), presents 25% fat in its dry weight while cyphomyrmex rimosus spinola (1853), has 11% (seal & tschinkel, 2007). in higher attini, a. sexdens rubropilosa queens have a great amount of lipids in their body reserves, about 30% (fujihara et al., 2012). during the foundational period the queen loses about 40 % of her weight, presenting the lowest body mass 4 months after the nuptial flight (atta sexdens and atta laevigata smith, 1858) (della lucia et al., 1995). probably, the entirety of this loss of body mass is due to the utilization of the resources in the queen’s activities, including oviposition. the mortality of colonies was high in 70th averaging 48 % of colonies. however, this value is lower than found by camargo et al. (2011), averaging 60% at 63rd days after nuptial flight due to digging effort during nest foundation. our study is the first to present co2 emission data in initial nests, in their claustral foundation under laboratory conditions. acknowledgements we are grateful to fundação de amparo à pesquisa do estado de são paulo (fapesp) for the financial support and stipends to the authors (grants 2007/04010-0 and 2007/070910). r.s. camargo thanks coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the postdoctoral fellowship. l cf thanks cnpq for the research assistance (301917/2009-4). references autuori, m. (1942). contribuição para o conhecimento da saúva (atta spp. – hymenoptera – formicidae). ii. o sauveiro inicial (atta sexdens rubropilosa, forel, 1908). arquivos do instituto biológico, 13: 67-86. barker, j.f. 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(2011). digging effort by leaf-cutting ant queens (atta sexdens rubropilosa) and its effects on survival and colony growth during the cloistered phase. insectes sociaux, 58: 17-22. chapman, r. f. the insects: structure and function (4th ed). cambridge university press, cambridge, uk ; new york, 1998. della lucia, t.m.c., moreira, d.d.o., oliveira, m.a. & araújo, m.s. (1995). perdas de peso da rainha de atta durante a fundação e o estabelecimento das colônias. revista brasileira de biologia, 55: 533-536. eidmann h. (1935). zur kenntnis der blattschneiderameise atta sexdens l., insbesondere ihrer okologie. zeitschrift fur angewandte entomologie, 22: 185-436. fowler, h.g., pereira-da-silva, v., forti, l.c. & saes, n.b. (1986). population dynamics of leaf-cutting ants: a brief sociobiology 63(1): 720-723 (march, 2016) 723 review, p. 123–145. in: fire ants and leaf-cutting ants: biology and management. lofgren, c.s. & vander meer, r.k. (eds). westview press, 435 p. fröhle, k. & roces, f. (2012). the determination of nest depth in founding queens of leaf-cutting ants (atta vollenweideri): idiothetic and temporal control. the journal of experimental biology, 215: 1642-1650. fujihara, r.t., camargo, r.s. & forti, l.c. (2012). lipid and energy contents in the bodies of queens of atta sexdens rubropilosa forel (hymenoptera, formicidae): pre-and postnuptial flight. revista brasileira de entomologia, 56: 73-75. hebling m.j.a., penteado c.h.s. & mendes e.g. (1992). respiratory regulation in workers of the leaf-cutting ant atta sexdens rubropilosa forel, 1908. comparative biochemistry part a: physiology, 101a: 319-322. keller, l. & passera, l. (1989). size and fat content of gynes in relation to mode of colony founding in ants (hymenoptera: formicidae). oecologia, 80: 236-240. kleineidam c & roces f. (2000). carbon dioxide concentrations and nest ventilation in nests of leaf cutting ants atta vollenweideri. insectes sociaux, 47: 241-248. mintzer, a. (1990). foundress female weight and cooperative foudation in atta leaf-cutting ants, p.180–183. in: applied myrmecology: a world perspective (westview studies in insect biology). r.k. van der meer, k. jaffe, a. cedeno, eds. bouder, westview press,741 p nielsen, m.g. & christian, k.a. (2007). the mangrove ant, camponotus anderseni, switches to anaerobic respiration in response to elevated co2 levels. journal insect physiology, 53: 505-508. nielsen, m.g., christian, k. & birkmose, d. (2003). carbon dioxide concentrations in the nests of the mud-dwelling mangrove ant polyrhachis sokolova forel (hymenoptera : formicidae). australian journal of entomology, 42: 357-362. nielsen, m.g., christian, k., henriksen, p.g. & birkmose, d. (2006). respiration by mangrove ants camponotus anderseni during nest submersion associated with tidal inundation in northern australia. physiological entomology, 31: 120-126. pereira-da-silva, v. (1979). dinâmica populacional, biomassa e estrutura dos ninhos iniciais de atta capiguara gonçalves, 1944 (hymenoptera: formicidae) na região de botucatu, sp; tese de doutorado, universidade estadual paulista 77 p. ribeiro, f.l. (1995). a escavação do solo pela fêmea da saúva (atta sexdens rubropilosa). revista de psicologia, 6: 75-93. roces, f. & lighton, j.r.b. (1995). larger bites of leafcutting ants. nature, 373: 373. schilman, p.e. & roces, f. (2005). energetics of locomotion and load carriage in the nectar feeding ants, camponotus rufipes. physiological entomology, 30: 332-337. schilman, p.e. & roces, f. (2008). haemolymph sugar levels in a nectar-feeding ant: dependence on metabolic expenditure and carbohydrate deprivation. journal of comparative physiology b, 178: 157-165. seal, j.n. & tschinkel, w.r. (2007). energetics of newlymated queens and colony founding in the fungus-gardening ants cyphomyrmex rimosus and trachymyrmex septentrionalis (hymenoptera: formicidae). physiological entomology 32: 8-15. stahel g, & geijskes dc. (1940). observations about temperature and moisture in atta nests. revista de entomologia, 11: 766-775. wilson, e.o. 1971. the insect societies. cambridge, harvard university, 548 p. 1323 daily rhythm of pollen production by apis mellifera l(hymenoptera: apidae) in sorriso, mato grosso state, brazil by evaldo martins pires1*; sandro tarcísio celmer1; joão alfredo marinho ferreira2; ana claudia ruschel mochko1; adriana garcia do amaral1; artur kanadani campos1 & marcus alvarenga soares3 abstract insect pollinators are important in reproduction of several plants species increasing the agricultural productivity and the quality of the food. apis mellifera (hymenoptera: apidae) is an important plant pollinator besides its fundamental role in the pollen based products often used in food and pharmaceutical industry. in the field of beekeeping, the palynolog y is important in botanical biodiversity studies, because pollen provides information about environmental quality. the understanding of the time frequency of pollen collection by a. mellifera can give additional information about the foraging behavior of this bee species in studies about daily and seasonal availability of pollen for plants, mainly during the periods of lower activity of the bee colony. the aim of this research was to study the time frequency of pollen production by a. mellifera in sorriso, mato grosso state, brazil and to observe the effect of the temperature, relative humidity and sunlight on this activity of this bee. the period with higher pollen production was between 10:00 am until 01:00 pm and this period can be characterized by higher sunlight and temperature with lower relative humidity periods. keywords: pollination, foraging activity, climatic effects. introduction the bee pollen is composed by the accumulation of several pollen grains 1federal university of mato grosso, campus of sinop, 78550-000 sinop, mato grosso state, brazil. 2departament of entomolog y, federal university of viçosa, 36570-000 viçosa, minas gerais state, brazil. 3federal university of vale do jequitinhonha and mucuri; 39100-000; diamantina – minas gerais state – brazil. * author for correspondence. 1324 sociobiolog y vol. 59, no. 4, 2012 daily collected by bees. they promote agglutination through the addition of salivary secretions and small amount of nectar (brasil 2001). the productivity of pollen varies according to some factor, such as geographical localization and the type of collector (barreto et al. 2005). the intermediate internal collector showed good results in the states rio grande do sul, (900 2200g/colony/month), santa catarina (930 4000/colony/ month), são paulo (2.000 3.500g/colony/month) and minas gerais (2.000 3.000g/colony/month) (barreto et al. 2005). it is estimated that the pollen production in brazil is around 200 ton/ yea, mainly in the states of bahia, paraná and santa catarina. however mato grosso, mato grosso do sul and ceará have high potential for pollen production (barreto et al. 2005) there are different compounds in the bee collected pollen, such as some fig. 1 (a) and (b). colonies of apis mellifera (hymenoptera: apidae) placed in an amazonian rain forest fragment (e 11°49’34” e sw e 55º48’46”), countryside of sorriso, mato grosso – brazil. 1325 author — dummy antioxidants that are important in the inhibition of free radicals (kroyer & hegedus 2001, nagai et al. 2002, campos et al. 2003). additionally they can be used as a food supplement because of they contain amino acids, carbohydrates, lipids, mineral substances, proteins and vitamins that are indicated to prevent pathologies (escribano et al. 1999, kroyer & hegedus 2001). studies about palynolog y have contributed on the environmental quality by providing the identification of harmful compounds that makes this technique important in bio monitor programs (conti & botre 2001). the pollination is one of the most important mechanisms for biodiversity maintenance and promotion because most of the plants depend on pollinators for the sexual reproduction. additionally, the flower resources are the main food sources for different group of animals (proctor et al. 1996, alvesdos-santos 2003, endress 1998). so, the plants developed some mechanisms to attract pollinators, such as aroma, color and shape of the structures to be pollinated (dafni 1992) and the bees are fundamental for this process (gallai et al. 2009). the bees also are important pollinators in agricultural crops, most of the times increasing the productivity (silva 2007) in brazil, the african bee has a great acceptability by the beekeepers because of its productivity, tolerance to diseases and adaptability in different environments (kerr 1967, gonçalves 1994). apis mellifera (hymenoptera: apidae) is very important in honey, wax, real jelly and propolis production, can contribute for the pollination (witter & blochetein 2003, vandermeer & perfecto 2006) and bigger fruit production and in higher quantity (kristjansson & rasmussen 1991, carvalho & marchini 1999, boti et al. 2005, vieira et al. 2008, richards 2001, lemasson 1987). the fly characteristics of the bees can allows the foraging up to 30 meters high but they are commonly found up to 5 meters high and 2000 meters far from the colony (moreti & marchini 1998, wiese 1985). the foraging activity can vary during the day and this variation is caused by changes in temperature, relative humidity, season of the year and day lightness (antonini et al. 2005, hilário et al. 2007, hilário et al. 2000, kajobe & echazarreta 2005, silva et al. 2011). it can also vary among insects but there is a pattern where bigger bee species (apidae and andrenidae) and ants forage during all day long. species of vespidae and lepidoptera prefer foraging 1326 sociobiolog y vol. 59, no. 4, 2012 under higher temperature and lightness, especially between 8 am – 3 pm o’ clock, with maximum activity around 12 pm o’clock (antonini et al. 2005, souza & teresinha 2011). the aim of this study was to evaluate the time frequency of the bee pollen by a. mellifera in sorriso, mato grosso state, considering that this information can improve the maintenance and manipulation techniques of bee colonies in the north of mato grosso state, brazil. material and methods this research was carried out in the “fazenda luana” apiary (e 11°49’34” e sw e 55º48’46”), in the countryside of sorriso, mato grosso state, brazil. the region is covered by small areas of ombrophylous tropical rain forest. the pollen grains were collected in samples between 13-15th of december 2011. this period is characterized by the higher productivity of bee products and production of larvae, increasing the foraging activities of the bees and consequently increasing the pollen production (dreller & page 1999). the dates were defined through weather forecast website (www.climatempo. com.br), observing and choosing the sunny days and with absence of any rain that are favourable conditions for foraging activities of hymenoptera insects (santos et. al. 2009). in this experiment 29 a. mellifera colonies weeplaced on a stand (70 cm high) and 2 meters far from each other (figure 1). for collecting the pollen .fig. 2 (a). internal or top pollen collectors. (b) collector tray of pollen. (c) detail of the amount of pollen grains collected per hour. (d). collection procedure of the pollen grains. (d) pollen grains collected and counted. 1327 author — dummy grains we used the one internal collector (top type) (figures 2a, b, c, d, e), (15 cm high, 48 cm length and 40 width) in each colony. this type of collector has a lateral tray where the pollen grains can be removed and is composed by four grids, where they can remove the pollen from the bees and prevent other insects to access the collected pollen. collector pollen were assembled in field 24 hours before the evaluation for the bees to adapt to the collectors. at 6 am o´clock the collectors were cleaned and removed all the pollen grains. one hour later was started sampling, collecting all the pollen grains every hour. at 6pm o´clock we collected the last sample and then finished the experiment. weather data (temperature, relative humidity and sunlight) were collected from a weather station of the institute of agricultural and environmental sciences of the federal university of mato grosso. the data were analysed using the system for statistical analysis (saeg 9.1). it was tested the effect of the time during the day, temperature, relative humidity and sunlight on the amount of pollen grains collected using analysis of variance (anova). all the significant effects were then tested by analysis of regression (p = 0.05). those parameters without significance were presented by descriptive analysis. results the time (f= 18,81; p= 0,0006), relative humidity (f= 7,08; p= 0,0120) and sunlight factors (f= 24,21; p= 0,0006) had significant effects on the amount of pollen grains collected by apis mellifera during the period of the experiment. in other hand, only temperature did not significantly affected the amount of pollen grains collected (f= 2,96; p= 0,1900). between 7 am 10 am o´clock the amount of pollen grains collected increased exponentially and around 10 am o´clock we collected the higher amount after 11 am o´clock the amount reduced until 2 pm o´clock. after this period the amount of grain pollen was regular until 6 pm o´clock but around zero (figure 3 a). the sunlight range between 300 600 w/m2, the relative humidity between 80 95% were the ranges with higher amount of pollen grains collected. sunlight lower than 100 w/m2 and relative humidity lower than 80% and 1328 sociobiolog y vol. 59, no. 4, 2012 fig.3. pollen grains collected per hour (a), in function of the sunlight (b) and the relative humidity (c) in an amazonian rain forest fragment in sorriso, mato grosso – brazil. 1329 author — dummy higher than 95% showed lower productivity of pollen grains by a. mellifera (figure 3 b and c). because of the fact that temperature did not affected the amount of pollen grains collected, we presented this data through descriptive analysis that allows this factor to contribute in the knowledge of the foraging dynamics and pollen collection by a. mellifera in sorriso. temperatures up to 26 oc and higher than 30 oc showed lower amount of pollen grains collected by a. mellifera. in other hand, temperatures between 27 – 29 oc showed higher activity of pollen collection (fig. 4). the sunlight, temperature and relative humidity dynamics showed that between 10am-01pm o´clock there were higher sunlight and temperature and lower relative humidity (figure 5). discussion one fact that could be associated to the dynamics of collecting the pollen grains is the foraging activity. some hymenoptera (e.g. andrenidae, apidae, formicidae and vespidae) forage almost during all day long, mainly between 8 am 3 pm o´clock. this period seems to be favourable for this taxon and fig. 4. pollen grains collected in function of temperature in an amazonian rain forest fragment in sorriso, mato – brazil. 1330 sociobiolog y vol. 59, no. 4, 2012 fig. 5. weather dynamics (sunlight, temperature and relative humidity) in an amazonian rain forest fragment in sorriso, mato grosso – brazil. 1331 author — dummy periods around 12 pm o´clock is the one with higher activity (antonini et al. 2005, souza & teresinha 2011). the temperature did not affect the amount of pollen grains collected. this may happen by the fact the temperature in sorriso region is almost constantly high, without significant variations. it is common temperatures higher than 24 oc after 7 am o´clock. the higher amount of pollen grains collected occurred under 29 oc of temperature and this result agrees with others researches carried out with a. mellifera (roubik 1989). so, temperatures around 29 oc maybe ideal for the pollen collection by a. mellifera also in sorriso (figure 2). the sunlight range observed showed that the higher amount of pollen grains collected is around the same range found in the literature on a. mellifera where the range between 200 799 w/m2 showed higher activity on the pollen collection (roubik 1989). the relative humidity range that showed higher amount of pollen grains collected was different from the ones in the current literature about the intensity of pollen collection by a. mellifera around 40 79% of relative humidity (roubik 1989). this means that this fact can related to the geographical position of the experimental site inside a amazonian rain forest fragment. the fragment is characterized by high relative humidity level during all day long mainly because of the plant respiration. the period between 10 am – 1 pm o´clock showed great conditions for the foraging activity and consequently the pollen grain collection. this range period can be considered as the period with higher activity of a. mellifera. besides the weather conditions on the pollen collection by a. mellifera, another important factor is the availability of the pollen by the plant that occurs with higher intensity during the morning and lower intensity during the afternoon (marchini & moreti 2003, heard 1994, vithanage & ironside 1986). according to our results about the daily period of higher activity or pollen grain production, we could conclude that the period between 10 am – 1 pm o´clock is the best period for the pollen collection by a. mellifera in sorriso, mato grosso. this period is also the one for higher activity of the bee colony. 1332 sociobiolog y vol. 59, no. 4, 2012 according to our results, the best period of managing the colonies is during the early mornings (7 am 9 am o´clock), with lower bee activities. references alves-dos-santos, i. 2005. comunidade, conservação e manejo: o caso dos polinizadores. revista tecnologia e ambiente 8(2): 35-57. antonini, y., h.g. souza, c.m. jacobi, f.b. mury. 2005. diversidade e comportamento dos insetos visitantes fllorais de stachytarpheta glabra cham. 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annals of botany 88(2): 165-172. roubik, d.w. 1989. ecolog y and natural history of tropical bees. new york, cambridge university. 514 p. universidade fedeeral de viçosa – ufv. sistema para análises estatísticas saeg, versão 9.1. viçosa, mg, 2007. santos, g.p., j.c. zanuncio, e.m. pires, f. prezotto, j.m.m pereira & j.e. serrão. 2009. foraging of parachartergus faternus (hymenoptera: vespidae: epiponini) on cloudy and sunny days. sociobiolog y 53(2b): 431-441. 1334 sociobiolog y vol. 59, no. 4, 2012 apidae) forrageia sob alta umidade relativa do ar? iheringia. série zoologia 101(1-2): 131137. souza, m. & d. teresinha. 2011. biodiversidade de polinizadores em passiflora cincinnata mast. (passifloraceae), em ribeirão preto, sp, brasil. zootecnia tropical 29(1): 17-27. vandermeer, j. & i. perfecto. 2006. a keystone mutualism drives pattern in a power function. science 311(5763): 1000-1002. vieira, g.c., l.c. marchini, b.a. souza & a.c. moreti. 2008. fontes florais usadas por abelhas (hymenoptera, apoidea) em área de cerrado no município de cassilândia, mato grosso do sul, brasil. ciência e agrotecnologia 32(5): 1454-1460. vithanage, v. & d.a. ironside. 1986. the insect pollinators of macadamia and their relative importance. the journal of the australian institute of agricultural science 52(3): 155-160. wiese, h. 1985. nova apicultura. porto alegre: agropecuária ltda. 493 p. witter, s. & b. blochetein. 2003. efeito da polinização por abelhas e outros insetos na produção de sementes de cebola. pesquisa agropecuária brasileira 38(12): 1399-1407. doi: 10.13102/sociobiology.v63i1.937sociobiology 63(1): 728-730 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 polybia (myrapetra) scutellaris (hymenoptera: vespidae) foraging on flies at carcasses of rattus norvegicus (rodentia: muridae) social wasps stand out due to their role in the trophic balance of ecosystems. this is the result of their generalist and opportunistic foraging features (hunt, 2007; prezoto et al., 2008; prezoto & souza, 2015), which comprises carbohydrates such as nectar, pollen, fruit pulp (clemente et al., 2012; barbosa et al., 2014) and also proteins. social wasps can obtain these substances through predation of other insects, such as lepidoptera, diptera, hymenoptera and from decomposing remains of vertebrates (o’donnell, 1995; lima & prezoto, 2003). thereby, the group shows potential as possible pest control agents (elisei et al., 2010). some species, such as polybia scutellaris (telleria, 1996), can also use pollen and nectar stored in their nests to produce honey. there are also researches focused on the capture of prey by some species of social wasps (prezoto et al., 2005; prezoto et al., 2006; bichara et al., 2009; brocanelli, 2015). these works show that predation, in certain circumstances, abstract social wasps stand out due to their role in the trophic balance of the ecosystems. these insects can get nutrients by preying on other insects, such as lepidoptera, diptera, hymenoptera and the decomposing remains of vertebrates. some species, such as polybia scutellaris, can also use pollen and nectar stored in their nests to produce honey. some studies lay emphasis on the prey captured by social wasps, showing that predation, in some conditions, is directed to lepidoptera larvae, such as for polybia occidentalis, polybia paulista and polybia ignobilis. other species like p. scutellaris focus on flies of the tabanidae, syrphidae, muscidae and anthomyiidae families. there are few studies with social wasps that feed on animal carcasses; this way, our study aims to report the predation on individuals of the sarcophagidae family, which use rattus norvegicus carcasses as a food source, by the social wasp polybia scutellaris. sociobiology an international journal on social insects as herdina1, gss bitencourt1, ra di mare1, bc barbosa2 article history edited by gilberto m. m. santos, uefs, brazil received 16 october 2015 initial acceptance 05 november 2015 final acceptance 11 february 2016 publication date 29 april 2016 keywords brachycera, epiponini, itaara, predation. corresponding author anita da silva herdina laboratório de biologia evolutiva, departamento de ecologia e evolução universidade federal de santa maria cep: 97105-900, santa maria rio grande do sul, brazil e-mail: anitabioufsm@gmail.com can be directed to lepidoptera larvae such as for polybia occidentalis (olivier, 1791) (gobbi et al., 1984), polybia paulista (ihering, 1896) (gobbi & machado, 1985) and polybia ignobilis (haliday, 1836) (gobbi & machado, 1986). furthermore, other species, such as p. scutellaris, seem to prefer capturing insects of the diptera order, particularly from the families tabanidae, syrphidae, muscidae and anthomyiidae (matsuura, 1984). there is a lack of knowledge on social wasps’ foraging in animal carcasses (o’donnell, 1995; gomes et al., 2007). this study aims to contribute to the knowledge of this behavior by reporting the predation of flies in carcasses of rattus norvegicus (berkenhout, 1769) by social wasp polybia scutellaris (white, 1984). the observations were made in august 2014 in the itaara town, rio grande do sul (29°36’56.1”s; 53°48’27.6” w). for that, were used shannon traps (fig 1a) with r. norvegicus’carcasses. these traps were placed in a forest area (fig 1b). short note 1 universidade federal de santa maria, rio grande do sul, brazil 2 universidade federal de juiz de fora, minas gerais, brazil sociobiology 63(1): 728-730 (march, 2016) 729 the foraging of p. scutellaris (fig 1c) was exclusively focused on sarcophagidae flies during the winter (august). by observing the trap’s contents, we conclude that the wasps first overfly the carcasses and then they capture the flies and remove them out the trap; lastly, they lacerate their prey in order to carry them to the colony. p. scutellaris showed four stages during the foraging of diptera (fig 2): i. prey capture using mandibles (fig 2a); ii. prey decapitation (fig 2b); iii. prey’s wings removal (fig 2c) and iv. thorax’s removal and transportation to the colony (fig 2d). fig 2. polybia scutellaris foraging flies of sarcophagidae’s family in carcasses of rattus norvegicus. a – fly captured by p. scutellaris using mandibles; b – p. scutellaris. decapitating the fly captured; c – p. scutellaris removing the wings; d – abdomen removed by wasp, holding the thorax to be transported to the nest. fig 1. a – shannon trap containing pet bottle of 500 ml, nylon pyramid, iron cage and wooden tray; b – trap in the forest area; c – lateral and dorsal view of polybia scutellaris. as herdina et al. – polybia scutellaris foraging on flies at carcasses730 the foraging pattern observed on this study is similar to the behavior of other social wasp species previously studied: mischocyttarus drewseni (saussure, 1857) (jeanne, 1972), polybia sericea (olivier, 1792) (richter & jeanne, 1991) and polybia ignobilis (gomes et al., 2013). we also observed the wasps’ opportunistic behavior, since they forage the prey caught in traps near animal carcasses. as a consequence, this behavior grants resource saving when compared to active searching preys in the natural environment. despite of the recent increase of forensic entomology studies in brazil (moretti et al., 2008; moretti et al., 2011; barbosa et al., 2015; maciel et al., 2015), there is no effective understanding of ecological interaction related to social wasps within the scavenger community yet. therefore, wasps foraging on forensic-important insects such as the calliphoridae and sarcophagidae families could influence on the abundance and diversity of these families’ species and, as a result, lead to an underestimation of the postmortem interval. references barbosa, b.c.; paschoalini, m.f; prezoto, f. (2014). temporal activity patterns and foraging behavior by social wasps (hymenoptera: polistinae) on fruits of mangifera indica l. (anacardiaceae). sociobiology, 61: 239-242. doi: 10.13102/ sociobiology.v61i2.239-242. barbosa, r.r., carriço, c., souto, r.n., andena, s.r., ururahy-rodrigues, a., & queiroz, m. m. (2015). record of postmortem injuries caused by the neotropical social wasp agelaia fulvofasciata (degeer) (hymenoptera, vespidae) on pig carcasses in the eastern amazon region: implications in forensic taphonomy. revista brasileira de entomologia, 59: 257-259. doi: 10.1016/j.rbe.2015.07.004 bichara-filho, c.c., santos, g.m.m., resende, j.j., dantas, c.j., gobbi, n. & machado, v.l.l. (2009). foraging behavior of the swarm-founding wasp, polybia (trichothorax) sericea (hymenoptera: vespidae): prey capture and load capacity. sociobiology, 53: 61-69 clemente, m.a., lange, d., del-claro k., prezoto f., campos, n.r. & barbosa, b.c. (2012). flower-visiting social wasps and plants interaction: network pattern and environmental complexity. psyche: a journal of entomology: 1-10. doi: 10.1155/2012/478431 elisei, t., nunes, j.v.e., ribeiro junior, c., fernandes junior, a.j. & prezoto, f. (2010). uso da vespa social polistes versicolor no controle de desfolhadores de eucalipto. pesquisa agropecuária brasileira, 45: 958-964. gobbi, n. & machado, v.l.l. (1986). material capturado e utilizado na alimentação de polybia (trichothorax) ignobilis (haliday, 1836) (hymenoptera: vespidae). anais da sociedade entomológica do brasil, 15: 117–124. gobbi, n., machado, v.l.l. & tavares-filho, j.a. (1984). sazonalidade das presas utilizadas na alimentação de polybia occidentalis occidentalis (olivier, 1791) (hymenoptera: vespidae). anais da sociedade entomologica do brasil, 13: 63-69. gomes, l., gomes, g., oliveira, h.g., morlin jr., j.j., desuó, i.c., silva, i.m., shima, s.n. & zuben, c.j.v. (2007). foraging by polybia (trichothorax) ignobilis (hymenoptera: vespidae) on flies at animal carcasses. revista brasileira de entomologia 51: 389-393. doi: 10.1590/s0085-56262007000300018 hunt, j.h. (2007). the evolution of social wasps. oxford university press, new york, 259 p. lima, m.a.p. & prezoto, f. (2003). foraging activity rhythm in the neotropical swarm-founding wasp polybia platycephala sylvestris richards, 1978 (hymenoptera: vespidae) in different seasons of the year. sociobiology, 42: 645-752. maciel, t.t., castro, m.m., barbosa, b.c., fernandes, e.f., santos-prezoto, h.h. & prezoto f. (2015) foraging behavior of fire ant solenopsis saevissima (smith) (hymenoptera: formicidae) in felis catus linnaeus (carnivora: felidae) carcass. sociobiology 62: 610-612. moretti, t. d.c., giannotti, e., thyssen, p.j., solis, d.r., & godoy, w.a.c. (2011). bait and habitat preferences, and temporal variability of social wasps (hymenoptera: vespidae) attracted to vertebrate carrion. journal of medical entomology, 48: 1069-1075. doi: 10.1603/me11068 moretti, t.c., thyssen p.j., godoy w.a.c. & solis d.r. (2008). necrophagy by the social wasp agelaia pallipes (hymenoptera: vespidae: epiponini): possible forensic implications. sociobiology, 51: 393-398. o’donnell, s. (1995). necrophagy by neotropical swarm founding wasps (hymenoptera: vespidae: epiponini). biotropica 27: 133-136. doi: 10.2307/2388911 prezoto, f. & braga, n. (2013). predation of zaprinus indianus (diptera: drosophilidae) by the social wasp synoeca cyanea (hymenoptera: vespidae). florida entomologist, 96: 670672. doi: 10.1653/024.096.0243. prezoto, f. & souza, c.a.s. (2015). a vida secreta das vespas. in: lima, m.s.c.s.; carvalho, l.s. & prezoto, f. (ed.). métodos em ecologia e comportamento animal (pp. 121-148). teresina: editora da ufpi. prezoto, f., cortes, s.a.o. & melo, a.c. (2008). vespas: de vilãs a parceiras. ciência hoje, 48: 70-73. prezoto, f., lima, m.a.p. & machado, v.l.l. (2005). prey captured and used in polybia platycephala (richards) (hymenoptera: vespidae, epiponini) neotropical entomology, 34: 849-851. doi: 10.1590/s1519-566x2006000500021 prezoto, f., santos-prezoto, h.h., machado, v.l.l. & zanuncio, j. c. (2006). prey captured and used in polistes versicolor (olivier) (hymenoptera: vespidae) nourishment. neotropical entomology 35: 707-709. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.7843sociobiology 69(4): e7843 (december, 2022) introduction blepharidatta wheeler (1915) is a neotropical ant genus in the attini tribe represented by only four species that nest in the soil and leaf litter and are distributed in the central and western amazon basin, in different localities in the cerrado, in the caatinga, and in the atlantic forest in brazil (brandão et al., 2015). wheeler (1915) described the genus and stated that these ants “evidently” belonged to the attini tribe. the blepharidattini tribe was proposed by wheeler and wheeler (1991) comprising the genera blepharidatta and wasmannia. however, this grouping is no longer valid since ward et al. (2015) placed it together with other tribes within the attini in its current conception. it is worthwhile to mention that blepharidatta specimens have the habit of keeping the remains of prey in the main chamber of the nest abstract blepharidatta is a rare neotropical ant genus in the attini tribe of the subfamily myrmicinae. it has only four valid species and among them blepharidatta delabiei was recently described and there is little knowledge on its biology. this study is the first cytogenetic characterization for the genus blepharidatta and also presents the biology of b. delabiei. cytogenetic analyses revealed a karyotype 2n = 28 with acrocentric and metacentric chromosomes and a karyotypic formula (m: metacentric, a: acrocentric): 2k = 16m + 12a. we observed that ants of this species have diurnal habits with higher foraging activity in the afternoon and are possibly omnivorous as they accepted the baits used. the distance between colonies varied from 5 to 7 meters. sociobiology an international journal on social insects laís l. lopes1, cléa s. f. mariano2, jacques h. c. delabie2,3,4, janisete g. silva1 article history edited by evandro nascimento silva, uefs, brazil received 22 februry 2022 initial acceptance 01 june 2022 final acceptance 12 october 2022 publication date 22 november 2022 keywords chromosome number, foraging, karyotype, morphology, natural history. corresponding author janisete gomes silva universidade estadual de santa cruz rodovia jorge amado, km 16 cep: 45662-900 ilhéus-ba, brasil. e-mail: jgs10@uol.com.br for a while. the observation that these carcasses are often covered by fungi led to the hypothesis that this behavior could represent one of the possible initial steps that led to the close symbiosis between ants and fungi observed today in the attina (diniz & brandão, 1997; diniz et al., 1998; rabeling et al., 2006). this hypothesis is further reinforced by the existence of ancestral groups (e.g. cyphomyrmex and myrmicocrypta) that use arthropod corpses to cultivate fungi as a source of food (hölldobler & wilson, 1990). out of the four valid species in this genus, blepharidatta brasiliensis (wheeler 1915), blepharidatta conops (kempf 1967), blepharidatta delabiei (brandão et al., 2015), and blepharidatta fernandezi (brandão et al., 2015), so far there is published data on the biology of only b. conops (diniz et al., 1998; brandão et al., 2008; pereira et al., 2014). some studies suggested that populations of b. conops found in the 1 universidade estadual de santa cruz, departamento de ciências biológicas, programa de pós-graduação em genética e biologia molecular, ilhéus, bahia, brazil 2 universidade estadual de santa cruz, departamento de ciências biológicas, programa de pós-graduação em zoologia, ilhéus, bahia, brazil 3 laboratório de mirmecologia, cepec/ceplac, itabuna, bahia, brazil 4 universidade estadual de santa cruz, departamento de ciências agrárias e ambientais, ilhéus, bahia, brazil short note first cytogenetic study through conventional staining of the ant genus blepharidatta wheeler, 1915 (hymenoptera: formicidae: attini) laís l. lopes, cléa s. f. mariano, jacques h. c. delabie, janisete g. silva – karyotype description of an ant of the genus blepharidatta2 brazilian caatinga and cerrado were actually two distinct species based mainly on the distinct morphology of the phragmotic queens (pereira et al., 2014; brandão et al., 2015). blepharidatta delabiei is the most recently described species within the genus (brandão et al., 2015). there is no published information on its biology so far, mainly due to the difficulty in locating colonies. this species is distributed in the leaf litter of atlantic forest remnants in the states of bahia and minas gerais (cassano et al., 2009, where blepharidatta sp. = b. delabiei; brandão et al., 2015). the analysis of mitotic chromosomes has been widely used for cytotaxonomic studies in some groups of eukaryotes (imai, 1983; lorite & palomeque, 2010; kretschmer et al., 2018; magalhães et al., 2020). the study of karyotypes allows inferences on species differentiation, degree of kinship, evolutionary processes, and the phylogenetic position of the taxa studied (guerra, 1988; mariano et al., 2019). an important aspect of chromosome evolution in ants is the haplodiploid nature of their reproductive system. as males are haploid, there is no meiosis to produce gametes (palomeque et al., 1990), which leads to the possibility that in formicidae as in the entire order hymenoptera, chromosomal alterations are much more tolerated than in other groups of organisms (gokhman, 2009). several chromosomal rearrangements can be tolerated by hymenopterans because aneuploids within this group are often viable and fertile and even though aberrations in meiosis are apparently more deleterious in males than in females, hymenopteran males lack this important checkpoint (gokhman, 2009). in formicidae, more than 800 species have been cytogenetically studied (lorite & palomeque, 2010, mariano et al., 2019, aguiar et al., 2020). the huge variation in chromosome number ranging from 2n = 2 to 2n = 120 (lorite & palomeque, 2010) may provide good models to investigate the role of chromosomal variation in speciation. the attini tribe alone currently comprises 48 genera with a total of 2.703 valid species, but there is cytogenetic information for only 21 genera (cardoso et al., 2018) with karyotypes ranging from 2n = 4 to 2n = 64 (alves-silva et al., 2014; murakami et al., 1998; barros et al., 2013; mariano et al., 2019; cardoso & cristiano, 2021). this means that the karyotypes of 56.25% of the attini genera, such as blepharidatta, are unknown. hence, to expand the knowledge on the genus blepharidatta, we investigated the karyotype of b. delabiei using classical cytogenetics and some aspects of its biology. material and methods colonies of b. delabiei were collected along the ilhéus/ una highway near águas de olivença, ilhéus, bahia, brazil (15º00’13.3”s 39º00’53.3”w). the collecting permit (sisbio number 63623-1) was issued to laís leal lopes by the instituto chico mendes de conservação da biodiversidade (icmbio). b. delabiei colonies were found near an indigenous reserve. nests were located with the help of bait distribution (bread and sardines) offered to foragers. three colonies were taken to the laboratory of social arthropods at the universidade estadual de santa cruz, ilhéus, where they were kept in artificial nests (delabie et al., 2021). the ants were identified by dr. j. h. c. delabie. mitotic metaphases were obtained following the protocol of imai et al. (1988) using prepupal cerebral ganglia. we analyzed 10 larvae from each of the 3 sampled colonies, totaling 30 samples and 117 metaphases. the best quality metaphases were captured and analyzed using an image capture system (olympus bx-51, a q-capture pro image capture camera, and the image pro plus software). the karyograms were assembled using the adobe photoshop® cs3 extended software following the nomenclature by levan et al. (1964). results the colonies of b. delabiei were found close to an indigenous reserve with well-preserved vegetation. considering the rarity of this species, we believe that this ant can be used as a biological indicator of environmental quality and a low degree of anthropic disturbance, which confirmed the findings by cassano et al. (2009). we observed that this species has diurnal habits with higher foraging activity in the afternoon. the distance between colonies varied from 5 to 7 meters. all colonies of b. delabiei were found in the leaf litter and were extremely discrete. the ants of this species do not recruit high numbers of workers and forage very slowly. thus, it was necessary to wait for the workers to carry the bait into the nest in order to locate the colony accurately. these ants are possibly omnivorous as they accepted the bait of both bread and sardines. we collected a total of 236 adult individuals from the three colonies (table 1) and immatures were present in all colonies. the number and morphology of chromosomes and the karyotypic formula were described using conventional staining (figure 1). the mitotic index was low (<10 metaphases/ slide) and in a large percentage of the prepared slides, no mitotic metaphases were observed. we analyzed 30 individuals (all workers, 10 individuals per colony) and a total of 117 metaphases. queens workers males immatures colony 1 95 1 >15 larvae colony 2 1 97 >20 larvae colony 3 1 28 >10 larvae table 1: demographics of blepharidatta delabiei colonies including immatures. sociobiology 69(4): e7843 (december, 2022) 3 the results of conventional staining revealed the karyotype 2n = 28 in all analyzed individuals with a constant karyotypic formula 2k = 16m + 8st + 4a (m: metacentric, st: subtelocentric, a: acrocentric) (figure 1). although morphometric studies were not performed, the analyzed metaphases showed chromosomes with a discrete size variation. discussion this is the first study carried out to cytogenetically characterize a population of b. delabiei. a total of 21 genera in the attini tribe were cytogenetically described. the range of recorded diploid chromosome numbers shows a remarkable variation from 2n = 4 in strumigenys louisianae (roger, 1863) (2n = 4) to 2n = 64 in mycetophylax lectus (forel, 1911) (= cyphomyrmex lectus) (alves-silva et al., 2014; murakami et al., 1998; barros et al., 2013; mariano et al., 2019; cardoso & cristiano, 2021). in attini, only the genera atta and acromyrmex have a karyotype considered constant of 2n = 22 and 2n = 38, respectively (cristiano et al., 2013; barros et al., 2014; barros et al., 2021), whereas there is intrageneric variation in the karyotype in the remaining genera within the clade (murakami et al., 1998, cardoso, et al., 2021; texeira et al., 2022). the closest genera to blepharidatta based on molecular analyses are wasmannia and allomerus (ward et al., 2015). the only species in the genus with a known karyotype, wasmannia auropunctata (roger, 1863), has a karyotype of 2n = 32 and the karyotypic formulae are 2k = 20m + 12a (souza et al., 2011) and 2k =16m + 10sm + 6st (aguiar et al., 2020) with acrocentric, metacentric, and telocentric chromosomes. allomerus is also considered a genus close to blepharidatta with only two species with a known karyotype, allomerus decemarticulatus (mayr, 1878) with 2n = 28 and karyotypic formula of 2k = 18m+6sm+2st+2a and allomerus octoarticulatus (mayr, 1878) with 2n = 44 and a karyotypic formula of 2k = 4sm+ 40a (aguiar et al., 2020). future discussions regarding karyotype evolution in this clade should include information on allomerus species since they cluster with blepharidatta and wasmannia in the study on the phylogeny and biogeography of the subfamily myrmicinae by ward et al. (2015). these authors obtained a grouping that places wasmannia closer to allomerus than to blepharidatta. however, regarding morphology and taxonomy, blepharidatta and wasmannia are closer (brown, 1953; wheeler; wheeler, 1991; longino; fernández, 2007). the point here is that karyotypic information on closely related taxa can provide additional evidence of their taxonomic affinities (sumner, 2003) and our results on b. delabiei reinforce the greater proximity between blepharidatta and wasmannia, both in chromosome morphology and karyotype. regarding its natural history, b. delabiei shares more characteristics with b. brasiliensis than with b. conops, both in colony structure and morphology. in a study on the behavioral ecology and natural history of b. brasiliensis, rabeling et al. (2006) noticed that this species is also omnivorous and nests in the leaf litter, but they did not find any queens with phragmotic cephalic discs as is seen in colonies of b. conops (brandão et al., 2001). the foraging schedule of b. delabiei is like that of b. conops, since b. delabiei workers forage preferentially during the day, with greater activity in the afternoon. the workers of b. conops also forage outside the nest during the day, avoiding the hottest period, even though some workers stay out of the nest all day long (brandão et al., 2001). however, b. brasiliensis foragers concentrate their activities predominantly at night, and at midday, this activity ceases and no other worker is observed until the late afternoon (rabeling et al., 2006). our results help to expand the knowledge on the genus blepharidatta regarding its natural history and karyotype. the variation in chromosome number and morphology between species with low and high numbers of chromosomes within the attini tribe are some of the examples that demonstrate the importance of cytogenetic studies since they can contribute to the systematics of ants. authors’ contribution lll: conceptualization, methodology, investigation, formal analysis, writing (original draft/review and editing) fig 1. blepharidatta delabiei karyotype 2n = 28 with chromosomes classified according to levan et al. (1964), where m: metacentric, st: subtelocentric, a: acrocentric. bar: 5 µm. laís l. lopes, cléa s. f. mariano, jacques h. c. delabie, janisete g. silva – karyotype description of an ant of the genus blepharidatta4 csfm: conceptualization, methodology, investigation, formal analysis, writing (original draft/review and editing) jhcd: conceptualization, writing (original draft/review and editing) jgs: conceptualization, methodology, investigation, writing (original draft/review and editing) acknowledgments we would like to thank josé raimundo maia and josé crispim soares do carmo (in memoriam) for their help with the fieldwork. we would like to thank carter robert miller for revising the manuscript. we also acknowledge coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the m.s. scholarship granted to the author. references aguiar, h.j.a.c., barros, l.a.c., silveira, l.i., petitclerc, f., etienne, s. & orivel, j. 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(1994). exotic ants: biology, impact, and control of introduced species. westview press, boulder, colorado. 332 p. _hlk112702564 _hlk112700282 _hlk112700412 _hlk107995668 _hlk112700993 _hlk112702244 _hlk112701941 _hlk112702780 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7620sociobiology 69(2): e7620 (june, 2022) epiponini is composed of swarming social wasps which include19 genera (carpenter & marques, 2001; carpenter, 2004; noll et al., 2021). the most widespread genus is polybia, with 56 species and 19 subspecies distributed across the neotropical region (jeanne, 1991; carpenter et al., 2000; noll et al., 2021). polybia nests are sessile or suspended, constructed with mud or vegetable fiber with a brown or gray envelope. nests have a circular entry located in the peripheral or in the lower region which is aligned to connect with internal combs (richards, 1978; wenzel, 1998). the secondary combs are sessile and attached to the preceding envelope, and the new cells are constructed in the lower portion (phragmocyttarous nests) (wenzel, 1998; somavilla, 2012; noll et al., 2021; barbosa et al., 2021). these social wasps are model organisms in studies of evolution and social behavior origin due to the specialization abstract social wasps are model organisms in studies related to evolution and social behavior origin. they show high degree of sinanthropism and due to their feeding habits, they play important ecological roles. however, wasps are considered dangerous, hence their nests are destroyed by humans. the aim of this study was to develop a technique for transferring the nests of some polybia (lepeletier, 1836) species located in human constructions to protected places. nests were removed in the morning blowing ether into the nest entrance and closing them with cotton. they were separated from the substrate with a spatula. nests were immediately attached to the new substrate with hot silicone glue and installed in the new place. transferred nests were monitored for one month to verify the efficiency of the technique. following the transference, individuals kept foraging and repairing/constructing new cells. workers performing colony tasks are evidence that the technique was efficient and that the colony was adapted to the new local. sociobiology an international journal on social insects amanda prato, rafael c. da silva, sidnei mateus, fabio s. do nascimento article history edited by evandro nascimento silva, uefs, brazil received 22 november 2021 initial acceptance 24 november 2021 final acceptance 11 april 2022 publication date 17 june 2022 keywords wasp management; wasp conservation; synanthropic wasps, nest transference. corresponding author amanda prato universidade de são paulo, faculdade de filosofia ciências e letras de ribeirão preto, departamento de biologia av. bandeirantes, 3900, vila monte alegre, cep: 14040-900 ribeirão preto são paulo, brasil. e-mail: amandaprato@usp.br expressed by workers (age polyethism) (carpenter, 1991). furthermore, they play important ecological roles in pollination, biological control, and as environmental bioindicators (prezoto & machado, 1999; urbani et al., 2006; rocha et al., 2010; brock et al., 2021). this is possible due to their different dietary habits throughout their life. larval stages consume mainly protein (caterpillars, adult forms of insects, or other arthropods), while adult wasps have a carbohydrate-based diet, such as nectar (akre & davis, 1978; hunt, 1991, 1998; spradbery, 1973). as they display these roles, wasps can be useful to humans (prezoto et al., 2007, 2011; brock et al., 2021); many species are well adapted and associated with anthropogenic environments, exhibiting a high degree of sinanthropism (fowler, 1983; lima et al., 2000; prezoto et al., 2007; da silva et al., 2019; barbosa et al., 2020, 2021). despite their universidade de são paulo, faculdade de filosofia, ciências e letras de ribeirão preto, departamento de biologia, ribeirão preto-sp, brazil short note a technique for transferring nests of polybia (hymenoptera: vespidae: epiponini) wasps in anthropized environment amanda prato, rafael c. da silva, sidnei mateus, fabio s. do nascimento – transferring nests of polybiawasps2 positive roles in the environment, wasps are considered dangerous (mainly because they sting and the misinformation about their importance), hence their nests are destroyed by humans (fowler, 1983; prezoto et al., 2007, 2011; sumner et al., 2018; brock et al., 2021; elisei et al., 2021). an alternative to avoid accidents between wasps and humans, or to prevent nest destruction, is to remove and transfer their nests to other localities (prezoto et al., 2007, 2011). however, few attempts have been made to develop techniques to transfer their nests (prezoto et al., 2016). there is still a lack of information regarding the basic biological aspects of many species of social wasps, this is likely associated with the fact that information is often difficult to be collected due to the inaccessibility of their nesting sites (barbosa et al., 2021). thus, the goal of this study was to develop an effective technique for transferring nests of some polybia species found in human constructions to protected places, enabling their use in behavior studies and avoiding their elimination. this work was performed at são paulo university, ribeirão preto campus, sp – brazil between 2016 and 2017. for this study, four nests of polybia occidentalis (olivier, 1792), four of polybia paulista (h. von. ihering, 1896), one of polybia ignobilis (haliday, 1836), and one of polybia fastidiosuscula de saussure, 1854 were used (fig 1) (barbosa et al., 2021; somavilla & carpenter, 2021). the nests were removed in the morning between 06:00 and 09:00 a.m. to perform the nest transference procedure we always wore latex gloves and beekeeper clothes. the first step was to blow ether into the nest entrance and close it with a cotton ball. the cotton can be moistened with ether; this is an alternative when it is not possible to blow the chemical compound (fig 2). to start the removal (one to two people to perform the procedure), the nests were carefully handled in a way to prevent the external envelope structure to be destroyed. then, using a spatula, the envelope was separated from them substrate where it was located. at last, the nests were immediately attached to the new substrate (piece of cardboard) with hot silicone glue (can be used hot glue gun or a lighter to heat the glue) (fig 2). all the nests were in masonry construction, only one of the p. paulista nests was in a metal structure and the p. fastidiosuscula was in a wood substrate. although not all the nests were attached to the same substrate, all of them were transferred in the same way. fig 1. nests that were transferred a, c, e, and g. nests after the transfer b, d, f, and h. nests of the four polybia species used in this work. a and b – polybia paulista; c and d – polybia fastidiosuscula; e and f – polybia ignobilis; g and d – polybia occidentalis. fig 2. steps performed to transfer nests. ablowing ether in the nest entrance; bremoving the nest from the old substrate; cputting glue in the nest base; dattaching the nest in the new supporting; etaking the nest to the new place, and fattaching the nest in the new place sociobiology 69(2): e7620 (june, 2022) 3 nests were then transferred to the new site (by walking or by car). the nests were transferred attached to the new substrate but without protection around. because of the ether, the individuals were not aggressive during the course. the cardboard with nests was attached to a place preferably protected from the rain with silver tape. the minimum distance of transference between the original site and the new one was 1000 meters and the maximum was 2500 meters. during this process, there was only a small loss of individuals, the ones that already were over the nest surface when the procedure started. the nests that were transferred were monitored for one month to verify the efficiency of the procedure; furthermore, the p. occidentalis nests were used inother experiments, hence they were followed for three months (prato, 2018; prato et al., 2021). following the transference procedure, wasps continued to perform their foraging activities, started to repair the damages, and build new cells. workers performing their usual activities are indicative that they were already used to the new place. thus, it was confirmed that this technique is efficient because of the minimal invasive manipulation done to the transferred nests. polybia nests are built directly in the substrate (only the top part of the nest is in contact with the nesting place), differently from other epiponini species, in which all the nest is attached to the structure, such as in the genera metapolybia and synoeca (wenzel, 1998; noll et all., 2021; kudô, 2021). this facilitates the nest transference process for polybia nests, as it is possible to separate their nests from the substrate without major damage. an important factor that must be considered is the type of environment where the nest is located and the new area where the nest will be placed because each species has different preferences for substrate or feeding habits (cruz et al., 2006; santos et al., 2007; almeida et al., 2014; da silva et al., 2019; barbosa et al., 2020, 2021). these features can directly affect the success of the colony (dejeanet al., 1998; santos et al., 2007). within polybia, some species are generalist regarding the nesting substrate (souza et al., 2012; oliveira et al., 2017; barbosa et al., 2020). furthermore, polybia is the genus of epiponini that are more frequently found in anthropic environments (detoni et al., 2018; da silva et al., 2019). this generalist feature of nesting and adaptation to urban areas increases the chances to be in contact with humans (and accidents). however, they also represent good candidates for biological control studies in different crops, including urban agriculture. at last, it is possible to use them for investigating the influences of urbanization on generalist insects in comparison with preserved places. thus, this technique may work as an alternative to help to preserve those wasp species, which are often found in high-risk areas, by transferring them to more protected localities. hence, the technique can be used as a tool to promote the conservation of them in anthropized environments. lastly, our method will facilitate future studies using nests that were initially present in sites that are hard to reach or even relocate colonies to perform experiments in different environmental contexts. declaration of competing interest none. authors' contributions conceptualization [amanda prato, rafael carvalho da silva and sidnei mateus]; methodology [amanda prato, sidnei mateus and rafael carvalho da silva]; formal analysis and investigation [amanda prato, rafael carvalho da silva, sidnei mateus and fabio s. nascimento]; writing-original draft preparation [amanda prato and fabio s. nascimento]; writing-review andediting [amanda prato, rafael carvalho da silva, sidnei mateus and fabio s. nascimento]. acknowledgments this study was financed by conselho nacional de desenvolvimento científico e tecnológico (cnpq process: 870320/1997-1, 405082/2018-5 and 307702/2018-9), coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) financecode 001 (rcs) and fundação de amparo à pesquisa do estado de são paulo (fapesp) undergrant aps [proc. nº 2016/11887-4], rcs [proc. nº 2016/08761-9 and 2018/22461-3], and fsn [proc. nº 2018/10996-0]. references akre, r.d. & davis, h.g. 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(2021). key to the genera of social wasps (polistinae) occurring in neotropics. in f. prezoto; f.s. nascimento, b.r, correa & a. somavilla (eds.), neotropical social: basic and applied aspects (pp. 327-336). springer, cham. de souza, a.r., de f.a. venâncio, d., prezoto, f. & zanuncio, j.c. (2012). social wasps (hymenoptera: vespidae) nesting in eucalyptus plantations in minas gerais, brazil. florida entomologist, 95: 1000-1002. spradbery, j.p. (1973). an account of the biology and natural history of solitary and social wasps. universidade washington press, seattle. sumner, s., law, g. & cini, a. (2018). why we love bees and hate wasps. ecological entomology, 43: 836-845. urbani, a., sparvoli, e. & turillazzi, s. (2006). social paper wasps as bioindicators: a preliminary research with polistes dominulus (hymenoptera: vespidae) as a trace metal accumulator. chemosphere, 64: 697-703. wenzel, j.w.a. (1998). generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae: vespinae, polistinae). american museum novitates, 3224: 1-39. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7123sociobiology 68(4): e7123 (december, 2021) introduction bees are the most effective pollinators of nature and thus ensure the maintenance of crops and wild plants (nabhan & buchmann, 1997; klein et al., 2007). they belong to the apoidea superfamily. it is estimated that there are more than four thousand genera and 25 to 30 thousand species distributed in different regions of the world (michener, 2007), of which 20.507 have been described (ascher & pickering, 2019). solitary bees comprise 85% of known species, and among them, females make their own nest, supply them with food, and die before the next generation emerges (batra, 1984; michener, 2007). these bees can use different substrates for nesting, and the ground is the most widespread among all groups (michener, 1964; 2007; harmon-threatt, 2020). on abstract we described the nesting behavior and ecology of diadasina riparia from an urban dry forest fragment. the nests of d. riparia were shallow with a circular entrance, closed by a mud plug, from which a straight vertical tunnel ending in one or more brood cells. most females need one day for construction and provisioning the nest. the natural enemies were flies of the anthrax genus, the cleptoparasitic bees leiopodus trochantericus, and mutillidae wasps. the feature about the life history, nest architecture, materials used, pollen provision behavior, and associated organisms of this species allows comparison with other emphorines species. sociobiology an international journal on social insects herbeson o. j. martins1, gilson p. amorim-junior2, william o. sabino3, vinina s. ferreira2 article history edited by evandro nascimento silva, uefs, brazil received 03 april 2021 initial acceptance 31 july 2021 final acceptance 08 october 2021 publication date 24 november 2021 keywords fossorial bee, caatinga, oligolectic bees, nesting behavior. corresponding author herbeson ovidio de jesus martins departamento de biologia, faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo av. bandeirantes nº 3900, cep 14040-901 ribeirão preto, são paulo, brasil. e-mail: herbeson.bio@hotmail.com the other hand, the number of studies focused on the groundnesting bees is still low compared to the guild of bees nesting in aboveground substrates. this scarcity of studies is mainly due to the difficulty of finding nests in the field. amid the ground-nesting solitary bees, diadasina (moure) is a genus of the emphorini tribe composed of eight species with distribution restricted to argentina, bolivia, brazil, panama, and paraguay (moure & melo, 2012). in brazil, this genus is represented by the species d. distincta (holmberg), d. monticola moure, d. paraensis (ducke), d. parahybensis (cockerell), and d. riparia (ducke) (moure & melo, 2012). the biology and nesting behavior of diadasina species is poorly known, and the studies are restricted to the neotropical species d. distincta (martins & antonini, 1994; hazeldine, 1997). 1 programa de pós-graduação em entomologia, faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, ribeirão preto, brazil 2 universidade federal do vale do são francisco, colegiado de ciências biológicas, petrolina, brazil 3 instituto tecnológico vale, pará, brazil research article bees nesting biology of the solitary ground-nesting bee diadasina riparia (apidae: emphorini) id https://orcid.org/0000-0002-9889-3104 herbeson o. j. martins, gilson p. amorim-junior, william o. sabino, vinina s. ferreira – nesting biology of diadasina riparia2 here we present data and observations on the nesting biology of d. riparia, a solitary bee species with distribution restricted to south america (moure & melo, 2012). information about the nest architecture, foraging behavior, life cycle, sex ratio, and natural enemies are provided. material and methods study site the study was carried out in a fragment of caatinga (seasonally dry forest) at universidade federal do vale do são francisco (9°19’44,2’’ s, 40°33’’30,1 w), municipality of petrolina, pernambuco state, brazil. the climate is bsh, according to the köppen classification. and it is characterized by low rainfall with an annual average precipitation of 433 mm. two well-defined seasons are observed: the dry season from may to october and the rainy from november to april (amorim-neto, 1989). data collection and analysis we exposed and measured the nest architectures using a digital caliper. after that, we drew them on the original scale utilizing the software adobe photoshop and adobe illustrator version cc 2018. we collected females of d. riparia at the beginning of the nesting process. to identify individuals, we marked them on the thorax with a non-toxic pen (uni posca ®; tokyo, japan) with different colors combinations. we conducted observations weekly, from october 2018 until july 2019. we monitored six females during the construction of nine nests from the initial digging through nest closure. we applied the focal animal and ad libitum methods to observe the following female activities to build their nests: time spent constructing each brood cell, provisioning resources trips, and the behavior near the nesting area. we measured the nest density in the area using a 1-m2 quadrat to count the number of nests during the nesting season. to obtain the sex ratio and time of emergence, we placed clear plastic cups over 200 nest entrances. we collected some specimens and sent them to the identification. the vouchers are deposited in the entomological collection padre jesus santiago moure of the department of zoology at universidade federal do paraná (dzup). nest structures are deposited in the entomology laboratory at universidade federal do vale do são francisco (univasf). a chi-square test was used to assess if the proportion of males and females that emerged in the laboratory differed from a standard 1:1 fisherian sex ratio. results nesting sites and seasonality we identified more than 800 nests. they were distributed in two sites 1500 meters apart. females constructed the nests in compact sandy soil and on the vertical surface of hard clay escarpments. (fig 1a, b). the majority of the nests were established on the vegetation-covered ground. overall, the species was active throughout the year, with two peaks of nesting activity, one from march to april and another from october to november. nests were not homogeneously distributed in the area, ranging from 1 to 9 nests/m2 (3.56 ± 1.53) in the dispersed area and 26 to 42 nests/m2 (33.6 ± 7.12) when aggregated. nest construction and provisioning behavior at the beginning of the nest building, females of diadasina riparia (n = 6) flew low close to the nest site and frequently landing on the ground. they initiated digging in multiple locations until selecting a definitive nest site and immediately started the nest building (fig 1). for excavation, females made field trips to collect water (fig 1d), which was regurgitated on the ground, making it soft, and then the soil was then excavated with the mouthparts and legs. the water collection sources were near the nesting sites, from three to 40 meters distance. two females built up two nests all over the observation period, three built up three nests, and one built four nests. the females began the nest construction in the middle of the morning (10:00-11:00 a.m.) and ended it the next day (10:0011:00 a.m.). females start the pollen-collecting trips in the afternoon (12:20-01:40 p.m.) immediately after the conclusion of the digging activity. the number of trips to supply each brood cell ranged from eight to 16 (fig 2a). pollen sources were close to nesting sites. the end of daily female activities occurred around 04:20 p.m., when the females obstructed the entrance of the nests with their abdomen, remaining in this position until the next morning. to finalize the nest building, the females made field trips to gather water. we also visualized the females collecting mud pellets from outside and deposited them inside the nests. the females (n = 6) spent between 169-305 min (245 ± 83.8 min) in indoor activities such as nest building, provision unloading, and oviposition and 42-115 min (74 ± 37.32 min) in outdoor activities such as pollen-collecting trips, water gathering and foraging (fig 2b). four monitored nests were not completed. one female attempted to usurp a neighbor’s nest but being expelled by the host females. we did not visualize the female leaving the following morning in one of these nests, suggesting that it died inside the nest. another female abandoned the nest after finding leiopodus trochantericus ducke there for the second time. nest description the nest entrances were circular, closed by a mud plug, from which a straight vertical tunnel was dug up, ending in one or more brood cells (fig 3). mud pellets were deposited around the nest entrance. the tunnel descended vertically to a depth of 24.77 to 45 mm (33 ± 7.36 mm; n = 5 nests) and then terminated in one or more brood cells, with most of the sociobiology 68(4): e7123 (december, 2021) 3 excavated nests having a single brood cell (fig 3a). the nests built upon hard clay escarpments had a slightly different structure. the entrance of the nests was arranged in the fig 1. general aspects of the nesting sites and nesting behavior of diadasina riparia; a. aggregation of diadasina riparia in flat soil. b. aggregation of diadasina riparia in vertical surface c. female in the begging of the nest construction. d. floating on the water surface during the water-collecting trip. horizontal position with a tunnel assuming vertical orientation (fig 3a). in some nests, the construction of septa along the main tunnel was observed (fig 3c). fig 2. nesting behavior of a single female of diadasina riparia to construct three nests; a. number of trips performed during different stages of each nest construction and their purpose. b. duration of time inside and outside the nest. herbeson o. j. martins, gilson p. amorim-junior, william o. sabino, vinina s. ferreira – nesting biology of diadasina riparia4 the brood cells were strongly adhered to the ground with vertical to sub-vertical orientation. the brood cells were closed by a slightly convex cap, with an internal surface in a spiral pattern. brood cells are urn-shaped with length of 7.85 to 11.1 mm (9.08 ± 1.11 mm; n = 10), minimum diameter ranging from 4.49 to 7.23 mm (6.09 ± 0.66 mm; n = 10) and maximum diameter of 5.86 to 7.87 mm (6.9 ± 0.99 mm; n = 10). inside the brood cells, a mass of orange and compacted pollen was observed. the whitish, elongated, and slightly curved eggs were placed on the bottom of the pollen mass (fig 3b). fig 3. nest architectures of diadasina riparia. a. nest from a vertical surface (1. nest plug in the entrance; 2. brood cell cap; 3. turret on nest entrance). b-c. nest from flat soil (4. main tunnel; 5. mud pellets deposited inside nest; 6. mud pellets deposited around the entrance; 7. septa; 8. egg; 9. spheroid pollen mass). life cycle and sex ratios from the 200 nests, 34 females (17%) and 41 males (20.5%) emerged with a sex ratio with no statistical difference of 1:1 (χ2 = 0.653, df = 1, p = 0.419). development time was 13 to 21 days for females (16.58 ± 3.06 days) and 13 to 45 days for males (17.04 ± 5.25 days). after eight months, we opened nine nests, in which no emergencies were registered. in these nests, we observed four mature larvae, probably in diapause, one brood cell with dry pollen mass and one fungal brood cell. natural enemies the most frequent natural enemies were the bombyliid fly anthrax sp. and the bees of the protepeolini tribe, l. trochantericus. females of l. trochantericus were the main natural enemies present in the aggregations. the kleptoparasite bees had the behavior of waiting near the entrance of the nests, in the soil, or on branches (fig 4b). shortly after the host females left their nests, l. trochantericus invaded and spent approximately 30 seconds inside them. a total of 13 adults l. trochantericus emerged from the nests. the development time of l. trochantericus ranged from 13 to 22 days (17.72 ± 3.37 days). fig 4. natural enemies associated with diadasina riparia. a. female of mutillidae wasp parasitizing a nest. b. leiopodus trochantericus females awaiting at the nest entrance. c. anthrax pupa. d. brood cell with a larva of a parasite feeding on a pupa of diadasina riparia. sociobiology 68(4): e7123 (december, 2021) 5 the bombyliid fly anthrax sp. was frequently seen patrolling the aggregation, flying over the nest entrance, and throwing eggs into the nests while the host females were out. further, the bombyliid fly larvae were frequently seen inside cells that were opened (fig 4c). the number of anthrax sp. that emerged from the nests was 13, with development time ranged from 16 to 37 days (28.63 ± 6.23 days). various female mutillids were observed at the nest site, entering open nests or digging closed nests (fig 4a). two mutillidae wasps emerged from the nests. we did not observe antagonistic behaviors from host females against parasites. discussion our study presented the first record on the nesting biology of diadasina riparia. the species is multivoltine with nesting activity in both the dry and rainy seasons, confirming the suspicions of aguiar and zanella (2005). this characteristic was also observed for d. distincta, which can undergo up to five generations a year (martins et al., 1990). the females’ nest in the ground is a common feature among the group of solitary bees (see in antoine & forrest, 2021). site selection is an important part of the nesting process. some locations for nests may be better than others concerning favorable edaphic conditions, proximity to food plants, and/ or reduced parasite loads (sabino et al., 2020). the more time a female spends searching for the ideal location and creating a nest, the less time she will have to provision and lay eggs in it or construct additional nests (antoine & forrest, 2021). the nesting sites of d. riparia are close to areas with frequent irrigation and flower availability throughout the year, which may be the main factor supporting the activity of the species, even in the dry season. nevertheless, larvae in nests opened after eight months also indicate a possible diapause in d. riparia within the same population, part of the offspring emerged in a short period [13-45 days]. in contrast, the other part entered a possible diapause of more than eight months. santos et al. (2019) did an ancestral reconstruction analysis of diapause types in bees. they showed that within apidae, a clade with d. rinconis and ptilothrix plumata smith has common ancestrality in developmental diapause, a characteristic preserved in bee species that did not evolve to eusociality. thus, it is plausible to infer that d. riparia shares the same evolutionary basis of diapause with other emphorini (and even other solitary bee species). other emphorini species have a diapause period as part of their developmental biology. martins and antonini (1994) observed diapause in d. distincta during the rainy season. prepupae of d. rinconis cockerell may stay in diapause from one reproductive season to the next one (neff & simpson, 1992). martins et al. (2001) reported the offspring of p. plumata undergoing diapause at the end of the breeding season, emerging in the following year. the nests constructed by d. riparia females are mostly single-celled, with a simple and short tunnel, vertically on the surface. this pattern is similar in many aspects to the nests of other emphorines species such as d. distincta (martins & antonini, 1994), p. plumata (martins et al., 1996; schildwer et al., 2009), and ptilothrix bombiformi (cresson) (rust, 1980). these similarities in nest architecture may reflect the phylogenetic relationship between the genera ptilothrix and diadasina (roig-alsina & michener, 1993). although already considered a subgenus of diadasia (michener, 1954), the genus diadasina was later considered the closest part to ptilothrix (roig-alsina & michener, 1993). we do not know exactly the benefits associated with building one cell per nest. there are costs associated with digging a new nest for each cell, but on the other hand, this behavior can avoid massive parasitism in one nest with multiple cells. pathogens and parasites can significantly reduce the survival of bees while nesting (linsley & mcswain, 1942), affecting more than 50% of nest cells in some species (linsley, 1958). however, a high number of parasitized nests was still observed in this study. this high number of parasitic nests suggests that the defense mechanisms adopted by the species proved to be inefficient. leiopodus trochantericus was the most frequent natural enemy in d. riparia nests. there is likely a strong association between this species and the genus diadasina. this species’s only record of parasitism is for nests of d. distincta (straka & bogusch, 2007). also, parasitism by the anthrax fly was frequent. this genus has been frequently registered associated with several groups of solitary bees; being also registered in nests of d. distincta (martins & antonini, 1994) and p. plumata (martins et al., 1996; schlindwein et al., 2009). besides the strategy to build one cell per nest, some structural features observed in the d. riparia nests, such as the closing of the nest and the presence of septa along the tunnel, are probably mechanisms to prevent parasitism of the nests. these mechanisms may guarantee the protection of the nests after their completion. however, they are inefficient against the two main parasites that attack the nests during their construction. during the observation period, we register evidence that the species is philopatric, nesting in the same site for at least three years (from 2018 to the present). philopatric behavior is expected in many groups of hymenoptera and has been associated with food resources available in the area and their resources, such as suitable soil for nesting (rosenheim, 1990). the tendency of the d. riparia offspring to nest near the places they emerged may be related to the availability of suitable soil areas and their proximity to specific floral resources since the species are considered oligolectic (schlindwein, 1998). antonini et al. (2000) reported that the high degree of philopatry observed in d. distincta was mainly related to soil texture at nesting sites. d. riparia prefers to nest in compacted soils, a scarce resource in the study area, limiting the dispersion of the nests. another behavior herbeson o. j. martins, gilson p. amorim-junior, william o. sabino, vinina s. ferreira – nesting biology of diadasina riparia6 performed by females was collecting water in their crops to soft the soil and allow the excavation of the nests, similar to that observed in most species of emphorine bees (linsley et al., 1956; laroca & almeida, 2009). in this way, the presence of puddles of water near the nesting sites can make these areas attractive, as the females use this resource to facilitate the excavation of the nests. although the knowledge on the nesting biology of many groups of emphorini bees is growing, the information is still fragmented and focused on a few species of certain genera, such as diadasina in which presents only a detailed study on nesting biology. the data available in the present study will contribute to expanding the knowledge about some fundamental aspects related to the biology of the genus diadasina. besides, our study showed that d. riparia shares several characteristics with species from close groups, such as the genus ptilothrix, evidencing the phylogenetic relationship between the genera. acknowledgments the authors thank the two anonymous reviewers for improving the quality of the initial manuscript with their helpful comments. we thank carlos martínez (usp) for identifying the bees and gabriel a. r. melo (ufpr) for confirming the identification. we also thanks carlos j. e. lamas (mz/usp) for identifying bombyliidae. we also are grateful to vashtir r. s. braga for the help in fieldwork. authors’ contributions hojm: conceptualization, investigation, methodology, writingoriginal draft preparation and review vsf: conceptualization, writing-original draft preparation and review gpa-j: investigation, methodology wos: writing-original draft preparation and review all authors have read, revised, and agreed to the final version of the manuscript. funding hojm is currently funded by a scholarships from conselho nacional de desenvolvimento científico e tecnológico – cnpq (134121/2019-8). disclosure statement the authors declare no conflicts of interest. references aguiar, c.m. & zanella, f.c. 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(2021). nesting biology of centris (paracentris) burgdorfi (apidae: centridini). journal of apicultural research, 60: 817-827. doi: 10.1080/00218839.2020.1717760 santos, p.k.f., arias, m.c. & kapheim, k.m. (2019). loss of developmental diapause as prerequisite for social evolution in bees. biology letters, 15: 20190398. doi: 10.1098/rsbl. 2019.0398 sarzetti, l.c. & genise, j.f. (2011). the nest architecture of diadasia hirta (jörgensen) (apidae: emphorini) from la rioja province, northwestern argentina. journal of the kansas entomological society, 84: 249-255. schlindwein, c. (1998). frequent oligolecty characterizing a diverse bee–plant community in a xerophytic bushland of subtropical brazil. studies on neotropical fauna and environment, 33: 46-59. doi: 10.1076/snfe.33.1.46.2168 schlindwein, c., pick, r.a. & martins, c.f. (2009). evaluation of oligolecty in the brazilian bee ptilothrix plumata (hymenoptera, apidae, emphorini). apidologie, 40: 106-116. doi: 10.1051/apido/2008067 straka, j. & bogusch, p. (2007). phylogeny of the bees of the family apidae based on larval characters with focus on the origin of cleptoparasitism (hymenoptera: apiformes). systematic entomology, 32: 700-711. doi: 10.1111/j.13653113.2007.00394.x doi: 10.13102/sociobiology.v63i2.981sociobiology 63(2): 800-803 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 first occurrence of the ant genus brachymyrmex mayr, 1868 (hymenoptera: formicidae) from the kingdom of saudi arabia introduction the ant genus brachymyrmex was established by mayr in 1868 for the type species b. patagonicus from argentina. the genus is included in the formicinae and the myrmelachistini (blaimer et al., 2015; ward et al., 2016). currently 61 species and subspecies are recognized (bolton, 2015). the genus is distributed in the nearctic (creighton, 1950) and primarily in the neotropical regions (brown, 2000; wild, 2007; ortiz & fernández, 2014). there are records from the afrotropical region as introductions including b. cordemoyi forel, 1895 from tanzania, and an unidentified species from gabon, south africa, são tome and principe, and the malagasy region (http://www.antweb.org/, accessed 20 september 2015). few treatments of the genus are presently available. santschi (1923) published the last revision of the genus. creighton (1950) reviewed the species for north america. abstract the formicine ant genus brachymyrmex mayr, 1868 is recorded for the first time from the kingdom of saudi arabia and from the arabian peninsula by the species brachymyrmex cordemoyi forel, 1895. a brief description and automontage images of the species are presented including ecological observations. sociobiology an international journal on social insects mr sharaf1, s salman1, hm aldhafer1, afa_yousef2, as aldawood1 article history edited by john lattke, ufpr, brazil received 12 january 2016 initial acceptance 18 march 2016 final acceptance 04 april 2016 publication date 15 july 2016 keywords new record, arabian peninsula, palearctic region, kingdom of saudi arabia, riyadh province, formicinae. corresponding author mostafa r. sharaf economic entomology research unit (eeru) plant protection department college of food and agriculture science king saud university riyadh 11451, p. o. box 2460 kingdom of saudi arabia e-mail: antsharaf@gmail.com quirán et al. (2004) and quiran (2005, 2007) have provided limited synopses of this genus for argentina, but much additional revisionary work is required for the neotropical region. recently, brachymyrmex and myrmelachista roger, 1863 were transferred from the plagiolepidini and placed into the resurrected tribe myrmelachistini by ward et al. (2016). the majority of species of brachymyrmex are considered generalized foragers with a broad range of nesting habitats including the soil, decaying wood, leaf litter, under loose bark at the base of tree trunks (macgown et al., 2007), in seeds, and trees or fallen fruits (brown, 2000). the genus includes species that are known as successful invasive such as b. patagonicus having successfully established in many areas of the united states and has become a serious pest in both natural and disturbed habitats (macgown et al., 2007). two key morphological features distinguish workers of brachymyrmex from other formicine genera, a 9-segmented 1 king saud university, riyadh, kingdom of saudi arabia 2 princess nora bint abd-elrahman university, riyadh, kingdom of saudi arabia research article ants sociobiology 63(2): 800-803 (june, 2016) 801 antennae and lacking a differentiated antennal club (bolton, 1994). herein, the ant genus brachymyrmex is recorded from the first time from the kingdom of saudi arabia (ksa) and from the arabian peninsula by the species b. cordemoyi. materials and methods a sifting tray was used to collect the specimens. digital color images of lateral and dorsal views of the entire body and full-face views of the head of each species were made at the california academy of sciences and were created using a leica dfc450 digital camera with a leica z16 apo microscope and las (v3.8) software. these images are also available online on antweb (www.antweb.org, accessed 29 december 2015) and are accessible using the unique identifying specimen code. measurements and indices: measurements: all measurements are in millimeters. eye length (el): maximum diameter of eye. head length (hl): length of head, excluding mandibles, measured from mid-point of anterior clypeal margin to midpoint of posterior head margin, in full-face view. head width (hw): maximum width of head in full-face view, measured behind eyes. interocular distance (iod): measured on the straight line between inner margins of eyes. mesosomal length (ml): diagonal length of mesosoma in profile from point at which pronotum meets the cervical shield to posterior base of metapleuron. pronotal width (pw): maximum width of pronotum measured in dorsal view. scape length (sl): maximum straight line length of antennal scape excluding basal constriction or neck to condylar bulb. total length (tl): outstretched body length from mandibular apex to gastral apex. indices: cephalic index (ci): hw x 100/hl. occular index (ei): el x 100/hw. scape index (si): sl x 100/hw. museum abbreviations. casc: california academy of sciences collection, san francisco, california, usa. ksma: king saud university museum of arthropods, king saud university, riyadh, kingdom of saudi arabia. results and discussion brachymyrmex cordemoyi forel, 1895 (figs 1-3) brachymyrmex patagonicus var. cordemoyi forel, 1895: 49 (w.) reunion, malagasy. raised to species: emery, 1906: 179. subspecies of brachymyrmex patagonicus: forel, 1908: 399; forel, 1912: 165; santschi, 1912: 533. revived status as species: wheeler, 1922: 1036; emery, 1925: 41. current subspecies: nominal plus brachymyrmex cordemoyi distinctus. fig 1. brachymyrmex cordemoyi, habitus. casent 0922067, photographer: michele esposito, available from http://www. antweb.org. accessed 01 december 2015. fig 2. brachymyrmex cordemoyi, lateral view, casent 0922067, photographer: michele esposito, available from http://www.antweb. org. accessed 01 december 2015. fig 3. brachymyrmex cordemoyi, frontal view, casent 0922067, photographer: michele esposito, available from http://www.antweb. org. accessed 01 december 2015. mr sharaf et al. – first occurrence of the ant genus brachymyrmex mayr saudi arabia802 material examined. saudi arabia, 8 workers, riyadh, king saud university campus, 24.71383°n, 46.62557°e, 02.ii.2014, 660 m (s. salman leg.); 6 workers, 20.ix.2014; 6 workers, 15.iii.2015, ksma; 1 workers, 14.iii.2015, casc, (casent0922067). all the above material has identical locality and collector information. diagnosis. worker. head distinctly longer than broad with a straight posterior margin and clearly convex lateral sides; eyes with nine ommatidia in the longest row; scapes when laid back from their insertions just reach posterior margin of head. mesosoma. metanotal groove impressed; propodeal dorsum short descending abruptly into long declivity; propodeal spiracle small, circular, situated at middle of propodeal declivity. sculpture. body smooth and shining. pilosity. cephalic dorsum with abundant appressed pubescence; two pairs of long setae on anterior and posterior clypeal margins; one pairs of setae on frontal carinae, one pair on posterior margin of head; mesosoma with one pair of long setae on pronotal and promesonotal dorsum; gaster with many scattered long setae and some appressed pubescence. color. head and body uniform brown with the antennae and legs yellowish. measurements: el 0.08–0.11, hl 0.40–0.55, hw 0.35– 0.51, iod 0.30–0.41, ml 0.34–0.51, pw 0.25–0.35, sl 0.34–0.45, tl 1.16–1.92, indices: ci 84–94, oi 20–28, si 87–100 (n=19). diagnostic notes: the short scapes of b. cordemoyi separate it from b. patagonicus. brachymyrmex obscurior forel, 1893 has short scapes and appressed pubescence on the gaster, but the appressed hairs are distinctly denser. ecological notes: this species was found nesting in soil at the base of a date palm tree (phoenix dactylifera l.) in king saud university campus (fig 4), riyadh. specimens were collected by sifting the soil which was a mixture of sandy clay, with much decaying organic material. workers were found about 8 cm deep in the soil. other ant species collected with b. cordemoyi included: solenopsis saudiensis sharaf & aldawood 2011, nylanderia jaegerskioeldi (mayr, 1904), tapinoma simrothi krausse, 1911, and cardiocondyla mauritanica forel, 1890. geographic range. neotropical (wild, 2007), and afrotropical (mauritius) (forel, 1907) regions. brachymyrmex patagonicus is an example of a successful invasive ant species. this species has the ability to survive in a wide range of habitats and the capacity to coexist with various dominant ant species (macgown et al., 2007). colonies of this species can be established in relatively small areas and are apparently transported easily by human activities from site to site. king saud university is the home for many students from different countries in africa (sudan, somalia, nigeria, mali, senegal and guinea), and perhaps b. cordemoyi was incidentally introduced by students with their belongings. another possibility is by the numerous imported cosmopolitan horticultural plants and irrigated lawns that are planted throughout the campus. this introduction may have recently occurred. the senior author has been collecting ants for ten years on the campus and in many other regions of ksa and this species has not been observed. additional studies will indicate the extent of the distribution of this ant species in ksa and the impact on native fauna. acknowledgements the project was funded by the national plan for science, technology and innovation (maarifah), king abdulaziz city for science and technology, kingdom of saudi arabia, award number 12-env2804-02. we are most grateful to two anonymous reviewers for valuable suggestions. the authors are grateful to dr. b. kondratieff for useful comments and to dr. brian fisher and mrs. michele esposito (california academy of sciences) for photographing the species. references blaimer, b. b.; brady, s. g.; schultz, t. r.; lloyd, m. w.; fisher, b. l.; ward, p. s. (2015). phylogenomic methods outperform traditional multi-locus approaches in resolving deep evolutionary history: a case study of formicine ants. bmc evolutionary biology, 15: 257. bolton, b. (1994). identification guide to the ant genera of the world. harvard university press,cambridge, mass., 222 pp. bolton, b (2015). an online catalogue of the ants of the world. http://antcat.org [accessed december, 20, 2015]. brown, w.l. jr. (2000). diversity of ants. pp 45–79. in: agosti et al. (eds.) ants. standard methods for measuring and monitoring biodiversity, biological diversity hand book series. smithsonian institution press, washington d. c. 280 pp. creighton, w.s. (1950). the ants of north america. bulletin of the museum of comparative zoology at harvard college, 104: 1–585. fig 4. habitat of brachymyrmex cordemoyi, king saud university campus, riyadh, kingdom of saudi arabia. (photo: s. salman). sociobiology 63(2): 800-803 (june, 2016) 803 emery, c. (1906). studi sulle formiche della fauna neotropica. xxvi. bullettino della società entomologica italiana, 37: 107–194. emery, c. (1925). hymenoptera. fam. formicidae. subfam. formicinae. genera insectorum, 183:1–302. forel, a. (1895). nouvelles fourmis de diverses provenances, surtout d’australie. annales de la société entomologique de belgique, 39: 41–49. forel, a. (1907). ameisen von madagaskar, den comoren und ostafrika. wissenschaftliche ergebnisse. reise in ostafrika, 2: 75–92. forel, a. (1908). ameisen aus sao paulo (brasilien), paraguay etc. gesammelt von prof. herm. v. ihering, dr. lutz, dr. fiebrig, etc. verhandlungen der kaiserlich-königlichen zoologischbotanischen gesellschaft in wien, 58: 340–418. forel, a. 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(1923). revue des fourmis du genre brachymyrmex mayr. anales del museo nacional de historia natural de buenos aires, 31: 650–678. ward, p.s., blaimer, b.b., fisher, b.l. (2016). a revised classification of the ant subfamily formicinae (hymenopter: formicidae), with the resurrection of the genera colbopsis and dinomyrmex. zootaxa, 40723: 343–357. wheeler, w. m. (1922). ants of the american museum congo expedition. new york: bulletin of the american museum of natural history, 1139. wild, a.l. (2007). a catalogue of the ants of paraguay (hymenoptera: formicidae). zootaxa, 1622: 1–55. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.8317sociobiology 69(4): e8317 (december, 2022) the genus metapolybia includes 18 species of social wasps of the tribe epiponini with distribution restricted to the neotropics (andena et al., 2019), 16 of which are recorded in brazil (cooper, 1999; andena & carpenter, 2011; andena et al., 2019; somavilla et al., 2021). wasps of this genus build colonies by swarming and build nests on different substrates (souza & zanuncio, 2012), without a pedicel and classified as astelocyttarus (richards & richards, 1951) and, for the most part, with a protective envelope (cooper, 1999). similarities between the chemical composition of social wasp nests and the plants on which they were built can provide information on the ideal physical conditions and safety and integrating the “chemical signature” of their colonies (sguarizi-antonio et al., 2021). colony defense strategies by social wasps are diverse, including repellent substances against ants (jeanne, 1970), abstract colony defense by social wasps includes aggressive behavior and camouflage with a high diversity of adaptation of their nests to the environment, including substrate choice. the objective of this study was to present new data on nesting and colony defense by the neotropical social wasps metapolybia cingulata and metapolybia docilis (hymenoptera: vespidae). field observations were made on january 4th and 5th, 2022 at the state park of iguaçu, state of paraná, brazil, on m. cingulata and m. docilis nests and information on nesting of these wasps obtained in 2014 at the pandeiros river wildlife refuge (revis), and in 2012 at the rio doce state park, state of minas gerais, brazil. the types of substrates used by m. cingulata and m. docilis reinforce the importance of nest camouflage as a defense strategy for their colonies. the selection of nesting sites by these species was very variable. sociobiology an international journal on social insects marcos m. de souza1, norma barbado2, josé adolfo m. de almeida2, gabriel teofilo-guedes3, josé c. zanuncio4 article history edited by alexandre somavilla, inpa, brazil received 30 june 2022 initial acceptance 17 july 2022 final acceptance 08 september 2022 publication date 28 december 2022 keywords aggressive displays, colony defense, nest architecture, wasp colony. corresponding author josé adolfo m. de almeida instituto federal de educação, ciência e tecnologia do paraná – ifpr campus umuarama, parque industrial, km 310, pr 323, cep: 87507-014 umuarama, paraná, brasil. e-mail: jose.almeida@ifpr.edu.br nest built with protective envelope or camouflage and aggressive behavior (chavarría-pizzarro & west-eberhard, 2010; barbosa et al., 2016; milani et al., 2021). defense strategies of metapolybia species include camouflage by homochromy and colony homotypy on tree trunks (somavilla et al., 2012; souza et al., 2020) and aggressive behavior in eventual disturbances, as reported for metapolybia cingulata (fabricius, 1804; souza et al., 2020). the objective of this study was to present new data on nesting and colony defense in neotropical social wasps metapolybia cingulata (fabricius, 1804) and metapolybia docilis (richards, 1978) (hymenoptera: vespidae). m. cingulata nests were recorded in 2010, in an evergreen forest in the rio doce state park (19°42’ s, 42°34’ w) and the others in deciduous forest in the pandeiros river wildlife refuge (revis) (15°88’ s, 45°95’ w), state of minas 1 instituto federal do sul de minas gerais, inconfidentes-mg, brazil 2 instituto federal do paraná, umuarama-pr, brazil 3 universidade estadual de campinas, campinas-sp, brazil 4 universidade federal de viçosa, viçosa-mg, brazil nest camouflage in metapolybia cingulata and nesting and colony defensive behavior in metapolybia docilis (vespidae: polistinae) in the brazilian atlantic forest short note mailto:jose.almeida@ifpr.edu.br marcos m. de souza, et al. – nesting and defensive behavior of colonies of the wasps 2 gerais in 2014. nests of m. docilis were recorded in september 2014 and 2021 and in january 2022 in the state park of iguaçu (25°22’s, 54°2’w), state of paraná, all in brazil. behavioral aspects of one colony of metapolybia docilis were observed and recorded in the state park of iguaçu using the ad libitum method (del-claro, 2010) for 50 minutes divided into 10-minute sessions with two at 9:30 a.m., two at 5:00 p.m. and one at 1:00 p.m. on january 08th, 2022. this colony was subjected to stress stimuli: 1) manipulating the nest; 2) seeking to catch the specimens; 3) removing part of the nest envelope to model the behavior related to the aggressiveness or docility of metapolybia. a part of the colony envelope (≈ 3 cm) was removed by exposing the comb (chavarría-pizzarro & west-eberhard, 2010). the height of the other colonies made these procedures impossible. this study complied with the norms of the sisbio (licence number: 76084-3). each nest of m. cingulata and m. docilis was located in different plant species and anthropic substrates (table 1). data species metapolybia cingulata metapolybia docilis number of colonies nine four locality state park of rio doce (one nest); revis rio pandeiros (eight nests) state park of iguaçu phytophysiognomy evergreen forest (one nest); deciduous forest (eight nests). evergreen forest nesting substrate euterpe edulis (arecaceae) (one nest); house construction lumber (eight nests). trunk of pliniarivularis (cambess.) myrtaceae (one nest); unidentified tree species (one nest), residential walls (two nests). height above the ground 1.5 m (one nest); 2 to 2.4 m (eight nests). 2 m (one nest); 3.5 m (one nest); 4 and 5 m (two nests). nest color light grey to dark gray, similar to substrate. light grey to dark grey, similar to substrate. table 1. data on numbers of colonies, locality, phytophysiognomy nesting substrate, height above the ground and nest color of metapolybia cingulata and metapolybia docilis (hymenoptera: vespidae) colonies. diversity of plant species used as substrate for nesting by m. docilis and m. cingulata were similar to those reported for polistes actaeon haliday, 1836 using from tree species, as aspidosperma cuspa (khunt), to shrubs, such as baccharis dracunculifolia dc., and for polybia platycephala richards, 1978 and protopolybia sedula (saussure, 1854), nesting in nine and 19 plant species, respectively (alvarenga et al., 2010; souza et al., 2014). the m. cingulata and m. docilis nesting, on anthropic substrates, evidences the plasticity of nest site selection in metapolybia species as reported for mischocyttarus, polistes and polybia (silva et al., 2019; barbosa et al., 2020) in timber and walls (oliveira et al., 2017; silva et al., 2019) and metapolybia miltoni andena & carpenter, 2011 (silva et al., 2019), metapolybia fraudator carpenter and andena, 2019 (andena et al., 2019) and metapolybia unilineata (ihering, 1904) (somavilla et al., 2012) nesting in wooden houses. nesting in human constructions can offer greater protection against environmental factors, such as rainfall, wind and temperature extremes (silva et al., 2022), which explains the use of these substrates near forested areas. the color of metapolybia nests, similar to the substrate, seems to be related to homochromy camouflage, since in all the records made, there is a similarity between the color of the nest and the substrate (fig 1), whether in a natural environment, using plant substrates, such as tree trunks or palm tree stems, and in anthropic environments, nesting on walls and wooden structures of houses, as already reported and suggested for species of the genera mischocyttarus and parachartergus (souza et al., 2020), metapolybia cingulata (souza et al., 2020) and chartergellus communis richards, 1978 (silva et al., 2022), which suggests that it constitutes a defense mechanism against visual predators, such as birds. the height of m. cingulata and m. docilis colonies on plant substrates was lower than those on anthropic substrates, the latter on roofs, and wooden or concrete structures with greater protection from the weather (barbosa et al., 2020). this condition may be associated with the fact that nesting on anthropic substrates, below 2 m, could facilitate the destruction of nests by humans (lópez et al. 2012), however, this assumption needs to be better evaluated. individuals of m. docilis, after stimulation by stress, behaved as follows: upon the first stimulus (touching with the hand or the handle of the entomological net), the individuals raised their heads and the first pair of legs, moving them intensely, but without attacking (fig 2a), characterizing bluff behavior (chavarría-pizzarro & west-eberhard, 2010). this may also characterize an alarm behavior, but no contraction of the gaster or wing movement was observed, as reported for polistes erythrocephalus latreille (1813) (west-eberhard, 1969), sociobiology 69(4): e8317 (december, 2022) 3 polybia occidentalis (olivier, 1791) (jeanne, 1981) and protopolybia exigua (saussure, 1854) (chadab, 1979). alarm behavior was recorded in the morning, but not in the late afternoon, when the sun was directly shining on the colony, increasing wasp activity with foraging and colony maintenance (giannotti et al., 1995; santos & presley, 2010). wasp individuals subjected to the second (collection of individuals) and third (attempt to remove the colony envelope) stimuli raised their heads and the first pair of legs, moving them intensely, but they flew away or remained in the surroundings without attacking (fig 2b). the absence of attack with only bluff behavior characterizes m. docilis as docile, increasing the importance of nest camouflage for this species (chavarría-pizzarro & west-eberhard, 2010). however, the absence of sting attempts by this wasp differs from that reported for m. cingulata (souza et al., 2020), showing that the colony defense behavior by metapolybia species involves camouflage and, also, some degree of aggressiveness (forsyth, 1981), as reported for synoeca wasps (chavarría-pizarro & west-eberhard, 2010). camouflage as a defense strategy is important to select nesting substrates by metapolybia cingulata and metapolybia docilis and shows the plasticity in the selection of anthropic substrates or plants for nesting by these wasps. fig 1. nest camouflage in metapolybia docilis (hymenoptera: vespidae) (a and b) at the state park of rio doce; metapolybia cingulata in the pandeiros river wildlife refuge (revis) (c and d) and m. docilis in the state park of iguaçu (d, e and f), brazil. marcos m. de souza, et al. – nesting and defensive behavior of colonies of the wasps 4 acknowledgements the authors thank ief and sisbio for providing license 76084-3. the taxonomist phd orlando tobias silveira, museu paraense emílio goeldi, who identified the collected specimens. the itaipu technological park foundation (fpti) for sponsoring this study, to the research sector of the iguaçu national park for hosting and to the chico mendes biodiversity institute (icmbio), especially its brigade members for their logistical support. to the brazilian institutions “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capesfinance code 001)”, “fundação de amparo à pesquisa do estado de minas gerais (fapemig)”, and “programa cooperativo sobre proteção florestal (protef) from instituto de pesquisas e estudos florestais (ipef)” for financial support. authors’ contributions mms: contributed on conceiving and designing the analysis, collecting the data, and writing both the draft and the final paper. nb: contributed on collecting the data, writing the draft and the final paper. jama: contributed on collecting the data, formatting, writing the draft and the final paper. gstg: contributed on writing the draft and the final version, formatting, translating and preparing the figures. jcz: contributed on designing the analysis, writing the draft and the final version, translating and supervising the manuscript writing. references alvarenga, r.d., de-castro, m.m., santos-prezoto, h.h. & prezoto, f. 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(1969). the social biology of polistine wasps. miscellaneous publications, museum of zoology, univ. of michigan, 101 p https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.30j0zll https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.1fob9te https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.3znysh7 https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.2et92p0 https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.tyjcwt https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.3dy6vkm https://docs.google.com/document/d/1vh-hmdsbyx543vqvqfhhh9ujvy0dgzbm/edit#bookmark=id.1t3h5sf https://doi.org/10.13102/sociobiology.v66i2.4303 doi: 10.13102/sociobiology.v67i4.5789sociobiology 67(4): 584-592 (december, 2020) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 introduction the amazon rainforest is the largest biome within the brazilian territory, covering an area of 4,196.943 km² (ministério do meio ambiente, 2018). many areas of brazil lack the most basic biodiversity studies, particularly in the case of invertebrates. in order to develop any effective conservation proposals, first of all, it is necessary to acquire knowledge of the species that occur in a particular area. for this reason, the thematic network ‘biodiversity of insects in the amazon’ is the first network among researchers of the brazilian amazon in terms of the increase of knowledge and provision of subsidies for the conservation of amazonian biodiversity, focusing on insects, and to disseminate this knowledge to different sectors of our society (see somavilla et al., 2020). the brazilian amazon rainforest is one of the greatest biodiversities hotspots in the world, including the greatest diversity of social wasps (silveira, 2002; somavilla et al., 2014a; barbosa et al., 2016; somavilla et al., 2020). knowledge on abstract the acre and rondônia states in brazil are part of the western amazon rainforest in brazil, an area harboring high biodiversity and high degree of endemisms. nevertheless, there are few studies on diversity of social wasps occurring in both states. this study presents a list of social wasps (vespidae: polistinae) collected using three modified malaise trap models: townes, gressitt and gressitt, and suspended traps in two localities in acre and two in rondônia states. a total of 60 species were collected, 20 of these species are new distribution records to acre state. meanwhile, 54 species were collected in rondônia state, 15 of which are new collecting records. some species are not commonly found in collections and lists of species, and some are recorded for the first or second time to brazil or the amazon region. currently there are 114 species (19 genera) for acre and 116 species (19 genera) for rondônia. this increase may be an indication that the polistinae richness is probably higher in the regions studied and that acre and rondônia may well contain a number of additional (as yet unrecorded) social wasp species. we also present an update on the geographic records of social wasps’ fauna in both states. sociobiology an international journal on social insects a somavilla, rnm de morais junior, pcs barroso, ml oliveira, ja rafael article history edited by marcel hermes, ufla, brazil received 14 august 2020 initial acceptance 06 novemebr 2020 final acceptance 10 november 2020 publication date 28 december 2020 keywords amazon biome, entomologists network, new records, hymenoptera, western amazon. corresponding author alexandre somavilla https://orcid.org/0000-0002-8174-7418 instituto nacional de pesquisas da amazônia coordenação de biodiversidade av. andré araújo nº 2936 cep 69067-375, manaus, amazonas, brasil. e-mail: alexandresomavilla@gmail.com social wasps remains from some few studies carried out in forest fragments. ducke (1904, 1907) conducted one of the first surveys of wasp fauna in the eastern region of the brazilian amazon, mainly in pará state. recently, similar works have been carried out in the brazilian amazon, such as in acre state (morato et al., 2008; gomes et al., 2018), amapá state (silveira et al., 2008; silveira et al., 2019), amazonas state (silveira et al., 2008; somavilla et al., 2015; somavilla et al., 2016; somavilla & oliveira, 2017; somavilla et al., 2019; somavilla et al., 2020), maranhão state (somavilla et al., 2014b), pará state (silveira, 2002; silva & silveira, 2009), rondônia state (gomes et al., 2020), and roraima state (raw, 1998; barroso et al., 2017). despite the contributions of these works, somavilla et al. (2014a) stated “there are many sample gaps in the amazon region and distribution and occurrence studies are necessary for improving upon this prior knowledge”. furthermore, for acre there are two works on social wasps: one in serra do divisor national park (morato et al., 2008) and another in three fragments instituto nacional de pesquisas da amazônia, coordenação de biodiversidade, manaus, amazonas, brazil research article wasps biodiversity of insecta in amazonia: updating the geographic records of social wasps (vespidae: polistinae) in acre and rondônia states, brazil sociobiology 67(4): 584-592 (december, 2020) 585 of the amazon rainforest, near to the city of rio branco (gomes et al., 2018), in addition to the previous records from richards (1978) and the taxonomic catalog of fauna of brazil – ctfb (hermes et al., 2015), reporting a sum of 94 species for this state. in rondônia, one recent work has been published by gomes et al. (2020) with samples from three fragments in northern rondônia, in addition to the previous records from richards (1978) and the taxonomic catalog of fauna of brazil – ctfb (hermes et al., 2015), reporting 101 species for this state. moreover, it is an area close to peru and bolivia, regions known for presenting endemic species for different groups of organisms (brown, 1991; hoorn et al., 2010), but the diversity of social wasps in this part of the amazon is poolry known, generating a gap in knowledge about the species occurring in the western amazon of brazil. in this way, a long term collection of one year was carried out within the network ‘biodiversity of insects in the amazon’ and we present here the social wasps results of two different areas sampled in acre and two in rondônia states. also, we present updated geographic records of social wasps’ fauna in both states. material and methods acre state areas the social wasps were collected in two areas in acre state: bujari, experimental farm antimary (09º20’01” s, 68º19’17” w) and senador guiomard, experimental farm catuaba (10º04’28” s, 67º37’00” w). the region where the two areas are located is characterized by open forest with the presence of gadua bamboos (locally known as tabocais), and palm trees (silveira, 2005). the predominant type of soil in the region is latosol and the landscape is slightly hilly (daly & silveira, 2008). the average annual temperature varies between 22 and 24º c. lowest temperatures occur in august (about 12 to 14 ºc) (mesquita & paiva, 1995; mesquita, 1996). average annual rainfall is 1,944 mm, varying between 1,566 and 2,425 mm. the climate is tropical wet, with well-defined hot/rainy (winter) and hot/dry (summer) seasons. the rainy period occurs from october to april and the driest period from june to august. may and september are transitional months between seasons (duarte, 2005). rondônia state areas the social wasps were collected in two areas in rondônia state: porto velho, forest fragment of universidade federal de rondônia (unir) (08º50’15.8” s, 63º56’17.5” w) and itapuã do oeste, floresta nacional of jamari (09º15’36” s, 62º54’46” w). the predominant vegetation in the region are of the dense rainforest and open rainforest types. the predominant type of soil in the region is latosol and the relief is slightly rugged with few depressions, the state is on an average of 240 m in altitude (schlindwein et al., 2012). the average annual temperature is about 25º c. average annual rainfall is 2,400 mm, varying between 2,200 and 2,600 mm. the climate is tropical wet, with well-defined hot/rainy (winter) and hot/dry (summer) seasons. the rainy period occurs from november to april and the driest period from june to september. may and october are transitional months between seasons (fernandes & guimarães, 2002). wasp collection and identification the wasps were collected in the forest fragments using three modified malaise trap models: 1. townes (1972) model 2-meter long; 2. gressitt and gressitt (1962) model 6-meter long with two collector vials in understory; and 3. suspended trap (rafael & gorayeb, 1982) model in the canopy. all of these trap models were distributed in each forest fragment and were active for twelve uninterrupted months, totaling a period of one year between july 2016 and june 2017, and every fifteen days only the collection bottles were replaced. the polistinae specimens were sorted and identified at the hymenoptera laboratory of the national institute of amazonian research (inpa). the vouchers were deposited into the inpa’s invertebrate collection and duplicates will be sent to other collections like mnrj (museu nacional do rio de janeiro, brazil, rio de janeiro) and mzusp (museu de zoologia da universidade de são paulo, brazil, são paulo), as recommended by the thematic network ‘biodiversity of insects in the amazon’. specimens were identified using the keys proposed by richards (1978), carpenter and marques (2001), carpenter (2004), somavilla and carpenter (2020) and were compared to previously identified species from the inpa invertebrate collection, and some type specimens images from the natural history museum (london), muséum national d’histoire naturelle (paris) and american museum of natural history (new york city). results a total of 114 species in 19 genera of social wasps were recorded for acre; we identified 60 species in our samples, 40 already reported in literature and 20 as new collecting records. there are still other 54 species known from literature not sampled in this study (richards, 1978; morato et al., 2008; gomes et al., 2018; andena et al., 2019; somavilla & andena, 2019; iunh, 2020) (table 01). for rondônia we listed the occurrence of 116 species in 19 genera; we identified 54 species in our samples, 39 already reported in literature and 15 as new records. for this state there are also 61 species from literature, not collected in this study (richards, 1978; gomes et al., 2020; iunh 2020) (table 1). about 50% of the species belong to three genera: polybia lepeletier (35 species), mischocyttarus de saussure (21 species) and agelaia lepeletier (18 species). the list also comprises polistes latreille (13), apoica lepeletier (09), brachygastra perty (09), protopolybia ducke (09), parachartergus r. von ihering (07), chartergellus bequaert (06), angiopolybia araujo (04), asteloeca raw (03), chartergus lepeletier (03), clypearia de saussure (03), metapolybia ducke (03), pseudopolybia de a somavilla, rnm de morais junior, pcs barroso, ml oliveira, ja rafael – social wasps in acre and rondônia states586 saussure (03), synoeca de saussure (03), leipomeles möbius (02), charterginus fox (01), epipona latreille (01) and nectarinella bequaert (01). nectarinella was not registered for acre and asteloeca for rondônia. protonectarina ducke was not registered for both states, and according to da silva et al. (2018) species of this genus occur widely in the atlantic rainforest and arboreal caatinga, being absent in the amazon region. from 60 species sampled, 20 represent new records for acre state: agelaia centralis, ag. flavipennis, ag. ornata, apoica albimacula, ap. gelida, ap. pallens, brachygastra bilineolata, chartergellus afoveatus (first time in brazil), c. amazonicus, leipomeles spilogastra, mischocyttarus adolphus, polistes canadensis, polybia belemensis, poly. depressa, poly. ignobilis, poly. micans, poly. occidentalis, poly. singularis, synoeca chalibea and s. surinama. for rondônia, the numbers, although smaller, are also noteworthy: from 54 species sampled, about a quarter of them (15) represent new records for rondônia state: agelaia brevistigma, angiopolybia pallens, apoica pallens, chartergellus nigerrimus, leipomeles spilogastra, mischocyttarus drewseni, nectarinella manauara, polistes carnifex, poli. geminatus, poli. pacificus, polybia batesi, poly. minarum, poly. simillima, poly. velutina and protopolybia fuscatus. discussion for acre state, richards (1978) recorded the occurrence of 52 species, of which 29 were not collected in this study. morato et al. (2008) recorded the occurrence of 20 species in serra do divisor national park and gomes et al. (2018) recorded the occurrence of 36 species in three areas and just 20 species not collected in our work. the other 20 species are recorded in our samples and are new occurrences for acre. for rondônia state, only the original descriptions and species recorded by richards (1978) were known. recently, gomes et al. (2020) recorded the occurrence of 72 species in three rondônia areas. richards (1978) and previous studies recorded the occurrence of 101 species in rondônia, and 28 species were not collected in this study. the other 15 species are recorded in our study and are new occurrences for rondônia. a sampling of a large diversity of polistinae can be attributed to more than one factor. one of them is the location of the collection areas in the western amazon, known for harboring great biodiversity (brown, 1991; hoorn et al., 2010). another factor are the methodology and effort applied. usually only one method is used to sample social wasps, which can limit the number of species sampled. in our study, we used a set of traps: two different malaise trap models and suspended traps to collect wasps for an uninterrupted year. regarding species composition, silva and silveira (2009), somavilla et al. (2014a) and somavilla et al. (2020) showed that fast inventories were efficient for sampling the most abundant species, recording three genera: polybia, mischocyttarus and agelaia. herein, we found the same most speciose genera, which constituted about 50% of the species collected. specimens of polybia have a very active foraging behavior, which facilitates the collection of the specimens by interception flight traps, adding to the fact it is the genus with the largest number of species within epiponini. mischocyttarus is the genus with the higher number of species within social wasps (around 250), of which more than 120 occur in brazil, supporting the high diversity in this study (silveira, 2002). agelaia species usually form large colonies with millions of individuals (zucchi et al., 1995), and, consequently, are more likely to be captured, and probably due to the habits of this genus that presents generalist and opportunistic species in relation to food and resource choices (silva & silveira, 2009; somavilla et al., 2014a) (silveira, 2002; somavilla et al., 2014a; somavilla et al., 2020). the amazon region has the highest diversity of polistinae species (richards, 1978; carpenter & marques, 2001; silveira, 2002; barbosa et al., 2016; somavilla et al., 2020). in the brazilian amazon, 20 genera and more than 250 species have been recorded, which represents about two thirds of the brazilian diversity of social wasps (silveira, 2002; hermes et al., 2015; somavilla & oliveira, 2017; somavilla et al., 2020). nevertheless, this impressive number surely does not yet represent the region’s mega diversity, since there were only two studies carried out on acre state (morato et al., 2008; gomes et al., 2018) and only one study in rondônia state (gomes et al., 2020). the present study presents new occurrences of 20 social wasp species for acre and 15 new occurrences for rondônia. our findings extend the species distributions and increase the number of species recorded to 114 and 116 species for acre and rondônia, respectively. this increase may be an indication that the richness is probably higher in the regions studied and that acre and rondônia may well contain a number of additional (as yet unrecorded) social wasp species. nevertheless, large gaps still remain in the geographic distribution for many species of social wasps in brazil, mainly in the amazon region, and more comprehensive studies are needed in order to increase the knowledge of wasp species in acre and rondônia. in a region of great diversity but also high indexes of deforestation, as western amazon, further studies become necessary and urgent. acknowledgments we sincerely thank two anonymous referees and marcel g. hermes for suggestions. to elder morato for support in collections in acre. to fundação de amparo à pesquisa do estado do amazonas for the postdoctoral scholarship (fapeam – fixam, process number 062.01427/2018)) to a somavilla and doctoral scholarship (fapeam posgrad/ inpa 006/2020) to pcs barroso. to cnpq research productivity fellowship of ja rafael and ml oliveira. specimens were collected during a project coordinated by ja rafael and supported by the conselho nacional de desenvolvimento científico e tecnológico (cnpq, process: 407623/2013-2) through the program “rede bia – biodiversidade de insetos na amazônia”. sociobiology 67(4): 584-592 (december, 2020) 587 table 1. species of social wasps from acre and rondônia states, listed by richards (1978), morato et al. (2008), gomes et al. (2018), gomes et al. (2020), web catalog from natural history laboratory of the ibaraki university (iunh 2020), and in the present work. 1 new distribution record for acre; 2 new distribution records for rondônia; * distribution records only in the descriptions of the species. ac=acre; ro=rondônia; buj=bujari; seg=senador guiomard; ido=itapuã do oeste; pvh=porto velho. taxon richards (1978) morato et al. (2008) gomes et al. (2018) gomes et al. (2020) iunh web catalog (2020) present work, locality collected agelaia acreana silveira & carpenter, 1996 ac agelaia angulata (fabricius, 1804) ac, ro ac ac ro ac, ro ac: buj, seg; ro: ido, pvh agelaia baezae (richards, 1943) ac agelaia brevistigma (richards, 1978) 2 ac ro: pvh agelaia cajennensis (fabricius, 1798) ac ro ac ac: seg agelaia centralis (cameron, 1907) 1 ro ac: buj, seg; ro: ido agelaia flavipennis (ducke, 1905) 1 ro ro ac: seg agelaia fulvofasciata (degeer, 1773) ac, ro ac ac ro ac, ro ac: buj, seg; ro: ido, pvh agelaia hamiltoni (richards, 1978) ac, ro ro ac, ro ac: seg agelaia lobipleura (richards, 1978) ac ro agelaia melanopyga cooper, 2000 ro agelaia myrmecophila (ducke, 1905) ac ac ro ro: pvh agelaia ornata (ducke, 1905) 1 ro ac: buj, seg; ro: ido, pvh agelaia pallidiventris (richards, 1978) ro agelaia pallipes (olivier, 1792) ac ac ro ac, ro ac: buj, seg; ro: ido, pvh agelaia pleuralis cooper, 2000 ac ac: seg agelaia testacea (fabricius, 1804) ac, ro ac ro ac, ro ac: buj, seg; ro: ido, pvh agelaia timida cooper, 2000 ro ro: ido angiopolybia pallens (lepeletier, 1836) 2 ac ac ac ac: buj, seg; ro: ido angiopolybia paraensis (spinola, 1851) ac, ro ac ro ac, ro ac: seg; ro: ido angiopolybia obidensis (ducke, 1904) ac angiopolybia zischkai richard, 1978 ac ro apoica albimacula (fabricius, 1804) 1 ac: buj apoica ambracarina pickett, 2003 ac apoica arborea de saussure, 1854 ac, ro ac, ro ro: pvh apoica flavissima van der vecht, 1972 ac, ro ac, ro apoica gelida van der vecht, 1972 1 ro ac: buj apoica pallens (fabricius, 1804) 1,2 ac: buj, seg; ro: ido, pvh apoica pallida (olivier, 1792) ac, ro ac, ro ac: buj, seg; ro: pvh apoica strigata richards, 1978 ac ac ac: buj apoica thoracica du buysson, 1906 ac, ro ac, ro ac: seg, ro: ido, pvh asteloeca lutea carpenter, 2004 ac asteloeca traili (cameron, 1906) ac ac asteloeca ujhelyii (ducke, 1909) ac ac: buj brachygastra albula richard, 1978 ro ac ro ro a somavilla, rnm de morais junior, pcs barroso, ml oliveira, ja rafael – social wasps in acre and rondônia states588 brachygastra augusti (de saussure, 1854) ac ac ro ac ro: pvh brachygastra bilineolata spinola, 1841 1 ro ro ac: buj, ro: ido brachygastra cooperi (richards, 1978) ro brachygastra lecheguana (latreille, 1824) ac ro ro ac: seg; ro: pvh brachygastra moebiana (de saussure, 1867) ac ac ro ac: seg brachygastra propodealis bequaert, 1943 ac ro brachygastra scutellaris (fabricius, 1804) ac, ro ro ac, ro ac: buj, ro: ido brachygastra smithii (de saussure, 1854) ac ac chartergellus afoveatus cooper, 1993 1 ac: seg chartergellus amazonicus richard, 1978 1 ro ac: seg chartergellus communis richards, 1978 ro chartergellus flavoscutellatus somavilla, 2019 * ac: buj, seg chartergellus nigerrimus richards, 1978 2 ac ac ro: pvh chartergellus zonatus (spinola, 1851) ro charterginus fulvus fox, 1898 ac ro ac: buj; ro: pvh chartergus artifex (christ, 1791) ac, ro ac: buj, seg chartergus globiventris de saussure, 1854 ro chartergus metanotalis richards, 1978 ac ac clypearia duckei richards, 1978 ac ac clypearia nigrior richards, 1978 ac ac ac: buj clypearia sulcata (de saussure, 1854) ac, ro ac, ro epipona tatua (cuvier, 1797) ac, ro ac, ro ro: ido leipomeles dorsata (fabricius, 1804) ac ro ac: buj, seg; ro: ido leipomeles spilogastra (cameron, 1912) 1,2 ac: seg; ro: ido metapolybia acincta richards, 1978 ro metapolybia cingulata (fabricius, 1804) ac ac metapolybia fraudator carpenter and andena, 2019 * mischocyttarus acreanus silveira, 2006 ac mischocyttarus adolphi zikán, 1949 1 ac: buj mischocyttarus alfkenii (ducke, 1904) ac ac mischocyttarus artifex ducke, 1914 ac mischocyttarus carbonarius (de saussure, 1854) ac ac: buj mischocyttarus drewseni de saussure, 1857 2 ro: ido mischocyttarus duckei (du buysson, 1908) ac mischocyttarus flavicans (fabricius, 1804) ac ro ro: ido mischocyttarus gomesi silveira, 2013 ro mischocyttarus heliconius richards, 1941 ac mischocyttarus imitator (ducke, 1904) ro mischocyttarus interruptus richards, 1978 ro mischocyttarus labiatus (fabricius, 1804) ac ac ro ac: seg; ro: pvh mischocyttarus lecointei (ducke, 1904) ro table 1. species of social wasps from acre and rondônia states, listed by richards (1978), morato et al. (2008), gomes et al. (2018), gomes et al. (2020), web catalog from natural history laboratory of the ibaraki university (iunh 2020), and in the present work. 1 new distribution record for acre; 2 new distribution records for rondônia; * distribution records only in the descriptions of the species. ac=acre; ro=rondônia; buj=bujari; seg=senador guiomard; ido=itapuã do oeste; pvh=porto velho. (continuation) taxon richards (1978) morato et al. (2008) gomes et al. (2018) gomes et al. (2020) iunh web catalog (2020) present work, locality collected sociobiology 67(4): 584-592 (december, 2020) 589 table 1. species of social wasps from acre and rondônia states, listed by richards (1978), morato et al. (2008), gomes et al. (2018), gomes et al. (2020), web catalog from natural history laboratory of the ibaraki university (iunh 2020), and in the present work. 1 new distribution record for acre; 2 new distribution records for rondônia; * distribution records only in the descriptions of the species. ac=acre; ro=rondônia; buj=bujari; seg=senador guiomard; ido=itapuã do oeste; pvh=porto velho. (continuation) mischocyttarus metathoracicus (de saussure, 1854) ac mischocyttarus prominulus richards, 1941 ac mischocyttarus rotundicollis (cameron, 1912) ro mischocyttarus socialis (de saussure, 1854) ac mischocyttarus surinamensis (de saussure, 1854) ac ac ac, ro ac: seg mischocyttarus synoecus richards, 1940 ac mischocyttarus tomentosus zikán, 1935 ac, ro ro ac, ro ro: pvh nectarinella manauara silveira & nazareno jr. 2016 2 ro: ido parachartergus colobopterus (lichtenstein, 1796) ro ro parachartergus flavofasciatus (cameron, 1906) ac ro ac: seg; ro: ido parachartergus fraternus (gribodo, 1892) ro parachartergus lenkoi richards, 1978 ro ro: ido parachartergus pseudoapicalis willink, 1959 ro parachartergus smithii (de saussure, 1854) ro ro: pvh parachartergus weyrauchi willink, 1959 ac ac, ro polistes bicolor lepeletier, 1836 ac ac polistes canadensis (linnaeus, 1758) 1 ro ac: seg; ro: ido polistes carnifex (fabricius, 1775) 2 ro: pvh polistes deceptor schulz, 1905 ac polistes geminatus fox, 1898 2 ro: pvh polistes erythrocephalus latreille, 1813 ac ac polistes lanio (fabricius, 1775) ac, ro ac polistes occipitalis ducke, 1904 ro polistes pacificus fabricius, 1804 2 ac ac: seg; ro: ido polistes rufiventris ducke, 1904 ro ro polistes testaceicolor bequaert, 1937 ac, ro ac ro: ido polistes torresae silveira, 1996 ro polistes versicolor (olivier, 1792) ac ac polybia affinis du buysson, 1908 ro ro polybia batesi richards, 1978 2 ro: ido polybia belemensis richards, 1970 1 ac: seg polybia bifasciata de saussure, 1854 ac ro ac polybia bistriata (fabricius, 1804) ac, ro ro ac, ro polybia catillifex möbius, 1856 ac ac ro polybia depressa (ducke, 1905) 1 ro ac: buj polybia diguetana buysson, 1905 ro polybia dimidiata (olivier, 1792) ro ac ro ro ac: buj polybia dimorpha richards, 1978 ac ac: seg polybia eberhardae cooper, 1993 ac ro polybia emaciata lucas, 1879 ac, ro ac ro ac, ro ac: buj, seg; ro: ido, pvh polybia furnaria von ihering, 1904 ac, ro ac, ro taxon richards (1978) morato et al. (2008) gomes et al. (2018) gomes et al. (2020) iunh web catalog (2020) present work, locality collected a somavilla, rnm de morais junior, pcs barroso, ml oliveira, ja rafael – social wasps in acre and rondônia states590 polybia gorytoides fox, 1898 ac ro ac: buj, seg; ro: ido, pvh polybia ignobilis (haliday, 1836) 1 ro ro ac: seg polybia jurinei de saussure, 1854 ac, ro ac ro ac, ro ac: buj; ro: pvh polybia juruana von ihering, 1904 ac ac ac: buj polybia liliacea (fabricius, 1804) ac, ro ac ro ac, ro ac: buj, seg; ro: ido, pvh polybia micans ducke, 1904 1 ro ac: buj, seg; ro: ido polybia minarum ducke, 1906 2 ro: ido polybia occidentalis (olivier, 1792) 1 ro ro ac: buj, seg; ro: ido polybia parvulina richards, 1970 ro polybia platycephala richards, 1951 ac ro ac, ro polybia procellosa dubitata ducke, 1910 ro polybia quadricincta de saussure, 1854 ac ro ac, ro polybia rejecta (fabricius, 1798) ac, ro ac ac ro ac, ro ac: buj, seg; ro: ido, pvh polybia rufitarsis ducke, 1904 ac ro ac: buj, seg; ro: ido polybia scrobalis richards, 1970 ac, ro ro ac, ro polybia sericea (olivier, 1792) ro ro ro ro: ido polybia simillima smith, 1862 2 ac ac: seg; ro: ido polybia singularis ducke, 1909 1 ro ro ro ac: buj, seg polybia spinifex richards, 1978 ro polybia striata (fabricius, 1787) ac, ro ac ro ac, ro polybia tinctipennis fox 1898 ac ro polybia velutina ducke, 1907 2 ac ac ac: seg; ro: ido protopolybia acutiscutis (cameron, 1906) ro ro protopolybia alvarengai richards, 1978 ro ro protopolybia amarella bequaert, 1944 ac ac protopolybia chartergoides (gribodo, 1892) ac, ro ac ro ac, ro ac: seg; ro: ido protopolybia emortualis (de saussure, 1855) ac protopolybia exigua (de saussure, 1854) ac ac protopolybia fuscatus (fox, 1898) 2 ro: ido protopolybia minutissima (spinola, 1851) ro ro protopolybia rotundata ducke, 1910 ro pseudopolybia compressa (de saussure, 1854) ro pseudopolybia difficilis (ducke, 1905) ro pseudopolybia vespiceps (de saussure, 1863) ac ro ro ac: buj; ro: pvh synoeca chalibea de saussure, 1852 1 ro ac: seg synoeca surinama (linnaeus, 1767) 1 ro ro ro ac: seg synoeca virginea (fabricius, 1804) ac ac ac ro ac, ro ac: seg; ro: ido, pvh table 1. species of social wasps from acre and rondônia states, listed by richards (1978), morato et al. (2008), gomes et al. (2018), gomes et al. (2020), web catalog from natural history laboratory of the ibaraki university (iunh 2020), and in the present work. 1 new distribution record for acre; 2 new distribution records for rondônia; * distribution records only in the descriptions of the species. ac=acre; ro=rondônia; buj=bujari; seg=senador guiomard; ido=itapuã do oeste; pvh=porto velho. (continuation) taxon richards (1978) morato et al. (2008) gomes et al. (2018) gomes et al. 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(1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of new york entomological society, 103: 129-137. doi: 10.13102/sociobiology.v63i1.904sociobiology 63(1): 724-727 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 opportunistic predation of a colony of polybia platycephala (richards) (hymenoptera: vespidae) by labidus praedator (smith) (hymenoptera: formicidae) the main function of social wasp nests is to provide a microenvironment conducive to the development of offspring (starr, 1991), providing protection from vertebrate and invertebrate predators (o’donnell & jeanne, 2002). swarming wasps have the habit of building an envelope to cover the combs, thus limit the entrance to the nest to one or a few holes. exceptions are the genera apoica (lepeletier, 1836) and agelaia (lepeletier, 1836) that do not present this habit (wenzel & carpenter, 1994). nests of polybia platycephala (richards, 1978) are fixed directly to the substrate without the presence of apetiole and are made up of several overlapping combs covered by an envelope over the single access hole into the colony (richards, 1978; rocha, 2011). p. platycephala is a social wasp that begins its nests by swarming. it is distributed in suriname, peru, south and north of brazil, with endemism in the southeast of the latter country (hermes & kohler, 2006; richards, 1978). army ants are one of the main insect predators (monteiro et al., 2008; powell & baker, 2008). the species labidus praedator (smith, 1858), popularly known as the army ant, abstract social wasps have developed several defense mechanisms, especially against ants. predator attacks are the main threat to their nests. the strategy adopted by the wasps, when attacked by ants, is to abandon the nest, thus preserving the adult population for future nesting. the present study reports in detail the predation of a colony of polybia platycephala by labidus praedator. sociobiology an international journal on social insects tt maciel, bc barbosa, f prezoto article history edited by gilberto m. m. santos, uefs, brazil received 28 august 2015 initial acceptance 01 march 2016 final acceptance 08 march 2016 publication date 29 april 2016 keywords army ants, ecitoninae, foraging, social wasp. corresponding author tatiane tagliatti maciel universidade federal de juiz de fora laboratório de ecologia comportamental e bioacústica labec, campus universitário bairro martelos, cep: 36036-900 juiz de fora, minas gerais, brazil e-mail: tatitagliatti@hotmail.com is a generalist predator, that lives in colonies consisting of millions of individuals and plays an import ant role instructuring invertebrate communities, covering large areas due to its nomadic habit. although rettenmeyer (1963) states that l. praedator shows strong preference for plant matter, these ants are usually observed carrying a mixture of ants and other arthropods, including spiders, scorpions, cockroaches and isopods, demonstrating diet plasticity (monteiro et al., 2008; powell & baker, 2008). although basic aspects of nomadic behavior and group predation of army ants are well known, few studies have been carried out about their specific foraging behavior and the impact of their attacks. the objective of this study was to report in detail the predation of l. praedator on a colony of p. platycephala and to describe the behaviors presented by both species. the observations were made in december 2012, the rainy season being a favorable period for the occurrence of a large number ofspecies of insects, especially termite swarms, in the botanical gardens of the federal university of juiz short note universidade federal de juiz de fora, minas gerais, brazil sociobiology 63(1): 724-727 (march, 2016) 725 de fora (21°43’28”s 43°16’47”w 800m asl). the site is located in a fragment of seasonal semideciduous montane forest (ibge,2012), located in the urban area of juiz de fora, which features warm subtropical climate with a dry winter and rainy summer (cwa), according to the classification of köppen (sá-júnior et al., 2012). the area, which covers 84 acres in length was classified by santiago et al. (2014) as a complex of expressive richness, diversity and floristic heterogeneity of woody vegetation with endangered species and a predominance of pioneer plants, in addition to the considerable presence of exotic species, maciel & barbosa (2015) suggest this area as novel ecosystem. the post-emergent colony (jeanne, 1972) of p. platycephala was conical in shape, with approximate dimensions of 15cm x 9cm, and was located on the a baxial part of a leaf of monstera deliciosa liebm. (araceae) at about 130cm above the ground (fig 1a). when they detected the presence of the ants, adult wasps that were inside the nest assumed a defensive posture characterized by semi-open wing sand contraction of the abdomen. as the number of ants increased, wasps left the nest and remained flying nearby, unable to defend themselves. it was observed that adult wasps were only attacked when returning from foraging and, as a typical behavior of foragers, entered the colony directly without displaying any defensive behavior, being immediately attacked by the ants present in the nest. ants exhibit two behaviors: (i) defensive posture of the soldiers, which remained standing with their jaws open and pointing upwards and (ii) foraging and transport by workers of eggs, larvae, pupae and stored resources, such as termite wings, described by prezoto et al. (2005) as a food source, regularly stored in p. platycephala colonies. there was no division of foraging items and these were transported whole (figs 1b and c). the action of the ants lasted about17min and during the attack, wasps gathered on other leaves (fig 1d), with no re-occupation of the nest. the wasps then aggregated to form a new colony. the foraging time shown by the ants corroborates the results of rettenmeyer (1963), which affirm that l. praedator attacks usually last between a few minutes to about an hour, before the ants gather underground, sometimes re-emerging from the soil shortly after, in a nearby area. it is important to note that prey of labidus are mostly species that occur in the soil, with some individuals being able to forage at a height ofup to 130cm (monteiro et al., 2008). for this reason, the reports about army ant attacks on social wasps are opportunistic and occasional, as in the work of monteiro et al. (2008) who, in addition to foraging time, also recorded a social wasp trying to seize a caterpillar that was being transported by ants and ended up being preyed upon by them. o’donnell and jeanne (1990), in costa rica, reported an attack on a colony of agelaia yepocapa (richards, 1978) located in the hollow of a tree trunk, facilitating the chance meeting by the ants. in this study, the p. platycephala nest was in a slope and the ants came from a higher level, thus facilitating the nest meeting and the opportunistic attack (fig 1 a). fig 1. a nesting place of polybia platycephala in the abaxial part of a monstera deliciosa leaf in a gully. b and c labidus praedator transporting eggs, larvae and pupae. d aggregation of wasps during the attack of the colony by the ants. tt maciel, bc barbosa, f prezoto – predation of a colony of polybia platycephala by army ants726 attacks by predators are a major threat to the integrity of social wasp nests (jeanne,1975; judd, 1998). those wasps have developed various defense mechanisms, especially against ants, such as the construction of a protective envelope that surrounds the entire surface of the nest, reducing access to the interior to a single hole, as in the case of p. platycephala (jeanne, 1975; wenzel& carpenter, 1994). other adaptations include the fixing of the substrate to the nest by a single petiole, hindering the entry of non-flying insects; secretion of an ant repellent by the gland in the fifth gastral sternite by species of independent foundation; vibrating wings, pumping abdomen, opening and closing of the jaw, and stinging (jeanne, 1995; togni,2008). the capability of wasps to detect army ants by sight and smell, as suggested by chadab (1979) as a result of coevolution, is quite advantageous because, when the ants find a colony of wasps, they are unable to defend the attack, besides which, the speed of escape is crucial since the ants attack suddenly and in large numbers (chadab & rettenmeyer, 1975). thus, a strategy adopted by wasps of the tribe epiponini, when attacked by army ants, is the immediate abandonment of the nest by the adult population (naumann, 1975; jeanne, 1979), confirmed by observations in this study. the success of this strategy is due to the fact that the queens of epiponini are quite numerous and poorly differentiated, allowing them to easily fly away and thus making possible the re-establishment of the colony in a new location (mateus, 2005). however, experiments conducted by mateus (2005) showed that there is a high energy expenditure during an abandonment which involves the search for a new nest site and chemical marking of the route. yet it should be noted that due to difference in size and slender body shape, the wasps are unable to sting ants, especially smaller ones like l. praedator. thus, the present report contributes to the understanding of the defense strategies presented by social wasps against ants, as well as opportunistic predatory behavior of the army ant. acknowledgements the authors thank michelle tagliatti maciel the contribution made by the making of the illustration of the location of polybia platycephala colony. references chadab, r. 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(1991). the nest as the locus of social life in: ross, k.g. & matthews, r.c. (eds) the social biology of wasps, cornell university press, pp 520-539 togni, o.c. & giannotti, e. (2008). nest defense behavior against ant attacks in post emergent colonies of wasp mischocyttarus cerberus (hymenoptera, vespidae). acta ethologica, 11(2):43-54. doi: 10.1007/s10211-008-0040-7 wenzel, j.w. & carpenter, j.m. (1994). comparing methods: adaptive traits and tests of adaptation in: p. eggleton & r.i. vane-wright (eds). phylogenetics and ecology, london: academic press, pp 79101 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7691sociobiology 69(2): e7691 (june, 2022) social wasps are insects that adopt different substrates for nesting (da cruz et al., 2006; alvarenga et al., 2010; souza et al., 2014), including native vegetal species with varied habits such as herbaceous and arboreal (francisco et al., 2018), exotic as eucalyptus (de souza et al., 2012) and coffee (milani et al., 2020), besides human buildings (oliveira et al., 2017). different biotic and abiotic factors influence the social wasps nesting, such as the ecosystem conservation degree (souza et al., 2010), fragmentation (bueno et al., 2019), preying (henrique & palma, 1998), food availability (santos et al., 2007) and the necessity for camouflage (souza et al., 2020a; milani et al., 2021). studies developed aiming to elucidate social wasps nesting has a high ecological relevance, above all concerning their relationship with the food webs, once social wasps prey on many agricultural pests (prezoto et al., 2018), such abstract social wasps use different substrates for nesting, such as plants, rocks, and human buildings, and may adopt different strategies to protect their colony and brood. here, we report the nesting behavior of polistes versicolor in the deciduous forest, northern minas gerais state, brazil. the occurrences were recorded during fieldwork from february to december 2021. we found 30 colonies in eight plant species. the statistics demonstrate a preference of this social wasp for the plant cereus jamacaru cactaceae, which is used for nestings over a large area. also, we report the first known record of p. versicolor colonies on the acacia plumosa, commiphora leptophloeos, quiabentia zehntneri, and ziziphus joazeiro, all of them with thorns. from the preference of p. versicolor for plants with thorns, we conclude this study to reinforce the role of these vegetal structures in protecting the colonies. sociobiology an international journal on social insects paola a. moura¹, gabriel c. jacques ², gabriel s. teofilo-guedes³, marcos m. souza4 article history edited by evandro nascimento silva, uefs, brazil received 16 december 2021 initial acceptance 19 march 2022 final acceptance 11 june 2022 publication date 24 june 2022 keywords polistinae; nesting; colony protection; dry forest. corresponding author gabriel teofilo-guedes universidade estadual de campinas cidade universitária zeferino vaz barão geraldo, campinas-sp, brasil. e-mail: gabrielteofiloguedes@gmail.com as polistes versicolor (olivier, 1791). this species mainly prays on caterpillars (elisei et al., 2010; prezoto et al., 2006) such as hedylepta indicata (fabricius, 1775), elasmopalpus lignosellus (zeller, 1848) (pyralidae), spodoptera frugiperda (je smith, 1797), heliothis virescens (fabricius, 1977), pseudoplusia includens (walker, 1857) (noctuidae), chlosyne lacinia saundersii (doubleday & hewitson, 1849) (nymphalidae), heraclides anchysiades capys (hübner, 1809) (papilionidae) e automeris sp. (saturniidae) (camposfarinha & pinto, 1996; marques, 1996; marques et al., 2005; rodríguez et al., 2012). p. versicolor presents a wide geographic distribution in brazil (richards, 1978), occurring in different biomes, including cerrado and atlantic forest (souza et al., 2020b, c), which act as a synanthropic species (oliveira et al., 2017), live near agricultural environments (jacques et al., 2015). nonetheless, there is no information on this species nesting 1 instituto de educação, ciência e tecnologia do sul de minas gerais – campus inconfidentes, inconfidentes, brazil 2 instituto de educação, ciência e tecnologia de minas gerais – campus bambuí, bambuí, brazil 3 universidade estadual de campinas, campinas, brazil 4 instituto federal de educação, ciência e tecnologia do sul de minas gerais inconfidentes, campus inconfidentes, inconfidentes, brazil short note polistes versicolor (olivier, 1791) nesting in a deciduous forest, northern minas gerais state, brazil (vespidae, polistinae) id mailto:gabrielteofiloguedes@gmail.com https://orcid.org/0000-0002-9619-6065 paola a. moura et al. – polistes versicolor nesting in a deciduous forest2 in the deciduous forest (brunismann et al., 2016; francisco et al., 2018). this way, here we report the nesting behavior of p. versicolor in the dry forest state park, aiming to interpret their apparent preference for specific species. this study was carried out in the dry forest state park (14º97’02’’s, 43º97’02’’w and 14º53’08’’s, 44º00’05’’w), northern minas gerais, which houses the biggest remanent of dry forest in the world, in the domain of the atlantic forest (oliveira et al., 2006) (figures 1a to 1d), characterized by the fall of leaves of more than 50% of the vegetation in the dry season (belém & carvalho, 2013). the fieldwork was developed from february to december 2021, with 20 sampling days from 9 am to 5 pm, amounting to 160 hours with a field effort of four researchers. identifying the plants was only possible for those fertile species being executed in the field, with the help of identification guides and the dry forest state park workers. to verify significant differences in p. versicolor nesting preferences, we made the kruskalwallis (kw) h test and, pairwise among the plants, the mann-whitney u test from the past 4.03 software, according to hammer et al. (2005). for identifying p. versicolor, we adopted dichotomous keys (richards, 1978; carpenter & marques, 2001) and made comparisons to the social wasps collection (cbvs) of minas gerais federal institute (ifsuldeminas) campus inconfidentes, where the specimens were deposited. sisbio and ief provided the needed licenses for this work (icmbio: 76140-1 and ief: 038/2020). we recorded 30 colonies of p. versicolor on eight different plant species (table 1). the kruskal-wallis test demonstrated that there was a nesting preference of the social wasp (p = 0,0009), which was specified by the mann-whitney u test, revealing a preference for cereus jamacaru de candolle (figure 2a) (p< 0,005), on which we found 21 colonies. the cactaceae c. jamacaru presents rectilinear branches of greenish color with the presence of yellowish and radial thorns, which may achieve 9 to 30 cm long. this species does not present leaves; its flowers are white, laterally projected, and subapical. its fruit is edible and has an elliptical shape (leal sales et al., 2014). due to its long thorns, this cactus may offer protection for the social wasp colony, such as p. versicolor. also, in caatinga environments, these social insects prefer to nest in trees or cactus, both of them with the presence of thorns (santos et al., 2007). such a fact is true for different species of the polistes genus (santos & gobbi, 1998; santos et al., 2007), and it is probably related to the absence of a protective casing, so becoming more susceptible to predation (jeanne, 1975), including by birds (almeida, 2015; van fig 1. study area. a. open area of rock outcrop. b. forest environment with a temporary dry stream channel. c. forest environment without a stream channel. d. open area of a marginal lake margin. sociobiology 69(2): e7691 (june, 2022) 3 bergen, 2019) and mammals (ying et al., 2010). therefore, thorns could constitute protection, supporting previous studies (richards, 1978; detoni et al., 2021). additionally, cactus may serve as a food source, as demonstrated by santos et al. (2007), whose work relates p. versicolor collecting food in c. jamacaru, pilosocereus catingicola (gurke), harrisia adscendens (gurke), pilosocereus gounellei (f.a.c. weber) and melocactus bahiensis (britton and rose). also, we recorded the first occurrence of a p. versicolor colony occurrence on acacia plumosa lowe (figure 2c), quiabentia zehntneri (britton and rose), and ziziphus joazeiro martius. these species are characterized by presenting thorns (zappi & taylor, 2020), which may favor the protection of p. versicolor colonies, as discussed before. still, we recorded for the first time p. versicolor nesting on commiphora leptophloeos (mart.), the imburana. this one is an arboreal, heliophila, and deciduous species constituted by tortuous branches. also, it presents a smooth rhytidome that detaches in thin orangish layers when senile, exposing its green stem (pareyn et al., 2018; barrus, 2020). vegetal species family number of colonies acacia plumosa fabaceae 1 cereus jamacaru cactaceae 21 commiphora leptophloeos burseraceae 2 quiabentia zehntneri cactaceae 1 ziziphus joazeiro rhamnaceae 1 sp. 01 asteraceae 2 sp. 02 1 sp. 03 1 table 1. number of colonies of polistes versicolor found in each plant species in the dry forest state park. fig 2. colonies of polistes versicolor in (a) cereus jamacaru, (b) commiphora leptophloeos and (c) acacia plumosa. therefore, due to the rhytidome projections, we interpret the occurrence of p. versicolor nesting on imburana due to the possibility of providing nest camouflage (figure 2b). such a possibility of camouflage has been raised before by souza et al. (2020). they demonstrated the colors of the vegetal structures, such as the tree bark, flowers, and leaves, provide camouflage for mischocyttarus anthracinus richards, 1945, parachartergus smithii (de saussure, 1854) and parachartergus wagneri buysson, 1904. from the field data and the statistics, we conclude that p. versicolor prefers to nest in c. jamaracu in deciduous forests, which reinforces the hypothesis that plants with thorns provide a favorable environment for the protection of social wasps colonies, which was already reported for the polistes genus. paola a. moura et al. – polistes versicolor nesting in a deciduous forest4 author’s contribution pam: investigation; writing-original draft gcj: conceptualization; formal analysis; resources; writing-review & editing gtgs: investigation; writing-original draft mms: supervision; methodology; resources; writingreview & editing acknowledgments we thank the sisbio and ief for providing the needed licenses for this work (icmbio: 76140-1 and ief: 038/2020), besides the field team of ief workers, the logistics, and infrastructure during the fieldwork. also, we thank minas gerais federal institute (ifsuldeminas, campus inconfidentes, and ifmg, campus bambuí) for ensuring the structure for the field trip. references almeida, s.m. & anjos-silva, e.j.d. 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(2020). cactaceae in flora do brasil 2020. jardim botânico do rio de janeiro. https://doi.org/10.34019/2596-3325.2020.v21.29157 https://doi.org/10.1007/s13744-021-00910-1 https://doi.org/10.1590/s0101-81752007000400023 https://doi.org/10.12741/ebrasilis.v13.e929 http://doi.org/10.13102/sociobiology.v67i2.4958 https://dx.doi.org/10.2139/ssrn.3522364 doi: 10.13102/sociobiology.v63i1.739sociobiology 63(1): 688-692 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 apitoxin harvest affects population development but not the hygienic behavior of african-derived honey bees introduction apitoxin is a product that bees can offer to humans, the word originating from the latin apis (bee) and toxikon (venom.) this substance is produced by the acid glands of worker bees, and characterized as being clear, colorless, and highly soluble in water (cruz-landim & abdalla, 2002). apitoxin toxicity is attributed to three proteic components: enzymes (phospholipase a2 and hyaluronidase), large peptides (melittin, apamin, and mast cell degranulating [mcd] peptide) and small molecules (peptides and biogenic amines), all substances that show pharmacological and allergenic activities. the allergenic factors are antigenic proteins such as phospholipases, hyaluronidases, lipases, and phosphatases, that are injected during the sting, and which initiate immune responses in hypersensitive individuals (cardoso et al., 2009). while the apitoxin can cause allergic and hypersensitivity reactions, small doses and fractions of its purified abstract the biological properties of apitoxin have prompted its production for use in human and animal health applications. however, the apitoxin harvest triggers a defense reaction in honeybee colonies, which includes the release of alarm pheromones (isopentyl acetate and 2-heptanone), which cause stress and could cause behavioral changes that influence the routine activities of the colony. considering the lack of data in the literature describing the effects of the prolonged harvesting of apitoxin, the present study conducted over a period of one year, aimed to investigate whether the apitoxin harvest influences population development and hygienic behavior of african-derived apis mellifera (l.). we observed that apitoxin harvest affected the uncapped brood area of the colonies during the months of april, may, and june, and affected the capped brood area in july. the hygienic behavior of the colonies was not affected. furthermore, we observed that during the study year, there was loss by abandonment of nine of the colonies subjected to apitoxin harvesting. we conclude that under the conditions of this study, the apitoxin harvest can negatively influence the development of the colony population during certain times of the year, without affecting the hygienic behavior of the colonies. sociobiology an international journal on social insects p onari, r zaluski, ts bovi, ro orsi article history edited by evandro nascimento silva, uefs, brazil received 10 december 2014 initial acceptance 24 february 2016 final acceptance 25 february 2016 publication date 29 april 2016 keywords beekeeping, venom harvest, management, brood areas. corresponding author ricardo de oliveira orsi faculdade de medicina veterinária e zootecnia, fmvz campus botucatu estrada alcídes soares, s/nº cep: 18611-661, botucatu-sp, brazil e-mail: orsi@fmvz.unesp.br components have been used in the treatment of diseases such as arthritis, arthrosis, osteoarthritis, bursitis, tendonitis, multiple sclerosis, lupus, lower back pain, and sciatica (ali, 2012). apitoxin components have also been studied during the development of some cancer treatments (orsolic, 2012) and for use against the human immunodeficiency virus (hiv) (hood et al., 2013). thus, the biological properties of apitoxin have stimulated its commercial production (leite & rocha, 2005). for the utilization of apitoxin without the direct application of a sting, electric collectors are placed at the hive entrance, allowing the harvest of crude apitoxin without causing bee mortality (benton et al., 1963; leite & rocha, 2005). the bee venom harvest triggers a massive defense reaction in the colony, because when bees bump against the electric collector, they release a quantity of apitoxin and alarm pheromones (isopentyl acetate and 2-heptanone), which prompts other bees to do likewise. although electric collectors research article bees universidade estadual paulista (unesp), botucatu, são paulo, brazil sociobiology 63(1): 688-692 (march, 2016) 689 of apitoxin do not promote the bees’ death, it is not known how much the prolonged harvest of apitoxin can influence colony activities. the assumption is that the prolonged harvest of apitoxin can promote acute and chronic stress, causing behavioral changes that interfere with the routine activities of the colonies. this can cause a decrease in the collection of resources by the colony, harming the development of broods and negatively affecting colony maintenance. it may also have a detrimental effect on the hygienic behavior of bees. considering that the harvesting of apitoxin is a growing part of beekeeping, and the lack of data describing its effects on colonies, the present study aims to investigate if harvesting apitoxin influences the population development and hygienic behavior of african-derived apis mellifera (l.) over a period of one year. materials and methods the experiment was conducted at the beekeeping production area of lageado experimental farm, faculty of veterinary medicine and animal science, unesp, botucatu, são paulo, brazil, 22°50′30.16′′s; 48°25′41.90′′w, with a humid subtropical (cfa) climate and an average elevation of 623 m. we used ten colonies of african-derived a. mellifera housed in wooden langstroth hives, each containing six brood frames, three frames of food and one empty frame. during the trial period, hives were checked on a weekly basis, and when necessary each hive received sugar syrup (50% water; 50% sugar) using individual boardman feeders. the colonies were free of diseases or parasites and each had a naturally mated queen, raised naturally by workers three months before the start of the experiment. before the start of the experiment, we performed defensive behavior tests on all of the colonies, according to the methodology described by stort (1972) and brandeburgo and gonçalves (1990), in order to assess whether the colonies used showed uniform defensive behavior. the time to first sting and the number of stings left in a suede ball were recorded. the tests were performed in triplicate. subsequently, the colonies were randomly divided into two groups: control, without apitoxin harvest; and treatment, with a biweekly harvest of apitoxin. the apitoxin harvest was always conducted in the morning, beginning at 09:00 am and continuing for an hour, by using an electric collector. in experiments carried out by our group, it was found that this timing of the harvests showed the best rates of toxin production, and exerted less stress on the colonies (as estimated by the expression of genes related to stress) (data in press). at the end of each apitoxin harvest, the collectors were removed and sent to the laboratory. the glasses containing the poison were kept at room temperature and shielded from exposure to light, to evaporation of the volatile phase. then, the apitoxin was scraped out with a stainless steel spatula, weighed, stored in an eppendorf tube, and kept in a freezer at -10 °c. after each harvest, the glasses were washed in running water and sanitized with 70° gl alcohol. the population development of colonies was evaluated according to methodology adapted from al-tikrity et al. (1971). for this purpose, two frames containing posture and/ or uncapped brood areas were removed from the colonies, labeled, and placed in a holder, with a grid comprised of squares of 2 cm × 2 cm. the number of squares with posture and/or uncapped brood areas was counted, and the frames were then returned to the hives. fifteen days later, the labeled frames had their brood area reanalyzed (to verify the quantity of capped brood present). this process was repeated every two weeks throughout the trial period. the hygienic behavior of all colonies (control and treatment) was analyzed by the method of drilling of capped brood, in accordance with the method described by garcia et al. (2013). in treatment colonies, after apitoxin harvest, a frame with capped brood areas of each colony was selected, and two different areas containing approximately 100 cells (area a and b) were marked. one area of capped brood (area a) was drilled with an entomological pin, and the frame was returned to the hive and re-evaluated after 24 h. for analysis, the total number of capped brood subjected to drilling was counted (area a), and the total number of empty cells was subtracted from the number of cells drilled, to obtaining the number of pupae removed by hygienic workers. in area b, we calculated the factor of correction z described by moretto (as cited in abdel-rahman, 2014). z is the natural taxa of removed pupae in a corresponding area, which is subtracted from the value of removed pupae in the area a. the result was considered if the value of the factor correction (area b) was equal to or less than 10%. the z value was calculated using the formula: z = (y × 100)/a, where a = the number of pupae in the area a and y = the number of empty cells where the pupae were removed naturally. the value of y is given by y = c ˗ b; where c = number of empty cells from control after the frame has been subjected to a hygienic behavior test and b = the number of empty cells in the control, before the frame has been subjected to a hygienic behavior test. the hygienic behavior rate (hbr) of each colony was calculated via the formula hbr = (cv1 − cv × 100 − z)/co; where cv1 = the number of empty cells 24 h after drilling, cv = the number of empty cells before drilling, co = the number of cells capped before drilling, and z = the correction factor obtained in the control area. data analysis was performed by anova, followed by the unpaired student’s t-test for comparing means, and considered significant when p < 0.05 (zar, 2010). results the time to first sting was 3.12 ± 2.10 s for the swarms of the control group, and 3.12 ± 3.04 s for the treatment group. the number of stings left in the suede ball in the control p onari et al. – effects of apitoxin production on a. mellifera colonies690 colonies was 32.87 ± 18.81 and 36.12 ± 20.15 in the treatment colonies. these results for the defensiveness parameters did not differ significantly between the control and treatment colonies (p > 0.05; student’s t-test). the population development of the colonies demonstrated significant reduction in uncapped brood areas during the table 1. means and standard deviations of the uncapped and capped brood areas (cm2) of african-derived apis mellifera (l.) colonies without (control) and with the harvest of apitoxin (treatment), between april 2013 to march 2014. months control treatment april 84.63 ± 13.86 79.95 ± 6.85 may 81.26 ± 14.74 71.95 ± 17.22 june 83.65 ± 7.13 80.76 ± 10.97 july 88.96 ± 11.77 87.82 ± 11.56 august 79.23 ± 9.36 75.54 ± 11.47 september 91.57 ± 5.12 84.34 ± 8.56 october 90.25 ± 6.37 74.35 ± 14.86 november 82.00 ± 12.40 92.64 ± 6.76 december 72.10 ± 30.20 91.13 ± 1.55 january 80.22 ± 3.86 69.90 ± 22.14 february 84.63 ± 4.86 73.64 ± 11.44 march 86.12 ± 6.14 71.33 ± 16.84 no significant differences were observed in hygienic behavior between treatment and control colonies. student’s t-test (p > 0.05). n = 5 colonies. months uncapped brood capped brood control treatment control treatment april 60.93 ± 12.45 34.22 ± 9.311 165.91 ± 30.72 147.54 ± 17.93 may 122.75 ± 19.73 78.22 ± 18.631 131.62 ± 44.24 87.61 ± 15.05 june 70.94 ± 18.45 53.36 ± 12.721 115.12 ± 65.14 32.73 ± 14.04 july 39.84 ± 19.63 30.82 ± 8.45 108.70 ± 20.56 23.74 ± 19.301 august 27.22 ± 10.15 20.33 ± 4.06 25.74 ± 8.43 20.32 ± 4.07 september 22.35 ± 9.54 14.13 ± 5.05 120.83 ± 71.55 107.34 ± 56.55 october 52.35 ± 23.54 25.93 ± 15.62 97.52 ± 71.85 158.26 ± 34.56 november 36.23 ± 9.45 32.76 ± 15.63 119.13 ± 17.21 173.42 ± 41.05 december 63.44 ± 17.32 42.05 ± 13.32 101.94 ± 65.26 126.56 ± 52.24 january 40.24 ± 21.42 77.94 ± 40.23 128.05 ± 58.07 221.40 ± 40.22 february 43.34 ± 19.25 65.15 ± 12.82 148.51 ± 62.35 151.71 ± 37.43 march 50.86 ± 7.83 75.91 ± 32.96 134.05 ± 42.02 146.46 ± 15.36 1significantly decreased compared with control (p < 0.05 student’s t-test). n = 5 colonies. months of april, may, and june in colonies where apitoxin was harvested. additionally, the areas of capped brood were reduced during july (table 1). the hygienic behavior test did not show a significant difference between colonies with or without the harvest of apitoxin (p > 0.05; student’s t-test) (table 2). table 2. means and standard deviations of the hygienic behavior test (%) of african-derived apis mellifera (l.) colonies without (control) and with the harvest of apitoxin (treatment), between april 2013 to march 2014. sociobiology 63(1): 688-692 (march, 2016) 691 discussion to ensure a similar degree of defensiveness in colonies used in our study, we evaluated the defensive behavior of all colonies before the start of the experiment. we noted that the colonies showed similar patterns; however, this differed from reports in the literature (nascimento et al., 2008). these results could be ascribed to environmental factors (temperature, humidity, and atmospheric pressure) and/or genetic factors (garcia et al., 2013). therefore, it is arguable that the similarities in the defensive behaviors seen in the colonies used here allow this study to avoid possible genetic effects that could influence our results. it was verified that the harvest of apitoxin decreases the area of uncapped brood during the months of april to june, and decreases the area of capped brood in the month of july, when compared with the controls. this decrease in brood area could be associated with the release of alarm pheromones that may change honeybee behavior, mainly by interfering with the flux of information transmitted by other pheromones in the colonies (kastberger et al., 2008). thus, the collecting of apitoxin could interfere with brood care, the queen’s oviposition of worker eggs, and with resources collection by worker bees in some periods. one important observation made by this study is the difficulty of maintaining colonies that are submitted to the harvest of apitoxin. between the months of august and january, nine colonies from which apitoxin was collected abandoned their hives, and had to be replaced with new colonies showing similar defensive behaviors. this abandonment behavior is common in african-derived honey bees and is characterized by all in the colony bees abandoning the hive (freitas et al., 2007). it is usually observed during stress conditions, during periods of climatic change or when resources are scarce, which are factors that can threaten the survival of the colony. it is suggested that the high rate of abandonment observed in our study could be caused by the collection of apitoxin, as the abandonment behavior was not observed in the control hives. we noted that the harvest of apitoxin did not affect the hygienic behavior of the colonies, suggesting that the release of alarm pheromones does not interfere with this activity. the removal of sick or dead bees from the hive is associated with the social immunity of bees, as it prevents the spread of pathogens and increases the bees’ resistance to diseases (wilson-rich et al., 2009). african-derived bees exhibit more intense hygienic behavior than european bees, which helps fight parasites and pathogens that cause disease in bees (aumeier et al., 2000; guerra, 2000). on the basis of our results, future studies must consider the effects of apitoxin harvest at different time intervals, in order to develop further sustainable methods of apitoxin harvest that may be carried out throughout the year without affecting the development of hives and to avoid abandonment of colonies. thus, we conclude that apitoxin harvest negatively affects population development during certain times of the year, although it does not affect the hygienic behavior of colonies. acknowledgments to fapesp (fundação de amparo à pesquisa do estado de são paulo) for the scholarship (process number 12/17112-3). references abdel-rahman, m.f. 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(2008). agressividade de abelhas africanizadas (apis mellifera) associada à hora do dia e a umidade em mossoró-rn. acta veterinaria brasilica, 2: 80-84. orsolic, n. (2012). bee venom in cancer therapy. cancer metastasis review, 31: 173-194. doi: 10.1007/s10555-011-9339-3. stort, a.c. (1972). estudo genético da agressividade da apis mellifera. ciência e cultura, 24: 208. wilson-rich, n., spivak, m., fefferman, n.h., starks, p.t. (2009). genetic, individual, and group facilitation of disease resistance in insects societies. annual review of entomology, 54: 405−423. doi: 10.1146/annurev.ento.53.103106.093301. zar, j.h. (2010). biostatistical analysis. new jersey: pearson prentice hall, 944 p. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.8151sociobiology 69(3): e8151 (september, 2022) introduction hymenoptera is one of the most diverse insect orders with more than 150,000 described species (aguiar et al., 2013). members of this group are known for their important ecological role as pollinators and regulators of arthropods populations through parasitism and predation (michez et al., 2019). the hymenopteran pollinators belong mainly to the superfamily anthophila which has a crucial role in the reproduction of most flowering plants. for example, 70% of cultivated plants worldwide directly depend on the anthophila (apoidea) (klein et al., 2007). anthophila is represented worldwide by more than 20,000 described species belonging to seven families (michener, 2007; ascher & pickering, 2021). despite its diversity and its significance in the biodiversity and the abstract based on a review of the available literature, the state of the art and a checklist of the fauna of the apidae family of tunisia is presented. the first list of the species of the family is given. 184 species and subspecies belonging to 19 genera, 12 tribes and three subfamilies were listed. distribution of recorded taxa from tunisia and from the world is provided. apinae is the subfamily with the highest species richness with 89 species. nomada has the highest number of species represented by 62 taxa. the tunisian east center is the least speciesdiversified regions with only 16 species and subspecies reported so far. five species are endemic to tunisian fauna. eight nomada and one anthophora species were collected from tunisia, but their identity should be re-confirmed. the presence of thyreomelecta sibirica (radoszkowski, 1893) in tunisia is doubtful and a re-examination and confirmation are needed. sociobiology an international journal on social insects hassib ben khedher, mohamed braham, ikbal chaieb article history edited by felipe vivallo, museu nacional, brazil received 28 april 2022 initial acceptance 23 may 2022 final acceptance 15 august 2022 publication date 07 september 2022 keywords apidae, distribution, fauna-checklist, hymenoptera, tunisia. corresponding author hassib ben khedher research laboratory lr21agr03 regional research center for horticulture and organic agriculture (crrhab) chott meriem, sousse university, 4042 sousse-tunisia. e-mail: benkhedherhassib@gmail.com terrestrial ecosystems, studies on this group, particularly within apidae, are scarce in tunisia. most species records in tunisian ecosystem were outdated (schulthess, 1924; dusmet & alonso, 1928, 1932; daly, 1983; sonet & jacob-remacle, 1987). luckily, during the last years have been conducted some studies focusing on pollination of wild and cultivated plants (rjiba, 2014; chouchaine, 2015; ben abdelkader et al., 2020). some records of species belonging to apidae were cited by chouchaine (2015) which reported 10 genera of apidae, indicating the genus eucera with a high number of species. also, rjiba (2014) reported the presence of nomada, eucera and anthophora in sousse and bizerte provinces. the study conducted by ben abdelkader et al. (2020) in northern tunisia (goubellat, beja province) indicated the presence of two families of bees, apidae represented by apis mellifera linnaeus, 1758 and megachilidae represented by megachile sp. research laboratory lr21agr03, regional research center for horticulture and organic agriculture (crrhab) at chott meriem, sousse university, sousse, tunisia research article bees the state of the art of the tunisian apidae fauna (hymenoptera: anthophila) hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)2 these studies are still seldom and do not determine the complete identity of captured specimens, in addition to have been carried out on a regional scale, not covering the entire country. here in, we provide an up to date list based on the screening of different literature review on the occurrence of species of apidae recorded in tunisia. materials and methods available data existing in old and recent literature about apidae family in tunisia (fig 1) were examined and summarized in a list as follows: for each species or subspecies, its valid name followed by the reference to its original description and its distribution in tunisia and in the world were given. the classification and nomenclature follow michener (2007) with some updates, as the case of the genus tetraloniella ashmead, 1900 which was synonymized under tetralonia spinola, 1838 by michener (2007) and subsequently treated as a junior synonym of eucera by dorchin et al. (2018). general distribution follows mainly ascher and pickering (2021). species and subspecies within genera were ordered alphabetically. references of the distribution in tunisia in published data were provided chronologically from the oldest to the most recent. “no specific locality” was mentioned when no reference about the excat location have been provided. abbreviations used in the list are: m.r.: main road; s.r.: secondary road; n: north; s: south; e: east; se: southeast; sw: southwest; nw: northwest and km: kilometer. results in this study and based on published data a total of 184 species and subspecies belonging to 19 genera, 12 tribes and three subfamilies of apidae are recorded in tunisia. the most species-rich subfamilies were apinae (89 species; 10 genera; six tribes), nomadinae (81 species; seven genera; four tribes) and xylocopinae (14 species; two genera; two tribes). the most diverse genus is nomada with 62 species, followed by eucera and anthophora with 33 and 26 species, respectively (fig 2). fig 1. provinces of tunisia: 1. bizerte; 2. mannouba; 3. ariana 4. ben arous; 5. zaghouan; 6. tunis; 7. nabeul; 8. beja; 9. jendouba; 10. le kef; 11. siliana; 12. sousse; 13. monastir; 14. mahdia; 15. sfax; 16. kairouan; 17. sidi bouzid; 18. kasserine; 19. gabes; 20. medenine; 21. gafsa; 22. tozeur; 23. kebili; 24. tataouine. sociobiology 69(3): e8151 (september, 2022) 3 subfamily: apinae latreille, 1802 tribe: anthophorini dahlbom, 1835 genus: amegilla friese, 1897 amegilla (amegilla) ochroleuca (pérez, 1879) anthophora ochroleuca pérez, 1879. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: france, greece, italy, morocco, russia, tunisia, and ukraine (ascher & pickering, 2021). amegilla (amegilla) quadrifasciata (de villers, 1789) apis quadrifasciata de villers, 1789. distribution in tunisia: ben arous (hammam lif), gafsa, kairouan, kasserine (sbeitla), sfax (schulthess, 1924 as anthophora quadrifasciata); sousse (ascher & pickering, 2021). general distribution: albania, algeria, austria, bulgaria, myanmar, china, croatia, czech republic, egypt, france, georgia, germany, greece, hungary, india, iran, iraq, israel, italy, japan, kazakhstan, kyrgyzstan, libya, luxemburg, malta, mongolia, montenegro, morocco, pakistan, poland, portugal, romania, russia, serbia, slovakia, slovenia, south africa, spain, switzerland, syria, tajikistan, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). amegilla (micramegilla) andresi (friese, 1914) anthophora andresi friese, 1914. distribution in tunisia: gabes (schwarz & gusentleiner, 2001). general distribution: egypt, israel, libya, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). amegilla (micramegilla) fasciata (fabricius, 1775) apis fasciata fabricius, 1775. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: egypt, france, italy, libya, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). amegilla (zebramegilla) albigena (lepeletier, 1841) anthophora albigena lepeletier, 1841. distribution in tunisia: sfax (schulthess, 1924 as anthophora albigena); le kef (ascher & pickering, 2021). general distribution: albania, algeria, austria, bulgaria, croatia, czech republic, egypt, france, georgia, greece, hungary, iran, israel, italy, jordan, lebanon, libya, macedonia, malta, morocco, portugal, romania, russia, serbia, slovakia, slovenia, spain, switzerland, syria, tunisia, turkey, and ukraine (ascher & pickering, 2021). amegilla marqueti (pérez, 1895) anthophora marqueti pérez, 1895. distribution in tunisia: tozeur (schulthess, 1924 as anthophora marqueti). general distribution: north africa (ascher & pickering, 2021). genus: anthophora latreille, 1803 anthophora (anthophora) canescens brullé, 1832 anthophora canescens brullé, 1832. distribution in tunisia: kairouan (schulthess, 1924 as anthophora nigrocincta); ariana, tunis (ascher & pickering, 2021). general distribution: algeria, croatia, cyprus, egypt, greece, israel, italy, libya, malta, morocco, spain, and tunisia (ascher & pickering, 2021). anthophora (anthophora) crinipes smith, 1854 anthophora crinipes smith, 1854. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). fig 2. apidae species number per genus in tunisia. hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)4 general distribution: algeria, austria, bulgaria, czech republic, egypt, france, georgia, germany, greece, iran, iraq, israel, italy, hungary, kyrgyzstan, morocco, portugal, romania, russia, spain, slovakia, slovenia, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). anthophora (anthophora) fulvitarsis brullé, 1832 anthophora fulvitarsis brullé, 1832. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, china, france, georgia, germany, iran, italy, kazakhstan, kyrgyzstan, morocco, pakistan, portugal, russia, spain, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). anthophora (anthophora) plumipes (pallas, 1772) apis plumipes pallas, 1772. distribution in tunisia: zaghouan (thuburbo majus ruins, 2 km n al fahs) (ascher & pickering, 2021). general distribution: afghanistan, azerbaijan, belarus, china, cyprus, czek republic, denmark, england, finland, germany, greece, hungary, iran, italy, japan, kazakhstan, lithuania, morocco, poland, portugal, russia, spain, syria, tunisia, and turkey (ascher & pickering, 2021). anthophora (anthophora) senescens lepeletier, 1841 anthophora senescens lepeletier, 1841. distribution in tunisia: kairouan, kasserine (sbeitla), tunis (carthage) (schulthess, 1924); medenine (zarzis) (ascher & pickering, 2021). general distribution: algeria, china, france, israel, italy, morocco, spain, and tunisia (ascher & pickering, 2021). anthophora (caranthophora) pubescens (fabricius, 1781) anthophora pubescens fabricius, 1781. distribution in tunisia: gafsa, kasserine (sbeitla) (schulthess, 1924). general distribution: china, germany, israel, morocco, russia, slovakia, slovenia, spain, tunisia, and ukraine (ascher & pickering, 2021). anthophora (heliophila) bimaculata (panzer, 1798) apis bimaculata panzer, 1798. distribution in tunisia: sfax, kairouan (schulthess, 1924). general distribution: algeria, austria, bulgaria, china, croatia, czech republic, england, estonia, france, georgia, germany, hungary, iran, ireland, israel, italy, kazakhstan, kyrgyzstan, libya, lithuania, morocco, netherlands, poland, portugal, romania, russia, slovakia, spain, switzerland, tajikistan, tunisia, turkey, and turkmenistan (ascher & pickering, 2021). anthophora (heliophila) humilis (spinola, 1838) saropoda humilis spinola, 1838. distribution in tunisia: tozeur (schulthess, 1924 as anthophora albocinerea). general distribution: algeria, egypt (ascher & pickering, 2021). anthophora (lophanthophora) dispar lepeletier, 1841 anthophora dispar lepeletier, 1841. distribution in tunisia: tunis (carthage) (schulthess, 1924); medenine (zarzis), tunis (ascher & pickering, 2021). general distribution: algeria, egypt, france, germany, israel, italy, libya, malta, morocco, portugal, spain, syria, and tunisia (ascher & pickering, 2021). anthophora (lophanthophora) hispanica (fabricius, 1787) apis hispanica fabricius, 1787. distribution in tunisia: tunis (carthage) (schulthess, 1924). general distribution: egypt, israel, libya, morocco, portugal, spain, syria, tunisia, and turkey (ascher & pickering, 2021). anthophora (lophanthophora) mucida gribodo, 1873 anthophora mucida gribodo, 1873. distribution in tunisia: tunisia: no specific locality (maidl, 1922 as podalirius mucidus). general distribution: bulgaria, egypt, france, greece, italy, kazakhstan, morocco, portugal, romania, spain, switzerland, turkey, and ukraine (ascher & pickering, 2021). anthophora (paramegilla) astragali morawitz, 1879 anthophora astragali morawitz, 1879. distribution in tunisia: tozeur (nefta) (schulthess, 1924). general distribution: armenia, azerbaijan, georgia, and russia (ascher & pickering, 2021). anthophora (paramegilla) blanda pérez, 1895 anthophora blanda pérez, 1895. distribution in tunisia: kairouan, kasserine (sbeitla), tunis (carthage) (schulthess, 1924). general distribution: algeria and israel (ascher & pickering, 2021). anthophora (paramegilla) planca pérez, 1895 anthophora planca pérez, 1895. distribution in tunisia: sfax (schulthess, 1924). general distribution: algeria (ascher & pickering, 2021). anthophora (paramegilla) quadricolor (erichson, 1840) megilla quadricolor erichson, 1840. distribution in tunisia: tunis (carthage) (schulthess, 1924). general distribution: france, italy, morocco, portugal, and spain (ascher & pickering, 2021). anthophora (petalosternom) calcarata lepeletier, 1841 anthophora calcarata lepeletier, 1841. distribution in tunisia: ben arous (hammam lif), kairouan, kasserine (sbeitla), tunis (carthage) (schulthess, 1924). general distribution: algeria, france, italy, morocco, spain, and tunisia (ascher & pickering, 2021). sociobiology 69(3): e8151 (september, 2022) 5 anthophora (petalosternom) rivolleti pérez, 1895 anthophora rivolleti pérez, 1895. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, egypt, israel, and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) albosignata (friese, 1896) podalirius albosignatus friese, 1896b. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: egypt, greece, italy, libya, morocco, syria, and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) atriceps pérez, 1879 anthophora atriceps pérez, 1879. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: egypt, iran, israel, libya, morocco, portugal, and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) atroalba lepeletier, 1841 anthophora atro-alba lepeletier, 1841. distribution in tunisia: tunis (cockerell, 1920); tunis (centre, carthage), kairouan (schulthess, 1924). general distribution: algeria, croatia, france, greece, india, iran, italy, lebanon, libya, morocco, pakistan, portugal, spain, tunisia, turkey, and ukraine (ascher & pickering, 2021). anthophora (pyganthophora) caroli pérez, 1895 anthophora caroli pérez, 1895. distribution in tunisia: kasserine (sbeitla) (schulthess, 1924). general distribution: algeria, palestine, and tunisia. anthophora (pyganthophora) facialoides brooks, 1988 anthophora facialoides brooks, 1988. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: egypt and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) holoxantha pérez, 1895 anthophora holoxantha pérez, 1895. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) libyphaenica gribodo, 1893 anthophora libyphaenica gribodo, 1893. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, israel, libya, morocco, and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) scopipes spinola, 1838 anthophora scopipes spinola, 1838. distribution in tunisia: sfax (schulthess, 1924). general distribution: algeria, egypt, israel, morocco, and tunisia (ascher & pickering, 2021). anthophora (pyganthophora) ventrilabris lepeletier, 1841 anthophora ventrilabris lepeletier, 1841. distribution in tunisia: tunisia: no specific locality (schulthess, 1924; ascher & pickering, 2021). general distribution: algeria, greece, italy, morocco, spain, and tunisia (ascher & pickering, 2021). anthophora sp. distribution in tunisia: tozeur (schulthess, 1924). genus: habropoda smith, 1854 habropoda oraniensis (lepeletier, 1841) anthophora oraniensis lepeletier, 1841. distribution in tunisia: tunisia: no specific locality (schwarz & gusentleitner, 2001); medenine (zarzis) (ascher & pickering, 2021). general distribution: algeria, egypt, libya, and tunisia (ascher & pickering, 2021). tribe: ancylaini michener, 1944 genus: ancyla lepeletier, 1841 ancyla brevis dours, 1873 ancyla brevis dours, 1873. distribution in tunisia: tunis (friese, 1922 as ancyla punica). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). ancyla oraniensis lepeletier, 1841 ancyla oraniensis lepeletier, 1841. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, greece, morocco, and tunisia (ascher & pickering, 2021). tribe: apini latreille, 1802 genus: apis linnaeus, 1758 apis mellifera linnaeus, 1758 apis mellifera linnaeus, 1758. distribution in tunisia: everywhere. general distribution: worldwide (ascher & pickering, 2021). tribe: bombini latreille, 1802 genus: bombus latreille, 1802 bombus (bombus) terrestris (linnaeus, 1758) apis terrestris linnaeus, 1758. distribution in tunisia: tunisia: no specific locality (schulthess, 1924 as var. with scopa red on hind tibiae); jendouba (hotel les chines, 5 km south ain draham) (ascher & pickering, 2021). hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)6 general distribution: afghanistan, algeria, argentina, australia, belarus, brazil, canary islands, chile, china, georgia, iceland, iran, israel, japan, kazakhstan, kyrgyzstan, libya, mongolia, morocco, new zeeland, palestine, russia, south korea, syria, tajikistan, tunisia, turkey, united states, and uzbekistan, very common in europe (ascher & pickering, 2021). bombus (megabombus) ruderatus (fabricius, 1775) apis ruderata fabricius, 1775. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, argentina, austria, czech republic, belarus, belgium, bosnia, bulgaria, canary islands, chile, czech republic, england, france, germany, greece, hungary, iceland, italy, luxemburg, morocco, new zeeland, norway, poland, portugal, romania, russia, spain, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). bombus (thoracobombus) laesus morawitz, 1875 bombus laesus morawitz in fedtschenko, 1875. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, azerbaijan, belarus, bulgaria, china, czech republic, estonia, france, georgia, germany, greece, hungary, iran, kazakhstan, kyrgyzstan, lithuania, macedonia, morocco, poland, romania, russia, serbia, slovakia, spain, switzerland, tajikistan, tunisia, turkey, ukraine, and uzbekistan (ascher & pickering, 2021). bombus (thoracobombus) ruderarius (müller, 1776) apis ruderaria müller, 1776. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: algeria, azerbaijan, georgia, iran, kazakhstan, kyrgyzstan, russia, tunisia, and turkey, very common in europe (ascher & pickering, 2021). tribe: eucerini latreille, 1802 genus: eucera scopoli, 1770 eucera (eucera) alopex risch, 1999 eucera alopex risch, 1999. distribution in tunisia: medenine (30 km n medenine, zarzis, 20 km s zarzis, 30 km s zarzis), nabeul (hammamet, grombelia) (risch, 1999). general distribution: tunisia (ascher & pickering, 2021). eucera (eucera) dimidiata brullé, 1832 eucera dimidiata brullé, 1832. distribution in tunisia: kairouan (dusmet & alonso, 1928). general distribution: afghanistan, algeria, cyprus, egypt, greece, hungary, iran, iraq, israel, morocco, saudi arabia, tunisia, and turkey (ascher & pickering, 2021). eucera (eucera) ferruginea lepeletier, 1841 eucera ferruginea lepeletier, 1841. distribution in tunisia: jendouba (10 km e ain draham) (ascher & pickering, 2021). general distribution: afghanistan, algeria, morocco, tunisia (ascher & pickering, 2021). eucera (eucera) grisea fabricius, 1793 eucera grisea fabricius, 1793. distribution in tunisia: kairouan, kasserine (sbeitla), (schulthess, 1924; dusmet & alonso, 1928). general distribution: algeria, egypt, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). eucera (eucera) interrupta bäer, 1850 eucera interrupta bäer, 1850. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: albania, algeria, austria, azerbaijan, bulgaria, china, france, georgia, germany, greece, hungary, italy kazakhstan, macedonia, poland, romania, russia, slovenia, slovakia, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). eucera (eucera) nigrilabris lepeletier, 1841 eucera nigrilabris lepeletier, 1841. distribution in tunisia: tunis (carthage) (schulthess, 1924); kairouan (dusmet & alonso, 1928); gabes (10 km se matmata), kasserine (bouchebka) (ascher & pickering, 2021). general distribution: azerbaijan, algeria, france, germany, greece, iran, israel, kazakhstan, morocco, portugal, romania, russia, spain, tajikistan, tunisia, turkey, turkmenistan, and uzbekistan (ascher & pickering, 2021). eucera (eucera) numida lepeletier, 1841 eucera numida lepeletier, 1841. distribution in tunisia: kairouan (dusmet & alonso, 1928); jendouba (tabarka), tozeur (oasis tozeur) (ascher & pickering, 2021). general distribution: algeria, france, italy, malta, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). eucera (hetereucera) algira brullé, 1840 eucera algira brullé, 1840. distribution in tunisia: tozeur (oued melah between gafsa and tozeur) (schulthess, 1924); kairouan, tunis (dusmet & alonso, 1928). general distribution: algeria, greece, israel, italy, malta, morocco, and spain (ascher & pickering, 2021). eucera (hetereucera) atricornis fabricius, 1793 eucera atricornis fabricius, 1793. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: iran, morocco, tunisia and turkey (ascher & pickering, 2021). sociobiology 69(3): e8151 (september, 2022) 7 eucera (hetereucera) bequaerti alfken, 1914 eucera bequaerti alfken, 1914. distribution in tunisia: jendouba (tabarka, 15 km s jendouba), kasserine (20 km s thala), nabeul (hammamet, grombelia), tunis (risch, 2001). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). eucera (hetereucera) clypeata erichson, 1835 eucera clypeata erichson, 1835. distribution in tunisia: jendouba (4 km s ain draham, 10 km e ain draham) (risch, 2003). general distribution: afghanistan, albania, algeria, azerbaijan, bulgaria, czech republic, france, georgia, germany, greece, iran, iraq, italy, jordan, kazakhstan, kyrgyzstan, macedonia, moldova, morocco, pakistan, portugal, romania, russia, serbia, slovakia, spain, syria, tajikistan, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). eucera (hetereucera) elongatula vachal, 1907 eucera elongatula vachal, 1907. distribution in tunisia: sfax (ascher & pickering, 2021). general distribution: algeria, france, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). eucera (hetereucera) genovefae vachal, 1907 eucera genovefae vachal, 1907. distribution in tunisia: gafsa (15 km n metlaoui, 20 km n metlaoui, 20 km n gafsa, sidi boubaker), kasserine, medenine (30 km sw medenine), tataouine (55 km s tataouine, 56 km s tataouine, ksar hadada) (risch, 2001); kasserine (bouchebka), tozeur (8 km s tamerza) (ascher & pickering, 2021). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). eucera (hetereucera) notata lepeletier, 1841 eucera notata lepeletier, 1841. distribution in tunisia: kasserine (sbeitla), tunis (schulthess, 1924); tunis (ascher & pickering, 2021). general distribution: algeria, egypt, germany, italy, libya, morocco, portugal, spain, tunisia, and turkey (ascher & pickering, 2021). eucera (hetereucera) obliterata pérez, 1895 eucera obliterata pérez, 1895. distribution in tunisia: beja (15 km se teboursouk, 10 km s beja), jendouba, kairouan (20 km n kairouan, 17 km n kairouan), kasserine (bouchebka), kebili (douz), le kef (tajerouine, 30 km s le kef), siliana (makthar), sousse (msaken), zaghouan (mograne, 20 km nw zaghouan, 30 km s zaghouan) (risch, 2003). general distribution: algeria, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). eucera (hetereucera) oraniensis lepeletier, 1841 eucera oraniensis lepeletier, 1841. distribution in tunisia: kasserine (bouchebka), tataouine (5 km e djouama) (ascher & pickering, 2021). general distribution: algeria, malta, morocco, spain, and tunisia (ascher & pickering, 2021). eucera (hetereucera) polita pérez, 1895 eucera polita pérez, 1895. distribution in tunisia: gafsa (40 km nw gafsa), kasserine (10 km nw thelepte) (ascher & pickering, 2021). general distribution: libya, morocco and tunisia (ascher & pickering, 2021). eucera (hetereucera) saundersi friese, 1899 eucera saundersi friese, 1899b. distribution in tunisia: tunis (friese, 1899b; schulthess, 1924; dusmet & alonso, 1928). general distribution: north africa (lhomme et al., 2020). eucera (hetereucera) seminuda brullé, 1832 eucera seminuda brullé, 1832. distribution in tunisia: kairouan (schulthess, 1924 as eucera trivittata). general distribution: armenia, austria, azerbaijan, bulgaria, croatia, cyprus, greece, hungary, macedonia, czech republic, russia, slovakia, turkey, and ukraine (ascher & pickering, 2021). eucera (hetereucera) spatulata gribodo, 1893 eucera spatulata gribodo, 1893. distribution in tunisia: beja (10 km s beja, 15 nw teboursouk, 15 km se teboursouk, 5 km nw teboursouk), gabes (matmata), jendouba (fernana, 15 km s jendouba, 10 km n jendouba, 21 km n jendouba, 10 km s tabarka), medenine (djerba, houmt souk 5 km s richtung midoun), nabeul (5 km nw grombelia, korba, nabeul), siliana (15 km sw makthar, 15 km n makthar), sousse (msaken), tunis (ne tunis jebel amarbei jabbes), gafsa (bou hedma), zaghouan (30 km se zaghouan, 20 km nw zaghouan) (risch, 2003). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). eucera (hetereucera) vachali pérez, 1895 eucera vachali pérez, 1895. distribution in tunisia: beja (10 km nw teboursouk), gabes (5 km se matmata, 10 km se matmata, matmata), kairouan (10 km w sbikha), kasserine (10 km nnw thelepte), medenine (zarzis, s djerba, 14 km se houmt souk, 20 km sw medenine), nabeul (hammamet, grombelia), sfax (30 km sw sfax), sidi bouzid, siliana (makthar), sousse (msaken), tataouine (ksar hadada, chenini), tunis (la marsa?), zaghouan (20 km nw zaghouan) (risch, 2003). general distribution: algeria, morocco, tunisia (ascher & pickering, 2021). hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)8 eucera (hetereucera) warnckei risch, 1999 eucera warnckei risch, 1999. distribution in tunisia: medenine (30 km e medenine), nabeul (hammamet, grombelia) (risch, 1999); gabes (50 km w matmata, 10 km se matmata), nabeul (ascher & pickering, 2021). general distribution: morocco and tunisia (ascher & pickering, 2021). eucera (pteneucera) eucnemidea dours, 1873 eucera eucnemidea dours 1873. distribution in tunisia: tunisia: no specific locality (schulthess, 1924; risch, 1997); gafsa (oasis gafsa) (dusmet & alonso, 1928). general distribution: algeria, armenia, france, italy, kazakhstan, libya, malta, morocco, portugal, russia, spain, syria, tunisia, and turkey (ascher & pickering, 2021). eucera (pteneucera) nigrifacies lepeletier, 1841 eucera nigrifacies lepeletier, 1841. distribution in tunisia: beja, mannouba (tebourba) (schulthess, 1924); tunisia: no specific locality (risch, 1997); jendouba (10 km e ain draham) (ascher & pickering, 2021). general distribution: algeria, azerbaijan, bulgaria, france, georgia, germany, greece, hungary, iran, israel, italy, jordan, kazakhstan, macedonia, morocco, portugal, romania, russia, serbia, slovakia, slovenia, spain, syria, tajikistan, tunisia, turkey, and uzbekistan (ascher & pickering, 2021). eucera (synhalonia) cuniculina klug, 1845 eucera cuniculina klug, 1845. distribution in tunisia: tozeur (schulthess, 1924 as eucera (macrocera) cuniculina) general distribution: egypt, israel, jordan, libya, morocco, spain, and tunisia (ascher & pickering, 2021). eucera (synhalonia) mediterranea friese, 1895 eucera mediterranea friese, 1895. distribution in tunisia: tunisia: no specific locality (schulthess, 1924). general distribution: greece, israel, italy, spain, and turkey (ascher & pickering, 2021). eucera (synhalonia) metallescens (morawitz, 1888) tetralonia metallescens morawitz, 1888. distribution in tunisia: tozeur (oued melah between gafsa and tozeur) (schulthess, 1924). general distribution: pakistan and turkmenistan (ascher & pickering, 2021). eucera (synhalonia) ruficollis (brullé, 1832) macrocera ruficollis brullé, 1832. distribution in tunisia: gafsa, kasserine (sbeitla), tunis (carthage) (schulthess, 1924). general distribution: greece, france, italy, kazakhstan, romania, russia, syria, tajikistan, turkey, turkmenistan, and uzbekistan (ascher & pickering, 2021). eucera (synhalonia) tricincta erichson, 1835 eucera tricincta erichson, 1835. distribution in tunisia: gafsa, kasserine (sbeitla) (schulthess, 1924) general distribution: afghanistan, algeria, azerbaijan, egypt, greece, hungary, iran, israel, italy, jordan, kazakhstan morocco, portugal, romania, russia, slovakia, spain, tajikistan, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). eucera (tetralonia) alticincta (lepeletier, 1841) macrocera alticincta lepeletier, 1841. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021 as tetraloniella alticincta). general distribution: albania, bulgaria, czech republic, france, germany, greece, hungary, italy, kyrgyzstan, macedonia, morocco, romania, russia, serbia, slovakia, slovenia, switzerland, tajikistan, tunisia, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). eucera (tetralonia) cinctella saunders, 1908 eucera (macrocera) cinctella saunders, 1908. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021 as tetraloniella cinctella). general distribution: algeria, portugal, spain, and tunisia (ascher & pickering, 2021). eucera (tetralonia) dentata germar, 1839 eucera dentata germar, 1839. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021 as tetraloniella dentata). general distribution: algeria, armenia, austria, belarus, bulgaria, china, croatia, czech republic, estonia, france, germany, iran, israel, italy, kazakhstan, kyrgyzstan, macedonia, mongolia, morocco, pakistan, poland, portugal, serbia, slovakia, slovenia, spain, romania, russia, syria, tajikistan, tunisia, turkey, ukraine, and uzbekistan (ascher & pickering, 2021). eucera (tetralonia) fulvescens (giraud, 1863) tetralonia fulvescens giraud, 1863. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021 as tetraloniella fulvescens). general distribution: albania, algeria, austria, azerbaijan, bulgaria, china, croatia, france, greece, hungary, italy, kazakhstan, morocco, portugal, romania, russia, slovakia, spain, switzerland, tunisia, turkey, and uzbekistan (ascher & pickering, 2021). tribe: melectini westwood, 1840 genus: melecta latreille, 1802 sociobiology 69(3): e8151 (september, 2022) 9 melecta (melecta) aegyptiaca radoszkowski, 1876 melecta aegyptiaca radoszkowski, 1876. distribution in tunisia: kasserine (feriana), tunis (carthage) (lieftinck, 1980). general distribution: algeria, czech republic, egypt, france, greece, hungary, israel, italy, morocco, portugal, slovakia, spain, syria, and tunisia (ascher & pickering, 2021). melecta (melecta) albifrons (forster, 1771) apis albifrons forster, 1771. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, croatia, denmark, egypt, england, france, georgia, germany, greece, hungary, iran, libya, macedonia, montenegro, morocco, poland, portugal, russia, spain, sweden, switzerland, syria, tunisia, turkey, and ukraine (ascher & pickering, 2021). melecta (melecta) duodecimmaculata (rossi, 1790) nomada duodecimmaculata rossi, 1790. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, china, croatia, cyprus, egypt, greece, iran, iraq, israel, italy, kazakhstan, kyrgyzstan, libya, malta, morocco, portugal, romania, russia, spain, tajikistan, tunisia, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). melecta (melecta) grandis lepeletier, 1841 melecta grandis lepeletier, 1841. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: algeria, egypt, israel, italy, libya, morocco, spain, tunisia (ascher & pickering, 2021). melecta (melecta) italica radoszkowski, 1876 melecta italica radoszkowski, 1876. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: algeria, croatia, cyprus, egypt, france, greece, italy, jordan, libya, macedonia, malta, morocco, portugal, spain, tunisia, and turkey (ascher & pickering, 2021). melecta (melecta) leucorhyncha gribodo, 1893 melecta luctuosa var. leucorhyncha gribodo, 1893. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: albania, algeria, croatia, cyprus, egypt, france, greece, italy, jordan, libya, malta, morocco, spain, tunisia, turkey, and ukraine (ascher & pickering, 2021). melecta (melecta) luctuosa (scopoli, 1770) apis luctuosa scopoli, 1770. distribution in tunisia: tunis (schulthess, 1924). general distribution: albania, algeria, austria, azerbaijan, belarus, belgium, bulgaria, czech republic, denmark, england, france, georgia, germany, greece, hungary, iran, israel, jordan, kazakhstan, lithuania, macedonia, malta, montenegro, morocco, pakistan, poland, portugal, romania, russia, serbia, slovakia, spain, sweden, switzerland, ukraine, uzbekistan, turkey, and turkmenistan (ascher & pickering, 2021). melecta (paracrocisa) guilochei dusmet & alonso, 1915 melecta guilochei dusmet & alonso, 1915. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: greece, morocco and tunisia (ascher & pickering, 2021). genus: thyreus panzer, 1806 thyreus affinis (morawitz, 1874) crocisa affinis morawitz, 1874. distribution in tunisia: kasserine (feriana) (pérez, 1895 as crocisa tunensis). general distribution: albania, algeria, azerbaijan, bulgaria, croatia, cyprus, egypt, france, greece, iran, italy, kazakhstan, kyrgyzstan, libya, montenegro, morocco, pakistan, portugal, romania, russia, slovakia, spain, tunisia, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). thyreus elegans (morawitz, 1878) crocisa elegans morawitz, 1878. distribution in tunisia: tozeur (lieftinck, 1968). general distribution: algeria, azerbaijan, cyprus, egypt, eritrea, iran, kyrgyzstan, libya, mauritania, morocco, pakistan, saudi arabia, sudan, tunisia, turkey, turkmenistan, and united arab emirates (ascher & pickering, 2021). thyreus hirtus (de beaumont, 1939) crocisa hirta de beaumont, 1939. distribution in tunisia: le kef (ascher & pickering, 2021). general distribution: algeria, france, greece, italy, morocco, portugal, russia, spain, tunisia, and turkey (ascher & pickering, 2021). thyreus histrionicus (illiger, 1806) melecta histrionica illiger, 1806. distribution in tunisia: kairouan (schulthess, 1924 as crocisa major), nabeul (hammamet) (lieftinck, 1968). general distribution: albania, algeria, azerbaijan, belarus, bulgaria, china, cyprus, czech republic, egypt, france, germany, georgia, greece, iran, israel, italy, kazakhstan, kyrgyzstan, libya, macedonia, malta, morocco, poland, portugal, romania, russia, saudi arabia, slovakia, spain, switzerland, syria, tunisia, turkey, tajikistan, ukraine, and uzbekistan (ascher & pickering, 2021). thyreus orbatus (lepeletier, 1841) crocisa orbata lepeletier, 1841. distribution in tunisia: tunis (carthage), kairouan, hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)10 kasserine (sbeitla) (schulthess, 1924 as crocisa orbata), tunis (lieftinck, 1968). general distribution: algeria, israel, libya, morocco, russia, tunisia, and turkey (ascher & pickering, 2021). thyreus ramosus (lepeletier, 1841) crocisa ramosa lepeletier, 1841. distribution in tunisia: kairouan, gafsa (schulthess, 1924 as crocisa ramosa); monastir, tunis (carthage) (lieftinck, 1968); bizerte (25 km s bizerte) (ascher & pickering, 2021). general distribution: albania, afghanistan, algeria, austria, belgium, bulgaria, croatia, egypt, france, georgia, germany, greece, hungary, india, iran, iraq, israel, italy, kazakhstan, kyrgyzstan, libya, macedonia, moldova, morocco, oman, pakistan, portugal, romania, russia, slovakia, spain, sudan, switzerland, syria, tajikistan, tunisia, turkey, turkmenistan, united arab emirates, ukraine, and uzbekistan (ascher & pickering, 2021). thyreus tricuspis (pérez, 1883) crocisa tricuspis pérez, 1883. distribution in tunisia: nabeul (hammamet) (schulthess, 1924 as crocisa tricuspis). general distribution: algeria, israel, italy, libya, morocco, syria, and tunisia (ascher & pickering, 2021). genus: thyreomelecta rightmyer & engel, 2003 thyreomelecta sibirica (radoszkowski, 1893) crocisa sibirica radoszkowski, 1893. distribution in tunisia: gafsa (oasis gafsa) (meyer, 1921 as crocisa sibirica (doubtful identity)) general distribution: armenia, china, russia and turkey (ascher & pickering, 2021). subfamily: nomadinae latreille, 1802 tribe: ammobatini handlirsch, 1925 genus: ammobates latreille, 1809 ammobates (ammobates) assimilis (warncke, 1983) pasites assimilis warncke 1983. distribution in tunisia: jendouba (10 km n jendouba) (warncke, 1983 as pasites assimilis). general distribution: tunisia (ascher & pickering, 2021). ammobates (ammobates) hipponensis pérez, 1902 ammobates hipponensis pérez, 1902. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria and tunisia (ascher & pickering, 2021). ammobates (ammobates) minor (pérez, 1902) phileremus minor pérez, 1902. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). ammobates (ammobates) rufiventris latreille, 1809 ammobates rufiventris latreille, 1809. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, italy, portugal, spain, and tunisia (ascher & pickering, 2021). ammobates (ammobates) syriacus friese, 1899 ammobates syriacus friese, 1899a. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, morocco, israel, jordan, and tunisia (ascher & pickering, 2021). ammobates (ammobates) vinctus gerstäcker, 1869 ammobates vinctus gerstäcker, 1869. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: austria, bulgaria, croatia, france, greece, italy, portugal, romania, russia, serbia, slovakia, spain, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). ammobates (euphileremus) muticus spinola, 1843 ammobates muticus spinola, 1843. distribution in tunisia: kairouan (schulthess, 1924 as ammobates carinatus). general distribution: algeria, france, italy, libya, morocco, portugal, romania, spain, and tunisia (ascher & pickering, 2021). ammobates (euphileremus) oraniensis (lepeletier, 1841) phileremus oraniensis lepeletier, 1841. distribution in tunisia: tunis (belvédère) (schulthess, 1924); tunis (ascher & pickering, 2021). general distribution: algeria, armenia, egypt, greece, iran, israel, italy, jordan, libya, morocco, romania, spain, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). ammobates (xerammobates) minutissimus mavromoustakis, 1959 ammobates minutissimus mavromoustakis, 1959. distribution in tunisia: medenine (djerba) (mavromoustakis, 1959). general distribution: tunisia (ascher & pickering, 2021). genus: pasites jurine, 1807 pasites maculatus jurine, 1807 pasites maculatus jurine, 1807. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). sociobiology 69(3): e8151 (september, 2022) 11 general distribution: afghanistan, algeria, azerbaijan, belarus, bulgaria, china, egypt, france, greece, hungary, iran, italy, kazakhstan, korea, kyrgyzstan, morocco, pakistan, poland, portugal, romania, russia, serbia, slovakia, slovenia, spain, switzerland, syria, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). genus: spinopasites warncke, 1983 spinopasites spinotus (warncke, 1983) pasites spinotus warncke 1983. distribution in tunisia: gabes (10 km s gabes) (warncke, 1983 as pasites spinotus). general distribution: tunisia (ascher & pickering, 2021). tribe: ammobatoidini michener, 1944 genus: ammobatoides radoszkowski, 1867 ammobatoides abdominalis (eversmann, 1852) phileremus abdominalis eversmann, 1852. distribution in tunisia: tunisia: no specific locality (maidl, 1922 as phiarus abdominalis). general distribution: albania, armenia, azerbaijan, belarus, bulgaria, croatia, cyprus, czech republic, france, germany, georgia, greece, iran, israel, italy, kazakhstan, kyrgyzstan, macedonia, moldova, romania, russia, syria, tajikistan, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). ammobatoides schachti schawarz, 1988 ammobatoides schachti schawarz, 1988. distribution in tunisia: kasserine (10 km e kasserine) (schwarz, 1988a). general distribution: morocco, tunisia (ascher & pickering, 2021). genus: schmiedeknechtia friese, 1896 schmiedeknechtia oraniensis friese, 1896 schmiedeknechtia oraniensis friese, 1896b. distribution in tunisia: jendouba (10 km n jendouba), tunis (schwarz, 1993); beja, jendouba, tunis (ascher & pickering, 2021). general distribution: algeria, spain and tunisia (ascher & pickering, 2021). schmiedeknechtia verhoeffi mavromoustakis, 1959 schmiedeknechtia verhoeffi mavromoustakis, 1959. distribution in tunisia: medenine (djerba) (mavromoustakis, 1959). general distribution: egypt, israel and tunisia (ascher & pickering, 2021). tribe: epeolini robertson, 1903 genus: epeolus latreille, 1802 epeolus aureovestitus dours, 1873 epeolus aureovestitus dours, 1873. distribution in tunisia: jendouba (south tabarka), ben arous (hammam lif) (bogusch, 2021). general distribution: algeria, portugal, spain and tunisia (ascher & pickering, 2021). epeolus collaris pérez, 1884 epeolus collaris pérez, 1884. distribution in tunisia: bizerte (ghar el melh, east bizerte), tunis (ezzahra, 15 km s tunis) (bogusch, 2021). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). epeolus fallax morawitz, 1872 epeolus fallax morawitz, 1872. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932); jendouba, tunis (bogusch, 2021). general distribution: algeria, france, germany, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). epeolus flavociliatus friese, 1899 epeolus flavociliatus friese, 1899a. distribution in tunisia: kebili (blidet), tozeur (nefta, tozeur), tataouine (bogusch, 2021). general distribution: algeria, egypt, iran, mauritania, morocco, pakistan, spain, and tunisia (ascher & pickering, 2021). tribe: nomadini latreille, 1802 genus: nomada scopoli, 1770 nomada accentifera pérez, 1895 nomada accentifera pérez, 1895. distribution in tunisia: jendouba (ain draham) (schwarz, 1977); tunis (ascher & pickering, 2021). general distribution: morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada agrestis fabricius, 1787 nomada agrestis fabricius, 1787. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932); kasserine (bouchebka) (ascher & pickering, 2021) general distribution: algeria, egypt, france, greece, iran, iraq, israel, italy, jordan, libya, morocco, portugal, spain, syria, tunisia, and turkey (ascher & pickering, 2021). nomada algira mocsáry, 1883 nomada algira mocsáry, 1883. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932 as nomada platyzona); tunis (carthage), kairouan (schwarz, 1978 as nomada platyzona). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). nomada barbilabris pérez, 1895 nomada barbilabris pérez, 1895. hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)12 distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932 as nomada dido); ben arous (hammam lif), zaghouan (fahs, thuburbo majus) (schwarz, 1965 as nomada cabrerai); tunis (carthage) (schwarz, 1977 as nomada dido). general distribution: algeria and morocco (ascher & pickering, 2021). nomada basalis herrich-schäffer, 1839 nomada basalis herrich-schäffer, 1839. distribution in tunisia: tunis (belvédère) (schulthess, 1924 as nomada flavomaculata var. tripunctata); tunisia: no specific locality (dusmet & alonso, 1932 as nomada astarte). general distribution: albania, algeria, austria, bosnia and herzegovina, bulgaria, croatia, czech republic, france, greece, hungary, iran, iraq, israel, italy, kazakhstan, kyrgyzstan, macedonia, montenegro, morocco, portugal, romania, russia, serbia, slovakia, spain, switzerland, syria, tajikistan, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). nomada beaumonti schwarz, 1967 nomada beaumonti schwarz, 1967. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, france, greece, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada biblica schwarz, smit & ockermüller, 2019 nomada biblica schwarz, smit & ockermüller, 2019. distribution in tunisia: jendouba (tabarak), kasserine (thala 20 km s), medenine (30 km zarzis), tataouine (55 km s foum tataouine), tozeur (15 km w nefta) (schwarz et al., 2019). general distribution: jordan, morocco and tunisia (ascher & pickering, 2021). nomada bifasciata olivier, 1811 nomada bifasciata olivier, 1811. distribution in tunisia: gabes (matmata), kasserine (bouchebka) (ascher & pickering, 2021). general distribution: algeria, austria, belgium, bulgaria, czech republic, france, germany, hungary, libya, liechtenstein, macedonia, malta, morocco, netherlands, portugal, romania, slovakia, spain, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada bispinosa mocsáry, 1883 nomada bispinosa mocsáry, 1883. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: austria, bosnia and herzegovina, bulgaria, croatia, czech republic, france, germany, greece, iran, macedonia, montenegro, morocco, poland, portugal, romania, spain, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada blepharipes schmiedeknecht, 1882 nomada blepharipes schmiedeknecht, 1882. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, bulgaria, czech republic, france, greece, hungary, israel, italy, kazakhstan, morocco, portugal, romania, russia, slovakia, switzerland, spain, tunisia, ukraine, and uzbekistan (ascher & pickering, 2021). nomada brevis saunders, 1908 nomada brevis saunders, 1908. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932 as nomada meyeri); tataouine (10 km se foum tataouine), tunis (ascher & pickering, 2021). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). nomada carnifex mocsáry, 1883 nomada carnifex mocsáry, 1883. distribution in tunisia: medenine (djerba, se houmt souk) (ascher & pickering, 2021). general distribution: algeria, france, italy, libya, morocco, portugal, spain, switzerland, and tunisia (ascher & pickering, 2021). nomada carthaginensis dusmet & alonso, 1932 nomada carthaginensis dusmet & alonso, 1932. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932); tunisia: no specific locality (alexander & schwarz, 1994: 240). general distribution: algeria, libya and tunisia (ascher & pickering, 2021). nomada chrysopyga pyrosoma dours, 1873 nomada pyrosoma dours, 1873. distribution in tunisia: kairouan (schulthess, 1924 as nomada scutellata and as nomada umbripennis); tunis (schwarz, 1977 as nomada nigrita). general distribution: north africa (ascher & pickering, 2021). nomada conjungens herrich-schäffer, 1839 nomada conjungens herrich-schäffer, 1839. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, belarus, belgium, bosnia and herzegovina, bulgaria, croatia, czech republic, england, france, germany, greece, hungary, kazakhstan, lithunaia, macedonia, netherlands, poland, portugal, romania, russia, slovakia, spain, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada coronata pérez, 1895 nomada coronata pérez, 1895. distribution in tunisia: tunis (carthage) (schwarz, 1977). sociobiology 69(3): e8151 (september, 2022) 13 general distribution: algeria, france, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada cristata pérez, 1895 nomada cristata pérez, 1895. distribution in tunisia: tunis (schwarz, 1977; ascher & pickering, 2021). general distribution: algeria, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada discedens pérez, 1884 nomada discedens pérez, 1884. distribution in tunisia: le kef (schulthess, 1924). general distribution: france, germany, morocco, portugal, and spain (ascher & pickering, 2021). nomada discrepans schmiedeknecht, 1882 nomada discrepans schmiedeknecht, 1882. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932). general distribution: algeria, france, hungary, israel, italy, morocco, portugal, romania, slovakia, slovenia, spain, tunisia, turkey, and turkmenistan (ascher & pickering, 2021). nomada distinguenda morawtiz, 1874 nomada distinguenda morawtiz, 1874. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932). general distribution: afghanistan, albania, algeria, austria, belgium, bulgaria, croatia, czech republic, denmark, france, georgia, germany, greece, hungary, iran, italy, lebanon, macedonia, malta, montenegro, morocco, netherlands, poland, portugal, romania, russia, serbia, slovakia, spain, switzerland, syria, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). nomada dolosa mocsáry, 1883 nomada dolosa mocsáry, 1883. distribution in tunisia: zaghouan (fahs, thuburbo majus) (schwarz & gusenleitner, 2013). general distribution: algeria, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada duplex smith, 1854 nomada duplex smith, 1854. distribution in tunisia: tunis (schwarz, 1977 as nomada cirtana). general distribution: algeria, france, italy, morocco, portugal, and spain (ascher & pickering, 2021). nomada fallax pérez, 1913 nomada fallax pérez, 1913. distribution in tunisia: medenine (djerba, se houmt souk) (ascher & pickering, 2021). general distribution: algeria, france, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada felici schawarz, 1977 nomada felici schawarz, 1977. distribution in tunisia: tunis (schwarz, 1977). general distribution: greece and tunisia (ascher & pickering, 2021). nomada femoralis morawitz, 1869 nomada femoralis morawitz, 1869. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932). general distribution: albania, algeria, austria, azerbaijan, belarus, belgium, bulgaria, croatia, czech republic, france, germany, greece, hungary, israel, italy, kyrgyzstan, lithuania, macedonia, morocco, poland, portugal, romania, russia, slovakia, spain, switzerland, syria, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada fenestrata lepeletier, 1841 nomada fenestrata lepeletier, 1841. distribution in tunisia: tunisia: no specific locality (schwarz, 1965 as nomada vicarioi); kasserine (10 km nw thelepte) (ascher & pickering, 2021). general distribution: afghanistan, algeria, egypt, iran, iraq, israel, jordan, lebanon, morocco, pakistan, portugal, spain, tunisia, and turkey (ascher & pickering, 2021). nomada ferruginata linnaeus, 1767 nomada ferruginata linnaeus, 1767. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932). general distribution: austria, belarus, belgium, bulgaria, czech republic, denmark, finland, france, germany, hungary, italy, england, lithuania, netherlands, poland, romania, russia, serbia, slovakia, sweden, switzerland, spain, and ukraine (ascher & pickering, 2021). nomada flavoguttata kirby, 1802 nomada flavoguttata kirby, 1802. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932). general distribution: algeria, austria, azerbaijan, belarus, belgium, bosnia and herzegovina, bulgaria, china, croatia, czech republic, denmark, england, estonia, finland, france, germany, georgia, greece, hungary, india, ireland, israel, italy, japan, kazakhstan, korea, kyrgyzstan, lithuania, macedonia, morocco, norway, poland, portugal, romania, russia, serbia, slovenia, slovakia, spain, switzerland, tajikistan, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). nomada fulvicornis maroccana pérez, 1895 nomada maroccana pérez, 1895. hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)14 distribution in tunisia: tunis (bardo) (schwarz, 1965 as nomada pintosi). general distribution: northwest africa (ascher & pickering, 2021). nomada glaucopis pérez, 1890 nomada glaucopis pérez, 1890. distribution in tunisia: kairouan (schulthess, 1924 as nomada pusilla); tunis (la marsa) (dusmet & alonso, 1932 as nomada pusilla). general distribution: algeria, czech republic, france, germany, greece, hungary, israel, italy, kazakhstan, libya, morocco, portugal, romania, russia, spain, tunisia, and turkey (ascher & pickering, 2021). nomada ibanezi dusmet & alonso, 1915 nomada ibanezi dusmet & alonso, 1915. distribution in tunisia: tataouine (10 km se foum tataouine) (ascher & pickering, 2021). general distribution: morocco and tunisia (ascher & pickering, 2021). nomada inermis pérez, 1895 nomada inermis pérez, 1895. distribution in tunisia: tataouine (ascher & pickering, 2021). general distribution: tunisia (ascher & pickering, 2021). nomada integra imperfecta schwarz, 1967 nomada cinctiventris ssp. imperfecta schwarz, 1967. distribution in tunisia: tunisia: no specific locality (alexander & schwarz, 1994). general distribution: algeria, israel and tunisia (ascher & pickering, 2021). nomada judaica schwarz & smit, 2018 nomada judaica schwarz & smit, 2018. distribution in tunisia: tataouine (schwarz & smit, 2018). general distribution: algeria, israel, jordan, syria, tunisia, and turkey (ascher & pickering, 2021). nomada keroanensis pérez, 1895 nomada keroanensis pérez, 1895. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932); kairouan, tunis (schwarz, 1977); kairouan, kasserine (10 km nw thelepte) (ascher & pickering, 2021). general distribution: morocco and tunisia (ascher & pickering, 2021). nomada kohli schmiedeknecht, 1882 nomada kohli schmiedeknecht, 1882. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932). general distribution: albania, algeria, austria, bosnia and herzegovina, bulgaria, croatia, czech republic, france, germany, greece, hungary, israel, italy, macedonia, malta, morocco, portugal, romania, russia, serbia, slovenia, slovakia, spain, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada lathburiana kirby, 1802 nomada lathburiana kirby, 1802. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: austria, belarus, belgium, bulgaria, croatia, czech republic, denmark, estonia, england, finland, france, germany, georgia, hungary, italy, kazakhstan, latvia, netherlands, norway, poland, portugal, romania, russia, slovakia, spain, sweden, tajikistan, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada linsenmaieri schwarz, 1974 nomada linsenmaieri schwarz, 1974. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, france, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada litigiosa gribodo, 1893 nomada litigiosa gribodo, 1893. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: algeria, italy and tunisia (ascher & pickering, 2021). nomada maculicornis pérez, 1884 nomada maculicornis pérez, 1884. distribution in tunisia: kasserine (bouchebka) (ascher & pickering, 2021). general distribution: algeria, france, italy, morocco, portugal, and spain (ascher & pickering, 2021). nomada marshamella kirby, 1802 nomada marshamella kirby, 1802. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: austria, azerbaijan, bulgaria, croatia, czech republic, england, estonia, denmark, finland, france, germany, greece, hungary, ireland, italy, kazakhstan, lithuania, macedonia, montenegro, norway, poland, portugal, romania, russia, serbia, slovakia, spain, sweden, switzerland, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada mauritanica lepeletier, 1841 nomada mauritanica lepeletier, 1841. distribution in tunisia: medenine (schwarz, 1977 as nomada poecilonota); kairouan, kasserine (10 km nw thelepte) (ascher & pickering, 2021). sociobiology 69(3): e8151 (september, 2022) 15 general distribution: algeria, austria, azerbaijan, bulgaria, chad, croatia, czech republic, egypt, france,, greece, hungary, iraq, israel, italy, kazakhstan, libya, morocco, romania, russia, serbia, slovenia, slovakia, spain, switzerland, syria, tajikistan, tunisia, turkey, turkmenistan, united arab emirates, ukraine, and uzbekistan (ascher & pickering, 2021). nomada numida lepeletier, 1841 nomada numida lepeletier, 1841. distribution in tunisia: sfax (schulthess, 1924 as nomada mauritanica manni) general distribution: algeria, croatia, cyprus, egypt, france, greece, hungary, iraq, israel, italy, libya, montenegro, morocco, portugal, romania, serbia, slovakia, spain, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada melanura mocsáry, 1883 nomada melanura mocsáry, 1883. distribution in tunisia: kairouan (schwarz, 1976 as nomada gracilipes). general distribution: algeria, japan and morocco (ascher & pickering, 2021). nomada mideltiaca schwarz, smit & ockermüller, 2019 nomada mideltiaca schwarz, smit & ockermüller, 2019. distribution in tunisia: kebili (bir soltane, 46 km e douz) (schwarz et al., 2019). general distribution: morocco and tunisia (ascher & pickering, 2021). nomada minuscula noskiewicz, 1930 nomada minuscula noskiewicz, 1930. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, croatia, france, germany, greece, hungary, italy, morocco, poland, portugal, serbia, slovakia, switzerland, spain, tunisia, and ukraine (ascher & pickering, 2021). nomada mutabilis morawitz, 1870 nomada mutabilis morawitz, 1870. distribution in tunisia: tunis (ascher & pickering, 2021). general distribution: algeria, austria, azerbaijan, belarus, bulgaria, croatia, czech republic, france, georgia, germany, greece, hungary, india, iran, israel, italy, kazakhstan, kyrgyzstan, lithuania, macedonia, morocco, nepal, netherlands, poland, portugal, romania, russia, serbia, slovakia, spain, switzerland, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). nomada nobilis herrich-schäffer, 1839 nomada nobilis herrich-schäffer, 1839. distribution in tunisia: jendouba (15 km e tabarka) (ascher & pickering, 2021). general distribution: albania, algeria, austria, bulgaria, croatia, czech republic, france, germany, greece, hungary, iran, israel, italy, macedonia, montenegro, morocco, poland, portugal, romania, russia, serbia, slovenia, slovakia, spain, switzerland, syria, tunisia, turkey, and ukraine (ascher & pickering, 2021). nomada panurgina morawitz, 1869 nomada panurgina morawitz, 1869. distribution in tunisia: tunisia: no specific locality (maidl, 1922 as nomada julliani); kairouan, kasserine (sbeitla), tunis (carthage) (schulthess, 1924 as nomada julliani). general distribution: algeria, croatia, france, italy, morocco, spain, switzerland, and tunisia (ascher & pickering, 2021). nomada panurginoides saunders, 1908 nomada panurginoides saunders, 1908. distribution in tunisia: tunis (la marsa) (dusmet & alonso, 1932); gabes (matmata) (ascher & pickering, 2021). general distribution: algeria, morocco, tunisia (ascher & pickering, 2021). nomada pectoralis morawitz, 1877 nomada pectoralis morawitz, 1877. distribution in tunisia: jendouba (1 km s ain draham) (ascher & pickering, 2021). general distribution: azerbaijan, bulgaria, croatia, france, greece, hungary, iran, morocco, romania, russia, slovakia, spain, tunisia, turkmenistan, and ukraine (ascher & pickering, 2021). nomada pleurosticta herrich-schäffer, 1839 nomada pleurosticta herrich-schäffer, 1839. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: austria, belgium, bulgaria, france, germany, greece, hungary, iran, italy, netherlands, portugal, russia, slovakia, spain, switzerland, tunisia, and turkey (ascher & pickering, 2021). nomada podagrica gribodo, 1893 nomada podagrica gribodo, 1893. distribution in tunisia: tunisia: no specific locality (alexander & schwarz, 1994). general distribution: algeria and tunisia (ascher & pickering, 2021). nomada pruinosa pérez, 1895 nomada pruinosa pérez, 1895. distribution in tunisia: kairouan, kasserine (10 km nw thelepte) (ascher & pickering, 2021). general distribution: algeria, egypt, israel, libya, morocco, spain, and tunisia (ascher & pickering, 2021). nomada rubiginosa pérez, 1884 nomada rubiginosa pérez, 1884. distribution in tunisia: tunisia: no specific locality (dusmet & alonso, 1932: 233; alexander & schwarz, 1994). hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)16 general distribution: algeria, france, israel, italy, morocco, portugal, spain, tunisia, and turkey (ascher & pickering, 2021). nomada sabulosa radoszkowski, 1876 nomada sabulosa radoszkowski, 1876. distribution in tunisia: kasserine (10 km nw thelepte) (ascher & pickering, 2021). general distribution: algeria, egypt, israel, morocco, spain, and tunisia (ascher & pickering, 2021). nomada sanguinea smith, 1854 nomada sanguinea smith, 1854. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, france, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). nomada sexfasciata panzer, 1799 nomada sexfasciata panzer, 1799. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, azerbaijan, belgium, bulgaria, croatia, czech republic, england, france, germany, greece, hungary, iran, israel, italy, kazakhstan, liechtenstein, luxemburg, montenegro, morocco, netherlands, poland, portugal, romania, russia, serbia, slovakia, spain, switzerland, sweden, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). nomada sheppardana (kirby, 1802) apis sheppardana kirby, 1802. distribution in tunisia: tunisia: no specific locality (schwarz, 1988b as nomada microsticta). general distribution: algeria, austria, belgium, bulgaria, croatia, czech republic, denmark, egypt, england, france, germany, greece, hungary, ireland, japan, morocco, netherlands, poland, portugal, romania, russia, slovakia, spain, switzerland, and tunisia (ascher & pickering, 2021). nomada stigma fabricius, 1804 nomada stigma fabricius, 1804. distribution in tunisia: jendouba (11 km s tabarka, kataria meadwo) (ascher & pickering, 2021). general distribution: algeria, austria, azerbaijan, belgium, bulgaria, czech republic, croatia, denmark, finland, france, germany, greece, hungary, italy, kazakhstan, kyrgyzstan, latvia, macedonia, netherlands, portugal, romania, russia, slovakia, spain, sweden, switzerland, tajikistan, tunisia, turkey, ukraine, and uzbekistan (ascher & pickering, 2021). nomada succincta panzer, 1798 nomada succincta panzer, 1798. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, austria, azerbaijan, bulgaria, croatia, cyprus, czech republic, denmark, estonia, finland, france, germany, greece, hungary, israel, italy, jordan, kazakhstan, lithuania, macedonia, morocco, netherlands, poland, portugal, romania, russia, spain, switzerland, syria, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). nomada tridentirostris dours, 1873 nomada tridentirostris dours, 1873. distribution in tunisia: jendouba (11 km s tabarka, kataria meadow) (ascher & pickering, 2021). general distribution: algeria, austria, bosnia and herzegovina, bulgaria, croatia, czech republic, france, greece, hungary, iran, israel, italy, macedonia, morocco, poland, portugal, russia, slovakia, slovenia, spain, switzerland, tunisia, and turkey (ascher & pickering, 2021). subfamily: xylocopinae latreille, 1802 tribe: ceratinini latreille, 1802 genus: ceratina latreille, 1802 ceratina (ceratina) cucurbitina (rossi, 1792) apis cucurbitina rossi, 1792. distribution in tunisia: ben arous (hammam lif) (schulthess, 1924); beja (teboursouk, 1 km s on m.r. 5; teboursouk, dougga ruins on s.r. 74; nefza, djebel abiod, 12 km s on s.r. 52), bizerte (utique ruins, jefna on m.r.7; jefna, 2.1 km w on m.r. 7), jendouba (ain draham, 12.4 km s on m.r. 17; ain draham, 5.6 km s on m.r. 17; ain draham, beni métir dam on s.r. 65; tabarka, 14 km e on m.r. 7; tabarka, 3.5 km s on m.r. 17; tabarka, 16.5 km s on m.r. 17; ain sebaa, 6.2 km e on m.r. 7), kasserine (thala, on m.r. 17), le kef (le kef, 26 km ne on m.r. 5), nabeul (grombalia, 1 km nw on m.r. 1; kélibia, 4 km ne on s.r. 27; menzel temime, 1.4 km sw on s.r. 27), sousse (bouficha, 10.4 km n on m.r. 1), siliana (makthar, north city limits; makthar, 16 km e on m.r. 12 or 0.5 km e junction la kesra road), tunis (la marsa), zaghouan (pont du fahs, 23 km ne on m.r. 3; pont du fahs, 19 km sw on m.r. 4; pont du fahs, 20 km sw on m.r. 4) (daly, 1983). general distribution: albania, algeria, azerbaijan, bosnia, and herzegovina, bulgaria, france, germany, greece, hungary, iran, israel, italy, jordan, libya, macedonia, montenegro, morocco, poland, portugal, russia, romania, serbia, slovenia, slovakia, spain, switzerland, syria, tunisia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). ceratina (dalyatina) parvula smith, 1854 ceratina parvula smith, 1854. distribution in tunisia: jendouba (ain draham, 7 km s on m.r. 17 and 2 km ne on s.r. 65; tabarka, 14 km e on m.r. 7), zaghouan (pont du fahs, 23 km ne on m.r.3) (daly, 1983). general distribution: algeria, bulgaria, croatia, cyprus, egypt france, greece, italy, libya, macedonia, montenegro, morocco, portugal, spain, syria, tunisia, turkey, and turkmenistan (ascher & pickering, 2021). ceratina (euceratina) acuta friese, 1896 ceratina acuta friese, 1896a. sociobiology 69(3): e8151 (september, 2022) 17 distribution in tunisia: tunisia no specific locality (friese, 1896a, 1901; maidl, 1922). general distribution: albania, armenia, austria, bulgaria, croatia, czech republic, germany, georgia, greece, iran, israel, kazakhstan, lebanon, macedonia, romania, russia, serbia, slovakia, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). ceratina (euceratina) albosticta cockerell, 1931 ceratina dallatorreana albosticta cockerell, 1931. distribution in tunisia: bizerte (jefna on m.r. 7), jendouba (ain draham, 5.6 km s on m.r.17; ain draham, 12.4 km s on m.r. 17), nabeul (grombalia, 1 km nw on m.r1; kélibia, 4 km ne on s.r. 27), siliana (makthar, north city limits), tunis (la marsa) (daly, 1983). general distribution: algeria, morocco, spain and tunisia (ascher & pickering, 2021). ceratina (euceratina) callosa (fabricius, 1794) apis callosa fabricius, 1794. distribution in tunisia: ben arous (hammam lif), tunis (carthage) (schulthess, 1924); beja (teboursouk, 1 km s on m.r. 5; teboursouk, dougga ruins on s.r. 74), gafsa (ksour), kairouan (kairouan, 17 km nw on m.r. 3), le kef (le kef, 22.5 km sw on m.r. 17; le kef, 26 km ne on m.r. 5), nabeul (grombalia, 1 km nw on m.r.1), siliana (makthar, 13 km e on m.r. 12), zaghouan (pont du fahs, 23 km ne on m.r. 3; pont du fahs, 19 km sw on m.r. 4; draa ben jouder, junction m.r.3/s.r. 123) (daly, 1983). general distribution: algeria, austria, france, germany, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). ceratina (euceratina) chalybea chevrier, 1872 ceratina chalybea chevrier, 1872. distribution in tunisia: jendouba (ain draham, 5.6 km s on m.r.17; ain draham, 4.4 km s on m.r.17; tabarka, 16.5 km s on m.r. 17), nabeul (menzel temime, 1.4 km sw on s.r. 27), siliana (makthar, north city limits; makthar, 16 km e on m.r. 12 or 0.5 km e junction la kesra road) (daly, 1983). general distribution: albania, algeria, austria, azerbaijan, bulgaria, croatia, france, georgia, germany, greece, iran, italy, jordan, macedonia, montenegro, morocco, portugal, romania, russia, slovakia, switzerland, syria, spain, tunisia, turkey, turkmenistan, and ukraine,(ascher & pickering, 2021). ceratina (euceratina) cyanea (kirby, 1802) apis cyanea kirby, 1802. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: albania, algeria, austria, azerbaijan, belarus, bosnia and herzegovina, bulgaria, croatia, czech republic, england, france, germany, georgia, greece, iran, italy, kazakhstan, libya, lithuania, malta, montenegro, netherlands, norway, portugal, romania, russia, serbia, spain, sweden, switzerland, turkey, turkmenistan, and ukraine (ascher & pickering, 2021). ceratina (euceratina) dallatorreana friese, 1896 ceratina dallatorreana friese, 1896a. distribution in tunisia: beja (nefza, djebel abiod, 12 km s on s.r. 52), bizerte (jefna on m.r.7; jefna, 2.1 km w on m.r. 7), jendouba (ain draham, 5.6 km s on m.r. 17; ain draham, 12.4 km s on m.r. 17; ain sebaa, 6.2 km e on m.r. 7; fernana, 3.1 km nw on s.r. 65; tabarka, 14 km e on m.r. 7; tabarka, 3.5 km s on m.r. 17), le kef (le kef, 26 km ne on m.r. 5), nabeul (grombalia, 1 km nw on m.r. 1; menzel temime, 1.4 km sw on s.r. 27; kelibia, 4 km ne on s.r. 27), siliana (makthar, north city limits; makthar 16 km e on m.r. 12 or 0.5 km e junction la kesra road), sousse (bouficha, 10.4 km n on m.r. 1), tunis (la marsa) (daly, 1983). general distribution: albania, algeria, bulgaria, cyprus, france, georgia, greece, iran, israel, italy, kyrgyzstan, malta, montenegro, morocco, portugal, romania, slovenia, spain, syria, tunisia, turkey, turkmenistan, ukraine, united states, and uzbekistan (ascher & pickering, 2021). ceratina (euceratina) dentiventris gerstäcker, 1869 ceratina dentiventris gerstäcker, 1869. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: croatia, france, georgia, germany, greece, israel, italy, morocco, portugal, romania, spain, tunisia, and turkey (ascher & pickering, 2021). ceratina (euceratina) maghrebensis daly, 1983 ceratina maghrebensis daly, 1983. distribution in tunisia: jendouba (tabarka), kairouan (kairouan, 17 km nw on m.r. 3), kasserine (sbeitla), zaghouan (pont du fahs, 19 km sw on m.r. 4; pont du fahs, 23 km ne on m.r. 3; pont du fahs, 20 km sw on m. r.4) (daly, 1983). general distribution: algeria, morocco and tunisia (ascher & pickering, 2021). ceratina (euceratina) mocsaryi friese, 1896 ceratina mocsaryi friese, 1896a. distribution in tunisia: jendouba (ain draham, 7 km s on m.r. 17 and 2 km ne on s.r. 65; ain draham, 15 km s on m.r. 17; ain draham, 7 km s on m.r. 17 and 2.8 km ne on s.r. 65; ain draham, 5.6 km s on m.r. 17; ain draham, 12.4 km s on m. r.17; ain draham, 4.4 km s on m.r. 17; ain draham, beni métir dam on s.r. 65; tabarka, 14 km e on m.r. 7; tabarka, 16.5 km s on m.r. 17; ain sebaa, 6.2 km e on m.r. 7; fernana, 11.3 km nw on s.r. 65), nabeul (grombalia, 1 km nw on m.r. 1), siliana (makthar, 16 km e on m.r. 12 or 0.5 km e junction la kesra road) (daly, 1983). general distribution: algeria, armenia, france, italy, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)18 ceratina (euceratina) saundersi daly, 1983 ceratina saundersi daly, 1983. distribution in tunisia: gabes (3 km sw matmata) (ascher & pickering, 2021); beja (nefza, djebel abiod, 12 km s on s.r. 52), bizerte (jefna on m.r. 7), jendouba (ain sebaa, 6.2 km e on m.r. 7, fernana, 3.1 km nw on s.r. 65; tabarka, 16.5 km s on m.r. 17), kasserine (thala, 13 km se on m.r. 17), le kef (le kef, 26 km ne on m.r. 5), nabeul (grombalia, 1 km nw on m.r. 1), siliana (makthar, north city limits; makthar, 16 km e on m.r. 12 or 0.5 km e junction la kesra road); sousse (bouficha, 10.4 km n on m.r. 1), tunis (la marsa), zaghouan (pont du fahs, 19 km sw on m.r. 4; pont du fahs, 20 km sw on m.r. 4) (daly, 1983). general distribution: algeria, italy, libya, morocco, portugal, spain, and tunisia (ascher & pickering, 2021). tribe: xylocopini latreille, 1802 genus: xylocopa latreille, 1802 xylocopa (copoxyla) iris (christ, 1791) apis iris christ, 1791. distribution in tunisia: tunis (quamart) (vicidomini, 2005). general distribution: afghanistan, albania, algeria, austria, azerbaijan, bosnia and herzegovina, croatia, france, bulgaria, georgia, germany, greece, hungary, iran, israel, italy, kazakhstan, libya, macedonia, moldova, morocco, portugal, romania, russia, serbia, slovenia, slovakia, spain, switzerland, tajikistan, tunisia, turkey, turkmenistan, ukraine, and uzbekistan (ascher & pickering, 2021). xylocopa (xylocopa) violacea (linnaeus, 1758) apis violacea linnaeus, 1758. distribution in tunisia: tunisia: no specific locality (ascher & pickering, 2021). general distribution: algeria, azerbaijan, egypt, georgia, indonesia, iran, iraq, israel, kazakhstan, libya, morocco, russia, syria, tajikistan, tunisia, turkey, and turkmenistan, very common in europe (ascher & pickering, 2021). discussion our study states that the tunisian fauna of apidae is composed of 184 species and subspecies belonging to 19 genera. the reported fauna is rich in terms of genera but not in species compared to moroccan fauna where have been reported 15 genera and 241 species (lhomme et al., 2020). an uncertain identification for some species were noticed leading to the need for a re-examination of the specimens such as of nomada femorata reported from tunis province (belvédère, carthage) and anthophora sp. from tozeur (schulthess, 1924). also, dusmet & alonso (1932) reported nomada species from tunisia (nomada discrepans schmiedeknecht, 1882 var.?, nomada n. sp. ? (furva panz. var. ?), nomada sp. ? (immaculata mor. ?)) but without a clear identification. this may correspond to species already mentioned and for this reason they are excluded from the present list. some researchers (maidl, 1922; schulthess, 1924; dusmet & alonso, 1932; ascher & pickering, 2021) reported some species and subspecies from tunisia, but without mentioning the specific locality. the total number of these taxa were 54 and the confirmation of their presence in tunisia may be confirmed. geographically, tunisia is divided into three main regions: north, center and south regions (fig 1). the north region is the most diverse in apidae species with about 80% of the total number of species in the country (fig 4). this may be related to edaphic, climatic and vegetation conditions characterizing this region which are favorable to the biology of those species. the east center part of tunisia including the tunisian coast is the least rich area with only 16 species and subspecies. future investigations in this area are required to contribute to knowledge of biodiversity of the apids in tunisia (figs 3, 4). a total of species recorded, only five are endemic to tunisian fauna (table 1). fig 3. apidae species number per province in tunisia. sociobiology 69(3): e8151 (september, 2022) 19 table 1. endemic and near endemic apidae species in tunisia. the symbol “+” means presence. species algeria libya morocco tunisia ammobates latreille, 1809 a. assimilis (warncke, 1983) + a. hipponensis pérez, 1902 + + a. minor (pérez, 1902) + + + a. minutissimus mavromoustakis, 1959 + ammobatoides radoszkowski, 1867 a. schachti schawarz, 1988 + + anthophora latreille, 1803 a. holoxantha pérez, 1895 + + + ancyla lepeletier, 1841 a. brevis dours, 1873 + + + ceratina latreille, 1802 c. maghrebensis daly, 1983 + + + epeolus latreille, 1802 e. collaris pérez, 1884 + + + eucera scopoli, 1770 e. alopex risch, 1999 + e. bequaerti alfken, 1914 + + + e. genovefae vachal, 1907 + + + e. polita pérez, 1895 + + + e. spatulata gribodo, 1893 + + + e. vachali pérez, 1895 + + + e. warnckei risch, 1999 + + nomada scopoli, 1770 n. algira mocsáry, 1883 + + + n. brevis saunders, 1908 + + + n. carthaginensis dusmet & alonso, 1932 + + + n. ibanezi dusmet & alonso, 1915 + + n. inermis pérez, 1895 + n. keroanensis pérez, 1895 + + n. mideltiaca schwarz, smit & ockermüller, 2019 + + n. panurginoides saunders, 1908 + + + n. podagrica gribodo, 1893 + + spinopasites warncke, 1983 s. spinotus (warncke, 1983) + fig 4. apidae species number per region in tunisia. hassib ben khedher, mohamed braham, ikbal chaib – tunisian apidae fauna (hymenoptera: anthophila)20 this list represents the first attempt to review apidae fauna of tunisia. more investigations on wild bees are necessary to improve our knowledge to this fauna in the country. acknowledgments we would like to thank anonymous referees and the editor, who significantly improved the quality of the paper and all our colleagues, specifically dr. faten abdelkader who gave us some old references. references aguiar, a., deans, a.r., engel, m.s., forshage, m., huber, j.t., jennings, j.t., johnson, n., lelej, a.s., longino, j.t., lohrmann, v., miko, i., ohl, m., rasmussen, c., taeger, a. & yu, d.s. 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(1983). zur kenntnis der bienengattung pasites jurine, 1807, in der westpaläarktis (hymenoptera, apidae, nomadinae). entomofauna, 4: 261-347. _hlk108721798 _hlk101262703 _hlk107898058 _hlk107898191 _hlk107898237 _hlk107898260 _hlk107898290 _hlk107898353 _hlk107898386 _hlk107898521 _hlk107898544 _hlk107898572 _hlk107898591 _hlk107898617 _hlk107898631 _hlk107898657 _hlk107898689 _hlk107898706 _hlk107898737 _hlk107898782 _hlk107898820 _hlk107898840 _hlk107898874 _hlk101247916 _hlk107898901 _hlk71593830 _hlk107898919 _hlk101247942 _hlk107898977 _hlk107899042 _hlk107899075 _hlk107899095 _hlk107899116 _hlk101249223 _hlk107899134 _hlk107899149 _hlk101247971 _hlk107899224 _hlk107899255 _hlk107899275 _hlk101248046 _hlk107899299 _hlk107899316 _hlk107899356 _hlk101248061 _hlk101248678 _hlk107899402 _hlk107899435 _hlk101248079 _hlk107899474 _hlk101248101 _hlk101248752 _hlk107899499 _hlk107899513 _hlk107899540 _hlk107899553 _hlk107899568 _hlk107899582 _hlk107899602 _hlk107899625 _hlk107899641 _hlk107899665 _hlk107899693 _hlk107899707 _hlk107899722 _hlk107899751 _hlk107899771 _hlk107899793 _hlk107899809 _hlk107899832 _hlk107899849 _hlk107899864 _hlk107899892 _hlk107899931 _hlk107899947 _hlk101249027 _hlk101248359 _hlk107902706 _hlk101248118 _hlk107902733 _hlk107902748 _hlk107902766 _hlk107902780 _hlk101249052 _hlk107902809 _hlk101249067 _hlk107902842 _hlk107902857 _hlk101248802 _hlk107902874 _hlk107902891 _hlk107902912 _hlk107902936 _hlk101248135 _hlk107902954 _hlk107902970 _hlk107902988 _hlk107903014 _hlk101248150 _hlk107903036 _hlk107903060 _hlk107903088 _hlk107903144 _hlk107903183 _hlk107903276 _hlk107903288 _hlk101248221 _hlk107903312 _hlk107903329 _hlk101248479 _hlk107903350 _hlk107903375 _hlk107904907 _hlk107904921 _hlk107904936 _hlk107904950 _hlk107904964 _hlk107904980 _hlk107905002 _hlk107905018 _hlk107905044 _hlk107905072 _hlk107905097 _hlk101248828 _hlk107905113 _hlk101249133 _hlk107905137 _hlk107905159 _hlk101248840 _hlk107905183 _hlk107905199 _hlk107905214 _hlk107905237 _hlk107905250 _hlk107905265 top _hlk107905348 _hlk101248858 _hlk107905379 _hlk107905410 _hlk107905440 _hlk107905464 _hlk107905480 _hlk101248243 _hlk107905501 _hlk107905517 _hlk107905532 _hlk101248412 _hlk107905570 _hlk107905588 _hlk107905606 _hlk107905619 _hlk107905632 _hlk107905645 _hlk107905658 _hlk107905679 _hlk107905781 _hlk107905798 _hlk107905814 _hlk107905833 _hlk107905849 _hlk107905864 _hlk107905878 _hlk107905894 _hlk107905915 _hlk101248271 _hlk107905932 _hlk107905960 _hlk107905975 _hlk107906010 _hlk107906025 _hlk108720885 _hlk95990957 _hlk108686907 _hlk108685604 doi: 10.13102/sociobiology.v62i2.266-269sociobiology 62(2): 266-269 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 new records and distribution for the neotropical ant genus ochetomyrmex mayr (hymenoptera: formicidae) introduction recently reclassified in the myrmicinae tribe attini (ward et al., 2014), the neotropical ant genus ochetomyrmex mayr, 1878 (formicidae,ward et al., 2014) includes two valid species: o. semipolitus mayr, 1878 and o. neopolitus fernández, 2003 restricted to the neotropical region. both species are comparatively small, monomorphic and cryptobiotic, found nesting in the leaf litter of tropical lowland forests east of the andes (fernández, 2003). limited information is available about where they are found, as little is known about ochetomyrmex biology. this study presents an update to known distribution of ochetomyrmex, adding recent information available from several sources (antweb, 2014; baccaro et al., 2010, 2012; boscardin et al., 2012; delabie et al., 2009; groc et al., 2009, 2013; miranda et al., 2012, 2013; santos et al., 2008; souza et al., 2012; vittar, 2008; wild, 2007; wilkie et al., 2007, 2009). unpublished records from the collection of formicidae cocoa research center (cpdc) and samples collected in pantanal of mato grosso state of brazil were added. abstract new records and known distribution from species of the genus ochetomyrmex (mayr) in the neotropics are presented, emphasizing the first occurrence of ochetomyrmex neopolitus (fernández) in the mato grosso pantanal region, a sub-region of cáceres, brazil. keywords biogeography, distribution, pantanal. sociobiology an international journal on social insects e meurer¹, ld battirola2, mi marques1, jhc delabie3 article history edited by rodrigo m. feitosa, ufpr, brazil. received 03 december 2014 initial acceptance 01 january 2015 final acceptance 20 january 2015 corresponding author eliandra meurer instituto de biociências universidade federal de mato grosso av. fernando corrêa da costa, nº 2.367, boa esperança, 78060-900, mato grosso, cuiabá, mt, brazil e-mail: eliandrameurer@gmail.com ochetomyrmex mayr, 1887: new records (cpdc collection): ochetomyrmex neopolitus fernández, 2003 brazil: acre, porto walter, #12670, #12764, #13202, 08015’31”s 72046’37”w, 05.xi-17.iv.1997, j. caldwell; amazonas, manaus, #4832, rs1301, 14.xii.1993, a.b. casimiro; manaus, #4642, br 174 km 72, iii.1990, h.l. vasconcelos; manaus, #72i, faz. porto alegre br 174, 20.vi.1996, h.l. vasconcelos; rio jaú, #f-40i, 01057’s 61049’w, 17.vii.1996, h.l. vasconcelos & j.m. vilhena; bahia, arataca, mata a47, 15016’49”s 39023’31”w, 25.v.1999, j.r.m. dos santos; arataca, mata a48, 15015’54”s 39016’00.6”w, 23.v.1999, j.r.m. dos santos; boa nova, joão-mata, 13.viii.2003, j.r.m. dos santos & j.c. do carmo; canavieiras, 15039’s 38058’ w, 07.xi.1997, j.r.m. dos santos; ibicaraí, km 41, 14053’75”s 39029’01”w, 21.xi.1998, j.r.m. dos santos; igrapiúna, reserva da michelin, #5704, v-ix.2012, s.l.s. varjão; itambé, leôncio-mata, 14038’87”s 40020’23”w, 08.viii.2003, j.r.m. dos santos; itororó, c área, 14057’31”s 40002’33”w, 08.viii.2000, j.r.m. dos santos; pará: goianésia, faz. rio capim, v-vi.2003, a.m. elizabeth; research article ants 1 universidade federal de mato grosso, cuiabá, mt, brazil 2 universidade federal de mato grosso. sinop, mt, brazil 3 centro de pesquisa do cacau, convênio uesc/ceplac. itabuna, ba, brazil sociobiology 62(2): 266-269 (june, 2015) 267 marituba, ceplac, 01022’s 48020’w, 21-22.x.2004, j.r.m. dos santos; oriximiná, porto trombetas, #4552, 01.viii.1992, j. majer; oriximiná, porto trombetas, platô bacaba, #5426, 03.viii.2004; j.c. santos; oriximiná, flona de saracá-taquera platô bacaba, #5542, 09-10.x.2007, j.c. santos; paragominas, faz. rio capim empresa cikel, 03033’s 48038’w, vii-viii.2002, a.m. elizabeth; tailândia, faz. santa marta empresa juruá, 30001’s 49016’w, 10-28.v.2002, a.m. elizabeth; tailândia, faz. santa marta, 13.v-08.viii.2003, a.m. elizabeth; rondônia: parque estadual guajará mirim, #5248b, 17.ii.1998, j.r.m. dos santos; ecuador: cuyabeno, #10441, #10427, 12.x-05. xi.1994, j.p. caldwell; french guiana: maripasoula, vi.2000, s. durou, j. delabie, a. dejean & m. gibernau; maripasoula, vii.2001, a. & a. dejean; petit saut, winkler, 04059’n 03008’w, vii.2000, s. durou & a. dejean; petit saut, v.2003, j. orivel & j. le breton; petit saut, forêt de basse-vie, 04.vii.1999, s. durou; petit saut, forêt de bassevie, vi-vii.2000, s. durou, j. delabie, a. dejean & m. gibernau; petit saut, montagne plomb, 25.vi.2000, s. durou, j. delabie, a. dejean & m. gibernau. ochetomyrmex semipolitus mayr 1878: brazil: minas gerais, bom despacho, #5301, vi-ix.2000, l.s. ramos & c.g. marinho; paraopeba, cerrado, iii.2001. c.r. ribas; santana do riacho, 19.ii.2001, s.m. soares; french guiana: hautitany, ix.1994, r. garrouste. here we provide the first record of o. neopolitus in the pantanal of brazilian state of mato grosso. during a survey for epigaeic ants assemblages using traps (see bestelmeyer et al., 2000 see bestelmeyer et al., 2000 and adis, 2002), at the baía de pedra ranch, cáceres, mato grosso (16º28’49”s 58º08’25”w), 195 individuals were collected. of those, 99 in deciduous seasonal forest, 52 in a relatively closed savanna, 31 in an open savanna, and the remaining in open areas. all specimens are deposited in the reference collection of the laboratório de ecologia e taxonomia de artrópodes (leta) of the biosciences institute of the federal university of mato grosso (ufmt) and the cpdc collection, under reference number #5574. the occurrence of o. neopolitus in the pantanal of mato grosso is possibly a consequence of amazonian vegetation in the floristic composition of the study area, as the vegetation here is strongly influenced by adjacent phytogeographic areas, including the amazon and chaco biomes (adámoli, 1982; silva et al., 2000; alho & gonçalves, 2005; junk et al., 2006). although other studies were carried out in this biome (e.g. marques et al., 2010, 2011; silva et al., 2013, corrêa et al., 2006), this species has not yet been documented in other regions of the pantanal. the genus ochetomyrmex is endemic to south america and it is rarely collected at east of the andes, between 6º n and 30º s (fernández, 2003). o. neopolitus occurs in guiana, suriname, french guiana, brazil (acre, pará, amazonas, mato grosso, tocantins, and bahia), bolivia, peru, ecuador and colombia. on the other hand, o. semipolitus seems to be more common in the amazon and cerrado and is not found in the atlantic forest of southeastern brazil. it is found in guyana, suriname, french guiana and brazil (acre, amazonas, rondônia, mato grosso, goiás, minas gerais, pará, rio grande do sul, and roraima), argentina, peru, bolivia, ecuador and colombia. the transition between the forest fragment and riparian, and terra firma forests appears as a gradient of occurrence for several ant species, particularly odontomachus spp., pachycondyla spp., camponotus rapax, and ochetomyrmex semipolitus. finally, the habitat quality of the pristine, terra firma forest was mostly marked by the diversity of the genus pheidole, and the occurrence of other species such as o. semipolitus (delabie et al., 2009). the distribution of ochetomyrmex in brazil and in the neotropics is possibly underestimated in the literature, because their morphology is rather close to pheidole, frequently provoking taxonomical confusion between genera. fig 1. distribution of ochetomyrmex neopolitus and ochetomyrmex semipolitus in the neotropics, based on information from literature, new records from the collection of formicidae cocoa research center (cpdc), and samples collected in pantanal of mato grosso. acknowledgements we thank the coordenação de aperfeiçoamento de pessoal de nível superior (capes), and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for a phd grant to eliandra meurer, and a research grant to jacques c. h. delabie. further financial and logistic support e meurer, ld battirola, mi marques1, jhc delabie new records and distribution of the genus ochetomyrmex268 came from the programa de pós-graduação em ecologia e conservação da biodiversidade at the instituto de biociências of ufmt, and the centro de pesquisas do pantanal (cpp). 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(2003), based on the heterogeneity of montane habitats and their endemic species, presenting different types of rocky woody vegetation, from grasslands to low, dense forests (alves et al., 2014). this ecosystem is in a transition abstract the rupestrian complex is a montane transitional vegetation type between the brazilian savanna (‘cerrado’) and the atlantic forest, frequently threatened by human activities. in this study, we evaluated the recovery to fire disturbance of ant fauna in an environment evolved under fire regime. we confirmed that the ant diversity recovers quickly after the fire. however, our results show that ant assemblage in burned areas presented greater foraging activity, here detected as higher abundance. the ant composition changed over time, being that species turnover lead to a strikingly different species composition comparing burned to unburned areas after 16 months of recovering. in fact, both areas changed their ant composition through species turnover, but we believe that the mechanisms that act in species turnover are different in each area. along time, in burned areas the fauna maintained a constant species diversity but dramatically changed species assemblage due to appearance of several species not found in the unburned area. sociobiology an international journal on social insects dv anjos1, rbf campos1,2, sp ribeiro1 article history edited by gilberto m. m. santos, uefs, brazil received: 01 december 2014 initial acceptance: 31 march 2015 final acceptance: 01 may 2015 keywords natural succession, burn, rupestrian complex, species substitution. corresponding author renata bernardes faria campos sala 01 – pva – campus ii univale av. israel pinheiro 2000, bairro universitário, governador valadares-mg, brazil e-mail: rbfcampos@gmail.com between brazilian savanna (‘cerrado’) and atlantic rainforest being many plant species common to these two biomes (harley, 1995; viana & filgueiras, 2008), thus it is composed by species adapted and not adapted to fire. this ecosystem is frequently threatened by human activities (see fernandes et al., 2014), and little is known about the effects of fire on communities associated with rupestrian complex vegetation, increasing the importance of the present study. fire may reduce the faunal diversity directly through mortality or evasion, and indirectly through habitat change and food shortage (frizzo et al., 2011; fagundes et al., 2015). however, the effects of fire can be specific and some taxa can benefit (frizzo et al., 2011) while others perish (andersen & muller, 2000; vasconcelos et al., 2009). ant diversity is directly affected by vegetation structure, mostly by the occurrence of plants producing extrafloral nectar, hosting honeydew-hemipterans and providing cavities for nesting (farji-brener et al., 2002; parr et al., 2004; ratchford et al., 2005; fagundes et al., 2015). in environments with vegetation adapted for and more resilient to burning, one may expect a research article ants 1 universidade federal de ouro preto, ouro preto, mg, brazil 2 universidade do vale do rio doce, governador valadares, mg, brazil dv anjos, rbf campos, sp ribeiro ant temporal turnover of species recovering from fire disturbance390 quick recovery of plants (hoffmann et al., 2003; simon et al., 2009) and consequently, a quick ant assemblage recovery (parr et al., 2004; ratchford et al., 2005). in fact, many studies did not detect decrease in ant diversity after fire (parr et al., 2004; andersen et al., 2006; arnan et al., 2006), and the fire effects may disappear after two years of recovering (vasconcelos et al., 2009). thus, we can predict that the ant assemblage might quickly return to an unburned conduction, especially in ecosystem adapted to fire (ratchford et al., 2005). ants may be important species in the beginning of the ecological succession after fire, because many species are generalists and tolerant to intense fluctuations of abiotic conditions (andersen & muller, 2000; campos et al., 2007; costa et al., 2010). thus, ants act as pioneers in the secondary succession by performing several ecological functions (fowler et al., 1991) affected by the fire (andersen & muller, 2000). in this context, we evaluated the ant fauna recover after a fire in an ecotonal zone between grassland and montane low forest, within a typical rupestrian complex vegetation. since the studied sites present a long term history of disturbance by fire, we expect that this is a fire resistant ecosystem. we tested the hypothesis that the ant diversity recovers quickly after the fire, considering that the ant assemblage may show a faster recovery than the vegetation. we also tested the hypothesis that the succession dynamics of ant assemblage must be different between areas, so we expected a higher species turnover along time in burned than in the unburned areas, where we expect to find an ant species composition nestedness pattern. material and methods study area we performed the study in the parque estadual do itacolomi (peit) (20°22’30” s and 43°32’30’’ w), located in ouro preto, minas gerais state, brazil. the park was created in 1967 with an area of 7,543 ha, and the vegetation is composed of seasonal forests, gallery forests, forests of the candeia tree [eremanthus erythropappus (dc.) macleish (asteraceae)], araucaria angustifolia forest remnants, and ferruginous and quartzite grasslands (pedreira & souza, 2009). we conducted the survey in a rupestrian ecosystem which is a complex mosaic of old secondary forests and areas where early successional stages of a forest are kept as grasslands possibly due to a long term human impact, along with natural grasslands, also depauperate of species if compared with other rupestrian grasslands. even if, peit is not highly diverse as typical rupestrian grasslands from other places as serra do cipó (giulietti& pirani, 1988). gastauer et al. (2012) evidences the park’s classification as a local hotspot of biodiversity. the climate presents mild temperate with dry and warm summers; the average annual temperature ranges from 17.4 to 19.8 °c and the average annual precipitation ranges from 1,100 to 1,800 mm. the relief is mountainous, with altitudes ranging from 900–1,772 m a.s.l. (itacolomi peak summit). we surveyed an area of four hectare of burned rupestrian complex vegetation (hereafter “burned area”) with a record of frequent fire in the last decade and compared it to the sampled ant fauna with a vicinal portion of this area, with unburned vegetation considered as our reference (hereafter “unburned area”). both areas are situated above 1200 m a.s.l, have low declivity, similar soil structure and vegetation dominated by grass and herbs with many shrubs sparsely distributed. in each area, we marked one 15 m x 20 m grid. the grids were 300 m apart in order to reduce effects of environmental variations (vasconcelos et al., 2008). within each grid, we demarcated four lines with five pitfalls trap 5m apart each other. the pitfall was a 20ml plastic cup with 3cm diameter and 10cm deep filled with a solution of water, salt (5%) and a few drops of detergent to break the water tension. the pitfalls stayed in the field for 96 hours. the first sample was four months after burning (october/2010), in the beginning of the rainy season. we did 12 samples monthly. the ants were identified according to fernández et al. (2003) and by comparison with morphotypes of the entomology collection of the departament of environment, evolution and biodiversity of federal university of ouro preto, ouro preto, brazil. voucher specimens were deposited in this collection. data analysis we first plot curves of accumulated species richness to compare burned and unburned areas, to allow us to assess if our 16 months of species survey were sufficient to reach completeness (a plateau). since pitfall traps measure active ants, we used species frequency of occurrences per sampling (pitfall trap), on the x axis. for the subsequent analyses, we considered the monthly sum of workers (abundance) and ants species number (richness) collected in the 20 pitfalls of each area as repeated measures (12 samples per area). we tested the difference in ant abundance and ant richness (dependent variables) between burned and unburned areas (fixed factor: area, which corresponds to treatment) using analysis of variance (repeated measures anova) considering the months as random blocks to prevent time effect. finally, we tested the temporal changes of the ant fauna composition through the ‘seriation’ model. this model orders the species throughout a predetermined gradient (recovery time, in our case) using the algorithm proposed brower & kile (1988). then, the program runs a monte carlo simulation creating 30 random matrices from the original data with the same number of occurrences for each taxon. thus, the model test if the observed gradient in the original data is different from a random one. we also verify temporal changes of beta diversity as described by baselga (2010) and baselga et al. (2012) sociobiology 62(3): 389-395 (september, 2015) 391 to find what mechanism (turnover or nestedness) determine the beta diversity at different scales, spatial and temporal. in this study, this analysis was used to understand what is the main mechanism influencing the beta diversity temporally in burned and unburned areas. so we expected a higher temporal turnover in burned areas and a temporal nestedness in the unburned areas. seriation test was carried out using past v3.01 and all other analyses were carried out in r (r development core team, 2006). results we collected 1706 of ants belonging to 55 species. the burned area present 1203 (70.5%) ants from 46 species, while the unburned area had 503 (29.5%) ants from 39 species (table 1). the mean abundance of ants (ants per month) was higher in the burned area compared to unburned area (anova: f1, 11 = 11.1. p = 0.007) (fig 1a). on the other hand, the mean species richness did not differ between areas (f1, 11 = 0.6, p> 0.5) (fig 1b).the mean abundance (f1, 11 = 9.2, p = 0.0005) and mean richness (f1, 11 = 7.2, p = 0.002) of ants decreased over the time (fig 1a, b). the curve of accumulated species richness indicate we have a plateau in both areas, and we have a separation from each other considering the last samples added only (fig 2). from the total of 56 sampled species, 31 (56%) were common in both areas. the burned area presented 16 (30%) exclusive species while unburned area presented 8 (14%) (table 1appendix). we observed a temporal turnover of species in the burned area is nonrandom (seriation: monte carlo, z = 2.46, p = 0.01), differently from the unburned area (z = 0.4, p = 0.7) (fig 3). therefore, in the burned area there was a directional variation in species composition than in the unburned area, where the change in the composition did not differ from that expected by chance. our results shows that in both areas temporal turnover contributed strongly to the beta diversity. turnover explains 98.7% of beta diversity from burned area (βsor = 0.991: βsim = 0.978, βnes = 0.012) and 98.6% of beta diversity from unburned area (βsor = 0.992: βsim = 0.978, βnes = 0.013). fig 1. total ant richness and abundance in studied areas along 12 months. burned area was impacted four months before the beginning of the study, while the unburned area was not burned since the last decade. the two areas did not differ significantly with respect the richness (a), while the abundance is lower in the unburned area (b). dv anjos, rbf campos, sp ribeiro ant temporal turnover of species recovering from fire disturbance392 responses regarding the ant abundance (andersen & yen, 1985; andersen, 1991; parr et al., 2004) we demonstrate that burned areas presented higher abundance than unburned area in the short term. moreover, our results indicated that the ant assemblage’s species composition changed over time after the fire as intensively as in the unburned area. however, in the former this had a trend into a new fauna instead of being a random variation in sampled ant species. still, in both areas species changes were defined by substitution and not by nestedness patterns (i.e., loss of part of an expected fauna from global data). although this mechanism explains the change within both studied areas, we emphasize that changes in the burned area differed from that expected just by chance while this change in the unburned area is random, as indicated by seriation test. there are two likely explanations to the maintenance of mean species richness after fire. first, the ant species richness was not affected by fire impacts. this statement sounds unlikely because the fire has direct effects on mortality of species that are foraging at the moment and indirect effects in habitat, indicating that any effect on ant community must exist (gillon, 1983; farji-brener et al., 2002; frizzo et al., 2011). nevertheless, ants with nests underground may have higher tolerance to fire since soil protect the nest from burning, than other ground-nesting species (miranda et al., 1993). ants nesting below-ground would recover rapidly because they could re-enter the above-ground habitats after disturbance from their protected nests in the soil (campos et al., 2007). secondly, in case of having severe fire effects, it is possible that the ant diversity had recovery four months after the fire passage only. since the impressive ant resilience to fire (parr et al., 2004, barrow et al., 2007), we believe this short time was enough for the ant diversity recovery. for instance, there is data suggesting that diversity of ground ants in the rupestrian complex vegetation recovers faster than in the savanna (parr et al., 2004; vasconcelos et al., 2009). however, these effects depend on the intensity and frequency of disturbance, habitat susceptibility and community resilience (farji-brener et al., 2002; ratchford et al., 2005; barrow et al., 2007). regardless the absence of difference in ant mean species richness, the ant mean abundance was higher in the burned area than in the unburned area. maravalhas & vasconcelos (2014) also did not found significant differences in the ant species richness between distinct regimes of fire. the lack of effects of fire on the ant assemblage species richness is contradictory to common sense, as many studies have shown null or positive effects of it (parr et al., 2004; andersen et al., 2006; arnan et al. 2006) and as it is visually a severe impact. nonetheless, an actual fire effect exists, but influences foraging and dominance. both increasing ant abundance in the burned area. the ant abundance may increase after the fire for two possible and additive reasons: immediately after fire, the lack of vegetation layers increases the foraging activity on the ground, as usually seen in open environment (andersen & yen, 1985; whelan, 1995); secondly, after fig 2. curve of accumulated species richness per point over 12 months sampling, where the shaded area corresponds to the standard error. in dark gray the accumulated richness in burned area four months before the first sampling and in light gray accumulated richness in the unburned area. fig 3. temporal changes in the ant species fauna of both studied areas through the model ‘seriation’. a gradient of species replacement over time was observed in the burned area (right panel) and which did not occur in the unburned area (left panel). the species names are in abbreviations, which grey for exclusive species and white for represent common species. discussion in this study, we refuted the hypothesis that fire changes the local ant mean species richness immediately after the burning, but we suggest that the effect acts in ant foraging rates, based on abundance data, such as found in andersen et al. (2006). although some studies have showed contrasting sociobiology 62(3): 389-395 (september, 2015) 393 the fire impact, vegetation grew up and germinated quickly, increasing availability of resources (alves-silva & del-claro, 2013; fagundes et al., 2015). this result corroborates with parr & andersen (2008) who also found that over the first four months after fire there was a considerable increase in the abundance of ants. fagundes et al. (2015) found higher abundance of cavity-nesting ants, which usually foraged in the ground, in the burned than unburned area. fire may affect the natural succession process creating opportunities for different species over time, whereas some species are favored and other are not (andersen & muller, 2000). this succession dynamics was perceptible at any sampled month, due to the species turnover. such high species/abundances changes along short periods of time are in support of our view that our sample design is not ecologically pseudo-replicated, as it detects a relevant ecological process. moreover, it is important to consider that sampling began in the rainy season when environmental conditions (high humidity, precipitation and temperature) also stimulate forage activity (kaspari& weiser, 2000), and this effect may be more evident in burned area (arnan et al., 2006). beyond that, the ant’s reproduction period also occur in the rainy season. the high colonization rate associated with low competition may be important to explain the observed species turnover being the principal mechanism related to beta diversity along time. in other hand, we unexpectedly found the same pattern in the unburned area, suggesting that species turnover may result from temporal partition of activity between species. anyway, after disturbance, the species richness may recover and different species composition may take place, depending on which species reinvade the litter patch, as occur in litter ant communities in forests (campos et al., 2007), leading to a stronger species changes. in other hand, in the unburned area, we suggest that the ground ants are always changing because the litter is a very perishable environment and in theunburnedarea may have suffered natural and random disturbances (campos et al., 2007). it is worth to notice that disturbance in litter communities occurs frequently because this microhabitat receives a constant input of vegetal and animal detritus, and also because this organic material is constantly subject to decomposition and incorporation into the soil (facelli & pickett, 1991). in fact, litter ant assemblages in tropical forests may attain a high diversity due to a small-scale patch dynamics model, since these communities are naturally and constantly disturbed (campos et al., 2007). in summary, the recovery of rupestrian complex vegetation to fire disturbance occur by at least three distinct ways. first, the ant fauna can be highly resistant but weakly resilient, slightly changing after the fire but becoming extremely different over time. secondly, the ant fauna may be little resistance and resilient, presenting great changes after the fire and slow long-term recovering (andersen, 1991). finally, as our results shown, the ant fauna can be very resistance and resilient to fire, showing little or no change after burning and rapid recovery (parr et al., 2004; vasconcelos et al., 2009). recently, fagundes et al. (2015) found high rates of new ant’s colonies foundation in vegetation in this study site. changes in fire regime in rupestrian complex vegetation can lead to habitat modification, as observed for rainforests, creating opportunities for many species and compensates species loss (cochrane et al., 1999; silvério et al., 2013). despite the fact that both areas have the same mechanism of ant composition changing along time, we found a considerable percentage of exclusive species (30%) in the burned area than in the unburned one (14%). our results showed that in the rupestrian complex vegetation ant fauna recover before the vegetation. parr et al. (2004) and andersen et al. (2014), suggest that little intense fire does not have effect on ground ant fauna, and our results indicate that the ant fauna in rupestrian complex vegetation are highly resistant and resilient to fire disturbance. nevertheless, we observed species turnover over time, common in both areas but possibly due to different causes. in fact, we did conclude that the ant fauna in burned area presented composition changes higher than expected by chance and higher abundance, indicating increased activity of foraging, than in unburned area. acknowledgments we are grateful to dr. r fagundes for suggestions which improved the quality of the manuscript; dr. fs neves and his helpful in statistics analysis; j. aidan manubay from gw university for his help with english language. we thank instituto chico mendes (icmbio) and instituto estadual de florestas (ief) for permission to research, federal university of ouro preto and laboratório de ecologia evolutiva de insetos de dossel e sucessão natural for logistic support. we also thank propp/ufop, capes and cnpq for financial support. spr is a cnpq researcher granted. references alves, r.j.v., silva, n.g., oliveira, j.a. & medeiros, d. 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(1995) the ecology of fire. united kingdom: cambridge university press. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i1.1-9sociobiology 62(1): 1-9 (march, 2015) availability of food and nesting-sites as regulatory mechanisms for the recovery of ant diversity after fire disturbance introduction food sources and nesting-sites are important factors limiting the development of ant colonies (mcglynn, 2006; peeters & molet, 2010). this limitation reduces the population growth and changes the species composition of the community (mcglynn, 2006; shik & kaspari, 2010). the availability of nesting-sites is particularly important for species that nest in hollow twigs, the cavity-nesting ants (ribas et al., 2003; philpott & foster, 2005; houdeshell et al., 2011). on the ground, these ants use cavities in dead branches or trunks, and curled dry leaves to nest (kaspari, 1996; philpott & foster, 2005; friedrich & philpott, 2009). while in the foliage, living and dead hollow abstract nest-site is an important resource for cavity-nesting ants, what limits colony establishment and structures ant community composition through competition. in ecosystems frequently disturbed by fire continuous establishment of new colonies is crucial to the process of natural succession. based on this perspective, we tested the hypothesis that fire reduces the amount of cavities for nesting (e.g., hollow branches, dry leaves curled, and galls), with negative impact on ant biodiversity. we searched for natural cavities and added artificialnests to assess the occupancy rate and its consequences for colony growth. we also evaluated the availability of food sources for ants (efn plants, honeydew-hemipterans and preys). we found that burned areas had less diverse and structurally simple vegetation. the occupation of natural and artificial nests was the same between the areas, but the reduced availability of nesting-sites in the burned area indicates higher limitation after the fire. this effect was even stronger in foliage habitat compared to the ground. in fact, most of the 11 cavity-nesting species found were typically arboreal. species richness was lower in burned area, possibly due to lower nesting-sites availability, but the abundance was higher, which may be explained by the greater availability of food resources, mainly efn-bearing plants. the high food availability may also explain the bigger colony size in burned area, since nectar and honeydew boosts colony growth and low richness prevents competition. in summary, our results show that changes in the availability of nesting sites and food resources may be key mechanisms by which fire changes the ant fauna, specifically cavity-nesting ants in the brazilian savanna. sociobiology an international journal on social insects r fagundes1,2, dv anjos2; r carvalho3, k del-claro1 article history edited by gilberto m. m. santos, uefs, brazil received 09 october 2014 initial acceptance 29 november 2014 final acceptance 07 february 2015 keywords limiting resources, ant nest, competition, cerrado, environmental disturbance. corresponding author roberth fagundes souza laboratório de ecologia comportamental & interações, instituto de biologia universidade federal de uberlândia uberlândia, minas gerais cep 38400-920, brazil e-mail: roberthfagundes@gmail.com twigs, and domatias are the main nesting-sites (philpott & foster, 2005; rico gray & oliveira, 2007; friedrich & philpott, 2009). the durability of these nesting-sites is low in both habitats, leading to constant migrations of ant colonies (sagata et al., 2010). this dynamic colonization makes the amount, size, availability and diversity of nesting-cavities important limiting factors of ant distribution (sagata et al., 2010). environmental disturbances, such as fire, increase the effect of the limiting factors of ant diversity and distribution (philpott & foster, 2005; lach et al., 2010). fire removes vegetation, destroys the litter layer (important habitat for ants), reduces the availability of prey, alters the chemical composition of the soil and enhances the effect of stressful research article ants 1 universidade federal de uberlândia, uberlândia, minas gerais, 2 universidade federal de ouro preto, ouro preto, brazil 3 universidade federal de lavras, lavras, brazil r fagundes, dv anjos; r carvalho, k del-claro mechanisms of fire impacts on ant diversity2 climatic conditions (mistry, 1998; lach et al., 2010; frizzo et al., 2011). in fact, fire reduces the availability of nestingsites leading to loss of ant diversity (philpott & foster, 2005; friedrich & philpott, 2009). this is particularly harmful for species of tree-dwelling ants, that require branches and trunks to build its nests, and ground-dwelling ants that nest in hollow cavities (i.e. leaves and twigs; friedrich & philpott, 2009). thus, in recently burned areas, one can predict that the availability of nesting-sites can be a limiting factor for recovery of the ant fauna, once species that require specific locations for nests cannot colonize (morais & benson, 1988). the cavity-nesting species may be the main group affected by the fire because they lose their habitat, forage areas and their nesting-site, which are often specific (yamamoto & del-claro, 2008; houdeshell et al., 2011). nesting-sites may be a limiting factor for ant diversity when: a) ants occupy high proportion of available nesting-sites; b) ants occupy artificialnests introduced in the environment, which reflects a saturation of natural nesting-sites; and c) when ants compete for nestingsites and steal cavities of other ants (byrne, 1994; philpott & foster, 2005; sagata et al., 2010; houdeshell et al., 2011). however, knowledge about nesting-site limitation remains scarce (philpott & foster, 2005; houdeshell et al., 2011). recent studies have shown that, in general, fire reduces the ant richness and abundance (vasconcellos et al., 2009; lach et al., 2010; frizzo et al., 2011). however, the mechanisms behind the effect of such disturbances on community structure have received little attention (andersen et al., 2006; houdeshell et al., 2011). in this study, we tested the hypothesis that fire regulate ant diversity changing food and nest-site availability. we predict that the reduction in vegetation diversity and structure (i.e. size) caused by fire leads to a reduced number of nesting-sites in burned areas. we also predicted, that new cavities will be more intensely and quickly occupied by ants in burned areas. we tested it using artificial nests as proposed by friedrich and philpott (2009). moreover, we tested the hypothesis that colonies in burned areas grow less, due to reduced availability of food resources. at last, we hypothesized that nest-site occupancy may reflect in higher colonization success, thus, we predict that ant species with higher occupancy rate would present higher local abundance. materials and methods study area we performed this study in an area of rocky montane savanna, known as rupestrian fields (campos rupestres, alves et al., 2014) between october 2012 and february 2013. the area belongs to the parque estadual do itacolomi (peit) (20°22’30” s and 43°32’30’’ w), located in ouro preto city, in the state of minas gerais, brazil. rupestrian fields are a type of cerrado ecosystem, the brazilian tropical savanna, composed of wet grass fields with patches of shrubs; the vegetation grows in quartzite or iron rock outcrops in elevation higher than 1000m (giuletti et al., 1997; alves et al., 2014). the climate in peit is temperate humid with 1700 mm of mean annual rainfall and mean annual temperature of 18.5° (giuletti et al., 1997). to perform the study, we used two sites within a rupestrian field area (proximately 10 ha), one area burned eighteen months prior the experiments (hereafter burned area) and one unburned area (hereafter control area). fire events are very common in the brazilian savanna directly interfering in biodiversity and interactions (alves-silva & del-claro, 2014). rupestrian fields, as well as the brazilian cerrado, is often disturbed by fire (kolbek & alves, 2008; alves et al., 2014). in fact, it is an ecosystem with many plant species adapted to resist fire (rapini et al., 2008). therefore, a suitable environment for studying the impacts of fire on biodiversity both due to recurrence of fire (increased by human activity) but also because of the urgency of conservation (jacobi & carmo, 2008; alves et al., 2014). effects of fire in the environment we started by evaluating the effect of fire in the environment in october of 2012. we drew one 300x1m transect in the burned area and one in the control area to evaluate differences in the vegetation. we identified all shrubs inside the transect line. additionally, we used one at each 10m in the transect line to evaluate differences in plant morphology (30 shrubs total). we measured plant height, crown diameter and number of branches. we also evaluated differences in the food availability. thus so, using the same transects, we quantified the number of plants with extrafloral nectaries (efns) and honeydew-producer hemipterans, since nectar and honeydew are important items in ant diet in the brazilian tropical savanna (del-claro & torezan-silingardi, 2009; lach et al., 2010; byk & del-claro, 2011; lange & del-claro, 2014). additionally, we used pitfall traps to evaluate the number of preys available for ants. we draw four 20x1m transects separated by 5m. in each transect we installed a pitfall trap every 5 m, one in the ground and one in the foliage (1m height). the pitfall consists of a 20 ml cup with 3 cm diameter filled with a solution of water, salt (5%) and a few drops of detergent to break the water tension. the pitfalls remained on the field for 96 hours. after that, the arthropods collected were separated in ants and nonants specimens. from the last group, we quantified potential ant preys by counting all coleoptera, hemiptera, diptera, collembola and larvae sampled. we used the ants sampled to quantify ant diversity by abundance and richness. natural-nests sampling we searched in the field for cavity-nests and identified the ant species found in these nests. we searched the nests in three 100x1m transects in each area by scouring the litter on sociobiology 62(1): 1-9 (march, 2015) 3 the ground and searching for hollow twigs and curled leaves; we also assessed the foliage to search for live or dead hollow twigs, domatias and abandoned galls. all twigs were broken and considered as occupied by a colony when we found brood, workers or queens. sometimes we could not assure the presence of a queen, but we considered as a colony whenever at least brood was present (friedrich & philpott, 2009). then, we quantified the number of nesting-sites occupied and collected some individuals for taxonomic identification. we used this information to separate the ants collected by the pitfalls in cavity-nesting ants and other ants (those not found nesting in cavities). artificial-nests sampling we evaluated the limitation of nesting-sites using artificial nests to quantify the number of occupied artificial-nests (new nests formed) as well as the size of the established colony (number of individuals, sensu friedrich & philpott, 2009). in each area, we draw two transects of 100m separated by 500m. we installed one artificial nest in the soil and one in the vegetation 10m apart (1.5 m height; total of 40 nests per area). the artificial nest constituted of falcon® test tubes of 30ml, with 10cm length and 2cm diameter, to mimic a cavity, lined inside with a 10x10cm paper cardboard to keep dark, and a cotton ball in the tube bottom to keep moisture. the height and diameter of the tube were chosen to maximize the efficiency of the trap, reducing the sampling effort by dismissing inefficient types of artificial-nests (philpott & foster, 2005; cobb et al., 2006; friedrich & philpott, 2009). there is no difference in results compared to artificial-nests made with bamboo (natural materials) or test tubes (artificial material), considering the pipe size used in this study (friedrich & philpott, 2009). since we are not testing preferences for types of nests, in fact we wanted to reduce this effect, we preferred to use plastic tube for standardization of artificial-nests. artificial nests remained in the field for four months (philpott & foster, 2005; friedrich & philpott, 2009) during the rainy season (october-january 2012/2013) to coincide with the period of ant’s reproduction, when the establishment of new colonies is higher. the artificialnests were placed declined (10°) so that rainwater does not accumulate inside the artificial-nests. however, this has proven to be unnecessary because the ants closed the openings of the tubes using carton material, a mixture of crushed dried plant material and saliva commonly used in building nests by arboreal ants (hölldobler & wilson, 1990), avoiding water entrance. at the end of the sample period, we collected the artificial-nests and then quantified the ant colonization. we also quantified the number of workers, immatures (larvae and pupae) and eggs found inside each artificial nest. for the analysis, these numbers were divided by the number of days the artificial-nest was occupied by the colony (from the queen arrival to the collection day) to remove time effect. data analyses we compared the plant diversity using diversity t-test based on shannon index. we used multiple analysis of variance (manova) to compare the size of the plants. we considered height, crown diameter and number of branches as dependent variables and the areas (burned and control) as fixed factor. we compared the number of preys between the areas and the habitats (ground and foliage) using two-way analysis of variance (anova). we compared the number of efnbearing plants and hemipteran-host plants in the flora between the areas using chi-square test. we compared the percentage of cavity-nesting ants (transformed by square-root arcsin), over the total ant abundance and richness per plant between the areas and habitats using two-way anova. we compared the percentage of occupied natural and artificial-nests between areas and habitats using general linear model (glm), accepting binomial distribution of error and logit link function. we compared the size of the colony between the areas and the habitats using glm with poisson distribution and log-linear link function. we used this approach to compare the number of eggs, immature and workers per colony between the areas and habitats. we evaluated the relation between the number of nests and the local ant abundance (continuous factor) in both areas (fixed factor) using glm, accepting poison distribution and log link function. all statistical tests were performed in past statistical software version 2.16. results effects of fire in the environment the burned area presented less plant diversity (20 species, h’ = 2.15) than the control area (31 species, h’ = 2.62; diversity t-test: t = 2.75; d.f. = 203.86; p = 0.007) even after 18 months of recovery. moreover, the plants in the control area were higher (mean ± standard deviation: 1.25 ± 0.37m, n = 30), had more branches (77.07 ± 61.63, n = 30) and wider crowns (0.7 ± 0.21 m, n = 30) than in burned area (0.57 ± 0.22m, 24.07 ± 15.17, 0.55 ± 0.15m, n = 30; manova: f3, 56 = 27.24, p < 0.001). thus, fire reduced the potential cavities to ant nests by simplification of vegetation structure. burned areas also had less prey for ants (0.65 ± 0.15, n = 20) than control area (1.85 ± 0.31, n = 20) (anova: f1, 76 = 13.25, p < 0.001). no difference in prey availability was found between ground and vegetation in any area (f1, 76 = 2.91, p = 0.09). on the other hand, the number of extrafloral nectar sources was higher in the burned area (32 efn-bearing plants, 30%) than in the control area (10%) (χ2 = 11.28, d.f.=1, p<0.001). we found no difference between burned (29 plants with hemipterans, 30%) and control area (34, 35%) in the number of honeydew sources (χ2 = 0.33, d.f.= 1, p=0.57). we do not found efn-bearing plants hosting hemipterans. r fagundes, dv anjos; r carvalho, k del-claro mechanisms of fire impacts on ant diversity4 natural-nests sampling we found 11 ants species nesting in cavities (table 1). the burned area had less species (6) than control area (9), with two exclusives species against four in the control area. moreover, we observed two-fold more species nesting on foliage (burned: 6, control: 7) compare to the ground (3, 4) in both areas. camponotus rufipes and c. crassus were the most common species nesting in the ground, while cephalotes pusillus was the most common nesting in the foliage. according to the pitfall samples, cavity-nesting ants represented 49% of 140 sampled ants and 29% of the 38 species collected in both areas. the ant fauna in the burned area presented higher ant abundance per pitfall (f1, 76 = 6.78, p = 0.01) but lower percentage of cavity-nesting ants (f1, 76 = 11.63, p = 0.01) (figure 1a). we also found higher richness in burned area (f1, 76 = 9.88, p = 0.002), but lower richness of cavity-nesting species (f1, 76 = 10.36, p = 0.002) (fig 1b). foliage showed higher percentage of cavity-nesting ants regarding the total abundance (f1, 76 = 6.25, p = 0.01) and richness (f1, 76 = 5.86, p = 0.02) of ants, in both areas (f1, 76 = 1.45, p = 0.23; f1, 76 = 0.93, p = 0.34) (fig 1). we found 66 nesting-sites in the burned area (19 colonized) and 82 in the control area (26 colonized). however, there was no difference in the occupancy (χ² = 0.15, d.f. = 1, p = 0.7). apparently, the number of nesting-sites found in the ground (burned: 42; control: 32) was similar to that found in the foliage (40; 34) in both areas. however, the nesting rate in the foliage (burned: 44%, control: 43%) was higher than in the ground (13%, 21%) in both areas (glm: χ² = 8.04, d.f. = 1, p = 0.001; χ² = 4.2, d. f. = 1, p = 0.04; fig 1). nests abundance formicidae subfamily dolichoderinae linepithema micans forel 1908 2 7 subfamily formicinae brachymyrmex sp.1 2 2 camponotus alboannulatus mayr 1887 1 3 camponotus crassus mayr 1962 16 14 camponotus rufipes fabricius 1775 7 10 camponotus senex smith 1858 1 1 subfamily myrmicinae cephalotes pusillus klug 1824 19 14 crematogaster goeldii forel 1903 3 1 crematogaster sp.1 1 1 crematogaster sp.2 5 5 solenopsis sp.1 3 1 table 1. ant species nesting in cavities found in burned and unburned areas in a brazilian savanna area. columns show nest quantity assessed with trap-nests and active search and local abundance assessed using pitfall traps. artificial-nests sampling we found 15 colonies occupying artificial-nests with the presence of queens, workers, soldiers, eggs and pupae. only two of the 11 cavity-nesting species occupied the artificial-nests: c. crassus and c. pusillus (table 1). the general nesting rate was 18.8%. the burned area had 22.5% occupied nests, while the control area had 15%. we found no difference between the rate of nesting in burned and control areas (glm χ² = 0.83, d.f. = 1, p = 0.36). however, nesting was higher in the vegetation (30% occupancy) than in the soil (7%) (glm χ² = 6.88; d. f. = 1; p = 0.01) (fig 2). we found differences between colonies of burned and control areas. the number of queens was always one per colony, but colonies had more individuals in burned than control areas (glm: eggs: χ² = 33.7, d.f. = 1, p < 0.001; immatures: χ² = 47.13, df = 1, p < 0.001, workers: χ² = 10.11, d.f. = 1, p < 0.001; figure 2). however, this difference was greater for colonies in soil, which hardly grew in the control area, as in initial fig 1. contrasts between burned and unburned areas of cerrado for ant diversity. ants presents higher abundance (p = 0,01) in the burned area but lower percentage (p = 0,01) of cavity-nesting ants (a). the same is true for ant richness (p = 0,002) and percentage of cavity-nesting species (p = 0,002; b). sociobiology 62(1): 1-9 (march, 2015) 5 fig 2. number of natural (a) and artificial (b) nesting-cavities placed on the ground and foliage, occupied by ant colonies in burned and recovered areas of cerrado. the burned area presents lesser availability of nest-sites, but the proportion of occupied nests was not different between burned and non-burned habitats for natural (p = 0.04) and artificial (p = 0.36) cavities. the proportion of occupied nests was higher in vegetation for natural (p = 0.04) and artificial (p = 0.01) cavities. fig 3. differences in size (number of eggs [a], immature [b] and workers [c]) of colonies established in artificial cavities placed on the ground and foliage of burned and recovered areas of cerrado. colonies established in cavities placed on the ground grew less in control areas (p = 0.001 for all variables), but no difference occur for nests in cavities on the foliage. in general, colonies of burned areas present higher number of individuals (p = 0.0001 for all variables). state had few workers and immature (glm: eggs: χ² = 42.8, d.f. = 2, p < 0.001; immature: χ²: 262.5, d.f. = 2; p = 0.0001; workers: χ² = 116.8; d.f. = 2; p < 0.001; fig 2). regarding vegetation nests, the size of the colonies was almost the same between the areas (fig 3). at last, we found a positive relation between the number of occupied nests and the local abundance (glm: χ² = 11.6, d.f. = 1, p = 0.006) consistent in burned and unburned area (glm interaction: χ² = 0.13, d.f. = 1, p = 0.72). discussion in this study, we found that the availability of nestingsites might be an important limiting factor for the diversity of cavity-nesting ants in a brazilian savanna, mainly for treedwelling species. although nesting rate was similar between burned and control areas, burned areas presented plants with small size, less number of stems and reduced crown, then with a reduced number of potential cavities to be used by ants. thus, we confirmed our hypothesis that nesting-sites would be a more limiting resource in burned areas. this effect was stronger on foliage because the nesting rate was higher than in the ground, but the number of nesting-sites available was similar. moreover, the abundance of cavity-nesting ants was higher in burned areas, but the number of colonies and richness of species r fagundes, dv anjos; r carvalho, k del-claro mechanisms of fire impacts on ant diversity6 was lower. this scenario may reflect higher competition for nesting-sites leading to low coexistence between species, but lower competition for food, leading to higher abundance. burned areas presented higher number of efn-bearing plants, and nectar is a primary food resource for cavity-nesting ants, leading to intense colony growth and higher abundance (e.g. byk & del-claro, 2011). our results surpassed the expected in the literature for ants nesting in natural or artificial cavities (philpott & foster, 2005; ambrecht et al., 2006; cobb et al., 2006; davidson et al., 2006; fagundes et al., 2010; houdeshell et al., 2011). we found 43% occupancy rate of natural-nests, and 19% occupancy of artificial-nests. in rupestrian fields the vegetation is short (giuletti et al., 1997; alves et al., 2014), which may prevent the construction of nests in the canopy, and the soil is shallow and sandy (giuletti et al., 1997; alves et al., 2014), which could hindering the formation of large terrestrial nests, so we supposed that ants may rely mostly in natural cavities to build nests. in addition, many ants possess nest territories that avoid other ants to colonize nesting-sites near the established colonies (lach et al., 2010). so, even when a nesting-site is empty, it cannot be occupied due to territorial interference. we believed that, even with less than 50% of the available nesting-sites occupied, nesting-sites are a scarce resource and the artificial nest occupancy reflects the limiting effect of this resource. we found that burning alters the vegetation structure and then reflects in the nesting-sites availability. first, the burned area had fewer plant species than control areas and most are grasses and herbs, which provides no cavities for nesting (friedrich & philpott, 2009). second, the vegetation structure is simpler in burned areas with smaller and lesser branched plants, which provides few suitable twigs for nesting, since ants prefer twigs thicker than 10mm (philpott & foster, 2005; friedrich & philpott, 2009). the cavities used for nesting come from parts of living or dead plant, thus the availability of this resource is directly related to the vegetation structure (armbrecht et al., 2004). low availability of nesting-sites and then high nesting-site limitation is associated with areas with less complex vegetation structures (armbrecht et al., 2004; philpott & foster, 2005; marquis & lill, 2010). moreover, vegetation diversity is positively related to the occupancy of nesting-sites and composition of cavity-nesting species (armbrecht et al., 2004; philpott & foster, 2005). the availability of nesting-sites is naturally less for arboreal ants, since space is limited to hollow cavities of dead branches or cavities created by beetles and gall, which is scarce and unpredictable (mcglynn, 2006; houdeshell et al., 2011). small shrubs mostly compose the vegetation of rupestrian fields, which prevents the construction of nests with soil or carton in the foliage (yamamoto & del-claro, 2008). thus, natural cavities are essential for the establishment of tree-dwelling ant species (philpott & foster, 2005; yamamoto & del-claro, 2008). this becomes even stronger in burned areas, because vegetation is small and dominated by grasses and herbaceous plants, providing little structure for nesting and preventing colonization (morais & benson, 1988; mistry, 1998). in burned areas, the arboreal ant species that also nest on the ground need to compete for space with the ground ant species, which increases the limiting effect of nesting-sites (frizzo et al., 2011). we found low richness of ants in artificial-nest than natural nests, and this was unexpected (phillpot & foster, 2005; houdeshel et al., 2011). of the 11 species nesting on natural cavities, c. pusillus (on the foliage) and c. crassus (on the ground) occupied most of the artificial nesting-sites. these species are very common and abundant in rupestrian fields, mainly in the tree layer (fagundes et al., 2012; lopes et al., 2012; viana-silva & jacobi, 2012). camponotus species are commonly found nesting in artificial-nests (ambrecht, 2004, cobb et al., 2006, sagata et al., 2010). camponotus crassus nests in soil chambers in the ground having satellite nests in dead hollow branches and collect food more intensely in trees, especially extrafloral nectar of plants and exudates of hemiptera (silvestre et al., 2003; marques & del-claro, 2006; fagundes et al., 2012). cephalotes pusillus build nests on live branches and trunks in the foliage where it forages for the same food of camponotus, besides pollen and animal excrements (byk & del-claro, 2010). camponotus species are fire resistant and have increased in abundance in recently burned areas (frizzo et al., 2011; alves-silva & del-claro, 2013), but cephalotes is considered to be vulnerable to fire (morais & benson, 1988), although we do not found less abundance of c. pusillus in the burned area. reduced availability of nesting-sites in burned area reflected in lower richness of cavity-nesting ants compared to control area. on the other hand, the overall abundance of cavity-nesting ants was higher in the burned area particularly in the foliage. this can be explained in part by the availability of food, once the cavity-nesting ants are adapted to feed on sugary secretions of animals and plants (davidson et al., 2004), which in turn tend to be higher in burned areas (alves-silva & del-claro, 2013). the consumption of extrafloral nectar increases the growth of the colony compared to a predatory diet (byk & del-claro, 2011), and provides energy for intensively forage (davidson et al. 2004), which concentrates on the foliage (blüthgen et al., 2000). dominant ants are commonly associated with the consumption of extrafloral nectar and exudates of hemipteran (bluthgen et al., 2000; davidson et al., 2004). in the study area, c. crassus and c. pusillus are the main species collecting extrafloral nectar (dáttilo et al., 2014). this dominance of highly energetic food sources can provide the energy needed for the dominance of the nesting-sites and explain why those two were the main species occupying the artificial-nests. we did not found difference in the size of colonies grown in burned areas compared to the control areas. colonies in burned areas grew more than in control area, but only for colonies founded on the ground. two hypotheses may explain this scenario. first, as we have shown, the burned area possesses sociobiology 62(1): 1-9 (march, 2015) 7 higher quantity of food resources for arboreal cavity-nesting ants (i.e. nectar and honeydew), especially on the foliage leading to higher colony growth (byk & del-claro, 2011), since food is not limited. second, the competition for preys and space may be lower in burned areas, because predatory ground ant species are few and may not completely have recovered yet. indeed, most of the species occupying natural ground-nesting-sites in burned area consisted of tree-dwelling species, while grounddwelling myrmicinae species occupy cavities in the control area. additionally, ground-nesting species were more abundant in the control area. in short, we believed that competition for nesting-sites between ground-dwelling and tree-dwelling species may have suppressed the growth of colonies founded on the ground of the control area, along with the competition for food resource, since control area had few efn-bearing plants and more preys, favoring predatory ants. more importantly, ants need to found new nest-sites to expand population size and even though with plenty of food resources in burned areas and proper colony growth, the population growth and distribution remain limited by nesting-site availability. the mechanisms of how fire alters the ant fauna are poorly known, and increased competition for resources between species of the same guild such as cavity-nesting ants may be a key mechanism (frizzo et al., 2011; houdeshell et al., 2011; powell et al., 2011). the reduced nesting-site availability helps to explain the low richness in burned areas (powell et al., 2011), while the higher amount of sugary resources help to explain the higher abundance of cavity-nesting ants (blüthgen et al., 2000; fagundes et al., 2012; alves-silva & del-claro, 2013, 2014). however, further studies may focus in how species interacts to determinate which species will dominate the nesting-site, or even if the occupation is opportunistic, and the temporal dynamic of these occupation. including different types of nesting-sites must be also important to reduce effect of ant selective nesting behavior (mallon et al., 2001). cavity-nesting ants may be key species to understanding community structure by competitive mechanisms and the effect of disturbances (fonseca, 1999; del-claro & torezan-silingardi, 2009; houdeshell et al., 2011), especially for species that nest in plants (fonseca, 1999; philpott & foster, 2005; philpott, 2009; powell et al., 2011). some studies show that the effect of fire can be specie-specific, and even though the overall ant fauna may not change after fire (vasconcellos et al., 2009), some species may be damaged (bess et al., 2002; underwood & christian, 2009; houdeshell et al., 2011). the study of disturbance effects in specifics taxa may provide quick answers about disturbance mechanisms. this is even more important in high endemic ecosystems such as rupestrian fields, which are threatened by mining activity and urge for conservation politics (jacobi & carmo, 2008). studies that transcend simple comparisons between areas with different impacts and focus in ecological mechanisms of disturbances should be priority in environmental conservation (lewinsohn et al., 2005; jacobi & carmo, 2008; del-claro & torezan-silingardi, 2009). acknowledgments we thanks to d. lange and e. alves-silva for comments in early version of the manuscript. we are grateful to instituto chico mendes (icmbio) and instituto estadual de florestas (ief) for permission to research (uc:064/11, icm: 28125-1). we thank universidade federal de ouro preto and laboratório de ecologia evolutiva de insetos de dossel e sucessão natural for logistic support. we thank propp/ufop and ufu for financial support. capes (r. fagundes; 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(2008). natural history and foraging behavior of the carpenter ant camponotus sericeiventris guérin,1838 (formicinae, camponotinini) in the brazilian tropical savanna. acta ethologica, 11: 55-65. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.786sociobiology 62(4): 607-609 (december, 2015) natural parasitism in acromyrmex landolti forel (hymenoptera: formicidae) in pastures of bahia, brazil leaf-cutting ants from the atta and acromyrmex genera (myrmicinae: attini) are general pests in agricultural crops, forestry and pastures. the acromyrmex genus is endemic in the americas and its distribution occurs from california (usa) to patagonia (argentina), being present in mexico, central america, as well as cuba and the lesser antilles (trinidad, tobago, corriacou, curaçao and guadeloupe) and in all countries of south america, except chile (gonçalves, 1961; weber, 1972; delabie et al., 2011). some acromyrmex species, like a. balzani (e.) and a. landolti (f.), have specialized in cutting monocots, occurring in high densities of nests and causing severe damage to pastures due to the fact that they cut the grass very close to the ground (mariconi et al., 1963; amante, 1967a, b). in pastures in the municipality of itapetinga, state of bahia, in brazil, the occurrence of a. landolti is common, reaching average density of 260 nests ha-1 (silva júnior et al., 2013). the control of leaf-cutting ants can be accomplished by mechanical, cultural, biological and chemical methods, and chemical control is the only one used on a large scale. abstract this study aimed to evaluate the occurrence of natural parasitism in acromyrmex landolti forel, to identify parasitoids associated to the species and to estimate parasitism rates. the experiment was conducted from january to may 2012 in itapetinga, ba. a sample comprised of 20 nests was studied. nests were excavated, eggs, larvae, pupae and adults were removed for counting and parasitism evaluation, isolating adult parasitoids and parasitized ant larvae. parasitism by mimopria sp. (hymenoptera: diapriidae: diapriini) occurred in nine nests, with an average rate of 12% of parasitized larvae. it is the first record of parasitism of mimopria holmgren in an attini species. sociobiology an international journal on social insects ms rodrigues1, ma castellani1, aa moreira1, ms d’esquivel2, eb ribeiro1, lc forti3, s lacau2 article history edited by evandro nascimento silva, uefs, brazil received 24 march 2015 initial acceptance 04 june 2015 final acceptance 07 august 2015 keywords attini, biological control, diapriidae, mimopria. corresponding author maria aparecida castellani department of plant and animal science state university of southwest bahia c.p. 95, cep: 45.083-900 vitória da conquista, bahia, brazil e-mail: castellani@uesb.edu.br the natural biological control by predators, parasitoids and pathogens is an important factor in regulating the populations of these insects. however, there are knowledge gaps for the species of leaf-cutting ants specialized in cutting grasses. among the parasitoids of leaf-cutting ants, a small group of hymenoptera species is known, especially from the diapriidae family. this family has 121 species in 34 genera, which were collected in association with ants, and 26 species in seven genera were recorded with parasitoid behavior (lachaud & pérez-lachaud, 2012). the species doliopria myrmecobia (k.), szelenyiopria pampeana (l.) and trichopria formicans (l.), belonging to diapriini tribe, were recorded in argentina in association with acromyrmex lundii (g.m.) and a. lobicornis (e.) (loiácono et al., 2013). the present study was conducted to evaluate the occurrence of parasitism in colonies of a. landolti, identify the species of parasitoids associated and estimate parasitism rates. the experiment was conducted between january and may 2012, in the lagoa alagoinhas farm (15°21’s/40°17’w), municipality of itapetinga, southwest region of the state of short note 1 universidade estadual do sudoeste da bahia, vitória da conquista-ba, brazil 2 universidade estadual do sudoeste da bahia, itapetinga-ba, brazil 3 universidade estadual paulista, botucatu-sp, brazil ms rodrigues et al. – parasitism in acromyrmex landolti forel in pastures of bahia, brazil608 bahia, brazil. the property comprises the countryside dominated by extensive cattle raising, within an area of pasture (brachiaria sp., poaceae) with 2.7 ha and sub-humid to dry climate, with average annual rainfall and temperature of 800 mm and 25.4°c. a. landolti nests were located, marked with stakes and georeferenced with a gps (global position system), four groups consisting of five nests each were established, named as nuclei, totaling twenty plots, selected at random. nests were excavated by following the procedures described by moreira et al. (2003, 2004a, b), with adaptations. as the nest was dug, the eggs, larvae, pupae, workers and queen were collected with the aid of suction devices (oral suction device) and spoons. the material collected was immersed in ethanol hydrated to 70% in properly identified plastic containers, sent to the laboratory of animal biosystematics lbsa/uesb, for screening and identifying of parasitoids as to their genus according to naumann (1982). after identification, individuals were counted and isolated, considering the nest of origin for subsequent calculation of mean and standard deviation and estimate of parasitism rates. all specimen of wasps found in association with the leaf-cutting ants a. landolti belong to the mimopria genus (hymenoptera: diapriidae). in the excavated nests, adults of the mirmopria sp. were found and parasitized larvae of a. landolti. mimopria (h.), according to masner and garcia (2002), has its distribution in several countries in south america, in tropical lowland areas, from argentina to southern venezuela. in the few studies of parasitism in leafcutting ants, no references were found to describe the action of parasitoid species of the mimopria genus. lachaud and pérez-lachaud (2012), reviewing the diversity of species and behavior of parasitoids (hymenoptera) of ants, do not include in their records the existence of parasitism by mimopria. nine in 20 nests excavated in this work (45 %) presented a. landolti larvae parasitized by mimopria sp. (table 1). considering the total population of larvae of the 20 nests in relation to the total parasitized larvae of a. landolti collected, a parasitism rate of 12% was obtained, with a mean of 16.1 ± 26.9 parasitized larvae per nest (table 1). however, evaluating only the nine parasitized nests, there was a 20.1% rate of parasitism, averaging 35.7 ± 30.4 parasitized larvae per nest (table 1). seven winged adult parasitoids were found and a total of 321 parasitized larvae, the rate of parasitism per nest ranging from zero to 51.6 % (fig 1). table 1. parasitism rate, average and standard deviation of mimopria sp. in larvae and nests of acromyrmex landolti. itapetinga, ba, 2012. fig 1. parasitism rate of mimopria sp. in relation to larvae per nest of acromyrmex landolti. itapetinga, ba, 2012. nests larvae not parasitized (nº) parasitized larvae (nº) total larvae (nº) average parasitized larvae per nest (nº) standard deviation parasitism nests (%) parasitism larvae (%) escavated n=20 2347 321 2668 16.1 26.9 45 12.0 parasitized n=09 1273 321 1594 35.7 30.4 100 20.1 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 pe rc en ta ge o f pa ra si tis m acromyrmex landolti nest larvae parasitized larvae not parasitized 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 pe rc en ta ge o f pa ra si tis m acromyrmex landolti nest larvae parasitized larvae not parasitized the works published on the mimopria genus do not present data regarding parasitism rates in relation to the ants studied. however, fernández-marín et al. (2006) showed parasitism rates by acanthopria spp. of 16.6 ± 2.4% and 34.3 ± 3.3 % for the total number of cyphomyrmex minutus (m.) and cyphomyrmex rimosus (e.) larvae, respectively, when observing the infestation of four morphospecies of acanthopria and one of mimopriella. comparing the data presented, it appears that, for the total number of larvae in parasitized nests, there was a higher incidence of parasitism in c. rimosus, than that occurred for mimopria sp. in a. landolti, however, in c. minutus, smaller quantities of larvae egg-layed by acanthopria sp. were found. thus, the discovery of the unprecedented parasitism of mimopria sp. in a. landolti paves the way for new studies needed to understand the factors related to this association and to the development of management techniques of leaf-cutting ants, considering the possibility of being potential biological control agents of some species of the attini tribe. for the first time, the occurrence of natural parasitism in larvae of a. landolti is recorded, with parasitoids of the mimopria genus. sociobiology 62(4): 607-609 (december, 2015) 609 references amante, e. (1967a). saúva tira boi da pastagem. coopercotia, 23: 38-40. amante, e. (1967b). a saúva atta capiguara, praga das pastagens. instruções práticas dpa 41, 12p. delabie, j.h.c., alves, h.s.r., reuss-strenzel, g.m., carmo, a.f.r. & nascimento, i.c. (2011). distribuição das formigascortadeiras acromyrmex e atta no novo mundo. in t.m.c. della lucia (ed), formigas-cortadeiras da bioecologia ao manejo (pp.80-101). viçosa: editora ufv. fernández-marín, h., zimmerman, j.k. & wcislo, w.t. (2006). acanthopria and mimopriella parasitoid wasps. (diapriidae) attack cyphomyrmex fungus-growing ants (formicidae, attini). naturwissenschaften. 93: 17-21. doi: 10.1007/s00114-005-0048-z. gonçalves, c.r. (1961). o gênero acromyrmex no brasil (hymenoptera, formicidae). studia entomologica. 4: 113-180. lachaud, j.p. & pérez-lachaud, g. (2012). diversity of species and behavior of hymenopteran parasitoids of ants: a review. psyche. 24p. doi: 10.1155/2012/134746. loiácono,m.s., margaría, c.b.& aquino, d.a. (2013). diapriinae wasps (hymenoptera: diaprioidea: diapriidae) associated with ants (hymenoptera: formicidae) in argentina. psyche. 11p. doi: 10.1155/2013/320590. mariconi, f.a.m., zamith, a.p.l., castro, u.p. & joly, s. (1963). nova contribuição para o conhecimento das saúvas de piracicaba (atta spp.) (hymenoptera: formicidae). revista agrícola. 38: 86-93. masner, l. & garcía, r.j.l. (2002). the genera of diapriinae (hymenoptera: diapriidae) in the new world. bulletin of the american museum of natural history. 268:1-138. doi: 10.1046/j.1439-0418.2003.00719.x. moreira, a.a., forti, l.c., boaretto, m.a.c., andrade, a.p.p. & rossi, m.n. (2003). substrate distribution in fungus chambers in nests of atta bisphaerica forel, 1908 (hymenoptera: formicidae). journal of applied entomology. 127: 96-98. doi: 10.1046/j.1439-0418.2003.00719.x. moreira, a.a., forti, l.c., andrade, a.p.p., boaretto, m.a.c. & lopes, j.f.s. (2004a). nest architecture of atta laevigata (f. smith, 1858) (hymenoptera: formicidae). studies on neotropical fauna and environment. 39: 109-116. doi: 10.1080/01650520412331333756. moreira, a.a., forti, l.c., boaretto, m.a.c., andrade, a.p.p., lopes, j.f.s. & ramos, v.m. (2004b). external and internal structure of atta bisphaerica forel (hymenoptera: formicidae) nests. journal of applied entomology. 128: 204211. doi: 10.1111/j.1439-0418.2004.00839.x. naumann, i.d. (1982). systematics of the australian ambositrinae (hymenoptera: diapriidae), with a synopsis of the non-australian genera of the subfamily. australian journal of zoology. 85: 1-239. doi: 10.1071/ajzs085. silva júnior, m.r., castellani, m.a., moreira, a.a., d’esquivel, m., forti, l.c. & lacau, s. (2013). spatial distribution and architecture of acromyrmex landolti forel (hymenoptera: formicidae) nests in pastures of southwestern bahia, brazil. sociobiology. 60: 20-29. doi: 10.13102/sociobiology.v60i2.162-168. weber, n.a. (1972). gardening ants: the attines. philadelphia: american philosophical society. xvii+146pp. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7746sociobiology 69(2): e7746 (june, 2022) introduction one of the eight millennium development goals of the united nations is to ensure sustainable development (unpd, 2012). this implies changing the way food is produced today because what is currently happening is, for the most part, a process of intensifying land usage to increase productivity, which consequently amplifies the negative effects on biodiversity abstract organic farming is becoming more popular as there is a greater demand for pesticide-free food. pest control in organic agricultural production requires a set of skills, including the identification of effective predators and land-use practices. predation by selected coleopteran, dipteran, and hemipteran insects and araneae is well established, whereas the predatory role of ants (hymenoptera: formicidae) has received little attention in the rashad district, sudan. this study was carried out to assess the predation rates of belenois solilucis eggs and the impact of the land use type around the properties on these rates. an experiment involving predation tests on belenois solilucis eggs and fauna sampling were conducted in 18 areas of organic agriculture in the rashad district. the study showed that ants can reduce the eggs population by 26.8% per day. at the same time, other predator taxa, primarily coleoptera, from coccinellidae and staphylinidae families, removed only 13% of the eggs. ant species with the most significant recruiting power were axinidris acholli, tapinoma carininotum, and technomyrmex moerens. ant genera such as linepithema, dorymyrmex, and camponotus ants were also frequently observed. the proportion of the planted area within a 500-meter radius, in addition to the interaction of other landscape categories, had a minor influence on predation, but only when the predators were not ants. the landscape does not affect predation by predators in general, including ants, or on ant predation in particular. sociobiology an international journal on social insects khalid a.e. eisawi1,2, indra prasad subedi3, christine dakele yode4, hong he1 article history edited by evandro nascimento silva, uefs, brazil received 22 january 2022 initial acceptance 23 may 2022 final acceptance 09 june 2022 publication date 24 june 2022 keywords biodiversity, biological control, disturbance, land use, population. corresponding author hong he college of forestry, northwest a & f university, yangling, shaanxi 712100, china e-mail: hehong@nwafu.edu.cn (maitima et al., 2009; taube et al., 2014). on the other hand, increasing land-use intensification is not the only way to increase productivity. according to yanoviak et al. (2009), land use intensifies complex natural systems toward managed and simpler ecosystems, dependent on artificial sums, mechanization, and frequent human intervention. in addition to the loss of species due to intensification, there is a reduction in ecological services 1 college of forestry, northwest a & f university, yangling, shaanxi 712100, china 2 college of forestry and rangeland, university of east kordofan, rashad, sudan 3 central department of zoology, tribhuvan university, kathmandu, nepal 4 felix houphouët boigny university, (ufr biosciences), natural environment and biodiversity conservation laboratory, abidjan, côte d’ivoire research article ants ants (hymenoptera: formicidae) increase predation of belenois solilucis (lepidoptera: pieridae) eggs in organic agriculture production systems: a multiple-site field study at rashad, sudan id mailto:hehong@nwafu.edu.cn https://orcid.org/0000-0001-8267-3855 khalid a.e. eisawi et al. – ants increase pest egg predation in sudanese organic agricultural systems2 such as pollination (nicholls & altieri, 2007), biological control (mori et al., 2013), and this increases the dependence of these systems on human intervention. however, other alternatives, such as organic production (settle & garba, 2011), reduce this impact; although some uncertainties remain about its true environmental benefits (davis et al., 2015). in this type of agricultural production, we highlight the production of organic vegetables, which has a lower “ecological footprint” than grain and meat production (jin et al., 2020) and has the added benefit of being associated with healthier foods owing to the absence of pesticides (popp et al., 2013). the transition from conventional to organic agriculture requires management adjustments that pose specific risks. organic production depends on the restoration and conservation of many ecological services, making sustainable agricultural production viable (röös et al., 2018). therefore, one of the critical needs of farmers is connected to phytosanitary issues with insect pests, which cannot be controled with pesticides in organic systems. as a result, biological control is used (bielza et al., 2020). for example, the use of insects that are parasitoids of agricultural pests is currently a commercially available tool in which the parasitoid efficiently limits the population size of its hosts (georgis et al., 2006). the most widely used and investigated strategy in recent decades to boost biological control while not favoring insects pests has been the increase in plant diversity at scales ranging from the crop plot to the diversity of the agricultural landscape (tuomisto et al., 2012; tuck et al., 2014). an increase in plant diversity at the first trophic level, in general, tends to have a positive influence on natural enemies but a detrimental one on herbivores (de siqueira neves et al., 2011). on the other hand, increased plant diversity leads to increased habitat complexity, which reduces the chance of insect pests being excluded from this environment while decreasing the probability of the population explosion of these insects (karp et al., 2018). these have been studied regarding agricultural landscapes to explain why some areas host more predators and have greater levels of predation on insect pests (morandin et al., 2014). according to these researches, agricultural landscapes with an increase in the complexity or proportion of native areas have more species (reviewed by moser et al., 2002) and higher rates of predation and productivity. however, this impact is also related to the taxon, and some studies did not detect landscape effects (bianchi et al., 2006; rusch et al., 2016). the most studied beneficial organisms within organic areas are coccinellids, often known as ladybugs (coleoptera: coccinellidae) (jones & gillett, 2005), and microhymenopterans. however, in addition to predatory behavior and eusocial organization, referred to as a superorganism (gowdy & krall, 2014), ants possess all the characteristics required to be termed as biological control agents. according to elizalde et al. (2020), ants of the genus oecophylla reduce pests’ abundance and damage while increasing financial gains in a variety of crops. their effectiveness was deemed even better than that of chemical pesticides, with additional advantages of being low cost. based on this information, we sought to assess the contribution of ants to the natural biological control of herbivorous insects in agro-systems and determine which factors influence predation rates in organic areas. specifically, we attempted to know: (1) if ants are a group that effectively reduces the herbivore population by predating on eggs of belenois solilucis butler, 1874; and (2) if these predation rates are explained by the abundance of predators (including taxa other than ants), the occurrence of ants, and land use around and within properties. material and methods study areas this study was conducted in the rashad district from february to june 2020 and february to may 2021, covering an area of 7,872 km² (located in the center of the kordofan between latitudes 10°, 13° n and longitudes 29°, 33° e), in 18 areas of organic agriculture production (eisawi et al., 2021) (fig 1). the distance between the locations was 1.5 km, with each region having a distinct land use. the crops cultivated in each site vary, but mainly include eggplants, okra, carrots, yams, corn, zucchini, tomatoes, and peppers. the cultivation methods are also diverse. some locations have a single type of cultivation, while others have many cultivation lines of different vegetable species. finally, in the same line of cultivation, certain properties combine more than one type of vegetable. in addition, types of agronomic management vary from one property to the next – such as fertilization or the use of natural products to combat pests – making it hard to assess how this affects biological control due to the lack of repetition and gradient of change across properties. sample design the stakes are strong wooden or metal post with a point at one end, driven into the ground to support a tree, form part of a fence, act as a boundary mark, etc. in each location, sixty stakes were installed, including 40 predation stakes, and 20 sampling stakes, totaling 1,080 in 18 location. the stakes were distributed following the cultivation lines, at a distance of 7 m from each other, in the following order: sampling stakes, predation stakes with cards containing 1 to 10 eggs, predation stakes with cards containing 11 to 90 eggs, and so on repeatedly. therefore, 20 sampling and 40 predation stakes were installed in each location, showed in a 413-meter-long straight line. however, because most plots are less than 40 meters long, a second line was installed seven meters away parallel to this one. finally, care was taken to alternate predation stakes with low and high egg densities to avoid eggs concentration in a certain region of the locality and skewing the results. sociobiology 69(2): e7746 (june, 2022) 3 to measure predation rates, a 24-hour experiment was developed, for which a device (called predation stakes) that simulates a plant (fig 2a) was crafted, in which eggs of belenois solilucis (lepidoptera: pieridae), a plague insect of various crops (especially soybean), raised in the laboratory, were made available for use in research. the predation stakes had a height of 25 to 30 cm, and six “branches” on its top, which hold, at the tips, rectangles of green cardboard with approximately 5 x 3 cm, simulating leaves. each of these devices contained: a) a branch with a leaf containing eggs that could be accessed by all possible predators – this leaf was called treatment; and b) a second branch covered with lithium grease (also known as white grease), an odorless, insoluble substance that prevents the transit of unwinged animals, primarily ants, and, at the tip of this branch, a leaf, called control. eggs were also made available in this second leaf, with the care of laying several eggs equal to or nearly the treatment pack in each predation stakes. due to the variation in eggs density from the rearing of belenois solilucis, the age of the colony and the number of eggs available in each pack fig 1. map of the study area showing distribution of sampling sites. southeren kordofan state rashad district khalid a.e. eisawi et al. – ants increase pest egg predation in sudanese organic agricultural systems4 ranged from 1-90 to prevent this from influencing the results. stakes with low density (≤20 eggs) were interspersed with high-density stakes (20 eggs). in addition, to simulate the natural laying conditions of most insect pests, all eggs were made available on the bottom surface of the leaf (face down). finally, before going into the field, the number of eggs in each card was counted and written on the card itself (fig 2b). as a result, the number of predated eggs may be determined immediately after the cardboard is exposed in the field. in addition to these devices to measure predation, sampling stakes were established to sample the fauna that accesses the predation stakes (fig 2c). also, with a height of 25 – 30 cm and universal collector of 100 ml was installed, which was filled up to half its volume by water with detergent in the field. a small amount of honey was poured around the edge of the collector as an attractive. in addition to this collector, there was a branch isolated by lithium grease, with a 5 x 5 cm piece of green cardboard, at its tip containing at least 20 eggs of belenois solilucis and a 5 x 3 cm piece of yellow adhesive trap (fig 2d). as a result, the fauna that rises in the cuttings could be seen, distinguishing the one that possibly accesses the eggs of the treatment (captured in the universal collector) or control (caught in the yellow adhesive trap). fig 2. images of the methodology used in the 18 locations of organic vegetable production in the rashad, sudan, from february to june 2020 and february to may 2021. (a) photograph of one of the 720-predation stakes in the field, where two cards with eggs of belenois solilucis were available. (b) photo of the egg cartons after 24 hours in the field, with the number of initial eggs written in pencil, and the predation of all eggs on the left cartouche occurred. (c) photograph of one of the 320 sampling stakes, containing a universal collector and a yellow adhesive trap card. (d) photo of one of the sampling cards that remained in the field, with a strip containing eggs as an attractant on top and covered by transparent plastic for storage. arrows indicate the locations in (a) and (c) that have white grease preventing ants’ passage. sociobiology 69(2): e7746 (june, 2022) 5 data analysis all analyses were performed in the r program (pinheiro et al., 2020). initially, a paired t-test was used to determine if the ants effectively contributed to the increase in predation rates in the treatment cards vs control, with the stake serving as the pairing factor. we have chosen models to verify if the landscape influences egg predation rates. in this analysis, three response variables were evaluated: (1) the number of eggs predated in the treatment (predation by ants and other predators), (2) the number of eggs predated in control (predation by alated arthropods, excluding ants) and (3) the number of eggs predated in the treatment minus the control (predation attributed to ants). the explanatory variables were areas of the landscape categories (tree cover, planting, without planting and construction) and all possible interactions between two of these variables. for each of these response variables, four models were constructed to evaluate landscape effect. landscape was delimited as a buffer and we developed a model for each buffer (1,000 m, 500 m, 250 m and 125 m). we applied the “backward” process using the lm function of r (pinheiro et al., 2020), to perform the model selection procedure. the modeling began with a model containing all the variables and then we sequentially removed the variables with the highest p obtained in the model until only significant variables remained. in addition, we used function r to compare each new model with a variable removed from the previous one to verify that it undergoes significant changes. finally, to determine the best model, the akaike criterion was applied, with the aic function of r (sakamoto et al., 1986). to determine the number of preyed eggs related to the number of predators, three simple regressions were performed: (a) the number of eggs pre-given in the treatment according to the total number of predators; (b) the number of eggs pre-given in control as a function of predators excluding ants; and (c) the number of eggs pre-given in the treatment minus control according to the number of occurrences of ants. specimens were identified using a single key to identify subfamilies, a series of keys to identify genera and a series of keys to identify species (bingham, 1903; holldobler & wilson, 1990; mathew & tiwari, 2000), and comparison with voucher specimens housed in the museum of natural science, department of biological sciences, louisiana state university, comparison with type images available at antweb (www.antweb.org), antwiki (www.antwiki.org). results a total of 3,631 individual arthropods were collected, (fig 3) among them 406 were predators (fig 4). the most common predators were coleopterans (178 individuals), particularly from the families staphylinidae (105) and fig 3. proportion of the 3,875 individuals captured according to the functional group to which they belong. the collections were carried out in 18 localities of organic vegetable production in the rashad, sudan, from february to june 2020 and february to may 2021, using 360 universal collectors and 360 yellow adhesive trap cards. *for the predator group, ants were counted in number of occurrence (244) instead of number of individuals (1,831)., **for the indeterminate group, of the 1,814 records, 1,558 were of the order diptera, that is, 86%. captured functional groups http://www.antwiki.org khalid a.e. eisawi et al. – ants increase pest egg predation in sudanese organic agricultural systems6 species number of locations with occurrence number of captured individuals occurrence cumulative percentage of occurrence axinidris acholli weber, 1941 11 371 44 18% tapinoma carininotum weber, 1941 10 245 33 32% technomyrmex moerens santschi, 1913 7 154 22 41% camponotus bayeri forel, 1913 9 26 22 50% camponotus brutus forel, 1886 8 57 20 58% pheidole megacephala forel, 1913 7 493 14 64% tetraponera bifoveolata 5 82 14 69% pheidole punctulata santschi, 1937 7 104 12 74% pheidole rugaticeps emery, 1881 5 102 8 77% solenopsis orbula forel, 1905 4 44 8 81% camponotus acvapimensis donisthorpe, 1945 6 13 6 83% bothroponera pachyderma santschi, 1920 4 47 5 85% pheidole aeberlii emery, 1901 3 14 4 87% camponotus aegyptiacus santschi, 1926 3 7 4 89% pheidole rugaticeps arabs emery, 1881 1 9 2 89% monomorium abeillei collingwood, 1996 1 8 2 90% pheidole termitophila forel, 1911 2 7 2 91% pheidole sinaitica forel, 1907 2 7 2 92% bothroponera crassa emery, 1877 2 6 2 93% atopomyrmex mocquerysi santschi, 1924 2 6 2 93% brachyponera sennaarensis santschi, 1921 2 2 2 94% leptogenys crustosa weber, 1942 1 6 1 95% camponotus etiolipes bolton, 1995 1 4 1 95% crematogaster latuka weber, 1943 1 3 1 95% pheidole speculifera santschi, 1930 1 3 1 96% camponotus cinctellus santschi, 1939 1 2 1 96% camponotus kersteni gerstäcker, 1871 1 1 1 97% camponotus maculatus weber, 1943 1 1 1 97% camponotus tricolor bolton, 1995 1 1 1 98% cardiocondyla fajumensis weber, 1952 1 1 1 98% pheidole crassinoda ruspolii emery, 1897 1 1 1 98% pheidole decarinata santschi, 1929 1 1 1 99% pheidole jordanica stitz, 1917 1 1 1 99% leptogenys maxillosa emery, 1895 1 1 1 99% solenopsis punctaticeps weber, 1943 1 1 1 100% table 1. list of ant species potentially predatory on belenois solilucis eggs captured in the 360 sampling cuttings. the collections were carried out in 18 locations of production of organic vegetables in the rashad, sudan, from february to june 2020 and february to may 2021. coccinellidae (71). the order diptera was the second most prevalent among predators, mainly from the family dolichopodidae (128). still, these are predominantly predators of animals in flight and small animals (harterreiten souza, oral communication), not being observed in the field directly preying eggs of belenois solilucis, so they were not included in the analyses of predators. the other predators found were hemiptera-heteroptera (35), araneae (27), hymenoptera – vespoidea (25), neuroptera (6), psocoptera (2), pseudoscorpionida (1), coleoptera-melyridae (1) and coleopteracarabidae (1). the use of white grease to inhibit the passage of ants proved to be effective, although not infallible (fig 4). overall, 1,831 ant individuals were captured in 244 occurrences (table 1) belonging to 35 different species which have potential to act as predators. the most frequent ant species were axinidris acholli, tapinoma carininotum and technomyrmex moerens, all from the subfamily dolichoderinae. however, the species with the most individuals was pheidole megacephala, sociobiology 69(2): e7746 (june, 2022) 7 a myrmecine ant. in general, the ten most frequent ant species accounted for more than 80% of the occurrence of ants. the number of eggs pretested between control and treatment (fig 5) indicates that ants were its main predators (t-test, t = 12.445, df = 717, p < 0, 0001). on average, approximately 39.8% of offered eggs were consumed per day, with ants accounting for 26.8% and other predators accounting for 13%. however, there are substantial variations in the properties, both in total predation (ranging from 9.4% to 63.2% eggs), as in predation only by ants (from 5.3% to 52.4%) and other predators (4.2% to 21%). the analysis of linear models shows that the total number of predating eggs and eggs pressed by ants in each organic locality is not influenced by the landscape (table 2). the planted area within 500 meters, on the other hand, positively influences the predator eggs in control and interactions with the rest of the landscape. the regressions between the number of pre-given eggs and the number of predators were significant (fig 6). the number of eggs perused in the treatment is positive and significantly related to the total number of predators (number of pre-given fig 4. proportion of predators taxon captured in the yellow adhesive trap (170 records) and in the universal collector (352 records). the collections were carried out in 18 localities of organic vegetable production in the rashad, sudan, from february to june 2020 and february to may 2021, using 360 universal collectors and 360 yellow adhesive trap cards (a. predators shown in the yellow sticky trap, b. predators shown in universal collector). in this figure, the family dolichopodidae, with 128 individuals, has been suppressed. *for the formicidae family, ants were counted in number of occurrence (244) instead of number of individuals (1,831). khalid a.e. eisawi et al. – ants increase pest egg predation in sudanese organic agricultural systems8 eggs = 129.4 + 4.878 x number of predators, r² = 0.41, p = 0.004). as well as the control (number of predating eggs = 49.8 + 2.6 x number of non-ant predators, r² = 0.27, p = 0.025) and the treatment minus the control (number of pre-given e ggs = 110.5 + 5.185 x number of occurrence of ants, r² = 0.30, p = 0.017). finally, ants showed a correlation between their occurrence and species richness (t = 7,5254, r = 0,88, p < 0,001). discussion in this study, it was possible to isolate the effects of winged predators from those of non-winged predators by using white grease, as also performed by östman et al. (2003) (fig 4). although this isolation is not complete, we can consider it effective since 12 ants were also found in yellow sticky traps. ants were not the only predators isolated during the experiment; besides these, in principle, spiders, nymphs, and predator larvae may have been isolated, contributing to predation rates in the treatment. however, the occurrence of these predators was similar to the control and negligible when compared to ants. therefore, the data strongly suggest that ants are the main predators of belenois solilucis on vegetables and shrubs in organic agriculture (fig 5). other studies in different countries found similar results such as in southern mexico, where habel et al. (2010) measured an ant predation rate of 30.5% on the coffee borer (hypothenemus hampei) in five days, and kaushal and vats (1983) found a predation rate of 65.5% to 71.8% on belenois eggs, during 22 hours on golf courses. rahman et al. (2021) reviewwith studies from oceania and southeast asia, shows that, out of 17 different publications, ants of the genus oecophylla did not reduce the pest population size in only one of the studies. among the ant species sampled, it is evident that few are responsible for most of the predation (table 1). surprisingly, the three most frequent species captured in the cuttings belong to the genera linepthema and dorymyrmex (subfamily dolichoderinae), recognized as a group that presents thermal tolerance to high temperatures and preference for open places (grimaldi & agosti, 2002). generally, studies have identified the genus pheidole and solenopsis as the primary predators in neotropical agricultural areas (groc et al., 2017). the ability of species from the genera dorymyrmex and linepithema, as well as secondarily the genus camponotus, to climb plants places them as the primary predators of belenois solilucis eggs on vegetables. however, the ants of the myrmecinae subfamily, which account for 24% of the occurrences and are represented mainly by the genus pheidole, had the highest means of individuals recruited per occurrence (mean of 13.4 individuals) compared to the subfamily dolichoderinae (7.5) and formicinae (2,6). complementing the role played by ants, table 2. linear model that explains the influence of landscape on predation rates on belenois solilucis eggs. the experiment was carried out in 18 localities of production of organic vegetables in the rashad, sudan, sudan, from february to june 2020 and february to may 2021. factor and spatial scale model estimates r² adjusted aic p value a) predator eggs (total) buffer 1000m (none) <0,05 buffer 500m (none) <0,05 buffer 250m (none) <0,05 buffer 125m (none) <0,05 b) new predator by ants buffer 1000m (none) <0,05 buffer 500m (none) <0,05 buffer 250m (none) <0,05 buffer 125m (none) <0,05 c) predator eggs in control buffer 1000m (none) <0,05 buffer 500m planting construction x tree cover planting x construction tree cover x no planting planting x no planting 2.306e-03 2.533e-09 -3.550e-08 -1.577e-09 -2.402e-09 0.3003 189,7 0,0103 0,0361 0,0142 0,0065 0,0337 buffer 250m (none) <0,05 buffer 125m (none) <0,05 sociobiology 69(2): e7746 (june, 2022) 9 fig 5. number of belenois solilucis eggs available (dark grey) and preyed (light grey) on the control cards (without access to ants) and treatment (with access to ants). the experiment was carried out in 18 locations within the rashad, sudan, from february to june 2020 and february to may 2021. bars represent standard deviation and different letters indicate significant differences (p < 0.05). fig 6. linear relationships between abundance, predation or richness, such that: (a) eggs predated on the cards treatment in relation to all predators; (b) eggs predated on control cards in relation to predators except ants; (c) eggs predated in the treatment minus the control (predation attributed to ants) in relation to the occurrence of ants; and (d) correlation between ant richness and its occurrence. for all regressions and correlation, the results were significant (p<0.05). these data are based on the experiment carried out in 18 locations within the rashad, sudan, from february to june 2020 and february to may 2021. a meta-analysis with studies in agrosystems found that generalist predators control the abundance of herbivores in 79% of analyzed publications. in 65% of the studies, they reduce plant damage or increase production yield (panwar et al., 2016). the existence of commercial organic crops is only possible due to predators that control the occurrence of pests (ratnadass et al., 2012). in addition, other predators belonging to the orders coleoptera, dermaptera, neuroptera and diptera also contribute substantially to this ecological service (sáenzromo et al., 2019). khalid a.e. eisawi et al. – ants increase pest egg predation in sudanese organic agricultural systems10 when analyzing pest control in agricultural systems, it is essential to emphasize that the pest’s identity affects its ability to be controlled by a particular predator taxon. for example, ants are excellent egg predators, as seen here. still, winged pests such as adults of the whitefly (kenis et al., 2009), in theory, would hardly be preyed upon by ants due to their small size and ability to fly, this being the preferred target of some predators of the orders hemiptera, neuroptera and coleoptera (rosenheim et al., 1995). another example is the presence of aphids, a phytophagous that can coexist with ants while being protected by them (bishop et al., 2014). ladybugs (coccinellidae) would therefore serve as complementary predators for aphids and redundant for eggs (munyai & foord, 2015). to better demonstrate how much predation can be beneficial to producers, we can simulate leaf herbivory calculations. according to hlongwane (2019), throughout the larval stage of belenois solilucis, it consumes an average of 89 cm² of the soybean leaf area (varying between types of cultivar). assuming that all predated eggs in this experiment generated larvae that devoured 89 cm²/individual leaf area, each location would suffer damage of 2.4m²/day of leaf area loss on average. non-winged predators and the remainder avoided one-third of this leaf loss by ant predation. many studies have shown that the landscape is critical; it provides biodiversity and can increase the presence of predators of multiple taxa and consequently increase the ecological, biological control service (eisawi et al., 2022). however, in this study, it is observed that the landscape does not influence the total number of preyed eggs and those preyed only by ants (table 2). as seen in other studies, the influence and importance of each landscape factor vary according to the taxon studied (bishop et al., 2015). according to netshilaphala et al. (2005), it is possible to maintain the biological control of pests in organic systems, regardless of the landscape, through pest management. however, this management is dependent on the species of pest, predators, and their interaction. the ant community in forest settings is made up of a distinct set of species than those found in agrosystems. however, another study showed that the presence of scattered trees in agricultural systems increases the local richness of ant species (sithole et al., 2010), which correlates with ants’ occurrence (fig 6) and may result in an increase in predation rates, although this remains unproven scientifically. for predators, excluding ants, tree cover influences predation rates by interacting with other landscape categories (table 2). according to maurice kouakou et al. (2018), forest formation increases the diversity of both herbivores and predators in the surrounding crops, serving as a source of natural enemies. the presence of forest formations in the landscape is associated to an increase in alternative resources and the availability of a refuge site during periods of low resource availability for both predators and pests (ratnadass et al., 2012). the presence of planting in the landscape, on the other hand, significantly increases predation rates by winged predators (groc et al., 2017), most likely because it is linked to the hypothesis of resource concentration (hlongwane et al., 2019) and because it maintains an already regional set of species adapted to agrosystems. nonetheless, it is essential to note that very high proportions of conventional planting in the landscape – above 31.2% of the landscape, as analyzed here – have had a detrimental impact on biodiversity and its services (mauda et al., 2018). furthermore, as shown in table 2, there are several interactions between landscape categories, indicating that the rest of the landscape also influences egg predation by winged predators, despite the fact that these interactions are complex patterns and with a model that relatively poorly fitted to the data (fitted r2=0.30). however, in order to explain predation rates, significant relationships were found with the abundance of all predators, in addition to winged predators and ants (fig 6). this is most likely related to the resource-consumer hypothesis (yanoviak et al., 2008), which states that the more resources are available, the more consumers there are. in this situation, a larger concentration of resources on a property implies a greater number of predators and, consequently, higher predation rates. this suggests success in properties with high predation rates to increase the number of predators, but it was not possible to determine what causes this greater abundance. in the literature, the availability of floral resources is an alternative food for predators such as ladybugs (fotso kuate et al., 2015). in addition, farms can be managed to create an adequate “ecological infrastructure” to enhance biological control (mauda et al., 2018). conclusion despite farmers’ ignorance, ants are an important group of predatory insects capable of reducing the eggs population of a belenois solilucis pest on organic farm plants by an average of 26.8% per day. it is also noteworthy that other predators sampled mainly consist of two families of the order coleoptera (coccinellidae and staphylinidae), which, together with other predator taxa, removed 13% of the available eggs. the landscape has a limited effect solely on predation carried out by winged predators, being positively influenced by planting areas and interactions with the remainder of the landscape within a 500-meters radius. finally, a positive relationship was found between the abundance of predators and their respective predation rates. this indicates that the successful conservation of biological control within organic areas is associated with the ability of these systems to manage to maintain a large number of predators. compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest. acknowledgements i would like to express my deep gratitude to professor he hong, my research supervisors, for their patient guidance, sociobiology 69(2): e7746 (june, 2022) 11 enthusiastic encouragement and useful critiques of this research work. we also thank the northwest a & f university and university of east kordofan for financing field data collection and providing financial and other material support during the study. authors’ contributions conceptualisation, study design, field data collection, data analysis and interpretation: kae eisawi. writing-initial draft: ip subedi. supervision, study design: h he. writing revisions: cd yode. all authors read and approved the final manuscript. references bianchi, f.j., booij, c.j.h. & tscharntke, t. 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(2016). agricultural landscape simplification reduces natural pest control: a quantitative synthesis. agriculture, ecosystems and environment, 221: 198-204. doi: 10.1016/j.agee.2016.01.039 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.879sociobiology 63(1): 699-704 (march, 2016) the disappearance of eggs and larvae from the nests of the mischocyttarus (monogynoecus) montei zikán (hymenoptera: vespidae), especially in autumn and winter: can it mean an adaptive cannibalism to ensure the survival of colonies? introduction cannibalism is a behavioral trait which occurs in many group of animals, although the most of the reports are based on behavioral observations of laboratory (fox, 1975). further, according to the same author, the intraspecific predation can be a control agent for homeostasis of the population. in a review study, polis (1981) described the intraspecific predation in about 1300 species. for this author, the cannibalism is important for the population structure, history of life, competition for sexual partners or resources, and several behavioral aspects of these species. in social wasps, studies have suggested that cannibalism on larvae is caused when food is scarce (kudô & shirai, 2012). larval cannibalism of brood can be induced by poor nutrition of adult individuals in social wasps (hunt, 1991). in independentfounding wasps, the ingestion of immature individuals from abstract the aim of this study was to verify the disappearance of eggs and larvae from the nests of the mischocyttarus (monogynoecus) montei, especially in autumn and winter, and at the same time, to discuss about the cannibalism as the main reason for survival of the colonies during the unfavorable climatic conditions. forty one colonies were studied during the seasons of the year, period from march in 1999 to september 2000. a total of 314 disappearances of immature individuals was counted, corresponding to 95 eggs (27,1 %) and 229 larvae (72,9 %). the results showed that the disappearance of eggs and larvae were significantly higher during the autumn and winter . in addition, the disappearance rate of larvae increases with the falling of rainfall and of temperature, in autumn and winter. there were no relation between the disappearance of eggs and any climatic factors. from the total number of disappearances, small larvae disappeared from the cells at a higher frequency (55,9%, n = 128) than medium (18,3%, n = 42) and large (25,8%, n = 49) size larvae. it is probably that these immatures (eggs and larvae) were cannibalized due to scarcity of prey and nectar under unfavorable climatic conditions, especially during the cold and dry periods of the year. sociobiology an international journal on social insects asn murakami, st sakamoto, sn shima article history edited by gilberto m. m. santos, uefs, brazil received 02 august 2015 initial acceptance 02 december 2015 final acceptance 11 february 2016 publication date 29 april 2016 keywords food, brood, intraspecific predation, season, mischocyttarini. corresponding author andré sunao nishiuchi murakami universidade estadual paulista (unesp) av. 24 a, 1515 bela vista cep: 13506-900, rio claro-sp, brazil e-mail: sunamigobio@yahoo.com.br the same colony has been related frequently as the result of conflicts among females to ensure the individual fitness. oliveira et al. (2012) have reported that, in m. montei, the occurrence of cannibalism on eggs and larvae resulted in the replacement of dominant female or in the colonial fission, after the intense conflict among females of the colony. in m. drewseni, cannibalism on eggs has occurred at higher frequency after the emergence of first larvae and, the females ingested their own eggs in four of five times, whereas in another occasion, one egg was ingested by other female (jeanne, 1972). prezoto et al. (2004) have verified that the dominant female of m. cassununga monopolizes the reproduction doing cannibalism on eggs of her subordinate workers. litte (1977) reported that many eggs and larvae disappeared from the nest after the queen removal of m. mexicanus, moreover, about one week after the queen removal, one female started to lay her eggs and became the queen of colony. intracolonial cannibalism of research article wasps universidade estadual paulista, rio claro-sp, brazil asn murakami et al. – disappearance of eggs and larvae from the nests of the mischocyttarus (monogynoecus) montei zikán (hymenoptera: vespidae)700 eggs was reported in m. cerberus styx by giannotti (1999) and intercolonial cannibalism in m. mexicanus by clouse (1995). brood cannibalism also occur in species of the genus polistes, as reported by giannotti (1994) in p. simillimus; giannotti & machado (1999) in p. lanio lanio; tannure & nascimento (1999) in p. versicolor and p. ferreri; zara & balestieri (2000) in p. versicolor and torres et al. (2009) in p. canadensis canadensis. furthermore, there are the classic studies of deleurance (1963), gervet (1964) and westeberhard (1969) which showed the occurrence of differential oophagy in the genus polistes. in metapolybia aztecoides, a swarm-founding wasp from epiponini tribe, the same behavior was reported by west-eberhard (1982). studies about the influence of climatic factors and availability food on the disappearance of brood from the nest of mischocyttarus species are still non-existent. however, some studies in polistes species revealed that young larvae of p. dominulus were preferentially cannibalized in limited conditions of food, ensuring a fast production of workers in initial development of colony (mead et al., 1994). more recently, the relationship between availability of food and larval cannibalism of the foundress of p. chinensis antenalis was studied by kudô and shirai (2012), in laboratorial conditions. according to this study a higher frequency of cannibalism occurred under limited conditions of food, i.e., young larvae were cannibalized at higher frequency during limited conditions of prey whereas old larvae were cannibalized at higher frequency during limited conditions of honey. mischocyttarus is the only genus of the tribe mischocyttarini (carpenter, 1993) and represents a large group in the subfamily polistinae of social wasp, with 245 species described in 9 subgenera (silveira, 2008) distributed almost exclusively in south america (richards, 1978). the genus mischocyttarus, polistes and most part of the genus ropalidia found nests by haplometrosis (only one female) or by pleometrosis (a group of females) (jeanne, 1972; richards, 1971, 1978). colonies are usually characterized by absence of age polietism (jeanne, 1980), the social hierarchy is established through behaviors of dominance and subordination (richards, 1971, 1978; jeanne, 1972; noda et al., 2001; prezoto et al., 2004; murakami & shima, 2006, 2010; costa-filho et al., 2011; oliveira et al., 2012), there are no morphological differentiation between female castes (murakami et al., 2009) and all adult individuals show a plasticity on the social role in the colony (richards, 1971; jeanne, 1972; cowan, 1991; murakami & shima, 2006, 2010; costa-filho et al., 2011; murakami et al., 2013). in m. montei there are only studies about some social behavior aspects which were realized by richards (1978) and by oliveira et al. (2012). the present study was aimed at investigating the disappearance of eggs and larvae from cells of the nest of m. montei, especially during the autumn and winter, discussing this disappearance as an adaptive strategy for colony development during the unfavorable climatic conditions. materials and methods forty one colonies were mapped during the preand post-emergence phase, from twice to four times per week, since 10th march 1999 until 06th february 2000, at campus of the são paulo state university, rio claro/sp, brazil (22°24’36”s, 47°33’36”w) with an average elevation of 612 meters. the disappearance of eggs and larvae was considered when a cell, previously with an egg or a larva, was found empty or the position of the egg had changed during the mapping of the nest. the frequencies of eggs and larvae removal were inferred when previously filled cells with eggs and larvae were empty and /or when the positions of the eggs in cells were changed. larvae were classified as large, medium or small size, according to murakami and shima (2006). the data collection was made after the establishment of social hierarchy in the colonies, i.e., when there was no conflict among females and, thus, discarding the hypothesis of the disappearance of immature individuals caused by differential cannibalism to ensure the individual fitness of the dominant female in the colony. this point was important because it is very common the occurrence of a differential oophagy or larviphagy by the queen or dominant female in independent-founding wasps. in our study, there was not observed an occurrence of a hierarchical conflict related to differential oophagy or larviphagy because there was no aggression or any aggressive behavior that would indicate the existence of a hierarchical dispute in the colony until the end of the behavioral observations. according to oliveira et al. (2012), colonies of m. montei are usually founded by an association of females in the pre-emergence and it may contribute to the success of association during pre-emergence, once it tends to reduce conflicts during initial phases of colonial development. on the other hand, the occurrence of conflicts tends to be more intense, usually involving more physical contacts during the post-emergence phase. from 41 studied colonies, the parasitoidism was detected in 7 cases, i.e., only 1 egg, 6 larvae and 9 pupae were parasited. thus, due to the occurrence at low frequency, the hypothesis that the disappearance of brood was influenced by parasitoidism rate was discarded. the higher frequencies of disappearance of immature individuals were detected during the autumn and winter, thus it is impossible to suppose that the disappearance of eggs and larvae occurred due to removal caused by parasitoidism or some disease. furthermore, all colonies had continued its natural development even under unfavorable climatic conditions (especially during the cold and dry periods). the climatic factors as temperature, relative humidity and rainfall index were obtained from ceapla (centro de análise e planejamento ambiental) located at campus of são paulo state university, rio claro/sp, where the present study was realized. chi-square test for a contingency table (p < 0,05) was applied to verify the relationship between disappearance rates of brood and the months (seasons) of the years, whereas sociobiology 63(1): 699-704 (march, 2016) 701 spearman correlation test (p < 0,05) were applied to measure the relationship between disappearance rates of brood and the climatic factors (zar, 1999). results the frequencies of disappearance of eggs and larvae from the nests during the four seasons (in months) of the year according to temperature, relative humidity and rainfall index is showed at table 1 and figure 1. the total of disappearance of eggs and larvae was 85 (27,1 %) and 229 (72,9 %), respectively (table 1). the highest frequency occurred during the winter (a total of 31 eggs and 103 larvae), in june and july, when the average temperatures 18,3o and 18,0o, respectively. the second highest frequency of disappearance was observed during the autumn (27 eggs = 37,0 % and 73 larvae = 63,1 %), more specifically in april. in spring, the total frequency of disappearance was 46, being 19 eggs and 27 larvae. the disappearance at lowest frequency occurred during the summer when a total of 8 eggs and 26 larvae removed from the cells (table 1 and figure 1). chi-square test for contingency table showed that the frequency of disappeared larvae significantly differed (p < 0,05) according to the month of the year (seasons) (x2larva= 443,15; g.l.larva= 3; clarva= 0,81). the analysis of spearman showed a significant and strong negative correlation (p < 0,05) between the frequency of disappearance of larvae and average temperature (r = -0,6165), as well as between this frequency and rainfall index (r = -0,7133). the spearman coefficient (r) between the disappearance of larvae and relative humidity was -0,1189, indicating a weak correlation without a significance (table 2). although the contingency fig 1. graphical interface of absolute frequency (abs) of disappearance of eggs and larvae from cells of m. montei nests, temperature (° c), relative humidity and rainfall rate (mm) according to the months and seasons from march 1999 to september 2000. season month eggs removal larvae removal temperature relative humidity rainfall abs % abs % fall mar 0 0 19 100 24,2 74,2 33,2 apr 27 37 46 63,1 22,1 69,1 32,1 may 0 0 8 100 19,1 67,9 50,7 total 27 27 73 73 21,80 70,40 38,67 winter jun 3 8,3 33 91,7 18,3 72,2 73,3 jul 24 33,3 48 66,7 18 68,3 25 aug 4 15,4 22 84,6 19,6 60,4 32 total 31 23,1 103 76,9 18,63 66,97 43,43 spring sep 6 25 18 75 21,5 63,6 109,4 oct 7 70 3 30 22,4 62 50,8 nov 6 50 6 50 23 60,2 77,6 total 19 41,6 27 58,4 22,30 61,93 79,27 summer dec 6 26,1 17 73,9 24,7 68 169,7 jan 1 33,3 2 66,7 24,5 72,9 323,8 feb 1 12,5 7 87,5 24,2 76,3 219,4 total 8 23,5 26 76,5 24,47 72,40 237,63 total amount 85 27,1 229 72,9 table 1. absolute frequency (abs) and percentage (%) of eggs and larvae removed from the cells of m. montei’s nests according to the months and seasons, considering the average temperature (° c), relative humidity and rainfall rate (mm) from march 1999 to september 2000. asn murakami et al. – disappearance of eggs and larvae from the nests of the mischocyttarus (monogynoecus) montei zikán (hymenoptera: vespidae)702 coefficients indicated that the disappearance of eggs was significantly different (p < 0,05) according to months (seasons) of the year (x2egg= 15,53; g.l.egg= 3; cegg= 0,38), there were no significant correlations between the disappearance of eggs and any climatic variables (table 2). in table 3 it is possible once the most part of larvae was cannibalized, it is probably that the cannibalism of small larvae would be advantageous to the colony. small larvae need a higher demand of time and forage activity to complete their development in comparison to medium and large larvae. thus, adult wasps can feed larger larvae through the cannibalism of smaller larvae to obtain a faster production of adult individuals for colony. as observed by kudô and shirai (2012) in laboratorial conditions, the cannibalism occurred more frequently under preyand honey-limited conditions. however, young larvae were cannibalized at high frequency under prey-limited conditions whereas old larvae under honey-limited colonies. the authors concluded that a limited condition of nutrition induces the larval cannibalism and/or the production of first workers in the colony. mead et al. (1994) analyzed the frequency of cannibalism during the nest foundation of p. dominulus and they verified that young larvae were more frequently cannibalized under preylimited condition when compared to colonies with availability of prey. furthermore, in his work, it was observed that adult wasps preferentially feed old larvae in the colony. although it was not possible to observe the behavior of females after removals of eggs and larvae of m. montei, this phenomenon probably occurred in the studied colonies. the following facts support the hypothesis of larval cannibalism as an adaptive strategy to overcome unfavorable climatic conditions: 1stour hypothesis about the cannibalism of eggs and larvae to ensure the individual fitness is not considered here because the collection of data was made without the existence of conflict among females which can do the differential cannibalism (pardi, 1948, west-eberhard, 1969, jeanne, 1972, oliveira et al., 2012); 2ndthe disappearance of eggs and larvae did not occur due to parasitoid attacks on the nests. this fact was observed in low frequencies (7 colonies from 41 studied colonies totalizing 1 egg, 6 larvae and 9 parasited pupae); 3rd cannibalism in the social wasps was related under induced conditions with poor nutrition of adult individuals of the colony (hunt, 1991), when the resource is limited (mead et al., 1994; kudô & shirai, 2012) and in unfavorable environment eggs removal larvae removal temperature relative humidity rainfall temperature relative humidity rainfall r -0,2089 -0,4099 -0,3039 -0,6165 0,1189 -0,7133 p-value 0,5148 0,1857 0,3369 *0,0328 0,7162 *0,0121 table 2. spearman’s correlation coefficient (r) and p-value (* p <0.05) between the absolute frequencies of eggs and larvae removed from the cells of m. montei’s nests in relation to average temperature (° c), relative air humidity and rainfall rate (mm) according to the four seasons (fall, winter, spring and summer). total frequency (n=229) small larvae medium larvae large larvae abs % abs % abs % 128 55,9 42 18,3 59 25,8 table 3. absolute frequency (abs) and percentage (%) of removed small according to size (larvae, medium and large) in m. montei. to observe the absolute frequency of the disappearance of eggs and larvae according to all months of the year. the frequency of disappearance of small larvae from the cells were higher (n= 128; 55,9 %) than medium larvae (n= 42; 18,3 %) and large larvae (n= 59; 25,8 %) (table 3). discussion and conclusion studies about the intraspecific cannibalism of brood and its relationship with climatic factors are scarce. on the other hand, there are many studies about the cannibalism of eggs and larvae to ensure the individual fitness among female wasps (caused by a competition in the dominance hierarchy of the colony), especially in the independent-founding wasps (west-eberhard, 1969, 1982; richards, 1971, 1978; litte, 1977; hunt, 1992; tannure-nascimento & nascimento, 1999; oliveira et al., 2012; and others). close (1995) reports that conspecific predation was more frequent after the usurpation of nests of m. mexicanus, which were founded by haplometrosis. giannotti (1999) observed that larvae of m. cerberus styx were removed from cells by workers with her jaws and first pair of legs, and then, this food resource could be divided among other adult females and brood of the nest. this behaviour also was described in polistes lanio by giannotti and machado (1999). based on the analysis of spearman correlation our results showed that, the adverse climatic conditions in autumn and winter (low temperature and low index of rainfall) influenced the disappearance of brood through the cannibalism. this behaviour probably occurred due to scarce resource of food (especially in winter) and difficulties for foraging (for example, as happened in cold and rainy days). according to giannotti (1994), a high frequency of cannibalism after dispersion of aggregate was related to the absence of food resource during the foundation of colonies in the polistes simillimus. our study also shows that small larvae disappeared from the cells at higher frequency than medium and large larvae. sociobiology 63(1): 699-704 (march, 2016) 703 conditions; and 4ththe similarity between our results under natural conditions and the results obtained by mead et al. (1994) and by kudô and shirai (2012) under manipulated conditions showed that it is possible to hypothesize the high disappearance of brood caused by scarce of food resource in adverse climatic conditions during the cold and dry period of the year. thus, we believe that the cannibalism of small larvae, which occurred more frequently than cannibalism of medium and large larvae, represents an adaptive strategy of m. montei to ensure the development of the colony cycle, especially during unfavorable climatic conditions (during the autumn and winter). references carpenter, j.m. (1993). biogeographic patterns in the vespidae (hymenoptera): two views of africa and south america. in p. goldblatt (eds.) biological relationships between africa and south america (pp.139-155). new haven and london, yale university press. clouse, r.m. 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(1999). biostatistical analysis. prentice hall. new jersey, 663p. 1401 larger seeds are dispersed farther: the long-distance seed disperser ant aphaenogaster famelica prefers larger seeds by satobu takahashi1 & takao itino1, 2* abstract seed dispersal by ants (myrmecochory) is an important life phase in flowering herbs of temperate deciduous forests, and long dispersal is basically preferable in terms of plant fitness. although it is known that larger ants transport seeds farther, previous studies conflicted whether larger seeds are preferred by large ants. we examine how seed size and ant body size influence removal rate of seeds and their dispersal distance by ants. the obligate myrmecochorous, large-seed bearing viola hondoensis and ant-ballistic diplochorous, small-seed bearing viola spp. and corydalis pallida were comparatively investigated. 1) ant preference: attraction rate and transportation rate of large v. hondoensis seeds by ants were consistently high regardless of the disperser ant size whilst those rates of small diplochorous seeds were conspicuously lower for large aphaenogaster ants than for small pheidole ants, suggesting small seeds are not adapted to be carried by large ants. 2) dispersal distance: large v. hondoensis seeds were transported for a long distance because they were carried by both large and small ants while the small diplochorous seeds were transported for a short distance because they were carried only by small ants. these results suggest that large seeds are advantageous to attract large ants and to be farther dispersed as far as ant-mediated dispersal is concerned. on the other hand, the diplochorous small seeds do not attract large ants, and so are transported short by ants although they disperse their seeds by ballistic dispersal. key words: myrmecochory, plant-animal interaction, pheidole, seed dispersal, viola 1department of biolog y, faculty of science, shinshu university, 3-1-1 asahi, matsumoto, nagano 390-8621, japan. e-mail: itinot@ shinshu-u.ac.jp 2institute of mountain science, shinshu university, 3=1=1 asahi, matsumoto, naggano 390-8621 japan 1402 sociobiolog y vol. 59, no. 4, 2012 introduction seed dispersal by ants is called myrmecochory. myrmecochorous plants have seeds that have caruncles or arils that act as elaiosomes which attract ants. ant workers take such seeds and transport them to their nest. after the elaiosome part is consumed by the ants, the seed itself may remain in the nest or be discarded on the midden or farther away (gorb & gorb 2003). the seed, thus, can disperse from their mother plant. howe & smallwood (1982) reviewed advantages to seed dissemination and grouped them into three hypotheses. first, escape hypothesis proposes that dispersal may represent escape from disproportionately high seed and seedling mortality near the parent due to competition and/or predation. second, colonization hypothesis assumes that seed dissemination may allow parent plants to establish their offspring in vacant sites. and the third hypothesis is termed directed dispersal, in which animals are assumed to consistently deliver seeds to special sites critical for establishment. in the cool temperate forests of japan, the first and second hypotheses have been shown important (higashi et al. 1989; ohkawara & higashi 1994). apparently, the two hypotheses predict that long distance dispersal is essentially advantageous in terms of plant fitness. to get seeds dispersed farther, myrmecochorous plants have two alternative tactics. one is to associate with ‘good’ long-distance transporting ants, and the other is to adopt diplochorous (ballistic long-distance and ant-mediated short-distance) dispersal (ohkawara & higashi 1994). ness et al. (2004) demonstrated that seed dispersal distance generally increases with ant body size in three ant subfamilies that include most of seed-dispersing ants. so, as far as ant dispersal distance is concerned, plants should hire large ants as seed dispersers for long-distance dispersal. then, what kind of seed character is desirable in order to attract larger ants? beattie et al. (1979) found that large-sized ant species tended to take larger diaspores, and hughes & westoby (1992b) reported that diaspore size increased removal rates of seeds by pheidole ants. on the contrary, boulay et al. (2007) showed the seed size did not affect ant visitation rate to a myrmecochorous plant, helleborus foetidus. in japanese temperate deciduous forests, two types of myrmecochorous herbs are encountered: obligate myrmecochorous herbs and ant-ballistic diplochorous herbs. as the former have no way to disperse their seeds other 1403 takahashi, s. & t. itino — larger seeds are dispersed farther than ant dispersal, we hypothesize that they have ‘desirable’ seeds to attract large, ‘good’ disperser ants. the aim of the present study is to investigate the effect of seed size on attraction rate, transportation rate, and dispersal distance by ants in both obligate myrmecochorous and ant-ballistic diplochorous herbs. additionally, we compared the effect between small-sized and large-sized seed disperser ant species. our hypothesis is that larger seeds are attractive to large ants and are transported farther. to address this issue, we investigated (1) preference of two different-sized ant species for seed size, and (2) relationship between dispersal distance and seed size. materials and methods study site and materials the study site is a temperate deciduous forest at fujii glen, nagano, central japan (alt.: c.700m). it is an oak-pine forest, the dominant tree species being quercus acutissima, q. serrata and pinus densiflora. field work was carried out from april to october 2005 along footpaths through the forest (c. 10m x 400m). the potential seed-dispersing ants of the forest were aphaenogaster famelica: body length 3.5-8mm (large), pheidole fervida: 2-2.5mm (small), formica japonica: 4.5-6mm (large), lasius japonicus: 2.5-3.5mm (small), camponotus obscuripes: 7-12mm (large), pristomyrmex pungens: 2-2.5mm (small), and paratrechina flavipes: 2-2.5mm (small), among which a. famelica, p. fervida, and p. flavipes were conspicuously abundant and were selected for the study material. the density of ants (all the ant species included) was c. 10-30 walking ants and several ant nests per 1m2 forest floor. myrmecochorous herbs of the study site include viola spp., corydalis pallida and erythronium japonicum. these plants are encountered at similar microhabitats in the forest floor. generally, viola and corydalis are diplochorous plants that disperse their seeds in two ways, ballistic and ant dispersal. a matured capsule opens and ejects seeds in a moment. additionally, tiny elaiosomes are attached to the pole of the diaspores and attract ants when the seeds are dropped on the ground. the myrmecochorous plants we studied are as follows. viola hondoensis: obligate myrmecochorous, have especially large seeds. they do not disperse the seeds ballistically, seed size: 2.6-4.1mm (large), elaiosome size: 1.0-1.8mm. v. mirabilis: ant-ballistic diplochorous dispersal, 1404 sociobiolog y vol. 59, no. 4, 2012 seed size: 2.7-3.3mm (large), elaiosome size: 0.6-1.1mm. v. tokubuchiana: ant-ballistic diplochorous dispersal, seed size: 2.0-2.2mm (small), elaiosome size: 0.6-0.8mm. v. yedonensis: ant-ballistic diplochorous dispersal, seed size: 1.5-2.0mm (small), elaiosome size: 0.4-0.5mm. corydalis pallida: ant-ballistic diplochorous dispersal, seed size 1.5-2.2mm (small). ant preferences for seed size we observed the behavior of ants that collected myrmecochorous seeds at 13 depots situated randomly within the study area of 10m x 400m on the forest floor. each depot included fifteen seeds: six depots contained “large” seeds (v. hondoensis) and seven depots “small” seeds (mixtures of c. pallida, v. tokubuchiana, and v. yedoensis seeds). the “small” seeds were grouped together irrespective of the species because they showed no recognizable differences in ant preferences. we observed each depot for 15 minutes and recorded behavior of each ant walking near the depot (nearer than the ant body length). within the 15 minutes, no depletion of the seeds or no aggressive behavior of the ants for the seeds was observed. ant behavior was classified as follows. (1) ignore: ignore or do only antennation, (2) try to transport: try to grasp the seed with mandible but failed, and (3) succeed to transport: succeed to transport the seed at least five times longer than the ant body length. from the frequencies of each behavior, we calculated ‘attraction rate’, ‘success rate’, and ‘transportation rate’. attraction rate is the proportion of ants that was attracted to the depot and tried to transport a seed among total observed ants. success rate is the proportion of ants succeeded to transport a seed among the attracted ants. transportation rate is the proportion of ants succeeded to transport a seed among total observed ants. transportation rate is a product of attraction rate x success rate. simple equations can be written thus: attraction rate = (t + s) / (i+ t + s) [1] success rate = s / (t+ s) [2] transportation rate = s / (i + t + s) [3] where i, t, and s are the frequencies of observation for ignore, try to transport, and succeed to transport, respectively. 1405 takahashi, s. & t. itino — larger seeds are dispersed farther dispersal distance from june to august 2005, we collected ripen seeds of v. hondoensis, v. tokubuchiana, v. yedoensis, v. mirabilis and c. pallida from the plants, measured major axis of the seeds (including elaiosomes), put them one by one (up to 15 seeds per observation) at the base of the plants, and waited for ants to transport them. thirteen observations were carried out at randomly selected sites in the forest floor (10m x 400m). the fallen seeds around the plants were cleared away beforehand. when an ant began to carry a seed, it was tracked until it brought the seed into the ant nest. all the transporting ants under observation were not lost in the way. then the dispersal direct distance was measured. totally, 72 tracking observations were made. again, the “small” seeds were grouped together irrespective of the species because they showed no recognizable differences in dispersal distance. results ant preferences for seed size to small seeds (viola spp. and c. pallida), large ant species, a. famelica, was consistently less attracted than small ant species, p. fervida, i.e., large ants tried to carry the small seeds less often (table 1). large ants were also less successful in transporting the small seeds than small ants, i.e., even when large ants tried to carry, they failed to transport the small seeds (table 1). on the contrary, to large seeds (v. hondoensis), the large ants and the small ants were similarly attracted, and they were similarly successful in transportation (table 1). dispersal distance large a. famelica ants transported seeds for a long distance, while small p. fervida ants did for a short distance (fig. 1). the dispersal distance of large ant species (128.6±13.3 cm, mean ± se) was longer than that of small ants (21.8±2.5 cm, ttest, p<0.01). large ants exclusively transported large seeds while small ants transported both large and small seeds (fig. 1). discussion the results indicate that the interaction between myrmecochorous herbs and ants in japanese deciduous forest is asymmetric, i.e., large ants are associated only with large seeds whilst large seeds of obligate myrmecochorous plants 1406 sociobiolog y vol. 59, no. 4, 2012 table 1. response of ants to myrmecochorous seeds. large ant: aphaenogaster famelica (body length: 3.5-8mm), small ant: paratrechina fervida (2-2.5mm), large seed: viola hondoensis (seed size: 2.64.1mm), small seed: corydalis pallida (c. 1.8mm), v. tokubuchiana (2.0-2.2mm), and v. yedoensis (1.5-2.0mm). attraction rate is the proportion of ants attracted to the depot among total observed ants. success rate is the proportion of ants succeeded to transport a seed among the attracted ants. transportation rate is the proportion of ants succeeded to transport a seed among total observed ants. i, t and s indicate ignore, try to transport, succeed to transport, respectively. p–values are based on chi-square test. * p<0.05; ** p <0.01; ns not significant. 　 　 seed size large ant small ant p attraction rate large 0.75 (9/12) 1.00 (5/5) ns (t + s) / (i+ t + s) small 0.40 (19/48) 1.00 (23/23) * success rate large 0.67 (6/9) 0.40 (2/5) ns s / (t+ s) small 0.26 (5/19) 0.96 (22/23) * transportation rate large 0.50 (6/12) 0.40 (2/5) ns s / (i + t + s) small 0.10 (5/48) 0.96 (22/23) ** fig. 1. scatterplots of the relationship between seed dispersal distance by ants and seed size. each ccircle indicates an ant individual of large ant species (a. famelica), and each triangle indicates that of small ant species (p. fervida). large seeds of v. hondoensis (2.5mm-4mm) were transported by large and small ants, while small seeds of viola spp. and c. pallida (1.5mm-2.5mm) were transported by only small ants. 1407 takahashi, s. & t. itino — larger seeds are dispersed farther are associated with large and small ants. this study confirms that these sizedependent specificities in seed-disperser interaction are maintained through ant preference for seed size. ant preferences for seed size attraction rate, success rate, and transportation rate of large seeds (v. hondoensis) were consistently high regardless of the disperser ant size (table 1). contrarily, those rates of small seeds (c. pallida, v. tokubuchiana and v. yedoensis) were low for large ants, suggesting they are not adapted to be carried by large ants (table 1). additionally, in the result of the dispersal distance experiment (fig. 1), the small seeds were, again, rarely transported by large ants. an ant usually grips the elaiosome when it transports a myrmecochorous seed (byrne & levery 1993). when large a. famelica ants tried to carry small seeds, they could not grip it because the elaiosome of small seeds appeared too small for large ants to grip. gorb & gorb (1995) similarly observed that elaiosome and seed bodies of small seeds are too small for manipulation by large ant (formica polyctena), especially by large individuals. in addition, small seeds are usually hard and spherical, which makes their manipulation even more difficult for large ants. on the other hand, large seeds were carried by both large and small ants. large ants usually lift a large seed whereas small ants usually drag one. hughes & westoby (1992b) emphasized the elaiosome/seed ratio rather than just the seeds size as the trait that might have been selected to increase removal rate by beneficial ant partners. however, we observed that the large ants often slipped past small seeds without checking their elaiosome/seed ratio even when they walked within c. 1cm of the seeds (table 1, counted as ‘i: ignore’). this suggests that absolute size of seed is primarily important to attract large ants. of course, once ants notice a seed, they may assess whether it is worth transporting or not. the assessment may depend on the cost and benefit of the transportation, i.e., the elaiosome/seed ratio and absolute elaiosome size (hughes & westoby 1992b). as ants need a larger inducement to remove larger seeds, elaiosome size should increase with seed size (edwards et al. 2006). for the plants studied here, the seed size of each plant species well reflects the elaiosome size (see materials and methods) so that the assessment 1408 sociobiolog y vol. 59, no. 4, 2012 by the ants is supposed to depend basically on seed size. dispersal distance large ants dispersed seeds farther (fig. 1), which is consistent with the results of gómez & espadaler (1998) and ness et al. (2004). as larger ant species usually have larger territories, they can transport seeds for a longer distance on average. such long-distance dispersing partner is basically advantageous for plants because they disperse far enough to reduce distance-related and density-dependent costs for the plants such as parent-offspring conflict and sibling competition. as the overall fitness gain for plants resulting from myrmecochory may depend on the identity of the seed disperser, plant traits that affect the attraction of beneficial dispersers are likely to be under strong selection pressure (giladi 2006). in this study, as large seeds attracted large ants and enabled longer dispersal on average (fig. 1), large seeds are supposed to be selected for, especially in the obligate myrmecochorous v. hondoensis, which depends their seed dispersal entirely on ants. the large seeds of v. hondoensis, however, were not necessarily dispersed for a long distance (fig. 1). there are two reasons for this. first, large seeds were attractive not only to large ants but also to small, short-distance disperser ants (table 1). second, dispersal distance can not be longer than the distance between the seed and ant nest (gómez & espadaler 1998). even when a large ant disperses a seed, dispersal distance will be short if the seed is near the ant nest. for these two reasons, large seeds are not necessarily dispersed for a long distance. on average, however, the dispersal distance is longer in large seeds than in small ones. for ant-ballistic diplochorous, small-seed bearing viola spp. and corydalis pallida, there may be a tradeoff for the seed size. smaller seeds are supposed to disperse farther by ballistic dispersal mechanisms, while they disperse shorter afterwards by ant dispersal. although we did not measure the distance by ballistic dispersal, ohkawara & higashi (1994) reported that diplochorous v. selkirkii and v. verecunda ejected their seeds for 38cm and 56cm on average, respectively. this means that the small seeds are already dispersed far enough, and they need not be dispersed for a long distance by ants. to get in ant’s nests may be the sole purpose for the diplochorous small seeds to bear elaiosomes because deposition of seeds in nests or refuse piles by ants reduces 1409 takahashi, s. & t. itino — larger seeds are dispersed farther losses to aboveground seed predators (ohkawara & higashi 1994; pizo & olivia 2001; vander & longland 2004). factors affecting seed size attractiveness and dispersal distance of myrmecochorous seeds were size-dependent: small seeds were not attractive to large ants, and so were not carried for a long distance while large seeds were carried by large and small ants so were dispersed farther (table1, fig. 1). as the obligate large seeds of v. hondoensis do not disperse ballistically, they must rely on ants for long distance dispersal. as large ants consume much energ y to work per se (fewell et al. 1996), they would prefer large foods to collect. therefore, not only the elaiosome/seed ratio but also absolute seed (elaiosome) size is important to attract large ants (mark & olesen 1996). on the other hand, ant-ballistic diplochorous plants do not need to have large seeds in order to be dispersed farther by ants. the distance of ballistic dispersal is negatively correlated with the weight of the seed (lisci & pacini 1997), and seed size and number are negatively correlated (stearns 1992). so, ant-ballistic diplochorous plants may have rather been selected for to have many small seeds. interactions between myrmecochorous plant species and disperser ant species were size-specific (table 1). our results agree with the prediction of giladi (2006) and boulay et al. (2007), that myrmecochory is not as diffuse as it is frequently presented. such size-dependent specificity is explained by the dichotomy of the plant strateg y to get dispersed for a long distance: produce large seeds to be transported by large ants, or produce small seeds to flip farther. then, which strateg y should a plant embrace? predation pressure of vertebrate and invertebrate predators may influence the choice of plant strateg y. ballistic and ant dispersal diplochorous plants must lift their fruits previous to flip the seeds for a long distance. such lifted fruits are conspicuous so that seed predators like birds often eat them (takahashi s personal observation, although there is a possibility that the seeds are capable of surviving ingestion by birds and are ‘dispersed’ by birds). on the other hand, obligate ant dispersed seeds of v. hondoensis bear fruits under the leaves which are not found by birds but easily accessible by ground beetle predators and mammals. comparison of seed mortality between diplochorous plants and 1410 sociobiolog y vol. 59, no. 4, 2012 obligate ant dispersed plants will shed light on the evolution of dichotomy of plat dispersal strategies. acknowledgments we thank j.h. ness and i giladi for their helpful comments on the manuscript, u ban and k komatsu for their advices on the experimental design, and n morishita for his help in the statistical analyses. references beattie, j.a., c.d. culver & j.r. pudlo 1979. interactions between ants and the diaspores of some common spring flowering herbs in west virginia. castanea 44: 177-186. boulay b., j. coll-toledano, j. manzaneda & (cerdá x). 2007. geographic variations in seed dispersal by ants: are plant and seed traits decisive? naturwissenschaften 94: 242-246. bymee m.m. & d.j levery. 1993. removal of seeds from frugivore defecations by ants in a costa rican rain forest. vegetatio 107/108: 363-374. edwards w., m. dunlop & l. rodgerson 2006. the evolution of rewards: seed dispersal, seed size and elaiosome size. journal of ecolog y 94: 687-694. fewell h.j., f.j. harrison, r.b.j. lighton, & d.m. breed 1996. foraging energetics of the ant, paraponera clavata. oecologia 105: 419-427. giladi i. 2006. choosing benefits or partners: a review of the evidence for the evolution of myrmecochory. oikos 112: 481-492. gómez c. & x. espadaler 1998. seed dispersal curve of a mediterranean myrmecochore: influence of ant size and the distance to nests. ecological research 13: 347-354. gorb s. & e. gorb 1995. removal rates of seeds of five myrmecochorous plants by the ant formica polyctena (hymenoptera: formicidae). oikos 73: 367-374. gorb e. & s. gorb 2003. seed dispersal by ants in a deciduous forest: ecosystem mechanisms, strategies and adaptations. kluwer academic publishers, dordrecht. howe f.h. & j. smallwood 1982. ecolog y of seed dispersal. annual review of ecolog y and systematics 1: 327-356. higashi s., s. tuyuzaki, m. ohara & f. ito 1989. adaptive advantages of ant-dispersed seeds in the myrmecochorous plant trillium tschonoskii (liliaceae) oikos 54: 389-394. hughes l. & m. westoby 1992a. fate of seeds adapted for dispersal by ants in australian sclerophyll vegetation. ecolog y 73: 1285-1299. hughes l. & m. westoby 1992b. effect of diaspore characteristics on removal of seeds adapted for dispersal by ants. ecolog y 73: 1300-1312. lisci m. & e. pacini 1997. fruit and seed structural characteristics and seed dispersal in mercurialis annua l. (euphorbiaceae). acta societatis botanicorum poloniae 66: 379-386. 1411 takahashi, s. & t. itino — larger seeds are dispersed farther mark s. & j.m. olesen 1996. importance of elaiosome size to removal of ant-dispersed seeds. oecologia 107: 95-101. ness j.h., j.l. bronstein, a.n. andersen & j.n. holland 2004. ant body size predicts dispersal distance of ant-adapted seeds: implications of small-ant invasions. ecolog y 85: 1244-1250. ohkawara k. & s. higashi 1994. relative importance of ballistic and ant dispersal in two diplochorous viola species (volaceae). oecologia 100: 135-140. pizo m.a. & p.s. olivia 2001. size and lipid content of nonmyrmecochorous diaspores: effects on the interaction with litter foraging ants in the atlantic rain forest of brazil. plant ecolog y 157: 37-52. stearns s.c. 1992. the evolution of life histories. oxford university press, oxford. vander w.s. & w.s. longland 2004. diplochory: are two seed dispersers better than one? trends in ecolog y and evolution 19: 155-161. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7208sociobiology 68(4): e7208 (december, 2021) introduction swarming is a strategy of colony reproduction in honey bees characterized by the parent colony separating into one or more offspring colonies, with at least one queen bee and a proportion of worker bees in each new colony. the swarming process includes two stages: 1) drone cell building to rear drone broods (free, 1967; avitabile & kasinskas, 1977), and queen cell building to rear queen bees (walrecht, 1961); 2) abstract this paper describes the organization and structure of the swarm queen cells of apis cerana cerana in spring, summer, and autumn in kunming, yunnan province, china. we measured the following indices to reveal the organization rule of swarm cells: number of swarm cells built by each colony during different seasons; the shortest distance between two adjacent swarm cells on the comb; distance between swarm cell base and bottom bar of movable frame. we revealed the swarm cells structural characteristics using the following indicators: maximum diameter of swarm cell, the length between mouth and bottom of swarm cell, depth between maximum diameter and bottom of swarm cell, and the ratio of maximum diameter to depth between maximum diameter and bottom of swarm cell. regarding seasonal differences, results indicated a significant variation in the distance between the swarm cell base and the bottom bar of the movable frame. still, no such effect was observed in the shortest distance between two adjacent swarm cells. the maximum swarm cell diameter was not considerably influenced either, while the distance between the maximum diameter and the bottom of the swarm cell had substantial variation. the detected ratio of the maximum diameter to the depth between the maximum diameter and the bottom of the swarm cell indicated seasonal changes in the bottom shape of the swarm cell. this study clarifies the temporal and spatial distribution and structure of swarm cells of a. c. cerana. it establishes the basis for predicting the time and position of appearing swarm cells, thus allowing for a more precise determination of the shape and size of queen-cell punch and the ideal position of a cell cup on the bar of queen cup frames in artificial queen rearing. sociobiology an international journal on social insects shunhua yang, dandan zhi, xueyang gong, yiqiu liu, wenzheng zhao, kun dong article history edited by evandro nascimento silva, uefs, brazil received 30 april 2021 initial acceptance 17 august 2021 final acceptance 14 november 2021 publication date 19 november 2021 keywords honey bee, diameter, length, swarming, swarming season, swarm cell. corresponding author wenzheng zhao yunnan provincial engineering and research center for sustainable utilization of honeybee resources, eastern bee research institute, college of animal science and technology, yunnan agricultural university, kunming 650201, china e-mail: rurosezwz@163.com the queen bee of the parent colony leaves the original colony with half of the colony worker bees and drones, and chooses a new location as a new home (winston, 1987). in the first stage, when the drone broods emerge as adult drones from the drone cells, workers begin to build queen cell cups that are different from the hexagonal cells used to rearing workers or drone broods. these queen cell cups are always hung freely along the bottom of the comb, are vertically oriented, and open downward (simpson, 1959; lensky & seifert, 1975). eastern bee research institute, college of animal science and technology, yunnan agricultural university, kunming, china research article bees seasonal variations in the organization and structure of apis cerana cerana swarm queen cells shunhua yang, dandan zhi, xueyang gong, yiqiu liu, wenzheng zhao, kun dong – seasonality in the structure of swarm queen cells2 the queen lays eggs in the queen cell cups, which hatch into queen larvae within three days, while the worker bees secrete royal jelly to feed them. this reproductive behavior ensures that queen larvae continue to develop and grow appropriately in their cells. as queen larvae grow in body size, the worker bees increase the diameter and length of the queen cells (simpson, 1959). after five days, the worker bees seal the cell mouth with beeswax to form a sealed queen cell when the queen’s larval stage is complete. then, the queen larva will proceed to the next stage of its development, the pupal stage, thus making the total development time eight days (jay, 1962). at the end of this period, the emerging queen bites off the cap of the queen cell and starts its adult stage (jay, 1963). some studies have shown that honey bee swarming generally exhibits clear seasonality. among the western honey bee species, the natural swarming of apis mellifera ligustica begins in spring and ends in autumn. this period was reported to start at the end of may and end at the beginning of august in southern england (simpson, 1959); according to gary and morse (1962), the time of swarming of a. m. ligustica in the northern united states usually occurred in may and june. in contrast, it fell from may to september in the central new york (ithaca) area (burgett & morse, 1974, fell et al., 1977). the number of queen cell cups of a. m. ligustica varies considerably among different colonies each year, and the factors governing the number of queen cups per colony have not been well-defined. this number for a. m. ligustica colonies in aberdeen, scotland, ranged from four to forty-five (mean twenty-four) in 1961, from seven to sixty-two (mean twenty-two) in 1962, from one to eleven (mean four) in 1963, and from one to forty-eight (mean fourteen) in 1964 (allen, 1965). in maryland, usa, the number of queen cell cups had obvious seasonal variation; queen cups were present from april throughout the summer season, but their numbers were greatest from late may to early july (caron, 1979). the shape of the a. m. ligustica swarm cell varies considerably, is about 20-30 mm in length (robinson, 1982), and its diameter from the base to the mouth gradually decreases. its wall is thick with its surface carved into wrinkles, resembling a peanut pod, and the opening is always downward (hamdan, 2010). the natural swarming of eastern honey bee species, such as apis cerana cerana or asiatic honey bee (also known as chinese honey bee, from now on is referred to as chinese bee), also begins in spring and ends in autumn. this period for a. c. cerana in the fujian province of china was shown to occur in spring from march to may. with 3-7 swarm cells produced in each colony, the swarm cells were stout and straight in shape (liu, 1964). in addition to the period from march to may, the natural swarming of the same subspecies also occurred in summer from june to july and in september in guizhou province, while the swarm cell numbers were not determined (xu, 1987). in guangdong province, a. c. cerana natural swarming was reported to occur in spring with 3-9 swarm cells per colony and six cells on average from march to april, with 3-7 (average of 5) swarm cells in each colony in summer from may to june, and no known swarming events in autumn (liu & lai, 1990). the phenomenon of natural swarming, which is one of the inherent biological characteristics of honey bee colonies, may cause economic losses to beekeepers if the swarm is not found and captured in time. as natural swarming has obvious characteristics, such as seasonality or preparation process from breeding drones to rearing queens, beekeepers can use these characteristics to predict swarming in the bee colony and take appropriate beekeeping management measures to prevent the associated economic losses. kunming is an important apiculture development area in yunnan province, one of the main beekeeping areas within china with a high density of chinese honey bee colonies. natural swarming that often occurs in these colonies is difficult to predict, which brings many difficulties to the management of the bee colony. therefore, this study aimed to determine the natural swarming pattern of the eastern honey bee, a. c. cerana, in three different seasons (spring from march to may, summer from june to august, and autumn from september to november) in kunming, yunnan province, china, and to reveal the temporal and spatial patterns of organization of swarm cells and their structure. accordingly, we propose the following hypotheses: hypothesis 1: in kunming, yunnan province, the natural swarming phenomena of chinese honey bee colonies occur in spring, summer, and autumn; hypothesis 2: the position of swarm queen cells show a seasonal difference; hypothesis 3: there is a seasonal difference in the structure of swarm queen cells. the following variables were measured in the spring, summer, and autumn of 2020 to test these hypotheses: (1) the number of swarm cells built within a. c. cerana colonies; (2) the position of swarm cells on the comb; (3) the structure and size of swarm cells. the results will provide a basis for predicting the time and position of swarm cell production and help determine the shape and size of queen-cell punch and the position of cell cup on the bar of the queen cup frames in artificial queen rearing. materials and methods statistics of swarm queen cup numbers in colonies the experiment was carried out from february to november 2020, investigating 40 colonies in the spring, 47 in the summer, and 51 in the autumn. according to the beekeeping requirements of each season, certain highquality virgin queens were selected and retained to replace old queens, or single colonies were artificially divided into several colonies. some of these newly established colonies were selected as investigation colonies for the next season. the number of swarming queen cups was determined every seven days in all three seasons to obtain nsp for spring, nsu for summer, and nau for autumn. the shortest distance (sd) measured between two adjacent swarm cells on each comb sociobiology 68(4): e7208 (december, 2021) 3 was sdsp for spring, sdsu for summer, and sdau for autumn. in addition, the distance between the swarm cell base and the bottom bar (dbb) of the movable frame was measured to give dbbsp for spring, dbbsu for summer, and dbbau for autumn. the above indices were used to reveal the organization rule of swarm cells. collection of swarm cell shells and preparation of cell molds queen cells were fitted with protectors to prevent them from being destroyed by workers. the cell shells were collected immediately once the queens have emerged from their cells (fig 1a). crystal glue solution was prepared by mixing epoxy resin, and its curing agent in a volume ratio of 1:1 extracted using a disposable syringe and then injected into the queen cell shells. the amount of crystal glue solution injected into a shell was adjusted to keep the fluid level with the shell mouth. the solution was left to solidify in the shell at room temperature for 12 hours to form a fixed-shape molding reflecting the shape and size of each shell. during the curing process, no effect of the crystal glue solution on the shape or volume of shells was observed, including shell expansion or shrinkage due to heat; shells retained their original shape and structure. measurement index of queen cell mold the queen cells containing the mold were soaked in hot water at 90 ℃ to melt the beeswax on the queen cell surface to obtain clean queen cell molds. the acquired queen cell molds had above 80 hd hardness, which did not deform in 90 ℃ hot water. therefore, the shape and size of obtained queen cell molds resembled the actual structure and size of collected queen cells (fig 1b). finally, the maximum diameter of queen cell molds was measured using vernier calipers (accuracy ± 0.01 mm) to obtain dsp for spring, dsu for summer, and dau for autumn. the length of queen cell molds, i.e., the distance from the cell mouth to the bottom of the cell, was also measured to obtain lsp for spring, lsu for summer, and lau for autumn. the parameters of dpsp for spring, dpsu for summer, and dpau for autumn were measured as the depth between the maximum diameter of the swarm cell mold and the bottom of the swarm cell mold. the above indicators were used to reveal and evaluate the structural characteristics of swarm cells. statistical analysis one-way analysis of variance (anova) was conducted to determine the influence of the season on 1) the shortest distance (sd) between two adjacent swarm cells on the comb in different seasons; 2) the distance (dbb) between the base of swarm cell and the bottom bar of the movable frame; 3) the maximum diameter (d) of the swarm cells constructed by colonies; 4) the length (l) of the mouth to the bottom of the swarm cell built by bee colonies; 5) the depth between the maximum diameter and the bottom of the swarm cell (dp) of the colony. the ratio (w) of the maximum diameter (d) to the depth between the maximum diameter and the bottom of the swarm cell (dp) was calculated as wsp for spring, wsu for summer, and wau for autumn, and the shape of the bottom of the swarm cell was determined using these ratios. all data obtained in the experiments were statistically analyzed using the statistical analysis system (sas v8.0) software provided by sas institute inc., north carolina, usa. fig 1. (a) swarm cells of the eastern honey bee, apis cerana cerana. (b) the molds of swarm cells for the eastern honey bee, a. c. cerana. top, queen cells in spring; middle, queen cells in summer; bottom, queen cells in autumn. shunhua yang, dandan zhi, xueyang gong, yiqiu liu, wenzheng zhao, kun dong – seasonality in the structure of swarm queen cells4 results statistics of queen cup numbers in different seasons results showed that 95% of colonies produced queen cups in the spring, 44.7% in the summer, and 9.8% in the autumn. during the investigation period, the number of queen cups per colony ranged from 3 to 23, averaging 8.59. in the spring, the summer, and the autumn, the average number of queen cups per colony averaged 8.05 (4-15), 8.52 (3-17), and 13.00 (4-23), respectively. seasonal variation of shortest distance (sd) between two adjacent swarm cells on the same comb the mean shortest distance (sd) of two adjacent swarm cells on the comb was 41.73 mm ± 3.05 mm (n = 191). the results of one-way anova showed that seasons had no significant influence on the shortest distance between two adjacent swarm cells on the comb of the eastern honey bee, a. c. cerana (f = 1.53; df = 2, 188; p = 0.22 > 0.05). the mean shortest distance between two adjacent swarm cells on the comb in the spring, the summer, and the autumn was 49.19 mm ± 4.40 mm (n = 64), 38.70 mm ± 5.96 mm (n = 56) and 37.40 mm ± 5.40 mm (n = 71), respectively. the multiple comparisons showed no significant differences between sdsp, sdsu, and sdau (p > 0.05) (fig 2). that the season had a significant effect on the distance (dbb) between the base of the swarm cell and the bottom bar of the movable frame on the comb (w = 21.61; df = 2, 151.4; p < 0.05). the mean distance between the base of the swarm cell and the bottom bar of the movable frame on the comb was 56.82 mm ± 3.88 mm (n = 88), 28.86 mm ± 1.82 mm (n = 70), and 36.76 mm ± 2.74 (n = 82) mm in the spring, the summer and the autumn, respectively. the results of multiple comparisons showed that dbbsp was significantly higher than dbbsu and dbbau (p < 0.05), whereas the difference between dbbsu and dbbau was insignificant (p > 0.05) (fig 3). fig 2. seasonal variation in the shortest distance (sd) between two adjacent swarm cells on the comb. notes: all values in the figures are represented as mean ± standard error. the same lowercase letter among different columns indicates that the difference is not significant (p > 0.05), while different lowercase letters indicate statistical significance (p < 0.05), the same rule applies to the graphs below. fig 3. seasonal variation in distance (dbb) between base of swarm cell and bottom bar of movable frame. comparison of maximum diameter of swarm cells among seasons the average maximum swarm cell diameter was 9.54 mm ± 0.04 mm (n = 144), ranging from 8.20 mm to 11.19 mm. the results of one-way anova showed no significant seasonal effect on the maximum diameter of natural swarm cells (f = 1.22; df = 2, 141; p = 0.30 > 0.05). the average maximum diameter of swarm cells in the spring, the summer, and the autumn was 9.60 mm ± 0.06 mm (n = 55), 9.56 mm ± 0.08 mm (n = 46), and 9.45 mm ± 0.06 mm (n = 43), respectively. the results of multiple comparisons showed that the differences among dsp, dsu, and dau were not significant (p > 0.05) (fig 4). comparison of length between mouth and bottom of swarm cell the average length of swarm cells was 17.38 mm ± 0.14 mm (n = 144), ranging from 13.58 mm to 23.91 mm. the welch’s anova test results showed a significant seasonal effect on the length of a. c. cerana natural swarm cells (w = 10.19; df = 2, 93.95; p < 0.05). the average length of swarm cells in the spring, the summer, and the autumn was 17.77 mm ± 0.25 mm (n = 55), 17.69 mm ± 0.21 mm (n = 46), and 16.55 mm ± 0.20 mm (n = 43), respectively. the results of multiple comparison of distance (dbb) between swarm cell base and bottom bar of the movable frame the mean distance (dbb) between the swarm cell base and the movable frame’s bottom bar was 41.81 mm ± 1.93 mm (n = 240). the results of welch’s anova test showed sociobiology 68(4): e7208 (december, 2021) 5 comparisons showed significantly smaller lau than lsp and lsu values (p < 0.05), while the difference between lsp and lsu (p > 0.05) was insignificant (fig 5). comparison of depth between maximum diameter and bottom of swarm cell (dp) the average depth between the maximum diameter and the bottom of swarm cells (dp) was 4.74 mm ± 0.05 mm (n = 144). the results of welch’s anova test showed that the season significantly affected the depth between the maximum diameter and the bottom of a. c. cerana swarm cells (w = 33.35; df = 2, 90.81; p < 0.05). in the spring, the summer, and the autumn, the average depth between the maximum diameter and the bottom of swarm cells was 5.12 mm ± 0.08 mm (n = 55), 4.68 mm ± 0.10 mm (n = 46), and 4.32 mm ± 0.06 mm (n = 43), respectively. multiple comparisons showed that the differences among dpsp, dpsu, and dpau all reached a significant level (p < 0.05) (fig 6). calculation of ratio (w) of maximum diameter (d) to depth between maximum diameter and bottom of swarm cell (dp) the mean ratio (w) of maximum diameter (d) to depth between the maximum diameter and the bottom of swarm cells (dp) was 2.05 ± 0.02 (n = 144), ranging from 1.47 to 3.55. the average ratio was 1.89 ± 0.02 (n = 55), 2.08 ±0.04 (n = 46) and 2.21 ± 0.04 (n = 43), with ranges of 1.47-2.29, 1.57-2.80 and 1.70-3.55 in the spring, the summer and the autumn, respectively. fig 6. comparison of depth (dp) between maximum diameter and bottom of swarm cell. fig 4. comparison of maximum diameter of swarm cells among seasons. fig 5. comparison of length between mouth and bottom of swarm cell. discussion seasonality of natural swarming of chinese bee it was established in the present study that, in kunming, yunnan, china, the natural swarming of the eastern honey bee, a. c. cerana began in early march and ended in early november, which corresponds to obvious seasonality with spring, summer, and autumn swarming periods. concerning the temporal swarming pattern of the chinese bee, natural swarming was found to occur mainly in spring, followed by summer, and in a few colonies in autumn. the number of queen cells in colonies in spring and summer was the same, but significantly less than in autumn. therefore, our first hypothesis was confirmed. in both the spring and the summer, the average number of queen cups per colony was eight, while that in the autumn was thirteen. the number of swarm cells in the autumn was higher than in the spring, or the summer, which may have occurred as the temperature in the autumn was falling, and the daily temperature variation was large. especially after shunhua yang, dandan zhi, xueyang gong, yiqiu liu, wenzheng zhao, kun dong – seasonality in the structure of swarm queen cells6 august, the weather gradually turned cooler. therefore, the number of swarm cells in the autumn was more conducive to selecting a high-quality virgin queen and thus improved the mating success rate (zhou, 1995; yang et al., 2020). liu and lai (1990) found out that the average number of queen cups constructed by a. c. cerana in spring and summer in guangzhou, guangdong province was six and five, respectively. our survey results for kunming, yunnan province (with eight queen cups in both spring and summer) were lower, most likely due to the climate differences between regions. some studies have reported significant differences in the number of queen cells produced between chinese bee colonies, with the same colony strength in beijing and fuzhou, which cities have significantly different climates (yang, 1974; yang et al., 1981; liu, 1993). the spatial position of natural swarm cells the shortest distance between two adjacent swarm cells on the comb is defined as the minimum distance to keep between two adjacent cell cups of beeswax on the bar of queen cup frames in artificial queen rearing (liu, 1962, 1966; song, 1987; li, 1993). our results showed that this distance was not affected by the season, suggesting that beekeepers should keep a minimum of 42 mm distance regardless of the season. honey bee queen cells are purposefully built outside the normal comb area to accommodate the larger queen (simpson, 1959, lensky & seifert, 1975, hepburn et al., 2014). these cells hang freely along the bottom of the comb, are vertically oriented, and open downward (seeley & morse, 1976). the position of the queen cups on the comb is measured by the linear distance between the base of the queen cup and the bottom bar of the movable frame. in our study, the distance between the queen cup and the bottom bar of the frame was longer in the spring and the autumn. in the summer, when the temperature is higher, the queen cell is placed closer to the bottom bar of the frame, where heat easily dissipates, favoring the development of the queen. previous studies have indicated that placing artificial cell cups near the beehive entrance can improve the survival rate during artificial queen rearing in summer (gong & ning, 1992; bi & li, 2003; yang et al., 2020). in summary, the rules of swarm cells’ temporal and spatial organization revealed no seasonal difference in the shortest distance between two adjacent swarm cells and a seasonal difference in the linear distance between the swarm cell base and the bottom bar of the movable frame. the linear distance from the bottom bar of the movable frame was short in the summer and long in the spring and the autumn. to predict the time and position of the swarm cells occurring in the colony, more than 90% of the bee colonies will produce swarm cells in spring, over 40% of the bee colonies will produce swarm cells in summer, and less than 10% of the bee colonies will produce swarm cells in autumn. in general, swarm cells are located on the lower edge of the comb, and they are closer to the bottom bar of the movable frame in summer when compared with spring and autumn. accordingly, our second hypothesis was supported. therefore, to prevent natural swarming in the colony, we recommend beekeepers take appropriate management steps every five to seven days in spring in all bee colonies. as for the summer and autumn, beekeepers could perform these steps in bee colonies with more than five frames to promote colony strength. structure of natural swarm cell in this paper, the structure of swarm cells was quantified for the eastern honey bee (a. c. cerana). indices of maximum diameter, length, depth from maximum diameter to bottom of the cell, and the ratio of maximum diameter to depth between maximum diameter and bottom of the cell, were measured to determine the actual structure of swarm cells. the data provide parameters for defining the structure of artificial queen cups. no seasonal variation was shown in the maximum diameter of swarm cells. still, seasons significantly impacted the cell length and depth between the maximum diameter and the bottom of the swarm cell. therefore, our third hypothesis was partially confirmed. since the swarm cell length values in the spring and the summer were significantly longer than in the autumn, the body length of queens reared by beekeepers in spring and summer may be larger than that of queens raised in autumn. here we pose the question of whether the depth of artificial queen cups should be determined according to the season and whether it is necessary to use different sizes of queen-cell punches in beekeeping at different times of the year. answering these questions requires further experimental verification. the bottom shape of a natural swarm cell the mean ratio (w) of the maximum diameter (d) to the depth between the maximum diameter and the bottom of a. c. cerana swarm cells (dp) was 2.05 ± 0.02. the mean ratios in the spring, the summer, and the autumn were 1.89 ± 0.02 (1.47-2.29), 2.08 ±0.04 (1.57-2.80), and 2.21 ±0.04 (1.703.55), respectively. these results show that the bottom shape of the swarm cell was a vertical semi-ellipsoid in the spring, a hemisphere in the summer, and a horizontal semi-ellipsoid in the autumn. herein, the average maximum diameter of the swarm cell was 9.54 mm ± 0.04 mm, which was greater than that reported by fang et al. (1995) (9.10 mm) or ren et al. (2012) (9.02 mm). the average depth between the maximum diameter and the bottom of the swarm cell was 4.74 mm ± 0.05 mm, which was smaller than the result of fang et al. (1995) (6.08 mm) or ren et al. (2012) (6.41 mm). whether this is explained by regional differences or by different ecotypes of a. c. cerana remains a subject of further studies. however, it is worth noting that the study area of fang et al. (1995) was fuzhou, fujian province, and the swarm cells built by a. c. cerana of fujian were of the southern chinese bee population. ren et al. (2012) study area was chongqing municipality, and swarm cells built by a. c. cerana of chongqing were of the central chinese bee population. meanwhile, the experimental sociobiology 68(4): e7208 (december, 2021) 7 area of this study was kunming, yunnan province, and the swarm cells built by a. c. cerana of yunnan were of the bee population of yun-gui plateau. based on the findings of the current study, we suggest that chinese beekeepers in kunming make different shapes of queen-cell punches according to seasonal differences. in artificial queen rearing in spring, the shape of the queen-cell punch should be a vertical semi-ellipsoid, while in summer and autumn, it should be a hemispherical and a horizontal semi-ellipsoid, respectively. the diameter of the queen-cell punch should be no less than 8.0 mm and no more than 9.5 mm. the depth of artificial queen cups in spring, summer, and autumn is suggested to be 5.1 mm, 4.7 mm, and 4.3 mm, respectively. the queen cup frame should be equipped with three bars, and each bar should de equipped with 8-9 cups, the distance between two adjacent cups on the bar should be 47-53mm. conclusions this paper established that the natural swarming of the eastern honey bee a. c. cerana in kunming, yunnan province, begins in early march and ends in early november, corresponding to spring, summer and autumn, and showed obvious seasonality. the shortest distance between two adjacent swarm cells on the comb showed no seasonal variation, while the distance between the base of the swarm cell and the bottom bar of the movable frame differed among seasons. although the time of year was not a factor influencing the maximum diameter of swarm cells, it did affect swarm cell length, the depth between the maximum diameter to the bottom of the swarm cell, and the shape of the bottom of the swarm cell. therefore, it is concluded that both the temporal and spatial organization of swarm queen cells of the chinese bee exhibit seasonal variations. based on the findings, recommendations were made to enhance current beekeeping practices to lessen the losses caused by natural swarming. authors’ contribution k.d.: conceptualization, visualization, writing-review and editing s.y.: conceptualization, methodology, investigation, formal analysis, visualization, writing-original draft preparation, writingreview and editing k.d.: methodology, investigation, d.z.: investigation, x.g.: investigation, y.l.: investigation, w.z.: investigation, writing-review and editing all authors have read and agreed to the published version of the manuscript. disclosure statement we declare no conflict of interest and no competing or financial interests. funding this study was supported by grants from the national natural science foundation of china (nos. 32060241 and 31572339), the china agriculture research system of the ministry of finance and the ministry of agriculture and rural affairs(cars-44-kxj13), and the reserve talents training program for young and middle-aged academic and technical leaders in yunnan (no.2018hb041). acknowledgments we express our gratitude to our research assistant zhiwen liu for assisting us with the experimental bee colony management. we would also like to thank mrs. zhou and mrs. liu for recording the experimental data, and topedit (www.topeditsci.com) for the english language editing of this manuscript. references allen, m.d. 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(1995). seasonal management of chinese bee (apis cerana cerana) in yunnan plateau (in chinese). apicultural science and technology, 1: 9-11. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.175-180sociobiology 62(2): 175-180 (june, 2015) two new strumigenys f. smith (hymenoptera: formicidae: myrmicinae) from montane forests of ecuador introduction the genus strumigenys, with 836 valid species worldwide (bolton, 2014), is one of the most species rich ant genera, with the majority concentrated in tropical forests, of which a little under 25% are to be found in the neotropical region. they are predators of other arthropods and many are trap-jaw hunters specializing in stalking collembola, a key group in vegetational litter decomposition and formation of soil microstructure (rusek, 1998). despite a recent monumental revision (bolton, 2000) it is clear that the diversity of the group still has much to be discovered. recent sampling in high altitude forests in southern ecuador has revealed two unknown species of strumigenys, both of which are presently described. materials and methods the specimens were studied using a zeiss stemi 2000-c stereocope with a 1x objective lens and 10x ocular lens. images were taken using the same stereoscope fitted with a zeiss axiocam mrc5 camera and the image analysis software axiovision by zeiss was used for measuring the specimens. the drawings abstract two new species from the myrmicine ant genus strumigenys found in cloud forests above 2000 m in southern ecuador are described and illustrated. s. lojanensis n. sp. is a member of the gundlachi complex described from 8 workers and 2 queens. s. madrigalae n. sp. is a member of the schulzi group described from 1 worker and 1 queen. characters that permit separation from closely appearing species are presented. the gundlachi group of strumigenys now has at least 5 species known from andean cloud forests above 2000 m in northern south america. sociobiology an international journal on social insects je lattke; n aguirre article history edited by rodrigo m. feitosa, ufpr brazil received 20 november 2014 initial acceptance 01 january 2015 final acceptance 13 january 2015 keywords ants, cloud forests, northern andes, biodiversity. corresponding author john e. lattke universidad nacional de loja, dirección de investigación, programa biodiversidad, bosques y servicios ecosistémicos ec 110101 loja, ecuador e-mail: john.lattke@miza-ucv.org were made using images taken with the camera plus direct comparison with specimens. for comparative purposes, besides the cited references, images of strumigenys posted on antweb (www.antweb.org) were extensively consulted. mandibular denticles are best seen using background lighting and a dorsaloblique view, especially for s. lojanensis. measurements and indices follow bolton (2000). the following collections are mentioned in the text as repositories of specimens. cisec colección de invertebrados del sur del ecuador, universidad técnica particular de loja, loja, ecuador. dzup coleção entomológica padre jesus santiago moure, departamento de zoologia, setor de ciências biológicas, universidade federal do paraná, curitiba, paraná, brazil. icn instituto de ciencias naturales, universidad nacional de colombia, bogotá d. c., colombia mczc – museum of comparative zoology, harvard university, cambridge, massachusetts, usa. mzsp museu de zoologia da universidade de são paulo, são paulo, brazil rbins royal belgian institute of natural sciences, recent invertebrates collection, brussels, belgium. qcaz quito catholic zoology museum, pontificia universidad católica de ecuador, quito, ecuador. research article ants universidad nacional de loja loja, ecuador je lattke and n aguirre – two new strumigenys species from ecuador176 results descriptions of new species strumigenys lojanensis n. sp. figs 1 – 3. strumigenys lojanensis n. sp. worker. scale bar = 0.25mm. pilosity omitted. diagnosis. mandibles straight with small relatively samesized 5-7 preapical denticles along anterior half; mandibular apex with two minute intercalary denticles that appear midway between upper and ventral apical teeth. disc of postpetiolar dorsum weakly reticulate-punctate, spongiform processes of postpetiole well-developed. head, most of mesosoma, coxae, femora and tibiae densely reticulate-punctate; katepisternum mostly smooth and shining. type material. holotype. ecuador, loja, reserva el madrigal, 4.04655°s 79.17583°w, 6.3 km sse of loja, 2350m, 28 august 2014, j. lattke 3590-14. holotype (worker) deposited in qcaz. paratypes. (1) one worker and one queen from the same sample as the holotype also deposited in qcaz. (2) same locality data and date as the holotype: sample lattke 3590-15 1w in rbins. (3) same locality data and date as the holotype: sample lattke 3590-03 1w in dzup, 1w in icn, 1w in mczc, 1w in mzsp. (4) ecuador, loja, estación fundación arco iris, loja – zamora road, km 23, 3.98846°s 79.09326°w, 12.12k e of loja, 2105m, 18 august 2014, j. lattke 3573. from sifted leaf litter sample. one dealate queen and 1 worker deposited in cisec. worker description. holotype (paratypes, n=4): hl 0.62(0.60-0.63), hw 0.47(0.46-0.50), ml 0.49(0.48-0.50), sl 0.43(0.40-0.43), pw 0.31(0.31-0.34), al 0.67(0.66-0.69) mm; ci 76(74-79), mi 79(76-82), si 91(0.82-93). posterior cephalic margin deeply concave, occipital lobes well developed; surface of cephalic dorsum transversely convex; anterior clypeal margin transverse, slightly sinuate with low median convex lobe. dorsolateral margin of head with flagellate hair just posterad of eye level; cephalic dorsum with two pairs of slightly spatulate erect hairs, highest pair close to posterior cephalic border whilst shorter pair close to eye level. leading edge of scape with slender hair at base that arches apicad, followed by 5 thicker, spatulate hairs: second hair from base arches apicad and is more slender than hairs 3-6, hairs 3-4 arch basad and hairs 5-6 usually arch apicad, though some specimens may have hair 6 arching towards base. apical lobes of labrum clearly visible with mandibles closed, short and converge towards each other, slightly wider at base than at blunt apex; trigger hairs extend anterad to midpoint of mandibular length. mandible straight, external margin straight to weakly convex, internal margin weakly sinuate, basally convex and apically concave, basal lamella convex; preapical denticles 5-6 on left mandible and 5-7 on right mandible with mandible pointing towards observer; denticles closest to apex usually larger though minute denticles or stubs may be present. mandibular apex with two minute intercalary denticles that appear midway between upper and ventral apical teeth. mesosoma in lateral view with pronotal margin broadly convex, mesonotal to propodeal margin relatively flat. transverse section of pronotal dorsum broadly convex; pronotal humeral hair fine and flagellate, sometimes forming apical loop; mesonotum with single pair of flagellate hairs; propodeal spiracle separated from posterior edge of propodeal lamella by over single fig 1. strumigenys lojanensis n. sp., head dorsal view. fig 2. strumigenys lojanensis n. sp., head lateral view. fig 3. strumigenys lojanensis n. sp., body lateral view. elongated shape on mesopleuron outlined by broken line defines area of smooth and shining sculpturing. sociobiology 62(2): 175-180 (june, 2015) 177 width; propodeal tooth triangular, apex pointed. propodeal tooth triangular, sharply pointed and slightly higher than basal length; metapelural lobe bluntly triangular, not higher than propodeal tooth. petiole lacking spongiform processes, ventrum with low anterior lamella that extends posterad to midlength; spongiform sculpturing well-developed on postpetiolar ventrum and posterolaterally, vestigial strands of spongiform sculpturing usually present along anterior margin of abdominal sternite iv. postpetiolar tergite with narrow lamella along anterior margin. dorsum of abdominal segments ii – v with series of straight to weakly arched hairs, all semi-erect over cuticular surface. petiolar node with posterior pair, postpetiole with 3 pairs, two dorsal pairs and a lateral pair. anterior border of abdominal tergite iv with transverse arched crest that parallels posteriorly projecting lamella of postpetiole, a few brief longitudinal costulae may be present along this crest; most of tergite smooth and shining with 20-24 erect, slightly arched, spatulate hairs and no appressed pubescence. head, most of mesosoma, petiole, postpetiole, coxae, femora and tibiae densely reticulate-punctate, sculpturing attenuated on discal area of postpetiolar node and along its anterior margin; katepisternum mostly smooth and shining with strip of reticulate-punctate sculpture along anteroventral and ventral margins, propodeal declivity mostly smooth and shining posteriorly; abdominal tergite iv smooth and shining. body mostly dark brown, almost black; mandibles, antennae, and legs brown. gyne. (gyne 1 gyne 2): hl(0.62-0.64), hw(0.5-0.51), ml(0.51-0.48), sl(0.41-0.41), pw(0.38-0.39), al(0.77-0.77) mm; ci(81-80), mi(82-75), si(82-80). besides the expected morphological differences the gyne is quite comparable to the workers except for the better developed petiolar ventral lamella, although still low it bears more resemblance to spongiform sculpturing whilst in the worker it is simply a brief, low lamella. etymology. the specific epithet “lojanensis” is derived from the name of the ecuadorian state in which the specimens were found, loja. discussion. this species fits quite well within the gundlachi complex as defined by bolton (2000:176) but the spongiform processes of the postpetiole are much more developed in s. lojanensis. within the gundlachi complex, s. lojanensis fits comfortably into the gundlachi species cluster, with the only difference consisting in the weaker reticulate sculpturing of the postpetiolar dorsum. using the key to neotropical pyramica in bolton (2000:137) specimens of s. lojanensis will flow directly to s. enopla (bolton). using the description of s. enopla (bolton, 2000: 185) and images of a paratype from antweb (casent0900177) it was possible to identify a number of discrete differences between the two species: the cephalic subdecumbent pilosity is not as arched and high as in s. enopla; the subdecumbent hairs on the mesosomal dorsum are also higher and more arched in s. enopla, as well as the standing hairs on petiole, postpetiole and gaster. the mesosomal dorsal margin of s. lojanensis is straight in lateral view, whilst in s. enopla it is distinctly sinuate and the propodeal denticles are stouter in s. lojanensis than in s. enopla. there are 2 standing hairs on the petiolar dorsum of s. lojanensis, compared with 4 in s. enopla. in s. enopla the posterior edge of the spongiform sculpturing on the postpetiole is convex, but in s. lojanensis there is a distinct low median lobe. the reticulate-punctate sculpture of the postpetiole is mostly strong and comparable to that of the petiole, covering most of it but weakens posteromedially on the dorsum, so it could be difficult to decide which way to go at couplet 7 of bolton’s key. if the user decides for first option then the specimen will become stranded at couplet 9 due to the number of denticles, exceeding the maximum amount for either option. if the modification of bolton’s key proposed by rigato and scupola (2008: 481) is used, then the specimen will key directly to s. osellai (rigato & scupola). in this case differences in the preapical dentition and metapleural sculpturing will permit easy separation: s. lojanensis bears only small denticles and never a large pair of denticles, plus the metapleuron in s. osellai is smooth and shining, but not so in s. lojanensis. the arco iris site is located within podocarpus national park along the road that joins loja with zamora within dense montane cloud forest, bearing a canopy not more than 20 m high upon steep slopes. it is within the catchment area of the río san francisco, with an estimated average annual rainfall of 3500-4000 mm, relative cloud cover of 70-80% (richter et al. 2013), and an estimated mean annual temperature of 15-16°c (wilcke et al., 2008). the reserva madrigal locality is a private reserve that neighbors podocarpus national park approximately 7 km sse from loja. it was previously a dairy farm and has been undergoing restoration towards forest during the last ten years. the specimens were taken from several samples along a 200 m long transect in mostly secondary vegetation, ranging from trees approximately 15 m high forming a loose canopy to more open bracken (pteridium sp.) dominated understory with scattered trees and shrubs. neighboring slopes to the sampling site across the stream bear dense forest with a canopy apparently not more than 15 m high. the area has an estimated average annual rainfall of 3000-2500 mm (richter et al., 2013). with the discovery of s. lojanensis it is possible to discern a group of 5 northern andean species of the gundlachi complex with a preference for cold forests above 2000 m altitude, where the presence of most ants is negligible (longino, 2014). the other species are s. enopla, known from altitudes between 1900 and 2200 m in sw colombia, s. nubila lattke and goitía, sampled from altitudes between 2000 and 2500 m in colombia and venezuela, s. vartana (bolton), a colombian species known from altitudes between 1800 and 2530 m and s. heterodonta (rigato & scupola) which was recently described from 2940 m altitude in ecuador (rigato & scupola, 2008). are these cold weather specialists closely related? do they form a monophyletic group? bolton recognizes two complexes of species within the gundlachi-group based upon the mandibular dentition and the development of the apical lobes of the labrum je lattke and n aguirre – two new strumigenys species from ecuador178 and their accompanying trigger hairs: the crassicornis complex, and the gundlachi complex. bolton (2000) informally divides the gundlachi complex into four clusters of apparently related species using the mandibular index, number and position of intercalary denticles of the mandibular fork, number and position of the preapical mandibular dentition, configuration of the main pilosity, and sculpture of the postpetiolar node. in his scheme both s. enopla and s. lojanensis fit in the gundlachi cluster, where they are the only cloud forest specialists, though widely distributed species such as s. denticulata (mayr), and s. gundlachi (roger) also have broad altitudinal ranges that include cloud forests but none approaching 2000 m (bolton, 2000; lattke & goitia, 1997). s. vartana lies in the laevipleura cluster, which includes s. gemella kempf recorded from cloud forests in colombia close to 1700 m (bolton, 2000). s. nubila shares the nubila cluster along with s. lalassa (bolton), a species found from sea level to 1500 m from costa rica to ecuador (bolton, 2000). rigato and scupola (2008) described s. heterodonta as part of the gundlachi complex, and opted for placing it in fifth species cluster as they were not satisfied with its affinities to any of the other species. nevertheless s. heterodonta is quite comparable with the nubila cluster as it fits most of the traits used by bolton, the differences being more of a degree than categorical. most of the seven species of the subedentata cluster are found in low to medium altitudes except for s. connectens (kempf), known from forests close to 1500 m in ecuador (bolton, 2000), and s. paniaguae (longino) found in costa rica between altitudes of 500-1500 m (longino, 2006). a brief overview of the ten species making up the crassicornis complex (bolton, 2000) shows most of them to inhabit low to medium altitudes except for s. pasisops (bolton), found in cloud forests of 1500 m, so it seems that the cold weather (>2000 m) specialists are, for the while being, restricted to the gundlachi complex, implying some close relationship. a phylogenetic analysis is in order to get a better grip on the issue but it should include not only the species as they are presently known but also representative populations from different sites for each species, especially the more widespread ones with broad geographical and altitudinal coverage such as s. gundlachi or s. denticulata. upon discussing morphological variability within s. gundlachi, bolton (2000) points out the distinctness of some high altitude specimens from colombia, which could represent a different species. a similar situation repeats itself when bolton discusses variability within s. denticulata and some high altitude specimens from sw colombia. longino (2006) found and described three species apparently closely related to s. subedentata mayr that form an elevational succession along the slopes of his sampling sites in costa rica with each occupying a determined altitudinal range. a similar situation is then conceivable for s. gundlachi and s. denticulata, or perhaps other species. more sampling and alpha taxonomy is needed, especially in the mountains of colombia, ecuador, peru, and bolivia, before we may have a better picture of the distribution and relations of the gundlachi complex cold weather specialists. climate change and the ever increasing destruction and fragmentation of the andean cloud forests actively work against such a research agenda. strumigenys madrigalae n. sp. figs 4 – 5. strumigenys madrigalae n. sp. worker. scale bar = 0.25mm. pilosity omited. fig 4. strumigenys madrigalae n. sp., head dorsal view. fig 5. strumigenys madrigalae n. sp., body lateral view. elongated shape on mesopleuron outlined by broken line defines area of smooth and shining sculpturing. diagnosis. mandibular dentition with 5 acute teeth apicad of basal lamella, the basal 3 largest; teeth followed by 4 minute preapical denticles, and apical tooth; no diastemma between teeth and lamella, lamella higher than longest teeth. postpetiolar dorsum smooth. type material. holotype: ecuador, loja, reserva el madrigal, 4.04655°s 79.17583°w, 6.3 km sse of loja, 2350m, 28 august 2014, j. lattke 3590-17. holotype (worker) deposited in qcaz. paratype: one dealate queen with the same locality data and date as the holotype but from sample lattke 3590-14 is also deposited in qcaz. worker description. holotype: hl 0.61, hw 0.45, ml 0.13, sl 0.31, pw 0.29, al 0.61mm; ci 74, mi 21, si 69. posterior cephalic margin has shallow median concavity sociobiology 62(2): 175-180 (june, 2015) 179 with head in dorsal view, occipital lobe weakly expanded laterally; posterolateral cephalic margin convex, with curved decumbent spatulate hairs, apical scrobal hair absent. four standing hairs present between highest point of vertex and occipital margin. eye oval-elongate with 5 ommatida in the longest axis. scape external margin with appressed spatulate hair close to base; also with three erect spatulate hairs, one basad of basal angle; three spatulate hairs that curve apicad present close to scape apex. anterolateral clypeal margins convex throughout, clypeal dorsum with appressed squamate hairs. mandibular dentition with 5 acute teeth apicad of basal lamella, the basal 3 are largest; followed by 4 minute preapical denticles, and apical tooth; no diastemma between teeth and lamella, lamella higher than longest teeth. promesonotal dorsal margin broadly convex in lateral view, metanotal groove broad and shallow, propodeal dorsal margin straight, slightly elevated above metanotal groove and forming obtuse angle with declivitous margin. pronotal humeral hair absent, mesonotum with two hairs, erect and slightly arched medially. anterior pronotal margin with transverse crest bearing row of short spatulate hairs. propodeal tooth short, sharply pointed with lamella extending posterad towards low metapleural lobes; propodeal spiracle directed posterolateally, separated from posterior margin of lamella by not more than one diameter. petiolar node forms convex dome with two erect hairs close to posterior margin, hairs slightly inclined posterad, anterolaterally with two pairs of curved decumbent hairs; postpetiole with three pairs of standing hairs; anterad, posterad, and laterad. petiole with narrow fringe of spongiform tissue along posterior margin, laterally and dorsally, none ventrally; postpetiole with well-developed ventral and lateral spongiform tissue which continues onto dorsoposterior margin, narrow fringe present on anterior margin of abdominal tergite iv; none present on abdominal sternite iv. abdominal tergite iv with sparse erect hairs, each slightly inclined posterad; mostly smooth and shining with short, basal longitudinal costulae, not longer than the maximum metatibial width. profemur dorsum with elongate bullae, rice grain shaped. outer surface of meso and metatibia with decumbent arched hairs, each hair about as long as respective maximum tibial width or slightly shorter. head, mesosoma, and petiole densely and finely reticulatepunctae; mesopleuron mostly smooth medially; postpetiolar dorsum smooth. coxae, femora and tibiae densely reticulatepunctate. body mostly ferruginous brown, dorsum of head and thorax dark brown, gaster black. gyne. hl 0.71, hw 0.51, ml 0.15, sl 0.34, pw 0.41, al 0.82mm; ci 72, mi 21, si 67. gyne comparable with the worker and differing in expected traits such as the greater development of the mesosoma and presence of ocelli. etymology. the specific epithet “madrigalae” is derived from the name of the nature reserve in which the specimens were found, madrigal. discussion. this species fits within the schulzi group as defined by bolton (2000:214) but the dental arrangement in s. madrigalae is unlike any of the other species in the group as it lacks a couple of teeth after the first five that follow the basal lamella, for a total of 10 teeth compared with the 12 teeth of other species. using the key for neotropical pyramica in bolton (2000:137), this species will key smoothly to couplet 65, where it coincides with the characteristics of s. microthrix, a species known from costa rica and colombia. using the original description of s. microthrix by kempf (1975:422) as well as that of bolton (2000: 185), and images of a specimen (inbiocri001283688) available from antweb, it was possible to identify several discrete differences between the two species. the occipital lobes in s. microthrix are more strongly expanded and its mandible has only 5 acute teeth between the minute preapical denticles and the basal lamella. the hairs laterally bordering the clypeus are more slender in s. madrigalae. s. microthrix lacks standing hairs on the mesosomal and petiolar dorsum, and the anterior margin of its petiolar node forms an abrupt angle with peduncle. the postpetiole has a negligible amount of ventral spongiform tissue. the postpetiolar node has one pair of erect hairs and very sparse erect hairs on the gastral dorsum. the postpetiolar dorsum is densely sculpted in s. microthrix. please see the discussion for s. lojanensis for information on the type locality of s. madrigalae. acknowledgements this research was mostly financed by the prometeo project of the secretary for higher education, science, technology, and innovation (senescyt) of the republic of ecuador. additional support was provided by the program for biodiversity, forests, and ecosystem services of the universidad nacional de loja. gabriela piedra, maria tuza, cristina gomez, and manual velez provided valuable effort in field and lab. prof. josé ramirez permitted the use of his lab and equipment. elsa & beatriz tapia castro, representatives of the madrigal private reserve, granted permission to carry out field work on the premises. references beck, e. & richter, m. (2008). ecological aspects of a biodiversity hotspot in the andes of south ecuador. in: s.r. gradstein, j. homeier & d. gansert (eds.), biodiversity and ecology series 2: the tropical mountain forest – patterns and processes in a biodiversity hotspot (pp 195–217). universitätsverlag göttingen, göttingen, germany, 217pp. richter, m., beck, e., rollenbeck. r. & bendix, j. (2013). the study area. in j. bendix, e. beck, e. bräuning, f. makeschin, r. mosandl, s. scheu & w. wilcke (eds.), ecosystem services, biodiversity and environmental change in a tropical mountain ecosystem of south ecuador. ecological studies 221 (pp. 3-17). springer-verlag, berlin, germany. doi: 10.1007/978-3642-38137-9_1. je lattke and n aguirre – two new strumigenys species from ecuador180 bolton, b. (2000). the ant tribe dacetini. memoirs of the american entomological institute, 65: 1-1028. bolton, b. (2014). an online catalog of the ants of the world. http://antcat.org. (accessed: 25 september 2014). kempf, w. w. (1975). report on neotropical dacetine ant studies (hymenoptera: formicidae). revista brasileira de biologia, 34: 411-424. [1974]. lattke, j., w. goitía. 1997. el género strumigenys en venezuela. caldasia, 19:367-396. longino, j. 2006. new species and nomenclatural changes for the costa rican ant fauna (hymenoptera: formicidae). myrmecologische nachrichten, 8: 131-143. longino, j.t, branstetter, m.g. & colwell. r.k. (2014). how ants drop out: ant abundance on tropical mountains. plos one, 9(8): e104030. doi:10.1371/journal.pone.0104030. rigato, f. & scupola, a. (2008). two new species of the pyramica gundlachi-group from ecuador (hymenoptera: formicidae). memoirs on biodiversity, 1: 477-481. rusek, j. (1998). biodiversity of collembola and their functional role in the ecosystem. journal of biodiversity and conservation, 7: 1207-1219. doi: 10.1023/a%3a1008887817883. wilcke, w., oelmann, y., schmitt, a., valarezo, c., zech, w. & homeier, j. (2008). soil properties and tree growth along an altitudinal transect in ecuadorian montane forest. journal of plant nutrition and soil science, 171: 220-230. doi: 10.1002/jpln.200625210. doi: 10.13102/sociobiology.v62i4.762sociobiology 62(4): 474-480 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 toxicities comparison of rotenone and acetone extract of tephrosia vogelii and derris trifoliata against solenopsis invicta introduction the red imported fire ant (rifa), solenopsis invicta buren (hymenoptera: formicidae), is one of over 280 species in the widespread genus solenopsis. rifa is a voracious consumer of numerous other arthropod species and often is the most abundant predaceous arthropod in crop fields throughout the united states, new zealand, and australia (nattrass, 2001; ascunceet et al., 2011). rifas were introduced to mainland china in 2005 (zheng et al., 2007) and became widely distributed in south china, eventually causing severe damage to humans, animals, and the environment. rifa stings humans, pets, farm animals, and wildlife and can damage farms, electrical equipment, and irrigation systems. many botanical insecticidal compounds exhibit good toxicity abstract the high rotenone content and the rotenone crude extract of tephrosia vogelii and derris trifoliata were evaluated for its efficacy in the control of red imported fire (rifa), solenopsis invicta under both laboratory and field conditions. the acetone extracts of d. trifoliata roots and t. vogelii leaves exhibited strong toxicity to macroergate and micrergate of rifa. when active ingredients of the crude extracts were convert to rotenone, the activity of the acetone extracts were higher than that of rotenone technical material. at the same time, the extracts showed significant inhibitory effect on walking ability and grasping ability of worker ants and stronger than the effect of 98.6% rotenone technical material. under field conditions, the 0.01% rotenone-bait, formulated with the acetone extract of d. trifoliata roots and t. vogelii leaves, had the same control effect on rifa as that of 0.01% fipronil-bait when treated after 30 d. the bait formulated with the extract of d. trifoliata exhibited quicker and higher effect on rifa than that of rotenone technical material. it was showed that the acetone extracts of d. trifoliata roots and t. vogelii leaves are able to control s. invicta under both laboratory and field conditions and can be used as an effective agent against rifa. sociobiology an international journal on social insects dm cheng1, cl huang2, ws li2, yq tian2, rq mao4, zx zhang2,3 article history edited by evandro do nascimento silva, uefs, brazil received 03 february 2015 initial acceptance 09 august 2015 final acceptance 20 september 2015 received initial acceptance final acceptance keywords solenopsis invicta, rotenone, tephrosia vogelii, derris trifoliata, extract, toxicity. corresponding author zhixiang zhang key lab. of nat. pesticide and chemical biology, ministry of education south china agricultural university guangzhou, 510642, china e-mail: zdsys@scau.edu.cn against rifa, and botanical insecticides can potentially be used to control rifa (li et al., 2014). rotenone-based bait is a good botanical insecticide used to control rifa. tephrosia vogelii and derris trifoliata are resource plants containing rotenone that can be used to control rifa. t. vogelii is native to west africa but is also found in india, asia, and other tropical regions (dalziel, 1937; lambert et al., 1993). t. vogelii was introduced into china in 1986 and has been planted over a large area in guangdong province (huang et al., 2006). the leaf macerate has purgative and emetic properties (walker, 1961; burkill., 1995). the acetone extracts of the t. vogelii leaf exhibited insecticidal activity against pieris rapae (shin, 1989). the volatile compounds released from the mashed fresh bean pods, branches, and leaves research article ants 1 zhongkai university of agriculture and engineering, guangzhou, guangdong province, china 2 south china agricultural university, guangzhou, china 3 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 4 guangdong entomological institute, guangzhou, china sociobiology 62(4): 474-480 (december, 2015) 475 of t. vogelii exhibited high toxicity against the rifa workers and evidently reduced the walking and grasping abilities of the rifa workers. d. trifoliata is an ever green mangrove plant belonging to the family fabaceae (leguminosae), which is commonly known as the legume family. d. trifoliata is a perennial climber or a highly branching climbing shrub that can reach a length of 8 m or less. the leaves of t. vogelii and the roots of d. trifoliata contain rotenone, a poison that kills a wide range of creatures from insects to earthworms and fish. t. vogelii and d. trifoliata are commercially cultivated as sources of insecticidal rotenoids in china. d. trifoliata is used to produce rotenone in commercial quantities as an insecticide. the insecticidal raw material of rotenone-based bait is usually pure rotenone. raw material can also be obtained from t. vogelii leaf extract and d. trifoliata root extract, which are cheaper. this study aimed to demonstrate which one of the above mentioned three sources is more applicable to be used as the raw material of rotenone based bait. materials and methods insects s. invicta colonies were collected from the campus of south china agricultural university. ant workers were reared in plastic containers (50 cm diameter × 20 cm height) coated with teflon emulsion on the top under ambient conditions (temperature 25 ± 2º c and a relative humidity of 60% to 80%). a test tube (25 mm × 200 mm) partially filled with 10% honey water and plugged with cotton was used as the water source. the tested ants were fed with ham sausage. the tested macroergates ranged from 5 mm to 6 mm in length, and the micrergates ranged from 3 mm to 4 mm in length. plant t. vogelii (family: legume, papilionacea; genus: tephrosia) was planted in the sample region of insecticidal plants at south china agricultural university in april 2012. the size of the plant area was 20 m×20 m. the height of the trees was about 2 m, and the basal stem diameter was about 3 cm. the leaves were cut off in february 2014 and immediately sent to the laboratory. d. trifoliata (family: fabaceae, subfamily: faboideae, genus: derris) was planted in may 2010 in fengshun county, meizhou city, guangdong province, china. the size of the plant area was 90 m×20 m. the roots were collected in february 2014. apparatus and chemicals high-performance liquid chromatography (hplc): lc20 a equipped with uvd and an auto sampler, interfaced to lc solution data processing software (shimadzu, japan). zorbax tc-c18column (250 mm4.6 mm id) with film thickness of 5 μm (agilent, american) was used. rotenone technical material (98% purity) was purchased from tangxijiaxing welfare chemical factory (fengshun county, meizhou city, guangdong province, china). rotenone standard substance (99.5% purity) was purchased from sigma co. ltd., united states (www.sigmaaldrich.com). hplc-grade acetonitrile and acetone were purchased from guangzhou chemical reagent factory, china (www.chemicalreagent.com). fipronil technical material (96% purity) was purchased from bayer crop science china co., ltd (www.bayercropscience.co.th). preparation of plant extracts the dried leaves of t. vogelii and roots of d. trifoliata were powdered by using an electric blender. dried plant powder (200 g) was prepared by passing the leaves through a 1 mm sieve and extracted thrice (60 min each time) with 3000 ml of acetone. the extracts were concentrated using a rotary vacuum evaporator at 40º c, and the residue obtained was stored at 0º c. the rotenone content was analyzed using reverse-phase hplc (zhou et al., 2014). preparation method of rotenone or fipronil-based bait rotenone or fipronil was mixed with maize powder, peanut oil, fishbone powder, and carbohydrates and was prepared into granules (700 mm to 1500 mm particles) according to the method previously described by lekhnath (2010) and zhong (2012), with slight modifications. rotenone, fipronil, and the acetone extracts of t. vogelii leaves and d. trifoliata roots were dissolved separately with small amounts of acetone. the solution was mixed uniformly with the bait. the rotenone-or fipronilbased bait was prepared after the acetone volatilized completely. insecticidal toxicity bioassay the method was similar with those described by zeng (2006). a water test tube, 0.5 g of rotenone-based bait, and worker ants were placed in a disposable plastic cup (top/ bottom/height: 62 mm/40 mm/60 mm) whose vertical wall was coated with a fluon emulsion. each treatment was replicated three times, and each replicate included 30 worker ants. the contrast group was the acetone-based bait. the workers were maintained at 24º c to 26º c and 60% to 80% relative humidity. during the tests, the mortalities were recorded at 24, 48, and 72 h after treatment. behavior observation on walking ability of worker ants worker ants were treated as insecticides toxicity bioassay. behavioral observation on walking ability of worker ants was performed using methods similar with those of li (2014) and zheng (2007). worker ants were placed on a square graph paper (side:20 cm), and the traveling paths were filmed using canon zx zhang et al. – toxicity of tephrosia vogelii and derris trifoliata extract against solenopsis invicta476 digital camera (eos6d), after which the camera was connected to a computer. the total distance covered by the worker ant was calculated. if the worker ants could walk continuous for 10 cm without falling, they were regarded to possess walking ability. the following equation was used for calculations: grasping rate (%) = (number of workers possessing walking ability/number of workers per replicate)x100 behavior observation on grasping ability of worker ants worker ants were treated as insecticides toxicity bioassay. worker ants were placed in the disposable plastic cup, and the cup was shook gently. after 10 s, when the ants were distributed uniformly in the bottom, after which the cup was turned 180 degrees gently. the ants were regarded to possess grasping ability if they would not fall from the cup. the following formula was used for calculation: grasping rate(%)=(number of workers possessing grasping ability/number of workers per replicate)x100 control effect of rotenone-based bait against rifa in the field to determine the control effect of 0.01% rotenonebased bait against rifa in the field, a method previously reported by zhong (2012) was used with some modifications. the field studies were conducted in the lawn of zengcheng farm, south china agricultural university, guangzhou city. the experiment was designed in single factor random block. the following five treatments were employed: d: 0.01% rotenone-based bait, formulated with the acetone extract of d. trifoliata roots; e: 0.01% rotenone-based bait, formulated with the acetone extract of t. vogelii leaves; f: 0.01% rotenonebased bait, formulated with 98.6% rotenone technical material; g: 0.01% fipronil-based bait as control treatment; h: the blank bait mixed with only acetone was used as a negative control treatment. rifa mounds that were scattered randomly in the lawn were selected, and each treatment had three replicates. poison bait (10 g) was placed in the periphery of each active rifa mound. the rifa population numbers of each treated mound were investigated 1 d before treatment and 1, 5, 10, 20 and 30 d after treatment. the mounds were designated as active when at least30 adult workers exited the mounds of excavated soil when probed with a metal rod (5 mm in diameter) (furman et al., 2006). the control effect was determined using the following equation: p= [(sck-streatment)/ (100-sck)] ×100 statistical analysis data were reported as means ± se based on three independent experiments. the percentage values were transformed into arc sin of square root of the percentages prior to the analysis and three-factor anova. worker size and concentration were considered as the main effects. moreover, the differences between the data were assessed using duncan’s multiple range test (sas 1989), with p < 0.05 regarded as statistically significant. the figures were produced using microsoft excel 2007. results rotenone contents of t. vogelii and d. trifoliata the rotenone contents of t. vogelii leaves and d. trifoliata roots were measured by hplc method (fig 2 and 3). compared with the hplc figure of rotenone standard substance (fig 1), the rotenone content of the acetone extract of d. trifoliata roots was 18.14%, whereas that of t. vogelii leaves was 2.11% (table 1). when the rotenone content was converted into the percentage of dry plant powder, the obtained values were 1.86% and 0.19%, respectively. plant material rotenone content (%) dry plant powder acetone extracts derris trifoliata roots 1.86±0.25 18.14±2.37 2.11±0.30tephrosia vogelii leaves 0.19±0.11 fig 3. the hplc figure of rotenone in acetone extractof tephrosia vogelii leaves. fig 1. the hplc figure of rotenonestandard substance. fig 2. the hplc figure of rotenone in acetone extract of derris trifoliata roots. table 1. determination of rotenone content. sociobiology 62(4): 474-480 (december, 2015) 477 the toxicity of the extract toxicities of rotenone, extracting solution of d. trifoliata, and t. vogelii against rifa were determined (table 2 and 3). the acetone extracts of d. trifoliata roots and t. vogelii leaves exhibited strong toxicity to macroergates of rifa and were treated for 24 h. the lc50 values were 30.27 and 48.63 mg/kg, which are higher than those of 98.6% rotenone technical material. the toxicity of the acetone extracts and rotenone technical material increased as treatment time increased. the lc50 of d. trifoliata roots extract and t. vogelii leaf extract were respectively 19.28 and 31.91 mg/kg when treated for 48 treatment time regression equation lc50(mg/kg) 95% fiducial limit correlation coefficient a 24h y=3.5233+0.9970x 30.27 18.32~50.03 0.9945 b 24h y=3.1531+1.0948x 48.63 31.35~75.43 0.9918 c 24h y=2.3158+1.2592x 135.41 91.89~199.54 0.9928 a 48h y=3.5911+1.0963x 19.28 12.36~30.06 0.9930 b 48h y=3.0618+1.2887x 31.91 20.91~48.70 0.9959 c 48h y=2.1394+1.4465x 94.99 66.39~135.89 0.9893 a 72h y=3.5294+1.2607x 14.67 9.57~22.49 0.9863 b 72h y=3.0477+1.3733x 26.40 16.99~41.01 0.9874 c 72h y=2.0125+1.5476x 85.20 59.89~121.21 0.9907 a：the acetone extract of d. trifoliata roots; b：the acetone extract of t. vogelii leaves；c：98.6% rotenone technical material. active ingredients in rotenone. table 2. lc50(mg/kg) of d. trifoliata roots estract, t. vogelii leaves extract, and rotenone against macroergates of rifa. treatment time regression equation lc50(mg/kg) 95 % fiducial limit correlation coefficient a 24h y=3.7725+1.0309x 15.52 9.49~25.36 0.9917 b 24h y=3.0373+1.3826x 26.28 18.41~37.50 0.9913 c 24h y=2.7892+1.2184x 65.24 43.11~98.73 0.9915 a 48h y=3.9148+1.0883x 9.94 6.37~15.51 0.9926 b 48h y=3.2200+1.4239x 17.79 12.27~25.78 0.9922 c 48h y=3.0368+1.1537x 50.30 33.11~76.41 0.9946 a 72h y=3.8289+1.3937x 6.92 4.63~10.36 0.9932 b 72h y=3.3601+1.4934x 12.53 8.19~19.17 0.9946 c 72h y=2.9653+1.2990x 36.85 24.78~54.79 0.9868 a:the acetone extract of d. trifoliata roots; b: the acetone extract of t. vogelii leaves; c: 98.6% rotenone technical material. active ingredients in rotenone. table 3. lc50 (mg/kg) of d. trifoliata roots extract, t. vogelii leaves extract, and rotenone against micrergates of rifa. hand were 14.67 and 26.40 mg/kg when treated for 72 h. the bioactivities of the two extract baits to macroergates of rifa were apparently higher than that of rotenone technical material. experimental results indicated that the toxicity of d. trifoliata root extracts and t. vogelii leaf extracts was higher than that of 98.6% rotenone technical material under the same treatment time. the lc50 values of the two extracts to microergates of rifa were respectively 15.12 and 26.28 mg/ kg when treated for 24 h; 9.94 and 17.19 mg/kg when treated for 48 h; and 6.92 and 12.53 mg/kg when treated for 72 h. the lc50 values of 98.6% rotenone technical material were 65.24, 50.30, and 36.85 mg/kg, respectively. behavior observation on walking ability and grasping ability of worker ants the extracts of d. trifoliata roots and t. vogelii leaves exhibited good toxic activity against the rifa workers and at the same time showed significant inhibitory effect on walking ability and grasping ability of worker ants (tables 4 and 5), which is stronger than the effect of 98.6% rotenone technical material. the macroergates and microergates showed poor walking and grasping abilities after treatment with the extracts and rotenone. the walking and grasping abilities of rifa workers significantly decreased as treated time increased. the walking rates of microergates of rifa treated by t. vogelii leaf extracts were 43.33%, 21.67%, and 5.00%, and the grasping abilities were 20.00%, 8.33%, and 0.00%, respectively, which were significantly lower than the walking ability and grasping ability of d. trifoliata root extracts and rotenone. compared with the control, the treatments by extracts and rotenone could reduce the walking ability and grasping ability of microergates of rifa. within these treatments, t. vogelii leaf extract, d. trifoliata root extract, and rotenone had the same effect on the walking ability and grasping ability of macroergates of rifa. zx zhang et al. – toxicity of tephrosia vogelii and derris trifoliata extract against solenopsis invicta478 control effect of rotenone-based bait on rifa in the field the 0.01% rotenone-based bait, formulated with the acetone extract of d. trifoliata roots, showed significant controlling effect on rifa in the field and worked faster than the other three treatments. the control effect was 57.78% for 5 d after treatment. the control effect sequence of other three treatments was 0.01% for fipronil-based bait, 0.01% for rotenone-based bait (formulated with the acetone extract from t. vogelii leaves), and 0.01% for rotenone-based bait (formulated with 98.6% rotenone technical material) 5 d after treatment. the control effect of 0.01% rotenone-based bait (formulated with the acetone extract of d. trifoliata roots) was higher than the other treatments 10 d after treatment and approached 100% within 20 d. at the end of the study (30 d), all four treatments achieved perfect controlling effect. treatment rotenone content (mg/kg) walking ability (%) grasping ability (%) 1 d 3 d 7 d 1 d 3 d 7 d a 5 51.67±4.41c 28.33±4.41c 10.00±2.89c 35.00±5.77c 11.67±3.33c 3.33±1.67c b 10 43.33±3.33d 21.67±4.41c 5.00±2.89c 20.00±2.89d 8.33±1.67c 0.00±0.00d c 20 81.67±1.67b 63.33±6.01b 28.33±4.41b 70.00±2.89b 40.00±2.89b 16.67±4.41b ck 0 100.00±0.00a 100.00±0.00a 96.67±1.67a 100.00±0.00a 98.33±1.67a 95.00±2.89a a：the acetone extract of d. trifoliata roots；b the acetone extract of t. vogelii leaves；c：98.6% rotenone technical material. active ingredients in rotenone. table 4. influences of d. trifoliata roots extract, t. vogelii leaves extract, and rotenone on walking and grasping abilities of micrergates of rifa (mean ± se, %). treatment rotenone content (mg/kg) walking ability (%) grasping ability (%) 1 d 3 d 7 d 1 d 3 d 7 d a 5 66.67±6.01c 45.00±2.89c 23.33±1.67c 56.67±4.41c 35.00±2.89c 16.67±3.33c b 10 50.00±5.77d 33.33±3.33d 15.00±2.89d 36.67±4.41d 25.00±2.89d 10.00±2.89c c 20 91.67±3.33b 76.67±1.67b 46.67±4.41b 83.33±3.33b 63.33±4.41b 41.67±4.41b ck 0 100.00±0.00a 98.33±1.67a 95.00±2.89a 98.33±1.67a 96.67±1.67a 91.67±4.41a a：the acetone extract of d. trifoliata roots；b the acetone extract of t. vogelii leaves；c：98.6% rotenone technical material. active ingredients in rotenone. table 5. influences of d. trifoliata root extract,t. vogelii leaf extract, and rotenone on walking and grasping abilities of macroergates of rifa (mean ± se, %) discussion scientists have previously reported that natural products derived from plant extracts were tested in a discovery program for effective, environmental-friendly pesticide control agents. among the natural products, rotenoids is a type of important plant source pesticide and has been widely applied for insect control (zhang et al., 2005). rotenone from natural sources easily degrades and has high environment compatibility that can be used in organic agriculture. in china, the rotenone is extracted using different organic solvents, such as dimethylbenzene, methylbenzene, and the major product of rotenone, is an emulsifiable concentrate (xu et al., 2001). the extraction process and traditional product application could harm the environment and endanger public health, thereby lowering the merit of the rotenone. the results obtained in this study demonstrate that the acetone extract with rotenone of d. trifoliata roots showed higher biological activity than rotenone technical material against rifa at same doses and treatment times. results of the study showed that rotenone crude extracts could be directly used to control pests and serve as a powerful insecticide for controlling rifa. treatment dose (g/nest) average nest number before treatment control effect (%) 1 d 5 d 10 d 20 d 30 d d 10 15±0 22.22±2.22a 57.78±2.22a 93.33±3.85a 100.00±0.00a 100.00±0.00a e 10 15±0 17.78±2.22a 35.56±2.22b 68.89±4.44b 88.89±4.44b 95.56±4.44ab f 10 15±0 20.00±3.85a 31.11±4.44c 40.00±3.85d 73.33±3.85c 88.89±4.44b g 10 15±0 22.22±2.22a 40.00±3.85b 53.33±3.85c 86.67±3.85b 97.78±2.22a note: d:0.01% rotenone bait, formulated with the acetone extract of d. trifoliata roots. e: 0.01% rotenone bait, formulated with the acetone extract t. vogelii leaves. f: 0.01% rotenone bait, formulated with 98.6% rotenone technical material. g:0.01%fipronil bait. table 6. the control effect of rotenone-based bait on rifa. sociobiology 62(4): 474-480 (december, 2015) 479 resource plants containing rotenone and its analogues belong to the legume family, including derris, tephrosia, lonchocarpus, and millettia (xu, 2001). derris plant is one of the most famous insecticidal plants and has a long history in china. some of the more common derris plants are d. trifoliata, d. elliptica, d. malanccensis, and d. ferruginea. various plants of tephrosia with rotenone exist, and t. vogelii leaves are rich in rotenone and its analogues (li et al., 2010). high activity and rich plant resources of rotenone are conducive to develop new products by crude extract to control rifa. the d. hancei root power mixture had good field control efficacy against rifa (tian et al., 2010). the control efficacy of worker ants reached up to 98.25%, and the nest elimination averaged 95.32% 21 days after applying the powder mixture that contains 20% d. hancei root power. after which, all ants within nests eventually died. the results indicated that plant dry power containing rotenone could be directly used to control red fire ants. the toxicity bait with the active ingredient of rotenone showed an obvious trapping and killing effect on pheidole yeensis (wang et al., 2010). li et al (2014) had studied the effect of volatile compounds from the mashed fresh bean pods, as well as the branches and leaves of t. vogelii on the behavior of s. invicta workers. results showed that the volatile compounds possess high toxicity against the rifa workers and can reduce the walking and grasping abilities of these workers. compared with dry matter, the mashed branches and leaves of t. vogelii exhibit greater potential for producing insecticides. the results of this study showed that rotenone can be developed into a new variety that is effective against s. invicta. aside 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(2014). effect of two formulations on the decline curves and residue levels of rotenone in cabbage and soil under field conditions. ecotoxicology and environmental safety, 104: 23-27. doi: 10.1016/j.ecoenv.2014.02.014 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v61i4.355-368sociobiology 61(4): 355-368 (december 2014) investigating eusociality in bees while trusting the uncertainty introduction during the last century, research on bee behavior and morphology has largely become increasingly comparative, incorporating the diversity of traits found in the variety of species of these insects (e.g., michener, 1944, 1974, 2007; engel, 2011; danforth et al. 2013). remarkably detailed investigations had been previously made on bees (e.g., schmid & kleine, 1861; girdwoyn, 1876; macloskie, 1881; briant, 1884; carlet, 1884; bordas, 1894; abonyi, 1903; hommell, 1904, 1905; arnhart, 1906; snodgrass, 1910, 1935, 1942, 1956), particularly with apis mellifera linnaeus, 1758 the honeybee. this has long been a species with close proximity of mankind (the species of apis are distributed all over the old world), with economic interest because of honey production (pollination became a hotly debated topic more recently, e.g., oldroyd, 2007; cameron et al., 2010; potts et al., abstract phylogenetic hypotheses and estimates of divergence times have already been used to investigate the evolution of social behavior in all lineages of bees. the interpretation of the number of origins of eusocial behavior and the timing of these events depends on reliable phylogenetic hypotheses for the clades in which these lineages are nested. three to six independent origins of eusocial behavior are interpreted to have occurred in bee taxa that differentiated in the late cretaceous, or much later in the paleogene. only two groups of bees exhibit the behaviors that qualify their members to be considered obligate (i.e. ‘fixed-caste’) eusocial, the honey bees (apini) and the stingless bees (meliponini). the evolutionary history of corbiculate bees remains uncertain in many respects, but phylogenetic research has been paving the path for comprehensive comparative approaches likely to shed light on the origin of diversity of forms and behaviors of these bees. in total, corbiculate bees encompass about 1,000 species, roughly 5% of the described species diversity of bees. these bees are rather heterogeneous in terms of social organization, particularly stingless bees and orchid bees, which display a fascinating range of behavioral variation. using phylogenetic tools, it has been possible to infer that caste polymorphism, division of labor and other traits of corbiculate bees probably started evolving over 80 million years ago. phylogenetic hypotheses must be interpreted as more or less uncertain scenarios for studying the biological diversity, but when trusted they can provide powerful tools to investigate the evolution of social behaviors. sociobiology an international journal on social insects eab almeida, ds porto article history edited by denise araujo alves, esalq-usp – brazil received 26 october 2014 initial acceptance 25 november 2014 final acceptance 09 december 2014 keywords apini, comparative biology, meliponini, divergence time, phylogeny, social behavior. corresponding author eduardo a. b. almeida laboratório de biologia comparada e abelhas (lbca), depto. de biologia faculdade de filosofia, ciências e letras de ribeirão preto (ffclrp) universidade de são paulo avenida bandeirantes, 3900. 14040-901 ribeirão preto,sp, brazil e-mail: eduardo@ffclrp.usp.br 2010; albrecht et al., 2012; bartolomeus et al., 2013; garibaldi et al., 2013), and certainly because of its fascinating social organization. along with ants, bumble bees, termites, social wasps, and unusual species of mammals (mole-rats: jarvis [1981]), honey bees have had a prime importance for the understanding of the cohesion observed among organisms of some species that allow them to live close together, interact, share tasks related to survival and reproduction. arthropods comprise about half of the described biodiversity, and the total number of known origins of “eusociality” is approximately 12 in this clade (wilson, 1971; wilson & hölldobler, 2005). sociality, in this way, is a very rare condition in nature, indicating that it is not a frequently acquired trait, particularly because it involves reproductive division of labor (wilson, 1971) and all potential conflicts related to this (e.g., trivers & hare, 1976; reeve & keller, 1999; tóth et al., 2004). review universidade de são paulo, ribeirão preto, sp, brazil eab almeida, ds porto eusociality in corbiculate bees 356 if the evolution of sociality was elementary, one might ask why there are relatively few eusocial species. although the number of social species is reduced in comparison to the known biodiversity, sociality can be viewed as ubiquitous because social organisms, particularly insects, are everywhere and tend to be very common. some estimates indicate that social organisms, particularly termites and ants, might comprise almost 30% of the biomass of a tropical rainforest (fittkau & klinge, 1973); and that the number of workers in a single colony of ants may reach more than 20 million (hölldobler & wilson, 1990; wilson & hölldobler, 2005). among termites, this number can be as high as 13 million individual workers (lee, 2002), honey bees are documented to have colonies comprising between 10 to 100 thousand workers (bourke, 1999) and nests of eusocial stingless bees of the genus scaptotrigona (meliponini) may encompass 50 thousand workers (lindauer & kerr, 1960). these huge colonies account for the ubiquity and the heavy biomass of social organisms. the study of the honey bee and research of all the traits related to its social behavior have been a challenge posed by researchers of several fields over the centuries. in addition to external and internal morphology (e.g., snodgrass, 1910, 1942, 1956), there were several landmarks in ethological investigation made with a. mellifera (the most famous must be karl von frisch’s 1946 study that deciphered the “waggle dance” of honey bees — see also the excellent summaries by winston, 1987; seeley, 1995, 2010; page, 2013); genetics (a. mellifera mitochondrial genome sequencing [crozier & crozier, 1993]; discovery of important insulin/tor signaling pathways and receptors related to queen caste determination [patel et al., 2007]; discovery of the royalactin by kamakura, 2011]); physiology (food contents versus caste determination in a. mellifera; hormones and pathways related to social behaviors, queen dominance, etc. [e.g. weaver, 1955; laidlaw et al., 1956; hartfelder & engels, 1998; hartfelder, 2000; sullivan et al., 2000]); and more recently genomics (honeybee genome sequencing consortium, 2006). a natural progress in the research of social behavior is to make it comparative, to unravel similarities and differences among the diversity of organisms that display some level of sociality. there exist intricate physiological (hormonal/ neurobiological) interactions of different signaling pathways that accounts for the complexly self-organized colony of a. mellifera, perhaps just a fortuitous result of the evolutionary odds, perhaps a set of specific mechanistic components interacting with each other, responsible for ordering colonylevel functions. one of the exciting challenges resulting from the enormous body of knowledge about a. mellifera is the understanding of how social interactions would be expressed in other bee species that also exhibit complex behaviors related to the self-organization of their societies (e.g., page, 2013), and how comparative research could help elucidate the evolution of social behaviors. eusociality among the various levels of intraspecific interactions that can be interpreted as social interactions, this work will focus on the eusocial behavior of bees, more specifically on obligate eusociality sensu crespi and yanega (1994; commonly referred to as “advanced eusocial” or “highly eusocial” behavior). michener (1974) presented a comprehensive classification of kinds of social behaviors documented among bees in his chapter 5 “kinds of societies among bees”. eusocial organisms may be characterized by life in “colonies, which are family groups”, “consisting of individuals of two generations, mothers and daughters”; and by “division of labor, with some individuals functioning as egg layers or queens and others as workers, that is with more or less recognizable castes” (michener, 1974: p.46). “eusociality” has been defined in slightly different ways over the years, but the presence of castes seems to be a main trait for the recognition of this phenomenon (crespi & yanega, 1994). other behaviors that are often cited as necessarily co-occurring in eusocial organisms are the superposition of generations and the cooperation in the tasks necessary for survival and reproduction. one important distinction between eusocial bees refers to the morphological polymorphism between females of reproductive and non-reproductive castes, and the capacity of gynes (i.e., potential or actual reproductive individual: queen) of surviving alone (michener, 1974). when the morphological distinction is more clearly marked and a gyne is not able to start a nest by herself (michener 1974, 2007), hence neither caste can be said to be totipotent (crespi & yanega, 1994), there is a phenomenon defined as dependent colony foundation, which was neatly reviewed by cronin et al. (2013). lack of gyne totipotency thus is key for the recognition of “advanced eusocial” or “highly eusocial”, or “obligate eusocial” behavior. in this paper we choose to favor the term “fixed-caste eusociality”, similar to that proposed by crespi and yanega (1994), because it transmits a less teleological notion than the former two. michener (1974: p.47) remarked that it “is tempting to look at the sequence of increasingly complex societies (...) as an evolutionary sequence”, but in fact multiple evolutionary changes toward simpler as well as more complex behaviors have occurred throughout bee history. terms with ability to introduce misconceptions, such as “primitive” and “advanced” should, hence, be avoided and replaced by “totipotent-caste” and “fixed-caste” when possible. there are at least five independent origins of eusociality among bees, as discussed below, but obligate eusociality is rare and can only be found in the honey bees (apini: apis) and the stingless bees (meliponini). phylogenetic research and the understanding of social evolution recent insights given by phylogenetic work have allowed estimation of divergence ages between distinct lineages of social organisms (e.g., cardinal & danforth, 2011). the two most distantly related eusocial taxa, mammals and insects, diverged between the precambrian and the early cambrian, at the early sociobiology 61(4): 355-368 (december 2014) 357 split of the common ancestor of bilaterian animals that gave rise to protostomia and deuterostomia (blair, 2009; edgecombe et al., 2011). focusing on the eusocial insects, termites diverged from the lineage that gave rise to hymenoptera in the beginning of the mesozoic or earlier (ware et al., 2010; cardinal & danforth, 2011), and began their differentiation and diversification about 235 mya, during the late triassic. within hymenoptera the origin of eusocial behavior in ants probably occurred in the early cretaceous, which occurred independently from vespid wasps (some time in the cretaceous: grimaldi & engel [2005], brady et al. [2006a], moreau et al. [2006], hines et al. [2007]); and bees, which comprise three to six independent origins of eusocial behavior in taxa that differentiated in the late cretaceous, or much later in the paleogene (brady et al., 2006b; chenoweth et al., 2007; cardinal & danforth, 2011; gibbs et al., 2012; danforth et al., 2013). these discrepancies in divergence ages bring one certainty and several possible questions. we can say we are confident that eusociality is not a homologous condition that can be properly used in a comparative (i.e., historical) context. therefore, caution must be exercised at all times when comparisons are made between taxa showing social behaviors because their traits may not be directly comparable (i.e., homologous; nixon & carpenter, 2012). a more radical interpretation of sociality, as a nonhomologous trait, is its interpretation as an emerging property or epiphenomenon resulting from the co-occurrence of complex suite of traits related to the coexistence and certain interactions among individuals of a species. given that eusociality surely is not a homologous characteristic, there are various interesting queries that can be posed about the evolution of social behaviors, such as the following: (a) when can we assume that at least some of the traits comprised in the definition of “eusociality” to be homologous?; (b) do the defining traits of “eusociality” appear together or they evolve in a stepwise manner?; (c) is it possible to relate multiple evolution of “eusocial” behaviors with general evolutionary factors or they are mostly intrinsic properties of the organisms that possess them? it is very possible that many of those questions will be confidently answered in the years to come partly because of the empirical knowledge that is fundamental for depiction of life’s diversity, as well as the growing phylogenetic basis necessary for historical comparative research. below we are going to discuss the relative confidence and uncertainty regarding the phylogenetic history of bees (and its relevance for comparative research); the most diverse taxon of obligate eusocial bees: the meliponini; and the importance of the growing body of knowledge about orchid bees to the understanding of behaviors in corbiculate bees. relative uncertainty of phylogenetic hypotheses available for bees our understanding of bee phylogeny has been improving (michener, 1944, 2007; roig-alsina & michener, 1993; alexander & michener, 1995; danforth et al., 2013) and, with it, a growing comparative framework for understanding the evolution of social behavior is being constructed. the higher-level (i.e., familylevel) classification of bees has become very remarkably stable over the last decades particularly because of intense phylogenetic research within apoidea (e.g., roig-alsina & michener, 1993; alexander & michener, 1995; melo, 1999; danforth et al., 2006a, b, 2013; michener, 2007; engel 2011; debevec et al., 2012; cardinal & danforth 2013; hedke et al., 2013). there are still portions of the tree of bees that can be represented by incongruent competing hypotheses (e.g., alexander & michener, 1995; engel, 2011; danforth et al., 2013), but the accumulated scientific progress is nevertheless clear. phylogenetic hypotheses and estimates of divergence times have been used to investigate the evolution of social behavior in all lineages of bees (reviewed by schwarz et al., 2007; cardinal & danforth, 2011; danforth et al., 2013). the interpretation of the number of origins of eusocial behavior and the timing these events depends on reliable phylogenetic hypotheses for the clades in which these lineages are nested. a summary of bee phylogeny is presented in figure 1, depicting in particular detail clades where eusocial behavior is present fig 1. phylogenetic relationships within bees (hymenoptera: apoidea: anthophila). the overall relationships depicted are those summarized by danforth et al. (2013). bee lineages are represented at family-level resolution for taxa not comprising eusocial representatives, or in more detail in cases where eusocial species are present (indicated by solid circles, stars indicate the bee taxa considered obligate eusocial) — see michener (2007) and schwarz et al. (2007) for details. unresolved nodes (i.e., polytomies) and gray shaded branches indicate regions of the tree of bees upon which uncertainty and conflicts have been most noticeable in studies published during the last decade. branch lengths are proportional to the evolutionary time, and a ruler scaled in million years before the present is given beneath the tree (divergence ages and the estimates for clade ages were taken primarily from cardinal & danforth [2013], and complemented by estimates by brady et al. [2006b], cardinal et al. [2010], almeida et al. [2012], gibbs et al. [2012], and martins et al. [2014]). eab almeida, ds porto eusociality in corbiculate bees 358 in all of its component taxa or part of them. it is worth noticing that hypotheses about the factors and processes favoring the evolution of social interactions (e.g., michener, 1974, 1985; wcislo & tierney, 2009) can be dissociated from a number and timing of evolutionary events, although there are obvious advantages in jointly investigating these two fields. whereas the hypotheses of phylogenetic relationships among the eusocial representative taxa of allodapine bees (apidae: xylocopinae) and halictinae (halictidae) have become more stable over the years (brady et al., 2006b; chenoweth et al., 2007; schwarz et al., 2007; gibbs et al., 2012), relationships among the corbiculate bees have remained largely uncertain based on phylogenetic investigations and discussions published since the 1970’s. nevertheless, two points must be made about the systematics of corbiculate bees: (a) there is almost no doubt about the naturalness (i.e., monophyly) of the corbiculatebee clade (e.g., roig-alsina & michener, 1993; cardinal & danforth, 2011); and (b) there is little or no controversy over the natural boundaries of the four most distinctive corbiculate lineages, which are classified as the tribes apini, bombini, euglossini, and meliponini. the most recent common ancestor of corbiculate bees is estimated to have begun differentiating from the closest apid lineages at about 95-72 million years ago, during the late cretaceous (cardinal et al., 2010; cardinal & danforth, 2011, 2013; martins et al., 2014). the group comprises the well-known honey bees (apis, 11 species — apini), bumble bees (bombus, approximately 260 species — bombini), orchid bees (5 genera, approximately 240 species — euglossini), and stingless bees (ca. 60 genera, over 500 species — meliponini) (michener, 2007; rasmussen & cameron, 2010; camargo & pedro, 2012; moure et al., 2012; ascher & pickering, 2014). in total, corbiculate bees encompass about 5% of the described species diversity of bees known to this day, and the group is far from homogeneous in terms of social organization (particularly meliponini and euglossini). in contrast, the closest relatives to the corbiculate clade are the bee genera centris and epicharis (cardinal & danforth, 2011; martins et al., 2014), which do not display any kind of social behavior (michener, 2007; cardinal & danforth, 2011; martins et al., 2014). it should be a rather straightforward problem the search for the phylogenetic relationships among apini, bombini, euglossini, and meliponini, because there are only three possible unrooted tree topologies, based on the mathematical properties of trees (felsenstein, 1978). for each of these unrooted networks, there are five possible rooted tree topologies, as shown in figure 2. after comparing the empirical support each of the 15 possible cladograms received over the history, it is amazing to realize that nine of them have had at least one paper in their favor (fig 2), although some of them are supported by many results (hypotheses h1-c, h1-d, h2-c, h2-e shown in fig 2). the conflicting empirical support received by the various phylogenetic hypotheses of corbiculate bees has been hard to explain. the gap between hypotheses listed as h1 and those portrayed as h2 in figure 2 is clearly related to the source of the data employed for the investigation, as molecular datasets largely support the former, and phenotypic characters (behavior and morphology) favor the latter in most cases. potential convergence in parts of the phenotype and artifacts related to molecular evolution have been pointed as the most likely causes for the contention (winston & michener, 1977; michener, 1990; ascher et al., 2001; cameron & mardulyn, 2001; lockhart & cameron, 2001; kawakita et al., 2008), but this has not resolved the controversy about how apini, bombini, euglossini, and meliponini are related to each other. the early diversification of corbiculate bees probably happened in ways that make the ancient phylogenetic signal hardly detectable. in part, this might be explained by the extinction of various lineages of corbiculate bees since the cretaceous (e.g., engel, 2001a, b; engel et al., 2009) and possibly by rapid divergence among some lineages. choice of outgroups is a sensitive issue when working with corbiculate bee relationships (e.g., canevazzi & noll, 2014), which is illustrated by the unstable placement of different lineages of apidae near or within the corbiculate clade (table 1 of cardinal & packer, 2007). finally, it is worth speculating the rooting is also an issue when selecting one of the alternative rooted tree topologies (right column of fig 2) from one of the unrooted trees shown on the left of the same figure. character data will support one of the three topologies on the left, whereas rooting (more specifically, the placement of the root-node) will be decisive for the proposal of one rooted tree-topology as the most likely scenario for the evolutionary connections among the four corbiculate-bee lineages. the consequences of the instability of placement of the root-node are far from trivial, as for example, in h2 the relationships can vary from apini as sister-group of all remaining corbiculates (fig 2, hypothesis h2-a), meliponini as sister to the other three apine tribes (fig 2, hypothesis h2-b), or meliponini as sister to apini (fig 2, hypotheses h2-c and h2-e). these are the possibilities only within the realm of the hypothesis termed ‘h2’ in figure 2, but there are also three possible rootings accepted for h1 and two possibilities within h3, as can be seen in the same figure. phylogenetic uncertainty and behavioral evolution within the corbiculate clade. how do the incongruences and the uncertainty about the phylogeny of this group of bees affect our understanding of the evolution of obligate sociality in the corbiculate clade? if the phylogenetic hypotheses sustained by the majority of morphological and behavioral data obtained so far is correct, thus we should assume that the direct ancestor of apini+meliponini also presented some (or all) the traits necessary to be considered obligate eusocial too (fig 3a, b). alternatively, it is possible that the immediate ancestor of apini+meliponini+bombini (fig 3c) or of all four taxa (fig 3d) already presented some or all the traits observed in obligate eusocial bees. although the debate about sociobiology 61(4): 355-368 (december 2014) 359 fig 2. the corbiculate bee clade comprises four monophyletic groups: apini, bombini, euglossini, and meliponini. there are only three possible unrooted tree topologies that enable the representation of alternative scenarios for these four taxa, which are illustrated by the diagrams on the left (h1-h3). on the right column, the five possible rooted topologies are given to the corresponding three diagrams. published hypotheses supporting each of the rooted tree topologies are given inside circles and can be tracked by the following reference numbering system: <01> cameron (1991); <02> koulianos et al. (1999); <03> schultz et al. (1999); <04> cameron & mardulyn (2001); <05> kawakita et al. (2008); <06> cardinal et al. (2010); <07> cardinal & danforth (2011); <08> cardinal & danforth (2013); <09> sheppard & mcpheron (1991); <10> cameron (1993); <11> koulianos et al. (1999); <12> mardulyn & cameron (1999); <13> kerr (1987); <14> winston & michener (1977); <15> kimsey (1984); <16 >sakagami & maeta (1984); <17> michener (1974); <18> michener (1990); <19> schultz et al. (1999); <20> serrão (2001); <21> michener (1944); <22> maa (1953); <23> michener (1990); <24> prentice (1991); <25> roig-alsina & michener (1993); <26> chavarría & carpenter (1994); <27> schultz et al. (1999); <28> ascher et al. (2001); <29> engel (2001a, b); <30> noll (2002); <31> cardinal & packer (2007); <32> payne (2013); <33> canevazzi & noll (2014); <34> plant & paulus (1987); <35> peixoto & serrão (2001); <36> pereira-martins & kerr (1991). placement of outgroup taxa (i.e., non-corbiculate bees) was ignored when accounting for the support of published hypotheses in relation to the 15 possible rooted cladograms. all unrooted tree topologies were favored by at least one published study as noticeable by references listed in the figure; and nine out of the fifteen possible rooted tree topologies were favored by at least one study (black trees on the right of the figure). three points are worth highlighting when evaluating the support given by the various types of data and/or authors about these alternative phylogenetic scenarios: (1) there is a clear prevalence of molecular data supporting hypotheses shown in h1; (2) there is a clear prevalence of phenotypic genotypic data supporting hypotheses shown in h2; (3) scenarios shown in h3 have received lower support by empirical data than h1-2. eab almeida, ds porto eusociality in corbiculate bees 360 single versus multiple origins of obligate eusociality has generated enthusiastic discussions over the years, it must be pondered that this is an over-simplification of a more complex case. it is far less informative because the relation of homology being sought and investigated is not about “obligate eusociality” itself, but the suite of traits that evolve and are the requisites for this condition to emerge. if research programs are targeted on obligate eusociality, growth of knowledge about the evolution of this epiphenomenon will be likely hindered. an innovative examination of key traits most directly related to the appearance of eusociality in bees was that by cardinal and danforth (2011), who reconstructed states of phenotypic characters that co-occur in eusocial bees. this resulted in the reconstruction of evolutionary paths taken by the following five characters in the corbiculate clade: (a) castes/division of labor, (b) adults of two generations, (c) morphologically distinct gynes, (d) progressive feeding, and (e) swarming. a simplified version in which levels of sociality are treated as character-states was also considered, but it is way less informative than the former (cardinal & danforth, 2011: fig 1c, their “traditional” model). in all scenarios evaluated, totipotentcaste eusociality was the most likely condition estimated for the most recent common ancestor of all corbiculate bee species (cardinal & danforth, 2011). worth noting too is the extensive behavioral comparison by noll (2002), who expanded the term “social” into no fewer than 42 characters! the continued detailed investigations of morphological and behavioral changes, as well as the genetic basis for eusociality to appear (e.g., toth et al., 2007; woodard et al., 2011) will be key for the advancement in this field. fascinating behavioral variation and the need of comparative research: stingless bees and orchid bees (apidae: meliponini and euglossini) when comparing the four major lineages of corbiculate bees, the meliponini occupy a very special position in all measures of their diversity. there are about 500 described species of stingless bees, which are distributed in various tropical regions of the planet (the distribution was wider in the past as documented by the fossil fig 3. schematic representation of four commonly proposed hypotheses of phylogenetic relationships among the four tribes of corbiculate bees (apidae). hypotheses a and b are favored by the several datasets comprising phenotipical characters (fig 2: hypothesis h2-e and h2c), whereas hypotheses c and d are recurrent results when these relationships are inferred using molecular datasets (fig 2: hypothesis h1-e and h1-c). numbers at each of the terminal branches represent conditions of sociality: 1: fixed-caste eusociality; 2: totipotent-caste eusociality; 3: social behaviors present, but not eusociality. tree topologies a-c correspond to hypotheses h2-e, h2-c, h1-e, h1-c (fig. 2), respectively. record: e.g., engel [2011]), there is a great morphological diversity, which is reflected by the approximately 60 genera currently accepted, and great behavioral variation. the diversity of biologies of stingless bees has been documented by several researchers, many of which made important contributions to document it in comparative manners (e.g., ihering, 1903, 1930; kerr, 1948, 1969, 1987; sakagami, 1982; engels & imperatriz-fonseca, 1990; imperatrizfonseca & zucchi, 1995; faustino et al., 2002; tarpy & gilley, 2004; tóth et al., 2004; santos-filho et al., 2006). observations, experiments and predictions exist for attributes such as queenpolicing, mode of queen production, worker egg-laying and cost of worker reproduction, relatedness between sisters in a colony (e.g., engels & imperatriz-fonseca, 1990; imperatrizfonseca & zucchi, 1995; tóth et al., 2004), making stingless bees a bionomically diverse and with an incredibly open field for comparative research. many comparisons made in the past lacked an explicit phylogenetic framework, which have the potential of illuminating which evolutionary scenarios are more likely to explain the observed phenotypic diversity. one notable exception was the evaluation of the diversity in composition of the cuticular chemistry within the meliponini using an explicit phylogenetic framework (leonhardt et al., 2013). different authors contributed to the understanding of phylogenetic relationships within meliponini (e.g., schwarz, 1948; moure, 1961; wille, 1979; michener, 1990; camargo & pedro, 1992a, b; costa et al., 2003; rasmussen & cameron, 2010). a summary of the three most comprehensive phylogenetic hypotheses (in terms of taxon sampling) is presented in fig 4. the great diversity of forms, questionable proposals for generic delimitation, and wide geographical distribution certainly made the task of studying all stingless bees very challenging. the comparison of three hypotheses proposed independently along the last 24 years (fig 4a-c) makes it clear that certain portions of the tree are very stable for example, trigonisca, leurotrigona and related lineages have been placed relatively distant from the remaining neotropical genera, whereas placement of certain taxa have varied widely with the analysis. the genus melipona, for example, is placed as sister to the majority of neotropical meliponine genera in one hypothesis (fig 4a; rasmussen & cameron, 2010), sister of a smaller neotropical clade (fig 4b; camargo & pedro, 1992a), or sister of all other stingless bee genera (fig 4c; michener, 1990). the neotropical genus melipona has had an important place in the discussion of behavioral evolution of stingless bees, because of its rather unique morphological and bionomical characteristics. some authors have argued for a classification of stingless bees in which melipona would be clustered in its own subgroup (i.e., meliponini s.str.), apart from the trigona-group (trigonini), comprising all remaining genera of meliponini (e.g., moure, 1961). melipona is the sole stingless bee genus where polygynic colonies (multiple queens) are known (bego, 1989; alves et al., 2011); these bees are also unique for the breeding queens, males and workers in identical cells, instead of having distinctly larger royal cells as sociobiology 61(4): 355-368 (december 2014) 361 in the other meliponini (michener, 1974; engels & imperatrizfonseca, 1990); and the occurrence of worker parasitism related to male production (alves et al., 2009) and alien queens infiltrate colonies whose own queen had recently died (wenseleers et al, 2011). a robust phylogenetic framework for the meliponini can make possible the investigation of the unique traits of melipona, which might be autapomorphic for these bees, or be better interpreted as retention of plesiomorphic states in some cases. behaviors related to the evolution of obligate eusociality will then be reinterpreted in historical terms. orchid bees (euglossini) are another key-group for discussing the evolution of social interactions in the corbiculate clade (e.g., soucy et al., 2003). whereas research focused on the other corbiculate tribes (apini, bombini, meliponini) mainly clarify the maintenance of elaborate social behaviors, investigation on euglossini species are especially important because they would be able to potentially shed light on issues concerning the appearance of these social behaviors. two genera are strictly cleptoparasitic on other euglossine bees, aglae and exaerete, whereas species of euglossa, eulaema, and eufriesea are solitary, communal or “primitively social” (zucchi et al., 1969; dressler, 1982; garófalo, 1985; cameron, 2004; augusto & garófalo, 2009). although orchid bees are closely related to the eusocial honey bees, bumble bees, and stingless bees, no species of euglossini has been reported to display eusocial behaviors comparable to what is known for the remaining corbiculate tribes. some species in euglossa, however, present clusters of behavioral traits that are somewhat similar to those observed in bombini. the decision to classify them as totipotent-caste eusocial in this case would be, perhaps, arbitrary. unfortunately, little is known about the nesting biology and social interactions of most of the species of euglossini. if we consider all the described species, only a small amount of them fig 4. summary of the phylogenetic relationships among the genera of stingless bees (apidae: meliponini) as proposed by (a) rasmussen and cameron (2010) – molecular dataset; (b) camargo and pedro (1992a), accompanied by a more detailed account of relationships among neotropical genera later studied by camargo and pedro (2003) on the left – both morphological datasets; and (c) michener (1990) – morphological dataset – showing two alternative scenarios for the inner relationships of one of the clades. the genus-level classification was standardized to match rasmussen and cameron’s classification and thus make the three hypotheses readily comparable. when one of the three analyses did not include a given genus, a putative position was defined for it and dashed lines represented the placement of this genus. for example, the following five genera were not originally included in the analysis by rasmussen and cameron (2010): camargoia, cleptotrigona, meliwillea, papuatrigona, and paratrigonoides; putative placement of these in this summary tree was based on hypotheses and comments by various authors (michener, 1990; roubik et al., 1997; camargo & pedro, 2004; camargo & roubik, 2005), as previously indicated by rasmussen and cameron (2010). eab almeida, ds porto eusociality in corbiculate bees 362 (< 20%) has their nests described in some detail (dressler, 1982; kimsey, 1982; garófalo, 1985, 1992; garófalo et al., 1998; cameron, 2004). the genus euglossa is the best-studied group and probably the most interesting taxon for sociobiological investigation in euglossini, with the nesting behavior described for at least six species (garófalo, 1985, 1992; augusto & garófalo, 2009; andrade-silva & nascimento, 2012). the interest on this genus is largely explained by the usual occurrence of multifemale nests (mfn) with some overlap of generations and task allocation with reproductive dominance (augusto & garófalo, 2009, 2011). mfn’s are commonly attained by nest reactivation by the daughters in the presence of the mother (matrifilial associations), although it can be potentially accomplished also by two or more sisters of a same generation (i.e., sororal associations). when a nest is reactivated, the mother or older sister could act as the dominant (ramírez-arriaga et al., 1996; pech et al., 2008; augusto & garófalo, 2009, 2011). in addition to euglossa, there are reports of task allocation and reproductive dominance (division of labor) in the genus eulaema (bennett, 1965; dodson, 1966), which is not verified in communal nests of eufriesea (dressler, 1982; kimsey, 1982). in this context, a phylogenetic framework is indispensable to fully understand the evolution of social-related traits in euglossini (e.g., ramírez et al., 2010), and corbiculate bees as a whole, without making erroneous assumptions on the homology of behavioral complexes. noll (2002) remarked that many components of what are called “primitive” and “advanced” social behavior should be viewed as sets of independent characters. in this way, analyses of complex traits related to sociality should be done very carefully as in the following comparisons: (a) single-female nests (solitary) vs. multi-female nests (mfn), and matrifilial or sororal associations; (b) communal nests vs. existence of task allocation (e.g., guarding, provisioning) and evidence of reproductive dominance (i.e., oophagy, threatening/aggressive behavior). the knowledge about the co-occurrence of these traits and the instances that this occurs in the euglossine phylogeny is central for grasping the fine aspects of social evolution. nonetheless, phylogenetic relationships among the five genera of euglossini remain highly uncertain (see cameron, 2004; cardinal & danforth, 2011; ramírez et al., 2011 for a sample of conflicting results). better understanding of these relationships together with a much needed accumulation of behavioral data for orchid bees (particularly euglossa) will provide the confidence needed for the resolution of enigmas regarding social evolution within euglossini and the corbiculate clade as whole. according to the resulting ancestral state reconstructions by cardinal and danforth (2011), the most likely ancestral state for corbiculate bees was facultative eusociality. perhaps more relevant than the accuracy of the reconstruction itself is the indication of a strong signal of social interactions as pervasive in the early stages of evolution of corbiculate bees. complex forms of communication, caste polymorphism and division of labor, and construction of complex nests could have begun its unique path of evolution in corbiculate bees in the cretaceous, over 80 million years ago (cardinal & danforth, 2011). acknowledgements we dedicate this review to professor charles d. michener, who has contributed to the comparative research of bees for more than 70 years. his genuine interest for the broadest and most and most general questions regarding bee morphology, behavior, classification, and evolution in general laid the basis that allow comparative research to be on the right track presently in bee research. his enthusiasm has also served as inspiration and encouragement for several generations of bee researchers who have been answering and opening avenues for the future of melittology. we would like to thank carlos a. garófalo, aline andrade-silva, denise a. alves, and túlio m. nunes for discussions and for providing useful literature for this paper. we are grateful to luis roberto faria junior for being so kind as to carefully read and make suggestions to the manuscript, as well as for the feedback provided by three anonymous reviewers and by denise a. alves. this project was partly supported by grant # 2011/09477-9, são paulo research foundation (fapesp) to e.a.b.almeida; 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(1969). biological observations on a neotropical parasocial bee, eulaema nigrita, with a review on the biology of euglossinae (hymenoptera: apidae). a comparative study. j. fac. sci. hokkaido univ., zool. 17: 271-380. doi: 10.13102/sociobiology.v63i1.944sociobiology 63(1): 712-719 (march, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 task division and age polyethism in colonies of mischocyttarus latior social wasps (fox) (hymenoptera: vespidae) introduction in polistini and mischocyttarini wasps, called “primitively eusocial” which present independent foundation, by haplometrosis (solitary) or pleometrosis (addition of co-founders), there is no morphological differentiation of castes and determining the hierarchyis made based on aggressive interactions (jeanne, 1972; gadagkar, 1991). giannotti (1999) registered 24 behavioral categories for mischocyttarus cerberus styx (r.), 19 categories for founders (in the pre-emergence), 20 for queens and 18 for workers (postemergence). considering the founders and queens, the first appear to be more active, since they carry all the maintenance tasks of the nest and forage more frequently, while the queens remain unmoved more often. torres et al. (2012) studying the behavioral repertoire related to division of labor in mischocyttarus consimilis z. registered 30 behavioral acts, of which 23 were executed by queens and 29 workers, and 22 abstract this paper aimed to study the division of labor between queen and workers, division of labor by age range and sequence of behavioral patterns exercised by females and males from mischocyttarus latior (f.) colonies. behaviors of 13 dominant females (queens), 68 subordinate (workers) from emergence until they are 77 days and 12 males in 37 colonies were quantified during 545 hours. there were 23 behavioral acts registered divided into five categories. both dominant females as the workers showed a behavioral repertoire of 23 items, while males only 15. dominants remain longer on the nest than subordinates and exercise exclusively the behavior of oviposition and rubbing gaster on the nest. dominants also share 21 behaviors with the workers. males foraged nectar, preys and water, and exercise other behaviors, mostly related to social activity. all behaviors can be performed by any potentially worker at any age. sociobiology an international journal on social insects dss cecílio1, aa da rocha2, e giannotti1 article history edited by gilberto m. m. santos, uefs, brazil received 05 november 2015 initial acceptance 11 february 2016 final acceptance 09 march 2016 publication date 29 april 2016 keywords ethogram, social organization, polistinae, mischocyttarini. corresponding author agda alves da rocha universidade federal da bahia campus anísio teixeira rua rio de contas, quadra 17, lote 58 bairro candeias, cep: 45.029-094 vitória da conquista-ba, brazil e-mail: rocha.agda@gmail.com acts common to both castes. the act of starting new cells was the only behavior unique to queens. behaviors of submission are usually related to workers (jeanne, 1972; giannotti, 1999; giannotti & machado, 1999; torres et al. 2009). males in social wasps colonies usually perform, mostly, behaviors related to their own survival, although they may perform other activities such as caring for the larvae (spradbery, 1965; jeanne, 1972; raposo-filho, 1981; o’donnell, 1999; sinzato et al. 2003; giannotti, 2004). giannotti (2004) considers the possibility that males play a greater number of tasks within the colony in wasp species which they are morphologically similar to the females. mischocyttarus latior (f.) is a social wasp present in the midwest region and common in the southeast region of brazil (richards, 1978; elpino-campos et al. 2007; lima et al. 2010). their larvae present five instars and the mean duration of the immature stages, from egg laying to adult emergence, is 67.38 ± 9.41 days (cecílio et al. 2015). the males present length research article wasps 1 universidade estadual paulista júlio de mesquita filho (unesp), rio claro-sp, brazil 2 universidade federal da bahia (ufba), vitória da conquista-ba, brazil sociobiology 63(1): 712-719 (march, 2016) 713 of the wing and a similar staining pattern to the females although the legs and lower portion more brownish (richards, 1978). the work aimed to study the division of labor between queen and workers, division of labor by age range and sequence of behavioral patterns exercised by females and males from colonies of m. latior. materials and methods the study was conducted in the campus of universidade estadual paulista júlio de mesquita filho (unesp) and in the distrito (district) ferraz city of rio claro, são paulo, brazil (22º24’36’’ s 47º33’36’’ w ), from march 1994 to august 1995. prior to the performance of observations, all subjects of the 37 colonies were collected with the help of an insect net and marked on the thorax with model airplanes ink. the same was done with the females as soon as they emerged. to quantify the behavioral categories of the subjects, daily observation sessions of one hour were done, in the hottest periods of the day and hence of greater activity of the wasps, for 77 days. in them, the behavioral acts were recorded for each adult subject (dominant and subordinate females in addition to males) at five minute intervals, totaling 545 hours. it was also registered the position occupied on the nest (on the cells, the side of the nest, behind the nest, in the substrate, out of the nest). the χ2 test was applied for two independent samples (siegel, 1979), in order to verify the significance of the behavioral differences between queens and workers. this procedure also allowed the study of the variation of the different tasks performed by individuals in the colony according to their age range. results and discussion the behaviors of 13 dominant females were recorded (queens), 68 subordinate (workers) and 12 males, as well as the spatial position occupied on the nest. spatial position. through fig 1, we can observe the spatial position occupied by wasps. the dominant females remained much longer on the cells (87.2%) that workers, 38.6% (c2 = 432.42; p < 0.001; g.l. = 1) which indicates the dominance of the first over the subordinates (workers). but behind the nest, there is an inversion and the workers come to occupy 13.3% of the time and queens, only 1.6% (c2 = 97.32; p < 0.001; g.l. = 1). regarding the position in the substrate, the workers were 0.8% and queens did not occupy this place at any time. when it is analyzed the location on the side of the nest, it is apparent that the workers remained 19.2% and queens, 4.3% (c2 = 108.48; p < 0.001; g.l. = 1), showing once more the dominant / subordinate relationship. the workers remained more time out of the nest: 28.1% of the time and queens for only 6.9% (c2 = 149.65; p < 0.001; g.l. = 1). on the other hand, the males remained 13.0% on the cells, behind the nest 6.4%, 0.5% on the substrate, 17.3% at the side of the nest and 62.9%, outside the nest. behavioral repertoire. table 1 shows the 23 behavioral acts, exercised by individuals of m. latior colonies, distributed over five categories, described according to giannotti (1999), as follows: i. social activity 1. check cells. the wasps insert their heads on the nest, fumbling with antennas its contents. 2. check the cells rubbing the gaster against the nest. according to gamboa and dew (1981) this behavior may be related to dominance, and represents a kind of communication between adults and larvae. 3. trophallaxis adult-adult (receiver). is the receiving of ingluvial liquid among adults (usually nectar of flowers or other sugary substances). as described by west-eberhard (1969), there is a clear difference in the donor (subordinate) postureto the receiver (dominant), then considered, as two distinct behaviors. 4. trophallaxis adult-adult (donor). is the passage of ingluvial liquid among adults (usually nectar of flowers or other sugary substances). 5. trophallaxis larva-adult. is the passage of liquid feed (secretion of the salivary gland) from the larva to the adult. it is perceived when the adult, checking cells, makes a longer insertion in a cell containing a larva. 6. feed larvae. it occurs after chewing, when adults provide a protein juice, which is the principal source of food for larvae. 7. physically dominate. a wasp in a higher position in the social hierarchy of the colony slightly nibbles or even climbs up on the subordinate. 8. being physically dominated. involves the act of the individual to stand passively before the one which dominates or even retreat or fly out of the nest. ii. oviposition 9. lay eggs in an empty or newly built cell. the female inserted the gaster in the cell and laid eggs. this behavior lasts 4.5 minutes on average. fig 1. positions of females (subordinates and dominants) and males on the nests of mischocyttarus latior (fox, 1898). dss cecílio, aa da rocha, e giannotti – task division and age polyethism in colonies of social wasps714 iii. nest maintenance activity 10. add material to the cells. as the larvae develop, wasps deposit material in the cell, increasing its height. 11. ventilate cells. there is a flutter of wings in order to cool the nest. may or may not be associated with the collection of water. 12. giving alarm. wasps take a high posture, lifting and opening the wings and directing the antennas to the source of disturbance. usually they perform a “back-andforth” movement with the gaster, which is probably related to the dispersion of alarm pheromone. 13. chewing prey. wasps chew a piece of solid feed. 14. apply oral secretion to the nest. the application of this secretion has been observed in the stem and over the top of the nest, making the surface more darkened and shiny, resembling a varnish. this secretion offers more rigidity to these structures and is a protection against parasitoids. 15. apply gastral glands secretion to the nest pedicel. as studied by jeanne (1972) and turillazzi and ugolini (1979), wasps rub the gaster against the nest pedicel, particularly in the pre-emergence stage, dispersing a secretion that has repellent properties to ants (predators of wasps nests). depending on the intensity of the stimulus, the wasps vibrate their wings and walk on the nest in fast-moving movements, producing characteristic noises and only after that they attack the disturbing agent. iv. foraging activity 16. unsuccessful foraging. it occurs with the outing of the wasp and subsequent return, without bringing anything to the nest. 17. prey foraging. it is the act when the wasp leaves the nest to go in search of food, in general caterpillars. when the wasp reaches the nest, it can share or not the material collected with a companion before chewing the prey and give to the larvae. 18. foraging nectar. the foraging returns without anything visible in the mouth, but always makes trophallaxis with other wasps. 19. foraging water. the foraging comes back with a visible droplet between the jaws and deposits iton the cells, without dividing it with another wasp. it was observed in warmer weather days, as it is associated with the maintenance of the nest temperature. 20. wood pulp foraging (nest building material). after an average of 7.5 minutes period, the foraging returns to the nest with a wood pulp acorn in the jaws to add material to the nest cells. v. individual behaviors 21. immobile on the nest. it occurson the nest or in the substrate, apparently at rest. 22. self-grooming. the wasp rubs the legs against each other, against the antennae, head, chest, wings, abdomen and finally licks the front legs. it could be partial or total. this behavior is exhibited, mostly, after performing any task on the nest. 23. walk. walking on the nest or in the substrate. dominant and subordinate behavior. both the dominant females as the workers showed a behavioral repertoire of 23 items, while the males only 15 (c2 = 827.75; p > 0.001; g.l = 3). the number of quantified behavioral categories for some species of social wasps can be variable: polistes chinensis antennalis (f.) presented 34 to 36 behavioral categories in colonies in the stages of pre and post-emergence, respectively (kasuya, 1983a,b); 37, in polistes fuscatus variatus (f.) (post et al., 1988.); 31 in polybia occidentalis (o.) (jeanne et al., 1988.); 27, in polistes dominulus (c.) (theraulaz et al., 1990), 28, in polistes lanio lanio (f.) (giannotti, 1992), 24 in mischocyttarus cerberus styx (r.) (giannotti, 1999), 28 in p. canadensis canadensis (l.) (torres et al. 2009), 30 in mischocyttarus consimilis z. (torres et al., 2012) and 42 behavioral acts performed by mischocyttarus parallelogrammus z. (togni, 2014). standing still occurs in the same way for workers and queens. it is common in other species of the genus that females remain still for a long time on the nest (giannotti, 1999; torres et al., 2012). the individual suddenly stops, remaining so for a very variable time, and always with the wings on the gaster, never under. the m. latior queens were the ones that remained more time standing still on the nest (42.1%), while the workers stayed 39.2% of the time (c2 = 1.79; p < 0.05; g.l. = 1). these figures show that, despite the workers having a more active life than queens, it cannot be said that there is a tendency in one or another class to carry out this behavior more times. the subordinate cleaned up less often than the dominant (c2= 58.40; p < 0.001; g.l. = 1). these figures show that queens perform more this behavior as they remain longer on the nest, while the workers are more active, that is, spend a great deal of time on foraging activities. the behavioral sequence of cleaning is equal to queens and workers. the wasp stands immobile on the nest and performs the self-grooming. the average time in queens was 18.41 seconds ± 36.13 (1.57 – 131.82) and for workers, 12.77 ± 15.20 (1.64 – 101.41). more specifically, this cleaning can occur only in the antennae (passing the front legs in the mouthparts and over the same). this latter behavior had an average duration of 6.0 seconds ± 12.81 (3.46 – 36.09) for the workers and 5.03 seconds ± 2.97 (1.57 – 10.71) for the queens. for only in the front legs (rubbing one leg against each other), the average time for workers was 12.68 seconds ± 15.45 (1.64 – 61.41) and for queens, 67.75 seconds ± 34.04 (43.68 – 91.82). for only the rear legs and wings (rubbing one leg against the other, passing over and under the gaster, rubbing the leg again and then passing over the wing, repeating this sequence many times). the average time for workers was 4.75 seconds ± 3.82 (2.37 – 11.50) and for queens, 12.59 seconds ± 16.05 (4.03 – 45.00). a clean sweep, in which all the above described sequences are performed, the average duration for the workers was 28.26 seconds ± 22.46 (2.41 – 42.97) and for the queens, 16.41 seconds ± 10.41 (4.90 – 33.84). sociobiology 63(1): 712-719 (march, 2016) 715 walking through the nest or in the substrate was a behavior observed mostly for queens and in less number for workers (c2 = 13.74; p < 0.001; g.l. = 1) demonstrating that as the queens are longer on the nest, they perform more this behavioral act of walking, which may represent a type of patrolling against parasitoids, as ichneumonidae wasps, which have been observed surrounding the nest. however, this observation would need to be deepened. considering the social activities carried out by m. latior wasps, there was a greater behavioral frequency for the queens checking cells than to the workers (c2 = 35.93; p < 0.001; g.l. = 1). this demonstrates that the dominant has greater parental care, besides the fact that it spends most of the time on the nest. checking cells was a behavior that consisted in the same sequence for both dominant and subordinate, as follows: wasps pass their antennas through the opening of the cell, insert their head in it (sometimes also the thorax) and remove quickly. the duration of this action is very fast, being approximately 1 second. although for gamboa and dew (1981) the behavior of verifying cells rubbing the gaster against the nest may be related to the dominance, besides representing a kind of communication between adults and larvae, in m. latior, there was no significant difference in the frequency of this behavior between queens and workers (c2 = 1.53; p > 0.05; g.l. = 1). with respect to trophallaxis, taking into account the receiver behavior, it was found that queens receive a greater amount of food than workers (c2 = 36.31; p < 0.001; g.l. = 1), confirming the dominant/subordinate relationship. in relation to the donation behavior, it was observed that both the workers and the queens, donate food (c2 = 0.21; p > 0.05; g.l. = 1), with no significant difference. both for dominant and subordinate the behavior sequence to adult-adult trophallaxis as receiver was the same. the individual walked on the nest, quickly invested in the wasp that was effecting the donation, performed an antennal beat on the head of those who donated (probably to whoever was donating enter the proper position), acquired a dominant stance on it (who received was up and who donated below). thus performed the exchange of materials and generally cleaned-up (mainly antennas), for approximately 2.99 seconds ± 1.08 (2.06 – 4.96) for queens and 5.49 seconds ± 2.99 (2.10 – 10.46) for workers. for the latter, this value was considered for both the receiving behavior as for the donation. to queens it was not verified the sequence of donation behavior to others. there was no significant difference in frequency of the trophallaxis larva-adult between subordinates and dominants (c2 = 0.16; p > 0.05; g.l. = 1). both castes receive “larval salivary” and, likewise, it was found that both varieties feed larvae (workers, 1.5%, and queens 0.7%), with no significant difference (c2 = 1.66; p > 0.05; g.l. = 1). the individual walked on the nest, checked the contents of the cell with antennas, placed the head and chest in it and performed trophallaxis with the larva. the average time was 5.83 seconds ± 5.57 (2.14 – 22.96). for queens and workers, the behavior sequence to perform trophallaxis between larva and adult was the same. they approach the opening of the cell, make an antennal recognition, insert a part of the body (head and thorax), perform trophallaxis with the larva and usually clean the mouth parts after the act. for the dominants, the average time was 7.10 seconds ± 5.43 (2.13 – 26.47) and for the workers it was 4.97 seconds ± 3.55 (1.87 – 15.58). in relation to dominance, as we have already seen, the queens dominate a larger number of times (12.5%) and the workers only 1.1% (c2 = 330.80; p < 0.001; g.l. = 1), confirming what was found in the literature (pardi, 1948; west-eberhard, 1969; giannotti & machado, 1999; torres et al. 2012), emphasizing that such behavior is used as a “method” of domination. and as expected, it was found that the workers are physically dominated in a greater number of times, while this behavior in queens almost never happens (0.6%) (c2 = 25.10; p < 0.001; g.l. = 1). when it occurs, the subordinate does so only investing against the dominant without showing too much aggression. this action usually occurs when there is competition for dominance in the colony. it is seen that in m. latior queens, dominance behavior occurs in two ways: it can walk towards the worker and then nibble it, or walk towards it and attack, though without physical contact (this assault can happen more than once to the same individual in the same act to master). among workers, this behavior occurs in much the same way; the only difference is the less aggressiveness with which the action is carried by these. considering the nest maintenance activities, ventilate cells and chew prey were the most executed by both queens and workers. the dominants presented 0.4% of the behavior to ventilate cells and the subordinate 0.4% (c2 = 0.00; p > 0.05; g.l. = 1), so this is not significant. ventilate cells occurred once for dominants by 2.75 seconds. the behavioral sequence was as follows: they vibrated frantically the antennas, directing the body towards the cells and intensely shaking the wings. for workers, the act occurred, on average, for 0.82 seconds ± 0.16 (0.60 – 1.00). for them, the sequence occurred without vibration of the antennas and sometimes between the cleaning-up behavior. it was observed a greater number of queens (0.8%) chewing preys than workers (0.4%), but the values were not significant (c2 = 2.19; p > 0.05; g.l. = 1). as the larvae were developing, it was seen that both the queens added material into the cells (1.7%) as the workers (0.3%) (c2 = 28.42; p < 0.001; g.l. = 1). considering the defensive behavior, “to alarm” was the same for both castes (0.1%) (c2 = 0.03; p > 0.05; g.l. = 1). apply oral secretion on the nest was the only task performed exclusively by workers (0.1%). as for applying gastral glands secretion on the nest pedicel, this behavior was observed in both queens (0.2%) and workers (0.0002%) (c2 = 4.31; p < 0.05; g.l. = 1), suggesting a behavior related to the permanence time in the colony. the alarm behavior occurs in the same way for both workers and queens. when individuals perceive a threat against the nest, they perform the dss cecílio, aa da rocha, e giannotti – task division and age polyethism in colonies of social wasps716 following behavioral sequence: immobile on the nest, raise their wings, raise the body, flap their wings causing a loud noise, move through the colony flapping wings and legs against the nest, what causes the sound to be even more intense, and finally attack the disturbing agent. the average time for this behavior (beginning of the act until rest) was 13.78 seconds ± 4.36 (10.69 – 16.87). with respect to behavioral acts related to the foraging activity, the workers foraged prey much more often than queens (c2 = 33.45; p < 0.001; g.l. = 1) since this activity requires more time out of the nest and therefore the queen rarely exercises. forage nectar is also a much more frequent behavior for workers than for queens (c2 = 165.87; p < 0.001; g.l. = 1), thus proving the high rate of this activity by workers. foraging water was a behavior most often done by workers than queens (c2 = 54.25; p < 0.001; g.l. = 1), showing again the high rate of foraging activity of workers within a colony. the value found for wood pulp collection was 1.8% for the workers and 2.5% for queens (c2 = 1.87; p > 0.05; g.l. = 1) and although it most frequently occurs among the queens, such behavior was not significant. giannotti and machado (1999) considered that the collection of pulp is the main foraging activity exercised by queens of p. lanio lanio. according to west-eberhard (1969) the dominant females are in charge of the construction of new cells, some of them laying eggs right in sequence, similar to what was observed for p. canadensis. for the author, the occasional foraging trips are aimed at collecting material for nest building (pulp and water). giannotti (1999) noted that in wasps of the species m. cerberus styx, both founders as queens, collected wood pulp more intensively than the workers. these data differ from those observed by torres et al. (2009) to p. canadensis canadensis, whose queens only collected prays and still in a much smaller proportion than the workers. on the contrary, polistes versicolor o. in the foundation phase collected all the features except prey, having an equal proportion between queens and workers in wood pulp collection (sinzato & prezoto, 2000). the return to the nest without success was another prevalent behavior in the workers, and little seen in queens (c2 = 35.44; p < 0.001; g.l. = 1) since the workers go out of the nest more often to forage and therefore are more subject to an unsuccessful output. there was no any particular behavioral sequence for the act of foraging. the individual went out flying straight from the nest, or it happened to walk on the colony and fly, or walked off the substrate and then flew. it has been observed only once, a worker making two wing beats prior to leaving for the field. in one of the leavings of a dominant for foraging nectar, the time observed was 139.52 seconds and a leaving for water foraging of a worker, this time was 9.21 seconds. it was also verified for the subordinates, the average times of 12.97 seconds ± 2.49 (10.10 – 14.55) to collect nectar and 0.28 seconds ± 0.12 (0.19 – 0.36) for unsuccessful foraging. it was observed that there occurs an induction via dominance for the leaving for foraging. this usually occurred after the foraging worker performed trophallaxis with other workers or with the queen and after feeding the larvae and cleansed. after these acts, the dominant or even other workers (hierarchically superior to the foraging worker), invested on it and practically forced it to make a new exit to the field. the oviposition and rubbing of the gaster on the nest were unique behaviors of the dominant females, which presented 21 behaviors in common with the workers. this differs from m. consimilis, whose queens had just the act of starting new cells as the sole activity and the workers had seven unique behaviors (torres et al., 2012). as for the males, they played no unique behavior (table 1). this shows that there is not a clear division of labor between castes, but between the genders. male behavior. it was observed that males foraged nectar, prays, water, and that many times there was an unsuccessful return (table 1). only the foraging of pulp to build new nest cells was not observed in males. other behaviors observed for males of the present species involved social activity: check the cells, adult/adult trophallaxis (donor), larva/adult trophallaxis, adult/adult trophallaxis (receiver), feeding larvae, being physically dominated (and physically dominate. they performed an activity related to the maintenance of the colony (apply oral secretions to the nest, besides standing still, performing self-grooming of the body and walk the nest. the males are usually food receivers, both adults and larvae (giannotti, 2004). however, it is noteworthy that there was adult/adult trophallaxis behavior (donor), even if a small percentage. o’donnell (1995) reported the collection of nectar and later trophallaxis by males of polistes instabilis s., even considering that the activities carried out on the nest by them have been at low rates. it is likely that this behavior is related to the fact that there is a need for food donations to females so they continue being accepted in the colony. according to o’donnell (1995), production costs of males are compensated, in part, for any investments that they can make through their work. in the colonies, males get food from various sources, including regurgitation of workers and salivary secretions of the larvae (spradbery, 1965). the males of mischocyttarus drewseni s. and mischocyttarus mastigophorus r. also request, for their own consumption, nectar and macerated insects from the foraging workers that are returning (jeanne, 1972 and o`donnell, 1999 respectively). the mischocyttarus extinctus z. males also request proteic and glucidic food fromthe foraging workers. it was found that the males of this species leave the colony to forage; this fact could be followed because the colony was two meters away from the forage area. they frequented these areas because, probably, it was the place where we found matings, in addition to foraging (raposo-filho, 1981). in addition to requesting food from foragers, m. latior males play a greater participation in the activities of the nest, as it occurs with p. lanio lanio (giannotti, 2004). however, m. latior also plays foraging activities, not happening with this species. sociobiology 63(1): 712-719 (march, 2016) 717 behavior according to age. table 2 shows the frequency of 17 behaviors performed by workers of m. latior at intervals of 1 to 11 weeks (up to 77 days): one week range (1 to 7 days): it is verified that the most accomplished behavior by subordinates was to immobile on the nest (50.19%) and secondly, the forage (32.27%). two weeks range (8 to 14 days): it was observed a high foraging index (71.43%), showing that this is a typical behavior for workers of this age, as they gained more experience within the colony. three weeks age range (15 to 21 days): the activity of forage is the most accomplished behavior by workers, with 46.56% of the time, although there was a drop compared to the previous range. second, the body self-grooming behavior was shown, with 13.30%, perhaps by the fact that the wasp had remained longer in the colony. four-week age range (22 to 28 days): it was found the highest percentage of foraging behavior (76.89%), reaffirming what has been said above that the experience is an important factor for the exit of workers to field. these data are similar to those found for workers of p. lanio lanio in a study by giannotti and machado (1994), where it was found that the foraging begins only in the second week and reaches its peak only in the fifth. the difference is that in this case, m. latior starts to perform foraging activities in the first week, with the peak of foraging in the fourth week. five weeks age range (29 to 35 days): there is a decrease in the foraging activity (45.74%) over the previous; still remained the most accomplished work, followed by theimmobile on the nest (31.91%). six weeks age range (36 to 42 days): there is a big drop in foraging activity (6.58%) and an increase in the standing still activity (59.87%), which was the most frequently performed. table 1. ethogram of females (subordinates and dominants) and males of mischocyttarus latior (fox, 1898). behavioral acts individuals in the colony subordinates (n=68) dominants (n=13) males (n=12) n (%) n (%) n (%) i social activity 1. check cells (cc) 182.0 4.2* 92.0 8.8* 26.0 3.9 2. rub gaster on nest (rn) 1.0 0.0 2.0 0.2 0.0 0.0 3. trophallaxis adult-adult (receiver) (tr) 59.0 1.3* 44.0 4.2* 9.0 1.3 4. trophallaxis adult-adult (donor) (td) 85.0 1.9 1.0 0.1 12.0 1.8 5. trophallaxis larvae-adult (tl) 90.0 2.1 49.0 4.7 12.0 1.8 6. feed the larvae (fl) 65.0 1.5 7.0 0.7 7.0 1.0 7. dominate physically (dp) 49.0 1.1* 131.0 12.5* 6.0 0.9 8. be physically dominated (bd) 156.0 3.6* 6.0 0.6* 12.0 1.8 ii oviposition 9. oviposit (ov) 0.0 0.0 1.0 0.1 0.0 0.0 iii nest maintenance activity 10. add material to cells (am) 15.0 0.3 18.0 1.7 0.0 0.0 11. ventilate cells (vl) 17.0 0.4 4.0 0.4 0.0 0.0 12. give alarm (ga) 5.0 0.1 1.0 0.1 0.0 0.0 13. chew prey (cp) 18.0 0.4 8.0 0.8 0.0 0.0 14. apply buccal secretion to the nest (ab) 6.0 0.1 0.0 0.0 2.0 0.3 15. apply gastral glands secretion (ag) 14.0 0.3 1.0 0.1 0.0 0.0 iv foraging activity 16. unfruitful foraging (uf) 175.0 4.0* 3.0 0.3* 155.0 23.1 17. foraging preys (fr) 175.0 4.0* 4.0 0.4* 39.0 5.8 18. foraging nectar (fn) 861.0 19.6* 19.0 1.8* 234.0 34.9 19. foraging water (fw) 351.0 8.0* 15.0 1.4* 7.0 1.0 20. foraging pulp (fp) 80.0 1.8 26.0 2.5 0.0 0.0 v individual behaviors 21. immobile on the nest (in) 1718.0 39.2 441.0 42.1 92 13.7 22. self-grooming (sg) 200.0 4.6* 114.0 10.9* 39.0 5.8 23. walk (wa) 63.0 1.4* 61.0 5.8* 18.0 2.7 total 4385.0 100.0 1048.0 100.0 670.0 100.0 *values presenting significant difference among behaviors of dominants and subordinates. dss cecílio, aa da rocha, e giannotti – task division and age polyethism in colonies of social wasps718 the second most performed behavior was the performance of adult/adult trophallaxis as donor (7.24%), probably by the fact that the subordinates are reducing the foraging and increasing the work inside the colony, as they are not so young anymore, tending to have dominant behaviors in the colony. seven weeks age range (43 to 49 days): a high value is observed in standing still (60.75%), despite the frequency of foraging behavior have increased (32.80%). eight weeks age range (50 to 56 days): the foraging rate decreases to zero, while the standing still in the colony was the second highest among all intervals, with 78.95% of the time. at this age, the workers performed only just four tasks: checking cells, adult/adult trophallaxis (donor), adding materials to the cell and walking, all these with a frequency of 5.26%. nine weeks age range (57 to 63 days): the largest value of standing still within the colony occurred in this interval (79.03%), while forage and being physically dominated was observed in only 6.45% of the time. ten weeksage range (64 to 70 days): there was an increase in self-grooming jobs and checking cells, both with 7.69%; these behaviors probably occurred due to the subordinated being for a longer time on the nest. eleven weeks age range (71 to 77 days): there was an increase in foraging activity (35.71) and a decrease in standing still behavior (45.92%). the checking of cells was the behavior more frequently found among all other studied intervals. it can be seen that there was a greater distribution of behaviors within the colony in the first three intervals analyzed and, by means of these data, it is verified that all behaviors can be potentially performed by any worker at any age, as observed in p. lanio lanio (giannotti & machado, 1994). thus, for a also eusocial basal wasp as m. latior, this behavioral plasticity that exists between the workers is essential for the survival of small colonies of social wasps such as mischocyttarus and polistes (giannotti, 1999). conclusion dominants remain longer on the nest than subordinates and exercise exclusively the behaviors of oviposition and rubbing gaster on the nest. they share most of the behaviors with the workers and the main differences between the frequency of behaviors are related to foraging activity and social activity. the males developed various activities unrelated to their own survival and all behaviors can be potentially performed by any subordinate of m. latior at any age. acknowledgements the authors are grateful to the fundação de amparo à pesquisa do estado de são paulo – fapesp for the sponsorship. references cecílio, d. s. s., da rocha a. a. & giannotti, e. 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(1969). the social biology of polistine wasps. miscellaneous publications, museum of zoology, university of michigan, 140: 1-101. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7276sociobiology 68(4): e7276 (december, 2021) social parasitism has evolved several times and in different forms among ants, showing amazing morphofunctional, chemical and behavioral adaptations to this peculiar habit (visicchio et al., 2001; buschinger, 2009). inquilinism is considered the most extreme of such forms: inquiline social parasites produce few to no workers while they concentrate their efforts on the production of reproducers which their hosts raise (hölldobler & wilson, 1990; buschinger, 2009; degueldre et al., 2021). as a result, inquiline ant species are rarely detected during field surveys. abstract we conducted a survey on the alpine fauna of one of the largest natural park of the italian alps (stelvio national park) in the framework of a broad ecological monitoring of alpine biodiversity. a two-years standardized sampling employing pitfall traps along a 1200 m altitudinal gradient led to the discovery of the inquiline social parasite ants myrmica myrmicoxena forel, 1895 and m. microrubra seifert, 1993. myrmica myrmicoxena, which is classified as vulnerable according to the iucn red list, was so far known from only three sites across a narrow geographic range between italy and switzerland. our data support the previous hypothesis over its ecology and host association. myrmica microrubra is considered an incipient species of high evolutionary interest, sometimes regarded as an intraspecific form of m. rubra. while having a wide distribution in europe, its presence in italy was hitherto known only from a single site. our record extends its altitudinal distribution limit in europe upwards by about 600m. sociobiology an international journal on social insects enrico schifani1, cristina castracani1, fiorenza a spotti1, daniele giannetti1, martina ghizzoni1, mauro gobbi2, valeria lencioni2, luca pedrotti3, donato a grasso1, alessandra mori1 article history edited by evandro nascimento silva, uefs, brazil received 18 june 2021 initial acceptance 25 october 2021 final acceptance 14 november 2021 publication date 24 november 2021 keywords inquiline social parasitism, rare species, alpine insects. corresponding authors enrico schifani department of chemistry, life sciences & environmental sustainability, university of parma, parco area delle scienze 11/a, 43124 parma, italy. e-mail: enrico.schifani@unipr.it cristina castracani department of chemistry, life sciences & environmental sustainability, university of parma, parco area delle scienze 11/a, 43124 parma, italy. e-mail: cristina.castracani@unipr.it there is an underdeveloped understanding of their biology, ecology, and conservation status (lópez-soria, 1991; schifani, 2017). however, a large number of the european inquiline ants were classified as vulnerable on the iucn red list (social insects specialists group, 1996). the genus myrmica latreille, 1804 counts about 190 species distributed across the holarctic region (bolton, 2021), and it includes a remarkable variety of inquiline social parasites: in the west-palearctic region, over 30 myrmica species are recognized (radchenko & elmes, 2010; seifert, 2018), 1 department of chemistry, life sciences & environmental sustainability, university of parma, parma, italy 2 section of invertebrate zoology and hydrobiology, muse science museum of trento, trento, italy 3 stelvio national park, bormio, italy social parasite ants in the alps: a new site of the vulnerable myrmica myrmicoxena and new uppermost altitudinal limit for m. microrubra short note mailto:enrico.schifani@unipr.it enrico schifani et al. – m myrmica microrubra and m. myrmicoxena in the alps2 one quarter of which is made of obligate or facultative social parasites of other congeneric species (radchenko & elmes, 2003; 2010). most of them are inquilines, which evolved multiple times, independently in the genus (radchenko & elmes, 2003; jansen et al., 2010; seifert, 2018). unlike in other ant genera (e.g., plagiolepis, see degueldre et al., 2021), most parasite myrmica spp. do not follow the strict version of emery’s rule (emery, 1909), as their host species are not always their closest evolutionary relatives (jansen et al., 2010). in particular, parasite myrmica species with a long evolutionary history seemed to have developed the ability to exploit a wider and less closely related set of congeneric species as hosts (jansen et al., 2010; seifert, 2018). some of the free-living species of the m. scabrinodiscomplex are the hosts for the overwhelming majority of the west-palearctic social parasite myrmica (radchenko & elmes, 2003; 2010; seifert, 2018): these range from the widespread facultative parasite m. vandeli bondroit, 1920, to more specialized species with narrower geographic ranges (m. bibikoffi kutter, 1963, m. hirsuta elmes, 1978, and m. laurae (emery, 1907)), to forms of extreme morpho-functional adaptation to inquilinism (m. lesmanei bernard, 1967, m. kabylica cagniant, 1970, and m. karawajevi arnol’di, 1930) (radchenko & elmes, 2010; seifert, 2018). host species range from only one to several, with m. karawajevi exploiting the widest amount of different hosts, probably due to a longer evolutionary history (jansen et al., 2010; seifert, 2018). outside the m. scabrinodis-complex, only two parasite myrmica species are found in the west-palearctic. the alpine endemic m. myrmicoxena is a workerless inquiline collected only in three sites within a narrow altitudinal range for 150 years. m. lobulicornis (from the m. lobicornis-group) is considered to be likely its only host species (forel, 1895; glaser et al., 2010). on the other hand, m. microrubra seifert, 1993, widely distributed in europe, is either considered as an incipient species or as an intraspecific parasitic form of m. rubra (linnaeus, 1758) (pearson, 1980; 1981; seifert, 1994; 2018; seppä & pamilo, 1995; steiner et al., 2006; schär & nasch, 2014; leppänen et al., 2011; 2015; 2016). here we treat it as a good species following the latest review of the european ant fauna (seifert, 2018). from 2018 to 2020, terrestrial arthropods were sampled by 150 pitfall traps in 30 sites across an altitudinal and habitat gradient in the lombardy sector of the stelvio national park. the traps were built with plastic glasses of c. 6 cm of diameter and c. 7 cm of height, buried in the ground, filled with c. 150 ml cc of an attractive and a preserving mixture of white vinegar, sodium chloride, and a drop of detergent as a surfactant, as described by gobbi (2020). a total of 1800 samples were taken during six sampling sessions per year (150 x 6 x 2 = 1800). ants were identified under a zeiss stemi 508 stereoscopic microscope and measured with the aid of an axiocam erc 5 s and zeiss zen core software according to the keys provided by radchenko & elmes (2010) and seifert (2018). this effort led to a significant database of over 1700 alpine ant records and interesting incidental discoveries, such as the first finding of ergatandromoph individuals of m. lobulicornis in 2018 (schifani et al., 2020). in 2019, two rare social parasite myrmica species of particular interest were collected, namely m. microrubra and m. myrmicoxena (fig 1). both were previously known from only one locality in italy (glaser, 2003; glaser et al., 2011), and for both, the new occurrence sites yielded new important ecological or distributional information. collecting data of the two species are as follows: m. microrubra (1♀) alongside m. rubra (103☿☿ 1♀): valle messi, sondrio, 46.296930, 10.503609, 1588 m asl, southfacing slope, peat bog, 24.ix.2019 (plot 6.1.4). m. myrmicoxena (1♀) alongside formica lemani bondroit, 1917 (5 ☿☿), f. lugubris zetterstedt, 1838 (13☿☿), m. lobulicornis (12☿☿): sobretta-valle del gavia, sondrio, coordinates hidden in conformity with glaser et al. (2011), 2175 m asl, northfacing slope, pasture/shrubland, 04.x.2019 (plot 4.5.2). the geographic range of m. microrubra is relatively wide but does not cover the entire distribution of its host m. rubra (seifert, 2018). records of m. microrubra around the alps often come from sites below 700 m asl (e.g., wagner, 2020). in italy, m. rubra records are mostly distributed north of the apennines and especially common in the po plain and alps (baroni urbani, 1971; mei, 1984; le moli & zaccone, 1995; glaser, 2003; 2004; sielezniew et al., 2010; glaser et al., 2012; scupola, 2018; castracani et al., 2020). the first discovery of m. microrubra in italy was published by glaser (2003) and made in south tyrol (980 m asl). since our record also comes from the alpine region, it is still uncertain where the species’ southernmost distribution limit stands. during our survey, m. rubra was detected in four sites, mostly occurring from 1400 to 1600 m asl, with a single finding at 1900 m asl. a total of 1848 m. rubra workers were found in 113 samples from pastures or peat bogs. ecological data on m. microrubra are relatively few and scattered; however, to the best of our knowledge, our record significantly extends its altitudinal limit upwards by about 600 m. on the other hand, ecological data over m. myrmicoxena mostly come from only two sites (glaser et al., 2011), as very little information is contained in its original description (forel, 1896). our data are consistent with these previous two findings regarding altitudinal preferences (m. myrmicoxena was previously found at 1700 and 2213 m asl), habitat selection (alpine pastures), north-facing slopes, and host species. the new site is geographically placed between the two swiss sites and the italian site reported by glaser et al. (2011) (fig 2). we collected m. lobulicornis, possibly the exclusive host of m. myrmicoxena, on 151 samples distributed in 15 sites from 1600 to 2400 m asl, resulting in 962 workers. sites above 1900 m mostly represented open habitats, while a few sites at lower altitudes were coniferous and deciduous forests or peat bogs. sociobiology 68(4): e7276 (december, 2021) 3 in comparison, the related m. lobicornis, which appears to be the only other species that could potentially serve as a host for m. myrmicoxena (glaser et al., 2011), was collected between 1400 (the lowest sampled altitude) and 2000 m asl in 12 sites. given the extreme scarcity of data on m. myrmicoxena and the conservation concerns expressed by previous authors, this additional discovery significantly reinforces the ecological and biological hypotheses formulated so far (glaser et al., 2011). the italian distribution of m. lobulicornis currently appears poorly documented, and future investigations may contribute to either confirming or dismissing m. myrmicoxena as an alpine endemic ant (a rare condition only shared with f. paralugubris seifert, 1996 – see seifert, 2018). myrmica microrubra and m. myrmicoxena were caught only in 0.7-0.9% of the samples in which their respective host species were found. during the two-years survey, only a single specimen of another inquiline social parasite (formicoxenus nitidulus (nylander, 1846) (also considered vulnerable by the iucn, social insect specialists group, 1996) was found in about 980 samples containing its host species (a queen was found in the same locality of m. myrmicoxena in a trap retrieved on 16.vii.2018). this collection number represents a lower rate compared to the parasite myrmica spp. despite literature data suggesting this species is frequent in the italian alps (baroni urbani, 1971). inquiline social parasites are difficult to detect during standardized field surveys with a generic focus on ants (e.g., glaser, 2004; glaser et al., 2012; fig 1. myrmica microrubra (left) and m. myrmicoxena (right) from the stelvio national park. scale bars: 0.5 mm. enrico schifani et al. – m myrmica microrubra and m. myrmicoxena in the alps4 spotti et al., 2015), and pitfall trapping is not a proper sampling method useful to detect inquiline parasites. yet, our findings demonstrate that even on these ants, important information can still be recovered thanks to the extensive use of this sampling tool in studies with a broader ecological aim. in both our findings, the parasites were found in the same sample along with several individuals of their respective host species, which suggests they may have been caught during colony relocation. finally, insect decline and its severe potential outcomes on ecosystems functioning has recently attained much attention showing the need for a deeper commitment in field survey, the development of effective monitoring systems, and biodiversity database implementation in the face of global changes and contexts with different anthropic impact (campanaro et al., 2011; gibb et al., 2017; leather, 2017; homburg et al., 2019). acknowledgments this work was supported by the stelvio national park as part of the mid-term monitoring of alpine faunistic biodiversity concerning climate change, carried out in coordination and with the same survey design of the other three italian alpine national parks and in cooperation with musescience museum of trento (italy). the work was granted by the italian ministry for environment, land and sea protection and co-financed by the autonomous province of trento. alessandro gugiatti and paolo belotti supervised and managed fieldwork for the survey of epigean arthropod fauna. teresa boscolo and michael bernasconi (muse-science museum of trento, italy) sorted the formicidae collected by pitfall traps, and alessandra franceschini (muse -science museum of trento, italy) managed the ant collection. this work was also supported by grants from the university of parma (fil-2020) assigned to a. mori and benefited from the equipment and framework of the comphub initiative, funded by the “departments of excellence” program of the italian ministry for education, university and research (miur, 2018–2022). authors’ contributions es: conceptualization, methodology, investigation, data curation, writing-original draft, writing-review & editing, visualization. cc: conceptualization, methodology, validation, resources, data curation, writing-review & editing, supervision, project administration. lp: methodology, validation, resources, data curation, supervision, project administration, funding acquisition. mgo: methodology, validation, resources, data curation, writing-review & editing, supervision, project administration, writing-review & editing. vl: validation, resources, data curation, supervision, project administration, funding acquisition. fas: validation, resources, writing-review & editing. am: resources, writing-review & editing, supervision, project administration, funding acquisition. dg: visualization, writing-review & editing. dag: resources, writing-review & editing, supervision, project administration, funding acquisition. mgh: resources, writing-review & editing. mg: funding acquisition. references baroni urbani, c. 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(myrtaceae) in fragments of atlantic forest in south brazil introduction the brazilian atlantic forest is one of the world’s most diverse biomes, one of those most threatened by anthropic action (myers et al., 2000), and one of 34 world hotspots and priority areas for conservation (conservation international do brasil et al., 2000). within the atlantic forest biome the mixed ombrophilous forest formation (forest with araucaria) is an area of great interest given its high biodiversity (silveira et al., 2002). deforestation impacts, habitat fragmentation, exotic species introduction and irrational agriculture practices are the likely main causes of diminishing native populations of pollinators (kevan & phillips, 2001; kremen et al., 2002; steffan-dewenter et al., 2006). this can affect reproduction of native plants and eventually lead to local extinction of plant populations, as well as of the animals which depend on them abstract eugenia uniflora l. (myrtaceae) is pollinated mostly by bees and there are no restrictions for pollen collection in their flowers. this stimulated us to study the bee diversity on its flowers in two forest fragments in southern brazil, in august and september, 2012. a total of 826 bees belonging to 39 species and four subfamilies were captured with entomological nets. halictinae presented the greatest richness, followed by apinae, colletinae and andreninae. apis mellifera was the only common species and the most abundant, followed by scaptotrigona bipunctata and melipona obscurior. seven species were classified as intermediate and 31 as rare. highest numbers of bees were collected from 10 to 11:30 h. the species richness of flower visiting bees was much higher than that of previous studies with first records of plebeia remota and anthrenoides paolae. eugenia uniflora is a food source for exotic and native bees and possibly contributes to the conservation of these bees in forest fragments. in return, the bees probably act in maintaining this plant native of the atlantic forest. sociobiology an international journal on social insects mer diniz, mlt buschini article history edited by anne zillikens, university of tuebingen, germany received 12 january 2016 initial acceptance 25 february 2016 final acceptance 12 august 2016 publication date 25 october 2016 keywords species richness, halictinae, anthrenoides paolae, apis mellifera, scaptotrigona bipunctata. corresponding author mary ellen dos reis diniz universidade estadual do centro-oeste departamento de ciências biológicas programa de pós-graduação em biologia evolutiva rua simeão camargo varela de sá, n. 3, cascavel cep 85040-080 guarapuava, paraná, brasil e-mail: maryreisdiniz@yahoo.com.br (pinheiro machado & silveira, 2006). in tropical areas the bees are considered the main pollinators (ramirez & brito, 1992) and the animals best adapted to pollination (faegri & pijl, 1979). bees and other pollinators seem to be declining globally (potts et al., 2010) and habitat loss appears to be the most important factor driving the decline of bees (brown & paxton, 2009). myrtaceae is one of the largest plant families with thousands of species and approximately 140 genera, considered important in several neotropical ecosystems (johnson & briggs, 1984; kawasaki & landrum, 1997; wilson et al., 2001). it is one of the families most often found in the different vegetation formations of brazil (silva et al., 2001). eugenia, a genus with approximately 1,000 species, is one of the largest genera, with distribution mostly in central and south america (merwe et al., 2004). in brazil, eugenia predominates with 388 species of which 302 are endemic (sobral et al., 2015). universidade estadual do centro-oeste, guarapuava-pr, brazil research article bees mailto:maryreisdiniz@yahoo.com.br sociobiology 63(3): 982-990 (september, 2016) 983 eugenia uniflora, known commonly as “pitangueira”, is a native species from mixed ombrophilous forest, and a tree that can be used in landscaping or cultivated in home orchards (lorenzi, 2000; lorenzi, 2002). it has monoecious and generalist flowers, and pollen grains as the only floral reward to pollinators. it is therefore classified as a “pollenflower”, which is characteristic of many species in this genus (romagnolo & souza, 2006). it has a prominent role in the natural regeneration of mixed ombrophilous forest, through seed replacement and by providing food for birds (aguiar et al., 2013). eugenia uniflora grows under different shading conditions, and propagates easily by seeds, these being the determining factors in the regeneration process (scalon et al., 2001; callegaro et al., 2012). silva & pinheiro (2007) report that its flowers are visited by a wide variety of insects, including bees, which are the most common visitors. during the anthesis period, which starts at the sunrise, remaining until the end of the day (silva & pinheiro, 2007), the pollen grains are totally exposed, without any restrictions to collection, a common trait of generalist flowers (faegri & pijl, 1979; endress, 1994). the mass flowering, a common feature in myrtaceae (lughadha & proença, 1996), makes the flowers stand out and gives a white aspect to the plants that can be considered as a strategy to attract pollinators (gentry, 1974; o’brien & calder, 1993). the pollination in myrtaceae is highly diversified and its species are visited by a large variety of animals, including bees, wasps, flies, birds and even mammals, but bees are the main pollinators (beardsell et al., 1993; proenca & gibbs, 1994; lughadha & proença, 1996). in the neotropical region, the most common visitors of myrtaceae are apinae (lughadha & proenca & 1996). in a review on the flower visitors of myrtaceae gressler et al. (2006) reported bees the subfamilies apinae and halictinae only. in the early hours of the morning, the flowers exhale a smooth and sweet odor (silva & pinheiro, 2007), a strategy directly related to the attraction of bees (faegri & pijl, 1979, endress, 1994; proença & gibbs 1994; maués & couturier, 2002). eugenia uniflora produces fruit after cross-pollination and self-fertilization (franzon et al., 2011) however, self-fertilization in a natural population may result in inbreeding depression (keller & waller, 2002), which reduces fertility, viability of the seeds, vigor, among others (mettler & gregg, 1973; falconer & mackay, 1996). according to franzon et al. (2010) for the preservation of the e. uniflora is necessary to preserve them in the original forest fragments to ensure sufficient variability. this study sought to document the diversity of the flower visiting bees of e. uniflora in fragments of mixed ombrophilous forest in south brazil. materials and methods research area this research was conducted in two areas, located in the guarapuava municipality, paraná state (25°23’36”s; 51°27’19”w) in the central-south region of the state. according to the köppen geiger classification the climate in this region is humid subtropical, without a dry season, with the occurrence of severe frosts, average annual temperature around 22°c and average annual precipitation of 1961 mm. the two study areas are fragments of mixed ombrophilous forest with similar plant physiognomy.the areas are located around 25°22’05’’s/51°32’27,16’’w, with 8 ha, and 25°23’43’’s/51°25’29’’w, with 60 ha. the areas are 12 km distant from each other and are surrounded by other fragments of this same formation and crop fields. sampling design the visits for the monitoring of flowering phenology and the collection of flower visitors were performed between the end of august and during september 2012. in total we selected 51 trees to monitor the flowering and to collect the bees. during the whole flowering period bees were captured on e. uniflora flowers daily. the collection days alternated between the two study areas. in 30-minute intervals per hour, the bees that visited e. uniflora flowers were captured using entomological nets, by one collector. all collected individuals were sorted into morphospecies. some specimens were sent for identification to gabriel a. r. melo, at universidade federal do paraná (ufpr). the rest of the obtained specimens were deposited at the entomological collection of bees and wasps of the biology and ecology laboratory, of universidade estadual do centro-oeste (unicentro). data analysis the indices of shannon-wiener, margalef and pielou were used to assess the diversity, richness and evenness of the bees. those indices were obtained and calculated using the past® software (paleontological statics), version 1.98 (1999 – 2010) (hammer et al., 2001). the frequency of occurrence (fo) and the species dominance were calculated for each bee species obtained. frequency of occurrence is the percentage of the number of collections with a given species and was calculated as fo= (f/n) x100 (silveira neto et al., 1976), where “f” is the number of collections with the species and “n” is the total number of collections performed. the bee species were classified as primary (fo > 50%), secondary (fo = 25% 50%), or accidental (fo < 25%). the species dominance of bees (d) was calculated as d= (d/n) x100 (palma, 1975), where “d” is the abundance of a specific species and “n” the total abundance. the species were classified as dominant (d > 5%), accessory (d = 2.5% 5%), or accidental (d < 2.5%). according to palma (1975), the fo and d indices when used together group and determine the species as common, intermediary or rare. mer diniz, mlt buschini – diversity of flower visiting bees of eugenia uniflora l. (myrtaceae)984 results the flowering occurred from august 25 to september 14, 2012. during this period, 17 collections were performed for a total of 168 hours. due to the similarity of the collection areas the bees obtained at both locations were grouped in only one sample. a total of 826 bees belonging to 39 species and the four subfamilies andreninae, colletinae, apinae and halictinae were captured. the most abundant species was apis mellifera (337 individuals), followed by scaptotrigona bipunctata (293) and melipona obscurior (74). fifteen species were recorded by one specimen only (table 1). the subfamily with the highest number of species was halictinae (23 species); andreninae was represented by just one species (anthrenoides paolae). the bees were captured from 9:00 to 16:30, with highest numbers collected in the intervals between 10:00 and 10:30, and 11:00 and 11:30 and with highest number of species in the interval from 11h to 11:30 (table 2). subfamily tribe genus/species number of individuals fo (%) d (%) denomination andreninae protandrenini anthrenoides paolae urban, 2005 15 47.05 1.82 intermediary colletinae hylaeini hylaeus aff. geminus (vachal, 1910) 1 5.8 0.12 rare hylaeini hylaeus aff. vachali meade-waldo, 1923 3 17.64 0.36 rare hylaeini hylaeus cf. asper (vachal, 1909) 1 5.8 0.12 rare colletini rhynchocolletes albicinctus moure, 1943 1 5.8 0.12 rare halictinae augochlorini augochlora sp. smith, 1853 5 29.42 0.61 rare augochlorini augochloropsis cf. cognata moure, 1944 6 29.41 0.73 intermediary augochlorini augochloropsis chloera (moure, 1940) 1 5.8 0.12 rare augochlorini augochloropsis cupreola (cockerell, 1900) 2 11.76 0.24 rare augochlorini augochloropsis imperialis (vachal, 1903) 5 23.52 0.61 rare augochlorini augochloropsis notophos (vachal, 1903) 3 17.64 0.36 rare augochlorini augochloropsis sparsilis (vachal, 1903) 1 5.8 0.12 rare augochlorini augochloropsis sympleres (vachal, 1903) 3 17.64 0.36 rare augochlorini ceratalictus psoraspis (vachal, 1911) 1 5.8 0.12 rare augochlorini halictillus loureiroi (moure, 1941) 3 11.76 0.36 rare augochlorini neocorynura cf. chapadicola (cockerell, 1901) 2 5.8 0.24 rare augochlorini paroxystoglossa andromache (schrottky, 1909) 2 11.76 0.24 rare augochlorini paroxystoglossa cf. barbata moure, 1960 1 5.8 0.12 rare augochlorini paroxystoglossa sp. moure, 1941 4 17.64 0.48 rare halictini (caenohalictina) caenohalictus tesselattus (moure, 1940) 1 5.8 0.12 rare halictini (halictina) dialictus bruneriellus (cockerell, 1918) 2 11.76 0.24 rare halictini (halictina) dialictus pabulator (schrottky, 1910) 1 5.8 0.12 rare halictini (halictina) dialictus picadensis (strand, 1910) 2 11.76 0.24 rare halictini (halictina) dialictus sp.1robertson, 1902 1 5.8 0.12 rare halictini (halictina) dialictus sp.2 robertson, 1902 2 11.76 0.24 rare halictini (halictina) dialictus sp.3 robertson, 1902 1 5.8 0.12 rare halictini (halictina) dialictus sp.4 robertson, 1902 1 5.8 0.12 rare halictini (halictina) dialictus sp.5robertson, 1902 1 5.8 0.12 rare table 1 species of flower visiting bees collected on eugenia uniflora l. in fragments of mixed ombrophilous forest (atlantic forest) in south brazil. indices of frequency of occurrence (fo), dominance (d) and the denomination for each species of floral visitor bee. sociobiology 63(3): 982-990 (september, 2016) 985 table 1. species of flower visiting bees collected on eugenia uniflora l. in fragments of mixed ombrophilous forest (atlantic forest) in south brazil. indices of frequency of occurrence (fo), dominance (d) and the denomination for each species of floral visitor bee. (continuation) subfamily tribe genus/species number of individuals fo (%) d (%) denomination apinae apini apis mellifera linnaeus, 1758 337 100 40.80 common exomalopsini exomalopsis (diomalopsis) bicellularis michener & moure, 1957 13 52.94 1.57 intermediary exomalopsini exomalopsis (phanomalopsis) aureosericea friese, 1899 2 11.76 0.24 rare meliponini melipona (eomelipona) obscurior moure, 1971 74 82.35 8.96 intermediary meliponini plebeia emerina (friese, 1900) 2 11.76 0.24 rare meliponini plebeia remota (holmberg, 1903) 21 70.58 2.54 intermediary meliponini scaptotrigona bipunctata (lepeletier, 1836) 293 88.23 35.47 intermediary meliponini schwarziana quadripunctata (lepeletier, 1836) 7 35.29 0.85 intermediary meliponini tetragonisca angustula (latreille, 1811) 1 5.8 0.12 rare xylocopini (ceratinina) ceratina cf. (ceratinula) biguttulata (moure, 1941) 3 11.76 0.36 rare xylocopini (xylocopina) xylocopa (dasyxylocopa) bimaculata friese, 1903 1 5.8 0.12 rare the margalef richness index (dmg) for the bee sample collected in this study was 5.658, and the shannon-wiener diversity index (h’) was 1.686.the pielou evenness index (j) was 0.4601. the obtained fo and dvalues classified only a. mellifera as common visitors of e. uniflora flowers.seven species were classified as intermediary: m. obscurior, s. bipunctata, a. paolae, schwarziana quadripunctata, exomalopsis bicellularis, plebeia remota and augochloropsis cf. cognata.the other 31 bee species were considered rare visitors of e. uniflora (table 1). discussion the richness and diversity of bee visitors of e. uniflora was much higher than the 11 species recorded by silva and pinheiro (2007) in the western part of the municipality of rio de janeiro (rio de janeiro state, brazil).high values of richness and diversity indicate, in most cases, a well-structured community, with many rare species (costa et al., 1993). according to michener (1979), southern brazil it is one of the regions in the world with the highest richness of apoidea. regarding the species richness per subfamilies, our sample from the guarapuava (pr) region also had a higher numbers of species of apinae visiting e. uniflora flowers than recorded in rio de janeiro state, but we must consider that both studies were conducted in totally different phytophysionomies. the greatest activity of bees on e. uniflora flowers occurred during the hottest hours of the day. bees’ foraging activity is influenced by weather conditions, mainly temperature because maintaining high body temperature during the flight is energetically costly (roubik, 1989). flower visitors of the subfamilies colletinae and andreninae are new records for e. uniflora.in the southern region of brazil, the most diverse subfamilies are apinae and halictinae (alves-dos-santos, 2007), and we also found the greatest number of speciesfor halictinae among the flower visitors or e. uniflora, demonstrating that this pattern is also reflected in our bee sample. silva and pinheiro (2007) considered species of the genus xylocopa to be among the pollinators of e. uniflora. it is therefore possible that x. bimaculata, even though it was a rare visitor, is also able to pollinate those flowers. bees of the genus xylocopa are also floral visitors of other myrtaceae species (schlindwein et al., 2003; siqueira et al., 2012). out of all the species captured in this study, only a. mellifera was classified as common, which was also observed in e. uniflora flowers by both pelacani et al. (2000) in são paulo state (brazil), and by silva and pinheiro (2007). although a. mellifera is an exotic species in brazil, it has become the most common floral visitor in the neotropics (roubik, 2000). apis mellifera has considerable interference in the reproduction of many plant species and can facilitate or hamper their reproductive success, influencing directly or indirectly on the foraging of native pollinators (paton, 1993; vaughton, 1996; villanueva-g, 2002). according to silva and pinheiro (2007) the high number of a. mellifera in the early anthesis can impair the pollen supply for native visitors. this may be one of the reasons for the high number of species considered rare visitors of e. uniflora, because there is a lower amount of pollen available when native bees start the foraging. depletion of pollen is also indicated by the sharp decrease in the number of pollinators after 12:30 h. of the species belonging to the genus scaptotrigona only s. xanthotricha moure, 1950 was registered visiting e. uniflora flowers in rio de janeiro state (silva & pinheiro, 2007). other species, namely s. depilis (moure, 1942) and mer diniz, mlt buschini – diversity of flower visiting bees of eugenia uniflora l. (myrtaceae)986 subfamily tribes genus/species hour of the day total of individuals9h 10h 11h 12h 13h 14h 15h 16h andreninae protandrenini anthrenoides paolae urban, 2005 2 7 2 3 1 15 colletinae colletini rhynchocolletes albicinctus moure, 1943 1 1 hylaeini hylaeus aff. geminus (vachal, 1910) 1 1 hylaeini hylaeus aff. vachali meade-waldo, 1923 1 1 1 3 hylaeini hylaeus cf. asper (vachal, 1909) 1 1 halictinae augochlorini augochlora sp. smith, 1853 1 3 1 5 augochlorini augochloropsis cf. cognata moure, 1944 1 2 1 2 6 augochlorini augochloropsis chloera (moure, 1940) 1 1 augochlorini augochloropsis cupreola (cockerell, 1900) 1 1 2 augochlorini augochloropsis imperialis (vachal, 1903) 2 1 1 1 5 augochlorini augochloropsis notophos (vachal, 1903) 1 1 1 3 augochlorini augochloropsis sparsilis (vachal, 1903) 1 1 augochlorini augochloropsis sympleres (vachal, 1903) 1 2 3 augochlorini ceratalictus psoraspis (vachal, 1911) 1 1 augochlorini halictillus loureiroi (moure, 1941) 2 1 3 augochlorini neocorynura cf. chapadicola (cockerell, 1901) 2 2 augochlorini paroxystoglossa andromache (schrottky, 1909) 1 1 2 augochlorini paroxystoglossa cf. barbata moure, 1960 1 1 augochlorini paroxystoglossa sp. moure, 1941 2 2 4 halictini (caenohalictina) caenohalictus tesselattus (moure, 1940) 1 1 halictini (halictina) dialictus picadensis (strand, 1910) 1 1 2 halictini (halictina) dialictus bruneriellus (cockerell, 1918) 2 2 halictini (halictina) dialictus pabulator (schrottky, 1910) 1 1 halictini (halictina) dialictus sp.1 robertson, 1902 1 1 halictini (halictina) dialictus sp.2robertson, 1902 2 2 halictini (halictina) dialictus sp.3robertson, 1902 1 1 halictini (halictina) dialictus sp.4robertson, 1902 1 1 halictini (halictina) dialictus sp.5robertson, 1902 1 1 apinae apini apis mellifera linnaeus, 1758 96 86 62 43 19 15 12 4 337 exomalopsini exomalopsis (diomalopsis) bicellularis michener & moure, 1957 2 3 4 1 2 1 13 exomalopsini exomalopsis (phanomalopsis) aureosericea friese, 1899 1 1 2 table 2. dial pattern of the number of flower visiting bees captured on eugenia uniflora l.,in fragments of mixed ombrophilous forest. sociobiology 63(3): 982-990 (september, 2016) 987 s. fulvicutis (moure, 1964) were found as abundantflower visitors of myrtaceae in mato grosso do sul and amazonas states in brazil (marques-souza et al., 2007; ferreira et al., 2010). scaptotrigona bipunctata is among the most common species of meliponini found in paraná state (paraná, 2009), which explains the large number of individuals collected in this work. this species displays highly defensive behaviour (nogueira-neto, 1970), which could explainits abundance and its frequency found here, when compared to the presence of other species. melipona obscurior bees are polylectic foragers (kleinertgiovannini & imperatriz fonseca, 1987) and there are records of their occurrence in bahia state, as well as in southern and southwestern brazil (roubik, 1989; nogueira-neto, 1997; silveira et al., 2002). bees in this genus (melipona scutellaris latreille, 1811) were reported to pollinate psidium guajava l. (myrtaceae) (castro & araújo, 1998). the findings that deserve highlighting in this work is the unprecedented recording of plebeia remota in e. uniflora flowers. plebeia remota is not commonly observed as a floral visitor or pollinator of other myrtaceae species. another unprecedented record was the presence of a. paolae visiting e. uniflora flowers, given that no floral visitation or pollination of this species to any myrtaceae has been observed before. apis mellifera is possibly the most important potential pollinator of e. uniflora because it was the most frequent floral visitor, followed by s. bipunctata and m. obscurior. this study reveals that e. uniflora is an easily obtainable pollen source for exotic and native bees, and thus contributes to the conservation of these bee species in forest fragments helping in the maintaining the biodiversity of the bees in these fragments. at the same time, these bees are probably acting in the preservation of this native plant and assist in the regeneration of the highly fragmented atlantic forest. acknowledgments we thank capes (coordenação de aperfeiçoamento de pessoal de nível superior) for the scholarship and the programa de pós-graduação em biologia evolutiva of universidade estadual do centro-oeste (unicentro). we also thank gabriel a. r. melo of universidade federal do paraná (ufpr) for bee identification and cláudia i. silva for the help with sample design. references aguiar, r.v., cansian, r.l., kubiak, g.b., slaviero, l.b., tomazoni, t.a., budke, j.c., mossi, a.j. 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(2001). myrtaceae revisited: a reassessment of intrafamilial groups. american journal of botany, 88: 2013-2025. ole_link1 ole_link2 doi: 10.13102/sociobiology.v62i3.793sociobiology 62(3): 426-438 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 global elevational, latitudinal, and climatic limits for termites and the redescription of rugitermes laticollis snyder (isoptera: kalotermitidae) from the andean highlands introduction taxonomic diversity declines for most organisms as climatic temperatures decrease either from increasing elevation (rahbek, 1995) or increasing latitude (willig et al., 2003; mittelbach et al., 2007). termites also show reduced diversityas elevation increases above ca. 800 m (inoue et al., 2006; palin et al., 2011; gathorne-hardy et al., 2001) and as latitude becomes extra-tropical (emerson, 1952; cancello et al., 2014). termite diversity is overwhelmingly greatest in the tropics (≤23.5° n and s) where several hundred genera abstract we compile, map, and discuss global elevational, latitudinal, thermal, and rainfall extremes of termite localities from literature sources and unpublished records. rugitermes laticollis from ecuador and bolivia occurs at higher elevation (2700-3600 m) than any other termite species. termites span the globe from 54.3°n (zootermopsis angusticollis in british columbia (b.c.), canada) to 48.9°s (porotermes quadricollis in magdalena, chile). the coldest locality supporting termites (reticulitermes sp.) is at churn creek, b.c., where the mean annual temperature is 4°c. lake havasu city, arizona, where heterotermes aureus and gnathamitermes perplexus occur, has the highest recorded temperature maximum (52°c) for a termite locality. cryptotermes brevis and neotermes chilensis are endemic to the pacific coast of peru and chile where rain is essentially absent. we further provide locality extremes for six termite families from six zoogeographical regions. in addition, the winged imago of ru. laticollis is redescribed and the soldier is described for the first time. sociobiology an international journal on social insects rh scheffrahn1, aj mullins1, j krecek1, ja chase2, jr mangold2, t myles3, t nishimura4, r setter5, ra cannings6, rj higgins7, bs lindgren8, r constantino9, s issa10, and e kuswanto11 article history edited by gilberto m. m. santos, uefs, brazil received 14 april 2015 initial acceptance 18 june 2015 final acceptance 11 august 2015 keywords termites, andean highlands, rugitermes laticollis snyder, redescription. corresponding author rudolf h scheffrahn fort lauderdale research & education center 3205 college avenue, davie 33314, florida, usa e-mail: rhsc@ufl.edu are recorded compared to five found in the temperate regions (≥40° n and s, jones & eggleton, 2011). in addition to elevational and latitudinal extremes, some termite species also occupy environments in hot or arid deserts and in nearfreezing climates (emerson, 1936; emerson, 1955). among t.e. snyder’s last taxonomic publications was a note on a new termite from bolivia (snyder, 1957). therein, he briefly describes winged adults of rugitermes laticollis from a sample which he “found” in the smithsonian collection where he worked. what is especially noteworthy was snyder’s account of the type locality of his new species as research article termites 1 fort lauderdale research & education center, davie, florida, usa 2 terminix international, memphis, tennessee, usa 3 city of guelph, building services, ontario, canada 4 basf corporation, north carolina, usa 5 integrated dna technologies, inc., 1710 iowa, eua 6 royal british columbian museum, victoria bc, canada 7 department of biological sciences, thompson rivers university, british columbia, canada 8 university of northern british columbia, british columbia, canada 9 universidade de brasília, brasília, df, brazil 10 universidad simón bolívar, caracas, venezuela 11 school of life sciences and technology, bandung institute of technology,ganesa, indonesia sociobiology 62(3): 426-438 (september, 2015) 427 follows: “la paz, bolivia. described from 6 winged adults, 4 males and 2 females, collected at the type locality by r. pérez alcalá, 1947.” unaware of ru. laticollis, one of the authors (ajm) traveled to quito, ecuador, after departing from a 2010 termite expedition to the ecuadorian amazon. after reuniting, ajm reported finding a kalotermitid species in a tree at a city park in quito. having found very few termites in the neotropics above 1600 m, we were astonished to learn of ajm’s find at an elevation of 2800 m! on our return, we determined that the quito sample was indeed ru. laticollis snyder. we now surmise that snyder (1957) did not realize the altitudinal significance of the la paz locality for ru. laticollis which is even more elevated than quito by some 400 to 1400 m. furthermore, after reviewing the uf collection holdings, a third high-elevation sample of ru. laticollis was identified from luribay, bolivia (2700 m). prompted by our unexpected collection of ru. laticollis and the lack of a compilation of termite localities at the limits of termite habitation, we herein review published records and provide new data on the elevational, latitudinal, thermal, and rain fall limits of representative termite taxa worldwide. furthermore, we describe for the first time, the soldier of ru. laticollis and redescribe its winged imagos. materials and methods elevation and locality coordinates (appendix) were taken from published data or from our database (university of florida (uf) termite collection, davie, florida) listed as “current paper” in appendix. most specimens in the uf collection were taken during week-long expeditions in which the goal was to acquire maximum regional diversity. in some cases, published elevation, geographic coordinates, and locality names were confirmed or corrected using google earth and web resources. climatic data were taken from http://www.weatherbase.com/. rugitermes laticollis localities and those reported or selected for other termite taxa were mapped (fig 1) using arcgis desktop 10.2 software. figures 2 and 3 were taken as multilayer montages using a leica m205c stereomicroscope controlled by leica application suite version 3 software. montage specimens were taken from 85% ethanol and positioned in purell® hand sanitizer contained within a transparent plastic petri dish bottom. field photographs of ru. laticollis (figs 4 and 5) were taken with a nikon 7sc digital camera in macro flash (fig 4) or non-macro (fig 5) mode. measurements (tables 1 and 2) were taken with an olympus szh stereomicroscope with an ocular micrometer. results elevational maxima in most of the world’s tropics, termite diversity and abundance are greatest from sea level to about 1200 m (inoue et al., 2006). palin et al. (2011) reported a decrease in termite diversity and abundance with increased elevation in peru and found no termites above 1550 m. atkin and proctor (1988) found no termites at 1500m or above on a costa rican volcano. on mount giting-giting, philippines, thomas and proctor (1997) found a few termite species at 1240 m elevation and none at 1540 m. collins (1980) reported the upper limit of termites at 1850 m on the west ridge of gunung (mount) mulu, sarawak. gathorne hardy et al. (2001) found the least termite diversity (five species) at 1400 m compared to 11-35 species at lower elevations on the island of sumatra. in our new world sampling efforts, scheffrahn et al. (unpublished data) have observed a similar elevational decline in termite diversity. the appendix provides a summary of 33 reported and 24 new global elevational maxima for selected termite taxa. the most elevated locality reported for any termite is 3900 m for “kunwari pass”, chamoli state, india, for the damp wood termite, archotermopsis wroughtoni (roonwal et al., 1984). this elevation is 1100 m higher than the next highest of the 35 localities reported by these authors. the name and elevation of this western himalayan site, however, could not be confirmed on google earth, the web, or maps of northern india. when queried for “kunwari pass”, internet resources only yielded the town of joshimath (1860 m) which is at the trail head of “kauri pass”. shedding further doubt on the “kunwari pass” locality, imms (1920) is quoted: “in june, 1910, i first came across this insect in a decaying fallen trunk of pinus excelsa, in a forest area situated between the kuari pass and ramni, at an altitude of about 8,500 feet, in the himalayas of british garwhal.” imms (1920) further table 1. measurements of rugitermes laticollis winged imagos (n=7). table 2. measurements of rugitermes laticollis soldiers (n=10). mean range head length to lateral mandible base 3.08 2.50-3.33 head widthmax. 2.06 1.83-2.43 head height with gula max. 1.64 1.40-1.83 pronotum length 1.08 1.00-1.17 pronotum width 2.21 1.93-2.76 no. antennal articles 15 14-16 females (n=3) males (n=4) mean range mean range head width max. (without eyes) 1.73 1.50-2.00 1.64 1.60-1.75 pronotum width 1.96 1.88-2.06 2.02 1.83-2.13 eye diam. max 0.37 0.36-0.38 0.38 0.37-0.39 body length 8.89 8.66-9.00 8.93 8.75-9.10 right forewing length 9.35 9.05-9.75 9.54 9.25-9.90 body length with wings 11.77 11.30-12.25 12.20 12.00-12.65 rh scheffrahn et al. – global elevational, latitudinal, and climatic limits for termites428 f ig 1 . d is tr ib ut io n m ap fo r r ug ite rm es la tic ol lis a nd te rm ite g eo gr ap hi ca l l im its re fe re nc ed b y nu m be r f ro m t ab le 1 . sociobiology 62(3): 426-438 (september, 2015) 429 notes that n.c. chatterjee provided him with material taken at 2743 m in deoban, uttar pradesh. deoban, is the second highest ar. wroughtoni site listed at 2800 m by roonwal et al. (1984) and is very likely the same sample referenced by imms (1920). google earth gives the elevation of deoban village at over 400 m lower (2280 m). this leaves the highest confirmed locality of material examined by roonwal et al. (1984) as gulmarg, jammu and kashmir province (2700 m). although the exact locality in la paz from which snyder’s (1957) original ru. laticollis sample was collected cannot be determined, the elevational range for this city is between 3400-4000 m (http:// travel.state.gov/travel/ cis_pa_tw/cis/cis_1069.html) with the old central plaza at 3600 m. the recorded elevational mean and range for ru. laticollis iscurrently more elevated and narrower (2700-3600 m) than that of ar. wroughtoni (1000-2743 m, roonwal et al., 1984). elevational records for the afrotropics are limited to the higher termites (family termitidae) which reach a maximum elevation in the highlands surrounding the great rift valley of ethiopia and kenya. here, all afrotropical subfamilies except nasutitermitinae have been recorded at 1900 m or above (appendix). the epigeal mounds of cubitermes and odontotermes colonies are conspicuous in these more barren highlands. in ethiopia, cowie et al. (1990) reported that “above 2000m the fauna is very restricted, but odontotermesis found in addis ababa (ca. 2300 m) and a single worker of a completely subterranean odontotermes species was seen (but not collected) near debre birhan at about 3200m”. google earth, however, gives the elevation of the debre birhan plateau at 2800 m. sjöstedt’s 1905 record of odontotermes apollo at 2600 m from eburru, kenya, is the elevational maximum for the family termitidae. donovan et al. (2002) collected 11 genera of termitidae, many of which are soldierless soil feeders in the subfamily apicotermitinae, at 1900 m on the nyika plateau of malawi. the nasutitermitine genus trinervitermes, although reported to be very rare in the area, was recorded at 1650m at adami tullu, ethiopia (debelo & degaga, 2014). the only significant elevational records for the australian region include the family stolotermitidae (porotermes adamsoni and stolotermes vitoriensis) from the brindabella range near canbarra (1300 m, lacey et al., 2010). the highest report for termites in papua new guinea is that of pericapritermes cf. schultzei collected in the bismarck range at 1650 m (bourguignon et al., 2008). in addition to the elevation records for ar. wroughtoni noted above, the himalayan foothills also support odontotermes distans at 2250m (thakur, 1981). elevational records in the eastern indomalayan region include two nasutermitine genera, bulbitermes and longipeditermes, from sumatra (1400 m) and bulbitermes from malaysia (1860 m, appendix). since 2004, the neotropical mainland has been the subject of largely unpublished termite biodiversity surveys (cf. uf collection) with the kalotermitidae generally occupying higher elevations than other families of the region (appendix). aside from ru. laticollis and the exotic cryptotermes brevis, members of the kalotermitid genera comatermes, glyptotermes, incisitermes, marginitermes, and neotermes have been recorded above 1500 m in the highlands of guatemala and honduras (appendix). a new species of neotermes was collected at 1831 m from a damp log in parque nacional de yacumba, venezuela. coptotermes testaceus and heterotermes convexinotatus (rhinotermitidae) have also been found at elevations above 1500 m in the central american highlands. in south america, mostly termitidae have been recorded above 1400 m including anoplotermes turricola and procornitermes lespesii (bolivia) and nasutitermes guayanae and na. octopilus (peru, palin et al., 2011). the highest elevation for termites in the west indies is currently at 1239 m for glyptotermes liberatus in the blue mountains of eastern jamaica. we expect, however, that the west indian elevational record actually lies in the poorly accessible cordillera central of the dominican republic which rises to pico duarte at 3098 m. in the nearctic region, only reticulitermes sp. (rhinotermitidae) and zootermopsis sp. (archotermopsidae) have been found above 2000 m (appendix). a colony of the western drywood termite, incisitermes minor, was sampled from a pine log in the davis mountains of texas at an elevation of 1772 m. gnathamitermes nr. perplexus and amitermesnr californicus [not a synonym of am. wheeleri (desneux) scheffrahn unpubl. obs.] were collected near i. minor at slightly lower elevation. tenuirostritermes tenuirostris, the only nasutitermitine of the nearctic, was collected at 1765 m in the chiricahua mountains of southeastern arizona. winter snowfall accumulations are common in these mountains. in the palearctic, both elevational records are from afghanistan. anacanthotermes septentrionalis is reported at 2000 m in jija, a record for the family hodotermitidae. microcerotermes gabrielis was collected at 1850 m in kabul (weidner, 1960). it should be mentioned that two termite species have been found along the red sea where elevation below sea level is a global minimum. angulitermes quadriceps was collected around jericho and sdom, israel, at about -252 m (harris, 1964), and microcerotermes palestinensis was found in ein gedi, israel, at about -282 m (spaeth, 1964). latitudinal maxima the northern global limits of termite distribution are reported by vickery and kevan (1985) who recorded zootermopsis angusticollis at 54.3°n (prince rupert, british columbia) and z. nevadensis at 53.3°n (dunkley, bc, near quesnel). coastal southern alaska needs to be surveyed for the possibility of a more northerly record for zootermopsis sp. (r.a. cannings pers. comm.). a reticulitermes sp. was collected as far north inland as 51.3°n (churn creek protected area near dog creek, bc; coll. rjh) and near kamloops, british columbia (50.7°n, vickery & kevan, 1985;).the rh scheffrahn et al. – global elevational, latitudinal, and climatic limits for termites430 southern limits for termites are ca. 48.9°s for porotermes quadricollis (magallanes province, chile, constantino, 1998) followed by stolotermes ruficeps at ca. 46°s (“widespread throughout new zealand”, bain & jenkin 1983). for a higher termite, onkotermes brevicorniger holds the record at 43.3°s (rawson, chubut province, argentina, constantino et al., 2002). precipitational and thermal extremes the pacific coast of peru and northern chile receives the least rainfall of any nonpolar region on earth. however, due to riparian habitats nurtured by andean snowmelt, both the peruvian and the atacama deserts support woody growth and termites. this remarkably cool and humid desert coastline is the endemic habitat of cryptotermes brevis (scheffrahn et al., 2009) and neotermes chilensis, the latter occurring in both dead wood and inside living trees (scheffrahn pers. obs.).amitermes lunae was discovered in soil at a moche archeological site near trujillo, peru (scheffrahn & huchet, 2010) which receives a mere 0.5 cm of rainfall annually. termites are common to very wet circum-tropical climates. the village of cherrapunji at the base of the himalayas in assam province, india, receives more rain, at 10.6 m per annum, than any place on earth. roonwal and chhotani (1962) report four termitid genera from cherrapunji (appendix). the termite localities with the highest recorded temperature maxima include lake havasu city, arizona (52°c, heterotermes aureus and gnathamitermes perplexus), imperial california (51°c, marginitermes hubbardi), and ghat, libya (51°c, psammotermes hybostoma, harris, 1966). the coldest annual mean (record minima) where termites occur include churn creek, b.c. 4°c (-40°c ) for reticulitermes sp.; new meadows, idaho,5°c (-45°c) for z. angusticollis; dunkley, b.c. 5.5°c (-47°c) for z. nevadensis; and amidon, north dakota, 6°c (-40°c), for r. tibialis (emerson, 1936). discussion the nesting and foraging habits of most termites should protect colonies from excesses of air, soil, or substrate temperatures found at elevational or latitudinal extremes. however, only two genera, zootermopsis and reticulitermes, live in climates where the mean annual temperatures ranges between 4-6°c, while porotermes quadricollis and stolotermes ruficeps occur at 8 °c (puerto aisen, chile) and 10° c (intercargil, new zealand) respectively. cabrera and kamble (2001) showed that reticulitermes flavipes foragers avoid the coldest periods by harboring in warmer soil refugia. cook and smith (1942) reported that the metabolism of the protist symbionts of zootermopsis were similar at 9° to 29°c, but stopped at 4°c leading to starvation of the termites. lacey et al. (2010) have recently shown that trehalose is used as a cryoprotectant in both porotermes and stolotermes in australian climates which are much warmer than those of either southern chile or new zealand. it is unclear why termites, especially reticulitermes, do not reside naturally in northern europe or the british isles (harris, 1962) where mean/minimum temperatures (e.g. warsaw, poland, 8°/-30° c; edinburgh, scotland, 9°/-17°c; and london, england, 10°/-13°c) are above the temperatures where reticulitermes and zootermopsis occur in the northern united states and canada. termites appear to inhabit all geographic regions with high thermal maxima or mean temperatures as long as food and ground moisture are sufficiently available. the overwhelming majority of termites live in humid tropical environments (jones & eggleton, 2011). in seasonally hot regions termites move into cooler, deeper soils to avoid periods of excessive maximum surface temperatures (heterotermes aureus and gnathamitermes perplexus, collins et al., 1973) or into cooler deeper or lower wood substrata (incisitermes fruticavus rust, rust et al., 1979). paraneotermes simplicicornisis the only example of a kalotermitid that has adapted to hot seasonal climates by evolving a completely subterranean habit (light, 1937). regarding elevation (e.g., cr. brevis in bogota colombia, 2600 m), anthropogenic introductions of exotic termites within buildings often exceed latitudinal limits. for example, a na. corniger colony was observed building foraging tubes from a potted plant near the swimming pool of a fitness club in east kilbride, scotland (57.213°n, 5.997°w, scheffrahn et al., 2002). the plant had been imported from barbados. cryptotermes brevis was collected in anchorage, alaska (61.21n, -149.89w), from a bookcase originally transported from hawaii (scheffrahn unpubl. data). worldwide, z. angusticollis shows the greatest adaptation of any termite to climatic and geographic variability. this species occupies a 93°c temperature range from new meadows, idaho (-45°cmin. record) to riverside, california (48°c max. record). in addition, the vast elevational range of z. angusticollis from 2124 m in the sierra nevada mountains of california (weesner, 1965) to sea level (los angeles, banks and snyder 1920; santa monica ca, scheffrahn unpublished), a large precipitation range (197 cm tatoosh island, washington state, thorne et al., 1993) to riverside, ca, (26 cm) and a vast geographical range extending 3,000 km from guadeloupe is., mexico (light, 1933), to prince rupert, b.c., attest to the remarkable climatic tolerance of this species. taxonomic summary rugitermes laticollis snyder 1957 description imago (modified from snyder, 1957, figs 2, 4; appendix). head capsule very dark brown to black except for dark ferruginous border around antennal sockets. dorsum of body, including pronotum, concolorous with head capsule. postclypeus yellowish, labrum light brown. compound eyes sociobiology 62(3): 426-438 (september, 2015) 431 fig 2. rugitermes laticollis.oblique lateral and dorsal views of the imago head and pronotum. and ocelli very dark gray; compound eyes small; occupying mid one-third of lateral head capsule aspect. pronotum wider than head capsule. antennae with 15 articles; basal articles concolorous with labrum, becoming darker toward apex. head, pronotum, and wing scale generously covered with both long and short setae. setae widely dispersed on abdominal tergites, and sternites. pronotum with scattered long and short setae; anterior margin weakly concave, posterior emarginate in middle. compound eyes subcircular; eye margins broadly subrectate along antennal sockets. ocelli hyaline, slightly protruding, suboval; well separated from eyes. wing membrane smoky brown with bronze and greenish prismatic sheen in live specimens (fig 4); membrane covered with tiny punctuations; sclerotized veins dark brown. venation as in fig 13 of krishna (1961). in forewing, all major veins emerge from scale independently; sclerotized median vein joins radial sector about one-eight distance to wing tip; cubitus unsclerotized and running parallel with radial sector to tip of wing. hind wing without median vein. arolia present. comparisons. unlike many species of rugitermes that are bicolored (usually pronotum and/or wings contrast with head coloration), the imago of r. laticollis is evenly and very darkly concolorous. the imago of rugitermes niger oliveira 1979 is also uniformly very dark (dark brown to black) but it is much smaller (head width 1.26-1.34 mm). soldier (figs 3, 4; table 2). monomorphic. head capsule rectangular; lateral margins parallel in dorsal view. antenna with 14 articles 2<3>4=5; third article clavate and twice the length of second. mandibles orange-brown near base, grading to castaneus before first marginal tooth and black to apical tooth. pronotum pale yellow at posterior margin grading to reddish orange from anterior fourth to anterolateral margins. mandibles project about one-half length of head capsule; without basal hump. anterolateral corners of genae prominent; forming angular corners when viewed from above. antennal ridge robust over antennal sockets but gives way to weakly rugose concavity formed by frons. head capsule slightly wider than pronotum.pronotum collar-like; anterior margin evenly concave; posterior margin evenly convex with small concavity in middle. gula slender in middle, about one-third width of anterior portion. fig 3. rugitermes laticollis.dorsal, oblique, lateral, and ventral views of the soldier head and pronotum. fig 4. habitus of a soldier, imagoes, and nymphs of rugitermes laticollis. rh scheffrahn et al. – global elevational, latitudinal, and climatic limits for termites432 fig 5. collection site of rugitermes laticollis in quito, ecuador. comparisons. soldiers of ru. laticollis cannot be confidently separated from congeners using the soldier caste alone, however, for those species with uniformly dark imagoes, the soldiers of ru. laticollis are the largest. material examined. bolivia, depto. de la paz: luribay (-17.05994 lat., -67.66363 long.; elev ca. 3600 m), dec 1986, col. r. subieta, uf collection no. sa1, (2 soldiers and pseudergates; ex. live branch of peach (prunus) tree). ecuador, distr. pichincha: quito, parque la carolina, (-0.18845, -78.48595; elev 2780 m), 3 jun 2011, col. a mullins, ec1465 (alates, soldiers, nymphs; ex. dead portion of live tree, fig 5).adjacent quito locality (-0.18879, -78.48556) 4 jun 2011, col. krecek, mullins, scheffrahn; ec1466 (alates, soldiers, nymphs), ex. dead portion of live tree fig 5).adjacent quito locality (-0.18879, -78.48556) 4 jun 2011, col. krecek, mullins, scheffrahn; ec1467 (soldiers and nymphs), ex. dead portion of live tree. biology. rugitermes laticollis colonizes live or dead wood occasionally exposed to freezing temperatures in the upper montane zone of andes (2300 to 3630 m as defined by young & keating, 2001). alates in quito were found in june but the timing of flight season is uncertain. the dark coloration suggests daytime flight which may improve alate mobility compared to cool nighttime mountain temperatures. although known from only three locations, ru. laticollismay well occur over a large area of andean highlands which support woody growth. acknowledgements we thank tiago f. carrijo, john warner, and ben gillenwaters for their reviews. termites collected under permit no. 06-2011-fau-dpap-ma, ministerio del ambiente, quito ecuador. references atkin, l., & proctor, j. 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.7 30 0 w ill ia m s 19 66 4 el ev a fr ic ot ro pi ca l te rm iti da e m ac ro te rm iti na e m ac ro te rm es su bh ya lin us r am bu r 20 00 et hi op ia bi sh oft u 8. 75 00 38 .9 80 0 ru el le 1 97 0 6 el ev a fr ic ot ro pi ca l te rm iti da e m ac ro te rm iti na e o do nt ot er m es sp . 24 00 ke ny a ch er an ga ni h ill s 1. 22 00 35 .4 30 0 d ar lin gt on 1 98 5 5 el ev 2 a fr ic ot ro pi ca l te rm iti da e m ac ro te rm iti na e o do nt ot er m es sp . 28 00 et hi op ia nr . b eb re bi rh an 9. 68 00 39 .5 30 0 co w ie e t al . 1 99 0 7 el ev a fr ic ot ro pi ca l te rm iti da e n as uti te rm iti na e tr in er vi te rm es sp . 16 50 et hi op ia a da m i t ul lu 7. 86 38 .7 07 d eb el o & d eg ag a 20 14 8 el ev a fr ic ot ro pi ca l te rm iti da e te rm iti na e o do nt ot er m es ap ol lo s jö st ed t 26 00 ke ny a eb ur ru -0 .6 50 0 36 .2 70 0 sj ös te dt 1 90 5 9 el ev a us tr al ia n st ol ot er m iti da e po ro te rm es ad am so ni f ro gg att 12 98 a us tr al ia , a ct br in da be lla ra ng e nr ca nb er ra -3 4. 22 80 14 8. 98 80 la ce y et a l. 20 10 10 la t a us tr al ia n st ol ot er m iti da e st ol ot er m es ru fic ep s br au er 10 0 n ew z ea la nd so ut h is la nd -4 6. 00 00 16 9. 00 00 ba in & je nk in 1 98 3 9 el ev a us tr al ia n st ol ot er m iti da e st ol ot er m es vi to ri en si s h ill 12 98 a us tr al ia , a ct br in da be lla ra ng e nr ca nb er ra -3 5. 38 00 14 8. 80 00 la ce y et a l. 20 10 4 11 el ev in do m al ay an a rc ho te rm op si da e a rc ho te rm op si s w ro ug ht on i d es ne ux 27 43 in di a d eo ba n 30 .7 5 77 .8 5 im m s 19 20 12 el ev 3 in do m al ay an a rc ho te rm op si da e a rc ho te rm op si s w ro ug ht on i 27 00 in di a g ul m ar g 34 .0 30 0 74 .9 50 0 ro on w al & c hh ota ni 1 98 9 13 el ev in do m al ay an rh in ot er m iti da e pa rr hi no te rm es bu tt el -r ee pe ni h ol m gr en 14 00 in do ne si a su m at ra , ke m ir i 3. 82 30 97 .5 18 0 g at ho rn eh ar dy e t al . 2 00 1 13 el ev in do m al ay an rh in ot er m iti da e pa rr hi no te rm es sp . 14 00 in do ne si a su m at ra , ke m ir i 3. 82 30 97 .5 18 0 g at ho rn eh ar dy e t al . 2 00 1 14 el ev in do m al ay an te rm iti da e m ac ro te rm iti na e o do nt ot er m es di st an s h ol m gr en & h ol m gr en 22 50 in di a ku m ao n h ill s 29 .4 10 0 79 .5 50 0 th ak ur 1 98 1 15 w et in do m al ay an te rm iti da e m ac ro te rm iti na e o do nt ot er m es ka pu ri r oo nw al & ch ho ta ni 13 11 in di a ch er ra pu nj i 25 .3 00 0 91 .7 00 0 ro on w al & ch ho ta ni 1 96 2 13 el ev in do m al ay an te rm iti da e n as uti te rm iti na e bu lb ite rm es co ns tr ic to id es (h ol m gr en ) 14 00 in do ne si a su m at ra , ke m ir i 3. 82 30 97 .5 18 0 g at ho rn eh ar dy e t al . 2 00 1 16 el ev in do m al ay an te rm iti da e n as uti te rm iti na e bu lb ite rm es sp . 18 60 m al ay si a g un un g m ul u, sa ra w ak 4. 05 00 11 4. 90 00 co lli ns 1 98 0 a pp en di x. e le va tio na l, la tit ud in al , t he rm al a nd p re ci pi ta tio na l g eo gr ap hi c lim its fo r t er m ite s. rh scheffrahn et al. – global elevational, latitudinal, and climatic limits for termites436 m ap no . ca te go ry re gi on 1 fa m ily su bf am ily g en us sp ec ie s m et er s co un tr y, pr ov in ce lo ca lit y la ti tu de lo ng it ud e re fe re nc e 13 el ev in do m al ay an te rm iti da e n as uti te rm iti na e lo ng ip ed ite rm es ki st ne ri a kh ta r an d a hm ad 14 00 in do ne si a su m at ra , ke m ir i 3. 82 30 97 .5 18 0 g at ho rn eh ar dy et a l. 20 01 15 w et in do m al ay an te rm iti da e n as uti te rm iti na e n as uti te rm es ch er ra en si s ro on w al an d ch ho ta ni 13 11 in di a ch er ra pu nj i 25 .3 00 0 91 .7 00 0 ro on w al & ch ho ta ni 1 96 2 15 w et in do m al ay an te rm iti da e te rm iti na e pe ri ca pr ite rm es du rg a ro on w al & ch ho ta ni 13 11 in di a ch er ra pu nj i 25 .3 00 0 91 .7 00 0 ro on w al & ch ho ta ni 1 96 2 15 w et in do m al ay an te rm iti da e te rm iti na e ps eu do ca pr ite rm es tik ad ar r oo nw al & ch ho ta ni 13 11 in di a ch er ra pu nj i 25 .3 00 0 91 .7 00 0 ro on w al & ch ho ta ni 1 96 2 18 co ld n ea rc tic a rc ho te rm op si da e zo ot er m op si s an gu sti co lli s h ag en 11 80 id ah o n ew m ea dow s 44 .9 69 0 -1 16 .2 84 0 cu rr en t pa pe r 19 el ev n ea rc tic a rc ho te rm op si da e zo ot er m op si s an gu sti co lli s 21 24 u .s ., ca lif or ni a si er ra n ev ad a m ts . 38 .3 00 0 -1 19 .8 00 0 w ee sn er 1 96 5 17 la t n ea rc tic a rc ho te rm op si da e zo ot er m op si s an gu sti co lli s 20 ca na da pr in ce r up er t 54 .3 00 0 -1 30 .3 00 0 v ic ke ry & k ev an 19 85 20 el ev n ea rc tic a rc ho te rm op si da e zo ot er m op si s la tic ep s ba nk s 21 20 u .s ., a ri zo na ro se c an yo n la ke 32 .3 90 0 -1 10 .7 10 0 th or ne e t al . 1 99 3 21 el ev n ea rc tic ka lo te rm iti da e in ci si te rm es m in or (h ag en ) 17 72 u .s ., te xa s d av is m ts . 30 .6 96 0 -1 04 .0 80 0 cu rr en t pa pe r 23 el ev n ea rc tic ka lo te rm iti da e m ar gi ni te rm es hu bb ar di (b an ks ) 12 22 u .s ., a ri zo na pa te go ni a 31 .5 37 0 -1 10 .7 59 0 cu rr en t pa pe r 22 ho t n ea rc tic ka lo te rm iti da e m ar gi ni te rm es hu bb ar di -5 0 ca lif or ni a im pe ri al 33 .0 45 1 -1 15 .5 00 3 cu rr en t pa pe r 24 ho t n ea rc tic rh in ot er m iti da e g na th am ite rm es pe rp le xu s (b an ks ) 20 9 a ri zo na la ke h av as u ci ty 34 .5 54 4 -1 14 .3 57 8 cu rr en t pa pe r 24 ho t n ea rc tic rh in ot er m iti da e h et er ot er m es au re us (s ny de r) 20 9 a ri zo na la ke h av as u ci ty 34 .5 54 4 -1 14 .3 57 8 cu rr en t pa pe r 25 el ev n ea rc tic rh in ot er m iti da e re tic ul ite re m es tib ia lis (b an ks ) 21 33 u .s . c ol or ad o “n or th er n” 40 .7 00 0 -1 05 .0 00 0 w ee sn er 1 96 5 26 el ev n ea rc tic rh in ot er m iti da e re tic ul ite rm es fla vi pe s ko lla r 21 34 u .s ., a ri zo na pi ne ry c an yo n ro ad 31 .9 32 0 -1 09 .2 71 0 cu rr en t pa pe r 27 el ev n ea rc tic rh in ot er m iti da e re tic ul ite rm es he sp er us b an ks 17 19 u .s ., ca lif or ni a sa n be rn ar di no m ts . 34 .2 34 0 -1 17 .1 87 0 ba nk s an d sn yd er 19 20 28 co ld n ea rc tic rh in ot er m iti da e re tic ul ite rm es sp . 12 00 ca na da ch ur n cr ee k pa rk 51 .3 00 0 -1 22 .3 00 0 h ig gi ns p er s. c om m . 29 co ld n ea rc tic rh in ot er m iti da e re tic ul ite rm es tib ia lis 88 0 n . d ak ot a a m id on 46 .4 80 0 -1 03 .3 20 0 em er so n 19 36 26 el ev n ea rc tic te rm iti da e n as uti te rm iti na e te nu ir os tr ite rm es te nu ir os tr is (d es ne ux ) 17 65 u .s ., a ri zo na s pa ra di se 31 .8 66 9 -1 09 .2 16 8 cu rr en t pa pe r 21 el ev n ea rc tic te rm iti da e te rm iti na e a m ite rm es nr . c al ifo rn ic us ba nk s 17 27 u .s ., te xa s d av is m ts . 30 .7 47 0 -1 04 .1 62 0 cu rr en t pa pe r 21 el ev n ea rc tic te rm iti da e te rm iti na e g na th am ite rm es nr . p er pl ex us 17 27 u .s ., te xa s d av is m ts . 30 .7 47 0 -1 04 .1 62 0 cu rr en t pa pe r a pp en di x. e le va tio na l, la tit ud in al , t he rm al a nd p re ci pi ta tio na l g eo gr ap hi c lim its fo r t er m ite s (c on tin ua tio n) . sociobiology 62(3): 426-438 (september, 2015) 437 m ap no . ca te go ry re gi on 1 fa m ily su bf am ily g en us sp ec ie s m et er s co un tr y, pr ov in ce lo ca lit y la ti tu de lo ng it ud e re fe re nc e 30 el ev n eo tr op ic al ka lo te rm iti da e co m at er m es pe rf ec tu s kr is hn a 16 46 bo liv ia es pi a -1 6. 50 00 -6 7. 30 00 sn yd er 1 92 6 31 dr y n eo tr op ic al ka lo te rm iti da e cr yp to te rm es br ev is (w al ke r) 49 ch ile ca ld er a -2 7. 06 95 -7 0. 81 93 sc he ff ra hn e t al . 20 09 32 el ev n eo tr op ic al ka lo te rm iti da e cr yp to te rm es br ev is 26 00 co lo m bi a bo go ta (e xo tic ) 4. 60 00 -7 4. 10 00 sc he ff ra hn e t al . 20 09 33 el ev n eo tr op ic al ka lo te rm iti da e g ly pt ot er m es lib er at us (s ny de r) 12 39 ja m ai ca h ol le yw el l p ar k 18 .0 86 0 -7 6. 72 60 cu rr en t pa pe r 34 el ev n eo tr op ic al ka lo te rm iti da e g ly pt ot er m es n. s p. 16 68 g ua te m al a bi ot op o q ue tz al 15 .2 13 0 -9 0. 21 70 cu rr en t pa pe r 35 el ev n eo tr op ic al ka lo te rm iti da e in ci si te rm es m ar gi ni pe nn is ( la tr ei lle ) 15 24 m ex ic o la v en ta 20 .7 00 0 -1 03 .4 00 0 li gh t 19 33 37 el ev n eo tr op ic al ka lo te rm iti da e in ci si te rm es ni sh im ur ai s ch ef fr ah n 16 58 h on du ra s p. n . l a ti gr e 14 .2 20 0 -8 7. 08 30 sc he ff ra hn 2 01 4 34 el ev n eo tr op ic al ka lo te rm iti da e m ar gi ni te rm es ca cti ph ag us m yl es 16 53 g ua te m al a m at an za s 15 .1 16 0 -9 0. 17 50 cu rr en t pa pe r 38 el ev n eo tr op ic al ka lo te rm iti da e n eo te rm es n. s p. 18 31 ve ne zu el a p. n . y ac am bu 9. 70 70 -6 9. 65 10 cu rr en t pa pe r 39 dr y n eo tr op ic al ka lo te rm iti da e n eo te rm es nr . c hi le ns is (b la nc ha rd ) 36 pe ru ch ac ra y m ar -1 1. 60 80 -7 7. 23 94 cu rr en t pa pe r 36 el ev n eo tr op ic al ka lo te rm iti da e pr on eo te rm es pe re zi (h ol m gr en ) 12 77 g ua te m al a e cu ila pa 14 .2 54 0 -9 0. 12 60 cu rr en t pa pe r 40 el ev la ti n eo tr op ic al ka lo te rm iti da e ru gi te rm es la tic ol lis s ny de r 27 00 bo liv ia lu ri ba y -1 7. 06 19 -6 7. 66 08 cu rr en t pa pe r 41 el ev la ti n eo tr op ic al ka lo te rm iti da e ru gi te rm es la tic ol lis 36 00 bo liv ia la p az -1 6. 50 00 -6 8. 20 00 sn yd er 1 95 7 42 el ev la ti n eo tr op ic al ka lo te rm iti da e ru gi te rm es la tic ol lis 28 00 ec ua do r q ui to -0 .1 88 8 -7 8. 48 56 cu rr en t pa pe r 37 el ev n eo tr op ic al rh in ot er m iti da e co pt ot er m es te st ac eu s (l in na eu s) 16 07 h on du ra s za m or an o 14 .0 36 0 -8 7. 07 50 cu rr en t pa pe r 36 el ev n eo tr op ic al rh in ot er m iti da e h et er ot er m es co nv ex in ot at us (s ny de r) 15 33 g ua te m al a w s an je ro ni m o 15 .0 69 0 -9 0. 26 80 cu rr en t pa pe r 37 el ev n eo tr op ic al rh in ot er m iti da e pr or hi no te rm es si m pl ex ( h ag en ) 99 1 h on du ra s a m ar at ec a 14 .2 25 0 -8 7. 37 70 cu rr en t pa pe r 43 la t n eo tr op ic al st ol ot er m iti da e po ro te rm es qu ad ri co lli s (r am bu r) 13 0 ch ile re g. m ag da lle na -4 8. 90 00 -7 3. 90 00 co ns ta nti no 19 98 35 el ev n eo tr op ic al te rm iti da e a pi co te rm iti na e a no pl ot er m es ca . f um os us 15 24 m ex ic o la v en ta 20 .7 00 0 -1 03 .4 00 0 li gh t 19 33 34 el ev n eo tr op ic al te rm iti da e a pi co te rm iti na e a no pl ot er m es n. s p. 14 08 g ua te m al a a lta v er ap az 15 .5 67 0 -9 0. 14 30 cu rr en t pa pe r 44 el ev n eo tr op ic al te rm iti da e a pi co te rm iti na e a no pl ot er m es tu rr ic ol a s ilv es tr i 19 20 bo liv ia el f or tin -1 8. 17 87 -6 3. 82 14 cu rr en t pa pe r 38 el ev n eo tr op ic al te rm iti da e a pi co te rm iti na e ru pti te rm es si lv es tr ii (e m er so n) 12 20 ve ne zu el a sa na re a re a 9. 79 01 -6 9. 64 20 cu rr en t pa pe r 37 el ev n eo tr op ic al te rm iti da e n as uti te rm iti na e n as uti te rm es co rn ig er ( m ot sc hu ls ky ) 11 25 h on du ra s se s an ju an ci to 14 .2 29 0 -8 7. 05 01 cu rr en t pa pe r 34 el ev n eo tr op ic al te rm iti da e n as uti te rm iti na e n as uti te rm es ep hr at ae (h ol m gr en ) 14 08 g ua te m al a a lta v er ap az 15 .5 67 0 -9 0. 14 30 cu rr en t pa pe r 45 el ev n eo tr op ic al te rm iti da e n as uti te rm iti na e n as uti te rm es gu ay an ae (h ol m gr en ) 15 00 pe ru sa n pe dr o cl ou d fo re st -1 3. 04 90 -7 1. 53 70 pa lin e t al . 2 01 1 a pp en di x. e le va tio na l, la tit ud in al , t he rm al a nd p re ci pi ta tio na l g eo gr ap hi c lim its fo r t er m ite s (c on tin ua tio n) . rh scheffrahn et al. – global elevational, latitudinal, and climatic limits for termites438 m ap no . ca te go ry re gi on 1 fa m ily su bf am ily g en us sp ec ie s m et er s co un tr y, pr ov in ce lo ca lit y la ti tu de lo ng it ud e re fe re nc e 45 el ev n eo tr op ic al te rm iti da e n as uti te rm iti na e n as uti te rm es oc to pi lis b an ks 15 00 pe ru sa n pe dr o cl ou d fo re st -1 3. 04 90 -7 1. 53 70 pa lin e t al . 2 01 1 44 el ev n eo tr op ic al te rm iti da e sy nt er m iti na e pr oc or ni te rm es le sp es ii (m ue lle r) 14 02 bo liv ia p. n . l os v ol ca ne s -1 8. 11 96 8 -6 3. 60 88 1 cu rr en t pa pe r 46 dr y n eo tr op ic al te rm iti da e te rm iti na e a m ite rm es lu na e sc he ff ra hn 57 pe ru h ua ca d e la l un a -8 .1 35 0 -7 8. 99 10 sc he ff ra hn & h uc he t 20 10 36 el ev n eo tr op ic al te rm iti da e te rm iti na e m ic ro ce ro te rm es se pt en tr io na lis l ig ht 12 77 g ua te m al a e cu ila pa 14 .2 54 0 -9 0. 12 60 cu rr en t pa pe r 47 la t n eo tr op ic al te rm iti da e te rm iti na e o nk ot er m es br ev ic or ni ge r (s ilve st ri ) 10 a rg en tin a nr . r aw so n -4 3. 30 00 -6 5. 10 00 co ns ta nti no 19 98 38 el ev n eo tr op ic al te rm iti da e te rm iti na e te rm es fa ta lis l in na eu s 12 20 ve ne zu el a sa na re a re a 9. 79 01 -6 9. 64 20 cu rr en t pa pe r 48 el ev o ce an ia n te rm iti da e te rm iti na e pe ri ca pr ite rm es c f. sc hu lt ze i (h ol m gr en ) 16 50 pa pu a n ew g ui ne a ea st er n h ig hla nd s, a si ra ng ka -6 .3 00 0 14 5. 90 00 bo ur gu ig no n et al . 2 00 8 49 el ev pa le ar cti c h od ot er m iti da e a na ca nt ho te rm es se pt en tr io na lis 20 00 a fg ha ni st an jij a 33 .7 00 0 64 .2 00 0 w ei dn er 1 96 0 50 ho t pa le ar cti c rh in ot er m iti da e ps am m ot er m es hy bo st om a 67 0 li by a g ha t 24 .9 60 0 10 .1 80 0 h ar ri s 19 66 51 la t pa le ar cti c rh in ot er m iti da e re tic ul ite rm es gr as se i c lé m en t 85 fr an ce ch ar en te 46 .0 00 0 -0 .0 20 0 a us tin 2 00 2 52 la t pa le ar cti c rh in ot er m iti da e re tic ul ite rm es sp er at us ( ko lb e) 13 0 ja pa n h ok ka id o 45 .0 00 0 14 2. 00 00 m or i e t al . 2 00 2 53 el ev pa le ar cti c te rm iti da e te rm iti na e m ic ro ce ro te rm es ga br ie lis w ei dn er 18 50 a fg ha ni st an g ol ba gh (k ab ul ) 34 .4 00 0 69 .1 00 0 w ei dn er 1 96 0 1 as d efi ne d by u dv ar dy (1 97 5) . 2 co w ie e t a l. re po rt 3 20 0 m (s ee te xt ). 3 k un w ar i p as s (n ot k au ri p as s) ” lis te d at 3 90 0 m . th is lo ca lit y co ul d no t b e co nfi rm ed (s ee te xt ). 4 c oo rd in at es o f 3 4. 22 °s , 1 48 .9 8° e gi ve n by l ac ey e t a l. 20 10 in co rr ec t. a pp en di x. e le va tio na l, la tit ud in al , t he rm al a nd p re ci pi ta tio na l g eo gr ap hi c lim its fo r t er m ite s (c on tin ua tio n) . open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.818sociobiology 62(4): 467-473 (december, 2015) effects of environmental factors on life cycle regulation in lasius japonicus santschi (formicidae) introduction in the temperate region, insects commonly undergo winter diapause. even when environmental conditions are suitable for growth and reproduction, insects anticipate the occurrence of unfavorable conditions and enter diapause by responding to specific environmental stimuli, such as short day length or low temperature (lees, 1955; danilevsky, 1961; tauber et al., 1986; danks, 1987; košťál, 2006). in warm temperate regions or at higher latitudes, ants enter winter diapause according to the criteria delineated by kipyatkov (2001). in relatively warm climates, low temperature is the primary environmental stimulus for the induction of diapause, although day length is critical for many other insect orders (kipyatkov, 1993, 2001). ants inhabiting higher latitudes often enter diapause after a certain period of reproduction and development, regardless of environmental stimuli, and even abstract this study investigated environmental factors that regulate oviposition by queens and colony development in lasius japonicus santschi. newly mated queens were collected from fields in okayama, japan. the insects were maintained outdoors or under a long or short-day photoperiod at 30 °c, 25 °c, 20 °c, or 15 ± 1 °c. all queens died within 50 days at 30 °c, whereas all or most of the queens survived at lower temperatures. at 25 °c, many pupae were observed approximately 1 month after the onset of oviposition. diapause in either queens or workers was not induced at 25 °c. at 20 °c, many larvae did not pupate, indicating that larval diapause was induced. at 15 °c, hatching was not recorded and eggs disappeared. low temperatures may induce reproductive diapause in queens. there were no significant differences between long-day (ld 16:8 h) and short-day (ld 12:12 h) conditions at any temperature. under outdoor conditions, when summer temperature was moderate in 2005, queens started their nuptial flights in june, and pupation was recorded three times. however, when summer temperature was high in 2006, pupation occurred 1–2 times before winter, with queens making their nuptial flights as late as mid-july. eggs and pupae disappeared in most colonies before the arrival of winter. only queens, adult workers, and larvae were observed during winter. the present study showed that queens of l. japonicus founded and developed their colonies for as long as environmental conditions remained favorable, by responding to changes in temperature. sociobiology an international journal on social insects s kamitani, k asakura, k nakamura article history edited by kleber del-claro, ufu, brazil received 09 may 2015 initial acceptance 04 july 2015 final acceptance 20 july 2015 keywords colony foundation, colony develop-ment, diapause, photoperiod, temperature. corresponding author keiji nakamura department of biosphere-geosphere system science, faculty of informatics okayama university of science, okayama 700-0005, japan e-mail: nakamura@big.ous.ac.jp when they were kept under stable warm conditions in the laboratory (kipyatkov, 1993, 2001). workers have been shown to influence the regulation of diapause in queens or larvae. for instance, larvae tend to emerge as workers without entering diapause if they are reared by workers that have been exposed to low temperatures. in contrast, larvae tend to enter diapause if they are reared by workers that have passed the winter or experienced low temperature in the laboratory (e.g., brian, 1955; weir, 1959; kipyatkov et al., 1996, 1997; kipyatkov & lopatina, 1999). reproductive workers of the japanese queenless ant, pristomyrmex pungens, lay eggs during the long-day photoperiod, whereas they enter diapause in response to the short-day photoperiod (muramatsu & numata, 2002). in contrast, the second to fifth instar larvae of amblyopone silvestrii wheeler are found year-round, but the eggs, first instar larvae, pupae, and callow adults are mainly found in research article ants okayama university of science, okayama, japan s kamitani, k asakura, k nakamura – life cycle adaptation in lasius japonicus468 summer (masuko, 2003a). for example, cocoons were found from july to october, with the greatest numbers occurring in august (masuko, 2003a). however, detailed experimental studies on the seasonal adaptations of japanese ants have not been conducted in other species. claustral colony founding is common in many ant species. this strategy involves a newly mated queen starting a colony without any help from workers by using nutrient reserves (hölldobler & wilson, 1990). after the workers emerge, the development of the colony is highly dependent on the social conditions of the nest, such as the size and structure of the population and the age of workers (e.g., brian, 1953, 1957a, 1957b; schneirla, 1957; tschinkel, 1988; børgesen & jensen, 1995; masuko, 2003b). although social effects influence the development of a colony, such effects might also hinder direct investigations of how environmental factors affect the physiological status of queens. lasius japonicus santschi is found in the japan islands, korean peninsula, and taiwan (jadg, 2003). it was previously assumed to be lasius niger (linnaeus), which is widely distributed in the palearctic region, but was formally identified as l. japonicus by seifert (1992). oviposition of l. niger queens collected from russia is not affected by photoperiod, but is strongly controlled by temperature. the queens stop ovipositing soon after being transferred to a temperature of 20 °c or less (kipyatkov, 2001). induction and termination of diapause are partly affected by the physiological state of workers. specifically, workers in the spring physiological state influence larval diapause when exposed to low temperatures (kipyatkov et al., 1996). furthermore, as in other ants found at high latitudes, diapause induction is obligatory at the colony level. specifically, colony development ceases within a certain period after the onset of oviposition by queens, even at optimal temperatures (kipyatkov, 2001). these seasonal regulations of queen oviposition and colony development are adaptive for high latitudinal regions, where the seasons that are favorable for growth and reproduction are short. however, in southwestern japan, the climate is moderate; thus, insects are able to develop and reproduce for a longer period. therefore, we hypothesized that l. japonicus has evolved physiological adaptations to the warm climate, and will exhibit more intensive colony development than l. niger. in the present study, we collected l. japonicus santschi queens immediately after their nuptial flight, and maintained them under various photoperiod and temperature combinations to determine the effect of environmental factors on colony founding. the results of this study are expected to provide novel insights about the life cycle adaptations of this species under warm temperate climate. materials and methods within 3 hours of their nuptial flight, more than 300 queens of l. japonicus were collected from okayama city (34.7°n, 133.9°e), japan, between late june and early august in 2005 and 2006. queens that shed their wings were separately reared in plastic cases (70 × 40 mm, 20 mm in depth). the bottom of the cases was covered by plaster, and water was provided ad libitum. the insects were maintained outdoors or under a longor short-day photoperiod at 30 °c, 25 °c, 20 °c, or 15 ± 1 °c. we used a long-day light:dark (ld) photoperiod of 16:8 h, which is slightly longer than the day length at the summer solstice in okayama, and is also the length often used to examine the life cycle of insects in southwestern japan (e.g., musolin & numata, 2003; shintani & numata, 2010). a short-day photoperiod of ld 12:12 h was also used. this photoperiod was equivalent to the day length slightly after the autumnal equinox, including the twilight portion of the day. because l. japonicus exhibits claustral colony founding, food was not provided until the first worker, or minim, emerged as an adult. after the workers emerged, dried bloodworm and artificial diet for adult beetles (fujikon co. ltd., nose, osaka, japan) were supplied as food twice a week. in this study, 21 queens were reared individually in 21 cases for each experimental condition (168 queens in total). mortality and oviposition of queens were recorded daily until day 100 and 180 at 25 °c and 20 °c, respectively. we recorded the pre-oviposition period and the daily number of pupae to assess colony development in this study. because eggs and small larvae were attached together by queens, we did not record the number of them. in addition, we did not record the number of fast-moving workers. experiments in the laboratory were discontinued at day 100 and 180 at 25 °c and 20 °c, respectively, which was when counting the number of pupae became too difficult. queens at 15 °c were transferred to either a longor short-day photoperiod at 25 °c at 100 days after collection, when they were considered to be in diapause. after the transfer to 25 °c, queens were kept another 100 days, at which point the experiments were discontinued. some queens were reared under natural conditions. specifically, plastic cases were placed in cages that could not be reached by direct sunlight on the campus of okayama university of science. the daily average temperature data recorded at the meteorological station closest to the university (about 3 km south of the university) was obtained from the japan meteorological association (http://www.data.jma. go.jp/obd/stats/etrn/index.php). in february 2006, 5 queens maintained under outdoor conditions were dissected to determine whether they were in diapause, by assessing the development of ovarioles under a stereoscopic microscope. results all queens died without producing workers when reared at 30 °c under ld 16:8 h. the median longevity was 33 days (range, 23-45). however, at lower temperatures, queens survived for 100 days, both under ld 16:8 h and 12:12 h (table 1). at 25 °c, 5 queens died within a few days after collection under the long-day photoperiod. sociobiology 62(4): 467-473 (december, 2015) 469 however, all the other 16 queens started laying eggs about 3 days after collection. under ld 12:12 h, all 21 queens started laying eggs soon after collection. also at 20 and 15 °c, all queens started oviposition under any experimental conditions, indicating that they have mature eggs at nuptial flight. under the long-day photoperiod, 6 queens which had started oviposition died before the emergence of pupae, whereas no queen died under the short-day photoperiod. pupation was recorded approximately 25 days after the beginning the experiment at 25 °c (table 1, fig 1a, b). there was no significant difference in the average pre-pupation period (from the collection of queens to the appearance of pupae) between longand short-day conditions (p > 0.05, t-test). average numbers of pupae increased sharply, and peaked at about day 40. average number (± s.d.) of pupae was 23.2 ± 2.0 (n = 10) and 22.5 ± 7.5 (n = 21) on day 40 under ld 16:8 h and 12:12 h, respectively. there was no significant difference in the number of pupae between longand short-day conditions (p > 0.05, t-test). subsequently, the number of pupae decreased and adult workers emerged. when workers of the first batch started emerging, many pupae and a few well-grown larvae, but not small larvae, were found in the colonies. second pupation initiated at around day 50, and the number of pupae increased until approximately day 80. the second peak in pupation was less clear than the first peak, because of less synchronization among colonies. at 20 °c, queens started laying eggs approximately 5 days after collection (table 1). there was no significant difference in the pre-oviposition period between the 2 photoperiods (p > 0.05, t-test). eggs hatched at about 30 days after the collection of queens. although all queens, except one under ld 16:8 h, survived 180 days, pupae did not emerge in 1 and 4 colonies under ld 16:8 h and ld 12:12 h, respectively. pupation started at about 50 days after the collection of queens. there was no significant difference in the average pre-pupation period between longand short-day conditions (p > 0.05, t-test). pupation peaks were observed twice on days 40-80 and days 100-150, regardless of the photoperiod (fig 2c, d). at day 60, the average number (± s.d.) of pupae was 7.3 ± 7.9 (n = 19) and 4.2 ± 4.0 (n = 17) days under ld 16:8 h and 12:12 h, respectively. there was no significant difference in the average number of pupae at day 60 between the 2 photoperiods (p > 0.05, t-test). in addition, larvae of different instars were always present until the end of the experimental period, even though their sizes and numbers were not recorded. at 15 °c, all queens started oviposition under both ld 16:8 h and 12:12 h, respectively, but hatching was not observed. thereafter, the eggs disappeared until day 100, even though most queens were still alive (table 1). because no dead or rotted eggs were found in the container, it was assumed that they had been eaten by the queens. the queens resumed egg laying 8.5-9.2 days on average after being transferred to 25 °c at day 100 (table 2). there was no significant difference in the period from the transfer to the resumption of oviposition table 1. effects of temperature and photoperiod on queen survival and colony development in lasius japonicus. number of queens starting to oviposit is shown in parentheses after the average pre-oviposition period. number of colonies in which pupae emerged is shown in parentheses after the average pre-pupal period. fig 1. effects of photoperiod and temperature on the mean number of lasius japonicus pupae. n = 16–21. temperature photoperiod initial number of colonies percentage of queen survival at day 100 average (± s.d.) preoviposition period (days) average (± s.d.) prepupal period (days) 25 oc ld 16:8 h 21 47.6 3.4 ± 1.5 (16) 25.0 ± 1.0 (10) ld 12:12 h 21 100 3.3 ± 1.0 (21) 24.0 ± 2.0 (21) 20 oc ld 16:8 h 21 95.2 5.3 ± 4.0 (21) 48.9 ± 11.9 (19) ld 12:12 h 21 100 5.0 ± 3.6 (21) 49.3 ± 13.8 (17) 15 oc ld 16:8 h 21 85.7 5.0 ± 1.6 (21) ld 12:12 h 21 100 5.8 ± 3.6 (21) s kamitani, k asakura, k nakamura – life cycle adaptation in lasius japonicus470 among experimental treatments (two-way anova, p > 0.05; table 3). larvae hatched from these eggs. although some queens died after the transfer to 25 °c, pupation was observed in 3-8 colonies. the periods from the transfer of queens to the onset of pupation were 29.8-36.9 days in the 4 experimental treatments, which was 5-12 days longer than the periods for colonies transferred directly from the field to 25 °c (see table. 1). under natural conditions, the rearing experiment was started in late june, 2005. summer temperatures were not very high, with a monthly average of 25.8 °c and 26.7 °c in july and august, respectively (fig 2). all 21 queens laid eggs soon after collection, and pupation initiated in late july (fig 3). table 3. summary of two-way anova testing for the effects of photoperiods before and after transfer from 15 to 25 °c on the pre-oviposition period in lasius japonicus. fig 3. mean number of lasius japonicus pupae under quasi-natural conditions. arrows indicate the timing of the nuptial flight of queens. n = 21 in 2005 and 17 in 2006. table 2. effects of photoperiod on colony development after temperature rising in lasius japonicus. number of queens starting to oviposit is shown in parentheses after the average pre-oviposition period. number of colonies in which pupae emerged is shown in parentheses after the average pre-pupal period. the greatest number of first-brood pupae occurred in early august. secondand third-brood larvae pupated in september and october, respectively. approximately half of the colonies had produced 3 broods by the end of the growing season, while the remaining colonies produced 2 broods. the number of pupae in the second brood was similar to that in the first, but was higher than that in the third brood. the third pupation peak was unclear in the figure because of less synchronization among colonies. in winter, the pupae disappeared in most colonies. the queens, workers, and the larvae of several instars were found in the colonies. in the end of january 2006 when rearing experiment was discontinued, 19 of 21 queens were still alive. some of them were dissected in february, but no mature eggs were found in the ovaries. in 2006, when spring temperature was less than that in 2005 (see fig 2), the nuptial flight started in mid-july. however, the 2006 summer temperatures were higher (about 2-3 °c difference) than those of 2005. four queens died within a few days of the collection, and 17 queens started oviposition under outdoor conditions. pupation of the first-brood larvae occurred in august (fig 3). however, in september or later, considerably fewer pupae were produced in 2006 compared to 2005, with no production of second-brood pupae occurring in approximately half of the colonies in 2006. fig 2. change in 5-day mean temperature under quasi natural conditions from april to december in 2005 and 2006 (japan meteorological agency, 2015). source of variation sum of squares d.f. ms f p photoperiod before transfer 75.383 1 75.38 1.817 0.191 photoperiod after transfer 63.965 1 63.97 1.541 0.228 interaction 18.334 1 18.33 0.442 0.513 error 912.899 22 41.50 　 　 rearing conditions before transfer rearing conditions after transfer initial number of colonies average (± s.d.) pre-oviposition period (days) average (± s.d.) pre-pupal period (days) 15 oc, ld 16:8 h 25 oc, ld 16:8 h 9 9.0 (9) 34.7 ± 5.8 (3) 25 oc, ld 12:12 h 9 9.2 ± 0.7 (9) 29.8 ± 8.3 (8) 15 oc, ld 12:12 h 25 oc, ld 16:8 h 11 8.5 ± 1.5 (11) 36.9 ± 4.1 (7) 25 oc, ld 12:12 h 10 9.6 ± 1.0 (10) 31.6 ± 6.1 (8) sociobiology 62(4): 467-473 (december, 2015) 471 discussion queen mortality and colony development of l. japonicus were highly dependent on temperature. all queens died without producing workers at 30 °c. yet, during summer, the daily mean temperature often exceeds 30 °c in okayama (japan meteorological agency, 2015). thus, l. japonicus nests are usually constructed underground (jadg, 2003), where changes in temperature are more moderate than those on the ground surface. however, a solitary queen may not construct a nest deep underground soon after mating. the broods of solenopsis invicta buren develop faster in sunny, warmer sites; yet, total production and queen survival are lower at sunny sites compared to shady sites (tschinkel, 1993). our results confirm the importance of environmental temperature during colony foundation. at 25 °c, the number of pupae noticeably increased at 30–40 days after oviposition (fig 1a, b). synchronized pupation indicates that l. japonicus larvae from okayama do not undergo diapause at this temperature. moderate temperature is considered to be suitable for colony development. the number of pupae decreased once, but increased again on days 60-70, approximately 30 days after the pupation of the first brood. the interval between the 2 successive pupation peaks indicates that queens started laying the eggs of the second brood when pupation of the first brood had initiated. similar cyclic egg-laying is also exhibited in other ant species, such as myrmica rubra linnaeus (brian, 1951, 1957b), eciton hamatum fabricius, and eciton burchelli westwood (schneirla, 1957). the reproductive activity of queens in the army ants, e. hamatum and e. burchelli, is highly dependent on the cycle of colony development. specifically, when larvae reach maturity, workers feed both the larvae and the queen, which results in cyclic ovarian maturation (schneirla, 1957). in the present study, second oviposition began before the emergence of workers. therefore, in l. japonicus, egg-laying of the second brood may initiate without feeding by workers. in a. silvestrii, oophagy by the firstand second-instar larvae results in cyclic egg production. in this species, the number of eggs increases when the larvae molt into the third instar (masuko, 2003b). although we found egg cannibalism in l. japonicus, its impact on the egg population has not been evaluated. further studies might be necessary to elucidate the mechanism of cyclic oviposition. the number of l. japonicus pupae at 20 °c was considerably lower than that at 25 °c (fig 2c, d). furthermore, different instar larvae were always present in most of the colonies throughout the experiments. the suppression of pupation indicates that many larvae were in diapause. among warm temperate ant species, larval diapause is often induced by low temperature (kipyatkov, 2001). our results indicate that l. japonicus larval diapause is regulated by temperature, as in other ant species. at 15 °c, eggs disappeared from the colony without hatching, although most of the queens survived for 100 days of the experimental period (fig 1). thus, this temperature might be too low to allow the completion of regular embryonic development. the queens needed a certain period of time to resume oviposition after they were transferred from 15 °c to 25 °c. both the pre-oviposition and pre-pupation periods were longer than those in colonies where queens were directly transferred from the field to constant 25 °c. this delay indicates that ovarian development was arrested at 15 °c. furthermore, queens that were reared outdoors had no mature eggs in their ovaries during winter. these results indicate that low temperatures induce queens to enter reproductive diapause. in l. niger from russia, colony development is known to be suppressed by low temperatures. in this species, queens stop ovipositing and enter diapause soon after they are transferred to 20 °c or 17 °c under both longand shortday photoperiods (kipyatkov, 1993). however, l. japonicus colonies continuously developed during the 180 days of experiments at 20 °c, indicating that the queens of this species are less sensitive to low temperature than those of l. niger. therefore, l. japonicus has become adapted to the warm climate of okayama, because queens are able to develop the colony for a longer period. in many ants of temperate regions, photoperiod does not influence the induction of diapause, even though it is the most important environmental stimulus in many other insect orders (kipyatkov, 1993, 2001). the queens and larvae of l. niger from russia do not show a photoperiodic response for the induction of diapause (kipyatkov, 1993). our findings confirm that photoperiod has a limited effect on the induction of diapause, even in l. japonicus. in some ant species, larvae and queens that were reared by workers without exposure to low temperature tended to enter diapause (e.g., brian, 1955; weir, 1959; kipyatkov et al., 1996, 1997; kipyatkov & lopatina, 1999). in the present study, worker of the first brood emerged in the experimental treatments at 25 °c and 20 °c. however, there was no clear difference in the number of pupae between the first and second broods (fig 1). even though the average number of pupae tended to be greater in the first brood compared to the second brood, this difference was considered to be due to the less synchronization among colonies. thus, workers do not induce diapause in worker larvae and queens, even if they are not exposed to low temperatures. this phenomenon is considered to be an adaptation to warmer climate, in which more than 1 brood may be produced. these responses to environmental factors provide a reasonable explanation for the seasonal development of colonies under natural conditions. queens make the nuptial flight in early summer when the temperature rises to around 25 °c, which is suitable for the rapid development of colonies (fig 2). in 2005, as many as 3 broods were produced before the end of the growing season (fig 3). temperatures in july and august of 2005 were lower and more suitable than those of the following year, allowing the development of second and third broods. in comparison, in 2006, queens produced broods s kamitani, k asakura, k nakamura – life cycle adaptation in lasius japonicus472 once or twice when the nuptial flight occurred in mid-july or later. in august, the number of second-brood pupae was small under high summer temperatures of approximately 30 °c. this near-lethal temperature may suppress the development of colonies. similarly, environmental temperature has been shown to markedly affect colony founding by s. invicta (tschinkel, 1993). in our study, the number of pupae decreased after mid-october, and almost none were found in the winter of both years, indicating that larval diapause is induced by low temperatures. the present study showed that l. japonicus is well adapted to the warm temperate climate of okayama. queens founded and developed their colonies for as long as environmental conditions remained favorable, by responding to changes in 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(1959). the influence of worker age on trophogenic larval dormancy in the ant myrmica. insectes sociaux, 6: 271290. doi: 10.1007/bf02224411. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.908sociobiology 63(1): 705-711 (march, 2016) wasps are better plant-guards than ants in the extrafloral nectaried shrub ouratea spectabilis (ochnaceae) introduction in the brazilian savanna, many plant species are severely attacked by a wide range of herbivorous insects, which feed and inflict damage on virtually every plant part, especially leaves and flowers (price et al., 1995; bächtold et al., 2012; vilela et al., 2014). however, many plants present biotic protection against herbivores, in the form of aggressive and predatory ant-guards, which feed on extrafloral nectaries (efns) and protect the plant against assorted types of insects, especially folivores (koptur et al., 1998; bronstein et al., 2006; alves-silva, 2011; heil, 2015). although many studies provide evidence that ants can suppress herbivore populations and in turn increase plant fitness (vesprini et al., 2003; rosumek et al., 2009; del-claro et al., 2016), some studies point out that ants are not effective against all types of abstract in the cerrado, many plants are patrolled by ants, but in some cases, predatory wasps may also play an important role in herbivore deterrence. here we examined the effects of the wasp brachygastra lecheguana and the ant pseudomyrmex gracilis on the predation of endophytic weevils which develop in the flower buds of ouratea spectabilis trees. we (i) compared frequency and the abundance of weevils preyed upon by wasps; (ii) possible synergic effects of ants and wasps in decreasing weevil population; and (iii) whether wasps preyed upon weevils in more visible, easy-access inflorescences, i. e. those located on the edges of canopy. in contrast to ants, wasps were observed significantly more frequently on o. spectabilis and were responsible for 88% of the weevil larvae preyed upon. plants visited by both b. lecheguana and p. gracilis had a substantial reduction in weevil larvae compared to plants visited by b. lecheguana only. this indicates a complementary effect of predators with distinct skills in deterring the weevil population; however, ants were responsible for only a small portion of weevils preyed upon. weevil larvae occurred evenly in both visible and hidden inflorescences; nonetheless, wasps predominantly visited inflorescences located on the edges of the canopy. in this ecological system, predatory wasps played a substantial part in weevil deterrence and can be considered a better plant-guard than ants. sociobiology an international journal on social insects e alves-silva, k del-claro article history edited by gilberto m. m. santos, uefs, brazil received 09 september 2015 initial acceptance 11 february 2016 final acceptance 01 march 2016 publication date 29 april 2016 keywords anthonomus, brachygastra, ant-plant mutualism, pseudomyrmex, trophic cascade. corresponding author estevão alves-silva rua ceará, s/nº, bloco 2d campus umuarama, cep: 38400-902, uberlândia, minas gerais, brazil e-mail: estevaokienzan@yahoo.com.br herbivores, as some insects occur in structures inaccessible to ants (mody & linsenmair, 2004; nogueira, et al., 2012; alvessilva & del-claro, 2015). furthermore, predator behavior can be affected by plant structural complexity, which confers more hiding places for herbivores (lawton, 1983), or restrains the foraging activity of herbivores’ natural enemies (hopper, 1984; andow & prokrym, 1990). as shown by del-claro et al., (1997) the presence of natural enemies and/or the likelihood of being preyed upon, influence the behavior of herbivores, and they may seek habitats that confer some type of protection and less risk of being encountered by predators (bächtold & alves-silva, 2013). as well as ants, wasps are also important natural enemies of herbivores (pereira & trigo, 2013; bächtold et al., 2014), especially endophytic weevils (torezan-silingardi, 2011). for instance, the social polistinae wasp brachygastra lecheguana latr. (hymenoptera: vespidae) is the main predator research article wasps instituto de biologia, universidade federal de uberlândia, uberlândia, minas gerais, brazil e alves-silva, k del-claro – wasps prey on endophytic beetles and benefit the plant706 of a group of anthonomus species (coleoptera: curculionidae) in malpighiaceae (alves-silva et al., 2013), but this wasp is also able to capture a wide range of other herbivores (see souza & zanuncio, 2012). in general, predatory insects such as wasps and ants are bounded by the seasonal availability of their prey and as such, variations in herbivore prey are expected to directly affect the occurrence of their natural enemies in the field (see mooney & tillberg, 2005; mody et al., 2011). ouratea spectabilis (mart.) engl. (ochnaceae) is an extrafloral nectaried shrub that supports almost 30 ant species; however, studies showed that ants are unable to protect the plant against certain herbivores such as thrips, caterpillars and curculionid beetles whose larvae are endophytic (byk & del-claro, 2010; bächtold et al., 2012). nonetheless, in recent observations, a particular ant species, pseudomyrmex gracilis (fabr.) (formicidae: pseudomyrmicinae), was observed lacerating flower buds and preying on endophytic weevil larvae (e.a-s. personal observation). such behavior is very similar to that of predatory wasps (alves-silva et al., 2013) and may indicate that ants and wasps have a cumulative negative effect on the population of weevils, thus ultimately benefiting the plant. in this study we aimed to investigate (i) the temporal variation of p. gracilis, b. lecheguana and anthonomus in o. spectabilis according to the plant’s flowering season; (ii) the abundance of weevils preyed upon by wasps and ants; (iii) possible synergic effects of ants and wasps in decreasing weevil population; and (iv) the influence of inflorescence location on the abundance of anthonomus and on the prey finding ability of wasps. we hypothesized that wasps and ants would be more abundant following an increase in weevil larvae availability; plants supporting both wasps and ants would present higher reductions in weevil population, but wasps would play a major role in weevil predation; and easy-access inflorescences (i.e. those on the edges of the plant canopy) would be more visited by wasps. material and methods study area fieldwork was carried out in a sensu stricto cerrado area (18°59’ s, 48°18’ w) in uberlândia city, brazil, from august to september 2012 and for the same period of 2013, which corresponds to the flowering period of o. spectabilis. the cerrado covers about 230 ha and is dominated by herbaceous plants; the height of shrubs and trees ranges between 2 and 4 m. the climate is markedly seasonal with two well established seasons: a dry winter (may to september) and a rainy summer (october to april) (réu & del-claro, 2005). study species ouratea spectabilis is a common sclerophylous tree species (2-5 m in height) in the cerrado savanna. its leaves may reach up to 80 mm in length and 50 mm in width and the efns occur at the stipules, close to the leaf base (bächtold et al., 2012). flowers are pentamerous and yellow, and blooming starts in august and lasts until october. flower buds (~0.7 mm in length) are greenish and conical with an acute apex (fig 1a), and are attacked by species of anthonomus beetles (weevils), whose larvae develop inside the flower buds (byk & del-claro, 2010). these beetles are a complex of three species with very similar behavior and natural history (torezan-silingardi, 2011), but their recognition in the field conditions is unpractical, thus we studied anthonomus as a group, for the sake of clarity. wasp predation towards beetles in order to examine the temporal abundance of wasps, ants and beetles in the field, in 2012 we tagged 20 o. spectabilis plants with approximately the same phenological status (i.e. presence of flower buds in abundance). we also took care to select short plants (< 2 m in height) to enhance the observers’acuity. fieldwork was conducted for three weeks, at the peak of flower bud production. in the first week of observation, adult weevils were frequently observed engaged in mating behavior in inflorescences of o. spectabilis, so we considered that the following week was the period in which weevil larvae might be under development inside flower buds. this second week also coincided with the peak of flowering in o. spectabilis. from the third week onwards, the plants started to cease production of flower buds and blooming prevailed, so we ended our fieldwork, as these weevils develop exclusively in flower buds (torezan-silingardi, 2011). during each field trip, we stood carefully in front of each tagged plant for 20 minutes and performed focal observations to register the number of visits of b. lecheguana to the plant and the frequency with which weevil larvae were successfully captured and preyed upon by wasps (adapted from pereira & trigo, 2013). ‘visits’ were considered as whenever a given individual wasp landed on the plant; ‘successful predation’ was the act of capturing and preying upon a weevil larva (alves-silva et al., 2013). we also scanned the plant for p. gracilis individuals, and their predation upon weevils was also registered. all the observations were conducted once a week on sunny days between 08:00h and 13:00h, since in preliminary observations wasps were commonly noted visiting the plants during this period. our total sampling effort together with fieldwork accounted for approximately 65 hours. an experimental factorial procedure, examining ‘antpresent/excluded’ and ‘wasp-present/excluded’ and its variations, is often impractical since the exclusion of wasps from plants is a major issue (pereira & trigo, 2013). so we attained to compare weevil larvae preyed upon in plants where wasps and ants were seen together with plants where only wasps were observed. this might give us a good estimate of the role of wasps alone and wasps+ants in deterring the weevil population in o. spectabilis. sociobiology 63(1): 705-711 (march, 2016) 707 temporal variation in weevil larvae the temporal abundance of weevil larvae in o. spectabilis was examined in a further 10 individual plants in the same weeks of the observations described above. weevil larvae could not be estimated in the tagged o. spectabilis plants, as we might affect the visitations of predatory wasps and ants. from each of the 10 trees we randomly collected 20 flower buds, which were taken to the laboratory and dissected to look for weevil larvae. plant phenology the phenology and intensity of flower bud production was examined in two inflorescences for each individual plant (n = 20 plants). inflorescences were taken from opposite sides of the canopy and we also took care to select structures which had no previous signs of herbivore attack, fungus or other type of damage. the percentage of flower buds per plant was obtained by counting all buds and dividing the total by the number of flowers. therefore, the intensity of bud production is presented in percent values (see alves-silva & del-claro, 2015). wasp visitation and inflorescence location in order to examine whether inflorescence location influenced wasp visitation to o. spectabilis, we randomly chose one pair of inflorescences per plant (n = 10 plants) in 2013. inflorescences were located ~1 m from each other and, whenever possible, located in the same horizontal plane of plant symmetry. each pair consisted of an inflorescence located and/ or growing towards the edge of the plant canopy, and another inflorescence at the core or growing towards the interior of the canopy. the former inflorescences were characterized by constant sun exposure, lack of surrounding leaves and high visibility; while the latter received little or no sunlight, were surrounded by leaves from neighboring branches and were less conspicuous (fig 1a, b). for the sake of clarity, each inflorescence will hereafter be referred to as a “visible inflorescence” or “hidden inflorescence”. wasp visitation to these inflorescences was examined on two non-consecutive days; each plant was examined for 30 minutes between 08:00h and 12:00h, and we recorded the frequency with which weevils in each inflorescence type were captured. at the same period, other pairs of inflorescences with the same characteristics described above were collected from a further 10 individual plants, taken to the laboratory and dissected to investigate the weevil infestation. we intended to examine whether weevil larvae were more abundant in visible or hidden inflorescences and relate it to the visits of wasps. statistical analysis quantitative data are presented as the mean ± se. parametrical statistical tests were given priority whenever data fitted the assumptions of normality (bell-shaped distribution and p> 0.05 in the normality test). otherwise, non-parametrical tests were used instead. the variation of weevils (adult and larvae) and wasps during the study was examined with kruskal-wallis tests. the relationship between the abundance of adult anthonomus and parasitism in flower buds (per plant) was examined with a linear regression test. in this test, we used the mean abundance of flower bud parasitism during the three weeks of the study. the relationship between weevil larvae consumed and wasp visits to o. spectabilis was also examined with a linear regression test. in this test, we used the cumulative abundance of wasp visitations and anthonomus larvae consumed during the study. the difference in the number of weevils captured by wasps and ants was examined with a wilcoxon test. individuals of p. gracilis were not observed on o. spectabilis as frequently as b. lecheguana, so we decided to compare the number of weevil larvae preyed upon in plants where only wasps were observed and in plants where both ants and wasps were registered. this comparison was made with a student’s t test. the visits of wasps to visible and hidden inflorescences, as well as the abundance of weevil larvae in such inflorescences, were compared with g-tests. results the number of adult weevils was higher at the beginning of the study, during the weevil reproductive season (fig 2a), but as the season progressed and flower buds became scarce (fig 2b), the abundance of adult anthonomus gradually decreased (h3 = 37.2098; p< 0.0001). weevil larvae, on the other hand, were much more abundant at the end of the study (h3 = 9.1386; p< 0.05) (fig 2a). the infestation of flower buds was on average 11.25% (n = 45 of the 400 buds examined). a positive and significant relationship between the abundance of adult anthonomus and the infestation of larvae (per plant) was found (f18 = 12.1572; r² = 0.4031; p< 0.01; regression coefficients: constant = 2.1708; slope = 0.7177). fig 1. predator-prey interactions in ouratea spectabilis in a brazilian tropical savanna. (a) a visible inflorescence; (b) hidden inflorescence; (c) brachygastra lecheguana and (d) pseudomyrmex gracilis capturing weevil larvae (arrows); (e) pachycondyla villosa biting an adult weevil. (photo e by alexandra bächtold). scale bars = 10 mm. e alves-silva, k del-claro – wasps prey on endophytic beetles and benefit the plant708 the abundance of b. lecheguana followed the same trend as weevil larvae (fig 2c). for instance, wasp visits to the plants at the end of the study were five times higher than at the beginning of the study (h3 = 19.6225; p< 0.0001). as shown in fig 2a-c, there was an inverse association between the abundance of adult anthonomus and wasps, and a positive increase in wasp visits to the plants following the high levels of weevil larvae on flower buds. p. gracilis was observed only at the end of the study and its abundance was low (n = 6 observations). the relationship between the number of weevil larvae consumed (2.25 ± 0.29; n = 45 larvae preyed upon) and wasp visits to the plants (3.0 ±0.31; n = 60 visits to 20 plants) was positive and statistically significant (f18 = 21.7181; r² = 0.5468; p< 0.001; constant = 0.1667; slope = 0.6944). wasps (fig 1c) landed on inflorescences and wandered on flower buds, very often migrating from buds. antennation on flower buds was commonplace and whenever an endophytic weevil larva was found, the wasp started to lacerate the external layers of the flower bud with its mandibles to reach the larva inside. as soon as the flower bud was opened, the wasp pulled the weevil larvae out with the mandibles and started to eat it immediately (complete behavior described in alves-silva et al., 2013). pseudomyrmex gracilis was also observed walking on o. spectabilis inflorescences and lacerating flower buds to capture anthonomus larvae (fig 1d). however, unlike b. lecheguana, which consumed the larvae on the plant, the ants took the larvae to their nest. the number of weevils captured by p. gracilis (n = 6 larvae preyed upon) was much lower than the abundance of weevils captured by b. lecheguana (n = 45) (wilcoxon test = 7.0648; p< 0.0001). plants that were visited by both b. lecheguana and p. gracilis (n = 6 plants) had a substantial reduction of weevil larvae compared to plants visited by b. lecheguana only (n = 14 plants) (t18= 2.9686; p< 0.01) (fig 3a). in these plants, however, each ant preyed upon only one weevil larvae, while wasps were responsible for the major part of weevils captured (2.83 ± 0.48 individuals). it is also worth mentioning that on one single occasion, in a sporadic observation in the field, an individual of pachycondyla villosa (fabr.) (ponerinae) managed to capture and bite an adult weevil (fig 1e). wasps predominately visited inflorescences located on the edges of the canopy (g = 9.7891, df = 1, p< 0.01). nonetheless, weevil larvae occurred evenly in visible and hidden inflorescences (g = 0.3620, df = 1, p> 0.05) (fig 3b). discussion main findings confirming our main hypothesis, wasps and ants reduced the infestation of endophytic weevils in o. spectabilis, so both predators can be considered as important biotic defenses for the plant. brachygastra lecheguana was the main predator of anthonomus larvae, while p. gracilis was less abundant and was responsible for 12% of the total weevil larvae preyed upon. moreover, weevil’s abundance was bounded by plant reproductive phenology, which ultimately affected the whole ecological system (trophic cascade), because as bud production ceased, so did weevil reproduction and wasps, which fed on weevil’s larvae. weevil infestation weevil infestation in o. spectabilis (11.25%) was lower compared to another cerrado plant, banisteriopsis malifolia (20.7%) (alves-silva et al., 2013), but regardless of the level of parasitism, weevil development in flower buds is negatively related to plant fitness, as larvae consume the whole fig 2. (a) abundance of adult and larvae of anthonomus (curculionidade) in ouratea spectabilis in a brazilian tropical savanna; (b) phenological events of o. spectabilis; (c) temporal variation in the abundance of brachygastra lecheguana. figures a and c – mean ± se; p< 0.0001 and p< 0.05 indicate statistical significant differences (kruskal-wallis test). fig 3. (a) abundance (mean ± se) of weevil larvae preyed upon in plants where only brachygastra lecheguana were observed and in plants where both wasps and pseudomyrmex gracilis were noted. p< 0.01 indicates statistical significant differences, student’s t test; (b) wasps visited predominantly inflorescences located on the surrounding of canopy, but weevils occurred evenly in both visible and hidden inflorescences. sociobiology 63(1): 705-711 (march, 2016) 709 of the internal part of buds. thus, the predatory behavior of both b. lecheguana and p. gracilis towards weevils can be reflected in higher flower abundance in future events of o. spectabilis reproductive phenology. the role of ants in plant defence pseudomyrmex gracilis forages in a diverse array of plants, regardless of the presence of other ants (stefani et al., 2000; santos & del-claro, 2001) and is pointed out as one common ant species in ant-plant ecological networks in cerrado vegetation (lange et al., 2013). unfortunately, the natural history of this ant species is not well understood, most likely because of the low number of individuals found on plants compared to other ants (rodrigues et al., 2008). despite that,some studies (dansa et al., 1989; fagundes et al., 2013) found that p. gracilis has a wide feeding flexibility, consuming both arthropods and plant exudates (extrafloral nectar). in the study by byk and del-claro (2010), the authors did not record interactions between ants and anthonomus, but p. gracilis was not the focus of their investigation. in this context, our findings in the present research are important by showing that an overlooked ant species acts as an herbivore (weevil) deterrent for o. spectabilis. nonetheless, its role in weevil predation was low compared to the quantity of weevils captured by wasps. to our knowledge, this is the first record of p. gracilis preying on endophytic weevils, and given the widespread frequency of both anthonomus and p. gracilis in the cerrado (torezan-silingardi, 2007), the potential interaction between these parties could be pervasive. predatory wasps and weevil predation brachygastra lecheguana predation upon weevils was intense, but wasps were more abundant in the last two weeks of the study, during the period of weevil development. according to the data presented in fig 2, there was an inverse association between the abundance of flower buds in the field, and the abundance of both wasps and weevil larvae. that might have occurred because as the phenology of o. spectabilis advanced, fewer buds were available in the field, thus leading to the densification of weevil larvae in the remaining buds. hence, the higher abundance of wasps at the end of the study might be a reflection of the high density of weevil larvae in the flower buds. this temporal variation in wasp behavior is expected, as new evidence (unpublished data) indicates that b. lecheguana visits several plant species according to the flowering phenology and availability of prey (see also torezan-silingardi, 2007; alves-silva et al., 2013). in crops, where b. lecheguana attacks leaf-miners, the presence of wasps can also be related to the abundance of its prey, as pests occur at the specific time of leaf maturation (see perioto, et al. 2011). our observations also revealed that adult weevils can be preyed upon by p. villosa, but these ants are infrequent visitors to o. spectabilis (byk & del-claro, 2010), so their role in weevil deterrence is negligible. plant architecture, weevils and wasps predatory wasps can use chemical and olfactory cues to locate their prey (richter & jeane, 1985), as damaged plants metabolize blends of volatile compounds which attract herbivores’ natural enemies, especially wasps (turlings et al., 1995; moraes et al., 1998; paré & tumlinson, 1999). furthermore, environmental cues can also play an important role in the prey finding ability of wasps (turlings et al., 1993). the foraging activity of b. lecheguana was concentrated on the inflorescences located predominantly on the edges of o. spectabilis canopy. these inflorescences were not shaded by leaves or in contact with any other structure of the plant, in contrast to the hidden inflorescences. previous evidence (alves-silva et al., 2013) suggests that b. lecheguana may use visual and mechanical/olfactory cues to find their weevil prey as plants with plentiful inflorescences were significantly more visited; and antennation on parasitized flower buds was frequent. in o. spectabilis, the weevil parasitism in hidden and visible inflorescences was similar, thus the higher visit rates of wasps to the latter might indicate that b. lecheguana is guided by visual cues. however, chemical signals emitted by the plant in response to weevil herbivory cannot be ruled out yet, as in our case, hidden inflorescences may restrict the range of such chemicals, in contrast to buds located on the edges of the canopy. conclusion as shown in other studies, b. lecheguana has a wide feeding flexibility, as it can consume beetles, lepidopterans, extrafloral and floral nectar, and honey (gusmão et al., 2000; mussury et al., 2003; aguiar & santos, 2007;alves-silva et al., 2013). therefore, its actual host range may be enormous and this wasp can visit several plant species, acting as an herbivore deterrent. further studies may reveal that this wasp species has an important impact on herbivore population while indirectly benefiting plants. o. spectabilis is patrolled by a diverse community of predators (e.g. ants, wasps and spiders, see byk & del-claro, 2010; bächtold et al., 2012; nahas et al., 2012), but only p. gracilisand (especially) b. lecheguana are able to capture the endophytic weevils. this reveals that the outcomes of ant–herbivore–plant systems are highly conditional, and that wasps might play a more important role in herbivore deterrence than extrafloral nectar-drinking ants. acknowledgments we would like to thank the staff of the clube de caça e pesca itororó de uberlândia, where the study was carried out; three anonymous reviewers for the effort in making substantial improvements in the text; capes (coordenação de aperfeiçoamento de pessoal de nível superior) and cnpq (conselho nacional de desenvolvimento científico e tecnológico) for funding. e alves-silva, k del-claro – wasps prey on endophytic beetles and benefit the plant710 references aguiar, c.m.l. & santos, g.m.m. 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(2014). conditional outcomes in ant–plant–herbivore interactions influenced by sequential flowering. flora, 209: 359-366. doi:10.1016/j.flora.2014.04.004 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.7340sociobiology 69(1): e7340 (march, 2022) introduction the mite varroa destructor (anderson & trueman, 2000) is one of the most studied insect parasites worldwide. formerly, this species was considered v. jacobsoni oudemans, which is only found on asian honey bee apis cerana f. (anderson & trueman, 2000). however, unlike v. jacobsoni, v. destructor has adapted to parasitize apis mellifera l. it has quickly spread, becoming a great concern in worldwide apiculture (anderson & trueman, 2000; rosenkranz et al., 2010; thoms et al., 2019). in a. mellifera, v. destructor generally shows a wide variation in infestation dynamics and colony damage (mortensen et al., 2016). although there is no consensus, genetic variation of this mite and infested bee race are considered the main factors for this variation (guzmannovoa et al., 2012; mortensen et al., 2016). anderson and trueman (2000) have evaluated the morphology and abstract the mite varroa destructor is one of the most studied parasites in apiculture, and its genotype variation is a key factor for the severity of infestation in bee colonies. here we report the genetic and reproductive profile of mites from 14 brazilian states with different geographic and climatic conditions. we performed pcr to amplify a fragment of the coi gene and differentiate the haplotypes using restriction enzymes. the k haplotype was widely prevalent in the studied sites, while the j haplotype was found only in four municipalities. we also observed both haplotypes (j and k) coexisting in the same colony, a fact unprecedented in brazil. infestation levels were low (0.33 to 15.3%). the reproductive potential showed wide variation (0 to 1.5), indicating that even with the massive presence of k haplotype, environmental and biotic factors related to africanized honeybees may be responsible for maintaining the mite under low levels in brazil. sociobiology an international journal on social insects fábio a pinto¹, érica w teixeira², lubiane guimarães-cestaro¹, marta f martins³, maria luisa t m f alves², dejair message4 article history edited by evandro n. silva, uefs, brazil received 07 august 2021 initial acceptance 24 december 2021 final acceptance 17 february 2022 publication date 22 april 2022 keywords acari, apidae, bee pathology, parasitic mite, varroosis. corresponding author lubiane guimarães-cestaro av. prof. manoel césar ribeiro, 192, santa cecília, cp: 07, cep: 12411010 pindamonhangaba, são paulo, brasil. e-mail: lubi.guimaraes@gmail.com genetics of mites from several world regions, identifying 18 mitochondrial genotypes, of which only two (koreanrussian or k and japanese-thai or j) could be found in apis mellifera. in this same study, the authors could identify relevant differences in reproductive potential between these two haplotypes, which could explain why the mite is more virulent than others in some regions. besides the direct effects on bees, v. destructor mites are also vectors of several bee viruses (levin et al., 2019), such as deformed wing virus (dwv), which affects colony health and demands the use of acaricides to control infestations in countries such as usa and argentina (rosenkranz et al., 2010). in brazil, these mites are easily found in colonies; however, infestation levels are low, causing no damage to local apiculture (de jong & gonçalves, 1998; pinto et al., 2011; 2015; guimarães-cestaro et al., 2017a; 2017b). previously it was believed that low levels of infestation found in brazil are 1 graduate program in entomology, department of entomology, federal university of viçosa-mg, brazil 2 laboratory specialized in beekeeping health, biological institute / apta / saa, pindamonhangaba-sp, brazil 3 embrapa dairy cattle research center, molecular genetics laboratory, juiz de fora-mg, brazil 4 pvns/capes fellow, graduate program in animal science, ufersa, mossoró-rn, brazil research article bees varroa destructor in africanized honey bees in brazil: genetic and reproductive profile fábio a. pinto et al. – varroa destructor in brazil: genetic and reproductive profile2 due to the wide presence of j haplotype mites in africanized honey bees (ahb) used in brazilian apiculture (anderson & trueman, 2000). however, in a study with honey bee colonies from the south and southeast region of the country, garrido et al. (2003) have identified predominance of k haplotype and increasing mite reproductive rates, which urges caution about the bee health situation in the country. in addition, local beekeepers and field researchers have reported increasing mite infestation and virus infections in africanized honey bees. considering the relevance of v. destructor on worldwide bee health, exhaustive investigations on different aspects of mite biology are necessary to infer the influence of this mite on these recent colony losses. few records about reproductive dynamics and the genetic profile of v. destructor can be found in brazil. until now, only small studies considering specific regions have been carried out to identify genetic and biological aspects of interactions between the mite and ahb. in this context, we have evaluated the genetic and reproductive profile of v. destructor from 14 brazilian states with different geographic and climatic conditions. given the importance of this mite to worldwide apiculture, a wider genetic and reproductive profile can be highly useful to know the current situation of v. destructor in brazil and understand differences in infestation rates between regions and bee races. methods sampling samples were collected in 58 municipalities distributed in 14 brazilian states (table 1). samples from 3 colonies were obtained from 1-3 apiaries for each municipality, each consisting of a piece (3 x 10 cm) of capped worker brood comb (16th day) obtained from colonies in langstroth nests. in addition, ~200 adult nurse bees were obtained from each colony to increase the chances of finding mites. adult mites and offspring found in bee combs were quantified and stored in 1,5 ml microtubes with 70% ethanol until use. the dna of three adult females obtained from different brood cells was extracted for each sample. in samples where it was not possible to find mites in combs, we used mites found on nurse bees from the same colony. along with brazilian samples, mites from two argentinean regions were used only in genetic comparisons. genetic profile of v. destructor mites for genetic identification, a fragment of coi gene from v. destructor mites was amplified using the primers f 5’-tacaaagagggaagaagcagcc-3’ e r 5’-gcccctattcttaatacatagtgaaaatg-3’, described by navajas et al. (2002). the pcr conditions were as follows: (1) 92°c for 4 min, one cycle; (2) 92°c for 60 s, 52°c for 90 s, 72°c for 90 s, 35 cycles; and (3) 72°c for 90 s, one cycle. amplified products were separated by electrophoresis and photo-documented under uv transillumination. after fragment amplification, to distinguish the mite species and haplotypes, 5 µl-aliquot of each amplified pcr product was used in two separated reactions with the enzymes xhoi to distinguish v. destructor from v. jacobsoni; and saci for differentiation between haplotypes k and j of v. destructor (anderson & fuchs, 1998). upon incubation for 3 h at 37ºc, the digested products were also separated by electrophoresis and photo-documented under uv transillumination. according to the results of restriction enzymes, pcr products of samples related to each haplotype (k and j) were purified and sequenced by the sanger method. the sequences were edited and organized by the lasergene® software suite. the edited sequences were subsequently identified and compared with sequences from both haplotypes present on genbank® by ncbi’s blast® tool (national center for biotechnology information). lastly, the amount of divergence between v destructor haplotypes (k and j) and v. jacobsoni was calculated using the software mega v 6.0. infestation rates and reproductive potential to evaluate mite reproductive potential and infestation levels, 100 capped worker brood cells were inspected per comb piece, and all adult mites and their offspring were quantified (de jong et al., 1982; dietemann et al., 2013). with comb inspections, it was evaluated (per sample) the number of adult female mites (total), infested brood cells (%), brood cells infested with offspring (%), viable offspring (%), and reproductive potential (number of viable offspring/number of adult females) (table1). to identify the viability of offspring, it was considered the age of the analyzed honey bee brood (brown eyes pupa, 16th day) and the time till they reach the adult phase. this information made it possible to establish that only female mite offspring from the deutonymph phase could be considered viable offspring (martin, 1994). data analysis to evaluate the location effect on mite infestation rates and reproductive potential, the municipalities and states were used such as explanatory variables and the five evaluated parameters (% of infested cells, % of infested cells with offspring, total number of adults, viable offspring, and reproductive potential) were considered as response variables. all parameters were analyzed through “generalized linear models” (glm) with standard errors (crawley, 2002), followed by anova (α < 0.05). all statistical analyses cited above were conducted using r software (r development core team, 2012). all analyses were carried out in the laboratory specialized in beekeeping health. results genetic profile of v. destructor mites among the 158 samples analyzed, 474 mites presented pcr products with approximately 376 bp (table 1 and fig 1). all samples presented cleavage site on the presence of xho i, sociobiology 69(1): e7340 (march, 2022) 3 generating two fragments of ~150 and ~226 bp, which indicates that all belong to v. destructor species (fig 2). in the presence of sac i, 152 samples did not present any cleavage, which indicates the k haplotype (fig 2). in turn, six samples presented cleavage in the presence of sac i generating two fragments of 124-128 and 252-256 bp indicating j haplotype. fernando de noronha island (pernambuco) was the sole location where only the j haplotype could be found. both haplotypes could be found in bananal (são paulo) and areias (são paulo) samples but in different colonies. in turn, both haplotypes could be found in the same colony in samples from rio claro (rio de janeiro). two other samplings were carried out in 6 months in the same colonies to confirm this coexistence. ten adult mites per colony were individually analyzed for each sampling, totaling 90 evaluated mites on the three evaluated colonies. in all samplings and colonies, it was possible to find both haplotypes in the same colonies, but not sharing the same brood cell (fig 1) fig 1. representative map from evaluated regions. dots represent each municipality where samples were taken to genetic characterization of mites. blue dots represent places where only k haplotype was found. green dot represents where only j haplotype was found. red dots represent where both haplotypes were found coexisting in a same colony or in a same sampling site. fig 2. results from restriction fragment analysis (pcr-rflp) of partial coi region (376 bp). m = marker; c = control (pcr result without any enzyme); saci = restriction enzyme with cleavage site for haplotype differentiation (k and j); xhoi = restriction enzyme with cleavage site for v. destructor differentiating it from v. jacobsoni. fábio a. pinto et al. – varroa destructor in brazil: genetic and reproductive profile4 municipality country/state nº samples af ic co vo rp hap buenos aires arg/b. a 0 £ k mar del plata arg/b. a 0 £ k salvador bra/ba 3 7.3±4.0 6.7±2.9 6.7±2.9 10.0±4.4 1.4±0.2 k fortaleza bra/ce 3 11.0±8.2 9.6±6.0 8.0±6.0 5.7±2.0 0.3±0.3 k domingos martins bra/es 1 6.0 6.0 2.0 0.0 0.0 k goiânia bra/go 1 8.0 7.0 6.0 12.0 1.5 k maracaçumé bra/ma 3 10.3±14.4 9.3±12 6.7±9 5.7±6.2 0.7±0.3 k maranhãozinho bra/ma 3 8.6±5 8.7±5 8.7±5 13.7±8.5 1.5±0.2 k junco do maranhão bra/ma 3 3.3±0,8 3.3±0,8 3.0±0.0 3.0±0.0 9.0±0,3 k muzambinho bra/mg 3 8.0±6.0 8.0±6.0 7.0±5.5 14.6±15.6 1.3±1.1 k são joão evangelista bra/mg 3 2.0±2.6 2.0±2.6 1.7±2.9 0.7±1.2 0.1±0.2 k viçosa bra/mg 3 10.7±12.0 8.3±8.0 6.7±7.4 8.3±11.0 0.8±0.3 k guaraciaba bra/mg 3 7±4,4 6,7±3,8 6,3±3,2 7,3±4,2 1,1±0,4 k janaúba bra/mg 3 1.3±1.5 1.3±1.5 1.3±1.5 2.0±2.0 1.1±1.0 k bocaiuva bra/mg 3 1.0 1.0 1.0 2.0±2.0 0.3±0.6 k r. machados bra/mg 3 1.3±2.3 1.3±2.3 0.7±1.1 0 0 k sete quedas bra/ms 1 21.0 19.0 17.0 24.0 1.1 k campo grande bra/ms 3 3.0±3.6 2.7±3.0 2.3±2.5 3.7±3.5 1.0±1.0 k sinop bra/mt 3 1.7±1.5 1.3±1.5 1.3±1.5 1.0±1.0 0.4±0.5 k fernando de noronha bra/pe 0 £ j irati bra/pr 3 19,6±13.2 15.3±9.2 3.0±2.6 4.7±4.5 0.3±0.4 k tamarana bra/pr 3 20.0±8.0 13.6±5.3 2.3±2.3 6.3±3.2 0.4±0.3 k rio claro bra/rj 3 4.3±2.5 4.3±2.5 3.3±1.1 0.7±1.1 0.2±0.3 k/j** são gabriel bra/rs 3 13.7±3.5 12.3±4.0 14.0±3.6 17.0±5.3 1.2±0.2 k balneário bra/sc 3 17.0±5.5 14.0±4.5 9.6±4.0 5.7±2.0 0.3±0.2 k bom retiro bra/sc 3.0 1.3±1.5 1.0±1.0 1.0±1.0 2.0±2.7 0.9±0.8 k caçador bra/sc 1.0 0.6±0,5 1.0 0,6±0,5 0 0 k criciúma bra/sc 3.0 6.0±1.7 5.6±1.5 5.0±1.7 5.0±1.0 0.9±0.5 k garuva bra/sc 0 £ k florianópolis bra/sc 3.0 17.6±10.0 15.0±7.0 13.0±6.5 11.3±2.9 0.7±0.3 k itainópolis bra/sc 3.0 7.3±7.5 5.0±3.4 3.0±4.3 0.0 0.0 k matos costa bra/sc 3.0 5.6±4.0 5.3±3.5 4.0±4.3 4.0±4.0 0.9±1.0 k mafra bra/sc 1.0 1.0 1.0 1.0 1.0 1.0 k orleans bra/sc 3.0 0.3±0.5 0.33±0.55 0.3±0.5 0.3±0.6 0.3±0.6 k papanduva bra/sc 1.0 53.0 16.0 0.0 0.0 0.0 k rio negrinho bra/sc 3.0 4.0±0.0 3.5±0.7 1.5±0.7 1.5±2.1 0.4±0.5 k são josé bra/sc 3.0 5.0±4.2 5.0±4.2 3.5±2.1 2.5±0.7 0.7±0.4 k urupema bra/sc 2.0 0.5±0.71 0.0 0.5±0.71 0.0 0.0 k xanxerê bra/sc 3.0 3.3±5.7 3.0±5.1 3.0±5.1 1.3±2.3 0.1±0.2 k bocaina do sul bra/sc 3.0 2.7±1.2 2.7±1.2 0.7±1.2 0.3±0.6 0.1±0.1 k araras bra/sp 2.0 1.0 1.0 1.0 1.5±0.7 1.5±0.7 k areias bra/sp 3.0 10.0±6.0 9.3±5.5 2.3±3.2 3.7±6.3 0.3±0.5 k/j* bananal bra/sp 3.0 7.0±10.4 6.6±9.8 5.3±8.3 3.3±5.7 0.2±0.3 k/j* botucatu bra/sp 3.0 2,3±0,5 2.0±1.0 1.0±1.0 1.0±1.7 0.3±0.6 k corumbataí bra/sp 3.0 2.3±2.3 2.3±2.3 1.6±1.1 2.0±1.0 1.4±1.3 k cunha bra/sp 3.0 7.3±2.8 8.3±4.0 5.3±5.8 2.3±1.5 0.3±0.2 k table 1. reproductive and genetic parameters of worker brood cells infested with varroa destructor (mean ± sd): af – adult female mite (total), ic – infested brood cells (%), co – brood cells infested with offspring (%), vo – viable offspring (total), rp – reproductive potential (total) and hap – haplotype. sociobiology 69(1): e7340 (march, 2022) 5 the mitochondrial sequences related to the k haplotype were identical to the complete sequence of the mitochondrial genome published by navajas et al. (2002) (mites from avignon, france; embl accession number aj493124). in turn, sequences related to j haplotype were 98% identical to j haplotype sequences from taiwan and usa (ncbi accession number aj784872; solignac et al. (2005). the alignment of k and j sequences with v. jacobsoni sequence (ncbi accession number af106909.1; anderson &trueman 2000) presented nucleotide divergences of 12.7 and 10.9%, respectively. in turn, both sequences presented a nucleotide divergence of 1.3% between each other (table 2). infestation rates and reproductive potential the aspects of v. destructor mites infestation were evaluated in 154 colonies. the levels of infested honey bee brood cells varied from 0.33 ± 1.55 % to 15.3 ± 9.2 %. infested cells hosting offspring ranged from 0 to 13 %. in turn, considering only viable offspring, the reproductive potential varied from 0 to 1.5 offspring per female mite. significant differences were observed on five parameters analyzed between municipalities evaluated (table 1). there was no difference in reproductive potential compared to the haplotype pattern in colonies with both haplotypes in the municipality of rio claro-rj (p > 0.05), with low reproductive levels regardless of the genetic profile of the mite. significant differences were observed between 58 municipalities distributed in 14 states of brazil in all parameters evaluated regarding the infestation by the mite v. destructor in brood combs. this study covered municipalities with regional climatic differences both in temperature and rainfall located in a tropical zone, where varroa infestations are less harmful (de jong et al., 1984) and have different infestation rates, higher in the colder regions (moretto et al., 1991). the climate has a strong influence on the rate of mite infestation. (de jong et al., 1984; moretto et al., 1991; mondragon et al., 2006). unfortunately, it is impossible to make an objective comparison with several studies on mite reproduction due to the lack of uniformity of the variables. in brazil, mite infestation rates are considered low and may be related to defense mechanisms carried out by africanized bees, such as hygienic behavior (carneiro et al., 2014). no differences in infestation and reproductive parameters related to mite haplotypes were observed. discussion our genetic screening of v. destructor in different brazilian regions could identify the massive predominance of the k haplotype. the high prevalence of this haplotype could also be identified in other south-american countries where the mite is considered a great threat in apiculture. k haplotype j haplotype v. jacobsoni k haplotype 1,3% 12,7% j haplotype 1,3% 10,9% v. jacobsoni 12,7% 10,9% table 2. level of divergence (in percentage) between the different varroa destructor coi haplotypes and varroa jacobsoni. this table includes the korean haplotype (widely found in this study), the japanese haplotype (found only in four brazilian municipalities) and the v. jacobsoni sequence (vj, accessions af106909.1). municipality country/state nº samples af ic co vo rp hap descalvado bra/sp 3.0 4.3±5.8 3.3±4.9 0.6±0.1 0.7±1.1 0.1±0.1 k lagoinha bra/sp 3.0 5.6±3.0 4.6±2.5 3.0±2.6 1.0±1.7 0.1±0.2 k m. lobato bra/sp 3.0 10.0±4.5 9.0±4.0 4.0±3.6 4.7±4.1 0.5±0.5 k paraibuna bra/sp 3.0 9.6±8.3 9.0±7.2 3.0±4.3 3.3±5.0 0.2±0.2 k pindamonhangaba bra/sp 3.0 5.0±1.0 5.0±1.0 1.3±1.5 1.0±1.7 0.2±0.4 k pirassununga bra/sp 3.0 4.3±3.5 3.6±2.5 3.0±3.0 1.7±1.5 0.3±0.2 k redenção bra/sp 3.0 2.6±1.5 2.6±1.5 1.0±1.7 0.0 0.0 k rio claro bra/sp 3.0 5.0±0.0 4.6±0.5 3.0±1.0 4.0±1.0 0.8±0.2 k são francisco xavier bra/sp 3.0 6.3±2.9 5.3±2.5 4.3±2.5 2.3±1.5 0.4±0.1 k são luiz do paraitinga bra/sp 3.0 5.3±3.8 5.0±3,4 4±2,6 2,3±2,3 0,6±0,4 k são josé dos campos bra/sp 3.0 4.6±0.6 4.6±0.5 2.3±2.3 1.0±1.0 0.2±0.2 k taubaté bra/sp 3.0 3.0±0 3.0 1.0±1.7 0.0 0.0 k tremembé bra/sp 3.0 5.3±3.2 5.0±2.6 2.3±4.0 0.7±1.1 0.1±0.1 k ubatuba bra/sp 3.0 5.0±0.0 4.6±0.5 4.3±0.5 5.7±1.1 1.1±0.2 k £ = there was no brood comb samples, but only mites collected from adult bees for genetic evaluations. k/j* = both haplotypes found in a same city, however on different apiaries. k/j** = both haplotypes found in a same samples/colony table 1. reproductive and genetic parameters of worker brood cells infested with varroa destructor (mean ± sd): af – adult female mite (total), ic – infested brood cells (%), co – brood cells infested with offspring (%), vo – viable offspring (total), rp – reproductive potential (total) and hap – haplotype. (continuation) fábio a. pinto et al. – varroa destructor in brazil: genetic and reproductive profile6 in a related study in argentina, 100% of mites from six provinces were identified as k haplotype (maggi et al., 2012), and ten other provinces were recently sampled (muntaabski et al., 2020). other studies have also identified related results with more than 95% of mites belonging to k haplotype in chile (solignac et al., 2005) and 100% in different areas of uruguay (mendoza et al., 2020). previously it was believed that the j haplotype, which was generally found in areas with low infestation rates (anderson & trueman, 2000), was the most prevalent genotype of the mite in brazil. however, with multiple entries of colonies from different regions of the world to south america, the genetic profile of mites could be changed. garrido et al. (2003) identified that the j haplotype, widely found in samples from the 1990s in ribeirão preto (são paulo) and florianopolis (santa catarina), was excluded from mite populations after 2001. as in our results, strapazzon et al. (2009) has also found only k haplotype in municipalities from santa catarina, south of brazil, reinforcing this haplotype replacement. recently, the k haplotype was also observed in samples collected in the vale do ribeira region, including municipalities bordering the state of são paulo and the state of paraná (guimarãescestaro et al., 2017a). on the other hand, the j haplotype apparently could be found in specific isolated areas where geographic barriers or the absence of migratory beekeeping prevents the entry of the k haplotype. thus, the geographic isolation can explain the exclusive presence of haplotype j in fernando de noronha island (de jong & soares, 1997; strapazzon et al., 2009). colonies of european honey bees were introduced to this island in the 1970s (malagodi et al., 1986) when only haplotype j mites were established in south america. besides fernando de noronha, haplotype j mites were also found in colonies from municipalities of the extreme east of são paulo and south of rio de janeiro (fig 1). considering the proximity of municipalities where j haplotype mites were found, we believe this is a specific endemic region where the remnant populations can still be found. besides the isolation characteristics of this region (apiaries surrounded by fragments of atlantic rain forest), it is not clear why the j haplotype is still present and coexisting with the k haplotype in some sites, even at the same colony. such an event is reported for the first time in brazil. solignac et al. (2005) admitted the possibility of the coexistence of both haplotypes. however, due to reproductive advantages and isolation factors, a strong tendency to k prevalence and j exclusion could be expected. such mechanisms could explain the wide prevalence of the k haplotype found in this study, which gradually could have excluded the j haplotype from honeybee colonies after its introduction in brazil. our study could not detect mites from different haplotypes sharing the same brood cell, and reproduction possibilities between haplotypes were not identified. in a study with k/j infested colonies, solignac et al. (2005) identified heterozygosis between haplotypes but in a very low percentage of mites due to the necessity of multiple infestations in brood cells to achieve cross reproduction. as observed here, the infestation levels of v. destructor in ahb are generally low, and multiple infestations in a single cell are rare events (fuchs & langenbach, 1989; martin, 1995). moreover, all colonies where we found both haplotypes were monitored for six months and, even confirming the coexistence, we could not find k and j mites in the same brood cell and offspring with heterozygosis. considering the necessity of an operculated brood for evaluations, all samplings were obtained during months with high pollen availability, a factor that stimulates brood production. in this colony stage, female mites finish the forethic period and invade brood cells to reproduce and increase the mite population. then, the infested colony faces a weakening in the following months when food availability is low (rosenkranz et al., 2010). however, in tropical/subtropical countries such as brazil, the dynamics of mite infestation tend to be different. due to the absence of well-defined seasons, rain occurrence and its effect on food availability may be more related to infestation rates. each brazilian region has specific rainfall characteristics throughout the year, which drastically affects bee flora and brood production, consecutively affecting mite reproductive dynamics (moretto et al., 1997; pinto et al., 2011; correia-oliveira et al., 2018). besides environmental effects, a variation in biological features on bees among studied regions may also affect the relation between host and parasite. due to crossbreeding between a. mellifera scutellata with european races, there is great genetic variability among bee populations in brazil (moretto et al., 1991; sheppard et al., 1999; collet et al., 2006). for example, bees from south and southeast states, where several samples were taken for this study, have highly distinctive genotypes (collet et al., 2006). due to this, genetically based behaviors directly involved in v. destructor control, such as hygienic behavior and grooming, also present great variation between colonies (spivak, 1996; ibrahim & spivak, 2006; guzman-novoa et al., 2012; pinto et al., 2012; oddie et al., 2021). despite the high variation in infestation rates between the regions evaluated (table 1), the values were lower than those found in european honey bees during summer/spring without acaricide treatment: bulgaria, 18-49% (kanchev et al., 1989); southern england, 15-40% (martin, 1994); and the united kingdom, 6-42% (martin, 2001). however, closer values were found when comparing the results obtained in other southeast regions of brazil: 3.3-11% in the mata mineira zone, minas gerais state (bacha-júnior et al., 2009); 1.1 to 11.6% and 5.0 to 14.0% in the vale do ribeira region, são paulo state (pinto et al., 2011 and guimarães-cestaro et al., 2017a, respectively); 2.83 to 9.48% in the centraleastern region of são paulo state (guimarães-cestaro et al., 2017b) and 7.2 % ± 3.3% in southeastern region of the country (peixoto et al., 2021). although the levels of infestation were generally low, they were high in some southern regions, such sociobiology 69(1): e7340 (march, 2022) 7 as the states of paraná (19.6±13.2 and 20.0±8.0) and santa catarina (17.0±5.5 and 17.6±10.0) (table 1). in the usa, where the mite causes great economic losses every year, and acaricide tolerance starts to show off, a recent selection of v. destructor-tolerant bee lineages has been shown promising results (gupta et al., 2014). thus, considering the high number of v. destructor-tolerant ahb colonies in brazil, a selection program focused on bee health and production could provide great results, avoiding future problems. in the same way as the other infestation parameters, mite fertility also presented great variation between regions, but low levels were generally found. this result was expected considering the direct relationship between infestation levels and reproductive potential, as observed in several studies (martin, 1994; martin & medina, 2004; rosenkranz et al., 2010). these variations and low levels found in ahb are generally attributed to high levels of offspring mortality, including the only male produced by the first laid egg (mondragon et al., 2006). in addition, this fact, together with the rarity of multiple invasions in a single cell, may be responsible for the high number of infertile mites found in most of our samples. a few years ago, increasing v. destructor reproductive potential was reported in some regions, but due to the absence of proper monitoring, it isn’t easy to track possible causes (garrido et al., 2003; carneiro et al., 2014). in municipalities of são paulo state, where we also found contrasting values, garrido et al. (2003) found increases of 52 to 83% in the number of fertile females during evaluations between 1998 and 2001. it is unknown if reproductive changes can be attributed to the mite’s genetic profile. however, once considered the main one responsible for high infestation levels, haplotype k also presented great variation between the studied regions. the set of geographic characteristics and honey bee genetic variability in brazil promotes this wide variation of the dynamics of v. destructor infestation. the korean-russian haplotype presented wide prevalence and great variation in reproductive potential, indicating that other factors apart from genotype could be responsible for mite tolerance in ahb. in turn, we observed that haplotype j, once highly prevalent in brazil, has been drastically affected by the k haplotype’s introduction and is now restricted to only a few isolated areas. authors contribution fap: investigation, writing-original draft, writing-final draft. ewt: investigation, formal analysis, resources, writing original draft, writing-final draft. lgc: investigation, formal analysis, writing-original draft, writing-final draft. mfm: methodology, formal analysis, writing-original draft, writing-final draft. mltmfa: investigation, formal analysis, writing-original draft, writing-final draft. dm: supervision, writing-original draft, writing-final draft. acknowledgments we are grateful for the support from lasa/ib/apta (specialized honey bee health laboratory/biological institute/ são paulo state agribusiness technology agency) for support to this study. this work was funded by the national council for scientific and technological development (cnpq) and the ministry of science and technology (cnpq/mapa/ edital 64 grant – e.w.t.). fábio pinto thanks the office to coordinate improvement of university personnel (capes) for the scholarship and the graduate program in entomology of the federal university of viçosa. we thank the official veterinary services of all states cited in this work, as well as the beekeepers that opened their apiaries for us, except for the state of pernambuco, in which the sample was kindly provided by prof. g. moretto (blumenau regional university). conflict of interest the authors declare that they have no potential conflict of interest concerning the study in this paper. references anderson, d.l. & fuchs, s. 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(2019). beekeeper stewardship, colony loss, and varroa destructor management. ambio: 48, 1209-1218. doi: 10.1007/ s13280-018-1130-z https://doi.org/10.1080/00218839.2021.1960746 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7757sociobiology 69(2): e7757 (june, 2022) introduction sunflower (helianthus annuus l.: asteraceae) represents the 3rd source of raw matter in the world, contributing with more than 13% of the total edible oil production (ramulu et al., 2011). likewise, it contributes 16% in domestic edible oil production in pakistan. therefore, pakistan still has a deficit in edible oil production and imports 1.98mt of edible crude oil by spending about 45 billion rupees (khan et al., 2017; ali et al., 2008). abstract sunflower (helianthus annus l.) is a highly cross-pollinated crop dependent on insect pollinators to provide a good quality edible oil worldwide. different sunflower hybrids vary in terms of dependence on insect pollinators. previously few studies have been conducted regarding the role of insect pollinators in hybrid sunflower seed production in pakistan. therefore, the current study was planned to explore the abundance and diversity along with foraging behavior (visitation rate and stay time) of native insect pollinators as well as to study the effect of different pollination treatments (free insect visits vs. no insect visits) on the reproductive success of different hybrids of sunflower. two sunflower hybrids were grown at the research farm of mns university of agriculture, multan, under the randomized complete block design (rcbd). in our study, the pollinator community consisted of honeybees (apis dorsata, a. mellifera, and a. florea), solitary bees (pseudapis sp., megachilidae sp. and xylocopa sp.), and syrphid flies (eristalinus aeneus and e. megacephalus). furthermore, the relative abundance of pollinators was high in the h4 (bird resitant) having a flat head with 45º head angle from the stem hybrid, while the least abundance was observed in h3 (bird susceptible 180º head angle from stem). h4 proved to be a better hybrid among the hybrids regarding the number of seeds and seed weight. both bees, i.e., solitary bees and honeybees, are crucial for pollinating sunflower. comparative results of free insect visits and no insect visit treatments showed that the maximum number of seed weight, number of seeds, and seed diameter was observed in free insect visits compared to no insect visit treatment. therefore, conserving the diversity of the native insect pollinators will lead to a higher yield of sunflower hybrids and other cross-pollinated crops. sociobiology an international journal on social insects shahid iqbal1, mudssar ali1, fawad z. a. khan1, naeem iqbal1, fahim nawaz2 article history edited by evandro nascimento silva, uefs, brazil received 30 january 2022 initial acceptance 19 march 2022 final acceptance 09 june 2022 publication date 05 july 2022 keywords visitation rate, stay time, abundance and diversity, reproductive success corresponding author mudssar ali institute of plant protection: muhammad nawaz shareef university of agriculture multan, pakistan. e-mail: mudssar.ali@mnsuam.edu.pk in ecosystems, pollination plays a vital role in crop production. nearly 84% world’s crops are pollinated by insects (bareke & addi, 2019). insect pollinators are the major contributors to about 5% of global food production (carper et al., 2016). according to an estimate, the role of pollinators in the worldwide economy is usd 577 billion (potts et al., 2016), and it contributes 1.59 billion dollars to the economy of pakistan (ahmad & aziz, 2017). sunflower is an allogamous plant that requires insects in the flowering stage, particularly honeybees, to increase seed 1institute of plant protection: muhammad nawaz shareef university of agriculture multan, pakistan 2 department of agronomy, mns university of agriculture, multan, pakistan research article bees linking bird resistant and susceptible sunflower traits with pollinator’s fauna and seed production shahid iqbal et al. – comparing pollinator effect on seed production in bird resistant and susceptible sunflower2 production (chambo et al., 2011). floral cues, shape, size, flowering timing, odor, color, and arrangement are vital in helping to attract pollinators to plants because flowers provide nectar and pollen (nakata et al., 2021; chittka & walker 2006: berjano et al., 2009). research shows that sunflower has olfactory cues, including floral volatiles and a quantity of nectar and pollen that attracts bees to the flowers (painkra & kumaranag, 2019; mallinger & prasifka, 2017). some investigations also described that insect pollinators, especially the bees (wild and managed), increased seed yield by 18-65% on average in open pollination (where insects visited) (degrandi-hoffman & chambers, 2006; tamburini, lami, & marini, 2017; stein et al., 2017). previously, some studies have reported managed honeybees (a. mellifera) as the most abundant and effective pollinators due to their contribution towards seed weight, filled seed per head, and seed yield per head of hybrid sunflower (rajasri et al., 2012; abrol, 2012; perrot et al., 2019). however, some other studies have reported solitary bees as more effective pollinators than honeybees (mallinger et al., 2019). but some studies showed that honeybees’ effectiveness increased with the presence of wild bees. in the absence of wild bees, nearly three seeds were produced by the single visit of honeybees, while in the presence, pollination efficiency increased up to 15 seeds on an average (greenleaf & kremen, 2006). studies have revealed that the flower angle affects pollination and increases pollination in zoophilous plants (haverkamp et al., 2019). some studies showed that plants with horizontal oriented flowers head enhance insect pollination compared to upward oriented flowers (nakata et al., 2021; wang et al., 2014; ushimaru et al., 2009). at the same time, the upward orientation enhances the nocturnal (hawkmoth) pollination (campbell et al., 2016). bird predation is a major problem in sunflower crop, resulting in seed loss. breeders have developed such bird resistant sunflower hybrids with special characteristics (convex and flat shape, distance between head and stem that is higher than 15 cm, and horizontally oriented head), which makes the sunflower unfavorable for birds (yasumoto et al., 2012, khaleghizadeh, 2011, prakash et al., 2010, tarimo, 2000). on the contrary, bird susceptible cultivars have concave or flat shape capitula, the distance between the head and stem lesser than 15 cm, and vertically oriented heads as characteristics that enhance bird predation (hladni et al., 2017; khaleghizadeh et al., 2009; parfitt, 1984). previously, seed loss due to bird damage in susceptible sunflowers was 25-70% higher than in bird-resistant cultivars (tomaz et al., 2019). to the best of our knowledge, no work has been reported on bird resistant and bird susceptible varieties response to insect pollinators. keeping in view this gap in research, our study aimed to test the following hypothesis: a) pollinator visiting increases sunflower reproductive success, leading to higher number of seeds and seed weight per head; b) sunflower plant architecture traits in susceptible and resistant bird predation hybrids affect: b1) insect pollinator abundance; b2) foraging behavior (visitation rate and stay time). materials and methods study site the studies were carried out at the research farm of mns university of agriculture, multan. two sunflower hybrids (bird resistance and bird susceptible) were grown on an area of 0.25 acres in february for growing seasons (2021). the sunflower was sown on 0.762-meter ridges. the experimental plot and sowing of sunflower were homogeneously designed to avoid any variation that could affect the analytical comparisons of the experiment. berseem (trifolium alexandrinum: leguminoceae) surrounded the crop in the south, maize (zea mays: poaceae) in the north, and quinoa (chenopodium quinoa: amaranthaceae) in the west; moreover, perennial trees including shisham (dalbergia sissoo: fabaceae), kikar (acacia karoo: fabaceae) and mulberry (morus alba: moraceae) were also present near sunflower fields. cultivars abbreviated cross bird resistant cultivar h4 br.11 x br.82 bird susceptible cultivar h3 bs.16 x r36 the climatic condition of the study area was subtropical with cold winters and hot summers; the daily mean minimum and maximum temperatures ranged from 8 to 12 °c and 38 to 50 °c, respectively, and the mean monthly rainfall of summer was ca. 18 mm. the sunflower hybrids were grown in rcbd experimental design (randomized complete block design). there were two treatments, and each treatment was replicated three times. plant to plant distance was 0.22 m, replication to replication distance was 3.04 m, and plot to plot distance was 1.52 m was maintained. plant characteristics plant characteristics data, i.e., head shape, head angle, plant height, chaff length, the distance of the head from the stem, and head positioning, were recorded according to khaleghizadeh (2011). abundance and diversity the data of the abundance and diversity of insect pollinators in different hybrids of sunflower was recorded during clear sunshine days when the pollinators are fully active. the total number of sunflowers in each plot was 60, which was thrice replicated. for abundance, when 10% flowering started, ten sunflower heads were selected for each hybrid during each data census. each sunflower head was observed for one minute to record the different abundance and diversity of varying insect pollinators (jadhav & prasad, 2011). insect specimens were preserved for later taxonomic identification. https://www.sciencedirect.com/science/article/pii/s1439179118300975?casa_token=plvanag82q8aaaaa:i-iqi90mzddywd-afj9hnb7onosdfqgqj8wu7a9wne4x1shachebz2tofowmyfqvv6dencgi89u#bib0060 https://www.sciencedirect.com/science/article/pii/s1439179118300975?casa_token=plvanag82q8aaaaa:i-iqi90mzddywd-afj9hnb7onosdfqgqj8wu7a9wne4x1shachebz2tofowmyfqvv6dencgi89u#bib0185 https://www.sciencedirect.com/science/article/pii/s1439179118300975?casa_token=plvanag82q8aaaaa:i-iqi90mzddywd-afj9hnb7onosdfqgqj8wu7a9wne4x1shachebz2tofowmyfqvv6dencgi89u#bib0185 https://pbgworks.org/sites/pbgworks.org/files/randomizedcompleteblockdesigntutorial.pdf https://pbgworks.org/sites/pbgworks.org/files/randomizedcompleteblockdesigntutorial.pdf https://pbgworks.org/sites/pbgworks.org/files/randomizedcompleteblockdesigntutorial.pdf sociobiology 69(2): e7757 (june, 2022) 3 diversity indices diversity indices are considered as those mathematical measures which refers to the diversity of species with respect to species richness (no. of species recorded from a particular area) and abundance (total number of individuals belonging to given species) from a community (purvis and hector, 2000; schleuter et al., 2010). in this research, diversity indices are calculated with respect to type of impressions explored from the insect pollinator’s species. diversity indices were calculated individually from the bird resistant and bird susceptible cultivars. species richness and species evenness are calculated with the help of shannon diversity index (h), shannon evenness measure (eh), simpson’s diversity index (d), and simpson’s measure of evenness (ed). shannon diversity index (h) analyze the data through statistical information by keeping in view of diversity principle and is used globally to calculate the ecological diversity of species (shannon, 1948). simpson’s diversity index (d) is a measure of dominance that includes the most abundant species recorded from a sample and least sensitive compared to species richness (magurran, 2013). the formula for species richness and species evenness is given below: h = σs i=1 pilnpi eh = h/lns d = 1/σs i=1 pi2 ed = d × 1/s where h = impression-based shannon diversity index, eh = impression-based shannon evenness measure, s = richness of impression types, pi = proportion of individual impressions that are of the impression type, ln = natural logarithm, d = impression-based simpson diversity index, ed = impression-based simpson evenness measure. foraging behavior foraging behavior (stay time, visitation rate) of abundant insect pollinators in different hybrids of sunflower was recorded. for visitation rate and stay time, five sunflower heads were selected for each plot, and a total of thirty values were taken of five species of pollinators. visitation rate was observed in terms of the number of sunflower heads visited by a single pollinator in one minute per plot, while stay time was recorded in seconds spent by a pollinator on a single sunflower head (mehmood et al., 2018). pollination treatments two pollination treatments, i.e., free insect visits (open pollination) and no insect visits (caged pollination), were used to compare insect pollinators’ effectiveness in the reproductive success of sunflower. at the bud stage, before opening any floret, 10 sunflower heads per plot and 30 per treatment were selected and tagged randomly as open heads accessible to visitors (open pollination). at the same time, nine heads per plot and 27 seven heads per treatment were caged randomly by white nylon fine mesh bags (1mm mesh width) to exclude insect visitors, allowing only selfpollination (caged pollination). after harvesting, reproductive success parameters, i.e., head diameter, head weight, number of seeds per flower head, and seed weight per flower head, were compared between open and cage pollinated plants. reproductive success following parameters of reproductive success were recorded, i.e., head diameter, the number of seeds per head, and seed weight per head (tamburini et al., 2016). thirty heads (20 from open and 10 from caged) of sunflower were harvested from each sunflower hybrid and measured diameter in centimeters. after the measurement of head diameter, the number of the seed of each harvested head were counted manually. the weight of seeds was measured with digital weight balance after the sanitation of seeds per head. for this purpose, we harvested thirty sunflower heads from each treatment. data analyses the data were analyzed using statistical analysis system (sas) software (sas institute, 2013). the shannon and simpson diversity indices and evenness data were subjected to t-test using proc ttest in sas (α = 0.05). the pollinator visitation time and the stay time of pollinators on two cultivars were square root transformed and tested using t-test (proc ttest) in sas (α = 0.05). the difference among different species’ visitation and stay time was tested using proc glimmix. the tukey’s test was used to see differences among the mean values. the data for head weight, head diameter, seed weight and numbers of seeds per head were subjected to proc univariate to check the normality. the data were then subjected to square root transformation to achieve the normality. the transformed data were subjected to proc ttest (α = 0.05). the analyses were preformed individually for open and caged condition, followed by the combined (open + caged) plant parameters. results plant characteristics both sunflower hybrid h4 and h3 have flat heads. according to table 01 head angle of h4 was 90º while h3 had 180º while h4 had head positioning (8), and h3 had head positioning (9). plant height, chaff length, distance of the head from the stem was observed higher in the h4 sunflower hybrid followed by the h3 sunflower hybrid (table 1). shahid iqbal et al. – comparing pollinator effect on seed production in bird resistant and susceptible sunflower4 abundance and diversity during the flowering season of sunflower, six bee species (three solitary bees and three honeybees) belong to hymenoptera, two syrphid flies belonging to diptera and three lepidopterans species were observed. the overall abundance of pollinators was high in bird resistant h4 (65%) hybrid as compared to bird susceptible h3 (35%) cultivars (table 2). honeybees include apis dorsata, a. mellifera, a. florea while in solitary bees xylocopa sp., pseudapis sp and megachilidae sp, in syrphid flies include eristalinus aeneus and e. megacephalus and in lepidopteran include pseudaletia unipuncta, utethesia cardui and pieridae rapae. a. mellifera was the most abundant among all pollinators, followed by a. dorsata, while pseudaletia unipuncta was the least abundant (table 2). diversity indices according to the outcomes of this research, in june and september 2019, values recorded for shannon’s and simpson’s diversity and evenness indices in bird resistant and bird susceptible cultivars were not statistically different with respect to the impression types investigated on diversity of insect pollinators. greater values were seen for species evenness from bird resistant as compared to that of bird susceptible (table 3). sunflower hybrids h4 h3 head shape flat flat head angle 45° 180° chaff length (inch’s) 17.48 14.4 distance of head from the stem (cm) 5.2 5 head positioning 8 9 plant height 159.41 148.7 table 1. plant characteristics of bird resistant and susceptible in sunflower cultivars. order family genus/species h3 h4 hymenoptera apidae apis dorsata 24 67 apis mellifera 94 149 apis florea 30 52 xylocopa sp. 4 17 halectidae pseudapis sp 22 37 megachilidae megachilidae sp. 10 22 diptera syrphidae eristalinus megacephalus 19 47 eristalinus aeneus 32 41 lepidoptera noctuidae pseudaletia unipuncta 2 4 nymphalidae utethesia cardui 3 5 pieridae pieris rapae 7 16 total 247 (35%) 457 (65%) table 2. abundance and diversity of pollinators in bird resistant and susceptible sunflower cultivars. variety h eh d ed h3 1.553 ± 0.201 0.758 ± 0.100 4.235 ± 0.567 0.610 ± 0.080 h4 1.759 ± 0.137 0.833 ± 0.031 5.106 ± 0.545 0.565 ± 0.057 t, df -0.92, 18 -0.92, 18 -1.06, 18 -0.01,18 p 0.371 0.3714 0.304 0.989 table 3. means (± se) of shannon diversity index (h), shannon’s equitability (eh), simpson’s diversity index (d), and simpson’s equitability (ed) for pollinator diversity recorded on sunflower cultivars. foraging behavior (visitation rate and stay time) in this experiment, no significant difference was found for the visitation rate of the different pollinator species on the two hybrids. however, on h4 hybrid significantly higher number of a. dorsata and psedapis sp. visited the flowers as compared to a. florea and a. mellifera, while no significant differences among different species were found on h3 hybrid (table 4). the stay time of a. mellifera, a. florea, and e. aeneus was not significantly different in both cultivars while a. dorsata presented a significantly different stay time in h4 hybrid than in h3. a. mellifera had the highest stay time fallowed by pseudapis sp. while the least stay time was observed in a. florea among all pollinators (table 4). sociobiology 69(2): e7757 (june, 2022) 5 reproductive success in sunflower hybrids the reproductive success in both sunflower hybrids h4 and h3 in terms of head diameter and head weight have no significant difference while seed weight and the number of seeds have significant difference in all conditions i.e. open, caged and both open + caged (table 5). in open pollination, seed weight in h4 cultivar was higher (16%) than in h3 cultivar. the number of seeds per head in h4 cultivar was higher (46%) than in h3 cultivar. in open pollination, plants reached the maximum head diameter, i.e., 27% greater in h4 and 16% in h3 in comparison to caged heads (table 5). apis florea apis dorsata apis mellifera pseudapis sp. eristalinus aeneus f, df p visitation rate h3 0.52 ± 0.26 2.83 ± 0.49 1.93 ± 0.21 2.533 ± 0.40 2.36 ± 0.35 1.3, 4, 116 0.272 h4 1.86 ± 0.19b 3.10 ± 0.56a 1.67 ± 0.20b 2.26 ± 0.28a 2.03 ± 0.21ba 4.2, 4, 116 0.004 t, df 1.65, 58 -0.27, 58 1.00, 58 0.47, 58 0.64, 58 p 0.10 0.79 0.32 0.64 0.52 stay time h3 50.45 ± 6.49c 48.70 ± 6.59bc 67.71 ± 7.14b 74.03 ± 9.19a 69.82 ± 8.79 ba 65.5, 4, 116 <.001 h4 67.19 ± 7.45c 99.44 ± 13.52aa 92.91 ± 10.59a 55.47 ± 3.54d 84.14 ± 7.56b 122.5, 4, 116 <.001 t, df -1.84, 58 -3.11, 58 -1.90, 58 1.72, 58 -1.48, 58 p 0.07 0.00 0.06 0.09 0.14 table 4. mean (± se) number of pollinators visiting and the pollinator stay time on bird-susceptible and bird-tolerant cultivars. condition variety head diameter (cm) head weight (g) seed weight (g) no. of seeds open h3 17.91 ± 0.38b 326.02 ± 22.43 70.32 ± 4.25 663.20 ± 41.41b h4 19.16 ± 0.38a 337.04 ± 14.60 86.11 ± 4.15a 1108.57 ± 46.21a t, df -2.30, 58 -0.66, 58 -2.57, 58 -7.07, 58 p 0.02 0.51 0.012 <.001 caged h3 18.07 ± 0.40a 190.05 ± 16.67 30.061 ± 1.06b 341.66 ± 14.95b h4 16.44 ± 0.38b 192.35 ± 12.71 39.96 ± 39.96a 524.11 ± 26.63a t, df 2.95, 52 -0.26,52 -4.72, 52 -6.19, 52 p 0.04 0.79 <.001 <.001 open + caged h3 17.98 ± 0.27 261.61 ± 16.75 51.25 ± 3.52b 510.89 ± 31.25b h4 17.87 0.32 268.50 ± 13.67 64.25 ± 3.86a 831.71 ± 47.52a t, df 0.32, 112 -0.53, 112 -2.57, 112 -5.73, 112 p 0.7 0.59 0.01 <.001 table 5. mean (± se) head diameter, head weight, seed weight, and number of seeds recorded for two sunflower cultivars under the open, caged and combined (open + caged) conditions. discussion the current study showed that the overall abundance of pollinators was higher in bird resistance h4 cultivar compared to bird susceptible h3 cultivar. among all the pollinator species visiting sunflower cultivars, a. mellifera was the most abundant pollinator followed by a. dorsata and a. florea. no significant differences were found in the visitation rate of the different pollinator species on the two hybrids. a. dorsata stayed for significantly greater time on h4 cultivar than on h3. significant differences in seed weight and number of seeds were observed in all pollination treatments, i.e., open, caged and combined (open + caged) conditions. apis spp. were found to be abundant species visiting the sunflower cultivars. visitation of a. mellifera and a. dorsata as dominant pollinators, followed by the solitary bee trigona iridipennis, has been recorded on sunflower hybrids (jadhav et al., 2011; rajasri et al., 2012). in another study, a. mellifera was the most frequent visitor in different sunflower hybrids (oz et al., 2009; hoffman & chambers, 2006; kasina et al., 2007; mallinger & prasifka, 2017). for the oilseed crops honeybees were found to be abundant in mustard (brassica juncea: brassicaceae) (shakeel et al., 2019; delaplane et al., 2013; parbat, 2019), canola brassica napus: brassicaceae) (akhtar et al., 2018; amro, 2021), and sesame (sesamum indicum: pedaliaceae (das et al., 2019; pashte et al., 2013). previous studies from pakistan indicated a. mellifera as an abundant and efficient pollinator of sunflower (akhtar et al., 2018; ali et al., 2015). the abundance of the apis spp. indicates pollinator preference to forage on sunflower. shahid iqbal et al. – comparing pollinator effect on seed production in bird resistant and susceptible sunflower6 pollinator species showed significant differences for visitation rate on bird resistant sunflower cultivar (h4). visitation rate is an important parameter for evaluating the efficiency of insect pollinators, especially native species (albano et al., 2009). apis mellifera has been reported as a frequent visitor on sunflower crop in multiple studies (estravis barcala et al., 2019 & nderitu et al., 2008). similarly, wild bees like melissodes spp. and andrena helianthus have also a frequent visiting activity on the sunflower (mallinger et al., 2019; mallinger et al., 2015 and greenleaf & kremen, 2006). in another study, the frequency of a. mellifera ranged from 2.27 to 2.94 bees per sunflower head (chambó et al., 2011). overall, the visitation frequency could also be linked with the body size of bee species, in addition to other factors (everaars et al., 2018). the number and weight of seeds were higher in bird resistant cultivar (h4). pollinator species, like a. mellifera have been reported to induce better seed setting in terms of number and weight of seeds (jadhav et al., 2011; rajasri et al., 2012; martin et al., 2016), while in the case of pollinator restricted arenas, 18-25% less seed set was observed for sunflower hybrid (hoffman & chambers, 2006; mallinger & prasifka, 2017). so, the pollinator activity leads to a better seed health and seed set, as found in the current study. to conclude, the cultivar doesn’t change the structure of the flower-visiting guilds (richness and abundance) but the change in the total abundance might lead to a higher oilseed yield. the selection of sunflower cultivars is an important pollination factor of sunflower in all aspects, i.e. abundance and diversity and foraging behavior (visitation rate, stay time) of insect pollination which may ultimately increase or decrease the sunflower yield. the sunflower bird resistant cultivar has been found leading to an increase of bee abundance with a coupled effect in reproductive success and yield increase. further research should focus on screening multiple sunflower cultivars for pollinator activity with reference to the flower head angle. acknowledgements thanks to s. rauf for providing the sunflower hybrid seeds. special thanks to m. a. ahmad, m. zubair, m. talha, m. daud. n. nizam, m. a. tahir, m. nasir for helping in farm operations. authors’ contribution si: conceptualization, data curation, investigation and writing: original draft. ma: conceptualization, funding acquisition, planning experiment, investigation, supervision. fzak: formal analysis, writing: original draft. ni: writing: review & editing. fn: writing: review & editing. references abrol, d.p. 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(2012). growth, yield and quality of bird-resistant sunflower cultivars found in genetic resources. plant production science, 15: 23-31. doi: 10.13102/sociobiology.v62i4.770sociobiology 62(4): 494-505 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 effect of fire on ant assemblages in brazilian cerrado in areas containing vereda wetlands introduction the brazilian cerrado is a mosaic of vegetation types, with interspersed savanna formations, woodlands, grasslands, gallery forests and wetland vegetation, whose composition is mainly determined by edaphic conditions, although heavily influenced by fire (eiten, 1972; vasconcelos et al., 2009). fire in cerrado is an ancient phenomenon, evidenced by the presence of charcoal samples dated between 27,100 to 41,700 years b.p. (vicentini, 1993). fire is considered as one of the most influential factors defining the cerrado’s physiognomy (sato, 2003). however, the influence of fire is not necessarily negative, and van-wilgen (2009) pointed that a good deal of vegetation heterogeneity in cerrado may be positively influenced by fire and other intermediate disturbance regimes. abstract cerrado is a biome whose evolution is intimately influenced by constant fire events. although many species are capable of dealing with this predictable impact, many others may be negatively affected, resulting in community changes after fire. using ants as bioindicators of changes in biodiversity and environmental conditions, this study evaluated the effects of fire in two cerrado vegetation types: “cerrado” sensu stricto, a xeric savanna, and wetland “veredas”, a mesic vegetation on floodable soils, where water concentrates and ultimately flows towards rivers. we examined the effects of fire on both habitats in two independent sites, but with special consideration to the wetlands, which are not fully adapted to fire. ant sampling was conducted twice before and twice after a fire event, using 288 baits and 416 pitfall traps (soil and arboreal), and 16 hand collections along three random replicate transects per area. ant species richness and abundance were resilient to fire, and exhibited a remarkably consistent seasonal variation at unburned and burned sites. on the other hand, the fire markedly changed the ant species composition. in the wetlands, the fire spread underground due to the high concentration of peat. the impact on ant assemblages was substantial and visually perceptible for some species like camponotus rufipes, which suffered a considerable reduction in the number of individuals after fire in this habitat. in the cerrado, a similar result was observed for crematogaster nr. obscurata, which disappeared after fire. the wetland vegetation having little adaptation to fire, plus low resilience in the ant community resulted in a severely changed fauna, both in guild predominance and species composition, and return to an original state is uncertain. sociobiology an international journal on social insects cb costa-milanez1, ff ribeiro1; pta castro1, jd majer2, sp ribeiro1 article history edited by kleber del-claro, ufu, brazil received 02 march 2015 initial acceptance 11 june 2015 final acceptance 12 july 2015 keywords mesic environments, bioindication, formicidae, wetlands. corresponding author cinthia borges da costa milanez department of geology universidade federal de ouro preto 35.400-000, ouro preto-mg, brazil e-mail: cborgesdacosta@gmail.com the intensity and frequency of fire are determinants of its actual effect on the ecological community. the more frequent the fire, the less intense it is, because there is less accumulation of fuel on the ground. nevertheless, a mild fire pattern consumes herbaceous substrate and seedlings, which tend not to reach adulthood in sites constantly burned. fire drastically alters microclimate and habitat heterogeneity, leading to modifications in community dynamics, along with changes in species diversity and composition (kapos, 1989). on the other hand, the total exclusion of fire in fire-prone ecosystems could reduce resilience, once the accumulation of fuel enables favorable conditions for very intense fires (ramos-neto & pivello, 2000). despite the long-term adaptation of cerrado communities to fire, such as through thick rhytidome, tubers, bulbs, corms and underground rhizomes, it could have a research article ants 1 universidade federal de ouro preto, ouro preto, minas gerais, brazil 2 curtin university, perth, australia sociobiology 62(4): 494-505 (december, 2015) 495 negative impact at an ecological time scale. for instance, there are records of 97% plant biomass destruction, local extinction of tree species that are only recruited by seeds, and severe release of greenhouse gases into the atmosphere (cortês et al., 2011). moreover, the impact of fire on seed dispersers has hardly been studied (hoffmann, 1996; camargos et al., 2013). changes caused by fire on ant communities may result in substantial indirect impacts on the vegetation at a mid-term scale (carvalho et al., 2012; beaumont et al., 2013). research on ant community responses to the impact of natural or prescription fire suggests that ants are resilient and resistant to vegetation burning (knoechelmann & morais, 2008; andersen et al., 2012; frizzo et al., 2012; alves-silva & del-claro, 2013). however, to our knowledge no study has taken into consideration ant communities inhabiting ecosystems such as veredas that are sensitive and with low resilience to the fire. the effect of fire can be more pronounced in ecosystems that are sensitive to change and which have little capacity for regeneration, such as wetland communities (alves & silva, 2011). this is the case of veredas situated inside the cerrado domain, which are wetland ecosystems formed on sandy soils with high concentrations of peat, and which are responsible for recharging important aquiferous reservoirs of the central brazilian plateau. veredas are ecologically and socially important wetland systems (oliveira & ferreira, 2007; ferreira, 2009), initially colonized by an endemic species of palm, the buriti (mauritia flexuosa l. f.). the extent of the impacts of fire on the cerrado is well known (coutinho, 1978; 1982; 1990; hoffmann, 1996; alves & silva, 2011; cortês et al., 2011), but the effect of fire on veredas and its entomofauna is poorly understood. there is an urgent need to improve our knowledge of these impacts, as fire frequency escalates yearly due to climate change (i.e. longer dry seasons, smaller time interval between el niño phenomena) and the expansion of agriculture and cattle farming. although soil fauna in cerrado is mostly hypogeal (coutinho, 1978; castanheira de morais & benson, 1988), and thus well protected from mild fires, the direct effect of fire on the arboreal fauna, or the indirect effect on soil fauna due to litter destruction, has hardly been studied. ants are a taxonomic assemblage that exhibits substantial responses to impacts such as fire (philpott et al., 2010) and have also been extensively studied in the cerrado, but their responses to fire in terms of abundance and species composition are minimally understood (frizzo et al., 2007; frizzo et al., 2012). we studied the effects of fire in the wetland vereda and cerrado sensu stricto using ants as tools to evaluate changes in biodiversity and environment conditions. we predict that ant response will be stronger in the wetlands than in the cerrado, due to their lower resilience to loss of heterogeneity, along with lack of plant species adapted to fire events. we further predict that with the death of certain ant colonies previously present, generalist ant species, especially those nesting in soil, can quickly colonize the burned sites from nests outside the directly affected area. with the local extinction of rare and sensitive species, an increase in foraging activity by generalist species is expected, leading to an impoverishment of the fauna. material and methods study area the area of cerrado studied in this project is located in the northwest region of minas gerais state. the veredas are located in the municipalities of são gonçalo do abaeté (18°29’10.61”s and 045°40’03.01”w) and andrequicé (a district of três marias 18°27’08.69”s and 044°50’51.12”w. the climate in the region of the high–mid san francisco river basin is characterized aw, according to köppen’s climatic divisions, being tropical and seasonal, with an average temperature of 24°c, prevalent rainfall in summer, dry winters, and an average rainfall of 1000 mm to 1800 mm (ribeiro, 2007). the field work was carried out in two independent locations: a preserved vereda habitat (curral das éguas, control site) with an adjacent area of cerrado habitat (sensu stricto); and a vereda habitat recently disturbed by fire (são josé, burned site) with a neighboring cerrado habitat (sensu stricto) area which was also burnt at the same time. both vereda habitats are geomorphologically classified as hillside (boaventura, 1978), and have vegetation predominantly composed of plants from the alismataceae, arecaceae, asteraceae, cyperaceae, and melastomataceae. the fire occurred during the prolonged and intense dry season of 2011, under the influence of an el niño year. within each habitat unit we established three replicated transects (fig. 1). hereafter, the two habitat types are referred to as: we – wetland habitats, under the direct influence of yearly floods, and ce – cerrado habitats surrounding the wetland. ant sampling design ant sampling was conducted in may 2010 and february 2011, before the fire, then in october 2011 and march 2012, after the fire (as the fire was accidental, we do not know the exact fire date or if it lasted for more than one day). three replicated 20 m linear grids, with 10 m between replicates, were established in each habitat (wetland and cerrado; see fig. 1). the distance of 10 m between transects was set in order to establish independent replicates, as in ribeiro et al. (2013). we used three complementary sampling methods, namely baits (soil 192 and arboreal 96 making a total of 288), pitfall traps (soil 288 and arboreal 128 making a total of 416), and hand collections (a total of 16, one of each habitat for sampling). pitfall traps was placed so as to alternate with baits, 5 m apart from each other along the linear grid (in each line grid three pitfall traps and two baits in soil). the ground pitfall traps were adapted from the method used by holway (2005), but traps were left in the ground for 3 days. in order to cb costa-milanez et al. – effect of fire on ant assemblages496 minimize affects of water flow, which hinders the installation of the soil pitfalls traps during the rainy season, the traps were placed on non-flooded sediments when close to the wetland. arboreal pitfall traps followed the methodology proposed by majer (1983) and installed only in buritis (wetland). the baits followed the method used by espírito santo et al. (2012), the arboreal baits was installed only in cerrado. the hand collection was performed for 1 hour in each habitat, as in costa et al. (2010). sampled ants were sorted and identified to genus level, and then separated into morphospecies or species by consulting a specialist. the reference collection is stored at the department of biodiversity and evolution, iceb, federal university of ouro preto (ufop), brazil. data analysis ant species richness and abundance were calculated from the combined transect pairs, using all the information from all sampling methods together. the ant species richness and abundance data were compared by a nested analysis of variance. we considered “vereda” (with levels control and burned), and “habitat” (with levels cerrado and wetland) as fixed factors, the effect “fire” (with levels before and after), and the transects considered as nested fixed and random blocks respectively. using ant species presence/absence data, non-metric multi-dimensional scaling (nmds) was performed with a jaccard similarity matrix to produce ordinations. analyses of similarities (anosim – one way) were performed to test for differences in ant composition for “habitat” (with levels cerrado and wetland), “vereda ” (with levels control and burned), and “fire” (with levels before and after). similarity percentages (simper) identified the contribution of ant species to the dissimilarity between selected factors. the above analyses were performed using past version 2.04 (hammer et al., 2001). in order to further investigate the change in species composition, we performed cochran’s q test, which takes the number of common and unique species among treatments, comparing the factors “habitat” (with levels cerrado and wetland), “vereda” (with levels control and burned), and “fire” (with levels before and after). hence, this analysis verifies the influence of rare (singletons and exclusive) species distribution among the sites, while anosim focuses on general patterns caused by dominant species. results a total of 20,519 ants were collected from all methods, belonging to seven subfamilies, 33 genera and 141 species (table s1). the ant assemblages sampled were predominantly composed of species from the subfamilies myrmicinae and formicinae, with 72 (50%) and 30 (21%) species, respectively. before fire, 10,609 ants were collected, distributed in 113 species (79%). after fire, 9,910 ants were collected, distributed in 110 species. overall ant species richness did not differ significantly when comparing unburned and burned veredas (anova, f(1,20) = 0.204; p = 0.655; fig 2 a; table 1), and neither did abundance, which followed a similar pattern to richness (anova, f(1,20) = 1.098; p = 0.307; fig 2 b; table 1). however, ant species richness tended to increase after the period when fire occurred, but the pattern was observed both in the unburned and burned sites, suggesting a response to season (anova richness x season, f(1,20) = 8.269; p=0.009; table 1). before the fire, the most abundant species in the cerrado was crematogaster nr. obscurata, and in the wetland habitat it was pheidole gertrudae. after fire, in both unburned and burned cerrado habitats, crematogaster torosa and cr. acuta were the most abundant species, while in the unburned and fig 1. layout of transects for ant sampling in the veredas, each comprising paired 20 m transects set of 10 m apart (without scale). sociobiology 62(4): 494-505 (december, 2015) 497 burned wetland habitat atta sexdens and solenopsis invicta were the most abundant species, respectively. after the fire this increased to 71 species, with 44 species being common to samples taken before and after the fire. some opportunist species, such as cr. acuta, cr. torosa and forelius maranhaoensis appeared after the fire. therefore, the fire affected the ant species composition in the cerrado and in the wetland (figure 3 a and b, tables 2 and 3). for both habitats, there were significant differences in species composition between unburned and burned sites, whenever comparing before or after the time when fire occurred (anosim, cerrado: r = 0.64; p = 0.0001; wetlands: anosim, r = 0.34; p = 0.0001). hence, those sites were distinct in fauna regardless of fire, and continued to be so after it. on the other hand, the species composition between unburned sites before and after the time when the fire occurred did not differ significantly, while it was significantly different before and after fire for the burned sites of both cerrado and wetlands (nmds cerrado: stresss 0.23, fig. 3 a; nmds wetland: stress 0.34, fig. 3 b). fig 2. richness (a) and abundance. (b) of ants in wetland and cerrado habitats for each vereda sampled before and after the fire. bars are standard deviations. 0 5 10 15 20 25 30 35 dry wet dry wet dry wet dry wet before fire after fire before fire after fire cerrado vereda r ic hn es s (m ea n +/ s ta nd ar d er ro r) control burned 0 100 200 300 400 500 600 dry wet dry wet dry wet dry wet before fire after fire before fire after fire cerrado vereda a bu nd an ce (m ea n +/ s ta nd ar d er ro r) control burned a b source sum of squares df f – statistics p value richness*fire 0.0061 1 0.20491 0.65565 abundance*fire 0.2487 1 8.26964 0.00934 season*fire 0.1490 1 1.09807 0.30718 error 20 table 1. analysis of variance for richness and abundance of ants in control and burned veredas. ant species richness in the vereda wetlands before the fire reached 56 species, while after the fire the number of species decreased to 38, with only 26 species represented in both dates. after the fire, a drastic reduction in abundance of camponotus rufipes was observed and colonization by species such as s. invicta occurred in the wetland habitat. by contrast, the cerrado had 68 species before the fire, but -0,25 -0,2 -0,15 -0,1 -0,05 0 0,05 0,1 0,15 0,2 0,25 -0,4 -0,2 0 0,2 0,4a xi s 2 axis 1 control site before burned site before control site after burned site after -0,3 -0,2 -0,1 0 0,1 0,2 0,3 0,4 -0,4 -0,2 0 0,2 0,4 0,6 a xi s 2 axis 1 control site before burned site before control site after burned site after fig 3. distribution of (a) cerrado habitat and (b) wetland habitats on the nmds ordination, based on jaccard similarity, derived using ant presence/absence data. a b cb costa-milanez et al. – effect of fire on ant assemblages498 the most influential species in discriminating cerrado habitats before fire were wasmannia auropunctata (1.82%), pheidole sp.27 (1.56%), and a. sexdens (1.32%). after the fire, the major discriminating species were pheidole sp.34 (1.94%), pheidole sp.27 (1.65%), and ectatomma edentattum (1.65%). a similar effect could be detected up to five months after the fire in our study. species composition changed dramatically, with an increasing number of generalist species offsetting the loss of community components. despite the invasion of generalist species and disappearance of sensitive ones in all burned sites, the cerrado vegetation manintained a higher proportion of its original fauna compared with the wetlands (65% in the cerrado against 46% for wetlands). as a consequence, the overall summing up of species in cerrado was positive, with an addition of five percent against an overall loss of 38% of species diversity in the wetlands. the soil of cerrado is comprised of quartz sands with a high concentration of iron and aluminum (gomes, 2006), which are features that prevent the fire from spreading underground, and this may be related to the well known resilience of cerrado communities to fire. the vegetation has several adaptations to fire, such as the possession of thick rhytidome, and xylopodium, tubers, bulbs, corms and underground rhizomes, which assist plants to survive the fire (bond & keeley, 2005; silva & batalha, 2010). the species of ants with colonies no deeper than 5 cm in the soil have lower chances of surviving fire, due to exposure to flames and associated high temperatures (castanheira de morais & benson, 1988), which may range from 85°c to 840°c in the air, and 29°c to 55°c in the ground at 1 cm depth. nevertheless, ants with deeper nests are more likely to survive, as natural fires do not cause large elevations in the underground temperatures (coutinho, 1978). this is the case with the attines (atta and acromyrmex), which build deep nests and, due to their architecture, are able to stabilize fluctuations in temperature and humidity (farji-brener, 2000; carvalho et al., 2012). a few other noteworthy cases may contribute to clarifying overall fire effects. for instance, in the present case ca. rufipes suffered a considerable reduction in the number of individuals, especially in the wetland habitat. this species nests on the ground, forming shallow and tall mounds of litter and fine debris (similar to the temperate formica rufa l.) that are highly inflammable. however, the colony may survive due to arboreal satellite nests, as described by espírito santo et al. (2012). such strategies may reflect the general adaptation of typical savanna species to survive common fire events. however, after fire a further decline in the colonies may be observed, in part due to the reduction of food resources used for this ant, or because the fire actually burns most of the colonies or the cavity-nesting ant sites (fagundes et al., 2015). similar fire effects may have been related to the observed decline of the genus crematogaster. crematogaster nr. obscurata almost disappeared from the cerrado vegetation, which was then colonized by cr. acuta and cr. torosa. the genus crematogaster is a cosmopolitan lineage of myrmicinae, and the majority of species nests on trees (hölldobler & wilson, 1990), which reduces insect herbivore attacks (del-claro & marquis, 2015). however, cr. torosa and cr. acuta occur primarily in open and dry areas, which are often highly disturbed, and nest rvalues / p – values unburned site before burned site before unburned site after burned site after unburned site before 0/0 burned site before 0.478/0.0014 0/0 unburned site after 0.276/0.0094 0.862/0.0023 0/0 burned site after 0.860/0.0021 0.738/0.0024 0.827/0.0026 0/0 table 2. comparison of the variation of similarities (anosim) of ant species composition among the cerrado sites before and after fire. the species which contributed strongly to this separation in the wetland habitat in burned sites before fire were pheidole sp.4 (2.15%), camponotus melanoticus (1.81%) and p. gertrudae (1.61%), and after the fire they were dorymyrmex jheringi (2.01%), pheidole sp.24 (2.00%) and pseudomyrmex termitarius (1.95%). finally, when taking into consideration the relative importance of singletons and exclusive species in a comparison between the sites, only the ant species composition of wetlands was significantly affected by the fire (cochran q test: q = 7.714; p = 0.005). r values / p – values control site before burned site before control site after burned site after unburned site before 0/0 burned site before 0.221/0.0346 0/0 unburned site after 0.185/0.0618 0.450/0.0024 0/0 burned site after 0.652/0.002 0.225/0.0258 0.570/0.0023 0/0 table 3. comparison of the variation of similarities (anosim) of ant species composition among the wetland site before and after fire. discussion fire promotes changes in biomass, species diversity and energy flux (peterson et al., 1998). in australia, york (1994) showed that one week after a fire, generalist and opportunistic ant species moved into the disturbed areas, thereby increasing the number of species and individuals. on the other hand, fire reduces the abundance of other arthropod groups, such as collembolans, mites, and beetles, which are killed or forced to disperse to unburned sites (gillon, 1983). sociobiology 62(4): 494-505 (december, 2015) 499 in dead wood (antweb, 2012), a behavior that clearly may favor their colonization after fire. in contrast, ca. melanoticus, another species typical of the brazilian savanna, did not change in abundance after the fire. therefore, the fire can interfere with the survival rate of certain species, destroying their basic niche requirements, but can confer advantage to other species better adapted to live in more xeric conditions (frizzo et al., 2011). the fire has a strong potential to damage wetlands, where the plant species tend to have no mechanisms for fire protection (maillard et al., 2009). furthermore, the wetland’s soils are rich in peat, which can lead to underground fires, with high temperatures causing severe damage to plant roots, which can further increase the rate of local extinction of many species (cortês et al., 2011). in our work, the impact of fire was more substantial in the wetland habitat, resulting in a more intense post-fire colonization by generalist ant species such as p. gertrudae, d. jheringi and s. invicta. these genera live in various habitats, ranging from the humid tropics to deserts (naves, 1985; helms & vison, 2002), frequent the litter and soil, have populous colonies, and are aggressive, competitive and omnivorous. the combination of these characteristics makes them effective at colonizing many habitats and excluding competitors (mcglynn, 1999; ramos et al., 2003). nonetheless, a dramatically negative fire effect was detected on the genus ectatomma. after fire in the wetlands, seven species of this genus disappeared, with e. brunneum smith being the only species of ectatomma to be sampled again in a burned wetland site. this species lives in secondary forest, is a generalist predator of terrestrial arthropods, and has preference for termites and other species of ants (tofolo & giannotti, 2005). in contrast, in the more resilient cerrado habitat four species of this genus survived the fire. it is worth noting that e. edentatum was hardly affected by the fire. the fact that e. edentatum is an aggressive, generalist predator or scavenger, foraging in vegetation or on the ground (silvestre et al., 2003), may have contributed to the maintenance of its abundance after the fire event. our results suggest that the wetlands are less resilient than the surrounding cerrado to the impact of fire. the lack of plants adapted to fire in this vegetation, along with the soil traits that favor intense fire on and beneath the ground, may have reduced soil meso and macro-fauna. fire promotes the formation of a surface crust, which reduces the porosity and water infiltration into the soil, leading to drying out of the wetland veredas, with immediate consequences to the soil fauna (spera et al., 2000). although some species benefit from the fire, some unique, sensitive, species may disappear without substitutes for their vacant niches. further, ecological functions may be seriously affected by increasing populations of dominant, generalist species (andersen et al., 2012). acknowledgments we thank flávio de castro and rodrigo feitosa for identifying the ants sampled in this project. roberth fagundes for the help in statistics. glênia lourenço-silva, diego anjos, bruno gomes and carlos augusto were particularly helpful in field. financial support for cb costa-milanez was provided by conselho nacional de desenvolvimento científico e tecnológico (cnpq), and by fundação de amparo à pesquisa do estado de minas gerais (fapemig). sp ribeiro and pta castro are granted researcher by cnpq. ibama provided the collect permits. references alves, r.j.v. & silva, n.g. 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(1994). the long-term effects of fire on forest ant communities: management implications for the conservation of biodiversity. memoirs of the queensland museum, 36: 231-239. cb costa-milanez et al. – effect of fire on ant assemblages502 species areas unburned site burned site before after before after we ce we ce we ce we ce dolichoderinae azteca sp.1 6 4 1 4 dolichoderus bispinosus olivier 1 12 dolichoderus germaini emery 6 dolichoderus lutosus smith 13 dorymyrmex jheringi forel 424 31 18 730 2 60 45 dorymyrmex sp.1 5 20 dorymyrmex sp.2 6 2 45 1 12 192 forelius brasiliensis forel 2 forelius maranhaoensis cuezzo 1 1 239 gracilidris pombero wild & cuezzo 2 2 linepihema anathema wild 3 10 linepithema humile mayr 79 16 5 6 17 9 10 6 linepithema micans forel 17 26 28 14 5 1 1 6 ecitoninae labidus praedator smith 1 5 neivamyrmex pseudops forel 1 6 neivamyrmex sp.2 3 ectatomminae ectatomma brunneum smith 46 102 24 26 10 25 16 ectatomma edentatum roger 2 2 6 52 52 ectatomma lugens emery 2 2 1 1 4 ectatomma opaciventre roger 5 1 11 19 ectatomma permagnum forel 3 5 1 1 4 1 ectatomma planidens borgmeier 37 1 1 1 54 27 ectatomma nr. suzanae almeida 3 2 gnamptogenys acuminata emery 1 1 gnamptogenys striatula mayr 1 1 formicinae brachymyrmex sp.1 10 35 33 17 28 10 brachymyrmex sp.2 2 55 1 2 2 2 brachymyrmex sp.3 7 camponotus arboreus smith 38 12 11 1 20 20 4 camponotus atriceps smith 4 10 39 20 7 22 28 35 camponotus blandus smith 103 157 73 479 4 19 62 camponotus bonariensis mayr 1 camponotus cameranoi forel 1 camponotus cingulatus maur 1 11 1 5 10 2 7 camponotus crassus mayr 42 179 91 77 42 521 34 172 camponotus leydigi forel 2 4 3 16 1 6 3 7 camponotus melanoticus emery 8 15 11 128 1 20 4 25 camponotus rufipes fabricius 25 6 3 2 183 10 13 8 camponotus sericeiventris guérin-méneville 7 2 4 appendix. ant species sampled in theunburned and burned sites (before and after the fire) in wetland (we) and cerrado (ce) habitats. sociobiology 62(4): 494-505 (december, 2015) 503 species areas unburned site burned site before after before after we ce we ce we ce we ce formicinae (continuation) camponotus sexgutattus fabricius 2 camponotus trapezoideus mayr 4 1 1 camponotus (myrmobrachys gr.) sp.1 51 186 51 306 4 19 61 camponotus (myrmothrix gr.) sp.2 7 33 13 32 1 37 2 15 camponotus (myrmothrix gr.) sp.3 2 camponotus (tanaemyrmex gr). sp.1 1 2 camponotus sp.13 2 3 camponotus sp.15 9 36 4 camponotus sp.16 1 camponotus sp.19 1 camponotus sp.20 4 4 1 1 20 camponotus sp.22 1 camponotus sp.23 5 camponotus sp.24 1 nylanderia nr. fulva mayr 10 2 3 myrmicinae acromyrmex balzani emery 11 3 38 1 79 acromyrmex crassispinus forel 3 1 5 3 apterostigma sp.1 1 1 atta laevigata smith 1 atta sexdens linneaus 104 56 177 16 1 94 12 124 cephalotes atratus lineaus 117 1 cephalotes borgmeieri kempf 13 cephalotes cristopherseni andrade & baroni urbani 5 cephalotes eduarduli forel 1 22 15 cephalotes maculatus smith f. 3 1 17 6 cephalotes pusillus klug 25 44 2 17 2 53 1 43 crematogaster acuta fabricius 14 398 18 1412 crematogaster limata smith f. 3 1 crematogaster torosa mayr 10 316 797 211 1022 crematogaster nr. obscurata emery 1255 15 1680 1 65 crematogaster sp.2 2 crematogaster sp.3 17 cyphomyrmex rimosus spinola 4 4 3 1 1 cyphomyrmex peltatus kempf 1 1 21 cyphomyrmex sp.4 1 mycocepurus goeldii forel 2 4 21 7 29 myrmicocrypta sp.1 1 8 nesomyrmex spininodis mayr 1 pheidole gertrudae forel 666 23 39 4 pheidole (flavens gr.) sp.1 1 20 2 13 15 1 5 pheidole (flavens gr.) sp.3 1 6 appendix. ant species sampled in the sites unburned and burned (before and after the fire) in wetland (we) and cerrado (ce) habitats (cont.). cb costa-milanez et al. – effect of fire on ant assemblages504 species areas unburned site burned site before after before after we ce we ce we ce we ce myrmicinae (continuation) pheidole sp.1 7 5 pheidole sp.6 1 3 2 17 4 pheidole sp.7 2 3 2 pheidole sp.19 4 33 1 24 6 31 5 39 pheidole sp.21 1 16 pheidole sp.22 4 11 24 7 pheidole sp.23 5 1 23 50 10 pheidole sp.24 69 75 22 41 16 44 17 79 pheidole sp.25 9 pheidole sp.26 15 10 16 158 37 19 pheidole sp.27 3 33 239 pheidole sp.28 18 8 pheidole sp.29 5 pheidole sp.31 2 pheidole sp.32 2 12 18 pheidole sp.33 3 pheidole sp.34 1 15 8 pheidole sp.35 1 19 1 10 3 pheidole sp.36 6 pheidole sp.37 175 pheidole sp.38 3 pheidole sp.39 5 1 63 pheidole sp.40 1 11 pheidole sp.41 28 4 pheidole sp.42 1 pheidole sp.43 1 7 30 34 pheidole sp.44 17 pheidole sp.45 15 62 23 15 9 pheidole sp.46 4 pheidole sp.47 1 pheidole sp.48 1 2 9 29 1 pogonomyrmex naegelii emery 1 5 3 1 35 rogeria sp.1 1 1 2 4 sericomyrmex parvulus forel 1 solenopsis invicta buren 317 13 9 582 152 165 solenopsis (geminata gr.) sp.1 3 3 solenopsis sp.1 2 1 5 1 2 111 25 solenopsis sp.8 6 trachymyrmex compactus mayhé-nunes e brandão 1 1 trachymyrmex dichrous kempf 1 7 trachymyrmex fuscus emery 2 4 17 1 appendix. ant species sampled in the sites unburned and burned (before and after the fire) in wetland (we) and cerrado (ce) habitats (cont.). sociobiology 62(4): 494-505 (december, 2015) 505 species areas unburned site burned site before after before after we ce we ce we ce we ce myrmicinae (continuation) trachymyrmex (opulentus gr.) sp.1 12 trachymyrmex sp.1 1 3 trachymyrmex sp.3 2 2 wasmannia auropunctata roger 471 183 7 16 50 1 7 wasmannia rochai forel 2 ponerinae anochetus (inermis gr.) sp1 1 odontomachus haematodus emery 1 odontomachus meinerti forel 1 3 1 pachycondyla ferruginea smith 1 1 pachycondyla obscuricornis emery 20 15 4 12 10 pachycondyla stigma fabricius 1 pachycondyla villosa fabricius 1 4 2 pseudomyrmecinae pseudomyrmex acanthobius emery 1 1 1 pseudomyrmex gracillis fabricius 1 1 pseudomyrmex termitarius smith 5 7 18 27 2 1 pseudomyrmex nr. urbanus smith 3 1 2 2 pseudomyrmex oculatus gr. sp.1 5 1 pseudomyrmex sp.2 1 5 pseudomyrmex sp.3 1 1 4 1 pseudomyrmex sp.8 1 appendix. ant species sampled in the sites unburned and burned (before and after the fire) in wetland (we) and cerrado (ce) habitats (cont.). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.740sociobiology 62(4): 519-526 (december, 2015) the search rate of the african weaver ant in cashew introduction cashew (anacardium occidentale l.) is the fourth most valuable export crop after coffee, cotton, and tea in tanzania (martin et al., 1997). the crop is attacked by a variety of insects that feed on shoots, panicles, and fruits. among insect pests the mosquito bug (helopeltis schoutedeni reuter), the leaf-footed bug (pseudotheraptus devastans, dist.), and the coreid bug anoplocnemis curvipes (f.) are most important (dwomoh et al., 2009). the damage caused by the bugs reduces the cashew nut quality and yields (martin et al., 1997), and consequently the farmers’ incomes (peng, 2008). as most ant species, the canopy dwelling weaver ants, oecophylla sp., require animal protein and honeydew in their diet (way, 1954a, lokkers, 1990). they are generalist predators preying upon a great variety of insects, some of which are pest species (vanderplank, 1960; way & khoo, 1992). the weaver ants occur in many fruit orchards in tropic asia, australia and africa where they are important biological abstract oecophylla longinoda is a species of eusocial colony living ants that prey upon other insects to feed their larva. many of these insects are considered pests. an ecosystem model of the interactions between an o. longinoda colony and its potential prey is under construction by the team behind this article, and it is unknown which functional response equations are useful for eusocial insect colonies. we investigated the search rate of o. longinoda using artificial feeding experiments in a tanzanian cashew (anacardium occidentale l.) orchard to determine the search efficiency of the ants, and to assess which functional response equation can be used for eusocial insects. artificial feeding experiments consisted of providing each of ten colonies 50 pieces of sardine (175 mg dry weight in average) in cashew trees at time 0 and counting the remaining food items at four intervals of 45 minutes during a seven day period. the equations of gutierrez-baumgärtner, lotka-volterra, and nicholson-bailey were assessed and the nicholson-bailey equation was found to be most suitable. the gutierrez-baumgärtner equation is useful if the demand for storage can be assessed. a large variation in search rates was found between the observed colonies and this problem is discussed in relation to oecophylla sp. efficacy as a biological control agent. sociobiology an international journal on social insects s henriksen1, ja axelsen1, kh lemming1, m mwatawla2, j offenberg1 article history edited by gilberto m. m. santos, uefs, brazil received 23 december 2014 initial acceptance 27 may 2015 final acceptance 28 october 2015 keywords oecophylla longinoda, biocontrol, eusocial insect. corresponding author jorgen aagaard axelsen institute of bioscience aarhus university vejlsovej 25, dk-8600 silkeborg, denmark e-mail: jaa@dmu.dk control agents in fruit trees, such as mango and cashew (way & khoo, 1992; peng et al., 1999a; offenberg et al., 2004; peng & christian, 2006; dwomoh et al., 2009) dwomoh et al. (2009) showed that o. longinoda was as effective in protecting cashew against pest insects as insecticides such as l-cyhalothrin and cypermethrin + dimethoate. several studies suggest that with appropriate management weaver ants can significantly increase fruit yield and reduce the use of pesticides (van mele, 2008). for instance, research done on vietnamese citrus showed that farmers who managed o. smaragdina in their orchards spent on average half the amount of money on pesticides compared with those who did not (van mele & cuc, 2000). this suggests a significant economic potential of using weaver ants in pest control. in australia, o. smaragdina has been used to control major cashew pests (peng et al., 1999a) with increases in grower revenue by 1500 aud/year per ha. (peng et al., 2004). based on the similarities of the two oceophylla sp., it is reasonable to believe that o. longinoda can provide similar research article ants 1 aarhus university, aarhus c, denmark 2 sokoine university of agriculture, morogoro, tanzania s henriksen et al. – the search rate of the african weaver ant in cashew520 benefits to cashew producers in africa. two oecophylla species are known, o. longinoda in africa and o. smaragdina in asia and australia and it has been suggested that they can be regarded as one species in many respects (way, 1954a; way & khoo, 1992). according to peng et al. (2013), it takes about three years for a weaver ant colony to reach an adequate size to be used successfully in biocontrol. the growth of a colony is affected by the number of founding queens (peng et al., 2013), augmenting colony size by transplanting pupae from other nests (peng et al., 2013), by the availability of prey, and by the capacity of the colony to capture the prey. one of the factors affecting the capacity of the colony to acquire prey is the search capacity of the colony. thus the search rate is a crucial parameter in the understanding of population dynamics of a colony and its capacity as biocontrol agent. axelsen, ja et al. (unpublished) developed a physiologically based metabolic pool simulation model (gutierrez, 1996) to simulate the growth of weaver ant colonies as a function of temperature, prey availability and artificial feeding and this model was the starting point for our field study. in predator–prey models the functional response equation is a core element in simulating the interactions between predator and prey populations, and the search efficiency parameter is an important factor. information on the functional response and search efficiency of eusocial insects such as o. longinoda is crucial in modeling the trophic interactions. the concept of functional response (holling, 1959) was based on the actions of an individual consumer and may not be applicable to eusocial insects as their behaviour do not only reflect the demand of the single ant, but the demand of the entire colony (schmid-hempel et al., 1993) which can be regarded as a super-organism (hölldobler & wilson, 2009). the objective of this study was to investigate the prey search rate of o. longinoda colonies in a cashew orchard in tanzania in order to identify the most appropriate functional response equation to model the food acquisition by o. longinoda colonies. this was done by following the removal of experimentally provided small pieces of dried sardines that were used to simulate prey from trees in the cashew orchard, and using these data to estimate the search rate using the three different functional response equations, gutierrez-baumgärtner (gutierrez et al., 1984), lotka – volterra (lotka, 1925; volterra, 1931), and nicholsonbailey (nicholson & bailey, 1935). methods and materials the experiment was carried out in a cashew orchard at the naliendele agricultural research station, mtwara, tanzania (10o21’09.60”s, 40o10’16.24”e). the area is situated on sandy soils and is characterized by an equatorial climate with two rainy seasons each year and an annual rainfall of 900 – 1200 mm. the surrounding area is mainly used for agriculture where cassava is the main crop and cashew is the main cash crop. the cashew trees used in this study were on the average 5.9 m tall (se = 0.4), had an average crown diameter of 10.9 meters (se = 0.7), and an average stem circumference of 1.6 meters (se = 0.1). most adjacent trees had overlapping branches. ten mature weaver ant colonies (based on the presence of large worker individuals that are not present in young colonies) were identified and used in the experiment. a weaver ant colony usually consists of many nests scattered in the tree canopy, and a colony may cover more than one tree. fifty pieces of bait (simulating a prey item) were placed on a single tree per colony. prey items consisted of small pieces of dried sardines of approximately equal size, with an average dry mass of 87.5 mg. five pieces of duct tape were wrapped around 5 different branches in each tree, with the sticky surface pointing outwards. the tape was 1.6 cm wide and its length depended on the thickness of the branch. on each piece of tape we placed 10 pieces of fish, giving a total of 50 pieces of fish per tree. each piece of tape was placed on the same location in the tree throughout the observation period. the tape was only replaced when needed (when it was wet or when filled with fish scales), so not all pieces of tape were replaced at the same time. data collection consisted of counting the number of fish pieces on tapes in each of the 10 trees at intervals of 45 minutes for 3 hours giving data from 4 intervals per day. the experiment was repeated 7 times on different days between 10 am and 2 pm at the beginning of the long rainy season from the 29th of february to the 8th of march 2012. the background disappearance rate of prey items was investigated on five randomly selected trees kept free of ants. the remaining prey items were counted using the method described above. other predators beside o. longinoda or rain/wind were assumed to have removed the prey. calculation of the search rate (s) was done by the gutierrez-baumgärtner functional response model (gutierrez, 1996) (1) (1) which can be rearranged to (2) where m’ is the available biomass of prey, m* is the food collected by predator, d is the predator demand for prey (not including sugar), and s is the search rate of predator (gutierrez., 1996). all biomasses were dry weights. the search rate (s) was estimated by equation (2) at fixed time intervals of 45 min., m’ was controlled (biomass of fish provided), m* was measured (biomass of fish removed), and d was estimated through calculation of the daily colony demand (equation 3): (3) sociobiology 62(4): 519-526 (december, 2015) 521 dgrowth depends on the amount of brood present. brood consists both of larvae and pupae but since pupae do not grow (hölldobler & wilson, 1990) and was excluded from this calculation. dgrowth was calculated according to equation (4). (4) where mlarvae,t was the mass of larvae at the time t, nants,t was the estimated number of ants in the colony at time t (table 1 and table 3), ratiol/w was the proportion of larvae compared to workers, mlarvae was the average dry weight of larvae, rg was the maximum growth rate per unit of physiological time, and δt was the time in physiological time units per day (taylor, 1971). the expression in the parenthesis of equation 4 calculated the mass of larvae in the colony. the number of ants was estimated according to lim (2007) who have presented a non-destructive method to estimate the abundance of o. smaragdina within a nest, equation (5): (5) where nants is the estimated number of ants in a nest, and v is the estimated volume of a nest. although the equation of lim (2007) concerned o. smaragdina, it was adopted for o. longinoda as the two species are very similar in many respects (way, 1954a; way & khoo, 1992). number of nests per tree was estimated by dividing each tree into sections, and counting all visible nests from the ground. the numbers of nests in each section of the tree were then added up to give the estimated number of nests in each tree. only nests built in the surveyed trees were included in the data collection. many nests were situated high in the tree crowns and it would have been very time consuming to measure their sizes precisely. therefore, the average volume (v) of nests from the plantation was used. the estimation of average nest volume of the plantation was based on 20 randomly picked nests from the investigated trees. the volume was calculated by assuming the nests to be spherical (lim 2007) using equation (6) to calculate the volume of the sphere. (6) where γ (= ½ diameter) is the radius of the nest. the diameter was calculated as the average of length, width and height. dreproduction was expressed only through the reproduction of the queen, as in this species she is the only individual of the colony producing fertilized eggs (hölldobler & wilson, 1990). calculation of this was done by multiplying the total number of ants by the larvae-worker ratio (ratiol/w), divided by the developmental time in days. this gave an estimate of the number of new larvae added to the colony from eggs per day, and the input of new larvae were assumed even with the oviposition rate. multiplying this rate by the dry weight of an egg resulted in the daily demand for reproduction (7): (7) where megg was the average dry weight of an egg and td was the development time. drespiration refers to the respiration of the colony, and because ants mainly meet these demands through sugar sources (caroll & jansen, 1973; way, 1954b) and not through protein this parameter was ignored in the calculation of the demand for prey. dstorage refers in the gutierrez-baungärtner equation primarily refers to the fat reserve of the predator, but in the case with a colony of eusocial insects it refers to the amount of food being stored within the colony for later use. in calculating the prey search rate according to the gutierrezbaumgärtner equation this parameter ignored, as the demand for food to be stored can presently not be quantified. results several ants (5-10 individuals) were observed carrying single food items together from the tape back to the nest, which demonstrated that the ants were capable of removing the prey items from the tape. likewise it was observed that two or more ants often worked together in detaching the food items from the tape by forming a bridge. there was a tendency for higher prey removal (table 1) at the beginning of the observation period compared to the end but the difference was not significant (wilcoxon test, df = 6, p = 0.0895). colony 1, 2, and 9 showed variable prey-removal rates and had days with very low prey-removal compared to the other colonies. the background disappearance rate was 0.0027g prey per hour (1.46% disappearing per hour). grams dry weight date colony 1 colony 2 colony 3 colony 4 colony 5 colony 6 colony 7 colony 8 colony 9 colony 10 average (se) 02.29.2012 2.28 0.96 4.29 3.24 2.80 1.75 1.14 3.68 0.09 2.45 2.27 (0.41) 03.01.2012 0.44 0.61 2.8 2.89 3.85 1.23 1.93 2.71 4.29 4.29 2.50 (0.45) 03.02.2012 0.18 0.44 3.33 3.24 2.36 0.79 1.14 2.71 4.29 3.41 2.19 (0.46) 03.05.2012 0 0.7 2.45 1.84 3.94 1.66 1.31 2.71 0.44 2.8 1.79(0.39) 03.06.2012 0.79 0.26 2.98 2.1 3.68 1.58 0.88 2.71 0.18 3.41 1.86 (0.41) 03.07.2012 0 0 2.71 1.84 3.41 1.66 1.49 0.61 0.09 0.96 1.28(0.37) 03.08.2012 0 0.18 1.49 2.1 2.45 0.35 1.66 0.79 0.18 0.44 0.96 (0.28) total 3.69 3.15 20.05 17.25 22.49 9.02 9.55 15.92 9.56 17.76 12.84 (2.14) table 1. daily removed prey items in grams (dry weight) per experimental period (180 minutes) in each tree. s henriksen et al. – the search rate of the african weaver ant in cashew522 the study trees had different numbers of nests, and the estimated ant density per tree varied from about 1900 to about 27000 (table 2). large workers there are still 980 to continue searching, making the lack of the 20 ignorable. leaving out the handling time in hollings disc equation reduces it to the classical lotka-volterra equation giving a simple type i functional response. thus, based on the holling’s disc equation a new and simple equation (8) to calculate the prey search efficiency was generated (8) where m* was food collected by predator per unit time, m’ is the available biomass of prey, n was the number of workers in a colony, and s was the search rate of predator during the observation period (45 min). another alternative to calculate the search rate was the nicholson-bailey equation (nicholson & bailey, 1935), (9) where m* was food collected by predator per unit time, m’ was the available biomass of prey, n is the number of workers in a tree, and s(δt) was the ants’ search rate per unit time. this equation is not dependent on the demand rate and assumes that predators are searching randomly and that a group of predators may search overlapping areas. the search rate for an experimental period (45 minutes) using the lotka-volterra equation (8) ranged from 0.01 to 8.29 × 10-3 ant-1 and the average search rate (s) was 1.29 × 10-3 ant-1 (table 3), which gives an hourly search rate of 1.72 × 10-3 h-1 ant-1. the search rate for the same period of time determined by the nicholson-baily equation (9) ranged from 0.0 to 8.4 × 10-5 ant-1, an average search rate (s) of 2.3 × 10-5 ant-1 (table 4), and an hourly search rate of 3.1 × 10-5 ant-1. the lower removal of prey items during the last two days was not reflected in a significantly lower search rate (wilcoxon, df = 6, p = 0.597 and p = 0.209 calculating s according to the lotka-volterra and nicholson bailey equations, respectively) but a paired t-test between the first and the last date revealed a significant difference using s-values calculated using the nicholsom-bailey eqution, indicating an effect of date which may have been obscured in the wilcoxon test by the large variance. furthermore, there was a highly significant difference in search rate between the weaver ants in the investigated trees (wilcoxon, df = 9, p < 0.0001) using s-values from both equation. numbers tree nr. nests ants per tree 1 11 3592 2 24 7836 3 31 10122 4 18 5877 5 21 6857 6 9 2939 7 20 6530 8 19 6204 9 6 1959 10 82 26774 table 2. estimated number of worker ants in 10 different trees. it was impossible to calculate the search rate in four out of the ten colonies using the rearranged gutierrez-baumgärtner equation (2) as the acquired prey exceeded the estimated colony demand. this situation caused the argument of the natural logarithm function in equation 2 to return negative values, which is not defined. this made it clear that the gutierrez-baumgärtner equation is not suitable because it is implicitly assumed that the predator does not acquire more than demanded, and hence it is necessary to use a functional response equation that does not rely on colony demand. as an alternative to using the gutierrez-baumgärtner equation in calculating the search rate, a derivation of the holling’s disc equation was tried. hollings disc equation, like the gutierrez-baumgärtner equation, produces a type ii functional response that shows the relationship between the number of prey items eaten during a period of time and prey density, leaving out demand (d), but including handling time (holling, 1959). handling time was in this case ignored as there are so many ants in a colony that the few individuals taking care of prey handling are insignificant and therefore this parameter was ignored. for instance, if 20 ants are handling a prey in a colony housing 1000 table 3. the estimated search rates (×10-3) per observation period (45 min) for each colony on each day using the lotka-volterra functional response equation. search rates (lotka-voltera equation) colony 1 2 3 4 5 6 7 8 9 10 average (se) 02/29 0.48 0.46 4.56 1.80 1.20 0.35 0.47 2.02 0.01 5.45 1.68 (0.59) 03/01 0.09 0.28 2.26 1.60 2.31 0.23 0.83 1.30 0.05 5.90 1.49 (0.56) 03/02 0.04 0.20 2.96 1.92 1.02 0.14 0.47 1.27 0.48 8.27 1.68 (0.79) 03/05 0.00 0.33 1.84 0.86 2.43 0.33 0.55 1.33 0.05 5.90 1.36 (0.56) 03/06 0.17 0.12 2.41 0.99 2.08 0.29 0.35 1.26 0.02 8.29 1.60 (0.79) 03/07 0.00 0.00 2.01 0.86 1.82 0.33 0.64 0.23 0.01 1.61 0.75 (0.25) 03/08 0.00 0.08 0.99 1.01 1.08 0.06 0.73 0.30 0.02 0.69 0.50 (0.14) average (se) 0.11 (0.07) 0.21 (0.06) 2.43 (0.42) 1.29 (0.17) 1.71 (0.22) 0.25 (0.04) 0.58 (0.06) 1.10 (0.24) 0.09 (0.07) 5.16 (1.12) 1.29 sociobiology 62(4): 519-526 (december, 2015) 523 discussion in this paper we present the first attempt to estimate the prey search efficiency of a eusocial insect species. additionally, we present the choice of a functional response equation useful for eusociale insects, which has not been attempted before. the gutierrez-baumgärtner equation generally works well with solitary living insects (gutierrez, 1996), where the demand is a readily quantifiable parameter. in this case the equation could not be used, as o. longinoda is a eusocial colony living insect, were colony demand is difficult to quantify, as the maximum demand for storage seems to be virtually unknown. the storage may be very large as ants are known to carry prey items too large to be consumed immediately to their nests (hölldobler & wilson, 2009), and are capable of concealing large prey items by covering them with leaves (rastogi, 2000). additionally the workers can collect and store food for a short period in their crop, but eventually they digest it (eisner, i957; eisner & brown, i958). workers are also capable of laying trophic eggs serving as a food reserve that can be eaten in times of need (carbaugh & judd, 2011). however, we do not know how much food they conserve in these manners, and a quantification of dstorage will be very difficult with known methods, and therefore the search rate for these eusocial colony living insects can presently not be calculated by the gutierrez-baumgärtner functional response equation. as long as the colony demand is unknown it is necessary to use a functional response equation that does not rely on estimates of the colony demand for storage, and the nicholson –baily equation (equation 9) is a good possibility. the nicholson-baily functional response equation assumes that the population of predators searches their habitat randomly and that they do not affect each other, which may cause their searched areas to overlap. ants are known to communicate on attracting other colony members to larger preys (hölldobler, 1983; wojtusiak et al., 1995; deneubourg et al., 2002) causing an aggregated response to larger prey items (crawley, 1992), and in that case the two assumptions fail. however, this aggregated response only seems to apply to larger prey items that cannot be carried by one or a few ants, and does therefore not apply to our situation where single or a few ants were observed to carry the food items. most o. longinoda prey items are smaller insects that can be carried by single ants (lynegaard et al., 2014). on the other hand, situations where the ants generally feed on larger food items such as larger beetles, cockroaches, dragon flies, larger moths, butterflies, lizards or dead mammals may occur, and in such cases aggregated responses can be expected and the assumptions behind the nicholson-bailey equation fail. in these cases, the only useful functional response equation is the lotka-volterra equation (equation 8). the acceptance of both equation (8) and (9), which are linear models, as useful to calculate the prey search rate, is supported by (fast et al, 2015) whose results suggest o. longinoda to follow a functional response type i (a linear response), as it was not possible to safely identify a saturation point of the functional response curve. the value of the average search rate cannot be compared with the results from other investigations, as we have found no similar attempts to estimate this parameter for eusocial insects in the literature. instead, it should be possible to compare with estimates of search rates from solitary insects, but this is problematic because estimated search rates depend to a large extend on the experimental arena and may come out in different units (e.g. de kranker et al., 2001; madadi et al., 2011; sentis et al., 2012; mrosso et al., 2013). no or at least very few investigations have been dealing with estimating the search rate under field conditions. therefore, the outcomes presented here are valuable as they were obtained under field conditions and, furthermore, on a eusocial insect species. there is a long list of papers on o. smaragdina and o. longinoda used in pest control (see review of van mele, 2008; offenberg, 2015) but there is virtually no knowledge on search rates (nicholson-baily equation) colony 1 2 3 4 5 6 7 8 9 10 avg. (se) 02/29 5.0 0.8 6.2 5.7 3.7 4.3 1.2 7.4 0.3 0.9 3.5 (0.7) 03/01 0.7 0.5 2.5 4.6 7.7 2.8 2.2 3.9 1.3 1.0 2.7 (0.4) 03/02 0.3 0.3 3.5 5.7 2.8 1.7 1.2 3.9 1.3 1.4 2.2 (0.3) 03/05 0.0 0.6 2.0 2.3 8.4 4.1 1.4 3.9 1.3 1.0 2.5 (0.5) 03/06 1.4 0.2 2.8 2.8 6.7 3.8 0.9 3.9 0.5 1.4 2.4 (0.4) 03/07 0.0 0.0 2.4 2.3 5.5 4.1 1.6 0.6 0.3 0.2 1.7 (0.3) 03/08 0.0 0.1 1.0 2.8 3.0 0.7 1.8 0.8 0.5 0.1 1.1 (0.2) avg. (se) 1.1 (0.6) 0.4 (0.1) 2.9 (0.6) 3.7 (0.5) 5.4 (0.8) 3.1 (0.5) 1.5 (0.2) 3.5 (0.8) 0.8 (0.2) 0.8 (0.2) 2.3 (0.1) table 4. the estimated search rates (×10-5) per observation period (45 min) for each colony on each day using the nicholson-baily functional response equation. s henriksen et al. – the search rate of the african weaver ant in cashew524 factors that control their efficacy, except ant density (peng et al., 2013) and boarder fights (peng et al, 1999b). knowledge on other factors affecting efficacy as for instance the highly variable search rates between colonies found in this paper suggests that there are some important factors controlling the predatory behavior of a weaver ant colony. these factors may for instance include the amount of available sugar, which is crucial as fuel for ant colonies (holldöbler & wilson, 1990), and the colony search effort for insect prey may depend on the colony´s access to sugar. sugar sources are usually honeydew excreted from hemipterans (aphids, scale insects, mealy bugs, etc.), floral nectaries, and extra-floral nectaries as found on newly produced leaves, inflorescences and young nuts of cashew (peng et al., in prep). therefore, the satiation of a weaver ant colony with sugar must depend on the presence of these sources, which in the case of floral nectar will depend on flowering, and the production of extra-floral nectar in cashew will depend on the flushing period when trees produce new leaves and inflorescences. populations of hemipterans may also vary over the season and between trees. therefore, access to sugar is very likely to show seasonal fluctuations as well as differences between colonies. also the amount of growing larvae in the colony may affect the demand for protein and in turn the prey search effort. if there is little brood in the colony there is less reason to gather prey as the larvae are the primary consumers of protein in ant colonies (sanders et al., 1992). it is of great importance for the possibilities of modeling weaver ant efficiency as biocontrol agents to have good estimates of the search rate and to know which factors are controlling it, as the search rate is essential in the modeling of predator – prey relationships. with the knowledge presented in this paper, modelers now have a figure of the search rate to use in model parameterization. the results presented here have, however, raised some new questions on the possible seasonal variability in search rate, but our results are a first step towards a better understanding of the predation efficiency of weaver ant colonies. a yet unpublished model (axelsen ja, unpublished) has been constructed to simulate the colony development of weaver ants (oecophylla sp.) depending on available amounts of sugar and protein. as there is no information available to simulate the amount of available sugar from natural sources in cashew trees the modeled daily amount of available sugar in the model can be regarded sugar feed to the colony by cashew growers. similarly, the model has been constructed to simulate what feeding the colony with meat as a protein source means to the colony growth. by simulating the colony growth based on manipulations of sugar and protein sources by the cashew grower it becomes possible to simulate how quickly a colony can be brought to a size large enough for effective biocontrol. according to peng et al. (2004) it takes at least two years for a colony to grow to an efficient size, but supplementing with artificial feeding this is likely to be achievable much faster. the link between the colony size and its functioning in biocontrol is the search rate, and the figure on the search rate from this paper makes it possible to simulate how much prey is captured in a cashew tree at a given prey density. this prey may include pest species, and therefore, the results on the search rate presented here are very important for linking colony size to biological pest control in cashew plantations. acknowledgements the authors thank sokoine university of agriculture for providing facilities for the investigation. this study was supported by danida through project dfc no. 10-025. references carbaugh, j.s. & judd, t.m. (2011). dominance hierarchy and nutrient acquisition in the slave-making ant protomognathus americanus (hymenoptera: formacidae). sociobiology, 58: 457-472. crawley, m. j. 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(1992). role of ants in pestmanagement. annual review of entomology, 37: 479-503. wojtusiak j., godzinska e. j. and dejean a., 1995. capture and retrieval of very large prey by workers of the african weaver ant, oecophylla longinoda (latreille 1802). tropical zoology, 8: 309-318. 531 performance of altriset™ (chlorantraniliprole) termiticide against formosan subterranean termites, coptotermes formosanus shiraki, in laboratory feeding cessation and collateral transfer trials, and field applications by robert t. puckett1, t. chris keefer1, & roger e. gold1* abstract chlorantraniliprole represents the first compound to be registered as a termiticide by the environmental protection agency (epa) in over a decade. this novel termiticide is currently registered as a ‘reduced-risk pesticide’ by the epa. laboratory and field trials were conducted to quantify mortality of formosan subterranean termites (fst), coptotermes formosanus shiraki resulting from chlorantraniliprole treated soil, the degree to which the termites curtail feeding intensity post-exposure to chlorantraniliprole treated soil, collateral transfer of chlorantraniliprole among nest mates, and the effectiveness of chlorantraniliprole as a remedial treatment against structural infestations of fst. termites which were exposed to chlorantraniliprole treated soil consumed significantly less paper than unexposed fst. the mean percent mortality of those termites exposed to chlorantraniliprole treated soil was significantly greater than that of unexposed fst. depending on donor:recipient ratios, the mean mortality of recipients ranged from 14.65 – 90.00 % in the collateral transfer trials. there was a positive correlation between increased donor density and recipient mortality. through 24 mo post-treatment, 27.3% of the structures which were treated in field trials were observed to have infestations of termites that required re-treatment; however, no fst were observed during the 30 and 36 month post-treatment inspections. additionally, a novel scoring rubric was developed that will allow standardization of field study sites with respect to dissimilarity in site variables, and will allow for more consistent comparison of results across disparate field experiments. an explanation for 1department of entomolog y, texas a&m university, 2143 tamu, college station, tx 778432143. 1*corresponding author e-mail: r-gold@tamu.edu 532 sociobiolog y vol. 59, no. 2, 2012 the lack of successful remediation of many of the structures involved in the field trial is proposed and is based on our novel scoring system. key words: coptotermes, chlorantraniliprole, termiticide, reduced-risk, invasive species introduction formosan subterranean termites (fst) coptotermes formosanus shiraki (isoptera: rhinotermitidae), are an invasive insect pest in the united states and elsewhere. while this termite species is endemic to mainland china (kistner 1985), their introduction into the hawaiian archipelago is believed to have been the result of maritime activity originating from the island of taiwan (formerly formosa), with subsequent introductions to continental united states originating from hawaii (su and scheffrahn 1998; cabrera et al. 2000; hawthorne et al. 2000; howell et al. 2000; scheffrahn et al. 2001; jenkins et al. 2002). however, austin et al. 2006 suggests two distinct fst introductions to hawaii, and then the continental united states, both originating from mainland china. fst represent one of the most economically important pest insects in the united states. estimates indicate that the cost associated with monitoring, control, and repair of damages caused by fst exceeds $1 billion per year (paudel et al. 2010). fst control methods include physical and chemical barriers to prevent termites from gaining access to structures, biological controls (including nematodes, bacteria, fungi, and botanical extracts), chemical treatment of soil and wood, and baits (verma et al. 2009). the general public is educated regarding many of these termite control tactics, but when surveyed they cite (in descending order of acceptability) perimeter treatment with a liquid termiticide, bait treatment, and liquid + bait treatments at the most preferred methods of termite control (paudel et al. 2010). however, there is an increasing emphasis towards the development of least-risk chemical treatments, as well as non-chemical tactics for termite control (lewis 1997). belonging to a new class of chemical insecticides, chlorantraniliprole (altriset™) was recently developed and marketed by dupont crop protection. chlorantraniliprole is currently classified as a ‘reduced-risk pesticide’ by the evironmental protection agency (epa, 2008). the compound is an anthranilic diamide, and exhibits a novel mode of action in which insect ryanodine 533 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus receptors are activated, resulting in rapid paralytic muscle dysfunction (hannig et al. 2009, cordova et al. 2006, cordova et al. 2007, lahm et al. 2005, and lahm et al. 2007). regulation of the release of internal cell calcium is affected by activation of ryanodine receptors. the downstream physiological effect of this disruption of calcium homeostasis results in feeding cessation, and eventual death of the insect (teixeira et al. 2008). the effectiveness of this compound has been demonstrated in mortality trials against a variety of insect species belonging to the orders coleoptera, diptera, and hemiptera (kuhar et al. 2008, palumbo 2008, and schuster 2007). we designed laboratory trials to study the efficacy of chlorantraniliprole on fst feeding rates, collateral transfer of chlorantraniliprole among fst nestmates, and field trials to determine the effectiveness of chlorantraniliprole to control infestations of fst in structures and to protect those structures from reinvasion through time. gautam & henderson (2011) described the effects of chlorantraniliprole on fst in laboratory trials. our work represents a synthesis of data related to the effectiveness of this new compound on fst in field applications correlated with laboratory trials. material and methods laboratory trials: termite feeding cessation fst were field-collected from beaumont, tx approximately 2 weeks prior to the initiation of this study. sandy-loam soil was prepared for these trials by treating it with chlorantraniliprole using the following procedures. a 1,000 ppm stock solution of chlorantraniliprole and water was made by adding 0.10 g of technical grade chlorantraniliprole to 100 ml of deionized water. next, a serial dilution of the stock solution was accomplished by adding 15 ml of deionized water to 15 ml of stock solution. this final solution was added to 270 g of soil and distributed by mixing the soil with a stir rod within a 750 ml plastic beaker. the treated soil was allowed to rest for a period of 24 hrs. glass test-tubes (10 cm in length and open at both ends) served as arenas for this experiment and followed the design of gold et al. 1996 (fig. 1). after preparation, the tubes were stored vertically in test-tube racks (untreatedagar end at top). tubes were stored at 25˚c overnight to allow moisture to become uniformly distributed in the matrix. there were 10 replications 534 sociobiolog y vol. 59, no. 2, 2012 of each treatment and untreated control groups. after the 24 hr period had elapsed, 20 fst workers and 5 soldiers per replication were randomly selected from laboratory stock and introduced into the “untreatedagar end” of the test-tube arenas, and the introduction time was recorded. the time at which the tunneling termites reached the treated soil was recorded as the exposure ‘start’ time. cohorts of termites were allowed to tunnel in the soil for four different time periods (1, 2, 4, & 8 hr). these four time periods represent four distinct treatments. after the termites had tunneled for the pre-determined time period, the rubber stoppers were removed and termites and soil were carefully tapped out through the untreated end of the arena into a clean petri-dish. to measure feeding intensity, pre-feeding digital images of 5 x 5 cm pieces of brown paper towel (cormatic-georgia pacific, atlanta, ga) were taken for comparison to post-feeding images at the end of the trial. using soft forceps, the termites were then carefully moved to a smaller petri dish containing the paper towel, half of which was covered by 50 g of play sand, moistened with 10 ml of water. this arrangement of paper towel and moistened sand provided ambient humidity within the arenas, as well as constantly moist paper towel, on which the termites fed. petri dishes were sealed with parafilm®, transferred to an environmental chamber, and maintained at 25 ± 5° c and 85 ± 5% rh. untreated control tubes were constructed in the same manner as the treatment tubes, but the soil remained untreated. termite cohorts were allowed to tunnel for the same time as the treatment groups periods (1, 2, 4, and 8 h). termites were transferred to fig. 1. schematic showing components of glass-tube arenas used in fst feeding cessation experiment. 535 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus identical feeding dishes, and feeding rates were then calculated in the same manner as in the treatment groups. these control groups are referred to as ‘tunneling controls’ (tc). an additional set of controls which were not allowed to tunnel, were established in feeding dishes (as above) to compare the amount of feeding for the same time period as the treatment and tc groups. twenty termite workers and 5 soldiers were introduced directly into these petri dishes without subjecting them to tunneling, and observation periods were identical to the treatment and untreated control groups. this control group is referred to as ‘feeding controls’ (fc). termite mortality was recorded daily for 12 d post-exposure. at the end of the trial, the remaining paper towel was allowed to dry in the laboratory, after which, digital images of the paper were taken with a canon eos 50d 15.1 megapixel digital camera fitted with a 28-135 mm lens (canon u.s.a., inc. lake success, ny). pre-feeding and post-feeding images were then compared using sigmascan pro v.5.0 photo-editing software, and the surface area (cm2) differential was calculated and statistically analyzed (calibration of the photography technique was made for each image prior to area measurement). collateral transfer as in the feeding cessation study detailed above, termites used in this study were field-collected from beaumont, tx approximately 2 weeks prior to the initiation of the trial. ten replications of each treatment (donor:recipient ratios), and untreated controls were conducted for this trial. arenas consisted of a 15 cm petri dish, each with a a 7.6 x 7.6 cm piece of brown paper towel (cormatic-georgia pacific, atlanta, ga) placed on the floor of the petri dish and moistened with 8-10 droplets of water from a 25 ml samco scientific corporation pipette (san fernando, ca). the paper served as food and harborage for termites. donor:recipient ratios in this trial included 0:20 (untreated controls), 1:19, 5:15, 10:10, 15:5, 19:1, and 20:0. donor fst were marked using orange rust-oleum marking paint (vernon hills, il). the methodolog y used to mark the the donors was similar to that described by forschler 1994. a stock solution of 50 ppm was made using formulated chlorantraniliprole. donor fst were treated on the thorax with 0.3 µl of 50 ppm chlorantraniliprole using a hamilton 700 series micro syringe pipette (reno, nv). two untreated fst soldiers were added to each arena. addition536 sociobiolog y vol. 59, no. 2, 2012 ally, two sets of post-treatment observations were made to observe mortality at 4 h, then daily through 7 d. feeding intensity was measured in these trials by taking pre-feeding images of the 7.6 x 7.6 cm brown paper towel and postfeeding images at the end of these trials (7 d after treatment). as described above, preand post-feeding images were compared using sigmascan pro v.5.0 photo-editing software, and the surface area (cm2) differential was the metric used for statistical analysis. calibration of photography was made for each image prior to area measurement. a separate trial was established simultaneously to determine if marking had a significant deleterious effect on the termites. this trial included ten replications each of untreated-unmarked controls and untreated-marked controls. field trials: for the purposes of this field trial, center for urban and structural entomolog y (cuse) and dupont personnel jointly inspected and agreed upon 11 structures with monolithic slabs or pier and beam construction, each of which had at least one active fst shelter tube on the exterior of the structure. the minimum area of any structure included in the trial was 78.97 m2. pre-treatment inspections also included the interior of structures and bath traps. a diagram of each structure was prepared by cuse personnel, who measured the size and recorded the shape of the foundation, and areas of termite activity. live termites were collected from each structure, preserved in 100% ethanol, and stored as voucher specimens. all of these structures were located in southeast texas. chlorantraniliprole treatments were made by a pest management professional at each property according to the manufacturer’s label directions. these treatments were overseen by cuse personnel. all structures were treated between may and july 2008. nine of the structures were of monolithic slab construction, and the two were pier and beam. post-treatment exterior (and interior when possible) inspections were made on or about 2 wk, and then at 1, 3, 6, 12, 18, 24, 30, and 36 months. this monitoring schedule is more robust than that which would generally be incorporated into professional pest management protocols. if active termites were discovered during post-treatment inspections, termite samples were collected and preserved in 100% ethanol as voucher specimens. dupont authorized personnel were notified of any post-treatment activity before any supplemental treatments (st) or re-treatments (rt) were performed. 537 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus a supplemental treatment (st) in this study is defined as: (a). chlorantraniliprole spot treatments to active termite areas that were not originally treated at the initial application. this is not a failure of chlorantraniliprole, since the termiticide was not applied to that area, and termites penetrated through an untreated zone. (b). treatment of infested elements of construction where termites survived due to conducive conditions, such as leaking pipes, unusual construction elements that did not allow application to reach the infested area, or an isolated above ground colony with no soil contact. (c). treatment where a conducive condition existed which contributed to or allowed termites to remain active and penetrate the treated zone. thus, it was not considered a chlorantraniliprole failure. a re-treatment (rt) in this study was defined as: the application of chlorantraniliprole as a spot treatment to active termite areas that were originally treated at the initial application. this was considered a failure of chlorantraniliprole. that is, termites were able to penetrate through the treated zone; however, if that area of penetration had conducive conditions (or other issues as above) that allowed termites to penetrate, and the condition was not corrected, then this was considered a supplemental treatment (st) as described above. in this study, all structures were ranked based on several parameters related to the difficulty of the structure-specific treatment procedures. the parameters used to populate the ranking rubric included: termite species (1-desert termite, 2drywood termite, 3-reticulitermes, and 6-coptotermes); number of mud tubes and location; the number of conducive conditions present at each structure; and, construction type (1 monolithic slabs, 2 pier and beam, and 3 floating slabs). the total difficulty score (tds) of each structure was calculated by summing all points assigned for each category. it is presumed that difficulty to control termites at structures is positively correlated with higher tds. results termite feeding cessation consumption: all treatment cohorts that were exposed to chlorantraniliprole treated soil (regardless of exposure time) consumed significantly 538 sociobiolog y vol. 59, no. 2, 2012 less paper (f = 21.37; df = 8,89; p < 0.01) than the ‘tunneling controls’ (tc) and ‘feeding controls’ (fc) (fig. 2). additionally, the mean amount of paper consumed by fst after exposure to chlorantraniliprole treated soil was negatively correlated with time of exposure (fig. 2). a similar trend was not observed in the tc (fig. 2). mortality: with the exception of the 1 and 4 hr tunneling control groups, the mean % mortality of fst remained below 10% in the untreated controls through 11 d after exposure to untreated soil (fig. 3). at and beyond 3 d post exposure, the mean % mortality of all treatment cohorts that were exposed to chlorantraniliprole treated soil (regardless of exposure time) was significantly greater (f = 9.64; df = 8,89; p < 0.01) than the tc and fc (fig. 3). additionally, with the exception of the 12 d observation period, the mean % mortality of treatment cohorts that were exposed to chlorantraniliprole treated soil was positively correlated with time of exposure. fig. 2. mean paper consumed (cm2) by fst through 12 d after exposure to chlorantraniliprole treated soil, untreated soil, or no soil. treatments were replicated 10 times. bars with the same letter are not significantly different using analysis of variance (anova) and tukey’s hsd mean separation test at p < 0.05. 539 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus collateral transfer mortality percent donor mortality in all of the treatment ratios was significantly different (f = 17.73; df = 8,89; p < 0.01) than that of the 0:20 donor:recipient ratio (untreated controls) at 7 d post-treatment (table 1). however, there were no significant differences in donor mortality between the different treatment ratios at the 7 d observation period, and mean mortality ranged from 50.00 – 76.00 % (table 1). there were significant differences (f = 17.73; df = 8,89; p < 0.01) in recipient mortality levels among the different treatment ratios at 7 d post-treatment, and mean mortality ranged from 14.65 – 90.00 % (table 1). regarding total mortality (donors and recipients) for each donor:recipient ratio, there were significant differences (f = 7.58; df = 8,89; p < 0.01) at the 7 d post-treatment observation period (table 1). the greatest percent total mortality (74.5 %) occurred in the 15:5 ratio at 7 d, followed by 67.5% mortality in the 19:1 donor:recipient ratio (table 1). mortality in the untreated and unmarked group was 9.00 %, and was not significantly different from that of the untreated and marked group (21.00 %) starting at 24 h thru 120 h post-treatment (f = 0.067, df=19, p = 0.31). consumptionthere were significant differences in consumption of paper among the different treatment groups and the 0:20 (untreated control) fig. 3. mean accumulated fst % mortality through 12 d of observation. 540 sociobiolog y vol. 59, no. 2, 2012 table 1. mean percent mortality at 7 d post-treatment of fst donors and recipients when donors were treated with 50 ppm chlorantraniliprole. % mortality total ratio donor recipient % mortality 0:20 (untreated control) 0.00 (a) 42.50 (bcd) 42.50 (abcd) 1:19 70.00 (b) 48.93 (cd) 47.50 (bcd) 5:15 76.00 (b) 14.65 (abc) 29.50 (abc) 10:10 68.00 (b) 62.00 (de) 65.00 (cd) 15:5 69.98 (b) 88.00 (e) 74.50 (d) 19:1 65.76 (b) 90.00 (e) 67.50 (bcd) 20:0 50.00 (b) 0.00 (a) 50.00 (bcd) *means followed by the same letter(s) in the same column are not significantly different (tukey’s hsd, p=0.05) fig. 4. mean amount of consumption of substrate (cm2) by fst (donors and recipients) in each donor:recipient ratio through 7 d post-treatment. bars with the same letter are not significantly different using analysis of variance (anova) and tukey’s hsd mean separation test at p < 0.05. 541 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus donor:recipient group (f = 15.33; df = 8,89; p < 0.01) (fig. 4). the greatest consumption occurred in the 0:20 ratio, followed by the 5:15, and then the 10:10 (fig. 4). field trial the mean number of pre-trial exterior termite mud tubes per structure was 3.91, and ranged from 1 – 10 (table 2). the mean volume of finished solution applied to structure exteriors was 302.15 l, and 10.33 l on the interiors (table 2). during the first 24 mo of the 36 mo trial duration, 27.27% of the structures were infested with fst and required re-treatment (rt), and 18.18% required supplemental treatment (st). this includes structure #3 in which fst were not completely controlled until a re-treatment (rt) was made after the 1 mo inspection, when fst were discovered on the exterior at the original site of infestation (fig. 5 and table 3). the re-treatment (rt) was performed with 15.14 l of chlorantraniliprole. due to damage caused by hurricane ike, access to eight of the structures was limited at the 3 mo posttreatment inspection, and only three of the eleven structures were inspected. no termite activity was found at those three structures. at the 6 mo inspection, two structures were found to have termite activity (fig. 5 and table 3), including structure #2, which had active termites on the exterior and received a re-treatment (rt) with 15.14 l of chlorantraniliprole. additionally during the 6 mo inspection, active termites were found swarming from an interior wall within structure #9. after further investigation of the swarm, a previously unknown cold joint was discovered and this structure received a supplemental treatment (st) with 37.85 l of chlorantraniliprole. at the 12 mo inspection, structure #8 had active fst swarming from a previously treated bath trap. upon further examination, it was determined that there was a water leak in table 2. mean, standard deviation, and range of exterior and interior mud tubes, perimeter length, and amount of chlorantraniliprole applied to the exterior and interior of structures associated with the field study during initial observations and treatment. exterior mud tubes interior mud tubes perimeter length (m) product applied to exterior (l) product applied to interior (l) mean and s.d. 3.91 (3.28) 0.18 (0.40) 57.05 (55.90) 302.15 (25.37) 10.33 (1.62) range 1 10 0 1 30.48 – 91.44 151.42 454.25 0.00 -18.93 542 sociobiolog y vol. 59, no. 2, 2012 the bath trap area, this leak was repaired, and the area received a supplemental treatment (st) using 22.71 l of chlorantraniliprole. no subterranean termite activity was found during the 18 mo inspections. at 22 mo post-treatment, structure #9 homeowners notified us of fst swarming again from the same cold joint, but in a different area. this area received a supplemental treatment table 3. data-populated scoring rubric developed for, and used in these trials to standardize the total difficulty score (tds) associated with treatment of each structure. structure # termite species # of mud tubes construction type # of conducive conditions total difficulty score (tds) # of st’s # of rt’s 1 c. formosanus=6 10 exterior monolithic=1 1 18.0 0 0 2 c. formosanus=6 10 exterior monolithic=1 1 18.0 0 2 3 c. formosanus=6 1 exterior 1 interior monolithic=1 1 10.0 0 1 4 c. formosanus=6 3 exterior pier & beam=2 2 13.0 0 0 5 c. formosanus=6 2 exterior 1 interior pier & beam=2 1 12.0 0 1 6 c. formosanus=6 4 exterior monolithic=1 1 12.0 0 0 7 c. formosanus=6 5 exterior monolithic=1 1 13.0 0 0 8 c. formosanus=6 3 exterior monolithic=1 1 11.0 1 0 9 c. formosanus=6 2 exterior monolithic=1 1 10.0 2 0 10 c. formosanus=6 2 exterior monolithic=1 1 10.0 0 0 11 c. formosanus=6 1 exterior monolithic=1 1 9.0 0 0 mean & (s.d.) 6 (0.00) 3.91 (3.24) 1.18 (0.40) 1.09 (0.30) 12.36 (3.07) 0.27 (0.64) 0.36 (0.67) note: all structures were ranked on the difficulty of the treatment based on several parameters which included, termite species, number of mud tubes and location, and the number of conducive conditions present at each structure. the table details the ranking of termite species based on colony size: 1 desert termite, 2 drywood termite, 3 reticulitermes, and 6 coptotermes. each structure received one point for every mud tube located on the structure, and one point for each conducive condition found to be present at the structure. construction type was also given a treatment difficulty ranking as follows: 1 monolithic slab, 2 pier and beam, and 3 floating slab. the total difficulty score (tds) of each structure was calculated by summing all points assigned for each category. 543 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus (st) with 189.27 l of chlorantraniliprole. this st is included in the 24 mo post-treatment observation period on fig. 5. also during the 24 mo inspection, active fst were discovered at structures # 2 and 5 (fig. 5 and table 3). both structures had active termites on the exterior, and both were re-treated (rt) with 15.14 l of chlorantraniliprole. no subterranean termite activity was found on any of these structures at the 30 or 36 mo inspection. after ranking, using the rubric described in the material and methods section, the mean total difficulty score (tds) of the structures in this study was 12.36, and ranged from 9 – 18 (table 3). at least one st was required in 18.18% of the structures, and all were made to structures that ranked below the mean tds. of the re-treated structures (rt), all but one occurred in structures which ranked above, or just below the mean tds (table 3). fig. 5. mean % of re-treated (rt) structures infested with fst after a post-construction treatment with chlorantraniliprole through 36 mo post-treatment. nomenclature above bar: individual structure number and rt (re-treatment). note: active termites were found at structure #3 at 2 weeks posttreatment, but no corrective action was taken until 1 month post-treatment. 544 sociobiolog y vol. 59, no. 2, 2012 discussion in laboratory trials, exposure of fst to chlorantraniliprole treated soil resulted in significantly greater mortality and significantly less consumption of food than either; 1) fst exposed to untreated soil, and 2) fst which were not exposed to soil at all. these data suggest that chlorantraniliprole could be considered an appropriate soil barrier-treatment for use against fst. however, it should be noted that the minimum termite exposure time investigated in these trials was 1 hr. it is not known if it would be realistic to presume that foraging termites would remain in a ‘treatment zone’ for this period of time in actual situations in which chlorantraniliprole is used to provide perimeter protection to a structure against subterranean termite infestations. thus, we intend to initiate further investigations to determine the minimum temporal exposure threshold required for significant mortality and consumption cessation. the significantly greater mortality of chlorantraniliprole-exposed termites relative to that of the tc and fc is a noteworthy aspect of this study. however, it is important to note that mortality of termites which were exposed to chlorantraniliprole ranged from a minimum of 51.31% (1 hr of exposure) to a maximum of 85.51% (4 hrs of exposure) after 12 d of post-treatment observation. it is likely that the mortality exhibited by these termites was partially the result of deprivation of nutrients resulting from feeding cessation. this effect of treatment is significant, and demonstrates a potential mode of action of chlorantraniliprole, and subsequent cascade of physiological alterations in termites that is more nuanced than that of many other classes of termiticides. no attempt was made in these trials to determine the minimum concentration required to illicit the observed feeding inhibitory effects. we intend to design experiments to investigate this aspect of chlorantraniliprole. additionally, the effects of chlorantraniliprole on other important subterranean termite species should likewise be investigated. the positive correlation (with exception of 5:15 donor:recipient ratio) between increased donor numbers and recipient mortality provides evidence of the transfer effect of chlorantraniliprole (altriset™) among fst nestmates, and suggests that this compound could affect mortality of fst in 545 puckett, r.t. et al. — performance of altriset termiticide against c. formosanus field applications via insect-to-insect transfer. this density-dependent effect is dependent on the density of chlorantraniliprole-exposed insects (donors) relative to the density of recipients. this of course calls into question the feasibility and efficiency of reliance on this insect-to-insect transfer effect in order for the use of this product to provide effective remedial or prophylactic treatments on fst populations. that is, the density of chlorantraniliproleexposed foragers (donors) in field treated colonies would likely not reach a level which would be analogous to the donor:recipient ratios which were required in these trials to significantly effect the density of recipients, and thus the colony. interestingly, mortality of the donor population remained lower than expected and, on average (66.62 % overall mean mortality of donors) was similar to that observed in fst after exposure to chlorantraniliprole treated soil for 4 and 8 hrs in the feeding cessation trial. when considered in their totality, these results suggest that the chlorantraniliprole concentration to which fst were exposed in these trials (50 ppm) may be less than that required for acceptable termite control in field applications. however, altriset™ is labeled for application at 500 ppm, or 10 times the concentration used in this laboratory trial. evidence of the slow acting nature of chlorantraniliprole when applied to infestations of fst is provided by the fact that several of the structures (27.27%) which were treated and monitored over the course of these trials were observed to have continuing infestations of fst. these structures required re-treatment (rt) during the first 24 mo of the monitoring phase of the study. in one case, rt became necessary twice within the same structure. however, by 30 months post-treatment, no fst activity was observed at any of the structures. this suggests that while chlorantraniliprole effectively controlled fst at structures in these trials, the compound requires a longer period of time to reduce populations of structure-infesting fst than alternative termiticides, but the end result of structure protection is the same. comparisons of field experiment results (such as those discussed in the field trial section of this document) across treatment environments are greatly complicated by the myriad variations associated with treatment scenarios and structure variables (french 1988; french and ahmed 2005). differences in treatment sites such as termite species, degree and location of infestation, degree and location of conditions conducive to termite infestation, foundation 546 sociobiolog y vol. 59, no. 2, 2012 type, construction materials, and soil type result in great difficulty in standardization of the finite number of treatment environments that are available to researchers. we have proposed a rubric for scoring treatment environments which allows for enhanced ability to compare and assess data related to field experimentation such as that documented herein. careful examination of the results of the utilization of this rubric in this work revealed an interesting bifurcation of the mean total difficulty scores (tds) of those structures that required either a supplemental treatment (st) or re-treatment (rt). that is, the tds of all st’s fell below the mean of 12.37, and a trend was noted in which rt’s scored greater than or just below the mean tds. this more thorough characterization and comparison of our treatment environments provides a refined and more complete picture of the challenges involved with treatment for fst in all study structures, and presents explanatory variables that we believe led to the lack of successful remediation and protection of rt structures with regards to fst. it is our hope that others will attempt to use this system in future work, and that after subsequent model refinement, eventually this will provide a consistent method to compare results across disparate, but related field research involving subterranean termites. acknowledgments we thank mrs. laura nelson for her helpful reviews and comments on earlier drafts of this document. additionally, we would like to thank mr. bryan springer (bevis pest control, texas city, tx) for his technical assistance with treatment of structures. the center for urban and structural entomolog y also thanks dr. clay scherer (dupont) for his support of this work. references austin, j.w., a.l. szalanski, r.h. scheffrahn, m.t. messenger, j.a. mckern, and r.e. gold. 2006. genetic evidence for two introductions of the formosan subterranean termite, coptotermes formosanus (isoptera: rhinotermitidae), to the united states. fla. entomol. 89:183-193. cabrera, b.j., p.g. koehler, f.m. oi, r.h. scheffrahn, and n.-y. su. 2000. the formosan subterranean termite. eny-126, florida cooperative extension service, ifas, university of florida. 7pp. cordova d., e.a. benner, m.d. sacher, j.j. rauh, j.s. sopa, g.p. lahm. 2006. anthranilic diamides: a new class of insecticides with a novel 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10.13102/sociobiology.v63i1.764sociobiology 63(1): 623-627 (march, 2016) geographic range of pachycondyla harpax (fabricius) (hymenoptera: formicidae) introduction ponerine ants (subfamily ponerinae) include >1000 species (bolton 2015), many of which are well known for their potent sting. for example, brachyponera sennaarensis (mayr) (formerly pachycondyla sennaarensis) is a widespread species of sub-saharan africa and the middle east that is well known for its powerful sting, which sometimes leads to anaphylactic shock in humans and even death (wetterer, 2013). brachyponera chinensis (emery) (formerly pachycondyla chinensis) is an east asian species now spreading through the eastern us that has a painful sting that can also induce severe allergic reaction (guénard & dunn, 2010). here, i examine the geographic range of the ponerine ant pachycondyla harpax (fabricius). wittenborn and jeschke (2011) included p. harpax on a list of exotic ant species established in north america. based on wittenborn and jeschke (2011), miravete et al. (2013; now retracted) listed p. harpax as neotropical species from south abstract pachycondyla harpax (fabricius) is a widespread and conspicuous new world ponerine ant (subfamily ponerinae). to examine the geographic distribution of p. harpax, i compiled and mapped published and unpublished specimen records from >1600 sites. i documented the earliest known p. harpax records for 28 geographic areas (countries, west indian islands, and us states), including four for which i found no previously published records: the islands of guadeloupe, margarita, and tobago and the us state of georgia. pachycondyla harpax has been recorded from every country in south and central america except chile and uruguay. pachycondyla harpax is also now known from six west indian islands: grenada, guadeloupe, jamaica, margarita, trinidad, and tobago. the known continental range of p. harpax appears to be essentially continuous, extending from rio grande do sul, brazil in the south (31.8°s) to wood county, texas in the north (32.8°n), including the continental islands of margarita, tobago, and trinidad. isolated island populations of p. harpax on grenada, guadeloupe, and jamaica may be exotic, introduced through human commerce. in the us, it is unclear why p. harpax populations are only known from texas, louisiana, and georgia, when there would appear to be suitable habitat for this species all along the gulf coast of alabama, mississippi, and florida. sociobiology an international journal on social insects jk wetterer article history edited by flavia esteves, cas, usa evandro nascimento silva, uefs, brazil received 05 february 2015 initial acceptance 07 april 2015 final acceptance 04 september 2015 publication date 29 april 2016 keywords biogeography, native range, native species, ponerinae, west indies. corresponding author james k. wetterer wilkes honors college florida atlantic university, 5353 parkside drive, jupiter, fl, 33458, usa e-mail: wetterer@fau.edu america that is an introduced and established exotic species in the us. pachycondyla harpax is a large (up to 10 mm in length), conspicuous new world ponerine ant (fig 1). pachycondyla harpax has an exceptionally broad geographic range in south, central, and north america (wheeler, 1900) and occupies a great diversity of natural and disturbed environments. mackay and mackay (2010) wrote that p. harpax is “found in a wide variety of habitats, ranging from dry forest, late dry season gap forest, urban environments, parks, grassy areas, coffee, cacao and banana plantations, cypress swamps, to oak forests, oak riparian forests, rocky wet quebradas, arid scrub, palm thorn forests, tropical deciduous forests, tropical evergreen forests, second growth tropical forests, transitional bamboo/ cloud forest, old growth dry tropical forest, steep rocky forest slopes, ridge forest, wet mountain forests, riparian rain forest, montane evergreen forest, lowland forests, cloud forest and riparian tropical rain forests.” longino (2001) wrote: “i have research article ants wilkes honors college, florida atlantic university, jupiter, florida, usa jk wetterer – geographic range of pachycondyla harpax (fabricius)624 never found a nest of this common species. the nest must be subterranean. if they nested in the leaf litter or in dead wood, nests would be more frequently encountered.” taxonomy and identification is absent on the dorsum of the meso soma and the petiole is rectangular shaped with a distinct dorsal face and the posterior lateral margin forms a sharp carina, which is barely evident as it passes to the anterior edge of the petiole.” brown (1950) suggested that p. harpax may actually be a species complex rather than a single species. schmidt (2013) determined that the genus pachycondyla, as formerly constituted, was polyphyletic. in schmidt and shattuck’s (2014) revision, however, p. harpax remained in the genus pachycondyla along with ten other species. methods using published and unpublished records, i documented the worldwide range of p. harpax. i obtained unpublished site records from museum specimens in the collections of the museum of comparative zoology (mcz) and the smithsonian institution (si). in addition, i used online databases with collection information on specimens by antweb (www.antweb.org), and the global biodiversity information facility (www.gbif.org). i received unpublished collection information of p. harpax records from c. alatorrebracamontes (mexico) and e. mendoza (el salvador). finally, i collected p. harpax specimens from costa rica, el salvador, guadeloupe, margarita, trinidad, and tobago. i obtained geo-coordinates for collection sites from published references, specimen labels, maps, or geography web sites (e.g., earth.google.com, www.tageo.com, and www. fallingrain.com). if a site record listed a geographic region rather than a “point locale,” and i had no other record for this region, i used the coordinates of the capital or largest town within the region or, in the case of small islands and natural areas, the center of the region. published records usually included collection dates, but when this was not the case, i was sometimes able to determine the approximate date based on information on the collector’s travel dates or limit the date by the collector’s date of death. for example, nils holmgren visited in peru and bolivia in 1904-1905, thus bracketing the date he collected p. harpax specimens examined by wheeler (1925). henry t. vanderford (1901-1990), who collected the only known p. harpax specimen from georgia (identification confirmed by s.p. cover), worked for the georgia department of agriculture until at least 1967 (williams, 1986). results i compiled and mapped published and unpublished specimen records from >1600 sites. i documented the earliest known p. harpax records for 28 geographic areas (countries, west indian islands, and us states), including four for which i found no previously published records: the islands of guadeloupe, margarita, and tobago and the state of georgia. pachycondyla harpax has been recorded from every fig 1. pachycondyla harpax. a) head and b) lateral view of worker from costa rica (casent0249149; p.s. ward leg.; photos by r. perry from antweb.org). fabricius (1804) described formica harpax (= p. harpax) from south america. junior synonym include pachycondyla harpax concinna wheeler (from bolivia, brazil, and peru), pachycondyla harpax dibullana forel (from colombia), pachycondyla harpax irina wheeler (from colombia, costa rica, guatemala, guyana, and peru), pachycondyla montezumia smith (from mexico), pomera amplinoda buckley (from texas), and pachycondyla orizabana norton (from mexico) (see brown, 1950). mackay and mackay (2010) wrote that the p. harpax: “worker can be separated from most other species in the genus, as it lacks the malar carina, the eye is small but is located less than one maximum diameter from the anterior edge of the head. the pronotal carina is poorly developed but forms a shiny raised line (usually), the metanotal suture sociobiology 63(1): 623-627 (march, 2016) 625 country in south and central america except chile and uruguay (table 1). pachycondyla harpax is also now known from six west indian islands: grenada, guadeloupe, jamaica, margarita, trinidad, and tobago (table 2). most records of p. harpax come from intact forest habitats. in 2015, antweb.org recorded that p. harpax was “found most commonly in these habitats: 382 times found in mature wet forest, 169 times found in tropical moist forest, 152 times found in tropical rainforest, 120 times found in tropical wet forest, 106 times found in secondary lowland rainforest, 106 times found in lowland wet forest, 104 times found in montane wet forest, 77 times found in secondary wet forest, 51 times found in lowland rainforest, 50 times found in mesophil forest.” i have collected p. harpax at 42 sites, 39 of which came from inland forest sites on west indian islands: guadeloupe (4), margarita (1), tobago (10), and trinidad (24). the three others came from central america: costa rica (beach vegetation in tortuguero national park) and el salvador (two gardens in urban san salvador). discussion wheeler (1900) wrote that p. harpax ranged from “texas through mexico, central america and brazil to bolivia and paraguay.” more recent records extend that range to include louisiana and georgia in the north, argentina in the south, plus several west indian islands (table 1). the native continental range of p. harpax appears to be essentially continuous from rio grande do sul, brazil (31.8°s) in the south to wood county, texas (32.8°n) in the north (fig 2). although there are numerous apparent gaps in this continental range (e.g., in central brazil), it is likely that much of this is an artifact of insufficient sampling. it is unclear why p. harpax populations in the us are only known from texas, louisiana, and georgia when there would appear to be suitable habitat for this species below 31°n all along the gulf coast of alabama and mississippi and into florida. populations of p. harpax on the west indian islands of margarita, trinidad, and tobago represent part of the continuous continental range because these are continental islands that were connected to south america when sea levels were >100 m lower 15,000-30,000 years ago (lambeck et al., 2014). the west indian islands of grenada, guadeloupe, and jamaica, however, have never been connected to the adjacent continent. it is unclear how p. harpax population came to colonize grenada, guadeloupe, and jamaica. it is possible that the populations of p. harpax on these islands are exotic, introduced through human commerce. on all three islands, however, p. harpax was collected almost exclusively in intact native forest, an atypical locale for an exotic species. genetic analyses could be useful in determining whether p. harpax is native or exotic on grenada, guadeloupe, and jamaica. i found no evidence indicating that p. harpax is exotic to continental north america. in addition to p. harpax, wittenborn and jeschke (2011) appear to have misclassified numerous other ant species as exotics to north america that are actually natives. for example, gnamptogenys hartmani (wheeler), labidus coecus (latreille), and leptogenys elongata (buckley), all have distributions in the southern us that appear south america ≤1804 (fabricius, 1804) mexico ≤1858 (smith, 1858 as p. montezumia) texas ≤1866 (buckley, 1866 as pomera amplinoda) colombia ≤1870 (mayr, 1870) guatemala ≤1883 (forel, 1899) venezuela 1887-1888 (emery, 1890a) costa rica 1889 (emery, 1890b) bolivia 1885-1893 (emery, 1894) brazil ≤1895 (forel, 1895) guyana ≤1895 (forel, 1895) paraguay ≤1895 (forel, 1895) nicaragua ≤1899 (forel, 1899) peru 1904-1905 (wheeler, 1925 as p. harpax irina & p. harpax concinna) belize 1905-1906 (wheeler, 1907) panama 1911 (w.m. wheeler, mcz): ancón louisiana 1913 (j.r. horton, si): nairn honduras 1920 (mann, 1922) ecuador 1922 (f.x. williams, si): baños el salvador 1959 (n. krauss, si): santa ana surinam 1959 (kempf, 1961) argentina ≤1978 (kusnezov, 1978) +georgia ≤1990 (h.t. vanderford, si): eastman french guiana 1996 (g.d. alpert & m. moffett, mcz): paracou forest table 1. earliest known records for pachycondyla harpax in south, central, and north america. + = no previously published records. mcz = museum of comparative zoology. si = smithsonian institution. jamaica 1909 (wheeler, 1911) grenada 1912-1913 (r. thaxter, mcz): grand etang trinidad 1913 (wheeler, 1916) +tobago 2003 (j.k. wetterer, mcz): gilpin trace +guadeloupe 2008 (j.k. wetterer, mcz): carbet falls road +margarita 2010 (j.k. wetterer, mcz): se of san sebastian table 2. earliest known records for pachycondyla harpax on west indian islands. jk wetterer – geographic range of pachycondyla harpax (fabricius)626 to be the northern end of continuous native ranges and give no indication of being exotic to north america. other species that wittenborn and jeschke (2011) most likely misclassified as exotics include cephalotes varians (smith), a widespread arboreal species in cuba, the bahamas, and florida (andrade & baroni urbani, 1999) and leptogenys manni (wheeler), a species endemic to florida (trager & johnson, 1988). mark deyrup (pers. comm.), who has encyclopedic knowledge of the ants of florida, confirmed that he knows of no reason why these two species would be that considered exotic in florida (the only us state where they occur). regrettably, wittemborn and jeschke (2011) did not provide evidence to support their classification. there is some danger that if native us species, such as p. harpax, are erroneously considered to be invasive exotic species, they may be treated as such and exterminated, rather than valued and protected. acknowledgments i thank m. wetterer for comments on this manuscript; s. cover and j. longino for ant identification; s. cover (mcz), t. schultz (si), and m. deyrup (abs) for help with their respective ant collections; c. alatorre-bracamontes and e. mendoza for unpublished records; w. o’brien for gis help; d.p. wojcik and s.d. porter for compiling their valuable formis bibliography; r. pasos and w. howerton of the fau library for processing so many interlibrary loans; florida atlantic university and the national science foundation (des-0515648) for financial support. references andrade, m. l. de and baroni urbani, c. 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(2011). characteristics of exotic ants in north america. neobiota 10: 47-64 (appendix 1). doi: 10.3897/neobiota.10.1047.app1 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v68i4.7220sociobiology 68(4): e7220 (december, 2021) introduction stingless bees tetragonula iridipennis (apidae: hymenoptera), are eusocial, corbiculate with perennial colonies, polylectic, no/rudimentary sting, amenable for conservation and colony maintenance, forager recruitment behaviour, ability to store more food resources in hive unlike honeybees (roubik, 1984; leonhardt et al., 2007; kumar et al., 2012). they construct their nests in hollows of tree trunks, stone walls, mud walls, corners of walls, crevices, termite mounds and other concealed places with proper insulation (muthuraman & thirugnanasambantam, 2003; rasmussen & camargo, 2008; suriawanto et al., 2017). stingless bees are reported to be efficient pollinators of many crops like sunflower, strawberry, cherry tomato, cucumber, egg plant and sweet pepper and could be viably utilised for pollination abstract the trap occupancy rate and colony development parameters of swarms of stingless bee, tetragonula iridipennis in coconut shell traps was studied in the research farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus karnataka, india. the trap occupancy rate by the stingless bees was 44.87% in a time period of 13.40 ± 4.38 days. new cells were constructed by the bees in 12.10 ± 2.13 days. the number of honey and pollen pots filled was 15.60 ± 3.92 and 6.61 ± 2.95, respectively. the brood cells were constructed 89.50 ± 6.07 days after acceptance of the shell traps with an average of 67.70 ± 20.83 brood cells per trap. the foragers preferred foraging for nectar, resin and pollen during the 15, 30 and 45 days, respectively, after acceptance of the coconut shells for nesting. coconut shell traps are easiest and economic way of trapping the swarming population of stingless bees. sociobiology an international journal on social insects amala udayakumar, arakalagud n shylesha, timalapur m shivalingaswamy article history edited by celso f. martins, ufpb, brazil received 17 may 2021 initial acceptance 08 october 2021 final acceptance 18 october 2021 publication date 15 december 2021 keywords traps, coconut shells, stingless bees, swarms. corresponding author amala udayakumar indian council of agricultural research national bureau of agricultural insect resources h a farm post, pb no 2491, hebbal bengaluru 560024, india. e-mail: amala.uday@gmail.com of crops grown under protected cultivations (free, 1993; slaa et al., 2000; nicomedo et al., 2003; del sarto et al., 2005; kishantej et al., 2017). the maintenance of colonies of stingless bees in different structures viz., mud pots, arecanut culms, bamboo culms and box hives were well studied under indian conditions. different hive structures viz., mango wood hive, bamboo stem hive and box hives also been studied for their efficacy (karthick et al., 2018). reproduction of stingless bees occurs through swarming that starts with the location of new nesting site by the scout bees (sommeijer et al., 2013). swarming occurs in a gradual phase in stingless bees when few of the workers fly out to locate a new nesting site. after locating the new site, the foragers start building the nest by transporting the materials from the mother nests, actively forage for resources and build the storage pots in the new nest (engels & imperatriz-fonseca, indian council of agricultural research – national bureau of agricultural insect resources, bengaluru, india research article bees coconut shell traps: easiest and economic way to attract stingless bees (tetragonula iridipennis) smith amala udayakumar, arakalagud n shylesha, timalapur m shivalingaswamy – coconut shell traps to attract swarms of stingless bees2 1990). nests found by swarming process will be of perennial nature usually founded by sterile workers and queen (roubik, 2006). installation of traps is reported to be a viable method to trap the swarms of different species of stingless bees like scaptotrigona sp (alvarenga, 2008), tetragonula angustula (malkowski et al., 2006) and plebeia nigriceps (witter et al., 2007). oliveira et al. (2012) reported the trapping of 61 swarms of nine different stingless bee species in large plastic containers of 3 litre capacity coated with wax and propolis extract. inoue et al. (1985) reported foraging of 70-80%, 10-20% and < 10% percent of three species of stingless bees for nectar, pollen and resin material, respectively, from disturbed forest areas in sumatra, indonesia. dispersion of colony resources in the hive is a significant factor in the nesting biology of stingless bees (nogueira-neto, 1997). unlike honeybees, the swarming behaviour in stingless bees in less explored and there is a paucity of data in the traps used for attracting the swarms. utilisation of different trap nests for stingless bees is practised in some parts of kerala (india). however, there are hardly any published reports on the use of empty coconut shells to attract the swarms of stingless bees and colony development parameters under indian conditions. the present study was designed to understand the nesting characteristics of swarming foragers, colony development and foraging behaviour of stingless bees in empty coconut shells. materials and methods study site the present study was carried out in the experimental farm of icar-national bureau of agricultural insect resources (nbair) bengaluru, yelahanka campus (13.096792 n, 77.565976e) from april 2020 to march 2021. the study area comprised cultivated croplands with various annual crops like cereals and pulses, orchard blocks of mango, sapota, and cherimoya. also, there were two patches of pollinator gardens of about 1.5acres with over 100 plant species of diverse plant families. this research campus is situated right in the heart of a rapidly growing high-tech-city and capital of the southern indian state of karnataka. the mean maximum and minimum temperature during the flowering period were 27.8 °c and 19 °c, respectively, with rainfall of 51.4 mm. we maintained a two years-old strong colony of t. iridipennis in the study site. preparation of coconut shells used empty coconut shells (split into half) of uniform size (approximately 100-110mm of inner diameter and total height of 110-120mm) were collected from the households of bengaluru were employed for the study. holes if any observed in the shells were plugged using the propolis obtained from the nests of t. iridipennis. in the lower half of the shell, a hole (5 mm diameter) was made using hand drill and a flexible rubber tube of 2 cm length was fitted inside the hole in such a way that half of the of the tube protruded out. a thick layer of bee propolis and resin mixture was applied at the outside the protruding end of the tube to attract the swarming bees to the nest. later the two halves of the coconut shell were joined together tightly using cellotape so that they could be separated for observation of the colony over time. we installed 25 traps in close proximity of the foragers and observed for the acceptance of these traps. the traps were tied singly and fastened using galvanized iron wire on to a stand. the nest site seeking foragers of t. iridipennis were observed near the study site. observations on the number of days taken by the bees to occupy the shells was recorded. the occupancy of the shell traps by the bees was confirmed by observing the foragers entering the shell through the nest entrance. the occupied shell traps were monitored on weekly basis by gently opening the two halves of the shell trap to observe the colony establishment and construction of storage pots and brood cells. the number of days taken to construct new cells, brood cells and storage pots were recorded. the density of brood cells, density of pollen and honey pots per three fourth of cubic inch was measured after construction of the cells by the bees. the length and width of pollen and honey pots was also recorded. after establishment of the colonies, the foraging trips of the bees was recorded for a period of five days. the number of bees entering the nest and departing bees per hour was recorded from 8.00 am till 6.00 pm. the reward carried by the foragers to the nest viz., mud, pollen and resin was also recorded. the foraging preference of the founding bees for different resources viz., pollen, nectar and resin in the just accepted nests was recorded at 15, 30 and 45 days after the nest acceptance. results colony development parameters the colony development parameters of stingless bees in the coconut shell trap are presented in table 1. out of the 25 traps of coconut shells installed, 11 traps were successfully accepted by the swarms with an average trap occupancy rate of 44.87%. the coconut shell traps were accepted by the stingless bees in a time period of 13.40 ± 4.38 days. the time taken for the construction of new cells was 12.10 ± 2.13 days. storage pots appeared egg shaped, honey pots were dark brown in colour, pollen pots pale were yellow in colour arranged in clusters. the number of days taken to construct the storage pots was 7.50 ± 3.06 days. the number of honey and pollen pots filled was 15.60 ± 3.92 and 6.61 ± 2.95, respectively. brood cells appeared elliptical in shape, surrounded by sheaths of bitumen with multiple layers of wax and cerumen. newly formed broods appeared dark brown in colour and matured brood cells turned pale yellowish white in colour. the length and width of brood cells was 4.40 ± 0.52 mm and 2.90 ± 0.21 mm respectively while that of honey pots was 6.79 ± 0.35 and 6.35 ± 0.67 respectively. the brood cells sociobiology 68(4): e7220 (december, 2021) 3 were constructed 89.50 ± 6.07 days after acceptance of the shell traps. on an average, the bees constructed 67.70 ± 20.83 brood cells per trap. foraging behaviour of the bees during the initial 15 days after acceptance of the nest by the bees, 40% of the bees foraged for nectar followed by resin foragers (34%) and pollen foragers (26%) (fig 1). resin foraging bees were found to be dominant (41.34%) at 30 days after acceptance of the nest followed by pollen foragers (21.82%) and nectar foragers (19.97%). at 45 days after nest acceptance, the pollen foraging bees were the highest (34.11%) followed by nectar (31.06%) and resin foragers (11.78%). increased nectar foraging trips during the initial days of nest acceptance might be due to the requirement of sugar reserves in the storage pots for the bees engaged in initial hive maintenance work. as resin is an important hive construction material for the bees, greater abundance of resin foragers was noticed during the early nest founding days. the requirement of pollen for provisioning the broods might be the reason for increased foraging of pollen at 45 days as the brood cluster formation started 53.60 days after nest acceptance. active foraging of the bees was recorded few days after acceptance of the coconut shells. the average number of bees entering and departing the nest to the traps were 32.81 and 26.83 per day, respectively. around 30.60 foragers carried nectar load, 14.20 foraging bees carried pollen load, 10.80 bees carried resin load and 7.20 bees carried mud load during an active foraging day (fig 2). parameters mean ± sd number of days taken for acceptance of the shell traps 13.40 ± 4.37 trap occupancy rate 44.87% number of days taken for construction of new cells after acceptance 7.50 ± 3.06 number of days taken for initiation of filling of storage pots 29.23 ± 8.97 number of honey pots observed after initiation of filling of storage pots 15.60 ± 3.92 length of honey pots 6.79 ± 0.35 mm width of honey pots 6.35 ± 0.67 mm number of pollen pots observed after initiation of filling of storage pots 6.63 ± 2.95 length of pollen pots 5.83 ± 0.91 mm width of pollen pots 4.04 ± 0.53 mm number of days taken for construction of brood cells 89.50 ± 6.07 density of brood cells / three fourth cubic inch 67.71 ± 20.83 cells length of brood cell 4.42 ± 0.51 mm width of brood cell 2.92 ± 0.21 mm table 1. colony development parameters of tetragonula iridipennis in shell traps. fig 1. foraging activity of bees after acceptance of coconut shell traps. amala udayakumar, arakalagud n shylesha, timalapur m shivalingaswamy – coconut shell traps to attract swarms of stingless bees4 fig 2. forage resource partitioning during 15 (a), 30 (b) and 45 (c) days after acceptance of traps by stingless bees discussion the colony development parameters and foraging behaviour of the swarming population of stingless bees in the coconut shells were studied (fig 3a-c). the swarms accepted the traps in a two weeks time period. guard bees were often sighted at the nest entrance soon after acceptance of the nest by the bees. the nest entrance guards has been reported to guard the in-nest resources like food stores and developing broods (holldobler & wilson, 2009). the filling of storage pots was observed at the end of the three weeks after acceptance of the traps. honey was filled initially compared to pollen in the storage pots. during the early period of nest acceptance by the swarms, there was a significant increase in the number of nectar foraging bees as compared to other resources’ foragers resulting in filling of honey pots first. the honey pots appeared pale brown in colour made of cerumen and were similar in size and shape. further the honey pots were built closer to the nest entrance as compared to the pollen pots. pollen pots appeared yellowish brown in colour with their shape and size slightly smaller than honey pots. the preparation of storage pots soon after acceptance of the traps indicated the preparedness of the foragers for the brood provisioning in the hive. previously, mounika et al. (2019) reported that newly manually divided colonies of t. iridipennis established in a time period of 40 to 107 days and storage pots were constructed in a time interval of 8-17 days after colony division. this difference in the time period of colony establishment and construction of storage pots might be due to the ready resources provided to the foragers during manual division of colonies unlike the natural swarming population. swarming occurs in a gradual phase in stingless bees where in scouts move out of the hive in small numbers seeking new nest site (van veen & sommeijer, 2000) unlike honeybees wherein swarming occurs as a singular event in mass numbers of whole colony (winston, 1987). the bees after accepting the trap started constructing the involucrum inside the trap. there were drops of propolis deposits in the fig 3a-c: development of stingless bee colony in coconut shell. a. foragers in the accepted coconut shell traps. b. honey pots formed during the initial days of acceptance of shell traps. c. fully established colony. sociobiology 68(4): e7220 (december, 2021) 5 accepted traps within few days after acceptance of the traps by the bees. propolis is a substance collected by the bees from the plant parts or wounded trees (cherbuliez, 2013) used for hive construction and protection of the colony from microbial infections (bankova et al., 2003). the swarming scouts were observed seeking new nest site at a relatively closer vicinity of two years older strong colony of stingless bees maintained in the study site. in addition to nest entering foragers, the outgoing foragers were observed to carry resin materials in their corbicula from the mother colony. the scouts of the stingless bees in a swarm were reported to seek a new nesting site closer to mother nest (kazhuiro et al., 1999). the scouts were also reported to carry resources like resin from mother nest to new site of nesting for easier establishment (vijayakumar et al., 2013). the foragers constructed honey pots filled with honey at the bottom layer which was intermingled with few brood cells during the early stage of acceptance of the trap. stingless bees use wing fanning mechanism to thermoregulate the inside hive temperature (hazelhoff, 1954; moritz & crewe, 1988). greater hive space also has a negative role in maintenance of co2 balance inside the hive by the workers during the initial phase of nest establishment (kronenberg & heller, 1982). smaller nesting space could help in successful and easier establishment of stingless bees as less energy is to be spent in thermoregulation of nesting space in smaller hives. bamboo and wooden trap nests of 2 litres capacity attracted the swarming population of stingless bee, trigona (tetragonula) minangkabau with 6% occupancy rate of the traps for nesting (inoue et al., 1993). keeping stingless bee colonies in coconut shells is a common practice adopted in kerala in india, but utilisation of coconut shell traps to attract the swarms is being reported for the first time through this study. trapping the swarms is also a viable method for conserving the natural population of stingless bees. the results of the present study indicated that coconut shells could be easily used for trapping stingless bees and maintain their colony in the urban households. empty coconut shells find a new value addition as ‘stingless bee nest’ apart from other uses. t. iridipennis was reported to construct its nests in relatively unusual sites in varied rural/urban households, wherein such shell traps can be suitably used for trapping the swarming bees. the well-established trap nests of coconut shells can be employed in pollination of crops under protected cultivation. author’s contribution au conceptualised, recorded field observations and analysed the data. tms provided technical guidance in conduct of the study. ans aided in design of the experiments. all the authors read and approved the manuscript. references alvarenga, p.e.f. 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(2007). meliponiculture in rio grande do sul: contribution to the biology and conservation of plebeia nigriceps (friese, 1901) (apidae, meliponini). biosciences journal, 23:134-140. _goback doi: 10.13102/sociobiology.v63i2.987sociobiology 63(2): 848-850 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 age components of queen retinue workers in honeybee colony (apis mellifera) honeybees are widely acknowledged as a model insect for the studies of social organization, social behaviour and neurology (robinson et al., 2005; he et al., 2015; liu et al., 2015). it is known that the age-dependent division of labor in honeybee colonies is one of the most typical features of this social insect (robinson et al., 2005). newly hatched workers undergo the task of cleaning during their first few days (kryger et al., 2000). later, they begin to consume protein-rich pollen. during the age of 5-15d, their hypopharyngeal glands fully develop and most of them play the role of nursing the queen and larvae (pan et al., 2013). then, they begin to build comb by secreting wax, cleaning the hive and compacting pollen. after 15-20 days of age, some workers begin to undertake tasks that are out of the nest whilst other workers remain inside to hive to act as receivers and distributors (kryger et al., 2000). however, this division of labor is not immutable. environmental changes can accelerate, delay, or even reverse the transition of nurse bee and forager bee (robision 1992; huang & robision 1996). furthermore, polyethism in honeybee colonies might be affected by genotypical variability (page & robinson 1991; kryger et al., 2000; wang et al., 2012). abstract it’s known that elaborate age is closely associated with polyethism in honeybee colonies, and the circle composed of queen retinue workers is a usual phenomenon in honeybee colonies. in this study, we showed that the age-bracket of retinue workers is 2-23d, but mainly 6-18d by marking newly hatched workers in two colonies. sociobiology an international journal on social insects y yi, y li, zj zeng article history edited by evandro nascimento silva, uefs, brazil received 21 january 2016 initial acceptance 17 may 2016 final acceptance 17 may 2016 publication date 15 july 2016 keywords queen, retinue workers, age. corresponding author zhi jiang zeng honeybee research institute jiangxi agricultural university nanchang, jiangxi, 330045, china e-mail: bees1965@sina.com the queen retinue pheromone (qrp) released by a honeybee queen attracts workers that we name queen retinue workers to surround beside her (slessor et al., 2005). within the hive, it can be easily observed that there is a circle consisting of queen retinue workers, however, little is known about them as few studies have been performed on them. in this study, we intend to understand how the age-bracket of queen retinue workers is determined by marking newly hatched workers. two honeybee (apis mellifera) colonies (colony a and b) were maintained at the honeybee research institute, jiangxi agricultural university, nanchang, china (28.46 °n, 115.49 °e). both colonies consisted of a new comb for the queen to lay eggs, and also a honey comb. additionally, we placed extra brood combs, which contain sealed honeybee brood that will emergence during the night, into the queen spawn controller at 5 pm (all apertures of the queen bee spawn controller were too small for workers to pass through). three hundred newly hatched workers were labeled with dyes at 9am the next day. following this, they were put into their natal colonies. in total 6000-8000 worker bees were present in jiangxi agricultural university, nanchang, jiangxi, p. r. of china short note sociobiology 63(2): 848-850 (june, 2016) 849 each observation hive. we used colors to represent their date of birth and identify their age. every two days, we marked workers with different color. in total we marked bees ten times and then began recording videos to monitor their behaviour. we recorded the queen’s behavior and the forming process of the queen retinue workers’ circle in an open transparent awning at 9:00-11:30am and 2:00-4:30pm. in order to minimize the impact of frequent interruptions on colonies, we began recording 2 days after bees were placed into the hive. we then continued recording until there were no more marked bees around the queen, a process which took about 20 days. after recording, we counted the number of marked workers in each color by watching the videos. we were then able to come to a conclusion regarding the age-bracket of queen retinue workers. a frame from a video is shown in fig 1. we analyzed the differences in the number of retinue workers of each age in the two colonies with the z-test (spss statistics 17.0). the nest (kryger et al., 2000). thus, we indicate that the agebracket of queen retinue workers is 2-23 day, but mainly 6-18 day. the honeybee queen releases qrp to encourage queen retinue workers to feed and groom her, and also to acquire and distribute her pheromone messages to other workers throughout the colony (keeling et al., 2003). fig 1. a circle composed of queen retinue workers surrounding a feeding queen. different colors on worker bee thoraxes stands represent different ages. it was easy for us to see a circle composed of queen retinue workers when the queens were laying eggs, standing still or being fed in their respective colonies during the recording period. the footage showed that there were many queen retinue workers within the age-bracket of 2-23 days. additionally, there was only one worker at the age of 29d in colony a, and three 28d workers and one 30d worker in colony b (fig 2). however, we deduce that this data should be disregarded for the reason that the number of queen retinue workers aged over 23d old was negligible. in addition, fig 3 showed that two spots (ages 6 and 8) in colony a and four spots (ages 6, 8, 16 and 18) in colony b were higher than branch line (the number stands for the age of marked workers in colonies). it is known that some workers aged 5-15d play the role of nurse to feed the queen and small larvae (pan et al., 2013). after 15-20 days of age, some workers begin to undertake tasks that are outside of fig 2. the number of queen retinue workers at each age. most queen retinue workers were within the age-bracket of 2-23d. fig 3. the z values of same aged queen retinue workers in colony a and b. the z-values of two spots in colony a and four spots in colony b were higher than 1.65. acknowledgements we thank dr. qiang huang and ms. lianne meah for revising the paper. this work was supported by the earmarked fund for the china agriculture research system (no.cars45-kxj12). all experimental procedures outlined in this work were performed in accordance with current chinese laws on animal experimentation. y yi, y li, zj zeng – age components of queen retinue workers850 references he, x.j., tian, l.q., wu, x.b. & zeng, z.j. (2016). rfid monitoring indicates honeybees work harder before a rainy day. insect science, 23:157-159. doi: 10. 1111/1744-7917.12298. huang, z.y. & robinson, g.e. (1996). regulation of honey bee division of labor by colony age demography. behavioral ecology and sociobiology, 39: 147-158. keeling, c.i., slessor, k.n., higo, h.a. & winston, m.l. (2003). new components of the honey bee (apis mellifera l.) queen retinue pheromone. biochemistry, 100: 4486–4491. doi: 10.1073/pnas.0836984100. kryger, p., kryger, u. & moritz, r.f.a. (2000). genotypical variability for the tasks of water collecting and scenting in a honey bee colony. ethology, 106: 769-779. liu, h., wang, z.l., zhou, l.b. & zeng, z.j. (2015). quantitative analysis of the genes affecting development of the hypopharyngeal gland in honey bees (apis mellifera l.). sociobiology, 62: 412416. doi: 10.13102/sociobiology.v62i3.760. page, r.e. & robinson, g.e. (1991). the genetics of division of labour in honey bee colonies. advances in insect physiology, 23:117-169. pan, q.z., lin, j.l., wu, x.b., zhou, l.b., zhang, f., yan, w.y. & zeng, z.j. (2013). research and application of key technique for mechanized production of royal jelly ( ⅳ)-design and application of a machine for gathering royal jelly. acta agriculturae universitatis jiangxiensis, 35: 1266-1271. robinson, g.e. (1992). regulation of divition of labor in insect societies. annual review of entomology, 37: 637-665. robinson, g.e., grozinger c.m. & whitfield, c.w. (2005). sociogenomics: social life in molecular terms. nature reviews genetics, 6: 257-270. doi:10.1038/nrg1575. slessor, k.n., winston, m.l. & le, c.y. (2005). pheromone communication in the honeybee (apis mellifera l.). journal of chemical ecology, 31: 2731-2745. doi:10.1007/ s10886 -0057623-9. wang, h., zhang, s.w., zhang, f. & zeng, z.j. (2012). analysis of lifespans of workers from different subfamilies in a honeybee colony. chinese journal of applied entomology, 49: 1172-1175. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.331-333sociobiology 62(2): 331-333 (june, 2015) first record of the genus mycetarotes (emery, 1913) (formicidae: myrmicinae) from colombia abstract we report the first record of fungus-growing ant genus mycetarotes (formicidae: myrmicinae) in colombia, with the species m. parallelus (emery, 1906). keywords fungus growing ant, biodiversity, ants, oil palm, colombia llanos. sociobiology an international journal on social insects af sánchez1,2, ca medina3, gw prescott4 article history edited by john lattke, universidad nacional de loja, ecuador received 01 december 2014 initial acceptance 20 february 2015 final acceptance 21 february 2015 corresponding author andrés fernando sánchez restrepo fundación para el estudio de especies invasivas bolivar 1559 (b1686efa), hurlingham, buenos aires, argentina e-mail: andrescp@gmail.com of fungus-growing ants by the presence of tridentate occipital corners (in frontal view the occipital spines project between the outer spines of the occipital margin and supraocular spines), subparallel frontal carinae that terminate near the occipital margin, lateral margins of frontal lobes little expanded laterally and bluntly angular or rounded, and the absences of clearly delimited antennal scrobes. their mandibles are finely and longitudinally striolate, with 5-6 teeth on the masticatory margin (kempf, 1960; mayhénunes & brandão, 2006). kempf (1960) carried out the first revision of the genus and recognized two species m. senticosus and m. parallelus. before this, it was initially described as a subgenus of cyphomyrmex by emery (1913). forel (1913) questioned the validity of this taxonomic rank, and borgmeier (1950) elevated it to genus. mayhé-nunes & brandão (2006) reviewed the genus, with synoptic descriptions of workers and known reproductives of the four known species and updated information about the distribution records, nest architecture, and biology. here we observe for the first time the presence of the myrmicine ant genus mycetarotes in colombia. short note 1 fundación para el estudio de especies invasivas (fuedei), buenos aires, argentina 2 universidad de buenos aires, buenos aires, argentina 3 instituto de investigación de recursos biológicos alexander von humboldt, villa de leyva, colombia 4 university of cambridge, cambridge, uk introduction to date in colombia there are about 330 species of myrmicine ants, and of these 40 are fungus-growing ants (fernández & sendoya, 2004). members of the fungusgrowing ants belong to the atta-genus group or “attine ants” in old sense (after ward et al., 2014) and its geographical distribution range covers central and south america and southern north america (mayhé-nunes & jaffé, 1998). they characterized by having a mutualistic relationship with a fungus (in most cases a leucoprineaceae), where obligately depend on the cultivation of the fungus for food and the fungus, in return, gain protection (mehdiabadi & schultz, 2009). these are some of the most ecologically important ants, and include among their number some of the dominant herbivores inthe neotropics (wilson & hölldobler, 1990). the ant genus mycetarotes is represented by four species, all neotropical: m. acutus mayhé-nunes 1995; m. carinatus mayhé-nunes, 1995; m. paralellus (emery, 1906), and m. senticosus kempf, 1960. the workers are monomorphic and can be distinguished from other members af sánchez, ca medina, gw prescott – first record of the genus mycetarotes from colombia332 material and methods the material examined comes from a study comparing the biodiversity of several taxa in forest, oil palm plantations, and cattle pastures in the colombian llanos (giloryet al., 2014). we sampled ants using pitfalls traps, following agosti et al. (2000). we based our measures on those used by mayhénunes and brandão (2006), and carried them out using a leica (80x) stereomicroscope at 60x magnification with an ocular micrometer. we measured: tl, total length; hl, head length (except mandibles); hw, head width (including eyes); ifw, inter frontal width (distance between the lateral margins of frontal lobes); scl, scape length; wl, weber’s length (alitrunk length); ci, cephalic index (hw/hl x 100); and fli, frontal lobes index (ifw/hw x 100). results and discussion subfamily myrmicinae mycetarotes parallelus (emery, 1906) material examined: 2 workers, colombia, meta, cabuyaro, 4°17’36.3”n 72°57’38.1”w, 189m, 12-feb2013, pitfall trap in oil palm plantation, g.w. prescott leg. [iavh-e-143891]. deposited and preserved point mounted, in the entomology collection of the “instituto de investigación de recursos biológicos alexander von humboldt (iavh)” villa de leyva, colombia. the workers analyzed share all the diagnostic characters listed by kempf (1960) and mayhé-nunes and brandão (2006) for the genus mycetarotes. we identified it as m. parallelus, which can be clearly distinguished by the following characters: two pairs of spines on the mesonotum (where other members of the genus have three pairs), well developed petiolar spines, postpetiole without a tooth near each side of the lateral anterior margin, weakly marked outer frontal carinae branches, and a deep circular impression on the dorsal surface of the postpetiole (mayhé-nunes & brandão 2006). additionally the specimen has an opaque integument, reddish-brown head color, yellowish legs and mesosoma, dark gaster with erect hairs absent and sparse appressed hairs. we futher corroborated the identification with photographs of the type species provided by the online database antweb v5.9 (antweb, 2014). the measurements taken from one of the workers (in mm) are: tl= 3.4; hl= 0.80; hw= 0.91; ifw=0.26; scl= 0.85; wl= 1.02; ci= 113.75; fli= 28.58. in general these measures are within the expected range or very close, except for the ci index which is a bit higher and fli index is more lower than that reported by mayhé-nunes & brandão (2006). all published records of mycetarotes to date have been from south america, with most records from brazilian amazon to southeastern brazil and northern argentina (solomon et al., 2004; mayhé-nunes & brandão, 2006). in additional to their distribution in brazil (described by mayhé-nunes & brandão, 2006), the genus has recently been recorded in venezuela (goitía & jaffé, 2009), french guyana (diasferreira et al., 2012), ecuador (salazar & donoso, 2013) and guyana (schultz & sosa-calvo, 2006). our record of mycetarotes is the first report from colombia and matches an expected pattern of distribution, where the records in venezuela and colombia possibly correspond to the northern boundary of genus. the m. parallelus is the most widespread and common of all species of the genus (mayhé-nunes & jaffé, 1998), in contrast to the other species of the genus which are uncommon and whose distributions are restricted to specific conditions (mayhé-nunes, 1995; mayhé-nunes & lanziotti, 2004). unlike other mycetarotes species m. parallelus commonly lives in open habitats, gallery forest, secondary forest, and disturbed habitats (solomon et al., 2004). this trend is corroborated by our discovery of mycetarotes workers in a mature oil palm plantation, located in the colombian llanos (a seminatural system dominated by grasslands interspersed with wet and dry forests) (gilory et al., 2014). the ant fauna of colombia is very rich (see fernández & sendoya 2004; pérez et al., 2009; vergara-navarro & serna, 2013), but more comprehensive sampling will be needed to fully understand its richness and distribution across colombia. the presence of mycetarotes is a clear example of what remains to be known. acknowledgments we thank the staff of the instituto de investigación de recursos biológicos alexander von humboldt for their logistical support. fernando fernández for help with identification of the ants. plantation managers at palumea ltda, copalma ltda, and guajcaramo s.a. for allowing access to their land, and to apolinar rojas of asohumea for helping us to coordinate our research. finally, we thank two anonymous reviewers who made valuable comments and useful suggestions that improved the quality of the manuscript. references agosti, d., majer, j., alonso, e. & schultz, t.r. (eds.) (2000). ants: standard methods for measuring and monitoring biodiversity. biological diversity handbook series. smithsonian institution press. washington d.c., usa. antweb. http://www.antweb.org/. (accessed date: 21 april 2014). borgmeier, t. (1950). bemerkungenzu dr. creighton’s werk “the ants of north america”. revista de entomologia, 21: 381-386. dias-ferreira, f., maurice madkaud, j., freyre, p. (2012). description of ant communities in dead wood ofthe french guiana rainforest and impact of disturbances on diversity. sociobiology 62(2): 331-333 (june, 2015) 333 université des antilles et de la guyane. http://www.ecofog. gf/img/pdf/ants.pdf (accessed date: 2 feb 2015). emery, c. (1906). studi sulle formiche della fauna neotropica. xxvi. bullettino della società entomologica italiana, 37: 107-194. emery, c. (1913). études sur les myrmicinae.v-vii. annales de la société entomologique de belgique, 57: 250–262. fernández, f. & sendoya, s. (2004). list of neotropical ants (hymenoptera: formicidae). biota colombiana, 5(1): 3-109. forel, a. (1913). fourmis d’argentine, du brésil, du guatémala & de cuba. bulletin de la société vaudoise des sciences naturelles, 49: 203-250. gilroy, j.j., prescott, g.w., cardenas, j.s., castañeda, p.g.d.p., sánchez, a.f., rojas-murcia, l. e., medina uribe, c.a., haugaasen, t. & edwards, d.p. (2014). minimizing the biodiversity impact of neotropical oil palm development. global change biology, doi: 10.1111/gcb.12696 goitía, w. & jaffé, k. (2009).ant-plant associations in different forests in venezuela. neotropical entomology, 38:7-31. doi: 10.1590/s1519-566x2009000100002 hölldobler, b.; wilson, o.e. (1990). the ants. harvard university press. usa. 732 p. kempf, w. (1960). a review of the ant genus “mycetatores” emery (hymenoptera, formicidae). revista brasileira de biologia, 20: 277-283. mayhé-nunes, a. j. & jaffé, k. (1998). on the biogeography of attini (hymenoptera: formicidae). ecotropicos, 11: 45-54. mayhé-nunes, a.j. (1995). sinopse do gênero mycetarotes (hym.,formicidae), com a descrição de duas especies novas. boletín de entomologia venezolana, 10: 197-205. mayhé-nunes, a.j. & brandão, c.r. (2006). revisionary notes on the fungus-growing ant genus mycetarotes emery (hymenoptera, formicidae). revista brasileira de entomologia, 50: 463-472. doi: 10.1590/s0085-56262006000400005. mayhé-nunes, a.j. & lanziotti, a.m. (2004). description of the female and male of mycetarotes carinatus (hymenoptera: formicidae). revista de biologia tropical, 52: 109-114. doi: 10.15517/rbt.v52i1.14758. mehdiabadi, n.e., & schultz, t. (2009). natural history and phylogeny of the fungus-farming ants (hymenoptera: formicidae: myrmicinae: attini). myrmecological news, 13: 37-55. pérez, l.g., pérez, g.a., echeverri-rubiano, c., sánchez, a.f., durán, j. & pedraza, l.m. (2009). riqueza de hormigas (hymenoptera: formicidae) en várzea y bosque de tierra firme de la region amazónica colombiana. boletín de la sociedad entomológica aragonesa, 45: 477-483. salazar, f., & donoso, d.a. (2013). new ant (hymenoptera: formicidae) records for ecuador deposited at the carl rettenmeyer ant collection in the qcaz museum. boletín técnico 11, serie zoológica, 8: 150-175. schultz, t. r., & sosa-calvo, j. (2008). chapter 2 – ants of southern guyana a preliminary report (pp. 31-32). in: alonso, l.e., mccullough, j., naskrecki, p., alexander, e. & wright, h.e. (eds.). a rapid biological assessment of the konashen community owned conservation area, southern guyana. rap bulletin of biological assessment 51. conservation international, arlington, va, usa. solomon, s.e., mueller, u.g., schultz, t.r., currie, c.r., price, s.l.; oliveira da silva-pinhati, a.c.; bacci jr., m. &vasconcelos, h.l. (2004). nesting biology of the fungus growing ants mycetarotes emery (attini, formicidae). insectes sociaux, 51: 333-338. doi: 10.1007/s00040-004-0742-4. vergara-navarro, e.v., & serna, f. (2013). a checklist of the ants (hymenoptera: formicidae) of the department of antioquia, colombia and new records for the country. agronomía colombiana, 31: 324-342. ward, p.s., brady, s.g., fisher, b.l., & schultz, t.r. (2014). the evolution of myrmicine ants: phylogeny and biogeography of a hyperdiverse ant clade (hymenoptera: formicidae). systematic entomology, 40, 61-81. doi: 10.1111/syen.12090. doi: 10.13102/sociobiology.v62i2.132-162sociobiology 62(2): 132-162 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 mapping continental ecuadorian ant species introduction ecuador is a neotropical country occurring along the equator line, for which it is named. ecuador is bordered by colombia to the north, peru to the east and south, and the pacific ocean to the west. the country has an astonishing variety of ecosystems provided by its tropical climate (ecuador is surrounded by wet forests on the north and east sides), oceanic influence (the humboldt current provides southwest ecuador drylands) and an altitudinal gradient (i.e. the andes mountains) that cuts the continental territory in halves. responding to these conditions, species richness of amphibians, birds, mammals, and plants peak in ecuador (bass et al., 2010). most previous ecuadorian ant research has focused on invasive ants of the galapagos islands (herrera & albelo, 2007; herrera & causton, 2010; herrera & longino, 2008; herrera et al., 2014). available information on continental ants is much more scattered and little is known about the abstract ecuador is considered a diverse country but information on the distribution and conservation of its ant species is scarce and scattered through the literature. here we review 150 years of published literature to assemble the first comprehensive species list of continental ecuadorian ants (excluding the galapagos islands). our main goal is to serve as a reference to the various research initiatives currently being done in the country. we found 2,124 ant records of 679 ant species from 180 localities reported in 149 articles. we used a subset of this database (i.e. 1,125 records left after removal of duplicates and records with no locality information) to review the ecuadorian regions, provinces, and national parks covered by the list. for a tropical country, both the number of records per ant species (mean=1.8, sd=1.9) and the number of ant species per locality (mean=6.2, sd=29.7) are extremely low. moreover, the ant records in our list are biased towards three provinces (orellana, 410 ant records and 378 ant spp.; sucumbios, 212 and 177; pichincha, 129 and 92), one region (oriente, 779 records and 487 ant species) and non-protected areas (777 ant records and 510 ant spp.). endemic ants are poorly covered by the ecuadorian system of protected areas. this study highlights the gaps and opportunities in ant research for the country. sociobiology an international journal on social insects f salazar1, f reyes-bueno2, d sanmartin3 & da donoso3 article history edited by rodrigo m. feitosa, ufpr, brazil received 02 january 2015 initial acceptance 20 january 2015 final acceptance 16 february 2015 keywords formicidae, ecuador, yasuni, otongachi, protected areas. corresponding author david a. donoso museo de colecciones biológicas mutpl departamento de ciencias naturales universidad técnica particular de loja san cayetano alto s/n, loja, ecuador e-mail: david.donosov@gmail.com species richness and distribution of the continental ant fauna (kempf, 1972; brandao, 1991; fernandez & sendoya, 2004). the first publications on continental ants date back to the 19th century and were focused on the valley of quito. mayr (1866) reported hypoclynea ursus mayr 1866 [currently known as dolichoderus abruptus (smith 1858)] in quito, pichincha province. orton (1872) reported again dolichoderus abruptus and neoponera carbonaria (smith, 1858) in the valley of quito. cameron (1891) published a list of six ant species (camponotus atriceps (smith, 1858), camponotus silvicola forel, 1902, cylindromyrmex whymperi (cameron, 1891), ectatomma brunneum smith, 1858, odontomachus haematodus (linnaeus, 1758), and pheidole cameroni mayr, 1887) gathered by e. whymper in their famous travels across the andes. recently, ant research has focused on amazonian diversity. for example, the research group lead by j. traniello (k. ryder and a. mertl) provided us with a better glimpse of the true alpha diversity of ants in tiputini, orellana review 1 museo de zoología qcaz, sección invertebrados, escuela de ciencias biológicas, pontificia universidad católica del ecuador, quito, ecuador 2 departamento de ciencias naturales, universidad técnica particular de loja, loja, ecuador 3 museo de colecciones biológicas mutpl, departamento de ciencias naturales, universidad técnica particular de loja, loja, ecuador sociobiology 62(2): 132-162 (june, 2015) 133 province (mertl et al., 2010; ryder et al., 2010). salazar & donoso (2013) gathered museum specimens from limoncocha (sucumbios province) collected by c. rettenmeyer’s research group. these studies suggest that alpha diversity in the amazon may well reach >400 ant species. nowadays, ecology-focused research has made a larger impact (and has filled more museum cabinets) on our understanding of ecuadorian ant biodiversity and its ecological functions (kaspari et al., 2003, kaspari & o’donnell, 2003, o’donnell et al., 2005, o’donnell et al., 2007, mertl et al., 2012, delsinne et al., 2013, donoso et al., 2013, clay et al., 2015). this study aims to 1) provide a first comprehensive review of published literature on ecuadorian continental ants, and current local initiatives to document the ant species living in ecuador; 2) update published species names and locality information for all records; 3) explore the geographic patterns of the records, reviewing the ecuadorian regions, provinces, and national parks covered in the list; and 4) provide recommendations for future collection efforts. materials and methods ant records for continental ecuador were extracted from peer-reviewed journal articles (e.g. taxonomic literature, checklists, ecological manuscripts) found in our research libraries and through the internet using standard tools (google scholar) and databases (isi web of knowledge, antcat.org and antweb.org). information included in phd theses, reports, museum and online databases were excluded from the list. we relied on published literature because we wanted to provide a working baseline of ants in continental ecuador, and not a comprehensive review of unpublished museum material. given our focus on the distribution of ant species, records from all previous regional catalogues (e.g., kempf, 1972; brandao, 1991; fernandez & sendoya, 2004) that did not include locality information were excluded from analysis. however, species reported in the regional catalogues elsewhere for ecuador with no specific locality info was included in the list as locality “unknown”. we standardize all taxonomic information using bolton (2014) available in the antcat.org online tool. we georeferenced all specimen records in the list by using google earth and country-level gazetteers. no georeferencing error was estimated. all locality names were updated, correcting for misspelling (common among non-spanish speaker collectors) and the latest changes in political divisions of ecuador. three provinces (guayas, napo and pichincha) have recently been split into 7 provinces (guayas and santa elena; napo and sucumbios and orellana; pichincha and santo domingo de los tsachilas). we plotted these records against standard maps of provinces (instituto nacional de estadísticas y censos, 2012), national regions (universidad del azuay, 2003), and ecuadorian protected areas and national parks (ministerio de ambiente del ecuador, 2014). results we found a total of 149 articles that report continental ecuadorian ants. in total, these articles provided us with 2,124 ant records from 679 ant species (table 1). these ant species were distributed among 180 localities. we found 68 ant species currently endemic to ecuador. the number of duplicate records (i.e. museum specimens reviewed in more than one publication) and records with no specific locality info was high; only 1,125 ant records were counted after removal of duplicate records and records with no locality information. the full dataset used for analysis is provided as supplemental material (available at http:// periodicos.uefs.br/ojs/index.php/sociobiology/rt/suppfiles/744/0; doi:10.13102/sociobiology.v62i2.132-162.s976). the average number of records per ant species was 1.8 (sd=1.9) and the average number of ant species per locality was 6.2 (sd=29.7). the majority of records are from three provinces (orellana, 410 ant records and 378 ant species; sucumbios, 212 and 177; pichincha, 129 and 92) from the northeast of the country (fig 1 and table 2). one region (i.e. “oriente” or amazon basin) has the highest quantity of records (779 records and 487 ant species). consequently, only 10% of the 1,125 records in our gazetteer fall within the coast region (fig 2). the ecuadorian system of national parks and protected areas are not well represented in our database. nonprotected areas registered 777 ant records and 510 ant species. the same pattern applied to endemic ant species, which were poorly covered by the ecuadorian system of protected areas. most endemic ant records are towards the pichincha and santo domingo de los tsáchilas provinces (at the center of the country) near the major airport (fig 3). only 8 (out of 68) endemic ant records fall within conserved areas (fig 4). fig 1. distribution of ant locality records in the 24 ecuadorian provinces. provinces have been colored by the number of ant locality records in each. notice the trend in the accumulation of records from the northeast (more records) to southwest (less records) of the country. f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species134 fig 4. distribution of endemic ant locality records regarding protected areas and national parks, which have been color coded to show if they protect endemic species or not. only 8 (out of 68) endemic ant locality records fall within such areas. fig 2. distribution of ant locality records in the three ecuadorian regions (coast, highlands and amazon). regions have been colored by number of ant locality records in each. only 10% of the 1,125 records in our gazeetter fall within the coast. fig 3. distribution of endemic ant locality records in the 24 ecuadorian provinces. provinces have been colored by thenumber of endemic ant locality records in each. most endemic ant records are towards the pichincha and santo domingo de los tsachilas provinces (at the center of the country) near the major airport. discussion we present the first list of continental ecuadorian ants 150 years after the first record of an ant in the country. the 149 papers report 1,125 unique ant records distributed in 180 unique localities. consideration of this list shows an incomplete and skewed picture of the diversity and species richness of ecuadorian ants. the total number of species in the all species records endemic species records province specimens species specimens species azuay 1 1 1 1 bolivar 3 3 1 1 cañar 2 2 0 0 carchi 4 3 4 3 chimborazo 4 3 2 1 cotopaxi 17 17 1 1 el oro 2 2 1 1 esmeraldas 12 9 1 1 guayas 32 27 5 4 imbabura 13 10 3 2 loja 0 0 0 0 los ríos 53 50 4 4 manabí 9 8 0 0 morona santiago 37 31 2 2 napo 82 56 4 4 orellana 410 378 3 2 pastaza 29 26 2 2 pichincha 129 92 14 13 santa elena 3 3 0 0 santo domingo de los tsáchilas 40 37 15 14 sucumbios 212 177 2 2 tungurahua 10 10 3 3 zamora chinchipe 7 5 0 0 table 2. distribution of georeferenced ant records across ecuadorian provinces. data are provided for ant specimen and ant species in our dataset. records for both, all ant species and endemic ant species, are given. sociobiology 62(2): 132-162 (june, 2015) 135 references albuquerque, n.l. & brandão, c.r.f. 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(1982). ground-cuckoos (aves: cuculidae) as army ant followers. revista brasileira de biologia, 42: 753–756. wilson, e.o. (2003). pheidole in the new world. a dominant, hyperdiverse ant genus. cambridge, mass.: harvard university press, [ix] + 794 pp. wygodzinsky, p. (1982). description of a new species of trichatelura (insecta, thysanura, nicoletiidae) from ecuador. sociobiology, 7: 21-24. young, a.m. & hermann, h.r. (1980). notes on foraging of the giant tropical ant paraponera clavata (hymenoptera: formicidae: ponerinae) in costa rica and ecuador. journal of the kansas entomological society, 53: 35-55. f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species142 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table. species provinces cited in acanthognathus acanthognathus brevicornis smith 1944 orellana [67] acanthognathus teledectus brown & kempf 1969 pichincha, s.domingo [33], [115] acanthoponera acanthoponera minor (forel 1899) orellana, s.domingo [16], [119], [127] acanthoponera mucronata (roger 1960) s.domingo [115] acanthoponera peruviana brown 1958 orellana [119] acanthostichus acanthostichus fuscipennis emery 1895 sucumbios [96] acanthostichus quadratus emery 1895 orellana, sucumbios [96], [117], [119] acromyrmex acromyrmex aspersus (smith 1858) s.domingo [122] acromyrmex coronatus (fabricius 1804) orellana, sucumbios [106], [119], [120] acromyrmex coronatus andicola emery 1924 loja [44], [70] acromyrmex hystrix (latreille 1802) s.domingo [122] acromyrmex niger (smith 1858) guayas [54] acromyrmex octospinosus (reich 1793) guayas, s.domingo [58], [122] acropyga acropyga decedens (mayr 1887) napo, orellana [79], [106], [119] acropyga donisthorpei weber 1944 orellana [106], [119] acropyga exsanguis (wheeler 1909) orellana [104] acropyga fuhrmanni (forel 1914) cañar, orellana [79], [106], [119] acropyga goeldii forel 1893 sucumbios [120] acropyga guianensis weber 1944 manabi, napo, orellana, sucumbios [79], [106], [119], [120] acropyga hirsutula lapolla 2004 napo [79] adelomyrmex adelomyrmex cristiani fernández 2003 pichincha [92] adelomyrmex myops (wheeler 1910) unknown [92] adelomyrmex striatus fernández 2003 napo [92] adelomyrmex tristani (menozzi 1931) unknown [92] allomerus decemarticulatus (mayr 1878) sucumbios [120] allomerus octoarticulatus mayr 1878 sucumbios [120] anochetus anochetus bispinosus (smith 1858) orellana, sucumbios [119], [120] anochetus diegensis forel 1912 napo, orellana [21], [67], [106], [118], [119] anochetus mayri emery 1884 orellana [106], [119] anochetus simoni emery 1890 guayas, manabi, pastaza, pichincha [21] anochetus targionii emery 1894 sucumbios [21] sociobiology 62(2): 132-162 (june, 2015) 143 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in apterostigma apterostigma auriculatum wheeler 1925 m.santiago, orellana [83], [106], [118], [119] apterostigma bolivianum weber 1938 m.santiago [83] apterostigma callipygium lattke 1997 sucumbios [83] apterostigma dentigerum wheeler 1925 esmeraldas, los ríos, manabi [83] apterostigma depressum lattke 1997 napo, pastaza [83] apterostigma epinotale weber 1937 sucumbios [120] apterostigma ierense weber 1937 los ríos, sucumbios [83] apterostigma mayri forel 1893 orellana, sucumbios [67], [120] apterostigma tholiforme lattke 1997 los ríos [83] apterostigma urichii forel 1893 orellana, sucumbios [67], [120] atta atta cephalotes (linnaeus 1758) napo, orellana, pichincha, s. domingo, sucumbios [25], [28], [44], [54], [59], [60], [115], [120], [130] atta sexdens (linnaeus 1758) unknown [44] azteca azteca alfari emery 1893 sucumbios [120] azteca constructor emery 1896 los ríos [126] azteca flavigaster longino 2007 los ríos [89] azteca forelii emery 1893 guayas, sucumbios [89] azteca trigona emery 1893 sucumbios [120] azteca velox forel 1899 sucumbios [59], [120], [128] basiceros basiceros conjugans brown 1974 orellana, sucumbios [12], [17], [106], [119] basiceros manni brown & kempf 1960 orellana [106], [119] basiceros militaris (weber 1950) orellana [106], [119] brachymyrmex brachymyrmex cavernicola wheeler 1938 orellana [106], [117], [118], [119] camponotus camponotus abscisus roger 1863 orellana [119] camponotus ager (smith 1858) sucumbios [120] camponotus arboreus (smith 1858) orellana [119] camponotus atriceps (smith 1858) guayas, orellana, s.domingo, sucumbios [26], [106], [118], [119], [120], [122] camponotus bidens mayr 1870 orellana [119] camponotus bispinosus mayr 1870 orellana [118], [119] camponotus branneri (mann 1916) orellana [119] camponotus brevis forel 1899 orellana [119] camponotus cacicus emery 1903 orellana, sucumbios [118], [119], [120] camponotus callistus emery 1911 orellana [119] camponotus callistus bradleyi wheeler 1934 orellana [119] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species144 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in camponotus camponotus chartifex (smith 1860) napo, sucumbios [47], [120] camponotus claviscapus forel 1899 orellana [106], [119] camponotus constructor forel 1899 orellana [119] camponotus emeryodicatus forel 1901 orellana [119] camponotus eurynotus forel 1907 orellana [104], [119] camponotus excisus mayr 1870 orellana [119] camponotus extensus mayr 1876 s.domingo [122] camponotus femoratus (fabricius 1804) orellana, sucumbios [106], [117], [118], [119], [120] camponotus formiciformis forel 1885 orellana [119] camponotus helleri emery 1903 orellana [119] camponotus hippocrepis emery 1920 orellana [119] camponotus holzi forel 1921 pichincha [60], [70] camponotus integellus forel 1899 orellana [44], [106], [118], [119] camponotus latangulus roger 1863 orellana [119] camponotus linnaei forel 1886 orellana [119] camponotus macrochaeta emery 1903 orellana [119] camponotus melanoticus emery 1894 s.domingo [122] camponotus mocsaryi forel 1902 orellana [119] camponotus mus roger 1863 orellana [119] camponotus nidulans (smith 1860) napo, orellana, sucumbios [47], [118], [119], [120] camponotus nitidior (santschi 1921) orellana [119] camponotus novogranadensis mayr 1870 orellana, sucumbios [119], [120] camponotus orthocephalus emery 1894 orellana [119] camponotus picipes (olivier 1792) pichincha [122] camponotus planatus roger 1863 orellana [106], [119] camponotus planus peregrinus emery 1893 guayas [70] camponotus rapax (fabricius 1804) orellana, sucumbios [106], [117], [119], [120] camponotus reburrus mackay 2013 napo, orellana, sucumbios [100] camponotus renggeri emery 1894 sucumbios [120] camponotus rudigenis emery 1903 s.domingo [122] camponotus senex (smith 1858) orellana [106], [119] camponotus sericeiventris (guérin-méneville 1838) pichincha, s.domingo, sucumbios [33], [120], [122] camponotus sericeiventris rex forel 1907 unknown [58] camponotus sericeiventris satrapus wheeler 1931 unknown [44] camponotus sexguttatus (fabricius 1793) orellana, pichincha, sucumbios [60], [119], [120] camponotus sexguttatus albotaeniolatus forel 1921 pichincha [70] camponotus silvicola forel 1902 chimborazo, pichincha, sucumbios [26], [120], [122] camponotus simillimus (smith 1862) pichincha [122] camponotus simillimus indianus forel 1879 pichincha [57] camponotus taeniatus roger 1863 s.domingo [122] camponotus wheeleri mann 1916 sucumbios [120] camponotus wytsmani emery 1920 orellana [119] sociobiology 62(2): 132-162 (june, 2015) 145 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in cardiocondyla cardiocondyla minutior forel 1899 orellana [32] cardiocondyla wroughtonii (forel 1890) loja [32] carebara carebara angulata fernandez 2004 orellana, sucumbios [49], [106], [119] carebara anophthalma (emery 1906) imbabura [49] carebara globularia fernandez 2004 sucumbios [49] carebara panamensis (wheeler 1925) orellana [106], [119] carebara paya fernández 2004 orellana [106], [117], [119] carebara urichi (wheeler 1922) orellana [106], [117], [118], [119] centromyrmex centromyrmex alfaroi emery 1890 orellana [117], [119] cephalotes cephalotes atratus (linnaeus 1758) m.santiago, napo, orellana, pichincha, sucumbios [24], [27], [44], [60], [119], [120] cephalotes basalis (smith 1876) s.domingo [27] cephalotes cordatus (smith 1853) orellana [119] cephalotes depressus (klug 1824) sucumbios [27], [120] cephalotes ecuadorialis de andrade & baroni urbani 1999 imbabura, pichincha [27] cephalotes laminatus (smith 1860) orellana [119] cephalotes maculatus (smith 1876) m.santiago, orellana [27], [119] cephalotes manni (kempf 1951) orellana [119] cephalotes marginatus (fabricius 1804) orellana [119] cephalotes minutus (fabricius 1804) orellana [106], [119] cephalotes opacus santschi 1920 orellana, sucumbios [27], [119], [120] cephalotes pallidus de andrade & baroni urbani 1999 orellana [119] cephalotes pavonii (latreille 1809) orellana [119] cephalotes peruviensis de andrade & baroni urbani 1999 orellana [119] cephalotes porrasi (wheeler 1942) los ríos [27] cephalotes pusillus (klug 1824) unknown [27], [44] cephalotes ramiphilus (forel 1904) napo, orellana [27], [119] cephalotes scutulatus (smith 1867) esmeraldas [27] cephalotes spinosus (mayr 1862) orellana, pastaza, sucumbios [27], [44], [72], [119], [120] cephalotes umbraculatus (fabricius 1804) orellana, sucumbios [119], [120] cerapachys cerapachys neotropicus weber 1939 sucumbios [72], [120] cheliomyrmex cheliomyrmex andicola emery 1894 orellana [107], [108], [137] cheliomyrmex audax santschi 1921 unknown [4], [137] cheliomyrmex morosus smith 1859 unknown [123] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species146 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in crematogaster crematogaster acuta (fabricius 1804) orellana, sucumbios [118], [119], [120] crematogaster brasiliensis mayr 1878 orellana, sucumbios [87], [118], [119], [120] crematogaster brevispinosa mayr 1870 el oro [121] crematogaster bryophilia longino 2003 unknown [87] crematogaster carinata mayr 1862 orellana [87], [106], [118], [119] crematogaster crinosa mayr 1862 sucumbios [87], [120] crematogaster crucis forel 1912 orellana [119] crematogaster curvispinosa mayr 1862 orellana, sucumbios [87], [119], [120] crematogaster distans mayr 1870 unknown [87] crematogaster egregior forel 1912 orellana, sucumbios [119], [120] crematogaster erecta mayr 1866 orellana, sucumbios [87], [106], [118], [119], [120] crematogaster flavomicrops longino 2003 orellana [87], [106], [118], [119] crematogaster foliocrypta longino 2003 orellana [119] crematogaster jtl-022 longino morphospecies orellana [119] crematogaster jtl-026 longino morphospecies orellana [119] crematogaster jtl-034 longino morphospecies orellana [119] crematogaster levior longino 2003 orellana [87], [106], [117], [118], [119] crematogaster limata smith 1858 orellana, sucumbios [87], [106], [118], [119], [120] crematogaster longispina emery 1890 pichincha, sucumbios [60], [87], [120] crematogaster longispina naumannae forel 1921 pichincha [70] crematogaster mancocapaci santschi 1911 el oro [70], [87], [122] crematogaster montezumia smith 1858 guayas [44], [58], [87] crematogaster nigropilosa mayr 1870 orellana [87], [106], [119] crematogaster rochai forel 1903 orellana [119] crematogaster sotobosque longino 2003 orellana [87], [106], [118], [119] crematogaster stollii forel 1885 orellana, sucumbios [87], [106], [118], [119], [120] crematogaster tenuicula forel 1904 orellana [106], [119] cryptopone cryptopone guianensis (weber 1939) los ríos [99] cylindromyrmex cylindromyrmex godmani forel 1899 orellana [18], [59], [119] cylindromyrmex striatus mayr 1870 guayas, los ríos [18], [44] cylindromyrmex whymperi (cameron 1891) guayas [26] cyphomyrmex cyphomyrmex bigibbosus emery 1894 sucumbios [120] cyphomyrmex cornutus kempf 1968 cañar, los ríos [129] cyphomyrmex costatus mann 1922 orellana [106], [119] cyphomyrmex kirbyi mayr 1887 guayas [123] cyphomyrmex laevigatus weber 1938 orellana, sucumbios [106], [118], [119], [120] cyphomyrmex muelleri schultz & solomon 2002 esmeraldas [125] cyphomyrmex salvini forel 1899 los ríos [129] sociobiology 62(2): 132-162 (june, 2015) 147 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in daceton daceton armigerum (latreille 1802) orellana, sucumbios [3] dinoponera dinoponera longipes emery 1901 pastaza [86] discothyrea discothyrea denticulata weber 1939 orellana [106], [119] discothyrea horni menozzi 1927 orellana [106], [119] discothyrea sexarticulata borgmeier 1954 orellana [106], [119] dolichoderus dolichoderus abruptus (smith 1858) pichincha, sucumbios [70], [95], [102], [120], [128] dolichoderus attelaboides (fabricius 1775) orellana, sucumbios [24], [119], [120] dolichoderus baenae mackay 1993 pichincha [110] dolichoderus bidens (linnaeus 1758) orellana, sucumbios [119], [120] dolichoderus bispinosus (olivier 1792) orellana, sucumbios [67], [120] dolichoderus decollatus smith 1858 orellana, sucumbios [119], [120] dolichoderus diversus emery 1894 orellana, sucumbios [119], [120] dolichoderus gagates emery 1890 sucumbios [120] dolichoderus germaini emery 1894 sucumbios [120] dolichoderus imitator emery 1894 orellana, sucumbios [106], [118], [119], [120] dolichoderus inpai (harada 1987) orellana [118], [119] dolichoderus lamellosus (mayr 1870) orellana, sucumbios [119], [120] dolichoderus laminatus (mayr 1870) orellana [119] dolichoderus lobicornis (kempf 1959) orellana [119] dolichoderus lugens emery 1894 sucumbios [110], [120] dolichoderus lutosus (smith 1858) orellana, sucumbios [119], [120] dolichoderus quadridenticulatus (roger 1862) orellana, sucumbios [119], [120] dolichoderus rosenbergi forel 1911 esmeraldas [59], [70], [128] dolichoderus rugosus (smith 1858) orellana, pichincha, sucumbios [44], [60], [106], [118], [119], [120] dolichoderus septemspinosus emery 1894 sucumbios [120] dolichoderus shattucki mackay 1993 orellana, pichincha [110], [119], [128] dolichoderus validus (kempf 1959) orellana [119] dolichoderus varians mann 1916 orellana, sucumbios [119], [120] dolopomyrmex dolopomyrmex ktrw-001 kari ryder morphospecies orellana [117] eciton eciton burchellii (westwood 1842) el oro, los ríos, napo, orellana, z.chinchipe [23], [24], [40], [66], [68], [107], [108], [146] eciton burchellii foreli mayr 1886 guayas, m.santiago [137] eciton drepanophorum smith 1858 unknown [8], [44], [60], [137] eciton dulcium forel 1912 sucumbios [40], [148] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species148 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in eciton eciton hamatum (fabricius 1782) guayas, los ríos, orellana, pastaza, pichincha, sucumbios [36], [39], [44], [60], [73], [106], [108], [109], [119], [137], [148] eciton jansoni forel 1912 unknown [137] eciton lucanoides emery 1894 sucumbios [42], [148] eciton mexicanum roger 1863 orellana, sucumbios [24], [39], [40], [109], [148] eciton rapax smith 1855 orellana, sucumbios [19], [40], [108], [109], [137] eciton uncinatum borgmeier 1953 chimborazo [8], [45], [70], [137] eciton vagans (olivier 1792) orellana, sucumbios [74], [106], [119] ectatomma ectatomma brunneum smith 1858 guayas [26], [44] ectatomma edentatum roger 1863 orellana, sucumbios [106], [118], [119], [120] ectatomma goninion kugler & brown 1982 esmeraldas, los ríos, pichincha, s.domingo, sucumbios [24], [78], [120] ectatomma lugens emery 1894 orellana, sucumbios [67], [106], [118], [119], [120] ectatomma opaciventre (roger 1861) napo [2] ectatomma ruidum (roger 1860) guayas, pichincha, sucumbios [16], [33], [54], [78], [112], [120], ectatomma tuberculatum (olivier 1792) orellana, sucumbios [67], [106], [119], [120] eurhopalothrix eurhopalothrix alopeciosa brown & kempf 1960 sucumbios [72], [120] gigantiops gigantiops destructor (fabricius 1804) orellana, sucumbios [106], [118], [119], [120] gnamptogenys gnamptogenys aculeaticoxae (santschi 1921) sucumbios [120] gnamptogenys acuminata (emery 1896) sucumbios [120] gnamptogenys acuta (brown 1956) pastaza [82], [85] gnamptogenys alfaroi (emery 1894) guayas [82], [85] gnamptogenys andina lattke 1995 bolívar [82], [85] gnamptogenys annulata (mayr 1887) los ríos, m.santiago, napo, pichincha, s.domingo [16], [33], [44], [82], [85], [122] gnamptogenys banksi (wheeler 1930) guayas [82], [85] gnamptogenys bisulca kempf & brown 1968 pichincha, s.domingo [33], [82], [85], [115] gnamptogenys concinna (smith 1858) orellana, sucumbios [119], [120] gnamptogenys continua (mayr 1887) orellana, pichincha, s.domingo [67], [82], [85], [115] gnamptogenys extra lattke 1995 pichincha, s.domingo [82], [85] gnamptogenys falcifera kempf 1967 sucumbios [81] gnamptogenys fernandezi lattke 1990 m.santiago [82], [85] gnamptogenys haenschi (emery 1902) napo, orellana, sucumbios [16], [44], [70], [82], [85], [106], [117], [119], [120] gnamptogenys horni (santschi 1929) el oro, esmeraldas, los ríos, m.santiago, orellana [82], [85], [104], [106], [118], [119] gnamptogenys kempfi lenko 1964 orellana [106], [119] sociobiology 62(2): 132-162 (june, 2015) 149 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in gnamptogenys gnamptogenys lanei kempf 1960 sucumbios [120] gnamptogenys laticephala lattke 1995 guayas [82], [85] gnamptogenys mecotyle brown 1958 napo, sucumbios [85], [120] gnamptogenys mediatrix brown 1958 orellana [106], [119] gnamptogenys mina (brown 1956) napo, orellana [85], [106], [119] gnamptogenys minuta (emery 1896) el oro, los ríos, napo, orellana, pichincha, sucumbios [13], [33], [81], [85], [106], [119] gnamptogenys moelleri (forel 1912) cotopaxi, orellana, sucumbios [82], [85], [104], [106], [118], [119] gnamptogenys mordax (smith 1858) m.santiago, napo [85], [120] gnamptogenys perspicax kempf & brown 1970 el oro, los ríos [82], [85] gnamptogenys pleurodon (emery 1896) napo, orellana, sucumbios [82], [85], [106], [119], [120] gnamptogenys porcata (emery 1896) pichincha, s.domingo [82], [85] gnamptogenys regularis mayr 1870 el oro, los ríos, orellana, s.domingo, sucumbios [44], [54], [59], [82], [85], [119], [120] gnamptogenys simulans (emery 1896) orellana [106], [119] gnamptogenys striatula mayr 1884 m.santiago, orellana, sucumbios [82], [85], [104], [106], [118], [119] gnamptogenys strigata (norton 1868) bolívar [76] gnamptogenys sulcata (smith 1858) los ríos, orellana, pichincha, s.domingo [82], [85], [106], [119] gnamptogenys teffensis (santschi 1929) sucumbios [120] gnamptogenys tortuolosa (smith 1858) m.santiago, pichincha [16], [60], [82], [85], [120] gnamptogenys triangularis (mayr 1887) el oro, los ríos [32], [82], [85] gnamptogenys vriesi brandão & lattke 1990 m.santiago [12], [13], [81], [127] hylomyrma hylomyrma balzani (emery 1894) napo [64] hylomyrma blandiens kempf 1961 orellana [106], [118], [119] hylomyrma dolichops kempf 1973 orellana [106], [118], [119] hylomyrma immanis kempf 1973 orellana [106], [118], [119] hylomyrma longiscapa kempf 1961 sucumbios [72] hylomyrma praepotens kempf 1973 orellana [67], [106], [119] hylomyrma sagax kempf 1973 orellana [106], [119] hypoponera hypoponera distinguenda (emery 1890) pichincha, s.domingo, sucumbios [33], [115], [120] hypoponera perplexa (mann 1922) orellana [106], [118], [119] hypoponera trigona (mayr 1887) pichincha [33] kempfidris kempfidris inusuale (fernandez 2007) sucumbios [50], [51], labidus labidus coecus (latreile 1802) orellana, pichincha, s.domingo, sucumbios [8], [33], [35], [44], [66], [67], [106], [108], [117], [119], [122], [137] labidus coecus (latreille 1802) guayas, s.domingo [54], [58], [59], [122] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species150 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in labidus labidus curvipes (emery 1900) unknown [137] labidus praedator (smith 1858) esmeraldas, orellana, sucumbios [24], [35], [36], [37], [41], [42], [68], [107], [108], [109], [118], [119], [137] labidus spininodis (emery 1890) orellana, pichincha [33], [66], [67], [108], [118] lachnomyrmex lachnomyrmex pilosus weber 1950 m.santiago, sucumbios [46], [120] lachnomyrmex scrobiculatus wheeler 1910 orellana [106], [119] lenomyrmex lenomyrmex foveolatus fernández & palacio 1999 pichincha, s.domingo [33], [115] lenomyrmex inusitatus fernandez 2001 z.chinchipe [29] lenomyrmex wardi fernandez & palacio 1999 pichincha [53] leptanilloides leptanilloides caracola donoso, vieira & wild 2006 cotopaxi [34] leptanilloides erinys borowiec & longino 2011 napo [9] leptanilloides improvisa brandão, diniz, agosti & delabie 1999 cotopaxi [13] leptanilloides nomada donoso, vieira & wild 2006 cotopaxi [34], [45] leptanilloides nubecula donoso, vieira & wild 2006 cotopaxi, s.domingo [34], [115] leptogenys leptogenys amazonica borgmeier 1930 m.santiago, orellana [84] leptogenys ciliata lattke 2011 pichincha [84] leptogenys cuneata lattke 2011 m.santiago [84] leptogenys famelica emery 1896 los ríos [84] leptogenys gaigei wheeler 1923 los ríos, m.santiago, orellana [84], [106], [119] leptogenys gorgona lattke 2011 los ríos [84] leptogenys imperatrix mann 1922 orellana [106], [119] leptogenys langi wheeler 1923 m.santiago [84] leptogenys nigricans lattke 2011 m.santiago, orellana, sucumbios [84], [106] leptogenys phylloba lattke 2011 sucumbios [84] leptogenys pucuna lattke 2011 pichincha [84] leptogenys quadrata lattke 2011 los ríos [84] leptogenys rasila lattke 2011 los ríos [84] leptogenys ritae forel 1899 orellana [84], [106], [119] leptogenys unistimulosa roger 1863 m.santiago, orellana, sucumbios [84], [120] linepithema linepithema angulatum (emery 1894) napo, pichincha, tungurahua [144], [145] linepithema fuscum mayr 1866 napo [144] linepithema humile (mayr 1868) pichincha [32], [134], [139], [142], [144] linepithema iniquum (mayr 1870) pichincha, s.domingo, tungurahua [144], [145] sociobiology 62(2): 132-162 (june, 2015) 151 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in linepithema linepithema neotropicum wild 2007 napo [144], [145] linepithema piliferum (mayr 1870) napo, pastaza, pichincha [144], [145] linepithema tsachila wild 2007 los ríos, orellana, pichincha, s.domingo [144], [145] mayaponera mayaponera constricta (mayr 1884) napo, orellana, pastaza, sucumbios [67], [99], [106], [118], [119], [120] megalomyrmex megalomyrmex balzani emery 1894 orellana [106], [119] megalomyrmex bidentatus fernandez & baena 1997 pichincha, s.domingo [33], [115], [133] megalomyrmex brandaoi boudinot, sumnicht & adams 2013 napo [10] megalomyrmex caete brandão 1990 guayas [11] megalomyrmex cuatiara brandão 1990 orellana, sucumbios [104], [106], [119], [133] megalomyrmex cupecuara brandão 1990 s.domingo [11] megalomyrmex cyendyra brandão 1990 imbabura [133] megalomyrmex drifti kempf 1961 cotopaxi, el oro, los ríos, sucumbios [10], [11] megalomyrmex foreli emery 1890 napo, orellana, sucumbios [11], [65], [67], [106], [117], [118], [119] megalomyrmex glaesarius kempf 1970 cotopaxi, napo [11], [133] megalomyrmex incisus smith 1947 m.santiago, orellana, pastaza, s.domingo [10], [11], [106], [115], [119] megalomyrmex leoninus forel 1885 napo, orellana [133] megalomyrmex modestus emery 1896 cotopaxi [133] megalomyrmex mondabora brandão 1990 orellana [106], [119] megalomyrmex mondaboroides longino 2010 orellana, s.domingo [91] megalomyrmex piriana brandão 1990 s.domingo [11] megalomyrmex silvestrii wheeler 1909 los ríos, orellana, pastaza, pichincha, sucumbios [11], [33], [106], [118], [119] megalomyrmex staudingeri emery 1890 pastaza [133] megalomyrmex tasyba brandão 1990 sucumbios [11] megalomyrmex timbira brandão 1990 orellana [106], [119] monomorium monomorium floricola (jerdon 1851) orellana [32], [119] monomorium pharaonis (linnaeus 1758) orellana, pichincha [32] mycetarotes mycetarotes acutus mayhé-nunes 1995 orellana [106], [119] mycetarotes senticosus kempf 1960 orellana [67] mycocepurus mycocepurus smithii forel 1893 orellana [106], [118], [119] myrmelachista myrmelachista plebecula menozzi 1927 guayas [88] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species152 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in myrmica myrmica rubra buckley 1866 s.domingo [122] myrmicocrypta myrmicocrypta longinoda weber 1938 orellana [106], [119] neivamyrmex neivamyrmex adnepos (wheeler 1922) unknown [137] neivamyrmex cristatus (andré 1889) orellana, sucumbios [39], [44], [107], [108], [137] neivamyrmex diana (forel 1912) unknown [137] neivamyrmex emersoni (wheeler 1921) unknown [137] neivamyrmex falcifer (emery 1900) unknown [44], [137] neivamyrmex gibbatus borgmeier 1953 orellana [109], [137] neivamyrmex halidaii (shuckard 1840) unknown [137] neivamyrmex legionis (smith 1855) unknown [38] neivamyrmex planidens borgmeier 1953 bolívar [8], [45], [70], [137] neivamyrmex pseudops (forel 1909) orellana [106], [119] neivamyrmex punctaticeps (emery 1894) orellana [106], [117], [119] neivamyrmex rosenbergi forel 1911 esmeraldas [8], [59], [137] neoponera neoponera aenescens (mayr 1870) m.santiago, orellana, pichincha [99], [119] neoponera apicalis (latreille 1802) guayas, los ríos, m.santiago, napo, orellana, pastaza, pichincha, s.elena, s.domingo, sucumbios [33], [44], [67], [99], [106], [118], [119], [120], [122], [143] neoponera bugabensis (forel 1899) guayas, pichincha, s.domingo [99] neoponera carbonaria (smith 1858) carchi, imbabura, pichincha [56], [70], [99], [111], [122] neoponera carinulata (roger 1861) orellana, pastaza, pichincha, sucumbios [99], [119], [120] neoponera cavinodis mann 1916 orellana, sucumbios [99], [119], [120] neoponera chyzeri (forel 1907) cotopaxi, imbabura, pichincha [33], [99] neoponera commutata (roger 1860) orellana, pastaza, pichincha, sucumbios, tungurahua [24], [44], [60], [99], [120], [141] neoponera cooki (mackay & mackay 2010) sucumbios [99] neoponera crenata (roger 1861) orellana, pastaza, s.domingo, sucumbios [99], [119], [120] neoponera curvinodis (forel 1899) sucumbios [99] neoponera donosoi (mackay & mackay 2010) pichincha [99] neoponera eleonorae (forel 1921) pichincha, tungurahua [60], [70], [99] neoponera fauveli (emery 1895) napo, pastaza, pichincha, tungurahua [44], [55], [99] neoponera foetida (linnaeus 1758) los ríos, orellana, sucumbios [99], [119] neoponera globularia (mackay & mackay 2010) orellana, pichincha, sucumbios [99] neoponera goeldii forel 1912 napo, sucumbios [99] neoponera hispida (mackay & mackay 2010) cotopaxi [99] neoponera inversa (smith 1858) los ríos, napo, orellana, pastaza [99], [106], [119] neoponera laevigata (smith 1858) m.santiago, orellana [99], [106], [118], [119], [141] neoponera marginata (roger 1861) orellana [118], [119] neoponera oberthueri (emery 1890) orellana, sucumbios [99], [119], [120] sociobiology 62(2): 132-162 (june, 2015) 153 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in neoponera neoponera obscuricornis (emery 1890) esmeraldas, los ríos, napo, orellana, pichincha, sucumbios [60], [67], [99], [118], [119], [120], [143] neoponera rostrata (emery 1890) orellana [99], [119] neoponera rugosula emery 1902 orellana [99] neoponera schoedli (mackay & mackay 2006) cotopaxi, pichincha [97], [99], [127] neoponera striatinodis (emery 1890) orellana [119] neoponera unidentata (mayr 1862) m.santiago, napo, orellana, pastaza, pichincha, s.domingo, sucumbios [44], [99], [119], [120] neoponera verenae forel 1922 los ríos, napo, orellana, pichincha, s.domingo, sucumbios [33], [99], [106], [118], [119], [143] neoponera villosa (fabricius 1804) m.santiago, napo, orellana, pastaza, pichincha, sucumbios, tungurahua [44], [60], [99], [119], [120] nesomyrmex nesomyrmex argentinus (santschi 1922) orellana [119] nesomyrmex asper (mayr 1887) orellana [119] nesomyrmex brasiliensis (kempf 1958) orellana [119] nesomyrmex costatus (emery 1896) orellana [119] nesomyrmex echinatinodis (forel 1886) orellana [119] nesomyrmex pleuriticus (kempf 1959) orellana [119] nesomyrmex rutilans (kempf 1958) orellana [119] nesomyrmex spininodis (mayr 1887) orellana [119] nomamyrmex nomamyrmex esenbeckii (westwood 1842) orellana [106], [107], [108], [118], [119] ochetomyrmex ochetomyrmex neopolitus fernandez 2003 orellana, sucumbios [48], [106], [118], [119], [120] ochetomyrmex semipolitus mayr 1878 orellana [106], [117], [118], [119] ochetomyrmex subpolita (wheeler 1916) sucumbios [72] octostruma octostruma ascrobicula longino 2013 manabi [93] octostruma balzani (emery 1894) orellana [104] octostruma batesi (emery 1894) unknown [93] octostruma iheringi (emery 1888) orellana [106], [119] octostruma onorei (baroni urbani & de andrade 2007) tungurahua [6], [93] octostruma stenoscapa palacio 1997 unknown [93] odontomachus odontomachus bauri emery 1892 esmeraldas, pichincha, s.domingo, sucumbios [19], [24], [33], [115], [120] odontomachus biumbonatus brown 1976 orellana, sucumbios [19], [106], [117], [119], [120], [127] odontomachus bradleyi brown 1976 napo [20] odontomachus brunneus (patton 1894) sucumbios [120] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species154 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in odontomachus odontomachus caelatus brown 1976 sucumbios [19], [120], [127] odontomachus cornutus stitz 1933 guayas [19], [70], [75], [131] odontomachus erythrocephalus emery 1890 unknown [19] odontomachus haematodus (linnaeus 1758) guayas, orellana, sucumbios [19], [26], [44], [54], [59], [67], [106], [118], [119], [120], odontomachus hastatus (fabricius 1804) esmeraldas, orellana, sucumbios [24], [119], [120] odontomachus laticeps roger 1861 sucumbios [120] odontomachus mayi mann 1912 orellana, sucumbios [119], [120] odontomachus meinerti forel 1905 orellana, sucumbios [19], [67], [104], [106], [118], [119], [120] odontomachus mormo brown 1976 guayas, los ríos, pichincha [12], [19], [20], [127] odontomachus opaciventris forel 1899 unknown [59] odontomachus panamensis forel 1899 orellana [67], [106], [118], [119] odontomachus ruginodis smith 1937 sucumbios [120] odontomachus scalptus brown 1978 napo [12], [20] odontomachus yucatecus brown 1976 orellana [106], [119] oxyepoecus oxyepoecus ephippiatus albuquerque & brandão 2004 orellana [106], [119] oxyepoecus quadratus albuquerque & brandão 2004 sucumbios [1] pachycondyla pachycondyla crassinoda (latreille 1802) m.santiago, orellana, pastaza, pichincha, sucumbios [24], [60], [67], [99], [106], [117], [118], [119], [120] pachycondyla fuscoatra (roger 1861) s.domingo [122] pachycondyla harpax (fabricius 1894) imbabura, los ríos, m.santiago, orellana, pastaza, pichincha, s.elena, s.domingo, sucumbios, tungurahua, z.chinchipe [33], [44], [67], [99], [106], [115], [118], [119], [120] pachycondyla impressa (roger 1861) cotopaxi, esmeraldas, guayas, imbabura, los ríos, napo, orellana, pichincha, s. domingo, sucumbios, tungurahua [24], [33], [99], [117], [119] pachycondyla striata smith 1858 orellana [67] pachycondyla vieirai mackay & mackay 2010 los ríos [99] paraponera paraponera clavata (fabricius 1775) orellana, s.domingo, sucumbios [24], [44], [62], [106], [119], [120], [122], [149] paratrechina paratrechina longicornis (latreille 1802) azuay, s.domingo [32], [115], [122] pheidole pheidole ademonia wilson 2003 orellana [67], [104], [105], [106], [119], [147] pheidole allarmata wilson 2003 napo, orellana [67], [103], [104], [105], [106], [119], [147] sociobiology 62(2): 132-162 (june, 2015) 155 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in pheidole pheidole alpestris wilson 2003 pichincha [147] pheidole amazonica wilson 2003 napo, orellana [103], [104], [105], [106], [117], [118], [119], [147] pheidole araneoides wilson 2003 orellana [105], [118], [119] pheidole astur wilson 2003 orellana [67], [105], [106], [118], [119], [147] pheidole biconstricta mayr 1870 orellana [67], [104], [105], [106], [117], [118], [119] pheidole bufo wilson 2003 orellana [67], [147] pheidole bulliceps wilson 2003 napo [147] pheidole cameroni mayr 1887 bolívar, carchi, chimborazo, pichincha, s.domingo [26], [70], [121], [122], [147] pheidole camilla wilson 2003 pichincha [147] pheidole caracalla wilson 2003 orellana [147] pheidole cardiella wilson 2003 manabi, orellana, pichincha [67], [147] pheidole cataractae wheeler 1916 orellana [103], [104], [105] pheidole chalcoides wilson 2003 el oro [147] pheidole cramptoni wheeler 1916 orellana [67], [103], [104], [105], [106], [119] pheidole crinita wilson 2003 m.santiago [147] pheidole cursor wilson 2003 orellana [105] pheidole deima wilson 2003 orellana [105], [106], [118], [119] pheidole diligens (smith 1858) orellana [67] pheidole ectatommoides wilson 2003 unknown [147] pheidole ecuadorana wilson 2003 pichincha [147] pheidole embolopyx brown 1868 orellana [67], [103], [104], [105], [147] pheidole epetrion wilson 2003 pichincha [147] pheidole exigua mayr 1884 orellana [106], [119] pheidole exquisita wilson 2003 pichincha [147] pheidole fimbriata roger 1863 orellana [67], [105], [106], [117], [118], [119] pheidole fissiceps wilson 2003 orellana [103], [104], [105] pheidole flavens roger 1863 orellana [105] pheidole fracticeps wilson 2003 orellana [67], [105], [106], [118], [119], [147] pheidole fullerae wilson 2003 m.santiago [147] pheidole gagates wilson 2003 orellana, sucumbios [67], [103], [104], [105], [118], [119], [147] pheidole geminata wilson 2003 pichincha [147] pheidole gilva wilson 2003 orellana [119] pheidole globularia wilson 2003 pastaza [147] pheidole gnomus wilson 2003 pichincha [147] pheidole guayasana wilson 2003 guayas, los ríos [147] pheidole haskinsorum wilson 2003 orellana [105], [147] pheidole hazenae wilson 2003 los ríos [147] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species156 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in pheidole pheidole horribilis wilson 2003 napo, orellana [67], [103], [104], [105], [106], [118], [119], [147] pheidole huacana wilson 2003 orellana [105] pheidole inversa (forel 1901) unknown [122] pheidole jivaro wilson 2003 napo [147] pheidole laelaps wilson 2003 cañar [147] pheidole laidlowi mann 1916 orellana [105], [118], [119] pheidole laselva wilson 2003 guayas [89], [90], [147] pheidole lemnisca wilson 2003 orellana [103], [104], [105], [106], [119] pheidole lupus wilson 2003 orellana [105] pheidole meinerti forel 1905 orellana [67], [147] pheidole melastomae wilson 2003 pichincha [147] pheidole mendicula wheeler 1925 orellana [67], [147] pheidole metana wilson 2003 orellana [103], [104], [105], [106], [119] pheidole micridris wilson 2003 orellana [104], [105] pheidole midas wilson 2003 orellana [67], [104], [105], [106], [118], [119], [147] pheidole minutula mayr 1878 unknown [147] pheidole napoensis wilson 2003 napo [147] pheidole nitella wilson 2003 orellana [67], [117], [118], [119], [147] pheidole palenquensis wilson 2003 los ríos [147] pheidole peckorum wilson 2003 pastaza [147] pheidole peltastes wilson 2003 orellana [67] pheidole peruviana wilson 2003 orellana [67], [105], [106], [117], [118], [119] pheidole pholeops wilson 2003 orellana [67], [103], [104], [105], [106], [119], [147] pheidole plato wilson 2003 pastaza [147] pheidole praeusta roger 1863 s.domingo [122] pheidole pubiventris mayr 1887 orellana [119] pheidole riveti santschi 1911 azuay, carchi [70], [121], [122], [124], [147] pheidole rotundiceps wilson 2003 s.domingo [147] pheidole sabella wilson 2003 orellana [106], [119] pheidole sagax wilson 2003 orellana [104], [105], [106], [117], [118], [119] pheidole sarcina (forel 1912) guayas [147] pheidole sarpedon wilson 2003 napo, orellana [103], [105], [106], [119], [147] pheidole scalaris wilson 2003 orellana [103], [105], [106], [119] pheidole scolioceps wilson 2003 orellana [104], [105], [106], [119] pheidole sospes (forel 1908) orellana [104], [105] pheidole stigma wilson 2003 tungurahua [147] pheidole subarmata mayr 1884 orellana [67] pheidole tobini wilson 2003 orellana [105], [106], [119] pheidole triplex wilson 2003 orellana [105], [106], [118], [119] pheidole tristicula wilson 2003 orellana [105], [106], [118], [119], [147] pheidole unicornis wilson 2003 cañar [147] pheidole viriosa wilson 2003 napo [147] pheidole vorax (fabricius 1804) orellana [103], [105], [106], [118], [119] pheidole xanthogaster wilson 2003 orellana [105], [118], [119] sociobiology 62(2): 132-162 (june, 2015) 157 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in pheidole pheidole zeteki smith 1947 pastaza [147] platythyrea platythyrea angusta forel 1901 m.santiago, orellana [18], [119] platythyrea sinuata (roger 1860) el oro, sucumbios [18], [120] platythyrea zodion brown 1975 pastaza [12], [18] pogonomyrmex pogonomyrmex theresiae forel 1899 guayas [52], [54], [70] prionopelta prionopelta amabilis borgmeier 1949 orellana, pichincha, s.domingo [33], [104], [106], [115], [119] prionopelta antillana forel 1909 sucumbios [120] prionopelta modesta forel 1909 s.domingo [115] prionopelta punctulata mayr 1866 orellana [67] probolomyrmex probolomyrmex petiolatus weber 1940 orellana [106], [119] proceratium proceratium ecuadoriense baroni urbani & de andrade 2003 los ríos, pichincha, s.domingo [5], [6] proceratium micrommatum (roger 1863) unknown [22] procryptocerus procryptocerus attenuatus (smith 1876) orellana [119] procryptocerus belti forel 1899 los ríos [94] procryptocerus coriarius (mayr 1870) orellana [119] procryptocerus hirsutus emery 1896 sucumbios [94] procryptocerus hylaeus kempf 1951 orellana [119] procryptocerus impressus forel 1899 orellana [119] procryptocerus mayri forel 1899 pichincha [33] procryptocerus nalini longino & snelling 2002 orellana [119] procryptocerus paleatus emery 1896 orellana [119] procryptocerus pictipes emery 1896 guayas, los ríos, orellana, [72], [94], [119] procryptocerus spiniperdus (forel 1899) unknown [69], [72] procryptocerus subpilosus (smith 1860) napo [94] procryptocerus virgatus kempf 1964 pastaza [70] protalaridris protalaridris armata brown 1980 pichincha, s.domingo [12], [22], [33], [115] pseudomyrmex pseudomyrmex atripes (smith 1860) orellana, sucumbios [119], [120] pseudomyrmex colei (enzmann 1944) orellana [119] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species158 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in pseudomyrmex pseudomyrmex curacaensis (forel 1912) sucumbios [135] pseudomyrmex dendroicus (forel 1904) m.santiago, napo, sucumbios [136] pseudomyrmex duckei (forel 1906) orellana [119] pseudomyrmex eculeus ward 1999 napo [136] pseudomyrmex eduardi (forel 1912) orellana [119] pseudomyrmex elongatus (mayr 1870) guayas, orellana, s.domingo [119], [135] pseudomyrmex ethicus (forel 1911) orellana [119] pseudomyrmex faber (smith 1858) orellana [119] pseudomyrmex filiformis (fabricius 1804) orellana [119] pseudomyrmex flavidulus (smith 1858) sucumbios [120] pseudomyrmex gracilis (fabricius 1804) guayas, orellana [32], [44], [58], [119], [138] pseudomyrmex laevifrons ward 1989 orellana [119] pseudomyrmex oculatus (smith 1855) orellana [119], [135] pseudomyrmex psw-37 ward morphospecies orellana [119] pseudomyrmex psw-52 ward morphospecies orellana [119] pseudomyrmex psw-58 ward morphospecies orellana [119] pseudomyrmex psw-59 ward morphospecies orellana [119] pseudomyrmex psw-161 ward morphospecies orellana [119] pseudomyrmex pupa (forel 1911) orellana [119] pseudomyrmex rochai (forel 1912) orellana [119] pseudomyrmex sericeus (mayr 1870) orellana [119] pseudomyrmex simplex (smith 1877) orellana [119] pseudomyrmex spiculus ward 1989 orellana [119] pseudomyrmex subater (wheeler & mann 1914) orellana [119] pseudomyrmex tenuis (fabricius 1804) orellana [106], [119] pseudomyrmex tenuissimus (emery 1906) unknown [135] pseudomyrmex terminalis (smith 1877) orellana [119] pseudomyrmex triplaridis (forel 1904) napo, pastaza, sucumbios [136] pseudomyrmex triplarinus (weddell 1850) m.santiago [43], [136] pseudomyrmex ultrix ward 1999 napo [14], [136] pseudomyrmex unicolor (smith 1855) orellana [119] pseudomyrmex urbanus (smith 1877) orellana, s.domingo [119], [135] pseudomyrmex viduus (smith 1858) napo, orellana, sucumbios [119], [136] pseudoponera pseudoponera gilberti (kempf 1960) orellana [99], [106], [119] pseudoponera gilloglyi (mackay & mackay 2010) pichincha [99] pseudoponera stigma (fabricius 1804) esmeraldas, guayas, imbabura, los ríos, napo, orellana, pichincha, s.domingo, sucumbios [32], [44], [99], [120], [122] pseudoponera succedanea (roger 1863) cotopaxi, guayas, imbabura, los ríos, manabi, napo, pastaza, pichincha, s. domingo [99] sociobiology 62(2): 132-162 (june, 2015) 159 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in rasopone rasopone arhuaca (forel 1901) napo, orellana, sucumbios [99], [106], [118], [119], [120] rasopone becculata (mackay & mackay 2010) cotopaxi, pastaza, pichincha, s.domingo [99] rasopone cernua (mackay & mackay 2010) sucumbios [99] rasopone ferruginea (smith 1858) los ríos, pichincha [99] rasopone lunaris (emery 1896) orellana [99], [106], [119] rasopone pergandei (forel 1909) esmeraldas, los ríos, manabi, orellana, pastaza, pichincha [67], [99] rhopalothrix rhopalothrix ciliata mayr 1870 pichincha [6] rogeria rogeria belti mann 1922 s.domingo [115] rogeria besucheti kugler 1994 orellana [67] rogeria blanda (smith 1858) orellana, sucumbios [77], [106], [119], [120] rogeria ciliosa kugler 1994 orellana, sucumbios [77], [106], [119] rogeria gibba kugler 1994 los ríos, manabi, pichincha, s.domingo [77] rogeria lirata kugler 1994 orellana [106], [119] rogeria merenbergiana kugler 1994 pichincha [77] rogeria micromma kempf 1961 orellana [106], [119] rogeria scobinata kugler 1994 napo, orellana, pastaza, sucumbios [77], [106], [119] rogeria subarmata (kempf 1961) orellana [119] rogeria tonduzi forel 1899 orellana [106], [119] rogeria unguispina kugler 1994 orellana [106], [119] simopelta simopelta jeckylli (mann 1916) orellana [98] simopelta longirostris mackay & mackay 2008 cotopaxi [98] simopelta manni wheeler 1935 pastaza [61], [70], [98], [140] simopelta vieirai mackay & mackay 2008 cotopaxi [98] simopelta williamsi wheeler 1935 chimborazo, guayas [61], [70], [98], [140] solenopsis solenopsis geminata (fabricius 1804) orellana, sucumbios [32], [114], [120] solenopsis stricta emery 1896 napo, pichincha [113] solenopsis virulens (smith 1858) orellana, sucumbios [32], [106], [117], [118], [119], [120] stegomyrmex stegomyrmex connectens emery 1912 orellana [106], [119] stegomyrmex manni smith 1946 orellana [106], [118], [119] stenamma stenamma alas longino 2005 s.domingo [15] stenamma felixi mann 1922 cañar [15] stenamma schmidti menozzi 1931 manabi [15] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species160 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in stigmatomma stigmatomma lurilabes (lattke 1991) s. domingo [80], [115] strumigenys strumigenys aequinoctialis de andrade 2007 pichincha [6] strumigenys alberti forel 1893 esmeraldas, los ríos, sucumbios [7], [71], [72], [120] strumigenys beebei (wheeler 1915) orellana [106], [119] strumigenys biolleyi forel 1908 cotopaxi, los ríos, pichincha, s.elena [7] strumigenys cosmostela kempf 1975 cotopaxi, orellana [7], [106], [119] strumigenys decipula (bolton 2000) orellana [7], [67], [106], [119] strumigenys denticulata mayr 1887 orellana, s. domingo, sucumbios, tungurahua [7], [67], [106], [118], [119], [120] strumigenys depressiceps (weber 1934) napo, orellana [7], [106], [119] strumigenys dolichognatha weber 1934 orellana [118], [119] strumigenys doryceps (bolton 2000) s.domingo [7] strumigenys eggersi emery 1890 orellana, sucumbios [106], [119], [120] strumigenys elongata roger 1863 los ríos, orellana, sucumbios [7], [67], [106], [119], [120] strumigenys epinotalis weber 1934 orellana [119] strumigenys fairchildi brown 1961 sucumbios [120] strumigenys formicosa bolton 2000 pichincha, s. domingo [7] strumigenys glenognatha (bolton 2000) orellana [106], [119] strumigenys godmani forel 1899 los ríos [7] strumigenys gundlachi (roger 1862) orellana, pichincha [7], [106], [119] strumigenys heterodonta (rigato & scupola 2008) pichincha [116] strumigenys incuba bolton 2000 orellana [118], [119] strumigenys lalassa (bolton 2000) pichincha [7] strumigenys longimala de andrade 2007 orellana [6] strumigenys longispinosa brown 1958 orellana, s. domingo [7], [67] strumigenys louisianae roger 1863 esmeraldas, sucumbios [7], [120] strumigenys metopia (brown 1959) orellana [106], [119] strumigenys moera bolton 2000 s.domingo [7] strumigenys nageli baroni urbani & de andrade 2007 esmeraldas [6] strumigenys necopina bolton 2000 s.domingo [7] strumigenys obliqua bolton 2000 m.santiago [7] strumigenys onorei baroni urbani & de andrade 2007 tungurahua [6] strumigenys osellai (rigato & scupola 2008) pichincha [116] strumigenys perparva brown 1958 orellana [106], [119] strumigenys precava brown 1954 napo, orellana [7], [106], [119] strumigenys rogeri emery 1890 unknown [32] strumigenys schulzi emery 1894 orellana [106], [119] strumigenys skia bolton 2000 tungurahua [7] strumigenys smithii forel 1886 orellana [106], [119] strumigenys subedentata mayr 1887 orellana, sucumbios [106], [119], [120] strumigenys tococae wheeler & bequaert 1929 orellana [119] strumigenys trinidadensis wheeler 1922 orellana, sucumbios [67], [106], [119], [120] sociobiology 62(2): 132-162 (june, 2015) 161 table 1. species list of ant species known to occur in continental ecuador, arranged alphabetically by genus. endemic species names are underlined. abbreviated references are provided for all [citation numbers] listed below table (continuation). species provinces cited in strumigenys strumigenys trudifera kempf & brown 1969 orellana [106], [119] strumigenys umboceps (bolton 2000) s. domingo [7] strumigenys urrhobia (bolton 2000) napo, orellana, pastaza [7], [67], [106], [119] strumigenys vartana (bolton 2000) pichincha [7], [116] strumigenys vilhenai bolton 2000 orellana [119] strumigenys villiersi (perrault 1986) orellana [106], [119] strumigenys vivax bolton 2000 napo [7] strumigenys zeteki (brown 1959) orellana [106], [119] tapinoma tapinoma melanocephalum (fabricius 1793) orellana [32] tatuidris tatuidris tatusia brown & kempf 1968 cotopaxi, pichincha, s. domingo, z.chinchipe [31], [33], [63], [115], [132] tetramorium tetramorium bicarinatum (nylander 1846) s. domingo, sucumbios [32] tetramorium lucayanum wheeler 1905 napo, s. domingo [32] thaumatomyrmex thaumatomyrmex atrox weber 1939 s. domingo [115] trachymyrmex trachymyrmex diversus mann 1916 orellana [106], [119] trachymyrmex farinosus (emery 1894) orellana [67], [106], [118], [119] trachymyrmex isthmicus santschi 1931 el oro, guayas [101] trachymyrmex levis weber 1938 orellana [67] trachymyrmex ruthae weber 1937 orellana [106], [118], [119] trachymyrmex zeteki weber 1940 el oro [101] tranopelta tranopelta gilva mayr 1886 orellana, sucumbios [48], [117], [119] tranopelta subterranea (mann 1916) orellana, sucumbios [48], [106], [117], [118], [119], [120] typhlomyrmex typhlomyrmex pusillus emery 1894 orellana, pichincha [33], [67], [106], [119] typhlomyrmex rogenhoferi mayr 1862 orellana, sucumbios [106], [119], [120] wasmannia wasmannia auropunctata (roger 1863) orellana, pichincha, s. domingo, sucumbios, z. chinchipe [30], [32], [33], [67], [104], [106], [115], [117], [118], [119], [120] wasmannia iheringi forel 1908 orellana [119] wasmannia rochai forel 1912 orellana [119] wasmannia scrobifera kempf 1961 orellana [106], [119] f salazar, f reyes-bueno, d sanmartin & da donoso – equadorian ant species162 table 1 notes. citation numbers as follows [1]albuquerque & brandão (2004); [2]almeida (1986); [3]azorsa & sosa-calvo (2008); [4]baroni urbani (1977); [5]baroni urbani & de andrade (2003); [6]baroni urbani & de andrade (2007); [7]bolton (2000); [8]borgmeier (1953); [9]borowiec & longino (2011); [10]boudinot et al. (2013); [11]brandão (1990); [12]brandão (1991); [13]brandão et al. (1999); [14]brandão et al. (2010); [15]branstetter (2013); [16] brown (1958); [17]brown (1974); [18]brown (1975); [19]brown (1976); [20]brown (1978a); [21]brown (1978b); [22]brown (1980); [23]brown (1997); [24]brown (2000); [25]brown (2001); [26]cameron (1891); [27]de andrade & baroni urbani (1999); [28]della lucia (2003); [29] delsinne & fernández (2011); [30]delsinne et al. (2012); [31]donoso (2012); [32]donoso et al. (2014); [33]donoso & ramón (2009); [34] donoso et al. (2006); [35]elzinga (1982a); [36]elzinga (1982b); [37]elzinga (1989); [38]elzinga (1990); [39]elzinga (1993); [40]elzinga (1994); [41]elzinga (1995); [42]elzinga & rettenmeyer (1974); [43]enzmann (1944); [44]escalante (1993); [45]esteves et al. (2011); [46]feitosa & brandão (2008); [47]fernández (2002); [48]fernández (2003); [49]fernández (2004); [50]fernández (2007); [51]fernández et al. (2014); [52] fernández & palacio (1998); [53]fernández & palacio (1999); [54]forel (1899); [55]forel (1901a); [56]forel (1901b); [57]forel (1904); [58] forel (1907); [59]forel (1911); [60]forel (1921); [61]gotwald & brown (1967); [62]hermann & young (1980); [63]jacquemin et al. (2014); [64]jansen & savolainen (2010); [65]jones et al. (1999); [66]kaspari & o’donnell (2003); [68]kaspari et al. (2011); [67]kaspari et al. (2003); [69]kempf (1964); [70]kempf (1972); [71]kempf (1975a); [72]kempf (1975b); [73]kistner & davis (1989); [74]kistner & jacobson (1990); [75] kugler (1979); [76]kugler (1991); [77]kugler (1994); [78]kugler & brown (1982); [79]lapolla (2004); [80]lattke (1991); [81]lattke (1992); [82] lattke (1995); [83]lattke (1997); [84]lattke (2011); [85]lattke et al. (2004); [86]lenhart et al. (2013); [87]longino (2003); [88]longino (2006); [89]longino (2007); [90]longino (2009); [91]longino (2010); [92]longino (2012); [93]longino (2013); [94]longino & snelling (2002); [95] mackay (1993); [96]mackay (1996); [97]mackay & mackay (2006); [98]mackay & mackay (2008); [99]mackay & mackay (2010); [100]mackay & barriga (2013); [101]mayhé-nunes & brandão (2007); [102]mayr (1866); [104]mertl et al. (2009); [105]mertl et al. (2010); [103]mertl & traniello (2009); [106]mertl et al. (2012); [107]o’donnell et al. (2005); [108]o’donnell et al. (2007); [109]o’donnell et al. (2009); [110]ortiz & fernandez (2011); [111]orton (1872); [112]ouellette et al. (2006); [113]pacheco & mackay (2013); [114]plowes et al. (2009); [115]ramón et al.(2013); [116]rigato & scupola (2008); [117]ryder et al. (2007); [118]ryder et al. (2009); [119]ryder et al. (2010); [120]salazar & donoso (2013); [121]santschi (1911); [122]santschi (1913); [123]santschi (1921); [124]santschi (1923); [125]schultz et al. (2002); [126]schupp (1986); [127]scott-santos et al. (2008); [128]shattuck (1994); [129]snelling & longino (1992); [130]solomon et al. (2008); [131]stitz (1933); [132]vieira (2004); [133]vieira (2005); [134]vogel et al. (2010); [135]ward (1989); [136]ward (1999); [137]watkins (1976); [138]wetterer (2010); [139]wetterer et al. (2009); [140]wheeler (1935); [141]wheeler (1936); [142]wild (2004); [143]wild (2005); [144]wild (2007); [145] wild (2009); [146]willis (1982); [147]wilson (2003); [148]wygodzinsky (1982); [149]young & hermann (1980). open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.7878sociobiology 69(3): e7878 (september, 2022) introduction habitat transformation and the consequent increase in species extinction rates bring enormous damage to biodiversity knowledge (tedesco et al., 2014). furthermore, abstract habitat transformation and species loss bring enormous environmental damage, whereas establishing protected areas promotes more sustainable use of environmental resources through biodiversity conservation. in this study, we aimed to point out gaps in ant knowledge and provide a species checklist that contributes to biodiversity conservation in the transition areas between cerrado and caatinga biomes, constantly threatened by land use changes. this checklist integrates data from previous studies developed at “área de proteção ambiental do rio pandeiros” (apa pandeiros), minas gerais, brazil, involving ant diversity and their contribution to ecological processes accessed and described in the studies. we showed and discussed how authors managed and provided information regarding methodologies and habitats sampled. we listed 143 ant species formally named. pheidole, camponotus and cephalotes were the most speciose genera, with more than ten species each. among ants involved in ecological processes, 40 are linked to diaspore removal (part of seed dispersal) and 30 to carcass interaction (part of the decomposition process). unbaited pitfall traps, epigeic stratum and cerrado sensu stricto, were the top sampling method, stratum, and habitats among ant studies. we presented proposals for the best management and integration of data from surveys in apa pandeiros (e.g., sharing the results of the studies with the apa managers, creating a database, and the local community). these surveys are fundamental for understanding biodiversity and ecological processes and provide valuable information to conservation biology. therefore, neglecting the importance of the cerrado-caatinga transition can lead to irreparable setbacks for scientific knowledge and biodiversity. sociobiology an international journal on social insects antônio césar m. queiroz1, lívia p. prado2,3, rafael a. casarino1, graziele s. santiago1,4 cynthia v. oliveira1,5, mariana a. rabelo1,6, carla r. ribas1,7 article history edited by eduardo soares calixto, university of florida, usa received 13 march 2022 initial acceptance 19 march 2022 final acceptance 29 july 2022 publication date 12 september 2022 keywords ant inventories, ecological role, open vegetations, neotropical savannas, tropical dry forest. corresponding author antonio c. m. queiroz laboratório de ecologia de formigas, programa de pós-graduação em ecologia aplicada, departamento de ecologia e conservação, instituto de ciências naturais, universidade federal de lavras – ufla campus universitário, cep: 37200-900 lavras, minas gerais, brasil. e-mail: queirozacm@gmail.com recent studies show that species loss exceeds early estimates (barlow et al., 2016). on the other hand, public policies such as creating protected areas are adopted to minimize biodiversity losses and conserve the ecosystems (assad et al., 2017; venter et al., 2017). 1 laboratório de ecologia de formigas, programa de pós-graduação em ecologia aplicada, departamento de ecologia e conservação, instituto de ciências naturais, universidade federal de lavras – ufla, campus universitário, lavras-mg, brazil 2 laboratório de morfologia e ecologia funcional de formigas – antmor, coordenação de ciências da terra e ecologia, museu paraense emílio goeldi/mctic, belém-pa, brazil 3 laboratório de hymenoptera, museu de zoologia da universidade de são paulo, são paulo-sp, brazil 4 instituto federal do norte de minas gerais – ifnmg, campus pirapora, pirapora-mg, brazil 5 laboratório de ecologia aplicada à conservação – leac, universidade estadual de santa cruz – uesc, ilhéus-ba, brazil 6 fundação jardim botânico de poços de caldas, poços de caldas-mg, brazil 7 lancaster environment centre, lancaster university, lancaster, uk research article ants ants (hymenoptera, formicidae) of apa pandeiros: a perspective from a decade of research in an environmental protection area in the cerrado-caatinga transition id https://orcid.org/0000-0002-7434-9796 antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research2 in brazil, most protected areas occur in the amazon biome (~70%) (vieira et al., 2019). therefore, there is a need to increase protection in non-forest environments, neglected in protection policies in recent years. two of these neglected non-forest biomes, cerrado and caatinga, have less than 10% of protected areas each (vieira et al., 2019) and are increasingly threatened by changes in land use (e.g., conversion of habitats for production of agricultural commodities) (overbeck et al., 2015). the cerrado is one of the world’s biodiversity hotspots, as it has many endemic species and high anthropogenic pressure (myers et al., 2000). the cerrado biome, also known as brazilian savanna, has a rich myrmecofauna (vasconcelos et al., 2018) due to its great diversity of habitats, edaphic and climatic conditions (oliveira & marquis, 2002). in the cerrado, ants forage and/or nest in different strata and have an intimate relationship with many plant species, so we find a great diversity of these insects, whether in the ground or vegetation. the caatinga, an arid environment and the only uniquely brazilian biome, is a center of plant species diversity (overbeck et al., 2015). however, the ant fauna in the caatinga is not as diverse as in cerrado (leal et al., 2018). endemic ant species are rarely found in caatinga, which helps to characterize this group as an impoverished formicidae fauna of cerrado (leal et al., 2017). since knowledge about the diversity of ants in the caatinga is incipient, it is expected that the same occurs with ant-plant interactions, which are present in this biome (câmara et al., 2018; leal et al., 2018; oliveira et al., 2019). cerrado and caatinga are rich in terrestrial invertebrates, but there is a discrepancy in knowledge about terrestrial invertebrate species in these biomes. if, on the one hand, studies in the cerrado are growing increasingly (borges et al., 2015), on the other hand, we know very little about the caatinga (lewinsohn et al., 2005; ganem et al., 2017), a pattern that repeats itself if we consider only ants (divieso et al., 2020). thus, more studies on non-forest biomes are essential for discovering and describing new species, their interactions and ecological functions in brazil, a center of neotropical ant diversity (fernández et al., 2021; feitosa et al., 2022; schmidt et al., 2022). the rio pandeiros environmental protection area (área de proteção ambiental do rio pandeiros – apa pandeiros) is the largest protected area in minas gerais state and one of the protected areas that cover the transition of the cerradocaatinga biomes with vegetation types of both biomes. “apa” is one of the categories of conservation units (ucs) existing in brazil (iucn – international union for conservation of nature – category v). as a sustainable use conservation unit, the apa pandeiros presents fewer restrictions, allowing human occupation, scientific research, and sustainable use of natural resources (brasil, 2000). located north of minas gerais state, this area covers the entire pandeiros river basin, an important tributary of the são francisco river, and is present in the municipalities of bonito de minas, januária and cônego marinho (nunes et al., 2009; ief-mg, 2022). queiroz-dantas et al. (2011) published the first study on ant diversity regarding apa pandeiros. it showed ant richness differences across habitats and strata. ant diversity varies across habitats, vegetation types, strata, and disturbances (philpott et al., 2010; queiroz et al., 2020). since then, different research groups have performed studies with distinct aims and methodologies evaluating ant diversity and their contribution to ecological processes in the pandeiros region. almost ten years later, santiago et al. (2020) reported ants removing diaspores in apa pandeiros. ants act by influencing plant distribution and environment structure dispersing plant seeds that facilitate their establishment. these insects also play essential roles in nutrient cycling via decomposition through environments by removing organic matter or altering the microbial community (del-toro et al., 2012). furthermore, ants participate in several other vital processes for ecosystem functioning (e.g., nutrient cycling, pollination, bioturbation, and biological control) (elizalde et al., 2020). however, despite many studies in this region, we still did not have a study that compiled information about the species in this conservation unit. in this study, we aimed to integrate previous studies involving ants at apa pandeiros to formulate a species checklist and discuss their novelties and inconsistencies. this list demonstrates a compilation of original and published data from studies that tried to understand ant assemblages and their contribution to ecological processes. these studies described and accessed the ecological processes: diaspore removal (part of seed dispersal) and carcass interaction (part of the decomposition process). in addition, we point out which are the most common habitats, methods, and strata in samplings. finally, we identify, fill, and point out gaps in ant knowledge that contribute to biodiversity conservation in the transition areas between cerrado and caatinga biomes. material and methods the ants of apa pandeiros the “área de proteção ambiental do rio pandeiros” (apa pandeiros), northern minas gerais state, brazil (fig 1) is a conservation unit with 396,060.407 hectares (ief-mg, 2022) and is in the transition between cerrado and caatinga biomes (rizzini, 1997). this ecotonal region is semiarid, with a mean temperature ranging from 21 to 24ºc and two welldefined seasons: a dry winter, from april to september, and rainy summer, from october to march. the area is under many environmental pressures involving anthropogenic activities, such as vegetation loss, fire, monocultures (eucalyptus spp.), pasture, and charcoal production (nunes et al., 2009). we compiled original and published data from studies focused on understanding ant assemblages and their contribution to processes such as diaspore removal and carcass interaction (e.g., neves et al., 2013; santiago et al., 2020; rabelo et al., 2021). once we aimed to provide a broad inventory of sociobiology 69(3): e7878 (september, 2022) 3 the ant fauna by compiling records from the datasets, we detailed the projects implemented at apa pandeiros since 2008, regarding references, sampling methods used, habitats, and location. ant diversity samplings were performed with pitfall traps (baited or not) (table 1). diaspore removal was evaluated using artificial diaspores with beads and attractive paste (made with sugars, protein and fat), and the interaction with the carcass was collected through the installation of the decaying carcass (chicken feet). details on species identification were extracted from studies or interviews with researchers. all specimens collected during research are deposited at centro de coleções, biodiversidade e patrimônio genético (icn-ufla), coleção zoológica da universidade federal de uberlândia (ufu), coleção entomológica padre jesus santiago moure da universidade federal do paraná (dzup-ufpr), and coleção de formicidae do centro de pesquisas do cacau (uesc-ceplac). all occurrences in the literature and original data were compiled and classified in a table (with data on the study area, habitat, collection method, and sampled stratum). we estimated the number of redundant species by counting the minimum and the maximum number of species that could occur if we had the identification of all morphospecies from the databases. for example, if we had found three camponotus sp.1 in three different databases, we considered these works showed a minimum of one species not formally named and a maximum of three camponotus sp.1 species. however, suppose we found two camponotus sp.1 in two databases and just one camponotus rufipes (fabricius, 1775) in the other. in this case, we considered a minimum of zero new species (among these morphospecies, once the morphospecies could be c. rufipes) and a maximum of two. we adopted this method because we do not have the compatibility of morphospecies among the studies. table 1. references, species and morphospecies richness, sampling methods, strata, habitats and geographic coordinates related to datasets (ds). a and b are parts of the same datasets published or considered separately: (d1) pct-hidro “dinâmicas de organismos associados aos ambientes de matas ciliares, cerrado e floresta estacional decidual, no médio são francisco, norte de minas gerais”. (cnpq grant ed. 35/2006, no. 555978/2006-0); (d2) “rede de pesquisa biota do cerrado – isoptera e hymenoptera” (cnpq grant 457407/2012-3) and (fapdf, projeto pronex 563/2009); (d3, d4, d5) “desenvolvimento de ferramenta para priorização de descomissionamento de pequenas centrais hidrelétricas (phc) no estado de minas gerais e estudo de caso para a pch pandeiros” (fapemig apq-03593-12). css = cerrado sensu stricto,tdf = tropical dry forest, rif = riparian forest, ant = anthropogenic habitats. data references species and morphospecies richness sampling method strata habitats coordinates d1 queiroz-dantas et al., 2011; neves et al., 2013 73; 113 baited pitfalls; baited pitfalls, beating hypogeic, epigeic, arboreal; hypogeic, epigeic, arboreal, canopy css, tdf, rif 15°30’26.2” s, 44°45’21.3” w d2 vasconcelos et al., 2018 (pandeiros) 102 baited and unbaited pitfalls epigeic, arboreal css 15º29’54” s, 44º42’29” w d3 rabelo et al., 2021 11 hand collecting (diaspores removal) epigeic css 15º26’00” s, 44º49’19” w d4 santiago, unpublished data; santiago et al., 2020 174; 37 unbaited pitfalls, hand collecting (diaspores removal); hand collecting (diaspores removal) epigeic css, tdf, ant; css 15º29’18.3” s, 44º45’30.5” w 15°30’24.5” s, 44°45’30.5” w 15°30’47.5” s, 44°45’12.6” w; 15º29’18.3” s, 44º45’30.5” w d5 santiago, unpublished data 251 unbaited pitfalls, hand collecting (diaspores removal and carcass interaction) epigeic css 15°28’2.1” s, 44°49’53.2” w 15°41’25.1” s, 44°34’18.8” w results ant samplings at apa pandeiros are concentrated to the south, in or surrounding the state wildlife refuge of pandeiros river (revs do rio pandeiros) (fig 1). we obtained a list with 470 ant species records from 66 genera and eight subfamilies. this list can present ~120 redundant records based on the number of morphospecies due to differences in specimen identification in the projects. in this way, we estimate that the sampled richness was of at least 350 species. in total, 143 species (from 470: 30.4%; from 350: 40.9%) were formally named (table 2). myrmicinae was the most speciose subfamily, with 51% identified species, followed by formicinae (15.4%) and pseudomyrmecinae (7%). antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research4 the most speciose of the 51 genera were pheidole (22), camponotus (17), and cephalotes (11). on the other hand, the highest richness among the morphospecies without confirmed identification is from the subfamilies: myrmicinae (46.6%), formicinae (9.8%), and dolichoderinae (7.2%) (table 3). among genera, pheidole (24.7%), camponotus (6.6%), and solenopsis (4.9%) dominated the group of morphospecies that need a description or confirmation of identification. ectatomma edentatum roger, 1863 was sampled in all datasets, areas, strata, with all sampling methods and fig 1. map of minas gerais state (gray), in brazil, with área de proteção ambiental do rio pandeiros – apa pandeiros (red) (a). map of apa pandeiros in (red) and revs do rio pandeiros (pink), riparian forest (rif, represented by green triangles), anthropogenic habitats (ant, represented by white dots), tropical dry forest (tdf, represented by blue pentagons), and cerrado sensu stricto (css, represented by yellow diamonds) sampled areas (b). performing both processes (diaspore removal and carcass interaction). in total, ten species belonging to three genera (camponotus, pheidole and strumigenys) and two subfamilies (formicinae and myrmicinae) were recorded for the first time in minas gerais (see table 2). among these, one species was also a new record for brazil (p. gigaflavens wilson, 2003) and another species was recorded for the first time for south america (p. caribbaea wheeler, 1911). we have two different ant profiles among the ants that performed diaspore removal and interacted with the carcass. sociobiology 69(3): e7878 (september, 2022) 5 from 40 ant species that interacted with diaspores, pheidole (35%), camponotus (15%), and ectatomma (12.5%) dominate. among the species, 87.5% interacted positively by removing the diaspores (e.g., e. permagnum forel, 1908 and odontomachus bauri emery, 1892), 45% partially consumed the diaspores (e.g., forelius brasiliensis (forel, 1908) and linepithema cerradense wild, 2007), and 32.5% interacted from both ways (e.g., c. blandus (smith, f. 1858) and e. edentatum roger, 1863). regarding 30 ant species interacting with the carcass, camponotus and pheidole genera represented 50% of the total. as examples of ants captured interacting with the carcass, we have nomamyrmex esenbeckii (westwood, 1842) and sericomyrmex sp.1. when we considered just species formally named, among sampling methods, unbaited pitfall traps captured more species and a higher number of exclusive species, followed by baited pitfall traps, hand collection, and beating (that did not present exclusive species) (fig 2a). pitfall and hand collection seem to be the best complementary sampling methods (fig 3a). the highest richness is in the epigeic, followed by arboreal, canopies and the hypogeic stratum, respectively (fig 2b). canopy does not present a specific fauna and the hypogeic stratum presents only one exclusive species (fig 3b). the cerrado sensu stricto (css) is, by far, the vegetation type with the highest number of identified species, followed by tropical dry forest (tdf), anthropogenic habitats (ant), and the riparian forest (rif) (fig 2c). the rif does not present a specific fauna, sharing almost the total species richness with tdf and css (fig 3c). fig 3. diagrams representing the number of shared and exclusive species according to: (a) sampling methods, (b) strata and (c) habitats. different colors represent different methods, strata, and habitats. bt = beating, hc = hand collection, bp = baited pitfall traps, up = unbaited pitfall traps, rif = riparian forest, ant = anthropogenic habitats, tdf = tropical dry forest, css = cerrado sensu stricto. x represents that is no occurrence of ants in the intersection. fig 2. graphs representing the number of exclusive (grey) and total (black) species according to: (a) sampling methods, (b) strata, and (c) habitats. bt = beating, hc = hand collection, bp = baited pitfall traps, up = unbaited pitfall traps, rif = riparian forest, ant = anthropogenic habitats, tdf = tropical dry forest, css = cerrado sensu stricto. antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research6 discussion we obtained a list of 470 species and morphospecies of ants by compiling species records from published and unpublished sources that used different sampling methods in different habitats and strata. our checklist provides the basis for talking about state of the art and the challenges and opportunities related to a decade of studies at apa pandeiros that will contribute to biodiversity conservation in the transition areas between cerrado and caatinga biomes. protected areas are essential to limit the loss of biodiversity, as they prevent the increase in deforestation and maintain high levels of biodiversity and endemism (oliveira et al., 2017). although inventories in protected areas are considered a priority for understanding and preserving biodiversity, recently published works have drawn attention to the scarcity of records of ants in protected areas (e.g., prado et al., 2019; divieso et al., 2020). our survey points to an expressive ant richness in this protected area, at the transition between cerrado and caatinga biomes, compared to other surveys (ulysséa & brandão, 2011; camacho & vasconcelos, 2015; costa et al., 2015; leal et al., 2017). apa pandeiros was created in 1995 (ief-mg, 2022), and the diversity of formicidae at the site only started to be investigated over a decade later. regardless of whether research on ant diversity was carried out before the publication of the “revs do rio pandeiros” management plan (ief-mg, 2019), there is just one mention of ant studies in the document. this neglect contrasts with ants’ ecological and cultural importance, which play vital roles in different environments (del-toro et al., 2012), arousing curiosity and getting people’s attention (queiroz et al., 2021). profile of ant species and morphospecies diversity the richness distribution between genera and subfamilies in apa pandeiros follows the proportion expected for these groups of ants in brazil, with camponotus, pheidole and solenopsis belonging to the subfamilies formicinae and myrmicinae, as the most recurrent (baccaro et al., 2015). despite efforts to identify challenging genera, these three still dominate the list of morphospecies in our checklist. sixty years ago, kempf (1961) already drew attention to the difficulty of identifying part of these genera, indicating the complexity of these groups and the need to advance in their taxonomy. the study of these morphospecies will contribute to understanding their biodiversity and role in the environment, increasing the number of new occurrences, and may lead to the discovery of new species. another 41 genera had species not identified at a specific level. although minas gerais is one of the best-sampled states concerning ants (schmidt et al., 2022; silva et al., 2022), further efforts and investments are needed to identify and describe possible new species (camacho & vasconcelos, 2015). the new distribution records revealed in apa pandeiros belong to camponotus, pheidole and strumigenys. these genera are highly diverse and taxonomically challenging, with many described (over 800 species) and undescribed species (baccaro et al., 2015). in this sense, our checklist helps to fill gaps in species distribution. we found both widely distributed species in south america (p. exigua) and surprising records (p. caribbaea), which, until then, was only known from a few specimens from jamaica (janicki et al., 2016). in addition, we highlight the species e. edentatum, from the subfamily ectatomminae, also widely distributed in brazil (see baccaro et al., 2015; delabie et al., 2015), as the most common species in studies carried out at apa pandeiros. this species has reasonably deep nests in the ground, with trash disposal that favors better seed germination (delabie et al., 2007). in the cerrado, e. edentatum is also frequently found consuming nectar or honeydew provided by homopterans, acting in plant defense (marques et al., 2015). furthermore, this species may play a fundamental role in biological control (delabie et al., 2007). profile of ants that contribute to ecological functions the diaspore removal, part of the secondary seed dispersal by ants, was carried out in apa pandeiros mainly by species of the most common genera, more prominently in poneromorphic species, such as e. edentatum, e. permagnum and odontomachus bauri. poneromorphs play a crucial role in seed dispersal in the cerrado and caatinga (christianini, 2015). these ants, considered high-quality dispersers, remove many seeds and disperse them over greater distances (magalhães et al., 2018), in contrast to species of the genus pheidole, considered low-quality dispersers (leal et al., 2014), who mainly clean the seeds, which can sometimes reduce fungal infections and favor germination and survival. dolichoderinae from open habitats in the cerrado and caatinga, such as forelius brasiliensis and linepithema cerradense (wild, 2007; leal et al., 2017), present in apa pandeiros, can also be considered poor quality dispersers. such species have generalist habits and remove particles from diaspores without carrying them (padilha, 2013). therefore, due to the large proportion of ants that remove and clean diaspores (70%), we saw that the contribution of these ants to the restoration of degraded areas by dispersing seeds could be high, especially in pandeiros that presented many abandoned sites that recovered to cerrado in the last years (guimarães-silva, 2019). despite the contribution of ants to decomposition (del-toro et al., 2012), studies on this process are still unusual. in this study, we saw that most of the species that contributed to the process were camponotus and pheidole. the ants participating in decomposition are mostly generalists (tabor et al., 2005). however, nomamyrmex esenbeckii and sericomyrmex sp.1 were found exclusively interacting with the carcass. nomamyrmex often follows and attacks other ant species (baccaro et al., 2015). in experiments, this ant may have been collected occasionally. sericomyrmex genus is a fungus-growing ant whose biology is not well known (baccaro sociobiology 69(3): e7878 (september, 2022) 7 et al., 2015). the fact that this species is removing putrefying organic matter indicates that the sources for the fungus may be broader than previously documented. therefore, these records reinforce the need to know the species’ biology that acts in each process. methodologies, strata and habitats sampled focusing on ants in apa pandeiros the differences found in species richness, especially between sampling methods and studies, are directly related to the sampling effort (e.g., exposure time, number of samples, habitats and microhabitats sampled) and the capture approach (e.g., micro-habitat to be sampled). it is natural that the studies that used manual collections to evaluate processes presented lower richness than the capture of ants with pitfall traps. still, we found more exclusive species captured through unbaited pitfall traps. since ground pitfalls capture larger numbers of ants per sampling point (agosti & alonso, 2000; wang et al., 2001) it is not surprising that this technique sampled the highest proportion of species. however, in this study, we saw that adding baits helps to capture more ants in other strata, such as the arboreal (10 arboreal ants exclusively sampled with baited pitfalls). it is also possible that pitfall trapping is the most efficient technique for capturing insects in more open environments with fewer litter cover. for instance, the vegetation types of non-forest environments such as the cerrado and caatinga. finally, we found that the beating adds a small number of species, but we emphasize that the methods used are, in a way, complementary and the use of one or more methods depends on the need and objectives of the study. the greatest ant richness was found in the cerrado sensu stricto and in the epigeic stratum, the best-sampled vegetation type and stratum present in all studies. in neves et al. (2013), the cerrado sensu stricto showed similar richness compared to other vegetation types, but the epigeic stratum had the highest ant richness, composed mainly by species of the genera camponotus and pheidole (neves et al., 2013; schmidt et al., 2013). however, we emphasize that the caatinga of northern minas gerais is still a poorly studied area (lessa et al., 2019), and ants’ studies in tropical dry forests of the region (e.g., marques & schöereder, 2013) can bring valuable new information. as seen in the map (fig 1b), the areas sampled and mentioned above are concentrated to the south of the apa. probably, researchers focused their work on these areas for logistical reasons. thus, it is necessary to point out that sampling ants in other apa pandeiros regions and habitats can present us with many other novelties concerning ant diversity. recommendations and conclusions in this study, we provided a list of 143 ant species. with this list, we can reinforce the importance of poneromorph ants to the seed dispersal process that helps seedling establishment and growth and, consequently, the increment in plant cover (christianini, 2015). still, we need to know how low-quality diaspore dispersers can contribute to ecological restoration in apa pandeiros. we also found that little is known about the ecology and biology of ants involved in the decomposition process. furthermore, the collection of the highest number of species in the cerrado sensu stricto, epigeic stratum, and unbaited pitfall traps is due to the number of samples and studies with these characteristics. based on the information compiled here, we propose in the short term: 1) sharing studies products and creating a database to strengthen the dialogue among academia, apa managers and the local community, at the most appropriate time, highlighting positive aspects of ants (queiroz et al., 2021). 2) use the information presented here to suggest priority areas for conservation in the region (places with high species diversity, endemic species, endangered species and species that are essential to ecological processes). 3) the publication and permanent updating of the apa pandeiros ant species database in repositories, at least from projects of the same groups. some actions have been incorporated and improved on recent projects in apa pandeiros (e.g., activities with children’s books and presentations in local primary schools). in the long term, we also recommend that future studies with ants at apa pandeiros implement standardized approaches in other areas of the apa for further excellent knowledge of diversity in the conservation unit. preferably, at the north of “revs do rio pandeiros”. insects’ surveys, especially ants, in protected areas are fundamental for understanding biodiversity and ecological functions. this type of survey can contribute to the management and monitoring of areas by using ants as bioindicators of environmental quality and ecosystem services. however, the decrease in funding for studies in brazil, provided mainly by public agencies (mcmanus & neves, 2021) in little-known regions, such as the cerrado-caatinga transition areas, can lead to irreparable setbacks for scientific knowledge and its biodiversity (overbeck et al., 2018; feitosa et al., 2022). if not, we will observe the advance of new agricultural frontiers, mining, dam and reservoir projects in these regions with rapid habitat transformation and consequent species loss, many before they are formally described. acknowledgements we thank r.m. feitosa, j.h.c. delabie, a.c. ferreira, g. camacho, f. fernández, t. schultz, j. sosa-cavo, a. jesovnik, f.s. neves, h. vasconcelos and many myrmecologists involved in these ant identifications and surveys. this study was funded by cemig/programa de pesquisa e desenvolvimento da aneel p&d gt 611 – descomissionamento da pch pandeiros: uma experiência inédita na américa do sul. acmq has a postdoctoral fellowship from cemig/programa de pesquisa e desenvolvimento da aneel p&d gt 611 “descomissionamento da pch pandeiros: uma experiência inédita na américa do sul”. lpp was funded by são paulo research foundation (fapesp) grant #2022/01974-8. antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research8 subfamilies genus/species habitats amblyoponinae fulakora fulakora armigera (mayr, 1887)2 css prionopelta prionopelta punctulata mayr, 18665 css dolichoderinae azteca azteca instabilis (smith, 1862)1 css, rif dolichoderus dolichoderus bispinosus (olivier, 1792)1,4 ant, rif dolichoderus diversus emery, 18941,2 css, rif, tdf dolichoderus lamellosus (mayr, 1870)2 css dorymyrmex dorymyrmex brunneus forel, 19082,3,4,5 ant, css, tdf dorymyrmex goeldii forel, 19042 css forelius forelius brasiliensis (forel, 1908)3 css gracilidris gracilidris pombero wild & cuezzo, 20061,2,4,5 ant, css, rif linepithema linepithema cerradense wild, 20072,3 css dorylinae acanthostichus acanthostichus serratulus (smith, 1858)1 tdf labidus labidus coecus (latreille, 1802)1,2,4,5 ant, css, rif labidus mars (forel, 1912)1 css labidus praedator (smith, 1858)1,4,5 css, tdf neivamyrmex neivamyrmex minensis (borgmeier, 1928)4,5 css, tdf neivamyrmex pseudops (forel, 1909)2,4,5 ant, css nomamyrmex nomamyrmex esenbeckii (westwood, 1842)5 css ectatomminae acanthoponera acanthoponera goeldii forel, 19125 css acanthoponera mucronata (roger, 1860)1 css ectatomma ectatomma brunneum smith, 18583,4,5 ant, css ectatomma edentatum roger, 18631,2, 3,4 ant, css, rif, tdf ectatomma muticum mayr, 18701,2 css, rif, tdf ectatomma opaciventre (roger, 1861)1,2,3,4,5 css ectatomma permagnum forel, 19082,4,5 ant, css ectatomma planidens borgmeier, 19393,4,5 ant, css ectatomma tuberculatum (olivier, 1792)1,2,4,5 css gnamptogenys gnamptogenys sulcata (smith, 1858)5 css holcoponera holcoponera striatula (mayr, 1884) css table 2. taxa recorded for apa pandeiros. species indicated with an asterisk (*) represent new records for the state of minas gerais, two asterisks (**) represent new records for brazil, and three asterisks (***) represent new records for south of america. the numbering provided after the species name represent different datasets: (1) queiroz-dantas et al., 2011 and neves et al., 2013; (2) vasconcelos et al., 2018; (3) rabelo et al., 2021; (4) santiago, unpublished data from santiago 2015 and santiago et al., 2020; (5) santiago, unpublished data from santiago 2019. non-numbered species represent data included in this work. css = cerrado sensu stricto, rif = riparian forest, ant = anthropogenic habitats, tdf = tropical dry forest. sociobiology 69(3): e7878 (september, 2022) 9 formicinae acropyga acropyga fuhrmanni (forel, 1914)2 css brachymyrmex brachymyrmex heeri forel, 18741 css, rif, tdf brachymyrmex patagonicus mayr, 18681,2 css, rif, tdf camponotus camponotus ager (smith, 1858)4 css camponotus arboreus (smith, 1858)1,2,4,5 css, rif, tdf camponotus atriceps (smith, 1858)1,2,4,5 css, tdf camponotus blandus (smith, 1858)2,3,4,5 ant, css, tdf camponotus bonariensis mayr, 18682 css camponotus cingulatus mayr, 18621 css, rif, tdf camponotus crassus mayr, 18623,4,5 css camponotus fastigatus roger, 18631,2,4,5 ant, css, tdf camponotus leydigi forel, 18864,5 ant, css camponotus melanoticus emery, 18941,2,4,5 ant, css, rif, tdf camponotus mus roger, 18634,5* css, tdf camponotus novogranadensis mayr, 18702 css camponotus renggeri emery, 18941,2,4,5 ant, css, rif, tdf camponotus rufipes (fabricius, 1775)1 css, rif, tdf camponotus senex (smith, 1858)1,2 css, rif, tdf camponotus substitutus emery, 18942 css camponotus vittatus forel, 19043 css myrmelachista myrmelachista nodigera mayr, 18872 css paratrechina paratrechina longicornis (latreille, 1802)4 ant myrmicinae atta atta laevigata (smith, 1858)1,5 css, rif atta sexdens (linnaeus, 1758)1,4,5 ant, css, tdf blepharidatta blepharidatta conops kempf, 19671,2,3,4,5 ant, css, rif, tdf cardiocondyla cardiocondyla emeryi forel, 18814 ant carebara carebara brevipilosa fernández, 20044 css carebara urichi (wheeler, 1922)1 css, rif cephalotes cephalotes atratus (linnaeus, 1758)1,5 css, rif, tdf cephalotes betoi de andrade & baroni urbani, 19922 css cephalotes clypeatus (fabricius, 1804)2,5 css cephalotes grandinosus (smith, 1860)1 css, tdf cephalotes maculatus (smith, 1876)1 tdf cephalotes minutus (fabricius, 1804)1,2,5 css, tdf cephalotes notatus (mayr, 1866)1 css, rif, tdf cephalotes pallidoides de andrade, 19992 css cephalotes pavonii (latreille, 1809)1 css, tdf cephalotes persimilis de andrade, 19992,5 css cephalotes pusillus (klug, 1824)1,2,4,5 css, rif, tdf crematogaster subfamilies genus/species habitats t ab le 2 . t ax a re co rd ed fo r a pa p an de ir os . ( c on tin ua tio n) antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research10 myrmicinae crematogaster abstinens forel, 18991,5 css, rif crematogaster chodati forel, 19215 css crematogaster crinosa mayr, 18621 css, rif, tdf crematogaster erecta mayr, 18661 css, rif, tdf crematogaster obscurata emery, 18954,5 css, tdf crematogaster rochai forel, 19035 css crematogaster stollii forel, 18853 css cyatta cyatta abscondita sosa-calvo et al., 20134 css cyphomyrmex cyphomyrmex transversus emery, 18942 css hylomyrma hylomyrma reitteri (mayr, 1887)4,5 css kalathomyrmex kalathomyrmex emeryi (forel, 1907)4,5 ant, css megalomyrmex megalomyrmex silvestrii wheeler, 19094 ant monomorium monomorium pharaonis (linnaeus, 1758)4 ant mycetarotes mycetarotes parallelus (emery, 1906)4,5 ant, css, tdf mycetomoellerius mycetomoellerius dichrous (kempf, 1967)5 css mycetomoellerius urichii (forel, 1893)1 rif mycetophylax mycetophylax lectus (forel, 1911)2 css mycocepurus mycocepurus goeldii (forel, 1893)2,4,5 ant, css mycocepurus smithii (forel, 1893)2,4,5 ant, css nesomyrmex nesomyrmex costatus (emery, 1896)1 css nesomyrmex asper (mayr, 1887)1 css, rif, tdf ochetomyrmex ochetomyrmex semipolitus mayr, 18782 css paratrachymyrmex paratrachymyrmex bugnioni (forel, 1912)2 css pheidole pheidole aberrans mayr, 18684,5 ant, css pheidole biconstricta mayr, 18704,5 * css pheidole capillata emery, 19063 css pheidole caribbaea wheeler, 19114,5*** ant, css pheidole cyrtostela wilson, 20032 css pheidole exigua mayr, 18844,5* ant, css, tdf pheidole fracticeps wilson, 20032,4,5 ant, css, tdf pheidole gigaflavens wilson, 20034** ant pheidole jelskii mayr, 18844,5 css pheidole microps wilson, 20035* css pheidole obscurithorax naves, 19854,5 ant, css, tdf pheidole oxyops forel, 19082,5 css pheidole radoszkowskii mayr, 18842 css subfamilies genus/species habitats t ab le 2 . t ax a re co rd ed fo r a pa p an de ir os . ( c on tin ua tio n) sociobiology 69(3): e7878 (september, 2022) 11 myrmicinae pheidole rochai forel, 19125* css pheidole scapulata santschi, 19234,5 css pheidole schwarzmaieri borgmeier, 19392 css pheidole subarmata mayr, 18845 css pheidole susannae forel, 18864 ant, tdf pheidole triconstricta forel, 18862,4,5 ant, css, tdf pheidole valens wilson, 20034 tdf pheidole vallifica forel, 19015* css pheidole zelata wilson, 20034** ant, css pogonomyrmex pogonomyrmex naegelii emery, 18781,5 css, rif solenopsis solenopsis globularia (smith, 1858)5 css solenopsis invicta buren, 19724 ant, tdf solenopsis substituta santschi, 19252,4,5 css solenopsis tridens forel, 19113,4 ant, css, tdf strumigenys strumigenys borgmeieri brown, 19545* css strumigenys grytava (bolton, 2000)2 css strumigenys infidelis santschi, 19194,5 css tetramorium tetramorium simillimum (smith, 1851)4 ant wasmannia wasmannia auropunctata (roger, 1863)2,4,5 css, tdf wasmannia lutzi forel, 19082 css ponerinae anochetus anochetus inermis andré, 18892 css centromyrmex centromyrmex brachycola (roger, 1861)2 css neoponera neoponera laevigata (smith, 1858)5 css neoponera villosa (fabricius, 1804)1,2,4 css, tdf odontomachus odontomachus bauri emery, 18922,4,5 css, tdf odontomachus brunneus (patton, 1894)1 css, tdf odontomachus chelifer (latreille, 1802)4 css odontomachus haematodus (linnaeus, 1758)4 css thaumatomyrmex thaumatomyrmex mutilatus mayr, 18871,4 ant, rif, tdf pseudomyrmecinae pseudomyrmex pseudomyrmex curacaensis (forel, 1912)2 css pseudomyrmex elongatus (mayr, 1870)1,2 css, rif, tdf pseudomyrmex gracilis (fabricius, 1804)1,2,4,5 ant, css, rif, tdf pseudomyrmex oculatus (smith, 1855)1 rif, tdf pseudomyrmex schuppi (forel, 1901)4 tdf pseudomyrmex tenuis (fabricius, 1804)1,4,5 css, rif, tdf pseudomyrmex tenuissimus (emery, 1906)2 css pseudomyrmex termitarius (smith, 1855)4,5 css pseudomyrmex unicolor (smith, 1855)2,4 css pseudomyrmex urbanus (smith, 1877)2 css subfamilies genus/species habitats t ab le 2 . t ax a re co rd ed fo r a pa p an de ir os . ( c on tin ua tio n) antônio c. m. queiroz et al. – ants of apa pandeiros: a decade of research12 number of morphospecies/dataset genus d1 d2 d3 d4 d5 acromyrmex 2 1 anochetus 1 apterostigma 1 1 atta 3 azteca 1 1 brachymyrmex 3 1 4 6 camponotus 5 1 1 11 21 cardiocondyla 1 carebara 1 cephalotes 1 crematogaster 1 5 4 8 cyphomyrmex 1 2 dolichoderus 1 dorymyrmex 1 2 5 9 eciton 1 1 ectatomma 1 2 4 eurhopalothrix 1 forelius 3 2 1 5 5 gnamptogenis 3 hypoponera 1 1 labidus 1 linepithema 1 2 2 megalomyrmex 1 1 mycetophylax 1 2 mycetosoritis 1 mycocepurus 1 myrmicinae (subfamily) 1 myrmicocrypta 1 2 2 neivamyrmex 2 3 3 nesomyrmex 1 1 nylanderia 2 1 1 1 ochetomyrmex 1 1 odontomachus 1 oxyepoecus 1 5 pheidole 23 13 1 34 64 pseudomyrmex 2 4 2 rogeria 2 sericomyrmex 1 1 solenopsis 7 5 9 11 strumigenys 2 1 3 1 tapinoma 1 1 3 mycetomoellerius/paratrachymyrmex 6 1 5 5 wasmannia 3 3 6 xenomyrmex 1 morphospecies richness 65 41 6 108 181 table 3. morphospecies recorded for apa pandeiros regarding publications or databases. the morphospecies follows the alphabetical order. the number in parentheses refers to the richness recorded. as the species recorded in the literature were not examined, the genus identified as “trachymyrmex” was updated to mycetomoellerius/paratrachymyrmex. the morphospecies are not standardized among the datasets (ds). sociobiology 69(3): e7878 (september, 2022) 13 authors’ contribution acmq, rac conceived and designed the research and wrote the first draft; acmq and lpp organized the data; gss, mar helped with non-published data, all authors contributed writing and editing the manuscript. references agosti, d., majer, j.d., alonso, l.e. & schultz, t.r. 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(2007). taxonomic revision of the ant genus linepithema (hymenoptera: formicidae) (vol. 126). university of california press. _hlk109926680 _hlk109926965 _hlk109922484 _hlk105156932 _hlk105157081 _hlk109917580 doi: 10.13102/sociobiology.v63i2.946sociobiology 63(2): 804-812 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 risk of local extinction and genetic diversity of melipona quadrifasciata (apidae: meliponini) in a possible northeastern limit of its distribution in brazil introduction the stingless bees have an important ecological role, especially in the pollination of a large fraction of plants forming brazilian forests. the stingless bee m. quadrifasciata is considered a generalist species, acting as a pollinator of several plant species and playing an important ecological role in the ecosystem (ramalho et al., 2004; antonini et al., 2006). in melipona genus, the bee m. quadrifasciata is one of the best known species found in brazil and is divided into two subspecies m. quadrifasciata quadrifasciata (lepeletier) and m. quadrifasciata anthidioides (lepeletier) (waldschmidt et al., 2002). m. quadrifasciata quadrifasciata was initially abstract rapid loss of genetic diversity among eusocial bees, and extinction of their local population has become a major world concern. populations of m. quadrifasciata have increasingly declined due to predatory extractivism and destruction of their habitat. knowledge of their local population could give insights on the strategies for monitoring and conservation of this species. in this study, initially, 14 colonies from the northern limit of the northeastern sandbank of the atlantic forest were analysed employing geometric morphometrics techniques. then the cytochrome b gene sequences and issr primers were utilized for molecular analysis of these colonies and the results were compared with 15 m. quadrifasciata colonies found in the semiarid caatinga region. the morphometrics and the molecular analysis suggested the formation of disjunct populations between the studied geographical zones. all the 14 colonies analysed from the são francisco river region were found on the southern margin of the são francisco river and they all belonged to one single haplotype. no colonies were found on the northern bank of the river. we suggest that the são francisco river is an effective geographical barrier for the distribution of this species and propose an urgent need for the development of a conservation program for the population of m. quadrifasciata as it represents a unique haplotype in the region. also their nesting habit exclusively in the diseased coconut trees which probably will be replaced by new trees, create the risk of population extinction due to the lack of nesting site. sociobiology an international journal on social insects ed araujo1, rg oliveira1, hcm calazans1, ccs frança1, v santos1, gt ribeiro1, s jain1, mva batista1, la nunes2 article history edited by cândida m. l. aguiar, uefs, brazil received 09 november 2015 initial acceptance 17 december 2015 final acceptance 20 april 2016 publication date 15 july 2016 keywords coconut trees, nesting site, geometric morphometrics, stingless bee, molecular marker. corresponding author edilson divino de araujo federal university of sergipe são cristóvão-se, brazil e-mail: edaraujo@ufs.br registered in colder biomes located in rio grande do sul and southeast brazil, with patterns of yellow continuous tergal bands, and m. quadrifasciata anthidioides is found in northeastern são paulo along the east coast of brazil until the state of paraíba with patterns of interrupted tergal bands. there is a zone of hybridization of this species in the southeastern brazil between the states of são paulo and minas gerais (silveira et al., 2002; camargo & pedro, 2007; tavares et al., 2013). waldschmidtet al. (2000) reported that individuals with a pattern similar to the continuous tergal bands of m. quadrifasciata quadrifasciata can be found in warm climates. this fact was confirmed by batalha-filho et al. (2009) who found 1 federal university of sergipe, são cristóvão-se, brazil 2 state university of southwestern bahia, jequie-ba, brazil research article bees sociobiology 63(2): 804-812 (june, 2016) 805 specimens with standard tergal bands of m. quadrifasciata quadrifasciata in the semiarid region in the states of bahia and sergipe, in the northeast of brazil. these authors concluded that existence of more than two groups should be considered within this subspecies according to their phylogeographical pattern, and suggest that the level of genetic variation is too low for their division into different subspecies. according to kerr et al. (1996), the changes caused by anthropogenic disturbances have promoted changes in habitats, compromising the diversity of stingless bees due to the reduction in nesting sites and the availability of trophic resources, unfavourable for the maintenance of the natural colonies. the reduction of these resources promotes both the local disappearance of certain species, as well as forces nesting in unusual sites (souza et al.,2009). the first scientific record of a population of m. quadrifasciata, using hollow coconut trunk (cocus nucifera l.) as nesting site, at the mouth of the river são francisco in the state of sergipe was made in by souza et al. (2006). these authors also called attention to the conflict between shortage of nesting sites in the region, habitat fragmentation and the dependence of this species on nesting sites located in trunks of the economically doomed coconut trees attacked by the weevils. in the beginning of the colonization of brazil by the portuguese, in the sixteenth century, the atlantic forest covered practically the entire brazilian coast. in the same century, in the year of 1587, the first records of the introduction of coconut (cocus nucifera l.) were described in brazil. since then, coconut cultivation has proliferated due to its high suitability to the climate conditions and sandy soils of the brazilian coast in atlantic rainforest biome (siqueira et al., 2002). in the late twentieth century, the coconut crop was spread over an area of 247,000 ha with a production of about 1.1 billion fruits, with over 70% of production concentrated in the northeastern brazil (cuenca & nazario, 2004). the incidence of pests in coconut trees is an important limiting factor accounting significantly for the economic losses of the plantations and reduced productivity. beetles of the species rhinostomus barbirostrise (fabricius, 1775) are among the principal coconut palm borers. cutting and burning of the heavily infested coconut trees is usually recommend by the area supervisors in order to reduce the spread of the beetle (araújo et al., 2015). ribeiro et al. (2009) in a study on the brazilian atlantic forest revealed a serious situation with respect to the forest fragmentation, with more than 80% of the fragments <50 ha and almost half the remaining forest <100 m from its edges. they also reported the presence of large average distance (1440 m) between the fragments, the fact that the são francisco river region has only 4.7% of forest cover and that the main land use in this area is associated with plantations of coconut. extensive forest fragmentation results in the isolation of bee population, compromising the genetic diversity of bees and reduced availability of the trophic resources. this can lead to the loss of natural colonies, and local species extinction (carvalho et al., 1995). the decrease of forest areas, forming fragments, reduces the size of the populations that live and depend on this habitat. these processes directly affect eusocial bees that use tree hollows for nesting, isolating small populations and leading them to inbreeding, genetic drift and local extinction (beaumont & bruford, 1999; araújo et al., 2004). in the case of the stingless bees, genetic drift is an extremely important factor in the isolation of small local populations. araújo et al. (2000) suggested that the populations of melipona are particularly sensitive to the effect of genetic drift due to homozygosity in the xo sex determination locus. carvalho et al. (1995) documented the loss of alleles and the extinction of local populations due to low population number and kerr and vencovsky (1982) described that a meliponini population should contain a minimum of 44 colonies to lower the risks of rapid extinction. in addition to the morphological analysis, the use of molecular markers and geometric morphometrics can be used to clarify questions regarding hybridization, dispersal and geographic distribution, genetic identity and risks of extinction (moritz & hills, 1996; chakraborty & kimmel, 1999; waldschmidt et al., 2002; araújo et al., 2004; nascimento et al., 2010). this study aimed to evaluate the genetic diversity and the risk of local extinction of a population of m. quadrifasciata that uses coconut trunks predominantly as nesting site in an area used for coconut plantation at the mouth of the são francisco river in the northeastern brazil. for this, first geometric morphometric analysis was carried out to identify the river specie found at the mouth of the são francisco and then molecular analyses were carried out to compare this population with another natural population of m. quadrifasciata with the same pattern of continuous tergal bands found in the caatinga biome which is the closest biome where this species occurs naturally. material and methods samples coconut plantation from an area south of the mouth of the são francisco river (brejo grande, state of sergipe), from the north of the mouth of the river são francisco river (piaçabuçu, state of alagoas), and from an island (criminosa island) formed by the são francisco river between the states of alagoas and sergipe were visually examined for the presence of bee nests. these three areas of coconut plantations totalled an area of approximately 37 ha (table 1). the island had a distinct physiognomy than the other two areas of sandbank. the sandbanks were primarily composed of wetlands characterized as floodplain and used for coconut cultivation in elevated regions, and covered by aningas (montrichardia arborescens l.) in the lower regions. the search for nests was conducted randomly, traversing the ed araujo et al. – risk of local extinction and genetic diversity of melipona quadrifasciata806 coconut trees and other potential nesting sites along the path, using the same sampling profile (three persons in each area). all the tree trunks were numbered and, when a colony was identified, it was marked and registered, specimens were collected with the help of an entomologist and characteristics of their activity, morphology and height of the nests were recorded. 10 workers per nest were captured. the captured bees were placed in numbered containers, containing 70% alcohol for preservation of the material. for molecular analysis, apart from 140 workers from brejo grande (s 10° 28’47” and w 36°26’21”), 150 workers from the municipality of nossa senhora da glória, sergipe (semiarid caatinga region s 10º13’06” and w 37º25’13”) were also used. the standard specimens m. q. anthidioides, m. q. quadrifasciata and m. mandacaia utilized were provided by the federal university of reconcavo of bahia, cruz das almas, ba. geometric morphometrics the left forewing of all the workers from river são franciscoregion were mounted on the slides and placed under disecting microscope. photomicrographs of the forewings were organized in tps util software and 13 anatomical landmarks were marked using the tpsdig2 software version 2.10 (rohlf, 2006), as illustrated in figure 1. the anatomical landmarks were then transformed into arrays of coordinates. these coordinates were adjusted using orthogonal superimpositions of least squares criterion using morphoj software (klingenberg, 2010). after adjusting the size, position and orientation, and generation of an array of covariance, principal component analysis (pca) was performed. besides the formation of the groups, the degree of population diversity was observed by the analysis of canonical variables. to identify the difference between the groups for the size and shape of individuals, analysis of variance (anova) of procrustes was performed. an analysis of cross-validation between the scores of principal components and canonical variables was performed with r statistical software which verified the correlation of the individuals with their respective nests. the distance matrix of procrustes were used for the cluster analysis by upgma method that resulted in arrangement of wing distances between the colonies. values of the coefficient of cophenetic correlation equal to or greater than 0.7, using the ntsyspc software (rohlf, 2000) were considered for analysis. molecular analysis extraction of dna was performed as described by waldschmidt et al. (1997). adult workers preserved in 70% ethanol and stored at -20 ºc were used. dna was precipitated with ethanol and resuspended in 25 μlte and stored at -20 ºc. quality of the dna extraction was checked by agarose gel electrophoresis and dna was quantified using qubit®. 2.0 fluorometer (invitrogen). issr markers and cytochrome b gene sequences (cyt bsequences) were used to study the genetic variability. the dna extracted was amplified using the following issr primers: (ca)8ry, (ga)8ry, ca8g (tg)7rg, (gata)2(caga)2, ubc 807, ubc 808, ubc 811, ubc 834: r ubc 836, ubc 848, ubc 856 and ubc 857 (zietkiewicz et al., 1994; diniz et al., 2004, 2005; chen et al., 2005; nascimento et al., 2010). for amplification and sequencing of the cyt b region, universal primers, 5’-cyt b tatgtactacc-atgaggacaaatatc-3 ‘(forward) and 5’-attacacctcctaatttattaggaat 3’ (reverse) (crozier et al., 1991), were utilized. pcr was in a final volume of 20 μl using 10 μl oftaq master mix red (amplicon®), 2 μl of 10 μm each primer, and 1 μl of extracted dna with an approximate concentration of 35 ng/μl using the following conditions: hold at 94 ° c for 5 min and 40 cycles of 94 °c for 30 sec, varied annealing temperature according to the melting temperature of each primer as described in table 2, extension at 74 °c for 45 sec and a final extention at 74 °c for 10 min. electrophoresis was performed using 0.8% agarose gel, stained with sybr green (sybr green i, biotecnologia lcg) and visualized under uv.(obs: these amplification conditions were used both issr and paracytb?) pcr amplified cyt b fragments were purified using nucleo spin®gel and pcr clean-up kit (macherey-nagel, germany), quantified and sequenced. for sequencing, the big dye terminator v.3.1 cycle @ sequency kit was used in the abi 3500 sequencer (applied biosystems). analysis of the results obtained by issr primers were carried out using multivariate statistics. the past 3.4 software (hammer et al., 2001) was used to evaluate the genetic diversity index measured by the jaccard. values obtained from the measurement of genetic distance were used for upgma cluster analysis (unweighted pair-group method with arithmetic mean) and amova (analysis of table 1 – geographical location of sample areas in the region of the mouth of são francisco river. local latitude (s) longitude (w) number of plants observed area (ha) brejo grande-se 10° 28’47” 36°26’21” 200 13.5 criminosa island 10 ° 28’47 “ 36º24’68” 270 9 piaçabuçual 10 ° 26’96” 36º24’81” 280 14.5 fig 1. left forewing showing the anatomical landmarks used in morphometric analysis. sociobiology 63(2): 804-812 (june, 2016) 807 molecular variance). using alerquim version 3.5, fst index was calculated and the values obtained were used to estimate the genetic distance between the populations (excoffier et al., 2005). in addition, the software structure v.0.6.9 (evanno et al., 2005) was used to estimate the genetic structure of populations collected from the river são francisco and the caatinga region. staden package version 2.0 (staden et al., 2001) was used to verify the quality of the cyt b gene sequences. only sequences with phred values above 30 were considered for subsequent analysis. the cyt b sequences were aligned using the clustalw algorithm embedded in the software mega (molecular evolutionary genetics analysis) version 6.0.(tamura et al., 2013). dnasp 5.0 (librado & rozas, 2009) was used to identify haplotypes and to estimate haplotype (h) diversity among and within the populations. in order to observe the relationship among the haplotypes, analysis using the median-joining network (network 4.612) was also carried out (bandelt et al., 1999). results and discussion after screening approximately 37 ha, constituting three sampling areas fromthe mouth of the river são francisco, all the trees found with possible nesting sites for the bees of the genus melipona were of coconut. a total of 750 trees were numbered and analyzed, all featuring hollow generated by the pests, and in majority consisting of old coconut trees with low productivity. the sampling from the south of the river são francisco (municipality of brejo grande) resulted in 14 nests of stingless bees from 200 trees analysed. this is the same region where souza et al. (2006) described the first scientific records of the occurrence of the nests of m. quadrifasciata in the coconut palms. we verified that about 7% of potential nesting sites in this area were occupied. all colonies found were quite active, with large flow of workers foraging at the entrance of the nest, with some nests having more than one entry, and in these cases only one nest per tree was considered. considering the high level of activity of thecolonies and the existence of this population for several years, hardiness and resistance of thesebees to isolation and in breeding must be considered, a situation contrary to the literature on the melipona bees, but in agreement with the information published by alves et al. (2011) who had described the successful maintenance of melipona scutellaris colonies for a decade despite severe genetic bottleneck. in the other two sampling sites, located in the ilha da criminosa and north of the river são francisco, despite a greater sampling effort and screening of about 23.5 hectares, totaling 550 trees with potential nesting sites, no m. quadrifasciata nests were found (fig 2). the group of bees found at the mouth of the river são francisco showed tergal band pattern similar to that found in subspecies m. quadrifasciata quadrifasciata. an analysis of identity for this group of bees was performed using geometric morphometrics and compared with the standard specimens of m. quadrifasciata quadrifasciata and m. quadrifasciata anthidioides. also was utilized standared specimen of m. mandacaia (smith 1863) which is the sister species of m. quadrifasciata (ramírez et al., 2010) as negative control. the result of canonical variate analysis (cva) clearly defined the group in which the population under study belongs. in figure 3 one can easily see the separation between the three reference groups, with the group of bees collected from the river são francisco region overlapping with the subspecies m. quadrifasciata quadrifasciata, result in agreement with the yellow continuous tergal bands observed in them. designation sequences of primers (5”-3”) tm reference (ca)8ry cacacacacacacacary 56°c zietikiewicz et al, 1994 (ga)8ry gagagagagagagagary 56°c zietikiewicz et al,1994 (ca)8g cacacacacacacacag 56°c chen et al , 2005 (tg)7rc tgtgtgtgtgtgtgrc 56ºc zietikiewicz et al, 1994 (gata)2(gaca)2 gatagatagacagaca 56°c diniz et al, 2004, 2005 ubc 807 agagagagagagagagt 54 ºc wang 2011 ubc 808 agagagagagagagagc 54 ºc wang 2011 ubc 811 gagagaggagagagac 56 °c nascimento et al, 2010 ubc 834 agagagagagagagagyt 48 ºc nascimento et al, 2010 ubc 836 agagagagagagagagagya 54 ºc nascimento et al, 2010 ubc 848 cacacacacacacacarg 56 ºc nascimento et al, 2010 ubc 856 acacacacacacacacya 48 ºc nascimento et al, 2010 ubc 857 acacacacacacacacyg 50 ºc nascimento et al, 2010 cytb f: tatgtactaccatgaggacaaatatc r: attacacctcctaatttattaggaat’ 54 °c crozier et al, 1991 table 2primers utilized in this study for the analysis of genetic variability. ed araujo et al. – risk of local extinction and genetic diversity of melipona quadrifasciata808 analyses of genetic diversity a total of 195 issr loci with good reproducibility were found in the two populations of m. quadrifasciata, collected from the river são francisco region, and from sergipe semiarid region. the two populations showed 143 and 119 polymorphic loci respectively and their values of the polymorphisms were 73% and 61% respectively. fig 2. sampling area and nesting sites of m. quadrifasciata in coconut groves of the river são francisco region. am. quadrifasciata; b-detail of tergal bands of the collected workers; csampling sites: site 1 where 14 colonies of m. quadrifasciata were found, site 2: criminosa island and site 3: left margin, state of alagoas. our results using issr demonstrated that the population of m. quadrifasciata from the mouth of the river são francisco is significantly different from the population of m. quadrifasciata from sergipe semiarid region. the upgma analysis showed a high cophenetic correlation coefficient (r=0.97), and together with the fst index (0.52) they confirm the genetic divergence between the two populations compared (fig 4). also the bayesian algorithm implemented in structure software fig 3. analysis of the canonical variables showing comparison of 140 samples form 14 colonies found at the mouth of the river são francisco with the standard specimens melipona quadrifasciata quadrifasciata, melipona quadrifasciata anthidioides and melipona mandacaia. overlapping of the samples with the standard specimen m.quadrifasciata quadrifasciata can be seen. cv1: canonical variable 1; cv2: canonical variable 2. sociobiology 63(2): 804-812 (june, 2016) 809 using the delta k test (evanno et al. 2005) showed a clear formation of two distinct clusters (fig 5). analysis of molecular variance (amova) provided additional support to the evidence of population differentiation between the two populations under study, with 52% and 48% of the total genetic variability distributed among and within the populations, respectively. the comparison between cyt b sequences of m. quadrifasciata from caatinga and brejo grande (south of the mouth of the river são francisco) showed the presence of a higher genetic variability in the population of caatinga with three distinct haplotypes, one being more frequent than the others, while in the population of brejo grande from the north of the river são francisco region, only one haplotype circulating in the population was found, which is different from haplotypes circulating in the population of the caatinga (fig 6). the subject of this study was a population of stingless bees found in 2006 nesting exclusively in coconut trees in a region extreme northeast of its distribution. despite morphological identification, no record of the presence of this species exists in the scientific literature. in this manuscript we carried out morphometric and molecular analyses of this population of stingless bees found at the mouth of the river são francisco and confirmed that it belonged to m. quadrifasciata quadrifasciata. also, all nests found at the mouth of the river san francisco were found in the state of sergipe, and even with greater collection effort, no nests were found north of the river. acknowledging the fact that the confirmation of the absence of a species is more difficult than the proof of its presence, and that the absence of a finding gets stronger with higher sampling effort, we suggest that this species does not reach the state of alagoas via the coastal region (atlantic forest biome) and also that the river são francisco represents a geographic barrier strong enough for limiting the distribution of this species in the coastal region, south of the river san francisco. our main arguments that support this hypothesis are not based only in the sampling effort but also in the behaviour of the foundation of the new colonies and in the foraging behaviour of the stingless bees. according to araujo et al. (2004) the flight distance in stingless bees is associated with the body size, and the size of the workers of m. quadrifasciata present a maximum flight fig 4. dendrogram upgma based on issr markers. cophenetic correlation index: 0.97. a: population of m. quadrifasciata from the semiarid region (caatinga). b: population of m. quadrifasciata found in coconut palms of the mouth of river são francisco, coastal area of brejo grande, sergipe. fig 5. population of m. quadrifasciata from the mouth of the river são francisco (light grey) and from the semiarid region (dark grey) showing the formation of two distinct clusters using baysian algorithm ed araujo et al. – risk of local extinction and genetic diversity of melipona quadrifasciata810 distance of 2 km radius from their colony of origin. from the standpoint of the multiplication of the colonies, restriction is even greater, since in stingless bees, reproduction and foundation of a new colony are carried out by a group of bees (queen and the workers) and generally occurs in a progressive manner, with long period of contact between the mother and the daughter colonies. this feature limits the formation of new colonies closer to the nesting site of the maternal nest (batista et al., 2003). são francisco is the main river in the northeastern brazil and the distance between its margins at the mouth, is around 2 km (figure 2). thus the river can really be considered as an effective barrier of geographic isolation for the population of this species. following the same reasoning, the greater possibility is that the m. quadrifasciata population from the mouth of the river san francisco region is derived from the populations of atlantic forest biome. however, the nearest population with morphology similar to the tergal bands of the subspecies m. quadrifasciata quadrifasciata recorded in this region lies in the semiarid caatinga biome in sergipe (batalha-filho et al., 2009), and the bees of this species are not generally found in the coastal region of the northeastern brazil. the genetic diversity found among populations analysed indicates low or absence of inter-population gene flow and / or divergent evolutionary pressures, especially considering that the populations compared belong to different biomes, although geographically separated by a linear distance of less than 150 km. according to araújo et al. (2000) the fixation of an allele can occur by genetic drift and depends on its initial frequency in the population. it is a process that occurs in all finite populations, and is faster in smaller populations. thus, checking the number of haplotypes circulating in a population is essential for the genetic evaluation of the population. the sequencing analysis of the mitochondrial cytochrome b region showed that the two populations have a small genetic divergence, but may belong to the same subgroup identified by the continuous tergal bands as found in the subspecies m. quadrifasciata quadrifasciata. however, it is noteworthy that the existence of similar tergal band patterns does not determine that this group belongs to this subspecies, and perhaps the divergence between the groups in m. quadrifasciata is so small that it does not even justify its separation into subspecies, as argued batalha-filho et al. (2009). our results point to some important scenarios for the local population from the mouth of the river são francisco. first it highlights a great risk of local extinction of this species. in the genus melipona, the species are very sensitive to the reduction in genetic variation which may increase the probability of the loss of colonies by the loss the of alleles at the xo locus consequently producing diploid males (carvalho et al., 1995; araújo et al., 2000). linked to the lack of genetic variability, the risk of extinction is also directly associated with the isolation of this population in an area where practically all the nesting sites depend on the existence of pests that damage the trunks of coconut trees. improved cultural practices, economic subsidies or improve technical assistance to the coconut growers may result in a massive replacement of diseased coconut trees by new plants, drastically reducing the potential nesting sites and simultaneously destroying the nests of m. quadrifasciata housed in these plants. the cytochrome b sequence analysis also showed that the haplotype found in the são francisco region is unique, and thus the loss of this population could represent a permanent loss of this genetic lineage that seems to be quite resistant to inbreeding, since all the nests encountered showed excellent level of activity despite the lack of genetic variability. conservation actions are urgently needed, including increasing the supply of nesting sites and the development of beekeeping directed towards the multiplication of the colonies and maintenance of this haplotype in the region. acknowledgements we thank the members of the association of brejograndense de criadores de abelhas e artesãos (abeca) and fapitec-se for the financial support, and capes and cnpq for the scholarships to the students related to this project. conflict of interest the authors declare that they have no conflict of interest. fig 6. haplotype network generated by the comparison of dna sequences from the cytochrome b region. population of m. quadrifasciata from semiarid region (caatinga) and from the mouth of river são francisco are represented by dark grey and light grey respectively. sociobiology 63(2): 804-812 (june, 2016) 811 references alves d.a., imperatriz-fonseca v.l., francoy t.m., santosfilho p.s., billen j, wenseleers t. 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(1994). genome frigerprinting by simple sequence (ssr) anchored polymerase chain reaction amplification. genomics, 20: 176-183. doi:10. 1006/geno.1994.1151. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.894sociobiology 63(2): 831-840 (june, 2016) combinatorial potential of bait matrix against subterranean termites under lab and field conditions introduction termites are economically important pests because they destroy wood products of human homes, building materials, forests, agriculture crops and other commercial products (monica et al., 2009). baiting has been promoted as a desirable method of termite pest control. it is lauded as environmentally sound as it uses very small amounts of insect specific toxicants that are administered in localized baits that are targeted at the pest species (i.e. not large amounts of toxicants spread over large areas around a house). however, in order for baiting to work successfully, termites must find and consume the bait matrix and for the toxicant contained therein to be transferred back to the nest. since the introduction of baiting systems for managing subterranean termites in many parts of the world, various approaches have been used to evaluate the bait matrix and transfer the toxicant back to the nest. these approaches, among others, included the incorporation of additives, attractants, and phagostimulants into the bait matrices. attractants such as abstract two bait matrices with different treatments were evaluated against two termite species i.e. odontotermes obesus and coptotermes heimi both under laboratory and field conditions. mean wood consumption in laboratory bioassays was investigated for 2, 4 and 6 weeks with maximum consumption after 4 weeks. while, field experiments were conducted for 24 weeks and there was greater consumption of the loosely bound bait matrix compared to the tightly bound matrix. however, feeding was comparatively high in combinations with attractants i.e. feeding stimulants (agar and sugarcane bagasse). overall, treated colonies experienced a 90-95% decrease in population size after 24 weeks of baiting. the queen in the royal chamber of the mound was found dead. sociobiology an international journal on social insects kz rasib, h ashraf article history edited by evandro nascimento silva, uefs, brazil received 12 august 2015 initial acceptance 19 january 2016 final acceptance 27 may 2016 publication date 15 july 2016 keywords populus euramericana, odontotermes obesus, coptotermes heimi, metarhizium anisopliae, bait, combinatorial. corresponding author khalid zamir rasib biology department forman christian college university lahore -54600, pakistan e-mail: khalidrasib786@gmail.com fungal extracts (esenther et al., 1961; cornelius et al., 2004; su, 2005) and carbon dioxide at10-12 mmol/mol (bernklau et al., 2005) has been shown to improve the attractiveness of bait to termites. as a means to increase consumption, phagostimulants that made the bait more palatable, such as urea (waller, 1996) amino acids (chen & henderson, 1996), and sugars (morales-ramos & rojas, 2003a; waller & curtis, 2003; saran & rust, 2005, 2008), were also explored. termites fed more on baits with additives than those without. nutritionally enhanced bait such as a commercially available product, summon preferred food source, was more preferred by coptotermes formosanus (shiraki) than standard cardboard disks. summon bait aggregated more termites, which resulted in higher consumption compared with standard cardboard disks (cornelius & lax, 2005). there are different types of baits that were made from cellulose material like wood, cardboard or tissue which attract termites and used for forecast infestation or control. however, not all termites’ species prefer the same bait matrix. this may be due to the nature of matrix which is not preferred by these termite species. forman christian college university, lahore, pakistan research article termites kz rasib, h ashraf – combinatorial potential of bait matrix against subterranean termites832 baiting technology involves placing in-ground monitoring stations that contain a cellulose material in the soil at regular intervals along the perimeter of the structure. these stations are checked at regular time intervals for foraging termites. there are commercially available baits that eliminate the prebaiting period by initially installing in-ground stations that contain active ingredient. individual termites consume the bait and share it with other individuals within the colony after through feeding and grooming behaviors, by which the bait is distributed and eliminate the colony. in social insects, horizontal transfer usually involves interactions where both the donors and the recipients are alive and actively interacting. key among these mechanisms is trophallaxis, which facilitates the spread of bait toxicants (hu et al., 2005), and mutual grooming which facilitates the spread of liquid spray insecticides (soeprono & rust, 2004). control of subterranean termite was transfigured when first termite bait product was registered by dow agrosciences llc (formerly known as ~ow~lanco) (su, 1994). su (1994) was the pioneer to use hexaflumuron as baits in field trials, which lead to the development of sentriconb. termite baiting takes the advantage of social nature and foraging behaviour of subterranean termites where food sharing among the workers and nestmates via trophallaxis could enable the transfer of slow-acting toxicant to the whole colony (lee & chung, 2003). a new generation of baits includes high-moisturecontent matrices formulated to be nutritionally attractive to termites. one of these bait matrices, developed by usdaars researchers, was based on the feeding preferences and nutritional requirements of the formosan subterranean termite (morales-ramos & rojas, 2003a; rojas & morales-ramos, 2001; rojas et al., 2003). the purpose of this formulation was to increase bait consumption by subterranean termites in order to enhance assimilation of active ingredients within colonies, consequently reducing the time required by treated termites to attain lethal doses (morales-ramos & rojas, 2003b; rojas, 2002a,b; rojas & morales-ramos, 2003). these bait matrix formulations in combination with feeding stimulants and masking agents (rojas et al., 2004) allow incorporation of less palatable but more widely available active ingredients, such as diflubenzuron, without compromising bait consumption by the termites (morales-ramos & rojas, 2003a). the current research focusing on the effect of bait design and applications by employing combinatorial treatments on the survival and consumption of wood by highly destructive pakistani termites odontotermes obesus and coptotermes heimi. materials and methods collection of termites o. obesus and c. heimi were collected from different areas of district lahore including changa manga forest, jallo park, wagha border, ravi siphon, jinnah garden, agricultural crops and fallen wooden logs infested under natural conditions. they were also collected through the installation of different woods as stakes and supplemented by sugarcane stalks to aggregate the maximum number of termites. collection was made by using bucket traps /wetted toilet rolls/ cardboards packed in plastic bottles with small holes at the base to permit the entry of termites, as they visit to the feeding stations. augmentation of termites the workers and soldiers of o.obesus and c. heimi which were collected from different locations were established in a transparent test apparatus made up of acrylic sheet with specific dimensions (length× height× width: 30× 35×30cm) supplied with sterilized moist soil, cellulose powder, pieces of moistened corrugated cardboards and tissues rolls which serves as source of food for termites, under laboratory conditions. the termites were handled with a moist paint brush. all the cages were then covered with black polythene/cloth to minimize the effects of light and placed in the controlled room at 26±1°c, rh 75%. moisture in the containers was kept judiciously and checked twice a month. 2.3. isolation and extraction of fungal toxins metarhizium anisopliae was isolated from different sources. cultures were maintained on potato dextrose agar (pda). conidial suspensions were prepared by lightly scraping the surface and suspending the conidia in 100ml distilled sterile along with 0.01% of tween 80. the conidial concentration of the suspensions was determined using a haemocytometer. isolates of m. anisophilae were cultured in 500 ml erlenmeyer flasks containing 250 ml of sterile potato dextrose liquid medium the flasks were incubated separately for 7-10 days in the dark at 27-30oc without agitation. twenty five ml of chloroform were added to lyse the cells in order to recover mycelia and allow it to rotate for 10 min on a shaker. the flasks contents were filtered (whatman no. 1) and the filtrate was used for toxin extraction. the filterate was transferred quantitatively to a separatory funnel and extracted successively with 100 ml of chloroform to partition the chloroform and aqueous layers. the procedure was repeated three times with lower transparent chloroform layer collected in a new flask. the chloroform was evaporated at 100oc by a vaccum rotatory evaporator to obtain the crude extract of each fungus according to mallek et al. 1993. the extracts were finally weighed and kept in refrigerator at 4oc until further use. chemicals commercial formulation of two insecticides used for the bioassays were, fipronil (fiprostar® 25 ec starlet international,pakistan) and imidacloprid (mirage® 5% sc ali akbar enterprises, pakistan) at lower concentration of 0.3ppm. sociobiology 63(2): 831-840 (june, 2016) 833 bait application under lab and field conditions locations for bait installation this experiment was designed to test whether bait station design affected bait matrix removal, not whether bait design affected bait discovery. therefore it was decided that bait stations should be placed onto active foraging sites and thereby be encountered immediately. two locations were selected for bait installation which was previously monitored for termite foraging activity. mounds/nests at wagha border were selected for o. obesus and lahore canal bank in the vicinity of fc college dominated by p. euramericana plantation was selected for c. heimi (fig 1 (a-b). bait matrix the most palatable wood, populus euramericana (manzoor et al., 2009; aihetasham & iqbal, 2012) was selected as bait matrix. the other components of bait matrix including: agar, sugarcane molasses, fungal conidial suspension (1*107) and 0.3ppm of imidacloprid and fipronil. 100ml of suspension is prepared for each treatment. wood blocks (length× height× width: 30× 35×30cm) were soaked for 72 hours in each treatment in order to achieve leaching of suspension in wood blocks. the formulation was evaluated against o. obesus and c. heimi workers separately under laboratory conditions. all wood blocks were pre-weighed before start of experiment (fig1(c-e). bait treatments under laboratory and field bioassay five treatments were prepared for bait application under lab and field conditions which are as follow: control (populus euramericana) (a) populus euramericana + 1*107conidia/ml (b) populus euramericana + 0.3ppm of fipronil (c) populus euramericana + 0.3ppm of imidacloprid (d) combinatorial bioassay using populus euramericana + 1*107 conidia/ml + 0.3ppm of fipronil + feeding stimulantants (agar 2g/100ml + 2ml sugarcane bagasse) (e) combinatorial bioassay using populus euramericana + 1*107 conidia/ml + 0.3ppm of imidacloprid + attractant (agar 2g/100ml + 2ml sugarcane bagasse) for laboratory bioassay all these treated combinations of wood blocks were conducted in three replicates i.e. in three petri dishes with 148 worker and 2 soldiers of either odontotermes obesus and coptotermes heimi with a control parallel to determine the feeding on wood blocks under no choice experiment for two, four and six weeks. the lab no choice study was done by calculating the weight of each petri plate alone and with the wood sample+matrix being given to the termites and as well as the bait is provided in a form of matrix so it was weighted along with wood sample and all the calculations were done before the placement of final value in the table. treatment and control materials were preweighed prior to the introduction of termites. the mortality data was added in the paper as it was calculated but not added in article because we think we are more concern with consumption as well as field data was also related to consumption that’s why we skip it but now it is a part of updated paper as well as control data was also added. however, for field bioassay all these treated blocks of p. euramericana were tied with copper wire into a bundle separately with three replicates of each and placed into mound buried up to 30 cm at different active places of the nest. control with untreated woods was also driven into the nest for comparison. the bioassay duration lasted for 24 weeks which is effective from ist july 2014 -31st jan 2015, and follow up studies for the remaining period up to march 2015. monthly inspection of stations will be carried out. three replicates (n=3 i.e. indicates “to repeat” (a scientific experiment) to confirm findings or ensure accuracy) of each bait treatment were prepared both for laboratory and field trial. while, five termites colonies were selected in field for each treatment. statistical analysis when no-choice laboratory bioassay was conducted, data on wood consumption (%) were subjected to the analysis of variance (one-way anova). data obtained from laboratory and field bioassays were statistically analyzed using tukey’s test. results bait treatments under lab conditions using no-choice bioassay different bait treatments were used to observe the consumption by o. obesus and c. heimi workers and soldiers under no choice laboratory bioassay for 2, 4 and 6 weeks. in both termites highest consumption was noted on combinatorial treatments with feeding stimulants. in case of o. obesus, after 4 weeks the baits with feeding stimulants showed significantly higher consumption i.e. 38.5±0.1 mg and 45.4±0.2 mg in comparison to other treatments and control which gives 33.6±0.1 mg respectively. however, the rate of mortality after 4 weeks was 38.5±0.1 and 45.4±0.2% in contrast to control which showed 35.7±0.4% respectively. consumption of bait after 2 and 6 weeks were not significantly different from each other. after 2 weeks maximum consumption was 23.5±0.3 and 26.5±0.1mg with 27.3±0.1 and 30.2±0.2% mortality whereas, 27.5±0.2 and 29.4±0.1mg consumption and 55.2±0.1 and 56.2±0.3% mortality were observed after 6 weeks. however the minimum consumption was noted in pe+ma treatment 13.5±0.1, 15.4±0.2 and 14.9 ±0.3mg with 36.3±0.2, 40.3±0.1 and 62.2±0.1% mortality was noted after 2, 4 and 6 weeks respectively (table 1). same results were observed for c. heimi where maximum consumption was noted after 4 weeks. 30.5±0.1 and 40.2±0.3 mg consumption was observed in bait with feeding stimulants however, the rate of mortality was 42.4±0.1 and 44.2±0.2% kz rasib, h ashraf – combinatorial potential of bait matrix against subterranean termites834 respectively as compare to control and other treatments which showed 39.8±0.2 mg consumption with 37.8±0.2% mortality after 4 weeks. after 2 and 6 weeks, high rate of mortality was indicated with low consumption of bait. 24.4±0.4 and 36.4± 0.3 mg consumption and 28.3±0.2 and 31.8±0.1% mortality was measured after 2 weeks whereas, at the end of 6 weeks bait with feeding stimulants was consumed to be 26.5±0.5 and 38.5±0.3 mg with 54.2±0.2 and 56.3±0.1% death rate respectively. while the lowest consumption was detected on pe+ma treatment i.e.14.6±0.1, 19.4±0.3 and 17.2±0.2 mg with33.8±0.3, 52.1±0.1 and 64.2±0.1% mortality at various weeks (table 2). bait treatments under field conditions under no choice bioassay the mean wood consumption by o. obesus and c. heimi at the end of the experiment is an important measure against which all bait treatments should be compared under no choice field bioassay for 24 weeks. combinatorial treatments with feeding stimulants showed higher consumption in comparison to other treatments. as well as, bait which was loosely bound indicates greater termite activity as compared to tightly bound. in case of o. obesus, bait with feeding stimulants gives 42.2±0.2 and 44.2±0.1mg in tightly bound wood blocks whereas, fig 1 (a-b). showing bait matrix treated blocks tied with copper wire (a-b) site selection at wagha border and installation of bait delivery matrix inside mounds of odototermes obesus under no choice field trials (c-e) queen of odontotermes obesus with physogastry (f) (magnification=100x). sociobiology 63(2): 831-840 (june, 2016) 835 table 1. shows mean wood consumption and mortality (±s.d) under no choice laboratory bioassay by odontotermes obesus for 2, 4 and 6 weeks. (n=3) table 3. shows mean wood consumption (±s.d) for tightly and loosely bound wood blocks under no choice field bioassay by odontotermes obesus for 24 weeks. treatments mean wood consumption (mg) tightly bound loosely bound control(pe) 40.1±0.2 a 75.3±0.1 bc pe+ma 39.6±0.3c 50.7±0.2ab pe+fip 20.4±0.1d 32.4±0.3c pe+imi 28.5±0.2cd 43.8±0.4b pe+fip+ma+att 42.2±0.2b 76.4±0.1a pe+imi+ma+att 44.2±0.1b 80.4±0.3a treatments mean wood consumption (mg) 2 weeks 4 weeks 6 weeks control(pe) 21.8±0.1c 33.6±0.1b 22.30.2a pe+ma 13.5±0.1e 15.4±0.2e 14.9±0.3e pe+fip 15.4±0.4e 18.3±0.5d 16.7±0.3de pe+imi 18.2±0.2d 22.4±0.1c 19.6±0.2d pe+fip+ma+att 23.5±0.3c 38.5±0.1b 27.5±0.2c pe+imi+ma+att 26.5±0.1c 45.4±0.2a 29.4±0.1c mean mortality (%) control(pe) 20.2±0.1a 35.7±0.4c 50.4±0.2b pe+ma 36.3±0.2c 40.3±0.1b 62.2±0.1b pe+fip 38.3±0.4c 58.9±0.4a 80.2±0.3c pe+imi 40.2±0.3c 60.7±0.3cd 85.5±0.2d pe+fip+ma+att 27.3±0.1d 40.5±0.2a 55.2±0.1c pe+imi+ma+att 30.2±0.2e 41.2±0.1ab 56.2±0.3e note: means followed by different letters within a column differ significantly at 0.05 (p < 0.05). populus euramericana + 1*107 conidia/ml (metarhizium anisopliae) (pe+ma), populus euramericana+0.3ppm of fipronil (pe+fip), populus euramericana + 0.3ppm of imidacloprid (pe+imi), populus euramericana +1*10 conidial/ ml (metarhizium anisopliae) + 0.3ppm of fipronil + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+fip+att) and populus euramericana + 1*107 conidial/ml (metarhizium anisopliae) + 0.3ppm of imidacloprid + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+imi+att). treatments mean wood consumption (mg) 2 weeks 4 weeks 6 weeks control(pe) 24.6±0.1a 39.2±0.2b 32.30.1c pe+ma 14.6±0.1d 19.4±0.3c 17.2±0.2cd pe+fip 15.4±0.2d 20.3±0.5c 19.6±0.5c pe+imi 18.5±0.5c 28.2±0.2b 22.4±0.1bc pe+fip+ma+att 24.4±0.4b 30.5±0.1ab 26.5±0.5b pe+imi+ma+att 36.4± 0.3a 40.2±0.3a 38.5±0.3a mean mortality (%) control(pe) 28.4±0.1a 37.8±0.3c 52.2±0.2b pe+ma 33.8±0.3c 52.1±0.1c 64.2±0.1b pe+fip 41.2±0.1a 62.9±0.3c 83.2±0.1bc pe+imi 45.1±0.1e 63.8±0.2c 85.5±0.1d pe+fip+ma+att 28.3±0.2c 42.4±0.1cd 54.2±0.2a pe+imi+ma+att 31.8±0.1a 44.2±0.2ab 56.3±0.1a table 2. shows mean wood consumption and mortality (±s.d) under no choice laboratory bioassay by coptotermes heimi for 2, 4 and 6 weeks. (n=3) note: means followed by different letters within a column differ significantly at 0.05 (p < 0.05). populus euramericana + 1*107 conidia/ml (metarhizium anisopliae) (pe+ma), populus euramericana+0.3ppm of fipronil (pe+fip), populus euramericana + 0.3ppm of imidacloprid (pe+imi), populus euramericana +1*10 conidial/ ml (metarhizium anisopliae) + 0.3ppm of fipronil + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+fip+att) and populus euramericana + 1*107 conidial/ml (metarhizium anisopliae) + 0.3ppm of imidacloprid + feeding stimulants ttractant (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+imi+att). 76.4±0.1 and 80.4±0.3 mg was observed in loosely bound bait matrix as compared to control which showed 40.1±0.2 and 75.3±0.1 mg consumption respectively. however, in other treatments wood consumption in tightly and loosely bound wood blocks are 39.6±0.3, 20.4±0.1 and 28.5±0.2 mg and 50.7±0.2, 32.4±0.3 and 43.8±0.4 mg, respectively (table 3). note: means followed by different letters within a column differ significantly at 0.05 (p < 0.05). populus euramericana + 1*107 conidia/ml (metarhizium anisopliae) (pe+ma), populus euramericana+0.3ppm of fipronil (pe+fip), populus euramericana + 0.3ppm of imidacloprid (pe+imi), populus euramericana +1*10 conidial/ ml (metarhizium anisopliae) + 0.3ppm of fipronil + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+fip+att) and populus euramericana + 1*107 conidial/ml (metarhizium anisopliae) + 0.3ppm of imidacloprid + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+imi+att). while with respect to c. heimi similar results was analyzed bait treatments with feeding stimulants showed maximum consumption in loosely bound wood blocks i.e. 80.7±0.1 and 86.6±0.2 mg in contrast to tightly bound which showed 43.4±0.3 and 45.2±0.1 mg. however, control treatment signifies 42.2±0.2 and 78.3±0.3 mg consumption in tightly and loosely bound woods respectively. similarly, in other treatments the mean wood consumption observed was 38.5±0.1, 22.7±0.4 and 32.4±0.5 mg in tightly bound and 54.3±0.2, 43.3±0.1 and 47.8±0.3 mg was calibrated in loosely bound bait matrix (table 4). changes recorded in mound/ nest after bait application after 24 weeks the mounds of o. obesus and nests of c. heimi were dissected. over the course of the experiment, all bait stations were infested by termites. in general, termites covered the top surface of the bait station and termite trap with mud whereas, bait matrix was consumed. the consumption data under controlled as well as treated conditions were analyzed. both in mound and nest of termites revealed interesting results. in case of mound, although king caste was unable to be detected, whereas, queen of the mound was present in the royal chamber found dead (figure 1f). similarly, in nest of c. heimi we could not locate both of reproductive castes, however, different workers and soldiers were found in aggregated form as dead in the dissected nest. a strong malodor/smell was emitted from the inner nest kz rasib, h ashraf – combinatorial potential of bait matrix against subterranean termites836 eger et al. (2012) and mckern-lee et al. (2010) studied that durable bait matrix will reduce the costs associated with frequent monitoring, reduce the disturbance for sensitive species and potentially provide the opportunity for baiting across large areas. hamm et al. (2013) worked on different termites and observed that consumption of the blank durable bait matrix was significantly higher than consumption of a blank preferred textured cellulose matrix (ptc) when both contained the active ingredient noviflumuron. all bait treatments resulted in significant mortality relative to the untreated controls. agar as major components of termite bait, agar has been used by a number of researchers (spragg & fox, 1974; patton & miller, 1980; spragg & paton, 1980; su et al., 1982; easey, 1983, 1985; french & robinson, 1984; holt & easey, 1985; easey & holt, 1988, 1989; miller, 1990; su, 1994). maximum bait consumption and termite tunneling activities recorded with 3% concentration. this may be due to the fact with the increasing agar concentrations, the bait becomes more solid and termites prefer semi solid medium than soft medium. baiting systems may provide long lasting control by suppressing termite activity. studies testing the efficacy of different bait materials in managing o. obesus proved that sugarcane bagasse was more attractive to o. obesus and also rendered the colony weak (rajavel et al., 2007). su et al. (1984) while evaluating insecticides noted that higher concentration of agar stimulated tunneling behavior in termites. huang et al. (2006) used fipronil, which is a neurotoxin and normally considered to be fast acting, in baits against odontotermes formosanus in wuhan, china. the baits ‘suppressed’ termite populations in 3-4 months, which is not particularly fast (nb ‘suppressed’ meant no active termites in their stations for 1012 months in two of three sites, which other authors consider to be ‘eliminated’). the longer time to control o. formosanus is probably because it is a fungus growing termite, and delivery of toxicants in food to these termites is more complex than for the wood-feeding rhinotermitids. of the two attributes tested in the field study, related to compaction were consistently important in affecting bait consumption. more of the wood that binds loosely was consumed more as compared with that which was tightly bound. highest consumption in different bait treatments by o. obesus was observed in combinatorial treatments (pe+fip+ma+att and pe+imi+ma+att) with feeding stimulants (42.2mg) and (44.2 mg) in tightlty bind and 76.4 and 80.4mg in loosely bind treated woods; same results were obtained in case of c. heimi for these treatments with 43.4 and 45.2mg for tightly, 80.7 and 86.6mg loosely bound woods. however in other treatments the rate of consumption was comparatively low. the success of loosely bound wood has one simple explanation; the greater surface area of wood immediately available for gnawing. the results of the present study showed that changes in the presentation of materials in the bait station can efficiently effects on bait matrix consumption. evidently, much more work on food and foraging preferences is required and mound due to the presence of dead and decaying termite cadavers in a number of spots inside, especially near the nursery zone. small numbers of workers and soldiers were observed, whereas none of the immature castes (e.g., larvae and nymphs) were found inside the nest. overall, treated colonies experienced a 90-95% decrease in population size after 6 months of baiting. fast-growing fungus was found growing on the carton material in the nursery zone or growing on termite cadavers. the queen was dark yellow in color, flaccid and physogastric. discussion results from this study show that bait systems can be used effectively to reduce subterranean termite population’s area wide. consumption by termites on treated baits in ascending order was documented as: pe+ma= pe+fip <pe+imi <pe+ fip+ma+att = pe+imi+ma+att. consumption of the pe+ma bait matrix was significantly higher than other bait treatments by o. obesus and c. heimi at 6 and 8 weeks. reduced consumption seen on treatments containing imidacloprid and fipronil only, is due to the mortality occurring more rapidly as compared to treatments with feeding stimulants. the substrate is the same between the pe+ma bait and other baits matrices, this shows that the matrix can have an influence on the amount of termite consumption. termite species-specific behaviors such as speed and amount of consumption may influence the time to mortality after consumption of the bait (wood, 1978; delaplane & lafage, 1989). mortality was achieved over time, and the slightly delayed toxicity may provide additional time for trophallaxis to occur, which in turn may lead to better overall colony elimination by keeping insecticides in the colony longer. for a bait to be successful, it must be palatable and toxicologically active across termite species. treatments mean wood consumption (mg) tightly bound loosely bound con(pe) 42.2±0.2a 78.3±0.3a pe+ma 38.5±0.1b 54.3±0.2ab pe+fip 22.7±0.4d 43.3±0.1b pe+imi 32.4±0.5c 47.8±0.3b pe+fip+ma+att 43.4±0.3b 80.7±0.1a pe+imi+ma+att 45.2±0.1b 86.6±0.2a table 4. shows mean wood consumption (±s.d) for tightly and loosely bound wood blocks under no choice field bioassay by coptotermes heimi for 24 weeks. note: means followed by different letters within a column differ significantly at 0.05 (p < 0.05). populus euramericana + 1*107 conidia/ml (metarhizium anisopliae) (pe+ma), populus euramericana+0.3ppm of fipronil (pe+fip), populus euramericana + 0.3ppm of imidacloprid (pe+imi), populus euramericana + 1*10 conidial/ ml (metarhizium anisopliae) + 0.3ppm of fipronil + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+fip+att) and populus euramericana + 1*107 conidial/ml (metarhizium anisopliae) + 0.3ppm of imidacloprid + feeding stimulants (agar 2g/100ml + 2ml sugarcane bagasse) (pe+ma+imi+att). sociobiology 63(2): 831-840 (june, 2016) 837 to design more efficacious baiting protocols. according to evans et al. (2006) for wood-eating termites, bait stations should be designed to encourage termite presence and to maximize their consumption of bait matrix in order to expedite control in minimal time. they examined the effect of bait size, compaction, and composition on termite presence for four months and suggested that all three factors were significant, with bait size the most important factor, followed by compaction and then composition. wang and henderson (2012) studied the consumption efficiency of two commercially used termite bait materials, southern yellow pine wood and cardboard, and one potential bait material, maize (zea mays) cob, against the formosan subterranean termite, coptotermes formosanus (isoptera: rhinotermitidae), under laboratory conditions. they observed that in no-choice test, the consumption of wood and cob was similar and significantly more than cardboard while in the two-choice test, the consumption was cob > wood, wood > cardboard, cob = cardboard whereas, in the three-choice test, no significant difference was detected in consumption. it is difficult to compare previous baiting studies as these normally had the aim of demonstrating that elimination was possible, whereas the current study aimed to determine the time to elimination. the high variability in time to control (24 weeks) reported in previous studies would depend on toxicants, concentrations, termite species, colony sizes, environment geography, season, and so forth; for example, wood eating reticulitermes or coptotermes species (family: rhinotermitidae) in single structures or smaller areas (su, 1994; forschler & ryder, 1996; tsunoda et al., 1998; su et al., 2000; cabrera & thomas, 2006), to multiple structures in larger areas (su et al., 2002; su & hsu, 2003; rojas & morales-ramos, 2003; ripa et al., 2007) to fungus culturing odontotermes and macrotermes species (family termitidae, subfamily macrotermitinae) termites in dams (huang et al., 2006; wang et al., 2007). the current study indicates the efficacy of termite baits against rhiniotermitids and termitids. peters et al. (2008) reported successful colony suppression and elimination of termitids including a higher termite macrotermes gilvus with chlorfluazuron baits. in the current study, on the average o. obesus and c. heimi ingested toxicants with different treatments both under laboratory and field conditions for six months baiting period, but one of colony of o. obesus was completely suppressed and eliminated and c. heimi colony was found suppressed. but with regards to field consumption data, contact activity of toxicants is also important with the elimination or suppression of colony. this finding demonstrates the complexity and challenges faced when trying to manage fungus growing termites using baiting systems. although a maximum amount of bait was removed by termites, but toxicant was not shared entirely and digested among nest mates in case of c. heimi colony. the bait matrix (p. euramericana) was detected in food stores and fungus comb, indicating that toxicants were integrated into the food processing pathway of termites. the bait matrix was picked up by termites approximately a week and subsequently excreted as fecal pellets. this lag of toxicants distribution to the termite castes explains why imidacloprid especially take longer to affect termites than wood feeding rhinotermitids. the colonies were moribund in termitids colony lost their reproductive capacity, as evidenced by the unhealthy and dead queen and absence of newly produced off springs. this could happen as early as a month after the baiting began, as large number of workers in the combs was observed at this point. two possible explanations may account for the low worker output in the colony. firstly, toxicant along with other treatment combination could have indirectly caused the worker population decline over time. furthermore, foraging activity of workers declined because of number of workers present was insufficient to initiate foraging activity. second, the queen appeared unhealthy. this likely reflects the fact that queen receives less food, nursing, and grooming by workers following the drastic decrease in the worker population, which in turn reduced their reproductive capacity. this finding is also reported by other termite investigators in other parts of the world (peppuy et al., 1998; rajos & morales-ramos, 2004; haagsma & rust, 2005). nevertheless, we don’t deny the possibility of toxicants eliminating or suppressing termites both lower and higher colonies if the test colonies are given more time to accumulate up to lethal level. the results help explain the mixed performance of bait against termites both rhinotermitids and termitids group. one of the factors that limit the widespread collective impact is the unique caste developmental pathways and incorporation of other effects (fungus, additives like agar and attractants) pose a great challenge in termite management programs. our results suggested that colonies require intensive baiting efforts to allow sufficient active ingredient diffuse into the colony, either by installing more bait stations or extending the baiting period. in addition, it is also believe that bait toxicants and soil treatment to find out ways to target both the immature stages and the worker population. such treatments are needed badly to increase the chances of successful termite colony elimination in lower as well as higher termites. acknowledgements the grant for this work was provided by pakistan science foundation (psf). it is fully acknowledged and appreciated. references aihetasham, a. & iqbal, s. 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(ed): production ecology of ants and termites: cambridge university press, cambridge, pp. 55–80. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.795sociobiology 62(4): 527-532 (december, 2015) ants (hymenoptera: formicidae) associated with pig carcasses in an urban area introduction the most important aspect of forensic entomology is the study of cadaveric fauna, which is based on the successional patterns of insects that feed on decomposing carcasses (catts & goff, 1992). the most commonly utilized insects for analysis of a crime scene are flies and beetles (sousa & linhares, 1997; carvalho et al., 2000; pujol-luz et al., 2006; biavati et al., 2010). however, although less studied, ants and other insects also belong to the cadaveric fauna, actively participating in the fractionation and decomposition of vertebrate carcasses (cruz & vasconcelos, 2006; moretti & ribeiro, 2006). because some ant species display necrophagous affinities, these social insects have potential to aid in crime solving in forensic entomology studies. although studies of ants and their relationship with decomposition of cadavers appear among the oldest in forensic entomology in brazil abstract the study of cadaver fauna is the most important aspect of forensic entomology, and it is based on successional patterns of insects that feed on decaying carcasses. many ant species have scavenger lifestyles, and thus have the potential for use in forensic entomology studies. there are no previous studies of ant fauna as forensic entomology tools in state of maranhão. here we characterize ants found on domestic swine carcasses at different stages of decomposition, with the goal of initiating a forensic entomology database in the state. we collected ants between november and december 2012 on two pig carcasses. a third carcass with no ant collection was used as a control to assess the influence of ant removal on decomposition rate. we captured 1,692 formicidae in total, distributed among 10 genera and including 17 species. the most abundant species was solenopsis saevissima, with 55.6% of relative abundance. solenopsis was present at various stages of decomposition either feeding on carcass tissues or exudates, or preying on larval, pupal and newly emerged adult flies. ants associated with this environment had high species richness and abundance in all stages of decomposition. we suggest that these insects influence the decomposition process either as facilitators (i.e., by laceration and fragmentation of tissue) or as hindering agents (i.e., due to predation upon other scavenging organisms). sociobiology an international journal on social insects j andrade-silva1,2, ekc pereira2, o silva2, clc santos2, jhc delabie3, jmm rebêlo1,2 article history edited by gilberto m. m. santos, uefs, brazil received 14 april 2015 initial acceptance 11 august 2015 final acceptance 30 october 2015 keywords forensic entomology, necrophagy, solenopsis saevissima, dolichoderus lutosus. corresponding author joudellys andrade silva universidade federal do maranhão departamento de patologia praça madre deus nº 02 65025-560, são luís, maranhão, brazil e-mail: andradejoudellys@yahoo.com.br (luederwaldt, 1911; freire, 1914), studies of their role and importance in the vertebrate decomposition process have been neglected (payne, 1965; nuorteva, 1974). the role of the hymenoptera, especially ants, has taken a secondary position in forensic entomology. despite the strong presence of ant species throughout the decomposition process cadavers, few studies in brazil suggest their use in forensic entomology (moura et al., 1997; moretti & ribeiro, 2006; moretti et al., 2013). it is also known that these insects can contribute to estimation of the post-mortem interval (win & goff, 1997), including those with sarco-saprophagous habits (cornaby, 1974). some forensic studies show that ants display omnivorous behavior, feeding as much on decaying cadavers as other associated insects (carvalho et al., 2004; cruz & vasconcelos, 2006). consequently, their relationship with vertebrate carcasses can vary significantly, from predatory when feeding upon research article ants 1 programa de pós-graduação em biodiversidade e conservação, universidade federal do maranhão, são luís, maranhão, brazil 2 universidade federal do maranhão, são luís, maranhão, brazil 3 cepec-ceplac, itabuna-ba/ universidade estadual de santa cruz, ilhéus, bahia, brazil j andrade-silva et al. – ants associated with pig carcasses528 eggs, larvae, pupae, or adults of other insects, to scavenging when feeding the decomposing tissues or exudates (early & goff, 1986). this behavior influences criminal investigations because scavenging ants can produce spots on the body that are occasionally mistaken for mutilation or injury (patel, 1994); predatory species can delay cadaver decomposition by decreasing the population of dipteran larvae active on the body, especially those in families calliphoridae and sarcophagidae (wells & greenberg, 1994; carvalho et al., 2004; moretti & ribeiro 2006). forensic entomology research in maranhão, brazil, is still in early stages, with, to our knowledge, only a single study of dipterans existing in caxias (silva, 2012), and no mention of hymenopteran species. the purpose of this study was to investigate the ant species present at different stages of cadaver decomposition, in order to implement a forensic entomology database for state of maranhão. we characterized ants occurring on pig carcasses in various stages of decomposition, analyzing species richness, frequency, abundance, and feeding habits. material and methods study area the study took place on são luís island (2º31’ s, 44º18’ w), in maranhão, brazil. the climate is hot and humid tropical, with a rainy season (january to june), with an average rainfall of 1,954 mm, and a dry season (july to december) with only 6% of the annual precipitation. the average temperature is approximately 26.1 °c (carvalho, 2015). the study area is located on the banks of a secondary vegetation fragment amazon located within the federal university of maranhão campus (02°33’36” s, 44°18’33” w). the environmental characteristics and dynamics result from a high degree of human intervention in the original tropical rain forest vegetation (icmbio, 2014), which was reduced to a low, secondary growth forest, with a high degree of anthropogenic change due for the installation of an area intended for higher education. field procedure ants were collected during the dry season of 2012. we collected daily in the first 15 days, and every other day thereafter. three pigs (sus scrofa l.), average weight 25 kg, were sacrificed by a qualified expert via shot to the head using a pistol. the carcasses were placed roughly 40 meters apart. ants were collected exclusively from first (p1) and second (p2) carcasses. the third carcass (pc) was used as a control, in which ants were not collected, and instead observations were made for comparison of rates of decomposition. we opted for pig carcasses because they are generally accepted as a model organism for experimental research of forensic insect fauna, due to similarities to human cadavers with respect to the amount of hair, dermal constitution, and torso/limb relationship (payne, 1965; catts & goff, 1992). all carcasses were enclosed within metal cages, which allowed the entry of insects and prevented access by medium and large-sized animals. the determination of decomposition stage followed those described by payne (1965), as follows: fresh, bloated, active decay, advanced decay, dried and remains. ant collection and laboratory procedures we use six pitfall-type traps (for details see bestelmeyer et al., 2000) placed approximately 1.5 m from the carcasses to capture attracted ants. collection was aided by use of entomological forceps. collected ants were fixed and preserved in vials containing 70% ethanol. ants were identified at the myrmecology laboratory of the cocoa research center (ceplac) in ilheus, bahia, brazil. samples were deposited in the myrmecology laboratory collection (cpdc), convention uesc/ceplac at the cocoa research center, bahia. the project was approved by the ethics committee on animal use of federal university of maranhão (protocol number: 23115.004032/2012-34). results we captured 1,692 formicids in total, belonging to 17 species and 10 genera (table 1). the camponotus and pheidole genera had the greatest diversity, with four and three species, respectively (table 1). ectatomma brunneum smith and solenopsis saevissima smith were the most common species (present in 80% of the traps). the most abundant species was s. saevissima (54.5% ra), followed by dorymyrmex brunneus forel (8.5%), solenopsis globularia smith (8.2%) and e. brunneum (8%). both pig carcasses (p1 and p2) underwent the five stages of decomposition at roughly the same pace: fresh, lasted for one day; bloated, one day; active decomposition, five days; advanced decomposition, five days; dried and remains, 18 days. ants were present at all stages of decomposition, though abundance strongly varied. the two solenopsis species (s. globularia and s. saevissima) were seen preying on fly larvae and causing injury to carcasses (p1 and p2). dolichoderus lutosus smith were seen feeding on exudates of the decomposing carcass; we did not observe predation upon other organisms, nor damage to carcasses resulting from this activity. to our knowledge, this is the first report of d. lutosus associated vertebrate cadaver decomposition in the neotropics. the last three stages of decomposition had the highest species richness in both carcasses, which may be related to the longer duration of these phases. overall, there was a gradual increase in ant abundance throughout the different stages of decomposition. sociobiology 62(4): 527-532 (december, 2015) 529 a nt s pe ci es c ar ca ss 1 c ar ca ss 2 to ta l fr b l a c t a d v d r n fr b l a c t a d v d r n n % d ol ic ho de ri na e d ol ic ho de ru s lu to su s (s m ith , 1 85 8) 23 23 23 1, 3 d or ym yr m ex b ru nn eu s fo re l, 19 08 5 16 64 85 2 3 7 47 59 14 4 8, 5 e ct at om m in ae e ct at om m a br un ne um (s m ith , 1 85 8) 30 3 7 41 81 28 2 2 23 55 13 6 8, 0 e ct at om m a tu be rc ul at um (o liv ie r, 17 92 ) 1 4 10 15 1 1 16 1, 0 fo rm ic in ae c am po no tu s bl an du s (s m ith , 1 85 8) 6 6 1 1 2 8 0, 5 c am po no tu s m el an ot ic us e m er y, 1 89 4 6 10 6 48 70 4 1 14 19 89 5, 3 c am po no tu s ru fip es (f ab ri ci us , 1 77 5) 6 3 25 34 7 7 24 38 72 4, 2 c am po no tu s se ne x (s m ith , 1 85 8) 5 1 9 15 3 11 14 29 1, 7 m yr m ic in ae a cr om yr m ex r ug os us (s m ith , 1 85 8) 1 1 8 10 3 16 19 29 1, 7 c re m at og as te r vi ct im a (s m ith , 1 85 8) 3 7 10 1 11 12 22 1, 3 p he id ol e gr up o f al la x sp . 3 2 7 12 12 0, 7 p he id ol e ra do sz ko w sk ii m ay r, 18 84 1 1 3 3 4 0, 3 p he id ol e sy na rm at a w ils on , 2 00 3 2 13 15 1 1 14 16 31 1, 9 so le no ps is g lo bu la ri a (s m ith , 1 85 8) 17 13 44 74 25 2 38 65 13 9 8, 2 so le no ps is s ae vi ss im a (s m ith , 1 85 5) 28 72 27 5 37 5 1 63 99 14 7 23 7 54 7 92 2 54 ,5 po ne ri na e o do nt om ac hu s ba ur i e m er y, 1 89 2 14 14 14 0, 8 ps eu do m yr m ec in ae p se ud om yr m ex s ch up pi f or el , 1 90 1 2 2 2 0, 1 a bu nd an ce 30 10 10 6 11 7 55 3 81 6 45 67 14 1 17 7 44 6 87 6 16 92 10 0 ta bl e 1. a bs ol ut e an d re la tiv e fr eq ue nc y of f or m ic id s ca pt ur ed in d if fe re nt s ta ge s of p ig c ar ca ss d ec om po si tio n in th e dr y se as on ( n ov em be r an d d ec em be r 20 12 ) in a n ur ba n ar ea in th e ci ty o f sã o l uí s, m ar an hã o st at e, n or th ea st b ra zi l. fr : f re sh ; b l : b lo at ed ; a c t: a ct iv e d ec ay ; a d v : a dv an ce d d ec ay ; d r ; d ry a nd r em ai ns . j andrade-silva et al. – ants associated with pig carcasses530 discussion ant species richness in the current study was the greater than in many other studies of decaying organisms in brazil (luederwaldt, 1911; monteiro-filho & penereiro, 1987; moura et al., 1997; moretti et al., 2007; gomes et al., 2007, 2009; santos et al., 2014). in one forensic study using visual observation only, luederwaldt (1911) reported nine ant species distributed among nine genera. monteiro-filho and penereiro (1987) found five species, and moretti et al. (2007) found four genera, three of which were also present in this study (camponotus, crematogaster and dolichoderus). gomes et al. (2009) highlighted e. brunneum, as well as pheidole and camponotus in his study of the insect fauna on pig carcasses. the high number of species in our study seems to be due to the strong anthropogenic disturbance in the area. it is noteworthy that many of the species recorded here exhibit mass recruitment behavior (see fowler et al., 1991; silvestre & silva, 2001) or some affinity for impacted environments (brown, 1973; overal, 1986; piva & campos-farinha, 1999; fernandes et al., 2001; barbosa & fernandes, 2003). pheidole and camponotus are the genera with the greatest geographical distributions, species diversity, and adaptations in the whole neotropical region, and even in the world (wilson, 1976, 2003). furthermore, one of the most abundant species of camponotus in this study, c. rufipes has already been noted as having medical and legal importance in forensic studies in brazil (moura et al., 1997). the “fresh” period of decomposition hosted predominantly e. brunneum, one of the most common species in this study. according to hölldobler and wilson (1990), species in this genus show aggressive behavior, and may inhibit the population growth of less aggressive species, or species less adapted for resource competition (fernandes et al., 2000). the presence of this genus in the remaining stages of decomposition might be linked to the presence of other arthropods, since ectatomma species are largely predacious upon other arthropods (agosti et al., 2000). the “active and advanced” stages of decomposition showed a significant increase in the number of ant species compared to previous stages. liquids associated with decomposition and other carcass resources are exposed in these stages, which with advancing decay diffuses odors at greater distances, acting as an additional attractant for formicids (goff, 2009). the majority of ants was collected in the final stages of decomposition (i.e., dry and remains). however, abundance and species richness may have been overestimated in these final stages due to the greater number of days in this stage compared to other decomposition stages in the collection period. in addition to species richness, abundance was also high and constant in the vicinity of carcasses, which has been observed by other authors (horenstein et al., 2005; chin et al., 2009; bonacci et al., 2011). s. saevissima abundance, for example, can be explained by the typical behavioral dominance of the solenopsis genus. this genus shows massive behavioral recruitment and is capable of rapidly colonizing the exploited resource (delabie & fowler, 1995), and further, it behaves aggressively with both other ants and other animal species (fowler et al., 1991). solenopsis was found at various stages of cadaver decomposition, feeding on carcass tissues or exudates, or preying on larvae, pupae and newly emerged adult flies, behavior previously noted by oliveira-costa et al. (2007). the prevalence of s. saevissima on our carcasses may have inhibited the proliferation of other ant species. predation on dipteran larvae and carcass damage caused by solenopsis species in this study suggests that this taxon may influence both corpse decomposition rate and the number of flies that reach adulthood on the corpse. this notion has also been suggested in other studies (early & goff, 1986; stoker et al., 1995). we report dolichoderus lutosus workers feeding on vertebrate carcasses for the first time in the neotropical region. dolichoderus has worldwide distribution, but most species are neotropical and arboreal (silvestre et al., 2003). failure to observe predation on other organisms or damage to carcasses corroborates moretti et al. (2013), which indicates that dolichoderinae are generally attracted by the release of carcass exudates. this behavior justifies the classification of these ants as opportunistic scavengers (early & goff, 1986) as well as future actions to study them in more detail on the role in and influence upon cadaver decomposition. even though few studies exist on the subject in brazil, hymenoptera seem to influence vertebrate carcass decomposition. bees, for example, can be present in all phases of the corpse decomposition and can feed upon exudates (santos et al., 2014). the wasp epiponini agelaia pallipes (olivier) visit s. scrofa carcasses to feed on body fluids and collect carcasss pieces to feed their larvae (gomes et al., 2007). in this same study, the only ant species observed was neoponera obscuricornis (emery), preying upon larval and adult blowflies. our study shows high species richness and abundance of ants associated with pig carcasses. the presence of ants in all stages of corpse decomposition as well as the observed feeding and predatory behaviors suggest that these insects can influence the decomposition process in two opposite ways: as facilitators, through the disruption and fragmentation of tissue, or as hindering agents, through predation of invertebrates (insect larvae, especially) that effectively contribute to decomposition. consequently, these insects are of great importance for forensic entomology in brazil, and henceforth should be given more attention in this field. acknowledgements we thank the fundação de amparo à pesquisa e ao desenvolvimento científico e tecnólogico do maranhão (fapema) for financing support, and the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for research grants awarded to jhcd and jmmr. sociobiology 62(4): 527-532 (december, 2015) 531 references agosti, d., majer, j.d., alonso, l.e. & shultz, t.r. 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(2003). pheidole in the new world: a dominant, hyperdiverse ant genus. cambridge, harvard university press, 794 p. doi: 10.13102/sociobiology.v63i2.909sociobiology 63(2): 762-769 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 composition and richness of arboreal ants in fragments of brazilian caatinga: effects of secondary succession introduction there is a consensus that along a gradient of secondary succession, environments in late stages of regeneration tend to have greater species richness of arthropods than more disturbed habitats (in early stages of succession). late succession environments generally leads to a greater richness and density of plant species, increasing availability of food resources and nesting sites (bazzaz, 1975; vasconcelos, 1999; klimes et al., 2012). the idea that more arthropods species can be find in environments with high complexity (i.e. high species richness abstract ecological succession is a complex process involving mainly some changes in the structure of plant community, with subsequent changes in resource availability as well as in the interactions among organisms which are also important factors determining the structure of arboreal ant assemblages, but little is known about the consequences of secondary succession on ant assemblages in tropical dry forests (tdfs), such as the brazilian caatinga. here we investigate the effects of ecological succession on the richness and species composition of arboreal ants in fragments of caatinga, testing the following hypothesis: i) the species richness of arboreal ants increases along a gradient of secondary succession, in response to habitat complexity and season; ii) species composition of arboreal ants differs among stages of secondary succession due to differences in vegetation structure in theses stages and seasonality. this study was conducted in 15 plots distributed in three areas with different stages of secondary succession (early, intermediate and late). tree species richness and density were used as surrogates of habitat complexity. ants were sampled using the technique of beating the foliage and baited pitfall traps, where five trees were sampled per plot, totaling 75 individual trees sampled. we sampled 37 species of ants, distributed in 16 genera and five subfamilies. ant species richness differed among stages of succession and seasons, with higher number of species in the late succession and rainy period, also increasing with habitat complexity. we conclude that the structure of arboreal ants assemblage was related to ecological succession of vegetation, mainly in the rainy season. fifteen years of secondary regeneration are enough to increase species richness and maybe to restore species composition of arboreal ants but secondary habitats are also important to biodiversity maintenance of this group in caatinga. sociobiology an international journal on social insects l sousa-souto1, pmg figueiredo1, bg ambrogi1, acf oliveira1, gt ribeiro1, fs neves2 article history edited by kleber del-claro, ufu, brazil received 10 september 2015 initial acceptance 07 october 2015 final acceptance 18 april 2016 publication date 15 july 2016 keywords tropical dry forest, habitat structure, bioindicators, ant diversity. corresponding author leandro sousa souto programa de pós-graduação em ecologia e conservação universidade federal de sergipe são cristóvão-se, brazil e-mail: leandroufv@gmail.com and/or abundance of trees) along a successional gradient has been demonstrated in studies using different groups of insects such as beetles, termites, butterflies (romero-alcaraz & avila, 2000; tewset al., 2004; quesada et al., 2009; neves et al., 2010a; vasconcellos et al., 2010; neves et al., 2014). among these groups, ants have been the subject of several studies that seek to understand how species richness and composition has been influenced by vegetation structure (leal, 2003; ribas et al., 2003; corrêa, 2006; ribas & schoereder, 2007; vargas et al., 2007; neves et al., 2010b; gomes et al., 2014; klimes et al., 2012). 1 universidade federal de sergipe, são cristóvão-se, brazil 2 universidade federal de minas gerais, belo horizonte-mg, brazil research article ants sociobiology 63(2): 762-769 (june, 2016) 763 ants represent an important group of terrestrial organisms due to several factors, such as their high taxonomic diversity (erwin, 1989), their contributions to ecological processes (hölldobler & wilson, 1990) and their sensitiveness to variations of landscape structure (lindsey & skinner, 2001; ribas et al. 2003). ant species richness has usually been positively associated with habitat structure and its association is more pronounced at local scale, mainly at sites with higher resource availability, diversity of microhabitats and a greater number of nesting sites (ribas et al., 2003; marques & del-claro, 2006; ribas & schoereder, 2007; pacheco & vasconcelos, 2012; fagundes et al., 2015). arboreal ants, however, commonly show a strong territorial behavior (schoener, 1970; bernstein, 1975; espírito-santo et al., 2009; ribeiro et al., 2013a; dáttilo et al., 2015), and changes in vegetation structure due to ecological succession may result in an increase of aggression and monopolization of space, leading a shift in the species composition. tropical dry forests (tdfs) cover significant land areas in brazil and provide a habitat for a diverse number of species. the brazilian caatinga is a mosaic of scrub vegetation and patches of dry forest and covers over 800,000 km2, of the brazilian territory (sampaio, 1995). this biome undergo 7-11 months of dry season (prado et al., 2003), and such seasonal variation has a negative impact in the production and maintenance of the leaves, with most tree species losing their leaves during the dry months, diminishing connectivity between tree canopies. in these periods, when the resources are scarce and the abiotic conditions are stressful, arboreal ants could have different survival strategies, such as changing daily activity (e.g. diurnal to nocturnal pattern) (dáttilo et al., 2015), expand the foraging area to alternative strata (e.g. vertical stratification) (dejean et al., 2003; campos et al., 2008; silva et al., 2014), or move to more favorable adjacent habitats to search for resources (neves et al., 2010b). for example, trees in early successional stages would be occupied by both ground-dwelling and arboreal ants, whereas exclusive arboreal ants predominate in late-stage trees (dejean et al., 2003). however, effects of seasonality and environmental changes in areas of tdfs, along a successional gradient on the structure of ant assemblages have been poorly documented (lewinsohn et al., 2005; neves et al., 2010b). thus, the aim of this study was to investigate the effects of ecological succession on the structure of arboreal ant assemblages in areas of caatinga, testing the following hypotheses: i) the species richness of arboreal ants increases along a gradient of secondary succession, in response to season (dry and rainy), tree species richness and/or density; ii) species composition of arboreal ants differs among seasons and stages of secondary succession. material and methods study area the study was conducted in the state of sergipe, northeastern brazil encompassing three fragments of caatinga with different stages of plant regeneration surrounded by a matrix of active or abandoned pastures (ribeiro et al., 2013b). caatinga is a tropical dry forest that covers approximately 9% of the brazilian territory, characterized by a tropical semiarid climate (bshi), annual rainfall ranging from 250 to 900 mm and mean temperature of 24°c and 26°c (andrade-lima, 1981; sampaio, 1995; pennington et al., 2000). according to oliveira et al. (2013) the soil type is classified as eutrophic litholic neosol. the stages of succession were divided in early, intermediate and late. the early stage (20 ha) is characterized by a abandoned pasture with three years of plant recovery, composed of sparse patches of trees with open canopy and 2 meters of high. intermediary area is located 2.5 km apart from the early stage within a conservation unit (monumento natural grota do angico), in the municipality of poço redondo (9º 41’s and 38º 31’w) covering 2,183 ha. this area is composed of deciduous trees 2-4 m in height and with approximately 12-15 years of regeneration. the late successional area is a forest fragment (115 ha) with more than 40 years of forest regeneration, located 30 km far away from the other two areas (10° 02’s 37° 24’w). the fragment is characterized by taller deciduous trees which form a canopy 4-15 m high, on a private farm. sampling design sampling areas consisted of 15 plots with 50 m x 20 m (0.1 ha each), being five plots per stage of succession, with a minimum distance of 200 m between each plot. tree species richness and abundance (circumference at breast height cbh > 6 cm) were used as surrogate of vegetation structure. in each plot, we arbitrarily chose five trees (cbh > 6 cm, regardless of the species), at least 10 m apart from each other as sampling points. sampling of ants was conducted in six sample periods: february/2011, march/2012 and november/2013 (dry season), may/2011, july/2012 and june/2013 (rainy season). ant sampling was made using a entomological umbrella and pitfall traps. the chosen trees were beaten over the umbrella with a stick. we standardized the sampling effort according to the number of secondary branches per tree, being 30 beats for trees with 1–5 branches (n = 60), including all branches. for the other trees with 6–10 branches (n = 15), we selected three sections of the tree canopy and performed 10 beatings in each section, totaling 30 beatings per tree. thus, each tree was beaten 30 times, regardless of its size (sousasouto et al., 2014). in addition, we used pitfall traps on the same five trees. these traps consisted of plastic cups (350 ml) with water and detergent (2%) baited with sardine and honey (see ribas et al., 2003). these traps were left in the field for a period of 48 hours. traps were set with a nylon thread on tree trunks at breast height. ants were identified with taxonomic keys (bolton, 1994; fernández, 2003) and by comparison with specimens from the collection of the laboratório de mirmecologia of the cepec/ ceplac, ilhéus, bahia, brazil. voucher specimens of all species are deposited in laboratório de entomologia of the federal university of sergipe. l sousa-souto et al. – arboreal ants in brazilian caatinga764 data analysis to compare the species richness of ants among the three successional stages (early, intermediate and late) and between seasons, we used liner mixed effects (lme) due to the pseudoreplication of plots in different seasons (dry and wet). thus, ant species richness was treated as fixed effects, while plots and seasons were the random effects. in addition, we tested the response of ant species richness to richness and abundance of trees using generalized linear models (glm’s). in these models, ant species richness was used as the response variable and the species richness and abundance of trees were treated as explanatory variables. the minimum adequate models (mam) were obtained by extracting non-significant terms (p > 0.05) from the full model (crawley, 2013). we tested if accumulated species composition of arboreal ants was affected by secondary succession, season, tree species richness and density using a permanova — permutational multivariate analysis of variance (anderson, 2001). a non-metric multidimensional scaling (nmds) was used to represent the results of the permanova (k = 2). in these analyses, the ordination of species composition was undertaken using the jaccard index. all statistical analyses were conducted with the r software (r development core team, 2015). results a total of 37 species of ants were sampled, belonging to 16 genera and five subfamilies (table 1). myrmicinae was the subfamily with the largest number of species, representing approximately 55% of all species collected. pheidole and camponotus were the richest genera, with seven and six species, respectively. only 12 species were common to all successional stages: camponotus arboreus, c. atriceps, c. cingulatus, c. melanoticus, c. senex, c. vittatus, crematogaster stolli, c.victima, pheidole radoszkowskii, solenopsis tridens, pseudomyrmex gracilis, and pseudomyrmex rochai. in the initial stage area16 species were sampled, where three were exclusively sampled in this stage of succession. in the same way, intermediate stage had 16 species sampled being just one exclusively found in that habitat. in the late stage of succession, 33 species were collected, with 17 species sampled exclusively in that environment (table 1). the species richness of arboreal ants differed among stages of succession (f = 8.3; p < 0.01) as well as between seasons (f = 72.1; p < 0.001). in the lme model, interaction between secondary succession and season was also significant (f = 22.4; p < 0.001), indicating that the response of ant species richness was different depending of the stage of succession and season. ant species richness was higher in wet than dry season in intermediary and late forests fragments, while species richness did not differ among stages in the dry season (fig 1). besides, differences in habitat structure (tree species richness and density) had affected positively the species richness of arboreal ants (f = 5.8; p = 0.03 and f = 17.1; p = 0.02, respectively) (fig2). fig 1. species richness of arboreal ants in response to season (dry and wet) and among three forest fragments of caatinga, with different stages of succession. different letters above bars indicate significant differences amongst successional stages, while asterisks indicate differences between seasons within a same stage. fig 2. species richness of arboreal ants in response to (a) tree species richness and (b) tree density among 15 plots in fragments of caatinga. regarding species composition, arboreal ants presented differences among successional stages and these differences were in response to changes of tree density, tree species richness and season (permanova; table 2; figure 3). sociobiology 63(2): 762-769 (june, 2016) 765 subfamily species treatment code early intermediary late formicinae brachymyrmexsp.1 sp1 0.1 camponotus arboreus sp2 0.1 0.5 0.1 camponotus atriceps sp3 0.5 0.1 0.5 camponotus cingulatus sp4 0.8 0.1 0.3 camponotus melanoticus sp5 0.2 1.0 0.3 camponotus senex sp6 1.0 1.0 0.6 camponotus vittatus sp7 0.9 0.9 0.7 ectatomminae ectatomma suzanae sp8 0.1 0.6 gnamptogenys sulcata sp9 0.3 gnamptogenys moelleri sp10 0.6 myrmicinae cephalotes minutus sp11 0.4 0.9 cephalotes persimilis sp12 0.1 cyphomyrmex transversus sp13 0.2 crematogaster chodati sp14 0.3 crematogaster distans sp15 0.6 crematogaster sp.1 sp16 0.1 crematogaster stolli sp17 0.4 0.5 0.1 crematogaster victima sp18 0.3 0.7 0.6 nesomyrmex spininodis sp19 0.1 pheidole (gr. diligens) sp.1 sp20 0.1 0.2 pheidole (gr. fallax) sp.2 sp21 0.5 pheidole (gr. fallax) sp.3 sp22 0.4 pheidole (gr. fallax) sp.4 sp23 0.1 pheidole sp.1 sp24 0.1 pheidole sp.2 sp25 0.3 pheidole radoszkowskii sp26 0.8 0.5 0.8 solenopsis tridens sp27 0.1 0.3 0.2 solenopsis globularia sp28 0.4 strumigenys sp.1 sp29 0.2 wasmannia auropunctata sp30 0.3 pseudomyrmecinae pseudomyrmex gracilis sp31 0.7 0.4 0.3 pseudomyrmex rochai sp32 0.4 0.4 0.1 pseudomyrmex symplex sp33 0.1 pseudomyrmex termitarius sp34 0.1 0.1 dolichoderinae dorymiyrmex thoracicus sp35 0.1 dolichoderus voraginosus sp36 0.1 0.1 tapinoma melanocephalum sp37 0.1 table 1 relative frequency of arboreal ants collected in three areas with different stages of secondary succession (early, intermediary and late succession) in areas of caatinga of sergipe. l sousa-souto et al. – arboreal ants in brazilian caatinga766 richness and changes in species composition along the gradient of forest regeneration. the species richness of arboreal ants sampled in this study (n = 37) can be considered low compared to other studies done in cerrado or atlantic forest, using a similar sampling method. in fragments of atlantic forest, for example, species richness may vary from 69 to 153 (resende et al., 2013; vasconcelos et al., 2014), whereas in areas of brazilian savanna (cerrado) species richness of arboreal ants ranges from 37 to 95 (ribas et al., 2003; frizzo et al., 2012). however, for tropical dry forests areas, such as caatinga, the number of arboreal ants sampled is generally low, ranging from 24 to a maximum of 43 species (neves et al., 2010c; neves et al., 2013; silva et al., 2014), indicating that caatinga environments, may naturally harbor a smaller ant richness when compared to other environments, probably due to habitat limitations such as drastic variation in resources and conditions during the season and between years (quesada et al., 2009). of all ant species sampled, seventeen were unique of the area of late succession, indicating that these species could be bioindicators of advanced stages of forest regeneration in caatinga. however, w. auropunctata was also found in areas of early forest regeneration in caatinga (neves et al., 2010b) as well as two species (c. distans and c. transversus) were also found in fragments with early forest regeneration in atlantic forest fragments in sergipe (gomes et al., 2014), suggesting that the occurrence and dispersion of some potential species used as bioindicators may vary depending on the scale and environment. the results presented in figures 1 and 2 confirm some studies showing a general pattern that areas of advanced stages of forest regeneration tend to harbor greater species richness when compared to simpler environments (bazzaz, 1975; vasconcelos, 1999; klimes et al., 2012; gomes et al., 2014), due to environmental factors such as high habitat complexity and high resource availability (kalacska et al., 2004; quesada et al., 2009). in the present study, we found that areas considered at late stages of succession have greater species richness of arboreal ants compared with environments in recent forest regeneration. this result indicates that changes in habitat structure, promoted by the increment of variables such as richness and abundance of trees, along a gradient of secondary succession favor directly and indirectly the increase of species richness of ants, corroborating other studies (lassau & hochuli, 2005; ribas et al., 2003; vargas et al., 2007; klimes et al., 2012). seasonality in our study area was also an important driver of the biodiversity of arboreal ants, accounting for a decrease of 40 to 50% in species richness of ants in the dry season in the intermediate and late successional stages, respectively. this reduction in species richness was due to the drastic loss of leaves in most tree species during the dry season, diminishing connectivity between tree canopies with a consequent reduction of resource availability in these areas. those differences can be a result of variations in the exploitation of resources between response variable terms of the model df f-model r2 p ant composition stage of succession 2 3.52 0.17 0.001 plant species richness 1 1.83 0.05 0.04 plant abundance 1 5.10 0.12 0.001 season 1 3.54 0.08 0.001 table 2 permanova main tests for differences in assemblage composition of arboreal ants among successional stages, season and habitat structure (plant species richness and density), in a semiarid caatinga, state of sergipe, brazil. discussion considering that secondary succession is a process, several factors that arise as a consequence of this ecological process may act individually or together modifying the structure of ant assemblages along the successional stages. in the present study we tested the influence of two of these factors: habitat complexity (herein referred to as species richness and density of trees) and seasonality as drivers of change in the diversity of ants in areas of caatinga and we found that the factors above contribute to the increase in species richness of ants as well as for modification of its composition along the successional gradient, with strong influence of the rainy season. thus, for arboreal ants, the ecological succession in areas of caatinga has the same patterns already known for other brazilian biomes that are the increase of species fig 3. nmds ordination of the 37 ant species along three successional gradients (early, intermediate and late) in a caatinga environment, between dry and wet season. sociobiology 63(2): 762-769 (june, 2016) 767 seasons and due to vertical migration of some ant species from soil and shrub layer to canopy. further studies, however, are necessary to test how of these factors (variation in the use of resources or vertical migration) are determinant in caatinga, since there are few information about the effects of seasonality on the ant activity and structure of communities in tropical dry forests (neves et al., 2010b). differences in species richness of ants between dry and rainy seasons, however, did not occur in the initial succession area. this may reflect a higher occurrence of non-specialist ants in the arboreal stratum, with predominance of species adapted to search for food in the ground or shrub vegetation. thus, in a scenario of high unpredictability of resource supply, is common that generalist ground-nesting ants start to climb the trees more often, and start to monopolize resources (trees) (martinez, 2015). this behavior was previously reported by martinez (2015) and may explain the low diversity of ants in caatinga, considering that in our study area some predominantly ground-nesting ants were sampled in the trees, such as cyphomyrmex transversus, strumigenys sp.1, gnamptogenys sulcata and g. moelleri. as the forest regeneration occurs, gradual changes take place in the composition, abundance and richness of vegetation, thus driving changes in species composition in the assemblages of organisms associated (bazzaz, 1975; neves et al., 2014; sousa-souto et al., 2014). these changes become clear since we observe the differences in species composition of arboreal ants between different successional stages. similar results were observed in previous studies (arnan et al., 2011; neves et al., 2013; sousa-souto et al., 2014), indicating that changes in habitat structure tends to affect the species composition of ants. this turnover of ant species can be determined by factors specific to each successional stage (e.g. microclimatic gradients, especially moisture and temperature) or each individual tree species (i.e. architectural tree characteristics), since resource specialization performed by some ant species can influence the distribution of less competitive species in the canopies (ribas & schoereder, 2002; campos et al., 2006; neves et al., 2010b). this study strengthens the evidence for a regional ecological pattern, in which changes of the vegetation along the ecological succession have direct and indirect consequences for changes in the fauna of arboreal ants with regards of differences in species richness and composition of ant assemblages in dry forests, with pronounced effects in the rainy season. besides, this study was the first to assess the effects of ecological succession on the assemblage of arboreal ants in area of caatinga in sergipe. in conclusion, habitat complexity and heterogeneity resulting of ecological succession can be considered an important factor in determining patterns of richness and species composition of arboreal ants in caatinga, where each area tends to have specific characteristics. based on these results it can be stated that the conservation of areas with different stages of forest regeneration (with a progressive focus on late successional areas) is crucial, since such areas would preserve most ant species associated with vegetation. acknowledgments the authors are grateful to jacques hc delabie for his help in the identification of species and to allow the comparison with voucher species of the cepec/ceplac collection; 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(2014). evaluating sampling sufficiency and the use of surrogates for assessing ant diversity in a neotropical biodiversity hotspot. ecological indicators, 46: 286-292. doi: 10.1016/j.ecolind.2014.06.036 doi: 10.13102/sociobiology.v62i4.845sociobiology 62(4): 538-563 (december, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 revision of the african ants of the bothroponera pumicosa species complex (hymenoptera: formicidae: ponerinae) introduction the afrotropical ants of the genus bothroponera are a highly diverse group of formicidae that belongs to the subfamily ponerinae, tribe ponerini. little information is known about their behavior, biodiversity, richness, biology, ecology, biosystematics and evolution. the most common species in the afrotropics are b. talpa and b. pachyderma of talpa species complex and b. crassior, b. silvestrii and b. soror of the sulcata species complex among 43 taxa of bothroponera that are distributed in the afrotropics and southern asia. the species of pumicosa species complex are mostly restricted to south africa with the exception of b. cariosa that was collected in mozambique (emery, 1895), tanzania and gabon (ant web, 2013), b. granosa that was collected in zimbabwe (arnold, 1926) and b. pumicosa that was collected in cameroon (wheeler, 1922; stitz, 1910). abstract african ants are poorly known, especially afrotropical ants of the subfamily ponerinae, despite recent advances. the genus bothroponera (ponerinae) includes the pumicosa, talpa and sulcata species complexes. we here present a revision of members of the pumicosa species complex. these ants can be characterized by having coarsely foveolate sculpture and by having either a raised a “v” or “u” shaped anterior medial border of the clypeus (anteclypeus), with or without a carina. members of this complex lack the metatibial gland on the anterior side of the lower metatibia. species in the bothroponera pumicosa species complex are mainly distributed in the southern part of africa. they include: bothroponera aspera arnold, 1962 (stat. nov.), b. berthoudi (forel, 1901) (= variolosa syn. nov.), b. cariosa emery, 1895, b. cavernosa (roger, 1860), b. granosa (roger, 1860), b. laevissima (arnold, 1915), b. montivaga arnold, 1947 (stat. nov.), b. pumicosa (roger, 1860), b. strigulosa emery, 1895, and b. umgodikulula joma and mackay, 2013. a key to the workers with diagnoses and comparisons is provided, together with illustrations of each species and colored photographs of the species, as well as maps and the distributions of each species. sociobiology an international journal on social insects ama joma1, 2, wp mackay2 article history edited by gilberto m. m. santos, uefs, brazil received 05 june 2015 initial acceptance 11 august 2015 final acceptance 02 december 2015 keywords afrotropical, conservation, biodiversity. corresponding author abdulmeneem m. a. joma department of biological sciences 500 west university avenue el paso, tx 79968, usa e-mail: alnoure69@hotmail.com in this paper, we focus on the members of the bothroponera pumicosa species complex from africa to reorganize their taxonomy, which is part of a revision of this conspicuous and ecologically important group of ants, in order to improve the knowledge of african bothroponera and provide keys for the identification of species. materials and methods museums and collections: the specimens of the african bothroponera species complexes were obtained from the following museums. : naturhistorisches museum, basel, switzerland (nhmb). muséum d’histoire naturelle, geneva, switzerland (mhng). iziko south african museum, south africa (iziko). research article ants 1 the university of sebha, sebha, libya 2 university of texas, el paso, texas, usa sociobiology 62(4): 538-563 (december, 2015) 539 the mackay collection, the university of texas at el paso, usa (cwem). british natural history museum, london, uk (bmnh). museum für naturkunde, berlin, germany (mfn). museum nationale d’histoire naturelle, paris, france (mnhn). museo civico di storia naturale, genova, italy (mcsn). american museum of natural history, new york, usa (amnh). los angeles county museum of natural history, california, usa (lacm). museum of comparative zoology, cambridge, massachusetts, usa (mczc). measurements and abbreviations used: the specimens were examined with a zeiss binocular microscope with an ocular micrometer. all measurements are in millimeters. head length (hl), in full face view, the maximum length of the head excluding the mandibles, from the midpoint of the anterior clypeal margin to the mid-point of the posterior margin of the head. head width (hw), in full face view, the maximum width of the head from the extreme side of head to the other extreme side excluding the eyes. mandible length (ml), the distance from the mandible’s base to the apex of the apical tooth. eye length (el), the maximum diameter of the eye as seen from the side. eye width (ew), the maximum distance of the eye from the anterior edge to the posterior edge as seen from the side. scape length (sl), the maximum length of the scape from the proximal to the distal extremes, excluding the basal constriction. funiculus length (fl), the measurement of the distal 11 segments of the antenna including the club and all of the funicular segments. weber length (wl), the length in lateral view, from the anterior edge of the pronotum to the end of posterior margin of the propodeal lobes. petiole length (pl), in lateral view, the maximum distance of the petiole from the anterior face to the posterior edge, excluding the helcium. petiole width (pw), in dorsal view, the maximum side to side thickness of the petiole, generally at the posterior edge since it has the largest width. petiole height (ph), in lateral view, the maximum length from the lower point of the sternopetiolar process excluding the petiolar teeth, to the highest point at the apex of the petiolar node. cephalic index (ci), hw/hl x 100. ocular index (oi), el/hw x 100. mandibular index (mandi), ml/hl x 100. scape index (si), sl/hw x 100. petiolar index (peti), pw/pl x 100. in each specimen we measured the hair length, the total body length, the malar space length (from lower edge of the eye to the base of the mandible) and the length of the side of the head from the upper margin of the eye to the highest point of the posterior lateral corner of the head (side view). in some cases, we measured the frontal lobe width and the gaster length. there are other characters that were taken into account including the shape of the head, size of the eyes (large or small), characteristics of the pronotum, mesopleuron, propodeum, petiole and postpetiole. the shape of the pronotal shoulder, lower margin of the pronotum, basalar sclerite, and propodeal spiracle are also important. the entire body color including the antennae, clypeus, mandibles and legs were included as well. figures 1 and 2 show the various details and morphological characters of genus bothroponera that were used to identify taxa in this genus. the morphological terms are from serna and mackay (2010) and keller (2011). illustrations were completed using the typical methods such as a compound microscope, microscopic grids and a micrometer. photos were taken in the museum of comparative zoology (mczc) using an automontage photosystem provided with computer software (leica mz 7.5 stereomicroscope, canon camera eos 7d 18 megapixel digital slr, helicon focus software and photoshop). the ant website was the alternative source to obtain ant photos. maps of the distribution of african bothroponera were completed using golden software mapviewer version 3.0. the terrestrial ecoregions map (map 1) was used to display information about ecological and biological nature of the plant community distribution in africa. google earth was also used to characterize the ant localities. the longitudes and latitudes of the specimen localities were determined using fuzzy gazetteer (isodp.hof-university.de/fuzzyg/query/). lectotypes and paralectotypes were named in order to establish the identity of the specimens. fig 1. the full view of the head of an african bothroponera. a, frons; b, clypeus; c, frontal lobe; d, scape; e, malar space; f, posterior lateral corner of the head; g, mandible; h, eye. ama joma; wp mackay – bothroponera pumicosa species complex540 results family formicidae genus bothroponera mayr, 1862 worker: large ants, with maximum total length of 5 16 mm; head subquadrate in most species (excluding mandibles), suborbicular in some species, posterior border generally concave; mandibles narrowed or triangular-shaped in most species, shorter than head length with teeth number ranging from 6 to 9; anterior medial margin of clypeus convex, often sharply angled or straight to slightly concave with medial raised area; frontal lobes divided by well-developed frontal furrow; scape shorter, nearly reaches or extends slightly past posterior border of head; compound eyes vary from relatively small to large; sculpture smooth or slightly rough to punctate or foveolate; pronotal shoulder squared or rounded, but without lateral margin or carina in all species of bothroponera; mesonotum and propodeum poorly separated by notopropodeal groove; mesometapleural suture well developed; propodeum rounded between faces, mesonotal basalar sclerite rounded or oval-shaped, propodeal spiracle elongate or slightly ovalelongate; petiole subquadrate, rounded antero-posteriorly, usually wide with definite dorsal face; stridulatory file present on second acrotergite of gaster; hairs scarce on body and usually short; color mostly dark brown or black. female: head subquadrate or suborbiculate; pronotum rounded anteriorly, pronotal shoulder lacking carina or lateral margins; scutum wide anteriorly, reaches same width as pronotum, narrowed posteriorly to same width as scutellum; metanotum slightly elevated, narrowed, well separated from propodeum and scutellum; mesopleuron divided by anapleural sulcus to form ventral katepisternum and dorsal anepisternum; mesometapleural suture well defined; mesonotal basalar sclerite oval or round shaped, propodeal spiracles elongate or subrectangular in some species; petiole rounded anteriorly, vertical with slightly concave posterior face in some species; postpetiole rounded or subquadrate anteriorly; postpetiole and remainder of gaster larger than mesosoma; short to moderately long (up to 0.40 mm) erect golden hairs scattered on dorsum of pronotum, scutum, scutellum, metanotum, propodeum, petiole and postpetiole; short (up to 0.15 mm) erect golden hairs on head; surfaces mostly brown, dark brown or black. male: head excluding mandibles rounded or elongated; eyes large, cover most of side of head; scape shorter and thicker than second segment of funiculus; pronotum triangular, scutum usually with notauli; scutellum elevated, triangular in dorsal view, metanotum slightly raised between scutellum and propodeum, mesopleuron divided by anapleural sulcus into ventral katepisternum and dorsal anepisternum; dorsopropodeum gradually sloping downward posteriorly to reach insertion of petiole; petiole small, apex rounded, width and height less than those of propodeum and postpetiole; postpetiole rounded or squared anteriorly; color mostly black or dark brownish. bothroponera pumicosa species complex description worker: the workers of the b. pumicosa species complex are very similar to each other. workers large, head shape excluding mandibles subquadrate or suborbiculate, slightly narrowed anteriorly; posterior border concave; mandibles triangular, shorter than head length, with 7 8 teeth, smooth, coarsely punctate or covered with fine striae in some species; anterior medial margin of clypeus convex, with single medial longitudinal carina (clypeal carina) in some species, “v” or “u” shaped margin anteriorly, sharp or blunt; carinal disc rounded, often divided by longitudinal furrow; lower margins of frontal lobes smooth and shiny; scape barely reaches or slightly passes posterior lateral corner of head; compound eyes relatively large; pronotal shoulder rounded anteriorly; basalar sclerite oval or rounded; mesonotum and propodeum fused (dorsal view); mesometapleural suture well developed; propodeum fig 2. the lateral view of an african bothroponera. 1, head; 2, mesosoma; 3, petiole; 4, gaster; a, frons; b, clypeus; c, frontal lobe; d, scape; e, malar space; f, posterior lateral corner of the head; g, mandible; h, eye; i, funicular segment; j, propleuron; k, pronotum; k1, anteroinferior pronotal process; k2, inferior pronotal process; k-m, promesonotal suture; l, mesopleuron; l1, epicnemial process; l-p, mesometapleural suture; m, mesonotum; n, propodeum; o, basalar sclerite; p, lateropropodeum; q, metapleural gland; r, propodeal spiracle; s, posteropropodeum; t, sternopetiolar process; u, petiole; v, sternopostpetiolar process; w, postpetiole; x, sting; (ii second, iii third, iv fourth, v fifth, vi sixth and vii seventh abdominal segments). sociobiology 62(4): 538-563 (december, 2015) 541 map 1: the terrestrial ecoregions of africa (burgess et al., 2004) used with permission from island press, washington, d.c. angulate, quadrate or rounded posteriorly, propodeal spiracle elongated; petiole well developed with petiolar spiracles and developed sternopetiolar process; sternopostpetiolar process well developed; metatibial gland absent; generally head punctate or coarsely foveolate; edges and bottom of frontal lobes shiny; head, body, legs, antennae, mandibles shiny or weakly striated; dorsum of pronotum, mesonotum, propodeum, petiole, postpetiole usually more coarsely sculptured than sides; entire body covered with scattered or moderately abundant short or long erect golden hairs, denser on dorsum than on sides and longer on mesosoma than on head; frontal lobes covered with fine hairs; color mostly black or dark brown. the females and males are unknown. key to the afrotropical bothroponera species complexes 1. metatibial gland present (fig 3); scape extends at least length of first funicular segment past posterior lateral corner of head; lower margin of anterior medial area of clypeus convex; frontal lobes subquadrate ......................................... ...................................................... b. sulcata species complex metatibial gland absent; scape shorter, barely reaches posterior lateral corner of head or extends past less than length of first funicular segment; lower margin of anterior medial area of clypeus convex, straight or slightly concave; frontal lobes rounded .......…….…………………………………………. 2 ama joma; wp mackay – bothroponera pumicosa species complex542 the legend for map 1 (the terrestrial ecoregions of africa, burgess et al., 2004), used with permission from island press, washington, d.c. 2(1). anterior medial margin of clypeus convex, “u” or “v” shaped; eyes relatively large (eye width 0.30 – 0.45 mm, eye length 0.30 – 0.50 mm and malar space area length 0.38 – 0.75 mm) .....……………………… b. pumicosa species complex anterior margin of clypeus straight or slightly concave or convex but not “v” or “u” shaped; eyes relatively small (eye width 0.10 – 0.35 mm, eye length 0.11 – 0.45 mm and malar space area length 0.12 – 0.51 mm).....b. talpa species complex. fig 3. the metatibial ggland on the posterior leg of bothroponera crassior. f, femur; ti, tibia; mg, metatibial gland; ps, pectinate spur; ta, basitarsus. sociobiology 62(4): 538-563 (december, 2015) 543 key to the bothroponera pumicosa species complex based on the workers 1. hairs on entire mesosoma and gaster long (up to 0.55 mm) curly, anterior medial raised area of clypeus “u” shaped without carina ………………………………….... pumicosa hairs on entire surface relatively short (0.10 – 0.25 mm) erect moderately long, straight (not curly); anterior medial raised area of clypeus “u” or “v” shaped with or without carina ………………...........………………………………. 2 2(1). anterior border of clypeus “u” shaped, broadly rounded ………………………….……………..............................… 3 anterior border of clypeus “v” shaped with sharp anterior medial point …….....................….............……………..…. 6 3(2). anterior medial area of clypeus raised from surface (best seen in side view) to form sharp carina, which extends from between frontal lobes to anterior border of clypeus ...... 4 anterior medial area raised but does not form sharp carina, if carina partially present, not complete as described above or not sharp ………….…….……..…………………….……...…. 5 4(3). posterior border of petiolar node (seen from above) with deep medial depression; mandibles with several deep coarse grooves ...............…………..…………………cariosa posterior border of petiolar node with little evidence of impression; with smooth shiny mandibles .....……. strigulosa 5(3). body smooth, shiny, black, fourth abdominal segment smooth, shiny …………….…… laevissima body strongly sculptured by dense foveolae, fourth abdominal segment foveolate .....…………....……. berthoudi 6(2). anterior medial area of clypeus raised to form sharp carina …………………………........................……. granosa anterior medial area raised but does not form carina..…....7 7(6). propodeal spiracle nearly horizontal; fourth abdominal segment smooth, partially glossy....................... umgodikulula propodeal spiracle nearly vertical, usually nearly parallel with posteropropodeum; fourth abdominal segment smooth to slightly rough or sculptured …….................................…. 8 8(7). head and mesosoma with sparse punctures, moderately shiny, black ……………………..............................… aspera head and mesosoma sculptured with dense foveolae ….... 9 9(8). scapes longer, extending slightly past posterior lateral corner of head (si 78), short (0.10 up to 0.20 mm) erect golden hairs cover entire surface ………………........…... cavernosa scapes barely reaching posterior lateral corner of head (si 79.59 – 81.25), short (0.10 – 0.15 mm) erect silver hairs cover entire surface .....………………….… montivaga list of species of the bothroponera pumicosa species complex bothroponera aspera arnold 1962 (stat. nov.), b. berthoudi forel, 1901 (= variolosa syn. nov.), b. cariosa emery, 1895 b. cavernosa roger, 1860 b. granosa roger, 1860 b. laevissima arnold, 1915 b. montivaga arnold, 1947 (stat. nov.), b. pumicosa roger, 1860 b. strigulosa emery, 1895 b. umgodikulula joma and mackay 2013 species accounts of members of the afrotropical bothroponera pumicosa species complex bothroponera aspera arnold, stat. nov. figures 4, 5 and plate 1; map 2. bothroponera laevissima var. aspera arnold, 1962: 844 (w) south africa, saldanha bay; joma and mackay, 2013: 3; schmidt and shattuck: 2014: 76; pachycondyla laevissima var. aspera: brown, in bolton, 1995: 303. diagnosis: the worker of bothroponera aspera is large (total length 12 13 mm). the mandibles are triangular, shorter than the head length, and smooth. the anterior medial margin of the clypeus is convex, with a single raised medial carina, the anterior margin of the clypeus is “v” shaped. the scape reaches the posterior lateral corner of head or surpasses it by a short distance. the lower margin of the pronotum is straight, rounded anteriorly (anteroinferior pronotal process) and posteriorly (inferior pronotal process). in general, the head is shiny, but rough with dense, shallow punctures. the pronotum, mesonotum, propodeum, mesopleuron, petiole, and postpetiole are shiny, but rough with dense, shallow punctures. the terga of the fourth seventh abdominal segments are mostly smooth and glossy while the entire remainder of the body is sculptured. worker description: (n=2 for measurements), hl 2.55 2.75, hw 2.30 2.50, ml 1.35 1.65, ew 0.35 0.40, el 0.40 0.45, sl 1.90 2.00, fl 2.35 2.80, wl 3.50 3.75, wpl 4.35 4.70, pl 1.10, pw 1.15 1.30, ph 1.40 1.45, ci 90.19 90.90, oi 17.39 18.00, mandi 52.94 60.00, si 80.00 82.60, peti 104.54 118.18. head suborbiculate; mandibles with 7 teeth; maximum clypeal length 2.15 mm; maximum frontal lobe width in full face view 1.00 mm; malar space from side 0.55 mm, length from upper margin of eye to upper margin of occipital lobe 1.25 1.30 mm; basalar sclerite oval shaped; propodeum rounded posteriorly; propodeal spiracle elongated, obliquely vertical; petiole rounded anteriorly, posterior face vertical, slightly concave posteriorly; pronotum, mesonotum, propodeum, mesopleuron, petiole, postpetiole shiny, densely punctulate; tergum of second gastral segment mostly smooth, glossy, entire remainder of body sculptured; ama joma; wp mackay – bothroponera pumicosa species complex544 entire body and head covered with scattered or moderately abundant short erect silver hairs (0.07 0.10 mm), erect hairs on petiole and postpetiole range from 0.10 0.15 mm, denser on dorsum than on sides, longer than on head, scape covered with short erect silver hairs (up to 0.07 mm); body black; legs, antennae, mandibles brownish. comparison: the worker of b. aspera is similar to the worker of b. laevissima; however, there are two main differences between them. the first difference is the body sculpturing, which is partially sculptured in b. aspera while it is less sculptured and glossy in b. laevissima. the head of b. aspera is shiny with dense punctures whereas it is shiny with few scattered shallow punctures in b. laevissima. the pronotum, mesonotum, propodeum, mesopleuron, petiole and postpetiole are shiny and densely punctulate in b. aspera. on the other hand, in b. laevissima, the pronotum, mesonotum, propodeum, mesopleuron, and petiole are rough and shiny with a few scattered punctures, but the postpetiole and the 4th abdominal segment along with 5th to 7th abdominal segments are smooth and glossy. the tergum of the second gastral segment is mostly smooth and glossy in b. aspera as well. secondly, the clypeal structure is different in the two species. the clypeus is “v” shaped in b. aspera and does not form a carina on the medial raised area, which is sculptured on the sides and without a grooved beak on the lower medial margin of the posteroclypeus whereas the clypeus in b. laevissima is “u” shaped. the lower margin of the medial raised area of the clypeus of b. laevissima does not form a carina, but the grooved beak on the lower margin of the clypeus is present. other than that, it is easy to distinguish both b. aspera and b. laevissima from the rest of the b. pumicosa species complex members. the black, smooth, and shiny surface with punctures is found only in b. aspera and b. laevissima. the other b. cavernosa species are characterized by having coarse foveolae on the body surface; however, the 4th abdominal segment is similar in some species to that of b. aspera and b. laevissima, such as b. umgodikulula, b. cavernosa and b. montivaga. there is a specimen from the south africa museum that was identified as bothroponera aspera which is quite similar to the paratype specimen of b. aspera. this specimen and the paratype of b. aspera are from the same locality (ysterfontein area), but the labels do not indicate if they are from the same nest. we distinguished it from the paratype specimen of b. aspera because it does not have the typical clypeal shape. this excluded specimen is possibly a new species of bothroponera that belongs to b. pumicosa species complex. it has broad and slightly convex lower margin of the clypeus. the anterior medial area of the clypeus is completely lacking the “v” and “u” shapes of the anterior medial area of the clypeus that were obvious in all of the other b. pumicosa complex members. however, further specimens are needed to evaluate this taxon. material examined type material: south africa: western cape province, ysterfontein, farmstead, 33°1’0’’ s; 18°9’0’’ e, dr. a. j. hesse and mr. thom; bothroponera laevissima var. aspera, det. g. arnold, south africa museum ex. national museum bulawayo 1981; sam-ent, 9:60 (1 w #11519, paratype [designator not specified] iziko). non-type material: south africa: western cape province, ysterfontein, 33°1’0’’ s; 18°9’0’’ e; bothroponera laevissima var. aspera, det. g. arnold; s. a. m. 9:60, possible new species (1 w #coo11519, iziko). distribution: bothroponera aspera is known only from saldanha bay, south africa. biology and habitat: bothroponera aspera has been collected from saldanha bay area, western cape province. the individuals were living in holes in the ground at ysterfontein (yzerfontein), in the southern part of the saldanha bay area (arnold, 1962). the habitat is characterized by fynbos biome vegetation. the bay is one of the richest areas in biodiversity in the western cape province. there are assemblages of several groups of organisms including benthic, intertidal, marine and plant species (anchor environmental consultants, 2006, 2012). this habitat has unique distinct flora and fauna that are identified as endemic species to the area, as well as organisms in need of conservation (schils et al., 2001; anchor environmental consultants, 2006, 2012). the other members of the b. pumicosa species complex that can be found in this province include b. laevissima, b. cavernosa, b. granosa and b. montivaga. plate 1: bothroponera aspera, paratype worker. sociobiology 62(4): 538-563 (december, 2015) 545 the sculpturing of the head is shown only on the right or left side, to allow the illustration of the hairs on the other side of the head. bothroponera berthoudi (forel) figures 6, 7 and plate 2; map 3 pachycondyla (bothroponera) berthoudi forel, 1901: 344 (w), south africa, valdezia, transvaal; emery, 1911: 76; forel, 1913a: 306 (m), willwomore, colonie du cap; pachycondyla (bothroponera) pumicosa berthoudi: forel, 1913b: 109 (w), willowmore [willwomore], cap; bothroponera pumicosa race berthoudi: arnold, 1952: 460, considered berthoudi to be a junior synonym of strigulosa; pachycondyla berthoudi: brown in bolton, 1995: 303; bothroponera berthoudi: joma and mackay, 2013: 3. bothroponera variolosa arnold, 1947: 131 (w), south africa, mariepskop, transvaal; bothroponera cariosa variolosa: arnold, 1960: 452; bothroponera variolosa: joma and mackay, 2013: 3; schmidt and shattuck, 2014: 77; pachycondyla variolosa: brown, in bolton, 1995: 311 (syn. nov.). diagnosis: the worker of bothroponera berthoudi is a relatively large ant (total length 9.60 – 12.00 mm). the mandibles are hairy and coarsely covered with punctures. the anterior medial margin of the clypeus is “u” shaped and slightly bent ventrally, with the disc has a raised smooth area with striae. the upper part of the raised area, between the frontal lobes is rough with a few punctulae, and with a small clypeal carinae. the clypeal wings are punctulate and obliquely striate. the scape barely reaches the posterior lateral corner of the head. worker description: (n=8 for measurements), hl 2.00 2.61, hw 1.75 2.25, ml 1.15 1.45, ew 0.30 0.35, el 0.30 0.45, sl 1.40 1.85, fl 2.10 2.60, wl 2.75 3.85, wpl 3.60 4.90, pl 0.90 1.30, pw 0.95 1.35, ph 1.30 1.60, ci 86.20 87.50, oi 17.14 20.00, mandi 57.50 55.56, si 80.00 82.22, peti 103.84 105.56. mandibles smooth with about 7 teeth; head subquadrate; maximum transversal clypeal length 1.60 mm; compound eyes relatively large; length of malar space 0.40 mm; length from upper edge of eye to edge of posterior lobe 1.00 mm; maximum frontal lobes width 0.75 mm; surface of head, pronotum, mesonotum, mesopleuron, propodeum, lateropropodeum, metapleuron, densely foveolate, moderately shiny; petiolar and postpetiolar surfaces densely covered with larger foveolae than those of mesosoma, moderately shiny; cheek, sides of head, area posterior to eyes, frons covered with weakly defined striae; dorsum of fourth abdominal segment covered with shallow foveolae and striae; fifthseventh abdominal segments smooth, shiny; basalar sclerite oval in depressed surrounding area; pronotal shoulder rounded, lower margin straight (lateral view), anteroinferior pronotal process angled, inferior pronotal process rounded; mesometapleural suture developed; mesopleural-coxal excavation developed and continued with mesometapleural suture; antennae, legs, lower edges of frontal lobes, mandibles shiny; anterior face of petiolar node from dorsal view rounded, slightly narrowed anteriorly, posterior face vertical in side view, slightly concave with slight depression on medial upper margin; metapleuron rough, covered with fig 4. the lateral view of a paratype worker of bothroponera aspera (iziko). fig 5. the head of the paratype worker of b. aspera (iziko). fig 6. the head of the worker of b. berthoudi from south africa, eastern cape province, algoa-bay (mfn). fig 7. the lateral view of a worker of b. berthoudi from south africa, eastern cape province (mfn). map 2. the distribution of b. aspera. ama joma; wp mackay – bothroponera pumicosa species complex546 striae, foveolae; posteropropodeum rough, slightly concave; dorsum of postpetiole densely covered with large foveolae and striae; surface of 4th abdominal segment rough, covered with large foveolae; 5th to 7th abdominal segments moderately shiny, covered with fine striae; head, dorsum of pronotum, mesonotum, propodeum covered with fine moderately long golden erect hairs; hairs moderately long (0.12 mm up to 0.25 mm) hairs on mandibles, legs, scapes; length of hairs on pronotum, mesonotum, propodeum slightly longer (0.20 0.25 mm); dorsum of petiole, dorsal and ventral surfaces of postpetiole, and 4th to 7th abdominal segments covered with longer erect golden hairs (0.30 0.35 mm); head, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster black; legs and antennae brownish black; mandibles reddish brown. comparison: arnold (1952) considered b. berthoudi to be a race of b. pumicosa, but it is clear that this species is different from b. pumicosa in that b. pumicosa has long hairs that cover the entire body, which is not the case in b. berthoudi. there are other slight differences between the two species. the anterior medial raised area of the clypeus of b. pumicosa forms a partial carina on the posterior part and a smooth narrowed area on the grooved anterior part. the same character is found in b. berthoudi, but the upper part forms striae instead of a partial carina, the lower part is smooth but wider than that of b. pumicosa. the mandibles have 7 teeth in b. berthoudi, similar to the other b. pumicosa species complex members, whereas b. pumicosa has 8 teeth. bothroponera laevissima has the same “u” shaped clypeus as does b. berthoudi, but is easily recognized by the unique sculpture: shiny with scattered punctures. the mandibles have 7 teeth in b. berthoudi and b. laevissima, similar to the other b. pumicosa species complex members. the body surface of b. berthoudi is densely foveolate, and the fourth abdominal segment is densely covered with foveolae and striae. basically, the members of the type series consistently differ from the rest of b. pumicosa species complex species in having long erect hairs on most surfaces (except the head) and in lacking a well-defined medial clypeal carina. the specific epithet “berthoudi” was first used by forel (1890) as a name for ophthalmopone berthoudi from south africa. later, he used it again to identify and describe the male of ophthalmopone berthoudi. wheeler and wheeler (1971) used the same name to identify larvae of o. berthoudi. in 1901 forel used the same specific epithet to describe pachycondyla (bothroponera) berthoudi. pachycondyla (bothroponera) berthoudi has been considered to be a secondary homonym of pachycondyla (ophthalmopone) berthoudi, as they were both placed in the same genus (bolton, 1995), which has caused considerable confusion. in this project, we clearly distinguish between o. berthoudi and b. berthoudi as these now belong to two different genera (schmidt & shattuck, 2014). the worker of b. berthoudi is identical to the worker of b. variolosa, which is considered a synonym. they have the lower medial margin of the clypeus “u” shaped with a medial raised area and lack the sharp carinae. bothroponera cariosa and b. strigulosa have the same “u” shaped lower medial anterior margin of the clypeus, but this area forms a sharp clypeal carinae in both species, which is lacking in b. berthoudi. plate 2: bothroponera berthoudi worker. material examined type material: south africa: mpumalanga province, salique, a. r. i. pretoria a. c. u. p. 314 (forests), 24°36’0’’ s; 30°54’0’’ e, vii-1944, donated by univ. pretoria, 14 mar 1995, bothroponera variolosa (arnold, 1947) (1w paratype # c008009, sam-hym [iziko]); limpopo province, marieps, a.r.i. pretoria a. c. u.p. 367 (farmstead and forests), 24°35’0’’ s; 30°52’0’’ e, vii-1944, bothroponera variolosa n. sp. (arnold, 1947), (1w paratype, sam-ent [iziko # 0011524]); mariepskop transvaal, (farmstead), 24°35’0’’ s; 30°52’0’’ e, vii-1945, j. c. faure, bothroponera variolosa sociobiology 62(4): 538-563 (december, 2015) 547 (arnold, 1947), (2w # 0011524 paratypes, sam-ent [iziko], british museum 1947-250, bmnh (e) 1015515), antweb casent 0902470 (2w paratypes, bmnh). non-type material: south africa: eastern cape province, algoa-bay, capland, 33°50’0’’ s; 25°50’0’’ e, dr. h. brauns, forel det. 1922, that deposited in the berlin museum was designated by forel, (labeled pachycondyla (bothroponera) berthoudi forel, 1w # 6692, mfn), but this specimen is broken into two parts (head with the pronotum is one part and the second part includes the mesonotum, mesopleuron, propodeum, petiole, postpetiole and the 2nd to 5th gastral segments). it is not clear if this specimen is the type or not. limpopo province, mariepskop, 4000 transvaal (farm), 26°42’0’’ s; 29°53’0’’ e, vii-1944; south africa museum ex. national museum bulawayo 1981, (labeled bothroponera variolosa, 1w # 11524 sam-ent); some of the b. berthoudi specimens that were collected by h. brauns, paul berthoud and g. arnold were considered to be subspecies of b. strigulosa. distribution: the species is known from salique in the mpumalanga province, mariepskop and valdezia in the limpopo province and from algoa-bay in the eastern cape province of south africa. bothroponera berthoudi specimens were collected from other localities in south africa such as valdezia (locality of the type specimen), cape willowmore (forel, 1913a, 1913b), cape nordhoek and east griqualand (arnold, 1952). biology and habitat: the type specimen was collected from valdezia, limpopo province, which is far from the eastern cape province where the other material was located. the habitat in limpopo province is mainly covered with savanna biome (mucina & rutherford, 2008; dubel integrated environmental services, 2009). the additional material examined of b. berthoudi was collected from algoa bay, which is located at the east of the cape of good hope, on the southeastern coast of south africa. the bay area is characterized by two seasons of rain, winter and summer (goschen & schumann, 1988). the fynbos and thicket biomes are the major vegetation types that cover the algoa bay area. the collection site is in mpumalanga province close to the limpopo province that are both covered mainly with savanna. the mariepskop, limpopo province includes two continuous nature reserves: blyde river and motlatse canyon provincial nature reserves. they are covered with grassland and savanna biomes in both provinces, close to the kruger national park, the vast area that shared between south africa and mozambique. savanna is the major biome of limpopo province where the type specimens were collected (goschen and schumann, 1988; mucina and rutherford, 2008; dubel integrated environmental services, 2009). these specimens were mainly collected from farms, farmsteads and forest habitats (information from labels and from google maps). map 3. the distribution of b. berthoudi. fig 8. the lateral view of the holotype worker of b. cariosa (mcsn). fig 9. the head of the holotype worker of b. cariosa (mcsn). fig 10. the head of the holotype worker b. cavernosa showing sculpture on the left side of the head (mfn). fig 11. the lateral view of the holotype worker of b. cavernosa (mfn). bothroponera cariosa emery figures 8, 9 and plate 3; map 4 bothroponera cariosa emery, 1895: 20 (w), mozambique, delagoa-bay; wheeler, w.m. 1922: 769; wheeler, g. c. and wheeler, j. 1971: 389 (l, semipupa); joma and mackay: 2013: 3; schmidt and shattuck, 2014: 76; pachycondyla (bothroponera) cariosa, emery, 1901: 45 list; arnold, 1915: 59 (w); pachycondyla cariosa: brown in bolton, 1995: 303. ama joma; wp mackay – bothroponera pumicosa species complex548 diagnosis: the total length of the worker b. cariosa is 10.15 11.50 mm. the head is subquadrate. the anterior medial border of the clypeus is “u” shaped with the medial longitudinal clypeal area raised to form a sharp carina. the mandibles are partially covered by weakly defined striae with scattered coarse punctures. the scape does not reach the posterior lateral border of the head. worker description: (n=2 for measurements), hl 2.25 2.50, hw 1.95 2.15, ml 1.25 1.40, ew 0.30, el 0.35 0.40, sl 1.45 1.60, fl 2.45 2.75, wl 2.90 3.35, wpl 3.80 4.40, pl 1.15 1.25, pw 1.20 1.45, ph 1.25 1.50, ci 86 87, oi 17.94 18.60, mandi 55.56 – 56.00, si 74, peti 104 – 116. maximum clypeal width 1.90 mm; mandibles with 7 teeth, partially covered by weakly defined striae with scattered coarse punctures; maximum frontal lobe width 0.95 mm; length of malar space 0.40 0.60 mm, length from upper edge of eye to edge of posterior lobe 1.20 1.55 mm; compound eyes relatively large; head coarsely foveolate; lower margin of pronotum straight with rounded inferior and anteroinferior pronotal processes; basalar sclerite oval-shaped; antennae, legs, mandibles shiny; petiole thick, anterior face and apex rounded, seen from above slightly narrowed anteriorly with depression on upper medial margin between two edges posteriorly (seen from above), posterior face vertical (side view), slightly concave; sternopetiolar process developed with one tooth pointed ventrally; entire surface covered with fine scattered suberect to erect hairs, including mandibles, scapes; head covered with short (0.05 0.10 mm) erect golden hairs, pronotum, mesonotum, propodeum covered with short (0.05 0.15 mm) erect golden hairs, petiole, postpetiole, entire gaster covered with longer (0.15 0.25 mm) erect golden hairs; head, entire mesosoma (pronotum, mesonotum, mesopleuron, propodeum), petiole, postpetiole, entire gastral segments black; appendages (legs, antennae, mandibles) reddish brown; clypeus dark-brown; funiculus brown. comparison: bothroponera cariosa is similar to many other species of the b. pumicosa species complex with a “u” shaped anterior medial margin of the clypeus (e.g. b. berthoudi, b. strigulosa, b. pumicosa, and b. laevissima), but the anterior medial area of the clypeus is developed into a sharp longitudinal carina, which is similar to that of b. strigulosa. the sharp carina is partially present in b. pumicosa while it is absent in b. berthoudi and b. laevissima. bothroponera granosa is the other species in the b. pumicosa species complex that has the clypeus raised to form a sharp carina, but the anterior medial margin of the clypeus is “v” shaped. the “v” shaped clypeus is also present in b. aspera and b. umgodikulula, but both b. aspera and b. umgodikulula lack the carina and the surface of the disc is smooth and rounded. this area is formed into partial carinae in b. montivaga and b. cavernosa. the petiole viewed from above is similar to that of b. berthoudi, but is definitely indented posteriorly in b. cariosa, but nearly straight and not indented in b. berthoudi. bothroponera cariosa has total length about 11.50 mm, which is longer than that of b. berthoudi (9.60 12 mm) while it is smaller than that of b. cavernosa (12 mm), b. montivaga (12.20 12.65 mm), b. granosa (13.75 14.50 mm), b. strigulosa (12.20 mm), b. umgodikulula (14.80 15.65 mm), b. laevissima (12 13.00 mm), and b. aspera (12 13 mm). the total length of bothroponera pumicosa ranges from 11.00 11.65, which overlaps the total length of b. cariosa. material examined type material: mozambique: delagoa bay, mountains, 25°59’0’’ s; 32°42’0’’ e, type, bothroponera cariosa emery, holotypus pachycondyla cariosa emery 1895, museo geneva coll. c. emery (1w holotype, mcsn). non-type material: south africa: eastern cape province, grahamstown, 33°18’0’’ s; 26°32’0’’ e, f. jacotguillarmod, highlands rd, w. grahamstown grassy grove, b. cariosa em. wlb 1973, compared with type (1w, bmnh). distribution: bothroponera cariosa is distributed in south africa, tanzania, gabon (ant web, accessed may 2013) and mozambique (emery, 1895). workers were collected from the cape province; transvaal area, south africa by g. arnold (arnold, 1915). biology and habitat: the bothroponera cariosa holotype was collected from delagoa bay, mozambique. this area is located at the southeast coast of mozambique, near the south african border, on the coast of the indian ocean, east africa. plate 3: holotype worker of b. cariosa. sociobiology 62(4): 538-563 (december, 2015) 549 delagoa bay is the former name of maputo bay. the climate in the south of mozambique is semi-arid and subtropical while it is tropical in the north; the southern areas of the country are generally drier than the northern areas and have fluctuations in temperature and rainfall (country briefs web page, accessed may 2013). the country has one rainy and one dry season per year. the habitat in mozambique is characterized by forest ecosystems that increase in elevation, especially close to zimbabwe border and are also characterized by grassland ecosystems. the mangroves grow in the swamps and there are palm trees on the coast. these types of ecosystems most likely hold various species of ants, including bothroponera (based on bolton, 1994, 1995, 2012; ant web, accessed january 2012; ant wiki, accessed may 2013). mainly, this species can be found in habitats that are characterized by high humidities and wet soils, which is the typical environment of the tropical and subtropical areas. it builds nests underground or under stones to form colonies with a small number of individuals (wheeler, 1922; wheeler & wheeler, 1971). the ant biodiversity in mozambique is high where we can find the following species: megaponera crassicornis, paltothyreus tarsatus delagoensis, bothroponera strigulosa, b. kruegeri, b. talpa besides b. cariosa (bolton, 1994, 1995, 2012; ant web, accessed january 2012). the recent ants collected from mozambique, by dr. gary alpert, are deposited in the mczc. this collection includes variable species that belong to several genera of subfamily ponerinae such as bothroponera, hypoponera, leptogenys, megaponera, mesoponera, odontomachus, platythyrea, paltothyreus and plectroctena (based on a personal visit to the mczc in 2013). bothroponera cariosa is also found in cape province, transvaal. the transvaal area is located at the north of vaal river and extends to the borders of botswana, zimbabwe, mozambique and swaziland. bothroponera cavernosa (roger) figures 10, 11 and plate 4; map 5 paraponera cavernosa roger, 1860: 288 (w) south africa, kaffernlande; pachycondyla (bothroponera) cavernosa: emery, 1901: 45 (list); arnold, 1915: 60 (w) cape; bothroponera cavernosa: mayr, 1862: 717; wheeler, 1922: 769, south africa, caffraria, cape province (w); joma and mackay: 2013: 3; schmidt and shattuck, 2014: 76; pachycondyla cavernosa: brown, in bolton, 1995: 304. diagnosis: the main distinguishing character of the worker b. cavernosa is the lack of foveolae on the second tergum of the gaster, which is rough and covered with short hairs. the worker is large (total length 12 mm). the anterior margin of the clypeus is “v” shaped and covered with fine striae. the anterior medial area of the clypeus is raised, covered with longitudinal striae and coarsely punctate on the sides with an incomplete clypeal carina. the mandibles are rough, moderately shiny and covered with striae. the scape extends slightly past the posterior lateral border of the head. the frontal lobes are sculptured and covered with striae. the frons is weakly striated. the propodeal spiracle is parallel to the posteropropodeal margin. the petiole is rounded and slightly narrowed anteriorly while it is vertical, slightly concave posteriorly (seen from above). worker description: (n=1), hl 3.00, hw 2.70, ml 1.45, ew 0.40, el 0.45, sl 2.10, fl 3.60, wl 4.15, wpl 4.70, pl 1.15, pw 1.45, ph 1.75, ci 90, oi 17, mi 48.33, si 78, pi 126.08. head suborbiculate; mandibles covered with fine striae, with 7 teeth; clypeus covered with striae, anterior medial area raised to form discontinuous carina, coarsely punctate and rough on sides, clypeal length 2.35 mm; scape extends slightly past posterior border of head; maximum frontal lobe width 1.10 mm; length of malar space 0.55 mm; length from upper edge of eye to edge of posterior lobe 1.40 mm; pronotal shoulder rounded anteriorly, lower margin of pronotum straight with rounded anteroinferior pronotal process, pointed inferior pronotal process; basalar sclerite oval shaped; head mostly foveolate; antennae rough, scape covered with tiny shallow punctures, legs shiny; entire dorsum of mesosoma foveolate and rough; mesopleuron, lateropropodeum foveolate; metapleuron, lateropropodeum covered with coarse striae and grooves orientated perpendicular to posteropropodeal margin; dorsum of petiole and postpetiole coarsely foveolate and more punctate than other body parts; metapleuron and lateropropodeum covered with striae that have perpendicular orientation with posteropropodeal lateral margin; entire head, pronotum, mesonotum, propodeum, petiole, postpetiole covered with short erect golden hairs (up to 0.20 mm), on head, antennae, mandibles (0.03 0.08 mm in length), on pronotum, mesonotum, propodeum (up to 0.10 mm length), on petiole, postpetiole (0.13 0.15 mm in length); sternopostpetiolar process and 4th to 7th abdominal segments covered with relatively short (0.20 mm) erect golden hairs; head, mesosoma, petiole, 3rd 7th abdominal segments black; mandibles, clypeus, appendages brownish red.map 4. the distribution of b. cariosa. ama joma; wp mackay – bothroponera pumicosa species complex550 comparison: bothroponera cavernosa is very easy to recognize as it is one of the five species in the b. pumicosa species complex with a specific form of the tergum of the 4th segment of the abdomen (2nd gastral tergite), that is rough and covered with short hairs. the other species are bothroponera laevissima, b. aspera, b. umgodikulula and b. montivaga. the 2nd gastral segment of b. umgodikulula is mostly smooth and glossy, similar to that of b. aspera and b. laevissima, while in b. montivaga it is smooth with few shallow scattered punctures and is moderately shiny. the head shape of b. cavernosa, b. aspera, and b. laevissima is suborbicular while it is subquadrate in b. umgodikulula and b. montivaga. the other important differences between b. cavernosa, b. umgodikulula and b. montivaga compared to b. laevissima and b. aspera is that the body surface is heavily sculptured with foveolae in b. cavernosa, b. umgodikulula and b. montivaga while it is black, nearly smooth and shiny in b. laevissima and b. aspera with a few scattered punctures in b. aspera. bothroponera cavernosa and b. umgodikulula both share all of the characteristics of b. montivaga except for the propodeal spiracle, which is obliquely vertical in b. montivaga and b. cavernosa while it is horizontal in b. umgodikulula. the anterior medial area of the clypeus is raised and does not form a complete clypeal carina in b. cavernosa (it is partially carinated). on the other hand, the anterior medial area of the clypeus of b. umgodikulula and b. montivaga is mostly smooth, and does not form carinae and in some specimens of b. montivaga the carina is only on the upper part of the anterior medial raised area of the clypeus while the lower part is smooth. material examined type material: south africa: eastern cape province, caffraria drege, farm, 27°48’0’’ s; 25°7’0’’ e, bothroponera cavernosa roger, mayr (1 w # 7165 gbif-d/ focol 0955, holotype, mfn). non-type material: although we requested material from several collections, the holotype was the only specimen available for this study. distribution: south africa. biology and habitat: the type specimen is known from kaffernlande, south africa. kaffernlande is the former name of what is known today as the transkei and ciskei regions (transkei district), eastern cape province (dr. worden, personal communication). the habitat in this area is characterized by three types of biomes: grassland, savanna and thicket. this indicates the high biodiversity and different habitats that are available to the organisms in the area, which could result in high speciation rates. in fact, most of the species in the b. pumicosa species complex species were found in south africa. caffraria or kaffraria also is a descriptive name that was given to the southeast part of what is called today the eastern cape of south africa. the material examined was collected in a farmland area in caffraria. wheeler (1922) and wheeler and wheeler (1971) reported that this species, as well as b. pumicosa and b. cariosa, are usually found in colonies with a small number of individuals under stones in humid habitats and wet soils. plate 4: bothroponera cavernosa, holotype worker. map 5. the distribution of b. cavernosa. sociobiology 62(4): 538-563 (december, 2015) 551 bothroponera granosa (roger) figures 12, 13 and plate 5; map 6 ponera granosa roger, 1860: 290 (w) south africa, cape of good hope; bothroponera granosa: mayr, 1862: 717; dalla torre, 1893:36; wheeler, w. m. 1922: 770; joma and mackay: 2013: 3; schmidt and shattuck, 2014: 76; pachycondyla (bothroponera) granosa: emery, 1901: 45; arnold, 1915: 61(w); pachycondyla granosa: arnold, 1926: 201 (m); brown, in bolton, 1995: 305; bolton, 2013. diagnosis: the head of the worker is large and subquadrate. the mandibles are covered with hairs and partially by fine weakly defined striae and scattered coarse punctures. the anterior medial margin of the clypeus is “v” shaped with a raised sharp medial longitudinal carina. the scape reaches the posterior lateral corner of the head. the lower margin of the pronotum is straight with a strongly rounded angle at the anteroinferior pronotal process (lateral view), rounded inferior pronotal process and sometimes forming a sharp angle that is pointed posteroventrally. the petiole (dorsal view) is rounded and slightly narrowed anteriorly while it has a slight concavity on the upper medial margin between the two posterior angles of the petiolar apex. the posterior edge of the petiole is vertical (side or top view) and slightly concave (dorsal view). the sternopetiolar process is developed with a single tooth pointed ventrally. worker description: (n= 20), hl 2.90 3.10, hw 2.40 2.75, ml 1.40 1.75, ew 0.30 0.40, el 0.40 0.50, sl 1.90 2.35, fl 2.90 3.50, wl 3.90 4.45, wpl 4.80 5.40, pl 1.20 1.30, pw 1.40 1.60, ph 1.50 1.80, ci 83 89, oi 17 18.18, mandi 48.27 56.45, si 79.16 85.45, peti 117 – 123. total length 13.75 14.50 mm; mandibles with 7 teeth; clypeal length 1.70 2.35 mm; malar space length from lower edge of eye to base of mandible 0.40 0.65 mm; from upper edge of eye to edge of posterior lobe 1.35 1.70 mm; frontal lobe width 0.90 1.15 mm; metapleuron rough and weakly punctate; head covered with small dense punctures; sides of head covered with fine striae; dorsum of pronotum, mesonotum, mesopleuron, lateropropodeum and propodeum moderately punctate, moderately shiny; metapleuron rough, weakly punctate; petiole covered with deeper sparse punctures and foveolae than postpetiole; postpetiole covered with shallow sparse punctures, foveolate, with weakly defined fine striae; dorsum of second gastral segment covered with shallow foveolae and striae; remaining gastral segments shiny; lower margin of pronotum straight with strong angle at anteroinferior pronotal process, rounded inferior pronotal process; antennae, legs, lower edge of frontal lobes, mandibles shiny; entire surface of b. granosa worker covered with fine short (up to 0.15 mm) silver hairs, denser on second seventh gastral segments (up to 0.20 mm); entire surface covered with scattered erect golden hairs, including mandibles, scapes; head, pronotum, mesonotum; propodeum covered with short erect golden hairs (0.10 0.15 mm); dorsum of petiole covered with short erect golden hairs (0.15 mm); postpetiole, entire gaster covered with longer erect plate 5: bothroponera granosa, worker. (from www.antweb.org. accessed 23 february 2015). golden hairs (0.15 0.20 mm); hairs on sternum of postpetiole, hairs on other gastral segments reach about 0.30 mm. head, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster black; legs, antennae, clypeus, mandibles brownish black. comparison: the worker of b. granosa can be recognized by the anterior medial raised area of the clypeus (“v” shaped anterior border) that forms a sharp longitudinal carina, which differs from most of the other b. pumicosa species complex members (except b. cariosa and b. strigulosa). the anterior medial area of the clypeus is also “v” shaped in b. cavernosa, b. montivaga, b. aspera and b. umgodikulula, but without a carina in b. aspera and b. umgodikulula while it is partially carinate in b. cavernosa and b. montivaga. the other species, including b. cariosa, b. strigulosa, b. pumicosa, b. laevissima and b. berthoudi have an anterior medial raised area of the clypeus with a “u” shaped anterior border, however b. cariosa and b. strigulosa have a sharp carinae similar to b. ama joma; wp mackay – bothroponera pumicosa species complex552 granosa. the surface of b. granosa is more likely to be rough with moderately scattered punctures than that of the other b. pumicosa species complex taxa, which are always coarsely foveolate except for b. aspera and b. laevissima, which are shiny black with punctate sculpture. the petiolar shape of b. granosa is unique among the b. pumicosa species complex individuals even if the petiolar indices seem not to be separable. the petiole is rounded and slightly narrowed anteriorly (dorsal view) in all species of the b. pumicosa species complex including b. granosa, but the posterior face is deeply depressed from the upper edge to form two rounded apices found only in b. granosa (best seen from above). the petiolar indices vary among the b. pumicosa species complex members in that the smallest peti recorded were for b. aspera (104.54 118.18), and b. berthoudi (105.55), whereas the largest peti registered was for b. montivaga (130.00). the other species have intermediate peti, b. umgodikulula (115 126), b. granosa (117 123), b. strigulosa (117.39), b. laevissima (118.18 121.05), b. pumicosa (120.00 125.00), b. cariosa (104 116), and b. cavernosa (126.08). material examined type material: the types were not found, therefore, we used specimens determined by forel and bolton to define this species. south africa: kwazulu-natal province, natal, broughton, (farm) 29°26’0’’ s; 30°27’0’’ e, wm. m. wheeler collection, pachycondyla (bothroponera) granosa roger (1 worker, amnh, one worker specimen from mfn determined by forel in 1922. south africa: george [cgcorge kocysica browns], pachycondyla (bothroponera) granosa roger, forel det. 1922, zool. mus. berlin (1w mfn and two specimens from lacm that were determined by bolton in 1977). eastern cape province, highland rd., w. grahamstown grassy grove, 33°18’0’’ s; 26°32’0’’ e, 22x-1966, f. jacot-guillarmod, collection of w. s. creighton purchased by lacm 1974 (2 workers # 315919 lacm). one specimen (1 worker # 315920) from the los angeles county museum determined by forel with no further information. this specimen was in collection of w. s. creighton, but was purchased by the los angeles county museum in 1974. non-type material: south africa: eastern cape province, highlands rd. w., grahamstown grassy grove, 33°18’0’’ s; 26°32’0’’ e, f. jacot guillarmod (3w mczc, 1w cwem), coldsprings, grahamstown, under stone, 33°18’0’’ s; 26°32’0’’ e, 9-viii-1964, c. jacot-guillarmod (1w mczc), near highlands farm sw of grahamstown c. p., 33°18’0’’ s; 26°32’0’’ e, 22-x-1966, l. h. weatherill, anic ants vial 14.164, ent. 315917, 315918 (4w lacm), 27 km nw of cathcart, 32°18’0’’ s; 27°8’0’’ e, n. g. robertson, 16-ix1985, c46, h. rock (2w bmnh); kwazulu-natal province, estcourt natal, 29°0’0’’ s; 29°53’0’’ e (r.c.w.) 1914, g. arnold, arnold coll. b. m. 1934-354, pachycondyla pumicosa roger det. b. bolton 1977 (1w # 315925 lacm). natal, drakensberg, 29°0’0’’ s; 29°0’0’’ e, 2200m, 1983, c. peeters, giant’s castle dra (p) io (3w, bmnh). distribution: most of the specimens of bothroponera granosa were collected from south africa, including the eastern cape and kwazulu-natal provinces. they were also collected from the cape of good hope (roger, 1860), natal area (forel, 1901) and from cape knysna and cape majuba nek areas (arnold, 1926). some specimens were collected from victoria falls, zimbabwe (arnold, 1926). biology and habitat: bothroponera granosa inhabits the grassy grove areas of west grahamstown in south africa, under stones. it can also be found in rocky habitats such as the area north west of cathcart city in south africa (label information). the three specimens from natal, drakensberg and that from kwazulu-natal province, estcourt natal, south africa were misidentified as b. pumicosa. arnold (1926) reported that b. granosa was also taken at victoria falls located on the border between zimbabwe and zambia, which is far from the south africa collection sites. the habitat at victoria falls is similar to that at the town of knysna which has the knysnarivier stream (river) and the areas are covered with deciduous forests. the stream is connected with the indian ocean at the extreme southern shores of south africa. the material examined was collected from a grassy grove and farmland habitats (information from labels and google earth maps). one specimen was collected from cape province, south africa by f. jacot-guillarmod, misidentified in the mczc as b. cariosa. the western province, kwazulu-natal province and eastern province include several other species that belong to the b. pumicosa species complex: b. montivaga, b. aspera and b. laevissima found in the western cape province, b. umgodikulula, b. cariosa, b. granosa, b. cavernosa, b. berthoudi and b. pumicosa found in eastern cape province. bothroponera granosa was also collected from knysna, western cape province and majuba nek, eastern cape province (2 workers and one male) as material examined (arnold, 1926). map 6. the distribution of b. granosa. sociobiology 62(4): 538-563 (december, 2015) 553 bothroponera laevissima (arnold) figures 14, 15 and plate 6; map 7 pachycondyla (bothroponera) laevissima arnold, 1915: 58 (w) south africa, cape province, saldanha bay; bothroponera laevissima: wheeler, w.m. 1922: 73; joma and mackay: 2013: 3; schmidt and shattuck, 2014: 76; pachycondyla laevissima: brown, in bolton, 1995: 306 diagnosis: the workers of bothroponera laevissima are large (total length 12 13 mm) shiny black ants. the mandibles are shorter than the head length and covered with fine striae. the anterior medial raised area of the clypeus is convex, but lacks a longitudinal carina; the anterior border is “u” shaped. the lower margin of the clypeus has a short grooved beak on the lower margin of the posteroclypeus. the metapleural area is compressed in some specimens. the head is smooth and shiny with few punctulae scattered on the surface. the pronotum, mesonotum, mesopleuron, propodeum, petiole and postpetiole are smooth and shiny with a few scattered punctulae. the petiole is more sculptured than the other body parts. the second gastral tergite is smooth and glossy. worker description: (n=9), hl 2.90 3.10, hw 2.50 2.65, ml 1.55 1.70, ew 0.35, el 0.35 0.40, sl 1.95 2.15, fl 2.95 3.10, wl 3.65 3.95, wpl 4.40 4.85, pl 0.95 1.10, pw 1.15 1.30, ph 1.35 1.55, ci 85.48 86.20, oi 14.00 15.09, mandi 53.44 54.83, si 78.00 81.13, peti 118.18 121.05. head suborbiculate; mandibles with 7 teeth; clypeal length 2.15 2.35 mm; frontal lobe width 0.90 1.05 mm; scape nearly reaches posterior lateral corner of head; compound eyes relatively large; malar area length 0.60 0.75 mm, length from upper margin of eye to upper margin of posterior corner of head 1.25 1.45 mm; area around basalar sclerite depressed; basalar sclerite oval; propodeum rounded; propodeal spiracle elongated, diagonal on lateropropodeum; sternopostpetiolar process well developed; edges and anterior part of frontal lobes shiny; entire body covered with scattered or moderately abundant short erect silver hairs (0.07 0.10 mm), hairs on dorsum denser than those on sides, longer than those on head, similar hairs on petiole, postpetiole, range from 0.10 0.15 mm; entire body black; legs, antennae and mandibles brownish. comparison: the worker of b. laevissima can be recognized as a shiny black ant. the general characters of the worker of b. laevissima are similar to those of the worker of b. aspera, but the lower margin of the anterior medial raised area of the clypeus has a “u” shape in b. laevissima while it has “v” shape in b. aspera. the lower margin of the clypeus forms a grooved beak in b. laevissima, which is not found in b. aspera. despite that they both have a shiny surface and black color, b. laevissima is characterized by a smooth head with few scattered punctulae, similar to the pronotum, mesonotum, mesopleuron, propodeum, petiole and postpetiole. the petiole is more sculptured than other body surfaces; the second gastral tergite is smooth. conversely, b. aspera is recognized as rough with dense shallow punctulae on the body surface (head, pronotum, mesonotum, mesopleuron, propodeum, petiole and postpetiole), the tergum of the second-fifth gastral segments are mostly smooth and glossy. due to the mostly polished sculpture, b. laevissima would not be confused with any other species except b. aspera. bothroponera laevissima was collected from saldanha bay, western province area of south africa, where b. aspera also occurs, which further suggests they are separate species. material examined type material: south africa: western cape province, saldanha bay, 33°1’0’’ s; 17°57’0’’ e, ix-1912, l. p., rhodesian museum, south africa museum ex. national museum bulawayo 1981, pachycondyla laevissima g. arnold, sam-ent 11518, 11517, bothroponera laevissima (4w, lectotype marked with red dot and 3 paralectotype worker without dot [here designated], iziko). leg. f. peringuey; ix 1912; pachycondyla laevissima, g. arnold; arnold coll. b. m. 1934-354 (bmnh (e) 1015515) antweb casent 0902471, bothroponera laevissima (2w, syntypes, bmnh). non-type material: south africa: western cape province, saldanha bay, 33°1’0’’ s; 17°57’0’’ e, sept. 1912, l. p., arnold determ., pachycondyla laevissima g. arnold, sam-ent 0011517, bothroponera laevissima (3w, iziko). fig 12. the lateral view of worker of b. granosa (amnh). fig 13. the head of worker of b. granosa (amnh). fig 14. the head of the lectotype worker of b. laevissima (iziko). fig 15. the lateral view of the lectotype worker of b. laevissima (iziko). ama joma; wp mackay – bothroponera pumicosa species complex554 distribution: known from saldanha bay, south africa. a b. laevissima worker was collected from jacobsbai, 32°58’0’’ s; 17°53’0’’ e, cape province by h. g. robertson and c. peeters (the ants of africa website, accessed march 2014). biology and habitat: the western cape province includes the cape floristic region (cfr), which is considered one of the global biodiversity hotspots that needs priority conservation attention. it is small area, but includes high plant species richness, especially plants considered as endemics to cfr. it also includes several endemic species of birds, amphibians, insects and a few invertebrates (giliomee, 2003). ants play an important role in maintaining this ecosystem and they use different habitat such as under litter, on the ground, in logs, inside dead trees and on tree branches. the nest of b. laevissima from jacobsbai, cape province, south africa was found in sandy soil (the ants of africa web, accessed may 2014). the main vegetation characterizing the western province are fynbos and succulent karoo biomes with high floral diversity accompanied by a moderate to high biodiversity of ant species. although these ecosystems have their own biodiversity and richness of organisms resembling similar global ecosystems, they contain less ant biodiversity and richness than rainforest habitats (braschler et al., 2012). both b. laevissima and b. aspera, with b. cavernosa, b. montivaga and b. granosa were collected from the western cape province. the various vegetation types and biodiversity in the western cape province has apparently led to high speciation in the area which may happen only in this and similar areas in south africa. the cape provinces in south africa include eastern cape, western cape, and northern cape and includes about half of the b. pumicosa species complex species. for example, b. strigulosa is found in the northern cape, b. berthoudi in the eastern cape with the previous five species in the western cape province. the cape provinces are unique because they also hold the majority of the south africa area and all types of biomes, including forest, nama karoo, fynbos, thicket, savanna and succulent karoo. map 7. the distribution of b. laevissima. plate 6: bothroponera laevissima, lectotype worker. bothroponera montivaga arnold, stat. nov. figures 16, 17 and plate 7; map 8 bothroponera cavernosa var. montivaga arnold, 1947: 132 (w) south africa, steenberg mountains, cape peninsula; joma and mackay: 2013: 3; schmidt and shattuck 2014: 76; pachycondyla cavernosa var. montivaga: brown, in bolton, 1995: 307. diagnosis: the worker is large, total length 12.20 12.65 mm. the 2nd gastral segment of the b. montivaga worker is smooth and moderately shiny with a few shallow scattered punctures. the anterior border of the clypeus is convex, “v” shaped with a smooth anterior medial raised area and with a carina on the posterior half. worker description: (n=2), hl 2.80 2.90, hw 2.40 2.45, ml 1.50, ew 0.35 0.40, el 0.45, sl 1.95, fl 2.90, wl 3.80 3.75, wpl 4.50 4.55, pl 1.00, pw 1.30, ph 1.55 1.50, ci 84.48 85.71, oi 18.36 18.75, mandi 51.72 53.57, si 79.59 81.25, peti 130. head subquadrate; maximum clypeal length 2.00 2.05 mm; mandibles weakly striate with few scattered punctures, 7 teeth; scape nearly reaching posterior border of head; anterior margins of frontal lobes smooth, posterior part punctate; maximum width of frontal sociobiology 62(4): 538-563 (december, 2015) 555 lobes 0.95 1.00 mm; length of malar space (0.50 mm), length from upper edge of eye to edge of posterior lobe 1.25 mm; propodeal spiracle sloping vertically; antennae, legs, mandibles shiny; petiole rounded and slightly narrowed anteriorly (top view), slightly concave posteriorly; sternopetiolar process developed with one tooth pointed ventrally; head covered with short (0.10 mm) erect silver hairs; pronotum, mesonotum, propodeum covered with short (0.10 0.15 mm) erect silver hairs; petiole and postpetiole covered with similar hairs (0.15 mm); head, pronotum, mesonotum, propodeum, mesopleuron, lateropropodeum, metapleuron, petiole and postpetiole coarsely foveolate. metapleuron and lateropropodeum covered with vertical striae with upper part of posteropropodeal margin with coarse vertical nearly parallel grooves with posteropropodeal margin on lower part;second segment of the gaster smooth, slightly shiny and with tiny scattered punctures; mandibles, antennae and legs shiny; petiole rounded and slightly narrowed anteriorly, slightly concave posteriorly; head, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster black; legs, antennae, mandibles brownish black; clypeus dark-brown. comparison: the workers of b. montivaga are nearly identical to those of b. cavernosa and b. umgodikulula. they differ from b. cavernosa and b. umgodikulula in the following ways: the surface of the 4th abdominal segment is smooth, but slightly less shiny, with tiny scattered punctures in b. montivaga while it is somewhat rough and shiny in b. umgodikulula and b. cavernosa. the anterior medial area of the clypeus is raised, but does not form a complete clypeal carina in b. montivaga, b. umgodikulula and b. cavernosa. the lower medial margin of the clypeus is “v” shaped without a carina in b. montivaga similar to that in b. umgodikulula, b. cavernosa and b. aspera, but this shape is with a longitudinal sharp carina in b. granosa. this character separates b. montivaga from the other b. pumicosa species complex members in that all species including b. berthoudi, b. cariosa, b. laevissima, b. pumicosa and b. strigulosa have an “u” shaped anterior medial margin of the clypeus. the propodeal spiracle is nearly vertical, leaning slightly anteriorly in b. montivaga resembling that in b. cavernosa and the other species in this complex, but not horizontal as in b. umgodikulula. the scape nearly reaches the posterior lateral corner of the head in b. montivaga similar to that in b. berthoudi, b. cariosa, b. granosa, b. laevissima, b. pumicosa and b. strigulosa. conversely, the scape slightly exceeds the posterior lateral border of head in b. cavernosa and b. aspera, but just reaches or slightly exceeds it in b. umgodikulula. the sculpture of b. montivaga is foveolate identical to that of b. berthoudi, b. cavernosa, b. pumicosa, b. strigulosa, b. cariosa and b. umgodikulula while it is mostly smooth in both b. laevissima and b. aspera with few punctures in b. aspera. the sculpture is somewhat less foveolate in b. granosa. the head is subrectangular of b. montivaga, but it is suborbicular in both b. laevissima and b. aspera. plate 7: bothroponera montivaga, lectotype worker. map 8. the distribution of b. montivaga. material examined type material: south africa: western cape province, steenberg mountains, cape peninsula, 34°4’0’’ s; 18°28’0’’ e, ii-1946, c. pearson b. cavernosa v. montivaga (arnold, 1947), 1 lectotype worker [here designated, marked ama joma; wp mackay – bothroponera pumicosa species complex556 bothroponera pumicosa (roger) figures 18, 19 and plate 8; map 9 ponera pumicosa roger, 1860: 290 (w) south africa, kaffernlande; bothroponera pumicosa: mayr, 1862: 717 (w); joma and mackay: 2013: 3; schmidt and shattuck 2014: 76; bothroponera pumicata: raffray, 1887: 21 [misspelled name for b. pumicosa (bolton, 2012)]; pachycondyla (bothroponera) pumicosa: emery, 1901: 45; stitz 1910: 130; arnold, 1915: 62; santschi, 1914: 4 pachycondyla pumicosa: forel, 1901: 344 (m); wheeler, 1922: 771; wheeler, g. c. and wheeler, j. 1971: 390 (l); brown, in bolton, 1995: 308 diagnosis: the worker of bothroponera pumicosa is characterized by a large total length (1112 mm). the main distinguishing character of the b. pumicosa worker is the long (up to 0.50 mm or more) golden hairs that are distributed on the entire body including the mandibles, clypeus, and legs. the hairs on the scape are long, at least as long as greatest diameter of the scape. the mandibles are hairy and smooth. the anterior medial margin of the clypeus is convex, “u” shaped, with a raised smooth medial clypeal area on the lower part and is partially carinate on the upper part between the frontal lobes. the lower part of the medial raised area tends to form a groove. the scape does not reach the posterior lateral corner of the head. worker description: (n=5), hl 2.50 2.75, hw 2.05 2.15, ml 1.30 1.40, ew 0.30 0.35, el 0.40, sl 1.75 1.80, fl 2.35 2.60, wl 3.25 3.70, wpl 4.10 4.30, pl 1.00, pw 1.20 1.25, ph 1.00 1.55, ci 82.00 78.18, oi 19.51 18.60, mandi 51.00 52.00, si 81.39 85.36, peti 120 – 125. head subquadrate; mandibles with about 8 teeth, covered with hair (0.20 mm in length); maximum clypeal length 1.85 2.00 mm; scape not reaching posterior lateral corner of head; maximum frontal lobes width 0.90 0.95 mm; frontal furrow well developed; length of malar space from lower edge of eye to base of mandible 0.50 mm; length from upper edge of eye to edge of posterior lobe 1.20 mm; frontal furrow well developed; lower margin of pronotum straight with anteropronotal area forming strongly curved angle, rounded inferior pronotal angle; basalar sclerite oval shaped; mesometapleural suture developed; anterior face of petiole from dorsal view rounded, slightly narrowed, posterior face vertical, slightly concave posteriorly (side view); posterior edge of petiole with slight depression (seen from above); mandibles shiny; antennae, legs, edges of frontal lobes, surface of head densely punctate; pronotum, mesonotum, propodeum, lateropropodeum, and metapleuron densely foveolate; mesopleuron rough with few scattered punctae and foveolae; petiolar and postpetiolar surfaces covered with larger foveolae than those of mesosoma; postpetiolar dorsum partially covered with striae; dorsum of second segment of gaster covered with shallower foveolae than those of petiole and postpetiole, covered with large striae; gastral segments rough, shiny; long golden hairs (up to 0.50 mm or more) distributed on entire body including mandibles, fig 16. the lateral view of the lectotype worker of b. montivaga (iziko). fig 17. the head of the lectotype worker of b. montivaga (iziko). fig 18. the head of the holotype worker of b. pumicosa (iziko). fig 19. the lateral view of the holotype worker of b. pumicosa (iziko). with red dot] and 1 paralectotype worker without dot (# 11516), collection south africa museum ex. national museum bulawayo 1981 sam/ent (iziko). non-type material: none. distribution: only known from south africa, cape province. biology and habitat: bothroponera montivaga specimens were collected from the steenberg mountains of the cape peninsula of south africa. this area is located in southern cape town city opposite the region with b. cavernosa, which is the northern cape town. the habitat is similar in both areas; they are covered mostly with fynbos and thicket biomes (picker & samways, 1996). the cape peninsula is rich with very high percentage of endemic fauna and flora, especially for species that inhabit caves and mountains. the area is considered as a distinct hotspot that provides relictual habitats for organisms. human activities, disturbances, introduction of alien species and fragmentation increase the importance of conservation priority for species in the cape peninsula (picker & samways, 1996). sociobiology 62(4): 538-563 (december, 2015) 557 clypeus, legs; hairs on scape long, at least as long as greatest diameter of scape; erect and suberect hairs on body surface as following: head, funiculus, mandibles with long hairs (0.20 mm), hairs on scape about 0.20 mm on far end, about 0.40 mm on near area of scape’s base, hairs on legs about 0.25 0.30 mm, on pronotum, mesonotum, propodeum (0.40 0.45 mm), on petiole, postpetiole, entire gaster (0.50 0.55 mm); head, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster black; femora, mandibles brownish black; tibia, tarsi, antennae, edges of frontal lobes brown. comparison: the worker of b. pumicosa has a similar “u” shaped anterior margin of the clypeus as those of b. strigulosa, b. cariosa, b. berthoudi, and b. laevissima. the upper part of the raised area of the clypeus forms a partial carina while it forms a complete longitudinal sharp carina in both b. strigulosa and b. cariosa, but b. berthoudi, and b. laevissima lack the carina. the long hairs (up to 0.50 mm or more) separate b. pumicosa from all of the other members of the b. pumicosa species complex. the hair length of the other b. pumicosa species complex species is less than 0.20 mm for the head; less than 0.25 mm for the pronotum, mesonotum, and propodeum; and less than 0.35 mm for the petiole and postpetiole. the traits of b. pumicosa are very similar to those of b. berthoudi and b. strigulosa, but it can be separated by the longer hairs and the form of the anterior medial border of the clypeus. the length of the hairs on the head of b. strigulosa is 0.07 0.13 mm with a few hairs up to 0.16 mm. the length of the hairs of the head of b. berthoudi is 0.20 mm. the hairs on the dorsum of the pronotum, mesonotum and propodeum are 0.25 mm, and 0.07 0.16 mm in b. berthoudi and b. strigulosa respectively. the length of the hairs on the petiole and postpetiole in b. strigulosa is 0.15 0.18 mm, and those of b. berthoudi less than 0.30 mm. the hairs length comparison of these species with b. pumicosa shows that b. pumicosa has the longest hair length among those mentioned above. the hairs on the entire body of b. berthoudi are up to 0.35 mm in length, but the hairs of b. pumicosa are longer, up to 0.45 0.55 mm. among the 5 specimens of b. pumicosa, there is one specimen, a worker # 315926, from the lacm, that appears to be different from the others based on hair length. this specimen was collected from south africa and determined by forel without any further information. the hairs on the head measure from 0.07 to 0.11 mm, on the mandibles from 0.10 to 0.20 mm, on the scape from 0.14 to 0.21 mm, hairs on the legs range from 0.15 to 0.22 mm, on the anterior part of the pronotum from 0.20 to 0.22 mm, posterior part of pronotum, sides of pronotum, mesonotum and propodeum from 0.05 to 0.15 mm, on edges of posteropropodeum up to 0.22 mm, on the petiole from 0.05 to 0.13 mm, on the postpetiole up to 0.10 mm, on the sides of postpetiole up to 0.22 mm, hairs between the gastral segments (ventrally) from 0.36 to 0.44 mm, on the pygidium up to 0.22 mm and ventrally (hypopygium) up to 0.30 mm. the other characters for this specimen are quite similar to those of b. pumicosa. it is possible it could be a new species, but when more specimens are collected, it can be reevaluated. material examined type material: south africa: kaffernlande, transkei district, 31°30’0’’ s; 29°0’0’’ e, bothroponera pumicosa type (roger, 1860) (1 w holotype, #11522.) sam-ent (iziko). non-type material: south africa: eastern cape province, grahamstown, 33°18’0’’ s; 26°32’0’’ e, 21-iv1986, n. g. robertson, c154, u. stone (3w bmnh). south africa: no further information, forel det., collection of w. s. creighton purchased by lacm 1974, pachycondyla pumicosa roger, det. forel (1w # 315926 lacm). distribution: bothroponera pumicosa is known from the cape province of south africa (wheeler 1922), the cape of good hope (roger, 1860), the natal, province of kwazulunatal (forel, 1901; santschi, 1914: 4) and some other workers were collected from a nest in burntkraal, cape province (the ants of africa website, accessed march 2014). this species collected also from cameroon (wheeler, 1922) and mundame, cameroon (stitz, 1910).plate 8: bothroponera pumicosa, holotype worker. ama joma; wp mackay – bothroponera pumicosa species complex558 biology and habitat: the type specimen was collected from kaffernlande, former name of the transkei and ciskei regions, both in the transkei district (per. comm. dr. worden) and the eastern cape province, south africa. the type specimens of b. cavernosa were also collected from kaffernlande. the habitat in transkei district is covered with three types of biomes: grassland, savanna, and thicket biomes (map 1). these species, with b. cariosa, are recognized by their behavior in that they build small colonies under stones in moist clay soils. they are mainly specialized to feed on termites (wheeler, 1922; wheeler and wheeler, 1971). the worker and male of b. pumicosa were collected from the cape province, natal, south africa (forel, 1901: arnold, 1915). diagnosis: the head of the worker is subquadrate and the mandibles are smooth and covered with hairs. the anterior medial margin of the clypeus is convex and forms a “u” shaped edge, and the clypeus has a raised medial sharp carina which extends from the base of the frontal furrow to the lower medial margin of the clypeus. worker description: (n=1), hl 2.50, hw 2.10, ml 1.35, ew 0.35, el 0.45, sl 1.65, fl 2.50, wl 3.60, wpl 4.50, pl 1.15, pw 1.35, ph 1.45, ci 84, oi 21.42, mandi 54, si 79, peti 117.39. total length 12.20 mm; mandibles triangular with 7 teeth, smooth, moderately covered with hairs (0.10 0.20 mm long); clypeal length 1.85 mm; scape nearly reaches posterior lateral corner of head; malar space from lower edge of eye to base of mandible 0.38 mm; length from upper edge of eye to edge of posterior lobe 1.10 mm; surface of head coarsely foveolate; frontal lobes rounded, smooth, shiny with width of 0.85 mm; pronotal shoulder rounded; two sharp angles on anterior (anteroinferior pronotal process) posterior (inferior pronotal process) ends of lower margin of pronotum (lateral view); basalar sclerite rounded; lower part of mesopleural suture well developed with mesopleuralcoxal excavation; petiole in dorsal view rounded, slightly narrowed anteriorly, anterior face vertical (side view), slightly concave posteriorly (side view) with slight depression on upper medial margin (top view); mesosoma 3.4 mm, gaster length 4 mm; antennae, edges of frontal lobes, mandibles and legs shiny; pronotum, mesonotum, propodeum, lateropropodeum, metapleuron densely foveolate, punctate; posteropropodeum rough, slightly concave; petiolar and postpetiolar surfaces densely covered with larger foveolae than those of mesosoma; postpetiolar dorsum partially covered with striae; second fifth gastral segments densely covered with foveolae, punctures that become smaller, shallower posteriorly; postpetiolar dorsum partially covered with striae; dorsum of 2nd 5th segments of gaster covered with fine striae; short (0.05 – 0.22 mm) fig 20. the lateral view of the holotype worker of b. strigulosa (mcsn). fig 21. the head of the holotype worker of b. strigulosa (mcsn). fig 22. the head of the holotype worker of b. umgodikulula (mczc). fig 23. lateral view of the holotype worker of b. umgodikulula (mczc). map 9. the distribution of b. pumicosa. bothroponera strigulosa emery figures 20, 21 and plate 9; map 10 bothroponera strigulosa emery, 1895: 19 (w) south africa, kimberley; wheeler, w.m. 1922: 72 in key; joma and mackay: 2013: 3; schmidt and shattuck, 2014: 76; pachycondyla (bothroponera) strigulosa: emery, 1901: 45 list; arnold, 1915: 61 (w); wheeler, w.m. 1922: 773; pachycondyla strigulosa: brown, in bolton, 1995: 310. sociobiology 62(4): 538-563 (december, 2015) 559 golden hairs distributed on entire body including mandibles, clypeus, head, scape, legs; top of the head covered with short (0.05 0.13 mm) golden erect hairs; dorsum of pronotum, mesonotum, propodeum covered with short golden erect hairs (0.07 0.13 mm, a few up to 0.18 mm); petiole (0.08 0.22 mm), postpetiole covered with moderately short erect hairs (0.12 0.18 mm); sternopetiolar and sternopostpetiolar processes and 3rd to 7th abdominal segments covered with moderately long hairs (0.11 – 0.22 mm); ventral surface of gastral segments and between segments (0.15 0.32 mm); pygidium and hypopygium (up to 0.30 mm); head, scape, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster, legs black or dark-brown; mandibles, funiculus, frontal lobes reddish brown. comparison: bothroponera strigulosa is similar to b. cariosa, and b. pumicosa, as they all have a “u” shaped anterior medial margins of the clypeus. bothroponera strigulosa is quite similar to b. cariosa, but it can be distinguished because the mandibles of b. strigulosa are smooth and shiny while in b. cariosa they are covered with fine striae. the mandibles in b. pumicosa are hairy and smooth. the hairs are long (from 0.20 up to 0.55 mm) in b. pumicosa while they are short in b. strigulosa (from 0.05 up to 0.32 mm) and b. cariosa (from 0.05 up to 0.25 mm). the characters of bothroponera strigulosa are similar to those of b. berthoudi. the only apparent differences between them are that the raised medial area of the clypeus of b. berthoudi is smooth and the clypeal carina is not present, but b. strigulosa has a clypeal carina (it partially forms a carina in b. pumicosa). the erect golden hairs on most surfaces are slightly longer in b. berthoudi than those of b. strigulosa. plate 9: bothroponera strigulosa, holotype worker. map 10. the distribution of b. strigulosa. material examined type material: south africa: northern cape province, kimberley, 1230 m [4040 ft] (gr. w.), 28°44’0’’ s; 24°46’0’’, e, e. simon 1893, bothroponera strigulosa (emery, 1895), museo geneva coll., emery (dono 1925) (1w holotype, mcsn). non-type material: none. distribution: known from the type locality of kimberley, south africa. bothroponera strigulosa was collected from vaalwater, northern province, south africa (the ants of africa website, accessed march 2014). biology and habitat: kimberley, northern cape province, south africa is a large city located almost in the center of south africa, close to the free state province. the summer climate is hot and wet, the annual maximum temperature is 26.05 ºc, the annual minimum temperature is 10.8 ºc (kimberley website 1 and kimberley website 2). it rains an average of 42.0 cm/year while the winter climate is dry to moderately dry (kimberley website 1 and kimberley website 2). the area is considered as a dry or semi-arid region, which is the typical environment for the northern cape province. mokala national park, one of the 20 national parks ama joma; wp mackay – bothroponera pumicosa species complex560 in south africa, is located south-southwest of kimberley. the main vegetation in this park is the savanna biome with kameeldoring trees or camel thorn trees acacia erioloba, one of the major tree species of the desert regions (kimberley website 3). this park is also one of the protected areas that include several endangered species and wild animals. the northern cape province is characterized by three types of biomes, succulent karoo, nama karoo, and savanna biomes. bothroponera umgodikulula joma and mackay figures 22, 23 and plate 10; map 11 bothroponera umgodikulula joma and mackay 2013: 1 8 (w) south africa, whittlesea; schmidt and shattuck: 2014: 77. diagnosis: the worker of b. umgodikulula can be diagnosed by several morphological characters, such as the lack of sculpture on the tergum of the fourth abdominal segment (second gastral segment), which is mostly smooth and glossy. the propodeal spiracle is unusual in being nearly horizontal on the lateropropodeum. the worker of b. umgodikulula is also characterized by the largest body size among bothroponera species, which is 14.80 15.65 mm. worker description: hl 3.00 3.10, hw 2.85 2.95, ml 1.50 1.70, ew 0.40 0.45, el 0.45, sl 2.35 2.40, fl 3.65 3.75, wl 4.20, wpl 5.00 5.50, pl 1.30 1.35, pw 1.50 1.70, ph 1.75 1.80, ci 95.00 95.16, oi 15.78 15.25, mandi 50.00 54.83, si 82.45 81.35, peti 115 – 126. head subquadrate; mandibles triangular, shorter than head length, smooth and glossy with scattered elongated coarse punctures and about 7 teeth; clypeus convex, “v” shaped, covered with striae, except medial area; anterior medial area raised, coarsely punctate on sides, smooth, glossy medially; scape reaches or extends slightly past posterior border of head; compound eyes relatively large; lower margins of frontal lobes smooth, upper part punctate; maximal frontal lobe width 1.10 1.20 mm; head coarsely foveolate; length of malar space on side of head (0.65 0.70 mm), length from upper edge of eye to edge of posterior lobe 1.35 1.50 mm. pronotal shoulder rounded; petiole rounded, slightly narrowed anteriorly, slightly concave posteriorly; pronotum, dorsum of mesonotum, dorsum of propodeum coarsely foveolate, rough; dorsum of petiole, postpetiole coarsely foveolate, punctate; mesopleuron, lateropropodeum coarsely grooved, covered with striae, foveolae, punctures; antennae, legs, posterior edge of each gastral tergite shiny. entire head, pronotum, mesonotum, propodeum, petiole, postpetiole covered with short (0.03 0.10 mm) fine golden hairs; hairs on underside of head range from 0.25 0.50 mm in length; ventral surface of postpetiole, fourth–seventh abdominal segments covered with relatively long (0.20 0.25 mm) golden suberect hairs. head, pronotum, mesonotum, mesopleuron, propodeum, petiole, postpetiole, entire gaster black; legs, antennae, mandibles red; clypeus dark-brown. comparison: the worker of bothroponera umgodikulula is easily recognized by the horizontal propodeal spiracle on the lateropropodeum, while it is obliquely vertical in all of the other african bothroponera species. the 4th abdominal segment (second gastral segment) is smooth and glossy in b. umgodikulula, conversely, the 4th abdominal segment of b. cavernosa is rough, moderately shiny with few scattered hairs and fine poorly defined striae; this structure is moderately smooth and shiny (less than b. umgodikulula) with a few scattered punctures in b. montivaga. the other taxa that can be confused with b. umgodikulula are b. laevissima and b. aspera, which both have a 4th abdominal segment that is smooth and shiny, similar to b. umgodikulula. the unique sculpture of these three species simplifies their separation. the surface from the head to the postpetiole is smooth and shiny with few scattered punctulae in b. laevissima and is shiny, rough with dense, shallow punctures in b. aspera, but is coarsely foveolate in b. umgodikulula. the total length of b. umgodikulula is large (14.80 15.65 mm) compared to b. cavernosa (11.90 mm) and b. montivaga (12.20 12.65 mm). in fact, b. umgodikulula has the largest body size among plate 10: bothroponera. umgodikulula, holotype worker. (photo by michele esposito/from www.antweb.org. accessed 23 february 2015). sociobiology 62(4): 538-563 (december, 2015) 561 the other species of the b. pumicosa species complex (e.g. b. granosa 13.75 14.50 mm, b. strigulosa 12.20 mm, b. laevissima 11.80 13.00 mm, b. aspera 11.70 12. 70 mm, b. pumicosa 11.00 11.65 mm, b. cariosa 11.50 mm and b. berthoudi 9.60 12.75 mm). the anterior medial margin of the clypeus is “v” shaped in b. umgodikulula similar to that of b. granosa, b. cavernosa, b. montivaga and b. aspera, conversely, the anterior medial margin of the clypeus is “u” shaped in b. cariosa, b. strigulosa, b. pumicosa, b. laevissima and b. berthoudi. the anterior medial raised area of the clypeus of b. umgodikulula is completely smooth (lacking a carina) shiny, but sculptured and punctate on the sides of the medial raised area. the anterior medial raised area of the clypeus of b. granosa has a sharp clypeal carina whereas it is partially carinate in b. cavernosa and b. montivaga. in this area (rouget et al., 2004), and it is one of the threatened ecosystems in south africa (farrier et al., 2013). the ecological importance of the hopefield area results from the soil structure, water permeability, climatic influence and vegetational cover. the area is characterized by spreading of several alien invasive plants such as the alien wattles acacia cyclops (rooikrans), a. longifolia (long-leaf wattle), a. saligna (port jackson), a number of eucalyptus species, manitoka (myoporum montanum) and introduced prickly pear cactus (opuntia sp.). also many endemic and threatened plant taxa are present (department of environmental affairs & development planning 2011). this type of mixed habitat is likely to include many species of insects such as tropical ants. acknowledgements we would like to thank the curators and museums who provided us with ant specimens including dr. claire villemant (mnhn), dr. bernhard merz (mhng), drs. stefan cover, jignasha rana, linda ford, philip perkins, jack boyle, charles whittemore farnum (mczc), drs. frank koch, viola richter (mfn), drs. roberto poggi, giuliano doria, maria tavano (mcsn), drs. isabelle zürcher-pfander, daniel burckhardt (nhmb), drs. gavin broad, suzanne ryder (bmnh), drs. james carpenter, christine lebeau (amnh), drs. weiping xie, laura mcgover (lacm) and drs. dawn larsen and aisha mayekiso (iziko). special thanks are due to dr. gary alpert who offered to take photos of the bothroponera species and allowed lodging during the study period at the museum of comparative zoology. dr. alain dejean provided several publications on african ants, drs. hamish robertson and robert taylor provided their ant publication websites, dr. brigitte braschler offered useful information about african ant richness and biodiversity and dr. nigel worden offered historical information about south africa. we also would like to thank dr. barry bolton, who suggested useful comments for ant character descriptions and identifications. dr. brian fisher allowed me to use figures from antweb (http://www. antweb.org/), and photographed b. umgodikulula. dr. brian taylor allowed me to use the information from his website, the ants of africa (http://antsofafrica.org/). dr. sulaiman abushagur offered advice during our study and made important suggestions for the research. dr. jung w. kim, who offered to take photos of the bothroponera species. we also would like to thank dr. robert kirken, chairman of the department of biological sciences and now dean of the college of sciences of the university of texas at el paso who facilitated procedures to borrow specimens from iziko, dr. elizabeth walsh, doctoral advisor, who encouraged us and assisted with paperwork, and also supported the senior author economically, as well as the doctoral committee members dr. carl lieb, dr. eli greenbaum, dr. michael moody, dr. mahesh narayan. thanks also due to dr. shawn dash and dr. jerry johnson, for assistance and useful suggestions. map 11. the distribution of b. umgodikulula. material examined type material: south africa: eastern cape province, bulhoek, klaver-clanw [whittlesea], bulhoek at 32°10’0’’ s; 26°49’0’’ e, mus. expd. oct. 1950, identified as bothroponera cavernosa roger, 1860, f. w. g. (1 w holotype, mczc) and (1 w paratype, # c005835 iziko). non-type material: south africa: western cape province, hopefield, 33°03′56″s 18°21′03″e, identified as bothroponera cavernosa roger, det. g. arnold (1w bmnh). distribution: whittlesea and hopefield areas in south africa. biology and habitat: the type specimens were collected in whittlesea city in south africa. this area is located in the eastern cape province, but the additional material examined (one specimen) was collected in hopefield city in the western cape province. hopefield is a small village situated 90 miles north of cape town and about 24.14 km [15 miles ] east of saldanha bay (singer, 1954). the fynbos biome is dominant ama joma; wp mackay – bothroponera pumicosa species complex562 this research was supported by the libyan ministry of higher education, libyan-north american scholarship program, the canadian bureau for international education, the ernst mayr fund of the museum of comparative zoology, harvard university, a generous donation from the estate of maxie groce templeton and the dodson research grant from the university of texas at el paso. references anchor environmental consultants. 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(1922). ants of the american museum congo expedition. bulletin of the american museum of natural history, 45: 1-1004. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 sociobiology 60(1): 123-124 (2013) biological notes on the parasitism of apoica flavissima van der vecht (hymenoptera: vespidae) by seminota marginata (westwood) (hymenoptera: trigonalidae): are social paper wasps primary or secundary hosts of trigonalidae? ef santos1, fb noll2 trigonalidae comprises species of diurnal parasitic wasps that lay their eggs on the foliage, depending on a herbivorous primary host to ingest their eggs together with plant material (weinstein & austin, 1991). trigonalids may have a secondary host, such as wasps or tachinid fly larvae, which are predators or parasites of the primary host, and ingest eggs (gauld & bolton, 1988; weinstein & austin, 1991). mechanical and chemical stimuli from the host mandibles lead to the eclosion of trigonalid egg (clausen, 1931), and larvae at the first larval instar penetrates the gut lining into the primary host haemocoel (weinstein & austin, 1991). although primary hosts are not known for social wasps, it is assumed that trigonalid larvae infect their larvae after ingestion of tissues of the primary host (usually caterpillars) by trophallaxis, or they may penetrate directly through the cuticle of the secondary host to gain access to the haemolymph (weinstein & austin, 1991). several trigonalidae have been recorded as parasiabstract hosts of trigonalidae include larvae of social paper wasps, which have been considered secondary hosts, supposedly following predation of the primary host (usually caterpillars) by adult wasps. this study provides observations on biological aspects of the parasitism of apoica flavissima van der vecht by seminota marginata (westwood), and suggests that social wasps may be both primary and secondary hosts, whereas they extract and chew vegetable fiber. sociobiology an international journal on social insects 1 faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, ribeirão preto, sp, brazil 2 universidade estadual paulista, são josé do rio preto, sp, brazil article history edited by: gilberto m. m. santos, uefs brazil received 07 november 2012 initial acceptance 03 december 2012 final acceptance 17 december 2012 keywords epiponini, polistinae, facultative host, neotropical. corresponding author eduardo fernando dos santos faculdade de filosofia, ciências e letras de ribeirão preto, univ. de são paulo. avenida bandeirantes, 3900, bairro monte alegre, 14040-901, ribeirão preto, sp, brazil. e-mail: efs.wasp@gmail.com toids of social paper wasps (polistinae and vespinae). species of seminota spinola have been recorded as parasitoids of polistes latreille, mischocyttarus de saussure, apoica lepeletier, pseudopolybia de saussure, and parachartergus r. von ihering (weinstein & austin, 1991). seminota marginata was recorded parasitizing polistes versicolor (olivier), p. cinerascens de saussure, p. melanosoma de saussure, p. canadensis (linnaeus), pseudopolybia vespiceps (de saussure), and possibly apoica pallida (olivier) (weinstein & austin, 1991). apoica flavissima van der vecht, like other species of apoica, are nocturnal wasps that build their nest using plant material, more specifically plant hairs (wenzel, 1998). their nests are circular with only one comb, lacking an envelope (wenzel, 1998). foragers of apoica are generalists, preying upon invertebrates, mainly lepidopteran larvae, regurgitating their crop contents to feed their own brood. specimens of seminota marginata westwood emerged or were taken off from nest cells of a apoica flavisshort note ef santos, fb noll biological notes on the parasitism of apoica flavissima124 sima nest, which was collected in indiaporã (19°58’54’’s 50°17’48’’w), são paulo state, brazil, on may 16th of 2010. a. flavissima, like other species of apoica, are nocturnal wasps that build their nest using plant material, more specifically plant hairs (wenzel, 1998). the nest was well developed and the eleven cells with parasitoids were at the center of the nest. we recovered one adult trigonalid male, one larva, two pupae in early development and two in late development from the a. flavissima nest on may 21th, while four males emerged on may 27th, and one male on june 9th of 2010. two adults were deposited in the mzusp’s hymenoptera collection, and four adults and three immatures, preserved in alcohol 70%, were deposited in the hymenoptera collection of the departamento de botânica e zoologia of the instituto biociências, letras e ciências exatas da universidade estadual paulista (ibilce – unesp). parasitized cells were easily recognized because unlike regular apoica larvae that spin their cocoons above the cell margin, s. marginata larvae spin their cocoons about one third the cell height below the margin. parasitism of apoica species by trigonalidae was recorded by bertoni (1912), who found s. marginata parasitizing larvae of a. pallida van der vecht in a nest sampled in puerto bertoni, paraguay. a. pallida is a valid name, but in the past the name was used for several other species of apoica, among them a. flavissima and angiopolybia pallens (fabricius) (pickett & wenzel, 2007). bertoni (1912) highlights the similarity among the specimens identified as apoica pallens from guyana and as a. pallida from paraguay. thus, possibly bertoni’s (1912) record is for angiopolybia pallens. paper wasps have been considered the secondary host of trigonalidae, although no primary host has been associated with the host wasp species (weinstein & austin, 1991). the first host, in this case, would be a lepidopteran larvae, one of the main preys of apoica and other epiponini. however, considering that polistines use plant material to build their nests, it is possible that species of apoica, like other social paper wasps, could be facultative primary hosts of seminota. weinstein & austin (1995) observed larvae of perga leach, which are herbivorous, as facultative primary hosts. species of apoica possess shorter and more ventrally curved mandibles with a higher ventral tooth, and a long and high dorsal tooth. sarmiento (2004) suggests that mandibular structure is strongly adaptaded for a fine manipulation of plant fibers. then, adults of polistinae may ingest trigonalidae eggs while collecting fibers to build their nests. similarly to other primary hosts, the mechanical action of the mandible and digestive secretions, present in the adult oral cavity, would lead to egg hatch, and consequently larval emergence. according to sarmiento (2004), short mandibles, like those found in apoica, are efficient in fiber management and may be less effective in prey chewing. as in the secondary host condition, parasitoid larvae pass from adult to host larvae by trophallaxis. if the adult ingests the parasitoid eggs collecting vegetable fibers, the parasitoid may be transferred to the host larvae indirectly by trophallaxis or directly by penetrating the cuticle of polistinae larvae from the nest. in this regard, apoica as well as other polistinae and species of vespinae may be facultative secondary hosts of trigonalidae (weinstein & austin, 1991). acknowledgments we thank dr. david carmean for help in species identification, and dr. james carpenter and dr. marcel hermes for the critical reading of an early version of the manuscript. references bertoni, a. w. (1912). contribucción a la biología de las avispas y abejas del paraguay (hymenoptera). an. mus. nac. hist. nat. de buenos aires, 22: 97-146. carmean, c. & kimsey, l. (1998). phylogentic revision of the parasitoid wasp family trigonalidae (hymenoptera). system. entomol., 23: 35-76. clausen, c. p. (1931). biological notes on the trigonalidae (hymenoptera). proc. of the entomol. soc. of wash., 33: 7281. gauld, i. & bolton, b. (1988). the hymenoptera. london: british museum. jeanne, r. l. (1970). descriptions of the nests of pseudochartergus fuscatus and stelopolybia testacea, with a note on a parasite of s. testacea (hymenoptera, vespidae). psyche, 77: 54-69. pickett, k. m. & wenzel, j. w. (2007). revision and cladistic analysis of the nocturnal social wasp genus, apoica lepeletier (hymenoptera: vespidae; polistinae, epiponini). am. mus. nov., 3562: 1-30. sarmiento, c. e. (2004). a test of adaptive hypotheses: mandibular traits, nest construction materials, and feeding habits in neotropical social wasps (vespidae, polistinae). insectes soc. 51: 387-391. doi: 10.1007/s00040-004-0756-y weinstein, p; austin, a. d. (1991). the host relationships of trigonalyid wasps (hymenoptera: trigonalyidae), with a review of their biology and catalogue to world species. j. nat. hist., 25: 399-433. weinstein, p. & austin, a. d. (1995). primary parasitism, development and adult biology in the wasp taeniogonalos venatoria riek (hymenoptera: trigonalyidae). aust. j. zool., 43: 541-455. doi: 10.1071/zo9950541 wenzel, j. w. (1998). a generic key to the nests of hornets, yellowjackets, and paper wasps worldwide (vespidae: vespinae, polistinae). am. mus. nov., 3224: 1-39. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.824sociobiology 63(2): 735-743 (june, 2016) ant community (hymenoptera: formicidae) associated with callisthene fasciculata (spr.) mart. (vochysiaceae) canopies in the pantanal of poconé, mato grosso, brazil introduction the forest canopy is responsible for mechanisms that regulate key processes in ecosystems (basset et al., 2002), such as high primary productivity (lowman & nadkarni, 1995). this productivity makes a high variety of resources possible, especially food, for different taxa, including arthropods (novotny et al., 2002a,b), which represent the most abundant and diverse taxon associated with tropical forest canopies in all regions of the world (stork & grimbacher, 2006). in this way, tree canopies are characterized as a diverse environment and of considerable importance due to nutrient cycling processes and the ecological interactions among the species that live in them (adis, 1997; adis et al., 2010). abstract ants act in different trophic levels and are important due to their abundance, distribution and diversity in a variety of habitats, exercising influence on many different organisms and ecosystems. thus, this study compared temporal variation on the structure and composition of the ant community in canopies of callisthene fasciculata (spr.) mart. (vochysiaceae) during high water and dry periods, in the pantanal of poconé, mato grosso, brazil. ant sampling was performed on 12 specimens of c. fasciculata, in 2010 and 2011, using canopy fogging with insecticide, in a total of 120m² of sampled canopy. altogether, 2,958 ants were collected. the 2,943 adults were distributed in four subfamilies, 12 genus and 26 species. myrmicinae (18.7 ind./m2) was the most representative taxon, followed by formicinae (3.2 ind./m2), dolichoderinae (2.2 ind./m2) and pseudomyrmecinae (0.3 ind./m2). the community is made up of six trophic groups, in which omnivorous (23.5 ind./m2) were the most prevalent, followed by minimum hypogeical generalists (0.5 ind./m2) and arboreal generalist predators (0.3 ind./m2). although the results showed that ant community richness, associated to c. fasciculata canopies, does not represent a significant difference among the seasonal periods, there are differences as to species distribution on the host plant over the seasonal periods, indicating the influence of temporal variation, and therefore, habitat conditions on this community. sociobiology an international journal on social insects l yamazaki1, j dambros1, e meurer2, vf vindica1, jhc delabie3, mi marques2, ld battirola1 article history edited by kleber del-claro, ufu, brazil received 14 july 2015 initial acceptance 01 march 2016 final acceptance 09 may 2016 publication date 15 july 2016 keywords biodiversity, canopy, temporality, monodominance. corresponding author lúcia yamazaki universidade federal de mato grosso campus universitário de sinop sinop-mt, brazil e-mail: lucia_yamazaki_ly@hotmail.com among the arthropods, formicidae is an important component of the communities inhabiting the tree layer of tropical forests, using same as habitat, foraging area and nesting (corrêa et al., 2006). this association also allows plants to benefit because the ants provide materials which are rich in nitrogen, food waste and useful metabolic waste to plants (delabie et al., 2003), as well as reducing herbivory activity by organisms that feed on these plants, influencing the structuring of the communities in these habitats (hölldobler & wilson, 1990). ants are predators and herbivores which are significant in many habitats, mainly due to their abundance, omnipresence and diversity (fowler et al., 1990; kaspari, 2003). in tropical forests, seasonality is a factor that determines the structure of 1 universidade federal de mato grosso, sinop-mt, brazil 2 universidade federal de mato grosso, cuiabá-mt, brazil 3 universidade estadual de santa cruz, ilhéus-ba, brazil research article ants l yamazaki et al. – ant community associated with callisthene fasciculata canopies736 their communities (simberloff & dayan, 1991). the majority of the species appear to be opportunistic foragers, feeding on nectar, seeds, leaves and live or dead animals, while some may be specialized in their feeding habits (kaspari, 2003). studies carried out in the pantanal have demonstrated that ants are an important component of the arthropods communities, participating in ecological processes, developing different survival strategies and social behaviors in these areas (adis et al., 2001; battirola et al., 2005; castilho et al., 2007; santos et al., 2008; marques et al., 2010, 2011; soares et al., 2013). considering the importance of conserving wetlands for the maintenance of biological diversity and the role played by this taxon in food chains associated with these habitats, this study evaluated the temporal variation in the structure and composition of the ant community in the canopy of callisthene fasciculata (spr.) mart. (vochysiaceae), during periods of high water and dry season in the pantanal of poconé, mato grosso, brazil. material and methods study area samples were collected in the pantanal of poconé, specifically on the fazenda alvorada (16°26’s and 56°24’w), porto cercado road, in poconé, mato grosso, brazil. the local climate is characterized as aw, according to the köppen classification. this region has well defined seasons, with the rainy season between october and march, and the dry season between april and september, defining its water cycle in four distinct seasonal periods (high water, receding water, dry and rising water) (heckman, 1998). the samples were obtained in a monodominant, seasonally flooded forest, with predominance of c. fasciculata, locally known as “carvoal”, which can reach 4 to 15m in height when mature and has dark, thick and rough bark (pott & pott, 1994). the flowering period is between september and october, occurring together with seed dispersal generated in the previous year (custódio et al., 2014). it is a tree that is characterized as deciduous and with dormancy absent during the rising water season until the beginning of the dry season, partial dormancy in the dry season until the beginning of the rising water season, and total dormancy at the end of the dry season (corsini & guarim-neto, 2000). field procedures insecticide was applied by way of fogging on 12 c. fasciculata canopies in the high water season (tree numbers 1-6) and in the dry season (tree numbers 7-12), in 2010 and 2011, respectively. in each seasonal period six individual c. fasciculata were randomly selected keeping a minimum distance of 10m between each sample according to adis et al. (1998) and battirola et al. (2004). initially, the entire diameter at the base of these trees was surrounded by nylon funnels (1m² in diameter each), distributed according to the scope and architecture of the canopy, totaling 120m2 of sample area (10m² per sampled tree). at the base of each collecting funnel a plastic bottle collector was installed with 92% alcohol which remained suspended at about 1m from the ground by means of cords tied to surrounding trees. during the high water period funnels were suspended at 1.5m above the soil due to flooding of part of the forest (water depth ranging between 0.1 and 0.3m). the fogging procedure was carried out for ten minutes on each tree, using the synthetic pyrethroid lambda-cyhalothrin (icon®) at 0.5%, diluted in two liters of diesel at a concentration of 1% (20ml), associated with the synergist (ddvp) 0.1% (2ml). the fogging used was the swingfog sn50 model, which produces a strong jet of smoke that is directed from the ground to all parts of the canopy. these procedures took place, always, around 6:00am when the air circulation is less intense, allowing the insecticide cloud to rise slowly through the canopy and not disperse. in each sampled tree a fogging and a collection took place. the collection was carried out two hours after application of the insecticide, the recommended time for its action on arthropods (adis et al., 1998), when the walls of the funnels were manually shaken and washed with the aid of sprayers containing alcohol at 92% so that the material could be gathered from the existing collector bottles at the basis of the hoppers. laboratory procedures all the material coming from the samples was transported to the acervo biológico da amazônia meridional (abam) of the universidade federal de mato grosso, sinop mt. here all the ants were quantified and identified according to fernández (2003a,b) and bolton (2003, 2014). then the ants were compared with the reference collection from the laboratório de ecologia e taxonomia de artrópodes (leta) of the instituto de biociências of the universidade federal de mato grosso in cuiabá, mt. the determination of trophic guilds was carried out according to rojas and fragoso (2000), silvestre et al. (2003) and brandão et al. (2009) and the material is deposited in the abam/ufmt/sinop entomological collection. data analysis the estimate for species richness was evaluated by bootstrap and jackknife 1 estimators. richness comparison between periods of high water level and dry was carried out using the t test. to assess the composition of species and guilds between the periods of high water and dry indirect sorting was carried out by non-metric multidimensional scaling (nmds) using one axis for analysis. the sorting was carried out using quantitative data and the bray-curtis similarity measure. the vegan (oksanen et al., 2013) package was used for nmds analysis and richness estimates, and all analyzes were performed using the r software, version 3.0.1 (r core team, 2013). sociobiology 63(2): 735-743 (june, 2016) 737 results community composition a total of 2,958 ants was collected in c. fasciculata canopies during the high water and dry periods in the pantanal of poconé, mato grosso, with a density of 24.6 ind./m2. of these individuals, 2,943 (99.5%; 24.5 ind./m2) are adults, and only 15 (0.5%; 0.1 ind./m2) are immature (larvae). the adults are distributed into four subfamilies, 12 genera and 26 species. myrmicinae (2,250 ind.; 76.5%; 18.7 ind./m2) was the dominant group, followed by formicinae (391 ind.; 13.3%; 3.2 ind./m2), dolichoderinae (264 ind.; 9.0%; 2.2 ind./ m2) and pseudomyrmecinae (37 ind.; 1.3%; 0.3 ind./m2) (fig 1). crematogaster lundi, 1831 and cephalotes latreille, 1802 were the most diverse genus, with five and four species, respectively (table 1). temporal variation during the high water period 1,549 individuals (52.4%; 12.9 ind./m2) were collected and 1,409 individuals (47.6%; 11.7 ind./m2) were collected in the dry period. the number of species between high water and dry periods did not present any difference (t-test = -0.136; gl = 9.131; p = 0.894). the indirect ordering of data as to species occurrence (nmds) between high water and dry periods resulted in a stress = 0.35. the result of the t-test, which compares the scores of the axis, showed a significant difference (t-test = -4.557; gl = 9.943; p = 0.001) between species distribution for the two periods (fig 3). among the 26 species living in canopies of c. fasciculata, camponotus melanoticus emery, 1894, crematogaster arcuata forel, 1899, all belonging to the cephalotes genus, were found only during the high water period. all species of azteca forel, 1878, crematogaster quadriformis roger, 1863, pseudomyrmex termitarius (f. smith, 1855) and tapinoma sp. 2 occurred exclusively during the dry period (table 1), and 13 species are common to both seasonal periods evaluated. although there was no significant difference in the richness of the ant community between seasonal periods, variations are seen in the distribution of species, individually, however w. auropunctata, camponotus (myrmaphaenus) sp. 1, tapinoma sp. 1, crematogaster sp. 1, brachymyrmex heeri forel, 1874, and pseudomyrmex pallidus (f. smith, 1855) occurred in high abundance in the high water period (906 ind.; 59.0%; 15.1 ind./m2) compared to the dry season (176 ind.; 12.5%; 2.9 ind./m2). crematogaster sp. 3, crematogaster sp. 2, solenopsis globularia (smith, 1858) e dolichoderus voraginosus mackay, 1993 appear with high abundance in the dry period (1,064 ind.; 75.6%; 17.7 ind./m2) when compared to the high water period (555 ind.; 36.1%; 9.2 ind./m2). fig 1. proportion of the number of formicidae individuals spread over subfamilies in c. fasciculata canopies, between the periods of high water and dry season in the pantanal of poconé, mato grosso. fig 2. species richness observed (rarefaction curve) and estimated (bootstrap and jackknife 1) for the ant community in c. fasciculata canopies, during high water (trees 1-6) and dry season (trees 7-12) in the pantanal of poconé, mato grosso. the community was represented by 26 species. the expected number according to the bootstrap diversity estimator is 29 species and 32 for jackknife 1, of which the sampled richness corresponded to 90% and 81% respectively of the species expected for the community (fig 2). camponotus (myrmaphaenus) sp. 1, wasmannia auropunctata roger, 1863, crematogaster sp. 2 and crematogaster sp. 3 corresponded to the species with the highest frequency of occurrence on the c. fasciculata individuals (table 1). myrmicinae was the subfamily with greater richness (13 spp.), followed by dolichoderinae (6 spp.), formicinae (4 spp.) and pseudomyrmecinae (2 spp.). crematogaster sp. 3 (1,196 ind.; 40.6%; 10.0 ind./m2) was the most abundant species of the community and also among the myrmicinae, followed by w. auropunctata (459 ind.; 15.6%; 3.8 ind./m2) and crematogaster sp. 2 (346 ind.; 11.8%; 2.9 ind./m2). l yamazaki et al. – ant community associated with callisthene fasciculata canopies738 ta xa h ig h w at er d ry s ea so n to ta l f re qu en cy t ro ph ic n % in d. /m 2 n % in d. /m 2 n % in d. /m 2 % g ui ld s d ol ic ho de ri na e d ol ic ho de ri ni a zt ec a in st ab ili s f. s m ith , 1 86 2 3 0. 2 0. 1 3 0. 1 <0 .1 16 .7 o a zt ec a sp . 1 11 3 8. 0 1. 9 11 3 3. 8 0. 9 41 .7 o a zt ec a sp . 2 4 0. 3 0. 1 4 0. 1 <0 .1 16 .7 o d ol ic ho de ru s vo ra gi no su s m ac ka y, 1 99 3 3 0. 2 <0 .1 17 1. 2 0. 3 20 0. 7 0. 2 58 .3 o ta pi no m a sp . 1 12 1 7. 9 2. 0 2 0. 1 <0 .1 12 3 4. 2 1. 0 33 .3 o ta pi no m a sp . 2 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 o fo rm ic in ae b ra ch ym yr m ic in i b ra ch ym yr m ex h ee ri f or el , 1 87 4 88 5. 7 1. 5 15 1. 1 0. 3 10 3 3. 5 0. 9 58 .3 o c am po no tin i c am po no tu s ar bo re us (f . s m ith , 1 85 8) 7 0. 5 0. 1 5 0. 4 0. 1 12 0. 4 0. 1 41 .7 o c am po no tu s m el an ot ic us e m er y, 1 89 4 43 2. 8 0. 7 43 1. 5 0. 4 50 .0 o c am po no tu s (m yr m ap ha en us ) s p. 1 16 6 10 .8 2. 8 67 4. 8 1. 1 23 3 7. 9 1. 9 10 0. 0 o m yr m ic in ae a tti ni c ep ha lo te s at ra tu s (l in na eu s, 1 75 8) 2 0. 1 <0 .1 2 0. 1 <0 .1 16 .7 p c ep ha lo te s gr an di no su s (f . s m ith , 1 86 0) 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 p c ep ha lo te s m in ut us (f ab ri ci us , 1 80 4) 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 p c ep ha lo te s pa vo ni i ( l at re ill e, 1 80 9) 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 p w as m an ni a au ro pu nc ta ta r og er , 1 86 3 38 4 25 .0 6. 4 75 5. 3 1. 3 45 9 15 .6 3. 8 91 .7 o c re m at og as tr in i c ar eb ar a an op ht ha lm a (e m er y, 1 90 6) 3 0. 2 0. 1 3 0. 2 0. 1 6 0. 2 0. 1 25 .0 h g c re m at og as te r ar cu at a fo re l, 18 99 5 0. 3 0. 1 5 0. 2 <0 .1 8. 3 o c re m at og as te r qu ad ri fo rm is r og er , 1 86 3 29 2. 1 0. 5 29 1. 0 0. 2 25 .0 o c re m at og as te r sp . 1 11 7 7. 6 2. 0 11 0. 8 0. 2 12 8 4. 3 1. 1 66 .7 o c re m at og as te r sp . 2 12 1 7. 9 2. 0 22 5 16 .0 3. 8 34 6 11 .8 2. 9 75 .0 o c re m at og as te r sp . 3 42 8 27 .9 7. 1 76 8 54 .6 12 .8 1, 19 6 40 .6 10 .0 91 .7 o n es om yr m ex s p. 1 12 0. 8 0. 2 7 0. 5 0. 1 19 0. 6 0. 2 50 .0 m s so le no ps id in i so le no ps is g lo bu la ri a (s m ith , 1 85 8) 3 0. 2 0. 1 54 3. 8 0. 9 57 1. 9 0. 5 66 .7 m g h ps eu do m yr m ec in ae ps eu do m yr m ec in i p se ud om yr m ex p al lid us (f . s m ith , 1 85 5) 30 2. 0 0. 5 6 0. 4 0. 1 36 1. 2 0. 3 75 .0 g a p p se ud om yr m ex te rm ita ri us (f . s m ith , 1 85 5) 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 g a p in de te rm in at ed in de te rm in at ed 1 0. 1 <0 .1 1 <0 .1 <0 .1 8. 3 to ta l a du lts 1, 53 6 99 .2 25 .6 1, 40 7 99 .9 23 .4 2, 94 3 99 .5 24 .5 10 0. 0 to ta l l ar va es 13 0. 8 0. 2 2 0. 1 <0 .1 15 0. 5 0. 1 41 .7 g e n e r a l t o ta l 1, 54 9 10 0. 0 25 .8 1, 40 9 10 0. 0 23 .5 2, 95 8 10 0. 0 24 .6 10 0. 0 t ab le 1 . t ax a, n um be r o f i nd iv id ua ls (n ), re la tiv e ab un da nc e (% ), de ns ity (i nd ./m 2 ) a nd fr eq ue nc y of o cc ur re nc e pe r t re e (% ) o f f or m ic id ae o bt ai ne d in c . f as ci cu la ta c an op ie s, d ur in g hi gh w at er a nd d ry s ea so n in th e pa nt an al o f p oc on é, m at o g ro ss o, a nd it s ca te go ri za tio n in tr op hi c gu ild s (o m ni vo ro us (o ); p ol lin iv or es (p ); m in im um s pe ci al is ts (m s) ; h yp og ei ca ls g en er al is ts (h g ); m in im um g en er al is ts h yp og ei ca ls (m g h ); g en er al is t a rb or ea l p re da to rs (g a p) . sociobiology 63(2): 735-743 (june, 2016) 739 trophic guilds ants associated with c. fasciculata are distributed in six groups of trophic guilds. in total, the omnivorous were dominant (2,818 ind.; 18 sp.; 95.7%; 23.5 ind./m2) over the general minimum hypogeicals (57 ind.; 1 sp.; 1.9%; 0.5 ind./ m2), general arboreal predators (37 ind.; 2 sp.; 1.3%; 0.3 ind./ m2), minimum specialists (19 ind.;1sp; 0.6%; 0.2 ind./m2) and pollinivores (5 ind.; 4sp; 0.2%; < 0.1 ind./m2). the indirect ordering of the community in relation to the trophic guilds (nmds) between the high water and dry periods resulted in a stress = 0.29. the result of the t-test, comparing the scores of axis, did not show any difference (t-test = -0.797; gl = 8.844; p = 0.446) between the distribution of guilds for the two periods. as for the distribution of the guilds over the high water and dry periods, omnivores also predominated, 96.5% and 94.9%, respectively. as for the other trophic groups little variation occurred in relation to abundance in both seasonal periods. it is observed that the ants with general nutritional habits, such as those belonging to the omnivores, general minimum hypogeicals and general arboreal predators groups are more distributed among the trees and over the seasonal periods, while the pollinivores were restricted to the high water period. discussion in canopies of the c. fasciculata myrmicinae was the dominant subfamily, both in abundance as in species richness. this predominance was observed by battirola et al. (2005) in the pantanal of poconé, mt, corrêa et al. (2006), soares et al. (2013) in the pantanal of mato grosso do sul, and by castaño-meneses (2014), in a dry tropical forest in mexico. for ribas et al. (2003), dominance behavior in ants can be observed according to the heterogeneity of host plants and availability of resources. a low availability of resources causes the exclusion of some species due to competition, while a greater availability reduces competition and encourages the coexistence of a greater number of species, especially among non specific or generalist groups such as those existing on c. fasciculata canopies. fernández (1998) pointed out that the high abundance associated with a moderate diversity characterized communities of tropical tree ants. such a pattern may be associated, according to kaspari (2003), to the fact that, in the area around the dominant species, richness and density are often reduced because the subordinate species generally formed small colonies with low recruiting capacity and are located on the outskirts of the territories controlled by the dominant species. arboreal communities generally exhibit high richness of associated species in comparison with other habitats in the same areas. in the c. fasciculata canopies, 26 species were sampled, divided into four subfamilies. a similar result, using the same sampling methods was obtained in the study of ant community in the canopies of attalea phalerata mart. (arecaceae) in the same region, with register of 29 species and 6 subfamilies (battirola et al., 2005), including, inclusively, typically soil species such as atta sp.. however, methodological variations influence these results, as observed by conceição et al. (2014) in a study on cocoa plantations (theobroma cacao l., malvaceae) of different ages in ilhéus, ba, where 113 species of ants were obtained using sardine baits and honey, entomological net and manual collection in trunks and foliage. in mexico, castaño-meneses (2014) while collecting ants using thermal fogging, verified the presence of 21 morphospecies. in costa rica, longino and nadkarni (1990) obtained 21 species on different host plants, while schonberg et al. (2004) sampled 27 species in a comparison study between primary and secondary forest and grassland. floren and linsenmair (1997) captured 61 species on aporusa lagenocarpa a. shaw and a. subcaudata merr. (euphorbiaceae) in malaysia, and stork (1991) recorded 32 species in tree canopies in borneo. it appears, therefore, that richness is similar in arboreal communities from different regions, despite the different sampling efforts, however, the compositions of the communities are extremely variable. cephalotes and crematogaster were richer genus on c. fasciculata. in a. phalerata, solenopsis westwood, 1840 and camponotus mayr, 1861 predominated (battirola et al., 2005). pheidole, camponotus and ectatomma smith, 1868 were the most diverse genres in trees in flooded and nonflooded areas in the pantanal de miranda, ms (soares et al., 2013). pheidole, ectatomma and camponotus were the most diverse groups in clumps of dry matter in the pantanal, ms (côrrea et al., 2006). crematogaster and pheidole presented greater presence in ilhéus, ba (conceição et al., 2014). it is observed that camponotus and pheidole showed a significant proportion of species presence in several local communities (brandão et al., 2009), probably because they are generalist species. cephalotes despite being one of the genus with the greatest presence in number of species in this study fig 3. comparison of scores on nmds axis, generated from the distribution of 26 species of formicidae in c. fasciculata canopies between the high water and the dry season in the pantanal of poconé, mato grosso. l yamazaki et al. – ant community associated with callisthene fasciculata canopies740 showed low abundance, which can be explained by brandão et al. (2009), when affirming that these arboreal species nest exclusively within living or dead branches, making sampling by applying insecticides difficult (adis et al., 1998). the pantanal is a stochastic environment, characterized by its seasonality, thus influencing the structure of the landscape and biological characteristics. studies of arthropods in this region have indicated that their communities can vary over the seasonal periods due to environmental changes imposed the hydrological regime in the region (battirola et al., 2004, 2005, 2007, 2009, 2014; marques et al., 2006, 2007, 2011, 2014; soares et al., 2013). the community of ants in c. fasciculata canopies did not show significant differences in the number of species between high water and dry periods. this may be related to the dominance exercised by some species, such as the crematogaster, which totaled 56.7% of the community. in addition to dominance, factors such as the stability, availability, quantify and quality of resources, and nesting sites present in the habitats can affect the structuring of these communities (e.g. ribas et al. 2003). however, there was a significant difference (t-test = -4.557; gl = 9.943; p = 0.001) in the distribution of species between c. fasciculata individuals during both seasonal periods evaluated. the frequency of occurrence obtained for camponotus (myrmaphaenus) sp. 1, w. auropunctata, crematogaster sp. 2 and crematogaster sp. 3 shows that these species occur in many c. fasciculata individuals, indicating a close interaction with this habitat (table 1). it is observed that, individually, w. auropunctata in the high water season, was the second most abundant species, representing 25.0% of the c. fasciculata community with 384 individuals, and in the dry season, accounted for only 5.3%, with 75 representatives. extensive areas of undergrowth are used as natural habitat by this species, but individuals can secondarily move to the treetops, acquiring characteristics of a dominant arboreal species (majer & delabie, 1993; majer et al., 1994), which would explain the significant variation in their occurrence in this habitat. the greater abundance of w. auropunctata in c. fasciculata canopies in the high water period may indicate a possible vertical migration as a survival strategy of this species. adis et al. (2001) observed soil shifts to tree trunks during periods of high water in the pantanal de mato grosso for acromyrmex lundi carli santschi, 1925 (fomicidae: myrmicinae) shifting their ground nests to tree trunks. solenopsis saevissima (f. smith, 1865) also feature specific survival strategies, which in addition to vertical displacement, also move horizontally over the water, following the high water line in the pantanal. the displacement between the soil, trunks and tree canopies associated with the flooding cycle was also observed for polyxenida (diplopoda) (battirola et al., 2009) and plusioporus salvadorii silvestri, 1895 (diplopoda: spirostreptida) (adis et al., 2001). a dominant species often tolerates a number of nondominant or subdominant species, creating positive associations between them (majer et al., 1994; delabie et al., 2007), which may explain the few changes that have occurred in the structure and composition of the community in the two seasonal periods of this study. castaño-meneses (2014) verified that the abundance in tree canopies in the biological station of chamela, mexico, is greater during the dry period because many plant species flourish in this period. they also have epiphytes, which provide resources such as shelter, water and debris to be used by the ants. these results differ from those obtained in c. fasciculata, because during the high water season, the c. fasciculata foliage matures, while in the dry season, the plant has partial dormancy (corsini & guarim-neto, 2000), changing probably the availability of resources. it appears therefore, that the phenology of c. fasciculata did not influence directly on the number of individuals and species of the ant community, but indirectly, the falling leaves can change the habitat conditions, influencing the distribution of some species. ants can explore a wide variety of resources due to their diversity of eating habits (castaño-meneses, 2014), this fact favors the predominance of omnivorous ants in c. fasciculata canopies and in the most tree species canopies (castaño-meneses, 2014). the disturbed areas are dominated by opportunistic or generalist species, as the same present a competitive advantage due to the varying resources (hoffmann & andersen, 2003), which may explain the large amount of omnivorous in this study, indicating the existence of a few specialized groups as well as the predominance of generalist groups. rojas and fragoso (2000) collected ants in the soil and on vegetation in the chihuahuan desert, mexico, and 72% of the sampled individuals belonged to omnivorous and granivorous guilds, followed by predators, pollinivores and fungi cultivators, facts related by the authors to the availability of resources. castaño-meneses (2014) found in their collections groupings of omnivores (60%), granivores (17%), predators (14%), nectarivores (7%) and herbivores (2%), unlike the study in c. fasciculata, where granivores and nectarivores were separated from herbivores. they also found that predators were more abundant in the rainy months, herbivores were restricted to this same period and omnivores were more abundant during the dry season. changes in the pattern of ant’s composition at different times occur due to variations in eating habitats and in the specie’s ability to use distinct resources (meyer et al., 2010; cook et al., 2011; castaño-meneses, 2014). the results of this study showed that the temporal variation not influence the richness of ant arboreal community, however they influence the distribution of species between c. fasciculata canopies over the assessed seasonal periods. the richness of species in this habitat is similar to that obtained in another study of arboreal communities in the pantanal (battirola et al., 2005). it can be noted, therefore, that the c. fasciculata canopies are an important habitat, contributing to the maintenance of biological diversity in the pantanal of mato grosso. sociobiology 63(2): 735-743 (june, 2016) 741 acknowledgements our thanks to the programa de pós-graduação em ciências ambientais (ppgcam/ufmt/sinop) for the opportunity to carry out this study and to the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the scholarship to the first author for the development of his masters. to the fundação de amparo à pesquisa do estado de mato grosso (fapemat process no. 737641/2008.), and to nebam núcleo de estudos da biodiversidade da amazônia mato-grossense for logistical support. references adis, j. 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(2006). beetle assemblages from an australian tropical rainforest show that the canopy and the ground strata contribute equally to biodiversity. proceedings of the royal society b, 273: 1969-1975. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.899sociobiology 63(2): 841-844 (june, 2016) polymorphic microsatellite loci in the independent-founding wasp polistes versicolor (hymenoptera: vespidae) in recent years, many microsatellite primers have been developed for various animals and plants and the sequences of these primers have been deposited in data banks such as genbank. microsatellite primers developed for a given species are sometimes useful for other species in the same genus (henshaw et al., 2001; kudô et al., 2005; katada et al., 2007; komatsu et al., 2012), so it is possible to search for preexisting suitable primers in the data banks. polistes versicolor olivier is an independent-founding wasp that builds nests formed by a single, uncovered comb attached to the substratum by a single petiole (richards, 1978). in this species, colonies show different development stages throughout the year and are influenced by climatic and ethological factors (gobbi & zucchi, 1980, 1985), which characterize an asynchronous biological cycle. strassmann et al. (1989) and junior et al. (2010) estimated genetic relatedness among nestmates in p. versicolor using enzymes, but no study has been made to evaluate colony genetic structure in this species using microsatellite markers. in this study, we examined whether existing primers developed for polistes could be used for p. versicolor. abstract microsatellite primers developed for a given species are sometimes useful for another in the same genus, making possible to search for pre-existing suitable primers in the data banks such as genbank. we examined whether existing primers developed for polistes wasps could be used for the independent-founding wasp polistes versicolor. we tested 50 microsatellite primers from three polistes species and found that five microsatellite loci show polymorphism in size in p. versicolor. these five loci were highly polymorphic, having four to 10 alleles in p. versicolor with an expected heterozygosity of 0.530–0.836. these loci can be used to study parameters concerning genetic relatedness such as social interactions in colonies and genetic conflicts of interest among nestmate individuals. sociobiology an international journal on social insects y takahashi1, a yamada1, s mateus2, r. zucchi2, fs nascimento2, k kudô1 article history edited by gilberto m. m. santos, uefs, brazil received 17 august 2015 initial acceptance 07 april 2016 final acceptance 26 april 2016 publication date 15 july 2016 keywords corresponding author kazuyuki kudô laboratory of insect ecology, faculty of education, niigata university niigata, 950-2181 japan e-mail: kudok@ed.niigata-u.ac.jp eleven colonies of p. versicolor were collected at a farm near the town of cajuru (47º15’s; 21º26’w) in the state of são paulo, brazil, in late march 2011. all insect samples were kept in 99.5% ethanol until dna extraction. we tested 50 microsatellite primers from three polistes species, 22 from polistes chinensis antennalis (f.) (tsuchida et al., 2003, saigo & tsuchida, 2010), 10 from polistes rothneyi (cameron) (takahashi & yamasaki, 2007), and 18 from polistes bellicosus (cresson) (strassmann et al, 1997), using 222 females of p. versicolor from 11 colonies (mean ± se: 20.18 ± 5.58) (table 1). genomic dna of p. versicolor was extracted from two hind legs. the hind legs were homogeneized with pestles in 1.5 ml tubes, and then mixed with 50 µl extraction buffer containing 150 mm nacl, 10 mm tris-hcl (ph 8.0), 1.0 mm edta, 10 µg proteinase k, and 40 µg chelex 100 (bio-rad laboratories). samples were kept at 56°c for 2 h and at 99.9°c for 3 min (walsh et al., 1991). each solution was then precipitated with ethanol and maintained in te buffer at 4°c. pcr was performed using 1 µl diluted genomic dna (≈1ng) in a mixture consisting of 1 µl primer mix (2.5 mm), 0.1 µl of 10 mm dntp mix, 0.05 µl taq polymerase (5 1 niigata university, niigata, japan 2 faculdade de filosofia, ciências e letras, universidade de são paulo, ribeirão preto-sp, brazil short note y takahashi et al. – polymorphic microsatellite loci in polistes versicolor846 u/ml takara ex taq; takara bio, inc.), 1 µl of 10´ pcr buffer (provided with the polymerase, containing 1.5 mm mgcl2), and 6.85 µl dh2o in a total volume of 10 µl. pcr was carried out using a thermal cycler (gene amp pcr system 2700; applied biosystems) programmed for an initial denaturation for 4 min at 94°c, followed by 24–35 cycles of 1 min at 94°c, 1 min at the annealing temperature of each primer set (described in the table 1), and 45 s at 72°c, with a final extension for 7 min at 72°c. the pcr products obtained were electrophoresed in 8% polyacrylamide gels and visualized by silver staining (bassam et al., 1991). genotype scoring and data entry were conducted independently by two people, and their scores were compared. discrepancies were rechecked and if necessary the sample was rerun. statistical analyses were carried out using the program package arlequin ver. 3.11 (excoffier et al., 2005). we found that five microsatellite loci for which the primer sets were originally developed in independent-founding paper wasps, p. chinensis antennalis (pc25 and pc76), p. bellicosus (pbe411) and p. rothneyi (pr4 and pr10), show polymorphism in size in p. vericollor (genbank accesion numbers, pc25: ab190311; pc76: ab190314; pbe411: af120410; pr4: ab281175; pr10: ab281181). the number of alleles per locus ranged from four to ten with a mean of 6.6, and the observed heterozygosity ranged from 0.593 to 0.743, with a mean of 0.661 (table 1). in all five loci, we successfully detected pcr products for all males (n=134) and therefore null alleles could be ruled out. each locus was tested for hardy-weinberg equilibrium, but none of all loci did not significantly deviated. each locus was also tested for genotyping linkage disequilibrium using arlequin, where we randomly selected one adult female from each colony for calculation. none of the tested loci showed significant linkage disequilibrium (all p-values > 0.05 after bonferroni correction for multiple comparisons). the five microsatellite loci provide a powerful means to examine the social and genetic structure of p. versicolor colonies and will enhance our ability to answer important questions regarding genetic conflicts of interest among nestmate individuals. acknowledgments the authors thank j. m. carpenter for the identification of the species. we also thank k. komatsu and y. yamaguchi for their kind help during the field collection. this study was supported in part by a grant-in-aid for young scientists (b) (no. 21770017) to k. kudô. annealing size temperature number of locus (bp) ( c) cycles alleles he ho references pc25 185 50 28 4 0.737 0.743 tsuchida et al., 2003 pc76 223 50 28 7 0.584 0.608 saigo & tsuchida, 2010 pbe411 173 50 28 4 0.530 0.593 strassmann et al., 1997 pr4 211 50 28 8 0.736 0.635 takahashi & yamasaki, 2007 pr10 206 50 28 10 0.836 0.725 takahashi & yamasaki, 2007 he: expected heterozygosity; ho: observed heterozygosity table 1 characteristics of five microsatellite loci in poliste versicolor. references bassam, b. j, caetano-anolles, g. & gresshoff, p. m. (1991). fast and sensitive silver staining of dna in polyacrylamide gels. analytical biochemistry, 196: 80-83. excoffier, l. g., laval, g. & schneider, s. (2005). arlequin ver. 3.0: an integrated software package for population genetics data analysis. evol bioinform online, 1: 47-50. gobbi, n. & zucchi, r. (1980). on the ecology of polistes versicolor versicolor (olivier) in southern brazil (hymenoptera, vespidae, polistini). i. phenological account. naturalia, 5: 97-104. gobbi, n. & zucchi, r. (1985). on the ecology of polistes versicolor versicolor (olivier) in southern brazil (hymenoptera, vespidae, polistini). ii. colonial productivity. naturalia, 10: 21-25. henshaw, m. h., strassmann, j. e. & queller, d. c. (2001). swarm-founding in the polistine wasps: the importance of finding many microsatellite loci in studies of adaptation. molecular ecology, 10: 185-191. doi: 10.1046/j.1365-294x. 2001.01172.x. junior, k. n., simokomaki, k., gruber, c. v. & del lama, m. a. (2010). sociogenetic structure of polistes (aphanilopterus) versicolor olivier, 1791 colonies (hymenoptera, vespidae, polistini). genetics and molecular biology, 33: 669-675. doi: 10.1590/s1415-47572010005000081. katada, s., suzuki, t. & tsuchida, k. (2007). application of microsatellite primers for the social wasp polistes to another social wasp genus, parapolybia, to estimate genetic relationships among nestmates. entomological science, 10: 1-5. doi: 10.1111/j.1479-8298.2006.00192.x. komatsu, k., mateus, s., zucchi, r., nascimento, f. s. & kudô, k. (2012). application of microsatellite primers developed for polistes in the independent-founding wasp polistes satan sociobiology 63(2): 841-844 (june, 2016) 847 bequaert (hymenoptera: vespidae). neotropical entomology, 41: 204-206. doi: 10.1007/s13744-012-0035-y. kudô, k., ishiguro, n., tsuchida, k., tsujita, s., yamane, sô. & zucchi, r. (2005). polymorphic microsatellite loci for the swarm-founding wasp polybia paulista (hymenoptera: vespidae). entomological science, 8: 5-7. doi: 10.1111/j.14798298.2005.00093.x. richards, o. w. (1978). the social wasps of the americas, excluding the vespidae. london: british museum (natural history) 580pp. saigo, t. & tsuchida, k. (2010). nine newly designed polymorphic microsatellite loci for the japanese paper wasp, polistes chinensis antennalis (hymenoptera: vespidae). applied entomolgy and zoology, 45: 575-577. doi: 10.1303/aez.2010.575. strassmann, j. e., hughes, c. r., queller, d. c., turillazzi, s., cervo, r., davis, s. k. goodnight, k. f. (1989). genetic relatedness in primitively eusocial wasps. science, 342: 268-270. strassmann, j. e., barefield, k., solís, c. r., hughes, c. r. & queller, d. c. (1997). trinucleotide microsatellite loci for a social wasp, polistes. molecular ecology, 6: 97-100. takahashi, j. & yamasaki, k. (2007). isolation and characterization of 10 microsatellite loci in the paper wasp polistes rothneyi (hymenoptera; vespidae). molecular ecology notes, 7: 821-823. doi: 10.1111/j.1471-8286.2007.01715.x. tsuchida, k., saigo, t., tsujita, s., takeuchi, k., ito, n. & sugiyama, m. (2003). polymorphic microsatellite loci for the japanese paper wasp, polistes chinensis antennalis (hymenoptera: vespidae). molecular ecology notes, 3: 384386. doi: 10.1046/j.1471-8286.2003.00457.x. walsh, s. p., metzger, d. a. & higuchi, r. (1991). chelex100 as a medium for simple extraction of dna for pcr based typing from forensic material. biotechniques, 10: 506–513. doi: 10.13102/sociobiology.v62i2.276-280sociobiology 62(2): 276-280 (june, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 second record and dna barcode of the ant tyrannomyrmex rex fernández (hymenoptera: formicidae: myrmicinae) introduction the ant genus tyrannomyrmex (fig 1) is relatively small and endemic to tropical broadleaf wet forests of south and southeast asia (indomalaya biogeographic region, udvardy, 1975; olson et al., 2001). the genus was described from peninsular malaysia based on a single worker, which was designated as t. rex (fernández, 2003). since then, two new species have been described: t. dux, from india (borowiec, 2007), and t. legatus, from sri lanka (alpert, 2013), both known from a single worker. the genus has been recently collected in borneo (fisher et al., in press), and it is possible that it has been found in the philippines as well. indeed, a single male collected in 1965 in the philippines has been putatively placed in the genus based on the similarity of petiole shape and mandibular teeth with the worker described by fernández (2003), but this placement needs to be confirmed (general & alpert, 2012; alpert, 2013). abstract tyrannomyrmex is a rarely collected ant genus from old world tropical forests comprising only three described species, all of them known from a single worker. here we report the discovery of a second worker of tyrannomyrmex rex from a selectively logged primary forest of singapore, increasing the known distribution range of the species to nearly 250 km south-east. we also provide a dna barcode for the species and a partial sequence of the wingless gene. although insufficient evidence prevents us to draw any firm conclusion, the genus seems to be restricted to pristine or relatively undisturbed forests and, as a result, could be highly sensitive to habitat degradation. sociobiology an international journal on social insects j jacquemin1, g sonet1, t bourguignon2, 3, ta evans2, t delsinne1,4 article history edited by rodrigo m. feitosa, ufpr, brazil. received 26 december 2014 initial acceptance 30 january 2015 final acceptance 12 february 2015 keywords solenopsidini, singapore, primary forest, leaflitter ant, dna barcoding, scanning electron microscopy. corresponding author justine jacquemin od nature (ateco), royal belgian institute of natural sciences, 29 rue vautier, b-1000 brussels, belgium e-mail: jjacquemin@naturalsciences.be tyrannomyrmex is placed in the myrmicinae. the principal traits of this rarely collected and documented genus are mandibles with only two apical teeth, and inner margin of the edentate masticatory border of the mandibles with setae. these setae are modified in t. rex (i.e. thick and cylindrical) (fig 2c., d.) but simple and similar to other setae of the body in t. dux and t. legatus. the three species have small eyes reduced to a few ommatidia (fig 2a.), 11-segmented antennae with an ill-defined 3-segmented club (fig 1a., b., c.) and an apparent palpal formula 2,2 (the formula could not be reliably determined in t. dux). also, the mesosoma does not present any groove or suture, forming a single broad and continuous convexity in side view (fig 1c) and the metapleural gland seems to be lacking (fernández, 2003; borowiec, 2007; fig 1d., 2e., f.). the reliable placement of tyrannomyrmex among the myrmicinae was tentative based on morphological data only. research article ants 1 royal belgian institute of natural sciences, brussels, belgium 2 department of biological sciences, national university of singapore 3 czech university of life sciences, prague, czech republic 4 universidad técnica particular de loja, loja, ecuador sociobiology 62(2): 276-280 (june, 2015) 277 based on morphological characters (i.e. presence of transformed setae along internal border of mandibles), fernández (2003) suggested that the genus was close to the adelomyrmecini and to the solenopsidini tribes (according to the taxonomy of that time). alpert (2013) suggested that tyrannomyrmex was close to monomorium, a member of the solenopsidini, by using the comprehensive morphology-based treatment of the formicidae described by bolton (2003). a recent phylogeny of the myrmicinae, based on 11 nuclear gene fragments, confirmed that tyrannomyrmex belongs to the solenopsidini, albeit this clade was somehow redefined (ward et al., 2015). according to this work, tyrannomyrmex legatus is the sister taxon to monomorium latinode + m. brocha, two species distributed mostly in tropical asia, similar to tyrannomyrmex. it should be noted that m. brocha has been transferred to the genus epelysidris and a new genus under description by barry bolton will be erected for m. latinode (ward et al., 2015). here, we report the discovery of a second worker of t. rex, which becomes therefore the first duplicate (species known from two individuals) of the genus. the specimen is documented by high resolution pictures and sem photographs and its dna barcode is provided. materials and methods we collected the ant specimen in december 2012 in macritchie reservoir, singapore (n 01°20’30’’, e 103°49’43’’), during a biodiversity survey of the ants from singapore. it was found in a selectively logged primary forest not exploited for decades, comprising primary forest species at an intermediate stage of regeneration. while the plant species composition was that of a primary forest, it lacked large emergent trees present in adjacent unlogged primary forest. the site was located near an old growth primary forest. altogether, we collected 5525 ant specimens in six sites and 877 specimens in the site in which t. rex was collected. the sampling consisted of 25 samples of leaflitter (1 m² each) collected in each site and placed in berlese extractors. the extractors consisted of a plastic funnel heated with a 100 watt light bulb which we kept running for two to five days, until the leaf litter was completely dried out. we preserved all specimens in 80% ethanol until identification. the t. rex individual was mounted for photography and a hind leg was dissected immediately afterwards for dna analysis. fig 1. high resolution pictures of the tyrannomyrmex rex worker collected in singapore. dorsal (a.), frontal (b.) and lateral (c.) views, and a close-up lateral view of the propodeum, petiole and postpetiole (d.). j jacquemin, g sonet, t bourguignon, ta evans, t delsinne new record of tyrannomyrmex rex278 we took high resolution digital images using a leica mc170 camera attached to a leica s8apo stereomicroscope. a series of images was taken by focusing the sharpness on different levels of the specimen, using the leica application suite v38 (2003-2011) and combined with the stacking software combine zp (hadley, 2010). final editing of the images was done in adobe photoshop cs5. sem photographs of the specimen were taken using a scanning electron microscope fei quanta 200. the specimen is deposited at the royal belgian institute of natural sciences, brussels, belgium (spm_id 4904401; rbins general inventory 33045). we extracted dna from one hind leg using the commercial nucleospin tissue kit (macherey-nagel, germany) and performed the pcr amplification of the 5’ end of the mitochondrial cytochrome c oxidase subunit i (coi) marker (standard dna barcode region) and a fragment of the wingless gene (wg). pcr amplification and bidirectional dna sequencing were executed according to the protocol described by delsinne et al. (2012) and using the primer pairs lco1490 / hco2198 (folmer et al., 1994) for coi, and wg578f (ward & downie, 2005) / wg1032r (abouheif & wray, 2002) for wg. using the blast algorithm (altschul et al., 1990), we searched through genbank and the barcode of life data system (ratnasingham & hebert, 2007) for best matches with the two sequences obtained here. finally, the same two sequences were compared to all coi and wg sequences available for the tribe solenopsidini in genbank. alignments, pairwise uncorrected p-distances (proportion of nucleotide sites at which two sequences differ) and a neighbor-joining tree were calculated using mega 6.06 (tamura et al., 2013). results and discussion the coi barcode of t. rex (genbank accession number: kp294328) has never been sequenced before and is markedly different from all coi sequences available in genbank and bold (< 86% similarity). the best matches involve species of the genera pheidole and monomorium. the genbank sequence that matches the best with our t. rex wg (genbank accession number: kp294329) is the single wg sequence already available for the genus tyrannomyrmex (accession number: kj861941; species: tyrannomyrmex legatus). on the basis of this marker, the distance between t. rex and t. legatus is 2.8% whereas the inter-generic distances between t. rex and any other solenopsidini sequence range from 3.4 to 9.6% (fig 3). fig 2. close-up sem pictures of tyrannomyrmex rex illustrating the eye composed of 6 ommatidia (a.), the very short appressed hairs located within foveae on the pronotum (b.), the modified stout and cylindrical setae on the inner margin of the masticatory border of the mandible (dorsal view c., lateroventral view d.), the large and round propodeal lobe (e.), and an oblique view of the metapleuron (f.). note the absence of metapleural gland orifice on (e.) and (f.). fig 3. unrooted neighbor-joining tree based on pairwise p-distances among all solenopsidini wingless sequences available in genbank. sociobiology 62(2): 276-280 (june, 2015) 279 the discovery of a second specimen of t. rex expands the known range of the species to nearly 250 km to the southeast of the only other known location in malaysia. it also expands the known altitudinal range of the genus, from sea level (24 m; this study) to 1300-1500 m (t. dux) (t. legatus was discovered at intermediate elevation, between 432 and 571 m). it is worth mentioning that tyrannomyrmex rex seems to be rare at the scale of the country. indeed, the specimen mentioned is the single one discovered despite our recent efforts to document leaf-litter ant assemblages from other singaporean forested sites, representative of both degraded and pristine habitats (six sites; 25 berlese samples per site). however, this apparent rarity could result from a methodological artifact since the morphology of the genus suggests subterranean habits and the berlese extraction of leaf-litter fauna is not the more efficient method for documenting hypogean ants. interestingly and as anteriorly noted (fernández, 2003; borowiec, 2007), the genus seems to lack a functional metapleural gland (fig 1d., fig 2e., f.), a structure unique to ants and playing a key role in sanitation and chemical defense (yek & mueller, 2011). this gland has been secondarily lost is some ant lineages, mostly in social parasites and arboreal formicines, probably because their life histories render them less exposed to pathogens than ground-dwelling species (yek & mueller, 2011 and references therein). its absence in tyrannomyrmex, a probable hypogean genus, suggests either that these ants could be social parasites or that they possess other protections against pathogens as observed for instance in some camponotus (walker & hughes, 2011) and polyrhachis species (graystock & hughes, 2011). additional material and anatomical studies are obviously required to test these hypotheses. the unique specimen of t. legatus was found in an undisturbed and unlogged lowland dipterocarp forest in sri lanka, and the t. rex individual studied by fernández (2003) was collected in a forest reserve. we found the second t. rex worker in a selectively logged primary forest unexploited for decades, very similar in terms of vegetation composition to the primary forest nearby. although information about the habitat where t. dux was collected is lacking, available data suggest that this rarely collected genus occurs in pristine or little disturbed tropical forests of asia. singapore has lost more than 95% of its original forest over the last two centuries leading to a massive extinction of its biodiversity (brook et al., 2003). fortunately, efforts were recently made to preserve the remaining pristine areas of the island, to improve the biodiversity value of secondary forests and urban areas, and to increase the connectivity among green patches (fam et al., 2014; tan & hamid, 2014). the presence of tyrannomyrmex rex, a species suspected to be restricted to well-preserved tropical forests, points out that efforts for maintaining singaporean biodiversity are worth being pursued. ackowledgments we warmly thank julien cillis (rbins) for sem photographs and lee gang for field assistance. this work was supported by the lhk fund of the national university of singapore; by the singapore-mit alliance for research and technology; by the belgian federal science policy office (belspo) through an action 1 impulse for research and by the senescyt through its prometeo grant to td (http:// prometeo.educacionsuperior.gob.ec/); and by the internal grant agency of the faculty of forestry and wood sciences, czech university of life sciences (registration number a08/14). references abouheif, e. & wray, g.a. 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(2011). the metapleural gland of ants. biological review, 86: 774-791. doi: 10.1111/j.1469185x.2010.00170.x doi: 10.13102/sociobiology.v63i3.988sociobiology 63(3): 998-1004 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 external morphology of immatures during the post-embryonic development of mischocyttarus nomurae richards (hymenoptera: vespidae) introduction the species mischocyttarus nomurae was described by richards in 1978 from two females collected in fortaleza, ceará state (ce), brazil; it was included in the mischocyttarus cerberus group in a review published by silveira (2004). the individuals are black, with many pale-yellowish gaster markings, with distinct stripes and frontal margin of the proepisternum distinctly elevated (richards, 1978). the males of that species were described by silveira (2004) from samples collected in lençóis (chapada diamantina), bahia state (ba), brazil. among other features, the clypeus is covered by dense recumbent hairs with silvery reflections; the antenna is spirally rolled at the apex, with the last three segments elongated. souza et al. (2015) recently recorded the occurrence of specimens of m. nomurae in montes claros, minas gerais state (mg), brazil, captured in traps using passion fruit bait. these are the only references to the species in the literature. abstract we describe here the immatures of mischocyttarus nomurae richards from twenty colonies collected in the municipality of rio de contas in the chapada diamantina, bahia state, brazil. we determined the number of larval instars in post-embryonic development and measured 145 eggs, 353 larvae, 12 prepupae, and 59 pupae. egg lengths varied from 0.80 to 1.33 mm and their widths from 0.33 to 0.50 mm. the average rate of growth of the larvae was 1.48. cephalic capsule widths varied from 0.20 – 0.43 mm in the 1st instar larvae, from 0.47 – 0.63 mm in the 2nd instar, 0.67 – 0.83 mm in the 3rd instar, 0.87 – 1.20 mm in the 4th instar, and 1.37 – 1.73 mm in the 5th instar larvae. the cephalic capsule of the 1st instar larva was smaller than the width of the egg. the median widths of the 5th instar larvae and the pre-pupae cephalic capsules did not differ significantly. the species has two abdominal lobes that are highly projected forward, and the diameter of first thoracic spiracle is 3.1 times greater than the second. m. nomurae shows development patterns typical of the genus. sociobiology an international journal on social insects aa rocha1, e giannotti2 article history edited by marcel g. hermes, ufpr, brazil received 05 february 2016 initial acceptance 17 july 2016 final acceptance 15 september 2016 publication date 25 october 2016 keywords abdominal lobe, spiracles, larvae, social wasps, polistinae. corresponding author agda alves da rocha, universidade federal da bahia campus anísio teixeira rua rio de contas, quadra 17, lote 58, bairro candeias, cep: 45.029-094 vitória da conquista ba, brasil e-mail: rocha.agda@gmail.com richards (1978) characterized the larvae of mischocyttarus de saussure representatives as having the first abdominal sternite with one, two or three lobes strongly projected, first thoracic spiracle twice as long as the others, and mandibles with only a single elongated tooth. dias-filho (1975) studied the 5th instar larvae of several wasp genera and characterized six species of the genus as having the labrum twice as wide as the mentum, tentorial pits located below the level of the antennae, and mandibles ending in a fine, single, curved tooth. most of the species studied showed larvae with five instars (giannotti & fieri 1991; giannotti & trevisoli 1993; cecílio et al., 2015) and two showed four instars (raposo-filho, 1981; silva, 1984). we describe here the external morphologies of the eggs and immatures of mischocyttarus nomurae richards during their post-embryonic development, and determine the number of larval instars of that species. 1 universidade federal da bahia (ufba), campus anísio teixeira, vitória da conquista ba, brazil 2 universidade estadual paulista júlio de mesquita filho (unesp), rio claro sp, brazil research article wasps sociobiology 63(3): 998-1004 (september, 2016) 999 material and methods the present study was conducted near the town of rio de contas in the chapada diamantina ecoregion, in southcentral bahia state, brazil (sei, 2011). the municipality of rio de contas covers 1082 km2 (13º34’44’’s x 41º48’41’’w) and is located 612 km from the state capital of salvador (ibge, 2015). the regional climate is sub-humid to dry, with a mean annual temperature of 19.1 ºc and mean annual rainfall rate of 813.2 mm (sei, 2011). twenty colonies were collected in february/2013 without adults, and were subsequently maintained in plastic flasks. following their sacrifice at low temperatures, the immatures were removed from the nest cells (using tweezers and a desk magnifying glass) and then fixed in dietrich solution for 48h and preserved in 70% alcohol. eggs, larvae, pre-pupae and pupae were measured under a stereoscopic microscope, with the aid of an ocular micrometer, in the zoology laboratory of the federal university of bahia, campus anísio teixeira (ims-cat-ufba). the following aspects were examined: 1) the largest widths and lengths of the eggs; 2) the greatest widths of cephalic capsules of the larvae, pre-pupae and pupae; and 3) the diameters of the first two spiracles of the last instar larvae (giannotti & silva, 1993; giannotti, 1995; 1998; prezoto & gobbi, 2005). photographic records of immatures and the cephalic capsules were made using a digital camera coupled to a stereoscopic microscope in the zoology department of the júlio de mesquita filho state university of são paulo (unesp-rio claro). scanning electron microscopy of prepared samples of larvae of 5th instar was undertaken in december/2014 at the gonçalo moniz research center of the oswaldo cruz research foundation (fiocruz) in salvador, bahia (ba), brazil. the samples were prepared according to standard protocols used at that research center: five colony samples were fixed in a 2% glutaraldehyde solution in 0.1 m sodium cacodylate buffer (ph 7.4) for 2h at room temperature, and subsequently rinsed in 0.1 m sodium cacodylate buffer (ph 7.4) (3 rinses of 5 minutes each, at room temperature). the samples were then post-fixed in 1% osmium tetroxide in 0.1 m sodium cacodylate buffer for 1h at room temperature, and subsequently rinsed again with 0.1 m sodium cacodylate buffer (ph 7.4) (3 rinses of 10 minutes each, at room temperature). the samples were then dehydrated in an ethanol series (30%, 50%, 70%, 90% and 100% for 40 min. each) and then submitted to critical point drying. after this procedure, the specimens were mounted on stubs, sputter coated with gold, and observed using a jeol 6390lv scanning electron microscope. the growth rates of the larvae were calculated (dyar, 1890), and the non-normally distributed quantitative variables were compared among eight groups using the nonparametric kruskal-wallis test, followed by dwass-steel-critchlowfligner post hoc test to obtain the adjusted p-values for each pairwise comparison in multiple-group analyses. normality was assessed by visual inspection of histogram plots and using the shapiro-wilk normality test. all statistical analyses were performed with r statistical software (r development core team, 2011). a p-value of <0.05 was considered to be statistically significant, and all reported p-values are two-sided. results and discussion morphometry and larval instars. 145 eggs, 353 larvae, 12 pre-pupae, and 59 pupae from twenty colonies were measured and characterized. the eggs, with narrow and transparent chorion, had an average length of 1.03 ± 0.12 mm (n= 145, 0.80 – 1.33 mm) and an average width of 0.40 ± 0.03 mm (n= 145, 0.33 – 0.50 mm) (table 1). the 1st instar larvae differed in width from the eggs, being smaller (p<0.0001; dwass-steel-critchlow-flignertest) (table 1, fig 1). this pattern was similar to that reported for p. lanio (fabricius) (giannotti, 1995) and differed from three other species of the genus that showed 1st instar larvae with dimensions similar to egg diameters: m. cassununga (giannotti & fieri, 1991), m. drewseni (giannotti & trevisoli, 1993), and m. latior (cecílio et al., 2015). the eggs were attached either to the side wall of the cell (giannotti & silva, 1993), or at the angle formed between two walls (giannotti, 1995 and prezoto and gobbi, 2005). the larvae specimens showed five instars, with differences in the median widths between the five groups (p<0.0001, kruskal-wallis test) (table 1, fig 1, fig 2). this is similar to the situation observed with m. cassununga (von ihering) (giannotti & fieri, 1991), m. drewseni saussure (giannotti & trevisoli, 1993), and m. latior (fox) (cecílio et al., 2015). other species of other genera likewise follow this pattern, such as agelaia lepeletier (giannotti, 1988), brachygastra perty (machado et al., 1988), polistes latreille (giannotti, 1995; 1997; prezoto & gobbi, 2005), polybia lepeletier (tech & machado, 1989; carvalho & silva, 1975; solis et al., 2012), and protopolybia ducke (silveira, 1994). giannotti and fieri (1993) associated high numbers of instars to the long post-embryonic development of social wasps. it is immatures n mean sd 95% ci median iqr* min max egg 145 0.40 0.03 (0.39; 0.40) 0.40 (0.40; 0.40) 0.33 0.50 l1 90 0.33 0.04 (0.32; 0.34) 0.33 (0.33; 0.33) 0.20 0.43 l2 46 0.51 0.04 (0.50; 0.53) 0.50 (0.50; 0.53) 0.47 0.63 l3 54 0.72 0.05 (0.71; 0.74) 0.71 (0.67; 0.77) 0.67 0.83 l4 53 1.03 0.07 (1.01; 1.05) 1.03 (1.00; 1.07) 0.87 1.20 l5 110 1.60 0.08 (1.58; 1.61) 1.60 (1.57; 1.67) 1.37 1.73 pp 12 1.58 0.07 (1.53; 1.62) 1.60 (1.52; 1.63) 1.47 1.67 p 59 1.85 0.07 (1.83; 1.87) 1.83 (1.80; 1.93) 1.67 2.03 *interquartile range table 1 – descriptive statistics of the widths (mm) of the eggs and the cephalic capsules of the larvae (l1 – l5), pre-pupae (pp), and pupae (p) of mischocyttarus nomurae (hymenoptera, vespidae). aa rocha, e giannotti – external morphology of immatures of mischocyttarus nomurae1000 therefore probable that the development time of m. nomurae is longer than that of m. atramentarius z. (silva, 1984) and m. extinctus z. (raposo-filho, 1981), which develop four instars, although that has not been directly studied. the average growth rate of the larvae was 1.48 (1.57 between l1 and l2, 1.38 between l2 and l3, 1.43 between l3 and l4, and 1.55 between l4 and l5). the highest rate, between l1 and l2, is similar to that observed in m. latior (cecílio et al., 2015). the average growth rate is in accordance with dyar’s rule. the species showed a higher average growth rate than the other species of the genus. the ascending order of growth was:1.37 in m. latior (cecílio et al., 2015), 1.38 in m. drewseni (giannotti & trevisoli, 1993), 1.42 in m. atramentarius and m. cassununga (silva, 1984; giannotti & fieri 1991, respectively), and 1.46 in m. extinctus (raposo-filho, 1981). no differences were observed in the median width values among 5th instar and pre-pupae cephalic capsules (p= 0.957; dwass-steel-critchlow-fligner test). the elongated 5th instar larva is considered pre-pupae, not an instar. the data on the widths of the pupae cephalic capsules are shown in table 1 and fig 1. (that served as the attachment to the cell wall) (figs 2-b and 3-h). this differs from the l5 larvae, which are fixed to the cell wall by the dorsal crests and lateral humps. the head, formed by the cephalic capsule and buccal pieces, is more sclerotized in l5 larvae. it has a median suture and a pair of temporal bands that are seen in all instars (fig 2-a). the tentorial pits are located below the level of the antennae sockets and in the upper portion of the lateral margins of the clypeus (fig 2-c), as was observed by dias-filho (1975) for other species of mischocyttarus. the fig 1. box plots representing the widths (mm) of the eggs and the cephalic capsules of the larvae (l1 – l5), pre-pupae (pp), and pupae (p) of mischocyttarus nomurae (hymenoptera: vespidae). general morphology. like the other species of wasps studied by dias-filho (1975), the vermiform larvae of m. nomurae had a light-yellow color, thorax with three segments, and abdomen with 10 segments. the abdomen showed a pair of dorsal crests on each segment, ventral lobes, and lateral humps arranged below the spiracle line (fig 2-a: l5). the anal slot, which is transversal, is found on last segment (the anal segment) (fig 3-g). the l1 kept the chorion of the egg and the l2 to l4 larvae kept the exuviae of the previous instar fixed to the final portion of the abdomen fig 2. dorsal (a), lateral (b) views of larvae and frontal view of cephalic capsules (c) of mischocyttarus nomurae (hymenoptera, vespidae): (l1 – l5) – 1stto 5thinstar larvae, respectively. al – abdominal lobe, ar – antenna ring, dc – dorsal crest, la – labrum, li – labium, lp – labial papilla, me – mentum, mp – maxillary papilla, ms – median suture, mt – mandible tooth, mx – maxilla, ps – pleurostome, tb – temporal band, tp – tentorial pit. scale bar: 1 mm. sociobiology 63(3): 998-1004 (september, 2016) 1001 mandible is covered by the bilobate labrum in all of the instars, with only a single tooth, this being elongated, falcate, and thin, as is typical for the genus (dias-filho 1975; giannotti & silva, 1993). both the pair of maxilla and the labium have a pair of elongated brownish papillae (fig 2-c and 3-b). the ten spiracles can be seen in fig 3 (larva l5). the first spiracle of the l5 larvae (located between the prothorax and the mesothorax) has a diameter of 0.26 mm ± 0.04 (n=47, 012 – 032 mm), being 3.1 times larger than the second thoracic spiracle (between the mesothorax and the metathorax; 0.08 ± 0.01 mm [n=47, 0.06 – 0.09 mm] (fig 3-a to 3-d). it is therefore twice the diameter of the other spiracles (richards, 1978). the other spiracles have similar diameters, with the third to the tenth being located in the 1st to the 8th abdominal segments respectively (fig 3a, 3g). the species shows two abdominal lobes on the first abdominal sternite that are strongly projected forward. they can be seen as small protuberances in l3, still little developed in l4, and fully developed in l5 (fig 2-b). according to silveira (2008), this character is widely observed in several fig 3. larva 5 (a to g) and larva 4 (h) of mischocyttarus nomurae (hymenoptera, vespidae): a – side view of the frontal region; b – ventral view of the frontal region; c – detail of the spiracles; d – detail of the 1st spiracle; e – basal portion of the right side abdominal lobe; f – apical portion of the right side abdominal lobe; g – detail of posterior region; h – detail of posterior region. al abdominal lobe, as – anal slot, br – bristles of lobes, cc – cephalic capsule, ex – exuviae; la – labrum, li – labium, lp – labial papilla, mt – mentum, mx – maxilla, sp1 – spiracle between the prothorax and the mesothorax, sp2 – spiracle between the mesothorax and the metathorax, sp3 – spiracle of the 1st abdominal segment, sp9 – 9th spiracle, sp10 – 10th spiracle, tb – temporal band, tn – taenidia. numbers in black indicate the abdominal segments. scale bar: a, b, g, h = 500µm; c, e, f = 100 µm; d = 20 µm. aa rocha, e giannotti – external morphology of immatures of mischocyttarus nomurae1002 subgenera of mischocyttarus. the fact that m. nomurae bears two lobes differs it from m. latior, which has only a single lobe on the first abdominal sternite – this being a character of the subgenus kappa. these lobes are vestigial in the second instar larvae of m. latior, and also in the third instar of m. drewseni (giannotti & trevisoli 1993; silveira, 1998; cecílio et al., 2015). the lobes show ornamentations on their cuticles and bristles that increase in density from the base to the apex, what could indicate a sensorial function (fig 3-a, 3-b, 3-e, 3-f). giannotti and silva (1993) disagreed with the suggestions of reid (1942) and wheeler and wheeler (1979) that the lobes could be used to hold food before its ingestion, as they observed that the workers of m. cassununga delivered already macerated food directly to the buccal parts of the larvae. jeanne (1972) reported larvae of m. drewseni using those lobes to help pump larval saliva to adults that had solicited it. hunt (1988) designated this behavior “lobe erection”. detailed studies of lobe ultrastructure have not yet been undertaken that could shed more light on its function(s). as observed in m. cassununga and m. drewseni, the transparent cuticle of the elongated pre-pupae allows the thoracic appendixes of the pharate pupa (giannotti & silva, 1993; giannotti & trevisoli, 1993), and sometimes their compound eyes (giannotti, 1995), to be seen. the lobes are now strongly projected rearwards as the larva prepares to metamorphosis (fig 4-a). fig 4. lateral view of a pre-pupa (a) and dorsal view of a pupa (b) of mischocyttarus nomurae (hymenoptera, vespidae). al – abdominal lobe, ey – compound eye, l1 – protothoracic leg, l2 – mesothoracic leg, l3 – metathoracic leg, wg – wing. scale bar: 1 mm. sociobiology 63(3): 998-1004 (september, 2016) 1003 the pupae become graded in color from milky white to dark as they became older, also becoming more sclerotized. darkening initiated in the head and then advanced towards the thorax, while the eyes likewise darkened from reddish to dark brown, as had been reported for p. lanio (giannotti, 1995). the wings, which were shaped like filaments below the gaster, then became free and positioned over the gaster (fig 4-b). thus, with the exception of the 1st instar larva, which is much smaller than the egg, m. nomurae shows development patterns typical of the genus: with the presence of abdominal lobes strongly projected forward, but only fully developed in the 5th instar; mandibles with a single elongated tooth; and the first spiracle twice the diameter of the others, as reported by richards (1978). acknowledgements the authors thank fabio akashi hernandes for his valuable support in the photographic registrations of the larvae, and for lending us the digital camera and stereoscopic microscope set up; frederico moreira for statistical support; and the team at fiocruz, in salvador, adriana lanfredi rangel, maria lúcia vieira moreno, and cláudio pereira figueira for their attention and guidance in performing the scanning microscopy. references carvalho, g.g.& silva, m.t. 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(1979). larvae of the social hymenoptera. in h.r. hermann (ed.), social insects (pp. 287-338). new york: academie. _goback open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.7881sociobiology 69(4): e7881 (december, 2022) introduction drywood termites (kalotermitidae) occupy unique niches not available to other xylophagous termites (evans et al., 2013). unlike many subterranean termite species that attack wood above the ground by carrying moisture upwards into the timber, drywood termites have evolved to thrive at the low wood moisture contents typical of these environments (evans, 2010). these adaptations allow drywood termites to attack timber with no direct soil contact, making them difficult to detect and costly to control (lewis et al., 2014). among the most important drywood termites globally is the west indian drywood termite cryptotermes brevis (walker). although first described in the caribbean, the species is thought to have originated from deserts in chile or peru, where it evolved to attack dry wood at low moisture abstract reliable drywood termite detection in structures is challenging but is critical for effective management. a microwave-based non-destructive method was evaluated for detecting termite activity. this study evaluated factors affecting the ability of this device to reliably detect cryptotermes brevis in timber. the device displayed a high probability of successfully detecting c. brevis in naturally infested boards. the system detected termites 97% of the time when used at the highest sensitivity level, while producing few false positives. the number of termites did not affect detection ability, and detectable signals were produced even when a single termite was present. detection success decreased with both increasing wood density and testing perpendicular to the grain in abrupt transition timber species. the device detected termites to a maximum depth of 45 mm in southern pine (pinus spp.), but sensitivity declined with increased wood density with the detection limit declining to only 20 mm in denser tasmanian oak (eucalyptus spp). the device could only detect termites in samples with densities between 392 to 511 kg/m3 in 38 mm thick radiata pine samples. the results support the ability of microwaves to reliably detect c. brevis in timber. sociobiology an international journal on social insects janet mcdonald1, christopher fitzgerald1, barbar hassan2, jeffrey morrell2 article history edited by og desouza, fuv, brazil received 14 march 2022 initial acceptance 16 march 2022 final acceptance 14 october 2022 publication date 28 december 2022 keywords west indian drywood termite, microwave detection, termatrac™, detection depth, wood density. corresponding author jeffrey morrell university of th sunshine coast sunshine coast, queensland, australia. e-mail: jmorrell@usc.edu.au contents (<12-15% emc) (evans et al., 2013; scheffrahn et al., 2009). cryptotermes brevis has since been transported globally in various wood articles and is considered invasive in many countries. the species is currently found in multiple locations in southeast queensland, australia (peters, 1990). their cryptic nature, relatively slow rate of colony growth, and small colony sizes make c. brevis detection difficult in the early stages of an infestation. the traditional method for eliminating c. brevis infestations has been wholehouse fumigation, usually with sulfuryl fluoride (lewis et al., 2014). the process is costly and does not prevent reinfestation. heating wood to achieve internal temperatures of at least 50 °c for an hour or more has been proposed as an alternative treatment method (lewis & haverty, 1996). however, wood is an excellent insulator, and whole-house heating requires substantial energy inputs. a complicating 1 queensland department of agriculture and fisheries, queensland, australia 2 university of the sunshine coast, queensland, australia research article termites non-destructive detection of an invasive drywood termite, cryptotermes brevis (blattodea: kalotermitidae), in timber janet mcdonald, christopher fitzgerald, barbar hassan, jeffrey morrell – cryptotermes brevis detection in timber2 factor in many older structures is the absence of exterior insulation that might increase heating efficiency, especially in the sub-tropical locations where this termite is found, such as southeast queensland. spot treatment with injectable pesticides also has potential, but requires the ability to accurately detect infestations. while c. brevis infestations can be extensive in some structures, most are smaller colonies present in distinct building assemblies. these infestations might be more easily controlled using directed heating systems instead of the whole-house approach or by injection of insecticides in nests. spot treatment could markedly reduce the costs of dealing with minor infestations (perry, 2019; scheffrahn & su, 1997; woodrow & grace, 1997); however, it requires the ability to reliably detect colonies. detecting even advanced infestations can be challenging, with inspectors searching for frass expelled through kick holes or sounding surfaces to detect cavities beneath (evans, 2002). the use of localized treatments will depend on accurately detecting active infestations in timbers in many different configurations without causing excessive damage to the substrate. a variety of non-destructive tools such as acoustic monitoring have been evaluated for detecting termite infestations, but most are of limited value because they are affected by wood variables such as density or grain direction (lewis, 2009). another non-destructive approach uses microwaves (radar) for detecting movement, a temperature sensor for detecting surface temperature, and moisture sensors for detecting changes in moisture levels (termatrac™, ormeau, qld, australia, taravati, 2018). temperature and moisture sensing are likely to be less useful in this application since the termites do not appreciably alter moisture content and any possible temperature changes will be buffered by the surrounding wood. however, radar has the potential to detect c. brevis movement within cavities. the radar emits a stream of microwave signals into the timber which are then reflected back to the device. a moving termite can be detected via changes in the reflected waves which are interpreted on a screen in the form of a moving bar and line graph (recil et al., 2016). this technology is increasingly used for detecting subterranean termite infestations in structures within a variety of substrates (evans, 2002). active subterranean termite attack; however, is characterized by higher degrees of activity with more workers in a given area and moisture content increases as workers transport moist soil into the timber, sharply increasing the probability of detection. conversely, c. brevis colonies are often small, ranging from as few as ten to upwards of several hundred and they are less active in the wood compared to subterranean termites. these characteristics may make them more difficult to detect. the purpose of this study was to evaluate factors affecting the ability of the radar function to detect c. brevis activity. we hypothesized that wood characteristics related to density and grain orientation as well as termite abundance would affect detection capability. materials and methods termite infested timber: timber containing c. brevis was collected from a building in maryborough, qld. the timber consisted primarily of aged hoop pine floorboards (araucaria cunninghamii ait. & d. don) but also included plywood, door panels, and stiles, as well as millwork. because c. brevis is listed as a notifiable pest within the queensland biosecurity act 2014, the infested boards were cut into shorter lengths and placed into sealed containers (40 ×20×25 cm) for safe transport to a secure laboratory in brisbane. the materials were stored at 26 °c and 75% relative humidity and removed for assessment as needed. while moisture content has been shown to affect microwave sensitivity (recil et al., 2016), the moisture levels in this study were low (~12-15 %) and consistent, making them less likely to affect results. detection of c. brevis in infested boards: all tests were performed using a termatractm t3i unit which provided data directly to a mobile phone (termatractm, ormeau, qld, australia). the ability of the device to detect termites in the timber was assessed in a series of tests on both naturally infested boards as well as boards with artificial galleries to which a specific number of termites were added. in some cases, it was necessary to agitate boards prior to testing to stimulate termite movement. depending on the size of the board, a grid consisting of ~30-36 cm squares was marked on one face of each board. boards were placed, one at a time, on a laboratory worktable. the device was placed with the sensor directly on a given grid section for at least 15-20 seconds per reading. an additional three boards were examined by moving the instrument in 30 mm overlapping increments with a 15 second pause between movements. termite activity, as indicated by motion detection on either the line graph or bar output on the display screen, was recorded by board location. the device can be used at intensity levels (gain) ranging from 1 to 10, with ten being the most sensitive. evaluations were performed at intensity levels 2, 6, and 10. each piece of timber was carefully dissected using a chisel and hammer immediately after a reading to confirm the presence or absence of termites in each section of the board. if present, termites were collected and counted. the results from the evaluation and termite collections were used to determine four possible outcomes: 1. negative: termites absent and not detected 2. false positive: termites absent but detected 3. false negative: termites present but not detected 4. positive: termites present and detected effect of termite numbers on detection: the small size of most c. brevis colonies makes detecting even a few termites important. the ability of the instrument to detect differing numbers of termites at increasing depths in the wood was assessed by drilling a 4 mm deep hole in the wide faces of three radiata pine boards (pinus radiata) (20×50 ×80 mm) to sociobiology 69(4): e7881 (december, 2022) 3 create a small chamber to which 1, 5 or 10 pseudergates were added. a five mm thick wafer of eucalyptus nitens (5×50×100 mm) was clamped on top of the radiata pine board, and the instrument, set at the maximum gain, was placed on top of the e. nitens board directly over the chamber with the termites. the ability of the device to detect termites was assessed, and then additional 5 mm thick layers were sequentially added until the device was unable to detect termite activity. each termite number/detection depth was assessed on three samples. in the second trial, pseudergates were placed on roughened filter paper in an open, clear plastic container (diameter: 4 cm; height: 5.5 cm), and a 5 mm thick e. nitens wafer was placed directly on top. the instrument was placed on the wafer directly beneath the active termites. additional 5 mm thick wafers were sequentially added and tested until activity was no longer detectable (fig 1a). the test was repeated three times with 1, 5 or 10 termites in the plate. finally, 5 mm thick wafers were sequentially added to the top of an infested board where the termatrac™ indicated that termites were present in 1 or 2 locations (fig 1 b). the device was assessed at these locations as 5 mm thick e. nitens wafers were sequentially added until activity could no longer be detected. ability to detect c. brevis galleries: the ability of the device to detect galleries within the wood in the absence of live termites was determined by oven drying three infested boards for 3 hours at 102 °c to kill any termites that might still be present. the boards were divided into a grid of 35 one cm squares that were examined using the termatrac™. control boards with no prior infestation were similarly assessed. the infested boards were then dissected, and the tunnels were mapped. effect of wood density on detection ability: the device claims to be capable of detecting termites up to 40 mm from the surface but makes no mention of the possible effects of wood density on detection. wood density varies widely between and even within a given species. the effect of wood density on detection was evaluated by placing five termites on roughened filter paper in an open, clear plastic container as previously described and clamping boards of the same thickness but with densities ranging from ~ 400 to 800 kg/m3 to the container. the device was placed directly over the termites and readings were taken at the maximum gain setting. each density was assessed three times. in a second trial, 38 mm thick p. radiata boards of varying densities were selected. each board was placed on the top of the container with termites. the device was placed directly over the termites and readings were taken at the maximum gain setting. each density was assessed three times using fresh termites. fig 1. setup used to determine detection depth and effect of c. brevis number on device signal strength (a) (side view) and an example of the sampling pattern on a naturally infested board used to determine depth detection showing presence (√) and absence (x) of c. brevis in different sections (b) (top view). janet mcdonald, christopher fitzgerald, barbar hassan, jeffrey morrell – cryptotermes brevis detection in timber4 in the third trial, 38 mm thick p. radiata sapwood samples with densities of ~390 kg/m3 were clamped to the container with termites, and the device was placed directly over the active termites. additional layers were added until the device no longer detected the termites effect of wood grain orientation on detection ability: wood sawyers commonly use two methods to cut trees into boards, each revealing a different type of grain. wood is flat sawn when grain angle is ∼45º from the wide face. in contrast, the grain is angled ∼ 90º to the board face in quarter sawn wood. grain direction may be especially important in hardwoods with large earlywood pores resulting in low density in that region or in softwood boards with an abrupt transition that creates a dense band of latewood. the effect of grain orientation on performance in an abrupt transition softwood was assessed using 35 mm thick p. radiata blocks cut so that the growth rings were parallel or perpendicular to the microwave signal. readings were taken at the maximum gain setting, and the experiment was repeated three times using fresh termites each time. data analysis: the data essentially indicates the presence or absence of termites. as a result, the data were percentages of successful detection under a given set of test conditions. the relationship between wood density and maximum detection depth was determined using simple linear regression analysis in origin pro software. results detection of c. brevis in naturally infested wood: the device successfully detected termites whenever they were present, even when subsequent dissection revealed the presence of only one pseudergate (table 1). although only one sample was inspected that contained no termites, the device also successfully indicated that absence. the ability to detect relatively low numbers of termites is especially important with c. brevis since colony sizes tend to be small and activity levels lower than those found with subterranean termites. it is also notable that the number of positive readings tended to increase with increasing numbers of termites. the results indicated that the device had a high probability of successfully detecting infestations coupled with a low risk of false positives. results also showed that the device was unable to detect c. brevis galleries without active termites. board # board area (cm2) positive/negative readings /board termites collected/board by life stage positive negative pseudergates larvae soldiers reproductives total 1 755 2 8 8 2 2 12 2 560 6 2 5 7 1 4 17 3 560 8 0 112 34 3 14 163 4 900 10 0 42 18 4 7 71 5 660 7 0 2 17 7 26 6 700 5 4 6 3 9 7 840 2 10 1 1 8 266 0 4 0 9 960 10 6 113 47 4 8 172 10 936 7 5 38 7 2 47 11 1120 6 10 23 17 1 5 46 12 504 4 11 2 18 1 6 27 13 1080 15 3 96 52 4 5 157 table 1. ability of the termatrac™ to reliably detect c. brevis in different boards in relation to the number of life stages detected by subsequent destructive sampling. effect of signal intensity on instrument performance: the device can be operated at different intensities (gain levels) that do not increase the intensity of the radar signal, but only amplify the results. the use of low intensity levels creates the risk that termites will not be detected, while high intensities increase the risk of false positives, leading to unnecessary treatments. this factor can be important if there is a lot of extraneous vibration. increasing the gain from 2 to 6 resulted in an increase from 10 % to 62% in the ability of the device to detect actual infestations (table 2). increasing the intensity to 10 resulted in a further 35 % increase in successful detection. conversely, using the device at level 2 intensity resulted in only ~3 % false positives, but that coupled with only 10% detection of actual infestations suggests that this level would be too low to be useful for this species. increasing the intensity to 6 resulted in 12 % false positives, while increasing the intensity to 10 resulted in 22 % false positives. while detection remains the primary goal of inspection, the results illustrate the trade-off. inspectors would need to determine which level of false detection was acceptable. sociobiology 69(4): e7881 (december, 2022) 5 effect of colony size on detection: chambers with one to eleven termites were assessed using the device. termites were consistently detected when only a single termite was present, although the chamber had to be disturbed by slight banging to get the termite to move (figure 2). this would be relatively easily accomplished in practice by tapping the wood ahead of placing the device. termite detection declined with depth in the wood, but a single termite was still detectable up to 20 mm inward from the wood surface. these results were consistent with subsequent thickness tests and illustrated the ability of the device to detect small numbers of termites near the wood surface. effect of wood density and grain orientation: the device could detect termites to a maximum depth of 43 mm in southern pine, but this figure declined steadily with increased wood density to a maximum depth of only 20 mm in e. obliqua which had a density approaching 650 kg/m3 (table 3). the termites used in these studies were primarily in hoop pine, which has relatively low density (~530 kg/m3). it is unclear whether they can attack denser timbers, but the results indicate that the device has a limited ability to detect infestations in denser timbers. while this would be a limitation in larger timbers and poles (brodie et al., 2021), it poses less of an issue in building components. table 2. reliability of termatrac™ detection of c. brevis as determined by frequencies of true and false readings for termite infested boards. instrument sensitivitya frequency (%) true positive false positive true negative false positive 10 97.2 22.0 78.0 2.8 6 62.5 12.1 87.9 37.5 2 10.2 2.9 97.1 89.8 asensitivity as determined by the amplification of the signal fig 2. examples of termatrac™ output as affected by the number of termites and their depth in the wood. lines above the baseline denote termite movement (i.e. detection). note the decline in signal with increasing depth with a given number of termites but no signal change with increasing termite numbers. subsequent trials using 38 mm thick radiata pine samples with densities ranging from 392 to 781 kg/m3 showed that the device could only detect termites in samples with densities less than or equal to 512 kg/m3 (table 4). the maximum depth of detection in p. radiata was 45 mm when the density of tested samples was 392 kg/m3. increasing wood density was negatively correlated with detection illustrating the potential detection limitations associated with wood density (figure 3). grain orientation of the radiata pine block markedly affected the detection ability of the device. the device failed to detect termites through 35 mm thick radiata pine blocks when the test was performed perpendicular to the growth rings (i.e. radially) but successfully detected the same termites when the microwave beam was transmitted tangentially. these results indicate that care needs to be taken when using the device on timbers with an abrupt earlywood to latewood transition. sr. no. density (kg/m3) maximum detection depth (mm) wood material 1 432 43.6 southern pine (pinusspp) 2 511 35 shining gum (eucalyptus obliqua) 3 548 33 qld maple (flindersia breyleyana) 4 600 30 tasmanian oak (e. obliqua) 5 660 29 shining gum (e. nitens) 6 776 20 tasmanian oak (e. obliqua) table 3. maximum termatrac™ detection depth in wood species with different densities. janet mcdonald, christopher fitzgerald, barbar hassan, jeffrey morrell – cryptotermes brevis detection in timber6 it would also be helpful to ensure proper training of inspectors since interpretations can vary among operators (zahid et al., 2012). further studies are recommended on timbers with other anatomical features that might affect the signal. discussion effective control of drywood termites in structures is highly dependent on accurate inspection results since these species nest exclusively in wood and can be in any part of a structure (lewis & lemaster, 1991). currently, the pest control industry relies heavily on visual signs or destructive methods to locate drywood termites (evans, 2002). an effective non-destructive inspection device must reliably detect infestations with a minimum of false detections that lead to unnecessary damage when applying treatments. our test successfully detected termites whenever they were present. true positives and true negatives were the most common results found at the maximum sensitivity level. although the highest sensitivity level improved the ability to detect termites, it also increased the probability that other factors such as exterior vibrations could affect output, creating false positives. the successful detection rates were higher than results from previous studies for subterranean and drywood termites (duarte et al., 2014; evans, 2002). separate studies showed that the device also performed better in detecting drywood termites than either acoustic emission or termite dogs, although the dogs only produced a < 1% false positive response (brooks et al., 2003; lewis & lemaster, 1991). wood thickness was also an important contributor to signal strength, while the termite number did not affect detection ability. the device detected even a single termite in artificial galleries or naturally infested boards, and termite detection declined with the increased thickness of the wood. these results are consistent with the previous studies on other drywood termite species (taravati, 2018). similar observations were recorded previously for a stored grain insect pest where the device output signals were not dependent on insect numbers under observation (mankin, 2004). single termites in our tests tended to move more compared to groups of termites that tended to aggregate. this may help explain the higher signal strength observed when using one termite. the factors affecting detection of small vs large numbers merit further study. detecting termites in relatively thin wood samples should be simple, but many building elements are thicker and may contain multiple layers. the manufacturer claims the device detects termites up to 40 mm from the surface. however, wood density, grain orientation, moisture content, and wood species can affect microwave propagation through the wood. maximum detection depth in previous studies has been reported to be 35 and 50 mm in pine and western hemlock (tsuga heterophylla) or douglas-fir (pseudotsuga menziesii), respectively which have densities similar to hoop pine (lewis et al., 2009; taravati, 2018). wood density affected detection ability in our study and the device could only detect termites in samples with densities ≤511 kg/m3 with similar thicknesses (38 mm). moreover, grain orientation also affected detection ability. similar results were also reported in previous studies (lewis et al., 2009). the very high rate of positive c. brevis detection makes this device a valuable tool for detecting this termite species in structures. however, in-house or building conditions may be quite different from controlled laboratory conditions with regard to wood thickness, wood species, paints, wall layers, wood density, moisture conditions, and external noise factors (taravati, 2018). further in-house or building studies are recommended to validate the laboratory results. conclusions cryptotermes brevis infestations in structures are very difficult to detect due to their cryptic nature, small colony size, and wood nesting habit. the termatrac™ successfully detected termites in wood and appears to be useful for detecting infestations for subsequent remedial treatments. 400 450 500 550 600 650 700 750 800 0 5 10 15 20 25 30 35 40 45 50 d e te ct io n d e p th ( m m ) density (kg/m3) r= -0.97 r2= 0.95 fig 3. relationship between maximum detection depth and density of different wood species. density (kg/m3) detection 392 yes 453 yes 512 yes 560 no 596 no 641 no 781 no table 4. ability of the termatrac™ to detect c. brevis through 38 mm thick pinus radiata samples of increasing density. sociobiology 69(4): e7881 (december, 2022) 7 conflicts of interest the authors declare no conflicts of interest. author contributions the first two authors conceptualized, provided resources, and developed the methodology. the third author performed the experiments and the fourth author assisted with method development and validation. all authors contributed to the analysis of the data and preparation of the manuscript. data availability the authors will provide all data upon request. references brodie, g., thanigasalam,d.b., farrell, p., kealy, a., french, j.r.j. & ahmad (shiiday), b. 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(2012). assessment of technologies and dogs for detecting insect pests in timber and forest products. scandinavian journal of forestry research, 27: 492-502. http://www.pestboard.ca.gov/howdoi/research/ucbfinal.pdf http://www.pestboard.ca.gov/howdoi/research/ucbfinal.pdf doi: 10.13102/sociobiology.v62i3.746sociobiology 62(3): 356-363 (september, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nestmate recognition in the amazonian myrmecophyte ant pseudomyrmex concolor smith (hymenoptera: formicidae) introduction in social insects, altruistic behavior towards relatives is a direct result of nestmate recognition, which is the ability to discriminate members of their own colony from non-members (breed & bennett, 1987; breed, 2014). the same pattern is repeated in other social arthropods (tizo-pedroso & del-claro, 2007; del-claro & tizo-pedroso, 2009). nestmate recognition behavior is a central feature for maintaining the colonial cohesion in eusocial insects, since it allows not only altruistic acts towards relatives but also territorial-environmental resources abstract nestmate recognition is fundamental to colonial cohesion in social insects, since it allows altruistic behavior towards relatives, recognition of intruders, territorial monopoly and resources defense. in ants, olfactory cues is a key factor in this process. pseudomyrmex concolor is a highly aggressive ant that defends their host plant tachigali myrmecophila against herbivores. however, this defense depends on the ant ability to discriminate in order to treat differentially between members of their own colony and intruders . in this study we investigated “whether” and “how” p. concolor recognizes nestmates from non-nestmates. we hypothesized that p. concolor is skillful in recognizing nestmates and tested it in field with experiments using nestmates and non-nestmates. additionally, to test the efficiency of resident ants against intraspecific competition during colony foundation, we simulate the plant occupation by a competitor queen, introducing nonnestmates queens in plants previously occupied by p. concolor. for the issue of the “how”, we hypothesized that the main cue used by this ant in nestmate recognition is olfactory signal. thus, we tested adaptive threshold model, which predicts that, if the individual odor and colony’s internal template are discrepant enough, the resident nestmate will behave aggressively towards incoming individuals. in this case, we confined nestmates with non-nestmates odors, and then, we reintroduced them in its host plants. in each experiment the frequency of aggressive behaviors were recorded and compared. results showed that p. concolor recognize and discriminate nestmates from non-nestmates workers (biting and stinging them) and exclude potential competitors queens. workers reintroduced in their own colony after impregnated with non-familiar odor were treated as non-nestmates. the adaptive threshold hypothesis was confirmed, the main cue used by this ant species in nestmate recognition is olfactory signals. sociobiology an international journal on social insects psmjr. pacheco 1,2, k del-claro3 article history edited by gilberto m. m. santos, uefs, brazil received 07 january 2015 initial acceptance 27 march 2015 final acceptance 02 april 2015 keywords amazonia, social insects, chemical, cues, aggressiveness, kin selection. corresponding author kleber del-claro instituto de biologia universidade federal de uberlândia rua joão naves de ávila, 2121 campus umuarama, bloco 2d sala 28 38400-902, uberlândia, mg, brazil e-mail: delclaro@ufu.br defense, parasites avoidance and intruders recognition (wilson, 1971; crozier & pamilo, 1996; bos & d’ettorre, 2012). thus, the studies about nestmate recognition are fundamental to understand the adaptive success in eusocial insects (hamilton, 1964a, 1964b; hamilton, 1972; sturgis & gordon, 2012). this mechanism consists in the expression and detection of recognition signals, as well as the assessment of these cues and the behavioral responses which arise during the process (beecher, 1982; sherman & holmes, 1985; starks, 2004). during the evolutionary process, the cuticular hydrocarbons, which are waterproof waxes that cover the external surface of research article ants 1 university of são paulo, ribeirão preto, sp, brazil 2 federal university of amapá, macapá, ap, brazil 3 federal university of uberlândia, minas gerais, brazil sociobiology 62(2): 356-363 (september, 2015) 357 insects, have an important role in the communication system of social insects (wilson, 1971; lockey, 1988; nunes et al., 2009). these substances play a central role in ants such as recognition of species, sexual pheromones, marking of territories and parental recognition (hölldobler & wilson, 1990; van zweden & d’ettorre, 2010). there are several evidences that olfactory cues (cuticular hydrocarbons) are responsible for recognition and discrimination between nestmates and non-nestmates in ants (hölldobler, 1995; sudd & franks, 1987; carlin, 1988; errard & hefetz, 1997; vander meer & alonso, 1998; astruc et al., 2001; katzav-gozansky, 2008; van zweden & d’ettorre, 2010; newey, 2011; bos & d’ettorre, 2012; sturgis & gordon, 2012; nascimento et al., 2013). the individuals are recognized as nestmates when their recognition cues are congruent with internal patterns, template, of their own colony (vander meer & alonso, 1998; sturgis & gordon, 2012). in the adaptive threshold model, if the difference between odor of individual and internal template of colony is sufficiently discrepant, the resident nest member will aggress the intruder (sherman et al., 1997). nestmate recognition in social insects is mainly based on olfactory cues and, thereby, as predicts adaptive threshold model, ants should accept nestmates and reject nonnestmates. the rejection behavioral response, generally agonistic, is a consequence of this process and is used as a conspicuous sign for nestmate recognition in eusocial insects (carlin & hölldobler, 1983; bos & d’ettorre, 2012). ants are highly aggressive to intraand inter-specific intruders, displaying a series of agonistics behaviors towards non-members of the colony (carlin, 1988; crosland, 1990; johnson et al., 2012). this phenomenon is called discrimination mechanism, which is the display of different behaviors to nestmate and non-nestmates according to their recognition (barnard & aldhous, 1991). thereby, nestmate recognition plays a central role during the discrimination. in the north of the amazon region, the stinging ant pseudomyrmex concolor (smith, 1860) nests inside hollow rachis (domatia) of the leaves of tachigali myrmecophila ducke (fabaceae: caesalpinioideae) (wheeler, 1921). pseudomyrmex concolor is a highly aggressive ant, attacking incoming individuals that cause any disturbance and thus, defending their host plant against herbivores. plants from which p. concolor colony was experimentally removed are more susceptible to herbivory with the leaf longevity and apical growth almost twice smaller than plants without ants (fonseca, 1994). additionally, seedlings of t. myrmecophila in the early developmental stages are exclusively nested by p. concolor, revealing great competitive ability of this ant. these results suggest that p. concolor is skillful to defend its host plant, thus, it avoids the invasion and the competition of intruders. however, that defense depends upon the ability of p. concolor to recognize nestmates and non-nestmates, since natural selection favored kin recognition systems in order to detect effectively relatives, discriminating and avoiding potential competitors (sherman et al., 1997). although abundant, ant-plant mutualistic relationships have been poorly studied in the amazon region regarding the behavioral ecology, especially from behavior recognition perspective and aggression towards aliens mediated by olfactory cues. studies about nestmate recognition system (jaffé et al., 1986; mintzer, 1982; starks et al., 1998) and kinship (mintzer & vinson, 1985) in pseudomyrmex genus are scarce and little is known about how the neotropical pseudomyrmex ants recognize nestmates. here, we investigated the nestmate recognition system in the stinging amazon ant p. concolor. at the first time we tested the hypothesis that p. concolor is skillful in recognizing and discriminating nestmates from non-nestmates workers. in order to test the hypothesis that colonies of ants are effective in preventing intraspecific competition, we simulated the plant occupation by a competitor queen, introducing intraspecific non-nestmates queens in host plants previously occupied by p. concolor. lastly we also hypothesized that the main cue used by this ant species in kin recognition is olfactory signal and tested whether and how olfactory signals can influence the nestmate recognition ability in p. concolor. based in adaptive threshold model, we predicted that p. concolor workers will behave aggressively towards nestmates impregnated with olfactory cues distinct of its own colony. material and methods study area field experiments were carried out during june and july 2013 and may to july 2014 in the terra firme amazonian forest environments of river curiaú reserve about 20 km north of macapá, amapá, brazil, located between 00o 15’n and 51o 00’ w. the vegetation is mainly evergreen and has a variable canopy about 30-50m in height and presents short frequency deciduousness in the driest season with flowering and fruiting throughout the wet season. the regional climate is classified, according to köppen, as wet tropical, characterized mainly for high annual rainfall rate and the average annual temperature is 27,6o c, with 31oc maximum and 23oc minimum. the average annual rainfall is 2.850mm, being one of the rainiest places in brazil (alvares et al., 2013). nestmate recognition experiments and behavioral observations twenty plants of tachigali myrmecophila (between 0.70 m and 2.17m) colonized by pseudomyrmex concolor were paired according to height, number of leaves and leaflets. we tagged each plant pair with letters (a to j) and each plant received a numerical designation (e.g a1 and a2 composing a pair). plants separated by a minimum distance of 7 meters were selected to compose the pair, but most plants distanced more than 15 meters from each other. after pairing, we performed behavioral experiments based on two experimental groups: a) control group removing and reintroducing an ant of the same colony (e.g. a1 x a1; a2 psm pacheco jr., k del-claro – nestmate recognition in the amazonian myrmecophyte ant pseudomyrmex concolor358 x a2;...) and b) treatment group introducing ant of another colony (e.g. a1 x a2; a2 x a1; ...). we performed twenty introductions for each behavioral test. nestmate and non-nestmates were randomly collected with entomological feather-weight forceps, marked with acrylic white ink docrafts artistepara® and kept confined for ten minutes in glass tubes (7,5cm x1,0cm x0,8cm; new and clean) in order to reduce the handling of the collection. then, a single related worker of p. concolor (control group) or unrelated (treatment group) was introduced on the distal leaflet of one t. myrmecophila leaf randomly chosen. furthermore, in order to simulate interspecific competition and the presence of a potential herbivore we performed twenty introductions of azteca sp. (formicidae) and termites (termitidae) in each tested plant respectively. ants of azteca genus is a reasonable model for interspecific competition test, since it participates of ant colony replacements during ontogenetic succession of tachigali genus in amazon forest (fonseca & benson, 2003) and termites were used in order to simulate the presence of herbivores in host plant (oliveira et al., 1987). additionally and similarly, aiming to simulate the plant occupation by a competitor queen, we introduced fifteen intraspecific non-nestmates queens in fifteen different plants. behavioral interactions were observed for five minutes, after the first contact resident-intruder, and we recorded absence (no aggression) or presence of bites and/or stings (aggression) since these behaviors are very conspicuous and easy to record and indicate discrimination between individuals. the data were subsequently analyzed according to the all occurrence sampling method (altmann, 1974). all introductions were independent, in other words, no individual introduced was used twice and each plant was tested only once for each experimental group. finally, to test the influence of olfactory cues in the nestmate recognition ability of p. concolor, five nestmate or non-nestmate ants and other ant species (azteca sp.) were accommodated in a clean new vial glass (42mm x 21mm x 7mm) for 60 minutes in order to impregnated the bottles with their smell. then workers of p. concolor were collected, marked and confined for 60 minutes in the vials containing just odors of nestmates (control group), odors of intraspecific non nestmates (treatment 1) and odors of other ant species; azteca sp. (treatment 2). after confinement, nestmates were reintroduced in their host plants. we performed twenty introductions for each experimental group. statistical analyses behavioral experiments showed discreet and independent variables data, being categorized on the occurrence and absence of aggression and assessed through ranking testing within two positions. thus, for data analyses we followed breed model (breed, 2003), where chi-square test was used to compare differences between treatment and control group in experiments about nestmate recognition. results pseudomyrmex concolor workers discriminate introduced non-nestmates from nestmates. during the experiments we recorded 80% of aggression towards intraspecific non-nestmates and just 10% of reintroduced nestmates were attacked by resident ants. aggressive behaviors such as bites and stings were targeted significantly to workers of treatment group (χ2=19.79, p<0.001, n=20) (fig 1). unsurprisingly, all azteca workers and also termites introduced in the plant were beaten by p. concolor. thus, the aggression frequency was similar between interspecific ants and termites. on the other hand, 60% of nestmate workers were allowed to enter the colony (χ2=13.78, p<0.001, n=20) (table 1). thereby, workers of p. concolor treat both intraand interspecific nonnestmates as potential competitors or herbivores. as expected, workers of p. concolor were effective in preventing competing queens to establish in the host plant. all introduced intraspecific queens non-nestmates (n=15) were attacked, chased and/or removed from the plant by resident workers. table 1. comparison on the frequency of nestmates and non-nestmates allowed entering the domatia of tachigali myrmecophila (fabaceae) colonized by pseudomyrmex concolor (pseudoyrmecinae) ants in the amazon forest. enter inside the domatia yes no χ2 p nestmate 12 60% 8 40% 13.78 <0.001 pseudomyrmex concolor non-nestmate 1 5% 4 95% fig 1. occurrence of aggression between nestmate and nonnestmate (χ2=19.79, p<0.001 n=20) in pseudomyrmex concolor (pseudoyrmecinae) ants observed during recognition experiments conducted in the amazon forest host plant tachigali myrmecophila (fabaceae). sociobiology 62(2): 356-363 (september, 2015) 359 finally, we confirmed the adaptive threshold model in p. concolor. after 1h of intraspecific non-nestmates odors exposition (treatment 1), ants reintroduced in their own host plant were attacked in 30% of introductions by its nestmates. on the other hand when ants were introduced into their colonies after contact with nestmates odors (control), aggression rate was just 5% (χ2=4.32, p<0.05, n=20). fifty-five percent of nestmates that came into contact with odors of azteca workers (treatment 2) were bitten (χ2=11.9, p<0.001, n=20). there was no significant difference of aggression frequency between ants confined in vial glass with intra or interspecific odors (30% vs 55%, χ2=2.55, p>0.10) (fig 2). therefore, contact with disparate odors between internal template and individual odors provided interference in nestmate recognition ability in p. concolor. we recorded 21 behavioral acts, ranging from non-aggressive to aggressive ones recorded and described in table 2 and table 3. the analysis of recorded behaviors enable us to suggest a sequential behavioral pattern to the two occasions, when workers are and are not recognized by nestmates (fig 3). fig 2. frequency of aggression towards workers ants of pseudomyrmex concolor (pseudomyrmecinae) which remained in contact with odor of its nestmates (control, n=20), intraspecific non-nestmates odors (treatment 1, n=20) and interspecific ant odor (treatment 2, n=20). control vs. treatment 1: χ2=4.32, p<0.05; control vs. treatment 2: χ2=11.9, p<0.001 and treatment 1 vs. treatment 2: χ2=2.55, p>0.10. to accept introduced ant is accepted and allowed to enter inside the foreign plant domatia to lick resident ant touched with its palps the gaster of the introduced individual to ignore introduced individual did not stimulate any behavior in the resident antennation reciprocal antennation between resident and introduced individual to inspect to touch with its antennas, the gaster, thorax and head of the introduced individual. this behavior can be performed by several resident ants, and the introduced remained still during the inspection self-grooming introduced ant rubbed parts of its body. this behavior can be performed between antennas and forelegs, as well as between hind legs and gaster gaster vibration semi-rotation movements of gaster shacking it avoidance introduced or resident ant changed in 90o or 180o the axis of its body before interaction assault resident ant changed its course towards the introduced individual, touching it with its antennas in the gaster or head of the intruder to chase resident ant followed the intruder for more than three seconds touching it with its antennas in the gaster of the intruder to scape introduced or resident ant changed in 90o or 180o the axis of its body after interaction nibbling biting without trapping the intruder’s body with the jaws biting trapping and compressing the jaws in parts of the intruder’s body, such as legs, antennae, gaster and jaws. the intruder was bitten by one or more residents simultaneously full attack resident moves towards the intruder and bites it without any prior interaction to drag resident bites the introduced individual (antennae, legs and petiole) and moved them beyond its initial position to carry resident ant bites and lifts the intruder above the level of its body to expulse resident ant threw the intruder from its host plant or removed its corpse after death tug of war resident ants were biting and pulling the intruder in the opposite directions from its body axis. the intruder was immobilized because its bodily appendages were pulled in different directions, thus, it was unable to escape fighting residents and intruders were engaged in mutual bites. they were entwined and stuck with their mandibles, trapping and compressing their opponent. when fights were performed with interspecific ants, residents used the sting as pparatus attack. sometimes, the intruder remained attached in the body of the resident even after death to sting resident ant ventrally doubled its gaster towards the intruder, leaned on the two pairs of hind legs and then, inserted the sting in the body of the intruder to kill after bites and/or stings the intruders stopped its body movements table 2. behavioral acts exhibited by the ant pseudomyrmex concolor (pseudomyrmecinae) during nestmate recognition experiments conducted in the amazon forest host plant tachigali myrmecophila (fabaceae). psm pacheco jr., k del-claro – nestmate recognition in the amazonian myrmecophyte ant pseudomyrmex concolor360 fig 3. behavioral pattern identified in pseudomyrmex concolor (pseudomyrmecinae) ants observed during nestmates recognition experiments conducted in the amazon forest host plant tachigali myrmecophila (fabaceae). in brackets, the frequency of behaviors observed during the interactions (n=80) between nestmate (grey label) and non-nestmates (black label). behaviors control (n = 20) conspecific (n = 20) interspecific ant (n = 20) termites ( n = 20) to accept 13 1 0 0 to lick 11 0 0 0 to ignore 0 0 1 3 antennation 19 9 4 3 to inspect 15 8 3 5 self grooming 18 8 0 0 gaster vibration 0 1 0 0 avoidance 0 1 3 4 assault 3 0 0 0 to chase 0 10 0 0 to scape 0 6 1 0 nibbling 1 1 0 0 biting 0 16 20 20 full attack 0 11 15 16 to drag 0 7 11 5 to carry 0 2 6 1 to expulse 0 1 4 1 tug of war 0 1 0 0 fighting 0 5 13 2 to sting 0 0 18 13 to kill 0 0 16 3 table 3. results of interactions between the resident ant pseudomyrmex concolor (pseudoyrmecinae) and introduced nestmates (control group), intraspecific non-nestmates (azteca sp.) and termites, in the amazon forest host plant tachigali myrmecophila (fabaceae). the line below “avoidance” separate between non(above) and aggressive (below) behaviors. discussion the manipulation experiments confirmed the hypothesis that p. concolor is skillful in recognizing nestmates and discriminating non-nestmates according to its host plant. the adaptive threshold hypothesis was also confirmed; results demonstrated that the main cue used by this ant species in nestmate recognition is olfactory signal. workers of p. concolor were significantly more aggressive towards non-nestmates, both intra and interspecific, than against nestmates. similar results were observed to other ant species (bos & d’ettorre, 2012; sturgis & gordon, 2012) and social arthropods (breed & bennett, 1987; jungnickel et al., 2004; nunes et al., 2008; tizo-pedroso & del-claro, 2014). similar to other formicidae, p. concolor workers also recognize and avoid, chasing or attacking all alien introduced queens. the second hypothesis that workers prevent a second nesting in previously colonized plants, reducing intraspecific competition, was also corroborated. newly fertilized queens searching for nesting sites are often killed or removed by pseudomyrmex ants that establish obligatory mutualistic relationships with myrmecophyte plants (janzen, 1967; janzen, 1973). the low frequency of t. myrmecophila trees unoccupied in similar sites in the amazon forest (fonseca & benson, 2003), suggest that the competition for this plant species by p. concolor queens is strong. thus, the defense of the host plant by resident ants against herbivores and any other incoming individual is imperative, not arising from the success of this mutualism, but as a result of the highly effective colonial defense due to nestmate recognition. the behaviors recorded in p. concolor revealed a common behavioral pattern during the nestmate recognition mechanism in social insects (wallis, 1970; wilson, 1971; carlin & hölldobler, 1983; carlin & hölldobler, 1986; carlin & hölldobler, 1987; hölldobler & wilson, 1990; breed & sociobiology 62(2): 356-363 (september, 2015) 361 page, 1991;), systematized in figure 3. during behavioral interactions, the first contact of the resident ant was a touch with its antennas on the introduced individual’s body or mutual antennal touches between them. after this inspection the individual introduced could be tolerated, ignored or attacked by the resident ants. however, the physical contact has not occurred often before the aggressive interactions. in this case, the resident ant threw himself towards the incoming individual before there was any body-to-body contact, biting legs, antennas, mandibles and petiole. this aggression, which occurs even without investigation, reduces the time that the intruder could remain on the plant. recognition is the ability of the identify individuals, while discrimination is the differential treatment for another individual based on recognition (hepper, 1986; barnard & aldous, 1991). thus, the reduced time between recognition and discrimination demonstrated in p. concolor combined with its highly aggressive behavior and constant patrolling in the host plant provides an efficient defense against incoming individuals and competitors. the same is also observed in other arthropods (del-claro & tizo-pedroso, 2009). behavioral tests about the influence of odor during nestmate recognition demonstrated that workers of p. concolor introduced in their colonies, after contact with both interspecific and intraspecific non-familiar odors, were treated differentially by their nestmates. the one-hour exposure to non-familiar odors sets off aggressive behaviors such as biting and threats to those introduced ants. it’s suggest that transfers of olfactory cues used in recognition through contact with the unfamiliar odor may have occurred. the contact with discrepant odors from the cuticular pattern interferes in the nestmate recognition in this ant species. thus, workers of p. concolor utilize olfactory similarity cues during recognition and discrimination process. as expected by the adaptive threshold model, differences between the individual odor and colony’s internal template triggered aggressive behaviors for non-nestmates. ants in contact with unfamiliar odors were often assaulted by their nestmates. thus, it is clear that p. concolor, as well as p. ferruginea (mintzer, 1982; mintzer & vinson, 1985), uses chemical similarity to discriminate between nestmates and non-nestmates. the difference of the frequency of aggression between nestmates confined in vials impregnated with interspecific and intraspecific odor was not significant. these ants recognize olfactory discrepancies derived both interspecific and intraspecific in a similar way, since workers from two treatments were attacked. therefore, workers of p. concolor perceive individual variations of cuticular hydrocarbons with their internal template and thus behave appropriately for nestmates and non-nestmates. during the evolutionary process, natural selection must have favored ants which were more and more efficient in recognizing nestmates and discriminating non-nestmates. this feature is critical for maintaining obligatory mutualism between ants and myrmecophyte plants. thus preventing intruders is a benefit not only to the colony but also to the host plant, since an efficient colony in recognizing and discriminating between nestmates and intruders should also be efficient in expelling herbivores and defending their host plant. therefore, individuals with discrepant olfactory cues of the internal colonial pattern should be avoided, battered or forced out by p. concolor workers that nest in t. myrmecophila. in this study we clearly demonstrated that p. concolor ant is skilled in recognizing and discriminating nestmates from non-nestmates using highly aggressive behaviors, being effective in the defense of the colony against competitors and their plant against herbivores. during this process, workers of p. concolor perceive olfactory signals from other individuals and so, compare these cues with its odoriferous identity and the colony’s internal template. according to chemical similarities assessed ants may accept or reject, through aggression, individuals present in their host plant. however, more studies (eg: chemical analysis) are required to confirm that nestmate recognition behavior is directly related to the chemical compounds present on the surface of the body of p. concolor. furthermore, it is possible that p. concolor uses other information such as visual cues, common mechanism for members of the subfamily pseudomyrmecinae (see ward & downie, 2005) and other social insects (see sheehan & tibbetts, 2011; 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(2010). nestmate recognition in social insects and role of hydrocarbons. in blomquist, g.j (eds.), insect hydrocarbons biology, biochemistry, and chemical ecology (pp. 222-243). england: cambridge university press. vander meer, r.k.v. & alonso, l.e. (1998). pheromone directed behavior in ants. in r.k. vander meer, m.d. breed, k.e. espelie & m.l.p. winston (eds.), pheromone communication in social insects (pp.159-191). usa: westview press. wallis, d.i. (1970). aggression in social insects. in c.h. southwick, (eds), animal aggression: selected readings (94102). new york, usa: van nostrand reinhold company, ward, p.s. & downie, d.a. (2005). the ant subfamily pseudomyrmecinae (hymenoptera: formicidae): phylogeny and evolution of big-eyed arboreal ants. systematic entomology, 30: 310-335. wheeler, w.m. (1921). the tachigali ants. zoologica, 3: 137-168. wilson, e.o. (1971). the insect societies. cambridge: belknap press, harvard university. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.910sociobiology 62(4): 481-483 (december, 2015) first record of the ant-loving cricket myrmecophilus fuscus stalling, 2013 (orthoptera: myrmecophilidae) in mainland spain introduction social insect nests are a rich source of biodiversity (kistner, 1982). arthropods are the main organisms inhabiting the galleries and spaces of ant nests (passera & aron, 2005). antloving crickets are rarely observed and seldom collected, although we believe their occurrence in the field is greatly underestimated. accordingly, myrmecologists and orthopterologists should collaborate to determine the distribution of these species and share knowledge and techniques. one ant-loving cricket species, myrmecophilus fuscus stalling, 2013, was recently described from mallorca, balearic islands, spain (stalling, 2013) and was subsequently found in several countries within the mediterranean basin (stalling, 2015). we present the first records of m. fuscus from the spanish mainland. two additional myrmecophilus species are presently known in mainland spain: myrmecophilus acervorum (panzer, [1799]) and myrmecophilus ochraceus fischer, 1853. abstract we present the first published records of myrmecophilus fuscus stalling, 2013 from the spanish mainland. we observed the species at various localities in catalonia and in the city of madrid. six ant species are known hosts for m. fuscus in mainland spain: camponotus lateralis (olivier, 1792), crematogaster scutellaris (olivier, 1792) and formica cunicularia latreille, 1798, as well as three ant species of the genus lasius, two native and one exotic: l. grandis forel, 1909, l. cinereus seifert, 1992, and the invasive l. neglectus van loon, boomsma & andrásfalvy, 1990. sociobiology an international journal on social insects t stalling1, x espadaler2, noz carrón3 article history edited by evandro do nascimento silva, uefs, brazil received 11 september 2015 initial acceptance 14 september 2015 final acceptance 14 september 2015 keywords myrmecophilus fuscus, myrmecophilidae, distribution, new records, spain. corresponding author thomas stalling möndenweg 26, d-79594 inzlingen, germany e-mail: stalling@gmx.de materials and methods during routine myrmecological surveys, ant nests located beneath stones were checked for ant-loving crickets by xavier espadaler and nilo ortiz de zugasti carrón. all myrmecophilus specimens were caught, preserved in ethanol, and subsequently pinned and dried. additional specimens collected by hervé brustel were examined. most of the records from previous years from the barcelona region were published as m. acervorum (panzer, [1799]) by espadaler and olmo-vidal, (2011) and the specimens were re-examined in 2015, after the description of m. fuscus. specimen identification was performed according to the criteria of stalling (2013) and by direct comparison with specimens of the type series of m. fuscus. the species differs from other myrmecophilus species in spain by the following characteristics: the coloration is dark ochreous with pale ochreous posterior margins of the pronotum, mesonotum research article ants 1 biologist (private), inzlingen, germany 2 universitat autònoma de barcelona, bellaterra, spain 3 biologist (private), cáceres, spain t stalling, x espadaler, noz carrón – myrmecophilus fuscus from the spanish mainland482 and tergites 1–3 (dark reddish-brown coloration with pale ochreous posterior margins of the pronotum and mesonotum in m. acervorum), the valvae of female (lateral view) are double-pointed (rounded in m. ochraceus), the hairs on the front and antennae are short and inconspicuous (long, distant, and bushy in m. ochraceus), and the first segment of the basitarsus has two or sometimes three subapical spines (only one spine in m. ochraceus). results we detected myrmecophilus fuscus at the following localities (fig 1, all specimens in coll. t. stalling). latreille, 1798; alcover, mas de flores, 41°15’ 60” n, 001°07’ 55” e, 05.v.2014, 2 adult males and 3 adult females in nest of crematogaster scutellaris (olivier, 1792) and 4 female nymphs in nest of camponotus lateralis (olivier, 1792). spain, madrid (all leg. n. ortiz de zugasti carrón): madrid, 40°25’40.7” n, 003°43’54.1” w, 590 m.a.s.l., 25.vii.2014, 3 ad. males, 2 ad. females (fig 2), 2 female nymphs and 3 nymphs of unknown sex in nest of lasius grandis. madrid, 40°26’17.0” n, 3°43’29.28” w, 641 m.a.s.l., 03.vii.2015. 2 adult females, 1 adult male and 8 nymphs in a nest of l. grandis. the first nest, observed in 2014, was established under a small concrete slab of 60 × 60 × 6 cm. the location is watered periodically, although the irrigation frequency has greatly diminished over the past few years owing to drought in the madrid region, resulting in a loss of the green lawn that surrounded the nest. however, all of the trees in the area remain. the slab is located underneath three cypress trees where the ants actively forage as well as a fruit tree (prunus sp.), and is in proximity to robinia pseudoacacia. despite the dry and dusty surrounding ground, there is sufficient atmospheric humidity owing to the manzanares river, which is within approximately 25 m (with a phreatic level of about -3 m underground). some cricket nymphs were found (but not collected) in other l. grandis nests (under typical rocks) within 300 m of this nest. the second nest, detected in 2015, was established under a similar concrete slab in a permanently watered lawn with abundant poplar trees; this site is located within the terrain of the principalship buildings of complutense university. some nymphs were also detected (july, 2015) under rocks along the artificial stream of the “parque del oeste” (western park), within 500 m of the second location and also in permanently watered lawn. considering together all sexually identifiable specimens from mainland spain (16 males, 21 females) the sex ratio is not different from 1:1 (chi-square = 0.67; p = 0.58). fig 1. distribution of myrmecophilus fuscus in spain. black dots: new records from the spanish mainland; white dot: locus typicus published by stalling (2013). the map is based on a map from виктор, © creative commons license. spain, catalonia, barcelona province (all leg. x. espadaler, most specimens were published as m. acervorum by espadaler & olmo-vidal, 2011): bellaterra, universitat autònoma de barcelona campus, 41º29’54.1” n, 002º06’34.7” e, approximately 115 m.a.s.l., 25.v.2012, 1 ad. male in nest of lasius grandis forel, 1909; la roca del vallès, 41º35’ n, 002º19’ e, approximately 100 m.a.s.l., 12.v.2003, 2 ad. males and 3 nymphs of unknown sex in nest of lasius cinereus seifert, 1992; matadepera, 41º36’33.6” n, 002º00’56.1’ e, 400 m.a.s.l, 22.x.2009, 1 ad. female, 3 ad. males and 3 nymphs of unknown sex in nest of lasius neglectus van loon, boomsma & andrásfalvy, 1990; riera de fuirosos, la batlloria, 41º42’58.55” n, 002º34’29.60” e, 85 m.a.s.l., 27.x.2008, 1 ad. female in nest of lasius cinereus; sant cugat del vallès, 41º28’ n, 002º04’ e, approximately 150 m.a.s.l., 14.x.2014, 4 ad. males, 3 adult females and 2 female nymphs in nest of lasius grandis. spain, catalonia, tarragona province (all leg. h. brustel): alcover, vall del glorieta, 41°16’ 03” n, 001°07’ 43” e, 25.xii.2012, 1 adult female in nest of formica cunicularia fig 2. myrmecophilus fuscus, female, july 25, 2014, spain, madrid. scale bar: 1 mm. sociobiology 62(4): 481-483 (december, 2015) 483 discussion we recorded myrmecophilus fuscus on the spanish mainland. prior to its description in 2013, the species was not distinguished from m. acervorum and some specimens were therefore erroneously identified as m. acervorum (espadaler & olmo-vidal, 2011). m. acervorum is also found in the barcelona region. the observation of the species in madrid greatly extends the known range of m. fuscus in spain. we detected the crickets on both banks of the manzanares river at sites separated by a distance of 3 km. the presence of the species in artificially watered gardens indicates a possible accidental introduction. camponotus lateralis, crematogaster scutellaris and formica cunicularia, as well as three lasius species, two native and one exotic, are known hosts for m. fuscus in mainland spain. l. grandis “… is the most abundant species of the subgenus on the iberian peninsula…” (seifert, 1982) and l. cinereus is common in eastern spain (espadaler et al., 2011). the invasive l. neglectus in iberia is restricted to limited localities in catalonia and the basque country. camponotus lateralis, crematogaster scutellaris and formica cunicularia are widely present in the mediterranean region, with continuous distribution across the iberian peninsula (gómez & espadaler, 2007). accordingly, m. fuscus could be expected to occur in the wide area between madrid and barcelona. portugal is a candidate country for m. fuscus based on the presence of known ant hosts in the country. acknowledgments many thanks to hervé brustel (toulouse, france) for providing specimens. references espadaler, x., casevitz-weulersse, j. & imbert, e. (2011). lasius cinereus seifert, une espèce nouvelle pour la france (hymenoptera: formicidae), sa distribution en espagne et en france et remarques sur sa biologie. revue française d’entomologie (n.s.), 32: 105-112. espadaler, x. & olmo-vidal, j.m. (2011). the myrmecophilic cricket myrmecophilus in spain (orthoptera: myrmecophilidae). sociobiology, 57: 321-328. gómez, k. & espadaler, x. (2007). hormigas.org. http:// www.hormigas.org/mapas/index.php (accessed date: 8 september, 2015). kistner, d.h. (1982). the social insects’ bestiary. in: h.r. hermann (ed.). social insects. vol. 3 (pp1-244). academic press. passera, l. & aron, s. (2005). les fourmis: comportement, organisation sociale et évolution. ottawa: les presses scientifiques du cnrc. 480 p. seifert, b. (1992). a taxonomic revision of the palaearctic members of the ant subgenus lasius s. str. (hymenoptera: formicidae). abhandlungen und berichte des naturkundemuseums görlitz, 66: 1-66. stalling, t. (2013). a new species of ant-loving cricket from mallorca, balearic islands, spain (orthoptera: myrmecophilidae). graellsia 69(2): 153-156. doi:10.3989/ graellsia.2013.v69.073 stalling, t.( 2015). new records of the ant-loving cricket myrmecophilus fuscus stalling, 2013 from the mediterranean basin (orthoptera: myrmecophilidae). articulata, 30: 105-108. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.7667sociobiology 69(3): e7667 (september, 2022) introduction formicidae is the major living group in many habitats, accounting for up to 15% of the total animal biomass, 94% of the individuals, and 50% of the arthropod biomass in the tropical forest canopy (hölldobler & wilson, 1990). ants play a diverse role in terrestrial ecosystems acting as predators, scavengers, herbivores, detritivores, and granivores (hölldobler & wilson, 1990) and participate in an astonishing array of associations with plants and other insects (hölldobler & wilson, 1990; jolivet, 1996). their ubiquity and importance in the ecosystems explain the repeated evolution of many types of interactions, sometimes very specific and specialized (lebas et al., 2016). some ant species are useful and play an important role in maintaining a certain biological balance as predators or parasites, while other species are considered pests, especially in agriculture (bernard, 1968). ants play a crucial ecological role in agrosystems through their regulatory action of phytophagous insects, seed dispersal, and symbiosis with abstract the present work studied the diversity of formicidae in two agrosystems of bejaia, a lemon orchard located at inraa and an orange orchard located at amizour. we used the transect method by combining three (3) sampling methods: barber pots or pitfall, bait, and manual capture. we identified 18 ant species representing 11 genera and three subfamilies: dolichoderinae (tapinoma magnum), formicinae (cataglyphis, camponotus, lasius, and plagiolepis), and myrmicinae (messor, aphaenogaster, crematogaster, pheidole, tetramorium, and temnothorax). the species richness was 13 species in the oued ghir’s site and 12 species in amizour’s site. at inraa, dolichoderinae and myrmicinae have the same proportions (43.95 and 43.17%, respectively) followed by formicinae (12.88%). in amizour, formicinae showed the higher frequency (40.28%), followed by dolichoderinae and myrmicinae (30.47 and 29.25%, respectively). in the inraa lemon orchard tapinoma magnum was the dominant species with a relative abundance of 43.95%, whereas in amizour the predominance is attributed to the cataglyphis viatica with a relative abundance of 39.05%. sociobiology an international journal on social insects anissa henine-maouche, wissam guergouz, thiziri moudache article history edited by evandro nascimento silva, uefs, brazil received 10 december 2021 initial acceptance 13 february 2022 final acceptance 19 july 2022 publication date 07 september 2022 keywords diversity, dominance, abundance, myrmecofauna. corresponding author anissa henine-maouche laboratoire de zoologie appliquée et d’ ecophysiologie animale faculté des sciences de la nature et de la vie université de bejaia, bejaia, algeria e-mail: anissa.maouche@univ-bejaia.dz plants and are at the same time accidental pollinators (ramón & donoso, 2015; diame et al., 2015). they also play an important role in soil enrichment and aeration, thus improving the soil quality by mixing it and accelerating the transport and recycling of organic matter (lyford, 1963; wagner & jones, 2006). in algeria, the formicidae family is only known from the works of bernard (1968, 1976, and 1983) and cagniant (1966, 1968, 1969, 1970, and 1973). since then, the systematics and diversity of this taxonomic group have not undergone any major revision except recently with the works of barech et al. (2011, 2014, 2015, 2016, and 2017), chemala et al. (2017), henine-maouche et al. (2019), henine-maouche et al. (2020) and henine-maouche (2020). information on ant community structure in agrosystemsis still unknown in algeria, except for the works such as dehina (2004). seeking to fill in this information gap, the objective of our study was to analyze antcommunity diversity in two agrosystems in bejaia’s wilaya. research article ants ant community diversity in two agrosystems in bejaia wilaya (northern algeria) laboratoire de zoologie appliquée et d’ecophysiologie animale, faculté des sciences de la nature et de la vie, université de bejaia (algérie) mailto:anissa.maouche@univ-bejaia.dz anissa henine-maouche, wissam guergouz, thiziri moudache – ant community diversity of two agrosystems in bejaiawilaya (algeria)2 methods the investigations were carried out in bejaia’s northern region (north-east of algeria) where two experimental stations were chosen. these are a lemon orchard located at inraa in oued ghir (fig 1) and an orange orchard located in amizour (fig 2). the national institute of agronomic research of algeria in oued ghir (36°42’22.03 “n 4°57’23.66 “e) is located on the rn 12, 10 km southwest of bejaia’s main town. the inraa station covers a total land area of 22.06 ha of which: 15.50 ha of useful agricultural area, 5.50 ha are uncultivated land, and 1.5 ha has built areas.the studied station vegetation is composed of citrus×limon trees regularly spaced and weeds such as scolymus hispanicus, sonchus asper, cynodon dactylon, and lolium perenne. the amizour station (36°40’9.76 “n 4°54’58.55 “e) is located 24 km southwest of bejaïa. it is a citrus agrosystem “young orange orchard” where the vegetation is composed of (citrus × sinensis) regularly spaced weeds such as lolium perenne. next to this orchard is a vineyard (vitis vinifera) where the soil is well worked. the station is bordered by arundo donax windbreaks. fig 1. geographical location of the inraa's station (google earth). fig 2. geographical location of the amizour's station (google earth). sociobiology 69(3): e7667 (september, 2022) 3 the simultaneous use of several sampling methods and their combination is the best approach to biodiversity assessment (marshall et al., 1994).the sampling took place during the ants’ activity period. the first field work took place on 19 may 2021 for amizour and on 24 may 2021 for inraa. the second field work took place on 1 july 2021 for amizour and on 13 july 2021 for inraa. a well-defined protocol was followed combining three sampling methods which are pitfall traps or barber pots, baiting, and manual capture for a complete sampling. we followed the sampling plan of abdi-hamecha et al. (2021) (fig 3): we subdivided the ten (10) barber pots into two transects, each segment consisting of five (5) trap pots spaced at 15 meters. each barberpot is surrounded by four baits at a distance of 5 meters, making a total of forty (40) baits. an area of 1750 m2 was covered. the baits consisted of tuna oil, honey, and jam, and were left in place for 3 hours, but probes were made every 30 minutes to collect the ants. the barber pots were left for 24 hours during the first visit and 48 hours during the second. the manual capture lasted 4 hours, from 9 am to 1 pm. the identification of collected ants was made by referring to various identification guides (bernard, 1968; cagniant, 1968; 1970; 1997; 2005; 2006 and 2009) and reference websites (www.antarea.com and www.antweb.com). diversity indices were calculated, and parametric statistics (student test) were used to evaluate the results of the two communities. the assessment of the sampling effort was obtained by calculating non-parametric diversity estimators (jackknife 1, jackknife 2, and bootstrap) using the estimates version 9.1.0 software. the observed species richness (sobs) and the estimated species richness (sest) are used to produce the accumulation curve. the ratio of observed species richness and estimated species richness allows the calculation of the sampling completeness rate by the following formula: sampling efforts = x100 fig 3. sampling plan developed for the two sites at amizour and oued ghir according to abdi-hamecha et al. (2021). results our study identified 18 ant species representing 11 genera and three subfamilies: dolichoderinae (tapinoma magnum), formicinae (cataglyphis, camponotus, lasius and plagiolepis) and myrmicinae (messor, aphaenogaster, crematogaster, pheidole, tetramorium, and temnothorax) (table 1). the sampling result allowed us to note that the inraa site is slightly richer in species (13 species) than the amizour site (12 species). some species are only present in one of the two agrosystems, such as lasius flavus, plagiolepis atlantis, crematogaster scutellaris, temnothorax sp, tetramorium maurum, and tetramorium semilaeve, which were only found at inraa. while camponotus ruber, cataglyphis diehlii, tetramorium biskrense, tetramorium forte and aphenogaster subterranea were only sampled in amizour. tapinoma magnum, camponotus spissinodis, cataglyphis viatica, aphenogaster sardoa, aphenogaster testaceo-pilosa, messor barbarus, and pheidole pallidula belong to both agrosystems. at the inraa, the frequencies of dolichoderinae and myrmicinae subfamilies are quite identical (43.95 and 43.17%, respectively) followed by the formicinae with a frequency of 12.88% (fig 4). in amizour, the formicinae are in the lead with a frequency of 40.28%. dolichoderinae and myrmicinae follow with frequencies of 30.47 and 29.25%, respectively (fig 4). the histogram below (fig 5) shows the proportions of the seven genera of formicidae recorded during the sampling of the two agricultural environments (inraa and amizour). these genera are: tapinoma, plagiolepis, lasius, camponotus, cataglyphis, tetramorium, aphaenogaster, messor, pheidole, crematogaster and temnothorax. it can be observed that at inraa the genera tapinoma is largely dominant with a frequency of 43.95% followed by aphaenogaster with a frequency of 31.49%. lasius is far http://www.antweb.com anissa henine-maouche, wissam guergouz, thiziri moudache – ant community diversity of two agrosystems in bejaiawilaya (algeria)4 behind with a frequency of 10.23%. on the other hand, in amizour it is the cataglyphis genera which was the most recorded with a frequency of 39.75%, followed by tapinoma with a frequency of 30.45%. messor only represents 14.36%. in amizour orange orchard, the cataglyphis viatica species was dominant with a relative abundance of 39.05% followed by tapinoma magnum with 30.47%. these are the two most abundant species on this site. messor barbarus follows far behind with 14.36%. aphaenogaster testaceopilosa and pheidole pallidula do not exceed 6% (5.60 and 4.55%, respectively). in the inraa lemon orchard, tapinoma magnum is the most abundant species with 43.95%, followed by aphenogaster testaceo-pilosa, lasius flavus, and pheidole pallidula with relative abundances of 29.80, 10.23, and 6.02%, respectively. the other species do not exceed 3%. table 1. number and relative abundance (ra%) of formicidae sampled in the two study stations (amizour and inraa oued ghir). subfamilies species amizour inraa ni ra% ni ra% dolichoderinae tapinoma magnum mayr 174 30.473 730 43.95 formicinae plagiolepis atlantis santschi 29 1.75 lasius flavus (fabricius) 170 10.23 camponotus spissinodis forel 2 0.35 1 0.06 camponotus ruber emery 1 0.18 cataglyphis diehlii (forel) 4 0.70 cataglyphis viatica (fabricius) 223 39.05 14 0.84 myrmicinae tetramorium biskrense forel 3 0.53 tetramorium forte forel 10 1.75 tetramoriumm aurum santschi 1 0.06 tetramorium semilaeve andré 15 0.90 aphaenogaster subterranea (latreille) 4 0.70 aphaenogaster sardoa mayr 10 1.75 28 1.69 aphaenogaster testaceo pilosa (lucas) 32 5.60 495 29.80 messor barbarus (linnaeus) 82 14.36 41 2.47 pheidole pallidula (nylander) 26 4.55 100 6.02 crematogaster scutellaris (olivier) 25 1.51 temnothorax sp 12 0.72 total 18 571 100 1661 100 fig 4. frequency (%) by subfamily for each study station. sociobiology 69(3): e7667 (september, 2022) 5 comparison of the two outputs of each station the two histograms below (fig 6) represent the numbers of each species for each station and each trap. it is noted that for the amizour site, some species are present in the first sampling and not in the second and vice versa. other species are present in both samplings. ten species were recorded in the first sampling andeight species in the second. the same was true at the inraa site, where 10 species were sampled on the first sampling and 11 on the second. at amizour, the tapinoma magnum and cataglyphis viatica species were less present in the first sampling with 59 individuals each, whereas in the second sampling they were more abundant (155 and 158 individuals, respectively). in contrast, messor barbarus was more abundant in the first sampling with 73 individuals sampled compared to only nine individuals in the second sampling. camponotus spissinodis, tetramorium forte, and pheidole pallidula were only sampled on the first sampling with 2, 10, and 26 individuals, respectively. while camponotus ruber and tetramorium biskrense were only caught on the second sampling with only one and three individuals. to find out if there is a significant difference between the abundance of each species over the two sampling periods (may and july), we used student’s t-test, which confirmed that the species numbers between the two periods are different (t = 0.46, df = 11, p = 0.6515). at inraa, we find species present during both periods such as tapinoma magnum present in large numbers, 368 and 362 individuals, respectively. on the first sampling, the plagiolepis atlantis and aphaenogaster sardoa numbers were 27 individuals per species compared to two and one individuals on the second sampling. aphaenogaster testaceopilosa was represented by 310 individuals in the first sampling compared to 185 individuals in the last. we used the same student’s t-test to comparethe abundance of each species over the two sampling periods and the result of the test shows us that, again, the two samplings are different (t = 0,17, df = 13 et p = 0,8662). fig 5. frequency (%) by formicidae's genera recorded in the two study stations. other species were only found on the first sampling such as tetramorium semilaeve with 15 individuals and temnothorax sp with 12 individuals. others were found in the second sampling such as lasius flavus with 170 individuals, camponotus spissinodis, and tetramorium maurum with only one individual each. the shannon-weaver index shows that both orchards have relatively good myrmecological diversity with almost identical values, 2.28 bits for amizour and 2.23 bits for inraa. the piélou’s equitability index’s values were very close for the two stations: 0.64 at amizour and 0.60 at inraa. this indicates that the species frequency (number of individuals) at these stations tends to be relatively similar to each other (table 2). the calculated jaccard index reveals a difference in species composition between the two stations. these stations (amizour and inraa) together share up to 0.39 of the common species. this makes these agrosystems different in terms of species composition. table 2. species richness, shannon-weaver diversity index, maximum diversity index and piélou equitability index for comparing the ant communities recorded at two study stations. stations amizour inraa species richness 12 13 shannon-weaver diversity index 2.28 2.23 maximum diversity index 3.58 3.70 piélou equitability index 0.64 0.60 anissa henine-maouche, wissam guergouz, thiziri moudache – ant community diversity of two agrosystems in bejaiawilaya (algeria)6 richness estimation for the orange orchard, the jackknife 1, jackknife 2, and bootstrap indices predict higher specific richness (13.99, 14.97 and 12.99, respectively). this means that there would be an average of 2 to 3 species of ants left to discover at the site. the completeness rate of the inventory calculated based on the average of these three estimators is 85.9%, which suggests that the sampling effort was satisfactory and this is confirmed by the growth of the species accumulation curve (sobs) and its convergence with those of the estimators (fig 7). for the lemon orchard, the jackknife 1, jackknife 2, and bootstrap indices predict higher specific richness (16.31; 19.55 and 14.38, respectively). this means that there would be an average of 2-6 species of ants left to discover at the site. the completeness rate of the inventory calculated based on the average of these three estimators is 78.86%, which means that only three-quarters of the species were sampled. fig 6. number of individuals collected per species during the two sampling periods in the two study stations. sociobiology 69(3): e7667 (september, 2022) 7 the lowest completeness rate was recorded with the jackknife 2 estimator (66.49%) and this can be seen in figure 7 where the species growth curve (sobs) diverges from that of the estimator. the efficiency comparison of the three sampling methods used in myrmecology, no sampling method allows the collection of all species in an environment. in our study, the number and percentage of species sampled by the three sampling methods used are listed in table 3. for amizour, the highest number of ant species were collected by manual capture (75 ℅ of the total number of species sampled); bait and pitfall traps were relatively effective (50 and 58.33% of the species’ total number, respectively). concerning the inraa station, pitfall traps were the most efficient and captured 76.92% of the species’s total number; followed by baiting (69.23 ℅ of the species’ total number) and hand catching (46.15 ℅ of the total number of species sampled). in this study, we decided to improve the pitfall protocol by leaving the traps in the field for additional 24 hours (i.e. 48 hours) as recommended in the protocol of bestelmeyer et al. (2000). to know if the change of protocol was effective, we used the student’st-test and the results show that for the inraa station, the increase in pitfall trap duration was effective (t = 0,3; df = 9 et p = 0,7699). indeed, five-fold more ant species were caught compared to the first sampling. environments sampling methods species’s numbers species’s percentage amizour capture manual baits pitfall traps 9 6 7 75% 50% 58.33% inraa capture manual baits pitfall traps 6 9 10 46.15% 69.23% 79.92% table 3. number of species and percentage of total species according to sampling methods. fig 7. species accumulation curves, richness estimators with all sampling methods combined. a: orange orchard, b: lemon orchard. anissa henine-maouche, wissam guergouz, thiziri moudache – ant community diversity of two agrosystems in bejaiawilaya (algeria)8 whereas in amizour, the increase in trap duration was not more effective (just three more species compared to the first sampling). discussions the survey that we performed using the three combined sampling methods (barber pots, baits, and manual capture), allowed us to record 18 species of formicidae in the two agrosystems: 13 species in the lemon orchard located at the inraa of oued ghir and 12 species in the “young” orange orchard located at amizour, divided into three subfamilies, namely; dolichoderinae, formicinae, and myrmicinae. our results are superior to djioua’s (2011), where she was able to record 10 species in oued aissi (tiziouzou) in her study on formicidae from some forest and agricultural environments, those of dehina et al., (2007) who recorded 11 species during two years (2006 and 2007) at the national agronomic institute of el-harrache and those of ait said (2005). mohammedi-boubekka (2006) reports a richness of eight species in his study on the bioytmatics of aphidae and their place in the orange trees’ entomofauna in the metidja plain. in the same station, dehina (2004), noted a total species richness of nine species in the heuraoua region (the mitidja). she noted seven species in the citrus orchard, and four species in the vegetable crops. chemala (2013), in his myrmecological study of the sahara’s areas, reported in the djamâa area 13 species in the palm grove and 11 species in cultivated areas.the three subfamilies identified during our sampling are represented with different proportions at the two sites. at inraa, the dolichoderinae and myrmicinae dominate over the formicinae. on the other hand, at amizour, the subfamily formicinae dominates, followed by the dolichoderinae and myrmicinae. according to djioua’s study (2011), on the myrmecofauna of some forest environments and two agricultural orchards, the myrmicinae predominate with more than 50% in the two agricultural environments, followed by the dolichoderinae with more than 30% and finally the formicinae with only 20%.concerning the genera, 11 genera were noted at inraa, of which only seven are present at amizour with very distinct frequencies. the tapinoma genus is the best represented at the oued ghir site and predominates with a frequency of 43.95%, followed by aphaenogaster with a frequency of 31.49%. at amizour, it is the cataglyphis and tapinoma genera that dominate (39.79 and 30.47% respectively). djioua (2014) recorded 12 genera in his study in the two orchards of oued aissi. nadji et al. (2017) were able to record 11 genera in some crescia orchards in sidi slimane in his study on the predation impact by cataglyphis viatica (insecta, formicidae) about trophic availability in agricultural and natural environments in the algerian sahel. mohammediboubekka (2006) sampled eight genera in his study on the aphidae’s biosystematics and their place in the orange trees’ entomofauna in the mitidja plain. some common species are not distributed in the same way in the two sites and over the two sampling periods.  tapinoma magnum: this species prefers watered or flooded areas. it is fond of clayey and humid soils on river banks, coastal dunes, and scrubland (lebas et al., 2016). t. magnum is highly adaptable, tolerating both very wet and very dry soils. it maintains aphids and mealy bugs on plants (bernard, 1968). in this study, the tapinoma magnum is dominant at inraa. indeed, we observed in the field that this station’s lemon trees are invaded by aphids and this is due to the very rare use of pesticides and insecticides by the managers of the institute (personal communication dr. boussad b.). in contrast, the amizour orange trees have been treated with phytosanitary products and are free of all parasites. at inraa, we noted the same numberof individuals during the two samplings (368 individuals and 362 individuals, respectively), whereas at amizour, the highest number of t. magnum was noted in july (155 individuals). this can be explained, probably, by an irrigation system installation by the orchard’s owner, which was not present during the may sampling, whereas at inraa the orchard is permanently irrigated.  cataglyphis viatica: it is more abundant in amizour because these antsnest in open habitats (large clearings, mountain pastures, and steppes) (cagniant, 2009). indeed, the amizour station is more open than the inraa station where a lot of vegetation remains in the lemon orchard due to the absence of plowing. at amizour, the species was more abundant during our second sampling in july with 158 individuals compared to 59 in may. this is explained by its scavenging and thermophilic nature, which collects the corpses of other arthropods that have fallen victim to the heat and stress of their hostile environment (wehner et al., 1983). the high temperatures in july probably increased the death of arthropods.  aphaenogaster testaceo-pilosa: this species colonizes all environments. cagniant (1973) considers that this species is indifferent to the vegetation cover. during our sampling at the inraa lemon orchard, we noted a higher number of individuals in may (310) compared to 158 in july.  lasius flavus: according to bernard (1968), l. flavus prefers cool, moist, open, mostly granitic, gently sloping ground and tolerates swampy facies more than most other ants. most crops and pastures give an advantage to lasius. the inraa station is, therefore, favorable to its establishment.  messor barbarus: it is the most spectacular granivorous species. although it is common in the mediterranean region, it is adapted to warm and dry environments and is more frequent in open, herbaceous ecosystems such as meadows and scrublands (lebas et al., 2016). at inraa, the number of individuals was not very different during the two samplings, whereas at amizour, the number of individuals decreased from 71 in may to only 9 in july (although this is the period sociobiology 69(3): e7667 (september, 2022) 9 when it should be present). it is thought that land plowing between the two samplings destroyed the m. barbarus nests, reducing their numbers.  pheidole pallidula: this is an omnivorous species with a wide distribution in the mediterranean region (bernard, 1983). in north africa, it occupies humid environments, especially in sandy and clayey soils. in algeria, it prefers flat land (bernard, 1968). it is the typical interior ant, a generalist species, very common, adaptable, and tolerant to human intervention. at inraa’s sampling, more than 64 individuals were recorded in july compared to 36 in may. in amizour, however, it was absent from our records during the second sampling without any apparent explanation.  crematogaster scutellaris: the crematogaster are among the most important warm-region genera in the world (bernard, 1968). it is very common in the mediterranean and commonly called the cork ant because it establishes its nests in aging oak trees causing their death. it is very flexible in its nesting behavior and can settle in cavities in trees, shrubs, soft wood, or walls, dry stone walls, various cracks, crevices, and cavities in friable rocks (casevitz-weulersse, 1972). it is a pastoral species that seeks out and exploits arborealaphids colonies. it can also nest in fruit or ornamental trees and various pines (villemant & fraval, 2002; cagniant, 2005). in this study, its capture at the inraa station is explained by the fact that the latter remained in its wild state as well as by the presence of aphids on lemon trees (it was on these trees that we captured them). in djioua’s (2011) study on agricultural environments, she noted that tapinoma simrothi is the most abundant species, in an orchard, with a frequency of 32.24%, followed by aphaenogaster testaceo-pilosa (17.72%), pheidole pallidula (14.90%), cataglyphis bicolor (9.79%), tetramorium biskrense (7.30%), and messor barbarus (5.75%). the same observation was made by the author in a second orchard with a dominance of tapinoma simrothi (ar=30.39%), followed by aphaenogaster testaceo-pilosa (18.38%), pheidole pallidula (16.86%), cataglyphis bicolor (10.61%), tetramorium biskrense (6.62%), and messor barbarus (4.32%). in northern algeria, ait said (2005) noted that the tapinoma simrothi abundance was 62.3% in staoueli. similarly, dehina (2004) working in the heuraoua region (mitidja), noted the dominance of tapinoma simrothi in the citrus orchard (ar% = 41%) and at the market gardening station (ar% = 82.3%). chemala (2009) mentions the monomorium salomonis dominance at three stations in djamaa (el-oued) with an abundance of 68.8% in palm groves, 54.8% in natural environments, and 29.9% in cultivated environments. guehef et al. (2018) show that in oued souf, messor arenarius (ar% = 36.76%) is the most abundant species in the potato station and pheidole pallidula (ar% = 54.17%) in another potato station. the diversity index carried out for the two orchards (inraa-lemon and amizour-orange) gives the following values: 2.23 bits for the first orchard and 2.28 bits for the second. these values are fairly close and reveal a good formicidae diversity. these values are higher than those reported by djioua (2011) (h’ = 0.86 bits for the orange and peach orchards (oued aissi). chemala (2009) in a study carried out in three stations in djamâa (el-oued), diversity indices of 1.62 bits in the palm grove, 2.20 bits in the natural environment, and 2.42 bits in the cultivated environment. toukali (2016), at the pear orchard in sidi embarek, noted that the shannon diversity index value is very high and equal to 4.64 bits. guehef et al (2018), recorded in oued souf, values of 2.32 bits in two stations. the equitability index values indicate 0.60 for the inraa orchard and 0.64 for the amizour orchard. the values are quite similar and indicate that in both stations, the number of individuals among the species of each community tends to be relatively similar. our results are lower than those found by djioua (2011) in oued aissi in both orchards (respectively e = 0.73 and 0.86) reflecting an equilibrium between the abundance of species sampled. chemala (2013) noted, in the region of el-oued, an equitability value of 0.93 for the palm grove, 0.91 for the natural environment, and 0.79 for the cultivated environment. chemala (2009), noted, in oued righ, an equitability value of 0.4 in the palm grove and 0.28 in the cultivated environment. we also noted that formicidae nests are very rare in the amizour station (exception for tapinoma magnum and messor barbarus). the ants collected on this station are only transient (they come to hunt, collect corpses of other insects or collect seeds). in contrast, at inraa, nests are present in large numbers between the lemon trees. it seems that the main factors determining ant distribution are firstly insecticide use and secondly the environment nature (absence or not of plowing). indeed, we found that at inraa, certain species have higher abundance because it is favorable to their installation, contrary to amizour where the land is continuously worked, which disturbs the establishment of ant species. the formicidae have a significant role in agricultural environments as they are front-line plowmen through their role in the regulation of phytophagous insects, seed dispersal, and symbiosis with plants and sap-sucking insects. in the future, we should be interested in the relationship between plants and ants and the importance of the latter in agricultural environments. author`s contributions anissa henine-maouche conceived the idea presented. wissam guergouz, thiziri moudache carried out the sampling and laboratory work, assisted by anissa heninemaouche for the identification. anissa henine-maouche carried out the calculations and statistical analyses and supervised the results of this work. all authors discussed the results and contributed to the final manuscript. anissa henine-maouche, wissam guergouz, thiziri moudache – ant community diversity of two agrosystems in bejaiawilaya (algeria)10 acknowledgments we are very grateful to the director of the national institute of agronomic research of algeria, mrs. oudjiane, for accepting our request and for welcoming us warmly to her institute. references abdi-hamecha, l., barech, g., khaldi, m., sadoudi, d.a.a., salem, s., zazgad, i. & cagniant, h. 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(1983). foraging strategies in individually searching ants cataglyphis bicolor (hymenoptera: formicidae). fischer. stuttgart _hlk108888933 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.948sociobiology 63(2): 777-782 (june, 2016) estimation of nuclear genome size of three species of camponotus (mayr, 1861) (hymenoptera: formicidae: formicinae) and their cytogenetic relationship introduction the genus camponotus (mayr, 1861) is widely distributed. this includes the so-called carpenter ants and, nowadays, is the richest ant genus in respect to the number of species, with 1099 described species in 46 subgenera (bolton, 2014). molecular phylogenetic studies indicate that this genus is paraphyletic (brady et al., 2000), and the taxonomic relationship between different species of the genus camponotus is even more critical. the neotropical subgenus myrmothrix is considered monophyletic (brady et al., 2000; hashmi, 1973), and it presents only 10 valid species (bolton, 2014). however, due to the morphological variations within some species, this number is still under discussion. abstract the chromosome variability among ant species is remarkable, and the processes generating such variation are still under discussion since polyploidy has been observed in some distinct taxa. the chromosome number of species belonging to the camponotus, subgenera myrmothrix and myrmobrachys, are highly different, whereas, the first subgenus has double the number of chromosomes of the second. in order to test the hypothesis of chromosome number doubling through polyploidy, the genome sizes of camponotus (myrmothrix) rufipes, camponotus (myrmothrix) renggeri and camponotus (myrmobrachys) crassus were estimated by flow cytometry. the chromosome number of specimens from the nests studied was also defined. no significant variation was noted in the genome size among them. the mean haploid genome size value (1c) of workers for the three species was 286.16 mpb (0.29 pg). the polyploidy hypothesis can be ruled out as an evolutionary step linking the karyotype variations among the three studied species since the genome size of c. crassus with 2n = 20 chromosomes was the same as that of c. rufipes and c. renggeri with 2n = 40. the lack of variation in the amount of dna between the related species c. rufipes and c. renggeri also demonstrate that flow cytometry is not an adequate approach to distinguish them. our results highlight the importance of combining distinct methods, dna quantification, and cytogenetics from the same colony. understanding the path of chromosome evolution of three species with distinct degrees of relatedness should provide further information in enriching our knowledge about the minimum interaction theory. sociobiology an international journal on social insects hjac aguiar1,2, lac barros1,2, faf soares1, cr carvalho1, sg pompolo1 article history edited by yi-juan xu, south china agricultural university, china received 23 march 2016 initial acceptance 21 april 2016 final acceptance 09 may 2016 publication date 15 july 2016 keywords flow cytometry, chromosome number, genome size, evolution, ants. corresponding author hilton jeferson alves cardoso de aguiar rodovia br 156, nº 3051 oiapoque-ap, 68980-000, brazil e-mail: hilton.aguiar@hotmail.com two species of subgenus myrmothrix, camponotus rufipes (fabricius, 1775) and camponotus renggeri emery, 1894, have been the subject of much discussion, primarily due to their morphological similarities. since the morphological techniques have limitations (seifert, 2009; schlick-steiner et al., 2010), the use of integrative approach is very useful in distinguishing these species, as indicated by a recent study of ronque et al. (2015). cytogenetics has drawn the attention of myrmecologists over recent years (delabie et al., 2012), and it can be a useful tool in integrative taxonomy. cytogenetic data are available for 71 taxa of camponotus belonging to different countries in the world (revised in mariano et al., 2003) showing that the chromosome number in camponotus ranges from 2n = 1 universidade federal de viçosa, minas gerais, brazil 2 universidade federal do amapá, oiapoque, amapá, brazil research article ants hjac aguiar – genome size of three species of camponotus778 18 to 2n = 50. within the monophyletic neotropical subgenus myrmothrix, cytogenetic data are available only for camponotus punctatus (goñi et al., 1983), c. atriceps, c. cingulatus (mariano et al., 2001), and c. rufipes (goñi et al., 1983; mariano et al., 2001), all presenting 2n = 40 chromosomes, and similar chromosome morphologies. cytogenetic studies on more than 500 species of ants allowed imai et al. (1988; 1994) to develop the minimum interaction theory (mit) that explains how the chromosomes were modified in the course of evolution of formicidae. this theory suggests a selective pressure that leads to a reduction in the size of the chromosomes, avoiding interactions that can reduce the fitness of individuals. reduction in the size of chromosomes occurs mainly through centric fissions, although other types of chromosomal rearrangements that may not modify the size of the chromosomes, such as inversions and translocations, were also noted. one consequence of this process is the increase in the chromosome number. this theory suggests that the increase in terminal heterochromatin after centric fissions rearrangements is a part of the chromosomal evolution, which is correlated with chromosome stability (imai et al., 1994). the chromosome configuration of camponotus (myrmobrachys) crassus (mayr, 1862) is in contrast with that of the species of camponotus species inserted in the myrmothrix subgenus (mariano et al., 2001). the karyotype of c. (myrmobrachys) crassus possesses one of the lowest diploid chromosome numbers described for the genus, with 2n = 20, all with metacentric morphology (mariano et al., 2001). since the myrmothrix species have double the number of chromosomes (2n = 40) as that found in c. (myrmobrachys) crassus, with all the chromosome centromeres positioned at the edges, the manner in which the chromosomes have modified can be superficially associated with the centric fission processes or chromosomal translocations. the use of the karyographic method by mariano et al. (2003) indicated the presence of chromosomal rearrangements of the centric fission type in the karyotype evolution of camponotus spp. however, there is a second possibility: through a single polyploidization event, followed by secondary chromosome modifications, the number of chromosomes can jump from 20 to 40 chromosomes. this is a real possibility since ants reproduce through arrhenotokous parthenogenesis, where fertilized eggs give rise to diploid females and unfertilized eggs originate haploid males (reviewed in normark, 2003). studies associating chromosome numbers and genome size to test the hypothesis of polyploidization in the genus were not yet evaluated. karyotype-dna content comparison is, therefore, a useful tool to understand the genome size evolution in ants. data concerning the genome size in formicidae are restricted to 73 species (tsutsui et al., 2008; ardila-garcia et al., 2010; cardoso et al., 2012; gregory, 2015). three different techniques have been used to quantify the genome of ants as: flow cytometry (fcm), feulgen image analysis densitometry (fiad), and, more recently, through genomics (reviewed in nygaard & yannick, 2015). in general, ants have smaller genomes than other insects (tsutsui et al., 2008). the genome size in formicidae ranges from 210.7 mb (0.22 pg) in cerapachys edentata, to 690.4 mb (0.71 pg) in ectatomma tuberculatum (tsutsui et al., 2008). the species apterostigma dentigerum and e. tuberculatum showed dna content which was twice higher than that of the most closely related species, however karyological information is not available, or is not related to the same nest. studies concerning dna content of the genus camponotus are available for c. (camponotus) pennsylvanicus, c. (tanaemyrmex) castaneus (tsutsui et al., 2008), and c. (myrmothrix) floridanus (ardila-garcia et al., 2010; bonasio et al., 2010), all from the usa (table 1), and are not related with karyology. camponotus spp. mean genome size (1c) (pg – mbp) chromosome number method reference c. (myrmothrix) rufipes 0.29 – 286.16 2n = 401,2 fcm present study c. (myrmothrix) renggeri 0.29 – 286.16 2n = 402 fcm present study c. (myrmobrachys) crassus 0.29 – 286.16 2n = 201,2 fcm present study c. (myrmothrix) floridanus 0.23 – 224.94* nd fiad ardila-garcia et al. (2010) c. (myrmothrix) floridanus 0.245* – 240† nd genome sequencing bonasio et al. (2010) c. (tanaemyrmex) castaneus 0.31 – 304.2 nd fcm tsutsui et al. (2008) c. (camponotus) pennsylvanicus 0.33 – 322.8 nd fcm tsutsui et al. (2008) 1: mariano et al. (2001), 2: present study, nd: not defined. fcm: flow cytometry; fiad: feulgen image analysis densitometry. * converted values from the available published data. † corresponding to more than 90% of the genome size sequenced. table 1 – chromosome number and genome size values of the studied camponotus species. sociobiology 63(2): 777-782 (june, 2016) 779 considering the lack of genome size and cytogenetics combined data in camponotus spp., this study aimed to estimate the dna content of c. rufipes, c. renggeri and c. crassus for a better understanding of the chromosome evolution of species of the genus camponotus. the main basis of the mit suggests small chromosome modifications usually with little variation in the amount of dna. therefore, the hypothesis of doubling the number of chromosomes among the species studied through polyploidization was tested, bringing further subjects to future discussions about the well accepted mit in explaining chromosome variability among ant species. materials and methods biological materials samples of c. rufipes and c. crassus were collected in viçosa, state of minas gerais, brazil (20°45’s, 42°51’w), and c. renggeri was collected in nova mutum, state of mato grosso, brazil (13°49’s, 56°05’w). the colonies were maintained in plastic recipients at the laboratório de citogenética de insetos, departamento de biologia geral, universidade federal de viçosa and fed with tenebrio molitor (coleoptera, tenebrionidae) larvae and honey from bees in order to obtain pupae, which were utilized for the flow cytometer analysis. the sampling authorization was provided by the instituto chico mendes de conservação da biodiversidade (icmbio) (n° 34567-4), released by sisbio for hilton j.a.c. de aguiar. adult specimens were identified by dr. jacques h. c. delabie and were deposited as vouchers in the reference collection of the laboratório de mirmecologia, centro de pesquisas do cacau (cepec/brazil) #5755. genome size by fcm the fcm analysis were performed at the laboratório de citogenética e citometria, departamento de biologia geral, universidade federal de viçosa (ufv). the nuclear dna content of three female pupae (workers and therefore diploid) of each species was measured by using an internal standard of the female pupae bee scaptotrigona xantotricha (2c = 0.88 pg), as described by lopes et al. (2009). for preparation of fcm nuclei suspensions, the cerebral ganglia nuclei of the standard and sample were excised in physiological solution (0.155mm nacl). the materials were simultaneously crushed 10 times with a pestle in a tissue grinder (kontes glass company®) with 100 µl of otto-i lysis buffer (otto, 1990) containing 0.1 m citric acid (merck®), 0.5% tween 20 (merck®), and 50 µgml−1 rnase (sigma-aldrich®), ph = 2.3. the suspension was adjusted to 1.0 ml with the same buffer, filtered through 30 µm nylon mesh (partec®), and centrifuged at 100g in microcentrifuge tubes for 5 min. the pellet was then incubated for 10 min in 100 µl of otto-i lysis buffer and stained with 1.5 ml of otto ii solution (loureiro et al., 2006a; b) supplemented with 75 µm propidium iodide (pi, sigma® – excitation/emission wavelengths: 480–575/550–740 nm (shapiro, 2003) and 50 µgml−1 rnase (sigma-aldrich®), ph = 7.8. the nuclear suspension was filtered through 20 µm diameter mesh nylon filter (partec®) and maintained in the dark for at least 30 min. the nuclear suspension was analyzed by using a partec pas® flow cytometer (partec®) equipped with a laser source (488 nm). flowmax® software (partec®) was used for data analyses. the standard nuclei peak was set to channel 200 and more than 20.000 nuclei were analyzed. three independent replications were conducted and histograms with a coefficient of variation (cv) above 5% were rejected. the mean genome size (pg) of each female hymenopteran sample was measured according to the formula adapted from doležel & bartos (2005) and their values were subsequently converted to megabase pairs (1 pg = 978 mbp) (doležel et al., 2003). the 2c dna content was converted to c-values, presented in this study here in picograms and megabase pairs. chromosome preparation mitotic metaphases were obtained from cerebral ganglia of the larvae from the same nests which were studied by fcm using the protocol of imai et al. (1988) the metaphases were observed and photographed using a bx 60 microscope with a 100x objective, coupled with a q-color3 olympus® image capture system to confirm the chromosome number of the same nests studied by means of fcm. results in this study, the genome sizes of c. rufipes, c. renggeri and c. crassus were measured by fcm. the analyses of the nuclei suspensions generated histograms with peaks corresponding to the average relative dna content of the g0/ g1 nuclei (2c dna amount), and a minor peak representing nuclei in g2 (4c dna amount) of the three studied species, and s. xantotricha, the comparative internal standard (fig. 1). the histograms revealed appropriate levels of resolution and cv ranging from 3.8% to 4.9%. the mean genome size (1c) values of c. rufipes, c. renggeri and c. crassus was approximately 286 mbp (0.29 pg) (table 1). the chromosome number observed were identical for c. rufipes and c. renggeri with 2n = 40 chromosomes (4sm + 34st + 2t). c. crassus presented 2n = 20 chromosomes (18m + 2sm) (fig. 2). discussion the three ant species c. rufipes, c. renggeri and c. crassus presented the same genome size and values that fell within the already known range for camponotus species (table 1) (tsutsui et al., 2008; ardila-garcia et al., 2010). the dna content data for the two species of subgenus myrmothrix with double the chromosome number in comparison with hjac aguiar – genome size of three species of camponotus780 those of c. (myrmobrachys) crassus, give support to the conclusions acquired by the karyographic method (mariano et al., 2003). the present study rules out the possibility of polyploidy in the studied species of camponotus subgenera myrmobrachys and myrmothrix, including species with different chromosome numbers (2n = 20 and 2n = 40). this is the first time the karyotype of c. renggeri is available. further studies including chromosome banding and molecular cytogenetics are in preparation. the results are in accordance with the mit, because we can observe the increase in the chromosome number of the species studied and the decrease in the chromosome size without alteration in the content of dna. the genome size investigation associated with the chromosome number of individuals from the species studied revealed its importance due to the possibility of intraspecific population variation as the presence of aneuploidy or b chromosomes. h. aguiar observed one colony of c. crassus collected in rio de janeiro, brazil, with the presence of supernumerary chromosomes (personal observation, july, 2012). this could influence its genome size. the ant colony collected from minas gerais in this study, presented 2n = 20, without supernumerary chromosomes, corroborating the previous cytogenetic surveys of mariano et al. (2001) from the same locality. studies conducted in formicidae indicated greater interspecific variations in the dna content between subfamilies (58%) and genera (36%), and interspecific variations are less likely among species (3.8%) (tsutsui et al., 2008). however, these authors suggested that this small variation may be due to the small number of species studied. fig 1. histograms of relative nuclear dna content from suspension obtained from cerebral ganglia cells after flow cytometric analysis with propidium iodide stained nuclei isolated of the ant camponotus (myrmothrix) rufipes. g0/g1 peak of unknown sample and the female bee scaptotrigona xantotricha (2c = 0.88 pg), which served as internal reference standard, are clearly visible. 2c nuclear dna content was determined based on the ratio of g0/g1 peak positions. the ants c. (myrmobrachys) crassus and c. (myrmothrix) renggeri presented indistinguishable results. nuclei peak of female c. rufipes at channel 132 (2c = 0.58 pg, 1c = 0.29 pg) and nuclei peak of female s. xantotricha at channel 200 (internal standard 2c = 0.88 pg, 1c = 0.44 pg). fig 2. metaphases and karyotypes of camponotus species studied by means of flow citometry. a) camponotus (myrmobrachys) crassus (2n=20), b) camponotus (myrmotrix) renggeri (2n=40), c) camponotus (myrmotrix) rufipes (2n=40). m = metacentric; sm = submetacentric; st = subtelocentric; t = telocentric. bars = 5 µm. sociobiology 63(2): 777-782 (june, 2016) 781 the fungus-growing ants, mycetophylax morschi and mycetophylax conformis, showed the same genome size and chromosome number. only mycetophylax simplex differed from their congeneric species, which may be due to the difference in the heterochromatin content (cardoso et al., 2012). although m. morschi shows variation in the chromosome number, differences in the dna content within its variants were not observed (cardoso et al., 2012). the stingless bees with high heterochromatin content melipona rufiventris and melipona mondury showed the same chromosome number, with little difference in the genome size; this observation suggests difference in the amount of heterochromatin (lopes et al., 2009). the recent study conducted by ronque et al. (2015) based in an integrative approach showed very useful in understanding taxonomic issues of c. rufipes and c. renggeri. the lack of variation in the amount of dna between these two camponotus ants indicated that these species could not be distinguished based solely on genome size, highlighting the importance of additional approaches. on the other hand, c. (myrmobrachys) crassus also showed a genome size of 286 mbp, revealing that comparative investigation about the amount of dna of phylogenetically distant camponotus spp. may have limitations. the presence of supernumerary chromosomes could influence the dna content, however, it can be ruled out for all three species in the present study, because the karyotype study was conducted for the same colonies analyzed by fcm. several ant species with the estimated genome size do not have basic cytogenetic data. the species c. floridanus has been taken for a model species in different fields of biological knowledge and, as a consequence, information about its genome size is already available (ardila-garcia et al., 2010; bonasio et al., 2010). unfortunately, there is no data regarding cytogenetics of this ant. both morphological (hashmi, 1973) and molecular observations (brady et al., 2000) suggest that this species is closely related to c. atriceps, but there is no cytogenetic data available to support this finding. the c. floridanus chromosome number availability could enrich the discussion about the chromosome and also genome size evolution within this group. different methods were used to estimate the genome size of a species belonging to a few taxa. the study of the wasps aphidius ervi, polistes fuscatus and sceliphron caementarium revealed differences with the data from fcm and fiad (ardilagarcia et al., 2010). genome size estimation using fiad revealed lower values for the three species. the dna content of the ant solenopsis invicta also showed discrepancies probably due to the methodology employed, differences in the examined cell type, or other genetic differences between the studied populations (wurm et al., 2010). the genome size of the main neotropical malaria vector anopheles darlingi obtained by fcm was similar in comparison with the value obtained by sequencing the genome after estimation and addition of repetitive portions as centromeres, telomeres and other portions of the genome rich in repetitive dna sequences (marinotti et al., 2013). the methodology employed to estimate the genome size of c. floridanus was fiad, which may have underestimated the value (ardila-garcia et al., 2010). this value differs from that obtained by bonasio et al. (2010) through partial genome sequencing. both c. (tanaemyrmex) castaneus and c. (camponotus) pennsylvanicus do not belong to myrmothrix, and the standardized fcm data obtained revealed values closer to those of the camponotus species of the present study. these findings highlight the importance of method and the cell type standardization to study insect genome size (ardilagarcia et al., 2010), allowing comparisons for consistent evolutionary studies. the present study represents the first data relating to the genome size and chromosome number of camponotus, allowing more robust observations with respect to the evolution of genome size-karyotypes of these species. the estimation of the genome size of two related species c. rufipes and c. renggeri, and a third distant species c. crassus, have its importance in demonstrating the chromosome evolution, pointing that the transition between 2n = 20 and 2n = 40 did not involve genome duplication. however, further information from additional fields of knowledge is necessary for better understanding of the taxonomic relationship of c. rufipes and c. renggeri. acknowledgments we would like to thank lucio a.o. campos for providing scaptotrigona xantotricha pupae, jacques h.c. delabie for identifying the camponotus species, danúbia r. alves and gisele a. teixeira for their field support. this research was part of the dsc. thesis of the first author, and was supported by fundação de amparo à pesquisa de minas gerais (fapemig) process no. apq-00259-13. the authors wish to thank the conselho nacional de pesquisa (cnpq) for the scholarship granted to hjaca and fafs, and the research grant to crc. we also acknowledge coordenação de aperfeiçoamento de pessoal de nível superior (capes/ pnpd) the postdoctoral grant provided to lacb. references ardila-garcia, a.m., umphrey gj and gregory tr (2010). an expansion of the genome size dataset for the insect order hymenoptera, with a first test of parasitism and eusociality as possible constraints. insect molecular biology 19: 337-346. doi: 10.1111/j.1365-2583.2010.00992.x. bolton b (2014). an online catalog of the ants of the world. available from http://antcat.org. 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(2010) the genome of the fire ant solenopsis invicta. proceedings of the national academy of sciences of the united states of americ, 108(14): 5679-5684. doi: 10.1073/ pnas.1009690108. doi: 10.13102/sociobiology.v63i2.848sociobiology 63(2): 744-747 (june, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 trophic ecology of the ant pachycondyla crassinoda (formicidae: ponerinae) in a lowland neotropical forest introduction ants are the main predators of land invertebrates in most of the world, withwide variation in foraging habits and the degree to which these are specialized (hölldobler & wilson, 1990; lach et al., 2010; traniello, 1989). initial scouting for food sources is most commonly by separate individuals, but in many species a scout who finds a good source then recruits nestmates to aid in its exploitation. in some species large food items are typically retrieved by several nestmates. food-source recruitment, where it exists, can take several forms, including tandem running (a nestmate follows the scout closely back to the food source, maintaining constant physical contact) and laying odor trails. the latter method can be extremely efficient, quickly bringing a large mass of nestmates to the food source and potentially allowing the colony to monopolize it. pachycondyla is a largepantropical genus of ponerine ants. p. crassinoda (latr, 1802) is the largest member of its genus in the new world, the robust, all-black workers having a body length of almost 20 mm (mackay & mackay, 2010). abstract pachycondyla crassinoda (latr, 1902) is one of the largest ant species in the new world tropics with a worker body length of about 18 mm. we studied its foraging habits in the field in a lowland forest in trinidad, west indies, with supplemental observations in the laboratory. the estimated density of colonies at our study site, 144/ha, is probably exceptionally high for this species. like other members of its genus, p. crassinoda forages on the forest floor, apparently never climbing trees or other plants. foraging is mostly limited to periods when the ground is relatively dry and is largely close to the nest. ants searched under fallen leaves at a high frequency and preyed mostly on small arthropods. they seldom stung prey, never unless the prey struggled vigorously. the only form of food-source recruitment observed was tandem running, with a maximum of two nestmates following the scout. sociobiology an international journal on social insects ae tudor, ck starr, k mohammed article history edited by gilberto m. m. santos, uefs, brazil received 14 june 2015 initial acceptance 11 august 2015 final acceptance 10 april 2016 publication date 15 july 2016 keywords tandem running, foraging, predation. corresponding author christopher k. starr dep’t of life sciences university of the west indies st augustine, trinidad & tobago e-mail: ckstarr@gmail.com it is the second largest of all ants (after paraponera clavata fabr.) in much of its broad range in south america east of the andes and north of the southern cone, while in trinidad it is the largest. it nests in the soil, typically under rotting logs or between tree roots (henriques & moutinho, 1994). the workers are commonly encountered on the surface of lowland forests. our preliminary observations indicated that foragers very often briefly looked under fallen leaves on the forest floor and that if disturbed they typically hid under leaves. the workers’ sting is initially sharp and painful, fading within a few minutes (pers. obs. of cks). for such a conspicuous, widely distributed species, p. crassinoda has been surprisingly little studied. in the main study to date, based on three laboratory colonies, henriques and moutinho (1994) reported very small colony sizes, a simple nest structure and social organization similar to that of other studied pachycondyla species, and food-source recruitment by tandem running. they concluded that the 33 behavior patterns recorded from workers were probably close to the complete repertory. silveira-costa and moutinho (1993) described the behavior of nestmate recognition in p. crassinoda. university of the west indies, st augustine, trinidad & tobago research article ants sociobiology 63(2): 744-747 (june, 2016) 745 our purpose here is to describe the foraging habits of p. crassinoda with a view to understanding its place in the ecosystem. given its very large size and powerful stinger, we hypothesized that it preys mainly on unusually large invertebrates beyond what other sympatric ant species can subdue. materials and methods all field study was at the arena forest reserve (10°34’n 61°14’w) in northeastern trinidad, west indies.the soil is sandy and well drained, with an undulating surface fragmented by many streams. the well-developed secondary forest has a closed canopy. as in the rest of trinidad, there is a moderately pronounced dry season from about mid-january to the end of may. our field observations, amounting to about 200 personhours, were from september 2012 to january 2013, and september 2014 to january 2015. laboratory observations were from november 2014 to march 2015. preliminary observations suggested that foraging activity (measured as number of workers seen on the forest floor) is greatest during the warmest part of the day from late morning to early afternoon. accordingly, our field observations were conducted from about 09:00 to about 12:00. preliminary observations likewise suggested that foraging activity is sharply reduced during and soon after rain. the study area was mapped with the aid of measuring tape, compass and flagging tape. it comprised 0.325 ha bounded by two broad trails and a stream (fig 1). within this area was a small, shallow pooland a small, low peninsula (marked “floodplain”) that was flooded when the stream was exceptionally high. our principle method in locating colonies was by laying out baits (fragments of cooked pork fat) throughout the study site and following foragers back to their nests (agosti et al., 2000), which we then marked with numbered stakes. we continued this for several weeks until very few of the nests we were finding were novel. a few nests also came to our attention by the typical tumulus of excavated soil at the entrance. baiting also allowed us to observe recruitment and retrieval of food items.we spent a significant minority of field time observing the behaviour of workers outside of the study site. in order to study how ground moisture affects foraging activity when not raining, one of us (aet) undertook paired walking transects at about 09:00 and 11:00. each transect followed a standard route for 30 min at a moderate walking pace, enumerating the workers on the trail or within about half a meter of its edge. these were done under three conditions: a) ground mostly dry, b) much of the ground moist with no significant standing water, and c) ground mostly wet with many spots of standing water. in order to determine whether foragers search over a wide area, we followed individuals for a total of 10 min each, estimating to the nearest meter the distance between their positions at the beginning and end of the period. at the same time, we counted the number of fallen leaves under which the worker at least put her head during the 10 min. we studied food preferences in the field by means of a simple cafeteria experiment. five kinds of food (moistened sugar, finely-chopped fresh fruit, crumbled salt crackers, ground cooked meat, and freshly-killed grasshopper) were placed on a dry fallen leaf within a meter of at least one nest on three relatively dry days. we then recorded instances of retrieval from the cafeteria. we collected five colonies for laboratory observation, beginning by collecting ants coming to baits placed near the nest entrance until very few new ones appeared, and then excavating. we failed to find the queen of three colonies, although collection appeared to be close to complete.the queenless colonies continued in apparent good health for several weeks. we provided laboratory colonies with a variety of live arthropods (grasshoppers, cockroaches, termites, beetles, large ants) similar to those found on the forest floor and whole or fragmented earthworms. these were categorized into three size classes: a) large (about the size of a worker ant, n=136), b) medium (about the size of an ant’s head, n=288), and c) small (distinctly smaller than the head, n=112). results within the study area we identified 48 colonies in the study site, presumably not far short of the true total. without attempting a dispersion analysis, the appearance is of a modest degree of clumping (fig 1), presumably due to unevenly suitable substrate. not only were there no colonies on the floodplain but we never observed a forager there.the six colonies that we were able to census apparently nearly completely had between 52 and 86 known workers. in our many hours of observing workers in unhindered activity outside the nest, we never saw any individual climbing a tree trunk or other plant. all indications are that foraging activity is strictly at the soil surface. fig 1. study area in the arena forest reserve at 10°34’41”n 61°14’35”w. circles indicate sites of known pachycondyla crassinoda nests. several of theseeach represent two apparently distinct colonies very close to each other. ae tudor, ck starr, k mohammed – pachycondyla crassinoda foraging746 the transectstudy recorded 112 foragers during two pairs of dry-condition transects, versus 12 during one pair of moist transects and nine during two pairs of wet transects. of the 20 workers whose movements were monitored for 10 min, 12 (60%) were one meter from the starting point at the end of observation. seven of the others showed a net displacement of either zero or two meters, and one ant was six meters away. the search pathway did not give the impression of being a random walk, as there was a great deal of turning back, and it was not uncommon to see an ant walk over the very same spot more than once during monitoring. the total distance traveled in each case was evidently several times the net displacement. the 20 ants searched under a mean of 27.3 (range 1246) fallen leaves in the course of 10 min. on two occasions we observed an ant returning from under a leaf with a small prey in her mandibles and proceeding directly away in the manner as ants returning to the nest with baits. there is, then, striking difference between a worker scouting for food and one retrieving. we observed 21 instances of tandem running, mostly to baits. of these, 17 involved two ants; the other four involved three ants. on the other hand, we never observed two or more ants in group retrieval of a food item, despite ample opportunity if this does occur. most baits were at least the size of a worker ant when first laid out, and if too large for a single ant to carry she would chew off a smaller piece. given the overall density of nests, it was to be expected that foragers from different colonies often contact each other. workers from different colonies often appeared at the same baits, commonly coming into direct contact. in no case did we observe any apparent antagonism or attempt to repel other workers. however, they were antagonistic to the social wasps (angiopolybia pallens (lep.)) that frequently came to baits. the cafeteria experiment yielded 24 observations of retrieval. of these, 21 were of grasshoppers or fragments of grasshoppers, the other three of cooked meat. that is, they spurned the fresh fruit, salt cracker, and even concentrated sugar solution. on the one occasion we found that the baits we had brought to the field showed evident decay (judge by smell). the ants accepted these apparently as readily as they did freshly-cooked bait. we recorded just 12 preys in the field. insofar as they could be identified, these were a variety of immature and adult arthropods. one was roughly the same size as the ant, the others much smaller, roughly the size of the ant’s head.in the laboratory we recorded no instance of a forager stinging a medium or small prey, and they stung large prey only when these struggled vigorously. in several cases, two or more workers attacked and stung an especially difficult large prey before cutting it into pieces for individual retrieval. on one occasion in the field we observed several ants at three open wild nutmeg (virola surinamensis) (myristaceae) fruits that had fallen to the ground. they were collecting and transporting fragments of the bright red ariloid. discussion the known density of colonies in the study area was 144/ha. because the study site was chosen for its apparent wealth of p. crassinoda foragers, this is probably close to the maximum density for the species, at least toward the northern end of its range. foraging at the ground level only is typical of pachycondyla (j.t. longino, pers. comm.). differences in number of foragers observed on transects during differing ground conditions are striking enough that no sophisticated statistical analysis is needed. the ants are clearly more active when the ground is drier (χ² test, p<0.01). it is known that p. crassinoda workers distinguish nestmates from other conspecifics (silveira-costa & moutinho, 1993), so that the lack of antagonism between non-nestmates at large, valuable baits is notable. given a choice of food, it prefers live or freshlykilled arthropod prey without specializing, although it also takes carrion and vegetable matter on occasion. against our working hypothesis, p. crassinoda appears to prey mostly on animals just a fraction its own size and seldom stings them. the ariloids of virola surinamensis appear to have a strikingly high lipid content (horvitz, 1981; maya et al., 2006). this, along with the ants’ strong foraging at cooked pork fat, suggest that the ants normally have little access to lipids. a tendency not to sting prey is consistent with the hypothesis that venom is metabolically expensive and best reserved for defense of the colony or self-defense against large predators. the picture emerging from our observations is of an ant that searches solitarily close to the nest and mostly under fallen leaves for small prey, avoiding wet ground conditions, with little recruitment of nestmates. acknowledgements we thank luke rostant and lee ann beddoe for assistance with various aspects of this project, and these and alda henriques, jack longino, danielle morong and the journal’s reviewer for helpful comments. rakesh bhukal provided very valuable field assistance. references agosti, d.m., alonso j. and schultz, t. (eds). (2000). ants: standard methods for measuring and monitoring biodiversity. washington: smithsonian institution press, 280 p. henriques, a. and moutinho, p.r.s. (1994). algumas observações sobre a organização social de pachycondyla crassinoda latreille, 1802 (hymenoptera: formicidae: ponerinae). revista brasileira de entomologia, 38: 605-611. translation at http://ckstarr.net/ cks/henriques1994.pdf horvitz c.c. (1981). analysis of how ant behaviors affects sociobiology 63(2): 744-747 (june, 2016) 747 germination in a tropical mymecochore calathea microcephala (p. & e.) koernicle (marantaceae): microsite selection and aril removal by neotropical ants, odontomachus, pachycondyla, and solenopsis (formicidae). oecologia, 51: 47-52 hölldobler, b. and wilson, e.o. (1990). the ants. cambridge: harvard university press 732 p. lach, l., parr, c. and abbott, k.(eds.)(2010).ant ecology. new york: oxford university press 424 p. mackay, w.p. and mackay, e. (2010). the systematics and biology of the new world ants of the genus pachycondyla (hymenoptera: formicidae). lewiston, ny: edwin mellon 642 pp. maya, k.m., zachariah, j.,krishnamurthy, k. rema, j and b. krishnamoorthy (2006). fatty acids and leaf amino acids in myristicafragrans houtt. andrelated taxa. indian journal of horticulture, 63: 316-318. silveira-costa, a.j. and moutinho, p.r.s. 1993. observações preliminaries sobre a reconhecimento entre operárias de duas colȏnias de pachycondy lacrassinoda latr (hymenoptera: formicidae: ponerinae). sociedade entomologica do brasil, 22: 619-622. traniello, j. (1989). foraging strategies of ants. annual review of entomology, 34: 191-210. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.882sociobiology 63(1): 637-644 (march, 2016) bacterial communities in the midgut of ponerine ants (hymenoptera: formicidae: ponerinae) introduction insects act as hosts for microorganisms, with which they share a wide range of interactions. the orders blattaria, hymenoptera, coleoptera and hemiptera are typically involved in symbiotic relationships with microorganisms (boursaux-eude & gross, 2000). among the symbionts of these species, bacteria have received the most attention because they determine the ecological success of the insect host, influencing insect development and the immune response (shoemaker et al., 2000), reproduction (giorgini et al., 2010), behavior (dillon et al., 2002) and particularly abstract symbiotic microorganisms are directly related to the ecological success of host insects, influencing many aspects of their biology. the present study is the first to investigate the microbiota associated with ants of the subfamily ponerinae and aims to identify the bacterial midgut communities of dinoponera lucida, neoponera curvinodis, pachycondyla striata, odontomachus brunneus and odontomachus bauri relied on culture-dependent technique, particularly 16s rrna sequencing. the greatest species richness was observed in o. bauri, with 15 otus, followed by d. lucida with five otus, o. brunneus, with four otus, and n. curvinodis and p. striata, both with three otus. there were representatives of the phyla actinobacteria, proteobacteria, tenericutes and firmicutes, including the genera bartonella, mesoplasma, mesorhizobium, spiroplasma, wolbachia and serratia in the guts of the studied ponerine ants. spiroplasma and mesoplasma were found to be prevalent in the studied ants and they were the only genera of bacteria found in more than one of the analyzed ant species suggesting they might be beneficial symbionts. the low microbial diversity observed given the predatory trophic habits of the species studied suggests that there is selection for these microorganisms, predominantly preserving symbionts with functional roles that are able to colonize this environment. it is also valid to infer that the identified bacteria are predominant in the gut and exhibit mutualistic functions that are important mainly for immunity, but also to reproduction and nutrition; moreover, a subset may be parasites that could have considerable impacts on the studied ants. sociobiology an international journal on social insects tb de oliveira2, m ferro2, m bacci júnior2, dj de souza3, r fontana1, jhc delabie4, a silva1 article history edited by evandro nascimento silva, uefs, brazil received 04 august 2015 initial acceptance 06 march 2016 final acceptance 11 march 2016 publication date 29 april 2016 keywords 16s rrna, dinoponera, odontomachus, pachycondyla, symbionts. corresponding author aline silva universidade estadual de santa cruz departamento de ciências biológicas laboratório de microbiologia rodovia jorge amado, km 16, salobrinho cep 45662-900, ilhéus-ba, brazil e-mail: asilva1@uesc.br nutrition (eilmus & heil, 2009; feldhaar et al., 2007; jaenike et al., 2010; van borm et al., 2002). the gut is an environment with a high incidence of symbiotic partnerships. there are reports of the existence of endosymbionts throughout the gastrointestinal tracts of insects (dillon & dillon, 2004; dunn & stabb, 2005). the bacterial diversity in these environments is related to the ph, redox potential, digestive enzymes in the gut and type of food, among other factors (dillon & dillon, 2004). social insects, such as ants (hymenoptera: formicidae), are interesting models for studies involving symbiotic relationships with microorganisms and for studies on 1 universidade estadual de santa cruz (uesc), ilhéus-ba, brazil 2 universidade estadual paulista (unesp), rio claro-sp, brazil 3 universidade federal do tocantins (uft), gurupi-to, brazil 4 uesc/laboratório de mirmecologia, ceplac, ilhéus-ba, brazil research article ants tb de oliveira et al. – bacterial communities in the midgut of ponerine ants638 coevolution. however, little is currently known about these interactions. in the tribe camponotini, the omnivorous ants of the genus camponotus have established an association with the intracellular bacteria blochmannia and it has been a fundamental partner by improving colony growth and the host immune system (souza et al., 2009). the congruence in the topology of bacteria and ant phylogeny suggests it has initiated in a common ancestral followed by the coevolution between the partners (sameshima et al., 1999; sauer et al., 2000). moreover, genomic analysis of blochmannia from host divergent lineages has showed differential loss of genes that affect cellular functions and metabolic pathways and this variation have been linked to distinct host-associated pressures (williams and wernegreen, 2015). it was also observed the symbiosis between tetraponera ants and nitrogen-fixing root-nodule bacteria which might be related to recycling nitrogen-rich metabolic waste (borm et al., 2002). nitrogen-fixing bacteria were also observed in a great diversity of ants and it is has been linked with the evolution of herbivory in ants (russel et al., 2009). in this sense, many researches have attempted to characterize microbial communities associated to ants and their putative role to improve host fitness (eilmus & heil, 2009; ishak et al., 2011; anderson et al., 2012; he et al., 2014). in the subfamily ponerinae, although the existence of bacteria in the midgut of odontomachus bauri emery 1892 has been reported using an ultrastructural approach (caetano et al., 2008; caetano et al., 2010), the identity of these microorganisms remains to be determined. although these ants exhibit plesiomorphic behavioral and morphological characteristics, such as very small colonies, reduced fertility rates and a poor capacity for dispersal and colonizing new areas, they present a wide geographical distribution (bolton, 2003; martins et al., 2007; werren et al., 2008), which could be related to the presence of symbiotic microorganisms. as a first step toward understanding the mutual benefits of symbiotic microorganisms in these ants, the purpose of the present study was to describe the bacterial communities in the midguts of the ants dinoponera lucida (currently included in the red list of endangered species in brazil (ministério do meio ambiente, 2003), neoponera curvinodis, pachycondyla striata, odontomachus brunneus, odontomachus bauri and to discuss the putative functional roles of bacteria-host interactions. materials and methods biological material and collection individuals of the ponerine species d. lucida, n. curvinodis, p. striata, o. brunneus and o. bauri. for each species, approximately 15 individuals were collected from a single nest. the individuals collected were processed immediately to avoid the possible alteration of their intestinal microbiota. the collected specimens were identified and deposited in the collection of the myrmecology laboratory of the cocoa research center (centro de pesquisas do cacau – cepec) under accession number # 5676. dna extraction and amplification of 16s rrna a total of 8 to 13 individuals of each species were used to increase the amount of dna extracted. the ants were anesthetized by freezing at -20°c for 2 min and then immersed in 70% ethanol, followed by washing in 0.9% nacl for external disinfection. the dissection was conducted under a dissecting microscope, with the materials placed on a glass slide containing a drop of 0.9% nacl. then, the midgut was washed three times in 0.9% nacl, and dna extraction was performed using standard proteinase k digestion in tnes buffer (250 mm tris, 2 m nacl, 100 mm edta and 2.5% sds, ph 7.5) and incubated in 1.5 ml tubes for 3 h at 55°c with 5 µl of proteinase k (20 mg/ml); then incubated at 37°c for 30 min after addition of 3 µl of rnase a (4 mg/ml). proteins were precipitated with 200 µl of 5-molar nacl and centrifugation; dna was precipitated from the supernatant using 600 µl of isopropanol and washing with 70% ethanol. samples were re-suspended with 30 µl of te (10 mm tris, ph 8; 1 mm edta ph 8) and extraction was confirmed in 1% agarose gel. for n. curvinodis, the amplification of the bacterial 16s rrna region was conducted via pcr, which was performed using the puretaq ready-to-go pcr beads kit (ge healthcare). each reaction contained 1.0 µl of dna (~ 30 ng) and 5 pmol of each of the universal bacterial primers 27f (5´-agagtttgatca/ctggctcag) and 1492r (5´-tacggt/ctaccttgttacgactt) (lane, 1991) in a final reaction volume of 25 ml. for all of the other species, the reactions consisted of 3 µl of 1x buffer ((nh4)2so4), 2.4 µl of mgcl2 (2 mm), 1.5 µl of dntps (0.2 mm), 0.3 µl of each primer (10 pmol), 1 µl of dna (~ 30 ng) and 0.2 µl of taq polymerase (1 u) (fermentas) in a final reaction volume of 30 µl. the pcr program consisted of an initial denaturation at 95°c for 5 min, followed by 35 cycles at 95°c for 1 min, 50°c for 1 min and 72°c for 3 min for n. curvinodis; there was an additional final extension at 72°c for 10 min for the other species. the primers amplified a region of approximately 1.5 kb. the pcr products obtained from n. curvinodis were purified using the gfx pcr gel band and dna purification kit (ge healthcare). for the other species, the purification was performed using a nucleic acid and protein purification kit (macherey-nagel). cloning and sequencing purified pcr products from p. curvinodis were ligated into pjet vectors using the clone jet pcr cloning kit (fermentas). competent e. coli dh10β cells were transformed via heat shock. subsequently, 100 µl of cells was inoculated into petri dishes containing lb agar and ampicillin (20 µg/ml), then incubated overnight at 37°c. for all of the other species, the purified pcr products were ligated into pgem vectors using sociobiology 63(1): 637-644 (march, 2016) 639 the pgem-t easy vector systems kit (promega). competent e. coli jm109 cells were transformed via thermal shock. subsequently, 100 µl of cells was inoculated into petri dishes containing lb agar, ampicillin (20 µg/ml) and x-gal (45 µl/plate) and incubated overnight at 37°c. the obtained recombinant colonies were transferred to 96-well plates containing lb agar (1 ml) and ampicillin (20 µg/ml) and grown at 37°c for approximately 22 h. the extraction of the plasmid dna was performed following the protocol described by vettore et al. (2001). the results of the extraction were visualized on 2% agarose gels, and only those clones that contained an insert of the expected size were used in the subsequent stages. the sequencing reactions were performed on microplates using the abi bigdye terminator cycle sequencing kit (version 3.1), 0.5 to 1 µg of dna and 5 pmol of the pjet1.2_forward primer (5’-cgactcactatagggagagcggc) for n. curvinodis and the m13_forward primer (5’-gttttcccagtcacgac) for the other species. after purification, the samples were run in an abi 3500 automated sequencer (applied biosystems). analysis of 16s rrna sequences the obtained 16s rrna sequences were pre-processed using the egene automated pipeline generation system (durham et al., 2005). at this stage, the sequences were screened for primer and vector sequences (pjet1.2 and m13), which were then removed. the base quality was also checked, with 90% considered to be the threshold for good bases within a window (phred value > 20), using a window size of 200 bp. subsequently, the sequences were filtered based on a minimum size of 200 bp and aligned using the clustalw tool (thompson et al., 1994), followed by manual refinement. using the distance matrix generated in dnadist, the sequences were assigned to operational taxonomic units (otus) using mothur (version 1.8.0) (sanchez-contretas & vlisidou, 2008). the obtained frequency data were used to construct rarefaction curves and to calculate the chao1 richness estimator and the shannon and simpson diversity indices. the sequences were assigned to phylogenetic classes using the classifier tool of the ribosomal database project (rdp) (xie et al., 2010) and compared with related sequences in the genbank database using the blastn tool (http://www.ncbi.nlm.nih.gov/genbank/index.html). results after pre-processing, 603 high-quality sequences were obtained; 129 were from d. lucida, 76 from p. curvinodis, 159 from p. striata, 125 from o. brunneus and 116 from o. bauri. these sequences were classified into known bacterial phyla using rdpclassifier from the ribosomal database project public database. using the 97% similarity criterion, the sequences were grouped into otus, and a representative sequence for each otu was compared with sequences deposited in genbank. the following phyla were detected: proteobacteria (in d. lucida, n. curvinodis, p. striata and o. bauri), tenericutes (in d. lucida, n. curvinodis, p. striata and o. brunneus ), actinobacteria (in n. curvinodis and o. bauri) and firmicutes (in o. bauri) (tables 1, 2 and 3). phylum sequence database match (organism; % identity; genbank accession no.) accession no. otu no. of clones tenericutes spiroplasma velocicrescens; 98%; nr_025713 km503191 1 45 spiroplasma diminutum; 96%; cp005076 km503192 2 72 proteobacteria mesorhizobium sp.; 97%; fj827045 km503193 3 2 uncultured mesorhizobium; 97%; dq303307.1 km503194 4 2 uncultured bartonella; 97%; dq113413 km503195 5 8 table 1. the phylogenetic grouping of the 16s rrna sequences from the midguts of the ant dinoponera lucida. table 2. the phylogenetic grouping of the 16s rrna sequences from the midguts of the ants of the genus neoponera and pachycondyla. ant species phylum sequence database match (organism; % identity; genbank accession no.) accession no. otu no. of clones neoponera curvinodis proteobacteria wolbachia pipientis; ay026912; 98% jq957016 1 35 actinobacteria micrococcus sp.; jn602241; 98% jq957017 2 1 tenericutes mesoplasma sp.; gq275130; 96% jq957018 3 40 pachycondyla striata proteobacteria bartonella vinsonii; eu295657.1; 98% jq957014 1 1 tenericutes spiroplasma velocicrescens; nr025713.1; 97% jq957013 2 69 spiroplasma sp.; ay189317.1; 96% jq957015 3 89 tb de oliveira et al. – bacterial communities in the midgut of ponerine ants640 ant species bacterial phylum sequence database match (organism; % identity; genbank accession no.) accession no. otu no. of clones odontomachus brunneus tenericutes spiroplasma leucomae; ab681166.1; 99% jq957019 1 43 uncultured mesoplasma; gq275130.1; 97% jq957020 2 22 spiroplasma atrichopogonis; ab681165.1; 99% jq957021 3 42 uncultured mesoplasma; hm996788.1; 98% jq957022 4 18 odontomachus bauri actinobacteria brevibacterium paucivorans; eu086796; 99% jq957026 1 1 propionibacterium acnes; jf277163.1; 99% jq957033 2 2 uncultured propionibacterium sp.; jf893681.1; 99% jq957027 3 1 leifsonia xyli; dq232616; 99% jq957028 4 1 microbacterium aurum; gu441767.1; 98% jq957031 5 1 schumannella luteola; nr041637.1; 95% jq957034 6 1 rubrobacter xylanophilus; cp000386.1; 99% jq957035 7 1 firmicutes bacillus thuringiensis; jq004436.1; 99% jq957025 8 1 dolosigranulum pigrum; nr026098; 98% jq957037 9 2 proteobacteria pseudochrobactrum kiredjianiae; nr042519.1; 99% jq957024 10 1 agrobacterium larrymoorei; eu741094.1; 99% jq957036 11 1 serratia marcescens; aj550467; 98% jq957023 12 55 aeromonas veronii; jf920563.1; 100% jq957029 13 1 enterobacter cancerogenus; jn644583.1; 98% jq957030 14 1 serratia marcescens; ab681729.1; 98% jq957032 15 46 table 3.the phylogenetic grouping of the 16s rrna sequences from the midguts of the ants of the genus odontomachus. in d. lucida were detected five otus, in which were detected two species of the genera spiroplasma (table 1). in n. curvinodis, three otus were found, with a predominance of the genera wolbachia and mesoplasma. three otus were also detected in p. striata, in which two spiroplasma species were predominant (table 2). in these three species of ants, otus were found that showed less than 97% similarity with homologous genbank sequences, indicating that they potentially represented new species of the genera spiroplasma and mesoplasma. four otus were detected in o. brunneus and 15 in o. bauri (table 3). bacteria of the mesoplasma and spiroplasma genera were found in o. brunneus, whereas in o. bauri, there was a predominance of species from the genus serratia as well as a large number of otus represented by single or only a few sequences from other genera. rarefaction analysis showed no significant differences in the richness of the bacterial communities present in d. lucida, n. curvinodis, o. brunneus and p. striata whereas o. bauri showed a significantly greater richness compared with the other species (figure 1). the curves reached plateaus at 3% difference, except for o. bauri, between sequences (95% confidence), indicating that the number of clones was sufficient to cover the diversity of bacteria in the midgut of these ants. in addition, according to the chao1 values, the highest species richness is expected in the bacterial community in o. bauri, sociobiology 63(1): 637-644 (march, 2016) 641 followed by d. lucida and o. brunneus, and there was no difference in the bacterial species richness between the two pachycondyla and neoponera (table 4). in terms of diversity, according to the shannon index, the odontomachus and dinoponera samples differed significantly from the pachycondyla samples. however, using the simpson index, differences were detected both between genera and between the odontomachus species studied, but there was no significant difference when compared with dinoponera. and are known to be present in a number of ant species (funaro et al., 2011; ishak et al., 2011; sapountzis et al., 2015). however, previous studies (funaro et al., 2011) have found that these bacteria are not always present in different subcastes within a single ant colony or different colonies from the same population, indicating that these microorganisms are not essential for the development of the host species. nevertheless, in certain cases, there is specificity between associated bacteria and ant species. sapountzis et al. (2015) suggest that their function might be related to the processing of chitin, the main component of the cuticles of insect prey. in the present study, spiroplasma and mesoplasma were found to be prevalent in the studied ants and they were the only genera of bacteria found in more than one of the ant species analyzed, and more than one species from the same genus was present in several cases, indicating that ants of this tribe is a natural reservoir of this group of microorganisms. it was observed that in the ant species in which both of these genera occurred (n. curvinodis, p. striata and o. brunneus), the diversity of other phyla was reduced. in the presence of these bacteria, there is likely a mechanism for the inhibition of other prokaryotes. spiroplasma has been reported to be responsible for causing the death of males originating from infected females (anbutsu & fukatsu, 2003; hurst & jiggins, 2000). this effect distorts the sex ratio, increasing the number of females in the population (wang et al., 2007). on the other hand, spiroplasma was reported as a mutualistic symbiont in drosophila associated with an increased tolerance against infection by nematodes and parasitic attack. moreover, no other symbiont bacteria was detected, and possibly this is the only symbiont responsible for defense against parasites (jaenike et al, 2010; xie et al, 2010) what could be essential for these ants considering their predatory habits which can bring several entomopathogenic microorganisms. another relatively common bacterium found in o. bauri in the present study was the enteropathogenic species serratia marcescens. this bacterium has been used as a model in studies addressing the biological control of insects due to its low to moderate pathogenicity and its ability to infect a wide variety of insects (connick et al., 2001; dillon et al., 2005). bacteria from the genus wolbachia were found in n. curvinodis. this genus includes intracellular bacteria that are widely distributed in arthropods. it is estimated that more than 20% of insect species are infected by a strain of wolbachia (russel et al., 2009). this occurrence is even more widespread in ants and may be related to their mode of colony formation, as wolbachia are more common in species in which the queen depends on workers to found the colony (watts et al., 2009). wolbachia belongs to the order rickettsiales (phylum proteobacteria, class alphaproteobacteria), and its members are considered to be reproductive parasites because they induce reproductive changes in their hosts, such as the death of males, the feminization of males, thelytokous parthenogenesis fig 1. rarefaction analyses of the dinoponera lucida, neoponera curvinodis, pachycondyla striata, odontomachus bauri and o. brunneus midgut 16s rrna clone libraries. the predicted numbers of otu’s were calculated at the 3% level of sequence divergence. sample s richness estimate diversity indices chao shannon simpson d. lucida 5 6 (±0) 1.03 (±0.15)a 0.43 (±0.15)a,b,c o. bauri 15 33 (±13.86) 1.31 (±0.23)a 0.37 (±0.05)b o. brunneus 4 4 (±0) 1.33 (±0.06)a 0.27 (±0.03)a p. striata 3 3 (±0) 0.72 (±0.06)b 0.51 (±0.02)c n. curvinodis 3 3 (±0) 0.76 (±0.01)b 0.51 (±0.03)c table 4. the richness of otus (s), estimated richness of otus and diversity indices for the 16s rrna clone libraries. the values that are followed by different letters are significantly different (95% confidence interval). discussion studies on the occurrence and functional role of bacterial symbionts in ants are still very scarce, making it difficult to understand how these bacteria affect their hosts or how host diet and intestinal physiology and structure affect the bacterial community. a predominance of species from the genera mesoplasma, spiroplasma, wolbachia and serratia was observed in the guts of the ponerine ants studied. mesoplasma and spiroplasma are genera of intracellular bacteria from the order entomoplasmatales (phylum tenericutes; class mollicutes) that are commonly found in the guts of insects tb de oliveira et al. – bacterial communities in the midgut of ponerine ants642 and cytoplasmic incompatibility (wenseleers & billen, 2000). in ants, cytoplasmic incompatibility is likely the main mode of action of these bacteria (wenseleers et al., 1998; wenseleers et al., 2002; watts et al., 2009). in other insects, it has been observed that wolbachia interfere with the expression of ferritin and with iron metabolism. these bacteria reduce the concentration of iron, protecting the cell from oxidative stress and apoptosis. this phenomenon has been observed both in species in which these bacteria occur as mutualists and in species in which they occur as facultative parasites (keller et al., 2001). the occurrence of wolbachia has also been related to supplementation with vitamin b (hosokawa et al., 2010). despite the high incidence of these bacteria in ants, there are no conclusive studies addressing their effects, and little is known about this topic (wenseleers et al., 1998; jaffe et al., 2001; rani et al., 2009; schloss et al., 2009). interestingly, the three species of ants found in this study to harbor bacteria that alter the reproduction or sex ratio, such as wolbachia and spiroplasma, are polygynous. this characteristic of their colonies could be evolved in the ants to reduce the effects of these bacteria by allowing uninfected queens to maintain the production of males in the nest. however, the opposite was observed to the ant solenopsis, where wolbachia infection was prevalent in monogynous colonies (ishak et al., 2011). species belonging to the order rhizobiales (class alphaproteobacteria, phylum proteobacteria) were found in the present analysis, such as mesorhizobium and bartonella in d. lucida, pseudochrobactrum and agrobacterium in o. bauri and bartonella in p. striata. rhizobiales includes known nitrogenfixing species. the presence of these bacteria has been reported in the guts of other ants, and it has been suggested that these microorganisms are responsible for dietary supplementation through nitrogen fixation or possibly through the recycling of nitrogen (reuter et al., 2005). the occurrence of micrococcus, which was found in n. curvinodis, has also been reported in the midgut of other insects based on dependent and independent culture methods (broderick et al., 2004; kremer et al., 2009; rafagopal, 2009). these bacteria are not pathogenic. their presence is related to the immune response via the production of compounds that act against fungal antagonists (cardoza et al., 2006; hillesland et al., 2008). overall, there was a low diversity of bacteria in the midguts of n. curvinodis, p. striata and o. brunneus; the genera spiroplasma and mesoplasma were always present in these species but not necessarily together. in contrast, the bacterial diversity found in o. bauri was high (13 genera) and included s. marcescens, an entomopathogenic species. in o. bauri, neither of the two bacterial genera mentioned above were detected. ishak et al. (2011) explored the microbiome of the ant solenopsis geminata, which has a more granivorous diet, using a massive parallel sequencing, and they found a high abundance of spiroplasma in workers and that is why they suggested it to be an important partner for this ant. therefore, considering that these bacteria infect ants with different diet it is possible to predict that spiroplasma and mesoplasma are likely mutualists and have more immunologic instead of nutritional role by inhibiting the occurrence of a greater diversity of bacteria, including potential pathogens, protecting the host ants against massive infection by other prokaryotic microorganisms. taking into account the predatory trophic habits of the studied species, the low microbial diversity observed suggests that there is selection of these microorganisms by the host ants, which primarily maintain symbionts that play functional roles and that are able to colonize this environment. feeding from other insects may bring entomopathogenic bacteria to the ant gut, thus it can also be concluded that the predominant identified bacterial species might exhibit mutualistic functions that are important mainly for immunity, but also to reproduction and nutrition. acknowledgements this work was supported by secti/fapesb/cnpq (process: fapesb/cnpq no. 020/2009-pronex mbj and jhcd acknowledge their cnpq research grants). references anbutsu, h., fukatsu, t. 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(2015) genome evolution in an ancient bacteria-ant symbiosis: parallel gene loss among blocmannia spanning the origin of the ant tribe camponotini. peerj, 3:e881. doi: 10.7717/peerj.881 wilson, e.o., hölldobler, b. (2005) the rise of the ants: a phylogenetic and ecological explanation. proceedings of the national academy of sciences of the united states of america, 102:7411-7414. doi: 10.1073/pnas.0502264102 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.7763sociobiology 69(3): e7763 (september, 2022) introduction multiple interacting factors including exposure to pesticides, attack of parasites and pathogens and habitat loss have been suggested as chronic sub lethal stress associated with the decline in the population of honeybees (bryden et al., 2013). a group of neurotoxic insecticides, the neonicotinoids, has been singled out from all the stress factors due to its wide use in crop protection (godfray et al., 2014). neonicotinoids abstract neonicotinoids are one of the major stresses contributing to the decline in the population of honeybees. worker bees are prone to various stress factors during foraging and are susceptible to imidacloprid due to the reduction in the number of genes encoding for the major enzyme families responsible for the detoxification of toxins. the present study worked on the hypothesis that the dietary supplementation of ascorbic acid (vit c) could reduce the peroxidative damage in the worker bees of apis cerana indica exposed to sublethal concentration of imidacloprid (imd). furthermore, we also evaluated the role and efficacy of vit c supplementation on the cytoarchitecture of midgut tissues on exposure to imd. colonies of honeybees were maintained by providing sugar syrup to the control group and sugar syrup supplemented with 0.2% vit c for the experimental group for six months. worker bees from both groups were randomly collected and exposed to 0.001 mg/ml imd. to study the peroxidative damage, the activities of various enzymes were analyzed. the activities of antioxidant enzymes including catalase, superoxide dismutase, glutathione s transferase, and glutathione peroxidase in the hemolymph and midgut tissues of worker bees were significantly decreased due to exposure to imd as a single agent. however, their activities showed a significant elevation under diet supplementation with vit c. histological examination revealed midgut tissue damage and the rupture of peritrophic membrane among the workers exposed to imd as compared with the control group. the damage to the midgut was alleviated and the peritrophic membrane was found to be intact in the worker bees supplemented with vit c. our results indicated that the dietary supplementation of vit c has the potential to maintain the redox status and thereby can offer protective potential against the peroxidative damages induced by the sub-lethal concentration of imd. sociobiology an international journal on social insects syama p. s., sreeranjit kumar c. v. article history edited by evandro nascimento silva, uefs, brazil received 01 february 2022 initial acceptance 23 may 2022 final acceptance 17 june 2022 publication date 07 september 2022 keywords neonicotinoids, ascorbic acid, antioxidant enzymes, peritrophic membrane, honey bees. corresponding author syama p. s. p. g. and research department of zoology, govt. victoria college palakkad, kerala. e-mail: syamaktp@gmail.com are synthetic analogs of nicotine which mimics the action of acetylcholine, the main excitatory neurotransmitter in the brain of honeybees (casida et al., 2013). neonicotinoids, which comprise imidacloprid, acetamiprid, clothianidin, thiomethoxam, thiacloprid, dinotefuran and nitenpyram were extensively used in seed treatments, soil applications and as foliar sprays to control crop pests (blacquiere et al., 2012). they target nicotinicacetylcholine receptors (nachr) and disrupt the functioning of the central nervous system p. g. and research department of zoology, govt. victoria college, palakkad, kerala, india research article bees evidence of diet supplementation with vitamin c protecting honeybees from imidacloprid induced peroxidative damage: a study with apis cerana indica syama p. s., sreeranjit kumar c. v. – vitamin c supplementation protects honeybees from peroxidative damages of imidacloprid2 by overstimulation (matsuda et al., 2001). this group of pesticides may cause behavioral problems in honeybees such as defective or delayed communication, navigation, homing and foraging (henry et al., 2012) and adversely affect their immunity (di prisco et al., 2013). as these are systemic insecticides, their traces are found in nectar and pollen, bee products like bee bread, honey and beeswax (blacquiere et al., 2012). imidacloprid (imd) is widely used against insect pests because of its diverse application methods and lower toxicity to non-target organisms (medrzycki et al., 2003). imd and its metabolites were detected in pollen, honey samples, and from different honeybee body parts such as hemolymph, midgut, thorax and rectum (suchail et al., 2004). sub lethal effects manifested in bees exposed to imd include inhibition in associative learning (decourtye et al., 2004), abnormal foraging behavior (yang et al., 2008), reduction in mobility and loss of communicative ability (medrzycki et al., 2003). considering the effect on metabolism, imd impairs brain metabolism (decourtye et al., 2004) and reduces mitochondrial activity (nicodemo et al., 2014). the midgut of honeybees is highly sensitive to poisonous substances (bielenin & ibek, 1980) and malnutrition (szymas & przybyl, 2007) due to the absence of chitinous lining. the midgut lumen has a protective covering called peritrophic membrane (pm) that surrounds the food (snodgrass, 2018). in the lumen, pm acts as a semipermeable membrane and protects the midgut epithelium from mechanical damage, attack of pathogens (lehane et al., 1997) and from toxic substances (bielenin & ibek, 1980). metabolic resistance is one of the principal mechanisms used by insects to escape the adverse effects of natural and synthetic toxins (esther et al., 2015). organisms are endowed with a wide variety of endogenous antioxidative enzymes that protect cells from peroxidative damage, such as superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione reductase (gsr) and glutathione s-transferase (gst). as the honeybees have a lower number of genes coding for antioxidant proteins in their genome, the ability of honeybees to defend against ros is strongly limited as compared to other insects (corona et al., 2006). the antioxidant enzymes in honeybees are efficient in detoxification of ros and sod acts as the first line defense against oxygen free radicals (corona et al., 2006). h2o2 is eliminated by the action of cat (aebi, 1984) and gpx (mannervik, 1985). gst conjugates the toxic compounds to glutathione to facilitate their removal from the tissues (hinton et al., 1995; jakoby, 1985; lee, 1991). vitamin c is an important water-soluble antioxidant which can neutralize ros and reduce oxidative stress (verma et al., 2007). it can work from both inside and outside the cells to combat free radical damage and may act as a source of electrons to free radicals such as hydroxyl and superoxide radicals in order to quench their reactivity (bendich et al., 1990; bindhumol et al., 2003). earlier studies showed that the dietary supplementation of vit c positively influences the antioxidant defense and in turn reduces the winter mortality rate (farjan et al., 2012; andi & ahmadi, 2014). the present study was designed to examine the protective effect of dietary supplementation of ascorbic acid on the antioxidant defense system and histological alterations in the midgut of honeybee workers under imd exposure. materials and methods 1. experimental protocol 1.1. field colonies used in the experiments the honeybee colonies of a. cerana indica used in the experiment were maintained in a domestic garden (palakkad district, latitude-10.89, longitude-76.4). experimental colonies were provided with 0.2 % vit c in 250 ml sugar syrup (1:1 sugar and water) while the control colonies were provided with 250 ml sugar syrup alone. they were fed once in a week, throughout the experiment over a period of six months. the colonies were periodically examined for the viability of the colony. 1.2. laboratory experiment worker bees were randomly collected from the brood of control colonies and were divided into two groups. out of this one group was maintained as control by providing sugar syrup alone (control) and the other group was administered with a sub lethal concentration of 0.001 mg/ml imidacloprid along with sugar syrup (imd). similarly, the vitamin c supplemented bees were also grouped into two. out of this one group was maintained as control with vitamin c supplementation (vit c control) and the other group was administered with imidacloprid at a sub lethal concentration of 0.001 mg/ml (vit c + imd) and all the groups were left undisturbed for an hour after treatment (the sub lethal concentration of imd was achieved by evaluating ld50 as per the guidelines mentioned in oecd, 1998). 2. enzyme assays ten worker bees were randomly selected from each treatment, and they were anesthetized by keeping them at 4 ºc for five minutes. the hemolymph was collected using microcapillary tubes after incising it dorsally between the 5th and 6th abdominal segment by using a sterile needle. the midgut was dissected out according to the protocol described by carreck et al. (2013). they were pooled and homogenized in saline solution, centrifuged at 10,000 rpm for 20 minutes at 4 ºc, then the supernatants were decanted and used for enzyme assays. the enzymatic activity of catalase (luck, 1974), peroxidase (reddy et al., 1995), superoxide dismutase (paoletti et al., 1986), glutathione s-transferases (habig et al., 1974), glutathione peroxidase (rotruck et al., 1973), glutathione reductase (david & richard, 1983) of the hemolymph and the midgut tissues were determined. the experiment was repeated three times. sociobiology 69(3): e7763 (september, 2022) 3 3. histological analysis the midgut of worker bees from each treatment were dissected out and fixed in bouin’s fixative for the histological studies. serial sections were dewaxed and dehydrated using different grades of alcohol and were double stained with hematoxylin and eosin. the histological analysis was done and photographs were taken by using a leica dm 750 light microscope. scaling and labelling were done by using image j software. 4. statistical analysis a probit analysis was done for the ld50 calculation. significant differences among treatments were identified by one way anova and pairwise analysis was carried out using dmrt (duncan multiple range test) at 5% (p < 0.05) level of significance with statistical software ‘r’ version 4.1.1. results 1.enzymes of antioxidant system 1.1. catalase activity the exposure of honeybees to imd significantly inhibited catalase activity in the hemolymph and midgut, since in this treatment the cat activity was 4.7 ± 2.5 and 57.9 ± 13.1, respectively. there was a clear decrease in the cat activity in comparison to the control (12.06 ± 3.38 and 101.1 ± 11.8, for hemolymph and midgut, respectively). when the diet was enriched only with vit c, the cat activity (34.9 ± 5.9 for hemolymph and 146.2 ± 0 for midgut) presented the highest and statistically significant effect. finally, the supplementation of vit c in the diet of honeybees exposed to imd contributed to suppress the negative effects the insecticide, reestablishing the cat activity to levels higher than in the control for the hemolymph and equal to the control for the midgut (fig 2). 1.2. superoxide dismutase activity superoxide dismutase (sod) activity was assessed in both hemolymph and midgut (fig 3). no significant change (p ≥ 0.05) was observed in the activity of sod in the hemolymph of worker bees, when they were exposed to imd (2.0 ± 0.005) compared to control (2.0 ± 0). in the midgut tissues, the imd treatment resulted in a significant (p ≤ 0.05) decrease of enzyme activity (2.01 ± 0.008). a significant increase in the sod activity in the hemolymph and midgut of worker bees was noticed in vit c supplemented treatment (2.06 ± 0.008 for hemolymph and 2.04 ± 0.02 for midgut) against the control (2.0 ± 0 for hemolymph and 2.03 ± 0.01 for midgut). when worker bees were exposed to imd but the diet was supplemented with vit c, the sod activity was found to control colony (not supplemented with vitamin c ) experimental colony (supplemented with 0.2% vitamin c ) experimental design control imd vit c vit c+imd feeder fig 1. control: workers from control colony, imd: workers from control colony exposed to imd at 0.001mg/ml, vit c: workers from experimental colony, vit c + imd: workers from experimental colony exposed to imd at 0.001mg/ml. syama p. s., sreeranjit kumar c. v. – vitamin c supplementation protects honeybees from peroxidative damages of imidacloprid4 be increased significantly in the hemolymph (2.03 ± 0.01) when compared to control, but no significant difference was observed in the midgut in comparison to the control. 1.3. peroxidase activity peroxidase (pod) activity was assessed both in the hemolymph and midgut tissues of worker bees (fig 4). no significant difference (p ≥ 0.05) was recorded in the activity of pod in the hemolymph of worker bees from treated groups and control. an increase of activity was recorded in imd fed bees (2846 ± 1223), but no significant difference (p ≥ 0.05) was found in other treatments. the imd treatment resulted in a significant elevation (p ≤ 0.05) of enzymatic activity in the midgut of worker bees (11660 ± 1725) as compared to the control treatment (2837 ± 18.02). a significant decrease of pod activity was observed in the midgut of worker bees from vit c treatment (3055 ± 212.3) and vit c + imd treatment (5670 ± 515) when compared to imd treatment (11660 ± 1725). fig 2. specific activity of cat in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. fig 3. specific activity of sod in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. sociobiology 69(3): e7763 (september, 2022) 5 1.4. glutathione s-transferases activity statistical analysis performed on the data showed that worker bees in the imd treatment had a significantly (p ≤ 0.05) lower gst activity both in the hemolymph (2.24 ± 0.6) and midgut (0.7 ± 0.3) compared to the control (6.4 5± 1.0 and 1.01 ± 0.3). the worker bees from the vit c treatment (11.4 ± 2.1 and 2.0 ± 0.3) and vit c + imd treatment (7.44 ± 0.9 and 2.6 ± 0.3) had a significantly (p ≤ 0.05) higher gst activity both in hemolymph and midgut as compared with the control (fig 5). fig 4. specific activity of pod in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. fig 5. specific activity of gst in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. syama p. s., sreeranjit kumar c. v. – vitamin c supplementation protects honeybees from peroxidative damages of imidacloprid6 1.5. glutathione peroxidase activity the activity of the enzyme glutathione peroxidase was measured in the hemolymph and midgut of worker bees (fig 6). the activity of this enzyme in the hemolymph was significantly higher with the treatments with exposure to imd and/or supplementation with vit c, in comparison with the control. regarding midgut tissues, the exposure of honeybees to imd did not affect the enzyme activity. however, the supplementation of the diet with vit c alone or combined with the exposure of honeybees to imd significantly increased the enzyme activity. 1.6. glutathione reductase activity gsr activity was measured in the hemolymph and midgut of worker bees (fig 7). the statistical analysis performed on all data showed an effect of vit c in increasing enzyme activity in the hemolymph (0.76 ± 0.09) of worker fig 6. specific activity of gpx in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. fig 7. specific activity of gsr in the hemolymph and mid gut tissues of worker bees (treatment means obtained with n = 6). significant differences with p-value ≤ 0.05 are marked with different letters. sociobiology 69(3): e7763 (september, 2022) 7 bees in comparison to the control (0.16 ± 0.02). the activity of the enzyme was found to be reduced when the worker bees were exposed to imd. 2. histological analysis the histological analysis of the midgut of honeybees from the control (figure 8-a) showed a typical morphology of epithelium which contains a thin gelatinous layer, multiple layers of peritrophic membrane (pm) and a wide lumen between pm and epithelium. in some regions, the midgut epithelium was higher and, in some places, it was not well defined. at the base of the epithelium, the nuclei are surrounded with lighter areas (hallo’s) with vacuolated cell cytoplasm. pollen grains were surrounded by numerous layers of peritrophic membranes. tearing of the pm from the gut epithelium was clearly visible in some regions. intestinal lumen is wider in the control group. in the vitamin c supplemented group (figure 8-b), the analysis of the midgut showed a well-defined epithelium with a thick gelatinous layer. the nuclei were covered with hallo’s and the cell cytoplasm was less vacuolated. the epithelium was higher in some places and the epithelial folds were not well defined. pollen grains present in the midgut region were covered with multiple layers of peritrophic membranes. tearing of the peritrophic membrane was seen at some regions. intestinal lumen was slightly wide. cyto-architectural analysis of the midgut of bees in the imd treatment (figure 8-c) showed adverse effects, where the peritrophic membrane was totally ruptured and the midgut contents were dispersed in the lumen. the gut epithelium was degenerated and without the gelatinous matrix, and the cells had large vacuoles. the severity of the damage to the histological architecture was comparatively less on vit c +imd treatment (figure 8-d). the thickness of the epithelial lining was found to be the same as in vit c treatment, but large vacuoles were present in the epithelium. the peritrophic membrane was found intact, degeneration of epithelial cells and loss of gelatinous matrix were not observed. a e l pm b e l pm c e l d e pm fig 8. histology of midgut cells of worker honeybee (a. cerana indica) stained with h & e. a-d includes, a. midgut cells of honeybee workers received sugar syrup presenting thin gelatinous layer of epithelium with wide lumen and intact peritrophic membrane. b. midgut cells of honeybee workers supplemented with vitamin c showing well defined epithelium, slightly wide lumen and multiple layers of peritrophic membrane. c. midgut cells of honeybee workers exposed to imd showing degenerated epithelial cells and completely ruptured peritrophic membrane. d. midgut cells of ascorbic acid supplemented worker bees exposed to imidacloprid presenting thick epithelium and multi layered peritrophic membrane. e epithelium; pm peritrophic membrane; l lumen. syama p. s., sreeranjit kumar c. v. – vitamin c supplementation protects honeybees from peroxidative damages of imidacloprid8 discussion oxidative stress refers to an imbalance in the redox status of the body due to the overproduction of free radicals beyond the capability of the antioxidant defense system to neutralize it. the uncontrolled scenario of free radical production may pose a serious threat to the survival of the body tissues due to the damage of the vital cellular components such as the membranes, lipids, nucleic acids, and proteins (hodgson & smart, 2001). aerobic organisms are endowed with protective mechanisms comprising enzymatic and nonenzymatic antioxidants that are habitually efficient in blocking the detrimental effects of free radicals, including ros. antioxidant enzymes such as superoxide dismutase (sod), catalase (cat), and glutathione peroxidase (gpx) are the key components to maintain the redox balance since they scavenge the excess of free radicals and can be the first line of defense against various types of such oxidative radicals. sod and cat are the enzymes which act as subsequent constituents in the antioxidant response system of the cells. cat converts superoxide (o2ˉ) into h2o2 and then sod converts the so formed h2o2 into h2o and o2. various enzymatic free radical scavengers including catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), peroxidase, and superoxide dismutase (sod) are reported to be present in vast quantities in bees (weirich et al., 2002; strachecka et al., 2014; strachecka et al., 2017). the elevation in the levels of antioxidant enzymes may therefore be an important indication of an organism’s endeavor to counter an induced oxidative stress. the present study brings forth new insights to the capability of dietary supplementation of vitamin c in diminishing the peroxidative damages generated by imd. the specific levels of antioxidant enzymes in the honeybee a. cerana indica were studied to understand the tolerance and detoxification strategies against imd toxicity. the present observations indicate that the exposure of honeybees to imd had a significant effect on the activities of antioxidant enzymes. the activation of antioxidant enzymes observed in the present study is supposed to be a defensive mechanism to scavenge the excess ros to alleviate the adverse effects induced by the pesticide. the primary function of catalase enzymes is the conversion of hydrogen peroxide (h2o2) into h2 and o2. h2o2 is one of the most stable reactive oxygen species and plays a key role in the pathologies of numerous diseases, including alzheimer’s disease. catalase is an antioxidant enzyme found in many cell types and is constituted by several sulf-hydryl groups (mainly of the amino acid, cysteine) in its internal structure. neutralization of free radicals by this antioxidant is through the expenditure of its sulf-hydryl groups (deisseroth & dounce, 1970; chance et al., 1979; egaas et al., 1999; milton, 2004; enayati et al., 2005; kirkman & gaetani, 2007). in the present investigation we observed a significant reduction in the activity of catalase enzyme in both the midgut and hemolymph regions of worker bees exposed to imd. down regulation of catalase activity is coupled with augmented defenselessness to oxidative stress. uncontrolled free radical production may be due to the saturation of the available sulf-hydryl groups of catalase enzyme. this imbalance in the redox balance may in turn attack the catalase enzyme and can cause denaturation of its structure. the overall result is the damage to the cell membranes by h2o2 induced oxidative damages (goth et al., 2004; ho et al., 2004). vitamin c as an adjuvant was found to elevate the levels of catalase enzyme significantly in both the midgut and hemolymph regions of worker bees. the protective potential of this vitamin is supposed to be due to its capability to reduce the free radical induced damages by either reducing the levels of free radicals or by stimulating the activity of catalase enzyme. superoxide dismutases (sods) refer to a group of metalloenzymes that are present in all kingdoms of life. sods serve as the front-line defense mechanism against tissue injuries due to reactive oxygen species (ros). these proteins catalyze the dismutation of superoxide anion free radical (o2) into molecular oxygen and hydrogen peroxide (h2o2) and thereby decreasing o2 levels which otherwise can damage the cells at excessive concentrations (younus, 2018). our study showed a reduction in the activity of sod in the hemolymph and midgut tissues of honeybees exposed to imd. the reduced activity of the scavenging enzymes observed in the study reflects the excess of ros generation due to the exposure to imd. our findings are in line with kapoor et al., (2010). on the other hand, vitamin c supplementation was found to enhance the activity of sod in the corresponding group in the present study. the elevated level of sod is supposed to be due to the boosting of the dismutation process by imd which in turn can facilitate the elimination of ros. the major purpose of the peroxidase enzyme is the breakdown of h2o2 to nontoxic components (thangudu & su, 2021). a significant elevation in pod levels was observed in honeybees exclusively exposed to imd. this elevation in peroxidase levels is an indication of the enhanced synthesis of peroxidase enzyme to counter the increased volume of free radicals in the tissues. vitamin c as an adjuvant was found to cause a significant reduction in peroxidase enzyme levels and this probably indicates the capability of this vitamin in alleviating the stress generated by oxidative stress. glutathione peroxidase refers to an enzyme family endowed with peroxidase activity which is assigned with the biological role of protecting the organisms from oxidative damage. gpx defends free radical attacks at the expense of gsh. el-gendy et al. (2010) reported that the main function of gpx is to reduce lipid hydroperoxides to their consequent alcohols and to reduce free hydrogen peroxide, thereby regulating the redox balance in the body. in the present study, we observed a significant reduction in gpx levels in the midgut of worker bees exposed to imd. this is an indication of the probable damage of midgut cell membranes due to the enhanced production of hydroperoxides and other free radicals because of the failure of gpx in neutralizing them. sociobiology 69(3): e7763 (september, 2022) 9 our observations are in line with the findings of kapoor et al. (2010) who also reported a reduction in gpx activity on imd exposure. the enhanced level of gpx in the hemolymph in imd treated bees indicates the probable defense mechanism against the enhanced production of hydroperoxides, which in turn can reduce the damage of cell membranes. the different mechanisms of action of gpx in the midgut and the hemolymph may be due to the difference in basal concentrations of this key free radical scavenging enzyme in the two regions. vitamin c supplementation was found to boost the activity of gpx in both the midgut and hemolymph in worker bees. this is an indication of the capability of vitamin c in enhancing gpx production in various tissues, irrespective of the basal concentrations. this clearly indicates that the administration of vitamin c in the diet of beehives subject to foraging in areas or agricultural landscapes with imd application can alleviate the toxic aspects of this insecticide in various tissues. this line of investigation can be of great importance to the practice of apiculture and the prevention of colony decline or even collapse. our study presents interesting results in support of diet supplementation as a strategy to counteract the deleterious effects of imd in the metabolic defenses of honeybees against toxic agrochemical compounds. glutathione s-transferases (gsts) comprise a family of phase ii detoxification enzymes that are assigned with the function of safeguarding the different cellular macromolecules from attack by reactive electrophiles. gsts regulate the levels of an enormous range of electrophiles by conjugating them with glutathione (gsh). glutathione conjugation is the primary step in the mercapturic acid pathway that facilitates the eradication of toxic compounds (townsend & tew, 2003). observations from our study indicate the significant reduction of gst levels in the hemolymph and midgut tissues of worker bees exposed to imd. this may be due to the enhanced utilization of gsh in neutralizing the larger volumes of electrophilic toxic compounds produced due to imd administration. on the other hand, vitamin c as an adjuvant was found to present a different scenario with a significant enhancement of gst levels in the respective groups. the enhancement of gst activity may be due to the alleviation of the levels of oxidative radicals by vitamin c, which in turn resulted in the reduced usage of gsh. this elevated concentration of gsh can offer protection against cellular stress by scavenging the excess free radicals. glutathione reductase (gsr) is a key enzyme that is involved in the redox metabolic cycle of gsh along with gpx, which in turn catalyzes the oxidation of gsh (reduced form) to gssg (oxidized form). once gssg is formed it is reduced by gsr, which utilizes nadph as the reducing factor (jefferies et al., 2003). a significant reduction in gsr level was observed in the hemolymph and midgut tissues exposed to imd alone. since the concentration of gsr in cells is much lower than that of gpx, any variation in the redox balance due to enhanced oxidative stress can cause a significant reduction in these enzyme levels. it can be noted that vitamin c supplementation was effective in enhancing the activities of these antioxidant enzymes in the hemolymph and midgut tissues. gsr also follows the same pattern of activity. such a positive up regulation of antioxidant status along with the down regulation of oxidative stress may be the reason for maintaining the proper redox status in the hemolymph and midgut tissues. the digestive system is the element of contact with pathogens and poisonous substances and is responsible for the detoxification of harmful substances (higes et al., 2013). histological analysis of the midgut epithelium may reveal the toxicity of ingested xenobiotics (han et al., 2012). the midgut epithelial cells of bees fed with sugar syrup elicited normal appearance, the peritrophic membrane (pm) appeared visible and intact with normal thickness (figure 8-a). the midgut cells of honeybees supplemented with vitamin c elicited well defined epithelium, slightly wide lumen and multiple layers of the peritrophic membranes, indicating normal tissue morphology (figure 8-b). microscopic examination of the midgut cells exposed to imd reveals degenerated epithelial cells and completely ruptured peritrophic membrane, indicating damage to the tissue membranes. the damage may be due to the irregular redox balance in tissues due to the enhanced production of free radicals on exposure to imd (figure 8-c) (balieira et al., 2018; yucel & kayis, 2019). peritrophic membrane plays an important role in protecting epithelial tissues from the adverse effects of chemical and microbial components in food (terra, 1988). any alterations in the midgut epithelial cells and pm can be considered as an indicator of environmental stress (pawert et al., 1996). histological observations of the midgut epithelium of vitamin c treated bees exposed to imd showed a multilayered peritrophic membrane. this indicates the capability of ascorbic acid supplementation in protecting the tissues from imd induced toxic effects by the proper maintenance of the cellular redox balance (figure 8-d). multiple layers of peritrophic membrane in bees are associated with better utilization of nutrients (szymas et al., 2012; crailsheim, 1988). the modifications in the cyto-architecture of gut epithelium give supplementary evidence for the protective role of vit c against imd toxicity. conclusion the present study revealed the potential of imidacloprid in adversely affecting the redox balance in the midgut tissues and hemolymph of honeybees. the redox imbalance thus generated has resulted in the accumulation of free radicals in the midgut tissues, which in turn resulted in the building up of oxidative stress. the scavenging of electrons from the cell membranes by the free radicals has resulted in the altered morphology of tissues as evidenced from the histological syama p. s., sreeranjit kumar c. v. – vitamin c supplementation protects honeybees from peroxidative damages of imidacloprid10 analysis. on the contrary, vitamin c supplementation as an adjuvant was found to properly regulate the antioxidant defense system in the tissues as evidenced from the activities of scavenging enzymes. the maintenance of normal morphology of the tissues by vitamin c serves as strong visual evidence for such regulation. hence, we suggest that vitamin c can be used as an effective natural supplement in relieving the toxic aspects of pesticide induced oxidative stress. acknowledgements we are grateful to the university grants commission (ugc) of india for funding this study. authors’ contributions sps: conceptualization, methodology, formal analysis, investigation, writing and editing. scv: conceptualization, methodology, supervision, writing and editing. references aebi h. 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itvds) editors for this special issue sociobiology an international journal on social insects open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.989sociobiology 63(2): 855-857 (june, 2016) suitability of substrate for laboratory studies with the subterranean termite reticulitermes grassei clément (isoptera: rhinotermitidae) reticulitermes grassei clément is a subterranean termite species native to the northern, western and southern of the iberian peninsula and the southern-west france (clement et al., 2001). like other subterranean termites, r. grassei colonises both natural and urban environments. in human habitats, it causes damage affecting all type of constructions and represents a major pest problem (gaju et al., 2002) because of its wide-ranging biology and the lack of implementation of effective treatments (alcaide, 2010). in most iberian forests r. grassei is very common but it has never been considered as a pest. nevertheless, gallardo et al. (2010) described lesions in cork oaks (quercus suber l) as consequence of the feeding activity of these insects and indicated that nests and foraging areas appeared to be associated with sites having a high abundance of cork oaks (cárdenas et al., 2012). in a current research addressed to investigate the feeding response of r. grassei to different oak woods, the question of selecting the most suitable substrate for laboratory studies with this species prompted a prior essay to elucidate which of the two most frequently substrates on laboratory rearing abstract two substrates, sand and vermiculite, were tested in rearing laboratory experiments with reticulitermes grassei at different culture periods (2, 4 and 6 weeks). although information is available regarding procedures to keep species of reticulitermes in lab, none of them is referred to the mediterranean termite r. grassei. the suitability of substrate was assessed in terms of survival of termites. no statistically significant differences were obtained in the survival rate in the experiments with sand and vermiculite when short-time duration tests are performed, concluding that it seems to be indifferent using any of these substrates for shorter test than 8 weeks. sociobiology an international journal on social insects p gallardo, am cárdenas, r murillo article history edited by astrid kleinert, usp, brazil received 27 january 2016 initial acceptance 16 march 2016 final acceptance 21 june 2016 publication date 15 july 2016 keywords reticulitermes grassei, rhinotermitidae, suitability of substrate, termites survival rate. corresponding author ana m cárdenas talaverón department of zoology, campus rabanales, e-14071, university of córdoba, spain e-mail: ba1cataa@uco.es species of reticulitermes, sand and vermiculite (haverty, 1979; haverty & howard, 1981; perkin, 2012; en 118, 2013), was more adequate in the particular case of r. grassei. in order to obtain successfully results on bioassays, it is crucial to know the optimal conditions to keep the selected species of termite, not only as far as the temperature and moisture requirements are concerned, but also the suitability of the substrate (lenz, 2005). research done on this subject (haverty, 1979; fougerousse, 1973; lenz, 2005; gaju et al., 2010) suggests that both vermiculite (laminar aluminium-iron-magnesium silicate) and sand, are suitable substrates for experimenting with reticulitermes species, but none of these surveys deals with r. grassei. for this species, gaju et al. (2010) conducted bioassays to measure the effectiveness of different termiticides using vermiculite as substrate; however, other researchers recommend sand as preferable substrate (nobre et al., 2007; en 118, 2013). research aimed to elucidate which of recommended substrates is, really, the most suitable for laboratory studies with r. grassei are necessary in order to optimize the results of rearing experiments on this species of subterranean termite. university of córdoba, campus rabanales, córdoba, spain short note p gallardo, am cárdenas, r murillo – suitability of substrate for bioassays with reticulitermes grassei856 similar studies to test the suitability of substrate for laboratory essays for other species of reticulitermes (r. flavipes kollar and r. virginicus banks) have been conducted by haverty (1979). however, there are no studies analyzing this aspect in the case of r. grassei. this research has been addressed to test the suitability of both substrates in short duration tests with r. grassei. termites used on this study were collected in april 2014 from the surrounding of sta. mª de trassierra (central sierra morena mountains; southern iberian peninsula). the oak fragments of timber in which the insect were found were transported to the laboratory in plastic perforated containers. once there, the termites were manually and carefully extracted. the insects used in these experiments were undifferentiated larvae (workers) of at least the third stage. two experiments were performed to determine the suitability of the substrate (haverty, 1979; ho & kirton, 2007). for each experiment, 15 rearing containers measuring 18x15x7 cm were used. these containers corresponded to an experimental design with three treatments (of 2, 4 and 6 weeks duration) and 5 replications each. as humidity is a decisive factor in the survival of these insects, the proportions of water/substrate used for these experiments were: 1 volume of water to 4 volumes of sand, and 3 ml of water to 1g of vermiculate, proportions recommended in literature for reticulitermes species (nobre et al., 2007; munizaga, 2007; munizaga et al., 2008; arinana et al., 2012; en 118, 2013). eight sapwood blocks (approximately 2x2x1cm in size) of pine (pinus pinea l.) were provided as food. the wood of pine is considered the most adequate for laboratory studies with termites (perkins, 2012; smythe & carter, 1970). a total of 150 termite workers randomly selected were introduced into each container, which were kept in a rearing chamber in permanent darkness at a constant temperature of 26±1 ºc and at 80±5% relative humidity. after 2, 4 and 6 weeks, the sapwood blocks were removed and the surviving termites were censused. the survival rate was estimated as percentage of surviving workers found for each treatment (table 1). to jointly analyse the results and to elucidate possible interaction between the variables substrate and time, a factorial anova was performed using the spss statistical software (spss 20.0, 2011). the results of the statistical analysis are displayed on table 2. from this, it could be observed that the factorial anova did no find significant differences between variables, or in their interaction. that’s to say, the survival rate of the different treatments of the experiment with vermiculite does no vary significantly from those obtained in the experiment using sand as substrate. however, if the values of the survival rate in the two experiments are examined (table 1), it can be observed that in sand this value decreases over time, and that the worst results are obtained after six weeks exposure (but lacking of statistical significance). this trend has not been observed in vermiculite, and could be related with the moisture level, because the sand has a minor water-holding capacity throughout time that vermiculite. our results are consistent with those provided by haverty (1979) for closer species as r. flavipes and r. virginicus, who suggested that it seems to be indifferent the use of vermiculite or sand as substrate for shorter test than 8 weeks. nevertheless, in experiments of longer duration (12 weeks or more) performed with other reticulitermes species, the use of vermiculite resulted to be the most appropriate substrate (haverty, 1979). more research is necessary in order to know the response to the kind of substrate of r. grassei, in long-term tests. survival rate vermiculite sand t1 t2 t3 t1 t2 t3 r ep lic at io ns 1 89.33 56.67 30 84.67 84.67 66 2 49.33 82.67 72.67 89.33 93.33 30.67 3 86.67 68 96 94.67 90 81.33 4 97.33 84.67 80.67 92 79.33 81.33 5 65.33 48 84 83.33 85.33 84.67  77.60 68 72.67 88.80 86.53 68.80 ds 19.74 15.98 25.29 4.79 5.36 22.52 table 1. survival rate (%), survival rate mean () and standard deviation (sd) of five replications of each treatment (t1, t2 and t3: 2, 4 and 6 weeks duration, respectively) for the substrates tested (vermiculite and sand). statistics values variable f p substrate 1.815 0.191 time 1.266 0.300 interaction substrate-time 1.061 0.362 table 2. results of the factorial anova of comparison of survival rates between the variables substrate, time of treatment, and their interaction. p: probability for α = 0.05; f: values of factorial anova. dependent variable: survival rate; r2= 0.212 (adjusted r2= 0.048). acknowledgements this research has been financially supported by acuaes (ministry of agriculture, food and environment, government of spain) and by ingeniería y gestión del sur, s.l. (grupo ig-ipa). references alcaide, m.e. (2010). estudio del control integral de una plaga urbana de termitas en el sur de españa. dissertation, universidad de córdoba, españa. sociobiology 63(2): 855-857 (june, 2016) 857 arinana, l., tsunoda, k., herliyana, e.n. & hadi, y.s. (2012). termite-susceptible species of wood for inclusion as a reference in indonesian standardized laboratory testing. insects, 3: 396-401. doi: 10.3390/insects3020396. cárdenas, a.m., moyano, l., gallardo, p. & hidalgo, j.m. (2012). field activity of reticulitermes grassei (isoptera: rhinotermitidae) in oak forests of the southern iberian peninsula. sociobiology, 59: 493-509. doi: 10.13102/sociobiology.v59i2613. clément, j.l., bagnères, a.g., uva, p., wilfert, l., quintana, a., reinhard, j. & dronnet, s. (2001). biosystematics of reticulitermes termites in europe: morphological, chemical and molecular data. insectes sociaux, 48: 202-215. doi: 10.1007/pl00001768. en 118 (2013). wood preservatives: determination of preventive action against reticulitermes species (european termites) (laboratory method). european committee for standardization, bsi standards publication, cen-cenelec, brussels, belgium. fougerousse, m. (1973). some aspects of laboratory and field testing methods of antitermite wood preservatives. holzforsch, 27(4): 137-142. doi: 10.1515/hfsg.1973.27.4.137. gaju, m., notario, m.j., mora, r., alcaide, e., moreno, t., molero, r. & bach, c. (2002). termite damage to buildings in the province of córdoba, spain. sociobiology, 40: 75-85. gaju, m., moyano, l., alcaide, e., patiño, c., diz, j., nunes, l., bach, c. & molero, r. (2010). laboratory study of fipronil baits against reticulitermes grassei (isoptera: rhinotermitidae). proceeding of 41st annual meeting the international research group on wood protection, biarritz, france. gallardo, p., cárdenas, a.m. & gaju, m. (2010). ocurrence of reticulitermes grassei (isoptera: rhinotermitidae) on cork oaks in the southern iberian peninsula: identification, description and incidence of the damage. sociobiology, 56: 675-687. haverty, m.i. (1979). selection of tunnelling substrates for laboratory studies with three subterranean termite species. sociobiology, 4: 315-320. haverty, m.i. & howard, r.w. (1981). production of soldiers and maintenance of soldier proportions by laboratory experimental groups of reticulitermes flavipes (kolar) and reticulitermes virginicus (banks) (isoptera: rhinotermitidae). insectes sociaux, 28: 32-39. doi: 10.1007/bf02223620. ho, t.h. & kirton, l.g. (2007). effects of different substrates and activated charcoal on the survival of the subterranean termite coptotermes curvignathus in laboratory experiments (isoptera: rhinotermitidae). sociobiology, 50: 479-497. lenz, m. (2005). laboratory bioassays with termites-the importance of termite biology. proceeding the 2ª conference of pacific rim termite research group on wood protection, bangkok, thailand. munizaga, m.j. (2007). estudio de alimentación y de sustancias tóxicas potenciales para reticulitermes flavipes (kollar) (isoptera: rhinotermitidae). dissertation, university of chile. munizaga, m.j., araya, j.e., karsulovic, t. & bozo, a. (2008). ¿afecta la exposición previa a ninfas el consumo de madera de pino radiata de reticulitermes flavipes (kollar) (isoptera: rhinotermitidae)? boletín de sanidad vegetal plagas, 34: 533-536. nobre, t., nunes, l. & bignell, d.e. (2007). tunnel geometry of the subterranean termite reticulitermes grassei (isoptera: rhinotermitidae) in response to sand bulk density and the presence of food. insect science, 14: 511-518. doi: 10.1111/j.17447917.2007.00180.x perkins, g.d. (2012). reticulitermes feeding preference and experimental design. dissertation, university of georgia. smythe, r.v. & carter, f.l. (1970). feeding responses to sound wood by coptotermes formosanus, reticulitermes flavipes, and r. virginicus (isoptera: rhinotermitidae). annals of the entomological society of america, 63: 841-847. doi: 10.1093/ aesa/63.3.841. spss inc. (2011). spss 20.0 for windows use manual (version 20.0). open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v60i4.441-445sociobiology 60(4): 441-445 (2013) population growth characteristics of incipient colonies of the eastern subterranean termite, reticulitermes flavipes (kollar) (isoptera: rhinotermitidae) ma janowiecki, sc jones, jl bryant introduction subterranean termites are the most economically important structural pests in the united states, and approximately $11 billion is spent annually for prevention and treatment measures and repair of termite damage (su, 2002). reticulitermes flavipes (kollar) is the most widespread and economically important native species. better insights into initial colony growth will improve our understanding of colony establishment and the spread of pest species. the termite colony’s life cycle begins with the dispersal of winged alates to colonize new resources. the alates pair, shed their wings, and establish a new nest. the castes are determined during post-embryonic development, and any larva (first or second instars) can become a worker, soldier, or reproductive (lainé & wright, 2003). abstract we investigated growth of inbred monogamous reticulitermes flavipes (kollar) colonies monthly during their first year, thereby assessing colony growth at a much finer temporal scale than previous studies. detailed data were obtained on important growth parameters, including numbers of each caste and/or developmental stage in each colony and their biomass. growth of 180 r. flavipes incipient colonies was documented via destructive sampling of 15 unique colonies each month for 12 months. king and queen biomass greatly decreased from the 1 mo to 3 mo census coinciding with egg production and colony foundation, but their biomass remained relatively constant thereafter. three cohorts of eggs were produced, with the greatest number of eggs (mean = 14.5) being produced during the first month. larvae were present at 1 mo, and workers were first observed at 2 mo. the survival rate of the initial brood was less than 100%, most likely due to cannibalism. a single soldier was first noted in some colonies at 6 mo. offspring biomass was equal to that of the reproductive pair at the 2 mo census, double at 3 mo, and quadruple at 11 mo. one-year-old colonies ranged in size from 20 to 40 individuals, with a mean of 28.9 individuals; mean total colony biomass was 39.8 mg. the growth rate of these r. flavipes colonies was much slower than previously reported, but our observations are consistent with previous models of colony growth. our detailed baseline information on r. flavipes colony growth is expected to allow for more refined hypotheses and growth model development in future studies of this economically important termite. sociobiology an international journal on social insects department of entomology, the ohio state university, columbus, ohio, usa. research article termites article history edited by: og desouza ufv, brazil received 11 february 2013 initial acceptance 17 july 2013 final acceptance 11 september 2013 keywords colony growth, caste differentiation, inbreeding, subterranean termites, reproduction corresponding author susan c. jones, ph.d. department of entomology, rothenbuhler research lab, 2501 carmack rd., columbus, oh 43210-1065 e-mail: jones.1800@osu.edu early colony development has been studied in various rhinotermitids. for example, alate-founded colonies have been studied for the formosan subterranean termite, coptotermes formosanus shiraki (king & spink, 1974, 1975; fei & henderson, 2003; morales-ramos & rojas, 2003; sun, 2007); the western subterranean termite, reticulitermes hesperus banks (light & weesner, 1955); and the eastern subterranean termite, r. flavipes (snyder & popenoe, 1932; thorne et al., 1997; grube & forschler, 2004). in addition, development of new colonies from isolated groups of workers and nymphs has been studied in r. urbis bagneres (ghesini & marini, 2009). r. flavipes is an economically significant pest species that is widely distributed in the eastern united states. incipient colony growth has been studied by various researchers. snyder and popenoe (1932) observed laboratory-reared ma janowiecki, sc jones, jl bryant growth of incipient colonies of reticulitermes flavipes442 colonies and documented the abundance of castes at various times, but they did not conduct a rigorous census. thorne et al. (1997) used a destructive sampling technique to census 2-yr-old colonies, noting the abundance of castes and colony size range. grube and forschler (2004) studied incipient colony growth of monogamous and polygynous colonies during the first 2 yr. they censused a total of 36 monogamous colonies, inbred and outbred, during the first year of colony growth. these colonies were assigned to three groups of 12 colonies, which were censused at 4, 8, and 12 mo, but the researchers provided no colony size averages, only ranges. in spite of this important study, information is needed on colony growth at a much finer temporal scale. more frequent inspection intervals would be useful to better understand r. flavipes colony growth during the first year. the objective of our descriptive study was to investigate growth dynamics of inbred monogamous colonies of r. flavipes during the first year through the parameters of numbers, castes and/or developmental stages, and biomass. this detailed baseline information on colony growth will allow for more refined hypotheses and growth model development. materials and methods experimental setup thousands of r. flavipes swarming alates were collected on may 20, 2011, in orient, oh (39°46'19.99"n, 83°09'22.30"w). the swarm was from a presumably single large colony and it originated in a barn on a rural farm. the termites were individually sexed by examining the posterior abdominal sternites with the aid of a dissecting microscope (stemi sv11 apo, zeiss, germany). in the isoptera, female alates lack styli and have a noticeably elongated seventh abdominal sternite compared to the males (jones & la fage, 1980; zimet & stuart, 1982; roisin & lenz, 1999). a male and female reproductive pair was placed into a plastic “nest” container (3.8 cm ht x 5.4 cm dia, with lid; pioneer plastics, inc., north dixon, ky) that had been lined with a moist filter paper pad and provisioned with a combination of moist softwood and hardwood mulch. approximately 800 colonies were maintained in the lidded containers in an environmental chamber in the dark at 26 ºc with 85-90% relative humidity. water was added to the colonies as needed throughout the study. census methods only established colonies headed by a single queen and king were censused. during a 1-yr period, 15 different colonies were randomly selected each month from the initial pool of ~800 colonies, providing a grand total of 180 censused colonies. the census procedure involved careful removal and inspection of small portions of the mulch until aggregated termites were located (often near the nuptial chamber). at that point, any remaining mulch and filter paper was placed on a large tray. all termites and eggs were individually removed by hand from the mulch using a finetip paint brush. immature termites were examined using a dissecting microscope and categorized as larvae, workers, nymphs, or soldiers (see thorne 1996), then the biomass of each caste and/or developmental stage was obtained using an analytical balance (±0.01 mg, sartorius ag, model cpa225d, goettingen, germany). the primary reproductives were microscopically examined to determine the queen and king, then each was individually weighed. each colony was re-established after censusing, but no colony was re-sampled during the study due to the extensive disturbance caused by the indepth inspection process. results and discussion throughout the first year of colony development, the number of individuals varied dramatically from month to month (fig. 1). egg production was intermittent, with the greatest number of eggs (mean = 14.53; se = 0.95; n = 15 colonies) observed during the first month (fig. 1). there were three cohorts of eggs--the first occurred during the first and second month, the second occurred in the sixth and seventh month, and the third spanned months nine through twelve (fig. 1). larvae were observed at 1 mo, and workers were first observed at 2 mo (fig. 1). the number of larvae in the colony remained between 0 and 3 throughout the first year, with the exception of the initial cohort which consisted of 7.6 larvae at 2 mo (fig. 1). one-year-old colonies ranged in size from 20 to 40 individuals (all stages), with an average of 28.9 individuals. workers constituted the majority of colony members from the second month onward, and 1-yrold colonies had an average of 18 workers (range 13-27). no nymphs were observed during the first year of colony development, which is consistent with grube and forschler’s (2004) observation that nymphs were first found in 24-moold colonies. there was an initial peak in total colony members that decreased dramatically from an average of 22.3 termites and eggs at 2 mo to 13.9 at 5 mo (fig. 1). this sharp decline indicates that not every egg hatched and not all offspring survived. snyder and popenoe (1932) observed cannibalism of eggs, larvae, and workers in incipient colonies, with healthy young being eaten despite adequate food, and this may have been a contributing factor. it is uncertain whether trophic eggs contributed to the decline in numbers observed in our study since there are no published reports of trophic eggs in r. flavipes, and yamamoto and matsuura (2011) indicated that trophic eggs did not occur in a closely-related species, r. speratus (kolbe). however, buchli (1950) indicated that r. sociobiology 60(4): 441-445 (2013) 443 lucifugus rossi sometimes produced unfertilized eggs, which were cannibalized. at the 6 mo census, a single soldier was observed in each of two colonies with total populations of 12 and 13 individuals. a soldier was present in 47% of the colonies at the 12 mo census and comprised 1.6% of the colony (fig. 1). similarly, soldiers were present during the first year in grube and forschler’s (2004) study, but they occurred earlier (4 mo) and comprised 3% of the colony at 1 yr. in snyder and popenoe’s (1932) study, soldiers were first observed in 7-mo-old colonies. the appearance of soldiers later in the first year for r. flavipes is consistent with a closely related rhinotermid, r. hesperus. light and weesner (1955) observed that groups of r. hesperus colonies produced soldiers either in the initial cohort or in the second cohort. the biomass of both the king and queen greatly decreased from the 1 mo to 3 mo census (fig. 2), which corresponds with the first cohort of eggs and care of larvae (fig. 1). this is the first report of this phenomenon in r. flavipes. the dramatic decrease in biomass of both primary reproductives is likely due to their use of fat stores to foster the developing young. the reproductives’ biomass continued to decrease until 4 mo when relatively large numbers of workers (mean = 12.67; se = 0.67; n = 15 colonies) were present in the colonies (fig. 1). the reproductives’ biomass remained relatively constant thereafter through the 1 yr census (fig. 2), which is consistent with grube and forschler (2004). eventually, as the colony grows, brood care tasks and nest construction will be taken over by workers, then egg production may become continuous. grube and forschler (2004) noted that r. flavipes queens experience significant weight gain during the second year of colony development. the total biomass of each colony gradually increased over time, and total biomass of 1-yr-old colonies averaged 39.8 mg (fig. 2). offspring biomass was equal to that of the reproductive pair at the 2 mo census, double at 3 mo, and quadruple at 11 mo (fig. 3). the growth rate of r. flavipes incipient colonies observed in our study was much slower than observed by grube and forschler (2004). regional diversity among termite colonies is expected to be an important factor influencing colony growth rates (reviewed by lenz 2009). we expect that the genetic diversity of our inbred colonies, all headed by reproductives from a single location in ohio, would be somewhat limited. for example, at the 12 mo census, there was some variation among our 15 sampled colonies, which ranged from 13-27 workers, but nothing as extreme as observed by grube and forschler (2004) whose smallest r. flavipes colony had 15 workers and largest colony had 259 workers (colony averages not provided). grube and forschler’s study involved alates collected from numerous sites in georgia, usa (university of ga, athens, ga [33°56’52.82”n, 83°22’38.36”w]; whitehall forest, athens, ga [33°53’05.42”n, 83°21’27.57”w]; westbrook farm, kennesaw, ga [33°59’24.92”n, 84°41’56.50”w]), fig. 1. numbers of each caste and/or developmental stage (mean±se) per colony at monthly intervals during the first year of incipient colony growth in reticulitermes flavipes. total colony size includes the founding pair of primary reproductives. a grand total of 180 colonies was examined, with 15 unique colonies dismantled each month for 12 months. fig. 2. mean biomass of each caste and/or developmental stage in reticulitermes flavipes incipient colonies (n=15 colonies per monthly observation) during the first year of colony growth. fig. 3. ratio of offspring to reproductives’ biomass at monthly intervals in reticulitermes flavipes incipient colonies (n=15 colonies per monthly observation) during the first year of colony growth. ma janowiecki, sc jones, jl bryant growth of incipient colonies of reticulitermes flavipes444 and data were combined for inbred and outbred colonies because no differences in colony growth parameters were evident (b.t. forschler, professor of entomology, university of georgia, usa, personal communication, july 25, 2013). fei and henderson (2003) specifically studied the effect of mate relatedness in a total of 338 sibling and nonsibling incipient colonies of c. formosanus, and they found that outbred colonies had significantly increased fecundity compared to inbred colonies. hence, inbreeding may be an important factor in the slower growth rate of our r. flavipes colonies compared to some other studies. rearing conditions (food type, temperature, relative humidity, container size, etc.) also are expected to influence colony growth rates (reviewed by lenz 2009). for example, grube and forschler (2004) used sawdust as a food source for newly founded colonies compared to our use of a mixture of soft and hardwood mulch, which is more coarse and may be more difficult for termites to consume. they used a slightly lower temperature (24 ºc) than we did, but lower temperatures would be expected to result in slower growth rates; both studies involved comparable relative humidity (85%). furthermore, our colonies were maintained in their original small container (80.7 cm3) whereas grube and forschler’s colonies were moved from 87.9 cm3 containers to much larger containers (1312.5 cm3) between 6 and 18 mo when they had consumed more than 50% of their sawdust allotment. these larger containers were provisioned with a much larger food supply, including solid pine wood and sawdust. these conditions may have contributed to increased colony growth rates. our study is the first to provide extensive monthly numerical data to track growth trends in r. flavipes. however, general colony trends are paralleled in related studies of r. flavipes (snyder & popenoe, 1932, grube & forschler, 2004). our study also reveals similar growth trends as observed in rhinotermitids such as r. hesperus (light & weesner, 1955) and c. formosanus (king & spink, 1974, 1975; fei & henderson, 2003; morales-ramos & rojas, 2003; sun, 2007). in conclusion, the growth rate of our r. flavipes colonies was much slower than observed by grube and forschler (2004), but our results are consistent with previous models of colony growth. several distinct colony growth trends are revealed in our study. biomass of the king and queen greatly decreased from the 1 mo to 3 mo census, coinciding with egg production and colony foundation, but the reproductives’ biomass remained relatively constant thereafter. egg production was intermittent throughout the first year of r. flavipes colony development, with three distinct cohorts of eggs as well as periods without eggs, implying that the king and queen partition their initial investment between reproduction and initial brood care. the survival rate of the initial brood was less than 100%, most likely due to cannibalism. the total biomass of each colony gradually increased over time and 1-yr-old colonies consisted primarily of the worker caste, with an occasional soldier. offspring biomass was equal to that of the reproductive pair at the 2 mo census, double at 3 mo, and quadruple at 11 mo. one-year-old colonies ranged in size from 20 to 40 individuals, with an average of 28.9 individuals; 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(2011). queen pheromone regulates egg production in a termite. biol. lett., 7: 727-729. doi: 10.1098/rsbl.2011.0353 zimet, m. & stuart, a.m. (1982). sexual dimorphism in the immature stages of the termite, reticulitermes flavipes (isoptera: rhinotermitidae). sociobiology, 7: 1-7. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7883sociobiology 69(2): e7883 (june, 2022) ants prey on social wasps (maciel et al., 2016) affecting the evolution of nest architecture (andena et al., 2009), defense behaviors (milani et al., 2021), and selection of nesting sites of these insects (corbara et al., 2009). in addition, they attack and loot larvae and pupae in wasp colonies (o’donnell & jeanne, 1990; bouwma et al., 2007), as reported for eciton burchelli (westwood, 1842) (hymenoptera: formicidae) preying on larvae and pupae of agelaia yepocapa (richards, 1978) (hymenoptera: vespidae), even in colonies with thousands of individuals of this wasp (hunt et al., 2001). the importance of social wasps in natural and agricultural ecosystems (souza & zanuncio, 2012), especially in the biological control (prezoto et al., 2019), justifies studies on predation of these insects by ants. the objective of this abstract ants prey on social wasps affecting the evolution of nest architecture, defense behaviors, and selection of nesting sites of these insects. the importance of social wasps in natural and agricultural ecosystems, especially in biological control, justifies studies on predation of these insects by ants. the objective of this work is to report the colony predation of the social wasp agelaia vicina (de saussure, 1854) and the nest occupation of parachartergus pseudapicalis willinck, 1959 (hymenoptera: vespidae) by the ant camponotus renggeri emery, 1894 (hymenoptera: formicidae) in the atlantic forest biome. the records occurred in 2015 and 2018 in southern minas gerais state, brazil. this is the first record of c. renggeri preying on and occupying social wasp nests. this relationship between social wasps and ants constitutes an adaptation for the survival of these latter insects, and the limited number of records increases the need for research on the relevance of this phenomenon to the ecology of atlantic forest hymenoptera. sociobiology an international journal on social insects marcos m. de souza1, lidiane a. junqueira1, gabriel c. jacques2, gabriel s. teófilo-guedes3, josé c. zanuncio4 article history edited by evandro nascimento silva, uefs, brazil received 15 march 2022 initial acceptance 24 april 2022 final acceptance 13 june 2022 publication date 30 june 2022 keywords ant; polistinae; social wasp, predation. corresponding author gabriel de castro jacques instituto de educação, ciência e tecnologia de minas gerais campus bambuí, varginha farm bambuí-medeiros roadway km 05 minas gerais, brasil. e-mail: gabriel.jacques@ifmg.edu.br work is to report the colony predation of the social wasp agelaia vicina (de saussure, 1854) and the nest occupation of parachartergus pseudapicalis willinck, 1959 (hymenoptera: vespidae) by the ant camponotus renggeri emery, 1894 (hymenoptera: formicidae) in the atlantic forest biome. camponotus renggeri preyed on an a. vicina nest and occupied a p. pseudapicalis nest in 2015 and 2018, respectively, in the atlantic forest in the municipality of inconfidentes (22.3174° s, 46.3255° w), minas gerais state, brazil. information about the environment, year season, the substrate used for nesting, and height of the social wasps’ nests from the ground were recorded. specimens of social wasps and ants were collected and identified by dichotomous keys (richards, 1978; carpenter & marques, 2001) or by 1 instituto federal de educação, ciência e tecnologia do sul de minas, inconfidentes, minas gerais, brazil 2 instituto federal de educação, ciência e tecnologia de minas gerais, campus bambuí, bambuí, minas gerais, brazil 3 universidade de campinas, campinas, são paulo, brazil 4 universidade federal de viçosa, viçosa, minas gerais, brazil camponotus renggeri (formicidae) predated agelaia vicina (vespidae) nest and occupied parachartergus pseudapicalis (vespidae) nest id short note mailto:gabriel.jacques@ifmg.edu.br https://orcid.org/0000-0002-9619-6065 marcos m. de souza et al. – camponotus renggeri predated and occupied social wasps’ nests2 the taxonomists dr. orlando tobias da silveira (emílio goeldi museum, belém, pará) and dr. rodrigo m. feitosa (laboratory of systematics and biology of ants, federal university of paraná). the collected material was deposited in the biological collection of ifsuldeminas, campus inconfidentes, tumble number 04798-2018, 04786-2018, 06531 2018 and 06532-2018. the plants used by wasps for nesting were identified in the field or based on photographs by professor laércio loures from ifsuldeminas campus inconfidentes. camponotus renggeri preyed on individuals of a. vicina in a nest built in the tree species erythrina falcata (fabaceae) at about 1.5 m above the ground in late autumn, the dry season in the research region. individuals of this ant tried to enter through the hollow of the tree, accessing the combs fixed inside the trunk in the colony of a. vicina, and the wasps, agitated, tried to defend the colony, apparently by biting the ants, as reported with polybia occidentalis olivier, 1791 on camponotus atriceps smith, 1858 (bowman et al., 2007) and a. yepocapa on e. burchelli (o’donnell & jeanne, 1990). in addition to this defensive behavior, wasps can also detect and eliminate pheromone foci left by ants, reducing access signaling to their colonies (bouwma et al., 2007). on the other hand, wasps can prey on ants, as demonstrated by the analysis of food stored in colonies of p. occidentalis and polybia paulista ihering 1896 (michelutti et al., 2014). fig 1. camponotus renggeri (formicidae) (a, b and c) occupied a nest parachartergus pseudapicalis (vespidae) (d and e). sociobiology 69(2): e7883 (june, 2022) 3 camponotus renggeri (figure 1a, 1b and 1c) occupied a nest of p. pseudapicalis (figure 1d) nesting in tibouchina granulosa (desr.) cogn. (melastomataceae), 1.9 m above ground in late spring, rainy season, apparently abandoned by this wasp. this occurred one week after the registration of the social wasp colony. no information on whether the ants looted and preyed on the wasps or simply occupied the abandoned nest (figure 1e) and the lack of wasps prevented the ethological record between c. renggeri and p. pseudapicalis. these records are important, as ants can be opportunistic, preying on offspring without necessarily confronting adult wasps (lacey, 1979). the importance of nest height for social wasps is poorly studied (de souza et al., 2012; almeida et al., 2014), but the proportion between nest height and aggressiveness as defense strategies of these insects seems to be inversely correlated (jeanne, 1975). the height of agelaia spp. nests varies because these wasps use natural and artificial cavities, such as tree hollows to nest, commonly without a protective envelope (zucchi et al., 1995). the seasonality of occurrences and the opportunistic nesting and foraging behavior (silvestre et al., 2003; yamamoto & del-claro, 2008; lange et al., 2019) of ants of the genus camponotus suggests that the attack in late autumn on the a. vicina nest is associated to the reduction of resources in the environment due to drought. on the other hand, the occupation of the p. pseudapicalis nest at the beginning of the rainy season may be associated with the search for refuge and, eventually, for resources. camponotus renggeri, widely distributed in south america, feeds mainly on liquid resources of extrafloral nectaries and trophobionts and builds its nests underground and on dead branches and trunks or plant roots (ronque et al., 2018). camponotus atriceps smith, 1858, camponotus rectangularis emery, 1890, and other species of this genus preyed on and occupied nests of the social wasp polybia occidentalis (bouwma et al., 2007). this is the first record of c. renggeri preying on and occupying social wasp nests. this relationship between social wasps and ants constitutes an adaptation for the survival of these latter insects, and the limited number of records increases the need for research on the relevance of this phenomenon to the ecology of atlantic forest hymenoptera. author’s contribution laj: investigation; writing-original draft mms: conceptualization; project administration; resources gcj: resources; writing-review & editing; visualization gstg: investigation; writing-original draft jcz: supervision; writing-review & editing acknowledgments to taxonomists dr. orlando tobias da silveira (emílio goeldi museum), dr. rodrigo m. feitosa (federal university of paraná) and professor laércio loures (ifsuldeminas), for the insects and plants identification. to the brazilian institutions “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capes finance code 001), “fundação de amparo à pesquisa do estado de minas gerais (fapemig)” and “programa cooperativo sobre proteção florestal (protef) do instituto de pesquisas e estudos florestais (ipef)” for financial support. references andena, s.r., carpenter, j.m. & noll, f.b. 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(2008). natural history and foraging behavior of the carpenter ant camponotus sericeiventris guérin, 1838 (formicinae, campotonini) in the brazilian tropical savanna. acta ethologica, 11: 55 -65. doi: 10.1007/s10211-008-0041-6 zucchi, r., sakagami, s.f., noll, f.b., mechi, m.r., mateus, s., baio, m.v & shima, s.n. (1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.920sociobiology 62(4): 615-619 (december, 2015) expanding the distribution of the remarkable ant gnamptogenys vriesi brandão & lattke (formicidae: ectatomminae): first record from brazil the ant genus gnamptogenys roger, 1863 is distributed worldwide, with about 140 valid species (bolton, 2014; feitosa, 2015) found from southern united states to northern argentina and from southeast asia to northern australia (lattke, 2003; 2004; lattke et al., 2007). they have relatively small colonies and can nest on the ground, in fallen rotten logs, or even in trees (lattke, 1994; lattke et al., 2008), and associated with epiphytes (gobin et al., 1998; delabie et al., 2010). most species of gnamptogenys are generalist predators, whereas several species may have varying degrees of specialization (lattke, 1995; 2003; brandão et al., 2009; brandão et al., 2015), preying on a large diversity of arthropods, such as other ants, beetles and millipedes (brown, 1993; brandão et al., 2009; brandão et al., 2015). in gnamptogenys, the minuta-group comprises nine species (g. bufonis (mann, 1926), g. caelata kempf 1967, g. falcifera kempf 1967, g. fieldi lattke 1990, g. minuta abstract gnamptogenys vriesi brandão and lattke is a rarely collected ant, which was originally described based on a worker and a dealated queen collected in morona-santiago province, ecuador. after the original description, only a few specimens were collected in the ecuadorian territory. in this paper, we report the first record of g. vriesi in brazil, which results in a significant extension of its distribution previously considered restricted to ecuador. in addition, we provide images and a distribution map for the species. sociobiology an international journal on social insects re vicente1, lp prado2, rcl santos1 article history edited by rodrigo m. feitosa, ufpr, brazil received 24 september 2015 initial acceptance 30 october 2015 final acceptance 09 december 2015 keywords amazon, ants, biodiversity, faunistic inventory, cryptic, myrmecology. corresponding author ricardo eduardo vicente laboratório de ecologia de comunidades do instituto de biologia universidade federal de mato grosso av. fernando corrêa da costa, nº 2367 boa esperança, 78060-900 cuiabá-mt, brazil e-mail: ricardomyrmex@gmail.com (emery, 1896), g. petiscapa lattke 1990, g. simulans (emery, 1896), g. striolata (borgmeier, 1957) and g. vriesi brandão & lattke 1990) distributed only in the neotropics (lattke, 1992). gnamptogenys vriesi is a rarely collected species of the minuta-group (lattke, 1992). biological and behavioral traits of this species are still unknown, but the foveolae that covers its body surfaces are usually filled with earth, as found in basiceros schulz 1906 and stegomyrmex emery 1912, suggesting that the species presents cryptobiotic habits (hölldobler & wilson, 1986; brandão & lattke, 1990; feitosa et al., 2008). since the only known specimens of gnamptogenys vriesi are a single worker (holotype) and one dealated queen (paratype) collected in ecuador, the species has been considered endemic so far (salazar et al., 2015). in this paper, we provide the first record of g. vriesi from brazil. a single worker of g. vriesi was collected by a pitfall trap in february, 2015, in a forest remnant in the municipality short note 1 universidade federal de mato grosso (ufmt), cuiabá-mt, brazil 2 museu de zoologia da universidade de são paulo (mzsp), são paulo-sp, brazil re vicente, lp prado, rcl santos – first record of gnamptogenys vriesi from brazil616 of juara, state of mato grosso, brazil (fig 1). the municipality has an area of 22,666 km² with 34,87% of its original area deforested (instituto nacional de pesquisas espaciais, 2015). according to the köppen classification, the climate is tropical humid (am) with an annual average temperature above 24°c and precipitation ranging from 2,800 to 3,100 mm year (alvares et al., 2013). there are two well-defined seasons: a rainy season, which occurs between november and april; and a dry season, that occurs between may and october (vicente et al., 2014). the region is characterized by the transition of cerrado-amazon areas (avila & kawashita-ribeiro, 2011), and the predominant vegetation consists of submontane open ombrophilous forest, submontane dense ombrophilous forest and ecotonal areas between seasonal forest and dense ombrophilous forest (instituto brasileiro de geografia e estatística 2004). the single specimen of g. vriesi collected is deposited at the ant collection of the museu de zoologia da universidade de são paulo (mzsp). the specimen was compared to the holotype worker, and its identity was confirmed by specialists in the group (see acknowledgments). both specimens (the newly collected and the holotype) were photographed using a leica application suite v3 system, at the mzsp hymenoptera lab. the images were processed in adobe photoshop 7.0. all measurements (in mm) were obtained under 60x magnification with a leica mz95 stereomicroscope. abbreviations used throughout the text are: ml (mandible length): the straight line length of the mandible at full closure. hl (head length): the length of the head capsule excluding the mandibles, in full face view. wl (weber’s length): the diagonal length of mesosoma in profile. pl (petiole length): measured from anterior to posterior inflections of petiole node, in profile. gl (gaster length): the maximum length of gaster excluding sting in profile. tl (total length): the sum of ml, hl, wl, pl and gl. previous geographic distribution (holotype worker): hl 2.2, ml 1, wl 3.2, pl 1.2, gl 4.7, tl 12.3; fig 2): ecuador, morona-santiago province, los tayos, 03°08´s, 78°14´w, 03.vii.1976, tjitte de vries col. (worker and dealated queen deposited in mzsp). new record (worker): hl 1.8, ml 1, wl 3.0, pl 0.9, gl 4.5, tl 11.2; fig 2): brazil, mato grosso state, municipality of juara, m1l1p7, 11°5’24.73”s 57°43’6.55”o, ii-2015, santos r.c.l. col. (worker deposited in mzsp). the specimen studied in this work presents the same diagnostic characters that define g. vriesi according to brandão & lattke (1990), such as wl>3,00mm; anterior border of clypeus concave; more than 20 ommatidia in the greatest diameter of eye; metanotal groove deep; and declivity of propodeum strongly impressed and concave. the worker collected in brazil is morphologically similar, but the size is comparatively smaller (tl 11,2) than the holotype (tl 12,3); the coloration and sculpturing of the surface integument of the scape of antennae and legs are shiny and slightly darker; the anterior portion of the frontal lobe is weakly striate while in the holotype worker it is smooth; the region below the propodeal spiracles is smooth while in the holotype, this region is superficially foveolate. this new record increases to 34, which is the number of gnamptogenys species known from brazil (feitosa, 2015), and to 12, which is the number of species of the genus for mato grosso state (economo & guénard, 2015). as g. vriesi has only been collected to date in the ecuadorian territory, this work extends the distribution of this species to more than 2,000 km. therefore, the distribution of this species is much broader than was previously known. in fact, few inventories have been conducted between the extremes of the g. vriesi occurrence (e.g. souza et al., 2015; miranda et al., 2012). little is known about the distribution of most ant species due to the scarcity of inventories performed in most biomes (brandão et al., 2008). in the amazon, most of these inventories have been concentrated in the eastern and central regions (vasconcelos et al., 2006; 2010; oliveira et al. 2009; bastos & harada, 2011; baccaro et al., 2012). therefore, efforts should be made in the southern amazon rainforest, as well as in mato grosso state, brazil where the ant fauna is practically unknown (vicente et al., 2011; 2012), and historically the region has faced strong pressure from the agricultural frontier and intensive anthropogenic disturbances.fig 1. distribution map of gnamptogenys vriesi. sociobiology 62(4): 615-619 (december, 2015) 617 acknowledgments we are thankful to dr john lattke and dr carlos roberto f. brandão for his help with the confirmation of the ant species. to vinicius s. ferreira for reviewing a previous version of the manuscript and three anonymous reviewers for pertinent remarks that improved the manuscript. rev and rcls thanks the coordenação de aperfeiçoamento de pessoal de nível superior, brazil (capes) for the doctoral fellowships in ecologia e conservação da biodiversidade postgraduate program – ufmt. is publication 51 in the nebam technical series. references alvares, c.a., stape, j.l., sentelhas, p.c., de-moraesgonçalves, j.l. & sparovek, g. 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(2014). differential recruitment of camponotus femoratus (fabricius) ants in response to ant garden herbivory. neotropical entomology, 43: 519-525. doi: 10.1007/s13744-014-0245-6 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i2.7422sociobiology 69(2): e7422 (june, 2022) introduction foraging behavior is the main form to reach food resource by ant colonies. but environmental factors such as temperature, humidity, rainfall and light (abril et al., 2007; asfiya et al., 2016) can affected its success. variations in temperature or water strees, for example, can increase the energy costs of foraging or use of ant worker time (traniello, 1989). furthemore, temperature has an important role in many ecological processes, as in circadian rhythms, communication of individuals, and foraging schedules (jayatilaka et al., 2011; van oudenhove et al., 2011). abstract the study of foraging dynamics is essential to understanding the way organisms arrange themselves to reduce the effects of competition in the most diverse natural systems. the analysis of temporal foraging patterns is an important tool for understanding how ant communities respond to different environmental conditions. thus, to verify how complexity of the vegetation and abiotic factors can influence ground-dwelling ants communities, we evaluated the foraging temporal patterns in three types of landscapes (grassland, arboreal caatinga, shrub caatinga) in an area of dry seasonal rainforest. these environments were characterized by abundance of plant life forms. the ants were collected by pitfall trap, arranged in six rows each with five traps. the pitfalls were inspected every hour from 7:00 am to 6:00 pm, and temperature and humidity data were taken at the same time. the foraging structure of ant communities presented a nested pattern between the phytophysiognomies, but with variation in the observed metric values. for less complex environments, foraging activity was restricted to preferential times, demonstrating a temporal niche partition. despite more complex environments have a greater richness of species foraging throughout the day, we found greater diversity in environment with intermediate complexity. temperature influences the richness of foraging ants throughout the day, but we found no effect on diversity. our results indicate that, although temperature may influence the temporal dynamics of ground-dwelling ant communities, changes in the structural complexity of the environment affect the foraging activity among species, influencing ant-mediated ecological processes. sociobiology an international journal on social insects josieia t. santos1, emanuelle l. s. brito2, gilberto m. m. santos3 article history edited by kleber del-claro, ufu, brazil received 16 september 2021 initial acceptance 22 september 2021 final acceptance 06 may 2022 publication date 05 july 2022 keywords habitat complexity, semi-arid region, niche partitioning, complex networks, competition. corresponding author josieia teixeira santos programa de pós-graduação em zoologia – ppgzoo universidade estadual de santa cruz rod. jorge amado, km 16 salobrinho cep: 45662-900 ilheus, bahia, brasil. e-mail: josieiabiologa@gmail.com the existence of a gradient of tolerance to different environmental factors allows species to avoid superimposing their period of activity on the same time scale (bernstein, 1979; traniello, 1989; delsinne et al., 2007), creating distinct temporal patterns in foraging schedules (fellers, 1987; cros et al., 1997). due to the tolerance gradient for abiotic factors, species are able to forage very close to their critical limits. this behavior diretly affects the ability to discover and dominate various resource (cerda et al., 1998). thus, from the behavior point of view, more efficient ants (see fowler et al., 1991) can monopolize the resource, pressing other species to forage in schedules where dominant competitors are absent (sanders & gordon, 2000; richards, 2002). 1 programa de pós-graduação em zoologia – ppgzoo, universidade estadual de santa cruz (uesc), ilheus, bahia, brazil 2 programa de pós-graduação em ecologia e evolução, universidade federal de goiás (ufg), goiânia, goiás, brazil 3 departamento de ciências biológicas, universidade estadual de feira de santana (uefs), feira de santana, bahia, brazil research article ants the role of vegetation structure and abiotic factors affecting the temporal dynamics of ant foraging josieia t. santos, gilberto m. m. santos, emanuelle l.s. brito – temporal dynamics of ant foraging2 ants can show multiple responses to the influence of abiotic factors on foraging and according the organization of temporal dynamics in communities. temporal niche partitioning is commonly reported in behavioural studies of a range of species (e.g. pianka, 1973; santos & presley, 2010; carvalho et al., 2013). is an important mechanism allowsing coexistence between individuals with similar niches, by reducing the frequency of encounters between species potentially competing (davidson, 1977; laurance et al., 2002; kronfeld-schor & dayan, 2003; dunn et al., 2007) and promote an increase in local diversity. morphological (retana & cerdá, 2000; cerdá et al., 2002) and physiological aspects (cerda et al., 1997; boyle et al., 2021), as well as diet (houadria et al., 2015) and competitive interactions (bluthgen & fieldler, 2004) are factors that drive temporal nest partitioning in ant communities. in addition, the close relationship with plant community structure has a direct effect on the temporal dynamics and foraging behavior of ants (majer, 1983; andersen, 1991, 1997; andersen et al., 2004; gomes et al., 2010; chen et al., 2014; cross et al., 2016; sousasouto et al., 2016). the structure of the local plant community can act as an environmental filter in the composition, richness and diversity of ant communities, or on the selection of functional traits in these groups (gibb & parr, 2013; nooten et al., 2019). in addition, they affect the occurrence and distribution of organisms (gardner et al., 1995; ribas et al., 2003; chen et al., 2014; stirnemann et al., 2015) influencing the behavior of species (bernstein, 1975; palmer et al., 2003; meurer et al., 2015). vegetation gradients promoting different levels of complexity of habitat and environments with greater plant diversity imply a higher carrying capacity, due to increase the variety of resources, nesting sites, shading, and shelter against predators (hampton, 2004; santos et al., 2007; tadu et al., 2014). a higher richness and diversity of ants is also found in more complex environments (de la mora et al., 2013; martins et al., 2022), as well as a range of functional traits within the communities (silva & brandão, 2014). thus, in response to different levels of vegetation complexity, the organization of ant communities and multiple interactions between species can adjust to distinct temporal dynamics (dejean et al., 2015; costa et al., 2018; neves et al., 2021) generating activity rhythms, foraging patterns and renewing the turnover of the community for each situation (andersen, 1986; cros et al., 1997; gibb & parr, 2010). for these reasons, understanding how this dynamic occurs on small scales allows us to investigate patterns that indicate the main mechanisms that drive the temporal organization of ant communities. thus, in this study, we verified the influence of vegetation structure on local temporal foraging patterns in ground-dwelling ants communities. our hypothesis is that variations in the level of plant complexity of the environment create distinct temporal patterns of foraging. therefore, we expected that: i) in environments with a large vegetation complexity the temporal structure of foraging are more generalist, with greater overlap in forraging times between species throughout the day and with greater connectivity between invididuals within the community. for environment with low vegetation gradients, we expect to find “ temporal windows” between foraging times and fewer active species throughout the day. furthemore, we expected to find greater richness and diversity of species foraging throughout the day in more complex environments. material and methods study area the climate of the study area is semi-arid tropical, and is inserted in the caatinga domain. the vegetation is a mosaic of thorny bushes and seasonally dry forests that covers most of the northeastern region of brazil. the biome holds an area of about 735.000 km2 (leal et al., 2005). fieldwork was conducted from may to july 2016 in retiro farm (14°00’24.1”s/42°52’40.9”w), municipality of guanambi, southwestern bahia, brazil. this area comprises a continuous fragment of different phytophysiognomies of the caatinga biome and agricultural system. we selected three different types of phytophysiognomies: grassland, shrub caatinga and arboreal caatinga. the grassland environment presents predominantly herbaceous vegetation, with little diversity of plants, exposed grounds, bushes and few trees. the shrub caatinga is composed exclusively of herbaceous and shrubby strata, with scattered shrubs and sub-shrubs, whose vegetation presents sparse trees. arboreal caatinga is a mosaic of vegetation that includes true forest, medium-sized trees and patches of forest. experimental design for each type of phytophysiognomy (grassland, shrub caatinga and arboreal caatinga) three plots of 8.000 m2 each were selected. for characterization of the vegetation structure, we followed the class system of raunkiaer (1934). this system uses the position of the growth bud to establish types of life form or functional role of the plant at different stages of development (figure 1). to analyze the rhythm of temporal activity of grounddwelling ants communities, we use pitfall type traps, as they are more efficient for this type of sampling. thirty pitfall traps were installed in each of the three areas. the traps consist of a 300 ml cup containing only a solution of detergent and salt. the traps were arranged in six rows with five pitfall traps each (6x5), and a distance of 20m was left between points, totaling 90 traps per phytophysionomy. in each trap, we installed two plastic cups at ground level. we used the outer cup as a base and the inner cup as a receiver for the capture solution. the traps were activated on the field from 7:00 am to 6:00 pm, and were inspected every hour. sociobiology 69(2): e7422 (june, 2022) 3 at each inspection, the inner cup was removed, the ants collected and the cup was returned to the trap with only the capture solution. specimens caught by the traps were fixed in 70% alcohol. therefore, the sampling effort totalize 33 hours per phytophysiognomy. the samples were transferred to state university of feira de santana (uefs), where they were screened and morphotyped to the lowest possible taxonomic level. posteriorly, the ants were identified by dr jacques hubert delabie, mirmecology laboratory, cepec/ceplac, and voucher specimens of each species were deposited in the johann becker collection at uefs and the collection of mirmecology laboratory (cpdc), cepec/ceplac at ilheus, bahia, brazil. in order to verify the influence of abiotic factors on the rhythm of temporal activity of the ground-dwelling ant communities, climatic data on temperature and humidity were collected every hour between 7:00 am to 6:00 pm, together with the ant fauna per phytophysiognomy. the temperature and humidity data were collected with the support of a digital thermo-hygrometer (jumbo incoterm, model 7663). statistical analysis community framework to characterize the studied phytophysiognomies, we used the abundance of each plant life form per plot as a surrogate for complexity structural. we compare the structural complexity of environments through an analysis of variance (anova), using plant life form abundance values as the dependent variable and phytophysiognomy as the independent variable. afterwards we performed tukey hsd post-hoc test for pair-wise comparisons between phytophysiognomies. fig 1. vegetal life forms in accordance to raunkiaer (1934). from to left to right: (1) phanerophytes, the surviving buds or shoot-apices are borne on negatively geotropic shoots that project into the air; (2,3) chamaephytes, the surviving buds or shootapices are borne on shoots very close to the ground; (4) hemicryptophyte, the surviving buds or shoots-apices are situated in the soil-surface; (5,6) cryptpphytes, the surviving buds or shoot-apices are buried in the ground at a distance from the surface that varies in the different species; hydrophyte, the surviving buds or shoots-apices are rest submerged under water (7,8). we used two analysis of covariance (ancova) to test the hypothesis that the average richness and diversity of ants foraging throughout the day varied among the three phytophysiognomy types. considering the influence of abiotic factors on the behavior of the species, we use the variation of air temperature and air humidity during the day as covariables. thus, our ancova model treated richness and diversity as dependent variables, phytophysiognomy as a categorical fixed effect and temperature and humidity as a random effect (covariates), followed by tukey hsd post-hoc test for pair-wise comparisons between phytophysiognomies. temporal patterns and foraging schedules to analyze the temporal dynamics and foraging patterns in ground-dwelling ant communities, we use a complex network approach. for this, data from ants collected at each hour period in different environments were used to generate incidence matrices x/i e x/j, where x is the ant species, i is the phytophysiognomy and j is the collection hour. the matrices were used to build temporal networks and generate metric values for network properties. we used the metrics of connectance (see jordano, 1987), nestedness (see josieia t. santos, gilberto m. m. santos, emanuelle l.s. brito – temporal dynamics of ant foraging4 almeida neto et al., 2008), specialization (see blüthgen et al., 2006), robustness (see burgos et al., 2007) and overlap, to check the properties of networks temporal. niche overlap was calculated by null models, using the pianka index, with 1000 randomizations and at a significance level of α <0.05. we tested if the values observed in the metrics (except for the niche overlap) differ much to what one would expect by chance, using null models, with 1000 randomization and a 95% confidence interval. we estimated the significance level by t-test (with all its assumptions). for this analysis we used the “bipartite” (dormann et al., 2008) and “ecosimr” packages (gotelli & ellison, 2013) of r software (r development core team, 2015). the frequency of each species in networks was calculated by the percentage of individuals of each species in relation to the total number of individuals in the sample. these calculations were determined by the formula: pi = ni / n * 100, where ni is the number of individuals of species i collected and n is the total number of all species collected (silveiraneto et al., 1976). the constancy of species occurrence was calculated using the bodenheimer (1938) formula: c = (p x 100) / n where p = number of samples where the species occursand n = total number of samples. according to the percentages obtained, the species ants can be separated into three categories: constant species (w), occurring in more than 50% of the collections; accessory species (y), which occur in 25% to 50% of the collections and accidental species (z), occurring in less than 25% of collections. results the environment complexity there were significant differences in the abundance of plant life forms between the three phytophysiognomies analyzed (f(2,12)= 5.014, r 2 = 0.45, p = 0.02; figure 2). the results of the tukey hsd test indicated a difference in the average abundance of plant life forms only between arboreal caatinga and grassland environments (figure 2). according to these results, the environments were classified into three levels of complexity, with grassland considered as the less complex environment, shrub caatinga as an intermediate environment and arboreal caatinga as the more complex environment. fig 2. variation in the abundance of plant life forms between the phytophysiognomies of caatinga biome, bahia state, brazil. grassland (low complexity), shrub caatinga (medium complexity), arboreal caatinga (high complexity). different letters indicate significant difference between the phytophysiognomies (p ≤ 0.05). community framework a total of 7.072 individuals of ants were collected from 27 species, 15 genera, and 6 subfamilies. the subfamily myrmicinae was the most common (52% of the species), followed by formicinae (22%), dolichoderinae (11%), ponerinae (7%), pseudomyrmecinae and ectatomminae (4% each) (table 1, suplementary material). we found a high negative correlation between the abiotic variables temperature and humidity (t = 16.15, df = 97, p < 0.001). thus, only temperature was used in the co-variance analyses. the covariate temperature was significantly related to richness (ancova; f(1,93) = 17.54, p < 0.001, figure 3c) unlike diversity (ancova; f(1,93) = 1.15, p = 0.28, figure 3d) of ants foraging throughout the day. there was also a significant effect of the phytophysiognomy in the average richness (ancova; f(1.93) = 4.27, p < 0.05, figure 3a) and the average diversity (ancova; f(2.93) = 6.22, p < 0.01, figure 3b) of ants foraging throughout the day after controlling for the effect of temperatura. sociobiology 69(2): e7422 (june, 2022) 5 temporal patterns the temporal networks of foraging rhythm in the phytophysiognomies show nested patterns (figure 4), but the properties of the network differed between environments. in general, the networks presented high robustness, in grassland (r = 0.81, p < 0.05), shrub caatinga (r = 0.74, p < 0.05) and arboreal caatinga (r = 0.71, p < 0.05) (table 2, supplementary material). for grassalnd, we found greater specialization (h2’= 0.38, p < 0.05) and niche overlap (pianka = 0.49, p < 0.05). the foraging activity of ground-dwelling ants shows clear distribution limits between foraging times, with preferential times in different ant groups. more than 70% of species were absent from 9:00 to 14:00 (figure 5a). the more constancy species throughout the day were forelius maranhaoensis (72%), forelius brasiliensis (69%) and dorymyrmex brunneus (69%) (table 1, supplementary material; figure 5a). for the shrub caatinga environment, the connectance metric was high (c = 0.53, p < 0.05), but the niche overlap level does not differ from model null (pianka = 0.30, p > 0.05; table 2, supplementary material). the foraging activity of grounddwelling ants showed arbitrary limits in relation to the temporal distribuition the temporal distribution (figure 5b). in shrub caatinga phytophysiognomy, camponotus crassus, although it is less frequent (f = 12%), maintained very constancy throughout the day (75%), instead of solenopsis substituta, which was more frequent (28%), but had a low constancy (33%) (table 1, supplementary material). for the arboreal caatinga environment the temporal structure of foraging was more generalist, presented less niche overlap level (pianka = 0.29, p < 0.05) and low specialization (h2’ = 0.26, p < 0.05; table 2, supplementary material). the foraging activity of ground-dwelling ants exhibited a diffuse pattern, with a larger variation in the foraging activity schedule (figure 5c). the ant c. crassus showed higher constancy (87%), followed by dorymyrmex bicolor (72.7%), which was also the most frequent species (72.3%) (table 2, supplementary material). discussion the results partially corroborate our hypothesis. we found differences in the richness and diversity of ants foraging throughout the day, between distinct phytophysiognomies (figure 3a, b). our results show a negative effect of temperature on the richness, but not on the diversity of the ants which forage throughout of the day (figure 3c, d). furthermore, the metrics considered in the network analysis show a different topology than our predictions (table 2, supplementary material). the influence of abiotic variables, like temperature, over foraging behavior is common for many groups of insects (e.g. resende et al., 2001; santos & presley, 2010). despite the negative effect of temperature in richness ants throughout the day, the fig 3. average richeness (a) and average diversity (b) species of ants foraging throughout the day (7 am to 6 pm) among different phytophysiognomies of caatinga biome, bahia state, brazil. different letters indicate significant difference between the phytophysiognomies (p≤0.05). influence of temperature in average richness (c) and average diversity (d) foraging throughout the day (7 am to 6 pm). josieia t. santos, gilberto m. m. santos, emanuelle l.s. brito – temporal dynamics of ant foraging6 fig 4. temporal networks of foraging activity of ground-dwelling ants in the evaluated environments. black left boxes are the foraging times and and the color of the boxes on the right represent the phytophysiognomies: grassland (red); shrub caatinga (green); tree caatinga (blue). the thickness of the lines between the boxes corresponds to the abundance of individuals. the code of species is: sp1: azteca sp; sp2: blepharidatta conops; sp3: brachymyrmex admotus; sp4: brachymyrmex heeri; sp5: camponotus cameranoi; sp6: camponotus crassus; sp7: camponotus melanoticus; sp8: cephalotes pusillus; sp9: crematogaster victima; sp10: dinoponera quadríceps; sp11: dorymyrmex bicolor; sp12: dorymyrmex brunneus; sp13: ectatomma muticum; sp14: forelius brasiliensis; sp15: forelius maranhaoensis; sp16: kalathomyrmex emeryi; sp17: odontomachus bauri; sp18: pheidole diligens; sp19: pheidole fallax; sp20: pheidole fimbriata; sp21: pheidole meinerti; sp22: pheidole obscurithorax; sp23: pheidole radoszkowskii; sp24: pseudomyrmex urbanus; sp25: solenopsis substituta; sp26: solenopsis globularia; sp27: solenopsis tridens. difference between grassland and arboreal caatinga suggest that the structure of vegetal community is especially important for temporal dynamic of ants communities ground-dwelling. the presence of distinct plant strata, as observed in the arboreal caatinga, favors shading and decreases the incidence and reflection of sunbeams. also contributes to maintain lower soil temperature with a weaker thermic amplitude and creating microclimates that allow species to expand foraging time (resende et al., 2001; vilani et al., 2006). the increase of foraging time was observed in dorymyrmex bicolor (sp. 11) and dorymyrmex brunneus (sp. 12), species absent from 10:00 to 13:00, period with higher temperature, in the phytophysiognomy with less environmental complexity (grassland) (figure 5a). however, in a more complex environment (arboreal caatinga), these ants were constantly present throughout the day, reinforcing the idea that the forested environment offers a more favorable microclimate for ants foraging. furthermore, the greatest species richness found in arboreal caatinga is a response to the environment complexity because the increased abundance of the different plant life forms enables a greater number of realizable niches, thus supporting a richer ant fauna. as expected, in environments with a less structural complexity, we found “temporal windows foraging” throughout the day. in grassland, more than 70% of the ants were not active during periods with high temperatures (from 10:00 to 13:00 hours). this condition can be seen like as niche temporal partitioning, likely influenced by physiological constraints. sociobiology 69(2): e7422 (june, 2022) 7 the occurrence and abundance of f. brasiliensis (sp. 14) and f. maranhaoensis (sp. 15) at time with high temperatures, instead of other species, suggest temporal complementarity. studies show that in networks where complementarity is evident, the species can be highly redundantly, with large overlaps of activity (blüthgen & klein, 2011). the thermal tolerance of species that live in dry environments, such like the caatinga biome, may be the product of physiological specialization for different temperature ranges (e.g. bestelmeyer, 1997; cerda et al., 1998; bestelmeyer & wiens, 2001). species of genus forelius were “key species” in temporal network structuring. they kept cohesion and robustness, suggesting a strong coexistence between species and increasing the stability of the network. because of this, the temporal network pattern in this environment seems less susceptible to random events (like competition or predation) and may be influenced by directional events, such as climate changes (blüthgen & klein, 2011). thus, we infer that in low complex environments, the physiological constraints are the mechanism that explains ant foraging schedules while the temperature can be a factor regulating the temporal structure in these environments although we found more richness of species foraging throughout the day in arboreal caatinga, the temporal network was less stable, with less co-occurrence patterns between species. in this environment, the pattern of the network is more susceptible to stochastic events such as competition or predation (bastolla et al., 2009). in this study, competitive interactions are certainly the factor that explains the topology of the network in arboreal caatinga. camponotus crassus (sp. 6) and dorymyrmex bicolor (sp. 11) were the most constant species (central species), and both of them can have a negative fig 5. matrices of occurrence of ant species at each hour of the day in the studied phytophysiognomy: (a) grassland; (b) shrub caatinga; (c) arboreal caatinga. the gray scale represents the abundance of individuals by species. the code of species: sp1: azteca sp; sp2: blepharidatta conops; sp3: brachymyrmex admotus; sp4: brachymyrmex heeri; sp5: camponotus cameranoi; sp6: camponotus crassus; sp7: camponotus melanoticus; sp8: cephalotes pusillus; sp9: crematogaster victima; sp10: dinoponera quadríceps; sp11: dorymyrmex bicolor; sp12: dorymyrmex brunneus; sp13: ectatomma muticum; sp14: forelius brasiliensis; sp15: forelius maranhaoensis; sp16: kalathomyrmex emeryi; sp17: odontomachus bauri; sp18: pheidole diligens; sp19: pheidole fallax; sp20: pheidole fimbriata; sp21: pheidole meinerti; sp22: pheidole obscurithorax; sp23: pheidole radoszkowskii; sp24: pseudomyrmex urbanus; sp25: solenopsis substituta; sp26: solenopsis globularia; sp27: solenopsis tridens josieia t. santos, gilberto m. m. santos, emanuelle l.s. brito – temporal dynamics of ant foraging8 effect on the presence/abundance of other species (peripheral species). this division (central and peripheral species) shows an interaction relationship with the resources (foraging time) as an indicative of dominance (dáttilo et al., 2014a). the network patterns found in this environment can reflect a difference in competitive ability of species, by discovery, use and monopoly of resources (dáttilo et al., 2014b). thus, competitive interactions and the ability to explore the environment would be the mechanisms that explain the ants foraging schedule in these environments. in the shrub caating environment, the scenario seems in opposite to the other phytophysiognomies. the diffuse foraging pattern show several species active at different times of day. this pattern can be seen as a combination of three distinct factors. first, the temperature has less effect over ground-dwelling ant communities, as opposed to extreme environments (grassland and arboreal caatinga). secondly, the species presented low constancy and frequency throughout the day (table 1; figure 5b), and they seem o reduce the effects of competitive pressure in this phytophysiognomy. third, the species collected in this phytophysiognomy belong to distinct trophic niches (see silvestre et al., 2003). the relationships between these three factors favor an intermediate environment, enabling a diffuse distribution of species and greater diversity for this phytophysiognomy. our network analyses support this idea, and the value of metrics for this environment were interposed, in contrast with other phytophysiognomies. thus, we emphasize that for intermediary environments there is a “reduction of the force” that control the foraging patterns in extreme environments (grassland and arboreal caatinga). to conclude, our study reinforces the importance of the structure of the plant community on the foraging activity and the temporal organization of ground-dwelling ant assemblages. however, further investigations will be necessary to understand how the combined effects of vegetation structure, abiotic factors, and interactions between species may affect the 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(2006). sazonalidade da radiação, temperatura e umidade em uma floresta de transição amazônia/cerrado. revista brasileira de meteorologia, 21: 331-343. _hlk106829491 ole_link1 381 effect of temperature and humidity on survival of coptotermes formosanus and reticulitermes flavipes (isoptera: rhinotermitidae) by b. a. wiltz1 abstract two subterranean termite species were subjected to combinations of six temperatures (10°, 15°, 20°, 25°, 30°, or 35°c) and five relative humidities (rh) (55, 65, 75, 85, or 99%) to determine optimum conditions for survival. when small groups of the formosan subterranean termite coptotermes formosanus shiraki or the eastern subterranean termite reticulitermes flavipes (kollar) were exposed to all 30 combinations of temperature and rh, survival times were significantly influenced by temperature, rh, and their interaction. for both species, survival times were longest at low temperatures and high rh. maximum survival of small groups of c. formosanus and r. flavipes workers and soldiers occurred at the combination of 10°c and 99% rh c (lt 50 = 28.2 d, lt 50 = 18.1 d, respectively). survival of paired c. formosanus dealates was evaluated at combinations of 20°, 25°, or 30°c and 55, 65, 75, 85, or 99% rh. survival was strongly influenced by temperature and humidity. longest survival times until 50% mortality occurred at 99% rh and 20° or 25°c (lt 50 = 2.5 d, lt 50 = 3.0 d, respectively). at all temperatures, mortality occurred too quickly for lt 50 values to be determined when rh was 55% or 65%. introduction temperature and moisture are key factors affecting termite survival, activity, and geographic distribution. effects of soil moisture on feeding and tunneling activity have been extensively studied (su and puche 2003, arab and costa-leonardo 2005, green et al. 2005, mcmanamy et al. 2008). in addition, several studies have linked relative humidity to the survival and 1 usda-ars, srrc, 1100 robert e. lee blvd., new orleans, la 70124 this article presents the results of research only. mention of a commercial or proprietary product does not constitute endorsement or recommendation by the usda. 382 sociobiolog y vol. 59, no. 2, 2012 activity of arid climate species (smith and rust 1993a,b, cabrera and rust 1994). nakayama et al. (2004) determined optimum temperature-humidity combinations for feeding by two japanese subterranean termite species and kulis et al. (2008) investigated the effects of moisture and relative humidity at a single temperature (28°c) on survival and feeding of the asian subterranean termite, coptotermes gestroi. termites are well adapted to regulating moisture within the nest, thus limiting the possibility for direct effects of rh on colony survival. sponsler and appel (1990) found that nest materials from two subterranean termite species had moisture contents ranging from 16.3% – 67.7% and interstitial spaces near saturated rh levels. humidity may indirectly influence subterranean termite success by affecting the ability of soil and wood to retain moisture. changes in moisture content of wood occur much more slowly than changes in air temperature and relative humidity, but wood moisture eventually stabilizes at an equilibrium moisture content dictated by average relative humidity (smulski 1996). outdoors, daily fluctuations in temperature and relative humidity do not have much affect on wood moisture. however, inside homes, the relative humidity of outdoor air is altered by heating and cooling, resulting in seasonal changes in wood moisture content. more directly, humidity and temperature can affect the survival of alates, dealates, aerial populations, and colony fragments transported to new locations, thus having important implications for termite dispersal. in most of its current united states range, the formosan subterranean termite coptotermes formosanus shiraki is sympatric with the eastern subterranean termite reticulitermes flavipes (woodson et al. 2001, messenger 2003) and both are important economic pests. the purpose of this study was to determine the combined effects of temperature and relative humidity on survival of these two species. materials and methods termites termites from two field colonies each of c. formosanus and r. flavipes were used in laboratory bioassays. c. formosanus were collected from colonies located in mcneill, ms and new orleans, la using underground open-bottom bucket traps (su and scheffrahn 1986). r. flavipes were collected from logs 383 wiltz, b.a. — effect of temperature and humidity on termites in picayune, ms. termites were maintained on stacked, moistened spruce (picea spp.) (15.5 x 2.5 x 0.5 cm) in plastic containers (12 x 17 x 6.5 cm) in constant darkness and tested within 30 d of collection. c. formosanus alates and dealates were collected on the evening they swarmed in 6 locations in new orleans and metairie, la. following their flights, termites were collected from surfaces using soft forceps or damp paper towels. temperature and humidity treatments for each termite species, survival and feeding assays were conducted at each combination of six temperatures (10, 15, 20, 25, 30, and 35° c) and five relative humidities (55, 65, 75, 85, and 99%). humidity chambers were created using covered plastic boxes (12 x 17 x 6.5 cm) containing a 2 cm layer of one of five saturated salt solutions: mg(no 3 ) 2 ·6h 2 o (55% rh), nacl + sucrose (65% rh), nacl (75% rh), kcl (85% rh), or k 2 so 4 (99% rh). salts were selected for their stability across a range of temperatures and relatively low toxicity and solutions were prepared according to the methods of winston and bates (1960). a data logger (model 42270, extech instruments, waltham, ma) was attached inside the lid of each box to record temperature and humidity and monitor conditions without opening the humidity chambers. covered petri dishes (55 mm diameter) containing pea pebbles were placed in the bottom of each box to elevate assay dishes from the salt solutions. before starting experiments, humidity chambers were placed in incubators (model 1-36 vl, percival scientific) at the appropriate temperature and the salt solutions were adjusted when necessary to achieve the desired humidity. survival assays for assays conducted on small groups of termites, filter paper circles (whatman #4, 42.5mm) were oven-dried for 24 h, weighed, and placed in 55 mm diameter petri dishes in the humidity chambers. after 2 days, filter papers were re-weighed to determine the moisture gain at each humidity level. twenty termites (18 workers and 2 soldiers) were placed in each petri dish and the dishes were returned to the covered, incubated humidity boxes and maintained in 24 h darkness. mortality was recorded daily until all termites were dead. after all of the termites in a dish were dead, the remaining filter paper was weighed, oven dried and re-weighed to determine moisture content 384 sociobiolog y vol. 59, no. 2, 2012 and consumption. for each termite species, ten experimental units (2 colonies x 5 replicates) were used at each temperature-humidity combination. assays using c. formosanus dealates were conducted at 20, 25, and 30°c and all five rh levels. a filter paper circles (whatman #4, 42.5mm) was placed in each 55 mm diameter petri dishes in the humidity chambers at least 2 days before beginning the experiments. on the mornings following swarms, one male and one female reproductive were placed in a petri dish in one of the humidity chambers. because few termites were collected some evenings, termites were assigned to treatments on a rotating basis, allowing all treatments to have a similar number of replicates (7 or 8 replicates per treatment). mortality was recorded daily until all termites were dead. data analysis data were analyzed separately for each termite species and for c. formosanus dealates. for each temperature-rh combination, lethal times (lt 50 and lt 90 ) were determined using probit analysis for correlated data (throne et al. 1995), executed in mathematica (wolfram research, inc., champaign, il). non-overlapping confidence intervals were used to determine significant differences among mortality times. to evaluate temperature and humidity effects, survival data were arcsine of the square root transformed and analyzed using repeated measures analysis, with temperature and rh as the between subject effects and time as within subject effect. analyses were performed using sas proc mixed (sas institute inc. cary, nc) (littel et al. 1996). results measurement of filter paper consumption was attempted for 20, 25, and 30° r. flavipes treatments. all changes in dry weight were negligible; therefore, filter paper weighs were not recorded for the remaining treatments. of the filter papers that were weighed, final moisture contents (mean ± sd) were 4 ± 1%, 5 ± 1%, 5 ± 2%, 7 ± 2%, and 12 ± 3% in the 55, 65, 75, 85, and 99% rh treatments, respectively. in all treatments, c. formosanus mortality reached 100% within 52 d. at each temperature, survival times increased with relative humidity (figs. 1-6, table 1). survival was significantly affected by the interaction between temperature and rh over time (f = 56.76, p < 0.0001), by temperature over time 385 wiltz, b.a. — effect of temperature and humidity on termites (f = 452.07, p < 0.0001), and by rh over time (f = 572.44, p < 0.0001). in separate repeated measures anovas conducted at each rh, effects of temperature on mortality were always highly significant (all p < 0.0001). likewise, relative humidity effects were significant at all temperatures (p < 0.0001). the importance of humidity to c. formosanus survival was most dramatically observed at 30° and 35°c. at these temperatures, mortality in the 55-85% rh treatments occurred too rapidly to permit the calculation of lt 50 and lt 90 values. table 1. lethal times (days) and lower and upper confidence limits for small groups of c. formosanus exposed to combinations of six temperatures and five relative humidities. °c % rh lt 50 (95% cl) lt 90 (95% cl) χ2 slope 10 55 6.5 (5.8-7.1) 9.4 (8.6-10.4) 30.4 0.44 65 7.7 (7.2-8.3) 11.2(10.5-12.0) 24.1 0.37 75 10.2 (9.1-11.3) 16.2 (14.9-17.9) 37.5 0.21 85 14.3 (11.9-16.8) 23.5 (20.6-27.7) 108.9 0.14 99 28.2 (24.5-32.0) 44.7 (40.3-50.8) 132.0 0.08 15 55 4.8 (3.5-6.2) 7.5 (6.1-10.2) 80.6 0.49 65 6.0 (5.1-6.9) 8.8 (7.8-10.4) 43.1 0.46 75 5.7 (3.9-7.5) 9.9 (8.0-13.2) 98.5 0.31 85 9.6 (4.5-14.6) 20.4 (15.3-31.2) 229.5 0.12 99 32.3 (25.5-39.4) 48.2 (40.8-62.7) 328.2 0.08 20 55 3.0 (2.5-3.4) 4.7 (4.2-5.4) 17.2 0.76 65 4.5 (3.9-5.0) 6.2 (5.6-7.2) 27.4 0.72 75 5.5 (5.1-5.9) 7.6 (7.1-8.2) 17.5 0.62 85 9.4 (8.1-10.8) 13.0 (11.6-15.4) 120.8 0.36 99 17.1 (14.2-20.0) 26.5 (23.3-31.3) 156.7 0.14 25 55 1.4* 2.1* 6.4 1.70 65 2.0* 2.9* 20.0 1.50 75 2.5(1.7-3.4) 3.7 (3.0-5.6) 41.3 1.08 85 3.7 (3.4-4.1) 5.4 (5.0-5.9) 10.6 0.77 99 9.4 (8.5-10.4) 11.9 (10.8-13.6) 70.5 0.53 30 55 65 75 85 99 5.7 (4.3-7.0) 7.3 (6.2-11.0) 44.9 0.79 35 55 65 75 85 99 2.8 (2.1-3.6) 3.7 (3.1-5.5) 18.2 1.44 probit analysis did not produce lt values because of rapid mortality * confidence limits are undefined 386 sociobiolog y vol. 59, no. 2, 2012 fig. 1. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 10°c and varying relative humidities. fig. 2. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 15°c and varying relative humidities. 387 wiltz, b.a. — effect of temperature and humidity on termites table 2. lethal times (days) and lower and upper confidence limits for small groups of r. flavipes exposed to combinations of six temperatures and five relative humidities. °c % rh lt 50 (95% cl) lt 90 (95% cl) χ2 slope 10 55 3.5 (2.1-4.9) 6.5 (5.1-9.0) 79.9 0.44 65 5.2 (4.3-5.9) 8.2 (7.3-9.5) 39.3 0.41 75 7.2 (6.5-7.8) 11.3 (10.4-12.3) 23.5 0.31 85 9.7 (8.1-11.4) 16.2 (14.2-18.9) 65.6 0.20 99 18.1 (16.3-20.0) 28.9 (26.7-31.7) 80.1 0.12 15 55 2.1 (1.7-2.4) 3.4 (3.0-4.0) 8.2 0.93 65 3.1 (1.9-4.1) 5.5 (4.4-7.5) 60.8 0.53 75 3.7 (3.0-4.4) 6.2 (5.4-7.3) 34.9 0.52 85 6.5 (5.1-8.0) 11.1 (9.5-13.6) 83.2 0.28 99 18.8 (17.1-20.6) 28.8 (26.7-31.5) 65.3 0.13 20 55 3.1 (2.8-3.4) 4.2 (3.8-4.8) 16.7 1.18 65 3.7 (3.3-4.0) 5.0 (4.6-5.5) 14.9 0.95 75 5.0 (3.6-6.5) 7.7 (6.3-10.6) 84.7 0.49 85 6.8 (6.2-7.3) 8.3 (7.7-9.3) 59.2 0.81 99 14.4 (12.2-16.8) 20.7 (18.1-25.1) 112.6 0.20 25 55 65 2.2* 2.9* 9.4 1.9 75 3.0 (2.5-3.4) 3.8 (34.-4.7) 9.2 1.45 85 3.8 (3.7-3.9) 5.0 (4.8-5.2) 7.9** 1.08 99 8.7* 16.4* 508.2 0.17 30 55 65 75 85 99 4.1 (3.7-4.5) 5.4 (4.9-6.1) 16.3 0.95 35 55 65 75 85 99 1.8 (1.7-1.9) 3.0 (2.8-3.2) 2.6** 1.07 probit analysis did not produce lt values because of rapid mortality * confidence limits are undefined ** p > 0.05 complete mortality of r. flavipes was recorded after 44 d (10°c, 99% rh). as with c. formosanus, r. flavipes survival increased with rh at each of the temperatures evaluated (figures 1-6, table 2). survival of r. flavipes was significantly affected by the interaction between temperature and rh over time (f = 33.98, p < 0.0001), by temperature over time (f = 280.13, p < 0.0001), and by rh over time (f = 426.78, p < 0.0001). in one-way repeated measures anova conducted at each rh, effects of temperature on mortality were always highly significant (all p < 0.0001) and rh effects were significant when evaluated at each individual temperature (p < 0.0001). 388 sociobiolog y vol. 59, no. 2, 2012 fig. 3. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 20°c and varying relative humidities. fig. 4. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 25°c and varying relative humidities. 389 wiltz, b.a. — effect of temperature and humidity on termites fig. 5. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 30°c and varying relative humidities. fig. 6. survival of small groups (18 workers + 2 soldiers) of c. formosanus and r. flavipes at 35°c and varying relative humidities. 390 sociobiolog y vol. 59, no. 2, 2012 all c. formosanus dealates died within 9d, with high mortality occurring within the first day in most treatments (figure 7). survival after 24h exposure was recorded for all rh levels only at the lowest temperature tested (20°c). at 30°c, 2d survival occurred only at 99% rh. rapid mortality allowed the calculation of lt 50 and lt 90 for only six of the treatments (lethal times, followed by lower and upper confidence limits, if available): 20°c/75% rh (lt 50 = 1.1d (0.0-1.9), lt 90 =2.7d (1.9-5.5)), 20°c/85% rh (lt 50 = 1.5d, lt 90 =4.6d), 20°c/99% rh (lt 50 = 2.5 d (0.0-4.6), lt 90 =6.9d (4.8-12.4)), 25°c/85% rh (lt 50 = 1.8 d (0.8-2.6), lt 90 =3.5d (2.7-5.5)), 25°c/99% rh (lt 50 = 3.0 lt 90 =6.2), 30°c/99% rh (lt 50 = 3.1 d (2.6-3.6), lt 90 =4.2d (3.7-5.4)). fig. 7. survival of c. formosanus alates at a) 20°, b) 25°, and c) 30°c. 391 wiltz, b.a. — effect of temperature and humidity on termites discussion the results presented here concur with those of other studies, finding that, within a certain range of conditions, subterranean termite success increases at low temperature and high rh (smith and rust 1993b, nakayama et al. 2004, kulis et al. 2008), but provide data on the most extensive range of temperature and rh combinations tested for both c. formosanus and r. flavipes. subterranean termites are extremely susceptible to desiccation (nakayama et al. 2004). at lower temperatures, termites have lower metabolic rates (smith and rust 1993b) and lower body water loss (sponsler and appel 1990). reduced susceptibility to desiccation at lower temperatures makes seasonal humidity patterns more important than average humidity. in addition to high humidity increasing the suitability of hotter climates, it is critical for the survival of alates and dealates because they are the stages least protected from fluctuations in environmental conditions. in the united states, the formosan subterranean termite has been reported in 11 states (woodson et al. 2001; scheffrahn and su 2005; messenger et al. 2002; hu and oi 2004; brown et al. 2007; sun et al. 2007). ongoing surveys indicate the fst distribution is spreading. the physiological limitations affecting the spread of c. formosanus are not as well understood as they should be. r. flavipes is found throughout much of the eastern united states, but has also recently been reported in nevada, california, and oregon (austin 2005, mckern et al. 2006). the oregon populations, as well as r. hageni in that state are believed to be the result of anthropogenic introductions (mckern et al. 2006) and their potential western distribution is unknown. winter temperature has long been considered the primary environmental factor affecting termites’ northern distribution (kofoid 1934; abe 1937). for many species, rainfall is another climatic variable closely associated with distribution. however, because subterranean termites are highly susceptible to desiccation, rainfall cannot be used as the sole predictor of available moisture. the amount of moisture available at any given time is a critical factor for survival and reproduction. precipitation and irrigation provide moisture, while soil texture, groundcover, and relative humidity influence retention and availability of moisture in soil and food sources. of these factors, seasonal patterns of humidity and temperature may be the most useful in determining 392 sociobiolog y vol. 59, no. 2, 2012 potential regional distributions of fst. in addition, fst is able to establish aerial colonies with no ground contact. this is only possible in food sources with sufficient moisture. data presented here will be valuable in the development of models for potential distribution of c. formosanus. specifically, rh should be considered as a possible limiting factor when temperatures are high and for the establishment of new colonies from nuptial flights. in addition, survival times for c. formosanus were at least as high as those of the native species r. flavipes. this suggests that rh is probably not a factor that would exclude c. formosanus from areas currently occupied by r. flavipes. references abe, y. 1937. on the distribution of the oriental termite, coptotermes formosanus shiraki in japan. the science reports of the tohoku imperial university, fourth series, biolog y 11: 463-473. 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tunneling activity of subterranean termites (isoptera: rhinotermitidae) in sand with moisture gradients. journal of economic entomolog y 96: 88-93. su, n.-y., and r. h. scheffrahn. 1986. a method to access, trap, and monitor field populations of the formosan subterranean termite (isoptera: rhinotermitidae) in the urban environment. sociobiolog y 12: 299-304. sun j. -z., m. e. lockwood, j. l. etheridge, j. carroll, c. z. hollomon, c. e. h. coker, and p. r. knight. 2007. distribution of formosan subterranean termite (isoptera: rhinotermitidae) in mississippi. journal of economic entomolog y 100: 1400-1408. throne, j. e., d. k. weaver, v. chew & j. e. baker. 1995. probit analysis of correlated data: multiple observations over time at one concentration. journal of economic entomolog y. 88: 1510–1512. 394 sociobiolog y vol. 59, no. 2, 2012 winston, p. w., and d. h. bates. 1960. saturated solutions for the control of humidity in biological research. ecolog y 41: 232-237. woodson, w. d., b. a. wiltz, and a.r.lax. 2001. current distribution of the formosan subterranean termite (isoptera: rhinotermitidae) in the united states. sociobiolog y 37:661-671. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.972sociobiology 63(2): 783-791 (june, 2016) insecticidal effect of volatile compounds from plant materials of murraya exotica against red imported fire ant workers introduction red imported fire ant (solenopsis invicta buren 1972 (myrmicinae), rifa), native from parana river basin of south america, is a voracious consumer of numerous dead animals, such as arthropods, earthworms and vertebrates (nattrass & vanderwoude, 2001). rifas have become a pest in southern united states, australia, new zealand, thailand, taiwan, and the philippines, and they were introduced to southern chinese provinces of guangdong, guangxi, and fujian in 2005 (ascunce et al., 2011; zhang et al., 2007). in several highly infested areas, rifas have caused the decline of native ant species by up to 90% through displacement (porter & savignano, 1990). rifas have caused severe damage to human, animals, agriculture, and environment. furthermore, abstract the effect of volatile compounds from the mashed fresh, fallen, and dried leaves of murraya exotica on the behavior of red imported fire ant (solenopsis invicta, rifa) workers was investigated by fumigation toxicity bioassay. the volatile compounds from different mashed leaves (fresh, fallen, and dried leaves) of m. exotica were collected by solidphase microextraction and identified by gas chromatography–mass spectrometry. β-caryophyllene, α-cedrene, α-copaene, β-cubebene, and germacrene d were identified as major components of the volatile compounds. in exposure time from 1 d to 9 d, the mortality of rifa increased from 5.00% to 100.00% (fresh leaves), 11.67% to 93.33% (fallen leaves), and 15.00% to 83.33% (dried leaves) in minor workers, whereas in major workers, the increases were from 13.33% to 93.33% (fresh leaves), 6.67% to 83.33% (fallen leaves), and 10.00% to 60.00% (dried leaves). the volatile compounds reduced the walking and grasping abilities and aggregation rate of rifa workers. results indicate that mashed leaves of m. exotica have potential for controlling rifa. sociobiology an international journal on social insects rl huang1, zh li1, sy wang1, jt fu1, dm cheng 1, 3, zx zhang 1, 2 article history edited by jacques h. c. delabie, ceplac, brazil received 17 december 2015 initial acceptance 21 april 2016 final acceptance 09 may 2016 publication date 15 july 2016 keywords volatile compounds, murraya exotica, solenopsis invicta, activity. corresponding author zhixiang zhang, key laboratory of natural pesticide and chemical biology ministry of education south china agricultural university 510642, guangzhou, china e-mail: zdsys@scau.edu.cn these ants negatively affect the local biodiversity and cause approximately us $5 billion losses yearly in urban and agricultural areas in the united states (cheng et al., 2008). traditional methods such as insecticides and baits are used for managing s. invicta; however, these methods cause pollution to the environment. therefore, new methods such as natural and environment-friendly insecticides should be found to control rifas (vogt et al., 2002; appel et al., 2004). murraya exotica belongs to the family rutaceae and is an evergreen shrub or occasionally a small tree, which is mainly distributed in the west and southeast of china. its flowers are few, white and fragrant (zhang et al., 2008). previous studies have reported the chemical compositions of essential oils from flowers, leaves, and stems of m. exotica (pino et al., 2006; raina et al., 2006; rout et al., 2007; olawore et al., 1 south china agricultural university, guangzhou, china 2 state key laboratory for conservation and utilization of subtropical agro-bioresources, guangzhou, china 3 zhongkai university of agriculture and engineering, guangzhou, china research article ants r. l. huang et al. – insecticidal effect against red imported fire ant workers784 2005; negi et al., 2005). yu et al. (2001) reported that m. exotica extract on some fungi has significant inhibitory effect. li et al. (2001) found the essential oils of m. exotica have good control effect on stored grain pests. moreover, luo et al. (2005) found that the extracts of m. exotica indicated very strong antifeedant activities against the third instar and fifth instar larvae. however, no research has yet been conducted on the insecticidal activity of the essential oil of m. exotica against rifas. this study is the first to report on the toxicity of the volatile compounds released from the mashed leaves of m. exotica against rifas. moreover, the present study investigates the effects of the volatile compounds from the mashed leaves of m. exotica on the rifa workers. materials and methods plant materials the leaves of m. exotica were collected from the insecticidal botanical garden at the south china agricultural university. three different leaves of m. exotica were used for the test. the fresh leaves were cut off and immediately sent to the laboratory. the fallen leaves were collected from the ground below m. exotica. the dried leaves were gained by putting the fresh leaves in baking box with 40 ℃ for 5 h. insects s. invicta colonies were obtained from a suburb in guangzhou and stored in the laboratory for bioassays in plastic containers at 25 ± 2 °c and 60% to 80% rh. a test tube (25 mm × 200 mm) partially filled with 10% honey water and plugged with cotton was used as a water source, and a petri dish (8.5 cm × 1.5 cm) containing the larvae of tenebrio molitor l. (coleoptera tenebrionidae) was used as a food source. rifas were kept in a dry indoor environment at 25 ± 2 °c until the experiment was over. fumigation toxicity bioassay fresh, fallen, and dried leaves of m. exotica were crushed thoroughly in a blender for 5 min. about 30 g of mashed leaves was placed on the bottom of a 1000 ml breaker. twenty minor workers (body length = 2.6 mm to 3.0 mm, head width = 0.5 mm to 0.6 mm) and 10 major workers (body length = 4.2 mm to 4.6 mm, head width = 1.0 mm to 1.2 mm) were placed on the bottom of a 100 ml breaker. the breaker was coated with fluon emulsion inside vertical wall to prevent the ants from escaping and placed on the bottom of a 1000 ml breaker without covering the mashed leaves. the 1000 ml beaker was covered with plastic film. the ants were placed on the laboratory maintained at 25 ± 2 °c and 65% ± 5% relative humidity. all treatments were replicated thrice. the contrast was the absence of mashed leaves in the 1000 ml beaker. physiological index observation of rifa workers the mortalities of the workers were observed 1, 3, 5, 7, and 9 d after adding the 100 ml beaker to the leaves. behavioral observation on the grasping ability of the workers was determined 1, 3, 5, 7, and 9 d after adding the 100 ml beaker to the leaves. the workers were placed on an a4 paper (made from plant fibre, 210 mm × 297 mm), which was slowly turned over to 180 degrees for 1 min. if the ants would not fall down from the a4 paper, they were regarded as possessing grasping ability. the formula was as follows: grasping rate = number of worker ants possessing grasping ability/number of worker ants per replicate × 100. behavioral observation on the walking ability of the workers was determined 1, 3, 5, 7, and 9 d after testing. the workers were placed on an a4 paper. the ants were regarded as possessing walking ability if they could walk continuously for 10 cm without falling down from the a4 paper. the formula was as follows: walking rate = number of worker ants possessing walking ability/number of worker ants per replicate × 100. behavioral observation on the aggregation of the workers was determined 1, 3, 5, 7, and 9 d after testing. the aggregating level based on the method employed by depickere et al. (2004) and devigne et al. (2011). the workers were regarded as aggregating if over two workers gathered, and the distance between each other was less than 0.5 cm. the formula was as follows: aggregation rate = number of worker ants in aggregate mass/number of worker ants per replicate × 100. extraction of volatiles by solid-phase microextraction fresh, fallen, and dried leaves were mashed with a high-speed organization stamp mill for 5 min, and then 30 g of mashed leaves was placed into a 250 ml glass erlenmeyer flask covered with silver paper. the manual solid-phase microextraction device equipped with a 100 μm polydimethylsiloxane fiber (supelco) was employed in this study. the fiber was activated at constant temperature (250 ℃) for 30 min before use. the activated fiber was then pushed into the erlenmeyer flask contained mashed leaves to absorb the volatiles for 40 min. fiber was then poured into the injection port of the gas chromatography– mass spectrometry (gc–ms) with a temperature of 250 ℃ for 3 min, and the volatiles were analyzed by gc–ms. chemical analysis by gas chromatography–mass spectrometry the sample was detected by an agilent 6890 gas chromatograph equipped with an agilent mass spectrometer detector. a db-5 capillary column (30.00 m × 0.25 mm; film thickness by 0.25 mm) was held at 50 ℃ for 1 min, raised to 200 ℃ at the rate of 3℃/min for 2 min, and raised to 230 ℃ (10 ℃/min) for 2 min. the injection temperature was set at 230 sociobiology 63(2): 783-791 (june, 2016) 785 ℃. the detector was operated at 280 ℃. helium was used as a carrier gas at a flow rate of 1 ml/min. the compounds were identified by retention times, kovats indices, and mass spectra. statistical analysis data were transformed into arcsine square root values for a three-way analysis of variance (anova) to determine the significance of the effects of plant material, exposure time, mortalities, grasping rate, walking rate, and aggregation of the minor and major workers as well as various interactions. furthermore, the differences in the data were assessed using duncan’s multiple range test, with p < 0.05 considered statistically significant. the figures were generated using microsoft office excel 2007. results results of anova the mortalities, grasping rate, walking rate, and aggregation rate of workers may vary significantly according to plant material, exposure time, and worker size (anova, p < 0.05). the results reveal that the three main effects and the partial interactions are significant (table 1). however, the mortalities, grasping rate, walking rate, and aggregation rate of rifas had no difference between with the interaction exposure time × worker size (p=0.2626, 0.2544, 0.2431, 0.3612) and the interaction plant material × exposure time × worker size (p=0.1496, 0.4848, 0.4598, 0.0922). fumigation toxicity bioassay according to fig 1, both major and minor workers exposed to volatiles from mashed leaves of m. exotica show increased mortality over time. after 1 d exposure, the volatile compounds of fresh, fallen, and dried leaves of m. exotica lead to 5%, 11.6%, and 10% mortality in minor workers as well as 13.33%, 6.67%, and 15.00% mortality in major workers. however, the mortality rates are 100.00%, 93.33%, and 83.33% for factors df mortality grasping rate walking rate aggregation rate f values p values f values p values f values p values f values p values a 3 105.002 0.0001 105.558 0.0001 107.198 0.0001 14.0164 0.0001 b 4 104.588 0.0001 109.724 0.0001 113.959 0.0001 47.0154 0.0001 c 1 19.3056 0.0001 16.8368 0.0001 17.2425 0.0001 43.0126 0.0001 a×b 12 9.9821 0.0001 10.3586 0.0001 10.7487 0.0001 4.4143 0.0001 a×c 3 7.3649 0.0002 6.3701 0.0006 6.9493 0.0003 0.3572 0.784 b×c 4 1.3393 0.2626 1.3625 0.2544 1.3953 0.2431 1.1025 0.3612 a×b×c 12 1.479 0.1496 0.9694 0.4848 0.9967 0.4598 1.6587 0.0922 a: plant material, b: exposure time, c: worker size (p=0.05) table 1. anova for the main factors of the fumigation test affecting the behaviors of rifa. fig 1. mortalities of workers (a: major workers, b: minor workers) after treated with different mashed leaves of m. exotica. the minors as well as 93.33%, 83.33%, and 83.33% for the majors at 9 d of treatment. grasping, walking, and aggregation rate compared with the mortality of fumigation toxicity, the grasping, walking, and aggregation rates of rifas caused by volatiles from mashed leaves of m. exotica decrease over time (figs 2, 3, and 4). at exposure times ranging from 1 d to 9 d, the grasping abilities decrease from 86.7% to 6.7% (fresh r. l. huang et al. – insecticidal effect against red imported fire ant workers786 leaves), 93.3% to 16.7% (fallen leaves), and 96.7% to 40.0% (dried leaves) for the major workers and from 95.0% to 0.0% (fresh leaves), 88.3% to 6.7% (fallen leaves), and 85.0% to 16.7% (dried leaves) for the minor workers (fig 2,table 3); the walking abilities are reduced from 86.7% to 6.7% (fresh leaves), 93.3% to 16.7% (fallen leaves), and 90.0% to 40.0% (dried leaves) for the major workers, as well as from 95.0% to fig 2. effects on grasping ability of workers (a:major workers, b: minor workers) after treated with different mashed leaves of m. exotica. leaves workers mortality (%, mean±se) 1d 3d 5d 7d 9d fresh major 13.3±3.3 20.0±5.8 66.7±8.8 76.7±6.7 93.3±3.3 leaves minor 5.0±2.9 11.7±3.3 83.3±9.3 85.0±10.0 100.0±0.0 fallen major 6.7±3.3 10.0±0.0 56.7±12.0 66.7±8.8 83.3±8.8 leaves minor 11.7±4.4 53.3±19.2 73.3±10.9 81.7±9.3 93.3±4.4 dried major 10.0±3.3 16.7±3.3 30.0±10.0 33.3±8.8 60.0±5.8 leaves minor 15.0±5.0 30.0±0.0 51.7±11.7 76.7±8.8 83.3±9.3 ck major 0.0±0.0 3.3±3.3 10.0±0.0 13.3±3.3 16.7±3.3 minor 0.0±0.0 3.3±1.7 5.0±0.0 6.7±2.9 10.0±2.9 table 2. mortality of workers caused by mashed fresh, fallen, and dried leaves of m. exotica in the fumigation bioassay. fig 3. effects on walking ability of workers (a: major workers, b: minor workers) after treated with different mashed leaves of m. exotica. 0.0% (fresh leaves), 88.3% to 6.7% (fallen leaves), and 85.0% to 16.7% (dried leaves) for the minor workers (fig 3, table 4); the aggregation rate is reduced from 50.0% to 0.0% (fresh leaves), 86.7% to 16.7% (fallen leaves), and 50.0% to 40.0% (dried leaves) for the major workers and from 31.7% to 0.0% (fresh leaves), 68.3% to 3.3% (fallen leaves), and 51.7% to 13.3% (dried leaves) for the minor workers (fig 4, table5). sociobiology 63(2): 783-791 (june, 2016) 787 leaves workers grasping rate (%, mean±se) 1d 3d 5d 7d 9d fresh major 86.7±3.3 80.0±5.8 33.3±8.8 23.3±6.7 6.7±3.3 leaves minor 95.0±2.9 85.0±5.8 16.7±9.3 15.0±10.0 0.0±0.0 fallen major 93.3±3.3 90.0±0.0 43.3±12.0 33.3±8.8 16.7±8.8 leaves minor 88.3±4.4 63.3±16.9 26.7±10.9 16.7±8.8 6.7±4.4 dried major 90.0±5.8 80.0±5.8 70.0±10.0 66.7±8.8 40.0±5.8 leaves minor 85.0±5.0 70.0±0.0 48.3±11.7 23.3±8.8 16.7±9.3 ck major 100.0±0.0 96.7±3.3 90.0±0.0 86.7±3.3 83.3±3.3 minor 100.0±0.0 96.7±1.7 95.0±0.0 93.3±1.7 90.0±2.9 leaves workers walking rate (%, mean±se) 1d 3d 5d 7d 9d fresh major 86.7±3.3 80.0±5.8 33.3±8.8 23.3±6.7 6.7±3.3 leaves minor 95.0±2.9 88.3±3.3 16.7±9.3 15.0±10.0 0.0±0.0 fallen major 93.3±3.3 90.0±0.0 43.3±12.0 33.3±8.8 16.7±8.8 leaves minor 88.3±4.4 63.3±16.9 26.7±10.9 16.7±8.8 6.7±4.4 dried major 90.0±5.8 80.0±3.3 70.0±10.0 66.7±8.8 40.0±5.8 leaves minor 85.0±5.0 70.0±0.0 48.3±11.7 23.3±8.8 16.7±9.3 ck major 100.0±0.0 96.7±3.3 90.0±0.0 86.7±3.3 83.3±3.3 minor 100.0±0.0 96.7±1.7 95.0±0.0 93.3±1.7 90.0±2.9 table 3. effects on grasping rate of workers after treated with mashed fresh, fallen, and dried leaves of m. exotica. table 4. effects on walking rate of workers after treated with mashed fresh, fallen, and dried leaves of m. exotica. fig. 4 effects on aggregation of workers (a: major workers, b: minor workers) after treated with different mashed leaves of m. exotica. chemical compositions of mashed leaves of murraya exotica figure 5 shows the gc–ms total ion chromatograms of extracts of fresh, fallen, and dried leaves of m. exotica. the result demonstrates that the volatile compounds from the mashed fresh, fallen, and dried of m. exotica contain 20, 28, and 21 major constituents, respectively (tables 6, 7, and 8). the major components of fresh leaves comprising 89.82% of the total volatile compound are identified as β-caryophyllene (40.20%), (+)-b-himachalene (17.76%), linalool (8.81%), 1,3-cyclohexadiene, 5-(1,5-dimethyl-4hexenyl)-2-methyl, [s-(r*,s*)]-(7.00%), germacrene d (6.28%), (e)-b-farnesene (3.67%), a-copaene (3.09%), and humulene (3.01%). for fallen leaves, the 67.31% major components of the total volatile include β-caryophyllene (25.30%), α-cedrene (14.01%), curcumene (10.56%), trans-α-bergamotene (9.06%), germacrene d (6.90%), (1r)-(+)-α-pinene (5.00%), and α-copaene (3.38%). the major components of dried leaves comprising 77.14% of the total volatile compound are α-cedrene (25.05%), β-caryophyllene (23.43%), germacrene d (9.13%), b-cubebene (8.79%), benzene, 1-(1,5-dimethylhexyl)4-methyl-(7.56%), trans-α-bergamotene (6.38%), and α-copaene (3.18%). r. l. huang et al. – insecticidal effect against red imported fire ant workers788 fig. 5 gc-ms total ion chromatograms of mashed fresh(x), fallen(f), and dried(d) leaves of m. exotica. number composition relative retention time (min) percentage (%) 1 artemisia triene 20.343 0.76 2 4-ethenyl-4-methyl-1-(propan-2-yl)3-(prop-1-en-2-yl) cyclohe xene 20.454 0.57 3 α-cubebene 20.842 0.36 4 α-copaene 21.810 3.09 5 (-)-b-bourbonene 22.050 0.18 6 linalool 22.239 8.81 7 (z,e)-α-farnesene 22.742 0.41 8 bicyclo[3.1.1] hept-2-ene,2,6-dimetyl-6-(4-methyl3-pentenyl)23.125 0.66 9 β-caryophyllene 23.381 40.20 10 (e)b-farnesene 23.665 3.67 11 (-)-b-santalene 24.086 0.27 12 humulene 24.370 3.01 13 alloaromadendrene 24.477 0.60 14 b-copaene 24.951 0.09 15 germacrene d 25.170 6.28 16 (+)-b-himachalene 25.640 17.76 17 1,3-cyclohexadiene,5-(1,5-dimethyl-4-hexenyl)-2-methyl-, [s-(r*,s*)]25.689 7.00 18 b-bisabolene 25.978 0.88 19 d-cadinene 26.270 0.72 20 cyclohexene,3-(1,5-dimethyl-4-hexenyl)-6-methylene-, [s-(r*,s*)]26.464 1.73 leaves workers aggregation rate (%, mean±se) 1d 3d 5d 7d 9d fresh major 50.0±5.8 43.3±8.8 26.7±14.5 20.0±10.0 0.0±0.0 leaves minor 31.7±6.0 25.0±5.8 10.0±10.0 5.0±5.0 0.0±0.0 fallen major 86.7±8.8 66.7±12.0 30.0±5.8 23.3±14.5 16.7±8.8 leaves minor 68.3±8.3 25. 0±7.6 8.3±4.4 15.0±8.7 3.3±3.3 dried major 50.0±10.0 53.3±3.3 43.3±6.7 36.7±3.3 40.0±10.0 leaves minor 51.7±4.4 35.0±5.8 21.7±10.9 13.3±1.7 13.3±8.8 ck major 46. 7±3.3 43.3±8.8 53.3±3.3 50.0±5.8 43.3±8.8 minor 50.0±2.9 45.0±2.9 40.0±2.9 26.7±1.7 16.7±1.7 table 5. effects on aggregation rate of workers after treated with mashed fresh, fallen, and dried leaves of m. exotica. table 6. chemical compositions from mashed fresh leaves of m. exotica. sociobiology 63(2): 783-791 (june, 2016) 789 discussion our results have shown that the volatile compounds released from the mashed fresh, fallen, and dried leaves of m. exotica evidently reduce the grasping and walking abilities and aggregation rate of the rifa workers. furthermore, volatile compounds of the leaves of m. exotica have been showed to be insecticidal. plant insecticides are relatively safe, degradable, and readily available in many regions of the world; hence, these insecticides could replace some traditional chemicals. li et al. (2014) reported that volatile compounds of tephrosia vogelii exhibited high toxicity against the rifa workers. the essential oils of m. exotica possessed fumigant toxicity against sitophilius zeamais, and tribolium castaneum adults with lc50 values of 8.29 and 6.84 mg/l, respectively. according govindarajan et al. (2012), the essential oils from mentha spicata (linn.) exhibited larvicidal activity against three mosquito species, aedes aegypti, anopheles stephensi, and culex quinquefasciatus. the natural product of the leaves of boenninghausenia albiflora was active against plecoptera reflexa, clostera cupreata, and crypsiptya coclesalis at different concentrations varying from 1.0% to 5% w/v (sharma et al. 2006). the acetone extract of m. exotica leaves number composition relative retention time (min) percentage (%) 1 (1r)-(+)α-pinene 6.880 5.00 2 β-pinene 8.216 0.72 3 α-phellandrene 9.135 0.36 4 d-limonene 9.898 0.49 5 eucalyptol 10.005 2.05 6 linalool 12.350 0.61 7 artemisia triene 20.330 0.89 8 4-ethenyl-4-methyl-1-(propan-2-yl)3-(prop-1-en-2-yl) cyclohe xene 20.442 0.48 9 α-cubebene 20.829 0.89 10 α-copaene 21.781 3.38 11 (-)-β-bourbonene 22.033 0.52 12 germacrene d 22.185 6.90 13 bicyclo[3.1.1] hept-2-ene,2,6-dimethyl-6-(4-methyl3-pentenyl)22.668 0.53 14 (z,e)α-farnesene 23.014 1.69 15 β-caryophyllene 23.241 25.30 16 trans-α-bergamotene 23.636 9.06 17 alloaromadendrene 23.768 0.42 18 (-)-β-santalene 24.057 0.76 19 (e)-β-farnesene 24.255 2.61 20 humulene 24.321 2.11 21 aromadendrene 24.448 1.64 22 curcumene 25.149 10.56 23 α-cedrene 25.574 14.01 24 l-β-bisabolene 25.949 1.83 25 d-cadinene 26.246 0.63 26 cyclohexene,3-(1,5-dimethyl-4-hex enyl)-6-methylene-, [s-(r*,s*)]26.427 2.02 27 espatulenol 27.981 0.50 28 caryophyllene oxide 28.133 1.50 number composition relative retention time (min) percentage (%) 1 camphene 20.339 1.11 2 4-ethenyl-4-methyl-1-(propan-2-yl)3-(prop-1-en-2-yl) cyclohe xene 20.458 1.59 3 α-cubebene 20.838 0.47 4 α-copaene 21.794 3.18 5 germacrene d 22.210 9.13 6 (z,e)-α-farnesene 22.684 0.50 7 bicyclo[3.1.1] hept-2-ene,2,6-dimetyl-6-(4-methyl3-pentenyl)23.039 1.22 8 β-caryophyllene 23.278 23.43 9 trans-α-bergamotene 23.649 6.38 10 cis-β-farnesene 23.871 0.38 11 epi-β-santalene 24.073 0.51 12 1,3-cyclohexadiene,5-(1,5-dimethyl-4-hexenyl)-2-methyl-, [s-(r*,s*)]24.271 2.59 13 humulene 24.337 1.97 14 alloaromadendrene 24.461 1.12 15 β-cubebene 25.145 8.79 16 α-cedrene 25.619 25.05 17 benzene, 1-(1,5-dimethylhexyl)-4-m ethyl25.648 7.56 18 β-bisabolene 25.969 1.39 19 d-cadinene 26.258 0.73 20 cyclohexene,3-(1,5-dimethyl-4-hexenyl)-6-methylene-, [s-(r*,s*)]26.452 2.26 21 α-guaiene 27.985 0.25 table 8. chemical compositions from mashed dried leaves of m. exotica. table 7. chemical compositions from mashed fallen leaves of m. exotica. r. l. huang et al. – insecticidal effect against red imported fire ant workers790 showed an antifeedant activity against the early third-stage larvae of spodoptera litura (wang et al. 2009). the results of the fumigation toxicity bioassay have shown that the fresh leaves of m. exotica is more active against rifas than fallen and dried leaves. the volatile compounds of mashed leaves of m. exotica are β-caryophyllene, α-cedrene, α-copaene, β-cubebene, and germacrene d. this finding is similar to that reported by jiang et al. (2009). according to the results of gc–ms, β-caryophyllene is the major component of the leaves of m. exotica. the content of β-caryophyllene is highest in fresh leaves (40.20%) among in the fallen (25.30%) and in the dried leaves (23.43%). these observations show that the significant activity of m. exotica leaf may be due to the presence of major chemical constituents such as β-caryophyllene. several studies reported that β-caryophyllene in other plants possessed insecticidal activity (zhu & tian, 2011; krishnamoorthy et al., 2015; venturi et al., 2015; salleh et al., 2015). m. exotica as a popular hedge plant well adapted for topiary work is widely distributed in southern china and several tropical and subtropical regions of asia. in these m. exotica growing areas, the plant need to trim every year and there are a lot of fresh leaves cut from it. therefore the fresh leaves cut from m. exotica can be crushed thoroughly and used to control rifas. the volatile compounds from the mashed fresh of m. exotica possess high insecticide activity against the rifa workers. these facts indicate that the leaves of m. exotica have potential for controlling these pest ants. fresh leaves of m. exotica could be collected and used as raw materials to control rifa. acknowledgment this study were funded by the talent development program on joint training of postgraduates demonstration base in guangdong province, china (2013jdxm11), science and technology planning project of guangdong province (2014a020208114) and science and technology planning and scientific research project of guangzhou city (201510010299). references appel, a. g., gehret, m. j. & tanley, m. j. 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(2007). an overview of the red imported fire ant (hymenoptera: formicidae) in mainland china. florida entomologist, 90: 723-731. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i3.805sociobiology 62(3): 401-411 (september, 2015) cryptic lineages in the cardiocondyla sl. kagutsuchi terayama (hymenoptera: formicidae) discovered by phylogenetic and morphological approaches introduction although most classic taxonomic techniques based on external morphology require a great deal of skill and are laborious, molecular markers based on dna sequences have become a convenient and powerful genetic tool (bickford et al., 2007) to unveil novel interspecific relationships, intraspecific variations, gene flow and adaptive mutations (besansky et al., 2003; molbo et al., 2003; hebert et al., 2004; murray et al., 2008; cheng et al., 2011). among such genetic concerns, detection of cryptic species can help us togain a deeper understanding of species interactions. in the field of myrmecology, this convenient tool has been used to detect several cryptic species among species complexes whose species status had been determined based mainly on external morphology (schlick-steiner et al., 2006; ross et al., 2010; yashiro et al., 2010; bernasconi et al., 2011). the ant genus cardiocondyla (formicidae: myrmicinae), with approximately 70 described species (bolton, 2014), abstract the taxonomy of ant species in the genus cardiocondyla is very confused due to the extreme difficulty in separating many species based on morphology alone. in japan, one group of the species complex cardiocondyla sl. kagutsuchi has both winged and wingless worker-like (ergatoid) males (dimorphic) whereas others have only ergatoid males (monomorphic). the presence of both groups prompted us to hypothesize that c. sl. kagutsuchi presumably includes several independent species with differences in their male wing morphologies and modes of reproduction. however, whether any species boundary actually exists between the male groups has remained unsolved over the 10+ years since the previous revision of this genus. in this study, using discriminant analysis, we compared the worker caste morphology of this species complex among lineages detected by phylogenetic analyses. in addition, we examined the number of sexuals present in field colonies. our results revealed the existence of at least three morphological and phylogenetic groups within this species complex. sociobiology an international journal on social insects i okita1, m terayama2, k tsuchida1 article history edited by gilberto m. m. santos, uefs, brazil received 07 may 2015 initial acceptance 22 july 2015 final acceptance 04 august 2015 keywords cardiocondyla, mitochondrial dna, morphology, taxonomy. corresponding author ichiro okita the united graduate school of agricultural science gifu university, 1-1 yanagido, gifu, 501-1193, japan e-mail: okkie0626@yahoo.co.jp displays male wing polymorphism (bolton, 1982; kugler, 1983; seifert, 2003; heinze et al., 2005; oettler et al., 2010). some species of this genus have both winged and wingless worker-like (ergatoid) males (dimorphic), while others have only ergatoid males (monomorphic). winged males disperse from their natal nests using functional wings to seek mating partners when access to a virgin queen is unavailable within the nest. in contrast, ergatoid males stay in their natal nests and mate with virgin queens for the entirety of their lives (kinomura & yamauchi, 1987; yamauchi & kinomura, 1993; heinze et al., 1998). the ergatoid males have well-developed mandibles and lifelong spermatogenesis. they engage in lethal fighting against other ergatoid rivals and monopolize virgin queens in their nests. in contrast, winged males are docile, have limited sperm supply, chemically mimic female sexuals and mate with virgin queens without being attacked by their wingless rivals (kinomura & yamauchi, 1987; yamauchi & kawase, 1992; yamauchi & kinomura, 1993; heinze et al., 1998; cremer et al., 2002). colonies of the male dimorphic research article ants 1 gifu university, gifu, japan 2 university of tokyo, tokyo, japan i okita, m terayama, k tsuchida – phylogenetic and morphological groups of cardiocondyla kagutsuchi 402 species are polygynous and have only a single ergatoid male as the result of lethal fighting (kinomura & yamauchi, 1987; yamauchi & kinomura, 1993; heinze et al., 1998; boomsma et al., 2005). in colonies of male monomorphic cardiocondyla species, this single ergatoid male system seems to be the rule also for some species (oettler et al. 2010), whereas not for others: a polygynous species, cardiocondyla mauritanica, sometimes contains more than one ergatoid male, which kill only callow rivals and do not engage in lethal fighting against full-grown rivals. some monogynous species, e.g., cardiocondyla batesii, cardiocondyla elegans and cardiocondyla ulianini, usually contain more than one ergatoid male which are mutually tolerant (boomsma et al., 2005, schrempf et al., 2005, lenoir et al., 2007). cardiocondyla kagutsuchi sensu seifert (2003), hereafter simply referred to as cardiocondyla sl. kagutsuchi, is native to east and southeast asia, and has both monomorphic and dimorphic male groups (seifert, 2003; heinze et al., 2005; yamauchi et al., 2005; okita et al., 2013). the presence of both groups prompted us to hypothesize that c. sl. kagutsuchi presumably includes several independent species with differences in their male wing morphologies and modes of reproduction (yamauchi et al., 2005; seifert, 2008; yamane, 2010). however, whether any species boundary actually exists between the male groups has remained unsolved over the 10+ years since the previous revision of this genus (seifert, 2003). the male dimorphic group of c. sl. kagutsuchi is widely distributed from east asia to south-east asia, including japan, whereas the male monomorphic group of the species occurs in japan and polynesian islands including hawaii (yamauchi et al., 2005; okita et al., 2013; frohschammer and heinze, 2009). the male monomorphic group in japan has more than one ergatoid male, whereas the male dimorphic group in japan has only a single ergatoid male (yamauchi & kinomura, 1993). the two groups in japan were originally thought to be two distinct species because of the differences in male morphology (terayama, 1999), but were later considered to be a single species because of the high morphological similarity between the worker castes of the two groups (seifert, 2003). however, our previous phylogenetic analyses showed that the two groups can be classified into distinct lineages (okita et al., 2013). furthermore, the monomorphic group consisted of at least two lineages, with one of the monomorphic lineages at a considerable genetic distance from the other, i.e., the substitution rate of mtdna coi/ii was 3.7% (33bases/890, okita et al., 2013). these phylogenetic analyses strongly suggest that worker caste morphology within the species complex c. sl. kagutsuchi should be compared among the lineages detected by phylogenetic analyses as well as between the male groups. in this study, using additional samples of c. sl. kagutsuchi collected in japan, we re-analysed the phylogenetic relationships and examined morphological differences in the worker caste among the lineages detected by phylogenetic analyses. in addition, we examined the number of sexuals (males and females) present in field colonies especially to reveal whether each male monomorphic lineage of c. sl. kagutsuchi in japan has multiple males and/or females or not in a single colony. materials and methods sample collection we collected colonies of c. sl. kagutsuchi in honshu, shikoku, kyushu and the ryukyus from early summer to late autumn (may – november). as terayama (1999) reported that this ant inhabits open areas, we searched for colonies on the seashore, parks and vegetable fields. when colonies were found in the soil, we collected the whole colony by carefully digging up the soil using a scoop. when whole colonies were difficult to access (e.g., several colonies were found in the gaps between tiles and stones), we collected at least 10 workers from each colony at the colony entrances. in total, we studied specimens from 79 c. sl. kagutsuchi colonies, including one colony previously analysed by heinze et al. (2005) and 20 from our previous study (okita et al., 2013). among the 79 colonies, 38 from 37 sites were collected from honshu, shikoku and kyushu (temperate zone), 16 from 15 sites were from okinawa island (subtropical zone) and 25 from 18 sites were from ishigaki and iriomote islands (subtropical zone). ants were preserved in 99.5% ethanol or brought live to our laboratory after field collection. we counted the numbers of sexuals (winged males, ergatoid males, winged females and wingless females) present in field colonies if applicable. for morphological analysis, we also examined paratype workers (n = 4) of c. sl. kagutsuchi, and nontype material of cardiocondyla strigifrons viehmeyer, 1922 (one colony). note that the colony of c. strigifrons was collected by dr. yamauchi in indonesia, and it was identified as c. strigifrons in seifert (2003) and treated as c. strigifrons in previous phylogenetic analyses (heinze et al., 2005; okita et al., 2013). phylogenetic analysis an 829-bp fragment of the coi/ii regions deleting the trna leucine region between coi and coii was used for phylogenetic analysis. dna was extracted from individual workers using landry’s method (cheung et al., 1993). the dried dna pellet was suspended with autoclaved distilled water (dw2) and preserved at –20°c until further analysis. we amplified coi/ii using the primers c1-j-2195 and c2n-3661, respectively (simon et al., 1994). polymerase chain reaction (pcr) was performed in mixtures containing 2 μl of template dna suspended with dw2, 0.55 μm of each primer, 0.65 u of ex taq hot start version (takara, otsu, shiga, japan), 10× pcr buffer (100 mm tris–hcl, ph 8.3, 500 mm kcl, 15 mm mgcl2) and 0.25 mm of each deoxynucleoside triphosphate in a thermal cycler (dice ver. iii tp600; sociobiology 62(3): 401-411 (september, 2015) 403 takara). pcr consisted of an initial denaturation at 95°c for 4 minutes followed by 34 cycles of denaturation at 95°c for 1 minute, annealing at 59°c for 1 minute and extension at 72°c for 2 minutes. amplified dna was purified using a pcr purification kit (qiagen, hilden, germany). all products were sequenced using a bigdye™ terminator v 3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) and the primers c1-j-2195 (simon et al., 1994) and 3f (okita et al., 2013) designed as an internal primer between c1-j2195 and c2-n-3661 with the abi prism 3100 (applied biosystems). sequencing reactions consisted of one cycle of 1 minute at 96°c, 25 cycles of 10 s at 96°c, 5 s at 50°c and 4 minutes at 60°c, then stopped by cooling to 4°c. we obtained sequences of all 79 colonies of c. sl. kagutsuchi from japan, among which the sequences of 21 colonies had already been obtained in previous studies and consisted of only four haplotypes (heinze et al., 2005; okita et al., 2013; accession numbers: dq023082, ab723724, ab723725, ab723726, ab723727; dq023082 was the same haplotype as ab723724). for phylogenetic analysis, we obtained an additional 25 sequences (heinze et al., 2005) from genbank (accession numbers: ay754686, dq023061, dq023064, dq023068, dq023071, dq023080, dq023081, dq023083, dq023084, dq023085, dq023086, dq023087, dq023088, dq023089, dq023091, dq023094, dq023095, dq023096, dq023102, dq023105, dq023107, dq023108, dq023109, dq023113, dq023118). eight of the 25 sequences were those of c. sl. kagutsuchi detected in hawaii, indonesia and malaysia, and 15 sequences were those of other cardiocondyla species, including cardiocondyla atalanta, c. batesii, c. bulgarica, c. elegans, c. koshewnikovi, c. mauritanica, c. minutior, c. nigra, c. obscurior, c. parvinoda, c. stambuloffi, c. strigifrons, c. tjibodana, c. ulianini and c. wroughtonii. the other two sequences of monomorium pharaonis and harpagoxenus sublaevis were used as outgroups according to previous phylogenetic studies (heinze, 2005; moreau, 2006). phylogenetic relationships were inferred from the sequences aligned using clustalw (thompson et al., 1994) with distance, maximum likelihood and bayesian methods. a neighbour-joining tree with kimura 2-parameter distance method (kimura, 1980) and a maximum likelihood tree applying a general time-reversible model with an invariable and a γ-distributed substitution rate (gtr+i+g) model selected by bayesian information criterion (bic) scores were constructed using mega5.2 (tamura et al., 2011), with bootstrap values estimated from 5000 replicates. bayesian inference was performed using mrbayes ver. 3.2.2 (ronquist et al., 2012) with the gtr+i+g model selected by akaike information criterion (aic) scores in mrmodeltest 2.3 (nylander, 2004). two runs with four chains of markov chain monte carlo iterations were performed for 1,000,000 generations. trees were kept every 100 generations, and the latest 75% of trees were used to calculate the 50% majorityrule trees and to determine the posterior probabilities. morphological analysis we randomly selected five workers from each of the 79 colonies of c. sl. kagutsuchi originating in japan and compared the worker morphologies of each lineage based on our phylogenetic analyses. we measured six body parts (fig 1) of the workers, including the paratypes (n = 4) of c. sl. kagutsuchi, using a stereomicroscope (szx7; olympus, tokyo, japan) connected to a digital camera (dp21; olympus) and digital imaging software (cellsens standard; olympus) or a scanning electron microscope (sem; jsm-6610la; jeol, tokyo, japan). the measured parts were selected according to seifert (2003). the following explanations of measured body parts are the same as those of seifert (2003), with some minor modifications: 1. head length (hl): maximum head length in the median line; excavation of the posterior margin of the head reduces the hl. 2. head width (hw): maximum head width including the eyes. 3. metanotal groove (mgr): depth of the metanotal groove or depression in lateral view, measured from the tangent connecting the dorsalmost points of the promesonotum and propodeum. 4. spine height (sph): height of the propodeal spine in lateral view, measured from the tangent connecting the dorsalmost points of the propodeum and propodeal spine to the meniscus between the propodeum and propodeal spine. 5. petiole length (pel): diagonal maximum length of the petiole in lateral view, measured from the anterior corner of the subpetiolar process to the posterodorsal corner of the petiole. 6. petiole height (peh): maximum petiole node height. fig 1. six measured parts (hl, hw, mgr, sph, pel, peh) of cardiocondyla sl. kagutsuchi workers for morphological analysis. these figures were drawn from the sem photographs of the specimens. the left figure represents a dorsal view of the head, the middle shows a lateral view of the mesosoma and that on the right is a lateral view of the petiole. hl, hw, mgr, sph, pel and peh represent the head length, head width, depth of the metanotal groove, height of the propodeal spine, petiole length and petiole height, respectively. following the measurements, canonical discriminant analysis (da) was performed using spss v. 22 (spss inc., chicago, il, usa) to determine whether the mtdna lineages would be compatible with differences in the external morphology of the workers. using a stepwise method, we selected characteristic measurements that minimized the overall wilk’s λ and used them for the da. after running “leave-one-out cross-validation” analysis, we examined the compatibilities i okita, m terayama, k tsuchida – phylogenetic and morphological groups of cardiocondyla kagutsuchi 404 between mtdna lineages and external morphology. in cases in which compatibility was detected, we applied the obtained discriminant functions to the morphological data on paratype workers (n = 4) of c. kagutsuchi and determined whether the paratypes could be classified to one of the lineages. furthermore, our previous phylogenetic study (okita et al., 2013) showed that the genetically closest species to c. sl. kagutsuchi was c. strigifrons, which was distinguished from c. sl. kagutsuchi by the dorsal mesosomal sculpture of the worker caste (seifert, 2003). therefore, in this study, we randomly selected one worker from each colony of the lineage that was genetically closest to c. strigifrons, as well as five workers from the colony of c. strigifrons, and examined whether the selected workers of c. sl. kagutsuchi could be distinguished from these five workers of c. strigifrons based on the dorsal mesosomal sculpture using a stereomicroscope or sem. the ratio of “head length” to “head width” (hl/ hw) of c. strigifrons workers is known to be larger than that of c. sl. kagutsuchi (seifert, 2003). therefore, we examined the significance of differences between the mean hl/hw value of the selected five workers of c. strigifrons and that of all measured workers of the lineage genetically closest to c. strigifrons by the t test. fig 2. neighbour-joining tree based on the mitochondrial coi/ii genes (829 bp) of the ant genus cardiocondyla and two other genera. bootstrap support values in 5,000 pseudoreplicates for distance and likelihood analyses are shown by the left and right numbers at the node, respectively. the genera names are as follows: c—cardiocondyla, m—monomorium and h—harpagoxenus. geographic origins of c. sl. kagutsuchi and c. strigifrons are shown following their species names. accession numbers of lineages a, b, c and d of c. sl. kagutsuchi were ab723724, ab723726, ab723727 and ab723725, respectively. lineage d was the same haplotype as dq023088 (hawaii). accession numbers of other lineages of c. sl. kagutsuchi were as follows (*: dq023083 and dq023084, *1: dq023081, dq023085, dq023086 and dq023087. *2: dq023080). white circles indicate that both winged and ergatoid males were present, half-white and half-filled circles represent both short-winged and ergatoid males and filled circles denote ergatoid males only (heinze et al., 2005; yamauchi et al., 2006; okita et al., 2013). male morphology of lineage b was based on okita’s personal observations. no males were found for c. atalanta, c. parvinoda and c. tjibodana. the number of colonies and sampling sites of each of the four c. sl. kagutsuchi lineages in japan are as follows: lineage a, 33 colonies in 32 sites; lineage b, 15 colonies in 14 sites; lineage c, 10 colonies in 3 sites; lineage d, 21 colonies in 21 sites (fig 4). results phylogenetic analyses and lineage distribution in previous studies (heinze et al., 2005; okita et al., 2013), four haplotype sequences in the mtdna coi/ii regions were detected in c. sl. kagutsuchi from japan. in this study, we obtained the same four haplotypes in 79 colonies of c. sl. kagutsuchi from japan, including 58 newly collected colonies. that is, no new haplotypes were detected in this study. briefly, c. sl. kagutsuchi from japan was divided into four lineages (a, b, c and d), which corresponded to haplotypes 1, 3, 4 and 2, respectively, in okita et al. (2013), with accession numbers ab723724, ab723726, ab723727 and ab723725 (figs 2, 3). in the phylogenetic trees, c. sl. kagutsuchi was divided into two distinct clades; one clade included lineages a, b and c, and the other included lineage d. the former clade was further divided into two subclades; one subclade included lineages a and b, and the other included lineage c. these results suggested that c. sl. kagutsuchi in japan was likely divided into three genetic groups: the first consisting of lineages a and b, the second of c and the third of d. based on these analyses, lineage d was genetically distant from the others. in fact, the substitution rates sociobiology 62(3): 401-411 (september, 2015) 405 a b c c. strigifrons c. kagutsuchi a 4.0 b 0.2 3.7 c 1.2 1.2 3.7 d 3.9 3.6 3.9 2.6 table 1. substitution rates (%) of mtdna coi/ii regions (829 bp) among four mtdna lineages (a, b, c and d; figs 2 and 3) of cardiocondyla sl. kagutsuchi in japan and cardiocondyla strigifrons. fig 3. majority rule consensus tree based on the mitochondrial coi/ii genes (829 bp) of the ant genus cardiocondyla and two other genera from bayesian analysis (1,000,000 generations). the posterior probabilities are shown at the node. the genera names are as follows: c—cardiocondyla, m—monomorium and h—harpagoxenus. geographic origins of c. sl. kagutsuchi and c. strigifrons are shown following their species names. accession numbers of lineages a, b, c and d of c. sl. kagutsuchi were ab723724, ab723726, ab723727 and ab723725, respectively. lineage d was the same haplotype as dq023088 (hawaii). accession numbers of other lineages of c. sl. kagutsuchi were as follows (*: dq023083 and dq023084, *1: dq023081, dq023085, dq023086 and dq023087. *2: dq023080). white circles indicate that both winged and ergatoid males were present, half-white and half-filled circles show both short-winged and ergatoid males and filled circles denote ergatoid males (heinze et al., 2005; yamauchi et al., 2006; okita et al., 2013). male morphology of lineage b was based on okita’s personal observations. no males were found for c. atalanta, c. parvinoda and c. tjibodana. the number of colonies and sampling sites of four c. sl. kagutsuchi lineages in japan were as follows: lineage a, 33 colonies in 32 sites; lineage b, 15 colonies in 14 sites; lineage c, 10 colonies in 3 sites; lineage d, 21 colonies in 21 sites (fig 4). between lineage d and the other three (>3.5%; table 1) were higher than those of three cardiocondyla species pairs, i.e., 0.6% between c. obscurior and c. wroughtonii, 1.6% between c. minutior and c. tjibodana, and 3.1% between c. batesii and c. nigra. furthermore, the substitution rates between lineage d and the other three were higher than those between lineage d and c. strigifrons (2.6%; table 1). fig 4. distribution map of cardiocondyla sl. kagutsuchi lineages a, b, c and d in japan. lineages a and d were distributed widely in the temperate and subtropical zones of japan from honshu (main island) to ishigaki and iriomote islands, lineage b was distributed in the subtropical zone of japan from okinawa island to ishigaki and iriomote islands and lineage c was distributed only in the subtropical zone of japan on ishigaki island (fig 4). i okita, m terayama, k tsuchida – phylogenetic and morphological groups of cardiocondyla kagutsuchi 406 morphological analyses phylogenetic analyses (figs 2, 3) showed that c. sl. kagutsuchi in japan was clearly divided into four lineages. therefore, we measured and analysed six body parts (fig 1) of workers of the following samples: lineage a, 165 workers (33 colonies) in 32 sites; lineage b, 75 workers (15 colonies) in 14 sites; lineage c, 50 workers (10 colonies) in 3 sites; lineage d, 105 workers (21 colonies) in 21 sites (fig 4). these data are available from the online supplementary material. the means ± se (μm) of the six measurements (fig 1) of the four lineages are shown in table 2. lineage hl hw mgr sph pel peh a 556.6 ± 2.0 465.1 ± 1.4 8.0 ± 0.2 4.7 ± 0.1 303.7 ± 1.3 157.3 ± 0.7 b 563.3 ± 2.7 467.0 ± 2.2 7.8 ± 0.2 4.8 ± 0.2 306.5 ± 1.5 158.9 ± 0.8 c 563.6 ± 3.2 472.1 ± 2.7 9.1 ± 0.4 7.8 ± 0.3 308.0 ± 2.4 149.5 ± 1.2 d 566.8 ± 2.2 482.7 ± 1.9 12.0 ± 0.3 10.2 ± 0.2 308.2 ± 1.5 165.0 ± 0.8 table 2. metric traits (mean ± se, μm) of six measured parts (fig 1) obtained from the four mtdna lineages (a, b, c and d) of cardiocondyla sl. kagutsuchi workers in japan. hl, hw, mgr, sph, pel and peh designate the head length, head width, depth of the metanotal groove, height of the propodeal spine, petiole length and petiole height, respectively. table 3. classification of predicted lineage for lineages a, b, c and d of cardiocondyla sl. kagutsuchi workers in japan using discriminant analysis (da). numerical values are the number of individuals that da classified into each of the four lineages. table 4. discriminant scores (mean ± se) of each lineage (a, b, c and d) of cardiocondyla sl. kagutsuchi in japan. a plus or minus sign on the discriminant score of each lineage shows that the first function discriminates lineage d and the second function discriminates lineage c. lineage predicted lineage a b c d total correct (%) a 70 76 9 10 70/165 (42.4) b 38 30 6 1 30/75 (40.0) c 3 1 45 1 45/50 (90.0) d 0 3 0 102 102/105 (97.1) lineage function 1 2 a –0.924 ± 0.080 –0.572 ± 0.072 b –0.924 ± 0.111 –0.657 ± 0.124 c –0.543 ± 0.108 +2.165 ± 0.117 d +2.495 ± 0.105 –0.294 ± 0.109 the second function discriminated lineage c. using the first and second functions, the discriminant scores of all individual workers are plotted in fig 5. briefly, our da showed that c. sl. kagutsuchi, distributed from honshu to ishigaki and iriomote islands, consisted of three morphological groups compatible with the mtdna lineages: lineages a and b could be compiled as the first group, lineage c as the second group and lineage d as the third group (fig 5). the standardized discriminant function coefficients (table 5) showed that the third group (lineage d) had larger sph, peh and mgr and smaller pel than the other two groups, and the second group (lineage c) had a larger pel and smaller peh than the other two groups with regard to worker morphology. sem photographs (fig 6) show these morphological characteristics in the three groups. in addition, we applied the obtained discriminant functions to the morphological data on worker paratypes (n = 4; data are available from the online supplementary material) of c. sl. kagutsuchi, and all of these paratypes were assigned to the second group (lineage c; fig 5) with high probabilities (0.963, 0.935, 0.911, 0.996). 　 function 1 2 sph 0.806 0.424 mgr 0.425 0.195 peh 0.852 –1.688 pel –0.970 1.346 table 5. standardized coefficients of the two discriminant functions. the first function discriminates lineage d and the second function discriminates lineage c of cardiocondyla sl. kagutsuchi in japan (table 4). sph, mgr, peh and pel designate the height of the propodeal spine, depth of the metanotal groove, petiole height and petiole length, respectively. selected characteristic measurements using a stepwise method in the da included sph, peh, pel and mgr in order of selection, and these characteristic measurements contributed significantly to the discrimination (wilk’s λ = 0.169, f12,1026.843 = 81.993, p < 0.001). the rate of individual workers correctly assigned to lineage a by the da was 42.4%, that of lineage b was 40.0%, that of lineage c was 90.0% and that of lineage d was 97.1% (table 3). these rates showed that our da correctly discriminated each of lineages c and d from the other three lineages, whereas they failed to discriminate between lineages a and b (table 3). two canonical discriminant functions that significantly discriminated between the lineages were obtained: the first function (wilk’s λ = 0.169, d.f. = 12, χ2 = 693.422, p < 0.001 was + 0.175 × mgr + 0.104 × peh – 0.062 × pel – 1.731, and the second function (wilk’s λ = 0.547, d.f. = 6, χ2 = 235.308, p < 0.001) was + 0.080 × mgr – 0.207 × peh + 0.086 × pel + 4.238. the first function explained 73.1% of the variance, while the second function explained 26.6%. the average discriminant scores of each lineage (table 4) showed that the first function discriminated lineage d and sociobiology 62(3): 401-411 (september, 2015) 407 our phylogenetic studies showed that the lineage genetically closest to c. strigifrons was lineage d (table 1, figs 2, 3), and we examined whether lineage d was morphologically distinguishable from c. strigifrons. visual inspection of the deviating dorsal mesosomal sculpture (a morphological criterion used by seifert (2003) to separate c. strigifrons from c. kagutsuchi) of lineage d and c. strigifrons revealed that all workers of lineage d examined could be clearly discriminated from c. strigifrons; the circular sculptures (foveolae) of the dorsal promesonotum of c. strigifrons were apparently shallower than in lineage d. furthermore, the number of sculptures of c. strigifrons was considerably less than that of lineage d (fig 7). the mean value of hl/hw for all measured workers of lineage d (mean ± se, 1.175 ± 0.002, n = 105) was significantly different from that of c. strigifrons (1.274 ± 0.010, n = 5; tcal = –10.13, d.f. = 108, p < 0.001; morphological data are available from the online supplementary material). fig 5. scatter plot of the discriminant scores of individual workers of cardiocondyla sl. kagutsuchi in japan using the first and second functions: lineage a (open circle, n = 165 workers), lineage b (filled circle, n = 75 workers), lineage c (diamond, n = 50 workers), lineage d (triangle, n = 105 workers) and paratypes (filled square, n = 4 workers). ellipses are 95% confidence limit lines. fig 6. sem images of the workers of group consisting of lineages a and b (i and ii) of cardiocondyla sl. kagutsuchi, those of lineage c (iii and iv) and those of lineage d (v and vi). pictures on the left display a lateral view of the mesosoma and pictures on the right show a lateral view of the petiole. each scale bar is 100 μm. number of sexuals per colony we obtained a total of 24 colonies containing sexuals; seven colonies were from lineage a, one colony was from lineage b, eight colonies were from lineage c and nine were from lineage d (table 6). among these 24 colonies, four contained no female sexuals, probably due to incomplete sample collection. irrespective of incomplete sample collection, our data (table 6) showed that nearly half of the colonies from lineages a and d contained more than one ergatoid male (lineage a: 3/7, lineage d: 4/9) and wingless females (lineage a: 5/7, lineage d: 6/9). with regard to male morphology, our previous study (okita et al., 2013) showed that lineages a and d were monomorphic (only ergatoid males) and lineage c was dimorphic (winged and ergatoid males). the results of this study (table 6) agreed with those reported previously. we obtained only one colony containing sexuals in lineage b. this colony produced several ergatoid males and no winged males for more than 1.5 years under laboratory conditions (okita, personal observation). discussion this study showed that c. sl. kagutsuchi in japan consisted of three groups that differed in both mtdna sequences and worker morphology: lineages a+b, c and d. originally, c. kagutsuchi terayama, 1999 was the species name given to the endemic ant collected from ishigaki island known to have both winged and ergatoid males (terayama, 1999). with regard to the distribution area and male morphology of our samples, lineage c has so far been collected only from ishigaki island (fig 4), and most of the examined colonies (6/8) contain winged males or both winged and ergatoid males (table 6) as mentioned by terayama (1999). furthermore, our das showed that all paratypes of worker c. kagutsuchi (n = 4) were assigned to lineage c (fig 5) with high probabilities (0.963, 0.935, 0.911, 0.996). thus, lineage c is likely assigned to c. kagutsuchi terayama,1999 for the following three reasons: i) a high morphological similarity i okita, m terayama, k tsuchida – phylogenetic and morphological groups of cardiocondyla kagutsuchi 408 between lineage c and the paratype of c. kagutsuchi; ii) the presence of male dimorphism in lineage c as mentioned by terayama (1999); and iii) an overlapping distribution between lineage c and the paratypes. the substitution rates between the group of lineage d and the other two groups consisting of lineages a+b and c were more than 3.5% (table 1), which was higher than those of three cardiocondyla species pairs (figs 2, 3). schlick-steiner et al. (2006) considered certain lineages of tetramorium ants in europe as new species, in which the interspecific substitution rates of the mtdna coi region were between 1.6% and 8.5%. the observed substitution rates between the group of lineage d and the other two groups consisting of lineages a+b and c (>3.5%, table 1) showed a fair amount of overlap with the interspecific substitution rates of the european tetramorium ant species pairs (between 1.6% and 8.5%), suggesting that lineage d is a distinct species from the other two groups. furthermore, with regard to morphology, 97.1% of the workers in lineage d were correctly assigned to the same lineage by the da (table 3). this morphological analysis strongly suggested that lineage d is a distinct species. as the previous revision of this genus (seifert, 2003) showed that c. strigifrons, which was genetically the closest species to lineage d (figs 2, 3), was distinguished from c. sl. kagutsuchi by the dorsal mesosomal sculpture and head shape of the worker caste, we easily distinguished lineage d from c. strigifrons lineages based on sculpture (fig 7) and the head shape of workers. furthermore, the rate of mtdna substitution between c. strigifrons and lineage d (2.6%) was higher than those of the two cardiocondyla species pairs (0.6% between c. obscurior and c. wroughtonii, 1.6% between c. minutior and c. tjibodana), and the lower extreme of those of the european tetramorium ant species pair (1.6%; schlick-steiner et al., 2006). these evidences strongly suggest that lineage d is a distinct species from c. strigifrons. in this study, we showed that three phylogenetic and morphological groups (lineages a+b, c and d) exist within c. sl. kagutsuchi in japan. furthermore, our previous study (okita et al., 2013) showed that lineages a and d were male monomorphic, while lineage c was dimorphic. that is, male monomorphic c. sl. kagutsuchi in japan includes at least two lineages differing in worker morphology. table 6. number of sexuals present in 24 field colonies of cardiocondyla sl. kagutsuchi in japan. we counted the number only if collected colonies contained sexuals. four colonies did not have any female sexuals, possibly due to incomplete sample collection. lineage number of month of collection male sexuals female sexuals winged ergatoid winged wingless a 0 0 0 2 may 0 0 1 0 july 0 3 0 0 july 0 2 13 19 august 0 1 1 3 september 0 1 3 5 november 0 4 5 4 november b 0 2 0 1 june c 2 1 2 0 may 1 0 0 1 june 1 0 0 1 june 1 0 0 0 june 1 0 0 0 june 0 0 1 13 june 0 0 0 1 june 1 1 3 1 june d 0 3 0 1 june 0 2 3 0 june 0 1 14 4 august 0 1 0 0 august 0 3 22 9 august 0 6 65 58 september 0 0 4 7 september 0 0 2 4 september 0 1 1 2 october fig 7. sem images of the dorsal promesonotum of the worker caste. the top is lineage d of cardiocondyla sl. kagutsuchi and the bottom is cardiocondyla strigifrons. each scale bar is 50 μm. sociobiology 62(3): 401-411 (september, 2015) 409 supplementary material data set available at: http://periodicos.uefs.br/ojs/index.php/sociobiology/rt/ suppfiles/805/0 doi: 10.13102/sociobiology.v62i3.805.s902 references bernasconi, c., cherix, d., seifert, b. & pamilo, p. 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(2010). on the evolution of the species complex pachycondyla chinensis (hymenoptera: formicidae: ponerinae), including the origin of its invasive form and description of a new species. zootaxa, 2685: 39-50. open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.8536sociobiology 69(4): e8536 (december, 2022) introduction invasive alien species (hereafter ias) represent one of the main causes of biodiversity loss with impacts on ecosystem function and services (vilà & hulme, 2017; blackburn et al., 2019). alien species also negatively affect societies, economies as well as both human and animal health and well-being on a global scale (mazza & tricarico, 2018; chinchio et al., 2020; pyšek et al., 2020). more than 14,000 alien species are currently distributed across europe, including 65 alien ant species (easin, 2022). among them, four species have been identified as ias of union concern, namely the fire ants solenopsis geminata (fabricius, 1804), s. invicta buren, 1972 and s. richteri forel, 1909 as well as the abstract native to the neotropics, wasmannia auropunctata (roger, 1863) has been unintentionally introduced around the world, heavily impacting native ant biodiversity, societies, and economies as well as human and animal health due to its potentially dangerous stings. herein we report on the first record of w. auropunctata in cyprus. specimens were collected from plant nurseries and tourist facilities in paphos and limassol district. wasmannia auropunctata is believed to spread via the horticultural pathway to locations with sufficient humidity. further research is necessary to determine the distribution and assess possible negative impacts of w. auropunctata to native biodiversity, society, the economy and human health in cyprus. sociobiology an international journal on social insects jakovos demetriou1,2, christos georgiadis3, helen e. roy4, angeliki f. martinou1,2,5, lech borowiec6, sebastian salata6 article history edited by enrico schifani, university of parma, italy received 13 september 2022 initial acceptance 15 september 2022 final acceptance 26 september 2022 publication date 28 december 2022 keywords alien ants, biological invasions, first record, non-native species, island invasions, little fire ant. corresponding author sebastian salata university of wrocław, department of biodiversity and evolutionary taxonomy, myrmecological laboratory przybyszewskiego 65, 51-148 wrocław, poland. e-mail: sebastian.salata@uwr.edu.pl little fire ant wasmannia auropunctata (roger, 1863) (eu regulation 1203/2022). furthermore, four alien ant species have been identified among the 100 of the world’s worst invasive alien species (gisd, 2022), namely the argentine ant linepithema humile (mayr, 1868), the recently revised african big-headed ant pheidole megacephala (fabricius, 1793) (salata & fisher, 2022), the red imported fire ant s. invicta, and w. auropunctata. these species have been considered responsible inter alia for the displacement of local ant species, economic losses in agriculture and have been reported as human nuisance (gisd, 2022). wasmannia auropunctata also called the “electric ant” and “little or small fire ant” is native to the neotropical zoogeographic realm and currently spread throughout the 1 joint services health unit cyprus, bfc raf, akrotiri, cyprus 2 enalia physis environmental research centre, nicosia, cyprus 3 section of zoology and marine biology, department of biology, national and kapodistrian university of athens, greece 4 uk centre for ecology & hydrology, oxfordshire, united kingdom 5 climate and atmosphere research centre/ care-c, the cyprus institute, nicosia, cyprus 6 university of wrocław, department of biodiversity and evolutionary taxonomy, myrmecological laboratory, wrocław, poland research article ants one of the world’s worst invasive alien species wasmannia auropunctata (hymenoptera: formicidae) detected in cyprus mailto:sebastian.salata@uwr.edu.pl jakovos demetriou et al. – first record of wasmannia auropunctata in cyprus2 world (wetterer, 2013; janicki et al., 2016; guénard et al., 2017). within its invaded range, it has been found to reduce native arthropod biodiversity (lubin, 1984; roque-albelo et al., 2000; wetterer & porter, 2003; mbenoun-masse et al., 2017), affect the fecundity and fertility of vertebrates (i.e. tortoises and birds) (hayashi, 1999; roque-albelo & causton, 1999; nishida & evenhuis, 2000) but also to pose a significant health risk to humans due to its stinging that may cause anaphylactic shocks to allergic people or harm domestic animals (i.e. cats and dogs) (wetterer et al., 1999; nishida & evenhuis, 2000; wetterer & porter, 2003; kidon et al., 2022). in europe and the mediterranean basin, w. auropunctata has been reported indoors in the united kingdom (donisthorpe, 1908), germany (geiter et al., 2002) and the netherlands (boer & vierbergen, 2008). the species was recorded once in 1996 in italy (lisa blanca island) (jucker et al., 2008), although upon further investigations “w. auropunctata is to be considered absent from lisca bianca where it was probably recorded erroneously” (mr enrico schifani, personal communication, september 23, 2022; see schifani, 2022). in israel (vonshak et al., 2009, 2010; vonshak & ionescu-hirsch, 2009) and spain (espadaler et al., 2018, 2020) w. auropunctata has established viable populations and spread into natural habitats. in cyprus, nine species of alien ants have been identified (salata et al., 2019). nevertheless, the presence of cardiocondyla mauritanica forel, 1890, monomorium pharaonis (linnaeus, 1758) and solenopsis geminata (fabricius, 1804) on the island have been supported only by single literature records (emery, 1909; collingwood et al., 1997; wetterer, 2010). in this publication, w. auropunctata is reported for the first time in cyprus. materials and methods material examined cyprus: ● paphos, veronica hotel [34.749580on, 32.428932oe], alt. 20 m, 24.iv.2022, leg. and ident. l. borowiec and s. salata, habitat details: hotel parking area. ● paphos, kato paphos, [34.736479, 32.435402], alt. 6 m. 29.iv.2022, leg. and ident. l. borowiec and s. salata, habitat details: collected from sea shore at night. ● paphos, kissonerga lemba [34.813211, 32.410112], alt. 85 m, 30.iv.2022, leg. j. demetriou, c. georgiadis, l. borowiec and s. salata, ident. l. borowiec and s. salata, habitat details: in plant nursery on plastic sheets placed on the soil. ● paphos, kissonerga lemba [34.813211, 32.410112], alt. 85 m, 07.iχ.2022, leg. j. demetriou, ident. j. demetriou, habitat details: in plant nursery on plastic sheets placed on the soil. ● paphos, kissonerga [34.8333632, 32.405394], alt. 109 m, 30.iv.2022, leg. j. demetriou, c. georgiadis, l. borowiec and s. salata, ident. l. borowiec and s. salata, habitat details: in plant nursery on the soil. ● paphos, chlorakas [34.7979, 32.3960], alt. 20 m, 6+23.viii.2022, leg. j. demetriou, ident. j. demetriou, habitat details: on pedestrian path with ornamental surrounded by luxury villas. ● limassol, kato polemidia [34.6811, 33.0051], alt. 38 m, 26.iii.2022, leg. j. demetriou, ident. l. borowiec, habitat details: urban park, nest under rock close to pistacia atlantica. ● limassol, savvas savva park [34.6805, 33.0357], alt. 15 m, 31.v.2022, leg. j. demetriou, ident. l. borowiec and s. salata, habitat details: collected from pavement bordering urban park, shaded, predominantly with large ficus microcarpa l. trees (fig 1, 2). specimens were collected by hand and stored in ≥70o ethanol at the myrmecological laboratory, department of biodiversity and evolutionary taxonomy, university of wroclaw, poland and jd’s personal collection. species identification was performed using the identification keys of bolton (1994) and longino and fernández (2007) as well as through comparison with available photographic material on antweb (2022). (https://www.antweb.org/images.do?subfa mily=myrmicinae&genus=wasmannia&species=auropunct ata&rank=species&project=allantwebants). the competent authorities were informed upon the identification of collected specimens. fig 1. habitus of worker of wasmannia auropunctata (roger, 1863), dorsal and lateral, collected from cyprus (locality kissonerga): scale bar 0.5 mm (photographed by l. borowiec). maps maps were created using qgis version 3.18.2 free and open source geographic information system (https:// qgis.org/en/site/). results and discussion specimens of w. auropunctata were collected in urban habitats of limassol and paphos such as a hotel’s parking area, two plant nurseries, and urban parks; representing the https://qgis.org/en/site/ https://qgis.org/en/site/ sociobiology 69(4): e8536 (december, 2022) 3 ornamental and aromatic plants grown in well-irrigated plant nurseries and subsequently planted in parks and tourist facilities with sufficient moisture. in neighbouring israel, it was suspected that “w. auropunctata was first established in irrigated gardens in the warm climate of the jordan valley, and only afterwards spread into less favourable habitats […] through commercial transport of chopped wood, logs, and potted plants” (vonshak et al., 2010). “as initial eradication efforts failed the ants widely spread mainly by commerce in flowerpots” (dr armin ionescu, personal communication, september 5, 2022). as a result, the species currently occurs predominantly in irrigated habitats or near natural water sources with frequent human activity, enabling its survival in both warmer and drier habitats than those of its native range (vonshak et al., 2010). in addition, the species has been found entering households during the warm summer months in search of habitats with sufficient moisture (vonshak et al., 2010), a much needed resource that is considered to facilitate the species spread (meier, 1994; vonshak et al., 2010). its adverse environmental impacts on the abundance, species richness, and community composition of native arthropod biodiversity in israel (vonshak et al., 2010) as well as the related human health risks connected with a reported case of severe anaphylactic allergic reaction (kidon et al., 2022), confirm the need for further research on its origin, distribution, pathways of spread and impacts on cyprus. fig 2. current known distribution of wasmannia auropunctata (roger, 1863) in the mediterranean basin and cyprus (inset). first records for cyprus and supplementing our knowledge around the distribution of this invasive alien ant in europe and the mediterranean (fig. 2). opportunistic material surveys in larnaca, famagusta and nicosia yielded no specimens. as biological invasions have been found to pose an important threat to island ecosystems (reaser et al., 2007; russel et al., 2017), the presence of w. auropunctata in cyprus raises concerns regarding its possible spread to natural habitats and threat towards native biodiversity. this omnivorous alien species is predicted to expand its range in cyprus over the next decades because of its ability to colonize and establish in a wide range of disturbed and undisturbed habitats (longino & fernández, 2007; mbenoun masse et al., 2017; cabi, 2022), construct polygynous nests in a variety of substrates (longino & fernández, 2007) and tolerate a wide range of climatic and environmental conditions (i.e. altitude and humidity) (longino & fernández, 2007; vonshak et al., 2010; cuezzo et al., 2015). horizon scanning exercises for ias not yet present on the island, ranked w. auropunctata amongst the top 50 most likely ias to reach cyprus and potentially threaten human health and the island’s economy (peyton et al., 2020). peyton et al. (2020) mention organic packing material, ships, containers and natural dispersal across borders as the most prominent introduction pathways for the species. based on our study, the w. auropunctata is presumed to have spread through the horticultural pathway, hidden in the soil of jakovos demetriou et al. – first record of wasmannia auropunctata in cyprus4 furthermore, cyprus has similar climatological conditions to israel therefore we expect that the spread of w. auropunctata could follow the same patterns observed in israel causing negative impacts to biodiversity, human health and socioeconomic parameters. the use of molecular markers could help identify the origin and number of introductions of w. auropunctata in cyprus. further research is necessary to assess both the native and alien myrmecofauna of cyprus, which are relatively understudied. structured material surveys in plant nurseries and touristic facilities are required to study pathways of introduction and further spread of w. auropunctata. additionally, sampling in natural habitats could enhance early detection efforts in protected areas and establish effective management strategies for its rapid eradication. acknowledgements we are very thankful to dr armin ionescu (the steinhardt museum of natural history, tel aviv university) and section editor msc enrico schifani (department of chemistry, life sciences and environmental sustainability, università degli studi di parma) for their valuable provided information on the distribution of w. auropunctata in israel and italy, respectively. we are also thankful to the anonymous reviewers for their comments, suggestions and corrections upon the manuscript. we would also like to thank the uk government through darwin plus (dplus0124), for funding this project and material surveys of jakovos demetriou. authors’ contributions conceptualization: jd, ss; methodology: jd, cg, lb, ss; software: jd; validation – verification: lb, ss; formal analysis: jd; investigation: jd, cg, afm, her, lb, ss; resources: jd, cg, afm, her, lb, ss; data curation: jd; writing-original draft: jd, ss; writing-review & editing: jd, cg, afm, her, lb, ss; visualization: jd, lb; supervision: afm, her, lb, ss; project administration: ss; funding acquisition: afm, her, lb, ss. references antweb. 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(1999). wasmannia auropunctata (roger) (hymenoptera: formicidae), a highly destructive tramp ant, in wildlife refuges of gabon, west africa. african entomology, 7: 292-294. https://www.tau.ac.il/lifesci/zoology/members/dayan_files/articles/merav_ziv_2006.pdf https://www.tau.ac.il/lifesci/zoology/members/dayan_files/articles/merav_ziv_2006.pdf https://www.tau.ac.il/lifesci/zoology/members/dayan_files/articles/merav_ziv_2006.pdf https://doi.org/10.1007/s10530-009-9593-2 https://doi.org/10.1163/18749836-06001068 https://doi.org/10.1163/18749836-06001068 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i2.321-327sociobiology 62(2): 321-327 (june, 2015) morphological differentiation between species of myrmelachista roger (formicidae: formicinae) in atlantic forest areas of the alto tietê (são paulo) introduction myrmelachista is an arboreal genus with 69 described species (longino, 2006; bolton, 2013). its distribution is restricted to the neotropical region, and 41% of the species occur in brazilian territory (kempf, 1972; fernández & sendoya, 2004). it is characterized by a visible antennal club with one or two segments, a character that is absent in the remaining neotropical formicinae (fernández, 2003). myrmelachista species have antennae with nine or ten segments; most species have nine-segmented antennae and abstract myrmelachista is an exclusively neotropical ant genus. the ants from this genus nest and forage in live or dead trees, but data on its life cycle are relatively scarce. the taxonomy of this genus is considerably complex. the morphological characters of taxonomic importance are not well defined, and combining characteristics from the largest possible number of castes is thus essential. the goal of the present study was to present the results of multidimensional morphological analyses conducted based on morphometric measurements of workers and alates to help with the identification of myrmelachista species. for that purpose, we gathered data from 277 specimens (workers, males and gynes) from eight species cohabiting atlantic forest areas. eighteen morphometric variables were measured on workers and 20 on reproductive ants. measurements related to eye size and mandible width were the variables more strongly associated with the two morphological clusters obtained based on the morphology of workers. however, the morphospace described by workers cannot be used to delimit the species of myrmelachista. mandible width, petiole height and eye size (eye length and ocelli width) were the most informative variables associated with the four clusters of morphology that clearly delimit males of m. arthuri, m. ruszkii sp.7, and in part, m. catharinae. finally, petiole length and head length were the best descriptors of the six morphological clusters recognized for gynes. m. catharinae, m. sp.7, m. nodigera and m. gallicola were clearly delimited in the gyne morphological space. m. ruszkii showed high phenotypic variability, and this species was classified in two morphological clusters based on gyne specimens. species identification employing multiple gyne morphological traits exhibited the best results. our results show that morphospace analysis can be useful for delimitation of myrmelachista species. sociobiology an international journal on social insects ma nakano1, rr silva2 vfo miranda3, rm feitosa4, msc morini1 article history edited by kleber del-claro, ufu, brazil received 09 march 2015 initial acceptance 26 june 2015 final acceptance 28 june 2015 keywords twigs, arboreal ants, morphometric variables, multivariate analysis, taxonomy. corresponding author maria santina de castro morini umc – núcleo de ciências ambientais laboratório de mirmecologia av. dr. cândido xavier de almeida souza, 200 08780-911 mogi das cruzes, sp, brazil e-mail: mscmorini@gmail.com are found mainly in central america (only two have been described in south america), whereas ten-segmented species are mainly found in south america (only three species have been described in central america and mexico) (longino, 2006). workers have been observed in association with 20 families and 53 species of angiosperms (see review by nakano et al., 2013) and in leaf litter (suguituru et al., 2011, 2013). nests are located in the trunks of live trees (longino, 2006). colonies with immature and reproductive ants also occupy twigs dispersed on the leaf litter (nakano et al., 2012, research article ants 1universidade de mogi das cruzes, mogi das cruzes, sp, brazil 2museu paraense emílio goeldi, belém, pa, brazil 3universidade estadual paulista, jaboticabal, sp, brazil 4universidade federal do paraná, curitiba, pr, brazil ma nakano, rr silva vfo miranda, rm feitosa, msc morini – morphological differentiation of myrmelachista (formicidae) species322 2013). the genus myrmelachista is characterized by forming mutualistic associations with some myrmecophytes (renner & ricklefs, 1998; edwards et al., 2009), where workers use formic acid to protect the host plant (frederickson, 2005), as well as with coccidae and pseudococcidae (kusnezov, 1951; stout, 1979; ketterl et al., 2003; longino, 2006). although some studies on this genus have been published recently (longino, 2006; nakano et al., 2012, 2013), information on the biology of myrmelachista species is still scarce. ants from this genus feed on extrafloral nectaries (frederickson, 2005; mcnett et al., 2010), but workers may take plant, fungal and animal fragments (eggs, feces and larvae) into the nests (torres, 1984). the taxonomy of this genus is considered extremely complex (wheeler, 1934; snelling & hunt, 1975; longino, 2006). morphological characters of taxonomic importance are not well defined, and it is necessary to combine information from all castes (workers and reproductive ants). nakano et al. (2014) showed that some species may be separated using molecular characters, despite their high morphological similarity (nakano, 2010). however, no detailed morphological study has been performed to evaluate species delimitation in myrmelachista. it is well known that many species can be difficult to diagnose and delimit, especially when using single operational criteria such as morphological characters or dna markers (cardoso et al., 2009; ezard et al., 2010). morphological data are useful for delimiting species, and arguments exist to refine approaches to species delimitation (barr et al., 1985; ezard et al., 2010; seifert et al. 2014a). some studies have suggested that multivariate analysis based on morphology provides further insights into species alpha-taxonomy and distribution of organisms (seifert, 2002; seifert et al. 2014ab). in this study, we present the results of multidimensional morphological analyses performed based on morphometric measurements of workers and alates (males and females) to help identify myrmelachista species. materials and methods collection sites and species collections were performed at three sites: (1) francisco affonso de mello municipal natural park, 23◦31’22” s, 46◦11’16” w, 807–1140 m, in the municipality of mogi das cruzes; (2) barragem de ponte nova, 23◦31’85” s, 45◦50’77” w, 783 m, in the municipality of salesópolis; and (3) nascentes do tietê state park, 23◦34’19” s, 45◦44’10” w, 1027 m, in the municipality of salesópolis. all sites were located in the alto tietê region, which belongs to the brazilian atlantic forest and is characterized by dense ombrophilous forest (fiaschi & pirani, 2009; colombo & joly, 2010). eight species of myrmelachista can be found in this region (suguituru et al., 2015), corresponding to 11% of the species recorded in the neotropical region and to 29% of the brazilian species. therefore, our study area exhibited high local species richness of the genus myrmelachista, which is adequate to evaluate species discrimination tools, considering that these species are seldom recorded (deyrup, 2003; observations on different published lists of ant species recorded in brazil). the species analyzed were myrmelachista arthuri forel, 1903, m. catharinae mayr, 1887, m. gallicola mayr, 1887, m. nodigera mayr, 1887, m. reticulata borgmeier, 1928, m. ruszkii forel, 1903, myrmelachista sp.4 and myrmelachista sp.7 (suguituru et al., 2015). a more detailed description of the collection sites, methods used, and specimen identification can be found in nakano et al. (2012, 2013, 2014). species (or morphospecies) were identified by comparing the examined specimens with specimens deposited in the reference collection of the museum of zoology of the university of são paulo. vouchers were deposited in the myrmecofauna collection of the alto tietê myrmecology laboratory of the university of mogi das cruzes and in the museum of zoology of the university of são paulo. number and morphology of specimens the number of specimens per species is shown in table 1. the morphological parameters were measured using a micrometer ruler coupled to a stereoscopic microscope. eighteen morphological parameters were selected for workers and 20 for reproductive ants according to longino (2006) and silva and brandão (2010). species number total/specie number of nests worker/nest males gynes myrmelachista arthuri 6 5 7 37 myrmelachista catharinae 10 5 7 9 66 myrmelachista gallicola 5 5 2 27 myrmelachista nodigera 5 5 10 35 myrmelachista reticulata 1 5 5 myrmelachista ruszkii 5 5 3 19 47 myrmelachista sp.4 5 5 25 myrmelachista sp.7 4 5 4 11 35 table 1. total number of specimens per myrmelachista species subjected to morphological analysis. sociobiology 62(2): 321-327 (june, 2015) 323 data analysis we used a method developed to delimit species morphometrically in a multivariate space without using an a priori definition. this approach for species delimitation identifies clusters in the morphospace when two or more well-separated groups are a better way of describing a given sample than one group (ezard et al., 2010). the approach to species delimitation consists of four steps (based on ezard et al., 2010): (1) extraction of orthogonal axes with robust covariance estimators (croux et al., 2007); (2) reduction of the dimensionality of the orthogonal axes to only those with significant explanatory power (jackson, 1993); (3) identification of the optimal number, shape and orientation of groups within the rotated dimension-reduced data (fraley & raftery, 2002). in step 1, each morphometric variable was centered on the median and scaled by percentile variability prior to reducing the dimensionality (i.e., the rotation was based upon the robust covariance estimates). in step 2, the number of components retained was determined using the broken stick criterion (peres-neto et al. 2005). in step 3, the dimension-reduced space was split into groups using cluster analysis, with optimal delimitation by k-means approaches (klingenberg & froese, 1991) combined with methods to estimate the shape and orientation of clusters in the morphospace, and gaussian mixture models and a bayesian approach were used to estimate the support for particular arrangements of clusters using iterative expectation-maximization methods for maximum-likelihood (fraley & raftery, 2002). the volume and shape can be equal or variable among axes (elliptical, round, diagonal or univariate), and the choice between competing models is made through the bayesian information criterion (bic). all analyses were performed in r version 3.1.1 (r core team, 2014) and used the mclust (fraley & raftery, 2002), pcapp (filzmoser et al., 2014) and mvoutlier (filzmoser & gschwandtner, 2014) packages. results workers the robust pca of 18 morphometric variables from 205 worker specimens from the atlantic forest populations revealed a continuum of variation but no discrete clusters within the myrmelachista morphospace (fig 1). the clustering on the dimension-reduced morphospace found weak evidence to reject the null hypothesis of homogeneous data in the worker measurements. the best model found two clusters of morphologies (ellipsoidal, varying volume, shape, and orientation) and did not delimit the worker individuals in the data set. the broken stick criterion suggested that two principal components should be retained, which represented 94% of the variation in the original data (table 2). as expected, the first component is a size component, with variables loading males the robust pca of 20 morphometric variables from 21 male specimens suggested that five groups can be characterized mostly by their distinctive size and shapes in the plane of the first and second pca axes (fig 2). the clustering on the dimensionreduced morphospace found evidence in male measurements to reject the null hypothesis of homogeneous data in favor of the alternative hypothesis of more than one species of males. the best model found five clusters of morphology (univariate, equal variance) and delimited mainly males of m. catharinae (71%; n=7) and m. sp.7 (100%, n=4). m. arthuri was split in two groups, although very close in the morphospace (fig 2). by contrast, individuals of m. sp.7 and m. ruszkii were equal in the morphospace; two individuals of m. catharinae were classified as a particular group. the broken stick criterion suggested that one principal component should be retained; pci and pcii represented 89% of the variation in the original data (table 2). the first component is a size component, with variables loading relatively weakly and positively into this component. mandible width and petiole height were the variables with higher loads in axis i. principal component ii showed some allometry with positive loading of head width, maximum eye length, scape length, size, petiole size (length and width), femur length, distance between ocelli and ocelli width (the latter being strongly associated with pc ii) (table 2). fig 1. point clouds for workers of myrmelachista species in the brazilian atlantic forest. the two clusters identified in the robust pca are shown in different colors. symbols: art= m. arthuri, cat = m. catharinae, gal= m. gallicola, rus= m. ruszkii, sp.4 = m. sp.4, sp.7= m. sp.7. relatively weakly and positively into this component. in the second component, mandible width and eye size loaded positively and relatively strongly with axis ii (table 2). ma nakano, rr silva vfo miranda, rm feitosa, msc morini – morphological differentiation of myrmelachista (formicidae) species324 gynes the robust pca of 20 morphometric variables from 51 gyne specimens showed that the species can be distinguished from each other with different degrees of consistency (fig 3). the clustering on the dimension-reduced morphospace found evidence for more than one species in the gyne dataset. the best model found seven clusters of morphology (ellipsoidal, equal volume, shape and orientation) and delimited the gynes of m. catharinae (88%; n=9), m. sp.7 (100%, n=11), m. nodigera (100%, n=10) and m. gallicola (100%, n=2); individuals of m. ruszkii (n=19) were classified into two clusters suggesting high morphometric variability. the broken stick criterion suggested that two principal components should be retained, which represented 95% of the variation in the original data (table 2). the first and second components explain 53% and 42%, respectively. the first component showed some allometry because petiole length loaded positively and relatively strongly (compared to the other variables loading negatively). petiole length was the variable with higher loads in the first principal component; and head length and petiole length were the variables with higher loads in the second principal component (table 2). fig 2. point clouds for males of myrmelachista species in the brazilian atlantic forest. the five clusters identified in the robust pca are shown in different colors. symbols: art= m. arthuri, cat = m. catharinae, rus= m. ruszkii, sp.7= m. sp.7. table 2. the loadings onto the robust principal components for workers, males and gynes specimens of myrmelachista; larger absolute values indicate more influential traits on the dimension-reduced space. the first or second higher loadings in each axis were highlighted in bold. workers: 18 variables and 205 specimens; males: 20 variables and 21 specimens; gynes: 20 variables and 51 specimens. workers males gynes component i component ii component i component ii component i component ii standard deviation 3.43 2.57 3.91 2.35 4.24 3.76 proportion of variance 0.60 0.34 0.66 0.24 0.53 0.42 cumulative proportion 0.60 0.94 0.66 0.90 0.53 0.95 trait head width 0.29 0.236 0.109 -0.251 head length 0.243 0.188 0.454 mandible length 0.357 -0.209 0.35 mandible width 0.577 0.454 -0.357 -0.158 0.122 distance between mandibles 0.279 0.136 -0.266 scape length 0.162 0.234 0.13 -0.113 0.223 eye length 0.577 0.195 0.556 -0.229 0.151 eye width 0.577 0.255 -0.155 -0.142 0.105 distance of eye to mandible insertion 0.324 0.249 0.199 interocular distance 0.288 0.286 -0.252 pronotum width 0.229 0.178 -0.278 0.181 weber’s length 0.285 0.123 -0.238 0.123 hind tibia length 0.217 0.124 -0.227 hind femur length 0.203 0.118 -0.218 petiole width 0.211 0.219 0.11 -0.108 petiole length 0.265 0.386 0.487 0.642 petiole height 0.166 0.489 -0.152 0.175 gaster width 0.244 0.125 -0.246 ocelli width 0.452 -0.267 distance between ocelli 0.324 0.182 sociobiology 62(2): 321-327 (june, 2015) 325 fig 3. point clouds for gynes of myrmelachista species in the brazilian atlantic forest. the seven clusters identified in the robust pca are shown in different colors. symbols: symbols: cat = m. catharinae, gal= m. gallicola, nod = m. nodigera, rus= m. ruszkii, sp.7= m. sp.7. discussion the myrmelachista species analyzed in the present study exhibit ten-segmented antennae, a characteristic associated with a group considered to be very heterogeneous (snelling & hunt, 1975). our results indicate that workers can not be identified based only on multivariate morphometric analyses. separation of workers using morphometric data is a complex task because some studies show that the morphology of individuals of the same species originating from different colonies may vary to the point of being considered different species (wheeler, 1934; snelling & hunt, 1975). surprisingly, the set of variables used to describe the morphometry of gynes was efficient at separating groups of species. the resulting groups of species were largely in agreement with the identification by comparison with worker specimens deposited in biological collections, the identity of which was confirmed through direct comparisons with type specimens. workers of m. catharinae and m. arthuri are genetically close (nakano et al., 2014). in our analyses, these species exhibited high morphological overlap, similarly to workers from m. gallicola and m. nodigera. other studies have also reported morphological (quirán & martinez, 2006), behavioral (nakano et al., 2013) and genetic (nakano et al., 2014) similarities between these species. genetic analysis of m. catharinae workers revealed two groups (nakano et al., 2014), which was not confirmed by morphometry. however, males of m. catharinae were classified into two groups, which suggests some phenotypic variability retrieved in our morphometric analyses. the multivariate analysis indicated that characters such as head length and the sizes of the eye, ocelli and petiole may help with the delimitation of some species. in terms of function, these morphometric variables are mainly related to predation and locomotion (see review by silva & brandão, 2010). m. arthuri workers have been observed in the field preying on soldiers and minor workers of atta sexdens linnaeus, 1758, as well as on small beetles from different species (see image in suguituru et al., 2015; ghp castro personal communication), although this type of behavior is not usually reported for myrmelachista species. our results clearly show that, unlike workers, the analysis of morphometric characters of males and especially of gynes enables the distinction of morphological groups corresponding to five of the named species in the atlantic forest sites studied (tab. 1). our observation that gynes of m. nodigera, m. catharinae and m. sp.7 are morphologically different corroborates the molecular data from nakano et al. (2014). in contrast, the observation of different morphological groups for workers and gynes of m. ruszkii differs from the results of the molecular analyses (nakano et al., 2014). the morphometric analysis indicated high phenotypic variability of m. ruszkii, and of males of m. arthuri. longino (2006) stated that myrmelachista species are more easily identified using reproductive ants, which is in accordance with our results. however, males and gynes of myrmelachista are rarely recorded in studies of ant fauna, and the data available on myrmelachista reproductive biology are still scarce (nakano et al., 2012, 2013), hindering the taxonomic resolution of this genus. our results also indicate that the eyes, petiole and ocelli are important characters for species distinction when reproductive ants are collected. the overlapping of morphometric characters of m. catharinae and m. arthuri workers could lead to the classification of these species under the same species name, which would then represent a morphologically and biologically highly variable species. this may also be the case for m. nodigera and m. gallicola. in both cases, the morphometric and molecular analyses produce consistent results. acknowledgements the authors wish to thank the national council for scientific and technological development (conselho nacional de desenvolvimento científico e tecnológico cnpq) for the scholarships granted to ma nakano (ref. 132485/2008-7) and msc morini (ref. 302363/2012-2) and to thank the support fund for research and teaching (fundação de amparo à educação e pesquisafaep) for their financial support. ma nakano, rr silva vfo miranda, rm feitosa, msc morini – morphological differentiation of myrmelachista (formicidae) species326 references barr, c., van boven, j. & gotward, jr., w. h. 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(hymenoptera: apidae) have great importance for crop pollination (potts et al., 2016), and beekeeping allows the production of several bee products and provides pollination service to many crops (maderson & wynne-jones, 2016). beekeeping can be divided into two main types: stationary and migratory. the first one consists of keeping the apiary permanently in the same place. thus, colony production is limited by the supply of resources (nectar, pollen, resin, and water) determined by the local floristic potential (crane, 1999). the second modality is based on the displacement of colonies to specific places where blossoms are distributed throughout the year to maximize honey production and the pollination of crops (whynott, 1991). abstract migratory beekeeping can harm the bee colonies if not executed properly. here, colonies of apis mellifera were transported (for one or two hours) or not, following proper technical standards. to analyze gene expression (defensin-1, abaecin, and hsp70), forager bees were collected immediately, 24, and 72 hours after transportation. bee mortality and population growth were measured before and after transportation. this study concludes that transporting honey bee colonies for 2 hours promotes immune system gene expression, although there are no significant changes in bee mortality and population growth of the colonies. sociobiology an international journal on social insects maurice f. scaloppi2, samir m. kadri2, daniel d. mendes2, paulo eduardo m. ribolla3, ricardo o. orsi1 article history edited by evandro nascimento silva, uefs, brazil received 18 october 2021 initial acceptance 12 october 2022 final acceptance 03 december 2022 publication date 28 december 2022 keywords beekeeping, colony transportation, stress, transportation management, welfare. corresponding author ricardo de oliveira orsi departamento de produção animal e medicina veterinária preventiva faculdade de medicina veterinária e zootecnia da unesp, campus de botucatu rua prof. dr. walter maurício correa, s/nº cep: 18618-681 botucatu, são paulo, brasil. e-mail: ricardo.orsi@unesp.br however, colony transportation in beekeeping can be a stressor for the colonies, along with other factors such as pests, diseases, poor diet, and pesticide exposure (even et al., 2012; goulson et al., 2015). colonies that undergo migratory beekeeping for commercial pollination in the summer showed a reduced capacity to perform thermoregulation during winter (glenny et al., 2017). colony transportation can also affect hypopharyngeal gland development, impairing the ability of nurse bees to feed future generations (ahn et al., 2012). moreover, it can reduce worker bees’ life span, leading to increased levels of oxidative stress (simone-finstrom et al., 2016) and the prevalence and abundance of pests and diseases (zhu et al., 2014; alger et al., 2018). nevertheless, some procedures aim to minimize the stressful effects of colony transportation, such as the use of screened lids to promote ventilation; closing the hive 1departamento de produção animal e medicina veterinária preventiva, faculdade de medicina veterinária e zootecnia, universidade estadual paulista, botucatu-sp, brazil 2faculdade de medicina veterinária e zootecnia, universidade estadual paulista – unesp, botucatu-sp, brazil 3instituto de biotecnologia, faculdade de medicina veterinária e zootecnia, universidade estadual paulista – unesp, botucatu-sp, brazil research article bees colony transport affects the expression of some genes related to the apis mellifera l. immune system ricardo o. orsi et al. – honeybees colonies transportation and welfare2 entrance at dusk, when the entire population is inside the hive, avoiding its exit; and performing the transportation during the night, with milder temperatures, preventing the colony from overheating. meanwhile, studies showing the impact of colony transportation on bees individually or at the colony level still need to be made available. a possible effect of this management may occur on the bees’ immune system, affecting their ability to combat pathogens and pests (mason et al., 2013). the bees’ immune system is composed mainly of innate systems. the immune response against pathogenic organisms can be divided into two mechanisms that work together: cellular and humoral responses (nation, 2015; iwasaki & medzhitov, 2015). the humoral response consists of a battery of antimicrobial peptides that are synthesized in the presence of pathogenic organisms or situations of stress (yang & cox-foster, 2005; antúnez et al., 2009). honey bees have six antimicrobial peptides, including defensin-1 and abaecin. defensin-1 is a cysteine-rich cationic peptide, is the primary defense system of many organisms. it acts mainly against gram-positive bacteria, permeating its cytoplasmic membrane, and is up-regulated upon bacterial infection (evans, 2004; randolt et al., 2008). in turn, abaecin is a proline-rich peptide and has a broad spectrum of action against bacteria (casteels et al., 1990; lourenço et al., 2018). its expression is up-regulated very quickly in response to bacterial infection (richard et al., 2012; aronstein & saldivar, 2005). in addition to antimicrobial peptides, other proteins act in stressful situations. hexamerins are highly conserved molecules that act as molecular chaperones in biotic or abiotic stresses, such as overheating, the presence of toxic compounds and pathogens (sahebzadeh & lau, 2017). constituted by proteins of different molecular masses, including hsp70, they perform functions in cell differentiation and regulation and embryonic development (elsik et al., 2014). stress caused by colony transportation can have effects both on antimicrobial peptides and proteins that compose the bees’ immune system and also on bee mortality and the colony’s population growth. therefore, this work aims to evaluate the effects of honeybee colony transportation for different time lengths on immune system gene expression and bee mortality, and population growth of the colonies. material and methods colony preparation and transportation twenty-one honey bee colonies kept in standard langstroth nuclei were used, standardized in the number of brood and food frames. the colonies were submitted to three treatments of 7 colonies each: treatment 1 (t0), control, where the colonies were not transported; treatment 2 (t50), where the colonies were transported for 1 hour, which corresponded to about 50 km; treatment 3 (t100), where the colonies were transported for 2 hours, corresponding to approximately 100 km of distance. the colonies were transported during the night, once, on may 25th, 2018. for transportation, a screened transport lid was placed, allowing the hive to be ventilated. the entrance was closed with foam at the time of the transportation and the colonies were placed side by side in the back of a truck. the colonies left an apiary located at the geographical coordinates 22°49’14.9” s and 48°23’23.8” w at an average altitude of 502 m; circulated by local roads for the specified time lengths, and arrived at an apiary located at the geographical coordinates 22º50’28” s and 48º25’42” w and at an average altitude of 730 m, where the control colonies already were. the distance between the two sites was approximately 4.6 kilometers. the colonies were placed on individual stands, the entrances opened, and the transport lids were replaced by conventional lids. the region’s climate is characterized as cfa by the köppen classification (da cunha & martins, 2009). gene expression analysis for gene expression analysis, before transportation, a frame containing capped brood was removed from the colonies of each treatment, placed in a screened tissue, returned to the original colony, and kept inside the hive for 24 hours. then, approximately 100 newly emerged bees were marked on the dorsal part of the thorax (pronotum) with a non-toxic marker and reintroduced into their original colonies. after 22 days, the colonies were transported in their determined time lengths. at 0, 24, and 72 hours after transportation, 10 marked bees were collected from each colony to evaluate the effects of transportation as foragers. the same protocol was used for the control colonies, and the bees were collected in the same periods described above. the collected bees were immediately anesthetized on ice and subsequently frozen at -80 ºc. total rna was extracted from a pool of 5 heads (schlüns & crozier, 2007) following the methodology proposed by scharlaken and collaborators (2008) using 500 µl of trizol® reagent (life technologies, carlsbad, ca), according to the manufacturer’s instructions. the investigated genes were defensin-1, abaecin, and hsp70. the oligonucleotides sequences and details are described in table 1. the extraction product was visualized on a 1% agarose gel and quantified using a nanodrop device (spectrophotometer nd-1000). then, cdna synthesis was prepared with superscript® iii reverse transcriptase kit (invitrogen, carlsbad, ca), according to the manufacturer’s protocol. gene expression determination was performed by realtime polymerase chain reaction (rt-pcr) in triplicates, using the actin gene as an endogenous control (scharlaken et al., 2008). negative control was used for each reaction, consisting of a mixture of reagents and water. they were performed on abi 7500 fast device (applied biosystems, foster city, ca) using a sybr® green pcr master mix kit (applied sociobiology 69(4): e7522 (december, 2022) 3 biosystems, foster city, ca) under the following conditions: one cycle at 50 ºc for 2 minutes; one cycle at 94 ºc for 10 minutes, followed by 40 cycles of 94 ºc for 15 seconds and 60 ºc for 1 minute. the dissociation curve was obtained as follows: 95 °c for 15 seconds, 60 °c for 30 seconds, and 95 °c for 15 seconds. oligonucleotides efficiency (e) was calculated using a standard curve from four dilutions of the cdna samples: 1: 5, 1:25, 1:125, and 1:625, using the formula e = 10 (-1/slope). genes relative quantification (r) was determined by the ratio of the expression of the target gene to the endogenous gene, in which the cp (crossing point) is defined as the point at which the detected fluorescence is appreciably above the background fluorescence, according to the equation below (pfaffl, 2001): etarget being the efficiency of the target gene; eref the efficiency of the reference gene; δcptarget is the cp of the target gene of the control treatment cp of the target gene of the transported treatments; δcpref is the cp of the control treatment cp of the reference gene of the transported treatments. bee mortality evaluation to assess bee mortality after transportation, under basket-dead bee boxes (accorti et al., 1991) were installed in all experimental colonies. the boxes were positioned immediately after transportation and maintained for 7 days, with bee mortality being assessed daily and expressed in the number of dead bees per box. population growth evaluation for population growth assessment, open and closed broods of the central frame of each experimental colony were evaluated. the evaluation was carried out one week before transport, one day before transport, and four weeks after transport, according to the methodology adapted from al-tikrity and collaborators (1971). the pictures were positioned in a structure in which its sides are composed of a 2 x 2 cm grid. in this way, the two sides of the selected frame were photographed, and the areas (cm²) were measured using coreldraw® x8 (corel, ottawa, on). for the proposed assessment, the results were grouped into two periods: before and after transportation. statistical analysis the data obtained for population development and gene expression were first tested for normality (anderson-darling test) and homogeneous variances (levene’s test). when significant deviations (p < 0.05) from these assumptions were detected, the data were compared using the non-parametric mann-whitney test, and the median and interquartile intervals (q1_q3) were presented. when no significant deviations from normality or homoscedasticity were detected, the data were analyzed with one-way anova, and the mean ± standard deviation values were presented. p-values below 0.05 were considered significant. all statistical analyses were performed using the statistical software minitab. results the results of the relative expression analysis of defensin-1, abaecin, and hsp70 genes are shown in graphics 1a, 1b, and 1c, respectively. for the defensin-1 gene, upregulation was observed in treatment t50 right after transport, with a significant difference compared to the control and treatment t100. then, t50 showed down-regulation 24 hours after transport, returning to expression values similar to the beginning 72 hours after. for treatment t100, downregulation was observed 0, 24, and 72 hours after, with a significant difference, compared to the period immediately after transport. down-regulation observed 72 hours after transport in t100 also showed a significant difference compared to the treatment t50 in the same period (figure 1a). regarding abaecin gene, treatment t50 presents upregulation immediately after transport, with a significant difference compared to the control. then, there is downregulation 24 hours after transport, with a significant difference concerning the 0 hour period. finally, 72 hours after transport, there is a new up-regulation with a difference between the t100 treatment for the same period. in treatment t100, up-regulation is also observed right after transport, with a significant difference in relation to the control and the period 72 hours after, which shows down-regulation (figure 1b). gene number gene bank primer sequence 5’-3’ actin ab023025 tgccaacactgtcctttctg agaattgacccaccaatcca defensin-1 u15955 ctgcacctgttgaggatgaa gcgcaagcactgtcattaac abaecin af442147 cagcattcgcatacgtacca gaccaggaaacgttggaaa hsp70 martins et al., 2008 caaagttgtaagcgacggcggaa tgtctccggctgtggagcgca table 1. oligonucleotides initiating immune system genes (defensin-1, abaecin, and hsp70) and constituent (actin) of honeybees submitted to different transportation time lengths. ricardo o. orsi et al. – honeybees colonies transportation and welfare4 fig 1. relative expression of defensin-1 (a), abaecin (b) and hsp70 (c) genes in honey bees submitted to transport for 1 hour (t50) and 2 hours (t100) and collected at 0, 24 and 72 hours after. *represents difference in relation to the control; distinct letters represent differences between the means: upper case between the treatment t50; lower case between treatment t100. for the hsp70 gene, treatment t50 is down-regulated right after transport, with a significant difference concerning the control and the periods 24 and 72 hours after, which have up-regulation. in treatment t100, down-regulation is also observed right after transport, but with a significant difference only 72 hours after. in both treatments where transportation occurred, up-regulation observed 72 hours afterward presents a significant difference compared to the control (figure 1c). the results of bee mortality are shown in figure 2. there was no significant difference between treatments t50 and t100 compared to the control (p = 0.6149). the average mortality was obtained during seven evaluation days, observing a similar pattern between treatments. the results of population growth are shown in figure 3. for the closed brood area, a reduction in all treatments is observed, being significant only for the control (p = 0.0809 and p = 0.3938, for t50 and t100, respectively). evaluating sociobiology 69(4): e7522 (december, 2022) 5 the treatments among themselves in each of the bee collection periods, control colonies presented a closed brood area larger than treatment t100 before transport. after transport, the areas were not statistically different in any of the treatments (p = 0.4325). open brood areas did not show significant differences in any of the treatments before and after transport (p = 0.7634; p = 0.8999; p = 0.6009, for t0, t50, and t100, respectively). there was also no significant difference between these areas before (p = 0.1088) and after transport (p = 0.3181). as for the food areas (nectar, honey, and pollen), there was an increase in all treatments after transport, but there was no significant difference between treatments (p = 0.3195, p = 0.0733, before and after transport, respectively). however, there was a significant difference in the evaluation of treatments between t0 and t50 (p = 0.1671 for t2). fig 2. average daily mortality of honey bees not transported (t0) and submitted to transport for 1 hour (t50) and 2 hours (t100). fig 3. open brood and sealed brood (cm²) before and after transport for not transported (t0) and submitted to transport for 1 hour (t50) and 2 hours (t100). discussion the results obtained in the experimental conditions of this work show that colony transportation promotes defensin-1, abaecin, and hsp70 gene modulation. relative gene expression analysis of the antimicrobial peptides defensin-1 and abaecin are valid indicators of effects on the bees’ immune system (siede et al., 2012). colonies transported for 1 hour showed similar patterns in the expression of these two genes, with up-regulation right after transport, followed by down-regulation after 24 hours, and up-expression again 72 hours later, at a level close to the one observed in the collection made shortly after transportation. for colonies transported for two hours, the defensin-1 gene is downregulated in all evaluated periods and the abaecin gene 72 hours after transport. these results suggest that there is a suppressive effect on the bees’ immune system at the time lengths considered for ricardo o. orsi et al. – honeybees colonies transportation and welfare6 transport, with the 24 hours being the most vulnerable for colonies transported by one hour, where there is an immunosuppressive effect for both genes (casteels et al., 1990; lourenço et al., 2018). for colonies transported for two hours, this occurs more intensely 72 hours later, when there is down-regulation for the two antimicrobial peptides. however, this is also observed for defensin-1 24 hours after transport. although colony transport can be considered acute stress by lasting less than four hours (even et al., 2012), its effects lasted for at least 72 hours. thus, transport in both treatments caused up-regulation in antimicrobial peptide genes defensin-1 and abaecin, as well as down-regulation, indicating an immunosuppressive effect. immune system suppression can occur due to several factors, such as pesticides (mason et al., 2013; sánchezbayo et al., 2016), pests (navajas et al., 2008; chaimanee et al., 2012), and mechanical injuries (koleoglu et al., 2017). in turn, immunosuppression can promote several effects, such as greater susceptibility to diseases and pests (nazzi & pennacchio, 2014); down-regulation of vitellogenin and juvenile hormone genes, both related to labor differentiation of worker bees (bordier et al., 2017); and also, impairment of foraging activity, affecting pollen collection and consequently the colony’s nutritional balance (bordier et al., 2018). for the hsp70 gene, up-regulation was observed 24 and 72 hours after transport when performed for one and two hours, respectively. hexamerins are considered important storage proteins, acting as molecular chaperones in thermal stress situations, assisting in refolding proteins that denature with temperature increases and preventing their erroneous binding to other compounds (sahebzadeh & lau, 2017). thus, transport may have caused the expression of this gene related to the physiological regulation of body temperature variations. also, the period considered in the experiment was not enough to verify the necessary time to restore normal levels of hsp70 for the transported treatments. bee mortality and population growth analysis did not change in transported colonies compared to control colonies. for bee mortality, this result indicates that the method used to prepare the colonies for transportation minimizes a stressful effect, which is the death of bees and the consequent population decrease. finally, for population growth, despite a decrease in the control and t50 colonies for closed brood areas, there was no change in the colonies of the t100 treatment. this may also indicate a positive effect of the colony preparation method. however, as migratory beekeeping aims to move colonies to areas with a high abundance of floral resources, the stressful effect of this management can be masked by the increase in food availability (zhu et al., 2014). this can be seen by the increase in the area of stored food for both control and t50 treatments since the colonies were transported to the same apiary where the control colonies were placed. colony transportation for both pollination and honey production is a common practice in beekeeping worldwide. nevertheless, different transportation protocols are used by beekeepers to make it a fast and practical operation, only sometimes prioritizing the well-being of bees, which could promote stress in the colonies. for instance, transporting colonies during the day, when temperatures are higher, can promote an increase in the colony’s internal temperature, in addition to the loss of worker bees in the foraging stage if the colonies are not closed at night. likewise, transporting colonies with closed entrances helps to reduce the colonies’ population loss and, consequently, their population growth. in this work, colony transportation was carried out to minimize possible stressful effects on the colonies, using a screened lid for ventilation, closed entrance, and transport at night. thus, it can be inferred that when colonies are transported in an attempt to maintain the minimum conditions of well-being for the bees, the losses of this management can be reduced. however, some physiological changes can be observed. conclusion colony transportation for up to two hours promotes modulation of immune system gene expression, although there are no significant changes in bee mortality and population growth in the colonies. authors’ contribution mfs: conceptualization, methodology, investigation and formal analysis. smk: conceptualization, methodology, investigation, formal analysis and writing & editing. ddm: conceptualization, methodology and formal analysis. pemr: investigation and writing & editing. roo: conceptualization, methodology, investigation, formal analysis and writing & editing. funding this work was supported by coordenação de aperfeiçoamento de pessoal de nível superior brazil (capes), financial code 001. references accorti, m., luti, f. & tarducci, f. 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(2014). transportation and pollination service increase abundance and prevalence of nosema ceranae in honey bees (apis mellifera). journal of apicultural research, 53: 469-471. doi: 10.3896/ibra. 1.53.4.06 https://doi.org/10.14411/eje.2017.024 https://doi.org/10.1016/j.dci.2008.01.010 https://doi.org/10.1016/j.dci.2008.01.010 https://doi.org/10.1111/j.1365-2583.2007.00768.x https://doi.org/10.3896/ibra.1.51.4.05 https://doi.org/10.1038/srep32023 https://doi.org/10.1038/srep32023 https://doi.org/10.1073/pnas.0501860102 https://doi.org/10.3896/ibra.1.53.4.06 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i1.7715sociobiology 69(1): e7715 (march, 2022) 1. background it is an honor to be invited to present an account of my career as a biologist, and i thank the editors of sociobiology for this opportunity to do so. the following narrative will, i hope, give the reader an idea of the kinds of questions i like to ask and how i came to answer some of them. many of those remain only partially answered, others completely unanswered. i call attention to some of these. because this is more of a memoir than a topical review, i will limit citations largely to my own work and that of my students, with reference to other works where relevant; other sources will be found cited in our papers. to explain how i got here, let me to go back to my earliest days. my mother, ruth, was a part-time private music teacher, and my father, armand, was a self-trained electrical engineer working at bell labs in new york city on the development of telephone switchboard components. although we lived on suburban long island, new york, my family owned a modest summer home on 60 acres in southwestern new hampshire. we spent our summers there, starting when i was around three years old and lasting through high school. sociobiology an international journal on social insects robert l. jeanne article history edited by gilberto m. m. santos, uefs, brazil received 04 january 2022 initial acceptance 24 january 2022 final acceptance 25 january 2022 publication date 22 april 2022 keywords social wasps, ethology, evolution, colony cycle, research hypotheses corresponding author robert l. jeanne professor of entomology (emeritus) university of wisconsin madison, wisconsin, usa e-mail: rljeanne@wisc.edu my younger brother, richard, and i had the run of the fields, woods, ledges, and waterways. mom especially encouraged us to explore nature, make collections, ask questions. those summers connected us to the natural world and made us feel comfortable in the outdoors. spheksophobia was never a serious issue for us. in the tenth grade i had the good fortune to have an unusually engaging teacher of biology. harold turkel was then a graduate student in botany at harvard university who was teaching during the school year to support himself. he took us students on field trips to local natural areas and encouraged us to make collections, mainly of plants. his enthusiasm was contagious, so when in the fall of 1960 i enrolled as a freshman at denison university in granville, ohio, without hesitation i declared a major in biology. university of wisconsin, madison, wisconsin, usa review my life as a curious naturalist photo: dr. robert l. jeanne/publicity robert l. jeanne – my life as a curious naturalist2 during my college years i took courses in botany, comparative anatomy, ecology, and more (regrettably, i could never schedule entomology!). but the class that steered me toward my future career was bob haubrich’s course called plant and animal behavior. haubrich was another of those unusual teachers who inspire their students to see and think in new and different ways. plant behavior was his take on plant ecology, but what most excited me were the sections on animal behavior. i always enjoyed academics and got good grades, so there was never a doubt that i would continue on to graduate school. but first there was an interlude. in addition to meeting the requirements for the bs degree in biology at denison, i took several classes in scientific german under guy stern. i recall one semester when my weekly assignment was to read and translate an article appearing in die naturwissenschaften. back then most of the papers in that journal were in german. (the journal is now titled the science of nature and publishes exclusively in english.) stern tipped me off to the rotary foundation and its scholarships in international understanding, which were designed to enable recent college graduates to study in foreign countries. with the sponsorship of the local rotary club, i applied and was awarded a one-year fellowship to study at the justus liebig-universität in giessen, in what was then west germany. it was a horizon-broadening year. i attended lectures in literature, history, and even took a beginning class in russian, taught in german. the anchoring course in biology, though, was the zoologisches grosspraktikum, a year-long lab course taught in the old way of a taxonomic survey. we students spent many hours each week, first learning the zoological procedures of the fixing, staining, and mounting of preserved specimens, and the use of the microscope. the second semester was devoted to drawing in intricate detail the parts of preserved animals representing all the major phyla. i became quite good at this. while in germany i wrote to the university of california at berkeley and to harvard, expressing my interest in doing graduate work in behavior. a very positive response came back from donald griffin at harvard. griffin was well-known for his discovery of echolocation in bats. his kind of research matched my interests well, so i chose to go there. before i left germany, however, griffin wrote again to explain that he was leaving harvard to go to the rockefeller university in new york. but don’t worry, he said, there was an up-and-coming young professor i could work with, and that was e. o. wilson. i arrived at harvard in the fall of 1965 and quickly settled in with wilson as my advisor. his first advice to me was to choose a taxonomic group to specialize on. wilson, of course, worked with ants. by the end of my first semester i had narrowed my interest from the entirety of the animal kingdom down to the social insects. meanwhile, the requirement of a reading knowledge of two foreign languages was still in place at phd-granting institutions around the country, and wilson personally tested his grad students’ proficiencies. by the end of my year in giessen i was dreaming in german, so i chose to get that exam out of the way as soon as possible. for my test, wilson opened a book by karl von frisch and had me translate a page. then his telephone rang, and by the end of the 5-minute call i had the translation down pat: a pass. french was another matter. although my immigrant paternal grandmother, who lived with us as richard and i grew up, was a native french speaker, she taught us boys only two words: pomme de terre and moutarde. what to do? the massachusetts institute of technology, on the other side of cambridge, offered a class in french for science students, so i signed up. i did well on the midterm exam and took the result to wilson, who said, “if it’s good enough for mit, it’s good enough for me,” and passed me on french. these days my reading knowledge those two languages is rarely needed. for my work, portuguese, spanish, and the bit of italian i picked up later are more frequently called to duty. my russian never made it past the eins-oh-eins class i took in giessen. the next step was choosing a group of social insects to focus on. it was clear to me that the ants were getting plenty of research attention, as were the honey bees, so i chose the relatively little-studied social wasps. as a graduate student developing an interest in insect ethology, i delved into the research and writings of niko tinbergen and karl von frisch, among others. i was particularly inspired by tinbergen’s four questions about animal behavior: two ‘how’ questions (mechanism and development) and two ‘why’ questions (function/adaptation and phylogenetic history) (tinbergen, 1963). [the title of this memoir is a nod toward niko.] von frisch’s work on color vision in honey bees and on the function of the waggle dance were particularly inspiring examples of how well-designed experiments can quickly reveal how a given behavior works. john platt’s paper on the role of logic in designing good experiments was also influential (platt, 1964). (see fudge, 2014, for a 50-year retrospective.) throughout my career, i revisited platt from time to time and attribute my success in discovering publishable results in part to his case for the application of logic to developing hypotheses. in addition, i have always found it helpful to keep a journal in which i have conversations – and arguments – with myself about new ideas, recently published research, hypotheses i’ve come up with, and developing the logical pathways to experiments that can support or disprove them. not surprisingly, in recent decades my journaling has gone digital. i have often found that when i am struggling with developing hypotheses, designing an experiment, or solving some other biological problem, if i think about it before falling asleep at night my mind will have cleared itself of other things and an insight often pops into my head. lest it pop out again before morning, i routinely keep a pad and pen on my bedside table. in the summer of 1966, i took the organization for tropical studies course in tropical ecology in costa rica. it was my first introduction to the tropics. i became intrigued with tropical social wasps and did a small project on polistes sociobiology 69(1): e7715 (march, 2022) 3 at one of the research stations. that experience led to my setting my sights on tropical social wasps as a group to focus on. i selected the genus mischocyttarus because it was much less known biologically than polistes. 2. toward the phd back at harvard that fall i made plans to return to costa rica to do the field work for my phd dissertation. i began to learn spanish. but in the summer of 1967, i had a chance encounter with dr. paulo vanzolini, director of the zoology museum in são paulo, brazil, who at the time was visiting harvard’s museum of comparative zoology (vanzo had earned his phd in herpetology under e. e. williams there). he convinced me that i should really go to brazil instead. so, i changed my plans, hurriedly learned a bit of portuguese, and booked a flight to belém, pará. i still remember ed wilson’s words of advice as i prepared to head off for months of field work: “keep twenty irons in the fire,” that is, have multiple projects on your to-do list. some will dependably lead to results with routine data collection. these will be the meat of the dissertation. others are more of a longshot, but those that pay off could lead to significant discovery and a paper in a major journal. those are the gravy. in belém i took up residence in an apartment on the grounds of the museu paraense emilio goeldi. the museu occupies an entire city block and is enclosed by high walls. inside the walls are a botanic garden, a small zoo, and numerous laboratory buildings and museum collections. my plan was to study the behavior of the single most common species of mischocyttarus i could find nesting on the grounds. over the next few weeks i found numerous active nests on plants, rock walls, and buildings, and i began marking the wasps with paint spots for individual recognition, and recording their behavior on their nests. but my plan did not go well. many of the little colonies did not last long. the grounds crew seemed to be operating under instructions to remove any wildlife that might pose harm to visitors to the grounds, and that included wasps. so, after an unproductive few weeks i changed plans. i was able to arrange museum transportation to utinga, a nature reserve a few kilometers outside of belém. there i found several species of mischcocyttarus, along with a variety of epiponines. utinga afforded the protection and privacy i needed for the long-term survival of the colonies i was studying, but in the end, this did not work out either, because my rides to the preserve were only available a few days a week, and often a scheduled ride would not show up. as the 1967 christmas holidays approached, i took a break and travelled by bus from belém to brasília and then on to são paulo, where i visited vanzolini at the zoology museum. my most vivid memory of that time was the holiday party he hosted at his house. i remember being impressed with the diversity of the guests. they were not just colleagues from the museum, but musicians, performers, and television personalities, whom vanzolini knew through his avocation of writing and recording samba music. vanzo confided to me that he would never have been able to put up with a faculty position in the u.s. – too much pressure to publish, leaving no time to cultivate the rest of a life. i went on to spend new year’s eve in rio, where i joined local celebrants to cheer as 1968 washed up onto ipanema beach at midnight. i returned to belém determined to find a better location to conduct my research, a place that had undisturbed wasp colonies right outside my door. in february, 1968, i traveled up the amazon in search of such a site. my first stop was the field station at the reserva florestal adolfo ducke in manaus. i spent a few days there but found the social wasp population surprisingly depauperate. what i needed was a population of at least a few nests of a single species. what i found at the reserva was lots of species diversity, but often no more than a single colony of any one species. based on a suggestion from scientists at the reserve, i next took a river boat from manaus down the amazon to a logging camp on the rio curuá una, east of the city of santarém. my hosts were very accommodating, offering to provide me with free food and lodging should i decide to work there. but again, i found no wasp species common enough to meet my needs. as chance would have it, on the boat from manaus were some researchers from the museum in são paulo. they had been tracking down and visiting localities for some of the museum’s collections that had been made over the decades. one of these was fazenda taperinha, a plantation dating back to the 19th century that was abandoned after brazil outlawed slavery in 1888. in the early 20th century, gottfried hagmann, a newly minted phd zoologist from switzerland, bought the place. he raised his family there and over the next decades hosted numerous botanists and zoologists who collected extensively and deposited their specimens in museums, both in brazil and around the world. it was just upriver of the logging camp, so i hitched a ride on a barge loaded with logs heading for the mill in santarém. they dropped me off at santana, where i hired a boy and his canoe to paddle me up the rio ituquí to taperinha. at that time taperinha was owned and managed by donas erica and violeta hagmann, twin daughters of the late gottfried hagmann. it took me just a few days to recognize that this place was ideal for what i wanted to do. one of the most abundant social wasps was mischocyttarus drewseni, a large, dark species that builds fairly large nests hung from remarkably long petioles. its nests were most common on the two main buildings on the property, but also occurred on the low vegetation scattered about the extensive grounds surrounding the houses. i had found my dream field site! here i would do my phd research on the social biology of m. drewseni. when i indicated my desire to move my field work there, the sisters welcomed me most warmly, graciously inviting me to stay as long as i wished, with free room and board. robert l. jeanne – my life as a curious naturalist4 i returned to belém in march to tie up loose ends and prepare for the move. but first, at vanzo’s encouragement i traveled to ribeirão preto, são paulo state, where dr. warwick kerr invited me to stay with him and his wife while i visited his lab. i spent a couple of weeks there, looking in on all the research going on, mostly with bees at the time, and learning some of the techniques they were using. i have fond memories of my interactions with kerr, joão camargo, and ronaldo zucchi, among many others. on returning to belém, i packed up my belongings and arrived back at taperinha in may of 1968, to settle in for a long-term stay. i lived as a guest of the hagmann sisters for the next year, except for a two-month hiatus in 1968, when i returned home for the last days of my father, who was dying of cancer. the local m. drewseni population numbered 10-15 colonies at any one time. that was sufficient for my goal, which was a long-term intensive study of the social behavior on individual colonies. using paint-dot-coded numbers, i marked all the adults on each colony for individual recognition, then recorded their behavior over their lifetimes. close observation inevitably leads to questions, both tinbergen’s ‘how’ and ‘why.’ one of the most irresistible ‘why’ questions related to an odd and conspicuous rubbing behavior. several times per hour the founding female would move to the top of the comb, raise her gaster, and rub the ventral side of its terminal segment up and down against the length of that long nest petiole and around its attachment to the top of the comb. on puzzling over what the function of this behavior could be, i came up with several hypotheses, some highly speculative, even outlandishly improbable. one idea was that it applied a compound that acted as an epoxy to harden the labial gland secretion that was applied to the petiole by licking. when i fastened small bits of glass microscope slide covers to the petiole and let the wasps lick them, but not rub them with the gaster, the secretion nevertheless darkened and hardened to look just like the black material of the petiole, suggesting that no second compound was needed. meanwhile, i had noticed that small scouting-and-recruiting ants were everywhere and often approached the attachments of my nests to the substrate, but never made it down the petioles to the comb. this generated the hypothesis that the rubbing behavior served to apply an ant repellent to the petiole. the several experiments i did to test this idea provide strong support, which ultimately led to a paper in science (jeanne, 1970). gravy. this episode reinforced for me the value of experiments that could be done in the field. later, david post and i went on to show the same function for the behavior in temperate-zone polistes (post & jeanne, 1981). it has since been supported for the other three genera of independent-founding wasps. ed wilson’s initial letters to me while i was at taperinha were lengthy and full of encouragement and advice. as time passed, his letters got shorter and shorter, until the last one, which said essentially, “you have enough for a phd. come back.” i returned to harvard in may of 1969 to compile my results into a dissertation, which i successfully defended in late spring of 1970. the defense was a pleasant affair, ed wilson presiding. not long into it, frank carpenter, an esteemed and congenial member of my phd committee, sidetracked us into a discussion of insect photography, a special interest of his, and this took up most of the rest of the 45-minute meeting. i remember wondering by the end of the exam if any of my committee members had actually read my dissertation, as there were virtually no questions raised about it and little discussion of it. back in those times post-docs were not as all-butrequired for attaining a faculty position as they are today, so after completing my degree i accepted a one-year visiting professor position in biology at the university of virginia in charlottesville for the 1970-1971 academic year. while handling my teaching assignments there, i revised my thesis for publication (jeanne, 1972). from here on in this memoir i’ll organize things by topic, with apologies for demoting chronological order to second place. 3. regulation of the colony cycle one of the aspects of m. drewseni’s social life that caught my interest was the colony cycle and how it is regulated. in the relatively aseasonal environment at taperinha, colonies are initiated in all months of the year, but last for only 5-8 months from founding to decline. i wondered why colonies decline when the environment is still benign. the queen’s running out of sperm or eggs or becoming senescent were not the answer, because queens on my colonies were frequently superseded by younger females, some of them daughters of the foundress. it soon became clear that the regulation was imposed by the state of the whole colony itself. when the colony is young and the ratio of mouths to feed (nonworkers + males + larvae) to the rate of food supplied by workers is still low, an up-and-coming superseder will take over the colony, lay eggs, and the colony will continue to grow. later in the cycle, with the emergence of males and non-working females (gynes), the (nw+m+l)/worker ratio rises, increasing the demand on the food foragers. because the nw and m adults get first dibs on incoming food, the larvae increasingly go unfed. as the ratio of mouths to feed vs. supplies continues to increase, even the non-worker adults are not fully fed, and they begin eating the larvae. aspiring young dominant females have two options: (1) take over the egglaying role from the existing queen or (2) leave and initiate their own colonies. option (1) is not viable at this stage because of the high (nw+m+l)/worker ratio; any eggs they lay would be eaten. thus, females emerging at this time are increasingly likely to leave and start (or join) new colonies, dooming their natal colony to decline. whether this scenario applies to other tropical independent founders, or even to swarm founders, would sociobiology 69(1): e7715 (march, 2022) 5 make a nice comparative study. in temperate zones, the limited nesting season imposes the need for colonies to carefully time their production of gynes and males ahead of the end of the warm season. how they do this has not been fully worked out, neither for polistines nor vespines. 4. natural enemies and defense 4.a. arthropod predators i returned to taperinha several times in the 1970s and focused on other species that were abundant there. one of the phenomena i became curious about was the construction of multiple combs by colonies of polistes canadensis. the number of combs in a mature colony can exceed 30, closely clustered together, but not touching. most of the combs are small, averaging about 30 brood cells, and are used to rear just one generation of brood before the attending adults chew through the petiole and let the comb drop to the ground. emptied cells are rarely reused. my pursuit of why they do all this led to a likely answer: predation on the brood by the larvae of a small tineid moth, antipolistes anthracella, which appear to be attracted by the meconia in cells recently vacated by eclosing adults (jeanne, 1979). i found that in many of the combs the moth larvae had chewed their way into neighboring occupied cells, where they fed on the larvae or pupae there. rearing a second generation of brood in such an infested comb would be futile. cutting and dropping them to the ground ensures that ants will make quick work of the moth larvae. this work gave rise to several questions that i was unable to address. one is why p. canadensis colonies in particular are attractive to a. anthracella when mischocyttarus and other polistes species in the same habitat are not. another is whether the comb-cutting behavior is innate or is expressed only if moth larvae are detected in the cells. a third is whether the multiple combs reported for several polistes spp. at scattered locations from veracruz, mexico, to são paulo, brazil, is a response to the same moth, or to different sets of natural enemies. 4.b. ants working on m. drewseni’s defensive behavior impressed on me the importance of ants as natural enemies of social wasps, especially in the tropics. all social wasps rear their brood in open cells, subject to predation by ants. from my next faculty position (boston university, 1971-1976) i obtained u.s. national science foundation funding to measure the levels of ant-predation pressure at five localities from the equator to 40o north latitude. i used live wasp larvae as baits set out in both forest and field habitats and in a variety of microhabitats in each, then recorded the rates of survival of the defenseless baits in the face of ant predation. the results showed clearly that ant predation pressure was higher in the tropics and subtropics than in the temperate zone, and that the diversity of the ant species attracted to the baits increased markedly toward the equator (jeanne, 1979). although such ecological work has its own rewards, i found the 12/7 routine of checking and restocking baits and collecting ants mindnumbing. i would much prefer to sit in front of a colony of wasps observing their behavior, pondering how and why they do what they do, and dreaming up experiments to test my ideas. yet that 1979 study is one of the most frequently cited of my papers. but then, ecology is a bigger field than is the ethology of social wasps. in 1976 louise grenville bluhm and i married, and shortly thereafter we moved to madison, wisconsin, where i had accepted an assistant professorship in the department of entomology at the university of wisconsin. there, my graduate students and i went on to explore other aspects of defense in both tropical and temperate-zone social wasps. we pursued the chemistry of the ant repellent in polistes fuscatus far enough to identify methyl palmitate as one of the active components (post et al., 1984), work that later led to a u.s. patent for its potential as a commercial ant repellent. we also expanded our work on defensive behavior to other genera of social wasps, exploring what has turned out to be quite a diversity of mechanisms. ant predation in particular has selected for a variety of defenses in the social wasps. the independent-founding and the swarm-founding wasps have evolved different strategies of defense against scouting-and-recruiting ants. the former rely on repellent secretions applied to the petiole and upper parts of the comb, whereas the latter largely depend on their numerous workers for active detection and physical removal of ants that reach the nest. my graduate student karen london showed that the evolutionary loss of chemical defense against ants was associated with the evolution of swarm founding and not the evolution of covered nests (london & jeanne, 2000). army ants are a horse of a different color, able to overrun repellents, sticky traps, and even the most robust of active defenses mounted by species with some of the largest colonies. ed wilson referred to them as the huns and tartars of the insect world. wasps that nest in association with largecolony ants such as azteca spp. gain virtually complete protection against them. in the neotropics at least 35 polistine species in 10 genera have been recorded nesting in trees occupied and defended by azteca. most of these are probably commensals, gaining protection via their hosts but providing nothing in return. an exception appears to be the mutualistic relationship entered into by polybia rejecta, a notoriously aggressive wasp. it gains protection from army ants while providing protection to its host in the form of stinging attacks against ant-eating birds and mammals (jeanne, 2021). this wasp typically nests within a few centimeters of the azteca nest, close enough that it will attack birds and mammals that attempt to breach the ant nest. such close contact is in rejecta’s self-interest: if they nested elsewhere in the tree it would leave the azteca colony at risk of being weakened or killed by vertebrate predators, thereby exposing themselves to eciton predation. robert l. jeanne – my life as a curious naturalist6 interestingly, in the varzea (flooded-forest) habitat at taperinha p. rejecta nests in tight, intraspecific clusters of up to 23 in a single small tree and not in association with any territorially dominant ant species (jeanne, 1978). clearly, the azteca-rejecta mutualism has broken down there, but what’s missing? is it eciton, or azteca? or both? the varzea and the terra firme habitats can be viewed as two naturally occurring experimental treatments, each with a distinct array of wasp predators. the two environments could be exploited to unravel some of the defensive adaptations among social wasps, and perhaps discover some new ones. another mode of defense against eciton is nestentrance blocking. synoeca septentrionalis appears to be at least sometimes successful at this by deploying several workers to block their large entrances with their bodies. early on at taperinha my curiosity was aroused by chartergus artifex, which narrows the inter-comb passageways in its phragmocyttarous nest to the size of a single brood cell, so that access to the upper, brood-containing combs can potentially be blocked by a single worker (jeanne, 1991). the extremely tough outer nest carton likely prevents ants from chewing through it to gain access to the brood. the lower two or three combs have no brood cells; entrances to those combs, as well as the outer entrance, are much larger, suggesting that these chambers house a contingent of older workers that can quickly exit to defend against an attacking vertebrate. whether these wasps actually do block the upper passageways when ants enter the nest has never been documented, but with a bit of effort one or more nests could be carefully transplanted to an area subject to army ant raids. if the colony survives unscathed after a raid passes over its nest it would be circumstantial evidence that blocking was effective. adding ingenuity to the effort, chloroform could be injected into a nest during an army ant raid via preplaced hypodermic needles in the lower chamber (accessible to ants via the large nest entrance) and each of the next 4-5 chambers above it. subsequent dissection of the nest would show whether ants were able to get through the narrower inner passageways and the chloroform may even freeze in place the wasp blocking the access to the broodfilled combs. with advanced technology, it may be possible to record the actual behavior during a raid via a small video camera and light placed in the lower chamber and aimed at the narrow passageway to the chamber above. learning plays a role in response to some ants. in polybia occidentalis, for example, while recognition of the odor of eciton appears to be innate, recognition of the odor of scouting-and-recruiting ants is learned upon encountering them when they attempt to invade the nest (london & jeanne, 2005). because the nest is often exposed to just one or two ant species occupying the shrub bearing the nest, learning their odors may enhance the speed of mounting a defense against repeated attempts to break through the wasps’ defense. there is still much to be discovered about learned vs. innate recognition of the natural enemies faced by wasp species. 4.c. parasitoids the evolution of the nest envelope appears to have been effective in reducing the exposure of the brood not only to ants but also to parasitoids. the brood in the covered combs of the swarm-founding wasps are subject to parasitism by fewer than 25% as many species as are the independent founders, and the levels of infestation are much lower (jeanne, 2021). karen london showed experimentally that the envelope of polybia occidentalis significantly reduces access to the brood by phorid flies, a potentially serious natural enemy (london & jeanne, 1998). the parasitoids that do get in, such as mantispids, often have evolved indirect and intricately stealthy means of getting their propagules in past the narrow, guarded nest entrance (and getting their progeny out). these behaviors are challenging to study and have been little investigated. 4.d. vertebrates the first line of defense against vertebrate predators is of course the sting. the intensity of a stinging defense mounted by a colony responding to an attack on its nest varies tremendously across species and mostly correlates with species-typical colony size. but it also varies widely within species, roughly increasing with the colony’s investment in the brood. while working with swarm emigration in polybia sericea at taperinha, i frequently forced active colonies to form absconding swarms by methodically dismantling their nests, comb by comb, over several minutes. each colony pugnaciously defended its nest until all but the last one or two combs had been removed. then, almost as if a switch had been flipped, the aggressiveness dramatically disappeared. somehow, within seconds, the adult population had sensed that the remaining brood were no longer worth defending. but how does a colony know that its brood population has been lost and how does the information spread so quickly to and among the workers? karen london directly addressed the relationship between brood biomass and defense in p. occidentalis. she provoked newly founded colonies p. occidentalis to attack an artificial target in response to both mechanical and chemical (alarm pheromone) stimuli. her results were clear: as the colonies’ brood biomass grew over the 30-day pre-emergence period, the intensity of their defense increased linearly, in spite of the strong decline in numbers of workers due to postfounding attrition (london & jeanne, 2003). thus, the degree of a colony’s defensive response is closely correlated with its investment in brood in the nest. these results mean that a colony knows not only whether it has brood or not, it knows the current size of its investment in brood. it has recently been shown that honey bees sense the size of their worker population via the density of workers in the hive (smith et al., 2017), but the wasp case sociobiology 69(1): e7715 (march, 2022) 7 must involve some kind of cue or signal passed from the brood to the adults. identifying what this is poses a fascinating challenge that we were unable to pursue. a volatile chemical emitted by the brood, a kind of “defend-me pheromone,” is one possibility, but there are others. perhaps only one stage of brood – larvae or pupae – produces the signal. generating alternative hypotheses and designing clever experiments to disprove all but one would be a worthy phd topic. the nest itself may play a major role in defense against vertebrates. just in the genus polybia, for example, the species that build mud nests appear to rely on the hard structure as a first line of defense. rather than mounting a stinging attack in the face of a mechanical disturbance, p. emaciata adults retreat inside, coming out to attack only after sustained disturbance. in contrast, p. occidentalis, which builds a flimsier carton nest, always responds to the slightest disturbance with an immediate stinging attack (o’donnell & jeanne, 2002). we still have little idea of the range of vertebrate predators these species face and how differences may influence selection for defensive strategies. perhaps p. emaciata, which builds its ceramic-like mud nests on thin twigs and lianas, is more exposed to predation by bats (jeanne, 1970) or small, nocturnally foraging rodents, against which a mud nest may be an effective defense. in contrast to the many solitary wasps that build with mud, the road to eusociality in the wasps was paved with paper. paper may initially have been an adaptation that met the need for a light and flexible nesting material that allowed the ancestor on the threshold of sociality to remove its brood comb from direct contact with the substrate by suspending it on a narrow petiole that could be defended chemically against ants. the reversion to mud, which apparently occurred twice within the genus polybia, was an option available only to the lineages that lost that petiolar comb support and evolved an attachment to the substrate broad enough to support greater weight, i.e., astelocyttarous and phragmocyttarous nests. once these architectures had evolved, a reversion to the use of mud may have been a relatively easy evolutionary step. in that regard, i once amused myself by offering small pellets of mud to p. occidentalis builders engaged in expanding their nest. these were accepted without hesitation and were worked into the otherwise carton nest structure. although mud is probably cheaper to collect than wood pulp, it may require more oral section to bind it together than does wood fiber. this should be testable as part of a cost-benefit analysis of nest architectures. i’ll return to the economics of nest design again in section 9 below. a possible example of a wasp that has evolved a way to filter its vertebrate predators is parachartergus colobopterus. the color of its carton matches that of the tree trunks on which it nests, and the shallow dome of its envelope eliminates most shadows, reducing its profile for birds or bats, even from a short distance. this may narrow the range of the vertebrate predators they face to small insect-gleaning birds or mammals that forage on tree trunks. as in p. emaciata, when the colony is disturbed most of the adults rush inside, while those on the outside respond to large approaching objects not by stinging, but by spraying a mist of venom at them (jeanne & keeping, 1995). a spritz of sticky, irritating venom in the eyes may be a more effective defense against small birds and rodents than would be one or two stings. a take-home lesson from this is that the sting’s not always the thing. clypearia weyrauchi has apparently independently evolved a similarly cryptic nest, but its defensive behavior has not been investigated (jeanne, 1979). as described above, nesting in association with other species is a common defensive adaptation of both independent – and swarm-founding wasps. mischocyttarus rufidens (=immarginatus) often nests in close association with polybia occidentalis and other swarm founders, thereby presumably gaining some protection from bird predation. we showed that founding females of m. rufidens nest close to their host’s nest, but not so close that the nest can be reached and the brood eaten by the occidentalis workers (london & jeanne, 1997). the threshold distance is about 18mm. the fact that the rufidens nests were not always built distally on the same twig as the occidentalis nests suggests that the association is maintained by vertebrate predation, not by the defense against ants provided by occidentalis. most predation on adult wasps takes place away from the nest, but the array of predators they face has been little investigated. the flying social hymenoptera – bees and wasps – have more protein (in their flight muscles) and lipids in their bodies than do worker ants, and so may be subject to predation by a wider range of predators. large arthropod predators such as mantids lucky enough to find a p. occidentalis nest can station themselves a few centimeters away from it and spend days snatching slow-flying, coming-and-going foragers with relative impunity (personal observation). on the other hand, dragonflies appear to avoid taking larger wasps, perhaps due to the risk of being stung in the neck while grasping them with their legs (jeanne, 1972). müllerian mimicry is common among tropical social wasps and must have evolved primarily in response to predation on foragers away from the nest. it appears to me that virtually every species of mischocyttarus is a mimic of one or another sympatric epiponine wasp, but to my knowledge this has not been the subject of a systematic investigation. selection for mimicry may come from visually hunting arthropods such as mantids, dragonflies, and jumping spiders as well as from vertebrates (o’donnell, 1996). any such study would need to bear in mind that the visible spectra of birds and insects differ from ours. we know next to nothing about how nest architecture, nesting site, colony size, and other features of social wasps filter the dizzying array of predators they face. much of the variation in nest-site selection, ferociousness of defense, body size, nest crypsis, and more may best be understood by the set of predators each species is exposed to, but has been littleexplored in any rigorous, systematic way. it remains fertile ground for exploration and experimentation. robert l. jeanne – my life as a curious naturalist8 5. communication 5.a. swarm emigration my last field trip to taperinha was in 1980. obtaining the necessary research visas and collecting permits from the brazilian authorities had become so arduous and unreliable by then that i moved my field work to costa rica, starting in 1982. i found an ideal field site in centro ecológico la pacífica (later las pumas) in the northwestern province of guanacaste. at the time, the property was a resort, restaurant, cattle ranch, and animal rescue facility owned and operated by werner and lilly hagnauer. the property was ideal habitat for polybia occidentalis, which nested in abundance in the low trees dotting the pastures and along the hedgerows, so i focused much of my tropical work on that species. i began with a study of communication. back in 1973, while working on the ant-predation project at the estación de biologia los tuxtlas in veracruz, mexico, i had observed a large swarm of agelaia areata on the move to a new nest site. i saw that scouts conspicuously dragged their gasters on projecting vegetation along the emigration route. shortly thereafter marty naumann (1975) published his own observations of similar behavior in emigrating swarms of several other species, and speculated that its function was to deposit scent marks that guide the swarm along the route selected by scouts. a few years later, in a field experiment with polybia sericea at taperinha, i obtained experimental evidence strongly supporting the chemical trail hypothesis (jeanne, 1981). in most species it is sternal glands that produce the trail pheromone. yet some species lack these glands, raising the question of how they guide their swarms to new nest sites. experimental investigation of the function of this gland in other carefully targeted species may well turn up surprising results. chemical analysis of the products of these glands needs to be followed by bioassays done in the field to determine the active components. working with p. occidentalis in costa rica, peter sonnentag and i showed that both chemical and mechanical signals are involved in the coordination of swarm emigration. this species uses a scent-marked trail to lead the swarm to the new site. by closely observing behavior on the swarm clusters, we became proficient enough at reading early cues of the direction of the consensus nest site the scouts were converging on to locate it long before the swarm arrived. but we were primarily curious about how the scouts inform their nestmates back in the swarm that a consensus on a new site had been reached and that they should take flight and follow the chemical trail. we used videorecording of behavior at swarm clusters of p. occidentalis to show that returning scouts move about on the swarm surface, bumping into inactive wasps in the swarm. as the rate of bumping behavior increases, it becomes contagious, with frequently bumped swarm-mates now themselves becoming active in spreading the behavior, culminating in the swarm members taking off over several minutes and following the scout-laid chemical trail to the new site (sonnentag & jeanne, 2009). we obtained information about each individual’s behavior preceding her flight toward the new site by playing our videotapes backwards from her take-off. using existing technology in innovative ways can be just as informative as applying new technology to old research problems. do all species of swarm-founding wasps chemically mark their emigration routes and initiate swarm emigration in the same manner? we know that apoica, at least, is an exception. we found that emigrating swarms use a calling display while in flight to guide the swarm to the new nest site (howard et al., 2002). at least two hypotheses come to mind regarding how the new site is decided on. one is that in the hours before lift-off, scouts settle on a single site, possibly marking it chemically, then guide the swarm there while keeping the wasps together in-flight via the calling pheromone. the other is that the swarm lifts off before a site has been chosen, then lands on arbitrarily selected sites a short distance away. cleverly designed observations and/or experiments could decide between these two hypotheses – or disprove both in favor of a third i haven’t thought of. 5.b. alarm recruitment alarm recruitment is another context in which pheromones play a central role. at the beginning of the 1980s alarm pheromones had been demonstrated only in vespines and had been reported to be absent in the one polistes species that had been tested. in 1980, in one of the last investigations i did at taperinha, i showed experimentally that the venom of polybia occidentalis contains an alarm pheromone (jeanne, 1981). i went on to show the same for polistes canadensis (jeanne, 1982), the first for the genus. graduate students david post and holly downing later demonstrated the existence of alarm pheromones in two north american species (post et al., 1984). in a later collaboration with a team of biochemists we identified spiroacetal (e,e)-2,8-dimethyl1,7-dioxaspiro[5.5] undecane as the active component in the venom of p. occidentalis and p. sericea (dani et al., 2000). to my knowledge, it still has not been experimentally demonstrated that release of venom is contagious, i.e., that release by one worker elicits release by others in a chain reaction, although i provided circumstantial evidence for it in p. occidentalis (landolt et al., 1998). in separate work, david post showed that the venom of two species of polistes also contains a sex pheromone that attracts males and stimulates sexual behavior (post & jeanne, 1983, 1984, 1985). in some epiponine species non-pheromonal signals appear to play a role. in several species, workers in an alarmed colony move onto the envelope, or even onto the intruder, and display their flexed gasters vertically, a behavior we called gaster flagging (o’donnell et al., 1997). in some species, the whole gaster is more conspicuously colored sociobiology 69(1): e7715 (march, 2022) 9 than the rest of the body (in others it is just the ventrum), suggesting that the behavior has a visual-signaling function. how this adaptation functions have not been worked out. in other species mechanical signals are used to communicate alarm (see section 5.e.). in sum, the evidence for releasers of alarm behavior in wasps include chemical, mechanical, and possibly visual signals, and ranges from being experimentally demonstrated in a few species to speculation that needs experimental follow-up. 5.c. foraging and recruitment to food sex pheromones were likely among the earliest chemical signals to evolve, no doubt long pre-dating the evolution of eusocial behavior. after sociality evolved, alarm recruitment via a chemical releaser likely soon followed. recruitment to food sources, on the other hand, is much rarer among social species. nevertheless, some form of trail-laying to food sources has evolved in termites, ants, and bees. laying a chemical trail is not hard if you’re a cursorial insect, but for flying hymenoptera it’s a different story. nevertheless, the apparent failure of social vespids to evolve a similar adaptation is puzzling, given the very large colony sizes attained by some epiponine species, and that the exploitation and defense of point food sources such as carrion, utilized by a number of species, ought to favor some sort of efficient recruitment ability. some species of stingless bees have evolved chemical food-recruitment trails, and it doesn’t seem much of a stretch to envision that the emigration-trail scent marking that is widespread in the epiponines could be commandeered for use in recruitment to food, especially in species with very large colonies. yet so far, we have come up empty. monica raveret richter, a masters student with me, showed that foragers of polybia sericea use visual and chemical cues to locate prey, but she found no evidence that they recruit nestmates to food sources (raveret richter & jeanne, 1985). later, malcolm keeping, jim hunt, and i tested for recruitment to carrion in agelaia multipicta and a. hamiltoni in venezuela and peru and found no evidence for it (jeanne et al., 1995). but absence of evidence is not evidence of absence. we later pursued the question with species more accessible to us. several species of yellowjacket (dolichovespula and vespula) are common in southern wisconsin. stephanie overmyer, a masters student in my lab, showed for v. germanica that foragers do not scent-mark rich food sources, but that naïve nestmates learn the odor of a rich food brought in by others and will leave the nest in search of its source (overmyer & jeanne, 1998). jennifer jandt, a later masters student, confirmed this and found no evidence that a behavioral signal is produced by successful foragers when they return to the nest (jandt et al., 2005). what she did find is a cue-based recruitment system in which the odor of the food brought into the nest is alone enough to recruit others to leave the nest and search for the source of the odor (jandt & jeanne, 2005). ben taylor, a phd student, subsequently showed that v. germanica foragers search preferentially for the richer of two food resources they experience in the nest (taylor et al., 2010). that is, resource quality matters (taylor et al., 2012). he later showed that the rate of rhythmic gastral drumming by workers on the comb (1) increases when colonies experience an increase in the inflow of food and (2) causes increased rates of movement, trophallaxis, and departures from the nest. we concluded that gastral drumming is a nest-based recruitment signal, the first reported for a social wasp (taylor & jeanne, 2018). in a series of studies in costa rica, taylor and teresa schueller, also a phd student, tested whether polybia occidentalis has a similar nest-based food-recruitment system, and found that just as in vespula, naïve foragers in the nest depart and hunt for the source of the scented resource they experienced in the nest (schueller et al., 2010, schueller & jeanne, 2012, taylor et al., 2012) and they also do not scent-mark food resources (taylor et al., 2011). all these species thus appear to lack off-nest signals that can guide nestmates to a rich food source, such as traillaying, direction and/or distance-indicating dances, or even scent-marking the food source. but it would be premature to conclude from the few species investigated so far that all eusocial wasps lack off-nest food-recruitment signals. p. occidentalis and v. germanica form colonies of a just few hundred to a few thousand, at the low-to-medium range of the spectrum of colony sizes in the epiponines. candidate species abound. one is certainly agelaia vicina, with its huge colonies, but another is pseudopolybia difficilis, which has an outsized richards’s gland (jeanne et al., 1983), a tantalizing clue suggesting that it may be used to recruit to food. such species have been waving their hands for years, in effect shouting, “test me, test me!” neither species occurs at my field site in costa rica, so the opportunity never arose. 5.d. exocrine gland morphology our work with allomones and pheromones, particularly those produced by sternal glands on the gaster, led us to look into their morphology and their distribution among the social wasps. in the 1980s david post, holly downing, and i published several studies of the gland histology and associated surface specializations in males and females of a number of polistine species (jeanne & post, 1982, downing & jeanne, 1983, jeanne et al., 1983, post & jeanne, 1983, downing et al., 1985). in recent years our work in this area has been expanded and improved on by others. 5.e. vibrational signaling as prevalent as pheromones are, mechanical signals may be equally widespread. i became aware of vibrational robert l. jeanne – my life as a curious naturalist10 signaling early on in my field work, long before ben taylor’s discovery of the signal function of gastral drumming in vespula germanica, described above. in my thesis work i described gastral drumming behavior in m. drewseni (i called it abdominal vibration) (jeanne, 1972). females drum their gasters dorso-ventrally against the nest for a fraction of a second before entering each cell to feed liquids to the resident larva. another vibratory behavior that sometimes follows a round of feeding is what i called pecking. a female, usually the queen, would insert her head partway into a cell, then rapidly and vigorously shake her whole body forward and back longitudinally, her head contacting the cell to produce an audible rattle. at the time i had no idea of its function. several authors in the first half of the 20th century working with polistes spp. and belonogaster junceus described various forms of vibrational behavior performed while visiting larvae. some reported that it occurred in the context of feeding liquid to larvae, while others concluded that it stimulated larvae to release their salivary secretion. in most polistes species the drumming is produced by trilling the antennae on the rim of a brood cell, what we called antennal drumming. michel pratte and i were the first to describe in detail the form and context of antennal drumming in polistes (pratte & jeanne, 1984). we showed that it is performed by the queen in the context of regurgitating prey liquid (imbibed while masticating prey) to the larvae and concluded that its function is to signal the larva that it is about to be fed liquid and that it should withhold the release of its salivary secretion, which is attractive to the adults. this conclusion stood for 25 years. until we disproved it. i began to doubt the function pratte and i had assigned to antennal drumming when it was reported for polistes dominula that vibratory behavior is performed in the same context, but only during the first half or so of the colony cycle (brillet et al., 1999). in a 2009 book chapter i laid out additional reasons why the data don’t support our salivary-inhibition idea, and to hypothesize instead that antennal drumming functions as a mechanical switch to bias the development of larvae into worker-like adults (jeanne, 2009). the default assumption at the time was that developmental differences in social wasps are solely nutritionally based. my phd student sainath suryanarayanan obtained experimental support for a role of drumming (jeanne & suryanarayanan, 2011, suryanarayanan et al., 2011). later, in a collaboration with amy toth at iowa state we showed that both vibrational signals and nourishment levels experienced by developing larvae influence the expression of genes that modulate the molecular pathways related to diapause and metabolism (jandt et al., 2017). this taught me a lesson about the need for open-mindedness and creativity when generating hypotheses – john platt again. when michel pratte and i did our study in the 1980s, it simply never occurred to us that antennal drumming could have an effect on larval development. in retrospect, though, we can take some consolation from the fact that it hadn’t occurred to anyone else, either. on the plus side, i took a definite pleasure in overturning my own hypothesis, even if it took 25 years to do so. it was a good example of karl popper’s dictum that science advances by disproof of hypotheses. several questions around this phenomenon remain unanswered. we speculated that the development-modulating drumming directed at the larvae may not be the only casteinfluencing mechanical signals employed among the independent-founding wasps. do the physical dominance attacks, lateral vibration, and abdominal wagging that are so widespread among the independent-founding wasps have a similar effect on gene expression that in turn influences the reproductive physiology and behavior of adult females? a study correlating level of dominance received with gene expression across individuals could answer it. although our 2017 study showed that nourishment levels also play a role, our post-emergence treatment colonies were prevented from foraging for half the day for up to five days per week, imposing what is probably an unnaturally low food supply. still to be determined is the relative importance of vibrational vs. nutritional inputs during the pre-emergence period. with regard to the more advanced eusocial wasps, do the vespines and the epiponines employ analogous but perhaps more subtle mechanical signals that similarly influence caste development? or have pheromonal signals completely replaced mechanical ones? mechanical signals are used in contexts other than caste development. our investigations of parachartergus colobopterus showed that the species lacks an alarm pheromone, and instead workers signal alarm by drumming the tips of their gasters on the nest carton (jeanne & keeping, 1995). in this species not only has the venom lost its dual function of alarm pheromone and toxin to become a sticky eye irritant (described above), but the sting apparatus has evolved from a hypodermic injector into an atomizer. these changes very well may have necessitated the evolutionary change from a chemical alarm signal in the venom to a mechanical one. such reports are suggestive of the diversity that likely exists among the 245-odd species of epiponini. there are two takeaways from this. one is that mechanical signals are often overlooked; not all produce sound as a byproduct. the second is that in many cases these signals may not function in ways we usually think of, that is, as releaser stimuli that elicit an immediate behavioral response, but as primers that trigger physiological changes via changes in gene expression that modify future responses to environmental stimuli or even influence developmental pathways. the science of biotremology as applied to wasps and other social insects has a wide-open future. 6. organization of work my interest in this topic began after i moved my field work from taperinha to costa rica and began working on sociobiology 69(1): e7715 (march, 2022) 11 polybia occidentalis. i became fascinated with how these colonies organize the task of nest construction. the operation involves a cooperative interaction among three groups of specializing workers: water foragers, pulp foragers, and builders. water foragers fill their crops at a water source, then distribute their loads to pulp foragers and builders back at the nest. pulp foragers use the water to moisten the wood source as they scrape together a pulp wad. back at the nest, they distribute it to builders, who add it to the nest. i coined the term ‘task partitioning’ to describe the subdivision of resource handling into the foraging of materials in the field vs. processing them at the nest. i showed that organizing work in this way–series-parallel (oster & wilson, 1978) – has costs as well as benefits. the main cost arises from the need for foragers to transfer their loads to willing receivers, the builders. this can sometimes take many seconds before the forager is relieved of the last of her load, freeing her up to make her next trip. likewise, builders face periods of idleness as they wait for the next load of water or pulp to arrive. a major benefit of this form of task partitioning is that it allows temporal polyethism, or age specialization. because the riskier task of foraging is performed by older workers, colonies gain a demographic advantage through an increase in the workers’ mean lifespan, thus maximizing the lifetime contribution to the colony made by the average worker (jeanne, 1986, tofilski, 2002). individuals make the switch from nest work to foraging suddenly and with little overlap between old and new tasks (jeanne et al., 1988, jeanne, 1991). the age at which workers switch from nest work to foraging ranges from 5 to 36 days of age. part of this variability is explained by worker body size–smaller workers switch at older ages (o’donnell & jeanne, 1995) – and may also be influenced by lipid content (o’donnell & jeanne, 1995). we showed that the stinging response to a vertebrate threat, another risky task, is also performed by older workers (older than 10 days) (jeanne et al., 1992). methoprene, an analog of juvenile hormone (jh), when applied topically to young workers, accelerates their transition to foraging, suggesting that age polyethism is under jh control (o’donnell & jeanne, 1993). phd student cristie roberson did a detailed study of age polyethism in vespula germanica and found that it is extremely weak compared to p. occidentalis, despite the fact that mature colony sizes are similar in the two species (hurd et al., 2007). this species difference still begs a coherent explanation. another advantage of task partitioning accrues to the foragers, who can collect and carry loads much larger than they would be able to build with on the nest. this in turn means fewer foraging trips and less foraging time per unit of material. i found that the benefit:cost ratio was higher in larger colonies, where the queuing times are reduced to a few seconds. by quantifying the number of worker-minutes it takes to collect and add one unit of nest material to the nest, i was able to show that large colonies are indeed much more efficient – in terms of worker-minutes – at nest construction than are small ones (jeanne, 1986). my phd student sean o’donnell later showed that forager success in this species increases with experience, another benefit of specializing, since fewer individuals need to learn the location of nearby and reliable resources (o’donnell & jeanne, 1992). but is the series-parallel (sp) system of organizing work seen in p. occidentalis actually more efficient than the parallel-series (ps) system used by the independentfounders? the latter employ little or no task partitioning when it comes to nest construction. the queen or a worker leaves the nest, flies to a place to collect water and from there to a pulp source, where she collects a small pulp load. she then returns and adds her load to the nest. which system – sp or ps – is more efficient in terms of worker-minutes required to accomplish the same amount of work? table 1 breaks down the components of input, in seconds, required by each of the three specialists in the sp system of p. occidentalis to process an average pulp-forager load (0.66mg dry wt.), resulting in a total of 1,205.6 worker-seconds. a actor b seconds per task cycle c multiplier (acts/mean pulp load) d seconds per sp unit of pulp (b x c) pulp forager 223.2 0 223.2 water forager 77.5 1.35 104.6 builder 143.9 6.1 877.8 total 1,205.6 table 1. series-parallel (sp) system of nest construction. for each actor (column a), the time required per task cycle (column b) is multiplied by the number of cycles (column c) of that task required to process one standard load brought in by a pulp forager. the result is the number of seconds required by that actor to service the standard load (column d). times are in seconds and include times in queue on the nest. data from jeanne (1986, table 2, large colonies). if p. occidentalis were instead to operate via the ps system of polistes and mischocyttarus, each worker would perform all three tasks in sequence, flying first to a water source, then a pulp source, then back to the nest to apply its load. we can again use the values i reported (jeanne, 1986), with the following estimated (and admittedly crude) adjustments (table 2). the size of the pulp loads the worker collects is limited by what she can build with back at the nest: 1/6.1 = 0.164 of an average p. occidentalis (sp) load. likewise, she need imbibe and carry only the amount of water required to collect and build with that reduced load: 0.164, multiplied by 1.35 again, to yield 0.22 of a normal water load. off-nest time for water foragers averaged 42.9 seconds (jeanne, 1986, table 2). if we assume that 10 seconds of that is required to fly to and from the source and start to collect, then 32.9 seconds is spent on water uptake. since the ps worker needs to collect only 0.22 of an sp water specialist’s load, she takes 32.9 x 0.22 = 7.2 seconds to obtain what she needs. she then flies to robert l. jeanne – my life as a curious naturalist12 a pulp source and collects 0.164 of a normal sp load. again subtracting 10 seconds flying/searching time to and from the pulp source from the 188.8 seconds mean off-nest time for pulp foragers leaves 178.8 for collecting the load. again, the ps worker needs to collect only 0.164 of a load, so she spends 178.8 x 0.164 = 29.3 seconds collecting her wad and returns to the nest. upon arrival, she builds right away; there is no transfer delay. builders spend a mean of 61.9 seconds adding a load to the nest, which is the same for both systems, since builder-load sizes are the same. so, the total time per cycle of the ps system is 7.2 + 29.3 + 61.9 = 98.4 seconds. adding back in 5 seconds for each of the three flights on the circuit gives 113.4 seconds. multiplying this by 6.1 to get us back up to the full-sized sp pulp load yields 691.7 seconds (table 2). this is much less than the 1,205.6 seconds required for the sp system because it omits the lengthy queuing times experienced by the latter. we can conclude that the ps system of the independent founders is more efficient in terms of worker-minutes than is the sp system. if the ps system requires much less time to add a unit of pulp to the nest, what is the adaptive advantage of evolving the more time-costly sp system that appears to characterize most, if not all, of the epiponines? the answer may be that energy is the more relevant currency than is time (jeanne, 1986). since flight is the most energetically costly activity for social wasps, the number of foraging trips can be used as a reasonable proxy for energy expenditure. again taking the mean dry weight of pulp collected by a polybia occidentalis pulp forager (0.66mg) as a standard unit for comparison, by the sp system of p. occidentalis, each foraged load of pulp requires 1 trip by a pulp forager and 1.35 trips by water foragers, for a total of 2.35. the ps system, in contrast, requires 6.1 trips, or 2.6 times as many as the sp system p. occidentalis actually uses, and potentially 2.6 times as much energy. there is also the demographic advantage of the sp system of p. occidentalis. in addition to being costly, foraging is the riskiest activity social wasps engage in. the 2.6-fold greater number of foraging trips required by the ps system exposes them to nearly a 2.6-fold increase in risk (slightly less because their trips take less time if their water and pulp sources are in the same direction from the nest). moreover, because there is no task partitioning, younger workers on average will be engaging in the risky task of foraging, and as explained above this will decrease workers’ mean lifespan, imposing a demographic cost on the colony. these arguments suggest that the reason taskpartitioning, age polyethism, and the sp system of organizing work are adaptive is not a saving in time – there is a loss – but because they confer both energetic and demographic advantages. whether this hypothesis holds up will require quantitative measurements of demographic and energetic costs. among the unanswered questions are (1) why have the independent-founding wasps, especially the vespines, largely failed to evolve the sp system of organizing work? (2) do the gains shown in p. occidentalis for pulp foraging (larger load size, fewer trips/mg pulp; similar for water) also occur for prey foraging? if so, does it lead to a wider range of prey sizes and types taken by swarm-founders compared to independent founders? (3) have any of the epiponines that form extremely large colonies evolved an even more efficient way of partitioning tasks than has p. occidentalis? (4) has the evolution of task partitioning and the enabling of specialists led to associated morphological/anatomical changes? for example, is the crop capacity of workers of swarm founders significantly larger, relative to body size, than among the independent founders? crop volume can readily be measured by letting foragers imbibe water (for water foragers) or sugar water from a calibrated capillary tube (jeanne & taylor, 2020). 7. evolution of swarm founding in a recent paper (jeanne, 2020) i raised the question of how swarm founding among the wasps could have evolved from independent-founding ancestors. unlike the enormous amount of attention accorded to the origins of eusocial behavior in insects, this later step, taken at least four times in the vespidae, has hardly been addressed. i proposed one plausible sequence of evolutionary steps. i also pointed out some of the consequences of evolving the swarm-founding mode. one of the most intriguing is that of small body size. epiponine wasps are significantly smaller than are mischocyttarus and polistes. i proposed four possible hypotheses to explain this (and there table 2. parallel-series (ps) system. times required if p. occidentalis were to use the ps system of nest construction. tasks are performed in sequence by a forager-builder. time at task (column b) is time off-nest for foragers and time spent building for builders (derived from data for large colonies (jeanne, 1986, table 2)). columns c-f are adjustments made for the ps system. times in seconds. see text for detailed explanation. a tasks (in sequence by each forager) b time at task c flying/ searching time d net time at task (b – c) e multiplier f flying/searching time (ps) g seconds per ps unit of pulp [(d x e) + f] water foraging 42.9 10.0 32.9 0.22 5.0 12.2 pulp foraging 188.8 10.0 178.8 0.164 10.0 39.3 building 61.9 0 61.9 0 61.9 total time per trip 113.4 adjustment to the equivalent of an sp-sized pulp load: 113.4 x 6.1 691.7 sociobiology 69(1): e7715 (march, 2022) 13 may well be others). creative minds will come up with ways to test these. a curious aspect of the epiponine size-frequency distribution is that it is left-end truncated, i.e., there appears to be a wall at 4mm wing length below which natural selection cannot take them. i suggested that wasps smaller than this lack the size and mandible strength to collect and manipulate vegetable fibers into a nest. one testable prediction of this hypothesis is that wasps of this size construct their nests of fibers that are soft and easily collected, such as plant hairs. undoubtedly, others will come up with additional hypotheses. 8. colony efficiency my demonstration that task-integration efficiency in terms of work accomplished per worker-minute is greater in larger colonies of p. occidentalis (jeanne, 1986) raised the question of whether this translates into greater overall efficiency at the colony level. my grad students and i investigated this in both p. occidentalis and parachartergus fraternus by following growth from colony founding (by absconding swarms) to a fixed number of days just short of the emergence of the first offspring. this time frame ensured that our measures of each colony’s output were solely the product of the females in the founding swarm. we measured the two forms of output: the biomass of brood in the nest and the size of the nest in weight and number of cells. when converted to per-capita productivity and regressed on colony size, the results were mixed, some datasets showing increases with colony size (jeanne & nordheim, 1996), while others showed no effect of colony size (bouwma et al., 2005, bouwma et al., 2006). meanwhile, phd student kenneth howard showed that large colonies of p. occidentalis rear their brood significantly faster than do small colonies (howard & jeanne, 2004). taken together, these results call into serious question the dogma known as michener’s paradox, that as colonies of social hymenoptera grow larger they become less efficient at rearing reproductives in terms of output per worker (michener, 1964). unlike the studies michener analyzed, many of which used proxies that did not measure total productivity, our research explicitly set out to measure colony efficiency based on total output – the nest structure and the standing crop of brood. several co-authors and i have recently completed a review and meta-analysis of data bearing on this issue (jeanne et al., submitted). several unanswered questions arise from these results. what is the shape of the per-capita-productivity curve along an intraspecific colony-size gradient? it is probably a decelerating curve, rising to approach an asymptote, but what is that asymptote? was it effectively reached by the larger colonies (500-600 adults) in my 1986 study, or do taskintegration efficiencies continue to rise beyond that? ken howard’s (2004) result suggests that efficiency at the colony level continues to rise through colonies of 3,000 adults. what explains those gains at the individual-performance and taskintegration levels? more generally, what factors other than ergonomic efficiency select on species-specific colony size in wasps? these issues are central to such questions as the regulation of the colony cycle and how natural selection acts to determine species-specific colony size. 9. economics of nest architecture despite all the attention devoted to nest architecture in social wasps, little has been paid to the economics of nest design. the data reported by jeanne and nordheim (1996) suggest that the phragmocyttarous nest of p. occidentalis is relatively expensive to build on a per-cell basis. the fraction of the mass of the nest that is invested in brood cells (vs. supporting structures + envelope) is 0.29, regardless of the size of the nest. that is, the cost of adding each additional cell is the same in large and small nests. whether this is true of all nest types remains to be determined. one advantage of the phragmocyttarous type is that nest expansion does not require tearing down any existing part of the structure, unlike in vespines and some epiponines (e.g., parachartergus, synoeca). other architectural types do appear to have a much higher fraction of material invested in brood cells and are therefore cheaper to build. the gymnodomous nests of agelaia, for example, have no envelope and the only supporting materials are the petioles connecting the combs. stelocyttarous and astelocyttarous nests have non-supporting envelopes that are relatively thin and cheap. the phragmocyttarous nest type, which appears to be the most expensive to build, evolved relatively late from a stelocyttarous ancestor (noll et al., 2020). what adaptive advantages does it confer to offset the higher cost? details of the economics of nest type and construction behavior may help explain the evolution of nest architecture. the huge diversity of nest types among the epiponines invites a quantitative analysis of the relative economics of nest architectures. 10. explosive nest construction another interesting aspect of nest construction is the pacing of it. in a 2004 paper andy bouwma and i called attention to two divergent patterns among the social wasps (jeanne & bouwma, 2004). the independent founders and some of the epiponines engage in continuous nest construction: the rate of cell construction is gradual and oviposition rates closely follow, leaving few if any empty cells as the nest grows. in contrast, some groups of epiponines are characterized by explosive construction: the nest is built rapidly to large size in the first days of colony founding at a pace that far exceeds the rate of oviposition, and then is not expanded for weeks or months. we proposed five factors that could favor the latter. kevin loope tested four of these and found support for two: first, that explosive construction minimizes exposure of the brood to natural enemies and desiccation, robert l. jeanne – my life as a curious naturalist14 and second, that rapid construction promotes competition among queens by providing a superabundance of empty cells for oviposition, thereby facilitating the selecting out of the less fecund of the multiple reproductive females (loope & jeanne, 2008). this is far from a settled issue. there are numerous swarm-founding polistines that do not appear to engage in explosive construction, despite being polygynous (jeanne & bouwma, 2004, table 2). there is also some correlation with nest architecture: the majority of species that appear to use explosive construction build phragmocyttarous nests, compared to no phragmocyttarous nests among species engaging in continuous construction. reaching an understanding of the adaptive advantage of explosive nest construction, its taxonomic distribution, and its correlation with nest architecture will require better data for carefully selected key species along with a new round of creative hypothesizing. as niko tinbergen emphasized (tinbergen, 1963), a thorough comparative study of carefully selected species can often test among hypotheses where experimentation is not feasible. esther cullen’s classic study of nesting adaptations in gulls remains an excellent example of the comparative approach (cullen, 1957). 11. regulation of nest construction in epiponines swarm-founding wasps are complex societies (jeanne, 2003). their often-large worker forces specialize by age, with older individuals taking on the risky tasks of foraging and defense (see above). at a finer level, an individual worker may specialize in a way that is based on her recent experience and the current demands of her colony. her specialization may be temporary, lasting for a few days or even just a few hours. responses requiring the cooperation of two or more groups of specialists are self-organizing and self-regulating. one of the most fascinating and appealing manifestations of these emergent phenomena is nest construction (nc). as described above, it requires the coordinated interaction of three groups of transitory specialists – water foragers (w), pulp foragers (p), and builders (b). for the curious biologist, the beauty of nc as a subject for investigation is that it can be activated at will simply by removing a part of the nest envelope, triggering a bout of nc to repair the damage. moreover, most of the activity takes place in full view, where it can be videotaped. the many questions one may ask about how nc works can be sorted into at least three categories. one focuses on the round of nest construction once it is up and running. it asks how the levels of activity of the three groups are kept in optimal proportions so as to minimize the queuing delays the workers experience during transfer of materials. the second asks what determines the overall level of the nc response. the third asks how the whole operation is started up and what brings it to a stop. only some of these questions have ever been addressed. this is not the place for a thorough review of the subject, but i will describe two competing hypotheses on mechanism relating to the first question. working with p. occidentalis, i proposed and tested three hypotheses on the sources of information used by each of the three groups, p, b, and w, to adjust its level of activity vis à vis the others (jeanne, 1996). i perturbed the up-andrunning nc operation in two ways. first, i experimentally increased the amount of raw materials – water and wood pulp – flowing into the operation. second, i removed some of the workers in each of the three groups in turn. i concluded that the b receive information about the nest damage directly from the nest. feedback among b appears to determine the level of the group’s building activity, which in turn sets the size of the entire repair operation, although that may be modulated downward by negative feedback from the p and w. the level of pulp-foraging activity is set by information about the demand for pulp received by p from the b. the w group similarly adjusts its activity rate in response to feedback received from p and b about the demand for water. these results support the hypothesis that information flows from the nest damage to the b and from them to the w and p; that is, information flows in a direction opposite that of the materials. thus, it is a demand-driven operation. there is good evidence that time in queue (waiting time) to give (or receive) a building material cues information about the demand (or supply) of the material and that workers respond to the information by continuing with their task, switching to one of the other tasks, or quitting altogether. istvan karsai and co-authors (karsai & wenzel, 2000, karsai et al., 2020) have taken issue with this informationflow model on several grounds. one is their claim, made without explanation, that it’s a runaway model. it is not. the maximum rate of nc is limited by the size of the damage being repaired. a second criticism is that i identified predetermined classes or workers identified with single behaviors. i did not. i focused on the performed tasks, not castes; the identity of the performers was secondary. a third critique is that in my model the specialists simply switch off and on instead of flexibly changing to other roles. again, this is incorrect. workers in each group frequently switch from one task to another; i reported this behavior in the form of transition frequencies among the three tasks (jeanne, 1986). karsai et al.’s fourth criticism, that they found no evidence for the feedback mechanisms i claim, is also unwarranted. i proposed and tested three explicit hypotheses on the sources of information used by p, w, and b, and provided evidence for rejecting some of them and supporting others. they provided no arguments for refuting those conclusions, performed no alternative tests leading to different conclusions from mine, nor did they put forward an alternative set of testable hypotheses. at bottom, they found no evidence in support of my model because they didn’t look for any. karsai has proposed a somewhat more complicated model based on his observations of two species of metapolybia sociobiology 69(1): e7715 (march, 2022) 15 (karsai & wenzel, 2000). while metapolybia colonies are typically smaller than those of p. occidentalis, he showed that the activity of nc is partitioned into the same three groups of tasks – p, b, and w. however, he suggested that there is a fourth worker group, a set of mostly inactive individuals on the nest that act as water tankers to provide an on-nest reservoir of water that he calls the social stomach or communal crop. (the former term is a misnomer – water is stored in the crop, not the stomach – so i will refer to this group by the latter term, abbreviated here as cc.) in support of postulating cc as a separate class, karsai remarked that he never saw w regurgitate water directly to a p, supporting his notion that water foragers unload exclusively to the cc. in my study of p. occidentalis direct w-to-p transfers were a common occurrence (personal observation). i suspect that karsai’s failure to see it in his metapolybia study is a result of the low rates of nc reported in his small colonies, combined with a low total number of hours of observation. his model states that the need for repair is perceived not by b inspecting the damage as in my model, but is sensed by the entire colony via the changes in light, humidity, and/or co2 levels caused by the removal of the envelope. w presumably begin foraging in response to the same general physical cues and unload their water to the cc group. karsai posits that the total volume of water in the cc correlates to the size of the nest damage, and that this somehow acts as an information center to set the level of activity. that is, information flows from the nest to the cc, and then to the w, p, and b. in this sense it is a supply-driven system. in several publications (see karsai et al., 2020, and cited papers) karsai and co-authors have developed various computer models of the process. left unexplained is how members of the cc group, stationed on the front of the nest (at least in p. occidentalis) and away from the nest damage, are better informed of the current size of the damage than are the builders (jeanne model). it is possible that my model is correct for polybia and karsai’s is correct for metapolybia. i think that is unlikely. the idea that the cc serves as a center for gathering and dispensing information about demand level adds a level of complexity to the nc system that in my view is unlikely to be adaptive. in effect, karsai proposes a supply-driven model as an alternative to my simpler demand-driven one. nevertheless, the karsai proposal is a welcome addition to the effort to understand the details of how nc functions, because now there are two competing models to test. the way forward is to test predictions made by each. mathematical models and computer simulations have their place, but they don’t help us to evaluate each alternative. what’s needed are observations and empirical tests on real colonies in the field. here are a few suggestions for how to move forward on this. 1) is the cc category real? the karsai model predicts that members of the cc group have little or no overlap with those in the p and b task groups. the jeanne model predicts that these workers are merely temporarily idle p and b workers who are waiting to receive enough material for their next task. test: during ongoing nc mark members of the cc group and determine if they engage in any of the p, w, or b tasks. if not, it would support the notion of a separate fourth group, the karsai model. 2) who obtains the initial information about the nest damage? the karsai model suggests that it is the whole colony; that is, the information comes in the form of changes to the physical conditions inside the nest and that no contact with the damaged parts is necessary. the jeanne model suggests that those involved in repair – b, and perhaps p and w as well – inspect the damage directly. test: after a day or two of marking p, w, and b as they engage in nc, allow the repairs to be completed. early the next morning (by 6:30 or so, before nc begins and when relative humidity is high), remove a moderate amount of the nest envelope. videorecord which marked workers inspect the damage as repair gets underway. the jeanne model predicts it will be primarily b, perhaps p and w, but not a large contingent of unmarked workers. 3) who takes the first step toward initiating nc at the beginning of the day? the karsai model predicts that it starts with a good amount of water-foraging to stock the cc reservoir, then once the level approaches saturation, p and then b will begin. the jeanne model predicts that the first foraging trip will often be by a pulp forager. on several occasions before nc begins in the early morning, i have observed that the first workers to depart the nest go to nearby leaves for water (dew), then continue on for pulp. after a short time, w become active, followed by more p as the system gains speed. this suggests that there is not a communal store of water on the nest, or the stocking of one by water foraging. test: same as for (2) above, but record the sequence of arrivals of water and pulp loads. if water predominates, it would support the karsai model. even after these issues are resolved, others will remain to tantalize. we have a reasonable idea of how the operation responds to experimental perturbation of different kinds and this has provided clues to the sources of information used by the various groups of workers (jeanne, 1996). what is less clear is what determines the scale of the process. i have proposed that the maximum level of nc activity is set by the amount of space available for b to work; if a b cannot find room to apply its load of pulp it is likely to quit building. the consequent feedback to p and ultimately to w in the form of fewer receivers to accept their loads will regulate the whole system slightly downward. rates in the early mornings of the first day or two of new nest construction appear to approach this maximal level (personal observation). but it is clear that the levels of nc are often far lower. lower levels may be imposed if the number of workers in the colony is small in relation to the size of the damage, or if the number of workers whose response thresholds are exceeded by the stimulus of the damage is small. it can also be limited by environmental conditions, notably relative humidity. we do know that nc rates slow then stop as the morning progresses, and that this robert l. jeanne – my life as a curious naturalist16 is likely due to the increasing cost per pulp load in the form of a rising w/p ratio imposed by decreasing rh (loope & jeanne, 2008). cost-benefit analyses of this dynamic process have yet to be done. then there is the question of the cues conveying the positive and negative feedback that must take place in setting activity levels. can a builder, for example, indicate through its mouth-to-mouth contact with a nestmate that it is seeking a load of pulp and not water? can it sense, short of contact, what a nearby nestmate has to offer? finally, as karsai and collaborators point out, repair work is one thing, but none of the research done so far sheds much light on how new nest construction is initiated, or how a colony decides when to expand its nest. these are questions that ought to challenge ambitious and innovative experimental naturalists. teasing out answers will take ingenuity and persistence. 12. conclusion the most motivating questions for me have typically started with “i wonder why…” or “i wonder how…” many animals, including social wasps, also show curiosity, but for them the question “i wonder what that object that just appeared over there (or on my nest) is” generally leads to one of three possible responses: chase it and/or eat it, flee from it, or mate with it. their curiosity is largely an adaptation that enhances survival and/or reproduction. at a fundamental level it may have been the same for our ancestors: natural selection may have favored curiosity leading to a knowledge of the natural world around them. with us moderns, it’s different: we can turn a question about a particular wasp structure or behavior into a burning need to know why and how that has not the slightest connection to our surviving or reproducing – just the immense satisfaction we gain after a long, curiositydriven pursuit of how one part of the natural world works. paraphrasing karl von frisch, nature is like a magic well: the more answers we draw from it, the more questions it generates. paraphrasing george mallory, we sociobiologists study social insects because they’re there. i retired from the university of wisconsin at the end of 2009, but my curiosity hasn’t retired. i continue to be inspired by the wonderful diversity and intricacy of insect social behavior, and i even still publish a little bit about it every now and then. acknowledgments i thank christine roberson and louise jeanne for reading a draft of the manuscript and offering helpful suggestions for improvement. references bouwma, a.m., howard, k.j. & jeanne, r.l. 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(2002). influence of age polyethism on longevity of workers in social insects. behavioral ecology and sociobiology, 51: 234-237. _hlk85808472 _hlk90202153 open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i2.900sociobiology 63(2): 755-761 (june, 2016) whiteflies provide honeydew to camponotus ants without receiving reciprocal favor introduction the mutualism between tending ants and honeydewproducing hemipterans has been widely recognized (way, 1963; völkl et al., 1999; flatt & weisser, 2000; queiroz & oliveira, 2001; oliveira & freitas, 2004; del-claro et al., 2006, muller et al., 2016). nevertheless, although many hemipterans are known to interact with tending ants more attention has been given to the aphid-ant mutualism (floate & whitham, 1994; kaplan & eubanks, 2002; stadler & dixon, 2005; muller et al. 2016). some attention has also been given to ant-membracids interactions in the neotropics (del-claro & oliveira, 1999; del-claro, 2004; fagundes et al., 2013). in general, various hemipteran species produce an aminoacidpoor, but carbohydrate-rich excretion namely honeydew, which is the main driver of these mutualistic relationships (buckley, abstract the notion that tending ants provide protection to honeydew-producing hemipterans is widely accepted. nevertheless, there have been debates about whether or not this protection can always disrupt the biological control of hemipterans. although various hemipteran species interact with tending ants, most studies have focused on the mutualism between ants and aphids. woolly whitefly aleurothrixus floccosus (maskell) is an important pest of citrus whose nymphs are frequently tended by ants such as camponotus. however, it is unknown whether or not ants in this genus alone can disrupt biological control of woolly whitefly by protecting this pest’s nymphs from natural enemies. we investigated the impact of camponotus ants on the biological control of woolly whitefly in the field by excluding or allowing the access of ants to whitefly nymph colonies in different tangerine trees. furthermore, in a laboratory study we also assessed the behavior of camponotus ants in response to woolly whitefly’s common predator cues (visual and scent). in summary, this fieldlaboratory study suggests that there is no mutualism between tending camponotus ants and the whitefly a. floccosus; rather it indicates commensalism as an alternative interaction. interactions as this may provide more benefits for the host plant, whereby camponotus ants can reduce sooty mold by removing honeydew from the leaves and favor pest biological control by leaving the whiteflies unprotected. sociobiology an international journal on social insects rm jesus1, rc paes junior1, gj carmo1, d mota1, lm gontijo1,2, ma oliveira2 article history edited by gilberto m. m. santos, uefs, brazil received 17 august 2015 initial acceptance 08 april 2016 final acceptance 26 april 2016 publication date 15 july 2016 keywords tending ants, camponotus, woolly whitefly, honeydew, citrus, biological control. corresponding author lessando m. gontijo universidade federal de viçosa campus florestal rodovia lmg 818, km 06, cep: 35690-000 florestal-mg, brazil e-mail: lessandomg@ufv.br 1987; hölldobler & wilson,1990). tending ants can provide benefits to hemipterans by protecting them from predators, parasitoids and fungal infections (banks, 1962; way, 1963; samways, 1983;völkl, 1992; kaplan & eubanks, 2002; delclaro & oliveira, 2000; byk & del-claro, 2010; zhang et al., 2013), thus suggesting a potential to disrupt biological control of such hemipterans. the magnitude of the mutual benefits between ants and hemipterans is highly influenced by the ecological settings in which they occur (cushman & whitham, 1989, 1991; cushman & addicott, 1991; del-claro & oliveira, 2000). more specifically, the level of ant attendance can be influenced by both the host plant and hemipteran species (hendrix et al., 1992). ants respond most intensively to honeydew containing high amounts of melezitose (kiss, 1981; völklet al.,1999). for example, fischer & shingleton (2001) reported that 1 instituto federal goiano, morrinhos-go, brazil 2 universidade federal de viçosa, florestal-mg, brazil research article ants rm jesus et al. – interactions between woolly whiteflies and tending ants756 aphids chaitophorus populialbae (boyer de fonscolombe) were often tended by ants because its honeydew had high proportions of melezitose, whereas the species c. populeti (panzer) were untended because of its melezitose-poor honeydew. furthermore, the intensity of ant-hemipteran mutualism is positively correlated with the quantity of honeydew required by the ants (hölldobler & wilson, 1990; bristow, 1991; bonser et al., 1998), suggesting that ants with high sugar demand provide longer attendance and protection to honeydew-producing hemipterans, which in turn may mediate the disruption of biological control. extrafloral nectar can also attract ants to plants, which can mediate some plant protection whereby the ants ward off some herbivores (heil & mckey, 2003). nevertheless, extrafloral nectar is known to attract more generalist ants whereas honeydew attracts more specialized ants (bluthgen et al., 2000), which have a more narrow relationship with the honeydew-producing insects. woolly whitefly aleurothrixus floccosus (maskell) (hemiptera: aleyrodidae) is an important pest of citrus. the sessile nymphal stages produce wax filaments and occupy the undersurface of the leaves. the nymphs also excrete honeydew which favors the growth of sooty mold and the attraction of tending ants such as the argentine ant (debach & rose, 1976; belay etal., 2011). other ants that have been documented to often tend woolly whitefly nymphs in the neotropics are camponotus spp. (queiroz & oliveira, 2001; rodrigues & cassino, 2011; alves et al., 2015). however, it is still unknown whether ants in this genus alone can significantly disrupt woolly whitefly biological control by protecting them from natural enemies. a week prior to the beginning of our experiments, an unsprayed citrus orchard located at the instituto federal goiano, campus morrinhos, go, brazil, was noticed to be highly infested with woolly white flies. at that same time great numbers of camponotus ants were observed either walking within the tree canopies or tending the woolly whitefly colonies on every infested citrus tree. thus, thereafter we set up a field experiment in that citrus orchard to investigate whether or not camponotus ants could disrupt the biological control of the woolly whitefly nymphs by protecting them from natural enemies. furthermore, in a laboratory study we also assessed the behavior of camponotus ants in response to woolly whitefly’s predator cues. materials and methods field experiment this experiment was conducted in a 5-year old unsprayed citrus orchard composed of tangerine trees cultivar ‘ponkan’, and located at instituto federal goiano campus morrinhos, in the state of goias, brazil. the orchard had 465 trees spaced 5m along row and 7 m between rows. there was also an irrigation system of micro sprinklers running along the tree rows (south-north lengthwise). this field experiment had two treatments and ten replicates, which were set up in a completely randomized design. treatments were comprised by i) woolly whitefly nymph colonies freely accessible to ants and natural enemies (with ants), and ii) woolly whitefly nymph colonies inaccessible to ants, but accessible to natural enemies (without ants). each replicate was composed of a 1-m long middle branch infested with woolly whitefly nymphs. for treatment ii, the base of each branch on the tree was spread weekly with tangle foot to prevent ants reaching the nymph colonies, and thus allowing access only to parasitoids and flying predators. prior to the experiment each experimental tree branch (from all treatments) was trimmed to avoid connectivity with other branches, which was specifically important to prevent ants from other parts of the tree reaching the nymph colonies in treatment ii. in addition, before the experiment the number of woolly whitefly nymphs (all stages) in each branch was adjusted to 400 individuals distributed among 5-10 leaves close to each other. the possible presence of woolly whitefly eggs was not investigated because of the difficulty of doing so in the field. the number of woolly whitefly nymphs, ants and natural enemies was evaluated weekly in each replicate during the course of six weeks (5 october 9 november, 2013). to do so, each evaluator approached the branch with care and looked first for motile forms of natural enemies, which were visually identified to taxon level and recorded. thereafter, the number of ants and whitefly nymphs was counted on the infested leaves. however, after the experiment started we did not control for adult woolly whiteflies that might eventually come to the experimental branch and oviposit there contributing to an increase in nymph density. we did so because the initial 400 nymphs (all stages) might hatch in a short period of time not allowing enough time for predation to occur on repeated dates. in addition, we conducted weekly a targeted search on non-experimental trees for ants associated with woolly whitefly colonies, which were collected and placed in a glass vial containing preserving alcohol 70% for later identification. behavior experiment in the laboratory in this experiment we assessed the behavior of ants in response to visual/scent and visual cues from predators. the predators used were adults of cycloneda sanguinea(l.) (coleoptera: coccinellidae). this predator was chosen because it appeared to be one of the most common species during a random search on non-experimental trees infested with woolly whitefly. this lab experiment had three treatments and 15 replicates, which were carried out at five different dates (n = 3 replicates of each treatment/date). the treatments were as follow: i) no predator cue (no coccinellid), ii) visual + scent cues from predator (live coccinellid), and iii) visual cue from predator (drawn coccinellid). each replicate consisted of a clear-plastic cup (13 x 10 cm: h x d) closed with a lid that had an orifice (3 cm in diameter) covered by organza fabric, which allowed ventilation. each cup (arena) had inside an 8 sociobiology 63(2): 755-761 (june, 2016) 757 cm-long citrus stem with four leaves infested with woolly whitefly nymphs placed inside a small glass vial (50 ml) with water and sealed with cotton. in addition, all replicates had a two-hour starved camponotus sp. ant. the ants were collected manually with the aid of a camel brush from citrus leaves infested with woolly whitefly nymphs two hours prior to the experiment and kept individually in plastic vials in the absence of food and water. likewise, adults of c. sanguinea (both sexes) were collected manually from infested trees twenty four hours prior to the experiment and kept in plastic cups containing a small cotton ball wet with a honey/water solution (30% honey). for treatment ii, two adult c. sanguinea were released at the bottom of the cup (arena) three minutes prior to the experiment, which was expected to provide both scent and visual cues to the ant. for treatment iii, the figures of three adult ladybeetles were accurately and equidistantly painted on the inner walls of each cup using water soluble paint (dark red and black), which were expected to provide the predator visual cue. this experiment was carried out in the laboratory at temperature of 27 °c, r.h. of 70% and under fluorescent lights. during the experiment each ant was carefully placed on an infested leaf with the aid of a fine paint brush and allowed two minutes to settle. ants that did not stay on the leaves for these first two minutes were discarded. thereafter, the following ant behavioral categories were measured during 10 minutes: number of times it left the citrus stem/leaves to explore the arena, number of times it attacked the coccinellid, time spent resting on citrus stem/leaves and arena, time spent moving on citrus stem/leaves and arena. resting was defined as the time that the ant remained stationary; moving represented the time spent walking, regardless of direction. these measurements were taken by direct observation, where one person informed the beginning and the end of each behavioral category while another person recorded the time in which that occurred. subsequently, the broken times (s) were summed for each behavioral category. statistical analyses field experiment: differences in woolly whitefly nymph density between treatments were assessed using repeated measures analyses of variance (procmixed). covariance structures for the mixed model repeated measures were constructed, and baysian information criterion (bic)were used to assess that the un covariance structure provided the best fit for the data. date was considered a random factor in these models. behavior experiment: treatments were blocked through time and 3 replicates of each treatment were run per day for a total of 15 replicates at the end of the 5-day experiment. however, the data were pooled for all dates at the time of analyses. ant behavioral data were organized for analys into the following categories: (1) time spent walking on the citrus stem/leaves; (2) time spent resting on the citrus stem/leaves; (3) time spent walking on the arena; (4) time spent resting on the arena; (5) total time walking; and (6) total time resting, regardless of place. thereafter, a separate manova was carried out for each data category. for the data regarding the number of times each ant left the infested leaves to explore the arena a simple analysis of variance (proc glm) was carried to test for differences among treatments. all analyses were carried out using the software sas (sas, 2008). results field experiment there was no significant treatment (f = 0.30, p = 0.60), time (f = 1.47, p = 0.25) or treatment*time interaction effect (f = 0.90, p = 0.49) on the number of whitefly nymphs per replicate during the course of the experiment. in general, it was noticed only a slight increase trend for whitefly nymph density towards the last three weeks of the experiment in both treatments (fig 1) suggesting that adult whiteflies continued to reproduce on the replicates (citrus branches) after the experiment had started. the fact that whitefly nymphs did not reach higher densities in treatment i (with ants) indicates that these ants may not be able to significantly disrupt the biological control of this pest by inhibiting natural enemy attack. in fact, it was noticeable that biological control was able to maintain the whitefly nymphs at a very slow rate of increase in both treatments, therefore preventing further outbreaks (fig 1). despite the fact that the field evaluations constituted a “snapshot” in time, the presence of camponotus ants tending whitefly nymphs in treatment i (with ants) was very common during the weekly evaluations (table 1). likewise, a diverse group of generalist predators was found associated with the nymph colonies during the evaluations. the main predator taxa encountered (larvae and adults) were coccinellids, lacewings, spiders and syrphids (table 1). there was no sign of nymph parasitism nor the presence of adult parasitoids associated with whitefly nymph colonies. the species of ants associated with the whitefly nymphs identified by dr. marco antonio oliveira were camponotus femoratus (fabricius, 1804) and camponotus blandus (smith, 1858). fig 1. number of woolly whitefly nymphs per replicate (citrus branch) at different dates. rm jesus et al. – interactions between woolly whiteflies and tending ants758 behavior experiment there was no significant treatment effect on time spent by ants walking on the stem/leaves (f = 1.36, p = 0.27) or arena (f = 0.77, p = 0.47). likewise, there was no significant treatment effect on time spent by ants resting on the stem/leaves (f = 0.74, p = 0.48) or arena (f = 1.76, p = 0.19). in general, ants spent more time on the arena compared to the stem/leaves (fig 2), suggesting little engagement in whitefly nymph protection. there was no treatment effect on the total amount of time spent by ants walking (f = 1.64, p = 0.20) or resting (f = 2.30, p = 0.11), regardless of place (fig 3). nonetheless, it appears that ants tended to spend more time in activity than resting (figs 2 and 3). in addition, there was no significant treatment effect on the number of times the ants left the stem/leaves to explore the arena (f = 2.00, p = 0.15). the ants left the stem/leaves in the treatment ‘live coccinellid’ 3.53 ± 0.70 times, in treatment ‘drawn coccinellid’ 2.50 ± 0.62 times, and in the treatment ‘no coccinellid’ 4.80 ± 1.10 times. ten of all experimental ants were observed to feed on whitefly nymph honeydew during the experiment. only one experimental ant was observed to attack a live ladybeetle during the experiment. five of all experimental ants inspected the drawn (painted) ladybeetles by moving its antennae to and fro upon the drawing. discussion it has been widely accepted that certain ant species associated with honeydew-producing hemipterans can disrupt the biological control of the latter (vinson & scarborough, 1991; queiroz & oliveira, 2001; kaplan & eubanks, 2002). however, the magnitude of mutual dependence between ants and hemipterans plays an important role in the equilibrium of this relationship, which in turn may indirectly affect biological control. in this study we investigated whether or not camponotus ants that feed on honeydew can disrupt the biological control of the woolly whitefly aleurothrixus floccosus. furthermore, we assessed the behavior of tending camponotus ants in response to woolly whitefly’s predator cues. our results show that the field population of woolly whiteflies increased equally little in both treatments during the course of six weeks, suggesting that the ants were not able to disrupt biological control when they had free access to whitefly nymph colonies. although the presence of camponotus ants associated with the experimental whitefly nymph colonies was common in treatment i (with ants),there were times during the evaluation when ants were absent from some of the replicates in this treatment. this suggests that these ants may not have a very narrow relationship with the woolly whitefly, date 12 oct. 19 oct. 26 oct. 2 nov. 9 nov. treatment with ant no ant with ant no ant with ant no ant with ant no ant with ant no ant ants 22 0 12 0 3 0 14 0 11 0 coccinellid 1 1 0 0 1 0 0 1 0 0 lacewing 1 2 0 2 1 0 1 3 0 0 spider 0 2 0 0 0 0 0 0 0 0 syrphid 1 2 1 0 1 0 2 1 0 0 natural enemies, including both larval and adult stages. table 1 total number of ants and predators found associated with woolly whitefly nymphs on citrus at different dates. fig 2. time spent walking and resting by camponotus ants on the stem/leaves and arena (cup) of three treatments: live coccinellid (scent and visual cues), drawn coccinellid (visual cue only), and no coccinellid (no predator cue). fig 3. total time spent walking and resting by camponotus ants in three treatments: live coccinellid (scent and visual cues), drawn coccinellid (visual cue only), and no coccinellid (no predator cue). sociobiology 63(2): 755-761 (june, 2016) 759 and thus do not take turns or stay continuously with the nymph colonies. because camponotus ants are omnivorous (fowler & roberts, 1980, cannon & fell, 2002; feldhaar et al., 2007), and honeydew is a suboptimal and ephemeral food source (wäckers et al., 2008) it may be more advantageous for them not to attend continuously the whitefly nymphs, and had better forage at additional locations for alternative food. moreover, the honeydew quality depends on the host plants species, whereby nutritionally-poor plants may mediate lower levels of ant attendance to hemipteran colonies (nemec & starý, 1990; hendrix et al., 1992). however, this has yet to be empirically investigated for our particular scenario. a study conducted by alves et al. (2015) reports that camponotus ants were observed warding off stingless bees that would come to feed on the honeydew of the whiteflies aleurothrixus aepim (goeldi) attacking cassava plants. however, unlike in our study they did not quantify the biological control of whiteflies nor assessed the response of the ants to other natural enemies (e.g., predators). the main generalist predators found associated with the whitefly nymph colonies in our study were coccinellids, syrphids, lacewings, and spiders. with the exception of spiders, all the other three predators have been documented to feed on whiteflies (ghahhari & hatami, 2000; queiroz & oliveira, 2001). because the predators we surveyed were in or near the whitefly colonies, we have a reasonable degree of confidence that they were preying on whitefly nymphs. however, our samples were taken only during the day, so nocturnal predators such as earwigs and others may have escaped notice. the more mobile predators (i.e., coccinellidae) may also have been underrepresented in relation to the syrphidae, which are relatively sessile once settled in a colony. the relatively low encounter rate of predators in association with the nymph colonies may not, therefore, reflect the impact of predators; these samples constitute a “snapshot” in time, rather than the cumulative effect of predation. there was no significant difference among treatments in regards to the number of times each ant left the infested stem/ leaves to explore the arena during the behavior experiment. in general, the ants spent more time on the arena than on stem/ leaves, regardless of experimental treatment. this suggests that neither scent nor visual cues from coccinellids were able to induce a protection behavior on ants for guarding the whitefly nymphs. this may be so because guarding whitefly nymphs and attacking predators such as coccinellids require a substantial amount of energy and implies a risk of getting intoxicated from coccinellid chemical defense, therefore not justifying the necessity of attending continuously the whitefly nymphs. nevertheless, this has yet to be empirically demonstrated. in any case, coccinellids are known to defend themselves from hemipteran-tending ants by using behavioral, physical and chemical mechanisms (pasteels et al., 1973; richards, 1985; majerus et al., 2007). because we tested only coccinellids, it is unknown whether or not these camponotus ants could respond in the same way to other predator species. most of the studies regarding ant-hemipteran association have been done in temperate regions, and these have focused mostly on aphids (bretton &addicott, 1992; floate & whitham, 1994; flatt & weisser, 2000; kaplan & eubanks, 2002; stadler & dixon, 2005). by contrast, most of the work regarding anthemipteran interactions in the neotropics involves plant hoppers (wood, 1984; dansa & rocha, 1992; del-claro & oliveira, 1999, 2000; del-claro, 2004; fagundes et al., 2013), whereas very few concern white flies (queiroz & oliveira, 2001; rodrigues & cassino, 2011; alves et al., 2013). in general, studies of this nature have congruent results indicating that tending ants can disrupt the biological control of honeydew-producing hemipterans by protecting them from predators and parasitoids. for example, queiroz & oliveira (2001) observed higher densities of whiteflies aleurothrixus aepim goeldi on croton floribundus (euphorbiaceae) when these plants were freely accessed by ants, possibly because these ants protected the whiteflies from its natural enemies. by contrast, our results show that the biological control of the woolly whitefly a. floccosus is not significantly affected by camponotus ants. this difference between our studies may be due to the fact that we investigated a different whitefly and plant species, and moreover, besides camponotus ants their experiment had also the presence of other ant species (e.g., fire ants). our results raise the question whether the biological control disruption could be an exception rather than the rule, which has been advocated by many insect ecologists. in summary, this field-laboratory study provides an interesting piece of natural history information that suggests that there is no mutualism between tending camponotus ants and the woolly whitefly a. floccosus attacking citrus plants; 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(2013).the ecological effects of ant-aphid mutualism on plants at a large spatial scale. sociobiology, 60: 236-241. doi: 10.13102/sociobiology.v60 i3.236-241 doi: 10.13102/sociobiology.v64i1.995sociobiology 64(1): 18-25 (march, 2017) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 yearlong association of apis dorsata and apis florea with flowering plants: planted forest vs. agricultural landscape introduction honey bees play a vital role in crop production and in preserving the wild habitats. they have coevolved with native flora and sometimes this coevolution results in specialized relationships. because of their diverse nesting strategies and specific host plant biochemical relationships, honey bees are regarded as the most suitable social insect for the monitoring of environmental processes (nathan et al., 1999). there are three native honey bee species in pakistan i.e. the wild bee or rock bee, apis dorsata (fabricius, 1793); the little bee or dwarf bee a. florea (fabricius, 1787) and domesticated bee, a. cerana (fabricius, 1793) whereas, the exotic italian bee, apis mellifera (linnaeus, 1758) has been successfully domesticated since 1977. apis cerana occurs in northern and western hills and foot-hills in some parts of kpk, punjab, baluchistan and kashmir, while the a. dorsata and a. abstract the yearlong association of two native honey bee species (apis dorsata and a. florea) with 49 plant species was recorded in a planted forest and adjacent agricultural landscape at multan, pakistan. the study resulted in 588 interactions of a. dorsata with 40 plant species and 454 interactions of a. florea on 38 plant species. the most visited plants species by a. dorsata included helianthus annuus, citrus reticulata, trifolium alexandrinum, moringa oleifera and calotropis procera, while the most visited plant species by a. florea included c. procera, mangifera indica, t. alexandrinum, coriandrum sativum and h. annuus. the peak abundance of bees and floral resources (i.e. number of plant species in flowering and abundance of floral units) was recorded during early march to late may followed by a gradual decline until december. monthly abundance of both bee species was positively related to the floral resources, negatively related to relative humidity while it was not significantly related to temperature. the current study may serve as a baseline to track the degradation in ecosystem service of cross pollination and making new conservation strategies at local scale while future research should focus on tempospatial variations in foraging preferences, floral constancy and effect of foraging competition on crop pollination in different ecological regions of pakistan. sociobiology an international journal on social insects a sajjad1, m ali2, s saeed2 article history edited by gilberto m. m. santos, uefs, brazil received 20 february 2016 initial acceptance 05 february 2017 final acceptance 06 february 2017 publication date 29 may 2017 keywords honey bees, floral host plants, seasonal variations, conservation. corresponding author asif sajjad department of entomology the islamia university of bahawalpur bahawalpur, pakistan e-mail: asifbinsajjad@gmail.com florea occur in the foot hills and plains of pakistan. although asia is not rich in bee diversity compared with some other biogeographical regions e.g. neotropics (michener, 1979) however, social bees (apis sp.) – in terms of their abundance – play a critical role in maintaining the vital ecological process of cross pollination (momose et al., 1998; roubik et al., 2005; devy & davidar, 2006). both, a. dorsta and a. florea tend to migrate locally in response to the availability of floral resources and have the ability to increase rapidly in number in response to flowering events (itioka et al., 2001). honey bees require carbohydrates, proteins, fats, minerals, vitamins, and water for the maintenance, growth and development of their colonies (loper & berdel, 1980). these elements are sourced by flowering plants in shape of pollen and nectar. however, the foraging activity of honey bees is greatly influenced by the weather conditions and availability of nectar and floral resources (neupane & thapa, 2005). 1 department of entomology, university college of agriculture and environmental sciences, the islamia university of bahawalpur, pakistan 2 department of entomology, muhammad nawaz shareef university of agriculture, multan, pakistan research article bees sociobiology 64(1): 18-25 (march, 2017) 19 both apis bees have already been reported as the most abundant and efficient pollinators of various economically important crops in pakistan e.g. canola; brassica napus (ali et al., 2011), onion; allium cepa (saeed et al., 2008; sajjad et al., 2008), fodder; sesbania sesban (sajjad et al. 2009), fodder alfalfa; medicago sativa (ahmad 1976) and bitter gourd; momordica charantia (saeed et al., 2012). using bioeconomical methods irshad and stephen (2013) quantified economic value of pollination in pakistan. they estimated production value of pollination dependent crops at us$1.59 billion i.e. 62% fruits, 20% vegetables, 9% nuts, 8% oilseed and 0.25% spices. a sharp decline in number of honey bee combs has been reported during last two decades in neighboring india (sihag, 2014) however, for pakistan, there is no study in hand based on systematic observations. the decline is probably due to the increase in pesticide usage and destruction of natural habitats (kremen et al., 2002). khan et al. (2002) reported the direct annual loss of 9.91 million rupees on account of a loss of 5661 mt of honey while net annual loss of 6.55 million rupees in sunflower production in the selected nine districts of the cotton zone of punjab, pakistan. the associations between plant diversity, social bee abundance, honey production and local livelihoods have poorly been documented for pakistan. the flora visited by honeybees remains scarce in most parts of the beekeeping areas for a fairly long period during the year (ahmad, 1984; aziz, 2015). moreover, the strong turnover in quality and quantity of floral resources are known to influence the behavior and foraging strategy of pollinator species (subbareddi & reddi, 1994; murugan et al., 1997). site list of floral host plants, their temporal dynamics in providing floral resources and ways they interact with honey bees temporally, serve as baseline studies without which it is difficult to track the degradation in ecosystem service of cross pollination and making new conservation strategies (mark et al., 2002). several preferred plant species for managed honey bees (a. mellifera) have been documented from himalayan foothills and northern pakistan (noor et al., 2009; partap, 1997) however no such data is available for the plain areas of southern punjab. the purpose of this study was to enlist the floral host plants and identify the apis most loving plant species for the first time from southern part of punjab, pakistan. moreover, we hypothesized that seasonal population fluctuations of apis is influenced by abiotic (relative humidity and temperature) and biotic (number of plant species in flowering and total floral abundance) factors. materials and methods the study was conducted in a planted forest of 20 hectares and an adjacent agricultural farm at bahauddin zakariya university campus multan (30.255°n; 71.513°e; 114±6 meter above sea level), pakistan from january to december, 2008. the planted forest is comprised of trees/ shrubs of the species prosopis juliflora, moringa oleifera, eucalyptus camaldulensis, tamarix aphylla, albizia procera, ziziphus jujuba, dalbergia sissoo, capparis decidua, leucaena leucocephala and acacia nilotica. besides planted trees, there also grows a variety of natural vegetation including annual wild plants and perennial shrubs (table 1). some seasonal crops and fruit trees are grown in adjacent agricultural landscape including helianthus annuus, trifolium alexandrinum, coriandrum sativum, momordica charantia, brassica campestris, daucus carota, allium cepa, citrus reticulata, grewia sub inaequalis and mangifera indica. climate of the area is sub-tropical with a long hot summer and short cold winter. the mean daily maximum and minimum temperatures are in the range of 30 to 35°c and 15 to 20°c, respectively with the mean monthly summer rainfall of ca. 18mm. the highest temperature (45 to 51°c) is recorded in may and june while the lowest (3 to 0°c) is recorded in january (khan et al., 2010). a variety of annual wild plants and perennial shrubs naturally grow in the forest. there were eight hives of a. dorsata while the number of hives of a. florea was uncertain as they are smaller in size and mostly build combs in thick vegetation. as different plant species had different type of inflorescences, we defined the floral units for each plant species separately and each time recorded observations on those floral units. floral abundance was estimated by randomly tagging 15 plants of each plant species and counting total floral units at every two weeks interval. during each census, we performed random walks in forest and selected15 plants of each plant species at their flowering stage. for agricultural crops, 15 plants were selected randomly from the margins of the field. each plant was observed for 60 seconds in its floral units for any visit of honey bees. in this way there was a total of 15 minutes of observation per plant species in one census. only those plant species were selected which were in the phase of flowering. for each plant we counted the number of visiting individuals of both apis species by visual observation. visiting bees other than apis species were also recorded during the census. the census of each flowering plant species was carried out at two weeks interval throughout the flowering period. the observations were taken on clear sunny days, while rainy or cloudy days were avoided. to avoid the phenomenon of floral constancy i.e. bees tend to visit single plant species even in the presence of many other flowering plant species in that particular area (gruter et al., 2011), we selected wild plants of a particular species at a considerable distance from each other (>50m). we used rank-abundance plot with fitted curve as a way to visualize the overall visitation pattern of bees on 36 plant species (magurran, 2004). frequency distribution test was applied to identify various classes of plant species based on abundance of apis bees. we used linear regression a sajjad, m ali, s saeed – yearlong association of honey bees with plants20 table 1. monthly abundance of bees, on 49 plant species at bahauddin zakariya university campus, multan pakistan during january to december, 2008. plant species family flowering period flower color a. dorsata a. florea total class boundary lower-upper (contribution to ᵡ² ) calotropis procera asclepiadaceae jul-sep white+purple 36 93 129 103-129 (13.82) helianthus annuus asteraceae feb-mar yellow 59 24 83 77-103 (1.63)trifolium alexandrinum fabaceae apr-may pale 42 39 81 citrus reticulata rutaceae feb-mar white 56 6 62 52-77 (1.66)coriandrum sativum apiaceae feb-mar white 27 30 57 prosopis juliflora fabaceae mar-june pale 21 22 43 26-52 (3.15) melilotus indica fabaceae apr-aug yellow 41 41 moringa oleifera moringaceae apr-jun white 41 41 grewia subinaequalis malvaceae mar yellow 34 2 36 eucalyptus camaldulensis myrtaceae apr-may pale 34 1 35 launaea nudicaulis asteraceae feb-sep yellow 15 17 32 raphanus sativus brassicaceae feb-mar white+pink 12 18 30 momordica charantia cucurbitaceae apr-jul yellow 6 24 30 cirsium arvense asteraceae jan-mar purple 19 10 29 brassica campestris brassicaceae nov-jan yellow 8 19 27 tamarix aphylla tamaricaceae jun-oct pink 11 15 26 albizia procera fabaceae jun-aug white 21 1 22 ageratum conyzoides asteraceae mar-may purple 13 7 20 tribulus terrestris zygophyllaceae jun-aug yellow 9 7 16 haloxylon recurvum amaranthaceae sep-oct green 1 14 15 ziziphus jujuba rhamnaceae jun-jul green 14 1 15 daucus carota apiaceae feb-mar white 1 13 14 salsola baryosma amaranthaceae aug-oct green 13 1 14 launaea procumbens asteraceae feb-sep yellow 12 12 pulicaria crispa asteraceae mar-april yellow 11 1 12 dalbergia sissoo fabaceae mar-april pale 9 3 12 1-26 (31.59) trianthema portulacastrum aizoaceae sep-oct white+pnik 3 9 12 malvastrum coromendelianum malvaceae round the year yellow 8 3 11 capparis decidua capparaceae mar-apr pink 11 11 convolvulus arvensis convolvulaceae round the year white 8 8 parkinsonia aculeata fabaceae mar-may yellow 7 1 8 leucaena leucocephala fabaceae jun-nov pale 7 7 achyranthes aspera amaranthaceae oct-nov purple 6 1 7 sonchus oleraceus asteraceae mar-apr yellow 7 7 heliotropium europaeum verbenaceae apr-jun white 4 2 6 carthamus oxycantha asteraceae mar-may yellow 4 2 6 spergula arvensis caryophyllaceae mar-apr white 5 5 acacia nilotica mimosaceae jun-nov yellow 4 4 sonchus asper asteraceae feb-sep yellow 2 2 lantana camara verbenaceae round the year yellow+pink 2 2 physalis peruviana solanaceae july-oct yellow+brown 2 2 oxystelma esculentum asclepiadaceae dec white+purple 2 2 cleome viscose cleomaceae july-sep yellow 2 2 euphorbia helioscopia euphorbiaceae mar-apr green 1 1 oxalis corniculata oxalidaceae mar-dec yellow 1 1 allium cepa liliaceae mar-apr white 1 1 suaeda fruticosa amaranthaceae dec-jan green 1 1 chenopodium album amaranthaceae mar-apr green 1 1 portulaca oleracea portulacaeae aug-dec yellow 1 1 sociobiology 64(1): 18-25 (march, 2017) 21 analysis to explore the relationship between abundance of bees and abiotic (relative humidity and temperature) and biotic (number of plant species in flowering and total floral abundance) factors. results a total of 72 flowering plant species in 30 families were observed for recording the visitation of a. dorsata and a. florea. the total sampling efforts of 121 hours yielded 1049 interactions of both bee species (constituting 44.36% of total native bee visits) on 49 plant species in 26 families (table1). twenty three plant species in 18 families were not visited by any of the two apis species (table 2). a total of 588 individuals of a. dorsata were recorded on 40 plant species in 21 families while 454 individuals of a. florea were recorded on 38 plant species in 19 families. eleven plant species were exclusively visited by a. dorsata while 13 by a. florea. twenty five plant species were visited by both the bees (table 1). the rank abundance curve shows only a few plant species with higher bee abundance while many plant species showed lower bee abundance (fig 1). the frequency distribution test identified five classes of plant species based on visitation frequency of both bee species. calotropis procera (class 1) was visited by the maximum number (129) of bees followed by helianthus annuus, trifolium alexandrinum (class 2) and citrus reticulata and c. sativum (class 3). class 4 included ten plant species while class 5 included 34 plant species (table 1). the plant species most visited by a. dorsata included h. annuus, c. reticulata, t. alexandrium, moringa oleifera and c. procera, while the most visited plant species by a. florea included c. procera, m. indica, t. alexandrium, coriandrum sativum and h. annuus (table 1). plant species family flower color flowering period sesuvium sesuvioides aizoaceae purple dec mangifera indica anacardiaceae green+white mar-apr asphodelus tenuifolius asphodelaceae white nov-apr conyza canadensis asteraceae green+white may-jun sisymbrium irio brassicaceae yellow mar-may stellaria media caryophyllaceae white apr-aug chenopodium murale chenopodiaceae green jan-july convolvulus sp. convolvulaceae white mar-jun cucumis prophetarum cucurbitaceae yellow jun-july phyllanthus niruri euphorbiaceae green aug-sep cajanus cajan fabaceae yellow jul-aug medicago sativa fabaceae purple may-sep lathyrus sativus fabaceae pruple+white feb-mar vicia sativa fabaceae purple jul-aug alhagi graecorum fabaceae pink mar fumaria indica fumariaceae pink marjun centaurium pulchellum gentianaceae pink may-jun abutilon indicum malvaceae yellow aug-dec abelmoschus esculentus malvaceae yellow jun-aug melia azedarach meliaceae white+ purple mar-apr ranunculus muricatus ranunculaceae yellow mar-apr solanum surattense solanaceae purple apr-aug torilis japonica apiaceae white feb-mar table 2. plant species which were not visited by a. dorsata and a. florea during january to december, 2008. 0 20 40 60 80 100 120 140 0 10 20 30 40 50 po pu la ti on o f b ee s rank of plant species fig 1. rank abundance curve based on population of a. dorsata and a. florea on 49 plant species at bahauddin zakariya university campus, multan, pakistan during january to december, 2008. the seasonal dynamics revealed the peak activity of both apis bees during early march to late may. almost no activity was recorded in the months of november, december and january (fig 2). similarly, the maximum availability of floral resources (abundance of floral units and number of plant species in flowering) was also recorded in the spring season (march to may) followed by a gradual decline until december (fig 3). average monthly temperature and relative humidity is shown in fig 4. 0 50 100 150 200 250 jan feb mar apr may jun jul aug sep oct nov dec o ve ra ll ab un da nc e apis dorsata apis florea fig 2. monthly abundance of a. dorsata and a. florea at bahauddin zakariya university campus, multan, pakistan during january to december, 2008. a sajjad, m ali, s saeed – yearlong association of honey bees with plants22 monthly abundance of both bee species was positively related to the floral resources i.e. number of plant species in flowering (y = 10.43x-15.81, r² = 0.338, p = 0.047) and abundance of floral units (y = 0.029x 31.76, r² = 0.382, p = 0.032). the monthly abundance of bees had a significant negative relationship (y = -5.674x + 474.9, r² = 0.409 p = 0.025) with relative humidity while no relationship was verified with temperature. availability of food in general while livelihoods of local communities in specific (devy & davidar, 2006). therefore, in order to maintain honeybee populations, it is important to ensure the conservation and management of sufficient forage resources within agricultural and surrounding natural landscapes (zhang et al., 2007). these resources include both suitable nesting habitat and sufficient floral resources i.e. nectar and pollen (kremenet al., 2007). in various parts of india, a. dorsata, a. cerana, a. florea and a. mellifera are the most effective pollinators (chandel et al., 2004). apid bees probably play an important role in pollinating crop plants in india, although quantitative data are lacking (potts et al., 2003). in our study, both honey bee species visited 49 species across the year. honey bees are generalist feeders and visit several plant species for getting nectar and pollen (michener, 1990) yet they prefer the flowers with exposed nectar, abundant pollen production, zygomorphic nature and compact umbels (free & ferguson, 1983; koul et al.,1989; diederichsenn, 1996). that is why in our study, their higher abundance was recorded mostly on open shaped flowers (c. procera, h.annus) along with zygomorphic (m. indica, t. alexandrium) and umbelliferous flowers (c. sativum). previous studies have also reported honey bees as the frequent visitors of calotropis sp. (betz et al., 1994; fishbein & venable, 1996), h. annus (jadhavet al., 2011) and c. sativum (abou-shaara, 2015). twenty five plant species were visited by both apis species. there was evidence of ecological niche overlapping among them i.e. value of pianka’s index was 0.66 (simulated indices mean = 0.66, simulated indices variance = 0.000, p (observed >expected) = 0.122) at alpha 0.05. different floral nectar compositions are preferred by different species (abrol, 2011). for example, a. florea prefers flowers having low caloric rewards (sihag & rathi, 1992) whereas a.dorsata prefers flowers with high caloric rewards. similarly, in our studies, a. dorsata did not visit two of the most preferred host plants of a. florea i.e. m. indica; c. sativum which contain low nectar rewards. twenty three plant species were not visited by both of the apis species. however, some of these plant species have already been reported as good source of pollen and nectar for a. mellifera i.e. sisymbrium irio, cucumis prophetarum, medicago sativa (taha, 2015). besides this, there seems no specific reason of avoiding some other very good sources of nectar and pollenin this study like mangifera indica, conyzacan adensis, cajanus cajan, abutilon indicum and torilis japonica etc. this might be due to the marked behavior of floral constancy among bees (amaya-marquez, 2009). floral constancy is the behavior of restricting visits largely to a single floral type (waser, 1986) yet little is known about the reasons and thereby no any general theory is in place that can explain all kinds of floral constancy (amaya-marquez, 2009). this constraint suggests taking into account rather a large geographical area when attempting quantification of floral host plants of bees in order to avoid this behavior. 0 5 10 15 20 25 0 2000 4000 6000 8000 10000 jan feb mar apr may jun jul aug sep oct nov dec n um be r of fl ow er in g pl an t s pe ci es a bu nd an ce o f f lo ra l u ni ts floral abundance flowering plant species fig 3. availability of floral resources (number of flowering plant species and floral abundance) at bahauddin zakariya university campus, multan, pakistan during january to december, 2008. fig 4. average monthly temperature and relative humidity at bahauddin zakariya university campus, multan, pakistan during january to december, 2008. 40 50 60 70 80 90 5 15 25 35 45 jan feb mar apr may jun jul aug sep oct nov dec t em pe ra tu r ° c r el at iv e hu m id ity % temperature relative humidity discussion in this study, we recorded 1049 visits of both apis species which constituted 44.36% of the total pollinating bee visits. this high abundance of honey bees signifies the importance of semi-natural habitats (i.e. forest in our case) in maintaining the viable population of pollinators (johannsmeier & mostert, 2001). moreover, the size and quality of seminatural habitats have been reported to impact significantly on the bee abundance in adjacent agricultural landscapes (liow et al., 2001). since insect pollination contributes 75% of total global crop pollination while honey bees constitute 7080% of insect pollination (klein et al., 2007), loss of honey bees therefore can significantly impact the biodiversity and sociobiology 64(1): 18-25 (march, 2017) 23 the seasonal dynamics revealed the peak activity of both apis bees during early march to late may (summer months) while almost no activity was recorded from november to january (winter months). this could be due to the availability of high foraging resources in summer months compared to the winter months as it is clear from the significant positive relationship of monthly bee abundance with the floral resources (number of plant species in flowering and availability of floral units) in our data. hussein et al. (1992) also reported the maximum honeybee foraging activities during the summer months (july to september) and minimum in winter months (november to january). whereas, it has also been studied that a. dorsata migrates locally in response to the availability of floral resources and its abundance showed positive relationship with the floral resources (itioka et al., 2001; sihag, 2014). the monthly bee abundance was also found to have significant negative relationship with relative humidity while it had no relationship with the temperature. the previous studies have also reported a decreased daily activity of honey bees on the days characterized by maximum temperature and highest humidity (nargis et al., 2001; kumar et al., 2002; mordago et al., 2002). high humidity, heavy rain fall, wind and low temperature had negative influence on sunflower inflorescences visits. knowledge of the honeybee flora of an area is a basic tool for the development and sustainability of apiculture as well as commercial agricultural crop pollination (dimou et al., 2006). the availability of adequate forage resources positively impact the honeybee colony health and beekeeping profitability leading to improved crop pollination (van engelsdorp & meixner, 2010). the current study may serve as a baseline to track the degradation in ecosystem service of cross pollination and making new conservation strategies at local scale while future research should focus on tempo-spatial variations in foraging preferences, floral constancy and effect of foraging competition on crop pollination in different ecological regions of pakistan. references abou-shaara, h.f. 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(2010). a historic review of managed honey bee populations in europe and the united states and the factors that may affect them. journal of invertebrate pathology, 103: 580-595. doi: 10.3896/ ibra.1.49.1.03 waser , n.m. (1986). flower constancy: definition, cause, and measurement. the american naturalist, 127: 593-603. zhang, w., ricketts, t.h., kremen, c., carney, k. & swinton, s.m. (2007). ecosystem services and dis-services to agriculture. ecological economics, 64: 253-260. doi: 10.1016/j.ecolecon. 2007.02.024 (2007) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.975sociobiology 63(1): xx-xx (march, 2016) m s 97 5 p r o o f opportunistic occupation of nests of microcerotermes spp. silvestri (termitidae: termitinae) by partamona seridoensis camargo & pedro (apidae: meliponini) in the brazilian tropical dry forest social bees have evolved a diversity of strategies to protect their colonies from environmental stressors, mainly climatic adversities and predators. whereas the majority of bee species makes use of natural structures, such as cavities in trees or in the ground, some occupy animal-constructed shelters, often sill inhabited by the builders (wilson, 1971; roubik, 2006). among these, particularly invitingfor many kinds of inquilines are active termite nests due to the stable climatic conditions of the intranidal environment (greaves, 1964; redford, 1984; korb & linsenmair, 2000; dechmann et al., 2004). the reduced thermal variations inside of termite nests is probably the main attractive for colonies of stingless bees (apidae, meliponini),a group of highly eusocial bees comprising more than 500 species distributed in the tropical and southern subtropical regions around the globe (michener, 2013). due to a limited capacity to actively regulate the nest temperature, abstract social bees make use of natural or animal-built structures to protect their colonies from environmental stressors. here, particularly attractive shelters are active termite nests because they provide a stable climatic environment for inquilines. several social bee species form obligatoryassociations with termites, among these the stingless bee partamona seridoensis (apidae: meliponini), whose distribution is limited to the tropical dry forest in the brazilian northeast. so far, colonies of this meliponine species have been found mainly in arboreal nests of the termite constrictotermes cyphergaster, which suggests a tight relationship between these two social insect species. the present study was conducted in an area of the tropical dry forest in the brazilian state of rio grande do norte, where p. seridoensis naturally occurs albeit the absence of c. cyphergaster.we registered 14 colonies of p. seridoensis, all occupyingactive arboreal nests of termites of the genus microcerotermes. the only other termites with arboreal nests present in the study area, nasutitermes corniger, never housed p. seridoensis. thisselective preference of the bees for microcerotermesnests might be due to differences between termites concerning defense mechanisms or concerning thermal stability within the nests. our study indicates that the occurrence of p. seridoensis is not restricted to the presence of c. cyphergaster in the brazilian tropical dry forest, and suggests that the bees opportunistically use the best nesting substrates available in the environment. sociobiology an international journal on social insects tffn oliveira, ll silva, m hrncir article history edited by evandro nascimento silva, uefs, brazil received 03 january 2016 initial acceptance 06 march 2016 final acceptance 07 march 2016 keywords stingless bees, termitophily, defense mechanisms, opportunistic association, thermoregulation, caatinga. corresponding author michael hrncir universidade federal rural do semi árido, rodovia br-226, s/nº, 59900-000 pau dos ferros rn, brazil e-mail: michael@ufersa.edu.br these bees depend on nesting substrates that provide a stable microclimate to guarantee successful brood development and colony survival (roubik, 2006; jones & oldroyd, 2007). several meliponine species obligatorily use termite nests as nesting substrate, among these partamona seridoensis camargo &pedro 2003 (camargo & pedro, 2003; roubik, 2006), whose occurrence is restricted to the brazilian tropical dry forest, the caatinga, in northeastern brazil (camargo & pedro, 2013). the climate of this biome is characterized through rare and irregular rainfall, high solar radiation, and elevated ambient temperatures throughout the year, with averages varying from 26 to 34 °c (andrade-lima, 1981). these abiotic conditions are among the principal causes for the reduced occurrence of social bees in the caatinga as compared to the adjacent ecoregions, the atlantic rainforest, the tropical savanna (cerrado), and the amazon rainforest short note universidade federal rural do semi-árido, mossoró, rn, brazil tffn oliveira, ll silva, m hrncir – opportunistic occupation of termite nests by meliponine bees2 m s 97 5 p r o o f (zanella, 2000). nesting inside of termite nests, which provide a stable microclimate, might be a selective advantage for meliponine species facilitating colony survivalin the brazilian tropical dry forest (carvalho et al., 2014). to this day, colonies of p. seridoensis have been found mainly in arboreal nests of constrictotermes cyphergaster silvestri 1901 (termitidae, nasutitermitinae) (lorenzon et al., 1999; barreto & castro, 2007; fernandes, 2011; miranda et al., 2015), despite the abundant occurrence of two other termite generawith the same nesting habit, nasutitermes dudley 1890 (termitidae, nasutitermitinae) and microcerotermes silvestri 1901 (termitidae, termitinae), in the caatinga (martius, 1999; vasconcellos et al., 2010; viana junior et al., 2014). this fact suggests a tight relationship between p. seridoensis and c. cyphergaster. however, what happens if c. cyphergasterdoes not occurat a certain location, either owing to the lack of adequate feedingand nesting substrates (leite et al., 2011; bezerra-gusmão et al., 2013), or due to its exterminationin man-modified environments? do the associated bees also not occur at these locations? or do they opportunistically switch to other host species? the present study was performedto answer this question. surveys were conducted between february and november of 2015 at the experimental field-station rafael fernandes of the brazilian federal university at mossoró (ufersa), rio grande do norte, brazil (5º 03’ s, 37º 24’ w).the field-station comprises a total area of 416 ha, divided into a central area for experimental agriculture surrounded by a belt of native caatinga vegetation, partly in regeneration after destruction by fire in the 1980ies. in the course of a bee survey at the field-station, various specimens of p. seridoensis had been collected at flowers (airton torres carvalho, personal communication, april of 2012), indicating the presence of colonies of this meliponine species in the vicinity. we located colonies of p. seridoensis through active search in the areas with native caatinga vegetation (total sampling effort: 60 hours). the termite nests housing meliponine colonies were identified to the species-level based on speciesspecific external morphological characteristics of the nests (constantino, 1999). in total, we found 14 colonies of p. seridoensis, all nesting in active arboreal nests of termites of the genus microcerotermes, 13 associated with microcerotermes indistinctus mathews 1977 and one with microcerotermes strunckii sörensen 1884. to evaluate whether this observed association between p. seridoensis and microcerotermes spp.was related to the dominance of this termite genus in the study area, we estimated the abundance of arboreal nests of all termite species present in the area. for this, we chose randomly five transects of 200 m each within the area covered by native caatinga vegetation. all arboreal termite nests within 10 m to the left and right of the transect (total sampling area = 20,000 m²) were identified to the species level according to constantino (1999). in total, we located 34 nests of m. indistinctus and 5 nests of nasutitermes corniger motschulski 1855. the estimated abundance of m. indistinctus nests in the study area (17 nests/ha) was almost seven times that of n. corniger nests (2.5 nests/ha), pointing to a strong dominance of microcerotermes at the university’s field-station. we found no nests of c. cyphergaster within the sampling area. our study is the first report of an association between p. seridoensis and termites of the genus microcerotermes, therewith demonstrating that the occurrence of this meliponine species is not at all restricted to the local availability of c. cyphergaster. apparently, the bees occupy nests of other termite species, selecting opportunistically the best nesting substrates available in the respective environment. despite the presence of n. corniger in the study area, the absence of p. seridoensis nests associated with this termite species suggests that some feature other than availability of arboreal termite nests affects the choice of the nesting substrate by the bees. here, differences between termites concerning their defense mechanisms might impose different levels of difficulty for nest invaders. the defense secretions of nasutitermes soldiers together with the pheromone-mediated coordination of aggressive attacks compose a much more efficient defense strategy than that of microcerotemes, which relies mainly on biting and piercing the opponents with the mandibles (prestwich, 1979; prestwich, 1984; leponce et al., 1999; quinet et al., 2005). second, it might be more difficult for invaders to isolate themselves successfully from the termites in nasutitermes nests, given that the openings between chambers are much wider than in nests of microcerotermes (deligne & pasteels, 1982).these facts taken together suggest that invasions by p. seridoensis may be easier in microcerotermes nests than in nasutitermes nests. additionally to differences in nest defense, differences between microcerotermes and nasutitermes concerning the physical nest characteristics might influence the choice by p. seridoensis. owing to the bees’ need for nesting substrates that minimize the thermal stress of the outside environment (jones & oldroyd, 2007), differences between termite nests concerning their quality of thermal insulation may bias the bees’ nest-site selection.whereas nasutitermes build carton nests using wood particles in the construction, microcerotermes use mainly soil and sand particles for building their nests (emerson, 1938). this inclusion of mineral material results in an increased material density (amelung et al., 2002) and, consequently, in an increased heat capacity of the material used for nest construction (john et al., 2005). hence, in environments with elevated ambient temperatures, as is the case in the brazilian tropical dry forest, microcerotermes nests may be more attractive for inquilines owing to the fact that more thermal energy is necessary to heat-up the building materialas compared to nasutitermes nest. thus, the observed selective preference for microcerotermes over nasutitermes nestsby p. seridoensis in the study area may be a result of both the ease of invasion and thermal stability. sociobiology 63(1): xx-xx (march, 2016) 3 m s 97 5 p r o o f acknowledgements the authors thank elder assis miranda for his help with identifying the bee species. this work was supported by the brazilian funding agencies conselho nacional de desenvolvimento científico e tecnológico (cnpq) under grants 481256/2010-5, 309914/2013-2, 404156/2013-4, and coordenação de aperfeiçoamento de pessoal de nível superior (capes) under grant mi nº 55/2013 – pró-integração, auxpe: 3168/2013. references amelung w., martius, c., bandeira, a.g., garcia, m.v.b., & zech, w. 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(1984). the termitaria of cornitermes cumulans (isoptera, termitidae) and their role in determining a potential keystone species. biotropica, 16:112-119.doi: 10.2307/2387842 roubik, d.w. (2006). stingless bee nesting biology. apidologie, 37: 124-143. doi: : 10.1051/apido:2006026 vasconcellos, a., bandeira, a.g., moura, f.m.s., araújo, v.f.p., gusmão, m.a.b., & constantino, r. (2010). termite assemblages in three habitats under different disturbance regimes in the semi-arid caatinga of ne brazil. journal of arid environments, 74: 298-302. doi: 10.1016/j.jaridenv.2009.07.007 viana junior, a.b., souza, v.b., reis, y.t. & marquescosta, a.p. (2014) termite assemblages in dry tropical forests of northeastern brazil: are termites bioindicators of environmental disturbances? sociobiology, 61: 324-331. doi: 10.13102/sociobiology.v61i3.324-331 wilson, e.o. (1971). the insect societies. cambridge: belknap press, 548 p zanella, f.c.v. (2000). the bees of the caatinga (hymenoptera, apoidea, apiformes): a species list and comparative notes regarding their distribution. apidologie, 31: 579-592. doi: 10.1051/apido:2000148 _goback open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v62i4.807sociobiology 62(4): 513-518 (december, 2015) fast food delivery: is there a way for foraging success in leaf-cutting ants? introduction one of the most important aptitudes of life involves motility. from intracellular molecular transport to flight of birds, movement is one of life’s central attributes (chowdhury et al., 2005). this assumption is strikingly evident for leaf-cutting ants who travel along trails hundreds of meters long (lewis et al., 2008). walking long distances through a trail system is an intrinsic feature of leaf-cutting ants. the main function of this trail system is to guide foragers between the nest and the resource patch (shepherd, 1982; fowler & stiles, 1980). foraging trails arise from the recruitment process, which involves outbound scouts, who are the first workers to leave the nest seeking food. once they find it, the scouts return to the nest laying chemical cues, which leads forager workers to the resource (jaffe & howse, 1979). after collecting leaves, foragers return to the nest also laying the trail pheromone. as a result, a positive feedback occurs, in which the more intense the pheromone trails, the more ants that are recruited and so on (sumpter, 2006). this huge flow of individuals can abstract walking long distances through a trail system is an intrinsic feature of the leafcutting ants. workers travel hundreds of meters to forage by using well-defined physical routes that are cleared of vegetation or obstacles. despite the costs of construction and maintenance, cleared trails should promote more benefits than non-physical ones, especially related to the leaf supply for the colony. here, the leaf delivery rate in constructed and non-constructed trails was compared by counting the foraging flow and travel time of workers. in addition, we measured the length and width of trails. the leaf delivery rate was almost 70% higher for foragers who were walking along physical trails. the forager walking speed on physical trails was 86.10% higher. these significant increments might be related to the truly narrow corridor present inside physical trails that are leaf litter-free, and thereby chemically stronger and smoother than non-physical ones. the speed improvement could induce the construction of longer trails, which guide the workers more efficiently to the foraging patch. thus, physical trails have an important role in foraging efficiency as they allow workers to go quickly and further to forage, since they limit a path and congregate more individuals, raising the leaf delivery rate. this study provides additional information about foraging trails of leaf-cutting ants. sociobiology an international journal on social insects ta sales, in hastenreiter, ng almeida, jfs lopes article history edited by kleber del-claro, ufu, brazil received 06 may 2015 initial acceptance 23 june 2015 final acceptance 13 october 2015 keywords atta sexdens, physical trails, leaf delivery rate. corresponding author tatiane archanjo de sales rua josé lourenço kelmer, s/nº campus universitário, são pedro 36036-900, juiz de fora – mg, brazil e-mail: tatiane.archanjo@gmail.com assure a greater food supply to the colony, since more forager workers are directly related to increase of material collection. however, the forager flow along a trail can be established in various environments with different physical characteristics, such as rugosity (moll et al., 2010), and in the presence of leaf litter (bruce & burd, 2012), which may or may not facilitate the forager traveling (bollazzi & roces, 2011). some ant species, such as those which belong to the genera atta, are able to construct true corridors by cleaning vegetation and debris from the trails. ant-constructed trails (from now on “physical trails”) have inherent costs, such as the construction effort (lugo et al., 1973; shepherd, 1982) and reduction of the foraging flow due to increase of head-on collisions among foragers traveling on the same path (burd & aranwela, 2003). on the other hand, the intrinsic benefits of physical trails involve increased velocity, improved traillaying properties (shepherd, 1982) and even physical memory of foraging (lewis et al., 1974; shepherd, 1982; farji-brener & sierra, 1998). another extremely profitable circumstance to workers is that physical trails may remain inactive in the field research article ants universidade federal de juiz de fora, mg, brazil ta sales et al. – foraging success in physical and non-physical trails 514 up to eight months without regrowth of vegetation (rockwood & hubbell, 1987) and it can be reactivated with almost no cost. thus, considering the advantages and disadvantages of constructed trails it is probable that the benefits of physical trails are greater than non-physical trails, especially related to the leaf supply of the colony. here, we compared in the field the leaf delivery rate and travel time of forager workers in physical and nonphysical trails of atta sexdens (linnaeus, 1758) colonies, as well as some physical features of the trails such as length and width. the investment in physical trail construction and maintenance is offset by worker walking speed improvement (johnson & hubbell unpublished data, cited in rockwood & hubbell, 1987), thus it has been argued if the leaf delivery rate in physical trails may also be higher than when physical trails are not present. also, physical trails may be longer and narrower since their construction is actively modulated by workers. material and methods the study was carried out in rio preto state park, minas gerais, brazil (18°9’15”s 43°20’50”w), located in the cerrado biome. this park is a conservation unit with approximately 12 ha. data was taken in august 2014, during four days between 7:00 and 11:00 pm, with assistance of a white flashlight filtered with red cellophane paper. we selected three physical and three non-physical trails located at least 100-m apart from each other. physical trails were those free of debris and/or litter and visible soil surface (fig 1), while non-physical trails were those in which it was not possible to see the soil surface, but leaf transport by workers was observed. in each trail, we established a 20 cmtrail section 2m apart from the foraging hole. at this section, we also established a fixed point for foraging flow register. to assess the foraging flow we counted loaded and unloaded inbound foragers at three consecutive 1-minute intervals every 5 minutes for 20 minutes. thus, it was possible to obtain 12 replicates of 1-min foraging flow/trail/day. we repeated this procedure for four days in all six selected trails. therefore, there are 144 replicates for physical trails and 144 for non-physical trails. flow data was used to calculate leaf delivery rate (ld)/minute by the formula (dussutour et al., 2007): ld= li/(li+ui), where: li= loaded inbound foragers; ui = unloaded inbound foragers. we registered the travel time of 20 loaded inbound medium workers along the 20cm-trail section in each one of the six trails during the same four days. medium workers were selected due to this size class being likely to act as foragers (wetterer, 1999). in addition, as they belong to the same size class, it is possible to infer that they were probably carrying leaf fragments of the same size (weber, 1972). in order to compare trail features, we measured the length of the trails from the nest entrance to the foraging patch while width was taken every 50 cm, beginning from the foraging patch. we repeated this procedure daily for nonphysical trails, as they were not marked over the soil surface. the anova test was used to analyse the response of leaf delivery rate under effect of the categorical variable type of trail (physical and non-physical) and the influence of type of trail in the continuous variable travel time. trail width variances were not homogeneous (bartlett’s k-squared: t = 236.00, df = 1, p <0.001), thus this variable was compared with the mann-whitney-wilcoxon test. in the same way, the trail length variance homogeneity was tested (bartlett’s k-squared: t = 2.95, df = 1, p = 0.085) and now the t test was more appropriate to compare physical and non-physical trail length. statistical analyses were performed and graphs generated in the r environment (r core team 2013). fig 1. atta sexdens physical trail. parque estadual do rio preto mg, brazil. results leaf delivery rate was higher in physical trails than in non-physical ones (f = 87.33, df = 1 and 474, p < 0.001) (fig 2) (physical: = 0.39 ±0.22un/min, maximum value = 1.00, minimum value = 0.03; non-physical: = 0.23 ±0.17, maximum value = 0.75, minimum value = 0.02). leaf delivery rate also represents the proportion of laden foragers in relation of the total flow of inbound foragers, so it indicates that there were proportionally more laden foragers in physical trails than in non-physical ones. travel time of forager workers was shorter on physical trails than along non-physical ones (f = 206.41, df = 1 and 474, p < 0.001) (fig3), indicating that foragers walked faster in these trails and thus physical trails facilitate the foragers’ travel. in other words, across the 20cm observed, the travel time of laden foragers on clean paths were 48.33% lower than for workers on non-physical trails (physical: = 20.14 ±7.32s, non-physical: = 38.98 ±19.10s). in other words, the forager walking speed raised in average 86.10% in physical trails. sociobiology 62(4): 513-518 (december, 2015) 515 physical trails were longer than non-physical ones (t = -5.444, df = 2.242, p = 0.025), which allow workers to explore resources farther from the nest (fig 4a). the average length of physical trails was 21m (maximum value = 24.3m, minimum value = 16.2m) and for non-physical trails was 8m (maximum value = 10.4m, minimum value = 5.4m). furthermore, physical trails were narrower than non-physical ones (w = 7127, p < 0.001), promoting a directed flow as workers travel along a delimited path (fig 4b). the average width of physical trails was 6.72 cm (maximum value = 10cm, minimum value = 3cm) while for non-physical trails was 11.11 cm (maximum value = 75cm, minimum value = 3cm). discussion the present study has indicated that the leaf delivery rate was on average 69.56% higher in physical trails when compared with non-physical ones. inbound laden workers walking in these trails took less time to travel up to the nest compared with those traveling in non-physical ones, which could explain the construction of longer and narrower trails. several works have well documented the widespread feature of ant species with regards to physical trails and their benefits in terms of foraging efficiency (shepherd, 1982; hölldobler & wilson, 1990; anderson & mcshea, 2001) and the impact of rough surfaces on walking speed (seidl & wehner, 2008; bernadou et al., 2011). clear trails enable workers to forage in a straight line, almost without obstacles, increasing their walking speed. physical trails seems to be an important attribute to leaf-cutting ants (rudolph & loudon 1986; wetterer, 1990; roces & hölldobler, 1994; burd, 1996), since it increases the leaf delivery rate of the colony. nevertheless, the presence of leaf litter along the path could make the traffic flow of forager ants difficult, resulting in a lower leaf delivery rate on non-physical trails. even in physical trails, there is an edge-trail effect provoked by the vegetation on trail edges that disturb the forager flow (bruce & burd, 2012). once non-physical trails are formed over leaf litter, forager workers must be under this edge-trail effect all along the trail path. in this way, a higher disturbance on the traffic flow in non-physical trails would be expected when compared with physical ones. physical trails were the narrow ones and those with high leaf delivery rate, inferring that narrow trails improve leaf delivery rate. a similar negative relationship between trail width and foraging efficiency has already been identified for atta colombica (guérin-méneville, 1844) (dussutour et al., 2007). fig 2. relationship between leaf delivery rate and the two types of trails physical and non-physical. fig 3. relationship between travel time of forager workers and the two types of trails physical and non-physical. fig 4a. comparison of foraging trail features physical and nonphysical. a) average and standard deviation of physical and nonphysical trail length. ta sales et al. – foraging success in physical and non-physical trails 516 this relationship can be explained by the ontogeny of physical trails. since non-physical trails are predecessors of the physical ones, they are chemically weak, confused and the pheromone marking is dispersed, whilst the physical ones become chemically stronger (edelstein-keshet et al., 1995) through a positive feedback mechanism (sumpter, 2006), in which several forager workers will follow and reinforce the pheromone cues. in the case of intense flow persistence, these trails now chemically stronger will delineate straight lines over time (edelstein-keshet et al., 1995). this process allied to path clearing by workers molds physical trails. physical trails are narrower and more attractive to the ants due the high pheromone concentration (edelstein-keshet et al., 1995). undisputed is the fact that the construction and maintenance of physical trails incur costs (lugo et al., 1973; shepherd, 1982; howard, 2001). these costs can be variable depending on environmental conditions as the potential gains differ according with distance, quality and availability of different food sources. in this way, some features that influence the workers travel time, like wind and surface rugosity (lewis et al., 2008; seidl & wehner, 2008), could also be considered here. cleared trails create barriers unintentionally at both sides of the trail. one can therefore assume that foragers may be protected from some abiotic factors, for instance the wind. high wind speed could unbalance the laden foragers by tilting them laterally and making them slower. inside the trail, however, workers may be less affected by this force. likewise, physical trails are leaf litter-free and thereby are smoother than non-physical ones. this evident difference at soil surface rugosity leads a striking improvement in travel speed of workers in these type of trails. data showed that the walking speed was 86.10% higher in physical trails when comparing with non-physical ones. in a field study comparing cleared soil trails and foraging trails over fallen branches, farji-brener et al. (2007) found that due to lower rugosity, foragers have higher speed over fallen branches than those walking along cleared trails on the forest floor. this intrinsic feature of physical trails leads to a shorter travel time and a higher leaf delivery rate beyond the workers having a physical foraging trail memory (lewis et al., 1974; shepherd, 1982; farji-brener & sierra, 1998). our results highlight one more advantage to physical trail construction that surpasses their costs, because when foragers travel along longer trails they spend much time traveling from the nest to the foraging patch. the additional time needed for this round trip causes a delay of laden workers arriving to the nests (bruce & burd, 2012). here we verified that physical trails, which are longer than the non-physical ones, had a higher leaf delivery rate due to being cleared and thus reducing the travel time. the travel speed of forager workers seems to be an important attribute of the foraging process (wetterer, 1990; roces & hölldobler, 1994; burd, 1996; farji-brener et al., 2007). furthermore, long physical trails allow workers to enlarge their foraging area (howard, 2001). the same occurs for camponotus sericeiventris. for this species, lianas and plant branches can function as physical trails, increasing their life area (yamamoto & del-claro, 2008). physical trails allow workers to move faster and, as they become longer and narrower, the foraging flow becomes greater, allowing both the discovery of new and more distant unexplored food sources as well as the maintenance of stable routes to permanent and already known food sources. we showed that physical trails are more profitable to the colony than non-physical ones as they decrease the time travel of laden inbound workers and thus increase leaf delivery rate to the colony even for more distant food sources. moreover, physical trails are narrower, mobilizing a huge quantity of workers rapidly through chemical recruitment. physical trails have such a great role in foraging efficiency as they (i) enlarge colony foraging territory; (ii) speed-up forager workers along their trip; (iii) limit a path in the external environment and so focus more individuals; and finally (iv) raise the leaf delivery rate, making the foraging even more efficient. this study contributes to explain the investment in physical trail construction, filling gaps for the comprehension of the complex and successful foraging system of leaf-cutting ants. acknowledgments the authors are grateful to professor dr. ricardo ildefonso campos and the reviewers for their comments, which improved the quality of this manuscript; to universidade federal de juiz de fora and conselho de aperfeiçoamento de pessoal de nível superior (capes) for the grants; to programa de pós-graduação em ecologia ufjffor the opportunity to participate of the curso de campo, offered by universidade federal de ouro preto, universidade federal de viçosa and universidade federal de juiz de fora. fig 4b. minimum, medium and maximum width of non-physical and physical trails. sociobiology 62(4): 513-518 (december, 2015) 517 references anderson, c. & mcshea, d.w. 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(2008). natural history and foraging behavior of the carpenter ant camponotus sericeiventris guérin, 1838 (formicinae, campotonini) in the brazilian tropical savanna. acta ethologica 11:55-65. doi:10.1007/s10211-0080041-6. open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v63i1.854sociobiology 63(1): 693-698 (march, 2016) morphological differences between reproductive and non-reproductive females in the social wasp mischocyttarus consimilis zikán (hymenoptera: vespidae) introduction insect societies are characterized by the division of labor between the reproductive and non-reproductive castes, which is essential for the success of their societies (robinson, 1992). in advanced eusocial species, castes are determined pre-imaginally and there is a clear physical divergence between queen and worker (bourke, 1999). however, in less-advanced eusocial species, morphological divergences between females are less pronounced, and the factors affecting caste differentiation are not well known (smith et al., 2011; murakami et al., 2009; 2013; jandt et al., 2014; montagna et al., 2015). nevertheless, evidence suggests that the morphological attributes, such as body size and ovarian state, can play an important role in establishment of the reproductive division of labor in independent-founding (if) polistine wasps (tibbetts & dale, 2004; tibbetts, 2006; cervo et al., 2008). abstract phenotypic divergence plays an important role in establishment of the reproductive division of labor among castes in eusocial insects; however, little is known about this subject in social wasps. we tested the hypothesis that alpha foundressesin colonial foundations of the independent-founding polistine wasp mischocyttarus consimilis zikán show greater body size and ovarian development than auxiliary and solitary foundresses. the hypothesis was also tested that females produced in the late post-emergence stage (gynes) are larger than those produced at the beginning (workers) of the colony cycle of this species. our results showed that the body size of alpha foundresses was significantly greater than that of auxiliary foundresses, but did not differ from that of solitary foundresses. in addition, the alpha had greater ovarian development than the auxiliary and solitary. we also demonstrated that gynes were significantly larger than workers. these results therefore suggest that the phenotype of females emerging in colonies of m. consimilis can vary with the progress of the colony cycle, and that body size and ovarian state are closely linked to the function performed by the foundress. sociobiology an international journal on social insects ts montagna1, wf antonialli-junior1,2 article history edited by kleber del-claro, ufu, brazil received 22 june 2015 initial acceptance 28 january 2016 final acceptance 16 march 2016 publication date 29 april 2016 keywords caste determination, division of labor, dominance hierarchy, neotropical wasp, polistinae, reproductive status. corresponding author thiago santos montagna prog. de pós-graduação em entomologia e conservação da biodiversidade universidade federal da grande dourados cep: 79804-970, dourados-ms, brazil e-mail: thiagomontag@yahoo.com.br if polistine wasps are an important model for study of the division of labor in less-advanced eusocial species. colonies of if polistine wasps can be initiated individually (haplometrosis), when a reproductive female unaccompanied by other females starts the colony; or by association (pleometrosis), when two or more reproductive females start the colony (westeberhard, 1969; reeve, 1991). in haplometrosis, the foundress performs all the intraand extra-nidal tasks until the workers emerge (jeanne, 1972; gadagkar, 1991). in pleometrosis, the colony tasks are shared among the foundresses, although the reproductive function is performed by one female, who dominates the others (röseler, 1991; keeping, 1992; prezoto et al., 2004). hence, females with a greater capacity for dominance are more likely to perform the colony reproductive function (cervo et al., 2008; zanette & field, 2009). since physical attributes are linked to the capacity for dominance, it has been suggested that body size could influence the 1 universidade federal da grande dourados, dourados-ms, brazil 2 universidade estadual de mato grosso do sul, dourados-ms, brazil research article wasps t. s. montagna; w. f. antonialli-junior – morphological caste in the social wasp mischocyttarus consimilis694 reproductive strategies in pleometrotic foundations (fukuda et al., 2003; tannure-nascimento et al., 2008). in tropical regions, colonies of if polistine wasps are started at any time of the year, so different colonial stages can occur in the same population in the same period. this nesting cycle pattern is termed asynchronous (poltronieri & rodrigues, 1976; o’donnell & joyce, 2001; giannotti, 1998; torres et al., 2011). the workers are produced at the beginning of the post-emergence stage, and they are mainly dedicated to defending the colony and foraging for food and nest material (jeanne, 1972; torres et al., 2011). in contrast, gynes are produced in the late post-emergence stage and generally invest in their own reproduction, contributing little to the maternal colony (jeanne, 1972; torres et al., 2011). it is therefore possible that the conditions for the development of immature vary with the progress of the colony cycle, due to fluctuations in the work force, which may be reflected in differences in the body size of emerging females (o’donnell, 1998; schmidt et al., 2012). mischocyttarus consimilis zikán is an if polistine wasp belonging to the subgenus phi, one of the largest groups in the tribe mischocyttarini, with the most extensive geographic distribution. in m. consimilis, the caste system is based on the division of tasks between the reproductive and non-reproductive females, in which the behaviors of oviposition, physical dominance, and nest presence characterize the reproductive caste, while behaviors such as alarm and resource foraging characterize the non-reproductive caste (torres et al., 2012). in order to investigate morphological differences among castes in m. consimilis, we measured the body size and ovarian development of foundresses from haplometrosis and pleometrosis foundations, as well as the body size of females emerging in different phases of the colony cycle. materials and methods field procedures and data collection colonies of m. consimilis were collected from the eaves of vacant houses situated in rural areas in the municipality of dourados (22º13’ s, 54º48’ w) in mato grosso do sul, from march 2011 through february 2012. to prevent the loss of individuals, colonies were collected in the early evening when foraging activity had ceased. all the individuals collected were killed by freezing (-20°c), prior to any manipulation procedure. a total of 59 foundresses from 29 foundations were evaluated for body size and ovarian development. the foundresses were classified as: alpha (female performing the reproductive function in a pleometrotic foundation); auxiliary (each female subordinate to the alpha); and solitary (female of a haplometrotic foundation). alpha foundresses were identified by direct observation of the oviposition behavior. we encouraged oviposition by the alpha by removing a larva with the aid of tweezers, leaving an empty cell in the comb. our study did not require an ethics statement; however, we endeavored to minimize the stress caused by manipulation. additionally, we evaluated the body sizes of a total of 90 newly-emerged females (<24 h old), from 47 colonies. the newly-emerged females were classified as: worker (female created only by the foundresses); intermediate (female created by foundresses and workers); and gyne (female emerging in the colony reproductive phase). the reproductive phase in colonies of m. consimilis occurs in the final post-emergence, when males appear among the offspring (torres et al., 2011). measurements of body size and ovarian development the measurements of body size, obtained according to gunnels (2007), were: head height; maximum distance between the inner margins of the eyes; minimum distance between the inner margins of the eyes; mesonotum length; mesonotum width; thorax length; mesopleura height; tibia length; and forewing length. for analysis of ovarian development, the gasters were fixed by immersion in dietrich’s solution, stored in 70% ethanol, and dissected in a petri dish containing physiological saline solution. the ovarian development index was obtained for each female, based on the mean length of the six largest oocytes (sledge et al., 2004). all measurements of body size and ovarian development were obtained with the aid of a stemi 2000c stereomicroscope (carl zeiss microscopy, oberkochen, germany) fitted with a micrometer eyepiece. statistical analyses all statistical analyses were carried out using r statistical environment software (r development core team, 2013), with significance level of 0.05. morphometric data were subjected to principal component analysis (pca), and the first pca component was used as the body size index (gunnels, 2007). in all cases, the data for body size were normally distributed (shapiro-wilk test, p>0.05) and showed homogeneity of variance (bartlett’s test, p>0.05), so the differences among the categories of females were evaluated by parametric tests, including analysis of variance (one-way anova) and tukey’s test (hsd) for multiple comparisons. the ovarian development data were not normally distributed (shapirowilk test, p<0.05), so differences among groups were assessed by nonparametric tests, including the kruskal-wallis and steel-dwass test for multiple comparisons. results the pca with the morphometric data for the foundresses recovered most of the variation among the groups, with the first principal component explaining 82.9% of the total variation and being positively associated with all the measured variables. the second principal component explained an additional 6.7% of the variation. considering the first principal component as the explanatory variable in the analysis of variance, it was demonstrated that the foundresses differed in body size (one-way anova, f2.56=9.26, p<0.001, fig 1). sociobiology 63(1): 693-698 (march, 2016) 695 fig 1. variation in body size index (mean ± sd) for foundresses of mischocyttarus consimilis. different letters indicate statistical significance (p<0.05). the analysis comparing the groups demonstrated that alpha foundresses were significantly larger than auxiliary (tukey’s hsd, p<0.01, fig 1), but did not differ from solitary (p=0.78, fig 1). in addition, solitary foundresses were significantly larger than auxiliary (p<0.01, fig 1). the three categories of foundresses also differed significantly in the degree of ovarian development (kruskalwallis, h=38.20, df=2, p<0.001, fig 2). comparison of fig 2. variation in ovarian development index for foundresses of mischocyttarus consimilis. box-plot represents the median, 25% 75% percentiles and minimum/maximum range. different letters indicate statistical significance (p<0.05). fig 3. correlation analyses between the body size index and ovarian development index for foundresses of mischocyttarus consimilis (r=0.41; p<0.01). fig 4. variation in body size index (mean ± sd) for newly-emerged females in colonies of mischocyttarus consimilis. different letters indicate statistical significance (p<0.05). the groups showed that alpha foundresses had greater ovarian development than auxiliary (stell-dwass, p<0.001, fig 2) and solitary (p=0.01, fig 2), while solitary foundresses had greater ovarian development than auxiliary (p<0.001, fig 2). there was a significant positive correlation between body size and ovarian development when the foundresses were evaluated together (pearson correlation coefficient; r=0.41, p<0.01, n=59, fig 3). the pca performed with the morphometric data for the newly-emerged females recovered most of the variation among the groups. the first principal component was positively associated with all the variables and explained 75.8% of the total data variation. the second principal component explained an additional 5.6% of the variation. considering the first principal component as the explanatory variable in the analysis of variance, significant differences in body size were found among the categories of newly-emerged females (one-way t. s. montagna; w. f. antonialli-junior – morphological caste in the social wasp mischocyttarus consimilis696 anova, f2.87=6.86, p<0.01, fig 4). comparison of the groups showed that the gynes were significantly larger than the workers (tukey’s hsd, p<0.01, fig 4) and the intermediates (p=0.02, fig 4). however, the body size of the workers did not differ significantly from that of the intermediates (p=0.79, fig 4). discussion the results showed that foundresses of m. consimilis performing the reproductive function in pleometrotic foundations were significantly larger than their auxiliary. several studies have shown that in pleometrotic foundations of if polistine wasps, the colony reproductive function is aggressively disputed among potential females, and larger females have increased chances of subduing smaller competitors (cant & field, 2001; tannure-nascimento et al., 2008). under these conditions, the reproductive organization is highly dependent on a dominance hierarchy, with larger females occupying higher positions in this hierarchy (turillazzi & pardi, 1977; cervo et al., 2008). larger females are therefore more likely to assume colony reproductive functions, suggesting that attributes established during immature development are relevant for the adult behavioral phenotype (zanette & field, 2009). recent studies have shown that preimaginal caste determination can occur in if polistine wasps, and that subtle morphological differences can be decisive in establishing the reproductive division of labor among foundresses (fukuda et al., 2003; judd et al., 2015; montagna et al., 2015). these results provide evidence that body size affects the reproductive organization in pleometrotic foundations of m. consimilis. it was also shown that the solitary foundresses were similar in size to the alpha. in a study with m. consimilis, torres et al. (2011) demonstrated that haplometrotic foundation is the most common strategy adopted by the foundress. our results suggest that large females with high reproductive potential invest in direct reproduction using the strategies of either fighting for the alpha position in pleometrotic foundations, or founding colonies individually. in addition, individual foundation could be an alternative strategy for large females who refrainfrom disputing the reproductive function in pleometrotic foundations. nevertheless, these hypotheses need to be evaluated, considering the origins of the females that found colonies alone. alpha foundresses had a significantly greater degree of ovarian development, compared to the auxiliary. furthermore, a significant positive correlation was found between body size and ovarian development, when the foundresses were evaluated together. these data suggest that the degree of ovarian development is closely relatedto both body size and the function performed by the foundress. other studies have shown that foundresses performing reproductive functions (i.e., alpha and solitary), must invest more in ovarian development (turillazzi & pardi, 1977; dropkin & gamboa, 1981; fukuda et al., 2003; tannure-nascimento et al., 2008). moreover, auxiliary foundresses performing activities with high energy costs, such as foraging for resources, are likely to undergo ovarian regression (keeping, 2000; cervo et al., 2008). it was also found that gynes were significantly larger than workers and intermediates, in agreement with other studies that evaluated differences in the body size of offspring females in if polistine wasps (west-eberhard, 1969; haggard & gamboa, 1980; miyano, 1983). for example, females of polistes fuscatus (fabricius) produced in the late post-emergence stage, which were possibly gynes, were larger than workers produced at the beginning of this stage (west-eberhard, 1969). since the adult body size in holometabolous insects is fixed before emergence, it is possible that in if polistine wasps it could be influenced by pre-imaginal trophic factors (miyano, 1998; karsai & hunt, 2002). differential feeding is known to be the main factors influencing caste determination in the honey bee, apis mellifera, where larvae fed a highly nutritious diet (based on royal jelly) develop into queens (winston, 1987). the effect of differential feeding in caste determination has also been demonstrated in the bumblebee, bombus terrestris, where larvae that have access to greater amounts of food emerge as queens (cnaani et al., 2002). in the case of if polistine wasps, judd et al. (2010) showed that the development of larvae of polistes metricus into either workers or gynes was significantly influenced by the levels of nutrients in the hemolymph, including lipids, carbohydrates, and proteins, and that the differences were associated with possible variations in nutritional quality. in studies of the same species, judd et al. (2015) showed that an increase in the quality of larval nutrition led to the development of larger females. in m. consimilis, workers and gynes emerge in different phases of the colony cycle, so the conditions for development of the immature are different (torres et al., 2011). these findings suggest a link between body size and colonial stage, which needs to be further investigated. however, in if polistine wasps, other factors are associated with the caste ontogeny, which vary as the colony grows. for example, larvae of p. fuscatus developing in the pre-emergence stage are subjected to higher levels of vibration, compared to larvae that develop in the later postemergence stage (suryanarayananet al., 2011). the vibration is produced by adults using their antennas to touch the walls of cells occupied by larvae. adult females subjected to high levels of vibration in the larval stage show a worker-like phenotype, while those subjected to low levels of vibration show a gyne-like phenotype (suryanarayanan & jeanne, 2008; suryanarayanan et al., 2011). this form of manipulation of the caste ontogeny is essential for the success of the colony, because it allows adults to control the caste production throughout the colony cycle. the results presented in this study suggest that individual attributes such as body size and ovarian development are linked to the function performed by the foundresses in m. consimilis. in addition, it is clear that gynes are larger than workers, suggesting that factors associated with the pre-imaginal caste determination vary with the development of the colony. knowledge of the caste ontogeny in if polistine wasps sociobiology 63(1): 693-698 (march, 2016) 697 sheds light on individual attributes that increase the chances for direct reproduction, in the context of disputes, and also helps to understand the conditions under which the castes are produced in less-advanced eusocial wasps. acknowledgements the authors acknowledge the coordenação de aperfeiçoamento de pessoal de nível superior (capes) for a scholarship granted to the first author. the fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul (fundect) provided financial support (proc. no. 23/200.767/2012). wfaj thanks the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for research productivity scholarship. references bourke, a.f.g. 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(2009). cues, concessions, and inheritance: dominance hierarchies in the paper wasp polistes dominulus. behavioral ecology, 20: 773-780. doi: 10.1093/beheco/ arp060 911 contrasting “carrasco” and forest ant communities in the chapada diamantina, bahia, brazil by henrique jesus de souza¹ & jacques hubert charles delabie²* abstract this study contrasts the structure of ant communities of the “carrasco” (deciduous forest) and forest (semideciduous forest) remnants in the buffer zone of the chapada diamantina national park, bahia, brazil. our aim was to compare the richness, composition, vertical partitioning and guild organization at the level of leaf-litter, ground-surface and lower vegetation between the habitat types. ants were sampled at six sites within each habitat by manual extraction in the leaf-litter, pitfall traps at ground level and baited pitfall traps on tree-trunks and shrubs 1.5 m above the soil surface. a total of 132 ant species was collected belonging to 34 genera and seven subfamilies. according to the analyses performed, the habitat types had equivalent species richness and a distinct species and functional composition at the vertical strata level. furthermore, a greater vertical partitioning was observed in the forest ant community than in the carrasco ant community. the results indicated a distinct biogeographical association between the ant fauna of the two habitat types and suggested that ant communities in carrasco and forest remnants in the buffer zone of the chapada diamantina national park are especially interesting for conservation and ecological research. key words: ant communities, “carrasco” vegetation, chapada diamantina. introduction in the central region of the chapada diamantina, bahia, brazil, “carrasco” (deciduous forest) and forest (semideciduous forest) remnants occur as isolated fragments surrounded by other vegetation formations, especially those associ¹ programa de pós-graduação em ecologia e biomonitoramento, instituto de biologia, universidade federal da bahia. rua barão de geremoabo, s/n. ondina, cep. 40170-000, salvador, bahia, brasil. ² centro de pesquisas do cacau, laboratório de mirmecologia, convênio uesc/ceplac, cep. 45600-970, itabuna, bahia, brasil. *corresponding author. e-mail: jacques.delabie@gmail.com 912 sociobiolog y vol. 59, no. 3, 2012 ated with “caatinga” (tropical dry forest) and “cerrado” (tropical savanna) biomes, and by an anthropogenic matrix, mainly dominated by agricultural activities (mma 2007). they are of high biogeographic and conservation interest because their peculiar physiognomy and floristic composition are so dramatically different from that in other vegetation formations in brazilian semi-arid domains (funch et al. 2005, queiroz et al. 2005). apparently carrasco and forest support a highly distinctive ant community because the habitat structure differs markedly between them, while in the carrasco the aspect of more open vegetation should create an arid microclimate for the ant assemblages of different strata, favouring the persistence of tolerant species, similar to those found in warm and open habitats (andersen 2000). in the forest, the greater vegetation stratification, with well-defined canopy and understory, should soften the microclimate on the ant foraging surface, favouring the existence of an ant community more sensitive to the higher insolation levels of the habitat. furthermore, considering the fact that ant species in local communities may differ markedly in their preferences with regard to food and nesting and foraging substrates (yanoviak & kaspari 2000), it is expected that differences in ecological properties of the vertical strata between the carrasco and forest range constrain the types of ants and guilds that may occur in them. this study contrasts the structure of ant communities of the carrasco and forest remnants in the buffer zone of the chapada diamantina national park, bahia, brazil. our aim was to compare the richness, composition, vertical partitioning and guild organization at the level of leaf-litter, ground-surface and lower vegetation between the habitat types. the following hypotheses were tested: 1) the species richness of ants in the vertical strata differs between the carrasco and the forest; 2) the vertical strata of the habitat types have a distinct species composition of ants; 3) in the forest, due to higher habitat structural heterogeneity, there is greater vertical partitioning in ant community; 4) the guild composition of ants in the vertical strata differs between the habitat types. methods study area the study was carried out at 12 sites located in cascavél, county of ibicoara, in the buffer zone of the chapada diamantina national park, state of bahia, 913 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil brazil (13°10’-13°16’s 41°21’-41°23’w). the sites studied currently comprise the legal reserves of two farms in the region (caraíbas farm and londres farm), which are characterized by the occurrence of two vegetation types: carrasco (deciduous forest) and forest (semi-deciduous forest), both at an interface between the caatinga (tropical dry forest) and the cerrado (tropical savanna) biomes. the climate of the region is tropical semi-arid, ranging from subhumid to dry and is marked by two defined seasons: dry (may-september) and rainy (october-april). the mean annual rainfall is approximately 1.062 mm and the average annual temperature is 20°c (mma 2007). carrasco is a shrub-dominated vegetation type of the brazilian semi-arid domain, which is derived from a natural process of ecological succession slowly adjusted to the xeric environment, in line with climate changes, possibly from the pliocene (fernandes 1998). it can be characterized as a deciduous, closed, and unistratified shrubland intermingled with lianas, with an irregular canopy and sparse emergent trees rarely reaching 6-8 m (araújo & martins 1999, queiroz et al. 2005). the trees eremanthus capitatus (spring.) macleish, bowdichia virgilioides kunth, jacaranda irwinii a.h. gentry, himatanthus lancifolius (müll. arg.) and pouteria ramiflora (mart.) radlk. are characteristic of this vegetation formation in the region. the forest studied is part of a group of remnants of semi-deciduous forest associated with the brazilian atlantic forest biome that occurs in this region, especially at altitudes above 800 m, where the moist and sandy soils favour the occurrence of canopy trees generally larger than in other vegetation formations of the chapada diamantina (10-20 m) (funch et al. 2005). in general, these remnants have diverse floristic composition and most species have wide geographical distribution, occurring in various formations, from northern south america to southern brazil (e.g. aspidosperma discolor a.dc., copaifera langsdorffii desf., tapirira guianensis aubl., schoepfia obliquifolia turcz. and terminalia brasiliensis cambess.). sampling ants were collected in april 2010, at the end of the rainy season in the region. within each habitat type, six sites were sampled at least 500 m distant from each other to ensure the independence of the data collected. in addition, 10 sampling points were established for each site at 50 m intervals to 914 sociobiolog y vol. 59, no. 3, 2012 facilitate the sampling of individuals from different colonies on the leaf-litter, ground-surface and lower vegetation. for the sampling of the leaf-litter ants, 0.5 m² samples were manually extracted from the soil surface. the collected material was placed in plastic bags for the manual processing of each sample. the screening was performed using sieves of 55 mm mesh attached to white plastic containers to facilitate the detection of organisms captured that were later removed with forceps and brushes (sarmiento 2003). the non-particulate material retained in the sieves (e.g. leaves, branches and twigs) was transferred to other containers and inspected in detail in order to enhance the effectiveness of the screening. ground-foraging ants were sampled using pitfall traps installed at ground level. the traps consisted of standard volume and diameter plastic containers partially filled with ethylene glycol to preserve the organisms collected. the containers were covered with plastic dishes supported by toothpicks to prevent flooding by rain and were left in the field for 24 h (andersen 1990). arboreal-foraging ants were sampled using baited pitfall traps on treetrunks and shrubs 1.5 m above the soil surface. smaller plastic containers were attached inside the pitfall traps containing small quantities of honey and sardine as an ant attractive (bestelmeyer et al. 2000). the larger compartment of the containers also was partly filled with ethylene glycol to preserve the specimens collected. the baited pitfall traps operated in parallel with the pitfall traps installed at ground level. the ants were identified to specific level according bolton (1995, 2003). species that could not be confidently named were identified to morphospecific level. voucher specimens were deposited at the hymenoptera collection of the museum of zoolog y, federal university of bahia (mzufba) and at the myrmecolog y laboratory of the cocoa research center (cpdc) in brazil. data analysis the observed species richness in the carrasco and forest was determined by the rarefaction curve (mao tau) (colwell et al. 2004), and expected species richness was estimated using the 1st order jackknife estimator (heltshe & forrester 1983), calculated with program estimates version 7.5.2 (50 randomizations) (colwell 2006). the effects of habitat type on the mean 915 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil species richness of ants in the vertical strata were examined using the student t-test in the bioestat version 5.0 (ayres et al. 2007). the effects of habitat type on the ant species composition in the vertical strata were examined using analysis of similarity (anosim) in primer version 5.0 (clarke & gorley 2001). the similarity percentage procedure (simper) was then used to determine which species were good difference discriminators among the sample groups analyzed (clarke & warwick 2001). the presence/absence data of ant species in the vertical strata of each site were submitted to analysis of non-metric multidimensional scaling (nmds) using the bray-curtis index as a measure of association to order the ant assemblages of habitat types in a two-dimensional space. the relative abundance of ant species in the vertical strata of each habitat type (leaf-litter: n = 60, ground-surface, n = 60, lower vegetation, n = 60) was measured from the species occurrence frequency in the sample sets. the number of individuals was not used as a measure of abundance because this estimate is greatly influenced by the patterns of nesting and foraging strategies of ant species (bestelmeyer et al. 2000). the vertical partitioning of ant communities was characterized by obtaining the proportion of species that occupied an only one stratum and the proportion of species that occupied more than one vertical stratum in the set of sites for the same habitat type. anosim was used to examine the similarity in ant species composition among samples from different vertical strata. in addition, a cluster analysis was carried out using the bray curtis index as a measure of association to produce a similarity dendrogram. the functional composition of ant communities was compared by assigning species to guilds based on the model proposed by brandão et al. (2009). in this model, species that share the same food resources and use the same strategies to occupy their niches are grouped in the same guild based on a series of ecological, morphological and behavioral attributes adopted by the authors in range habitat types of the neotropics (e.g. body size, diet, foraging activity and substrate nesting ). the adoption of this classification approach has allowed a greater understanding of the structure of ant communities and the factors that determine their organization in habitats of different biogeographic regions (andersen 1995, brandão et al. 2009, silvestre et al. 2003). the ant guild organization was characterized by calculating the percentage amplitude (the proportion of the total number of species) in the vertical 916 sociobiolog y vol. 59, no. 3, 2012 strata of each habitat type. the functional similarity was assessed using the functional similarity index, which considers how many guilds are in each location and how many are common between them, the number of species recorded by location and number of shared species within the guilds, by observing the minimum number of species in each guild in each locality (silvestre et al. 2003). results a total of 132 ant species belonging to 34 genera and seven subfamilies was collected in the 12 sites sampled, including two exotic species, tetramorium simillimum and paratrechina longicornis (appendix 1). the most species-rich genus was pheidole with 26 species (19.7% of the total) and camponotus with 20 (15.2%). in the carrasco, 83 species of 30 genera were observed against 81 species of 25 genera in the forest. despite the equivalence in the ant species richness between the carrasco and the forest, only 32 species were common fig. 1. percentage of ant species for subfamilies in the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil. stacked bars show cumulative proportions of total species records from different subfamilies of ants. 917 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil to both habitats. it means that 75.7% of the total species collected in this study occurred in a single habitat type. the carrasco and forest showed little variation in the percentage distribution of ant species by subfamilies. myrmicinae was the dominant subfamily with approximately 50% of the species collected, while ecitoninae had the lowest percentage of species, occurring only in the forest (fig. 1). despite considerable sampling effort in this study, the species accumulation curves for the two habitat types showed no indication of reaching a plateau, and therefore many more species are likely to be found in them (fig. 2). the observed and expected ant species richness was similar between the carrasco and the forest. furthermore, with the exception of lower vegetation, where a statistically significant difference was observed, the mean species richness of fig. 2. observed (mao tau) and expected ( jack1) richness curves of ant species in the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil. a total of 180 samples (60 samples of 0.5 m² of leaf-litter, 60 ground pitfall traps and 60 baited pitfall traps) were collected in each habitat type, april 2010. 918 sociobiolog y vol. 59, no. 3, 2012 ants in the vertical strata did not differ among the habitat types (leaf-litter: t = -0.72, df = 10, p = 0.49; ground-surface: t = 0.59, df = 10, p = 0.57; lower vegetation: t = 2.51, df = 10, p = 0.03; and all strata combined: t = 0.91, df = 10 , p = 0.38) (fig. 3 a-d). multivariate analysis according to the ant presence/absence data showed a clear separation between the carrasco and forest sites. vertical strata of habitat types showed a distinct species composition of ants, particularly at the lower vegetation level (anosim, leaf-litter: global r = 0.48, p = 0.002; ground-surface: global r = 0.41, p = 0.002; lower vegetation: global r = fig. 3. mean (± sd) species richness of ants in the vertical strata (a: leaf-litter, b: ground-surface, c: lower vegetation, d: all strata combined) per site in the carrasco (n = 6, in each case) and forest (n = 6, in each case) in the buffer zone of the chapada diamantina national park, bahia, brazil, april 2010. 919 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil 0.73, p = 0.002; and all strata combined: global r = 0.56, p = 0.001). the ordinations produced by nmds showed that ant assemblages sampled in sites of the same habitat type are more similar to each other than those from other habitat types (fig. 4 a-d). simper analysis indicated that the ant species that contributed most to the dissimilarity in the assemblage compositions among habitat types were: odontomachus chelifer, crematogaster distans and tetramorium simillimum at the leaf-litter level; pheidole sp.18 gp. flavens, solenopsis sp.3 and odontomachus chelifer at the ground-surface level; and pheidole sp.18 gp. flavens, linepithema pulex and ectatomma muticum at the lower vegetation level (table 1). within the two habitats, more than half the species were found in only one stratum (carrasco 67.4% and forest 70.5%), and the number of species exclusive to the ground-surface level was greater than in the lower vegetation fig. 4. non-metric multidimensional scaling (nmds) showing differences in the species composition of ants of the vertical strata sampled (a: leaf-litter, b: ground-surface, c: lower vegetation, d: all strata combined) in the carrasco sites and forest sites in the buffer zone of the chapada diamantina national park, bahia, brazil, april 2010. 920 sociobiolog y vol. 59, no. 3, 2012 and leaf-litter, respectively (table 2). anosim based on presence/absence data showed a greater similarity in the species composition between the vertical strata of the carrasco (global r = 0.38, p = 0.001) than of the forest (global r = 0.51, p = 0.001). furthermore, the cluster analysis showed that table 1. species contributing most to percentage dissimilarity in the species composition of the vertical strata between the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil. vertical strata species percentage contribution to dissimilarity leaf-litter odontomachus chelifer 8.37 crematogaster distans 4.76 tetramorium simillimum 4.20 solenopsis sp.1 4.12 pheidole sp.8 gp. fallax 4.12 ground-surface pheidole sp.18 gp. flavens 3.13 solenopsis sp.3 2.51 odontomachus chelifer 2.20 linepithema neotropicum 2.19 wasmannia auropunctata 2.09 lower vegetation pheidole sp.18 gp. flavens 4.35 linepithema pulex 4.35 ectatomma muticum 4.35 crematogaster victima 3.78 wasmannia auropunctata 2.98 table 2. number and percentage of stratum-specialists species of ants in the vertical strata of the carrasco and forest at the buffer zone of the chapada diamantina national park, bahia, brazil. vertical strata carrasco forest leaf-litter 5 (6.0%) 5 (6.4%) ground-surface 30 (36.1%) 35 (44.9%) lower vegetation 21 (25.3%) 15 (19.2%) total 56 (67.4%) 55 (70.5%) 921 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil fig. 5. similarity dendrogram (bray curtis index) showed the similarity relations in the species composition of ants of the vertical strata in the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil, april 2010. fig. 6. percentage of species for ant guilds in the vertical strata of the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil, april 2010. stacked bars show cumulative proportions of total species records from different ant guilds: (aa) army ants, (afp) arboreal feeding on pollen, (ap) arboreal predators, (da) dominant arboreal, (dp) dacetini predators, (egp) epigaeic generalist predators, (fc) fungus collectors, (ge) generalists and (hgp) hypogaeic generalist predators. 922 sociobiolog y vol. 59, no. 3, 2012 the ant species composition on the ground-surface and lower vegetation was more similar in the carrasco than in the forest (fig. 5). on the other hand, the ant species composition in the lower vegetation and leaf-litter was more similar in the forest than in the carrasco (fig. 5). in both habitat types, the vertical strata varied markedly in guild composition (fig. 6). the most frequently recorded guilds were generalists (mostly species of pheidole, dorymyrmex and linepithema) in the leaf-litter and ground-surface, and dominant arboreal (mostly species of camponotus and crematogaster) on the lower vegetation. furthermore, the ant community functional organization in terms of vertical strata differed markedly between the carrasco and forest. the functional similarity index for the leaf-litter ant assemblages was 21.4%, while for the ground-surface ant assemblages it was 35.3% and 34.7% for the lower vegetation ant assemblages. discussion although the sampling methods used in this study did not provide exhaustive information on ant communities in the habitats studied, they were enough to perform comparative analysis. the adoption of complementary collecting methodologies (bestelmeyer et al. 2000, sarmiento 2003) and planning of a larger sampling period (delabie et al. 2000) are necessary to obtain a more complete and exhaustive picture of the ant communities from the carrasco and forest remnants in the buffer zone of the chapada diamantina national park, bahia, brazil. the myrmicinae taxonomic dominance can be attributed to the fact that this subfamily has the greatest generic and specific diversity between subfamilies of ants in the neotropics (brown 2000). pheidole, for example, comprises one of the ant genera with the greatest number of species around the world, a phenomenon known as hyperdiversity (wilson 2003). some of the more well-founded explanations for the existence of this phenomenon are related to a successful combination of small size, population factors and an appropriate set of adaptations to occupy new niches and exclude competitors (wilson 2003). the occurrence of exotic species in the habitats studied alerts to the danger of biological invasions, which have produced significant impacts on the eco923 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil logical integrity of many natural habitats (mikissa et al. 2008). tetramorium simillimum and paratrechina longicornis are species from the old world that are widely distributed in savannas and forests in brazil (delabie et al. 2007, silvestre et al. 2003). paratrechina longicornis also been recorded in grassland in mexico (gove et al. 2009) and in australian savannas (andersen et al. 2007). although these taxa have shown a low relative abundance in the collection sites, it is of fundamental importance to carry out an effective ecological monitoring to assess the expansion of these species in the region. the results of this study showed that ant communities inhabiting the carrasco and forest remnants have equivalent species richness, contradicting the results of the literature from different biogeographic regions, which have suggested that the species richness for these taxa differs markedly between vegetation formations with deciduous and semi-deciduous features (vasconcelos & vilhena 2006, andersen et al. 2007). in spite of this equivalence, the greater number of ant species found in the lower vegetation of the carrasco can be attributed to greater availability of the food supply and nesting sites for ants in this stratum, due to the high density of shrub and emergent trees (ribas et al. 2003). in fact, the carrasco has a dense shrubland capable of supporting a greater number of ant species in the lower vegetation than the forest, where the greater stratification of the vegetation, mainly in the upper strata, usually maintains an arboreal ant fauna more associated with the canopy (brühl et al. 1998, delabie et al. 2007). despite the differences in the sampling effort and collecting methods employed, the diversity of ants observed in the forest sites was lower than that found by martins et al. (2006) in four remnants of semi-deciduous forest in the same region, and by surveys carried out in the atlantic forest in southern bahia, where the ant fauna inhabiting especially the leaf-litter is much greater (alves et al. 2008, delabie et al. 2007, delabie & majer 1999). on the other hand, the ant diversity observed in the carrasco sites was greater than that documented by other authors in open habitats, such as in vegetation types related to the caatinga biome in the brazilian semi-arid region (leal 2003, santos et al. 1999, soares et al. 2003). in this study, several species presented a strong association with a particular habitat. in fact, the carrasco and forest shared only about one-quarter of the ant species. this result, associated with the discontinuity in species composi924 sociobiolog y vol. 59, no. 3, 2012 tion observed in the multivariate analysis, indicated a distinct biogeographical association between the ant fauna of the two habitat types. while in the carrasco there was a higher prevalence of open habitat taxa of ants related with the semi-arid zone (e.g. ectatomma, dorymyrmex, pseudomyrmex and solenopsis), in the forest, a considerable prevalence of forest-associated fauna related with remnants of brazilian atlantic forest was observed (e.g. strumigenys, pachycondyla, labidus and crematogaster). the greater overlap of species between vertical strata within the carrasco was due to the presence of a group of species with generalist and tolerant habits. for example, ectatomma brunneum, odontomachus chelifer, camponotus cingulatus, pheidole sp.18 gp. flavens and linepithema pulex had favored their distributions probably due to better competitive strategies in obtaining food resources and occuping nesting substrates (brandão et al. 2009, fernández 2003, lattke 2003, silvestre et al. 2003). thus, the plasticity of foraging and nesting of these species contributed to homogenizing the composition of ant assemblages between the vertical strata. the data obtained by vasconcelos & vilhena (2006) and campos et al. (2008) in savannas reinforce the results of this study that in simplified structure habitats the ant species composition varies less between the vertical strata, especially between the ground-surface and lower vegetation. in contrast to this pattern, in the forest, the ant community was characterized by a greater vertical partitioning, with more than 70% of species associated with only one habitat stratum. this pattern was similar to that reported by brühl et al. (1998) in a primary forest in malaysia, suggesting that greater vegetation stratification in forests provides a broad spectrum of permanent habitats for ant species allowing a greater vertical partitioning. analysis performed applying the guild model indicated that the functional composition of ant communities in terms of vertical strata was significantly different between the carrasco and forest remnants in the same region. this result suggested that possible differences in the intrinsic properties of the strata of the habitat types may determine the amplitude and species composition of ants within the guilds. for example, the dominant arboreal ant guild presented high percentage amplitude in the forest lower vegetation that might limit the ant species distribution in this stratum of other guilds. this 925 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil pattern is well documented in tropical forests, where ant colonies with high numbers of individuals can become dominant, because of the potential to raise territory defense (dejean et al. 2007). on the other hand, in the carrasco lower vegetation, the persistence of other ant guilds typical of vegetation (e.g. arboreal predators and arboreal feeding on pollen) together with the expansion of ant guilds typical of the ground-surface (epigaeic generalist predators) in this stratum, seem to decrease the percentage amplitude of the dominant arboreal ant guild. correlation tests between different ant guild traits and ecological properties associated with habitat vertical strata are encouraged in future studies to assess the real contribution of these properties in determining species patterns and functional composition in ant communities. for example, supply and distribution of extrafloral nectaries in the habitat and the dominant arboreal ant guild amplitude; availability and variety of prey and the epigaeic generalist predator, specialists and dacetini predator ant guild amplitude. although the lack of information about the insect diversity of the brazilian semi-arid region has not yet attracted the attention of most researchers (bravo & aguiar 2005), the findings of this study suggest that ant communities in carrasco and forest remnants in the buffer zone of the chapada diamantina national park have great potential for conservation and ecological research. acknowledgments the authors acknowledge the administrators of the caraíbas and londres farms for facilities for the field study; thanks to tatiana cabral, alexandre camanho and josé rodrigues for help collecting the ant data, to adrienne hurley toledo for improving the english manuscript; they also acknowledge their grants from capes and cnpq. references alves, h.s.r., j.h.c. delabie, i.c. nascimento, j.r.m. santos, r.m. oliveira & m.s. moreau. 2008. uso de formigas para identificação de mini-corredores ecológicos na apa itacaré/ serra grande, bahia, brasil. revista sitientibus série ciências biológicas 8:71-79. 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(ed.). introducción a las hormigas de la región neotropical. instituto de investigación de recursos biológicos alexander von humboldt, bogotá 201-210. silvestre, r., c.r.f. brandão & r.r. silva. 2003. grupos funcionales de hormigas: el caso de los grêmios del cerrado. in: fernandéz, f. (ed.). introducción a las hormigas de la región neotropical. instituto de investigación de recursos biológicos alexander von humboldt, bogotá 113-148. soares, i.m.f., a.a. santos, d. gomes, j.h.c. delabie & i.f. castro. 2003. comunidades de formigas (hymenoptera: formicidae) em uma “ilha” de floresta ombrófila serrana em região de caatinga (ba, brasil). acta biologica leopoldensia 25: 197-204. vasconcelos, h.l. & j.m.s. vilhena, 2006. species turnover and vertical partitioning of ant assemblages in the brazilian amazon: a comparison of forests and savannas. biotropica 38:100-106. wilson, e.o. 2003. la hiperdiversidad como fenómeno real: el caso de pheidole. en: fernandéz, f. (ed.). introducción a las hormigas de la región neotropical. instituto de investigación de recursos biológicos alexander von humboldt, bogotá 363-370. yanoviak, s.p. & m. kaspari. 2000. community structure and the habitat templet: ants in the tropical forest canopy and litter. oikos 89:259–266. 929 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil appendix appendix 1. frequency of occurrence of ant species in the vertical strata of the carrasco and forest in the buffer zone of the chapada diamantina national park, bahia, brazil. a total of 180 samples (60 samples of 0.5 m² of leaf-litter, 60 ground-surface pitfall traps and 60 baited pitfall traps in the lower vegetation) were collected in each habitat type. the guild of each species is also given: (aa) army ants, (afp) arboreal feeding on pollen, (ap) arboreal predators, (da) dominant arboreal, (dp) dacetini predators, (egp) epigaeic generalist predators, (fc) fungus collectors, (ge) generalists, and (hgp) hypogaeic generalist predators. species carrasco forest guild leaf litter ground surface lower vegetation leaf litter ground surface lower vegetation dolichoderinae azteca sp. da 0 0 3 0 0 0 dorymyrmex thoracicus gallardo, 1916 ge 0 0 0 0 3 0 dorymyrmex sp.1 ge 0 0 0 0 2 0 dorymyrmex sp.2 ge 0 0 0 0 1 0 dorymyrmex sp.3 ge 0 1 0 0 0 0 dorymyrmex sp.4 ge 0 1 1 0 0 0 dorymyrmex sp.5 ge 0 0 0 0 1 0 linepithema aztecoides wild, 2007 ge 0 7 1 0 0 0 linepithema cerradense wild, 2007 ge 0 1 1 0 0 0 linepithema pulex wild, 2007 ge 0 15 0 0 33 2 linepithema neotropicum wild, 2007 ge 0 0 0 0 4 0 ecitoninae labidus praedator (fr. smith, 1858) aa 0 0 0 0 2 0 labidus coecus (latreille, 1802) aa 0 0 0 0 2 0 ectatomminae ectatomma brunneum smith, 1858 egp 1 19 13 8 1 0 930 sociobiolog y vol. 59, no. 3, 2012 ectatomma muticum mayr, 1870 egp 0 36 7 9 0 0 ectatomma opaciventris roger, 1861 egp 0 18 1 0 0 0 ectatomma tuberculatum (olivier, 1792) ap 0 0 1 0 0 0 gnamptogenys moelleri (forel, 1912) egp 0 0 0 1 0 0 gnamptogenys regularis mayr, 1870 egp 0 1 0 0 0 0 gnamptogenys sulcata (fr. smith, 1858) egp 0 0 1 0 0 0 formicinae brachymyrmex heeri forel, 1874 ge 3 0 0 0 0 0 brachymyrmex patagonicus mayr, 1868 ge 0 0 0 0 0 1 brachymyrmex sp.1 ge 0 0 0 0 0 0 brachymyrmex sp.2 ge 0 0 0 0 1 0 brachymyrmex sp.3 ge 0 0 0 1 1 0 camponotus agra (f. smith, 1858) da 0 0 0 0 0 3 camponotus arboreus (f. smith, 1858) da 0 0 1 0 0 1 camponotus blandus (f. smith, 1858) da 0 1 0 0 0 0 camponotus cingulatus mayr, 1862 da 0 3 15 0 0 1 camponotus crassus mayr, 1862 da 0 0 3 2 2 3 camponotus fastigatus roger, 1863 da 0 2 6 0 0 0 camponotus leydigi forel, 1886 da 0 1 3 0 1 0 camponotus melanoticus emery, 1894 da 0 2 7 0 0 0 camponotus novogranadensis mayr, 1870 da 0 6 2 0 0 0 camponotus punctulatus andigenus mayr, 1868 da 0 1 0 0 0 0 931 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil camponotus renggeri emery, 1894 da 0 2 8 0 2 10 camponotus rufipes (fabricius, 1775) da 0 0 2 0 0 0 camponotus (myrmaphaenus) sp.1 da 0 0 1 0 0 0 camponotus (tanaemyrmex) sp.2 da 0 0 3 0 0 1 camponotus (myrmaphaenus) sp.3 da 0 0 1 0 0 0 camponotus (myrnothrix) sp.4 da 0 0 0 1 2 1 camponotus sp.5 da 1 0 0 0 0 0 camponotus sp.6 da 0 0 0 0 0 1 camponotus sp.7 da 0 0 0 0 0 3 camponotus sp.8 da 0 0 0 0 0 3 myrmelachista sp.1 ge 0 0 1 0 0 0 myrmelachista sp.2 ge 0 0 0 0 8 1 myrmelachista sp.3 ge 0 0 0 1 0 1 nylanderia fulva (mayr, 1862) ge 0 2 0 0 0 0 nylanderia guatemalensis (forel, 1885) ge 0 0 0 0 1 0 nylanderia sp.1 ge 0 0 0 0 2 0 nylanderia sp.2 ge 0 0 0 0 1 0 nylanderia sp.3 ge 0 0 0 0 2 0 paratrechina longicornis latreille, 1802 ge 2 0 0 0 0 0 myrmicinae acromyrmex balzani emery, 1890 fc 0 1 0 0 0 0 acromyrmex subterraneus molestans santschi, 1925 fc 0 2 0 0 0 0 atta sexdens (linnaeus, 1758) fc 0 1 0 0 0 0 cephalotes atratus (linnaeus, 1758) afp 0 0 0 0 0 4 cephalotes minutus (fabricius, 1804) afp 0 0 1 0 0 0 932 sociobiolog y vol. 59, no. 3, 2012 cephalotes pallidus andrade, 1999 afp 0 0 0 0 1 0 cephalotes pavonii (latreille, 1809) afp 0 0 4 0 0 0 cephalotes pusillus (klug, 1824) afp 1 3 22 0 2 3 cephalotes sp. afp 0 0 0 1 0 0 crematogaster curvispinosa mayr, 1862 ad 1 0 1 1 0 1 crematogaster distans mayr, 1870 da 4 2 2 4 0 1 crematogaster victima smith, 1958 da 1 1 9 0 0 0 crematogaster sp.1 da 0 0 0 0 0 3 crematogaster sp.2 da 0 0 0 1 0 0 crematogaster sp.3 da 0 0 0 2 0 0 cyphomyrmex salvine forel, 1899 fc 2 0 0 0 0 0 cyphomyrmex sp.1 gp. strigatus fc 0 3 0 0 0 0 cyphomyrmex sp.2 fc 0 0 0 0 3 0 hylomyrma balzani (emery, 1894) fc 0 0 0 1 3 0 mycetophylax sp. fc 0 0 0 0 1 0 nesomyrmex sp. ge 0 0 1 0 0 0 oxyepoecus sp.1 ge 0 2 0 0 0 0 oxyepoecus sp.2 ge 0 3 0 0 0 0 pheidole sp.1 gp. diligens ge 0 1 0 0 1 0 pheidole sp.2 gp. diligens ge 0 0 0 0 0 1 pheidole sp.3 gp. diligens ge 0 8 0 0 13 1 pheidole sp.4 gp. diligens ge 0 18 3 0 9 11 pheidole sp.5 gp. diligens ge 0 4 10 0 3 0 pheidole sp.6 gp. diligens ge 1 1 0 0 0 0 pheidole sp.7 gp. flavens ge 0 7 0 1 2 0 pheidole sp.8 gp. fallax ge 0 11 0 2 6 1 pheidole sp.9 gp. fallax ge 0 1 0 0 0 0 pheidole sp.10 gp. diligens ge 0 0 1 0 0 0 933 de souza, h.j. & j.h.c. delabie — contrasting ant communities in brazil pheidole sp.11 gp. diligens ge 0 0 0 0 1 0 pheidole sp.12 gp. tristis ge 0 6 0 0 0 0 pheidole sp.13 gp. fallax ge 0 0 0 0 1 0 pheidole sp.14 gp. diligens ge 0 1 0 0 0 1 pheidole sp.15 gp. diligens ge 0 2 0 0 0 0 pheidole sp.16 gp. fallax ge 0 1 0 0 2 0 pheidole sp.17 gp. diligens ge 0 5 0 0 1 0 pheidole sp.18 gp. flavens ge 1 17 2 1 2 0 pheidole sp.19 gp. tristis ge 0 4 1 1 9 0 pheidole sp.20 gp. flavens ge 0 2 0 0 5 0 pheidole sp.21 ge 0 0 0 0 1 0 pheidole sp.22 ge 0 0 0 0 0 1 pheidole sp.23 gp. diligens ge 0 0 0 0 3 0 pheidole sp.24 gp. diligens ge 0 0 0 1 4 0 pheidole sp.25 gp. diligens ge 0 0 0 0 1 0 pheidole sp.26 ge 0 0 0 0 1 0 pogonomyrmex naegelii (forel, 1878) ge 0 1 0 0 2 0 procryptocerus sp. da 0 0 0 0 0 1 sericomyrmex sp. fc 0 2 0 0 0 0 solenopsis globularia (smith, 1858) ge 0 1 0 0 0 0 solenopsis sp.1 ge 0 2 1 3 2 9 solenopsis sp.2 ge 0 0 0 3 0 1 solenopsis sp.3 ge 0 0 0 0 5 1 solenopsis sp.4 ge 0 0 0 0 1 0 strumigenys appretiatus (borgmeier, 1954) dp 0 1 0 0 0 0 strumigenys denticulata (mayr, 1887) dp 0 0 0 0 1 0 934 sociobiolog y vol. 59, no. 3, 2012 tetramorium simillimum (f. smith, 1851) ge 3 0 0 2 0 0 trachymyrmex fuscus emery, 1834 fc 0 10 0 0 0 0 wasmannia auropunctata roger, 1863 ge 3 7 2 0 2 0 wasmannia rochai forel, 1912 ge 2 4 0 4 1 0 ponerinae anochetus oriens kempf, 1964 egp 0 1 0 0 0 0 hypoponera sp.1 hgp 2 0 0 0 0 0 hypoponera sp.2 hgp 0 0 0 1 0 0 hypoponera sp.3 hgp 0 0 0 0 1 0 odontomachus chelifer (latreille, 1802) egp 0 4 2 4 39 0 odontomachus meinerti forel, 1905 egp 3 1 0 0 0 0 pachycondyla bucki (borgmeier, 1927) egp 0 0 0 0 2 0 pachycondyla harpax (fabricius, 1804) egp 0 3 0 0 1 0 pseudomyrmecinae pseudomyrmex gracilis (fabricius, 1804) ap 0 0 7 0 0 0 pseudomyrmex oculatus (f. smith, 1855) ap 0 0 9 0 0 0 pseudomyrmex schuppi (forel, 1901) ap 0 0 1 0 0 0 pseudomyrmex tenuis (fabricius, 1804) ap 0 3 0 0 0 0 pseudomyrmex termitarius (f. smith, 1855) ap 0 0 0 1 1 0 pseudomyrmex sp.1 gp. pallidus ap 0 0 1 0 0 0 pseudomyrmex sp.2 gp. pallidus ap 0 0 2 0 0 1 sociobiology 62(2): june (2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 editorial special issue on taxonomy, morphology, and phylogeny of ants formicidae (ants), a family of ~13,000 described species, are a critically important component of biodiversity in nearly every terrestrial habitat. in the neotropics, for example, ants are among the most abundant insect taxa, with a combined biomass greater than all vertebrates. they thus not only provide food for many vertebrates and invertebrates, but also play major roles in community organization and ecosystem processes. they are abundant in subterranean, epigaeic, and arboreal habitats, with several species living in close symbiosis with plants or fungi. ants fill virtually every important ecosystem function and every available niche. they influence bioturbation, superficial groundwater flow, nutrient cycling, seed dispersal, and decomposition, stabilize food webs, control and regulate pests, and form and modify habitats (ecosystem engineers). some are major defoliators in natural and agroecosystems (e.g., leafcutter ants), whereas others are major predators of invertebrates and vertebrate prey (e.g., army ants). several ant species are major agricultural pests, causing billions of dollars in losses annually. ants are used as model organisms in many ecological and evolutionary studies, but they have also gained prominence in conservation biology since they are sensitive to anthropogenic disturbance,and can be also readily grouped into functional groups. unfortunately, the lack of taxonomic knowledge for many groups may limit their potential for use in the areas mentioned above, since many tropical species are undescribed or new to science, greatly slowing morphological species identification. in this special issue, almost twenty articles provide a sampling of how important morphology-based studies and ant inventories really are. the origin of ant soldiers and the use of this term in ant literature are reviewed, underlining the distinction between “soldiers” and “major workers”. in another excellent contribution, twelve fossil ant species and an entirely new genus are described from the middle eocene (ca. 46 myo), providing a new window into eocene ant diversity and the tempo of ant early evolution. in a series of very detailed and pleasingly illustrated contributions, authors describe five new ant species belonging to four different genera. also, different castes and sexes are described for the first time for several known species. new records are made for a series of ant taxa, considerably extending their known distributions in different regions of the world. updated lists based on exhaustive ant surveys highlight the importance of south america as a megadiverse region, with more than 3,000 records and almost 1,000 ant species registered for different localities in argentina, brazil, and ecuador. other contributions examine in detail ant morphology, including exocrine glands and the effect of nematode infestation in ant phenotypes. assessing ant biodiversity at various levels will lay the baseline for continued monitoring of ecosystem health and biodiversity, especially under the present scenario of climate change, and may guide conservation decisions by allowing rapid and efficient appraisal of ecosystems. the study of ant morphology, diversity and distribution will also provide the framework for the identification of tropical pest species and thus will provide the basis for applied research of international importance. sociobiology, a recently reformulated journal, is aware of the importance of ant diversity for the different ecosystems of the world and thanks all authors and reviewers for this rich and important special issue. rodrigo m. feitosa (universidade federal do paraná, brazil) john e. lattke (universidad nacional de loja, ecuador) ted r. schultz (smithsonian institution, usa) editors for this special issue sociobiology an international journal on social insects _goback open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.7729sociobiology 69(3): e7729 (september, 2022) introduction the spanish word ganado (livestock) is directly derived from the verb ganar (to gain) and has several definitions, available in the tercentenary edition (the most updated one) of the spanish language dictionary. among these definitions, livestock is: “a group of beasts that feed and move together”, such as sheep, goats, and cattle. in its second definition, current since 1803, livestock is also defined as the group of bees in a beehive (rae, 2017). abstract the knowledge of the different plant species that make up the feeding diet of animals is highly important to develop more efficient strategies. this research aimed to characterize the food potential available for the cuban stingless bee livestock of the matanzas and mayabeque provinces. palynological analysis was done using 60 g of pollen from sealed pots and 80 ml of honey from the ten randomly selected beehives (five in each province). the results showed that in the honey collected in matanzas province, the most represented family was amaranthaceae, followed by myrtaceae and fabaceae. meanwhile, for mayabeque, the most represented ones were the families fabaceae and myrtaceae. regarding the stingless bee pollen of matanzas provenance, the family fabaceae prevailed, followed by burseraceae and myrtaceae. the pollen corresponding to mayabeque coincided in showing fabaceae as the most representative. in addition, pollen grains of small size (from 10 to 25 µm) were collected, with a marked representation of the pollen type of mimosa pudica in the mayabeque honey. it was concluded that the cuban stingless bee livestock of the matanzas and mayabeque provinces had a specialist feeding behavior because a low number of plant taxa made up its diet. sociobiology an international journal on social insects leydi f carballo1, david r de la cruz2,4, josé s. sanchez2,4, estefanía s. reyes2, walberto l peña3 jorge d lorenzo3, maykelis d solares5, dariel m querol1 article history edited by evandro nascimento silva, uefs, brazil received 12 january 2022 initial acceptance 04 march 2022 final acceptance 16 july 2022 publication date 07 september 2022 keywords honey, stingless bee pollen, melipona beecheii, melissopalynology. corresponding author leydi fonte carballo estación experimental de pastos y forrajes “indio hatuey” central españa republicana, matanzas, cuba. e-mail: leydis.fonte@ihatuey.cu taking the previous concept as a reference, lóriga (2015) defines the cuban stingless bee livestock as all the managed beehives of the bee species melipona beecheii bennett, 1831 (apidae: meliponini), included in a census or not. according to sáenz et al. (2000), during the foraging process, bees visit flowers in search of food, then pollen grains adhere to their body and later become part of honey. the palynology branch studying these grains to know the botanical origin of the different products elaborated or stored in the hives of diverse groups of bees is called melissopalynology. 1 estación experimental de pastos y forrajes “indio hatuey” central españa republicana, matanzas, cuba 2 grupo de palinología y conservación vegetal (gpcv), instituto hispano luso de investigaciones agrarias (ciale), universidad de salamanca, españa 3 universidad agraria de la habana, facultad de medicina veterinaria. carretera tapaste y autopista nacional, san josé de las lajas, mayabeque, cuba 4 departamento de botánica y fisiología vegetal, universidad de salamanca, españa 5 independent researcher, usa research article bees cuban stingless bee livestock exhibit specialized floral resource use: a palynological study on honey samples from matanzas and mayabeque provinces mailto:leydis.fonte@ihatuey.cu fonte-carballo et al. – cuban stingless bees with the specialized resource use2 with the use of this discipline, the study of the floral sources visited by bees is facilitated to a large extent (sánchez, 2001). the antecedents of using trophic resources by bees allow the evaluation of the plant taxa that contribute nectar and/or pollen to the bee diet. and consequently, from these data, the elaboration of floristic lists and phenological calendars can be done, contributing to better management of the beehives by farmers (flores et al., 2015). due to the above-exposed facts, knowing the main plant species that make up the diet of these animals is a priority in developing efficient feeding strategies that guarantee higher production with an optimum health level. this research aimed to characterize the food potential available for the cuban stingless bee livestock of the matanzas and mayabeque provinces and its possible application to other territories with beehives of this species. materials and methods study sites the study was conducted in two cuban stingless bee apiaries in april 2018. the first one is located in the matanzas municipality, of matanzas province, at 23°02’25’’ north latitude and 81°30’58’’ west longitude, at an altitude of 14 m.a.s.l.; and the second one, in the san nicolás municipality, belonging to the mayabeque province, located at 22°47’13’’ north latitude and 80°55’05’’ west longitude, at an altitude of 34 m.a.s.l. the characteristic climate of the studied area is classified as tropical, with marked seasonality of the rains in two periods corresponding to the dry season (ds), which extends from november to april, and the rainy season (rs), which lasts from may to october (academia de ciencias de cuba, 1989). during the sampling period, the matanzas locality showed a warm climate that caused minimum temperature values and positive anomalies of 0.8ºc. the recorded cumulative rainfall was 22.8 mm, lower than the historical mean (52.6 mm). regarding relative humidity, it was 74%, over the historical values. in the case of mayabeque, it was also warm, conditioned by the influence of high oceanic pressure that weakly influenced the territory during most of the month. this situation is responsible for the cold air masses not reaching the territory, which provoked temperature values with significant positive anomalies of 1.7 ºc compared with the historical average. the minimum temperatures averaged 19.9ºc. regarding rainfall, the cumulative exceeded the historical mean of 171.3 mm, and the mean air relative humidity was 75%, slightly higher than the historical average. floristic inventory of each stingless bee apiary knowing the foraging area of bees is important to understand the use of floristic resources. studies on species of the genus melipona illiger‎, 1806‎, found flight radios close to 2 km, with the studied species having body sizes larger than m. beecheii (nunes silva et al., 2020). however, a flight radius of 1 km was estimated for m. beecheii, similar to that of species of the genus trigona jurine, 1807 (araújo et al., 2004), since they share similar biometric parameters (fonte, 2007). considering that both stingless bee apiaries were located in urban areas, where gardens and house backyards prevail, the main existing plant species potentially contributing with pollen and nectar were determined: native, naturalized, and ornamental plants (pérez-piñeiro, 2017). in addition, to determine the representativeness of the species in the environment of stingless bee apiaries, four categories (abundant, frequent, little frequent, and rare or scarce) and their corresponding biological form (tree, shrub, and herbaceous) were established, adapting the methodology proposed by garcíaabad (2015) to urban environments, which are less diverse from the botanical point of view (wittig & becker, 2010). construction of the palynotheque from each inventoried species, floral buds were taken and preserved in a universal fixator preservative composed of a mixture of 95º ethylic alcohol, glacial acetic acid, 3740% formaldehyde, and distilled water. afterward, they were dissected with the aid of a surgical blade, extracting the anthers, which were placed in conical test tubes of 10 ml capacity, and the protocol used for the preparation of the samples for the palynological and melissopalynological qualitative analysis was followed. the photographic shot of each grain from the previously identified botanical species of reference was done through a camera (leica dfc290) coupled to the optical microscope (nikon optiphot-i) with a magnification of 400. the leica application suite, version 3.3.1 for windows xp, was used for this purpose. the obtained photographs and bibliographic references (aira et al., 2018, pérez, 2016) served as a basis for determining pollen types found in the honey and pollen samples. sample collection from the ten beehives under study in each province, five were randomly selected in each locality. in mayabeque, beehives 14, 2, 48, 5, and 6 were chosen, while in matanzas, beehives 1, 9, 11, 12 and 13 were selected. sixty grams of stingless bee pollen from sealed pots and 80 ml of honey in each one of them were taken. sampling was done in april 2018. melissopalynological qualitative analysis the protocol used for sample preparation was that of honey analysis proposed by the honey work group belonging to the spanish society of palynology (aple, for its initials in spanish), recently constituted (sánchez et al., 2018, gonzálezporto et al., 2018). the protocol is based, with modifications, on garcía et al. (2001), consisting of the previous treatment sociobiology 69(3): e7729 (september, 2022) 3 of the samples with 5% acidulated water with their respective washings and centrifugations to obtain sediment that is in turn deposited on the microscope slide and one drop of glycerin jelly with fuchsin is applied before taking it to the microscope for its later analysis. palynological qualitative analysis of stingless bee pollen one gram of sample was weighed and was subject to the above-explained protocol, with modifications in the volume of 5% acidulated water that was added. this methodology was used to liken it to the one used in the processing of honey samples, also considering the collection conditions of stingless bee pollen, with different load sizes, variable from small masses of 5-6 mm to dust particles. the reading of stingless bee honey and pollen samples was done under the optical microscope with a magnification of 400, identifying a minimum number of 300 to 500 pollen grains to guarantee correct botanical and geographical characterization. the samples were done in quadruple. the observed pollen types were placed in the different class frequencies according to loveaux et al. (1978), as shown below:  dominant pollen (>45%)  companion pollen (16-45%)  important isolated pollen (4-15%)  rare or sporadic pollen (1-3%)  present pollen (<1%) the shannon-weaver diversity index was used to determine the browsing strategy deployed by m. beecheii from the pollen diversity found in the honey samples (shannon & weaver, 1949). where: h’= shannon-wiener index. pi = proportion of each pollen type found. ln = natural logarithm. meanwhile, the browsing uniformity of the bees was determined through pielou’s evenness index (pielou, 1984). where: j’= browsing uniformity of the bees. h’= shannon-wiener index. h’ max = napierian logarithm of pollen richness. the diversity (h’) and evenness (j’) indices were determined through the utilization of the software diversity species & richness 3.02 (henderson & seaby, 2002). results the results of the floristic inventory in both localities show that in the flight environment of stingless bees, there are 16 plant species in common. we identified 42 plants belonging to 27 families in matanzas (table 1). alternanthera sessilis (l.) r. br. ex dc, mangifera indica l., adonidia merrillii (becc.) becc, bidens pilosa l., bursera simaruba (l.) sarg, senna spectabilis (dc.) h.s. irwin & barneby, persea americana mill., musa x paradisiaca l., psidium guajava l. and coccoloba uvifera (l.) l. were the most abundant. table 1. floristic inventory of the matanzas locality, cuba, 2018. matanzas meliponary family scientific name biological form appearance frequency amaranthaceae alternanthera sessilis (l.) r. br. ex dc. herbaceous abundant anacardiaceae mangifera indica l. tree abundant anacardiaceae anacardium occidentale l. tree rare or scarce annonaceae annona muricata l. tree frequent annonaceae annona squamosa l. tree little frequent arecaceae cocos nucifera l. tree frequent arecaceae roystonea regia (kunth) o.f. cook tree little frequent arecaceae adonidia merrillii (becc.) becc. tree abundant asteraceae bidens pilosa l. shrub abundant asteraceae viguiera dentata spreng. shrub little frequent bignoniaceae crescentia cujete l. tree little frequent boraginaceae cordia collococca l. tree rare or scarce boraginaceae bourreria virgata (sw.) g. don shrub frequent boraginaceae cordia alliodora (ruiz & pav.) oken tree frequent burseraceae bursera simaruba (l.) sarg tree abundant cactaceae nopalea auberi (pfeiff.) salm-dyck tree rare or scarce fonte-carballo et al. – cuban stingless bees with the specialized resource use4 calophyllaceae calophyllum antillanum britton tree little frequent combretaceae terminalia catappa l. tree frequent commelinaceae tradescantia pallida (rose) d.r. hunt herbaceous frequent convolvulaceae ipomoea triloba l. herbaceous frequent convolvulaceae turbina corymbosa (l.) raf. herbaceous frequent cucurbitaceae cucurbita pepo l. herbaceous little frequent fabaceae mimosa pudica l. herbaceous little frequent fabaceae vachellia farnesiana (l.) wight & arn. tree little frequent fabaceae senna spectabilis (dc.) h.s. irwin & barneby tree abundant fabaceae tamarindus indica l. tree rare or scarce fabaceae trifolium incarnatum l. herbaceous frequent fabaceae dichrostachys cinerea (l.) wight & arn. tree frequent lamiaceae tectona grandis l. f. tree rare or scarce lauraceae persea americana mill. tree abundant malvaceae hibiscus rosa-sinensis l. shrub frequent malvaceae hibiscus elatus sw. tree rare or scarce meliaceae swietenia mahagoni (l.) jacq. tree rare or scarce moraceae morus alba l. tree rare or scarce moringaceae moringa oleifera lam. tree frequent musaceae musa x paradisiaca l. herbaceous abundant myrtaceae psidium guajava l. tree abundant passifloraceae passiflora edulis sims herbaceous little frequent polygonaceae coccoloba uvifera (l.) l. tree abundant rutaceae citrus x aurantium l. tree frequent rutaceae citrus x limon (l.) osbeck tree frequent sapotaceae manilkara zapota (l.) p. royen tree frequent table 1. floristic inventory of the matanzas locality, cuba, 2018. (continuation) matanzas meliponary family scientific name biological form appearance frequency in mayabeque (table 2), a lower number of plant species (33) was identified, grouped in 22 families. in the category of abundant are mangifera indica l., bursera simaruba (l.) sarg, ipomoea triloba l., vachellia farnesiana (l.) wight & arn., gliricidia sepium (jacq.) kunth, persea americana mill., psidium guajava l., gouania polygama (jacq.) urb. in general, in the two localities, the biological form that prevailed was the tree, followed by shrubs and herbaceous. in the honey samples from matanzas (table 3) higher richness of pollen forms is observed, with a mean of 11.2 and a maximum of 12 (beehives 9 and 11). in the analyze honey from this locality, the predominant pollen percentage was that of alternanthera forssk., with a mean of 38.26%, with the dominant pollen (>45%) in three of the five samples, followed by psidium guajava l. (17.50%) and the genus trifolium l. (16.07%). the most represented family was amaranthaceae, followed by myrtaceae, and finally fabaceae because only one sample (beehive 9) showed pollen of mimosa pudica l., justifying its inclusion in the companion category, being lower than in the other beehives. meanwhile, in the samples of the mayabeque provenance (table 4), the quantity of pollen forms or types is considerably lower, with a mean value of 7 and a maximum of 13 for the case of the honey belonging to beehive 6. it should be highlighted that the pollen from mimosa pudica prevailed in all the examined honey samples, with a mean percentage of 74.05%, besides being the dominant pollen (>45%) in the five studied beehives. fabaceae and myrtaceae, represented by the species psidium guajava, stand out as companion pollen (16-45%) of the honey obtained from beehives 2 and 48, with a mean of 23.21%. when analyzing the foraging strategy of m. beecheii (fig 1), we found that in the matanzas honey, the mean value of the diversity index (h´) was 1.34, with a maximum value in beehive 11 (1.88). on the contrary, mayabeque showed a lower mean (0,84). these results are in correspondence with the ones exposed in table 4. regarding the evenness index (fig 1), in matanzas, the mean value was 0.45, and again in beehive 11, the highest value was recorded (0.63). for mayabeque, this indicator had a mean value of 0.28. sociobiology 69(3): e7729 (september, 2022) 5 mayabeque meliponary family scientific name biological form appearance frequency anacardiaceae mangifera indica l. tree abundant arecaceae cocos nucifera l. tree frequent arecaceae roystonea regia (kunth) o. f. cook tree frequent arecaceae adonidia merrillii (becc.) becc. tree frequent asteraceae bidens pilosa l. shrub frequent boraginaceae cordia gerascanthus l. tree little frequent burseraceae bursera simaruba (l.) sarg tree abundant cactaceae dendrocereus nudiflorus (engelm. ex sauvalle) britton & rose tree little frequent convolvulaceae ipomoea triloba l. herbaceous abundant convolvulaceae turbina corymbosa (l.) raf. herbaceous frequent ericaceae lyonia myrtilloides griseb. tree little frequent fabaceae mimosa pudica l. herbaceous frequent fabaceae vachellia farnesiana (l.) wight & arn. tree abundant fabaceae gliricidia sepium (jacq.) kunth tree abundant lauraceae persea americana mill. tree abundant malvaceae ceiba pentandra (l.) gaertn. tree little frequent meliaceae trichilia hirta l. tree little frequent moringaceae moringa oleifera lam. tree little frequent myrtaceae psidium guajava l. tree abundant nyctaginaceae pisonia aculeata l. shrub frequent oxalidaceae averrhoa carambola l. tree rare or scarce polygonaceae antigonon leptopus hook. & arn. herbaceous frequent rhamnaceae gouania polygama (jacq.) urb. shrub abundant rosaceae prunus domestica l. tree frequent rosaceae pyrus communis l. tree little frequent rubiaceae coffea arabica l. shrub little frequent rutaceae citrus x aurantium l. tree frequent rutaceae citrus x limon (l.) osbeck tree frequent sapindaceae melicoccus bijugatus jacq. tree frequent sapindaceae nephelium lappaceum l. tree rare or scarce sapindaceae serjania diversifolia (jacq.) radlk. herbaceous little frequent sapotaceae manilkara zapota (l.) p. royen tree frequent sapotaceae pouteria dictyoneura (griseb.) radlk. tree little frequent table 2. floristic inventory of the mayabeque locality, cuba, 2018. fig 1. values obtained in the diversity (h´) and evenness (j´) indices of the matanzas (1, 9, 11, 12, 13) and mayabeque honeys (2, 14, 48, 5, 6). fonte-carballo et al. – cuban stingless bees with the specialized resource use6 according to terrab et al. (2004), pollen grain size is one of the factors influencing higher or lower representation of plant species pollen in honey. pollen size can be defined by the length of its polar axis and equatorial diameter, and considering the major axis, they are differentiated into several types (erdtman, 1952). the results indicate that the cuban beehives dominant pollen (>45%) companion pollen (16-45%) important isolated pollen (4-15%) sporadic or rare pollen (1-3%) present pollen (<1%) 1 psidium guajava (65,96%) mimosa pudica (13,98%) citrus sp. (6,07%) alternanthera (2,9%) moringa oleífera (2,37%) mimosa pigra (1,58%) cariofilaceae (1,85%) acasia farnesiana (0,26%) trifolium (0,26%) bidens pilosa (0,53%) unknown (0,53%) 9 alternanthera (54,11%) mimosa pudica (25,44%) trifolium (7,23%) psidium guajava (5,49%) citrus sp. (2,49%) moringa oleífera (1,5%) acasia farnesiana (0,75%) bursera simaruba (0,99%) bidens pilosa (0,75%) unknown (0,25%) mimosa pigra (0,75%) cariofilaceae (0,25%) 11 trifolium (55,97%) mimosa pudica (14,68%) alternanthera (10,95%) acasia farnesiana (4,48%) bursera simaruba (4,73%) psidium guajava (2,49%) citrus sp (1,24%) moringa oleífera (1,74%) bidens pilosa (1,74%) cariofilaceae (1,49%) albizia lebbeck (0,25%) unknown (0,25%) 12 alternanthera (64,79%) mimosa pudica (9,26%) psidium guajava (10,66%) trifolium (8,25%) acasia farnesiana (2,21%) bursera simaruba (1,41%) moringa oleífera (1,21%) bidens pilosa (1,81%) albizia lebbeck (0,2%) mimosa pigra (0,2%) 13 alternanthera (58,53%) mimosa pudica (9,71%) acasia farnesiana (6,82%) trifolium (8,66%) bursera simaruba (4,2%) psidium guajava (2,89%) moringa oleífera (1,05%) bidens pilosa (1,81%) albizia lebbeck (1,84%) citrus sp. (2,36%) coccoloba uvifera (1,31%) table 3. pollen types identified in the honeys from the beehives of matanzas, cuba, 2018. table 4. pollen types identified in the honeys from mayabeque beehives. beehives dominant pollen (>45%) companion pollen (16-45%) important isolated pollen (4-15%) sporadic or rare pollen (1-3%) present pollen (<1%) 14 mimosa pudica (71,29%) psidium guajava (11,88%) mimosa pigra (4,95%) cariofilaceae (4,95%) acasia farnesiana (3,96%) moringa oleífera (2,97%) 2 mimosa pudica (64,11%) psidium guajava (22,6%) moringa oleífera (5,52%) acasia farnesiana (1,54%) mimosa pigra (2,45%) bursera simaruba (3,68%) 48 mimosa pudica (70,24%) psidium guajava (23,81%) mimosa pigra (4,76%) acasia farnesiana (1,19%) 5 mimosa pudica (78,03%) mimosa pigra (9,85%) psidium guajava (6,44%) acasia farnesiana (1,14%) moringa oleífera (3,41%) cariofilaceae (1,14%) 6 mimosa pudica (86,6%) mimosa pigra (2,9%) psidium guajava (6,5%) acasia farnesiana (0,4%) moringa oleífera (0,4%) coccoloba uvifera (0,9%) arecaceae tipo adonidia (0,2%) nopalia auberi (0,3%) pristimera coriacea (0,2%) mangifera indica (0,3%) myrtaceae tipo syzygium (0,6%) unknown (0,3%) unknown (0,2%) sociobiology 69(3): e7729 (september, 2022) 7 stingless bee livestock of both provinces has a predilection for collecting pollen grains of small size (varying from 10 to 25 µm) and explain the highest representation of the species m. pudica in the mayabeque honey (it measures 10 µm) (figure 2). the main floral resources used by the livestock of matanzas province (fig 3) were, first, m. pudica, with a mean percentage of 55,24%, dominant in three of the five samples, followed by b. simaruba (31,11%) and p. guajava (25,50%). fabaceae was the most represented family in matanzas and mayabeque (fig 4). fig 2. pollen grains of the prevailing types identified in the stingless bee honey and pollen samples of both provinces seen with a magnification of 400. genus trifolium (a); genus alternanthera (b); mimosa pudica (c); bursera simaruba (d) and psidium guajava (e). scale bar: 20 µm. fig 3. main floral resources identified in the pollen collected from matanzas province. dominant pollen (>45%), companion or secondary pollen (16-45%), isolated pollen (4-15%). fonte-carballo et al. – cuban stingless bees with the specialized resource use8 discussion if the criterion expressed by pérez de zabalza (1989) is followed, the studied honey would be classified as of low palynological richness because they represent less than 20 pollen forms. nevertheless, this is a parameter established for european bee (apis mellifera linnaeus, 1758) honey in a mediterranean environment which largely differs from the tropics. studies by imperatriz-fonseca et al. (1989) and ramalho (1990) in stingless bees showed some general foraging patterns for meliponini, proving that, despite foraging on the wide diversity of available floral resources in the area, they are concentrated only on a few species. this would explain the obtained results. ramalho (2004) observed that the bees of the genus melipona visit flowers of different shapes, sizes, and colors to obtain their food. however, there was a preference for small flowers with radial symmetry and open corollas in which access to pollen and nectar is relatively easy. studies conducted in 18 colonies of three species of the meliponini tribe found a prevalence of small pollen grains, suggesting the preference of these bees for small-size flowers with short pistils (vossler, 2015). guimarães et al. (2021) studies confirm the results obtained in the mayabeque honey regarding the abundance of m. pudica pollen grains (10 µm). these authors detected high representativeness of the mimosa type in the pollen content of honey samples of melipona seminigra pernigra moure and kerr, 1950, ascribable, according to ferreira and absy (2017), to its release in large quantities, their configuration, and the small size of pollen grains (zappi et al., 2018). in the case of m. beecheii, it has been reported that the type of opening or dehiscence of the anthers (vossler, 2015) influences the preference for pollen types among the flora in the foraging area. such preference favors collecting pollen types from fabaceae, melastomataceae, and myrtaceae, which have poricidal openings (biesmeijer, 1997). hence, the higher representation of these families in the evaluated honey of both provenances coincides with the observation made by novais and absy (2015) and costa et al. (2017) in the melionini tribe. the results of the diversity index (h´) for the matanzas honey (fig 1) coincide with the value range from 1.23 to 2.00, obtained by pérez-sato et al. (2018) in a. mellifera honey produced in the high plateau of the puebla state in mexico. however, they are below the ones reached by soto (2007) for the stingless bee species scaptotrigona pectoralis dalla torre, 1896, in guatemala; and the ones reported by rezende et al. (2020) in three melipona species in brazil. for this same index, the mayabeque provenance was shown in correspondence with those obtained by novais et al. (2013) for the stingless bee species tetragonisca angustula illiger, 1806, in brazil, with ranges comprised from 0.36 to 2.55. the diversity index (h´) is a tool that determines the breadth of the trophic niche bees through the number of botanical taxa present in the honey samples. ferreira and absy (2017) concluded that the breadth of the niche is determined by the movements of massive foraging of bee species with a higher number of workers, which consequently collect more trophic resources. on the other hand, colonies that have less population tend to compete with lower intensity for trophic resources, as we observed for m. beecheii in matanzas (table 2). yet, both localities show a breadth of trophic niches in correspondence with the results of hilgert-moreira et al. (2013) for the genus melipona illiger, 1806. the evenness index (j´) values were low in matanzas (0.45) and mayabeque (0.28). according to espinoza-toledo fig 4. main floral resources identified in the pollen collected from the mayabeque province. dominant pollen (> 45%), companion or secondary pollen (16-45%), isolated pollen (4-15%). sociobiology 69(3): e7729 (september, 2022) 9 et al. (2018), these results can be explained by the dominance of some species that causes (j´) to show a strong trend toward zero, as observed in the analyzed honey. this fact is markedly proven in the mayabeque honey (table 4), where the dominant species in all the samples was mimosa pudica. in addition, the values reached for this index indicate that the bees from both localities showed oligolectic behavior. according to biesmeijer (1997), bees classified as oligolectic or specialists behave by visiting and collecting pollen from a very restricted group of plants, which applies to m. beecheii. on the other hand, polylectic or generalist bees utilize pollen and nectar from different plant species in their feeding. in turn, ramalho et al. (1990) and rezende et al. (2020) ascribe this characteristic to the fact that the beehives of this stingless bee species are composed of a small number of individuals compared to other species. that is why they focus on few available resources and exploit them intensely. the most represented family in matanzas and maybeque (figure 4) was fabaceae. similar results were obtained by ferreira and absy (2013) and barquero-elizondo et al. (2019) in stingless bee species. they ascribe this fact to the relevance of this family within the ecosystem for its diversity (small-size plants, lianas, shrubs, and trees) and abundant flowerings throughout the year. within this family (fabaceae), the species that stood out was mimosa pudica, reported as a pollen source for bees of the melipona genus in el salvador (biesmeijer, 1997), colombia (rodríguez, 2005) and other neotropical habitats. in cuba, in previous studies conducted by leal-ramos and sánchez (2013), they observed a high frequency of the pollen type of m. pudica in pollen samples obtained from the species m. beecheii. conclusions a specialist feeding habit characterizes the cuban stingless bee livestock of the matanzas and mayabeque provinces. it has a diet based on a few plant species, among which bursera simaruba (l.) sarg., mimosa pudica l., psidium guajava l., trifolium spp., and alternanthera spp. stand out. in addition, a marked preference for small pollen grains from 10 to 25 µm was observed, especially those of mimosa pudica in the mayabeque honey. acknowledgments the authors acknowledge the support of the swiss cooperation and development agency (sdc) for its contribution to funding this research through the project biomas-cuba, in its stage iii. likewise, they thank the pastures and forages research station indio hatuey, the university of matanzas, and the palynology and plant conservation group (gpcv) of the instituto hispano luso de investigaciones agrarias (ciale) of the university of salamanca. authors’ contribution lfc: conceptualization, methodology, investigation, software, validation, formal analysis, resources, data curation, writingoriginal draft, writing-review & editing, visualization, supervisión, project administration. wlp: conceptualization, investigation, resources jdl: conceptualization, investigation, resources mds: conceptualization, investigation, resources dmq: conceptualization, investigation, software, resources. drc: methodology, investigation, software, validation, formal análisis, resources, writing-original draft, writing-review & editing. jss: methodology, investigation, validation, resources. esr: methodology, investigation, validation¸ resources. references academia de ciencias de cuba. 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(2010). the spontaneous flora around street trees in cities – a striking example for the worldwide homogenization of the flora of urban habitats. floramorphology, distribution. functional ecology of plants, 205: 704-709. zappi, d.c., gastauer, m., ramos, s., nunes, s., caldeira, c. f., souza-filho, p.w., guimarães, j.t.f., giannini, t.c., viana, v.p.l., lovo, j., mota, n.f.o. & siqueira, j.o. (2018). plantas nativas para recuperação de áreas de mineração em carajás. belém, pa: instituto tecnológico vale (itv). 282 p. doi: 10.13102/sociobiology.v62i1.56-64sociobiology 62(1): 56-64 (march, 2015) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 topological structure of plant-bee networks in four mexican environments introduction one of many goals in ecology is to understand how different mechanisms shape ecological interactions within natural communities (loreau et al., 2001; dyer et al., 2010). using a network approach several studies have described complex, hyperdiverse communities (bascompte et al., 2003; lewinsohn et al., 2006; hagen et al., 2012; bascompte & jordano, 2014). most studies have primarily focused on mutualistic interactions between plants and animals (mainly pollinators and seed dispersers) (e.g. vázquez & aizen 2004; olesen et al., 2008; dupont et al., 2009 donatti et al., 2011; mello et al., 2011). furthermore, in tropical regions, reproduction (pollination, fruit and seed dispersal) of more than 90% of shrubs and trees depends on animals (e.g., insects, birds, bats) (bascompte & jordano, 2008). abstract we analyzed the topological structure (e.g., links per species, connectance, core-periphery analyses, specialization, nestedness, modularity) of plant-bee interactions of four areas in mexico. we used qualitative data (binary networks). mexico exhibits a complex geography and community variation that can affect bee networks. network architecture is variable within large spatial scales, thus our results should vary according to site characteristics (la mancha and totula in veracruz, carrillo puerto in quintana roo, and the tehuacancuicatlan valley, in puebla), type of vegetation, endemism, altitude, size of area sampled. network topology varied among sites, and the presence of nested or modular patterns was analyzed for robustness to simulated species extinctions. the lowest species richness was recorded for the quintana roo site (15 plant, 25 bee species), and the highest for the tehuacan-cuicatlan valley site (88 plant, 231 bee species). there was a tendency to have more connected networks when species richness was low and networks with greater species richness had a higher number of interactions. the distribution of interactions differed between environments but not due to network size and all were significantly nested. the robustness to cumulative extinctions showed a different pattern at each site; the most robust network was at carrillo puerto, which also was the site with less species. sites with more endemic species (e.g. tehuacan) had more specialized interactions, and were more susceptible to extinction. sociobiology an international journal on social insects va ramírez-flores1, r villanueva-gutiérrez2, dw roubik3, ch vergara4, n lara-rodríguez5, w dáttilo1, meb ferrer6, v rico-gray1 article history edited by gilberto m. m. santos, uefs, brazil received 19 january 2015 initial acceptance 20 february 2015 final acceptance 22 february 2015 keywords bee, plant, network, endemism, mexico. corresponding author dr. victor rico-gray instituto de neuroetologia universidad veracruzana calle dr. luis castelazo s/n, col. industrialanimas, xalapa, veracruz 91190, mexico e-mail: vricogray@yahoo.com traditionally, plant-pollinator systems have been studied as small groups of species (stephens et al., 2009; santos et al., 2012). however, modern network tools allow simultaneous analysis of species over different spatial and/or temporal scales (stephens et al., 2009; lange et al., 2013). such analysis allows the study of complex systems and mega-diverse communities (jordano et al., 2009; hagen et al., 2012). although several communities defy analysis based on few, incomplete samples, as the behavior of the system is often very different from the sum of its parts (jordano et al., 2009). for instance, when most interactions are not reached by a study, the ‘system’ remains, essentially, unknown. nonetheless, the conservation and management of the studied communities, as well as of pollination services, can receive useful information through these efforts (burkle &alarcón, 2011). research article bees 1 universidad veracruzana, xalapa, veracruz, mexico; 2 colegio de la frontera sur, ecosur-unidad chetumal, mexico; 3 smithsonian institution, washington, dc., u.s.a.; 4 universidad de las américas, puebla, mexico; 5 universidad de alicante, san vicente del raspeig (alicante), spain; 6 universidad complutense, madrid, spain sociobiology 62(1): 56-64 (march, 2015) 57 understanding the ecological and evolutionary causes and consequences of spatial and/or temporal variation in plant/ floral visitor systems is very important when formulating basic and applied questions on the structure and function of a community, evolution of floral characteristics (e.g. hernández-yáñez et al., 2013), and development of optimal conservation strategies (e.g. burkle & alarcon, 2011). although landscape heterogeneity may augment the number of interactive species, these may not constantly share the same habitat (jordano et al., 2009; clemente et al., 2013). for instance, olesen and jordano (2002) analyzed plant-pollinator networks from different geographical areas and found that interaction patterns co-varied with factors such as latitude, altitude and habitat isolation (islands). also, plant species are more generalized (i.e. have more interactions) at higher latitude and in lowland habitats. in oceanic islands, for instance, networks are often specialized (i.e. have less interactions), pollinators are scarce, and a low proportion of animals exists, relative to plant species, which may add to the idea of higher specificity in the tropics that at higher latitudes (olesen & jordano 2002). based on high species numbers and the abundance of certain species, bees are probably the insect group best-adapted to floral biology and flower visits. this group is essential for pollination and sexual reproduction of most flowering plants (including many cultivars) (roubik, 1992; michener, 2000). the latter is very important in terms of securing future food supplies (deguines et al., 2014). specific studies (reviewed by hagen et al., 2012) of plant-pollinator networks consider all floral visitors of different taxonomic groups (e.g. bees, flies, butterflies, beetles, wasps, birds, and bats), and have evaluated how different factors (biotic, abiotic) structure networks. however, despite the general growth of knowledge on network topology, little is known about how plantfloral visitor networks are structured and how they vary over space when only one taxonomic group in considered. mexico exhibits a complex geography and community variation, both of which should affect plant-bee networks. thus, network architecture (e.g. links per species, connectivity, core-periphery analyses, specialization, nestedness) is likely change within the country so that results should vary according to site characteristics (e.g. type of vegetation, endemism, altitude, size of area sampled). here we describe the topological structure of plant-bee interactions in four distinct areas in mexico. in particular, we searched for the presence of modular patterns and analyzed the pattern of secondary extinctions and stability of the networks in the range sampled. material and methods study area and field work field work was accomplished in four mexican sites: 1) tehuacan-cuicatlan valley, puebla; 2) surroundings of carrillo puerto, quintana roo; 3) la mancha, veracruz; and 4) a traditional management coffee plantation in the montane forest in totutla, veracruz (table 1). due to the number of researchers involved, field work differed among sites, although entomological nets were used throughout. the surveys in the tehuacan and totutla sites were guided by c. vergara, the survey in quintana roo was guided by villanueva and roubick, and the survey at the la mancha site was guided by n. lara and v. rico-gray. the number of different areas per site was given by the vegetation changes (see table 1). all accounts of visits by potential bee pollinators were determined by observations of bees on flowers and not from previous knowledge of the interactions. in the tehuacan-cuicatlan valley the vegetation is a xerophytic shrubland (rzedowski 1978), and zavala-hurtado (1982) described four vegetation types: shrubs with spines (‘matorral espinoso’), tetechera, cardonal and izotal (yucca spp.). even though some of the vegetation types are in spanish, the latter names are based on dominant plant species (see also table 1). the biota of the valley is characterized by a high number of endemic species, the highest density known in mexico (méndezlarios et al., 2004). sampling was accomplished with monthly observations (3-5 days per visit) between july 1996 and june 1997. five areas with similar plant composition were repeatedly surveyed (total area of site was 10,000 km2), which included the main vegetation associations and the border of agricultural fields. plants with flowers were selected and bees were collected, killed and mounted the same day, and plants collected (see vergarabriceño, 1999). bees and plant vouchers are deposited in the collection at universidad de las americas. in carrillo puerto, quintana roo field work was carried out in areas near the carrillo puerto-valladolid highway and the road from carrillo puerto to vigia chico, which is within the sian ka’an biosphere reserve, an area of a half million hectares (e.g. ciqro, 1983). five collecting sites were selected along a 48 km transect, which were visited every two months between june 2008 and september 2009. bees were collected in each site within a 1 km radius, then killed, mounted and identified. bees and plant vouchers are deposited in ecosur. two sites were sampled in the state of veracruz: centro de investigaciones costeras la mancha (cicolmala mancha) and in the vicinity of totutla. at la mancha, bees were collected monthly between march 2007 and march 2008 (five days per visit) along established trails that included different environmental types (see rico-gray, 1993). bees and plants were collected, identified using the collections of instituto de ecologia, a.c. (iexa, xal) and deposited in those collections. the ranch “finca el mirador” is located in the vicinity of totutla. bees were collected every month in october-november 2005 and january through september 2006 in three sites within the ranch: el desengaño, loma araucarias and loma ventura. a part of this site has been converted to an organic coffee plantation. bees and plants were collected and plants were identified in xal herbarium of instituto de ecologia, a.c. and bees were identified by c. vergara (udla-p), with the help of terry griswold (usda bee laboratory, logan ut, u.s.a.) and michael engel (biodiversity institute, university of kansas, lawrence, ks, u.s.a.). plants and bees are deposited in c. vergara’s collection. va ramírez-flores et al. bee-plant networks 58 si te a lti tu de ( m ) c lim at e te m pe ra tu re o c pr ec ip ita tio n (m m ) v eg et at io n l a m an ch a 19 º 3 6’ n –9 6º 2 2’ w 20 w ar m s ub hu m id w ith d ry p er io d, w et in s um m er , a nd a c ol d fr on t s ea so n in w in te r 24 -2 6 15 00 d un e pi on ee rs , s an d du ne s cr ub , t ro pi ca l d ry an d de ci du ou s fo re st s, m an gr ov e an d fr es h w at er s w am ps c ar ri llo p ue rt o 19 o 42 ’ 1 9o 2 5’ n 87 o 52 ’ – 8 8o 0 6’ w 30 w ar m s ub hu m id w ith s um m er r ai n 25 .8 11 58 su bd ec id uo us tr op ic al f or es t a nd s ec on da ry ve ge ta tio n te hu ac an -c ui ca tla n 18 o 14 ’ – 1 8o 2 2’ n 97 o 26 ’ – 9 3o 3 4’ w 60 030 00 w ar m s ub hu m id , s um m er r ai n, m ar ke d dr y pe ri od d ur in g su m m er r ai n 21 40 0 sr ub s w ith s pi ne s, c ol um na r ca ct i a ss oc ia tio ns , a nd y uc ca s p. a ss oc ia tio ns to tu tla 19 º 1 2’ n –9 0º 5 3’ w 10 10 te m pe ra te , h um id w ith s um m er r ai n, ha rd ly a ny th er m ic o sc ill at io n 20 18 25 r us tic c of fe e pl an ta tio n, m on ta ne f or es t t ab le 1 . c ha ra ct er is tic s pe r si te ( g ar cí a 19 81 ; i n e g i 19 88 ; v al ie nt e b an ue t 1 99 8; d ía zc as te la zo 2 00 4; c as til lo -c am po s et a l. 20 06 ; d el oy a et a l. 20 07 ). a ls o, s ee th e m et ho ds s ec tio n (“ st ud y si te s an d fie ld w or k” ). network analyses the manner in which networks were sampled, only allows us to build binary (qualitative) networks. we defined each sampling site as an adjacency matrix c, in which cij represents the number of interactions between bee species i and plant species j (bascompte et al., 2003). we then calculated the following metrics derived from the network theory to describe the bee-plant networks in each of the four sites (tehuacan-cuicatlan valley, quintana roo, la mancha, and totutla): links per species, connectance, network specialization, nestedness, modularity, and robustness to species extinction. we used these metrics mainly because they provide a way to describe the organization of our networks in a way that allows direct comparison with previous studies on plant-pollinator networks and among our four sites. we calculated the average degree for bees and plants using the arithmetic means of degrees (number of interactions in which each species was involved) of all species. we calculated the (c) connectance, defined as the fraction of registered interactions with respect to the total number of possible interactions: c= i (b x p) where, i is the total number of interactions observed, b is the number of bees species, and p is the number of plant species (jordano, 1987). we calculated the degree of specialization (h2’) for each network (blüthgen et al., 2006). the index ranges from zero (extreme generalization) to one (extreme specialization). the index is derived from the shannon index (h’ is not dependent on sample size), rare species are not equally presented by the index and is robust to changes in sampling intensity and the number of interacting species in the network (poole, 1974; blüthgen et al., 2006). we used the package bipartite (dormann et al., 2009) in “r” to perform network-specialization analysis. we used the nodf metric (nestedness metric based on overlap and decreasing fill) (almeida-neto et al., 2008) to estimate nestedness in each network. to calculate nestedness we used aninhado 3.0 (guimarães & guimarães, 2006). under perfect nestedness, generalist species interact with each other, while the specialist species interact with generalist species (bascompte et al., 2003). we tested the significance of nestedness for each network through 1,000 simulated networks generated by the null model ii (ce) (bascompte et al., 2003), which allowed us to determine if the degree of nestedness was higher than those expected when compared to random patterns of interactions. in this null model, the probability that a bee-plant interaction occurs is proportional to the mean number of interactions of both species (bascompte et al., 2003). due to differences of sampling, and species richness, in our study sites, we also calculated nestedness controlling potential differences in richness and heterogeneity of interactions among our four sites by using a z-score, which normalizes the deviation from a mean value (almeida-neto et al., 2008). we also tested whether there were groups of bee species that interacted more frequently with a particular group of plant species (mello et al., 2011). we assessed modularity (m) within each network based on simulated annealing (sa) (guimerà et al., 2004; guimerà & amaral 2005) using the sociobiology 62(1): 56-64 (march, 2015) 59 software modular (marquitti et al., 2013). the index ranges from zero to one, and high values of m indicate that different bees and plants species are grouped by modules that are semi-independent, within the network (olesen et al., 2007). the significance of m also was estimated with null model ii, described above. if a network was significantly modular, we also tested within each module for a nested pattern. we calculated network robustness for each site, and to species extinction based on the cumulative random removal of species (sensu burgos et al., 2007). we also used the robustness index (r) to estimate the area under the extinction curve (burgos et al., 2007; dáttilo, 2012). this index ranges from zero to one, and values of r close to one come from robust network curves, because their extinction curve decreases slowly (burgos et al., 2007). when compared to other curves, a robustness index is not as sensitive to curve shape (burgos et al., 2007). the generalist core of species per network (gc), and peripheral species, were categorized using core-periphery components (dáttilo et al., 2013). when gc >1 the species are those with the greatest number of interactions when compared with other species in the same trophic level. they constitute the generalist species core. in contrast, when gc <1 the species exhibit a low number of interactions related to other species in the same trophic level, and form the periphery of the network (dáttilo et al., 2013). results our results show that network topology among sites varied widely (fig 1). the lowest species richness was recorded for the quintana roo site (15 plant and 25 bee species), and the highest was tehuacan-cuicatlan (88 plant and 231 bee species). the networks of la mancha and totutla had intermediate species richness (la mancha: 65 plant and 43 bee species; totutla: 56 plant and 156 bee species, table 2). networks with lower species richness were more connected: the quintana roo site exhibited c= 0.26 and tehuacancuicatlan c= 0.03 (table 2). greater species richness was linked to a higher number of interactions: tehuacan-cuicatlan, 764 beeplant interactions; totutla, 615 bee-plant interactions; la mancha 134 bee-plant interactions; and the quintana roo site 98 bee-plant interactions. however, the distribution of interactions was different among the four environments, apparently unrelated to network size. totutla exhibited the highest mean number of interactions per species (3.18), followed by the quintana roo site (2.45) and tehuacan-cuicatlan (2.39). the network from la mancha had the least number of interactions per species (1.24) (table 2). the totutla network had the lowest specialization value (h2’= 0.39); the other specialization values were: the quintana roo site (h2’= 0.40), la mancha (h2’= 0.69), and tehuacancuicatlan (h2’= 0.58). moreover, the four networks were significantly nested (p < 0.05) (z-scorenodf values: quintana roo site= 1.68, la mancha= 5.74, tehuacan-cuicatlan = 15.28, and totutla= 24.33). additionaly, the tehuacan-cuicatlan network exhibited significant modularity (m= 0.469) (p= 0.02), with only four of the resulting eight modules significantly nested (table 2). table 2. network descriptors for bee-plant networks studied in four sites of mexico. network descriptor tehuacancuicatlan valley la mancha carrillo puerto totutla plant richness 88 65 15 56 bee richness 231 43 25 156 connectance 0.03 0.04 0.26 0.08 network specialization (h2’) 0.58 0.69 0.40 0.39 links per species 2.39 1.24 2.45 3.18 no. of interactions 764 134 98 616 nestedness (nodf) 11.76* 14.62* 38.39* 48.38* nestedness (z-scorenodf) 15.28 5.74 1.68 24.33 modularity 0.46* 0.61 0.31 0.34 robustness to cumulative plant extinctions (r) 0.32 0.49 0.86 0.49 robustness to cumulative bee extinctions (r) 0.49 0.25 0.65 0.26 no. of species in the generalist core (% of total) 15 (17.04%) 10 (15.38%) 4 (26.66%) 6 (10.71%) no. of species in the generalist core (% of total) 24 (10.38%) 5 (11.62%) 4 (12%) 16 (10.25%) * represents p-values < 0.05 fig 1. ecological network involving bees (right) and plants (left) sampled in four different sites of mexico: (a) totutla, veracruz (b) la mancha, veracruz (c) carrillo puerto, quintana roo and (d) tehuacancuicatlan valley, puebla. each node or rectangle represents one plant or bee species, and lines represent bee–plant interactions. rectangle size represents the number of interactions of a certain species, for instance, a larger rectangle is equal to more interactions by that species. the robustness to cumulative extinctions showed a different pattern at each site. the most robust network was the quintana roo site, where plants exhibited a robustness of r= 0.86 and bees r= 0.65. the bees at la mancha were the most vulnerable (r= 0.25), while plants (r= 0.49) exhibited a similar value that for tehuacan-cuicatlan. the totutla va ramírez-flores et al. bee-plant networks 60 discussion we found that in the four areas bees exhibit a nested pattern of foraging, independent of variation in local and landscape environmental factors or species composition. plant– pollinator networks have been evaluated in different types of habitats around the world, and in most cases they exhibit a highly nested pattern (olesen & jordano, 2002; bascompte et al., 2003; jordano et al., 2003). despite differences in habitat structure among areas, we observed that our networks were significantly nested, suggesting that different populations of plants and bees interact in a similar way independent of variations in local and landscape environmental factors, and are highly stable as predicted by previous studies (bascompte et al., 2003; vázquez et al., 2009; hagen et al., 2012). biologically, the nested pattern decreases interspecific competition within mutualistic networks and increases species coexistence, generating a stable pattern of plant-animal interactions (bastolla et al., 2009). based on this perspective, we show that our nested networks have great stability to simulated species loss when compared with structurally different networks (e.g. highly compartmentalized) (mello et al., 2011; dáttilo, 2012; clemente et al., 2013). however, when we evaluated network robustness to simulated species extinction, we observed that areas with greater specialization (e.g. tehuacan-cuicatlan valley and la mancha) tend to be less robust. this probably occurred because environments with a high number of specialist interactions tend to have lower functional redundancy, and therefore, when a species is extinct in a trophic level only a few species from another trophic level can “dampen” the system (mouchet et al., 2010; joner et al., 2011). due to the high specialization of bee-plant networks in the tehuacan-cuicatlan valley and la mancha generated by different mechanisms, it is expected that these areas are functionally less redundant and resilient to disturbances (e.g. sánchez-galván et al., 2012). this was confirmed when species were removed from these networks, the extinction curves declined faster compared with other networks, because deletion of one species necessarily causes the deletion of other partner species. our results demonstrate that environments with many specialized interactions are highly susceptible to loss of species and this pattern should occur in other systems of ecological networks with a high degree of specialization (wasp-plant and ant-myrmecophyte) (dáttilo et al., 2012; clemente et al., 2013). it has been noted that as species richness increases in the community, the number of registered interactions also increases; however, connectance decreases exponentially (jordano, 1987; blüthgen, 2010). our results corroborate these studies, since the most species-rich networks (tehuacan-cuicatlan and la mancha) were the least connected networks. our results suggest that this occurred because bees and plants within these networks had fewer interactions per species, when compared with the other study sites. this result indicates that most interactions in less connected networks tend to be pairwise (fonseca & ganade, network exhibited very similar values to those of la mancha (bees r= 0.26, plants r= 0.49). finally, the plants in tehuacancuicatlan were most vulnerable to species extinctions (r= 0.32), while bees exhibited higher robustness to species loss (r=0.49) (table 2). the quintana roo site network exhibited the most robust species core (26.66% of total plant species in the network), while only three bee species (12% the total, see appendix) were included in the network core. compared to the other three sites, the number of plant species in the core of the totutla network was the smallest (10.71%) (n= 6 species, see appendix), with a similar result for core bee species (10.25%) (n= 16 species) (figure 2). the la mancha plant species core was the second smallest (15.38%), however, more species were included (n= 10 species, see appendix), the la mancha bee core exhibited an intermediate value (11.62%) (n= 5 species, see appendix). core bees for tehuacan-cuicatlan were fewer (10.38%), although composed by 24 species (see appendix). the plant core exhibited intermediate size (17.04%) and was composed by 15 species (see appendix). overlap is meaningless among core species (plants, bees): 1 plant species between sites 1 and 2, and 1 plant species between sites 2 and 3; 1 bee species between sites 1 and 4, and 2 bee species between sites 1 and 2. fig 2. robustness to cumulative species removal of plants and bees in (a-b) totutla, veracruz (c-d) la mancha, veracruz (e-f) carrillo puerto, quintana roo and (g-h) tehuacan-cuicatlan valley, puebla. the images on the left (a-c-e-g) simulate the extinction of plant species, and the images on the right (b-d-f-h) simulate the extinction of bee species. the x-axis axis represents the proportion of primary extinctions of a trophic level, and the y-axis represents the proportion of species in the other trophic level still alive. sociobiology 62(1): 56-64 (march, 2015) 61 1996), which could generate a high degree of specialization within such networks, as found in our networks. why were the tehuacan-cuicatlan and la mancha networks the most specialized? we believe that different mechanisms could be responsible for the high degree of specialization found in these areas. in la mancha, sampling was conducted in locations with different environmental complexities, and therefore, so that certain groups of plants and bees could be habitat specialists (i.e. collected in few sites) and cause more specialized interaction profiles (mcpeek, 1996; warren et al., 2001). the high degree of specialization in tehuacan-cuicatlan possibly occurred due to the extreme endemism that occurs in the area. for instance, areas with large numbers of endemic species tend to have more specialists and rare interactions (olesen et al., 2002; hansen et al., 2002), which is due to the great geographical isolation over evolutionary time and may lead to specialized processes among species (thompson, 2005). interestingly, when data on floristic richness and endemism is compared with that of other natural areas in mexico, it becomes evident that tehuacan-cuicatlan is highest for endemic species (méndezlarios et al., 2004). therefore, based on the differences between tehuacan-cuicatlan and la mancha, it is important to consider that structural characteristics of the vegetation and endemism may be important factors shaping the ecological interactions involving bees and plants. several studies have demonstrated that pollination networks can be highly modular (olesen et al., 2007; ramos-jiliberto et al., 2010; hernández-yáñez et al., 2013). these structures only refer to the presence of groups of species (plants, animals) that interact more intimately with other species in their group rather than with other species in the network (olesen et al., 2007). however, these studies have evaluated networks considering different taxonomic groups together. here we only considered bees, and we should expect a scarce presence of modules, because having only one type of organism a certain historical evolution is shared. in fact, the presence of a modular pattern depends on several factors, from evolutionary to spatial and temporal (olesen et al., 2007; mello et al., 2011). our results only show a modular pattern for the plant-bee network collected in tehuacan-cuicatlan valley. it is possible that through evolutionary time, the high endemism found in this area contributed to the convergence of traits in phylogenetically related bee groups, which currently may visit a group of common plants. finally, part of the totutla forest has been converted to an organic coffee plantation, altering the interactions naturally found there (steffan-dewenter et al., 2001; tylianakis et al., 2007). our results suggest that the abundance of bidens pilosa (compositae, asteraceae), a common secondary plant species, could be explained due to the type of management used. in summary, our results show that structural characteristics of the environments over space are extremely important factors that shape interactions involving plants and bees. moreover, we found that areas with endemic species tend to have more specialized interactions, and that the nature of this characteristic can also influence the patterns of interactions among organisms and the susceptibility to extinction of species. therefore, our results reinforce the role of environmental variation in species maintenance and biodiversity in hyperdiverse systems. acknowledgments we wish to thank the following organizations that allowed us to either accomplish field work or support the analyses and writing of this manuscript: universidad de alicante (nlr), universidad veracruzana (vrg, wd), and the smithsonian institution (dwr, rvg). for tehuacan-cuicatlan, funding was provided by the comision nacional para el conocimiento y uso de la biodiversidad (conabio, h278 apoidea (hymenoptera) del valle de zapotitlan de las salinas, puebla), and by consejo nacional de ciencia y tecnologia (conacyt, 2635-pn, “diversidad, fenologia y flora visitada por apoideos del valle de zapotitlan de las salinas, (puebla)”. the study in totutla was supported by a grant from mexico’s environmental ministry (semarnatconacyt 2002-c01-0194) to cv. the dean office of instituto de investigacion y posgrado (inip) at universidad de las americaspuebla, provided funding and logistic support. we thank wilberto colli-ucán and margarito tuz-novelo for their help in field work in carrillo puerto region. for the totula study we wish to thank jorge a. mueller grohmann for allowing us to work in his property. for the tehuacan-cuicatlan study rhonda and terry griswold and robert w. brooks participated in the collection of specimens; 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(1982). estudios ecológicos en el valle semiárido de zapotitlán, puebla y clasificación numérica de la vegetación, basada en atributos binarios de presencia o ausencia de las especies. biótica, 7: 99-120. appendix species (plants, bees) in the core of the networks per sampled site la mancha, veracruz plants bees bauhinia divaricata apis melifera bidens pilosa lasioglossum sp1 randia laetevirens trigona nigra turnera diffusa ceratina sp1 lantana camara euglossa viridissima waltheria indica palafoxia lindenii hyptis suaveolens tecoma stans piscidia piscipula carrillo puerto, quintana roo plants bees gymnopodium floribundum centris sp. cassia fistula augochlora sp. croton campechianus trigona fulviventris parthenium hysterophorus totutla, veracruz plants bees bidens pilosa augochloropsis sp1 elephantopus molis ceratina sp2 smallanthus maculatus bombus medius sida rhombifolia lasioglossum sp1 marsypianthes chamaedrys paratetrapedia apicalis cyperaceae sp1 apis melifera ceratina sp1 trigona fulviventris lasioglossum sp8 augochlora aurifera ceratina nautlana melissodes tepaneca lasioglossum costale trigona corvina trigona nigerrima tehuacan-cuicatlan valley, puebla plants bees salvia polystachya plebeia frontalis ipomoea pauciflora agapostemon cockerelli sanvitalia fruticosa melissodes gilensis crenata echinocactus platyacanthus augochlorella neglectula hechtia podantha lasioglossum sp16 gymnosperma glutinosum centris atripes tecoma stans lasioglossum sp6 wigandia urens halictus ligatus flaveria pringlei paraugochloropsis vesta prosopis laevigata ashmeadiella zapotitlana viguiera dentata macrotera azteca verbesina neotenoriensis megachile zapoteca beaucarnea gracilis paraugochloropsis metallica macroptilium atropurpureum anthidium maculifrons nissolia microptera anthophora californica exomalopsis pueblana agapostemon sp1 lasioglossum sp13 centris transversa ceratina sp3 ceratina nautlana lasioglossum sp18 megachile otomita melissodes tepaneca open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.7894sociobiology 69(4): e7894 (december, 2022) introduction bees, wasps and ants are insects that take part in various ecological interactions; among them, flower-visitation. the visitation of flowers by hymenopterans may result in plant pollination, although other phenomena, such as nectar robbing and competition with real pollinators, may also be observed. (vizentin-bugoni et al., 2018). bees are key pollinators, responsible for the pollination of several botanical species and the absence or population decline of these animals is substantially risky for crops and the native flora, because, abstract the hymnoptera order includes several flower-visiting insects (e.g. ants, bees, and wasps) and the coexistence of many different species in the same community can generate interspecific competition. notwithstanding shared communities, research which evaluates how these taxonomic groups influence a whole community of flower-visiting hymenoptera is lacking. moreover, abiotic factors can also impact these floral visits, because each organism responds differently to climatic variations. the goal of this study is to evaluate abiotic factors, specifically relative air humidity and air temperature, which may be able to impact the number and the frequency of interactions between hymenopterans and flowers and to assess the composition and niche organization, by making use of interaction networks, of the entire community of flower-visiting hymenoptera at the botanical garden of the universidade federal rural do rio de janeiro. for the duration of a year, we took samples in that botanical garden, compartmentalizing the collections temporally in accordance with the time of the insects’ shift (morning or afternoon). we observed a positive influence of air temperature on the number of ant interactions and visits. it is also possible to observe that most of these interaction networks exhibited a nested and nonmodular pattern and an average level of network specialization. in addition, bees stood out as the species with the highest frequency of visits and with the most generalist behavior. this study demonstrates how a botanical garden can sustain a diverse community of floral visiting hymenoptera in an urban environment and why it consists in an important tool for biodiversity conservation. sociobiology an international journal on social insects mariana r. menezes¹, bianca f.s. laviski¹, adriano p.l. dos santos², eder c.b. de frança²,³, mariane s. moreira² ricardino da conceição-neto¹, jarbas m. queiroz² article history edited by wesley dáttilo, instituto de ecología a.c., mexico received 28 march 2022 initial acceptance 14 april 2022 final acceptance 29 august 2022 publication date 28 december 2022 keywords hymenoptera, abiotic factors, pollination, niche overlap. corresponding author mariana r. menezes universidade federal rural do rio de janeiro, instituto de floresta rodovia br 465, km 07 zona rural cep: 23890-000 seropédica-rj, brasil. e-mail: mariana_romenezes@hotmail.com since many species depend on these pollinators for sexual reproduction, it can lead plant species to extinction and affect food production (de marco jr. & coelho, 2004; klein et al., 2007). on the other hand, the role of wasps as plant pollinators may be considered unusual because certain wasps visit flowers to prey on other arthropods, to collect or to steal nectar collected by other hymenopterans or because they are attracted by hormones released by the plant (torezansilingardi, 2012; nagasaki, 2021). the importance of ants as plant pollinators is still a matter of debate while some ant species can contribute to the pollination process (gonzálvez 1 programa de pós-graduação em biologia animal, icbs, ufrrj, seropédica-rj, brazil 2 departamento de ciências ambientais, if, ufrrj, seropédica-rj, brazil 3 programa de pós-graduação em entomologia, ufpr, curitiba-pr, brazil research article social arthropods flower visitation by bees, wasps and ants: revealing how a community of flower-visitors establish interaction networks in a botanical garden mailto:mariana_romenezes@hotmail.com mariana r menezes et al. – hymenoptera flower-visitors in a botanical garden2 et al., 2013; wang et al., 2015), other species can chase away potential pollinators (villamil et al., 2018; nogueira et al., 2021). notwithstanding shared communities, research which evaluates how these taxonomic groups influence the composition and the organization of a whole community of flower-visiting hymenoptera is lacking. when focusing on the interactions of hymenoptera and flowers, several factors can impact the amount of these interactions and their frequency. some of them are abiotic, such as relative air humidity. in another study on a community of floral-visitors, barbosa et al. (2016) observed a negative correlation between relative air humidity and flower visits by hymenoptera, which may be related to the fact that nectar changes its concentration and viscosity according to relative air humidity (winkler et al., 2009). another factor possibly related to the hymenopteran-flower interaction is air temperature. air temperature can modulate the foraging behavior of hymenopterans, as the energy cost of foraging varies in accordance with changes in air temperature (classen et al., 2015; kovac et al., 2015). in warm environments, hymenopterans tend to increase their foraging and, consequently, their floral visitation, what can change the community’s characteristics (e.g. specialization, diversity, richness, resource supply) (classen et al., 2015; classen et al., 2020; luna et al., 2021). however, because this community consists of organisms biologically and behaviorally unique, these factors can affect each of these taxonomic groups differently: ants are expected to respond better to increases in temperature once they are more resistant to high temperatures than bees and wasps are (heinrich, 1993; kovac et al., 2015). therefore, assessing how abiotic factors relate to each taxonomic group separately may be a proper strategy to understand how these factors influence interactions in the community of flower-visitors. when different species of flower-visitors interact with the same group of plants, they can be functionally redundant and end up increasing interspecific competition, which would reduce the diversity and richness of these visitors (blüthgen & klein, 2011). the co-occurence of many species in the same community of flower visitors can be facilitated by the specialization of some groups and by the differentiation of niches (blüthgen & klein, 2011; watts et al., 2016). to help us to understand how this particular community's organization and species composition, we can use a tool that has been widely employed to study insectplant interactions: interaction networks (e.g. dáttilo & ricogray, 2018). measures of specialization on the network level can show us the degree of niche division among species of floral visitors in the community studied (blüthgen et al., 2006). moreover, some other network metrics can help us understand community organization. to assess whether a group of selective species interact with a set of plants visited by generalist floral-visitors, we can calculate the nestedness of networks in that community (fortuna, 2010; dehling, 2018). and to assess the tendency of a subset of species to relate more frequently to each other than to other species, constituting modules, we can calculate the modularity of the networks in that community (fortuna, 2010; dehling, 2018). we must be careful when treating insect-flower interactions as mutualistic networks because some visits have negative effects on plants (vizentinbugoni et al., 2018). what usually happens in mutualistic plantinsect networks (e.g. flower visitors, extrafloral nectaries or seed removers) is that, due to the insects’ behavior, they display low specialization, high nestedness and no modularity (camposnavarrete et al., 2013; lange & del-claro, 2014; anjos et al., 2018; laviski et al., 2021). although all hymenopteran networks follow the assumptions for insect-plant mutualistic networks, metric values are expected to be quite different for networks with each group of organisms, because they are different taxonomic groups (campos-navarrete et al., 2013). bees, for instance, are responsible for the pollination of many botanical species and tend to be more generalist when it comes to flower visitation (de marco jr. & coelho, 2004; klein et al., 2007; torezan-silingardi, 2012). thus, we expect the interaction networks which present bees to be more nested, less specialized and not to feature modularity (blüthgen et al., 2006; fortuna, 2010; dehling, 2018). while specialization indicates a niche division in a community, identifying its generalist species is of utmost importance, because they are crucial to the stability and operation of community organization, mainly because the core species interact with nearly all species in the community (bascompte et al., 2003; guimarães et al., 2006; dáttilo et al., 2013a). we can evaluate the most generalist species in a community based on the analysis of core species in the network (dáttilo et al., 2013a). as stated earlier, bees usually present generalist flower-visiting behavior, therefore they are commonly the species with the highest frequency of interactions in flower-visiting hymenopteran networks (campos-navarrete et al., 2013). another factor for assessing similarities or differences between niches is time: observing the species composition and activities during a day, during a year or even over decades allows us to compare the species that interact during each interval and calculate the overlap of these temporal niches (díaz-castelazo et al., 2013; dáttilo et al., 2014a). on an even smaller scale, we can evaluate how the temporal niche divides itself throughout the day. some bees exhibit a peak behavior of flower visitation at different times (tschoeke et al., 2015) and ant species can also have different peaks of activity in a 24-hour period (fellers, 1989), as well as wasp species (brito et al., 2020). however, there are no studies which consider the overlap of temporal niches during the day for a floral-visitor community with more than one hymenopteran group. in this work, we used samplings of floral-visitors of a botanical garden’s community collected over a year, both in the morning and in the afternoon, to understand how some abiotic factors correlate with the number and frequency sociobiology 69(4): e7894 (december, 2022) 3 of floral visitation interactions and also to comprehend the organization and composition of this community by using floral-hymenopterans networks. regarding the influence of abiotic factors on increased foraging and how these factors correlate with the number and frequency of floral visitations, we hypothesized that relative air humidity would negatively impact the number and frequency of floral visits, while air temperature would positively affect the interactions. regarding the organization and composition of this community of floralvisitors, we also hypothesized that interaction networks would show patterns similar to other insect-plant mutualistic networks: they will be nested, lack modularity and have low specialization. in addition, we presumed that bee species would be most prevalent network core species. we also expect communities to be different according to the daily temporal niche: in the morning, with a higher frequency of bees in interactions, we expect the networks to be more nested, less specialized and non-modular; in the afternoon, with a higher frequency of ants, we expect the networks to be less nested, more specialized and non-modular. material and methods study area we conducted the study at the botanical garden of the universidade federal rural do rio de janeiro (22°45’56”s; 43°41’33”w), a 16.5 hectare area, located in seropédica, rio de janeiro state, brazil. the site is located in a degraded region of the atlantic forest, where some botanical species, native and exotic, were planted, aiming to create a living botanical collection; the area has also a pond, a small secondary forest fragment and a separate area for crops (cysneiros et al., 2011). seropédica is located 26 meters above sea level, has an annual precipitation of 1294 mm and an average annual temperature of 23.9 °c (oliveira jr. et al., 2014). according to köppen (1948), the region’s climate is classified as ‘aw’: tropical, with drier winters and rainy summers. the garden’s local collection includes 125 dicotyledonous species, of which 94 are native (cysneiros et al., 2011). it is important to highlight that botanical gardens as urban parks are key instruments to preserve biodiversity in urban areas and maintain the community previously established in that area (maruyama et al., 2019; marín et al., 2020); also, they are unvaluable for scientific investigations (chen & sun, 2018), such as this one. sampling we did the sampling of flower-visiting hymenopterans from december 2018 through december 2019, twice a month, when the weather conditions were favorable. we collected from 8:30 am to 11:00 am and from 1:30 pm to 3:00 pm, in accordance with the method established by sakagami (1967). we chose these periods because the organisms studied are more active in them (silveira et al., 2002; barbosa et al., 2016). we surveyed sixty-nine plant species inside the botanical garden. on the day before the sampling, we monitored flowering plants, regardless of each one’s flowering stage. then, we collected samples from the previously observed plants with an entomological net, tweezers and brush. from each plant individual, we sampled visiting hymenoptera for ten minutes. in the case of ant collecting, an animal that recruits to dominate resources (hölldobler & wilson, 1990), we collected only one specimen per plant. at the beginning of each sampling period, we measured the air temperature and the relative air humidity at the time with a portable digital thermo hygrometer, thar-300 instrutherm. we took the collected insects to the laboratory for mounting and subsequent identification. we considered an interaction as every relationship between a flower-visiting hymenoptera and a plant species, while visits are the frequency of the interactions. data analysis for each sample, we correlated the number of visits and interactions of bees, wasps and ants with the relative air humidity and air temperature at the time, separately, at the moment of sampling. we discarded the data relative to when there were no visits. thereafter, we had 39 replicates for bee visits, 31 for wasp visits and 29 for ant visits. with the kendall’s rank correlation coefficient, we assessed the influence of relative air humidity and air temperature, independently from one another, on the number of visits and interactions, also independently from one another, for each sample. we previously tested the assumptions of normality and homogeneity of variances. we performed the test on the software r, version 4.1.0 (r core team, 2020). with the data of all the collected hymnopterans and all the sampled plants, we built 12 networks of flower-visiting hymenopteran interactions. there were four groups: one consisting of ants and bees, another consisting of bees and wasps, another consisting of ants and wasps and another consisting of the three taxonomic groups. for each of those groups, we built three networks: one for the morning period, one for the afternoon period and one for the period of a whole day. we built the networks in this way to check how each group impacts the community of hymenopterans. for each network, we used weighted matrices to analyze specialization and discover the central species and binary matrices to analyze nestedness and modularity. primarily, on the software r version 4.1.0 (r core team, 2020), we evaluated specialization by employing the specialization index h₂’, which ranges from 0 (low specialization, generalist network and many overlapping interactions) to 1 (high specialization, specialist network and low or no overlapping interactions) (blüthgen et al., 2006). making use of null models, we calculated the significance of specialization by looking at the actual value of specialization mariana r menezes et al. – hymenoptera flower-visitors in a botanical garden4 and comparing it to a sequence of values randomized a thousand times. the p-value reflects the position of the observed value in the probability distribution which was plotted (mello et al., 2016). next, to assess non-random patterns of hymenopteran-flower interaction, we measured nestedness. we calculated the nestedness of the network using the nodf metric in the aninhado software (guimarães & guimarães, 2006), using 1000 networks generated by null model ii. values range from 0 (not nested) to 100 (perfectly nested) (bascompte et al., 2003). then, we assessed whether there were groups of hymenoptera that only associate with one plant set and are extremely specialized; to do this, we calculated the modularity of the networks. we used modular software (guimerà & amaral, 2005) to verify the modularity of the networks. the modularity index ranges from 0 (no modules) to 1 (completely separated modules). we used a thousand networks randomized by the null model ii with constant total margins (bascompte et al., 2003). thereafter, we used the methodology described by dáttilo et al. (2013a) to discover the core species in that community. this allowed us to verify the flower visitors’ species with the higher frequency of interactions, in other words, the most generalist species in the community. we also discovered the plant core species. moreover, to measure the turnover in the hymenopteran community composition between the morning and the afternoon periods, we calculated the temporal niche overlap. we used jaccard’s similarity for the networks of mixed assemblages and the networks of all communities; as follows: a/(a + b + c), where a is the number of flowervisitor species shared between the two sampling periods, b is the number of flower-visitor species present only during the morning and c is the number of flower-visitor species present only during the afternoon (dáttilo et al., 2014a). results in our study, we found 10 different bee species, 9 different wasp species and 18 different ant species (table 1) interacting with twenty different plant species (table 2). the total number of visits by bees recorded over the whole sample period was 231, resulting in 49 different interactions with 17 plant species; visits by wasps were 77, distributed in 34 interactions with 13 plant species; and ants accounted for 175 recorded visits and 65 interactions with 16 plant species. average relative air humidity in samplings was 69.01 ± 7.56%. the relative air humidity was not shown to impact the amount of visits or interactions of any hymnopteran. neither the number of bee visits (p = 0.2243, τ = -0.1427) nor the amount of bee interactions were influenced by relative humidity of air (p = 0.2717, τ = -0.1571), as were not the number of wasp visits (p = 0.7777, τ = -0.0391) and the amount of wasp interactions (p = 0.3538, τ = -0.1293) or the number of ant visits (p = 0.1359, τ = -0.1982) and interactions (p = 0.1488 τ = -0.1937). id family hymenoptera species #01 apidae plebeia sp1 #02 apidae plebeia sp2 #03 apidae apis mellifera linnaeus, 1758 #04 halictidae augochlora sp1 #05 halictidae augochloropsis sp1 #06 apidae trigona spinipes (fabricius, 1793) #07 apidae scaptotrigona sp1 #08 apidae nomada sp1 #09 apidae bombus sp1 #10 colletidae actenosigynes sp1 #11 vespidae charterginus sp1 #12 vespidae polybia paulista von ihering, 1896 #13 vespidae angiopolybia sp1 #14 vespidae polybia sp1 #15 vespidae ceramiopsis gestroi zavattari, 1910 #16 vespidae polybia sp2 #17 vespidae polybia sp3 #18 vespidae charterginus sp2 #19 vespidae charterginus sp3 table 1. the flower-visiting hymenopterans at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from december 2018 to december 2019. #20 formicidae pseudomyrmex sp1 #21 formicidae pseudomyrmex sp2 #22 formicidae brachymyrmex heeri forel, 1874 #23 formicidae solenopsis invicta buren, 1972 #24 formicidae crematogaster sp1 #25 formicidae camponotus crassus mayr, 1862 #26 formicidae wasmannia auropunctata (roger, 1863) #27 formicidae cephalotes atratus (linnaeus, 1758) #28 formicidae solenopsis sp1 #29 formicidae brachymyrmex sp1 #30 formicidae camponotus sp1 #31 formicidae brachymyrmex admotus mayr, 1887 #32 formicidae crematogaster curvispinosa mayr, 1862 #33 formicidae crematogaster limata smith, f., 1858 #34 formicidae pseudomyrmex sp3 #35 formicidae camponotus novogranadensis mayr, 1870 #36 formicidae brachymyrmex sp3 #37 formicidae brachymyrmex sp2 id family hymenoptera species sociobiology 69(4): e7894 (december, 2022) 5 average air temperature during the sampling period was 27.65 ± 3.83 °c. ambient temperature did not affect the number of bee visits (p = 0.6062, τ = -0.0605) or interactions (p = 0.3514, τ = -0.1094) or the number of wasp visits (p = 0.6592, τ = -0.0610) or interactions (p = 0.7618, τ = -0.0422), but it did positively influence the number of ant visits (p = 0.0010, τ = 0.4360, fig 1) and the number of interactions (p = 0.0010, τ = 0.4420, fig 2). fig 1. kendall regression of temperature and number of ant visits to flowers at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from january 2019 to december 2019. the x-axis represents temperature and the y-axis represents the number of visits. fig 2. kendall regression of temperature and number of ant interactions with flowers at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from january 2019 to december 2019. the x-axis represents temperature, and the y-axis represents the number of interactions. mariana r menezes et al. – hymenoptera flower-visitors in a botanical garden6 in august, bees had the highest average number of visits and the highest average number of interactions (fig 3; fig 4), whereas the highest average number of wasp visits was observed in may and august (fig 3), especially in the former (fig 4); for ants, march had the highest average number of visits and interactions (fig 3; fig 4). in december 2019, the lowest average numbers of visits and interactions by bees (fig 3; fig 4) and no wasps foraging on any flowers were observed, while no ants visited any flowers in the period from september through october of that same year (fig 3; fig 4). the networks of the community of flower-visiting hymenoptera were nested during the morning (nodf = 23.17, p = 0.03), the afternoon (nodf = 19.96, p = 0.01) and all day (nodf = 25.41, p < 0.001). these networks exhibited no modularity and showed significant specialization (morning: h₂’ = 0.35, p < 0.001; afternoon: h₂’ = 0.39, p < 0.001; all day: h₂’ = 0.37, p < 0.001) (table 3). the hymenoptera core species were: in the morning apis mellifera, plebeia sp.1, fig 3. the average number of visits by groups of flower-visitors at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from december 2018 to december 2019. in red, the number of ant visits; in green, the number of bee visits; in blue, the number of wasp visits. the x-axis represents the average number of visits, and the y-axis represents the month of the sample. plebeia sp.2, trigona spinipes, and camponotus crassus; in the afternoon apis mellifera, plebeia sp.2, trigona spinipes, camponotus crassus, and wasmannia auropunctata; on the whole day apis mellifera, plebeia sp.1, plebeia sp.2, trigona spinipes, camponotus crassus, and wasmannia auropunctata (table 3). the plant core species were: in the morning, in the afternoon and on the whole day, respectively: stifftia chrysantha, clerodendrum x speciosum, callistemon viminalis, and antigonon leptopus (table 3). the networks for ants and bees assemblages were nested during the afternoon (nodf = 19.31, p = 0.05) and during the whole day (nodf = 14.64, p < 0.001), but were not nested during the morning. these networks showed no modularity and displayed significant specialization (morning: h₂’ = 0.38, p < 0.001; afternoon: h₂’ = 0.43, p < 0.001; whole day: h₂’ = 0.40, p < 0.001) (table 3). the hymenoptera and plant core species in these nets were the same as the core species obtained for the community nets (table 3). sociobiology 69(4): e7894 (december, 2022) 7 fig 4. the average of interactions by groups of flower-visitors at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from december 2018 to december 2019. in red, the number of ant visits; in green, the number of bee visits; in blue, the number of wasp visits. the x-axis represents the average number of interactions and the y-axis represents the month of the sample. id family plant species n p01 anacardiaceae schinus terebinthifolia raddi 7 p02 arecaceae phoenix sp1 1 p03 asteraceae stifftia chrysantha mikan 9 p04 boraginaceae cordia superba cham. 4 p05 cactaceae cactaceae sp1 1 p06 chrysobalanaceae microdesmia rigida (benth.) sothers & prance 3 p07 ericaceae rhododendron sp1 2 p08 fabaceae bauhinia variegata l. 3 p09 fabaceae cassia fistula l. 3 p10 fabaceae paubrasilia echinata (lam.) gagnon & lewis 1 table 2. plants involved in interactions of floral-visiting hymnopterans at the botanical garden of ufrrj, in seropédica, rio de janeiro, brazil; from december of 2018 to december of 2019. p11 lamiaceae clerodendrum x speciosum w. bull 5 p12 lamiaceae clerodendrum speciosissimum drapiez 4 p13 lecythidaceae couroupita guianensis aubl. 4 p14 malvaceae hibiscus sp1 5 p15 malvaceae hibiscus sp2 1 p16 melastomataceae tibouchina granulosa (desr.) cogn. 1 p17 myrtaceae callistemon viminalis g. don ex loud. 6 p18 polygonaceae antigonon leptopus hook. & arn. 6 p19 sapindaceae sapindus sp1 1 p20 verbenaceae duranta repens l. 2 id family plant species n mariana r menezes et al. – hymenoptera flower-visitors in a botanical garden8 the networks for bees and wasps assemblages were nested during the morning (nodf = 16.89, p = 0.01), the afternoon (nodf = 16.25, p = 0.02) and the whole day (nodf = 17.35, p = 0.02). these networks exhibited no modularity and the same specialization in every period (h₂’ = 0.29, p < 0.001) (table 3). the hymenoptera core species were: in the morning and on the whole day – apis mellifera, plebeia sp.1, plebeia sp.2, and trigona spinipes; in the afternoon – a. mellifera, plebeia sp.2, and t. spinipes (table 3). the plant core species were: in the morning, in the afternoon and on the whole day, respectively – stifftia chrysantha, callistemon viminalis, and antigonon leptopus (table 3). at last, the networks for wasps and ants assemblages were nested during the afternoon (nodf = 11.43 p = 0.02) and during the whole day (nodf = 17.35, p = 0.01), but weren’t nested during the morning. these networks showed no modularity and significant specialization (morning: h₂’ = 0.37, p < 0.001; afternoon: h₂’ = 0.42, p < 0.001; all day: h₂’ = 0.43, p < 0.001) (table 3). the hymenoptera core species were the same in all networks: angiopolybia sp1, camponotus crassus, and wasmannia auropunctata. the plant core species were: in the morning – clerodendrum x speciosum, and a. leptopus (table 3); in the afternoon and on the whole day – cordia superba, clerodendrum x speciosum, and a. leptopus (table 3). h₂’ modularity nestedness hymenoptera core plant core all groups morning 0.35 (p < 0.001) 0.30 (p = 0.88) 23.17 (p = 0.03) #01; #02; #03; #06; #25 p03; p11; p17; p18 afternoon 0.39 (p < 0.001) 0.33 (p = 0.90) 19.96 (p = 0.01) #02; #03; #06; #25; #26 p03; p11; p17; p18 all day 0.37 (p < 0.001) 0.26 (p = 0.91) 25.41 (p < 0.001) #01; #02; #03; #06; #25; #26 p03; p11; p17; p18 ants + bees morning 0.38 (p < 0.001) 0.41 (p = 0.30) 13.11 (p = 0.11) #01; #02; #03; #06; #25 p03; p11; p17; p18 afternoon 0.43 (p < 0.001) 0.46 (p = 0.32) 19.31 (p = 0.05) #02; #03; #06; #25; #26 p03; p11; p17; p18 all day 0.40 (p < 0.001) 0.35 (p = 0.65) 14.64 (p < 0.001) #01; #02; #03; #06; #25; #26 p03; p11; p17; p18 bees + wasps morning 0.29 (p < 0.001) 0.37 (p = 0.66) 16.89 (p = 0.01) #01; #02; #03; #06 p03; p17; p18 afternoon 0.29 (p < 0.001) 0.38 (p = 0.65) 16.25 (p = 0.02) #02; #03; #06 p03; p17; p18 all day 0.29 (p < 0.001) 0.33 (p = 0.86) 17.35 (p = 0.02) #01; #02; #03; #06 p03; p17; p18 wasps + ants morning 0.37 (p < 0.001) 0.45 (p = 0.27) 12.65 (p = 0.11) #13; #25; #26 p11; p18 afternoon 0.42 (p < 0.001) 0.46 (p = 0.32) 11.43 (p = 0.02) #13; #25; #26 p04; p11; p18 all day 0.43 (p < 0.001) 0.36 (p = 0.79) 14.15 (p = 0.01) #13; #25; #26 p04; p11; p18 the organism code in the hymenoptera central species column is in table 1. the organism code in the plant central species column is in table 2. table 3. network metrics of all matrices obtained based on the observation of a community of flower-visiting hymenopterans at the botanical garden of the ufrrj, in seropédica, rio de janeiro, brazil. during december of 2018 to december of 2019. specialization = h₂’. hymenoptera core species = hymenoptera core. plant core species = plant core. the jaccard similarity index of the networks of all taxonomic groups was 0.73, which means that 73% of observed hymenopteran species are the same in the morning and in the afternoon periods; networks of bees and wasps and of wasps and ants exhibited a higher similarity (jaccard similarity index = 0.76; 0.79, respectively); ants and bees networks displayed the highest composition similarity among all networks (jaccard similarity index = 0.80). the morning network of all groups indicates a predominance of interactions made by ants; however, the number of visits made by bees was higher. because of that, bee visits were the most often, mainly the visits made by apis mellifera and trigona spinipes (fig 5a). the most visited plants in the morning were, respectively, antigonon leptopus and callistemon viminalis (fig 5a). in the afternoon network, a. mellifera occupied the position with the highest number of visits, in other words, a greater frequency of interaction, followed by the ant species camponotus crassus (fig 5b). it is remarkable that in the afternoon the frequency of ants became higher and they were still the group with more interactions. the plants with the highest number of visits also changed during the afternoon: species that occupied prominent positions were c. viminalis and stifftia chrysantha (fig 5b). the all-day network showed similarities with both nets, in different aspects: regarding flower visitors, the all-day net was similar to the afternoon network, with a greater frequency of interaction of the species a. mellifera, but with a large frequency of interaction of ants; regarding the plants visited, the all-day sociobiology 69(4): e7894 (december, 2022) 9 network was closer to the result obtained by the morning network, with the species a. leptopus and c. viminalis being the plants with the highest number of visits (fig 5c). discussion in this study, we correlated the number of observed visits and interactions of each group of organisms with two abiotic factors – air temperature and relative air humidity. our results showed that relative air humidity did not affect the number of visits or interactions of any of the assemblages and that air temperature did not affect the number of visits or interactions of bees or wasps; however, the higher the temperature, the more visits and interactions ants made. we also used the networks constructed from the floral-visitor community to assess the interaction pattern and composition of this community and the influence of each group of organisms on the community in different temporal niches. our results showed that most networks exhibited the same interaction pattern in the morning and in the afternoon, except for the ant-bee and wasp-ant networks: these networks were nested, non-modular and of low specialization. on the other hand, in all networks, the core hymenopteran species changed when comparing the morning and the afternoon periods, which means that the composition of this community differed according to temporal niche. the assemblages also showed that the entire community of floral-visitors displayed different compositions and interaction patterns: the networks with bees displayed greater nestedness, while the networks with ants exhibited greater specialization; the morning ant-bee and wasp-ant networks were not nested; in addition, the central plant species visited in the wasp-ant network differed from the central plant species visited in the other networks. relative air humidity, despite influencing nectar composition (winkler et al., 2009), did not impact the number of floral visits or interactions in any communities. elements of the study area may have contribuited to the the irrelevance of this factor to visits and interactions, such as the presence of a lake, irrigation and plant management (cysneiros et al., 2011). irrigation is one of the anthropogenic tools which can alter the microclimate, influencing not only plants but also the distribution of insect species (federman et al., 2013). air temperature had a positive role on the number of ant visits and interactions, exhibiting a strong correlation. visits tended to be more frequent between 30-33° celsius, which is the air temperature range of highest insect activity (paaijmans et al., 2013; carvalho et al., 2014). air temperature shapes the foraging behavior of ants and these are very heat resistant organisms, with activity recorded at air temperatures above 60 °c (heinrich, 1993; luna et al., 2021). thus, a positive correlation of air temperature with ant visits and interactions had been expected and was duly confirmed, whereas air temperature had no effect on the number of bee and wasp visits and interactions. bee and wasp visits and interactions may be affected by other abiotic factors which were not analyzed fig 5. networks of all groups of floral visiting hymenopterans at the ufrrj botanical garden, in seropédica, rio de janeiro, brazil; from december 2018 to december 2019. the network is organized according to the frequency of interactions (visits). on the left are the visiting insect species and on the right are the plant species. in yellow are highlighted visits by bees, whereas visits made by wasps are highlighted in blue and visits made by ants are highlighted in red. a: morning network; b: afternoon network; c: all-day network. mariana r menezes et al. – hymenoptera flower-visitors in a botanical garden10 in this work, such as rainfall, wind speed and solar radiation (simões et al., 1985; loyola & martins, 2006; oliveira et al., 2012; kovac et al., 2015; marinho & vivallo, 2020). almost all networks showed low specialization, nestedness and no modularity, what points to the presence of highly generalist species which used the resources of several plants (blüthgen et al., 2007; blüthgen & klein, 2011; blüthgen, 2012). low specialization also indicates that this network has a functional ecological niche redundancy; this may be due to the nestedness of niches among the present species or the fact that the species interacted with many different plant species, displaying generalism. networks which present niche redundancy usually are observed in communities which are more stable in the face of adversity; however, as many species exploit the same resources, that can generate an increase in interspecific competition (blüthgen & klein, 2011; blüthgen, 2012). several factors may explain the nestedness and nonmodular patterns of insect-plant community networks; some are abiotic (e.g. air temperature, rainfall, soil ph, elevation) and others are biotic (e.g. flower-visitor size, animal behavior, nectar characteristics) (chamberlain et al. 2010; rico-gray et al., 2012; lange et al., 2013; dáttilo et al., 2013b; dáttilo et al., 2014a; santos et al., 2014; giannini et al., 2015; petanidou et al. 2017; adedoja et al., 2018). regarding the community studied, what possibly explains the nested pattern is the presence of super-generalist and dominant species (such as apis mellifera, trigona spinipes, camponotus crassus, and wasmannia auropunctata), which visit most of the plant species in the area and also interact with the set of plants visited by peripheral species (dáttilo et al., 2014a; dáttilo et al., 2014b; giannini et al., 2015). moreover, it is important to note that the ant-bee and wasp-ant networks did not achieve nestedness during the morning period, probably because the frequency of visitation of important super-generalist species (such as camponotus crassus, and wasmannia auropunctata) decreased, what promoted nestedness in the afternoon and allday networks (díaz-castelazo et al., 2013), and because of a greater presence of peripheral species (díaz-castelazo et al., 2010; lange & del-claro, 2014). the likely explanation for the non-modular pattern of these networks, besides a small specialization and a mostly generalist community, is the behavior of the flower-visiting hymenoptera (dáttilo et al., 2014a; dáttilo et al., 2014b). wasps and ants do not use only floral resources in their feeding; these organisms are occasional floral-visitors, often associated with the predation of other arthropods, the exploitation of other botanical resources (e.g. extrafloral nectary, fruits) and the consumption of other types of food sources, what makes their interaction more generalized and does not facilitate the constitution of modules in the networks (brodmann et al., 2008; rico-gray & oliveira, 2008; mello et al., 2011; dáttilo et al., 2014a; brock et al., 2021; lavisky et al., 2021). bees, while exclusively floralvisitors, interact with different types of plants according to availability, which can even affect the density and abundance of these organisms (ebeling et al., 2011; grass et al., 2016). the low diversity of vegetation in a botanical garden can affect these patterns too since vegetation structure is important for the composition of insect communities (clemente et al., 2012; junker et al., 2012; rico-gray et al., 2012). although non-modular networks are a common pattern in generalist mutualistic networks (del-claro et al., 2018), the small number of plant species sampled may have impacted the result of this metric, because pollination networks with less than 50 plant species tend to lack modularity (olesen et al., 2007). when we looked at the metrics of the combined taxonomic group network, we found that most core species in the nets were bees and that the nestedness of bee-wasp and bee-ant networks was higher, except for the morning period of the bee-ant network. this may be explained by the behavior of the bees. bees receive much attention for being crucial for the pollination of a large number of plants; their body is adapted to collect and store floral resources (imperatrizfonseca et al., 2012) and they are the only animals capable of consuming all available floral resources (torezan-silingardi, 2012). oligolectic bees visit either a restricted group of plants or only one type of plant, being highly specialized and performing pollination with great efficiency (schlindwein, 2004; imperatriz-fonseca et al., 2012). no bee species found in the locality of this study are classified as oligolectic, which is probably caused by the low plant diversity, since no plants pollinated by these bees are found in the study area (schlindwein, 2004). that can also explain why the specialization is lower in nets combined with bees. a higher specialization value in networks which feature ants may also be explained by the presence of peripheral species, but the value obtained was lower than that obtained for ant-plant interaction networks observed in other studies (guimaraes et al., 2006; junker et al., 2012). the networks were not highly specialized, as specialization is one of the strategies which may be adopted by organisms involved in floral visitation to decrease competition (junker et al., 2012); another strategy which they can adopt is to perform visits at different times (tschoeke et al., 2015). the similarity of community composition between the morning and the afternoon periods may be considered high, possibly indicating that this is not a strong strategy found in this community. however, there were differences between the morning and the afternoon networks and those differences may be greater according to the size of the community, the study area and the time discrepancy between niches (díaz-castelazo et al., 2013; dáttilo et al., 2014). the difference which stands out the most is the change of some core species organisms when comparing the periods. on the community level, the core species in the all-day network are four bee species and two ant species; although the greater presence of bees in this position is already expected, it is important to understand the biology and behavior of these animals to comprehend the important characteristics of the community and the study site. in the morning period, sociobiology 69(4): e7894 (december, 2022) 11 there was a higher presence of bees as core species; in the afternoon, the frequency of visits by ants increases, adding one more ant species to the group of core species. apis mellifera, a generalist bee, with an average size of 11-13 mm, which has a high foraging and pollination efficiency, having a very large foraging range for an arthropod (beekman & ratnieks, 2001; costa et al., 2015) was firstly observed as the species with the highest number of visits, being the main key species of the community studied. despite being an important species in the network, linked to several plant species, this bee is an exotic species, which has been pointed out as a risk to the conservation of native species (paini, 2004; russo et al., 2021). the genus plebeia had two species pointed as core species. the genus belongs to the meliponini tribe of stingless bees and includes species native to brazil. the bees of the genus are small, with an average size of 3-4 mm, generalists and responsible for the pollination of several native plants (pick & blochtein, 2002; wittmann, 2008). trigona spinipes was also considered a key bee species. it is also from the meliponini tribe, measuring approximately 7 mm, a native pollinator capable of pollinating several plant species, supergeneralist and associated with improved fruit production (chalegre et al., 2020; tschoeke et al., 2015). highlighting the core species of ants, camponotus crassus belongs to the formicidae subfamily and is a species which measures 20 mm and has no metapleural gland, being unable to produce harmful substances in the pollen (del-claro et al., 2019); it is a dominant forager in vegetation, exploits resources which do not come from flowers alone (lange et al., 2019) and has already been associated with effective pollination (de vega et al., 2014; del-claro et al., 2019). finally, wasmannia auropunctata is an ant from the subfamily myrmicinae; the smallest of the key species, measuring 1.5 mm, it is invasive, commonly found in urban areas, often involved in cases of environmental impact and may have medical importance (azevedo et al., 2022; gruber et al., 2022). there are no studies relating this species to pollination, so it competes with pollinators once it recruits massively to dominate resources (azevedo et al., 2022). wasps had occasional interactions with flowers and were not often observed (except for angiopolybia sp.1) having a greater presence as a peripheral species (díazcastelazo et al., 2010; lange & del-claro, 2014); therefore, no wasp species was considered core in the all-group networks. in almost all the other networks, the species core remained the same, except for the wasp-ant networks, which featured a wasp as a key species. angiopolybia is one of the genera of polistinae and presents native representatives from brazil; this genus is abundant in atlantic forest areas and, despite adaptations to the necrophagic habit, consumes other resources (lima et al., 2010; togni et al., 2014), as observed in this study. in addition to the core species of floral visitors, the core species of plants visited also reveal to us important characteristics of this community. in the community nets and the ant-bee nets, the core plant species were the same, not varying according to time of day. in the bee-wasp networks, the composition was very similar to the general network, minus one core plant species – clerodendrum x speciosum. however, for the wasp-ant networks, the composition was different from the community network and there were differences between the most visited plants according to the time of day. only in the networks with bees, stifftia chrysantha and callistemon viminalis were part of the core species, indicating a greater interaction of bees with these species. s. chrysantha is a native tree, which can reach 5 meters in height, has inflorescence of tubular flowers, displays shades of orange and is most frequently pollinated by hummingbirds, while rarely by bees (nishida et al., 2014; lorenzi, 2020; gobatto et al., 2021). c. viminalis is an exotic species, one of the several honey trees pollinated by bees; it reaches up to 7 meters in height, has terminal, pendulous and spike-like inflorescence and flowers of numerous red stamens (latif et al., 2016; lorenzi, 2018; guallpa-calva et al., 2019). in the case of clerodendrum x speciosum, it is a core species only in networks with ants. it is a woody shrub, a hybrid of exotic species, with inflorescence in terminal racemes and red flowers whose genus’ species are commonly pollinated by bees; herbivorous ants, plunderers and attendant ants have already been associated with these plants (carver et al., 2003; rohitash, 2010; lorenzi, 2015; groutsch et al., 2018; mukhopadhyay & quader, 2018). it is noticeable that in the wasp-ant networks there was a smaller amount of plants in the core species and also a plant which was not present in any core species of any other networks: cordia superba. this native tree can reach up to 10 meters high, has large white flowers, is pollinated by small insects and bees and is not uncommonly visited by nectar pillagers (agostini & sazima, 2003; vale et al., 2013; silva & rossaferes, 2016; lopes et al., 2015; lorenzi, 2020; gobatto et al., 2021). one of the plants was in the core species of all the community networks, indicating that it is a well-visited plant by all three organisms: antigonon leptopus, a semiherbaceous climber from mexico with inflorescence of many durable flowers in pink or white colors (lorenzi, 2015); it is a flower species visited frequently by bees, decently by wasps and rarely by ants (raju et al., 2001; lorenzi, 2015; gobatto et al., 2021; lima et al., 2021). studies on plant-pollinator networks in the tropics have been conducted more often in forests than in open habitats, with sampling concentrated over a single season (vizentin-bugoni et al., 2018). however, deforestation in the tropics has created an increasing proportion of open habitats (e.g. in the atlantic forest domain) (hirota, 2003) and it is crucial to assess information about ecological interactions in this kind of habitat, now predominant in most landscapes. the loss of green areas can impact the richness and abundance of flower-visitor communities in cities, what can jeopardize the biodiversity and functional richness of that community (spiesman & inouye, 2013; geslin et al., 2016). controlled environments may be critical for the preservation of flowermariana r menezes et al. – hymenoptera flower-visitors in a botanical garden12 visiting species in urban areas (smith, 2006; larson, 2014; twerd & banaszak-cibicka, 2019; twerd et al., 2021), what demonstrates that even small green areas are important for insect conservation. however, urban administrators should ideally plan to create urban parks which support, attract and maintain flower-visitors and have a large vegetation cover and a high variety of plant species, preferably with different morphologies (garbuzov & ratnieks, 2014; banaszak-cibicka et al., 2016; hall et al., 2017). in this context, botanical gardens stand out as a good tool for the conservation of flower-visitor communities, as they serve as ecological corridors, shelter and foraging sites (hall et al., 2017; maruyama et al., 2019; marín et al., 2020; gobatto et al., 2021). our study endorses that a botanical garden can sustain a diverse community of flower-visiting hymenoptera in an urban environment (ito et al., 2001; mazzeo & torretta, 2015; marinho & vivallo, 2020), being an important tool for biodiversity conservation (hall et al., 2017; maruyama et al., 2019; marín et al., 2020; gobatto et al., 2021). when it comes to botanical gardens in atlantic forest areas, especially in the state of rio de janeiro, it is clear that our results corroborate other studies already conducted, as the botanical garden studied clearly supports a diverse community of floral-visiting hymenoptera, whose composition, which includes many pollinating species, is very similar to communities observed by other researchers (pimentel & rangel, 2017; santos et al., 2017; silva et al., 2018; marinho e vivallo, 2020; gobatto et al., 2021). being close to an area of forest fragments, the botanical garden studied has the potential to be an ecological corridor, as well as the botanical garden of rio de janeiro (gobatto et al., 2021). however, future work is necessary to prove that this urban park is effectively an ecological corridor. moreover, the current study can serve as a comparison for following works that investigate the response of flower-visiting hymenoptera to climate change (hofmann et al., 2018). from the above, our study can contribute by showing that there is a correlation between the number and frequency of ant floral interactions and ambient temperature and by strengthening data on network patterns of flower-visitor communities. moreover, it highlights the importance of botanical gardens for the maintenance of flower-visiting hymenopteran communities in urban environments. future studies can further explore whether pollination is performed by floral-visitors and their niche in their communities. acknowledgments we are grateful to dr. marcelo da costa souza for his help with botanical identification. apls and msm would like to thank the proverde program (jardim botânico ufrrj), and mrm and rc are thankful to capes (coordenação de aperfeiçoamento de pessoal de nível superior; 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(2018). plant-pollinator networks in the tropics: a review. in w. dáttilo & v. rico-gray (eds.), ecological networks in the tropics (pp. 29-42). springer international publishing. wang, y., deangelis, d.l., & nathaniel holland, j. (2015). dynamics of an ant-plant-pollinator model. communications in nonlinear science and numerical simulation, 20: 950-964. doi: 10.1016/j.cnsns.2014.06.024 watts, s., dormann, c.f., martín gonzález, a.m., & ollerton j. (2016). the influence of floral traits on specialization and modularity of plant-pollinator networks in a biodiversity hotspot in the peruvian andes. annals of botany, 118: 415429. doi: 10.1093/aob/mcw114 winkler, k., wäckers, f.l., kaufman, l.v., larraz, v., & van lenteren, j.c.(2009). nectar exploitation by herbivores and their parasitoids is a function of flower species and relative humidity. biological control, 50: 299-306. doi: 10.1016/j. biocontrol.2009.04.009 wittmann, d. (2008). nest architecture, nest site preferences and distribution of plebeia wittmanni (moure & camargo, 1989) in rio grande do sul, brazil (apidae: meliponinae). studies on neotropical fauna and environment, 24: 17-23. doi: 10.1080/01650528909360771 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.8251sociobiology 69(4): e8251 (december, 2022) abstract this novel review of analytical methods for pot-honey research was intended to provide concise references to a 35-day post-harvest experiments at 30 °c, in an integrated study. diverse methods were selected from specialized literature, from the aoac (association of official analytical chemists), and the international honey commission. besides the geographical and seasonal origin, the pot-honey i.d. consists of entomological and botanical identifications, the latter performed by acetolyzed or natural melissopalynology. the methods of this integrative study included: 1. physicochemical analysis (aw, color, moisture, ph, free acidity, lactone acidity, total acidity, hydroxymethylfurfural (hmf), and sugars by highperformance liquid chromatography hplc), 2. targeted proton nuclear magnetic resonance 1h-nmr metabolomics (sugars, ethanol, hmf, aliphatic organic acids, amino acids, and botanical markers), 3. biochemical composition (flavonoids, polyphenols), 4. antioxidant activity (abts 2,2'-azino-bis(3-ethylbenzothiazoline6-sulfonic acid-free radical scavenging assay, dpph 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric reduction assay frap), 5. microbial counts (aerobic plate, yeast and mold, bacillus, and lactic acid bacteria count), 6. honey microbiome profiling via independent-culture method: high-throughput bacteria and fungi based on amplicon sequencing approaches, 7. sensory evaluation (odor, aroma, taste, persistence), and 8. honey authenticity and biosurfactant tests by an interphase emulsion. a further section was included to provide basic information on the results obtained using each method. this was needed to explain the interacting components derived from pot-honey processing within the stingless bee nest and post-harvest transformations. sociobiology an international journal on social insects patricia vit1, bajaree chuttong2, norhasnida zawawi3, maria diaz4, jane van der meulen4, hajar f. bin ahmad5, francisco a. tomas-barberán6, gina meccia1, khanchai danmek7, j. enrique moreno8, david w. roubik8, ortrud m. barth9, dirk w. lachenmeier10, michael s. engel11,12 article history edited by evandro nascimento silva, uefs, brazil received 14 may 2022 initial acceptance 10 july 2022 final acceptance 11 september 2022 publication date 28 november 2022 keywords antioxidant activity, biosurfactant activity, 1h-nmr, honeymicrobiome, flavonoids, meliponini, melissopalynology, sensory analysis corresponding author patricia vit apiterapia y bioactividad facultad de farmacia y bioanálisis universidad de los andes merida 5101, venezuela. e-mail: vitolivier@gmail.com 1 universidad de los andes, merida, venezuela 2 chiang mai university, chiang mai, thailand 3 universiti putra malaysia, selangor, malaysia 4 quality services international gmbh, bremen, germany 5 universiti malaysia pahang, pahang, malaysia 6 cebas-csic, campus universitario de espinardo, murcia, spain 7 university of phayao, phayao, thailand 8 smithsonian tropical research institute, balboa, ancon, republic of panama 9 instituto oswaldo cruz, rio de janeiro, brazil 10 chemical and veterinary investigation agency, karlsruhe, germany 11 university of kansas, lawrence, kansas, usa 12 american museum of natural history, new york, usa a novel integrative methodology for research on pot-honey variations during post-harvest review patricia vit et al. – standard and innovative integrative research of pot-honey2 introduction stingless bees (tribe meliponini) are a diverse lineage of highly eusocial bees, currently with more than 600 species distributed in the tropical and subtropical habitats of the globe. this diversity is not evenly distributed and the greatest concentration of species is found in south and central america, accounting for more than 500 species. in the new world, stingless bees are found from mexico to northern argentina, while in the old world they are found throughout sub-saharan africa and madagascar, and throughout the indian subcontinent, southern and southeastern asia, through the indomalayan and papuasian regions, and into australia (roubik & vergara, 2021). meliponini are ancient, with species scattered in various cenozoic deposits throughout the world (engel et al., 2021a), as well as a singular fossil from the end of the cretaceous period (engel, 2000). accordingly, stingless bees have been producing honey for at least 70 million years and have evolved complex chemistries with associated microbiomes throughout this span of time, and through dramatic changes in global environments. the common descriptor of “stingless” is a seemingly sensational moniker for any honey-making bees (michener, 2013), and certainly, the usual method of stinging as a defense of the nest is hampered by the vestigial sclerites associated with the sting apparatus in meliponini (michener, 2007, 2013). nonetheless, stingless bees are quite capable of defending themselves from predation, by the construction of inaccessible nests and sometimes alternative means of deterring predation (e.g., painful defensive secretions in species of oxytrigona cockerell) (michener, 2007; engel & rasmussen, 2021; melo, 2021). as honey-making bees, indigenous peoples have a long history of exploiting stingless bee nests and products, as well as their own forms of meliponiculture. aside from the use of honey as food or for ethnomedicinal applications, various nest components such as resins and propolis are also employed in crafts (ayala et al., 2013; bhatta et al., 2020). stingless bees process and store their honey in cerumen pots rather than in the hexagonal prismatic waxen cells of honey bees (michener, 1974), and the differences in honeys are not merely those of the architectural form of the storage media. pot-honeys produced by stingless bees differ considerably in many properties from the comb-honey produced by honeybees (tribe apini, extant sister group to meliponini). this distinction was emphasized by schwarz (1948), carefully explored by gonnet et al. (1964), mentioned in diverse articles and reviews, and ultimately became the focus of a book where the term pot-honey was coined and highlighted (vit et al., 2013).the effect of storage was studied on honey produced by thai stingless bees (chuttong et al., 2015). in biological studies, specialized techniques are required for understanding the biological, botanical, chemical, microbial, and physical components of honey, and their interactions. methods used for the determination of a given honey physicochemical characteristic (moisture, ash or electrical conductivity, free acidity, hmf, diastase activity, and sugars), microbiological counts (aerobic plate, yeast and mold, bacillus, and lactic-acid bacteria), sensory evaluation (color, odor, aroma, taste, persistence), and melissopalynological composition for routine quality control are basically the same as those for any biological investigation. post-harvest variations were monitored by routine analysis and further metabolites by hplc, 1h-nmr, and recent approaches to determine the honey-microbiome used to identify microbes associated with stingless bees, involved in the biotransformation of the honey, as well as an interphase emulsion test as a preliminary indicator of biosurfactant activity. the scope of this article is to describe the analytical methods used in the integrated post-harvest study of pothoney produced by tetragonula laeviceps smith 1857, and lepidotrigona flavibasis (cockerell 1929) from chiang mai, thailand; geniotrigona thoracica moure 1961, and heterotrigona itama (cockerell 1918) from selangor, malaysia; lepidotrigona terminata (smith 1878) from kubang kerian, kelantan, malaysia; tetragonisca angustula (latreille 1811) from costa rica; and scaptotrigona vitorum engel 2022 honey from ecuador. variations in most parameters were monitored weekly at 7 day-intervals for 6 times (days: 0-7-14-21-28-35), or 5 times excluding the first measurement due to transportation to international laboratories. the following methods presented here were required: 1. to identify the origin of the pot-honey, and 2. to describe the standard and innovative methods for pot-honey analysis, integrated to study post-harvest transformations. sterile pothoney sampling was needed for the microbiological quality and for the honey microbiome assessment. therefore, a sterile sampling protocol was followed to collect one batch of honey produced by each stingless bee species, homogenized, and distributed laterto all participating laboratories. 1. identification of the entomological and botanical origin of pot-honeys the identification of the pot-honeys was done by entomological and botanical origins, the countries of origin were known, and the habitats and seasons were also recorded for the harvest date of year 2022. this i.d. of each honey was the starting point for the integrated multiparametric postharvest study along 35-day storage at 30 °c. 1.1 entomological identification of the stingless bees a study of pot-honey characterization initiates with the mandatory identification of the taxon involved by an authority in this field. this is not a trivial effort as taxonomic research is a challenging and time-consuming process requiring considerable training and resources, ideally including the significant investment in an authoritatively identified reference collection. accordingly, taxonomists must be acknowledged sociobiology 69(4): e8251 (december, 2022) 3 in the materials and methods as well as acknowledgements, their grants should be cited, and when developing grant proposals or projects it is ideal to incorporate the necessary funding to support the costly preparation and identification process. furthermore, it is ideal to also invite taxonomists as coauthors given the responsibility, costs, and labor necessary for contributing to the identification of the stingless bees, as this is a basic component of the overall research. their knowledge of species biology may also provide additional insights into comparative studies of pot-honey. modern photographic technology and photographer expertise produce scientific details of taxonomic interest and natural beauty. for example, the face of scaptotrigona vitorum engel, 2022 in fig 1 shows many features such as the pattern of integumental coloration; the color, density, length, and plumosity of the fine hairs and bristles; the relative breadth and width of the head; the length of the antennal scape and proportions of the flagellomeres; the dentition of the mandible; the relative length of the malar space; the distance between the antennal sockets; and the sculpturing of the integument (e.g., density of punctures, smooth versus patterned, shiny versus matte). naturally, more information is required relative to other body structures, but the figure highlights a range of features that can be observed easily from sufficiently detailed photographs of particular views. stingless bee identification was undertaken by shipping specimens along with their associated collecting-event data (georeferenced coordinates, locality data, date of collection, elevation in m.a.s.l., collector, contact, and any ancillary data) to a research taxonomist. the specimens were then mounted, labeled, and digitized according to standard entomological procedures. the mounted specimens were then sorted initially into genus and morphotypes. the resulting morphotypes were then either run through existing identification keys in the case of those genera for which such keys exist or were pointby-point compared with authoritatively identified material in the bee research collections of the university of kansas biodiversity institute, american museum of natural history, as well as against original and subsequent descriptions existing in the literature. where needed, males were dissected to examine their hidden sterna and genitalia, and workers dissected to examine the vestigial sting sclerites. ultimately, specimens were identified either as existing species known in the literature or were determined to represent taxa hitherto undescribed. when possible, formal taxonomic descriptions were prepared and published in order to provide a name for use in subsequent research articles on such species. the processes of preparation and taxonomic identification can take days to many months (and sometimes much longer if new species must be described) depending on the taxa involved, the number of specimens requiring identification, the condition of the material provided, the availability of funds for these tasks, and various other factors (e.g., teaching and curation responsibilities). the degree to which taxonomists are made full partners in research grants and publications can go a long way to easing the process of identification and supporting the science of taxonomy more broadly (engel et al., 2021b). 1.2 botanical identification by melissopalynology melissopalynology is the palynological study of honey. pollen grains are well preserved in honey, as such acetolysis is not required, but is often necessary for species identification. acetolyzed pollen is used by some researchers who have a wider reference collection for ecological studies. some preserved features in natural pollen techniques such as oils are used in the identification process (haidamus et al., 2019). the diverse characteristics of the pollen exine are used in both methods to identify the botanical taxa to species, genus, or family visited by the honey-making bees. pollen grains are quantified, and their percentage may be used for a unifloral protected denomination of origin (pdo). 1.2.1 acetolyzed pollen residual pollen grains present in honey, to be analyzed and identified, require cleaning and preparation with chemical and physical methods. without them, light microscopy, especially in species-rich botanical areas, has quite limited value. fortunately, pollen coming directly from flowers or bee nests requires minimal treatment, compared to pollen in soil or other sediments (brown, 1960; kearns & inouye, 1993). because pollen is extremely resistant to strong acids (e.g. hydrofluoric acid, hydrochloric acid, sulfuric acid, acetic acid) while sensitive to oxidizing agents (nitric acid, potassium hydroxide, sodium hydroxide, and acetolysis, explained below, see erdtman, 1952; faegri & iversen, 1950) we use a combination of chemicals and apply a special protocol to improve our identification of pollen. spore tablets of lycopodium, with a known number of spores per tablet fig 1. scaptotrigona vitorum engel 2022, from el oro, ecuador, a protagonist of pot-honey making stingless bees. photo: michael s. engel. patricia vit et al. – standard and innovative integrative research of pot-honey4 (stockmarr, 1971), are sometimes added to samples for which the quantification of different species proportions is desired, especially among multiple microscope slide preparations from a given sample. this is a straightforward method to quantify the relative portion per weight or volume of each pollen species (roubik & moreno, 2009). after the lycopodium spores were included, the acetolysis method was the same as without those spores, as follows: 1. one lycopodium tablet into a 10 to 50 cc of honey sample is dissolved in water and sieved with mesh (250 mm), to remove nest material, bees or large impurities. next, samples were concentrated at 2,700 rpm for 5 min, and the supernatant was discarded. the honey was dissolved and a homogeneous sample the residue was obtained. 2. the residue was dried with glacial acetic acid and concentrated at 2,700 rpm for 5 min, and the supernatant was discarded. 3. an acetolysis solution (a mixture of 1:9 of sulfuric acid and acetic anhydride) was added and heated for 5 min to destroy all cellulose content. samples were concentrated at 2,700 rpm for 5 min and the supernatant was discarded. samples were washed twice with distilled water and the residue was concentrated. 4. ethanol, used as a dehydrating agent, was added and samples were concentrated at 2,700 rpm for 5 min. ethanol was discarded and some drops of glycerol were added. 5. the sediment was collected with a pipette and deposited on a slide. 6. finally, permanent microscope preparations were prepared using glycerin jelly as the mounting medium and paraffin as the coverslip sealant. to identify all pollen grain types (morphotypes, species, genera, or families), transects of slide preparations were examined at 400x magnification using a nikon eclipseni binocular scope. counts of grains were obtained at 400x magnification (barth, 1970a,b). biological daylight and differential interference contrast (dic) microphotographs were obtained at 1000x magnification using a nikon ds-ri1 camera system attached to the nikon scope. palynological descriptions were based on the terminology established by punt et al. (2007). botanical names were derived by comparisons with pollen atlases and collections (moreno et al., 2014; roubik & moreno, 1991). the taxonomic status of botanical names was updated by consulting tropicos (missouri botanical garden online database). all work was carried out at the center for tropical paleoecology and archaeology (ctpa) of the smithsonian tropical research institute (stri) in panama. 1.2.2 natural pollen zander (1935) published the first research on the natural pollen of honey, latter updated by louveaux et al. (1978). it consisted of preparing a slide with the honey sediment by removing the sugars of honey. besides pollen, this method permitted the detection of other elements of the sediment such as oils, fungi hyphae, yeast, insect fragments, and organic matter (haidamus et al., 2019). kerkvliet & meijer (2000) observed unusual residual rings fingerprinting sugar cane origin in sediments of adulterated honey. a honey dilution was prepared with 10 g of honey weighed into a 100 ml beaker, 20 ml distilled water was added, stirred with a glass rod, microwaved if needed for complete dilution, distributed in two conical 15 ml centrifuge tubes, centrifuged at 1,500 rpm for 15 min, the supernatant was discarded keeping the tube inverted for approximately 30 sec, 5 ml glycerin:distilled water (1:1) were added to resuspend the pellet, let 30 min, centrifuged again at 1,500 rpm for 15 min, the supernatant was discarded, draining the inverted tube by touching an absorbent paper, the sediment of each tube was used to prepare a microscope slide in a 2.4x2.4 cm2 area, a drop of glycerine jelly was added, the slide heated gently to avoid bubbles, a coverslip on the mounted sediment was sealed with paraffin. the honey sediment slides were kept in slide boxes. microscopic observations always started at 100x, increased to 200x, and were done at 400x magnification, also at 1000x when needed (barth et al., 2021). reference pollen slides from regional or country vegetation and images of melissopalynology for natural (barth, 1989; vit, 2005) and acetolyzed pollen (roubik & moreno, 1991) were used for pollen grain identifications and further counts of 300 pollen per slide. 2. standard and innovative methods for pot-honey analysis a glass bottle (duran) was selected to store the pothoney at 30 °c in the dark. the size of the container was at least double the volume of the pot-honey we placed in it. a separate honey sample was organized for each day of analysis, taken bottle by bottle for the planned measurements every seven days until 35 days post-harvest. 2.1 physicochemical analysis all measurements were performed in triplicate for each pot-honey sample. 2.1.1 water activity (aw) aw was determined using a water activity meter (alta novasina aw-center novasina aw-box, switzerland). a total of 5 g of honey was filled into the chamber of the aw-box and the value of water activity was read (cavia et al., 2004). 2.1.2 color (pfund) the color of honey was measured using a pfund classifier. homogeneous honey samples devoid of air bubbles were transferred into a cuvette with a 10 mm light path. the cuvette was inserted into a color photometer (hi 96785, hanna instruments, cluj county, romania). color grades were expressed in millimeter (mm) pfund scale as compared to an analytical-grade glycerol standard. sociobiology 69(4): e8251 (december, 2022) 5 2.1.3 moisture moisture content was determined using a portable digital refractometer (atago pal-2 model, atago co., ltd tokyo, japan) (aoac, 2012) that could be thermostated at 20 °c, regularly calibrated with distilled water or with another certified reference material (bogdanov, 2004). 2.1.4 ph, free acidity, lactone acidity, and total acidity the determination of ph and acidity (free, lactone, and total) was performed according to the method described by aoac (2012). total acidity was obtained by examining free and lactone acidity. to analyze free acidity, 10 g of a sample was weighed and diluted in 75 ml of distilled water. the free acidity was then calculated by titration with 0.05 n naoh until the solution reached a ph of 8.5. the lactone acidity was obtained by adding 10 ml of 0.05 n naoh to the initial solution and titrating with 0.05 n hcl until the ph reached 8.3. 2.1.5 hmf by hplc hydroxymethylfurfural (hmf) was determined by high-performance liquid chromatography (hplc) waters e2695 with waters 2489 uv/visible detector, following the international honey commission (2009) method. the column used was inertsustain c18 (4.6 x 150 mm, 3 µm) and operated at 40 °c. using an isocratic method, the mobile phase was 10% methanol and 90% deionized water (v/v) with a flow rate of 1.0 ml/min. the solution was filtered through a 0.22 µm membrane filter before use. the detector was set at 280 nm wavelength. samples were prepared using the solid phase extraction (spe) method. we weighed 4 g of honey and diluted it with acidified water (hcl ph 2). bond elut c18 column cartridge was used for sample purification and proceeded with pre-condition the cartridge with methanol and hcl (ph 2) two times. then, the sample was loaded into the cartridge and released slowly. we added 100% methanol to wash the sediment absorbed in the sorbent bed (desired fraction). the collection fraction was dried using a vacuum pump (nitrogen). the dried sample was weighed and hydrated with 1 ml of 60% methanol. then, the sample was transferred into hplc vials and the injection volume used was 10 µl. 2.1.6 sugars by hplc the sugar contents of fructose, glucose, sucrose, maltose, and trehalulose in honey were determined following fletcher et al. (2020) with minor modifications using highperformance liquid chromatography (hplc) waters e2695 with waters 2424 evaporative light scattering detector (elsd). the column used was inertsustain nh2 (4.6 x 250 mm, 5 μm) and operated at 40 °c. using an isocratic method, the mobile phase was 85% acetonitrile and 15% deionized water (v/v) with a flow rate of 2.5 ml/min. the solution was filtered through a 0.22 μm membrane filter before use. the elsd was set at 60 °c for drift tube temperature, gain 1, and pressure at 60 psi. sample preparation was done according to ms2683:2017 where 2 g of honey were mixed with deionized water (40 ml) and transferred into a 100 ml volumetric flask. later 25 ml of methanol was added and deionized water was filled up to the 100 ml mark. the volumetric flask was shaken well to ensure the homogenization of the mixture solution. the honey stock solution was filtered into the vial using a nylon syringe filter 0.22 μm before injection. 2.2 targeted nmr metabolomics this is a multiparametric method. identification and quantification of sugars, ethanol, hmf, aliphatic organic acids, amino acids, and botanical markers were done. in nuclear magnetic resonance (nmr) spectroscopy, the chemical shift is the resonant frequency of a nucleus relative to a standard in a magnetic field. often the position and number of chemical shifts are diagnostic of the structure of a molecule. nmr spectral tables aid to remember structural positions according to the solvent. for proton nmr, which is 1h-nmr, the kinds of honey were diluted in deuterated water d2o. 2.2.1 nmr reference sample and honey sample preparation all chemicals used in nmr were of analytical grade (>99% purity). the hl 44518 quantrefa-nmr-tube 5 mm, 600 µl (reference sample for quantification in food applications) was used for the nmr mixture analysis. this reference sample was used with wilmad 507-pp-7 nmr-tubes in combination with bruker’s honey-profiling. deuterium oxide (d2o) was supplied from sigma-aldrich (steinheim, germany), 2,2,3,3-d(4)-3-(trimethylsilyl)propionic acid sodium salt (tsp) by alfa aesar (karlsruhe, germany), potassium dihydrogen orthophosphate (kh2po4), sodium hydroxide (naoh), and hydrogen chloride (hcl) from merck (darmstadt, germany), sodium azide (nan3) by fluka (steinheim, germany), and deionized water was supplied by th. geyer (renningen, germany). the nmr profiles were obtained for functional groups of honey compounds: sugars, organic acids, amino acids, alkaloids, and alcohols. the pot-honey sample preparation method was adapted from brukerbiospin (brukerbiospin, ettlingen, germany). the homogenized honey samples (5 g) were diluted in 17.5 ml nmr-buffer (15.7 g kh2po4, 0.05 g nan3 in 1l deionized water). for the neutralization of organic acids, the ph was adjusted to 3.1 with 1m hcl and 1m naoh. then, 900 µl of the homogenized honey solution were mixed with 100 µl standard solution (deuterium oxide d2o containing 0.1% tsp 2,2,3,3-d(4)-3-(trimethylsilyl) propionic acid sodium salt), centrifuged at 14,000 rpm for 10 min, and 600 µl of supernatant were transferred into a 5 mm × 178 mm nmr-tube for direct measurement. 2.2.2 targeted quantitative 1h-nmr spectra acquisition and processing all nmr spectra were acquired immediately after honey dilutions on a bruker ascend tm 400 mhz food screener (bruker biospin, ettlingen, germany) equipped with a patricia vit et al. – standard and innovative integrative research of pot-honey6 5 mm pa bbi 400si h-bb-d-05 z probe and using bruker sample xpress (bruker biospin, ettlingen, germany) for automatic sample change. 1h-nmr-spectra were recorded at 300 k using the pulse program noesygppr1d (1d spectra with water presaturation at 4.8 ppm) and jresgpprqf (2d j-resolved spectra, displaying chemical shift and spin-spin coupling information). for 1d spectra, 32 scans and 4 dummy scans of 64 k points were acquired with a spectral width of 20.55 ppm, a receiver gain of 16, and an acquisition time of 3.99 s. the 2d spectra were performed using 4 scans and 16 dummy scans of 8 k (f2-axis) and 40 k (f1-axis) points. the spectral widths were 16.7 ppm (f2) and 0.19 ppm (f1), receiver gain of 16, and acquisition times were 0.6 s (f2) and 0.3 s (f1). the noesy (nuclear overhauser effect spectroscopy) spectra were used for quantification. the jres (j-resolved spectrum) was used to verify the compound identifications. all spectra were standardized, automatically phased, baseline-corrected, and calibrated using 2,2,3,3-d4-3-(trimethylsilyl) propionic acid sodium salt (tsp) as a reference at 0.0 ppm. the compounds were quantified using the honey-profiling routine (release version 3.0, brukerbiospin, ettlingen, germany) for compound quantification by automatic integration of the peak area calculated with external standards (spraul et al., 2009). 2.3 biochemical analysis 2.3.1 flavonoids by hplc/pad/esi-ms the pot-honey flavonoids were identified and quantified by high-performance liquid chromatography/ photodiode-array detection/electrospray ionization ion trap mass spectrometry (hplc/pad/esi-ms), according to truchado et al. (2015). five grams. of pot-honey were dissolved with five parts of distilled water, and adjusted to ph 2 with hcl, up to a 50 ml volume, filtered through a sep-pak c18 cartridge, previously activated with methanol and water successively. the cartridge was washed with 10 ml distilled water and the phytochemical compounds were eluted with 2 ml methanol. the methanolic fraction was filtered through a 0.45 μm filter and stored at -20 °c until further analysis. a lichrocart column (250 × 4 mm, rp-18.5 μm particle size, lichrospher®100 stationary phase, merck, darmstadt, germany) protected with a lichrocart guard column (4 × 4 mm, rp-18.5 μm particle size, merck) was used for chromatographic analysis. the mobile phase consisted of two solvents: water/formic acid (1%) (a) and methanol (b), starting with 20% b, and using a gradient to reach 30% at 40 min, 60% at 45 min, 80% at 50 min. the flow rate was 1 ml/min, and the injection volume was 20 μl. spectral data from all peaks were accumulated in the range of 240-400 nm, and the chromatograms were recorded at 280, 320, and 360 nm. the hplc/pad/ esi-msn analyses were carried out in an agilent hplc 1100 series equipped with a photodiode-array detector and mass detector in series (agilent technologies, waldron, germany). the hplc system consisted of a binary pump (model g1312a), an autosampler (model g1313a), a degasser (model g1322a), and a photodiode-array detector (model g1315b). the hplc system was controlled by chem station software (version 08.03, agilent). the mass detector used an ion trap spectrometer (model g2445a) with an esi interface, controlled by lcmsd software (version 4.1, agilent). ionization was adjusted at 350 °c and 4 kv for capillary temperature and voltage. the nebulizer pressure and flow rate of nitrogen were 65.0 psi and 11 l/min. the ms analyses covered the range m/z 100 up to 1500. ms data were acquired in the negative ionization mode. msn was carried out automatically on the more abundant fragment ion in ms (n -1). fragment ions were assigned using the glycoconjugates nomenclature (domon & costello, 1988). ions k,lxj, y n j, z n j represent those fragments still containing the flavonoid aglycone, where j is the number of the interglycosidic bond broken, counted from the aglycone, n represents the position where the oligosaccharide is attached to the aglycone, and k and l denote the cleavage within the carbohydrate rings. 2.3.2 total flavonoid content the total flavonoid content (tfc) was determined by the aluminum colorimetric method according to hagr et al. (2017) and tuksitha et al. (2018). quercetin was used as a standard, with concentrations of 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm, 3.125 ppm, 1.56 ppm, and 0.78 ppm. in total, 100 μl of the diluted honey sample was mixed with 100 μl 2% aluminum chloride (alcl3) in a 96-well plate. in total, 100 μl of each standard solution was combined with 2% alcl3. in total, 100 μl distilled water was mixed with 100 μl 2% alcl3, which acts as a negative control. in total, 200 μl ethanol was pipetted directly into a 96-well plate and served as a blank. the mixture was incubated for 10 min at room temperature. the absorbance of the mixture was measured at 450 nm using a benchmark plus microplate (bio-rad1706930, singapore). the results were expressed as micrograms of quercetin equivalents (qe) per gram of honey (mg qe/100 g honey). 2.3.3 total phenolics the total phenolic content was analyzed by the follinciocalteu method, according to hagr et al. (2017). a 100 μl diluted honey volume was mixed with a 500 μl follinciocalteu reagent, and then vortexed for 30 s. in total, 400 μl 7.5% (w/v) aqueous sodium carbonate was added, and vortexed again. the mixture was allowed to stand for 1 h, and incubated at 40 °c in the dark. in total, 200 μl of the supernatant mixture was pipette out and loaded into a 96-well micro-plate. methanol and distilled water acted as blank. the absorbance of the reaction mixture was measured at 765 nm using a microplate spectrophotometer (bio-rad, hercules, ca, usa). gallic acid was used as a standard, with concentrations of 400 ppm, 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm, and 3.125 ppm. the results were expressed as milligrams of gallic acid equivalents (gae) per gram of honey (mg gae/100 g honey). sociobiology 69(4): e8251 (december, 2022) 7 2.4 antioxidant activity 2.4.1 abts+ free radical scavenging assay the assay was done according to ramlan et al. (2021). abts+ radical 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid was prepared by mixing 3 ml of an aqueous 7 mm abts solution with 3 ml of an aqueous 2.5 mm solution of potassium persulfate. the reaction was kept in the dark for 16 h, and 4 ml of this solution was then dissolved in deionized water until it reached an absorbance of 0.700 ± 0.005 at 734 nm. the reaction consisted of mixing 20 μl of a sample with 200 μl of the final abts+ solution. after 30 min, the absorbance of the reaction was measured at 734 nm using absolute ethanol and water as a blank. the abts+ solution was prepared daily, and the reactions were carried out in the dark. all reactions were done in triplicate. the trolox concentrations of 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm, 3.125 ppm, 1.56 ppm, and 0.78 ppm were used as a standard. the percentage of the abts+ radical scavenging activity was calculated as % inhibition = [(blank absorbance sample absorbance)/blank absorbance] × 100. the results were expressed as the % of abts+ inhibition. 2.4.2 dpph radical scavenging assay the antioxidant activity of the honey was analyzed using a dpph 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, as described by hagr et al. (2017). the trolox concentrations of 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm, 3.125 ppm, 1.56 ppm, and 0.78 ppm were used as a standard. in total, 50 μl diluted honey and standard trolox were added to 195 μl dpph reagent into a 96-well plate. in total, 50 μl methanol was also mixed with 195 μl dpph reagent as a negative control. those mixtures were incubated at room temperature in dark conditions and were allowed to stand for 1 h. the absorbance was measured at 540 nm using a microplate spectrophotometer (bio-rad, us), with methanol as a blank. the percentage of the radical scavenging activity was calculated as % inhibition = [(blank absorbance sample absorbance)/blank absorbance] × 100. the results were expressed as the % of dpph inhibition. 2.4.3 frap ferric reducing antioxidant power assay the ferric reducing antioxidant power (frap) of each honey was analyzed using the method of hagr et al. (2017) and tuksitha et al. (2020). ferrous sulfate (feso4) was used as a standard, with concentrations of 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm, 3.125 ppm, 1.56 ppm, and 0.78 ppm. in total, 20 μl diluted honey was mixed with 220 μl frap reagent. the frap reagent was prepared by mixing 40 ml acetate buffer with 4 ml 2, 4, 6-tri (2-pyridyl)-striazine (tptz) solution and 4 ml ferric chloride (fecl3) solution. in total, 20 μl distilled water was mixed with the frap reagent, and acted as a negative control, while 240 μl methanol was a blank. the mixture was pipetted into the 96-well plate, and the absorbance of the mixture was measured at 593 nm using a benchmark plus microplate (bio-rad170-6930, singapore). the results were expressed as micrograms of feso4 equivalents per gram of honey (µmol feso4 .7h2o/100 g honey). 2.5 microbiological quality the microbiological quality was examined following the study described by lani et al., (2017). 2.5.1 pot-honey sample preparation and storage pot-honey samples were collected using surgery gloves after piercing the honey pot and by suction with a sterile syringe, the needle was replaced by a rubber tube. the liquid honey was then forced into a centrifuge tube. previously sterilized, blue-cap bottles were used to disperse a portion of pooled honey (~25 g/bottle). to avoid any honey degradation while being transported to and inside the lab, the honey samples were stored and protected from sunlight. samples were stored at a constant temperature (30 ± 2°c) in the dark until analysis. 2.5.2 aerobic plate count agar (pca) in merker (1998), an aerobic plate count was conducted in accordance with the guidelines in the bacteriological analytical manual of the food and drug administration. five grams of pot-honey samples were thoroughly mixed with 45 ml of either 0.1% sterile buffered peptone water (merck, germany) in a stomacher bag (huko, seoul, korea) using a sample mixer (3m, wellington, new zealand). appropriate dilution 0.1 ml of 1x10-4 dilution was spread plated on plate count agar (himedia, mumbai, india) and incubated at 35 °c for 24 h. the morphology and gram stain of each isolate was observed using a light microscope (motic b series, usa) at 400x after pure selected isolates were grown at 35 °c for 24 h. 2.5.3 yeast and mold counts five grams of pot-honey samples were homogenized in a blue-capped bottle with 45 ml of 0.1% sterile buffered peptone water (merck, germany). appropriate 0.1 ml of 1x10-4 dilution was spread plated on yeast malt agar (himedia, mumbai, india) and incubated at 30 °c for 24-48 h. a 48-h culture of each isolate was stained with lactophenol blue, and a light microscope (motic b series, usa) at 400x was used to observe the morphology. 2.5.4 bacillus count five grams of each honey sample were diluted with 45 ml of 0.1% sterile buffered peptone water (merck, germany), 0.1 ml of 1x10-4 dilution was spread plated onto mannitol egg yolk polymyxin agar (oxoid, uk) and incubated at 35 °c for 24-48 h. a 48-h isolate culture was examined with a light microscope (motic b series, usa) at 400x. patricia vit et al. – standard and innovative integrative research of pot-honey8 2.5.5 lactic acid bacteria (lab) count following the procedure described by aween et al. (2012), which included a pre-enrichment process before isolation on de man, rogosa, and sharpe (mrs) agar (oxoid, uk), lab were isolated from pot-honey samples. a fivegram sample of pot-honey was aseptically transferred to 45 ml of mrs broth (oxoid, uk), and it was then incubated for 24 h at 30 °c in a co2 incubator (model ico150, memmert, germany). a 100 µl dilution in 0.1% sterile buffered peptone water (merck, germany) was spread on three different media mrs, mrs agar with 0.8% caco3, and mrs agar with 1% glucose). the plates were then put in an anaerobic incubator (model ico150, memmert, germany) at 30 °c for 24-48 h. hypothetical lab were shown as a zone of clearance on mrs agar with 0.8% caco3. twenty-four h pure cultures were gram stained, and then examined for catalase activity using 4% h2o2. lab is a catalase-negative organism that prevents the cells from producing gas bubbles. 2.6 honey-microbiome 2.6.1 honey preparation for dna extraction to prepare the pot-honey sample for dna extraction, approximately 5 ml of honey were diluted with 5 ml of distilled water in a 15 ml centrifuge tube. the mixture was stirred until the honey was completely dissolved and centrifuged for 5 minutes at 13,500 rpm in a (witeg, cf-10, germany) centrifuge to remove excess water. the supernatant was discarded, and the procedure was repeated three times, to obtain sufficient pellet from approximately 15 ml honey, based on optimized procedures. 2.6.2 dna extraction, library preparation, and dna sequencing the total dna was extracted using dneasy powersoil pro kit (qiagen, germany) with a modified protocol as described by (tay et al., 2021). briefly, 250 mg of the collected pellet was transferred into power bead pro tube. 800 µl of solution cd1 and 25 µl of proteinase k were added into the tube and vortex briefly to mix. the subsequent extraction process was carried out in accordance with the manufacturer’s protocol. 2.6.3 library preparation the library preparation for bacteria characterization was done by targeting the v3 region of the 16s rrna gene using primers prba338fgc and prun518r (castro-mejía et al., 2020) in two-step polymerase chain reaction (pcr) methods followed by 2×200 bp pair-ended illumina iseq 100 (illumina, san diego, ca) sequencing performed according to standard protocol. for fungi characterization, the partial 18s and full length of its1-its2 region were amplified using its9ngs and its4 primers with nanopore partial adapter on the primer 5’ end (tedersoo et al., 2015). 200 fmol of pooled barcoded amplicons were used for lsk110 library preparation (oxford nanopore, uk) and the library was sequenced on nanopore flongle flowcell for 24 hours, as previously described (tay et al., 2020). 2.6.4 qiime2 raw sequences data processing the gene sequencing service is generally provided by private companies. the paired-end reads generated from sequencing were overlapped, error-corrected, and merged using fastp v0.21 (chen et al., 2018). the reads were demultiplexed and the primers were removed using cutadapt v1.18 (martin, 2011) before being imported into qiime2 v.2021.4 (bolyen et al., 2019) for denoising using dada 2 (callahan et al., 2016). taxonomic assignations of the amplicon sequence variant (asv) were made using a q2feature-classifier (bokulich et al., 2018) trained on the latest gtdb release r202 16s rrna database (trimmed to only retain the v3 hypervariable region) (parks et al., 2020). asvs with taxonomic assignment at least to the phylum level were selected for subsequent analysis. the raw nanopore reads were basecalled and demultiplexed using guppy v5.0.7 (high accuracy mode). the raw reads containing primer ends were retained using cutadapt (martin, 2011) and the identified primer sequences were removed using nanoclust (rodríguez-pérez et al., 2020). the taxonomic assignations of consensus non-chimeric its158s-its2 sequences were made using the q2-feature-classifier (bokulich et al., 2018) against the latest unite its v8.3 database (abarenkov et al., 2021; nilsson et al., 2019). the 18s v9 region of the unsuccessful classified sequences was extracted using itsx and searched against the silva 138 ssu database (quast et al., 2013) using qiime2 v2021.4 classifyconsensus-blast pipeline (bolyen et al., 2019). both the asv table and otu table generated from nanoclust cluster output were manually formatted to generate microbiomeanalystcompatible input (chong et al., 2020; dhariwal et al., 2017), with minor modifications according to siew et al. (2022). 2.7 sensory evaluation descriptive quantitative measurements were performed on odor, aroma, taste, and persistence (vit, 2008). each honey sample (5.0 ± 0.5 g) was presented in plastic capped cups, coded with three random digit numbers. honey samples were served at room temperature in a tray with a plastic spoon, a glass of water, a serviette, a piece of green apple to bite at the end of each tasting, and water to prepare the oral cavity for the next tasting. these four actions were taken: 1. follow the 18 tasting steps, 2. select the sensory family for odor and aroma. if possible select the subfamily, and if the descriptor is identified select it too. if only a family such as floral-fruity is perceived, write it in the evaluation form, 3. in the nose is perceived the odor, and 4. in the mouth is perceived the flavor with tasting buds, and the aroma with retronasal perception. the sensory families and sub-families used to identify odor perception in the nose and aroma perception in the mouth sociobiology 69(4): e8251 (december, 2022) 9 are listed below as the odor-aroma (o-a) references for the eight sensory families (acronyms in bold) and corresponding subfamilies. see the table with sensory descriptors (vit, 2008), which are needed to guide the sensory evaluation in related families. the expansion of sensory descriptors is desirable to identify different targets such as fruits and flowers familiar to particular countries but unknown to others. the 18 steps below were followed in that order for the sensory evaluation of each pot-honey, to retrieve descriptors and their intensities: this is the form used for the descriptive sensory evaluation of honey. the intensity of each attribute was perceived on a scale 0-3. • in this test, the intensity of each odor, each flavor, each aroma, and persistence is evaluated. mark with x. • odor and aroma are described in table o-a for pothoney. add descriptors as needed. • select with a circle the relationship intensity odor/ aroma • code sample the acceptance test was rated with icons for a 7-point hedonic scale anchored by: 1. ‘dislike it a lot’, 2. ‘moderately dislike’, 3. ‘slightly dislike’; 4. ‘indifferent, don’t like it neither dislike it’, 5. ‘slightly like’; 6. ‘moderately like’, and 7. ‘like it a lot’. 2.8 honey authenticity test by an interphase emulsion (hatie) this test is based on the number of phases observed after shaking a 1:1 honey:distilled water dilution with a double volume of ethanol, as described by vit (1998). the two phases are a signature feature of fake honey. genuine 1 uncap the plastic cup expose honey volatiles 2 mix honey with a plastic spoon revolve honey in circles three times with the spoon 3 smell c l o s e y o u r e y e s inhale the odorants of honey 4 annotate odor intensity (use intensityscale 0-3) 5 smell inhale again (approximately 15 sec later) 6 annotate odor description (use table o-a descriptors) 7 s m e l l i t a g a i n i f n e e d e d 8 tasting take half tea spoon honey aprox. (0.5 g) 9 open and close your mandible 3 times, to dilute honey with saliva, rub your tongue against your palate 10 c l o s e y o u r e e y e s taste the admixture of honey and saliva 11 slowly swallow 12 annotate intensity of each flavor flavors have intensity (use intensity scale 0-3) 13 annotate aroma intensity aromas have intensity (use intensity scale 0-3) 14 annotate aroma description describe aromas (use table o-adescriptors) 15 annotate intensity of persistence aftertaste has intensity (use intensity scale 0-3) 16 select relationship intensity (o-a) odor aroma higher intensity nose o > a equal intensity nose and mouth o = a higher intensity mouth a > o 17 t a s t e i t a g a i n i f n e e d e d 18 clean the palate with a bite of green apple and a sip of water 1ff floral-fruity: floral, citrus fruit, fresh fruit, processed fruit 2v vegetable: fresh, dry, aromatic 3f fermented: acetic, alcoholic, lactic 4w woody: wood, resins, spices 5n nest: stingless bees, apis mellifera 6m mellow: sugary, caramelized, cakery 7p primitive: animal, smoke, wet, sulfated, mineral, marine, oily 8ci chemical industrial: petrochemical, medicinal. scale 0 1 2 3 intensity absent mild medium strong patricia vit et al. – standard and innovative integrative research of pot-honey10 honey was initially reported as a three-phase pattern, with a distinctive intermediate emulsion (vit, 1998) caused by putatively minor protein and lipid contents of honey made by bees, not present in sugar-derived imitations. recently, the third pattern of one phase was discovered for a type of honey with surfactant activity, lowering the interphase tension and distributing the intermediate emulsion in the reaction volume (vit, 2022). in fig 2, the number of phases is illustrated with an image and a diagram showing the three patterns observed for the hatie, with genuine honey generating one and three phases, in contrast to fake honey with two phases. 2.9 statistical analysis data were analyzed by one-way anova, principal component analysis (pca), and hierarchical cluster analysis (hca), using version 26 ibm spss statistics evaluated attributes intensity scale 0 1 2 3 odor intensity description intensity of each flavor sour bitter astringent sweet piquant savory umami aroma intensity description other perceptions in the mouth persistence intensity relationship intensity odor/aroma odor < aroma odor = aroma odor > aroma vit (2022) fig 2. number of phases obtained after shaking a honey aqueous dilution with diethyl ether. one (left) phase for scaptotrigona vitorum honey, three phases (right) for genuine honeys, and two phases in the center for fake honey. a. image, and b. diagram. sociobiology 69(4): e8251 (december, 2022) 11 software (ibm corp., 2019). pearson correlation matrix (p < 0.05) and principal component analysis (pca) were conducted using xlstat software premium 2021. microsoft power point 2018 was used to prepare, integrate, and edit the figures composed from multiple sources. 3. particular information on the basics of each method, background arts 3.1 identifications of the entomological and botanical origins of pot-honeys 3.1.1 entomological identification of the stingless bee several articles on pot-honey lack entomological identification of the stingless bee that produced the analyzed honey, in the materials and methods section. others use a generic name as an ethnic name, for example, the use of the name trigona in some indian contributions, without the identification by a trained entomologist taxonomist, and made worse by the fact that this genus does not exist in india, but solely in the neotropics (rasmussen & cameron, 2008). some important omissions of the stingless bee identification in pothoney research are guerrini et al. (2009) with an ecuadorian stingless bee, published in food chemistry, and more recently thomas & kharnaior (2021) with an indian stingless bee published in the journal of apicultural research. a lack of precise identifications, despite the significant advances and taxonomic updates to stingless bees (roubik, 2013; melo, 2021) means that such published data are effectively useless as they are devoid of any link back to a biological entity and therefore the results cannot be repeated nor properly compared across the diverse fauna of stingless bees. the growing complexity of entomological identifications was clear for peruvian stingless bees (marconi et al., 2022). authors and editors must be insistent on obtaining proper identifications as this is a mandatory requirement for any exploration of pothoney in stingless bee research. 3.1.2 botanical origin by melissopalynology because so many flowering plants are not observable they are high aboveground in trees, epiphytes, and vines despite the labors of field biologists, we are still woefully ignorant of which are most important to the honey-making social bees (roubik, 1989). the honey and pollen samples collected from pots in tropical stingless bee nests offer the best option for confronting this challenge (iwama & melhem, 1979, louveaux, 1968; maurizio, 1975). the honey analysis is the most efficient in many ways. honey carries not only the signature ‘fingerprints’ of nectar flowers but also the pollen signature of nectar-less flowering plant species, which are frequently found in the honey of tropical bees (roubik & moreno, 2013, 2018). the outer wall or exine of pollen grains contains sporopollenin. it is one of the most resistant organic compounds known and preserves pollen as a fossil. the exine is a distinctive structure observed by melissopalynologists for the thickness, surface, ornaments, aperture type, and number, and especially the shape of the grain that has been classified according to relationships between the polar axis and equatorial axis, in the equatorial view, spheric p/e = 1, prolate p/e > 1 (1.34-2.00) and oblate p/e < 1 (0.54-0.74) (punt et al., 2007). fatty acids provide different colors to pollen grains, they are observable by the naked eye, and in natural melissopalynology. to assign a botanical origin of genuine honey, it is necessary to identify the types of pollen, represented by taxa at the level of family, genus, and even species for the bestknown pollen grains. this is achieved with pollen reference collections, visual comparisons with photomicrographs from scientific literature (e.g. barth, 1989, 2004; roubik & moreno, 1991; vit, 2005; moreno et al., 2014), and experience. it is also necessary to know the ecology, which plants are nectariferous and which are polliniferous, or if they offer both nectar and pollen rewards for pollinators. some plants such as the clusia genus offer floral resin. for example, if a high pollen count in honey is caused by contamination with pollen from nectarless flora, it should be removed since honey is made from nectar. in those cases, the following taxon is used to propose a botanical origin. examples of polliniferous plants are mimosa caesalpiniifolia, melastomataceae, myrcia, solanum, borreria verticillata, and piper. contamination with anemophilous pollen needs to be eliminated because it did not provide nectar for the analyzed honey. for example, some asteraceae genera, casuarina, cecropia, celtis, chenopodiaceae, cyperaceae, pinus, poaceae, podocarpus, trema. another very important detail is the representation of pollen in the nectar of a certain taxon in honey, since there may be hypoand hyper-represented pollen in honey. for example, the bombax ceiba flower produces high quantities of nectar and its pollen is large, which causes low pollen counts in unifloral honey of bombacaceae detected in ecuador (barth et al., 2015). the fine-tuning of pollen analysis, using acetolyzed material, of course, comes with experience. our experiences include the following, given here as general observations worthy of documentation: 1. dye is not needed with acetolysis, the oxidative darkening of all surface features is sufficient, 2. delicate exines of marantaceae, zingiberaceae, and heliconiaceae are destroyed by acetolysis, 3. tropical flora are composed of roughly 20% tricolporate, reticulate grains, of many families, 4. therefore, pore and exine structures are of particular importance and require immersion oil magnification at 1000x, and 5. good phenological data as to the flowering schedule can greatly improve possible identification. the apertures of the exine allow the pollen tube to exit the pollen grain and transport the sperm to the egg in the pistil for germination. apertures are thin or missing exine areas in pollen grains. according to their shape, pores and colpi are two types of apertures, and combinations of them. some aperture features are annulate and aspidate. the surface patricia vit et al. – standard and innovative integrative research of pot-honey12 of the exine is very distinctive, it may vary from smooth or psilate to very ornamented types such as baculate, brochate, clavate, echinate, echinulate, eureticulate, fenestrate, foveolate, gemmate, granulate, microreticulate, punctitegillate, reticulate, rugulate, scabrate, striate, and verrucate (vit, 2005). regarding the size, form, and fine surface structures of pollen grains of five botanical families in fig 3, the two preparations of the honey sediment differ in color: 1. acetolyzed pollen is amber, and 2. natural pollen is light yellow or gray. polar and equatorial views are taken as standards, focusing both on the equatorial or polar outlines, and the surface to have dual information. no.1 amaranthaceae has panporate spherical pollen grains with no polarity, a thick exine, and numerous circular pores separated by a granulated exine surface. both methodologies allow correct identification. no. 2 shows anacardiaceae in equatorial view, with three long colpi (longitudinal apertures), each one containing a central and transversally elongate additional aperture, the surface is striate-reticulate, the yellowish natural pollen grains are more rounded and the apertures and surface features are less highlighted than the acetolyzed pollen. no. 3 asteraceae in polar view possess three short colpi, hardly distinguishable in both methodologies of processing, due to the lack of spines. no. 4 euphorbiaceae shows eight images. the two upper acetolyzed pollen grains are in equatorial view and the two below in polar view. colpi have irregularly shaped fig 3. photomicrographs of acetolyzed (left) and natural (right) pollen grains (1000x) of tetragonisca angustula (latreille, 1811) honey from panama, of five botanical families: 1. amaranthaceae, 2. anacardiaceae, 3. asteraceae, 4. euphorbiaceae, and 5. lamiaceae. sociobiology 69(4): e8251 (december, 2022) 13 contours and the surface sculpture is finely reticulate. the form of the four natural pollen grains did not change, but the exine remained colorless because there was no acetolytic oxidation, and appears to be smoother than that of the acetolyzed pollen grains, the protoplasm was not destroyed; so the surface features are more or less indistinguishable. no. 5 lamiaceae in polar view has six short well-defined colpi and a reticulate-granulate surface after acetolysis, similar in form and size to natural pollen grains, whereas its natural aspect is like that in no. 4. nevertheless, the strongly hydrated protoplasm is protruding outside the apertures. the protoplasm inside the pollen grains of no. 1-3 is more or less translucent. with some practice in observing pollen grains prepared by both methodologies, it is possible to identify most of the bee plant families. photomicrographs of acetolyzed (amber color) and natural (grey color) arecaceae pollen of trigona pot-pollen from panama were also observed at 200x and 1000x in fig 4. this pollen pot was filled with one pollen type. arecaceae is a family of perennial flowering plants known as palms. this family has unique pollen with trichotomosulcate or a single sulcus (a furrow, visible both in the acetolyzed and natural preparations), thus a monosulcate pollen type. the exine is tectate, and the sexine psilate. it is an oblate pollen with a p/e variable according to the preparation of the sample. visually the acetolyzed pollen is more flattened with a p/e = 0.54 which is smaller than the natural pollen p/e = 0.60, measured in the 1000x images 1b and 2b, respectively. the presence of the protoplasmic membrane in natural grains allows the effect of turgidity. the use of glycerin jelly as mounting media is known to increase the volume of the pollen grains in both preparations. here natural grains have a 20% larger equatorial axis (width of a pollen grain at the equator, representing the maximum measurable distance perpendicular to the polar axis) than acetolyzed pollen (natural = 60 µm, acetolyzed = 50 µm). the protoplasm is the inner content of the pollen grain, and the pollenkit is the external component comprised of oils, proteins, etc. in the opinion of an acetolysis methodology expert, the melissopalynologist j.e. moreno patiño, both are present in natural pollen and may mask characteristics of apertures, sculpture, and structure of the exine, that are neat in acetolyzed pollen. despite the pollen being mounted in water or glycerin, this does not clean or remove the protoplasm and pollenkit. that is why the acetolysis process helps to clean the grains and highlight their characteristics. probably other chemical substances can help to achieve this goal (for example sodium hydroxide, potassium hydroxide, nitric acid) but never reach the efficiency of acetolysis since they are focused on destroying coarse organic fragments such as cuticles. according to o.m. barth, a natural melissopalynologist, pollen grains from angiosperms are monocolpate or 1-colpate. many colleagues call them monosulcate or 1-sulcate, just like fossils. o.m. barth does not agree with that. the monocotyledons are close to the dicotyledons, and far from the pteridophytes. in acetolyzed 1a and 1b images at 200x and 1000x respectively, pollen grains are very clean, translucent, and good for observing the exine structures, but pollen grains have a fossilized aspect. inside the honey, they are preserved like living pollen grains and give other information, mainly about the quality of honey, without doing chemical analyses. photomicrographs 2a and 2b are the natural pollen grains not acetolyzed. the granules visible at 1000x are lipids, some are inside the pollen grains, others outside. sometimes they are indicators. the suggested palynological processing would be to prepare natural honey sediment and continue with acetolysis when the botanical origin of the pollen is not achieved. these are the basic differences between the two avenues of melissopalynology based on acetolyzed and natural pollen preparations of the honey sediment. classifying sporomorphs conservatively at the species, genus, or family level is important to ensure reproducible identifications between samples and between researchers (mander & punyasena, 2014). 3.2 standard and innovative methods for pot-honey analysis 1. physicochemical analysis moisture is estimated by a refractometric index as a physical measurement transformed into the water percentage of honey, which is a chemical component. hydroxymethylfurfural (hmf) is the indicator of honey aging or heating. the ph and the free acidity are chemical indicators selected for quality control of a biological product like honey. the gravimetric method to estimate ash content was replaced by electrical conductivity, but for tropical honeys the correlation is not as good as for european honeys. the enzymes most frequently measured in honey are diastase and invertase. their biochemical nature is not exactly physicochemical but this was the setup of the honey standards. the color is a physical property derived from the chemical nature of the honey matrix. it is an instrumental measurement in addition to sensory analysis. aw is considered a physical property important to ascertain the water availability for microorganisms. 2. targeted 1h-nmr metabolomics this is a multiparametric method. the maximum compound concentration measured in the profiler is 5000 mm (/www.chenomx.com/support/). profiler works exclusively with clusters (groups of peaks), not individual peaks, it is possible to see cluster centers in ppm. there is no minimum level of absolute peak height that applies to every sample, it is fixed by the analyst. identifications and quantifications of 36 organic metabolites were grouped into sugars, ethanol, hmf, aliphatic organic acids, amino acids, and botanical markers. interpreting a proton nmr spectrum systematically starts with the knowledge of the molecule, how many different proton environments exist in the molecule, the chemical shift, how many protons exist in that environment, and the splitting pattern on the number of protons on carbons. patricia vit et al. – standard and innovative integrative research of pot-honey14 the 36 metabolites were listed in supplementary table s1, with their molecular formula, chemical structure, 1h-nmr ranges in ppm, and signal type. the nmr signals shift around depending on matrix, ph, solvent, temperature, etc. therefore, shifts can be compared directly under absolutely the same conditions. this explains the differences observed between experimental honey (ohmenhaeuser et al., 2013) and other matrices. concerning the signal type, the multiplicity typically stays constant, apart from some cases where the peaks might shift into each other. few spectra of these metabolites are illustrated in fig 5. the x-axis corresponds to the chemical shift (ppm), and the y-axis is generally omitted, based on the intensity of the signals in arbitrary units. the 1h-nmr spectra increase the chemical shift (ppm) from right to left. they need to be expanded for the identification of signal types that are otherwise overlapped in the main spectra (0-6 ppm). in the upper right corner, region 1.0-1.6 ppm, a) ethanol is the first metabolite with a triplet signal, and b) lactic acid is the second metabolite with a doublet signal. below is the following region 2.6-3.0 ppm with c) acetic acid and d) citric acid. in the upper left corner, the following region, 4.6-5.3 ppm has the sugar e) glucose, and the last expanded spectra below 7.8-8.6 ppm have a singlet of the aliphatic organic acid f) formic acid. 2.1 sugars ten sugars were identified and quantified by 1h-nmr. a further fructose/glucose ratio and fructose +glucose addition were estimated (pubchem, https://pubchem.ncbi.nlm.nih.gov/). fructose c6h12o6 is one of the three most common natural monosaccharides, a ketonic simple sugar found in fruits – originating its etymology –, some vegetables, and honey. high-fructose corn syrup (hfcs) is used to sweeten processed foods and beverages, and to adulterate honey. fructose is a fructopyranose, a d-fructose, and a cyclic hemiketal. fructose is a reducing sugar. fructose bonded to glucose is a component of sucrose. glucose c6h12o6 (from ancient greek γλῠκῠ́ς, glukús “sweet”) is one of the three most common natural monosaccharides, a simple sugar of six carbon atoms and an aldehyde group. linear glucose is in equilibrium with the four cyclic forms. glucose bonded to fructose is a component of sucrose. glucose is a reducing sugar. sucrose c12h22o11 is a disaccharide of glucose and fructose, a glycosyl glycoside formed by glucose and fructose units joined by an acetal oxygen bridge from the hemiacetal of glucose to the hemiketal of fructose. gentiobiose c12h22o11 is a disaccharide of glucose, a natural sugar found in gentianopsis virgate and gentianopsis crinit, originating its etymology. synonym 6-o-beta-d-glucopyranosyl-d-glucopyranose. fig 4. acetolyzed (1) and natural (2) arecaceae pollen at 200x (a) and 1000x (b). https://pubchem.ncbi.nlm.nih.gov/ sociobiology 69(4): e8251 (december, 2022) 15 maltose c12h22o11 is a disaccharide of glucose. its degradation produces hydroxymethylfurfural. maltotriose c18h32o16 is a trisaccharide in which the glucose residue at the reducing end is in the pyranose ring form and has an alpha configuration at the anomeric carbon atom. it is a human metabolite. mannose c6h12o6 (from the hebrew manna’ miraculous food’) is a monosaccharide with six carbon atoms and an aldehyde group. it is thus a d-aldohexose. melezitose c18h32o16 is a trisaccharide produced by insects such as aphids. it plays a role as an animal metabolite. raffinose c18h32o16 is a trisaccharide composed of galactose, glucose, and fructose. glucose is a bridge between both α and β glycosidic bonds with galactose and fructose. it belongs to a family of oligosaccharides α-galactosyl derivatives of sucrose. it is found in asparagus, beans, broccoli, cabbage, celery, and radish. turanose c12h22o11 is a glycosylfructose disaccharide. fructose/glucose this ratio is used as a predictor of honey crystallization. fructose + glucose. this addition is the major sugar fraction in apis mellifera honey but in certain pot-honey other major sugars such as trehalose are present. 2.2 hmf the hydroxymethylfurfural (hmf) is a degradation product of fructose caused by heating and aging, and increases in acidic environments like that of honey. 2.3 ethanol the presence of ethanol in pot-honey derives from the alcoholic fermentation of diluted sugars by yeast enzymes. a balanced chemical equation for alcoholic fermentation converts one mole of glucose into two moles of liquid ethanol and two moles of volatile carbon dioxide, producing two moles of atp in the process via two moles of pyruvate. c6h12o6 diluted in water 2c2h5oh(liquid) + 2co2(gas) 2.4 aliphatic organic acids (aoa) most of these organic acids participate in the krebs cycle, also called tricarboxylic acid cycle (tac) or citric acid cycle. they are easily transformed into each other by electron transfer reactions. in metabocards (e.g., for acetic acid //hmdb.ca/metabolites/hmdb0000042), the aoa are present in bacteria, plants, animals, and humans, for their universal role in the organic reactions of life. they are commonly known as their salts or anionic forms: acetate, citrate, formate, fumarate, lactate, malate, pyruvate, quinate, shikimate, and succinate. the aoa of honey was recently reviewed (vit & simova, unpublished data). acetic acid c2h4o2 is a monocarboxylic acid ch3cooh, the smallest short-chain fatty acid (scfa). it has a distinctive sour taste and acrid vinegar smell. it is the product of a two-stage fermentation: 1. anaerobic fermentation, fig 5. 1h-nmr spectra of metabolites present in honey: sugar, aliphatic organic acids, ethanol.  patricia vit et al. – standard and innovative integrative research of pot-honey16 the transformation of sugars into ethanol by yeasts, 2. aerobic fermentation, is the oxidation of ethanol into acetic acid, by acetic acid bacteria (aab). citric acid c6h8o7 is a tricarboxylic acid hoc(cooh)(ch2cooh)2 found in citrus fruits (grapefruit, lemon, orange, tangerine) and pineapple. formic acid ch2o2 is a monocarboxylic acid hcooh, the simplest carboxylic acid also called methanoic acid. its presence in honey originates from the social chemistry of bee communication and defense. fumaric acid c4h4o4 is a dicarboxylic ho₂ acid cch=chco₂h, a key intermediate metabolite in the tca used to produce cellular energy as adenosine triphosphate (atp). the fumarate salt has a fruitlike flavor and is useful as an acidity regulator, and tartness agent for food. lactic acid c3h6o3 is a monocarboxylic acid ch₃ch(oh)cooh produced by lactic acid bacteria (lab). it is found in fermented food such as buttermilk, kefir, kimchee, kombucha, sauerkraut, yogurt. the intense sourness of lactic acid at ph 3.5 is caused by 70% of undissociated acid compared with 30% of citric acid (dziezak, 2016). malic acid c4h6o5 is a dicarboxylic acid hoocch2ch(oh)cooh tart-tasting. the tart taste of wine and honey has a malic acid component. malate and fumarate are intermediates in the tca cycle. malate can be formed from pyruvate. malic acid is both derived from food, like apples and pears, and is bio-synthesized by the tca in the mitochondria. pyruvic acid c3h4o3 is a monocarboxylic acid ch3cocooh. quinic acid c7h12o6 is an astringent cyclic polyol from plant sources. cyclic acids are not represented by the linear molecular formula. quinic acid and shikimic acid are key intermediates in the biosynthesis of aromatic compounds in living systems, with important roles in the formation of phenolic compounds. citric, malic, and quinic acids are the major aoa in coffee. the sour perception of coffee has a component related to quinic acid. shikimic acid c7h10o5 is a cyclohexene, a cyclitol, and a cyclohexanecarboxylic acid derived from phenolics. the shikimate biosynthesis pathway produces precursors for the aromatic amino acids phenylalanine, tyrosine, and tryptophan. the physiology of the bee or metabolic associations with microbiota in the nest, also the bee diet, or their combination may originate shikimic acid in honey (santos-sánchez et al., 2019). succinic acid c4h6o4 is a dicarboxylic acid (ch2)2(cooh)2, whose name denotes amber from latin succinum. it is an electron donor in the tca cycle. succinate is a cell signaling molecule, that alters gene expression patterns, modulates epigenetics by hormone-like signaling functions, and is a flavor-enhancer (dziezak, 2016). 2.5 amino acids (aa) eight free amino acids were identified and quantified in pot-honey. alanine c3h7no2 is a nonessential amino acid, biosynthesized from pyruvate by transamination. it is a glucogenic amino acid that constitutes a high percentage of the amino acids in most proteins. it contains an amine group and a carboxylic acid group, both attached to a carbon atom with a methyl group side chain. it is active in breaking down tryptophan and vitamin b-6. it is a source of energy for muscles and the central nervous system. it is active in the immune system. aspartic acid c4h7no4 is a nonessential amino acid that plays an important role as general acids in enzyme active centers. glutamine c5h10n2o3 is a chargeneutral, polar amino acid, with a side chain similar to that of glutamic acid, except that the carboxylic acid group is replaced by an amide. leucine c6h13no2 is a non-polar aliphatic amino acid with a side chain isobutyl group. phenylalanine c₉h₁₁no₂ is an aromatic amino acid, like a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of the terminal hydrogen of alanine. proline c₅h₉no₂ is a proteinogenic amino acid, without the amino group -nh₂, but is like a secondary amine with nitrogen in the protonated nh₂⁺ form under biological conditions, while the carboxyl group is in the deprotonated −coo⁻ form. tyrosine c9h11no3 is a non-essential amino acid with a polar side group. l-tyrosine or tyrosine or 4-hydroxyphenylalanine is a non-essential amino acid. valine c5h11no2 is a non-polar aliphatic amino acid with an α-amino group, an α-carboxylic acid group, and a side chain isopropyl group, an essential amino acid in humans that must be obtained from the diet. 2.6 botanical markers an interest in discovering residual botanical markers of diverse chemical nature has attracted engaging research in the years. a recent review was conducted for botanical markers of twenty monofloral apis mellifera honeys (machado et al., 2020). for pot-honey, more recent contributions on entomological markers are those of the tomás barberán group on distinctive honey flavonoids of melipona favosa from venezuela (truchado et al., 2011) and tetragonula carbonaria from australia (truchado et al., 2015). from the six botanical markers included in the reference sample for quantification in food applications (quantrefa-nmr-tube), kynurenic acid was not detected. acetoin c4h8o2 is formed by the decarboxylation of two pyruvic acids c3h4o3. this natural volatile is used as a botanical marker for the australian eucalyptus species e. leucoxylon and e. melliodora (d’arcy et al., 1997). 2,3-butanediol c4h10o2 molecular formula (ch₃choh)₂ is classified as a vic-diol. it exists as three stereoisomers, a chiral pair, and ameso isomer. a fermentation pathway will ferment glucose and produce a 2,3 butanediol end product instead of organic acids. dihydroxyacetone (dha) c₃h₆o₃ is the precursor to methylglyoxal (smallfield et al., 2018). it is obtained from plant sources such as sugar beets and sugar cane, and by the fermentation of glycerin. kynurenic acid c 10 h 7 no 3 is a metabolic derivative of the amino acid l-tryptophan. methylglyoxal c 3 h 4 o 2 (mgo)ch 3 ccho is a reduced derivative of pyruvic acid. 3-phenyllactic acid c9h10o3 is a lactic acid with a phenyl substituent, it belongs to the class of organic compounds known as phenylpropanoic acids. sociobiology 69(4): e8251 (december, 2022) 17 3. biochemical composition 3.1 flavonoids flavonoids are secondary metabolites, a group of polyphenols biosynthesized by plants. most flavonoids in edible plants are β-glycosides, bound to one or more sugar molecules which are detached by exposure to microbial β-glucosidases. the aglycones are not bound to sugars, and glycosylated flavonoids have a sugar substituent. methoxylated forms also exist. according to their structure, flavonoids are divided into six major subclasses: 1. anthocyanidins, 2. flavanols, 3. flavanones, 4. flavones, 5. flavonols, and 6. isoflavones. the linus pauling institute has a micronutrient information center for the public, with useful information on flavonoids online. two measurements of flavonoids were done: 1. total flavonoids by spectrophotometry, and 2. flavonoid identification by high-performance liquid chromatography/ photodiode-array detection/electrospray ionization ion trap mass spectrometry (hplc/dad/esi-ms). this advanced technique was needed for the study of flavonoids having diverse chemical natures, and present in a complex food matrix like honey. the monoglycosides/diglycosides/triglycosides and the position of the o-glycosylation and the sugar moiety were identified by the study of the ms data. flavonoids have significant antioxidant properties, varying according to the chemical structure. some of them like the aglycone quercetin are scavengers of reactive oxygen species (ros) such as superoxide radicals, and singlet oxygen, and also inhibit the formation of lipoid hydroperoxide radicals. in fig 6 the chemical structures of the aglycone quercetin, and quercitrin, one of its glycosides, formed with the deoxy sugar rhamnose. the effects of quercitrin in vivo perhaps derive from the release of quercetin by intestinal microflora. both quercetin and quercitrin attenuate lps-induced macrophage inflammation and oxidative stress by anti-ros effect (tang et al., 2019). their theoretical calculation illustrated that the oxygen atom on b rings suggests a possible mechanism for the anti-inflammatory and ros scavenging effects. 3.2 polyphenols they are generally called phenolic compounds, and this term is accepted and widely used. a single phenolic such as hydroxybenzoic acid is not toxic at all. tyrosine, and essential amino acid, is a single phenolic. however, phenol (hydroxy-benzene) is toxic. polyphenols are metabolites that contain at least two phenolic rings in the structure as happens for flavonoids, stilbenes, lignans, hydrolyzable and condensed tannins, and some dimers of hydroxycinnamics (rosmarinic acid, curcumin). a more inclusive name to cover polyphenols and simple phenolics is (poly)phenols. in the scientific literature, polyphenols are frequently wrongly named “phenols”. this is a conceptual mistake considering that a polyphenol is healthy and a “phenol” is toxic. the plural of the toxic phenol molecule is not a synonym for polyphenol, meaning phenol structures inserted in one molecule, from the greek πολύς, polyis a prefix meaning “many”. 4. antioxidant activity three methods were selected to measure the antioxidant activity of pot-honey using spectrophotometric techniques: abts, dpph, and frap. the abts+and dpph free radical scavenging assays are based on the quenching of stable colored radicals abts•+ or dpph to show the radical scavenging ability of antioxidants in a complex matrix like honey. the frap ferric reducing antioxidant power is an antioxidant capacity assay that uses trolox as a standard to measure the antioxidant capacity of honey because it contains polyphenols. 5. microbiological quality pollen, nectar, the digestive tracts of bees, and the environment are the primary sources of microbial contamination in pre-harvest honey, which is very difficult to control. while post-harvest sources include air, and honey handling, and cross-contamination influences microorganisms’ levels in honey. honey’s moisture and temperature influence microbial growth. thus, low mixture has been considered fig 6. chemical structures of a. the aglycone quercetin, and b. the glycosylated quercitrin with the deoxy sugar rhamnose. the b ring with the –oh in position 3, the flavonol signature. a b patricia vit et al. – standard and innovative integrative research of pot-honey18 to prevent honey spoilage for a long time. yeast and sporeforming bacteria are commonly found in pot-honey. while these properties indicate safety and quality for commercial honey, the role of microorganisms in human illness is the most considered. for no particular reason, total plate counts from honey samples can range from zero to tens of thousands per gram. yeasts can be found in most honey samples. bacteria do not proliferate in honey, thus a high number of vegetative bacteria could indicate a recent secondary source of contamination. at low temperatures, specified vegetative bacteria can persist for years in honey. on the other hand, honey provides antimicrobial properties that prevent many microbes from growing or persisting. this was a classic approach to have counts on microbiological screening of total, yeast and mold, bacillus, and lactic acid bacteria. 5.1 aerobic plate count agar (pca) the aerobic plate count agar (pca) is used to determine how many microorganisms are present in a product. it is also known as the standard plate count, mesophilic count, or total plate count. the test is predicated on the idea that when each cell is mixed with the necessary nutrient agar, it will form a visible colony. it is a generic test only for organisms that grow aerobically at mesophilic temperatures (25 to 40 °c) not for the entire bacterial population. apc does not discriminate among bacterial types however, it can be used to determine hygienic quality, organoleptic acceptability, and conformity with good manufacturing procedures and safety indications. 5.2 yeast and mold counts in this test, the total yeast and mold population is counted, whereas no identifications are made. both yeasts and mold can produce various degrees of degradation and decomposition of honey. yeasts found in honey can resist high concentrations of acids and sugar, they are osmotolerant. the number of yeasts increases in relation to the moisture of honey. yeasts are classified as non-filamentous fungi, which require higher moisture than molds but less than bacteria for growth, and an optimum temperature range for growth is between 25 and 30 °c. they transform sugars into ethanol during fermentation with an ideal acid ph. additionally, filamentous fungi (mold) can produce substances including enzymes and acids. yeast and mold counts in many honey samples are below 100 colony-forming units per gram (cfu/g). 5.3 bacillus count bacillus spp. are pore-forming bacteria frequent in honey, their count is an indicator of microbiological quality. osmotolerant microbial contamination of honey has primary sources (pollen, digestive tracts of bees, dust, air, earth, nectar), and post-harvest secondary sources as in any food product (air, soil, water, food handlers, containers, crosscontamination, equipment, buildings). primary sources of contamination are very difficult to control, whereas secondary sources are controlled by good manufacturing practices. bacteria do not reproduce in honey, thus vegetative bacteria origin could be contaminated from a secondary source. the bee gut-microbiota contains 1% yeast, 27% gram-positive bacteria including bacillus, bacteridium, streptococcus, and clostridium spp; 70% gram negative or gram variable bacteria including achromobacter, citrobacter, enterobacter, erwinia, escherichia coli, flavobacterium, klebsiella, proteus, and pseudomonas (tysset et al., 1991). 5.4 lactic acid bacteria (lab) count acid-producing microbes are common in nature and can be found in honey. lactic acid bacteria (lab) are one of the most important types of acid-producing bacteria in the food industry. lab counts are used to determine the total levels of bacteria in honey. 6. honey-microbiome the sanger method is a first-generation sequencing (fgs) technology. recent culture-independent approaches for characterizing complex microbiome communities using next-generation sequencing (ngs) technologies have evolved over time. they provide fast and efficient identification of the microbial communities (mardis, 2008), and permit phylogenetic studies. these methods are not based on growing microbes in culture media. for prokaryotes characterization, the v3-region of the 16s rrna gene was selected as a marker for bacteria (fig 7). it combines highly conserved regions useful as nearly universal prokaryotic primers, while at the same time it has maximum nucleotide heterogeneity and discriminatory power for capturing bacterial species due to the hypervariable v3-region (castro-mejía et al., 2020). although the v3-region of 16s rrna was used to describe the prokaryote community, we applied the flexibility of the yeast ribosomal gene complex (fig 8) to fungal markers for the detection of fungi and eukaryotic organisms. for example, the internal transcribed spacer (its) regions were useful for the detection of the filamentous fungi and d1+d2 domain of large subunits (lsu) 16s ribosomal rdna for yeasts, using the primers nl1 and nl4. then, the species were identified by their genbank accession number, after sequencing the d1+d2 domain of large subunits (lsu) ribosomal rdna. microbial genomic dna approaches for identifying the microbiome of honey-making bees and their products in the nest have increased. for instance, the bee gut microbiome of apis mellifera has been reported to be a home for novel lactic acid bacteria (olofsson & vásquez, 2008). the lactic acid bacteria of bee pollen and bee bread were studied in apis mellifera (vásquez & olofsson, 2009). similarly, the bee gut microbiome has also been examined in both apini and meliponini (vásquez et al., 2012), and in kenyan stingless bees (tola et al., 2021). the cuticle microbiome was also used sociobiology 69(4): e8251 (december, 2022) 19 to compare sympatric australian stingless bees (leonhardt & kaltenpoth, 2014) where a lack of acetobacteriaceae in austroplebeia australis clearly differentiated it from tetragonula carbonaria and tetragonula hockingsi. the latter two process their sour honey and pollen with aab, and thus are rich in acetic acid (massaro et al., 2018). the honey yeast microbiome was investigated in 17 species of brazilian stingless bees, and their associations based on the entomological origin were confirmed (echeverrigaray et al., 2021). these authors identified 16 species of yeasts from nine genera: candida, debaryomyces, moniliella, rhynchogastrema, starmerella, torulaspora, wickerhamiella, wickerhamomyces, and zygosaccharomyces. generally, the alteration of the gut microbiota of honey bees is reportedly due to seasonal changes (kešnerová et al., 2020) and also influenced by geography (liu et al., 2021). however, differences in the hygienic habits of stingless bees were reflected in the bacteriome of the inner abellan-schneyder et al. (2021) fig 7. generalized linear diagram of a bacterial ribosomal gene complex (drawing not to scale). sequence variation among bacterial 16s rrna is known to be not uniformly distributed. nine hypervariable regions (v1-v9) were identified among bacteria, tagged with the location of the genes of each region. blue arrows denote the names of forward primers, and green arrows signify the names of reverse primers. surface of nests. several species of brazilian stingless bees with distinctive habits for collecting materials to build their nests were investigated: 1. the microbiome of frieseomelitta varia and tetragonisca angustula were associated with pseudomonas sp. and sphingomonas sp. from the phylloplane (surface of leaves) and flowers because these bees collect plant materials, 2. the microbiome of trigona spinipes was related to coliforms such as escherichia coli and alcaligenes faecalis that are abundant in mud and feces they collect, and 3. melipona quadrifasciata combined microbiome of the previous two groups was caused by the collection of both plant resins and mud-feces (de sousa, 2021). arbefeville et al. (2017) interestingly, another study showed that the diversity of the gut microbiome of the stingless bees was associated with host wing size (fig 8) where the t. carbonaria bees with larger wings from more southern populations had higher microbial diversity in their guts (liu et al., 2021). moreover, the core gut microbiome of these australian stingless bees consisted of a distinct group of bacterial taxa from that observed in honey bees and bumble bees such as acetobacteraceae, bombella, lactobacillus sp., and snodgrassella. the core microbiome of the stingless bee were aureobasidium pullulans, didymellaceae, malassezia globose, malassezia restricta, and monocilium mucidum. fig 8. generalized linear diagram of anuclear yeast ribosomal gene complex (drawing not to scale). the organization of this complex includes a sequence coding for the 18s rrna gene, an internal transcribed region 1 (its1), the 5.8s rrna gene coding region, another internal transcribed region 2 (its2), and the sequence coding for the 28s rrna gene. the 28s rrna is preceded by the d1+d2 hypervariable regions. patricia vit et al. – standard and innovative integrative research of pot-honey20 dendrograms of microbial taxa associated with stingless bees show graphic evolutionary relationships, they are linear or circular graphics clustering more related species together. this microbial composition influences chemical profiles of honey processing and post-harvest transformations, as well as tracing back pollen, nectar, and other materials origins, defense, hygiene, phylloplane, bee, and plant pathogens. host-specific stingless bee-aab and -lab associations may have phylogenetic and geographical components in their natural history, as observed for austroplebeia and tetragonula bees (leonhardt et al., 2014; leonhardt & kaltenpoth, 2014), like fungi and yeasts. 7. sensory evaluation descriptive quantitative measurements were done on odor, aroma, taste, and persistence of pot-honeys. their histograms show similarities and differences between sensory family compositions for odor and aroma perceptions, compared between stingless bee species processing differently the honey in the cerumen pots with their associated microbiome. for example, see a melipona honey in fig 9. “fermentation produces the majority of flavor that we recognize as ‘chocolate’. in addition to the production of acetic and lactic acid, ethanol, and minor sugars such as glycerol and mannitol, a variety of volatile compounds are also produced. more than 600 volatile compounds are found in fermented cacao.” //thechocolatelife.com/factoid-fermentation-producesthe-majority-of-flavor-that-we-recognize-as-chocolate-in/ similarly, in pot-honey, sensory complexity is added during the fermentation of thin honey in the warm and moist environment of the nest. why a microbiota type is selected by some stingless bee species and not others? this question has not an immediate origin of bee vectoring yeasts from floral nectars and other sweet resources like honeydew. evolutionary biology may fit micro-ecological pieces into place with important contributions to be deciphered from nests observed in the present. we could smell some of them from stingless bee colonies and their substrates but were not yet able to identify and explain these relationships or the reasons for their origins. for example, the blue cheese smell of the ecuadorian scaptotrigona vitorum nest was annotated as roquefort in fieldwork forms (vit, 2022). from another perspective, honey bees changed the microbiota harbored in brassica pollen collected and packed after adding regurgitated nectar to glue the pollen grains (prado et al., 2022). the bacterial diversity of pollen increased with honey bee symbionts such as bombella, frischella, gilliamella, and snodgrassella, and nectar-dwelling lactobacillus – having bee gut microbes as inoculum during corbicular pollen processing. 8. the honey authenticity test by interphase emulsion, became a biosurfactant test genuine pot-honeys tested before this integrated study had one, two, and three phases, as illustrated in fig 2 in the previous section. in fig 10, the result observed in the scaptotrigona vitorum honey was interpreted as a honey biosurfactant test (hbt), for the surfactant activity. this was a new ecuadorian scaptotrigona species described by engel (2022). fig 9. melipona honey histograms for odor and aroma descriptors of sensory families 1ff floral-fruity, 2v vegetal, 3 f fermented, 4 w wood, 5 n nest, 6 m mellow, 7 p primitive, and 8 ci chemical industrial. sociobiology 69(4): e8251 (december, 2022) 21 9. final remarks these methods were used to analyze pot-honeys during post-harvest and to create a database to follow the pothoney transformations along the established period of 35 days at 30 °c. the participating laboratories were led by experts in the field, who sometimes also coordinated the logistics for pot-honey harvest. preliminary studies were based on multiparametric targeted 1h-nmr of ecuadorian pot-honey (vit et al., 2022), but never compared before with classic microbiology and honey-microbiome. a missing data is on volatile organic compounds vocs measured with ptrtof-ms, a powerful method requiring a small quantity of honey for key information on odorants developed in biological processes. these integrated methods of analysis were proposed for the first time to follow a limited but substantial post-harvest period where major changes have been observed over the years by stingless bee keepers, consumers of their honey, and scientists. new data should provide a few answers and more questions for proposing future research avenues. supporting sustainable meliponiculture, pot-honey regulations, and quality schemes, start from the id of stingless bee species. for example, about 10,000 s. vitorum catiana hives are kept by communities of stingless bee keepers from el oro and loja provinces (s loayza and d. encalada, unpublished data). the approval of ecuadorian standards for pot-honey (inen, 2015), including scaptotrigona, is imperative, and could be based on s. vitorum research. the relevance of the entomological origins and ethnic names of pot-honey in a normative domain, would be also mandatory for protected designation of origin (pdo) e.g. the scaptotrigona honey called catiana in ecuador, and its guaranteed traditional specialty (gts) protecting the artisanal production method. the quality policy of the european union (eu) focuses on protecting names of unique products. these eu strategies benefit producers and consumers, and would visualize the biodiversity of tropical stingless bee honey worldwide. authors’ contributions pv: conceptualization, writing original draft, review, writing review & editing, data curation, methodology, visualization, investigation, formal analysis. bc: writing original draft, writing review & editing, methodology, investigation. nz: writing original draft, writing review & editing, methodology, investigation. md: writing review & editing, methodology, investigation. jm: writing original draft, writing review & editing, methodology, investigation. hfa: writing original draft, writing review & editing, methodology, visualization. fat-b: writing original draft, writing review & editing, methodology. gm: writing-review & editing, data curation. kd: writing original draft, writing review & editing, methodology. em: writing original draft, writing review & editing, data curation, methodology, visualization, investigation, formal analysis. dwr: writing original draft, writing review & editing, methodology, formal analysis. omb: writing original draft, writing-review & editing, methodology, formal analysis. dwl: writing original draft, writing review & editing, visualization, investigation, formal analysis. mse: writing original draft, writing review & editing, data curation, methodology, visualization, investigation, formal analysis. fig 10. diagram of the honey biosurfactant test (hbt). to the left, the surfactant activity causes a cloudy emulsion in the reaction volume. to the right, the honey-based emulsion is an interphase between the water (lower) and the ether (upper) phases. this test was done by b. chuttong and n. zawawi for geniotrigona thoracica and heterotrigona itama, and p. vit for scaptotrigona vitorum and tetragonisca angustula. patricia vit et al. – standard and innovative integrative research of pot-honey22 acknowledgments to stingless bee keepers of the world, anonymous reviewers, and appreciated editorial processing. to c. rasmussen from aarhus university, denmark, for improving the manuscript. to m. linkogel from quality services international gmbh, bremen, germany, and to a. dübecke from tentamus center for food fraud, germany, for their institutional support. to the memory of e. crane, j.m.f. camargo, and c.d. michener for their scientific legacy on stingless bees. to universitá politecnica delle marche, ancona, italy; 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(2013). introduction. in p. vit., s.r.m. pedro & d.w. roubik (eds.), pot-honey. a legacy of stingless bees (pp. xi-xv). new york, usa: springer. 654 pp. zander, e. (1935). beiträge zur herkunftsbestimmung bei honig, band i: pollengestaltung und herkunftsbestimmung bei blütenhonig mit besonderer berücksichtigung des deutschen trachtgebietes, berlin: verlag der reichsfachgruppe imker e.v. https://www.alanrevista.org/ediciones/1998/1/art-12/ https://www.alanrevista.org/ediciones/1998/1/art-12/ open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.8099sociobiology 69(3): e8099 (september, 2022) introduction metacommunities are a set of local communities that are linked by dispersal of multiple interacting species (wilson, 1992). local metacommunities are shaped by speciation, dispersal, abiotic conditions, biotic interactions, and ecological drift processes (vellend, 2010; 2016). such processes, which shape the structure and dynamics of local communities, may occurs as a result of habitat fragmentation (chase et al., 2020). habitat fragmentation isolate natural habitats, change their abiotic conditions, resource availability and quality (arroyo-rodríguez et al., 2017; 2020; fahrig, 2003; schmidt et al., 2013; sobrinho et al., 2003; sobrinho & schoereder, 2007). communities from isolated habitats abstract habitat fragmentation changes biological communities and its spatiotemporal dynamics – which may lead to either biotic homogenization or heterogenization along time and space. both processes can occur by addition, replacement or loss of species within communities, altering compositional similarity across the landscape. we investigated which of these two processes (biotic homogenization or heterogenization) occurs, and its possible underlying mechanism, over 15 years in an atlantic forest landscape using ants as model organisms. we sampled ants in 17 forest fragments across three different years, compared their composition similarity, species richness, and species richness of groups classified according to their habitat preferences. we sampled a total of 132 ant species. ant communities in fragments diverged over time, suggesting they experienced an idiosyncratic structuring process. this biotic heterogenization occurred through an additive process, as ant species richness increased over time, mainly due to an increase of generalist ant species, and a decrease of forest specialist ant species. these changes occurred despite the higher forest cover in the landscape along years. since different species can perform different functions in ecosystems, this biotic heterogenization may have implications for ecosystem functioning. investigating how disturbances structure biological communities over time, especially those performing important ecosystem functions, can shed light to our understanding of possible changes in ecosystem functions and consequently for forest regeneration. sociobiology an international journal on social insects vanessa s. ribeiro¹, lucas n. paolucci¹, josé h. schoereder¹, ricardo r. de c. solar² article history edited by wesley dáttilo, mexico received 25 april 2022 initial acceptance 26 april 2022 final acceptance 12 august 2022 publication date 07 september 2022 keywords biotic heterogenization, biotic homogenization, habitat fragmentation, community divergence, formicidae, tropical rain forest. corresponding author vanessa soares ribeiro department of general biology federal university of viçosa av. peter henry rolfs s/nº, campus universitário, cep: 36570 000 viçosa, minas gerais, brasil. e-mail: soaresribeirov@gmail.com have smaller populations, different migration patterns and distribution, and richness and composition (arroyo-rodríguez et al., 2020; bender et al., 1998; fahrig, 2019; lasmar et al., 2021; madureira et al., 2012; sobrinho & schoereder, 2007; vellend, 2010). these changes in species dynamics within fragments, may lead communities to become either more similar among each other or more dissimilar – i.e., they can cause a biotic homogenization (mckinney & lockwood 1999) or biotic heterogenization, respectively (laurance et al., 2007; socolar et al., 2016). biotic homogenization may occur due to two mechanisms: additive and subtractive. additive homogenization occurs when new species immigrate from other regions and become present in most fragments in the landscape. 1 department of general biology, federal university of viçosa, viçosa-mg, brazil 2 department of genetics, ecology and evolution, federal university of minas gerais, belo horizonte-mg, brazil research article ants divergence of ant communities over time in a fragmented atlantic rain forest landscape mailto:soaresribeirov@gmail.com vanessa s. ribeiro et al. – divergence of ant communities over time across fragments2 subtractive homogenization occurs when particular species with a distribution restricted to a few fragments disappear from all of them and consequently from the landscape (socolar et al., 2016). therefore, biotic homogenization occurs through immigration and extinction, which in turn impact species distribution and abundance across local sites (olden, 2006). lower connectivity in landscapes after fragmentation benefits species with a high dispersal ability, that can establish in isolated fragments and contribute to biotic homogenization (chase et al., 2020). changes in resource availability due to habitat fragmentation can benefit species that have more similar needs and are more adapted to disturbances (generalist species), altering local species composition due to invasion and extinction (mckinney & lockwood, 1999; olden et al., 2004; tabarelli et al., 2012). biotic heterogenization may also occur due to species introduction (additive mechanisms) or species loss (subtractive mechanisms) (socolar et al., 2016). contrary to homogenization, heterogenization is likely driven mainly by idiosyncratic events, and if so local communities diverge along time after disturbance (audino et al., 2014; laurance et al., 2007; socolar et al., 2016). for instance, additive heterogenization occurs when each fragment experiences a unique successional pattern, being colonized by different species from neighbouring fragments (arroyo-rodríguez et al., 2013) which depends on landscape connectivity and dispersal ability of organisms (mouquet & loreau, 2003). subtractive heterogenization occurs if certain species are more prone to extinction due to new abiotic conditions, different biotic interactions and stochastic events, favouring different species across fragments (socolar et al., 2016). in this case, changes in species groups which represent the role that species play in the ecosystem can also occur due to specialist species extinction and/or generalist species invasion (tabarelli et al., 2012). habitat fragmentation can thus shape community structure across space and time by changing migration and extinction patterns, and local conditions and resources (chase et al., 2020; dornelas et al., 2014; solar et al., 2015). to better conserve and restore impacted communities it is necessary to understand how and why community structuring vary over time (arroyo-rodríguez et al., 2013; chase et al., 2020), but it is still unclear how biotic homogenization and heterogenization contribute to shape biological communities in human-driven fragmented landscapes (arroyo-rodríguez et al., 2020; chase et al., 2020). available studies suggest that metacommunities can suffer both processes in human-altered landscapes (chase et al., 2020), and as a demand for conservation, it is necessary to understand how these processes contribute to community structuring in tropical forests, which are under high anthropogenic pressure. additionally, anthropic pressure can favor non-native species invasion and exclude native species in forests, which first leads to biotic heterogenization, as initially species invades few fragments, but these species spread and can lead to biotic homogenization (mckinney & lockwood, 1999). here we investigated processes driving ant community composition in a fragmented atlantic forest landscape, a hotspot brazilian biome that suffered a severe fragmentation process: there is about 15% of the original cover remaining, almost half of which is located < 100 m from the edge, and most remaining fragments are < 50 ha (ribeiro et al., 2009). we studied ants (hymenoptera: formicidae) because they are highly sensitive to habitat fragmentation (lasmar et al., 2021; vasconcelos et al., 2006), reflect impacts on other animal and plant species (majer et al., 1997), and constitute an important ecological group of insects in tropical forests due to their large biomass. additionally, ants play important roles in several ecological processes such as seed dispersal, invertebrate predation, soil bioturbation and herbivory (ribas et al., 2012). we studied 17 secondary fragments of atlantic forest in southeastern brazil throughout a 15-year interval. we investigated whether ant communities across these forest fragments experienced homogenization or heterogenization (or if neither process occurred), and which underlying mechanism (subtractive or additive) was responsible for such changes. we also evaluated whether generalist ant species are responsible for possible changes in species composition, by investigating whether species proportion of this group is higher in fragments. materials and methods study site we carried out this study in the atlantic forest of southeastern brazil, in the municipality of viçosa, minas gerais state (20°45’s, 42°50’w). according to landowners of the region and previous studies, the region was covered by forests up to the 19th century, but during the 1930’s and 1940’s an accelerated process of fragmentation has begun and lasted until the 1960’s (schmidt et al., 2013; sobrinho & schoereder 2007). most current fragments in the region resulted from natural recovery, and are between 20 and 60 years old, although some are up to 100 years old (ribon et al., 2003). we documented changes in land-use types in our study region to better understand how different land-use cover could influence our results. during the years in which the samples were collected, there was an overall increase in forest cover in the study region (table 1: supplementary material; mapbiomas, 2020). ant sampling we sampled ant species from 17 fragments (figure 1) in three years: 1995, 1996 and 2010 at the same points in all years, and always in the rainy season (from november to march). fragments varied in size from three to 300 hectares (detailed in table 2: supplementary material). we used pitfall traps of 19 cm in diameter and 11 cm high buried at sociobiology 69(3): e8099 (september, 2022) 3 the ground level, containing water and detergent. traps were baited with human feces and cattle carrion, and remained in the field for 48 h. these baits were used because, in addition to ants, samples were also intended to collect other invertebrates. although the use of bait is not common in ant community studies, it is known that at least 60% of total ant species are captured using this method (przybyszewski et al., 2020). furthermore, the same method was repeated in all sampled and years. each sampling point was constituted by a set of two traps, each with one bait type, three meters apart. according to schoereder et al. (2004a), we used a sampling effort proportional to each fragment size: in fragments larger than 93 hectares, 48 traps were placed, totaling 24 sampling points; in fragments between 30 and 61 hectares, 24 traps in 12 sampling points were used; and in fragments between 3 and 9 hectares, 8 traps at 4 sampling points were used. traps were placed at least 20 m from the edge of the fragments, and the distance between each sampling point was at least 30 m. ant species were identified to the smallest possible level and deposited in the ant collection of the laboratório de sistemática e biologia de coleoptera of the universidade federal de viçosa, brazil. to investigate whether different groups of ant species are responsible for possible changes in species composition, we categorized sampled ant species as generalists, forest specialists, open-habitat specialists or unclassified, according to environments in which species are typically found. accordingly, forest specialist species rarely or never occur in savanna, and open-habitat specialists rarely or never occur in forest. generalist species frequently occur in both types of habitats. this classification was only attributed for species where enough information is available. an ant taxonomist (julio m.c. chaul) provided the classifications. statistical analysis to test whether ant communities in the studied fragments experienced a process of biotic homogenization or heterogenization, we first compared species composition similarity across all pairs of fragments from each year separately, using the jaccard similarity index. afterwards we compared the average similarity across years using a glmm (generalized mixed model), with fragment pair identity as a random effect, and the year of sampling as a fixed effect. as we expected larger changes in the mean similarity indices between the years 1995 2010 than between 1995 1996, the years were included in the model as a continuous variable. we used a binomial error distribution because these data are strictly bounded which values are between 0 and 1 (crawley, 2013), and a contrast analysis to check if ant composition similarity differed among years. we also investigated whether the found process (i.e., biotic homogenization or heterogenization process) was driven by species addition or subtraction mechanisms by analyzing changes in ant species richness over time across fragments. changes in ant species composition driven by an increase in ant species richness in fragments suggest an additive mechanism. alternatively, a decrease in ant species richness in each fragment suggests a subtractive mechanism. for that, we built a glmm with poisson error distribution, using fragments as a random effect and the sampling year as a fixed effect. we used a contrast analysis to check for differences among years. we used the proportion of ant species richness from each group (generalists, forest specialists and open-habitat specialists species) as a response variable to test whether changes in ant species richness of these groups could have fig 1. map of sampled atlantic rain forest fragments in viçosa region, minas gerais state, brazil. the red crosses show the location of the 17 fragments sampled in 1995, 1996 and 2010. vanessa s. ribeiro et al. – divergence of ant communities over time across fragments4 caused biotic homogenization or heterogenization, through additive or subtractive mechanisms. the proportion of ant species from each group was obtained by dividing the number of species from each group by the total number of species sampled in each fragment and year. we adjusted a glmm with a binomial error distribution, using proportion of ant species richness from each group as the response variable, forest fragments as a random effect, and year as a fixed effect. we also used a contrast analysis to check for species richness differences among years. we used the r software (r-core team 2021) to perform all analyzes. we used the packages vegan 2.5-6 (oksanen et al., 2019) for species composition, lme4 1.1-26 (bates et al., 2015) to perform glmms, and dharma 0.3.3.0 (hartig, 2020) to perform residual analysis, check model suitability and distribution. results we sampled a total of 132 ant species, distributed in eight subfamilies: dolichoderinae; dorylinae; ectatomminae; formicinae; heteroponerinae; myrmicinae; ponerinae and pseudomyrmicinae (table 3: supplementary material). subfamilies dorylinae and heteroponerinae was the only subfamily that decreased the species number in the year 2010, when compared to 1995 and 1996. dorylinae had only one species, neivamyrmex alfaroi, which was sampled in 1995 and 1996. only two species were from the heteroponerinae: heteroponera dolo, which was sampled in all years, and heteroponera dentinodis, sampled only in 1996. subfamily myrmicinae had the highest number of species in all years, representing 44% of total species. the average similarity of ant species composition among fragments differed over time (χ² [2, n = 17] = 72.036, p < 0.001, figure 2). ant species richness was higher in 2010 than in 1995 and 1996 (χ² [1, n = 17] = 9.13, p = 0.002, figure 3), which did not differ each other. among the species sampled, 28% were classified as generalist, 34% forest specialist, 3% open-habitat specialist, and 35% of the species could not be classified into the three categories. fig 3. average ant species richness across studied fragments, located in southeastern brazilian atlantic rain forest. in 2010, ant species richness was higher than in 1995 and 1996 (p = 0.002). each point represents a fragment sampled in the correspondent year. the black lozenges represent the average and the bars represent the standard error. different letters represent significant differences at alfa = 0.05. fig 2. average similarity of ant species composition across studied fragments, located in southeastern brazilian atlantic rain forest. each point represents a pairwise comparison between fragments using the jaccard similarity index. to compare averages, we used a mixed model considering each forest fragment identity as a random effect (see main text). the black lozenges represent the average, and the bars represent the standard error. different letters represent significant differences at alfa = 0.05. fig 4. average proportion of generalist ant species richness across studied fragments, located in southeastern brazilian atlantic rain forest. in 2010 it was higher than when compared to 1995 and 1996 (p = 0.01). points represent the proportion of generalist ant species from each fragment in each year. the black lozenges represent the average and the bars represent the standard error. different letters represent significant differences at alfa = 0.05. sociobiology 69(3): e8099 (september, 2022) 5 the proportion of generalist species increased over time (χ² [2, n = 17] = 9.0914, p = 0.01, figure 4). however, the proportion of forest specialist species was higher in 1995 compared to 1996 and 2010 (χ² [2, n = 17] = 32.264, p < 0.001, figure 5), while open-habitat specialist species was similar among years (χ² [2, n = 17] = 1.76, p = 0.41, figure 6). discussion here we documented a 15-year long biotic heterogenization of ant communities across a fragmented rain forest landscape, suggesting ant communities experienced an idiosyncratic structuring process. the average ant species richness in fragments increased over time, so this biotic heterogenization probably occurred through an additive mechanism. important implications for conservation biology emerge from this idiosyncrasy, since each fragment may contain a different set of species, including those with key roles in the ecosystem, interactions, and also invasive species (laurance et al., 2007). biotic heterogenization occurred due to an increase of ant species richness in 2010, which was ~20% higher in comparison to 1995. in the same period, the forested area in our study landscape increased by 3% and agricultural area decreased by 4.2% (table s.2: supplementary material). habitat structure can be an environmental filter strongly related to changes in ant richness and composition in forest fragments (castro et al., 2020). the increase of forest cover probably causes a higher habitat availability and quality for ant species in our study region (schmidt et al., 2013; sobrinho et al., 2003), which in turn reduces the extinction risk to forest species, and allows species to establish across fragments. furthermore, forest recovery likely contributed to the biotic heterogenization we observed because it increased the proportion of different habitat (i.e., forest and agriculture) available in the landscape. as ant communities markedly differ across habitat types in our study region (schmidt et al., 2013), some ant species from agriculture can enter forest remnants (thomas, 2020) and alter their species composition (sobrinho et al., 2003). despite higher forest area in 2010, forest specialist ant species richness decreased over time. specialist ant species are more vulnerable to disturbed environments (andersen, 2019), and our results showed that these species were not reestablished in our fragments across the years. contrarily, the number of generalist species increased about 254% in 2010 compared to 1995 – for instance, species usually found in disturbed savannas as ectatomma planidens and linepithema humile (ramos et al., 2003) were sampled only in 2010, suggesting that generalist species and from open areas invaded fragment forests along years, despite the higher forest cover. this result supports recent findings that edge and matrix effects can increase ant species richness in local scales due to an invasion of generalist ant species (lasmar et al., 2021), which are less vulnerable to impacts due to their wider use of resources (andersen, 2019). invasions of ant species from open areas into forests can occur after other anthropogenic disturbances, as due to recurrent forest fires and consequent canopy opening (paolucci et al., 2017). in the studied fragments, one of the explanations for the invasion of generalist species in small fragments is the similarity between fig 6. average proportion of open-habitat specialist ant species richness across studied fragments, located in southeastern brazilian atlantic rain forest. open-habitat specialist species was similar among years in fragments located in southeastern brazilian atlantic rainforest (p = 0.41). points represent the proportion of open-habitat specialist ant from each fragment sampled in each year. the black lozenges represent the average and the bars represent the standard error. different letters represent significant differences at alfa = 0.05. fig 5. average proportion of forest specialist ant species richness across studied fragments, located in southeastern brazilian atlantic rain forest. in 1995 it was higher when compared to 1996 and 2010 in fragments located in southeastern brazilian atlantic rain forest (p < 0.001). points represent the proportion of forest specialist ant species from each fragment sampled in each year. the black lozenges represent the average and the bars represent the standard error. different letters represent significant differences at alfa = 0.05. vanessa s. ribeiro et al. – divergence of ant communities over time across fragments6 these fragments and the surrounding matrix (schoereder et al., 2004b), and here we show that such invasions can contribute to a biotic heterogenization of ant communities. we did not test what were the consequences of additive biotic heterogenization for the ecosystem functions performed by ants in this landscape, although the loss of functions due to changes in ant species composition has been found before (del toro et al., 2012). what we do know is that the environmental filters imposed by habitat fragmentation can change ant taxonomic diversity, but not necessarily the functional diversity (castro et al., 2020). thus, even if biotic heterogenization occurs in the fragments, ecosystem functions promoted by ants can still be maintained, but this is an area for further investigations. our results have important implications for decisions regarding rain forests conservation, since ants are an ecologically dominant group, that play several ecosystem functions, and also bioindicators that can represent changes in other taxonomic groups (andersen & majer, 2004). forest fragments under a natural recovery process can experience idiosyncratic trajectories regarding environmental conditions, human pressure and thus colonization / extinction of plant species (laurance et al., 2007), and our results point to a parallel process for the associated fauna. the knowledge about ant species composition changes in remaining fragments and also about how these can impact ecosystem functions performed by ants are important to understand natural forest recovery, given several functions performed by ants relate to this process, as seed dispersal, herbivory and predation of insect herbivores. in addition, we hope that identifying empirically that fragmentation drives communities divergence leads to further investigations on other types of disturbances and their consequences for biological communities over time. acknowledgments we thank a. c. maradini for her data survey assistance, j. chaul for assisting with ant species identification and habitat classification. c. ribas, l. rattis, t. sobrinho and i. gomes discussed and improved the first draft of the manuscript. two anonymous referees and the editor provided valuable suggestions that improved the manuscript. we thank the coordenação de aperfeiçoamento de pessoal de nível superior – brasil (capes) and fundação de amparo à pesquisa do estado de minas gerais (fapemig) for supporting this study. jhs was supported by a conselho nacional de desenvolvimento científico e tecnológico (cnpq) grant. authors contribution all authors contributed 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(1992). complex interactions in metacommunities, with implications for biodiversity and higher levels of selection. ecology, 73: 1984-2000. doi: 10.2307/1941449. https://doi.org/10.2307/1941449 sociobiology 69(3): e8099 (september, 2022) 9 supplementary material table s1. area size in hectare and percentage of total area (in parentheses) showing changes in land uses in viçosa municipality -mg, brazil, classified in 1995, 1996 and 2010. class / year 1995 1996 2010 forest 6874.023 (22.96%) 6844.791 (22.86%) 7782.602 (25.99%) agriculture 22046.48 (73.64%) 22040.04 (73.61%) 20794.64 (69.45%) unvegetated area 997.3359 (3.33%) 1033.261 (3.45%) 1340.006 (4.48%) water 22.10562 (0.07%) 21.85455 (0.07%) 22.69341 (0.08%) size total 29939.95 (100%) source: mapbiomas table s2. proportional size in hectare of fragments area, number of traps installed and points sampled in viçosa, state of minas gerais, brazil. geographic coordinates fragment size fragment size 1995 fragment size 2010 traps points 20º48’22.2’’s/42º51’10.9’’w > 93 hectares 298.92 350.14 48 24 20º49’31.0’’s/42º55’44.5’’w > 93 hectares 93.37 88.52 48 24 20º45’24.2’’s/42º51’38.8’’w 30 to 61 hectares 60.52 79.57 24 12 20º47’29.5’’s/42º53’26.9’’w 30 to 61 hectares 46.16 51.12 24 12 20º46’17.6’’s/42º52’11.7’’w 30 to 61 hectares 39.21 100.47 24 12 20º47’56.9’’s/42º50’41.3’’w 30 to 61 hectares 30.13 36.90 24 12 20º44’34.0’’s/42º49’53.3’’w 3 to 9 hectares 8.57 9.24 8 4 20º49’17.0’’s/42º53’51.4’’w 3 to 9 hectares 4.05 4.72 8 4 20º48’55.9’’s/42º52’49.9’’w 3 to 9 hectares 3.15 9.98 8 4 20º49’07.3’’s/42º55’58.9’’w 3 to 9 hectares 4.99 4.15 8 4 20º46’36.2’’s/42º50’28.2’’w 3 to 9 hectares 8.00 6.58 8 4 20º43’20.2’’s/42º51’40.7’’w 3 to 9 hectares 7.72 7.19 8 4 20º43’30.9’’s/42º51’42.5’’w 3 to 9 hectares 7.97 5.66 8 4 20º43’20.3’’s/42º49’33.3’’w 3 to 9 hectares 6.91 5.09 8 4 20º42’50.7’’s/42º49’11.6’’w 3 to 9 hectares 5.56 6.44 8 4 20º47’08.8’’s/42º53’33.4’’w 3 to 9 hectares 4.21 6.12 8 4 20º48’52.7’’s/42º51’56.0’’w 3 to 9 hectares 5.77 2.32 8 4 vanessa s. ribeiro et al. – divergence of ant communities over time across fragments10 subfamily / species 1995 1996 2010 dolichoderinae dolichoderus attelaboides fs x x x linepithema humile g x linepithema iniquum fs x x x linepithema leucomelas fs x x linepithema sp.1 x linepithema sp.2 x linepithema sp.5 x x linepithema sp.6 x linepithema sp.7 x linepithema sp.8 x linepithema sp.9 x linepithema sp.10 x dorylinae neivamyrmex alfaroi fs x x ectatomminae ectatomma edentatum g x x x ectatomma permagnum fs x x x ectatomma planidens oh x ectatomma sp.3 x x x ectatomma sp.4 x ectatomma sp.5 x gnamptogenys acuminata fs x gnamptogenys horni fs x x x gnamptogenys mina fs x x x gnamptogenys regularis fs x gnamptogenys striatula g x x x gnamptogenys sp.3 x x gnamptogenys sp.4 x x gnamptogenys sp.8 x gnamptogenys sp.9 x formicinae brachymyrmex depilis g x x brachymyrmex sp.1 g x brachymyrmex sp.2 g x brachymyrmex sp.4 g x brachymyrmex sp.5 g x brachymyrmex sp.6 g x brachymyrmex sp.8 g x formicinae camponotus atriceps g x x x camponotus crassus oh x x camponotus genatus g x x x camponotus melanoticus g x x x camponotus novogranadensis g x x camponotus rufipes g x x x camponotus sp.2 x x camponotus sp.7 x x x camponotus sp.10 x x x nylanderia fulva g x x x nylanderia sp.1 x x x nylanderia sp.2 x heteroponerinae heteroponera dentinodis g x heteroponera dolo fs x x x myrmicinae acanthognathus rudis g x x acromyrmex niger g x x x acromyrmex rugosus g x acromyrmex subterraneus g x x x acromyrmex sp.2 g x acromyrmex sp.3 g x acromyrmex sp.4 g x acromyrmex sp.5 g x acromyrmex sp.7 g x acromyrmex sp.10 g x x acromyrmex sp.15 g x acromyrmex sp.21 g x apterostigma jubatum fs x x x apterostigma sp.3 fs x x x apterostigma sp.4 fs x x x apterostigma sp.7 fs x atta sexdens oh x x x basiceros disciger fs x basiceros sp.1 fs x x x cardiocondyla emeryi oh x x cephalotes atratus fs x x x cephalotes ustus x table s3. ant species sampled in the years of 1995, 1996 and 2010. the letter g represents the species classified as generalists, letters fs as forest specialist and letters oh those species classified as open-habitat specialist. species that do not have any letter were unclassified. subfamily / species 1995 1996 2010 sociobiology 69(3): e8099 (september, 2022) 11 myrmicinae crematogaster distans g x crematogaster nigropilosa g x crematogaster ocuta g x crematogaster sp.1 g x crematogaster sp.5 g x eurhopalothrix sp.1 fs x hylomyrma balzani fs x x x hylomyrma reitteri fs x x x hylomyrma sp.1 x x x megalomyrmex drifti fs x megalomyrmex gnomus fs x megalomyrmex goeldii fs x x x megalomyrmex iheringi fs x x x megalomyrmex incisus fs x x megalomyrmex modestus fs x x megalomyrmex pusillus fs x x megalomyrmex sp.2 x octostruma rugifera fs x x x oxyepoecus plaumanni fs x oxyepoecus reticulatus fs x x procryptocerus convergens fs x procryptocerus marginatus fs x x x procryptocerus sp.2 x rogeria ciliosa fs x rogeria sp.1 x x x rogeria sp.2 x strumigenys apreciatta fs x x trachymyrmex aspersus g x x trachymyrmex oetkeri fs x trachymyrmex sp.1 x x x trachymyrmex sp.2 x x x trachymyrmex sp.4 x trachymyrmex sp.5 x trachymyrmex sp.6 x trachymyrmex sp.7 x trachymyrmex sp.8 x ponerinae hypoponera foreli g x hypoponera sp.1 x x x hypoponera sp.2 x x x hypoponera sp.3 x x hypoponera sp.4 x hypoponera sp.6 x x leptogenys sp.1 fs x x x neoponera lenis fs x x x neoponera magnifica fs x x x neoponera metanotalis fs x x x neoponera venusta fs x neoponera sp.4 x x neoponera sp.6 x neoponera sp.7 x x x neoponera sp.8 x odontomachus chelifer g x x x odontomachus minutus fs x x x odontomachus sp.2 x x pachycondyla harpax fs x x x pachycondyla impressa fs x x pachycondyla striata fs x x x pseudomyrmicinae pseudomyrmex tenuis g x x pseudomyrmex sp.1 x x x pseudomyrmex sp.2 x x x pseudomyrmex sp.3 x table s3. ant species sampled in the years of 1995, 1996 and 2010. the letter g represents the species classified as generalists, letters fs as forest specialist and letters oh those species classified as open-habitat specialist. (continuation) subfamily / species 1995 1996 2010subfamily / species 1995 1996 2010 _hlk106809476 _hlk106809635 _hlk106809976 _hlk106726296 _hlk106869915 _hlk106883699 _hlk106884382 _hlk106884047 _hlk63952304 _hlk106810693 _hlk106811609 move80695823 _hlk106816192 _hlk109056316 _hlk110601015 _hlk110601278 _hlk108794325 _hlk106816258 _hlk106886543 _hlk106816279 _hlk109057517 _hlk110602194 _hlk47515972 _hlk110602427 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.7775sociobiology 69(4): e7775 (december, 2022) introduction as a predominant group of invertebrates in terrestrial ecosystems, ants (hymenoptera: formicidae) are ubiquitous and abundant organisms, both in biomass and in species richness (hölldobler & wilson, 1990). currently, about 14.225 valid species of ants are registered worldwide, comprising 347 genera and 16 subfamilies (antwiki, 2022). studies on formicidae biogeography have been published at the levels of subfamily, genus, or species (mikissa et al., 2016) at local (delabie et al., 2007), regional (delabie et al., 1997; human et al., 1998; resende et al., 2010), or global (mcglynn, 1999) scales. in recent years, great advances have been made in this kind of study, allowing inferring the history of distribution abstract the state of the art of formicidae biogeographic studies using distribution modeling tools was reviewed. we aimed to evaluate how and for what purpose such tools were used in ant studies, as well as detecting modeling methods, algorithms, and variables selected for these studies. we analyzed papers published from 2001 to 2021 and focused on predicting invasion risks, conservation, and potential distribution of species. we also considered the mechanistic and correlative approaches, types of algorithms, and environmental variables. we observed that modeling is first used to predict invasion risks before conservation. the correlative approach was the most used, although it does not consider biotic or physiological aspects as the mechanistic approach does. the most used algorithm was maxent, combining data set of occurrences with climatic variables. nine studies used combinations of algorithms with consensual models. research using modeling has been conducted more and more. however, it remains still incipient, mainly regarding conservation, as the current distribution of most of the formicidae species is not well known. although not frequently used in ant studies, distribution modeling represents an important approach for research in biogeography, ecology, and related areas. certain perspectives could be useful, for example, for studying climatic changes, since possible variations in ant distributions, if anticipated, could suggest or guide further investigations or decision-making in public policies. sociobiology an international journal on social insects priscila s. silva1,2, elmo borges a. koch3, alexandre arnhold2,4, jacques hubert c. delabie2,5 article history edited by evandro nascimento silva, uefs, brazil received 10 february 2022 initial acceptance 19 august 2022 final acceptance 29 october 2022 publication date 28 december 2022 keywords correlative modeling, mechanistic modeling, conservation, invasive species, maxent. corresponding author priscila s. silva laboratório de mirmecologia, centro de pesquisa do cacau (ceplac-cepec) cep: 45600-970 ilhéus-ba, brasil. e-mail: priscilapitth@hotmail.com based on phylogenetic analyzes, such as on myrmicinae (ward et al., 2014). a range of taxonomic studies by biogeographical regions (ladino & feitosa, 2020), new occurrences, and records (dias & lattke, 2019; fernandes & delabie, 2019; franco, et al., 2019), invasive species (chen & adams, 2018), and diversity (koch et al., 2020; silva et al., 2020) can also be found. in addition, modeling distribution studies on formicidae have gained space, under the following names and techniques: potential distribution modeling (murphy & breed, 2007; koch et al., 2018), niche modeling (peterson & nakazawa, 2007), paleodistribution (cristiano et al., 2016), and projections of future scenarios (jung et al., 2017). all of these studies assess the potential distribution of species to infer information of several natures on biodiversity. 1programa de pós-graduação em ecologia e conservação da biodiversidade, universidade estadual de santa cruz (uesc), ilhéus-ba, brazil 2laboratório de mirmecologia, centro de pesquisas do cacau, ceplac, itabuna-ba, brazil 3programa de pós-graduação em ecologia e evolução, universidade estadual de feira de santana (uefs), feira de santana-ba, brazil 4centro de treinamento em ciências agroflorestais, universidade federal do sul da bahia (ufsb), ceplac, itabuna-ba, brazil 5departamento de ciências agrárias e ambientais (dcaa)/ universidade estadual de santa cruz (uesc), ilhéus-ba, brazil review a review of distribution modeling in ant (hymenoptera: formicidae) biogeographic studies priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies2 such biogeographical reconstructions presented a more diverse range of species in tropical climates regions (jenkins, 2003; guénard et al., 2010; moreau & bell, 2013). however, there are still many gaps regarding the information on the geographic distribution of ants, mainly due to the heterogeneous spatial distribution of experts on this important biological group and data collection bias all over the terrestrial biomes. thus, many samplings may have occurred in a given region while other zones have not been duly explored. unexplored regions may host certain species or genera, yet no sampling may have been done, nor data may have been published on them (guénard et al., 2010). thanks to zoological collections, such as the formicidae collection from the cocoa research center (cpdc) (delabie et al., 2020) and internet databases, such as the global biodiversity information facility (gbif – https://www. gbif.org/), antweb (https://www.antweb.org/), and antwiki (https://www.antwiki.org/wiki/welcome_to_antwiki), myrmecologists have access to a variety of information on species occurrences in regions that are incipiently known. such records are important to understand the range limits of the genera (guénard et al., 2010). predictions about potential species occurrences and possible distribution changes caused by different types of impacts, whether anthropic or climatic, have become common in the last 20 years (peterson et al., 2018). however, as more and more distribution data are being entered into biodiversity databases and made freely available on the internet (soberón & peterson, 2004), researchers need to be confident about the correct identification of the species. many records have not been correctly identified or georeferenced (peterson et al., 2011). furthermore, occurrence data collected with inaccurate information may provide incomplete information on their responses to environmental gradients since they may be spatially and environmentally biased (lobo et al., 2007; hortal et al., 2008). the species distribution modeling (sdm) tool uses species occurrence data in addition to abiotic information to estimate the potential distribution of species (correlative modeling) (peterson et al., 2011; 2015). when the information is added to physiological data, it is called mechanistic modeling, which aims to understand, through detailed biophysical modeling approaches, the environmental requirements that make up the niche of a species. this allows the development of a model of the environmental conditions under which the species may exist (kearney & porter, 2009; kearney et al., 2010; peterson et al., 2015). when information about organisms is used together with environmental variables, for example, the total environmental range (set of abiotic conditions with different tolerance rates) is estimated. in this space, a species can survive and reproduce even without ideal biotic conditions (guisan & zimmermann, 2000; rourapascual & suarez, 2008; elith & leathwick, 2009). currently, species distribution modeling (sdm) and ecological niche modeling (enm) (warren, 2012; peterson & soberón, 2012) are among the most productive and rising research branches in ecology (zimmermann et al., 2010), with applications in a variety of other disciplines such as biogeography, evolution, and conservation (guisan & thuiller, 2005). applications are found in historical biogeography studies, such as evolutionary processes, the discovery of unknown species, effects of climate change, disease transmissions, species invasions, and conservation (guisan & thuiller, 2005; peterson et al., 2011). species distribution predictions based on correlative models can help to understand spatial patterns of biological diversity (jiménez-valverde et al., 2008). the presence or absence of a given species in a habitat derives from a range of factors (pulliam, 2000), which means that biogeographic patterns are not just limited by abiotic factors, such as climatic factors, commonly considered in correlative models. these patterns can be shaped by many other elements, such as biotic factors, geographic barriers, anthropogenic effects, stochastic events, and historical factors, among others (pulliam, 2000; soberón, 2007). therefore, the potential distribution can only be discussed as an ideal scenario in which the species distribution is considered in conjunction with the environment, established by favorable abiotic conditions (jiménez-valverde et al., 2008). mechanistic, that is, process-based sdms can be integrated for additional advanced predictions (rougier et al., 2015). for example, the effect of temperature on physiological and demographic processes can be used to test a causal effect of temperature on species distribution (monahan, 2009). some studies have discussed important conceptual and methodological parameters of species distribution models, particularly the need for careful delimitation of the analysis coverage (soberón & peterson, 2005; soberón, 2007; peterson et al., 2011). the bam diagram (biotic, abiotic, movement) considers biotic factors, abiotic factors, and movement factors to delimitate the geographic distributions of species (figure 1). fig 1. the ‘bam diagram’ adapted from soberón and peterson (2005): area g represents the entire region considered, where the species respond to abiotic (a) and biotic (b) and movement/ dispersion (m) factors. the g0 intersection represents the actual distribution area of the species. the g1 intersection represents a region that has both biotic and abiotic conditions suitable for the species, which could potentially be invaded if m conditions change (potential distribution). sociobiology 69(4): e7775 (december, 2022) 3 m corresponds to the area within the dispersal capabilities of the species in question, corresponding to the geographic regions that were accessible to the species within a certain period (peterson et al., 2011). the intersection of a ∩ b ∩ m is the occupied distributional area and is the subset of the accessible region where abiotic and biotic conditions allow species to maintain populations (peterson et al., 2011). the bam diagram allows the researcher to focus on delimiting the area to be analyzed. although one of the main functions of modeling in ecology is to estimate yet undescribed diversity, due to insufficient information, global models of species diversity have seldom focused on insects (guénard et al., 2012), except for species of medical importance (see ahadji-dabla et al., 2020; moo-llanesa et al., 2020). most of the studies investigate relatively well-known groups, such as vertebrates (see freeman et al., 2019) or plants (see lópez tirado et al., 2018). however, studies involving ants have become more popular in recent decades. roura-pascual & suarez (2008) reviewed climate modeling studies, highlighting applications with correlative and mechanistic methodologies, specifically in forecasting studies on invasive ants, emphasizing future scenarios. bertelsmeier et al. (2016) evaluated the mechanisms by which climate changes could favor future ant invasions at the regional and global levels, as well as in biodiversity hotspots. both reviews focused only on studies about invasive ants. in this review, we present qualitative and quantitative approaches to scientific productions related to formicidae biogeography that used geographic distribution modeling as a tool from 2001 to 2021. we synthesized a diagnosis of the formicidae biogeography, its history, and degree of development, as well as the types of modeling and the contributions of these studies to the scientific debate. in addition, we analyzed the contribution of these researches to emerging conservation issues and areas in which further research is necessary. material and methods the search was conducted on the platforms google scholar, gbif, scielo, and portal de periódicos capes, in english, by using the keywords “formicidae” or “ants”, combined with “modeling”, “niche”, “climate change”, “geographic distribution”, “future scenarios”, “paleodistribution”, “potential distribution”, “paleogeography” “paleoclimatology”, and “bioclimatic envelope”. we considered the studies published from 2001 to 2021, to offer a descriptive analysis and to give an overview of the specific research conducted in this area, using categories such as aims of the study, mechanistic and correlative approaches, algorithms, and variables used. thus, we evaluated the use of modeling as a tool in ant biogeography studies, describing its applicability and aiming at projecting possible areas of invasion, occurrence, and conservation. we also described the mechanistic and correlative approaches and identified algorithms and variables. the papers were categorized in five-year periods (20012005, 2006-2010, 2011-2015, 2016-2021), and classified according to the objectives of the studies as per table 1. based on descriptive information, we focused on which aspects and dimensions have been highlighted over the years. results evaluation of the applicability of species distribution modeling as a tool in ant biogeography studies forty-four studies of formicidae published from 2001 to 2021, which used sdm as a tool, were selected. among these studies, 48% assessed the potential for invasive species to invade new areas (figure 2). the invasive ant species most studied were: solenopsis invicta buren, 1972, with projections to oklahoma (leavia & frost, 2004), global expansion fig 2. representativeness of studies (n = 33) using the formicidae modeling tool with a correlative approach. the codes refer to the different types of objectives, presented in table 1. c = conservation, ef = evaluate flaws in the modeling method, dp = global density prediction, pi = predict invasion, nr = niche requirements, pi = infer areas of occurrence/habitat/ potential distribution, sp = identify spatial pattern of species richness, and st = develop predictive model of soil temperature. priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies4 (peterson & nakazawa, 2007), and inferring potential areas in large portions of europe, asia, africa, australia (morrison et al., 2004) and south korea (sung et al., 2018; jung et al., 2021); and linepithema humile (mayr, 1868), with a global prediction (roura-pascoal et al., 2004), new zealand (hartley & lester, 2003; harris & barker, 2007) and iberian peninsula (roura-pascoal et al., 2006; 2009; abril et al., 2009). studies having as the main focus the inference about areas of possible occurrence, predicting habitat or potential distribution unrelated to invasive species, had a frequency of 23%. studies that used modeling as a conservation tool had a frequency of 11% and were limited to endemic species: formica exsecta nylander, 1846, palearctic region (seifert, 2000), atta robusta borgmeier, 1939 exclusively for coastal vegetation (restinga) in southeastern brazil (fowler 1995; teixeira et al., 2003; 2004), lasius balearicus talavera, espadaler and vila 2014, endemic to the balearic islands (spain) (talavera et al., 2014), and dinoponera lucida emery 1901, restricted to a small portion of the atlantic forest in parts of the states of bahia, minas gerais, são paulo, and espírito santo, in brazil (mariano et al., 2008; lenhart, et al., 2013; escarraga et al., 2017). other purposes, shown in fig 2, represented only 3%. methodological aspects – detecting the distribution modeling methods used to predict areas of invasion, occurrence, and conservation mechanistic versus correlative approaches we observed that, since 2006, correlative modeling studies with ants have increased while mechanistic studies have decreased considerably (fig 3). in addition, more recent studies have used correlative modeling to infer potential areas of occurrence (souza & delabie, 2013; cristiano et al., 2016; simões-gomes et al., 2017; koch et al., 2018; senula et al., 2019), and predict invasion (bertelsmeier et al., 2015; jung et al., 2017; sung et al., 2018; byeon et al., 2020). fig 2 presents the frequency of all-purpose correlative modeling studies with formicidae. in general, studies on formicidae aimed at assessing the invasive potential of exotic species (rourapascual et al., 2006; hartley et al., 2006; roura-pascual et al., 2009) used mainly correlative modeling. conservation studies also used correlative modeling, mostly (dáttilo et al., 2012; talavera et al., 2014; campiolo et al., 2015). only one study used the mechanistic approach (maggini et al., 2002). on the other hand, inference studies of occurrence, habitat, and areas of potential distribution (solómon et al., 2008; souza & delabie, 2013; cristiano et al., 2016; sánchez-restrepo et al., 2019) used only the correlative methodology. identification of algorithms twenty-five (i.e., 75.8%) out of 33 correlative approach studies used only a single software/algorithm (table 1). among the studies which opted for consensual models using more than a single algorithm, five of them used more than four software/algorithms (15.2% of the total). the most used software/algorithms/analysis were maxent (used in 15 studies), generalized linear model (glm) (6), genetic algorithm for rule-set prediction (garp) (6), artificial neural networks (ann), classification tree analysis (cta), support vector machines (svms) (used in four studies each), classification trees (ct), climex software, and generalized boosting model (gbm), three studies each (table 1). the other 14 fig 3. frequency of mechanistic and correlative modeling studies on formicidae from 2001 to 2021. software/algorithms/analysis were used in one or two studies. maxent (phillips et al., 2006) is the most frequently used algorithm in studies on formicidae, both to predict areas susceptible to invasion (ward, 2007; steiner et al., 2008; roura-pascual et al., 2009; bertelsmeier et al., 2013; 2015; kumar et al., 2015; coulin et al., 2019), and to infer areas of occurrence/ habitat/ potential distribution (solómon et al., 2008; souza & delabie, 2013; cristiano et al., 2016; simõesgomes et al., 2017; koch et al., 2018; sánchez-restrepo et al., 2019; senula et al., 2019), as well as in two conservation studies (dáttilo et al., 2012; talavera et al., 2014). the garp algorithm (stockwell & peters, 1999) has been used in some studies mainly to assess the risk of the ant l. humile invasion (roura-pascual et al., 2004; 2006; 2009), and was used once in a conservation study (campiolo et al., 2015), and another time to identify species richness patterns (chaladze, 2012). glm was chosen for invasion risk assessment (hartley et al., 2006; abril, et al., 2009; roura-pascual et al., 2009) and conservation studies (maggini et al., 2002; talavera et al., 2014). we consider that the studies classified here as inferences of area occurrence/ habitat/ potential distribution, without a conservative approach or risk of invasion assessment, aimed to fill in the gaps regarding species distribution. most of these sociobiology 69(4): e7775 (december, 2022) 5 y ea r a ut ho r (s ) f in al it y a n n c t c ta c l im e x g a r p g a m g l m m ax en t sv m o th er (s ) to ta l 20 04 l ea vi a & f ro st pr ed ic t i nv as io n (p i) x ( 1) 1 r ou ra -p as cu al e t a l. pr ed ic t i nv as io n (p i) x 1 r ou ra -p as cu al e t a l. pr ed ic t i nv as io n (p i) x 1 20 06 h ar tle y et a l. pr ed ic t i nv as io n (p i) x 1 20 07 h ar ris & b ar ke r pr ed ic t i nv as io n (p i) 1 m ur ph y & b re ed n ic he re qu ire m en ts (n r) 1 pe te rs on e t a l. e va lu at e fla w s in th e m od el in g m et ho d (e f ) x 1 w ar d pr ed ic t i nv as io n (p i) x 1 20 08 so ló m on e t a l. in fe r a re as o f o cc ur re nc e/ ha bi ta t/p ot en tia l d is tri bu tio n (o /h /p d ) x 1 st ei ne r et a l. pr ed ic t i nv as io n (p i) x 1 20 09 r ou ra -p as cu al e t a l. pr ed ic t i nv as io n (p i) x x x x x ( 1) 5 20 11 je nk in s et a l. g lo ba l d en si ty p re di ct io n (d p ) x 1 20 12 c ha la dz e id en tif y sp at ia l p at te rn o f s pe ci es ri ch ne ss (s p) x x ( 1) 2 d át til o et a l. c on se rv at io n (c ) x 1 fi tz ge ra ld e t a l. pr ed ic t i nv as io n (p i) 1 20 13 b er te ls m ei er e t a l. pr ed ic t i nv as io n (p i) x x x x 4 so uz a & d el ab ie in fe r a re as o f o cc ur re nc e/ h ab ita t/ po te nt ia l d is tri bu tio n (o /h /p d ) x 1 20 14 b er te ls m ei er & c ou rc ha m p pr ed ic t i nv as io n (p i) x x x x 4 ta la ve ra e t a l. c on se rv at io n (c ) x x x x x ( 4) 8 20 15 b er te ls m ei er e t a l. pr ed ic t i nv as io n (p i) x x x x x ( 1) 5 c am pi ol o et a l. c on se rv at io n (c ) 1 k um ar e t a l. r ev is io n (r ) x 1 20 16 c ris tia no e t a l. in fe r a re as o f o cc ur re nc e/ h ab ita t/ po te nt ia l d is tri bu tio n (o /h /p d ) x 1 20 17 si m õe sg om es e t a l. in fe r a re as o f o cc ur re nc e/ h ab ita t/ po te nt ia l d is tri bu tio n (o /h /p d ) x x x ( 3) 5 ju ng e t a l. pr ed ic t i nv as io n (p i) x 1 20 18 k oc k et a l. in fe r a re as o f o cc ur re nc e/ h ab ita t/ po te nt ia l d is tri bu tio n (o /h /p d ) x 1 su ng e t a l. pr ed ic t i nv as io n (p i) x x x x x ( 3) 7 20 19 c ou lin e t a l. pr ed ic t i nv as io n (p i) x 1 sá nc he zr es tre po e t a l. in fe r a re as o f o cc ur re nc e/ h ab ita t/ po te nt ia l d is tri bu tio n (o /h /p d ) x 1 se nu la e t a l. in fe r a re as o f o cc ur re nc e/ h ab ita t / p ot en tia l d is tri bu tio n (o /h /p d ) x 1 st rö er e t a l. in ve st ig at e ph yl og eo gr ap hi c pa tte rn s (p p ) x 1 20 20 b ye on e t a l. in fe r a re as o f o cc ur re nc e/ h ab ita t / p ot en tia l d is tri bu tio n (o /h /p d ) x 1 20 21 ju ng e t a l. d ev el op p re di ct iv e m od el o f s oi l t em pe ra tu re (s t ) x 1 to ta l 4 3 3 3 6 3 6 15 4 7 33 t ab le 1 . m ai n an al yz es a nd s of tw ar e us ed a cc or di ng to th e ai m s of c or re la tiv e m od el in g st ud ie s fo r a nt s. t he c od es re pr es en tin g ea ch o bj ec tiv e ar e pr es en te d in p ar en th es es . a rt ifi ci al n eu ra l n et w or ks (a n n ), c la ss ifi ca tio n t re es (c t ), c la ss ifi ca tio n t re e a na ly si s (c t a ), s up po rt v ec to r m ac hi ne s (s v m s) , c l im e x m od el , g en er al iz ed l in ea r m od el (g l m ), g en et ic a lg or ith m fo r r ul ese t p re di ct io n (g a r p) , g en er al iz ed a dd iti ve m od el ( g a m ). s of tw ar e/ an al ys is u se d in a m ax im um o f tw o st ud ie s (c on si de re d in “ o th er (s )” ): b io c l im , b io se c u r e , b io m od 2, b oo st ed r eg re ss io n t re es ( b r t ), d om ai n, f le xi bl e d is cr im in an t a na ly si s (f d a ), g en er al iz ed b oo st in g m od el ( g b m ), i d r is i 32 , m a t l a b , m ul tip le a da pt iv e r eg re ss io n sp lin es (m a r s) , o pe nm od el le r, r an do m f or es t ( r f) , a nd w hy w he re 2 .0 e co lo gi ca l n ic he m od el in g ap pl ic at io n. priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies6 studies used the maxent algorithm. only one of them (byeon et al., 2020) used the climex algorithm, and two generated consensual models from other algorithms, besides maxent, bioclim, domain, generalized linear model (glm), support vector machines (svm), and boosted regression trees (brt) (simões-gomes et al., 2017), and garp (koch et al., 2018). nine studies used more than a single algorithm, combining the different results, such as in talavera et al. (2014) who used eight consensual models (generalized linear model – glm, gbm, generalized additive model – gam, flexible discriminant analysis – fda, multiple adaptive regression splines – mars, classification tree analysis – cta, random forest – rf, maxent). variables and projections of the 44 studies evaluated, 23 used only climate variables (see for example chaladze, 2012; diamond et al., 2012; koch et al., 2018). the remaining (21) combined climatic variables with a set of further information, such as vegetation, topography, and soil temperature (peterson & nakazawa, 2007; roura-pascoal et al., 2009; jenkins et al., 2011; fitzgerald et al., 2012; senula et al., 2019; jung et al., 2021). climate bias, studies of potential distribution, and scenarios for the future were the majority, while 13 studies estimated the current potential distribution. seven studies inferred further climate change. a total of 10 studies merged current and further potential distributions, two merged paleodistribution and current potential distribution, one study examined transitions between past, present, and future, and, finally, one study produced only paleodistribution models, comparing with phylogeography (table 1). discussion evaluation of the applicability of species distribution modeling as a tool in ant biogeography studies modeling aims to infer the best explanation for a data set and to represent it in a precise and compact way, emphasizing probabilities of where the species may or may not be present (guisan & thuiller, 2005). it is a tool that has been widely used in studies that analyze the potential distribution of species, including the evaluation of the impact of global climate change on species distribution, areas susceptible to invasive species, the selection of suitable habitats, and species conservation, besides prioritizing suitable areas for conservation (guisan & zimmermann, 2000; siqueira & peterson, 2003; marini et al., 2009). approximately half of the studies on formicidae that used distribution modeling were conducted with invasive species (ward, 2007; bertelsmeier et al., 2013; jung et al., 2017; sung et al., 2018). ants are considered a group of organisms that settle easily outside their native distribution area due to their small size, large number of individuals, and being colonial (bertelsmeier et al., 2013). thus, anticipating changes in the distribution of invasive species would minimize their impacts. they can be transported by accident, for example, on fruits, ornamental plants, tourism, and trade exchanges, or with agricultural tools (lofgren, et al., 1975; bertelsmeier & courchamp, 2014). a good example is the argentine ant l. humile, the most studied invasive ant with modeling purposes (roura-pascual et al., 2004; 2006; 2009; hartley et al., 2006; harris & barker, 2007; abril, et al., 2009; fitzgerald et al., 2012; bertelsmeier et al., 2015). roura-pascual et al. (2006) compared native and invasive ecological niches of l. humile, while hartley et al. (2006) evaluated the uncertainty in the predictions of bioclimatic range, corroborating the potential distribution with the known distribution of l. humile, pointing out the important role of temperature and precipitation in the establishment of this species. another study evaluated the probable risk of invasion by twelve ant species in new zealand (harris & barker, 2007). these authors deduced that the chances of the temporary establishment of colonies of species such as solenopsis geminata (fabricius, 1804), and anoplolepis gracilipes (smith, 1857) could be ignored, as temperatures in new zealand are lower than they can survive (harris & barker, 2007). other invasive species widely studied were s. invicta 1972 (levia & frost, 2004; bertelsmeier et al., 2015; sung et al., 2018) and a. gracilipes (bertelsmeier & courchamp, 2014; bertelsmeier et al., 2015; jung, et al., 2017). native from south america (vinson & sorenson, 1986), s. invicta was first introduced and spread in the southern part of the usa and the caribbean (morrison et al., 2004), and then was dispersed throughout china, taiwan, australia, and mexico (valles et al., 2015). in addition, large areas in mexico, northern south, and central america, the caribbean islands, part of the mediterranean region, as well as some areas close to the black and caspian seas, are at high risk of invasion by this ant (morrison et al., 2004). there is no consensus on a. gracilipes original native distribution (vásquez-bolaños & wetterer, 2021). it may have originated in asia or africa (holway et al., 2002; wetterer, 2005). it is a species that can propagate in humid tropical areas (wetterer, 2005). as such, it was introduced into regions of africa, including south africa and tanzania; central, and south america, tropical asia, and australia (wetterer, 2005). it is a quarantine pest in the united states and the republic of korea but is not considered as invasive in north america (csurhes & hankamer, 2012). recently, the risk of invasion was analyzed for a. gracilipes and s. invicta (jung et al., 2017; sung et al., 2018, respectively). both studies modeled the potential distribution under current and future scenarios for south korea and found favorable climatic conditions for these invasive species. the introduction of a. gracilipes occurred in south korea through trade routes and s. invicta, although still not found in the country, is a cause of major concern there (jung et al., 2017; sung et al., 2018). countries that are on international trade ways which connect countries from the sociobiology 69(4): e7775 (december, 2022) 7 pacific ocean to the asian continent, such as south korea, are especially at high risk of species invasion (jung et al., 2017). the projection of the potential distribution is then justified to avoid widespread distribution, and to minimize the economic costs of such invasion. a range of terrestrial environments are climatically suitable for invasive ants, especially in biodiversity hotspots (bertelsmeier et al., 2015). one of the modeled species, the fire ant s. geminate, was also recently studied by byeon et al. (2020), who suggested that expected climate changes would decrease the size of the climatically favorable areas for the species. although scarce, studies to infer areas of occurrence or potential distribution are important tools for research on formicidae (solómon et al., 2008; souza & delabie, 2013; senula et al, 2019; byeon et al., 2020). souza and delabie (2013) suggested that the occurrence suitability may be an important and useful parameter in investigating the biogeography of rare ant taxa. furthermore, the applicability of ecological niche models in generating information on the geographic distribution of pests, providing useful tools for integrated management, has been described for both genera atta fabricius, 1804 and acromyrmex mayr, 1865 (sánchezrestrepo et al., 2019). on the other hand, studies that used modeling as a tool for the purpose of conservation (maggini et al., 2002; dáttilo et al., 2012; campiolo et al., 2015) are still incipient. among these few studies, modeling of the potential habitat distribution of f. exsecta was conducted in a conservation area in switzerland (maggini et al., 2002), in order to understand possible reasons for the local distribution of this species, as it was threatened by extinction. using generalized linear models (glm) (guisan et al., 2002), initial local models generated from 160 field samples were extrapolated to the country scale through a geographic information system (gis). the purpose of studies with maps of potential distribution is, in short, to indicate interesting areas for further sampling, possible areas for future colonization, or areas previously occupied, from where the species has disappeared (maggini et al., 2002). thus, adjusting a model with data sampled in a conservation area is an interesting approach, since it allows for meeting the accurate ecological requirements of the species (maggini et al., 2002). dáttilo et al. (2012) modeled the potential distribution of the leaf-cutting ant a. robusta, an endemic species with occurrence records only in restingas in rio de janeiro and espírito santo, brazil (teixeira et al., 2003). thus, with a restricted geographical distribution and climatic variables, the model generated results showing the probability of occurrence (varying from low to high) in regions that do not have occurrence records, such as in the states of paraná and são paulo, and in southern bahia (dáttilo et al., 2012). therefore, the authors suggest that the generated models can be used to choose areas where to direct collection efforts and define priority areas for conservation. this was the first study using modeling tools in brazil to predict the distribution of an ant species. also, the study demonstrated a probable gradient in the probability of occurrence from the coast towards inland, corroborating the endemicity characteristics of a. robusta in restinga (dáttilo et al., 2012). another conservation-focused study associated phylogenetic inference with modeling the distribution of the endemic ant l. balearicus in the spanish mediterranean, estimating current and future potential distribution (talavera et al., 2014). the latter is based on coupled global climate models cgcm2 and cgcm3 created by the canadian center for climate modeling and analysis (cccma) under three varied carbon emission scenarios for the years 2050 and 2080. the study found that the potential of l. balearicus to deal with climate change by varying its climate niche is low, added to the impossibility of dispersion due to its insular situation and altitude isolation, suggesting that l. balearicus is at risk of extinction in the short term and the inclusion of the species in the iucn red list of threatened species. another ant species that was included in the red book of threatened brazilian fauna and that was studied with the use of modeling was d. lucida. in this study, campiolo et al. (2015) used climatic variables to predict past, present, and future areas of suitability. the generated models demonstrated that the earlier suitability areas were larger than the current ones and that those areas would be reduced in the year 2050 due to climate change. modeling is a tool that can help in assessing the distribution of little-known species. however, regarding the applicability, we observed that modeling studies on formicidae have a greater focus on risk assessment in invasive species. the three most species are l. humile, s. invicta, and a. gracilipes. this focus on invasive species is due to the substantial damages caused by these ants to biodiversity, economy, and human health that follow invasions (bertelsmeier et al., 2015). in fact, ants should be better controlled during the early stages of the invasion, when the population is still relatively small and geographically limited (bertelsmeier & courchamp, 2014). thus, we understand the importance of these predictions for assessing areas at risk of invasion, anticipating variations in these distributions, with a view to the conservation of native species in the studied areas. since the financial and environmental costs of a prediction may prove to be wrong for an invasive species, precaution is probably the best policy (harris & barker, 2007). on the other hand, studying ant species with little-known distribution, in addition to species with unknown conservation status is also important, and could benefit from using the modeling tool. methodological aspects – detecting the distribution modeling methods used to predict areas of invasion, occurrence, and conservation. mechanistic versus correlative approaches depending on the purpose of the model, species distribution modeling can be conducted according to two priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies8 approaches: mechanistic or correlative. in the mechanistic approach, the morphological, physiological, and behavioral requirements are obtained experimentally (kearney, 2006), and, then, linked to the environmental variables to estimate the geographic distribution of the species (kearney & porter 2004; 2009). the formulation of conceptual models based on physiological processes is required. their predictions are assessed through the theoretical rigor that cause-and-effect relationships are addressed (guisan & zimmermann, 2000). although it is a promising approach, it requires detailed knowledge about the biological aspects (fitness) of species with the environment (kearney 2006; buckley et al., 2010). following a mechanistic approach, korzukhin et al. (2001) and sutherst and maywald (2005) on a regional scale, and morrison et al. (2004) on a global scale, estimated the expansion of the potential range of the invasive s. invicta, using dynamic models, and the ecophysiological aspect of colony growth, evaluating soil temperature. this species is currently spread over much of the southern united states, besides its natural distribution in south america. studies predicted its expansion to the north (korzukhin et al., 2001; sutherst & maywald, 2005), and large portions of europe, asia, africa, and australia (morrison et al., 2004), as the adaptation of their populations to cooler or drier climates could increase the area of their potential range (morrison et al., 2004). a mechanistic study, which measured air and soil temperature, was conducted with the argentine ant l. humile (hartley & leter, 2003), suggesting locations in new zealand that meet the appropriate conditions for the species. in another mechanistic study, diamond et al. (2012) observed that the vast majority of ant genera are within the region where their heating tolerance is lower. such mechanistic models incorporate relations between environmental conditions and the organisms’ performance (buckley et al., 2010). although there are still problems related to the adequacy of the limits established experimentally in view of the reality of species in nature, the mechanistic approach brings a better understanding of the factors that determine the patterns of species distribution at large spatial scales (deutsch et al., 2008; hofmann & todgham, 2009). models based on a mechanistic approach allow a more direct view of the fundamental ecological niche since they can be developed regardless of access restrictions or biotic environments (peterson et al., 2018). the low number of studies with mechanistic models is possibly because they are more laborious to generate than correlative models. in addition, they require the collection of a lot of physiological data, which may not be available. in contrast, according to the correlative approach, the environmental conditions of the species are estimated by the spatial superposition between occurrences and environmental variables (elith & leathwick, 2009). koch et al. (2018), for example, modeled the potential distribution of gracilidris pombero wild and cuezzo (2006), in order to identify probable areas of occurrences. in south korea, a country that presents a high risk of biotic invasion, since it connects countries in the pacific region with asia for international trade, two potential invasive species, namely a. gracilipes and s. invicta, were studied by applying species distribution models, (jung et al., 2017; sung et al., 2018). most studies on formicidae aiming to predict suitable areas for invasive species (roura-pascual et al., 2006; hartley et al., 2006; roura-pascual et al., 2009) used correlative modeling, considering only abiotic variables and omitting the effects that other species may have on their distribution. the same happens with conservation studies with correlative modeling (dáttilo et al., 2012; talavera et al., 2014; campiolo et al., 2015), in which only one used the mechanistic approach (maggini et al., 2002). on the other hand, all studies of inference of occurrence, habitat, and potential distribution areas (solómon et al., 2008; souza & delabie, 2013; cristiano et al., 2016; sánchez-restrepo et al., 2019) used the correlative methodology. studying l. humile, roura-pascual et al. (2009) calibrated the models differently from previous studies (roura-pascual et al., 2006; hartley et al., 2006), distinguishing native and invaded areas. thus, they focused on invaded area records, using consensual models, which are important in this case, since such records may overestimate or underestimate the potential range of occupancy by the ant. they also suggested that future studies should pay special attention to areas of maximum uncertainty between the different models, aiming to elucidate the determinants of species distribution. bertelsmeier and courchamp (2014) and bertelsmeier et al. (2015) also used correlative modeling to infer the potential distribution of invasive species, combining several kinds of predictions in a single consensus model. correlative modeling is the most used approach among the studies since it does not make use of prior knowledge of the fundamental niche of species (kearney, 2006). in addition, there is a range of occurrence information in databases, such as specieslink, gbif, and antweb, which favors the application of this modeling method for ants (pearson, 2010). even so, when it comes to insects, for most taxonomic groups, the geographic distributions are poorly known and have many gaps, due to the so-called wallacean deficit (bini et al., 2006). thus, in many cases, the choice of method can be restricted due to the lack of verified occurrences. in the correlative approach, the model is empirical. in other words, it does not have the attribution of a causeeffect relationship (guisan & zimmermann, 2000). there are limitations and criticisms regarding this model since physiological variables are not applied and possible biotic interactions are not considered (dormann, 2007; kearney et al., 2010; buckley et al., 2010). therefore, exploring the physiological mechanisms that establish geographic occurrence is not possible as it is in the mechanistic approach. although it presents only a statistical approximation of reality (guisan & zimmermann, 2000), the mechanistic approach contributes to the formulation of new hypotheses about the mechanisms sociobiology 69(4): e7775 (december, 2022) 9 that determine the distribution of species, inferring areas of distribution with a greater (or smaller) degree of environmental suitability (guisan & zimmermann, 2000). identification of algorithms currently, a variety of species distribution modeling techniques are available. good knowledge of the performance of these techniques becomes extremely important to help researchers to select the most appropriate approach for their particular purposes (jiménez-valverde et al., 2008). modeling studies should first test a set of algorithms, regarding their predictive capacity. studies that do not take this first step may use inappropriate algorithms (qiao et al., 2015). the algorithms used by the correlative models aimed to establish non-random relationships between species occurrence data and data on relevant environmental variables. in essence, the methods extrapolate associations between occurrence points and the set of environmental data to identify predicted areas of occurrence using maps (pereira & siqueira, 2007). the choice of an algorithm must be based on the availability of occurrence data (number of records and presence/absence data), as well as based on the study question. when only presence data exists, algorithms such as domain and bioclim can be used (see ward, 2007). when the data available are presence/absence, distribution modeling can be performed using statistical methods (guisan & zimmermann, 2000), such as the glm (jenkis et al., 2011; abril et al., 2019) and the gam (talavera et al., 2014; sung et al., 2018). however, absence data are difficult to verify and do not always reflect the true absence of the species at that location. the absence of the species may be due to poor sampling, unavailable records, low detectability of the method, or the impossibility of dispersing the species to the site, among other factors (peterson et al., 2011). therefore, the absence of data should be used with caution, as they may underestimate the occurrence of the species. the algorithms that stand out in the modeling studies in formicidae are firstly maxent and secondly garp. these algorithms show good results with a low number of occurrence points (wisz et al., 2008). both fall into an intermediate category with respect to occurrence points, as they use presence and pseudo-absence (background) data to generate the sdms (see stockwell & peters, 1999; phillips et al. 2006). maxent (phillips et al., 2006) is based on the principle of maximum entropy. this method generates predictions from incomplete information regarding the target distribution. overall, maxent has outperformed other modeling methods, hence it is more popular than others (elith et al., 2006; wisz et al., 2008). garp, the second most used method, makes use of a genetic algorithm to search for non-random associations between environmental variables and known occurrences, in contrast to the environmental characteristics of the general study area (roura-pascual et al., 2004; peterson & nakazawa, 2007; campiolo et al., 2015). peterson and nakazawa (2007) used garp to model the potential distribution of s. invicta and solenopsis richteri forel 1909. the authors depicted the effects of different environmental data sets on the model quality. however, they emphasized that using garp was only a methodological option and similar results were found in preliminary tests using maxent (phillips et al., 2006). roura-pascual et al. (2009) also used correlative modeling with the selection of consensual areas, but with five different modeling techniques: glm, gam, gbm, garp, and maxent. given the results, a lack of geographic congruence between predictions from different approaches is evident. they are also divergent about the usefulness of group predictions in identifying areas of uncertainty on the potential invasiveness of some species. ward (2007) used three algorithms: domain, bioclim, and maxent, to model the virtual distribution of invasive ants in new zealand. the study found that among the six modeled species, bioclim performance was worse than the other two modeling methods. the consensual areas indicate the environmental areas in which all models offer the conditions allowing the species occurrence. different consensus methods are currently available, such as pca, which focuses on different algorithms or environmental layers (araújo et al., 2006), weighted averages of the results obtained through the accuracy values (auc, tss or kappa) (thuiller et al., 2009), the combination of the resulting maps (diniz-filho et al., 2009), etc. the most used technique is ensemble forecasting, which consists of generating a consensus model from the results of different algorithms or different scenarios (araújo & new, 2007). the understanding of the use of consensual models is based on the criteria that single predictions are not reliable, as well as that the whole models are incomplete at some point, although they carry useful information (araújo et al., 2005; araújo & new, 2007). the practice of combining results should not be an alternative to the traditional approach to building ever more accurate models (araújo & new, 2007). however, combining results from different models still depends on individual predictions, although it may improve the quality of multiple predictions. therefore, if better individual predictions can be reached, a more confident consensus may occur (araújo et al., 2005). currently, there is a wide discussion in the literature regarding the factors that can affect the ability to develop robust predictive models (boria et al., 2016; peterson et al., 2018). two of these factors about ecological niche models used to anticipate possible distribution patterns are climate uncertainty and algorithmic uncertainty. the former concerns the current existing gcms, as they do not capture all future details. the latter covers many comparative studies, which combine results from algorithms with consensual models, to identify the more appropriate projections. however, there are criticisms regarding consensual models since the different priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies10 algorithms evaluate different parameters. thus, the consensus should be designed with a single algorithm, when the aim is to assess the variability after projecting the previsions for different periods with the same parameters (boria et al., 2016). possibly, the development of integrative models, which aggregate climatic, ecological, and evolutionary variables, would allow more accurate inferences about responses and suitability of species to climatic fluctuations. variables and projections although modeling studies can be developed with variables related to climate, such as soil, vegetation, and topography, among others, in recent years significant advances have occurred in methods that seek to estimate changes in the distribution of species considering climate changes (franklin, 2010). these advances also happened in formicidae studies. one of the central demands of modern ecology is to understand how current climate changes will affect species, and efforts have been made to predict and mitigate such effects (araújo et al., 2004; araújo & rahbek, 2006). the growing availability of environmental variables that report to the past (e.g., 6.000, 21.000 years ago), as well as to the future (e.g., in the years 2050, 2070), allows us to perform temporal transfers, and predict spatial responses of organisms to climate change (faleiro et al., 2013). an important research field in ant studies aims at predicting possible distributions for species under altered climatic conditions (peterson et al., 2018). such ‘bioclimatic’ variables are provided from databases such as worldclim (hijmans et al., 2005) and chelsa (karger et al., 2017). these databases provide free access to high spatial resolution global climate data, including climate information for various time periods, ranging from paleoclimatic periods to current or future scenarios. the climatic data result from global circulation models (gcms), which represent scenarios of emission levels, and greenhouse gas trajectories for the climate in the future, the representative concentration pathways (rcps) (van vuuren et al., 2011; aguilar et al., 2015). computational tools that include bioclimatic models have been developed to define the relations (also called correlative models) between data on the confirmed occurrence of species and their spatial variation in different environmental conditions (guisan & thuiller, 2005). these correlative models establish a relationship between species occurrence and climatic variables in space and time in order to redesign the species’ geographic distribution after climatic changes, based on the assumption that the species will stay in equilibrium with the environment (pearson & dawson, 2003; hartley et al., 2010). levia and frost (2004) assessed the climatic suitability for the expansion of s. invicta buren in oklahoma under the current climate and with the duplication of atmospheric co2 using three general circulation models (gcms) (gfdl r30, osu, ukmo). roura-pascual et al. (2004) drew ecological niche models from four general climate model scenarios for the future (horizon 2050), which strongly suggested the potential expansion of l. humile distribution in hot climates. the correlative studies conduct the calibration and evaluation of ecological niche models in the whole current species distribution and, subsequently, the transfer of the model to climatic conditions for the years 2050 2070 (peterson et al., 2018). climate transferability can also occur under scenarios with past climatic conditions, resulting in important information about the history of species distribution. although only a few studies have been conducted on this issue, paleodistribution was estimated for atta spp. (solómon et al., 2008), acromyrmex striatus (roger, 1863) (cristiano et al., 2016), odontomachus meinerti forel, 1905, octostruma spp., and strumigenys spp. (ströer et al., 2019). these correlative studies used maxent and, all of them, integrated modeling and phylogeography. solómon et al. (2008) used the last glacial maximum (lgm) to test their hypotheses about the biogeography of speciation in the amazon basin, suggesting that marine incursions into the miocene or climatic changes during the pleistocene shaped the population structure observed today in the three species evaluated. a pioneer paleoclimatic study of acromyrmex spp. was conducted by bigarella et al. (1975), who speculated about the paleoenvironmental conditions that characterized the brazilian pleistocene. recently, in their assessment of the paleodistribution of a. striatus to the last interglacial (lig) and the last glacial maximum (lgm), cristiano et al. (2016) found that, in general, the past potential distribution included the current potential distribution of the species, showing constancy over time. ströer et al. (2019) transferred the calibrated models to lgm and lig. their results support the traditional north/south division of the brazilian atlantic forest, in addition to substantial differences between species in the location of genetic divisions and patterns of genetic variation within areas. studies have shown that temperature and precipitation are the two factors that most influence ant diversity patterns (kaspari et al., 2003; dunn et al., 2009; sánchez-restrepo et al., 2019), and are relevant to explain the models. climatic variables were also used for mechanistic studies. annual maximum temperature, seasonality, and aridity were the strongest predictors in the analysis of the warming tolerance of ant assemblages (ctmax) (diamond et al., 2012). still at the assemblage level, predicting potential changes in their relative abundance, a trait-based community selection model (cats), was used to assess the relationship between temperature and uv-b (bishop et al., 2019). this last study suggests that many more species will be present in higher elevation sites in the future and highlights the importance of environmental analyzes mediated by characteristics such as body color and size, as these can have consequences on thermoregulation and protection (see bishop et al., 2016; 2019). tropical organisms are more vulnerable to climate warming than temperate ones, especially when other factors, such as phylogenetic history and ecological characteristics, are accounted for (diamond et al., 2012). sociobiology 69(4): e7775 (december, 2022) 11 some studies have suggested that the climate change effect can overcome habitat loss as the greatest threat to biodiversity (pearson & dawson, 2003; lorenzen et al., 2011). although the risk of invasion stems from biotic and abiotic factors, the climate seems to be primarily responsible for determining the distribution of ants on a global scale (sanders et al., 2007; jenkins et al., 2011, roura-pascual et al., 2011). ants are ectothermic organisms, sensitive to temperature, and humidity, requiring adequate climatic conditions for their establishment (diamond et al., 2012; bertelsmeier and courchamp, 2014). the argentine ant distribution, for example, seems to be influenced mainly by altitude, average temperature, and precipitation (roura-pascoa et al., 2009). for harris and becker (2007), the average annual temperature and precipitation would be sufficient factors to highlight invasion risks, suggesting that the chances of a successful establishment would be reduced in cases in which these parameters were close to their limits. however, the lack of data related to human beings in fine resolution prevents the approach of anthropogenic influences, which is perhaps a better indicator of the establishment and spreading of the argentine ant in some areas than the climatic features (rourapascual et al. 2006). the non-climatic variables used were mainly topography, soil temperature, vegetation, and soil inclination angle. as an example, solar radiation is considered one of the most important f. exsecta requirements (maggini et al., 2002). in this case, terrain slope can be used as a surrogate to give a good idea of solar radiation, as this variable is challenging to be measured accurately. models based on the vegetation index (ndvi) can predict wider potential distributions than models that include only topographic information (roura-pascual et al., 2006). the addition of non-climatic data sets, such as soil characteristics (soil temperature, see jung et al., 2021), landscape configuration, and land use/cover, would likely refine predictions considerably (peterson & nakazawa, 2007). the role that climate change has played in species diversification must be then assessed, but other mechanisms possibly synergetic must also be considered (solómon et al., 2008). conclusion a summary of the current panorama of species distribution modeling in formicidae is represented in figure 4. most studies on formicidae focused on invasive species, and how climate change can act on their distribution and occurrence. the correlative models were the most used to estimate changes in their potential ranges, also using different global warming scenarios. this useful tool makes these studies necessary for investigations aiming to mitigate the effect of invasive species on biodiversity. however, we suggest further studies be conducted, especially for conservation purposes, since the distribution of many ant species is incipiently known, as well as their conservation status. modeling is a tool that can be used in biological management and conservation strategies. however, it does not substitute the need for original field records, since they guarantee the construction of more robust, predictive models and their respective validation. in addition, in the future, fig 4. conceptual structure of how the distribution modeling tool can be used in different approaches on formicidae. the dotted lines indicate the different applications; thin arrows indicate approach with a few studies; the thick lines indicate approaches with a range of studies conducted. blue arrows indicate correlative models and green arrows mechanistic models. priscila s. silva, elmo b. a. koch, alexandre arnhold, jacques h. c. delabie – modeling in ant biogeographic studies12 the risk of extinction due to loss of habitat, likewise, may be inferred based on the area of the potential occurrence of species, as long as maps of native vegetation remnants are available. in this case, an interesting approach to be studied would be the approximation of real occurrence areas of species from those of potential occurrence. acknowledgment we are very grateful to the following institutions and funding agencies: fundação de amparo à pesquisa do estado da bahia (fapesb), for pss granting a scholarship, conselho nacional de desenvolvimento científico e tecnológico (cnpq), for jhcd research scholarship, and universidade federal do sul da bahia (ufsb), for financial support. authors’ contributions pss: conceptualization, methodology, conceptualization, methodology, writing and editing the manuscript jhcd: conceptualization, methodology, writing and editing the manuscript aa: conceptualization, methodology, writing and editing the manuscript ebak: formal analysis, writing and editing the manuscript. references abril, s. roura-pascual, n., oliveras, j. & gomez, c. 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(2010). new trends in species distribution modeling. ecography, 33: 985-989. doi: 10.1111/j.1600-0587. 2010.06953.x https://doi.org/10.1111/j.1472-4642.2008.00482.x doi: 10.13102/sociobiology.v63i3.778sociobiology 63(3): 976-981 (september, 2016) open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology issn: 0361-6525 nesting stingless bees in urban areas: a reevaluation after eight years introduction stingless bees (apidae, meliponini) are eusocial species with populous colonies varying from hundreds to thousands of individuals (michener, 1974; nogueira-neto, 1997). brazil contains roughly 244 valid species and 89 undescribed forms across 29 genera (pedro, 2014). as the majority of species feed on nectar and pollen, they play a key role in the pollination of many native plants and cultivated plants from tropical environments, and together represent one of the largest groups of visitors to flowering plants in the tropics. (kerr et al., 1996; heard, 1999; cruz et al., 2005). stingless bees can be found in urban environments if favorable conditions for survival exist. some stingless bees, such as nannotrigona testaceicornis (lepeletier) and tetragonisca angustula (latreille), adapt very well to urban conditions and are common in southeastern brazilian cities (pirani & cortopassi-laurino, 1994; batista et al., 2003; abstract studies of nesting ecology have proven to be extremely important for stingless bee conservation. these studies have rarely been conducted in urban landscapes, and even fewer have compared species diversity and abundances over time. we surveyed native stingless bee nests at the federal university of juiz de fora campus in minas gerais, southeastern brazil, from may 2008 to april 2009. we recorded the number of nests, nest height, species diversity, and nest substrate type (i.e., natural or artificial). we compared our results to those of a similar survey carried out in the same location eight years prior (2000/2001) in order to evaluate how urban expansion on campus has influenced the meliponini bee community. stingless bee abundance and richness were greater in the second survey. the use of natural substrates decreased, while the use of artificial substrates increased. this suggests that the increase in man-made structures on the ufjf campus has provided favorable sites for establishment of some stingless bee species. sociobiology an international journal on social insects km vieira, p netto, dlas amaral, ss mendes, lc castro, f prezoto article history edited by candida m. l. aguiar, uefs, brazil received 11 march 2015 initial acceptance 21 september 2015 final acceptance 01 july 2016 publication date 25 october 2016 keywords urban expansion, hollow trees, urban sprawl, richness, diversity. corresponding author fabio prezoto universidade federal de juiz de fora labec campus universitário rua josé lourenço kelmer, s/n são pedro cep: 36036-900 juiz de foramg, brasil e-mail: fabio.prezoto@ufjf.edu.br souza et al., 2005; zanette et al., 2005). some species are even found nesting in rock crevices and man-made structures (velthuis, 1997). their ability to succeed in urban environments depends on several factors, including food availability (e.g., nectar, pollen, and water), nesting site availability (e.g., mud, oil, resin), and intrinsic factors related to the geographic distribution of each species (batista et al., 2003; samejima et al., 2004; souza et al., 2005; antonini et al., 2012). stingless bees usually build cryptic nests that may remain at the same site for many years. however, sometimes they build exposed nests (as in trigona spinipes) (almeida & laroca, 1988) or occupy pre-existing cavities such as tree hollows, holes in the ground, or abandoned termite, ant, or bird nests (nogueiraneto, 1997, silveira et al., 2002). loss or alteration of nesting substrate may result in increased populations of some species and local extinctions of others, where the responses are driven by species differences in foraging and nesting habits (cane, 2001; antonini et al., 2012). universidade federal de juiz de fora, juiz de foramg, brazil research article bees sociobiology 63(3): 976-981 (september, 2016) 977 studies in urban areas are critical for gaining ecological information (e.g., species diversity, resource interactions, nest density, among others) to inform strategies for species management and conservation. a survey of stingless bee nests was conducted in 2000/2001 on the campus of the federal university of juiz de fora (sousa et al., 2002). the present study assessed variation in stingless bee nesting habits eight years after the initial survey, and addressed the following questions: 1) how does stingless bee diversity and abundance change in urban environments after area expansion? and 2) how do these variables correlate with nesting substrate type and availability? material and methods the study took place at the federal university of juiz de fora (ufjf) campus, juiz de fora, minas gerais, southeastern brazil (21°46’s and 43°21’w, 800 m a.s.l.). the ufjf campus has a total area of 123.41 ha, with urban infrastructure such as buildings, paved streets, and parking lots (infrastructure office database at ufjfproinfra/ufjf, personal communication, may, 2010). the street edges and parking lots are surrounded by atlantic forest fragments, as well as pinus spp. and eucalyptus spp., and some ornamental woody species (moreira & carvalho, 2013). regional climate is classified as tropical highland (cwa) according to the köppen system, with a warm, rainy season from october to march and a cold, dry season from april to september. nest searches were carried out once per week from march to april 2008, always between 9am and 12pm (total search time = 120 hours). five researchers carefully checked all buildings and adjacent vegetation (gardens and flower beds only) for active colonies. once per month from may 2008 to april 2009 (9am to 2pm), we searched for new colonies and monitored preexisting colonies (300 h total search time). we recorded nest gps coordinates, as well as height from the ground and substrate used. the substrates were categorized as artificial (openings and cracks in bricks, stones, or concrete) or natural (live trees, abandoned wasp nests). five bees were collected from each nest for species identification. voucher specimes were deposited in the laboratory of behavioral ecology and bioacoustics, ufjf. we used the shannon diversity index (h`) (ludwig & reynolds, 1988) to characterize changes in stingless bee community diversity between the 2000/2001 study (sousa et al., 2002) and our data (2008/2009). this index is particularly sensitive to the number of rare species in a community. we then used the pielou evenness index (pielou, 1966), to analyze uniformity in abundance among species. we compared those indices using a hutcheson t-test (1970) in bioestat 5.3. species accumulation curves were constructed using estimates 9.1.0 software (colwell, 2005) to verify that the sampling effort in both surveys was sufficient to have sampled most species that were detectable using the adopted methodology. data on the extent of urban construction vs. natural areas between surveys were provided by the proinfra/ufjf. the ufjf campus was divided into ten main areas for statistical analyses. the spatial distributions of the most common species in the study were calculated using a dispersion index: di = ϭ/μ (clapham, 1936), where “ϭ” is the sample variance and “μ” is the sample mean. a dispersion index value lower than one indicates nests that were distributed more or less evenly, a value equal to one indicates random distribution, and a value greater than one indicates aggregated distribution. results and discussion we recorded 69 stingless bee colonies belonging to seven genera, with seven species (table 1). nest density was 1.92 nests/ha. the shannon and pielou index scores were 0.628 and 0.74, respectively. species abundances were ordered as follows: n. testaceicornis (50.72% of all nests); t. angustula (17.4%); trigona spinipes (fabricius) (13.4%); partamona helleri (friese) (10.15%); scaptotrigona sp (4.34%); tetragona clavipes (fabricius) (2.9%); and melipona quadrifasciata (lepeletier) (a single nest, 1.45%) (table 1). in another study by kerr et al. (1996), n. testaceicornis and t. angustula were also found to be dominant in urban environments, and the authors suggested that these species typically make nests in pre-existing cavities in human structures, soil, or hollow trees. the ufjf campus has many buildings with wall crevices that are suitable for use as nesting sites. artificial substrate (n = 50) was used more frequently than natural substrate (n = 19), however the number of species using natural substrate was higher (7 species in natural vs. 3 in artificial) (table 1). the higher frequency of nests in artificial substrates may be due to greater availability, as the urban area on campus is approximately 38.31 times larger than the natural area (e.g., gardens, flower beds, forest fragments). further, the dominant species in this study (n. testaceicornis and t. angustula) have small colonies which allows them to occupy smaller spaces, perhaps allowing them to exploit the high availability of artificial cavities (lindauer & kerr, 1960; taura & laroca, 1991). n. testaceicornis and t. angustula nests were the most abundant in the former and current surveys (table 3), and artificial substrate was used more commonly in both studies. in the 2000/2001 survey, n. testaceicornis utilized artificial substrates for 88.65% of its nests; in the current survey that proportion increased to 94%. in the current study, t. angustula placed 92% of nests on buildings. although they also exhibited a preference for buildings in the previous survey (37.2%), they also used a variety of natural substrates at that time including trees, termite nests, and ground cavities. scaptotrigona sp., t. clavipes, and m. quadrifasciata nested exclusively in live trees. these species are dependent on tree hollows to nest, so the quantity of this substrate type is a limiting factor for these populations (oliveira et al., 1995; km vieira p netto – nesting stingless bees in urban areas 978 silveira et al., 2002; antonini & martins, 2003; werneck & faria-mucci, 2014). we conclude that the presence of these species in urban environments (as in the current study) is only possible when adequate vegetation exists where they can construct nests. majority of partamona species nest above ground in natural environments, where they often build nests in abandoned termite nests (camargo, 1980; nogueira-neto, 1997). in this study p. helleri nests were found in artificial substrate, and one colony was found in an abandoned wasp nest. the occupation of abandoned nests is a simple and efficient method because it is less costly in terms of time expenditure and effort (pinto, 2005). nest height in natural substrate was more variable than in artificial substrate. nests in natural substrate were frequently found at heights up to 14m, where 47.36% belonged to t. spinipes. souza et al. (2005) and aidar et al. (2013) also found a preference for high nest placement in this species, and wille and michener (1973) noted that nests of this species are found a minimum of four meters above the ground. nests in artificial substrates were found within 2m above ground and height was less variable, probably because most nests (88%) were from only two species, n. testaceicornis and t. angustula. these species made use of low-lying building features such as stairs, windows, and cracks in the walls as nesting substrates. the spatial distribution index (di) indicated the most abundant species n. testaceicornis and t. angustula had an aggregated distribution, with di values of 1.2 and 2.62, respectively. t. spinipes nests were regularly distributed (di<= 1 (0.84)). similar distribution patterns for this species were also found pirani and cortopassi-laurino, (1994) in another brazilian urban area. hubbel and johnson (1977) argue that the spacing between nests is mediated by aggressive encounters between colonies competing for new nesting sites. our results generally agree with this hypothesis because nests were closer for n. testaceicornis and t. angustula, which are smaller and less aggressive species. the spatial distribution of nests is also correlated with the distribution of nesting substrates, and species characteristics such as the typical distance searched to found a new nest (hubbel & johnson, 1977; serra et al., 2009). n. testaceicornis and t. angustula preferred nesting in artificial substrate such as cracks in the walls. to take advantage of the availability of such sites, their nests were necessarily spatially close. p. helleri also preferred artificial substrates (86% of nests) but is a more aggressive species that defends its territory during foraging. this probably contributed to the greater distances found between their nests. the di value for this species was only slightly greater than 1, indicating a distribution pattern close to even distributed (not aggregated). it was not possible to determine distribution patterns of other species due to the low sample numbers. the ufjf campus had a low nest density (0.52 nests/ha) compared to other surveys in urban areas (adair et al., 2013, 2.17 nests/ha; taura & laroca, 1991, 4.91 nests/ha) (table 2). these studies found most nests in trees, however in the present study many areas with trees were not accessible for searching. this may explain the low density of tree nests found. despite having lower nest density, we had higher species richness than other similar studies (see adair et al., 2013, taura & laroca, 1991, souza et al., 2005). this may have happened because although the campus is an urban site, it contains trees for the species that rely upon them (t. clavipes, t. spinipes, m. quadrifasciata) in addition to artificial substrates primarily used by n. testaceicornis, t. angustula, and p. helleri. nest abundance increased from 46 to 69 between the 2000/2001 (sousa et al., 2002) and 2008/2009 (the current survey) (50% increased), and species richness increased by 75% (from 4 to 7 species). diversity (e’) and evenness (j’) were significantly higher in the current study (table 3). all species found in both surveys (table 3) were more abundant in the 2008-2009 survey. t. clavipes, m. quadrifasciata, and scaptotrigona sp. were only found in the 2008/2009 survey (table 3). rarefaction curve showed asymptotic tendency, which indicates that the sampling effort was sufficient to compare species numbers (figure 1). substrate species observed n relative frequency (%) height (m) artificial natural melipona quadrifasciata 01 1.45 03 0 01 (100%) nannotrigona testaceicornis 35 50.72 1.11 (± 1.66) 33 (94%) 02 (6%) tetragonisca angustula 12 17.4 2.88 (± 1.77) 11 (92%) 01 (8%) trigona spinipes 09 13.04 11.86 (± 4.68) 0 09 (100%) partamona helleri 07 10.15 4.97 (± 1.47) 6 (86%) 01 (14%) scaptotrigona sp. 03 4.34 0.82 (± 1.19) 0 03 (100%) tetragona clavipes 02 2.9 1.97 (± 1.16) 0 02 (100%) total 69 100 table 1: number of nests (n), relative frequency, height (m) from the ground to the nest entrance, and substrates used for nesting by stingless bees in the campus of the federal university of juiz de fora, southeastern brazil. sociobiology 63(3): 976-981 (september, 2016) 979 eight years after the study by sousa et al. (2002) we found an increase of 7.94% (8,458.65 m²) in the constructed area of the ufjf campus and a 7.36% (7,840.76 m²) decrease in natural area. although there are some differences in sampling effort between the two studies (see materials and methods), the 2008/2009 survey showed a significant increase in species richness and nest abundance. martins et al. (2013) assessed the effects of urbanization in the past 40 years in an area of natural vegetation surrounded by urban or agricultural areas and found decreased bee diversity, including the loss of some species. the primarily occurred because intense urbanization deteriorated the bee foraging matrix. taura and laroca (2001) grouped data from surveys conducted in 1975, 1986/1987, and 1992/1993 in a green area located in downtown curitiba, paraná, brazil. they concluded that the urban sprawl was linked to pollution and tree removal, which led to a significant reduction in bee species richness and abundance. contrary to this expectation, urban expansion on the ufjf campus did not negatively affect stingless bee nest abundance or species richness. this may be due to the increase in nesting site availability for species that easily adapt to urban environments and that utilize artificial substrate, such as t. angustula, n. testaceicornis and p. helleri. further, some species that exclusively use trees were able to do so despite expansion of the urban area of the ufjf campus. we conclude that species preferences for natural or artificial substrate were relatively stable over time. species that easily adapt to changing environments and with flexibility in use of nesting substrate (t. angustula, n. testaceicornis and p. helleri) were not jeopardized by urban expansion. tree-nesting species exclusive to the 2008/2009 survey (scaptotrigona sp., t. clavipes, and m. quadrifasciata) were able to find habitat despite the great extent of urban area on campus. hence, maintenance of natural areas is important for population growth and conservation of stingless bee species on this urban area. study study area size of sampled area (ha) n number of nests/ha richness present study juiz de fora/mg 132.6 69 0.52 07 aidar et al. (2013) uberlândia/mg 23 50 2.17 07 souza et al. (2005) salvador/ba 57 94 1.64 05 taura & laroca (1991) curitiba/pr 5.7 28 4.91 05 table 2. comparison of nesting attributes of stingless bees in the campus of the federal university of juiz de fora and other studies in urban landscapes. fig 1. rarefaction curve and standard deviation of the estimated richness in the 2008/2009 study in the campus of the federal university of juiz de fora, southeastern brazil. attributes 2000/2001 2008/2009 total number of colonies nannotrigona testaceicornis nests tetragonisca angustula nests trigona spinipes nests partamona helleri nests scaptotrigona sp. nests tetragona clavipes nests melipona quadrifasciata nests total density total richness number of nests in artificial substrates number of nests in natural substrates constructed area in the campus natural area in the campus diversity index (h’) 46 63.93% 24.46% 6.74% 4.87% 0 0 0 3.84 nests/ha 4 27 19 11.6 ha 0.35 ha 0.42 69 50.72% 17.4% 13.04% 10.15% 4.34% 2.9% 1.45% 5.37 nests/ha 7 50 8 12.52 ha 0.32 ha 0.62 evenness index (j’) 0.69 0.74 diversity in 2001/2002 x diversity in 2008/2009 t= 9.1062; df=11; p <0.0001 abundance in 2001/2002 x abundance in 2008/2009 t= 5.8033; df=22; p <0.0001 (t): hutcheson t test; (df): degrees of freedom table 3. comparison of nesting attributes of stingless bees in the campus of the federal university of juiz de fora, southeastern brazil, between 2000/2001 and 2008/2009. acknowledgements we would like to thank cnpq for the financial support, dr. georgina maria de faria mucci for species identification and erick esteves for collaboration in the components of field and laboratory. km vieira p netto – nesting stingless bees in urban areas 980 references aidar, i. f., santos, a. o. r., bartelli, b. f., martins, g. a., nogueira-ferreira, f. h. 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(2005). effects of urbanization on neotropical wasp and bee assemblages in a brazilian metropolis. landscape and urban planning, 71, 105-12. doi: 10.1016/j.landurbplan.2004.02.003 http://dx.doi.org/10.5061/dryad.k54hk http://dx.doi.org/10.5380/abpr.v20i0.764 b03 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.8261sociobiology 69(3): e8261 (september, 2022) introduction rainforest fragmentation is been a prominent topic of discussion between members of the scientific community for years (skole & tucker, 1993; laurance et al., 200a; vedovato et al., 2016). in general, historic forest fragmentation has occurred due to the subdivision of large areas of continuous abstract rainforest fragmentation drastically affects biodiversity and species composition, mainly due to habitat loss. several studies have already shown the effects of forest fragmentation on plant and ant communities. to date, however, there is limited empirical knowledge of how forest fragmentation affects ant-plant interaction in networks. we investigated the effects of the configuration of rainforest fragments on the structure of ant-plant interaction networks mediated by extrafloral nectaries (efns). we carried out this study in ten forest fragments, ranging in size from approximately 5 to 3,000 ha, located in the brazilian amazon. in each fragment we established a plot of 6,250 m2, in which all ant-plant interactions were recorded, and calculated the following network descriptors: number of interactions, network size, network specialization, diversity of interactions, and nestedness. we used four explanatory variables to investigate the effects of forest fragmentation on these network descriptors: three metrics of the configuration of fragments (i.e., fragment area, edge irregularity, and connectivity) and the forest structure within each fragment, represented by canopy cover. we did not detect any effect of the explanatory variables on the network descriptors. the structural stability of the networks sampled in forest fragments with different configurations is possibly related to the observed constancy of ant species in the central core of highly interacting species. our results corroborate other studies highlighting the structural stability of these facultative ant-plant networks mediated by efns in different spatial and temporal gradients. nonetheless, the low constancy of plant species in the generalist core should be understood as a warning, mainly because the functionality of this protective mutualism (i.e., food secretions in exchange for protection against herbivory) remains unknown. sociobiology an international journal on social insects patricia n. miranda1,2, josé eduardo l.s. ribeiro2, erick j. corro3,4, izaias brasil5, jacques h.c. delabie6,7, wesley dáttilo3 article history edited by kleber del-claro, ufu, brazil received 24 may 2022 initial acceptance 25 may 2022 final acceptance 08 august 2022 publication date 29 september 2022 keywords community ecology; ecological networks; landscape ecology; plant-animal interactions; tropical rainforest. corresponding authors patricia nakayama miranda instituto federal de educação, ciência e tecnologia do acre avenida brasil, 920, xavier maia cep: 69903-068, rio branco-ac, brasil. e-mail: patricia.miranda@ifac.edu.br wesley dáttilo red de ecoetología, instituto de ecología ac carretera antigua a coatepec 351, el haya, xalapa, veracruz, c.p.: 91070, mexico. e-mail: wesley.dattilo@inecol.mx forest generating an altered landscape composed of forest remnants with different size, shape, and connectivity level (klingbeil & willig, 2009). this process is usually accompanied by habitat loss which involves the outright removal of habitat patches (bartlett et al., 2016), thus making forest fragmentation one of the main drivers of biodiversity loss (newbold et al., 2015). in the brazilian amazon, productive 1 instituto federal de educação, ciência e tecnologia do acre, campus rio branco, rio branco-ac, brazil 2 centro de ciências biológicas, universidade estadual de londrina, londrina-pr, brazil 3 red de ecoetología, instituto de ecología ac, xalapa, veracruz, mexico 4 facultad de ciencias biológicas y agropecuarias, universidad veracruzana, peñuela, veracruz, mexico 5 programa de pós-graduação em biodiversidade e biotecnologia da rede bionorte, universidade federal do acre, rio branco-ac, brazil 6 comissão executiva do plano da lavoura cacaueira, centro de pesquisas do cacau, laboratório de mirmecologia, ilhéus-ba, brazil 7 universidade estadual de santa cruz, ilhéus-ba, brazil research article ants structural stability of ant-plant mutualistic networks mediated by extrafloral nectaries: looking at the effects of forest fragmentation in the brazilian amazon mailto:patricia.miranda@ifac.edu.br patricia n. miranda et al. – ant-plant networks in fragmented landscapes2 activities such as logging, cattle ranching, and small-scale farming are the main types of land use responsible for this process of landscape change (laurance et al., 2001a; laurance et al., 2011). changes in landscape configuration (i.e., the spatial arrangement of habitat in a landscape), such as size, isolation and edge density (haan et al., 2020), can lead to changes in plant and animal communities (fischer & lindenmayer, 2007; laurance et al., 2011). for instance, forest area reduction may directly affect the rate of local species losses, with small forest remnants tending to lose species more quickly (stouffer et al., 2008), due to their inability to support viable populations of certain groups (didham et al., 1998). connectivity reduction between forest fragments and/or continuous forests also tends to negatively affect the maintenance of some populations, since it changes resource availability spatially and, in consequence, the foraging behavior or pollination success of these populations (haddad et al., 1999; martensen et al., 2008; hadley & betts, 2011). finally, the shape of forest fragments might also cause disruptions in community structure (desouza et al., 2001). for instance, more irregular forest fragments tend to present a higher proportion of edge in relation to the total area of the forest remnant, which can result in high proportions of pioneer species in regenerating and light demanding (hill et al., 2003). ants are frequently studied in forest fragments (vasconcelos et al., 2006; leal et al., 2012). in general, the population density, number and diversity of ant species decline with reductions in the area of forest fragments (bruhl et al., 2003; vasconcelos et al., 2006; hill et al., 2011). the ant species composition may also change due to habitat loss and fragmentation (vasconcelos et al., 2006; leal et al., 2012; ahuatzin et al., 2019), mainly due to local extinctions, or generalist or even invasive species colonization (i.e., biotic homogenization) (carvalho & vasconcelos, 1999; bruhl et al., 2003; holwaya & suarez, 2006; solar et al., 2015). however, the species richness and species composition do not seem to vary with shape complexity of fragments (sobrinho & schoereder, 2007). many plant species, mainly in the tropics, secrete a liquid rich in carbohydrates and amino acids through specialized and non-floral glands called as a whole extrafloral nectaries (efns), which attracts different ant species (koptur et al., 1998; marques et al., 2015). in exchange for the food provided by the plant, some ants can protect their host plants against herbivores (rico-gray & oliveira, 2007). at the community level, these mutualistic associations are usually evaluated using a network approach, in which different ant and plant species are depicted as nodes and their interactions as links (dáttilo et al., 2013a). this network approach has provided important information about the organization of these antplant interactions (lange et al., 2013; dáttilo et al., 2013a, b; dáttilo et al., 2014a). for instance, some studies have recently shown that ant-plant networks are highly nested, indicating that species with few links interact with a subset of interactive species with several interactions (del-claro et al., 2018). biologically, nestedness describes the organization of the niche breadth of an interactive community, in which more nested networks tend to have the highest niche overlap (blüthgen, 2010). in general, the structure of these ant-plant networks mediated by efns is relatively stable in terms of connectance, network specialization and nestedness, at different spatial (dáttilo et al., 2013a) and temporal gradients (díaz-castelazo et al., 2013; lange et al., 2013; dáttilo et al., 2014a), or even after perturbations caused by tropical hurricanes (sánchezgalván et al., 2012). the structural stability of these networks is related to the facultative character of these ant-plant interactions, due the low fidelity of ant species when foraging on efn-bearing plants (schoereder et al., 2010). furthermore, the ant species constituting the core of these networks (i.e., ant species that interact with several partner species at high frequencies) are mainly competitively superior species, which gives the network core certain spatial and temporal stability (dáttilo et al., 2014b). more specialized ant-plant interactions, such as the interactions mediated by domatia, tend to present networks structurally stable in the context of fragmentation (passmore et al., 2012), since the plant presence determines the ant presence. on the other hand, it is possible that ant-plant networks mediated by efns, even being stable in different natural environments, have their structure altered by forest fragmentation, since the associated species can be differently affected by this process. so we wonder if the structure of these ant-plant networks mediated by efns responds to a disturbance gradient generated by habitat loss and forest fragmentation. it is known that the structure of these ant-plant networks, for example, is influenced by the vegetation structure (dáttilo & dyer, 2014), which tends to be quite modified due to fragmentation (ewers & banks-leite, 2013). dáttilo and dyer (2014) found that the diversity of ant-plant interactions may also be positively affected by canopy cover reduction, since some plant species secrete larger amounts of nectar in environments with higher light availability (szabo, 1980; kersch & fonseca, 2005). therefore, smaller, more irregular and less connected forest fragments, would present a greater number of interactions, with consequent increase in the overlap of pairwise interactions and nestedness. this is because forest fragments with a higher proportion of edges tend to present a reduction in canopy cover, with a consequent increase in light availability (ewers & banks-leite, 2013). sugiura (2010), in a study conducted on japanese islands, showed that the connectance and nestedness values of antplant interaction networks mediated by efns tend to increase with decreasing island size. these results, although based on subtropical forest islands (government of japan, 2010), that present a matrix impossible to be used by ants and plants, at least suggested investigations on the effect of rainforest fragmentation on these ant-plant networks. sociobiology 69(3): e8261 (september, 2022) 3 the aim of this study was therefore to investigate the effects of configurations of rainforest fragments on the structure of ant-plant interaction networks mediated by efns in the brazilian amazon. we based our research questions on hypotheses related to the configuration of forest remnants and the forest structure within the fragments, here represented by canopy cover. specifically, we postulated that smaller, less connected, and more irregular forest fragments with lower canopy cover would present: (i) smaller network size, since less-preserved forest fragments tend to support fewer number of species (ahuatzin et al., 2019); (ii) higher number of interactions, since extrafloral nectaries are more active in higher light environments (radhika et al., 2010); (iii) less network specialization, since dominant ant species do not need to monopolize the resource (extrafloral nectar) when it is available in large amounts (dáttilo et al., 2014b); (iv) lower diversity of interactions, due to the lower number of species expected for these fragments (dátillo & dyer, 2014); and (v) greater nestedness, due to the greater overlap of pairwise interactions arising from the greater number of interactions expected (dáttilo et al., 2013b). materials and methods study area this study involved ten forest fragments, ranging in size from approximately 5 to 3,000 ha (fig 1 and supplementary material 1), situated in the west of the state of acre, in the brazilian amazon. all forest fragments result from human activities since the 1980s. most of the primary type of vegetation was converted to croplands and pasture from this decade onwards (acre, 2011). the matrix types (i.e., pasture, urban, agriculture) surrounding the forest remnants are decisive for the biodiversity in the landscapes (prugh et al., 2008). the matrices will be more efficient from the point of view of functional connectivity if they are more similar in structure to the habitat patches (prevedello & vieira, 2010). for this reason, as an attempt to control the effect of matrix type, we selected only forest fragments surrounded by pasture. according to the köppen (1936) climate classification, the region is classified as monsoon climate (am), with an average rainfall of 1,450 mm per year (macêdo et al., 2013) and marked seasonality, with most rainfall between november and march (acre, 2006). the average annual temperature is 24ºc (inmet, 2016) with a daily thermal amplitude of around 9ºc (acre, 2006). the region varies between 110 and 270 m.a.s.l. (acre, 2006). the predominant vegetation type is open tropical rainforest (i.e., open ombrophilous forest) dominated by native species of bamboo and/or palm trees (acre, 2006). sampling ant-plant interactions we established a plot of 6,250 m2 (250 x 25 m) in each forest fragment, at a minimum distance of 100 meters from the edge, except for the smallest forest fragment, which was fig 1. map of the study area showing all forest fragments sampled. patricia n. miranda et al. – ant-plant networks in fragmented landscapes4 a plot beginning at approximately 20 meters from the edge. we decided not to establish the plots in regions under direct effects of edges, because in these regions the microclimatic conditions, and community composition, tend to be highly altered in any forest fragment (laurance et al., 2002), which would make it difficult to detect structure changes in ant-plant networks resulting from a disturbance gradient caused mainly due to fragmentation. we recorded all interactions between ants and efn-bearing plants with a height accessible to the collector (ranging from 0.5 to 3 m) between 09:00 am and 03:00 pm for each plot (dáttilo & dyer, 2014). all efn-bearing plants were observed for five minutes. ants were considered to be feeding on nectar when they were immobile, with mouthparts in contact with nectar secreting tissues during the observation period (falcão et al., 2016). when the interaction was confirmed, we collected all foraging ants and a plant sample for further identification (see below). plants in which we did not detect the presence of efns, but which had immobile ants with mouthparts in contact with plant tissues, were also collected for later confirmation of the presence of efns through stereoscope observations and literature review. ants were manually collected with the aid of an entomological umbrella (bestelmeyer et al., 2000) so as to record those that drop from the plant at the slightest sign of disturbance (dáttilo & dyer, 2014). considering the temporal variation in ant-plant networks mediated by efns (i.e., phenological variation) (lange et al., 2013; falcão et al., 2016), we sampled each plot twice (june 2016 and february 2017). ants were identified by species or morphospecies using identification keys (fernández, 2003; wilson, 2003; bolton et al., 2006) and by comparison with specimens deposited at the laboratório de mirmecologia of the centro de pesquisas do cacau, brazil (cpdc collection), where voucher specimens were deposited. plants were identified at species or morphospecies level using identification guides (ribeiro et al., 1999; pennington et al., 2004) and by comparison with specimens deposited at the herbário do parque zoobotânico (hpz) of the universidade federal do acre, brazil. voucher specimens of plants were deposited at the herbário (fuel) of the universidade estadual de londrina, brazil. canopy cover to measure the canopy cover in the plots where the antplant interactions were sampled, we established a 250 m long transect within each plot, located along the longitudinal direction of the plot, and maintained the same distance from the sides (12.5 m). in each transect, we installed ten subplots of 100 m2 (10 x 10 m) at intervals of 15 m. the first and last 100 subplots were installed 7.5 m from the beginning and end of transects, respectively. we estimated canopy cover using a spherical convex densiometer. the measurements were taken at the center and at the four edges of the 100 m2 subplots. the mean of these five measurements was used to calculate the average canopy cover per sampling point. we used the average of all sampling points to characterize each fragment for canopy cover. landscape descriptors we used landsat8 satellite images (path/row: 002/ 067) with a 30 m resolution, acquired on 07/23/2016, in the qgis software 2.18.10 (qgis development team, 2016) to describe the configuration of the forest fragments. the images were classified into two categories: forested areas (primary and secondary forests) and non-forested areas (water bodies, built areas, and pastures). we described the configuration of each forest fragment through metrics: forest fragment area (ha), edge irregularity index, and connectivity index. the forest fragment area was calculated based on its polygon shape. patton’s diversity index (di) (patton, 1975) was adopted as the edge irregularity index, which evaluates the forest fragment regularity, defined as di = p/2(√πa), where p = perimeter (m) and a = area (m2). to measure the isolation and fragmentation degree around the forest focal fragments, we used the index of connectivity named proximity (prox) (gustafson & parker, 1992), calculated by the following equation: in this equation, aijs represents the area (m 2) of fragment ijs within specified neighborhood (m) of the focal fragment; and hijs represents the distance (m) between fragment ijs and the focal fragment based on fragment edgeto-edge distance. the connectivity index is the inverse of the isolation and fragmentation degree (bender et al., 2003). we define buffers of 500 m around the focal fragment, established from the respective edge, to define the neighboring fragments considered for the calculation of the index. we used this buffer size for two main reasons: i) this distance explains the greater variation in the patterns of richness and diversity of ant species at the community level (spiesman & cumming, 2008); and ii) it is equivalent to the greater observed distance of an ant gyne’s flight (hölldobler & wilson, 1990). all forest fragment metrics were calculated using qgis software 2.18.10 (qgis development team, 2016). ant-plant interaction networks initially, we accumulated the data of the two samples per plot on different dates, in order to maximize the number of efn-bearing plants species in our samples, considering the annual variation in the extrafloral nectar production by different plant species (falcão et al., 2016). to verify whether our sampling was adequate to describe the ant-plant networks of each plot studied, we then generated accumulation curves with the number of ant and plant species and the number of distinct pairwise of interactions, as a function of the number of plants sampled (falcão et al., 2016). we used the nonparametric bootstrapping estimator with 1000 replicates (gotelli & colwell, 2001) for all accumulation curves, to estimate the expected number of plant and ant species and interactions for each network. ∑ = = n s ijs ijs h a prox 1 2 sociobiology 69(3): e8261 (september, 2022) 5 the ant-plant interaction patterns were examined using an ecological network approach. the data of each plot was organized in a quantitative matrix in which the elements (aij) represented the frequency (number of times) that plant species i interacted with ant species j inside the plot (bascompte et al., 2003). to evaluate the constancy of the central core of highly interacting species among the different forest fragments sampled, we categorized ant and plant species according dáttilo et al. (2013a): gc = (ki kmean)/σk, where ki = mean number of links for a given plant/ant species, kmean = mean number of links for all plant/ant species in the network, and σk = standard deviation of the number of links for plant/ant species. when gc ≥ 1, the species presents a large number of interactions in relation to other species of the same trophic level, as a species constituting the generalist core. when gc < 1, the species presents a lower number of interactions in relation to other species of the same trophic level, such as species constituting the periphery of networks. we used three network descriptors established from a quantitative matrix (number of interactions, network specialization, and diversity of interactions), and two calculated from a binary matrix (network size and nestedness), to describe the patterns of ant-plant interactions in the fragment. we adopted binary matrices for nestedness to facilitate the discussion of our results compared to other published studies, since this data category has been widely used to assess the structure of ant-plant networks mediated by efns (rico-gray et al., 2012; díaz-castelazo et al., 2013; lange et al., 2013; falcão et al., 2016; dáttilo et al., 2014a; díaz-castelazo et al., 2020). furthermore, the nestedness values calculated with binary and quantitative data have been shown to be highly correlated (corso et al., 2015; miranda et al., 2019). in frequency matrices, as mentioned above, the elements represent the number of times in which a plant species interacted with an ant species within the plot, whereas in binary matrices the elements (1 or 0) represent the presence or absence of an interaction between a plant and an ant species within the plot. these binary-weighted descriptors are the most commonly used in studies dealing with ant-plant networks, and they cover a wide range of possible structures with complementary biological meanings (del-claro et al., 2016). network size was calculated by multiplying the number of plant species by the number of ant species. the number of interactions represents the total interactions observed in the network, considering all ant and plant species. the diversity of interactions was calculated using an index (h’) based on the shannon’s diversity index, which ranges from zero to infinity (bersier et al., 2002). we estimated specialization using the h2’ index, which describes how species restrict their interactions from those randomly expected based on a partner’s availability (blüthgen et al., 2006). in this index, the low specialization of an ecological network represents values close to h2’ = 0, and total specialization is h2’ = 1. nestedness, which evaluates whether selective ant species only visit a subset of plant individuals visited by the generalist ant species (dáttilo et al., 2014c), was calculated using the nodf-metric (nestedness based on overlap and decreasing fill) (almeidaneto et al., 2008) in the aninhado software (guimarães & guimarães, 2006), using binary matrices. these index values range from 0 (non-nested) to 100 (perfectly nested). the network specialization and diversity of interaction were calculated using the bipartite package (dormann et al., 2017) in r 3.2.3 (r core team, 2016). data analysis the nodf significance was estimated using p-values based on the null model ii (1000 randomizations). in this null model, the probability of an interaction occurring is proportional to the observed number of interactions of both plant and ant species (bascompte et al., 2003). to evaluate the effect of rainforest fragmentation on ant-plant networks, we related each network descriptor (network size, number of interactions, network specialization h2’, diversity of interactions h’ and, nestedness nodf) to the explanatory metrics of the configuration of forest fragments (fragment area, edge irregularity index, and connectivity index), and to canopy cover. initially, we verified multicolinearity among all explanatory variables (fragment area, edge irregularity index, connectivity index, and canopy cover) using the variance inflation factor (vif) value (dormann et al., 2013). the vif indicates the degree to which each explanatory variable is explained by another exploratory variable in the model. vif values greater than 10 indicate high multicolinearity. as none of our exploratory variables showed collinearity between them, we included all four in the model selection. we used generalized linear models for all response variables (i.e., network descriptors). we used a negative binomial distribution for number of interactions and network size, due to an overdispersion of residues from our data, which occurs when the deviance of the response is greater than expected by the chosen distribution (hinde & demétrio, 1998). we used a gaussian distribution for network specialization h2’, diversity of interaction h’, and nestedness nodf. for all response variables, candidate models were classified using the corrected akaike information criterion for small samples (aicc) (akaike, 1974). for each response variable, 16 competing models were used to explain the patterns, including a null model representing the lack of effect. we adopted balanced model sets (i.e., all explanatory variables were present in the same number of models) without considering the interaction effects between them. subsequently, the corrected akaike information criterion for small samples was estimated, the δaicc (difference between the aicc of each model in relation to the best model) and the akaike weight (waic) (the probability of a given model being the best among a set of competing models) (johnson & omland, 2004). models with δaicc <2.0 and waic> 0.1 were considered equally plausible to explain the patterns observed patricia n. miranda et al. – ant-plant networks in fragmented landscapes6 (burnham & anderson, 2003). model selections were made according to akaike’s information criterion (aic) using the mumin package (bartoń, 2016) and the model diagnostics were run with the rt4bio package (reis jr. et al., 2013) in r 3.2.3 (r core team, 2016). results we observed 930 pairwise interactions between ants and efn-bearing plants on the ten plots studied. we recorded a total of 56 ant species, distributed in 19 genera and seven subfamilies (supplementary material 2). the subfamily myrmicinae comprised 48.2% of the total ant species (n = 27 ant species), followed by formicinae (26.8%, n = 15) and dolichoderinae (12.5%, n = 7). myrmicinae was also the subfamily that showed higher interaction frequency (64.6% of the total interactions, n = 601 interactions), followed by ectatomminae (13.6%, n = 126) and formicinae (8.9%, n = 83). we recorded a total of 148 plant species, distributed in 44 genera and 25 families, where liana was the plant habit with the highest number of species (60.1% of the plant species, n = 89 species) followed by trees (34.5%, n = 51) (supplementary material 3). the fabaceae family comprised 37.8% of the total plant species (n = 56 plant species), followed by bignoniaceae (25.7%, n = 38) and malpighiaceae (6.8%, n = 10). fabaceae was also the family that had a higher interaction frequency (41% of the total interaction, n = 381), followed by bignoniaceae (16.7%, n = 155) and malpighiaceae (14.0%, n = 130). the generalist core of ant species in the ant-plant networks tends to be more constant than the generalist core of plant species, between forest fragments. we observed that the generalist core of ant-plant networks ranged from one to five ant species, and from two to five plant species per forest fragment. seven ant species were present in the generalist cores, taking into account all ten ant-plant networks evaluated, and the most common species was crematogaster brasiliensis (present in the generalist core in 70% of the networks, n = 7 forest fragments), followed by crematogaster carinata (60%, n = 6) and ectatomma tuberculatum (50%, n = 5) (table 1). twenty-one plant species were present in the generalist cores of the ant-plant networks evaluated, and the most common species were bauhinia sp.1 (90%, n = 9) followed by polygonaceae sp.1 (50%, n = 5) and senegalia sp.2 (50%, n = 5) (table 1). generalist core plant species occurrence frequency generalist core ant species frequency bauhinia sp.1 90% (n = 9) crematogaster brasiliensis 70% (n = 7) polygonaceae sp1 50% (n = 5) crematogaster carinata 60% (n = 6) senegalia sp.2 50% (n = 5) ectatomma tuberculatum 50% (n = 5) fridericia sp.8 40% (n = 4) crematogaster limata 40% (n = 4) hirtella racemosa 40% (n = 4) dolichoderus attelaboides 10% (n = 1) banisteriopsis sp.2 40% (n = 4) wasmannia auropunctata 10% (n = 1) inga sp.5 40% (n = 4) ochetomyrmex semipolitus 10% (n = 1) palicourea sp.1 40% (n = 4) senegalia sp.3 30% (n = 3) fridericia sp.19 20% (n = 2) memora sp.2 20% (n = 2) zygia sp.1 20% (n = 2) fridericia sp.6 20% (n = 2) senegalia sp.8 20% (n = 2) inga punctata 20% (n = 2) aparisthmium cordatum 10% (n = 1) fridericia sp.2 10% (n = 1) inga sp.16 10% (n = 1) senegalia sp.4 10% (n = 1) bauhinia sp.4 10% (n = 1) senegalia sp.7 10% (n = 1) table 1. frequency of ant and plant species in generalist core of ant-plant networks sampled in 10 rainforest fragments located in the state of acre, brazilian amazon, between june 2016 and february 2017, n= number of fragments where the species was part of the core of the network. sociobiology 69(3): e8261 (september, 2022) 7 according to the species and interactions accumulation curves estimated, we collected an average (mean ± sd) of 84.30 ± 0.02% of the ant species (n = 18.60 ± 3.60 species of the 22.04 ± 4.04 species estimated) and 80.60 ± 0.01% of the plant species (n = 31.90 ± 4.53 species of the 39.61 ± 5.81 species estimated). we observed 76.23 ± 0.01% of the interactions between ants and efn-bearing plants (n = 63.70 ± 11.06 interactions of the 83.57 ± 14.44 interactions estimated), indicating that we had sampled enough ant and plant species and interactions to describe the ant-plant networks of each plot. we describe the values of the network descriptors (network-size, number of interactions, network specialization – h2’, diversity of interactions – h’, and nestedness – nodf) for each plot in supplementary material 4. eighty percent (n = 8 fragments) of the ant-plant networks presented a significant value for nodf, when compared to null models (supplementary material 4). we did not detect any effect of the explanatory variables on the network descriptors. for number of interaction, network size, network specialization and diversity of interaction, the null model, which indicates no detectable effects, was the best selected model (table 2). for nestedness, the edge irregularity index was the best explanatory variable, however the null model was also equally plausible to explain the observed pattern (table 2).the results of all candidate models for each ant-plant network descriptor are in supplementary material 5. table 2. summary of plausible models fitted to explain each ant-network descriptor in response to landscape structure metrics and forest structure (canopy cover) at 10 rainforest fragments located in the state of acre, brazilian amazon. δaicc, df, and waicc indicate the difference in corrected akaike values, degrees of freedom of the model, and akaike weights, respectively. the values in bold represent the best model selected for each of the network descriptors evaluated. response variable model δaicc d.f. waic slope symbol number of interactions null model 0.00 2 0.39 network size null model 0.00 2 0.59 network specialization null model 0.00 2 0.51 diversity of interaction null model 0.00 2 0.52 nodf edge irregularity index 0.00 3 0.44 null model 1.20 2 0.247 discussion in general, we observed, contrary to our hypotheses, that the structure of ant-plant interaction networks mediated by efns remained stable in forest fragments with different landscape configurations. none of the five network descriptors we used (i.e., number of interactions, network size, network specialization, diversity of interactions, and nestedness) were affected by the metrics of the configuration of fragments and vegetation structure. we also did not find evidence that canopy cover shapes the structure of ant-plant interaction networks, suggesting that the self-organization of the networks is independent of local characteristics of canopy openness in our area of study. the generalist core tended to be more constant among the sampled plots as regards ants rather than plants. of the seven ant species observed in the generalist cores, four (57%) were core species in more than 40% of the networks. between them, three are competitively superior due to their numerical dominance (c. brasiliensis, c. carinata and crematogaster limata) (parr, 2008; baccaro et al., 2012) and one due to aggressive displacement of competitors (e. tuberculatum) (hossaert-mckey et al., 2001; bächtold & alves-silva, 2013). this competitive nature is a feature of ant species of the generalist core (dáttilo et al., 2014b), and is possibly one of the main factors responsible for the relative constancy of ant species in the studied networks. in addition, most of the ant species of our generalist cores belonged to the functional group generalist myrmicinae (silvestre et al., 2003; silva & brandão, 2010), and are highly tolerant of habitat modification (hoffmann & andersen, 2003), which allows them to a competitive advantage, even in forest fragments with a high disturbance level. of the 21 plant species observed in the generalist cores, 12 (57%) were classified as in the central core of highly interacting species in less than 20% of the networks. this lowest constancy in the generalist core of plant species is possibly related to changes in plant species composition due to forest fragmentation, since the reduction in size tends to increase the mortality rate of trees, especially near the forest edge (laurance, 1991; ferreira & laurance, 1997), favoring fast-growing pioneer trees and liana growth (laurance et al., 2001b; laurance et al., 2006; santo-silva et al., 2016). the plant species turnover in south american tropical forests is also relatively high, with forests 60 km distant from each other (i.e., the maximum distance between our forest fragments), sharing only some 35% of species (condit et al., 2002). when evaluating the effects of forest fragmentation on the structure of ant-plant networks, we found a structural stability in the face of perturbation. no network descriptor was related to the configuration of forest fragments. this patricia n. miranda et al. – ant-plant networks in fragmented landscapes8 stability is possibly related to the low fidelity of ant species when foraging on efn-bearing plants (rico-gray et al.; 1998, schoereder et al., 2010), and to the constancy of ant species in the generalist cores observed in our study, as previously discussed in the literature (dáttilo et al., 2013a; lange et al., 2013; dáttilo et al., 2014b). in fact, the ant-plant networks mediated by efns are shaped by a few generalist species, which are essential for the maintenance of network stability (díaz-castelazo et al., 2010; mello et al., 2011). dáttilo et al. (2013a) observed that the structure of ant-plant networks mediated by efns remains stable throughout space, as does the generalist core of species. other studies have also detected the structural stability of these ant-plant networks mediated by efns over time (i.e., 20 years) (díaz-castelazo et al., 2013), between day and night periods (dáttilo et al., 2014a), and after tropical hurricanes (sánchez-galván et al., 2012). more recently, fagundes et al. (2018) also observed the limited effects of fire disturbances on the structure of ant-plant interaction networks mediated by efns. corro et al. (2019) observed landscape effects on network specialization and on the diversity of interaction of ant-plant networks, but in this case, the authors used a conceptual framework based on cooccurrence records of ant-plant associations to build ant-plant co-occurrence networks. no network descriptors were related to vegetation structure (i.e., canopy cover). according to our hypotheses, we expected an increase in the number of interactions and, consequently, in nestedness, due to the canopy cover reduction. dáttilo et al. (2013b) found no effects of canopy cover on the nestedness in ant-plant networks mediated by efns, in a study also carried out in the brazilian amazon. our results corroborate the discussion presented by dáttilo et al. (2013b), that perhaps the presence of ants in efn-bearing plant species is not primarily determined by the amount of nectar, but rather by the nectar quality (i.e., concentration and composition) (blüthgen & fiedler, 2004; lach, 2005). the amount of nectar is positively influenced by the light availability (szabo, 1980; kersch & fonseca, 2005), but also by the water availability (carroll et al., 2001), which is an abundant factor in tropical rainforests, not directly related to canopy cover. on the other hand, nectar quality, directly related to soil nutrients (burkle & irwin, 2009), may influence the behavior and preference of animal species when recruiting this food resource (parachnowitsch et al., 2019), being therefore an interesting factor to be investigated in structuring of these ant-plant networks. the structural stability of these ant-plant networks mediated by efns evidences the resistance or resilience of these interactions in environments with different levels of disturbance. even small, more irregular, and isolated forest fragments were able to maintain the dynamics of these antplant interactions, and therefore should be included in the definition of conservation strategies. nonetheless, special attention should be directed to the low constancy of plant species in the generalist core. these small changes should be understood as a warning, mainly because the functionality of this protective mutualism (i.e., food secretions in exchange for protection against herbivory) in these areas remains unknown. moreover, habitat loss and fragmentation often reduce gene flow and genetic diversity in plant populations (browne & karubian, 2018), and possibly in ant populations, as gene flow in this group is mainly maintained by male dispersion (jaffé et al., 2009). this possible reduction in the gene flow, in turn, could negatively affect the long term maintenance of some populations and, consequently, the structure of antplant networks mediated by efns. it is important to mention that these results were found in open tropical forests (acre, 2006), and possibly greater effects of forest fragmentation and forest structure can be expected for dense tropical forests. furthermore, we limited our sampling to three meters high, and we recognize the potentially serious implications of this sampling limitation on our results. we thus suggest that further studies evaluate how ant-plant interactions are structured along a vertical stratification gradient within the forest and to test their vulnerability to fragmentation and habitat loss. in summary, we have provided some important insights into the effects of rainforest fragmentation on the structure of ant-plant interactions mediated by efns. in an interaction networks context, the ant species of the generalist core seem to be little affected by forest fragmentation compared to the plant species of the generalist core. our results regarding the network descriptors indicate that the structure of ant-plant networks mediated by efns remains relatively stable in the face of the forest fragmentation, possibly due to the constancy of ant species in the generalist cores. in short, this study makes a valuable contribution to biodiversity and conservation, mainly because we show the vulnerability and robustness of tropical species-rich habitats to forest fragmentation. acknowledgements the authors thank christian frenopoulo for the english review and dr. milton cezar ribeiro for his important suggestions for the statistical analyses used in this work. the first author thanks the capes research grant received during the period in which she stayed at the instituto de ecologia, a.c. 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(continuation) camponotus sexguttatus (fabricius, 1793) 0 1 0 0 0 0 0 0 0 0 1 formicinae camponotus sp.1 0 0 0 0 0 0 0 0 2 1 3 gigantiops destructor (fabricius, 1804) 0 0 0 1 0 0 0 0 0 1 2 nylanderia guatemalensis (forel, 1885) 0 0 0 0 0 1 0 0 0 0 1 myrmicinae cephalotes atratus (linnaeus, 1758) 0 1 0 0 0 0 0 0 0 0 1 cephalotes marginatus (fabricius, 1804) 0 0 0 0 0 0 1 0 0 0 1 cephalotes pinelii (guérin-méneville, 1844) 1 0 0 0 0 0 0 0 0 0 1 cephalotus opacus santschi, 1920 0 0 0 0 2 0 0 0 0 0 2 crematogaster brasiliensis mayr, 1878 10 2 0 24 27 23 0 69 19 8 182 crematogaster carinata mayr, 1862 0 5 26 40 16 12 69 1 0 9 178 crematogaster curvispinosa mayr, 1862 0 0 0 0 0 2 0 0 0 0 2 crematogaster erecta mayr, 1866 0 0 0 0 0 1 0 0 0 0 1 crematogaster flavosensitiva longino, 2003 0 3 0 0 5 2 0 0 0 0 10 crematogaster limata fr. smith, 1858 14 8 1 0 2 16 1 8 12 16 78 crematogaster longispina emery, 1890 2 0 0 1 2 0 9 0 0 0 14 crematogaster nigropilosa mayr, 1870 1 0 4 1 1 0 0 1 0 0 8 crematogaster sp.1 0 0 0 0 2 0 0 0 0 0 2 megalomyrmex balzani emery, 1894 0 0 0 0 2 0 0 0 0 0 2 ochetomyrmex semipolitus mayr, 1878 2 7 2 2 9 1 7 1 2 5 38 pheidole (gr. fallax) sp.1 1 0 0 1 0 0 0 0 1 0 3 pheidole (gr. fallax) sp.2 2 0 0 0 0 0 0 0 0 0 2 pheidole (gr. fallax) sp.3 0 0 1 0 2 0 1 1 0 0 5 pheidole (gr. fallax) sp.4 0 1 0 0 1 0 1 2 0 1 6 pheidole (gr. flavens) sp.1 0 1 0 0 0 0 0 0 0 0 1 pheidole radoszkowskii mayr, 1884 0 0 2 1 1 0 1 0 1 3 9 solenopsis globularia (smith, 1858) 0 0 1 0 0 0 1 0 0 0 2 solenopsis sp.1 0 6 2 0 0 0 0 1 0 0 9 solenopsis sp.3 1 3 0 0 0 0 1 0 4 0 9 wasmannia auropunctata (roger, 1863) 2 5 3 2 8 3 3 0 3 5 34 ponerinae neoponera carinulata (roger, 1861) 0 0 0 0 2 0 0 0 0 0 2 neoponera unidentada mayr, 1862 2 0 1 0 2 2 0 0 0 0 7 odontomachus haematodus (linnaeus, 1758) 1 0 0 0 0 7 2 0 0 0 10 odontomachus hastatus (fabricius, 1804) 0 0 0 0 0 0 0 0 0 1 1 pseudomyrmecinae pseudomyrmex oculatus (smith, 1855) 0 0 0 0 0 2 0 1 1 0 4 pseudomyrmex tenuis (fabricius, 1804) 3 1 0 2 0 1 3 2 1 1 14 subfamily ant species frequency total f7 f8 f4 f3 f10 f6 f1 f2 f9 f5 patricia n. miranda et al. – ant-plant networks in fragmented landscapes16 appendix s3. plant species present in the ant-plant networks sampled in 10 rainforest fragments located in the state of acre, brazilian amazon, between june 2016 and february 2017. habitat: t = tree; l = liana; h = herb; ? = undefined. family plant species frequency total habitat f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 anonaceae anonaceae sp.1 0 0 0 0 1 0 0 0 0 0 1 t araceae philodendron sp.1 0 0 0 0 0 0 0 0 0 1 1 l fridericia sp.1 0 0 0 0 0 0 1 0 0 0 1 l fridericia sp.2 0 8 7 0 0 0 3 0 2 0 20 l fridericia sp.3 3 0 0 0 0 0 0 4 0 0 7 l fridericia sp.4 0 0 0 0 1 0 0 0 0 1 2 l fridericia sp.5 0 0 1 0 2 1 0 4 0 2 10 l fridericia sp.6 0 0 0 0 0 0 0 5 0 0 5 l fridericia sp.7 0 0 0 0 0 0 1 0 3 0 4 l fridericia sp.8 10 6 0 1 1 11 0 1 0 0 30 l fridericia sp.9 1 0 0 0 1 0 0 0 2 0 4 l fridericia sp.10 1 0 0 0 0 0 0 0 0 0 1 l fridericia sp.11 2 0 0 0 0 1 0 0 0 0 3 l fridericia sp.12 1 0 0 0 0 0 0 0 2 0 3 l fridericia sp.13 1 1 2 1 0 0 0 0 1 0 6 l fridericia sp.14 2 0 0 0 0 0 0 0 0 0 2 l fridericia sp.15 1 0 0 0 0 1 0 0 0 0 2 l fridericia sp.16 0 3 0 0 0 0 0 0 1 0 4 l fridericia sp.17 0 0 0 0 0 1 0 0 1 0 2 l fridericia sp.18 0 0 0 0 0 1 0 0 0 0 1 l bignoniaceae fridericia sp.19 0 0 1 0 7 0 0 0 0 0 8 l fridericia sp.20 0 0 2 0 0 0 0 0 0 0 2 l fridericia sp.21 0 1 0 0 0 0 0 2 0 0 3 l fridericia sp.22 0 0 0 1 0 0 0 0 0 0 1 l fridericia sp.23 0 0 0 0 1 0 0 0 0 0 1 l fridericia sp.24 0 0 0 0 2 0 0 0 0 0 2 l fridericia sp.25 0 0 0 0 0 0 0 1 0 0 1 l fridericia sp.26 0 0 1 0 0 0 0 3 0 0 4 l fridericia sp.27 0 0 0 0 0 0 0 1 0 0 1 l fridericia sp.28 0 0 1 0 0 0 0 0 0 0 1 l fridericia sp.29 0 0 0 0 1 0 0 0 0 0 1 l fridericia sp.30 0 0 0 0 0 0 0 0 0 1 1 l fridericia sp.31 0 0 0 0 1 0 0 0 2 0 3 l fridericia sp.32 0 0 0 0 0 0 0 2 1 0 3 l fridericia sp.33 0 1 0 0 0 0 0 0 0 0 1 l fridericia sp.34 0 0 0 0 1 0 0 0 0 0 1 l fridericia sp.35 0 1 0 0 3 0 0 1 1 0 6 l memora sp.1 1 0 0 0 0 0 0 0 0 0 1 l memora sp.2 0 1 0 0 5 0 0 0 0 0 6 l memora sp.3 0 0 0 0 0 1 0 0 0 0 1 l sociobiology 69(3): e8261 (september, 2022) 17 chrysobalanaceae hirtella racemosa lam. 0 0 0 0 9 1 0 6 0 0 16 t hirtella sp.1 0 0 0 0 0 0 0 1 0 1 2 t hirtella sp.2 0 0 0 0 0 0 0 0 1 0 1 t combretaceae buchenavia sp.1 1 0 0 0 0 0 0 0 0 0 1 t convolvulaceae ipomoea philomega (vell.) house 0 0 0 0 0 0 0 0 1 0 1 l ipomoea regnellii meisn. 0 0 0 0 1 0 0 0 0 0 1 l costaceae costus scaber ruiz & pav. 0 1 0 1 0 0 1 1 0 2 6 h cucurbitaceae cucurbitaceae sp.1 0 0 1 0 0 0 0 0 0 0 1 l gurania sp.1 0 0 1 0 0 1 0 0 0 0 2 l euphorbiaceae acalypha sp.1 0 0 0 0 0 0 0 0 4 0 4 ? aparisthmium cordatum (a. juss.) baill. 0 10 0 0 0 0 0 0 0 0 10 t dalechampia sp.1 1 0 0 0 0 0 0 0 0 0 1 l omphalea diandra l. 0 0 0 0 0 2 2 0 0 2 6 l senegalia sp.1 3 0 1 1 3 0 3 1 1 3 16 l senegalia sp.2 7 4 2 1 0 6 9 3 8 1 41 l senegalia sp.3 3 7 0 1 2 1 1 0 6 0 21 l senegalia sp.4 1 0 1 11 0 0 3 0 0 0 16 l senegalia sp.5 0 0 0 0 0 0 0 0 1 0 1 l senegalia sp.6 0 0 0 0 1 0 0 0 0 0 1 l senegalia sp.7 2 0 0 4 0 0 1 0 0 0 7 l senegaliasp.8 1 0 1 3 1 0 3 1 5 0 15 l senegalia sp.9 0 1 0 0 0 0 0 0 0 0 1 l bauhinia sp.1 33 15 16 0 0 9 17 2 7 0 99 l bauhinia sp.2 0 0 0 1 0 0 0 0 0 0 1 l bauhinia sp.3 0 0 0 0 0 1 0 0 0 0 1 l bauhinia sp.4 0 0 0 3 0 0 0 0 0 0 3 l fabaceae bauhinia sp.5 0 0 0 0 0 0 0 1 0 0 1 l bauhinia sp.6 0 0 0 0 0 2 0 0 0 0 2 l centrosema sp.1 0 0 0 0 0 0 3 0 0 0 3 l centrosema sp.2 0 0 0 0 0 0 2 0 0 0 2 l centrosema sp.3 0 0 2 0 0 0 0 0 0 0 2 l erythrina sp.1 0 0 1 2 0 0 0 0 0 0 3 t fabaceae sp.1 0 1 3 0 0 0 0 0 0 0 4 ? inga acreana harms 0 0 0 0 0 1 0 0 0 1 2 t inga alba (sw.) willd. 0 1 3 1 1 0 0 0 0 0 6 t inga calantha ducke 0 0 0 0 1 0 0 0 0 1 2 t inga capitata desv. 1 1 0 0 0 2 0 0 0 0 4 t inga chartacea poepp. 0 0 1 0 0 0 0 0 0 2 3 t inga densiflora benth. 0 1 2 0 0 2 0 1 0 0 6 t appendix s3. plant species present in the ant-plant networks sampled in 10 rainforest fragments located in the state of acre, brazilian amazon, between june 2016 and february 2017. habitat: t = tree; l = liana; h = herb; ? = undefined. (continuation) family plant species frequency total habitat f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 patricia n. miranda et al. – ant-plant networks in fragmented landscapes18 inga edulis mart. 2 0 1 1 0 0 1 0 1 0 6 t inga heterophylla willd. 1 0 0 0 0 0 0 0 0 0 1 t inga lateriflora miq. 1 0 2 1 2 0 3 0 1 0 10 t inga laurina (sw.) willd. 6 2 2 4 0 0 0 0 0 4 18 t inga microcoma harms 0 0 0 0 1 0 0 0 0 0 1 t inga punctata willd. 0 0 0 1 0 0 0 0 0 5 6 t inga sertulifera dc. 0 0 2 0 3 3 0 0 0 0 8 t inga suaveolens ducke 1 0 0 0 0 0 0 0 0 0 1 t inga tenuistipula ducke 0 0 0 0 2 0 1 1 0 3 7 t inga sp.1 0 0 0 0 0 1 0 0 0 0 1 t inga sp.2 0 0 0 0 0 0 0 0 2 0 2 t inga sp.3 0 0 0 0 0 2 1 0 0 0 3 t inga sp.4 1 0 0 1 0 0 0 1 0 2 5 t inga sp.5 0 0 0 0 1 1 1 6 0 6 15 t fabaceae inga sp.6 0 0 0 0 0 0 0 0 0 1 1 t inga sp.7 0 0 2 0 0 1 0 0 0 0 3 t inga sp.9 1 0 0 0 0 0 0 0 2 0 3 t inga sp.10 0 0 0 1 0 0 0 1 0 0 2 t inga sp.12 0 0 0 2 0 0 0 0 0 0 2 t inga sp.13 0 0 0 2 0 0 0 0 0 0 2 t inga sp.14 0 0 1 0 0 0 1 0 1 0 3 t inga sp.15 0 1 0 0 0 0 0 0 0 0 1 t inga sp.16 0 0 3 0 0 0 0 0 0 0 3 t inga sp.18 0 0 0 0 0 1 0 0 0 0 1 t inga sp.20 0 0 0 0 0 1 0 0 0 0 1 t senna sp.1 0 0 0 0 1 0 0 0 0 0 1 t zygia sp.1 0 0 1 0 4 0 0 0 1 0 6 t zygia sp.2 0 3 0 0 0 0 0 0 0 0 3 t zygia sp.3 0 1 0 0 0 0 0 0 0 0 1 t zygia sp.4 0 1 0 0 0 0 0 0 0 0 1 t lecythidaceae gustavia augusta l. 0 0 0 0 2 0 0 0 2 0 4 t lecythidaceae sp.1 0 0 0 0 3 0 0 0 0 0 3 t loganiaceae strychnos panurensis sprague & sandwith 0 0 0 1 1 0 0 0 0 0 2 l malpighiaceae banisteriopsis sp.1 0 0 0 0 0 4 0 0 0 0 4 l banisteriopsis sp.2 2 0 0 3 0 44 3 2 0 41 95 l banisteriopsis sp.3 1 1 0 0 0 1 1 0 0 0 4 l banisteriopsis sp.4 0 1 1 0 0 0 0 0 0 0 2 l banisteriopsis sp.5 0 9 1 0 0 0 0 0 0 0 10 l banisteriopsis sp.6 0 0 0 0 3 0 0 0 0 0 3 l banisteriopsis sp.7 0 0 0 0 1 0 0 0 0 0 1 l banisteriopsis sp.8 0 0 0 0 0 0 0 0 0 1 1 l heteropterys sp1 0 1 0 0 2 0 0 0 0 0 3 l tetrapterys sp.1 1 0 0 0 0 0 2 0 0 4 7 ? appendix s3. plant species... (continuation) family plant species frequency total habitat f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 sociobiology 69(3): e8261 (september, 2022) 19 malvaceae byttneria benensis britton 0 0 0 0 0 5 0 0 1 0 6 l menispermaceae abuta sp.1 0 0 0 0 0 0 0 0 1 0 1 l abuta sp.2 0 0 0 0 0 0 0 1 0 0 1 l ochinaceae ouratea sp.2 0 0 0 0 3 1 0 1 0 0 5 t olacaceae heisteria sp.1 0 0 0 2 3 0 0 1 0 1 7 ? passifloraceae dilkea sp.1 0 0 1 0 0 0 0 0 0 0 1 l passiflora coccinea aublet. 1 0 2 0 0 0 0 0 0 0 3 l passiflora sp.1 1 0 0 0 0 0 0 0 0 0 1 l passiflora sp.2 0 0 0 0 0 0 1 0 0 0 1 l passiflora sp.3 0 0 0 0 0 1 0 0 0 0 1 l passiflora sp.4 0 0 0 0 0 0 0 1 0 0 1 l polygonaceae polygonaceae sp.1 26 38 31 0 2 1 2 8 11 0 119 ? polygonaceae sp.2 0 0 0 0 0 0 1 0 0 0 1 t rhamnaceae gouania frangulifolia radlk. 0 0 0 0 0 0 0 0 1 0 1 l rubiaceae tocoyena sp.1 0 0 1 0 0 0 0 0 0 0 1 t palicourea sp.1 0 3 2 0 1 5 2 6 2 5 26 t sapindaceae paullinia sp.1 0 0 0 0 0 0 2 1 0 0 3 l paullinia sp.2 3 0 0 1 1 0 0 0 0 1 6 l paullinia sp.3 3 0 0 0 0 0 0 0 0 0 3 l paullinia sp.5 1 0 0 0 0 0 0 0 0 0 1 l paullinia sp.6 0 0 0 0 0 0 0 0 1 0 1 l serjania clematidea triana & plach. 0 0 0 0 0 0 0 0 2 2 4 l solanaceae solanum sp.1 0 0 1 0 0 0 0 0 0 0 1 ? vitaceae cissus sp.1 0 0 0 0 0 0 0 1 0 0 1 l volchysiaceae qualea grandiflora mart. 0 0 2 0 0 0 2 0 0 0 4 t volchysia sp.1 0 0 0 0 0 1 0 0 0 0 1 t appendix s3. plant species... (continuation) family plant species frequency total habitat f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 appendix s4. network size, number of interaction, network specialization, diversity of interaction and nestedness (nodf) of the ant-plant networks sampled in 10 rainforest fragments located in the state of acre, brazilian amazon, between june 2016 and february 2017. fragment network size number of interactions network specialization diversity of interaction nodf 1 648 128 0.15 3.81 36.70* 2 580 125 0.19 3.70 37.32* 3 518 106 0.17 3.75 32.29* 4 312 52 0.26 3.54 22.78 5 798 81 0.15 4.24 16.40 6 816 118 0.15 4.21 27.57* 7 609 74 0.21 3.96 19.05* 8 576 72 0.20 4.04 20.15* 9 561 79 0.27 3.97 26.52* 10 525 94 0.21 3.78 29.60* mean ± sd 594.2 ± 136.28 82.90 ± 33.08 0.20 ± 0.04 3.90 ± 0.23 26.84 ± 7.27 *significant descriptor value (95% ci). patricia n. miranda et al. – ant-plant networks in fragmented landscapes20 appendix s5. model selection analysis to explain the five ant-network descriptors in response to landscape structure metrics and forest structure at 10 rainforest fragments located in the state of acre, brazilian amazon. the δaicc, df, and waicc indicate the difference in corrected akaike values, degrees of freedom of the model, and akaike weights, respectively. ed = edge irregularity index; co = connectivity index; ar = fragment area; cc = canopy cover. the values in bold represent the models selected for each of the network descriptors evaluated. response variable model δaicc d.f. waic slope symbol number of interaction null model 0 2 0.396 ed 0.75 3 0.273 co 2.91 3 0.093 cc 3.02 3 0.087 ar 3.04 3 0.087 ed+co 6.45 4 0.016 -ed+co ed+cc 6.55 4 0.015 -ed-cc ar+ed 6.72 4 0.014 -ar-ed cc+co 8.12 4 0.007 -cc-co ar+cc 8.33 4 0.006 -ar-cc ar+co 8.51 4 0.006 -ar-co ed+cc+co 15.31 5 0.000 -ed-cc+co ar+ed+co 15.4 5 0.000 -ar-ed+co ar+ed+cc 15.54 5 0.000 -ar-ed-cc ar+cc+co 16.91 5 0.000 -ar-cc-co ar+ed+cc+co 30.28 6 0.000 -ar-ed-cc+co network size null model 0 2 0.597 cc 2.75 3 0.151 co 4.2 3 0.073 + ar 4.27 3 0.070 ed 4.28 3 0.070 + cc+co 8.33 4 0.009 -cc+co ed+cc 8.42 4 0.009 ed-cc ar+cc 8.66 4 0.008 ar-cc ed+co 10.07 4 0.004 -ed+co ar+co 10.12 4 0.004 -ar+co ar+ed 10.26 4 0.004 -ar+ed ed+cc+co 17.32 5 0.000 ed-cc+co ar+cc+co 17.33 5 0.000 ar-cc+co ar+ed+cc 17.42 5 0.000 ar+ed-cc ar+ed+co 19.03 5 0.000 -ar-ed+co ar+ed+cc+co 32.32 6 0.000 -ar+ed-cc+co null model 0 2 0.514 ar 1.63 3 0.228 + ed 3.88 3 0.074 + co 3.91 3 0.073 + network specialization cc 4.16 3 0.064 + ar+ed 7.59 4 0.012 ar-ed ar+co 7.59 4 0.012 ar-co ar+cc 7.63 4 0.011 ar-cc sociobiology 69(3): e8261 (september, 2022) 21 appendix s5. model selection analysis to explain the five ant-network descriptors in response to landscape structure metrics and forest structure at 10 rainforest fragments located in the state of acre, brazilian amazon. (continuation) ed+co 9.85 4 0.004 ed+co cc+co 9.87 4 0.004 cc+co ed+cc 9.87 4 0.004 ed+cc network specialization ar+ed+co 16.59 5 0.000 ar-ed-co ar+ed+cc 16.59 5 0.000 ar-ed+cc ar+cc+co 16.59 5 0.000 ar+cc-co ed+cc+co 18.83 5 0.000 ed+cc+co ar+ed+cc+co 31.58 6 0.000 ar-ed+cc-co diversity of interaction null model 0.00 2 0.521 ed 2.81 3 0.127 + co 2.9 3 0.122 + ar 3.59 3 0.086 + cc 3.73 3 0.081 ed+cc 6.32 4 0.022 ed-cc cc+co 7.42 4 0.013 -cc+co ar+cc 8.54 4 0.007 ar-cc ed+co 8.69 4 0.007 ed+co ar+ed 8.74 4 0.007 ar+ed ar+co 8.79 4 0.006 ar+co ar+ed+cc 15.19 5 0.000 ar+ed-cc ed+cc+co 15.29 5 0.000 ed-cc+co ar+cc+co 16.15 5 0.000 ar-cc+co ar+ed+co 17.63 5 0.000 ar+ed+co ar+ed+cc+co 30.17 6 0.000 ar+ed-cc+co nodf ed 0.00 3 0.449 null model 1.20 2 0.247 co 2.38 3 0.137 ar 4.60 3 0.045 cc 5.09 3 0.035 ed+cc 5.84 4 0.024 -ed+cc ar+ed 5.96 4 0.023 ar-ed ed+co 6.00 4 0.022 -ed+co cc+co 8.34 4 0.007 -cc-co ar+co 8.35 4 0.007 -ar-co ar+cc 10.42 4 0.002 -ar-cc ar+ed+co 14.80 5 0.000 ar-ed+co ed+cc+co 14.83 5 0.000 -ed+cc+co ar+ed+co 14.96 5 0.000 ar-ed+co ar+cc+co 17.32 5 0.000 -ar-cc-co ar+ed+cc+co 29.80 6 0.000 ar-ed+cc+co response variable model δaicc d.f. waic slope symbol _goback open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i3.8265sociobiology 69(3): e8265 (september, 2022) introduction many areas in brazil lack the most basic biodiversity studies, particularly in the case of invertebrates. for any effective conservation proposal to be established, the knowledge of species occurring in a particular area must be enhanced (melo et al., 2012). in this context, such a taxonomic baseline is only achieved by conducting biodiversity surveys. the state of tocantins is one of these undersampled areas, a large gap in the brazilian territory regarding insect diversity. only a few taxonomic works or faunal surveys have been developed, mostly over the last decade and including only a few taxa: coleoptera (fernandes et al., 2020; polizei & fernandes, 2020; polizei et al., 2022), diptera (figueiro et al., 2012, 2014; krolow & vieira, 2016; lima et al. 2015; lima et al., 2018; da silva et al., 2022), embioptera (krolow & valadares, 2016; salvatierra, 2020), ephemeroptera (boldrini abstract tocantins state has high biodiversity and a high degree of endemism, nevertheless, there are no studies on the diversity of social wasps. this study introduces a survey of social wasps actively collected using entomological nets and malaise trap in addition to different light traps in sixteen sites in the amazon and cerrado biomes in tocantins state, brazil. we sampled a total of 1,013 social wasps distributed in 57 species of 13 genera. fifty of these species and nine genera represent new distribution records for the state. some species are not commonly found in collections and lists of species, and protopolybia picteti (de saussure, 1854) is newly recorded for brazil. such an increase of 714% may indicate that polistinae richness is probably higher in the studied regions and that the state of tocantins may well contain several additional (yet unrecorded) social wasp species. more comprehensive studies should be conducted to enhance the knowledge of wasp species in this state, contributing to our understanding on the biodiversity in northern brazil. sociobiology an international journal on social insects lucas b furtado1, alexandre somavilla2, tiago k krolow1 article history edited by marcel g. hermes, ufla, brazil received 31 may 2022 initial acceptance 30 june 2022 final acceptance 11 august 2022 publication date 12 september 2022 keywords amazon biome, cerrado biome, new records, northern brazil, survey. corresponding author alexandre somavilla coordenação de biodiversidade instituto nacional de pesquisas da amazônia av. andré araújo, 2936 cep: 69067-375, manaus-am, brasil. e-mail: alexandresomavilla@gmail.com & krolow, 2017; orlando et al., 2019, 2021), ept (andrade et al., 2020b), hymenoptera (andrade et al., 2021); megaloptera (andrade et al., 2020a), neuroptera (machado & krolow, 2016; alvim et al., 2019), plecoptera (rippel et al., 2019a, b), and psocoptera (lienhard et al., 2010). such a scenario is also the case for social wasps (vespidae: polistinae) until 2009, there were no records of social wasps for the state. the first recorded species, synoeca surinama (linnaeus, 1767), is derived from a series of specimens examined in a phylogenetic proposal (andena et al., 2009a). souza et al. (2020) recorded six other species for tocantins: apoica arborea saussure, 1854, apoica pallida olivier, 1792, apoica strigata richards, 1978, apoica thoracica du buysson, 1906, mischocyttarus flavicornis zikán, 1935, and polybia striata fabricius, 1787. in summary, only seven species and four genera of social wasps are recorded for tocantins. 1 programa de pós-graduação em biodiversidade, ecologia e conservação, universidade federal do tocantins, porto nacional, tocantins, brazil 2 coordenação de biodiversidade, instituto nacional de pesquisas da amazônia, manaus, amazonas, brazil research article wasps a checklist of social wasps (vespidae: polistinae) from tocantins state, brazil mailto:alexandresomavilla@gmail.com lucas b furtado, alexandre somavilla, tiago k krolow – social wasps (vespidae: polistinae) of tocantins state, brazil2 brazil shelters the greatest richness of polistinae worldwide, with 381 species recorded so far (somavilla et al., 2021a; hermes et al., 2022). considering the richness and distribution of social wasps in other bordering states, like pará, maranhão, mato grosso, and goiás (santos et al., 2020; somavilla et al., 2021a), these data are notoriously underestimated and show the lack of information about the richness of this taxon in tocantins. thereby, our goal was to perform a pioneer systematic work and present an update about the geographic records of the social wasp fauna in tocantins. material and methods the state of tocantins is located in the northern region of brazil and represents a transition area between the amazon and cerrado domains. although the other states in the region have a large portion of their territories inserted in the amazon domain, around 90% of the area of tocantins is in the cerrado domain (ibge, 2004). the climate of the state is classified as aw according to the köppen system. it has a warm and dry winter from may to september, and a hot and rainy summer from october to april (alvares et al., 2014). the mean annual rainfall in the state varies between 1,200 and 2,100 mm, and the mean annual temperature is between 26 °c and 29 °c (miranda & bognola, 1999). social wasps were sampled from 16 locations in tocantins (figure 1), two of them in the amazon region (araguaína and pium) and fourteen in the cerrado (arraias, babaçulândia, brejinho de nazaré, dianópolis, lagoa da confusão, mateiros, miracema, monte do carmo, natividade, palmas, pedro afonso, porto alegre do tocantins, porto nacional, and wanderlândia). these wasps were collected using different sampling methods, mainly malaise flight interception traps, lighted sheet, lighted tray, and pennsylvania light trap, in addition to the active collection with an entomological net. the material is derived from collections carried out over the last decade but without standardization of methods or sampling effort. all specimens/vouchers examined are deposited in the entomology collection of the federal university of tocantins (ceuft). we identified specimens to genus-level based on the keys proposed by richards (1978) as well as somavilla and carpenter (2021b), in addition to further specific identification keys, in the case ofthe species agelaia (cooper, 2000), angiopolybia (barroso et al., 2022), apoica (pickett & wenzel, 2007), brachygastra (andena & carpenter, 2012), epipona (andena et al., 2009a), mischocyttarus (silveira, 2008), polistes (somavilla et al., 2021c), pseudopolybia (somavilla et al., 2021d), protopolybia (junior et al., 2018; 2020a; 2020b), and synoeca (andena et al., 2009b), for example. dr. orlando tobias silveira (museu paraense emílio goeldi) was responsible for verifying the mischocyttarus species/morphospecies identifications. fig 1. map with 16 cities in the tocantins state: the localities in green have phytophysiognomies predominantly from the amazon biome, while the localities in yellow have phytophysiognomies from the cerrado biome. sociobiology 69(3): e8265 (september, 2022) 3 results we provide an updated list of polistinae species for tocantins, which shows an increase of 714% in the species richness of social wasps for the state, from seven to 57 species (table 1). regarding tribes, epiponini was the most diverse with 41 species and 11 genera, followed by mischocyttarini, with ten species, and polistini, with four species. thirteen genera of social wasps were recorded, corresponding to approximately 62% of the genera likely to be found in brazil (21 genera). the other genera (angiopolybia araujo, 1946, asteloeca raw, 1985, charterginus fox, 1898, chartergus lepeletier, 1836, clypearia de saussure, 1854, leipomeles möbius, 1856, nectarinella bequaert, 1938, and protonectarina ducke, 1910) were not collected in the state of tocantins so far, although they are likely to be registered in further collection efforts due to their confirmed distribution in other northern states. the genera polybia (16 species), mischocyttarus (ten species), apoica (six species), and agelaia (five species) presented the largest number of species. together, these genera are responsible for 67% of the species recorded herein. agelaia pallipes (olivier, 1792) was the most representative of all species collected, with 282 specimens representing about 28% of all specimens examined. the species was followed by apoica pallida (olivier, 1792) and apoica thoracica du buysson, 1906, with 110 and 107 specimens collected, respectively. in contrast, most of the species were represented by a few individuals, like the 17 species with a single specimen collected, and other seven species with two individuals. among the known species for tocantins, only two were not recorded in this survey, apoica strigata richards, 1978 and mischocyttarus flavicornis zikán, 1935 (listed in the table without the number of specimens collected (-)). the species in this survey include 50 new records for the state (table1), and protopolybia picteti (de saussure, 1854) is a new record for brazil. taxon / collected methods active malaise illuminated tray light trap pennsylvania total polistini polistes canadensis (linnaeus, 1798)* 1 1 1 3 polistes cinerascens de saussure, 1854* 1 1 polistes occipitalis ducke, 1904* 1 1 polistes versicolor (olivier, 1792)* 1 1 2 mischocyttarini mischocyttarus cerberus ducke, 1898* 1 1 mischocyttarus cf. tricolor richards, 1945* 3 3 mischocyttarus flavicornis zikán, 1935 mischocyttarus gr. injucundus (de saussure, 1854)* 1 1 mischocyttarus gr. surinamensis de saussure, 1854* 2 2 mischocyttarus labiatus (fabricius, 1804)* 10 10 mischocyttarus rotundicollis (cameron, 1912)* 7 7 mischocyttarus sp.1* 1 1 mischocyttarus sp.2* 1 1 mischocyttarus (artifex) sp.1* 1 1 mischocyttarus (megacanthopus) sp.1* 1 1 epiponini agelaia cajennensis (fabricius, 1798)* 3 33 5 41 agelaia flavippenis (ducke, 1905)* 1 1 agelaia fulvofasciata (de geer, 1773)* 2 2 agelaia pallipes (olivier, 1792)* 9 270 3 282 agelaia testacea (fabricius, 1804)* 6 9 1 16 apoica arborea de saussure, 1854 1 39 40 apoica flavissima van der vecht, 1972* 3 2 29 11 2 47 apoica gelida van der vecht, 1972* 2 1 21 7 5 36 table 1. species of social wasps (vespidae: polistinae) from the tocantins state. relationship between the wasp species recorded herein and the number of specimens collected in each sampling method. fields with “-” mean that the particular species was not collected by the respective collection method. species marked with “*” represent new records for the state of thetocantins. the species marked with “**” represents a new record for brazil. lucas b furtado, alexandre somavilla, tiago k krolow – social wasps (vespidae: polistinae) of tocantins state, brazil4 the malaise trap was the most successful collection method, both in collected specimens (516) and in recorded species (40), followed by the lighted tray (317 specimens and 22 species), lighted sheet (85 and 20), and pennsylvania light trap (46 and eight). despite not being successful in collecting many specimens, the active search obtained a total of 49 individuals, represented by 14 species. discussion agelaia is a relatively abundant genus in surveys, possibly making agelaia pallipes the most abundant species. agelaia usually forms large colonies with millions of individuals (zucchi et al., 1995). consequently, it is more likely to be captured, probably also due to the habits of its generalist and epiponini apoica pallens (fabricius, 1804)* 4 2 6 apoica pallida (olivier, 1792) 81 15 14 110 apoica strigata richards, 1978 apoica thoracicadu buysson, 1906 2 64 21 20 107 brachygastra mouleae olivier, 1978* 2 2 5 9 chartergellus communis richards, 1978* 6 6 epipona tatua (cuvier, 1797)* 2 2 metapolybia docilis richards, 1978* 8 6 14 parachartergus fraternus (gribodo, 1892)* 1 1 parachartergus lenkoi richards, 1978* 1 1 parachartergus smithii (de saussure, 1854)* 1 1 polybia chrysothotrax (lichtenstein, 1796)* 38 38 polybia dimidiata (olivier, 1792)* 1 1 2 polybia emaciata lucas, 1879* 34 4 1 39 polybia erythrothorax richards, 1978* 15 1 16 polybia ignobilis (haliday, 1836)* 1 12 1 14 polybia jurinei de saussure, 1854* 1 1 polybia liliacea (fabricius, 1804)* 7 2 2 1 12 polybia micans ducke, 1904* 1 10 11 polybia occidentalis (olivier, 1792)* 2 30 8 2 42 polybia platycephala richards, 1951* 5 2 7 polybia rejecta (fabricius, 1798)* 12 1 1 14 polybia ruficeps schrottky, 1902* 5 4 1 10 polybia scroballis richards, 1970* 1 1 polybia sericea (olivier, 1792)* 6 6 polybia singularis ducke, 1905* 2 2 polybia striata (fabricius, 1787) 2 2 protopolybia chartegoides (gribodo, 1892)* 1 1 protopolybia picteti (de saussure, 1854)** 1 1 protopolybia sedula (de saussure, 1854)* 5 2 7 pseudopolybia compressa (de saussure, 1854)* 6 1 7 pseudopolybia vespiceps de saussure, 1863* 3 3 synoecacyanea (fabricius, 1775)* 1 1 synoeca surinama (linnaeus, 1767) 4 14 1 19 total 49 516 317 85 46 1.013 table 1. species of social wasps (vespidae: polistinae) from the tocantins state. relationship between the wasp species recorded herein and the number of specimens collected in each sampling method. fields with “-” mean that the particular species was not collected by the respective collection method. species marked with “*” represent new records for the state of thetocantins. the species marked with “**” represents a new record for brazil. (continuation) taxon / collected methods active malaise illuminated tray light trap pennsylvania total sociobiology 69(3): e8265 (september, 2022) 5 opportunistic behavior concerning food and resource choices (somavilla et al., 2014). in contrast, apoica species have a nocturnal foraging behavior (pickett & wenzel, 2007), therefore, since more than one light trap method was used, a larger number of specimens were collected. in turn, most species were represented by very few specimens collected. despite the effort to identify all social wasps, a few specimens, subjected to a thorough characterization and examination by a specialist, could not be determined with certainty, probably representing new species (mischocyttarus sp.1 and mischocyttarus sp.2). other examined specimens belonging to the same genus were not identified at the species level, however, they were allocated to the respective subgenera or species groups. the lack of local studies on social wasps – this is the first comprehensive survey – provides very little information to attribute the wasp species collected in tocantins with the “endemic” or “threatened” conditions. however, most of the species have been registered for the first time in the state, therefore, their status should be regarded as insufficient information in terms of geographic distribution. they could also be assigned with the condition of rarity, therefore deserving special attention and further investigations. among the six states bordering tocantins, only piauí has a lower species richness than the one presented herein, with 21 species (santos et al., 2020). however, as seen in tocantins, until recently piauí has not been the target of taxonomic studies involving social wasps. in contrast, pará has one of the highest number of species of social wasps, with 190 species recorded (somavilla et al., 2021a), followed by mato grosso with 137 species (somavilla et al., 2021a). both states have undergone comprehensive systematic studies over recent decades (richards, 1978, silveira, 2002; silva & silveira, 2009). the richness of the other border states is relatively close to that of tocantins – bahia, with 86 species (santos et al., 2020), maranhão, with 77 (silva et al., 2011; santos et al., 2020), and goiás, with 74 (somavilla et al., 2021a). the states of maranhão and mato grosso have the closest phytophysiognomies to that found in tocantins, presenting fragments of cerrado and amazon within their territories and well-defined transition areas. therefore, it was expected that the number of species was closer. nonetheless, both states have been the object of studies involving the collection of social wasps, resulting in greater sampling efforts (barbosa et al., 2016). different methods are used in the sampling of social wasps; however, few studies have attempted to propose standardization of these methods or to establish comparable and adequate protocols to survey the fauna of a given site. it is important to use different methods and traps when sampling social wasp richness in an area, since different species generally have varied foraging behaviors (silveira, 2002; somavilla et al., 2014). active search with entomological nets is one of the best ways to collect polistines, but previous collections have not focused on these taxa (silveira, 2002; somavilla et al., 2014). however, it was important to use indirect methods like interception and light traps for collecting certain wasp groups; for example, most species of different genera are always collected using a malaise trap (somavilla et al., 2014), and the light trap is used for nocturnal foraging wasps (apoica) (pickett & wenzel, 2007). this study introduces 50 new occurrences of social wasp species in the state of tocantins. our findings increase the number of species recorded in the state to 57, however, some taxa deserve special attention since they could not be determined at a specific level. such an increase is significant, but it can be even greater by enhancing the sampling effort and carrying out further field expeditions in areas without surveys in the state. therefore, further comprehensive studies should be conducted to enlarge the knowledge of wasp species in tocantins. our results also highlight the importance of continuing taxonomic studies on these insects to expand our knowledge on their distribution and determine priority areas for environmental preservation, especially in transitional environments between the amazon and cerrado biomes. authors’ contribution lbf: conceptualization, methodology, identification, formal analysis, writing. as: conceptualization, identification, formal analysis, writing. tkk: supervision, conceptualization, methodology, formal analysis, writing. acknowledgments lfb would like to thank for the scholarship (process number 130800/2019-8). tkk would like to thank the conselho nacional de desenvolvimento científico e tecnológico for the research grant (cnpq, process number 310214/2021-1). as is supported by a cnpq-pci scholarship (process number 300740/2022-0). we would like to thank dr. orlando tobias silveira for mischocyttarus identification. references alvares, c.a., stape, j.l., sentelhas, p.c., gonçalves, j.l.m. & sparovek, g. 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(2020). ampliação da distribuição de espécies de vespas sociais neotropicais no brasil (hymenoptera: vespidae: polistinae). nature and conservation, 13: 1-6. zucchi, r., sakagami, s.f., noll, f.b., mechi, m.r., mateus, s., baio, m.v. & shima, s.n. (1995). agelaia vicina, a swarmfounding polistine with the largest colony size among wasps and bees (hymenoptera: vespidae). journal of the new york entomological society, 103: 129-137. _hlk110950780 _hlk104537565 open access journal: http://periodicos.uefs.br/index.php/sociobiology issn: 0361-6525 doi: 10.13102/sociobiology.v69i4.8268sociobiology 69(4): e8268 (december, 2022) termites are part of the most important fauna which contributes to the enhanced productivity in the tropical ecosystem (bourguignon et al., 2016). they are grouped into lower and higher termites based on the presence of flagellated protist symbionts in the hindgut of the former and absence in the latter. thus, the higher termites are included in the family termitidae while lower termites comprise the other families, which are dependent on cellulolytic flagellated protozoa (lo & eggleton, 2011). gregarines are common as parasites of invertebrates, especially arthropods (criado-fornelio et al., 2017). these unicellular organisms may reach a length of 10 mm (manwell, 1961) and belong to the phylum apicomplexa, class conoidasida and order eugregarinorida (adl et al., 2019). generally, the arthropod host becomes infected with these protozoa through ingestion of mature cysts, which abstract this is the first record of gregarine infection in the worker midgut of the neotropical termites ruptitermes pitan and ruptitermes reconditus. gregarines have already been described in the gut and hemocoel in workers of other termite species, and although these protozoa are not able to kill these insects, they may affect their fitness. in the present study, gregarines were only observed in the gut lumen or linked to the midgut wall of termite workers. histological sections of the worker midgut of ruptitermes spp. showed three phases of the gregarine life cycle with the same individual worker displayed up to 4 gamonts and 2 trophozoites. sociobiology an international journal on social insects vanelize janei, ana m. costa-leonardo article history edited by og desouza, ufv, brazil received 31 may 2022 initial acceptance 06 june 2022 final acceptance 27 september 2022 publication date 28 december 2022 keywords parasitism; midgut; ruptitermes pitan; ruptitermes reconditus. corresponding author ana maria costa-leonardo laboratório de cupins departamento de biologia geral e aplicada universidade estadual paulista – unesp av. 24a, nº 1515, cep: 13506-900 rio claro, são paulo, brasil. e-mail: ana.costa-leonardo@unesp.br liberate sporozoites that reach the hemocoel and target tissue (schimid-hempel, 1998). many researches showed a certain costs for hosts which harbor gregarines, for instance, in damselfly species, the presence of gregarines alters survival and egg production, while it increases susceptibility to a fungal pathogen in the cockroach blattella germanica (arcila & meunier, 2020; lopes & alves, 2005). these protozoan parasites affect the fitness of their hosts and can cause a delayed development, decreased survival and reduced reproduction (zuk, 1987; cordoba-aguilar, 2003). according to gigliolli et al. (2016), the lack of nutrients induced by parasitic gregarines cause the already mentioned effects to the host. however, besides parasitic relationships, gregarines also can develop mutualistic or neutral association with their hosts and the type of this interaction is highly species-specific (zuk, 1987; rueckert et al., 2019). laboratório de cupins, departamento de biologia geral e aplicada, instituto de biociências, universidade estadual paulista – unesp, rio claro-sp, brazil occurrence of gregarines (protozoa: apicomplexa) in the neotropical soldierless ruptitermes spp. (isoptera, termitidae, apicotermitinae) short note vanelize janei, ana m costa-leonardo – occurrence of gregarines (protozoa: apicomplexa)2 termites are social cockroaches and according to bouwma et al. (2005), the behavior of a social insect reflects a selection of parasites since society favors infection transmission among nestmates. however, termite societies have evolved mechanisms to limit or prevent disease agents within their colonies, making such observation unusual (chouvenc et al., 2011). previous records of termite gregarine infections were reported in the families hodotermitidae (uttangi & desai, 1962), rhinotermitidae (uttangi & desai, 1962; huger & lenz, 1976; kalavati & narasimhamurti, 1978; hall & hostettler, 1993; costa-leonardo et al., 2008), archotermopsidae (grassé, 1986) and termitidae (kalavati & narasimhamurti, 1978; kalavati & narasimhamurti, 1980; zhang et al., 2021). gregarines present a life cycle with an endogenous stage within the insect body and an exogenous stage in the external environment (fig 1). infections by gregarines occur when termites and other insects ingest the parasite oocysts (mature cysts), which initiate the infection via the digestive tract (logan et al., 2012). then, oocysts release sporozoites, which develop into large stages named trophozoites that are attached to the intestinal wall. later, when trophozoites mature and become gamonts, they are released from the gut wall and appear free in the gut lumen. the gamonts of opposite sex adhere to one another in a reproductive process called syzygy and form a gametocyst, which is a cyst that forms around gamont pairs. gametocysts are then expelled in the environment together with the termite feces (kolman et al., 2015). the termite genus ruptitermes is endemic in south america and includes 13 species and subspecies with several of them being litter-feeders (acioli & constantino, 2015; constantino, 2022). ruptitermes pitan and ruptitermes reconditus (apicotermitinae, termitidae) are soldierless species of termites which have subterranean nests and forage in the open on the surface litter (matews, 1977; acioli & constantino, 2015). the present study is the first record of gregarines in the neotropical genus ruptitermes, which is included in the poorly studied group neotropical apicotermitinae. foraging workers of ruptitermes reconditus (silvestri, 1901) and ruptitermes pitan acioli & constantino, 2015 were collected in different areas located at the são paulo state campus of university (unesp), rio claro, sp, brazil (22°23′s, 47°32′w). for histology, 8 abdomens from each termite species were fixed in faa (absolute alcohol, glacial acetic acid, 40% formaldehyde, in the proportion of 3:1:1) and dehydrated in increasing ethanol concentrations (70, 80, 90 and 95%). next, the samples were transferred to a leica historesin solution and stored for seven days in a refrigerator. the abdomens were embedded with historesin (leica) plus catalyzer for polymerization and sectioned (3μm thick) using a leica rm 2245 microtome. the sections were stained with toluidine blue/fuchsin and xylidine-ponceau and documented using a leica photomicroscope. the abdomen histological sections showed the occurrence of gregarines infecting the midgut in both species of ruptitermes (fig 1). although gregarines occupy a variety of niches inside their hosts, from extracellular cavities, as gut lumen and coelom, to epicellular and intracellular sites, these eukaryotes were not observed in the coelomic cavity or intracellularly in the gut of ruptitermes spp. workers. we found 4 workers of r. reconditus and two of r. pitan infected from 8 examined specimens of each species. gregarines have fig 1. schematic representation of the gregarine life cycle which infect workers of ruptitermes spp. workers (w) ingest oocysts, containing sporozoites which develop into large stages named trophozoites. the trophozoites will attach to the gut epithelial cells (ep) through a special structure known as epimerite. after, the trophozoites will detach from the epithelium and form free gamonts. these free gamonts mature and undergo syzygy, originating the gametocyst that will be released into the environment. sociobiology 69(4): e8268 (december, 2022) 3 already been described in the gut and hemocoel in workers of several termite species (grassé, 1982; costa-leonardo et al., 2008), and although these protozoa do not cause termite death, they may decrease their longevity (villanueva, 2004). studies with wasps of the species polybia occidentalis parasitized by gregarines indicated that infected individuals decrease their foraging rates and, consequently, compromise the productivity of the colony (bouwma et al., 2005). free gamonts were observed in the midgut lumen in both species of ruptitermes (figs 2a, 2b and 3) and trophozoites in the inner wall of the midgut in r. pitan (figs 2a and 2d). gamonts in syzygy were also observed in the midgut lumen in r. pitan (fig 2c). previous studies with gregarine infection also displayed some of the cycle phases of as gamonts, oocysts and trophozoites in the midgut of other termites (kalavati & narasimhamurti, 1978; 1980; hall & hostettler, 1993). additionally, the stages of oocysts and gametocysts have already been described in the hindgut of zootermopsis angusticollis and hemocoel of coptotermes gestroi (henry, 1933; costa-leonardo et al., 2008). free gamonts, gamonts in syzygy and gametocysts were observed only in the worker caste of the termitid macrotermes barneyi, being absent in soldiers and reproductives (zhang et al., 2021). although the stages of gametocyst and sporozoites were not observed in the present study, it was possible to detect up to 4 gamonts and 2 trophozoites in the same worker. the trophozoite was attached to the mesenteric epithelium through an epimerite, which is a characteristic elongated structure, observed in r. pitan (figs 2d and 2e). ruptitermes spp. feed on litter and r. reconditus workers store this food in subterranean nest galleries, a fact that should facilitate the ingestion of mature cysts and, consequently, increase the infection by gregarines. however, future studies on the ruptitermes genus will be needed to clarify this gregarine infection. fig 2. histological sections of the midgut of ruptitermes pitan worker stained with toluidine blue/fuchsin. a free gamonts (g) in the midgut lumen and trophozoite (t) attached to the mesenteric epithelium. b detail of the free gamont (g) in longitudinal and transversal section. c gamonts in syzygy (ga). d trophozoite connected to the epithelium (ep). e detail of the epimerite (arrow) which is a elongated structure used by the trophozoite (t) for connection to the in epithelium (ep). n = nucleus acknowledgments the authors thank to cnpq (conselho nacional de desenvolvimento científico e tecnológico) for financial support on their termite research. they also are grateful to iago bueno da silva and varlei josé janei for valuable help with the schematic diagram. authors’ contributions vj: investigation, visualization, writing-original draft; 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